Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.
ABSTRACT: Background:In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas. Results:Using comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/µl in vitro and 1.5 × 104 CFU ml-1 in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study. Conclusions:We demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.
Project description:Copper sprays have been widely used for control of endemic citrus canker caused by Xanthomonas citri subsp. citri in citrus-growing areas for more than 2 decades. Xanthomonas alfalfae subsp. citrumelonis populations were also exposed to frequent sprays of copper for several years as a protective measure against citrus bacterial spot (CBS) in Florida citrus nurseries. Long-term use of these bactericides has led to the development of copper-resistant (Cu(r)) strains in both X. citri subsp. citri and X. alfalfae subsp. citrumelonis, resulting in a reduction of disease control. The objectives of this study were to characterize for the first time the genetics of copper resistance in X. citri subsp. citri and X. alfalfae subsp. citrumelonis and to compare these organisms to other Cu(r) bacteria. Copper resistance determinants from X. citri subsp. citri strain A44(pXccCu2) from Argentina and X. alfalfae subsp. citrumelonis strain 1381(pXacCu2) from Florida were cloned and sequenced. Open reading frames (ORFs) related to the genes copL, copA, copB, copM, copG, copC, copD, and copF were identified in X. citri subsp. citri A44. The same ORFs, except copC and copD, were also present in X. alfalfae subsp. citrumelonis 1381. Transposon mutagenesis of the cloned copper resistance determinants in pXccCu2 revealed that copper resistance in X. citri subsp. citri strain A44 is mostly due to copL, copA, and copB, which are the genes in the cloned cluster with the highest nucleotide homology (? 92%) among different Cu(r) bacteria.
Project description:BACKGROUND: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. RESULTS: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. CONCLUSION: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.
Project description:BACKGROUND: Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. RESULTS: Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands. CONCLUSION: The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker.
Project description:Xanthomonas citri subsp. citri (Xcc) A strain causes citrus bacterial canker, a serious leaf, fruit and stem spotting disease of several Citrus species. X. alfalfae subsp. citrumelonis (Xac) is the cause of citrus bacterial spot, a minor disease of citrus nursery plants and X. campestris pv. campestris (Xc) is a systemic pathogen that causes black rot of cabbage. Xanthomonas spp. form biofilms in planta that facilitate the host infection process. Herein, the role of extracellular DNA (eDNA) was evaluated in the formation and stabilization of the biofilm matrix at different stages of biofilm development. Fluorescence and light microscopy, as well as DNAse treatments, were used to determine the presence of eDNA in biofilms and bacterial cultures. DNAse treatments of Xcc strains and Xac reduced biofilm formation at the initial stage of development, as well as disrupted preformed biofilm. By comparison, no significant effect of the DNAse was detected for biofilm formation by Xc. DNAse effects on biofilm formation or disruption varied among Xcc strains and Xanthomonas species which suggest different roles for eDNA. Variation in the structure of fibers containing eDNA in biofilms, bacterial cultures, and in twitching motility was also visualized by microscopy. The proposed roles for eDNA are as an adhesin in the early stages of biofilm formation, as an structural component of mature bacterial aggregates, and twitching motility structures.
Project description:BACKGROUND: Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. RESULTS: A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic bacteria. The assay was capable of detecting CBC-causing strains from several geographical origins and pathotypes. CONCLUSIONS: The CBC-LAMP technique is a simple, fast, sensitive and specific method for the diagnosis of Citrus Bacterial Canker. This method can be useful in the phytosanitary programs of the citrus industry worldwide.
Project description:Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), is a severe disease of citrus. Xcc presents broad spectrum of citrus hosts including economically important species whereas X. fuscans subsp. aurantifolii-type C (XauC) causes a milder disease and only infects Citrus aurantifolia. Trehalase catalyzes hydrolysis of the disaccharide trehalose, a sugar that has been reported to be related to Xcc pathogenicity. We expressed the recombinant gene product and assessed Xcc trehalase structural and kinetics data. The recombinant protein presented 42.7% of secondary structures in ?-helix and 13% in ?-sheets, no quaternary structure in solution, and Michaelis-Menten constant (KM) of 0.077 mM and Vmax 55.308 ?Mol glucose.min-1.mg protein-1 for trehalose. A Xcc mutant strain (Xcc?treA) was produced by gene deletion from Xcc genome. Enzymatic activity of trehalase was determined in Xcc, XauC and Xcc?treA cellular lysates, showing the highest values for XauC in in vitro infective condition and no activity for Xcc?treA. Finally, leaves of Citrus aurantifolia infected with Xcc?treA showed much more drenching and necrosis than those infected by wild type Xcc. We concluded that trehalase contributes to alleviate bacterial virulence and that inability for trehalose hydrolysis may promote higher Xcc infectivity.
Project description:The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase ? subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.
Project description:Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by Xanthomonas citri ssp. citri (X. citri); thus, new sustainable strategies to manage this disease are needed. Although all Citrus spp. are susceptible to this pathogen, they are resistant to other Xanthomonas species, exhibiting non-host resistance (NHR), for example, to the brassica pathogen X. campestris pv. campestris (Xcc) and a gene-for-gene host defence response (HDR) to the canker-causing X. fuscans ssp. aurantifolii (Xfa) strain C. Here, we examine the plant factors associated with the NHR of C. limon to Xcc. We show that Xcc induced asymptomatic type I NHR, allowing the bacterium to survive in a stationary phase in the non-host tissue. In C. limon, this NHR shared some similarities with HDR; both defence responses interfered with biofilm formation, and were associated with callose deposition, induction of the salicylic acid (SA) signalling pathway and the repression of abscisic acid (ABA) signalling. However, greater stomatal closure was seen during NHR than during HDR, together with different patterns of accumulation of reactive oxygen species and phenolic compounds and the expression of secondary metabolites. Overall, these differences, independent of Xcc type III effector proteins, could contribute to the higher protection elicited against canker development. We propose that Xcc may have the potential to steadily activate inducible defence responses. An understanding of these plant responses (and their triggers) may allow the development of a sustained and sustainable resistance to citrus canker.
Project description:Xanthomonas citri pv. aurantifolii pathotype B (XauB) and pathotype C (XauC) are the causative agents respectively of citrus canker B and C, diseases of citrus plants related to the better-known citrus canker A, caused by Xanthomonas citri pv. citri. The study of the genomes of strains of these related bacterial species has the potential to bring new understanding to the molecular basis of citrus canker as well as their evolutionary history. Up to now only one genome sequence of XauB and only one genome sequence of XauC have been available, both in draft status. Here we present two new genome sequences of XauB (both complete) and five new genome sequences of XauC (two complete). A phylogenomic analysis of these seven genome sequences along with 24 other related Xanthomonas genomes showed that there are two distinct and well-supported major clades, the XauB and XauC clade and the Xanthomonas citri pv. citri clade. An analysis of 62 Type III Secretion System effector genes showed that there are 42 effectors with variable presence/absence or pseudogene status among the 31 genomes analyzed. A comparative analysis of secretion-system and surface-structure genes showed that the XauB and XauC genomes lack several key genes in pathogenicity-related subsystems. These subsystems, the Types I and IV Secretion Systems, and the Type IV pilus, therefore emerge as important ones in helping explain the aggressiveness of the A type of citrus canker and the apparent dominance in the field of the corresponding strain over the B and C strains.
Project description:The plant-pathogenic bacterium Xanthomonas citri subsp. citri is the causal agent of Asiatic citrus canker, a serious disease that affects all the cultivars of citrus in subtropical citrus-producing areas worldwide. There is no curative treatment for citrus canker; thus, the eradication of infected plants constitutes the only effective control of the spread of X. citri subsp. citri. Since the eradication program in the state of São Paulo, Brazil, is under threat, there is a clear risk of X. citri subsp. citri becoming endemic in the main orange-producing area in the world. Here we evaluated the potential use of alkyl gallates to prevent X. citri subsp. citri growth. These esters displayed a potent anti-X. citri subsp. citri activity similar to that of kanamycin (positive control), as evaluated by the resazurin microtiter assay (REMA). The treatment of X. citri subsp. citri cells with these compounds induced altered cell morphology, and investigations of the possible intracellular targets using X. citri subsp. citri strains labeled for the septum and centromere pointed to a common target involved in chromosome segregation and cell division. Finally, the artificial inoculation of citrus with X. citri subsp. citri cells pretreated with alkyl gallates showed that the bacterium loses the ability to colonize its host, which indicates the potential of these esters to protect citrus plants against X. citri subsp. citri infection.