copLAB gene prevalence and diversity among Trinidadian Xanthomonas spp. black-rot lesion isolates with variable copper resistance profiles

BACKGROUND: There has been limited exploration of copLAB genotypes and associated copper resistance phenotypes in Xanthomonas spp. in the southern Caribbean region. An earlier study highlighted a variant copLAB gene cluster found in one Trinidadian Xanthomonas campestris pv. campestris (Xcc) strain (BrA1), with <90% similarity to previously reported Xanthomonas copLAB genes. With only one report describing this copper resistance genotype, the current study investigated the distribution of the BrA1 variant copLAB gene cluster and previously reported forms of copper resistance genes in local Xanthomonas spp. METHODS: Xanthomonas spp. were isolated from black-rot infected lesions on leaf tissue from crucifer crops at intensively farmed sites with high agrochemical usage in Trinidad. The identity of morphologically identified isolates were confirmed using a paired primer PCR based screen and 16s rRNA partial gene sequencing. MGY agar amended with CuSO(4).5H(2)O up to 2.4 mM was used to establish MIC’s for confirmed isolates and group strains as sensitive, tolerant, or resistant to copper. Separate primer pairs targeting the BrA1 variant copLAB genes and those predicted to target multiple homologs found in Xanthomonas and Stenotrophomonas spp. were used to screen copper resistant isolates. Select amplicons were sanger sequenced and evolutionary relationships inferred from global reference sequences using a ML approach. RESULTS: Only four copper sensitive/tolerant Xanthomonas sp. strains were isolated, with 35 others classed as copper-resistant from a total population of 45 isolates. PCR detection of copLAB genes revealed two PCR negative copper-resistant resistant strains. Variant copLAB genes were only found in Xcc from the original source location of the BrA1 strain, Aranguez. Other copper-resistant strains contained other copLAB homologs that clustered into three distinct clades. These groups were more similar to genes from X. perforans plasmids and Stenotrophomonas spp. chromosomal homologs than reference Xcc sequences. This study highlights the localisation of the BrA1 variant copLAB genes to one agricultural community and the presence of three distinct copLAB gene groupings in Xcc and related Xanthomonas spp. with defined CuSO(4).5H(2)O MIC. Further characterisation of these gene groups and copper resistance gene exchange dynamics on and within leaf tissue between Xcc and other Xanthomonas species are needed as similar gene clusters showed variable copper sensitivity profiles. This work will serve as a baseline for copper resistance gene characterisation in Trinidad and the wider Caribbean region and can be used to boost already lacking resistant phytopathogen management in the region.