El-Naggar, M.

Karaffa, Erzsébet Mónika

Kövics, György

Váczy Kálmán, Zoltán

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 98PYRIMETHANIL TOLERANCE OF BOTRYTIS CINEREA ISOLATES FROM EGYPT AND HUNGARY  Magdy El-Naggar1, György J. Kövics22, Kálmán Z. Váczy3 and Erzsébet Karaffa2 13 Kafr el-Seikh University, Faculty of Agriculture, Department of Agricultural Botany, Kafr El-Sheikh, Egypt 2 University of Debrecen, Department of Plant Protection, H-4032 Debrecen, Böszörményi 138, Hungary 3 Research Institute for Viticulture and Enology, H-3301 Eger, POBox 83, Hungary   Botryotinia fuckeliana (de Bary) Whetzel, anamorph Botrytis cinerea Pers.:Fr. causes severe damages in vineyards on the yield as well as on quality of grapes at harvest.1 Pyrimethanil (anilinopyrimidine fungicide class) have been used to control grey mould, the disease caused by this pathogen.2 The anilinopyrimidine were registered in Switzerland in 1995 to control grey mould in grapes.3 A dual mode of action has been described involving the inhibition of methionine biosynthesis4 and the inhibition of hydrolytic enzyme secretion.5 The anilinopyrimidines inhibit mycelial growth of fungus. The intensive use of anilinopyrimidines may result reduced sensitivity and loss of efficacy.6-8 Recently, a reduction in sensitivity to anilinopyrimidines in B. cinerea strains was also reported from France.9 In the last 35 years B. cinerea developed resistance to virtually all the specific fungicides used to control grey mould. Field resistance to benzimidazoles, phenylcarbamates and dicarboximides was detected shortly after their introduction.10 Therefore, an anti-resistance strategy should be introduced for current fungicides as well. This study was initiated to study the resistance of B. cinerea to one of the main important class of modern fungicides, the anilinopyrimidines viz. pyrimethanil. This information will be useful to provide alternative fungicide options to the growers.  Materials and Methods  Isolation of fungus Twenty three Botrytis cinerea isolates were used to study their tolerance against the fungicide pyrimethanil (Mythos). Twenty one isolates (Bc18, Bc22, Bc29, Bc30, Bc 31, Bc33, Bc34, Bc36, Bc39, Bc41, Bc43, Bc45, Bc46, Bc53, Bc54, Bc55, Bc57, Bc58, Bc59, Bc61 and Bc63) were isolated from diseased grape berries in vineyards located in Eger Grapevine Region (Northern-Hungary) in 2003. Two isolates (Bc3, Bc4) were isolated in Egypt from postharvest fruits of two different host plants, bean and strawberry, respectively. All strains were maintained on PDA.  Assay on mycelium The mycelial growth of B. cinerea isolates were measured on Botrytis Minimal Agar (BMA) (glucose 10 g litre-1, K2HPO4 1.5 g litre-1, KH2PO4 2 g litre-1, (NH4)2SO4 1.0 g litre-1, MgSO4                                                  2 Correspondence to: György J. Kövics, University of Debrecen, Department of Plant Protection, H-4032 Debrecen, Böszörményi 138, Hungary E-mail: kovics@agr.unideb.hu Contract/grant sponsor: Hungarian Scholarship Board (HSB) grant for Magdy El-Naggar Contract/grant sponsor: Hungarian Ministry of Agriculture and Rural Development, FVM 33013/2003 and 46024/2004 grants. Contract/grant sponsor: János Bolyai Scholarship for Erzsébet Karaffa  99(x 7H2O) 5.0 g litre-1, agar 20g litre-1). This solid medium amended with a range of the fungicide (pyrimethanil) concentrations (0.75, 0.375, 0.075 and 0.0075 mg litre-1), were poured into 9 cm diameter Petri dishes. Each Petri dish was inoculated with an inverted mycelium plug (10 mm diameter), cut from the margin of a 5-day-old colony. Three replicates were used per treatments and incubation took place at room temperature in the dark. The mycelial growth rate was evaluated by the diameter of fungal colonies, measured daily for 5 days. Tolerance was determined by assessing the concentrations causing a 50% reduction in the growth rate (EC50) by decreasing the relative growth rate (in percentage of control) against the logarithm of the fungicide concentrations.  Assay on conidia The action of the fungicide pyrimethanil upon the spore germination and germ-tube elongation of B. cinerea was tested as follows. The nutrient medium (10g glucose, 2 g K2HPO4, 2 g KH2PO4, and 10 g agar in 1 litre water) was used in tests involving pyrimethanil. This medium amended with a range of pyrimethanil concentrations (0.75, 0.375, 0.075 and 0.0075 mg litre-1) was poured into 9 cm in diameter Petri dishes. Then a conidial suspension of each B. cinerea isolate was dropped onto the surface of the agar plates. After 20-24 hrs incubation at room temperature in the dark, the percentage of spore germination (100-200 conidia for each treatment) and the length of germ-tubes (50-100 germinated conidia for each treatment) were estimated under a microscope supplemented with an ocular micrometer. As in the previous test, the dose response curves allowed determination of the concentrations causing 50% reduction in the germination of spores or in the length of germ tubes (EC50).   Resistance level Resistance levels were estimated as ratios EC50 of each isolate / EC50 of the most sensitive isolate.  Results  Effect of the fungicide pyrimethanil on mycelial growth of Botrytis cinerea On the base of in vitro sensitivity of mycelial growth of B. cinerea isolates against pyrimethanil (anilinopyrimidine fungicide class), three phenotypes were detected. They could be characterized as sensitive (S) (Bc3, Bc29, Bc30, Bc33, Bc34, Bc36, Bc41, Bc43, Bc45), moderately resistant (MR) (Bc4, Bc18, Bc22, Bc31, Bc39, Bc46, Bc54, Bc57, Bc58, Bc61), and highly resistant (HR) (Bc53, Bc55, Bc59, Bc63). Their respective resistance levels (RL) were less than 2.09 (S), more than 2.09 and less than 10.09 (MR), more than 10.09 (HR). Such three phenotypes show EC50 values less than  0.3 mg litre-1 (S), 0.3  EC50  1.44 mg litre-1 (MR) and 1.44  EC50  3.725 mg litre-1 (Table 1 and Fig. 1, 2). Hence, the frequency percentage of sensitive, moderately resistant and highly resistant isolates was 39.3, 38.96 and 21.74%, respectively.   100Table 1 Effect of the fungicide pyrimethanil on the mycelial growth of Botrytis cinerea isolates  Isolates EC50 (mg/l)   Resistance Level (RL) Resistance Bc3 0.2193 1.049 S Bc4 0.3294 1.576 MR Bc18 0.3797 1.817 MR Bc22 0.3084 1.476 MR Bc29 0.2126 1.017 S Bc30 0.2444 1.169 S Bc43 0.210 1.005 S Bc45 0.219 1.048 S Bc31 0.418 2.000 MR Bc33 0.154 0.737 S Bc34 0.204 0.976 S Bc36 0.144 0.689 S Bc39 0.413 1.977 MR Bc41 0.273 1.306 S Bc46 0.321 1.536 MR Bc53 3.689 17.659 HR Bc54 0.528 2.527 MR Bc55 3.679 17.611 HR Bc57 0.956 4.576 MR Bc58 1.444 6.912 MR Bc59 2.137 10.229 HR Bc61 0.869 4.159 MR Bc63 2.244 10.741 HR   101 Fig 1 Cumulative frequency baseline distribution for pyrimethanil (EC50 0 – 4 mg/l) and Botrytis cinerea  Fig 2 Isolate number baseline distribution for pyrimethanil (EC50 0 – 4 mg/l) and Botrytis cinerea   Effect of the fungicide pyrimethanil on spore germination of Botrytis cinerea The effect of pyrimethanil on spore germination shows that all isolates of B. cinerea were sensitive (S) except four ones, two of which (Bc18, Bc22) were moderately resistant (MR) and the other two isolates (Bc33, Bc59) were highly resistant (HR). Resistance levels (RL) for sensitive (S), moderately resistant (MR) and highly resistant (HR) isolates were less than 1.36, more than 1.36 and less than 1.82, more than 1.82, respectively. On the other hand, their respective EC50 values were less than 0.3 mg litre-1 (S), 0.3  EC50  0.4 mg litre-1 (MR), and 0.4  EC50  0.5 mg litre-1 (HR) (Table 2 and Fig. 3, 4). Hence, the frequency percentage of sensitive, moderately resistant and highly resistant isolates was 82.6, 8.7 and 8.7%, respectively.  B . c in erea p y rim eth an il0204060801001200 0 ,5 1 1 ,5 2 2 ,5 3 3 ,5 4E C 50 m g /lCummulative frequency %B. cinerea pyrimethanil024680 0,5 1 1,5 2 2,5 3 3,5 4EC50 mg/lNumber of isolates  102 Table 2 Effect of the fungicide pyrimethanil on spore germination of Botrytis cinerea isolates  Isolates EC50 (mg/l)   Resistance Level (RL) Resistance Bc3 0.2553 1.165 S Bc4 0.2219 1.013 S Bc18 0.326 1.488 MR Bc22 0.3799 1.734 MR Bc29 0.1998 0.912 S Bc30 0.2264 1.033 S Bc31 0.1998 0.912 S Bc33 0.4151 1.895 HR Bc34 0.2115 0.965 S Bc36 0.2038 0.930 S Bc39 0.2602 1.188 S Bc41 0.2102 0.959 S Bc43 0.2038 0.930 S Bc45 0.2143 0.978 S Bc46 0.202 0.922 S Bc53 0.2885 1.317 S Bc54 0.2219 1.013 S Bc55 0.2219 1.013 S Bc57 0.2059 0.940 S Bc58 0.2038 0.930 S Bc59 0.5336 2.436 HR Bc61 0.2097 0.957 S Bc63 0.2097 0.957 S  103 Fig 3 Cumulative frequency baseline distribution for pyrimethanil (EC50 0 – 0.7 mg/l) and Botrytis cinerea Fig. 4 Isolate number baseline distribution for pyrimethanil (EC50 0 – 0.7 mg/l) and Botrytis cinerea  Effect of the fungicide pyrimethanil on germ-tube elongation of Botrytis cinerea The effect of pyrimethanil on germ-tube elongation of B. cinerea showed four phenotypes which were characterized as sensitive (S) (Bc43, Bc45), low resistant (LR) (Bc3, Bc4, Bc18, Bc30, Bc31, Bc33, Bc34, Bc41, Bc55, Bc61), moderately resistant (MR) (Bc29, Bc53), and highly resistant (HR) (Bc22, Bc36, Bc39, Bc46, Bc54, Bc57, Bc58, Bc59, Bc63). Their respective resistance levels (RL) were less than 1.58 (S), more than 1.58 and less than 3.96 (LR), more than 3.96 and less than 5.28 (MR), and more than 5.28. On the  other hand, their EC50 values were less than 0.06 mg litre-1 (S), 0.06  EC50  0.15 mg litre-1 (LR), 0.15  EC50  0.2 mg litre-1 (MR) and 0.2  EC50  7.85 (Table 3 and Fig. 5, 6). Hence, the frequency percentage of sensitive, low resistant, moderately resistant and highly resistant isolates was 8.69, 43.47, 8.69 and 39.13%, respectively.  B. cinerea pyrimethanil0204060801001200 0,1 0,2 0,3 0,4 0,5 0,6 0,7EC50 mg/lCumulative frequency %B. cinerea pyrimethanil02468100 0,1 0,2 0,3 0,4 0,5 0,6 0,7Ec50 mg/lNumber of isolates 104Table 3 Effect of the fungicide pyrimethanil on germ-tube elongation of Botrytis cinerea isolates  Isolates EC50 (mg/l) Resistance Level (RL) Resistance Bc3 0.0688 1.8177 LR Bc4 0.1212 3.2021 LR Bc18 0.099 2.6155 LR Bc22 0.0917 2.4227 LR Bc29 0.1998 5.2787 MR Bc30 0.0917 2.4227 LR Bc31 0.0663 1.7516 LR Bc33 0.113 2.9854 LR Bc34 0.1023 2.7027 LR Bc36 0.2079 5.4927 HR Bc39 0.2079 5.4927 HR Bc41 0.0851 2.2483 LR Bc43 0.0474 1.2523 S Bc45 0.0283 0.7476 S Bc46 0.2277 6.0158 HR Bc53 0.1904 5.0303 MR Bc54 0.2153 5.6882 HR Bc55 0.0685 1.8097 LR Bc57 0.2218 5.8599 HR Bc58 0.2282 6.0290 HR Bc59 0.2144 5.6644 HR Bc61 0.0792 2.0924 LR Bc63 0.2709 7.1571 HR   105 Fig. 5 Cumulative frequency baseline distribution for pyrimethanil (EC50 0 – 0.3 mg/l) and Botrytis cinerea    Fig. 6 Isolate number baseline distribution for pyrimethanil (EC50 0 – 0.3 mg/l) and Botrytis cinerea  Discussion and Conclusions  The development of tolerance to fungicides is mainly due to selection pressure exerted on resident populations of the pathogen by heavy applications of a single fungicide.11 When fungal populations are subjected to fungicides, sensitive individuals are selectively killed. Those individuals that harbour a mutation for tolerance may multiply without computation from the normal population.12 This new tolerant population then becomes dominant, and crop losses can occur despite of continued fungicide application. The pyrimethanil tolerance by B. cinerea usually develops over time. Use of single fungicide is discouraged because the pathogen rapidly develops tolerance.13 Our results revealed an increase in frequency percentage of isolates resist to pyrimethanil against mycelial growth and germ-tube elongation. The frequency percentages of moderately resistant isolates (MR) and highly resistant ones (HR) against mycelial growth were 38.96 and 21.74%, respectively. In parallel, the frequency percentage of low, moderately and highly resistant isolates against germ-tube elongation were 43.47, 8.69 and 39.13%, respectively (Fig. 1 and 5). Such results support those which were experienced that B . c inerea  pyrim ethanil0204060801001200 0,05 0,1 0,15 0,2 0,25 0,3E C 50 m g/lCumulative frequency %B. cinerea pyrimethanil00.511.522.50 0.05 0.1 0.15 0.2 0.25 0.3EC50 mg/lNumber of isolates 106anilinopyrimidines inhibited the germ-tube elongation and mycelial growth of wild type strains (AniS) at low concentrations (EC50 values below 0.1 mg litre-1).14 On the other hand, a decrease in frequency percentage of isolates resisting to anilinopyrimidines, pyrimethanil, against spore germination was recorded. The frequency percentages of moderately and highly resistant isolates against spore germination were 8.7 and 8.7% (Fig. 3). This observation coincides with those variable results of anilinopyrimidine towards the spore germination.15 Consequently, this stage of fungal development should not be taken into consideration in further studies involving anilinopyrimidine-resistant strains. This effect on pyrimethanil efficacy might be due to the frequent treatments of pyrimethanil which result increase of the resistant population, which may eventually lead to failure of disease control. Altering usage of different classes of fungicides that have different modes of action and high activity against B. cinerea together with limiting the applied number of fungicide treatments might be suggested as an effective way to control B. cinerea.  Acknowledgement  This research work was carried out by G.J. Kövics, K.Z. Váczy and E. Karaffa with support of Ányos Jedlik Project (NKFP-A2-2006-0017)   References  1 Rüegg J, Lauber HP, Siegfried W, Viret O and Hilber UW, Experiences with anilinopyrimidines in Switzerland. Pestic Outlook 8:28-33 (1997). 2 Leroux P, Recent developments in the mode of action of fungicides. Pestic Sci 47:191-197 (1996). 3 Baroffio CA, Siegfried W and Hilber UW, Long-term monitoring for resistance of Botryotinia fuckeliana to anilinopyrimidine, phenylpyrrole and hydroxyanilide fungicides in Switzerland. Plant Dis 87:662-666 (2003). 4 Pillonel C and Meyer T, Effect of phenylpyrroles on glycerol accumulation and protein kinase activity of Neurospora crassa. Pestic Sci 49:229-236 (1997). 5 Miura I, Kamakura T, Maeno SS, Hayashi S and Yamaguchi I, Inhibition of enzyme secretion in plant pathogen by mepanipyrium a novel fungicide. Pestic Biochem Physiol 48:222-228 (1994). 6 Hilber UW and Schüepp H, A reliable method for testing the sensitivity of Botryotinia fuckeliana to anilinopyrimidines in vitro. Pestic Sci 47:241-247 (1996). 7 Hilber UW and Hilber-Bodmer M, Genetic basis and monitoring of resistance of Botryotinia fuckeliana to anilinopyrimidines. Plant Dis 82:497-500 (1998). 8 Forster B and Staub T, Basis for use strategies of anilinopyrimidines and phenylpyrrole fungicides against Botrytis cinerea. Crop Prot 15:529-537 (1996). 9 Leroux P, Chapeland F, Desbrosses D and Gredt M, Patterns of cross-resistance to fungicides in Botryotinia fuckeliana (Botrytis cinerea) isolates from French vineyard. Crop Prot 18:687–697 (1999). 10 Leroux P, Progress and problems in the control of Botrytis cinerea in grapevine. Pestic Outlook 6:13-19 (1995). 11 Ogawa JM, Manji BT and Chastagner GA, Field problems due to chemical tolerance of plant pathogens. Proc Am Phytopath Soc 3:47-53 (1976).  10712 Dekker J, Acquired resistance to fungicides. Ann Rev Phytopathol 14:405-428 (1976). 13 Miller MW, Fletcher JT, Benomyl tolerance in Botrytis cinerea from greenhouse crops. Trans Brit Mycol Soc 62:99-103 (1974). 14 Leroux P, Chapeland F, Giraud T, Brygoo Y and Gredt M, Resistance to sterol biosynthesis inhibitors and various other fungicides in Botrytis cinerea. in: Modern fungicides and antifungal compounds II. ed by Lyr H, Russel PE, Dehne HW and Sisler HD, Intercept, Andover, UK, pp 297-303 (1999). 15 Leroux P and Lagouarde P, Etude in vitro de la sensibilite de Botrytis cinerea vis a vis du pyrimethanil. in: 4th International Conference on Plant Disease. ANPP, Paris, France, pp 1409-1416 (1994).  PYRIMETHANIL TOLERANCE OF BOTRYTIS CINEREA ISOLATES FROM EGYPT AND HUNGARY  Magdy El-Naggar1, György J. Kövics2, Kálmán Z. Váczy3 and Erzsébet Karaffa2 1Kafr el-Seikh University, Faculty of Agriculture, Department of Agricultural Botany, Kafr El-Sheikh, Egypt 2 University of Debrecen, Department of Plant Protection, H-4032 Debrecen, Böszörményi 138, Hungary 3 Research Institute for Viticulture and Enology, H-3301 Eger, POBox 83, Hungary  Pyrimethanil (anilinopyrimidine group) fungicides have been used widely to control grey mould, Botrytis cinerea fungus causes severe damages in vineyards. Resistance development in B. cinerea populations for pyrimethanil resulted decreasing the effectiveness on these areas.  Twenty three isolates of B. cinerea were isolated from Hungary and Egypt, and presence of fungicide resistance development was studied in vitro. Different decreasing effects of pyrimethanil on mycelial growth, spore germination, and germ-tube elongation were observed. Effect on mycelial growth showed that 39.13% (9 isolates), 43.48% (10 isolates), and 17.39% (4 isolates) of the tested isolates were sensitive, moderately and highly resistant, respectively. While the effect on spore germination showed that 82% (19 isolates), 8.69% (2 isolates), 8.69% (2 isolates) were sensitive, moderately and highly resistant, respectively. On the other hand, the effect on germ-tube elongation showed 8.69 (2 isolates), 43.47 (10 isolates), 8.69 (2 isolates) and 39.13% (9 isolates) were sensitive, low resistant, moderately resistant and highly resistant, respectively.  The increased presence of pyrimethanil fungicide resistance of B. cinerea strains in both countries initiates to take into consideration the decreased fungicide effectiveness in the anti-resistance strategies as well as providing alternative fungicide options.   

Pyrimethanyl tolerance of Botrytis cinerea isolates form Egypt and Hungary

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