First Report of Moroccan watermelon mosaic virus in Zucchini Crops in Greece

In the summer of 2012, zucchini (Cucurbita pepo L.) plants of F1 hybrid Rigas showing very severe malformation and blisters in leaves and fruit were observed in the prefectures of Ilia and Messinia, Peloponnese, southwestern Greece. Over 100 samples were collected and only a few were found by double antibody sandwich (DAS)-ELISA to be singly or mixed infected with the commonly encountered Cucumber mosaic virus (CMV, genus Cucumovirus), Zucchini yellow mosaic virus (ZYMV, genus Potyvirus), and Watermelon mosaic virus (WMV, genus Potyvirus), to which Rigas is known to be tolerant. All affected plants were also tested by DAS-ELISA and RT-PCR (2) for the presence of Moroccan watermelon mosaic virus (MWMV; genus Potyvirus), a virus not previously reported in Greece, and were consistently found positive by both methods. Sap from plants in which MWMV was solely detected was used to mechanically inoculate Chenopodium quinoa Willd. and cucurbit species (zucchini, cucumber, melon, and watermelon). C. quinoa produced chlorotic local lesions, while cucurbits showed very severe mosaic and malformation of leaves. Zucchini plants of F1 hybrids Rigas, Golden (tolerant to WMV and ZYMV), and Elion (not exhibiting any tolerance) grown in a screenhouse produced equivalent severe symptoms on leaves and fruits. Furthermore, transmission experiments in a non-persistent manner using a clone of Myzus persicae Sulz. and zucchini plants of F1 hybrid Boreas as donor and test plants were carried out. Ten plants were used in each experiment (one aphid/plant) and this was repeated five times (50 plants in total). The transmission rate was high ranging from 75 to 90%. RT-PCR obtained amplicons of 627 bp were subjected to direct sequencing (GenBank Accession No KF772944), which revealed 99% sequence identity to the corresponding region of a MWMV Tunisian isolate (EF579955). In 2013, in addition to zucchini plants found MWMV positive, watermelon (Citrullus lanatus Thunb.) plants from the same region of Peloponnese showing leaf malformation and mosaic symptoms were found MWMV positive (4/30) by DAS-ELISA and RT-PCR, revealing the virus establishment and further spread. In the Mediterranean basin, the virus has already been reported in Morocco, Italy, France, Spain, Tunisia, and Algeria, where it has emerged recently from a common source, has quickly become established through rapid dissemination and is considered as an important emerging threat (4). Isolates from these countries, including the present one from Greece, are very closely molecularly related to each other, contrary to isolates from sub-Saharan Africa (South Africa, Sudan, Congo, Zimbabwe, Niger, Cameroon, Nigeria) that are much more divergent (1,3). To our knowledge, this is the first report of MWMV in Greece. References: (1) H. Lecoq et al. Plant Dis. 85:547, 2001. (2) H. Lecoq et al. New Dis. Rep. 16:19, 2007. (3) A. T. Owolabi et al. Int. J. Virol. 8:258, 2012. (4) S. Yakoubi et al. Arch. Virol. 153:775, 2008.

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First Report of Papaya ringspot virus and Zucchini yellow mosaic virus in Luohanguo (Siraitia grosvenorii) in China

Plant Disease ◽

10.1094/pd-89-0530a ◽

2005 ◽

Vol 89 (5) ◽

pp. 530-530 ◽

Cited By ~ 4

Author(s):

Y.-M. Liao ◽

X.-J. Gan ◽

B. Chen ◽

J.-H. Cai

Keyword(s):

Mosaic Virus ◽

Zucchini Yellow Mosaic Virus ◽

Viral Disease ◽

Ringspot Virus ◽

Yellow Mosaic Virus ◽

Watermelon Mosaic Virus ◽

Papaya Ringspot Virus ◽

Rt Pcr ◽

First Report ◽

Siraitia Grosvenorii

Luohanguo, Siraitia grosvenorii (Swingle) C. Jeffrey, is a perennial cucurbitaceous plant that is an economically important medicinal and sweetener crop in Guangxi province, China. Surveys conducted during the summer to fall seasons of 2003-2004 in northern Guangxi showed symptoms typical of a viral disease, including leaf mottling, mosaic, vein clearing, curling, and shoestring-like distortion in the field. Mechanical inoculation of sap from leaves of symptomatic plants collected from the surveyed areas caused similar symptoms on tissue culture-derived healthy Luohanguo plants. Two sequences of 0.7 and 1.6 kb with 88 and 97% identity to Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) were amplified using reverse transcription-polymerase chain reaction (RT-PCR) with purified flexuous viral particles or total RNA extracted from the symptomatic Luohanguo leaves as templates with conserved degenerate potyvirus primers (1). To confirm the results, primers specific for PRSV (PP1/PP2, genome coordinates 4064-4083/5087-5069, GenBank Accession No X97251) and ZYMV (ZP1/ZP2, genome coordinates 5540-5557/7937-7920, GenBank Accession No L31350) were used to perform RT-PCR from the same RNA templates. The expected 1.0- and 2.3-kb fragments were amplified and they were 90 and 95% identical to PRSV and ZYMV in sequence, respectively. Watermelon mosaic virus was not detected. To our knowledge, this is the first report of the occurrence of PRSV and ZYMV in Luohanguo. Reference: (1) A. Gibbs et al. J. Virol. Methods 63:9, 1997.

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First Report of Cucumber mosaic virus on Melon in Bosnia and Herzegovina

Plant Disease ◽

10.1094/pdis-02-13-0135-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1124-1124 ◽

Cited By ~ 1

Author(s):

V. Trkulja ◽

D. Kovačić ◽

B. Ćurković ◽

A. Vučurović, I. Stanković ◽

A. Bulajić ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Bosnia And Herzegovina ◽

Cucumis Melo ◽

Cucurbita Pepo ◽

Yellow Mosaic Virus ◽

Rt Pcr ◽

First Report ◽

Negative Controls ◽

Das Elisa

During July 2012, field-grown melon plants (Cucumis melo L.) with symptoms of mosaic, chlorotic mottling, and vein banding as well as blistering and leaf malformation were observed in one field in the locality of Kladari (municipality of Doboj, Bosnia and Herzegovina). Disease incidence was estimated at 60%. A total of 20 symptomatic plants were collected and tested with double-antibody sandwich (DAS)-ELISA using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) against four the most commonly reported melon viruses: Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ringspot virus (PRSV) (1,3). Commercial positive and negative controls were included in each assay. Only CMV was detected serologically in all screened melon samples. Sap from an ELISA-positive sample (162-12) was mechanically inoculated to test plants using 0.01 M phosphate buffer (pH 7.0). The virus caused necrotic local lesions on Chenopodium amaranticolor 5 days after inoculation, while mild to severe mosaic was observed on Nicotiana rustica, N. glutinosa, N. tabacum ‘Samsun,’ Cucurbita pepo ‘Ezra F1,’ and Cucumis melo ‘Ananas’ 10 to 14 days post-inoculation. All five inoculated plants of each experimental host were DAS-ELISA positive for CMV. The presence of CMV in all naturally and mechanically infected plants was further verified by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and used as template in RT-PCR. RT-PCR was carried out with the One-Step RT-PCR Kit (Qiagen) using primer pair CMVCPfwd and CMVCPrev (4), amplifying the entire coat protein (CP) gene and part of 3′- and 5′-UTRs of CMV RNA 3. Total RNAs obtained from the Serbian CMV isolate from Cucurbita pepo ‘Olinka’ (GenBank Accession No. HM065510) and healthy melon leaves were used as positive and negative controls, respectively. An amplicon of the correct predicted size (871 bp) was obtained from all naturally and mechanically infected plants as well as from positive control, but not from healthy tissues. The amplified product derived from isolate 162-12 was purified with QIAquick PCR Purification Kit (Qiagen) and sequenced directly using the same primer pair as in RT-PCR (KC559757). Multiple sequence alignment of the 162-12 isolate CP sequence with those available in GenBank, conducted with MEGA5 software, revealed that melon isolate from Bosnia and Herzegovina showed the highest nucleotide identity of 99.7% (100% amino acid identity) with eight CMV isolates originating from various hosts from Serbia (GQ340670), Spain (AJ829770 and 76, AM183119), the United States (U20668, D10538), Australia (U22821), and France (X16386). Despite the fact that CMV is well established in majority of Mediterranean countries and represents an important threat for many agriculture crops, including pepper in Bosnia and Herzegovina (2), to our knowledge, this is the first report of CMV infecting melon in Bosnia and Herzegovina. Melon popularity as well as production value has been rising rapidly and the presence of CMV may have a drastic economic impact on production of this crop in Bosnia and Herzegovina. References: (1) E. E. Grafton-Cardwell et al. Plant Dis. 80:1092, 1996. (2) M. Jacquemond. Adv. Virus Res. 84:439, 2012. (3) M. Luis-Arteaga et al. Plant Dis. 82:979, 1998. (4) K. Milojević et al. Plant Dis. 96:1706, 2012.

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First Report of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Bottlegourd (Lagenaria siceraria) in Serbia

Plant Disease ◽

10.1094/pd-90-0380a ◽

2006 ◽

Vol 90 (3) ◽

pp. 380-380 ◽

Cited By ~ 8

Author(s):

N. Dukić ◽

B. Krstić ◽

I. Vico ◽

J. Berenji ◽

B. Duduk

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Zucchini Yellow Mosaic Virus ◽

Yellow Mosaic Virus ◽

Watermelon Mosaic Virus ◽

Mechanical Transmission ◽

Lagenaria Siceraria ◽

Indicator Plants ◽

First Report ◽

Das Elisa

During a cucurbit disease survey in August 2004, severe symptoms resembling those caused by viruses were observed on bottlegourd (Lagenaria siceraria (Molina) Standl.) in the Vojvodina region of Serbia. Symptoms included stunting, mosaic, green veinbanding, blistering, yellowing, chlorotic spots, leaf deformation, and fruit distortion. Leaf samples from 25 symptomatic plants were collected from two localities for virus identification using mechanical transmission and serological testing. Crude sap extract from leaf samples was mechanically inoculated onto bottlegourd and pumpkin (Cucurbita pepo) under greenhouse conditions. Field-collected bottlegourd and inoculated plants were tested using double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA). Positive reactions were obtained on collected and inoculated plants with polyclonal antiserum (Loewe Biochemica, Sauerlach, Germany) to Zucchini yellow mosaic virus(ZYMV) in 23 samples, with antiserum to Watermelon mosaic virus (WMV) in eight samples, and with antiserum to Cucumber mosaic virus (CMV) in seven samples. Each of the three viruses was detected in single as well as in mixed infections with the other two viruses. Biological characterization of viruses detected in single infections was done on the following indicator plants: Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Cucumis melo, Citrullus lanatus, Nicotiana glutinosa, and N. tabacum cv. Samsun. The symptoms observed on indicator plants for each isolate corresponded to the results of DAS-ELISA (2,3). All three viruses are known to be important pathogens of cucurbit plants and were previously reported in pumpkin in Serbia (1). To our knowledge, this is the first report of ZYMV, WMV, and CMV in bottlegourd in Serbia. References: (1) N. Dukić et al. J. Agric. Sci. 47:149, 2002. (2) D. E. Lesemann et al. Phytopathol. Z. 108:304, 1983. (3) H. Rahimian and K. Izadpanah. Phytopathol. Z. 92:305, 1978.

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First Report of Watermelon mosaic virus in Zucchini Squash in Bosnia and Herzegovina

Plant Disease ◽

10.1094/pdis-08-13-0834-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 573-573 ◽

Cited By ~ 1

Author(s):

V. Trkulja ◽

J. Stojčić ◽

D. Kovačić ◽

I. Stanković ◽

A. Vučurović ◽

...

Keyword(s):

Mosaic Virus ◽

Bosnia And Herzegovina ◽

Zucchini Yellow Mosaic Virus ◽

Yellow Mosaic Virus ◽

Watermelon Mosaic Virus ◽

Post Inoculation ◽

Rt Pcr ◽

Sequence Comparisons ◽

First Report ◽

Negative Controls

Aphid-borne Watermelon mosaic virus (WMV; genus Potyvirus, family Potyviridae) is widely distributed in the Mediterranean area and is one of the most prevalent cucurbit viruses in the region (4). In July 2012, approximately 20% of zucchini squash (Cucurbita pepo L.) plants showing virus-like symptoms were observed in one field in Kukulje locality (region of Banja Luka), Bosnia and Herzegovina. Infected plants exhibited mild to severe mosaic, chlorotic mottling, and dark green vein banding, as well as puckering and leaf deformation. Symptoms mostly developed on leaves, while fruits usually only failed to develop a normal coloration. Leaves from 15 symptomatic zucchini squash plants were sampled and analyzed utilizing double-antibody sandwich (DAS)-ELISA kits (Bioreba, AG, Reinach, Switzerland) with commercial antisera specific for five commonly occurring cucurbit-infecting viruses: WMV, Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus (PRSV), Cucumber mosaic virus (CMV), and Squash mosaic virus (SqMV) (1,3,4). Commercial positive and negative controls were included in each test. WMV was detected serologically in all tested zucchini squash samples, while no presence of other tested viruses were found. Crude sap extracted from leaves of a serologically positive sample (307-12) using 0.01 M phosphate buffer (pH 7) was mechanically inoculated onto five plants of C. pepo ‘Ezra F1’ and severe mosaic accompanied by bubbling and leaf malformation was observed 14 days post-inoculation. Viral identification in all naturally and mechanically infected plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed using the One-Step RT-PCR Kit (Qiagen) with specific primers WMV 5′ and WMV 3′ (4), yielding a 402- to 408-bp fragment corresponding to the N-terminal part of the coat protein (CP) gene (2). Total RNAs obtained from the Serbian WMV isolate from oil pumpkin (GenBank Accession No. JF325890) and healthy zucchini squash leaves were used as positive and negative controls, respectively. A product of the correct predicted size was obtained in all naturally and mechanically infected plants as well as positive control. No amplicon was recorded in healthy control. After purification (QIAquick PCR Purification Kit, Qiagen) the amplicon obtained from one selected isolate 307-12 was sequenced directly in both direction, aligned and compared by MEGA5 software with WMV sequences available in GenBank. Sequence comparisons revealed that the zucchini squash isolate from Bosnia and Herzegovina (KF517099) showed the highest nucleotide identity of 100% with one isolate from Serbia (FJ325891) and two Slovakian WMV isolates (GQ241712 to 13), all belonging to the classical group of WMV isolates (4). To our knowledge, this is the first report of WMV infecting zucchini squash in Bosnia and Herzegovina. Since squash and other cucurbit species represent valuable crops in Bosnia and Herzegovina, with annual production close to US$8.5 million ( http://faostat.fao.org ) and rising rapidly, the presence of a devastating pathogen like as WMV could be a serious constraint for their production. References: (1) A. Ali et al. Plant Dis. 96:243, 2012. (2) C. Desbiez et al. Arch. Virol. 152:775, 2007. (3) S. Jossey and M. Babadoost. Plant Dis. 92:61, 2008. (4) H. Lecoq and C. Desbiez. Adv. Virus Res. 84:67, 2012.

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First Report of Cucumber mosaic virus Associated with Capsicum chinense var. Scotch Bonnet in Florida

Plant Disease ◽

10.1094/pdis-12-13-1276-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1016-1016 ◽

Cited By ~ 1

Author(s):

B. Babu ◽

H. Dankers ◽

M. L. Paret

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Yellow Mosaic Virus ◽

Hot Pepper ◽

Crystalline Inclusion ◽

Post Inoculation ◽

Capsicum Chinense ◽

Rt Pcr ◽

First Report ◽

Pcr Assays

Scotch bonnet (Capsicum chinense) is a tropical hot pepper variety that is grown in South America, the Caribbean Islands, and in Florida, and is an important cash crop. In Florida, scotch bonnet is grown on ~100 acres annually. Virus-like leaf symptoms including mosaic and yellow mottling were observed on scotch bonnet plants in a field at Quincy, FL, with a disease incidence of ~5%. Two symptomatic and one non-symptomatic plant sample were collected from this field for identification of the causal agent associated with the symptoms. Viral inclusion assays (2) of the epidermal tissues of the symptomatic scotch bonnet samples using Azure A stain indicated the presence of spherical aggregates of crystalline inclusion bodies. Testing of the symptomatic samples using lateral flow immunoassays (Immunostrips, Agdia, Elkhart, IN) specific to Cucumber mosaic virus (CMV), Potato virus Y (PVY), Pepper mild mottle virus (PMMoV), Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ringspot virus (PRSV), showed a positive reaction only to CMV. The sap from an infected leaf sample ground in 0.01 M Sorensons phosphate buffer (pH 7.0) was used to mechanically inoculate one healthy scotch bonnet plant (tested negative for CMV with Immunostrip) at the 2- to 3-leaf stage. The inoculated plant developed mild mosaic and mottling symptoms 12 to 14 days post inoculation. The presence of CMV in the mechanically inoculated plant was further verified using CMV Immunostrips. Total RNA was extracted (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) from the previously collected two symptomatic and one non-symptomatic scotch bonnet samples. The samples were subjected to reverse-transcription (RT)-PCR assays using SuperScript III One-Step RT-PCR System (Invitrogen, Life Technologies, Grand Island, NY), and using multiplex RT-PCR primer sets (1). The primers were designed to differentiate the CMV subgroup I and II, targeting the partial coat protein gene and the 3′UTR. The RT-PCR assays using the multiplex primers produced an amplicon of 590 bp, with the CMV subgroup I primers. The RT-PCR product was only amplified from the symptomatic leaf samples. The obtained amplicons were gel eluted, and directly sequenced bi-directionally (GenBank Accession Nos. KF805389 and KF805390). BLAST analysis of these sequences showed 97 to 98% nucleotide identities with the CMV isolates in the NCBI database. The isolates collected in Florida exhibited highest identity (98%) with the CMV isolate from tomato (DQ302718). These results revealed the association of CMV subgroup I with symptomatic scotch bonnet leaf samples. Although CMV has been reported from scotch bonnet, this is the first report of its occurrence in Florida. References: (1) S. Chen et al. Acta Biochim Biophys Sin. 43:465, 2011. (2) R. G. Christie and J. R. Edwardson. Plant Dis. 70:273, 1986.

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First Report of Bean yellow mosaic virus in Alaska from Clover (Trifolium spp.)

Plant Disease ◽

10.1094/pdis-94-3-0372a ◽

2010 ◽

Vol 94 (3) ◽

pp. 372-372 ◽

Cited By ~ 1

Author(s):

N. L. Robertson ◽

K. L. Brown

Keyword(s):

Coat Protein ◽

Host Range ◽

Mosaic Virus ◽

Bean Yellow Mosaic Virus ◽

Yellow Mosaic Virus ◽

Degenerate Primers ◽

First Report ◽

Chlorotic Spot ◽

Putative Coat Protein ◽

Das Elisa

In mid-June 2008, distinct mosaic leaves were observed on a cluster of clover (Trifolium spp.) with light pink and white flowers growing at the edge of a lawn in Palmer, AK. Virus minipurification from leaves of affected clover and protein extractions on a polyacrylamide electrophoresis implicated a ~35-kDa putative coat protein (CP). Subsequent western blots and ELISA with a universal potyvirus antiserum (Agdia Inc., Elkhart, IN) confirmed potyvirus identity. Total RNA extracts (RNeasy Plant Mini Kit, Qiagen Inc., Valencia, CA) from the same plant were used for reverse transcription (RT)-PCR. Three sets of degenerate primers that targeted potyvirus-specific genes, HC-Pro (helper component protease) and CI (cylindrical inclusion protein) and the genomic 3′-terminus that included a partial NIb (nuclear inclusion), CP (coat protein), and UTR (untranslated region), produced the expected PCR segments (~0.7, ~0.7, and ~1.6 kbp, respectively) on 1% agarose gels (1). Direct sequencing of the HC-Pro (GenBank No. GQ181115), CI (GQ181116), and CP (GU126690) segments revealed 98, 97, and 99% nucleotide identities (no gaps), respectively, to Bean yellow mosaic virus (BYMV)-chlorotic spot (CS) strain, GenBank No. AB373203. The next closest BYMV percent identity comparisons decreased to 79% for HC-Pro (GenBank No. DQ641248; BYMV-W), 79% for CI (U47033; BYMV-S) partial genes, and 96% for CP (AB041971; BYMV-P242). Mechanical inoculations of purified virus preparations produced local lesions on Chenopodium amaranticolor Coste & A. Reyn. (2 of 5) and C. quinoa Willd. (6 of 7), and mosaic on Nicotiana benthamiana Domin (5 of 5). BYMV was specifically confirmed on tester plants using a double-antibody sandwich (DAS)-ELISA BYMV (strain 204 and B25) kit (AC Diagnostics, Inc., Fayetteville, AR) as directed. The absence of another potyvirus commonly found in clover, Clover yellow vein virus (ClYVV), was verified in parallel DAS-ELISA ClYVV assays (AC Diagnostics, Inc). The BYMV isolate was maintained in N. benthamiana, and virion or sap extracts inoculated to the following host range (number of infected/total inoculated plants [verified by BYMV ELISA]): Cucumis sativus L. ‘Straight Eight’ (0/5), Gomphrena globosa L. (1/4), Nicotiana clevelandii A. Gray (4/7), Phaseolus vulgaris L. ‘Bountiful’ (1/3), Pisum sativum L. (Germplasm Resources Information Network Accession Nos. -PI 508092 (8/12), -W6 17525 (13/13), -W6 17529 (0/13), -W6 17530 (13/14), -W6 17537 (0/12), -W6 17538 (0/12), and -W6 17539 (0/21), Tetragonia tetragoniodes (2/2), Trifolium pretense L. ‘Altaswede’ (6/10), T. repens L. ‘Pilgrim’ (0/8), and Vicia faba L. (1/3). All infected plants had symptoms ranging from systemic mosaic (T. pretense, P. sativum) to leaf distortions (N. clevelandii, Tetragonia tetragoniodes). Interestingly, the host range and genomic sequences of the BYMV Alaskan strain resemble the BYMV-CS (chlorotic spot) strain that was originally isolated from a diseased red clover (T. pretense) plant in Japan more than 40 years ago (2). Although BYMV occurs worldwide and has a wide host range in dictoyledonous and monocotyledonous plants (3), to our knowledge, this is the first report of a natural occurrence of BYMV in Alaska. The incidence and distribution of BYMV in clover and other plant species are not known in Alaska. References: (1) C. Ha et al. Arch. Virol. 153:36, 2008. (2) H. Kume et al. Mem. Fac. Agric. Hokkaido Univ. 7:449, 1970. (3) S. J. Wylie et al. Plant Dis. 92:1596, 2008.

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First Report of Soybean mosaic virus on Soybean in North Dakota

Plant Disease ◽

10.1094/pdis-93-7-0760c ◽

2009 ◽

Vol 93 (7) ◽

pp. 760-760 ◽

Cited By ~ 1

Author(s):

B. D. Nelson ◽

L. L. Domier

Keyword(s):

North Dakota ◽

Mosaic Virus ◽

Soybean Mosaic Virus ◽

Oilseed Crop ◽

Rt Pcr ◽

High Concentration ◽

Grid Pattern ◽

First Report ◽

History Of ◽

Das Elisa

Soybean, Glycine max L, is grown on 1,420,000 ha in North Dakota and is the most important oilseed crop in the state. Viruses in soybean have not previously been reported from North Dakota (2). In July and August of 2007, 64 soybean fields in Cass, Richland, and Sargent counties in southeastern North Dakota were surveyed for Soybean mosaic virus (SMV). These counties have a high concentration of soybean hectares, a long history of soybean production, and soybean aphid infestations that were observed in 2004 and 2006. Fields were sampled with a grid pattern across the area with at least 8 km (5 miles) between fields. A transect of approximately 60 m through each field was made and 20 leaves were collected at random. Sap was extracted in phosphate buffer and stored at –80°C until tested first using double antibody sandwich (DAS)-ELISA with positive controls and reagents and protocol from Agdia Inc. (Elkhart, IN). Using DAS-ELISA, SMV was detected in 19 of the 64 soybean fields sampled. To confirm the presence of SMV, 12 samples that were positive for SMV by DAS-ELISA also were tested by reverse transcription (RT)-PCR. RNA was extracted from sap by a Qiagen RNeasy Plant Mini Kit (Germantown, MD), reverse transcribed, and amplified with SuperScrip III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen Inc., Carlsbad, CA) and SMV-specific primers (5′-TTCAGCACAATGGGTGAGGATG-3′ and 5′-AATTCTGTGTGGCTTGATGTTGC-3′) (1). Eight of the twelve ELISA-positive samples were positive for SMV by RT-PCR, confirming the presence of SMV in the samples. To our knowledge, this is the first report of SMV infecting soybean in North Dakota. References: (1) L. L. Domier et al. (Abstr.). Phytopathology 98(suppl.):S47, 2008. (2) B. D. Nelson and G. Danielson. (Abstr.). Phytopathology 95(suppl.):S164, 2005.

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First Report of Alfalfa mosaic virus Infecting Tedera (Bituminaria bituminosa (L.) C.H. Stirton var. albomarginata and crassiuscula) in Australia

Plant Disease ◽

10.1094/pdis-04-12-0378-pdn ◽

2012 ◽

Vol 96 (9) ◽

pp. 1384-1384 ◽

Cited By ~ 5

Author(s):

R. A. C. Jones ◽

D. Real ◽

S. J. Vincent ◽

B. E. Gajda ◽

B. A. Coutts

Keyword(s):

Mosaic Virus ◽

Alfalfa Mosaic Virus ◽

Plant Viruses ◽

Yellow Mosaic Virus ◽

Rt Pcr ◽

First Report ◽

Pasture Legumes ◽

Bituminaria Bituminosa ◽

Pcr Products ◽

Burr Medic

Tedera (Bituminaria bituminosa (L.) C.H. Stirton vars albomarginata and crassiuscula) is being established as a perennial pasture legume in southwest Australia because of its drought tolerance and ability to persist well during the dry summer and autumn period. Calico (bright yellow mosaic) leaf symptoms occurred on occasional tedera plants growing in genetic evaluation plots containing spaced plants at Newdegate in 2007 and Buntine in 2010. Alfalfa mosaic virus (AlMV) infection was suspected as it often causes calico in infected plants (1,2) and infects perennial pasture legumes in local pastures (1,3). Because AlMV frequently infects Medicago sativa (alfalfa) in Australia and its seed stocks are commonly infected (1,3), M. sativa buffer rows were likely sources for spread by aphids to healthy tedera plants. When leaf samples from plants with typical calico symptoms from Newdegate (2007) and Buntine (2010) were tested by ELISA using poyclonal antisera to AlMV, Bean yellow mosaic virus (BYMV) and Cucumber mosaic virus (CMV), only AlMV was detected. When leaf samples from 864 asymptomatic spaced plants belonging to 34 tedera accessions growing at Newdegate and Mount Barker in 2010 were tested by ELISA, no AlMV, BYMV, or CMV were detected, despite presence of M. sativa buffer rows. A culture of AlMV isolate EW was maintained by serial planting of infected seed of M. polymorpha L. (burr medic) and selecting seed-infected seedlings (1,3). Ten plants each of 61 accessions from the local tedera breeding program were grown at 20°C in an insect-proof air conditioned glasshouse. They were inoculated by rubbing leaves with infective sap containing AlMV-EW or healthy sap (five plants each) using Celite abrasive. Inoculations were always done two to three times to the same plants. When both inoculated and tip leaf samples from each plant were tested by ELISA, AlMV was detected in 52 of 305 AlMV-inoculated plants belonging to 36 of 61 accessions. Inoculated leaves developed local necrotic or chlorotic spots or blotches, or symptomless infection. Systemic invasion was detected in 20 plants from 12 accessions. Koch's postulates were fulfilled in 12 plants from nine accessions (1 to 2 of 5 plants each), obvious calico symptoms developing in uninoculated leaves, and AlMV being detected in symptomatic samples by ELISA, inoculation of sap to diagnostic indicator hosts (2) and RT-PCR with AlMV CP gene primers. Direct RT-PCR products were sequenced and lodged in GenBank. When complete nucleotide CP sequences (666 nt) of two isolates from symptomatic tedera samples and two from alfalfa (Aq-JX112758, Hu-JX112759) were compared with that of AlMV-EW, those from tedera and EW were identical (JX112757) but had 99.1 to 99.2% identities to the alfalfa isolates. JX112757 had 99.4% identity with Italian tomato isolate Y09110. Systemically infected tedera foliage sometimes also developed vein clearing, mosaic, necrotic spotting, leaf deformation, leaf downcurling, or chlorosis. Later-formed leaves sometimes recovered, but plant growth was often stunted. No infection was detected in the 305 plants inoculated with healthy sap. To our knowledge, this is the first report of AlMV infecting tedera in Australia or elsewhere. References: (1) B. A. Coutts and R. A. C. Jones. Ann. Appl. Biol. 140:37, 2002. (2) E. M. J. Jaspars and L. Bos. Association of Applied Biologists, Descriptions of Plant Viruses No. 229, 1980. (3) R. A. C. Jones. Aust. J. Agric. Res. 55:757, 2004.

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First Report of Watermelon mosaic virus causing a Mosaic Disease on Cucumis metuliferus in China

Plant Disease ◽

10.1094/pdis-12-20-2744-pdn ◽

2021 ◽

Author(s):

Qiang Gao ◽

Hai-long Ren ◽

Wanyu Xiao ◽

Yan Zhang ◽

Bo Zhou ◽

...

Keyword(s):

Mosaic Virus ◽

Direct Sequencing ◽

Rna Extraction ◽

Viral Disease ◽

Complete Sequence ◽

Mosaic Disease ◽

Shanxi Province ◽

Watermelon Mosaic Virus ◽

Rt Pcr ◽

First Report

Cucumis metuliferus, also called horned cucumber or jelly melon, is considered as a wild species in the Cucumis genus and a potential material for nematodes- or viruses-resistant breeding (Provvidenti, et al. 1977; Sigüenza et al. 2005; Chen et al. 2020). This species, originating from Africa, has been cultivated as a fruit in China in recent years. In July 2020, a mosaic disease was observed on C. metuliferus growing in five fields (approximately 0.7 hectare) in Urumqi, Xijiang, China, where more than 85~100% of the field plants exhibited moderate to severe viral disease-like leaf mosaic and/or deformation symptoms. Delayed flowering and small and/or deformed fruits on the affected plants could result in yield loss of about 50%. To identify the causal pathogen, the symptomatic leaf samples were collected from the five fields (five plants/points for each field) and their total RNAs were extracted using a commercial RNA extraction kit. The universal potyviral primers (Ha et al. 2008) and specific primers for a number of frequently-occurring, cucurbit crop-infecting viruses including Papaya ringspot virus (PRSV) (Lin et al. 2013), Cucumber mosaic virus (CMV) and Watermelon mosaic virus (WMV) were designed and used for detection by RT-PCR. The result showed that only the WMV primers (forward: 5’-AAGTGTGACCAAGCTTGGACTGCA-3’ and reverse: 5’-CTCACCCATTGTGCCAAAGAACGT-3’) could amplify the corresponding target fragment from the total RNA templates, and direct sequencing of the RT-PCR products and GenBank BLAST confirmed the presence of WMV (genus Potyvirus) in the collected C. metuliferus samples. To complete Koch’s postulates, the infected C. metuliferus leaves were ground in the sodium phosphate buffer (0.01 M, pH 7.0) and the sap was mechanically inoculated onto 30 four-leaf-stage C. metuliferus seedlings (two leaves for each seedling were inoculated) kept in an insect-proof, temperature-controlled greenhouse at 25~28℃. Twenty-five of the inoculated plants were observed to have apparent leaf mosaic similar to the field symptoms two weeks after inoculation, and positive result was obtained in RT-PCR detection for the symptomatic leaves of inoculated plants using the WMV primers aforementioned, confirming the virus as the pathogen of C. metuliferus in Urumqi. To our knowledge, this is the first report of WMV naturally infecting C. metuliferus in China. We obtained the full-length sequence of the WMV Urumqi isolation (WMV-Urumqi) by sequencing the RT-PCR amplicons from seven pairs of primers spanning the viral genome and the 5’RACE and 3’RACE products. The complete sequence of WMV-Urumqi (GenBank accession no. MW345911) is 10046 nucleotides (nt) long and contains an open reading frame that encodes a polyprotein of 3220 amino acids (aa). WMV-Urumqi shares the highest nt identity (95.9%) and aa identity (98.0%) with the Cucurbita pepo-infecting isolation (KX664483) from Shanxi province, China. Our findings provide a better understanding of the host range and genetic diversity of WMV, and a useful reference for virus-resistant breeding involving C. metuliferus.

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