Members of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) species complex cause bottom rot on lettuce (Latuca sativa) and yield losses up to 70% (Subbarao et al. 2017). Severe symptoms include necrosis, stem rot, and/or discoloration especially on the leaf midrib. In Vermont, vegetable farms are small (0.5-30 acres) and grow lettuce concurrently with other vegetable crops in the same field but the AG(s) that causes the disease in Vermont has not been determined. Isolates (n = 157) were collected from 31 fields with reported history of bottom rot between July 10 and October 8, 2019, across Addison, Caledonia, Chittenden, Franklin, Lamoille, and Orleans counties. Isolates were collected from lettuce tissue or potato (Solanum tuberosum), a common rotation crop, or uncropped soil baited using radish (Raphanus sativus). Pieces of tissue (5-10 mm) were cut from the leading margin of lesions, surface disinfested with 0.1% NaClO for 1 min followed by 2 rinses with sterile water, blotted dry, and plated onto acidified 2% water agar (0.085% lactic acid, pH 4.8). After incubation for 48 to 72 h, mycelia resembling Rhizoctonia were examined for morphological characteristics including hyphal branching at ca. 90o angles, a septum near the branching point, multiple nuclei per cell, and lack of both clamp connections and conidia (Sneh et al. 1991). Colonies were white to dark brown, and some produced small sclerotia. Koch's postulates were performed by inoculating nine 8-week-old (9 leaf pairs) romaine lettuce plants (Johnny's Seeds, Winslow, ME, cv. Monte Carlo) per isolate. Isolates were grown on 2% potato dextrose agar for 1 week, from which a 5-mm agar plug was placed on the adaxial leaf surface at the base of a petiole. Plants were enclosed in a plastic bag to maintain high humidity and grown under a 16-hour photoperiod at 24 °C. Disease severity was rated 4 days after inoculation (0: healthy, 1: isolated lesions, 2: lesions across multiple petioles, and 3: systemic disease). Putative AG were determined by Sanger sequencing of the internal transcribed spacer (ITS) region using the ITS1F and ITS4B primer pair (758 bp) (Gardes and Bruns 1993). Contigs were assembled using CAP3 software (Huang and Madan 1999). Taxonomy was assigned to each OTU via the NCBI BLASTn database with criteria as 0.0 E and nucleotide match of at least 97%. Of the 10 isolates sequenced with sufficient coverage (735 to 784 bp alignment length) and definitive resolution (96.7 to 99.9% identity), 5 were putative AG 1-IB (Genbank Accession HG934430.1), 2 AG 1-IC (Genbank Accession AF354058.1), 2 AG 3 (Genbank Accession AF354064.1), and 1 AG 4-HGII (Genbank Accession AF354074.1). Fasta files and metadata are archived at 10.6084/m9.figshare.20301324, 10.6084/m9.figshare.20301375. Putative AG 1-IB was highly virulent on lettuce plants whether it originated from potato (mean 2.6) or lettuce (mean 1.3 to 3). AG 4-HGII and AG 1-IC isolated from lettuce and radish, respectively, were moderately severe (mean 1.4 to 2.2) on lettuce with identical symptoms. The two potato isolates (AG3) were not pathogenic on lettuce. Similarly, higher incidence of AG 1-IB is reported on lettuce in Quebec (Wallon et al. 2021), Ohio (Herr 1993), and Germany (Grosch et al. 2004). Because AG vary in their host range (Sneh et al. 1991), knowing the AG will inform management decisions such as crop rotation and weed control. This is the first report of the causal agent of bottom rot of lettuce or any AG of R. solani in Vermont.
Keywords: Causal Agent; Crop Type; Etiology; Fungi; Subject Areas; Vegetables.