ABSTRACT
Abstract Cucumber mosaic virus (CMV) is a tremendous threat to vegetables across the globe, including in Pakistan. The present work was conducted to investigate the genetic variability of CMV isolates infecting pea and spinach vegetables in the Pothwar region of Pakistan. Serological-based surveys during 2016-2017 revealed 31.70% overall CMV disease incidence from pea and spinach crops. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) revealed that all the positive isolates belong to CMV subgroup II. Two selected cDNA from ELISA-positive samples representing each pea and spinach crops were PCR-amplified (ca.1100 bp) and sequenced corresponding to the CMV CP gene which shared 93.7% nucleotide identity with each other. Both the sequences of CMV pea (AAHAP) and spinach (AARS) isolates from Pakistan were submitted to GenBank as accession nos. MH119071 and MH119073, respectively. BLAST analysis revealed 93.4% sequence identity of AAHAP isolate with SpK (KC763473) from Iran while AARS isolate shared maximum identity (94.5%) with the strain 241 (AJ585519) from Australia and clustered with some reference isolates of CMV subgroup II from UK (Z12818) and USA (AF127976) in a Neighbour-joining phylogenetic reconstruction. A total of 59 polymorphic (segregating) sites (S) with nucleotide diversity (π) of 0.06218 was evident while no INDEL event was observed in Pakistani isolates. The evolutionary distance of Pakistani CMV isolates was recorded as 0.0657 with each other and 0.0574-0.2964 with other CMV isolates reported elsewhere in the world. A frequent gene flow (Fst = 0.30478 <0.33) was observed between Pakistani and earlier reported CMV isolates. In genetic differentiation analysis, the value of three permutation-based statistical tests viz; Z (84.3011), Snn (0.82456), and Ks* (4.04042) were non-significant. The statistical analysis revealed the values 2.02535, 0.01468, and 0.71862 of Tajima's D, Fu, & Li's F* and D* respectively, demonstrating that the CMV population is under balancing selection.
Resumo Cucumber mosaic cucumovirus (CMV) é uma tremenda ameaça aos vegetais em todo o mundo, inclusive no Paquistão. O presente trabalho foi conduzido para investigar a variabilidade genética de isolados de CMV infectando vegetais de ervilha e espinafre na região de Pothwar, Paquistão. Pesquisas com base em sorologia durante 2016-2017 revelaram 31,70% da incidência geral da doença por CMV em safras de ervilha e espinafre. O ensaio de imunoabsorção enzimática em sanduíche de anticorpo triplo (TAS-ELISA) revelou que todos os isolados positivos pertencem ao subgrupo II do CMV. Dois cDNA selecionados de amostras positivas para ELISA representando cada safra de ervilha e espinafre foram amplificados por PCR (ca.1100 pb) e sequenciados correspondendo ao gene CMV CP que compartilhou 93,7% de identidade de nucleotídeo um com o outro. Ambas as sequências de isolados de ervilha CMV (AAHAP) e espinafre (AARS) do Paquistão foram submetidas ao GenBank como nos de acesso. MH119071 e MH119073, respectivamente. A análise BLAST revelou 93,4% de identidade de sequência do isolado AAHAP com SpK (KC763473) do Irã, enquanto o isolado AARS compartilhou a identidade máxima (94,5%) com a cepa 241 (AJ585519) da Austrália e agrupada com alguns isolados de referência do subgrupo II de CMV do Reino Unido (Z12818) e EUA (AF127976) em uma reconstrução filogenética vizinha. Um total de 59 sítios polimórficos (segregantes) (S) com diversidade de nucleotídeos (π) de 0,06218 foi evidente, enquanto nenhum evento INDEL foi observado em isolados do Paquistão. A distância evolutiva de isolados de CMV do Paquistão foi registrada como 0,0657 entre si e 0,0574-0,2964 com outros isolados de CMV relatados em outras partes do mundo. Um fluxo gênico frequente (Fst = 0,30478 < 0,33) foi observado entre os isolados de CMV do Paquistão e relatados anteriormente. Na análise de diferenciação genética, os valores de três testes estatísticos baseados em permutação viz, Z (84,3011), Snn (0,82456) e Ks * (4,04042) não foram significativos. A análise estatística revelou os valores 2,02535, 0,01468 e 0,71862 de Tajima's D, Fu, & Li's F * e D * respectivamente, demonstrando que a população de CMV está sob seleção de balanceamento.
Subject(s)
Cucumovirus/genetics , Cucumis sativus , Pakistan , Phylogeny , Plant Diseases , Genetic Variation , Spinacia oleracea , Pisum sativumABSTRACT
The habanero chilli pepper, Capsicum chinense is an important crop in the Amazon Basin, mainly grown by small-scale producers. Capsicum chinense plants in an experimental field in the northern Brazilian state of Amazonas were found exhibiting characteristic symptoms of viral infection. Leaf sap from symptomatic plants examined under a transmission electron microscope revealed the presence of elongated flexuous particles and isometric particles. Using molecular assays, the viruses were identified as pepper yellow mosaic virus (PepYMV) and cucumber mosaic virus (CMV). Aphids, identified as Aphis gossypii, were found colonizing the C. chinense plants in the field and may be the vector for both PepYMV and CMV. We report the first occurrence of these viruses infecting C. chinense in the state of Amazonas.
A pimenta-de-cheiro, Capsicum chinense é uma cultura importante na Bacia Amazônica, cultivada principalmente por pequenos produtores. Plantas de C. chinense em um campo experimental localizado no norte do estado brasileiro do Amazonas, foram encontradas apresentando sintomas característicos de infecção viral. Extratos de amostras de folhas sintomáticas examinados ao microscópio eletrônico de transmissão revelaram a presença de partículas alongadas e flexuosas e de partículas isométricas. Análises moleculares permitiram identificar a presença do pepper yellow mosaic virus (PepYMV) e do cucumber mosaic virus (CMV). Pulgões, identificados como Aphis gossypii foram encontrados colonizando pimenteiras-de-cheiro neste campo experimental e podem representar o provável vetor de PepYMV e CMV. Este trabalho relata a primeira ocorrência desses vírus infectando C. chinense no estado do Amazonas.
Subject(s)
Capsicum/virology , Cucumovirus/pathogenicity , Microscopy, Electron, Transmission/instrumentation , Polymerase Chain ReactionABSTRACT
The habanero chilli pepper, Capsicum chinense is an important crop in the Amazon Basin, mainly grown by small-scale producers. Capsicum chinense plants in an experimental field in the northern Brazilian state of Amazonas were found exhibiting characteristic symptoms of viral infection. Leaf sap from symptomatic plants examined under a transmission electron microscope revealed the presence of elongated flexuous particles and isometric particles. Using molecular assays, the viruses were identified as pepper yellow mosaic virus (PepYMV) and cucumber mosaic virus (CMV). Aphids, identified as Aphis gossypii, were found colonizing the C. chinense plants in the field and may be the vector for both PepYMV and CMV. We report the first occurrence of these viruses infecting C. chinense in the state of Amazonas. (AU)
Subject(s)
Potyvirus , Cucumovirus , Microscopy, Electron, Transmission , VirusesABSTRACT
A baixa produtividade e a qualidade dos frutos de melancia estão quase sempre associadas à ocorrência de fitopatogenos, dentre estes destacam as doenças de etiologia viral. Visando o entendimento do complexo vírus/planta de melancia, este trabalho teve o objetivo de avaliar os danos e sintomas induzidos pelos vírus PRSV-W, CMV, ZYMV e WMV, quando inoculados em plantas de melancia, cv. 'Crimson sweet', aos 5, 12 e 19 dias após a emergência (DAE). Os vírus PRSV-W, CMV, ZYMV e WMV influenciaram significativamente (p<0,05) na redução do peso da matéria fresca da parte aérea, na coloração da polpa e redução no peso dos frutos da melancia. Assim como, no teor de sólidos solúveis (ºBrix). Sendo mais significativa em plantas inoculadas com CMV. De modo geral, os vírus PRSV-W, WMV, CMV e ZYMV afetaram o desenvolvimento, a produção da planta da melancia e consequentemente, limita a qualidade do fruto.
Watermelon is one of the most important vegetables grown in Brazil. However, there are several factors responsible for low productivity of crops. Among these stands out the occurrence of associated virus culture. Thus, this study aimed to assess the damage and symptoms of PRSV-W, CMV, ZYMV and WMV inoculated at 5, 12 and 19 days after emergence (DAE). According to the results, PRSV-W viruses, CMV, ZYMV and WMV influenced significantly (p <0.05) reduction in the fresh weight of shoots and on pulp and reduction of fruit weight of inoculated plants. As noted on pulp plants inoculated with PRSV-W, at 5 DAE. With regard to the soluble solids content (° Brix) was dramatically reduced by the virus and inoculation period, in this case being more significant fruits of plants inoculated with CMV presented Brix 3.67 to 5.11 depending on the inoculation period. In general, viruses PRSV-W, WMV, CMV, ZYMV and influence the development and consequently the production of watermelon. Thus, an important factor for the reduction in the quality of fruit produced.
Subject(s)
Plant Diseases , Cucumovirus , CitrullusABSTRACT
Cucumber mosaic virus [CMV] from the Bromoviridae family, is one of the most widespread plant viruses in the world. In the present study tomato fields in Guilan, Isfahan, Khorasan Razavi, Khuzestan and Tehran provinces were surveyed to determine the presence of CMV subgroups during 2011-2012. Out of 305 symptomatic leaf samples tested by Enzyme-linked immuno sorbent assay [ELISA], 147 samples [48.2%] were found to be infected by CMV with the highest percentage in Khorasan Razavi [67.4%] followed by Khuzestan [50.6%], Tehran [48%], Isfahan [38.2%] and Guilan [34.3%]. The coat protein [CP] gene in the 19 sequenced CMV isolates composed of 657 nucleotides [nt] in a size that encodes 218 amino acids. Phylogenetic analysis based on the nt CP gene showed that the ToKz1, ToKz2, ToKz3 and ToKz4 from Khuzestan fell into subgroup IB and the rest of the Iranian isolates including those sequenced in this study fell into subgroup IA. Subsequent analyses showed that the Iranian CMV isolates belonging to subgroup IA of CMV were most related phylogenetically to each other and they were distinct from the subgroup IB and subgroup II isolates. Bioassay on Nicotiana glutinosa and Solanum lycopersicum showed that the symptoms caused by subgroup IB isolates from Khuzestan were milder than those caused by CMV isolates from subgroup IA under this study. In Iran only subgroups IA and II have been reported, however for the first time this study shows the occurrence and phylogenetic relationships of CMV subgroup IB isolated from tomato fields in West Asia, Iran.
Subject(s)
Capsid Proteins/genetics , Cucumovirus/isolation & purification , PhylogenyABSTRACT
To gain insights into the function of potential post-translational modifications on the activity of the Cucumber mosaic virus (CMV)-encoded silencing suppressor protein 2b, one predicted phosphorylation site (S40) and two predicted ubiquitination/sumoylation sites (K22 and K39) in CMV-Q2b protein were individually or simultaneously mutated by site-directed mutagenesis methods. These Q2b mutants were inserted into plant expression vectors, expressed in plant leaves, and then analyzed for their silencing suppressor activities. The results showed that S40A mutation greatly impaired both the local and systemic silencing suppressor activity, and the K22R mutation has no significant effect on the suppressor activity, while the K22R/K39R double mutation reduced the systemic silencing suppressor activity. To test if the decrease of suppressor activity were due to protein accumulation changes, western blot were performed to monitor the protein level of Q2b mutants. The results indicated that mutations of both K22 and K39 to R or S40 to A all significantly reduced the accumulation of the Q2b protein in plants, while the single mutation of K22 to R did not alter the accumulation of Q2b protein, suggesting that two potential post-translational modification sites, K39 and S40, contribute to the suppressor activity and stability of 2b protein in plant cells.
Subject(s)
Cucumovirus , Genetics , Metabolism , Gene Expression Regulation, Plant , Gene Silencing , Mutagenesis, Site-Directed , Mutation , Protein Processing, Post-Translational , Protein Stability , Viral Proteins , GeneticsABSTRACT
Coat protein (CP) -mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.
Subject(s)
Capsid Proteins/genetics , Cucumovirus/genetics , Enzyme-Linked Immunosorbent Assay , Immunoblotting , Plants, Genetically Modified/genetics , Agrobacterium tumefaciens/genetics , Nicotiana/genetics , Transformation, GeneticABSTRACT
<p><b>OBJECTIVE</b>To study viruses infecting Pinellia ternata in China.</p><p><b>METHOD</b>Symptom observation, DAS-ELISA and RT-PCR detection were applied.</p><p><b>RESULT AND CONCLUSION</b>During a survey in early spring, SMV and CMV were both commonly distributed as main viruses infecting P. ternata collected from different areas in China. But DsMV was the virus which infected P. ternate in natural condition. The infection ratio of cultivated P. ternate by SMV and CMV were 71.4% and 14.3% respectively for 21 samples collected from Ningbo, Zhejiang province; 100% and 44.4% for 18 samples from Xiaoshan, Zhejiang province; 61.9% and 33.3% for 21 samples from Hebei province; 50.0% and 41.7% for 12 samples from Anhui province; 16.7% and 16.7% for 12 samples from Sichuan province; 31.3% and none for 16 samples from Beijing. And the infection ratio of 25 wild samples from different areas of China infected by SMV and CMV were both 20.0%.</p>
Subject(s)
China , Cluster Analysis , Cucumovirus , Genetics , DNA, Complementary , Chemistry , Genetics , Mosaic Viruses , Classification , Genetics , Pinellia , Virology , Plant Diseases , Virology , Plants, Medicinal , Virology , Sequence Analysis, DNAABSTRACT
A viral disease was identified on geraniums (Pelargonium spp.) grown in a greenhouse at the Institute of Himalayan Bioresource Technology (IHBT), Palampur, exhibiting mild mottling and stunting. The causal virus (Cucumber mosaic virus, CMV) was identified and characterized on the basis of host range, aphid transmission, enzyme linked immunosorbent assay (ELISA), DNA-RNA hybridization and reverse transcription polymerase chain reaction (RT-PCR). A complete coat protein (CP) gene was amplified using degenerate primers and sequenced. The CP gene showed nucleotide and amino acid homology up to 97%-98% and 96%-99%, respectively with the sequences of CMV subgroup II. The CP gene also showed homologies of 75%-97% in nucleotide and 77%-96% in amino acid with the CMV Indian isolates infecting various crops. On the basis of sequence homology, it was concluded that CMV-infecting geraniums in India belong to subgroup II.
Subject(s)
Amino Acid Sequence , Base Sequence , Capsid Proteins/chemistry , Cucumovirus/chemistry , Molecular Sequence Data , PhylogenyABSTRACT
Plants of Viola cornuta displaying typical virus symptoms were observed during spring 2003 in a plant nursery in Córdoba, central Argentina. Electron microscopic examinations of symptomatic leaf samples revealed the presence of isometric virus-like particles about 30 nm in diameter. Subsequent serological analysis allowed the identification of the pathogen as a subgroup 1 strain of Cucumber mosaic virus (CMV). These results were confirmed by antigen capture - reverse transcription - polymerase chain reaction with specific CMV primers, and digestion with a restriction enzyme. This is the first report of CMV infecting V cornuta in Argentina
Subject(s)
Cucumovirus , Plant Diseases/etiology , Plant Diseases/virology , Serology/methodsABSTRACT
Cucumber mosaic virus (CMV) causing mosaic, leaf distortion and stunting of vanilla (Vanilla planifolia Andrews) in India was characterized on the basis of biological and coat protein (CP) nucleotide sequence properties. In mechanical inoculation tests, the virus was found to infect members of Chenopodiaceae, Cucurbitaceae, Fabaceae and Solanaceae. Nicotiana benthamiana was found to be a suitable host for the propagation of CMV. The virus was purified from inoculated N. benthamiana plants and negatively stained purified preparations contained isometric particles of about 28 nm in diameter. The molecular weight of the viral coat protein subunits was found to be 25.0 kDa. Polyclonal antiserum was produced in New Zealand white rabbit, immunoglobulin G (IgG) was purified and conjugated with alkaline phosphatase enzyme. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was standardized for the detection of CMV infection in vanilla plants. CP gene of the virus was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR), cloned and sequenced. Sequenced region contained a single open reading frame of 657 nucleotides potentially coding for 218 amino acids. Sequence analyses with other CMV isolates revealed the greatest identity with black pepper isolate of CMV (99%) and the phylogram clearly showed that CMV infecting vanilla belongs to subgroup IB. This is the first report of occurrence of CMV on V. planifolia from India.
Subject(s)
Amino Acid Sequence , Capsid Proteins/chemistry , Cucumovirus/genetics , India , Molecular Sequence Data , Phylogeny , Plant Leaves/physiology , Sequence Homology, Amino AcidABSTRACT
An attempt was made to detect various viruses of Piper betle grown at Mahoba and Banthara in India. DAC-ELISA and RT-PCR tests were performed in leaf sap samples of betelvine for detection of a cucumovirus (Cucumber mosaic virus) and potyvirus (Bean yellow mosaic virus) using specific antibodies and universal primers of respective viruses. DAC-ELISA could detect only CMV. However, RT-PCR detected both cucumovirus and potyvirus infection in betelvine samples. Association of CMV with betelvine was observed for the first time in the present study.
Subject(s)
Antibodies, Viral/analysis , Base Sequence , Cucumovirus/genetics , DNA Primers , DNA, Viral/analysis , Enzyme-Linked Immunosorbent Assay , Potyvirus/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Plant species associated with commercial melon crops and surrounding areas were examined to identity the natural host plants of Aphis gossypii Glover. The study was conducted in two farms located in different melon production areas and plant life zones of Costa Rica. Plant species diversity, percent coverage and distribution over time were recorded during one year. Differences between locations were observed. A total of 86 plant species (49 families) and 72 plant species (40 families) were identified associated to the crop in farms A and B, respectively. In both farms a total of 24 species plants (16 families) were colonized by A. gossypii and 16 (10 families) are new reports of host plant species for this aphid. The new reports are: Justicia comata, Tetramerium nervosum, Alternanthera pubiflora, Cassia massoni, C. reticulata, Cleome viscosa, C. spinosa, Croton argenteus, Caperonia palustris, Chamaesyce gyssopilopia, Phyllantus amarus, Sida decumbens, Ludwigia erecta, Passiflora foetida, Guazuma ulmifolia and Corchorus orinocensis.
Subject(s)
Animals , Aphids/virology , Mosaic Viruses , Plants , Aphids/physiology , Costa Rica , Cucumovirus , Disease Vectors , Nepovirus , Plants , Host-Parasite InteractionsABSTRACT
Lethal necrosis or systemic stem necrosis followed by death of Nicotiana benthamiana, severe leaf deformations of N. tabacum cv. white burley and blister formations on N. tabacum cv. samsun NN symptoms were induced by experimental inoculations of CMV RNA preparations containing satellite RNA (sat-RNA). Inoculations of RNA preparations without sat-RNA did not induce that severe symptoms on these plants, only late mild mosaic was observed. It is suggested that sat-RNA of CMV isolate has a certain role for enhancing severity of symptoms in tobacco plants. Local and systemic lethal necrosis of N. benthamiana is due to sat-RNA present with genome of CMV isolate. It is the first report of lethal necrosis induced in N. benthamiana by CMV satellite.
Subject(s)
Cucumber Mosaic Virus Satellite/genetics , Cucumovirus/genetics , Necrosis , Plants, Toxic , VirulenceABSTRACT
Virus strains isolated from Ocimum sanctum and Zinnia elegans were identified as cucumber mosaic virus (CMV) strains on the basis of non-persistant aphid transmission, 28 nm particles, 26 kDa coat protein subunits and serological relationships with CMV and chrysanthemum aspermy virus. The strains showed some biological, serological and satellite RNA based differentiation with other CMV strains isolated earlier from chrysanthemum, petunia and tobacco.
Subject(s)
Cucumber Mosaic Virus Satellite/genetics , Cucumovirus/genetics , India , RNA, Viral/geneticsABSTRACT
Virus causing severe chlorosis/mosaic disease of banana was identified as a strain of cucumber mosaic virus (CMV). Association of CMV with the disease was established by Western immunoblot using polyclonal antibodies to CMV-T and slot blot hybridization with nucleic acid probe of CMV-P genome.
Subject(s)
Blotting, Western , Cucumovirus/isolation & purification , DNA, ComplementaryABSTRACT
RNA isolation from purified cucumber mosaic virus, CMV-U strain and cDNA synthesis was carried out. The coat protein gene (RNA4) region was amplified selectively by polymerase chain reaction (PCR) with CMV RNA4-specific primers. Double-stranded cDNA was cloned in PRT103 vector at Xho1/Kpn1 site and about 1kb insert obtained. The insert was partly sequenced which showed 50% sequence homology with CMV-Q, C and WL strains.