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.     

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.     

Microbial community responses associated with the development of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cucumerinum</Emphasis> after 24-epibrassinolide applications to shoots and roots in cucumber

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FO), is one of the major diseases in cucumber (Cucumis sativus) production. Root and foliar applications of 24-epibrassinolide (EBL), an immobile phytohormone with antistress activity, were evaluated for their effects on the incidence of Fusarium wilt and changes in the microbial population and community in roots of cucumber plants. EBL pre-treatment to either roots or shoots significantly reduced disease severity followed by an improved plant growth regardless of the treatment methods applied. EBL applications decreased the Fusarium population on root surfaces and in nutrient solution, but increased the population of fungi and actinobacteria on root surfaces. PCR-DGGE analysis showed that FO-inoculation had significant effects on the bacterial community on root surfaces as expressed by a decreased diversity index and evenness index, but EBL applications alleviated these changes. Moreover, several kinds of decomposing bacteria and growth-promoting bacteria were identified from root surfaces of FO-inoculated plants and EBL-pre-treated plants, respectively. Overall, these results show that the microbial community on root surfaces was affected by a complex interaction between phytohormone-induced resistance and plant pathogens.     

Seed transmission of<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp.<Emphasis Type="Italic">lactucae</Emphasis>

Twenty-seven seed samples belonging to the lettuce cultivars most frequently grown in Lombardy (northwestern Italy), in an area severely affected by Fusarium wilt of lettuce, were assayed for the presence ofFusarium oxysporum on a Fusarium-selective medium. Isolations were carried out on subsamples of seeds (500 to 1500) belonging to the same seed lots used for sowing, and either unwashed or disinfected in 1% sodium hypochloride. The pathogenicity of the isolates ofF. oxysporum obtained was tested in four trials carried out on lettuce cultivars of the butterhead type, very susceptible to Fusarium wilt. Nine of the 27 samples of seeds obtained from commercial seed lots used for sowing in fields affected by Fusarium wilt were contaminated byF. oxysporum. Among the 16 isolates ofF. oxysporum obtained, only one was isolated from disinfected seeds. Three of the isolates were pathogenic on the tested cultivars of lettuce, exhibiting a level of pathogenicity similar to that of the isolates ofF. oxysporum f.sp.lactucae obtained from infected wilted plants in Italy, USA and Taiwan, used as comparison. The results obtained indicate that lettuce seeds are a potential source of inoculum for Fusarium wilt of lettuce. The possibility of isolatingF. oxysporum f.sp.lactucae, although from a low percent of seeds, supports the hypothesis that the rapid spread of Fusarium wilt of lettuce observed recently in Italy is due to the use of infected propagation material. Measures for prevention and control of the disease are discussed. http://www.phytoparasitica.org posting Dec. 16, 2003.     

Characterization of biotin-autotrophic isolates derived from naturally occurring auxotrophic race 2 isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lactucae</Emphasis>

Race 2 isolates of Fusarium oxysporum f. sp. lactucae have been recognized as biotin auxotrophs and consequently have restricted growth on Puhalla's minimal medium (MM), which contains no biotin. Biotin-autotrophic isolates were raised from race 2 isolates through cultural mutation that grew as well on MM as they did on MM supplemented with biotin. These autotrophs were identical to the parental isolates in pathogenicity on race differential cultivars of lettuce (Patriot, Banchu Red Fire, and Costa Rica No. 4), and thus were designated as race 2. A vegetative compatibility test indicated that the autotrophic isolates fell into the same vegetative compatibility group as the parents. Culture filtrates of the autotrophs allowed abundant growth of the parental auxotroph on MM, and, through a competitive enzyme-binding assay, biotin was detected in the culture filtrates. These results suggest that biotin auxotrophy in the natural race 2 isolates has no direct relation to pathogenicity, qualitatively defined as physiological race, or to vegetative compatibility.     

Selective media for <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Selective media without pentachloronitrobenzene were developed for quantitative assays of Fusarium oxysporum in soils. Media Fo-G1 and Fo-G2 were effective for naturally infested soils, Fo-W1 and Fo-W2 for wild-type isolates in soils containing a nitrate-nonutilizing (nit) mutant, and Fo-N1 and Fo-N2 for nit mutants. Selective media were made using ammonium citrate dibasic, l-sorbose, econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder for soil dilutions of 100-fold or more (Fo-G1, FoW1 and Fo-N1) and 10-fold (Fo-G2, Fo-W2 and Fo-N2). Potassium chlorate was added to Fo-N1 and Fo-N2. The efficacy for selectively isolating F. oxysporum was confirmed using six soils naturally infested with one of six formae speciales of F. oxysporum and with soil dilutions containing conidia of wild-type strains or nit mutants from the six formae speciales. On Fo-G1 and Fo-G2, most colonies of F. oxysporum were compact and round with purplish or reddish pigment in the reverse. Cylindrocarpon sp. formed colonies as large as those of F. oxysporum but were distinguishable by their colony morphology. Other contaminants such as F. solani, F. moniliforme, and Trichoderma were suppressed by medium ingredients and colonies of F. oxysporum. On Fo-W1 and Fo-W2, colony morphology of F. oxysporum and contaminants corresponded to that on Fo-G1 and Fo-G2, although F. oxysporum failed to produce the pigment. On Fo-N1 and Fo-N2, nit mutants formed clear colonies from 100- and 10-fold soil dilutions, respectively, and contaminants seldom formed large colonies.     

Impaired purine biosynthesis affects pathogenicity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis>

The vascular wilt pathogen Fusarium oxysporum f. sp. melonis causes worldwide yield losses of muskmelon. In this study, we characterized a UV-induced non-pathogenic mutant (strain 4/4) of F. oxysporum f. sp. melonis, previously identified as a potential biological control agent. During comparative analysis of vegetative growth parameters using different carbon sources, mutant strain 4/4 showed a delay in development and secretion of extracellular enzymes, compared to the wild type strain. Amendments of the growth medium with yeast extract, adenine or hypoxanthine, but not guanine, complemented the growth defect of strain 4/4, as well as secretion and partial activity of cellulases and endopolygalacturonases, indicating that the strain is an adenine auxotroph. Incubation of strain 4/4 conidia in adenine solution, prior to inoculation of muskmelon plants, partially restored pathogenicity to the mutant strain.     

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.     

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.     

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.     

Visualisation of <Emphasis Type="Italic">hrp</Emphasis> gene expression in <Emphasis Type="Italic">Xanthomonas euvesicatoria</Emphasis> in the tomato phyllosphere

The plasmid pUFZ75 conferred constitutive GFP expression on the bacterial pathogen Xanthomonas euvesicatoria (syn. X. campestris pv. vesicatoria). Colonisation of the tomato phyllosphere and invasion of tomato leaves by X. euvesicatoria was examined using both fluorescence and confocal laser scanning microscopy. Xanthomonas euvesicatoria established a limited population on the tomato leaf surface, primarily occupying the depressions between epidermal cells and around the stomata, prior to invasion of the leaf via the stomata and subsequent growth within the substomatal chamber and the leaf apoplast. Additionally, hrp-gfp fusions were used to report on the temporal and spatial expression of hrp genes during epiphytic colonisation and invasion. Xanthomonas euvesicatoria cells carrying hrpG- and hrpX-gfp reporter constructs were not fluorescent in vitro on non-hrp-inducing LB agar but did exhibit a low level of fluorescence on the leaf surface within 24 h of inoculation, particularly in the vicinity of stomata. Cells carrying hrpG- and hrpX-gfp fusions exhibited high levels of fluorescence 72 h after inoculation in the substomatal chamber and the leaf apoplast. Cells carrying the hrpF-gfp fusion were slightly fluorescent on LB agar and showed no further increase in fluorescence on the leaf surface by 24 h after inoculation, but did show a significant increase in fluorescence 72 h after inoculation in the substomatal chamber and apoplast. The apparent low-level induction of the regulators hrpG and hrpX on the tomato leaf surface may suggest that some of the genes of the X. euvesicatoria HrpG/HrpX regulon are up- or down-regulated prior to invasion of the stomata while still on the leaf surface.     

Cytological events in tomato leaves inoculated with conidia of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f. sp. <Emphasis Type="Italic">hordei</Emphasis> and <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01

Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.     

Screening of cultivated and wild adzuki bean for resistance to race 3 of <Emphasis Type="Italic">Cadophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis>, cause of brown stem rot

Two diseases of adzuki bean, brown stem rot (BSR, caused by Cadophora gregata f. sp. adzukicola) and adzuki bean Fusarium wilt (AFW, caused by Fusarium oxysporum f. sp. adzukicola), are serious problems in Hokkaido and have been controlled using cultivars with multiple resistance. However, because a new race of BSR, designated race 3, was identified, sources of parental adzuki bean for resistance to race 3 were needed. Therefore, we examined 67 cultivars and lines of cultivated and wild adzuki bean maintained at the Tokachi Agricultural Experiment Station using a root-dip inoculation method. Consequently, nine adzuki bean cultivars, one wild adzuki bean accession and 30 lines (including two lines resistant to all the three races of BSR and AFW) were confirmed to be resistant or tolerant to race 3 of BSR, and we found a cultivar Akamame as well as a wild adzuki bean Acc2515 to be a new source for a resistance gene to the race 3. This cultivar also holds promise as a source of resistance against other races of BSR and AFW.     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.     

Infectivity of a Japanese isolate of <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01 to a European tomato cultivar resistant to <Emphasis Type="Italic">O. lycopersici</Emphasis>

The infectivity of a Japanese isolate of tomato powdery mildew, Oidium neolycopersici KTP-01, to tomato cultivars was examined using a resistant cultivar Grace bred in The Netherlands to O. lycopersici, which was recently proposed to be renamed O. neolycopersici. Grace was severely infected with KTP-01, and its susceptibility was similar to that on susceptible tomato cultivars Moneymaker and Ponderosa, suggesting that KTP-01 differs in pathogenicity on tomatoes from those of European and American isolates.     

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.     

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     

Genetic diversity and pathogenicity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolated from wilted Welsh onion in Japan

Thirty isolates of Fusarium oxysporum from wilted Welsh onion plants were examined for their diversity in nucleotide sequences of the ribosomal DNA (rDNA) intergenic spacer (IGS) region and for pathogenicity with regard to five Welsh onion cultivars. Phylogenetic analysis based on the IGS sequences revealed polyphyletic origins of the isolates and a relationship between phylogeny and pathogenicity; low virulence isolates differed genetically from those with high and moderate virulence. Mating type analysis revealed that all F. oxysporum isolates were MAT1-1 idiomorphs, suggesting that the pathogens may be clonal in the fields examined.     

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.