Complete Coat Protein Gene Research Articles

Phylogenetic and Recombination Analysis of Apple Stem Grooving Virus Isolates from Pears in Korea

The apple stem grooving virus (ASGV) is one of the most harmful latent viruses infecting pear orchards worldwide. To examine the genetic diversity of ASGV in Korean pear orchards, the complete coat protein (CP) gene of five ASGV isolates collected from various regions were identified. The five Korean ASGV isolates showed 88–96% nucleotide identity with the 11 isolates worldwide occurring elsewhere in the world. Phylogenetic analysis of five isolates, as well as the previously sequenced isolates, indicated that the ASGV clusters had no correlation with the host or geographical regions of origin. Recombination analysis showed that one of the five Korean isolates is a recombinant, with a recombination site in the CP gene region (nt 532–708). This study is the first report of natural recombination within the CP gene of ASGV isolates from pears grown in Korea.

First Report of the Association of Zygocactus virus X with Dragon Fruit (Hylocereus spp.) plants from Telangana, India.

Dragon fruit (Hylocereus spp.) a member of the family Cactaceae, is widely cultivated throughout the world, includingspan style="font-family:'Times New Roman'; letter-spacing:0.05pt; color:#333333"> India. During 2020-2021 crop growing season, mosaic symptoms were observed on the cladodes of dragon fruit plants (Purple Pink cultivar: 1-2% disease incidence) grown at a farmer's field of Telangana, India (Fig. S1 a). The symptomatic cladodes (n= 4), observed under leaf-dip electron microscopy (Zuchmaan and Zellnig, 2009) at Indian Agricultural Research Institute, New Delhi, revealed the presence of flexuous rod- shaped virus-like particles (Fig S1 b). Virus particles were of 580 x 13 nm size, corresponding to the genus Potexvirus. For further confirmation, the total RNA isolated from symptomatic cladodes using a NucleoSpin RNA Plant Mini kit (Macherey-Nagel). Subsequently, a reverse transcription polymerase chain reaction (RT-PCR) was performed using the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio). The cDNA was further amplified with the primers specific to coat protein (CP) gene of four different species of the genus Potexvirus known to infect members of Cactaceae family. Four sets of primers were used for detection, viz., Cactus virus X (CVX) (F, 5'-ATGTCTACTACTGGAGTCCA-3'; R, 5'-CTACTCAGGGCCTGGGAGAA-3'); Pitaya virus X (PiVX) (F, 5'-ATGGCTACTCAAACAGCACAA-3'; R, 5'-CTACTCTGGGGAGGGAAG-3'); Schlumbergera virus X (SchVX) (F, 5'-ATGTCGACCACTCCATCTTC-3'; R, 5'-TTATTCAGGGGATGGTAGTA-3') and Zygocactus virus X (ZyVX) (F, 5'-ATGTCTAACACTGCAGGAGT-3'; R, TCATTC GGGACCCGGTAGGA-3') (Duarte et al., 2008; Janssen et al., 2021; Parameswari et al., 2021), by following the PCR profile (Park et al., 2018). The species-specific primers of CVX, PiVX and SchVX did not amplify any amplicon, whereas the primers specific to ZyVX at nucleotide position 5841-6521 from complete CP gene have resulted in amplification of expected size (~680 base pairs) from all the samples. The gel-purified RT-PCR products were cloned into a pDrive cloning vector (Qiagen, Germany) and sequenced bi-directionally using Sanger sequencing. The resultant sequences (681 nt) of the CP gene showed 98% (nucleotide) and 100% (amino acid) sequence similarity with the CP gene sequence (Accession No: KY581590) of ZyVX. Hence, one representative sequence was deposited to the NCBI GenBank database as ZyVX-DPC isolate (Accession number- OK415019). The Neighbour Joining Phylogenetic Tree constructed using MEGA6 software (Tamura et al. 2013) showed grouping of Indian ZyVX-DPC isolate with the previously reported ZyVX isolates from Korea, Taiwan, China and Germany (Fig. S1c). These results confirmed the association of ZyVX with the symptomatic cladodes of dragon fruit plants collected from Telangana, India. Earlier studies revealed that ZyVX is a member of the genus Potexvirus known to infect dragon fruit plants from Brazil and China (Duarte et al., 2008). In India until now, anthracnose disease (Colletotrichum siamense) and CVX from Hylocereus spp. were reported (Abirami et al., 2019; Parameswari et al., 2021). To the best of our knowledge, this is the first report of ZyVX infection on dragon fruit in India. The draon fruit, being vegetatively propagated and with increasing cultivable area in India (Abirami et al, 2019), the present study gains significance. Further studies on mode of virus transmission, estimation of crop yield losses, host range studies and finding out source of resistance are essential.

A Previously Undescribed Polerovirus (Solemoviridae) Infecting Theobroma cacao Germplasm.

Cacao Theobroma cacao L. (Malvaceae) is an economically important crop cultivated in tropical climates for the bean from which chocolate and other products are made (Zarrillo et al., 2018). Virus-like symptoms consisting of discoloration, leaf distortion with downward rolling of leaves, and yellow speckling or mottling (Fig. S1), were observed in imported cacao germplasm at the USDA-ARS-SHRS cacao quarantine facilities in the fall of 2020. Total RNA was isolated from leaves collected from four symptomatic plants using silica RNA extraction method (Rott and Jelkmann, 2001). Ribosomal RNA (rRNA)-depleted RNA samples were used for cDNA library construction, followed by high throughput sequencing on Illumina® NovaSeq 6000 platform (Novogene Corp., Sacramento, CA). Quality-filtered, 150-bp paired-ended reads (2,601,293-3,104,474) were assembled de novo using SPAdes v.3.14.1 (Nurk et al., 2013). The contigs (200,799 to 276,851) were queried against the NCBI virus reference sequence (RefSeq) database using the discontiguous megablast algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). The resultant contigs (n=1,344) were 150-nt to 1463-nt in length (k-mer coverage from 6.3x to 26,721.7x) and shared their highest nucleotide (nt) identity with potato leafroll virus (PLRV; NC_001747; genus Polerovirus; family Solemoviridae), at 69.1%-72.8%. The contigs pooled from the four samples were assembled into 15 scaffolds. BLASTn analyses of the 15 scaffolds against the RefSeq database indicated the best matches were to thirteen other polerovirus species, with top hits to cereal yellow dwarf virus-RPV (D10206) and pepper vein yellows virus (LC528383), having similarity scores of 66.2% and 100% respectively. The 15 scaffolds matched to the 5' terminal end, ORF1-2, ORF3, ORF4 and ORF3-5 of the different polerovirus genomes. For confirmatory sequencing, total RNA was subjected to reverse transcription using SuperScript IV (Invitrogen, Carlsbad, CA), followed by RT-PCR amplification with general polerovirus primers PoconF/PoconcpR (Xiang et al., 2008) expected to yield an amplicon of ~1,400-bp located at the 3' end of the RNA-dependent, RNA polymerase (RdRp), including the complete coat protein (CP) and movement protein (MP) genes. Amplicons were ligated to pGEM-T Easy vector (Promega, Madison, WI) and sequenced bi-directionally by Sanger sequencing (Eton Bio, Research Triangle Park, NC). BLASTn analysis of the polerovirus-like nt sequences (GenBank accession nos. (ON745771-ON745774) indicated the closest relatives were potato leafroll virus (OK058524) and cucumber aphid-borne yellows virus (FJ460218), at 71% and 73%, respectively. The CP amino acid (aa) sequence shared the greatest similarity to cereal yellow dwarf virus RPV (NP_840023), at 53%, and the MP aa sequence shared the greatest aa similarity to wheat yellow leaf dwarf virus-GPV (YP_003029842), at 38%. These results provide robust support for the association of a previously undescribed polerovirus with symptomatic cacao trees, herein named, cacao leafroll virus (Solemoviridae; Polerovirus). Although Koch's postulates have not been completed to confirm causality, the presence of this virus in cacao germplasm undermine efforts to distribute pathogen-free germplasm and may pose a risk to cacao production in trees established from virus-infected plant material. To our knowledge, this is the first report of a polerovirus infecting cacao trees. All trees of these accessions at the quarantined facility in Miami, FL have been destroyed.

Occurrence and Genetic Characterization of Grapevine Pinot Gris Virus in Russia.

Grapevine Pinot gris virus (GPGV) is a widespread grapevine pathogen associated with symptoms of leaf mottling and deformation. In order to study the distribution and genetic diversity of GPGV in Russia, we tested 1347 grapevine samples from 3 regions of Russia–the Krasnodar Krai, Stavropol Krai, and Republic of Crimea—using duplex real-time RT-PCR. GPGV was detected in 993 grapevines, both symptomatic and asymptomatic. In 119 isolates, we sequenced complete movement protein (MP) and coat protein (CP) genes of the GPGV genome. The percentage of identity of the obtained nucleotide MP/CP sequences with the closest isolates from the GenBank was 97.75–99.56%. A phylogenetic analysis showed that these Russian GPGV isolates are mainly grouped with previously described representative asymptomatic isolates. New post-translational modifications of the MP and CP at the positions of polymorphisms in the genomes of Russian isolates were predicted. The present work is the first study on the distribution and genetic diversity of GPGV in Russia.

Molecular characterization of strawberry vein banding virus from China and the development of loop\u2011mediated isothermal amplification assays for their detection

Strawberry vein banding virus (SVBV) is one of the serious viral pathogens infecting strawberry worldwide. To understand the molecular characterization of SVBV from China, complete genome sequences of sixteen SVBV isolates were cloned and sequenced. Sequence comparison showed they shared high nucleotide sequence identity (93.6–99.5%) with isolates from China and Japan (96.6–98.4%), while relatively low identity with the isolates from Canada (91.9–93.7%) and USA (85.5–85.9%). Phylogenetic analyses based on the complete genome sequence or coat protein (CP) gene showed the SVBV isolates clustered into three clades correlated with geographic distribution. Recombination analyses identified 13 recombinants and 21 recombinant events, indicating frequent and multiple recombinations in SVBV evolution. Furthermore, a sensitive loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of SVBV isolates, which could be especially suitable for seedling propagation, virus-free culture and routine diagnostics in field investigation. This study offers new understanding of the molecular evolution and may help to improve the management of SVBV.

Genetic diversity of cucumber green mottle mosaic virus (CGMMV) infecting cucurbits

Cucumber green mottle mosaic virus (CGMMV), a well-known Tobamovirus, infects cucurbits across the globe. To determine its current status, molecular characterization, genetic recombination, gene flow and selection pressure, 10 districts from Punjab province of Pakistan were surveyed and a total of 2561 cucurbits samples were collected during 2019–2020. These samples were subjected to virus-specific double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for the detection of CGMMV. The results revealed that viral disease was prevalent in all surveyed districts of Punjab with an overall 25.69% disease incidence. ELISA positive samples were further confirmed through RT-PCR and sequencing of coat protein (CP) cistron. Sequence analysis showed that the present studied CGMMV isolates have 96–99.5% nucleotide and 94.40–99.50% amino acid identities with those already available in GenBank. Phylogenetic analysis also revealed that understudied isolates were closely related with South Korean (AB369274) and Japanese (V01551) isolates and clustered in a separate clad. Sequence polymorphisms were observed in 663 bp of sequence within 31 CGMMV isolates covering complete CP gene. Total number of sites were 662, of which 610 and 52 sites were monomorphic and polymorphic (segregating), respectively. Of these polymorphic, 24 were singleton variable and 28 were parsimony informative. Overall nucleotide diversity (π) in all the understudied 31 isolates was 0.00010 while a total of 1 InDel event was observed and InDel Diversity (k) was 0.065. Haplotype diversity analysis revealed that there was a total 29 haplotypes with haplotype diversity (Hd) of 0.993458 in all the 31 isolates which provide evidence of less diversity among Pakistani isolates. The statistical analysis revealed the values 2.568, 5.31304 and 4.86698 of Tajima's D, Fu, & Li’s F* and D*, respectively, which witnessed the population of CGMMV was under balanced selection pressure.

Genetic Structure of Cucumber Mosaic Virus From Natural Hosts in Nigeria Reveals High Diversity and Occurrence of Putative Novel Recombinant Strains.

Cucumber mosaic virus (CMV, Bromoviridae: Cucummovirus), one of the most widespread plant viruses with several hosts, causes huge losses in yield quality and quantity. The occurrence of various CMV strains and high genetic diversity within the virus complicate its management. We describe the population structure of CMV in Nigeria using partial RNA1 and RNA3 gene sequences from three natural hosts: pepper (Capsicum annuum), tomato (Solanum lycopersicum), and watermelon (Citrullus lanatus). One hundred and six leaf samples were obtained from 16 locations across Nigeria, and specific primers were used to amplify the two gene fragments using PCR. Twenty-four samples tested positive for CMV using RNA1 primers, and amplicons were sequenced from 12 isolates, revealing 82.94–99.80% nucleotide and 85.42–100% amino acid sequence similarities within the population. The partial RNA3 fragment, corresponding to the complete coat protein (CP) gene, was sequenced from seven isolates, with 95.79–97.90% and 98.62–100% nucleotide and amino acid intrapopulation similarities, respectively. The isolates belonged to subgroup IB and formed distinct phylogenetic clusters in both gene sets, indicating putative novel strains. Recombination signals, supported by phylogenetic inferences, were detected within the RNA1 dataset (P ≤ 0.05) and identified a recombinant isolate within the Nigerian sequences. No recombination was detected within the CP genes. Population genetics parameters established high diversity within the Nigerian population compared to other isolates worldwide, while selection pressure estimates revealed the existence of negative selection in both gene sets. Although CMV subgroup IB strains were postulated to originate from Asia, this study reveals their prevalence across several hosts from different locations in Nigeria. To our knowledge, this is the first comprehensive description of a recombinant CMV subgroup IB isolate from West Africa, which has implications for its robust detection and overall management.

First report of Apium virus Y infecting Petroselinum crispum in Turkey

Apium virus Y (ApVY) is one of the most important potyviruses that infect apiaceous crops. In January-February 2021, parsley plants (Petroselinum crispum) with mild mosaic and vein clearing symptoms on their leaves were observed during field studies in the Balıkesir province of Turkey (Figure 1). Leaf samples from ten different diseased plants from two commercial fields in the province of Balıkesir were sampled. The samples were brought to the laboratory in a cold box and stored on silica gel at 4°C. Total nucleic acid (TNA) isolation was performed using 100 mg of leaf tissue with the CTAB method (Li et al., 2008). The obtained TNA (adjusted to 1000 ng/μl) from each sample was tested by RT-PCR using universal potyvirus primers as described previously (Zheng et al., 2010). Fragments of the expected size (350 bp) belonging to the nuclear inclusion b (NIb) region of potyviruses were amplified from all ten samples. Amplicons from two randomly chosen samples, once from each field, were subjected to Sanger sequencing. BLASTN analysis of the nucleotide sequences (GenBank Accession nos. OL741312-OL741313) had the highest sequence identify (90%) with the corresponding region of an ApVY isolate (HM363516) from the USA. A new primer pair (ApVYCP-F 5'-CATCCAAAGACGTGTCAGAT-3') and (ApVYCP-R 5'-GCTATGGTGCAATTTGAACG-3') was designed to detect ApVY infection in all samples, producing a 1066 bp amplicon including the complete ApVY coat protein (CP) gene. Each PCR reaction was prepared in a total volume of 25 μl including 2 μl cDNA (50 ng/μl), 12.5 μl 2x EmeraldAmp MAX PCR Mastermix (Takara, Japan), 0.5 μl primer mix (stock concentration 20 pmol/μl) and 10 μl nuclease-free water. The PCR conditions were 94°C for 5 min for initial denaturation, 40 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C; and a final extension for 5 min at 72°C. Fragments of the expected size were obtained from all samples. Amplicons from four samples, two from each field, were sequenced bi-directionally. The assembled sequences were submitted to GenBank (OL741314-OL741317). The identity of the isolates from this study with each other and the isolates from other countries were determined at the nucleotide and amino acid level by ClustalW alignment in the Sequence Demarcation Tool V.1.2 software (Muhire et al., 2014). Sequence analyses using the obtained CP gene sequences revealed that Turkish ApVY isolates had 88–100% nucleotide and 95–100% amino acid identities between each other. Nucleotide and amino acid identities between the Turkish isolates and those from other countries were 88–100% and 93–100%, respectively (Figure 2). Phylogenetic analyses of the ApVY CP gene sequences of the four Turkish isolates in this study and isolates from other countries revealed that three Turkish isolates were closely related to Slovenian isolates and clustered together in the same branch of the phylogenetic tree, while one Turkish isolate grouped with the isolates from the USA (Figure 3). ApVY has been shown to be transmitted experimentally in 17 plant species in the family Apiaceae by mechanical inoculation, and by Myzus persicae with a transmission efficiency of 10–20 % (Xu et al. 2011). Seed transmission has not been shown (Xu et al. 2011; Koike et al. 2012) but we suspect it might be possible since aphid populations were not found in the ApVY-infected fields in this case. Alternatively, it is possible that some other insect vector is spreading this virus, thus further studies are needed to understand the transmission of this virus under Turkish conditions. To the best of our knowledge, this is the first report of ApVY in Turkey. This study was supported by a grant from TUBITAK-TOVAG (project # 120O802).

Occurrence of leek yellow stripe virus and onion yellow dwarf virus from edible Allium plants in the south Marmara region of Turkey.

The online version contains supplementary material available at 10.1007/s13205-021-03067-1.

Genetic diversity and evolutionary analyses of potyviruses infecting narcissus in Iran.

Potyviruses are among the most important pathogens of dicotyledonous and monocotyledonous ornamentals and crop plants. In this study, leaf samples were collected from symptomatic narcissus plants and weeds in Fars and Tehran provinces of Iran. Enzyme-linked immunosorbent assay using broad-spectrum potyvirus antibodies gave a positive reaction with 38 out of 61 narcissus samples tested (62.3%); the results were confirmed by reverse-transcription polymerase chain reaction using universal NIb primers, and for thirty samples, by sequencing and phylogenetic studies. The results suggested the infection of almost all positive samples with narcissus yellow stripe virus (NYSV); only one sample seemed to be infected with narcissus late season yellows virus (NLSYV). The 3'-end of the genome of the NLSYV isolate and six NYSV isolates, encompassing the complete coat protein gene, was amplified and sequenced using species-specific and universal potyvirus primers. Sequence analysis indicated the presence of NLSYV and NYSV, not previously identified from Western Asia. No evidence of recombination was found in Iranian isolates. Based on phylogenetic analyses, isolates of NLSYV and NYSV clustered into five and three phylogroups, respectively, where all the Iranian isolates fell into distinct subpopulations in groups NLSYV-I and NYSV-II. Multiple sequence alignments showed some phylogroup-specific amino acid substitutions for both viruses. Phylogroup IV and II populations had higher nucleotide diversities as compared with other populations of NLSYV and NYSV, respectively. Our findings revealed the presence of negative selection in the populations of both viruses. Almost no statistically significant gene flow was found between populations of these viruses.Supplementary informationThe online version contains supplementary material available at 10.1007/s42161-021-00985-0.

Genetic Variability and Evidence of a New Subgroup in Watermelon Mosaic Virus Isolates.

Watermelon mosaic virus (WMV) is one of the important Potyviruses that infect cucurbits worldwide. To better understand the population structure of WMV in the United States (U.S.), 57 isolates were collected from cucurbit fields located in nine southern states. The complete coat protein gene of all WMV isolates was cloned, sequenced and compared with 89 reported WMV isolates. The nucleotide and amino acid sequence identities among the U.S. WMV isolates ranged from 88.9 to 99.7% and from 91.5 to 100%, respectively. Phylogenetic analysis revealed that all the U.S. WMV isolates irrespective of their geographic origin or hosts belonged to Group 3. However, the fifty-seven isolates made three clusters in G3, where two clusters were similar to previously reported subgroups EM1 and EM2, and the third cluster, containing nine WMV isolates, formed a distinct subgroup named EM5 in this study. The ratio of non-synonymous to synonymous nucleotide substitution was low indicating the occurrence of negative purifying selection in the CP gene of WMV. Phylogenetic analysis of selected 37 complete genome sequences of WMV isolates also supported the above major grouping. Recombination analysis in the CP genes confirmed various recombinant events, indicating that purifying selection and recombination are the two dominant forces for the evolution of WMV isolates in the U.S.

First report of squash vein yellowing virus naturally infecting butternut squash (Cucurbita moschata) in Texas.

Virus diseases are major constraints to the production of cucurbits in the Texas Lower Rio Grande Valley. In September 2020, a ~8.1 ha butternut squash (Cucurbita moschata) field in Hidalgo County, Texas, was observed with virus-like symptoms of vein yellowing, leaf curl, mosaic, and foliar chlorosis. The proportion of plants with virus-like symptoms in this field was estimated at 30% and seven samples (symptomatic = 5; non-symptomatic = 2) were collected randomly for virus diagnosis. Initially, equimolar mixtures of total nucleic acid extracts (Dellaporta et. al. 1983) from two symptomatic samples from this field and extracts from 12 additional symptomatic samples from six other fields across south and central Texas was used to generate one composite sample for diagnosis by high throughput sequencing (HTS). The TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina) was used to construct cDNA library from the composite sample, which was then sequenced on the Illumina NextSeq 500 platform. More than 26 million single-end HTS reads (75 nt each) were obtained and their bioinformatic analyses (Al Rwahnih et al. 2018) revealed several virus-like contigs belonging to different species (data not shown). Among them, 6 contigs that ranged in length from 429 to 3,834 nt shared 96 to 100% identities with isolates of squash vein yellowing virus (SqVYV), genus Ipomovirus, family Potyviridae. To confirm the HTS results, total nucleic acid extracts from the cucurbit samples from all seven fields (n = 46) were used for cDNA synthesis with random hexamers and the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio). A 1-μL aliquot of cDNA was used in 12.5-μL PCR reaction volumes with PrimeSTAR GXL DNA Polymerase (Takara Bio) and two pairs of SqVYV-specific primers designed based on the HTS derived contigs. The primer pairs SqYVV-v4762: 5'-CTGGATTCTGCTGGAAGATCA & SqYVV-c5512: 5'-CCACCATTAAGGCCATCAAAC and SqYVV-v8478: 5'-TTTCTGGGCAAACAAACATGG & SqYVV-c9715: 5'-TTCAGCGACGTCAAGTGAG targeted ~0.75 kb and ~1.2 kb fragments of the cylindrical inclusion (CI) and the complete coat protein (CP) gene sequences of SqVYV, respectively. The expected DNA band sizes were obtained only from the five symptomatic butternut squash samples from the Hidalgo Co. field. Two amplicons per primer pair from two samples were cloned into pJET1.2/Blunt vector (Life Technologies) and bidirectionally Sanger sequenced, generating 753 nt partial CI specific sequences (MW584341-342) and 1,238 nt that encompassed the complete CP (MW584343-344) of SqVYV. In pairwise comparisons, the partial CI sequences shared 100% nt/aa identity with each other and 98-99% nt/aa identity with corresponding sequences of SqVYV isolate IL (KT721735). The CP cistron of TX isolates shared 100% nt/aa identity with each other and 90-98% nt (97-100% aa) identities with corresponding sequences of several SqVYV isolates in GenBank, with isolates IL (KT721735) and Florida (EU259611) being at the high and low spectrum of nt/aa identity values, respectively. This is the first report of SqVYV in Texas, naturally occurring in butternut squash. SqVYV was first discovered in Florida (Adkins et al. 2007) and subsequently reported from few other states in the U.S. (Adkins et al. 2013; Egel and Adkins 2007; Batuman et al. 2015), Puerto Rico (Acevedo et al. 2013), and locations around the world. The finding shows an expansion of the geographical range of SqVYV and adds to the repertoire of cucurbit-infecting viruses in Texas. Further studies are needed to determine the prevalence of SqVYV in Texas cucurbit fields and an assessment of their genetic diversity.

Phylogenetic and Population Analysis of Lettuce mosaic virus Isolates Based on the Coat Protein Gene

Lettuce mosaic virus (LMV) is one of the most important and destructive members of Potyviruses in the family Potyviridae that is transmitted by aphid and seed which cause economic damage and serious yield losses to different crops around the world. To explore phylogenetic relationships, population evolution, and the effect of selection forces on the complete coat protein (CP) gene of LMV isolates, 36 complete coat protein gene sequences retrieved from the GenBank database in several different countries in four continents: Central and East Asia (China, Taiwan, South Korea, and Turkey), Europe (France), Africa (Tunisia), and America (Chile and Brazil). The phylogenetic tree of LMV isolates was grouped into independent clades with the significance of FST values (>0.27). The ratio of dN/dS is calculated less than one and showed that the LMV-CP has been under negative selection. Statistical tests (Tajima’s D, Fu and Li’s D* and F*) were used to estimate non-significantly negative values for all clades and geographic populations except for the Brazilian population and Clade III in a phylogenetic group. The negatives values revealed that there is less polymorphism estimation. All geographic populations in four phylogroups of LMV seem to be at equilibrium because all neutrality test statistics were non-significant. The findings suggested that the dynamics of LMV molecular evolution may be dependent on mutation, recombination, and negative selection. No recombination events were observed in this part of the LMV genome. Therefore, this study provides the first time evolution and differentiation between populations of LMV isolates from around the world and suggested that they may be occurred by the transmission of the virus among lettuce plants by types of aphid species, migration in different geographical areas by infected seeds and plant material, and broad host range which seems that these events have played an important role in shaping the LMV population structure.

Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid.

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 μM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds.

First molecular characterization of pepper severe mottle virus infecting chili pepper crops in Colombia

A potyvirus infecting chili pepper (Capsicum spp) crops has been characterized. The complete coat protein (CP) gene was obtained. Pairwise comparisons showed that the CP shares 82.8% nucleotide sequence identity and 83.5% amino acid identity with pepper severe mottle virus - PepSMoV (KT896752), a putative potyvirus previously found in pepper crops in Venezuela. These results suggest that this isolate found infecting chili crops in Colombia belongs to Pepper severe mottle virus (PepSMoV) species. To confirm the presence of PepSMoV in three varieties of chili pepper leaf samples collected in Colombia, specific primers were designed. Mechanical inoculation assays were successful and PepSMoV was transmitted to chili pepper plants, which developed severe mosaic symptoms and leaf deformation. To our knowledge this is the first report of the molecular and biological characterization PepSMoV which affects pepper crops in Colombia.

First report of cucurbit chlorotic yellows virus infecting cucumber in South Korea.

In October 2018, cucumber plants showing yellowing and chlorotic mottle symptoms were observed in a greenhouse in Chungbuk, South Korea. The observed symptoms were similar to those caused by cucurbit aphid-borne yellows virus (CABYV), which has been detected on cucumber plants in the region since it was reported on melon in Korea in 2015 (Lee et al 2015). To identify the potential agents causing these symptoms, 28 samples from symptomatic leaves and fruit of cucumber plants were subjected to total RNA extraction using the Plant RNA Prep Kit (Biocubesystem, Korea). Reverse transcription polymerase chain (RT-PCR) was performed on total RNA using CABYV specific primers and protocols (Kwak et al. 2018). CABYV was detected in 17 of the 28 samples, while 11 symptomatic samples tested negative. In order to identify the cause of the symptoms, RT-PCR was performed using cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) specific primers (Wintermantel et al. 2019). Eight of the 28 samples were positive using the CCYV specific primers while seven samples were infected with only CCYV and one contained a mixed infection of CABYV with CCYV. None of the samples tested positive for CYSDV. The expected 373 nt amplicons of CCYV were bi-directionally sequenced, and BLASTn analysis showed that the nucleotide sequences shared 98 to 100% identity with CCYV isolates from East Asia, including NC0180174 from Japan. Two pairs of primers for amplification of the complete coat protein and RNA-dependent RNA polymerase (RdRp) genes (Wintermantel et al., 2019) were used to amplify the 753bp coat protein and 1517bp RdRp genes, respectively. Amplicons of the expected sizes were obtained from a CCYV single infection and ligated into the pGEM T- Easy vector (Promega, WI, USA). Three clones from each amplicon were sequenced and aligned using Geneious Prime and found to have identical sequences (Genbank accession nos. MW033300, MW033301). The CP and RdRp sequences demonstrated 99% nucleotide and 100% amino acid identity with the respective genes and proteins of the CCYV isolates from Japan. This study documents the first report of CCYV in Korea. Since CCYV was first detected on melon in Japan, it has been reported in many other countries including those in East Asia, the Middle East, Southern Europe, North Africa, and recently in North America. CCYV has the potential to become a serious threat to production of cucurbit crops in Korea, particularly due to the increasing prevalence of the whitefly, Bemisia tabaci, in greenhouse production systems. It will be important to continue monitoring for CCYV and determine potential alternate hosts in the region to manage and prevent further spread of CCYV in Korea.

Biological, epidemiological and population structure analyses of vitiviruses in Iran

The presence of three vitiviruses associated with grapevine rugose wood disease, i.e. grapevine virus B (GVB), grapevine virus E (GVE) and grapevine virus F (GVF), was studied in 650 grapevine samples collected from eight viticulture growing provinces of Iran. RT-PCR assays conducted on these samples showed that 69.5% of the grapevine material is infected at least with one virus; of which GVE was the prevailing virus (29.5%), followed by GVF (23.6%) and GVB (16.4%). The genetic variability and population structures of vitiviruses found in Iran were thoroughly analyzed by sequencing the complete coat protein gene of 29 isolates from different regions. BLAST analysis disclosed 81–96%, 77–80% and 87–96% identities at the nucleotide level and 91–98%, 80–85% and 93–97% identities at the amino acid level between Iranian GVB, GVE and GVF, respectively; and their corresponding reference isolates from the GenBank. The phylogenetic analyses showed two divergent evolutionary lineages for each vitivirus species that were related to the geographic origin of isolates. The high FST values found among GVE and GVF populations indicated that both viruses are under different evolutionary constraints in Iran, suggesting more than one origin. The neutrality analysis also suggested that GVE and GVF in Iran and in the world were inferred from the old presence of GVB, through gene flow and positive selection pressures that shaped the genetic structure of vitiviruses populations. The transmission trial conducted with Planococcus ficus under controlled conditions showed that GVE can be transmitted by the first-instar nymphs.

Complete Genome Characterization and Coat Protein Genealogy of Isolates of Maize dwarf mosaic virus from Johnsongrass and Maize in Oklahoma and Missouri.

Maize dwarf mosaic virus (MDMV) significantly affects maize production worldwide, including the United States. This study describes the distribution and biological and molecular characterization of MDMV isolates from Johnsongrass and maize. A total of 262 samples (symptomatic = 214, asymptomatic = 48) were collected in Oklahoma and Missouri during 2016, 2017, and 2019 growing seasons. Based on a dot-immunobinding assay (DIBA), the average incidence of maize dwarf mosaic disease varied from ∼71% (79/111) in 2016, ∼76% (81/106) in 2017, and 62% (28/45) in 2019. Sixty-five DIBA-positive samples for MDMV were further confirmed by RT-PCR, and the complete coat protein (CP) gene was cloned and sequenced. Phylogenetic analysis of 132 isolates (This study = 65; GenBank = 67) revealed two main groups (G1 and G2) of MDMV isolates. All 65 MDMV isolates contained a 39-nucleotide insertion in the N-terminal region of CP genes and clustered in G1 which were different from the isolates in G2, without 39-nucleotide insertion. The first complete genome (9,563 nucleotides) of a MDMV isolate (Bixby1) from Johnsongrass was sequenced, which was distantly related to eight previously reported MDMV isolates from maize. The dN/dS ratio showed mostly purifying selection on each of cistrons except 6K1 being subjected to the diversifying selection. Further analyses revealed three putative recombination events between MDMV-Bixby1 and MDMV isolates from other countries. The successful mechanical and aphid transmission of MDMV-Bixby1 onto maize cultivars was achieved. Altogether, this information showed that Johnsongrass harbors genetically diverse MDMV isolates, which could pose a threat to cultivated crops such as maize and sorghum.

Wisteria Vein Mosaic Virus Detected for the First Time in Iran from an Unknown Host by Analysis of Aphid Vectors.

The development of reverse transcription–polymerase chain reaction using degenerate primers against conserved regions of most potyviral genomes enabled sampling of the potyvirome. However, these assays usually involve sampling potential host plants, but identifying infected plants when they are asymptomatic is challenging, and many plants, especially wild ones, contain inhibitors to DNA amplification. We used an alternative approach which utilized aphid vectors and indicator plants to identify potyviruses capable of infecting common bean (Phaseolus vulgaris). Aphids were collected from a range of asymptomatic leguminous weeds and trees in Iran, and transferred to bean seedlings under controlled conditions. Bean plants were tested serologically for potyvirus infections four-weeks post-inoculation. The serological assay and symptomatology together indicated the presence of one potyvirus, and symptomology alone implied the presence of an unidentified virus. The partial genome of the potyvirus, encompassing the complete coat protein gene, was amplified using generic potyvirus primers. Sequence analysis of the amplicon confirmed the presence of an isolate of Wisteria vein mosaic virus (WVMV), a virus species not previously identified from Western Asia. Phylogenetic analyses of available WVMV sequences categorized them into five groups: East Asian-1 to 3, North American and World. The Iranian isolate clustered with those in the World group. Multiple sequence alignment indicated the presence of some genogroup-specific amino acid substitutions among the isolates studied. Chinese isolates were sister groups of other isolates and showed higher nucleotide distances as compared with the others, suggesting a possible Eastern-Asian origin of WVMV, the main region where Wisteria might have originated.

First Report of Cucurbit Chlorotic Yellows Virus Infecting Cucumber and Zucchini in Algeria

This work was supported by the Spanish Ministerio de Ciencia, Innovacion y Universidades, cofunded with FEDER funds (project nos. AGL2017-85563-C2-1-R and RTA2017-00061-0O3-03 [INIA]) and the programa para grupos de investigacion de excelencia from the Conselleria d'Educacio, Investigacio, Cultura i Esport, (Generalitat Valenciana) (Prometeo Program 2017/078). A. Kheireddine thanks the Erasmus+ Programme of the European Union for her mobility project (KA107 2018-20). C. Saez is a recipient of a predoctoral fellowship from Generalitat Valenciana, cofunded by the Operational Program of the European Social Fund (FSECV 2014-2020) (grant no. ACIF/2016/188). Plant Dis. 104: 1264, 2020; published online as https://doi.org/10.1094/PDIS-10-19-2091PDN.Accepted for publication 19 December 2019.