First Report of Lettuce chlorosis virus Infecting Bean in Spain

In September 2011, symptoms typically associated with Bean yellow disorder virus (BYDV) such as intervenal mottling and yellowing on middle and lower leaves combined with brittleness were observed in green bean (Phaseolus vulgaris L.) produced in commercial greenhouses from Granada and Almeria provinces, Spain. The affected plants were all observed in greenhouses infested with Bemisia tabaci. However, collected samples tested negative for BYDV using a specific RT-PCR test (4). Electrophoretic double stranded (ds) RNA analysis from symptomatic plants revealed the presence of a slightly diffused high molecular weight dsRNA band of ~8.5 kb, similar to that produced by the crinivirus Lettuce cholorosis virus (LCV) (3). The dsRNA was purified and used for cDNA synthesis and PCR by uneven PCR (1) using primers derived from LCV genome sequences (GenBank FJ380118 and FJ380119). Amplified DNA fragments were cloned in pGEM-T Easy vector (Promega, Madison, WI) and sequenced. Two different sequences were obtained and the nucleotide and amino acid sequences were analysed using BLAST. Both showed the highest identity with different regions of the LCV genome. The sequence of the first product had 92% nucleotide and 98% amino acid sequence identity with the polyprotein (Orf1a) homologue from RNA1 of LCV (KC602376). The sequence from the second product (KC602375) revealed the highest nucleotide and amino acid identity with the heat shock protein 70 homologue from LCV (90% and 99%, respectively). Based on these sequences, two sets of specific primers were designed (LCVSP 3-forward 5′-AGTGACACAAGTTGGAGCCGAC-3′, LCVSP 4-low 5′-CAGTGTTTGTTGGATATCTGGGG-3′) and (LCVSP 1-forward 5′-TGTTGGAAGGTGGTGAGGTC-3′, LCVSP 2-low 5′-CAGAGACGAGTCATACGTACC-3′) and each produced amplicons of the expected size (463 and 434 nt, respectively) when used in RT-PCR from the collected field samples. Subsequent field surveys from 2012 to 2013 in commercial bean greenhouses confirmed the presence of LCV that apparently had replaced BYDV. Groups of 15 to 20 adults of B. tabaci introduced in clip cages were fed for 24 h on 12 green bean plants infected with LCV and later transferred to six seedlings of bean and six of lettuce (Lactuca sativa L.). After 2 and 4 weeks, total RNA from the lettuce and bean plants was extracted using Plant RNA Reagent (Invitrogen) and subjected to RT-PCR analysis with the LCV-SP 1-2 and LCVSP 3-4 primer sets. All six plants of bean and none of lettuce showed positive for LCV-SP and a repeat experiment revealed identical results. We also seeded and produced lettuce plants within a bean greenhouse that was naturally infected with the virus and infested with B tabaci whiteflies. Under these conditions, we observed that whiteflies migrated freely from the infected bean plants to lettuce. After 4 and 6 weeks, lettuce plants neither produced symptoms nor tested positive for LCV by RT-PCR. This result confirms the existence of a new putative strain of LCV, Lettuce chlorosis virus-SP, unable to infect lettuce plants. To date, natural infections of LCV have not been reported outside California, where the virus failed to infect P. vulgaris (2). This is also the first report of LCV in Spain that infects members of the family Leguminosae. Green bean in southeast Spain was produced in ~9,000 ha of greenhouses until the introduction of BYDV a decade ago, causing considerable economic damage. The recent finding of LCV-SP has urged the local phytosanitary inspections to include this virus in lab tests and to emphasize disease management strategies based on whitefly control. References: (1) X. Chen and R. Wu. Gene 185:195, 1997. (2) J. Duffus et al. Eur. J. Plant Pathol. 102:591, 1996. (3) N. M. Salem et al. Virology 390:45, 2009. (4) E. Segundo et al. Plant Pathol. 53:517, 2004.

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Squash vein yellowing virus Identified in Watermelon (Citrullus lanatus) in Indiana

Plant Disease ◽

10.1094/pdis-91-8-1056b ◽

2007 ◽

Vol 91 (8) ◽

pp. 1056-1056 ◽

Cited By ~ 17

Author(s):

D. S. Egel ◽

S. Adkins

Keyword(s):

Citrullus Lanatus ◽

Amino Acid Sequences ◽

Rt Pcr ◽

Strain Vector ◽

First Report ◽

Pcr Product ◽

Field Samples ◽

Health Progress ◽

Vine Decline ◽

Yellowing Virus

During September 2006, moderate vine decline symptoms including vine collapse and wilt and root rot were observed on numerous watermelon plants growing in a commercial field in Sullivan County, Indiana. No symptoms were observed on the fruit. Six plants displaying typical vine decline symptoms were collected and assayed for potyvirus infection and subsequently for Squash vein yellowing virus (SqVYV) and Papaya ringspot virus type W (PRSV-W). SqVYV is a whitefly-transmitted member of the Potyviridae, recently shown to cause watermelon vine decline in Florida (1,4). Plants infected with SqVYV in Florida are also frequently infected with PRSV-W, although SqVYV is sufficient for watermelon vine decline. The six field samples harbored one or more potyviruses as determined by ELISA (Agdia, Elkhart, IN). Mechanical inoculation of squash (Cucurbita pepo) and watermelon with sap from three of the field samples induced mosaic symptoms in both that are typical of potyviruses. Vein yellowing in squash and plant death in watermelon typical of SqVYV (1) later developed in plants inoculated with one field sample. A coat protein gene fragment was amplified by reverse transcription (RT)-PCR with SqVYV primers (1) from total RNA of five of the six field samples and also from the symptomatic, inoculated plants. Nucleotide and deduced amino acid sequences of a 957-bp region of the RT-PCR product (primer sequences deleted prior to analysis) were 100% identical to SqVYV (GenBank accession No. DQ812125). PRSV-W also was identified in two of the five SqVYV-infected field samples by ELISA (Agdia) and by sequence analysis of a 3′ genome fragment amplified by RT-PCR with previously described degenerate potyvirus primers (3). No evidence for infection by other potyviruses was obtained. To our knowledge, this is the first report of SqVYV in Indiana and the first report of the virus anywhere outside of Florida. The whitefly (Bemisia tabaci, B strain) vector of SqVYV is relatively uncommon in Indiana and the cold winter temperatures make it unlikely that any SqVYV-infected watermelon vines or whiteflies will overseason, necessitating reintroductions of virus and vector each season. We feel that the moderate and restricted occurrence of SqVYV in Indiana observed in September 2006 should pose little or no threat to commercial watermelon production in Indiana and should not cause growers to alter their growing practices. The occurrence of SqVYV in Indiana does not appear to explain the similar symptoms of mature watermelon vine decline (MWVD) that has been observed in Indiana since the 1980s. In contrast with the insect vectored SqVYV, MWVD seems to be caused by a soilborne biological agent (2). References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) D. S. Egel et al. Online publication. doi:10.1094/PHP-2000-1227-01-HN. Plant Health Progress, 2000. (3) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (4) P. Roberts et al. Citrus Veg. Mag. December 12, 2004.

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First Report of Beet necrotic yellow vein virus on Red Table Beet in Brazil

Plant Disease ◽

10.1094/pdis-10-14-1045-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 423-423 ◽

Cited By ~ 4

Author(s):

J. A. M. Rezende ◽

V. M. Camelo ◽

D. Flôres ◽

A. P. O. A. Mello ◽

E. W. Kitajima ◽

...

Keyword(s):

Amino Acid ◽

Sugar Beet ◽

Hairy Root ◽

Contaminated Soil ◽

The United States ◽

Amino Acid Sequences ◽

Yellow Vein ◽

Polymyxa Betae ◽

Rt Pcr ◽

First Report

Beet necrotic yellow vein virus (BNYVV) is an economically important pathogen of sugar beet (Beta vulgaris var. saccharifera) in several European, and Asian countries and in the United States (3). The virus is transmitted by the soil-inhabiting plasmodiophorid Polymyxa betae and causes the rhizomania disease of sugar beet. In November 2012, plants of B. vulgaris subsp. vulgaris cv. Boro (red table beet) exhibiting mainly severe characteristic root symptom of rhizomania were found in a commercial field located in the municipality of São José do Rio Pardo, State of São Paulo, Brazil. No characteristic virus-inducing foliar symptom was observed on diseased plants. The incidence of diseased plants was around 70% in the two visited crops. As the hairy root symptom is indicative of infection by BNYVV, the present study aimed to detect and identify this virus associated with the diseased plants. Preliminary leaf dip analysis by transmission electron microscopy revealed the presence of very few benyvirus-like particles. Total RNA was extracted from roots of three symptomatic plants and one asymptomatic plant according to Toth et al. (3). One-step reverse-transcription–polymerase chain reaction (RT-PCR) was performed as described by Morris et al. (2) with primers that amplify part of the coat protein gene at RNA2. The initial assumption that the hairy root symptom was associated with BNYVV infection was confirmed by the amplification of a fragment of ~500 bp from all three symptomatic samples. No amplicon was obtained from the asymptomatic control plant. Amplicons were directly sequenced, and the consensus nucleotide and deduced amino acid sequences showed 100% identity. The nucleotide sequence for one amplicon (Accession No. KM433683) was compared with other sequences deposited in GenBank. The nucleotide (468 nt) and deduced amino acid (156 aa) sequences shared 93 to 100 and 97 to 99% identity, respectively with the corresponding nucleotide and amino acid sequences for other isolates of type A of BNYVV. The virus was transmitted to three of 10 red table beet plants inoculated with contaminated soil, and infection was confirmed by nested RT-PCR, as described by Morris et al. (1), and nucleotide sequencing. This is the first report on the occurrence of BNYVV in Brazil, which certainly will affect the yield of red table beet in the producing region. Therefore, mapping of the occurrence of BNYVV in red table beet-producing areas in Brazil for containment of the spread of the virus is urgent. In the meantime, precautions should be taken to control the movement of contaminated soil and beet roots, carrots, or any vegetable grown on infested land that might introduce the virus to still virus-free regions. References: (1) J. Morris et al. J. Virol. Methods 95:163, 2001. (2) D. D. Sutic et al. Handbook of Plant Virus Diseases. CRC Press, Boca Raton, Florida, 1999. (3) I. K. Toth et al. Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica (Pectobacterium carotovorum subsb. atrosepticum) on Potatoes: A Laboratory Manual. Scottish Crop Research Institute, Dundee, Scotland, 2002.

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First Report of Passiflora chlorosis virus in Bituminaria bituminosa in Europe

Plant Disease ◽

10.1094/pdis-93-2-0196a ◽

2009 ◽

Vol 93 (2) ◽

pp. 196-196 ◽

Cited By ~ 1

Author(s):

L. Cardin ◽

B. Moury

Keyword(s):

Amino Acid ◽

Green Peach Aphid ◽

Amino Acid Sequences ◽

Cdna Clones ◽

Coding Region ◽

Rt Pcr ◽

Leaf Extracts ◽

Chenopodium Amaranticolor ◽

First Report ◽

Bituminaria Bituminosa

Bituminaria bituminosa (L.) Stirton (pitch trefoil) is a perennial legume endemic to the Mediterranean Basin used as forage in arid areas and for stabilization of degraded soils. Mosaic and chlorotic ringspot symptoms have been observed in leaves of B. bituminosa in the Provence-Alpes-Côte d'Azur and Rhône-Alpes regions (France), Liguria (Italy), and Spain since 1975. In crude leaf extracts from more than 50 samples of diverse geographical origins, flexuous particles 680 to 720 nm long and 12 nm wide and pinwheel-like inclusions have been observed with the electron microscope, suggesting infection with a member of the family Potyviridae. The presence of a virus was confirmed by the use of potyvirus-polyvalent ELISA reagents (Potyvirus group test; Agdia, Elkhart, IN) and by the amplification of a DNA fragment of the expected size (≈1,650 bp) with extracts of isolates from different locations using reverse transcription (RT)-PCR with primers specific to members of the Potyviridae (3) corresponding to the 3′ end of the virus genome. The amplified fragment of an isolate from Coaraze (Alpes Maritimes Department, France) was cloned and two cDNA clones corresponding to this amplicon were sequenced (GenBank Accession Nos. EU334546 and EU334547). These two sequences facilitated development of new primers (5′-AAARGCRCCCTATATAGCAG-3′ and 5′-TATAAAGGTAACGCTAGGTGG-3′) to specifically amplify and sequence the coat protein (CP)-coding region of isolates of the virus from five additional French locations. The amino acid sequences of the CP amplicon were more than 96% identical among the French isolates. Comparison with other virus sequences with the BLASTn program revealed that these isolates belonged to the same species as the potyvirus Passiflora chlorosis virus (2), with 89 to 90% and 95 to 97% identity at the nucleotide and amino acid levels, respectively, for the CP-coding region (1). The host range of the virus was evaluated by manual inoculation with the Coaraze isolate and was found to be very narrow. No symptoms and no infections were obtained in Capsella bursa-pastoris, Capsicum annuum, Claytonia perfoliata, Cucumis melo, Cucumis sativus, Cucurbita pepo, Datura stramonium, Gomphrena globosa, Medicago sativa, Nicotiana benthamiana, N. glutinosa, N. tabacum, Ocimum basilicum, Petunia hybrida, Phaseolus mungo, Physalis peruviana, Pisum sativum, Psoralea glandulosa, Ranunculus sardous, Salvia splendens, Solanum lycopersicum, Trifolium repens, Vicia faba, Vigna unguiculata, or Zinnia elegans. Necrotic local lesions were observed in Chenopodium amaranticolor, C. quinoa, and in all eight cultivars of Phaseolus vulgaris tested. The virus was transmitted either manually or by the green peach aphid (Myzus persicae) to healthy B. bituminosa seedlings. Symptoms appeared in 10 to 15 weeks, and the virus was detected in the symptomatic plants by RT-PCR. To our knowledge, this is the first report of a virus infecting B. bituminosa. References: (1) M. J. Adams et al. Arch. Virol. 150:459, 2005. (2) C. A. Baker and L. Jones. Plant Dis. 91:227, 2007. (3) A. Gibbs and A. M. Mackenzie. J. Virol. Methods 63:9, 1997.

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The Cryptococcus neoformans GeneDHA1 Encodes an Antigen That Elicits a Delayed-Type Hypersensitivity Reaction in Immune Mice

Infection and Immunity ◽

10.1128/iai.68.11.6196-6201.2000 ◽

2000 ◽

Vol 68 (11) ◽

pp. 6196-6201 ◽

Cited By ~ 26

Author(s):

M. Alejandra Mandel ◽

Greg G. Grace ◽

Kris I. Orsborn ◽

Fredda Schafer ◽

Juneann W. Murphy ◽

...

Keyword(s):

Amino Acid ◽

Cryptococcus Neoformans ◽

Culture Filtrate ◽

Genomic Library ◽

Amino Acid Sequences ◽

Full Length ◽

Delayed Type Hypersensitivity ◽

Pcr Analysis ◽

Rt Pcr ◽

Fruiting Body Formation

ABSTRACT When mice are vaccinated with a culture filtrate fromCryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-lengthDHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5′ rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes(shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded byDHA1. Recombinant DHA1 protein expressed inEscherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.

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First Report of Saguaro Cactus Virus Infecting Gymnocalycium mihanovichii in South Korea

Plant Disease ◽

10.1094/pdis-04-21-0770-pdn ◽

2021 ◽

Author(s):

Mi Sang Lim ◽

Byoung-Eun Min ◽

Sun Hee Choi

Keyword(s):

Amino Acid ◽

South Korea ◽

Rna Extraction ◽

Amino Acid Sequences ◽

Rt Pcr ◽

Systemic Necrosis ◽

Genome Database ◽

Total Rna ◽

First Report ◽

Saguaro Cactus Virus

Saguaro cactus virus (SgCV, genus Carmovirus, family Tombusviridae) was first isolated from an asymptomatic giant saguaro cactus (Carnegiea gigantea) in Arizona, USA (Milbrath and Nelson, 1972). In November 2017, 30 asymptomatic grafted cactus plants (Gymnocalycium mihanovichii grafted onto Hylocereus trigonus) were randomly collected from a commercial market in Gyeonggi Province, South Korea. Total RNA was extracted from both the scions and rootstocks of the plants using an RNeasy Plant Mini Kit (Qiagen, Germany) then subjected to reverse transcription polymerase chain reaction (RT-PCR) using RevertAid reverse transcriptase (Thermo Scientific, USA), TaKaRa Taq (TaKaRa, Japan), and SgCV-CP primers (forward, 5′- ATGGACGCTAAGTATGCG-3′; reverse, 5′- TCAGAGCCTAGCAACATA-3′). A validated SgCV stock (PV 0734, DSMZ, Germany) was used as an RT-PCR positive control. Out of 30 samples each of the rootstocks and scions, 21 and 8 produced, respectively, an amplicon at the expected size of 1,035 bp. The amplicons from three samples were cloned into a pGEM-T easy vector (Promega, USA), and three clones of each sample were sequenced (Macrogen, South Korea). The amplicons shared 100 % sequence identity with each other. BLASTn analysis showed that the sequence shared the highest identity at 66.3% with SgCV isolate Arizona (GenBank U72332). For bioassay of the virus, sap from infected G. mihanovichii was mechanically inoculated on four indicator plant species. The virus induced local lesions in Chenopodium amaranticolor, C. quinoa, and Gomphrena globosa, and systemic necrosis including growth reduction in C. capitatum. These results are consistent with those reported on SgCV by Milbrath and Nelson (1972). For determination of the exact species of the virus, non-inoculated leaves of C. capitatum were harvested 21 days after mechanical inoculation and subjected to total RNA extraction using the RNeasy Plant Mini Kit (Qiagen). A cDNA library was prepared using TruSeq RNA sample preparation v2, and sequenced on a NovaSeq 6000 system sequencer (Macrogen, South Korea). A total of 137,393,766 raw reads were quality-trimmed, and assembled into 120,408 contigs with sizes ranging from 201 to 15,898 nt using the Trinity program (r20140717). The assembled contigs were screened against the NCBI viral genome database using BLASTn, and a single contig of 3,858 nt matched the SgCV (acc. number U72332, coverage 88%, identity 70.3%). The sequence was deposited in GenBank (SgCV-gm, MW590184) and contained five open reading frames (ORFs), which is consistent with those of SgCV reported by Weng and Xiong (1997). Using DNAMAN software (Lynnon Biosoft, Canada) the deduced amino acid sequences encoded by the ORFs were determined and their homology with respective ORF proteins of various carmoviruses was subsequently compared (Table S1). The deduced protein sequences shared the highest identity of 68.2 to 81% with those of the SgCV isolate Arizona. King et al. (2012) suggested respective artificial host range reactions and percentage of coat protein and polymerase amino acid sequence identities of less than 52% and 57% as criteria for species demarcation in Carmovirus. These features suggest that SgCV-gm should possibly be designated a new SgCV isolate. To the best of our knowledge, this is the first report of SgCV naturally infecting G. mihanovichii in South Korea. Further research is needed to gain more in-depth insight into the biological and pathological properties of this virus.

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First Report of Capsicum chlorosis virus Infecting Amaryllis and Blood Lily in Taiwan

Plant Disease ◽

10.1094/pdis-93-12-1346a ◽

2009 ◽

Vol 93 (12) ◽

pp. 1346-1346 ◽

Cited By ~ 4

Author(s):

C. C. Chen ◽

C. H. Huang ◽

Y. H. Cheng ◽

T. C. Chen ◽

S. D. Yeh ◽

...

Keyword(s):

Chenopodium Quinoa ◽

Amino Acid Sequences ◽

N Gene ◽

First Report ◽

N Protein ◽

Calla Lily ◽

Field Samples ◽

Local Lesions ◽

Plant Pathol ◽

Capsicum Chlorosis Virus

Capsicum chlorosis virus (CaCV), a thrips-transmitted, tentative species in the genus Tospovirus, family Bunyaviridae, was first identified in solanaceous crops, but also infects several ornamental crops such as orchid (4), gloxinia (3), and calla lily (1). From 2005 to 2007, virus-like yellow ringspots were observed on the leaves of amaryllis (Hippeastrum hybridum Hort.) and blood lily (Haemanthus multiflorus Martyn.) plants cultured in screenhouses and a private garden, respectively. Three of several hundred amaryllis plants in screenhouses from two places were observed as showing yellow ringspot symptoms and one of six blood lily plants was observed as showing similar yellow ringspot symptoms. Sap extracts from symptomatic leaves were inoculated to Chenopodium quinoa Willd. and the resulting local lesions were passaged three successive times to C. quinoa for virus isolation. Using the tospovirus genus-specific primers gL3637 and gL4435c designed from the conserved region in the L RNA (2), DNA fragments of the expected size of 800 bp were amplified by reverse transcription (RT)-PCR from field samples and local lesions from C. quinoa. Extracts from the diseased plants and local lesions of C. quinoa reacted strongly with antiserum against the nucleocapsid (N) protein of CaCV in ELISA and western blotting. To confirm the identity of this virus, we amplified the N gene from three amaryllis and one blood lily source using primer pair WN2328 and WN3534 designed from the S RNA of Watermelon silver mottle virus (1), and these products were cloned and sequenced. The sequence from each virus isolate was determined from three independent clones. The nucleotide and deduced amino acid sequences of N genes for the blood lily isolate (GenBank Accession No. EF101344) and three amaryllis isolates (GenBank Accession Nos. EF101343, EF137177, and FJ185170) had identities greater than 97% with that of a CaCV isolate infecting Capsicum spp. found in Australia (GenBank Accession No. AY036057). Phylogenetic analysis using maximum parsimony showed that these sequences clustered with CaCV. These results show that the virus identified from amaryllis and blood lily that were expressing yellow ringspot symptoms are isolates of CaCV. To our knowledge, this is the first report of CaCV naturally infecting amaryllis and blood lily and it could become an important threat to ornamental production in Taiwan. References: (1) C. C. Chen et al. Plant Dis. 91:1201, 2007. (2) F. H. Chu et al. Phytopathology 91:361, 2001. (3) H. T. Hsu et al. J. Gen. Plant Pathol. 66:167, 2000. (4) Y. X. Zheng et al. Eur. J. Plant Pathol. 120:199, 2008.

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Cloning and Characterization of the Zebrafish mll Ortholog.

Blood ◽

10.1182/blood.v110.11.1249.1249 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1249-1249

Author(s):

Blaine W. Robinson ◽

Giuseppe Germano ◽

Yuanquan Song ◽

Rita J. Balice-Gordon ◽

Carolyn A. Felix

Keyword(s):

Amino Acid ◽

Blot Analysis ◽

Temporal Pattern ◽

Southern Blot Analysis ◽

Early Embryogenesis ◽

Amino Acid Sequences ◽

Pcr Analysis ◽

Adult Zebrafish ◽

Wild Type ◽

Rt Pcr

Abstract Introduction: Zebrafish enable studies of early embryogenesis and hematopoiesis like no other animal models. Many zebrafish orthologs of hematopoietic genes have been identified, and zebrafish models of leukemia are emerging but the zebrafish ortholog of MLL, a critical oncogene disrupted by leukemogenic translocations, has not yet been studied. We cloned the complete zmll cDNA and characterized its temporal expression as a framework for further studies of MLL where current knowledge is still incomplete. Methods: Bioinformatic tools were employed to interrogate the existence and relationship of a zmll ortholog and synteny with human MLL. Degenerate RT-PCR was used to determine whether MLL amino acid sequences in domains highly conserved across species could identify the orthologous zebrafish transcript. Cross-species Southern blot analysis was performed to determine if a predicted zmll gene from restriction map simulations of a projected genomic sequence could be detected with a human cDNA probe for the MLL breakpoint cluster region (bcr). The full-length zmll cDNA was obtained using a combination of 5′ RACE PCR, long-range and conventional PCR. The corresponding protein was analyzed in a phylogram tree. The temporal pattern of zmll RNA expression was examined using quantitative (Q) RT-PCR. Results: Bioinformatic analysis using the human MLL protein as the reference sequence identified two putative “similar to MLL proteins” and predicted two proximal transcript sequences on zebrafish chromosome 15. Gene prediction tools suggested a single genomic structure matching both protein sequences. A conserved block of synteny containing several linked genes surrounded the predicted zmll. Furthermore, zmll and human MLL were in the same map order in an uninterrupted segment with the gene for ubiquitination factor E4A. Degenerate RT-PCR analysis of wild-type adult zebrafish RNA based on cross-species amino acid sequences from highly conserved PHD and SET domains amplified the predicted transcript. Cross-species Southern blot analysis with the human probe detected the projected zmll genomic fragments corresponding to the MLL bcr in adult zebrafish genomic DNA. RT-PCR analysis of wild-type adult zebrafish RNA determined that the two predicted “similar to MLL proteins” were from a single gene. The full length 12657 bp, 35-exon zmll cDNA cloned from wild-type 24 hpf zebrafish embryo RNA predicted a 4218 amino acid protein with CXXC, PHD, Bromodomain, FYRN, FYRC, and SET domains and taspase cleavage sites with 45.8% sequence identity and 57.3% similarity to human MLL. Phylogram tree analysis suggested evolutionary divergence of mammals from teleosts but nonetheless conservation of critical functional domains. QRT-PCR demonstrated maternally supplied zmll mRNA during the earliest embryonic developmental timepoints, and expression of zygotic zmll mRNA during embryogenesis and in the zebrafish adult. Conclusions: These results indicate that there is a single zmll gene with highly conserved functional similarity to human MLL. The temporal pattern of expression, including maternal supply of transcript to the embryo, indicates that zmll is important from early embryogenesis through the entire lifespan of the fish. The high evolutionary conservation of critical domains creates the framework to use zebrafish for studying MLL in hematopoiesis and leukemia.

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First Report of Catharanthus mosaic virus in Mandevilla in the United States

Plant Disease ◽

10.1094/pdis-09-14-0913-pdn ◽

2015 ◽

Vol 99 (1) ◽

pp. 165-165 ◽

Cited By ~ 4

Author(s):

D. Mollov ◽

M. A. Guaragna ◽

B. Lockhart ◽

J. A. M. Rezende ◽

R. Jordan

Keyword(s):

United States ◽

Amino Acid ◽

Mosaic Virus ◽

Consensus Sequence ◽

The United States ◽

Amino Acid Sequences ◽

Universal Primers ◽

Rt Pcr ◽

First Report ◽

Virus Particles

Mandevilla (Apocynaceae) is an ornamental tropical vine popular for its bright and attractive flowers. During 2012 to 2013, 12 Mandevilla sp. samples from Minnesota and Florida nurseries were submitted for analysis at the University of Minnesota Plant Disease Clinic. Plants showed mosaic symptoms, leaf deformation, premature leaf senescence, and vine dieback. Filamentous virus particles with modal lengths 700 to 900 nm were observed by transmission electron microscopy (TEM) in partially purified preparations from symptomatic leaves. Partially purified virions were obtained using 30% sucrose cushion centrifuged at 109,000 gmax for 2 h at 10°C (5). No other virus particles were observed in these samples, nor were any observed in non-symptomatic samples. One sample was submitted as potted plant (Mandevilla ‘Sunmandeho’ Sun Parasol Giant White) and was kept under greenhouse conditions for subsequent analyses. Total RNA (Qiagen) was extracted from this sample, and Potyvirus was detected using the universal primers Poty S (5′-GGN AAY AAY AGY GGN CAR CC-3′) and PV1 (5′-20(T)V-3′) (1) by reverse transcription (RT)-PCR (3). The amplified product was the expected ~1.7-kb, corresponding to the partial nuclear inclusion body gene, the coat protein (CP) gene, and the 3′ end untranslated region. The RT-PCR amplicon was cloned (NEB) and sequenced, and the 1,720-bp consensus sequence was deposited in GenBank (Accession No. KM243928). NCBI BLAST analysis at the nucleotide level revealed highest identity (83%) with an isolate of Catharanthus mosaic virus (CatMV) from Brazil (Accession No. DQ365928). Pairwise analysis of the predicted 256 amino acid CP revealed 91% identity with the CatMV Brazilian isolate (ABI94824) and 68% or less identity with other potyviruses. Two potyviruses are usually considered the same species if their CP amino acid sequences are greater than 80% identical (2). Serological analysis of the infected sample Mandevilla ‘Sunmandeho’ Sun Parasol Giant White using a CatMV specific antiserum (4) resulted in positive indirect ELISA reactions. CatMV has been previously reported in periwinkle (Catharanthus roseus) in Brazil (4). Based on the analyses by TEM, RT-PCR, nucleotide and amino acid sequence identities, and serological reactivity, we identify this virus as a U.S. Mandevilla isolate of CatMV. To our knowledge, this is the first report of Catharanthus mosaic virus both in the United States and in Mandevilla. References: (1) J. Chen et al. Arch Virol. 146:757, 2001. (2) A. Gibbs and K. Ohshima. Ann. Rev. Phytopathol. 48:205, 2010. (3) R. L. Jordan et al. Acta Hortic. 901:159, 2011. (4) S. C. Maciell et al. Sci. Agric. Piracicaba, Brazil. 68:687, 2011. (5) D. Mollov et al. Arch Virol. 158:1917, 2013.

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First Report of Groundnut bud necrosis virus in Tomato in Bangladesh

Plant Disease ◽

10.1094/pdis-02-12-0199-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 917-917 ◽

Cited By ~ 7

Author(s):

M. S. Akhter ◽

S. K. Holkar ◽

A. M. Akanda ◽

B. Mandal ◽

R. K. Jain

Keyword(s):

Amino Acid ◽

Rna Extraction ◽

N Gene ◽

Virus Species ◽

Rt Pcr ◽

Necrosis Virus ◽

F1 Hybrid ◽

First Report ◽

Field Samples ◽

Groundnut Bud Necrosis Virus

An unusual disease of tomato characterized by leaf mottling and necrotic streaks on veins, shortened internodes, necrosis of terminal buds, and concentric rings on fruits was observed during 2010 to 2011 surveys in tomato growing regions of Godagari Upzila, Rajshahi district, Bangladesh. Disease incidence in popularly grown F1 hybrid cultivars, which include Sobal, Abhiruchi, Salamat, Bangobir, and BARI hybrid tomato-5 and -6 in about 40 commercial fields, ranged from 40 to 90%. Extracts from the field samples (n = 10) reacted with polyclonal antiserum to Groundnut bud necrosis virus (GBNV) in direct antigen coated ELISA, suggesting the association of a tospovirus antigenically related to serogroup IV topsovirus (1). To identify whether the tospovirus was a distinct virus species, ELISA-positive samples were subjected to total RNA extraction with an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA) followed by reverse transcription (RT)-PCR with tospovirus-specific primers (5′-ATGGTTGAAAAGAGCAAGAATGATGC-3′) and degenerate primer (5′-CTTCTTATGAAGTGTACTCACCATAAGTCATCC-3′) derived from the conserved sequences of GBNV, Watermelon bud necrosis virus (WBNV), and Capsicum chlorosis virus (CaCV) (2). The RT-PCR product was cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced at Department of Biochemistry, University of Delhi, South Campus, Delhi, India (GenBank Accession No. JQ692083). The sequences of cloned fragments were assembled. Analysis of the 477-bp region of the nucleocapsid protein (N) gene revealed that the tomato tospovirus shared maximum identity both at the nucleotide (96%) and amino acid (97%) levels with the corresponding region of GBNV. In contrast, only 78 to 81% and 85 to 87% identity at nucleotide and amino acid levels, respectively, was observed with the corresponding region of the N genes of CaCV, WBNV, and Watermelon silver mottle virus. These results suggested the association of GBNV with the diseased tomato samples. To our knowledge, this is the first report of GBNV infecting tomato in Bangladesh and regular surveys are necessary to ascertain the prevalence and incidence of GBNV in other crops. References: (1) R. K. Jain et al. J. Virol. Methods 130:162, 2005. (2) M. Tsompana and J. W. Moyer. Tospovirus. Page 157 in: Encyclopedia of Virology. Academic Press, New York, 2009.

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First Report of a Rhabdovirus Infecting Alfalfa in Argentina

Plant Disease ◽

10.1094/pdis-10-10-0764 ◽

2011 ◽

Vol 95 (6) ◽

pp. 771-771 ◽

Cited By ~ 18

Author(s):

N. Bejerman ◽

C. Nome ◽

F. Giolitti ◽

E. Kitajima ◽

S. de Breuil ◽

...

Keyword(s):

Amino Acid ◽

Consensus Sequence ◽

Amino Acid Sequences ◽

Vector System ◽

Rt Pcr ◽

Degenerate Primers ◽

Production Chain ◽

First Report ◽

Pcr Assays ◽

Plant Rhabdovirus

Alfalfa (Medicago sativa L.) is a major forage crop in Argentina with an estimated cultivated area of 4 million ha in the 2009–2010 season, which constitutes a primary component for the animal production chain. In early summer of 2010, alfalfa plants showing virus-like symptoms were identified in 20 commercial fields in La Pampa Province with 95% disease prevalence. Diseased plants had shortened internodes, a bushy appearance, deformations, puckering, epinasty of leaflet blades, vein enations, and varying sized papillae on the adaxial leaflet surfaces. Similar symptoms were observed in alfalfa crops in Buenos Aires, Cordoba, Santa Fe, and Santiago del Estero provinces. Electron microscopy (EM) and molecular assays were performed on leaf tissue from one asymptomatic and two symptomatic plants. EM of ultrathin sections revealed membrane-bound bullet-shaped particles associated with the endoplasmic reticulum of phloem cells from symptomatic plants only. Total RNA was extracted from symptomatic and asymptomatic plants with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) for a template in one-step reverse transcription (RT)-PCR assays with the Access RT-PCR Kit (Promega, Madison, WI). RT-PCR assays employed degenerate primers targeting conserved regions of plant rhabdovirus polymerase (L) genes (2). An amplicon of approximately 1 kilobase pairs (detected only from symptomatic plants) was gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega), cloned into pGEM-T Easy Vector System (Promega), and sequenced. Three independents clones were sequenced in both directions at Macrogen Inc. (Korea Republic) to generate a consensus sequence (GenBank Accession No. HQ380230) and compared to sequences of other plant rhabdoviruses available on GenBank. The partial L gene sequence of the alfalfa-infecting rhabdovirus shared highest nucleotide (68.0%) and amino acid (76.5%) sequence identity with the cytorhabdovirus Strawberry crinkle virus (Accession No. AY331390). A phylogenetic tree based on partial amino acid sequences of the polymerase gene indicated the alfalfa-infecting virus was more closely related to cytorhabdoviruses than to nucleorhabdoviruses. Symptoms observed resembled those reported for alfalfa plants infected with a plant rhabdovirus named Alfalfa enation virus (1), which has never been fully characterized. Collectively, the data implicate the observed rhabdovirus as the etiological agent. To our knowledge, this is the first report in Argentina (and South America) of a rhabdovirus infecting alfalfa. Additional field surveys and monitoring of vector/s and yield losses need to be conducted. References: (1) B. Alliot and P. A. Signoret. Phytopathol. Z. 74:69, 1972. (2) R. L. Lamprecht et al. Eur. J. Plant Pathol. 123:105, 2009.

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