Molecular markers associated with resistance to squash leaf curl China virus and tomato leaf curl New Delhi virus in tropical pumpkin (Cucurbita moschata Duchesne ex Poir.) breeding line AVPU1426.

Virus diseases are a major production constraint for pumpkin. Recessive resistance to squash leaf curl China virus and tomato leaf curl New Delhi virus has been mapped in Cucurbita moschata (Duchesne ex Poir.) breeding line AVPU1426 to chromosomes 7 and 8, respectively. Molecular markers tightly associated with the resistance loci have been developed and were able to correctly predict resistance and susceptibility with an accuracy of 99% for squash leaf curl China virus resistance and 94.34% for tomato leaf curl New Delhi virus in F2 and back cross populations derived from the original resistance source AVPU1426. The markers associated with resistance are recommended for use in marker-assisted breeding.

Thermoresponsive C22 phage stiffness modulates the phage infectivity.

Bacteriophages offer a sustainable alternative for controlling crop disease. However, the lack of knowledge on phage infection mechanisms makes phage-based biological control varying and ineffective. In this work, we interrogated the temperature dependence of the infection and thermo-responsive behavior of the C22 phage. This soilborne podovirus is capable of lysing Ralstonia solanacearum, causing bacterial wilt disease. We revealed that the C22 phage could better infect the pathogenic host cell when incubated at low temperatures (25, 30 °C) than at high temperatures (35, 40 °C). Measurement of the C22 phage stiffness revealed that the phage stiffness at low temperatures was 2-3 times larger than at high temperatures. In addition, the imaging results showed that more C22 phage particles were attached to the cell surface at low temperatures than at high temperatures, associating the phage stiffness and the phage attachment. The result suggests that the structure and stiffness modulation in response to temperature change improve infection, providing mechanistic insight into the C22 phage lytic cycle. Our study signifies the need to understand phage responses to the fluctuating environment for effective phage-based biocontrol implementation.

Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus.

BACKGROUND: Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA). METHODS: Monoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard. RESULTS: A TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA. CONCLUSIONS: Our findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

The complete genomic sequence of the novel myovirus RP13 infecting Ralstonia solanacearum, the causative agent of bacterial wilt.

A novel lytic bacteriophage, Ralstonia phage RP13, was isolated from tomato fields in Pang Nga, Thailand. Electron microscopic observation showed it to have the features of a myovirus with a novel triangulation number (T = 21, dextro). The RP13 DNA appeared to be heavily modified. By applying RNA sequencing and RNA-sequence-mediated DNA sequencing, the whole genome of RP31 was determined to be 170,942 bp in length with a mean G+C content of 39.2%. A total of 277 ORFs were identified as structural, functional, or hypothetical genes in addition to four tRNA genes. Phylogenetic analysis suggested that RP13 is not closely related to any other known phages. Thus, we concluded that the RP13 is a novel phage infecting R. solanacearum strains and will be a useful biocontrol agent against bacterial wilt disease.

3D structure of three jumbo phage heads.

Jumbo phages are bacteriophages that carry more than 200 kbp of DNA. In this study we characterized two jumbo phages (ΦRSL2 and ΦXacN1) and one semi-jumbo phage (ΦRP13) at the structural level by cryo-electron microscopy. Focusing on their capsids, three-dimensional structures of the heads at resolutions ranging from 16 to 9 Å were calculated. Based on these structures we determined the geometrical basis on which the icosahedral capsids of these phages are constructed, which includes the accessory and decorative proteins that complement them. A triangulation number novel to Myoviridae (ΦRP13; T=21) was discovered as well as two others, which are more common for jumbo phages (T=27 and T=28). Based on one of the structures we also provide evidence that accessory or decorative proteins are not a prerequisite for maintaining the structural integrity of very large capsids.

C22 podovirus infectivity is associated with intermediate stiffness.

Bacteriophages have potential for use as biological control agents (biocontrols) of pathogenic bacteria, but their low stability is limiting for their utilization as biocontrols. Understanding of the conditions conducive to storage of phages in which infectivity is maintained over long periods will be useful for their application as biocontrols. We employed a nanomechanical approach to study how external environmental factors affect surface properties and infectivity of the podovirus C22 phage, a candidate for biocontrol of Ralstonia solanacearum, the agent of bacterial wilt in crops. We performed atomic force microscopy (AFM)-based nano-indentation on the C22 phage in buffers with varying pH and ionic strength. The infectivity data from plaque assay in the same conditions revealed that an intermediate range of stiffness was associated with phage titer that remained consistently high, even after prolonged storage up to 182 days. The data are consistent with the model that C22 phage must adopt a metastable state for maximal infectivity, and external factors that alter the stiffness of the phage capsid lead to perturbation of this infective state.

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

BACKGROUND: Tomato 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. METHODS: The 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. RESULTS: Two 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. CONCLUSIONS: TAS-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.

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.

Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages.

Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All ß- and ß'-subunits were detected in ϕRSF1, but one ß'-subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants.

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 strain's cytoplasm, as revealed by fluorescence microscopy with SYBR Gold-labeled phage particles.

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.

Ethylene and pollination decrease transcript abundance of an ethylene receptor gene in Dendrobium petals.

We studied the expression of a gene encoding an ethylene receptor, called Ethylene Response Sensor 1 (Den-ERS1), in the petals of Dendrobium orchid flowers. Transcripts accumulated during the young floral bud stage and declined by the time the flowers had been open for several days. Pollination or exposure to exogenous ethylene resulted in earlier flower senescence, an increase in ethylene production and a lower Den-ERS1 transcript abundance. Treatment with 1-methylcyclopropene (1-MCP), an inhibitor of the ethylene receptor, decreased ethylene production and resulted in high transcript abundance. The literature indicates two kinds of ethylene receptor genes with regard to the effects of ethylene. One group shows ethylene-induced down-regulated transcription, while the other has ethylene-induced up-regulation. The present gene is an example of the first group. The 5' flanking region showed binding sites for Myb and myb-like, homeodomain, MADS domain, NAC, TCP, bHLH and EIN3-like transcription factors. The binding site for the EIN3-like factor might explain the ethylene effect on transcription. A few other transcription factors (RAV1 and NAC) seem also related to ethylene effects.

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 ß-1,4-glucanase (Den-Cel1) in the abscission zone (AZ). The abundance of Den-Cel1 mRNA was highly correlated with ß-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.

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 (approximately 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 degrees 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.

Molecular characterization of tomato-infecting begomoviruses in Thailand.

Bipartite geminiviruses infecting tomatoes in Thailand were detected by polymerase chain reaction (PCR) using CPA5/CPA2 primers. Products derived from PCR-amplified full-length DNA-A and DNA-B of TYLCV collected from Chiang Mai, Nong Khai, and Sakon Nakhon were cloned and sequenced. DNA-A from Chiang Mai was 2747 nts long; Nong Khai, 2744 nts; and Sakon Nakhon, 2747 nts, and those of DNA-B from Chiang Mai were 2750 nts long; Nong Khai, 2749 nts; and Sakon Nakhon, 2749 nts. The genomes of these virus isolates were organized like those of other begomoviruses. The DNA-A had two ORFs in the virion sense and four ORFs in the complementary sense. The DNA-B had two ORFs in the virion sense and one ORF in the complementary sense. Nucleotide sequences of DNA-A of TYLCV from Chiang Mai, Nong Khai, and Sakon Nakhon were closely related to those of Tomato yellow leaf curl Thailand virus (TYLCTHV) and Tomato yellow leaf curl Thailand virus-[Myanmar] (TYLCTHV-[MM]) with nucleotide sequence identity ranging from 89% to 95%. Based on sequence comparisons and phylogenetic analyses, these three virus isolates studied were identified as new strains of TYLCTHV and named Tomato yellow leaf curl Thailand virus-Chiang Mai (TYLCTHV-[CM]The GenBank accession codes for DNA-A of TYLCTHV-[CM], -[NK], and -[SK] are , and , respectively. The GenBank accession codes for DNA-B of TYLCTHV-[CM], -[NK], and -[SK] are , , and , respectively.), Nong Khai (TYLCTHV-[NK] and Sakon Nakhon (TYLCTHV-[SK]).

Tomato leaf curl Karnataka virus from Bangalore, India, Appears to be a Recombinant Begomovirus.

ABSTRACT The genome of Tomato leaf curl virus (ToLCV) from Bangalore, India, a whitefly-transmitted geminivirus, was cloned (pIND9) and sequenced. The circular DNA of 2,759 nucleotides (U38239) is organized similarly to that of other begomoviruses with monopartite genomes. Comparison of the nucleotide sequence of pIND9 with other tomato-associated begomoviruses from India (Tomato leaf curl Bangalore virus [ToLCBV, Z48182]) and Tomato leaf curl New Delhi virus-Severe (ToLCNdV-Svr, U15015) showed moderate DNA sequence identities (82 to 87%) between capsid protein (CP) genes but low identities (66 to 67%) for the intergenic regions and the replication-associated protein (Rep) genes (75 to 81% identity). Phylogenetic trees generated with nucleotide sequences of the Rep and CP genes of 26 begomoviruses indicated that this ToLCV is distinct from other begomoviruses and that it may be a recombinant virus derived from at least three different viral lineages. Tomatoes (Lycopersicon esculentum) inoculated with the cloned DNA monomer of ToLCV (pIND9) via particle bombardment developed leaf curling and yellowing symptoms. The virus was transmitted by Bemisia tabaci biotype B from tomatoes infected via particle bombardment to healthy tomatoes and by sap inoculation from infected tomatoes to tomato, Nicotiana benthamiana and N. tabacum. This ToLCV is a distinct member of the genus Begomovirus from India that differs from the previously characterized Tomato leaf curl Sadasivanagar virus isolate Bangalore 1 (L12739), ToLCBV (Z48182), ToLCBV isolate Bangalore 4 (AF165098), and the bipartite ToLCNdV (U15015, U15016). Thus, this ToLCV is named Tomato leaf curl Karnataka virus (ToLCKV).