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Primary Industries and Energy Research and Development Act - Grape and Wine Research and Development Corporation and Grape and Wine Research and Development Corporation Selection Committee - Report - 1994-95

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Grape and Wine Research afid Development Corporation : ■ ■ ■ ' - ' V / ' :: ‘ / · ' r


Chairman's Letter to Minister 2

Chairman's Report 3

The Corporation's Mission 4

How the Corporation Operates 4

How Funds were Allocated 6

How Funds are Received 6

Objectives of the Corporation 7

R & D Priorities 7

1994 - 1995 Overview of Achievements & R & D Highlights 9

Appointment of Chairman 12

The Corporation's Selection Committee 13

The Corporation's - Powers 13

- Funding and Accountability 14

- Membership and Staff 15

Financial Statements 17

Research and Development Program 1994/95 29

Annual Reports on R & D Projects 1994/95 32

Planned Research and Development Program 1995/96 109

Organisation Codes 112


20 October 1995

Grape and Wine Research and Development Corporation

W ine Industry House 555 The Parade Magill SA 5072

Telephone (08) 364 2688 Facsimile (08) 364 3315 Mobile 018 842 700

Senator the Honourable Bob Collins Minister for Primary Industries and Energy Parliament House Canberra ACT 2600

Dear Minister

I have pleasure in presenting the Annual Report of the Grape and Wine Research and Development Corporation for the year ended 30 June 1995, prepared in accordance with Section 28 of the Primary Industries and Energy Research and Development Act 1989.

Yours sincerely

John S Keniry Chairman

A dvancing the q u a lity and com petitiveness of the A ustralian w in e industry


The year under review was, in many ways, an eventful one for the grape and wine industry in Australia. It was also a year in which the Corporation, in its fourth year, continued to make significant and demonstrable progress against its objectives. This progress is reflected throughout the report, but some developments are worthy of particular mention.

A new expanded Board was appointed to the Corporation in September 1994. This Board, comprising both re-appointed and newly appointed members, has focussed increased attention on developing contacts with industry personnel at all levels, aimed at facilitating wider

input into research directions and broader understanding by industry and research providers of the Corporation's objectives. A Viticultural Priorities Reference Group has been established to focus specifically on research needs in viticulture.

Resources have been allocated for a major new initiative, being undertaken jointly with the CRC for Viticulture, that w ill improve technology transfer and extension within the viticultural sector.

The drought that has impacted on much of Eastern Australia over the past few years made its impact felt quite severely on the 1995 vintage and demonstrated the need, in many grapegrowing areas, for more sophisticated approaches to the

monitoring of soil moisture and the provision and management of irrigation. In Australia, water for agriculture w ill almost always be a limited resource, and its optimum use, both commercially and environmentally, w ill require both economic and scientific inputs. To the extent that the science

is relatively well developed, the decreased vintage in many areas is clearly an indication of the benefits to be achieved by the industry from improved extension and uptake of the industry's research. This is particularly so when one considers the very large investments now under way in expanding the area under winegrapes.

Through the year the Corporation made two submissions to the Committee of Inquiry into the Winegrape and W ine Industry in Australia. In these submissions, the Corporation stressed the need for

long term industry planning as a basis, inter alia, for relevant objective setting by the Corporation, and the need for input from the R & D sector into the development of a realistic plan. The Corporation also acknowledged that the Inquiry, in considering

John 5 Keniry, Chairman

proposals for changes in Statutory organisational arrangements should consider any such changes in the context of benefits to the industry in terms of

cost efficiencies and enhance progress towards R & D objectives. Finally, during the year Australia's food legislation, as it relates to wine, was changed to bring it more into line with international practices. An important outcome of this and related changes w ill be simplified access for Australian wine to export markets, and vice versa. These changes simply underline the on-going need to retain and enhance the competitive strengths on which the

industry's success has been based. To this end, the Corporation plans to maintain the emphasis in its research on productivity and sustainability to deliver a clean, quality product.

John S Keniry Chairman


'To improve the production efficiency, the

competitiveness in domestic and international

markets and the profitability o f the Australian

grape and wine industry, by managing and

funding a research and development program to

reduce production costs and improve product

quality and purity'.

Background The Crape and Wine Research and Development Corporation was established as a statutory authority on 2 July 1991, under provisions of the Primary Industries and Energy Research and Development Act 1989.

The Corporation was established in order to achieve greater independence, flexibility, responsiveness and accountability to the grape and wine industries, and the Minister for Primary Industries and Energy, with respect to identification of R & D needs and priorities, and through making more effective use of resources, increasing returns to industry.

The Corporation provides a mechanism for achieving joint investment by industry and government in an R & D portfolio which recognises the complementary benefits of achieving public good outcomes and industry improvement.

Australia's grape and wine industries are valuable contributors to the domestic economy, and are of increasing significance in the export sector.

The industry comprises over 5000 independent grapegrowers and more than 800 wineries, ranging in size from very small to a few very large operations. Generally, even in larger operations, there is inadequate capacity to internally fund more than the relatively straightforward problem solving R & D. Difficult or protracted problems


and strategic R & D is more effectively undertaken by contracted R & D providers. This is funded by all eligible members of the grape and wine industries, via a statutory levy on tonnage grown and delivered to wineries, (the Grape Research

Levy), and on the tonnage converted to wine, (the W ine Research levy). Upon collection of these funds by the Levies Management Unit of the Department of Primary

Industries and Energy, the funds are transferred to the Corporation for investment in appropriate R & D in the grape and wine sectors. Funds are allocated to selected projects and

programs as identified in the Corporation's Annual Operational Plan and the five year R & D Plan which have been developed after close and intensive consultation. As a statutory body, the Corporation utilises its flexibility to ensure

interactive, professional responsiveness to industry problems and priorities, and is committed to ensuring projects are outcome oriented w ithin this framework. Accountability to industry and the

Parliament and Minister is of prime importance.

Interaction with R & D Contractors Potential R & D contractors are invited, by direct public advertisement, to submit potential R & D projects which address Corporation priorities. The Corporation may also commission work in areas where a priority exists but where no satisfactory

proposals have been received. The Corporation also convenes review and planning workshops to allow joint industry-provider interaction and discussion on high priority issues, and the facilitation of more effective project development.

Projects are contracted on an annual term, with tentative budget commitment for a 3 year period in most instances. Because levy income has the potential to vary significantly year to year, longer term contracts are avoided.

Most projects are awarded to bodies such as the Australian W ine Research Institute, the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Universities, State

Departments of Agriculture, and Commonwealth Bureaux such as Australian Bureau of Statistics (ABS), and Australian Bureau of Agricultural and Resource Economics (ABARE).

Contracted R & D providers are required to submit regular, six-monthly financial and progress reports. Review of progress is then based against initial project objectives, performance indicators

and milestones, and any required corrective action then applied. The Corporation is placing increasing emphasis on ensuring contractors effectively communicate

progressive and end of project results and recommendations to both industry and the Corporation, in a planned effort to improve uptake of the results of R & D.

Interaction with Industry The Corporation recognises the need for close consultation with industry to ensure continuing

relevance of programs and to enable a regular review- of priorities. To this end the Corporation has undertaken an extensive series of consultations with regional groups and individuals including attendance and presentations at the Annual General meetings of the Winegrape Growers' Council of Australia Inc, and the Winemakers' Federation of Australia Inc. Other discussions and presentations involved the Hunter Valley Vineyards Association, the South Australian Grape Advisory Committee, the South Australian Vine Improvement Committee, the Ovens Valley Viticultural Association and the Australian Council of Viticulture. Submissions were made to the Inquiry into the Winegrape and Wine Industry in Australia.

The Corporation has met in Nagambie, central Victoria, in addition to other meetings in Adelaide, has visited the Waite Campus (Plant Science Centre, The Australian Wine Research Institute) and has met with the Board of the Cooperative

Research Centre for Viticulture (CRCV) in the past year. Review' and planning activity w'ith industry and

providers has covered the subjects of Pest and Disease, Spray Application and Vine Borers.


H o w funds are Received J g g


The Grape R & D Portfolio consisted of 24 continuing and 12 new projects approved at the 1994/95 Budget meeting of the Corporation. Funds expended for 1994/95 totalled $999,099. Two projects directed towards optimal soil management were deferred until 1995/96 with the intention of integrating these into one larger project.

Allocation of funds between different program areas is illustrated in Figure 1.

Wine Expenditure on wine R & D for 1994/95 totalled $2,293,438 assigned to 25 projects. Funds allocated to The Australian Wine Research Institute (AWRI) totalled $2,197,846 or 96% of the total allocation.

5upport was maintained for a joint CSIRO/AWRI Liaison Viticulturist, (AWR24) and for a major coordinated project on agrochemical residues which linked the AWRI with the Department of Agriculture Victoria and Primary Industries SA (DAV 92/4).

Projects AWR 24 and DAV 92/4 were jointly supported between the Grape R & D Account and the Wine R & D Account. Distribution of funds in the W ine R & D portfolio is shown in Figure 2.

The Levies Management Unit of the Department of Primary Industries and Energy collects levies from all eligible grapegrowers and winemakers. The mechanism for collection of levies is described under the section Funding and Accountability.

Following actual expenditure of funds, matching contributions are claimed from the Commonwealth Government, to the lim it of that expenditure, provided it does not exceed the industry levy contribution.

Further detail is shown in the 1994/95 Overview of Achievements and in Annual Reports on R & D Projects 1994/95 below.

Grape quality 21%

Statistics, economics 7% Tech transfer

Vine improvement 4%

Pest & disease management 29%

Production efficiency 20%

Resource protection 7% Wine chemistry


Statistics, economics



Figure 7 Distribution o f grape R & D funds Figure 2 Distribution o f wine R & D funds


The major objectives of the Corporation are

• through the support for appropriate research and development, increase the economic, environmental or social benefits to members of the grape and wine industry and to the community in general by improving the efficiency of production, processing, storage, transport and marketing of grapes and wine.

• to achieve through better management, the sustainable use of natural resources.

• to make more effective use of the resources and skills of the community in general and the scientific community in particular.

• to facilitate the dissemination, adoption and commercialisation of the results of research and development in relation to the grape and wine industry.

• to improve the impact of research and development and the accountability for expenditure upon research and development activities in relation to the grape and wine



• Crape Improvement - use of molecular biology for definitive identification of grape varieties and the creation of transgenic varieties by incorpor­ ation of genes to control pests and parasites;

improved adaptation to stress and greater potential for flavour expression. Conventional breeding to develop improved rootstocks and their evaluation

• Management o f Pests and Pathogens - rapid diagnostic tests for disease; epidemiological studies; development of low input integrated pest management (IPM); evaluation of bio pesticides and low cost non-toxic chemical compounds

• Crape Quality - reduction in chemical residues; maximising grape and wine flavour; objective measurement of quality; management control of grape composition and wine style

• Resource and Vineyard Management - use of modelling and systems analysis for resource water, nutrient and soil and vine management; to improve productivity efficiency and to ensure sustainability of the production system

• Economic Prediction and Assessment - research priority assessment; supply and demand; predictions; assessment of options for expansion of

Table 1 Distribution of R & D Investment funds between provider organisations

Organisation $ %

CSIRO 282,362 8.5

ABS, ABARE 82,059 2.5

AWRI 2,197,846 66.2

Departments of Agriculture/Primary Industry 504,902 15.2

Universities 108,011 3.3

CRCV 39,808 1.2

Other 95,910 3.0

TOTAL 3,310,898 99.9


grape production to meet expected increases in domestic and export demand; possible effect of global climate change and the measurement of

impact through benefit-cost analysis

• Technology Transfer - communication of advances in R & D; facilitate of adoption of results from R & D; evaluation of outcomes for industry impact from R & D; identification of new problems for R & D or literature review and extension program development

optimisation of malolactic fermentation; flavour and aroma control; control of the products of fermentation.

• Analysis o f Chemical Residues and Contaminants - development of methodology for the rapid and cost effective quality assurance related to the objective of minimal levels of agrochemical residues and other contaminants in wine as determined by the international regulations controlling w'ine imports.


• Production Efficiency - cost reduction combined with an increase in wine yield.

• Wine Chemistry - better understanding of wine chemistry; role of secondary metabolites; flavour and style development; role of wine proteins and haze control; phenolics; oak flavour; minimisation of additives and processing aids.

• Wine Microbiology - better understanding of w'ine microbiology and characterisation of wine yeast physiology and biochemistry; improvement in wine yeasts and bacterial strains using molecular biology techniques; improved reliability of inoculation and the control of ferments;

• Technology Transfer and Extension - resolution of industry technical problems; provision of analytical services; communication of progress in R and D; winemaking advice and consultation; literature review and development of extension programs; maintenance of and improvement of analytical methods; evaluation of winemaking additives and processing aids; maintenance of information on domestic and international food law'.

• Wine Quality - improving the quality (flavour, aroma, style) of wine through improved winemaking techniques, by the optimisation of the use of additives and processing aids, and by the avoidance of environmental and other contaminants.


The year 1994/95 saw continued refinement in the balance of the R & D portfolio, and, as the first triennium of the CWRDC's operation draws to a close, the completion of several important projects

initiated by the Corporation. In reporting achievements through 1994/95 and in planning for 1995/96 and beyond, the Corporation has been mindful of the expectations of both industry and government. Particular attention has been placed on integrating public good elements with industry objectives in R & D, as highlighted by the attention given to:

• optimising use and management of water resources • achieving integrated and biological control of pests • reducing agrochemical inputs and residues in

product • improving quality and competitiveness of the Australian product • maintaining Australian R & D at a leading level.

Viticultural Priorities Reference Croup As well as selecting and supporting individual projects, the Corporation bears an important responsibility for setting the overall direction of

industry research and ensuring its relevance and efficacy. After extended negotiations with the industry peak bodies, a Viticultural Priorities

Reference Group was established in order to provide more formal screening of preliminary project submissions to assess likely relevance, adoption and potential benefit:cost. This mechanism w ill, in future, provide a basis for reviewing the R & D program's balance and achievement against industry objectives.

Statistics collection and analysis The wine and grape industries rely critically on the timely and accurate gathering of statistical information to guide production, marketing and

investment decisions. Following a substantial analysis of industry statistics collection and interpretation conducted in 1993/94, the Corporation, jointly w ith the DPIE Agribusiness

Program supported a Viticultural Statistics Search Conference in late 1993/94. Several recommendations were made relating to the database, collection schedule, structure and

content of the survey, and linkages between ABS collection and publication of viticultural statistics and the ABARE supply and demand projections. Several substantial improvements have been achieved including:

• a substantially increased and refined respondent’s list supplied from industry sources • a simplified standard collection form for all states • an earlier schedule for survey collection and

analysis of the Viticultural collection, allowing earlier publication • refined transfer of data between ABS and ABARE • an earlier schedule and data processing refinements by ABARE, allowing earlier publication of its Supply and Demand Projections for Winegrapes

Validation by DNA typing The role of correct product description in labelling and packaging has been afforded significant support by completion of a project on DNA typing of grapevines. This allows definitive identification of the grapevine and its products from the nursery to the fermentation process, and thereby provides a basis for quality assurance throughout the winemaking process, from nursery through to the winery. International collaboration in the use of this technique for vine improvement and

identification studies has been led by Australian workers. A commercial service is to be offered to Australian industry by AWRI/CSIRO in early


Wafer resources The emerging focus on water resource limitations has been anticipated by the Corporation in earlier commitments to research on vine water

management. Several projects have demonstrated substantial capacity to achieve greater water use efficiency, making it possible to sustain productivity and improve quality with lesser water

input. It has been show in some regions that water input may be reduced by as much as 40% without adverse impact on productivity. Promising progress is being achieved in other water management studies which should enable consolidation of management strategies to achieve more efficient and sustainable water use and to cope with diverse soil conditions and problems such as salinity.


Pest and disease management Pests and diseases cause considerable production and marketing problems, including risks of crop loss and quality reduction. Their management involves consideration of input costs, adverse environmental effects and the prospects of agrochemical residues in product. The Corporation has maintained an effective integrated, whole- system approach to these issues, which is continuing to demonstrate substantial gains.

In the final year of a major project on biological activity and degradation of 20 pesticides, recommendations have been made to industry on both the pattern and rate of use for a number of products. Cooperative development of improved residue trials involving Departments of Agriculture, the agrochemical industry and the National

Registration Authority has been achieved and a process to collectively review- annual recommendations for product use has been adopted. The need for greater knowledge of agrochemical product application has been recognised and a project to provide a series of review and negotiation meetings with chemical suppliers has been approved for commissioning in

1995/96. The focus w ill be on defining parameters of effective coverage and persistence, essential for fully functional integrated pest management. Further refinement of biological control principles has been achieved in the control of mites and in the use of the biological caterpillar control agent Bacillus thuringiensis, enabling more effective pest management with less agrochemical

input. This work has been complemented by the consolidation of knowledge on the role of vine canopy configuration in disease development and management.

Substantial effort has been applied to communicating these developments in disease and pest management. A highlight was the publication in late 1994 of the book Crape Production Series Number 1, Diseases and Pests. Nearly 3000 copies have been sold at this point. Several seminars on disease and pest management have also been supported, including an industry training program on phylloxera detection and management.

Wine Wine R & D tends to have longer time frames and less specific increments in knowledge, however several achievements have been noted this year.

Waste-water recycling A PhD project addressing land disposal of winery waste-water has provided a basis for effective re­ use on vineyards. Continuing activity over the next two years w ill see a collaborative industry-EPA

initiative to develop a standard procedure for waste-water management, enabling vineyards to be irrigated by winery waste-water.

Yeast and bacteria typing Considerable progress has been achieved in applying molecular techniques to the identification and differentiation of strains of yeast, an important tool in fermentation research and the characterisation of commercial strains. Similar progress has been achieved in the characterisation of malolactic bacteria.

Flavour research As an important step in understanding flavour in grapes, juices and wines, an assay has been developed for the measurement of total glycosides. Major secondary metabolites of flavour and colour are typically bound up as glycosides, and a measurement of this component offers a means to define potential flavour and colour of grape products. Research is under way to validate the assay procedure and automate it. Indications are that the method may be a valuable tool in predicting the wine styles produced from different batches of grapes. The so called C-C (glycosyl- glucose) assay should also be a valuable tool in viticultural research, reducing the time taken to evaluate the impact of different viticultural treatments. This would result in the more effective and efficient utilisation of grape products, and of the resources applied to R & D.

Wine stability Refinement of wine stability tests has led to a better understanding and more effective management of wine deposits in bottle. A predictive test has been developed for pigmented deposits in red wines and for deposit formation in cold storage.

Cooperage Assessment of the role of oak cooperage on wine composition has defined the important role that toasting of the oak wood plays in flavour development, demonstrating that variation in the

intensity and depth of toasting accounts for much


Grape R & D Highlights

of the flavour variation seen in wines from the same source.

International regulatory affairs Professor T H Lee, Director of The Australian Wine Research Institute, was elected President of the OIV Expert Group 'Code International des

Pratiques Oenologiques' and a member of the OlV's 'Comite Scientifique et Technique', the role of which is to ensure consideration of scientific

and technical aspects in the development of international standards and protocols.

Support for The Australian Wine Research Institute As part of its commitment to technology transfer, the AWRI has been supported in the appointment of a Quality Liaison Manager and the development of an information kit on Total Quality Management to be trialled in vineyards and wineries in late

1995. The Institute has conducted an extension 'roadshow' in South Australia, Victoria and Queensland, undertaking 12 seminars and involving over 300 people. Advisory enquiries

have increased 14% over 1993/94 and 40% over 1992/93 reflecting heightened interest from industry, government bodies, students and the

general public. Similarly, utilisation of information services remains at ? ,’1" μ 1

Industry Development Objective: To achieve effective planning support for industry, based on objective, reliable and credible statistical data, and its interpretation, such that industry is able to schedule vineyard development and the associated processing,

storage and marketing investments to meet its market development objectives.

• Publication of the ABS Viticultural Statistics Collection • Publication of the ABARE Projections o f Wine Crape Production and Winery Intake to i 996-97 • Implementation of a coordinated approach to

collection and analysis of improved national viticultural statistics

Vine Improvement Objective: To achieve availability o f grapevine material which is capable o f meeting market and industry criteria for flavour, composition and style, but which is adapted to environmental stresses and cultural management demands.

• Completion of development activity to allow the commercial offering of a DNA typing service for grapevine identification • International collaboration to link modern DNA

based methods with classical ampelography as a basis for international variety reference collections

Soil and Water Management Objective: To develop efficient, sustainable systems o f soil and water management in viticulture.

• Demonstrated capacity to reduce water inputs, maintain productivity and quality and improve water use efficiency • Commitment of major companies to improved

water scheduling techniques

Vineyard Pest Management Objective: To achieve a low input integrated approach to, and systems for, pest and disease management, which results in minimal adverse environmental impact, and the highest degree of

customer and market confidence.


• Definition of canopy configuration influence on botrytis bunch rot and powdery mildew development, enabling canopy choice as a disease management strategy • Preliminary assessment of spray coverage on

different vine canopies as a preliminary activity in developing more rational criteria for spray effectiveness • Assessment of reasons for the poor performance

of the biological pesticide Bacillus thuringiensis (Bt); these include different strains of light brown apple moth, exhibiting different susceptibility, low efficacy of Bt on grape compared with other plants and poor timing and spray application • A strategy has been developed and promoted to

minimise the impact of an inter-season carry over of powdery mildew • Demonstration that, if effectively applied, a variety of oil products have good protectant

activity against powdery mildew • Development of a draft Code of Practice for Phylloxera control and management • Demonstrated ability to use a chemical spray in

a winter dormant treatment to control over wintering Phomopsis viticola • Publication of Grape Production Series 1 Diseases and Pests • Production of a video on biological control of


W in e R & D Highlights

Wine Fermentation

• Effective metabolism, in conditioned soil, established for carbon load in winery and distillery wastes; guidelines defined for use of wastewater in vineyard irrigation • Molecular techniques defined for distinguishing

strains of yeast and bacteria

Wine Chemistry

• Assay procedure developed for glycosyl-glucose as an indicator of potential winegrape flavour • Role of coopering heat treatment as a significant determinant in wine flavour • W ine stability tests developed for prediction of

pigmented lacquer deposits and for more effective assessment of cold stability

Appointment of Chairman The Minister for Primary Industries and Energy approved the appointment of Dr John Keniry; Pymble, New South Wales as Chairman of the Grape and Wine Research and Development Corporation for the period to June 1997.


Establishment On 20 April 1994, the Minister for Primary Industries and Energy re-appointed Mr Don Dyer

as the Presiding Member of the Grape and Wine Research and Development Corporation (GWRDC) Selection Committee for a further period of six months, in accordance with Section 122 of the Primary Industries and Energy Research and Development Act 1989.

At the time of his appointment as Presiding Member, the Minister asked Mr Dyer to form a Selection Committee for the purpose of selecting and nominating four persons for membership of the Corporation.

At the request of the Presiding Member, the Minister agreed in May to extend the deadline for nominations to 25 July 1994.

On 31 May 1994, the Minister appointed four members to the Selection Committee. The members appointed were nominated for the positions by the Corporation's representative industry bodies. The members appointed were:

Mr Perry Gunner, Gilberton, South Australia Mr Brian Croser, Piccadilly, South Australia Mr Guy Darling, Whitfield, Victoria Mr John Harvey, Willunga, South Australia

The Selection Process As well as advertising in the national press, the Selection Committee actively sought applicants

from industry organisations and from people with specific relevant qualifications to seek membership of the Board of the Corporation, for its second

three year term. The Selection Committee recommended to the Minister that six persons be appointed to the Board, compared with four appointments in the previous term. The Minister appointed the following members to the Board:

Mr Barry Avery; Robinvaie, Victoria Ms Dianne Davidson; Hahndort, South Australia Dr Diana Day; Oyster Bay, New South Wales M r Anthony Devitt; South Perth, Western Australia M r Philip Laffer; Rowland Flat, South Australia M r Geoff Weaver; Mitcham, South Australia

Costs Expenditure incurred by the Selection Committee for the year ending 30 June 1995 was $15,484 comprising:

Fees: Presiding Member $7796 Selection Committee; travel, advertising $7688

The Corporation has the power to

1 Apply for patents or otherwise manage intellectual property;

2 Set up its own committees;

3 Employ staff and set terms and conditions for their employment;

4 Charge for services and information;

5 Enter into agreements or joint ventures for R & D activities;

6 Act as trustee for money or property;

7 Accept gifts or bequests;

8 Acquire real or personal property; and

9 Form Companies

10 Borrow or raise money with the written approval of the Minister


The Corporation manages funds sourced from statutory levies collected from winegrape growers and winemakers, and from matching Commonwealth funds supplied after expenditure has been made by the Corporation.

Crape Research and Development Levy Grape producers contribute to the Grape and W ine Research Trust Fund by means of a levy on fresh and dried grapes and grape juice delivered to an establishment for processing, including the manufacture of a beverage or other product, canning or extraction of juice. Wineries which grow their own grapes are required to pay a levy.

Levy is not payable on grapes and juice delivered to an establishment which processes less than 20 tonnes (fresh grape equivalent) in a year. The levy system for grape research is regulated by the following legislation:

• Crape Research Levy Act 1986

• Crape Research Levy Regulations

• Primary Industries Levies and Charges Collection Act 1991

• Primary Industries Levies and Charges Collection Regulations

The operative rate of the grape research levy at the start of the year was 90 cents per tonne (fresh grape equivalent), with the maximum rate permitted under the legislation $2.00 per tonne. Negotiations with industry peak bodies saw the levy increased to $1.10 per tonne from and including the 1995 vintage, provided increased investment was placed in the area of technology transfer.

For the purpose of setting grape research levy rates the relevant industry organisations which may make recommendations to the Minister are the Winegrape Growers' Council of Australia Inc and the Winemakers' Federation of Australia.

Wine Research and Development Levy Winemakers' contributions to the Fund are derived from a specific research component of a levy on

fresh grapes, dried grapes and grape juice used in manufacture of wine. (The remaining component of this levy finances the Australian Wine and Brandy Corporation). Following legislative amendments effective from 1 January 1995 wineries using less than 10 tonnes (fresh grape

equivalent) for this purpose in a year are no longer exempted from levy payment. The relevant legislation for this levy system is:

• Wine Crapes Levy Act 7 979

• Wine Crapes Levy Regulation

• Primary Industries Levies and Charges Collection Act 199 7

• Primary Industries Levies and Charges Collection Regulations

The operative rate of the wine research component of the levy is $1.90 per tonne (fresh grape equivalent). The maximum currently permitted under the legislation is $2.00 per tonne.

The Winemakers' Federation of Australia (WFA) is the peak wine industry body and makes recommendations to the Minister concerning the operative rate of research component of the wine grapes levy.

Commonwealth Matching Funds Matching Commonwealth funding of grape and wine research is achieved by a contribution to the Fund equal to half the expenditure from the Fund, but is limited to no more than either the total of industry levy contributions or to 0.5% of the gross value of production.

Interest from the investment of balances in the Fund, sales of assets and produce and money collected as penalties imposed in regard to the collection of levy charges are also paid into the Fund. Provision exists for other income generated by the research projects, and any additional income other than levy charges, to be paid into the Fund.

Two separate sub-accounts are maintained in the Fund for grape and wine research and development programs, respectively.

Accountability The Corporation is directly accountable to the Minister of Primary Industries and Energy to whom


an R & D Plan and an Annual Operational Plan must be submitted for approval. An Annual Report must also be submitted to the Minister, and as

soon as practicable after that, be supplied to each of the Corporation's representative industry organisations, these being are the Winegrape Growers' Council of Australia Inc and the Winemakers' Federation of Australia Inc. The Chairperson or his nominee also reports to the

Annual General Meetings of these organisations.

The Act provides for the Minister for Primary Industries and Energy to appoint the Chairperson, a government director and no fewer than 4 nor more than 6 other directors, as determined by the Minister to be appropriate for the Corporation. The nominated directors are appointed from persons nominated by a Selection Committee.

The Selection Committee is required to nominate persons for appointment to R & D Corporations on the basis of expertise in one or more of the fields of:

• commodity production

• processing or marketing

• natural resource conservation or management

• science, technology and technology transfer

• environmental and ecological matters

• economics

• administration of R & D; and

• finance or business management

The Executive Director is appointed by the Corporation

Membership of the Corporation for 1 994/95 comprised:

Chair Dr John S Keniry Chairman, Ridley Corporation Gladesville .

New South Wales

Directors Mr Barry C Avery Grapegrower Bonyaricall Vineyards

Robinvale Victoria

Ms Dianne M Davidson Consultant Dianne Davidson Consulting Services Pty Ltd



The Corporation Board: (from left) Peter Hayes, Tony Devitt, Dianne Davidson, D r John Keniry, Geoff Weaver, Barry Avery, D r Diana Day, Phillip Fitch and Philip Laffer

Dr Diana G Day Policy Analyst NSW Department of Water Resources Parramatta New South Wales

Mr Tony C. Devitt Principal Research Officer Department of Agriculture South Perth

Western Australia

Mr Philip L. Laffer Company Winemaker Orlando Wyndham Wines Pty Ltd

Rowland Flat via Tanunda South Australia

Government Director Mr Phillip G. Fitch Principal Executive Officer, W ine and Brandy Section, Horticulture Branch, Crops Division, Department of Primary Industries and Energy Canberra

Executive Director M r Peter F Hayes W ine Industry House 555 The Parade Magi 1 1 SA 5072

Telephone (08) 364 2688 Facsimile (08)364 3315

Mr Geoff A Weaver Grapegrower/Winemaker Staff

Stafford Ridge Winery Mrs Elizabeth J Ward, Office Manager

Mitcham Adelaide



Independent Audit Report

To the M inister for Primary Industries and Energy

Scope I have audited the financial statements of the Crape and W ine Research and Development Corporation

for the year ended 30 June 1995. The financial statements comprise:

• Statement of Financial Position

• Statement of Revenues and Expenses

• Statement of Cash Flows

• Notes to and forming part of the Financial Statements, and

• Statement by Corporation members.

The members of the Corporation are responsible for the preparation and presentation of the financial

statements and the information contained therein. I have conducted an independent audit of the

financial statements in order to express an opinion on them to the Minister for Primary Industries and


The audit has been conducted in accordance with Australian National Audit Office Auditing

Standards, which incorporate the Australian Auditing Standards, to provide reasonable assurance as to

whether the financial statements are free of material misstatement. Audit procedures included

examination, on a test basis, of evidence supporting the amounts and other disclosures in the financial

statements, and the evaluation of accounting policies and significant accounting estimates. These

procedures have been undertaken to form an opinion whether, in all material respects, the financial

statements are presented fairly in accordance w ith Australian accounting concepts and standards and

statutory requirements so as to present a view which is consistent with my understanding of the

Corporation's financial position, the results of its operation and its cash flows.

The audit opinion expressed in this report has been formed on the above basis.

Audit Opinion In accordance w ith section 63H(2) of the A udit A ct 1901, I now report that the statements are in

agreement w ith the accounts and records of the Corporation, and in my opinion:

• the statements are based on proper accounts and records;

• the statements show fairly in accordance w ith Statements of Accounting Concepts and applicable

Accounting Standards the financial transactions and cash flows for the year ended 30 June 1995

and the state of affairs of the Corporation as at that date; • the receipt, expenditure and investment of moneys, and the acquisition and disposal of assets, by

the Corporation during the year have been in accordance with the Primary Industries and Energy

Research and Development Act 1989; and

• the statements are in accordance with the Guidelines for Financial Statements of Public Authorities

and Commercial Activities.

Trevor Burgess Executive Director For the Auditor-General

Canberra, 9 October 1995

Statement of Revenues and Expenses

For the Year Ended 30 June 1995 Note 1995 1994

OPERATING REVENUES Commonwealth Contributions 5





Industry Contributions 4 2,085,786 1,703,527

Investment Interest 69,296 27,736

Other 0 1,248

Refund of unexpended grants 91,849 (9,797)

Total Operating Revenues 3,997,841 3,573,714

OPERATING EXPENSES Payments to Research & Development Organisations

Commonwealth Organisations Universities / Colleges State Departments Other Organisations

293,845 97,512 512,872 2,427,339

204,423 129,140 659,128 2,194,402

Total Research & Development Expenditure 3,331,568 3,187,093

Administrative Expenditure 6 343,964 300,627

Total Operating Expenses 3,738,801 3,550,990

Surplus of operating revenues over expenses $ 259,040 $ 22,723


Accumulated surpluses at beginning of reporting period Total available for appropriation

834,894 1,093,934

812,171 834,894

Accumulated surpluses at end of reporting period 1,093,934 834,894

The Statement of Revenues and Expenses is to be used in conjunction with the notes to and forming part of the financial statements.


Statement of Financial Position

As at 30 June 1995

Note 1995 1994

($) ($)


Cash 7 1,360,736 824,239

Total Current Assets 1,360,736 824,239

NON CURRENT ASSETS Property, Plant and Equipment 9 18,021 19,193

Total Non Current Assets 18,021 19,193

Total Assets 1,378,757 843,432

CURRENT LIABILITIES Creditors and Borrowings Provisions

70 77

277,193 7,630

4,700 3,838

Total Current Liabilities 284,823 8,538

Total Liabilities 284,823 8,538

Net Assets 1,093,934 834,894

EQUITY Accumulated Surpluses 1,093,934 834,894

Total Equity 1,093,934 834,894

The Statement of Financial Position is to be read in conjunction with the notes to and forming part of the financial statements.


Statement of Cash Flows

For the year ended 30 june 1995

1995 1994

($) ($)


Commonwealth Contributions 1,975,381 1,851,000

Industry Contributions 2,085,786 1,703,527

Interest Received 69,293 27,736

Unexpended Grants 91,849 36,390

Other - 1,248

4,222,309 3,619,901


Payments to R&D Organisations (3,331,568) (3,187,093)

Administrative Expenditure (286,189) (291,641)

Levy Collection Fees (63,269) (63,270)

(3,681,026) (3,542,004)

Net cash provided or used by operating activities 541,283 77,897


Proceeds on sale of asset - 552


Payments for property, plant & equipment (4,787) (4,700)

Net cash provided or used by investing activities (4,787) (4,148)

Net increase or decrease in cash held 536,496 73,749

Cash at beginning of reporting period 824,240 750,491

Cash at end of reporting period 7&12 1,360,736 824,240

The Statement of Cash Flows is to be read in conjunction with the notes to and forming part of the financial statements.


Notes to and Forming Part of the Financial Statements

1. Statement of Accounting Policies

The following accounting policies have been adopted in the preparation of the accounts:

(a) The accounts have been prepared under the historical cost convention and accrual accounting has been adopted.

(b) The Corporation is subject to all forms of Commonwealth and State taxation except income tax.

(c) Assets are valued at cost less depreciation.

(d) Depreciation is calculated so as to write off the cost of fixed assets over their expected useful lives on a straight line basis.

(e) inventories are valued at the lower of cost or net realisable value.

(f) All known or doubtful debts are written off or provided for at balance date.

(g) The Corporation claims matching Commonwealth contributions. The contributions are equal to half the expenditure of the Corporation up to a lim it of either total industry levy contributions or 0.5% of the gross value of production. Due to the limitations on the amount of the matching contributions able to be claimed in any year, the Corporation recognises the contribution as a receivable and as revenue only on receipt of the industry levy by DPIE. Outstanding claims for matching contributions for which levies have yet to be collected are included in the notes to the accounts as a Contingent Asset - Receivable (contingent upon receipt of the industry levy).

2. Guidelines for Financial Statements Issued by Minister for Finance

The Grape and Wine Research and Development Corporation is required to comply with the Guidelines for Financial Statements of Commonwealth Authorities issued by the Minister for Finance which incorporate Australian Accounting Concepts and Standards and

other madatory professional reporting requirements.

3. Industry Segment

The Grape and Wine Research and Development Corporation operates predominantly in the grape and wine industry principally to enable appropriate research and development. The Corporation operates predominantly in one geographical area, being Australia.


4. Industry Contributions

Industry contributions consist of the following:

Grape Research and Development Levy - a levy imposed by the Grape Research Levy Act 1986 in respect of fresh and dried grapes and grape juice and is collected and paid to the Grape and Wine Research and Development Corporation by the Department of Primary Industries and Energy.

W ine Research and Development Levy - a levy imposed by the Wine Grapes Levy Act 1979 in respect of fresh and dried grapes and grape juice used in the manufacture of wine. The research component is collected and paid to the Grape and W ine Research and Development Corporation by the Department of Primary Industries and Energy.

5. Commonwealth Contributions

Matching Commonwealth funding of grape and wine research is achieved by a contribution to the Corporation equal to half the expenditure from the Corporation, but is limited to no more than either the total of industry levy contributions or 0.5 percent of the gross value of production.

6. Administrative Expenditure

For the Year Ended 30 June 1995

Accounting Auditor's Remuneration* Advertising Bank Charges Cleaning Committee Selection Fees Computer Expenses Consulting Fees

Depreciation Electricity & Pleating Fringe Benefits Tax General Expenses Insurance Legal Fees Loss on sale of Assets Motor Vehicle Expenses Meetings

Planning & reporting Postage Promotional expenses Provision for Annual Leave Publications

1995 1994

($) ($)

7,072 10,424

6,800 6,800

1,064 0

4,563 3,366

710 560

15,485 2,612

45 (30)

0 2,620

5,958 4,756

861 478

2,782 1,543

2,752 2,304

2,047 1,844

1,233 1,682

0 1,556

10,653 9,131

5,076 790

0 2,787

3,025 2,312

346 1,060

3,792 (2,025)

12,711 12,452


Recruitment & Relocation Rent - Local Office Repairs & maintenance Salaries/wages-D i rectors/Staff Stationery & Office Supplies Super - PSS (Director) Super - AGEST (Off Mgr, Board) Training Telephone & Facsimile Travel, Carhire & Accom-Board Travel, Carhire&Accom-ED/Staff Travel & Accom - other Project GWR 93/5

1995 1994

($) ($)

0 1,729

13,149 13,221

725 779

174,543 154,271

4,817 4,513

8,638 9,158

6,052 3,764

623 805

7,902 7,445

30,178 17,178

8,051 3,845

2,311 65

0 16,832

Total Administrative Expenditure 343,964 300,627

*A charge was made for audit services provided by the Australian National Audit Office (ANAO) during the year. No other services were provided by the ANAO. The auditor received no other benefits.

7. Cash

Operating Bank Account 1,360,648 823,492

Petty Cash Float _______88 747

1,360,736 824,239

8. Contingent Assets

At 30th June 1995 the Corporation had outstanding claims of $1 28,282 for matching Commonwealth contributions. These claims are expected to be paid to the Corporation in October 1995 following the expected receipt of the industry levy.

At 30th June 1995, the Corporation expects to receive funds in relation to grants not fully expended. As the amounts concerned rely on the return of administration forms from those in receipt of grants, it is not possible to determine an accurate figure. The funds are expected to be received by November 1995 and have been estimated by management to be approximately



9. Property Plant and Equipment

1995 ($)

1994 ($)

Plant & Equipment - at Cost Less: Accumulated Depreciation

28,772 (14,745)

23,985 (9,341)

14,027 14,644

Furniture & Fittings - at Cost Less: Accumulated Depreciation

5,918 (1,923)

5,918 (1,369)

3,995 4,549

Total Property, Plant, and Equipment 18,022 19,193

10. Creditors and Borrowings

Current Accounts Payable - Administration Unearned income

52,722 224,471

4,700 0

277,193 4,700

11. Provisions


Balance 1 July Movement during year

3,838 3,792

5,863 (2,025)

7,630 3,838

12. Reconciliation of Cash

Cash Deposits at call

1,360,736 0

824,240 0

1,360,736 824,240


13. Reconciliation of Operating Results with Cash Flows from Operations

1995 1994

($) ($)

Operating result ' 259,040 22,723

Less: Prior year annual leave adjustments 0 (2,025)

Add: Provision for annual leave 3,792 0

Accrued expenses 48,021 4,700

Depreciation 5,959 4,756

Refunds of unexpended grants 0 36,390

W rite back of unexpended grant refunds provision 0 9,797

Loss on sale of assets 0 1,556

282,243 57,199

Net cash provided or used by operating activities 541,283 77,897

14. Insurance

Following an assessment of insurance risk, appropriate insurance cover is provided through the Department of Primary Industries and Energy.

15. Superannuation Commitments

The Grape and Wine Research and Development Corporation contributes to the Public Service Superannuation Scheme (PSS) for its Executive Director and to a private scheme for other employees.

The principal type of benefits provided for under the plans are defined retirement benefits.

The basis of contributions to the plan is as advised by the Minister for Finance for both directors and staff, together with the 3% productivity benefit for staff under the Superannuation (Productivity Benefit) Act 1988.

The amount of contributions paid of $14,690 (1994 $12,922) were contributed as follows:

1995 1994

($) ($)

Executive Director · 8,638 9,158

Staff 6,052 3,764


16. Related Parties

The names of directors in office during the financial year were:

P. Hayes T. Devitt P. Laffer C. Weaver D. Day

J. Keniry D. Davidson P. Fitch B. Avery

The aggregate amount of remuneration of these directors during the financial year was $152,481 (1994 $126,246).

All Board Members received remuneration in the band $0— $ 10,000, except for the Chairman, whose remuneration fell within the band $10,000-$20,000. The Government representative did not receive any fees. The amount of aggregate superannuation contributions paid on behalf of Directors, included in Corporation expenses above was $13,500.

17. Contingent Liabilities

As at 30 June 1995 the Corporation was not aware of any contigent liabilities which could affect its operations.

18. Grape Research Profit & Loss Statement

For the Year Ended 30 June 1995

1995 1994

($) ($)

OPERATING REVENUES Commonwealth Contributions 584,523 616,035

Industry Contributions 702,864 545,456

Investment Interest 55,434 20,994

Other 0 331

Refund of unexpended grants 51,683 (9,797)

Total Operating Revenues 1,394,504 1,173,019

OPERATING EXPENSES Payments to Research & Development Organisations Commonwealth Organisations 293,845 204,423

Universities / Colleges 16,520 46,335

State Departments 512,872 659,128

Other Organisations 193,165 175,547

Total Research & Development Expenditure 1,016,402 1,085,433

Total Administrative Expenditure 0 0

Levy Collection Fees 31,669 31,635

Total Expenditure 1,048,071 1,117,068

Operating Profit/(Loss) $346,434 $ 55,951


19. Wine Research Profit & Loss Statement

For the Year Ended 30 June 1995

1995 1994

($) ($)

OPERATING REVENUES Commonwealth Contributions 1,166,387 1,234,965

Industry Contributions 1,382,922 1,158,071

Investment Interest 13,859 6,742

Other 0 917

Refund of unexpended grants 40,166 0

Total Operating Revenues 2,603,334 2,400,695

OPERATING EXPENSES Payments to Research & Development Organisations Commonwealth Organisations 0 0

Universities / Colleges 80,992 82,805

State Departments 0 0

Other Organisations 2,234,174 2,018,855

Total Research & Development Expenditure 2,315,166 2,101,660

Total Administrative Expenditure 0 0

Less Levy Collection Fees 31,600 31,635

Total Expenditure 2,346,766 2,133,295

Operating Profit/(Loss) 256,568 267,400

20. Agreements Equally Proportionately Unperformed

Lease and Rental Commitments

Later than one year but not later than two years 48,323 46,949

Later than two years but not later than five years 43,057 41,683

21. Contingent Liabilities

As at 30 June 1995, the Corporation was not aware of any contingent liabilities which could affect

its operations.


Statement by Corporation Members

In accordance with the resolution of the Members of the Corporation we state:

(a) In our opinion:

(i) the Financial Statements and notes thereto are drawn up so as to show fairly the operating result for the year ended 30 June 1995, the financial position of the Grape and Wine Research and Development Corporation as at that date and the cash flows of the Corporation during the year ended 30 June 1995.

(ii) there are reasonable grounds to believe that the Corporation w ill be able to pay its debts as and when they fall due.

(b) These statements have been made out in accordance with the Guidelines for Financial Statements of Commonwealth Authorities and Commercial Activities and in accordance with Australian Accounting Concepts and applicable Accounting Standards and other mandatory professional reporting requirements.

Dated this 6th Day of October 1994

John S. Keniry Corporation Chairman

P.L. Laffer Deputy Corporation Chairman




Project Title Projections of winegrape production and winery intake to 1997-98

Project No BAE 94/1

Organisation ABARE

Location Edmund Barton Building, Broughton Street, Barton ACT

Supervisor Mr Robert Rees

Funding $45,376

Time Span 1/95 - 7/95

Objectives To provide annual projections of winegrape production and winery intake for major winegrape varieties and grapegrowing regions in Australia for the vintage seasons, 1996,1997 and 1998.

Progress In this report, detailed production and utilisation projections are presented for wine grapes for the four years 1994-95 to 1997-98. The production

projections are based on the detailed area and production statistics collected by the Australian Bureau of Statistics while the projections of future winery grape intake have been estimated from a survey of major wineries by ABARE.

The study this year has refined the changes in methodology that were made last year. These changes permitted a direct comparison of production and planned intake for the major varieties covered in the study. On the production side of the study, the ABS winery intake census data are to be used to ensure a more accurate analysis of winegrape production over the projection period. Also, refining of the modelling procedure w ill allow for future time savings to be made at the estimation stage. The planned intake of wine grapes by wineries for the projection period, by variety, is being assessed by a survey of wineries. Over 55 wineries were surveyed, including all of the wineries estimated to crush close to 75 per cent of the harvest each year.

Preliminary results from the grape production analysis indicate that grape production and winegrape production are projected to increase

steadily in 1996, 1997 and 1998. The proportion of premium grape varieties in each production year is projected to continue rising, as growers respond to higher prices offered for these grapes.

Preliminary indications from the winery survey show intended winegrape intake increasing steadily throughout the projection period. More detailed results from the winery survey w ill be available shortly.

Project Title Viticulture statistical collection

Project No WFA 94/1

Organisation Winemakers' Federation of Australia /Winegrape Growers' Council of Australia

Location Adelaide, SA

Supervisor P van der Lee

Funding $29,500 (Wine account/Grape account)

Time Span May 1994- December 1994

Objective To collect Australian production data for grape varieties, area of vines (bearing and non-bearing), and the production tonnages of grapes by their end use to assist grapegrowers, wine producers, and government agencies to make informed decisions and observations on future grape supply and varietal mixes.

Progress As with previous collections, survey data was published covering major regions by variety, bearing area, not yet bearing area, and production (tonnes). To achieve improved capture the scope of this collection was modified to a lower estimated value of agricultural operations (EVAO) of $5,000 in 1993. An improved listing of growers should improve capture, and use of a standard national survey forum is expected to provide more consistent data. An earlier release of data is projected in alignment with industry negotiated target dates.


Project Title National Statistics Planning

Project No WGC 94/1

Organisation Winegrape Growers Council of Australia Inc

Location Adelaide, SA

Supervisor Mr C Pritchard

Funding $5,000

Time Span June 1994 - July 1995

Objectives To conduct a facilitated 'search conference' as a basis for defining industry needs in national and regional statistics collection and to devise

mechanisms to achieve further refinement in the collection and interpretation of such data.

Progress With investment support from both the GWRDC and DPIE Agribusiness Program, a search conference was conducted with representatives from peak and regional industry bodies, key state Agriculture/Primary Industry providers, DPIE, ABS, AWBC and GWRDC.

As a result of the conference several key outcomes were achieved including • an improved schedule for collection and earlier publication of viticultural collection


• a uniform national collection or survey form • more complete listing of grower respondents, arising from the regional support by grower groups

• a more effective relationship between user groups, ABS, and ABARE with prospects for further streamlining of project input from both these bodies • agreement to conduct an annual meeting of a

W ine Industry Statistics Reference Group to monitor progress in refining scope and quality of viticultural industry statistics and analysis


Project Title National Grapevine Accreditation

Project No AVI 94/1

Organisation Australian Vine Improvement Assoc

Location Irymple, Victoria

Supervisor Mr Peter Murphy

Funding $7,345

Time Span Feb 95 - May 95

Objectives To establish procedures and systems that w ill allow the Australian Vine Improvement Association (AVIA) to provide the grape industry with assurances about the integrity and reliability of the process of plant propagation

Progress Following recommendations arising from the National Grapevine Accreditation Committee, AVIA assumed responsibility for further development and implementation of a quality assurance scheme. A consultancy was contacted to develop two draft documents addressing, in Stage 1, Vine Improvement Group Procedures and in Stage 2, Vine Nursery Procedures. Draft manuals for both stages have been developed and have been forwarded to all major industry bodies, vine improvement groups and major nursery operators for consideration and comment.

A trial implementation process of the Vine Improvement Procedures has been undertaken in the 1995 cutting season, with evaluation and modification to occur in 1995-96. Final version

manuals w ill be prepared in the light of experience and feedback from review of the draft documents.



Project Title Heterogeneous rootzone hydration - implications for vine vigour

Project No CSH 92/2

Organisation CSIRO, Division of Horticulture

Location Adelaide

Supervisor Dr B R Loveys

Funding $33,404 (1994/95)

Time span 1992/3 to 1994/5

Objectives To develop the concept of heterogeneous rootzone hydration (partial root drying) and to investigate the possibility of achieving control of vine vigour through application of this technique.

Progress Measurements during the final year of this project have been made at two sites; one on the

University of Adelaide Waite campus and one at Blewett Springs. The vines (Cabernet Sauvignon on Ramsey) were established at the beginning of the 1992/3 season and had divided root systems, the two halves being separated by a plastic sheet

buried to a depth of about 1.5 m. Irrigation to either side of the vines could be controlled independently. From fruit set to just before harvest water was applied to both sides of the control vines and to one side or the other of the treated vines. On average the period of irrigation to the one side of the treated vines was 14 days. After this time the irrigated side was reversed. This timing was arrived at from a knowledge, gained in years one and two of the project, of the kinetics of abscisic acid production by the drying roots, stomatal conductance responses of the canopy and by real-time monitoring of soil moisture. During the treatment period (fruit set to 2 weeks before harvest) control vines received water at the rate of 0.92 ML/ha and treated vines 0.56 ML/ha (data from Adelaide site). The effects of the treatment on canopy vigour were substantial. Shoot elongation, pruning weight and leaf area were reduced by 42%, 25% and 30% respectively. Because of the

more open canopy, bunch exposure, measured as

light reaching the bunch zone at veraison, was improved by 1 71%. Fruit on treated vines reached maturity (23°Brix) about one week earlier than control fruit. There was no effect of the treatment on yield, bunch number or berry weight but water use efficiency, expressed as g of fruit per L of irrigation, was increased by 46%. Fruit quality, as judged by pH, titratable acidity, colour, phenolics and glycosyl- glucose, was significantly improved in fruit from treated vines.

It is concluded that the concept of heterogeneous rootzone hydration, or partial root drying, has great potential for the reduction of vigour and it has the additional advantages of improved water use efficiency and fruit quality.


Project Title Development of a sustainable soil management system with improved lime and immobile nutrient cycling capacity.

Project No DAV 92/3

Organisation Department of Agriculture, Victoria

Location Tatura

Supervisor Mr J W hiting

Research Staff Mr S McNab, Dr J Tisdall

Funding $25,000

Time Span July 1992 - June 1995

Objectives 1. To determine the effect of earthworms and other soil macroflora on lime and phosphorus movement in acidic soils. 2. To determine the influence of organic mulches,

herbicide strips and cover crops on the establishment and maintenance of earthworm populations. 3. To develop an improved and sustainable soil

management system for established vines in acidic soils.


Progress Vineyards with established vines and soils with low pH (acidic soils) and low phosphorus were treated with combinations of lime, phosphorus, straw mulch and the addition of worms. The main aim of the project is to monitor the movement of

lime and phosphorus down the soil profile. During the past year the soils in the two main trials were sampled down to 50 cm approximately two years after the treatments were added.

The movement of surface applied lime as recorded by soil pH changes, was apparent down to the 10-20 cm zone in both trials. In a clay loam kraznozem soil, the surface soil (0-5 cm) pH was

increased by 0.6 units with the 10-20 cm zone pH being increased by 0.2 units over the control treatment. On the site with a sandy loam duplex soil the surface soil (0-5 cm) pH was increased by

1.4 units and the 10-20 cm zone pH increased by 0.3 units over the control treatment. The addition of earthworms has had little influence on the movement of lime at this stage.

Earthworm activity underneath mulch treatments was observed on both sites as shown by the presence of earthworm castings. The very dry winter and spring of 1994 would have reduced the activity of earthworms, thus limiting the expected benefits from them.

Mulching with straw has had an unexpected negative influence on soil pH. In both trial sites the soil pH under the mulch tended to be more acidic than without mulch. Although the differences were

rarely significant, the trend is undesirable and should be investigated further. Exchangeable aluminium, whilst low in both trial sites, was reduced in the shallow layers of the soil by the addition of lime.

Phosphorus movement from the surface was only significant down to the 5-10 cm layer in both trials. At this depth the phosphorus value was 9.5 pg/kg on treated plots compared with about 6.6

pg/kg on untreated plots. Mulching and the addition of worms had no significant effect on phosphorus movement. Petiole analysis at flowering showed only small differences between treatments. The vines have not

responded to the surface applied lime or phosphorus after 2 years. In 1995 there was a significant increase in yield on the sandy loam duplex soil site from mulching in a low rainfall year. This was due to more bunches per vine and heavier bunches. Berry

weights were increased by about 0.1 g and the sugar level was 0.35° Brix lower on the mulched vines. The addition of lime and phosphorus or worms had no influence on yield.

In the kraznozem soil site (Yarra Valley) the rainfall during the 1994/95 season was much higher than the sandy loam duplex soil site (Avoca). No significant yield response to mulching was detected and there was no influence of mulching on berry weight or sugar level.

Project Title To assess the response of Cabernet Sauvignon winegrapes to the application of different sources of nitrogen.

Project No DAW 3

Organisation Department of Agriculture, Western Australia

Supervisor B H Goldspink

Funding Nil

Time span 1991-1997

Objective To assess whether sources of nitrogen applied at budburst and at fruit set influence the uptake, yield and juice quality of Cabernet Sauvignon wine- grapes. To also assess the effect of sources of nitrogen on soil acidity.

Progress In 1994 there was a statistically significant impact of nitrogen treatments on yield, however in 1995, the yields were found to be statistically unaffected by treatment. The 1994 data showed ammonium nitrate to be the source with which the highest

response was achieved. Yields were increased by 5 tonnes per hectare when 300 kg/ha of nitrogen was applied to nitrogen deficient vines. At 75 kg N/ha, the increase was approximately 3 tonnes per

hectare. In 1995 the differential in yields was reduced to approximately one tonne per hectare, with the high rates of nitrogen being the most affected.

There may be a number of explanations for this decrease in yield. The first to consider must be seasonal. The vines had a very dry year in 1994, despite being irrigated, and again in 1995. In both years the rainfall finished early in September but the irrigation was not applied until after flowering


in late November. Shoot extension had almost stopped by the time water was applied in each year. Some response in growth was seen to the water and the fruit set application of nitrogen, however, the drought effect on bud fruitfulness can only be guessed at. W hile shoot numbers have yet to be counted for 1994-95, there appeared to be more blind buds on the canes this year than in past years. This was also noted on cane pruned vines in the district by other vineyard managers.

Petiole analyses have indicated that the high nitrogen treatments have reduced phosphorus uptake by the vine, to the extent that some of the vines could now be classified as phosphorus deficient. This can also be a contributing factor to the reduction in yield. This effect has been observed in other trials. Phosphorus had been applied to try to counteract this occurring. There was, however, little rain after the phosphorus application, giving it no chance of moving into the root zone. Earlier applications w ill be made this season at a significantly higher rate.

Phosphorus availability may also be reduced by the low pH observed in the soil samples collected from the ammonium nitrate and ammonium sulphate plots. The pH has been reduced from 5 (CaCI2) to as low as 3.8. At pH less than 4, it has been shown that extractable aluminium has increased. This could have the effect of 'fixing' the phosphorus in much the same way as excessive iron w ill, thus reducing availability to the vine. Lime cannot be applied to the site until the trial has been completed and the extent of the pH reduction determined, hence higher rates of phosphorus will be tried to alleviate the problem in the short term.

An extensive tissue sampling program was again undertaken in 1994-95. Blade and petiole samples were collected from opposite the bunch at regular intervals throughout the season. Youngest mature blades and petioles were also collected from the growing tip from flowering to harvest. The data w ill be used to determine whether there is a better time of sampling than at flowering, and also whether tip leaves can be used as an indicator of the nitrogen status of the vine

Responses to the nitrogen treatments were recorded in the ammonium and free amino nitrogen analyses of free run juice from berry samples, however, this year's data is showing some plateauing which may be due to the carry over of nitrogen in the soil from previous

applications. Wine has been made again from fruit picked from the ammonium nitrate plots The nil treatment took 17 days to dryness, where as the 50, 100 and 200 gN/vine treatments took 14, 11 and 11 days respectively. Wine was also made from the 100 gN/vine calcium nitrate treatment, which took 14 days to reach dryness. All wines w ill be judged later by a panel of industry show judges to determine the effect of the vineyard applied nitrogen on quality.


Project Title DNA fingerprinting of grapevine cultivars

Project No CSH 93/1

Organisation Commonwealth Scientific and Industrial Research Organisation (CSIRO)

Location Hartley Grove, Urrbrae, SA 5064

Supervisors Dr M R Thomas Dr N S Scott

Research Staff Dr M R Thomas, Dr N S Scott, Ms P Cain

Funding $45,170 (1994-95)

Time Span 1993/94 - 1994/95

Objectives • Reduce the complexity and cost of the DNA fingerprinting procedure to make it suitable for application through a standard laboratory

testing procedure. • Integrate DNA fingerprinting and computerised ampelographic techniques to introduce into the industry a vine certification scheme which

could be used to cover grapevine material from nursery stocks to wine must just prior to clarification, with an accuracy sufficient to detect contamination of 10% of one variety in another. • Investigate the possibility of providing a

field/weigh bridge based test for selected varieties.


Progress Following the scientific work establishing the viability of the DNA profiling procedure further development has concentrated on preparing the process for industry application.

Major wine producers and other relevant authorities were consulted to obtain a list of varieties 'essential' for inclusion in an Australian DNA profile data base. DNA was prepared from

nearly all of these cultivars and DNA profiles obtained. DNA isolation methods were simplified to allow DNA extraction from leaves, berries and wood. Additional DNA typing loci have been

identified for varietal identification. Some suspect DNA profiles remain to be re-tested and cultivars with similar DNA profiles remain to be analysed with new loci. W hile, as yet, no molecular technologies are available which routinely detects clonal differences we have detected differences between the clones Primitivo and Zinfandel.

Under laboratory conditions the DNA profiling procedure accurately detects mixed DNA samples containing 10% contamination of one variety in another. DNA has been successfully isolated from

runoff of a first crush and DNA profiles obtained. This represents the first step in providing a DNA typing test post-weigh bridge with further experimental work required to develop the method

into a standard laboratory procedure. A national/international database has been set up containing Australian data. Access is available through the Internet with restricted access to some sections of the database until it is determined how best to manage distribution of the information and technology.

Visits by overseas scientists occurred during the period. Under a CNR/CSIRO agreement an Italian visitor from the late Professor Eynard's laboratory - now managed by Dr Guiliana Gay - visited Adelaide for a second time and successfully typed

many more Italian grapevines. A similar visit occurred under a ISTAC grant with Dr. Patrice This from the French laboratory of Dr j.M . Boursiquot. The data obtained by these scientists w ill be

included in the international DNA profile database. The results obtained by both scientists demonstrated the strength of the technology and the potential of extending the technology to other

international groups. A joint publication has resulted from the Italian visit confirming the usefulness of the technology [1]. Following a successful visit to Europe by Dr Scott and Dr

Thomas to discuss future collaborations and the integration of the DNA profile data with ampelography data it was decided that another Italian visitor w ill visit in 1995. European colleagues are also investigating the possibility of

EC funding. Successful completion of this project w ill now see the grapevine DNA profile service offered to industry through the Australian Wine Research Institute during 1995/96 as a joint CSIRO, AWRI, CRCV collaboration and the service was workshopped at the 1995 Wine Industry Technical Conference.

The new CSIRO publication Wine Crapes of Australia contains a list of all cultivars that have been DNA profiled along with ampelograhic details. In addition to various conference presentations there have also been several scientific publications arising from this work and they are recorded below.

1. Botta R, Scott NS, Eynard I, Thomas MR: Evaluation of microsatellite sequence-tagged site markers for characterising Vitis vinifera cultivars. Vitis 34: 99- 102(1995) 2. Thomas MR, Cain P, Scott NS: DNA typing of

grapevines: A universal methodology and database for describing cultivars and evaluating genetic relatedness. Plant Mol. Biol. 25: 939-949 (1994). 3. Thomas MR, Scott NS: Identification of grapevine cultivars using microsatellite loci as hypervariable genetic markers. In Wetherall JD, M. CD (eds), Hypervariable genetic markers: Nature and Application, CRC Press, Inc., Boca Raton (1995) 4. Thomas MR, Scott NS: Microsatellite sequence tagged site markers: simplified technique for rapidly obtaining flanking sequences. Plant Mol. Biol. Rep.

12: 58-64 (1994). 5. Thomas MR A global approach to grapevine identification. In: Morell M, Gibbs A (eds) Biological barcoding for individuals and populations. Cambridge

University Press, Canberra, (in press)


Project Title Analysis of azinphos-methyl residues in Hunter Valley wine grapes

Project No DAN 93/1

Organisation NSW Agriculture

Location Gosford Horticultural Research & Advisory Station

Supervisor Mr C R Turkington

Research Staff Dr Stephen Goodwin Mr Nazir Ahmad

Funding $4460 (94/95)

Time Span December 1993 - June 1995

Objectives • To analyse azinphos-methyl residues on grapes and in wine following field application of insecticide by grower high-volume sprayer,

1993/94 • To analyse azinphos-methyl residues on grapes and in wine following application of insecticides by hand sprayer, 1994/95 • To investigate removal of azinphos-methyl

residues in wine

Progress Fig longicorn is a major pest of winegrapes in the Hunter Valley and has been the subject of an intensive research program funded by GWRDC. A recommendation, including the use of azinphos- methyl, has been developed. However, growers have expressed concerns over the possibility of residues of this insecticide in wine which may be exported.

In Australia, azinphos-methyl has an MRL for wine of 2 ppm, which is greater than some of our wine importing trading partners and, in particular, Sweden and Denmark, MRL 0.5 ppm.

Grower concerns have been reflected over use patterns for azinphos-methyl and this study was undertaken to measure azinphos-methyl residues on grapes and in wine. Azinphos-methyl wettable powder (WP) and suspension concentrate (SC) formulations were applied to a block of Shiraz grapes at three concentrations by commercial high-volume and hand applied sprayers in replicated trials in 1993/94 and 1994/95 respectively. Treatments were applied on two occasions, a week apart, and grape samples were taken at five-weekly intervals for analysis of

residue on grapes. A sample was also taken 2-3 weeks after the second application and made into wine for subsequent residue analysis. This reflected observance of the withholding period for azinphos- methyl in grapes.

A control sample was removed before the first treatment had been applied. Results showed that residue levels on grapes resulting from all treatments applied by high-volume sprayer in

1993/94 were below the Australian MRL one week after the final application and below the most stringent overseas MRL (0.5 ppm) four weeks after the final application at the recommended rate of azinphos-methyl (1.2 g/L WP; 2.4 mL/L SC).

In 1994/95, hand-sprayed treatments of both formulations at the recommended rate produced unacceptable residues on grapes five weeks after the final application. Residues in wine from grower sprayed treatments in 1993/94 were negligible and further analysis of this wine in 1994/95 resulted in the failure to detect these residues. Residues in wine from hand-sprayed treatments in 1994/95 demonstrated that, at the recommended rate for both formulations, levels were only 6-7% of the levels on grapes.

In a separate laboratory experiment, a strong correlation between residues accumulated by barley through root uptake, and the concentration of azinphos-methyl, confirmed the hypothesis of systemicity not previously recorded for this insecticide.

It was concluded that absorption of chemical by the leaves and translocation through the grapevine may have occurred which may explain variation recorded in the residue data.

On the basis of these data, it would seem reasonable to conclude that, despite the combination of surface and translocated residue, at the recommended rate of application and usage pattern for either formulation of azinphos-methyl applied by commercial spraying equipment, the MRL for wine in Australia is unlikely to be exceeded. Further, that storage at 21 °C would seem to be an appropriate method of eliminating minor residues of this chemical in wine.


Project Title The relationship between 'effective leaf area to fruit weight ratio' and grape and wine composition and wine style for the varieties Shiraz and Pinot Noir

Project No UA 92/1

Organisation The University of Adelaide

Location Adelaide

Supervisors PC Hand

Funding $10,500

Time Span 1994-1995

Objectives To determine ways to evaluate the effective leaf area to fruit weight ratio of grape vines and to relate this measure to grape and wine composition

Progress A new analytical assay for glycosylated secondary metabolites of grapes has been developed. It is

based on the amount of D-glucose released by acid hydrolysis of a 50% aqueous ethanol extract of an homogenate of the berries. The measure of the released glucose is referred to as the glycosyl glucose (total G-G) determination. The extract can

also be analysed for red colour (anthocyanin concentration), the glucose associated with these anthocyanins estimated and then subtraction of this colour G-G from the total G-G gives a new

measure termed the red-free G-G. This gives an estimate glycosylated secondary metabolites other than colour compounds. These measures have been determined on Shiraz grapes from an irrigation and bunch thinning trial conducted during the 1995 vintage. For the bunch thinning trial, yield was reduced by 25%, there was little change in berry weight, while sugar concentration, as degrees brix, increased by 4%, total G-G colour and red-free G-G on a per gram

berry weight basis increased by 16, 15 and 18% respectively. For the irrigation trial, changes in berry size had a dramatic effect on the composition of these glycosylated secondary metabolites when the concentration was expressed on a per gram berry weight basis.

Data from these experiments highlight that there are tw o approaches to achieving high

concentration of secondary metabolites in grapes on a per gram berry weight basis, one being how much of each component is accumulated in the berry and the second being how the effect of berry size can either concentrate or dilute these components.


Project Title Practical management strategy for bunch rot of grapes

Project No DAN 92/1

Organisation NSW Agriculture

Location Biological and Chemical Research Institute, Rydalmere, NSW

Supervisor N G (Tan) Nair

Funding $18,000(1994/95)

Time Span June 1992 to July 1995

Objective • To develop a practical management strategy for bunch rot of grapes • To develop models for flower and berry

infections for incorporation into the practical management strategy for bunch rot control • To validate the infection models in Australian vineyards in combination with the current

Botrytis and fungicide resistance monitoring practices

Progress Biological data for the construction of infection models were collected. These data show infection of flowers and berries as a function of time and temperature. The major difference between

requirements for infection of flowers and berries related to the wetness period; K time was 1.3 hours for flowers compared with 1 3.9 hours for

berries. This emphasis the vulnerability of wet flowers to Botrytis. Population studies of Botrytis showed a quantitative relationship between carryover inoculum and flower infection. A similar

relationship was found between flower infection and berry infection. This led to the establishment of a monitoring service for grapegrowers. This has


enabled the growers to predict disease risk and time the application of fungicides rationally. The development of infection models w ill complement the measurement of disease risk through Botrytis

monitoring and w ill assist the growers to manage bunch rot disease efficiently.

Project Title Cover crops for management of nematode infestations in vineyards

Project No DAN 92/2

Organisation NSW Agriculture

Location Biological and Chemical Research Institute, Rydalmere

Supervisor M r R W McLeod

Funding $13,901

Time Span July 1992 to July 1995

Objective 1. To determine the effect of various cover crops on nematode infestation 2. To assess the potential for nematode control of

cover crops selected because of reported nematode control potential.


Field observations Trial sowings of 13 covercrops in the Griffith and Hunter Valley districts were made from 1992 to 1994 on sites with infestations of rootknot (Meloidogyne incognita, M. javanica) or with citrus nematode (Tylenchulus semipenetrans). The crops included the oat varieties Cooba, Coolabah and Saia, the legumes Dun field pea, Languedoc vetch and Ultra lupin and the brassicas Arran forage rape, Barossa canola, Hobson forage rape, Humus green manure rape, Indian mustard, Rangi forage rape and Winfred forage rape.

This experience suggests that autumn sowing of brassicas is a feasible option w ithin the viticultural system. Most experience has been with Rangi forage rape, it was found to have many characteristics suiting it to use as a covercrop.

At Griffith there was evidence of rootknot nematode (M. incognita) reproduction on autumn sown Languedoc vetch. There was no reproduction on Barossa canola or Rangi forage rape, the brassica covercrops sown in autumn there. Ploughed-in residues of Cooba oats and Rangi

forage rape controlled citrus nematode more effectively than did residues of Hobson and Winfred forage rapes. In the Hunter Valley trials there was evidence of

rootknot nematode (M. javanica) reproduction by mid August on autumn sown Dun field pea and Ultra lupin crops. Some nematode development occurred also on Rangi forage rape sown in early April but there was no infection of brassicas sown the following year in late April.

Mustard seed meal is available as an industrial byproduct and is a concentrated source of glucosinolates, the progenitors of the volatile biofumigants released when brassica crop residues decompose in soil. Experiments have established an effect of mustard seed meal against root knot nematode. Experiments have shown also that mulching, and watering in, leaving cut green covercrop material on the soil surface and then watering it, provides promising control of root knot nematode.

The forage rape Rangi is showing promise for control of rootknot and citrus nematodes by biofumigation. It is however a moderately favourable host of rootknot nematodes. So there is a risk of nematode increase while this crop is growing, before the crop is ploughed in. In its final year DAN 92/2 has concentrated on investigating factors influencing nematode increase, with the objective of finding ways of preventing or minimising nematode reproduction while the crop growing. In an extensive series of experiments the following aspects have been investigated:

• Infection when field soil temperatures are below 20°C. • Importance of various covercrop management options in spring, at the end of the covercrop


• Significance of temperature, numbers of invading nematodes and age of invading nematodes to infection of crops of different host status. • Rates of maturation of nematodes in very

favourable, moderately favourable and just favourable hosts.

Two small experiments are in progress, experimental work is otherwise complete. Results are now being collated and interpreted for final reporting and publication. Project Title Effects of canopy management


practices on diseases and spray distribution

Project No DAV 92/1

Organisation Agriculture Victoria

Location Sunraysia Horticultural Centre, Mildura

Supervisor Dr Bob Emmett

Funding $39,575 (94/95)

Time Span July 1992-June 1995

Objectives To determine the effects of minimal pruning, mechanical hedging and spur pruning or cane pruning of cordon trained vines on the

development of Botrytis bunch rot and powdery mildew.To estimate the effects of canopy configurations created by these pruning practices on spray deposition and spray distribution.

Progress Field trials to assess the effects of canopy configuration on disease development were established in early 1992-93 at Coonawarra SA on Chardonnay and Shiraz, at Iraak Victoria on Chardonnay, at Koorlong Victoria on Cabernet Sauvignon and at Broke NSW on Chardonnay. Levels of overwintering disease inoculum in spring and the incidence and severity of diseases at harvest in 1993, 1994 and 1995 were assessed in relation to vine canopy characteristics, fruit quality and yield and also in relation to lightbrown apple moth incidence.

Higher leaf area per vine was recorded on minimal pruned and mechanically hedged vines of Chardonnay at harvest at Coonawarra than on cane-pruned and VSP mechanically hedged vines. Calculated surface area and volume per vine were

lower on VSP mechanically hedged vines than on vines of other treatments. The leaf area to volume ratio (a measure of canopy density) was higher at

harvest for mechanically hedged vines (with and without VSP) than for vines of other treatments. Bunches on minimal pruned vines were dispersed through the outer part of the canopy, and were more exposed, smaller, larger in number, less compact and slightly more delayed in maturity than bunches on cane pruned or mechanically

hedged vines. Although bunches on cane pruned and mechanically hedged vines were located

w ithin the inner canopy, the bunch zone on cane pruned vines was more congested. Less Botrytis rot developed when vine management practice produced small, loose, well-

dispersed bunches and in an open canopy. At Coonawarra and Iraak, bunch rot was least severe on minimal pruned vines and most severe on cane

pruned vines. Botrytis rot was also more severe in large bunches and was often associated with berry damage caused by lightbrown apple moth regardless of canopy type. In the lower Hunter

Valley, monitoring of Botrytis throughout the season indicated higher incidence in bunches on spur-pruned VSP vines than on more open Scott Henry trained vines.

The number of overwintering powdery mildew cleistothecia extracted from vine bark and leaf litter varied widely between sites and no ascospore infections arising from surviving cleistothecia were detected on vine leaves in spring. However, at some sites (e.g. Iraak), lower numbers of cleistothecia were recovered from minimal pruned vines than from mechanically hedged and cane

pruned vines. Although powdery mildew incidence on bunches at harvest was low, substantially less leaf disease was observed on minimal pruned vines.

Spray coverage studies were conducted on minimal pruned, mechanically hedged, cane- pruned and vertical shoot positioned Chardonnay vines at Coonawarra in December 1993 and 1994 and in January 1994 and 1995 when vine foliage was fully developed. Spray distribution from standard commercial air-blast sprayers was highly variable. W hile some exposed sides of bunches were over-dosed, sheltered sides were under­ dosed. On average, 30% of the area of vine foliage was covered by visible spray droplets and

coverage in the centre of large vines was less than that on the sides and upper parts of vines. In all tests coverage on the lower surface of leaves was less than on the upper surface and coverage on the

surface of bunches within all types of vine canopy was poor when vine foliage was fully developed.


Grape Q uality

Project Title Minimisation of pesticide residues • in grapevines by improving the

efficacy and application of the biological insecticide, Bacillus thuringiensis

Project No DAV 92/2

Organisation Agriculture Victoria

Location Melbourne

Supervisor Dr A M Smith (Institute for Horticultural Development, Knoxfield)

Research staff Mr P Cole (IHD)

Funding $15,000(1994/95)

Time span 1992-1995

Objectives 1. To minimise the use of synthetic insecticides, and their consequent residues in grapes and wine, through improved efficacy and

application of formulations of Bacillus thuringiensis (Bt) 2. To improve the coverage and adhesion of Bt formulations on the leaves and bunches using

spreaders and stickers, and thus increase the efficacy of Bt 3. To identify compatible and incompatible chemicals applied with Bt formulations by

screening common tank mixers, such as fungicides. 4. To inform the industry of the best and worst tank adjuvants and mixers, and ideal dose for

application of Bt on grapevines.

Progress Bacillus thuringiensis (Bt) is an essential component of the grape and wine industry's aim to control lightbrown apple moth, and maintain high

quality products with low chemical residues. Variable levels of insect control have discouraged some viticulturists from adopting Bt permanently. Agriculture Victoria researchers have identified reasons for the variable efficacy of Bt and have determined ways to increase its reliability.

Bt efficacy is variable because of: (a) insecticide 'resistance'. Different field populations of lightbrown apple moth displayed varying susceptibility to dose of Bt; (b) host plant. Caterpillars fed on equivalent doses of Bt on

grapevine leaves had lower mortality than those that fed on other host leaves. Low pH (3.5) and high tannin levels are implicated in reducing efficacy of Bt. (c) Poor timing and spray application. Monitoring of moth, egg and caterpillar numbers is essential for the correct timing of any insecticide. Bt is most effective against young caterpillars (< 5 mm long). Two or 3 sprays closely timed to cover the egg hatch period is the best tactic.

Incompatibility with fungicides and other tank mixers was thought to be another factor affecting Bt efficacy. We screened the fungicides Bayfidan, Mancozeb, Fulasin, Ronilan, Oxydul (copper oxychloride) and Blue Shield (copper hydroxide) and other mixes, Kumulus (wettable sulphur) and D-C Tron (mineral oil). |D-C Tron and Blue Shield had insecticidal activity on young caterpillars.] Bt efficacy was not reduced by these tank mixes.

One approach to overcome the potential difficulties with Bt efficacy is to increase the rate/ha. Spray application trials showed that the effective rate is greater than or equal to 375 g/ha of 'double strength' Bt product. In practice, some regular Bt users follow this rate while others are spraying at rates too low for their local situation.

Seven common adjuvants, Agral, Bond, Chemwett, Pulse, Tween, Latron and Citowett, were screened in laboratory bioassays to see if Bt efficacy was enhanced. Some products enhanced efficacy but the benefits were not consistent. Use of adjuvants above the recommended rate led to a significant drop in Bt efficacy.

Our current recommendations to improve Bt efficacy are to:(1) use rates greater than or equal to 375 g/ha of 'double strength' Bt, according to label recommendations; (2) add a wetter/sticker at the correct rate; (3) time sprays to cover egg hatch and kill young caterpillars; (4) get 'good' coverage with sprays.


Project Title Review of spray technology and pest and disease management plan

Project No GWR 94/1

Organisation Grape and Wine Research and Development Corporation

Location Adelaide, SA

Supervisor M r P Hayes

Funding $5,000

Time Span ju ly 1994 - June 1995

Objectives 1. To review to effectiveness and efficiency of current agrochemical application technologies and practices 2. To review the broad range of pest and disease

management studies and produce an overview summary of the R & D program 3. To initiate planning of future spray application R & D

Progress In cooperation with the Dried Fruits Research and Development Council (DFRDC) a two day schedule was developed to address the issues of Spray Application and Pest and Disease Manage­ ment Coordination w ith the Horticultural Research and Development Corporation (HRDC) allowed the HRDC to conduct a Spray Application Review day consecutively with the GWRDC/DFRDC activity.

Virtually all current and recent pest and disease projects were presented in summary forum, emphasising objectives, results and outcomes for industry. A summary document was made available to participants from the provider, industry and service groups and to industry organisations,

and has served as a useful reference mechanism. The spray application review led to several separate proposals for further study with a joint project being negotiated for commissioning in

1995-96. Key issues of poor definitions of performance para r . rs, variable and ill managed coverage, aspects or dose rates, and persistence of product were defined as being critical to the future success of integrated pest management and low input viticulture.


Project Title Adoption of strategies to reduce use of pesticides in winegrape production

Project No DAN 92/4

Organisation NSW Agriculture

Location Horticultural Research & Advisory Station, Gosford

Supervisor Mr R Turkington

Funding $15,000 (94/95)

Time Span June 93 - June 95

Progress The project, commenced in 1992/93 season was continued with the main emphasis on a large vineyard in the Riverina. The whole vineyard of

nearly 200ha operated under the low chemical management (LCM) program, developed in 1992/93 under the auspices of Riverina Clean. An additional Environdata weather station has

been purchased installed at Nericon in the Riverina for operation in 1995-96. Nericon is a group of farms located 20km north of Griffith and separate from the main vineyard areas. The weather stations and central computer at CSU, Wagga, were a little short on reliability. At times pest and disease control management decisions were made on the basis of vineyard monitoring, manual weather readings, satellite maps (accessed by polling fax) and weather forecasts. To improve downloading reliability, heavy duty modems have been installed.

The LCM program uses a protective fungicide approach for the control of downy mildew. Applications are timed in response to weather events and previous infections to ensure the vines are protected from infection. W hile the program allows for the use of the curative fungicide,

phosphorous acid, its use is restricted to occasions only when the vines may not have been adequately covered by a protective fungicide. Conditions were generally not favourable for the

development of fungal diseases for the critical first half of the growing season. Overall the spray program was confined to four applications for the early maturing varieties and five for the later


varieties. The program targeted phomopsis, downy mildew and powdery mildew. Pesticides used were confined to mancozeb, copper, and sulphur. Mite control was effected by the 'soft' pesticide program that allowed predatory mites, aided by a release of T. doreenae to operate. A build up of rust mite in February/March was eliminated by predators by April. The incidence of disease was minimal, although rain close to harvest triggered a minor outbreak of botrytis, accentuated in

Semilion with an undetected infestation of light brown apple moth.

The concept of reduced use of pesticides was promoted at a low input viticulture field day held near Griffith on 2 May 1995 and followed up with an article in the July issue of the Australian Crape Grower and Winemaker.

The 1995 wines made from grapes grown in the demonstration vineyard have already won awards in wine shows, including a Gold medal in the Griffith Show for Shiraz.

Project Title Extension initiatives

Project No GWR 94/2

Organisation Grape and Wine Research and Development Corporation

Location Adelaide, SA

Supervisor Mr P Hayes

Funding $11,800

Time Span July 1994 - June 1995

Objective To permit the support of discrete small initiatives which with appropriate seeding could lead to valuable regional or national impact.

Progress Activities supported have included

• a network of cooperating vineyards have implemented an evaluation and adoption process for RDI (regulated deficit irrigation) in Western Australia; a seminar has been conducted • seminar support to the Barossa Winegrape

Industry Advisory Committee, the seminar

focussed on quality of production • purchase of slide and overhead projection equipment for use in seminar presentation; several groups have used this equipment • production of slide material for use in a series

of disease management seminars • support for Mr Peter Clingeleffer to attend the South African Society of Enology and Viticulture, International Congress, and report

back to the Australian Industry. • distribution of the video on Phomopsis recognition and management • purchase of mineral nutrition disorders

posters/brochures for distribution at industry meetings

Project Title OIV - Expert Group and general assembly meetings

Project No GWR 94/3

Organisation Grape and W ine Research and Development Corporation

Location Adelaide, SA

Supervisor Mr P Hayes

Funding $13,510

Time Span July 1994 - June 1995

Objectives 1. To achieve input to the major intergovernment reference body on matters of vine improvement, cultural practices and pest and disease

management 2. To facilitate international collaboration in matters of fundamental viticultural research 3. To provide a strategic overview of international

production, marketing and environmental issues as a basis for planning and managing the Corporation's R & D program

Progress Activity in each of the Expert Groups was focussed as noted:

Vine Selection and Improvement: • harmonisation of selection and propagation rules • description of clones • conservation of genetic variability


• improved and genetic techniques in ampelography including international collaboration • role of gene technology in vine improvement

Vine Physiology: • role and effect of irrigation on quality and productivity (paper presented by Australia) • studies on delimitation of 'terroirs' • prediction of crop levels

Disease, Pests and Vine Protection: • overview of major diseases in world viticulture and programs of research • development of a compendium of major

diseases, pests and management strategies • role of stress proteins in disease or pest response • resistance mechanisms

Professional visits were made to the South of France (December 1994). the Tuscany region of Italy (June 1995) and Central Coastal California/Washington State (June 1995) in conjunction with attendance at the American Society of Enology and Viticulture International Clonal Symposium and Annual Meeting.

Discussions on issues of disease resistance to agrochemicals, the role of phytoplasmas, use of heat shock treatments in phytosanitary practices and the role of molecular techniques in vine

identification and improvement have been reported and also introduced into influencing local programs. Other arrangements have enabled access of Australian industry representatives to market oriented seminars conducted by the Lien de la Vigne group, and other linkages with that body have facilitated activity of an international type on powdery mildew and phylloxera.

Project Title Australian Wine Expo

Project No WFA 93/2

Organisation Winemakers' Federation of Australia Inc

Location Adelaide

Supervisor Mr I Sutton

Funding $12,500

Time Span 1993 - 1996

Objectives • To provide a resource base for the development of an exhibition of R & D activities and impact on industry, at the Wine Australia Exhibition

Progress The Corporation retains its commitment to this high profile consumer-oriented activity as a means of promoting the role of R & D in achieving a highly competitive, internationally oriented

industry. The Corporation's Executive Director is Convenor of a Technical Support Group established to ensure appropriate emphasis on technical matters and to source suitable display and demonstration material to reflect the

innovative nature of Australia's grape and wine industry. Liaison with the Board and Management of Wine Australia '96 has been regular and contacts have also been established with the Australian Society of Wine Educators which has a role complementing to the Technical Support Group.




Project Title Use of rootstocks to reduce chloride and sodium levels in export wines

Project No CSH 8

Organisation Commonwealth Scientific Industrial Research Organisation (CSIRO)

Location CSIRO Division of Horticulture, Merbein, Victoria

Supervisor Dr R R Walker

Funding $39,530 (1994/95)

Time Span July Ί 994 - June 1997

Objectives To evaluate a range of rootstocks with Shiraz and Chardonnay scions for ability to maintain low grape juice and wine Cl' and Na* concentrations under irrigation with high salinity water in the Barossa, Padthaway and Sunraysia regions

Progress A range of rootstocks, with Shiraz and Chardonnay as scions, are being evaluated for chloride and sodium exclusion ability at sites in three regions; Sunraysia (Merbein and Koorlong, Victoria); Barossa Valley (Rowland Flat and Nuriootpa, South Australia); and Padthaway (South Australia). The salinity of irrigation water at chosen sites in the Barossa and Padthaway regions falls into the high range, being greater than EC 2.3 dS/m. The Merbein site is irrigated with water of 2.0 dS/m and the Koorlong site with water of low salinity (0.25 dS/m).

Soil type and soil salinity differ across the sites. The Padthaway site had the highest winter time soil salinities. The Chardonnay and Shiraz plantings at Padthaway are on the same property, with the Chardonnay site having higher soil salinities than the Shiraz site. The Merbein site had relatively high surface salinities, decreasing with depth (N.B. samples were taken before the commencement of 2.0 dS/m irrigations), while the

Koorlong site had the lowest salinities of all the sites. The Barossa sites had low surface salinities, but high sub-surface salinities. Data for Shiraz vines on a range of rootstocks at

the Rowland Flat site (irrigation water EC 3.4 dS/m) in season 1994-95 demonstrated a strong correlation (R2 = 0.85) between bloom time petiole chloride and harvest time grape juice chloride. A similar correlation (R2 = 0.62) was evident for sodium. Crape juice of own-rooted vines and vines on K51-40 and 1202C rootstocks had chloride concentrations of 500, 585 and 353 mg/L, respectively. Vines on Ramsey, 1103 Paulsen, Ruggeri 140, Schwarzmann and 101-14 rootstocks all had grape juice chloride concentrations less than 110 mg/L. Free sodium concentrations (excess of sodium over chloride in juice) were for all rootstocks, less than 60 mg/L. Data for vines at the other sites were still being analysed at the time of preparing the report.

Chloride and sodium exclusion rankings showed some differences between sites. Two further seasons of sampling and analysis and vine performance measures are needed to establish a basis on which to develop recommendations for appropriate rootstocks to minimise chloride and sodium in grape juice and wine.

Project Title Detection of closteroviruses associated with grapevine leafroll disease in Australia.

Project No CSH 94/1

Organisation CSIRO Division of Horticulture

Location Waite Campus (Adelaide)

Supervisor Dr N Habili and Mr A J W Ewart

Funding $15,000

Time Span July 1994-June 1995

Objectives 1. To identify the types of leafroll-associated closteroviruses in selected grapevine cultivars by ELISA using commercially available antisera. 2. To investigate the use of serological and cDNA

hybridisation tests for monitoring the spread of leafroll in a clonal selection trial of Pinot Noir planted in Nuriootpa, South Australia. 3. To conduct leafroll transmission studies using the long-tailed mealybug.

Progress Symptom observations in a Pinot Noir clonal evaluation trial at Nuriootpa, South Australia


indicated a progressive spread of leafroll from infected vines to the neighbouring symptomless vines along the rows. The infection apparently initiated from three leafroll-infected clones, i.e., ANTAV 543, Geisenheim 20 and Bourgogne

HI 99A. The trial consisted of 1 3 clones in 8 replicates with each clone being randomly distributed within a replicate. The involvement of

grapevine leafroll-associated virus type 3 (GLRaV- 3), which is the only naturally transmissible leafroll type, was suspected. Vine samples were tested by ELISA and by nucleic acid probes to determine the type of leafroll virus involved. Using an antiserum to GLRaV-3, ELISA testing was carried out on all

104 samples from thirteen clones collected at the end of two consecutive seasons. The same samples were subjected to double-stranded RNA extraction and probed with a cDNA specific to GLRaV-3. The

results of slot blot hybridisation analysis confirmed those of serology and showed the spread of GLRaV-3 in the vineyard. From a total of 104 vines, 21 new infections were recorded by slot blot

hybridisation analysis in 1994. Further analysis in 1995 detected 6 additional infections. The newly infected clones include: D5V12 and Bourgogne

HMOAt'rom replicate 1, D5V12 and ANTAV 167 from replicate 2 and MV6 and ANTAV 542 from replicate 8. All these vines were located adjacent to infected vines and scattered throughout the vineyard.

The pattern of leafroll spread appears to involve neighbouring vines implicating the involvement of a slow moving vector. Although mealybugs spread GLRaV-3 in other parts of the world, these insects have not been observed in the Nuriootpa vineyard. A potted vine experiment was set up to establish whether transmission of leafroll occurs with Australian mealybugs. Analysis of vine samples for GLRaV-3 by ELISA and by nucleic acid

hybridisation gave negative results. A planting of Pinot Noir clone D5V12 at Sunraysia Horticulture Centre, Irymple, Victoria, showed spread of leafroll symptoms along the rows (G. Fletcher, personal communication). Results of ELISA testing on samples from these vines confirmed the involvement of GLRaV-3.

Further ELISA testing on selected samples from over 70 grapevine cultivars and clones showed the occurrence of GLRaV types 1 to 5 in Australia. In Sultana clones GLRaV types 1 ,4 , and 5, but not GLRaV-3, were detected. GLRaV-5 was also detected in samples of Emperor 3A grapes from

Western Australia. A low-yielding Cabernet Sauvignon from McLaren Vale contained GLRaV-1. A cDNA probe, pLR34, derived from a low-

yielding Sultana clone (B4L) reacted specifically to dsRNA from a number of grapevine cultivars infected with GLRaV-1, suggesting the probe is specific to GLRaV-1. Another cDNA clone, pLR44, obtained from a leafroll-infected low-yielding Sultana reacted with dsRNA extracts from two grapevine clones, whereas pH5-18 probe reacted with the extracts from a wide range of clones. The type of leafroll-associated viruses occurring in a

number of infected grapevine cultivars is not known.

Manuscript prepared:

Habili, N., Fazeli, C. F., Ewart, A., Hamilton, R., Cirami, R., Saldarelli, P., Minafra, A. and Rezaian, M.A. (in press). Natural spread and molecular analysis of grapevine leafroll-associated virus 3 in Australia.



Project Title Optimisation of quality winegrape production through modelling vine phenology, canopy architecture, light interception, water use and yield

Project No CSH 94/2

Organisation CSIRO Division of Horticulture, Agriculture Victoria (ISIA)

Location Merbein, Victoria

Supervisors K j Sommer, I Goodwin, D Godw'in

Funding $95,712(1994/95)

Time Span July 1994 - June 1997

Objective Develop and validate a dynamic simulation model for grapevine growth and water use. The model w ill use known information on climate and soils to

predict vine phenology, canopy development, light interception and water use of selected winegrape cultivars.


Progress ' Experimental sites were established at Merbein and Mt Helen, representing hot and cool climatic areas. At Merbein, experimental plots were established in a vineyard of grafted (Ramsey) minimal and spur pruned Cabernet Franc vines. At Mt Helen, an existing experiment comprising minimal pruned and 'Scott Henry' trained Rinot Noir vines was selected and two crop load and water use sub-treatments were established. At both sites neutron probe access tubes were installed for monitoring of soil moisture levels. In addition, TDR buriable wave guides were installed at Merbein and an Enviroscan instrument at Tatura. Both instruments are capable of continuous logging of soil moisture levels.

At Merbein, shoot and vine growth measurements from minimal and spur pruned vines were collected throughout the growth period. Spur pruned vines on average had 104 buds of which 89% burst and developed into shoots Minimal pruned vines had 956 buds of which 56% burst. Shoots under spur pruning developed 22, as compared with 11 leaves on vines under minimal pruning. Shoot growth declined sooner under minimal than cane pruning. Under both systems shoot growth had largely ceased by mid December. Appearance of leaves followed a near linear relationship with regard to heat sum. Early vine leaf area development was accelerated under minimal over spur pruning but both pruning systems had attained similar leaf areas per vine at flowering and remained similar until the end of the growth period. Because of a larger leaf area early

in the season minimal pruned vines initially intercepted more light than spur pruned vines but during later growth stages this was reversed. Soil water depletion between 20 and 60cm tended to

be higher for minimal pruned vines early in the growth period, until about the end of flow'ering but, for the remainder of the season was similar under both pruning treatments. An estimate of leaf area and dry matter production per shoot at flowering could reasonably predict the berry yield

per shoot for minimal pruned vines but for spur pruned vines prediction was poor. At Mt Helen, each trellis type (minimal pruned and Scott Henry) was split into two water stress treatments by either maintaining soil moisture close to field capacity or restricting irrigation and developing water stress as measured by a midday

leaf water potential difference of 2 bar. Each water

stress treatment was further split into two crop load treatments by hand thinning 50% of the bunches 4 weeks after flowering. In two replicates of each treatment soil moisture was measured at 20 cm

increments to 1.4 m depth every week with a neutron probe from a grid of seven access tubes across vine rows and between vines. The neutron probe data, irrigation amount and rainfall w ill be used to calculate water use. The Enviroscan soil moisture probe was installed in one replicate of each treatment to measure relative differences in daily water use from the drip wetting pattern. Weekly shoot growth, leaf number, leaf water potential and stomatal conductance were also measured. Light interception was continuously monitored in each trellis.

Modelling: The primary water balance model has been revised to remove some anomalies in the simulation of upward flow from a water table and in the simulation of salt effects on plant growth. Observations on soil water extraction from the above and additional experiments were used to establish the upper and lower limits of water extraction to be used in the model. A new scheme to provide for simulation of annual increments of root growth from a framework of overwintering roots has been devised and is undergoing evaluation. A leaf senescence routine which operates by changing the duration of different categories of leaf area as a function of prevailing stress has been developed and is awaiting evaluation.

Measurements as described above w ill be continued in the coming growth period. In addition, light profile measurements for the different canopy types w ill be recorded. Such measurements w ill assist in quantifying relationships of light exposure of the fruit zone and fruit maturation and composition. Furthermore, historical data sets w ill be employed to calibrate and refine existing model routines.

Project Title Objective design of trellis structures

Project No DAV 93/1

Organisation Agriculture Victoria (AV) and University of Melbourne (Uni Melb)

Location Sunraysia Horticultural Centre, Mildura

Supervisor Mr Robert Hayes (AV), Irymple,


Research Staff Dr Mahabubur Mollah (AV), Irymple Dr Graham Moore (Uni Melb) Mr John W hiting (AV), Bendigo

Funding $3,100(94/95)

Time Span January 1994 to December 1997

Objectives 1. To reduce the capital and maintenance cost of vine trellising

2. To optimise the mix of material and construction costs 3. To improve the integrity, stability and effective life of trellis structures

Progress Automatic Logging o f Dynamic Loads A novel data recording system comprising a computer, software, data acquisition cards, and sensors has been designed to record dynamic

loads on trellis structures, particularly loads caused by wind gusts and/or heavy rain. Loads imposed on the fruiting wires of a Marshall trellis supporting Sultana vines on Ramsey rootstock, as well as wind speed, wind direction and ambient temperature are being monitored. This system w ill enable collation of detailed information relating to the loads imposed on trellises, such as sideways

load, wire tension and force exerted on end assemblies during wind gusts or storms, and with increasing canopy and crop load. After further development, the system w ill be placed on trellises

in different regions of Victoria and South Australia. The information w ill be used to develop a software package for objective design of trellis structures.

Regional Post Trials Posts have been installed at 7 different sites in South Australia and Victoria to determine: a) the resistance of the posts to vertical lift b) the resistance of the posts to sideways loading c) the effect of consolidation on the resistance of the posts to vertical lift and sideways loading. The location of these test sites are as follows: Coonawarra (1); Nuriootpa (1); Great Western (2); Tatura (1) and Mildura (2). More posts w ill be

installed at these sites in November / December prior to testing.

Bending Strength o f Different Post Types for Trellising A comprehensive trial has been completed, determining the strength of 8 different trellis post types. The post types represented included different combinations of diameter class and the two commonly used wood preservatives i.e. either CCA or Creosote. The actual diameter, age of the wood, age of the post after treatment, wood density and moisture content were recorded for each post to determine their effects on the strength. Currently, the data is being analysed. It is expected that the results w ill be published by the end of

December 1995.

Project Title The role of inter-row ground covers to improve the management and sustainability of Australian vineyard soils

Project No SEE 93/1

Organisation SEEDCO ( South Australian Seedgrowers Co-operative Ltd. )

Location 78 Burbridge Road, Hilton, SA

Supervisor Mr Jonathan Sobels

Funding $57,355(1994/95)

Time Span 1994/95-1996/97

Objectives Identify and select the most suitable spp/cvs. of temperate pasture legumes, grasses, grain legume, cereal and fodder crops which can be used for cover crops in the major grape growing regions of South Eastern Australia, culminating in the

production of an extension kit.

Progress Four major sites were established in April/May 1995 at Mildura (private grower), Griffith (Orlando Wyndham grower), Rowland Flat (Orlando Wyndham) and Coonawarra (Mildara Blass). At each location, approximately 85 cultivars, covering over 40 species, were sown in two

blocks, permanent sod and green manure, each comprising three replicates. These trials were all sown using small plot cone seeders which enabled precise sowing rates, uniform plot length and

accurate seed placement. Secondary sites containing up to 20 cultivars were established at Clare (Southcorp), McLaren


Vale (Southcorp), High Eden (Pewsey Vale), Qualco (Oxford Landing Estate), Rutherglen (Pfeiffers), Great Western (Southcorp), and Wagga Wagga (CSU). Evaluation of these trials in collaboration with CRC staff w ill be conducted throughout the growing season.

Trial sowing was completed on schedule at all but the Great Western and Wagga Wagga sites where seeding was delayed due to excess rainfall. Above average rains have fallen in all areas and the Griffith site is now adversely affected, with the soil being at or beyond field capacity since sowing (however reasonable results are still expected). An isolated side effect of early sowing has been a lack of adequate weed control at Rowland Flat where oxalis, a perennial regional problem, grew vigorously after trial establishment, causing a blanketing effect. Subsequently, the entire permanent sod block was sprayed with Glyphosate in late May and was resown in mid June, whilst the green manure block was left to compete with the weeds.

The first round of assessments was completed in early July 1995 at each of the major sites. Crop vigour ratings and seedling densities were recorded, and the cultivars ranked accordingly. Two further assessments are planned for August and September/October, at which times sward botanical composition, biomass production, percentage ground cover and vigour ratings w ill be determined. Early assessments have identified a rape, a white mustard, oats ( 2 cultivars), a white pea, perennial ryegrass (3 cultivars), fenugreek, beans 0 cultivar), Medicargo polymorpha (1 cultivar), M. truncatula (2 cultivars) and M. scutellata (1 cultivar) as consistently high ranking cultivars across all sites, as well as several site specific high ranking species.

Project Title Root pruning for growth control of winegrapes

Project No UA 93/1

Organisation The University of Adelaide

Location Adelaide

Supervisor P R Dry

Funding $ 6020

Time Span 1 July 1993 - 30 June 1996

Objective To test the hypothesis that manipulation of root mass by root pruning can be used to control the vegetative growth of grapevines without any detrimental effect on yield and with associated benefits for fruit composition.

Progress This was the second season of this project. The Lyndoch site was used again in 1994/95 but the Kalimna site was replaced due to lack of vigour. A new experiment with high vigour Shiraz was set up in the Riverland; unfortunately, the vines at this site were affected by restricted growth in spring, after the application of the treatments, and the results were inconclusive.

At Lyndoch, vines were pruned back to 60 nodes per vine by hand in winter in order to stimulate shoot vigour (they had been previously hedge-pruned). Six treatments were applied this season: one- and two-side root pruning just before budburst; the same at flowering; 2-side root pruning last season, nothing this season; and control. All root pruning treatments reduced shoot length and pruning weight relative to the control with the '2-sided at budburst' treatment (24% decrease in pruning weight) having the most effect.

Root pruning also increased yield per vine, ranging from 10 to 36% increase relative to the control; surprisingly, this was due to a significant increase in bunch number per vine. There was no effect of root pruning on time of maturity nor total soluble solids; however, pH of fruit from root pruned vines was significantly lower than that from control vines. Analysis of colour and glycosyl-glucose w ill be completed in 1995. In summary, root pruning appears to have potential for decreasing shoot vigour. Further evaluation in a high vigour situation (Shiraz on Ramsey) w ill take place in

1995/96. The potential beneficial effect on yield, and in particular bunch number per vine, w ill be further investigated in 1995/96.



Project Title Response of grapevines to partial wetting of the root system

Project No CRC 92/1

Organisation Cooperative Research Centre for Viticulture, ISIA Tatura

Location Tatura, Victoria

Supervisor M r I Goodwin

Funding $ 22,260

Time Span June 1994 - July 1996

Objective To determine the effects of the size of the wetted fraction of the grapevine rootzone and the cycle

of water potentials in the wetted fraction of the grapevine rootzone on some important performance determinants and indicators in a commercial winegrape production system.

Progress The information derived from the experiments w ill be incorporated immediately into commercial water management strategies advocated by the collaborators and into the strategic water

management trials of the CRCV. This project has been successfully relocated to Dookie Agricultural College Mt Major vineyard. Three wetted volumes were implemented by either: on 4 L/hr dripper per vine (an 'onion' shaped wetting pattern), for 2 L/hr drippers per vine equally spaced alone the vine row (a

'sausage' shaped wetting pattern), and twenty 2 L/hr drippers per vine equally spaced within vine rows and across vine rows (a 'flood' wetting pattern). Each wetted volume was split into either a full irrigation or a deficit irrigation strategy.

Results showed no difference in berry size (fresh weight), soluble solids, pH or TTA between wetted volumes. The deficit treatment reduced berry size and increased soluble solids irrespective of wetted volume. Yield correlated with wetted volume was attributed solely to bunch number per vine and

berry number per bunch as a result of severe water stress during flowering on the smaller wetted volume treatments.

Additional data is currently being collected to contribute to the vine water use model under development by Dr Karl Sommer of the CSIRO and ISIA Tatura. This is referred to as a 'minimum data set' comprising: weather, irrigation, soil, phenology, vine size and trellising, root distribution, dry matter production, fruit yield and fruit maturity.

Dr B Loveys. Dr P Dry and Dr Helmut During visited the experimental site in February and discussed the implementation of their results. It is hoped this project could be expanded to

incorporate any useful treatments forthcoming from the work of Dr Loveys. A student from Dookie College utilised this for his final year project.

CRAPE QUALITY__________________

Project Title Improving wine quality by controlled deficit irrigation at particular berry growth stages

Project No DAS 11 GW

Organisation Primary Industries (South Australia)

Location Nuriootpa

Supervisor M G McCarthy

Funding $41,960

Time Span July 1994-June 1995

Objectives 1. To determine which fully specified controlled deficit irrigation schedule causes the greatest improvement in the quality of wine made from

Shiraz grown in the semi-arid continuously irrigated winegrowing regions of S.E. Australia. 2. To develop functions describing the relationship with plant water status indicators and soil water

availability and atmospheric evaporative demand

Progress The 1994/95 season was the most successful season of the experiment. The dry and hot weather

during most of the growing season resulted in all treatments being effectively applied. Shoot length measurements commenced at budburst in September 1994 and continued until shoot growth


ceased in January 1995. Weekly sampling for berry size commenced immediately after flowering and sampling for brix and glycosyl-glucose (CC) assay commenced in the second week of January 1995. Pre-veraison stress treatments had significant effects on vegetative growth, yield, maturity, colour and CC - in particular the period

immediately after fruit set appears to be a critical time. Mr P Hand (University of Adelaide) has again had a major involvement in the experiment. Through his CRC involvement additional canopy configuration measures have been taken as well as photosynthesis activity measures. In attempting to achieve the maximum collaboration with other interested groups the experimental site has been used by the South Australian Department of Environment and Natural Resources as a site to test new developments in aerial remote sensing technology. We hope to present this information in a poster at the 1995 Wine Industry Conference. A request has been also made by the Department of Horticulture/Viticulture & Oenology for samples for a post-graduate student project on grape berry proteins. AWRI is also using samples to examine the anthocyanin composition of ripening grape berries.

Harvest for small-lot winemaking by the University of Adelaide was completed on 7 March and weekly berry sampling on the 13 March 1995. There has been a major effort into completing CC analysis of all the 1994 samples and the 1995 samples were analysed as they were collected. Mr M. McCarthy spent two days per week at the Waite precinct during January, February and March 1995 assisting the two Technicians at AWRI processing the samples. Over 500 CC analyses have been completed and all samples w ill be processed by April 1995. There have been regular meetings of CRC-V program 2 and 3 staff to review the data as it has been generated. The first release of the data w ill be at a CRC-V Program 3 meeting

in mid-April 1995. We plan a major coordinated publication of this and other CC data at the 1995 Wine Industry Technical Conference. This w ill be a world first and we anticipate much interest.

Detailed soil sampling w ill commence soon after vintage and before any leaching winter rain to determine salt distribution in the soil profile in relation to the minimal leaching fractions applied.

Visitors to the experimental site during the past year have included:

• Dr Helmut During • Dr Peter May • CCW growers field trip • TAPE student tour

Information and results of the experiment were presented to: • The Grampians Crape-growers seminar & field day - November 1994

• The Cowra Grape growers seminar and field day - December 1994 • CCW growers seminar - Barmera - Spring 1994 As foreshadowed, in collaboration with Southcorp, two of the experimental treatments have been duplicated on 1 ha. sites adjacent to the experimental area. Extra probe tubes were installed and are read twice weekly (by Southcorp) and the data faxed to Nuriootpa for processing. Fruit from these blocks was harvested in late February 1995 and processed in commercial sized lots (20 t) for evaluation. There are significant differences in the wines with the Nov-Dec deficit treatment having more colour and flavour. Wine and grape samples have been kept for further analysis. We anticipate that this exercise w ill continue as this is an integral part of the commercial evaluation of the results from the experiment. Plans are underway to establish 'commercial' sites in other districts. The development of a structured extension program to transfer the results to a wider cross-section of the grape growing industry was discussed at a RiverLink meeting in June 1995. An 'expert irrigation group' of scientists and extension staff is proposed to coordinate the technology transfer of the results of this and other experiments.

In view of the now successful completion of the 1993/4 and 1994/5 seasons we propose to significantly reduce data collection in 1995/96. This w ill also allow time for the major task of collating, analysing and writing-up of data for extension articles, scientific journals, seminars and major conferences. We currently envisage the same irrigation treatments on all plots during

1995/96 but reduced data collection. Sufficient crop water/yield data w ill be collected as an additional data set for the vine growth model under development. Discussions have been held with CSIRO scientists regarding the development of a minimum data set for project CSH 94/2 (Optimisation of quality wine grape production through modelling vine phenology, canopy architecture, light interception and yield).The Waikerie experiment w ill provide a very


comprehensive set of data to help validate the model. This data set is currently being compiled. Discussions w ill be initiated later in 1995 regarding the long-term future of the experimental

site at Waikerie. There is significant capital infrastructure established (and unlikely to be duplicated elsewhere) and the opportunity exists to use this facility for other research. Vines w ithin the experimental area have been hand pruned for five seasons and there is now detailed information about vine-to-vine variability and performance. Scientists within both the CRC-Viticulture and CRC- Soil and Land Management have been

invited to participate in the planning of the future of the experimental site. The site could for instance be used for nutrient or pesticide leaching studies, crop water use of different canopy configurations, saline water use and drainage studies, soil

management/water use, water requirements of cover crops etc. As Mr McCarthy is also a sub­ program leader in CRC-V Program 2 any collaborative work with other projects w ill be encouraged.

Project Title Persistence of biological activity and degradation of residues of key pesticides used on grapevines

Project No DAV 92/4

Organisation Agriculture Victoria

Location Sunraysia Horticultural Centre, Mildura

Supervisor Dr Greg Buchanan

Research Staff Alison MacGregor, Bob Emmett, Trevor Wicks, SARDI Adelaide Greg Ruediger, AWRI Waite Campus Adelaide

Funding $115,000(94/95)

Time Span July 1991 to June 1995

Objectives 1. To review the literature on residue persistence in grapes and dried fruit; 2. To measure degradation of residues in grapes

and dried fruit by conducting residue trials; 3. To measure persistence of toxicity of pesticides against grape pests and their predators

Progress The persistence of 20 pesticides on fresh grapes has been measured in replicated trials on Sultana and Cabernet Sauvignon vineyards in Victoria and South Australia. Dried vine fruit and wine were made from treated grapes and the effect of processing on residues was measured. Bioassays tested the persistence of biological activity of six pesticides against longtailed mealybug and of 9 pesticides against the predator Cryptolaemus montrouzouri. Pesticides were ranked according to their persistence and toxicity to these insects.

During four seasons, trials have also investigated the relationships between residue levels measured on grapes and • grape maturity at spraying and berry growth

after spraying; • equipment used and spray coverage achieved; • multiple applications; • water volumes and rates of active ingredient

applied per hectare. The Grapevine Pesticides Working Party has planned and regularly reviewed progress of the trials. Nine chemical companies have contributed funding to the residue trials and have been

involved in discussions regarding their own products.

Outcomes Recommendations for chemical use in winegrape and dried vine fruit production are being reviewed to take into account the effects of processing on

residues in wine and dried vine fruit and the needs of export markets. Specific requirements for different markets are published in the AWRI 'Agrochemical Grid' Results w ill be extended to

growers as pest and disease control recommendations via the winery spray diaries ADFA spray diary, the Vine Pest and Disease Control Chart and other Departmental publications and extension media. Extended withholding

periods are proposed for some chemicals, where appropriate, to ensure residues in dried fruit do not exceed tolerance limits set by domestic or export markets.

An annual review of chemical recommendations for pest and disease control in grapevines has brought together members of the dried vine fruit, wine and table grape industries with staff from

Departments of Agriculture in Victoria and South Australia and NSW, the chemical industry and National Registration Authority (NRA). Protocols


for commercial residue trials in grapes are being discussed with AvCare and the NRA to ensure that future residue trials conducted by the chemical industry provide data relevant to the dried vine fruit and winegrape industries.

Trials involving chlorpyrifos, fluazinam, chlorothalonil with flusilazole, myclobutanil and fenarimol have been reported to respective companies. The reports have been forwarded to the NRA and to Codex Alimentarius to support applications for MRLs, and in one case to the USA seeking an import tolerance in the USA.

Issues of spray application highlighted in this project w ill be addressed in a three year project on vineyard spray application, commencing July 1995.

A final report w ill be provided to the Grape and W ine Research and Development Corporation in September.

Project Title Field assessment of selected rootstock hybrids for quality wine production

Project No CSH 94/3

Organisation Commonwealth Scientific Industrial Research Organisation (CSIRO)

Location CSIRO Division of Horticulture, Merbein, Victoria

Supervisor Mr P R Clingeleffer

Funding $20,000(1994/95)

Time Span July 1994 - June 1997

Objectives Identify rootstock hybrids with the greatest potential under Australian conditions for producing high yields and optimal grape juice composition for wine production

Progress The basis of the project is a rootstock trial which includes 54 CSIRO hybrids and five standard rootstocks, all w'ith Shiraz as scion. It was established in 1989, produced some crop in 1993 and reached full production in 1994. Pruning weights, bunch numbers and harvest measurements were recorded for season 1994/95. Samples for determining berry weights and for

analysis of juice composition and berry antho- cyanin were taken. ICP analysis was used to determine not only the juice potassium but other major elements. Because the project is dependent on small scale winemaking to complete the assessment of selected hybrid rootstocks in years 2 and 3, wines were also produced for the 5 standard rootstocks (with and without acid adjustment) to verify procedures.

The first detailed assessments showed a wide range between the rootstocks in Shiraz productivity, vigour, berry weight, maturity and, juice pH, titratable acidity and ion content.

Differences in Shiraz juice pH between rootstocks correlated positively with vine vigour, berry weight, sugar level, titratable acidity and potassium, phosphorous and sulphur levels in juice. Similarly, differences between rootstocks in juice potassium level were correlated with yield, vigour, berry weight, sugar level, titratable acidity, pH and phosphorous, sulphur and boron levels in juice.

The results, although preliminary, indicate that there is potential to develop for Australian conditions, new, highly productive rootstocks that have improved juice composition for quality wine production.


Project Title The role of chitinase and β-(Ί -3)- glucanase gene expression in the response of grapevines to fungal infection

Project No CSH 94/4

Organisation CSIRO Division of Horticulture

Location Urrbrae South Australia

Supervisors Dr N S Scott

Dr S P Robinson

Funding $33,546

Time Span April 95 - March 98

Objectives Identification of enzymes contributing to the defence of grapevines against mildew infections:


• the possibility of substituting natural defence mechanisms for sprays; • isolation of genes controlling defence enzymes; • eventual insertion of these genes into sensitive

grapevines; • experimental testing of management techniques to enhance endogenous resistance in sensitive

grapevine varieties.

Progress Report of progress April 1995 - June 1995. Following a late start to the project due to difficulty in finding a suitable applicant an appointment was made in March 1995.

The project was originally designed to isolate the chitinase and glucanase enzymes responsible for the effects observed on powdery mildew, by methods of protein chemistry. The second step was to isolate the genes for the active enzymes. After the late withdrawal of a highly qualified applicant for the position, it proved impossible to find an applicant suitably qualified in protein chemistry to

pursue this strategy. Consequently the new strategy based on isolation of the genes first and the proteins second, was designed and approved by the DFRDC and CWRDC in 1995 and an appointment made in March 1995. The genetic sequence of the known chitinase and glucanase enzymes have been used to design PCR primers to isolate defence genes that are active in young leaves of resistant and sensitive grapevines. Chitinase and glucanase assays are also being utilised to identify those leaves from vines with the highest activities of chitinase and glucanase. The first chitinase gene has now been

isolated and is being characterised and other candidates are in the process of isolation. At the same time preparations are being made to test experimentally if pre-induction of chitinase and glucanase activity in experimental vines w ill

protect against mildew infections. The first experiments are timed to take place in the spring of 1995.

Project Title Biological control of mites in Australian viticulture: implementation and sustainability

Project No. DAN 93/2

Organisation NSW Agriculture

Location Yanco Agricultural Institute

Supervisor Dr David G James

Research Staff Jennifer Whitney

Funding $56,595 (1994/95)

Time Span September 1993 - August 1996

Objectives Development and implementation of biological control strategies for grapevine mites in all major viticultural regions of southern Australia. Develop­ ment of management practices which enhance sustainability of biological mite management.

Progress During 1994/95 data were collected on the mite fauna of vineyards in the following viticultural regions; the Granite Belt, Upper Hunter, Great Western, Geelong, Yarra Valley, Margaret River, Swan Valley, Manjimup and Mount Barker.

Typhlodromus dossei was the dominant predator in most of these regions and was successful in a number of instances in controlling rust and blister mite populations. However, in general terms it appears to be less effective than Typhlodromus doreenae (DOREEN) in regulating mite populations possibly due to greater susceptibility to some fungicides or less ability for development and survival on non-mite food sources (e.g. pollen).

Difficulties were experienced in rearing T. dossei on pollen in the laboratory. However, preliminary laboratory tests on the toxicity of some fungicides to T. dossei indicated that mancozeb, zineb, dithianon, propiconazole and wettable sulphur are

not harmful. A large culture of T. doreenae (DOREEN) was established at Yanco-during winter 1994. Occupying a 3 x 2 m constant temperature room

up to 250,000 predators were produced weekly during October-November 1994. Releases of approximately 10,000 predators each were made

at 20 vineyards in the Upper Hunter,Sunraysia, MIA, Granite Belt and Western Australia. Unfortunately, these releases were curtailed by progressive contamination of the Yanco DOREEN culture by another phytoseiid predator,

Typhlodromus transvaalensis. This predator, identical to DOREEN except when viewed under a very high-powered microscope, ultimately dominated the culture which had to be destroyed. Attempts to restart the culture have so far been

unsuccessful due to continued invasion by T.


transvaalensis. T. transvaalensis is found occasionally on grapevines and probably also feeds on rust/blister mites. However, it does not appear to be very successful in developing large populations in vineyards (only in constant temperature rooms!).

Despite culturing problems, DOREEN was successfully established in three or four MIA vineyards with large populations present by the end of the season. Monitoring of these and other release sites w ill continue in 1995/96.

A video 'Wine, Women and Vineyard Mite Control with Doreen and Victoria' was released by the Communications Unit, NSW Agriculture in November, 1994. This video summarises the research and practical implications of the biological mite management program to date and provides a useful and practical guide for implementation.

Publications James, D.C. and Whitney, J.M. (1990) Biological control of Grapevine mites. Australian Crapegrower and Winemaker. 321, 37-43. James, D.G. and Whitney, J.M. (1991) Biological control

of Grapevine mites: An update. Australian Crapegrower and Winemaker. 328, 119-120. Whitney, J.M., James, D.G. and Warren, G.N. (1991) Detection and assessment of overwintering mite

populations on dormant grapevines using a microwave oven. Australian Crapegrower and Winemaker. 333, 46. James, D.G. and Whitney, J.M. (1991) Wine, Women

and vineyard mite control: The story of Doreen and Victoria. Australian Crapegrower and Winemaker. 333, 36. James, D.G. and Whitney, J.M. (1991) Biological control of grapevine mites. Proceedings o f the First National Conference o f the Australian Society o f Horticultural Science. 413-418. James, D.G. and Whitney, J.M. (1991) Biological control of grapevine mites in inland south-eastern Australia.

The Australian and New Zealand Wine Industry journal. 6, 210-214. James, D.G. and Whitney, J.M. (1992) Doreen and Victoria: Sex life exposed! Australian Grapegrower and

Winemaker. 340, 73-74. James, D.G. and Whitney, J.M. (1992) Biological control of mites in inland viticulture. Australian Crapegrower and Winemaker. 340, 127-128. James, D.G. and Whitney, J.M. (1992) Predators control

mites in vineyards. Good Fruit and Vegetables. 2,13. Whitney, J. and James, D.G. (1992) Doreen and Victoria: Viticultural supermites. Fifth Australian Applied Entomological Research Conference, Canberra. James, D.G. and Taylor, A. (1992) Effect of temperature

on development and survival of Amblyseius victoriensis (Womersley) (AcarhPhytoseiidae). International journal ofAcarology. 18:93-96.

James, D.G., Warren, G.N. and Whitney, J. (1992) Phytoseiid mite populations on dormant grapevines: Extraction using a microwave oven. Experimental and Applied Acarology. 14, 175-178.

James, D.G. (1993) How cumbungi and domatia w ill help Doreen and Victoria conquer grapevine mites. Australian Crapegrower and Winemaker. 352:1 30-1 32.

James, D.G. and Whitney, J.M. (1993) Cumbungi pollen: A laboratory diet for Amblyseius victoriensis and Typhlodromus doreenae (AcarhPhytoseiidae). Journal o f the Australian Entomological Society. 32:5-6. Taylor, A. and James, D.G. (1993) Effect of temperature

on development and survival of Typhlodromus doreenae. International journal o f Acarology. 19: James, D.G. (1993) Pollen, mould mites and fungi: Improvements to mass rearing of Typhlodromus

doreenae and Amblyseius victoriensis. Experimental and Applied Acarology. James, D.G. and Taylor, A. (1993) Predator population density influences oviposition rate in Amblyseius

victoriensis and Typhlodromus doreenae. International Journal o f Acarology. 19: James, D.G. and Whitney, J. (1994). Biological control of mites (Eriophyidae, Tenuipalpidae) in Australian

viticulture. IX International Congress o f Acarology, Columbus, Ohio. James, D.G. and Whitney, J. (1994) Vine mites: Development of biological control strategies for all

Australian viticultural regions. Australian Grapegrower and Winemaker Technical Issue pp. 106-108. James, D.G. and Whitney, J. (1994) Wine, women and vineyard mite control. I PM Networker 1:5. James, D.G, Whitney, J. and Rayner, M. (1995).

Phytoseiids (AcarhPhytoseiidae) dominate the mite fauna on grapevines in the Australian Capital Territory. Journal o f the Australian Entomological Society. 34:79­ 82. James, D.G. and Rayner, M. (1995). Toxicity of

viticultural pesticides to the Predatory mites, Typhlodromus doreenae and Amblyseius victoriensis. Plant Protection Quarterly. James, D.G. and Whitney, J. (1995). Biological control of

mites in Australian viticulture: State of the art. XIII International Plant Protection Congress, The Hague, The Netherlands. Whitney, J. and James, D.G. (1995). Biological control of mites in Australian viticulture: Implementation and sustainability. XIII International Plant Protection Congress, The Hague, The Netherlands. Whitney, J. and James, D.G. (1995). Biological control of mites in Australian viticulture: Implementation and sustainability. Ninth Australian Wine Industry Technical Conference, Adelaide. James, D.G. and Whitney, J. (1995). Grapevine mites: Development of biological control strategies for all Australian viticultural regions. IREC Farmers Newsletter No. 176:14-16. James, D.G. and Whitney, J. (1995). Mass-production and commercialisation of Doreen. Australian Crapegrower and Winemaker Technical Issue.


Project Title Strategies to reduce the incidence of cane and leaf blight-disease of grapevines

Project No DAN 94/1

Organisation NSW Agriculture

Location Biological and Chemical Research Institute, Rydalmere, NSW

Supervisor N G (Tan) Nair

Funding $51,760(1994/95)

Time Span June 1994 to July 1997

Progress 1. Development of winter dormant spray for Phomopsis.

The importance of winter dormant spray to reduce the carryover of the disease was recognised in our earlier research on this disease. Several chemicals

were, therefore, tested in the laboratory for their effect on the germination of pycnidia. Three different methods were used as follows:

a. In the first method, pieces of carnation leaves carrying the pycnidia were immersed in the chemical solutions for 24 hours and placed on unamended potato dextrose agar (PDA) for 72

hours. The germination of pycnidia was noted. b. Pieces (5 centimetre long) of one year old canes of grapevine bearing the pycnidia were sprayed with the different chemicals and then incubated

in a humid chamber at room temperature for seven days. Germination of pycnidia was counted as before. c. In this method, pieces of one year old canes

with the pycnidia were immersed in the different chemical solutions for three minutes and incubated in a humid chamber as before. Germination of pycnidia was determined as in

the previous methods. The chemicals tested were Shirlan, Benlate, Dithane, Delan, Bravo, Kocide, Citowet, Synatrol and summer mineral oil. Different concentrations ranging from 10 to 1 500 parts per m illion were tested.

Shirlan plus summer mineral oil was found to be the most effective in inhibiting the germination of Phomopsis pycnidia. This chemical was effective on taxon 1 and taxon 2. An interesting observation was that while delan was ineffective in preventing

germination of pycnidia of both taxa, dithane inhibited pycnidia of taxon 1 and not taxon 2 at 500 ppm. Dormant spray trials using Shirlan plus summer mineral oil (in vineyards with taxon 1 and taxon 2) and dithane (in vineyards with taxon 1) w ill be carried out in different vineyards in mid-winter and early spring of 1995.

The finding of a chemical capable of inhibiting pycnidia of Phomopsis is seen as a breakthrough in providing the growers with a winter dormant spray as a practical means to disease management.

What is equally interesting is that dithane (mancozeb) was effective in inhibiting pycnidial germination of only taxon 1 and not taxon 2 of Phomopsis at lower levels. This appears one possible cause of failure of dithane to control the disease of grapevines infected with taxon 2 in certain vineyards.

2. Vineyard trials - Trial sites representing grapevines infected with taxon 1 and taxon 2 of Phomopsis were selected in New South Wales, Victoria and South Australia. The main objectives are to study the symptoms characteristic of the different taxa of Phomopsis and to assess crop loss over a period of three years.

The trial sites were at Yenda, New South Wales (taxon 2); Sunrise, Mid-Murray, Victoria (taxon 2): Yarra Valley, Southern Victoria (taxon 1); and, Adelaide Hills, South Australia (taxon and taxon 2).

The occurrence of these taxa at the different sites was established by examining one year old canes collected in August 1994. The spring of 1994/95 was very dry at all the sites and, therefore, the development of the disease was retarded.

However, a schedule of spray applications was carried out and the trials w ill be assessed before harvest. The unfavourable weather conditions hampered

progress of the vineyard trials. Lack of recognisable levels of the disease meant that proper crop loss assessments could not be made. Low carry over of the disease inoculum is likely to affect disease

potential in 1995/96; hence, the vineyard trials. However, a change in weather pattern may see a significant increase in disease potential due to the chronic nature of the disease. These factors w ill be taken into account in our vineyard trials during



Project Title A reliable, cheap weather station as a predictor for improved disease and pest control

Project No DAS 92/2

Organisation Primary Industries South Australia

Location Loxton, SA

Supervisor P A Magarey

Funding $7,900

Time Span July 1992 to July 1996

Objective To encourage the development of a low cost, reliable, easily-used automatic weather station with capacity to incorporate disease and pest management intelligence, to be used by grapegrowers as a predictor to improve control of disease and pest events and to reduce chemical use in Australian viticulture.

Progress The initial design of a circuit board with an RS232 interface has been completed and a prototype weather station (AWS) constructed by Western Electronic Design from Coromandel Valley, S.A. The AWS has sensors to measure the temperature (-10°C to 60°C, ±0.5°C), relative humidity (from dry and wet bulb temperature, leaf wetness (resistive grid) and daylight/dark. The data scan interval of 1 minute provides data which are averaged over Ί0 minute intervals with (in default mode) AWS capacity to store data for 28 days or to communicate with wither central processing facilities or with personal computers.

The inclusion of a fax modem enables a variety of communication options to be used to download data via comma delineated ASCII code for more detailed processing as required. The prototype has

incorporated software with capacity to be programmed to predict the occurrence of disease and pest events. Low-power lights are inbuilt for signalling disease (and pests) events once models are installed.

The units are portable, robust and compact. They are powered by 6 AA sized batteries with replacement expected every 12 months. It is expected to have 5 units in field test-sites during the growing season 1995/96.One of these is to be

included in a collaborative test with the Apple and Pear Growers' Association of South Australia at

Lenswood Centre. The design of a low-cost rain gauge is nearing completion. The completed AWS is expected to market at $1000 including the cost of software to download data to personal computers.

The prototype is continuing to attract interest both within and outside Australian viticulture. The proliferation of interest in user-friendly weather stations and the recent advent of similar low-cost AWS, suggests this project w ill continue to a successful conclusion.

Project title Non conventional control of powdery mildew

Project code DAS 93/3

Organisation South Australian Research & Development Institute

Location Adelaide

Supervisor Trevor Wicks

Funding $31,000(1994/95)

Time span Sept 1994 - June 1995

Objectives To develop materials for the control of powdery mildew to reduce the use of traditional fungicides on grapes. This should reduce pesticide residues in wine and prevent the potential development of

powdery mildew resistance to fungicides.

Progress report Large scale field experiments were set up at the Lenswood, Nuriootpa, Loxton and Irymple Research Centres to evaluate the efficacy of treatments in different climatic areas. Although powdery mildew developed in all these areas the severity of the disease was very low and in most cases the treatments were not subjected to severe disease pressure. The unusually dry spring and early summer delayed disease development at most sites and resulted in little differences between treatments when bunch infection was assessed. However, assessment of disease on the basal leaves showed that although oils controlled powdery mildew they were not as effective as sulphur or Topas.

The two experiments set up at the Nuriootpa Research Centre to evaluate the efficacy of oil sprays applied in commercial air blast sprayers did not yield useful data due to the low incidence of


disease in the unsprayed plots. The poor development of disease in the 1994/95 season was demonstrated in the Crouchen planting at the Nuriootpa Research Centre where for the past 15 years bunches in the unsprayed plots have been completely covered with mildew by early summer. Bunch assessment in January 1995 showed only trace levels of mildew in the unsprayed plots.

Useful data was obtained from two experiments in Adelaide where the treatments were applied to bunches grown under pergolas. High levels of mildew developed in the unsprayed bunches at both sites whereas oil treatments suppressed the development of mildew.

Other progress with this project has been the establishment of cultures of the powdery mildew fungus on potted vines at the Plant Research Centre as well as the maintenance of disease free

plant material for laboratory evaluation of chemicals. Using a 'Potter's tower' to apply precise rates of chemical and a known level of inoculum, 9 of the 18 chemicals evaluated (mainly oils) showed good protectant activity against powdery

mildew on excised leaves. Overall the 1994/95 experiments have shown that oil formulations are potentially useful chemicals for the control of powdery mildew on grapes although they are less effective than conventional fungicides. However spray coverage

appears to be critical for effective control with oils. Further evaluation of these materials is needed particularly with use in air blast spray machines, rates of application, and integration with reduced rates of conventional fungicides in areas subjected to high levels of disease.

Project Title Development of a national management strategy for grape phylloxera

Project No DAV 93/2

Organisation Agriculture Victoria

Location Sunraysia Horticultural Centre, Mildura

Supervisor Dr Greg Buchanan

Research Staff John W hiting (AV), Ross Turkington (NSW Agric.), Julie Hawtin (AV)

Funding $27,550

Time Span January 1994-December 1996

Objectives • To train vineyard staff and government personnel in detection and diagnosis of phylloxera. • To coordinate surveys of phylloxera infested

areas of south east Australia. • To develop collaborative links with European and North American scientists working on phylloxera. • To develop a national policy and operational

plans for management of phylloxera.

Progress This project is closely linked with an HRDC- funded project investigating the biology and control of phylloxera.

The third in a series of practical, 'hands on’ workshops was held at Rutherglen in January 1995. It was attended by over 30 vineyard managers, who were able to examine live phylloxera on leaves and roots microscopically and to find phylloxera in vineyards. A comprehensive set of notes was provided to participants.

The value of training programs was well illustrated by the early detection of phylloxera by a grower in the King Valley. Surveys of 43 vineyards (covering 360 ha) were carried out, resulting in the declaration of a new King Valley Vine Disease

District. There are seven vineyards known to be infested in the King Valley. Aerial colour photographs of King Valley vineyards proved useful as a mapping tool, but did not replace the need for root inspection of vines. Detailed information of surveyed vineyards was obtained as the first step in creating a database.

An active program to maintain awareness of phylloxera has continued, with signs for vineyards, printing of revised colour brochures and a Code of practice, field days and seminars, and contributions to industry newsletters and journals. The awareness was further heightened by well- targeted reports of the King Valley outbreaks.

Ms Angela Corrie is to visit overseas scientists working on phylloxera during July 1995. The study tour includes laboratory work on DNA typing at University of California (Davis), field surveys of

phylloxera in eastern North America, and visits to research centres in France and Italy. The study tour w ill benefit work on DNA typing of phylloxera. Progress towards development of a national

policy includes the draft Code of Practice which is


already widely used, and the formulation of a discussion paper on uniform quarantine regulations between States. The discussion paper w ill be circulated in September 1995.

Project Title Reduction of the impact of powdery mildew in Australian viticulture

Project No DAV 94/1

Organisation Agriculture Victoria

Location Sunraysia Horticultural Centre, Mildura

Supervisor Dr R W Emmett

Funding $50,000 (1994/95)

Time Span July 1994-June 1996

Objectives To reduce the impact of powdery mildew in Australian viticulture through an international collaborative research and extension program aimed at:

1. increasing the adoption of improved disease management practices developed as a result of research in Australia and overseas; and 2. decreasing early season disease incidence by developing management procedures for reducing sources of overwintering inoculum in vineyards.

Progress At the Second International Workshop on Grapevine Mildew Diseases, discussions with overseas plant pathologists highlighted the

importance of controlling overwintering inoculum of powdery mildew. In Germany, in years when severe frosts in winter kill weakened buds infected with powdery mildew, flag shoots do not appear in spring, disease epidemics do not develop until after veraison and crop loss is greatly reduced.

Studies of flag shoot incidence in Australian vineyards have indicated that at least half of diseased shoots appear within less than 4m of vines with flag shoots in the previous season. Similar observations have been made in northern Italy where most flag shoots occur less than 2m from those in the previous season. Studies to determine the window of bud infection in Australian vineyards have been commenced.

In Germany, where vines are mostly vertical

shoot positioned and maximum bud numbers on pruned canes are limited to 12, a spray of a demethylation inhibiting (DMI) fungicide applied

when new shoots have 6-7 leaves appears to prevent most bud infection and the development of flag shoots. However, this approach may not be applicable to all Australian vineyards where vine

canopy management and pruning systems vary widely. Nevertheless, during evaluation of disease management strategies in a number of Australian vineyards, the above findings have been substantiated. In vineyards where a concerted effort was made to reduce initial inoculum loads

by concentrated early season spraying, and in one case, by the physical removal of the flagshoots, vineyard inoculum levels were reduced. This led to reduced disease pressure and has allowed a reduced spray program to provide good control. This approach w ill be tested further.

Field trials to study the effects of pre- and post­ harvest sprays of sulphur, oil and DMI fungicides on cleistothecium development were also conducted in 1994/95 and data is being analysed.

Arrangements have been made to hold the Australian Technical Workshop on Grapevine Powdery Mildew in Adelaide in September 1995. Invited participants w ill include key researchers, representatives of industry, and representatives of research funding providers involved with the development and application of management strategies for grape powdery mildew. Overseas contributors w ill include Dr D Gubler from the

University of California, Davis, USA and Dr H Denzer from Laimburg Research Centre, Auer, Italy. Key topics to be addressed include the industry perspective, a review and evaluation of recent research in Australian and overseas management strategies and their limitations, key control options for adoption by industry and future

research and extension priorities. Arrangements have also been made for international contributors to the workshop to participate in some district extension meetings especially those held in Sunraysia, Riverland, Barossa Valley and Southern Vales, Central Victoria and the Murrumbidgee Irrigation Area. Meetings in other districts of NSW, Victoria and South Australia w ill be conducted by project staff during the latter part of September, 1995. Some district meetings may also be conducted in Western Australia.




Project Title Production of a soil test interpretation manual

Project No ASP 94/1

Organisation Australian Soil and Plant Analysis Council

Location Melbourne Victoria

Supervisor Dr Ken Peverill

Funding $2,000 (94/95)

Time Span 1994/95 - 1996/97

Objectives To publish an Australian manual of soil testing and interpretation which w ill enable consistency in interpretation, terminology and recommendations.

Progress The project commencement date was delayed until 1 April 1995 owing to the inability of ASPAC to negotiate for a single body to co-ordinate the

project management. A two day in-person meeting involving key contributing scientists was convened in Melbourne on 17-28 April 1995. The meeting determined the publication format, subject matter and basis for acceptance of data w ithin the book. It also established that a network of state co-ordinators would act as key contact points for accessing

published and unpublished data and organising biometric analysis of soil data. Potential authors and key contributors for chapters and major sections were also nominated on the basis of the

relevant experience, current research/extension activities and proven ability to complete tasks. Initial planning with respect to choice of publisher copyright arrangements, number of copies etc. was

also discussed. During 1995/96 data collection and ongoing co-ordination of work w ill proceed.


Project Title Professor T H Lee - OIV Expert Group meetings - Paris

Project No AWR 94/1

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Time Span 25 November-5 December 1994

Funding $3,750

Report The report covers participation in the meetings of the Office International de la Vigne et du Vin

(OIV) Expert Groups in Paris on 28 November-3 December. A more comprehensive report of the business of these meetings has been made to the International Trade and Technical Advisory

Committee of the Australian Wine and Brandy Corporation, to the Technical Committee of the Winemakers' Federation of Australia and to the Institute Council, and has been published in the

Institute's February 1995 issue of Technical Review. The meetings attended included Technologie du Vin, Microbiologie du Vin, Code International des Pratiques Oenologiques; as a result of being elected President of the latter Expert Group for the

next three years the author became a member of the Comite Scientifique et Technique. Two papers, entitled Management of winery wastewater in Australia and Application o f biotechnology to the

improvement o f wine yeasts, were presented to the Expert Groups, Technologie du Vin and Microbiologie du Vin, respectively, on behalf of J A Chapman and Dr P A Ffenschke.

Project Title Professor T FH Lee— OIV Expert Group Meetings— Paris

Project No AWR 94/2

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Time Span 2-26 June 1995

Funding $10,530


Report The purpose of these visits was to attend meetings of the OIV Expert Groups, Technologie du Vin, Microbologie du Vin and Code International des Pratiques Oenologiques, on 12-14 June 1995, of the OIV Subcommission on Methods of Analysis on 8-10 June 1995, and of the Comite Scientifique et Technique on 15 June 1995 , A full report of the meetings has been made to the International Trade and Technical Advisory Committee of the Australian Wine and Brandy Corporation, to the Technical Committee of the Winemakers'

Federation of Australia and to the Institute Council, and has been published in the August 1995 issue of the Institute's Technical Review. Other visits were made to government organisations in the UK, Germany and The Netherlands and the author participated in a bilateral meeting with the European Union in Brussels on 20 June 1995.

Project Title Land disposal of winery and distillery wastewater

Project No UA 92/2

Organisation The University of Adelaide

Location Adelaide

Supervisor Professor J M Oades/J Chapman

Funding $ 1,500

Time Span July 94 - October 95

Progress The PhD program of Ms Jeanette Chapman is complete and the project UA 92/2 has terminated in August with the submission of the thesis.

The thesis includes a literature survey of the production of wastewater by the wine industry in Australia. The information was supplemented by analysis of the organic and inorganic materials in winery and distillery wastewaters. Most of the organics were soluble and were dominated by simple carboxylic acids, sugars and alcohols. Salinity and pH were highly variable depending on management procedures.

Using ,4C-lactic acid and l4C-glycerol the two most abundant chemicals in wastewaters the fate of organics in wastewaters added to soils was determined. A logistic model was used to quantify

the adsorption and microbial decomposition of the ,4C-organic compounds. The effects of soil texture, acclimatisation of microbial populations and organic loadings on the adsorption and mineralisation of the organic compounds were determined.

Experiments involving irrigation of undisturbed soil cores confirmed results obtained with disturbed soil samples and it was shown that soils acclimatised to winery wastewaters were able to mineralise organic compounds to CO, in 1 to 2 days after irrigation providing the addition of wastewater and its organic loading were controlled.

The results were used to develop a model for the management of soils irrigated with winery wastewaters which is the scientific basis for protocols to dispose of wastewaters by irrigating soils.

Implications for the management of wineries and distillers with respect to the production of wastewater are discussed.

Project Title Selection of wine yeasts and malolactic bacteria for desirable glycosidases and sensory enhancement of wines

Project No UCS 92/4

Organisation Charles Sturt University

Location Wagga Wagga

Supervisors Dr Bryan Todd (CSU) Dr Paul Henschke (AWRI)

Research Staff Charoen Charoenchai

Funding $15,000(1995)

Time Span May 1995-October 1995

Objectives 1. Survey of β-glycosidase activity in wine yeasts and malolactic bacteria originating from commercial sources and the culture collections

of CSU, AWRI, UNSW and UA. 2. Assess selected wine microorganisms for the ability to hydrolyse glycosidically bound flavour precursors isolated from grape juice. 3. Evaluate selected wine microorganisms for their



potential to enhance wine flavour and aroma. 4. Evaluate desirable glycosidases from selected wine microorganisms and assess their potential for releasing glycosidically bound flavour

components during the wine making process.

Progress Following approval of provisional funding to recommence research progress (May 1995), Mr

Charoen Charoenchai was appointed as a research assistant to investigate glycosidase activity in microorganisms associated with winemaking. Initially, research has been directed towards

validation of preliminary data that was obtained using a crude extract of grape glycosides as a carbon source in semi-defined media. W hile the preliminary work did indicate that increased growth was obtained by selected strains of

Leuconostoc oenos when grown on these extracts, it was not possible to conclusively demonstrate the existence of β-glycosidase activity in the microorganisms investigated. An agar medium was therefore developed which contained a β-glycoside analogue, 4-m ethylumbelliferyl^-D-gluco-

pyranoside, as a carbon source. The hydrolysis of this compound via β-glycosidase activity results in the formation of 4-methylumbelliferone, a fluorescent compound which can be observed

under long wave UV light. Using this medium, Mr Charoenchai has been able to demonstrate that a variety of wine microorganisms (yeasts and lactic acid bacteria) do possess such enzyme activity,

and that the level of expression is strain specific.

The wine yeasts examined included strains of Saccharomyces cerevisiae in addition to a selection of non-Saccharomyces yeasts (isolated from wines undergoing fermentation and belonging to the genera Kloeckera, Candida and

Hansenula). In general, strains of 5.cerevisiae produced little or no β-glycosidase activity in the medium without glucose present. However, the addition of 0.5% glucose to the medium appeared to stimulate β-glycosidase activity in these yeasts, although whether this is merely due to increased growth is not clear at this stage. Interestingly, some strains of non-Saccharomyces yeasts belonging to the genera Candida and Hansenula appeared to express higher levels of β-glycosidase activity than

strains of 5.cerevisiae.

In contrast, selected strains of lactic acid bacteria belonging to the species Leuconostoc oenos produced higher levels of β-glycosidase activity in agar media in the absence of glucose, suggesting that such expression may be inhibited by residual glucose during wine fermentation.

Quantitative studies in liquid cultures are currently being developed to confirm the preliminary data observed in agar media.


Microbiology Group

Project Title Yeast evaluation and optimisation of fermentation performance: influence of physical environment, grape juice composition and yeast

physiological properties

Project No AWR 2CW

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Dr P A Henschke

Funding $214,966

Time Span 1990/91-1994/95

Objectives • Evaluate yeast fermentation performance and winemaking characteristics under defined, standardised conditions. • Establish the important winemaking variables

on, and study physiological and biochemical aspects of yeast metabolism relating to, acetate, nitrogen and sulphur metabolism to provide practical advice on controlling volatile acidity,

incomplete fermentation and formation of sulphides. • Study the biochemistry of yeast physiological condition and evaluate predictive tests with the

aim of improving yeast activity and fermentation performance. • Investigate strains and conditions for improving


the efficiency of sparkling wine production. • Advise winemakers on yeast strain characteristics, handling and fermentation



Systematic comparative evaluation of wine yeast strains The background and previous developments in this ongoing program have been detailed in former GWRDC and AWRI annual reports.

7. S en sory e v a lu a tio n o f c o m m e rc ia l w in e yeasts The aim of this project was to determine the influence of yeast strain on the sensory properties of wine as determined by formal sensory analysis and to establish its chemical basis. Furthermore, the persistence of yeast-induced sensory characteristics in aging wine was quantified. Replicated batches of Chardonnay wines made with six yeasts, Maurivin AWRI 796, AWRI 835,

Lalvin EC1118, Lalvin ICV D47, Lalvin R2 and Avize, were stored at constant temperature, and sensory analysis performed after three and 18 months.

As stated in the previous report, wines made with the five yeasts, Maurivin AWRI 796, AWRI 835, Lalvin EC1118, Lalvin ICV D47 and Avize, were found to be significantly different (at the 5% level or better) from at least two other wines after aging for three months at 4°C. These wines were retested after an additional 15 months ageing at 4°C. O f the six pairs of wines tested, those made with AWRI 835 and Lalvin ICV D47 remained significantly different from that made with Avize. These results confirm the general view that yeast strains impart specific flavour characteristics to wine and that some of these characteristics quickly dissipate with time while others are more persistent in wine. It was not possible, however, to reliably describe the differences between the wines.

This work was carried out by Ms Patricia Jane for her higher degree studies in the Department of Horticulture, Viticulture and Oenology, The University of Adelaide. The work was supervised by Dr Paul Henschke and Professor Terry Lee.

2 . R o le o f ind igen ou s yeasts in w in e m a kin g

The yeasts associated with grapes are believed to play a variable role in the fermentation of grape

juice depending on grape condition, harvest conditions, must processing, the success of yeast inoculation and the physico-chemical and nutrient

conditions of fermentation. These yeasts are mainly strains of the species Hanseniaspora uvarum (Kloeckera apiculata), Torulaspora delbrueckii (Candida colliculosa), Candida stellata, Candida krusei, Kluyveromyces thermotolerans and Hansenula anomala. Although some information is becoming available on the metabolic activities of these yeasts, the importance of these yeasts to wine flavour and quality is poorly understood. This project w ill characterise the oenological properties of the non-Saccharomyces yeasts isolated from fermenting grape juice and w ill determine their suitability for producing wine.

A collection of non-Saccharomyces yeasts has been isolated from spontaneous wine fermentations conducted in wineries in California and Burgundy during the 1994 vintage. These yeasts were isolated and purified on Lysine agar medium, a medium which prevents the growth of Sacch. cerevisiae strains. Classical taxonomic identification of these isolates has been attempted on a BIOLOG identification system in Professor Graham Fleet's Laboratory at The University of

New South Wales. Type strains of the frequently reported non-Saccharomyces yeasts have been obtained from the culture collection at Delft for use as references for identification using the BIOLOG system and genetic techniques (see Project AWR 3GW).

Ms Alison Soden visited various winegrowing regions and research facilities in the USA and Europe during October-November 1994. This trip was funded by Lallemand. One of the aims of the trip was to view facilities in other laboratories and to discuss aspects of the project with wine

microbiologists, especially those with an interest in alternative fermentation yeasts and spontaneous alcoholic fermentation. An industry perspective was gained, especially in California, where there is great interest in non-inoculated fermentation. Some consider that complex and interesting wines can be made in this manner, and some wineries are in their fifth year of production using these techniques.

This project forms the basis of a PhD research program for Ms Soden, who is being supervised by Dr Henschke and Professor Lee. Funding is being provided by Lallemand Australia Pty Ltd and an Australian Postgraduate Research Award (Industry).


Yeast aldehyde dehydrogenase and regulation of acetic acid production Literature concerning the role of acetic acid and the acetate esters in yeast metabolism and the

sensory properties of fermented media has been reviewed. A conclusion, relevant to this project, is that acetic acid is produced, mainly during the yeast growth phase, as a regulator of the metabolic

redox balance needed for anabolic metabolism. During anaerobic fermentation, acetic acid is believed to form mainly by the oxidation of acetaldehyde. Three isoenzymes which demonstrate acetaldehyde oxidising ability have been reported in Sacch. cerevisiae. W hile tw o are repressed under fermentative conditions during growth on glucose, the other one is apparently active. The enzymes are located separately in the mitochondrion and the cytosol. To determine the

role of the cytosolic enzyme in acetic acid production, it is necessary to inhibit the mitochondrial enzymes. This has been done using a rho° Sacch. cerevisiae mutant, one which has no mitochondrial function. No aldehyde dehydrogenase enzyme activity other than the cytosolic enzyme was detected.

The rho° mutant can ferment a semi-synthetic YPD medium containing a high concentration of glucose, both aerobically and anaerobically. This yeast produces a significant amount of acetic acid under these conditions (up to 1 g/L), which suggests that the cytoplasmic aldehyde dehydrogenase is important in this pathway.

Furthermore, a preliminary experiment showed that the specific activity of this enzyme in yeast is correlated in a non-linear (sigmoidal) manner with the rate of acetic acid production during fermentation. These data suggest that, immediately

after inoculation and again at the end of exponential growth, factors other than the absolute enzyme activity are important in determining the rate of acetic acid production. The most likely factors involved are the size and availability of the

cofactor (NADP*) pool, and the carbon flow through each of the relevant pathways at these stages of growth. Future fermentation experiments w ill be conducted with a view to determining the

relative contribution of these factors to the regulation of acetic acid production. Fermentation experiments have also commenced to investigate whether vitamins have a direct or indirect role in regulating acetic acid production in the rho° mutant yeast. These studies require the

use of a minimal medium which can be supplemented with nicotinic acid and thiamin. To date, a suitable medium for supporting the growth of this fastidious mutant has not yet been defined.

This work is being performed at the Institute by Mr Jeffrey Eglinton as part of his higher degree studies with The University of Adelaide. The work is being supervised by Dr Peter Langridge, The

University of Adelaide, and Dr Henschke.

Nitrogen composition of grape juice: analytical methods Three commercial HPLC systems were evaluated for the quantification of physiological amino acids

in grape juice and wine. The systems considered were the Waters AccQ.Tag, CBC Aminomate and Hewlett Packard amino acid analysis system. Each system is based on prederivatisation of the amino acids before separation on a reverse phase column with fluorometric detection. The AccQ.Tag system uses AccQ-Fluor, the Aminomate uses FMOC and the HP system uses a combination of OPA and

FMOC as derivatising agents. Each system was evaluated with a standard amino acids reference mixture, and grape juice and wine samples, with and without added amino acids. Unlike the current system, these analysers offer quantification of all the amino acids including proline, and ammonium

in a single determination. The CBC Aminomate has been selected for purchase. Routine estimation of the assimilable nitrogen concentration of grape juices requires a rapid, reliable, and inexpensive technique. Several chemical and physical methods have been suggested, and these are being evaluated by reference to amino acid plus ammonium concentration. Crape juice samples were obtained from the 1995 vintage.

Selection of wine yeasts for producing lower alcohol wines The alcohol content of wine made under Australian conditions is essentially determined by the sugar content of the must at the time of

inoculation. All commercial Saccharomyces wine yeasts have been selected for efficient conversion of sugar to ethanol and carbon dioxide so consequently wines made by different strains have a similar alcohol content. There are several situations, however, under which a lower alcohol content than that determined by the sugar content

is desirable: overripe grapes due to unavoidable


delays in harvest; deliberately overripened grape as a means to develop riper fruit flavours; and a preference to produce wine with a lower alcohol content. Although physical techniques are successfully being used to modify alcohol content of wine, it is the objective of this study to investigate the development of lower efficiency fermentation yeasts using techniques in molecular biology.

7. Strategies The yeast Sacch. cerevisiae efficiently converts sugar to ethanol during grape must fermentation, with approximately half of the sugar being metabolised into ethanol. W ith assistance from Dr

Neil Brewster an extensive literature review was performed to ascertain which pathways of intermediary metabolism can be manipulated in order to redirect some of the sugar into other compounds that are compatible with wine. Some of the options are increasing yeast reserve compounds, such as trehalose and glycogen,

increasing yeast biomass formation, and increasing the production of organic acids or glycerol. Because of the limitations of the above strategies, the focus of the research is to sequester carbon by increasing glycerol production. Glycerol

is a three-carbon polyol synthesised by reduction and dephosphorylation of dihydroxyacetone phosphate, an intermediate in the synthesis of ethanol from glucose. The key enzyme in the synthesis of glycerol is glycerol phosphate dehydrogenase and it has been shown that, in at

least some conditions, the amount of glycerol produced is directly related to the level of this enzyme. Previous work performed in the laboratory by Ms Ruth Setiawati and Mr Jeffrey Eglinton provides a method for measuring the activity of this enzyme during fermentation. We are currently cloning the gene using the

polymerase chain reaction (PCR). The amplified product is being cloned into plasmids (developed by Dr Jenny Petering and Ms Renata Polotnianka) that can be transformed into wine yeast strains where the effect of overexpressing this gene on glycerol and ethanol production during fermentation w ill be analysed.

2. E x p e rim e n ta l A genetic screen for identifying mutants that over produce glycerol is being developed and evaluated by Dr Miguel de Barros Lopes. This screen, which

depends on the relationship between glycerol synthesis and osmotic stress, should lead to the isolation of strains that produce less ethanol during fermentation. Interestingly, preliminary results have demonstrated that laboratory strains of Sacch. cerevisiae are more resistant to osmotic stress than wine strains. Genetic aspects of this project are reported under Project AWR 3GW.

This project forms a major focus of work being performed by Dr de Barros Lopes who is employed as a postdoctoral fellow by the Institute through the Cooperative Research Centre for Viticulture. The work is being supervised by Dr Langridge, The

University of Adelaide, and Dr Henschke.

Biocontrol of indigenous yeasts during fermentation The aim of this project is to explore the potential of yeast zymocidal activities for controlling the proliferation of indigenous yeasts during the fermentation of grape must as a strategy for reducing the need for SO, addition to lim it yeast growth. Mr Nicholas Yap was recently awarded a CRC for Viticulture postgraduate scholarship to undertake studies on this project.

Metabolism and autolysis of yeasts during 'champenoise secondary fermentation' The experimental work of this project has been completed as indicated in the previous report. Mr

Bryan Todd was recently awarded the PhD degree by The University of New South Wales for his work on this topic. Professor Fleet and Dr Henschke supervised the work which was carried out at The University of New South Wales.

Project Title Selection and improvement of wine yeasts by application of molecular biology

Project No AWR 3GW

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Dr P A Henschke

Funding $101,826

Time Span 1990/91-1994/95


Objectives • Continue development and evaluation of techniques for identification and marking of strains.

• Study the biochemistry and genetics of strain characteristics targeted for strain improvement by molecular genetics techniques.

• Construct strains with enhanced winemaking properties by application of recently established recombinant DNA protocols. • Evaluate the performance of microorganisms

manipulated by molecular genetics techniques. • Establish the structural requirements of strains destined for restricted and unrestricted release.

Progress Genetic alteration of wine yeasts for producing lower alcohol wines Genetic manipulation of Saccharomyces cerevisiae wine yeast is being undertaken with a focus on

reducing ethanol production by increasing glycerol production, as indicated in previous reports and in Project AWR 2GW. The key enzyme in the synthesis of glycerol is glycerol phosphate dehydrogenase, and it has been shown that, in at

least some conditions, the amount of glycerol produced is directly related to the level of this enzyme. Previous work performed in the laboratory by Ms Setiawati and Mr Eglinton

provides a method for measuring the activity of this enzyme during fermentation. Dr de Barros Lopes is currently isolating the genes encoding glycerol phosphate dehydrogenase from wine yeast by using the polymerase chain

reaction (PCR). Two genes, GPD1 and CPD2, have been amplified, cloned into general vectors, and are presently being sequenced. Plasmids are being constructed that w ill allow controlled expression of the GPD1 and GPD2 genes in laboratory, and

ultimately, wine yeast. The physiological consequences of altered glycerol production are being investigated.

Identification of wine yeast strains by genetic techniques The realisation that the yeast strain used for fermentation plays an important role in determining the flavour and quality of wine has encouraged the need for the development of a

rapid, simple, routine method for yeast strain identification. In the 1989 annual report, we first reported the development of a method for the

unequivocal identification of Sacch. cerevisiae wine yeasts and the potential for applying the method to non-Saccharomyces yeasts. The method relied on the high polymorphisms that exist in yeast chromosome length. W hile the method has proved useful for yeast producers and researchers, the method is not suited to routine use. The aim of the present study is to develop a technique for the

rapid identification of both Saccharomyces and non-Saccharomyces yeast strains. Such a method would not only facilitate the verification of wine yeast strains but also facilitate ecological studies of grape must fermentation and improve the efficiency of yeast inoculation.

Several methods exist for identifying and differentiating yeasts, but these techniques have similar disadvantages as chromosome analysis, and are not sufficiently sensitive for differentiating strains of the same species. The polymerase chain reaction (PCR) technique is being investigated as the basis for an alternative method which should overcome these limitations. PCR relies on the choice of suitable oligonucleotide primers to provide DNA sequence information necessary to establish the identity of a strain. Many choices for the design of primers are possible— DNA sequences based on regions of DNA that are hypervariable in an evolutionary sense can generate the polymorphisms required for good strain differentiation. Initially this project w ill focus on DNA primers that recognise the intron-exon splice sites of yeast DNA. Introns are intervening sequences that separate the coding regions (or exons) of a gene and have been found in the genomes of all eukaryotes. The exact role of

introns is unknown but since they are not essential for gene function, their presence and size are variable. Sequences within the intron are,

however, required for their precise excision, and consequently are well conserved amongst the different yeast. Four oligonucleotide primers, that

anneal to these conserved sequences, have been designed and tested for generating polymorphisms between laboratory and wine Sacch. cerevisiae strains using PCR amplification.

Initial PCR screens were performed using purified yeast strain DNA. As this is a time consuming step, yeast pretreatment steps were investigated so that PCR could be performed directly on a small sample of yeast. Freezing and

boiling of the yeast sample produced the best results. The method has now been modified so that


reliable results can be obtained directly on yeast from agar plates. This makes the method suitable for routine use. The technique works most efficiently for cells in the stationary phase of growth. O f the four primers that have been screened, only one has shown good potential for the identification of Sacch. cerevisiae strains, and also appears to be useful for species identification.

Ms Soden, under the guidance of Dr de Barros Lopes and Dr Dara Whisson, is developing a similar PCR method for identifying non- Saccharomyces yeasts. Yeast DNA purification techniques were compared initially, and a suitable protocol for DNA purification was established. The quick freeze/boil technique developed for Saccharomyces yeast was found to be adequate for the yeasts tested. PCR conditions, such as extension step duration and magnesium ion concentration, were also examined and were optimised for greatest band resolution. The 19 type strains of non-Saccharomyces yeasts tested showed distinguishable banding patterns, allowing the differentiation of all these species and strains. Further PCR experiments are underway to improve the reproducibility of the results and look at the stability of banding patterns of successive cell generations. This w ill be of importance when using

PCR to follow yeast strain growth during fermentation experiments. When testing strains of Sacch. cerevisiae it was observed that these primers gave amplification products that were present in all strains of the species. By comparing these patterns with those of other closely related yeasts it was determined that the PCR amplification fingerprint of Sacch. cerevisiae is unique to this species even when compared to that of Sacch. bayanus, a yeast of the same genus. Other species studied could also be defined by a characteristic amplification pattern.

These results suggest that intron-exon primers can be use as a rapid yeast identification system. Using the technique, several of the unknown yeasts present in grape juice have been correctly identified at this point in time. These include yeasts from the species Sacch. cerevisiae, Kloeckera apiculata, Candida krusei and

Torulaspora delbrueckii. In addition the lack of a sexual cycle poses no barrier to identification. Approximately 40 different species of yeasts have reportedly been isolated from grapes, must and wine. By determining the amplification fingerprint for these species it should be possible to rapidly

identify the main yeasts of significance to the wine industry.

Purification and cloning of cytosolic aldehyde dehydrogenase Further progress has been made on the molecular biology of yeast aldehyde dehydrogenases, enzymes believed to be responsible for acetic acid metabolism during grape must fermentation. The aim of this work is to clone the aldehyde dehydrogenase gene so that it can then be used to verify its role in anaerobic acetic acid metabolism. If successful, the gene w ill be manipulated to give yeasts with altered acetic acid production.

The fermentation experiments, referred to previously and in Project AWR 2CW, have shown that, of the three AIDFH isoenzymes which demonstrate acetaldehyde oxidising ability in Sacch. cerevisiae, the cytosolic enzyme is the most important under vinification conditions. The genes for the two mitochondrial isoenzymes have

previously been identified and cloned by others. However, the gene which encodes for the cytosolic enzyme has not. Attempts to clone this gene(s) have, to date, been unsuccessful. Traditional methods, such as complementation, have been unsuitable since no mutants are available. As previously reported, Southern analysis has also yielded negative results. The present approach to identify the gene involved has been to utilise DNA amplification (PCR) techniques. Essentially, short pieces of DNA (primers), either highly specific or degenerate, are used to 'amplify' a larger piece of DNA which contains a part or the whole of the AIDF1 gene. In the present experiments, primers based on either the conserved regions of 21 AIDH enzymes of diverse organisms or the amino acid sequence from yeast AIDH, which was purified previously, have been used to amplify yeast DNA using PCR. A variety of sources of yeast DNA have been investigated. These were (1) chromosomal DNA, (2) an E. coli 'library' of yeast DNA, and (3) cDNA, which was prepared by reverse transcription of RNA made from the rho° Sacch. cerevisiae yeast during the stage of cell growth in which the yeast is known to contain a high enzyme activity. DNA, which has been isolated using PCR, has been transformed into bacteria in order to determine its nucleotide sequence. To date, one sequence has been obtained, which shows strong homology to a bacterial AlDH and lesser homology to AIDHs


from other organisms, principally Aspergillus. Sequence analysis of tw o further clones is underway. These results confirm the suitability of PCR and the synthesised primers for this work. Sequence analysis of isolated DNA w ill continue.

This work is being performed at the Institute and The University of Adelaide by Mr Eglinton as part of his higher degree studies with The University of Adelaide. The work is being supervised by Dr

Langridge, The University of Adelaide, Dr de Barros Lopes and Dr Henschke.

Proline metabolism Crape juices low in assimilable forms of nitrogen often contain a relatively high concentration of proline. W ine yeasts are, however, unable to

utilise proline as a source of nitrogen during normal anaerobic fermentation. The first aim of this project was to characterise the accumulation of proline and some of its metabolites by Saccharomyces wine yeasts; the results of this work were reported previously. The second aim is to identify and study an organism which can catabolise proline under anaerobic conditions as a prelude to genetic engineering studies with the aim of transferring this ability to wine yeast.

The physiological studies reported previously in Project AWR 2GW have identified that, while fermentation yeast are not able to catabolise either D- or L-proline anaerobically, suitable intermediate substrates which do not require mitochondrial

involvement have been identified. Studies are now in progress to identify organisms which are able to catabolise proline to yield suitable products without the involvement of oxygen metabolism.

A variety of algae, plant cells and bacteria associated with anaerobic conditions have been identified from the literature and several screened in the laboratory. The screening work was carried out in Dr John Brooker's laboratory in The

University of Adelaide. A potentially interesting enzyme, pyrroline carboxylate (P5C) reductase, has been identified in a plant species. P5C reductase, which normally reduces pyrroline-5- carboxylic acid to proline, has been reported, in the literature, to carry out the reverse reaction. This oxidation of proline, however, only occurs at high pH; this reaction has been confirmed in cell extracts of the plant species. P5C reductase has also been isolated in a crude cell extract of Sacch.

cerevisiae, but under similar experimental conditions to those used for the plant species, it

was not possible to demonstrate the oxidation of proline under conditions of high pH. Cloning of P5C reductase from the plant species into yeast is nearly completed.

This work forms the basis of a PhD research program for Mr Christopher Smyl in the CRC for Viticulture program and is supervised by Dr Langridge, The University of Adelaide, and Dr Henschke.

Control of gene expression during fermentation Work is continuing to improve our understanding of gene regulation in wine yeast. This w ill permit a more efficient expression of foreign genes selected to improve the oenological characteristics of

production strains. In addition, the effect of environmental factors, such as increasing sugar and alcohol concentration during fermentation, on gene regulation has been investigated. The present work has continued on the isolation of promoter regions which activate genes at specific stages of the alcoholic fermentation.

Several candidate sequences, an early time- specific and a late time-specific expression sequence, present in cDNA clones, have been shown to possess high homology with a ribosomal protein and lesser homology with a yeast cell wall protein, respectively. The regulator regions (promoter sequences) from these genes have been isolated. That from the ribosomal gene was isolated using PCR with a primer based on published information. The late time-specific promoter was isolated from a genomic DNA library using the characterised cDNA cloned sequence to design suitable probes. The function of the isolated promoter sequences is now being studied in yeast by integrating the promotor sequence into a yeast transformation vector containing the β-glucuronidase reporter gene. The activity of the promotor, and hence gene, was monitored by the convenient CUS assay which was previously developed for genetically marking wine yeasts. This assay system is being applied to yeast cells isolated from different stages of fermentation to verify the time specific activity of the two promotors.

This project is being carried out by Ms Polotnianka as part of her PhD program in the CRC for Viticulture program and is being supervised by Dr Langridge, The University of Adelaide, and Dr Henschke.


Project Title Optimisation of malolactic fermentation in wine and strain improvement by application of molecular biology

Project No AWR 4GW

Organisation The Australian W ine Research Institute

Location Urrbrae, SA

Supervisor Dr P A Henschke

Funding $173,481

Time Span 1990/91-1994/95

Objectives • Investigate the conditions for simultaneous induction of biological deacidification and alcoholic fermentation and evaluate practical

considerations. • Investigate the strain, and nature and time- course of flavour compounds elaborated in wine during the MLF. • Investigate methods for strain identification. • Study the use of mutant strains for modifying

the metabolic capability of bacteria in relation to wine composition and flavour. • Genetically characterise wine lactic acid bacteria, and investigate the possible

development of a transformation system.

Progress Induction protocols for MLF Many wineries continue to rely on naturally occurring strains of malolactic bacteria to carry out MLF, however growing evidence suggests that naturally occurring strains can reduce wine quality through histamine formation, mousy taint and other flavour defects, such as a high concentration of volatile acidity and diacetyl. Induction of MLF, usually post alcoholic fermentation, with proven strains can substantially reduce the incidence of wine spoilage by dominating the indigenous microt'lora. For many reasons, which are generally poorly understood, the inoculated strain all too often fails to induce fermentation. This has prompted the trialling of better strains and alternative induction techniques. These techniques involve inoculation prior to, concurrently with or following inoculation with yeast, when the nutrient and chemical conditions are more conducive to

bacterial growth and metabolic activity. Furthermore, the recent commercial availability of high cell density inocula is claimed to decrease starter culture preparation time, decrease MLF induction time and increase the rate of acid degradation.

A series of trials has been planned to investigate the importance of culture strain, method of culture production, timing of inoculation in relation to alcoholic fermentation and selected wine compositional parameters. Previous trials undertaken by Mr Holger Gockowiak concentrated on direct inoculation of wine with a preparation of

Leuconostoc oenos (Viniflora oenos; Christian Hansen Laboratory). The commercial inoculum was systematically evaluated in a panel of three young Australian wines which had been adjusted to span a wide range of pH (2.9 to 3.5) and ethanol concentration (12.5 to 14.5% vol.). The general finding was that fermentation response was related to bacterial growth response. High pH and low ethanol enhanced growth and hence malate degradation while low pH and high ethanol was

highly inhibitory. Most important, however, the actual wine used had a major influence on bacterial growth and fermentation. Initial studies, summarised in the previous report, are aimed at establishing the nature of the inhibitory effect(s). Further trials to determine the best time to inoculate, with direct inoculum preparations, during/after alcoholic fermentation have been completed. An additional trial to investigate whether the Condimenta Bitec reactivation protocol improves the induction of MLF by different strains is in progress.

7. D ire c t in o c u la tio n fo r M L F s im u lta n e o u sly w ith yeast in o c u la tio n o r a fte r th e c o m p le tio n o f

a lc o h o lic fe rm e n ta tio n

The aim of this laboratory-scale trial is to investigate the relative merits of directly inoculating commercial freeze-dried MLF bacteria either together with yeast into a grape juice or, after the completion of alcoholic fermentation, into a wine. The four cultures of Leuc. oenos chosen were Viniflora oenos (Chr. Hansen), Bitec vino (Condimenta), Lalvin 4X (Lallemand) and Lalvin M C W (Lallemand) together with Maurivin 796 and a sterile-filtered Semilion juice (pH 3.1; TA 5.5 g/L; L-malic acid 2.4 g/L; sugar 20.4°Brix). All four cultures were inoculated (equivalent inoculation rates were based on viability) directly as


recommended by Chr. Hansen for the Viniflora oenos culture (the remaining cultures are not specifically produced for direct inoculation). MLF progress was monitored by measuring culture viability and malic acid concentrations.

Viniflora oenos was the only culture to complete MLF for both early and late inoculation w ithin 34 days; the remaining cultures had metabolised little malate regardless of inoculation time. By 65 days, however, the other three cultures had completed MLF for both early and late inoculations. Rapid die-back, except for Viniflora oenos, upon

inoculation into either grape juice or wine, could account for the delayed completion of MLF by Lalvin 4X, M C W and Bitec vino. This result is to be expected since only Viniflora oenos is recommended for direct inoculation. It is not clear whether the success of Viniflora oenos lies in its genetic constitution, method of preparation or both of these.

2. In o c u la tio n o f c o m m e rc ia l M L F cu ltu res,

re a c tiv a te d using th e C o n d im e n ta Bitec

p ro c e d u re , e ith e r s im u lta n e o u s ly w ith yeast

in o c u la tio n o r a ft e r th e c o m p le tio n o f a lc o h o lic

fe rm e n ta tio n

The previous trial investigated the effect on MLF induction of directly inoculating commercial freeze-dried MLF bacteria together with yeast into a grape juice or after the completion of alcoholic fermentation. The aim of this trial was to repeat the

previous trial except that the MLF cultures were first reactivated using the Bitec reactivation procedure instead of being used as direct freeze- dried inocula. The cultures of Leuc. oenos were Chr. Hansen's Viniflora oenos, Condimenta Bitec

vino, Bitec D, Lalvin 4X and Lalvin M C W together with Maurivin 796 and a sterile-filtered Semilion juice (pH 3.1; TA 5.5 g/L; L-malic acid 2.4 g/L; Sugar 20.4°Brix). The first and last of the LAB cultures are single strains, the remaining ones are mixed cultures. All cultures were reactivated as

recommended by Bitec for their Bitec vino culture. Sampling for this trial has been completed. The results are showing that MLF was completed more rapidly by Viniflora oenos, Bitec vino and Bitec D than by Lalvin 4x and MCW, irrespective of whether they were inoculated together with the yeast or after completion of fermentation.

These results suggest that either some strains cannot be satisfactorily reactivated by the Bitec procedure or that the procedure used in the

commercial preparation of some strains is not compatible with the Bitec procedure. Whatever the reason, the Bitec procedure is apparently not universally applicable to all commercial

preparations of bacteria.

3 . Bitec D d ire c t in o c u la tio n tria l

A new malolactic culture, Bitec D, recommended for direct inoculation, has been released onto the Australian market for the 1995 vintage by Condimenta. It requires only rehydration in water of ambient temperature and dispersal into a small quantity of wine before inoculation.

A trial is in progress to compare the performance of this culture with Viniflora oenos under similar conditions. Two wines, previously used in the evaluation of Viniflora oenos, have been selected (Coonawarra Chardonnay and Drumborg Cabernet Sauvignon) and were adjusted to three conditions of pH and alcohol [(i) pH 2.9/14.5% vol. alcohol; (ii) pH 3.1/13.5% vol. alcohol; (iii) pH 3.5/12.5% vol. alcohol] to represent marginal and usual conditions for MLF in wine. MLF progress was

monitored by measuring culture viability and malic acid concentration. The results indicated that Bitec D behaved differently to Viniflora oenos in the wines tested. In both wines some MLF had occurred under the 'softest' conditions (pH 3.5/12.5% vol. alcohol) after 20 days and little malate had been degraded

under harsh conditions (pH 2.9/14.5% vol. alcohol). By contrast, Viniflora oenos failed to degrade malic acid present in the Chardonnay wine under any of the pH/alcohol conditions.

Bitec D, at least in these wines, appears to be more tolerant to low pH and high alcohol content and other wine inhibitory factors.

Chaining of Leu con ostoc oenos and viability determination Some strains of Leuc. oenos form long chains during growth in wine. This can result in

inaccuracies in the quantitation of the number of viable cells as determined by colony forming units (cfu) on agar plates. To overcome this often serious inaccuracy two techniques that disrupt the chaining of cells while preserving cell viability were evaluated. The first involved disruption of the

chains mechanically using ultrasonication. No significant effect on viable plate counts as expressed by cfu were observed after 0.1 to 20 min. The second technique involved incubating


bacteria with divalent cations, such as calcium and manganese, which are reported to induce dechaining of some species of lactobacilli under certain conditions. A stationary phase culture of Leuc. oenos was treated with various concentrations of manganese chloride (1 μΜ to

100 mM) for either 50 or 100 min before plating. This method was also unsuccessful in dechaining the culture.

Effect of wine pretreatment on MLF induction Laboratory MLF trials in commercial wines are preferably performed in the absence of indigenous strains. Sterile filtration is the method of choice but does alter wine composition by the removal of colloids which have an unknown effect on MLF cultures. Dimethyldicarbonate (DMDC) is a chemical cold sterilant that is reported to exhibit

broad microbiocidal activity. DMDC is unstable in wine and the products, methanol and CCX, are not expected to significantly affect MLF. Neither sterile-filtration or DMDC treatment (200 mg/L) had a significant effect on the growth rate of

Viniflora oenos in a rose wine that had not previously undergone MLF.

Failure of MLF induction in wine Wines may fail to support MLF for two principle reasons; either they may lack a critical nutrient or they may contain a growth inhibitory substance. A relatively quick and simple test is needed to predict problem wines and to indicate the type of problem present. A test which could identify wines that are not susceptible to induction of malolactic fermentation would prevent futile attempted induction. Mr Gockowiak has commenced work on devising a test using a complete nutrient supplement to test for a nutrient deficiency and a combination of adsorbants and heat to test for the presence of an inhibitory substance. Inhibitory substances or agents reported to interfere with bacterial growth include bacteriophage, bacteriocins, fatty acids, unknown fungal products, and certain pesticides.

The results of applying these tests to two wines (Coonawarra Chardonnay and Mudgee Carbernet Sauvignon), in which induction of MLF had failed, were reported previously. A third wine in which multiple induction of MLF had proved difficult was tested using Bitec D as the inoculum applied at normal and high rate. Preliminary results indicate that none of the treatments were successful at

normal inoculation rate. The higher inoculation rate improved the rate of MLF, but culture viability still declined over the test period despite treatments designed to improve nutrient status and to remove inhibitors. The results suggest that a nutrient requirement or inhibitor was present which was not affected by the treatments used. Work w ill continue to improve the test for rapidly predicting the MLF response of a wine.

Biogenic amines Two aspects of biogenic amine formation in wine are currently being investigated. Ongoing work by- Mr Peter Costello investigates the taxonomical relationship with ability to form amines. A new project being undertaken by Mr Duncan Jamieson at the Royal Melbourne Institute of Technology (RMIT) w ill survey the presence of lactic acid bacteria (LAB) in commercial wines at different stages of production and histamine accumulation.

1. L a b o ra to ry in vestig atio n s

Mr Costello has made further progress on the screening of LAB for production of biogenic amines. The aim of present work is to determine the spectrum of wine bacteria that contain amino acid decarboxylation/amine excretion activities, the mechanism believed to be responsible for the generation of amines in wine. The strains were screened in Amino Acid Decarboxylase Assay (DCA) Medium containing the respective precursor amino acids for the amines histamine, tyramine, cadaverine, ethylamine, isopentylamine, phenylethylamine, methylamine and putrescine. Amine analysis has been performed by Dr Rainer Wittkowski of BCVV, Berlin, who is collaborating on this project, and is using a newly developed

HPLC method. Overall, most of the 40 LAB strains tested have been found to lack the ability to produce a significant concentration of biogenic amines under the test conditions employed. Apart from one reference strain, which produced a high concentration of histamine, cadaverine and putrescine, only a low to moderate concentration of histamine (0-4.0 mg/L), tyramine (0-3.6 mg/L), cadaverine (0-2.1 mg/L) and putrescine (0-2.5 mg/L) was produced. Lactobacillus spp. and Leuconostoc spp. produced a slightly higher concentration of amines than Pediococcus spp. O nly four of the 23 Leuc. oenos strains tested produced greater than 1 mg/L histamine (maximum


4.0 mg/L). Eight commercial Leuc. oenos strains produced 0-1.5 mg/L histamine, 0-0.4 mg/L tyramine and an insignificant concentration of cadaverine and putrescine, with only one strain exceeding 1 mg/L histamine, while three of the eight strains failed to produce a significant concentration of any amine.

A study of some of the factors suspected to affect the formation of biogenic amines has commenced. In particular the influence of nutrient availability and media composition is being investigated.

Recent French reports (Lonvaud-Funel and Joyeaux 1994) suggest stressful growth conditions, for example minimal nutritional availability and absence of L-malate, could induce greater

histamine formation by Leuc. oenos compared to that produced under more optimal growth conditions. Five representative LAB strains from previous screening trials were cultured in two different Decarboxylase Assay media of limiting

nutritional content. The results to date suggest that amine formation by various LAB is not greatly influenced by the culture medium and,

specifically, that amine production is not stimulated by growth-limiting conditions. The French report showed, however, that a histidine- decarboxylating strain of Leuc. oenos produced a maximum concentration of histamine under

limiting growth conditions. In conclusion, these results support other literature reports that amino acid decarboxylase activity is not common amongst LAB— most of the strains tested in the present work are wine isolates. These results also suggest that commercial strains of Leuc. oenos are not likely to be a source of biogenic amines in wine. The low incidence of moderate histamine producing LAB strains would appear to be one reason for the low, sporadic occurrence of a moderate (3-10 mg/L) concentration found in wine. Screening for such strains should therefore be ongoing in order to detect the infrequent occurrence of high histamine

producing strains, w hich for the affected wine, could result in export difficulties to certain countries. Future work w ill confirm the importance of precursor substrate concentration on amine formation as part explanation for observed

sporadic occurrence of amines in wine. This project forms part of a PhD research program for Mr Costello in the Department of Horticulture, Viticulture and Oenology, The

University of Adelaide. The work is being

supervised by Dr Henschke and Professor Lee.

2. W in e ry in v e s tig a tio n

Mr Jamieson has recently enrolled as a Master of Science candidate in Food Technology at RMIT, Melbourne. Dr Terry Schreikowski, Head of Department, RMIT, and Dr Henschke are supervisors. The aim of Mr Jamieson's research

project is to establish the relationship between histamine, histidine and the bacterial microflora present during the production of red wine, and to demonstrate histamine production by selected strains in the laboratory. His project has commenced this vintage. Participating wineries in the Yarra Valley and Mornington Peninsula are providing samples taken from different stages of production for chemical and microbiological analysis. This project can be expected to shed light on the source of histamine in wine, and therefore provide a strategy for its control.

MLF and wine flavour The positive and negative affects of LAB on wine flavour have been recently reviewed and published. The growth and metabolism of LAB can

modify wine flavour by elaboration of metabolic products of sugars and other nutrients and by modification of grape metabolites to render them flavoured or non-flavoured. Some by-products of

LAB metabolism are volatile and have characteristic aroma or flavour notes. The major product is lactic acid with minor products including organic acids, alcohols and carbonyl compounds. The impact of these products at the concentration produced by malolactic bacteria is

not well described. Furthermore, the differences between strains, while well described in chemical terms, is poorly known in sensory terms. Two avenues of investigations are being pursued

by Dr Eveline Bartowsky. The first is to screen wine bacteria for strain-specific characteristics identified by sensory analysis and description of the factors that enhance these properties. Chemical analysis w ill be used to determine the chemical

basis for the dominant flavour properties. A laboratory method for the production of wine suitable for sensory analysis is being established. A reliable method for wine preparation, inoculation, fermentation and packaging is being defined. Second, the metabolism of dominant flavour compounds is being investigated with the aim of developing strains with enhanced flavour


characteristics. Strains identified with differences in metabolism by the screen w ill be studied in an attempt to understand the regulation of flavour compound formation. This information w ill then allow the modification of strains by conventional

or recombinant techniques for use by industry.

D ia c e ty l

A characteristic buttery flavour is one of the most recognised characteristics of the MLF, but limited information is available on the reliable control of this flavour. Diacetyl, a volatile carbonyl compound, is believed to be the significant compound that confers on certain wines the

characteristic buttery flavour. The concentration of diacetyl in wine is usually near its flavour threshold value so the impact of this compound is therefore dependent on the factors which affect its concentration. The main factors include the grape type and primary fermentation conditions used to produce the wine and the strain and conditions used during the malolactic fermentation.

The metabolic formation of diacetyl by bacteria is dependent upon the intermediate pyruvic acid, which can be formed from the degradation of grape sugars or from the metabolism of citric acid.

Diacetyl can spontaneously form from a-aceto- lactate or enzymically via acetaldehyde-TTP. Reactions also occur which further metabolise diacetyl to less volatile compounds; reduction to acetoin and finally to 2,3-butanediol or α-acetolactate is decarboxylated directly to acetoin. A method for the quantitation of diacetyl is currently being established.

Strain identification Predictable control of the MLF requires knowledge of which bacterial strains are present during the fermentation. Current practice is to use a starter culture to increase the chance of the selected strain dominating the fermentation together with microscopic examination to confirm the presence

of the species used. Induction of MLF is not yet reliable and no rapid techniques are available to unequivocally verify the presence of or to quantify the selected strain. A variety of complex methods has been demonstrated but are not suitable for routine use. O f these, techniques based on DNA

analysis offer the greatest specificity with the potential for rapid strain verification. A method which has received some recent attention is Restriction Fragment Length

Polymorphism (RFLP) analysis of bacterial DNA. This technique uses low frequency DNA cutting enzymes and analysis of the DNA fragments generated by pulsed field gel electrophoresis. W hile this technique has proven successful it is not suitable for routine studies. Current efforts are being directed towards the use of PCR-based techniques which potentially can analyse a large

number of strains rapidly. This technique relies on the choice of suitable short pieces of DNA (primers), which allow analysis of selected variable regions of the bacterial genome to yield the diagnosis. Dr Bartowsky has designed oligonucleotide primers and has investigated the conditions for performing the PCR analysis on purified bacterial DNA and directly on bacteria.

Five oligonucleotide primers based on the sequence of conserved and variable regions of the 165 rRNA gene of Leuc. oenos are being screened for their potential to differentiate between strains of Leuc. oenos. Two primers are designed to be genus specific for Leuconostoc and the other three were designed to be unique for Leuc. oenos. Initially, chromosomal DNA isolated from LAB was used to establish a reliable PCR method. Work has concentrated on screening the two genus specific primers on the three main genera of wine lactic acid bacteria (Lactobacillus, Pediococcus and Leuconostoc). By adjusting the PCR conditions, this set of primers is specific for Leuconostoc species (Leuc. oenos and Leuc. mesenteroides have been tested at this stage) but does not amplify the specific fragment in other lactic acid bacteria (Lb. hilgardii, Lb. brevis, P. cerevisiae). This test therefore results in the presence or absence of a specific band on a gel providing a quick diagnosis of Leuconostoc species.

The method was first developed using chromosomal DNA purified from laboratory grown cultures, and then tested on bacterial colonies which were picked off an agar plate. Testing bacterial colonies is especially quick. The colony is resuspended in water and the PCR reaction is performed on the suspension without the need for DNA isolation. Results were identical to those with purified chromosomal DNA. Bacteria cultured in a wine medium also gave good results. Ethanol was found, however, to affect PCR. All cultures were tested and gave the same results as with the bacterial colonies and purified chromosomal DNA. Finally the number of bacteria required for detection/amplification of the diagnostic fragment


was tested with dilutions of the wine grown culture of Leuc. oenos. Approximately 50-500 bacteria are needed to amplify the specific fragment—this means that this number could be detected in a bottle (ca. 1 cell/ 1-10 mL) or wine cask (ca. 1 cell/ 10-100 mL) if the whole contents were filtered to concentrate the bacteria. This method

has the potential to not only estimate the number of bacteria in a wine sample but to indicate the type of LAB strain present.

Project Title Microbiological analysis of industry technical problems

Project No AWR 5CW

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Dr P A Henschke

Funding $113,140

Time Span 1990/91-1994/95

Objectives • Contribute to the maintenance and improvement of the quality of wines produced in Australia by providing a service to the wine

industry for investigation of significant, sporadic microbiological problems. • Maintain and expand the Institute's collection of microorganisms with particular emphasis on

strains of spoilage yeast, bacteria and moulds. • Evaluate selective media and other methods for rapid characterisation and tentative identification of wine spoilage microorganisms. • Isolate, identify, characterise and maintain

microorganisms from wine for winemaking trials.

Progress The Microbiology Group assists with non-routine microbiological problems initially dealt with by the Technical Services Group. The major work

involves isolation and characterisation of the microorganisms present in wine samples and the formulation of recommendations for controlling

and preventing a recurrence of the problem. A small project may be undertaken when information is not available for solving a problem of general

interest to the industry. Current projects concern studies on formation of mousy taint in wine, late fermentation H:S production, restarting stuck

primary fermentations, yeast EC1118 and SO, formation, and monitoring the quality of dried yeast.

Mousy taint occurrence and formation in wine Quantitative analysis of compounds associated with mousy taint in wines and experimental fermentations made with spoilage yeast and bacteria is being carried out for the first time using the recently developed high sensitivity, quantitative analytical technique involving liquid/liquid extraction and high resolution gas chromatography-mass spectrometry (GC-MS). The suspected mousy taint compounds being quantified include 2-acetyltetrahydropyridine (ACTPY), which is present in two tautomeric forms, the newly discovered 2-acetyl-1-pyrroline (ACPY) and ' 110/111'. W ork is now being focused on completing the survey of mousy wines for the main taint compounds to establish which of the three compounds has the highest occurrence and whether any relationship exists with the producer organism or wine type. Investigations to determine the main factors that influence the production of these compounds in wine are also continuing. This work is being carried out in the Institute by Mr Costello and Mr Paul Grbin, as part of their PhD programs. The work is being supervised by Dr Andrew Markides, The University of Adelaide, Dr Henschke and Professor Lee.

1. L ac tic a c id b a c te ria

Work has continued on developing a laboratory method for screening bacteria for the production of the suspected mousy taint compounds, ACPY, ACTPY, and Ί 10/111'. Carr medium has been modified to give good growth of LAB and taint production as determined by sensory testing. Due

to the large time and effort required in the extraction and mass spectrometry analysis, only a limited number of strains can be screened with this method. Ten LAB, including five strains of Leuc. oenos (four commercial and one wine isolate), and two rod-shaped bacteria (probable Lactobacillus spp.) isolated from mousy wines have been screened. Furthermore, since it is becoming apparent that acetic acid bacteria (AAB) are also being isolated from wines with mousy taint, an AWR I strain of Cluconobacter oxydans, which


produces taint, was also screened. Another advance has been the successful formulation of a totally chemically defined model wine medium for testing the ability of wine bacteria to grow and produce mousy taint. Seven selected strains of LAB and AAB have been screened in this new medium and most strains were found to grow well and produce taint as indicated by alkaline test strip. This is believed to be the first report of such a medium, and it w ill now enable a systematic study of taint precursor compounds to be undertaken. Further experimentation into factors affecting taint formation by LAB has shown that strong taint formation can be rapidly induced by a heavy suspension of whole-resting cells in the synthetic medium. These results are shedding light on the possible mechanism of taint formation and the missing factor(s) that trigger taint formation in only some wines that contain lysine. This method is also being adapted to give a rapid (overnight) method for screening microorganisms for the ability to produce mousy taint.

2 . Dekkera (Brettanomyces) yeast

Work has advanced in two main areas, screening type strains of Dekkera yeast for the production of mousy taint compounds and the role of lysine as a precursor compound, and establishing the relationship between taint formation and yeast growth.

S u rv e y o f strains

Twelve type strains of Dekkera yeast have been screened for production of the suspected mousy taint compounds ACTPY, ACPY and '110/111' in a chemically defined fermentation medium. Saccharomyces cerevisiae AWRI 729 was used as a negative control for ACTPY production. All strains were grown in the presence or absence of the amino acid lysine (100 mg/L), the suspected precursor of ACTPY.

ACTPY was produced by all strains of Dekkera with or without lysine present; none was produced by Sacch. cerevisiae. In the absence of lysine ACTPY was produced in the range of trace to 8.5 pg/L (mean of 0.5 pg/L) whereas with lysine the range was 8.6 to 1245 pg/L (mean of 88 pg/L). Given an aroma threshold of the order 2 pg/L, a just detectable concentration of ACTPY was produced in the absence of added lysine. Lysine markedly stimulated ACTPY production, however,

up to 600-fold above the aroma threshold, indicating that lysine may be an important precursor and trigger for ACTPY production by yeast in wine. The species of yeast that have been reported to occur in wine produced ACTPY in the range trace to 4.5 pg/L in the absence of lysine and 41 to 1245 pg/L with lysine, indicating that some but not all species are capable of producing an excessive concentration of ACTPY. These results also indicate that ACTPY formation is a characteristic of members of the Dekkera genus (with respect to these conditions) and that exogenous lysine is not required for ACTPY production.

No ACPY was detected in these samples suggesting that either appropriate precursor(s) were not present in the test medium or that ACPY is a specific indicator of bacterial spoilage. The unknown compound Ί 10/111' was only detected in fermentations to which lysine had been added.

R e la tio n s h ip w ith g ro w th

The relationship between ACTPY production and the stage of growth of a Brettanomyces yeast was investigated in a chemically-defined medium supplemented with lysine. ACTPY accumulation in the fermentation medium was determined by sensory analysis using alkaline paper strips and by GC-MS. Preliminary results suggest that ACTPY accumulates in the medium during the cell growth phase. ACTPY then commenced to decrease slightly as cells entered the stationary phase, and continued to decrease at a low rate post fermentation. These results suggest that mousy taint formation is associated with active growth and is

not released by dying cells.

Restarting stuck primary fermentation Many reasons for stuck fermentation and difficulties in restarting them have been documented. Strategies for restarting fermentation have largely been developed empirically and involve acclimatising the yeast to alcohol and adding critical yeast nutrients. Despite this advance, failure to reinitiate fermentation is still a problem. The current experimental approach is to determine the critical aspects of restarting fermentation by studying a series of commercial stuck wines.

Studies have been carried out on a batch of Chardonnay wine which stuck at approx. 30 g sugar/L, contained a high population of acetic acid


bacteria, and had a VA of 1.8 g/L. The variables under investigation are preparation of the yeast starter culture, and nutrient addition, including air. Tall tube fermenters have been designed to estimate the problem of yeast settling during restarting fermentation and the role of agitation in preventing yeast sedimentation.

Restarting fermentation showed no response to nutrient addition, confirming chemical analysis that sufficient nutrients were present. A critical yeast nutrient is oxygen— fermentation response was markedly improved when the fermentations were aerated at a low rate after fermentation had recommenced. Continuous aeration, on the other

hand, reduced fermentation response. Comparison of the activity of freshly reactivated dried yeast and acclimatised yeast showed the latter yeast recommenced fermentation quickly and fermentation had nearly completed before the dried yeast had initiated fermentation. The rate of fermentation by the dried yeast was, however, greater than that of the acclimatised yeast once fermentation had commenced. Finally, agitation of the wine gave a more rapid initiation and a greater

rate of fermentation. Monitoring yeast cell concentration suggested that agitation worked by maintaining a higher population of yeast in suspension.

Hydrogen sulphide production at the end of fermentation Hydrogen sulphide accumulation in fermenting must occurs in two distinct phases: during the active phase of fermentation and at the end of fermentation when little or no fermentable sugar

remains. Previous studies have shown that unlike the first phase of H2S production which is ameliorated by DAP supplementation, the second

phase shows little response to DAP. Systematic investigations are being undertaken to identify the important factors. During the 1994 vintage, twenty samples of red wine collected during the final stage of fermentation from three wineries showed that no

relationship could be established between H2S production and either the concentration of assimilable nitrogen or S 0 2, or the proportion of dead yeast cells. W ild yeast numbers were not significant. During the 1995 vintage, empirical trials were undertaken to determine which treatments accessible to winemakers could

influence H2S production. Fermenting samples, at

the point of separation from skins (1-3°Be), were collected from wineries and brought back to the Institute for study. The samples were fermented in 150 m l lots with H 2S production monitored by

entrainment with nitrogen gas and entrapment with CdS04. The amount of CdS formed was measured by a colorimetric method. Initially, trials were conducted to demonstrate

that fermenting samples monitored in the laboratory were producing K2S in a manner similar to that obser\ed in the winery. Subsequently, trials investigated the effect of nutrient supplementation of the fermentation at 3-5°Be on subsequent H 2S

production. The treatments involved various combinations of aeration, vitamin and DAP addition. Aeration appeared to exert the greatest effect on late stage H,S production, that is, in all experiments a small to large reduction in

production was noted. Results were highly variable with vitamin (Cerev/f) supplementation, but a reduction in H2S production was usually noted in association with aeration. On the other hand, data were obtained which suggest that DAP addition followed by aeration actually exacerbated late stage H2S production. This supports a previous observation that while DAP is highly beneficial during active fermentation, it may not be desirable as fermentation completes. This observation,

however, raises the question of why should DAP exert an effect not generated by amino acids— this apparent dilemma needs careful investigation.

Production of sulphur dioxide by Sacch. c e re v is iae EC1118 wine yeast

Several winemakers have reported an association between the fermentation of grape musts with Sacch. cerevisiae strain EC1118, and related strains, and the inability to induce malolactic fermentation (MLF). These winemakers noted that analysis of the wines showed a significant increase

in the concentration of sulphur dioxide (S02), a compound known to inhibit MLF under some conditions. The incidence of this inhibition appeared to be unrelated to the winery, viticultural region, or grape variety, and could be described as 'sporadic'. Mr Eglinton has investigated the production of S02 by Sacch. cerevisiae strain EC111 8 under a range of oenological conditions.

Twelve grape juices from a range of white grape varieties, including Riesling, Chardonnay, Sauvignon Blanc and Semilion, were obtained at the end of the 1995 vintage from several


viticultural regions in South Australia. Some of the juices, had been sterile filtered, while others contained large amounts of grape solids. The sugar content varied from 195 to 260 g/L and the total concentration of SO, from 35 to 98 mg/L. The juices were not supplemented, and all sugar could

be completely fermented. The following variables were examined for their effect on the total concentration of SO, in the wines: grape variety, isolate of Sacch. cerevisiae EC1118, initial concentration of sugar, initial concentration of SOa, initial pH, presence of grape solids, and aeration, at both start and end of fermentation, compared to completely anaerobic fermentation.

The results showed that there was no significant increase in the concentration of SO, in any of the juices tested. In the majority of juices, a very small decrease in the concentration of SO, was observed. These data suggest that there is no single grape variety which is predisposed to this problem. The change in the concentration of SO, was not correlated with the initial concentration of sugar,

initial concentration of S 02, or initial pH. There was no increase in the concentration of SO, in the presence or absence of grape solids. During completely anaerobic fermentation, there was a decrease in the concentration of SO,, regardless of whether grape solids were present or not. These results suggest that aeration is possibly an important factor in SO, metabolism in EC1118, although it could not explain the observed inhibition of MLF.

Under typical winemaking conditions, white wines can be cooled at the end of fermentation, prior to further processing. This cooling could occur for days, or even weeks, depending on the wine style. Cold settling of wines, in the absence or presence of aeration, has been investigated for

its effect on the concentration of SO: after fermentation. Preliminary results suggest that there is no change in the concentration of SO, in wines in the presence or absence of aeration, at 4°C or

18°C, after fermentation. These wines have been examined up to 10 days after fermentation, and w ill be monitored up to two weeks after its completion.

Since it was possible that the isolate of Sacch. cerevisiae EC1118 could be an important factor, four isolates were tested for production of SO, in a filtered Riesling grape juice. One isolate was obtained from the Institute culture collection, and used as a liquid starter culture. Three ADWY, from

two commercial companies, were tested. One of these ADWY was provided by a winemaker, who had observed a dramatic increase in the concentration of SO_, in wines after fermentation with this isolate of EC1118. Under laboratory conditions, none of the yeasts produced more than 2.5 mg/L S 02, and there was no difference between the isolates. These results, although

limited to four isolates, suggest that there is little difference in EC1118 yeast isolates between companies in terms of SO, production, and that the observed inhibition of MLF is probably not caused by this variable.

These experiments represent a comprehensive testing of the production of SO_, by Sacch. cerevisiae EC1118, and indicate that the inhibition of MLF reported by winemakers is probably not the result of increased production of SO, by this yeast strain, although factors which can influence the production of SO, , and which have not been investigated, cannot be discounted. W ork is continuing to establish whether this yeast can elicit bacterial inhibitory activity under certain conditions.

Active dried yeast testing Before each vintage, a selection of dried yeast is tested for quality and performance. The most important parameters are fermentation activity (estimated by the Institute Sugar Utilisation Rate

test), viability (vital staining and plating methods) and microbial contamination levels. Seven batches of the Institute wine yeasts made under licence by Mauri Yeasts were independently tested by the Institute.

Evaluation of the fluorescent lectin wheatgerm agglutinin-fluoroisothiocyanate for Gram staining wine bacteria Gram staining is the most widely used taxonomic test for bacteria. An alternative method, claimed to be simpler, faster and less ambiguous than conventional Gram staining, is based on the principle of selective binding of the lectin wheat germ agglutinin to N-acetylglucosamine. Although

N-acetylglucosamine is found in all bacteria, it is located in the outer portion of the cell wall of gram-positive bacteria and is therefore accessible to the lectin. In Gram-negative bacteria it is not accessible to the lectin, being located in the peptidoglycan layer which is covered by the cell membrane. The wheat germ agglutinin is


conjugated to the fluorescent dye fluorescein isothiocyanate (WGA-FITC) for convenient detection of the assay by fluorescence microscopy. A preliminary trial was carried out on wine- grown strains of pediococci, leuconostocs,

lactobacilli, acetobacter and gluconobacter, which were tested together with Bacillus subtilis and Escherichia coli as controls. The pediococci and 8. subtilis Gram positive controls stained unambiguously. Leuconostocs, however, stained poorly or not at all, and lactobacilli stained in a variable manner. None of the Gram-negative

bacteria (acetobacter, gluconobacter and E. coli) were stained. These preliminary results suggest that wine grown bacteria are not reliably differentiated by this method.

Chemistry Group

Project Title Elucidation of varietal flavour characteristics of grapes

Project No AWR 6V

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Dr P J W illiams

Funding $204,019

Time Span 1990/91-1994/95

Objectives • Gain an understanding of the factors that influence the flavour of grapes. • Develop techniques that could assist viticultur­

ists and winemakers to exercise some control over the varietal flavour of fruit and of wines.

Progress Cabernet Sauvignon and Merlot flavour precursors— sensory studies From December, Dr Leigh Francis carried out further research on black grape flavour precursors

in the Department of Viticulture and Enology at the University of California, Davis (UCD). As part of this work, Cabernet Sauvignon and Merlot grapes from California vineyards have been pressed to obtain juice and skin samples. The skin samples were extracted with 12% aqueous ethanol for

seven days to mimic the extraction process that occurs during vinification. After centrifugation and filtration, glycosides were isolated from both the juice and skin extracts. Similar glycoside isolates that were obtained from Cabernet Sauvignon and

Merlot grapes harvested from Australian vineyards and processed at The Australian W ine Research Institute at the end of 1994 were taken to UCD for the current study. The glycosides from both the California and Australian fruits have been

hydrolyzed in a model wine medium (12% aqueous ethanol/pH 3.2 tartrate buffer) to release the volatile compounds; the hydrolysates were

added to a base wine and subjected to sensory descriptive analysis. By descriptive analysis, wines made from Napa Valley Cabernet Sauvignon and Merlot grapes were profiled as being of high intensity in berry, dried fig, chocolate and tobacco descriptors. The

same aroma notes were also produced by the hydrolysates of skin and juice glycosides extracted from the same grapes. The skin extract samples were generally more intense than the juice samples; there were also substantial differences due to the region of the grapes' origin, and due to variety.

W hile working in Professor Noble's laboratory at UCD, Dr Francis investigated multivariate statistical methods, including procrustes analysis and partial least squares (PLS) regression, for their usefulness in relating compositional data to sensory data, and in evaluating judges' performance. PLS in particular has great potential

in this work. A project on the role of glycosides in Zinfandel wine flavour was also commenced.

Measurement of total glycosides in grapes, juices and wines An assay procedure has been developed to quantify the glycosylated secondary metabolites of grapes, juices and wines through a determination of the glycosyl glucose (G-G).

The accuracy and precision of the G-G assay method was determined through a series of experiments using graded additions of a standard glycoside, for example, n-octyl glucoside, to grape

homogenates, juice and wine. These experiments demonstrated that the enzymatic determination of glucose in the last step of the assay suffered no interferences from the substrates analysed. In the course of further research aimed at optimising

methods for the preparation of fruit samples for the


assay, values for the C-C of black grape berries were obtained that were lower than the concentration of glucose calculated as being present in the anthocyanin pigments in the fruit. The latter were determined spectrophotometrically. This discrepancy necessitated an investigation into the cause and the development of a method to overcome an apparent phenolic interference. This

interference was particularly evident in samples from immature fruit or samples with low G-G concentration. A small modification to the existing G-G procedure was found to be necessary to overcome the phenolic interference in the enzymatic step of the assay. The modification involves the use of a second solid phase extraction step before the final analysis of glucose. Fortunately, the same C-18RP cartridge by which the glycosides were isolated in the first step of the assay can be reused for the clean up step in the modified procedure. Thus, the modification incurs no additional expense on the assay. An important outcome of these studies has been a lower detection lim it for the G-G assay.

Processing of grape samples from Program 2 of the Cooperative Research Centre for Viticulture (CRCV) has continued, and since January over 1500 samples, most of Shiraz grapes from 1995, have been analysed. Productivity has been optimised to 80 assays per week and this is limited by constraints of laboratory space and personnel. Such an analysis rate reduces to less than 60 assays per week if the analysts, Dr Wies Cynkar and Ms Mariola Kwiatkowski, rather than the CRCV researchers, prepare the fruit homogenates.

Difference data between G-G and berry colour, referred to as the red-free G-G, shows, for the first time, the sharp development in secondary metabolites in Shiraz fruit in the latter stages of soluble solids accumulation. The rate of formation of these important compounds with degree Brix, and also possibly with the time of maturity at which the development accelerates, is influenced by irrigation as well as by the growing season. These characteristics are observable only by this analytical method; no other measure of fruit composition, including colour, reveals these trends.

The possibility of using the G-G assay as an indicator of the exposure of a wine to high temperature during transport has also been investigated.

Investigations into the utility of laboratory

robotic equipment to allow greater sample throughput have progressed. A high speed robotics and process automation firm, Advanced Rapid Robotic Manufacturing (ARRM), have submitted a report on the feasibility of building an instrument for automated G-G analysis along with an initial quotation. Extensive negotiations involving,

initially, the Institute, CRCV, Grape and Wine Research and Development Corporation (GWRDC) and ARRM have taken place, and a contract for the design and building of an instrument has been drawn up and signed.

Other CRCV research Studies on the monoterpene flavour compounds in grafted grapes were undertaken by Mr Yoji Hayasaka in collaboration with M r Mansour Gholami of the Department of Horticulture, Viticulture and Oenology (DHVO), The University of Adelaide. Fruits of Muscat Gordo Blanco

(Gordo) and Shiraz were grafted onto shoots of each variety and grown to maturity. Isolated glycosides from each grafted fruit and from controls were enzymatically hydrolyzed and analysed by GC-MS. The results showed that there is no difference in flavourant composition between grafted and non-grafted fruits of both cultivars,

indicating that berries have the ability to synthesise flavour compounds independently. Constituents in phloem sap flowing through the bunchstems of Gordo, Sultana and Shiraz have also been analysed by Messrs Gholami and Hayasaka. Because of the result of the grafting experiment above, the presence or absence of monoterpene or other secondary metabolite glycosides in phloem sap is of critical importance in understanding the origin of flavour compounds in the fruit. The collection of phloem sap from the bunchstems was monitored by the presence of sucrose and amino nitrogen in the fractions collected. From extensive GC-MS analyses of fractions taken under different conditions, Messrs Gholami and Hayasaka were able to demonstrate that secondary metabolites were secreted from various cells in the bunchstem but not transported in the phloem sap.

Mr Mani Naiker, a PhD student from Charles Sturt University (CSU), spent six months with the Institute's Chemistry Group working on grape flavour precursors in Cabernet Sauvignon skins. An apparently new precursor of damascenone has been recognised in the skins. This dominant


precursor of the flavourant gives only damascenone on mild acid hydrolysis-and not other by-products as has been observed with damascenone precursors in other varieties. This phenomenon had been indicated from earlier hydrolysis results (reported in the Institute's 1994 Annual Report) of total glycoside fractions from Cabernet Sauvignon and Merlot. The absence of by-products has been emphasised in the DCCC- separated skin fractions which are being studied by Mr Naiker, and the nature of this new precursor is

being further investigated by FAB-MS/MS coupled with chromatographic and other spectral analyses. The potency of damascenone, and its evident importance to the aroma properties of Cabernet

Sauvignon and Merlot, make the elucidation of this new precursor a priority.

Project Title The influence of oak cooperage on wine composition

Project No AWR 7V

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Dr M A Sefton

Funding $166,237

Time Span 1990/91-1994/95

Objectives • Study the influence of timber origin, seasoning and usage on the chemical composition of flavourants and their precursors that are derived

from oak wood.

• Assess the phenolic and wood-derived volatile composition as well as the sensory properties of wines stored in various oak cooperage in comparison with wines held in stainless steel.

Progress Furfuryl ethyl ether and 5-methylfurfuryl ethyl ether have been successfully synthesised and shown to be present in wines as a result of barrel maturation. These compounds are believed to be derived from the oak components furfural and 5-

methylfurfural via microbiological reduction and subsequent ether formation. Studies are now underway to determine the sensory properties of

these two ethers, and also to determine the nature of the equilibria between these ethers and the corresponding furfuryl alcohols. A study of the solvolytic breakdown, in model

wine solution, of the oak-derived wine components, furfuryl alcohol, 5-methylfurfuryl alcohol and vanillyl alcohol has also been completed. 5-Methylfurfuryl alcohol is rapidly converted to the corresponding ethyl ether at wine pH, and both of these compounds are subsequently totally degraded (presumably to polymers) over a period of several weeks. Neither of these compounds are, therefore, of any

importance to wine flavour. In contrast, the analogous reactions of furfuryl alcohol are several orders of magnitude slower, and so furfuryl alcohol and furfuryl ethyl ether are likely to remain as wine components for a significant period of time. Vanillyl alcohol was converted to its ethyl ether at a rate similar to the formation of 5-methylfurfuryl ethyl ether from 5-methylfurfuryl alcohol. In contrast to the furan anologues, however, no degradation of vanillyl alcohol or vanillyl ethyl ether was observed.

A program to synthesise isotopically labelled vanillin, vanillyl alcohol, and vanillyl ethyl ether has been successfully completed. These compounds are now being used as internal standards for the rapid and accurate quantification of their respective natural analogues in all red, white and model wines sampled from barrels during the course of the trial. By using these standards, preparation time for individual samples

has been reduced from 4 days to 2-3 min, and compound concentration can be measured to an accuracy of 99% or better, regardless of the stability of the samples. Vanillin and vanillyl ethyl ether concentration has been determined for all

red, white and model wines (130 samples); vanillyl alcohol w ill be determined in the near future. The measurements on vanillin in the wines has shown that the process of barrel fermentation and

lees maturation of the white wines removed approximately two thirds of the vanillin that was extracted from the oakwood during the first 11 weeks of maturation. Following racking and re­

barrelling, the white wines were then matured for a further 44-week period. Additional vanillin extracted during this time was not transformed to other products, even though malolactic fermentation took place in some barrels during this time.


The red' wines had completed both alcoholic and malolactic fermentation prior to barrel maturation. Nevertheless, undefined micro­ biological activity (probably including, but not necessarily limited to, Brettanomyces activity) during the 93-week maturation period depleted the vanillin extracted from the oak barrels by approximately 50%.

Comparison of all the data from red, white and model wines has confirmed the earlier observation that, in spite of efforts by the participating coopers to ensure a uniform level of toast for all barrels, there was considerable variation in the level of toast between barrels. Because the heating process is an extremely important variable in determining oak-derived flavour in wines, particularly in white wines, this lack of uniform toast level explains much of the flavour variation observed in the wines.

Reports in the literature have suggested that yeast or lactic acid bacteria may have a fining action on oak derived volatile phenols such as eugenol and guaiacol. Experiments carried out in our laboratory have now shown that there is no such fining action.

Sensory evaluation Formal sensory evaluation of all 50 red and white wines used in this research program has been completed. Comprehensive difference testing between within-treatment replicates showed that those samples with the largest compositional differences were also those which could be distinguished by difference testing. Difference

testing has also been used to determine the threshold of the sensory panel for oak contribution to white wine flavour. The panel could just detect the oak contribution when an oaked wine was diluted twenty fold by an unoaked control.

A ranking procedure was used to distinguish wines from each other, according to a group of aroma descriptors, and also according to preference. Significant differences between samples were observed for most descriptors. Detailed statistical analysis of the sensory data is now underway to determine how the various treatments imposed on the experiment (for example oak origin, seasoning location, etc.) have

influenced the sensory profile of the wines, and also to determine correlations between sensory and compositional data. Although this statistical analysis is not yet complete, many interesting

correlations are already evident. Aroma variation according to oak origin and seasoning appears to be due mainly to the sensory impact of a single compound, the c/s-oak lactone. In contrast to some compounds that are formed by the coopering process and are extracted mainly during the first few weeks of maturation, the c/s­ oak lactone was extracted continuously over a two-year period. Since the red wines were matured for tw o years, compared to one year for the whites, the sensory impact of the c/s-oak lactone was greater with the former. In pure form, the c/s-oak lactone has a coconut-like aroma, and therefore it was not surprising to observe a strong correlation between this compound and the descriptor 'coconut' for both red and white wines.

In the past it has been assumed, albeit without any experimental evidence, that the vanilla character of oak-aged wines is due to extraction of the compound vanillin from oakwood. In this study, there was no such correlation. The descriptor 'vanilla' was, however, strongly correlated with the c/s-oak lactone in the red wines. Similarly, the 'berry' descriptor in the red wines, which might have been thought to be a fruit-derived character, was actually least intense

in the unoaked control wine and was also strongly correlated with the c/s-oak lactone. In retrospect, this result is not surprising, as structurally similar lactones are important aroma volatiles in many fruits.

Among the red wines, and also the white wines matured in the Tronfais and Vosges oak barrels, the c/s-oak lactone was also strongly correlated with preference. For white wines aged in barrels made from the American or Limousin oakwood, coopering heat and wine microbiology appears to have had the main influence in determining preference.

Although the concentration of c/s-oak lactone varied greatly according to oak origin, it does not always do so in a predictable way. The industry could consider oak quality assurance based on quantification of the c/s-oak lactone in wood samples as an alternative to relying on oak origin.

Some strong aroma trends were affected by variations in toasting heat during coopering. By quantifying compounds thought to arise in different amounts according to barrel heating technique, it has been possible to calculate a coopering intensity and coopering depth index for each barrel. There was no significant difference in


coopering intensity between the barrels produced by two coopers who participated in the oak project. There was, however, a significant difference in coopering depth, with one cooper apparently employing a heating technique which resulted in significantly more barrels achieving a relatively high level of heat penetration. Even this cooper, however, produced some barrels that were relatively shallowly heated.

In the red wines, coopering intensity was strongly and positively associated with the 'smoky' descriptor, and negatively correlated with the descriptor 'green apple'. The red wines also showed the affect of coopering intensity, although

in this case it was the 'coffee' descriptor that was positively correlated. Among the red wines, coopering depth was correlated with 'preference' while coopering

intensity was not. The influence of coopering depth was greater for the wines aged in American and Limousin barrels than for those aged in barrels made from Trongais and Vosges wood.

Although not intended, some microbiological activity took place during maturation of both red and white wines. As the extent of this activity varied between barrels, it has been possible to correlate microbiological action with the sensory characters of the wines.

Among the white wines, MLF was accompanied by increases in 'buttery' and 'green apple' character, and with decreases in the intensity of the descriptors 'smoky', 'pencil shavings' and 'all spice'. Thus, MLF appears to have substantial potential to modify some oak-derived aromas, particularly those that are affected by the toasting process.

The red wines all contained a varying concentration of 4-ethylphenol, a product of Brettanomyces activity. This type of micro­ biological activity is possible particularly when a

low level of sulphur dioxide is maintained during red wine maturation. An increase in 4-ethylphenol was accompanied by a lower intensity of 'allspice' and 'coffee', and by a lower preference.

Once the vanillyl alcohol concentration in wines has been determined and the sensory data have been fully analysed statistically, this w ill bring to a completion all the experimental work associated with the industry-supported oak research that has

been undertaken over the past eight years. It is intended to prepare a detailed report on the outcome of this research for distribution to levy payers during the following year.

Project Title Characterisation of unstable proteins involved in haze formation

Project No AWR 8V

Organisation The Australian W ine Research Institute

Location Urrbrae, SA

Supervisor Dr PJ Williams

Funding $241,800

Time Span 1990/91-1994/95

Objectives • Cain an understanding of the nature of unstable proteins. • Use this knowledge for the development of

effective stabilisation processes.

Progress The role of procyanidins in wine protein instability There has been long standing speculation on the

role of tannins in white wine protein instability. To resolve this, we have developed procedures which allow us to detect and quantitate these flavanoid compounds in protein samples. Two independent methods, a mass spectrometric technique and a chemical assay, were used to examine procyanidins in wine and protein haze samples.

The mass spectrometric technique provided evidence for the presence of the polyphenols. This method used pyrolysis to degrade the sample and electron impact mass spectrometry with daughter

ion analysis of selected fragment ions with m/z 136 and 152. These ions were shown to be characteristic fragment ions derived from grape seed procyanidin, epicatechin and catechin under our experimental conditions and are known to be characteristic fragment ions for other flavan-3-ols.

The chemical assay to detect procyanidins in w'ine protein and haze samples was a modification of a published method by which polymeric procyanidins are converted to cyanidin by oxidative acid hydrolysis. The concentration of

procyanidin in the samples was measured by the cyanidin peak in the HPLC analysis of the acid hydrolysis assays. Calibration was made by

hydrolyzing grape seed condensed tannin, a procyanidin similar to that expected in wine samples. Protein did not interfere with the assay, because under the hydrolysis conditions, protein,


even when present in a large excess, did not yield cyanidin or other compounds co-eluting with the anthocyanidin. This made the modified hydrolysis assay eminently suitable for determining the procyanidin content of wine proteins and wine hazes containing protein, even when the procyanidins were present at a low level.

A range of white wine protein hazes, when examined by the hydrolysis assay, all contained procyanidin but at a concentration of less than 5% w/w. In the case of heat-induced hazes, the procyanidins appear to be merely associated with the protein in haze, and not covalently bound. This is because the resolubilised protein fraction from a heat-induced Gordo haze, after gel permeation chromatography to separate it from lower molecular weight components, such as procyanidins, had no detectable procyanidin. If procyanidins were covalently bound to protein in the haze the phenolics would still be present in the protein fraction after such manipulations.

We have also examined the association between procyanidins and native soluble wine proteins, the precursors of insoluble wine protein haze. Soluble proteins from Gordo wine were preparatively separated by anion exchange chromatography and collected in 10 fractions. The procyanidin content of the separated protein fractions and of the crude protein starting material was determined by the acid hydrolysis procedure. The low but detectable level of procyanidin found in the starting material was evidently not the result of strong association of protein with procyanidin since, after anion exchange chromatography, a majority of the separated protein fractions contained no detectable procyanidin. The observation of procyanidin in the soluble starting protein and its absence in the proteins separated chromatographically was confirmed by mass spectrometry. Since we have also observed that procyanidins were not covalently bound to wine proteins in haze, the lack of covalent binding or even a strong association between the soluble proteins and procyanidins was consistent.

Although the observations are relatively limited, protein hazes with a higher content of procyanidin tended to be sourced from wines which were high in total flavonoids. The standard protein, bovine serum albumin (BSA), when heated in wines, also formed a haze which contained procyanidins.

However BSA did not precipitate or form insoluble complexes when heated in model wine even with

catechin, a monomeric flavanoid, present. A similar result was observed with the chromatographically separated wine protein fractions. All but one of the fractions gave no significant haze when heated in model wine, whereas all gave an obvious haziness and high turbidity readings on heating in an ultrafiltered Gordo wine. Thus, the presence of polymeric flavonoids, for example, procyanidins, appears to be implicated in the formation of haze from BSA and from natural wine proteins.

Neither phenolic association nor glycosylation are responsible for the proteolytic resistance of wine proteins The association of tannins and proteins has also been proposed as an explanation for the resistance of wine proteins to enzymatic degradation. Since we now have a procedure to determine the procyanidin content of protein fractions and access to wine protein fractions which were free of procyanidins, it was possible to test this hypothesis. We have shown that the wine protein fractions in a model wine (for example a phenolic free environment) still exhibited remarkable resistance to a cocktail of peptidases. The major protein fractions were not degraded at all. Only one of the minor protein bands was slowly degraded. In contrast, BSA was rapidly degraded by the peptidase cocktail under the same conditions, confirming the high activity shown previously by the enzyme preparation under winemaking conditions. Thus the reason for the resistance of wine protein to proteolysis can not be the association of wine proteins with phenolics. This is because both the protein fractions tested here and the medium in which they were tested were free of wine phenolics.

Another explanation for the resistance of wine proteins to proteolysis that has been suggested is that the proteins are glycosylated. However most of the wine protein fractions from Gordo wine examined here did not contain any sugar. O f the four protein fractions which did contain detectable carbohydrate, only one fraction contained a sufficient level (eight moles sugar per mole protein), for the protein to be considered glycosylated. The amount of sugar detected in the other three fractions equated to only one mole mannose per mole protein. It is difficult to envisage how such a low level of sugar could protect all of the peptide bonds in these particular


proteins from proteolysis. The proteins left residual in wine may well be a proteolytically resistant and acid stable subset of grape proteins, since these proteins have survived the winemaking process, and the presence of

natural grape and yeast peptidases. Although we have eliminated phenolic association and glycosylation as causes for the proteolytic resistance of wine proteins, the reasons for the resistance of wine proteins to proteolysis remain elusive and a priority for future research.

Haze protective polysaccharides The recent discovery of a natural haze protective factor (HPF) in wine may be a possible partial alternative to bentonite fining to ensure protein stability in white wines. To exploit the factor, it is

necessary to investigate further the composition of wine HPF, alternative sources of the polysaccharide and the nature of the HPF interaction with wine proteins.

1. S creen in g w in e yeasts fo r H P F

In one part of the project, conducted by Ms Isabelle Dupin for her higher degree studies with The University of Adelaide, work has concentrated on screening winemaking yeasts for production of haze protective mannoprotein. Preliminary to this work, it has been necessary to investigate the growth conditions that w ill give the greatest yield of yeast cells with which to begin extraction. The choice of medium was found to be important; for example, classical media have a low sugar concentration compared to that of grape juice, hence the yield of cells was much lower with, for example, Yeast Nitrogen Base media than with a synthetic grape juice medium. The stage of yeast growth at which the cells are harvested was also

important, as cell numbers increase during the fermentation. However, cells harvested at the end of fermentation, although available in greater quantity, are not as easily extracted as cells

harvested during exponential stage. Thus, the optimum conditions have proved to be harvesting during exponential stage after growth in synthetic grape juice medium.

Four extraction methods have been assessed, two physical methods designed to rupture the yeast cell wall, for example, by use of a French press, and by autoclaving— a chemical treatment

using the detergent, sodium dodecyl sulphate (SDS) and an enzymatic treatment using

Zymolyase, a proprietary enzyme preparation containing both proteolytic and glucolytic activity. No active extracts have been isolated from the French sparkling wine yeast, AWRI 275, by the

four methods of extraction. One extract from the flocculent yeast, AWRI 350, collected with a French press, did show haze protective activity whereas extracts obtained by the other three

methods were inactive. For the commonly used winemaking yeast (and one used to ferment wine from which HPF was previously isolated), for example, EC 1118, active extracts have been

isolated by either SDS or Zymolyase treatment. The ability of the yeast to produce HPF seems unaffected by the nature of the sugar in the growth medium. For example, Zymolyase treatment of

EC1118 grown either on mannose or glucose gave a similar amount of HPF. The possibility of extracting HPF from yeast cell wall preparations (yeast 'ghosts') has also been examined, but has so far proved unsuccessful.

2 . A n im p ro v e d assay fo r H P F a c tiv ity

Due to the large number of test samples generated in this project, the heat stability test has been modified to allow for many samples to be assessed quickly and at low concentration. The protein heat stability test (80°C, 6 h; 4°C, 16 h) had been previously modified to a small scale (1 m l) and made quantitative by measuring turbidity as A 540. However, even with this modification haze protective activity tests still required about 10 mg of material. This had created a bottle neck in the screening process, since large quantities of yeast cell wall fractions needed to be prepared, extracted and fractionated. A range of heating protocols at various temperatures and times, and various containers for the heating was assessed. The heat test is now performed on a volume of 100-200 pL in polycarbonate tubes in a 96 well format, allowing rapid transfer after heating (modified to 80“C, 1 h; ice, 1 h) with a multihead pipette to a microtitre tray. The turbidity of all 96 samples in the microtitre tray is then read by a plate reader in less than 1 min, and the data automatically transferred to a spreadsheet on the computer network. This procedure, compared to the former 'micro' heat test, allows many more samples to be assessed, in less than 3 h rather than 24 h, using a tenth of the volume, and avoids errors introduced by manually inputting data.


3 . S tru c tu ra l studies on H P F

In the other part of the HPF project, conducted by Ms Vanessa Stockdale for her higher degree studies with The University of Adelaide, work has concentrated on structural characterisation of the polysaccharide portion of haze protective mannoproteins. The information gained w ill be part of a larger study on structural and compositional analysis of HPF, to enable a better understanding of its functional properties and to aid in determining its identity in the yeast. Initially, methylation analysis is being used to determine structural properties of the polysaccharide portion of the haze protective mannoprotein through analysis of partially methylated alditol acetates. Methylation analysis is a multistep procedure involving initially, methylation of free hydroxy groups in the polysaccharide. Hydrolysis of the methylated polysaccharide gives a range of partially methylated monosaccharides which are then reduced to give partially methylated alditols. The free hydroxy groups on the partially methylated alditols (for example those which were formerly involved in a linkage to another monosaccharide) are acetylated to give partially methylated alditol acetates (PMAAs). The PMAAs are then analysed by CC-MS.

The methylation method selected employs suspended sodium hydroxide in dimethyl sulphoxide, an improvement on earlier methods using methylsulphinylcarbanion, in terms of safety and ease of use, and also in the completeness of the reaction. This latter aspect has been assessed with standard sugars and oligosaccharides,

including those prepared from mannoproteins, such as hen egg albumin and yeast invertase. Appropriate hydrolysis conditions have also been developed. This is an important step in the linkage analysis, because different sugar linkages show differing resistance to hydrolysis. This means that some sugars may be degraded after release under conditions where other sugar linkages resist hydrolysis.

These procedures have been validated using yeast invertase, an appropriate mannoprotein model for HPF, since it has a small amount of haze protective activity. Results are in agreement with those published in the literature.

The data with yeast invertase also demonstrate that in order to improve sensitivity, deproteination of the mannoprotein prior to methylation is required. Several procedures have been assessed,

and deproteination has been achieved by enzymatically degrading the protein portion of the mannoprotein with Pronase or hydrolyzing the bond between protein and polysaccharide with either Endo-B-N-acetylglucosaminidase H or peptide N-glycosidase F. After enzymatic treatment, the resulting digest is passed down a C,f Sep Pak cartridge using water or 20% MeOH— protein is retained on the cartridge while the polysaccharides are collected in the eluate. This purification step is much quicker than the previously used gel permeation chromatography and it also removes salts and detergents which adversely effect the methylation reaction.

Having developed the methods for linkage analysis with the model mannoprotein, for example, yeast invertase, methylation analysis has now been applied to crude fraction of HPF prepared by Ms Dupin. The polysaccharide portion of this crude fraction contained terminal, 2-, 3- and 6-linked mannose and 2,6-linked mannose. These linkage types are characteristic of yeast derived mannoproteins. Attention has now turned to purifying HPF with structural analysis at all stages of the purification.

Project Title Studies on grape and wine phenolics, including their roles in oxidation and wine instability

Project No AWR 9V

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Dr PJ W illiams

Funding $128,456

Time Span 1990/91-1994/95

Objectives • Investigate the variable role of oxygen in its influence on phenolic composition of wines. • Investigate the influence of phenolic

composition of white wines on protein instability.

• Diagnose and to correct the causes of phenolic instability in red wines.


Progress Oxidation of bottled white wines ■ The Institute's Technical Services Croup has been approached by an increasing number of industry

members who have observed the precocious development of bottled white wines under conditions of cellar storage. This phenomenon

manifests itself, in a proportion of the stock after six to 18 months storage, as an obvious browning of the wines accompanied by loss of SO, and ascorbate. Initially, the affected wines show a loss of freshness and ultimately they exhibit madeirised, oxidised aromas and flavours. Wood-aged white table wines appear to be most commonly affected,

however, this may result from the fact that these wines are held for sufficient time for the browning to occur and the use of clear glass bottles permits its observation. The incidence in other wines is

unknown, principally because of the use of darker bottles and shorter pre-release cellaring by the wineries concerned. It has been seen in wines stored either on their sides or upright during storage and is not due to loss of seal integrity of the cork. This premature aging is variable in its extent and has been seen in wines bottled by different companies under different conditions and at different locations. The estimated quantity of oxygen necessary to cause the observed antioxidant losses appears greater than that which could be present due to faulty bottling equipment. These data, and the observation that it is not shown by all bottles from one bottling run, suggest that the corks are responsible, either by containing compounds capable of oxidising phenolics and other wine components or by selectively allowing

ingress of oxygen. Preliminary experiments to examine post-bottling oxidation have demonstrated that corks are indeed capable of consuming SO,. These experiments comprised a cork (either peroxide or chlorine washed) suspended in a model wine solution that contained S 02 in a sealed vessel with an air

headspace, and incubated at either 45‘C for two weeks or 25°C for nine weeks. The S 02 consumption due to the air in the headspace was

significantly lower than that exhibited by the solutions containing corks; for example, all of the corks tested provided an agent of oxidation over and above that of the oxygen in the air headspace. This agent could have been either residual oxidants in the corks, such as peroxide or chlorite

residues, or cork-derived quinones. Although only

a limited number of corks was screened in these studies, none of the corks were capable of causing the extent of oxidative damage observed in the affected wines.

An alternative possibility that could account for the oxidative spoilage is that some corks allow the passage of oxygen. The oxygen permeability of corks has been studied in two ways.

The first series of experiments has been commenced in collaboration with the technical staff of a major wine company. This involves an examination of the effects on white wines of storage in bottles with different closures, for example, Stelvin vs cork, vs Stelvin plus cork. Both

peroxide- and chlorine-treated corks, each from different suppliers, have been used. The study, which w ill be completed only after prolonged cellar storage, w ill provide further information on the presence of residual oxidants in the cork, since such compounds w ill cause premature oxidation of wines sealed with a cork or a cork plus Stelvin closure. Oxygen permeability of the corks w ill affect only those wines that are bottled and sealed with a cork alone.

In the second experimental approach, selected bottles from batches of wines that have already suffered the oxidation problem, and specifically bottles containing wine showing early signs of browning, and those that have not yet browned, w ill be stored in containers under different gases. The time rate of brow'ning of wine in the bottles stored in an atmosphere of oxygen or nitrogen or air w ill be monitored. By focusing on batches of

bottles already affected it is anticipated that this approach w ill provide data on the gas permeability of the corks more expeditiously than the first experiment; it also has the advantage of not

interfering with the corks already in the bottles. Recognising that this spoilage problem is highly sporadic means that a large number of samples will need to be examined in these experiments to establish the cause of the oxidation.

Red wine lacquer-like bottle deposits 1. A p re d ic tiv e test fo r d ep osit fo rm a tio n

In recent years a new type of instability problem in red wines has become evident in the form of a hard, lacquer-like pigmented deposit that adheres to the inner glass surface of bottled red wines. Recent solid state ,3C NMR studies have shown that the deposit contains protein in addition to phenolic constituents. Work in progress on the


problem has involved the development of an assay to predict whether wines w ill give the deposit during bottle storage. The assay has been used to examine the influence of various winemaking parameters, such as oxidation and its protection, cold and heat stabilisation and fining agents, on the formation of the bottle deposit, and procedures to prevent such formation.

The assay involves a determination of the turbidity induced by oxidatively heating a red wine— several tests have been made at temperatures ranging from 45 to 95°C. Wines that

had already thrown a bottle deposit (classified as unstable) gave greater turbidity in the test than wines that did not have a bottle deposit (for example, stable). Turbidity was measured spectrophotometrically at 700 nm. The greatest difference in turbidity between stable and unstable wines occurred between 80 and 90°C when using a dry block heater, or at more than 90°C when using a water bath for the heating. Subsequent cooling of the heated samples also had an influence on the amount of turbidity produced. After evaluation of different variables, the recommended predictive assay conditions are: heating at 84°C (dry block heater) or 90°C (water bath) for 16 h, followed by cooling in running water and holding the sample at room temperature for at least 4 h before spectrometric analysis.

To validate the predictive heat test, 17 red wines were stored at room temperature in bottles filled to capacity. After seven months of storage, a lacquer­ like pigmented deposit formed and adhered to the

inner glass surface of some of the bottles. After quantifying the amount of deposit in each bottle by dissolution in dimethyl sulphoxide and by spectrophotometry, it was possible to demonstrate that the amount of the deposit formed was strongly correlated to the turbidity value from the heating test at the start of the storage.

The material that precipitates during the predictive heat test and that which is measured as turbidity has also been investigated. ,3C solid state NMR spectroscopy of the heat test precipitate showed that it has similar chemical composition to that of the bottle deposit— both contain protein and phenolic substances. Total colloidal nitrogen analysis of the heat test precipitate and supernatant demonstrated that the precipitate is enriched in protein. Thus, it is valid to consider that the amount of turbidity produced on heating is an indication of bottle deposit potential, since the

heat precipitated material is similar in composition to that of the bottle deposit. Further long term cellar storage trials of bottled red wines are also under way.

2 . C o ld s ta b ilisa tio n to a v o id b o ttle d ep o sit

fo rm a tio n

Other experiments have concentrated on finding a practical solution to the problem. Several processes presumed to affect the protein or

phenolic concentration of wines were assessed. None of the commonly used proteinaceous fining reagents, such as gelatin, egg white or PVPP, reduced the potential of wines to throw bottle deposits— bentonite fining, however, was capable

of achieving this. From the limited number of samples that have been assessed, the amount of bentonite required to stabilise the red wines was variable but in a similar range to that required to stabilise white wines against haze formation. Bentonite treatment also reduced the concentration of both flavanoids and anthocyanins by up to 20% and reduced colour density.

Cold storage of red wines was found to be successful in reducing the potential of a wine to throw a bottle deposit. On cooling of red wine, a layer of sludge-like precipitate forms and the supernatant wine has a greatly reduced potential to give a deposit as assessed by the heat test. When the chill-induced precipitate from an unstable wine was added to a stable wine, and the mixture assessed for its potential to give bottle deposit by heating, the turbidity of this mixed wine increased to a level deemed unstable. The supernatant wine from an unstable wine or a stable wine, as well as the chill-induced precipitate from a stable wine, were not capable of this. Thus, the material that precipitates on chilling unstable red wines appears to be responsible for the bottle deposit formation.

The chemical composition of the precipitates produced during cold storage of unstable wines was very similar to that of the bottle deposits. Both ,3C solid state NMR spectroscopy and nitrogen analysis showed that the cold precipitated material and the bottle deposits were enriched in protein compared to that of the untreated wine. ,3C solid state NMR spectroscopy also showed that potassium hydrogen tartrate, anthocyanins and tannins, but not polysaccharides, were precipitated by cold treatment. Nitrogen and amino acid analysis of the cold precipitated material showed that it was enriched in protein compared to the


supernatant wine. The supernatant wines showed a decrease in colour density, and about a 10% loss of anthocyanins and flavonoids. Various time and temperature protocols for cold stabilisation have been assessed. A treatment of 2°C for two weeks or -4°C for five days completely stabilised most of the red wines tested.

The effect of the cold stabilisation procedure on the sensory properties of wine was also investigated. A commercially bottled dry red wine, which was shown to be highly unstable by the predictive heat test, was treated for 14 days at 2°C. A control wine was held at room temperature.

Both lots of wine were centrifuged to remove insoluble material and then assessed by a panel of tasters using a duo-trio difference test. The panel were unable to detect any difference on nose and

palate between the cold treated wine and the control. The efficacy of cold stabilisation in preventing bottle deposit formation has been supported by

long term storage trials. Twelve commercial wines (10 of which had a bottle deposit) were subjected to cold storage at -4°C for 5 days. The supernatant wines from the cold treatment and untreated controls were placed in bottles and stored at room temperature. After 6 months, 11 of the untreated wines produced significant lacquer-like bottle deposits. For the cold-treated wines, however, eight showed no visible deposit and four had only slight deposits.

Project Title The prevention of cork taint in wines

Project No AWR 94/4

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Dr M.A. Sefton

Funding $55,758

Time Span January 1995-June 1995

Objectives • Assist the Australian wine industry to undertake a more effective quality control program for their cork purchases • Assist the industry in training tasters to screen

for these compounds which w ill aid industry in taking steps to reduce, or even eliminate cork taint occurrence • Investigate whether, and how, TCA can be formed from 2,4,6- trichlorophenol following closure whcih w ill show whether steps to prevent such formation need to be taken.

Progress 'Fungal must' cork-taint Analyses have been carried out to determine the cause of a 'fungal musty' (FM) character, assessed

by a levy payer as an important cause of cork taint. Tainted wines and individual tainted corks were supplied by the levy payer; these were extracted with pentane, and the pentane extracts analysed

by GC/Sniff. In every case, the taint associated with the wine or cork could be attributed to a single point on the chromatogram that did not correspond to any compound known or believed to be responsible for cork taint. Several standards were also run, allowing the precise location, on the chromatogram, of the compound causing the taint to be determined under a variety of operating conditions. One sample assessed as having a

particularly strong FM character was then analysed by GC/MS. The location in the chromatograms of the compound causing the taint was free of large peaks due to cork components. This region

contained trace quantities of four components. The spectra of these compounds were extremely weak, and further samples w ill need to be collected and combined to obtain better spectra. One of the compounds was shown to be 1,2-dichloro­ benzene, which had a GC retention time that was virtually identical to the compound responsible for the FM taint. However, GC/Sniff showed that this compound was not responsible for the taint. A second component in the taint region of the chromatogram appears to be a dialkyl benzene with a molecular weight of 134. Analysis of authentic samples of all three isomers of iso-

propyltoluene have shown that these compounds are also not responsible for the taint. Other dialkylbenzenes are currently being investigated. Two other compounds in the FM region of the chromatogram have identical spectra to each other which resemble that of camphor and of some

alkoxypyrazines. 2-lsopropylmethoxypyrazine, which is a cause of taint in several foodstuffs but is not known as a cause of cork taint, was investigated as a possible cause of the FM aroma,


but was shown to have a slightly longer retention time than the FM compound.

The contribution of TCA to cork-taint Synthesis of deuterated analogues of 2,4,6- trichlorophenol (TCP) and 2,4,6-trichloroanisole (TCA) has been successfully completed. Both compounds are required for experiments on the formation and transportation of TCA, and the

deuterated analogue of TCA is now being used as an internal standard for a more accurate and rapid analysis of TCA in wines. The Institute now has sufficient stocks of deuterated TCA for research

and analytical purposes to last for the next 10 years or more. In a survey of wines presented to participants in the Advanced Wine Assessment Short Course in April 1995, 18 (4.5%) out of 398 wines presented were assessed by at least 20% of the participants as being affected by cork taint. Fourteen of the 18 wines were dry whites, that is 7.7% of the dry whites presented were seen as tainted. These 18 wines were then analysed for chloroanisoles, using deuterated TCA as the internal standard. TCA was present at a concentration close to, or above the sensory detection threshold in every wine analysed. All cases of taint seen by the participants could be attributed to the cork, since bottle to bottle variation could be seen in every case. The corks from these bottles w ill also be analysed in the near future and the results should indicate whether the chloroanisoles in the corks are likely to be due to contamination of the cork bark in the forest, or whether the taint is the result of processing or transport of corks. The survey w ill be repeated during the Advanced W ine Assessment Short Course to be held in November/December

1995, and this w ill provide a large sample overall on which to base our conclusions. Three medium-term trials covering a period of 4-18 months have been established using the

recently synthesised deuterated analogues of TCA and TCP. The first two trials, which study 10 samples each of 12 different types of cork, w ill investigate to what extent TCA can pass through a cork from the outside and what is the nature of the equilibrium between the TCA in wine and corks. The third study, which utilises 6 different wine samples, each treated with varying amounts of antimicrobial agents, w ill investigate whether TCA can be formed in the bottle as has recently been claimed.

Technical Services Group

Project Title Technical problem solving and consultation

Project No AWR 10V

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Mr P A Leske

Funding $171,558

Time Span 1990/91-1994/95

Objectives Contribute to the maintenance and improvement of the quality of wines and grape spirit produced

in Australia by providing: • A technical advisory and problem solving service to the wine industry in the form of the Technical Services Laboratory and its staff with

support from research staff if necessary for more complex problems; and • A winemaking consulting service, primarily by the Group Leader, a qualified winemaker, with

support from the remaining extension and research staff if necessary.

Progress Technical advisory and problem solving service The provision of problem-solving analysis and advice to Australian winemakers represents a significant and growing proportion of the workload in the Group. Several hundred wine samples are submitted to the Service each year (Table 1), which are subjected to sensory evaluation and either routine or complex investigative analyses, in order to determine the cause of the problem. Winemakers are offered remedial and preventative advice based on the cumulative problem solving experience of the staff, and the Group Manager's practical winemaking experience.

Confidentiality is an important aspect of such services, and is strictly maintained in all cases. When a particular problem is thought to be of interest to the wider industry, the results of investigative work are made available through relevant publications, but under no circumstances is the name of the winery or any possible identifying reference revealed.


A summary of the number and type of samples received by the Group over the past three financial years is presented in Table 1. The problems are diverse in nature, but some trends are apparent from year to year. W hile the number of wines submitted with general microbiological problems and hazes increased markedly from 1992/93 to

1993/94, this trend was not sustained in 1994/95. The pleasing decline in the number of 'tainted' wines submitted from 1992/93 to 1993/94 was reversed in 1994/95. Many of these samples arise from equipment failure, such as hydraulic fittings on grape harvesters, and from faulty winery

refrigeration installations. In one such case, a sample of red wine, suspected to be contaminated with a methanol- based refrigerant brine, was submitted along with a reference sample of uncontaminated wine and a sample of the brine. The methanol content of the suspect wine was markedly higher than that of the control sample— on the basis of this the contamination was estimated to be 3.6% by volume. Two other cases of suspected refrigerant contamination were confirmed by GC/MS analysis.

In each case, the wine was found to contain traces of methyl /so-butyl ketone, added to ethanol-based brines as a denaturant. The number of samples submitted with cork related problems shows steady growth, which is of concern to the Group and the wider industry. Many such samples are submitted for the

investigation of suspected cork taint problems. In response to this issue, the Institute has re-initiated its research on the origin and behaviour of cork taint compounds, in order to develop better

methods of detecting and therefore rejecting tainted corks. Other cork-related problems include leakage and seepage, poor adhesion of the cork in the neck of the bottle, and the shedding of surface treatment materials and cork dust into the wine. Of

particular concern was the increase seen in the number of samples with the latter category of problems: up by 58% over those submitted the previous year. An example of the type of problem observed includes a wine that contained a sample of floating material, thought to be surface treatment material, based on its physical appearance. This was confirmed by infra-red spectroscopy. The quantity of treatment material on the cork was determined, and found to be in excess of 100 mg, compared to the expected quantity of approximately 20 mg per cork.

Table 1. Summary of the number and type of samples submitted to the Group for problem­ solving during the past three years

Samples received 92/3 93/4 94/5

Determination of haze, deposit, etc. 51 108 118 Microbiological investigations 20 54 38

Organoleptic assessments 66 44 77

Taint problems 83 49 109

Other investigative analyses 247 101 132 Cork assessments' NA 118 207

Total 467 474 681

1 Until 1993/94, cork assessments were recorded as Other'

Table 2. Enquiries received by Group advisory staff during the past three years

1992/3 1993/4 1994/5

Wineries 576 678 796

Government organisations 85 115 97

Other 354 394 420

Students' NA 56 39

Total 1015 1243 1420

' Until 1993/94, students were recorded as Other'

The cases of microbiological problems included several wines affected by mousy taint, including one exceptionally strongly tainted sample. In this case, the wine was irretrievably spoilt, but in several other instances, often when the taint was detected in young red wines soon after primary or secondary fermentation, the taint was removed by subsequent addition of sulphur dioxide.

Several samples of wines with physical instability were submitted by different wineries, each of which was subsequently identified as being affected by copper instability. These several cases are indicative of the variable nature of copper instability, and it is hoped that the ongoing work being conducted at The University of


Table 3. Field visits and other major external technical service activities during 1994/95

Date Region Staff Major activity

14 July Riverland, SA


Prof. Terry Lee, Present seminar, visit vineyards and wineries Dr Pat Williams, Dr Paul Henschke, Phil Spillman, Alex Sas, Peter Leske

26 July Clare, SA Prof. Terry Lee, Present seminar

Dr Pat Williams, Dr Paul Henschke, Holger Gockowiak, Phil Spillman, Alex Sas,

Peter Leske

27 July Barossa, SA Prof. Terry Lee, Present seminar

Dr Paul Henschke, Dr Elisabeth Waters, Holger Gockowiak, Phil Spillman, Alex Sas,

Peter Leske

4 August Coonawarra, SA Prof. Terry Lee, Present seminar, visit vineyards and wineries Dr Pat Williams, Dr Paul Henschke, Dr Mark Sefton, Alex Sas, Peter Leske

1 5 August McLaren Vale, SA Prof. Terry Lee, Present seminar Dr Paul Henschke, Dr Elisabeth Waters, Dr Mark Sefton, Holger Gockowiak, Alex Sas,

Peter Leske

20 August Mornington Pensinsula, Vic. (Mornington)

Prof. Terry Lee, Present seminar and simulated faulty wine Dr Paul Henschke, tasting, visit vineyards and wineries Alex Sas, Peter Leske

21-22 August Campbelltown, Tas. Prof. Terry Lee, Present seminar and simulated faulty wine Dr Paul Henschke, tasting, visit vineyards and wineries Alex Sas, Peter Leske


23 August Yarra Valley, Vic. Prof. Terry Lee, Present seminar and simulated faulty wine (Lilydale) Dr Paul Henschke,

Alex Sas, Peter Leske

tasting, visit vineyards and wineries

24 August Central Victoria Prof. Terry Lee, Present seminar, visit vineyards and wineries (Nagambie) Dr Paul Henschke,

Alex Sas, Peter Leske

25 August Rutherglen, Vic. Prof. Terry Lee,

Dr Paul Henschke, Alex Sas, Peter Leske

Present seminar, visit vineyards and wineries

5 September Sunraysia, Vic. Prof. Terry Lee, Present seminar, visit vineyards and wineries (Mildura) Dr Paul Henschke,

Alex Sas, Peter Leske

6 September Central-Western Vic. Prof. Terry Lee, Present seminar, visit vineyards and wineries (Moonambel) Dr Paul Henschke,

Alex Sas, Peter Leske

28 September McLaren Vale, SA Peter Leske Address seminar and present simulated faulty wine tasting

20 October Adelaide, SA Peter Leske Convene seminar and present paper

ASVO seminar: Corks and Closures

(co-authors: Nick Bruer and Dr Mark Sefton)

24-25 Nov. Adelaide, SA Greg Ruediger, Address symposium

CRCV Symposium Peter Leske

13-18 January Adelaide, SA Prof Terry Lee, Present a series of lectures Institute of Masters Mark Gishen of W ine training program

27-28 January Granite Belt, Qld Alex Sas, Present seminar and simulated faulty wine (Stanthorpe) Peter Leske tasting, visit vineyards and wineries

15-17 Feb Adelaide, SA: Peter Leske, Lead, tutor, conduct tastings, organisation

Advanced Wine Nick Bruer, Assessment Short Mark Gishen, Course John Hughes

21 February Mornington Peninsula, Vic Mark Gishen Present workshop

22 February Yarra Valley, Vic. Mark Gishen Present workshop

9 June Adelaide Hills, SA Peter Leske, Address seminar

(Lobethal) Mark Gishen


Melbourne w ill be of assistance in determining with greater surety the conditions under which it may occur. The composition of the various wines and conditions of the formation of the casse were all quite different: in one case the wine was heat stable (indicating that protein was unlikely to be implicated) and had a concentration of copper of only 0.15 mg/L, while another was a heat stable sweet wine, of which three samples had a concentration of copper of 0.63, 0.70 and 0.78 mg/L, respectively.

A further case involved a wine which had been recently bottled. The filters had been changed during the bottling run— samples taken before the change had a concentration of copper of 3.2 mg/L, while after the change the concentration was only 0.7 mg/L. The wine had presumably been contaminated by the filters or another piece of bottling equipment. The wine was also shown to be grossly heat unstable.

Novel, non-instrumental methods were devised for the analysis of the size and persistence of mousse and bead in several sparkling wines. The results of this assessment were compared with the quantity of total protein in the wines, in order to investigate the relationship between the two and confirm the impressions of quality formed by winery staff. Quite substantial differences in quality of bead were apparent, and a relationship between bead and the quantity of protein present was noted.

Several winemakers submitted white and red wines from the 1995 vintage, for the assessment of laccase activity. Positive activity was found in samples of both wine styles from Coonawarra, Padthaway and central Victoria. Winemakers were advised to heat-treat the wines after clarification to deactivate the enzyme.

Three different lots of wine containing a haze and/or deposit found to consist of a protein/polyphenolic complex were submitted. An improved test for the presence of polyphenolic material was used in the investigations. The test relies on the acid hydrolysis of condensed tannins to form cyanidin, which has a reddish colour. In one case, samples of the same wine with and without the haze were compared. A significantly

lower concentration of free and total SO, and of ascorbic acid was noted in the affected sample, suggesting that oxidation had occurred. The resultant polymerisation of the phenolics probably led to co-precipitation with protein, which was

confirmed by the observation that the concentration of flavonoid phenolics in the affected wine was approximately 80% of that in the stable wine.

Winemaking consultation The Technical Services Group provides a winemaking consultancy service principally through the Manager, a qualified and experienced oenologist. Most queries are technical in nature and arise from Australian winemakers, however the Manager, Viticulturist and Technical Services Specialist also receive many general queries from Government bodies, the general public, and secondary and tertiary students. Where appropriate, the query is answered over the telephone— more complex cases are solved with winery visits and the support facilities provided by research and Library staff. The analytical capacity of the Technical Services Laboratory plays an important role in some of these enquiries.

The Institute often acts as a referral service, having links to Australian and international wine research and political bodies. The vast store of information, both formal (in the John Fornachon Memorial Library) and informal, is a valuable resource to the wider industry.

A summary of the enquires received by the Group Manager, Technical Services Specialist, Quality Liaison Manager and the Director during 1994/95 is presented in Table 2. The steady growth in the number of requests for advice and assistance seen over the past several years continued, with wineries consistently being the main users of the service.

This consultation and advisory service is supported by vineyard and winery visits and seminar tours to all major wine growing regions, generally organised in conjunction with local vignerons' associations. The Institute aims to reach each major Australian viticultural region through such formal visits and tours every second year, with routine shorter visits by key staff as opportunities arise, frequently in conjunction with industry events such as capital city Wine Shows, and seminars held by other industry bodies.

The major focus of the program of external services was an extensive tour of South Australian and Victorian regions, where various Institute staff addressed over 300 industry personnel at 12 seminars in July, August and September, and a two-day workshop in the Granite Belt,


Queensland, in January. In addition, The fifth Advanced W ine Assessment Short Course was conducted by the Institute during the year. The Associate Judges for the Adelaide Wine Show were again drawn from the participants of the Course. Strong demand for further Courses continues. Several members of the Group assisted with the presentation of tastings and the running of the courses, led by Mr Geoff Weaver and the Group Manager.

The Director and the Quality Liaison Manager provided tutorship to students of the Institute of Masters of Wine program, which held its annual school at the Institute for the third consecutive year. The program is run by the Australasian Advisory Council to the Institute of Masters of Wine, and comprises several days of lectures in aspects of viticulture and winemaking, and

intensive sensory evaluation sessions. Table 3 lists the industry visits and seminar events in which Group staff participated in the year.

Project Title Evaluation of new analytical techniques and of processing aids for winemaking

Project No AWR 11V

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Mr P A Leske

Funding $126,412

Time Span 1990/91-1994/95

Objectives • Maintain and, where appropriate, improve current methods of analysis of wine and grape juice; and • Evaluate the suitability of a range of winery

processing aids for the benefit of the wine industry and the research group staff at the Institute.

Progress Pesticide residue laboratory The Residue Unit, formerly a part of the Director's Group, was incorporated into the Technical

Services Group at the beginning of 1994/95.

The three staff of the Laboratory, Messrs G.A. Ruediger, A.P. Pollnitz, and K.H. Pardon, continued their work on the development and application of methods of analysis of pesticide residues in grapes and grape products. A major achievement was the gaining of ΝΑΤΑ accreditation of the laboratory and registration of the gas chromatography/mass spectrometry multi-residue assay. The assay has since been subject to further development work, and currently provides rapid analysis of over 24 common fungicides, insec­ ticides and herbicides, of which 19 are ΝΑΤΑ

registered. A method was also developed for the analysis of fluazinam in grapes and wine, and used to provide residue data from application trials. Other development work included HPLC methods for the determination of dithianon and carbendazim, and extraction procedures for dried grapes, fresh vine

leaves and soil samples. The resources of the Laboratory were used to provide analytical support to participants in Program 2 of the Cooperative Research Centre for Viticulture (CRCV). Several hundred grape and wine samples were analysed and the results used

in the interpretation of trials of pest management, spray application, and canopy management techniques. Additionally, preliminary trials

investigating the effect of storage and various winemaking treatments on the rate of degradation of several agrochemicals in wine were conducted as part of CRCV program 3.

The methods were used to analyse 105 commercial red wines on behalf of the Australian Wine and Brandy Corporation, following the analysis of 50 white wines in 1993/94. These comprehensive surveys have shown that the

majority of Australian wines contain no detectable residue of common agrochemicals, and in all of the wines analysed, no residue has been detected at a concentration in excess of the relevant Australian maximum residue lim it (MRL). The

Laboratory also provided routine analysis on a commercial basis for many Australian wineries, analysing grape, wine, vine leaf and soil samples.

Evaluation of new analytical techniques and of processing aids for winemaking The Technical Services Laboratory maintains a continuing project in the area of the improvement and development of methods of wine analysis, and the evaluation of winemaking processing aids and


additives. The evaluations take one of two forms: the relative performance of commercially available products (market surveys of commonly used additives), and the evaluation of new materials marketed to the industry. The staff also provide analytical support to the Australian Wine and

Brandy Corporation and the industry through the coordination and conduct of surveys of aspects of wine composition.

The Group has coordinated surveys of the anion composition of Australian grape juices and wines since 1989. The former concluded after the analysis of 230 samples from the 1994 vintage, bringing the total number analysed since 1989 to 1243. The survey of wines w ill continue for several more years— to date 616 samples have been collected and analysed. The data collected through these surveys w ill be published in an appropriate journal, so that it may be a useful resource to the

industry, especially in the field of international regulatory issues. The laboratory participated in several collaborative studies in 1994/95, including a ΝΑΤΑ alcohol and specific gravity proficiency test, and the coordination of samples submitted to members of the Interwinery Analysis Group, Inc.

The project initiated in 1992, evaluating methods for the determination of cold stability in wines, concluded in 1994/95, with the collection and analysis of wines from the 1994 vintage, and samples subjected to long term storage in the Institute's cellar. The results of the work were summarised for presentation to the industry at the Ninth Australian Wine Industry Technical Conference.

The method devised by staff of the Institute for the detection of chlorogenic acid in wine was validated on the equipment of the Technical Services Laboratory. It was then applied to the routine analysis of over 100 commercial wines, on behalf of the Australian Wine and Brandy Corporation. None of the samples contained detectable chlorogenic acid.

Following a query from a winemaker with reported negative feedback from the marketplace, the Group re-initiated its work on the quantifi­ cation of biogenic amines by HPLC. Using pre­ column derivatisation with s-phthaldialdehyde and fluorescence detection, a detection lim it for the samples in question was established at 3 mg/L. There was no evidence that the amine composition of the wine was responsible for the alleged problem.

A rapid system of analysis by colorimetric 'strips' was evaluated. The results were promising for the determination of ascorbic acid and iron in white wine, and hydrogen peroxide in water (of potential value in assessing cork quality), but poor for S 02 in wine and ammonium in grape juice. Further evaluation is required to determine the applicability of the device for other wine components.

Quality Liaison Manager M r M Gishen was appointed as Quality Liaison Manager in October, following a steady increase in the number of queries from industry to the Group, regarding the application of total quality management (TQM) techniques in vineyards and wineries.

The Quality Liaison Manager took responsibility for the dissemination of information regarding TQM, and liaised with other organisations such as the South Australian W ine and Brandy Industry Association in the preparation of a kit to assist

industry members in the implementation of such techniques, to be presented in pilot form to the industry later in 1995. He also provided assistance to the existing analytical methods and wine composition surveys, and is responsible for the maintenance and development of the Group's internal quality management systems.

Project Title Provision of technical information

Project No AWR 12V

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Mr P A Leske

Funding $126,412

Time Span 1990/91-1994/95

Objective Provide a technical and regulatory information service to the Australian wine industry, Institute staff and other organisations by means of direct

contact, print medium and construction of computerised databases.

9 6

Progress The John Fornachon Memorial Library The John Fornachon Memorial Library (JFML) is the largest collection of technical wine literature in Australia. The Library's principal responsibility is to

provide technical information to the Australian wine industry and to the researchers of the Institute. The Library is also used extensively by other groups, such as students, government bodies

and private companies. The Library relocated to the newly-opened Woolhouse Library on the Waite campus of The University of Adelaide in early February. This was

achieved with minimal disruption to library services and without the loss of a single working day, thanks to a team of dedicated staff who assisted with the move. The Library's relocation

means that library staff are in an ideal position to take advantage of the University's computer network, with access to the Internet and links to a

range of electronic document delivery services, thus enabling the Library to keep pace with the changing requirements of the Australian wine industry. The Library's relocation has had no

negative effect on the provision of services to industry or staff of the Institute, and has facilitated the provision of technical information to new client groups, such as students, research staff and private companies, by making the collection more accessible within a wider reference setting.

Negotiations are currently underway with The University of Adelaide to enter the details of the JFML's books and journals as a separate catalogue, accessible through the University catalogue system. The JFML's internal specialist in-house

information databases w ill be maintained separately, servicing industry members and industry staff through the Technical Services Group staff.

Librarian, Ms S. Dimitriadis left the Library late

in 1994, and was replaced by Mrs C.G. Daniel. The Library Assistant, Ms R.A. Cottle, resigned in March 1995, and was replaced by Ms I.B.M. Oats. Both new staff have settled well into their roles.

Information and document delivery services The JFML continues to have excellent access to international databases, particularly in the fields of science, technology and medicine. This access is complemented by the Library's new CD-ROM searching facilities.

Many requests for information are met entirely from the Library's in-house databases, for which no charge is made to levy payers. If requested, the Librarian can carry out online searches on commercial databases on any topic. The cost of such searches depends on the complexity of the subject. Only costs directly incurred in carrying out an online search are passed on to the industry client.

Other free Library services include answering information queries and supplying copies of Institute staff papers and Technical Notes. Apart from Technical Review, the Library receives many enquires by facsimile, telephone, and in writing. A summary of information requests for 1994/95 is

presented in Table 1. The Library supplies either books or photocopies from its collection, or obtains such items for industry clients through the interlibrary loan

system. Patents or standards can also be ordered. Electronic ordering and delivery services mean that most interlibrary requests can be fulfilled within five days. Charges apply for the supply of some


Agrochemicals Grid The Librarian and the Viticulturist prepared the sixth edition of the Agrochemicals registered for use in Australian viticulture which is commonly

Table 1. Summary of information requests during 1994/95

Winemakers Institute staff Others Total

Technical Review requests 180 180

Technical Review reprints 916 916

Staff publications 314

Online database searches 20 90 5 115

Other information requests 205 613 391 1209


known as the 'Agrochemicals Grid'. All Wine Grape Levy payers receive a copy of the revised Grid automatically.

T e c h n ic a l R e v ie w

Technical Review is received by all Wine Grape Levy paying wineries in Australia and, through subscription, by government and other organisations, both in Australia and overseas.

Technical Review provides progress reports to the industry on the Institute's research programs and updates on relevant conferences, regulatory amendments and medical issues. Technical

Review's Current Literature section provides citation details and abstracts of recently published technical and scientific articles. Recipients of

Technical Review may order articles featured in Current Literature via a request form available within each issue. Demand for such articles continues to be high, averaging 76 articles per month during 1994/95.

Staff of the Library played a major role in the compilation of the first edition of the Technical Review Index, prepared in time for release at the Ninth Australian Wine Industry Technical Conference. The Index was constructed to facilitate the location of relevant papers on any topic published in the Technical Review since issue 28, February 1984. Library staff can supply copies of articles appearing in the Index or generate reports detailing the contents of articles indexed. This edition and subsequent revisions w ill be sent to all Wine Grape Levy-payers, free of charge.

Library collection One hundred and fifty-four monographs and 19 conference proceedings were added to the Library collection during this year. The Library subscribes to 51 technical journals, and receives approximately 80 annual reports, journals and newsletters through exchange and donation. The Library also maintains a collection of over 12 000 reprints.

Library databases In addition to a computer-based catalogue of books and journal holdings, the Library has several specialist in-house databases, indexing a range of material, such as 19 000 scientific and technical reprint articles and almost 1400 articles on the medical aspects of alcohol consumption. The in­ house database can also provide the registration and maximum residue limits of vineyard agrochemicals used in Australia's main export

markets, brief records detailing the wine regulations for permitted preservatives and processing aids in nearly 60 countries, and the bibliographic details of the library's collection of European Community wine legislation.

The Librarian provides reports, either on particular subjects or authors, listing the records retrieved from any of the Library's in-house databases. A summary of the size of the Library's catalogue and information databases is given in Table 2.

Table 2. Number of records on the Library's catalogue and information databases

Number of records

L ib ra ry c a ta lo g u e databases

BOOKFILE: books, conferences and theses 2535 PAPERS: scientific papers 12049

MEDIC: medical papers 1343

JOURNALS: journals, newsletters, statistics and annual reports 373

L ib ra ry in fo rm a tio n databases

PRESERVE: wine additives legislation 783 REGS: European Community wine legislation 214 AGROCFJEM: agrochemicals 1917

MAILNEW: contact names and addresses 1651

I SYS — fu ll-te x t re trie v a l d atab ase coverin g

• United States of America Federal Register 224 • W ine Practices Committee documents 36 • Technical Subcommittee documents 67 • International Trade and Technical Advisory

Committee 75

Project Title Chemical analysis of industry technical problems

Project No AWR 19V

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Dr M A Sefton

Funding $90,788

Time Span 1990/91-1994/95

9 8

Objective Provide a service to the wine industry for the solution of major technical problems, the diagnosis of which require the use of advanced chamical and instrumental analysis techniques.

Progress Analysis for suspected chloroanisole and chlorophenol taints in wines Chloroanisole contamination continues to account for the majority of taint problems investigated by the Institute. Following sensory investigation by the Technical Services Group, wines from four wineries, believed to be affected by cork taint,

were further analysed by GC/MS. The analyses showed that all wines assessed by an Institute sensory panel as tainted contained 2,4,6- trichloroanisole (TCA) at a concentration high enough to have a deleterious effect on the quality of the wines. The tainted wines from three of the wineries had a moderate level of TCA only, albeit occuring at a frequency much higher than that normally encountered in the Australian industry. The wines from the fourth winery had only been bottled for a few weeks. The wines from this winery were assessed as having varying degrees of taint, and were found to contain a correspondingly variable level of TCA. The corks from these wines,

however, all contained a high concentration of TCA, indicating that it would have been only a matter of time before all of the bottles became badly tainted.

A wine submitted by another levy payer had been assessed by an Institute panel as being strongly tainted by the cork, but the concentration of TCA in this wine was low (1.4 ng/L) and this compound could not be considered as a primary cause of the observed taint. This demonstrates the

importance of carrying out further research on compounds, other than TCA, which might cause musty off-odours in wine. Wines believed to have been tainted by contact with contaminated barrels were submitted by three different levy payers for chloroanisole analysis. The wines from two of the wineries contained 2,4,6- trichloroanisole at a low concentration, sufficient to have a just discernible impact on the wine for some tasters. The results were consistent with the

sensory assessment of these wines by an experienced panel from the Institute, who detected only a low level of taint in the samples. The wine from the third winery was contaminated with tetrachloroanisole (TeCA) which has similar sensory properties to TCA. The TeCA in the wine was possibly due to contamination of the barrel staves with 2,3,4,6-tetrachlorophenol, a commonly used biocide.

Three levy payers submitted wines for analysis for compounds that could cause taint. Wines from two of the levy payers were found to contain 2.4.6- trichloroanisole at a concentration high enough to cause the perceived taint. The wine from the third levy payer was assessed by an

Institute sensory panel as having little or no taint, and no chloroanisoles were detected in the wine. Another winery submitted two water and two wine samples for chloroanisole and chlorophenol

analysis. The water sample contained 2,4- and 2,6- dichlorophenol, together with 2,4,6- trichlorophenol. No tetra- or pentachlorophenols

were found, nor were there any chloroanisoles in the water samples. The wine samples contained 2,4- and 2,6-dichloroanisole together with pentachlorophenol. No other chloroanisoles or

chlorophenols were observed. The chlorophenol and two chloroanisoles were present in the wines at a concentration well above their threshold. The

taint in the wines could therefore be attributed to these compounds. Investigations of cork taint problems submitted to the Institute such as those described above

indicate that, while cork taint may be no more than a nuisance for wine producers and consumers on most occasions, contamination of corks by TCA can sometimes cause severe problems for

individual producers. Where such severe problems are being encountered at the Institute, samples of tainted wines and corks are being put aside for future more detailed investigation as part of the

Institutes' research program into cork taint.

Oil taint in wines Six instances of suspected oil taint, all from different levy payers, were investigated by chemical analysis. In two cases, sensory evaluation

indicated a strong hydrocarbon-like taint in the wines. However, chemical analysis of the two wines failed to show evidence of contamination, because the relevant peaks were obscured by the background of fermentation products. The juice from which one of these wines was made, was also available for analysis and in this case, trace quantities of aromatic hydrocarbons which have been previously shown to be components of


hydraulic or diesel oil, were detected. In two other investigations, grape juices, suspected of being contaminated with hydraulic oil, were shown to contain a group of aliphatic hydrocarbons, but these hydrocarbons were typical of naturally occurring grape compounds rather than of petroleum oils. Also, no aromatic hydrocarbons were detected.

A sample of bentonite, believed by a levy payer to be capable of imparting an oily taint to wine, was submitted for analysis. A second sample of bentonite, assessed by the levy payer as free of taint was also submitted for comparison as a control. An extract of the control bentonite sample contained mainly n-tetradecane, n-pentadecane and n-hexadecane at low concentration as well as trace amounts of other hydrocarbons. The extract of the tainted sample contained the same compounds as observed in the control, but, in addition, also contained a highly complex mixture of branched and unsaturated C11 to C18 hydrocarbons. The oily taint derived from the bentonite sample could be attributed to this complex mixture of hydrocarbons.

A sixth levy payer submitted a wine sample which had an oily film on the surface. The gas chromatogram of the solution of the oily film showed peaks of high intensity which probably account for most of the oil composition. The largest peaks in the chromatogram were assigned as octanoic, decanoic and dodecanoic acid, together with the corresponding ethyl esters of these compounds. Although these compounds are major wine volatiles, no other common wine volatiles were observed in the chromatogram. The chromatogram also contained a homologous series of n-alkanes from C14 to CI8, and there was a broad hump in the C22 - C24 n-alkane region of the chromatogram. Mass spectra in the region of this hump indicated that it was a complex mixture of hydrocarbons. The observation of this hump is consistent with the presence of a hydrocarbon- based oil, possibly a lubricant, in the sample. The composition of the oily film suggests that it contained mainly wine distillation components, possibly from using hoses and other equipment that had not been cleaned correctly, but that it also contained a hydrocarbon-based oil from an unknown source.

These and previous investigations carried out by the Institute have shown that, while oil contamination of juices can sometimes be

detected, such contamination can be difficult or impossible to detect once the juice has been fermented. As oil contamination problems are regularly encountered at the Institute, development of a new methodology to detect hydrocarbons in wine at low concentration should be developed.

Other taint and contamination problems A brandy sample which was strongly tainted, together with an untainted control, was submitted by a levy payer for analysis. The tainted sample contained a high concentration of naphthalene which was not present in the control. No other analytical differences between the tainted brandy and the control were evident.

Two levy payers submitted wine samples for analysis from tanks which were suspected of being contaminated with a cooling 'brine'. A component of the brine, 4-methylpentan-2-one, was found to be present in the wines, confirming that contamination had taken place in both cases.

A sample of wine which had been stored in a fibreglass tank and which had a strong plastic-like taint was submitted by a levy-payer for analysis. The gas chromatogram of the wine headspace contained a prominent peak due to styrene, which

is known to cause such a taint. This peak was among the largest in the chromatogram, being three times the size of the peak for the major fermentation volatile /so-amyl acetate, and one third of the size of the peak for ethyl hexanoate. The presence of this quantity of styrene, which is not a natural wine component, demonstrated gross contamination of the wine, most probably because the fibreglass had been improperly cured. The gas chromatogram of the wine headspace also showed the presence, in the wine, of ethylbenzene, three

isomers of xylene, and a series of methylethyl-, propyl- and trimethylbenzenes. Some of these compounds have been reported as impurities which co-occur with styrene and are also extractable from fibreglass tanks.

An investigation into an apparent taint thought to be caused by the plastic used for bag-in-the box packaging showed that, w hile the plastic did not contribute any significant contaminants to the wine, it was nevertheless able to adsorb a large proportion of the flavour volatiles from the wine, thereby altering the wine flavour. The general nature of this phenomenon across the wine industry is unknown and may well be worth further investigation.


A wine which had been stored in old oak barrels and which had an elastoplast/reduced sulphur character was submitted for analysis for 4- ethylphenol. The wine contained this compound at a low concentration only (30 pg/L). At this concentration, 4-ethylphenol would have had no significant sensory effect on the wine. It was not

responsible for the characters seen by the sensory panel. The number of industry problems requiring analysis by gas chromatography/mass spectrometry submitted to the Institute during the past 12 months has increased three-fold in comparison to the previous year.

Project Title To develop links between viticultural and oenological research

Project No AWR 24GW

Organisation The Australian Wine Research Institute




Time Span

Urrbrae, SA

Professor T.H. Lee and Dr R.R. Walker


January 1991-1994/95

Objectives 1. Participate in ongoing viticultural research on wine grapes in relation to wine quality 2. Assess and disseminate information from a

variety of sources where viticulture interacts with oenology. 3. Maintain an awareness of the oenological implications of viticultural techniques and the

requirements of winemakers.

Progress Agrochemicals A large proportion of the Viticulturist's time was

spent liaising with the wine industry, the chemical industry and various State and Commonwealth departments on issues related to agrochemicals. Several enquiries were received from both wineries and vineyard workers for information on

'safe' re-entry intervals following chemical application to a vineyard. Re-entry intervals have not been established for the majority of chemicals

used in Australian viticulture. In response, the Viticulturist and Dr Judy Calvin of the Hunter Area Health Service applied to Worksafe Australia/National Occupational Health and Safety Commission for funding for a six-month research

position. The appointee would formulate guidelines for re-entry periods for vineyard workers. The proposal is being reviewed by the Commission after acceptance of the preliminary application.

A submission on behalf of the grape industries to the National Registration Authority's (NRA) Existing Chemicals Review Program was compiled by the Viticulturist and Ms Alison MacGregor of Agriculture Victoria. The submission asked for the

review of the insecticides endosulphan and parathion-methyl. The NRA assessed all submissions and selected five chemicals for review: atrazine, endosulphan, mevinphos, parathion and parathion-methyl. Parathion (ethyl) is sometimes used in viticulture.

With the assistance of the Librarian, the 1994/95 version of Agrochemicals registered for use in Australian viticulture, more commonly known as the 'Agrochemicals Grid', was produced and

posted to all wine levy payers. The publication lists the agrochemicals that are registered for use in Australian viticulture and the maximum residue limits (MRLs) for these agrochemicals in Australia's

major wine export destinations. This information assists wineries to develop pest and disease control strategies for their growers. The Viticulturist, as a participant in Program 4 of the CRC for Viticulture, is also coordinating the further development of the AusVit Chemical

Database. The database contains label information of the various chemicals used in viticulture as well as notes on MRLs. The chemical industry and the Institute are developing a collaborative relationship to ensure that agrochemicals can be used effectively while

minimising residues in wine. For example, the Viticulturist has assisted in the design of a trial to determine the residue status of a particular company's product. Further evidence of productive collaboration between the wine industry, the chemical industry and government agencies is evident in the development of recommendations for the management of Botrytis using fungicides. In April, the Viticulturist and Dr Chris Green of the CRC for Viticulture called a meeting of these three groups to devise a uniform management strategy.


This has been done and w ill soon be published and distributed to grape growers.

Research Preliminary research indicated that grape juice from vines irrigated by overhead sprinklers using water of moderately high salinity (between 1600 and 2000 EC units) had a significantly higher sodium and chloride content than juice from vines irrigated by dripper. Furthermore, the chloride concentration in wines made from both the dripper and overhead irrigation treatments was significantly greater than that in the free run juice. It appears that the berry skin may contain a major pool of chloride which is then released into the wine during vinification. Therefore, to determine the contribution of the berry skin, pulp and seeds to the concentration of sodium and chloride in juice and wine, the Viticulturist has commenced research to: • evaluate methods of extraction and analysis for

sodium and chloride in the grape berry; • determine the effect of overhead irrigation on sodium and chloride accumulation in the berry skin and pulp; and • determine the contribution of berry absorbed

sodium and chloride to the concentration of the ions in grape juice and wine. This work relies significantly on the support of CSIRO Division of Horticulture, in particular Mr Peter Clingeleffer and Dr Rob Walker.

Experimental sites have been established in vineyards at Loxton and Padthaway, South Australia. The first season's samples have been collected and are being analysed.

Extension The Viticulturist participated in the Institute's extension visit to the wine regions of South Australia, Victoria and Tasmania. He and Mr Peter Leske also conducted a two-day workshop for the Queensland wine industry. The Viticulturist accompanied Mr Clingeleffer on visits to inspect canopy management trials in Griffith, NSW and the newly established vineyards in the Ovens Valley, Victoria.

In addition to addresses given by the Viticulturist, he has attended numerous meetings, seminars, field days and workshops, and produced articles for the rural media. He is also a member of the Planning Committee for the Ninth Australian Wine Industry Technical Conference.

Viticultural enquiries of a technical and general nature have been received from the following sectors:

Private individuals 39

Companies 318

Government agencies 134

Technical Conference 67

Total 558

The companies category includes wineries, vineyards, and chemical suppliers and manufacturers.

Project Title Preparation of information on wine and health issues

Project No AWR 25V

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Professor T H Lee

Funding $7,566

Time Span 1990/91-1994/95

Objectives • Compilation of a database of research on the health effects of wine. • Review of research on the health effects of


Progress Subscription to relevant medical and other journals has continued. The journals have been regularly scanned, the database of research on the health effects of wine has been added to and articles have

been prepared for inclusion in the Institute's publication, Technical Review. Papers reviewing the physiological benefits of regular, moderate wine consumption and the concentration of histamine in wine have been prepared, and papers reviewing the cardioprotective effects of wine, gender differences

in the pharmacology of wine and the health implications of lead in wine have been prepared and published. Lectures on the health aspects of wine have also been presented at Charles Sturt


University, a workshop was conducted at the Winter School in the Sun/Second W indow of Opportunity National Congress, and an interview was conducted with Ms Nikki Dwyer of the Channel 10 News on wine and health issues. In addition, the Information Manager attended a NFA workhop on Safe Food Handling in Australia and the Seventh Biennial Scientific meeting of the

International Society of Free Radicals.

Project Title Relationship between vineyard soil lead concentration and lead concentration in grape berries

Project No AWR 92/2

Organisation The Australian Wine Research Institute

Location Urrbrae, SA

Supervisor Professor T.H. Lee

Funding $28,135

Time Span 1992/93-1994/95

Objectives • Characterise vineyard soils to determine the concentration of lead available for uptake and the soil conditions, for example, pH, which

facilitate this uptake by the vine and thus the grape berry.

• Analyse the grape berry to determine the 'natural' or background level of lead in the grape berry in relation to soil lead status.

Progress A preliminary study of 1 3 Shiraz vineyard sites and a further study of 96 Shiraz vineyard sites in South Australia was conducted from 1991 to 1993. Soil and grape berry samples were collected by the

Institute in conjunction with The Penfold Wine Group, and analysed by graphite furnace atomic absorption spectroscopy (AAS) by the CSIRO

Division of Soils. The concentration of lead in soil from South Australian vineyards ranged from 1.9 to 90.7 mg/kg. The mean concentration of lead in the grape berries was 16.5 ± 9.5 pg/L and the mean concentration of lead in the grape juice was 1.84 ± 0.59 pg/L. This implies that during crushing, only a

minor proportion of the lead present in the berry is released into the juice. Subsequent studies have

shown that the Shiraz variety is not an unique grape variety with respect to lead uptake from the soil and lead compartmentalisation in the berry. Until now, it has been presumed that the lead

content of grapes is significantly reduced during winemaking, for example, that the majority of lead has precipitated out as lead tartrate during fermentation— the final product has, however, a

higher lead content than that of the raw material. The lead concentration observed may reflect the fining agents and the composition of winery equipment, for example, lead soldered copper

plumbing and brass fittings, encountered during vinification.

To determine this source of lead, the vinification process was monitored in two wineries, d'Arenberg Wines Pty Ltd and Wirra Wirra Vineyards. Shiraz and Riesling juice and wine samples were collected at different production stages, from crushing through to bottling and analysed by AAS by the CSIRO Division of Soils.

The preliminary results indicate that the concentration of lead increases and decreases throughout the vinification process. For example, the type of 'storage' container influences the concentration of lead. Juice or wine stored in bituminised concrete has a significantly higher concentration of lead compared to that stored in stainless steel or waxed wood. Fining with bentonite and filtering with diatomaceous earth also contribute to the concentration of lead in the wine while fermentation, both primary and secondary, removes lead from the wine.

Project Title Synergistic and inhibitory interactions between wine yeast and lactic acid bacteria

Project No UA 92/3

Organisations Australian Wine Research Institute, University of Adelaide

Location Adelaide, SA

Supervisors Dr A J Markides (University of Adelaide, Project Coordinator) Dr P A Henschke (AWRI), Dr G P Jones (University of Adelaide)

Research staff Glenn Borlace

Funding $24,445 (94/95)

Time span April 1994 - April 1997


Objectives • Identify combinations of wine yeast and lactic acid bacteria (LAB) that exhibit stimulatory or inhibitory effects on microbial growth and the

conduct of malolactic fermentation (MLF) • Isolate the metabolites responsible for these interactions • Quantify the effect of these metabolites on LAB

growth and the conduct of MLF • Test the stimulatory/inhibitory metabolites in wine environments

Progress The University of Adelaide structured program has been satisfactorily completed with the presentation of a departmental seminar (Feb 1995), the submission of an Outline of Proposed Research (April 1995) and the enclosed Literature Review and Research Proposal (June 1995).

A study of yeast-bacterial interactions has commenced with selected combinations of yeast and bacterial strains sourced from the AWRI Culture Collection. The screening program has commenced with the development of the plate interaction assay using both complex and chemically defined media. Preliminary testing of four Saccharomyces cerevisiae strains [AWRI 729, AWRI 796, AWRI 838 (EC111 8) and AWRI 853 (SIHA-4)] with four Leuconostoc oenos strains

[Lc5a (ML34), Lc5b (PSU-1), Lc5f (Leucostart) and Lc5g (Mayer)] in the complex synthetic medium of de Man, Rogosa and Sharpe (MRS), supplemented with 20% v/v apple juice, is currently in progress. O f particular interest is S. cerevisiae strain EC1118 which, through anecdotal evidence, has been associated with unsuccessful malolactic fermentations. Four additional strains of EC1118 have been selected and are under investigation in the plate interaction assay. Furthermore, the screening program w ill be expanded to include some non-Saccharomyces yeasts which have been implicated in the early stages of the primary alcoholic fermentation.

Preliminary results have indicated that the plate interaction assay can detect interactions between yeast and lactic acid bacteria (LAB). At this stage yeasts have exhibited a range of both stimulatory and neutral effects on LAB growth. Fast growing yeast strains such as AWRI 796 and AWRI 729 have stimulated LAB growth and slow growing yeast strains such as AWRI 853 have exhibited little or no interaction with the LAB strains.

Interestingly, the slowest growing LAB strain (Lc5g) has not been stimulated by any of the yeast tested against it. The synergistic combinations identified w ill be further examined in well-diffusion assays in MRS medium. No antagonistic combinations of yeast and bacteria have been detected in the plate

interaction assay and the method is currently being modified to increase the sensitivity of this assay to detect inhibitory interactions. After the stimulatory or inhibitory combinations of yeast and LAB have been identified in the plate

interaction assay, the metabolites responsible for these interactions are to be isolated. Two bio­ assays: a well diffusion bioassay and a liquid medium bioassay are being developed to test yeast fermentation fractions against LAB cultures for effects on rate of malic acid degradation as well as microbial growth. Decanoic acid and tomato juice factor (TJF) w ill be used as test solutions to determine the sensitivity of the assays to inhibitory (decanoic acid) and stimulatory (TJF) compounds.

Project Title Studies on the role of copper and iron in wine oxidation and haze formation

Project Code UM 92/1

Organisation The University of Melbourne

Location Melbourne

Supervisors Dr G R Scollary (University of Melbourne; Project Coordinator), Dr PJ W illiams (AWRI), Dr V A Vicente-Beckett (RMIT)

Research Staff Minerva M itri (PhD student)

Funding $26,366

Time Span January, 1993-December 1995

Objectives 1. To characterise the chemical forms of copper and iron in wine. 2. To propose a working model for copper-and

iron-induced oxidative spoilage. 3. To examine the role of copper and iron in haze formation

Progress Significant progress has been made in understanding the involvement of copper in

1 0 4

oxidative spoilage (Objective 2). The chemistry of copper in wine oxidation is extremely complex and consequently, studies to date have been carried out using a model wine system. Once the

chemistry of the model is understood, research w ill be directed towards characterising the speciation or chemical distribution of copper in commercially available wines (Objective 1). It is not expected that Objective 3 w ill be met within the timeframe of this project.

The model wine consists of a 12% aqueous ethanol solution, saturated with potassium hydrogen tartrate and adjusted to pH 3.2, to which varying concentrations of copper and catechin are

added. Caffeic acid and 'white tannin' have also been used as potentially oxidisable organic substrates. The behaviour of the copper in this model is monitored using the electrochemical technique of anodic stripping voltammetry (ASV). The advantage of ASV is that it provides an in situ

indicator of the availability (or lability) of the copper with minimum disturbance of the chemical equilibria in the system. Oxidation of the organic component of the model is monitored by recording the absorbance at 440 nm. This wavelength was chosen as it is close to the traditional value (420

nm) used in the 'browning' measurement in wine. In our work, 440 nm is used as it represents the peak value in the absorption spectrum of the model. The general procedure followed has been to mix the appropriate concentrations of copper and the organic component in the base solution and to note the changes in the copper and browning values as a function of time. The solution is allowed free access to oxygen and is

normally kept at an elevated temperature (45°C) to enhance the reaction rate. Figure 1 shows the variation of the copper concentration, as measured by ASV. It is apparent that the amount of available copper decreases

rapidly in the initial part of the plot and then steadily drops until a plateau concentration, approximating 20 to 25% of the initial

concentration, is reached. Interestingly, the total amount of copper in solution remains the same throughout each experiment. That is, as the reaction in the model solution proceeds, the

copper is being converted from an initial 100% ASV available form to about 20% ASV labile form. It has not been possible as yet to identify the chemical species into which the copper is converted as the reaction progresses. This point is

receiving considerable attention using a range of analytical techniques as knowledge of the converted copper species appears to be critical to the mechanism.

Associated with this decrease in the available copper concentration, there is an increase in the degree of browning as measured at 440 nm. At higher copper concentrations, a faster initial rate of browning is observed. However, with extensive browning, the rate of colour production appears to become independent of the amount of copper present in the model. This implies that two mechanisms may be involved and w ill be the subject of more detailed study in the next phase of the project.

If ascorbic acid is added to the copper/catechin system, a significant enhancement of the rate of browning is observed (Figure 2). This result mirrors observations made at the AWRI in a related project on wine oxidation. Given the increasing use of ascorbic acid in white wine vinification and finishing, the result implies that a re-evaluation of the anti-oxidant value of ascorbic acid is required.

It is already well established, and we have also confirmed, that in the presence of ascorbic acid, copper is instantaneously reduced from the +2 to the +1 oxidation state. On the basis of the observations presented in Figure 2, it is tempting to

propose that the active species is in fact copper(l) and not copper(ll). However, it has not been possible to prove conclusively that copper(l) is present in the three component copper/catechin/ascorbic acid system, as the catechin interferes with the assay for copper(l).

Studies in this area are continuing. One fundamental area which is still to be addressed is the role of free radicals in the oxidation process. Preliminary work has commenced using electron spin resonance spectroscopy to determine whether free radicals are involved. More extensive experiments are to be carried out in this area in the next phase of the



Project Title Chemical speciation of lead and aluminium in wine

Project Code UM 94/1

Organisation The University of Melbourne

Location Melbourne

Supervisor Dr G R Scollary (University of Melbourne; Project Coordinator)

Collaborator Ms C Stockley, AWRI

Research Staff Dr Tony McKinnon (until 31 December, 1994), Ms Alison Green (from 1 March, 1995)

Funding $5,000

Time Span 1 July 1994 - 30 June 1997

Objectives 1. To identify the chemical forms of lead and aluminium in wine. 2. To use lead isotope distribution patterns as

markers for lead contamination of wine. 3. To understand the chemical availability of lead and aluminium in wine as a basis for assessing the bioavailability of these metals.

Progress Significant progress has been made towards achieving Objective 1. The approach adopted to date has been to speciate the metals in wine on the basis of molecular size. To achieve this

speciation, ultrafilters of varying nominal molecular weight cutoff (NMWCO) have been used. The concentration of appropriate metal was determined in the filtrate, so that a decrease in the measured concentration implies that part of the metal did not pass through the filter which in turn implies that the metal must be bound to molecules which are too large to pass through the pores in the ultrafilter.

It is apparent that the size speciation patterns for lead and aluminium are quite different. For aluminium, there is essentially no variation in concentration as a function of filter size. This confirms previous observations made in our research group which suggests that aluminium is bound to tartaric acid (and possibility other organic acids). A study of aluminium/organic acid chemistry is about to commence in an attempt to confirm this suggestion (Objective 1).

The size speciation pattern for lead differs markedly from that observed for aluminium and also for iron and calcium. The major portion of the lead is removed from wine by an ultrafilter with a NMW CO value of 10,000. The same cutoff is observed for both red and white wines, although the effect is more pronounced in red wines. These data suggest that the lead is bound to large molecules in wine. To test this hypothesis, some preliminary experiments have been carried out using two additional ultrafilters with larger NMW CO values. The initial results suggest that the lead is in fact bound to molecules with NMWCO values between 100,000 and 30,000.

It has not been possible as yet to identify the molecules in wine which are capable of binding lead. Electrochemical techniques are presently being employed in an attempt to determine the nature of the lead binding agents. Preliminary experiments have shown that, at the natural pH of the wine, the lead is tightly bound and is gradually released as the wine is acidified. The next phase of the project w ill involve the study of the interaction of lead with various wine components in a model system so that the speciation behaviour of the lead can be understood.

Discussions have commenced with Professor Ramon Barnes (Department of Chemistry, University of Massachusetts, USA) on the development of protocols for the determination of lead isotope patterns both in wine and in model systems containing potential lead binding agents (Objective 2). The combination of the size speciation, electrochemical speciation and isotope distribution patterns w ill allow working models for the chemical availability of lead to be proposed (Objective 3). The results on lead from this study w ill be linked with those from the separately funded GWRDC project, AWR92/2.

1 0 6

Project Title Polyuronic acids from veraison to the finished wine: their role in calcium tartrate stabilisation

Project Code UM 94/2

Organisation The University of Melbourne

Location Melbourne

Supervisor Dr G R Scollary (University of Melbourne; Project Coordinator), Dr P J W illiams (AWRI)

Research Staff Ms Deborah Hampson (from 1 February, 1995)

Funding $13,933

Time Span 1 January 1995 - 31 December 1997

Objectives 1. To examine the influence of grape processing and winemaking procedures on the polyuronic acids levels in wine.

2. To determine the efficiency of the grape-derived polyuronic acids in stabilising wines with respect to calcium tartrate precipitation. 3. To identify the chemical composition of the

grape-derived polyuronic acids which affect calcium tartrate stability.

Progress The initial aspect of the project has been directed towards a comparison of the chemical parameters of Australian sparkling wines with those from other

countries. Particular emphasis was placed on evaluating the use of ionised calcium concentrations rather than total calcium concentrations as an indicator of potential calcium tartrate instability. W hilst there was a significant difference between the ionised and total calcium concentrations, the fraction of ionised calcium was essentially invariant in all sparkling wines studies,

irrespective of the country of origin. This observation was somewhat surprising, but again confirms that a simple test for calcium tartrate

instability is difficult to find. The present phase of the work involves developing procedures for the large scale isolation of the polyuronic acid fraction from wine, a

necessary precursor for Objective 2. Arrangements have been made with a sparkling wine producer to perform winemaking trials during

the 1996 vintage so that the variables relevant to Objective 1 can be assessed. Negotiations have commenced for part of the experimental work in this project to be carried out at the Laboratoire des Polymres et des Technique

Physico-Chemiques, Institut des Produits de la Vigne, Institut National de la Recherche Agronomique (INRA), Montpellier, France. The collaboration would involve Ms Hampson working at INRA, Montpellier, to learn the techniques for separation and characterisation of wine polyuronic acids. If these negotiations are successful, Ms

Hampson w ill study in Montpellier from April 1996 for approximately 10 months.

Project Title Viticultural control of grape flavour in Cabernet Sauvignon, Sauvignon blanc and Semilion

Project No





Time Span

UCS 92/1

Charles Sturt University-Riverina

Wagga Wagga, NSW

Dr M Allen

Nil during 1994/95 year

July 1992 - June 1994, and July 1995- June 1996

Objective To quantify the impact of vine management on herbaceous/vegetative (methoxypyrazine) fruit flavour components.

Progress Trials at Wagga Wagga in 1993 studied the effects of irrigation, cropping level, and nitrogen

availability on methoxypyrazine levels in the fruit of Cabernet Sauvignon vines adjusted to uniform shoot numbers. Cluster light measurement was used to select fruit of uniform exposure for sampling. Methoxypyrazine analysis showed that the treatment effects on methoxypyrazine level were much less than those for the influence of exposure. The treatment severity was limited by fungal infection at flowering and fruit set and

unusually wet summer conditions, and more extreme treatment conditions w ill need to be established, but the results point towards closer

study of the extent to which exposure differences can account for methoxypyrazine flavour variation


between different trellising systems. Vine training for such trials is in progress. In view of the unusual seasonal conditions in

1993, attention was also directed to two factors less influenced by those conditions. Firstly, study of the timing of methoxypyrazine development from fruit set, to clarify when viticultural influences

may have the greatest effect. Secondly, further study of the relative influence of fruit and shoot exposure. These investigations require analysis of small fruit quantities, including individual grape clusters, rather than the 6-10 clusters that may be

normally sampled. Development of the analytical procedure has been undertaken to allow this, focusing on techniques for manipulating

methoxypyrazine isolation on very small scale. This has increased the extraction efficiency and reduced the solvent volume to ca. 0.1 mL, prior to concentration, providing a ten-fold increase in methoxypyrazine signal to noise ratio in the analysis. This is expected to allow quantitative analysis of isobutyl methoxypyrazine on samples of less than a single cluster.

Surprisingly low methoxypyrazine levels were measured in 1993 in Semilion vines of a GWRDC soil management trial in Griffith, despite very high levels being found with the same clone on a different rootstock in the same research station.

Further samples were taken in 1994 to identify whether this variation was due to clonal/rootstock effects or analytical problems. During the 1994/95 year, the project was on hold as the project supervisor undertook study leave for six months to study flavour research at the Institute of Oenology of the University of


1 0 8

Grape Projects approved at the 11th Board Meeting 20/21 April 1995

Agency Project Title Leader Total


Grape Industry Development

ABS 95/1 Viticultural Statistics Collection M r P F Hayes 32,000

GWR 95/1 Wine Grapes: Projections of wine grape production and winery intake to 1998-99 M r P F Hayes 56,000

GWR 95/2 Issues paper on national water resource security, for vineyard development M r P F Hayes 10,000


Vine Improvement

CSH 95/1 Isolation of gene promoters required for controlling genes in grapevine Dr M Thomas 24,670

CSH 95/2 Development of an international grapevine identification system Dr M Thomas 8,000

CSH 8 Use of rootstocks to reduce Cl- and Na+ residues in export wines Dr R R Walker 45,301

CSH 94/3 Field assessment of selected rootstock hybrids for quality wine production Mr P R Clingeleffer 27,705 105,676

Vineyard Water Management

CRS 92/1 The response of grapevines to partial wetting of the rootzone Mr 1 Goodwin 23,658

CSH 95/3 Development of methods for the control of vine vigour and water use optimisation based on the concept of heterogeneous rootzone hydration

Dr B Loveys 56,942

CSH 94/2 Optimisation of quality wine grape production through modelling vine phenology, canopy architecture, light interception, water use and yield

Mr K j Sommer 87,055

DAS 11 Improving wine quality by regulated deficit irrigation Mr M G McCarthy 11,301


Vineyard Soil Management

CRS 95/1 Sustainable viticultural production,optimizing soil resources Dr A Cass 43,442

SEE 93/1 The role of inter row ground covers to improve the Mr R Porter 64,985

management and sustainability of Australian vineyard soils 108,427


Cultural Practices

AWR 24 Linkages between AWR I and CSIRO ProfT Lee 49,693

UA 93/1 Root pruning for growth control of grapevines Dr P R Dry 7,270

UA 95/1 The basis of variation in size and composition of grape berries Mr J D Cray 4,000

UCS 92/1 Viticultural control of grape flavour in Cabernet Sauvignon, Sauvignon Blanc and Semilion vines M S Allen 30,619

DAV 93/1 Objective design of trellis structures Dr M Mollah 16,100


Vineyard Pest Management

CRV 95/2 Strategic response to restricted spring growth syndrome Dr J Hardie 10,000 DAV 95/1 Integrated management of Botrytis bunch rot and Dr C Buchanan/ 120,000

Light Brown Aple Moth Dr R Emmett

DAV 95/2 A national program for the management of phytoplasmas in Australian grapevines' Dr D Eagling 33,485

CSH 95/4 The status of virus and virus-like and phytoplasma grapevines and their relevance to Australian Viticulture Dr N S Scott 16,030

CRS 95/3 Assessment of the use of induced systemic resistance to control of powdery mildew in grapevines Dr P Stephens 23,899

DAN 95/2 Screening of crucifer crops for glucosinolate (nematicide) levels by HPLC chemical analysis and field assessment in semilion plantings

R McLeod 16,542

DAN 93/2 Biological control of mites in Australian viticulture: Implementation and sustainability Dr D G James 62,000

DAN 92/1 Supplementary Funds - Practical management strategy for bunch rot of grapes Dr N G Nair 7,000

DAN 94/1 Strategies to reduce the incidence of cane and leaf blight disease of grapevines Dr N G Nair 70,385

DAV 94/1 Reduction of the impact of powdery mildew in Australian viticulture Dr R W Emmett 55,647

CSH 94/4 The role of chitinase and β- (1-3)- glucanase gene expression in the response of grapevines to fungal infection Dr N S Scott 31,045

DAS 92/2 A reliable, cheap weather station as a predictor for improved disease and pest control Mr P A Magarey 5,980

DAS 93/3 Non conventional control of powdery mildew Mr T Wicks 44,243

SPY 95/1 Optimization of vineyard spray application technology through integrated testing and evaluation Mr B W Young 79,869


(1) Subject to outcomes from CSH 95/4


Technology Transfer

DAV 93/2 Development of a national management strategy for grape phylloxera GWR 94/2 RDI demonstration Project GWR 95/3 OIV GWR 95/4 National Extension Facilitator

Dr G Buchanan 29,850

J M Campbell-Clause 5,000 Mr R Hayes 20,000

Mr P Hayes 97,750

Dr J Hardie


Wine Projects approved at the 11th Board Meeting 20/21 April 1995


CRS 95/2 Standardised protocol for environmental management of winery production waste Dr A Cass 29,000

UM 92/1 Studies on the role of copper and iron in wine oxidation and haze formation Dr G R Scollary 13,229

UM 94/1 Chemical speciation of lead and aluminium in wine Dr G R Scollary 5,000

UM 94/2 Polyuronic acids from veraison to the finished wine: their role in calcium tartrate stabilization Dr G R Scollary 30,807


AWRI Microbiology 547,271

AWR 2 Yeast evaluation and optimization of fermentation performance: influence of physical environment, grape juice composition and yeast physiological properties

Dr P A Henschke

AWR 3 Selection and improvement of wine yeasts by application of molecular biology Dr P A Henschke AWR 4 Optimization of malolactic fermentation in wine and

strain improvement by application of molecular biology Dr P A Henschke

AWR 5 Microbiological analysis of industry technical problems Dr P A Henschke

Chemistry 658,110

AWR 6 Elucidation of the varietal flavour characteristics of grapes Dr P J Williams AWR 7 The influence of oak cooperage on wine composition Dr PJ Williams AWR 8 Studies on unstable proteins involved in haze formation Dr P J Williams AWR 9 Studies on grape and wine phenolic compounds, including

their roles in oxidation and wine instability Dr P J Williams


E xtension 548,665

AWR 10 AWR 11

AWR 12

Technical problem and solving and consulting Mr P A Leske

Evaluation of new analytical techniques and of processing Mr R A Leske aids for winemaking Provision of technical information Mr P A Leske

D ire c to r's C ro u p 400,462

AWR 19 Chemical analysis of industry technical problems AWR 24 Links between viticulture and oenology research AWR 25 Preparation of information on wine health issues AWR 92/2 Relationship between vineyard soil lead concentration

and lead concentration in grape berries AWR 94/1 OIV General Assembly - Paris AWR 94/2 OIV Expert Group Meetings AWR 94/3 Code of winemaking practice AWR 94/4 Cork taint studies AWR 25 Preparation of information on wine health issues AWR 92/2 Relationship between vineyard soil lead concentration

and lead concentration in grape berries AWR 94/1 OIV General Assembly - Paris AWR 94/2 OIV Expert Group Meetings AWR 94/3 Code of winemaking practice AWR 94/4 Cork taint studies

Dr R F Simpson To be appointed Ms C S Stockley Ms C S Stockley

Prof T H Lee Prof T H Lee Ms C S Stockley Mr M Sefton Ms C S Stockley Ms C S Stockley

Prof T H Lee Prof T H Lee Ms C S Stockley Mr M Sefton

Organisation Codes


Australian Soil and Plant Analysis Australian Vine Improvement Assoc Inc Australian Wine Research Institute Australian Bureau of Agricultural and Resource Economics Cooperative Research Centre for Viticulture Commonwealth Scientific and Industrial Research Organization CSIRO Division of Horticulture NSW Department of Agriculture South Australian Department of Agriculture Victorian Department of Agriculture and Rural Affairs

Department of Agriculture W A Grape and Wine Research and Development Corporation Seedco Pty Ltd University of Adelaide Charles Sturt University-Riverina University of Melbourne Winemakers' Federation of Australia Inc Winegrape Growers' Council of Australia

Typesetting and production by Winetitles

Printed and bound by Hyde Park Press, Adelaide





ISSN 0727-4181