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D RNA 3Yalpha (D3Yalpha), a defective (D) RNA 3 derived from the Y strain of cucumber mosaic virus (CMV-Y), was further characterized in combination with different helper viruses in the genus Cucumovirus. Interestingly, Nicotiana benthamiana plants inoculated with CMV-D8 and D3Yalpha developed systemic symptoms which were different from those induced by CMV-D8. To elucidate the potential effects of D RNA 3 on virus infection on the basis of the original combination of CMV-Y and D3Yalpha, a point mutation was made in the coat protein gene, which determined symptoms, of either CMV-Y RNA 3 (Y3) or D3Yalpha. Symptoms induced on N. benthamiana and N. tabacum plants, and semi-quantitative RT-PCR revealed that the ratio of RNA 3 to D RNA 3 was associated with the differences of symptoms in the leaf tissues. Furthermore, analysis of in situ hybridization suggested that there were spatial effects between coat proteins of Y3 and D3Yalpha in the infected leaves.

A defective (D) RNA 3 naturally generated from the Y strain of cucumber mosaic virus (CMV-Y) was characterized using a biologically active cDNA clone. Sequencing of the clone revealed that the D RNA 3, named D RNA 3Yalpha, was derived from CMV-Y RNA 3 and contained a 10 nt deletion in the 5' untranslated region and a 162 nt deletion within the 3a open reading frame. Co-inoculation of D RNA 3Yalpha with CMV-Y derived from in vitro transcripts facilitated propagation of CMV-Y containing D RNA 3Yalpha in Nicotiana benthamiana or Nicotiana tabacum plants. CMV-Y, however, did not produce deletion mutants upon serial mechanical passages in the plants.

Nucleotide (nt) sequencing has contributed to the identification of virus species and has also proved diagnostically useful in the control of tomato-infecting begomoviruses disease. We determined the complete nt sequences of the DNA-A genome and its cognate DNAbeta satellite molecules in isolates of Tobacco leaf curl Japan virus, Honeysuckle yellow vein mosaic virus, Eupatorium yellow vein virus in Japan. Pairwise comparison analyses based on the nt sequences of DNA-A from the genetic group of these viruses tentatively named as TbLCJV, HYVMV and EpYVV (TbJV/HYV/EpV) revealed that this group had a significance threshold of 84 % identity. Phylogenetic relationship analyses of the nt sequences of DNA-A and DNAbeta revealed that their isolates were separated into a discrete Far East Asian clade, distinct from all other begomoviruses. This clade was divided into two distinct clusters comprising the subgroups TbJV/HYV and EpV. Furthermore, recombination analysis revealed that members of the TbJV/HYV/EpV group had the genetic variation indicative of many recombination events. Our study demonstrates that this group forms a unique species complex, but that members have discrete lineages depending on their natural perennial host plants.

A virus causing chlorotic ringspot, yellow mosaic and vein clearing symptoms was prevalent on mungbean plants around Taean, Korea. The isolate caused mosaic on Chenopodium quinoa, Nicotiana benthamiana, Phaseolus vulgaris and Vida laba but no symptoms on peanut plants. Inclusion bodies such as scroll, pinwheel and laminated aggregates induced by the virus in the host cells were similar to those produced by members of the Potyvirus subdivision III. Multiple alignment as well as cluster dendrograms of the 709 nucleotide region comprising part of the coat protein gene and 3`untranslated region (UTR) showed that the isolate belongs to the BCMV-PSt subgroup. Altogether, these results support the identification of the causal virus as peanut stripe strain of Bean common mosaic virus (BCMV-PSt).

A virus causing vein banding, sometimes yellow mosaic and rugose symptoms on peanut was prevalent around Suwon, Korea. A survey conducted in the area found disease incidence, depending on cultivar, to range from 79 to . The virus was found to be seed-transmissible in all the five peanut cultivars tested with transmission rates ranging from 2 to . Host range analysis failed to differentiate 9 field isolates collected from different peanuts cultivars showing various symptoms. Inclusion bodies such as scroll, pinwheel and long laminated aggregates induced by the virus in host plant cells were similar to those induced by members of the Potyvirus subdivision III. The virus showed homology with Bean common mosaic virus (BCMV), BCMV-BICMV/AzMV strains and only with Desmodium mosaic virus. Based on biological characterization, electron microscopy and molecular analyses of a Korean isolate (Daewon 1), the virus was identified as peanut stripe strain of BCMV.

Turnip mosaic virus (TuMV) is an infectious viral pathogen on the cruciferous crops, predominantly Chinese cabbage (Brassica campestris subsp. pekinensis) and radish (Raphanus sativus). On the basis of the symptom development in selective differential hosts from indicator host species, Chinese cabbage and Korean radish inbred lines, the representative eight isolates of TuMV were divided into two major groups/or six types. Group I includes Th 1, Ca-ad7, and Cj-ca2-1 isolates, while group II includes the other isolates (rg-pfl, r 9-10, Rhcql-2, Stock and Mustard). According to the molecular phylogenetic analysis, these isolates, however, divided into two groups and two independent isolates. Phylogenetic analysis indicated that four isolates (Tu 1, r9-10, Stock and Rh-cql-2) formed a distinct phylogenetic group, and the other two isolates (Ca-ad7 and Cj-ca2-1) also formed another group. Mustard and rg-pfl isolates did not seem to have any relationship with these two groups. Taken together, these results indicated that virulence differentiation on host plants, molecular phylogenetic analysis of the nucleotide and the deduced amino acid of TuMV coat proteins did not show any relationship. The multi-resistant lines, Wonyae 20026 and BP058 in Chinese cabbage represent valuable genetic materials that can be used for crucifer breeding programs on TuMV resistance, but not in Korean radish.

A virus isolate collected from infected paprika (Capsicum annuum var. grossum) was characterized as Potato virus Y (PVY) based on biological, serological, cytopathological, and molecular properties. In host range studies, the paprika isolate produced the mosaic symptom on some tobacco, tomato and pepper (Capsicum annuum). A new paprika isolate also infected potato cultivars which is different biological characteristic compared to the other popular potyvirus infecting paprika, Pepper mottle virus (PepMoV). Previously reported PVY strains, and did not infect pepper and typical PepMoV isolates did not infect potato. Distinctive inclusion patterns of the scroll, pinwheel, long laminated inclusions, and helper components in the cytoplasm of infected cells were also different to those observed by the typical PVY isolate infections. However, the paprika isolate reacted to the monoclonal antibody of strain with high absorbance readings. RT-PCR amplification, cloning, and sequencing of the 3` untranslated region and a part of coat protein gene also added additional evidence of the paprika isolate as the -related isolate. Multiple alignments as well as cluster dendrograms of PVY-paprika isolate revealed close phylogenetic relationship to the subgroup. Altogether, these results suggest that a new PVY isolate infecting paprika contained distinct characteristics compared to the other previously described PVY strains with closer relationship to the strain.

Kagoshima, Japan. The complete DNA-A Iike molecule of KG5Tob was determined and found to comprise 2762 nucleotides with a typical begomovirus genome organization. A comparison of the DNA-A sequences revealed that KG5Tob shared 87"/o with its closest relatives, Honeysuckle yellow vei7h mosaic virus (HWMV) and Tobacco leaf curl Yamaguchi virus (TbLCYV), however, on comparison of the intergenic region (IR) nt sequences, the isolate shared only 66 and 64"/o of the IR sequences with the former and the latter, respectively. Phylogenetic analysis of full length DNA-A component clustered KG5Tob in a lone cluster but close to HrvMV and TbLCrv. When the CPs were aligned, sintilar results were observed and KG5Tob was placed in a lone cluster. A comparison of the Other ORFs showed that different parts of the genome had different affinities, suggesting that recombination could have played a major role in the evolution of this isolate. Molecular data show that KG5Tob isolate is an isolate of HYVMV, although the isolate was obtained from tobacco.

mined and found to comprise 2763 and 1262 nucleotides (nt), respectively, with a typical begomovirus genome organization. The DNA~ molecule had the conserved ORF (~ C1) and satellite conserved region (SCR) typical of the Geminiviridae family containing a 5'-TMTATTAC-3' motif in the stem loop and the adenine-(A) rich region. Additionally, its entire sequence was interrupted at intervals wlth sequences that were not viral and whose origin was unknown. A comparison of the DNA-A sequences revealed that HY12 shared 740/0 with four other honeysuckle begomoviruses but 88-890/0 with tobacco begomoviruses found in Japan. The isolate shared up to 920/0 of the IR sequences with the honeysuckle begomoviruses but only 60-880/0 with those of tobacco. Phylogenetic analysis of full length DNA-A component as well as its CP clustered HY12 with the tobacco begomoviruses. uvl2 is an isolate of Howeysuckle yellow vein mosaic virus.

Seventy-three endophyiic bacterial isolates obtained from the rootstocks of Solawam sp., widely distributed in Hanoi, Vietnam, were applied to Vitek32 system for their identification. Out of these, 29 isolates were tentatively identified as Chryseobacterium indologenes, Pseudomonas fluorescens, Chryseomonas luteola, Bacillus pumilis, B. megaterium, B. sphaericus. Sphingomonas paucimobilis, Enterobacter cloacae, Pantoea agglomerans, and Staphylococcus auricularis with the sirnilarity ranging from 97 to 990/0 to the species covered in the system. Eight isolates with sirnilarity less than 970/0 were also tentatively put into the relevant genus. However, the remaining 36 isolates could not be identified by this system. Thirty-seven identifiable isolates were evaluated for i7~ vitro antagonism against Ralstonia solanacearum and disease suppression of bacterial wllt of tobacco. Thirteen isolates formed growih inhibition zones around their colonies placed on lawn of R. sola7~acearum as an indicator. The variation of anti-R. solauacearum activity spectrum within the endophytic bacterial species and isolates suggests that there could be different antibacterial substances participated in the antagonism. Most of the endophyiic bacterial isolates have not significantly shown disease suppression. There was no significant correlation between antibiotic activity i?e vitro and disease suppression. For example, the tobacco plants treated with the isolate KuTox708, which was the least inhibitory to R. solavtacearum i7h vitro, were significantly protected from bacterial wilt infection.

Two Cucumber mosaic virus (CMV) isolates, CMV-NA from Nicotiana affi?~is, and CMV-SM from Solawam mammosum (nipple fruit plant) inducing mild and severe systemic mosaic s~mptoms, respectively, on tobacco (Nicotiana tabacum cv. Xanthi-nc), were characterized on the basis of their nucleotide and amino acid seqtlences of RNA3, serology and host ranges. The complete nucleotide sequencing of RNA3s of CMV-NA and CMV-SM has been done. Multiple alignment analysis of the nucleotide sequences of RNA3 of the 5'noncoding region and amino acid sequences of the CP and MP revealed that, CMV-NA and CMV=SM belong to subgroup I. CMV-NA in'duced systemic infection on Cryptotae7~ia japbnica and

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.

A survey of Potato virus Y (PVY) was conducted in farmers' fields in 9 Korean provinces, Gyeonggi-do, Gangwon-do, Gyeongsangnam-do, Gyeongsangbuk-do, Jeollanam-do, Jeollabuk~io, Chungchcongnam-do, Chungcheongbuk-do and Jeju-do from 1998 to 2000. One hundred and fifty nine samples from potato and tobacco were collected and analyzed. Based on ELISA results, three samples PVY-tob, PVY-potlii and PVY-pot-chw representing PVYN and PVYo, respectively, were further subjected to biological, serological and molecular analysis. Host range studies showed that PVY-pot-chw is symptomatically distinguishable from PVY-tob and PVY-pot-j isolates, both of which were symptomatically indistinguishable. However, all the three isolates failed to infect 10 capsicum varieties systemically. Isolates PVY-tob and PVY-pot-j reacted strongly wlth monoclonal antibody (MAb), anti-PVYN while PVY-pot-chw failed to react wlth the MAb but reacted strongly wlth anti-PVYo'c. Interestingly, both PVY-tob and PVY-pot-j isolates did not react with anti-Pvyo,C indicating that isolate PVY-pot-chw is serologically distinguishable from both PVY-tob and PVY-potlii. In order to ascertain the taxonomic status of the three isolates, the 3' untranslated terminal regions (3'-UTR) were sequenced. Alignment of the sequences revealed 990/0 sequence identity between PVY-tob and PVY-pot-j, both of which had 780/0 nt sequences identical to PVY-pot-chw. Phylogenetic analysis of the aligned 3'-UTRs clustered PVY-tob and PVY-pot-j in the PVY~ subgroup, and PVY-pot~:hw in the PVYO subgroup.

The dynamics of virus interference in Cucumber mosaic virus (CMV) infection in cowpea were investigated by tissue-blotting and in situ hybridization. Using co-inoculation assays, we discovered that spatial competition between CMV-LE (subgroup I) and CMV-m2 (subgroup II) occurred in the inoculated leaves. Interestingly, competitive interactions between the two viruses also could be observed in the non-inoculated upper leaf tissues of the plants. Furthermore, the pattern of exclusive distribution was observed between challenge and protecting viruses in the serially inoculated leaves. Taken together, it is suggested that the dynamics of competitive interactions between the two subgroups could be characterized by exclusive infection and multiplication of the individual viruses in cowpea plants.

A potyvirus, for which the name Japanese hornwort mosaic virus (JHMV) is proposed, was isolated from Japanese hornwort plants (Cryptotaenia japonica) with mosaic disease symptoms. The virus was used to inoculate mechanically 34 plants belonging to 33 species of 10 families. Of these species seven from two families were infected. Faint chlorotic spots appeared on the inoculated leaves of Chenopodium quinoa and C. amaranticolor, but no systemic infection occurred in these plants. JHMV systemically infected only Umbelliferae plants; they did not infect 26 other species in eight families. JHMV was transmitted in a nonpersistent manner by aphids (Myzus persicae). The virus was a flexuous rod-shaped particle about 750 nm in length. Sequencing the nucleotides in the 3′ terminal region of JHMV revealed that the coat protein contains 280 amino acids with a molecular mass of 32.2 kDa. The nucleotide sequence of the coat protein of JHMV had the highest similarity with that of Zantedeschia mosaic virus (83.3%) compared to those of other potyviruses (57.0%–64.9%). An antiserum against JHMV reacted strongly with JHMV and weakly with Potato virus Y. These results indicate that JHMV is a new potyvirus.

Phenotypic and genetic characteristics of nine bacterial strains isolated from mulberry (Morus spp.), which were originally described as Erwinia carotovora ssp. carotovora (Ecc), were investigated. Based on the results of biochemical tests, these bacterial strains were divided into two different types, type 1 and type 2. Two strains of type 1 were similar to Ecc, whereas seven strains of type 2 were distinct from Ecc. A polyphasic study that included serological assay, specific PCR assay for E. carotovora ssp. atroseptica (Eca), PCR-RFLP of a pectate lyase (pel) gene and RAPD-PCR was performed on the type 2 strains, and the data were compared with those of related E. carotovora subspecies. The results of serological and specific PCR assays for Eca showed that the type 2 strains were distinct from Eca. In RFLP analysis of the pel gene using Sau3AI, the type 2 strains showed a unique RFLP pattern. On the basis of RAPD analysis, similarity of RAPD patterns within the type 2 strains was very high. A unique RAPD fragment was isolated from the type 2 strains and used as a probe for Southern hybridization. This probe hybridized only with PCR products from the type 2 strains. Based on phenotypic, serological and genetic characteristics, the type 2 strains isolated from mulberry may belong to a distinct E. carotovora subspecies other than Eca or Ecc.

A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR-restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S-23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α-methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia.

A strain of Cucumber mosaic virus (CMV-D8) systemically infects Japanese radish (Raphanus sativus), but the Y strain of CMV (CMV-Y) only infects the inoculated leaves. Both of these strains cause severe systemic mosaic on the plants after dual infection with Turnip mosaic virus (TuMV). Synergistic interactions on long-distance transport of CMV-Y and CMV-D8 with TuMV were analyzed using an immunobinding assay. Direct tissue blots probed with either anti-CMV-Y or anti-TuMV antiserum clearly showed that CMV-Y efficiently spread and accumulated in the tissues of noninoculated upper leaves and roots when co-inoculated with TuMV, and that long-distance movement of CMV-D8 was enhanced by the presence of TuMV.

The genetics of cucumber mosaic cucumovirus (CMV) and the pathogenicity of the virus for Raphanus sativus were analyzed using pseudorecombinants constructed from the infectious transcripts of two naturally occurring strains of cucumber mosaic cucumovirus (CMV-D8 and CMV-Y). CMV-D8, but not CMV-Y, could cause systemic infection of the plant. Viral accumulation and systemic movement in the plants was examined using immuno-tissue blot analysis, dot blot and Northern blot hybridization. Virus was equally distributed and CMV RNAs accumulated to similar levels in the inoculated cotyledons of radish irrespective of the pseudorecombinant, suggesting that there are no apparent differences in the ability of infection and viral accumulation between CMV-D8 and CMV-Y. We found, however, that both RNAs 2 and 3 of CMV-D8 are involved in determining the efficiency for the systemic infection of R. sativus. Co-operated interactions between genetic information of RNAs 2 and 3 would control the efficient translocation of virus from the inoculated leaves to the uninoculated upper leaves of radish plant.

Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR) was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract parasite DNA from diseased rice sheaths. 28S ribosomal DNA (rDNA) derived from fungal genomic DNA extracted by the alkaline method was specifically PCR-amplified. The results of PCR-RFLP analysis for DNA samples from artificially inoculated rice sheath tissues with eachRhizoctonia spp. and the corresponding culture on the medium using two restriction enzymes.HhaI andMspI, showed identical polymorphisms. PCR-RFLP analysis using DNA samples from naturally infected rice sheath tissues also revealed the possibility of direct diagnosis ofR. solani AG 1 IA,R. oryzae andR. oryzae-sativae.

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.

We examined the correlation of the amino acid at position 129 in the coat protein (CP) of cucumber mosaic virus (CMV) with the phenotype of the viral pathology in tobacco by using CP mutants in which several amino acid substitutions had been introduced. An exchange between Ser129 in CMV-Y, a chlorosis-inducing strain, and Pro129 of CMV-O, a green-mosaic-inducing strain, reciprocally altered the phenotypes of those virus strains on tobacco. Replacement of either Ser129 in CMV-Y or Pro129 in CMV-O with a Leu, as is found in a chlorosis-inducing strain, CMV-M, resulted in veinal necrosis. Furthermore, we created mutants that have a Phe or a Gly at position 129. Two Phe129 mutants induced necrotic lesions on the inoculated leaves, and a Gly129 mutant induced green mosaic symptoms. In inoculated protoplasts, the mutant viruses and the wild-type virus all replicated RNA well, and accumulated CP; however, infection with the Leu129 and Phe129 mutants yielded few virions. The Phe129 mutants lacked the capacity to move systemically in tobacco; by 2 weeks post-inoculation, the Phe129 mutants occasionally gave rise to revertants that elicited chlorosis, green mosaic or veinal necrosis. Sequence analysis revealed that one had reverted to the parental Y strain, and the others had additional single amino acid changes (positions 138, 144 or 147). We suggest that amino acids at specific sites affect the whole structure of the CP and affect virus assembly, virus transport and symptom expression.

The relationship between the phytotoxin (C7H7N5O2) produced by Pseudomonas glumae and its virulence was studied. All of the Phytotoxin productive strains were virulent to rice plants with a few exception, whereas all the phytotoxin non-productive strains were avirulent. A striking difference in the time course of phytotoxin production was observed between virulent strains and avirulent strains of P. glumae. The phytotoxin inhibited elongation of sprouts and roots of rice seedlings almost completely at 10μg/ml. Rice panicles, which were treated with the phytotoxin at concentrations of 2.5 or 25μg/ml by dipping for 10min at the flowering stage, produced a brown band on the palea and lemma, a characteristic symptom of the bacterial grain rot of rice. The phytotoxin was isolated not only from culture filtrates, but also from rice seedlings infected with P. glumae. These results suggest that the phytotoxin produced by P. glumae will be closely associated with its virulence.

The phytotoxin produced by Pseudomonas glumae, the causal agent of bacterial grain and seedling rot of rice, was purified and characterized. The ultraviolet and visible absorption spectrum of the phytotoxin exhibited two maxima at 260nm and 399nm in 80% aqueous methanol. In the infrared absorption spectrum, main bands at 2,800 3,00Ocm-‘, 1,68Ocm-‘, 1,540cm.’ and 1,05Ocm-’ were observed, suggesting the presence of a methyl group, a ketone group, a benzene ring and a carbon- hydrogen bond, respectively. The molecular weight of this substance was determined as 193 and the molecular formula was decided as C7H7Ns02 by a high resolution EI mass spectrometry. The phytotoxin was isolated noi only from the PS broth, but also from rice seedling infected with the causal bacterium. This phytotoxin might be related to the pathogenicity of P. glumae.

A satellite RNA (T73-satRNA) gave reduced symptom severity on tomato plants when coinoculated with an ordinary strain of cucumber mosaic virus (CMVO). cDNA for T73-satRNA was introduced into a binary vector (pTOK162) through a homologous recombination in an Agrobacterium tumefaciens cell, and then transferred to leaf disks of tomato. Stable integration and transcription of the cDNA in the regenerants were verified by Southern and northern blot hybridizations, respectively. Upon inoculation with CMV-O, the transformants exhibited very slight symptoms of CMV, grew normally, and finally set fruits, whereas untransformed wildtype tomato plants showed very severe symptoms, and their growth was retarded and formed few fruits. Agarose gel electrophoresis of total RNA from CMV-O-inoculated transformants detected RNA molecules corresponding to T73-satRNA.

A monomeric cDNA copy of cucumber mosaic virus (CMV) satellite RNA which is non-necrogenic to tomato was introduced into nuclear DNA of tobacco plants with the aid of the binary vector system of Agrobacterium tumefaciens. In order to check the stability of the introduced genes, kanamycin resistance and expression of biologically active satellite RNA upon infection with CMV were assayed up to the fifth generation of the homozygous progeny obtained from a primary transformant. The inserted cDNA in the progeny of the fifth generation was sequenced after cloning of the sequence amplified with the polymerase chain reaction. Neither loss nor mutations of the inserted genes were detected.

An inhibitor of plant virus infection was found in the extracts of Mirabilis jalapa L. The inhibitor, a protein designated as MAP (Mirabilis antiviral protein), showed highly potent activity against mechanical transmission of viruses, tobacco mosaic (TMV), cucumber green mottle mosaic, potato Y, turnip mosaic and cucumber mosaic. Almost complete inhibition was achieved when MAP at 0.8μg/ml was applied on the upper surface of leaves of Xanthi nc tobacco 24hr before TMV inoculation, and 50% inhibition when MAP at 10μg/ml was applied on the under surface of leaves. MAP induced systemic resistance to TMV infection in tobacco plants when it was applied on their basal leaves 24hr before TMV inoculation onto the upper ones. MAP showed no inhibition when it was applied 1hr after TMV inoculation. Antiserum against MAP gave positive reaction in the agar gel with leaf extracts of M. jalapa and did not with those of the other Chenopodiales plants such as Basella rubra, Boerhaavia diffusa, Bougainvillea sp., Chenopodium amaranticolor and Phytolacca americana. Concentration of MAP was successfully quantified using enzyme-linked immunosorbent assay. MAP content in the root tissue was exceedingly higher than in the other parts of the plant and that of an yellow-flower trait of M. jalapa was as high as 1.1mg/g fresh weight. M. jalapa seems to be a promising source of antiviral substances for the practical use.

cDNA clones covering full-length of cucumber mosaic virus Y strain (CMV-Y) RNA2 were synthesized and the complete nucleotide sequence of the RNA was determined. Specific restriction sites, PstI and NotI, wer eadded to the 5' and 3' end of cDNAs, respectively. It consisted of 3, 051 nucleotides and contained a long single open reading frame of 2, 571 nucleotides. GDD consensus sequence, which is thought to be a “polymerase site”, and a common motif surrounding it was found in the amino acid sequence deduced from the RNA. There was almost complete sequence homology between CMV-Y and Fny-CMV RNA2s (98.4% sequence homology at the nucleotide level and 99.5% at the amino acid level). By contrast, lower degree of sequence homology was observed between CMV-Y and CMV-Q RNA2s (71.4% sequence homology at the nucleotide level and 74.4% at the amino acid level). These results suggest that CMV-Y belongs to Subgroup I.

Four satellite RNA isolates of cucumber mosaic virus (CMV), S19-, T43-, T62- and T73-sat-RNAs were obtained from CMV-infected plants collected in Tochigi Prefecture of Japan. Their physical and biological properties were investigated. T43- and T62-satRNAs induced necrosis on tomato like Y-satRNA, whereas S19- and T73-satRNAs did not. T43-satRNA was found to be identical to T62-satRNA by nucleotide sequence determination. The sequences were compared with each other and with other satellite RNAs, revealing high degree of nucleotide sequence homology between S19-satRNA and Y-satRNA (91%) and between T43-satRNA and OY2-satRNA (94%). Further analyses of the three sequences indicate that no common open reading frame is present and thus, they do not seem to encode any protein products essential for their biological activity. Competition of the satellite RNAs with Y-satRNA was examined by mixed inoculations under the condition using tobacco as a host and CMV-O as a helper virus. The relative replication ability of T43-satRNA is superior to that of Y-satRNA while T73-satRNA is apparently similar to or a little better than Y-satRNA.

Accumulation of the coat proteins of three isolates of cucumber mosaic virus (CMV-D8 isolated from a Japanese radish plant showing mosaic symptoms, a melon isolate, CMV-KM and tobacco isolate, CMV-Y) in seven Japanese radish (Rhaphanus sativus) cultivars was compared with immunological detection by direct tissue blotting. In the inoculated leaves, viral antigen equally accumulated irrespective of CMV isolates and the radish cultivars. In contrast, viral antigen was detected in almost all of the upper leaves of the all cultivars inoculated with CMV-D8 whereas rarely detected in those of the plants inoculated with CMV-Y. Accumulation of CMV-KM in the upper leaves was intermediate between that of CMV-D8 and CMV-Y. The cultivars, Akidu-mari and Natsu were highly resistant against systemic infection with CMV-KM and CMV-Y, but Shijunichi and Shogoin were rather susceptible to systemic infection with the both isolates compared to the other radish cultivars.