Induction of systemic resistance byPseudomonas fluorescens Pf1 againstXanthomonas oryzae pv.Oryzae in rice leaves

When lower leaves of rice plants were inoculated with powder formulation of a saprophytic strain ofPseudomonas fluorescens, Pfl, upper leaves, in addition to the inoculated lower leaves, showed resistance to the rice bacterial blight pathogenXanthomonas oryzae pv.oryzae. When the leaves were challenge-inoculated withX. oryzae pv.oryzae 4 days afterP. fluorescens application on lower leaves, the disease intensity in upper leaves decreased from 6.7 to 1.1. When rice seeds were treated with the formulation ofP. fluorescens Pfl and sown, 30-day-old seedlings showed resistance toX. oryzae pv.oryzae and the disease intensity decreased from 6.8 to 1.2. The induced resistance was transient; leaves sprayed withP. fluorescens Pfl at 30 days after treatment and leaves of 60-day-old seedlings fromP. fluorescens-treated seeds did not show resistance to the pathogen. In field trials, seed treatment followed by foliar application of the powder formulation ofP. fluorescens Pfl effectively controlled rice bacterial blight and increased the yield. In the induced resistant leaves a sharp increase in lignification and activities of peroxidase, phenylalanine ammonia-lyase and 4-coumarate: CoA ligase was observed when the leaves were challenge-inoculated withX. oryzae pv.oryzae. An approximately threefold increase in lignin content, peroxidase activity and phenylalanine ammonia-lyase activity and a fivefold increase in 4-coumarate: CoA ligase activity were observed 5 days after challenge inoculation withX. oryzae pv.oryzae in rice leaves pretreated withP. fluorescens for 5 days. A similar increase in defense-related activities was not observed in susceptible interactions or inP. fluorescens-treated plants at later stages of interactions when no resistance to the pathogen was observed.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

Generation and characterisation of Tn5-tagged <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> mutants that overcome <Emphasis Type="Italic">Xa23</Emphasis>-mediated resistance to bacterial blight of rice

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23. The authors C.-L. Wang, A.-B. Xu contributed equally to this work; Y. Gao and Y.-L. Fan contributed equally to this work.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.     

Hpa1 secretion via type III secretion system in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In many Gram-negative plant pathogenic bacteria the type III secretion system (TTSS), encoded by hrp genes, is essential for pathogenicity in the host and induction of a hypersensitive reaction (HR) in nonhost plants. The expression of hrp genes has been suggested to be repressed in complex media, whereas it is induced in planta and under certain in vitro conditions. We recently reported that XOM2 medium allows efficient hrp expression by Xanthomonas oryzae pv. oryzae. In this study, we investigated hrp-dependent secretion of proteins by the bacteria in vitro. Using modified XOM2, in which bovine serum albumin was added and the pH was lowered to 6.0, we detected at least 10 secreted proteins and identified one as Hpa1. This is the first evidence of protein secretion via TTSS in X. oryzae pv. oryzae.     

Influence of rice development on the function of bacterial blight resistance genes

Disease resistance genes most commonly used in breeding programs are single, dominant genes with relative effectiveness that is sometimes influenced by plant developmental stage. Knowing the developmental stages at which a resistance gene is functional is important for disease management. In rice, resistance at the seedling stage is crucial, because wounding during transplanting increases the potential for bacterial blight disease, and not all bacterial blight resistance genes are effective at the seedling stage. Effectiveness of the bacterial blight resistance genes Xa4, xa5, and Xa7, all in a common genetic background, was evaluated at different developmental stages by measuring lesion length and bacterial numbers after inoculation with the bacterial pathogen, Xanthomonas oryzae pv. oryzae. The Xa4 and xa5 genes controlled disease at all growth stages. In contrast, Xa7 was not fully functional in very young seedlings, but was completely effective by 21 days after sowing (das). The effects of plant developmental stage on interactions of the Xa7 gene with X. oryzae pv. oryzae strains carrying different mutant avrXa7 alleles were also tested. If a partial or fully functional avrXa7 allele was present, Xa7 resistance was effective at all growth stages tested after the transplant stage (>21 das).     

华南水稻白叶枯病菌致病性分化检测与分析

为了解华南稻区水稻白叶枯病菌的致病性分化和变异动态,采集华南地区水稻白叶枯病病叶标样分离病原菌,应用中国鉴别寄主IR26、南粳15、爪哇14、特特普、金刚30和国际水稻已知抗病基因的近等基因系IRBB5、IRBB13、IRBB3、IRBB14、IRBB2、IR24两套鉴别寄主,在水稻孕穗期采用剪叶法接种,依据寄主和菌株的互作反应检测病菌的致病性分化。结果显示,参试菌株可划分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅸ六个致病型和R1、R2、R3、R4、R5、R8、R10七个致病小种。Ⅴ、Ⅳ致病型和R8、R5小种出现频率分别为27.40%、19.30%和44.67%、15.34%,为华南稻区优势种群。Ⅸ、Ⅴ、Ⅳ致病型和R8、R5小种对500份华南稻区品种资源的致病率依次为96.40%、95.00%、50.40%、62.00%和42.60%;Ⅸ致病型毒性最强且发展很快;强致病菌系Ⅴ型已替代Ⅳ型发展为华南优势致病菌系。     

Detection of Pseudomonas syringae pv. aptata in Irrigation Water Retention Basins by Immunofluorescence Colony-staining

Bacterial blight of cantaloupe (Cucumis melo) caused by Pseudomonas syringae pv. aptata was first observed in south-western France and has since spread to all cantaloupe-growing areas of this country. Use of pesticides registered for this disease has proved ineffective and no commercial cultivars of cantaloupe are resistant to this blight. To develop control strategies for this disease, the principal sources of inoculum were investigated. Among the different sources of inoculum studied, we report the isolation of P. syringae pv. aptata from irrigation water retention basins in south-western France using the immunofluorescence colony-staining (IFC) method. In this study, the pathogen was detected at a low concentration (12 and 70cful^–1) in two different retention basins. These results suggest that P. syringae pv. aptata can survive in water used to irrigate cantaloupe crops and could be a source of inoculum for epidemics of bacterial blight. To develop control strategies for this bacterial disease, the importance of water retention basins as sources of inoculum for bacterial blight of cantaloupe needs to be evaluated relative to other potential sources such as seeds, plants from nurseries and plant debris in the soil.     

Notes on the occurrence of pathogenic races of Xanthomonas oryzae pv. oryzae found in Hiroshima Prefecture during 2000–2003

The pathogenic races of 450 cultures of Xanthomonas oryzae pv. oryzae, isolated from eight locations in the inland mountainous area of Hiroshima Prefecture during 2000 to 2003, were determined with a set of Japanese differentials. The rice cultivars infected with the bacterium are thought to be in the Kinmaze group, which does not have any resistance genes. Five Japanese races IA, IB, II, V, and VII occurred across the area, although the composition of the races in each location altered during the surveyed 4 years.     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.     

Identification of Resistance to Common Bacterial Blight Disease on Bean Genotypes Grown in Turkey

Common bacterial blight (CBB) in edible beans (Phaseolus vulgaris), incited Xanthomonas campestris pv. phaseoli, reduces bean yields and seed quality. The main objective of this study was to determine resistance to common bacterial blight in bean genotypes. Twenty-two bean genotypes grown in Turkey including common and snap bean cultivars/lines were collected from different parts of Turkey and tested for resistance against to Xanthomonas campestris pv. phaseoli strain MFD-11. All the common and snap bean lines/cultivars tested were moderately susceptible, susceptible or highly susceptible, except AG-7117 which was found resistant to Xanthomonas campestris pv. phaseoli. This is the first report of a resistance source in a common bean line (AG-7117) against Xanthomonas campestris pv. phaseoli.     

Functional analysis of the 3′ end of avrBs3/pthA genes from two Xanthomonas species

The phytopathogens Xanthomonas oryzae pathovar (pv.) oryzae and Xanthomonas axonopodis pv. citri each contain several avrBs3/pthA family genes. Structural features of these genes important for avirulence and/or virulence functions include a central region of multiple direct repeats and three nuclear localization signals (NLSs) and an acidic activation domain (AAD) at the 3′ end. To identify other regions critical to function in the 3′ ends of these genes, we constructed several chimeras using apl1 and apl2 from X. axonopodis pv. citri and avrXa10 and avrXa7 from X. oryzae pv. oryzae and evaluated their functions by inoculation to citrus and rice. The apl1 and avrXa7 genes are major virulence determinants in citrus and rice, respectively, while the contributions of apl2 and avrXa10 to virulence are negligible or not measurable. Constructs that contained a 417 bp HincII-SphI fragment from the 3′ end of apl1 in combination with the repeats from avrXa7, avrXa10, and apl1 caused a canker phenotype on citrus. Interchange of the HincII-SphI fragment between avrXa7 and avrXa10 abolishes avrXa7 avirulence function and reduces its virulence but it does not affect avrXa10 avirulence function in rice. avrXa7 caused a hypersensitive response (HR) in citrus and replacement of it's 3′ end with that of apl1 resulted in loss of canker and induction of HR. Thus, the HincII-SphI fragment of the avrBs3/pthA gene family is important for avirulence and virulence functions in two different plant species, Oryza sativa and Citrus natsudaidai HAYATA.     

Population structure of <Emphasis Type="Italic">Eleusine</Emphasis> isolates of <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> and its evolutionary implications

Population structure of Eleusine isolates of Pyricularia oryzae (Magnaporthe oryzae) was examined using DNA markers. On the basis of rDNA sequences, Eleusine isolates were divided into two groups. One group clustered with Triticum isolates, while the other clustered with Eragrostis isolates. This grouping was supported by DNA fingerprinting with three repetitive elements: MGR586, MGR583, and grasshopper. These results suggest that the population of Eleusine isolates is composed of at least two groups that evolved independently from the original population of P. oryzae. Most of the isolates that were collected just after an outbreak of finger millet blast in the 1970s had almost identical fingerprint profiles although they were collected in distant prefectures. This result supports the idea that the outbreak was caused by seed transmission of a particular strain of Eleusine isolates.     

水稻品种对稻瘟病和白叶枯病抗性的遗传

选用稻瘟病(Pyricularia oryzae)生理小种ZB15和白叶枯病(Xanthomonas campesfris pv.oryzae)菌株98对3个水稻品种的抗性基因进行了遗传分析。结果表明IR2061和戈马列的稻瘟病抗性分别受非等位的2对重复显性基因所控制。IR29的抗性表现较为复杂,其抗性遗传尚待进一步研究。IR29和IR2061具有1对相同的抗白叶枯病显性基因,而戈马列则具有1对非等位的显性抗性基因,并且受珍汕97的1对隐性抑制基因所制约。     

Persistence of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">vignicola</Emphasis> in weeds and crop debris and identification of <Emphasis Type="Italic">Sphenostylis stenocarpa</Emphasis> as a potential new host

The survival of Xanthomonas axonopodis pv. vignicola, incitant of cowpea bacterial blight and pustule, in residues of infested cowpea leaves was studied in the field in the forest savanna transition zone of South Benin and under variable controlled conditions. The pathogen survived for up to 60 days when placed on the soil surface, and up to 45 days buried at depths of 10 and 20 cm. In the glasshouse, bacteria survived in residue mixed with soil for at least 2 months in dry soil and less than 2 months in moist soil. The pathogen survived at least 30 days in the field after spray-inoculation on the weed species Euphorbia heterophylla, Digitaria horizontalis and Synedrella nodiflora; 20 days on Panicum subalbidum; 10 days on Euphorbia hirta; and 5 days on Talinum triangulare. After leaf-infiltration under glasshouse conditions, the pathogen was detected after 90 days in D. horizontalis; 75 days in T. triangulare, P. subalbidum and S. nodiflora; 60 days in E. hirta, and 30 days in E. heterophylla. Among 12 legume species tested as alternative hosts of X. axonopodis pv. vignicola, only Sphenostylis stenocarpa (African yam bean) showed typical symptoms of cowpea bacterial blight in a glasshouse experiment following artificial inoculation. This is the first time this legume species has been identified as a potential host of X. axonopodis pv.vignicola. Crop residue and weeds are likely sources of primary inoculum when planting two consecutive cowpea crops per year and they probably play a role in dissemination of the pathogen during the cropping season. The alternate host may form a bridge for primary inoculum between cropping seasons.     

Taxonomic characterization of Pyricularia isolates from green foxtail and giant foxtail, wild foxtails in Japan

Twenty-eight Pyricularia isolates from two wild foxtails—green foxtail (Setaria viridis) and giant foxtail (S. faberii)—in Japan were taxonomically characterized by DNA analyses, mating tests, and pathogenicity assays. Although most of the isolates failed to produce perithecia in mating tests with Magnaporthe oryzae, a diagnostic polymerase chain reaction-restriction fragment length polymorphism phenotype of M. oryzae was detected in the beta-tubulin genomic region in all isolates. The pathogenicity assays revealed that host ranges of the isolates were similar to those of isolates from foxtail millet (S. italica), which were exclusively pathogenic on foxtail millet. In addition to the 28 isolates from wild foxtails, 22 Pyricularia isolates from 11 other grasses were analyzed by RFLP using single-copy sequences as probes. In a dendrogram constructed from the RFLP data, isolates that were previously identified as M. oryzae formed a single cluster. All the wild foxtail isolates formed a subcluster with foxtail millet isolates within the M. oryzae cluster. From these results, we conclude that Pyricularia isolates from the wild foxtails are closely related to isolates from foxtail millet and should be classified into the Setaria pathotype of M. oryzae.     

水稻白叶枯病菌酸性还原酮加双氧酶的鉴定分析

 酸性还原酮加双氧酶(ARD)催化很多原核和真核生物中的甲硫氨酸急救途径(MSP)倒数第二步。本研究鉴定了水稻白叶枯病菌Xanthomonas oryzae pv. oryzae (Xoo)中的酸性还原酮加双氧酶, 命名为xard。Xoo 菌株PXO99^A、MAFF311018和KACC10331中的xard核苷酸序列完全相同。xard基因突变菌株在甲硫基腺苷(MTA)为唯一硫源时不能正常生长。这一结果证明Xard在MSP中起作用。xard突变体和野生型菌株PXO99^A 接种水稻IR24后病斑长度数据表明该基因突变对Xoo在水稻上的毒性没有影响。