PLANT HEALTH PROGRESS Vol. 16, No. 3, 2015 Page 134

Plant Health Brief

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Brassica carinata in South Dakota

C. Tande, A. Gebreil, F. Mathew, K. Grady, and E. Byamukama, Department of Plant Sciences, South Dakota State University, Brookings 57007

Accepted for publication 31 August 2015. Published 10 September 2015

Tande, C., Gebreil, A., Mathew, F., Grady, K., and Byamukama, E. 2015. First report of charcoal rot caused by Macrophomina phaseolina on Brassica carinata in South Dakota. Plant Health Progress doi:10.1094/PHP-BR-15-0021.

In August 2014, diseased Brassica carinata (commonly known as Ethiopian mustard) plants growing in Hughes County, South Dakota (44°39.00′ N 099°99.00′ W), were found with symptoms typical for charcoal rot. The incidence of symptomatic plants in the field was <1%. Symptoms consisted of whitish-gray lesions girdling the stem near the soil line (Fig. 1, top), causing pre-mature death and lack of seed fill. When the stems were split longitudinally, black microsclerotia were observed in the vascular tissue of the plants (Fig. 1). To recover the causal agent, sympto-matic stem tissues from two plants were surfaced sterilized in a 10% sodium hypochlorite, rinsed in sterile distilled water, and then placed onto potato dextrose agar (PDA). The PDA plates were incubated at 30°C under 12 h of light and 12 h of darkness. After two weeks, the plated tissues produced fungal colonies that were identified as Macrophomina phaseolina (Tassi) Goid. based on colony color (charcoal black), colony morphology (agar pigmentation, growth characteristics, and microsclerotia size) and the production of microsclerotia. Microsclerotia ranged in size from 0.66 mm to 0.77 mm in diameter (Fig. 2).

DNA was extracted from the mycelium of the two isolates using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), and the ITS region was amplified and sequenced using primers ITS1 and ITS2 (White et al. 1990). BLASTn analysis of the approxi-mately 600-bp fragments (GenBank Accession Nos. KR815495 and KR815496) identified from the two isolates shared 99% homology with Macrophomina phaseolina (Accession No. GU046902) from soybean. To complete Koch’s postulates, a pathogenicity test was performed using a modified layer test method (Schmitthenner and Bhatt 1994). A styrofoam cup was 2/3 filled with vermiculite, and a M. phaseolina isolate that had fully colonized a PDA plate was placed on top of the vermiculite layer. An 8-g layer of vermiculite was added on top of the agar. Fifteen to twenty B. carinata seeds were then placed on top and more vermiculite (1 g) was added to barely cover the seeds. The control utilized a clean agar plate. Seedlings were kept in the greenhouse under normal light (12 h darkness and 12 h light) and moisture for two weeks. After two weeks, plants were transferred to the growth chamber and were subjected to water stress and warm temperatures (30°C) to induce symptoms of charcoal rot. After four weeks in the growth chamber, plants began to show

characteristic signs of charcoal rot. Greyish lesions on the lower stem of infected plants were surface sterilized and plated onto PDA, and M. phaseolina was re-isolated and confirmed based on morphology and production of microsclerotia. The pathogen was not isolated from the control plants. This report indicates that B. carinata is a host for M. phaseolina and if susceptible crops (such

FIGURE 1 Microsclerotia of Macrophomina phaseolina embedded inside the lower stem of Brassica carinata .

Corresponding author: E. Byamukama. Email: ebyamukama@gmail.com

doi:10.1094 / PHP-BR-15-0021 © 2015 The American Phytopathological Society

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as soybean and corn) are planted following B. carinata, presence of inoculum combined with drought conditions can favor charcoal rot development and cause yield loss. To the best of our know-ledge, this is the first report of M. phaseolina on B. carinata in South Dakota.

LITERATURE CITED

Schmitthenner, A. F., and Bhat, R. G. 1994. Useful methods for studying Phytophthora in the laboratory. OARDC Special Circ. 143. Ohio State University, Wooster.

White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA.

FIGURE 2 Microsclerotia of Macrophomina phaseolina in culture at 40× (top) and at 100× (bottom).