First report of garlic viruses A, B and C on garlic (Allium sativum L.) in Tunisia

Mite-borne viruses belonging to the genus Allexivirus (family Alphaflexiviridae) commonly occur on garlic in many parts of the world. There are usually asymptomatic and cause small damage to the plants, but often occur in mixed infection with potyviruses and carlaviruses, with synergistic effects reducing crop quality and leading to higher losses (Taglienti et al. 2017). Their occurrence on Tunisian garlic crops was studied here in the same garlic germplasm collection mentioned in a previous first report (Ayed et al. 2019). Leaf samples from a total of 66 garlic accessions were tested by DAS-ELISA using specific antibodies (DSMZ, Germany) against garlic virus A (GarV-A), garlic virus B (GarV-B) and garlic virus C (GarV-C). These serological tests showed individual virus incidence of respectively 56.4%, 67.7% and 10%. Our findings corroborate with the results of Chodorska et al (2012). In order to confirm the presence of these viruses, RT-PCR assays were performed using total RNAs extracted using two silica-capture extraction procedures according to Foissac et al (2005) and specific primers targeting the coat protein genes of the various viruses. These primers, designed for the present study are (GarV-A-F: 5' YCTYTTCTCHYTDGCHTGGACYTG 3' and GarV-A-R: 5' RCCYTTCCTAGACCARTTRGCRGG 3' for GarV-A; GarV-B-F: 5' TGGGCYTGYTACCACAAYGGATC 3' and GarV-B-R 5' TCTGCGCGVGTGGADACCATRTT 3' for GarV-B; GarV-C-F: 5' ARGAYCTYTTYTCMCTYGCRTGGGC 3' and GarV-C-R: 5' GGAGGYTCRTGAATYTGTTGTTG 3' for GarV-C). The viruses were detected by a two-step RT-PCR as described by Marais et al (2015). PCRs consisted of one cycle at 95 °C for 5 min; followed by 40 cycles of denaturation at 95 °C for 45 s, annealing at 45 °C for 45 s, and elongation at 72 °C for 45s; and a final extension step at 72 °C for 10 min. Products of the expected size (214 bp for GarV-A, 363 bp for GarV-B and 439 bp for GarV-C) were amplified from 58 (88%), 47 (71%) and 56 (85%) accessions, respectively. Forty three samples (65%) were co-infected by the three viruses. Higher numbers of positives revealed by RT-PCR especially in the case of GarV-C may reflect the higher sensitivity and efficiency of this technique compared to ELISA. Direct sequencing of selected amplicons of the expected size obtained for GarV-A, -B, and -C Tunisian isolates was performed and the sequences submitted to GenBank, validating the specificity of the three RT-PCR assays. The two sequenced GarV-A isolates (MK599147 and MN995836) shared 98% nucleotide (nt) sequence identity with each other, and 93-94% identity with the closest isolate in GenBank, the “G118” isolate from China (MN059320). The three sequenced GarV-B isolates (MN995829 to MN995831) shared 88-98% nt identity with each other. For “GarV-B 18.1” (MN995830) and “GarV-B 36.2” (MN995831) the closest isolate was “1109.1” (JX682828) from Spain (92-93% nt identity). For “GarVB 17.2” (MN995829), the closest isolate was “B-Sp-3” (LC97167) from Spain (90% nt identity). The sequenced GarV-C isolate (MN995834) showed the highest sequence nt identity (93%) with the “GarV-9” isolate (HQ724848) from Spain. To our knowledge this is the first report of the presence of GarV-A, -B and -C in Tunisia. The presence of these allexiviruses may pose a threat to the preservation of the Tunisian garlic germplasm and, more broadly, to garlic production in Tunisia. For this reason, the scrupulous identification of viruses occurring in garlic plants will help to use the appropriate strategy to decrease viral incidence in garlic growing area.

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

Detection and Characterization of Human Enteroviruses, Human Cosaviruses, and a New Human Parechovirus Type in Healthy Individuals in Osun State, Nigeria, 2016/2017

Viruses ◽

10.3390/v11111037 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1037 ◽

Cited By ~ 3

Author(s):

Folakemi Abiodun Osundare ◽

Oladele Oluyinka Opaleye ◽

Akeem Abiodun Akindele ◽

Samuel Adeyinka Adedokun ◽

Olusola Anuoluwapo Akanbi ◽

...

Keyword(s):

Healthy Individuals ◽

Coding Region ◽

Rt Pcr ◽

Human Parechovirus ◽

Full Genome Sequencing ◽

Three Samples ◽

Two Samples ◽

Stool Samples ◽

Pcr Assays ◽

Species Specific

Human enteroviruses and human parechoviruses are associated with a broad range of diseases and even severe and fatal conditions. For human cosaviruses, the etiological role is yet unknown. Little is known about the circulation of non-polio enteroviruses, human parechoviruses, and human cosaviruses in Nigeria. A total of 113 stool samples were collected from healthy individuals in Osun State between February 2016 and May 2017. RT-PCR assays targeting the 5′ non-coding region (5′ -NCR) were used to screen for human enteroviruses, human parechoviruses, and human cosaviruses. For human enteroviruses, species-specific RT-PCR assays targeting the VP1 regions were used for molecular typing. Inoculation was carried out on RD-A, CaCo-2, HEp-2C, and L20B cell lines to compare molecular and virological assays. Ten samples tested positive for enterovirus RNA with 11 strains detected, including CV-A13 (n = 3), E-18 (n = 2), CV-A20 (n = 1), CV-A24 (n = 1), EV-C99 (n = 1), and EV-C116 (n = 2). Three samples tested positive for human parechovirus RNA, and full genome sequencing on two samples allowed assignment to a new Parechovirus A type (HPeV-19). Thirty-three samples tested positive for cosavirus with assignment to species Cosavirus D and Cosavirus A based on the 5′-NCR region. Screening of stool samples collected from healthy individuals in Nigeria in 2016 and 2017 revealed a high diversity of circulating human enteroviruses, human parechoviruses, and human cosaviruses. Molecular assays for genotyping showed substantial benefits compared with those of cell-culture assays.

Download Full-text

First Report of Tomato torrado virus Infecting Tomato in Colombia

Plant Disease ◽

10.1094/pdis-11-11-1000 ◽

2012 ◽

Vol 96 (4) ◽

pp. 592-592 ◽

Cited By ~ 9

Author(s):

M. Verbeek ◽

A. M. Dullemans

Keyword(s):

Mosaic Virus ◽

Potato Virus ◽

Polyclonal Antibodies ◽

Sandwich Elisa ◽

Potato Virus X ◽

Rt Pcr ◽

Leaf Extracts ◽

Serological Tests ◽

First Report ◽

Initial Symptoms

Tomato (Solanum lycopersicum L.) plants grown in plastic greenhouses near Villa de Leyva, northeast of Bogota, Colombia showed necrotic spots on the leaves in September 2008. Initial symptoms were necrosis beginning at the base of leaflets that were surrounded by yellow areas. These symptoms resembled those described for Tomato torrado virus (ToTV; family Secoviridae, genus Torradovirus), which was first found in Spain (2). Other (tentative) members of the genus Torradovirus, Tomato marchitez virus (ToMarV), Tomato chocolate spot virus (ToChSV), and Tomato chocolàte virus (ToChV) (3) induce similar symptoms on tomato plants. One sample, coded T418, was stored in the freezer and brought to our lab in 2011. Serological tests (double-antibody sandwich-ELISA) using polyclonal antibodies (Prime Diagnostics, Wageningen, The Netherlands) on leaf extracts showed the absence of Pepino mosaic virus (PepMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus X (PVX), and Potato virus Y (PVY). Leaf extracts were mechanically inoculated onto the indicator plants Physalis floridana, Nicotiana hesperis ‘67A’, and N. occidentalis ‘P1’ (six plants in total) and were kept in a greenhouse at 20°C with 16 h of light. Necrotic symptoms appeared 4 to 5 days postinoculation and resembled those described for ToTV (2). Two dip preparations of systemically infected P. floridana and N. occidentalis leaves were examined by electron microscopy, which revealed the presence of spherical virus particles of approximately 30 nm. To confirm the presence of ToTV, total RNA was extracted from the original leaf material and an inoculated P. floridana and N. occidentalis plant using the Qiagen Plant Mini Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. ToTV-specific primer sets ToTV-Dp33F/ToTV-Dp20R (5′-TGCTCAATGTTGGAAACCCC-3′/5′-AGCCCTTCATAGGCTAGCC-3′, amplifying a fragment of the RNA1 polyprotein with an expected size of 751 bp) and ToTV-Dp1F/ToTV-Dp2R (5′-ACAAGAGGAGCTTGACGAGG-3′/5′-AAAGGTAGTGTAATGGTCGG-3′, amplifying a fragment on the RNA2 movement protein region with an expected size of 568 bp) were used to amplify the indicated regions in a reverse transcription (RT)-PCR using the One-Step Access RT-PCR system (Promega, Madison, WI). Amplicons of the predicted size were obtained in all tested materials. The PCR products were purified with the Qiaquick PCR Purification Kit (Qiagen) and sequenced directly. BLAST analyses of the obtained sequences (GenBank Accession Nos. JQ314230 and JQ314229) confirmed the identity of isolate T418 as ToTV, with 99% identity to isolate PRI-ToTV0301 in both fragments (GenBank Accession Nos. DQ388879 and DQ388880 for RNA1 and RNA 2, respectively). To our knowledge, this is the first report of ToTV in Colombia, and interestingly, since ToTV has been found only in Europe and Australia (1) so far, this is the first report of ToTV on the American continent. References: (1) C. F. Gambley et al. Plant Dis. 94:486, 2010. (2) M. Verbeek et al. Arch. Virol. 152:881, 2007. (3) M. Verbeek et al. Arch. Virol. 155:751, 2010.

Download Full-text

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.

Download Full-text

First Report of Tomato chlorotic dwarf viroid in Greenhouse Tomatoes in Arizona

Plant Disease ◽

10.1094/pdis-93-10-1075b ◽

2009 ◽

Vol 93 (10) ◽

pp. 1075-1075 ◽

Cited By ~ 18

Author(s):

K.-S. Ling ◽

J. Th. J. Verhoeven ◽

R. P. Singh ◽

J. K. Brown

Keyword(s):

Direct Sequencing ◽

Leaf Tissue ◽

Natural Occurrence ◽

Rt Pcr ◽

Nucleotide Substitutions ◽

Tomato Production ◽

First Report ◽

Sequence Identity ◽

Tomato Chlorotic Dwarf Viroid ◽

Plant Pathol

Tomato chlorotic dwarf viroid (TCDVd), a member of the genus Pospivroid, family Pospiviroidae, was first identified on greenhouse tomato (Solanum lycopersicum) in Canada (2). Since then, it has also been reported elsewhere, e.g., on tomato in Colorado (4). During 2006 in Arizona, tomato plants in a large greenhouse facility with continuous tomato production exhibited viroid-like symptoms of plant stunting and chlorosis of the young leaves. Symptomatic plants were often located along the edge of the row, indicating the presence of a mechanical transmissible agent. Approximately 4% of the plants in this greenhouse were symptomatic in 2008. Symptoms were distinctly different from those caused by Pepino mosaic virus (PepMV), a virus that was generally present in this greenhouse and also in our test samples. Other commonly occurring tomato viruses were ruled out by serological, PCR, or reverse transcription (RT)-PCR tests in multiple laboratories. RT-PCR with two sets of universal pospiviroid primers, PospiI-FW/RE and Vid-FW/RE (4), yielded amplicons of the expected sizes of 196 and 360 bp in three samples collected from symptomatic plants. Direct sequencing of the amplicons revealed that the genome was 360 nt and 100% identical to the type TCDVd from Canada (GenBank Accession No. AF162131) (2). Mechanical inoculation with leaf tissue extract from four samples to plants of the tomato ‘Money-Maker’ resulted in the same viroid-like symptoms and TCDVd was confirmed in these plants by RT-PCR and sequencing. In both 2007 and 2008, 18 samples were tested using primers PSTVd-F and PSTVd-R (1), which are capable of amplifying the full TCDVd genome. Analysis of the sequences from the amplicons revealed two genotypes of TCDVd. The first genotype (GenBank Accession No. FJ822877) was identical to the type TCDVd and found in 11 samples from 2007 and one from 2008. The second genotype (GenBank Accession No. FJ822878) was 361 nt, differing from the first by nine nucleotide substitutions, 2 insertions, and 1 deletion. This second genotype was found in 7 and 17 samples from 2007 and 2008, respectively, and showed the highest sequence identity (97%) to a Japanese tomato isolate (AB329668) and a much lower sequence identity (92%) to a U.S. isolate previously identified in Colorado (AY372399) (4). The origin of TCDVd in this outbreak is not clear. The genotype identified first could have been introduced from a neighboring greenhouse where the disease was observed before 2006 and where this genotype also was identified in 2007. The second genotype may have been introduced from infected seed since TCDVd has recently been shown to be seed transmitted in tomato (3). To our knowledge, this is the first report of natural occurrence of TCDVd in Arizona. References: (1) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (2) R. P. Singh et al. J. Gen. Virol. 80:2823, 1999. (3) R. P. Singh and A. D. Dilworth. Eur. J. Plant Pathol. 123:111, 2009. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

Download Full-text

First Report of Alfalfa mosaic virus in Viburnum lucidum in Spain

Plant Disease ◽

10.1094/pdis-92-7-1132b ◽

2008 ◽

Vol 92 (7) ◽

pp. 1132-1132 ◽

Cited By ~ 2

Author(s):

M. C. Cebrián ◽

M. C. Córdoba-Sellés ◽

A. Alfaro-Fernández ◽

J. A. Herrera-Vásquez ◽

C. Jordá

Keyword(s):

Mosaic Virus ◽

Natural Infection ◽

Alfalfa Mosaic Virus ◽

The United States ◽

Rt Pcr ◽

Water Control ◽

First Report ◽

Pcr Product ◽

Pcr Assays ◽

The Mediterranean

Viburnum sp. is an ornamental shrub widely used in private and public gardens. It is common in natural wooded areas in the Mediterranean Region. The genus includes more than 150 species distributed widely in climatically mild and subtropical regions of Asia, Europe, North Africa, and the Americas. In January 2007, yellow leaf spotting in young plants of Viburnun lucidum was observed in two ornamental nurseries in the Mediterranean area of Spain. Symptoms appeared sporadically depending on environmental conditions but normally in cooler conditions. Leaf tissue from 24 asymptomatic and five symptomatic plants was sampled and analyzed by double-antibody sandwich (DAS)-ELISA with specific polyclonal antibodies against Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany) and Alfalfa mosaic virus (AMV) (SEDIAG S.A.S, Longvic, France). All symptomatic plants of V. lucidum were positive for Alfalfa mosaic virus (AMV). The presence of AMV was tested in the 29 samples by one-step reverse transcription (RT)-PCR with the platinum Taq kit (Invitrogen Life Technologies, Barcelona, Spain) using primers derived from a partial fragment of the coat protein gene of AMV (2). The RT-PCR assays produced an expected amplicon of 700 bp in the five symptomatic seropositive samples. No amplification product was observed when healthy plants or a water control were used as a template in the RT-PCR assays. One PCR product was purified (High Pure PCR Product Purification Kit; Roche Diagnostics, Mannheim, Germany) and directly sequenced (GenBank Accession No. EF427449). BLAST analysis showed 96% nucleotide sequence identity to an AMV isolate described from Phlox paniculata in the United States (GenBank Accession No. DQ124429). This virosis has been described as affecting Viburnum tinus L. in France (1). To our knowledge, this is the first report of natural infection of Viburnum lucidum with AMV in Spain, which might have important epidemiological consequences since V. lucidum is a vegetatively propagated ornamental plant. References: (1) L. Cardin et al. Plant Dis. 90:1115, 2006. (2) Ll. Martínez-Priego et al. Plant Dis. 88:908, 2004.

Download Full-text

First Report of Cherry virus A and Little cherry virus-1 in Poland

Plant Disease ◽

10.1094/pdis.2004.88.8.909c ◽

2004 ◽

Vol 88 (8) ◽

pp. 909-909 ◽

Cited By ~ 12

Author(s):

B. Komorowska ◽

M. Cieślińska

Keyword(s):

Nucleotide Sequence ◽

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Germplasm Collection ◽

Mottle Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Primer Sets

Cherry virus A (CVA), a member of the genus Capillovirus, has been reported in sweet cherry in Germany, Canada, and Great Britain. No data are available on the effects of CVA on fruit quality and yield of infected trees. Little cherry disease (LChD) occurs in most cherry growing areas of the world. Symptoms on sensitive cultivars include discolored fruit that remain small, pointed in shape, and tasteless. Three Closterovirus spp. associated with LChD have been described (Little cherry virus-1 [LChV-1], LChV-2, and LChV-3). Diseased local and commercial cultivars of sour cherry trees were found in a Prunus sp. germplasm collection and orchards in Poland during the 2003 growing season. The foliar symptoms included irregular, chlorotic mottling, distortion, and premature falling of leaves. Some of the diseased trees developed rosette as a result of decreased growth and shortened internodes. Severely infected branches exhibited dieback symptoms. Because the symptoms were suggestive of a possible virus infection, leaf samples were collected from 38 trees and assayed for Prune dwarf virus and Prunus necrotic ringspot virus using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). RNA extracted from leaves was used in a reverse transcription-polymerase chain reaction (RT-PCR) with the One-Step RT-PCR with Platinum Taq (Invitrogen Life Technologies) and primer sets specific for CVA (1), LChV-1 (3), and LChV-2 (3). The RNA samples were also tested using RT-PCR for detection of Cherry mottle leaf virus (CMLV), Cherry necrotic rusty mottle virus (CNRMV), and Cherry green ring mottle virus (CGRMV) with specific primer sets (2). Amplification of a 397-bp coat protein gene product confirmed infection of 15 trees with CVA. A 419-bp fragment corresponding to the coat protein gene of LChV-1 was amplified from cv. Gisela rootstock and local cv. WVIII/1. To confirm RT-PCR results, CVA amplification products from local cv. WX/5 and LChV-1 from cvs. Gisela and WVIII/1 were cloned in bacterial vector pCR 2.1-TOPO and then sequenced. The sequences were analyzed with the Lasergene (DNASTAR, Madison, WI) computer program. The alignment indicated that the nucleotide sequence of cv. WX/5 was closely related to the published sequences of CVA (Genbank Accession No. NC_003689) and had an 89% homology to the corresponding region. The nucleotide sequence similarity between the 419-bp fragment obtained from cvs. Gisela and WVIII/1 was 87% and 91%, respectively, compared with the reference isolate of LChV-1 (Genbank Accession No. NC_001836). The sampled trees tested negative for LChV-2, CGRMV, CMLV, and CNRMV using RT-PCR. Some trees tested positive for PNRSV and PDV. To our knowledge, this is the first report of CVA and LChV-1 in Poland. References: (1) D. James and W. Jelkmann. Acta Hortic. 472:299, 1998. (2) M. E. Rott and W. Jelkmann. Eur. J. Plant Pathol. 107:411,2001. (3) M. E. Rott and W. Jelkmann. Phytopathology. 91:61, 2001.

Download Full-text

Effect of Two Allexivirus Isolates on Garlic Yield

Plant Disease ◽

10.1094/pd-90-0898 ◽

2006 ◽

Vol 90 (7) ◽

pp. 898-904 ◽

Cited By ~ 14

Author(s):

E. E. Cafrune ◽

M. C. Perotto ◽

V. C. Conci

Keyword(s):

Allium Sativum ◽

Negative Control ◽

Yield Losses ◽

Assay Condition ◽

First Report ◽

Virus Complex ◽

Significant Yield ◽

Virus C ◽

Garlic Virus ◽

Assay Conditions

Garlic (Allium sativum) is infected by numerous viruses forming a viral-complex, which is widely distributed in the garlic production regions of Argentina. This work is the first report of the effect of two Allexivirus isolates, Garlic virus A (GarV-A) and Garlic virus C (GarV-C), on garlic yield. Garlic cvs. Morado-INTA and Blanco-IFFIVE were used in the experiments, and four treatments were evaluated: plants inoculated with GarV-A only, GarV-C only, virus-free plants (negative control), and plants infected with the virus-complex. Assays were performed in anti-aphid cages and in the field during 2002 and 2003. GarV-A caused significant reductions in bulb weight (14 to 32%) and diameter (6 to 11%) compared with the negative control in the two cultivars under both assay conditions. GarV-C caused less damage than GarV-A (15% in weight and 5% in diameter) with respect to the negative control in cv. Blanco-IFFIVE, and did not produce significant yield losses in cv. Morado-INTA in either year or under either assay condition.

Download Full-text

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.

Download Full-text

First report of Coguvirus eburi infecting pear (Pyrus communis) in South Africa

Plant Disease ◽

10.1094/pdis-08-21-1630-pdn ◽

2021 ◽

Author(s):

Kayleigh Bougard ◽

Hans Jacob Maree ◽

Gerhard Pietersen ◽

Julia Christine Meitz-Hopkins ◽

Rachelle Bester

Keyword(s):

South Africa ◽

High Throughput Sequencing ◽

De Novo ◽

Rna Extraction ◽

Pyrus Communis ◽

Rt Pcr ◽

Disease Symptoms ◽

Total Rna ◽

First Report ◽

Pcr Assays

Coguvirus eburi is a member of the genus Coguvirus in the family Phenuviridae (Khun et al., 2020). The species Coguvirus eburi was established to include citrus virus A (CiVA), which is a negative-sense, single-stranded RNA virus that was first found infecting sweet orange in southern Italy via high-throughput sequencing (HTS) (Navarro et al., 2018). This virus was also found to infect pome fruits in France, such as pear (Svanella-Dumas et al., 2019). More recently CiVA infections have been associated with impietratura disease in citrus (Beris et al. 2021). In the summer of 2021, leaf samples were collected from a pear tree (Pyrus communis cv. Bosc, B175) in the Koue Bokkeveld, South Africa as part of a virus survey. Sample B175 displayed no visual disease symptoms. One gram of leaf petioles was used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). Ribo-depleted RNA was prepared (Ribo-Zero Plant kit) and a sequencing library constructed (Illumina TruSeq Stranded Total RNA). The RNA library was paired-end (2 × 100 bp) sequenced on an Illumina HiSeqX instrument (Macrogen, South Korea). A total of 47,750,152 reads were obtained. Raw data was trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly performed with CLC Genomics Workbench 11.0.1 (Qiagen) (Default parameters) using high quality reads yielded 75250 contigs. BLASTn analysis identified two viral contigs with high nucleotide (nt) identity to apple stem pitting virus (ASPV) and CiVA. The CiVA contig was 9400 nts and on closer examination, a concatemer of CiVA RNA1 and RNA2. The concatenation occurred due to the characteristic near-identical nucleotides shared at the 5’ and 3’ ends of RNA1 and RNA2 of these negative-stranded RNA viruses (Navarro et al., 2018). After splitting and curation, the RNA1 contig was 6664 nts and the RNA2 contig 2686 nts. A total of 51397 and 34820 reads were used to construct these contigs resulting in an average depth of coverage of 761 and 1281 for RNA1 and RNA2, respectively. The contigs had the highest nt identity to the complete CiVA GenBank accessions MT720885.1 (95.53%) and MW148460.1 (96.03%), spanning 99.6% and 98.1 % of the genomes of RNA1 and RNA2, respectively. These contigs were submitted as partial genomes to GenBank as accessions MZ463039 and MZ463040. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the presence of CiVA in sample B175. Two RT-PCR assays, directed at RNA1 and RNA2 respectively (Bester et al. (2021)) were used to generate amplicons. Amplicon sequences were confirmed with bi-directional Sanger sequencing. Twenty-one additional samples from the same orchard as B175 as well as other samples from the Koue Bokkeveld and Elgin areas, including cultivars Abate (10 samples), Forelle (10 samples), Early Bon Chretien (3 samples), Packham’s Triumph (12 samples) and Rosemarie (3 samples), were all surveyed for CiVA using the same RT-PCR assays as mentioned above. Thirty-six of the 59 samples tested were positive for CiVA, which further confirms the presence and wide-spread distribution of this virus in the limited survey conducted in pears in South Africa. However, no association with any disease symptoms or specific cultivar were identified. This is the first report of CiVA infecting pear in South Africa. This study therefore contributed to investigating the distribution of this virus and will assist the South African plant material certification scheme to assess the incidence of CiVA in South Africa.

Download Full-text