W. S. Tsai

First Report of the Occurrence of Tomato chlorosis virus and Tomato infectious chlorosis virus in Taiwan

Pronounced yellowing symptoms on the lower leaves of tomato plants, similar to those caused by nitrogen deficiency, were observed in the spring of 1998 in The Asian Vegetable Research and Development Center and in farmers fields in southern Taiwan. However, the brittleness of the discolored leaves, occasional upward leaf rolling, and abundance of whiteflies on these plants suggested the involvement of Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) that belong to the group of whitefly-transmitted, phloem-limited criniviruses (family Closteroviridae). Leaves of symptomatic and healthy plants were collected, and total nucleic acids were extracted from 0.2 g of leaf tissue (1). The total nucleic acids were precipitated by ethanol and dissolved in 160 μl of sterile water. Eight microliters of total nucleic acids were observed on positively charged nylon membranes (Roche Diagnostic GmbH, Roch Applied Science, Germany). Two digoxigenin-labeled riboprobes, transcribed from pTIC8-44 (complementary to the 3-end region of TICV RNA 1) and pToC 78 (corresponding to the coat protein region of ToCV RNA 2), were used in hybridization tests to detect TICV and ToCV, respectively (2). Six of seventeen symptomatic tomato plant samples were positive with the ToCV probe, whereas none of the 13 samples reacted with the TICV probe. Similar symptoms as described above for tomato were observed on zinnia plants in the same locations. Five of eight zinnia samples gave a positive reaction with the ToCV probe. One of the ToCV positive samples also gave a positive reaction with the TICV probe. Electron microscopic examination from leaf-dip preparations of ToCV-positive leaf tissues, stained in 1% uranyl acetate, showed the presence of flexuous filamentous particles approximately 800 to 850 nm long. To our knowledge, this is the first evidence of the presence of ToCV and TICV in zinnia and ToCV in tomato in Taiwan. References: (1) A. Hadidi et al. J. Virol. Methods 30:261, 1990. (2) G. C. Wisler et al. Phytopathology 88:402, 1998.

Emergence and diversity of begomoviruses infecting solanaceous crops in East and Southeast Asia.

Over the past three decades diseases caused by whitefly-transmitted geminiviruses (begomoviruses) have emerged to be important constraints to the production of solanaceous crops, particularly tomato (Solanum lycopersicum) and peppers (Capsicum spp.), in many tropical and subtropical regions of the world. The most studied of these is Tomato yellow leaf curl virus (TYLCV), which has spread to many other areas from its likely origin in the Mediterranean basin region. The virus is usually associated with the polyphagous and virus-vectoring-efficient B-biotype of its vector whitefly (Bemisia tabaci). However, in Southeast and East Asia, a wide variety of distinct local begomovirus species have been identified from tomato and pepper crops over this period, and TYLCV was detected in Japan only in about 1996, China in 2006 and Korea in 2008, despite B-biotype whiteflies being present in several of the countries of the region since at least the early 1990s. Continental Southeast Asia appears to be a major center of diversity for begomoviruses and some species may have spread across the region; Tomato yellow leaf curl Thailand virus (TYLCTHV) appears to have spread from the Thailand-Myanmar region into southern China and is now displacing the local tomato-infecting species in Taiwan, and Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) appears to have spread from the Thailand-Vietnam region to Java, Indonesia. Since many of the native tomato- or pepper-infecting begomoviruses and associated satellite DNAs have also been detected in local weed species, it seems likely that their ancestors originated in these weed hosts, but with the expansion and intensification of tomato and pepper production in the region, there was selection for recombinant or mutant forms with greater virulence on tomato and/or pepper. Expansion and intensification of these crops may also have resulted in increased populations of local, and if present, B- or Q-biotype whiteflies, aiding the increase and spread of local begomovirus species.

Molecular characterization of tomato and chili leaf curl begomoviruses from Pakistan.

Leaf curl or yellowing symptoms, typical of those caused by begomovirus infection, are commonly observed in chili (Capsicum annuum) and tomato (Lycopersicon esculentum) plantings in Pakistan. One chili sample with leaf curl symptoms was collected in 1998 in Multan (Punjab Province), and two tomato samples with leaf curl and yellowing symptoms were collected from Islamabad and Dargai (North West Frontier Province) in 2000 and 2001, respectively. Virus DNA was first amplified by polymerase chain reaction using the degenerate primer pair PAL1v1978/PAR1c715 (3). The expected 1.4-kb PCR products were obtained from the three samples. Based on the sequences of the 1.4-kb DNA products, specific primers were designed to complete each of the DNA-A sequences. Two primer pairs, DNABLC1/DNABLV2 and DNABLC2/DNABLV2, were used for the detection of DNA-B (2). The genome of the tomato leaf curl isolate from Islamabad contained a DNA-A of 2,739 nucleotides (GenBank Accession No. AF448059), a DNA-B of 2,728 nucleotides (GenBank Accession No. AY150304), and had 94% nucleotide identity in the common region. The genome of the tomato leaf curl isolate from Dargai contained a DNA-A of 2,740 nucleotides (GenBank Accession No. AF448058), a DNA-B of 2,686 nucleotides (GenBank Accession No. AY150305), and had 96% nucleotide identity in the common region. Each of the tomato isolates contained eight predicted open-reading frames (ORFs) (AV1, AV2, AV3, AC1, AC2, AC3, AC4, and AC5) in the DNA-A and two predicted ORFs (BV1 and BC1) in the DNA-B. The DNA-A nucleotide sequence identity of the Islamabad isolate and Dargai tomato isolate is 96% and that of DNA-B is 88%. Sequence comparisons with begomovirus sequences available in the GenBank sequence database showed that these two tomato virus isolates had the highest sequence identity with Tomato leaf curl New Delhi virus-Severe (GenBank Accession No. U15015) from northern India (more than 95% for DNA-A and less than 90% for DNA-B). The DNA-A of the virus associated with chili leaf curl from Pakistan (GenBank Accession No. AF336806) consists of 2,754 nucleotides, containing six predicted ORFs (AV1, AV2, AC1, AC2, AC3, and AC4). The chili virus was unrelated to the two tomato begomovirus isolates from Pakistan, with which it shares less than 75% nucleotide identity. Sequence comparisons show highest sequence identity (87%) with Tomato leaf curl Bangladesh virus (GenBank Accession No. AF188481). DNA-beta of 1.3 kb was detected in the chili begomovirus isolate using Beta01/Beta02 primers (1). There was no evidence for the presence of a DNA-B in the chili begomovirus isolate when tested by the two DNA-B specific primer pairs. Based on DNA sequence comparisons, the chili leaf curl virus from Pakistan, to our knowledge, constitutes a distinct, new monopartite begomovirus. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. K. Green et al. Plant Dis. 85:1286, 2001. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Resistance to a DNA and a RNA virus in transgenic plants by using a single chimeric transgene construct.

Tomato leaf curl Taiwan virus (ToLCTWV) and Tomato spotted wilt virus (TSWV) are two major tomato viruses that cause serious economic losses. In this study, a partial C2 gene from ToLCTWV and the middle half of the N gene of TSWV were fused as a chimeric transgene to develop multiple virus resistance in transgenic plants. This construct was introduced into Nicotiana benthamiana and tomato by Agrobacterium-mediated transformation. Several transgenic lines showed no symptom post agro-inoculation with ToLCTWV and displayed high resistance to TSWV. The detection of siRNAs indicated that the resistance was via RNA silencing. This study demonstrated that linkage of gene segments from two viruses with distinct genomic organization, one DNA and the other RNA, can confer multiple virus resistance in transgenic plants via gene silencing.

Mapping of QTLs in tomato line FLA456 associated with resistance to a virus causing tomato yellow leaf curl disease

Tomato (yellow) leaf curl disease (TYLCD) is a serious threat to tomato production in the tropics and subtropics. The genetics of resistance to Tomato yellow leaf curl Thailand virus Taiwan strain (TYLCTHV-[TW]) in a highly resistant tomato line FLA456 was studied through quantitative trait loci (QTL) analysis. Four QTLs named qTy4.1, qTy6.1, qTy10.1 and qTy11.1 were detected on chromosomes 4, 6, 10, and 11, respectively, through evaluation of an F6 recombinant inbred line (RIL) population derived from a cross between FLA456 (resistant) and CLN1621L (susceptible). Gene action of all QTLs was recessive based on disease reaction of the F1. The markers SINAC1 and SLM4-34 flanked qTy4.1 on chromosome 4, and SLM11-12 and SLM11-17 defined qTy11.1, which co-located with the previously identified Ty-5 and Ty-2 loci, respectively. qTy6.1 was flanked by the markers SLM6-55 and TES-0014, and qTy10.1 by the markers SLM10-80-SLM10-46 on chromosomes 6 and 10. The LOD values of the putative QTLs ranged from 2.79 to 13.76. The phenotypic variance explained by each QTL ranged from 7.1 to 31.9xa0%. The four QTLs collectively contributed about 60.5xa0% of the phenotypic variation in resistance against TYLCTHV-[TW]. Group mean severity scores of those RILs possessing three or four qTy were generally lower than RIL groups with only one or no qTy. Given the diversity of begomoviruses that cause TYLCD across the regions, the new QTLs from FLA456 would be valuable in tomato breeding for developing varieties with durable resistance. Two QTL intervals (qTy4.1 and qTy10.1) contained virus resistance candidate genes such as CTV.22 and eukaryotic translation initiation factor 4E.

Analysis of sequences from field samples reveals the presence of the recently described pepper vein yellows virus (genus Polerovirus) in six additional countries

Polerovirus infection was detected by reverse transcription polymerase chain reaction (RT-PCR) in 29 pepper plants (Capsicum spp.) and one black nightshade plant (Solanum nigrum) sample collected from fields in India, Indonesia, Mali, Philippines, Thailand and Taiwan. At least two representative samples for each country were selected to generate a general polerovirus RT-PCR product of 1.4xa0kb length for sequencing. Sequence analysis of the partial genome sequences revealed the presence of pepper vein yellows virus (PeVYV) in all 13 samples. A 1990 Australian herbarium sample of pepper described by serological means as infected with capsicum yellows virus (CYV) was identified by sequence analysis of a partial CP sequence as probably infected with a potato leaf roll virus (PLRV) isolate.

Identification and characterization of a mechanical transmissible begomovirus causing leaf curl on oriental melon

Oriental melon plants, Cucumis melo var. makuwa cv. Silver Light, showing virus-induced symptoms of mosaic, leaf curl and puckering were observed in the fields of eastern Taiwan in 2007. A virus culture, designated as SL-1, isolated from the diseased melon was established in systemic host plants, Nicotiana benthamiana and oriental melon, by mechanical inoculation. SL-1 did not react to the antisera against common cucurbit-infecting RNA viruses. Viral DNAs extracted from the diseased plant were amplified with the degenerate primers for begomoviruses. The full-length genomic DNA-A and DNA-B of SL-1 were sequenced and found to be closest, with 97.7% and 90.6% nucleotide identity, respectively, to Tomato leaf curl New Delhi begomovirus (ToLCNDV) cucumber isolate from a group of cucurbit-infecting begomoviruses. The virus SL-1 was designated as ToLCNDV oriental melon isolate (ToLCNDV-OM). The pathogenicity of ToLCNDV-OM was confirmed by agroinfection. Progeny virus from the agroinfected N. benthamiana plants was able to infect oriental melon by mechanical inoculation and caused symptoms similar to the original diseased melon in the field. The ToLCNDV-OM also infected five other species of cucurbitaceous plants by mechanical inoculation. This is the first report of a new ToLCNDV isolate causing severe disease on oriental melon in Taiwan.

Molecular diversity of poleroviruses infecting cucurbit crops in four countries reveals the presence of members of six distinct species

When 66 cucurbit samples with yellowing symptoms from fields in Mali, the Philippines, Thailand and Uzbekistan were screened by RT-PCR using universal polerovirus primers, 21 were identified as harboring polerovirus RNA. When these 21 samples were screened with specific primers for the known cucurbit-infecting poleroviruses, suakwa aphid-borne yellows virus and a recombinant strain of cucurbit aphid-borne yellows virus were detected for the first time in the Philippines and Thailand. However, seven polerovirus-positive samples did not react with any of the known species-specific primers. Sequencing of 1.4-kb universal polerovirus RT-PCR products revealed the presence of two poleroviruses that had not been described previously. These viruses, from Mali and Thailand, were provisionally named pepo aphid-borne yellows virus and luffa aphid-borne yellows virus, respectively.

Molecular Characterization of a New Begomovirus Associated with Tomato Yellow Leaf Curl and Eggplant Yellow Mosaic Diseases in Thailand

Leaf curl and yellowing symptoms on tomato, and yellow mosaic symptoms on eggplant, are frequently observed in Kanchanaburi Province, Thailand. DNA was extracted from leaves of 13 symptomatic tomato and six symptomatic eggplant samples by the method of Gilbertson et al (1). Viral DNA was amplified by polymerase chain reaction (PCR) using the begomovirus-specific degenerate primer pair PAL1v1978/PAR1c715 (3), which amplified a 1.4-kb fragment of DNA-A. All samples, except one eggplant sample, yielded the expected product. The 1.4-kb PCR products of one tomato and one eggplant sample were cloned and sequenced. Both begomoviruses from tomato and eggplant contained a DNA-B component, amplified using two degenerate primer pairs, DNABLC1/DNABLV2 and DNABLC2/DNABLV2 (2). Based on sequences of the DNA products amplified by the degenerate primer pairs, specific primers were designed to complete the DNA-A and DNA-B sequences. Computer-assisted sequence comparisons were performed with geminivirus sequences available in the laboratory at the Asian Vegetable Research and Development Center, Taiwan and in the GenBank sequence database. Both tomato (GenBank Accession Nos. AF511529 and AF511528) and eggplant (GenBank Accession Nos. AF511530 and AF511527) virus isolates contain the conserved geminivirus sequence-TAATATTAC on the DNA-A and B. Based on the high sequence identities of 99% DNA-A and 98% DNA-B, these two virus isolates are considered to be the same species. Although the genome organization of these two isolates was the same as Tomato yellow leaf curl virus Thailand (TYLCTHV; GenBank Accession Nos. X63015 and X63016), including six open reading frames (ORFs) on the DNA-A (AV1, AV2, AC1, AC2, AC3, and AC4) and two ORFs on the DNA-B (BV1 and BC1), sequence comparisons showed highest DNA-A sequence identity (73%) with Ageratum yellow vein virus from Singapore (GenBank Accession No. X74516) and highest DNA-B identity (77%) with the TYLCTHV (X63016). To our knowledge, these tomato- and eggplant-infecting viruses from Thailand constitute a distinctly novel bipartite Begomovirus species. References: (1) R. L. Gilbertson et al. J. Gen. Virol. 72:2843, 1991. (2) S. K. Green et al. Plant Dis. 85:1286, 2001. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Molecular Characterization of Pepper yellow leaf curl Indonesia virus in Leaf Curl and Yellowing Diseased Tomato and Pepper in Indonesia

Yellowing and leaf curl symptoms were observed in tomato and pepper fields near Bogor, Java, Indonesia in 2000. Samples were collected from one diseased tomato (Lycopersicum esculentum) and three diseased chili pepper (Capsicum annuum) plants. Viral DNA was extracted (2) and tested for the presence of geminiviral DNA-A, DNA-B, and associated satellite DNA using polymerase chain reaction (PCR) with previously described primers (1,3,4). The begomovirus DNA-A general primer pair PAL1v1978/PAR1c715 amplified the predicted 1.4-kb DNA fragment from the tomato and two of the chili samples. DNA-B and satellite DNA were not detected using PCR with DNA-B general primer pairs (DNABLC1/DNABLV2 and DNABLC2/DNABLV2) and satellite detection primer pair (Beta01/Beta02). The amplicons from the tomato and from one of the chili samples were cloned and sequenced. On the basis of the 1.4-kb DNA sequences, specific primers were designed to complete the DNA-A sequences. Following sequence assembly, the full-length DNA-A nucleotide sequences were determined as 2,744 nt (GenBank Accession No. DQ083765) for the tomato- and 2,743 nt (GenBank Accession No. DQ083764) for the chili-infecting begomoviruses. Sequence comparisons and analyses were conducted using the DNAMAN sequence analysis software (Lynnon Corporation, Quebec, Canada). The DNA-A of both begomoviruses contained six open reading frames, including two in the virus sense and four in the complementary sense, and the geminivirus conserved nanosequence-TAATATTAC in the loop of the hairpin structure of the intergenic region. Because of their high nucleotide sequence identities of 99%, the tomato- and chili-infecting begomovirus are considered the same virus. When compared by using BLAST with available gem-iniviral sequences in the GenBank database, the DNA-A sequences of the tomato and the chili isolates showed highest nucleotide sequence identity (95%) with the partially sequenced Pepper yellow leaf curl Indonesia virus (GenBank Accession No. AB189849) in the 1,842 nt to 660 nt region and in the 1,841 nt to 659 nt region, respectively. Comparisons with full-length DNA-A sequences of begomoviruses available in the GenBank database indicated high sequence identities of 76 and 77% for the tomato and chili isolates, respectively, with an eggplant isolate of Tomato yellow leaf curl Kanchanaburi virus (GenBank Accession No. AF511530) from Thailand. According to our knowledge, this is the first report of full-length DNA-A sequence of the Pepper yellow leaf curl Indonesia virus and its natural occurrence in tomato and pepper in the Bogor area of Indonesia. References: (1) R. W. Briddon et al. Virology 312:106, 2003. (2) R. L. Gilbertson et al. J. Gen. Virol. 72:2843, 1991. (3) S. K. Green et al. Plant Dis. 85:1286, 2001. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.