Search

The rDNA-ITS sequences of ten single-sporangium isolates of Olpidium virulentus (a noncrucifer strain of Olpidium brassicae), which transmits Mirafiori lettuce big-vein virus (MLBVV) and tobacco stunt virus (TStV), were compared with those of six single-sporangium isolates of O. brassicae. The sequence similarity within isolates of O. virulentus or O. brassicae was almost identical (98.5%–100.0%), but was low between the two species (79.7%–81.8%). In a phylogenetic analysis of the rDNA-ITS region, O. virulentus and O. brassicae fell into two distinct clusters, indicating that O. virulentus, a vector of MLBVV and TStV, is a distinct species rather than a strain of O. brassicae.

Lettuce big-vein virus (LBVV) is the type species of the genus Varicosavirus and is a two-segmented negative-sense single-stranded RNA virus. The larger LBVV genome segment (RNA1) consists of 6797 nt and encodes an L polymerase that resembles that of rhabdoviruses. Here, the nucleotide sequence of the second LBVV genome segment (RNA2) is reported. LBVV RNA2 consisted of 6081 nt and contained antisense information for five major ORFs: ORF1 (nt 210–1403 on the viral RNA), ORF2 (nt 1493–2494), ORF3 (nt 2617–3489), ORF4 (nt 3843–4337) and ORF5 (nt 4530–5636), which had coding capacities of 44, 36, 32, 19 and 41 kDa, respectively. The gene at the 3′ end of the viral RNA encoded a coat protein, while the other four genes encoded proteins of unknown functions. The 3′-terminal 11 nt of LBVV RNA2 were identical to those of LBVV RNA1, and the 5′-terminal regions of LBVV RNA1 and RNA2 contained a long common nucleotide stretch of about 100 nt. Northern blot analysis using probes specific to the individual ORFs revealed that LBVV transcribes monocistronic RNAs. Analysis of the terminal sequences, and primer extension and RNase H digestion analysis of LBVV mRNAs, suggested that LBVV utilizes a transcription termination/initiation strategy comparable with that of rhabdoviruses.

Severe spotting and blighting of leaves were found on bacopa (Sutera cordata), a scrophulariaceous ornamental, in greenhouses in Gunma Prefecture, Japan, from January through February 2007. After we isolated and identified the causal fungus as Colletotrichum destructivum and inoculated host plants with the isolate to confirm pathogenicity, we named this new disease anthracnose of bacopa.

The complete nucleotide sequence of RNA1 from Lettuce big-vein virus (LBVV), the type member of the genus Varicosavirus , was determined. LBVV RNA1 consists of 6797 nucleotides and contains one large ORF that encodes a large (L) protein of 2040 amino acids with a predicted M r of 232,092. Northern blot hybridization analysis indicated that the LBVV RNA1 is a negative-sense RNA. Database searches showed that the amino acid sequence of L protein is homologous to those of L polymerases of nonsegmented negative-strand RNA viruses. A cluster dendrogram derived from alignments of the LBVV L protein and the L polymerases indicated that the L protein is most closely related to the L polymerases of plant rhabdoviruses. Transcription termination/polyadenylation signal-like poly(U) tracts that resemble those in rhabdovirus and paramyxovirus RNAs were present upstream and downstream of the coding region. Although LBVV is related to rhabdoviruses, a key distinguishing feature is that the genome of LBVV is segmented. The results reemphasize the need to reconsider the taxonomic position of varicosaviruses.

A sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M r of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M r of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7·3 kb (ss-1) and 6·6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.

Severe wilt with spots and/or leaf and stem blight were found on a scrophulariaceous flowering plant, Nemesia strumosa, grown in Kagawa Prefecture, Japan, in February 1999. Wilted plants had numerous lesions and died early. A mitosporic fungus isolated repeatedly from the diseased plants was identified as Colletotrichum fuscum and was demonstrated to cause the disease. N. strumosa is a new host for C. fuscum, which has been known to attack foxglove (Digitalis spp.). The present disease was named “anthracnose of N. strumosa” as a new disease.

In October 1998, a disease causing mainly foliar necrotic lesions was found on African marigold (Tagetes erecta) and French marigold (T. patula) grown in Miyagi Prefecture, Japan. Similar lesions also developed on stems and flowers, resulting in early blight of the affected organs. Plants with numerous lesions withered rapidly. A mitosporic fungus isolated repeatedly from the diseased plants was identified as Alternaria tagetica and demonstrated to cause the disease. The disease, as well as the fungus, is new to Japan. We propose the name “hanten-byo”, which means leaf spot in Japanese, for this disease.

A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1·41 and 1·43 g/cm3 in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3′ poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa (‘23K’; ORF1) and 235 kDa (‘235K’; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa (‘108K’). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the Comoviridae. The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages.

To compare biological and serological reactions of 28 bean yellow mosaic virus (BYMV) isolates collected from different hosts and locations in Japan, 15 different bean (Phaseolus vulgaris) cultivars were inoculated with the 28 isolates. Four biological variations (pathotypes I-IV) were observed among the 28 BYMV isolates. Pathotype I systemically infected only one bean cultivar, Honkintoki, out of the 15 cultivars studied. Pathotype II infected systemically Honkintoki, Kentucky Wonder and four other cultivars. Pathotype III infected systemically Honkintoki, Kentucky Wonder, Master Piece and four other cultivars. Pathotype IV infected systemically all 15 bean cultivars. Pathotype II also showed higher seed-transmissibility in broad bean (Vicia faba) than other pathotypes. The 28 BYMV isolates also varied serologically in their reactions in TAS-ELISA to 16 monoclonal antibodies (MAbs) produced against a BYMV isolate and two clover yellow vein virus (ClYVV) isolates. These reactions corresponded, to some degree, to biological variability. MAb-1F3 reacted with pathotypes I, II and III, and with one ClYVV isolate. MAb-2C4 reacted only with pathotype II. MAb-5F2 reacted with all BYMV and ClYVV isolates. MAb-2B4, -2C5, -3F9, -3F11, -4G8 and -4H9 reacted with all BYMV isolates in pathotypes II and III, and with some BYMV isolates in pathotypes I and IV. MAb-1A2 and -2H8 reacted strongly with pathotype III and with two ClYVV isolates. On the other hand, the differences in reactivity of six polyclonal antisera against four BYMV isolates and two ClYVV isolates in DAS-ELISA were not observed clearly. Our results indicate that the BYMV population in Japan is biologically and serologically variable and can be grouped into four pathotypes.

A comparative study was made on the host reactions, serological properties, and nucleotide sequences of the coat protein (CP) gene of 10 clover yellow vein virus (C1YVV) isolates and one bean yellow mosaic virus (BYMV) isolate collected from different host plant species and locations in Japan. Two strains of C1YVV isolates, grouped on the basis of host reactions on Chenopodium amaranticolor, C. quinoa, Nicotianaclevelandii, N. benthamiana, Vicia faba, and Trifolium repens, corresponded to two serotypes determined by double-antibody sandwich- and triple-antibody sandwich-enzyme-linked immunosorbent assay using three polyclonal and nine monoclonal antibodies. These results were also confirmed by nucleotide sequence analysis of the CP gene. The CP gene of C1YVV isolates of strain 1, including the Australian isolate C1YVV-B, had 93 to 98% nucleotide identities and 97 to 99.6% amino acid identities. The CP of C1YVV isolates of strain 2, including the New Zealand isolate C1YVV-NZ, had 92 to 98% nucleotide identities and 95 to 98% amino acid identities. The nucleotide identities and the amino acid identities between the two C1YVV strains were 82 to 84%, and 90 to 94%, respectively. When compared with the CP sequences of 12 C1YVV isolates, the CP sequence of the BYMV isolate had 71 to 73% nucleotide identity and 73 to 77% amino acid identity. Amino acid sequence differences among C1YVV isolates from strains 1 and 2 were located mostly at the N-terminal regions of the CP. Our results indicated that the C1YVV isolates studied could be separated into two strains on the basis of host reactions, serology, and the nucleotide sequence of the CP gene.

A virus, designated NC, isolated from gentian plants (Gentiana spp.) with severe necrosis in Fukushima Prefecture, was characterized and compared with other isolates of clover yellow vein virus (ClYVV). When 26 species of plants from 13 families were mechanically inoculated with the NC isolate, 19 species in 10 families were infected, thus being similar to ClYVV in host range and symptomatology. The virus was serologically identical to ClYVV in SDS agar double-diffusion tests, but was different from bean yellow mosaic virus (BYMV). The nucleotide sequence of the 3' terminal region of the NC isolate RNAs was compared with that of ClYVV-NFU, an isolate from bean (Phaseolus vulgaris) in Fukushima Prefecture. The 3' non-coding regions of both isolates were identical. They also had higher nucleotide sequence homologies with other ClYVV isolates (93.3-99.4%) than with BYMV isolates (73.7-77.1%). In the coat protein gene, the amino acid sequence of the NC isolate also showed higher homology with ClYVV isolates (91.6-98.2%), especially with ClYVV-NFU, than with BYMV isolates (72.9-76.5%). Based on these results, the NC isolate which caused gentian necrotic dwarf was identified as ClYVV.

Apple fruit crinkle (AFC) is a graft-transmissible fruit disease of apple, so far found only in Japan. A viroid-like RNA was detected in association with this disease. Its molecular size was larger than that of apple scar skin viroid (ASSVd) and it did not hybridize with ASSVd-cDNA. In addition, it was transmitted to apple seedlings when they were inoculated by razor-slash method with its electrophoretically purified preparation. These results suggest that this viroid-like RNA is a novel apple viroid and that AFC is a new viroid disease distinct from apple scar skin. We propose to call this viroid apple fruit crinkle associated viroid.

The sequence of 5,028 nucleotides, including one open reading frame (ORF), of rice tungro bacilliform virus (RTBV) dsDNA was determined. The predicted translational product comprises 1,675 amino acids and has Mr of 194,134 (p194). The amino acid sequences of three tryptic fragments from the 32 k capsid protein of RTBV (p32) were found in the predicted translational product indicating that the ORF codes for the RTBV capsid protein.

The 2326 nucleotides of the 3′-terminal region of Carnation vein mottle virus (CVMV) RNA, which included part of the nuclear inclusion b gene, the complete coat protein (CP) gene and the entire 3′-noncoding region (3′-NCR) were determined. The region encoding the CP gene is 843 nucleotides long and the deduced protein consists of 280 amino acids. A search of the EMBL and PIR databases showed that the amino acid sequence of CVMV CP most resembled that of Plum pox virus with a similarity of 67.9%. The 3′-NCR of CVMV RNA is 541 nucleotides long, second longest in the genus Potyvirus. These results indicate that CVMV is closely related to Plum pox virus but is a distinct species in the genus Potyvirus.

Mirafiori lettuce big-vein virus (MLBVV) and Lettuce big-vein associated virus (LBVaV) are found in association with big-vein disease of lettuce. Discrimination between the two viruses is critical for elucidating the etiology of big-vein disease. Using specific antibodies to MLBVV and LBVaV for western blotting and exploiting differences between MLBVV and LBVaV in host reaction of cucumber and temperature dependence in lettuce, we separated the two viruses by transfering each virus from doubly infected lettuce plants to cucumber or lettuce plants. A virus-free fungal isolate was allowed to acquire the two viruses individually or together. To confirm the separation, zoospores from MLBVV-, LBVaV-, and dually infected lettuce plants were used for serial inoculations of lettuce seedlings 12 successive times. Lettuce seedlings were infected at each transfer either with MLBVV alone, LBVaV alone, or both viruses together, depending on the virus carried by the vector. Lettuce seedlings infected with MLBVV alone developed the big-vein symptoms, while those infected with LBVaV alone developed no symptoms. In field surveys, MLBVV was consistently detected in lettuce plants from big-vein-affected fields, whereas LBVaV was detected in lettuce plants not only from big-vein-affected fields but also from big-vein-free fields. LBVaV occurred widely at high rates in winter-spring lettuce-growing regions irrespective of the presence of MLBVV and, hence, of the presence of the big-vein disease.

The genome segment 12 (S12) of rice dwarf phytoreovirus (RDV) isolated from the Philippines (RDV-P) and of a variant (RDV-S-6) of RDV severe strain (RDV-S) migrated abnormally slower during polyacrylamide gel electrophoresis than that of the isolate maintained at Hokkaido University (RDV-H). Nucleotide sequence analysis revealed that rearrangement had occurred in these segments, affecting the open reading frame. A polypeptide encoded by S12 (Pns12) of RDV-P had a duplication of 28 amino acids while 1/3 of the carboxyl terminus of Pns12 was deleted in RDV-S-6 by premature termination due to a frameshift. RDV-S is always present in plants infected with the RDV-S-6 variant, suggesting that Pns12 of RDV-S-6 is defective. On the other hand, Pns12 of RDV-P was expressed and appeared to be functional in infected cells in spite of the duplication, as demonstrated by immunoblot analyses using antibody raised against Pns12 expressed inEscherichia coli.

DNA complementary to the ninth largest (S9) of the 12 genome segments of rice gall dwarf virus (RGDV) was cloned and its sequence was determined. It is 1202 nucleotides in length and contains one open reading frame which extends for 969 nucleotides from nucleotide 26. It encodes a polypeptide of 323 amino acids with an M r of 35560. The dinucleotide sequence at the 5′ end and the trinucleotide sequence at the 3′ end of the plus strand, 5′ GG—GAU 3′, which are present in the RNA of both wound tumour virus (WTV) and rice dwarf virus (RDV), were also found in RGDV genome segment S9. The nucleotide sequences in the non-coding region at the 5′ terminus and in the 15 nucleotides at the 3′ terminus, which form an imperfect inverted repeat of 10 bp together with the 5′ terminus, are approximately 70% homologous with those of the WTV genome segment S9, but only 30% and 50% homologous with the respective termini of RDV S9.

The polymerase chain reaction (PCR) was 103-104 times more sensitive for detecting rice tungro bacilliform virus (RTBV) DNA extracted from infected rice plants than was enzyme-linked immunosorbent assay (ELISA). The greater sensitivity of PCR enabled the detection of the virus in individual RTBV-exposed leafhoppers, Nephotettix virescens, a semipersistent vector of RTBV. Detection of RTBV in the vector by ELISA has been impossible. To evaluate resistance to RTBV, seedlings of rice cultivars Utri Merah, Utri Rajapan, or Balimau Putih, were inoculated with RTBV, and infection of the cultivars with the virus was indexed by PCR and ELISA [...]

Nucleic acids and proteins of rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV) were analyzed by gel electrophoresis. RTSV contains single-stranded RNA of 3.7×10 6 Da and two major proteins of 24 and 23 kDa. RTBV contains circular double-stranded DNA of 8.3 kbp and a single major protein of 32 kDa. The RTBV-DNA is 2.1-2.3 μm in length and has two discontinuities, one in each of the two strands, having different polarities. Endonuclease cleavage sites in the RTBV-DNA molecule are mapped (...)

木谷・木曽(1968)の方法によりイネ縞葉枯ウイルス純化を試みたところ,氏らが報告しような小球形粒子が分離された。しかし,これら粒子は形が不揃いでcapsid構造を欠き,ウイルス粒子とは考えられなかった。次にdirect negative染色法によりイネ病葉からウイルス粒子の検出を試みたところ,枝分れ構造を示す糸状粒子が発見された。この糸状粒子を純化するため,イネ病葉を0.2Mリン酸緩衝液で磨砕し,低速遠心上清を10%蔗糖クッション遠心し,その沈澱をクロロホルムとTriton X-100で処理し,次いで分画遠心した。さらに5～20%蔗糖密度勾配遠心し,最終的に30～60% D2O-蔗糖密度平衡遠心した。得られた試料中には枝分れ糸状粒子が多量に含まれていたが,球形粒子はまったく存在しなかった。この試料は核蛋白の紫外部吸収スペクトルを示し,ヒメトビウンカへの虫体内注射で感染性が証明された。これらの結果から,この枝分れ糸状粒子がイネ縞葉枯ウイルスであると結論した。高倍率で電顕観察した結果では,この糸状粒子は幅3nmの細い糸が絡み合い二重らせん構造となっており,幅約8nm,長さ(全長)約400nm,らせんのピッチは6nmであった。枝分れの仕方は様々であり,時に部分的にらせんがほどけ,ループ状を呈している粒子も観察された。

Solution of diethyl ether extracts from culture filtrate of Valsa ceratosperma, a causal agent of apple Valsa canker, grown in a medium containing apple bark components caused similar bark necrosis to the canker on naturally infected apple trees. Three toxic substances were purified from culture extracts by column chromatography and were identified by co-chromatography and by ultraviolet absorption spectra. The toxic substances included phloretic acid (p-hydroxypropionic acid), p-hydroxybenzoic acid and protocatechuic acid which are known to be breakdown products of phloridzin. In cut stem bioassay these compounds showed a toxicity only in high concentrations. The toxicities of the compounds were not host specific.

Two species of low molecular weight RNA have been associated with apple scar skin disease. One of the RNAs, ASSARNA 1 was detected in the bark tissue as well as in the fruit tissue of diseased apple trees. Inoculation of young seedlings with extracted RNA was performed by rubbing with carborundum or by slashing with a razor. Nucleic acid was extracted from the bark of inoculated seedlings and analysed by two cycles of polyacrylamide gel electrophoresis. ASSARNA 1 was detected in one of 13 seedlings after 28 months following inoculation by the rubbing method with phenol-extracted nucleic acid and in 8 of 14 seedlings after 13 months following inoculation by the razor-slash method with purified ASSARNA 1. The results indicate that ASSARNA 1 was transmitted to apple seedlings and multiplied in them. These facts provided additional evidence for the viroid etiology of the causal agent of apple scar skin disease. However, the pathogenecity of ASSARNA 1 and the role of another disease-associated RNA, ASSARNA 2, are still unknown.

The complete nucleotide sequence of apple scar skin viroid(ASSV) has been established, and a probable secondary structure is proposed. A single-stranded circular ASSV RNA consists of 330 nucleotides and can assume the rodlike conformation with extensive base-pairing characteristic of all the known viroids. ASSV shows low sequence homologies with other viroids and lacks the central conserved region. These indicate that ASSV should be allocated to a separate viroid group. However, homologous sequences with potato spindle tuber viroid(PSTV) in ASSV occur in limited and scattered regions of both viroids. These homologous regions fall within the particular domains in the viroid domain model which has been previously proposed by Keese and Symons(Proc. Natl. Acad. Sci. USA. 82, 4582-4586, 1985).