First occurrence of strobilurin-resistant isolates of Stemphylium vesicarium in an Italian pear orchard

Stemphylium vesicarium (Wallr) Simm. is the causal agent of brown spot, the main fungal disease of pear in Italy (Po valley) since the late 1970s, but it is also present in other European countries such as Spain, France, The Netherlands, Portugal and Belgium. Many fungicides (mainly dithiocarbamates and dicarboximides) have been applied in field to control it. From the late 1990s new products have been introduced and among them the strobilurins kresoxim-methyl (1998), trifloxystrobin (2002) and more recently pyraclostrobin+boscalid (2006). Till 2005 S. vesicarium sensitivity to strobilurin fungicides have not shown any shift (MIC<0.5 mg/l) from baseline values both for kresoxim-methyl and trifloxystrobin according to a good efficacy in field. During the season 2006, the first strobilurin-resistant isolates of Stemphylium vesicarium were detected in a commercial pear orchard in north-eastern Italy (Emilia-Romagna region) where a heavy control failure was observed. In vitro sensitivity tests on conidial germination were carried out on this population with the 3 strobilurins. The concentrations used were 0.5 and 2 mg a.i./l and the evaluations were made after 3, 5 and 24 hours after inoculation. Already after 3 hours of incubation the population showed germination greater than 74% referred to untreated control at both the concentrations tested and towards all strobilurins. Another sensitivity test on conidial germination was carried out using the formulated compounds Stroby (kresoxim-methyl, BASF) and Flint (trifloxystrobin, Bayer) at 2, 10 and 100 mg a.i./l. Also in this case after 3 hours of incubation the germination of the population was greater than 90% of the untreated control. Finally the mitochondrial DNA of the isolates was analysed. After DNA extraction PCR was performed to amplify a gene fragment codifying for the region containing the G143A mutation. The presence of this mutation was then confirmed. In conclusion both in vitro test and genetic analysis showed that these isolates of S. vesicarium were resistant to all strobilurin fungicides tested. This is the first time where a failure in pear brown spot control in field is demonstrated to be caused by the occurrence of resistant isolates.