SENSITIVITY OF Venturia inaequalis ISOLATES TO FUNGICIDES WITH DIFFERENT MODES OF ACTION

Goran Aleksi1, Tatjana Popovi1, Mira Starovi1, Slobodan Kuzmanovi1, Dragana Joi2, Nenad Dolovac1, Dobrivoj Poti1
1Institute

for Plant Protection and Environment, Belgrade,Serbia 2Institute for Soil Science, Belgrade,Serbia

Apple scab caused by Venturia inaequalis (Cooke) Winter (anamorph Spilocaea pomi Fr.) is the most important apple disease in the world. Chemical control with fungicides has been the main measure of disease control. During the growing season, in order to keep the trees scab free, more than 20 treatments may be needed. In order to protect apple trees from the apple scab, a wide spectrum of different chemical preparations is used from different chemical groups, with different mode of action (preventative, preventative-curative, curative and eradicative) which are often combined in order to enhance their effectiveness or avoid development of resistance.

Unfortunately, fungicide resistance has been observed in some orchards. Investigating occurrence of reduced sensitivity of pathogens to fungicides is of great importance in conditions of intensive crop protection.

Fungicide resistance in V. inaequalis is well documented worldwide for old fungicides such as dodine (Kller and Wilcox, 1999; BroniarekNiemiec and Bielenin, 2008), benzimidazole (Koenraadt et al., 1992), demethylation inhibitors (DMIs) (Sholberg and Haag, 1993; Kller et al., 1997), and newer fungicides such as strobilurins (Olaya and Koller, 1999; Fontaine et al., 2009) and anilinopyrimidines (Kng et al., 1999). There are no reports in the literature about the sensitivity to mancozeb, an old fungicide still used for apple scab control, either used alone or mixed mainly with DMIs as an anti-resistance strategy.

The objective of this research was to determine the sensitivity of V.inaequalis populations isolated from treated and untreated apple orchards to the difenoconazole, flusilazole, cyprodinil, pyrimethanil, kresoxim-methyl, captan and mancozeb fungicides.

MATERIAL AND METHODS The purpose of this trial is to test, in vitro, the effectiveness of some fungicides on the mycelia growth of Venturia inaequalis isolated from infected apple leaves collected from treated (isolate Morovic M and M1) and untreated (isolate Nestin N and N1) orchards during 2011. Isolations were carried out according to the method of Bori (1987) and Aleksi (1996). Isolates were grown for six weeks on PDA at 20C.

Commercial formulations of seven fungicides, representing different chemical families, were used in this study. The experiment was done using a method described by Popovi (2004). Appropriate volumes of each fungicide were added to the PDA at approximately 50C in the quantities needed to achieve the final concentrations at the rate of the registered dose and poured in sterilized 50 mm Petri plates.

TABLE 1. Fungicides on tested fungal cultures of Venturia inaequalis

Active ingredient Fungicide Tested concentrations Tested doses / litar

Difenoconazole Flusilazole Cyprodinil Pyrimethanil Kresoximmethyl Captan Mancozeb

1%

Score 250 EC Punch 40 EC Chorus 75 WG Pyrus 400 SC

Stroby DF Merpan 80

0.021 (0.005)2 0.011 (0.004)2 0.0251 (0.01875)2 0.151 (0.06)2

0.021 (0.01)2 0.21 (0.16)2

0.2 mL 0.1 mL 0.25 g 1.5 mL

0.2 g 2.0 g

Mankogal

0.251 (0.2)2

2.5 g

of fungicide 2 % of active ingredient

Mycelia plug (1 mm in diameter), obtained from the actively growing cultures, were transferred to fungicide amended plates. The Control was PDA plates without the addition of the fungicide. There were four replicates for each fungicide. The dishes were incubated at 20C in the dark and the diameter of each colony was measured twice perpendicularly.

RESULTS
Four isolates (M, M1, N1 and N) of V. inaequalis, originating from two localities on the teritory of Serbia (Morovi and Netin) were used for investigations in this work. Isolates from Morovi locality were sampled from commercial apple orchards area of 100 ha, in which intensive measures for apple scab control are implementing for many years. This orchard had applications of the entire range of fungicides registered for apple scab control. Isolates from locality Netin were taken from individual apple trees distant from any commercial orchards. No fungicides have been applied for apple scab control, and these isolates could be considered wild or organic isolates.

TABLE 2. Growth of four Venturia inaequalis isolates on PDA medium with tested fungicides
Active ingredient Fungicide Dose/ lit. Isolate (average growth in mm) M D.t. a a a a a a a M1 1.0 1.0 1.0 1.0 12.75 1.0 1.0 D.t. a a a a b a a N 1.0 1.0 1.0 1.0 1.0 1.0 1.0 D.t. a a a a a a a N1 1.0 1.0 1.0 1.0 1.0 1.0 1.0 D.t. a a a a a a a

Difenoconazole Score 250EC 0.2 mL 1.0 Flusilazole Cyprodinil Pyrimethanil Kresoximmethyl Captan Mancozeb Punch 40 EC 0.1 mL 1.0 Chorus75WG 0.25 g 1.0 Pyrus 400SC 1.5 mL 1.0 Stroby DF Merpan 80 Mankogal 0.2 g 2.0 g 2.5 g 1.0 1.0 1.0

Control

LSD0.05

19.8

b
0.44

20.13

c
0.61

21.88

b
2.82

29.38

b
0.33

Growth of four Venturia inaequalis isolates on PDA medium with tested fungicides

30
Difenoc.

25
Growth in mm

Flusil. Cyprod. Pyrimet. Kr.-methyl Captan Mancozeb Control

20 15 10 5 0
M M1 N N1

Isolates

FIGURE 1. Isolate M; K-control, D-difenoconazole, F-flusilazole, P-pyrimethanil, C-cyprodinil, S-kresoxim-methyl

FIGURE 2. Isolate M; K-control, D-difenoconazole, F-flusilazole, C-cyprodinil, P-pyrimethanil, S-kresoxim-methyl

DISCUSSION
V. inaequalis is the most important disease in apple production. About 75% of the total application of pesticides applied in the production of apples are for the fungal diseases control (of which 70% is used for apple scab control alone. A major problem in protecting apples from this pathogen is the fact that a small number of fungicides are being used in ever increased quantity. This fact contributes to the occurence of pathogen resistance or reduced susceptibility of the pathogen towards used fungicides, due to capacity to the pathogen for genetic variation (Aleksi et al., 2005). According to Kller (1988) most fungicides used to control pathogens have a specific mechanism of action (eg. DMI fungicides), and it was clear that there is a risk of resistance development.

According to the results of testing the biological efficacy of some fungicides (QoI and DMI), conducted during two growing periods, Aleksi (2006) found that the examined products showed high efficiency in both preventive and curative control of apple scab. Similar results have been announced Balaz and Knezevic (2003) examining the efficacy of newer fungicides in controlling apple scab and apple powdery mildew. In studies conducted during 1998, 1999 and 2002, the authors found that the best efficacy in controlling apple scab was demonstrated by a group of strobilurins (trifloxystrobin and kresoxim-methyl) They found no statistically significant differences in the efficiency of the the preparations tested in this group of compounds. They then tested a group of DMI fungicides Difenoconazole (product-Score 250 EC), which exhibited a lower efficacy in controlling apple scab. In our in vitro experiments, colony growth of V. inaequalis was registered only in the treatment with kresoxim-methyl, which leads to the conclusion that there was an appearance of pathogens resistance to this fungicide.

Recent studies by other authors identified reduced sensitivity of pathogen to the compounds of the strobilurin group (Kller et al., 2004) which is consistent with the results obtained in this paper. According to Kuck and Mehl (2003), the resistance to strobilurins in the field is noticed on several pathogens since 1998. Mutations of the G143A are mostly responsible for the occurence of this resistance. According to Brunelli et al. (2003), in the case of fungicides in the strobilurin group, the second generation fungicides (trifloxystrobin) exhibited greater efficacy than the previous generation (kresoksim-methyl).

The occurence of resistance in another very important group of fungicides DMI, was observed even twenty years ago. The problems with decreasing effectiveness of these fungicides can occur after several years of their intensive use. Among them there is no cross-resistance. The genetic basis of resistance to the DMI is complex and has not been sufficiently studied. It is polygenic and developed through a process of gradual decline in pathogen sensitivity to fungicides (Gaunt et al., 1996). According to Pscheidt (2004) resistance to DMI fungicides in the United States is noticed in several states and in several cultures. The powdery mildew and apple scab causal agents are the first pathogens in which the emergence of resistance observed.