Effect of Phenylpyrrole-resistance Mutations on Ecological Fitness of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and their Genetical Basis in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Mutants of Botrytis cinerea and Ustilago maydis highly resistant to fludioxonil were isolated at a high frequency, after nitrosoguanidine or UV mutagenesis, respectively, and selection on media containing fludioxonil. Tests on the response of mutant strains to high osmotic pressure resulted in the identification of two fludioxonil-resistant phenotypes (FLD_osm/s and FLD_osm/r), regarding the sensitivity to high osmolarity. Approximately 95% of fludioxonil-resistant mutants were found to be more sensitive to high osmotic pressure than the wild-type parent strains. Genetic analysis of phenylpyrrole-resistance in the phytopathogenic basidiomycete U. maydis, showed that fludioxonil-resistance was coded by three unlinked chromosomal loci (U/fld-1, U/fld-2 and U/fld-3), from which only the U/fld-1 mutation coded an osmotic sensitivity similar to that of the wild-types. Cross-resistance studies with fungicides from other chemical groups showed that the mutations for resistance to phenylpyrroles affect the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides, but not to the benzimidazoles, anilinopyrimidines, phenylpyridinamines, hydroxyanilides or the sterol biosynthesis inhibiting fungicides. A study of fitness parameters in the wild-type and fludioxonil-resistant mutants of B. cinerea, showed that all osmotic sensitive (B/FLD_osm/s) isolates had significant reductions in the characteristics determining saprophytic fitness such as mycelial growth, sporulation, conidial germination and sclerotial production. Contrary to that, with the exception of mycelial growth, the fitness parameters were unaffected or only slightly affected in most of the osmotic resistant (B/FLD_osm/r) isolates. Tests on cucumber seedlings showed that the osmotic-sensitive strains were significantly less pathogenic compared with the wild-type and B/FLD_osm/r strains of B. cinerea. Preventative applications of the commercial products Saphire 50 WP (fludioxonil) and Rovral 50 WP (iprodione) were effective against lesion development on cotyledons by the wild-type and the mutant strains of B. cinerea that were resistant to the anilinopyrimidine cyprodinil (B/CPL-27) and to the hydroxyanilide fenhexamid (B/FNH-21), but ineffective, even at high concentrations, against disease caused by the fludioxonil-resistant isolates (B/FLD) and a mutant strain resistant to the dicarboximide iprodione (B/IPR-1). Experiments on the stability of the fludioxonil-resistant phenotype showed a reduction of resistance, mainly in osmotic-sensitive isolates, when the mutants were grown on inhibitor-free medium. A rapid recovery of the high resistance was observed after mutants were returned to the selection medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain of B. cinerea, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of osmotic-resistant mutants was higher than the osmotic-sensitive ones. Furthermore, competition tests, in planta, showed a significant reduction of the frequency of both phenylpyrrole-resistant phenotypes, with a respective increase in the population of the wild-type strain of the pathogen.     

Vascular blackening of wasabi rhizomes caused by <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis>

Wasabi (Wasabia japonica) is grown for its highly-valued rhizome which is used as a condiment in Japanese food. Symptoms of vascular blackening in the rhizome were first observed in 2005 in plants grown in British Columbia, Canada. Microscopic observations and microbial isolation from infected tissues revealed that most of the xylem tracheid cells were blackened and bacteria were consistently associated with symptomatic plants. The bacterium most frequently recovered was identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) using BioLog™ and sequencing of a specific ~510 bp IGS region. Pathogen-free plants obtained using meristem-tip micropropagation were inoculated with a wasabi isolate of Pcc. Vascular blackening symptoms developed in the rhizome after 8 weeks when the rhizome was first wounded by stabbing or cutting, or if the roots were pre-inoculated with Pythium species isolated from rhizome epidermal tissues, followed by inoculation with Pcc at 1 × 10⁸ cells ml⁻¹. Xylem tracheid cells were blackened and Pcc was reisolated from all diseased tissues. The highest frequency of rhizome vascular blackening occurred at 22°C and 27°C and these tissues occasionally succumbed to soft rot at higher temperatures, but not when inoculated tissues were incubated at 10°C. The rooting medium used by growers for vegetative propagation of wasabi was shown to contain Pcc but the pathogen was not recovered from the irrigation water. Entry of Pcc through wounds on wasabi rhizomes and the host tissue response result in symptoms of vascular blackening.     

Visualization of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> cells during biocontrol of bacterial wilt disease in tomato with <Emphasis Type="Italic">Pythium oligandrum</Emphasis>

The biocontrol agent Pythium oligandrum (PO) can suppress bacterial wilt caused by Ralstonia solanacearum (RS) in tomato. To understand the primary biocontrol mechanisms of bacterial wilt by PO, we pretreated tomato plants with sterile distilled water or preinoculated them with PO, followed by inoculation with RS, then observed PO and RS in fixed sections of tomato tissues using a confocal laser-scanning microscope and fluorescence labeling until 14 days after the inoculation with RS. Horizontal and vertical movement of RS bacteria was frequently observed in the xylem vessels of roots and stems of tomato plants (cv. Micro-Tom) that had not been inoculated with PO. In plants that were preinoculated with PO, the movement of RS was suppressed, and bacteria appeared to be restricted to the pit of vessels, a reaction similar to that observed in resistant rootstocks. PO colonization was mainly observed at the surfaces of taproots, the junctions between taproots and lateral roots, and the middle sections of the lateral roots. PO was not observed near wound sites or root tips where RS tended to colonize. However, RS colonization was significantly repressed at these sites in PO preinoculated plants. These observations suggest that the induction of plant defense reactions is the main mechanism for the control of tomato bacterial wilt by PO, not direct competition for infection sites.     

Extracts from <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> induce effective resistance to the pathogen in both <Emphasis Type="Italic">Arabidopsis</Emphasis> and solanaceous plants

Plants defend themselves against microbial invasions by detecting conserved molecules, collectively called pathogen-associated molecular patterns (PAMPs). PAMPs-triggered basal resistance is the first inducible layer of plant defense. Here we found that Ralstonia solanacearum strain RS1002 can efficiently grow and cause disease in ecotype Columbia-0 of the model plant Arabidopsis thaliana in a manner dependent on the Hrp type III secretion system (T3SS). The extent of disease symptoms caused by R. solanacearum was reduced in plants pretreated with ΔhrpY mutant deficient in the functional Hrp T3SS. Pretreatment with a boiled extract (BE) from R. solanacearum had a similar inhibitory effect on disease development or bacterial multiplication in both Arabidopsis and several solanaceous plants. Simultaneous inoculations with BE and R. solanacearum did not induce BE-mediated resistance, nor did a BE treatment with proteinases. These results indicate that host plants recognize an unknown proteinaceous PAMP in the BE to induce disease resistance and that the Hrp T3SS of R. solanacearum can suppress it. From an analysis using Arabidopsis mutants lacking PAMP receptors, the elongation factor Tu of R. solanacearum was shown to partially contribute to BE-mediated basal resistance in Arabidopsis plants.     

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.     

Induced resistance against Fusarium diseases of <Emphasis Type="Italic">Cymbidium</Emphasis> species by weakly virulent strain HPF-1 (<Emphasis Type="Italic">Fusarium</Emphasis> sp.)

The mechanism by which Fusarium diseases of cymbidium plants are suppressed by a weakly virulent strain HPF-1 of Fusarium sp. was studied. Strain HPF-1 produced microscopic, necrotic local lesions on cymbidium leaves, causing minor damage to palisade tissues at the infection sites. This weakly virulent strain remained near the site of infection and did not develop further. It systemically and nonselectively suppressed some diseases of cymbidium such as yellow spot of leaves caused by Fusarium proliferatum and F. fractiflexum, bulb and root rot caused by F. oxysporum, and dry rot of bulbs and roots caused by F. solani. Because endogenous salicylic acid levels increased in cymbidium leaves inoculated with strain HPF-1, the mechanism of disease suppression is thought to be systemic acquired resistance.     

Colonisation and histological changes in muskmelon and autumn squash tissues infected by <Emphasis Type="Italic">Acremonium cucurbitacearum</Emphasis> or <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis>

Muskmelon (Cucumis melo cv. Temprano Rochet) and autumn squash (Cucurbita maxima) seedlings were inoculated either with Acremonium cucurbitacearum or Monosporascus cannonballus, two of the soil-borne fungi implicated in ‘melon collapse’. Inoculation was achieved in two different ways: by growing the plants in pots containing infested soil to study the histological changes produced in the infected tissues using light microscopy and by growing seedlings in Petri dishes together with fungal colonies in order to observe the colonisation of the plant tissues using scanning electron microscopy. Both muskmelon and autumn squash roots infected with A. cucurbitacearum showed a suberised layer in the epidermis and the outermost layers of the parenchymatic cortex, but these symptoms developed earlier in the muskmelon plants. Muskmelon plants infected by this fungus also presented hypertrophy and hyperplasia, which led to a progressive separation of the vascular bundles in the lower stems of the affected plants. This response was not observed in autumn squash during the study. On the other hand, few histological changes were observed in tissues infected with M. cannonballus and only a slight increase in the size of cortical intercellular spaces was noted in the lower stems of muskmelon plants, and infected autumn squash tissues remained free of these symptoms throughout the study. The scanning electron microscope observations revealed that both fungi were able to colonise the tissues of the two host plants which were studied. A. cucurbitacearum colonised the epidermis and cortex of both muskmelon and autumn squash. The hyphae grew both inter- and intracellularly, and the density of the colonisation decreased within the endodermis. The same colonisation of host plants was observed as a result of M. cannonballus infection. The xylem vessel lumina of both muskmelon and autumn squash showed hyphae and tylose formation as a result of both fungal infections. However, non-fungal structures were detected in the hypocotyl vascular tissues. The present study demonstrates that both fungi are capable of infecting the tissues of a species which is resistant (autumn squash) and a species which is susceptible (muskmelon) to melon collapse.     

Fusarium rot of hyacinth caused by <Emphasis Type="Italic">Gibberella zeae</Emphasis> (anamorph: <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic"> graminearum</Emphasis>)

Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium rot of hyacinth” as a new disease because only the anamorph, F. graminearum, was identified on the diseased host plant. The authors contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively.     

Ultrastructural and cell wall modifications during infection of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis> roots by a pathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain

Fusarium species are soil-borne fungal pathogens that produce a variety of disease symptoms when attacking crop plants. The mode of root colonization of Eucalyptus viminalis seedlings by a pathogenic F. oxyporum strain (Foeu1) at the ultrastructural level and changes in cell wall pectin during host pathogen interactions are described. Root systems of E. viminalis plants were inoculated with F. oxysporum in an in vitro model system. Hyphae of F. oxysporum adhered to the outer epidermal cell walls through fibrillar material, and after penetration they spread into the internal tissues. They developed intercellularly and intracellularly in the root cortex and invaded vascular tissues. Papillae were induced, and the host plasma membrane ruptured in colonized cells, causing rapid host tissue and cell damage. Changes in distribution and occurrence of nonesterified and methyl-esterified pectins were evaluated after root colonization by F. oxysporum using two monoclonal antibodies, JIM 5 and JIM 7, respectively. Nonesterified pectin in control roots was mainly localized in the epidermal cell walls and middle lamellae in parenchymal cortex, whereas methyl-esterified pectin accumulated more in primary cell walls of the cortex and phloem. Decreases in immunodetected nonesterified and methyl-esterified pectins were associated with extensive plant tissue degradation after root colonization by the pathogenic fungus.     

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.     

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

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

A new leaf blight disease of <Emphasis Type="Italic">Trifolium dasyurum</Emphasis> caused by <Emphasis Type="Italic">Botrytis fabae</Emphasis>

A new disease was observed on Trifolium dasyurum, with symptoms beginning as a halo spot and developing into a leaf blight. The causal organism was identified by microscopy and DNA sequence studies as Botrytis fabae. This strain of B. fabae was also demonstrated to cause disease on foliage of a range of pulse crops, including Vicia faba, Pisum sativum, and Lens culinaris. This study demonstrates the potential of this strain of B. fabae to not only pose a significant threat to T. dasyurum but also to pulses grown in rotation with T. dasyurum that are susceptible to this strain of B. fabae.     

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.     

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

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

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis> elicits defence response genes in roots of potato plantlets challenged by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Biological control of Rhizoctonia solani with Trichoderma harzianum has been demonstrated in several studies. However, none have reported the dynamics of expression of defence response genes. Here we investigated the expression of these genes in potato roots challenged by R. solani in the presence/absence of T. harzianum Rifai MUCL 29707. Analysis of gene expression revealed an induction of PR1 at 168 h post-inoculation (hpi) and PAL at 96 hpi in the plants inoculated with T. harzianum Rifai MUCL 29707, an induction of PR1, PR2 and PAL at 48 hpi in the plants inoculated with R. solani and an induction of Lox at 24 hpi and PR1, PR2, PAL and GST1 at 72 hpi in the plants inoculated with both organisms. These results suggest that in the presence of T. harzianum Rifai MUCL 29707, the expression of Lox and GST1 genes are primed in potato plantlets infected with R. solani at an early stage of infection. Mycothèque de l’Université catholique de Louvain of S. Cranenbrouck's affiliation is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).     

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     

Induced resistance to<Emphasis Type="Italic">Cladosporium cucumerinum</Emphasis> in cucumber by pectinases extracted from<Emphasis Type="Italic">Penicillium oxalicum</Emphasis>

Pectinases extract (PE) from the fermentation product ofPenicillium oxalicum BZH-2002 was tested for its ability to induce protection against scab caused byCladosporium cucumerinum on cucumber (Cucumis sativus L.) plants. Seedlings with one true leaf were sprayed with various concentrations of PE (10–200 units ml⁻¹) 3 days before inoculation withC. cucumerinum. Results showed that the induced local protection against the pathogen was dose-dependent when the concentrations of PE were between 20 and 120 units ml⁻¹; systemically induced resistance against the pathogen was not observed. Boiled PE had a slight effect on disease reduction. Commercial pectinases prepared fromAspergillus niger showed lower protection against scab compared with PE when they were used at the same concentration of enzyme activity. No inhibitory activity was observed on conidial germination or germ-tube growth ofC. cucumerinum. PE was further evaluated for its enhancement of defense-related enzymes. Peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased in cucumber seedlings after treatment with PE. PO and PPO remained at a higher level in PE-pretreated seedlings throughout the experiment period whether pathogen-inoculated or non-inoculated, whereas PAL activity began to decrease 2 days after PE treatment. http://www.phytoparasitica.org posting July 14, 2004.     

Partial characterisation of a novel <Emphasis Type="Italic">Tobamovirus</Emphasis> infecting <Emphasis Type="Italic">Actinidia chinensis</Emphasis> and <Emphasis Type="Italic">A. deliciosa</Emphasis> (Actinidiaceae) from China

Actinidia chinensis and A. deliciosa plants from China, showing a range of symptoms, including vein clearing, interveinal mottling, mosaics and chlorotic ring spots, were found to contain ~300 nm rod-shaped virus particles. The virus was mechanically transmitted to several herbaceous indicators causing systemic infections in Nicotiana benthamiana, N. clevelandii, and N. occidentalis, and local lesions in Chenopodium quinoa. Systemically- infected leaves reacted with a Tobacco mosaic virus polyclonal antibody in indirect ELISA. PCR using generic and specific Tobamovirus primers produced a 1,526 bp sequence spanning the coat protein (CP), movement protein (MP), and partial RNA replicase genes which showed a maximum nucleotide identity (88%) with Turnip vein clearing virus and Penstemon ringspot virus. However, when the CP sequence alone was considered the highest CP sequence identity (96% nt and 98% aa) was to Ribgrass mosaic virus strain Kons 1105. The morphological, transmission, serological and molecular properties indicate that the virus is a member of subgroup 3 of the genus Tobamovirus.