Characterization of Distinct Tombusviruses that Cause Diseases of Lettuce and Tomato in the Western United States.

A soilborne disease of lettuce, associated with necrosis and dieback, has been found with increasing frequency in California and Arizona over the last 10 years. An isometric virus, serologically related to Tomato bushy stunt virus (TBSV), was consistently isolated from lettuce plants with these disease symptoms. Back-inoculation to healthy lettuce plants and subsequent reisolation of the virus from symptomatic lettuce leaves suggested that this virus was the causal agent of this disease. A tombusvirus was also associated with a necrosis disease of greenhouse-grown tomatoes in Colorado and New Mexico. Complementary DNA representing the 3' end of viral genomic RNAs recovered from diseased lettuce and tomato plants had identical nucleotide sequences. However, these sequences were divergent (12.2 to 17.1%) from sequences of the previously described strains of TBSV, Petunia asteroid mosaic virus (PAMV), Artichoke mottled crinkle virus, and Carnation Italian ringspot virus. Additional tombusvirus isolates were recovered from diseased lettuce and tomato plants and these were most closely related to the TBSV-cherry strain (synonymous with PAMV) and to Cucumber necrosis virus based on comparison of 3'-end sequences (0.1 to 0.6% and 4.8 to 5.1% divergence, respectively). Western blot analysis revealed that the new tombusvirus isolated from diseased lettuce and tomato plants in the western United States is serologically distinct from previously described tombusvirus species and strains. Based on genomic and serological properties, we propose to classify this virus as a new tombusvirus species and name it Lettuce necrotic stunt virus.

A century of plant virus management in the Salinas valley of California, 'East of Eden'.

The mild climate of the Salinas Valley, CA lends itself well to a diverse agricultural industry. However, the diversity of weeds, crops and insect and fungal vectors also provide favorable conditions for plant virus disease development. This paper considers the incidence and management of several plant viruses that have caused serious epidemics and been significant in the agricultural development of the Salinas Valley during the 20th century. Beet curly top virus (BCTV) almost destroyed the newly established sugarbeet industry soon after its establishment in the 1870s. A combination of resistant varieties, cultural management of beet crops to provide early plant emergence and development, and a highly coordinated beet leafhopper vector scouting and spray programme have achieved adequate control of BCTV. These programmes were first developed by the USDA and still operate. Lettuce mosaic virus was first recognized as causing a serious disease of lettuce crops in the 1930s. The virus is still a threat but it is controlled by a lettuce-free period in December and a seed certification programme that allows only seed lots with less than one infected seed in 30000 to be grown. 'Virus Yellows' is a term used to describe a complex of yellows inducing viruses which affect mainly sugarbeet and lettuce. These viruses include Beet yellows virus and Beet western yellows virus. During the 1950s, the complex caused significant yield losses to susceptible crops in the Salinas Valley. A beet-free period was introduced and is still used for control. The fungus-borne rhizomania disease of sugarbeet caused by Beet necrotic yellow vein virus was first detected in Salinas Valley in 1983. Assumed to have been introduced from Europe, this virus has now become widespread in California wherever beets are grown and crop losses can be as high as 100%. Movement of infested soil and beets accounts for its spread throughout the beet-growing regions of the United States. Control of rhizomania involves several cultural practices, but the use of resistant varieties is the most effective and is necessary where soils are infested. Rhizomania-resistant varieties are now available that perform almost as well as the non-resistant varieties under non-rhizomania conditions. Another soil-borne disease termed lettuce dieback, caused by a tomato bushy stunt-like tombusvirus, has become economically limiting to romaine and leaf lettuce varieties. The virus has no known vector and it seems to be moved through infested soil and water. Heavy rains in the past 4 years have caused flooding of the Salinas River and lettuce fields along the river have been affected severely by dieback. Studies are now in progress to characterize this new virus and identify sources of resistance. Agriculture in the Salinas Valley continues to grow and diversify, driven by demands for 'clean', high quality food by the American public and for export. The major aspects of plant virus control, including crop-free periods, breeding for resistance, elimination of inoculum sources, and vector control will continue to be vital to this expansion. Undoubtedly, the advances in crop production through genetic manipulation and advances in pest management through biological control will eventually become an important part of agricultural improvement.

Particle Lengths of Whitefly-Transmitted Criniviruses.

An improved method for particle length measurement was used for six members of the genus Crinivirus. Particle measurements were conducted with a CCD-72S solid state camera, which was interfaced with a Zeiss EM 109 electron microscope, and analyzed using the analysis 2.1 Image Analysis Software. In comparisons of specimen preparation methods, the leaf dip method is more representative and reproducible than the antibody capture method or preparation from purified virions. Particle length (nm) ranges of whitefly-transmitted criniviruses are: Abutilon yellows virus (AYV), 800 to 850; Cucurbit yellow stunting disorder virus (CYSDV), 750 to 800; Lettuce chlorosis virus (LCV), 800 to 850; Lettuce infectious yellows virus (LIYV), 700 to 750; Tomato chlorosis virus (ToCV), 800 to 850; and Tomato infectious chlorosis virus (TICV), 850 to 900.

Specificity of TAS-ELISA for Beet Necrotic Yellow Vein Virus and Its Application for Determining Rhizomania Resistance in Field-Grown Sugar Beets.

Levels of beet necrotic yellow vein virus (BNYVV), as measured by triple-antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA), were compared with biological evaluations in representative commercial and experimental sugar beet cultivars developed for production in the United States and ranging in their reactions to rhizomania from uniformly susceptible to highly resistant. TAS-ELISA was specific for BNYVV and did not react with related soilborne sugar beet viruses. Differences in absorbance (A405nm) values measured in eight cultivars closely correlated with the dosage and frequency of the Rz allele, which conditions resistance to BNYVV. A diploid (Rzrz) hybrid had a significantly lower absorbance value (less virus) than a similar triploid (Rzrzrz) hybrid. Cultivars that segregated (Rzrz:rzrz) had higher absorbance values than uniformly resistant (Rzrz) hybrids, as was expected. For all cultivars, absorbance values decreased as the season progressed. Absorbance value was significantly positively correlated with rhizomania disease index score and negatively correlated with individual root weight, plot root weight, and sugar yield. This information should be useful in resistance-breeding and -evaluation programs and in the sugar industry when considering cultivar choice, inoculum production, and future crop rotations.

First Report of Tomato Bushy Stunt Virus Isolated from Lettuce.

In recent years a disease causing dieback and necrosis of Romaine and leaf lettuce has become increasingly important in California and incidence is becoming more widespread. This disease has been primarily found in areas where soil has been dredged from a river or in flooded land. Tomato bushy stunt virus (TBSV) isolates have been isolated from roots and leaves of symptomatic lettuce. The particles are isometric with a diameter of 30 nm. Double-stranded RNA (dsRNA) profiles are identical to the tomato and Prunus isolates of TBSV. However, spurs are formed in agar double diffusion tests when antisera to the tomato and Prunus isolates were used. A similar dieback disease of lettuce was observed in several counties of California during the mid-1980s. Symptoms of this disease are very similar to those described for the "brown blight" disease of lettuce reported in the 1920s (1), including severe stunting of plants and extensive chlorosis, mottling, and necrosis of older leaves. Plants infected early in their development may die. Although inoculation under greenhouse conditions has not reproduced the dieback disease in lettuce, TBSV has been consistently isolated from field-grown, symptomatic lettuce. The question of whether this new dieback disease of lettuce is caused only by lettuce isolates of TBSV or if some other viruses are also involved needs further studies. Reference: (1) I. C. Jagger. Phytopathology 30:53, 1940.

Comparison of Diagnostic Techniques for Detecting Tomato Infectious Chlorosis Virus.

A polyclonal antiserum prepared against purified virions of tomato infectious chlorosis virus (TICV) was used to evaluate serological tests for its detection, to determine its distribution in infected plants, to study relationships among isolates of this virus, and to detect it in field samples. A cRNA probe representing TICV RNA 1 and RNA 2 was used in dot blot hybridization tests. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was also developed for detection of TICV isolates. The comparative study of these four techniques indicated that RT-PCR was 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA), Western blot, and dot blot hybridization assays for TICV detection. TICV was detected in leaf, stem, flower, and root tissues of the infected tomato plants. However, the virus was not uniformly distributed throughout the infected tomato plants, and the highest viral concentration was observed in fully developed young tomato leaves at the onset of yellowing symptoms. The virus was detected by indirect ELISA, Western blot, dot blot hybridization, and RT-PCR assays in laboratory-infected tomato, tomatillo, potato, and Nicotiana clevelandii and in naturally infected tomato, petunia, and Ranunculus sp. plants obtained from commercial sources. These tests indicate that there are apparently no detectable serological or nucleic acid differences among four TICV isolates obtained from Orange and Yolo Counties of California or from North Carolina or Italy.

Characterization of the beet yellow stunt virus coat protein gene.

ABSTRACT The beet yellow stunt virus (BYSV) genome contains at least nine open reading frames (ORFs) that code for proteins ranging from 6 to 66 kDa. Based on amino acid sequence comparisons, the coat protein (CP) was previously identified as the product of ORF7. We expressed the product of ORF7 in bacteria and confirmed that ORF7 codes for the BYSV CP by immunoblotting. BYSV is a phloem-limited virus, and virus CP antigen of a quality sufficient for diagnostic antisera production has not been available. To produce BYSV antigen free of plant host contaminants, ORF7 was cloned into a pMAL bacterial expression vector. The resulting fusion protein was affinity-purified and used as an antigen to raise anti-BYSV CP antisera in rabbits and guinea pigs. Using these antisera, an indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA)-based diagnostic system was developed. This indirect DAS-ELISA format enabled reliable detection of BYSV in tissue extracts from virus-infected lettuce diluted up to 5,000 times. The diagnostic system developed may enable large-scale epidemiological studies of BYSV using simple serological techniques. The antisera raised had a titer exceeding 1 x 10(5) in immunoblots and easily detected the 23.7-kDa BYSV CP in virus-infected lettuce and sowthistle plants. In these two plant species, BYSV CP was detected as two closely migrating bands during electrophoresis, which may suggest posttranslational CP modifications. To further characterize the BYSV CP gene, the 5'-untranslated region (UTR) of the BYSV CP subgenomic RNA (sgRNA) was cloned and sequenced. The CP-encoding, approximately 1.9-kb sgRNA has an AT-rich, 66-nucleotide-long 5'-UTR colinear to the genomic sequence upstream of ORF7.

Tomato chlorosis virus: a new whitefly-transmitted, Phloem-limited, bipartite closterovirus of tomato.

ABSTRACT Tomato chlorosis virus (ToCV) is the second whitefly-transmitted, phloem-limited, bipartite closterovirus described infecting tomato. ToCV is distinct from tomato infectious chlorosis virus (TICV), based on lack of serological and nucleic acid cross-reactions and differences in vector specificity. TICV is transmitted only by the greenhouse whitefly (Trialeurodes vaporariorum), whereas ToCV is transmitted by the greenhouse whitefly, the banded-wing whitefly (T. abutilonea), and Bemisia tabaci biotypes A and B (B. argentifolii). Double-stranded (ds) RNA analyses of ToCV show two prominent dsRNAs of approximately 7,800 and 8,200 bp, with several small dsRNAs. Digoxigenin-11-UTP-labeled riboprobes derived from cDNA clones representing portions of RNAs 1 and 2 were used in Northern blot hybridizations to detect two large nonhomologous dsRNAs and a subset of smaller dsRNAs. These probes were used in dot blot hybridizations to detect ToCV in infected tomato. Inclusion bodies and cytoplasmic vesicles were consistently observed in phloem tissues of ToCV-infected Nicotiana clevelandii. Computer-assisted sequence analysis showed significant homology between ToCV clones that hybridize specifically with RNAs 1 and 2 and the lettuce infectious yellows virus methyltransferase of RNA 1 and the HSP70 heat shock protein homolog of RNA 2, respectively. Thus, ToCV is another member of the growing subgroup of bipartite closteroviruses transmitted by whiteflies.

First Report of Lettuce Chlorosis Virus Naturally Infecting Sugar Beets in California.

Sugar beet (Beta vulgaris) plants showing interveinal yellowing and thickened leaves were collected from two fields in Imperial County, CA, for disease assessment in January 1996. Yellowing symptoms were widespread in these fields during the winter of 1995 to 1996. Initial enzyme-linked immunosorbent assays (ELISA) with polyclonal antiserum (ATCC) for beet western yellows virus were consistently negative. Inoculations with Bemisia tabaci "B" biotype (B. argentifolii) whiteflies onto the indicator plants Chenopodium capitatum, C. murale, lettuce (Lactuca sativa), and sugar beet resulted in interveinal yellowing, reddening, and thickened leaves characteristic of whitefly-transmitted closteroviruses (1). Western blot (immunoblot) analyses were performed with antisera to the purified virions of lettuce chlorosis virus (LCV) and lettuce infectious yellows virus (LIYV). Tissue extracts from original beet plants representing two fields and from all subsequent whitefly-inoculated indicator plants consistently showed a single band at ca. 32 kDa, reported to be the molecular mass for LCV capsid protein. Corresponding Western blot analyses for LIYV with the same tissue extracts were negative. No reactions were observed in Western blot assays with tissue extracts from healthy plants. Although sugar beet is a host for LCV as shown by laboratory experiments (1), this is the first report of a natural infection of LCV in sugar beet. Reference: (1) J. E. Duffus et al. Eur. J. Plant Pathol. 102:591, 1996.

A New Report of Rhizomania and Other Furoviruses Infecting Sugar Beet in Minnesota.

Several fields planted in sugar beet (Beta vulgaris) in the Southern Minnesota Beet Sugar Cooperative growing area showed patches of pale greenish yellow foliage and upright leaves characteristic of rhizomania. Other symptoms included reduced root size and root proliferation. Samples taken from these areas during August of 1996 were evaluated for beet necrotic yellow vein virus (BNYVV), the cause of rhizomania, and for other sugar beet furoviruses. BNYVV was identified in 59 of 90 beet samples tested with enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses (molecular mass approximately 22 kDa) with specific (polyclonal antisera donated by K. Richards; monoclonal antisera donated by G. Grassi) and broadly reactive antisera produced at the USDA in Salinas, CA. Recovery by mechanical inoculation of Chenopodium quinoa and Beta macrocarpa confirmed identity. Beet leaves showing symptoms of vein clearing, vein banding, mosaic, and vein necrosis were all identified as being infected with beet soilborne mosaic virus (BSBMV). No systemic leaf symptoms of BNYVV were found in any sample. The BSBMV isolates were identical to one another based on symptomatology of indicator plants and molecular masses in Western blots (approximately 24 kDa), but symptoms were distinct from those of other members of the BSBMV serogroup isolates previously studied from Texas, Idaho, Nebraska, and Colorado. The beet soilborne virus (BSBV) was also recovered by mechanical inoculation and Western blot analysis (antisera donated by R. Koenig) in three samples of field-collected beets. This is a new report of BNYVV, BSBMV, and BSBV in Minnesota. The distribution of rhizomania in infested fields was not isolated to any general area, which indicates that the virus has been present and multiplying in previous sugar beet crops and was not detected. Severity of infection ranged from mild root symptoms with near normal yields and sugar content, to moderate and severe root symptoms with low yields and low sugar content.

Genomic characterization of phenotypic variants of beet curly top virus.

Full-length infectious DNA clones were constructed for four distinct phenotypic variants of beet curly top virus (BCTV). Southern hybridization assays indicated that each cloned BCTV genome shared sequence homology with pBCT-028, a full-length infectious DNA clone of a California isolate of BCTV previously characterized by others. Restriction endonuclease maps of the cloned BCTV genomes were distinct from one another. Infectivity assays determined that plasmids containing tandem repeats of BCTV genomes were generally more infectious than excised linear DNA inserts. Progeny virus, derived from plants inoculated with cloned DNAs, differed in their ability to infect sugarbeet, Beta vulgaris L., and the severity of symptoms produced in B. vulgaris and other experimental hosts.

Unstable infectivity and sedimentable ds-RNA associated with lettuce speckles mottle virus.

Infectivity associated with extracts of lettuce speckles mottle virus (LSMV)-infected tissues is unstable and is susceptible to short periods of aging in vitro or low concentrations of RNase. Infected leaves extracted using phenol yielded greater infectivity than comparable tissue extracted with buffer. No typical virus particles were observed in infected tissues or clarified preparations, but 50- to 70-nm spherical membranous particles were observed associated with the tonoplast in vacuoles of infected cells. Polyacrylamide gel electrophoresis of nucleic acids from infected tissue and LSMV-clarified preparations revealed abundant double-stranded RNA (ds-RNA) in both preparations. A species of LSMV-specific single-stranded RNA (ss-RNA) was also present in LSMV bentonite-clarified preparations. Infectivity was associated only with the ss-RNA fraction. Quantitative isolation of ds-RNA from whole tissue and clarified preparations showed the majority of the ds-RNA was recovered from the bentonite-clarified fractions, and this RNA appeared to be associated with a relatively small structure of a low buoyant density. These data suggest that LSMV lacks a functional coat protein and the sedimentable dsRNA fraction from LSMV-infected tissues more likely contains a viral-specific replication site and not true virus particles.