First Report of Cucumber vein yellowing virus on Cucumber, Melon, and Watermelon in Iran

Several viral diseases are responsible for significant economic losses in commercial cucurbit production worldwide. During a survey conducted in July 2002 in cucurbit growing areas in southern Iran, vein-clearing symptoms and leaf chlorosis on older leaves were observed on a cucumber plant near Jiroft (Kerman Province). These symptoms were similar to those caused by Cucumber vein yellowing virus (CVYV, genus Ipomovirus, family Potyviridae), a virus first described in Israel (1) and now widespread in cucurbit crops in the Middle East and Mediterranean Regions (2). The identification of CVYV was established through differential host range reaction and immunosorbent electron microscopy (IEM) experiments. Typical vein-clearing symptoms were observed following mechanical inoculation of cucumber and melon plantlets, but no symptoms were observed in Chenopodium quinoa, C. amaranticolor, Nicotiana tabacum, or Vigna sinensis. Numerous, slightly flexuous, elongated virus particles were observed in infected plant extracts. The particles were decorated by a polyclonal antiserum raised against a Sudanese isolate of CVYV. To confirm CVYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St. Louis, MO) were obtained from the original cucumber sample. Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using CVYV-specific primers CVYV-CP-5′: 5′-GCTTCTGGTTCTCAAGTGGA-3′ and CVYV-CP-3′: 5′-GATGCATCAGTTGTCAGATG-3′ designed according to the partial sequence of the coat protein gene of CVYV-Isr (GenBank Accession No. AF233429) (2). A 540-bp fragment corresponding to the central region of CVYV coat protein was obtained from extracts of infected plants but not from healthy plant extracts. Additional watermelon (n = 6) and melon (n = 4) leaf samples collected from plants growing in the same farm were tested for the presence of CVYV using RT-PCR. All samples reacted positively for CVYV. However, a sample of Citrullus colocynthis, a wild relative of watermelon growing nearby, was negative. CVYV was not detected using RT-PCR in 123 additional cucurbit samples collected from the eastern and central regions of Iran during a survey conducted in 2002. To our knowledge, this is the first report of the occurrence of CVYV in Iran. Additional surveys in southern regions where Bemisia tabaci, the vector of CVYV, is abundant are required to better estimate the prevalence of this virus in cucurbit crops in Iran. References: (1) S. Cohen and F. E. Nitzany. Phytopathol. Mediterr. 1:44, 1960 (2) H. Lecoq et al. J. Gen. Virol. 81:2289, 2000.

Download Full-text

Cytological and molecular evidence that the whitefly-transmitted Cucumber vein yellowing virus is a tentative member of the family Potyviridae

Journal of General Virology ◽

10.1099/0022-1317-81-9-2289 ◽

2000 ◽

Vol 81 (9) ◽

pp. 2289-2293 ◽

Cited By ~ 36

Author(s):

H. Lecoq ◽

C. Desbiez ◽

B. Delécolle ◽

S. Cohen ◽

A. Mansour

Keyword(s):

Plant Extracts ◽

Infected Plant ◽

Molecular Evidence ◽

Rt Pcr ◽

Sequence Comparisons ◽

Cytoplasmic Inclusions ◽

The Family ◽

Tentative Member ◽

Dna Fragment ◽

Yellowing Virus

Cucumber vein yellowing virus (CVYV) is widespread in cucurbits in the Middle East. CVYV has filamentous particles and is transmitted by Bemisia tabaci by the semi-persistent mode. It has not yet been assigned to a specific genus or family. Ultramicroscopic observations revealed numerous cylindrical cytoplasmic inclusions in melon and cucumber cells infected by CVYV isolates from Israel and Jordan. Depending on the section orientation, the inclusions appeared as pinwheels or as bundles. In addition, a 1·9 kb DNA fragment was amplified by RT–PCR from CVYV-infected plant extracts using primers designed to detect all potyvirids. Sequence comparisons with the amplified fragment indicated that CVYV is more closely related to Sweet potato mild mottle virus than to any other virus in the family Potyviridae. These results suggest that CVYV can be considered as a tentative new member of the genus Ipomovirus, family Potyviridae.

Download Full-text

First Report of Cucumber vein yellowing virus in Spain

Plant Disease ◽

10.1094/pdis.2001.85.3.336a ◽

2001 ◽

Vol 85 (3) ◽

pp. 336-336 ◽

Cited By ~ 30

Author(s):

I. M. Cuadrado ◽

D. Janssen ◽

L. Velasco ◽

L. Ruiz ◽

E. Segundo

Keyword(s):

Total Nucleic Acid ◽

Accession Number ◽

Rt Pcr ◽

Specific Primers ◽

First Report ◽

Vein Clearing ◽

Acquisition Period ◽

Greenhouse Cucumber ◽

And Control ◽

Yellowing Virus

In the autumn of 2000, an outbreak of a disease caused considerable losses in greenhouse cucumber crops in Almeria (Spain). Infected plants showed vein clearing followed by chlorosis in leaves and yellow/green chlorotic spots on fruits. These symptoms as well as the presence of Bemisia tabaci in the crops suggested the possible involvement of Cucumber vein yellowing virus (CVYV), a proposed member of the Potiviridae family, which was first described in 1960 in Cucumis spp. from Israel (1). B. tabaci populations and leaves from cucumber plants were collected from the greenhouses and analyzed by RT-PCR using specific primers (CV(+): 5′-AGCTAGCGCGTATGGGGTGAC-3′; CV(-): 5′-GCGCCGCAAGTGCAA-ATAAAT-3′) that we designed based on the partial sequence published for CVYV (2). Total nucleic acid extracts from both B. tabaci individuals and the collected plants yielded amplification products of the expected size (449 bp), which were cloned and sequenced (Genebank accession number AJ301640). The sequence was 95.6% identical to that previously reported for CVYV. Nonviruliferous B. tabaci whiteflies were given a 24-h acquisition period on symptomatic leaves and then placed in groups of 15 insects on each of 10 healthy cucumber plants at the 4 leaf-stage for a 24-h inoculation period. Inoculated and control plants were analyzed 1 week later and the infection with CVYV was confirmed (10/10) by RT-PCR. Doublestranded RNA extractions from field-collected samples and from plants inoculated under controlled conditions suggested that no dsRNA formation was associated with the infection. This is the first report of CVYV in Spain. References: (1) S. Cohen and F. E. Nitzany. Phytopathol. Medit. 1:44, 1960. (2) H. Lecoq et al. J. Gen. Virol. 81:2289, 2000.

Download Full-text

First Report of Cucumber vein yellowing virus in Melon in France

Plant Disease ◽

10.1094/pdis-91-7-0909c ◽

2007 ◽

Vol 91 (7) ◽

pp. 909-909 ◽

Cited By ~ 10

Author(s):

H. Lecoq ◽

O. Dufour ◽

C. Wipf-Scheibel ◽

M. Girard ◽

A. C. Cotillon ◽

...

Keyword(s):

Coat Protein ◽

Direct Sequencing ◽

Infected Plant ◽

Chenopodium Quinoa ◽

Polyclonal Antiserum ◽

Mild Mosaic ◽

Rt Pcr ◽

Differential Host ◽

Total Rna ◽

Yellowing Virus

During the fall of 2003, mild mosaic symptoms were observed in melon (Cucumis melo L.) plants grown in glasshouses near Eyragues (southeastern France) resembling those caused by the Bemisia tabaci transmitted Cucumber vein yellowing virus (CVYV, genus Ipomovirus, family Potyviridae). In addition, large numbers of B. tabaci were observed to be colonizing these crops. The identification of CVYV was established through differential host range reaction, immunosorbent electron microscopy (IEM), and reverse transcription (RT)-PCR experiments. Crude sap from symptomatic leaves was used to inoculate differential host plants. Mild mosaic symptoms were observed on melon, and cucumber developed vein-clearing symptoms typical of CVYV. No symptoms were observed in Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana, N. tabacum, and Vigna sinensis. Numerous, slightly flexuous, elongated virus particles were observed in infected plant extracts; these particles were decorated by a polyclonal antiserum raised against a Sudanese CVYV isolate. To confirm CVYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St. Louis, MO) were obtained from the original symptomatic melon tissues. RT-PCR was carried out using CVYV-specific primers CVYV-CP-5′: 5′-GCTTCTGGTTCTCAAGTGGA-3′ and CVYV-CP- 3′: 5′-GATGCATCAGTTGTCAGATG-3′ designed according to the partial sequence of the coat protein gene of CVYV-Isr (GenBank Accession No. AF233429) (2). A 540-bp fragment corresponding to the central region of CVYV coat protein was amplified from total RNA extracted from symptomatic but not from asymptomatic melon tissue. Direct sequencing was done on RT-PCR products (GenBank Accession No. EF441272). The sequence was 95 and 99% identical to that reported for CVYV isolates from Israel and Spain, respectively. CVYV was first described in Israel and has recently emerged as the cause of important diseases in Spain and Portugal (1,3). Shortly after detecting CVYV during 2003, efforts were made to eradicate the virus in susceptible crops. CVYV was not detected again during intensive surveys conducted in southeastern France during 2004, 2005, and 2006, suggesting that the CVYV detected during 2003 resulted from an accidental introduction and that the virus has not become established in France. References: (1) I. M. Cuadrado et al. Plant Dis. 85:336, 2001. (2) H. Lecoq et al. J. Gen. Virol. 81:2289, 2000. (3) D. Louro et al. Plant Pathol. 53:241, 2004.

Download Full-text

First Report of Malva vein clearing virus Naturally Occurring in Hollyhock in Germany

Plant Disease ◽

10.1094/pdis-94-2-0276b ◽

2010 ◽

Vol 94 (2) ◽

pp. 276-276 ◽

Cited By ~ 5

Author(s):

W. Menzel ◽

S. Winter ◽

K. R. Richert-Pöggeler

Keyword(s):

Host Range ◽

Direct Sequencing ◽

Amino Acid Sequences ◽

Rt Pcr ◽

Electron Microscopic ◽

First Report ◽

Vein Clearing ◽

Naturally Occurring ◽

Potyvirus Species ◽

Cloned Cdna

Hollyhocks are popular garden plants and selected cultivars of Alcea rosea (family Malvaceae) are widespread in Germany. In spring 2009, dozens of A. rosea plants displaying strong vein clearing and veinal yellowing symptoms were found in private gardens in Hannover, Lower Saxony. Electron microscopic examinations of negatively stained adsorption preparations of five randomly selected samples of symptomatic plants or their offshoots revealed flexuous filamentous particles resembling those of potyviruses. Sap extracts also reacted strongly positive in an antigen coated plate (ACP)-ELISA with the broad-spectrum potyvirus antiserum AS-0573/I (DSMZ, Braunschweig, Germany). RNA extracts (RNeasy Kit, Qiagen, Valencia, CA) of the above mentioned leaf samples were used as templates in reverse transcription (RT)-PCR assays with potyvirus specific primers (2) that have been shown to amplify the 3′ terminus of the genome of many potyvirus species. For extracts from symptomatic samples, this resulted in a consistent amplification of an ~1.6-kbp fragment, whereas no products were obtained from RNA extracts of asymptomatic plants. From one positive sample, the amplified fragment was cloned and one clone was partially sequenced. The nucleotide (nt) and amino acid sequences showed the highest identities (81 to 83% and 87 to 90%, respectively) to GenBank sequences FJ539084, FM212972, EU884405, and FJ561293 of the potyvirus Malva vein clearing virus (MVCM). On the basis of these identity values and according to the species demarcation criteria in the genus Potyvirus, the virus can be regarded as a German isolate of the recently sequenced MVCV (3,4). Direct sequencing of the 5′-end of the amplified RT-PCR fragment revealed sequences of only one potyvirus species. The virus isolate has been submitted to the DSMZ Plant Virus Collection (Braunschweig, Germany) under accession PV-0963 and the sequence obtained from the cloned cDNA is deposited in GenBank (GQ856544). In addition, sap from affected leaves was mechanically inoculated onto sets of herbaceous indicator plants (Chenopodium quinoa, C. foliosum, C. murale, C. amaranticolor, Datura stramonium, Nicotiana benthamiana, N. hesperis, Petunia hybrida, and Solanum lycopersicum) of which only C. quinoa plants became infected. Symptoms of weak chlorosis along and beside veins of inoculated leaves, but not systemic leaves, became visible 2 weeks postinoculation. Symptomatic leaves contained flexuous filamentous particles and ACP-ELISA and RT-PCR confirmed virus presence. The partially sequenced amplicon showed 99% nt identity to the sequence from the cloned cDNA. To our knowledge, this is the first report of a MVCV isolate naturally occurring in A. rosea and C. quinoa is the first host identified that does not belong to the plant family Malvaceae. In contrast, the MVCV isolate used in the host range study of Lunello et al. (4) did not infect A. rosea and C. quinoa, confirming previous host range descriptions by Brunt et al. (1). Since MVCV infections of hollyhocks seem to cause only leaf symptoms and do not noticeably affect growth or flowering of the plants, this will hopefully not impair the usability of this popular garden plant. References: (1) A. A. Brunt et al. Descriptions and Lists from the VIDE Database. Online publication. Version: 16th January, 1997. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) A. Hein Phytopathol. Z. 28:205, 1957. (4) P. Lunello et al. Virus Res. 140:91, 2009.

Download Full-text

First Report of Cucurbit aphid-borne yellows virus in Iran Causing Yellows on Four Cucurbit Crops

Plant Disease ◽

10.1094/pd-90-0526a ◽

2006 ◽

Vol 90 (4) ◽

pp. 526-526 ◽

Cited By ~ 7

Author(s):

K. Bananej ◽

C. Desbiez ◽

C. Wipf-Scheibel ◽

I. Vahdat ◽

A. Kheyr-Pour ◽

...

Keyword(s):

Citrullus Lanatus ◽

Healthy Plant ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Chain Reactions ◽

Reference Isolate ◽

Sigma Chemical ◽

International Mycological Institute ◽

Das Elisa

A survey was conducted from 2001 to 2004 in the major cucurbit-growing areas in Iran to reassess the relative incidence of cucurbit viruses. Severe yellowing symptoms were observed frequently on older leaves of cucurbit plants in various regions in outdoor crops, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) (1,2). Leaf samples (n = 1019) were collected from plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita sp.), and watermelon (Citrullus lanatus L.) showing various virus-like symptoms (mosaic, leaf deformation, yellowing). All samples, collected from 15 provinces, were screened for the presence of CABYV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with IgGs and alkaline phosphatase-conjugated IgGs against a CABYV reference isolate (1). Of the 1,019 samples tested, 471 were positive for CABYV using DAS-ELISA. Some of the positive samples had typical severe yellowing symptoms while symptoms in other samples were masked by mosaic or leaf deformations caused by other viruses frequently found in mixed infections (data not shown). During the entire survey, CABYV was detected by DAS-ELISA in 201 of 503 melon samples, 72 of 129 cucumber samples, 158 of 249 squash samples, and 40 of 138 watermelon samples. These results indicate that CABYV is widely distributed on four cucurbit species in the major growing areas of Iran. In order to confirm CABYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St Louis, MO) were obtained from 25 samples that were positive using DAS-ELISA originating from Khorasan (n = 4), Esfahan (n = 6), Teheran (n = 3), Hormozgan (n = 4), Azerbaiejan-E-Sharqi (n = 4), and Kerman (n = 4). Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using forward (5′-CGCGTGGTTGTGG-TCAACCC-3′) and reverse (5′-CCYGCAACCGAGGAAGATCC-3′) primers designed in conserved regions of the coat protein gene according to the sequence of a CABYV reference isolate (3) and three other unpublished CABYV sequences. RT-PCR experiments yielded an expected 479-bp product similar to the fragment amplified with extracts from the reference isolate. No amplification of the product occurred from healthy plant extracts. To our knowledge, this is the first report of the occurrence of CABYV in Iran on various cucurbit species. The high frequency (46.2%) with which CABYV was detected in the samples assayed indicates that this virus is one of the most common virus infecting cucurbits in Iran. References: (1) H. Lecoq et al. Plant Pathol. 41:749, 1992 (2) M. A. Mayo and C. J. D'Arcy. Page 15 in: The Luteoviridae. H. G. Smith and H. Barker, eds. CAB International Mycological Institute, Wallingford, UK, 1999. (3) H. Guilley et al. Virology 202:1012, 1994.

Download Full-text

First Report of a Carlavirus in Fuchsia spp. in New Zealand

Plant Disease ◽

10.1094/pdis-05-11-0443 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1484-1484 ◽

Cited By ~ 2

Author(s):

Z. Perez-Egusquiza ◽

L. W. Liefting ◽

S. Veerakone ◽

G. R. G. Clover ◽

M. Ciuffo

Keyword(s):

Electron Microscopy ◽

New Zealand ◽

Coat Protein ◽

Chenopodium Quinoa ◽

Plant Diseases ◽

Nucleotide Identity ◽

Impatiens Necrotic Spot Virus ◽

Rt Pcr ◽

First Report ◽

Genome Region

The genus Fuchsia has 110 known species and numerous hybrids. These ornamental plants with brightly colored flowers originate from Central and South America, New Zealand, and Tahiti, but a wider variety are now grown all over the world. Few viruses have been reported in Fuchsia spp.: a carlavirus, Fuchsia latent virus (FLV) (1–3), a cucumovirus, Cucumber mosaic virus (CMV) (3), and two tospoviruses, Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV) (4). In August 2009, five plants, each representing a different cultivar of Fuchsia hybrid, from home gardens in the Auckland and Southland regions of New Zealand, displayed variable symptoms including mild chlorosis, mild mottle, or purple spots on leaves. Plants tested negative for CMV, INSV, and TSWV using commercial ImmunoStrips (Agdia Inc., Elkhart, IN); however, flexuous particles of ~650 to 700 nm were found by electron microscopy in all samples. Local lesions were also observed on Chenopodium quinoa plants 4 weeks after sap inoculation. Total RNA was extracted from all plants with a RNeasy Plant Mini Kit (Qiagen Inc., Doncaster, Australia) and tested by reverse transcription (RT)-PCR using two generic sets of primers (R. van der Vlugt, personal communication) designed to amplify fragments of ~730 and 550 bp of the replicase and coat protein genes of carlaviruses, respectively. Amplicons of the expected size were obtained for all samples, cloned, and at least three clones per sample were sequenced. No differences within clones from the same samples were observed (GenBank Accession Nos. HQ197672 to HQ197681). A BLASTn search of the viral replicase fragment showed the highest nucleotide identity (76%) to Potato rough dwarf virus (PRDV) (EU020009), whereas the coat protein fragment had maximum nucleotide identity (70 to 72%) to PRDV (EU020009 and DQ640311) and Potato virus P (DQ516055). Sequences obtained were also pairwise aligned using the MegAlign program (DNASTAR, Inc., Madison, WI) and results showed that the isolates had 83 to 97% identity to each other within each genome region. Further sequences (HQ197925 and HQ197926) were obtained from a Fuchsia plant originating from Belgium, a BLASTn analysis showed high nucleotide identity (84 to 99%) to the New Zealand isolates. The low genetic identity to other Carlavirus members suggests that these isolates belong to a different species from those previously sequenced. On the basis of electron microscopy and herbaceous indexing, the isolates had similar characteristics to a carlavirus reported from Fuchsia in Italy (1) and FLV reported in Canada (2). The Italian carlavirus isolate was obtained and tested with the same primers by RT-PCR. Pairwise analysis of the Italian sequences (HQ197927 and HQ197928) with the New Zealand and Belgian sequences showed between 84 and 95% similarity within each genome region. These results suggest that the carlavirus infecting these plants is the same virus, possibly FLV. To our knowledge, this is the first report of this carlavirus infecting Fuchsia spp. in New Zealand, but the virus has probably been present for some time in this country and is likely to be distributed worldwide. References: (1) G. Dellavalle et al. Acta Hortic. 432:332, 1996. (2) L. J. John et al. Acta Hortic. 110:195, 1980. (3) P. Roggero et al. Plant Pathol. 49:802, 2000. (4) R. Wick and B. Dicklow. Diseases in Fuchsia. Common Names of Plant Diseases. Online publication. The American Phytopathological Society, St. Paul, MN, 1999.

Download Full-text

First Report of Beet necrotic yellow vein virus Infecting Spinach in California

Plant Disease ◽

10.1094/pdis-94-5-0640a ◽

2010 ◽

Vol 94 (5) ◽

pp. 640-640 ◽

Cited By ~ 4

Author(s):

H.-Y. Liu ◽

B. Mou ◽

K. Richardson ◽

S. T. Koike

Keyword(s):

Spinacia Oleracea ◽

Sandwich Elisa ◽

Chenopodium Quinoa ◽

Yellow Vein ◽

Mechanical Transmission ◽

Polymyxa Betae ◽

Rt Pcr ◽

First Report ◽

Vein Clearing ◽

Rigid Rod

In 2009, plants from two spinach (Spinacia oleracea) experimental fields in Monterey County and one commercial spinach field in Ventura County of California exhibited vein-clearing, mottling, interveinal yellowing, and stunting symptoms. For experimental fields, up to 44% of spinach plants have symptoms. With a transmission electron microscope, rigid rod-shaped particles with central canals were observed from plant sap of the symptomatic spinach. Analysis with a double-antibody sandwich-ELISA assay for Beet necrotic yellow vein virus (BNYVV) showed that all 10 symptomatic plants we tested were positive and 5 asymptomatic plants were negative. Symptomatic spinach from both counties was used for mechanical transmission experiments. Chenopodium quinoa, Tetragonia expansa, and Beta vulgaris (sugar beet) showed chlorotic local lesions and B. macrocarpa and spinach showed vein-clearing, mottling, and systemic infections. To further confirm the presence of BNYVV, reverse transcription (RT)-PCR was conducted. Total RNA was extracted from field- and mechanically inoculated symptomatic spinach plants using an RNeasy Plant Kit (Qiagen Inc., Valencia, CA) and used as a template in RT-PCR. Forward and reverse primers specific to the BNYVV RNA-3 P25 protein gene from the beet isolate were used (2). Amplicons of the expected size (approximately 860 bp) were obtained. Four RT-PCR products were sequenced and the sequences were identical (GenBank Accession No. GU135626). Sequences from the spinach plants had 97 to 99% nucleotide and 94 to 100% amino acid identity with BNYVV RNA-3 P25 protein sequences available in the GenBank. On the basis of the data from electron microscopy, indicator plants, serology, and cDNA sequencing, the virus was identified as BNYVV. BNYVV has been reported from spinach fields in Italy (1). To our knowledge, this is the first report of BNYVV occurring naturally on spinach in California. Since BNYVV is transmitted by the zoospores of the soil-inhabiting plasmodiophorid Polymyxa betae, it could be a new threat to spinach production in the state. References: (1) C. R. Autonell et al. Inf. Fitopatol. 45:43, 1995. (2) H.-Y. Liu and R. T. Lewellen, Plant Dis. 91:847, 2007.

Download Full-text

First Report of Little cherry virus 2 in Flowering and Sweet Cherry Trees in China

Plant Disease ◽

10.1094/pdis-10-10-0766 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1484-1484 ◽

Cited By ~ 4

Author(s):

W.-L. Rao ◽

F. Li ◽

R.-J. Zuo ◽

R. Li

Keyword(s):

Sweet Cherry ◽

Sequence Data ◽

Community Gardens ◽

Economic Losses ◽

Helicase Domain ◽

Rt Pcr ◽

Viral Origin ◽

First Report ◽

Flowering Cherry ◽

Cherry Trees

Many viruses infect Prunus spp. and cause diseases on them. During a survey of stone fruit trees in 2008 and 2009, flowering cherry (Prunus serrulata) and sweet cherry (P. avium) trees with foliar chlorosis and reddening, stem deformity, and tree stunting were observed in private orchards in Anning and Fumin counties of Yunnan Province. Some sweet cherry trees with severe symptoms yielded small and few fruits and had to be removed. Leaf samples were collected from 68 flowering cherry and 30 sweet cherry trees, either symptomatic or asymptomatic, from private orchards and community gardens in Kunming and counties Anning, Chenggong, Fumin, Jinning, Ludian and Yiliang. Total nucleic acids were extracted with a CTAB extraction method and tested by reverse transcription (RT)-PCR assay using virus-specific primers. Little cherry virus 2 (LChV-2), Cherry virus A (CVA), Prunus necrotic ringspot virus (PNRSV), and Prune dwarf virus (PDV) were detected and infection rates were 68.4, 16.3, 9.2, and 7.1%, respectively. Infection of LChV-2 was common in all counties except Ludian where the orchards were healthy. Of 68 infected trees, 29 were found to be infected with LChV-2 and CVA, PDV or PNRSV. LChV-2 was detected in this study by RT-PCR using a pair of novel primers, LCV2-1 (5′-TTCAATATGAGCAGTGTTCCTAAC-3′) and LCV2-4 (5′-ACTCGTCTTGTGACATACCAGTC-3′), in 59 flowering cherry (87%) and 8 sweet cherry (27%) trees, respectively. The primer pair was designed according to alignment of three available LChV-2 sequences (GenBank Nos. NC_005065, AF416335, and AF333237) to amplify the partial RNA-dependent RNA polymerase gene (ORF1b) of 781 bp. The amplicons of selected samples (Anning26 and Yiliang60) were sequenced directly and sequences of 651 bp (GenBank No. HQ412772) were obtained from both samples. Pairwise comparisons and phylogenetic analysis of the sequences show that the two isolates are identical to one another and share 92 to 96% at the amino acid (aa) sequence level to those of other isolates available in the GenBank database. The sequence data confirm that these isolates are a strain of LChV-2 and genetic variation among different strains is relatively high (2). Biological and serological assays are not available for the LChV-2 detection; therefore, the LChV-2 infections of these trees were further confirmed by RT-PCR using primer pair LCV2-5 (5′-TGTTTGTGTCATGTTGTCGGAGAAG-3′) and LCV2-6 (5′-TGAATACCCGAGAACAAGGACTC-3′), which amplified the helicase domain (ORF1a) of ~451 bp. The amplicons from samples Anning26 and Yiliang60 were cloned and sequenced. The 408-bp sequences (excluding primer sequences) were 92 to 98% identical at the aa sequence level to those of other isolates, confirming again their viral origin. LChV-2 (genus Ampelovirus, family Closteroviridae) (4) has been associated with little cherry disease (LChD), a widespread viral disease of sweet and sour cherries (1,3). The virus is transferred between geographic areas mainly by propagated materials. Ornamental and sweet cherries are important crops in China and LChD has the potential to cause significant economic losses. Thus, certified clean stock should be used to establish new orchards. To our knowledge, this is the first report of LChV-2 in cherries in China. References: (1) N. B. Bajet et al. Plant Dis. 92:234, 2008. (2) W. Jelkmann et al. Acta Hortic. 781:321, 2008. (3) B. Komorowska and M. Cieslińska, Plant Dis. 92:1366, 2008. (4) M. E. Rott and W. Jelkmann. Arch. Virol. 150:107, 2005.

Download Full-text

First Report of Sharka Disease Caused by Plum Pox Virus in Lithuania

Plant Disease ◽

10.1094/pdis.1998.82.12.1405c ◽

1998 ◽

Vol 82 (12) ◽

pp. 1405-1405 ◽

Cited By ~ 1

Author(s):

J. Staniulis ◽

J. Stankiene ◽

K. Sasnauskas ◽

A. Dargeviciute

Keyword(s):

Coat Protein ◽

Plant Protection ◽

Virus Disease ◽

Pcr Amplification ◽

Enzyme Linked Immunosorbent Assay ◽

Plum Pox Virus ◽

Rt Pcr ◽

First Report ◽

Das Elisa ◽

Sharka Disease

Plum pox (sharka) disease caused by plum pox potyvirus (PPV) is considered the most important virus disease of stone fruit trees in Europe and the Mediterranean region. Nearly all those countries that produce stone fruits are affected (3). The causal virus of the disease is a European Plant Protection Organization A2 quarantine pathogen. Symptoms of leaf mottling, diffuse chlorotic spots, rings, and vein banding of varied intensity characteristic for plum pox virus infection were observed in the plum (Prunus domestica) orchard tree collection of the Lithuanian Institute of Horticulture in Babtai in 1996. Presence of this virus in the diseased trees was confirmed by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with kits from BIOREBA (Reinach, Switzerland) and by polyclonal antibodies raised against a Moldavian isolate of PPV courtesy of T. D. Verderevskaya (Institute of Horticulture, Kishinev, Moldova). ELISAs with both sources of antiserum were positive for presence of PPV. Electron microscopy revealed the presence of potyvirus-like particles averaging 770 nm in extracts of mechanically inoculated plants of Chenopodium foetidum (chlorotic LL [local lesions]) and Pisum sativum cvs. Rainiai and Citron (mottling). For molecular diagnosis and characterization of this isolate, PPV-971, reverse transcription-polymerase chain reaction (RT-PCR) was employed. Total RNA from the leaves of infected pea was isolated as described (2). High molecular weight RNA selectively precipitated with 2 M lithium chloride was used for RT-PCR amplification of the coat protein encoding sequence by use of specific primers complementary to 5′ and 3′ parts of PPV coat protein L1 (GenBank accession no. X81081). Amino acid sequence comparison with GenBank data indicated 98.2% similarity with coat protein of PPV potyvirus isolated by E. Mais et al. (accession no. X81083) and 97.3% with PPV strain Rankovic (1).The specific DNA fragment, corresponding to predicted coat protein sequence size, was cloned into Escherichia coli pUC57 for DNA sequencing. Expression of the cloned sequence in bacteria and yeast expression systems is under investigation. The presence of PPV in plum trees in the 9-year-old collection at Babtai was confirmed by DAS-ELISA in 1997 and again in 1998. PPV was then detected in 20% of symptomatic trees of three cultivars. The Lithuanian PPV isolate reacted positively with “universal” Mab.5b and with a Mab (Mab.4DG5) specific for PPV-D. No reaction was observed with Mabs specific for PPV-M (Mab.AL), PPV-C (Mab.AC and Mab.TUV), and PPV-El Amar (Mab.EA24). PPV-971 seems to be a typical member of the less aggressive Dideron strain cluster of PPV (D. Boscia, personal communication). This is the first report of PPV in Lithuania and confirms the necessity for continuing the precautionary measures established in this country for indexing of nursery plum trees used for graft propagation. References: (1) S. Lain et al. Virus Res. 13:157, 1989. (2) J. Logemann et al. Anal. Biochem. 163:16, 1987. (3) M. Nemeth. OEPP/EPPO Bull. 24:525, 1994.

Download Full-text

Deteksi Virus-Virus pada Kentang di Jawa Barat dengan Menggunakan Teknik Molekuler

Jurnal Hortikultura ◽

10.21082/jhort.v25n2.2015.p171-179 ◽

2016 ◽

Vol 25 (2) ◽

pp. 171 ◽

Cited By ~ 1

Author(s):

Tri Asmira Damayanti ◽

R Kartika

Keyword(s):

Coat Protein ◽

Rt Pcr ◽

Dna Virus ◽

Vein Clearing ◽

Strain Virus

ABSTRAK. Gejala infeksi virus ditemukan bervariasi di sentra pertanaman kentang di Jawa Barat (Rancabali, Pangalengan, dan Bayongbong). Diagnosis virus berdasarkan gejala sulit untuk menentukan identitas suatu virus. Oleh karena itu penelitian ini bertujuan mendeteksi virus-virus pada tanaman kentang dengan teknik molekuler. Sebanyak 50 sampel daun dikoleksi secara acak dari tanaman kentang yang bergejala di setiap lokasi. Parameter yang diamati adalah gejala, kejadian penyakit, dan runutan DNA virus yang dominan ditemukan. Kejadian penyakit ditentukan dengan uji serologi menggunakan antiserum PVY, PVX, PVS, dan CMV, sedangkan deteksi asam nukleat dilakukan dengan RT-PCR dan perunutan DNA. Hasil penelitian menunjukkan bahwa gejala yang ditemukan bervariasi pada daun kentang, seperti vein clearing, vein banding, rugose, dan malformasi daun. Kejadian penyakit oleh PVY, PVX, PVS, dan CMV di Rancabali berturut-turut adalah 28%, 0%, 0%, dan 28%, di Pangalengan adalah 80%, 24%, 2%, dan 82%, serta di Bayongbong adalah 82%, 0%, 6%, dan 74%. RT-PCR menggunakan primer spesifik PVY dan CMV berhasil mengamplifikasi gen coat protein PVY dan CMV asal Bayongbong masing-masing berukuran ~800 pb dan ~650 pb. Homologi nukleotida dan asam amino PVY asal Bayongbong terhadap PVY dari negara lain berkisar 89,5–99,7% dan 92,0–100%. Homologi tertingginya yaitu dengan PVY-NTN asal Cina dan Jepang, sedangkan homologi nukleotida dan asam amino CMV asal Bayongbong terhadap CMV asal negara lain berkisar 87,6–96,9% dan 86,9–93,7%. Homologi tertingginya yaitu dengan CMV strain soybean stunt (S) asal Bogor (Indonesia). Kedua strain virus (PVY-NTN dan CMV-S) pertama kali terdeteksi pada kentang di Jawa Barat. Hasil penelitian menunjukkan bahwa teridentifikasinya kedua strain virus baru harus dipertimbangkan sebagai pembatas penting produksi kentang di Jawa Barat

Download Full-text