A serious stem blight disease of white trumpet lily occurred in Okino-erabu island, Kagoshima Prefecture, Japan. The symptoms were characterized by browning of stem and leaves, stem rot and wilting of whole plants. This was ascertained to be Phytophthora disease by the immunofluorescent method and ELISA. Pathogenicity of the fungus isolated to white trumpet lilies was proved by artificial inoculation. The fungus was re-isolated from wilted plants and identified as P. megasperma on the basis of its morphology and physiology. This is the first report of the disease of lily by P. megasperma in Japan and elsewhere.

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Production and diagnostic utility of monoclonal antibody (MCA) to zucchini yellow mosaic virus (ZYMV) in enzyme-linked immunosorbent assay (ELISA) are described. Four selected hybridomas produced MCA of the IgG_2b subclass, and the titre of these MCA in ascitic fluid ranged between 10⁷ and 10⁹ which were 1, 000 times higher than those in culture fluid. Of the six procedures of ELISA tested, the most sensitive result was obtained when the plates were coated with MCA and rabbit polyclonal antibody (PCA) was used as intermediate antibody (procedure 5). This assay was able to detect ZYMV in purified preparation at detection end point of 0.11ng/ml and in crude extracts of pumpkin leaves at dilution end point of 10⁶10⁷. The use of MCA ZYMV-45 in non-precoated I-ELISA could discriminate ZYMV and watermelon mosaic virus 2 (WMV-2). Thus, the result represented a major advance in the detection of ZYMV which the use of PCAs in the same assay could not discriminate these two viruses. This assay gave also strong positive reactions with all of ZYMV-infected samples that reacted with PCA in double-antibody sandwich ELISA, thereby providing specific diagnostic tool for ZYMV which is needed for guiding the management of potyviruses on cucurbits.

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A bacterial wilt of statice (Limonium sinuatum Mill.) plants was observed in Kochi Prefecture in 1985. Affected leaves showed unilateral chlorosis or entire wilting. The midvein and lateral veins often turned to pronounced red color in localized portions of affected leaves. The disease occurs severely in the hot season but not in winter. A non-fluorescent, aerobic, Gram-negative bacterium was isolated from the diseased plants. The bacteriological characteristics of 25 isolates of the bacterium coincided with those of the type strain and reference strains of Pseudomonas caryophylli which were isolated from carnations. The bacterium as well as reference strains attacked statices and carnations by artificial inoculations and it reproduced the similar symptoms to those of the natural infections. On the basis of bacteriological characteristics and pathogenicities, the bacterium was identified as Pseudomonas caryophylli (Burkholder 1942) Starr and Burkholder 1942. Bacterial wilt of statice was proposed for the name of the disease.

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The isolation and structural determination of fungitoxic compounds from roots of eggplant stocks, Taibyo VF, were performed. Three sesquiterpenes (solavetivone, lubimin and epilubimin) and five phenolic compounds [vanillin, ethyl ferulate, ethyl p-coumarate, ethyl caffeate, p-hydroxybenzoic acid, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone] were isolated. The content of solavetivone in the stock was about five times that of a eggplant.

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Linkage analysis was carried out on Kuroka blast resistance with pedigrees of Kamenoo of la, lg isogenic line×Kuroka of glume colored with Pr and C. An individual F₃ line was raised from each F₂ without practicing any selection for a particular trait. Selecting homogeneous genotype for ligule, 150 F₄ lines were promoted as follows; two lines from each F₃ of 69 lines and one from each of 12. Existence of a close linkage between lg⁺ and one of Kuroka's resistant genes designated as Pi-kur 1 was confirmed among F₂ by the spray inoculation method and its recombination value was estimated as 7.8% and 10.3%. The present gene was ascertained to be the same as the one previously studied by the translocation analysis. The close relation above mentioned was distinctly shown among F₄ lines, too. As 32.4% of lglg lines, 25.4% of susceptible lines, 48.7% of lg⁺lg⁺ lines and 51.7% of resistant lines belonged to colored glume ones, Pi-kur 1 was assumed to locate between lg and Pr, and very nearly Ph. By the sheath inoculation method, F₂ plants revealed a segregation of two genes. One designated as Pi-kur 2 is commonly effective to used isolates and closely linked with la and the other is a complementary gene particular to each isolate. Pi-kur 2 participated not so much part as Pi-kur 1 in resistance on nursery bed, but its additive effects reached at significant level. There were some F₄ lines evidently more resistant than others among the group susceptible to spray inoculation, and so it was reasonably understood that the breakdown of Kuroka's “rr” grade of field resistance in Japan was limited to intermediate “m” in Philippines.

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A fluorescence microscopic method was developed to directly assess the pathogenic activity of resting spores of Plasmodiophora brassicae. Resting spores were stained with a mixture solution of two fluorochromes, calcofluor white M2R (CFW) and ethidium bromide (EB), and were observed by fluorescence microscopy (phase contrast, UV filter set, oil immersion). All the spores exhibited intense blue fluorescence on the wall layer, and some spores displayed red fluorescence in the cytoplasm. Spores could be distinguished into two groups, spores with a non-fluorescing cytoplasm (blue spores) and spores with a red fluorescing cytoplasm (red spores). The fluorescent staining reaction of the spores was not affected by a staining period of less than 4hr. However, when the concentration of EB solution equally mixed with CFW solution was increased, an increase in the percentage of red spores was observed. Suitable conditions for the differentiation between blue and red fluorescence corresponded to a concentration of 10 to 50μg/ml of the EB solution using 100μg/ml of CFW solution. When spores were heated for 72hr at 40C, 50C or 60C, the percentage of blue spores was constant at 40C, but decreased both at 50C and at 60C with the time of incubation, suggesting that the blue spores are active spores. Disease severity in the plants infected with heat-treated spores was closely related to the percentage of blue spores among the treated spores. This correlation was also observed for the spores stored for different periods of time. The results obtained indicate that the pathogenic activity of the resting spores can be assessed by examining the fluorescent staining reaction of the spores. In this study, the variations in the percentage of blue spores were generally less pronounced than those in the disease severity. This method is rapid, convenient and precise.

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Effects of a fungicide, thiophanate methyl (Topsin-M) on the incidence of akakabi-disease (scab) and the contamination of trichothecene mycotoxins, nivalenol (NIV) and deoxynivalenol (DON) in wheat and barley were examined in 1982, 1983 and 1987 on the basis of three criteria: the damage of the disease in fields, Fusarium-infected grains and the mycotoxin levels in the harvested cereal grains. Application of Topsin-M twice at earing (April 15 to 22) and flowering (April 22 to 27) periods of the crops resulted in the remarkable decrease in not only the damage of the disease but also the contamination levels of the mycotoxins. The level of DON in the grains was highly correlated to that of NIV regardless of the application of Topsin-M, suggesting that the fungicide may equally inhibit the infection of F. graminearum producing either NIV or DON. Although the mycotoxins were found in harvested grains even when no incidence of the disease had been observed in fields, the co-occurrence of the toxins was efficiently depressed by the fungicide.

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When grapefruit cutin was added to spore suspension of Alternaria alternata Japanese pear pathotype, significant cutinase activity was observed in the spore-germinated fluid. Cutinase activity was also detected in cutin-containing liquid medium, but not in sucrose-containing medium. Cutinase was produced not only by virulent isolates of this pathotype, but also by the avirulent mutants and saprophytic A. alternata in their spore-germinated fluids. Several organophosphorus pesticides which strongly inhibited cutinase were found to have a strong inhibitory effect on lesion formation on pear leaves by the fungus. The cutinase in culture filtrate of the fungus was precipitated by ammonium sulfate, and the precipitate was chromatographed by gel filtration, chromatofocusing and cation exchange columns. The purified fraction expressed clear cutinase activity and appeared as a single protein band with a molecular weight of 32, 000 daltons on SDS-PAGE. These data suggested that cutinase production, in addition to AK-toxin production, is essentially required for infection and disease development by this fungus.

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The secretory activity of Golgi bodies in Japanese pear leaves treated with AK-toxin was examined with an electron microscope by staining sections with alkaline bismuth stain as a tracer of Golgi vesicles to discern the viability of the toxin-treated susceptible cells. The toxin caused the invaginations of plasma membranes and the loss of abundant membranous fragments from the invaginated plasma membranes in the susceptible cells. In the susceptible cells treated with the toxin for 1, 3, and 6hr, many Golgi vesicles which were positive to alkaline bismuth stain were present at the peripheral cytoplasm beneath invaginated plasma membranes and some of them fused with the invaginated plasma membranes. The accumulation and fusion of the Golgi vesicles were rarely found in cells of the necrotic susceptible leaves treated with the toxin for 10hr. However, the phenomena were not observed in the toxin-treated resistant cells and the water-treated susceptible ones. The susceptible cells were viable within 6hr after the toxin treatment because the cells had the high activity of Golgi bodies and the high ability of plasma membranes to fuse with many Golgi vesicles. On the contrary, the susceptible cells appeared not to be viable 10hr after the treatment because they had both the low activity of Golgi bodies and the poor ability of plasma membranes. The results indicate that the enhanced production of Golgi vesicles may be associated with the viability of cells in the toxin-treated susceptible leaves, and that the produced Golgi vesicles may be supplied for repairing the damaged plasma membranes.

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Lethal and mutagenic effects of four kinds of mutagens [N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), UV light (UV) and γ-ray] on Cochliobolus heterostrophus were studied. In MNNG and UV mutageneses, the number of surviving conidia decreased linearly as the dosage increased, but in EMS and γ-ray mutageneses, the number of survivors decreased non linearly. In γ-ray mutagenesis, survival of the conidia did not depend on the total dose but on the dose rate. Four different color mutants (white, salmon, brown and pale green) were obtained. The maximum frequency of color mutants among the survivors was 0.51% at 5.5% survival in MNNG treatment, 0.43% at 1.4% survival in EMS treatment, 0.05% at 13.2% survival in UV treatment and 0.07% at 2.6% survival in γ-ray treatment.

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A novel transcription vector, pUT118 which starts transcription to be directed to the natural 5' end of viral cDNA insert was constructed. After cleavage at a Xba I site just downstream of the cDNA insert, in vitro transcription can be performed by T7 RNA polymerase to produce full-length copies of viral RNAs. Biologically active CMV satellite RNA (Y strain) was successfully synthesized by inserting complete cDNA copy into a unique Nsi I site in pUT118 and transcribing the clone. To overcome the low transcription efficiency due to the modified T7 promoter in pUT118, pUT118GG was also created from pUT118 to add two extra G residues to the 5' end of the transcripts, allowing high efficiency of transcription initiation of the cDNA insert.

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The complete nucleotide sequence of cucumber mosaic virus RNA3 of Y strain has been determined and was compared with the reported sequence of RNA3 of Q strain. The sequence of Y strain provided the amino acid sequences of two proteins: the 5'-terminal 3A protein with 279 amino acids and the 3'-terminal viral coat protein with 218 amino acids, while 3A and coat proteins of CMV-Q have been reported to have 333 and 236 amino acids, respectively. The 3'-terminal region of Y strain can be arranged into the secondary structures similar to those proposed for that of Q strain, although there are considerable differences in sequence.

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In assaying soil samples from 35 locations (610 samples/Prefecture) in Shikoku Island for Pythium species by a trapping isolation method with cucumber seeds as a baiting substrate, one to four taxa/sample were isolated from 33 samples, but Pythium was not isolated from two samples from Kagawa Pref. A total of 335 isolates were identified into 12 taxa including H-Zs, a provisional Pythium group with zoospore formation from hypha-like sporangia, but without sexual organs. Among these fungi, 134 isolates of P. sylvaticum obtained from 29 locations were the most dominant, followed by P. ultimum and P. spinosum.

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Since 1975, a new root rot disease of dasheen has been occurring at the various nursery beds in northern Chiba Prefecture. The disease was severe in high humidity and soil temperature. The infected plant with decayed roots was stunted, but its tubers remained relatively healthy. Although Pythium myriotylum and Rhizoctonia spp. were isolated from the infected roots, only Pythium myriotylum was pathogenic.

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[in Japanese]

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[in Japanese]

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A selective medium for Corynebacterium michiganense pv. michiganense (SMCMM) was developed. The composition of the medium was as follows: peptone 5g, yeast extract 3g, K₂HPO₄ 2g, KH₂PO₄ 0.5g, MgSO₄⋅7H₂O 0.25g, glycerol 20g, LiCl 5g, K₂Cr₃O₇ 80mg, NaN₃ 2mg, nalidixic acid 20mg, cycloheximide 40mg, 70% tetrachloroisophthalonitrate hydrate 3mg, agar 15g in one liter of distilled water. Recovery of C.m. pv. michiganense on SMCMM from the pure culture was higher than on D₂ medium by Kado et al. and CNS medium by Gross et al. C.m. pv. michiganense grew well on SMCMM and developed to the characteristic fresh yellow colonies. The colony forming efficiency of the general microorganisms on the tomato leaves and soil were very low compared with nutrient agar medium.

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Two hundreds and fourteen isolates of Phytophthora nicotianae var. parasitica collected from rotted roots of strawberry seedlings on nursery beds in Saga prefecture were tested on the mating types, A¹ or A², by formation of sexual organs. Among them, 54 isolates were identified as type A¹ and the others were as type A². From the result of the mating type test, it was found that all isolates were heterothallic. The isolates from Saga city·Saga-gun, Kanzaki-gun and Kishima-gun were separated into type A¹ and A², whereas those from Karatsu city, Ogi-gun and Tosu city·Miyaki-gun were either type A¹ or A².

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[in Japanese]

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[in Japanese]

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