A monoclonal antibody (MoAb) API9-2, which reacts specifically to members of the genus Fusarium, was tested for serological detection of F. oxysporum in plant tissue. Mycelia of F. oxysporum in infected stems, crowns, and roots of tomato and Japanese honewort plants were effectively detected by immunofluorescence assay (IFA) with the combination of MoAb API9-2 and FITC-conjugated anti-mouse IgM-sheep IgG indicating that IFA is a useful tool for the observation of behavior of Fusarium spp. in plant tissue. As another immunological assay with this MoAb, direct tissue-blotted immunobinding assay (DT-IBA) was applied to transverse sections from stems or crowns of tomato and cucumber plants. In this assay, samples of plants infected with F. oxysporum showed positive reaction within 4hr. In addition, the pathogen residing in vessels of infected, but symptomless plants was observed within 14 to 21 days after inoculation. Thus, DT-IBA is thought to be a practical method for quick and accurate diagnosis of Fusarium spp.

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By combining a simple ELISA with a newly developed, effective two-step incubation method using a selective medium, S-PC-1, inspection of Pseudomonas cepacia (Pc) which is antagonistic to plant pathogens could be detected specifically from roots or rhizosphere soil of 6 kinds of plants collected from several field sites. When the suspension prepared from root or rhizosphere soil was spread onto S-PC-1 plates and incubated, first at 40°C for 4 days, followed by 28°C for 3 days, bacterial colonies, presumably Pc, were detected almost at the same concentration at ca. 10³-10⁴cfu per gram from all tested plants of soybean, barley, Chinese cabbage, lettuce and tobacco. Samples from Welsh onion contained a larger number of bacteria at ca. 10³-10⁶cfu/g. Combining heat treatment of samples with the direct ELISA was useful for detecting a low number of Pc at a concentration of 10²-10³cfu/g. More than 80% of bacterial isolates randomly selected from the S-PC-1 medium reacted with the antiserum specific to Pc. On the other hand, 80-100% of bacterial isolates from rhizosphere samples which showed a serologically positive reaction grew on S-PC-1 at 40°C. Forty-one isolates thus selected were identified as Pc on the basis of pathogenicity to onion and bacteriological properties. They were characterized by antagonistic activity in vitro toward bacterial and fungal plant pathogens, such as Rhizoctonia solani (AG-4), Phytophthora capsici, P. solanacearum and so on. They were also suppressive to damping-off of radish seedlings caused by R. solani (AG-4) by seed bacterization.

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Collapse of melon with brown-rotted and corky roots occurred in Shizuoka Prefecture, Japan in 1975. This disease was named root rot of melon by Sato et al. in 1976. Nodulisporium melonis Watanabe & Sato (1995) was predominantly isolated from rotted root tissues together with five other fungus genera. In inoculation tests, N. melonis was pathogenic to melon seedlings, but other fungal isolates were not pathogenic. Melon and five other cucurbitaceous crops tested were diseased by growing in the artificially infested soil with N. melonis, but healthy in uninfested soils. This fungus was not pathogenic to wax gourd, and other 18 crops inoculated.

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Nodulisporium melonis sp. nov., the causal fungus of root rot of melon in Japan is described and illustrated. Colonies of this fungus on PDA are white and homogeneous, subhyaline, cream to pale brown in reverse. The conidiophores are erect, hyaline, simple or branched verticillately or irregularly, 45-280×1.2-2.3μm, bearing single conidia or apical spore masses, 2-9 conidia per head on apical fertile portions, denticulate after detachment of conidia, often proliferated from conidia on conidiophores. Conidia are single, in short chains or aggregated, sympodulosporous, acropleurogenous, hyaline, 1-celled, obovoid, cylindrical, elliptical or irregular in shape, 2.5-13.8×1.2-3.0μm (5.9×2.5μm) with basal frill or cylindrical appendix, 3.0-10.0×0.4-1.0μm.

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Ice-nucleating active fungus isolated from the gut of larvae of the rice stem borer, Chilo suppressalis Walker was identified as Fusarium moniliforme Sheldon var. subglutinans Wollenweber et Reinking. Ice-nucleating activity (INA) around -5°C was detected in the mycelial suspension and cell-free culture medium of the fungus. INA was also detected in two strains of F. moniliforme, two of F. avenaceum and one of F. tricinctum among the 37 Fusarium strains tested. The activity of these five strains was similar to that of F. moniliforme var. subglutinans. However, F. moniliforme and F. avenaceum ceased producing ice nuclei within a short time after subculture in liquid medium.

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Alternaria rot of okra pods caused by Alternaria alternata was described as a new post-harvest disease. The disease was frequently found in the market place of Japan. The optimal temperature for the mycelial growth of the fungus was more than 20°C. But expression of the disease symptoms were greatly promoted at low temperatures and became evident after prolonged exposure at 10°C in the laboratory. Chilling injury or “Alternaria rot” of okra pods which had previously recorded in the literature might be the same symptoms as seen in this study.

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Infection sources of the post-harvest development of Alternaria rot of okra pod were investigated. In the field, Alternaria alternata attacked old leaves of okra plants and produced numerous conidia on dying or dead tissues. These conidia are considered to be major sources of airspora in okra fields. Some isolates of A. alternata from Chinese radish and a Japanese pear and one isolate of A. brassicicola from Chinese radish seed also produced same disease symptoms. Typical symptoms developed on okra pods which were inoculated in the field after harvesting and storaging at 10°C.

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Chromosome-sized DNAs of Fusarium species were prepared for pulsed-field gel electrophoresis without first generating protoplasts. The technique involves pre-treatment of intact, agarose-solidified microconidia with endo-β-1, 3-glucanase before treatment with protease in the presence of EDTA and SDS. By using four different electrophoretic conditions for the separation of four size ranges of chromosomes, we have succeeded in separating the chromosomes of 6 formae speciales (lycopersici, radicislycopersici, melonis, cucumerinum, fragariae and spinaciae) or Fusarium oxysporum in an agarose gel matrix by crossed field gel electrophoresis. The total number of chromosome-sized DNA molecules of these fungi varied from 9 to 15, depending on the formae speciales and, in some case, individual strains. The sizes of chromosomes ranged from 0.8 to approximately 7.4Mb which gave estimates of genome size of between 36.0 and 56.3Mb.

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The reports about negation of mycotoxin productivity of Microdochium nivale (syn. Fusarium nivale) led us to reinvestigate Japanese isolates of M. nivale. Thirty isolates were collected from different district of Japan. These were from 5 species of gramineous plants in 1988-1993 and analyzed on their mycotoxin productivity within 2 years. Trials were conducted for combinations of medium (rice and wheat grain) with culture temperature (0°C and 20°C). In addition, we examined samples inoculated artificially on cut-spikes of winter wheat and field samples of orchardgrass infested with M. nivale. As a result, we did not detect the known mycotoxins (deoxynivalenol, 3-acetyldeoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, neosolaniol, zearalenone) in all trials. Mycelial growth temperature of Fusarium sp. strain Fn-2B, from which nivalenol was firstly isolated, was higher than that of M. nivale. Fn-2B was not pathogenic to winter wheat under snow.

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Soybean leaves artificially inoculated with basidiospores of Thanatephorus cucumeris (AG-2-3 of Rhizoctonia solani) developed at first, primary lesions, then, secondary lesions and eventually, irregular large-sized lesions. The germ tubes of the germinating basidiospores formed appressoria and their infection pegs penetrated into the epidermal cells. A stroma-like structure was produced within the invaded tissues. Hyphae growing radially from this structure formed circular necrotic spots (≤1mm diam.) which corresponded to the primary lesions. After further incubation in a moist chamber, hyphae from the primary lesions continued to grow on the leaf surface and hyphal tips re-entered the leaves through the stomata, causing secondary lesions with circular to irregularly shaped areas of necrosis around the primary lesions. The large-sized irregular lesions developed from additional hyphal infection through stomata. Many fruiting hymenia of the pathogen were detected on the stems of winter wheat or soybean, especially in fields where soybean was intercropped with winter wheat. The severity of the soybean foliar blight lesions caused by basidiospores increased with the delay in wheat harvest or with the increase of the soybean planting density.

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Effects of the elicitor and the suppressor from a pea pathogen, Mycosphaerella pinodes, on the H⁺-translocating activity of ATPase in pea plasma membranes were examined. The plasma membrane ATPase was solubilized with Triton X-100 and partially purified by continuous glycerol density gradient centrifugation. The ATPase fraction was reconstituted into soybean phospholipid (asolectin) liposomes by a Triton-X 100 dilution method. Almost all of resultant proteoliposome vesicles had unimembranes in size from 50 to 200nm. The ATP-driven H⁺-pumping activity was measured by quinacrine fluorescence quenching in the presence of Mg²⁺-ATP. The activity was sensitive to orthovanadate, dicyclohexylcar-bodiimide (DCCD), verapamil and neomycin but it was insensitive to azide and nitrate as well as ATP-hydrolyzing activity. Proton gradient was collapsed by NH₄Cl or a channel-forming ionophore, gramicidin D. The H⁺-pumping activity in proteoliposomes was hardly affected by the elicitor. The finding suggests that a putative target molecule of or receptor for the elicitor might not associated with the solubilized ATPase or that the elicitor could not reach the target site. However, the suppressor markedly inhibited both activities in proteoliposomes, showing that the H⁺-translocating ATPase might be inhibited directly in vitro by the suppressor as reported previously and that the fungal suppressor may disturb the regulation of pH in the host cells. Both activities were increased by the addition of phosphatidylinositolbisphosphate, even if in the presence of the suppressor. However, PIP₂ could not completely negate the effect of the suppressor. These results suggest that the H⁺-pumping activity of plasma membrane ATPase is also regulated by polyphosphoinositide metabolism and that the action sites of the suppressor on the ATPase may be different from those of PIP₂.

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A representative strain of Pseudomonas syringae pv. eriobotryae, the causal agent of stem canker of loquat, was studied to determine whether plasmids are involved in its virulence. The strain NAE6 harbored three plasmids of approximately 25, 52, and 60 megadaltons (Mdal). The derivative strains which were cured of the 52 Mdal plasmid by culturing at a maximum growth temperature of 32°C were uniformly avirulent. The 52 Mdal plasmid DNA, which was isolated from agarose gels using agarase, was digested with BamHI, ligated into the BamHI cloning site of the broad host range cosmid pLAFR3, packaged into λ phage particles, and transduced into Escherichia coli HB101. By use of helper plasmid pRK2013, twenty-five recombinant plasmids were mobilized into an avirulent recipient P. s. pv. eriobotryae strain, which was cured of three plasmids. The transconjugants which received the recombinant plasmid pVIR6 containing ca. 23kb insert DNA regained virulence. Southern hybridization analysis indicated that the 23kb insert DNA originated from the 52 Mdal plasmid. These data demonstrate that the 52 Mdal plasmid of P. s. pv. eriobotryae is associated with virulence.

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Return-PAGE was reliable for diagnosis of HSVd in hop, CEVd in citron and ASSVd in apple, but was not reliable for HSVd in grapevine and citron, and ASSVd in pear, because of their low concentration. A disadvantage of return-PAGE was possible appearance of double or triple bands in case of mix-infecting samples such as hop or citron. Although DIG-labeled DNA probe was 2.5 to 25 times more sensitive than return-PAGE, the practical reliability of this method was not so superior to return-PAGE. DIG-labeled RNA probe was the most sensitive of the three methods examined in this experiment, which was 25-125 times and 100-625 times more sensitive than DIG-DNA probe and return-PAGE, respectively. Furthermore, DIG-RNA probe was sensitive enough to detect HSVd in grapevine, a symptomless host of the pathogen, although negative sample was emphasized to be re-examined by the other sensitive method such as cucumber bioassay or RT-PCR. DIG-RNA probe, however, is not sensitive enough to detect HSVd in citrus and ASSVd in pear. Based on the results obtained in this experiment, we proposed appropriate amounts of plant tissues for the successful diagnosis of the three viroids.

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[in Japanese]

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Biological mode of action of fluazinam (Frowncide^®) against clubroot of Chinese cabbage was studied. Germination of resting spores was inhibited in a suspension by fluazinam application at 1ppm or more. Root hair infection decreased when the soil was inoculated with resting spores previously incubated in a suspension with fluazinam. Both root hair infection and club formation were greatly inhibited in the presence of fluazinam in the inoculated soil. Fluazinam inhibited club formation when working in the soil until the stage of root hair infection, whereas it had no effect after cortex infection. These results suggest that fluazinam possesses fungicidal action to resting spores and inhibits root hair infection and cortex infection, thereby leading to the inhibition of club formation.

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Sensitivity of bean gray mold fungus collected in several districts of Hokkaido to fluazinam and other botryticides was investigated. Three hundred and two isolates of Botrytis cinerea were obtained from diseased pods of kidney bean or adzuki bean collected in 1990 and 1991. Inhibition of mycelial growth on PSA medium and preventive activity on kidney bean of fluazinam, procymidone, thiophanate-methyl and diethofencarb were measured. Difference in sensitivity to fluazinam was not observed among isolates tested; EC₉₅ value of mycelial growth inhibition and minimum effective concentration of preventive activity were less than 1ppm and less than 31ppm, respectively. There were considerable differences in the sensitivity to procymidone and thiophanate-methyl. Resistant isolates to procymidone and/or thiophanate-methyl which could cause the decrease of control efficacy at practical dosages existed. Furthermore, fluazinam showed high antifungal activity against isolates which were resistant to procymidone and/or thiophanate-methyl similarly as sensitive isolates. These findings suggest that fluazinam is effective against bean gray mold resistant to conventional botryticides.

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Seeds obtained from rice plants, which had been sprayed with oxolinic acid (5-ethyl-5, 8-dihydro-8-oxo [1, 3] dioxolo [4, 5-g] quinoline-7-carboxylic acid, Starner^®) at the concentration of 200μg/ml at heading time, were selected with a solution of ammonium sulfate with the specific gravity of 1.13 and were dipped in a solution of oxolinic acid at the concentration of 1000μg/ml for 24hr. Rice plants raised from these seeds were sprayed with oxolinic acid at heading time. This control system was highly efficacious in the control of bacterial seedling rot and bacterial grain rot of rice caused by Pseudomonas glumae. And populations of P. glumae on spikelets and grains decreased remarkably. These results demonstrated that this control system had a great influence on infection cycle of P. glumae.

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Cucumber mosaic virus (CMV) occurred widely in maize (Zea mays) and also rarely in teosinte (Euchlaena mexicana) in Kanto area, Japan. Isolates from maize plants were classified into two types (I and II) on their biological and serological properties. Isolate II, but not isolate I, infected to barley and wheat plants by sap inoculation. It suggested that the isolate I is closely to leguminous strain, but isolate II (tentatively named maize strain) may be a previously undescribed strain, on their host range and symptomatology. Isolates from teosinte plants appeared to be related to yellow (Y) strain on tabacco symptoms. We propose to designate teosinte necrotic mosaic for the disease.

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