First Report of Dry Root and Stem Rot of Cowpea Caused by Fusarium proliferatum in the United States

Recently, pathogenicity of Fusarium proliferatum was confirmed in legume crops (Arias et al. 2013; Chang et al. 2015); however, to our knowledge there is no record of F. proliferatum pathogenicity in cowpea (Vigna unguiculata (L.) Walp.). In this report, we confirm F. proliferatum as a causal agent of root rot, stem lesions, and abnormal seedling development in cowpea. Diseased ‘Iron & Clay’ cowpea field plants with symptoms of dry root rot and stem rot were collected from the University of Tennessee Organic Crops Unit, Knoxville, TN, in midsummer 2014. Fungal mycelia were isolated from surface-sterilized (10% commercial bleach, 1 min) root and stem parts of symptomatic plants on 1.5% water agar plates and transferred to potato dextrose agar amended with 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO) and 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) (aPDA). Genomic DNA was extracted from mycelia grown in potato dextrose broth using the Qiagen DNeasy Plant Mini Kit. The translational elongation factor 1-α (EF1α) region was amplified with PCR using EF-1 and EF-2 primers. PCR products were sequenced and the sequences were BLAST searched against sequences in the Fusarium-ID database and GenBank. BLAST using Fusarium-ID analysis indicated that the sequences had 99.7% identity with F. proliferatum isolates and GenBank BLAST revealed 99% identity with F. proliferatum. The resulting 712-bp EF1α sequence was deposited in GenBank (Accession No. KT376486). To complete Koch’s postulates, pathogen inoculum was produced on barley grains, incubated at room temperature for 14 days, and added to potting mix at 1:10 w/w. Fifteen surface-sterilized ‘Iron & Clay’ cowpea seeds (10% commercial bleach, 1 min) were planted in each of four replicate pots. Control pots, without inoculum, were included. The growth chamber experiment was a CRD. After six weeks, plants with severe stem lesions, root rot, and reduced seedling vigor were observed in inoculated pots. Infected plants were collected, surface sterilized, and plated on aPDA and carnation leaf agar (CLA) for morphological identification to confirm the species based on cultural, morphological, and molecular techniques. F. proliferatum isolated from infected plants produced abundant white mycelium with a dark violet pigmentation on agar plates. On aPDA, morphological characteristics of the pathogen were confirmed (Leslie and Summerell, 2006; Chang et al. 2015). Single-celled, club-shaped microconidia were abundant on aPDA and CLA plates along with aerial aggregates of microconidia [4.52 to 10.04 (6.66) × 1.9 to 3.72 (2.7) µm in size]. Macroconidia were sparse, slender and 2-3 septate [28.53 to 15.87 (21.57) × 4.52 to 10.04 (6.66) µm in size]. Molecular identification of isolates from plant tissue was as described above. Fusarium proliferatum has been reported on cowpea seed in association with production of the mycotoxin fumonisin (Kritzinger et al. 2003), and to our knowledge this is the first report confirming dry root and stem rot of cowpea caused by F. proliferatum in the United States. This disease could be potentially problematic if sufficient inoculum levels are present in the field due to postemergence damping off of seedlings. © 2016, American Phytopathological Society. All rights reserved.