Stability and fitness of anilinopyrimidine-resistant strains of Botrytis cinerea.

The fitness of anilinopyrimidine-resistant isolates of Botrytis cinerea compared with that of sensitive isolates, collected from vegetable crops in Greece during 2005, was investigated. Stability of resistance to anilinopyrimidine fungicides was determined after consecutive transfers of the fungal isolates on fungicide-free potato dextrose agar for 16 culture cycles or on fungicide-untreated cucumber seedlings for eight disease cycles. Results showed that after the consecutive transfers of the isolates either in vitro or in vivo sensitivity to cyprodinil was not changed significantly compared to the initial sensitivity in all the isolates tested, suggesting a stable genetically controlled trait. Fitness parameters measured were mycelial growth, spore production in vitro, osmotic sensitivity, virulence, spore production in vivo, percentage of spore germination, and competitive ability of the resistant isolates in four pairs with sensitive isolates both on artificial nutrient medium or on cucumber seedling plants. The measurements of the fitness components in individual isolates showed high variability within both sensitivity groups in all, except virulence, fitness components tested. As a group, resistant isolates showed significantly lower (P < 0.05) mycelial growth and virulence, while they were more osmotically sensitive than the sensitive isolates. In addition the resistant isolates showed higher (P < 0.05) spore production in vivo but there was no difference (P > 0.05) between the two sensitivity groups in spore production in vitro and in the percentage of spore germination. However, the correlation to test if there is any relationship between the values of each fitness component tested and the level of cyprodinil sensitivity of each isolate was for all, except the spore production in vivo, fitness components not significant (P > 0.05). This absence of significant correlation coefficient values suggests that the development of resistance to anilinopyrimidine fungicides did not affect the fitness of the resistant isolates. Competition of the resistant versus sensitive isolates was isolates-dependent, since in two of the isolate pairs the resistance frequency decreased significantly after five culture or disease cycles, while in the remaining two pairs resistance frequency increased significantly after five disease cycles or remained stable for one pair after five culture cycles on artificial nutrient media.

Baseline Sensitivity of Botrytis cinerea to Pyraclostrobin and Boscalid and Control of Anilinopyrimidine- and Benzimidazole-Resistant Strains by These Fungicides.

Fifty-five isolates of Botrytis cinerea collected from vegetable crops were used to determine the pathogen's baseline sensitivity to two new fungicides: boscalid, which inhibits the enzyme succinate dehydrogenase in the electron transport chain, and pyraclostrobin, which blocks electron transport between cytochrome b and cytochrome c1. Measurement of sensitivity to boscalid was based on both inhibition of mycelial growth and spore germination, while measurement of sensitivity to pyraclostrobin was based only on inhibition of spore germination. For both fungicides, the sensitivity distribution was a unimodal curve, with a mean EC50 value (effective concentration that reduces mycelial growth or spore germination by 50%) of 0.033 µg ml-1 for pyraclostrobin and 2.09 and 2.14 µg ml-1 for boscalid based on the inhibition of mycelial growth and spore germination, respectively. No cross-sensitivity relationship was observed between the two fungicides (r = 0.09). In addition, no cross-resistance relationship was observed between these two fungicides with other botryticides: cyprodinil, pyrimethanil, fenhexamid, fludioxonil, and iprodione. Moreover, the control efficacy of the two fungicides was tested against two anilinopyrimidine-resistant and two benzimidazole-resistant isolates, and two of wild-type sensitivity. Both pyraclostrobin and boscalid provided satisfactory control of all six isolates that was independent of the isolate sensitivity to benzimidazoles and anilinopyrimidines. In contrast, carbendazim failed to control sufficiently the benzimidazole-resistant isolates, while cyprodinil failed to provide satisfactory control of the anilinopyrimidine-resistant isolates.

Resistance of Botrytis cinerea Isolates from Vegetable Crops to Anilinopyrimidine, Phenylpyrrole, Hydroxyanilide, Benzimidazole, and Dicarboximide Fungicides.

During February 2005, 55 single-spore isolates of Botrytis cinerea were collected at the end of the season from vegetable crops grown in 18 greenhouses on the island of Crete, Greece. They were tested for sensitivity to the anilinopyrimidine fungicides pyrimethanil and cyprodinil, the hydroxyanilide fungicide fenhexamid, the phenylpyrrole fungicide fludioxonil, the dicarboximide fungicide iprodione, and the benzimidazole fungicide carbendazim. Results of the study showed the existence of benzimidazole- and dicarboximide-resistant strains at frequencies of 61.8 and 18%, respectively. Moreover, for first time, the development of resistance to anilinopyrimidine fungicides by B. cinerea was detected in greenhouse vegetable crops on the island of Crete. High resistance frequencies of 49.1 and 57.4% were observed for pyrimethanil and cyprodinil, respectively. In addition, one isolate was found to be resistant to the hydroxyanilide fungicide fenhexamid, while no strains resistant to the phenylpyrrole fungicide were detected. Among the 55 isolates tested, 13 were resistant only to carbendazim, 6 were resistant only to anilinopyrimidines, 3 were resistant to both benzimidazoles and dicarboximides, 17 were resistant to both benzimidazoles and anilinopyrimidines, 6 were resistant to both dicarboximides and anilinopyrimidines, 1 was simultaneously resistant to benzimidazoles, dicarboximides, and anilinopyrimidines, 1 was resistant to both anilinopyrimidines and hydroxyanilides, and 8 were sensitive to all fungicides tested. A strong cross-resistance relationship was found between the two anilinopyrimidine fungicides tested when log transformed EC50 values of the isolates were subjected to a linear regression analysis (r = 0.71). Despite the detection of several phenotypes with simultaneous resistance to chemically unrelated active ingredients, in none of the remaining possible fungicide pairs was there observed any kind of cross-resistance relationship.