Brown spot, caused by Stemphylium vesicarium, is the main fungal disease of pear in northern Italy where it may cause severe crop losses and it requires numerous fungicide applications. Monitoring work was performed by collecting fungal populations in Po valley between 1995 and 2003 in order to study the dicarboximides resistance already detected in the 1990s for procymidone as a result of control failures in field. Sensitivity tests showed that the resistant strains occur all over the monitored areas. Where present the efficacy of procymidone in field is completely lost in spite of what is observed often in other fungi. In most of the isolates (phenotype R 1 ), S. vesicarium resistance level to procymidone (Sialex) was shown to be very high (RF@3000) whereas it was lower towards the other dicarboximides iprodione (Rovral), vinclozolin (Ronilan) and chlozolinate (Serinal) (RF@10). Therefore the resistance is partially crossed even if a high level of resistance was rarely observed for all dicarboximides (phenotype R 2 ). At least two different mechanisms of resistance seem to be involved: one that may provide a moderate resistance and the other that may give a high resistance level. Monospore isolate sensitivity tests confirmed the qualitative response suggested by such high resistance factors.
In Stemphylium vesicarium, four phenotypes were recognized according to their in vitro responses to dicarboximide fungicides: S (sensitive), S + (low resistant to iprodione and procymidone but moderately resistant to vinclozolin), R 1 (moderately resistant to iprodione and vinclozolin but highly resistant to procymidone), R 2 (highly resistant to all dicarboximides). Cross-resistance was observed between dicarboximides and aromatic hydrocarbon fungicides in all cases while crossresistance to phenylpyrroles was only detected in R 2 phenotype. Moreover, no changes were noted in sensitivity to oxidative and osmotic stress inducers. An osmosensing histidine kinase gene, homologous to OS1 from Neurospora crassa, was sequenced from several field isolates of Stemphylium vesicarium. This gene is predicted to encode a 1,329 amino acid protein, comprising a conserved histidine-kinase domain in the C-terminal region and six tandem repeats of about 90 amino acids at the N-terminal end. In S + and R 1 phenotype isolates, a single amino acid substitution was observed in the first amino acid repeat; F267L and L290S respectively. For the R 2 isolates, the exchanges T765R or Q777R were located within the histidinekinase domain.
Up to 2005 the sensitivity of Stemphylium vesicarium (Wallr.) Simm., the causal agent of pear brown spot, to the strobilurin fungicides kresoximmethyl, trifloxystrobin and pyraclostrobin was still comparable with baseline values associated with good efficacy in the field. During 2006, the first resistant isolates were detected in two commercial pear orchards in the Emilia-Romagna region (Italy), one of which was affected by considerable control failure linked to strobilurin treatments as demonstrated in a field trial. In vitro sensitivity tests with 0.5 mg l −1 of kresoxim-methyl, trifloxystrobin and pyraclostrobin showed that in the population collected in the orchard with control failure the conidial germination was greater than 90% compared to an untreated control both in 2006 and in 2007, i.e. 1 year after the suspension of strobilurin applications. In the other orchard, where only a few symptomatic fruits were found and the strobilurins were still in use, the conidial germination was lower, about 50% in 2006 and 25% in 2007. The molecular analysis of mitochondrial cytochrome b gene of some monospore isolates with different levels of sensitivity confirmed the presence of the mutation causing G143A substitution in all the resistant isolates. In conclusion, both in vitro tests and molecular analysis confirmed the first occurrence of Stemphylium vesicarium resistance to all strobilurin fungicides tested.
In vitro sensitivity assays were able to discriminate the three population types with different scab management, while the qualitative PCR analysis (CAPS) was only partially reliable. High sensitivity differences among V. inaequalis populations with good and poor field control by strobilurins were observed.
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