Copper resistance genes from different xanthomonads and citrus epiphytic bacteria confer resistance to Xanthomonas citri subsp. citri

Genetic exchange is considered to be an important process in the selective adaptation of microorganisms to shifting and challenging environmental conditions. As a consequence of the copious use of copper bactericides, many species of plant pathogenic bacteria, including Xanthomonas citri subsp. citri (Xcc), have developed resistance to copper. This study assesses whether copper resistant (Cu^R) strains of other Xanthomonas species and citrus epiphytic bacteria pose a risk for the development of copper resistance in Xcc. Cu^R epiphytic bacteria were isolated on MGY agar from citrus leaves collected in two citrus groves treated with copper bactericides in Florida. Horizontal gene transfer of copper resistance genes was investigated within different Xanthomonas species and from citrus epiphytic bacteria to Xanthomonas. Cu^R epiphytic bacteria from citrus were screened for the presence of copper resistance genes homologous to copL, copA and copB genes from Xcc and characterized regarding tolerance to copper. Copper resistance determinants from a citrus epiphytic strain of Stenotrophomonas maltophilia (Stm) were cloned and expressed in Xcc and other Xanthomonas strains. Copper resistance genes in Xcc were determined to be present on a large (~300?kb) conjugative plasmid. Cu resistance was transferred via conjugation from two copper resistant citrus strains, Xcc and X. alfalfae subsp. citrumelonis (Xac), and two tomato pathogens, X. euvesicatoria (Xe) and X. perforans (Xp), to Xcc. PCR analysis revealed that two Cu^R strains from citrus, an epiphytic Xanthomonas ssp. and a strain of Stm, harboured homologs of the copper resistance genes found in Cu^R Xcc. The introduction of copLAB gene cluster from Stm into different xanthomonads conferred copper resistance to sensitive strains of Xcc, Xac, Xe and Xp. Based on these results there is a low, but significant, likelihood of horizontal gene transfer of copper resistance genes from other xanthomonads or epiphytic bacteria to Xcc in nature.     

Citrus black spot severity related to premature fruit drop in sweet orange orchards

Citrus black spot (CBS) may cause substantial yield loss in sweet orange orchards. Severity and distribution of CBS symptoms on fruit may be variable and not all affected fruit may fall. However, the relationship between CBS symptoms and yield loss has not yet been investigated. This study aimed to determine CBS characteristics associated with premature fruit drop. A set of fruit was removed from the tree to measure the detachment force and another set of dropped fruit was collected from Pera and Valencia sweet orange orchards in three municipalities in Sao Paulo state, Brazil. CBS severity, distance of the nearest lesion from the peduncle, and fruit peel colour were assessed in both fruit sets. CBS severity was consistently related with the force to remove fruit and hence associated to the premature fruit drop, irrespective of the cultivar and location. The segmented linear regression showed that the fruit detachment force was reduced abruptly, at a rate of 7% for every 1% increase in disease severity up to 4.2 ± 1.0% CBS severity and, above this breakpoint, the force decreased slightly at a rate of 1.5%. Taking into account all orchards, the proportion of dropped fruit with CBS severity higher than the breakpoint was approximately 90%. This study provides a better understanding of the relationship between CBS severity and premature fruit drop of sweet orange. CBS severity breakpoint may be used as a reference to anticipate harvest and reduce CBS-related yield loss in orchards intended for juice processing.     

A comprehensive analysis of the Asiatic citrus canker eradication programme in São Paulo state,Brazil, from 1999 to 2009

Asiatic citrus canker (ACC), caused by Xanthomonas citri subsp. citri, has been controlled by exclusion and eradication measures in São Paulo state (SP), Brazil, since it was first detected in 1957. Several protocols of eradication have been used since then, but the protocol applied from 1999 to 2009 was the most successful and well documented of these. During that time, if the incidence of trees with symptoms in a citrus block was ≤0.5%, the infected trees and the symptomless trees within a 30 m radius were removed. However, if the incidence was >0.5%, the entire block was eliminated. Subsequently, inspections were carried out to monitor the affected blocks during quarantine. Although this protocol did not eliminate the disease from SP, it suppressed the incidence of affected blocks to very low annual levels (<0.20%) for a decade. Of over 5000 foci detected, 65.1% were eliminated by removing the block, either when disease was first detected (47.8%) or at a subsequent detection (17.3%), and 28.4% were eradicated by removing trees within a 30 m radius. The latter method was not an effective stand‐alone measure for ACC eradication and the 0.5% threshold was the key factor in suppression of disease statewide. Currently, a re‐emergence of ACC is occurring in SP. The information presented here will aid the establishment of protocols for the eradication of ACC in other citrus‐growing areas with low incidence of the disease or at risk of pathogen introduction.     

Description of copper tolerant Xanthomonas citri subsp. citri and genotypic comparison with sensitive and resistant strains

The copper-based products widely used for control of citrus canker may lead to the development of Xanthomonas citri subsp. citri (X. citri) resistant to copper (Cu^R). However, the study of copper sensitivity of X. citri strains from Paraná state, Brazil, did not reveal the existence of Cu^R, but copper tolerant (Cu^T) strains. The aim of this study was to describe for the first time the existence of Cu^T X. citri and compare the genetic determinants that differentiate the Cu^T strains from the sensitive (Cu^S) and Cu^R strains. Cu^T strains supported intermediate concentrations of copper in comparison to Cu^S and Cu^R. Cu^T strains lack the gene clusters copLAB or copABCD responsible for copper resistance in Cu^R strains and the large plasmids (c. ≥200 kb) that normally carry these genes. The nucleotide sequences of chromosomal homologous genes cohLAB, involved in copper homeostasis, were 100% similar in strains of all phenotypes. Cu^T strains differed from Cu^S strains by the higher expression of the homologous chromosomal genes cohA and cohB in the presence of copper. Cu^T X. citri strains are not precursors of Cu^R strains and do not pose a threat to the efficient use of copper-based bactericides for management of citrus canker in citrus orchards. Copper resistance and tolerance are distinct phenotypes and should not be used as synonyms. The proper characterization of the sensitivity to copper leads to a more confident monitoring of the distribution of copper resistant populations of X. citri and adoption of containment measures only when necessary.     

Copper rate and spray interval for joint management of citrus canker and citrus black spot in orange orchards

European Journal of Plant Pathology - Citrus canker and citrus black spot are two of the most important diseases in the São Paulo (SP) citrus belt, Brazil. Canker control was based on an...     

Characterization of a unique copper resistance gene cluster in <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> isolated in Trinidad,West Indies

Whole genome sequencing of a copper resistant (Cu^R) black rot strain of Xanthomonas campestris pv. campestris (Xcc) isolated from a broccoli plant in Trinidad revealed a unique operon for copper resistance. The cop genes of strain Xcc-BrA1 were determined to be present on a 160 to 180 kb plasmid shown to be non-conjugative with other xanthomonads. While nucleotide comparison of a putative 8.0 Kbp copLABMGF gene cluster identified in Xcc-BrA1 genome did not reveal any homologous region with other known Cu^R Xanthomonas strains from diverse origins, the comparison of the translated amino acid sequence indicated similarity with X. citri, X. c. pv. citrumelonis and X. vesicatoria Cop proteins. Cloning of the copLAB gene cluster from Xcc-BrA1 conferred copper resistance to other copper-sensitive xanthomonads. Although Xcc-BrA1 harbors copLAB genes with similar sizes and organization and is able to grow on Cu-amended medium as other Cu^R xanthomonads, the phylogenetic analysis of nucleotide sequences indicates that the cop cluster in Xcc-BrA1 is unique and distantly related to other copLAB genes from Xanthomonas and Stenotrophomonas. The origin of copper resistance genes in Xcc-BrA1 is likely a result of horizontal gene acquisition from a still unknown phylloplane cohabitant. The findings of this study have implications for the management of crop diseases caused by Cu^R xanthomonads. Future studies could focus on and determining the distribution, overall importance and appropriate control measures for strains harbouring these unique genes.     

Annual and polyetic progression of citrus canker on trees protected with copper sprays

The effects of copper sprays on annual and polyetic progress of citrus canker, caused by Xanthomonas citri subsp. citri, in the presence of the Asian citrus leafminer (Phyllocnistis citrella), were evaluated in a study conducted in a commercial orchard in northwest Paraná state, Brazil, where citrus canker is endemic. Nonlinear monomolecular, logistic and Gompertz models were fitted to monthly disease incidence data (proportion of leaves with symptoms) for each treatment for three seasons. The logistic model provided the best estimate of disease progress for all years and treatments evaluated and logistic parameter estimates were used to describe polyetic disease dynamics. Although citrus canker incidence increased during each of the seasons studied, it decreased over the whole study period, more so in copper‐treated trees than in water‐sprayed controls. Copper treatment reduced disease incidence compared with controls in every year, especially 2004–2005, when incidence was ca. 10‐fold higher in controls than in treated plots (estimated asymptote values 0·82 and 0·07, respectively). Copper treatment also reduced estimated initial disease incidence and epidemic growth rates every year.     

Timing of copper sprays to protect mechanical wounds against infection by Xanthomonas citri subsp. citri,causal agent of citrus canker

European Journal of Plant Pathology - Copper sprays are the most important measure for control of citrus canker. Mechanical wounds caused by cultural practices are entry points for the causal...     

Monitoring for resistant populations of <Emphasis Type="BoldItalic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis> and epiphytic bacteria on citrus trees treated with copper or streptomycin using a new semi-selective medium

Streptomycin has been tested as an alternative to copper bactericides, which are routinely used for the control of citrus canker (Xanthomonas citri subsp. citri, Xcc) in citrus producing areas where the disease is endemic. A major concern is that excessive use of copper as a bactericide may lead to development of copper-resistant strains of Xcc. In this study, we developed a semi-selective medium to recover copper or streptomycin-resistant strains of Xcc from citrus leaves. The newly developed semi-selective medium was used to monitor the effect of a 21-day-interval copper or streptomycin spray program on Xcc for three consecutive seasons and on citrus epiphytic bacterial populations for two seasons in a commercial grapefruit grove. Although, no copper- or streptomycin-resistant strains of Xcc were isolated after three seasons, we observed a significant increase over time in the frequency of citrus epiphytic bacteria resistant to these chemicals. Overall, the proportion of epiphytic bacteria resistant to streptomycin on treated and untreated leaves was proportionally lower than the copper-resistant bacterial population. When application of each bactericide was suspended for the season, the proportion of bactericide-resistant bacteria in the epiphytic population decreased to that of the non-treated bacterial population. Availability of an alternative bactericide, such as streptomycin, to integrate into a copper-based program would reduce the amount of each bactericide sprayed in citrus orchards and possibly lower the selection pressure for bacterial resistance to these chemicals.