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VG114 The management of fungicide resistance in horticulture crops

R G O'Brien Qld Dept Primary Industries

danikah

Stamp

This report is published by the Horticultural Research and Development Corporation to pass on information as to horticultural research and development undertaken on the management of fungicide resistance in horticulture crops.

All expressions of opinion are published on the basis that they are not to be regarded as expressing the opinion of the Horticultural Research and Development Corporation or any authority of the Australian Government.

The Corporation and the Australian Government accept no responsibility for any of the opinions or the accuracy of information contained in this Publication and readers should rely upon their own enquiries in making decisions concerning their own interests.

Cover Price - $20

ISBN - 1 86423 050 9

Published and Distributed by:

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Horticultural Research & Development Corporation 7 Merriwa Street Gordon NSW 2072 Phone (02) 418 2200 Fax (02) 418 1352

® Copyright 1993

The research contained in this report was funded by a grant from the Horticultural Research and Development Corporation with the financial support of

QLD Fruit & Vegetable Growers

Agricultural & Veterinary Chemicals Association (AVCA)

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SUMMARY PAGE

(a) Industry summary 1

(b) Technical summary 1

RECOMMENDATIONS

(a) Extension/adoption by industry 2

(b) Directions for future research 2

(c) Financial/commercial benefits 3

TECHNICAL REPORT

(a) Introduction 3

(b) Development of testing procedures and expected dose-response data in 26 fungal pathogens 5

(c) Investigation of suspected cases of new fungicide resistant strains of pathogens 5

Peronospora parasitica — Phenylamides 5

(i) Fungicide sensitivities of a Queensland and Victorian isolate of P. parasitica

Methods Results

(ii) Control of P. parasitica with standard spray concentrations of five fungicides

Methods Results Discussion

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Peronospora destructor — Phenylamides 9


Sclerotium cepivorum — Dicarboximides 10

Methods Results/Discussion

Uromyces appendiculatus — DMI, Oxathiins 12


(d) Competitive ability of isolates of Botrytis cinerea 13


(e) Developing strategies for resistance 14

GENERAL DISCUSSION

ACKNOWLEDGEMENTS

PUBLICATIONS ARISING FROM THIS PROJECT

REFERENCES

APPENDICES

1. SUMMARY

(a) Industry summary

Modern fungicides are highly effective in controlling many important fungal pathogens of horticultural crops but they are prone to resistance problems. In this project we established standard methods of testing for fungicide sensitivity in 26 pathogens, recorded three new instances of fungicide resistance, developed guidelines for fungicide use and analysed several field samples for fungicide sensitivity.

The establishment of rapid testing methods for fungicide sensitivity is important so that disease control failure can be attributed to fungicide resistance or some other cause. A set of standard procedures and expected fungicide sensitivity data were developed for 26 fungal pathogens and the "at risk" fungicides used in their control. These have been assembled in a booklet 'Testing procedures and standards to evaluate the sensitivity of fungi to fungicides'. Many samples were received from field situations where disease control was poor. If resistance was confirmed, it was usually possible to recommend a satisfactory alternative treatment. This service will continue.

Strains of Peronospora destructor (onion downy mildew) and Peronospora parasitica (crucifer downy mildew) with resistance to phenylamide fungicides were recorded. One isolate of Sclerotium cepivorum with resistance to dicarboximide fungicides was identified. Fortunately, it was still sensitive to fungicides in the DMI group.

Fungicide resistance is encouraged by the over-use of a fungicides in the same group. Selection for resistance can be reduced by employing integrated control programs and a diversity of fungicidal activity groups. General guidelines as well as detailed systems to be used in the control of cucurbit powdery mildew, grey mould, onion downy mildew and lettuce downy mildew were developed and publicised. A spray warning system "Downcast" showed promise as a method to lower spray applications to onions for downy mildew control and is being further developed as a service to be used by growers in the Lockyer Valley.

(b) Technical summary

Fungicide sensitivity testing procedures were developed for 26 pathogens of horticultural crops and the fungicides which control them. For many pathogens simple germination or growth tests on solid media were adequate. Obligate pathogens required tests using seedlings or leaf discs with fungicides applied as sprays or in nutrient solution. Reference isolates were stored as freeze dried cultures or in liquid nitrogen. A technique of storing Sphaerotheca fuliginea in liquid nitrogen was developed. Previously this fungus was maintained by serial transfer on seedlings or leaf discs.

While fungicide resistance had been known to occur in several pathogen-fungicide combinations, three new instances of fungicide resistance were found. These were isolates of Peronospora destructor and P. parasitica with reduced sensitivity to phenylamides and an isolate of Sclerotium cepivorum with an EC50 for procymidone of 10 ug/mL compared with 0.3 ug/mL for sensitive isolates.

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Analysis of field samples showed phenylamide resistance in P. destructor was present in the Lockyer Valley, Queensland and Moriarty, Tasmania. Crucifer downy mildew was a widespread problem in nurseries in Queensland, New South Wales, Victoria and Western Australia. No metalaxyl-sensitive isolate of P. parasitica was found. Lettuce downy mildew (Bremia lactucae) was difficult to control and, in tests, infection occurred on plants sprayed with 100 ppm metalaxyl indicating resistance.

Field experiments with onion downy mildew confirmed that phenylamide resistant strains of P. destructor have been an important factor in control difficulties with this disease. Mancozeb gave best control while dimethomorph showed promise as a new highly active material. The disease forecasting system "Downcast" was evaluated and showed potential for reduced spray application. This system of forecasting sporulation-infection periods based on temperature, relative humidity, dew formation and rainfall is being developed as a service for Lockyer Valley onion growers.

2. RECOMMENDATIONS

(a) Extension/adoption by industry

The testing procedures developed in this project will allow assaying of samples suspected of being fungicide resistant to be conducted. Guidelines for the submission of samples have been drawn up in collaboration with Australian Fungicide Resistance Action Committee (AFRAC).

Identification of resistance in submitted samples allowed alternative control recommendations to be suggested. In the case of onion downy mildew, an intensive program of field experiments and extension activities were considered successful in implementing a more integrated, sustainable control program.

(b) Directions for future research

In order to minimise selection of resistant strains, farmers must be able to recognise cross-resistant fungicides and follow the principles of strategic spraying. There has been satisfactory adoption of proposed strategies for grey mould, cucurbit powdery mildew and onion downy mildew following resistance. More emphasis should be placed on the development and extension of strategies for "at risk" situations before resistance is recorded. The use of spray warning systems and other management techniques should be considered part of the strategy to reduce the intensity of spraying. Development of "Downcast" should be pursued.

Besides a "clearing house" for suspected cases of fungicide resistance, monitoring of several diseases considered "at risk" to resistance problems should be undertaken in several horticultural districts. Besides the advantages of early detection of resistance, this would also serve to increase growers' awareness of the problem.

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(c) Financial/commercial benefits

There are many aspects to the benefits of this work. The easiest to assess are the individual enquiries, e.g. Accession No. 3706 involved a severe outbreak of grey mould (Botrytis cinerea) in hydroponically grown continental cucumbers. Isolates were 100% resistant to the benzimidazole fungicides used by the grower but sensitive to dicarboximides. After changing fungicides the problem was brought under control. Accession No. 3700 concerned a bulk shipment of oranges affected by green mould (PenicilUwn digitatum) into a Queensland producing area. Local growers were concerned the fungus may be fungicide resistant and thereby pose a threat to their production. Tests showed isolates were fungicide sensitive.

On a wider scale industries have benefited. The identification of phenylamide resistance in P. destructor and the adoption of the recommended strategy led to the increased use of lower priced protectant fungicides for downy mildew control in onion. These had been shown to be effective in field trials. Further development of the spraying warning system "Downcast" may lead to a 30% decrease in applications. These savings should amount to between $100 000 to $200 000 per annum.

Benefits more difficult to define result from the anticipated extension of the useful life of fungicides through adoption of strategies. Development costs of fungicides are increasing so new fungicides are invariably higher priced than those they replace. It is also uncertain whether there are reliable replacements under development. Strategies, if successfully implemented, lead to a more integrated approach to disease control and lower fungicide usage.

3. TECHNICAL REPORT

(a) Introduction

Due to the intensive nature of horticultural crop production, fungal diseases are common. Those which attack foliage and fruit, in particular, are capable of rapid reproduction and may greatly lower productivity during periods when weather conditions suit them. While farmers are encouraged to lower the disease threat by genetic and cultural control methods, production of high quality fruit and vegetable crops often depends on fungicidal protection.

Fungicide evolution over the last quarter century has been away from protectant types which generally act as inhibitors of fungal respiration towards highly active systemic products with specific modes of action. Their fungicidal activity is usually due to the disruption of one or two steps in fungal metabolic pathways, e.g. with benzimidazole fungicides microtubule assembly in mitosis is disrupted while the phenylamides are highly specific inhibitors of ribosomal RNA synthesis in the Peronosporales (Sisler, 1988). The practical benefits of these fungicides are their high activity at low dosage rates and systemic mode of action which allow root or foliar uptake; is forgiving of minor discrepancies in spray coverage due to equipment or wet weather; and post infection

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activity ideally suited to spray warning systems. Their major disadvantage is that variability within populations of fungal pathogens often includes individuals with alternative metabolic pathways to those blocked by the fungicide. These are the fungicide resistant strains and with selection pressure created by the continued use of the fungicide will make up an increasingly higher proportion of the population.

The importance of fungicide resistance as an impediment to efficient production has been recognised world-wide and in 1988 there were over 60 fungal genera with resistant strains (Eckert, 1988). In Australia, resistance is known to occur in several pathogens including Sphaerotheca fuliginea (Peterson, 1973), Botrytis cinerea (O'Brien & Glass, 1986), Sclerotinia fructicola (Penrose et al 1979, 1985) and Bremia lactucae (Trimboli, pers. comm.). There are several instances of resistance reported elsewhere which have not been detected, including Pseudoperonospora cubensis (Reuveni et al 1980) and Phytophthora infestans (Davidse et al, 1981).

Failure to recognise fungicide resistance as the cause of poor disease control can lead to excessive use of chemicals in an effort to rectify the problem. This, in turn, intensifies selection pressure.

Most "at risk" fungicides belong to one of four groups, the Benzimidazoles, Dicarboximides, Demethylation Inhibitors or Phenylamides. Members of each group have similar chemistry and affect the same metabolic processes in fungi. Resistance to one is therefore resistance ("cross resistance") to all fungicides in that group. On a practical level, it is essential for growers to recognise groups of fungicides which are cross resistant since most strategies are based on the inter-use of unrelated products with different "modes of action".

Eckert (1988) proposed that resistant strains are due to spontaneous mutations which result in decreased target affinity for the fungicide. They are present at low frequencies before an "at risk" fungicide is used. Selection for resistance is most rapid when: the fungicide is used frequently over large areas for long periods of time; the fungus is efficiently dispersed with an effective sexual state and a high infection rate on a susceptible variety (Georgopoulos, 1988). In some cases fungicide resistant strains are not as competitive as sensitive strains (Davis and Dennis, 1981a,b). In other cases there is evidence they compete very well. Competitive ability is important in determining the likely outcome of fungicide use strategies and useful market life of fungicides.

Strategies to minimise resistance problems are aimed at reducing the selection pressure in as many ways as possible in the particular use situation (Wade, 1988). Strategies may limit the number of applications of the "at risk" product per crop, combine or alternate it with one or two other fungicides with different modes of action and recommend management practices which reduce the fungal population. A successful resistance strategy will often be one of integrated disease control practices and emphasise that chemical control is an option to be used discretely.

The core objective of this project was to develop methods of testing pathogens for fungicide senstivity and establish expected dosage response data to enable fungicide

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resistance problems to be recognised as such. Other objectives were to develop and promote resistance strategies and assess competitive ability in Botrytis cinerea.

(b) Development of testing procedures and expected dose-response data in 26 fungal pathogens

The objectives of this section of the project were to develop tests which were rapid but reliable; record expected response data for sensitive (and resistant) isolates and place reference isolates in long-term storage.

As a general rule, tests for non obligate parasites were based on germination or growth responses on media containing a range of fungicide concentrations. The tests with obligate parasites such as downy and powdery mildews were developed with plants which had been treated with fungicides. These required much more effort to develop as whole plant tests were compared with leaf discs, spray treatments of leaves were compared with the application of fungicides to roots and their distribution systemically to leaves.

It is not practical to detail the numerous experiments in this section since, in some cases, there were over 20 small experiments in the development of a test. The end result is the booklet 'Testing procedures and standards to evaluate the sensitivity of fungi to fungicides" (Appendix I). A summary of the combinations of fungi/fungicides which comprise these tests is shown in Table 1.

One highlight of this section was the development of a technique for the long-term storage of the obligate parasite Sphaerotheca fuliginea, the cause of cucurbit powdery mildew. This is a very variable pathogen existing as a number of races (Appendix IV) and the only method of maintaining these has been as living cultures on leaves or leaf discs. The successful technique involves drying conidia for 6 hours before long-term storage in liquid nitrogen.

(c) Investigation of suspected cases of new fungicide resistant strains of pathogens

Peronospora parasitica — Phenylamides

Crucifer downy mildew was severe in seedling production units when weather conditions were cool and humid. The fungicide in common use, metalaxyl, provided little or no control. The disease was most detrimental to young seedlings which often showed sporulation on the cotyledons within a week of germination. The problem was not localised and samples from Queensland, New South Wales, Victoria and Western Australia were received for screening. Experiments were carried out to determine the sensitivity of isolates to the phenylamide group as well as to other groups which may be suitable alternatives. Two such experiments are described below.

(i) Fungicide sensitivities of a Queensland and Victorian isolate of P. parasitica.

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Table 1. List of the tests devised for determining the sensitivity of fungi to groups of fungicides

Fungus Fungicide Group*

Fungus Ben D/C DMI Ph OP Hp A

Alternaria solani + +

Botrytis cinerea + +

Bremia lactucae +

Cercospora beticola +

Cercosporidium personatum + +

Fulvia fulva +

Mycosphaerella melonis (Didymella bryoniae) +

Mycosphaerella musicola + +

Oidium sp. +

Penicillium spp. + +

Peronospora destructor +

Peronospora parasitica +

Phytophthora nicotianae var. parasitica +

Phytophthora palmivora +

Pseudoperonospora cubensis +

Rhizoctonia solani +

Sclerotinia laxa, S. fructicola + + +

Sclerotinia minor + + +

Sclerotiorum sclerotiorum + + +

Sclerotium cepivorum + +

Sclerotium rolfsii + +

Sphaerotheca fuliginea + + + +

Thielaviopsis basicola + +

Thielaviopsis paradoxa + +

Uromyces appendiculatus + +

Ben = Benzimidazole D/C = Dicarboximide DMI = Demethylation Inhibitor Ph = Phenylamide OP = Organophosphate Hp = Hydroxypyrimidine A = Anilide

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Methods. Isolate 2791 (Victoria) and 2966 (Queensland) were maintained by regular transfer to seedlings of broccoli cv. Premium Crop.

The experiment was a factorial design of 4 fungicides x 6 concentrations x 3 replications. Seed of cv. Premium Crop was sown in small (50 mm diam) pots and thinned to 6 plants per pot after germination. The fungicides metalaxyl (Ridomil 25 WP), fosetyl-Al (Aliette 80 WP), propamocarb (Preview 72 SL) and dimethomorph (CME15103 50 WP) were prepared in concentrations of 0,0.1,1,10, 50 and 100 ug/mL and applied as foliar sprays at emergence and again three days later. Cotyledons were detached 24 h after the second spray and placed on water agar in Petri plates where they were inoculated with a single 10 JAL droplet of a conidial suspension. Inoculum was prepared by brushing conidia from infected leaves into distilled water. Spore concentrations were adjusted to approximately 2 x Mf/mL using a haemocytometer. After incubation for 7 days in a controlled environment cabinet (20°C; 12/12 light) leaves were rated on a scale of 0-3 depending on the extent of the lesion and sponilation intensity.

Results. The two isolates showed similar responses to the fungicide treatments. The most effective was dimethomorph with a Minimum Inhibitory Concentration (M.I.C.) > 1 < 10, Leaves treated with propamocarb showed infection reactions but sponilation was prevented by concentrations of 50 yig/mL and higher. Metalaxyl and fosetyl-Al did not reduce disease severity at any concentration.

(ii) Control of P. parasitica with standard spray concentrations of 5 fungicides.

Methods. Seedlings of broccoli cv. Premium Crop were grown in small pots until cotyledons were expanded. The leaves were then dipped in fungicide solutions (Table 3) and allowed to dry before being detached and placed on water agar in Petri dishes.

Conidial suspensions of an isolate from Victoria and one from Queensland were prepared and standardised to 2 x l(f/mL. Droplets were placed on each leaf and then incubated for 6 days. The severity of infection was rated

0 - no disease 1 - reaction spot 2 - sponilation < 50% leaf area 3 - sponilation > 50 < 75% leaf area 4 - sponilation > 75%

Results. Dimethomorph gave complete control while propamocarb allowed reaction spots but prevented sponilation (Table 3). All other fungicides applied at normal field concentrations failed to prevent infection and sponilation.

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Table 2 The sensitivities of 2 isolates of P. parasitica to 4 fungicides

Disease severity (0-3)+

Fungicide Cone yg/mL 2966(Q) 2791 (Vic)

Dimethomorph 0.1 1

10 50

100

2.3 2.0 0 0 0

3.0 1.3 0 0 0

Propamocarb 0.1 1

10 50

100

2.7 2.0 2.3 1.0 1.0

3.0 3.0 2.3 1.0 1.0

Fosetyl-Al 0.1 1

10 50

100

3.0 2.3 2.7 2.3 2.3

3.0 3.0 3.0 3.0 2.7

Metalaxyl 0.1 1

10 50

100

2.7 2.0 2.0 3.0 3.0

3.0 2.7 2.7 3.0 3.0

Check 2.3 3.0

+ 0 = no disease 1 = disease reaction, no spomlation 2 = spomlation moderate 3 = intense spomlation

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Table 3 The effect of five fungicides on disease severity of crucifer downy mildew following inoculation with a Queensland and a Victorian isolate of P. parasitica

Fungicide Disease severity (0-4)

Qld isolate Vic isolate

CUE 15103 (500 g/kg dimethomorph) Provicur N (722 g/L propamocarb) Aliette ( g/kg fosetyl Al) Galben (250 g/kg benalaxyl) Ridomil (250 g/kg metalaxyl) Check

0.54 g/L 5mL/L 4 g/L 0.8 g/L 0.8 g/L

0 0.95 2.05 2.05 2.45 2.35

0 0.85 2.45 2.40 2.35 2.65

Discussion. Experiments conducted in 1978 showed that metalaxyl was highly effective against P. parasitica (unpublished data). The results of the trials described above and additional experiments indicate sensitivity to phenylamide fungicides has declined to the extent that they are ineffective in the control of crucifer downy mildew.

The problem is not localised since in the course of these studies isolates from Victoria, New South Wales, Western Australia and Queensland were resistant. There are alternatives to the phenylamides such as dimethomorph and propamocarb which should be useful once registration procedures are completed.

Besides chemical control, there are cultural measures which will reduce disease severity. These include elimination of inoculum sources, improved aeration of plant houses and irrigating so that foliage dries as quickly as possible. These, combined with careful use of registered protectant fungicides, should provide reasonable control until the more effective systemic products are registered.

Resistance to phenylamides in P. parasitica has been recorded in the USA. Resistant strains occurred in California in 1985 and in Florida and Georgia two years later (Morton and Urech 1988).

Peronospora destructor — Phenylamides

Since 1987 control of downy mildew in onion crops in Queensland, Victoria and Tasmania has been difficult. While some of the problem was considered due to weather conditions being more favourable for the disease it was also considered likely that strains of P. destructor with resistance to phenylamides may have developed.

Methods. A series of experiments in the glasshouse and controlled environment cabinets was set up to determine whether there were differences between isolates in their sensitivities to phenylamide fungicides. Field trials were established at Gatton Research Station in 1989 and 1990 to compare fungicide treatments and, to a limited extent, spraying schedules based on disease forecasting. Complete details of procedures are in Appendix II "Control of onion downy mildew in the presence of phenylamide resistant strains of Peronospora destructor".

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Results. An isolate collected from unsprayed shallots in a home garden at Bundaberg was sensitive to the phenylamide fungicides metalaxyl, benalaxyl and oxadixyl with EC 50 values of 1.6, 6.0 and 12.1 mg a.i./L respectively. In comparative tests, an isolate from onions in the Lockyer Valley did not respond to increasing concentrations of these fungicides between 0.1 and 200 mg a.i./L. Both isolates were sensitive to the morpholine fungicide dimethomorph with EC 50 concentrations of 1.4 and 4.3 mg a.i./L respectively. These indications of resistance were supported by the field experiments in which there was no difference from the check in either disease severity or yield when metalaxyl was applied. It is assumed from this that phenylamide resistance was high in the Lockyer Valley following 10 years of use. In the field experiments, excellent control was obtained with mancozeb, a protectant fungicide while dimethomorph showed promise even at low application rates (270 g a.i./ha) (Table 4). This is the first report of resistance in P. destructor to phenylamides.

Several samples from Victoria and Tasmania were received for testing but with one exception were inviable. The viable test sample from Moriarty, Tasmania showed a similar level of phenylamide insensitivity as the sample from the Lockyer Valley.

Discussion. Confirmation of phenylamide resistance in two of Australia's major onion producing areas led to an intensive effort to persuade growers to diversify their disease control practices. Emphasis was placed on understanding the disease cycle, recognition of weather conditions suitable for sporulation and infection and strategic rather than regular use of systemic fungicides (Appendices V, VI, VII). Two presentations were made to field officers of chemical companies, resellers and growers.

Since overuse of fungicides is a common factor in the development of fungicide resistance, a disease forecasting system "Downcast" was evaluated in the 1989 season to determine whether it could lead to lower fungicide usage. Predictions based on temperature, relative humidity, length of dew period, speed of dew deposition and night rainfall were confirmed by observations of disease development in a series of trap plants placed in an unsprayed field. It was considered a maximum of five sprays would have been necessary in that season (about half the number applied on an 8-10 day schedule) (Appendix VI).

It is planned to further develop 'Downcast' as a district spray warning service during the 1993/94 seasons. This will involve evaluation of environmental variability through the Lockyer Valley; further verification of the system in accurately predicting sporulation — infection periods and an evaluation of the suitability of systemic fungicides in the phenylamide and morpholine groups for use.

Sclerotium cepivorum — Dicarboximides

White rot of onions occurs in all major producing areas of Australia. It is a soil-borne disease which spreads slowly through fields with cultural operations. Recently, fungicides of the dicarboximide and DMI groups have been registered for use as soil treatments.

Methods. Isolates of S. cepivorum were taken from a field in the Lockyer Valley where the response to dicarboximide fungicides had been poor. These were screened for sensitivity to procymidone and tebuconazole as representatives of the two groups using the testing procedure in Appendix I.

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•able 4. 1990 field experiment The effect of regular applications (n= 11) of 12 fungicidal spray treatme on the severity of onion downy mildew 18 and 21 weeks after sowing and the yield and quality (si of harvested onions.

Fungicide treatment Disease Severity (0-6)A Plot Yields '(kg)

jEpray concentration g/L 18wks 21 wks No. 1 large > 70mm diam

No. 1 > 40-70mm diam

Small 20-40mm diam

Total Yield

gZontrol 185 5.67 0.95 15.48 12.69 29.12

Copper hydroxide (Kocide) 1.54 g | - i . / L

3.37 5.60 0.45 10.15 13.65 24.24

Mancozeb (Dithane M45) 1.6 g a.

phlorothalonil (Bravo 500) l-5g a. i./L

1.17 2.15 7.73 28.74 11.17 47.64 Mancozeb (Dithane M45) 1.6 g a.

phlorothalonil (Bravo 500) l-5g a. i./L

2.72 5.38 0.50 13.51 12.87 26.76

Irfetalaxyl (Ridomil 25) 0.2 g.a. 1/L

2.55 5.22 0.65 14.86 14.10 29.59

Propamocarb (Previcur) 3 g a. j/L Fosetyl-Al (Aliette) 222 g a. i./L

2.50

2.62

5.42

5.13

0.76

0.68

14.41

13.66

13.23

13.69

28.21

28.04

fcimethomorph (CME 15103) 1.27 g a. i./L

227 3.56 2.17 21.56 1237 36.14

Metalaxyl + mancozeb (Ridomil MZ 720) 0.2 + 1.6 g a. i./L

Experimental product 2022D 4.5 J5/L

bimethomorph 0.27 g.a. i/L + Mancozeb 1.8 g a. i./L

1.40

2.42

1.22

232

5.13

0.91

6.67

0.58

8.45

27.16

14.02

28.90

11.71

13.26

9.43

45-55

27.72

46.81

Strategy (mancozeb 1.6 g a. i./L 1.55 236 6.49 28.19 9.84 44.52 *JS regular spray; metalaxyl 0.2 g a. i./L + mancozeb 1.6 g a. i./L lis strategic spray)

L.S.D. (P = 0.05) (P = 0.01)

0.78 1.04

0.48 0.64

3.02 3.97

6.26 8.23

2.84 NS

7.29 9.59

Disease severity; 0, no disease; 1,1-5% leaf area affected; 2,6-10%; 3, 11-25%; 4, 26-50%; 5, 51-75%; >75%

iimethomorph 0.27g a.i./L + Mancozeb 1.8 a.i./L

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Table 5. The sensitivities of 5 isolates of Sclerothun cepivorum to the fungicides procymidone and tebuconazole

Fungicide pg/ml

Growth as % of Control Fungicide

pg/ml 3402-1 3402-2 3402-3 3403 3402-2R

Procymidone 0 0.01 0.1 0.5 1 5 10 100

100 98 45 5 0 0 0 0

100 102 57

8 0 0 0 0

100 97 65 6 0 0 0 0

100 93 53 7 0 0 0 0

100 79 93 93 86 71 57

0

Tebuconazole 0 0.01 0.1 0.5 1 5 10 100

100 19 0 0 0 0 0 0

100 20

0 0 0 0 0 0

100 22

0 0 0 0 0 0

100 40

0 0 0 0 0 0

100 36 0 0 0 0 0 0

Results/Discussion. The results in Table 5 show one isolate had reduced sensitivity to dicarboxmides but was sensitive to the DMI fungicide. The isolate was comparatively slow growing and may not be a good competitor in the absence of fungicide selection pressure. Resistance to dicarboximides has been reported by Littley & Rahe (1984). Growers should make use of fungicides from both chemical groups to avoid resistance problems.

Uromyces appendiadatus — DMI, Oxatbiins

Bean rust was severe in the Bowen-Burdekin district where the systemic fungicides bitertanol and oxycarboxin seemed to be losing efficacy. Several collections were examined for fungicide sensitivity. The experiments below describe a culture plate test and an in vivo test to determine whether the observed senstivity differences were important in disease control.

Methods. Using the technique of uredospore transfer from single bean rust pustules to fungicide amended water agar, five pustules from six fields were compared for fungicide sensitivity. Fungicide concentrations were 0, 10 and 25 ng/mL for oxycarboxin and 0, 5 and 10 ng/mL for bitertanol. The percentage germination was assessed after 24 h. Spores of each pustule were also transferred to leaf discs of cv. Covey to allow future comparisons between pustules judged resistant/sensitive in the germination test.

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Seven pustules were selected for further study in the glasshouse experiment and were transferred to several leaf discs to allow inoculum increase. Bean seed cv. Labrador was sown in vermiculite in flats and transplanted 1 per 10 cm pot following germination. The potting medium was UC mix. Sprays were applied when shield leaves were half expanded. Sprays used were:

Bitertanol - 10, 25, 100 ug/mL bitertanol Oxycarboxin — 10, 25, 100 yLg/mL oxycarboxin Check - 0

There were 3 replications of each treatment. Inoculation was made 24 h after spray application. Inoculum of the 7 isolates was prepared by shaking the leaf discs in 15 mL water. (Tween 80 added to water - 1 drop per 200 mL). Spore concentration was adjusted to 1 x Mf/mL approximately. Inoculum was misted over plants and 12 mL was used to inoculate each of the groups of 21 plants.

Plants were enclosed in moist plastic bags overnight then returned to glasshouse benches. Eleven days later rust pustules had developed and these were counted.

Results. There were differences between pustules in their abilities to germinate on fungicide amended media. Five isolates were selected on the basis of being apparently more resistant to either bitertanol or oxycarboxin while 2 apparently sensitive isolates were also selected (Table 6)„

There was no positive correlation between the indication of fungicide sensitivity from the plate test and the results of the glasshouse test (Table 7). Isolates such as 1 & 4 which seemed resistant to bitertanol in the plate germination test were sensitive in this test. Similarly isolate 2 which appeared sensitive in the plate test had a comparatively high number of rust pustules on the fungicide sprayed plants.

Discussion. The general conclusion was that minor differences in sensitivity as detected by the standard test are not reproducable. The range of sensitivities found in rust collections to date fall in what could be classed as a broad sensitive range.

Factors which may be contributing to poor rust control include late application of fungicide programs and poor application techniques. In the case of bitertanol its reported lower efficacy than oxycarboxin may be due to differences in dosages of active ingredient. Although bitertanol appears slightly more active than oxycarboxin, on an active ingredient basis, this is probably more than compensated by the higher field application rate of oxycarboxin (750 Mg/mL vs 150 jxg/mL).

(d) Competitive ability of isolates of Botrytis cinerea

Characteristics of B. cinerea have been widely studied since dicarboxmide resistance was first recognised in laboratory studies in 1975 (Leroux et al, 1977). The fungus is heterokaryotic with multinucleate mycelium. Resistance of varying degrees is explained by the accumulation of resistance genes in the nuclei. This is encouraged by the selection pressure of fungicides. Many believe that the accumulation of resistance genes is associated with a weakened competitive ability (Davis & Dennis 1981, Hisada et al. 1981) due to slower growth and lower sporulation capacity. Several experiments were

14

conducted in glasshouse and controlled environment cabinets but the experimental techniques were not satisfactory. A major difficulty in studying these mixed populations over several generations was contamination by saprophytic organisms on the natural substrate, tomato flowers.

(e) Developing strategies for resistance

Jointly, with members of AFRAC, general strategies to avoid or delay resistance problems have been publicised (Appendices II, III, IV). Well defined strategies have been promoted for cucurbit powdery mildew, tomato grey mould, onion downy mildew and lettuce downy mildew (Appendices II, VII, VIII, IX).

A field experiment was set up to validate the grey mould strategy in a field trial at Bundaberg Research Station in 1991, but weather conditions were unsuitable for disease development.

15

Table 6 Sensitivity of 30 rust pustules to bitertanol and oxycarboxin

Site Pustule

Germination % Code No. for future work

Site Pustule Check 0

Bitertanol 5 10

Oxycarboxin 10 25

Code No. for future work

Bichel 1 2 3 4 5

12 16 8

52 4

4 0 8 0 0

0 0 8 0 0

20 28 40 88 40

0 0 0 4 0

James (F) 1A

2 3 4 5

92 92 96 64 44

0 40 20 4 8

0 0 4 0

12

84 96 60 72 4

0 0 0 0 0

2

1

Turner 1 2 3 4 5

48 44 44 76 68

8 0 0

12 0

4 4 0

20 4

68 16 60 8

16

0 0 0 0 0

4

Jones 1 2 3 4 5

80 0

40 96 64

8 8 0 8 4

4 0 0 4 0

68 44 84 64 80

4 4 0

20 12

5 7

James (R) 1 2 3 4 5

48 12 96 48 32

0 0

12 0 4

0 0 4 0 0

0 0

76 0

52

0 0 0 0 0

6

Price 1 2 3 4 5

20

36 68 96

0 0 0 0

16

0 0 0 0

12

4 0 0

36 60

0 0 0 0 0 3

16

Table 7 Development of rust pustules on leaves sprayed with several fungicide treatments

Isolate and fungicide sensitivity from plate

test

Number of rust pustules per shield leaf Isolate and fungicide sensitivity from plate

test

Bitertanol (ug/ml) Oxycarboxin (y. g/ml) Isolate and fungicide sensitivity from plate

test 0 10 25 100 10 25 100

1. Bitertanol resistant 51 0.2 0 0 2.2 0.2 0

2. Sensitive 9.5 6.5 5.2 0.8 8.8 4.7 0

3. Bitertanol resistant 52.2 0.5 2.3 2.5 9.2 13.3 1.0

4. Bitertanol resistant 20.3 0 0 0 5.2 2.5 0

5. Oxycarboxin resistant 11.8 8 1.5 2.2 9.5 2.2 0.2

6. Sensitive 18.3 7.2 0.7 4.5 11.8 4.8 0

7. Oxycarboxin resistant 46.2 4.5 2.2 5.3 8.3 4.8 0.5

17

4. GENERAL DISCUSSION

The procedures and standards for determining fungicide sensitivity in 26 fungal pathogens will be of value in analysing the cause of future crop failures due to disease. In some cases, standards were also set for experimental products in addition to those currently registered. Many growers benefited from the analysis of their samples.

The recognition of 3 new instances of fungicide resistance during the term of this project, illustrates the need for strategies to be developed and put in place.

Some aspects of the project were disappointing, in particular, the attempts to determine relative competative ability between resistant and sensitive isolates. Constraints of time did not permit the conduct of the additional experiments to develop satisfactory techniques.

One of the most encouraging signs of progress over the 3 years has been the wider recognition by growers and field officers of the fungicide resistance problem. The cooperation between all sectors of the industry has been commendable.

5. ACKNOWLEDGEMENTS

The Queensland Fruit and Vegetable Growers and the Australian Fungicide Resistance Action Committee of the Agricultural and Veterinary Chemicals Association supported this project over its 3 year term. Funding by the Australian Horticultural Research and Development Corporation in the final year was also appreciated.

Thanks are due to the many officers of the Agrochemical industry and State Departments of Agriculture who assisted in collecting samples. The technical assistance of Mr M. Weinert and J. Sanchez is gratefully acknowledged.

6. PUBLICATIONS ARISING FROM THIS PROJECT

Scientific

O'Brien, R.G. (1991). Control of onion downy mildew in the Lockyer Valley. Proceedings of 8th Australasian Plant Pathology Society Conference, Sydney, p. 22.

O'Brien, R.G. (1992). Control of onion downy mildew in the presence of phenylamide resistant strains of Peronospora destructor (Berk.) Caspary. Australian Journal of Experimental Agriculture 32, 669-74.

FitzGerald, S.M. and O'Brien, R.G. Validation of "Downcast" in the prediction of infection events of Peronospora destructor in the Lockyer Valley. Submitted for publication in Australian Journal of Experimental Agriculture.

18

Extension

Carson, C, O'Brien, R. and Collins, P. (1991). Become a resistance fighter! Ornamentals Update 6: (3), 6-9.

O'Brien, R.G. (1991). Controlling grey mould. Good Fruit & Vegetables 1(11), 21.

O'Brien, R.G. (1991). Fighting resistance to systemic fungicides. Good Fruit and Vegetables 2 (5), 14.

O'Brien, R.G. (1992). Fungicide resistance - threats and strategies. Department of Primary Industries Farm Note. Agdex 201/625. 3 pp.

O'Brien, R.G. & Kerr, J. (1991). Control options for downy mildew. Queensland Fruit and Vegetable News, August 15, 1991.

7. REFERENCES

Davidse, L.C, Looijen, D., Turkensteen, LJ. and Van der Wal, D. (1981). Occurrence of metalaxyl-resistant strains of Phytophthora infestans in Dutch potato fields. Netherlands Journal of Plant Pathology. 87, 65-8.

Davis, R.P. and Dennis, C. (1981a). Studies on the survival and infective ability of dicarboximide-resistant strains of Botrytis cinerea. Annals of Applied Biology. 98, 395-402.

Davis, R.P. and Dennis, C. (1981b). Properties of dicarboximide-resistant strains of Botrytis cinerea. Pesticide Science. 12, 521-35.

Eckert, J.W. (1988). Historical development of fungicide resistance in plant pathogens. In 'Fungicide Resistance in North America' (Ed. CJ. Delp) pp 1-3. APS Press, USA.

Georgopoulos, S.G. (1988). Genetics and population dynamics. In 'Fungicide Resistance in North America' (Ed. CJ. Delp). pp 12-13. APS Press, USA.

Hisada, Y., Takaki, H., Kato, T., Osaki, T. and Kawase, Y. (1981). Fitness of procymidone resistant Botrytis cinerea strains developed in vitro. Netherlands Journal of Plant Pathology. 87, 243-4.

Leroux, P., Fritz, R. and Gredt, M. (1977). Etudes en laboratoire de souches de Botrytis cinerea Pers. resistantes a la Dichlozoline, au Dichloran, au Quintozene, a la Vinclozoline et au 26019 RP (Glycophene). Phytopathology Z. 89, 347-58.

Littley, E.R. and Rahe, J.E. (1984). Specific tolerance of Sclerotium cepivorum to dicarboximide fungicides. Plant Disease. 68, 371-4.

19

Morton, H.V. and Urech, PA. (1988). History of the development of resistance to phenylamide fungicides. In 'Fungicide Resistance' in North America' (Ed. CJ. Delp). pp 59-60. APS Press, USA.

O'Brien, R.G. and Glass, RJ. (1986). The appearance of dicarboximide resistance in Botrytis cinerea in Qld. Australasian Plant Pathology. 15, 24-25.

Penrose, LJ., Davis, K.C. and Koffmann, W. (1979). The distribution of benomyl tolerant Sclerotinia fructicola (Wint.) Rehm. in stone fruit orchards in New South Wales and comparative studies with susceptible isolates. Australian Journal of Agricultural Research. 30, 307-19.

Penrose, LJ., Koffmann, K. and Nicholls, M.R. (1985). Field occurrence of vinclozolin resistance in Monilinia fructicola. Plant Pathology. 34, 228-34.

Peterson, R A (1973). Field resistance to benomyl in cucurbit powdery mildew. Australian Plant Pathology Society Newsletter. 2, 27-8.

Reuveni, M., Eyal, H. and Cohen, Y. (1980). Development of resistance to metalaxyl in Pseudoperonospora cubensis. Plant Disease. 64, 1108-9.

Sisler, H.D. (1988). Fungicidal action and fungal resistance mechanisms. In 'Fungicide Resistance in North America' (Ed. CJ. Delp). pp 6-8. APS Press, USA.

Wade, M. (1988). Strategies for preventing or delaying the onset of resistance to fungicides and for managing resistance occurrences. In 'Fungicide Resistance in North America'. (Ed. CJ. Delp). pp 14-15. APS Press, USA.

8. APPENDICES

I Testing procedures and standards to evaluate the sensitivity of fungi to fungicides.

II Fungicide resistance - threats and strategies.

UJ Fighting resistance to systemic fungicides.

IV Become a resistance fighter.

V Control of onion downy mildew in the presence of phenylamide-resistant strains of Peronospora destructor (Berk.) Caspary.

VI Validation of Downcast in the prediction of infection events of Peronospora destructor in the Lockyer Valley.

VII Control options for downy mildew.

VIII Controlling grey mould.

IX Downy mildew in lettuce - DPI's fightback package.

APPENDIX I

Testing procedures and standards

to evaluate the sensitivity of

fungi to fungicides

Testing procedures and

standards to evaluate

the sensitivity of

fungi to fungicides

R.G. O'Brien 1992

The procedures for fungicide sensitivity testing

descibed in this booklet were developed during 1989-92 as

part of a project funded by the Australian Fungicide

Resistance Action Committee, Queensland Fruit and

Vegetable Growers and the Horticultural Research and

Development Corporation. Emphasis was placed on

keeping the procedures as simple and rapid as possible.

For a few fungi (e.g. Uromyces appendiculatus), it is felt

that improvements to the tests can be made and work is

continuing. Finally, since the standards were developed

in 1989-92, and many of the fungicides have been in use

for several years, our 'sensitive' isolates may not be true

wild types.

INDEX

Alternaria solani

Botrytis cinera

Bremia lactuca

Cercospora beticola

Cercosporidium personatum

Fulvia fulva

Mycosphaerella melonis (Didymella bryoniae)

Mycosphaerella musicola

Oidium sp.

Penicillium spp.

Peronospora destructor

Peronospora parasitica

Phytophthora nicotianae var. parasitica

Phytophthora palmivora

Pseudoperonospora cubensis

Rhizoctonia solani

Sclerotinia laxa, fructicola

Sclerotinia minor

Sclerotinia sclerotiorum

Sclerotium cepivorum

Sclerotium rolfsii

Sphaerotheca fuliginea

Thielaviopsis basicola

Thielaviopsis paradoxa

Uromyces appendiculatus

ORGANISM Altemaria solani DISEASE Target spot

CROPS Tomatoes, Potatoes

FUNGICIDES Dicarboximide - Iprodione (Rovral) DMI - Tebuconazole (Folicur)

TEST METHOD 1. Isolates are cultured on Difco PDA. Spore production enhanced by cutting the colony into cubes and placing them on water agar for 2 days before inoculum required.

2. Test plates are prepared by adding fungicides to molten PDA (52°C) to give 0, 1 and 10 ug/ml.

Iprodione - Rovral 250 g/L S.C.

Add 0.4 ml Rovral to 100 ml sterile distilled water (stock solution)

Add 1 ml stock solution to 99 ml molten PDA - 10 ug/ml. Add 0.1 ml stock solution to 100 ml " " - 1 ug/ml.

Tebuconazole - Folicur 250 g/L S.C. Add 0.4 ml to 100 ml sterile distilled water - stock solution. Add 1.0 ml stock solution to 99 ml molten PDA - 10 ug/ml. Add 0.1 ml stock solution to 100 ml " " - 1 ug/ml.

3. Inoculation of test plates is made by

(i) transfering 5 mm diameter agar plugs from the margin of colonies of A solani to 4 sites on each plate. Agar plugs should be inverted so hyphae are in immediate contact with the test medium.

(ii) Spores are transfered by glass rod to the surface of the test medium. The rod should be lightly rubbed over an area of about 1 sq cm and the deposition site marked on the plate. Several (6-8) transfers of spores can be made to each plate.

4. Incubation, (i) Measure the radius of growth of colonies from agar cores after 5 days, (ii) Examine spore germination under the LP objective of a compound microscope between 24-48 h after transfer.

5. Interpretation. On fungicide amended plates there should be no growth on 10 ug/ml iprodione and only slighty deformed growth on tebuconazole. At 1 ug/ml growth should be reduced to 30% of the check for both fungicides.

Spore germination at 10 ug/ml occurs, but germ tubes are very short and abnormal. At 1 ug/ml germ tubes are shorter (about 50%) than those developing on plain PDA

STANDARD Isolate 3658 ex potato and 3722 ex tomato are stored under ISOLATE water and as freeze dried culture.

ORGANISM Botrytis cinerea DISEASE Grey mould

CROPS Several e.g. tomatoes, grapes, strawberries, glasshouse grown cucurbits and capsicum

FUNGICIDES Benzimidazoles - benomyl (Benlate) Dicarboximides - iprodione (Rovral)

TEST METHOD Spores are collected on cotton buds and transferred to PDA amended SUMMARY with benomyl lOpp and iprodione 4 and 10 ppm. Germination with

germ tube elongation indicates resistance.

TEST METHOD 1. Isolates are collected directly from the field on sterile cotton buds or isolated from diseased material onto PDA Cotton buds in autoclavable vials are convenient to use and easy to store. Gently dab the cotton bud in the spore mass without rubbing against the host tissue.

2. Test media

A For benzimidazole sensitivity Benlate 500g/kg D.F. or W.P. Add 0.2g to 100ml sterile distilled water (stock solution).

Add 1.0ml stock solution to 99ml molten PDA to give lOppm a.i. benomyl.

B. For dicarboximide sensitivity Rovral 250g/L S.C.

(i) Add 0.4ml to 100ml sterile distilled water (stock solution). Add 1.0ml stock solution to 99ml molten PDA to give lOppm a.i. iprodione

(ii) Add 0.4ml stock solution to 100ml molten PDA to give 4ppm a.i. iprodione

C. Control - plain PDA.

3. Spore transfer. Six to eight transfers can be made to each plate provided there is an identification code. Spores are transferred directly from the cotton bud by lightly touching different areas of the bud on the four media. Alternatively, transfer spores from culture plates by glass rod.

4. Incubation. On laboratory bench for 24 h.

Rating. Examine under the low power objective of a microscope. Note whether germination has commenced and if germ tubes have developed.

Interpretation. Benomyl sensitive isolates may show germ tube initiation on lOppm benomyl but will not develop. The germ tubes of resistant isolates will develop normally.

Dicarboximide sensitive isolates will not germinate on either 4 or 10 ppm iprodione. Isolates with low level resistance will germinate on 4 but not lOppm. Resistant isolates will germinate on lOppm although germ tubes may be a little shorter than those on plain PDA.

STANDARD ISOLATES

Sensitive Benomyl resistant Dicarboximide resistant

1508-11 1508-40

low high -1508-40

STORAGE Freeze dried culture. NOTES Water agar may be substituted for PDA if cultures are not required.

It is unusual to have dicarboximide resistance in the absence of benomyl resistance.

ORGANISM Bremia lactucae DISEASE Downy mildew

CROPS Lettuce

FUNGICIDES Phenylamides - metalaxyl (Ridomil)

TEST METHOD Lettuce seedlings are grown in PlantconR containers on a SUMMARY vermiculite medium to which a fungicide-nutrient solution has been

added. Inoculate, incubate at 15 °C rate for infection after 7-10 days.

TEST METHOD 1. Isolates are maintained on young seedlings or detached leaves or discs. Seedlings should be uncovered after incubation then enclosed when moculum is required in 7-10 days.

Test plants. Place 200ml vermiculite in each PlantconR container. Make up a nutrient solution e.g. Duet K 3ml/L + Duet L lml/L. Add Ridomil (250g/kg metalaxyl) to make up the range of concentrations. Add 0.4g per litre of nutrient solution - 100>ig/ml Add 10 parts of above to 90 parts nutrient soln - lOug/ml. Add 10 parts of above to 90 parts nutrient soln - ljjig/ml. Add 10 parts of above to 90 parts nutrient soln - 0. lug/ml.

Dispense 120ml of required solution to each Plantcon container. Sprinkle approx. 50 surface sterilized (lOOOppm Av.Cl) seed of Yatesdale on the surface and close container.

Place containers in a CEC at 15-16°C on a 12/12 light regime. Grow for 1 week before inoculation.

Inoculation. Prepare spore suspension of lxlOVml and spray 2ml over the seedlings in each container. Replace the lid and return to CEC for 7-10 days.

Incubation. In CEC 15-16 °C for 7-10d.

STANDARD ISOLATES

5. Rating. % seedlings with sporulation.

6. Interpretation. Sporulation % should be reduced at 0.0 lug/ml and completely prevented at 0.1 (U.K. standards). Resistant isolates will sporulate on seedlings treated with 10 and lOOug/ml. Tests with isolate 3340 resulted in reduced sporulation at lug/ml and complete prevention at lOjig/ml. There may be more sensitive isolates than 3340.

-Isolate 3340 stored under liquid nitrogen.

NOTES This method is derived from a standard test developed in the UK Alternative methods using metalaxyl sprays have not given consistent results.

ORGANISM Cercospora beticola DISEASE Leaf spot

CROPS Beetroot, silver beet

FUNGICIDES Benzimidazoles - benomyl (Benlate, 500g/kg)

TEST METHOD Add benomyl to molten Difco PDA to give 10 ug/ml. SUMMARY Transfer spores directly to culture plates. Check germ tube

elongation after 24-48h.

TEST METHOD 1. Isolates are collected directly from the field. Leaves with young lesions are washed in tap water then immersed in distilled water + chlorine for 5 min. (1000 ppm Av.Cl.). Leaves are rinsed in tap water for several minutes then immersed in sterile distilled water. Blot dry, cut out sections of leaf with sporulating lesions and place them in water agar in culture plates. Incubate in diffuse light for 24h.

2. Test media is prepared with Benlate (500g/kg benomyl) 0.2g Benlate added to 100ml sterile distilled water (stock solution). lml stock solution added to 99ml molten Difco PDA (10ug/ml). Control media is plain Difco PDA.

3. Inoculation. Using a fine sterile glass rod, touch the tip to the agar surface to moisten it, then to conidia on a test lesion. Transfer conidia to test plate. Sterilize rod. Etc. Up to 8 transfers can be made to each plate at marked positions.

4. Incubation. Incubate 24h on laboratory bench 20-24°C.

5. Ratine. Examine spores under LP objective. Note whether germ tubes have emerged and if so, their length.

6. Interpretation. Sensitive isolates will not germinate, or germ tubes will be short and abnormal compared with the check. Resistant isolates will show germ tubes of a similar length on both media.

STANDARD None kept ISOLATE

ORGANISM Cercosporidium personatum DISEASE Leaf spot

CROP Peanuts

FUNGICIDES Benzimidazoles - benomyl (Benlate) DMI - propiconazole (Tilt)

TEST METHOD Spores are transfered to fungicide amended media. SUMMARY Germination and growth checked after 16h.

TEST METHOD 1. Isolates are taken directly from sporulating lesions. It may be necessary to rinse leaves in chlorine solution (1ml per 100ml) for 2 min., rinse in running tap water then sterile distilled, blot dry and incubate overnight to produce fresh conidia.

Test media. Fungicides are added to water agar to give concentrations of 0, 0.1, 1 and lOpg/ml.

A. Benzimidazole sensitivity - Benlate (500g/kg benomyl) Add 0.2g Benlate to 100ml sterile water Add 1 ml to 99ml water agar - lOjag/ml Add 0.1ml to 100ml water agar - ly.g/ml Add 1 ml to 99ml sterile water Add 1 ml to 99ml water agar - O.ljag/ml.

B. DMI sensitivity - Tilt (250g/L propiconazole) Add 0.4ml to 99.5ml sterile water then dilute as above.

C. Control - plain water agar.

3. Spore transfer. Use a fine tipped glass rod to transfer conidia from individual spots to each test medium. Up to 8 transfers at marked positions can be made on each plate.

4. Incubation, laboratory bench for 16h.

5. Rating. Examine under the low power of a compound microscope. Measure the length of germ tubes particularly at l)ig/ml and 10ng/ml.

6. Interpretation. Benomyl sensitive isolates will show germ tube initiation at 1 & 10]ag/ml but no further development. Resistant isolates will show germ tubes of similar length on all media.

STANDARD ISOLATE

NOTES

DMI sensitive isolates will show germ tube initiation and some development at both 1 and lOpg/ml but germ tube length is shorter (AV15]xm) than on check plates (200}am). At 0.1jjLg/ml germ tubes have growth inhibited (AV.lOOum).

None kept.

If resistance is suspected it may be advisable to conduct tests using sprayed detached leaves in sand culture for confirmation. (Phytopathology 79:136-42).

ORGANISM Fulviafulva DISEASE Leaf mould

CROPS Tomatoes

FUNGICIDES Benzimidazoles - benomyl (Benlate)

TEST METHOD Prepare PDA plates containing 0, 0.01, 0.1 and 1 ug/ml benomyl. SUMMARY Transfer spores and check germination after 48 h. Sensitive isolates

have an MIC of approximately 0.1 )ig/ml.

TEST METHOD 1. Isolates. Isolates may be maintained on Difco PDA but it is DETAILS preferable to transfer spores directly from diseased tissue to test

plates.

2. Test media. Difco PDA containing benomyl 0, 0.01, 0.1 and 1 y.g/ml is required. Add 0.2 g Benlate (500 g/kg benomyl) to 100 ml sterile water. Add 100 ml of this solution to 90 ml sterile water (Soln 1).

A. Add 1 ml to 99 ml molten PDA - (1 jag/ml) B. Add 0.1 ml to 100 ml molten PDA - (0.1 ng/ml) Add 1 ml to 99 ml sterile water (Soln 2) C. Add 1 ml to 99 ml PDA - (0.01 ug/ml) D. Plain PDA

3. Inoculation. If lesions show fresh, uncontaminated sporulation use a sterile glass rod with a fine tip to transfer spores to sites on the test plates. If lesions are old or do not have fresh conidia, wash leaves gently under tap water, blot dry and incubate overnight in a humid chamber to encourage sporulation. Alternatively, isolate the fungus from surface sterilised leaf pieces from the edge of lesions.

4. Incubation. Incubate for 48 h on a laboratory bench 22-25°C.

5. Rating. Check germination under a low power objective and rate as follows: 0 - no germination or germ tubes aborted 1 - germination but fungal growth reduced compared with

check. No sporulation 2 - as above but some sporulation 3 - normal growth and sporulation

6. Interpretation. Sensitive isolates are rated as 3 at 0.01, 0 or 1 at 0.1 jig/ml and 0 at 1 ug/ml.

STANDARD Isolate is stored in liquid Nitrogen. ISOLATE

NOTES Resistance has not been observed even in glasshouse situations where the use of benzimidazoles has been intense.

ORGANISM Mycosphaerella melonis DISEASE Gummy stem blight

Didymella bryoniae

CROPS Cucurbits

FUNGICIDES Benzimidazoles - benomyl (Benlate, 500g/kg) TEST METHOD Add benomyl to molten Difco PDA to give 10ug/ml. SUMMARY

TEST METHOD 1. Isolates are kept as cultures on PDA. DETAILS

2. Test media is prepared with Benlate (500g/kg benomyl) 0.2g Benlate added to 100ml sterile distilled water (stock solution). Add 1 ml stock solution to 99ml Difco PDA (lOug/ml) Add 0.1. ml stock solution to 100ml PDA (lug/ml). Control plates are plain PDA. Pour test plates using 15 ml/Petri dish.

3. Inoculation. Cores (4mm) are taken from the margin of 4-6 day old colonies and placed upside down on the test media. There should be 4 replicates.

4. Incubation. Place test plates on a lab bench (20-24 °C) for 48h.

5. Rating. Measure radius of growth after 48h.

6. Interpretation. The EC50 of sensitive isolates is >0.1<1. There should be no growth at lOyg/ml.

STANDARD Isolate 2968 is stored in liquid nitrogen. ISOLATE

ORGANISM Mycosphaerella musicola DISEASE Leaf Spot

CROPS Bananas


- tridemorph (Calixin)

TEST METHOD Fungicides added to molten Difco PDA. Banana leaves incubated to SUMMARY produce conidia which are transferred to test media by glass rod.

TEST METHOD 1. Isolates. M. musicola is a slow growing fungus on nutrient agar. DETAILS Isolates may be obtained by incubating diseased leaf sections

and transfering fresh conidia by sterile needle to either PDA or transfering them directly to the test plates. For a rapid test, conidial inoculum is preferable. North Qld isolates produce ascospores and these may also be used.

2. Test media - Use 2% water agar for conidial transfer. A. Benzimidazoles - use Benlate (500g/kg benomyl). Add O.lg Benlate to 100ml sterile water. Add 0.5ml to 99ml Difco PDA - (2.5]ag/ml) Add 0.2ml to 99.5ml PDA - (lug/ml) Add 2ml to 98ml sterile water (Solution 2) Add 1ml solution 2 to 99ml PDA - (O.lng/ml)

B. DMI - Tilt (250g/L propiconazole) Dilute as above but use 0.2ml Tilt in the first step.

C. DMI - Calixin (750g/L tridemorph)

Dilute as for Benlate but add 0.067ml Calixin to 100ml sterile water in the first step.

D. Plain agar.

3. Inoculation. (i) Mycelium onto PDA + fungicide media.

After test isolates have produced colonies (4-6 wks) use a hooked needle to collect mycelial fragments and place these on the test plates. Check under microscope to confirm transfer.

(ii) Conidia. Leaf sections with young lesions are cut, soaked in chlorine solution (2 min in 2% sodium hypochlorite), rinsed under running tap water then rinsed in sterile distilled water. Free moisture is removed and leaf sections incubated in Petri dishes containing water agar. After 24 - 48h check for development of conidia which can be transfered by sterile needle or glass rod.

(iii) Ascospores. Rinse leaf sections as above but attach to moist filter paper discs (9cm) and suspend above test plates.

4. Incubation. Incubate at 22 °C for 48h. W spore germination test or 7-14d for mycelial fragment test.

5. Rating. (i) Mycelial test. Rate on a 0 - 3 scale

0 = no growth 1 = slight mycelial growth (up to 1/3 of check) 2 = 1 / 3 - 2/3 of check 3 = same as check.

(ii) Conidia. Check under microscope to determine whether germtube elongation is equal to or less than check. 1 - germination, but abnormal, stunted germtubes 2 - germination but development less than check 3 - same as check

(iii) Ascospores. as above.

6. Interpretation (i) Growth. Sensitive isolates have EC50's for all fungicides <0. lug/ml. The MIC is close to 0. lug/ml and less than l^g/ml.

(ii) Conidia and Ascospores. Development is greatly inhibited at 0.1jig/ml which is virtually the MIC.

STANDARD Test isolate supplied and stored under liquid Nitrogen by D. Jones. ISOLATE

NOTES Ascospores are produced readily by Nf Qld. isolates on old infected tissue but less readily by S.Qld. isolates. The techniques described above are based on standard procedures developed by Du Pont and Ciba Geigy to detect small shifts in sensitivity. The ascospore test has not been tried as yet.

ORGANISM Oidiumsp. DISEASE Powdery mildew

CROPS Peas

FUNGICIDES Tebuconazole (Folicur, 250g/kg, EC). Triadimefon (Bayleton, 250g/kg, EC).

TEST METHOD Pea plants (cv. Small Sieve Freezer) are grown in the glasshouse and SUMMARY sprayed twice, 5 days apart with test fungicides. The next day they are

spray inoculated with a spore suspension. Inoculated leaves are rated after 14-16 days.

TEST METHOD 1. Isolates are used directly from the field or maintained on DETAILS glasshouse raised plants of S.S.F.

2. Test plants - 2 plants (cv. S.S.F.) per 10cm pot, fertilized weekly with liquid fertilizer. Each isolate tested requires 16 pots. When plants have fully expanded leaves from 4 nodes they should receive the 1st spray.

3. Test fungicides and application These should be mixed to give concentrations of 0, 5, 25 and 100p.g/ml with the addition of a non-ionic wetting agent e.g. Topwet 0.2ml/L. Fungicides applied by hand atomiser to give complete coverage with minimal runoff to root zone. (4 reps each concentration).

Folicur (250 g/L tebucanazole)

Dilution - 0.4ml (f.p.) in 1000ml (100>ig a.i./ml) - 250ml + 750ml water (25ug a.i./ml) - 200ml + 800ml water (5*ig a.i./ml).

Bayleton (125 g/L triadimefon)

Use twice the amount of product as above.

The second application is made 5 days after the first. A small dab of dilute acrylic paint is placed on the node above the youngest fully expanded leaf to mark the limit of disease assessment.

4. Inoculation is made 24h after the second fungicide application. Young leaves showing heavy sporulation are shaken in a flask in sterile water + 1 drop Tween 80 per 200ml. The conidial suspension is filtered through cheese cloth and concentration adjusted by using a haemocytometer to give a concentration of approximately 2 x 104/ml. As quickly as possible the inoculum is applied to test plants with a hand atomiser to give thorough coverage but not full leaf wetness.

5. Incubation - After inoculation, test plants should be allowed to dry as rapidly as possible. Watering should be to the root zone rather than foliage.

6. Rating system - After 14-16 days, plants are examined for mildew which is rated as present or absent on each leaf. The proportion of infected/total represents the test data.

7. Interpretation of results - Sensitive isolates have an EC50 of approximately 5>xg/ml and an MIC slightly higher than 25jxg/ml. Resistant isolates would be expected to grow on plants receiving 100ng/ml.

STANDARD It has not been possible to develop a technique to store Oidium sp. in ISOLATES a viable condition.

ORGANISM Penicillium spp. (digitatum, DISEASE Blue/green expansum, italicium) mould

CROPS Citrus, apples, pears

FUNGICIDES Benzimidazoles - benomyl (Benlate) DMI - imazalil (Fungaflor)

TEST METHOD Test fungicides are incorporated into Difco PDA to give SUMMARY concentrations of 0, 1 and 10 u.g/ml for benomyl and 0, 0.1, 1 and 10

Ug/ml for imazalil.

TEST METHOD 1. Isolates. It is preferable to obtain test samples directly from DETAILS infested fruit. The fungi grow readily on PDA and can be

maintained on this medium.

2. Test media.

A. Benzimidazole - Benlate (500 g/kg benomyl) Add 0.2 g Benlate to 100 ml sterile water. Add 1 ml to 99 ml molten Difco PDA - (10 ug/ml). Add 0.1 ml to 100 ml PDA - (1 ug/ml).

B. DMI - Fungaflor (750 g/kg imazalil) Add 0.320 g to 100 ml sterile water (solution 1). Add 0.416 ml solution 1 to 100 ml PDA - (10 ug/ml). Add 1 ml solution 1 to 9 ml sterile water (solution 2). Add 0.416 ml solution 2 to 100 ml PDA (1 ug/ml). Add 1 ml solution 2 to 9 ml sterile water. Add 0.416 ml to 100 ml PDA (0.1 ug/ml).

C. Check - plain Difco PDA.

3. Inoculation. Dry conidia are transferred from sporulating colonies (on media plates or infected fruit) to 5 ml screw cap tubes containing 0.5 ml of 0.25% water agar and 0.01% Tween 20. (Tween and agar added to 100 ml sterile distilled water, steamed to melt agar then dispensed to tubes and sterilized. Inoculum is delivered to test plates as 5 u-L droplets by sterile loop.

4. Incubation. Seven days on a laboratory bench or incubated 22-25°C.

5. Rating. Check growth after 3 days, measure radius of growth after 7 days.

6. Interpretation. Sensitive isolates show no growth on benomyl 1 ug/ml or imazalil 0.1 ug/ml.

REFERENCE Isolates 3055 and 3294 are stored as freeze dried cultures. ISOLATES

NOTES Other fungicides tested (iprodione, vinclozolin, guazatine) allowed growth of isolates at 100 ng/ml).

REFERENCES 1. Brown, Eldon G. (1989). Baseline sensitivity of Florida isolates of Penicillium digitatum to Imazalil. Plant Disease: 73; 773-774.

2. Bus, Vincent G., Bongers, Anton, J., and Risse, Lawrence A. (1991). Occurrence of Penicillium digitatum and P. italicum Resistant to Benomyl Thiabendazole and Imazalil on citrus fruit from different geographic locations. Plant Disease: 75; 1098-1100.

3. Rosenberger, D.A. (1991). Pathogenicity and Benzimidazole resistance in Penicillium species recovered from flotation tanks in apple packinghouses. Plant Disease: 75; 712-715.

ORGANISM Peronospora destructor DISEASE Downy mildew.

CROPS Onions, shallots


TEST METHOD Two week old onion seedlings are sprayed with fungicide then SUMMARY inoculated with a spore suspension and incubated at 16 °C for 24h

then 18-20° C for 14 days. They are returned to high humidity for 24h and rated for presence or absence of sporulation.

TEST METHOD 1. Isolates are used directly from the field or maintained on DETAILS glasshouse raised plants of onion cv. Lockyer Early Brown.

Cultivar more useful in summer is Spring Onion or Salad Onion (Hendersons).

2. Test plants - About 50 seed (cv. Lockyer Early Brown) are sown in 4" pots of UC mix. Seed is covered by 1 cm vermiculite. Two weeks after sowing check germination is complete and seedlings are established with 2 leaves.

3. Test fungicide and application. Ridomil 25 WP (250g/kg metalaxyl). Add 0.4 g per litre of water - 100 }ig/ml Dilute 1 part in 10 - 10 ng/ml Check - 0

Spray seedlings to run off taking care not to allow spray to deposit on the root zone. Spray a second time five days after the first application. Inoculate 24 h later.

4. Inoculation. Fresh conidia are suspended in water (add 1 drop Tween 80 per 200 ml) and concentration adjusted to approximately 2 x 104/ml. Seedlings sprayed until droplets form on leaves.

5. Incubation. Pots enclosed in plastic bags and placed in CEC set at 16 °C for 24 h. Plants then removed from bags and either returned to glasshouse for 13 days or kept in C.E.C. at 18 -20 °C.

6. Rating system. Ten or more seedlings from each pot are examined in detail for signs of sporulation which is rated as present or absent. The examination should be made under a stereo microscope or with a magnifying head band.

7. Interpretation of results. Sensitive isolates should not sporulate on either 10 or 100 pLg/ml treated seedlings. Resistant isolates will sporulate on both.

STANDARD A standard sensitive isolate (3074) is stored under liquid ISOLATES nitrogen.

NOTES If building up inoculum for a test, the inoculation should take place a few days after seed is sown for test plants. Inoculum can be induced at any time from about 13 - 20 days after inoculation by providing moisture and 16 °C. Sporulation does not occur over a succession of days.

If inoculating older plants, wipe leaves with a tissue to remove waxy layer before inoculating.

ORGANISM Peronospora parasitica DISEASE Downy mildew

CROPS Brassicas


TEST METHOD Broccoli seedlings are grown in PlantconR containers on a venniculite SUMMARY medium to which a fungicide-nutrient solution has been added.

Inoculate, incubate at 15 °C rate for infection after 7-10 days.

TEST METHOD 1. Isolates are maintained on young seedlings or detached leaves or discs. Seedlings should be uncovered after incubation then enclosed when inoculum is required in 7-10 days.

2. Test plants. Place 200ml venniculite in each PlantconR container. Make up a nutrient solution e.g. Duet K 3ml/L + Duet L lml/L. Add Ridomil (250g/kg metalaxyl) to make up the range of concentrations. Add 0.4g per litre of nutrient solution - lOOug/ml Add 10 parts of above to 90 parts nutrient solu. - lO ig/mL Add 10 parts of above to 90 parts nutrient solu. - ljig/ml. Add 10 parts of above to 90 parts nutrient solu. - Q.lyg/mL

Dispense 120ml of required solution to each Plantcon container. Sprinkle approx. 50 surface sterilized (lOOOppm Av.Cl) seed of Yatesdale on the surface and close container.

Place containers in a CEC at 15-16°C on a 12/12 light regime. Grow for 1 week before inoculation.

3. Inoculation. Prepare spore suspension of lxlOVml and spray 2ml over the seedlings in each container. Replace the lid and return to CEC for 7-10 days.

4. Incubation. In CEC 15-16 °C for 7-10d.

5. Rating. % seedlings with sporulation.

6. Interpretation. Sporulation % should be reduced at lug/ml and completely prevented at 10 and lOOug/ml. Resistant isolates will sporulate on seedlings treated with 10 and 100iig/ml.

STANDARD ISOLATES

NOTES This method has not been fully tested as yet. It is derived from a standard test developed in the UK. Alternative methods using metalaxyl sprays have not given consistent results.

ORGANISM

CROPS

Phytophthora nicotianae var. parasitica

Tomatoes

DISEASE Root/stem/ fruit rot

FUNGICIDES Phenylamides - (metalaxyl)

TEST METHOD Metalaxyl added to molten Difco CMA to give concentrations of 0,0.1, SUMMARY 1 and 5 ug/ml.

TEST METHOD 1. DETAILS

2.

4.

5.

6.

Isolates are cultured on Difco Cornmeal agar (CMA)

Test media is prepared by adding Ridomil (250 g/kg) metalaxyl) to molten, sterile, Difco CMA. Add 0.2 g Ridomil to 100 ml sterile water Add 1 ml to 99 ml CMA - (5 ug/ml) Add 0.2 ml to 99.8 ml CMA - (1 ug/ml) Add 2 ml to 98 ml sterile water (Soln 2) Add 1 ml to 99 ml CMA - (0.1 ug/ml) Plain CMA - (0)

Inoculation. Agar discs (5 mm) are taken from the margin of 4 day old cultures of the test isolates and placed upside down on the test media. There should be 4 replicates.

Incubation. Incubate at 25-28°C for 4 days.

Rating. Measure radius of growth.

Interpretation. Sensitive isolates have EC50's of 0.03-0.25 jjg/ml metalaxyl. The MIC is approximately 1 ug/ml.

STANDARD ISOLATE

NOTES

REFERENCE

Isolates 2918 and 2919 are maintained under liquid Nitrogen.

There is a deal of variability between isolates. Several isolates of P. nicotianae var. nicotianae tested had EC50 values of 3-5 ug/ml while others were in the sensitive range.

FERRIN, D.M. and KABASfflMA, J.N. (1991). In vitro insensitivity to metalaxyl of isolates of Phytophthora citricola and P. parasitica from ornamental hosts in southern California. Plant Disease 75: 1041-44.

ORGANISM Phytophthora palmivora DISEASE Fruit rot

CROPS Papaw

FUNGICIDES Phenylamides - (metalaxyl)

TEST METHOD Metalaxyl added to molten Difco CMA to give concentrations of 0,0.1, SUMMARY 1 and 5 vig/ml a.i. Measure radial growth


2.

3.

4.

5.

6.

Isolates are cultured on Difco Cornmeal agar (CMA)

Test media is prepared by adding Ridomil (250 g/kg) metalaxyl) to molten, sterile, Difco CMA. Add 0.2 g Ridomil to 100 ml sterile water Add 1 ml to 99 ml CMA - (5 jag/ml) Add 0.2 ml to 99.8 ml CMA - (1 ng/ml) Add 2 ml to 98 ml sterile water (Soln 2) Add 1 ml to 99 ml CMA - (0.1 pig/ml) Plain CMA - (0)

Inoculation. Agar discs (5 mm) are taken from the margin of 4 day old cultures of the test isolates and placed upside down on the test media. There should be 4 replicates.

Incubation. Incubate at 25-28°C for 4 days.

Rating. Measure radius of growth.

Interpretation. Sensitive isolates have EC50 >0.1 <1. The MIC is about 1.

STANDARD ISOLATE

NOTES

Isolates 2947 is stored under liquid Nitrogen.

The EC50 for benalaxyl is 0.6 and propamocarb >3000. The EC50's of other species tested:

P. cinnamomi - metalaxyl - 0.05-0.4 ng/ml P. dreschleri - metalaxyl - 0.3 >ig/ml P. citrophthora - metalaxyl - 0.5 pig/ml

ORGANISM Pseudoperonospora cubensis DISEASE Downy mildew.

CROPS Cucurbits

FUNGICIDES Phenylamides - Metalaxyl (Ridomil, 250g/kg)

TEST METHOD Spray test plants twice. Detach leaves, place on water agar and SUMMARY inoculate each leaf with up to 5 different isolates of P. cubensis by

droplets of conidial suspensions.

TEST METHOD 1. Isolates are maintained on detached leaves of rockmelon cv. DETAILS Hales Best or cucumber cv. Crystal Salad on water agar.

2. Test plants of cv. Hales Best or Crystal Salad are germinated in flats then transplanted l/15cm pot and held in a CEC at 20 °C until the first true leaf is Vi expanded. Remove to a cooled glasshouse and commence spray application program.

3. Test fungicide and application Ridomil 25 WP (250g/kg metalaxyl). Add 0.4g per litre of water - 100y.g/ml Dilute 1 part in 10 - lOjag/ml Dilute 1 part in 10 - lug/ml Spray upper and lower leaf surfaces thoroughly. Shake to remove excess spray droplets around leaf margins. Repeat spray applications at weekly intervals.

4. Inoculation Fresh conidia are collected and standardized to a concentration of 2 x l^/ml in sterile distilled water to which 1 drop Tween 80 has been added per 200ml. The youngest expanded leaves are collected from each plant (1-2 per plant), 24h after spraying, and placed on water agar in petri dishes. The lower side of the leaf is uppermost. At five sites on each leaf, inoculation droplets (IOJJLL.) are placed. These may be of different origin or serve as replicates of a single isolate.

5. Incubation Plates are incubated in diffuse light on a laboratory bench or CEC at 20-22 °C. After 7 days check for symptoms, rate after 9 days.

6. Rating system Each inoculation site rated: 0 - no symptoms 1 - small chlorotic spot, no sporulation 2 - large chlorotic spot - sporulation if present not as profuse as on control leaves 3 - symptoms similar to control leaves with abundant sporulation.

7. Interpretation of results. Sensitive isolates should not show symptoms at 10 or 100jag/ml. At lug/ml there may be a slight chlorotic spot.

STANDARD A standard sensitive isolate (1550) is stored in liquid nitrogen. ISOLATE

NOTES The test may also be carried out by inoculating the undersurface of leaves of potted plants using IOJIL droplets.

ORGANISM Rhizoctonia solani DISEASE Stem rot

CROPS Several e.g. beans, potatoes, tomatoes

FUNGICIDES Organophosphate - tolclofos methyl (Rizolex).

TEST METHOD Add fungicides to molten Difco PDA to give concentrations of 0, 0.1, SUMMARY 1 and 10ng/ml a.i.

Measure radial growth of isolates.


2.

3.

5.

Isolates are grown on Difco PDA

Test plates are prepared by adding fungicides to molten Difco PDA (52°C) to give 0, 0.1, 1 and 10 ng/ml a.i.

Rizolex 500g/kg WP. Add 0.2g to 100ml sterile water - stock solution (A) Add 1 ml stock solution to 99 ml molten Difco PDA - 10 Mg/ml a.i. Add 0.1 ml stock solution to 100 ml PDA - 1 lag/ml a.i. Add 1 ml stock solution A to 99 ml sterile water - stock solution B Add 1 ml stock solution B to 99 ml PDA - 0.1 iig/ml

Inoculation of test plates is made by transfering 5mm diameter agar plugs from the margin of colonies of the test fungi to 4 sites on each plate. Agar plugs should be inverted so hyphae are in immediate contact with the test medium.

Incubation. Plates should be stored 22 - 25° C for 3 days before the radius of growth is measured.

Interpretation. Sensitive isolates have an EC 50 of between 0.1 and 1. Their MIC is 1 ug/ml.

STANDARD ISOLATE

NOTES Resistance to tolclofos-methyl can be induced by serial transfer see: Netherlands Journal of Plant Pathology. 90 (1984) 95 - 106.

ORGANISM Sclerotinia laxa DISEASE Brown rot Sclerotima fructicola

CROPS Deciduous fruit - peaches, plums, nectarines etc.

FUNGICIDES Benzimidazoles - benomyl (Benlate) Dicarboximides - iprodione (Rovral) DMI - propiconazole (Tilt)

TEST METHOD Fungicides are added to molten PDA to give concentrations of 0, 0.1, SUMMARY 1,10.

Measure radial growth.

TEST METHOD Isolates are collected from diseased fruit by spore transfer to PDA. SUMMARY

Test media

A Benzimidazoles - Benlate (500g/kg benomyl) Add 0.2g to 100ml sterile water Add 1ml to 99ml PDA - (lOug/ml) Add 0.1ml to 100ml PDA - ( W m l ) Add 1ml to 99ml sterile water (solution 2) Add 1ml solution 2 to 99ml PDA - (0-l)jLg/ml)

B Dicarboximides - Rovral (500g/kg iprodione) Dilutions as above.

C D.M.I. - Tilt (250g/L propiconazole) Add 0.4ml Tilt to 99.5ml sterile water Add 1ml to 99ml PDA - (lO^g/ml) Add 0.1ml to 100ml PDA - (lug/ml) Add 1ml to 99ml sterile water (solution 2) Add 1ml solution 2 to 99ml PDA - (0. lug/ml)

D Plain PDA

Inoculation. Plates are inoculated at 4 sites around the margin with 3mm diam. discs taken from the margin of growing test cultures.

Incubation. 72h on a laboratory bench 20-24 °C.

Rating. Measure radius of growth and calculate % inhibition due to fungicide.

Interpretation. Benzimidazole: sensitive isolates have an EC50 of approximately 0.1 and an M.I.C. of >0.1< lug/ml benomyl. Dicarboximide: Sensitive isolates have an EC50 > 0.1 < 1 and an MIC of about 4 ug/ml iprodione. DMI: Sensitive isolates have an EC50 of about 0.01 and an MIC of > 0.1 <lpLg/ml propiconazole.

STANDARD S. fructicola - 2761-2 ISOLATES S. laxa - 2759-3

are stored in liquid nitrogen.

NOTES A more rapid test can be conducted using conidial transfer directly from diseased fruit to test plates.

ORGANISM Sclerotinia minor DISEASE W h i t e m o u l d Sclerotinia rot

CROPS Several - mainly lettuce, peanuts

FUNGICIDES Benzimidazoles - benomyl (Benlate) Dicarboximides - iprodione (Rovral) DMI - tebuconazole (Folicur)

TEST METHOD Add fungicides to Molten Difco PDA to give concentrations of 0, 0.1 SUMMARY and 1 ug/ml a.i. Measure radial growth

TEST METHOD 1. Isolates are maintained on PDA DETAILS

2. Test media A Benzimidazole - Benlate (500 g/kg benomyl)

Add 0.2 g Benlate to 100 ml sterile water Dilute this 1 part in 10 (solution 1) Add 1 ml solution 1 to 99 ml PDA - (1 ug/ml) Add 0.1 ml solution 1 to 100 ml PDA - (0.1 ng/ml)

B. Dicarboximide - Rovral (500 g/kg iprodione) Follow above procedure

C. DMI sensitivity - Folicur (250 ml/L tebuconazole) Add 40 pL Folicur to 100 ml sterile water Add 1 ml to 99 ml PDA - (1 ug/ml) Add 0.1 ml to 100 ml PDA - (0.1 Mg/ml)

D. Control - plain PDA

3. Inoculation. Cores of agar from the edge of test colonies are placed around the margin of test plates. Cores are 3 mm diameter and 4 can be sited on each plate. Place cores so hyphae are in direct contact with test medium.

4. Incubation. 72 h on a laboratory bench 22-25°C.

5. Rating. Measure radial growth of each colony.

6. Interpretation. Sensitive isolates have EC50's of <0.1, 0.1 and <0.1 for benomyl, iprodione and tebuconazole. The MIC values for these fungicides are >0.1 <1 ng/ml.

REFERENCE Isolate 3227 is stored under liquid Nitrogen. ISOLATE

NOTE Use young isolates as growth rates may vary if isolates are producing sclerotes.

ORGANISM Sclerotinia sclerotiorum DlSHASii W h i t e m o u l d , Sclerotinia rot

FUNGICIDES Benzimidazoles - benomyl (Benlate) Dicarboximides - iprodione (Rovral) DMI - tebuconazole (Folicur)

TEST METHOD Add fungicides to Molten Difco PDA to give concentrations of 0, 0.1 SUMMARY and 1 ng/ml a.i. Measure radial growth

TEST METHOD 1. Isolates are maintained on PDA DETAILS

2. Test media

A Benzimidazole - Benlate (500 g/kg benomyl) Add 0.2 g Benlate to 100 ml sterile water Dilute this 1 part in 10 (solution 1) Add 1 ml solution 1 to 99 ml PDA - (1 ug/ml) Add 0.1 ml solution 1 to 100 ml PDA - (0.1 ng/ml)

B. Dicarboximide - Rovral (500 g/kg iprodione) Follow above procedure

C. DMI sensitivity - Folicur (250 ml/L tebuconazole) Add 40 uL Folicur to 100 ml sterile water Add 1 ml to 99 ml PDA - (1 ng/ml) Add 0.1 ml to 100 ml PDA - (0.1 ug/ml)

D. Control - plain PDA

3. Inoculation. Cores of agar from the edge of test colonies are placed around the margin of test plates. Cores are 3 mm diameter and 4 can be sited on each plate. Place cores so hyphae are in direct contact with test medium.

4. Incubation. 72 h on a laboratory bench 22-25°C.

5. Rating. Measure radial growth of each colony.

6. Interpretation. Sensitive isolates have EC50's of approximately 0.5, 0.1 and 0.1 for benomyl, iprodione and tebuconazole and MICs of 1 ug/ml.

REFERENCE ISOLATES

Isolates 2994 and 3043 are stored under liquid Nitrogen.

NOTE Use young test isolates since growth rates may vary if isolates are producing sclerotes

ORGANISM Sclerotium cepivorum DISEASE White rot

CROPS Onion, garlic

FUNGICIDES Dicarboximides - procymidone (Sumisclex) DMI - tebuconazole (Folicur)

TEST METHOD Add fungicides to molten Difco PDA SUMMARY Measure growth of test isolates

TEST METHOD 1. Isolates are made from diseased plant tissue or sclerotes and DETAILS cultured on PDA.

2. Test media

A. For dicarboximide sensitivity - Sumisclex 500 WP

(i) Add 0.2 g to 100 ml sterile distilled water (solution 1) Add 1.0 ml to 99 ml molten Difco PDA to give 10 ug/ml a.i. procymidone

(ii) Add 0.1 ml solution 1 to 100 ml PDA to give 1 ug/ml a.i. procymidone

(iii) Add 1 ml solution 1 to 99 ml sterile water (solution 2) Add 1 ml solution 2 to 99 ml PDA to give 0.1 ug/ml a.i. procymidone

B. For DMI sensitivity - Folicur 250 EW

(i) Add 40 iii to 100 ml sterile water (solution 1) Add 1 ml solution 1 to 99 ml PDA to give 1 ug/ml a.i. tebuconazole

(ii) Add 0.1 ml solution 1 to 100 ml PDA to give 0.1 ug/ml a.i. tebuconazole

(iii) Add 1 ml solution 1 to 99 ml sterile water (solution 2) Add 1 ml solution 2 to 99 ml PDA to give 0.01 ug/ml a.i. tebuconazole

C. Control - plain PDA

3. Inoculation of test plates is made by transferring 5 mm plugs from the margin of colonies of the test isolates to 4 sites on each test plate. Agar plugs should be inverted so hyphae are in immediate contact with the test medium.

4. Incubation Plates should be stored at 20-22°C for 3-4 days then radius of growth measured.

5. Interpretation

Isolates sensitive to procvmidone have an EC 50 of approximately 0.1 ug/ml and an MIC >0.5 <1 ng/ml.

Isolates sensitive to tebuconazole have an EC 50 <0.01 and an MIC >0.01 <0.1 jig/ml.

STANDARD Sensitive 3043-1 ISOLATE Resistant 3042-2

Stored in liquid Nitrogen

NOTES The experimental fungicides CGA173506 and tolclofos methyl have EC 50 values of 0.01 and 1.0 >ig/ml. Quintozene has an EC 50 of approximately 3.

ORGANISM Sclerotium rolfsii DISEASE Base rot

CROPS

FUNGICIDES

TEST METHOD SUMMARY

TEST METHOD DETAILS

Several e.g. tomato, capsicum, potato

Organophosphate - tolclofos methyl (Rizolex) DMI - tebuconazole (Folicur)

Fungicides are added to Difco PDA to give a range of concentrations. Radial growth of isolates is measured.

1. Isolates are maintained on PDA

2. Test media A. Rizolex (500g/kg tolclofos methyl)

Add 0.2g Rizolex to 100ml sterile distilled water. Add lml to 99ml PDA - (10iig/ml) Add 0.1ml to 100ml PDA - (lug/ml)

B. Folicur (250g/L tebuconazole) Add 0.4 ml Folicur to 100ml sterile distilled water (Solution A). Add lml to 99ml PDA - (lOug/ml) Add 0.1ml to 100ml PDA - (lug/ml)

Add lml Solution A to 99ml sterile distilled water (Solution B). Add lml Solution B to 99ml PDA (O.lug/ml)

STANDARD ISOLATE

NOTES

C. Control - plain PDA.

3. Inoculation of test plates is made by transfering 3mm diameter plugs from the margin of young colonies to 4 sites on each plate. Agar plugs should be inverted so hyphae are in immediate contact with the test medium.

4. Incubation. Plates should be kept at 20-22 °C for 4 days before the radius of growth is measured.

5. Interpretation. Isolates sensitive to tolclofos methyl have an EC50 of approx 1 and an MIC of <2.5ug/ml. Isolates sensitive to tebuconazole have an EC50 > 0.01 < 0.1 and anMIC>0.1<0.5jag/ml.

3028 in liquid Nitrogen.

Other fungicides screened gave the following results. Quintozene EC50 15ug/ml approx. MIC not det. Benomyl EC50 >25\xg/ml approx >25>xg/ml Procymidone EC50 >25ug/ml approx >25ug/ml CGA173506 EC50 >0.1 < l^g/ml approx >0.K lyig/ml

wrvvj/Ai'tioivi opttuciuinci~u juuguicu JLSJLO.ILJ/-&O.LJ i uvvuciy I U U U C W

CROPS Cucurbits

FUNGICIDES Benzimidazoles - benomyl (Benlate)

DMI - triadimefon (Bayleton)

Organophosphate - pyrazophos (Afugan) Hydroxy pyrimidine - bupirimate (Nimrod)

TEST METHOD A susceptible cucurbit species (pumpkin cv. Qld Blue; cucumber cv. SUMMARY Marketer, Crystal Apple; rockmelon cv. Hales Best) is grown in pots

and sprayed weekly with fungicides. After at least 2 applications leaf discs are taken, plated on water agar and inoculated with the test isolates. These are rated after 7 days.

TEST METHOD 1. Isolates are maintained on cotyledons of pumpkin cv. Qld. Blue or other susceptible species. (Sow seed in washed vermiculite 1 week before required. Flush with lg/L Aquasol after sowing. Remove cotyledons wash in beaker containing 5ml/L 10% chlorine solution and a few drops of detergent. After 1 minute wash under running tap water then sterile water. Blot dry, place on water agar, transfer conidia by Artists brush (No. 2). Incubate 20 °C for 7-10 days use conidia when fresh. Check for contamination e.g. Ampelomyces quisqualis, Fulvia sp. etc.

2. Test plants. Seed of pumpkin cv. Qld Blue; cucumber cv. Marketer, Crystal Apple or rockmelon cv. Hales Best are germinated in flats of vermiculite. After germination and expansion of cotyledons they are transplanted 1 plant per pot to UC mix in a glasshouse. Aquasol applied weekly. When the 1st true leaf is 1/3 - 1/2 expanded apply first fungicide spray.

3. Test fungicides and application

Benzimidazoles - Benlate 500g/kg D.F. or W.P. DMI - Bayleton 50g/kg WP Organophosphate - Afugan 295g/L SC Hydroxypyrimidine - Nimrod 250g/L S.C.

Benlate - Add 0.1 g to 1L - 50ug/ml. - Dilute 1 part in 5 - 10ng/ml.

Bavleton - Add 1.0 g to 1 L - 50ug/ml - Dilute 1 part in 5 - lOjag/ml

Afugan - Add 0.1 ml to 1 L - 30 ug/ml - Dilute 1 part in 3 - lOug/ml

Nimrod - Add 0.2 ml to 1 L - 50 ug/ml - Dilute 1 part in 5 - lOjig/ml

at weekly intervals. Spray to point of run off with complete coverage. Lightly shake foliage to remove marginal drips. Avoid root application.

4. Leaf discs 12-15mm diameter are taken when spray has dried (ca. 4h). Foliage should not be wet during this interval. Discs are taken from the last fully expanded leaf on the vine. Avoid margins where fungicides may be concentrated. Place discs on water agar in Petri dishes or 6 well culture plates. Inoculate 24h after spray application. Keep similarly treated discs together to avoid gas phase interference.

5. Inoculation. Conidia can be transfered by fine Artists brush or, if conidia are sparse, by droplet.

a) Brush technique. Touch brush to conidia then to centre of disc.

b) Droplet. Brush conidia into a few drops of water to which small amount of Tween 80 has been added (1 drop Tween per 200 ml). Place droplets (5-10piL) in centre of disc, wait 2 minutes then remove water with the edge of a piece of blotting paper. Keep plates uncovered in sterile cabinet until area of deposition appears dry.

6. Incubation. Examine after 5-8 days in C.E.C. at 20° C. Conidial chains should be present after 6 days.

7. Rating. Rate after 8 days. Examine under stereo microscope 0 - no mycelium on leaf 1 - mycelium on leaf but no sporulation 2 - sporulation but less than check 3 - sporulation same as check. With some fungicides e.g. hydroxy pyrimidines and tridemorph (Calixin) mycelium occurs but is abnormal.

8. Interpretation. Sensitive isolates should have an EC50 < 10]jig/ml and should not sporulate at that concentration. Sporulation on the higher concentrations discs confirms resistance.

STANDARD It has not been possible to store isolates over long periods of time. ISOLATES Isolates can be maintained on detached cotyledons.

A technique of storing under liquid Nitrogen is being developed.

1. Allow sporulating lesions to dry on lab bench for 4-6h. 2. Collect conidia or cut infected leaf area into thin strips and

place in Nunc vials. 3. Place in liquid Nitrogen.

ORGANISM Thielaviopsis basicola DISEASE Black root rot

CROPS Lettuce

FUNGICIDES Benzimidazoles - (benomyl) DMI - (propiconazole)

TEST METHOD Add fungicides to molten Difco PDA to give concentrations of, 0, 0.1 SUMMARY and lug/ml a.i. Measure radial growth or note germination of spores.

TEST METHOD 1. Isolates are maintained on PDA The carrot disc technique is DETAILS useful to bait Thielaviopsis sp. from plant tissue as well as soil. It

is sometimes difficult to directly isolate Thielaviopsis from plant tissue.

2. Test media

A For benzimidazole sensitivity - use Benlate (500g/kg benomyl). Add 0.2g Benlate to 100ml sterile distilled water. Further dilute by adding 10ml to 90ml sterile distilled water (Solution 1). Add lml solution 1 to 99ml PDA - (lug/ml) Add 0.1ml solution 1 to 100ml PDA - (0.1pg/ml).

B For DMI sensitivity - use Tilt (250g/L propiconazole). Add 0.4ml Tilt to 100ml sterile water. Add 0.1ml to 100ml PDA -(lug/ml).

C Plain PDA

3. Inoculation. Two methods may be used:

a) Spore transfer by fine glass rod b) Agar discs from the margin of colonies. Colonies of T. basicola

are comparatively slow growing and spore transfer is probably the better technique for a rapid test. Make 4 transfers per test plate.

4. Incubation. Store for 24-48h (spore transfer) or 7 days (disc) at 22-25 °G

5. Rating. Examine spores for germination. Measure radius of growth of colonies.

6. Interpretation. Spores: Sensitive isolates show no germination at l>ig/ml, some germination but little further mycelial growth at O.ljag/ml and germination + growth at 0.01}ig/ml.

Discs: Sensitive isolates have an EC50 for propiconazole of O.Olug/ml with an MIC close to 0.lug/ml. EC50 and MIC figures for benomyl are >0.01 <0.1jjLg/ml.

STANDARD ISOLATE

Isolate 2864 is stored in liquid N.

CROPS Pineapple


TEST METHOD Add fungicides to molten Difco PDA to give concentrations of 0, 0.1 SUMMARY and lug/ml a.i. Measure radial growth or note germination of spores.

TEST METHOD 1. Isolates are maintained on PDA

2. Test media

A For benzimidazole sensitivity - use Benlate (500g/kg benomyl). Add 0.2g Benlate to 100ml sterile distilled water. Further dilute by adding 10ml to 90ml sterile distilled water (Solution 1). Add lml solution 1 to 99ml PDA - (lug/ml) Add 0.1ml solution 1 to 100ml PDA - (O.lug/ml).

B For DMI sensitivity - use Tilt (250g/L propiconazole). Add 0.4ml Tilt to 100ml sterile water. Add 0.1ml to 100ml PDA -(lug/ml).

C Plain PDA

3. Inoculation. Two methods may be used:

a) Spore transfer by fine glass rod b) Agar discs from the margin of colonies. Make 4 transfers per

test plate.

4. Incubation. Store for 24-48h (spore transfer) or 3-4 days (disc) at 22-25 °C.

5. Rating. Examine spores for germination. Measure radius of growth of colonies.

6. Interpretation. Spores: Sensitive isolates show no germination at lug/ml, some germination but little further mycelial growth at O.ljig/ml and germination + growth at O.Olug/ml.

Discs: Sensitive isolates have an EC50 for propiconazole of O.Olug/ml with an MIC close to O.lug/ml. EC50 and MIC figures for benomyl are >0.01 <O.lug/ml.

STANDARD ISOLATE

Isolate 2959 is stored in liquid N.

CROPS Beans

FUNGICIDES Anilide - oxycarboxin (Plantvax) DMI - bitertanol (Baycor)

TEST METHOD Fungicides added to molten water agar to give concentrations of 0, 5 SUMMARY and lOyxg/ml for bitertanol and 0,10,25ng/ml for oxycarboxin.

Uredospores are transfered to the solidified agar and spore germination counts made after 24h.

TEST METHOD 1. Isolates. Tests are conducted on field collected material. If bulk collections of uredospores are made with a spore collector, there may be considerable variability. Transfers to all test plates can be made from individual pustules to limit variability.

Test plates A. Anilide sensitivity - Plantvax (750g/kg oxycarboxin)

Add 0.34g to 100ml sterile water Add 1ml to 99ml water agar - (25ug/ml) Add 0.4ml to 100ml water agar - (lOpg/ml)

B. DMI sensitivity - Baycor (250g/L bitertanol) Add 0.84ml to 100ml sterile water Add 0.4ml to 100ml water agar - (lO^g/ml) Add 0.2ml to 100ml water agar - (5jag/ml)

C. Water agar.

3. Inoculation. Using a fine tipped glass rod, transfer uredospores to marked positions on test plates.

4. Incubation. - 24h on laboratory bench 22-24 °C.

5. Rating. Examine 25 spores from each transfer under low power objective of compound microscope. Record no. which germinated.

6. Interpretation. Sensitive isolates show little or no germination on bitertanol 5y.g/ml and oxycarboxin 10)ig/ml. Germination of sensitive isolates is prevented by bitertanol lOug/ml and oxycarboxin 25u£/ml.

REFERENCE ISOLATES

NOTES Since the fungicides have been in general use for several years, it is suspected our tests have not been with genuine wild isolates. There has been much variability in collections of U. appendiculatus.

APPENDIX II

Fungicide resistance — threats and

strategies «

FUNGICIDE RESISTANCE threats and strategies

PL9201004

Introduction

R.G. O'Brien, Plant Pathology Branch

F46/APR 92 Agdex 201/625

Just over 20 years ago the first systemic fungicides were released to Australian horticulture. Products such as Benlate (benomyl) and Plantvax (oxycarboxin) offered new standards of control for diseases such as grey mould, powdery mildews and rusts. The mode of action of these systemic fungicides differed from that of older protectant fungicides. The new fungicides offered selective toxic activity, effective against certain life processes in the fungi. At normal rates of application they had no deleterious effect on plant tissue, and could be absorbed into the plant to exert a fungitoxic effect. This type of activity has been called 'systemic', 'eradicant' or 'kick-back' since it stops the progress of established infections.

Development of resistance

The selective toxic activity of these fungicides was not without drawbacks because all communities of a species have genetic diversity. Within the fungal communities there were individuals which could cope with disruption of the metabolic pathways targeted by the fungicides. With continued use of the fungicide their proportions increased and disease control decreased. The population had developed fungicide resistance.

Today, many of our most effective fungicides are at risk. Within the current framework of reduced application of pesticides we can ill-afford to lose those products which have selective toxicity to fungi, are effective at low dosages and have eradicant activity. For some diseases of horticultural crops, resistance has already occurred and appears irreversible in some instances. Resistance to benzimidazole fungicides in cucurbit powdery mildew and grey mould appears enduring once it is established.

Although it may not always be possible to

prevent the ultimate occurrence of a resistance problem, it should be possible to delay it by understanding the problem and implementing some basic strategies.

At risk fungicides The fungicides to which resistance may occur are from several chemical groups (see table 1). Within each group, the mode of action of all the member fungicides is usually very similar. Hence, resistance to one member usually signifies resistance to the others as well. This is called 'cross resistance'. If resistance has developed, changing fungicides within a group e.g. replacing Rovral with Ronilan will not improve the standard of disease control.

Selection pressure

In most cases, the greater the selection pressure placed on the fungal population, the more rapidly it will become resistant. If the fungicide is used at high rates and short intervals over a long period of time, selection pressure is high.

Fungal factors Sometimes, especially with fungi which become resistant to the dicarboximide and DMI fungicides, the resistant strains are not as 'fit' as the sensitive strains. If the fungicide is not used for a period, the population becomes more sensitive and good control can, once again, be obtained.

Fungi with short lifecycles which produce large numbers of air-borne spores e.g. rusts, powdery and downy mildews, have the ability to rapidly change towards resistance. Conversely, fungi with long lifecycles or less prodigious spore production e.g. soil-borne fungi, take longer for resistance to affect disease control.

Large fungal populations (high disease

Queensland Department of Primary industries • GPO Box 46 Brisbane Qld 4001 • ISSN 0155-305^

2

situations) allow a greater opportunity for the selection of resistance.

Crop management practices Cropping practices which help to reduce disease pressure play an important part in any strategy. Such things as early destruction of crop residues, crop rotation, use of resistant cultivars and wide separation of successive plantings should always be used in conjunction with fungicide application.

Strategies to prevent resistance The most effective strategy to prevent fungicide resistance is to keep the product on the shelf. The least effective is to apply it intensively. In between these two there are options which gain maximum benefit from the 'at risk' fungicides while minimising the resistance risk. Although the best strategy for a particular crop/disease/fungicide combination should be considered individually, some of the strategies which can be employed are:

Mixtures

Two or sometimes three fungicides from different groups are used in combination. In most cases a protectant e.g. mancozeb or copper is combined with the 'at risk' product. The protectant acts on the plant surface to prevent the germination of most fungus spores thus reducing the number of challenges for the systemic product.

Alternations of chemicals

The 'at risk' product is used in rotation with one or more different fungicides.

This strategy relies on the fact that it is much more difficult for the fungus to develop dual resistance than resistance to just one product.

Limiting the number of sprays

Reducing the total number of applications per season to, for example two to four, will reduce selection pressure. For practical reasons these applications will often be made at critical times when disease pressure may be high. Under these conditions the 'at risk' fungicide should be used in a mixture.

Limiting the time of application

If an 'at risk' fungicide is essential for a postharvest treatment, it may be necessary to withdraw its pre-harvest use.

Examples of strategies which have been promoted in Queensland crops are:

Cucurbit powdery mildew

Use the protectant fungicide Morestan until fruit set then alternate or mix Morestan with a registered systemic product. It is best to use more than one systemic (from different groups) during the season.

Grey mould in tomatoes

Use the protectant fungicide chlorothalonil (Bravo or Rover) until flowering then alternate with a registered dicarboximide fungicide. If disease pressure remains high use the fungicides in a mixture.

Onion downy mildew

Use a protectant (Antracol or mancozeb) until bulbing. If the disease is present in the district and two mornings of heavy dew occur, apply two sprays of either Galben M, Fruvit or Ridomil MZ 720, one week apart then return to a pptectant schedule.

Summary The most difficult aspect of establishing a

resistance strategy is for growers to recognise there is a potential problem and take action before resistance is apparent. It is a great temptation to always use the most effective product but it is one which has to be overcome to prolong the effective life of modern fungicides.

Table 1 Fungicide groups, target diseases in horticultural crops and known cases of fungicide resistan

Fungicide groups Target diseases

1 Benzimidazoles: Bavistin, Benlate, Delsene, Spin, Topsin M

2 Demethylation Inhibitors (DMI): Bayfidan, Bayleton, Fungaflor, Nustar, Rubigan, Saprol, Sportak, Tilt, Topas

3 Dicarboximides: Ronilan, Rovral, Sumisclex

4 Phenylamides: Fongarid, Fruvit, Galben, Recoil, Ridomil

5 Hydroxy pyrimidines: Milcurb, Nimrod

6 Organophosphates: Afugan, Curamil

7 Oxathiins: Plantvax, Vitavax

8 Phosphonates: Aliette, Fos-ject, Phosphorous acid

Powdery mildews, rusts, brown rot, Botrytis grey mould, various leaf spots (leaf spo

moulds) fruit), Sp cucurbit

Powdery mildews, rusts, brown rot, Sphaero leaf spots cucurbit

Brown rot, grey mould, Sclerotinia rots, apple scab

Downy mildews, damping off, late blight, root rots

Powdery mildews

Powdery mildews

Rusts, smuts

Downy mildews, damping off, root rots

Alternar Botrytis

Bremia Peronosp Peronos

Sphaero cucurbit

Sphaero cucurbit

APPENDIX III

Fighting resistance to systemic

fungicides

CHEMICALS FUNGICIDE RESISTANCE By ROB O'BRIEN Just over 20 years ago the first systemic fungicides were released to Australian horticulture. Products such as Benlate (benomyl) and Plantvax (oxycarboxin) offered new standards of control for diseases such as grey mould, powdery mildews and rusts.

The mode of action of these systemic fungicides differed from that of older protectant fungicides. They had selective toxic activity effective against certain life processes in the fungi. At normal rates of application they had no deleterious effect on plant tissue. They could therefore be absorbed in to the plant to exert a fungitoxic effect.

This type of activity has been called 'systemic', 'cradicant' or 'kick-back' since it stops the progress of established infections.

The selective toxic activity of these fungicides was not without drawbacks since within all commu nities of a species there is genetic diversity. Within the fungi there were some which could cope with disruption of the metabolic pathways at which the fungicides aimed.

With continued use of the fungicide they increased and disease control decreased. The population had developed fungicide resistance.

Today, many of our most effective fungicides are at risk. Within the

Fighting resistanc systemic fungicid current framework of reduced application of pesticides we can ill-afford to lose those products which have selective toxicity -to fungi, are effective at low dosages, and have eradicant activity.

For some diseases of horticultural crops, resistance has already occurred and appears irreversible in some instances. Resistance to Benlate (and other benzimidazole fungicides) in cucurbit powdery mildew and grey mould appears enduring once it is established in populations of the causal organisms.

Although it may not always be possible to prevent the ultimate occurrence of a resistance problem, it should be possible to delay it by understanding the problem and implementing some basic strategies.

The fungicides to which resistance may occur are from several chemical groups (Table 1). Within each group, the mode of action of all the member fungicides is usually

similar. Hence, resistance to one member also usually signifies resistance to the others. This is called 'cross resistance'. If resistance has developed, changing fungicides within a group, for example replacing Rovral with Ronilan, will not improve the disease control level.

In most cases, the greater the selection pressure placed on the fungal population, the more rapidly will it become resistant. If the fungicide is used at high rates and short intervals over a long period of time, selection pressure is high.

Sometimes, especially with fungi which become resistant to the dicarboximide and DM I fungicides, the resistant strains are not as 'fit' as the sensitive strains. If the fungicide is not used for a while, the population becomes more sensitive and control can be regained.

Fungi with short life cycles which produce large numbers of air-borne spores such as rusts, powdery and

downy milde rapidly chan

Conversely cycles or l production, s take longer disease contr

Large fung greater oppo of resistance

Cropping reduce disea important p Early destru crop rotatio cultivar and successive pl be used with

The most prevent fung keep the pro least effectiv sively. In be are options benefit from while minimi

LEFT: Cucurbit mildew: Testing the tivity of Pseudop spora cubensis to laxyl. Infection i vented by concent of Img/Iitre or hi RIGHT: Pea po mildew: Testing the tivity of Oidium triadimefon. Infec prevented by con tions of greater Sing/.litre. The in

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Fungicide Groups Target Diseases

1. Benzlmidazoles: Bavistin, Benlale, Detsene. Spin, Topsin M Powdery mildews, rusts, brown r mould various leaf spots

2. Demethylatlon Inhibitors (DMI): Bayfidan, Bayleton. FungaMor, Nustar, Rubigan, Saprol. Sportak, Tilt, Topas

Powdery mildews, rusts, brown rot

3. Dlcarboxlmldes: Ronllan, Rovral, Sumisclex Brown rot, grey mould, ScleroUn apple scab

4. Phonylamldes: Fongarld, Fruvit, Galben, Recoil, Ridomil Downy mildews, damping off, lat root rots

5. Hydroxy pyrlmldlnes: Mtlcurb, Nimrod Powdery mildews

6. Organophosphates: Afugan, Curamil Powdery mildews

7. Oxalhllns: Plantvax, Vitavax Rusts, Smuts

8. Phosphonates: Aliette, Fos-ject. Phosphorous acid Downy mildews, damping off. roo

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APPENDIX IV

Become a resistance fighter

tarnations - does the answer lie i the solution?

•avid Huett ropical Fruit Research Station, Alstonville .

L carnation nutrition experiment is being Dnducted on a commercial property near.-Jstonville in order ta evaluate nutrient Dlutions for Sim carnation production. Many ommercial growers believe that carnations rown in northern New South Wales and outhern Queensland are sensitive to nitrogen in tie ammonium form (NH4

+) and that nutrition an affect stem strength.

Irree solutions

^ standard carnation nutrient solution was idopted from the widely used European ormulation of Sonneveld and Straver, principally by removing the ammonium nitrogen. This formulation is being compared with one :ontaining 10% nitrogen as ammonium and a ligh calcium (Ca) solution. The response by :our popular Sim varieties is being evaluated.

The experiment commenced in December 1990 and both the ammonium and high calcium [ormulations have produced about 20% more first grade flowers than the standard formulation: There has been no effect from the formulation on'stem strength or vase life.

Typical response

The response to the ammonium nitrogen could just be a typical preference for some of this nitrogen form to be present in the nutrient solution. This is, in fact, the case for many species. The pH in the rockwool slabs with the ammonium nitrogen is about 6.5 compared with 7.3 for the standard solution.

The response to the high calcium solution is difficult to explain at this stage. The calcium concentration in the plant was higher and the potassium slightly lower.

The experiment will continue for eighteen months and it is still too early to draw reliable, conclusions.

BecomeTa resistance^ghter?!

Cynthia Carson, Rob O'Brien -and Pat Collins^ : • QDPI, Brisbane!

The story is familiar - your spray program is the' same as last year (and the year before) yet your pest infestation is worse than ever! The old sprays are not effectively combatting .once easily controlled • fungal diseases. Your pests and pathogens may have become resistant to the chemicals you have been using and you may need to equip yourself to become a resistance fighter. .

Eradicant fungicides attack their targets by interfering with:. .one or more essential biochemical pathways in the pathogen. Most '• conventional insecticides are neuro-toxins acting at one or more sites in the nerve pathways of insects or mites. In both cases death follows.

Resistance problems can arise from alternative biochemical pathways or the enhanced activity of breakdown emzymes in a few naturally occurring rare individuals. These resistant individuals survive and reproduce in the presence of chemicals that kill normal (susceptible) individuals. Selection occurs on the population as a whole. Individuals with the ability to survive and reproduce are favoured by continued,spraying, as illustrated in Figure 1. Resistance is not noticed at first because only a few individuals survive, but as the proportion of resistant organisms in the population increases, control failures begin to occur.

Resistance is most likely to occur where the organism reproduces prolifically and has a short life cycle. Twospotted mite and cabbage moth are two such examples from the insect world. Similarly Botrytis and powdery mildew are commonly encountered plant pathogens with resistant populations.

A number of strategies can be used to combat resistance problems. The aim should be to:

e reduce the number Of spray applications by using biological or cultural methods to lower the overall incidence and severity of pest infestation or disease, and

© ensure that chemical sprays are applied in the most effective way possible.

(a) before spraying (b) after the first spraying

(c) after the next generation w

Key: ©- susceptible

note increased resistant individuals after second spray

= resistant

Figure L Diagrammatic representation of a resistanrpopulation developing on a rose.

Reducing pests and diseases

This problem may be tackled by the use of appropriate cultural practices, plants naturally resistant to attack or biological control.

Practices such as good hygiene from propagation through to harvest (including the destruction of crop residues), isolating infected plants and environmental modifications (such as good ventilation, rainout covers and trickle irrigation) help to control disease.

The use of insect screens and the control of weeds adjacent to the planting can reduce the incidence of insect pests.

Biological control

Biological control agents have become an increasingly popular alternative for pest and

disease control. Disease-suppressive composts have been used to control root disease in the nursery industry. In Queensland, Pseudomonas cepacia has been successfully used to control Phytophihora cinnamomi in container grown proteas. A strain of Agrobacterium radiobacter is commonly used to control crown gall in peaches and roses.

The. bacterium Bacillus thuringiensis (Thuricide® or Dipel®) is registered for the control of certain caterpillars in ornamental crops and the predatory mite Phytoseiulus persimilis is widely used for twospotted mite control.

Pests and diseases often have naturally occurring antagonists whose development is favoured by an absence of chemical sprays. Australian native predatory mites are sometimes

Ornamentals Update 7 Volume 6 Number 3

encountered controlling twospotted mite on unsprayed plants. Intensive spray programs discourage any natural biocontrol agents that may be present. Reducing the amount of spraying may enhance these agents. ;

Getting the most from your spray programme

Poor application methods favour the survival of pests and pathogens necessitating further spraying. Sprays should be considered the second line of defence against pests and diseases when cultural practices and biological control are not workable alternatives. There are several points to consider in order to get the best from your spray programme:

• Application. Make sure that you are getting good coverage from your existing equipment and application methods. Check for nozzle wear, for even distribution of droplets within the plant canopy and for suitable depth of wetting front with soil drenches.

Ensure that you are not reducing the efficacy of sprays by using inappropriate tank mixes, (for example a foliar fertiliser, an insecticide and a fungicide) or excessively alkaline water. When a spray manufacturer recommends the use of a wetting agent, use the rate and application method recommended on the label.

• Monitoring. Monitor your plants for early signs of problems and establish damage levels above which you commence treatment. Do not wait until the problem has become severe. A knowledge of the biology of the pest or disease can allow you to treat the problem before it becomes difficult to control. For insects, a well timed spray at an early stage of the life cycle may kill pests even in resistant populations.

• Restricted use of eradicant fungicides and conventional insecticides. Protectant fungicides (such as mancozeb and the copper and zinc-based fungicides) and insecticides which have a physical action

on the cuticle of the insect (such as insecticidal soap or white oil) have continued to remain effective over many years. These fungicides and insecticides, along with biological control agents, have a low risk of resistance problems developing and should be used in ] preference to other chemicals.

Eradicant fungicides; arid conventional insecticides have been demonstrated to have a high risk of selecting for resistance after repeated use. This risk can be reduced by alternating your spray programme between high and low risk chemical groupings • or by combining a high risk chemical with a km risk one in the tank mix.. Only Vdo this with compatible chemicals. Tables 1 and 2 list high and low risk fungicide and insecticide groupings respectively.

Alternating between chemical groupings where eradicant fungicides and conventional insecticides are used. Within families of chemicals, resistance to one chemical will often result in 'cross-resistance' to other chemically related compounds. Therefore, if Botrytis is resistant to Benlate® (a benzimidazole) it may rapidly develop a similar resistance to Topsin® or Spin® from the same grouping. To counteract this, it is desirable to alternate between different chemical groupings. Therefore if Botrytis has become resistant to Benlate® use Rovral® from the dicarboximide group rather than another benzimidazole.

Table 1 lists the chemical groupings of fungicides commonly used in ornamental plant production and their relative risk factors. Table 2 does the same for insecticides. These tables do not list the pest or disease the chemicals are registered to control. Check this with the latest version of the QDPI's 'Chemicals for the Protection of Ornamental Plants and Turf.

Do not use chemicals for purposes other than that recommended on the label.

Ornamentals Update Volume 6 Number 3

Chemical grouping

Era roples (tradenames)

High risk

benzimidazoles

carbamates

Benlate, Topsin M, Tecto 90, Spin, Bavistin, Delsene

Previcur

demethylation Baycor, Bayleton, Rubigan, inhibitors (DMI's) Saprol, Triforine, Octave,

Sportak

hydroxypyrimidines Nimrod

organophosphates Afugan

oxathiins Plantvax

phenylamides Fongarid, Ridomil

thiazoles Terrazole

• Low risk -

phosphonates ' ••• Aliette, Fos-ject, phosphorous acid

chlorinated benzenes

Terrachlor, Purasoil

copper compounds Kocide, copper oxychloride, Bordeaux

dithiocarbamates Mancozeb, Dithane, Zineb, Ziram, Thiram

phthalonitrilcs Bravo

quinoxalines Mores tan

others wettable sulphur

Table 1. A list of chemical groupings of fungicides used in ornamental plants categorised according to the risk of resistance developing in target pathogens.

Chemical grouping

Examples (tradenames)

- Very high risk -

pyrethrins/synthetic pyrethroids

Pyrethrum, Ambush, Permasect, Mavrik, Baythroid

- High risk -

carbamates Carbamult, Carbaryl, Bugmaster, Sevin, Carbene, Septene,Pirimor, Lannate

organochlorines Endosulfan, Thiodan, Endopest, Kelthane

organophosphates Folimat, Rogor, . Pcrfekthion, Saboteur, Go-Mite, Roxion, Kilval, Disyston, Diazinon, Orthene, Ekatin, Metasystox, Malathon, Lebaycid, Supracide, Nitofol, Phosdrin, Lorsban .:

organosulphurs Ted ion, Omite

organotins Torque

other - for example hexylhiazox Calibre (cross-resistance

t i ' with Torque)

- Low risk -

Bacillus thuringiensis Thuricide, Dipel

petroleum oils white oil, Albarol

soaps insecticidal soap spray

others derris dust, sulphur

Table 2. A list of chemical groupings of insecticides and miticides used in ornamental plants categorised according to the risk of resistance developing in target pests.

The authors: Cynthia Carson is an Extension Officer (Ornamentals) based at Redlands, near Brisbane. Rob O'Brien is a Senior Plant Pathologist and Pat Collins is a Senior Entomologist - both are based at the QDPI's facility at Indooroopillyin Brisbane.

Ornamentals Update Volume 6 Number 3

APPENDIX V

Control of onion downy mildew in

the presence of phenylamide-

resistant strains of

Peronospora destructor

(Berk.) Caspary

Australian Journal of Experimental Agriculture, 1992, 32, 669-74 669

Control of onion downy mildew in the presence of phenylamide-resistant strains of Peronospora destructor (Berk.) Caspary R. G. O'Brien

Queensland Department of Primary Industries, Meiers Road, Indooroopilly, Qld 4068, Australia.

Summary. Two isolates of Peronospora destructor differed in their sensitivities to phenylamide fungicides. An isolate (3014) from the Lockyer Valley did not respond to increasing concentrations of metalaxyl, benalaxyl and oxadixyl between 0.1 and 200 mg a.i./L. An isolate (2967) from Bundaberg was sensitive to these fungicides, with EC50 values (concentrations which reduce disease severity by 50%) of 1.6, 6.0 and 12.1 mg a.i./L, respectively. Isolates 3014 and 2967 were both sensitive to dimethomorph, with EC50 values of 4.3 and 1.4 mg a.i./L, respectively.

Field trials compared the efficacy of fungicides for the control of onion downy mildew in the Lockyer

Introduction Downy mildew caused by Peronospora destructor

(Berk.) Caspary was first recorded in Queensland in 1896 (Simmonds 1966) and can be a damaging disease of onions when weather conditions are cool and humid with long dew periods. Affected crops are harvested prematurely, resulting in lower yields due mainly to a smaller average bulb size. There is also a reduction in keeping quality since the neck scales of immature onions are fleshy and prone to soft rots (Walker 1952).

The centre of onion production in Queensland is the Lockyer Valley (100 km west of Brisbane), where 30000 t are produced each winter from a crop area of about 900 ha. Since 1987, there has been an upsurge in the severity of downy mildew in the area. Efforts to control the disease with fungicides have been unsatisfactory, but this could be due to higher than average rainfall affecting the timing of fungicide applications or their residual activity. Protectant fungicides including copper compounds, chlorothalonil, propineb and mancozeb are registered, and the systemic phenylamide fungicide metalaxyl has been registered for use in onions since 1980. Initially (1980-82) this fungicide was used as Ridomil 25 WP (250 g metalaxyl/kg), then (1982-88) as Ridomil MZ (40 g metalaxyl + 640 g mancozeb/kg), and finally as Ridomil MZ 720 (80 g metalaxyl + 640 g mancozeb/kg).

Valley in 1989 and 1990. In both trials, treatments containing mancozeb gave the best control, resulting in yield increases of 60% in 1990. There was no difference from the check treatment in either disease severity or yield when metalaxyl was applied, suggesting that phenylamide-resistant strains were prevalent. Other fungicide treatments which did not significantly reduce the severity of downy mildew in the 1990 trial included copper hydroxide, chlorothalonil, fosetyl-Al, and propamocarb. The experimental compound dimethomorph, although less effective than mancozeb, significantly reduced disease severity and increased yield by 24%.

A second phenylamide fungicide, benalaxyl, was released as Galben M (80 g benalaxyl + 650 g mancozeb/kg) in 1989.

Reports of the efficacy of the phenylamide fungicides on onion downy mildew show consistently that they are highly effective (Teviotdale et al. 1980; Wilson 1980; Mir et al. 1987). There is also support for the use of copper (Timchenko 1978) and mancozeb (Smith et al. 1985). Chlorothalonil appears less suitable, sij|ce Smith et al. (1985) showed that it had a shorter effective residual life than mancozeb, while Stoffella and Sonoda (1982) reported that chlorothalonil sprays reduced yields. Other fungicides with demonstrated systemic activity against Phycomycete fungi include dimethomorph (Marks and Smith 1990), fosetyl-Al (Ryan 1977), phosphorous acid (Pegg et al. 1985), and propamocarb (Samoucha and Cohen 1990). If effective against P. destructor, they would be useful alternatives to the phenylamide fungicides.

Poor field control of foliar pathogens with protectant fungicides can often be explained by high inoculum levels before the start of the program, inadequate application techniques, or incorrect timing of applications. With systemic fungicides, another consideration is the presence of fungicide-resistant strains. Phenylamide-resistant strains are known to occur in some downy mildew fungi (Reuveni et al. 1980; Crute 1987), and similar strains

670 R. G. O'Brien

could develop in P. destructor. The studies reported here examine the sensitivities of isolates of P. destructor to 3 phenylamide fungicides and to dimethomorph, to determine whether fungicide- resistant strains are present. In field experiments, several protectant and systemic fungicides were compared as controls for onion downy mildew.

Materials and methods Fungicide sensitivity

Two isolates of P. destructor were collected, one (3014) from an onion crop in the Lockyer Valley and the other (2967) from unsprayed shallots in a home garden at Bundaberg, 400 km north of Brisbane. These were maintained and inoculum (2 X lO^mL) was produced by the technique of Abd-Elrazik and Lorbeer (1980), except that plants were grown from seed rather than bulbs. The cultivar Early Lockyer Brown was used for maintenance of isolates and in all experimentts. Long-term cryogenic storage of the isolates has been achieved by the technique described for Peronospora tabacina (P. hyoscyami) by Bromfield and Schmitt (1967). The experiment compared the sensitivity of the 2 isolates to 4 fungicides by inoculating plants previously sprayed with the fungicides, using a range of concentrations. The test plants were 4-week-old onion seedlings (40 seedlings/15-cm pot) grown in a peat-vermiculite medium. Each pot served as a plot and there were 4 replications.

The fungicide treatments metalaxyl (Ridomil, 250 g a.i./kg), benalaxyl (Galben, 250 g a.i./kg), oxadixyl (Recoil, 250 g a.i./kg), and dimethomorph (CME15103, 500 g a.i./kg) were applied to run-off as sprays at 0, 0.1, 0.5, 1, 5, 10, 50, 100 and 200 mg a.i./L. During spray application, pots were held at an angle to minimise fungicides draining to the root zone. Plants were inoculated 24 h later with spore suspensions of the 2 isolates of P. destructor and incubated overnight in moist plastic bags at 15-18°C. Bags were then removed and pots returned to an evaporatively cooled glasshouse for 12 days. Sporulation was then induced by again enclosing the pots in moist plastic bags overnight in a controlled environment cabinet at 16°C.

Ten plants were taken at random from each pot and closely examined for sporulation, which was recorded as present or absent. The probit method (Finney 1971) was used to derive EC50 values (fungicide concentrations which reduce disease severity by 50%).

Field experiments Experiments were conducted in 1989 and 1990 to

compare the efficacies of fungicides in controlling onion downy mildew. They were located at the Queensland Department of Primary Industries' Gatton Research Station in the Lockyer Valley.

Early Lockyer Brown was sown on 18 April 1989 and 30 April 1990. Standard district cultural practices were

followed during crop growth and included irrigation by overhead sprinklers as required. Both trials were established as randomised blocks with 4 replications, but heavy rain soon after sowing resulted in the loss of 1 replication in 1989. Plots were 1 m wide (4 rows) by 7 m in 1989, and 1 m by 10 m in 1990. The 2 central rows were used for data collection.

Fungicides were applied by a gas-pressurised rig with a hollow cone nozzle positioned over each row in a 1-m boom. The spray volume was 500 L/ha in 1989 and 1000 L/ha in 1990. A spray adjuvant, Nu-Film 17 (Miller Chemical and Fertilizer Corporation, U.S.A.), was added to all treatments at the rate of 0.4 mL/L. The first application was made 6 weeks after sowing. Subsequent applications were made on a 7-10-day schedule, although this was extended to 14 days on 3 occasions in 1989 and twice in 1990 due to wet weather. There were 10 spray applications in 1989 and 11 in 1990.

Fungicide treatments included in each trial are listed (see Tables 2 and 3). In each trial we included a treatment of strategic phenylamide spraying designed to prevent or delay the development of resistant strains. The basis of the strategy was reduction of the number of phenylamide applications by limiting the use of these fungicides to the latter part of the season. The practical spraying criteron based on length of dew period was adapted from the disease-forecasting system, 'Downcast', developed by Jesperson and Sutton (1987). Downcast was evaluated in the Lockyer Valley in 1989, and it successfully predicted sporulation infection periods (S. M. FitzGerald and R. G. O'Brien unpublished data). High relative humidity and rapid, heavy dew formation during the pre-dawn hours were the most critical predictive factors. For the strategic spraying treatment the regular spray was mancozeb; however, if downy mildew was present in the district, if the crop had advanced beyond the bulbing stage (about 10 vsfeeks from sowing), and there was prolonged heavy dew formation on 2 or more days, then 2 applications of metalaxyl + mancozeb were made 1 week apart. In the 1989 experiment, strategic phenylamide sprays were used 12 and 13 weeks after sowing, while in 1990 they were applied 14 and 15 weeks after sowing. The post-planting drench of phosphorous acid in 1989 was made 2 days after sowing as a broadcast application.

Disease severity was assessed by rating 15 randomly selected plants from each plot. Observations were restricted to the youngest 7 leaves of each plant, to avoid confusion between natural senescence of old leaves and leaf death due to downy mildew. In 1989 a rating scale of 0-5 was used (0, no downy mildew; 1,1-5% leaf area affected; 2, 6-10%; 3, 11-25%; 4, 26-50%; 5,>50%). In the following year, category 5 was changed to 51-75% and category 6 (>75%) was added. Plants were assessed at 20 weeks of age in 1989 and 18 and 21 weeks in 1990.

Phenylamide resistance in Peronospora destructor 671

Table 1. Sensitivity (EC50) of isolates 2967 and 3014 to four fungicides

EC50 is the concentration at which 50% test plants were infected relative to the control

Fungicide E C 5 0 5% fudicial (mg a.i./L) limitsA

Isolate 2967 Metalaxyl 1.6 0.65-3.6 Benalaxyl 6.0 4.4-8.2 Oxadixyl 12.1 6.8-22.6 Dimethomorph 1.4

Isolate 3014 0.6-2.8

Metalaxyl >200 — Benalaxyl >200 — Oxadixyl >200 — Dimethomorph 4.3 3.1-6.1

A 5% fiducial limits were determined by probit analysis.

In 1989, 25 bulbs were harvested from the centre of each plot and weighed, to indicate the effect of disease severity on mean bulb weight. In the 1990 experiment, plot yields were obtained from 2 rows by 8 m. Harvested onions were mechanically sorted into the commercial grades No. 1 large (>70 mm diameter), No. 1 (> 40-70 mm), and small (20-40 mm).

Results Fungicide sensitivity

Isolate 2967 was sensitive to the phenylamide fungicides metalaxyl, benalaxyl and oxadixyl, with EC50 values of 1.6, 6.0 and 12.1 mg a.i./L, respectively. The differences between the fungicides were significant (P>0.05). In comparison, isolate 3014 was resistant to these fungicides with EC50 values >200 mg a.i./L, the highest concentration tested (Table 1).

The expression of symptoms by both isolates was inhibited by comparatively low concentrations of dimethomorph, although its effect on isolate 2967 (EC50, 1.4 mg a.i./L) was greater than on isolate 3014 (EC50,4.3 mg a.i./L).

Field experiments In the 1989 experiment, all fungicide treatments,

except phosphorous acid applied as a drench and weekly spray, reduced the severity of onion downy mildew compared with the unsprayed control (Table 2). The mean bulb weight in plots sprayed regularly with mancozeb or metalaxyl + mancozeb was greater than in other plots.

In the 1990 experiment, differences in disease severity between treatments were apparent at 18 weeks but were greater at the second disease assessment 3 weeks later (Table 3). Downy mildew was well controlled in the 4 treatments where mancozeb was applied as a component of the spray. The combination of dimethomorph and mancozeb was particularly effective and showed less disease at the final assessment than any other treatment. There were no significant differences in disease severity between mancozeb, mancozeb in combination with metalaxyl, and the mancozeb-metalaxyl + mancozeb strategy. Several fungicides (i.e. copper hydroxide, chlorothalonil, metalaxyl, fosetyl-Al, 2022D, and propamocarb) were not effective and showed no reduction in disease severity compared with the control. The activity of dimethomorph was intermediate, being significantly higher than this group but lower than mancozeb.

Onion yields closely followed the observed differences in downy mildew severity. Highest yields were recorded in the 4 mancozeb treatments, while the yields from the ineffective fungicidal treatments were no different from the control. The yield in the

Table 2. Field experiment, 1989. Effect of regular applications (n = 10) of six fungicidal spray treatments on the severity of onion downy mildew and mean weight of onion bulbs

Disease severity: 0, no disease; 1, 1-5% leaf area affected; 2,6-10%; 3,11-25%; 4,26-50%; 5, >50%

Fungicide treatment Disease severity Mean bulb weight (g)

Control 4.60 73.6 Phosphorous acid (Foli-R-Fos 200) post-planting

drench (1 g a.i./m2) and weekly spray (1 g a.i./L) 4.41 68.7 Dimethomorph (CME 15103) weekly spray (0.25 g a.i./L) 2.35 87.1 Mancozeb (Dithane M45) weekly spray (1.6 g a.i./L) 2.31 108.0 Metalaxyl + mancozeb (Ridomil MZ 720) weekly spray (0.2+1.6ga.i./L) 1.45 117.3

Strategy* 1.78 104.0 l.s.d.(/> = 0.05) 1.31 34.3

A Mancozeb (1.6 g a.i./L as regular spray); metalaxyl (0.2 g a.i./L) + mancozeb (1.6 g a.i./L) as strategic spray.

672 R. G. O'Brien

Table 3. Field experiment, 1990. Effect of regular applications (n = 11) of 12 fungicidal spray treatments on the severity of onion downy mildew 18 and 21 weeks after sowing and the yield and quality (size) of harvested onions

Disease severity: 0, no disease; 1, 1-5% leaf area affected; 2, 6-10%; 3, 11-25%; 4, 26-50%; 5, 51-75%; 6, >75%

Fungicide treatment (g a.i./L) Disease severity Plot yields (kg) 18 weeks 21 weeks No. 1 large No. 1 Small Total yield

(>70 mm diam.) (>40-70 mm diam.) (20-40 mm diam.)

Control . 2.85 5.67 0.95 15.48 12.69 29.12 Copper hydroxide (Kocide), 1.54 g 3.37 5.60 0.45 10.15 13.65 24.24 Mancozeb (Dithane M45), 1.6 g 1.17 2.15 7.73 28.74 11.17 47.64 Chlorothalonil (Bravo 500), 1.5g 2.72 5.38 0.50 13.51 12.87 26.76 Metalaxyl (Ridomil 25), 0.2 g 2.55 5.22 0.65 14.86 14.10 29.59 Propamocarb (Previcur), 3 g 2.50 5.42 0.76 14.41 13.23 28.21 Fosetyl-Al (Aliette), 2.22 g 2.62 5.13 0.68 13.66 13.69 28.04 Dimethomorph (CME 15103), 0.27 g 2.27 3.56 2.17 21.56 12.37 36.14 Metalaxyl + mancozeb (Ridomil MZ 720), 0.2+1.6 g 1.40 2.32 6.67 27.16 11.71 45.55

Experimental product 2022D, 4.5 gA 2.42 5.13 0.58 14.02 13.26 27.72 Dimethomorph, 0.27 g + mancozeb, 1.8 g 1.22 0.91 8.45 28.90 9.43 46.81 Strategy8

l.s.d. (P = 0.05)

1.55 2.36 6.49 28.19 9.84 44.52 Strategy8

l.s.d. (P = 0.05) 0.78 0.48 3.02 6.26 2.84 7.29 (P = 0.01) 1.04 0.64 3.97 8.23 n.s. 9.59

A Experimental product is a mixture of fosetyl-Al and Bordeaux. B Mancozeb (1.6 g a.i./L) as regular spray; metalaxyl (0.2 g a.i./L) + mancozeb (1.6 g a.i./L) as strategic spray.

dimethomorph treatment was intermediate to the control and mancozeb treatments and significantly lower than the latter. The increase in yield due to effective fungicidal control of the disease by treatments containing mancozeb was 53-63%.

The other result of effective disease control was an increase in the proportion of the crop in the 2 larger grades. Since there were only minor differences in the yield of small onions, most differences in total yield were due to differences in the yields of No. 1 and No. 1 large grades.

Discussion With sensitive isolates of P. destructor, metalaxyl is

highly effective. Jesperson (1985) (cited in Jesperson and Sutton 1987) reported a protectant and systemic activity which is effective in preventing sporulation if applied up to 8 days before or 4 days after inoculation. Evidence from our glasshouse and controlled environment experiments clearly shows that phenylamide resistance has developed in P. destructor in the main onion-growing region of Queensland. An isolate (2967) from an area unsprayed by phenylamides was at least 100 times more sensitive to metalaxyl than an isolate (3014) from a commercial onion field in the Lockyer Valley. There was cross-resistance in isolate 3014 to all phenylamide fungicides but not to dimethomorph. In the field, lack of control with

metalaxyl (1990 experiment) indicates resistant isolates are present as a high proportion of the P. destructor population. In other cases of phenylamide resistance, resistance factors have been as high (>100) as we recorded for P. destructor. In addition, phenylamide-resistant isolates of Pythium sp. (Sanders 1984), Phytophthora infestans (Holmes and Channon 1984), and Bremia lactucae (Crute and Harrison 1988) have shown no reduction in competitive ability and are therefore persistent in field populations.

The selection of phenylamide resistance in the P. destructor population, to the stage where these fungicides exhibit no field control (Table 3), is probably due to their widespread adoption and intensive use since 1980. The strategy of using proprietry mixtures of a phenylamide plus a protectant to prevent or delay resistance has been used since 1982, but it has not been particularly successful in this case. The fungicidal mixture of mancozeb with metalaxyl (Ridomil MZ) appears ideal since mancozeb acts to prevent spore germination through its inhibition of fungal respiratory precesses (Kaars Sijpesteijn 1982), while metalaxyl is ineffective in preventing germination of P. destructor spores (R. G. O'Brien unpublished data) and probably acts from within the plant as an inhibitor of fungal RNA synthesis (Davidse 1988). Provided spray application results in a high degree of foliage cover, mancozeb should prevent most spores from germinating and

Phenylamide resistance in Peronospora destructor 673

challenging the systemic metalaxyl. Perhaps the major shortcomings in using the mixture strategy in onions were that on-farm spraying techniques were often not of a high standard, and that there was no limit to the number of applications made in a season. This allowed resistance selection during the entire cropping cycle.

In both field experiments, severe outbreaks of downy mildew occurred. Although mancozeb gave effective disease control, other standard protectant fungicides did not. Copper hydroxide and chlorothalonil are both reg i s te red for con t ro l of on ion downy mi ldew in Queensland, and their failure to control the disease was unexpected. One possible explanation was provided by Smith et al. (1985), who showed that spray deposits of chlorothaloni l on onion leaves had a much shorter effective per iod of res idual activity than those of mancozeb.

Dimethomorph was the only systemic fungicide with activity against P. destructor under field conditions, and as a mixture with mancozeb, it was the most effective treatment. Although phosphorous acid, phosetyl-Al and propamocarb have shown systemic activity against other P h y c o m y c e t e s , they were ineffec t ive aga ins t P. destructor with the concentrations and methods of application tested. It is not known whether this is due to an inability to penetrate the waxy cuticle of the onion leaf or to low efficacy.

These experiments highlight the need to modify our use of phenylamide fungicides, as well as modifying some aspects of onion production in the Lockyer Valley. Future strategies will depend upon an analysis of the P. destructor populat ion to determine the extent of res is tance and the compet i t ive abil i ty of resis tant isolates. If resistance is extensive and competitive ability is high, there will be little benefit in the continued use of phenylamide fungicides. If one or both is low, limited use of phenylamides should have economic benefits. Cur ren t ly , g rower s are adv i sed to use a s t ra tegy (as de ta i l ed for the f ie ld t r ia ls ) wh ich resu l t s in 0 -4 applications of a phenylamide containing fungicide per season.

Essential to the continued use of phenylamides and future systemic products, such as dimethomorph, is the effective application of their companion protective fung ic ide . Cu l tu ra l fac tors which may ass is t in controlling onion downy mildew include the reduction of carry-over inoculum by the early destruction of crop residues, lower plant populations to allow improved fungicide application, and wide separation of sequential plantings.

Acknowledgments The techn ica l ass i s tance of Mr R. J. Glass and

Mr M. Weinert and the cooperation of the field staff of Gatton Research Station is gratefully acknowledged.

Statistical analyses were carried out by Mr J. Mulder. The work was f inanced by a jo in t grant from the Committee of Direction of Fruit Marketing, Rocklea, B r i sbane , and the F u n g i c i d e Res i s t ance Ac t ion Committee of the Agricultural and Veterinary Chemicals Association of Australia.

References Abd-Elrazik, A. A., and Lorbeer, J. W. (1980). A procedure for

isolation and maintenance of Peronospora destructor on onion. Phytopathology 70, 780-2.

Bromfield, K. R., and Schmitt, C. G. (1967). Cryogenic storage of conidia of Peronospora tabacina. Phytopathology 57, 1133.

Crute, I. R. (1987). The occurrence, characteristics, distribution, genetics, and control of a metalaxyl-resistant pathotype of Bremia lactucae in the United Kingdom. Plant Disease 71, 763-7.

Crute, I. R., and Harrison, J. M. (1988). Studies on the inheritance of resistance to metalaxyl in Bremia lactucae and on the stability and fitness of field isolates. Plant Pathology 37,231-50.

Davidse, L. C. (1988). Phenylamide fungicides: mechanism of action and resistance. In 'Fungicide Resistance in North America.' (Ed. C. J. Delp.) pp. 63-71. (APS press: St Paul, MN, U.S.A.)

Finney, D. J. (1971). 'Probit Analysis.' 3rd Edn. (Cambridge University Press: London.)

Holmes, S. J. I., and Channon, A. G. (1984). Studies on metalaxyl-resistant Phytophthora infestans in potato crops in southwest Scotland. Plant Pathology 33, 347-54.

Jesperson, G. (1985). Management of onion downy mildew with fungicides timed according to weather variables. M.Sc. Thesis. University of Guelph, Guelph, Ontario, Canada.

Jesperson, G., and Sutton, J. C. (1987). Evaluation of a forecaster for downy mildew of onion (Allium cepa L.). Crop Protection 6, 95-103.

Kaars Sijpesteijn, A. (1982). Mechanism of action of fungicides. In 'Fungicide Resistance in Crop Protection.' (Eds J. Dekker and S. G. Georgopoulos.) pp. 32-45. (PUDOC: Wageningen, The Netherlands.)

Marks, G. C , and Smith, I. W. (1990). Control of experimental Phytophthora cinnamomi stem infections of Rhododendron, Leucadendron and Eucalyptus by dimethomorph, fosetyl-Al and metalaxyl. Australian Journal of Experimental Agriculture 30, 139-43.

Mir, W. M., Dhar, A. K., Khan, M. A., Dar, G. H., and Zargar, M. Y. (1987). Screening of fungicides for field control of downy mildew (Peronospora destructor) on onion. Indian Journal of Mycology and Plant Pathology 17(3), 321-2.

Pegg, K. G., Whiley, A. W., Saranah, J. B., and Glass, R. J; (1985). Control of Phytophthora root rot of avocado with phosphorous acid. Australasian Plant Pathology 14, 25-9.

Reuveni, M., Eyal, H., and Cohen, Y. (1980). Development of resistance to metalaxyl in Pseudoperonospora cubensis. Plant Disease 64, 1108-9.

Ryan, E. W., (1977). Control of cauliflower downy mildew (Peronospora parasitica) with systemic fungicides. In 'Proceedings of the 9th British Crop Protection Conference, Brighton.' Vol 1. pp. 297-300.

674 R. G. O'Brien

Samoucha, Y, and Cohen, Y. (1990). Toxicity of propamocarb to the late blight fungus on potato. Phytoparasitica 18, 27-40.

Sanders, P. L. (1984). Failure of metalaxyl to control Pythium blight on turfgrass in Pennsylvania. Plant Disease 68, 776-7.

Simmonds, J. H. (1966). 'Host Index of Plant Diseases in Queensland.' (Government Printer: Brisbane.)

Smith, R. W, Lorbeer, J. W, and Abd-Elrazik, A. A. (1985). Reappearance and control of onion downy mildew epidemics in New York. Plant Disease 69,703-6.

Stoffella, P. J., and Sonoda, R. M. (1982). Reduction of onion yields by chlorothalonil. HortScience 17 (4), 628-9.

Teviotdale, B. L., May, D. M., Harper, D., and Jorde, D. (1980). New fungicide apparently controls onion mildew. California Agriculture 34 (6), 22-3.

Timchenko, V. I. (1978). Diseases of onion. Zashchita Rastenii 6, 55. Abstract in Review of Plant Pathology (1979) 58,352. Abstract No. 4141.

Walker, J. C. (1952). 'Diseases of Vegetable Crops.' (McGraw-Hill Book Company: New York.)

Wilson, G. J. (1980). Downy mildew control in onions. New Zealand Commercial Grower 35,27.

Received 6 June 1991, accepted 16 January 1992

APPENDIX VI

Validation of Downcast in the

prediction of infection events

of Peronospora destructor in

the Lockyer Valley

VALIDATION OF DOWNCAST IN THE PREDICTION OF INFECTION EVENTS OF PERONOSPORA DESTRUCTOR IN

THE LOCKYER VALLEY

S.M. Fitz GeraldA and R.G. O'Brien3

A Division of Plant Protection, Department of Primary Industries, Innisfail QW4860

B Division of Plant Protection, Department of Primary Industries, Meiers Road, Indooroopilly Qld 4068

Short Title: Forecasting onion downy mildew

2

Summary. The disease forecasting system 'Downcast' was developed in Canada

to indicate conditions suitable for sporulation and infection events oiPeronospora

destructor in onions. During 1989, observations were made of infection in trap

plants and disease progress in an unsprayed plot of onions in the Lockyer Valley

and related back to predicted sporulation - infection periods. Over a period of

113 days, there were 20 days when sporulation and infection were predicted.

These were concentrated (14) during 4 weeks in July-August. Of 16 groups of

trap plants exposed in the field, each for 7 days, 7 were subjected to predicted

sporulation-infection periods. Two sporulation-infection periods early in the

season did not result in trap plant infection or field infection, probably due to

absence of inoculum. Subsequently, groups of trap plants exposed in the field

during predicted sporulation-infection periods became infected while those

exposed when weather was unsuitable remained healthy. Downcast shows

promise as a technique for more effective disease management.

3

Introduction.

Onion downy mildew caused by Peronospora destructor Berk, causes variable

losses in onions grown in the Lockyer Valley, Queensland. The sporadic nature

of the disease is widely recognised (Viranyi 1981) and has been the subject of

detailed studies of the way environmental factors affect the sporulation, survival,

germination and infection processes (Hildebrand & Sutton 1982, 1984a, 1984b,

• 1984c, 1985; Bashi & Aylor 1983; Leach, Hildebrand & Sutton 1982). The

disease cycle is characterised by long incubation periods (9-16 days) interspersed

with short periods of 1-2 days when the pathogen sporulates, is dispersed and

infects the leaves (Hildebrand & Sutton, 1982). Stepwise increase in disease is

often apparent as the pathogen repeats successive infection cycles.

From their detailed studies of the disease cycle of onion downy mildew,

Jesperson and Sutton (1987) proposed criteria to enable the identification of

sporulation and infection events in field grown onions. The set of criteria was

termed Downcast, an acronym for downy mildew forecaster. Under Canadian

conditions it was found valuable as an aid to recognise the early disease cycles.

Early recognition resulted in improved disease control if a systemic fungicide was

used to arrest young infections.

The study described below was carried out to determine whether Downcast,

which was developed to predict downy mildew cycles in summer grown, Canadian

onion crops would be useful for the same purpose in winter grown crops in the

Lockyer Valley of Queensland.

Methods

Sporulation-infection criteria

In Downcast, infection is predicted when conditions are suitable for

sporulation, spore dispersal, spore survival and infection. Conditions favouring

dispersal are assumed to occur each day.

4

Sporulation will occur in the pre-dawn hours if

(a) temperature during the previous day was <24°C. If the temperature

exceeds 24°C it must not be >27°C for >8hrs, >28°C for >4hrs or >29°C

for >2hrs.

(b) temperature during the night is within the range 4-24°C

(c) there is no rainfall (> 1 mm) between 2300-0400 h

(d) relative humidity is >95% for a continuous 4 hours period between the

time when 6 hours of darkness has accumulated and sunrise. (Typically a

4 hour period between 0000 and 0600).

Infection is predicted on the same day as sporulation if leaf wetness persists

until 3 hours after sunrise at 6-22°C, or 4 hours after sunrise at 23-26°C. Infection

is predicted on the night following sporulation if

(a) Dew deposition is rapid.

(b) Leaf wetness is maintained for >3 h at 6-22°C.

Infection is predicted on the second night following sporulation if little or no

dew occurred on the first night after sporulation (slow dew deposition causes

death of spores) and the criteria applying to infection on the night following

sporulation are satisfied.

Infection is predicted on the third night following sporulation provided there

was little or no dew on the previous 2 nights and criteria applying to infection on

the night following sporulation are satisfied. Spores are assumed to be non-viable

thereafter.

Field Observations

Downcast was evaluated by correlating the sporulation-infection periods which

it identified with (a) the development of downy mildew in trap plants exposed for

weekly periods in a field of unsprayed onions and (b) the development of the

disease within the plot of unsprayed onions.

5

The experiment was conducted in a 40 x 21 m plot of unsprayed onions (cv.

Early Lockyer White) located near a large commercial field of the same cultivar.

The plot was sown 15 May 1989 and normal cultural practices of fertiliser and

weedicide application were followed. Spray irrigation was applied when required.

The variables of temperature and relative humidity were recorded on 2

channels of a Grant Squirrel logger. Probes were positioned amongst onion

foliage 7 cm above the ground. Leaf wetness was chartered with a De Witt 7 day

recorder. As a measure of speed of dew formation and total deposition, the area

below the trace (ABT) for the first 5 h of dew deposition was measured each day.

Rainfall and irrigation were measured daily with a standard rain gauge.

Trap plants

Two-litre pots containing 3-4 onion plants were raised in a glasshouse for use

as trap plants. When 6-8 weeks old, 5 pots were placed in the experimental plot

for a 1 week period before returning to a controlled environment cabinet set at

18/16°C on a 12/12 temperature and light regime?" After 1 week, they were

enclosed each night (for a further 7 days) in plastic bags to provide high humidity

and rated for disease severity if sporulation occurred (0, no sporulation, 1, <5%

leaf area infected; 2, >5-25%; 3, >25-50%; 4, >50-75%; 5, >75%). A mean

figure for the 5 pots was determined. There was aortal of 16 sets of trap plants

which were in the field consecutively from 1 June to 22 September (Table 1).

Field disease

Field plants were examined each week when instruments were checked and

trap plants exchanged. A field assessment of disease severity was made 25 July,

1 week after the disease was first observed in the field. Thirty-five samples each

of 25 plants were examined and the % infected leaves determined. Subsequent

severity ratings made 24 August and 19 September used 35 samples each of 5

plants which were assessed as: 0, no infection; 1, <5% leaf area affected; 2, 5-

10%; 3, > 10-25%; 4, > 25-50%; 5, >50%.

6

Results

Applying the criteria of temperature and relative humidity, there were 24

mornings out of 113 when conditions were suitable for sporulation. Conditions

of leaf wetness were suitable for infection to occur on the same morning for 13

of these. Delayed infection was indicated for the spores resulting from an

additional 7 predicted sporulation events. Slow dew deposition was assumed to

have killed spores on 4 occasions. The rainfall criteria prevented prediction of

sporulation on a further 5 mornings when humidity was not limiting.

Temperatures during the observation period were always within the favourable

range, i.e. <24°C. The majority of the predicted infection events occurred in

groups of nearly consecutive days (Table 1).

Trap plants

Sporulation was first seen in the 5th group of trap plants which were in the

field 28 June - 5 July. In these, sporulation was induced 18 July. None of the

plants in the 6th, 7th or 8th sets of trap plants showed infection but the next 4

sets of trap plants, covering the period 26 July - 24 August, were infected. No

symptoms developed in any plant in the last 4 sets of plants.

Field disease

The first appearance of the disease in the field coincided with a recording on

the fifth set of trap plants and was rated as 7.4% of leaves infected. Distribution

was uneven with no disease being found in 9 of the 35 samples (each of 25

plants). Disease assessments 24 August and 19 September showed an increasing

severity of disease with almost half the total leaf area destroyed at the second

rating.

There was a close relationship between predicted sporulation-infection events

and disease development in trap plants. Trap plants which did not experience a

predicted sporulation-infection period did not develop disease symptoms.

7

Conversely 5 of the 7 sets of trap plants which were in the field during 1 or more

sporulation-infection periods later showed downy mildew symptoms.

Discussion

The results of our observations show Downcast predictions of onion downy

mildew sporulation-infection periods were accurate within the limits of the r experimental design. Since we could only verify whether or not infection occurred

within a weekly observation period, it is not possible to determine that each

predicted infection event was fulfilled. Conversely, we also could not determine

whether factors such as slow dew deposition always prevented infection. Of the

7 observation periods containing infection events, mildew activity was confirmed

in 5. The 2 which did not result in infected trap plants occurred early in the

season before downy mildew was seen in the experimental plot. It is most likely

that these 'failures' can be discounted due to the absence of inoculum.

Besides indicating when infection is likely to occur, it is considered equally

important from a disease management viewpoint that several sets of trap plants

did not become infected when there were no predicted infection events. This

occurred even when the disease had affected all plants in the experimental plot

and the average severity rating indicated about 25% of leaves had been destroyed.

Table 1. The number of infection events of P. destructor indicated by 'Downcast' and the deve

exposed to field conditions for approximately weekly periods during a crop season in

JUNE JULY AU

Date

AObservation periods

1-7

1

8-14

2

15-21

3

21-27

4

28-6

5

7-12

6

13-18

7

19-25

8

26-1

9

2-10

10

11-18

11

BInfection periods

1 0 1 0 4 0 0 0 6 1 6

^ r a p plant disease (0-5)

0 0 0 0 1 0 0 0 2 1 2

DField plant disease

7.4%

A The dates during which sets of trap plants were exposed to field conditions.

D The number of days when conditions were suitable for both sporulation and infection.

c Severity of disease in trap plants exposed in the period indicated.

D Severity of downy mildew in the field at the period shown - % infected plants and disease s

9

The main parameter controlling sporulation-infection events during this study

was pre-dawn relative humidity through its effect on sporulation. Day and night

temperatures were never limiting which is an indicator of the differences in

conditions between summer grown crops in Canada and winter production in

Queensland.

Onion downy mildew was severe in the experimental plot and at the end of

the observation period nearly 50% of leaves had been destroyed. Initial inoculum

from external sources caused infections when the crop had been sown 7 weeks.

This resulted in 7.4% of plants becoming infected. The disease severity escalated

following 4 consecutive periods (9-12) when 14 infection events occurred.

When infection events are concentrated into a small part of the growing

season, it seems there is an opportunity for effective and economical fungicide

use. Jesperson and Sutton (1987) found this was so, especially if sprays with an

eradicant mode of action, e.g. phenylamides, were used. It was also important to

identify the early stages of infestation and commence spraying during the first

disease cycle. In the Lockyer Valley, Downcast offers sufficient promise to be

further developed as an indication of when to spray. The knowledge that

phenylamide resistant strains of P. destructor are now present (O'Brien 1992) may

result in a lower response to disease forecasting, however, there is scope to

develop new programs which will utilise both protectants, e.g. mancozeb and

systemics to make most use of their attributes.

Acknowledgements

We appreciate the assistance of Mr T. Fitz Gerald for generously providing

the observation plot.

10

References

Bashi, E. and Aylor, D.E. (1983). Survival of detached sporangia of Peronospora

destructor and Peronospora tabacina. Phytopathology 73, 1135-9.

Hildebrand, P.D. and Sutton, J.C. (1982). Weather variables in relation to an

epidemic of onion downy mildew. Phytopathology 72, 219-24.

Hildebrand, P.D. and Sutton, J.C. (1984a). Effects of weather variables on spore

survival and infection of onion leaves by Peronospora destructor. Canadian

Journal of Plant Pathology 6, 119-26.

Hildebrand, P.D. and Sutton, J.C. (1984b). Relationships of temperature,

moisture and inoculum density to the infection cycle of Peronospora

destructor. Canadian Journal of Plant Pathology 6, 127-34.

Hildebrand, P.D. and Sutton, J.C. (1984c). Interactive effects of the dark period,

humid period, temperature and light on sporulation of Peronospora destructor.

Phytopathology 74, 1444-9.

Hildebrand, P.D. and Sutton, J.C. (1985). Environmental water in relation to

Peronospora destructor and related pathogens. Canadian Journal of Plant

Pathology 6, 323-30.

Jesperson, G.D. and Sutton, J.G. (1987). Evaluation of a forecaster for downy

mildew on onion (Allium cepa L.). Crop Protection 6, 95-103.

Leach, CM., Hildebrand, P.D. and Sutton, J.C. (1982). Sporangia discharge by

Peronospora destructor. Influence of humidity, red-infra red radiation and

vibration. Phytopathology 72, 1052-6.

O'Brien, R.G. (1992). Control of onion downy mildew in the presence of

phenylamide resistant strains of Peronospora destructor. Journal of

Experimental Agriculture 32, 669-74.

Viranyi, F. (1981). Downy mildew of onion In 'The Downy Mildews', (edited by

D.M. Spencer.) pp. 461-72. London, New York, Academic Press.

APPENDIX VII

Control options for downy mildew

MFTOUT - TECHNICAL;FEATURE

Control options for, downy mildew # By QDPI research and

technical staff.

The following article on recommendations for control of downy mildew in onions follows recent trial research conducted at the QDPI Gatton Research Station.

Onion downy mildew can be expected when days are warm and nights are cool.

These conditions provide long periods of high humidity at night and heavy dews in the morning which are essential for the spread of the disease.

During winter in the Lockyer Valley we of ten receive wester ly winds which prevent dew formation on leaves and such periods are "low risk" for mildew.

The first outbreaks of downy mildew generally occur in early plantings in low lying areas.

The disease may come from resting spores in the soil or be carried over on seed crops or volunteer onion plants. Once established in the district it can become widespread within about 4 weeks if conditions are favourable.

Spread from initial infections is by spores, which are produced in millions on infected leaves and carried by wind to other onion crops.

With suitable conditions, these will infect and form leaf spots in 12-14 days.

Watch crops after bulbing

Crops seem to be more susceptible after bulbing. .

This may be due to the increased difficulty of getting good spray coverage on larger plants, or the greater canopy may make conditions within the crop more humid and thus more favourable for the disease.

It is essential to take every care in the application of fungicides after bulbing.

Even protectant fungicides such as man-cozeb and propineb (Antracol) will control the disease if they are thoroughly applied at 7-10 day intervals.

The addition of a spray sticker - extender (eg. Nu Film - 17) will help to get good coverage.

Resistance to fungicides

Another factor which has increased the difficulty of control is the development of strains of the downy mildew fungus (Peronospora destructor) which are resistan t to sys temic fung ic ides of the

Thursday, August 15, 1991

Figure 1: Severe downy mildew substantially

phenylamide group (benalaxyl, metalaxyl and oxadixyl).

Although we know they are present, we do not know what proportion of the fungus population is resistant.

It is probable that the population is not completely resistant and the systemic fungicides will be useful if used sparingly. If they are used frequently, resistance will increase more rapidly.

Current Recommendations The recommendation for this season is

QUEENSLAND FRUIT AND VEGETABLE NEWS

reduces yeilds

that growers should use a protectant fungicide eg. Antracol or mancozeb at 7 - 10 day intervals until bulbing.

After bulbing, a product containing a systemic + a protectant fungicide eg. Fruvit, Galben M or Ridomil MZ should be used when weather conditions favour the disease.

Two applications, one week apart, are recommended before returning to the normal schedule using a protectant fungicide.

(Continued on page 12)

Page 1 !

MFTOUT - TECHNICAL FEATURE (Continued from page 11)

Conditions which favour disease are:

• Downy mildew is present in the district.

• Two or more mornings when heavy dews occur and leaves remain wet until 8-9 AM.

The recommendations suggested above were tested in a field trial at Gatton Research Station last season and found to be effective, increasing yields by more than 50%.

In this trial, some fungicides did not perform to this standard. These included Aliette, Bravo, Kocide, Phosphorous acid and Previcur.

• Art ic le prepared by Rob O'Brien, Plant Pa tho logy Branch , and J o h n Kerr, Agr icul ture Branch.

ONION DOWNY MILDEW TRIAL, 1990

CHECK

KOCIDE 2g/l •

PREVICUR 5ml/l

BRAVO 3ml/l

RIDOMIL WP25 0.8g/l

ALLIETTE 3g/l

STRATEGY

RIDOMIL MZ 2.5g/l

DITHANE M45 2g/l

1 2 3 4 5 DISEASE SEVERITY

40 30 20 10 YIELD (t/ha)

Figure 2: The relative performance of fungicides against downy mildew in a trial at Gatto Research Station in 1990.

The cashew industry in Australia # By QDPI research and

technical staff.

7h/s article outlines the development of the cashew industry in Australia and discusses its prospects for the future.

The Cashew is a native of the Amazon area of Brazil. It attracted the attention of the Spanish and Portuguese explorers in the period 1550-1600 AD. By 1570 the Portuguese had taken the cashew to Goa in Ind ia , their Afr ican colonies and Malaysia.

Cashews were also spread around the Caribbean. Although it is now regarded as native to that area, the records of the early Spanish and Portuguese explorers and traders do not mention it growing there.

We do not know when the cashew came to Australia. F.M. Bailey in his comprehensive Catalogue of Queensland Flora 1909 does not mention cashew.

The cashew is now widely distributed through Top End Australian settlements and has become naturalised at Forest Beach near Ingham and in other areas of Cape York.

The potential food value of the fruit of c a s h e w has b e e n a p p r e c i a t e d by European settlers for some 400 years. In 1641, the Dutch in their northern Brazil colonies passed a law prohibiting the cutting down of cashew trees. Penalty 100 florins.

In 1941 the Brazi l ian Government passed a similar law to preserve the remaining natural stands of cashew.

Page 12

World trade in cashew kernels started in the 1920's. African and Indian production was processed in India. India remained the only processor in the region till 1966 when factories started to be built in Africa. Brazil has always processed its own production.

Major product ion areas have been Brazil, tropical African countries and India. Now any tropical country with a climate having a long dry spring - mid summer period is growing some commercial areas of cashew.

The Australian scene - The Last Twenty Years

In 1971 seed material of a range of cashew var iet ies was obtained from Kenya by Br ian C u l l . Hor t icu l tu r is t Queensland Department of Primary Industries.

These seed l i ngs w e r e p lan ted at Kamerunga Horticultural Research Station and the surplus material was planted at Lancini's property at Forest Beach. This material has been evaluated and seed from the better trees has been dist r ibuted to al l cashew p lant ings in Australia.

At the same time Dr. J . Millington was doing collection and selection work at Broome in W.A. This material forms the basis of selections planted at the Voyager Enterprises Plantation at Kununurra.

In the 1980's varietal testing was done by Ian Baker of the Northern Territory Department of Primary Industries and Fisheries (NTDPI&F) at the Coastal Plains Research Station at Humpty Doo.


Bruce Toohill of the Western Australian Department of Agriculture (WADA) carried out selection work at the Kununurra Research Station.

Private enterprise pilot farms were also planted at Wildman River east of Darwin, Comaico at Weipa, the Bamaga community and two plots at Cooktown.

1985-1990 - The Present

This period has seen an upsurge of interest and planting of cashew across Top End Australia. Much research has been done.

Two commercial sized plantations of cashews have been established. These are Cashew Australia at Dimbulah. Q. and Voyager Enterprises at Kununurra, W.A. Both plantations are at an early bearing age.

A third younger plantation is being planted by Comaico at Weipa Q. Secondary to this, it is likely that at least part of the 1992 production will be processed in Australia.

There have been further imports of seed mater ia l f rom Braz i l and vegetat ive material from India.

NTDPI&F staff have done second stage varietal trials at Coastal Plains and are establishing pilot and budwood plots at various sites in N.T.

Grafting studies have been done at Ber-rimah. A large fertiliser rate x frequency of irrigation trial has been conducted in conjunction with Wildman River Plantation.

(continued page 13)


APPENDIX VII

Control options for downy mildew

{Hoi o'&Ztev /l«j,iAf

MFIO\n. - TECHNICAL FEAWiPE::

Control options for, downy mildew # By QDPI research and

technical staff.

777© following article on recommendations for control of downy mildew in onions follows recent trial research conducted at the QDPI Gatton Research Station.

Onion downy mildew can be expected when days are warm and nights are cool.

These conditions provide long periods of high humidity at night and heavy dews in the morning which are essential for the spread of the disease.

During winter in the Lockyer Valley we often receive wester ly winds which prevent dew formation on leaves and such periods are "low risk" for mildew.

The first outbreaks of downy mildew generally occur in early plantings in low lying areas.

The disease may come from resting spores in the soil or be carried over on seed crops or volunteer onion plants. Once established in the district it can become widespread within about 4 weeks if conditions are favourable.

Spread from initial infections is by spores, which are produced in millions on infected leaves and carried by wind to other onion crops.

With suitable conditions, these will infect and form leaf spots in 12-14 days.

Watch crops after bulbing

Crops seem to be more susceptible after bulbing. .

This may be due to the increased difficulty of getting good spray coverage on larger plants, or the greater canopy may make conditions within the crop more humid and thus more favourable for the disease.

It is essential to take every care in the application of fungicides after bulbing.

Even protectant fungicides such as man-cozeb and propineb (Antracol) will control the disease if they are thoroughly applied at 7-10 day intervals.

The addition of a spray sticker - extender (eg. Nu Film - 17) will help to get good coverage.

Resistance to fungicides

Another factor which has increased the difficulty of control is the development of strains of the downy mildew fungus (Peronospora destructor) which are resistan t to sys temic fung ic ides of the


Figure 1: Severe downy mildew substantially

phenylamide group (benalaxyl, metalaxyl and oxadixyl).

Although we know they are present, we do not know what proportion of the fungus population is resistant.

It is probable that the population is not completely resistant and the systemic fungicides will be useful if used sparingly. If they are used frequently, resistance will increase more rapidly.

Current Recommendations The recommendation for this season is


reduces yeilds

that growers should use a protectant fungicide eg. Antracol or mancozeb at 7 - 10 day intervals until bulbing.

After bulbing, a product containing a systemic + a protectant fungicide eg. Fruvit, Galben M or Ridomil MZ should be used when weather conditions favour the disease.

Two applications, one week apart, are recommended before returning to the normal schedule using a protectant fungicide.

(Continued on page 12)

Page 1 I

LIFTOUT - TECHNICAL FEATURE (Continued from page 11)

Conditions which favour disease are:

• Downy mildew is present in the district.

• Two or more mornings when heavy dews occur and leaves remain wet until 8-9 AM.

The recommendations suggested above were tested in a field trial at Gatton Research Station last season and found to be effective, increasing yields by more than 50%.

In this trial, some fungicides did not perform to this standard. These included Aliette, Bravo, Kocide, Phosphorous acid and Previcur.

• Art icle prepared by Rob O'Brien, Plant Pa tho logy Branch, and John Kerr, Agr icu l ture Branch.

ONION DOWNY MILDEW TRIAL, 1990

CHECK

KOCIDE 2g/l •

PREVICUR 5ml/l

BRAVO 3ml/l

RIDOMIL WP25 0.8g/l

ALLIETTE 3g/l

STRATEGY

RIDOMIL MZ 2.5g/l

DITHANE M45 2g/l

1 2 3 4 5 DISEASE SEVERITY

40 30 20 10 YIELD (t/ha)

Figure2: The relative performance of fungicides against downy mildew in a trialatGattt Research Station in 1990.

The cashew industry in Australia # By QDPI research and

technical staff.

This article outlines the development of the cashew industry in Australia and discusses its prospects for the future.

The Cashew is a native of the Amazon area of Brazil. It attracted the attention of the Spanish and Portuguese explorers in the period 1550-1600 AD. By 1570 the Portuguese had taken the cashew to Goa in Ind ia , their Afr ican colonies and Malaysia.

Cashews were also spread around the Caribbean. Although it is now regarded as native to that area, the records of the early Spanish and Portuguese explorers and traders do not mention it growing there.

We do not know when the cashew came to Australia. F.M. Bailey in his comprehensive Catalogue of Queensland Flora 1909 does not mention cashew.

The cashew is now widely distributed through Top End Australian settlements and has become naturalised at Forest Beach near Ingham and in other areas of Cape York.

The potential food value of the fruit of c a s h e w has been a p p r e c i a t e d by European settlers for some 400 years. In 1641, the Dutch in their northern Brazil colonies passed a law prohibiting the cutting down of cashew trees. Penalty 100 florins.

In 1941 the Brazi l ian Government passed a similar law to preserve the remaining natural stands of cashew.

Page 12

World trade in cashew kernels started in the 1920's. African and Indian production was processed in India. India remained the only processor in the region till 1966 when factories started to be built in Africa. Brazil has always processed its own production.

Major product ion areas have been Brazil, tropical African countries and India. Now any tropical country with a climate having a long dry spring - mid summer period is growing some commercial areas of cashew.

The Australian scene - The Last Twenty Years

In 1971 seed material of a range of cashew variet ies was obtained from Kenya by Br ian C u l l . Hor t icu l tu r is t Queensland Department of Primary Industries.

These seed l i ngs w e r e p lan ted at Kamerunga Horticultural Research Station and the surplus material was planted at Lancini's property at Forest Beach. This material has been evaluated and seed from the better trees has been dist r ibuted to al l cashew p lant ings in Australia.

At the same time Dr. J . Millington was doing collection and selection work at Broome in W.A. This material forms the basis of selections planted at the Voyager Enterprises Plantation at Kununurra.

In the 1980's varietal testing was done by Ian Baker of the Northern Territory Department of Primary Industries and Fisheries (NTDPI&F) at the Coastal Plains Research Station at Humpty Doo.


Bruce Toohill of the Western Australiar Department of Agriculture (WADA) car ried out selection work at the Kununurn Research Station.

Private enterprise pilot farms were alsc planted at Wildman River east of Darwin Comaico at Weipa, the Bamaga community and two plots at Cooktown.

1985-1990 - The Present

This period has seen an upsurge of interest and planting of cashew across Top End Australia. Much research has been done.

Two commercial sized plantations of cashews have been established. These are Cashew Australia at Dimbulah, Q. and Voyager Enterprises at Kununurra, W.A. Both plantations are at an early bearing age.

A third younger plantation is being planted by Comaico at Weipa Q. Secondary to this, it is likely that at least part of the 1992 production will be processed in Australia.

There have been further imports of seed mater ia l from Brazi l and vegetat ive material from India.

NTDPI&F staff have done second stage varietal trials at Coastal Plains and are establishing pilot and budwood plots at various sites in N T .

Grafting studies have been done at Ber-rimah. A large fertiliser rate x frequency of irrigation trial has been conducted in conjunction with Wildman River Plantation.

(continued page 13)

Thursday, August 15,1991

APPENDIX VIII

Controlling grey mould

&*£>]> pAvir r V£6. Af<?/c ff?r pl<

VEGETABLES TOMATOES

Grey mould on tomato fruit. Serious wastage can occur during cool, wet weather..

Controlling grey mould By ROB O'BRIEN

Grey mould of tomatoes, caused by the fungus Botrytis cinerea, has caused significant losses in recent years.

Good control has been difficult due to cool and wet weather, which favours the disease, and the development of resistance to fungicides.

Strategies are needed to contain this resistance and prolong the useful life of modern fungicides.

The older fungicides usually act as general toxicants, affecting many parts of the fungal growth cycle. In contrast, highly-effective modern chemicals act on only one essential step in the cycle.

This gives the fungus the opportunity to side-step the action of the fungicide. Although only one in a billion fungal spores may initially have this ability, selection through generations will see the resistant population increase.

Present strategies for fungicide use aim to keep the resistant population at a low level.

The fungicides which control grey mould and which are at risk belong to two chemical groups: benzimidazoles — benomyl (Benlate) and dicarboximides — iprodione (Rovral), vinclozolin (Ronilan), procymidone (Sumisciex).

Farm surveys have shown that when a small proportion (5%) of the Botrytis population is resistant to dicarboximides, high levels (50%) can be expected to be resistant after three or more sprays.

The level of resistance to benzimidazoles is so high that they should not be used for grey mould control.

The following strategy has been promoted to gain maximum benefit from dicarboximide sprays: 1. During times of low disease risk or early growth stages use chlorothalonil (Bravo). 2. During periods of high disease risk or later growth stages alternate dicarboximides with chlorothalonil.

3. If the disease pressure is severe, tank mix chlorothalonil with a dicarboximide fungicide.

This strategy should be used in conjunction with good farm management practices which reduce the overall level of disease. These include prompt eradication of old crops and separation of old and new plantings.

Growers have confirmed the results of a trial at Bundaberg during 1988 which tested this strategy.

Although a source of infection was close by during the early stages of growth, no grey mould developed until plants were mature (12 weeks old).

During weather favouring grey mould, the disease was moderate in the mancozeb (older, protectant fungicide) plot and very low in the chlorothalonil plot.

In the iprodione (Rovral) plot, only two lesions (areas of diseased tissue) were found.

No lesions were found in the plots sprayed by alternating or tank mixing chlorothalonil and dicarboximide fungicide.

In the mancozeb and chlorothalonil plot, the B. cinerea population initially had 20-25% of low level dicarboximide resistance which declined to 0-5% over three weeks. This suggests that dicarboximide resistance is short-lived.

Benzimidazole resistance was more stable, only declining from 20-25% to 10-15% during the same period...

These observations support the use of the fungicide application strategy to preserve the highly-active chemicals.

Fungicide resistance is a serious problem for both growers and chemical companies — development of replacement chemicals is costly and by no means certain.

The spraying strategy each grower uses primarily affects the fungal population on his farm.

Rob O'Brien is with the Plant Pathology Branch of Queensland Department of Primary fmuhtry at Indooroopilly.

rcadia t 'etch a |

By LISA HALVORSEN »

When produce wholesalers start to ask for a product by name, you know it has to be good. And that's exactly what's happened with a & newcomer on the fresh market tomato scene, 1 Arcadia. i

Credit for this "new" fruit goes to researchers at <s the Department of Agriculture in Victoria who 1 developed this commercial cultivar in 11 years of 1 breeding trials and selection. I

•The beauty of this particular variety is that it consistently offers firm, uniform, full red fruit that's as tasty after two weeks as the day it's picked.

Although producer acceptance has grown as more and more have tried the variety for themselves, wholesalers have needed no prodding to put in their orders for Arcadia. The tomato practically sells itself.

Perhaps the biggest attraction for the grower is that it can be picked at forward colour.

Pickers find it easy to harvest as the bush is compact and they only have to pick for colour. Packers like it as its firmness means it can be handled, packed and shipped with minimal wastage.

Produce that doesn't make the grade is packed as seconds or sent to the cannery for processing. In fact, many of the Goulburn Valley's larger fresh market growers claim a pack-out rate (from bush to box) of more than 85% compared with 50% for traditional varieties such as C33 and Floradade.

Arcadia commands a higher price per box than traditional varieties. At the peak of the season, many growers were getting $20 to S30 per box.

Department extension officer at Shepparton, .Ray Holland, says Arcadia has been popular with

''consumers as well. ,," "Although most consumers say they select

tomatoes for taste, what they actually look for in a tomato is firmness and colour," Mr Holland says.

One packing company that has been converted to Arcadia is CPA Packers at Harston, Victoria. It is run by the Scrimizzi brothers, Albert and Pat,

Boxes of the new variety Arcadia.

APPENDIX IX

Downy mildew in Lettuce — DPI's

fightback package

(_ >,-t I '_ . y J..S C I- ' \ J

iT.

ro IN ••£» I

r--

i i H-I— a:

i T .

' " • J i T ,

I 1

Downy mildew in lettu DPFs fightback packa By SUE HEISSWOLF ond ROB O'BRIEN.

Deportment of Primcry Industries GOOD Cfop man- ar.d uttered.

.igemenl, including the nmcly use of fungicides, is the key efe-irient in f ighting downy mildew in lettuce CTOps.

The cool, moist weather during autumn wil l favour Ihe disease, making control difficult. A be tier understanding c! 'he disease cycle and the role of fungicides in the management program wil l improve control.

DEVELOPMENT

Downy mildew is caused l>v the fun; Hrtmia fungus spots on the upperside of leaves while a dense w)»it fungal growth occurs on the undersides of leaves. particularly ;n 'TlOiSt WCZthci TllL.-spores which spieaci 'h: disease are procured on ihii, fungri) growth Older lesions become bio^n

"HP* This lactiuar.

causes yellow-

Die life cycle of Ihe fungus is well adapted Jo the fluciuaiions of daily conditions. Spoies are pioduccd on ihe infected areas of the leaf in about six hours during humid, dewy conditions between midnichi and sunrise.

As the atmosphere dues and aw movement increases during the morning, these spores are released in their millions and hi own over a wide atea. As the wind drops a: the end of the day they sink back to e»rfh.

If the spores land on a kii_ce leaf they will gei-n;>na:e when leaves become dewy and infect Ihe leaf. I! takes about 5-6 r.i\s before' the spot becomes visible and capable i-f producing spores. The bercftis of spraying »>!• fu isicidcs to prevent i".Ji-cUons inking place v. jjl not be seen for al'H-.i si-scn dns.

'Continued Page 15)

Farmers win under Tightback' package

FARMERS l iv ing in srr..ill businessman vn)i

FUNGICIDES REGISTERED FOR USE AGAJDNS DOWNY MILDEW IN LETTUCE IN QUEENSLA

Active Constituent

Common 'lYadt Name

Kocide

Zinox

Action

copper hydroxide

copper oxychloride + zineb

mancoicb

metalaxy) • mancozeb

mcliiam

Common 'lYadt Name

Kocide

Zinox

Pioieclant v

Protcciant

copper hydroxide

copper oxychloride + zineb

mancoicb

metalaxy) • mancozeb

mcliiam

Lithane. Mancozeb

Ridomil MZ

Polyram

Protectant

_. . . Systemic Protectant

Protectant

Egg industry under review THL" Queensland

Fg» Industry Council lias appointed managem e n t c o n s u l t a n t s KPMG 1'cat Marwick lo oiKlcn.ikf ?. review of ihe egg indusns, the Minister (or f 'n-m;iry Industries, F.ii Casey, s.iid tost week

The Minister said lie was pleased the industry had .idoplirl a icsponsiMe aniluitc and appointed .tn independent consultancy firm lo ^o the K V H W .

" T h i s Uovcrnmenl wanis to see Ihe Queensland erg industry on the liont loot — we want a picfiiable and viable in-dustrv." M" Cascv -il'f.

'"The industry 'MM,) :ivie« is >imibi •• 'in: p'uviss used in ,;ijii)s. jK-anul.s, navy beans .i"d HIT ami provides tV opportunity to set Ms ,->w.i) strategic direction with appropriate lcpsl. i t '"n"

The industry hj.l until lune \ * lo deli* . i the final re| ••>••

,04-92 15:@0 GflTTON DPI 074-623349 P0;

;•<•-?• ::i:?s-

(From page 15)

Infcc'ionS will no! i.il--. pl^cc and spores \»il! no; he produced if thr aln^s phere is dry o: irrti;v-;.i turcs Me high

11Z7 l/E

. * - » _ pump shop

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MANAGEMENT The weekly plantings

made by most lettuce growers provide conditions ideal for disease to spread from old lo new plantings. Old crops ahoulil be ploughed in immediately after harvest, to reduce this risk.

Avoid irrigating crops in the late afternoon. Morning irrigations will allow plants to dry off before nightfall and re cJuce the length of time :rMt leaves Hrc wet. Temperatures during the day do not favout genmina-'ion of spores. Planting un beds and reducing planting density will im-ptove air circulation around plants and allow leaves to dry more quickly.

FUNGICIDES

Fungicides work in two different ways. Protectant fungicides inter fere with the ability of the fungus to infect the leaf. The fungicide must he on the leaf before infection take* plttcc. Gtxvl plant coverage, particularly on the underside of leaves, is essential.

Systemic fungicides «e taken up by the plant jnd can kill the fungus wuhin the leaf Downy mildew cm develop resistance to systemic fungicides. Frequent use leads to resistance, so use

DAY

N / Air driei and air movement. increasei

MIDDAY

Spores releaied into atmosphere and blown over a wide area

Spores produced o o \ iofected leaf under dewy, humid conditions

Sporej on lettuce leal germinate trader dewy

condition*

NIGHT UIDNIt

THIS DIAGRAM SHOWS the life cycle of the fungus which couses do\*r> mildew in lettuce. The fungus u well odapted to the doily fluctuations m

temperature and humidify

systemic fungicides sparingly

Use « protectMnt for most .tpplications. The formulat ion , Ridomil MZ, is. a mixture of the systemic fungicide mcta-Idxyl and the protectant nuncozeb. Begin spraying when plants have 3-4 Hue leaves and continue at 7-10 day intervals. During wet weather or when dews are heavy, bpray every seven days.

Uood spray coverage of pUnts is essential and m ihc crop matures it becomes increasingly difficult to achieve adequate coverage of leaves. It is important to have complete control of the disease in the early stages of the crop to avoid a buildup near harvest. Late applications of fungicides to 'clean up" downy mil

dew usually have little success.

[f the risk of infection is high, i.e. cool, wc: weather, in the last !*.. weeks before harvt-i. PolyTam® ot Zinox' may be applied as clost as seven days brfo '. harvest.

RESISTANCE Lettuce v,->netii»v

ststant to downy min.i . arc available and ae worth assessing in iri > plantings.

THE ULTIMATE TRAIL or FARM BIKE

Kawasaki I KLR250

&&.. 5b»

P M B AUSTRALIA

PEANUT GROWERS

vg114 the management of fungicide resistance in ... · isolate of sclerotium cepivorum with...

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