Botrytis cinerea has previously been shown to consist of two sibling species, referred to as Group I and Group II, that can be differentiated by PCR-RFLP analysis of the Bc-hch gene, a vegetative incompatibility locus (1). Group I has recently been described as a new cryptic species, B. pseudocinerea (4). Gray mold caused by B. cinerea is a major postharvest disease of blueberries in the Central Valley of California. In 2012 and 2013, blueberry fruit were sampled at harvest from various locations in the region and stored at 0 to 1°C for 5 weeks, and fungi were isolated from decayed fruit. In total, 526 isolates of Botrytis spp. were obtained. Genomic DNA was extracted and PCR-RFLP of a fragment of the Bc-hch gene was performed. Four isolates showed the distinctive restriction band pattern associated with Group I (1). The identity of these four isolates was further investigated by sequencing portions of four genes: internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPBII), using the primers described previously (3,4). Sequences were deposited in GenBank (Accession Nos. KJ796643 to 58). BLAST analysis showed that sequences of all four genes for the four isolates were 99.8 to 100% similar to those of B. pseudocinerea. Morphological characteristics of the four blueberry isolates were examined as described previously (4). On potato dextrose agar, colonies were gray; the mycelial growth rate was 26 mm/day at 19°C in the dark. Conidiophores were simple and erect, and conidia were borne in grapelike clusters, one celled, hyaline, elliptical to ovoid, 6.5 to 15.7 × 5.6 to 9.8 μm (average of 7.4 × 10.1 μm). As reported previously, none of the morphological characters was able to differentiate between B. cinerea and B. pseudocinerea (4). To test pathogenicity, freshly harvested organic blueberry fruits were treated with 0.5% sodium hypochlorite for 2 min, rinsed with sterile water, wounded using a sterile needle, and inoculated by placing 1 μl of a conidial suspension (1.0 × 105 spores/ml) from each isolate into the wound with a pipette. Inoculated fruit (10 for each isolate) were incubated at 20°C for 5 days in the dark. Experiments were performed twice. All inoculated fruit developed rot, and no decay was observed on the noninoculated controls. All four isolates of B. pseudocinerea were pathogenic, and the fungus was re-isolated from decayed fruit. B. pseudocinerea isolates are known to be naturally insensitive to fenhexamid (1,4). Sensitivity of the four isolates to fenhexamid was examined in vitro as previously described (4). The EC50 values for fenhexamid for the four isolates ranged from 7.7 to 9.9 μg/ml and isolates were considered resistant to fenhexamid (1,4). Based on the morphological, physiological, and genetic characteristics, the four blueberry isolates were identified as B. pseudocinerea. It appeared that this species was present at very low frequency (0.76%) in blueberry fields in California. Previously, B. pseudocinerea has been reported from French, German, and New Zealand vineyards (1,2,4). To our knowledge, this is the first report of B. pseudocinerea causing gray mold in blueberry in California and in North America.
References: (1) E. Fournier et al. Mycologia 95:251, 2003. (2) P. R. Johnston et al. Plant Pathol. 63:888, 2014. (3) M. Staats et al. Mol. Biol. Evol. 22: 333, 2005. (4) A.-S. Walker et al. Phytopathology 101:1433, 2011.
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