First Report of Chilli veinal mottle virus Infecting Tomato (Solanum lycopersicum) in China

Chilli veinal mottle virus (ChiVMV), a potyvirus, is widespread over the world. In China, it was first reported in chili pepper (Capsicum annuum) in Hainan Province (south China) in 2006 (2). Subsequently, it was reported in tobacco (Nicotiana tabacum) in Yunnan Province (southwest China) in 2011 (1). Sichuan Province is one of the largest vegetable producing areas of China. In May 2012, tomatoes with leaves displaying virus-infected symptoms like mottling, mosaic, narrowing, or curling were observed in several fields of Chengdu, eastern Sichuan Province, southwest China. Of the 20 fields we investigated, four fields with 90% tomato plants were infected. During 2012 and 2013, six samples were collected from symptomatic tomato leaves based on different symptoms and locations. All six samples were assayed by western blotting using polyclonal antisera (Cucumber mosaic virus [CMV], Tobacco mosaic virus [TMV]) obtained from Agdia (Elkhart) and one antiserum to ChiVMV obtained from Yunnan Academy of Agricultural Science (China). Two samples from Pengzhou and one sample from Shuangliu exhibiting mosaic leaves were positive for TMV, one sample from Pixian exhibiting narrowing leaves was positive for CMV, and the other two samples from Shuangliu exhibiting mottle and leaf distortion were positive for ChiVMV. Total RNAs was extracted from all six samples and healthy tomato leaves using Trizol reagent (Invitrogen), First-strand cDNA synthesis primed with oligo(dT) by SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed using primer pairs ChiVMV-CP F (5′-GCAGGAGAGAGTGTTGATGCTG-3′) and ChiVMV–CP R (5′-(T)16AACGCCAACTATTG-3′), which were designed to direct the amplification of the entire capsid protein (CP) gene and 3′ untranslated region (3′-UTR) of ChiVMV (GenBank Accession No. KC711055). The expected 1,166-bp DNA fragment was amplified from the two tomato samples from Shuangliu that were positive for ChiVMV in the western blot tests, but not from the others. The obtained fragments were purified and cloned into the PMD18-T vector (TaKaRa) and sequenced. The sequencing results showed that the two ChiVMV isolates from tomato in Shuangliu were identical (KF738253). Nucleotide BLAST analysis revealed that this ChiVMV isolate shared ~84 to 99% nucleotide identities with other ChiVMV isolates available in GenBank (KC711055 to KF220408). To fulfill Koch's postulates, we isolated this virus by three cycle single lesion isolation in N. tabacum, and mechanically inoculated it onto tomato leaves. The same mottle and leaf distortion symptoms in systemic leaves were observed. Subsequent RT-PCR, fragment clone, and sequence determination tests were repeated and the results were the same. All the evidence from these tests revealed that the two tomato plants were infected by ChiVMV. To our knowledge, this is the first report of ChiVMV naturally infecting tomato in China. It shows that ChiVMV is spreading in China and is naturally infecting a new solanaceous crop in the southwest area, and the spread of the virus may affect tomato crop yields in China. Thus, it is very important to seek an effective way to control this virus. References: (1) M. Ding et al. Plant Dis. 95:357, 2011. (2) J. Wang et al. Plant Dis. 90:377, 2006.

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First Report of Tobacco mild green mosaic virus Infecting Capsicum annuum in Tunisia

Plant Disease ◽

10.1094/pdis-93-7-0761b ◽

2009 ◽

Vol 93 (7) ◽

pp. 761-761 ◽

Cited By ~ 5

Author(s):

M. I. Font ◽

M. C. Córdoba-Sellés ◽

M. C. Cebrián ◽

J. A. Herrera-Vásquez ◽

A. Alfaro-Fernández ◽

...

Keyword(s):

Mosaic Virus ◽

Capsicum Annuum ◽

Polyclonal Antibodies ◽

Plant Viruses ◽

Mottle Virus ◽

Tomato Mosaic Virus ◽

Rt Pcr ◽

Tomato Plants ◽

First Report ◽

Pepper Plants

During the springs of 2007 and 2008, leaf deformations as well as symptoms of mild green and chlorotic mosaic were observed on pepper (Capsicum annuum) plants grown in Monastir (northwest Tunisia) and Kebili (southeast Tunisia). With the support of projects A/5269/06 and A/8584/07 from the Spanish Agency for International Cooperation (AECI), symptomatic leaf samples were analyzed by transmission electron microscopy (TEM) of leaf-dip preparations. Typical tobamovirus-like particles (rigid rods ≈300 nm long) were observed in crude plant extracts. According to literature, at least six tobamoviruses infect peppers: Paprika mild mottle virus (PaMMV); Pepper mild mottle virus (PMMoV); Ribgrass mosaic virus (RMV); Tobacco mild green mosaic virus (TMGMV); Tobacco mosaic virus (TMV); and Tomato mosaic virus (ToMV) (1). Extracts from six symptomatic plants from Monastir and four from Kebili fields tested negative for ToMV, TMV, and PMMoV and tested positive for TMGMV by double-antibody sandwich (DAS)-ELISA using polyclonal antibodies specific to each virus (Loewe Biochemica GMBH, Sauerlach, Germany). To confirm the positive TMGMV results, total RNAs from 10 symptomatic plants that tested positive by ELISA were extracted and analyzed by reverse transcription (RT)-PCR using primers designed to specifically amplify a region of the coat protein gene (CP) of TMGMV (2). The 524-bp TMGMV-CP specific DNA fragment was amplified from all samples, but was not amplified from healthy plants or the sterile water used with negative controls. RT-PCR products were purified and directly sequenced. BLAST analysis of the obtained sequence (GenBank No. EU770626) showed 99 to 98% nucleotide identity with TMGMV isolates PAN-1, DSMZ PV-0113, TMGMV-Pt, and VZ1 (GenBank Nos. EU934035, EF469769, AM262165, and DQ460731, respectively) and less than 69% with PaMMV and PMMoV isolates (GenBank Nos. X72586 and AF103777, respectively). Two TMGMV-positive, singly, infected symptomatic pepper plants collected from Monastir and Kebili were used in mechanical transmissions to new pepper and tomato plants. Inoculated pepper plants exhibited mild chlorosis symptoms and tested positive for TMGMV only; however, inoculated tomato plants cv. Marmande were asymptomatic and tested negative as expected for TMGMV infection (1). To our knowledge, although C. annuum has been shown as a natural host for TMGMV (2), this is the first report of TMGMV in Tunisia. Reference: (1) A. A. Brunt et al. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20th August 1996. Online publication, 1996. (2) J. Cohen et al. Ann. Appl. Biol. 138:153, 2001.

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First Report of Peanut mottle virus Infecting Soybean in South Korea

Plant Disease ◽

10.1094/pdis-04-14-0356-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1285-1285 ◽

Cited By ~ 5

Author(s):

S. Lim ◽

Y.-H. Lee ◽

D. Igori ◽

F. Zhao ◽

R. H. Yoo ◽

...

Keyword(s):

Nucleotide Sequence ◽

South Korea ◽

Mosaic Virus ◽

Soybean Mosaic Virus ◽

Nucleotide Sequences ◽

Mottle Virus ◽

Post Inoculation ◽

Rt Pcr ◽

First Report ◽

Pcr Product

In July 2013, soybean (Glycine max) plants at the research field in Daegu, South Korea, showed virus-like symptoms, such as mosaic, mottle, yellowing, and stunting. Overall, there were approximately 1% of soybean plants that showed these symptoms. Sixteen soybean samples were collected based on visual symptoms and subjected to laboratory characterization. Total RNA was extracted from each sample with the Tri Reagent (Molecular Research Center, Cincinnati, OH) and cDNA was synthesized using random N25 primer with RevertAid Reverse Transcriptase (Thermo Scientific, Waltham, MA), according to the manufacturers' instructions. All samples were tested by PCR with Prime Taq Premix (2X) (GeNet Bio, Daejeon, Korea) and primer sets specific to Soybean mosaic virus (SMV; 5′-CATATCAGTTTGTTGGGCA-3′ and 5′-TGCCTATACCCTCAACAT-3′), Peanut stunt virus (PSV; 5′-TGACCGCGTGCCAGTAGGAT-3′ and 5′-AGGTDGCTTTCTWTTGRATTTA-3′), Soybean yellow mottle mosaic virus (SYMMV; 5′-CAACCCTCAGCCACATTCAACTAT-3′ and 5′-TCTAACCACCCCACCCGAAGGATT-3′), and Soybean yellow common mosaic virus (SYCMV; 5′-TTGGCTGAGAGGAGTGGCTT-3′ and 5′-TGCGGTCGTGTAGTCAGTG-3′). Among 16 samples tested, five were positive for SMV and two for SYMMV. Three samples were found infected by both SMV and SYMMV and four by both SMV and PSV. Since two of the symptomatic samples were not infected by viruses described above, a pair of primers specific to Peanut mottle virus (PeMoV; 5′-GCTGTGAATTGTTGTTGAGAA-3′ and 5′-ACAATGATGAAGTTCGTTAC-3′) was tested (1). All 16 samples were subjected to RT-PCR with primers specific to PeMoV. Four were found positive for PeMoV. Two of them were already found infected with SYMMV. In order to identify the complete nucleotide sequences of PeMoV coat protein (CP), another primer set (5′-TGAGCAGGAAAGAATTGTTTC-3′ and 5′-GGAAGCGATATACACACCAAC-3′) was used. RT-PCR product was cloned into RBC TA Cloning Vector (RBC Bioscience, Taipei, Taiwan) and the nucleotide sequence of the insert was determined by Macrogen (Seoul, Korea). CP gene of the PeMoV (GenBank Accession No. KJ664838) showed the highest nucleotide sequence identity with PeMoV isolate Habin (KF977830; 99% identity), and the highest amino acid identity with GenBank Accession No. ABI97347 (100% identity). In order to fulfill Koch's postulates, several G. max cv. Williams 82 were inoculated with the extracts of PeMoV-infected leaf tissue. At 14 days post-inoculation, plants showed systemic mottle symptoms. These symptomatic plants were subjected to RT-PCR, and the nucleotide sequences of the PCR product were found identical to that of the virus in the inoculum. To our knowledge, this is the first report of soybean-infecting PeMoV, a member of the genus Potyvirus in the family Potyviridae, in South Korea. Reference: (1) R. G. Dietzgen et al. Plant Dis. 85:989, 2001.

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First Report of Tomato torrado virus in Tomato Crops in France

Plant Disease ◽

10.1094/pdis-93-12-1352c ◽

2009 ◽

Vol 93 (12) ◽

pp. 1352-1352 ◽

Cited By ~ 14

Author(s):

E. Verdin ◽

P. Gognalons ◽

C. Wipf-Scheibel ◽

I. Bornard ◽

G. Ridray ◽

...

Keyword(s):

Mosaic Virus ◽

Sandwich Elisa ◽

Trialeurodes Vaporariorum ◽

World Intellectual Property Organization ◽

Tomato Mosaic Virus ◽

Healthy Plant ◽

Rt Pcr ◽

Tomato Plants ◽

First Report ◽

Test Plants

In June 2008, tomato (Solanum lycopersicum L.) plants cv. Fer De Lance (De Ruiter Seeds, Bergschenhoek, the Netherlands) grown in greenhouses near Perpignan (southern France) showed growth reduction and necrotic lesions on fruits, stems, and basal parts of the leaves. Tomato torrado virus (ToTV) was suspected on the basis of symptoms and its recent description in Spain (4). Primer set A (3), designed to ToTV RNA-2, was used for reverse transcription (RT)-PCR experiments on RNA extracted from four infected plants and allowed the amplification of a 493-bp fragment. No amplification was observed from healthy plant extracts. The RT-PCR product was directly sequenced (GQ303330) and a BLAST search in GenBank revealed 99.8- and 99.5%-nt identity with Polish (EU563947) and Spanish type strain (DQ388880) isolates of ToTV, respectively. Double-antibody sandwich-ELISA tests were conducted on these four samples to check for the presence of other viruses commonly found in tomato crops in France. Tomato spotted wilt virus, Parietaria mottle virus, Cucumber mosaic virus, Tomato mosaic virus, and Potato virus Y were not detected but Pepino mosaic virus (PepMV) was detected in all samples. ToTV was mechanically transmitted to Physalis floridana but PepMV was not. This plant was used to inoculate healthy tomatoes that served as a ToTV source for further experiments. Mechanical inoculation to test plants showed that Nicotiana benthamiana, N. clevelandii, N. debneyi, N. glutinosa, Capsicum annuum, Solanum melongena, and some tomato cultivars (including Fer De Lance), in which typical necrotic symptoms were observed, were systemically infected by the virus. Isometric particles ~28 nm in diameter were observed by electron microscopy in crude extracts of infected plants negatively stained with 1% ammonium molybdate, pH 7. To confirm ToTV identification, whitefly transmission experiments were performed with Trialeurodes vaporariorum and Bemisia tabaci. Adult whiteflies were placed in cages with infected tomato plants for 1-, 24-, or 48-h acquisition access periods (AAP) before transferring them by groups of ~50 on susceptible tomato plantlets placed under small containers (six plants per AAP). Forty-eight hours later, plants were treated with an insecticide and transferred to an insect-proof containment growth room. Ten days later, RNA preparation from all plants was tested by RT-PCR for the presence of ToTV. No transmission was observed with a 1-h AAP. With a 24-h AAP, transmission to four of six test plants was observed with both whitefly species, while at 48 h, AAP transmission to three and four plants of six was observed with T. vaporariorum and B. tabaci, respectively. Noninoculated control plants were all negative by RT-PCR. These experiments confirm T. vaporariorum and B. tabaci as natural vectors of ToTV as previously described (1,2). ToTV has been already reported in Spain, Poland, Hungary, and Australia, but to our knowledge, this is the first report of ToTV in France. Our detection of ToTV in April 2009 from the same area revealed 7 positive tomato plants of 17 tested. This observation suggests the persistence of the disease in the Perpignan Region. References: (1) K. Amari et al. Plant Dis. 92:1139, 2008. (2) H. Pospieszny et al. Plant Dis. 91:1364, 2007 (3) J. Van der Heuvel et al. Plant Virus Designated Tomato Torrado Virus. Online publication. World Intellectual Property Organization WO/2006/085749, 2006. (4) M. Verbeek et al. Arch. Virol. 152:881, 2007.

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First Report of Cucumber green mottle mosaic virus Infecting Greenhouse Cucumber in Canada

Plant Disease ◽

10.1094/pdis-09-13-0996-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 701-701 ◽

Cited By ~ 8

Author(s):

K.-S. Ling ◽

R. Li ◽

W. Zhang

Keyword(s):

Mosaic Virus ◽

Real Time ◽

Cucumis Sativus ◽

Disease Incidence ◽

The United States ◽

Rt Pcr ◽

First Report ◽

Sequence Identity ◽

Two Samples ◽

Greenhouse Cucumber

In early 2013, greenhouse cucumber growers in Alberta, Canada, observed virus-like disease symptoms on mini-cucumber (Cucumis sativus) crops (e.g., ‘Picowell’). Two types of symptoms were commonly observed, green mottle mosaic and necrotic spots. In the early infection, young leaves of infected cucumber plants displayed light green mottle and blisters. The infected plants were stunted in growth, with darker green blisters and green mottle mosaic symptoms on mature leaves. Disease incidence varied from one greenhouse to another. In some severe cases, diseased plants were widely distributed inside the greenhouse, resulting in 10 to 15% yield losses based on grower's estimation. Nine symptomatic samples were collected and subjected to total RNA isolation using the TRIzol reagent (Invitrogen, Carlsbad, CA). Laboratory analyses were conducted using real-time RT-PCR systems for Cucumber green mottle mosaic virus (CGMMV) (1), Melon necrotic spot virus (MNSV, Ling, unpublished), and Squash mosaic virus (SqMV) (3). All nine samples were positive for CGMMV and seven of them were in mixed infections with MNSV. Two samples were selected for validation for the presence of CGMMV using conventional RT-PCR (2) with a new primer set (CGMMVMP F1: 5′-ATGTCTCTAAGTAAGGTGTC-3′ and CGMMV3′UTR R1: 5′-TGGGCCCCTACCCGGGG-3′) and two previous online published primer sets, one for CGMMV MP (5′ TAAGTTTGCTAGGTGTGATC-3′, GenBank Accession No. AJ250104 and 5′ ACATAGATGTCTCTAAGTAAG-3′, AJ250105), and another for CGMMV CP (5′ ACCCTCGAAACTAAGCTTTC-3′, AJ243351 and 5′ GAAGAGTCCAGTTCTGTTTC-3′, AJ243352). The expected sizes of RT-PCR products were obtained and sequenced directly. Sequences from these three products overlapped and generated a 1,282-bp contig (KF683202). BLASTn analysis to the NCBI database showed 99% sequence identity to CGMMV isolates identified in Asia, including China (GQ277655, KC852074), India (DQ767631), Korea (AF417243), Myanmar (AB510355), and Taiwan (HQ692886), but only 92% sequence identity to other CGMMV isolates identified in Europe, including Spain (GQ411361) and Russia (GQ495274), and 95% to CGMMV isolate from Israel (KF155231). The strong sequence identity to the CGMMV Asian isolates suggests that the Canadian CGMMV isolate identified in Alberta was likely of Asian origin. In two bioassay experiments using one sample prepared in 0.01 M phosphate buffer, the similar green mottle mosaic symptoms were observed on systemic leaves in the mechanically inoculated plants and the presence of CGMMV, but not MNSV, was confirmed through real-time RT-PCR on four different cucurbits, including three Cucumis sativus cultivars (six plants in ‘Marketer,’ five plants in ‘Poinsett 76,’ six plants in ‘Straight 8’), seven plants of C. melo ‘Athena,’ six plants of C. metulifer (PI201681), and two plants of Citrullus lanatus ‘Charleston Gray.’ To our knowledge, CGMMV has only been reported in Asia, Europe, and the Middle East, and this is the first report of CGMMV in the American continents. CGMMV is highly contagious and is seed borne on cucurbits. With the increasing trend in growing grafted watermelon and other cucurbits in the United States and elsewhere, it is even more important now that a vigilant seed health test program for CGMMV should be implemented. References: (1) H. Chen et al. J. Virol. Methods 149:326, 2008. (2) K.-S. Ling et al. Plant Dis. 92:1683, 2008. (3) K.-S. Ling et al. J. Phytopathol. 159:649, 2011.

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First Report of Pepper veinal mottle virus in Tomato and Pepper in Taiwan

Plant Disease ◽

10.1094/pdis-93-1-0107a ◽

2009 ◽

Vol 93 (1) ◽

pp. 107-107 ◽

Cited By ~ 9

Author(s):

Y. H. Cheng ◽

R. Y. Wang ◽

C. C. Chen ◽

C. A. Chang ◽

F.-J. Jan

Keyword(s):

Amino Acid ◽

Mosaic Virus ◽

Polyclonal Antibodies ◽

Amino Acid Identity ◽

Mottle Virus ◽

Rt Pcr ◽

Acid Identity ◽

First Report ◽

Cp Gene ◽

Pepper Veinal Mottle Virus

In May of 2006, samples from tomato plants (Solanum lycopersicum cv. Known-you 301) exhibiting necrotic symptoms on stems, petioles, and leaves were collected from Chiayi County, Taiwan. Double-antibody sandwich-ELISAs were performed using Cucumber mosaic virus, Tomato mosaic virus, Potato virus Y, Watermelon silver mottle virus, and Chilli veinal mottle virus (ChiVMV) polyclonal antibodies. Three of eight samples reacted with antibodies against ChiVMV but not with the others. Using the potyvirus degenerate primers (Hrp 5/Pot 1) (2), an expected 1.5-kb DNA fragment including the 3′-end of the NIb gene, the complete coat protein (CP) gene, and the 3′-nontranslatable region of the virus was amplified from total RNA isolated from these three samples by reverse transcription (RT)-PCR. A homology search in GenBank indicated that the new tomato-infecting virus in Taiwan belongs to Pepper veinal mottle virus (PVMV) since they shared >90% amino acid identity in the CP gene. A virus culture (Tom1) isolated from one of the diseased tomatoes was then established in Chenopodium quinoa and Nicotiana benthamiana and the CP gene was amplified and sequenced (GenBank Accession No. EU719647). Comparisons of the 807-nt CP gene with those of five PVMV isolates available in GenBank showed 81.5 to 93.1% nucleotide and 90.0 to 97.8% amino acid identity. Tom1 induced irregular necrotic lesions on stems, petioles, and leaves of tomato while inducing only mild mottle symptoms on pepper. Serological cross reaction between ChiVMV and PVMV has been observed previously (1,3) and also found in this study. To differentiate these two potyviruses by RT-PCR, primer pair CPVMVup/dw (5′-TATTC(T/C)TCAGTGTGG(A/T/C)T(T/C)CCACCAT and 5′-(T/C)C(A/T)C(A/T)(A/T/G)(A/T)AA(A/G)CCATAA(A/C)(A/C)ATA(A/G)T(T/C)T) was designed on the basis of the comparison of the CP gene and the 3′-nontranslatable region of the PVMV and ChiVMV. DNA fragments of 171 and 259 bp are expected to be amplified from ChiVMV and PVMV, respectively, by RT-PCR with primers CPVMVup/dw. In a field survey done in 2006, samples from diseased peppers (Capsicum annuum) that reacted with the polyclonal antibodies against ChiVMV were further identified by RT-PCR with primers CPVMVup/dw, indicating that both ChiVMV and PVMV infected pepper crops (Capsicum spp.) in Taiwan. A pepper isolate (Pep1) of PVMV was obtained from Nantou County through three times of single lesion passages on C. quinoa and then propagated on N. benthamiana. The CP gene of Pep1 was amplified and sequenced (GenBank Accession No. EU719646) and found to share 99.1% nucleotide and 100% amino acid identity with that of Tom1. Pep1 caused mild mottle symptoms on leaves of both tomato and pepper. To our knowledge, this is the first report of the presence of PVMV in Taiwan as well as in East Asia. References: (1) B. Moury et al. Phytopathology 95:227, 2005. (2) S. S. Pappu et al. Plant Dis. 82:1121, 1998. (3) W. S. Tsai et al. Plant Pathol. 58:408, 2008.

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First Report of Cucumber mosaic virus in Taro Plants in China

Plant Disease ◽

10.1094/pdis-09-13-0916-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 574-574 ◽

Cited By ~ 6

Author(s):

Y. F. Wang ◽

G. P. Wang ◽

L. P. Wang ◽

N. Hong

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Central China ◽

Post Inoculation ◽

Rt Pcr ◽

Cmv Infection ◽

First Report ◽

Cp Gene ◽

Two Samples ◽

Pcr Products

Taro (Colocasia esculenta L. Schott) is an important crop worldwide. In China, the growing area and productivity of taro increased greatly in recent years. During the 2010 to 2013 growing seasons (from May to July), the incidence of Cucumber mosaic virus (CMV) in taro was determined. Leaf samples from 91 taro plants, including 26 plants of cv. Hongyayu grown in Jiangxi Province in eastern China, 33 plants of cv. Eyu no.1 grown in Hubei Province in central China, and 32 plants of cv. Baiyu grown in Guangxi Province in southwest China were collected randomly and tested for the presence of CMV by reverse transcription (RT)-PCR. Some sampled plants of cv. Hongyayu and Eyu no.1 showed leaf chlorosis or chlorotic spots, and most of the plants of these three cultivars showed feather-like mosaic symptom on their leaves, which was confirmed to be associated with the infection of Dasheen mosaic virus (DsMV) in our previous studies (3). Total RNA was extracted from leaves using CTAB protocol reported by Li et al. (1). Primer set forward 5′-ATGGACAAATCTGAATCAACC-3′/reverse 5′-TAAGCTGGATGGACAACCCGT-3′ (4) was used for the amplification of a 777-bp fragment, which contains the complete capsid protein (CP) gene of 657 bp. PCR products of the expected size were identified from 11 taro samples, including two samples of Hongyayu, three Eyu no.1, and six Baiyu plants. The result did not show any specific association between the symptoms observed and CMV infection. The obtained PCR products were cloned individually into the vector pMD18-T (TaKaRa, Dalian, China). Three independent clones derived from each product were sequenced by Genscript Corp., Nanjing, China. Pairwise comparison of CP gene sequences (Accession No. of one representation CP sequence: KF564789) showed 99.7 to 99.8% nucleotide (nt) and 99.1 to 99.5% deduced amino acid (aa) sequence identity among themselves, and 92.0 to 94.3% and 76.5 to 77.7% nt identities with corresponding sequences of CMV isolates in subgroup I and subgroup II (2), respectively. The maximum likelihood phylogenetic trees of nt and aa sequences generated by Clustal X v1.8 revealed that all these CMV isolates from taro in China fell into subgroup I. To further confirm the CMV infection, leaf saps of CMV infected taro plants of cv. Eyu no.1 were mechanically inoculated onto Pinellia ternate and Cucumis sativus. Plants of P. ternate showed local chlorotic lesions on the inoculated leaves and downward curl of newly grown leaves, and C. sativus showed local chlorotic lesions on the inoculated leaves and crinkle of newly grown leaves at 10 to 15 days post inoculation. The RT-PCR detection confirmed the CMV infection in those inoculated plants, and that the plants of P. ternate were also positive to DsMV, further complementing the results obtained above. To our knowledge, this is the first report of CMV occurrence in taro plants grown in China. Our results indicated that taro plants were widely infected by CMV isolates in subgroup I. This study provides important information for further evaluating the viral sanitary status of taro germplasm and improving the certification program of taro propagation materials in China. References: (1) R. Li et al. J. Virol. Methods 154:48, 2008. (2) P. Palukaitis et al. Adv. Virus. Res. 62:241, 2003. (3) S. M. Shi et al. Acta Hortic. Sin. 39:509, 2012. (4) P. D. Xu et al. Chinese J. Virol. 15:164, 1999.

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First Report of Maize chlorotic mottle virus on Sweet Corn in Taiwan

Plant Disease ◽

10.1094/pdis-06-14-0568-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1748-1748 ◽

Cited By ~ 23

Author(s):

T.-C. Deng ◽

C.-M. Chou ◽

C.-T. Chen ◽

C.-H. Tsai ◽

F.-C. Lin

Keyword(s):

Nucleotide Sequence ◽

Mosaic Virus ◽

Sweet Corn ◽

Mottle Virus ◽

Rt Pcr ◽

First Report ◽

Cp Gene ◽

Chlorotic Mottle Virus ◽

Maize Chlorotic Mottle Virus ◽

Chlorotic Mottle

In February 2014, a severe disease on maize (Zea mays L.) broke out in the fields of central and southwestern Taiwan and caused yield losses in sweet corn production. Chlorotic spots first appeared at the base of infected leaves and later developed into systemic mottling. Diffused necrotic patches were also found on leaves or husks of the diseased plants. Moreover, severe rosetting and stunting accompanied by abnormalities in ear production were observed on mature plants. Eighteen leaf samples from symptomatic plants were collected and submitted to our Plant Diagnostic Clinic for virus diagnosis. All of the samples were first tested by reverse transcriptase (RT)-PCR to detect Maize stripe virus (MSpV) and by indirect ELISA to detect Maize dwarf mosaic virus (MDMV) or Sugarcane mosaic virus (SCMV), which were endemic to this area (1). Only 2 out of 18 samples were positive for MDMV, SCMV, or mixed infection of both viruses. Sap inoculation tests conducted on seedlings of sweet corn cv. Honey 236 indicated that the MDMV- and SCMV-negative samples still had an unknown pathogen causing original symptoms in the receptor plants. The isolate from Yunlin county reacted only with the antibody to Maize chlorotic mottle virus (MCMV) (AC Diagnostics, Fayetteville, AR) in ELISA. For further identification, the MCMV-specific primers (forward: MCMVg3514F-GGGAACAACCTGCTCCA; reverse MCMVg4014R-GGACACGGAGTACGAGA) were designed from the nucleotide sequence of MCMV coat protein (CP) gene. In RT-PCR using the AccuPower RT/PCR PreMix kit (Bioneer, Daejeon, Korea), an expected 500-bp DNA fragment was observed. This PCR product was cloned and its nucleotide sequence was determined by Mission Biotech Co., Taipei, Taiwan. BLAST analysis of the CP gene of the MCMV-Yunlin revealed the maximum nucleotide identities (99%) with Chinese Sichuan isolates (GenBank Accession No. JQ984270) and 98% identities to four Chinese Yunnan isolates (GU138674, JQ982468, JQ982469, and KF010583) and one Kenya isolate (JX286709), compared with 97% to Kansas isolate (X14736) and 96% to Nebraska isolate (EU358605). Subsequently, the complete nucleotide sequence of the viral genome (KJ782300) was determined from five overlapping DNA fragments obtained from independent RT-PCR amplification. The virus isolate was infectious to sweet corn cultivars Bai-long-wang, Devotion, SC-34, SC2015, and Zheng-zi-mi, on which similar symptoms were developed after mechanical inoculation. During the spring of 2014, a total of 224 sweet corn samples were collected from the epidemic areas of Taichung, Yunlin, Chiayi, and Kaohsiung counties. Samples (n= 161) reacted positive for MCMV in ELISA and/or RT-PCR. In the field survey, more than 20 adult thrips might be observed on an MCMV-infected plant. Two species of Frankliniella were found on maize plants: F. williamsi Hood and F. intonsa Trybom. Maize thrips (F. williamsi), an occasional pest of maize occurring during winter and spring in Taiwan, was characterized by its abdominal sternite II on which 1 or 2 discal setae of equal length with posteromarginal setae were borne (2). Samples with 1, 5, 10, and 30 F. williamsi collected in the field were tested by RT-PCR; MCMV was detectable not only in the pooled crushed bodies but also in a single maize thrips. This is the first report of MCMV occurrence on maize in Taiwan and of the virus transmitted by maize thrips. References: (1) C. T. Chen et al. Taiwan Sugar 37(4):9, 1990. (2) C.-L. Wang et al. Zool. Stud. 49:824, 2010.

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First Report of Sweet potato chlorotic fleck virus Infecting Sweet Potato in Sichuan Province, China

Plant Disease ◽

10.1094/pdis-05-13-0578-pdn ◽

2014 ◽

Vol 98 (1) ◽

pp. 163-163 ◽

Cited By ~ 3

Author(s):

X.-G. Deng ◽

F. Zhu ◽

T. Zhu ◽

H.-H. Lin ◽

D.-H. Xi

Keyword(s):

Sweet Potato ◽

Crop Yields ◽

Eastern China ◽

Sichuan Province ◽

Western China ◽

Rt Pcr ◽

Putative Gene ◽

First Report ◽

Potato Viruses ◽

Two Samples

Sweet potato chlorotic fleck virus (SPCFV) is one of several viruses naturally infecting sweet potato (Ipomoea batatas L.), and it has recently been classified as a new member of the genus Carlavirus (family Flexiviridae) (1). However, SPCFV is distantly related to typical carlaviruses, as most of its putative gene products share amino acid sequence identities of <40% with those of typical carlaviruses (1). China is the largest sweet potato producing country in the world. So far, SPCFV has been reported in eastern China, such as Jiangsu, Anhui, Henan (3), and Guangdong (2) provinces, but no reports exist in western China. Sichuan Province, located in southwestern China, is the largest sweet potato producing area in the country. There are big differences between the environment and climate conditions between Sichuan and eastern China. During 2012, a survey was constructed to determine the genetic diversity and distribution of sweet potato viruses in Sichuan. Forty-seven sweet potato samples exhibiting virus-like symptoms were collected from four different geographic areas of the province. Western blotting using the antisera obtained from the International Potato Center showed that two samples were positive for SPCFV, whereas with reverse transcription (RT)-PCR, only one isolate of SPCFV was obtained from a sample exhibiting symptoms of chlorosis, leaf distortion, and vein clearing. Serological detection indicated that the plant was co-infected with SPCFV, Sweet potato feathery mottle virus (SPFMV), and Sweet potato virus G (SPVG). Total RNA was extracted from symptomatic leaves using Trizol reagent (Invitrogen) according to the manufacturer's protocol, and RT-PCR was performed by using primer pairs SPCFV-CP F (5′-ATGGCGGCGAAGGAGGCTGATA-3′) and SPCFV-CP R (5′-TCACTTGCACTTCCCATTAC-3′) corresponding to the entire coat protein (CP) gene of SPCFV. Expected DNA fragments of 900 bp were obtained from the symptomatic plant but not from control plants. The obtained fragments were purified and cloned into the PMD19-T vector (TaKaRa). Recombinant plasmids were then transformed into competent cells of Escherichia coli strain DH5α. Nucleotide BLAST analysis revealed that the 900-bp fragment (GenBank Accession No. KC414676) shared 87 to 91% nucleotide identities with other SPCFV isolates available in the GenBank database. To our knowledge, this is the first report of the co-infection between SPCFV and other sweet potato viruses including SPFMV and SPVG in China, and this is the first molecular report of SPCFV in Sichuan, western China. It shows that SPCFV is spreading to a new ecological area of China, and the spread of the virus may affect sweet potato crop yields in western China. Some measures must be carried out quickly to control the virus. References: (1) V. Aritua et al. Arch. Virol. 152:813, 2007. (2) V. Aritua et al. Plant Dis. 93:87, 2009. (3) Q. Wang et al. Crop. Prot. 29:110, 2010.

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First Report of a Natural Infection by Mexican Papita Viroid and Tomato Chlorotic Dwarf Viroid on Greenhouse Tomatoes in Mexico

Plant Disease ◽

10.1094/pdis-93-11-1216a ◽

2009 ◽

Vol 93 (11) ◽

pp. 1216-1216 ◽

Cited By ~ 17

Author(s):

K.-S. Ling ◽

W. Zhang

Keyword(s):

Mixed Infection ◽

Natural Infection ◽

Cdna Clones ◽

Diseased Plant ◽

Rt Pcr ◽

Tomato Plants ◽

First Report ◽

Two Samples ◽

Tomato Chlorotic Dwarf Viroid ◽

Plant Pathol

In early 2008, tomato plants (Solanum lycopersicum) grown in a large greenhouse facility located near Mexico City exhibited general stunting, leaf chlorosis at the top of the diseased plant that later turned bronze or purple, and reduced-sized fruits. Initially, diseased plants were confined to a 5-ha greenhouse, but the disease quickly spread to two additional 5-ha greenhouses in the summer of 2008. By the end of 2008, approximately 5% of tomato plants in 35-ha of greenhouse were infected. Sixteen diseased samples were collected, twelve in 2008 and four in 2009. Bioassays through mechanical inoculation with leaf extracts of diseased samples demonstrated the transmissibility of the causal agent to plants of tomato cvs. Horizon or Rutgers, which expressed symptoms that were similar to those on the source plants. Serological or PCR assays were negative for several commonly occurring greenhouse tomato viruses. However, an expected size product (~196 bp) was consistently detected by reverse transcription (RT)-PCR using pospiviroid-specific primers Pospil-RE and Pospil-FW (4) in all symptomatic samples or from the mechanically inoculated tomato plants. Preliminary analysis with sequences obtained from direct sequencing of amplicons revealed one dominant sequence with 94% identity to Mexican papita viroid (MPVd) (GenBank Accessions Nos. L78454 and L78456–L78463). However, further analysis of the cloned cDNAs indicated a mixed infection of two pospiviroids in two samples. Of 10 cDNA clones analyzed, 9 were MPVd-like sequences and one was sequence of Tomato chlorotic dwarf viroid (TCDVd). Further analysis using full genomic sequences obtained by RT-PCR with previously designed primers (2) or a new set of primers (MTTVd-F: 5′ GGG GAA ACC TGG AGC GAA CTG G, and MTTVd-R: 5′ GGG GAT CCC TGA AGC GCT CCT) revealed genetic diversity in this population. Eight of thirteen cloned cDNAs represented by the 359-nt sequence of isolate Mex8 (GenBank Accession No. GQ131572) had 93 to 94% nucleotide sequence identity to other MPVd isolates (L78454 and L78456–L78463). Five other cDNA clones represented by the 361-nt sequence of isolate HM2 (GenBank Accession No. GQ131573) were 99% identical to a TCDVd isolate recently identified in Arizona (GenBank Accession No. FJ822878) and 96 to 97% identical to TCDVd isolates from other areas (GenBank Accession Nos. AF162131 and AB329668). These results are the first evidence of a mixed infection of two viroids infecting tomatoes in Mexico. MPVd was first identified in Mexico on papita (S. cardiophyllum) in 1996 (1). The origin of TCDVd in this greenhouse was not determined, but TCDVd potentially can be seed transmitted in tomato (3). The close relationship between the Mexican and the U.S. isolates suggests that TCDVd in these two countries may share a common origin, likely from seed. To our knowledge, this is the first report of a natural infection of MPVd and TCDVd on tomatoes in Mexico. References: (1) J. P. Martinez-Soriano et al. Proc. Natl. Acad. Sci. U.S.A. 93:9397, 1996. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) R. P. Singh and A. D. Dilworth. Eur. J. Plant Pathol. 123:111, 2009. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

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First Report of Pepino mosaic virus Infecting Greenhouse Cherry Tomatoes in Greece

Plant Disease ◽

10.1094/pdis-09-10-0643 ◽

2011 ◽

Vol 95 (1) ◽

pp. 78-78 ◽

Cited By ~ 7

Author(s):

K. E. Efthimiou ◽

A. P. Gatsios ◽

K. C. Aretakis ◽

L. C. Papayiannis ◽

N. I. Katis

Keyword(s):

Mosaic Virus ◽

Leaf Tissue ◽

Tomato Mosaic Virus ◽

Sequencing Analysis ◽

Rt Pcr ◽

Pepino Mosaic Virus ◽

Tomato Plants ◽

First Report ◽

Negative Controls ◽

Das Elisa

Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae) is a mechanically transmitted virus that has emerged as a significant problem of greenhouse tomato crops in Europe and around the world during the past 10 years (1). In spring of 2010, mosaic symptoms were observed on leaves of cherry tomato (Lycopersicon esculentum var. cerasiforme) greenhouse crops (hybrids Shiren, Tomito, and Rubino top) in the areas of Drymos and Vonitsa, located at Aitoloakarnania Prefecture, in Greece. A total of 63 tomato samples (55 from symptomatic and 8 from asymptomatic plants) were collected from 11 greenhouses where disease incidence ranged from 10 to 20%. All samples were tested by double-antibody sandwich (DAS)-ELISA using polyclonal antibodies from BIOREBA, AG (Reinach, Switzerland) for the presence of PepMV, Cucumber mosaic virus (CMV), and Tomato mosaic virus (ToMV). Leaf tissue from PepMV-, CMV-, and ToMV-infected samples and virus-free tomato plants were included in all tests as positive and negative controls, respectively. Results showed that 53 symptomatic samples collected from all greenhouses were infected with PepMV and two were co-infected with PepMV and CMV. Total RNA was extracted from all infected plants with a commercially available kit (Qiagen, Hilden, Germany) and amplified by conventional and real-time reverse transcription (RT)-PCR, using previously reported protocols (2). Positive and negative controls were also included in each assay. The 200-bp amplified PCR fragments of Triple Gene Block 3 (TGB3) obtained from five infected samples were purified and both strands were sequenced. Sequencing data were analyzed, deposited in the GenBank, and compared with other reported sequences. In addition, leaf tissue from five samples infected with only PepMV was used for mechanical inoculation of four plants of Nicotiana glutinosa, N. benthamiana, and tomato (L. esculentum FA 179 hybrid) plants. As negative controls, two plants from each species were used. Sequencing analysis showed that all five PepMV sequences were identical (GenBank Accession Nos. FR686904 to FR686908) and possessed 100% identity PepMVstrain CH2 (DQ000985). Inoculation results showed that the virus was successfully transmitted to N. benthamiana and tomato plants which developed mosaic symptoms, and tested positive by DAS-ELISA and RT-PCR. N. glutinosa plants did not develop any symptoms and were found to be free of PepMV when tested by DAS-ELISA and RT-PCR. To our knowledge, this is the first report of PepMV in Greece. Further studies on the disease prevalence and incidence and its economic impact on tomato production are required. PepMV is currently under quarantine status in the EU and therefore new protective measures should be recommended to prevent the spread of PepMV to other regions of Greece. References: (1) I. M. Hanssen and B. P. H. J. Thomma. Mol. Plant Pathol. 11:179, 2010. (2) K. S. Ling et al. J. Virol. Methods 144:65, 2007.

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