Purification, Properties, and Diagnosis of Banana Bract Mosaic Potyvirus and Its Distinction from Abaca Mosaic Potyvirus

Using biochemical, serological, and cytopathological evidence, we have confirmed that banana bract mosaic virus (BBrMV) is a distinct member of the family Potyviridae. Virions of a Philippine isolate of BBrMV were purified from field-infected banana cv. Cardaba. Particles were approximately 725-nm long, banded at a density equivalent to 1.29 to 1.31 g/ml in cesium chloride equilibrium gradients, and had an A260/280 of 1.17. Yields of about 4 mg/kg were obtained from fresh or frozen leaf midrib or lamina tissue. Three major protein species with sizes of 31, 37, and 39 kDa were resolved from dissociated virions, and all reacted specifically with polyclonal antibodies to BBrMV. Infected leaf cells contained typical pinwheel inclusions. Virus-specific cDNA was amplified from field samples by reverse transcription-polymerase chain reaction (RT-PCR) assay using potyvirus degenerate primers. In plate-trapped antigen-enzyme-linked immunosorbent assay (ELISA), weak serological relationships were demonstrated between BBrMV and other members of the family Potyviridae, including abaca mosaic (AbaMV), dasheen mosaic, maize dwarf mosaic, sorghum mosaic, sugarcane mosaic, and wheat streak mosaic viruses. Despite similarities in the symptoms caused by the two viruses, AbaMV was serologically distinct from BBrMV and reacted only weakly, or not at all, with BBrMV antibodies in double-antibody sandwich (DAS)-ELISA. No cross reactions were observed when RT-PCR products from the two viruses were examined by Southern blot hybridization using BBrMV- and AbaMV-specific digoxigenin-labeled DNA probes. BBrMV was consistently associated with banana bract mosaic disease, as assessed by DAS-ELISA and Southern blot hybridization using DNA probes. The known geographical distribution of BBrMV was extended to include India (Kokkan disease) and Sri Lanka.

Download Full-text

Leaf Curl Disease of Carica papaya from India May Be Caused by a Bipartite Geminivirus

Plant Disease ◽

10.1094/pdis.1998.82.1.126a ◽

1998 ◽

Vol 82 (1) ◽

pp. 126-126 ◽

Cited By ~ 18

Author(s):

Sangeeta Saxena ◽

Vipin Hallan ◽

B. P. Singh ◽

P. V. Sane

Keyword(s):

Southern Blot ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Dna Probes ◽

Economic Losses ◽

Degenerate Primers ◽

Tomato Leaf Curl ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Leaf Curl

Papaya has considerable economic importance to agriculture in India. Papaya leaf curl disease was first reported in 1939 by Thomas and Krishnaswamy (3). This disease is of moderate incidence and widely distributed in India. Recent observations of papaya fields in India indicated that there has been a continued increase in the incidence of papaya leaf curl disease (as shown by symptoms), resulting in severe economic losses. The disease is characterized by downward curling and cupping of leaves followed by vein clearing and thickening. Enations develop in the form of frills on green veins. The affected leaves become leathery and brittle and the petioles become twisted in a zig-zag manner. Diseased plants may bear a few small fruits, which are distorted in shape and tend to fall prematurely. The disease could be transmitted by the whitefly Bemisia tabaci Genn. Therefore, possible involvement of a geminivirus was suspected. Three different cloned geminiviral DNAs, Indian tomato leaf curl virus (ITLCV) (2), tomato yellow leaf curl virus from Sardinia (TYLCV Sar), and tomato golden mosaic virus (TGMV), were used as probes (with radioactive labeling) to detect the presence of geminiviral DNA from infected papaya tissue in both slot-blot and Southern blot hybridization studies with high stringency washes. These DNA probes gave strong signals with DNA isolated from infected papaya tissue whereas they did not give any signals with DNA from healthy tissue. Further, successful polymerase chain reaction (PCR)-based amplification of fragments from both DNA-A and DNA-B components with geminivirus degenerate primers (1) was accomplished only from the DNA of infected papaya plants. The PCR-amplified DNA fragments gave positive signals in Southern blot hybridization with the three geminiviral DNA probes. These results suggest that the causal agent of papaya leaf curl disease is a bipartite geminivirus that may be provisionally called papaya leaf curl virus (PLCV). References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) K. M. Srivastava et al. J. Virol. Methods 51:297, 1995. (3) K. M. Thomas and C. S. Krishnaswamy. Curr. Sci. 8:316, 1939.

Download Full-text

Comparison of Diagnostic Techniques for Detecting Tomato Infectious Chlorosis Virus

Plant Disease ◽

10.1094/pdis.1998.82.1.84 ◽

1998 ◽

Vol 82 (1) ◽

pp. 84-88 ◽

Cited By ~ 24

Author(s):

R. H. Li ◽

G. C. Wisler ◽

H.-Y. Liu ◽

J. E. Duffus

Keyword(s):

Western Blot ◽

Blot Hybridization ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Dot Blot ◽

Tomato Plants ◽

Dot Blot Hybridization ◽

Field Samples ◽

Infected Tomato ◽

Tomato Infectious Chlorosis Virus

A polyclonal antiserum prepared against purified virions of tomato infectious chlorosis virus (TICV) was used to evaluate serological tests for its detection, to determine its distribution in infected plants, to study relationships among isolates of this virus, and to detect it in field samples. A cRNA probe representing TICV RNA 1 and RNA 2 was used in dot blot hybridization tests. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was also developed for detection of TICV isolates. The comparative study of these four techniques indicated that RT-PCR was 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA), Western blot, and dot blot hybridization assays for TICV detection. TICV was detected in leaf, stem, flower, and root tissues of the infected tomato plants. However, the virus was not uniformly distributed throughout the infected tomato plants, and the highest viral concentration was observed in fully developed young tomato leaves at the onset of yellowing symptoms. The virus was detected by indirect ELISA, Western blot, dot blot hybridization, and RT-PCR assays in laboratory-infected tomato, tomatillo, potato, and Nicotiana clevelandii and in naturally infected tomato, petunia, and Ranunculus sp. plants obtained from commercial sources. These tests indicate that there are apparently no detectable serological or nucleic acid differences among four TICV isolates obtained from Orange and Yolo Counties of California or from North Carolina or Italy.

Download Full-text

Chickpea chlorotic stunt virus: A New Polerovirus Infecting Cool-Season Food Legumes in Ethiopia

Phytopathology ◽

10.1094/phyto-96-0437 ◽

2006 ◽

Vol 96 (5) ◽

pp. 437-446 ◽

Cited By ~ 37

Author(s):

A. D. Abraham ◽

W. Menzel ◽

D.-E. Lesemann ◽

M. Varrelmann ◽

H. J. Vetten

Keyword(s):

Faba Bean ◽

Polymerase Chain Reaction Amplification ◽

Rabbit Antiserum ◽

Polyclonal Antiserum ◽

Enzyme Linked Immunosorbent Assay ◽

Coding Region ◽

Degenerate Primers ◽

Beet Western Yellows Virus ◽

Field Samples ◽

The Family

Serological analysis of diseased chickpea and faba bean plantings with yellowing and stunting symptoms suggested the occurrence of an unknown or uncommon member of the family Luteoviridae in Ethiopia. Degenerate primers were used for reverse transcriptase-polymerase chain reaction amplification of the viral coat protein (CP) coding region from both chickpea and faba bean samples. Cloning and sequencing of the amplicons yielded nearly identical (96%) nucleotide sequences of a previously unrecognized species of the family Luteoviridae, with a CP amino acid sequence most closely related (identity of ≈78%) to that of Groundnut rosette assistor virus. The complete genome (5,900 nts) of a faba bean isolate comprised six major open reading frames characteristic of polero-viruses. Of the four aphid species tested, only Aphis craccivora transmitted the virus in a persistent manner. The host range of the virus was confined to a few species of the family Fabaceae. A rabbit antiserum raised against virion preparations cross-reacted unexpectedly with Beet western yellows virus-like viruses. This necessitated the production of murine monoclonal antibodies which, in combination with the polyclonal antiserum, permitted both sensitive and specific detection of the virus in field samples by triple-antibody sandwich, enzyme-linked immunosorbent assay. Because of the characteristic field and greenhouse symptoms in chickpea, the name Chickpea chlorotic stunt virus is proposed for this new member of the genus Polerovirus (family Luteoviridae).

Download Full-text

Chromosomal Assignment of HepG2 3′-Directed Partial cDNA Sequences by Southern Blot Hybridization Using Monochromosomal Hybrid Cell Panels

Genomics ◽

10.1006/geno.1994.1353 ◽

1994 ◽

Vol 22 (1) ◽

pp. 127-136 ◽

Cited By ~ 5

Author(s):

Atsushi Fukushima ◽

Kousaku Okubo ◽

Hidehiko Sugino ◽

Naohiro Hori ◽

Ryo Matoba ◽

...

Keyword(s):

Southern Blot ◽

Hybrid Cell ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Chromosomal Assignment ◽

Cdna Sequences ◽

Partial Cdna ◽

Monochromosomal Hybrid

Download Full-text

Yellow Net of Triumffeta Is Caused by a Geminivirus: A First Report

Plant Disease ◽

10.1094/pdis.1998.82.1.127a ◽

1998 ◽

Vol 82 (1) ◽

pp. 127-127 ◽

Cited By ~ 2

Author(s):

Vipin Hallan ◽

Sangeeta Saxena ◽

B. P. Singh

Keyword(s):

Rainy Season ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Viral Disease ◽

Hybridization Experiment ◽

Dorsal Side ◽

Causal Agent ◽

Degenerate Primers ◽

First Report ◽

Initial Symptoms

Triumffeta rhomboidiaceae Jacq. (Tiliaceae family) is an annual rainy season weed that is commonly found throughout India. For the last 3 years, during the rainy season, several plants of T. rhomboidiaceae in and around the gardens of the National Botanical Research Institute have been found with vein yellowing symptoms. The initial symptoms were vein clearing but in later stages the veins became yellow and thickened. In severe cases, the chlorosis extends into interveinal areas, resulting in complete yellowing of the leaves. In a few cases, green leafy or thorny enations could be seen on the dorsal side of the leaf. The disease was investigated to identify the causal agent. Vector transmission studies showed that the causal agent is transmitted by the whitefly, Bemisia tabaci, from infected to healthy seedlings of T. rhomdoidiaceae. Since whitefly transmission of the disease is consistent with a geminivirus as the causal agent, the role of such a virus was investigated. DNA isolated from Triumffeta plants (both from the infected plants in the field as well as from those inoculated experimentally in the greenhouse) showing above mentioned symptoms was amplified with two sets of degenerate primers, PAL1v1978/PAR1c496 (set 1) and PAL1v1978/PCRc1 (set 2), that have been shown to be specific for DNA-A of whitefly transmitted geminiviruses (WTGs), in polymerase chain reaction (1). We could amplify DNA-A fragments of approximately 1.2 kb from set 1 and 0.7 kb from set 2, as expected (1). DNA isolated from healthy seedlings gave no amplification of such fragments. Identification of the amplified DNA fragments (from infected samples) to be of geminiviral in nature was confirmed by Southern blot hybridization carried out under high stringency conditions. DNA-A of Indian tomato leaf curl virus (2) was used as a general probe for WTGs for the above hybridization experiment. Therefore, Triumffeta yellow net disease is caused by a geminivirus. A review of literature revealed that there is no record of a viral disease affecting this weed and, therefore, this is the first report of a viral disease affecting this plant. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) K. M. Srivastava et al. J. Virol. Methods 51:297, 1995.

Download Full-text

Genome analysis of HTLV-I virus in PA positive and IF negative healthy donors by southern blot hybridization.

Journal of the Japan Society of Blood Transfusion ◽

10.3925/jjtc1958.34.50 ◽

1988 ◽

Vol 34 (1) ◽

pp. 50-52

Author(s):

Chieko Suzuki ◽

Noriko Tomita ◽

Eiichi Uchiyama ◽

Akio Ishii ◽

Takeshi Nishizaki ◽

...

Keyword(s):

Southern Blot ◽

Genome Analysis ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Healthy Donors

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

Detection of “Candidatus Helicobacter suis” in Gastric Samples of Pigs by PCR: Comparison with Other Invasive Diagnostic Techniques

Journal of Clinical Microbiology ◽

10.1128/jcm.38.3.1131-1135.2000 ◽

2000 ◽

Vol 38 (3) ◽

pp. 1131-1135 ◽

Cited By ~ 39

Author(s):

D. De Groote ◽

R. Ducatelle ◽

L. J. van Doorn ◽

K. Tilmant ◽

A. Verschuuren ◽

...

Keyword(s):

Southern Blot ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Epidemiological Studies ◽

Diagnostic Techniques ◽

Diagnostic Methods ◽

Giemsa Staining ◽

Rapid Urease Test ◽

Pcr Assay ◽

Urease Test

Recently, a new 16S ribosomal DNA-based PCR assay was developed for the specific detection of “Candidatus Helicobacter suis” (former “Gastrospirillum suis”) in porcine gastric samples. In the present study, this PCR assay was compared to three other invasive diagnostic methods (rapid urease test, immunohistochemistry, histologic analysis by Giemsa staining). Antral stomach samples from 200 slaughterhouse pigs from Belgium and The Netherlands were examined. Bacterial presence was determined in 77% (154 of 200) of the samples by PCR in combination with Southern blot hybridization, 56% (111 of 200) of the samples by immunohistochemistry, 61% (122 of 200) of the samples by urease testing (20 h postinoculation [p.i.]), 36% (71 of 200) of the samples by urease testing (3 h p.i.), and 33% (65 of 200) of the samples by Giemsa staining. The intrinsic specificity of the PCR assay was assessed by Southern blot analysis with an “Candidatus H. suis”-specific probe and sequencing of PCR products. Interassay sensitivity and specificity values were assessed for each test by pairwise comparisons between tests. Agreement between tests was evaluated by calculating Cohen's kappa coefficient. From that analysis, the PCR assay was considered the most reliable benchmark. Microscopic detection of immunohistochemically labeled or Giemsa-stained “Candidatus H. suis” cells in stomach sections proved to be highly specific (100%) but relatively insensitive (72 and 42%, respectively) compared to the PCR assay. A longer incubation time of the urease test improved its sensitivity considerably (74 versus 55%) but was accompanied by a loss of specificity (72 versus 93%). In conclusion, we found the “Candidatus H. suis”-specific PCR assay to be a sensitive and reliable diagnostic method for the detection of “Candidatus H. suis” in the stomachs of pigs and could prove to be a valuable tool for further epidemiological studies both for “Candidatus H. suis”- and for “Helicobacter heilmannii” type 1-related research.

Download Full-text