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

BACKGROUND

There has been limited exploration of genotypes and associated copper resistance phenotypes in spp. in the southern Caribbean region. An earlier study highlighted a variant gene cluster found in one Trinidadian pv. (Xcc) strain (BrA1), with <90% similarity to previously reported genes. With only one report describing this copper resistance genotype, the current study investigated the distribution of the BrA1 variant gene cluster and previously reported forms of copper resistance genes in local spp.

METHODS

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.5HO 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 genes and those predicted to target multiple homologs found in and 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 sp. strains were isolated, with 35 others classed as copper-resistant from a total population of 45 isolates. PCR detection of genes revealed two PCR negative copper-resistant resistant strains. Variant genes were only found in Xcc from the original source location of the BrA1 strain, Aranguez. Other copper-resistant strains contained other homologs that clustered into three distinct clades. These groups were more similar to genes from plasmids and spp. chromosomal homologs than reference Xcc sequences. This study highlights the localisation of the BrA1 variant genes to one agricultural community and the presence of three distinct gene groupings in Xcc and related spp. with defined CuSO.5HO MIC. Further characterisation of these gene groups and copper resistance gene exchange dynamics on and within leaf tissue between Xcc and other 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.