Cocculus hirsutus extract inhibits the Xanthomonas oryzae pv. oryzae,the bacterial leaf blight pathogen in rice

Plant-derived natural bactericides and their possible applications in agriculture to control plant bacterial diseases has intensified as this approach has enormous potential to inspire and influence modern agro-chemical research. Naturally occurring and biologically active plant products such as essential oils and organic extracts could be a source of alternative classes of natural biopesticides to serve as templates for new and more effective compounds in controlling plant pathogenic micro-organisms. In the present study, the efficacy of six plants extracts from different solvent system were tested for their antibacterial activity aganist Xanthomonas oryzae pv. oryzae both in vitro and in vivo. Among these extracts, Cocculus hirsutus leaf chloroform extract exhibits significant antibacterial activity against X. oryzae pv. oryzae. Data obtained from the experiments such as minimum inhibitory concentration, effect of C. hirsutus leaf chloroform extract on the incidence of X. oryzae pv. oryzae, phytotoxicity test and effect of C. hirsutus leaf chloroform extract on seed germination and seedling vigour, along with the in vivo experiments under greenhouse conditions showed significant improvement over controls. Thus, the present study demonstrated that the C. hirsutus leaf chloroform extract posses antibacterial activity against bacterial leaf blight pathogen of rice.     

Plasmid and pathogenicity in Xanthomonas oryzae pathovar oryzae, the bacterial blight pathogen of Oryza sativa

Xanthomonas oryzae pv. oryzae , the causative agent for bacterial leaf blight of rice, comprises diverse groups of strains differing in biochemical and pathological characteristics. A collection of X.o . pv. oryzae strains differing in geographical origin was screened for the presence of plasmids. Out of 17 isolates of X.o. pv. oryzae , 14 harboured plasmids of which two isolates (XoP5, XoC26) had two plasmids each and one isolate (XoR20) had three. The remaining isolates contained a single plasmid of identical mobility. Finger print analysis of plasmids was carried out using Eco RI for 10 isolates. The restriction fragment pattern was distinct for each isolate. They were classified under three groups based on cluster analysis using the unweighted pair group method with averages (UPGMA). Of the 18 plasmids, the plasmid pMA36 ( X.o. pv. oryzae XoC36) was further characterized. This plasmid was cured by acridine orange at the frequency rate of 10%. The cured strain was transformed with pMA36 at a frequency of 2.3 times 10² transformants μg^-1 of plasmid DNA. The plasmid-cured strain was virulent on rice but symptom development was delayed when compared to wild and transformed strains. The wild type strain ( X.o. pv. oryzae XoC36) was resistant to ampicillin, carbenicillin and rifampicin whereas the cured strain was resistant to carbenicillin and rifampicin but sensitive to ampicillin. The transformant was resistant to the three antibiotics indicating that the plasmid pMA36 codes for ampicillin resistance. The plasmid influenced the pathogenicity of X.o. pv. oryzae.     

Genetic diversity and pathogenicity of the grass pathogen Xanthomonas translucens pv. graminis

Genetic diversity of Xanthomonas translucens pv. graminis (X.t.g.), the causal agent of bacterial wilt in forage grasses, was assessed using 16S rDNA sequencing, AFLP analysis and pathogenicity screening on three Italian ryegrass (Lolium multiflorum) cultivars. The aim of this in depth analysis was to provide insight into geographic variation and race specificity of X.t.g. in order to develop strategies for plant protection and resistance breeding. 16S rDNA sequencing of 29 putative X.t.g. isolates allowed to assign 28 isolates to the pv. translucens while one isolate was identified as X.t. pv. arrhenatheri. AFLP analysis and UPGMA clustering resulted in two distinct clusters with a similarity of 90% based on Nei and Li's coefficient and 306 polymorphic markers. A significant effect of geographic location of the collection sites on genetic diversity was detected by redundancy analysis using AFLP markers and the explanatory variables longitude, latitude and altitude. The two groups identified with redundancy analysis were congruent to the major clusters identified with cluster analysis. All X.t.g. isolates identified as pv. graminis were pathogenic, showing mostly moderate to high pathogenicity. However, the three cultivars used for pathogenicity testing showed significant differences in susceptibility and a significant interaction between cultivars and isolates was observed, indicating an at least partial race-specific resistance. With the information provided, targeted selection of X.t.g. isolates may allow one to efficiently address specific tasks in resistance breeding.     

Differentiation between Xanthomonas campestris pv. oryzae, Xanthomonas campestris pv. oryzicola and the bacterial 'brown blotch' pathogen on rice by numerical analysis of phenotypic features and protein gel electrophoregrams

Thirty-five Xanthomonas campestris pv. oryzae, fourteen X. campestris pv. oryzicola strains and six 'brown blotch' pathogens of rice, all of different geographical origin, were studied by numerical analysis of 133 phenotype features and gel electrophoregrams of soluble proteins, %G + C determinations and DNA:rRNA hybridizations. The following conclusions were drawn. (i) The Xanthomonas campestris pathovars oryzae and oryzicola display clearly distinct protein patterns on polyacrylamide gels and can be differentiated from each other by four phenotype tests. (ii) Both pathovars are indeed members of Xanthomonas which belongs to a separate rRNA branch of the second rRNA superfamily together with the rRNA branches of Pseudomonas fluorescens, Marinomonas, Azotobacter, Azomonas and Frateuria. (iii) 'Brown blotch' strains are considerably different from X. campestris pv. oryzae and oryzicola. They are not members of the genus Xanthomonas, but are more related to the generically misnamed. Flavobacterium capsulatum, Pseudomonas paucimobilis, Flavobacterium devorans and 'Pseudomonas azotocolligans' belonging in the fourth rRNA superfamily. (iv) No correlation was found between the virulence, pathogenic groups or geographical distribution of X. campestris pv. oryzae or oryzicola strains and any phenotypic or protein electrophoretic property or clustering.     

A new semi-selective medium for the isolation of Xanthomonas axonopodis pv. dieffenbachiae, the etiological agent of anthurium bacterial blight

Aims:  Xanthomonas axonopodis pv. dieffenbachiae causes anthurium blight, which is regarded as the most threatening disease for the anthurium industry worldwide. The bacterium is listed as a quarantine pathogen in several regions, including Europe. We evaluated the use of Neomycin-Cephalexin-Trimethoprime-pivMecillinam 4 (NCTM4) medium for its isolation.
Methods and Results:  A total of 104 bacterial strains were inoculated onto NCTM4 and on the previously published Cellobiose-Starch (CS) and Esculin-Trehalose (ET) media. The strain collection included: the anthurium blight pathogen, Xanthomonas strains, for which false positive results are known to occur using serological identification-tests; other bacterial pathogens of anthurium; and representatives of bacteria that are commonly present in the anthurium phyllosphere. Media were evaluated following the ISO 16140 protocol for the validation of alternative methods.
Conclusion:  Growth of the anthurium blight pathogen was better on NCTM4 and ET media than on CS. NCTM4 provided a better repeatability. It also displayed a lower rate of false positive and false negative results when the pathogen was isolated from plant extracts.
Significance and Impact of the Study:  This study will lead to improved isolation protocols of the anthurium blight in official procedures. NCTM4 medium could also favourably be used in studies, which aim to further understanding of the biology and epidemiology of this pathogen.     

Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1, which causes citrus bacterial spot disease, and related strains provides insights into virulence and host specificity

Xanthomonas axonopodis pv. citrumelo is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing, and optical mapping were used to obtain a complete genome sequence of X. axonopodis pv. citrumelo strain F1, 4.9 Mb in size. The strain lacks plasmids, in contrast to other citrus Xanthomonas pathogens. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen X. campestris pv. vesicatoria 85-10, with a completely different host range. We also compared X. axonopodis pv. citrumelo to the genome of citrus canker pathogen X. axonopodis pv. citri 306. Comparative genomic analysis showed differences in several gene clusters, like those for type III effectors, the type IV secretion system, lipopolysaccharide synthesis, and others. In addition to pthA, effectors such as xopE3, xopAI, and hrpW were absent from X. axonopodis pv. citrumelo while present in X. axonopodis pv. citri. These effectors might be responsible for survival and the low virulence of this pathogen on citrus compared to that of X. axonopodis pv. citri. We also identified unique effectors in X. axonopodis pv. citrumelo that may be related to the different host range as compared to that of X. axonopodis pv. citri. X. axonopodis pv. citrumelo also lacks various genes, such as syrE1, syrE2, and RTX toxin family genes, which were present in X. axonopodis pv. citri. These may be associated with the distinct virulences of X. axonopodis pv. citrumelo and X. axonopodis pv. citri. Comparison of the complete genome sequence of X. axonopodis pv. citrumelo to those of X. axonopodis pv. citri and X. campestris pv. vesicatoria provides valuable insights into the mechanism of bacterial virulence and host specificity.     

Type three effector gene distribution and sequence analysis provide new insights into the pathogenicity of plant-pathogenic Xanthomonas arboricola

Xanthomonas arboricola is a complex bacterial species which mainly attacks fruit trees and is responsible for emerging diseases in Europe. It comprises seven pathovars (X. arboricola pv. pruni, X. arboricola pv. corylina, X. arboricola pv. juglandis, X. arboricola pv. populi, X. arboricola pv. poinsettiicola, X. arboricola pv. celebensis, and X. arboricola pv. fragariae), each exhibiting characteristic disease symptoms and distinct host specificities. To better understand the factors underlying this ecological trait, we first assessed the phylogenetic relationships among a worldwide collection of X. arboricola strains by sequencing the housekeeping gene rpoD. This analysis revealed that strains of X. arboricola pathovar populi are divergent from the main X. arboricola cluster formed by all other strains. Then, we investigated the distribution of 53 type III effector (T3E) genes in a collection of 57 X. arboricola strains that are representative of the main X. arboricola cluster. Our results showed that T3E repertoires vary greatly between X. arboricola pathovars in terms of size. Indeed, X. arboricola pathovars pruni, corylina, and juglandis, which are responsible for economically important stone fruit and nut diseases in Europe, harbored the largest T3E repertoires, whereas pathovars poinsettiicola, celebensis, and fragariae harbored the smallest. We also identified several differences in T3E gene content between X. arboricola pathovars pruni, corylina, and juglandis which may account for their differing host specificities. Further, we examined the allelic diversity of eight T3E genes from X. arboricola pathovars. This analysis revealed very limited allelic variations at the different loci. Altogether, the data presented here provide new insights into the evolution of pathogenicity and host range of X. arboricola and are discussed in terms of emergence of new diseases within this bacterial species.     

Sequence and molecular analysis of the rpoA cluster genes from Xanthomonas campestris pv. campestris

The Xanthomonas campestris rpsM (S13)-rpsK (S11)-rpsD (S4)-rpoA (alpha)-rplQ (L17) cluster, encoding RNA polymerase alpha-subunit and four ribosomal proteins, reside in a 3164-bp DNA region. The N-terminal sequence of the authentic alpha-protein determined chemically matches that predicted from the nucleotide sequence. rplQ is monocistronic, instead of being co-transcribed with the other genes as in Escherichia coli. Antiserum against the His-tagged alpha-protein cross-reacted with the E. coli alpha-protein.     

Biochemical and molecular identification of Xanthomonas translucens pv. undulosa causing bacterial leaf streak of wheat in Punjab,Pakistan

Xanthomonas translucens pv. undulosa, (Xtu.), causal agent of Bacterial leaf streak (BLS) of wheat, was characterised through pathogencity, hypersensitivity, biochemical and molecular assays. Fifty symptomatic leaves of wheat were collected from eight agro-ecological zones of Punjab out of which 25 were isolated and purified. Maximum incidence and severity in Faisalabad were followed by Multan and Rahim Yar Khan. The pathogen isolated from diseased leaves was identified on the basis of colonies pattern, colour, biochemical and pathogencity test as X. translucens pv. undulosa and confirmed its pathogencity through pathogencity test. For molecular characterization, the bacterial 16S–23S rDNA spacer fragments were amplified by PCR with conserved primers (C1 and C2) and then in combination with specific primers (T1 & T2). 300?bp product amplified by C1 and C2 primer pair confirmed the presence of Xanthomonas, while specific primers T1 and T2 amplified a product of 200?bp, confirmed the presence of X. translucens pv. undulosa. This work will be quite helpful for wheat pathologist and breeders for future management strategy for this disease.     

Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.     

Phylogenetic relatedness of Xanthomonas axonopodis pv. punicae, the causal agent of bacterial blight of pomegranate based on two loci, 16S rRNA and gyrB

Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a major disease in pomegranate (Punica granatum) cultivation in India. The Xap strains from three distinct geographical origins, Delhi, Maharashtra and Andhra Pradesh were studied for their genetic variability and phylogenetic relationship with other Xanthomonads targeting two important loci 16S rRNA and gyrB. All Xap strains showed 100 % sequence conservation in both the loci, suggesting that geographical origin does not necessarily reflect variation to genetic make-up of the Xap. Phylogeny derived from 16S rRNA gene revealed that two Xanthomonas species, Xanthomonas citri subsp. malvacearum DSM 3849 T and X. axonopodis pv. manihotis NCPPB1834 formed a single cluster along with Xap. Further, analysis in the gyrB locus indicated that X. citri subsp. malvacearum shared 99.4 % identity while pathovars X. axonopodis pv. manihotis shared only 95 % identity with the Xap strains. Thus, we established that gyrB was the preferred locus over 16S rRNA gene to discriminate the Xap strains from closely related Xanthomonas species type strains. Nevertheless, our study demonstrated for the first time that pomegranate bacterial blight pathogen is phylogenetically very close to Xanthomonas citri subsp. malvacearum infecting cotton.     

A duplex-PCR method for species- and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni

A PCR-based method was developed for the stone fruit quarantine pathogen Xanthomonas arboricola pv. pruni (Xap), which provides rapid, sensitive and specific in planta detection and isolate identification. Primers specific for Xap were identified using random amplified polymorphic DNA (RAPD). Simplex PCR with these primers had a limit of detection per PCR reaction of approximately 10 CFU for isolate cultures and 50 CFU for plant material when used on tenfold dilutions of isolate culture or genomic DNA extracted from spiked samples, respectively. The primers were adapted as a high-throughput single-step screening based on a digoxigenin-labeled DNA probe assay with a detection limit of 4 × 10² CFU from isolate cultures. A duplex-PCR method was designed that includes the pathovar-level with species-level primers based on species-specific regions of the quinate metabolic gene qumA, increasing diagnostic confidence and offering the first molecular test for all X. arboricola pathovars.     

Biochemical and molecular detection of Xanthomonas citri subsp. malvacearum,the causal organism of bacterial blight of cotton in the northern cotton zone of Nigeria

Xanthomonas citri subsp. malvacearum causes severe qualitative and quantitative losses to farmers in cotton growing areas of the world. Timely detection of the bacterial pathogen causing blight in cotton is extremely important in developing management strategies against the disease. Bacterial isolates were extracted from cotton seeds obtained from five ginneries located in northern Nigeria. Bacterial colonies were isolated and tested for nitrate reductase activity, oxidase reaction, catalase reaction, aesculin hydrolysis, utilisation of carbon from different sources and Tween 80 hydrolysis was assessed, PCR was also carried out on the isolates. The studied bacteria from the four locations reacted positively to Tween 80 hydrolysis and catalase test. Maltose digestion was negative in all samples, hydrolysis and digestion of arabinose, arabitol, cellobiose, lactate and acetate were positive. They were thus identified as members of the species X. citri based on the biochemical tests run. Molecular detection using PCR positively identified the pathogen.     

The ubiquitous plasmid pXap41 in the invasive phytopathogen Xanthomonas arboricola pv. pruni: complete sequence and comparative genomic analysis

The complete DNA sequence of the 41 102-bp plasmid pXap41 from the invasive plant pathogen Xanthomonas arboricola pv. pruni CFBP 5530 was determined and its 44 coding regions were annotated. Comparative analysis with 15 Xanthomonas plasmids and 19 complete genomes revealed that nearly one-fourth of this plasmid has high sequence identity to plasmid pXAC64 and an 8.8-kb chromosomal region of Xanthomonas axonopodis pv. citri strain 306 carrying genes that encode type III effectors and helper proteins. The presence of pXap41 in all X. arboricola pv. pruni genotypes was confirmed for eight strains by plasmid profiling and for 35 X. arboricola pv. pruni isolates with a new plasmid multiplex PCR assay. This plasmid was not detected in any other X. arboricola pathovars (n=12), indicating the potential for the application of the pXap41 PCR method as a pathovar-level detection and identification tool.