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First Report of Leaf Spot of Soybean Caused by Aristastoma guttulosum in China

In fall, 2008, leaf spots were observed during the flowering stage of the Zhong Huang 13 cultivar of soybean in the fields of Anhui Province, China. The leaf spots were irregularly shaped, necrotic, brown-black, and surrounded by yellow halos. Often, on a given leaf, several spots joined one another to form a large blighted area. Finally, those leaves turned yellow followed by defoliation. Damaged leaves showed scattered black spots (i.e., numerous pycnidia) on the lower side. Fresh material was collected from infected plants and a single spore of the putative causal pathogen was isolated on potato dextrose agar (PDA) and incubated at 25°C during a 12-h dark/light cycle. The isolate produced a white fungal colony and black pycnidia after 30 days. The pycnidia are characterized as globose, dark brown-black, and distinctly papillate, with ostiolar setae, and are more or less straight, unbranched, and tapered at the apex. The conidia are clavate, hyaline, mostly with three transverse septa per cell; conidia are either straight or slightly bent, obviously guttulate, and 16 to 29 × 2.5 to 3.5 μm. This pathogen is similar to other Aristastoma guttulosum Sutton (1964), but with the following differences: (a) it has more than 10 versus 4 to 9 setae; (b) conidia are 16 to 29 × 2.5 to 3.5 μm versus 32 to 42 × 3.9 to 4.6 μm as reported for A. guttulosum (1). Conidia of the Chinese isolate were used to inoculate leaves of soybean. Five soybean leaves from potted plants, 1 month old, were sprayed with a suspension of conidia in water. Conidia were harvested from PDA cultures and the suspension was adjusted to 3 × 10⁵ conidia/ml with a hemocytometer. Five leaves were sprayed with sterile distilled water as controls. Inoculated plants were kept in the greenhouse. All five of the inoculated leaves displayed the same symptoms observed in the fields. The symptoms developed initially as brown pinhead spots on the upper side of the leaves, gradually increasing to large brown spots. These spots were irregularly shaped, brown and necrotic in the center and surrounded by a yellow halo. Black pycnidia appeared after 1 week whereas the controls remained asymptomatic. The pathogen was reisolated from the inoculated soybean leaves according to standard Koch's postulates. Primers ITS1 and ITS4 were used in PCR reactions to amplify the internal transcribed spacer region (ITS) (3). Sequencing was performed using the same primers. The ITS sequence (GenBank Accession No. JF825548.1) for this pathogen (587 bp) was submitted to a BLAST search in GenBank. Since the ITS sequence of the genus Aristastoma has never been previously submitted, results did not show high similarity with any extant GenBank sequences. The genus Aristastoma Tehon (1933) was described by Tehon (2). Five of the species in this genus were described by Sutton (1). The number of septate conidium and lack of obvious guttulate within the conidium are the morphological basis to separate these five species. Morphological features of the pathogen from soybean leaves in China were slightly different from those of A. guttulosum. To our knowledge, this is the first report of leaf spot caused by A. guttulosum on soybean in China.

References: (1) B. C. Sutton. Mycological Papers. 97:10, 1964. (2) L. R. Tehon. Mycologia XXV. 25:249, 1933. (3) T. J. White et al. Academic Press, San Diego, 1990.