Application of RFLP analysis of recA, gyrA and rpoS gene fragments for rapid differentiation of Erwinia amylovora from Erwinia strains isolated in Korea and Japan

Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified fragments of recA, gyrA and rpoS genes was applied for the characterization of Erwinia amylovora and Erwinia strains, which cause fire blight and Asian pear blight in orchards. Primers, constructed on the basis of the published recA, gyrA and rpoS gene sequences of Erwinia carotovora, allowed us to amplify DNA fragments for RFLP differentiation of E. amylovora and E. pyrifoliae and finally to distinguish strains within these species and relate them to pear pathogens from Japan. Three to seven restriction endonucleases were applied for RFLP analysis of each gene fragment. The electrophoretic patterns generated after PCR–RFLP for each of the tested genes, were characteristic and specific for each species and allowed their differentiation. The data show that PCR–RFLP analysis of the recA, gyrA and rpoS gene fragments may be considered as a useful tool for the identification and differentiation of E. amylovora and E. pyrifoliae. Almost identical restriction patterns of the analyzed gene fragments indicated a high relationship of E. pyrifoliae strains from Korea and pear pathogens from Japan and a divergence to E. amylovora. For quick and effective differentiation of E. amylovora strains from Erwinia strains from Asia without nucleotide sequencing we recommend the amplification of recA and rpoS gene fragments and digestion of each of them with restriction endonuclease Hin6I.     

Molecular cloning of an endo-pectate lyase gene from Erwinia carotovora subsp. atroseptica

We report the construction of a clone library of the Erwinia carotovora subsp. atroseptica genome and the isolation of a gene for endo-pectate lyase. The library, inserted into the PstI site of plasmid pBR322, contains approximately 1700 clones. Five of these produce pectolytic enzymes as detected by a plate screening assay. Using a cloned endo-pectate lyase gene from the related bacterium, E. carotovora subsp. carotovora, as a probe, we found that one pectolytic E. carotovora subsp. atroseptica clone had strong homology to the probe. We characterized that clone by restriction endonuclease mapping and studied its pectolytic protein product. The purified enzyme is an endo-pectate lyase with a cofactor preference for Co²⁺. The molecular weight of the protein is 31 000 and it has an isoelectric focusing point of 9·2, corresponding to an endo-pectate lyase produced by E. carotovora subsp. atroseptica, but not to the protein product of the E. carotovora subsp. carotovora probe DNA, which has a pI of 9·5. Restriction endonuclease site polymorphisms in the two cloned endo-pectate lyase genes suggest substantial sequence divergence between these two loci.     

Simultaneous and selective detection of two major soft rot pathogens of potato: Pectobacterium atrosepticum (Erwinia carotovora subsp. atrosepticum) and Dickeya spp. (Erwinia chrysanthemi)

Dickeya spp. and Pectobacterium atrosepticum are major pathogens of potato. Current methods to detect these soft-rotting bacteria require separate identification steps. Here we describe a simple method allowing simultaneous detection of both pathogens based on multiplex PCR. The sensitivity of the primer sets was first examined on purified genomic DNA of the type strains Dickeya chrysanthemi 2048^T and P. atrosepticum 1526^T. The specificity and detection limits of the primer sets were successfully tested on 61 strains belonging to various Dickeya and Pectobacterium species, on artificially inoculated and on naturally contaminated potato plants. This new method provides a gain in time and materials, the main advantages for large-scale processes such as pathogen-free seed certification.     

Erwinia isolates from the bacterial shoot blight of pear in Japan are closely related to Erwinia pyrifoliae based on phylogenetic analyses of gyrB and rpoD genes

The phylogenetic relationships among Erwinia amylovora biovar 4 (the pathogen of bacterial shoot blight of pear in Japan), other biovars of E. amylovora, and Erwinia pyrifoliae were investigated using the sequences of 16S rRNA, gyrB, and rpoD genes. The tested isolates formed two distinct monophyletic groups in the phylogenetic trees constructed based on the gyrB gene, rpoD gene, or a combination of the three genes: group 1 contained E. amylovora biovars 1, 2, and 3; group 2 contained E. amylovora bv. 4 and E. pyrifoliae. This phylogenetic analysis showed that E. amylovora bv. 4 was more closely related to E. pyrifoliae than to other biovars of E. amylovora. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB242876 to AB242925.     

Pectate lyases of Erwinia chrysanthemi: Pel E-like polypeptides and pelE homologous sequences in strains isolated from different plants

Pel E, one of the four major pectate lyases produced by Erwinia chrysanthemi (Echr) strain EC16, was purified to homogeneity and was found to have an apparent molecular weight of 47 500 and a pI of 10. Antibodies produced against this preparation inhibited Pel E activity, but did not affect Pel A, Pel B or Pel C activities. Immunotitration revealed that Pel E accounted for a major fraction of the total extracellular Pel activity ranging from 40–60% in culture and potato tuber tissue. Isoelectric focusing of the extracellular Pels produced by various Echr strains indicated that while the Pel profiles of strains isolated from various hosts were different, the profiles of strains isolated from the same host were very similar. A significant proportion (ranging from 39 to 74%) of the Pel activity of these strains was inhibited by the anti-Pel E antibodies. DNA hybridization under stringent conditions indicated the presence of pelE homologous sequences in the genomes of E. chrysanthemi strains. We conclude that a Pel E-like enzyme occurs in all E. chrysanthemi strains examined.     

Molecular typing of Japanese strains of Ralstonia solanacearum in relation to the ability to induce a hypersensitive reaction in tobacco

The genetic diversity of 120 Ralstonia solanacearum strains isolated from a variety of host plants across Japan was assessed on the basis of hypersensitive response (HR) in tobacco leaves and phylogenetic analyses of endoglucanase gene egl, hrpB, and gyrB. Phylogenetic analysis of egl revealed that only three strains belonged to phylotype IV, and 117 strains belonged to phylotype I. Partial sequences of HrpB were identical among phylotype I strains except for one strain. Analyses using the partial nucleotide sequences of the gyrB and egl gene fragments grouped phylotype I strains into 11 gyrB and 8 egl types, respectively, whereas analyses using the partial amino acid sequences of GyrB and Egl grouped phylotype I strains into 4 GyrB and 5 Egl types, respectively. Using multilocus sequence typing of GyrB and Egl, we identified 10 unique sequence types within the Japanese phylotype I strains. Strains belonging to the GyrB42 or GyrB66 type caused wilt in tobacco, and strains belonging to GyrB2 or GyrB9 type elicited HR, demonstrating that HR induction in tobacco is genetically differentiated in the Japanese strains of R. solanacearum.     

Cultural characteristics, pathogenicity and genetic diversity of Fusarium oxysporum isolates from tobacco fields in Spain

Several formae speciales of Fusarium oxysporum are capable to produce disease in tobacco plants. Different authors have classified those isolates as a forma specialis or a race within on the basis of the severity of disease and host specificity. Fusarium wilt of tobacco plant in Extremadura (central Spain) tobacco fields have been recorded in the last years and F. oxysporum was isolated from symptomatic plants. The aim of our study was to characterize these F. oxysporum populations. For this purpose, the in vitro spore production and growth and the virulence (severity of disease) have been tested. Although all isolates behaved as pathogen, the virulence of isolates was different. The differences in growth could not be correlated with other characteristics but the two isolates with scarce spore production have also behaved as the weakest pathogen. We have analyzed intergenic spacer (IGS) region polymorphism of ribosomal DNA and random amplified polymorphic DNA (RAPD) markers to assess the genetic diversity within F. oxysporum isolates. These molecular analyses showed two major groups with different physiological capabilities that could reflect two different lineages. One group was characterized by medium–high sporulation, high virulence and the same IGS-RFLP pattern. The other group was more heterogeneous featuring low–medium sporulation and variable virulence and growth. This first experimental approach to pathogen population could be a good starting point for further studies including non-pathogenic isolates and a larger number of pathogen that could clarify if there are two or more genetic lineages.     

Effect of salicylate stress on the hypersensitive reaction of asparagus bean to tobacco necrosis virus

Treatments of primary leaves of asparagus bean (Vigna sesquipedalis Fruhw.) with salicylate had different effects on resistance to local lesion development caused by tobacco necrosis virus (TNV), depending upon the concentration used for treatment. At 1 m , salicylate slightly reduced lesion size but not virus accumulation, while at 3 m it significantly decreased lesion size and virus accumulation. Treatments with 5 m solutions appreciably increased both parameters, but also damaged the leaf tissue.The hypersensitive reaction of the leaves to virus inoculation was accompanied by the accumulation of five host-coded protein bands (VS₀to VS₄). However, three (or more) days after treatment with salicylate at any concentration between 1 and 5 m only one band accumulated and this band appeared to correspond to VS₁. This finding clearly questions the role of the VS₁ protein in limiting the spread and multiplication of TNV.The leaves responded rapidly to treatment with salicylate with stomatal closure, increased K⁺ leakage and ethylene production. These responses are generally considered to be general reactions to stress. None of them however, was correlated with any of the observed changes in TNV infection.     

Tomato spotted wilt virus triggers specific and shared defense mechanisms in hypersensitive and susceptible Solanaceae hosts

In situ and in vitro techniques were employed to investigate the metabolic changes caused by Tomato spotted wilt virus in hypersensitive and susceptible hosts; Petunia hybrida and Nicotiana tabacum, respectively. In petunia, H₂O₂ accumulation preceded increased peroxidase and shikimate dehydrogenase activity at local lesion sites. In systemic tobacco plants, peroxidase activity was induced prior to symptom onset and the activity of shikimate dehydrogenase was disrupted upon viral infection. Taken together, our data suggest that reactive oxygen species-based mechanisms of defense are shared by hypersensitive and susceptible hosts, although downstream components and regulatory mechanisms are distinct.     

The spore coat of the bean anthracnose fungus Colletotrichum lindemuthianum is required for adhesion, appressorium development and pathogenicity

The spores (conidia) of the bean anthracnose fungal pathogen, Colletotrichum lindemuthianum, adhere to the aerial parts of plants to initiate the infection process. In previous studies we have shown that the Colletotrichum spores are surrounded by a fibrillar spore coat, comprising several major glycoproteins. Previous evidence showed that a monoclonal antibody (UB20) that recognised these glycoproteins was able to inhibit adhesion of spores to a hydrophobic surface. In this paper we have further studied the role of the spore coat in adhesion, germination and fungal development by studying the effects of UB20 and protease treatment of spores. The latter treatment has previously been shown to remove the spore coat. Spores germinate on glass, polystyrene and water agar, however, appressoria only develop on glass or polystyrene, showing a requirement for a hard surface. Removal of the spore coat with protease inhibits adhesion at 30 min, before the secretion of ECM glycoproteins. Protease treatment also inhibits the development of appressoria and reduces pathogenicity on leaves. Incubation of spores with the MAb UB20 inhibits adhesion at 30 min, but does not affect appressorium formation or pathogenicity. The results suggest that an intact spore coat has two functions; it is required for adhesion to a hydrophobic surface and for the detection of a hard surface necessary for appressorium formation. We suggest that contact with a hard surface, rather than adhesion, is the key event leading to appressorium formation.     

Toxicity of p-aminophenol and p-nitrophenol to Chlorella vulgaris and two species of Nostoc isolated from soil

An examination was made of the effects of p-aminophenol (PAP) treatment individually and in combination with its parent compound, p-nitrophenol (PNP), on growth and metabolic activities of a microalga, Chlorella vulgaris, and two cyanobacteria, Nostoc linckia and Nostoc muscorum, all isolated from soil. Comparatively, the cyanobacteria were more sensitive to the phenol treatments. All but the lowest (2 μg ml⁻¹) PAP treatments inhibited cell number, chlorophyll a, and total carbohydrate production, ¹⁴CO₂ uptake, and nitrate reductase and nitrogenase activity. The algistatic effect in C. vulgaris caused by PAP could not be reverted even in the presence of acetate (0.1%). However, the inherent toxic effect of PNP established toward the alga and cyanobacteria was found alleviated in the presence of PAP only at lower concentrations. Transmission electron microscopy revealed many cytological abnormalities in Chlorella vulgaris under the influence of the selected phenols, indicating that the toxicants directly interfere with membrane properties and enzymes.     

Rwt4 , a wheat gene for resistance to Avena isolates of Magnaporthe oryzae , functions as a gene for resistance to Panicum isolates in Japan

Rwt4 (synonym of Rmg1), a temperature-insensitive gene for resistance to Avena isolates of Magnaporthe oryzae, was identified in wheat cultivar Norin 4 in a seedling assay. The significance of Rwt4 was evaluated using flag leaves of wheat cultivars. At high temperature, Norin 4 was completely resistant to Avena isolate Br58, while Chinese Spring, a noncarrier of Rwt4, was susceptible. Genetic analysis of F₂ plants derived from Norin 4 × Chinese Spring indicated that the resistance of flag leaves of Norin 4 to the Avena isolate is conditioned by a single major gene. Segregation analysis of F₃ seedlings derived from the F₂ plants showed that the major gene is actually Rwt4. These results suggest that Rwt4 is effective against Avena isolates throughout the growth stages. Furthermore, screening of Pyricularia isolates from various hosts suggested that Panicum isolates are possible carriers of the corresponding avirulence gene, PWT4. Segregation analyses of F₂ and F₃ seedlings showed that Panicum isolates actually carry PWT4, and, therefore, that Rwt4 is also effective against Panicum isolates. On the other hand, none of the Oryza, Setaria, Triticum, and Lolium isolates tested was a carrier of PWT4. The significance of this finding is discussed from the viewpoint of epidemics of blast disease on wheat.     

Prevalence, species composition, genetic variation and pathogenicity of clover rot (Sclerotinia trifoliorum) and Fusarium spp. in red clover in Finland

The species composition of a total of 173 red clover root fungal isolates from red clover roots from two established organic fields, a field in a transitional phase to organic and from two conventional fields was investigated based on morphology and molecular methods. Fusarium avenaceum was the most common Fusarium species overall but it occurrred less frequently in older organic fields. Gliocladium spp., Trichoderma spp. and Rhizoctonia spp. isolates were more common in the established organic clover fields, which had been under organic management for more than ten years and in one conventional field, than in a field still in the transitional phase. The taxonomical status of certain Fusarium, Alternaria and Sclerotinia isolates difficult to identify by morphological traits alone could be confirmed by species-specific primers and by comparing their ITS (internal transcribed spacer region) sequences to known sequences. The fingerprinting patterns of RAPD-PCR products could be used for the identification of fungal isolates and for studying the genetic variation among the isolates. Only one of the Fusarium isolates originating from apparently healthy red clover roots was clearly pathogenic to germinated red clover seedlings. In detached leaf experiments, the cvs Jokioinen and Ilte were more susceptible to one of the Sclerotinia trifoliorum isolates than cvs Betty and Bjursele, while all of them were equally susceptible to two other S. trifoliorum isolates. In further greenhouse experiments with intact plants it was possible to slow down the development of clover rot to some extent by means of one of the biological agents tested (Bacillus subtilis 10-VIZR, commercial name Alirin B), and almost completely by chemical control.     

The use of Verticillium dahliae and Diplodia scrobiculata to induce resistance in Pinus halepensis against Diplodia pinea infection

The pathogenic fungi Verticillium alboatrum and Diplodia scrobiculata were assayed for biological control of Diplodia pinea on Aleppo pine (Pinus halepensis) in Catalonia (north-eastern Spain). Young shoots were pre-treated with inoculations of either V. dahliae or D. scrobiculata, by placing colonized agar plugs on wounds made by removing a single needle fascicle. An inoculation with D. pinea was performed 15 days later. Two months after the shoot inoculations, the canker length on the stems was measured and the percentage of shoot dieback calculated. Verticillium dahliae and D. scrobiculata were found to significantly reduce the canker length of D. pinea (P < 0.05) when compared with positive controls. Diplodia pinea was slightly more sensitive to V. dahliae than to D. scrobiculata, but no significant differences (P > 0.05) were observed in the mean canker length between the two treatments. Trees pre-inoculated with V. dahliae resulted in 31.12% shoot dieback, while those pre-inoculated with D. scrobiculata resulted in 32.18% shoot dieback, compared with positive controls (42.85%).     

In vitro and in vivo antifungal activities of decursin and decursinol angelate isolated from Angelica gigas against Magnaporthe oryzae, the causal agent of rice blast

Blast is considered the most important fungal disease of rice due to its wide distribution and destructiveness under favorable conditions. Development of new effective and environmentally benign agents against the causal pathogen, Magnaporthe oryzae, is of great interest. In the course of a search for natural antifungal compounds in medicinal plants, we found that the methanol extract of Angelica gigas roots showed a potent control efficacy against rice blast caused by M. oryzae. We isolated antifungal coumarins from the extract, and they were identified as decursin and decursinol angelate. Antifungal activities of these compounds, along with kasugamycin, were tested on M. oryzae in vivo and in vitro. In an in vivo assay, the three compounds effectively suppressed the development of rice blast at concentrations more than 100 μg/mL. Coumarins showed relatively weak inhibitory effect on fungal mycelial growth when compared to kasugamycin. However, they strongly inhibited M. oryzae spore germination, which was not observed in kasugamycin treatments. This is the first report demonstrating that decursinol angelate can provide control against rice blast and that the two coumarins inhibit M. oryzae spore germination. In addition, the wettable powder formulation of the crude extract of A. gigas prohibited the development of blast symptoms on rice plants more effectively than liquid concentrate formulation of kasugamin, a commercial fungicide. Based on our study, we propose that coumarin compounds as well as the A. gigas root crude extract can be used as natural, benign fungicides for controlling rice blast.