Protein(s) from the Gram-Positive Bacterium Clavibacter michiganensis subsp. michiganensis Induces a Hypersensitive Response in Plants

ABSTRACT The gram-positive tomato pathogen Clavibacter michiganensis subsp. michiganensis induced a local necrotic response on four-o'clock (Mirabilis jalapa) and tobacco (Nicotiana tabacum) plants. This necrosis response was characteristic of the hypersensitive response (HR). The cell-free culture supernatant from strain CMM623 also induced a necrosis that was phenotypically similar to that induced by the bacteria. Inhibitors of plant metabolism suppressed the necrotic reaction of both M. jalapa and tobacco. The HR-inducing activity present in the supernatant was heat stable, sensitive to proteases, and had an apparent molecular mass in the range of 35 to 50 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The properties observed for the necrosis-inducing activity resembled harpin and PopA described from gram-negative phytopathogenic bacteria.     

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.     

Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization

ABSTRACT Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.     

Colonization of tomato seedlings by bioluminescent Clavibacter michiganensis subsp. michiganensis under different humidity regimes

Tomato bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis, is transmitted by infected or infested seed and mechanically from plant to plant. Wounds occurring during seedling production and crop maintenance facilitate the dissemination of the pathogen. However, the effects of environmental factors on C. michiganensis subsp. michiganensis translocation and growth as an endophyte have not been fully elucidated. A virulent, stable, constitutively bioluminescent C. michiganensis subsp. michiganensis strain BL-Cmm 17 coupled with an in vivo imaging system allowed visualization of the C. michiganensis subsp. michiganensis colonization process in tomato seedlings in real time. The dynamics of bacterial infection in seedlings through wounds were compared under low (45%) and high (83%) relative humidity. Bacteria multiplied rapidly in cotyledon petioles remaining after clip inoculation and moved in the stem toward both root and shoot. Luminescent signals were also observed in tomato seedling roots over time, and root development was reduced in inoculated plants maintained under both humidity regimes. Wilting was more severe in seedlings under high-humidity regimes. A strong positive correlation between light intensity and bacterial population in planta suggests that bioluminescent C. michiganensis subsp. michiganensis strains will be useful in evaluating the efficacy of bactericides and host resistance.     

不同来源番茄溃疡病菌致病力差异研究

采用打顶法接种、半选择性培养基再分离发病植株中的病原菌,以及特异性PCR验证方法,对来自3个国家9个不同地区的46株番茄溃疡病菌进行了致病性测定,以病情指数评价不同菌株的致病力。结果显示,分离自我国河北滦平县、内蒙古包头市等地的24株菌株的病情指数达到75以上,属于强致病力水平;11株菌株的病情指数为50~75,属于中等致病力;而9株菌株的病情指数为50以下,属于弱致病力;检测同时证实,有2株属于无致病力菌株。强致病力、中等致病力、弱致病力和无致病力菌株占供试菌株总数的比例分别为52.2%、23.9%、19.6%和4.3%,表明供试的46株番茄溃疡病菌存在不同程度的致病力差异。     

In Planta - Complementation of Clavibacter Michiganensis Subsp. sepedonicus Strains Deficient in Cellulase Production or HR Induction Restores Virulence

Clavibacter michiganensis subsp. sepedonicus, a Gram positive bacterium that causes bacterial ring rot of potato, was studied in eggplant, an alternate host, using strains that differed in phenotype. Two factors affecting virulence, the ability to induce a hypersensitive response (HR) and cellulase production, were studied. A plasmid-free isolate of C. michiganensis subsp. sepedonicus that causes HR on tobacco but is unable to produce cellulase multiplied efficiently in planta, but caused only weak symptoms. In contrast, a strain that is unable to induce HR on tobacco but produces cellulase was impaired in the ability to multiply in the host and caused no symptoms. When the two non-virulent strains were coinoculated into eggplants, typical disease symptoms developed. This enhancement was not due to formation of a new phenotype or significant increases in population density of either of the strains. Our results suggest that both cellulase production and the ability to induce HR are required for a successful infection process and disease induction by C. michiganensis subsp. sepedonicus. Our results additionally suggest that the ability to induce HR on non-host plants is required for multiplication in the host plant, whereas cellulase expression is necessary for induction of disease symptoms.     

Erwinia amylovora hrpN mutants,blocked in harpin synthesis,express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco

Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting many rosaceous plants and especially pear tree and apple tree. A protein named harpin, secreted through the Hrp secretion pathway and able to elicit an hypersensitive reaction (HR) on tobacco has recently been isolated. Mutants inhrpN, the gene encoding harpin were described as non pathogenic on immature pear fruit and unable to elicit an HR on tobacco [Weiet al., 1992; Wei and Beer, 1993]. In this paper, the phenotype on plant ofhrpN mutants was carefully determined.hrpN mutants expressed a weak but significant virulence on host plants. Furthermore, when infiltrated into tobacco leaf mesophyll, thehrpN mutants elicited varied responses that fluctuated from null reaction to full necrosis of the infiltrated area. These results show that harpin is not absolutely required neither for pathogenicity on host plant nor for elicitation of an hypersensitive reaction on tobacco. Furthermore, in all the tests performed, mutant blocked in harpin secretion remained non pathogenic and unable to elicit an HR on tobacco. This suggests that factor(s), different from harpin, involved both in pathogenicity and HR eliciting ability is (are) secreted through the Hrp secretion pathway.Abbreviations HR hypersensitive reaction - NSI necrosis severity index - CFU colonie forming units     

Infection of plant material derived from Solanum acaule with Clavibacter michiganensis ssp. sepedonicus: temperature as a determining factor in immunity of S. acaule to bacterial ring rot

The development of potato ( Solanum tuberosum ) cultivars immune (completely free of infective agents) to bacterial ring rot disease, caused by Clavibacter michiganensis ssp. sepedonicus , would be a significant step toward eradication of the disease. Earlier results suggested that the wild potato species Solanum acaule , acc. PI472655 (genotype 7-8), was immune to C. michiganensis ssp. sepedonicus . The goal of the present study was to investigate the possibility of transferring this desirable trait to cultivated potato through interspecific somatic hybridization. Eight different somatic hybrids between S. acaule and S. tuberosum , with three different genome ratios, were tested for susceptibility to infection in the glasshouse using two C. michiganensis ssp. sepedonicus strains. All the somatic hybrids expressed symptoms of ring rot and were susceptible to infection as determined by IFAS (indirect immunofluorescent antibody staining) tests. The genome compositions of the hybrids influenced bacterial titres. Most of the hybrids with a higher proportion of the S. acaule genome contained lower numbers of C. michiganensis ssp. sepedonicus cells than hybrids with lower proportions of the S. acaule genome. In growth chamber tests, temperature was found to be a determining factor in the expression of immunity in the tetraploid S . acaule 7-8 line. At 21°C, S . acaule 7-8 was immune to infection by C. michiganensis ssp. sepedonicus , but at 15°C, plants supported large populations of the pathogen. However, none of the S. acaule plants expressed disease symptoms. Thus, S. acaule exhibits temperature-dependent immunity to infection by C. michiganensis ssp. sepedonicus and should be considered tolerant rather than immune.     

A Pectate Lyase Homolog, xagP, in Xanthomonas axonopodis pv. glycines Is Associated with Hypersensitive Response Induction on Tobacco

ABSTRACT Xanthomonas axonopodis pv. glycines is the causal agent of bacterial pustule disease of soybeans. A transposon insertional mutant (KU-P-M670) of X. axonopodis pv. glycines derived from wild-type strain KU-P-34017 lost the ability to induce the hypersensitive response (HR) on tobacco and pepper but retained its HR induction capacity on cucumber, sesame, and tomato. The mutation also resulted in loss of ability to cause a potato soft rot and express pectolytic activity at pH 6.5. An approximate 1.4-kb DNA fragment carrying the transposon insertion contained a single open reading frame that showed high homology with PSTRU-3, a pectate lyase gene in X. axonopodis pv. malvacearum. Complemented KU-P-M670 regained HR induction on tobacco and also pectolytic activity. Treatment of plants with inhibitors of eukaryotic metabolism blocked HR induction by wild-type strains and by complemented KU-P-M670. The presence of the pectate lyase homolog, which we designated xagP, in 26 X. axonopodis pv. glycines strains was highly correlated with their ability to induce an HR on tobacco. To our knowledge, this is the first study indicating a role for a functional pectate lyase in induction of a plant HR.     

Silencing of host basal defense response-related gene expression increases susceptibility of Nicotiana benthamiana to Clavibacter michiganensis subsp. michiganensis

Clavibacter michiganensis subsp. michiganensis is an actinomycete, causing bacterial wilt and canker disease of tomato (Solanum lycopersicum). We used virus-induced gene silencing (VIGS) to identify genes playing a role in host basal defense response to C. michiganensis subsp. michiganensis infection using Nicotiana benthamiana as a model plant. A preliminary VIGS screen comprising 160 genes from tomato known to be involved in defense-related signaling identified a set of 14 genes whose suppression led to altered host-pathogen interactions. Expression of each of these genes and three additional targets was then suppressed in larger-scale VIGS experiments and the effect of silencing on development of wilt disease symptoms and bacterial growth during an N. benthamiana-C. michiganensis subsp. michiganensis compatible interaction was determined. Disease susceptibility and in planta bacterial population size were enhanced by silencing genes encoding N. benthamiana homologs of ubiquitin activating enzyme, snakin-2, extensin-like protein, divinyl ether synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase 2, and Pto-like kinase. The identification of genes having a role in the host basal defense-response to C. michiganensis subsp. michiganensis advances our understanding of the plant responses activated by C. michiganensis subsp. michiganensis and raises possibilities for devising novel and effective molecular strategies to control bacterial canker and wilt in tomato.     

A Variant of Cucumber mosaic virus Is Restricted to Local Lesions in Inoculated Tobacco Leaves with a Hypersensitive Response

A variant of Cucumber mosaic virus, CMV(Y/GM2), was isolated from a tobacco plant with mild green mosaic symptoms that was regenerated in vitro from a yellow strain of CMV [CMV(Y)]-infected tobacco leaves by tissue culture. CMV(Y/GM2) has two amino acid substitutions at 36 and 111 positions in the coat protein encoded on RNA3. CMV, assembled by mixing in vitro transcribed CMV(Y) RNA1 and RNA2 plus infectious RNA3 transcribed in vitro from cDNA to RNA3 of CMV(Y/GM2), was prepared and designated as CMV(Y/GM2)tr. When tobacco (Nicotiana tabacum cv. Xanthi nc) plants were inoculated with CMV(Y/GM2)tr, large necrotic local lesions in which the virus was localized, developed on the inoculated leaves. This host response unique to CMV(Y/GM2)tr was similar to the hypersensitive response (HR), which is a common resistance response to avirulent pathogens and was observed in five cultivars of Nicotiana tabacum and eight Nicotiana species. The revertant virus, however, accumulated to quite different levels in the various hosts. CMV(Y/GM2)tr induced pathogenesis-related 1 (PR-1) protein accumulation and systemic acquired resistance (SAR) which were generally observed in the HR. However, when tobaccos were inoculated with CMV(S36P)tr and CMV(V111I)tr, which have an amino acid substitution at either the 36 or 111 position in the coat protein of CMV(Y), respectively, CMV(S36P)tr was restricted to the primary infection site without necrotic local lesion formation and PR-1 protein and SAR induction. CMV(V111I)tr, however, systemically spread and induced mild green mosaic symptoms, while the host had the HR to CMV(Y/GM2)tr. The localization of CMV(Y/GM2)tr at the primary infection site may not only be caused by the HR, but also by the restriction of virus systemic movement resulting from the amino acid substitution at position 36 in the coat protein of CMV(Y). Received 15 December 1999/ Accepted in revised form 18 April 2000     

Mutations that Affect Agrobacterium vitis-Induced Grape Necrosis also Alter Its Ability to Cause a Hypersensitive Response on Tobacco

ABSTRACT Tn5-induced mutations in Agrobacterium vitis F2/5 resulted in both altered grape necrosis and tobacco leaf panel collapse phenotypes, suggesting that the underlying mechanisms of the reactions are related. The reaction on tobacco resembles the classical hypersensitive response (HR) caused by several plant pathogenic bacteria in that it is observable within 14 h, is inhibited by treatment of plants with metabolic inhibitors, and results in the inability to recover the pathogen from the necrotic zone. Strains of A. vitis differ with regard to their efficiency of causing the reaction on tobacco. An EcoRI fragment from one mutant, M6, which is necrosis-altered and HR-minus, was cloned and sequenced. Sequence analysis revealed that the Tn5 insertion occurred in a region that shares significant homology with genes involved in long chain fatty acid production by the marine bacteria Shewanella spp. and Moritella marina. Complementation of M6 with a cosmid clone from an F2/5 DNA library restored the tobacco HR and grape necrosis phenotypes.     

Production of DAPG and HCN by Pseudomonas sp. LBUM300 contributes to the biological control of bacterial canker of tomato

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is known to cause significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical containment methods, which are often inefficient and may leave adverse effects on the environment. However, only little headway has so far been made in developing biocontrol strategies against C. michiganensis subsp. michiganensis. To address this knowledge gap, we investigated the antagonistic capacity of PCA, produced by Pseudomonas sp. LBUM223, and DAPG and HCN, both produced by Pseudomonas sp. LBUM300, on C. michiganensis subsp. michiganensis under in vitro and in planta conditions. Nonsynthesizing isogenic mutants of the producer strains were also developed to further dissect the role of each individual metabolite on C. michiganensis subsp. michiganensis biological control. Novel specific quantitative polymerase chain reaction TaqMan assays allowed quantification of C. michiganensis subsp. michiganensis in tomato plants and rhizospheric soil. Pseudomonas spp. LBUM223 and LBUM300 significantly repressed C. michiganensis subsp. michiganensis growth in vitro, while their respective nonproducing mutants showed less or no significant antagonistic activity. In planta, only Pseudomonas sp. LBUM300 was capable of significantly reducing disease development and C. michiganensis subsp. michiganensis rhizospheric population, suggesting that the production of both DAPG and HCN was involved. In summary, simultaneous DAPG/HCN production by Pseudomonas sp. LBUM300 shows great potential for controlling bacterial canker of tomato.     

Cell interactions between a nonpathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain and root tissues of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis>

Nonpathogenic isolates of Fusarium oxysporum can be successful antagonists of pathogenic forms of the same fungal species that commonly attacks crop plants. The characteristics that distinguish nonpathogenic from pathogenic forms are not well understood. In this study, the mode of root colonization of Eucalyptus viminalis seedlings by a nonpathogenic F. oxysporum strain is described at the ultrastructural level. Root systems of E. viminalis plants were inoculated with nonpathogenic F. oxysporum strain Fo47 in an in vitro model system. Changes in the occurrence of nonesterified and methyl-esterified pectins in colonized E. viminalis roots were evaluated by in situ immunolabeling using two monoclonal antibodies, JIM 5 and JIM 7. Modes of penetration and root colonization patterns in E. viminalis seedlings by the nonpathogenic fungus were similar to those described for pathogenic forms of F. oxysporum. However, root interactions differed in that the nonpathogenic fungus did not induce host tissue damage. No papilla-like appositions were observed in host cells in response to invading hyphae, which did not disrupt the host plasma membrane in many cases, suggesting that a biotrophic relationship was established. Root colonization by the nonpathogenic strain did not induce alteration in JIM 7 labeling of methyl-esterified pectin in E. viminalis cell walls, whereas nonesterified pectin was detected to a significantly greater extent in cell walls of roots colonized by the fungus. Pectin components decreased slightly only at points of hyphal contact with host cells. Because nonpathogenic strains utilize pectin in pure culture, host control over enzyme activity or production by the fungi may at least partly explain their compatible interactions with host tissues.     

Comparative dynamics of adherent and nonadherent bacterial populations on maize leaves

ABSTRACT The dynamics of the adherent and nonadherent populations of three bacterial species on maize leaves were examined to identify the extent to which bacteria adhere to leaves and the importance of this adhesion to leaf colonization. Pantoea agglomerans strain BRT98, Clavibacter michiganensis subsp. nebraskensis strain GH2390, and Pseudomonas syringae pv. syringae strain HS191R all rapidly adhered to maize leaves following inoculation, but differed in the percentage of cells that adhered to the leaves. Immediately following inoculation, the percentage of adherent cells was highest for the saprophyte P. agglomerans (8 to 10%) and was much lower for the pathogens C. michiganensis subsp. nebras-kensis and P. syringae pv. syringae (2 to 3 and <1%, respectively), although the results for P. syringae pv. syringae HS191R were based on only one experiment. In the 4 days following inoculation, the percentage of the P. agglomerans populations that adhered to the leaves increased to approximately 70%. Similarly, the percentage of C. michiganensis subsp. nebraskensis and P. syringae pv. syringae cells that resisted removal steadily increased in the days following inoculation, although these increases probably reflected both adherence and localization to endophytic sites. Based on differences in the percentage of cells adhering to several cuticular wax mutants of maize, the rapid adherence of C. michiganensis subsp. nebraskensis cells to maize leaves was influenced by the cuticular wax properties, while the rapid adherence of P. agglomerans was not. Finally, bacterial adherence to leaves was advantageous to P. agglomerans survival and growth on leaves based on the finding that the nonadherent populations of the P. agglomerans strain decreased significantly more than did the adherent populations in the 24 h following inoculation, and increased much less than did the adherent populations over the next 3 days. Similar results with the C. michiganensis subsp. nebraskensis and P. syringae pv. syringae strains indicate that bacterial adherence to leaves, bacterial movement to endophytic sites, or both were advantageous to the survival and growth of these strains on leaves.     

Pathogenicity,host specificity and anastomosis groups ofRhizoctonia spp. isolated from soils in Israel

One hundred and twenty-nine isolates ofRhizoctonia spp. were obtained from soil samples in Israel and from culture collections in the U.S.A. and Japan. The isolates varied in host range and disease severity when tested on six to eleven different host plants. Approximately 30% of the isolates were nonpathogenic to all the host plants tested. Mycelial growth rate of the nonpathogenic strains did not differ significantly from that of the virulent isolates. The 107 Israeli isolates represented anastomosis groups (AG) 1, 2, 3, 4, 5 and 6 ofR, solani, two groups ofR. zeae, and three groups of binucleateRhizoctonia AG: A, F and K.     

Molecular typing and spread of Clavibacter michiganensis subsp. michiganensis in greenhouses in Japan

Clavibacter michiganensis subsp. michiganensis (CMM) strains collected between 2005-2008 from greenhouses in different locations in Okayama Prefecture, Japan, were fingerprinted by repetitive sequence-based polymerase chain reaction (rep-PCR) with ERIC and BOX primers. One hundred and eighty strains from eight different locations in Okayama were differentiated into four haplotypes (A to D) based on rep-PCR. Regardless of the year of isolation, location or cultivar of tomato, the strains in each greenhouse and location belonged to the same haplotype, suggesting the strains originated from the previous greenhouse population. Based on Morisita's index of dispersion ( I _δ ), the distribution of diseased plants in the greenhouses, where disbudding and defoliation using either scissors or by hand were carried out in the same direction to promote the spread of CMM, occurred in an aggregated distribution in a quadrant along a row of plants, but the distribution of diseased plants indicated a random distribution in a quadrant along a furrow of plants (two adjoining rows of plants). These results showed that disbudding and defoliation contribute highly to the secondary spread of bacterial canker in commercial greenhouses.     

An oligonucleotide array for the identification and differentiation of bacteria pathogenic on potato

ABSTRACT Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.     

Antagonistic Interactions Between Strains of Xanthomonas oryzae pv. oryzae

ABSTRACT The ability of some phytopathogenic bacterial strains to inhibit the growth of others in mixed infections has been well documented. Here we report that such antagonistic interactions occur between several wild-type strains of the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae. In mixed inoculations, a wild-type Philippine strain was found to inhibit the growth of a wild-type Korean strain. Furthermore, a nonpathogenic mutant of the Philippine strain maintained these antagonistic properties. Growth curve analysis indicated that both the wild-type Philippine strain and its nonpathogenic mutant inhibited the growth of the Korean strain 2 days after infection and prior to the onset of disease symptoms. When mixed with the nonpathogenic mutant, 10 out of 18 diverse wild-type X. oryzae pv. oryzae strains did not cause disease. Conversely, three of the strains that were not affected by the nonpathogenic mutant were found to inhibit the growth of both the wild-type and mutant Philippine strains, indicating that antagonism is widespread and strain specific. The observed growth inhibition occurred only in planta and did not correlate with bacteriocin activity in vitro. Antagonistic interactions also were found to affect resistance (R) gene-mediated resistance. The R gene Xa21 was capable of protecting rice plants coinoculated with nonantagonistic virulent and avirulent strains; however, when avirulent strains were coinoculated with virulent antagonistic strains, disease ensued. Taken together, these results indicate that X. oryzae pv. oryzae has evolved strategies to compete with rival strains in a fashion that allows virulent strains to evade R gene-mediated protection even when avirulent strains are present in the inoculum.     

Population diversity of Pseudomonas syringae subsp. savastanoi on olive and oleander

Twenty-one strains of Pseudomonas syringae subsp. savastanoi , isolated from knots on olive and oleander trees growing in close proximity to or in physical contact with one another, were evaluated for knot induction and for bacteriocin production. In addition, DNA preparations from the bacterial strains were tested for hybridization to probes containing the tryptophan monooxygenase (iaaM) and isopentenyl transferase (ipt) genes, which are involved in indole-3-acetic acid and cytokinin biosynthesis, respectively, in P.s. subsp. savastanoi.
The strains showed features characteristic of strains usually isolated from their respective host plants. For example, all 10 oleander strains were virulent both to oleander and olive, did not produce bacteriocins and harboured the iaaM gene on plasmids. In contrast, all 11 olive strains were virulent only to olive, 10 strains produced bacteriocins, and nine strains carried the iaaM gene on the chromosome. Two olive strains (OAll, OD21) harboured the gene coding for iaaM on plasmids. Furthermore, strain OD21 carried the iaaM gene on the same plasmid as the ipt gene. This is the first report both of a plasmid-borne iaaM in typical olive strains (virulent only to olive and bacteriocin producers), and of the presence of the ipt gene on the same plasmid. In olive and oleander strains the ipt gene was located either on plasmids or on the chromosome. These results suggest that under natural conditions the pathogen does not appear to spread from oleander to olive even when trees are growing in close proximity or in physical contact.
The location of the phytohormone genes on plasmids or on the chromosome is discussed in relationship to bacteriocin-production and knot-induction on the host species.