Detection of Sri Lankan cassava mosaic virus by loop-mediated isothermal amplification using dried reagents.

Recently, the widespread occurrence of Sri Lankan cassava mosaic virus (SLCMV), genus Begomovirus, family Geminiviridae, which causes a mosaic disease in cassava (Manihot esculenta Crantz) in South-East Asia have, become a serious economic issue. Since cassava is propagated through vegetative cuttings, a rapid virus diagnostic method is crucial for generating virus-free planting materials. In this study, a loop-mediated isothermal amplification (LAMP) assay using six primers was developed and validated for the rapid detection of SLCMV in cassava leaves. This SLCMV assay had a detection sensitivity that was up to 10,000 times higher than that of the conventional polymerase chain reaction assay and can detect the virus from symptomless stem cutting, which is a potential long-distance spreader of the virus. Furthermore, a practical LAMP protocol using stable dried reagents from a commercial kit was established so that the assay could be performed in the field by incubating the reactions in water at 60-65 °C instead of using a thermal cycler. The primer sequences and the LAMP protocol described here should be useful for the rapid and sensitive on-site detection of SLCMV.

Insights Into Natural Genetic Resistance to Rice Yellow Mottle Virus and Implications on Breeding for Durable Resistance.

Rice is the main food crop for people in low- and lower-middle-income countries in Asia and sub-Saharan Africa (SSA). Since 1982, there has been a significant increase in the demand for rice in SSA, and its growing importance is reflected in the national strategic food security plans of several countries in the region. However, several abiotic and biotic factors undermine efforts to meet this demand. Rice yellow mottle virus (RYMV) caused by Solemoviridae is a major biotic factor affecting rice production and continues to be an important pathogen in SSA. To date, six pathogenic strains have been reported. RYMV infects rice plants through wounds and rice feeding vectors. Once inside the plant cells, viral genome-linked protein is required to bind to the rice translation initiation factor [eIF(iso)4G1] for a compatible interaction. The development of resistant cultivars that can interrupt this interaction is the most effective method to manage this disease. Three resistance genes are recognized to limit RYMV virulence in rice, some of which have nonsynonymous single mutations or short deletions in the core domain of eIF(iso)4G1 that impair viral host interaction. However, deployment of these resistance genes using conventional methods has proved slow and tedious. Molecular approaches are expected to be an alternative to facilitate gene introgression and/or pyramiding and rapid deployment of these resistance genes into elite cultivars. In this review, we summarize the knowledge on molecular genetics of RYMV-rice interaction, with emphasis on host plant resistance. In addition, we provide strategies for sustainable utilization of the novel resistant sources. This knowledge is expected to guide breeding programs in the development and deployment of RYMV resistant rice varieties.

Complete genome sequence of aucuba ringspot virus.

A new badnavirus, aucuba ringspot virus (AuRV), was identified in plants of Aucuba japonica showing mild mosaic, vein banding, and yellow ringspot symptoms on the leaves. The complete nucleotide sequence of the AuRV genome was determined and found to be 9,092 nt in length, and the virus was found to have a genome organization typical of members of the genus Badnavirus. ORF3 was predicted to encode a polyprotein containing conserved movement protein, coat protein, aspartic protease, reverse transcriptase (RT), and RNase H domains. Phylogenetic analysis suggested that this virus is most closely related to codonopsis vein clearing virus but belongs to a distinct species, based on only 69.6% nucleotide sequence identity within the part of ORF 3 encoding the RT and RNase H domains. The vector of AuRV is unknown, but based on phylogenetic relationships, it is predicted to be a type of aphid.

Association of an alphasatellite with tomato yellow leaf curl virus and ageratum yellow vein virus in Japan is suggestive of a recent introduction.

Samples were collected in 2011 from tomato plants exhibiting typical tomato leaf curl disease symptoms in the vicinity of Komae, Japan. PCR mediated amplification, cloning and sequencing of all begomovirus components from two plants from different fields showed the plants to be infected by Tomato yellow leaf curl virus (TYLCV) and Ageratum yellow vein virus (AYVV). Both viruses have previously been shown to be present in Japan, although this is the first identification of AYVV on mainland Japan; the virus previously having been shown to be present on the Okinawa Islands. The plant harboring AYVV was also shown to contain the betasatellite Tomato leaf curl Java betasatellite (ToLCJaB), a satellite not previously shown to be present in Japan. No betasatellite was associated with the TYLCV infected tomato plants analyzed here, consistent with earlier findings for this virus in Japan. Surprisingly both plants were also found to harbor an alphasatellite; no alphasatellites having previously been reported from Japan. The alphasatellite associated with both viruses was shown to be Sida yellow vein China alphasatellite which has previously only been identified in the Yunnan Province of China and Nepal. The results suggest that further begomoviruses, and their associated satellites, are being introduced to Japan. The significance of these findings is discussed.

Characterization of Lagenaria mild mosaic virus, a New Potexvirus from Bottle Gourd in Myanmar.

A putative Potexvirus was detected from bottle gourd (Lagenaria siceraria) showing mosaic and mottle symptoms in Myanmar in 2007. The virus was designated Lagenaria mild mosaic virus (LaMMoV) and was further characterized. In artificial inoculation tests, infectivity of LaMMoV was limited to two families: Chenopodiaceae and Cucurbitaceae. The host range of LaMMoV differs from those of the two cucurbit-infecting potexviruses, Alternanthera mosaic virus (AltMV) and Papaya mosaic virus (PapMV). Sequence analyses of LaMMoV showed that the C-terminal 3,859 nucleotides, excluding the poly-A tail, includes the C-terminal region of an RNA-dependent RNA polymerase (RdRp), a triple gene block (TGB), a coat protein (CP), and a 3' untranslated region (UTR), all of which are typical of potexviruses. Although LaMMoV is related closely to AltMV and PapMV, its nucleotide sequences differ from those of other previously reported potexviruses. Therefore, we report LaMMoV as a new species of the genus Potexvirus that occurs in the cucurbit bottle gourd.

A point mutation changes the serotype of a potato virus Y isolate; genomic determination of the serotype of PVY strains.

A Syrian isolate of Potato virus Y (PVY), named PVY-12, reacted to two monoclonal antibodies that are specific to PVY(O,C) and PVY(N) strains, although its coat protein (CP) belongs to the PVY(N) strain. Analysis of the CP of PVY-12 revealed that a point mutation in its N terminus switched it from PVY(N)-like to PVY(O)-like at this position. This mutation changed the second nucleotide of the codon that encodes the 29th amino acid of the CP of PVY-12 from A to G, which resulted in one amino acid substitution from Glu(29 )to Gly(29). The role of Gly(29) in the binding of PVY-12 to PVY(O,C)-specific monoclonal antibody was confirmed by gene expression in Escherichia coli. The N terminus of the CP gene of PVY-12 and another PVY isolate of the N serotype with identical CP to PVY-12 except for one amino acid substitution from Gly(29 )to Glu(29) was cloned and expressed in E. coli using a pUC18 vector. Resulting antigens showed similar reactivity to the relevant antibodies as same as the native CPs of these two isolates. Further analysis of the CP of PVY isolates showed that Gly(29) was conserved in the CP of PVY(O), PVY(C), PVY(N)W, and non-potato isolates of PVY while Gln(17) and Glu(31 )were conserved in the CP of PVY(N/NTN). Therefore, these amino acids are characteristic of the CP for these strain groups and subgroups in agreement with the serotype and phylogenetic relationships previously determined.

Neonectria castaneicola and Neo. rugulosa in Japan.

Differences between Neonectria castaneicola, which causes stem and perennial canker of trees, and Neo. rugulosa have not been clearly shown in previous studies. In this study these two species were compared in detail using 17 Japanese isolates consisting of 10 strains of Neo. castaneicola and seven of Neo. rugulosa. Four-spored asci were constantly found in Neo. castaneicola and this species produced larger ascospores and macroconidia than Neo. rugulosa which produced eight-spored asci. The mating system of Neo. castaneicola was homothallic while Neo. rugulosa was heterothallic. Characters in each species, such as the number of ascospores in an ascus and mating system, were constantly transferred to the 3rd generation. Molecular analysis revealed that the 10 isolates of Neo. castaneicola and seven of Neo. rugulosa were differentiated using rDNA sequence data from the nuclear rDNA ITS region. Moreover, Neo. castaneicola and Neo. rugulosa were separated into different clades. From these results, it was concluded that Neo. castaneicola should be maintained as an independent species, separate from Neo. rugulosa. The isolates of Neo. rugulosa used in this study were the first reported in Japan and found on Castanea crenata, Castanopsis sp., Myrica rubra and Quercus acutissima.