The complete nucleotide sequence of a Pakistani isolate of Watermelon mosaic virus provides further insights into the taxonomic status in the Bean common mosaic virus subgroup.

Watermelon mosaic virus (WMV) is a potyvirus with a worldwide distribution, but is mostly found in temperate and Mediterranean regions. The complete nucleotide (nt) sequence of a Pakistani isolate of WMV (WMV-Pk) was determined and compared with French isolate (WMV-Fr) and other closely related potyviruses. WMV-Pk showed overall identities of 94.4% (nt) and 96% (amino acid; aa) with the WMV-Fr. However, variability was observed in the 5' UTR and P1 region. Although sequence identities over most of the genome were well above 90% at both the nt and aa levels, reaching 99.6% (aa) in the CP and 100% (aa) in the 6K1 and 6K2, thereby suggesting that WMV-Pk and WMV-Fr are identical strains, but the sequence identities in the P1 region were only 80.6% (aa) and 82.8% (nt), while that in the 5' UTR was 82%. These differences may be due to different mutation phenomena of a common ancestor virus or mutations caused by different selection pressures in two different agro-ecological zones. The sequence of WMV-Pk is very close to that of Soybean mosaic virus (SMV) over most of the genome, except for the N-terminal region, which is subject to recombination between SMV and Peanut stripe virus (PSV)/Bean common mosaic virus (BCMV), as revealed by Simplot and phylogenetic analyses of N- and C-terminal P1, HC-Pro, and 5' UTR regions of the genome.

A simplified method for obtaining plant viral RNA for RT-PCR.

An easy and fast procedure (named the simple-direct-tube (SDT) method) was developed for preparing plant virus RNA for cDNA synthesis. The SDT method can be completed in approximately 15min and does not require the use of antiserum, filtering or centrifugation. The procedure to grind plant tissues in phosphate-buffered saline containing Tween-20 (PBST) and to place the extract in a microfuge tube for a few minutes allow adsorption of the virus particles to the tube wall. The sap is then removed and the tube is washed with PBST before the addition of RNase-free water. This manipulation can be performed at room temperature. Using this method followed by reverse transcription-polymerase chain reaction (RT-PCR), infections by turnip mosaic virus, cucumber mosaic virus, and cucumber green mottle mosaic virus (CGMMV) were readily detected, indicating that the SDT method can be used in assays to detect different viruses. For the detection of CGMMV, it was necessary to heat the tubes before cDNA synthesis, suggesting that the immobilized CGMMV particles required disruption by heat treatment to release RNA.

An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein.

Turnip mosaic virus (TuMV, genus Potyvirus, family Potyviridae) infects mainly cruciferous plants. Isolates Tu-3 and Tu-2R1 of TuMV exhibit different infection phenotypes in cabbage (Brassica oleracea L.) and Japanese radish (Raphanus sativus L.). Infectious full-length cDNA clones, pTuC and pTuR1, were constructed from isolates Tu-3 and Tu-2R1, respectively. Progeny virus derived from infections with pTuC induced systemic chlorotic and ringspot symptoms in infected cabbage, but no systemic infection in radish. Virus derived from plants infected with pTuR1 induced a mild chlorotic mottle in cabbage and infected radish systemically to induce mosaic symptoms. By exchanging genome fragments between the two virus isolates, the P3-coding region was shown to be responsible for systemic infection by TuMV and the symptoms it induces in cabbage and radish. Moreover, exchanges of smaller parts of the P3 region resulted in recombinants that induced complex infection phenotypes, especially the combination of pTuC-derived N-terminal sequence and pTuR1-derived C-terminal sequence. Analysis by tissue immunoblotting of the inoculated leaves showed that the distributions of P3-chimeric viruses differed from those of the parents, and that the origin of the P3 components affected not only virus accumulation, but also long-distance movement. These results suggest that the P3 protein is an important factor in the infection cycle of TuMV and in determining the host range of this and perhaps other potyviruses.

Nucleotide sequence and genome organization of Cucumber yellows virus, a member of the genus Crinivirus.

The genome of Cucumber yellows virus (CuYV), isolated in Japan from cucumber (Cucumis sativus L.), was completely sequenced and shown to be bipartite. CuYV RNA1 consisted of 7889 nucleotides and encompassed seven open reading frames (ORFs), which is typical of the Closteroviridae, including a heat-shock protein 70 homologue, a coat protein and a diverged coat protein (CPd). CuYV RNA2 consisted of 7607 nucleotides and included two ORFs: ORF1a potentially encoded a polyprotein containing putative papain-like protease, methyltransferase and helicase domains, and ORF 1b potentially encoded an RNA-dependent RNA polymerase, which is probably expressed via a +1 ribosomal frameshift. The size and organization of the CuYV genome are similar to those of Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, indicating that CuYV is a member of that genus, although CuYV differed in several points from LIYV.

Improved Saunders method for the analysis of lateral shearing interferograms.

An interferogram obtained by use of ordinary interferometers, such as Fizeau and Twyman-Green interferometers, will show a contour map of the wave front under test. A lateral-shearing interferogram, however, will show a contour map of the difference between the wave front under test and a sheared wave front, that is, a contour map of the derivative of the wave front under test. Therefore one can reconstruct the shape of the wave front under test by analyzing that difference. Many methods for reconstructing a wave front have been proposed. The Saunders method reconstructs a wave front; rapidly however the wave-front data are reconstructed only at intervals of the amount of shear along the direction of the shear. Therefore the method has low spatial resolution. A method for reconstructing a wave front that is based on the Saunders method and has high spatial resolution is proposed. The method analyzes the differences that are produced by shearing of the wave front under test in many directions. This method requires a large number of interferograms for reconstructing the wave front. Here the method is described, and its validity is confirmed by simulation.

Antiserum raised against gyrase A of Acholeplasma laidlawii reacts with phytoplasma proteins.

Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector. The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli. The fusion protein was purified and used as an antigen for rabbit immunization. Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A. laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants. The antiserum could also detect phytoplasmas in infected plant sap. These results demonstrate that the gyrase A protein (GyrA) of A. laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.

Two Groups of Phytoplasmas from Japan Distinguished on the Basis of Amplification and Restriction Analysis of 16S rDNA.

Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.