First report of Rhizoctonia solani AG 4 causing brown bean of lima bean in Delaware.

Abstract

Lima bean production has been an economically valuable staple in Delaware agriculture for almost a century, with annual revenue approaching 8 million dollars (USDA-NASS, 2019; Evans et al. 2007). From 2019-2021, lima beans displaying symptoms of brown discoloration, referred to as "brown bean" were observed in the green baby lima variety 'Cypress' across multiple commercial and research fields. Symptoms were present in approximately 1-5% of beans and not visible until pods were opened for harvest. Thirty-seven symptomatic beans were collected and surface disinfested in 0.85% sodium hypochlorite for 30 s, rinsed in sterile deionized water for 30 s, sectioned into four pieces and plated onto potato dextrose agar (PDA) amended with 50 µg/ml penicillin G and streptomycin sulfate. Petri dishes were incubated at 23ºC and observed for colony morphology. Pure cultures were obtained with tan colonies that had mycelia with right angle branching and septations near the branch, consistent with the description of Rhizoctonia solani Kuhn (Sneh et al. 1991). DNA extraction and pathogen identification was confirmed by sequencing of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA using primers ITS4/ITS5 (White et al. 1990) for thirty-seven isolates collected in 2019 and 2020. Isolates were identified as Rhizoctonia solani AG 4 (99.9% sequence identity with GenBank Accession [MN106359.1].) A representative isolate was selected to complete Koch's postulates and the sequence was deposited in GenBank as accession number MW560551. To observe colonization ability, 10 detached pods were sterilized in 75% EtOH for 60 s, then rinsed in Milli-Q water. The detached pods were divided among two 150 mm Petri dishes containing a single 150 mm filter paper saturated with Milli-Q water. Five 1 mm2 agar plugs colonized with the representative R. solani isolate were placed 0.5 cm apart along the length of the pod. Plates were sealed with parafilm and left at room temperature. Control pods were kept in identical conditions but inoculated using clean agar plugs. The trial was repeated and a second trial was conducted on 12 attached asymptomatic pods from C-Elite Select lima bean plants at the succulent seed stage to complete Koch's Postulates. Pods were surface disinfested with 70% ethanol. Three attached pods were wounded with the tip of a sterile scalpel blade where a colonized agar plug was placed and loosely wrapped with a thin parafilm layer to maintain contact. Three attached pods not wounded were also inoculated with a colonized agar plug and wrapped by parafilm. Three wounded and non-wounded pods received clean agar plug controls. Both attached and detached pods were kept at room temperature for one week until symptoms began to manifest on the pod surfaces, at which point the beans from infected pods were removed and placed on PDA, three to a plate. In the attached assay, all beans of both wounded and non-wounded pods developed symptoms. The plates were stored in identical conditions and monitored for 5 days until tan colonies were observed. Culture morphology was consistent with the original isolate in all beans. Sequencing of the ITS region confirmed identity as R. solani AG-4. No symptoms were observed on control pods or seeds. Rhizoctonia solani is most frequently associated with symptoms of root rot (Sharma-Poudyal et al. 2015), but no stem symptoms are associated with the late season "brown bean" that has been observed throughout production in recent years. To our knowledge, this is the first report of Rhizoctonia solani AG 4 causing symptoms of brown bean of lima bean in Delaware. In preliminary observations, symptoms seem to be worse in pods that could have had contact with the soil directly or via rain splash. This disease cannot be detected until pods are split open, which has potential to reduce lima bean quality at harvest. Further monitoring should be conducted to quantify yield impacts and develop appropriate preventative and curative techniques.