Cloning and Detection of DNA from a Nonculturable Plant Pathogenic Mycoplasma-like Organism.

The ability to detect, quantify, and differentiate nonculturable mycoplasma-like organisms (MLOs) would greatly facilitate epidemiological and taxonomical studies of this unique group of plant and insect pathogens. DNA isolated from extracts of insects infected with the Western X-disease MLO was cloned in Escherichia coli. X-disease-specific clones, when labeled and used as probes, readily detected X-disease MLOs in infected plants and insects but did not hybridize with DNA from healthy plants or insects, or from several other plant pathogenic MLOs or spiroplasmas. These methods provide both a sensitive diagnostic tool and a basis for genetically differentiating MLOs.

Effects of citrus and avocado irrigation and nitrogen-form soil amendment on host selection by adult Homalodisca vitripennis (Hemiptera: Cicadellidae).

Host plant water status is thought to influence dispersal of the xylophagous leafhopper Homalodisca vitripennis Germar, especially where plants are grown under high evaporative demand. Preference by adult H. vitripennis for plants grown under different water deficit and nitrogen form fertilization regimens was studied under laboratory conditions. Leafhopper abundance and ovipositional preference were studied on potted 'Washington navel' orange and 'Haas' avocado in cage choice tests, and feeding rate was estimated using excreta produced by insects confined on plants. A similar study compared responses to citrus treated with 1:1 and 26:1 ratios of fertigated nitrate-N to ammonium-N. The insects were more abundant, oviposited, and fed significantly more on surplus-irrigated plants than on plants under moderate continuous deficit irrigation except avocado feeding, which was nearly significant. Plants exposed to drought became less preferred after 3 and 7 d in avocado and citrus, respectively. Citrus xylem fluid tension at this point was estimated at 0.93 MPa. A corresponding pattern of decline in feeding rate was observed on citrus, but on avocado, feeding rate was low overall and not statistically different between treatments. No statistical differences in abundance, oviposition, or feeding were detected on citrus fertigated with 26:1 or 1:1 ratios of nitrate-N to ammonium-N. Feeding occurred diurnally on both plant species. Discussion is provided on the potential deployment of regulated deficit irrigation to manage H. vitripennis movement as part of a multitactic effort to minimize the risk of disease outbreaks from Xylella fastidiosa Wells et al. in southern California agriculture.

Draft Genome Sequence of Xylella fastidiosa subsp. fastidiosa Strain Stag's Leap.

ITALIC! Xylella fastidiosasubsp. ITALIC! fastidiosacauses Pierce's disease of grapevine. Presented here is the draft genome sequence of the Stag's Leap strain, previously used in pathogenicity/virulence assays to evaluate grapevine germplasm bearing Pierce's disease resistance and a phenotypic assessment of knockout mutants to determine gene function.

Genetic and Phenotypic Variation of the Pepper golden mosaic virus Complex.

ABSTRACT Three isolates of the bipartite begomovirus Pepper golden mosaic virus (PepGMV) were characterized for genomic and biological properties. The complete nucleotide sequences of the DNA-A and DNA-B components were determined from infectious clones of PepGMV-Serrano (PepGMV-Ser), PepGMV-Mosaic (PepGMV-Mo), and PepGMV-Distortion (PepGMV-D). Nucleotide sequence identity among PepGMV components ranged from 91 to 96% for DNA-A and from 84 to 99% for DNA-B, with each PepGMV component most closely related to the corresponding component of Cabbage leaf curl virus (CaLCV). However, phylogenetic relationships among begomovirus components were incongruent because DNA-A of PepGMV and CaLCV share an inferred evolutionary history distinct from that of DNA-B. The cloned components of PepGMV-Ser, -Mo, and -D were infectious by biolistic inoculation to pepper but differed in symptom expression: PepGMV-Ser exhibited a bright golden mosaic, PepGMV-Mo produced a yellow-green mosaic, and PepGMV-D caused only a mild mosaic and foliar distortion followed by a "recovery" phenotype in which leaves developing after initial symptom expression appeared normal. Differences in symptoms also were observed on tomato, tobacco, and Datura stramonium. Progeny virus derived from clones of PepGMV-Ser and -Mo were transmitted from pepper to pepper by the B biotype of Bemisia tabaci; progeny virus derived from PepGMV-D clones was not transmissible by the B biotype. Reassortant genomes derived from heterologous DNA components of the three isolates were infectious in all possible pairwise combinations, with symptom phenotype in pepper determined by the DNA-B component. Collectively, these results indicate that the three virus isolates examined may be considered distinct strains of PepGMV that have the capacity to exchange genetic material.

Complete nucleotide sequence of the hypervirulent CFH strain of beet curly top virus.

The complete nucleotide sequence of the hypervirulent CFH strain of beet curly top geminivirus (BCTV) has been determined. The circular DNA genome of BCTV-CFH consists of 2,927 nucleotides and shares extensive sequence homology with the biologically distinct California strain of BCTV. Analysis of the CFH nucleotide sequence indicated that the rightward open reading frames (ORFs) R1, R2, and R3 are highly conserved (> 95% amino acid chemical similarity) in the CFH and California strains, although CFH ORF R2 was extended by 24 carboxy-terminal amino acid residues not present in the California strain. The CFH leftward ORFs L1, L2, and L3 shared varying levels of amino acid chemical similarity with the corresponding ORFs of the California strain (78.8, 66.5, and 86.7%, respectively). CFH ORF L4 was the least conserved ORF present in both strains, encoding a 9.9-kDa protein of 87 amino acid residues, which shares 57.6% chemical similarity with 85 carboxy-terminal amino acid residues of the 19.4-kDa ORF L4 of the California strain. The CFH DNA sequence also contained a unique 12.5-kDa ORF (R4); however, there is no evidence to suggest that R4 is expressed. Comparison of the CFH and California strain nucleotide sequences indicates that certain regions of the BCTV genome have diverged, and this divergence may account for differences in the pathogenic properties of the two strains.

Replication Specificity Elements of the Worland Strain of Beet Curly Top Virus Are Compatible with Those of the CFH Strain But Not Those of the Cal/Logan Strain.

ABSTRACT Cloned genomes of the CFH, Worland, and Cal/Logan strains of beet curly top virus (BCTV) served as helper viruses to trans-replicate defective (D) DNAs that are incapable of self-replication due to deletions within the C1 open reading frame encoding the replication initiator (Rep) protein. The Logan Rep protein could trans-replicate a Logan-derived D DNA in a transient replication assay conducted in Nicotiana benthamiana leaf disks. However, the Logan Rep protein was unable to trans-replicate D DNAs derived from the CFH or Worland strains. In contrast, the Rep proteins of the CFH and Worland strains could trans-replicate CFH or Worland D DNAs, but not a Logan D DNA. These results indicate that the cis- and trans-acting replication specificity elements of the CFH and Worland strains are compatible and that the three strains of BCTV may be divided into two groupings based upon replication specificity determinants. A comparison of amino acid sequences of the Rep protein for the three BCTV strains suggests that the trans-acting replication specificity element may reside in one or more of 12 amino acid residues that are identical; in two amino acid residues that are chemically similar among the CFH and Worland Rep proteins, yet are different in the Logan Rep protein; or in both. Properties including replication specificity, nucleotide sequence identity, and symptom expression were used as criteria to propose separate species designations for each of the three BCTV strains. In this proposal, the Cal/ Logan strain retains the name BCTV, CFH and the closely related Iranian isolate are designated beet severe curly top virus, and Worland is designated beet mild curly top virus.

Genotypic diversity of beet curly top virus populations in the Western United States.

ABSTRACT The genotypic diversity of beet curly top virus (BCTV) present in the western United States has been examined by the analysis of 58 field isolates and eight laboratory or nursery isolates of the virus. Full-length clones for each isolate have been characterized for genotype by restriction endonuclease mapping. The results indicate that most of the genotypes examined may be classified as variants of the CFH, Worland, or Cal/Logan strains of BCTV. Two genotypes were recovered that appear to share certain genotypic markers of both Worland and CFH strains. Genotypic variants of the CFH and Worland strains and the two genotypes sharing markers of both strains were recovered from field isolates collected during 1994 and 1995. In contrast, the Cal/Logan strain was recovered only from isolates maintained in laboratories or nurseries. Comparisons of restriction endonuclease maps of cloned BCTV genomes revealed considerable variability both within and between strains. Although a total of 43 distinct genotypes of BCTV were identified, only 36 (84%) were recovered from field isolates. Of 37 field isolates for which more than a single clone was recovered, 16 (43%) contained more than a single genotype of one strain, whereas 4 (11%) harbored mixed infections of the CFH and Worland strains. A phylogenetic analysis using 43 characters derived from restriction endonuclease mapping data supported the grouping of 41 genotypes into three taxa consistent with the three currently recognized strains of BCTV. The relationships of the two genotypes sharing genotypic markers of both the Worland and CFH strains to other BCTV genotypes was unresolved in the phylogenetic analysis. Based on the mild symptom phenotype of the isolates from which these two genotypes were recovered and the presence of Worland genotypic markers in portions of the genome containing both cis- and trans-acting elements determining replication specificity, these two genotypes were tentatively considered as variants of the Worland strain.

Phylogenetic relationships within the family potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus.

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5' leader and 149-nt 3'-untranslated region and is polyadenylated at the 3' end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5'-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3'). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus "Ipomovirus." In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.

Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus.

ABSTRACT Infectious RNA of wheat streak mosaic virus (WSMV) has been produced using a full-length cDNA clone as a template for in vitro transcription with SP6 RNA polymerase. Infectivity was dependent on the use of template plasmid DNA that had not undergone spontaneous rearrangement during amplification in Escherichia coli. The presence of WSMV in systemically infected wheat plants inoculated with in vitro transcripts was confirmed by reverse-transcription polymerase chain reaction of the WSMV P3 gene and by accumulation of WSMV coat protein as detected by immunoblotting. Maintenance of the full-length WSMV cDNA in the high copy number plasmid pUC18 was problematic because of spontaneous rearrangement of WSMV sequences during growth in liquid media for more than 8 h or if the clone was subcultured. Stability of the WSMV cDNA clone was improved by the use of the low copy number plasmid pACYC177, and it could be grown in large scale volumes (up to 1 liter) of liquid culture for 14 h without noticeable rearrangements. Both the original WSMV culture and the progeny virus derived from infectious in vitro transcripts were efficiently transmitted by the natural eriophyid mite vector Aceria tosichella. This is the first report of infectious in vitro transcripts for any eriophyid mite-transmitted plant virus and represents the only monopartite member of the family Potyviridae infecting monocotyledonous hosts for which infectious in vitro transcripts are available.

Chino del tomate virus:Relationships to Other Begomoviruses and Identification of A-Component Variants that Affect Symptom Expression.

Phylogenetic and distance analyses place Chino del tomate virus (CdTV) in the New World clade of begomoviruses and indicate that CdTV and Tomato leaf crumple virus (TLCrV) are closely related strains of the same virus. One cloned CdTV A component (pCdTV-H6), when inoculated to tomato with the B component (pCdTV-B52), produced mild symptoms and low DNA titers. Another cloned CdTV A component (pCdTV-H8), when coinoculated to tomato with the B component, produced moderate leaf curling and veinal chlorosis similar to that of TLCrV. Coinoculation of both CdTV A components and the B component to tomato produced wild-type chino del tomate (CdT) disease symptoms consisting of severe leaf curling, veinal and interveinal chlorosis, and stunting. The two CdTV A components were nearly identical, except at nucleotide positions 1,722 and 2,324. The polymorphism at nucleotide 1,722 resulted in a change at Rep amino acid 261. The second polymorphism at nucleotide 2,324 resulted in changes at Rep amino acid 60 and AC4 amino acid 10. Two chimeric A components constructed by reciprocal exchange of a fragment bearing the polymorphic site at nucleotide 1,722 were evaluated for symptom phenotype. One chimeric A component (pCdTV-H86) produced wild-type CdT symptoms when coinoculated to tomato with the B component. The reciprocal chimeric A component (pCdTV-H68), when coin-oculated to tomato with the B component, also produced severe leaf curling, veinal chlorosis, and stunting. However, pCdTV-H68 induced less obvious interveinal chlorosis than wild-type or pCdTV-H86. Examination of A component genotypes recovered from tomato coinoculated with pCdTV-H6 and pCdTV-H8 indicated that recombination occurred to produce a genotype identical to pCdTV-H86. These results indicate that subtle genotypic variation has significant effects on symptom expression and may explain phenotypic differences observed among isolates and cloned DNAs of CdTV and TLCrV.

Biotic, molecular, and phylogenetic characterization of bean calico mosaic virus, a distinct begomovirus species with affiliation in the squash leaf curl virus cluster.

ABSTRACT Bean calico mosaic virus (BCMoV), a whitefly-transmitted geminivirus from Sonora, Mexico, was purified, and the genome components were cloned and sequenced. Purified viral fractions and cloned genome components were infectious by biolistic inoculation to bean, completing Koch's postulates for both. The B biotype of the whitefly Bemisia tabaci efficiently transmitted both native virus and progeny virus derived from cloned DNA inoculum. Host ranges of native virus and of progeny virus derived from cloned DNA were identical based upon whitefly and biolistic mediated transmission, respectively. BCMoV has a relatively wide experimental host range among begomoviruses known to infect bean, encompassing genera and species within the Fabaceae, Malvaceae, and Solanaceae. BCMoV has a bipartite genome, as do other New World begomoviruses. BCMoV DNA-A shared highest nucleotide sequence identities with squash leaf curl virus-E strain (SLCV-E) and cabbage leaf curl virus (CaLCV) at 80.1 and 80.7%, respectively. BCMoV DNA-B shared highest nucleotide sequence identity with SLCV-E at 70.7%. The common region (CR) sequences of BCMoV and SLCV-E are 73 to 76% identical; however, modular cis-acting elements within the CR involved in replication origin function and recognition are 100% conserved. Phy-logenetic analysis indicated that BCMoV DNA-A shares a most recent common ancestor with the DNA-A of two viruses that also occur in the Sonoran Desert, SLCV-E and Texas pepper virus (TPV-TAM), and CaLCV from Florida. In contrast, a phylogenetic analysis indicated that BCMoV DNA-B shares a most recent common ancestor with SLCV-E; whereas DNA-B of CaLCV clustered in a separate clade with pepper hausteco virus. Collectively, biological and molecular characteristics indicate that BCMoV is a distinct begomovirus species with the northernmost distribution of any begomovirus isolated from bean in the Americas. Furthermore, the phylogenetic relationships of begomovirus cognate components are not necessarily identical, suggesting that DNA-A and DNA-B of some begomoviruses may have different evolutionary histories.

Substitution of conserved cysteine residues in wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite.

Substitutions in the amino-proximal region of wheat streak mosaic virus (WSMV) HC-Pro were evaluated for effects on transmission by the wheat curl mite (Aceria tosichella Keifer). Alanine substitution at cysteine residues 16, 46 and 49 abolished vector transmission. Although alanine substitution at Cys(20) had no effect, substitution with arginine reduced vector transmission efficiency. Random substitutions at other positions (Lys(7) to Asn, Asn(19) to Ile, and Arg(45) to Lys) did not affect vector transmission. These results suggest that a zinc-finger-like motif (His(13)-X2-Cys(16)-X29-Cys(46)-X2-Cys(49)) in WSMV HC-Pro is essential for vector transmission.

Genome sequences of Agropyron mosaic virus and Hordeum mosaic virus support reciprocal monophyly of the genera Potyvirus and Rymovirus in the family Potyviridae.

Assignment of mite-transmitted species to the genus Rymovirus (family Potyviridae) has changed several times, and the status of the genus has been questioned. To address this issue, complete genome sequences of the rymoviruses Agropyron mosaic virus (AgMV) and Hordeum mosaic virus (HoMV) were determined. AgMV (9540 nucleotides) and HoMV (9463 nucleotides) each encode a single polyprotein with proteinase cleavage sites demarcating protein products characteristic of monopartite species of the family Potyviridae. Of the described species of Potyviridae, AgMV and HoMV are most closely related to each other (68.5% nucleotide and 71.6% amino acid sequence identity) and equidistant (about 53% nucleotide and about 49% amino acid sequence identity) from a third rymovirus, Ryegrass mosaic virus (RGMV). Phylogenetic analyses by neighbor joining, maximum parsimony, and Bayesian inference each grouped the three Rymovirus species in an exclusive clade distinct from a clade containing 34 species of the genus Potyvirus. Because AgMV, HoMV, and RGMV share a reciprocal monophyletic relationship with species of the genus Potyvirus and are divergent in sequence and type of vector, the genus Rymovirus should be retained as a taxonomic unit within the family Potyviridae.

Strain-specific mobilization and amplification of a transgenic defective-interfering DNA of the geminivirus beet curly top virus.

Transgenic Nicotiana benthamiana plants have been constructed which bear integrated, tandemly repeated copies of a beet curly top virus (BCTV) defective-interfering (DI) DNA derived from the Logan strain. Transgenic DI-DNA plant lines challenge-inoculated with BCTV-Logan exhibited delayed and attenuated symptoms compared to nontransgenic plants. Infection of transgenic plants with the Logan strain resulted in the mobilization of the integrated DI-DNA sequence, which was subsequently amplified as an episome. The accumulation of Logan helper virus DNA forms was reduced in transgenic plants, relative to nontransgenic plants. In contrast, no delay or attenuation of symptoms was observed for transgenic plants challenge-inoculated with the BCTV strains CFH and Worland. Infection by the CFH and Worland strains did not result in mobilization or amplification of the integrated Logan DI-DNA sequence, and no consistent differences in the accumulation of CFH or Worland genomic viral DNA forms were observed among transgenic and nontransgenic plants. These results, and a comparison of putative DNA replication origin sequences, suggest that BCTV strains display specificity with respect to recognition of heterologous DNA replication origin cis-elements.

Complete nucleotide sequence of Oat necrotic mottle virus: a distinct Tritimovirus species (family Potyviridae) most closely related to Wheat streak mosaic virus.

The RNA genome (9346 nucleotides) of Oat necrotic mottle virus (ONMV) was cloned and sequenced. Complete genome comparisons indicated that ONMV, currently classified as a rymovirus, was most closely related ( approximately 73% nucleotide, approximately 79% amino acid identity) to the tritimovirus Wheat streak mosaic virus (WSMV). ONMV encoded a single polyprotein, with proteinase cleavage sites very similar to those of WSMV. Pairwise comparison of ONMV and WSMV cistrons revealed that P3 was most conserved ( approximately 79% nucleotide, approximately 86% amino acid), whereas HC-Pro was most divergent ( approximately 67% nucleotide, approximately 69% amino acid). In contrast, the ONMV sequence was distantly related ( approximately 40% nt, approximately 26% amino acid identity) to that of the rymoviru s Ryegrass mosaic virus, with highest sequence conservation noted within the NIb cistron ( approximately 47% nucleotide, approximately 41% amino acid identity). These results firmly establish that ONMV is not a rymovirus but is instead a distinct species of the genus Tritimovirus.

The strain-specific cis-acting element of beet curly top geminivirus DNA replication maps to the directly repeated motif of the ori.

Strains of beet curly top geminivirus (BCTV) possess distinct cis- and trans-acting replication specificity elements which are not separately interchangeable among strains. Analysis of the replication competency of chimeric BCTV genomes, in which portions of the origin of DNA replication (ori) were derived from heterologous BCTV strains, have permitted identification of an essential cis-acting element governing strain-specific replication in a subgroup II geminivirus. Our studies indicate that the cis-acting element responsible for strain-specific replication properties resides within the directly repeated motif of the BCTV ori. Transient replication assays conducted in leaf disks and complementation experiments conducted in whole plants indicated that the trans-acting replication specificity element, residing within the amino-terminal region of the C1 Rep protein, may recognize and replicate a chimeric BCTV genome containing a heterologous ori so long as all or portions of the core element of the directly repeated motif are derived from the same strain as the Rep protein. As Rep protein binding to the core element of the directly repeated motif has been demonstrated by others to be essential for replication of subgroup III geminiviruses, our results support the hypothesis that replication specificity of subgroup II viruses is governed by processes similar to that of subgroup III viruses. However, a second cis-acting element of the ori, which appears to contribute to subgroup III virus replication specificity, does not seem to be required for replication specificity among the subgroup II viruses examined. Nonetheless, a potential role for a second cis-acting element in the BCTV ori contributing to maximal replication cannot be excluded.

Strain-specific determinants of beet curly top geminivirus DNA replication.

The Logan and CFH strains of the geminivirus beet curly top virus (BCTV) possess cis- and trans-DNA replication factors which exhibit specificity and are not functionally interchangeable. We demonstrate that the cis-acting replication specificity element is entirely contained within a 82- to 97-bp fragment which includes most of the viral DNA origin of replication. We also demonstrate that the strain-specific trans-acting replication determinant is located within amino acid residues 3-89 of the BCTV C1 replication protein. Transient replication assays indicated that chimeric BCTV genomes containing reciprocally exchanged regions of the CFH and Logan genomes were replication competent when the cis- and trans-replication specificity elements were derived from the same strain. Two reciprocal chimeric viral genomes with heterologous cis- and trans-replication elements were incapable of self-replication, yet could trans-replicate one another in a coinoculation experiment. Only chimeric genomes possessing the Logan trans-replication element were capable of mobilizing and amplifying a transgenic Logan derived DI-DNA. DI-DNA mobilization and amplification occurred in transient replication assays even when the helper virus genome was incapable of self-replication, providing that the trans-replication element was derived from the Logan strain. These results genetically define specific regions of the BCTV C1 replication protein determining viral DNA replication origin recognition and provide clear evidence that strains of BCTV have evolved specific cis- and trans-replication factors which functionally define the Logan and CFH strains as distinct viral agents.

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.

Heterologous complementation by geminivirus AL2 and AL3 genes.

The geminivirus group is diverse and contains viruses which can be placed into three distinct subgroups on the basis of their genome organization and biological properties. However, most dicot-infecting gemiviruses possess AL1, AL2, and AL3 open reading frames. AL1 encodes the only viral protein that is absolutely required for replication, AL2 codes for a protein that transactivates the expression of virion sense promoters, and AL3 specifies a protein that enhances viral DNA replication. In the studies presented here, we examined the functional specificity of the AL1, AL2, and AL3 specifies gene products of subgroup II and subgroup III geminiviruses. Surprisingly, we found that all viruses tested were able to produce a gene product which complemented the reduced DNA replication phenotype of a tomato golden mosaic virus (TGMV) AL3 mutant. We also found that all bipartite subgroup III viruses tested produced a protein that could transactivate the virion sense promoters of a TGMV AL2 mutant, although a subgroup II virus did not. In contrast, the replication activity of AL1 protein proved to be virus specific. The data are discussed with regard to the functions of these proteins in viral replication and their practical significance for the development of crop protection strategies.

Recombinant beet curly top virus genomes exhibit both parental and novel pathogenic phenotypes.

Recombinant genomes derived from the Logan and CFH strains of the geminivirus beet curly top virus (BCTV) have been constructed and analyzed for pathogenicity on Nicotiana benthamiana and sugar beet (Beta vulgaris L.). Infectivity assays indicated that the latent period on N. benthamiana was primarily determined by a DNA fragment bearing the leftward open reading frames (ORFs) L1, L2, L3, and L4. Recombinants bearing leftward ORFs from the CFH strain were characterized as having a short latent period (mean = 6-11 days), while the reciprocal recombinants bearing leftward ORFs from the Logan strain had latent periods defined as long (mean = 16-22 days). Infectivity assays on sugar beet indicated that certain recombinant BCTV genomes exhibited novel pathogenic properties not common to either wild type strain, including the loss of systemic movement and replication competency, or asymptomatic systemic infection of sugar beet. The results indicate that N. benthamiana is a more permissive host than sugar beet with respect to heterologous combinations of BCTV genes, and that pathogenicity and virulence of BCTV in sugar beet requires the interaction of certain viral gene products and/or cis-elements that have coevolved in the same strain.