Building accelerated plant breeding pipelines: Screening to evaluate lima bean resistance to root-knot nematode in diverse inbred lines and segregating breeding populations.

Lima beans (Phaseolus lunatus) are a cornerstone crop of Delaware's processing vegetable industry. Root-knot nematodes (RKN; Meloidogyne spp.) cause galling of root systems which severely reduces yield. Durable host resistance is an effective management strategy for RKN, but availability of resistant lima bean cultivars is limited. To overcome these challenges, breeding pipelines must simultaneously advance pre-commercial lines and identify new resistance sources with potential for incorporation into the breeding program. Inoculated field trials were conducted in 2021-2022 to evaluate three M. incognita resistant, pre-commercial experimental lines for resistance traits and yield potential in comparison to commercial standards, 'Cypress' and 'C-elite Select'. DE1306635 had the highest yield and reduced galling and reproduction compared to 'Cypress', and is a candidate for commercial release. To continue the breeding pipeline, 256 lima bean inbred accessions from around the world were assessed from 2022-2023 in greenhouse and field screenings to identify novel sources of resistance in the lima bean gene pool. This method allows for evaluation and/or advancement of three generations per year. The full panel was initially evaluated for root galling and 60 accessions were selected for additional field and greenhouse screening: 25 large- and 25 small-seeded with the lowest gall ratings, 5 high-gall controls, and 5 commercial standards. Seven accessions with reduced M. incognita galling and reproduction were identified, including two known resistant lines and five newly identified genotypes. The resistance carried by these genotypes will be further characterized to assess their potential use in lima bean RKN resistance breeding.

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

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.

Genome assembly of a Mesoamerican derived variety of lima bean: a foundational cultivar in the Mid-Atlantic USA.

Lima bean, Phaseolus lunatus, is closely related to common bean and is high in fiber and protein, with a low glycemic index. Lima bean is widely grown in the state of Delaware, where late summer and early fall weather are conducive to pod production. The same weather conditions also promote diseases such as pod rot and downy mildew, the latter of which has caused previous epidemics. A better understanding of the genes underlying resistance to this and other pathogens is needed to keep this industry thriving in the region. Our current study sought to sequence, assemble, and annotate a commercially available cultivar called Bridgeton, which could then serve as a reference genome, a basis of comparison to other Phaseolus taxa, and a resource for the identification of potential resistance genes. Combined efforts of sequencing, linkage, and comparative analysis resulted in a 623 Mb annotated assembly for lima bean, as well as a better understanding of an evolutionarily dynamic resistance locus in legumes.