EPIDEMIOLOGY AND INTEGRATED MANAGEMENT OF BACTERIAL LEAF SPOT CAUSED BY PSEUDOMONAS SYRINGAE PV. APTATA IN TABLE BEET AND SWISS CHARD SEED PRODUCTION IN WESTERN WASHINGTON

Marilen Nampijja

doi:10.7273/000007433

Bacterial leaf spot (BLS), caused by Pseudomonas syringae pv. aptata (Psa), has become more prevalent in table beet (Beta vulgaris subsp. vulgaris Condivita Group) and Swiss chard (Beta vulgaris subsp. vulgaris Cicla Group) seed production in the Pacific Northwest (PNW). This disease has caused significant economic losses due, in part, to increased demand for seed to plant baby leaf beet and Swiss chard crops which have become more popular as a result of the convenience and perceived health of these leafy green products. The PNW region of the United States produces up to 40% of the global supply of beet and chard seed (Western Washington Seed Advisory Committee, personal communication). Psa is a seedborne and seed transmitted pathogen that is characterized by testing positive for levan production; negative for oxidase, pectinolytic activity, and arginine dihydrolase production; and positive for tobacco hypersensitivity (+---+) LOPAT reaction. The objectives of this study were to: 1(i) determine the prevalence and genetic diversity of Psa in table beet and Swiss chard seed crops in western Washington, and (ii) identify genetic factors that differentiate strains of P. syringae pv. aptata pathogenic to beet and chard from non-pathogenic strains of P. syringae, and that differentiate strains pathogenic to beet and chard vs. beet only; (2) investigate the colonization of table beet and Swiss chard plants by Psa in the vegetative and reproductive growth stages; and 3 (i) determine the location of infection of table beet and Swiss chard seed by Psa, (ii) evaluate hot water and bleach seed treatments for management of seedborne Psa, and (iii) evaluate potential use of decortication for control of seedborne Psa. BLS was present in 72% of the beet and chard seed crops surveyed in 2020 but absent in all seed crops surveyed in 2021 and 2023. Pathogenicity tests on beet and chard seedlings demonstrated variation in virulence among Psa isolates from the beet and chard crops surveyed, with some isolates more virulent on beet than chard. A wide diversity of Psa strains was detected in surveys of beet and chard seed crops, based on multi-locus sequence analysis (MLSA) and whole-genome sequencing (WGS) of 38 strains. However, non-pathogenic isolates of P. syringae of these crops also grouped with pathogenic isolates, with genetic evidence of possible horizontal gene transfer between pathogenic and non-pathogenic isolates. Of 192 isolates, there were 25 from table beet seed crops, and one isolate from a table beet fresh market crop that were 100% identical to the pathotype strain CFBP 1617PTof Psa based on MLSA. Based on WGS, Type III effectors of the Psa strains are located in the pathogenicity island comprised of the conserved effector locus (CEL), the hypersensitivity response and pathogenicity (hrp)/HR and conserved genes(hrc), and the exchangeable effector locus (EEL), with the EEL being the most variable of these three loci among strains. Greenhouse trials were completed with two strains of Psa tagged with a green fluorescent protein (GFP), GFP-Pap009, which causes BLS on table beet and Swiss chard; and GFP-Pap014, which causes BLS on table beet only. Both strains colonized both the epidermis and apoplast of inoculated table beet and Swiss chard leaves, contrary to the hypothesis that Pap014 is non-pathogenic on Swiss chard if it can only colonize the epidermis, not the apoplast, of chard leaves. However, GFP-Pap009 was recovered at significantly greater populations (~500-fold) than GFP-Pap014 by 72 h after inoculation. Confocal microscopy revealed that Psa colonization is concentrated along the guard cells of stomata and the walls of spongy mesophyll cells in the apoplast, indicating that stomata may be important entry points for infection. A table beet seed crop field trial was inoculated with a rifampicin resistant strain of Psa (rif-Pap010) in each of three years (2021-2023) to monitor development of Psa and BLS in the seed crop. Fluctuating environmental conditions over the duration of each season had a significant impact on Psa population and BLS development. The amount of rif-Pap010 recovered from samples in each table beet seed crop trial varied over the duration of the trial. The rif-Pap010 population increased following each inoculation but declined when conditions remained dry. In 2021, seedborne rif-Pap010 was detected on the harvested seed compared to the 2022 harvested table beet seed at ~200-fold greater population (mean of Log104.9 ± 0.1 vs. Log102.5 ± 0.4 CFU/g seed), respectively. The recovery of rif-Pap010 population from each of three asymptomatic weed hosts, lambsquarters (Chenopodium album), pigweed (Amaranthus retroflexus) and ladysthumb (Persicaria maculosa), also varied widely over the trial duration, but demonstrated that weeds could serve as reservoirs for the pathogen in seed crops. Rif-Pap010 was only recovered from the pericarps, not the embryos, of the seed lots harvested from the inoculated beet seed crop field trials in 2021, 2022, and 2023. Chlorine seed treatment (1.2% NaOCl for 5, 15, 25, 35 minutes) eradicated rif-Pap010 from the 2021 seed lot at all durations of treatment but not from the 2023 seed lot. Evaluation of hot water seed treatments using the 2021 and 2023 harvested seed lots from the field trials revealed that treatment at 55℃ for 40 or 50 minutes eradicated rif-Pap010 without affecting seed quality (germination) adversely. Although treatment at 60℃ was highly effective for eradication of Psa at all durations, treatment for >30 minutes reduced seed germination. For the decortication trial with 2021 seed lot, the highest amount of rif-Pap010 was recovered from the powder removed from the seed by decortication of the table beet seed samples (log104.9 ± 0.0 CFU/g powder) recovered compared to (log104.7 ± 0.1 CFU/g seed for the non-decorticated seed), and (log103.9 ± 0.1 CFU/g seed) for the decorticated seeds. This study unravels some of the complexities of the epidemiology, genetic diversity, and management of Psa in table beet seed production in the Pacific Northwest USA.

EPIDEMIOLOGY AND INTEGRATED MANAGEMENT OF BACTERIAL LEAF SPOT CAUSED BY PSEUDOMONAS SYRINGAE PV. APTATA IN TABLE BEET AND SWISS CHARD SEED PRODUCTION IN WESTERN WASHINGTON

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