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.

Pseudomonas syringae pv. phaseolicola Uses Distinct Modes of Stationary-Phase Persistence To Survive Bacteriocin and Streptomycin Treatments.

Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of their persistence in the presence of membrane-disrupting biological compounds is less well understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here, we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population in the redox and membrane permeability state. Intact cells were sorted into three fractions according to metabolic activity, as indicated by a redox-sensing reporter dye. Streptomycin persisters were cultured from the fraction associated with the lowest metabolic activity, but tailocin persisters were cultured from a fraction associated with an active metabolic signal. Cells from culturable fractions were able to infect host plants, while the nonculturable fractions were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct metabolic states associated with antibiotic persistence, tailocin persistence, and loss of virulence and demonstrates that tailocin is highly effective in eliminating dormant cells.IMPORTANCE Populations of genetically identical bacteria encompass heterogeneous physiological states. The small fraction of bacteria that are dormant can help the population survive exposure to antibiotics and other stresses, potentially contributing to recurring infection cycles in animal or plant hosts. Membrane-disrupting biological control treatments are effective in killing dormant bacteria, but these treatments also leave persister-like survivors. The current work demonstrates that in Pph, persisters surviving treatment with membrane-disrupting tailocin proteins have an elevated redox state compared to that of dormant streptomycin persisters. Combination treatment was effective in killing both persister types. Culturable persisters corresponded closely with infectious cells in each treated population, whereas the high-redox and unculturable fractions were not infectious. In linking redox states to heterogeneous phenotypes of tailocin persistence, streptomycin persistence, and infection capability, this work will inform the search for mechanisms and markers for each phenotype.