Chuji Hiruki

Amplification of 16S rRNA genes from culturable and nonculturable Mollicutes

Abstract Five polymerase chain reaction (PCR) primer pairs were synthesized on the basis of the aligned 16S-like rRNA sequences of eukaryotes or 16S rRNA sequences of eubacteria, Mollicutes, and intracellular organelles. These PCR primer pairs had high sequence homology to the conserved 16S rRNA genes of various culturable and nonculturable Mollicutes, but less sequence homology to the eukaryotic nuclear 16S-like rRNA or 16S rRNA genes of intracellular organelles. Full-length 16S rRNA genes and partial-length 16S rRNA genes of evolutionarily variable regions were successfully amplified when DNA preparations from culturable Mollicutes such as Mycoplasma flocculare and three Spiroplasma strains and nonculturable Mollicutes associated with various plant diseases were used as PCR templates. Amplifications were not detected when Escherichia coli genomic DNA and DNA preparations from healthy plants were used under high stringency annealing conditions in thermocycling. The results suggest the possibility that 16S rRNA genes of culturable and nonculturable Mollicutes can be amplified for detection and for a phylogenetic study using crude Mollicutes DNA preparations under appropriately controlled thermocycling conditions.

Genetic Determinants Distributed in Two Genomic RNAs of Sweet Clover Necrotic Mosaic, Red Clover Necrotic Mosaic and Clover Primary Leaf Necrosis Viruses

SUMMARY Three serologically distinct polyhedral viruses, sweet clover necrotic mosaic virus (SCNMV), red clover necrotic mosaic virus (RCNMV) and clover primary leaf necrosis virus (CPLNV), contain bipartite RNAs with approximate mol. wt. of 1-35 x 106 to 1.55 x 106 (RNA l) and 0.5 x 106 to 0-6 × 106 (RNA 2). The homologous and heterologous combinations of RNA 1 and RNA 2 of the three viruses were highly infectious, while individual RNA species were not. Electrophoretic mobility of genomic RNA from the pseudorecombinan ts showed that the progeny viruses maintained the heterologous RNA combinations identically with those in the original inocula. Serological specificity of the progeny viruses was determined by RNA 1. A mixture of isolated coat protein and RNA 1 was not infectious. RNA 1 of SCNMV was essential for systemic infection of sweet clover at 26 °C, while RNA 2 of SCNMV complemented RNA 1 of RCNMV in causing local infection in sweet clover at 26 °C. The heterologous combinations of CPLNV RNA 1 and RNA 2 of SCNMV or of RCNMV acquired the ability to infect white clover at 26 °C and also caused symptoms characteristically different from those induced by their parental viruses on Phaseolus vulgaris L. cv. Red Kidney.

Molecular detection of alfalfa witches’-broom phytoplasma in four leafhopper species associated with infected alfalfa plants

Abstract Alfalfa witches’-broom (AWB) phytoplasma was detected in four leafhopper species ( Aceratagallia sp., Neokolla hieroglyphica, Cuerna septentrionalis and Macrosteles fascifrons ) and infected alfalfa plant samples by PCR. The transmission results suggest that Neokolla and Cuerna species could be the vectors of AWB phytoplasma. The amplified DNAs yielded products of 1.5 kb (primer pair P1/P6), 196 bp (primer pair 1A/1B) and 1.2 kb (primer pair R16 F2/R2) by direct and nested-PCR assay. After RFLP analyses, using the four endonuclease enzymes, similar patterns were formed among the digested 1.2 kb PCR products of all the leafhopper and plant samples, indicating the presence of the same kind of a phytoplasma isolate. The RFLP analyses and nested-PCR assays, using the primer pair 1A/1B, revealed that this isolate was genetically related to the clover proliveration group

The Dianthoviruses: A Distinct Group of Isometric Plant Viruses with Bipartite Genome

Publisher Summary This chapter discusses the dianthoviruses, emphasizing their unique bipartite genome strategy. The structure of dianthoviruses is simple and their RNAs—a bipartite genome—are compatible not only among strains of a virus but also among member viruses in the group. Dianthoviruses share a unique feature with pea enation mosaic virus in that the coat protein genome resides on RNA-1, the viruses of the dianthovirus group sediment as a single component. Coupled with other attractive properties such as high virus yield and stability, the group offers an ideal experimental system to research basic problems related to virus structure, replication, and genome strategy. There are conceivable advantages and disadvantages of divided genome systems in dianthoviruses. Deletion of RNA nucleotides representing smaller genome parts is a possible disadvantage of certain divided genome systems. However, such a case has not yet been reported for dianthoviruses. Future studies must address the issues of the relevance of the dsRNAs to the viral replication process, the mode of genesis of subgenomic RNAs, determination of genome functions, and the nature of the viral translation products generated in vitro. The dianthoviruses can serve as an excellent system for such studies and will continue to yield many interesting results, which will help expand the scope of plant virology.

Highly sensitive digoxigenin-labelled DNA probe for the detection of potato spindle tuber viroid

A molecular probe pSPAv6.2(+), with concatameric insert representing 6.2-times repeated copy of potato spindle tuber viroid (PSTV) RNA, was labelled with digoxigenin and used to detect PSTV by dot-blot hybridization assay. The probe was highly sensitive and specific, detecting as little as 2.5 pg of PSTV RNA. Both severe and mild PSTV strains were detectable in 64-512-times diluted crude extracts from infected tomato leaves, and potato leaves, sprouts, and seeds. For extraction of plant tissue three buffers were compared to determine the lowest non-specific background and the highest sensitivity. The results showed that the digoxigenin-labelled probe is as sensitive as the 32P-labelled probe and can replace radioactive techniques in PSTV detection. With such high sensitivity, the probe is also potentially useful for detecting the viroid in composite samples of mass-indexing programs.

Molecular biology and epidemiology of dianthoviruses.

Abstract The genus Dianthovirus is one of eight genera in the family Tombusviridae. All the genera have monopartite positive-stranded RNA genomes, except the dianthoviruses which have bipartite genomes. The dianthoviruses are distributed worldwide. Although they share common structural features with the other Tombusviridae viruses in their virions and the terminal structure of the genomic RNAs, the bipartite nature of the dianthovirus genome offers an ideal experimental system with which to study basic issues of virology. The two genomic RNAs seem to use distinct strategies to regulate their translation, transcription, genome replication, genome packaging, and cell-to-cell movement during infection. This review summarizes the current state of our knowledge of the dianthoviruses, with its main emphasis on the molecular biology of the virus, including the viral and host factors required for its infection of host plants. The epidemiology of the virus and the possible viral impacts on agriculture and the environment are also discussed.

Soil-Borne Viruses of Plants

A virus is soil-borne if it infects plants via soil. This may occur either through the activity of a soil-borne vector or by mechanical means, and usually this implies virus transmission to the underground parts of plants. However, if leaves or other aerial parts were infected following contact with soil (2), this would also be included. Viruses excluded from this definition are those transmitted by direct root-to-root contact, such as potato virus X (111) and cucumber mosaic virus (147), or root grafts, such as apple mosaic virus (61). Thus, to be soil-borne, a virus should have an existence in soil outside of a living host plant.

Unilateral Compatibility of Genome Segments from Two Distinct Strains of Red Clover Necrotic Mosaic Virus

Summary Two strains of red clover necrotic mosaic virus (TpM 34 and TpM 48) were distinguished by their serological relationship, by the symptoms they induced in selected host plants and by Northern hybridization analysis. Purified RNA 1 and RNA 2 of the two strains were inoculated to the leaves of Chenopodium amaranticolor, Vigna unguiculata and C. quinoa in all possible combinations. It was demonstrated that a heterologous mixture containing RNA 1 of TpM 34 and RNA 2 of TpM 48 was infectious, resulting in lesion development whereas the reciprocal combination was not. The infectious pseudorecombinant was isolated by several single lesion transfers in C. quinoa and its genetic nature was confirmed by serology and Northern hybridization analysis. Inoculation of the pseudorecombinant and the two parental strains to five selected host plants revealed that symptom expression was determined by RNA 1.

Host specificity in transmission of tobacco stunt virus by Olpidium brassicae

Abstract Seventy-one species in 24 families were tested for susceptibility to tobacco stunt virus (TSV) by inoculating with viruliferous zoospores of Olpidium brassicae (Wor.) Dang. TSV was transmitted to 35 species by the fungus. Most of these species were further tested for susceptibility by mechanical inoculation with TSV obtained from Olpidium -free tobacco leaves ( Nicotiana tabacum L. ‘Bright Yellow’). The following 4 types were obtained regarding the infections with Olpidium and TSV: (1) susceptible to both Olpidium and TSV, (2) susceptible to Olpidium but not to TSV, (3) susceptible to TSV but not to Olpidium , and (4) susceptible to neither Olpidium nor TSV. Successful TSV transmission by Olpidium was obtained in 8 species which were resistant to TSV when inoculated mechanically. In 5 species, the association of Olpidium with TSV did not contribute to transmission or TSV was lost in the presence of Olpidium , though the plants were susceptible to TSV when inoculated mechanically. These results suggest that the interrelationships among host, fungus, and virus are rather complex, and indicate that host specificity to the fungus vector as well as the virus is involved in transmission and prevalence of the virus.

A comparative study of the RNA-2 nucleotide sequences of two sweet clover necrotic mosaic virus strains

The nucleotide sequences of the RNA-2s of two strains of sweet clover necrotic mosaic virus (SCNMV-38 and -59) have been determined. The RNA-2s of SCNMV-38 and -59 consist of 1446 and 1449 nucleotides, respectively, and both contain one major open reading frame (ORF) which potentially can encode polypeptides of 326 amino acid residues (about 36.5K), designated SC38P2 and SC59P2, respectively. The nucleotide sequences of SCNMV-38 and -59 RNA-2s show 93.2% similarity, and the amino acid sequences of SC38P2 and SC59P2 are 91.7% identical, although the identical nucleotides and amino acids are not distributed uniformly in RNA-2 and the encoded proteins. Two highly conserved regions (from positions 23 to 221 and 297 to 326) and a relatively divergent region (from positions 222 to 296) are found in the P2 proteins of these strains. A similar pattern is apparent on comparison of the nucleotide and deduced amino acid sequences of RNA-2 of these SCNMV strains with those of the Australian and Czechoslovakian isolates of red clover necrotic mosaic virus.