The changes in the activities of enzymes and proteins that may be involved in localized reactions of cucumber cotyledons to tobacco mosaic virus (TMV) were investigated. The peroxidase (PO) and ribonuclease (RNase) activity of the cotyledons increased with the progress of TMV infection, while no increase was observed in plants systemically infected with cucumber green mottle mosaic virus (CGMMV). A marked increase in PO and RNase activity was also observed in cotyledons in which TMV localization had been weakened by actinomycin D (AMD) treatment. In this case the profiles of peroxidase-acidic isozymes and RNase-acidic isozymes did not change. Little polyphenoloxidase activity was detected in TMV-infected cucumber cotyledons. These enzymes thus do not play an active role in the localization process of TMV. When cucumber cotyledons were infected with tobacco necrotic virus or treated with salicylic acid, two pathogenesis-related proteins (PRps) detectable by electrophoresis were induced in the cotyledons. These two proteins were not detected in the cucumber cotyledons infected with TMV. When the two PRps were induced by salicylic acid treatment of cucumber cotyledons, little change was observed in the amount of TMV and in the diameter of starch-lesions. Therefore the two PRps may not be related to the localization of virus. New induced protein bands (82 kDa and 86 kDa) were detected 24 hr after TMV-inoculation. These proteins disappeared when TMV localization was weakened by AMD treatment or by prior inoculation with zucchini yellow moxaic virus (ZYMV). Thus these proteins seem to be directly related to TMV localization in cucumber cotyledons.

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Sixty six strains of Pseudomonas solanacearum from 9 different host plants were tested for bacteriocin production using the same strains as indicator. Fifty percent of them produced bacteriocins in CPG agar medium but only several producer strains released detectable bacteriocin in liquid medium. The bacteriocins produced by potato strain POPS 8409 was heat labile, resistant to trypsin and sedimentable by centrifugation and can be observed as phage-tail like particles under electron microscope. These results indicated that this bacteriocin belonged to the sedimentable and high molecular weight group.

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Four polyclonal antibodies were prepared against 4 strains of Xanthomonas campestris pv. oryzae. Sixty-three strains, among which 58 came from China and 5 from International Rice Research Institute, were divided into 4 serovars (I, II, III and IV) by means of Ouchterlony double diffusion test and immunoelectrophoresis with these antibodies. Fifteen hybridoma cell lines which stably secrete monoclonal antibodies were produced through fusion of mouse myeloma cells (SP2/OAg14) and spleen cells derived from BALB/c mice immunized with whole cells of X. campestris pv. oryzae strains KS-6-6, OS-213, YZ-32 and YZ-34. The monoclonal antibodies produced by the selected 4 representative hybridoma cell lines differentiated 6 epitopes of this bacterium. By combination of these epitopes, the serovar I and II were further differentiated into 7 subserovars and 63 strains of X. campestris pv. oryzae were consequently grouped into 9 reaction types consisted of 4 serovars and 7 subserovars.

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One of 88 strains of Xanthomonas campestris pv. oryzae harboured a covalently closed circular DNA (cccDNA) of 7.1 kb and released a filamentous phage into culture fluid. This strain M8883 easily lost the 7.1 kb cccDNA by subculturing five times, and the cured strains lost phage productivity. The filamentous phage released from strain M8883 was different from Xf and Xf2 phages in host range, particle length and molecular size of the replicative form DNA (RF), and was designated Xf8883. The electrophoretic pattern of the 7.1 kb cccDNA digested with HinfI or MspI was identical to that of Xf8883-RF isolated from M8819 infected with Xf8883. Furthermore, strain M8819 transformed with the 7.1 kb cccDNA acquired the productivity of a filamentous phage which was identical with Xf8883. From these results, it is concluded that the 7.1 kb cccDNA may be RF of a new filamentous phage, Xf8883. Electrophoretic analysis of the HaeIII, RsaI and HinfI fragments of Xf8883-RF and Xf-RF suggested that the two phages, Xf8883 and Xf, were closely related but not identical.

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Water-soluble, extracellular materials from germlings of Erysiphe pisi (a nonpathogen of barley) suppressed infection by E. graminis (a pathogen of barley) in barley coleoptiles by about 50%. The suppressive activity was associated with a low molecular weight, heat-stable moiety. The active preparation was separated into three fractions each having suppressive activity. One fraction suppressed infection by E. graminis by about 95%. Treatment of coleoptiles with various concentrations of the fractions prior to, or after inoculation with E. graminis suggested that at least 6 hr was required before suppression of infection was measurable. The effect was diminished within 10 hr after it reached a level sufficient to suppress the infection by E. graminis by about 50%. The results suggested that water-soluble extract of E. pisi either enhanced inaccessibility of coleoptile cells (elicitor activity) or directly affected penetration by E. graminis.

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The localization of coat protein and putative viral replicase proteins (130 kDa and 180 kDa) of tobacco mosaic virus (TMV) in tobacco protoplasts was examined at different post-inoculation intervals, using specific antibodies and protein A-gold complex. Gold labels specific to antiserum against virus particle were first detected 4 hr postinoculation (p. i.) in small areas of the cytoplasm, where virus particles were not initially observed. At 6-15hr p.i. when virus multiplication was active, virus particles were accumulated and formed a paracrystalline array in the cytoplasm. Specific labels were found only on the crystalline aggregates and in the cytoplasm near the aggregates. Many virus aggregates were observed in each section of protoplasts at this stage. At 24 hr p.i., larger aggregates of virus particles were heavily labelled, while areas of the cytoplasm, where no virus particles were evident, were labelled scarcely. Nuclei, plastids and mitochondria were free of labels throughout the infection stages. Gold labels for 130 kDa protein were first detected on small TMV-specific inclusions in the cytoplasm at 4 hr p.i.: Thereafter, these inclusions grew both in number and size, and occurred frequently in the vicinity of virus aggregates. Labels were localized on the inclusions but not on virus aggregates throughout the infection stages. Those inclusions have dense labels and grew a little further in size, though virus replication at this stage declined to a low level. Gold labels for 180 kDa protein on the inclusions and other parts of protoplasts at each stage were observed similarly as the labels for 130 kDa protein. These observations indicate that replication of genomic RNA, synthesis of coat protein and subsequent virus assembly do take place at limited areas in the cytoplasm, where virus particles appeared and formed crystalline aggregate at later stages.

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The T-DNA region of the root-inducing (Ri) plasmid (pRi1724) in the mikimopine type Agrobacterium rhizogenes strain MAFF03-01724 isolated in Japan was detected and characterized by means of Southern blot hybridization with the T-DNA probe of the plasmid pRiA4b in the agropine type A. rhizogenes strain A4. Although the homologous sequences with TL-DNA of pRiA4b was found on pRi1724, no homologous sequence with TR-DNA was found. The presence of pRi1724 sequences was demonstrated in the genome of Ajuga reptans hairy roots by the infection with strain1724. The T-DNA of pRi1724 was mapped by means of cloned BamHI partial digests. The homologous sequences with virulence region of pRiA4b was also found.

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Histochemical reactions in root tissues of Japanese radish inoculated separately with two isolates of Peronospora parasitica were studied. The root tissues inoculated with Japanese radish downy mildew fungus (Jr isolate) showed little resistant reaction 24 hr after inoculation and well grown hyphae were found in the tissues. In the tissues inoculated with shepherd's purse downy mildew fungus (Sp isolate), developed haustorium formed by the Jr isolate was not found at all. Instead, small haustoria and many hypha-like structures (HLS) appeared in the tissues. Development of the haustorium and HLS stopped 24 hr after inoculation. Wall apposition at the site of HLS penetration by Sp isolate contained callose and lignin. The haustorial sheath of the Jr isolate contained callose but no lignin. The haustoria of the Jr isolate showed specific fluorescence of membrane-bound Ca²⁺ by chlorotetracycline staining. HLS of the Sp isolate, however, did not show any fluorescence. It is suggested that HLS cannot function as a haustorium. SITS (4-acetamido-4'-iso-thiocyanate-stilben-2, 2'-disulphonic acid) fluorescence was observed on the small haustorium of the Sp isolate but never on that of the Jr isolate. Haustorium formation of the Sp isolate in Japanese radish root tissues may be inhibited by some host defense reactions against the fungus. Differences in pathogenicity between Sp and Jr isolates seem to be due to the failure of haustorium formation by Sp isolate.

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[in Japanese]

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Two types of seed transmission of Corynespora melonis on cucumber were confirmed by the transmission tests used artificially inoculated and home-raised seeds. One is ectoparasitism or simple adhesion of the pathogenic fungus to the surface of seeds, the other is endoparasitism where the location of the fungus in the seed is inside the testa as resting mycelia. Such affected seeds occur inferior germination and occasionally damping-off. Cucurbit leaf beetle, Aulacophora femoralis Motschulsky, feeds on the surface of cucumber fruit and promotes infection of the fungus.

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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