Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

Detection, Survival and Transmission of Xanthomonas axonopodis pv. manihotis and X. axonopodis pv. vignicola, Causal Agents of Cassava and Cowpea Bacterial Blight, respectively, in/by Insect Vectors

Populations of Xanthomonas axonopodis pv. manihotis and X. axonopodis pv. vignicola, causal agents of cassava and cowpea bacterial blight, respectively, were quantified in insects. The pathogens were found in the faeces, the intestines, and on the legs and mandibles of Zonocerusvariegatus. Additionally, X. axonopodis pv. manihotis was localized in the insect gut by immunofluorescence microscopy. Xanthomonas axonopodis pv. manihotis survived at least 1 week in the insect intestines and at least 5 weeks in faeces kept under controlled conditions, while survival in faeces exposed to sunlight was <2 weeks. Five percentage [e.g. 5.8 × 10⁷ colony‐forming units (CFU)/g faeces] of the fed population of X. axonopodis pv. manihotis in cassava leaves were recovered viable in the faeces after passage through the insect. The transmission of cassava bacterial blight by pathogen‐contaminated insect faeces to intact, healthy cassava leaves was demonstrated for the first time. Xanthomonas axonopodis pv. vignicola was isolated from organs and faeces of the grasshopper Pyrgomorpha cognata, the Senegalese grasshopper (Oedaleus senegalensis), bee (Apis mellifera) and three Coleoptera (Ootheca mutabilis, Mylabris spp., Exochomus troberti) collected in bacterial blight‐infected cowpea fields. Cowpea belonged to the diet of 19 grasshopper species collected in cowpea fields as demonstrated by residues in their faeces. Pathogen‐contaminated Z. variegatus initiated an epiphytic population of 8.9 × 10⁴ CFU/g on healthy cowpea leaves. Spraying cassava and cowpea leaves with 10² and 10⁴ CFU/ml of their respective pathogen was sufficient to evoke symptoms. A possible role of insects in the transmission of X. axonopodis pvs. vignicola and manihotis is discussed.     

Biological Control of Black Rot (Xanthomonas campestris pv. campestris) of Cabbage in Tanzania with Bacillus strains

We evaluated the biocontrol efficacy of strains of Bacillus from Tanzania against the black rot pathogen, Xanthomonas campestris pv. campestris, in cabbage and the influence of the method of application under field conditions. The incidence and severity of black rot in the foliage, stems and heads of the highly susceptible cultivar, Copenhagen Market, were significantly reduced, especially when antagonists were applied through the roots as compared to application through the seeds or foliage (cotyledons). Promising antagonists included strains of B. cereus, B. lentimorbus and B. pumilus.     

Biocontrol potential of actinomycetes against Xanthomonas axonopodis pv. punicae,a causative agent for oily spot disease of pomegranate

India is a largest producer of pomegranate with high export value. The cultivation is affected with the oily spot disease caused by Xanthomonas axonopodis pv. punicae infection. The present study aims to control the disease with newer biocontrol methods. Thirty-six isolates of X. axonopodis were isolated from different varieties of infected pomegranates fruits from Maharashtra. Forty strains of actinomycete were also isolated from natural sources and screened for their antagonistic activity against X. axonopodis isolates. Eight strains of actinomycete were screened out for their high antagonistic activity and were optimized for maximizing antibiotic production. The extracted compound from A5 strain exhibited maximum inhibitory activity against all the pathogenic isolates with a MIC in the range of 0.625 to 1.25 mg mL^?1. It was identified as Streptomyces violaceusnige by 16SrRNA gene sequencing (Accession number KP208943). The extracted compound belonged to aminoglycosides with a molecular formula C₂₂H₂₈N₃O₆ determined by thin layer chromatography, high-performance liquid chromatography, gas chromatography-mass spectroscopy, nuclear magnetic resonance spectroscopy, fourier transform infrared spectroscopy and carbon hydrogen nitrogen ratio analysis. In vivo biocontrol studies with strain A5 and its extracted compound effectively prevented the growth 36 Xanthomonas isolates inoculated on pomegranate fruits, illustrating its biocontrol potential against the oily spot disease of pomegranate.     

Virulence and type III effector diversities of Xanthomonas citri pv. fuscans and X. phaseoli pv. phaseoli in Brazil

Common bacterial blight (CBB) is caused by four genetic lineages belonging to two species of Xanthomonas, namely Xanthomonas citri pv. fuscans (includes fuscans, NF2 and NF3 lineages) and X. phaseoli pv. phaseoli (lineage NF1). A collection of 117 strains of Xanthomonas isolated from common bean plants grown in several producing regions of Brazil, between 2007 and 2016 was established. For species and lineage identification, the following tests were performed: multiplex PCR with a set of four specific primer pairs, pathogenicity tests on susceptible cultivar BRS Artico and phylogenetic analysis based on housekeeping gene sequences. The presence of the two species were confirmed among the 117 strains, being 62 non-fuscans strains (NF1, NF2 and NF3) and 55 fuscans strains of X. citri pv. fuscans. To select a set of representative strains for the virulence assay, a PCR-based analysis of effector diversity was performed with 42 strains belonging to the two species. PCR with primers for xopL, avrBsT, xopE2 and xopE1 genes were positive for all strains, while for the other six effectors there was variation. Six distinct effector profiles were detected, and one strain representing each type was inoculated in 15 common bean cultivars with varying levels of resistance to CBB. The fuscans strains showed uniformity in their effector profiles and were the most virulent. The phylogenetic analyses of our strain collection revealed that all genetic variants of CBB pathogens (NF1, NF2, NF3 and fuscans) are present in Brazil, with significant variability in virulence to common bean cultivars.     

Transgenic expression of the rice Xa21 pattern‐recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern‐recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21‐mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar ‘Gonja manjaya’ (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.     

转鞭毛蛋白基因水稻细菌性条斑病抗性研究

该研究从生防菌枯草芽胞杆菌Bs-916中克隆了鞭毛蛋白基因,利用转基因载体pCAMBIA1300转入水稻,筛选得到98株阳性转基因植株。分子检测结果表明,有12个转基因株系可检测到目的基因的表达。随后抗病性鉴定表明,有3个转基因株系对水稻细菌性条斑病具有较高的抗性。该研究为目前水稻抗细菌性条斑病转基因研究拓宽了可应用基因资源的范围。     

Involvement of Gluconeogenic Pathway in Virulence of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases of rice. A phosphoenolpyruvate synthase (ppsA)‐disrupted mutant OSPAM was generated by homologous suicide plasmid integration. The mutant was unable to grow in medium with pyruvate or C₄‐dicarboxylates as the sole carbon source, compared with the wild‐type, indicating a disruption in ppsA function. The mutant showed a reduction in virulence on rice but still induced a hypersensitive response in tobacco. When the mutant was complemented, the response was recovered to wild‐type. These results suggested that X. oryzae pv. oryzae possesses only PPSA route in gluconeogenesis, which is necessary for virulence.     

Biological control of bacterial spot disease and plant growth-promoting effects of lactic acid bacteria on pepper

In our previous studies, we observed the biological control effect of lactic acid bacteria strains (LABs) KLF01, KLC02 and KPD03 against different plant pathogenic bacteria in vitro against Ralstonia solanacearum, and strains KLF01 and KLC02 against Pectobacterium carotovorum under greenhouse and field experiments, respectively. In this study, we observed the efficacy of these bacteria against bacterial spot pathogen (Xanthomonas campestris pv. vesicatoria) and their plant growth-promoting activities in pepper (Capsicum annuum L. var. annuum), under greenhouse and field conditions. LABs significantly (P < 0.05) reduced bacterial spot on pepper plants in comparison to untreated plants in both the greenhouse and the field experiments. The plant growth-promoting effect of LABs on pepper varied; some strains had a significant effect on growth promotion (P < 0.05) compared with untreated plants, while some showed no significant effect in the greenhouse and field experiments. Additionally, LABs were able to colonise roots, produce indole-3-acetic acid (IAA), siderophores and solubilise phosphate. These findings indicate that application of LABs could provide a promising alternative for the management of bacterial spot disease in pepper plants and could therefore be used as a healthy plant growth-promoting agent.     

Activity of a novel bactericide,zinc thiazole against Xanthomonas oryzae pv. oryzae in Anhui Province of China

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv oryzae, is one of the most serious bacterial diseases in China. Presently, bismerthiazol has been the major bactericide for the control of BLB, however, bismerthiazol‐resistant strains of X. oryzae pv. oryzae have appeared in the field in China. Zinc thiazole is a novel bactericide with strong antibacterial activity against Xanthomonas spp. In this study, sensitivity of 109 X. oryzae pv. oryzae strains to zinc thiazole was determined. The EC₅₀ values for zinc thiazole in inhibiting bacterial growth of the 109 X. oryzae pv. oryzae strains were 0.53–9.62 µg mL^?1 with the average EC₅₀ value of 4.82 ± 1.86 µg/ml. The minimum inhibitory concentration (MIC) values of zinc thiazole against the 109 X. oryzae pv. oryzae strains were assessed and the results showed that the MIC values of zinc thiazole for completely inhibiting the growth of these 109 strains ranged from 5.0 to 40.0 µg mL^?1. In the evaluation of protective and curative activity test, zinc thiazole exhibited great activity against BLB and provided over 88% control efficacy (at 300 µg mL^?1) 1 and 3 days before or after inoculations, which was also higher that that of bismerthiazol in the corresponding treatments. Our field trials showed that zinc thiazole at 375 g.a.i ha^?1 provided over 70% control efficacy in 2012 and over 80% control efficacy in 2013 at both sites. Moreover, in all the four field trials, zinc thiazole at 250 g.a.i ha^?1 provided higher control efficacy than that of bismerthiazol at 250 g.a.i ha^?1. Taken together, zinc thiazole is therefore an alternative tool for the management of BLB.     

Profiling Differentially Abundant Proteins by Overexpression of Three Putative Methyltransferases in Xanthomonas axonopodis pv. glycines

Methyltransferases (MTases) are enzymes that modify specific substrates by adding a methyl group using S‐adenosyl‐l ‐methionine. Functions of MTases have been extensively studied in eukaryotic organisms and animal pathogenic bacteria. Despite their importance, mechanisms underlying MTase function in plant pathogenic bacteria have not been studied in depth, as is the case of Xanthomonas axonopodis pv. glycines (Xag) that causes bacterial pustule disease in soybean crops worldwide. Here, the association between Xag proteome alterations and three MTase‐overexpressing strains, Xag(XgMT1), Xag(XgMT2), and Xag(XgMT3), compared to Xag carrying an empty vector, Xag(EV) is reported. Using label‐free shotgun comparative proteomic analysis, proteins are identified in all three biological replicates of the four strains and ranged from 1004 to 1082. In comparative analyses, 124, 135, and 134 proteins are differentially changed (over twofold) by overexpression of XgMT1, XgMT2, and XgMT3, respectively. These proteins are also categorized using cluster of orthologous group (COG) analyses, allowing postulation of biological mechanisms associated with three MTases in Xag. COGs reveal that the three MTases may play distinct roles, although some functions may overlap. These results are expected to allow new insight into understanding and predicting the biological functions of MTases in plant pathogenic bacteria. Data are available via ProteomeXchange (Identifier PXD012590).     

Functionality of lactic acid bacteria peptidase activities in the hydrolysis of gliadin‐like fragments

Aims: To evaluate the role of the peptidase activities from sourdough lactic acid bacteria (LAB) in the degradation of α‐gliadin fragments. Methods and Results: Different proline‐containing substrates were hydrolysed by LAB indicating pro‐specific peptidase activities. Lactobacillus plantarum CRL 775 and Pediococcus pentosaceus CRL 792 displayed the highest tri‐ and di‐peptidase activities, respectively. Lactobacillus plantarum strains hydrolysed more than 60%α‐gliadin fragments corresponding to the 31–43 and 62–75 amino acids in the protein after 2 h. None of the LAB strains alone could hydrolyse 57–89 α‐gliadin peptide; however, the combination of L. plantarum CRL 775 and P. pentosaceus CRL 792 led to hydrolysis (57%) of this peptide in 8 h. Conclusions: The capacity of LAB strains to degrade α‐gliadin fragments was not correlated to individual peptidase activities. Several strains separately degraded the 31–43 and 62–75 α‐gliadin fragments, while the 57–89 peptide degradation was associated with the combination of peptidase profiles from pooled LAB strains. This is the first report on the peptide hydrolase system of sourdough pediococci and its ability to reduce α‐gliadin fragments. Significance and Impact of the Study: This study contributes to a better knowledge of sourdough LAB proteolytic system and its role in the degradation of proline‐rich α‐gliadin peptides involved in celiac disease.     

Bacterial and plant natriuretic peptides improve plant defence responses against pathogens

Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP‐like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP‐A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over‐expressing XacPNP, lines over‐expressing AtPNP‐A and AtPNP‐A‐deficient plants were generated. Plants over‐expressing XacPNP or AtPNP‐A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP‐deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP‐A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over‐expressing XacPNP or AtPNP‐A were more resistant to Pst infection than control plants, whereas PNP‐deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non‐host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.     

A centenary for bacterial spot of tomato and pepper

Disease symptomsSymptoms include water‐soaked areas surrounded by chlorosis turning into necrotic spots on all aerial parts of plants. On tomato fruits, small, water‐soaked, or slightly raised pale‐green spots with greenish‐white halos are formed, ultimately becoming dark brown and slightly sunken with a scabby or wart‐like surface.Host rangeMain and economically important hosts include different types of tomatoes and peppers. Alternative solanaceous and nonsolanaceous hosts include Datura spp., Hyoscyamus spp., Lycium spp., Nicotiana rustica, Physalis spp., Solanum spp., Amaranthus lividus, Emilia fosbergii, Euphorbia heterophylla, Nicandra physaloides, Physalis pubescens, Sida glomerata, and Solanum americanum.Taxonomic status of the pathogenDomain, Bacteria; phylum, Proteobacteria; class, Gammaproteobacteria; order, Xanthomonadales; family, Xanthomonadaceae; genus, Xanthomonas; species, X. euvesicatoria, X. hortorum, X. vesicatoria.Synonyms (nonpreferred scientific names) Bacterium exitiosum, Bacterium vesicatorium, Phytomonas exitiosa, Phytomonas vesicatoria, Pseudomonas exitiosa, Pseudomonas gardneri, Pseudomonas vesicatoria, Xanthomonas axonopodis pv. vesicatoria, Xanthomonas campestris pv. vesicatoria, Xanthomonas cynarae pv. gardneri, Xanthomonas gardneri, Xanthomonas perforans.Microbiological propertiesColonies are gram‐negative, oxidase‐negative, and catalase‐positive and have oxidative metabolism. Pale‐yellow domed circular colonies of 1–2 mm in diameter grow on general culture media.DistributionThe bacteria are widespread in Africa, Brazil, Canada and the USA, Australia, eastern Europe, and south‐east Asia. Occurrence in western Europe is restricted.Phytosanitary categorizationA2 no. 157, EU Annex designation II/A2.EPPO codesXANTEU, XANTGA, XANTPF, XANTVE.     

Rapid Detection and Typing of Xanthomonas campestris pv. vesicatoria by Pyrosequencing

Taking Xanthomonas campestris pv. vesicatoria (Doidge) Dye, a pathogen with a wide geographical distribution, as a representative, pyrosequencing is shown for the first time to provide characteristic information of plant pathogenic bacteria strain‐specific sequences. Pyrosequencing‐based plant pathogen detection and typing technology is demonstrated to be rapid, highly specific and more sensitive than conventional technologies. The specificity of such assays has been validated by conventional DNA sequencing and metabolic fingerprinting. It is a starting point for the application and development of pyrosequencing in plant inspection and quarantine which underlie agricultural communication.     

Purification,crystal structure and antimicrobial activity of phenazine‐1‐carboxamide produced by a growth‐promoting biocontrol bacterium,Pseudomonas aeruginosa MML2212

Aim: To purify and characterize an antimicrobial compound produced by a biocontrol bacterium, Pseudomonas aeruginosa MML2212, and evaluate its activity against rice pathogens, Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Methods and Results: Pseudomonas aeruginosa strain MML2212 isolated from the rice rhizosphere with wide‐spectrum antimicrobial activity was cultured in Kings’B broth using a fermentor for 36 h. The extracellular metabolites were isolated from the fermented broth using ethyl acetate extraction and purified by two‐step silica‐gel column chromatography. Three fractions were separated, of which a major compound was obtained in pure state as yellow needles. It was crystallized after dissolving with chloroform followed by slow evaporation. It is odourless with a melting point of 220–222°C. It was soluble in most of the organic solvents and poorly soluble in water. The molecular mass of purified compound was estimated as 223·3 by mass spectral analysis. Further, it was characterized by IR, ¹H and ¹³C NMR spectral analyses. The crystal structure of the compound was elucidated for the first time by X‐ray diffraction study and deposited in the Cambridge Crystallographic Data Centre ( http://www.ccde.com.ac.uk ) with the accession no. CCDC 617344 . Conclusion: The crystal compound was undoubtedly identified as phenazine‐1‐carboxamide (PCN) with the empirical formula of C₁₃H₉N₃O. Significance and Impact of the Study: As this is the first report on the crystal structure of PCN, it provides additional information to the structural chemistry. Furthermore, the present study reports the antimicrobial activity of purified PCN on major rice pathogens, R. solani and X. oryzae pv. oryzae. Therefore, the PCN can be developed as an ideal agrochemical candidate for the control of both sheath blight and bacterial leaf blight diseases of rice.     

Genotypic Characterization of the Common Bean Bacterial Blight Pathogens, Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. phaseoli var. fuscans by rep-PCR and PCR–RFLP of the Ribosomal Genes

Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans is one of the most destructive diseases of common bean worldwide. The interrelatedness, genetic diversity and geographical distribution of the CBB pathogens was assessed using restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction amplified 16S ribosomal gene, including the 16S–23S intergenic spacer region and repetitive element PCR (rep‐PCR). RFLP profiles generated by the restriction endonucleases MboI, RsaI and HaeIII differentiated X. axonopodis pv. phaseoli from X. axonopodis pv. phaseoli var. fuscans and non‐pathogenic Xanthomonas species associated with common bean. Cluster analysis of rep‐PCR profiles revealed a high level of genetic differentiation (G_ST = 0.56) between the two CBB pathogens, showing that they are genetically distinct. Significant levels of genetic diversity were observed within each strain, indicating that the two bacteria are not clonal. More genetic diversity was observed in X. axonopodis pv. phaseoli (H = 0.134; I = 0.223) than X. axonopodis pv. phaseoli var. fuscans (H = 0.108; I = 0.184). However, no geographical differentiation was evident for either X. axonopodis pv. phaseoli var. fuscans (GST = 0.013) or X. axonopodis pv. phaseoli (G_ST = 0.017). This lack of geographical differentiation has important practical implications, as available host resistance genes are likely to be effective in controlling the disease in diverse geographical areas.