Abstract
In August 2023, disease symptoms were noted on mint plants (Mentha arvensis) in two southwest Idaho fields with 10 to 30% of the plants exhibiting dark brown stem lesions and root necrosis. To identify the causal agent, isolations from symptomatic tissue were conducted. The surface of the disease material was treated with sodium hypochlorite (0.6%) for 1 minute and washed three times using sterile water. Approximately 2 mm³ tissue sections were cultured on water agar supplemented with 0.02% penicillin and 0.08% streptomycin and incubated at room temperature for 3-5 days. Fungal colonies were tentatively identified as Rhizoctonia from right-angle branching and septate hyphal structures, slight constriction and septum near the branch base. Single hyphal tips were transferred to potato dextrose agar (PDA) and grown at room temperature. Approximately five isolates with a consistent macromorphology were observed. These isolates had light brown mycelia and produced 1-10 mm light brown to dark irregularly shaped sclerotia after a month at ambient temperature with no exposure to continuous light. A representative isolate, designated 23D360 was selected for sequencing and pathogenicity testing. Isolate 23D360 mycelia were removed with a scalpel after 7 days of growth for DNA extraction (Synergy Kit, OPS Diagnostics, N.J., USA) and sequencing of the rDNA internal transcribed spacer (ITS) region and translation elongation factor 1α (tef1α) (Sharon et al. 2008). For ITS a 693 bp amplicon was generated and the sequence was submitted to GenBank (Accession PQ068734). NCBI-BLAST indicated this sequence was 100% identical to an isolate previously identified as R. solani AG 5 (Accession MW999177). For tef1α a 708 bp amplicon was generated and the sequence was submitted to Genbank (Accession PV021964). This tef1α sequence also had a 100% identical match to a previously characterized R. solani AG 5 strain (Accession OP832176). Final confirmation of 23D360 was done using an AG 5 specific real-time PCR assay which targets the beta-tubulin gene (Budge et al. 2009). Pathogenicity testing was performed twice on thirteen 1-week-old mint transplants cultivar 'Black Mitcham Scottish' grown in premium potting compost (Scotts) in greenhouse conditions (30°C, 12h daylight). Barley seeds soaked in water overnight were autoclaved twice, inoculated with a mycelial plug of isolate 23D360 and incubated for 2 weeks at ambient room temperature (25°C). Barley seed inoculum (5 g) was placed around each transplant at the root and stem convergence point. Thirteen 1-week-old transplants were mock-inoculated with autoclaved barley seeds as a control. Approximately 23% damping-off incidence was observed on inoculated transplants five days post-inoculation, while control transplants remained healthy. At 21 days post-inoculation, plants were assessed for visible stem lesions and root necrosis. The incidence of stem lesions occurred on 80% of seedlings, with all transplants with stem lesions ranging from 1-4 cm in diameter and root necrosis. No inoculated transplants were free of stem lesions and root necrosis. R. solani was re-isolated from symptomatic tissue, with a 90% isolation frequency, and rDNA ITS sequencing was used to confirm identity as AG 5, thus confirming Koch's postulates. R. solani was reported in mint in Florida (Alfieri et al., 1984), Indiana (Green, 1961), North Carolina (Grand, 1985), the Pacific Northwest of Oregon (Horner, 1952), and Washington (Shaw, 1973) but not in Idaho. No information on anastomosis groupings was reported in previous literature. Nitzan et al. (2012) identified a new disease affecting Mentha longifolia, a species of mint produced and exported from Israel. Two isolates from symptomatic plants were identified as R. solani AG 1-IB and 4 HG-I. To our knowledge, the isolate 23D360 described here, is the first record of R. solani AG 5 in mint in Idaho. Idaho ranks second in U.S. peppermint production where much of the mint is concentrated in the southwest of the state, this report will create awareness among mint growers on the presence of this fungal disease.