Parasitism of <Emphasis Type="Italic">Trichoderma</Emphasis> on <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> and role of the gelatinous matrix

Trichoderma (T. asperellum-203, 44 and GH11; T. atroviride-IMI 206040 and T. harzianum-248) parasitism on Meloidogyne javanica life stages was examined in vitro. Conidium attachment and parasitism differed beween the fungi. Egg masses, their derived eggs and second-stage juveniles (J2) were parasitized by Trichoderma asperellum-203, 44, and T. atroviride following conidium attachment. Trichoderma asperellum-GH11 attached to the nematodes but exhibited reduced penetration, whereas growth of T. harzianum-248 attached to egg masses was inhibited. Only a few conidia of the different fungi were attached to eggs and J2s without gelatinous matrix; the eggs were penetrated and parasitized by few hyphae, while J2s were rarely parasitized by the fungi. The gelatinous matrix specifically induced J2 immobilization by T. asperellum-203, 44 and T. atroviride metabolites that immobilized the J2s. A constitutive-GFP-expressing T. asperellum-203 construct was used to visualize fungal penetration of the nematodes. Scanning electron microscopy revealed the formation of coiling and appressorium-like structures upon attachment and parasitism by T. asperellum-203 and T. atroviride. Gelatinous matrix agglutinated T. asperellum-203 and T. atroviride conidia, a process that was Ca²⁺-dependent. Conidium agglutination was inhibited by carbohydrates, including fucose, as was conidium attachment to the nematodes. All but T. harzianum could grow on the gelatinous matrix, which enhanced conidium germination. A biomimetic system based on gelatinous-matrix-coated nylon fibers demonstrated the role of the matrix in parasitism: T. asperellum-203 and T. atroviride conidia attached specifically to the gelatinous-matrix-coated fibers and parasitic growth patterns, such as coiling, branching and appressoria-like structures, were induced in both fungi, similarly to those observed during nematode parasitism. All Trichoderma isolates exhibited nematode biocontrol activity in pot experiments with tomato plants. Parasitic interactions were demonstrated in planta: females and egg masses dissected from tomato roots grown in T. asperellum-203-treated soil were examined and found to be parasitized by the fungus. This study demonstrates biocontrol activities of Trichoderma isolates and their parasitic capabilities on M. javanica, elucidating the importance of the gelatinous matrix in the fungal parasitism.     

Analysis of sources of oxolinic acid-resistant field strains of <Emphasis Type="Italic">Burkholderia glumae</Emphasis> based on rep-PCR analysis and nucleotide sequences of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Repetitive sequence-based polymerase chain reaction (rep-PCR) analysis using BOX and ERIC as primers showed a highly divergent phylogeny among field strains of Burkholderia glumae. To elucidate the sources of oxolinic acid (OA) resistance in field strains of B. glumae isolated from rice seedlings cultivated in Mie, Toyama, and Iwate prefectures, Japan, the amino acid at position 83 of GyrA (GyrA83), which is involved in OA resistance, and the DNA patterns from the rep-PCR and the partial nucleotide sequences of gyrB and rpoD from various strains were analyzed. The ten Mie strains, in which GyrA83 was isoleucine (Ile), were divided into two groups based on the band patterns in rep-PCR analysis, although the nucleotide sequences of gyrB and rpoD were identical among the strains. Based on the band patterns in the rep-PCR analysis and the gyrB and rpoD sequences, two highly OA-resistant Toyama strains, Pg-13 and Pg-14, for which GyrA83 was serine (Ser) and Ile, respectively, were in the same lineage. This suggests that the bacteria might acquire OA resistance faster than phylogenic diversity as determined with the repetitive sequences BOX and ERIC and with gyrB and rpoD. Furthermore, three Iwate strains (H95, H101, and H104), isolated from seedlings of different cultivars grown in different years and having Ile at GyrA83, are probably in the same lineage, suggesting that OA-resistant bacteria might be transferred among different cultivars.     

Inheritance of resistance to <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis> in two wild accessions of <Emphasis Type="Italic">Pisum</Emphasis>

Mycosphaerella pinodes is one of the most devastating pea pathogens. Pea cultivars with adequate levels of resistance to control the disease are not so far available. However, promising levels of resistance have been identified in wild accessions of pea. In the present investigation the inheritance of resistance to M. pinodes was studied in two crosses between the susceptible pea cv. ‘Ballet’ and the partially wild resistant accessions P665 (Pisum sativum subsp. syriacum) and P42 (P. sativum subsp. sativum var. arvense). Both additive and dominant effects were important in control of resistance and susceptibility dominated over resistance.     

Evaluation of the durability of the <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-resistant <Emphasis Type="Italic">Zhong ZH</Emphasis> and <Emphasis Type="Italic">TC14</Emphasis> wheat lines

Serial passage experiments (SPE) of a Barley yellow dwarf virus-PAV (BYDV-PAV) isolate were performed on Zhong ZH and TC14 wheat lines to evaluate the durability of their resistance to BYDV. At different passage numbers (from the 2nd to the 114th), biological properties of the produced isolates were recorded either by monitoring infection percentages and virus titers of the first 3 weeks of viral infection or by measuring their impact on yield components. Statistical analyses using the area under pathogen progress curves and the area under concentration progress curves demonstrated that these two resistant lines induce, after only a few passages, a selection of variant(s) with significantly modified infection abilities. Isolates resulting from SPE performed on these lines induced important decreases of yield components. These results indicate that the use of Zhong ZH and TC14 lines in BYDV-resistant breeding programmes should be approached with caution.     

Effect of seed treatment of<Emphasis Type="Italic">Arachis hypogaea</Emphasis> with<Emphasis Type="Italic">Bacillus subtilis</Emphasis> on nodulation in biocontrol of southern blight (<Emphasis Type="Italic">Sclerotium rolfsii</Emphasis>) disease

The employment of formulatedBacillus subtilis for peanut seeds (Arachis hypogaea cv. ‘Shulamit’) counteracted the destructive effects of the seedborne pathogenSclerotium (Athelia)rolfsii on the nodulation, leghemoglobin and nitrogenase activity of peanuts. Moreover, the changes in crop vigor index, total nitrogen content and survivability of bothRhizobium spp. andB. subtilis have been related to compatibility and even an occasional synergism between them. http://www.phytoparasitica.org posting Nov. 12, 2006.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Mechanisms of resistance in<Emphasis Type="Italic">Lycopersicon</Emphasis> germplasm to the whitefly<Emphasis Type="Italic">Bemisia argentifolii</Emphasis>

The silverleaf whiteflyBemisia argentifolii Bellows & Perring (Homoptera: Aleyrodidae) [also known as strain B of the sweetpotato whiteflyB. tabaci (Gennadius)] is a major pest of tomatoes due to both feeding damage and transmission of plant viruses. Certain wild species ofLycopersicon have demonstrated high levels of resistance to the pest. Greenhouse studies were undertaken to quantify the effects on whitefly behavior and mortality of individual, resistant plants selected from three accessions ofL. pennellii (Corr.) D’Arcy (LA 1340, LA 1674 and LA 2560), five accessions ofL. hirsutum f.typicum Humb. & Bonpl. (LA 386, LA 1353, LA 1777, PI 127826 and PI 127827) and one accession ofL. hirsutum f.glabratum C.H. Mull. (PI 126449). In no-choice experiments, fewer adults settled on leaflets of the wild species and deposited 75–100% fewer eggs compared to the cultivated tomato,L. esculentum Mill. Adult mortality ranged from 77–100% on wild accessions but was only 1% onL. esculentum. Most dead adults were trapped in glandular trichome exudates. The effects of these resistant accessions onB. argentifolii were mechanically transferable by appressing the trichome exudates onto the leaves of the susceptible tomato, indicating an association between the factors mediating the resistance and the glandular trichomes. Laboratory studies evaluated the repellent, fumigant and residual toxic effects of representative constituents of trichome exudates onB. argentifolii adults by using selected concentrations and probit analyses. RC₅₀ values (estimated concentration to repel 50% of the adults) and LC₅₀ values for fumigant and residual toxicity indicated that 2-tridecanone had low levels of repellent and residual toxicity activity; that 2-undecanone had high levels of repellent and fumigant activity; and that ginger oil (composed, in part, of sesquiterpene hydrocarbons) had high levels of repellent and residual toxicity activity. These studies suggest that multi-factor resistance exists in wild tomato germplasm. By combining genetically the observed chemical constituents of resistance into a single germplasm, the resulting resistance may be more difficult forB. argentifolii to overcome. http://www.phytoparasitica.org     

Characteristics of a <Emphasis Type="Italic">Plasmopara angustiterminalis</Emphasis> isolate from <Emphasis Type="Italic">Xanthium strumarium</Emphasis>

Leaves of Xanthium strumarium infected with downy mildew were collected in the vicinity of a sunflower field in southern Hungary in 2003. Based on phenotypic characteristics of sporangiophores, sporangia and oospores as well as host preference the pathogen was classified as Plasmopara angustiterminalis. Additional phenotypic characters were investigated such as the size of sporangia, the number of zoospores per sporangium and the time-course of their release. Infection studies revealed infectivity of the P. angustiterminalis isolate to both X. strumarium and Helianthus annuus. Inoculation of the sunflower inbred line, HA-335 with resistance to all known P. halstedii pathotypes, resulted in profuse sporulation on cotyledons and formation of oospores in the bases of hypocotyls. Infections of sunflower differential lines often led to damping-off. Molecular genetic analysis using simple sequence repeat primers and nuclear rDNA sequences revealed clear differences to Plasmopara halstedii, the downy mildew pathogen of sunflower.     

Competition between Solanum nigrum and pepper in the presence of Meloidogyne incognita

The influence of the root-knot nematode Meloidogyne incognita (Kofoid & White) Chitwood on the competitive relationships between pepper (Capsicum annuum L.) and Solanum nigrum L. (black nightshade) was investigated in a glasshouse experiment. Seven competition treatments were set up: two intraspecific for the crop and the weed and five interspecific treatments in which the emergence of S. nigrum plants was progressively delayed with respect to that of the pepper. Nematodes reproduced in every inoculated plant and their multiplication rates were high in both pepper and S. nigrum. The parasite reduced all growth and yield parameters of the crop, but did less harm to the weed. The negative effect of S. nigrum on pepper peaked in the treatment in which the weed and pepper plants emerged together. S. nigrum was a stronger competitor than pepper under both nematode-infested and nematode-free conditions. The effect of nematodes on pepper yield was less than that of competition, but both appeared to be additive.     

<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> and <Emphasis Type="Italic">P. peruviana</Emphasis>, new natural hosts of <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>

Tomato chlorosis virus causes yellow leaf disorder epidemics in many countries worldwide. Plants of Physalis ixocarpa showing abnormal interveinal yellowing and plants of Physalis peruviana showing mild yellowing collected in the vicinity of tomato crops in Portugal were found naturally infected with ToCV. Physalis ixocarpa and P. peruviana were tested for susceptibility to ToCV by inoculation with Bemisia tabaci, Q biotype. Results confirmed that ToCV is readily transmissible to both species. The infection was expressed in P. ixocarpa by conspicuous interveinal yellow areas on leaves that developed into red or brown necrotic flecks, while P. peruviana test plants remained asymptomatic. Infected plants of both P. ixocarpa and P. peruviana served as ToCV sources for tomato infection via B. tabaci transmission. This is the first report of P. ixocarpa and P. peruviana as natural hosts of ToCV.     

Use of sclerotia of<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> for efficient production of conidia of<Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in liquid culture

A study was conducted to determine the feasibility of using sclerotia ofSclerotinia sclerotiorum for producing conidia ofConiothyrium minitans in liquid culture. The medium (SST) was made of water containing 2.0, 1.5, 1.0 or 0.5% (w/v) ground sclerotia ofS. sclerotiorum and 100 μgl ⁻¹ thiamine hydrochloride (HCl). One ml of conidial suspension (2 × 10⁷ conidia ml⁻¹) ofC. minitans LRC 2534 was inoculated into 100 ml of SST medium or control (thiamine HCl in water) and incubated at 20 ± 2°C on a shaker at 200 rpm. Subsamples were removed periodically and examined under a compound microscope. Conidia in the SST media germinated within 24 h, developed into branched hyphae within 48 h, produced pycnidia after 3–4 days, and the pycnidia released mature conidia after 7 days. Production of conidia varied with the concentration of sclerotia in the SST medium. It was lower (3.6 × 10⁶ conidia ml⁻¹) at 0.5% but higher (1.2 × 10⁸ conidia ml⁻¹) at 2%. The new conidia were viable and the colonies developing from them showed the original morphological characteristics. It was concluded that using SST liquid medium as a substrate for mass production of conidia ofC. minitans has potential for use in commercial development of this mycoparasite as a biocontrol product. http:www.phytoparasitica.org posting Jan. 23, 2007.     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.     

Molecular characterization and PCR detection of the melon pathogen <Emphasis Type="Italic">Acremonium</Emphasis><Emphasis Type="Italic">cucurbitacearum</Emphasis>

Acremonium cucurbitacearum is a soil-borne pathogen that causes collapse of muskmelon and watermelon plants. Cluster analysis based on RAPD patterns, obtained from use of 25 primers, divided isolates of A. cucurbitacearum from Spain and USA into two major groups. Most isolates from the USA fell into group 1, however, genetic similarity was not highly correlated with geographical origins or with previously established VCG groups. Analysis of 5.8S-ITS sequences showed very little sequence variation among isolates of A. cucurbitacearum, most had identical 5.8S-ITS sequence. Nodulisporium melonis, previously reported to cause a similar disease in Japan, had a 5.8S-ITS sequence that was identical to that of isolate A-419 proposed as the type strain of A cremonium cucurbitacearum suggesting that the two fungal pathogens should be considered a single species. Phylogenetic analysis, based on the 5.8S-ITS region, indicated that A cremonium cucurbitacearum is a monophyletic taxon more closely related to Plectosphaerella cucumerina than to other species of the genus Acremonium. Based on the 5.8S-ITS nucleotide sequence, a polymerase chain reaction was designed and used for specific detection of A. cucurbitacearum in diseased plants.     

Lack of biocontrol capacity in a non-pathogenic mutant of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis>

The aim of this study was to assess the biocontrol capacity of rev157, a non-pathogenic mutant of a pathogenic strain of Fusarium oxysporum f. sp. melonis (Fom24). Inoculated in association with the virulent parental strain, the mutant rev157 did not protect the host plant (muskmelon) against infection by Fom24. Applied on flax, a non-host plant, the mutant rev157 was not able to protect it against its specific pathogen F. oxysporum f. sp. lini. On the contrary the parental strain Fom24 did protect flax as well as a soil-borne biocontrol strain (Fo47). Since the mutant rev157 was affected neither in its growth in vitro nor in its capacity to penetrate into the roots, it can be speculated that the mutation has affected traits responsible for interactions within the plant. In F. oxysporum the pair of strains Fom24/rev157 is a good candidate to identify genes involved in the biocontrol capacity of F. oxysporum and to test the hypothesis of a link between capacity to induce the disease and capacity to induce resistance in the plant.     

<Emphasis Type="Italic">Bchex</Emphasis> virulence gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis>: characterization and functional analysis

We previously identified Bchex as a highly expressed gene during filamentous growth in Botrytis cinerea. The gene encodes the principal protein of the Woronin body and has been shown to seal septal pores in response to cellular damage. In the present study, Southern blot analysis of genomic DNA indicated that the gene exists as a single copy in the B. cinerea genome. The gene was differentially expressed during various developmental stages: expression was high in germinating conidia and the mycelial stage and lower in resting conidia and the appressorial stage. For functional analyses, homologous recombination was used to obtain a ΔBchex knockout mutant. Growth of the mutant was strongly reduced growth in complete medium and in defined media with sucrose, fructose or pectin as the carbon source. After detached tomato leaves were inoculated with the Bchex mutant, lesion development was markedly reduced compared to the control, suggesting that Bchex participates in normal growth, germination and virulence of this fungus.     

Occurrence of<Emphasis Type="Italic">Rhyacionia frustrana</Emphasis> on<Emphasis Type="Italic">Pinus brutia</Emphasis> in the Aegean Region of Turkey

The Nantucket pine tip moth (Rhyacionia frustrana (Comstock) (Lep., Tortricidae)) has been found on Turkish red pine (Pinus brutia L.) in the Aegean Region of Turkey. http://www.phytoparasitica.org posting Nov. 12, 2006     

Genetic diversity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan based on phylogenetic analyses of rDNA-IGS and <Emphasis Type="Italic">MAT1</Emphasis> sequences

Twenty-eight isolates of Fusarium oxysporum f. sp. spinaciae (FOS; the causal agent of spinach wilt) collected from Japan were assessed for mating type and subjected to phylogenetic analysis. Mating type analysis revealed all isolates to be MAT1-2, suggesting that there is no sexual recombination within the population. Phylogenetic analyses based on nucleotide sequences of the ribosomal DNA intergenic spacer (IGS) and the mating type locus (MAT1) suggested that FOS is polyphyletic. The cluster analysis based on IGS showed four phylogenetic groups (S1–S4) among the isolates. Two distinct lineages, S1 and S3, included FOS isolates both of the vegetative compatibility group (VCG) types, 0330 and 0331, demonstrating that VCG differentiation in FOS may not necessarily reflect the phylogenetic relationships based on IGS and MAT1-2-1.     

Susceptibility and resistance of <Emphasis Type="Italic">Beta vulgaris</Emphasis> subsp. <Emphasis Type="Italic">maritima</Emphasis> to foliar rub-inoculation with <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis>

Four lines (designated MR0, MR1, MR2, and M8) from 13 accessions of Beta vulgaris subsp. maritima were selected on the basis of phenotypes produced after foliar rub-inoculation with Beet necrotic yellow vein virus (BNYVV). The susceptible phenotype developed bright yellow local lesions, whereas the resistant phenotype had symptoms ranging from no visible lesions to necrotic lesions at the inoculation site. MR1 and MR2 lines had a resistant phenotype depending on the isolate and the MR0 line was susceptible to all isolates of BNYVV tested. The M8 line was highly susceptible; the virus spread systemically and caused severe stunting. These plant lines will be useful for distinguishing BNYVV isolates having different pathogenicities, especially those controlled by RNA3 and/or RNA5.     

AFLP analysis of genetic diversity in populations of <Emphasis Type="Italic">Botrytis elliptica</Emphasis> and <Emphasis Type="Italic">Botrytis tulipae</Emphasis> from the Netherlands

The objective of this study was to assess the genetic diversity and to infer the mode of reproduction of Botrytis elliptica and B. tulipae in the Netherlands. First, three molecular typing methods were compared for their ability to differentiate isolates of B. tulipae, B. elliptica, and B. cinerea. The methods compared were multilocus sequencing, restriction analysis of the ribosomal intergenic spacer (IGS) region, and amplified fragment length polymorphism (AFLP) analysis. AFLP fingerprinting provided the most efficient method to differentiate isolates within each Botrytis species and therefore this method was used for population analyses of B. elliptica and B. tulipae. Isolates of both species were sampled during successive growing seasons in experimental field plots in Lisse and other locations in the Netherlands. Among 174 B. elliptica isolates, 105 genotypes could be discriminated and 87 genotypes were found only once, reflecting high genotypic variation. Clonal genotypes were found only within growing seasons and in one location. Linkage disequilibrium analyses indicated that between 9.4% and 19.3% of the loci in clone-corrected samples were linked. The multilocus association index provided no evidence for random mating. We conclude that sexual recombination occurs in the B. elliptica population. Among the 170 B. tulipae isolates, 25 genotypes could be discriminated and four genotypes were found only once, reflecting a low genotypic variation. Clonal genotypes were frequently found in different growing seasons and different locations. Linkage disequilibrium analyses indicated that between 25.2% and 48.6% of the loci in clone-corrected samples were linked. We conclude that the B. tulipae population is mainly clonal with some recombination.