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Full text release has been delayed at the author's request until December 19, 2024

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Investigating Potential Virulence Genes of Plant Pathogenic Bacterium Pseudomonas syringae pv. syringae Utilizing Whole-genome Sequencing Analysis and Identifying Novel Small Molecule Growth Inhibitors to Manage Pseudomonas Leaf Spot Disease on Pepper Seeds and Seedlings

Ranjit, Sochina
http://rave.ohiolink.edu/etdc/view?acc_num=osu1669921517118292

Abstract Details

2022, Master of Science, Ohio State University, Comparative and Veterinary Medicine.
Pseudomonas syringae pv. syringae (Pss) is an emerging seed-borne pathogen that causes Pseudomonas leaf spot (PLS) disease in bell peppers. It causes severe necrotic lesions on pepper leaves that can spread to 50-80% of the field under favorable environmental conditions. PLS can cause significant economic losses to pepper production if the disease is left uncontrolled. However, not much is known about the genes that Pss carries to be able to cause disease in peppers. It is important to understand the virulence genes that Pss carries so that appropriate measures can be developed to control Pss in peppers. Therefore, part of my research aimed to use comparative genomic analysis to understand the genes in Pss that are important for virulence in pepper seedlings. The Pss strains (n=16) evaluated showed varying levels of virulence (disease severity and Pss population) at 3-, 7-, and 14-days post-infection (dpi) on the susceptible 'California Wonder' pepper variety in a controlled growth chamber environment. The Pss strains also displayed varying growth, biofilm development, and motility in vitro in M9 minimal broth at 28˚C, however, the variation in in vitro performance did not explain the variation in the virulence of the Pss strains in pepper seedlings. Whole genome sequencing was performed on these Pss strains. The genes were functionally characterized, and core genomes were separated from the variable genomes between the Pss strains. A total of 812 genes were variable among the Pss strains including known virulence genes. Additionally, a multivariate correlation analysis identified 285 genes that were significantly correlated to the virulence of Pss in pepper seedlings (r2 of  0.5 to 0.675; P<0.01). The genes that were significantly correlated with the virulence of Pss strains included known virulence genes associated with motility (n=2), biofilm (n=5), and Type III and VI secretion systems (T3SS and T6SS) (n=9). Further, the two strains (SM156-18 and SM226-1) that were least pathogenic on pepper seedlings were missing the variable biofilm and T6SS genes while all other, more pathogenic strains on pepper seedlings carried these genes. In summary, this study showed that variation in gene content could explain variation in the virulence of Pss strains seen in pepper seedlings. This study also identified potential genes that are important for the virulence of Pss in pepper seedlings in a controlled environment growth chamber setting. To manage bacterial diseases like PLS in peppers, copper and streptomycin-based antimicrobials are primarily used. However, in recent years, due to the emergence of antimicrobial resistance genes, these control methods are becoming less effective. There is an urgent need to find an alternative to control PLS disease in peppers. Therefore, part of my research aimed to identify novel small molecules (SM) growth inhibitors of Pss, determine the toxicity of the SMs on pepper tissues (host) and eukaryotic models (honeybees and human cell line), and evaluate the efficacy of the SMs on pepper seeds and seedlings infected with Pss. A SM library of 4,182 SMs was screened at 200µM against a model Pss strain SM1042-14R, grown in M9 minimal broth. A total of 141 SMs inhibited the growth of Pss by at least 70%. The 10 SMs (P1-P10) that exhibited bactericidal activity during the primary screening were selected for further studies. A dose-response assay with 10 SMs showed minimal inhibitory concentration (MIC) ranging from 6.25 to 200µM and minimal bactericidal concentration (MBC) ranging from 12.5 to 200µM against all 10 Pss strains. The 10 SMs were also effective against pathogenic phytobacteria (n=22), and biofilm-embedded Pss, but did not impact the growth of beneficial phytobacteria (n=12). The 10 SMs showed minimal toxicity on pepper seeds, seedlings, and fruits at 200µM. Additionally, the SMs also showed minimal toxicity at 200µM on eukaryotic models including honeybees and human cell lines (Caco-2 cells). Overall, 10 SMs significantly decreased disease severity by 89.5% compared to DMSO-treated control in Pss-infected pepper seedlings at 7 dpi (P<0.01). Similarly, six SMs significantly reduced the bacterial load of Pss on pepper seedlings by up to 1.93 log CFU/g compared to the DMSO-treated control at 7 dpi. In Pss-infested seeds, eight SM significantly reduced Pss bacterial load at 30 dpi by up to 3.09 log CFU/g P<0.01), compared to the DMSO untreated control. Overall, eight SMs were effective against pepper seeds or seedlings infected with Pss. These novel SMs are promising alternatives to currently used antimicrobials to control Pss in peppers.
Gireesh Rajashekara (Advisor)
Sally Miller (Committee Member)
James Fuchs (Committee Member)
219 p.
Biology; Microbiology; Plant Pathology
Pseudomonas syringae pv. syringae (Pss), whole genome sequence analysis, pathogenicity in pepper, antimicrobial resistance, novel antimicrobials, small molecules

Recommended Citations

Citations

  • Ranjit, S. (2022). Investigating Potential Virulence Genes of Plant Pathogenic Bacterium Pseudomonas syringae pv. syringae Utilizing Whole-genome Sequencing Analysis and Identifying Novel Small Molecule Growth Inhibitors to Manage Pseudomonas Leaf Spot Disease on Pepper Seeds and Seedlings [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1669921517118292

    APA Style (7th edition)

  • Ranjit, Sochina. Investigating Potential Virulence Genes of Plant Pathogenic Bacterium Pseudomonas syringae pv. syringae Utilizing Whole-genome Sequencing Analysis and Identifying Novel Small Molecule Growth Inhibitors to Manage Pseudomonas Leaf Spot Disease on Pepper Seeds and Seedlings . 2022. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1669921517118292.

    MLA Style (8th edition)

  • Ranjit, Sochina. "Investigating Potential Virulence Genes of Plant Pathogenic Bacterium Pseudomonas syringae pv. syringae Utilizing Whole-genome Sequencing Analysis and Identifying Novel Small Molecule Growth Inhibitors to Manage Pseudomonas Leaf Spot Disease on Pepper Seeds and Seedlings ." Master's thesis, Ohio State University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=osu1669921517118292

    Chicago Manual of Style (17th edition)

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