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Phenotypic and Genetic Analyses of the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas Hortorum Pv. Vitians, and the Development of a Rapid Detection Technique.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Phenotypic and Genetic Analyses of the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas Hortorum Pv. Vitians, and the Development of a Rapid Detection Technique./
Author:	Martinez, Emma R.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2023,
Description:	243 p.
Notes:	Source: Dissertations Abstracts International, Volume: 85-11, Section: B.
Contained By:	Dissertations Abstracts International85-11B.
Subject:	Pathogens. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=31085766
ISBN:	9798382646701

Phenotypic and Genetic Analyses of the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas Hortorum Pv. Vitians, and the Development of a Rapid Detection Technique.
Martinez, Emma R.

Phenotypic and Genetic Analyses of the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas Hortorum Pv. Vitians, and the Development of a Rapid Detection Technique. - Ann Arbor : ProQuest Dissertations & Theses, 2023 - 243 p.

Source: Dissertations Abstracts International, Volume: 85-11, Section: B.

Thesis (Ph.D.)--The Pennsylvania State University, 2023.

Bacterial leaf spot (BLS) of lettuce, caused by Xanthomonas hortorum pv. vitians (Xhv), was first described in 1918 and it remains a significant threat to lettuce cultivation. Symptoms of disease include water-soaking and chlorosis in the leaves and small necrotic spots that merge to form larger lesions. Outbreaks can be devastating, with up to 100% crop loss, and sources of inoculum have been found in crop debris and weeds. The pathogen is also thought to be seedborne, though this has not yet been demonstrated. Integrated management strategies include the removal of crop debris and weeds, the use of clean seeds, and the use of resistant cultivars. Chemical application may be effective in preventing outbreaks when applied at the earliest detection of the pathogen, but they work best when applied prophylactically, which can be a large sunken cost in years that the pathogen does not appear. Further, bactericides vary in their effectiveness. Multiple research groups have tried to make PCR-based detection methods for the pathogen, but none yet specifically detect Xhv. Detection is complicated by the existence of three hypothetical races of the pathogen, defined by their host reactions on various lettuce cultivars and accessions. While Xhvpopulations are predominantly clonal, any observed phenotypic variation is likely the result of genetic recombination over mutation.The major knowledge gaps in this field of study include a full characterization of the host reactions of the three Xhv races, an understanding of the genotypes underlying Xhv race-specificity, and a set of detection methods for Xhv strains and to distinguish the races. Chapter 1 summarizes the literature that is foundational to the study of Xhv. Chapter 2 describes our characterizations of the Xhv race 1, 2, and 3 reactions on differential lettuce cultivars. Strains representing each hypothesized race were inoculated by syringe into all three of the previously determined differential cultivars. We confirmed previous findings that Xhv race 1 strains triggered the resistant hypersensitive response (HR) on L. sativa cv. Little Gem, Xhv race 2 strains triggered HR on L. serriola PI491114, and Xhv race 3 strains triggered HR on L. serriola ARM-09-161-10-1. HR is a plant's resistance strategy in which it kills off its own tissue and limits the spread of bacteria; differential host reactions like those in Xhv have been used to define races in other bacterial species. We also demonstrated that Xhv race 1 strains triggered HR in L. serriola ARM-09-161-10-1, and that two sets of strains collected in Florida and France did include Xhv strains. A multi-locus sequence analysis scheme that has been used previously to predict Xhvrace predicted that nearly all these strains belonged to Xhv race 1. Finally, we found that three plant introductions deposited at the USDA-ARS Germplasm Resources Information Network (GRIN) of the National Plant Germplasm System may also have race-specific resistance.In Chapter 3, we sought to identify race-specific DNA sequences that might underlie the race-specific host reactions. Whole genome sequences of eighteen Xhv strains representing the three hypothesized races, along with eight related Xanthomonas strains, were included in the analysis. A maximum likelihood phylogeny based on concatenated whole genome single nucleotide polymorphisms confirmed previous results describing two major lineages of Xhv strains. Gene clusters encoding secretion systems, secondary metabolites, and bacteriocins were assessed to identify putative virulence factors that distinguish the Xhv races. Genome sequences were mined for effector genes, which have been shown to be involved in race-specificity in other systems. Two effectors identified in this study, xopAQ and the novel variant xopAF2, were revealed as possible mediators of a gene-for-gene interaction between Xhv race 1 and 3 strains and wild lettuce L. serriola ARM-09-161-10-1. Transposase sequence identified downstream of xopAF2 and prophage sequence found within Xhv race 1 and 3 insertion sequences suggest that this gene may have been acquired through phage-mediated gene transfer. No other factors were identified from these analyses that distinguish the Xhvraces.For Chapter 4, whole genome sequence alignments allowed us to identify gene clusters that were specific to Xhv or to each of its races that could be used as targets for pathovar- or race-specific PCR-based detection methods. Four gene clusters were identified for possible Xhv, Xhv race 1, Xhv race 2, or Xhv race 3 detection, and many primers were designed and evaluated by touchdown PCR. We found that our pathovar specific primer set successfully detected 97% of the Xhv strains tested and did not detect any of the other six X. hortorum pathovars. However, it was also able to detect a close relative, X. hydrangea, from symptomatic lettuce tissue. While this means our method is also not Xhv-specific, we recommend the use of our protocol in tandem with a published LAMP assay developed for detection of Xhv, X. hortorum pv. gardneri, and X. hortorum pv. cynarae. The use of these two methods together should reveal whether an unknown sample is Xhv. Further work is necessary to develop a single Xhv-specific method, as well as the race-specific detection methods as each of these three methods resulted in false positives or false negatives.In Chapter 5, we share our progress in evaluating the roles of Xhv race 1 and 3 specific effector genes, xopAQ and xopAF2, in HR induction in L. serriola ARM-09-161-10-1. Effector genes function in promoting bacterial colonization, but their detection by plant host proteins have been shown to trigger HR. We aimed to delete each effector gene from the Xhv race 1 BS0347 genome and tested two methods: scarless deletion and marker exchange deletion with a gentamycin resistance cassette. The deletion constructs were designed from upstream and downstream regions flanking each effector, which were then synthesized into the nonreplicable plasmid pk18mobsacB. Movement of the constructs into a rifampicin resistant mutant of Xhv race 1 BS0347 and the occurrence of homologous recombination was expected to result in gene deletion from the chromosomal DNA and its transfer onto the plasmid, which would then be lost in counterselection on sucrose and rifampicin amended media. With the marker exchange method, the gentamycin resistance cassette would replace the effector gene at its site in the bacterial chromosome, and so double recombinants could be also selected for with additional gentamycin in the selective media. We have evidence of a successful xopAQ gene deletion. Expression constructs were also designed for xopAQ and xopAF2 expression in a rifampicin resistant Xhv race 2 strain, but their transformation into the Xhv race 2 strain has not yet been successful. We outline additional strategies for expressing the effector genes in Xhv race 2. Once produced, these altered Xhvstrains, the wild type strains, and a set of empty vectors will be inoculated into L. serriola ARM-09-161-10-1 to determine whether the genetic alterations impact the strains' ability to induce HR. Continuing this work is expected to reveal the genes involved in HR induction.The final chapter of this dissertation summarizes our findings, highlights the importance of the work, and suggests avenues for future study. Overall, this dissertation advances the field of plant pathology by defining the Xhvrace reactions on lettuce cultivars, predicting two genes that may be responsible for race-specific HR induction in a lettuce host, demonstrating methods that could be used for pathovar-specific PCR-based detection and showing progress made toward similar methods for race-specific detection, and finally showing progress toward demonstrating in planta the potential role of two race-specific genes in HR induction.

ISBN: 9798382646701Subjects--Topical Terms:

3540520
Pathogens.

Phenotypic and Genetic Analyses of the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas Hortorum Pv. Vitians, and the Development of a Rapid Detection Technique.
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520 $a Bacterial leaf spot (BLS) of lettuce, caused by Xanthomonas hortorum pv. vitians (Xhv), was first described in 1918 and it remains a significant threat to lettuce cultivation. Symptoms of disease include water-soaking and chlorosis in the leaves and small necrotic spots that merge to form larger lesions. Outbreaks can be devastating, with up to 100% crop loss, and sources of inoculum have been found in crop debris and weeds. The pathogen is also thought to be seedborne, though this has not yet been demonstrated. Integrated management strategies include the removal of crop debris and weeds, the use of clean seeds, and the use of resistant cultivars. Chemical application may be effective in preventing outbreaks when applied at the earliest detection of the pathogen, but they work best when applied prophylactically, which can be a large sunken cost in years that the pathogen does not appear. Further, bactericides vary in their effectiveness. Multiple research groups have tried to make PCR-based detection methods for the pathogen, but none yet specifically detect Xhv. Detection is complicated by the existence of three hypothetical races of the pathogen, defined by their host reactions on various lettuce cultivars and accessions. While Xhvpopulations are predominantly clonal, any observed phenotypic variation is likely the result of genetic recombination over mutation.The major knowledge gaps in this field of study include a full characterization of the host reactions of the three Xhv races, an understanding of the genotypes underlying Xhv race-specificity, and a set of detection methods for Xhv strains and to distinguish the races. Chapter 1 summarizes the literature that is foundational to the study of Xhv. Chapter 2 describes our characterizations of the Xhv race 1, 2, and 3 reactions on differential lettuce cultivars. Strains representing each hypothesized race were inoculated by syringe into all three of the previously determined differential cultivars. We confirmed previous findings that Xhv race 1 strains triggered the resistant hypersensitive response (HR) on L. sativa cv. Little Gem, Xhv race 2 strains triggered HR on L. serriola PI491114, and Xhv race 3 strains triggered HR on L. serriola ARM-09-161-10-1. HR is a plant's resistance strategy in which it kills off its own tissue and limits the spread of bacteria; differential host reactions like those in Xhv have been used to define races in other bacterial species. We also demonstrated that Xhv race 1 strains triggered HR in L. serriola ARM-09-161-10-1, and that two sets of strains collected in Florida and France did include Xhv strains. A multi-locus sequence analysis scheme that has been used previously to predict Xhvrace predicted that nearly all these strains belonged to Xhv race 1. Finally, we found that three plant introductions deposited at the USDA-ARS Germplasm Resources Information Network (GRIN) of the National Plant Germplasm System may also have race-specific resistance.In Chapter 3, we sought to identify race-specific DNA sequences that might underlie the race-specific host reactions. Whole genome sequences of eighteen Xhv strains representing the three hypothesized races, along with eight related Xanthomonas strains, were included in the analysis. A maximum likelihood phylogeny based on concatenated whole genome single nucleotide polymorphisms confirmed previous results describing two major lineages of Xhv strains. Gene clusters encoding secretion systems, secondary metabolites, and bacteriocins were assessed to identify putative virulence factors that distinguish the Xhv races. Genome sequences were mined for effector genes, which have been shown to be involved in race-specificity in other systems. Two effectors identified in this study, xopAQ and the novel variant xopAF2, were revealed as possible mediators of a gene-for-gene interaction between Xhv race 1 and 3 strains and wild lettuce L. serriola ARM-09-161-10-1. Transposase sequence identified downstream of xopAF2 and prophage sequence found within Xhv race 1 and 3 insertion sequences suggest that this gene may have been acquired through phage-mediated gene transfer. No other factors were identified from these analyses that distinguish the Xhvraces.For Chapter 4, whole genome sequence alignments allowed us to identify gene clusters that were specific to Xhv or to each of its races that could be used as targets for pathovar- or race-specific PCR-based detection methods. Four gene clusters were identified for possible Xhv, Xhv race 1, Xhv race 2, or Xhv race 3 detection, and many primers were designed and evaluated by touchdown PCR. We found that our pathovar specific primer set successfully detected 97% of the Xhv strains tested and did not detect any of the other six X. hortorum pathovars. However, it was also able to detect a close relative, X. hydrangea, from symptomatic lettuce tissue. While this means our method is also not Xhv-specific, we recommend the use of our protocol in tandem with a published LAMP assay developed for detection of Xhv, X. hortorum pv. gardneri, and X. hortorum pv. cynarae. The use of these two methods together should reveal whether an unknown sample is Xhv. Further work is necessary to develop a single Xhv-specific method, as well as the race-specific detection methods as each of these three methods resulted in false positives or false negatives.In Chapter 5, we share our progress in evaluating the roles of Xhv race 1 and 3 specific effector genes, xopAQ and xopAF2, in HR induction in L. serriola ARM-09-161-10-1. Effector genes function in promoting bacterial colonization, but their detection by plant host proteins have been shown to trigger HR. We aimed to delete each effector gene from the Xhv race 1 BS0347 genome and tested two methods: scarless deletion and marker exchange deletion with a gentamycin resistance cassette. The deletion constructs were designed from upstream and downstream regions flanking each effector, which were then synthesized into the nonreplicable plasmid pk18mobsacB. Movement of the constructs into a rifampicin resistant mutant of Xhv race 1 BS0347 and the occurrence of homologous recombination was expected to result in gene deletion from the chromosomal DNA and its transfer onto the plasmid, which would then be lost in counterselection on sucrose and rifampicin amended media. With the marker exchange method, the gentamycin resistance cassette would replace the effector gene at its site in the bacterial chromosome, and so double recombinants could be also selected for with additional gentamycin in the selective media. We have evidence of a successful xopAQ gene deletion. Expression constructs were also designed for xopAQ and xopAF2 expression in a rifampicin resistant Xhv race 2 strain, but their transformation into the Xhv race 2 strain has not yet been successful. We outline additional strategies for expressing the effector genes in Xhv race 2. Once produced, these altered Xhvstrains, the wild type strains, and a set of empty vectors will be inoculated into L. serriola ARM-09-161-10-1 to determine whether the genetic alterations impact the strains' ability to induce HR. Continuing this work is expected to reveal the genes involved in HR induction.The final chapter of this dissertation summarizes our findings, highlights the importance of the work, and suggests avenues for future study. Overall, this dissertation advances the field of plant pathology by defining the Xhvrace reactions on lettuce cultivars, predicting two genes that may be responsible for race-specific HR induction in a lettuce host, demonstrating methods that could be used for pathovar-specific PCR-based detection and showing progress made toward similar methods for race-specific detection, and finally showing progress toward demonstrating in planta the potential role of two race-specific genes in HR induction.
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