Indigenous and introduced potyviruses of legumes and <Emphasis Type="Italic">Passiflora</Emphasis> spp. from Australia: biological properties and comparison of coat protein nucleotide sequences

Five Australian potyviruses, passion fruit woodiness virus (PWV), passiflora mosaic virus (PaMV), passiflora virus Y, clitoria chlorosis virus (ClCV) and hardenbergia mosaic virus (HarMV), and two introduced potyviruses, bean common mosaic virus (BCMV) and cowpea aphid-borne mosaic virus (CAbMV), were detected in nine wild or cultivated Passiflora and legume species growing in tropical, subtropical or Mediterranean climatic regions of Western Australia. When ClCV (1), PaMV (1), PaVY (8) and PWV (5) isolates were inoculated to 15 plant species, PWV and two PaVY P. foetida isolates infected P. edulis and P. caerulea readily but legumes only occasionally. Another PaVY P. foetida isolate resembled five PaVY legume isolates in infecting legumes readily but not infecting P. edulis. PaMV resembled PaVY legume isolates in legumes but also infected P. edulis. ClCV did not infect P. edulis or P. caerulea and behaved differently from PaVY legume isolates and PaMV when inoculated to two legume species. When complete coat protein (CP) nucleotide (nt) sequences of 33 new isolates were compared with 41 others, PWV (8), HarMV (4), PaMV (1) and ClCV (1) were within a large group of Australian isolates, while PaVY (14), CAbMV (1) and BCMV (3) isolates were in three other groups. Variation among PWV and PaVY isolates was sufficient for division into four clades each (I-IV). A variable block of 56 amino acid residues at the N-terminal region of the CPs of PaMV and ClCV distinguished them from PWV. Comparison of PWV, PaMV and ClCV CP sequences showed that nt identities were both above and below the 76-77% potyvirus species threshold level. This research gives insights into invasion of new hosts by potyviruses at the natural vegetation and cultivated area interface, and illustrates the potential of indigenous viruses to emerge to infect introduced plants.     

Biological characterization and complete nucleotide sequence of a Tunisian isolate of Moroccan watermelon mosaic virus

Summary During a survey conducted in October 2005, cucurbit leaf samples showing virus-like symptoms were collected from the major cucurbit-growing areas in Tunisia. DAS-ELISA showed the presence of Moroccan watermelon mosaic virus (MWMV, Potyvirus), detected for the first time in Tunisia, in samples from the region of Cap Bon (Northern Tunisia). MWMV isolate TN05-76 (MWMV-Tn) was characterized biologically and its full-length genome sequence was established. MWMV-Tn was found to have biological properties similar to those reported for the MWMV type strain from Morocco. Phylogenetic analysis including the comparison of complete amino-acid sequences of 42 potyviruses confirmed that MWMV-Tn is related (65% amino-acid sequence identity) to Papaya ringspot virus (PRSV) isolates but is a member of a distinct virus species. Sequence analysis on parts of the CP gene of MWMV isolates from different geographical origins revealed some geographic structure of MWMV variability, with three different clusters: one cluster including isolates from the Mediterranean region, a second including isolates from western and central Africa, and a third one including isolates from the southern part of Africa. A significant correlation was observed between geographic and genetic distances between isolates. Isolates from countries in the Mediterranean region where MWMV has recently emerged (France, Spain, Portugal) have highly conserved sequences, suggesting that they may have a common and recent origin. MWMV from Sudan, a highly divergent variant, may be considered an evolutionary intermediate between MWMV and PRSV. Correspondence: H. Lecoq, INRA, Station de Pathologie Végétale, Domaine Saint Maurice, B.P. 94, 84143 Montfavet cedex, France     

Genetic variability of the coat protein sequence of pea seed-borne mosaic virus isolates and the current relationship between phylogenetic placement and resistance groups

Nucleotide sequences of complete or partial coat protein (CP) genes were determined for 11 isolates of pea seed-borne mosaic virus (PSbMV) from Australia and one from China, and compared with known sequences of 20 other isolates. On phylogenetic analysis, the isolates from Australia and China grouped into 2 of 3 clades. Clade A contained three sub-clades (Ai, Aii and Aiii), Australian isolates were in Ai or Aiii, and the Chinese isolate in Aii. Clade A contained isolates in pathotypes P-1, P-2 and U-2; clade B, one isolate in P-2; and clade C, only isolates in P-4.     

Analysis of the molecular and biological variability of Zucchini yellow mosaic virus isolates from Slovakia and Czech Republic

The diversity of ZYMV isolates was analysed by the biological and molecular characterisation of 11 isolates sampled from cucumber, squash and zucchini between 2001 and 2006 in various localities of Slovakia and Czech Republic. Analysis of the molecular variability targeting three separate genomic regions of the ZYMV genome [P1, P3 and (Cter)NIb-(Nter)CP] revealed a remarkable low level of nucleotide variability between isolates, despite their temporal and spatial distinction. Phylogenetic analysis based on the 5′-terminal part of the CP gene highlighted the close relatedness of Slovak, Czech and other central European isolates. Low level of genetic diversity within central European ZYMV isolates is in contrast to the diversity observed for isolates from other geographical regions, in particular Asia. No evidence of recombination in the ZYMV genome was detected. Sequence comparison between aggressive and moderate ZYMV isolates revealed one amino acid difference in the N-terminal part of the P3 protein, potentially involved in the tolerance breaking.     

Analysis of the biological and molecular variability of Watermelon mosaic virus isolates from Iran

Watermelon mosaic virus (WMV) is one of the most important viruses that causes different symptoms in Cucurbitaceae. WMV is a potyvirus with a worldwide distribution, but occurs most commonly in temperate and Mediterranean regions. Cucurbit species grown in Yazd, Esfahan, West Azerbaijan, Hormozgan, and Kerman provinces were surveyed for the relative incidence of WMV in 2004–2005. A total of 757 symptomatic cucurbit and 31 weed species were collected and assayed for infection with WMV. Of 788 leaf samples from cucurbit and weed plants, 190 samples were positive by double antibody sandwich ELISA (DAS-ELISA) using specific polyclonal antibody. Among the weed species tested only colocynth (Citrullus colocynthis) was found to be infected with WMV. The coat protein (CP) gene from 18 representative isolates was PCR amplified, cloned, sequenced, and compared with the sequences available in GeneBank. Phylogenetic analysis using 778 nucleotide long sequences of the coat protein gene showed that these isolates fell into two; groups I and II. Only one isolates (KER.JI.1) was classified in the group II. This isolate had a wider host range and infected Nicotiana debneyii and Datura metel. None of the other 17 isolates could infect these two species. Members of group I were divided into three subgroups; A, B, and C. The subgroup I_B appears to be a new subgroup comprising only of the Iranian isolates. Phylogenetic analysis based on 200 nucleotides coding for the N-terminal segment of the CP showed that all Iranian isolates except KER.JI.1 clustered with the previously reported WMV strains. All Iranian isolates had a DAG amino acid triplet which is involved in aphid transmissibility. This is the first report on sequence analysis of the nearly full-length CP cDNA clones of WMV isolates from Iran.     

Genetic variability in the coat protein gene of Potato virus S isolates and distinguishing its biologically distinct strains

The complete coat protein (CP) nucleotide sequences of 13 Potato virus S (PVS) isolates from Australia and three from Europe were compared to those of 37 others. On phylogenetic analysis, the Australian sequences were in PVS^O sub-clades III and IV, and the European isolates were in sub-clades I and VII. The European isolates invaded Chenopodium spp. systemically, but eight Australian isolates did not. Amino acid sequence differences at the N-terminal ends of the CPs were unrelated to the ability to invade Chenopodium spp. systemically. The acronym PVS^O–CS is suggested for isolates that invade Chenopodium spp. systemically but are not within clade PVS^A.     

Genetic variability in the coat protein genes of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus

Summary. Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.     

Identification and full sequence of an isolate of Alternanthera mosaic potexvirus infecting Phlox stolonifera

Summary. A potexvirus was isolated from creeping phlox (Phlox stolonifera) plants from a commercial nursery in Pennsylvania. The virus was serologically related to clover yellow mosaic virus, plantain virus X, potato virus X, and potato aucuba mosaic virus, and was most closely related to papaya mosaic virus (PapMV). The sequence of a PCR fragment obtained with potexvirus group-specific primers was distinct from that of PapMV; the coat protein (CP) gene and 3′ untranslated region (UTR) were closely related to Alternanthera mosaic virus (AltMV), previously reported only from Australia. The host range was similar to that of the Australian isolate (AltMV-Au), and the phlox isolate reacted strongly with antiserum to AltMV-Au. The full sequence of the phlox isolate was more closely related to PapMV throughout the genome than to any potexvirus other than AltMV-Au, for which only the CP and 3′UTR sequences are available. The phlox isolate was therefore named AltMV-PA (for Pennsylvania), and the full 6607 nt sequence is presented¹. Additional AltMV isolates from creeping phlox (AltMV-BR and AltMV-SP) and trailing portulaca (Portulaca grandiflora; AltMV-Po) were also isolated, suggesting that AltMV may be widespread, and may have been mis-diagnosed in the past as PapMV. AltMV has the potential to spread to other ornamental crops. ¹The full sequence of AltMV-PA has been deposited in GenBank as Accession Number AY863024; the 3′-terminal sequences of AltMV-PA, AltMV-BR, AltMV-SP, and AltMV-Po have been deposited under Accession numbers AY850929, AY850928, AY850931, and AY850930, respectively.     

Sequence Divergence of Four Soilborne Sugarbeet-Infecting Viruses

Soilborne viruses are among the most harmful pathogens of sugarbeet (Beta vulgaris L.ssp. vulgaris) but most of them lack information on genetic variability due to paucity of sequence data. Only one isolate of Beet soil borne virus (BSBV; genus Pomovirus), Beet virus Q (BVQ; genus Pomovirus) and Beet soil borne mosaic virus (BSBMV; genus Benyvirus) has been characterised for the coat protein (CP) gene. In this study, the CP gene sequences of three isolates each of BSBV and Beet necrotic yellow vein virus (BNYVV; genus Benyvirus) (France, Germany and USA), two isolates of BVQ (France and Germany), and one isolate of BSBMV (USA) were determined. Phylogenetic analyses including sequences from databanks indicated that the French BNYVV isolate of this study belongs to so-called P-type, the American isolate to A-type and the German isolate to B-type. The CP genes of the three BSBV isolates characterised in this study and the one available from databank were highly identical (98.4–99.0% at nucleotide level; one variable amino acid). The BSBMV isolate studied here differed from the previously characterised isolate for five nucleotides and four amino acids in the CP region. The two BVQ isolates characterised in this study contained three additional nucleotides resulting in an additional amino acid residue (arginine) at CP position 86, as compared to the only isolate available in databank.     

Molecular variability and genetic structure of the population of <Emphasis Type="Italic">Onion yellow dwarf virus</Emphasis> infecting garlic in Iran

Onion yellow dwarf virus (OYDV) is one of the most important viral diseases of garlic crops worldwide. This study surveyed the occurrence of OYDV in 26 garlic ecotypes collected from different regions in Iran during 2008–2009. Using an electron microscope, we detected filamentous particles with about 700–800 nm in length and 12 nm in width in five samples. These features are typical of the genus Potyvirus. The coat protein (CP) gene from 26 samples was PCR amplified, cloned, sequenced, and compared with the sequences available in GenBank. Phylogenetic analysis using 235 deduced amino acids of the CP gene showed that virus isolates fell into two groups, group A and group B. Members of group A were divided into two subgroups: A-I and A-II. The subgroup A-I appears to be a new subgroup comprising 17 Iranian isolates. The identity levels among the amino acid of 26 Iranian isolates ranged between 90 and 100%. The results indicated that the genetic diversity found in Iran is due to local OYDV populations rather than introduction from other geographical regions. This study is the first report about the molecular structure and geographically diverse range of OYDV populations in this country.     

Zucchini green mottle mosaic virus is a new tobamovirus; comparison of its coat protein gene with that of kyuri green mottle mosaic virus

Summary.  A novel virus we call zucchini green mottle mosaic virus (ZGMMV) was isolated from zucchini squash and its properties were determined. The size and shape of its virions, and other properties suggest that the virus is a tobamovirus. The coat protein (CP) genes of ZGMMV and kyuri green mottle mosaic virus (KGMMV), which also infects zucchini squash plants, were cloned and their nucleotides sequences were determined. The CP genes of ZGMMV and KGMMV are composed of 161 amino acid residues, and they share 77.6% amino acid identity. Western blot analysis showed that the two viruses are serologically related but not identical. Comparison of the sequences with those of sixteen other tobamoviruses revealed that the two viruses had much higher identity to cucumber green mottle mosaic virus (CGMMV), another tobamovirus infectious to cucurbit plants, than other tobamoviruses. The nucleotide and amino acid sequences of ZGMMV were from 29.5 to 78.4% and from 29.3 to 77.6% identical, respectively, to those of other tobamoviruses. The predicted virion assembly origins of the two tobamoviruses were located in the CP region of the genomic RNAs, and the predicted secondary structures were more similar to that of CGMMV than those of other tobamoviruses. The seventeen tobamo-viruses could be classified into three main subgroups based on the cphylogenetic tree analysis on the CP gene, and ZGMMV and KGMMV formed a third subgroup together with CGMMV and sunn-hemp mosaic virus (SHMV). These results show that ZGMMV is a previously unknown member of the Tobamovirus genus. Received February 14, 2000/Accepted May 11, 2000     

Comparison of the coat protein genes of Lettuce big-vein associated virus isolates from Australia with those of isolates from other continents

The complete coat protein (CP) nucleotide sequences of seven Lettuce big-vein associated virus (LBVaV) isolates from Australia were compared to those of 22 other LBVaV and five tobacco stunt virus (TStV) isolates. On phylogenetic analysis, clade I contained only LBVaV isolates from Europe, sub-clade IIa only Australian LBVaV isolates, IIb only Japanese LBVaV isolates, and IIc only TStV isolates from Japan. In the amino acid sequences deduced, the central region of the gene was most divergent. Mean Dn/Ds ratios were 0.283 and 0.124 for clades I and II, respectively. The suggestion that TStV is a strain of LBVaV was supported.     

Nucleotide sequences of the cylindrical inclusion protein genes of two Japanese zucchini yellow mosaic virus isolates

The nucleotide sequences of the cylindrical inclusion protein (CIP) genes of two Japanese zucchini yellow mosaic virus (ZYMV) isolates (ZYMV-169 and ZYMV-M) were determined. The CIP genes of both isolates comprised 1902 nucleotides and encoded 634 amino acids containing consensus nucleotide binding motif. The sequence similarities between the two isolates at the nucleotide and amino acid levels were 91% and 98%, respectively. When the CIP gene sequences of the Japanese ZYMV isolates were compared with those of previously reported ZYMV isolates, the nucleotide and amino acid sequence similarities ranged between 81% and 97%, and between 95% and 97%, respectively. Phylogenetic analysis of the deduced amino acid sequences of the CIP genes indicated that the Japanese ZYMV isolates were closely related to those of other ZYMV isolates.     

Zucchini tigré mosaic virus is a distinct potyvirus in the papaya ringspot virus cluster: molecular and biological insights

In recent years, three new potyviruses have been described in the papaya ringspot virus (PRSV) cluster. In addition, two types of PRSV are recognized, type W, infecting cucurbit plants, and type P, infecting papaya and also cucurbits. A third type, PRSV-T, was also partially described in Guadeloupe. Complete genome sequencing of four PRSV-T isolates showed that this virus is a related virus that is distinct from PRSV, and the name zucchini tigré mosaic virus (ZTMV) is proposed, in reference to the typical symptoms observed in zucchini squash. Eleven other viral isolates from different geographic origins were confirmed as ZTMV isolates using the complete sequence of the cylindrical inclusion (CI) coding region, whereas pairwise sequence similarities in the coat protein (CP) coding region did not unambiguously distinguish ZTMV isolates from PRSV isolates. The use of the CI coding region for species demarcation appears more suitable than the CP coding region for closely related viruses. Principal coordinates analysis based on the biological behavior of the viral isolates studied clustered PRSV-P, PRSV-W and ZTMV isolates into three different groups. Therefore, ZTMV is different from PRSV in its molecular and biological properties.     

Wheat streak mosaic virus genotypes introduced to Argentina are closely related to isolates from the American Pacific Northwest and Australia

Wheat streak mosaic virus (WSMV) was first detected in Argentina in 2002. Comparison of 78 WSMV coat protein sequences revealed that three Argentine isolates were closely related to isolates from the American Pacific Northwest (APNW) and Australia. Complete sequences were determined for one Argentine isolate, four APNW isolates, and three additional isolates from other regions of the USA. Comparison of these eight new sequences with five previously sequenced isolates of WSMV confirmed close affinity of WSMV from Argentina with APNW isolates. Collectively, these results indicate concurrent establishment of the same WSMV lineage in both Argentina and Australia.     

Minimising losses caused by Zucchini yellow mosaic virus in vegetable cucurbit crops in tropical, sub-tropical and Mediterranean environments through cultural methods and host resistance

Between 2006 and 2009, 10 field experiments were done at Kununurra, Carnarvon or Medina in Western Australia (WA) which have tropical, sub-tropical and Mediterranean climates, respectively. These experiments investigated the effectiveness of cultural control measures in limiting ZYMV spread in pumpkin, and single-gene resistance in commercial cultivars of pumpkin, zucchini and cucumber. Melon aphids (Aphis gossypii) colonised field experiments at Kununurra; migrant green peach aphids (Myzus persicae) visited but did not colonise at Carnarvon and Medina. Cultural control measures that diminished ZYMV spread in pumpkin included manipulation of planting date to avoid exposing young plants to peak aphid vector populations, deploying tall non-host barriers (millet, Pennisetum glaucum) to protect against incoming aphid vectors and planting upwind of infection sources. Clustering of ZYMV-infected pumpkin plants was greater without a 25m wide non-host barrier between the infection source and the pumpkin plants than when one was present, and downwind compared with upwind of an infection source. Host resistance gene zym was effective against ZYMV isolate Knx-1 from Kununurra in five cultivars of cucumber. In zucchini, host resistance gene Zym delayed spread of infection (partial resistance) in 2 of 14 cultivars but otherwise did not diminish final ZYMV incidence. Zucchini cultivars carrying Zym often developed severe fruit symptoms (8/14), and only the two cultivars in which spread was delayed and one that was tolerant produced sufficiently high marketable yields to be recommended when ZYMV epidemics are anticipated. In three pumpkin cultivars with Zym, this gene was effective against isolate Cvn-1 from Carnarvon under low inoculum pressure, but not against isolate Knx-1 under high inoculum pressure, although symptoms were milder and marketable yields greater in them than in cultivars without Zym. These findings allowed additional cultural control recommendations to be added to the existing Integrated Disease Management strategy for ZYMV in vegetable cucurbits in WA, but necessitated modification of its recommendations over deployment of cultivars with resistance genes.     

Complete genomic sequence analysis of a highly virulent isolate revealed a novel strain of <Emphasis Type="Italic">Sugarcane mosaic virus</Emphasis>

Sugarcane mosaic virus (SCMV) is the most prevalent virus causing maize dwarf mosaic disease in northern China. A SCMV isolate, BD8, was obtained from the maize showing dwarf and mosaic symptoms in Baoding, China. The complete genomic sequence of BD8 is 9,576 nucleotides (nt) excluding the poly(A) tail. It contains one single open reading frame of 9,192 nt and encodes a large polyprotein of 3,063 amino acids (aa), flanked by a 5′-untranslated region (UTR) of 148 nt and a 3′-UTR of 236 nt. The entire genomic sequence of BD8 shares identities of 79.1–80.8% with those of other 13 SCMV isolates available in the GenBank at nt level, while their CP genes share identities of 76.9–82.6 and 82.8–86.9% at nt and aa levels, respectively. Phylogenetic analysis of the complete genomic sequences reveals that SCMV can be clustered to four groups: group I includes isolates from maize, group II consists of isolates from sugarcane or maize, groups III and IV contain single isolate of AU-A (AJ278405) and BD8, respectively. Thus, BD8 represents a new strain of SCMV. Furthermore analysis of the CP gene sequences of more isolates shows that BD8 is clustered to a group with the isolates from Thailand and Vietnam, which implies that isolates of this strain have been distributed in South Asia. In the greenhouse, BD8 can cause severe symptoms in all the 12 maize varieties tested with high incidence, indicating that BD8 is highly virulent.     

Nucleotide sequence of a Singapore isolate of zucchini yellow mosaic virus coat protein gene revealed an altered DAG motif

A cDNA clone of zucchini yellow mosaic virus (ZYMV) RNA was mapped to the 3 terminal region. The nucleotide sequence revealed a single open reading frame of 1035 nucleotides followed by a 3 noncoding region of 215 nucleotides. The putative protease cleavage site for the release of coat protein (CP) was deduced to be between Glu-Ser (at amino acid position 66–67), which would result in a protein of 279 amino acids. This non-aphid-transmissible Singapore isolate of ZYMV showed a change of DAG to GAG triplet near the N-terminal of the CP. The CP gene was expressed as a protein fused to the -galactosidase inEscherichia coli and as an unfused protein inSaccharomyces cerevisiae.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X62662.     

Molecular characterisation of a complex mixture of viruses in garlic with mosaic symptoms in China

Summary.  Degenerate primers were used to detect and amplify cDNA of viruses of the genera Carlavirus, Allexivirus and Potyvirus from garlic plants with mosaic symptoms growing in Zhejiang province, China. Plants contained a complex mixture of viruses and strains. Three distinct stains of Garlic latent virus were detected; the most frequent one was completely sequenced and partial sequences were obtained for the other two. The complete sequence (8363 nt) was 76.4% identical to a Korean isolate. Two allexiviruses were detected and completely sequenced. One (8319 nt) was identified as Garlic virus X and comparisons showed that a published Korean isolate (which had 90.2% identical nucleotides) had an N-terminal deletion in the serine-rich ORF4. The other isolate (8451 nt), tentatively named Garlic virus E, appeared to be a new member of the genus. Phylogenetic analyses of the different viral proteins and distinctive conserved sequence motifs within the genus are discussed. This is the first report of allexiviruses from China. Using potyvirus primers, three distinct isolates of Onion yellow dwarf virus and one of Leek yellow stripe virus were detected and the 3′-terminal sequences of their genomes were determined. In a coat protein phylogenetic analysis, the new isolates were most closely related to other published isolates from Japan and China. Accepted June 1, 2001 Received February 20, 2001