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In vitro sensitivity of Eutypa lata to various fungicides
Eutypa dieback (tandpyn), caused by the fungus Eutypa lata, is a serious disease in South African vineyards. Infection occurs when ascospores (sexual spores) of the fungus enter fresh pruning wounds in mature wood. These ascospores germinate and grow into the healthy wood below the wound, eventually causing progressive dieback symptoms of the vine. At present, management of Eutypa dieback relies on sanitation, timing of pruning and wound protection with wound sealants or benomyl. No fungicide is registered for control of this disease in local vineyards. A laboratory study was therefore undertaken to investigate the effectiveness of fungicides from different chemical groups currently registered for use on grapevines.
Materials and Methods
Twelve fungicides
(Table 1)
were screened in vitro for mycelial inhibition of 12 E lata isolates according to the method of Munkvold & Marois (1993). The isolates were obtained from different vineyards in the Stellenbosch, Durbanville, Somerset West, Paarl and Wellington areas
(Table 2)
. Isolates were stored on potato-dextrose agar (PDA) slants at 40C and transferred to PDA in petri dishes for propagation when needed. Petri dishes were subsequently incubated at room temperature for one week, at which time there was sufficient growth to transfer to fungicide-amended media. The fungicides were suspended in sterile distilled water and added to molten PDA at approximately 500C in amounts to achieve final concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 ęg/ml. Mycelium plugs (4 mm in diameter), obtained from the margins of actively growing cultures, were transferred to fungicide amended plates. Three mycelium plugs (three different isolates) were placed at an equal distance from each other on each plate. There were three replicates of each fungicide concentration, and the experiment was repeated once. Concentrations of 50.0 and 100.0 ęg/ml were added for less effective fungicides when the experiment was repeated. The dishes were incubated for five days at 230C (12 h light per day), whereafter the diameter of each colony was measured (two measurements per colony).
Statistical analyses
The experimental design was completely randomised with a 12x12x8 factorial and three random replications. The factors were 12 isolates, 12 fungicides and eight concentrations. The percentage inhibition was calculated as follows:
% Inhibition = 100 x [(Isolate Diameter - 4)-(Control Diameter - 4)]/(Control Diameter - 4)
The percentage inhibition data of both experiments were pooled and linear regressions were fitted over concentrations for each isolate and fungicide separately after the extreme tail points were deleted.
The equations fitted were as follows:
% Inhibition = a + b x Concentration (a = intercept and b = slope)
from which the effective concentration at which 50% of the mycelial growth was inhibited (EC50), was calculated as follows:
EC50 = b/(50-a)
The EC50 values and the rate of change (slopes) were subjected to analysis of variance and the Student's t-LSD (Least Significant Difference) was calculated at a 5% significance level to compare fungicide means (SAS, 1990). The Shapiro-Wilk test was performed to test normality on residuals (Shapiro & Wilk, 1965). Isolates with outliers were discarded until the residuals were normally distributed.
Results and Discussion
Flusilazole, tebuconazole, benomyl, fenarimol and myclobutanil were the most effective fungicides with EC50 values of 0.005, 0.01, 0.19, 0.29 and 1.48 ęg/ml, respectively. The hydroxy-analide and strobilurin fungicides were least effective with EC50 values > 98 ęg/ml (Fig 1).
Benomyl proved to be the most effective fungicide on the rate of change in EC50 for a 1% increase in concentration (Fig 2) and would therefore be very effective at low concentrations. Except for flusilazole and tebuconazole with 53.97% and 32.10% rate of change, all other fungicides represented the least effective group with a rate of change less than 16.46%. These fungicides therefore need to be applied at much higher concentrations, which is not economically viable.
Although the objective of this study was to compare the efficacy of benomyl with newer fungicides with different modes of action, results clearly underline the efficacy of benomyl. Furthermore, resistance to benomyl was not detected in this study, despite the fact that it had been applied to pruning wounds for several years in some of the vineyards from which isolates were obtained. Flusilazole or tebuconazole could also be used as effective alternatives to benomyl for grapevine pruning wound protection. More fungicides as well as biological control agents, will be screened during the following months prior to field trials commencing in August 2001.
For further information contact Francois Halleen at telephone number (021) 809 3040, fax number (021) 809 3002 or e-mail: francois@infruit. agric.za.
Acknowledgements
The authors would like to thank Carine Vermeulen and Linda Nel for providing technical assistance.
References
Munkvold, G P & Marois, J J, 1993. The effects of fungicides on Eutypa lata germination, growth, and infection of grapevines. Plant Disease 77: 50 - 55.
SAS (1990), SAS/STAT User's Guide, Version 6, Fourth Edition, Volume 2. SAS Institute Inc, SAS Campus Drive, Cary, NC 27513.
Shapiro, S S & Wilk, M B, 1965. An analysis of variance test for normality (complete samples). Biometrika 52: 591 - 611.
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