Yellow stunt, an emerging disease of tobacco (Nicotiana tabacum), has become increasingly prevalent in tobacco-growing regions in southern Brazil. Major symptoms are moderate to severe stunting, yellowing of leaves, severe wilting, darkened roots, necrosis of stem tissue directly above the soil line, and plant death. Phytophthora glovera was first proposed in 1999 as the primary causal agent of yellow stunt (1), but since then, there has been no data or completion of Koch's postulates to support this. Fifty-six isolates of fungi and fungus-like organisms were obtained from stem and root samples of tobacco plants with typical symptoms of yellow stunt in the Brazilian States of Rio Grande do Sul, Santa Catarina, and Paraná during the growing seasons of 2004/05 to 2006/07. They were identified to species level by analysis of the morphological characteristics (2) and sequence of rDNA internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) (3). The isolates were identified as Pythium dissotocum (29), Fusarium oxysporum (10), P. graminicola (5), Rhizoctonia solani (4), F. solani (3), P. ultimum (3), P. deliense (1), and P. inflatum (1). The ITS sequences of the 29 isolates identified as P. dissotocum were identical. The nucleotide sequence of one isolate, LFM27/2005, has been deposited in GenBank (GQ495982). Analysis of ITS sequences alone was not sufficient to differentiate this isolate from other species in the Pythium subclade B2, such as P. coloratum, P. lutarium, P. marinum, or P. diclinum. However, the combination of morphological and cultural characteristics (2) and sequence data support our identification of LFM27/2005 and similar isolates as P. dissotocum. Colonies of LFM27/2005 on cornmeal agar had filamentous sporangia and formed slightly inflated, dendroid structures. Zoospores formed at 5°C. Daily growth rate on potato carrot agar was 13 mm at 25°C. The oogonia (22 μm in diameter) were nonornamented and either intercalary or terminal. Antheridia, commonly 1 to 2 per oogonium, were sessile, born on unbranched stalks, and either monoclinous or diclinous. Aplerotic or nearly plerotic oospores measured 20 μm in diameter with a smooth wall 2.5 μm thick. Pathogenicity tests for each pathogen were performed in a greenhouse at ~24°C in pots filled with pine bark substrate infested with inoculum at the time Burley tobacco plants showed five expanded leaves. Each test consisted of five plants and was repeated three times. Inoculum for one to three isolates representative of each pathogen was prepared by growing 2-month-old cultures at 28°C in the dark for 7 days on potato dextrose agar medium overlaid with three sterile oat kernels. Noninfested oat kernels were used for control plants. Forty days after inoculation, only plants inoculated with isolates of P. dissotocum exhibited all symptoms associated with yellow stunt. P. inflatum and R. solani did not induce yellow stunt symptoms and the others induced only wilting and root rot. P. dissotocum was recovered from an inoculated, symptomatic plant, fulfilling Koch's postulates. Its morphology was identical to isolates obtained from original field samples. The results demonstrate the association of isolates of P. dissotocum with tobacco yellow stunt in Brazil.
References: (1) H. D. Shew et al. Phytopathology (Abstr.) 89(suppl):S72, 1999. (2) A. J. van der Plaats-Niterink. Stud. Mycol. 21:1, 1981. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.
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