Mild Mosaic Symptoms Research Articles

Identification and characterization of a novel Tritimovirus species isolated from wild Trisetum flavescens L., family Poaceae

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3' end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).

The potential role of native weed species and bumble bees (Bombus impatiens) on the epidemiology of Pepino mosaic virus

Surveys of native weed species growing in and within 30 m of tomato greenhouses infected with Pepino mosaic virus (PepMV) identified Solanum dulcamara (climbing nightshade), Solanum nigrum (black nightshade), and Solanum ptycanthum (eastern black nightshade) as susceptible host plants. All plants exhibited mild mosaic symptoms and virus was back-inoculated and confirmed in tomato (Lycopersicon esculentum). In transmission tests using both mechanical methods and bumblebees (Bombus impatiens), S. ptycanthum, Solanum sarrachoides (hairy nightshade), and Datura stramonium (jimsonweed) were infected. Weak positive assays by ELISA or DRT-PCR for Amaranthus blitoides (prostrate pigweed), Chenopodium album (lamb’s-quarters), and Plantago lanceolata (narrow-leaved plaintain) were possibly a result of either a hypersensitive localized response to infection or low level virus contamination by bees since the virus could not be recovered by back-inoculation into tomato. Bumblebees exposed to PepMV infected D. stramonium, S. ptycanthum, and S. sarrachoides successfully transmitted the virus back into the tomato. No over wintering perennial weed species were found to be natural hosts of PepMV, and build up of inoculum in the field is unlikely. Weeds do not appear to represent a significant role in the epidemiology of this disease in Ontario.

Putative agents of fig mosaic disease in Turkey

Leaves from 58 fig tree accessions showing different severity of mosaic and necrotic symptoms were collected in 2 different regions of Turkey and analysed by transmission electron microscope (TEM) and RT-PCR to determine putative causal agents of fig mosaic disease (FMD). Ultrastructural studies showed that double membrane-bound bodies (DMBs) were mainly observed in the parenchyma and mesophyll cells of infected leaves of the cultivars Göklop and Morgüz and some seedlings showing only mosaic symptoms. These bodies were surrounded by a fibrillar matrix in most of the infected cells. Long, flexuous rod-shaped virus-like particles (LFPs) were also observed in vascular tissues of the Bursa Siyahı, Sarılop, Sarızeybek, and Yediveren cultivars, as well as seedlings that exhibited mosaic and necrotic symptoms together. Isometric virus-like particles (IVLPs) were mainly observed in mesophyll parenchyma cells of cv. Yeşilgüz, which showed very mild mosaic symptoms. Some phytoplasma-like bodies, as well as mosaic symptoms, were also observed in the phloem tissues of cv. Morgüz, Göklop, and Yediveren. These kinds of particles were not observed in the healthy plant leaves. RT-PCR analyses revealed that a PCR product of 3.0 kbp was obtained from virus-purified RNA of cvs. Bursa Siyahı, and Sarılop and an unknown cultivar (seedling) using degenerate fig-associated primers. When primers specific for FMD agent in Spain were used, a DNA fragment of 750 bp was amplified from cvs. Göklop and Yeşilgüz. However, no product was obtained by using DC-random primers.

A New Pathotype of Pepper mild mottle virus (PMMoV) Overcomes the L4 Resistance Genotype of Pepper Cultivars.

The biological, serological, and molecular characteristics of a newly isolated L4 resistance-breaking isolate of Pepper mild mottle virus (PMMoV) were studied. The new pathotype of PMMoV is closely related to the Israeli pathotypes P1,2 and P1,2,3 of the virus; however, the mosaic symptoms caused by this new pathotype on pepper plants with an L4 genotype were more severe than the mild mosaic symptoms caused by other common pathotypes of the virus in susceptible plants. The predicted amino acid sequence of the putative coat protein (CP) of the newly described pathotype has two amino acid mismatches when compared with the CP of pathotype P1,2, leucine to glutamine at position 47, and alanine to glycine at position 87. The CP of the new pathotype has one amino acid mismatch when compared with P1,2,3, having alanine instead of glycine at position 87. Based on its biological characteristics, the new pathotype was designated P1,2,3,4 of PMMoV-Is. A method is described for the differentiation among the three PMMoV pathotypes using restriction cleavage analysis of reverse-transcription polymerase chain reaction products made from virus-infected plants. An additional unique MnlI site in the CP gene of the newly isolated P1,2,3,4 allows its distinction from the other two isolates, while BglI cleaved only products of the P1,2 pathotype.

First Report of a New Potyvirus, Tricyrtis virus Y, and Lily virus X, a Potexvirus, in Tricyrtis formosana in the United States

Tricyrtis formosana (toad lily) is an herbaceous perennial in the family Liliaceae. Native to Asia, T. formosana is now used in the United States as an ornamental border plant in woodland and shade gardens. A T. formosana var. stolonifera plant showing chlorosis and mild mosaic symptoms obtained from a commercial grower in Columbia County, Oregon tested positive for potyvirus by ELISA using our genus Potyvirus broad spectrum reacting PTY-1 Mab (3). Electron microscopic examination of negatively stained leaf-dip preparations from symptomatic leaves showed a mixture of two sizes of flexuous rod-shaped particles, approximately 700 nm long (resembling potyviruses) and 470 nm long (resembling potexviruses). Total RNA extracts from symptomatic leaves were used in reverse transcription (RT)-PCR assays with potyvirus- or potexvirus-specific primers. The degenerate primers for the genus Potyvirus (2) direct the amplification of approximately 1,600-bp fragments from the 3' terminus of most potyviruses. Overlapping potexvirus cDNA clones were generated using degenerate genus Potexvirus replicase primers, and later, virus-specific primers in 3' RACE (4). The RT-PCR amplified fragments were cloned and sequenced. Analysis of the 1,688 nt potyvirus sequence (GenBank Accession No. AY864850) using BLAST showed highest identity with members of the Bean common mosaic virus (BCMV) subgroup of potyviruses. Pairwise amino acid comparisons of the CP region of the new potyvirus showed 78% identity to strains of Bean common mosaic necrosis virus, 77% identity with Soybean mosaic virus and Ceratobium mosaic virus, 72 to 76% identity to strains of BCMV, and only 50 to 64% identity with 54 other potyviruses. Additionally, similar pairwise analysis of the CP nucleotide sequence and 3'NCR of the new potyvirus generally revealed the same identity trend as described for the CP amino acid sequences, albeit with the highest nucleotide identities at less than 73% for CP and less than 66% for the 3'NCR. These results suggest that this virus is a new species in the genus Potyvirus (1), which we have tentatively named Tricyrtis virus Y (TrVY). BLAST analysis of the 3' terminal 3,010 nt potexvirus sequence (GenBank Accession No. AY864849) showed 89% nucleotide identity with Lily virus X (LVX). Pairwise amino acid comparisons of the putative gene products revealed 98, 95, 94 and 99% identity with LVX TGBp1, TGBp2, TGBp3-like, and CP, respectively, and 97% identity with the 108 nt 3'NCR. Homology with other members of the genus Potexvirus was less than 50% for these corresponding genes and gene products. ELISA and RT-PCR analysis for these two viruses in toad lily plants obtained from a grower in Illinois also revealed the presence of TrVY in three of seven cultivars and LVX coinfecting only one of the plants. The standard propagation method for T. formosana is plant division, which along with mechanical contact, provides efficient means for spread of both viruses. To our knowledge, this is the first description of this potyvirus and the first report of any potyvirus in T. formosana. LVX has been reported in Lilium formosanum, but to our knowledge, this is also the first report of LVX in T. formosana.

First report of Broad bean wilt virus 1 in Slovenia

) cvs Atol,Olimpus, Bjanca and Verbrana, showing virus-like symptoms, were observedin a commercial field in SE Slovenia. Discolorations, rings or dark bumpson fruits resulted in almost a 100% loss of the yield in some areas. Symptomson leaves were expressed as arabesques or concentric rings. In autumn,infected plants expressed less severe symptoms and produced symptomless fruits.Infected leaves and fruits, with various symptoms, were tested by ELISAfor the presence of BBWV 1 (Broad bean wilt virus 1), BBWV 2 (Broadbean wilt virus 2), CSNV (Chrysanthemum stem necrosis virus), CMV(Cucumber mosaic virus ), INSV ( Impatiens necrotic spot virus ) and TSWV(Tomato spotted wilt virus ). Positive results were obtained only for BBWV1 in all samples collected from the epidemic area. Immuno-serological elec-tron microscopy (ISEM) (antibodies from DSMZ, Germany) of crude sapextracts confirmed BBWV 1 infection. RT-PCR using BBWV 1 specificprimers B1/B2 (Rubio et al., 2002) produced an amplicon of approxi-mately the expected size (500 bp) from extracts from infected but nothealthy plants. The sequence of the amplicon was determined (GenBankAccession No. EF405826). BLAST analysis of a 334 bp segment, encom-passing part of the two coat proteins genes, showed approximately 80%nt identity with various BBWV 1 isolates. Amino acid identities rangedfrom 90 to 92% with BBWV 1 isolates and from 54 to 58% with BBWV2 isolates. Sap of plants with symptoms was used for mechanical inocula-tion of test plants. Local and systemic symptoms were observed on pepper,Chenopodium quinoa, Nicotiana rustica, and N. tabacum cv. WhiteBurley. No symptoms were observed on N. clevelandii, N.benthamianaand Lycopersicon esculentum . BBWV 1 was also identified in one out of sixweed species belonging to the family Cichoriaceae, expressing mild mosaicsymptoms and growing near the epidemic area. This is the first report ofBBWV 1 in Slovenia.AcknowledgementsWe thank the Slovenian PARS, D.-E.Lesemann (BBA, Germany), J.Th.J.Verhoeven (PPS, Netherlands), B. Frajman (UL, Slovenia) and N. Petrovi c (NIB, Slovenia) for their help.

First Report of Cucumber mosaic virus in Echium candicans in France.

Echium candicans (Linn.) Herb. Banks (Pride of Madeira or Viper's Bugloss), family Boraginaceae, is a perennial shrub used in gardens for the ornamental quality of its deep blue inflorescences, especially in coastal areas near the Mediterranean Sea. Mosaic symptoms were observed in leaves of E. candicans in the Alpes Maritimes Department of southeastern France, St Jean Cap Ferrat in 1994, Menton in 2002, and Antibes in 2005. Symptoms exhibited in a range of inoculated plants including Nicotiana tabacum cvs. Xanthi and Samsun, Chenopodium quinoa, C. amaranticolor, Vigna unguiculata cv. Black, and Cucumis sativus cv. Poinsett were typical of Cucumber mosaic virus (CMV). Occurrence of CMV in one sample from each of the three localities was confirmed by the observation of isometric particles (approximately 30 nm) with the electron microscope in crude sap preparations from the infected plants, positive reactions in double-antibody sandwich (DAS)-ELISA to polyclonal antibodies raised against CMV (1), and the nonpersistent transmission of the virus from infected Xanthi to virus-free Xanthi plants by Myzus persicae. In double-immunodiffusion analysis, the three isolates were shown to belong to the CMV subgroup II (1,2). To determine if CMV was responsible for the symptoms observed, the isolate from Antibes was multiplied in Xanthi plants after isolation from local lesions on V. unguiculata and mechanically inoculated to 3-year-old plants of E. candicans tested to be free from CMV before the mechanical inoculation. One month after inoculation, mild mosaic symptoms were observed in young leaves and CMV was detected by DAS-ELISA in 10 of 10 inoculated plants. To our knowledge, this is the first report of CMV in E. candicans.

2b ORFs encoded by subgroup IB strains of cucumber mosaic virus induce differential virulence on Nicotiana species

Cucumber mosaic virus (CMV)-encoded 2b protein from subgroup IA or subgroup II was shown to be a determinant of virulence in many solanaceous hosts. In this study, the virulence of 2b proteins from subgroup IB strains was analysed using four intraspecies hybrid viruses, which were generated by precise replacement of the 2b open reading frame (ORF) in subgroup IA strain Fny-CMV with the 2b ORFs of four subgroup IB strains, Cb7-CMV, PGs-CMV, Rad35-CMV and Na-CMV, generating FCb7(2b)-CMV, FPGs(2b)-CMV, FRad35(2b)-CMV and FNa(2b)-CMV, respectively. FCb7(2b)-CMV was more virulent than Fny-CMV, and was similar in phenotype to its parental virus Cb7-CMV on the three Nicotiana species tested. FNa(2b)-CMV also was virulent on these host species, equivalent to Fny-CMV or Na-CMV. However, FRad35(2b)-CMV only caused mild mosaic or undetectable symptoms on all the host species tested, and was less virulent than Fny-CMV or Rad35-CMV. FPGs(2b)-CMV infected all the host species systemically, and induced either mosaic or barely visible symptoms, demonstrating that the inability of PGs-CMV to infect these three Nicotiana species was not due to its 2b protein. The diverse virulence was shown to be mediated by the 2b proteins rather than the C-terminal overlapping parts of the 2a proteins, and was associated with the level of viral progeny RNA accumulation in systemically infected leaves, but not with the rate of long-distance viral movement in host plants. Through analysis of encapsidation of viral RNAs, there was an apparent correlation between the virulence and the high level of encapsidated RNA 2 in virions of Fny-CMV, FCb7(2b)-CMV and FNa(2b)-CMV.

First Report of Cucumber vein yellowing virus in Melon in France.

During the fall of 2003, mild mosaic symptoms were observed in melon (Cucumis melo L.) plants grown in glasshouses near Eyragues (southeastern France) resembling those caused by the Bemisia tabaci transmitted Cucumber vein yellowing virus (CVYV, genus Ipomovirus, family Potyviridae). In addition, large numbers of B. tabaci were observed to be colonizing these crops. The identification of CVYV was established through differential host range reaction, immunosorbent electron microscopy (IEM), and reverse transcription (RT)-PCR experiments. Crude sap from symptomatic leaves was used to inoculate differential host plants. Mild mosaic symptoms were observed on melon, and cucumber developed vein-clearing symptoms typical of CVYV. No symptoms were observed in Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana, N. tabacum, and Vigna sinensis. Numerous, slightly flexuous, elongated virus particles were observed in infected plant extracts; these particles were decorated by a polyclonal antiserum raised against a Sudanese CVYV isolate. To confirm CVYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St. Louis, MO) were obtained from the original symptomatic melon tissues. RT-PCR was carried out using CVYV-specific primers CVYV-CP-5': 5'-GCTTCTGGTTCTCAAGTGGA-3' and CVYV-CP- 3': 5'-GATGCATCAGTTGTCAGATG-3' designed according to the partial sequence of the coat protein gene of CVYV-Isr (GenBank Accession No. AF233429) (2). A 540-bp fragment corresponding to the central region of CVYV coat protein was amplified from total RNA extracted from symptomatic but not from asymptomatic melon tissue. Direct sequencing was done on RT-PCR products (GenBank Accession No. EF441272). The sequence was 95 and 99% identical to that reported for CVYV isolates from Israel and Spain, respectively. CVYV was first described in Israel and has recently emerged as the cause of important diseases in Spain and Portugal (1,3). Shortly after detecting CVYV during 2003, efforts were made to eradicate the virus in susceptible crops. CVYV was not detected again during intensive surveys conducted in southeastern France during 2004, 2005, and 2006, suggesting that the CVYV detected during 2003 resulted from an accidental introduction and that the virus has not become established in France.

Two Amino Acid Substitutions in the Coat Protein of Pepper mild mottle virus Are Responsible for Overcoming the L4 Gene-Mediated Resistance in Capsicum spp.

ABSTRACT The Capsicum spp. L genes (L(1) to L(4)) confer resistance to tobamoviruses. Currently, the L(4) gene from Capsicum chacoense is the most effective resistance gene and has been used widely in breeding programs in Japan which have developed new resistant cultivars against Pepper mild mottle virus (PMMoV). However, in 2004, mild mosaic symptoms began appearing on the leaves of commercial pepper plants in the field which possessed the L(4) resistance gene. Serological and biological assays on Capsicum spp. identified the causal virus strain as a previously unreported pathotype, P(1,2,3,4). PMMoV sequence analysis of the virus and site-directed mutagenesis using a PMMoV-J of the P(1,2) pathotype revealed that two amino acid substitutions in the coat protein, Gln to Arg at position 46 and Gly to Lys at position 85, were responsible for overcoming the L(4) resistance gene.

석량풋콩재배지에서의 동부모자이크바이러스병의 발생도 및 풋콩병해모니터링과 동부모자이크 바이러스의 순화

석량풋콩 품종의 콩밭에서 모자이크 증상이 약하게 나타난 186개의 잎을 채집하여, Cowpea mosaic virus(CPMV)에 대한 ELISA 검정을 하였다. 그 결과 93개의 시료가 양성반응을 보였으며, 이 가운데 55개의 시료는 매우 높은 바이러스 밀도를 나타내고 있었다. 이러한 결과는 석량풋콩 품종의 동부모자이크병은 ELISA 검정, 바이러스 순화, 전자현미경검경 결과와 바이러스 표피 단백질분석 결과 Cowpea mosaic virus에 의한 병해로 확인되었다. One hundred and eighty-six leaves of soybean cv. Seokryangputkong that showed mild mosaic symptoms were collected randomly and ELISA tests were conducted with those leaf samples to screen the presence of Cowpea mosaic virus (CPMV). Ninety-three out of 186 samples reacted positively to CPMV, but those samples did negatively to Soybean mosaic virus (SMV). At least, 55 leaf samples revealed higher values than that of positive control. The results strongly confirmed that CPMV occurred severely in soybean cv. Seokryangputkong. However, a question is raised on the primary reservoir and vector for transmission of this virus. Since the farmer changes seeds every year, seed transmission is excluded. The virus was also purified, the analysis of coat protein conformed the virus of cowpea mosaic virus and UV absorption pattern confirmed that the causal virus of mosaic disease in soybean putkong was cowpea mosaic virus.

SEROLOGICAL AND MOLECULAR PROPERTIES OF FIVE POTYVIRUSES INFECTING CALLA LILY

Calla lily (Zantedeschia spp.), an aroid plant species indigenous to Africa, is a popular ornamental crop in Taiwan and many other countries. Dasheen mosaic virus (DsMV) is the first potyvirus species reported to infect calla lilies. Besides DsMV, several potyviruses with distinct serological and molecular properties were isolated from calla lilies in Taiwan. In 2001, a potyvirus with serological and molecular properties distinct to those of DsMV was recognized and widely distributed in calla lily fields in Taiwan. The virus inducing mosaic symptoms similar to those caused by DsMV was provisionally designated as Zantedeschia mosaic virus (ZaMV). The ZaMV was later identified as a strain of Konjak mosaic virus (KoMV), a potyvirus infecting aroid plants reported from Japan. This was based on the analysis that they shared more than 90% of identity between their coat protein (CP) gene sequences. In 2002, Turnip mosaic virus infecting calla lilies causing yellow spot and stripe foliar symptoms was recognized. TuMV was found frequently occurring in calla lily fields adjacent to cruciferous crops. In addition, TuMV was also detected in calla lily bulbs imported from other countries including USA and Vietnam. The same year, another potyvirus discovered from calla lilies showing mild mosaic symptoms. It was evidently different from those already reported calla lily-infecting potyviruses, and named Zantedeschia mild mosaic virus (ZaMMV) based on its distinct symptoms. ZaMMV was identified coincidentally by two working groups novel molecular properties that a sequence of 38-39 repeated glutamine residues located on the N-terminal of the CP gene. The biological and molecular significance of the unusual glutamine repeats is not known. Finally, Calla lily latent virus (CLLV) was discovered and detected from a calla lily plant dually co-infected with DsMV and KoMV. It is so named for its inability to induce any visible symptoms on calla lilies. This characteristic was confirmed by the consistent observation in field surveys that none of the calla lily plants singly infected by CLLV developed symptoms. Serological and molecular studies reveal that CLLV is a new species of Potyvirus.

Occurrence of Chilli veinal mottle virus in Himalayan butterfly bush (Buddleja crispa)

Buddleja crispa , commonly known as Himalayan butterfly bush, grows in open rocky places. It is a deciduous or semi-evergreen, ornamental, perennial shrub with fuzzy white leaves on long, arching stems, bearing tiny pink, violet or purple flowers. The flowers are often fragrant, attractive and irresistible to butterflies, bees and other nectar-feeding birds and insects. Buddleja davidii , an allied species of B. crispa , has been reported to be a host of Tomato ringspot virus (Hughes & Scott, 2003), Alfalfa mosaic virus (Walter et al ., 1985) and Cucumber mosaic virus (Perkins, 1991). In the course of a study to assess virus incidence in natural plant resources of high-altitude areas in the western Himalayan region of India, some leaves of B. crispa with mild chlorosis and mosaic symptoms were collected from the Kinnaur area (altitude 2500 m asl) of Himachal Pradesh during 2003. The infected leaves were tested by ELISA for the presence of Tomato ringspot virus (antibody kit supplied by DSMZ, Braunschweig, Germany), Cucumber mosaic virus and potyviruses (antibodies from Agdia, Elkhart, USA). A positive result was obtained with the potyvirus group-specific antibodies. To confirm the identity of the virus detected, leaf samples were tested using a universal potyvirus primer pair (P9502 and CPUP), which amplify part of the coat-protein gene and 3 ′ - UTR of the viral genome (Van der Vlugt et al ., 1999). An amplification product of 800 bp was obtained after RT–PCR. The DNA product was cloned, sequenced and submitted to the EMBL Database (accession number AJ889836). The sequence showed 95% homology to Chilli vein-banding mottle virus, a strain of Chilli veinal mottle virus reported from Japan (GenBank accession number AB012221). This is a new report of this potyvirus occurring on B. crispa growing in nature, therefore this plant should be taken into account as a potential reservoir of Chilli veinal mottle virus infecting chilli crops.

Occurrence of Three Strains of Cucumber mosaic virus Affecting Tomato in Kuwait

Three strains of Cucumber mosaic virus (CMV) have been found to cause a lethal disease, referred to as fern leaf syndromes and mild mosaic symptoms in tomato (Lycopersicon esculentum Mill.) crops grown in Kuwait. CMV strains were detected and identified based on host range, symptomatology, serology, electron microscopy, and ribonucleic acid (RNA) electrophoresis in polyacrylamide gels. A high degree of viral genomic heterogeneity was detected among CMV strains isolated in Kuwait, with no apparent correlation to symptomatology in tomato host plants. Two different virus satellites of 'CMV associated RNA 5', designated CARNA 5, were detected in two virus strains that caused both lethal disease and mild symptoms, designated CMV-D1 and CMV-S1 respectively. CARNA5 was not detected in the third CMV strain that caused fern leaf syndromes designated CMV-F. All the three isolated strains were serologically indistinguishable from each other and may belong to one serotype according to Ouchterlony gel diffusion tests. These strains transmitted via aphids (Myzus persicae Sulz) in a non-persistent manner. Physical properties of the virus strains were very similar where thermal inactivation test showed that virus withstood heating for 10 min at <TEX>$70^{/circ}$</TEX>, dilution end point was <TEX>$10^{-4}$</TEX>, and the longevity in vitro at room temperature was less than 5 days for all virus strains. CMV-D1 and CMV-F were the most devastating diseases spreading in both greenhouse and field-grown tomato where aborted flower buds failed on fruit setting due to the viral infection. This is the first report to isolate three different strains of CMV in Kuwait.

Biological characteristics and nucleotide sequences of three Korean isolates of Zucchini yellow mosaic virus

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important viruses of cucurbit crops, causing severe mosaic, necrosis, and malformation. Three ZYMV isolates were obtained from pumpkins at Andong (ZYMV-PA), Euiryung (ZYMV-PE), and Suwon (ZYMV-PS), and their biological variability was determined on different hosts, including cucurbit crops as well as other indicator plants. ZYMV-PA caused the most severe symptoms, including severe mosaic, size reduction, and deformation, in oriental melon (Cucumis melo) and cucumber (Cucumis sativus) leaves. In contrast, ZYMV-PE and ZYMV-PS caused mild mosaic symptoms on oriental melon and cucumber. The nucleotide sequences of the genomic RNAs were determined and compared to the sequences of other potyviruses, including ZYMV isolates Reunion Island and TW-TN3. Each ZYMV Isolate had a genome of 9593 nucleotides, excluding the poly(A) tail, and contained 139 and 214 nucleotides in the 5′- and 3′-untranslated regions, respectively. Each had one large open reading frame encoding a protein of about 351 kDa. The nucleotide sequences of ZYMV-PA, ZYMV-PE, and ZYMV-PS were more than 96.0% and the deduced amino acid sequences were more than 98.1% identical. When compared with other ZYMV isolates in a phylogenetic analysis, these three viruses formed a distinct virus clade and were more distantly related to other potyviruses (43.5%–62.8% identity).

Natural infection of maize by Pennisetum mosaic virus in China

In mid-2002, a potyvirus was isolated from maize ( Zea mays ) showing mild mosaic symptoms in the Xinzhou District of Shanxi Province, China. This isolate was mechanically transmitted to maize (cultivar Ye-Dan no. 2) and Sorghum bicolour (four cultivars: Atlas, Xin-Liang nos 7 and 52, and Xiong-Yue 191). All inoculated plants developed systemic mosaic symptoms. In serological tests, the virus was distantly related to Sugarcane mosaic virus (SCMV), the prevalent potyvirus infecting maize across China (Fan et al ., 2003a), but closely related to the newly identified Pennisetum mosaic virus (PenMV) isolated from a perennial grass ( Pennisetum centrasiaticum ) (Fan et al ., 2003b). The nucleotide sequence of the putative coat protein (CP) gene of the virus isolated has been determined (accession number AY543166), comprising 909 nucleotides and encoding 302 amino acid residues. Sequence comparisons amongst all potyviruses reported to date showed that this isolate was most closely related to PenMV (accession number AY172336) (Fan et al ., 2003b). The sequence identity between the putative CP genes of the maize isolate and PenMV was 92%, while between the CPs it was 99% at the amino acid level, thus identifying the virus isolated from maize as PenMV. This is the first report of PenMV in maize, where before SCMV had been the only potyvirus infecting maize in China. SCMV occurs in most maize-growing areas of China, and its symptoms, on most cultivars of sorghum, are mosaic followed by reddening and necrosis of the lamina. In contrast, the distribution of PenMV appears limited, as maize from only one region of China has been found to be infected, and the symptoms it caused, especially on sorghum, were significantly milder when compared with those of SCMV.

Black and Hairy Nightshade, the First Weedy Hosts of Potato virus A in the United States

Potato virus A (PVA; genus Potyvirus, family Potyviridae) occurs wherever potatoes are grown and may reduce tuber yields as much as 40%. Its host range consists of six experimental hosts (Lycopersicon pimpinellifolium (Jusl.) P. Mill., Nicandra physalodes (L.) Gaertn., Nicotiana tabacum L., Solanum demissum Lindl., S. demissum × S. tuberosum, and Nicotiana debneyi Domin.) and two natural hosts (S. tuberosum L. and S. betaceae (Cav.) Sendt.) (1). N. physalodes and Nicotiana tabacum sometimes grow in nature in the eastern and southern United States (2) but have not been reported to be PVA sources in nature. PVA is known to survive winter in temperate climates only in tubers used for potato propagation and potato volunteers. We detected PVA in all six Black Nightshade (S. nigrum L.) and all six Hairy Nightshade (S. sarrachoides Sendt.) weeds expressing mild mosaic symptoms and growing in PVA-infected potato fields near Prosser, WA in September of 2002. PVA was not detected in any of the 24 Cutleaf Nightshade (S. trifolium) plants selected from the same fields. The virus was detected using double antibody sandwich enzyme-linked immunosorbent assay with a test kit obtained from Agdia, Inc. (Elkhart, IN) and by reverse transcription-polymerase chain reaction using primers designated 3 (gta-ctg-aac-tgg-aaa-agt-act) and 4 (ccc-tga-cag-ttg-aaa-cat-aag) (3). One positive control for these assays was a PVA virus isolate from the "Shultz Collection" provided by Robert Goth (USDA, ARS, Beltsville, MD) in 1986. The Agdia, Inc. test kit also provided a positive control. We also transmitted PVA from one of the Black Nightshade and one of the Hairy Nightshade weeds to greenhouse-produced Nicotiana megalosiphon Van Heurck & Mull. Agr., Nicotiana tabacum cv. Samsum NN, S. tuberosum cv. Russet Burbank, S. sarrachoides, and S. nigrum plants by Myzus persicae Sulzer in a stylet-borne manner. The virus was transmitted from the greenhouse test plants back to S. sarrachoides and S. nigrum plants where it produced mild mosaic symptoms identical to virus from the original sources. PVA was not transmitted from weedy sources to Datura tatula L., Physalis floridana Rydb., or S. trifolium. M. persicae readily colonized S. sarrachoides, S. nigrum, and S. trifolium in the field and greenhouse. Since black and hairy nightshade are frequently found in potato fields (4) and our findings show these weeds to be hosts of PVA, their role in the epidemiology of this disease should be more fully evaluated.

First Report of Bean yellow mosaic virus (Pea Mosaic Strain) in Verbena × hybrida.

Verbena × hybrida is an ornamental annual used in rock gardens as an edging plant and hanging baskets. It comes in a variety of colors and grows approximately 1.5 to 2.5 cm (6 to 10 inches) high. In the spring of 2002, verbena cv. Lavender Shades plants from California showing leaf mosaic symptoms tested positive for potyvirus using an antigen-coated plate enzyme-linked immunosorbent assay with our genus Potyvirus broad spectrum reacting PTY-1 monoclonal as the detecting antibody (3). The virus was transmitted mechanically to Nicotiana benthamiana by sap inoculation from infected verbena plants. Infected tobacco showed systemic mild mosaic symptoms. Total RNA extractions from infected verbena and tobacco leaves were used in reverse transcription-polymerase chain reaction (RT-PCR) assays with generic potyvirus-specific primers that amplify highly conserved 700-bp or 1,600-bp fragments from the 3' terminus of most potyviruses. This region includes the 3' noncoding region (3'NCR) and the potyviral coat protein (CP). The PCR-amplified fragments were cloned by using standard TA cloning procedures and sequenced using dye-terminator chemistry. The cloned nucleotide and putative coat protein amino acid sequences from the infected verbena and tobacco plants were compared with the corresponding regions of other potyviruses. Amino acid comparison of the CP region of the verbena po-tyvirus showed 95 to 96% identity to four pea mosaic strains (PMV) of Bean yellow mosaic virus (BYMV), 85 to 89% identity to 20 other strains of BYMV, 74 to 76% identity with six strains of Clover yellow vein virus (CYVV), and only 50 to 64% identity with 28 other potyviruses. Pairwise comparisons among and between the CP sequences of PMV, BYMV, CYVV, and other potyviruses revealed identities of 92 to 99% for BYMV∷ BYMV, PMV∷PMV, and CYVV∷CYVV; 84 to 89% for BYMV∷ PMV, 69 to 78% for BYMV∷CYVV and PMV∷CYVV, and 50 to 64% for all other potyvirus combinations. Additionally, similar pairwise analysis of the 3'NCR of the verbena potyvirus revealed 98 to 99% identity to PMV strains, 81 to 94% to other BYMVs, 68 to 75% to CYVVs, and 52 to 64% with other potyviruses. Other 3'NCR pairwise comparisons generally revealed the same identity trend as described for the CP. Further serological analysis with our panel of BYMV-specific, BYMV-subgroup, and potyvirus cross-reactive monoclonal antibodies (3) confirmed the designation of the verbena potyvirus isolate as a pea mosaic strain of BYMV. To our knowledge this is the first confirmed report of BYMV-pea mosaic strain in Verbena (1,2).

Symptom Variances in Mixed Infections of Six Turnip mosaic virus and One Ribgrass mosaic vims Isolates in Crucifers

Turnip mosaic Potyvirus (TuMV) and Ribgrass mosaic Tobamovirus (RMV) are major viruses infecting crucifer crops in Korea. RMV-FG22 was isolated from oriental cabbage. TuMV isolates were TuMV-CA7 from oriental cabbage, TuMV-TU and TuMV-TU2 from turnip, TuMV-RA from rape, TuMV-ST from stock, and TuMV-R9 from radish. The six isolates of TuMV were classified by symptom expression in inbred lines of crucifers. TuMV-CA7 and TuMV-TU isolates infected mostly oriental cabbages; TuMV-ST, TuMV-TU2, and TuMV-R9 infected radishes; and TuMV-RA infected both oriental cabbages and radishes. Crops used in six combinations of mixed infections were 'Tambok' cultivar resistant to TuMV,'SSD63' susceptible inbred line of oriental cabbage, pure line of leaf mustard, and‘Daeburyungyeorum’cultivar of radish. External symptoms in 'Tambok' and radish by each of the six single infections of TuMV showed similar results by bioassay. Synergistic response of necrotic death occurred within 1 week after inoculation in all combinations mixed with TuMV and RMV-FG22 on leaf mustard. In oriental cabbage 'SSD63' , synergism of necrosis occurred in four TuMV isolates, but not in TuMV-ST and TuMV-R9. In oriental cabbage 'Tambok' , synergism was expressed only in two combinations of RMV-FG22+TuMV-CA7 and RMV-FG22+TuV-TU, but other combinations had the same symptoms produced by RM-FG22. In radish‘Daeburyungyeorum’, only mild mosaic symptoms were induced by combinations of RMV-FG22+TuMV-CA7, RMV-FG22+TuMV-TU, RMV-FG22+TuMV-RA, and RMV-FG22+TuMV-R9. Mosaic and severe mosaic were induced in combinations of RMV-FG22 +TuMV-TU2 and RMV-FG22+TuMV-ST, respectively.

First Report of Peach latent mosaic viroid on Peach in Uruguay.

Peach latent mosaic viroid (PLMVd) (2) is widely distributed and causes yellow, chlorotic mosaics and delayed foliation, flowering, and ripening. Infected fruits display a cracked suture and are often dented, misshapen, frequently flattened, and discolored. In the greenhouse, PLMVd natural isolates are divided into severe or latent strains depending on whether they induce leaf symptoms on seedlings of the peach indicator GF-305. PLMVd was detected in 2001 during a survey in three locations in the Canelones Department, the main peach producing area in Uruguay. Fifty samples were tested for the presence of five viruses: Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern virus (APLPV), Plum pox virus (PPV) and Apple chlorotic leaf spot virus (ACLSV); samples were also tested for the viroids affecting stone fruits, Hop stunt viroid (HSVd) and PLMVd. The analyses were completed with molecular hybridization using specific nonisotopic riboprobes for each virus (4). PLMVd, undescribed in Uruguay, was detected in 9 of 50 samples in three peach cultivars, Scarlet Pearl, EarliGrande, and Barcelo. The PLMVd-positive sample for 'Scarlet Pearl' showed mild mosaic symptoms on leaves whereas the two PLMVd-positives of 'EarliGrande' showed clear calico type symptoms. The remaining PLMVd-positive samples belonged to 'Barcelo' and showed no symptoms or mild chlorosis. The first two cultivars were imported from the United States, a source with a high percentage of PLMVd infections in peach germ plasm (1). In five of nine PLMVd-positive samples, the viroid occurred with PNRSV and in one with PDV and PNRSV. PLMVd has previously been reported in Brazil (3), but to our knowledge, this is the first report of PLMVd in Uruguay. These results reveal the importance of following strict sanitary practices with plant material used for propagation. Molecular tools are available to prescreen scion and rootstock sources for PLMVd.