Characterization of an antibacterial substance produced by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> subsp. <Emphasis Type="Italic">carotovora</Emphasis> Ecc 32

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.     

<Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> can cause potato blackleg in temperate climates

It is well established that the pectinolytic bacteria Pectobacterium atrosepticum (Pca) and Dickeya spp. are causal organisms of blackleg in potato. In temperate climates, the role of Pectobacterium carotovorum subsp. carotovorum (Pcc) in potato blackleg, however, is unclear. In different western and central European countries plants are frequently found with blackleg from which only Pcc can be isolated, but not Pca or Dickeya spp. Nevertheless, tubers vacuum-infiltrated with Pcc strains have so far never yielded blackleg-diseased plants in field experiments in temperate climates. In this study, it is shown that potato tubers, vacuum-infiltrated with a subgroup of Pcc strains isolated in Europe, and planted in two different soil types, can result in up to 50% blackleg diseased plants.     

Multidrug-resistance plasmid of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis>: transfer to <Emphasis Type="Italic">Erwinia herbicola</Emphasis> on the phylloplane of mulberry and weeds

A mulberry epiphytic Enterobacter cloacae MUL1 harbors plasmid pMUL1 encoding five drug-resistance genes. This plasmid was examined upon its conjugal transfer into epiphytic Erwinia herbicola on the phylloplane of mulberry and 12 species of weeds. The plasmid was transferred into Er. herbicola at a frequency of 10^–5–10^–3/recipient in mulberry and Lolium multiflorum LAM. 1–8 days after wound inoculation with 10⁶–10⁸/ml suspensions. In Chenopodium album L. and C. album L. var. centrorubrum, however, it was transferred only after wound inoculation with a 10⁸/ml suspension, but not with 10⁷/ml or 10⁶/ml suspensions, owing to the weak epiphytic fitness of Ent. cloacae on these weeds. Transconjugants were also obtained for seven other species of weeds in the case of inoculation with a 10⁸/ml suspension. In contrast, when bacterial suspensions were sprayed on mulberry leaves with or without fresh wounds, transconjugants were obtained only in wounded leaves, which were considered suitable for bacterial conjugation. These findings suggest that epiphytic bacteria, including Ent. cloacae and Er. herbicola, may be carriers of drug-resistance genes distributed among plant pathogenic bacteria in nature.     

An enrichment microsphere immunoassay for the detection of <Emphasis Type="Italic">Pectobacterium atrosepticum</Emphasis> and <Emphasis Type="Italic">Dickeya dianthicola</Emphasis> in potato tuber extracts

An enrichment microsphere immunoassay (MIA) was developed, based on the Luminex xMAP® technology, for the simultaneous (duplex) detection of Pectobacterium atrosepticum (former name Erwinia carotovora subsp. atroseptica) (Pca) and Dickeya dianthicola (former name Erwinia chrysanthemi) (Dcd) in potato plant extracts. Target bacteria in the extracts were enriched for 48 h in a semi-selective broth containing polypectate under low oxygen conditions. Samples were subsequently incubated with antibody-coated colour-coded microspheres (beads) and with secondary antibodies conjugated with Alexa Fluor® 532, a reporter dye. Samples were analyzed with the Luminex analyzer, in which one laser identified each microsphere and another laser the reporter dye conjugated to the secondary antibodies. The assay required minimal sample preparation, could be completed in 1 h, was performed in 96 wells microtitreplates and required no wash steps. The limit of detection for the duplex enrichment MIA was 100–1000 cfu ml^?1, which was a hundred times lower than of an enrichment-ELISA. Without enrichment, the sensitivity of MIA and ELISA was largely similar and ranged between 10⁶ and 10⁷ cells ml^?1. No difference in sensitivity was found between a MIA in a single or duplex format. In a comparative test with non-infected potato plant extracts and extracts from plants infected with Pca or Dcd, results of the enrichment MIA correlated well with those of the enrichment ELISA and enrichment PCR. These results indicate that MIA can be reliably used for multiplex detection of soft rot Enterbacteriaceae in crude potato plant extracts. The technology is an attractive and cost-effective alternative to other detection methods, including ELISA.     

Characterization of <Emphasis Type="Italic">Dickeya</Emphasis> strains isolated from potato and river water samples in Finland

Plant pathogenic enterobacteria in the genera Pectobacterium and Dickeya (formerly classified as Erwinia) were isolated from diseased potato stems and tubers. The isolated bacteria were identified as P. atrosepticum, P. carotovorum and pathogens in the genus Dickeya with PCR tests. Furthermore, Dickeya strains were isolated from river water samples throughout the country. Phylogenetic analysis with 16S-23S rDNA intergenic spacer sequences suggested that the Dickeya strains could be divided into three groups, two of which were isolated from potato samples. Phylogenetic analysis with 16S rDNA sequences and growth at 39°C suggested that one of the groups corresponds to D. dianthicola, a quarantine pathogen in greenhouse cultivation of ornamentals, while two of the groups did not clearly resemble any of the previously characterised Dickeya species. Field trials with the strains indicated that D. dianthicola-like strains isolated from river samples caused the highest incidence of rotting and necrosis of potato stems, but some of the Dickeya strains isolated from potato samples also caused symptoms. The results showed that although P. atrosepticum is still the major cause of blackleg in Finland, virulent Dickeya strains were commonly present in potato stocks and rivers. This is the first report suggesting that Dickeya, originally known as a pathogen in tropical and warm climates, may cause diseases in potato in northern Europe.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454     

Behavior of <Emphasis Type="Italic">Erwinia ananas</Emphasis> transformed with bioluminescence genes on rice plants

Erwinia ananas, the causal agent of bacterial palea browning of rice, was transformed with bioluminescence genes to clarify their behavior on rice plants. Transformant CTB009T2 was used to inoculate rice plants, and the subsequent bioluminescence of CTB009T2 was observed using a two-dimensional luminometer. Luminous spots frequently appeared on anthers after flowering and on dead tissues such as leaf tips, lower leaf sheaths, and leaf blades. In spikelets that developed the disease symptom on the palea, luminous spots appeared 48h after flowering on stigmas, basal parts of ovaries, and lodicules. These results indicate that postflowering anthers and dead tissues on rice plants are important sites for a rapid increase in the pathogen population, and that the multiplication of the pathogen on internal tissues of spikelets after flowering is associated with the appearance of browning.     

<Emphasis Type="Italic">In vitro</Emphasis> Selection of Maize Rhizobacteria to Study Potential Biological Control of <Emphasis Type="Italic">Aspergillus</Emphasis> Section <Emphasis Type="Italic">Flavi</Emphasis> and Aflatoxin Production

The aims of this study were to select bacterial isolates from the non-rhizophere of maize soil and to examine their antagonistic activity against Aspergillus section Flavi strains. The first selection was made through ecophysiological responses of bacterial isolates to water activity (a_w) and temperature stress. Subsequently, an Index of Dominance test (I_D), ecological similarity and inhibition of the lag phase prior to growth, growth rate and aflatoxin B₁ accumulation were used as criteria. From the first assay nine bacterial strains were selected. They grew well at 25 and 30 °C, with growth optima between 0.982 and 0.955 a_W using 48 h of incubation. There was ecological similarity between the bacterial strains Bacillus subtilis (RCB 3, RCB 6), Pseudomonas solanacearum RCB 5, Amphibacillus xylanus RCB 27 and aflatoxigenic Aspergillus section Flavi strains at 0.982 at 25 °C. The predominant interaction between all selected bacteria and fungi in dual culture was mutual intermingling at 0.982. Mutual inhibition on contact and mutual inhibition at a distance was observed at 0.955 a_w, between only four bacteria and some Aspergillus strains. Bacillus subtilis RCB 55 showed antifungal activity against Aspergillus section Flavi strains. Amphibacillus xylanus RCB 27, B.␣subtilis RCB 90 and Sporolactobacillus inulinus RCB 196 increased the lag phase prior to growth and decreased the growth rate of Aspergillus section Flavi strains. Bacillus subtilis strains (RCB 6, RCB 55, RCB 90) and P. solanacearum RCB 110 inhibited aflatoxin accumulation. Bacillus subtilis RCB 90 completely inhibited aflatoxin B₁ accumulation at 0.982 a_W. These results show that the bacterial strains selected have potential for controlling Aspergillus section Flavi over a wide range of relevant environmental conditions in the stored maize ecosystem.     

Use of a diagnostic medium for<Emphasis Type="Italic">in situ</Emphasis> determination of the response of<Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains to bactericides

Erwinia amylovora, the causal agent of fire blight, is managed by application of bactericides to protect fruit tree blossoms from infection. Monitoring the response ofE. amylovora strains to bactericides is crucial for adequate disease management. The coliform agar medium produced by Merck was recently reported as an effective tool for rapid diagnosis ofE. amylovora (RD-medium). The objective of the present study was to examine the possibility of using the RD-medium forin situ determination of the response ofE. amylovora strains to oxolinic acid and streptomycin. The phenotypic response of 48E. amylovora strains isolated in 2002 to both bactericides was determined with the RD-medium and, for comparison, by a routine laboratory test. The results of 45 samples (93.7%) were in agreement with the findings of the routine laboratory test. Aχ ² test rejected the null hypothesis that the phenotypic characteristics as determined by the two respective methods differed significantly (P=0.389). Thein situ test was implemented on a national scale in 2003 and the results were in agreement with those obtained in laboratory tests, which suggests that this medium can be usedin situ for monitoring the appearance of resistance inE. amylovora populations. http://www.phytoparasitica.org posting Feb. 11, 2004.     

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.     

Resistance to <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">hirsutum</Emphasis> Dierckx in garlic accessions

Among the factors affecting the quality and yield of garlic production, blue mold caused by -- Penicillium spp. -- is responsible for economical losses in many countries. Allicin, present in garlic bulbs, has been suggested as having antifungal activity against some Penicillium species. This study was conducted to evaluate the response of garlic accessions against Penicillium hirsutum infection and to compare this response with bulb allicin content. Twelve garlic accessions were inoculated with P. hirsutum, and assayed in greenhouse and growth chamber experiments. Plant growth parameters and the fungal production of conidia were evaluated. Significant differences were found among the accessions. Accessions Castaño and Morado were most resistant whereas AR-I-125 and Fuego were always severely affected by the disease. A low correlation was found (r = 0.17) between allicin content and tolerance, indicating that allicin is not the main factor involved in the resistance against P. hirsutum.     

Differentiation of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains from Bulgaria by PCR-RFLP analysis

Fifty strains of Erwinia amylovora isolated in Bulgaria from different host plants and locations as well as in different years were analysed by RFLP analysis of the pEA29 PstI amplified fragment with HpaII. All the strains formed three well-resolved fragments (large—from 365 to 440 bp, medium—about 341 bp and small—about 180 bp).The strains were classified into three RFLP groups based on the polymorphism in the length of the largest fragment. This fragment was of intermediate size for 63% of the strains, and it was the longest (from 410 to 440 bp) for 29% of the strains. The variable region was sequenced for five strains. The DNA sequence analysis confirmed the different size of the largest fragment. Ten or more than ten SSRs were found for the strains in the group with the largest size of the largest fragment. Some correlation between the RFLP profiles and the origin of the strains was revealed. The RFLP profiles displayed stability in certain strains isolated from the same trees and orchards, but in different years. The number of SSRs was different in strains isolated from one and the same host plant, orchard and year, and also in strains isolated from the same host plant and orchard, but in different years. This could indicate that under natural conditions the fire blight symptoms might be caused by a mixture of E. amylovora strains with different SSR numbers, and so coexistence of distinguishable strains or a change in the population could be assumed.     

<Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> isolated from Amazon lily (<Emphasis Type="Italic">Eucharis grandiflora</Emphasis>)

Cucumber mosaic virus (CMV) was isolated from a mosaic diseased plant of Eucharis grandiflora. The virus caused mosaic symptoms on leaves and slight distortion of flower petals in E. grandiflora by either mechanical or aphid inoculation. The virus was identified as a strain of CMV subgroup I from its biological and serological characteristics.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.     

Subcellular localization of the protein encoded by virulence gene <Emphasis Type="Italic">psvA</Emphasis> of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">eriobotryae</Emphasis>

The plasmid-encoded virulence gene psvA was previously isolated from Pseudomonas syringae pv. eriobotryae and sequenced. The deduced protein of the psvA gene had no significant similarity to any other protein sequences in the database. To gain a better understanding of the function of the PsvA protein its subcellular localization was examined. To localize the PsvA protein within the bacteria, the cells were fractionated into cytoplasmic, inner membrane, and outer membrane components. The cell fractions and culture supernatant were analyzed by immunoblotting. The PsvA protein was predominantly detected in the outer membrane fraction. Immunoelectron microscopy also showed that the PsvA protein was located in the outer membrane.     

Cloning of the pathogenicity-related gene <Emphasis Type="Italic">FPD1</Emphasis> in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB110097     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

Anthracnose of <Emphasis Type="Italic">Enkianthus campanulatus</Emphasis> and <Emphasis Type="Italic">Rhynchosia acuminatifolia</Emphasis> caused by <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> (new occurrence)

In 2003–2004, anthracnoses of Enkianthus campanulatus and Rhynchosia acuminatifolia were found for the first time in Kanagawa Prefecture and Tokyo in Japan. These pathogens were identified as Colletotrichum gloeosporioides based on their pathogenicity, morphology and ribosomal DNA spacer sequences. Results were presented at the annual meeting of The Phytopathological Society of Japan in 2004.