Molecular Characterization of the 3'-Terminal Region of Turnip mosaic virus Isolates from Eastern China

Turnip mosaic virus (TuMV; genus Potyvirus, family Potyviridae) causes great losses to cruciferous crop production worldwide. The 3′‐terminal genomic sequences of eight TuMV isolates from eastern China were compared with those of 74 other Chinese TuMV isolates of known host origin in the GenBank and isolated during the past 25 years. The reported sequences of the eight TuMV isolates are 1125 or 1126‐nucleotides (nt) long excluding the poly(A) tail. They all contain one partial open reading frame of 912 nt, encoding 304 amino acids, followed by a stop codon and a non‐translated region of 209–210 nt. Results of phylogenetic analyses showed that Chinese TuMV isolates clustered into three groups: basal‐BR, Asian‐BR and world‐B. The ratios of non‐synonymous and synonymous substitutions and results of amino acid alignment provided evidence for purifying or negative selection in TuMV populations of China.     

Characterization of Turnip mosaic virus from the Asian‐BR Population in Iran

Brassicaceae crops in eight provinces of the North‐west Iran were surveyed for Turnip mosaic virus (TuMV) infection during 2011 and 2012. Many symptomatic plants (38%; 226 of 598) were found to be infected with TuMV. The highest frequency was in turnip (61%), followed by radish (55%), oilseed rape (38%), and brassica weeds including annual bastard cabbage (42%), small tumbleweed‐mustard (50%) and wild radish (45%), but not Brassica oleracea and Lepidium sativum. Using biological assays, Iranian TuMV isolates grouped in three [B], [B(R)] and [BR] host‐infecting types. Phylogenetic analysis using complete coat protein (CP) gene nucleotide sequences showed that the Iranian isolates belonged to the Basal‐B and Asian‐BR populations. No evidence of recombination was found in these isolates using different recombination‐detecting programmes. To our knowledge, our study shows for the first time the occurrence of TuMV Asian‐BR subpopulation in the mid Eurasian region of Iran. The data suggest that the Asian‐BR subtype population is found across southern Eurasia and might be a continuous population in East Asia (mostly Japan and China) and Minor Asia (Turkey), the places considered to be one of the origins of TuMV populations.     

Detection and Molecular Variability of Turnip mosaic virus (TuMV) in Shaanxi,China

Turnip mosaic virus (TuMV) is one of the most devastating threats to oilseed rape by causing serious crop losses. A total of 86 leaf samples of oilseed rape from eight different locations in Shaanxi, China, were tested by RT‐PCR for TuMV; the results revealed an infection level of 43% by TuMV. The complete coat protein (CP) gene of 32 TuMV isolates was cloned and sequenced. Analysis of the CP gene with sequences from the database allowed the genetic classification of 170 TuMV isolates or sequences. Four genetic clusters were obtained: MB (mostly Brassica isolates), MR (mostly Radish isolates), IBR (mostly Intermediate between Brassica and Radish clusters) and OBR (mostly outside Brassica and Radish clusters). All subgroups were slightly related to the hosts, but unrelated to geographical origins. Most of Shaanxi TuMV isolates were on separate branches, compared with the 138 known isolates originating from other parts of the world. Our results help provide a better understanding of the genetic diversity of TuMV isolates infecting oilseed rape in Shaanxi, China.     

Molecular Analysis of Zucchini yellow mosaic virus Isolates from Hangzhou, China

Isolates of Zucchini yellow mosaic virus were obtained from different cucurbit crops in Hangzhou city, China. The complete nucleotide sequences of four isolates and the 3′‐terminal sequences, including the coat protein coding region, of four others were determined and then compared with other available sequences. Phylogenetic analysis of the coat protein nucleotide sequences showed that these isolates fell into three significant groups, one of which (designated group III) consisted exclusively of Chinese isolates and is reported for the first time. Comparisons over the completely sequenced genomes showed that, typically for potyviruses, the 5′‐end of the genome was usually the most variable but that the group III isolate differed from the others most significantly in the N‐terminal part of the coat protein. Partially sequenced group III isolates also varied from other isolates in this region. Group III isolates appear to differ biologically from the other isolates because they do not cause symptoms in watermelon fruit but induce more severe symptoms on the watermelon leaves.     

Molecular Detection and Characterization of Chinese Yam Mild Mosaic Virus Isolates

An improved RT‐PCR was developed and validated for the detection of Yam mild mosaic virus (YMMV). Sequences of the coat protein core region of 19 Chinese isolates were obtained, and analysis indicated the presence of different genetic variants. Phylogenetic analyses showed that the Chinese isolates were divided into two distinct clusters. Complete genomic sequences of two distinct Chinese variants were determined to be 9527 and 9529 nucleotides long, excluding the 3′ poly (A) tail. Their genomic structure and organization were virtually identical to that of a Brazilian isolate. The two variants shared identity of 87.3% to one another and 83.9–84.6% to the Brazilian variant at the genomic sequence level. Phylogenetic analyses supported that they represented two distinct YMMV lineages.     

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit‐growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT‐PCR using designed CCYV‐specific primer pair (CCYV‐F/CCYV‐R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non‐Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.     

Coat Protein Gene of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island

Nucleotide and amino acid sequences of the coat protein (CP) of 12 isolates of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island (CyMV‐R) were compared with each other and with those of previously described Asian strains. Alignment revealed that CyMV‐R isolates were highly homologous, suggesting that one strain is prevalent in Reunion. This strain also showed high homology with the Korean CyMV‐K2 and Singapore CyMV‐S2 strains, but nucleotide additions resulted in the carboxy‐terminal ends of the CP sequences differing from those of the Korean CyMV‐K1 and Singapore CyMV‐K1 strains.     

Konjac mosaic virus Naturally Infecting Three Aroid Plant Species in Andhra Pradesh,India

The genomes of three potyvirus isolates from, respectively, naturally infected Colocasia esculenta, Caladium spp. and Dieffenbachia spp. in Andhra Pradesh, India, were amplified by RT‐PCR using degenerate potyvirus primers. Sequence analysis of RT‐PCR amplicons (1599 nucleotides) showed maximum identity of 97% with the KoMV‐Zan isolate of Konjac mosaic virus (KoMV) from Taiwan (A/C AF332872). The three isolates had a maximum identity of 99.4%. The length of coat protein (CP) gene of three isolates was 846 nucleotides encoding 282 amino acids with a deduced size of 32.25 kDa. The CP gene of the isolates had, respectively, 78.1–95.7% and 88.2–96.4% identity at nucleotide and amino acid levels with KoMV isolates. The CP gene of the three isolates had 93.1–100% (nucleotide) and 98.2–100% (amino acid) identity. The 3′‐UTR of the three isolates showed maximum identity of 91.1–100% identity between and with other KoMV isolates. In the CP amino acid–based phylogenetic analyses, the isolates branched as a distinct cluster along with known KoMV isolates. The three potyvirus isolates associated with mosaic, chlorotic feathery mottling, chlorotic spots, leaf deformation and chlorotic ring spots on three aroids were identified as isolates of KoMV for the first time from Andhra Pradesh, India.     

A mixture of begomoviruses in leaf curl-affected tobacco in Karnataka, South India

Tobacco leaf curl is widespread in several states in India including Andhra Pradesh, Gujarat, Karnataka, Bihar and West Bengal. Tobacco leaf curl virus (TbLCV) isolates collected from five different parts of India induced four distinct symptom phenotypes (group I, II, III & IV) on tobacco cultivars Samsun and Anand 119 (Valand & Muniyappa, 1992). PCR was performed on DNA extracted from group I and IV leaf curl‐affected tobacco from Karnataka, India using degenerate begomovirus‐specific primers. Subsequent cloning and sequencing of PCR products revealed preliminary evidence for the presence of at least three begomoviruses in the affected material following alignment of a 333 bp region of the coat protein gene (CP). The complete CP and common region (CR) of two putative begomoviruses, Tobacco leaf curl virus‐Karnataka1 (TbLCV‐Kar1) and Tobacco leaf curl virus‐Karnataka2 (TbLCV‐Kar2), were sequenced using PCR clones obtained with designed sequence‐specific primers. Phylogenetic analysis of the CP and CR of TbLCV‐Kar1 and TbLCV‐Kar2 placed them in the Asian Old World begomovirus cluster. The two viruses differed from each other significantly in both the CP gene and the CR (< 90% nucleotide sequence identity). This difference, in conjunction with distinct iterative sequences strongly suggests that these begomoviruses are distinct from one another. Group I and IV tobacco were also found to harbour a possible third begomovirus following the 333 bp CP alignment. Comparison of TbLCV‐Kar1 and TbLCV‐Kar2 with other geminiviruses, showed that both sequences shared high nucleotide sequence identity (> 90%) with other begomoviruses in either the CP or CR, thereby suggesting these viruses to be possible strains of other reported begomoviruses. Combined comparison of the CP and CR sequences however, suggests that the two viruses are not strains of other reported begomoviruses, but may be distinct begomoviruses that could have arisen through recombination events during mixed infections. Phylogenetic comparison demonstrated no significant homology between the Indian tobacco begomoviruses and a tobacco‐infecting begomovirus from Zimbabwe, again showing that as with other geminiviruses, there is a geographic basis for phylogenetic relationships rather than an affiliation with tobacco as a host.     

Genetic Diversity Among Iranian Isolates of Rhizoctonia solani Kühn Anastomosis Group1 Subgroups Based on Isozyme Analysis and Total Soluble Protein Pattern

Rhizoctonia solani is a destructive fungal pathogen with a wide host range. The R. solani complex species includes several divergent groups delimited by affinities for hyphal anastomosis. In this study, genetic variation among 20 isolates of R. solani anastomosis group 1 (AG1) subgroups (AG1‐IA and AG1‐IB) collected from Mâzandaran province, Iran, and standard isolates of these subgroups, was determined by isozyme analysis and total soluble protein profile. Mycelial protein pattern and isozyme analysis were studied using denaturing and non‐denaturing polyacrylamide gel electrophoresis, respectively. A total of 15 enzyme systems were tested, among which six enzymes including esterase, alkaline phosphatase, superoxide dismutase, octanol dehydrogenase, lactate dehydrogenase and mannitol dehydrogenase generated distinct and reproducible results. The soluble protein patterns were similar among the R. solani isolates examined; however, minor differences in banding pattern were observed between the two subgroups. In isozyme analysis, a total of 64 electrophoretic phenotypes were detected for all six enzymes used. Based on cluster analysis and similarity matrix, the fungal isolates were divided into two genetically distinct groups of I and II consistent with the previously reported AG1‐IA and AG1‐IB subgroups in AG1. Group I represented all isolates belonging to AG1‐IA subgroup, whereas group II represented all isolates belonging to AG1‐IB subgroup. Results from isozyme analysis suggest that the subgrouping concept within AGs is genetically based.     

Incidence and genetic diversity of raspberry bushy dwarf virus (RBDV) in Rubus spp. in Turkey

Raspberry bushy dwarf virus (RBDV), recently renamed to Idaeovirus rubi, is one of the most common viruses infecting Rubus species worldwide but there is still a limited number of genome sequences available in the GenBank database and the majority of the sequences include partial sequences of RNA-1 and RNA-2. The distribution and incidence of RBDV in main raspberry and blackberry growing provinces in Turkey were monitored during 2015–2019 and 537 Rubus spp. samples were tested by both DAS-ELISA and RT-PCR. Among the tested samples, 36 samples tested positive for RBDV by DAS-ELISA and 67 samples by RT-PCR. There was relatively low nucleotide diversity among the Turkish isolates. Turkish isolates shared 93%–97.7%, 84.3%–98.9%, and 85%–99.2% nucleotide sequence identities with available sequences in the GenBank, in partial RNA-1, movement protein (MP) and coat protein (CP) genes, respectively. In the phylogenetic tree constructed for RNA-1, MP, and CP sequences, all Turkish raspberry isolates were clustered in a distinct clade. However, the blackberry isolates showed considerable variation in nucleotide sequences and were placed in three distinct groups. The divergent blackberry isolates showed high variability in MP (84.5%–89.3%) and CP (85.5%–89.7%) regions and were placed in a distinct group. The rest of blackberry isolates clustered together with sweet cherry RBDV isolates adjacent to the grapevine clade or together with raspberry isolates. The comparative analysis conducted on three RNA segments of RBDV highlighted the high sequence diversity of Turkish RBDV isolates. This study also emphasizes the importance of regular monitoring of RBDV infections in Turkey, with special regard to those Rubus spp. and grapevine accessions employed in conservation and selection programmes. In particular, the presence of new RBDV genetic variants and infection of Rubus species must be taken into account to choose a correct detection protocol and management strategy.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Characterisation of resistance to turnip mosaic virus in oilseed rape (Brassica napus) and genetic mapping of TuRB01

Turnip mosaic virus (TuMV) is the major virus infecting Brassica crops. A dominant gene, TuRB01, that confers extreme resistance to some isolates of TuMV on Brassica napus (oilseed rape), has been mapped genetically. The mapping employed a set of doubled-haploid lines extracted from a population used previously to develop a reference RFLP map of the B. napus genome. The positioning of TuRB01 on linkage group N6 of the B. napus A–genome indicated that the gene probably originated from Brassica rapa. Resistance phenotypes were confirmed by indirect plate-trapped antigen ELISA using a monoclonal antibody raised against TuMV. The specificity of TuRB01 was determined using a wide range of TuMV isolates, including representatives of the European and American/Taiwanese pathotyping systems. Some isolates of TuMV that did not normally infect B. napus plants possessing TuRB01 produced mutant viruses able to overcome the action of the resistance gene. TuRB01 is the first gene for host resistance to TuMV to be mapped in a Brassica crop. A second locus, TuRB02, that appeared to control the degree of susceptibility to the TuMV isolate CHN 1 in a quantitative manner, was identified on the C-genome linkage group N14. The mapping of other complementary genes and the selective combining of such genes, using marker-assisted breeding, will make durable resistance to TuMV a realisable breeding objective. Received: 14 December 1998 / Accepted: 10 April 1999     

Coat protein-mediated resistance to Turnip mosaic virus in oilseed rape (Brassica napus)

Oilseed rape (Brassica napus) lines transformedwith the coat protein (CP) gene of Turnip mosaic virus(TuMV) were used to determine the effectiveness of resistance to TuMV mediatedby CP RNA or coat protein. Lines with one, two, or more copies of transgeneswere produced. T₂ and T₃ lines containing the CP genewitha functional start codon synthesised coat protein and showed high, but variablelevels of resistance to TuMV (21–96% resistant plants per line). TheT₁ and T₂ progeny of all lines carrying the CP gene withamutated start codon so that RNA but not protein was expressed, were assusceptible to TuMV as controls. Thus, in these experiments we were able toinduce CP-mediated resistance, but not RNA-mediated resistance.     

Demonstration of the Specific Involvement of Coat Protein in Tobacco Mosaic Virus (TMV) Cross Protection using a TMV Coat Protein Mutant

The DT-1G mutant of tobacco mosaic virus (TMV) which has no coat protein was used to study the specific involvement of coat protein in TMV cross protection in N. sylvestris. Leaves of N. sylvestris previously inoculated with the mutantor the common strain of TMV were challenged with either turnip mosaic virus (TuMV) or a strain of TMV (TMV-N). Both TuMV and TMV-N produce necrotic lesions on N. sylvestris. About one-half as many lesions were produced by TuMV and TMV-N on leaves, inoculated with the DT-1G mutant compared with lesions produced by the same inoculum on control leaves. When leaves of N. sylvestris previously inoculated with the common strain of TMV were challenged with either TuMV or TMV-N, TuMV produced about one-half as many lesions as on control leaves whereas TMV-N produced about one-tenth as many lesions as on control leaves. A high level of non-specific resistance was induced by the mutant without coat protein, but it did not specifically protect against TMV.     

Detection,Identification and Phylogenetic Analysis of Indian Ralstonia solanacearum Isolates by Comparison of Partial hrpB Gene Sequences

A species‐specific Polymerase Chain Reaction (sPCR) method was developed to identify and detect isolates of Ralstonia solanacearum, the cause of bacterial wilt disease in chilli. PCR primers for R. solanacearum were identified by alignment of hrpB gene sequences and selection of sequences specific for R. solanacearum at their 3′ ends. The primers were shown to be specific for R. solanacearum, as no PCR product was obtained when genomic DNA from other bacterial species including closely related Ralstonia species, were used as test species. Lone pair of primers (RshrpBF and RshrpBR) was designed using hrpB gene sequence, unique to R. solanacearum which amplified a predicted PCR product of 810 bp from 20 different isolates. Phylogenetic analysis was also attempted to understand the evolutionary divergence of Indian R. solanacearum isolates. Based on phylogenetic analysis, Indian isolates showed homology with the standard reference isolates from other countries but, interestingly, one new isolate showed complete evolutionary divergence by forming an out‐group.     

Cloning and Phylogenetic Analysis of Coat Protein of Barley yellow dwarf virus Isolates from Different Regions of Pakistan

Barley yellow dwarf virus (BYDVs) is an emerging threat for wheat and may seriously threaten its production, especially as climate change may result in increased infestation by aphids, the insect vectors of the virus. To assess the possibility of using pathogen‐derived resistance against the virus, the genetic diversity of BYDVs originating from different wheat‐growing areas of Pakistan where its incidence has been higher was investigated. Wheat samples with suspected symptoms of BYDVs were screened for the presence of Barley yellow dwarf and Cereal yellow dwarf viruses (B/CYDVs) subgroup 1 (Barley yellow dwarf virus‐PAV, BYDV‐MAV, BYDV‐SGV) and subgroup II (BYDV‐RPV, CYDV‐RPV, BYDV‐GPV) by PCR using basic multiplex oligonucleotides designed on coat protein (CP) of the virus. Of 37 samples tested, 13 were positive for BYDV subgroup I and only one sample was positive for BYDV subgroup II. Samples positive for subgroup I were further tested by PCR, and results showed that 10 samples were positive for BYDV‐PAV and three for BYDV‐MAV. DNA sequences of CP region of nine isolates (BYDV‐PAV) were determined and compared with available sequences in databases. Sequence analysis showed that three isolates (from Fatehjang, Nowshera and Attock districts) had maximum identity (92.8–94.6%) to BYDV‐PAS, and six isolates (from Peshawar, Islamabad Swabi and Faisalabad districts) had maximum identity (99.3–99.7%) to BYDV‐PAV. Thus BYDV‐PAV species may be dominant in northern wheat‐growing areas of Pakistan. The conserved nature of the BYDVs suggests that pathogen‐derived resistance strategies targeting the coat protein of the virus are likely to provide protection under field conditions.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Low Genetic Diversity of the Coat Protein Gene among Blueberry Red Ringspot Virus Isolates

Blueberry red ringspot virus (BRRSV) isolates have been investigated for genetic diversity. Nucleotide sequences of the coat protein (CP) gene of 19 isolates from Poland, Czech Republic, Slovenia and the United States were analysed. The nucleotide and amino acid sequence identity were 92–100% and 89–100%, respectively. Estimations of the distribution of synonymous and non‐synonymous changes indicated negative selection within the analysed CP gene and confirmed the genetic stability of the virus. At a capsid protein level, our results revealed BRRSV to be distinct from other, recombination‐prone pararetroviruses.