Date of Award
12-2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Plant and Environmental Science
Committee Chair/Advisor
Dr. Hehe Wang
Committee Member
Dr. Guido Schnabel
Committee Member
Dr. C. Nathan Hancock
Committee Member
Dr. Daniel J. Anco
Committee Member
Dr. Jose Payero
Abstract
Xanthomonas arboricola pv. pruni (XAP) is a phytopathogenic bacterium that causes bacterial spot of peach. It causes spotted lesions on leaves and fruit, leading to early defoliation and unmarketable fruit, respectively. The pathogen can overwinter in twig cankers, which serve as the primary initial inoculum source for the next season’s leaf and fruitlet infection. The current disease management practice mainly relies on routine application of chemical bactericides containing copper or oxytetracycline (OTC) in the growing season. However, disease incidence can still be high despite chemical control measures in warm, rainy seasons.
Frequent applications of copper and OTC in United States peach orchards are alarming for resistance development that copper tolerance and OTC resistance has previously been reported in peach orchards of South Carolina. In this study, we surveyed the prevalence of copper tolerant and OTC resistant XAP strains in South Carolina and Georgia peach orchards (Chapter 2), developed PMA-qPCR assay to quantify viable XAP (chapter 3), compared the overwintering capability of these resistant/tolerant strains with the sensitive XAP strains (Chapter 4), and identified the genetic determinants for the copper resistance in a XAP strain (Chapter 5).
From Orchard surveys during 2021-2024, 1,739 XAP isolates were collected from four South Carolina and three Georgia peach farms and screened on copper (0, 38, 44, and 51 µg/ml Cu2+) or OTC (25 µg/ml) amended nutrient agar (NA). Among the isolates tested, 95.6% were low copper tolerant (grown on NA amended with 38 µg/ml Cu2+), 5.8% were high copper tolerant (grown on NA amended with 51µg/ml Cu2+), and 24.9% were OTC resistant (grown on NA amended with 25 µg/ml of OTC). The resistant/tolerant XAP population has increased or been maintained over the years in these orchards.
To study the overwintering capability of XAP strains, a long-amplicon propidium monoazide (PMA)–quantitative PCR (qPCR) assay was developed. This assay enabled sensitive, specific, and accurate quantification of viable XAP cells as low as 103 CFU/ml with the presence of up to 107 CFU/ml of dead XAP cells, while suppressing the amplification of DNA from dead cells. With the PMA-qPCR assay, we studied the survival of XAP strains with different chemical sensitivities on peach trees after inoculation during bud set, after harvest, and leaf drop in fall. Resulted showed that the inoculation during leaf drop and after harvest led to significantly more twig canker and overwintered XAP on peach twigs than the bud set inoculation. No difference was observed in overwintering capability of XAP strains with different chemical sensitivities, suggesting that these OTC-resistant and copper-tolerant strains may persist and accumulate in peach orchards across seasons.
Genome sequence analysis of a copper resistant XAP strain revealed a ~249 kb plasmid carrying large duplicated clusters of copper resistant genes, including copL, copA, copB, copC,copD, copM, copG, copF, cusA and cusB. Gene functional analysis revealed that copL-D were the major contributors to copper resistance. Addition of copL doubled the copper resistance level compared to copA-D alone. Duplication of this copper resistance gene cluster increased the resistance level compared to the single copy, while deletion of both copies of this cluster caused the loss of copper resistance. The copper resistant genes and phenotypes were able to be transferred to Xanthomonas perforans via conjugation.
In conclusion, this study showed that OTC resistance and low level of copper tolerance is persistent in the peach orchards regardless of spray programs, indicating the need of new management options for peach production. Leaf drop and after harvest periods were identified as important time for XAP to establish in peach twigs for overwintering survival. These results, in combination with our first report on the copper resistance mechanism in XAP, provide new knowledge to improve our understanding of epidemiology and genetics of the chemical resistant/tolerant XAP population and will contribute to the future development of more sustainable disease management program to reduce XAP inoculum sources and minimize disease development.
Recommended Citation
Panth, Milan, "Exploring Mechanisms of Chemical Resistance in Xanthomonas Arboricola pv. Pruni and Strategies to Mitigate Resistance and Improve Disease Control" (2024). All Dissertations. 3789.
https://open.clemson.edu/all_dissertations/3789
Author ORCID Identifier
0000-0003-2837-2036