First Report of Fusarium brasiliense Causing Root Rot of Dry Bean in the United States

Michigan is the second largest dry bean (Phaseolus vulgaris L.) producer in the United States and is ranked first in the production of black beans. Stand establishment and root rots are constant constraints in dry bean cultivation owing to susceptibility of germplasm. To determine which pathogens are responsible, surveys were conducted on Michigan State University research farms and in commercial dry bean fields in Michigan during 2014 to 2017. Fungi were isolated from symptomatic dry bean roots on water agar amended with metalaxyl (15 µg/ml) and streptomycin (300 µg/ml) at room temperature (22°C) under laboratory lighting conditions for 7 to 10 days. Single spore isolates were obtained from sporodochia, which developed on cultured root tissue. Fusarium isolates F-14-42, F-15-46, and F-15-174 were collected from Montcalm, Huron, and Gratiot Counties in Michigan, respectively. Colony morphology and color of isolates grown on full-strength potato dextrose agar at 25°C under dark conditions for 10 days displayed undulated margins, with variations in color of white, with tints of yellow, pink, or greyish-blue. On spezieller nahrstoffarmer agar or carnation leaf agar medium at 25°C under dark conditions for 10 days, sporodochia were a yellowish-white to pale yellow, and macroconidia were falcate, mostly four-septate, apex rounded or hooked ventrally, and base with a distinct foot cell (45.8 to 52.0 × 5.0 to 6.4 μm on average). DNA of each isolate was extracted using a cetyltrimethylammonium bromide method (Cubero et al. 1999), and the ribosomal intergenic spacer (IGS) region and partial translation elongation factor (EF-1α) were amplified by polymerase chain reaction with primer pairs NL11/CNS1 (Aoki et al. 2003) and EF1/EF2 (O’Donnell et al. 1998), respectively. A BLAST search of IGS and EF-1α sequences from the three isolates showed they were 100 and 99% identical with Fusarium brasiliense NRRL 22678 (GenBank accession nos. FJ919501 and AY320158). Phylogenetic analysis of IGS sequences of Fusarium solani species complex clade 2 revealed that the sequences of three isolates and two strains of F. brasiliense (NRRL 22678 and NRRL 31757) were clustered together in a group, which was supported by a bootstrap value of 97%. Based on morphological and sequence analysis, the isolates were identified as F. brasiliense (Aoki et al. 2005). All sequences obtained in this study were deposited into GenBank (accession nos. MG967556 to MG967561). To determine pathogenicity of isolates to dry bean, a seedling pathogenicity screening was performed on dark red kidney (cv. Red Hawk) and black (cv. Zorro) seed types. Each isolate was grown on Nash–Snyder medium at room temperature under laboratory lighting conditions for 21 days and inoculated to sterilized sorghum grain and incubated for 4 weeks to generate inoculum. Twelve grams of infested inoculum mixed with 250 ml of vermiculite was poured into a pot, with an additional 70 ml of vermiculite placed over the inoculum mixture. Six bean seeds were sown in each pot and covered with 70 ml of vermiculite. After 14 days, roots were harvested and washed for disease severity rating and measurement of dried root mass. Roots infected by F. brasiliense exhibited a large lesion of rot on the tap root, 20 to 50% reduction of root mass, and a disease severity rating of 4 on a 1 to 7 scale. The experiment was conducted twice with five replicate pots (six seeds in each pot) per experiment. The fungus was reisolated from the diseased roots and confirmed to be F. brasiliense. Soybean sudden death syndrome caused by F. brasiliense has been reported in Brazil and Argentina (Aoki et al. 2005; O’Donnell et al. 2010) and South Africa (Tewoldemedhin et al. 2017). Significantly, this is the first record of F. brasiliense causing root rot of dry bean in the United States.