Oxytetracycline and Streptomycin Resistance Genes in Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot in Peach.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot, a major worldwide disease of Prunus species. Very few chemical management options are available for this disease and frequent applications of oxytetracycline (OTC) in the United States peach orchards have raised concerns about resistance development. During 2017-2020, 430 Xap strains were collected from ten peach orchards in South Carolina. Seven OTC-resistant (OTC R ) Xap strains were found in 2017 and 2020 from four orchards about 20-270 km apart. Interestingly, the seven strains were also resistant to streptomycin (STR). Six strains grew on media amended with ≤100 µg/mL OTC, while one strain, R1, grew on ≤250 µg/mL OTC. Genome sequence analysis of four representative OTC R strains revealed a 14-20 kb plasmid carrying tetC, tetR, and strAB in each strain. These three genes were transferable to Xanthomonas perforans via conjugation, and they were PCR confirmed in all seven OTC R Xap strains. When tetC and tetR were cloned and expressed together in a sensitive strain, the transconjugants showed resistance to ≤100 µg/mL OTC. When tetC was cloned and expressed alone in a sensitive strain, the transconjugants showed resistance to ≤250 µg/mL OTC. TetC and tetR expression was inducible by OTC in all six wild-type strains resistant to ≤100 µg/mL OTC. However, in the R1 strain resistant to ≤250 µg/mL OTC, tetR was not expressed, possibly due to the presence of Tn3 in the tetR gene, and in this case tetC was constitutively expressed. These data suggest that tetC confers OTC resistance in Xap strains, and tetR regulates the level of OTC resistance conferred by tetC. To our knowledge, this is the first report of OTC resistance in plant pathogenic xanthomonads.

Copper Tolerance in Xanthomonas arboricola pv. pruni in South Carolina Peach Orchards.

Bacterial spot of peach, caused by Xanthomonas arboricola pv. pruni, causes yield loss every year in southeastern U.S. peach orchards. Management is mainly driven by season-long applications of copper-based products, site location, and choice of cultivar. Although tolerance to copper has not been reported in X. arboricola pv. pruni in the United States, adaptation of populations from frequent use is a concern. We collected X. arboricola pv. pruni from shoot cankers, leaves, and fruit of cultivar O'Henry over 2 years from three conventional farms and one organic farm in South Carolina, one orchard per farm. The four farms had been using copper extensively for years to control bacterial spot. X. arboricola pv. pruni was isolated from four canker types (bud canker, tip canker, nonconcentric canker, and concentric canker) in early spring (bud break), as well as from leaf and fruit tissues later in the season at the phenological stages of pit hardening and final swell. X. arboricola pv. pruni was most frequently isolated from cankers of the organic farm (24% of the cankers) and most isolates (45%) came from bud cankers. X. arboricola pv. pruni isolates were assessed for sensitivity to copper using minimal glucose yeast agar and nutrient agar amended with 38 µg/ml or 51 µg/ml of Cu2+. Two phenotypes of copper tolerance in X. arboricola pv. pruni were discovered: low copper tolerance (LCT; growth up to 38 µg/ml Cu2+) and high copper tolerance (HCT; growth up to 51 µg/ml Cu2+). A total of 26 (23 LCT and 3 HCT) out of 165 isolates in 2018 and 32 (20 LCT and 12 HCT) out of 133 isolates in 2019 were tolerant to copper. Peach leaves on potted trees were sprayed with copper rates typically applied at the stages of delayed dormancy (high rate; 2,397 µg/ml Cu2+), shuck split (medium rate; 599 µg/ml Cu2+), and during summer cover sprays (low rate; 120 µg/ml Cu2+), and subsequently inoculated with sensitive, LCT, and HCT strains. Results indicated that the low and medium rates of copper reduced bacterial spot incidence caused by the sensitive strain but not by the LCT and HCT strains. This study confirms existence of X. arboricola pv. pruni tolerance to copper in commercial peach orchards in the southeastern United States, and suggests its contribution to bacterial spot development under current management practices.

Preservation of Monilinia fructicola Genotype Diversity Within Fungal Cankers.

Monilinia fructicola is a destructive pathogen causing brown rot on stone fruits worldwide. Though it is best known as a fruit rot pathogen, M. fructicola also causes blossom blight and, subsequently, twig cankers in the spring. Orchard management strategies often overlook cankers as an inoculum source, though they are an inoculum source of both blossom and fruit infections. In this study, we analyzed the role of cankers as storage structures for diverse genotypes of M. fructicola, examining whether multiple genotypes can be transmitted from blossom to canker. Fungal spores from blossoms, and 2 months later from their corresponding cankers, were collected from a conventional and an unsprayed orchard in 2015 and 2016. Simple sequence repeat markers were used to genotype 10 to 20 single spores from each of four blossom/canker pairs per orchard. Individual blossoms and cankers were detected containing up to four and five genotypes, respectively. The average number of genotypes in blossoms and corresponding cankers were not significantly different (P = 0.690) across both years and farms, showing that a bottleneck for genetic diversity was not generated during the transition from blossom to canker. The average number of genotypes unique to blossom or canker was not significantly different (P = 0.569) and no significant effect of farm (P = 0.961) or year (P = 0.520) was observed, although blossoms had a numerically greater number of unique genotypes in both cases. In conclusion, a single blossom may be infected by one or more genotypes of M. fructicola, and this diversity is being preserved in the corresponding canker. This information implicates M. fructicola cankers as diversity storehouses, and may also apply to other Monilinia spp. and fungal diseases that initiate in reproductive tissue.