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Gray Mold Caused by Botryotinia fuckeliana on Edible Pods of Pea in Brazil

Gray mold on edible pods of snow pea (Pisum sativum Lam. [Fabaceae]) was observed in greenhouse-cultivated pea (cvs. Luana Gigante and Gigante Flor Roxa) in the city of Pelotas (Rio Grande do Sul, Brazil) in September and October 2012. The incidence of diseased pods was high (∼25% of immature pods) after up to 3 cloudy and rainy days that hindered the ventilation inside the greenhouse resulting in high relative humidity. Infection occurred first on senescing petals adhered to the forming pods, leading to pod abortion or rotting that began at the contact site with the infected petal. The first symptoms on pods included water soaked tissue that quickly turned light brown and progressed to necrosis. Conidia and conidiophores produced on profuse gray mycelium could be easily seen on infected tissue 2 to 3 days after the appearance of symptoms. Conidiophores were smooth-walled, 400 μm to over 1.5 mm long, hyaline to pale brown, and branched in their upper part; each branch ended with a hemispherical or spherical swelling, 5 to 9 μm in diameter with minute sterigmata. Macroconidia were globose, ellipsoidal, smooth, hyaline to pale brown, usually with protuberant hila, 7 to 15 × 5 to 9 μm. Microconidia were not observed. On potato dextrose agar (PDA), colonies were fast-growing, white, low, covering entire 10 cm petri plates in 4 to 5 days when they turned gray to brownish-gray. Conidiophores and conidia were often formed in sectors. Shield-like, elliptical, lenticular to irregular, black, 1.5 to 6.0 × 1.0 to 4.0 mm sclerotia developed in 10-day-old colonies incubated at room temperature. Genomic DNA was extracted from conidia, conidiophores, and mycelium and used to amplify both the internal transcribed spacer (ITS) (ITS1-5.8s-ITS2) region and the β-tubulin gene using the ITS1/4 and Bt2a/b primers, respectively (1,4). The ITS (541 bp) and β-tubulin (467 bp) sequences were deposited in GenBank under accessions KC683713 and KC683712, respectively. BLASTn searches revealed similarity of 100% (EF207415) and 99% (FQ790278) with Botryotinia fuckeliana (De Bary) Whetzel for the ITS and β- tubulin sequences, respectively. Based on morphological characteristics and sequence analysis, the pathogen causing pod rot of peas was identified as B. fuckeliana. To fulfill Koch's postulates, 10 unwounded pods of P. sativum ‘Luana Gigante’ were inoculated by depositing PDA plugs (5 mm) colonized with fungal mycelium on their surface. Non-inoculated and mock-inoculated pods with sterile PDA plugs served as control. Inoculated and control pods were incubated inside a clear plastic box (11 × 11 × 3.5 cm) and over moistened filter paper under 12-h photoperiod at 25 ± 1°C. A surrounding water-soaked halo was visible only on pods inoculated with the fungus 48 h after inoculation (hai). Intense sporulation and necrosis were visible 96 hai. Botrytis spp. was previously detected, through standard blotter test, on seeds of P. sativum in Brazil, but without pathogenicity test nor its transmission through seeds (2,3). To our knowledge, this is the first report of B. fuckeliana causing epidemics on pea pods in Brazil. The high incidence of the disease in a protected environment has the potential to cause significant economic impact due to its damage to the pods, rendering them unmarketable.

References: (1) N. L. Glass and G. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) M. A. S. Mendes et al. Fungos em Plantas no Brasil. Embrapa-Cenargen, Brasília, 1998. (3) W. M. Nascimento and S. M. Cícero. Rev. Bras. Sementes 13:5, 1991. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.