A tomato field in Qianjiang County, Hubei Province, China, was surveyed for gray mold in April 2013. Diseased leaves with V-shaped lesions along the margin and masses of grayish hyphae and conidia on the surface were collected from different plants. Eight Botrytis isolates were obtained from eight symptomatic leaves by plating the conidia from each leaf onto potato dextrose agar (PDA). A representative isolate (No. 116) was compared to two reference isolates, B. cinerea B05.10 (from Z. H. Ma, Zhejiang University, China) and B. pseudocinerea 10091 (from A. S. Walker, INRA, France) for morpho-cultural and molecular features. On PDA at 20°C, isolate 116 grew 13.8 mm/day (n = 9), which was similar to those of isolates 10091 (13.7 mm/day), and B05.10 (14.6 mm/day). The isolates all formed black sclerotia of similar shape and size (2 to 13 × 1 to 7 mm). To induce conidia production, the isolates each were inoculated onto tomato fruit (cv. Hezuo 903, Jiangsu Seed Co., China) using colonized agar plugs (each 6 mm in diameter), with four plugs per fruit and four fruits tested per isolate. After incubation of the fruit for 10 days (20°C), abundant conidia were produced on the fruit surface. The conidial size of isolate 116 (6.8 to 14.3 × 6.1 to 10.2 μm) was similar to that of isolates 10091 (7.7 to 12.2 × 7.0 to 9.8 μm) and B05.10 (7.0 to 14 × 6.6 to 10.5 μm). The three isolates were indistinguishable morphologically. The sequences of each of four nuclear genes (Bc-hch, G3PDH, HSP60, and MS547) and the microsatellite Bc6 locus (1,4) were determined and analyzed for each isolate. DNA was extracted from mycelium of each isolate and used as a template to amplify each gene by PCR using specific primers (1,2,4). Bc-hch-RFLP genotyping of the 1,171-bp amplicon (2,4) showed that isolates 116 and 10091 had a 601-bp DNA product, whereas B05.10 had a 517-bp product. The G3PDH, HSP60, and MS547 sequences of isolate 116 (GenBank Accession Nos. KJ534270, KJ534271, and KJ534273, respectively) and those of B. aclada, B. calthae, B. cinerea, B. pseudocinerea, and Sclerotinia sclerotiorum (3) were used for phylogenetic analysis. Isolate 116 and eight B. pseudocinerea isolates formed a subclade with 100% bootstrap support. Furthermore, two DNA markers, 86 bp for isolates 116 and 10091 vs. 170 bp for B05.10 were identified at the Bc6 locus. These results suggest that isolate 116 belongs to B. pseudocinerea (1,4). Pathogenicity of each isolate was tested by inoculation of each of five newly expanded tomato leaves on a 50-day-old plant (cv. Hezuo 903, Jiangsu Seed Co) with a 20-μl droplet of a conidial suspension (1 × 105 conidia/ml), using a pipette. Five noninoculated control leaves were treated similarly with water. The plants were all maintained at 20°C and 100% RH for 72 h, and lesion diameter was then measured. While control leaves remained asymptomatic, leaves inoculated with isolates 116, 10091, and B05.10 developed necrotic lesions averaging 19 to 20 mm in diameter. A fungus re-isolated from the lesions on isolate-116–inoculated leaves formed colonies with morphology identical to that of the original isolate 116. To our knowledge, this is the first report of B. pseudocinerea on tomato in China. The remaining seven isolates were identified as B. cinerea based on Bc-hch-RFLP genotyping (data not shown), suggesting that B. pseudocinerea may infect tomato plants at a low frequency in this region of China.
References: (1) E. Fournier et al. Mol. Ecol. Notes 2:253, 2002. (2) E. Fournier et al. Mycologia 95:251, 2003. (3) P. R. Johnston et al. Plant Pathol. 63:888, 2014. (4) A. S. Walker et al. Phytopathology 101:1433, 2011.