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.     

Narrow host range phages associated with citrus canker lesions in Florida and Argentina

Bacteriophages were isolated from naturally infected citrus canker lesions from diverse locations in Florida and Argentina and characterized for host range using a world-wide collection of Xanthomonas citri subsp. citri (Xcc) strains. Sixty-seven bacteriophages isolated from citrus canker lesions in Florida (37 bacteriophages) and Argentina (30 bacteriophages) revealed little diversity. All 30 phages isolated from four locations in Argentina had identical host ranges (group ARG), while 37 phages from Florida made up two groups (FLA and FLB). ARG and the 31 FLA phages produced clear plaques and had nearly identical host ranges as phage CP2 of Japan in that they only reacted with typical A strains and none of the atypical A strains (A^* and A^W) or other Xanthomonas spp. FLB phages had a different host range from the other strains and produced turbid plaques. We used phage typing, fatty acid analysis and riboprinter analysis to classify citrus-associated xanthomonads. Phage typing using 12 phages isolated from Xcc, X. fuscans subsp. aurantifolii (Xfa), X. alfalfae subsp. citrumelonis (Xacm), and other sources proved useful for classifying all major Xcc pathotypes and/or strains (A, A*, Miami (MI), Manatee (MA) and Wellington (A^W)), as well as B and C types of Xfa. X. citri subsp. citri strains from a worldwide collection were diverse in phage susceptibility. The majority of Xcc strains, which originated from different regions of the world and which were typical “A” pathotype strains based on pathogenicity characteristics, was sensitive to most phages (including CP2, FLA and ARG), and had nearly identical phage sensitivity profiles. MA strains were quite unique in that they reacted with none of the phages; furthermore, they were different from the putative progenitor MA strain, ATCC 49118, which reacted with a group of phages. Fatty acid analysis revealed considerable variation in Xcc-A, Xfa-B, Xfa-C and Xacm strains. Using riboprinter analysis, we identified a unique riboprinter pattern for strains isolated from an etrog tree (Citrus medica) in Florida that were “A” pathotype strains based on pathogenicity characteristics. Phage typing and fatty acid analysis were useful in corroborating that the etrog strains represent a unique new Xcc strain in Florida.     

Biofilm formation and motility of Xanthomonas strains with different citrus host range

Xanthomonas citri subsp. citri (Xcc) strain A is the causal agent of citrus bacterial canker (CBC) on most Citrus spp. and close relatives. Two restricted host range strains of CBC, A^w and A*, from Florida and southwest Asia, respectively, infect Mexican lime. Several studies have linked biofilm formation by Xcc to bacterial colonization prior to and after plant ingress, but none have evaluated connections between biofilm formation and the behaviour of different strains of Xcc on citrus hosts and non‐hosts. In this study biofilm formation and swimming motility were evaluated for citrus pathogenic xanthomonads including wide and restricted host range strains of Xcc, X. alfalfae subsp. citrumelonis (Xac) (the causal agent of citrus bacterial spot) and X. campestris pv. campestris (Xc). Differential biofilm formation was observed in vitro and in planta among the Xanthomonas strains assayed. Minimal medium XVM2 increased biofilm formation, especially for those strains with a host range restricted to Mexican lime. In planta, strains produced more biofilm on leaves or fruits of their host than on non‐hosts. Scanning electron microscopy of biofilms on leaf and fruit surfaces revealed differences in structure of bacterial aggregates with respect to the strain's host range. In addition, swimming motility varied widely depending on the host range of the strain. It was concluded that biofilm formation in vitro and in planta for strains of Xcc and Xac was related to their host range, as these processes affect colonization at the early stages of the infection process.     

N‐acetylcysteine interferes with the biofilm formation,motility and epiphytic behaviour of Xanthomonas citri subsp. citri

Citrus canker is caused by Xanthomonas citri subsp. citri. Bacterial biofilm formation is important in the development of this disease because it is a factor in epiphytic bacterial survival on leaves and in infection. N‐acetylcysteine (NAC), in addition to having antibacterial properties, reduces biofilm formation by a variety of bacteria and was therefore tested for impairing biofilm formation by X. citri. Copper is currently the antimicrobial compound most commonly applied in agriculture to control citrus canker. Therefore, this study also evaluated a possible synergistic effect between NAC and copper to improve the strategy for controlling this phytopathogen. NAC was found to decrease biofilm formation, the production of extracellular polysaccharides and bacterial stickiness. Motility was also affected in the presence of NAC. The best combination of NAC and copper for controlling X. citri was application of NAC followed by copper 48 h later. The concentrations of 6 mg mL^?1 of NAC and 3·5 μg mL^?1 of copper were able to kill X. citri. NAC inhibited the epiphytic behaviour of X. citri on leaves, altering cell growth and the bacterial ability to form biofilms. The addition of copper to cells previously treated with NAC enhanced its bactericidal activity. In conclusion, NAC has antibacterial properties against X. citri, interfering with bacterial growth, motility and biofilm formation. Under epiphytic conditions, NAC made the cells more susceptible to copper by affecting X. citri biofilm formation. This study opens new possibilities for the use of NAC in combination with copper, possibly resulting in more sustainable management of citrus canker.     

Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a disease that affects almost all types of citrus crops. Production of particular Xcc pathogenicity factors is controlled by a gene cluster rpf, which encodes elements of a cell–cell communication system called quorum sensing (QS), mediated by molecules of the diffusible signal factor (DSF) family. Interference with cell–cell signalling, also termed quorum quenching, either by signal degradation or over‐production, has been suggested as a strategy to control bacterial disease. In this study, three bacterial strains were isolated from citrus leaves that displayed the ability to disrupt QS signalling in Xcc. Pathogenicity assays in sweet orange (Citrus sinensis) showed that bacteria of the genera Pseudomonas and Bacillus also have a strong ability to reduce the severity of citrus canker disease. These effects were associated with alteration in bacterial attachment and biofilm formation, factors that are known to contribute to Xcc virulence. These quorum‐quenching bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used to treat citrus canker disease.     

Unstable green fluorescent protein for study of Xanthomonas citri subsp. citri survival on citrus

Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus bacterial canker, an important disease for the citrus industry. Studies of Xac survival in environments outside of the lesion performed in the past may have underestimated the viable population because the recovery was based on the ability of the bacterium to grow on culture media. This study monitored survival of Xac that express green fluorescent protein (GFP) in two different forms: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Transformed strains of Xac were verified to be stable in their expression of GFP and to show no differences in virulence and fitness compared to wild type strains. Evaluation of protein stability confirmed that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal microscopy was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. Aggregation of viable bacteria in biofilms confirms their role in pathogen survival outside of lesions and protection from bactericide treatments in the field or in the fruit disinfection process.     

Epiphytic Curtobacterium flaccumfaciens strains isolated from symptomless solanaceous vegetables are pathogenic on leguminous but not on solanaceous plants

In Iran, during 2013–16, 16 Gram‐positive corynebacteria‐like strains were recovered from the epiphytic parts of solanaceous vegetables including eggplant, pepper and tomato. The strains were recovered accidentally as a result of monitoring for other bacterial pathogens in solanaceous fields. The strains were phenotypically different from Clavibacter michiganensis strains. Although none of the strains were pathogenic on their host of isolation or on any other solanaceous plants, 12 out of 16 strains were pathogenic on common bean, cowpea, mung bean and soybean. Colonization by strains was observed on maize, zucchini, faba bean, honeydew melon, rapeseed, sugar beet and sunflower plants under greenhouse conditions. In PCR tests, the primer pair CffFOR2/CffREV4, specific for Curtobacterium flaccumfaciens pv. flaccumfaciens, enabled the amplification of the appropriately sized fragment in 12 out of 16 strains, and all 12 strains were pathogenic on dry beans. Phylogenetic analysis, using the gyrB and recA genes, showed all 16 bacterial strains clustered within several pathovars of C. flaccumfaciens. A nonpathogenic yellow‐pigmented strain (Xeu15) was clustered with the type strains of C. flaccumfaciens pv. betae and C. flaccumfaciens pv. oortii. Bacteriocin profiling assays revealed no significant differences among the pathogenic and nonpathogenic strains. Host range and population dynamics of four representative strains on 17 plant species showed population build‐up of the strains only on common bean, cowpea, wheat and red nightshade plants. The results provide important insights into the possible role of nonhost plants as reservoirs of plant pathogenic bacteria, which has important implications in plant disease epidemiology and management.     

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database under the accession numbers AB469331to AB469354.     

Consequences of the taxonomic revision of Xanthomonas axonopodis pv. dieffenbachiae and of the analysis of its associated pathogenicity for the recommendation of regulation (EPPO A2 List)

Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of bacterial blight of Araceae (aroids), is a regulated pest in several countries and is included in the EPPO A2 List. Reference strains of Xanthomonas axonopodis pv. dieffenbachiae have recently been reclassified into the species Xanthomonas phaseoli, Xanthomonas citri and Xanthomonas euvesicatoria on the basis of different features, including multilocus sequence analysis, average nucleotide identity and homology in DNA–DNA hybridization analyses. Based on pathogenicity tests, Constantin et al. (2017) proposed naming the pathogens on aroids as X. phaseoli pv. dieffenbachiae, X. phaseoli pv. syngonii and X. citri pv. aracearum. Recommendations are made on how to deal with these changes for the group of pathogenic bacteria for Araceae. The name Xanthomonas axonopodis pv. dieffenbachiae on the EPPO List should be adjusted to the names proposed in the taxonomic study by Constantin et al. (2016). The current EPPO Diagnostic Standard is directed at strains pathogenic on Anthurium. They mainly belong to X. phaseoli pv. dieffenbachiae, but some also to X. citri pv. aracearum that are not detected by the EPPO Diagnostic Standard. Xanthomonas phaseoli pv. syngonii strains are also aggressive, but with a host range restricted to Syngonium. The pathogenicity specific to aroids of the bacterial isolates reclassified as Xanthomonas euvesicatoria was not confirmed and no pathovar epithet has been retained for these strains.     

Differentiation ofVerticillium dahliae populations on the basis of vegetative compatibility and pathogenicity on cotton

Complementary auxotrophic nitrate-nonutilizing (nit) mutants were used to investigate vegetative compatibility within 27 strains ofVerticillium dahliae isolated from several hosts originating from Africa, Asia, Europe and the United States. Using about 500nit mutants generated from these strains, three vegetative compatibility groups, 1, 2 and 4, were identified. Simultaneously, virulence of each strain was assessed on cultivars ofGossypium hirsutum, G. barbadense andG. arboreum, based upon Foliar Alteration Index (FAI) and Browning Index (BI) estimation. The strains in VCG1 were of both the cotton-defoliating pathotype and race 3 (on cotton) but were non pathogenic on tomato; those in VCG2 and VCG4 were of the nondefoliating pathotype and belonged to different races on cotton and on tomato. Hyaline mutants deriving from parental wild-type strain showed differences in pathogenicity but were always assigned to the parental VCG. A relationship was established between VCGs and the taxonomic position of host plants. Data fromnit pairings indicated that the sub-populations ofV. dahliae (VCGs) may not be completely isolated genetically.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.     

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.     

Pathogenicity and virulence gene content of Xanthomonas strains infecting Araceae,formerly known as Xanthomonas axonopodis pv. dieffenbachiae

Bacterial leaf blight of aroids is caused by a heterogeneous group of xanthomonads listed as Xanthomonas axonopodis pv. dieffenbachiae (Xad) on the EPPO A2 quarantine list. Recently, Xad strains were shown not to belong to X. axonopodis but to the species X. citri, X. phaseoli and X. euvesicatoria. Here, to verify the pathovar designation, 11 representative strains were tested for pathogenicity on six aroid genera. They had overlapping host ranges and only the strain isolated from Syngonium showed host specificity. The X. citri strains, isolated from various hosts, showed dissimilarity in virulence to the tested aroid genera. The X. phaseoli strains, isolated from Anthurium and Syngonium, were generally more virulent and, additionally, induced systemic infections. The X. euvesicatoria strains, isolated from Philodendron, were scored as not pathogenic on the tested aroids. Four representative strains were genome sequenced and showed a variable virulence‐associated gene content. Pathogenicity to aroids was correlated with the presence of three specific T3 effector genes and with a T6SS gene sequence. Together, the phylogenetic and pathogenic differentiation among Xad strains justifies the installation of three pathovar epithets for the pathogens on aroids: X. phaseoli pv. dieffenbachiae comb. nov. for the strains isolated from Anthurium; X. phaseoli pv. syngonii comb. nov. for the strain isolated from Syngonium; and X. citri pv. aracearum comb. nov. for the strains isolated from Aglaonema, Xanthosoma and Dieffenbachia. It is proposed that phytosanitary regulations for xanthomonads on aroids are restricted to these three pathovars.     

A molecular marker for the specific detection of new pathotype 5‐like strains of Plasmodiophora brassicae in canola

Clubroot of crucifers, caused by Plasmodiophora brassicae, is managed in canola (Brassica napus) by the deployment of resistant cultivars. Recently, however, new strains of P. brassicae have been detected in Alberta, Canada, that can overcome this resistance. Some of these strains are classified as pathotype 5 on the differential system of Williams, but are distinguished by their ability to overcome host resistance. In order to expedite the identification of these new pathotype 5‐like strains, three primer sets were developed based on the 18S‐ITS region of the pathogen. With primers P5XF3 and P5XR3, a 127 bp product was amplified from all new pathotype 5‐like strains following optimized PCR analysis. A TaqMan probe‐based quantitative assay was also developed. These protocols could be used to detect as little as 0.5 pg P. brassicae DNA, and as few as 10⁴ mL^?1 pathogen resting spores; infection of host tissues could be detected as soon as 4 days after inoculation. The PCR and qPCR assays described in this study represent useful tools for the rapid and reliable diagnosis and quantification of new pathotype 5‐like strains of P. brassicae.     

Spirodiclofen and spirotetramat bioassays for monitoring resistance in citrus red mite, Panonychus citri (Acari: Tetranychidae)

BACKGROUND: Citrus red mite, Panonychus citri (McGregor), is a key pest of San Joaquin Valley California citrus. Spirodiclofen was registered for mite control in 2007, and spirotetramat for scale control in 2008. Because of the potential for resistance to spirodiclofen to develop in spider mites, and cross‐resistance to spirotetramat used for other citrus pests, bioassay methods for resistance monitoring were developed. RESULTS: The responses of four populations of adult female, egg and larval stages of P. citri to spirodiclofen were compared to determine the most robust bioassay method for this pesticide. Adult females responded with a higher LC₉₉ and larval stages exhibited higher control mortality and a lower slope of response compared with the egg stage. Thus, the egg stage was found to be the most suitable stage for testing. Egg production and egg shape were significantly affected by spirodiclofen treatment of adult female mites. Bioassays with the related compound spirotetramat revealed that P. citri egg hatch was less affected by this compound, requiring the assessment of mortality to be extended to 11 days after treatment when the hatched larvae succumbed to the pesticide. Discriminating concentrations of 10 ppm for spirodiclofen and 31.6 ppm for spirotetramat in an 11 day bioassay were tested against eight field populations of P. citri, and 99–100% mortality resulted. CONCLUSION: These results provide a baseline for the response of P. citri to spirodiclofen and spirotetramat that will aid resistance management in California citrus. Copyright © 2011 Society of Chemical Industry     

A PCR-based assay for differentiating A- and A*-type strains of Xanthomonas citri subsp. citri, the causal agent of Asiatic citrus canker

By applying A- and A*-type strains of Xanthomonas citri subsp. citri (Xcc) in a repetitive sequence-based polymerase chain reaction (rep-PCR), two DNA amplicons, one unique to each strain, were evaluated as a probe against the DNA of Xcc strains. Two pairs of primers derived from these amplicons were tested in a PCR analysis. The results confirmed that primers Ms⁺/Ms^? are useful for differentiating A-type from A*-type strains of Xcc. Also, a multiplex PCR with both set of primers can be used to distinguish three groups in Xcc populations: A-type strains and two subgroups of A* strains including Iranian and Thai A* strains.     

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.     

Mutational analysis of type III effector genes from <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>

Xanthomonas citri subsp. citri, the causal agent of citrus canker, relies extensively on a type III secretion system for infection by delivering type III effectors into host cells. In the genus Xanthomonas, two major regulators, HrpG and HrpX, are involved in the expression of genes encoding the type III secretion system. Twenty-three candidate type III effectors were identified as targets for analysis. The involvement in pathogenicity of 20 candidate effector genes in X. citri strain 306 (Xcc-306) was investigated using site-directed mutagenesis. Pathogenicity assays in grapefruit of 19 genes using site-directed mutagenesis revealed that none of the mutants demonstrated to have reduced ability to cause canker disease. A mutation in the TAL effector pthA4 ^– resulted in loss of hypertrophy although no changes were observed in bacterial growth in leaves. Mutations in hrpG, hrpX, or hrpA genes displayed a complete loss of pathogenicity. Moreover, all mutants maintained the ability to trigger a hypersensitive response (HR) in non-host tomato. In contrast to previous studies, hrpG ^– , hrpX ^– and hrpA ^– mutants also retained the ability to elicit an HR in tomato, indicating the presence of an Hrp independent elicitor in Xcc-306.     

Genome‐wide identification of pathogenicity genes in Xanthomonas oryzae pv. oryzae by transposon mutagenesis

A transposon mutant library was constructed from the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) KACC10331 by Tn5 transposon mutagenesis. The susceptible rice cultivar Milyang 23 was inoculated with a total of 24 540 mutants resistant to kanamycin and 67 avirulent or reduced‐pathogenicity mutant strains were selected for study. Southern hybridization verified that 84 mutant strains had single‐copy insertions and their single‐transposon insertion sites were identified by sequencing analysis combined with thermal asymmetric interlaced (TAIL)‐PCR. The single‐transposon‐tagged sequences of 21 mutant strains belonged to pathogenicity‐related genes previously reported in Xanthomonas species, while the other 46 single‐transposon‐tagged sequences included diverse functional genes encoding, five cell‐wall‐degrading enzymes, three fimbrial and flagella assembly regulators, five regulatory proteins, 15 metabolic regulators and 18 hypothetical proteins, which were identified as novel pathogenicity genes of Xoo.