Orawan Chatchawankanphanich

Field Evaluation of Mungbean Recombinant Inbred Lines against Mungbean Yellow Mosaic Disease Using New Disease Scale in Thailand

Studies were conducted to identify the sources of resistance in mungbean recombinant inbred lines (RILs) in Thailand against mungbean yellow mosaic disease (MYMD). 146 mungbean RILs in series were evaluated in a field including resistant parent NM-10-12-1 and susceptible parent KPS 2 during summer 2008 under high inoculum pressure. The RILs were subsequently scored for disease symptom severity ratings (DSSR) using a new scale. Observations regarding DSSR and % disease index (%DI) showed that the tested RILs responded differently to the disease. A large number of RILs (132) were found highly susceptible, 12 were susceptible, 3 were tolerant and one was resistant. Overall screening results showed that three RILs, viz. line no. 30, 100 and 101 had minimum DSSR and % disease index thus they are good source of resistance to MYMD in spite of high disease pressure and can therefore be used directly as varieties to manage the disease in Thailand.

Detection of quantitative trait loci for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean (Vigna radiata (L.) Wilczek) in India and Pakistan

Yellow mosaic disease (YMD) is one of the major diseases affecting mungbean (Vigna radiata (L.) Wilczek). In this study, we report the mapping of the quantitative trait locus (QTL) for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean. An F8 recombinant inbred line (RIL) mapping population was generated in Thailand from a cross between NM10-12-1 (MYMIV resistance) and KPS2 (MYMIV susceptible). One hundred and twenty-two RILs and their parents were evaluated for MYMIV resistance in infested fields in India and Pakistan. A genetic linkage map was developed for the RIL population using simple sequence repeat (SSR) markers. Composite interval mapping identified five QTLs for MYMIV resistance: three QTLs for India (qYMIV1, qYMIV2 and qYMIV3) and two QTLs for Pakistan (qYMIV4 and qYMIV5). qYMIV1, qYMIV2, qYMIV3, qYMIV4 and qYMIV5 explained 9.33%, 10.61%, 12.55%, 21.93% and 6.24% of variation in disease responses, respectively. qYMIV1 and qYMIV4 appeared to be the same locus and were common to a major QTL for MYMIV resistance in India identified previously using a different resistant mungbean.

Glutamine synthetase mutation conferring target‐site‐based resistance to glufosinate in soybean cell selections

BACKGROUND Glufosinate-resistant soybean cells were achieved through direct selection of diploid cells in the suspension culture. Here, the mutations in the glutamine synthetase (GS) gene are described to understand the evidence pointing to the functional role of the GS gene in the herbicide sensitivity of the mutant cells. RESULTS Based on the I(50) values, dose-response experiments at the cell level showed that the resistance ratio of the resistant cell was 50-fold, whereas the in vitro inhibition of GS activity required a 4.56-fold greater concentration of glufosinate in the resistant cell than in the untreated control. Comparison of the nucleotide sequences identified nine point differences in the GS gene between the resistant and untreated cells, leading to eight amino acid substitutions in the deduced polypeptide sequence. Northern hybridization of the GS mRNA showed that the accumulation of GS gene mRNA transcript in resistant cells was higher than that in the untreated cells. CONCLUSION Changes in sensitivity to glufosinate have been related to mutations at the binding site of the herbicide on the glutamine synthetase. His(249) is one of the residues implicated in the binding domain for the substrate and inhibitor, and hence the exchange of this residue with tyrosine plays a role in lowering the sensitivity of the mutated enzyme.

Endogenous auxin regulates the sensitivity of Dendrobium (cv. Miss Teen) flower pedicel abscission to ethylene

Dendrobium flower buds and flowers have an abscission zone at the base of the pedicel (flower stalk). Ethylene treatment of cv. Miss Teen inflorescences induced high rates of abscission in flower buds but did not affect abscission once the flowers had opened. It is not known if auxin is a regulator of the abscission of floral buds and open flowers. The hypotheses that auxin is such a regulator and is responsible for the decrease in ethylene sensitivity were tested. Severed inflorescences bearing 4-8 floral buds and 4-6 open flowers were used in all tests. The auxin antagonists 2,3,5-triiodobenzoic acid (TIBA, an inhibitor of auxin transport) or 2-(4-chlorophenoxy)-2-methyl propionic acid (CMPA, an inhibitor of auxin action) were applied to the stigma of open flowers. Both chemicals induced high flower abscission rates, even if the inflorescences were not treated with ethylene. The effects of these auxin antagonists virtually disappeared when the inflorescences were treated with 1-methylcyclopropene (1-MCP), indicating that the abscission induced by the auxin antagonists was due to ethylene. Removal of the open flowers at the distal end of the pedicel hastened the time to abscission of the remaining pedicel, and also resulted in an increase in ethylene sensitivity. Indole-3-acetic acid (IAA) in lanolin, placed on the cut surface of the pedicel, replaced the effect of the removed flower. Treatments that promoted abscission of open flowers up-regulated a gene encoding a β-1,4-glucanase (Den-Cel1) in the abscission zone (AZ). The abundance of Den-Cel1 mRNA was highly correlated with β-1,4-glucanase activity in the AZ. The results show that auxin is an endogenous regulator of floral bud and flower abscission and suggest that auxin might explain, at least partially, why pedicel abscission of Dendrobium cv. Miss Teen changes from very ethylene-sensitive to ethylene-insensitive.

Genomic diversity of large-plaque-forming podoviruses infecting the phytopathogen Ralstonia solanacearum

The genome organization, gene structure, and host range of five podoviruses that infect Ralstonia solanacearum, the causative agent of bacterial wilt disease were characterized. The phages fell into two distinctive groups based on the genome position of the RNA polymerase gene (i.e., T7-type and ϕKMV-type). One-step growth experiments revealed that ϕRSB2 (a T7-like phage) lysed host cells more efficiently with a shorter infection cycle (ca. 60 min corresponding to half the doubling time of the host) than ϕKMV-like phages such as ϕRSB1 (with an infection cycle of ca. 180 min). Co-infection experiments with ϕRSB1 and ϕRSB2 showed that ϕRSB2 always predominated in the phage progeny independent of host strains. Most phages had wide host-ranges and the phage particles usually did not attach to the resistant strains; when occasionally some did, the phage genome was injected into the resistant strains cytoplasm, as revealed by fluorescence microscopy with SYBR Gold-labeled phage particles.

Detection of tomato yellow leaf curl Thailand virus by PCR without DNA extraction.

We report the simple and rapid method for detection of tomato yellow leaf curl Thailand virus (TYLCTHV) based on the direct capture of virus particles to the surface of a polymerase chain reaction (PCR) tube. This method allowed PCR without the time-consuming procedures of DNA extraction from infected plant tissue. A small amount of tomato tissue (∼10 mg) was ground in extraction buffer to release viruses from plant tissues. The constituents of the plant extract that might inhibit PCR activity were discarded by washing the tube with PBST buffer before adding the PCR mixture to the tube. This method was used for detection of TYLCTHV with plant sap solution diluted up to 1:20,000 and was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method. In addition, this method can be used for detection of TYLCTHV in viruliferous whiteflies. The PCR tubes with captured TYLCTHV could be used for PCR, after storage at 4°C for 4 wk. The method presented here was used for detection of begomoviruses in cucurbit and pepper. In addition, this method was effectively used to detect papaya ringspot virus in papaya and zucchini yellow mosaic virus in cucumber by reverse transcriptase (RT)-PCR.

Possible recombination of tomato-infecting begomoviruses in Thailand

Nucleotide sequences of the three distinct Tomato yellow leaf curl Thailand virus (TYLCTHV) strains (CM, NK, SK) were analyzed for recombination events. Recombination detection program analyses and a sequence alignment survey provided evidence of recombination between AC1 sequences of TYLCV, TYLCTHV-[MM], and TYLCTHV-[NK] as major parents and of ToLCLV, ToLCTWV, and TYLCTHV-[SK] as minor parents of TYLCTHV-[NK], -[SK], -[CM], respectively. The results further support the notion that interspecies recombination may play a significant role in geminivirus diversity and their emergence as important pathogens.

Replications of Two Closely Related Groups of Jumbo Phages Show Different Level of Dependence on Host-encoded RNA Polymerase

Ralstonia solanacearum phages ΦRP12 and ΦRP31 are jumbo phages isolated in Thailand. Here we show that they exhibit similar virion morphology, genome organization and host range. Genome comparisons as well as phylogenetic and proteomic tree analyses support that they belong to the group of ΦKZ-related phages, with their closest relatives being R. solanacearum phages ΦRSL2 and ΦRSF1. Compared with ΦRSL2 and ΦRSF1, ΦRP12 and ΦRP31 possess larger genomes (ca. 280 kbp, 25% larger). The replication of ΦRP12 and ΦRP31 was not affected by rifampicin treatment (20 μg/ml), suggesting that phage-encoded RNAPs function to start and complete the infection cycle of these phages without the need of host-encoded RNAPs. In contrast, ΦRSL2 and ΦRSF1, encoding the same set of RNAPs, did not produce progeny phages in the presence of rifampicin (5 μg/ml). This observation opens the possibility that some ΦRP12/ΦRP31 factors that are absent in ΦRSL2 and ΦRSF1 are involved in their host-independent transcription.

Development of a protocol for the identification of tospoviruses and thrips species in individual thrips

A protocol for identifying tospovirus and thrips species in an individual thrips sample was successfully developed. First, an individual thrips was soaked in an RNA stabilization solution to preserve protein and nucleic acids and ground in a carbonate buffer containing 0.2% sodium diethyldithiocarbamate. Initially, the thrips extracts were screened for tospovirus infection by dot blot analysis using antibodies to nucleocapsid (N) proteins of tospoviruses. Thrips extracts with positive results by dot blot analysis were further subjected to RNA extraction. Next, tospovirus species were identified by reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers for the N genes of four tospoviruses known to occur in Thailand, including Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV) and Watermelon silver mottle virus (WSMoV). The residual genomic DNA in the thrips RNA extract was used as a template to identify thrips species by PCR with species-specific primers to the internal transcribed spacer 2 regions of the rRNA of Ceratothripoides claratris, Frankliniella intonsa, Scirtothrips dorsalis and Thrips palmi. This protocol was initially validated against laboratory-reared thrips and then used to determine the occurrence of viruliferous thrips species collected from tomato, pepper, watermelon and cucumber fields in Thailand.

Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus

BackgroundTomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars.MethodsThe recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene.ResultsTwo MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72–81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78–96% identity with each other and 72–91% identity with those that were not detected by MAb D2.ConclusionsTAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of TYLCTHV and TbLCYnV, whereas TAS-ELISAs using the broad-specificity MAb D2 were highly efficient for the detection of TYLCTHV, TbLCYnV, ToLCNDV and SLCCNV. Both newly developed assays allow for sensitive, inexpensive, high-throughput detection of begomoviruses in field plant samples, as well as screening for virus-resistant cultivars.