Resistance to Pseudomonas syringae in a collection of pea germplasm under field and controlled conditions

A total of 242 Pisum accessions were screened for resistance to Pseudomonas syringae pv. pisi under controlled conditions. Resistance was found to all races, including race 6 and the recently described race 8. Fifty‐eight accessions were further tested for resistance to P. syringae pv. syringae under controlled conditions, with some highly resistant accessions identified. Finally, a set of 41 accessions were evaluated for resistance to P. syringae pv. pisi and pv. syringae under spring‐ and winter‐sowing field conditions. R2, R3 and R4 race‐specific resistance genes to P. syringae pv. pisi protected pea plants in the field. Resistance sources to race 6 identified under controlled conditions were ineffective in the field. Frost effects were also evaluated in relation to disease response. Results strongly suggest that frost tolerance is effective in lowering the disease effects caused by P. syringae pv. pisi and pv. syringae under frost‐stress conditions, even in the absence of disease resistance genes, although the highest degree of this protection is reached when frost tolerance and disease‐resistance genes are combined in the same genetic background.     

Identification of pathovars and races of <Emphasis Type="Italic">Pseudomonas syringae,</Emphasis> the main causal agent of bacterial disease in pea in North-Central Spain,and the search for disease resistance

A total of 298 bacterial isolates were collected from pea cultivars, landraces and breeding lines in North-Central Spain over several years. On the basis of biochemical-physiological characteristics and molecular markers, 225 of the isolates were identified as Pseudomonas syringae, either pv. pisi (110 isolates) or pv. syringae (112), indicating that pv. syringae is as frequent as pv. pisi as causal agent of bacterial diseases in pea. Most strains (222) were pathogenic on pea. Further race analyses of P. syringae pv. pisi strains identified race 4 (59.1% of the isolates of this pathovar), race 2 (20.0%), race 6 (11.8%), race 5 (3.6%) and race 3 (0.9%). Five isolates (4.6%) showed a not-previously described response pattern on tester pea genotypes, which suggests that an additional race 8 could be present in P. syringae pv. pisi. All the isolates of P. syringae pv. syringae were highly pathogenic when inoculated in the tester pea genotypes, and no significant pathogenic differences were observed. Simultaneous infections with P. syringae pv. pisi and pv. syringae in the same fields were observed, suggesting the importance of resistance to both pathovars in future commercial cultivars. The search for resistance among pea genotypes suitable for production in this part of Spain or as breeding material identified the presence of resistance genes for all P. syringae pv. pisi races except for race 6. The pea cultivars Kelvendon Wonder, Cherokee, Isard, Iceberg, Messire and Attika were found suitable sources of resistance to P. syringae pv. syringae.     

Effect of simultaneous and sequential inoculations of <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> and <Emphasis Type="Italic">Phomopsis vexans</Emphasis> on eggplant in sand mix and fly ash mix soil

Effects simultaneous and sequential inoculations of Meloidogyne incognita, Ralstonia solanacearum and Phomopsis vexans were studied on the growth, chlorophyll and carotenoid contents of eggplants grown in 25% fly ash and 25% sand mix soil. Plants grown in 25% fly ash mix soil had lesser plant growth than grown in 25% sand ash mix soil. Inoculation of M. incognita / R. solanacearum or P. vexans caused reduction in plant growth, chlorophyll and carotenoid contents in both types of soils but these pathogens in combination caused a greater reduction in than individual inoculation. Inoculation of M. incognita 20 days prior to R. solanacearum caused a greater reduction in plant growth than inoculation of M. incognita prior to P. vexans. Inoculation of P. vexans prior to R. solanacearum caused a lesser reduction in plant growth, chlorophyll and carotenoid contents than inoculation of P. vexans prior to M. incognita. Inoculation of R. solanacearum 20 days prior to M. incognita caused a greater reduction in plant growth, chlorophyll and carotenoid contents than inoculation of R. solanacearum prior to P. vexans. Galling and multiplication of M. incognita was higher in plants grown in 25% sand amended soil than with 25% fly ash soil. R. solanacearum and P. vexans had adverse effects on galling and nematode multiplication. Wilt and blight indices caused by R. solanacearum and P. vexans were 3 respectively. Wilt and blight indices were 4 when two pathogens were inoculated together.     

Differential Responses to Pea Bacterial Blight in Stems, Leaves and Pods Under Glasshouse and Field Conditions

Resistance to pea bacterial blight (Pseudomonas syringae pv. pisi) in different plant parts was assessed in 19 Pisum sativum cultivars and landraces, carrying race-specific resistance genes (R-genes) and two Pisum abyssinicum accessions carrying race-nonspecific resistance. Stems, leaves and pods were inoculated with seven races of P. s. pv. pisi under glasshouse conditions. For both race-specific and nonspecific resistance, a resistant response in the stem was not always associated with resistance in leaf and pod. Race-specific genes conferred stem resistance consistently, however, there was variability in the responses of leaves and pods which depended on the matching R-gene and A-gene (avirulence gene in the pathogen) combination. R2 generally conferred resistance in all plant parts. R3 or R4 singly did not confer complete resistance in leaf and pod, however, R3 in combination with R2 or R4 enhanced leaf and pod resistance. Race-nonspecific resistance conferred stem resistance to all races, leaf and pod resistance to races 2, 5 and 7 and variable reactions in leaves and pods to races 1, 3, 4 and 6.Disease expression was also studied in the field under autumn/winter conditions. P. sativum cultivar, Kelvedon Wonder (with no R genes), and two P. abyssinicum accessions, were inoculated with the most frequent races in Europe under field conditions (2, 4 and 6). Kelvedon Wonder was very susceptible to all three races, whereas P. abyssinicum was much less affected. The combination of disease resistance with frost tolerance in P. abyssinicum enabled plants to survive through the winter. A breeding strategy combining race-nonspecific resistance derived from P. abyssinicum with race-specific R-genes should provide durable resistance under severe disease pressure.     

Detection and identification of the crucifer pathogen, <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis>, by PCR amplification of the conserved Hrp/type III secretion system gene <Emphasis Type="Italic">hrcC</Emphasis>

The development of a rapid detection method for Xanthomonas campestris pv. campestris (Xcc) in crucifer seeds and plants is essential for high-throughput certification purposes. Here we describe a diagnostic protocol for the identification/detection of Xcc by PCR amplification of fragments from the pathogenicity-associated gene hrcC. Under stringent conditions of amplification, a PCR product of 519 bp from hrcC was obtained from a collection of 46 isolates of Xcc, with the exception of two isolates from radish. No amplicons were obtained from 39 pure cultures of the phytopathogenic bacteria Xanthomonas campestris pv. cerealicola, X. campestris pv. juglandis, X. campestris pv. pelargonii, X. campestris pv. vitians, X. arboricola pv. pruni, X. axonopodis pv. phaseoli, X. axonopodis pv. vesicatoria, X. vesicatoria, Pseudomonas syringae pv. phaseolicola, P. syringae pv. syringae, P. syringae pv. tomato, P. fluorescens, P. marginalis, Pectobacterium atrosepticum, P. carotovorum subsp. carotovorum. In addition, PCR reactions were negative for fifty unidentified environmental isolates purified from the surface of crucifers. The PCR fragment was obtained from four strains previously classified as X. campestris pv. aberrans, X. campestris pv. armorociae, X. campestris pv. barbarae and X. campestris pv. incanae using pathogenicity assays. Our PCR protocol specifically detected Xcc in inoculated leaves, seeds and naturally infected leaves of crucifers.     

Differentiation of some pathovars of Pseudomonas syringae with monoclonal antibodies1

Two stable hybridoma clones secreting antibodies specific for three pathovars of Pseudomonas syringae were obtained from a fusion of murine myeloma cells with spleen cells of BALB/c mouse immunized with P. syringae pv. savastanoi. Undiluted hybridoma culture medium reacted strongly in indirect ELISA tests with 20 strains of pv. savastanoi, 10 strains of pv. tomato, and 3 strains of pv. papulans. There were no reactions with 23 (of 24) strains of pv. glycinea, three strains each of pvs pisi and tabaci, two strains of pv. tagetis and one each of pvs lachrymans and aptata. Hybridomas also reacted positively with six of 16 strains of pv. syringae and with one of three strains of pv. phaseolicola.     

Characterization of the pathogenicity of strains of Pseudomonas syringae towards cherry and plum

Bacterial canker is a major disease of Prunus avium (cherry), Prunus domestica (plum) and other stone fruits. It is caused by pathovars within the Pseudomonas syringae species complex including P. syringae pv. morsprunorum (Psm) race 1 (R1), Psm race 2 (R2) and P. syringae pv. syringae (Pss). Psm R1 and Psm R2 were originally designated as the same pathovar; however, phylogenetic analysis revealed them to be distantly related, falling into phylogroups 3 and 1, respectively. This study characterized the pathogenicity of 18 newly genome‐sequenced P. syringae strains on cherry and plum, in the field and laboratory. The field experiment confirmed that the cherry cultivar Merton Glory exhibited a broad resistance to all clades. Psm R1 contained strains with differential specificity on cherry and plum. The ability of tractable laboratory‐based assays to reproduce assessments on whole trees was examined. Good correlations were achieved with assays using cut shoots or leaves, although only the cut shoot assay was able to reliably discriminate cultivar differences seen in the field. Measuring bacterial multiplication in detached leaves differentiated pathogens from nonpathogens and was therefore suitable for routine testing. In cherry leaves, symptom appearance discriminated Psm races from nonpathogens, which triggered a hypersensitive reaction. Pathogenic strains of Pss rapidly induced disease lesions in all tissues and exhibited a more necrotrophic lifestyle than hemibiotrophic Psm. This in‐depth study of pathogenic interactions, identification of host resistance and optimization of laboratory assays provides a framework for future genetic dissection of host–pathogen interactions in the canker disease.     

Identification of two serological flagellar types (H1 and H2) inPseudomonas syringae pathovars

Flagellar antigen specificity was studied for the speciesPseudomonas syringae, P. viridiflava andP. cichorii. After checking their motility, bacteria were reacted against six polyclonal antisera containing anti-O (LPS) and anti-H (flagellar) antibodies by indirect immunofluorescent staining. Two distinct flagellar serotypes (H1 and H2) were described. The distribution of H1 and H2 serotypes was then determined for a collection of 88 phytopathogenicPseudomonas strains. Serotype H1 was possessed byP. syringae pv.aptata (12 strains),P. s. pv.helianthi (2),P. s. pv.pisi (11), andP. s. pv.syringae (13). Serotype H2 was possessed byP. cichorii (2),P. s. pv.delphinii (1),P. s. pv.glycinea (4),P. s. pv.lacrymans (1),P. s. pv.mori (1),P. s. pv.morsprunorum (10),P. s. pv.persicae (1),P. s. pv.phaseolicola (8),P. s. pv.tabaci (10) andP. s. pv.tomato (1).P. viridiflava (5) revealed HI, H2 and untyped flagella. The following isolates were untypable by the H1/H2 system:P. corrugata (3),P. fluorescens (2),P. tolaasii (1). H1/H2 serotypes distribution is not linked toP. syringae O-serogroups. On the other hand, H1/H2 distribution seems remarkably linked to the new genospecies of theP. syringae group.Abbreviations CFBP French Collection of Phytopathogenic Bacteria, Angers, France - ICMP International Collection of Micro-organisms from Plants, Auckland, New-Zealand - NCPPB National Collection of Plant Pathogenic Bacteria, Harpenden, Great Britain     

Evaluation of methyl iodide as a soil fumigant in container and small field plot studies

Methyl iodide was evaluated as a soil fumigant as a potential replacement for the widely used soil fumigant methyl bromide. In container trials, methyl iodide was significantly more effective than methyl bromide against the plant parasitic nematodes Meloidogyne incognita, Heterodera schachtii and Tylenchulus semipenetrans and the plant pathogenic fungus Rhizoctonia solani. In small field plots, soil populations of root-knot nematodes were no longer detected after methyl iodide fumigation at an application rate of 112 kg ha^-1. However, after growing a susceptible lima bean host for two months, substantial root-knot galling occurred, while Rhizobium nodulation was absent. At 168 kg ha^-1 of methyl iodide, root-knot galling was reduced to less than 1%, and no Pythium propagules were recovered on selective detection media. These efficacy data support the conclusion that methyl iodide is a likely candidate for replacing methyl bromide as a soil fumigant. © 1998 SCI.     

Pseudomonas syringae pv. syringae from cool climate Australian grapevine vineyards: new phylogroup PG02f associated with bacterial inflorescence rot

During the period 2006–2011, Pseudomonas syringae pv. syringae caused a bacterial inflorescence rot (BIR) epidemic in an Australian cool climate viticultural region. Molecular multilocus sequence typing of ‘housekeeping’ genes (MLST), biochemical testing and analysis of molecular variance (AMOVA) were used to characterize the genotypes and phenotypes of P. syringae pv. syringae grapevine isolates. Comparison of the MLST data with exemplars of phylogroups available at PAMDB demonstrated that the BIR isolates formed a new clade within P. syringae pv. syringae phylogroup 2 (PG02): putatively designated PG02f. Analysis of the MLST and phenotypic data by AMOVA demonstrated some genetic differences between the BIR isolates and the general vineyard P. syringae pv. syringae population. Isolates positive for syringopeptin, syringomycin and tyrosinase, tobacco leaf hypersensitivity reaction (HR), ampicillin resistance and grapevine leaf pathogenicity were genetically distinct from those negative for these factors. This study has shown that, generally, the core genome of P. syringae pv. syringae is only weakly associated with the virulence-associated traits. As the new phylogroup PG02f consists of the epidemic BIR isolates and nonpathogenic grapevine isolates, these genetically similar isolates differ greatly in pathogenicity and most of the other tested phenotypic traits. However, within the PG02f group, tobacco leaf HR and presence of sylC (the gene for phytotoxin syringolin A) are associated with the BIR and bacterial leaf spot (BLS) isolates, and negative for the nonpathogens, indicating that these two virulence factors may be associated with vineyard pathogenicity within the new Australian phylogroup.     

Glomus intraradices, Pseudomonas alcaligenes, and Bacillus pumilus: effective agents for the control of root-rot disease complex of chickpea (Cicer arietinum L.)

The effects of Glomus intraradices, Pseudomonas alcaligenes and Bacillus pumilus on the root-rot disease complex caused by the root-knot nematode Meloidogyne incognita and the root-rot fungus Macrophomina phaseolina in chickpea was assessed by quantifying differences in the shoot dry mass, pod number, nodulation, and shoot content of chlorophyll, nitrogen, phosphorus and potassium. Inoculation of plants with G. intraradices, P. alcaligenes and B. pumilus alone and in combination significantly increased shoot dry mass, pod number, and content of chlorophyll, nitrogen, phosphorus and potassium in plants inoculated with pathogens over that in the uninoculated control plants. P. alcaligenes caused a greater increase in shoot dry mass, pod number, chlorophyll, nitrogen, phosphorus and potassium in plants with pathogens than did G. intraradices or B. pumilus. Combined application of G. intraradices, P. alcaligenes and B. pumilus to plants inoculated with pathogens caused a greater increase in shoot dry mass, pod number, nitrogen, phosphorus, and potassium than did an application of P. alcaligenes plus B. pumilus or of G. intraradices plus B. pumilus. Root colonization by G. intrardices was high when used alone, while inoculation with the pathogens reduced root colonization by G. intraradices. In the presence of P. alcaligenes and B. pumilus, root colonization by G. intraradices increased. In plants inoculated with just one antagonist, P. alcaligenes reduced galling and nematode multiplication the most, followed by G. intraradices, then B. pumilus. The greatest reduction in galling, nematode multiplication and root-rot was observed when both bacterial species and G. intraradices were applied together.     

Host suitability of Vitis rootstocks to root‐knot nematodes (Meloidogyne spp.) and the dagger nematode Xiphinema index,and plant damage caused by infections

The host suitability of commercial Vitis rootstocks commonly used in Spain (161‐49C, 41B, 1103P, 110R, 140Ru and SO4) to root‐knot nematodes (Meloidogyne arenaria, M. incognita, M. javanica) and Xiphinema index, and damage caused by nematode infection were determined under controlled conditions. The three root‐knot nematodes reproduced with a rate higher than one in all rootstocks, indicating that they are suitable hosts for these nematodes. Growth of rootstocks infected with the root‐knot nematodes was less vigorous than that of nematode‐uninfected controls in the majority of the rootstocks studied. Root infection resulted in moderate to severe root galling in all rootstocks. The shoot and main stem diameters appeared to be the most sensitive variables of damage caused by infection by Meloidogyne spp., with reduction rates from 36% and 53% in 161‐49C to 57% and 66% in 140Ru, respectively. The shoot height was not significantly affected by the root‐knot nematodes and the root fresh weight generally increased as a consequence of intensive galling. The nematode X. index caused significant root damage with a reproduction factor higher than one in all rootstocks. However, reproduction factor was significantly influenced by the rootstock and significantly decreased by about 12‐fold (5·7 to 18·1‐fold) with the increase in inoculum density from 100 to 1000 nematodes per plant. The root dry weight was reduced by X. index infections, and was the plant growth variable most affected by the nematode infection in all rootstocks at both inoculum densities. Meloidogyne arenaria, M. incognita, M. javanica and X. index, prevalent in many world vineyards, are all shown to have a damaging effect on the six tested rootstocks.     

The tomato Pto gene confers resistance to Pseudomonas floridensis,an emergent plant pathogen with just nine type III effectors

A newly discovered bacterial species, Pseudomonas floridensis, has emerged as a pathogen of tomato in Florida. This study compares the virulence and other attributes of P. floridensis to Pseudomonas syringae pv. tomato, which causes bacterial speck disease of tomato. Pseudomonas floridensis reached lower population levels in leaves of tomato as compared to the P. syringae pv. tomato strains DC3000 and NYT1. Analysis of the genome sequence of the P. floridensis type strain GEV388 revealed that it has just nine type III effectors including AvrPtoB_GEV388, which is 66% identical to AvrPtoB in DC3000. Five of these effectors have been previously reported to be members of a ‘minimal effector repertoire’ required for full DC3000 virulence on Nicotiana benthamiana; however, GEV388 grew poorly on leaves of this plant species compared to the DC3000 minimal effector strain. The tomato Pto gene recognizes AvrPtoB in race 0 P. syringae pv. tomato strains, thereby conferring resistance to bacterial speck disease. Pto was also found to confer resistance to P. floridensis, indicating this gene will be useful in the protection of tomato against this newly emerged pathogen.     

Surface antigens of Pseudomonas syringae pv. syringae are associated with pathogenicity

Antisera were raised against cell surface components of Pseudomonas syringae pv. syringae strain R32, the causal agent of brown spot disease of bush bean, and against a non-pathogenic Tn5 derived strain, PS9021. When the antiserum from strain R32 was purged against the non-pathogenic mutant PS9021, pathogenicity-specific antibodies (purged AB) were detected in the supernatant which agglutinated strain R32 but not the mutant. When the mutant, PS9021, was complemented with an intact wild type DNA fragment cloned in a cosmid vector, it was agglutinated with purged AB. When the mutant PS9021 was cured of this cosmid by introducing an incompatible plasmid no agglutination with purged AB was detected.Site-directed mutagenesis of P. syringae R32 with Tn5-containing homologous Pseudomonas DNA from the non-pathogenic mutant resulted in mutants that were indistinguishable from PS9021 with respect to either titre of purged AB or pathogenicity. The complementation of these mutants with cloned wild type DNA and their subsequent curing resulted in the same pathogenicity and purged AB behaviour as previously observed with PS9021.Cultivation of P. syringae at 30 °C, or higher temperatures, resulted in no agglutination with purged AB. These bacteria produced significantly reduced symptoms when inoculated into beans. Dot blot DNA-DNA hybridization revealed DNA homology between the pathogenicity coding region of P. syringae and DNA from several different plant pathogenic bacteria but not with naturally occurring non-pathogenic or unrelated pseudomonads. A correlation was found between the intensity of the hybridization and the titre of the purged AB of each individual Pseudomonas isolate.     

Defense responses of Arabidopsis thaliana inoculated with Pseudomonas syringae pv. tabaci wild type and defective mutants for flagellin (ΔfliC) and flagellin-glycosylation (Δorf1)

Flagellin, an essential component of the bacterial flagellar filament, is capable of inducing a hypersensitive response (HR), including cell death, in a nonhost plant. A flagellin-defective mutant (ΔfliC) of Pseudomonas syringae pv. tabaci lacks both the flagellar filament and motility, whereas a flagellin-glycosylation-defective mutant (Δorf1) retains the flagellar filament but lacks the glycosyl modification of flagellin protein. To investigate the role of flagellin protein and its glycosylation in the interaction with its nonhost Arabidopsis thaliana, we analyzed plant responses after inoculation with these bacteria. Inoculation with wild-type P. syringae pv. tabaci induced HR, with the generation of reactive oxygen species and cell death. In contrast, inoculation with either ΔfliC or Δorf1 mutant induced a low level of HR, and inoculated leaves developed a disease-like yellowing. These mutant bacteria multiplied better than the wild-type bacteria in A. thaliana. These results indicate that A. thaliana expresses a defense reaction in response to the bacterial flagellin with its glycosyl structure.     

Frost promotes the pathogenicity of Pseudomonas syringae pv. actinidiae in Actinidia chinensis and A. deliciosa plants

Frost occurs in all major areas of cultivation, presenting a threat for the production of kiwifruit crops worldwide. A series of experiments were performed on 1‐year‐old, potted plants or excised twigs of Actinidia chinensis and A. deliciosa to verify whether strict relationships exist between bacterial canker outbreaks from Pseudomonas syringae pv. actinidiae (Psa) attacks and the occurrence of autumn and winter frost events. The association between the occurrence of autumn frost and the sudden outbreak of bacterial canker in A. chinensis in central Italy has been confirmed. Both autumn and winter frosts promote Psa multiplication in the inoculated twigs of both species. The day after the frost, reddish exudates oozing from the inoculation sites were consistently observed in both species, and Psa was re‐isolated in some cases. During the thawing of both A. deliciosa and A. chinensis twigs, the 2‐cm upward and downward migration of Psa from the inoculation site was observed within 3 min, and the leaves were consistently colonized with the pathogen. A consistent brown discoloration, accompanied with a sour‐sap odour, was observed throughout the length of the excised twigs of both Actinidia species after Psa inoculation and winter frost. Psa inoculation induced a remarkably higher necrosis in excised twigs that were not frozen compared with P. s. pv. syringae inoculation. Antifreeze protection using irrigation sprinklers did not influence the short‐term period of Psa and P. s. pv. syringae multiplication in both A. deliciosa and A. chinensis twigs. Thus, the damage from frost, freeze thawing and the accumulation of Psa in Actinidia twigs promotes the migration of the pathogen within and between the orchards. Taken together, the results obtained in this study confirmed that A. deliciosa is more frost tolerant than A. chinensis, autumn frosts are more dangerous to these crops than winter frosts, and in the absence of Psa, young kiwifruit plants remain sensitive to frost.     

Biocontrol of Meloidogyne incognita on tomato using antagonistic fungi,plant‐growth‐promoting rhizobacteria and cattle manure

BACKGROUND: Biocontrol achieved by a single biocontrol agent is generally inconsistent under field conditions. The aim of the present study was to increase the competitiveness and efficacy of biocontrol agents by using them together with cattle manure. RESULTS: The effects of antagonistic fungi [Aspergillus niger v. Teigh., Paecilomyces lilacinus (Thom) Samson and Penicillium chrysogenum Thom] and plant‐growth‐promoting rhizobacteria (PGPR) [Azotobacter chroococcum Beijer., Bacillus subtilis (Ehrenberg) Cohn and Pseudomonas putida (Trev.) Mig.] were assessed with cattle manure on the growth of tomato and on the reproduction of Meloidogyne incognita (Kof. & White) Chitwood. Application of antagonistic fungi and PGPR alone and in combination with cattle manure resulted in a significant increase in the growth of nematode‐inoculated plants. The highest increase (79%) in the growth of nematode‐inoculated plants was observed when P. putida was used with cattle manure, followed by use of P. lilacinus plus cattle manure. Paecilomyces lilacinus resulted in a high reduction in galling and nematode multiplication, followed by P. putida, B. subtilis, A. niger, A. chroococcum and P. chrysogenum. The combined use of P. lilacinus with cattle manure resulted in a maximum reduction in galling and nematode multiplication. CONCLUSION: Application of P. lilacinus or P. putida with cattle manure was useful to achieve greater biocontrol of M. incognita on tomato. Copyright © 2009 Society of Chemical Industry