Pepper Huasteco Virus Research Articles

A Begomovirus Associated with Leaf Curling and Chlorosis of Soybean in Sinaloa, Mexico is Related to Pepper golden mosaic virus.

Since June 2001, symptoms of yellowing, leaf curling, crumpling, and stunted growth were observed on soybean (Glycine max Merr.) plants in Sinaloa, Mexico. These symptoms and the presence of whiteflies (Bemisia tabaci Gennadius) in the affected fields suggested a viral etiology. Samples from symptomatic plants were collected from commercial fields and analyzed for the presence of begomoviruses using DNA hybridization, and as a probe, the DNA A of Pepper huasteco virus at low stringency (2). Thirty-five positive samples were subsequently used for polymerase chain reaction (PCR) amplification with the degenerate primers RepMot and CPMot (1). These primers direct the amplification of a DNA A segment comprising the entire intergenic region (IR) and the first 210 bp of the coat protein (CP) gene, which is highly variable in size and nucleotide sequence among begomoviruses. PCR products were obtained for 25 of 35 samples and five of these were cloned into the pGEM-T easy vector (Promega, Madison, WI) and sequenced. The 571-bp DNA sequence (GenBank Accession No. AY905553) was compared with sequences of other begomoviruses in GenBank using the Clustal alignment method (MegAlign, DNASTAR software, London). The sequence was 74 and 70% identical to the Pepper golden mosaic virus (PepGMV; GenBank Accession No. U57457) and Cabbage leaf curl virus (CaLCuV; GenBank Accession No. U65529) sequences, respectively. Interestingly, the partial coat protein gene sequence (210 nt) of this soybean-infecting virus was 98% identical to the CP gene of Tobacco apical stunt virus (TbASV; GenBank Accession No. AF076855). Nonetheless, the known sequence of TbASV intergenic region (GenBank Accession No. AF077744) is very different from the homologous region of the soybean virus (34% of nucleotide identity). Analysis of the soybean virus intergenic region revealed that it harbors almost identical iterons (i.e., Rep-binding sites) to PepGMV, suggesting a close relationship between these two viruses. Soybean-infecting geminiviruses have been previously reported only from Asia; however, the partial sequence of a begomovirus isolated from soybean in Brazil was recently deposited in Genbank (Accession No. AY436328). Sequence comparisons between the Brazilian and Mexican isolates showed these viruses are less related with a nucleotide identity of 46%. Taken together, our data indicate that the virus identified in this study might be either a different strain of PepGMV adapted to leguminous plants or a new begomovirus species. To our knowledge, this is the first report of a begomovirus infecting soybean in Mexico.

Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco

The conserved late element (CLE) was originally identified as an evolutionarily conserved DNA sequence present in geminiviral intergenic regions. CLE has subsequently been observed in promoter sequences of bacterial (T-DNA) and plant origin, suggesting a role in plant and plant viral gene regulation. Synthetic DNA cassettes harboring direct repeats of the CLE motif were placed upstream from a -46 to +1 minimal CaMV 35S promoter-luciferase reporter gene and reporter activity characterized in Nicotiana species during both transient and stable expression. A single direct-repeat cassette of the element (2x CLE) enhances luciferase activity by 2-fold, independent of the element's orientation, while multiple copies of the cassette (4-12x CLE) increases activity up to 10- to 15-fold in an additive manner. Transgenic tobacco lines containing synthetic CLE promoter constructs enhance luciferase expression in leaf, cotyledon and stem tissues, but to a lesser extent in roots. Single nucleotide substitution at six of eight positions within the CLE consensus (GTGGTCCC) eliminates CLE enhancer-like activity. It has been previously reported that CLE interacts with the AC2 protein from Pepper Huasteco Virus (PHV-AC2). PHV-AC2 (also called AL2 or C2) is a member of the transcriptional activator protein, or TrAP, gene family. In transient and stable expression systems PHV-AC2 expression was found to result in a 2-fold increase in luciferase activity, irrespective of the presence of CLE consensus sequences within the reporter's promoter. These data suggests that the PHV-AC2 protein, instead of interacting directly with CLE, functions as either a general transcriptional activator and/or a suppressor of post-transcriptional gene silencing.

Inducible gene expression by Pepper huasteco virus in Capsicum chinense plants with resistance to geminivirus infections

To understand the interaction between pepper (Capsicum chinense) and Pepper huasteco virus (PHV), gene expression analyses using differential display reverse transcription – polymerase chain reaction (RT–PCR) were carried out with plants (C. chinense accession BG-3821) previously identified as resistant to single and mixed geminivirus infection. In vitro propagated virus-free C. chinense plants were used in our work to reduce variability among plants. With this strategy, 45 differentially expressed RT–PCR fragments were identified, and two of them were further confirmed by Northern analyses as differentially induced when the plants were PHV infected. Sequencing of these RT–PCR fragments and comparison with GenBank showed some similarity with a bacterial methyl transferase gene (CbiL) and an NADP-malic enzyme of Sorghum bicolor. Aspects of the possible role of these genes in the interaction between C. chinense and PHV are discussed.

IDENTIFICATION OF RESISTANCE TO SINGLE AND MIXED INFECTIONS OF PEPPER GOLDEN MOSAIC VIRUS (PepGMV) AND THE HUASTECO PEPPER VIRUS IN CHILI PEPPERS (Capsicum chinense Jacq.).

In this study, resistance to single and mixed infections to pepper golden mosaic virus (PepGMV) in Capsicum chinense Jacq. individuals were evaluated. Plant inoculation was carried out by biolistics and grafting. To 140 plants from seven accessions of C. chinense Jacq., inoculations were preformed with PepGMV. Of these plants, 99 were asymptomatic, although the virus was detected by ELISA and PCR techniques. Additionally, resistance to geminivirus (PepGMV and Pepper huasteco virus or PHV) mixed infections, was evaluated in these accessions. Thirty-one plants from the accessions with the best characteristics of resistance to single infections by PepGMV were evaluated, and 15 individuals displayed resistance in different degrees. Resistance to geminivirus mixed infections was grouped as: delayed symptoms, symptom remission and asymptomatic plants. DNA levels of both geminiviruses correlated positively with symptom severity.

Resistance to Geminivirus Mixed Infections in Mexican Wild Peppers

Screening for resistance to mixed infections with pepper huasteco virus (PHV) and pepper golden mosaic virus (PepGMV) was carried out on plants representing wild pepper accessions collected in different states of México. One accession collected in Yucatán (BG-3821) corresponded to Capsicum chinense Jacq., and three collected from Michoacán (BG-3818), Tamaulipas (BG-3820), and Sinaloa (BG-3819) were identified as C. annuum L. Forty-eight plants were initially inoculated with a 1:1 mix of PHV and PepGMV DNAs by a biolistic method. Those plants that did not show typical symptoms after the biolistic method, were inoculated by grafting. Half of the plants (24) were highly susceptible, while the other half expressed different degrees of resistance. Of the resistant individuals, eight plants were asymptomatic and viral DNA of both viruses was detected in low levels. Two individuals showed delayed symptoms 34 days after symptom expression in the control plants. This delay was correlated with an increase in PHV DNA levels when plants became symptomatic. The remaining 14 plants showed symptom remission in newly developed leaves at 31 days postinoculation, and this asymptomatic effect was correlated diminished PHV DNA within the plants. Our results suggest that the resistance shown by some individuals to geminivirus mixed infections (PHV and PepGMV) is likely due to constrains in viral movement.

Interactions Between Geminiviruses in a Naturally Occurring Mixture: Pepper huasteco virus and Pepper golden mosaic virus

ABSTRACT Pepper huasteco virus (PHV) and Pepper golden mosaic virus (PepGMV) are found in mixtures in many horticultural crops in Mexico. This combination constitutes an interesting, naturally occurring model system to study several aspects of virus-virus interactions. Possible interactions between PHV and PepGMV were studied at four levels: symptom expression, gene expression, replication, and movement. In terms of symptom expression, the interaction was shown to be host-dependent because antagonism was observed in pepper, whereas synergism was detected in tobacco and Nicotiana benthamiana. PHV and PepGMV did not generate viable pseudorecombinant viruses; however, their replication is increased during mixed infections. An asymmetric complementation in movement was observed because PHV was able to support the systemic movement of PepGMV A whereas PepGMV did not support the systemic distribution of PHV A. Heterologous transactivation of both coat protein promoters also was detected. Several conclusions can be drawn from these experiments. First, viruses coinfecting the same plant can interact at several levels (replication, movement) and in different manners (synergism, antagonism); some interactions might be host dependent; and natural mixed infections could be a potential source of geminivirus variability by generating viable tripartite combinations that could facilitate recombination events.

First Report of a Geminivirus Inducing Yellow Mottle in Okra (Abelmoschus esculentus) in Mexico.

Okra is an annual vegetable species native to Africa. In Mexico, the states of Tamaulipas, Guerrero, and Morelos contain the most important okra-producing areas. Viral-like diseases have recently affected the fruit production. In the field, the most common symptoms encountered include yellow streak, distortion of fruits, and foliar yellow mottle. Total DNA extracts from symptomatic okra plants were used as a template for polymerase chain reaction (PCR)-based detection using begomovirus-specific primers. RepMot and CPMot primers (1) were used for the amplification of DNA fragments that included the Rep and coat protein (CP) genes of begomoviruses. PCR results suggested the presence of a begomovirus in symptomatic plants. Southern and dot blot hybridization analysis were performed using a DNA fragment containing the CP gene of Pepper huasteco virus as a probe. Hybridization conducted under low stringency conditions confirmed the presence of a geminivirus. Additionally, transmission by grafting and biolistic (total DNA extracts from symptomatic plants) inoculations induced consistently severe streak fruits and yellow mottle symptoms in okra plants. Cloning of the PCR products (approximately 632-bp fragment) was performed in the PCRTopo vector (Invitrogen, San Diego, CA). Cloned viral inserts were sequenced (Genbank Accession No. AF349113). Nucleotide sequence comparisons were performed using the Clustal Method (MegAlign, DNAStar software, Madison, WI) with the GenBank database. Analysis of the PCR products confirmed the begomovirus nature of the sequence. The first 64 amino acids of the CP had 89% identity with Squash leaf curl virus while the intergenic region had 85% identity with Sida golden mosaic virus. To our knowledge, this is the first report of a begomovirus infecting okra in Mexico. Reference: 1) J. T. Ascencio et al. Plant Dis. 86:692, 2002.

First Report of Rhynchosia golden mosaic virus (RhGMV) Infecting Tobacco in Chiapas, Mexico.

After a tobacco virus outbreak associated with whiteflies in Chiapas, Mexico, we conducted a survey to detect the presence of begomoviruses. Previously, two tobacco-infecting geminiviruses were reported in the same geographical area: Texas pepper virus-Chiapas and Tobacco apical stunt virus (TPV-CPS and TbASV, respectively) (2). DNA extracts from symptomatic tobacco plants (yellow mosaic, severe foliar distortion, and dwarfing) were used to biolistically inoculate tobacco plants (1). After symptom expression, the viruses were analyzed by polymerase chain reaction (PCR) and sequencing. For the first PCR procedure, the primers used (RepMot: 5'GAGTCTAGAGGATANGTRAGGAAATARTTCTT GGC3' and CPMot: 5'CGCGAATTCGACTGGACCTTACATGGNCCTT CAC3') were designed from conserved regions of the Rep and CP genes, and directed the amplification of a fragment that includes the intergenic region and varies in size from 600 (for New World begomoviruses) to 750 bp (Old World begomoviruses). Cloning of the PCR products (approximately 600 bp) was performed in the pCRII vector (Invitrogene, San Diego, CA), and viral inserts derived from different symptomatic plants were sequenced. Nucleotide sequence comparisons were performed using the Clustal method (MegAlign, DNAStar software, Madison, WI) with GenBank databases. Analysis of the PCR products allowed the identification of two types of viral sequences. The first virus identified was 98% identical to TPV-CPS, whereas the second virus was clearly related to Rhynchosia golden mosaic virus (RhGMV; 91% identity in the amplified region), and 65% identical to Pepper Huasteco virus (PHV). To disclose the identity of the second virus, another set of primers was used, p260 and p261 (4). These primers are located back-to-back in a conserved region of the CP gene, and direct the amplification of a full-length DNA-A from circular templates. The resulting PCR fragment (2.6 kb) was cloned in pCRII and fully sequenced (GenBank Accession No. AF408199). Analysis showed that this tobacco-infecting geminivirus is a strain of the recently described RhGMV from Honduras (3) (overall DNA A sequence identity, 94%; protein similarities: CP, 98.4%; AL1, 93.6%; AL2, 92.8%; and AL3, 91.7%). Comparative analysis of the intergenic regions of RhGMV-Tob, TPV-CPS, and TbASV showed that these viruses display different Ori-associated iterative motifs (iterons): RhGMV-Tob (GGTRT/G), TPV-CPS (GGAGTC), and TbASV (GGTAT). Since iterons are critical determinants of replication specificity, this observation indicates that those viruses are probably unable to form infectious pseudorecombinants in nature. To date, at least three different geminiviruses have been identified from symptomatic tobacco samples in Chiapas (2), showing how complex a geminiviral outbreak can be in a permissive environment. To our knowledge, this is the first time that the presence of RhGMV has been reported in Mexico and also the first time that this virus has been associated with an economically important crop.

Pepper huasteco virus and Pepper golden mosaic virus are Geminiviruses Affecting Tomatillo (Physalis ixocarpa) Crops in Mexico.

Whitefly-transmitted geminivirus diseases cause important losses in several horticultural crops in all areas in Mexico (1). Tomatillo is important in the Mexican diet since it is widely used to prepare many types of salsas and other dishes. As a result, tomatillo, also known as tomate verde (green tomato), is cultivated in 29 of 32 states in Mexico, with the main production areas located in the states of Morelos, Puebla, and Michoacán. Leaf samples of 105 tomatillo plants exhibiting yellowing, yellowing mosaic, leaf curl, bunchy top, and stunting were collected from the states of Puebla, Morelos, Estado de México, and Sinaloa. Symptomatic plants were associated with the presence of whiteflies in many fields and suggested a viral etiology. Total DNA extracted from symptomatic tomatillo plants was used as a template in a polymerase chain reaction (PCR)-based geminivirus detection procedure. MP16 and MP82 primers (2) were used to direct the amplification of a segment from the stem-loop structure in the intergenic region (IR) to a conserved region in the coat protein (CP) of begomoviruses (2). Sixty-nine percent (72/105) of the samples produced the expected PCR fragment (400 to 450 bp). Similar results were obtained with a dot-blot hybridization procedure using as a probe the component A of Pepper huasteco virus (PHV) under low stringency conditions. More than 50 PCR products were cloned and sequenced. Sequence analysis (nucleotide level for the IR; amino acid level for the CP) revealed that the tomatillo-infecting geminiviruses clustered into two main groups. The first group showed a high percent identity (average of 95.3% at the CP N terminus) to PHV, whereas the second showed a similarly high percent (average 93.8%) identity to Pepper golden mosaic virus (PepGMV, previously called Texas pepper geminivirus. Both PepGMV and PHV were found in all sampled areas. Although mixed infections (differentiated by the respective IR probes) of PHV and PepGMV were common (61%), single infections were also detected (PHV 27%; PepGMV 10%). The presence of begomoviruses in tomatillo crops has been previously reported (1); however, their identity as PHV and PepGMV was not confirmed.

Screening wild plants of Capsicum annuum for resistance to pepper huasteco virus (PHV): Presence of viral DNA and differentiation among populations

Plants collected in thirteen wild populations of Capsicum annuum from Northwest Mexico were tested for resistance to the pepper huasteco begomovirus (formerly subgroup III) (PHV) that is transmitted by the white fly Bemisia tabaci Genadius. Plants were inoculated using both grafting and biolistic methods. Presence of viral DNA was detected by dot blot hybridization and densitometry. Populations varied in their resistance to PHV. Plants of only two of the populations either did not develop disease symptoms or showed very light symptoms after inoculation. In some cases, symptoms appeared several days after inoculation. In plants of these populations viral DNA was detected by dot-blot hybridization assays but they appear to be a good source of resistance (symptomless) for use in breeding programmes.

First Report of Tomato Yellow Leaf Curl Geminivirus in Yucatán, México

Geminiviruses are probably the most important viral pathogen affecting tomatoes and other crops in the Caribbean region. In addition to losses previously caused by native virus populations, the introduction of tomato yellow leaf curl virus (TYLCV) into the area has become a major concern for tomato growers (1). Since the detection of TYLCV in Cuba, and later in Florida (2,3), we have been monitoring the tomato- and pepper-growing areas of the Yucatán Peninsula, México, for TYLCV. We also have reanalyzed samples previously collected. Other geminiviruses (pepper huasteco virus [PHV], Texas pepper virus [TPV], and tomato mottle virus [ToMoV]) in the area can cause symptoms similar to those induced by TYLCV, which led us to refine our analysis of samples, using a polymerase chain reaction (PCR) procedure that can differentiate between monopartite and bipartite begomoviruses based on the size of the amplification product, 750 and 600 bp, respectively. One advantage of using this set of primers is that the PCR product, which includes the amino terminus of the Rep protein, intergenic region, precoat protein, and amino terminus of the coat protein, can be sequenced completely with only one sequencing reaction from each end. Using the primer set, we analyzed samples collected from tomato and pepper fields (as well as from weeds surrounding the fields) from December 1996 until March 1999. In most cases, samples were taken from plants that showed yellowing, curling, and stunting symptoms. Most of the samples that were positive for geminiviruses came from plants infected with PHV or TPV. However, three tomato samples collected during two seasons in Dzidzantun and Yobain counties (northeast of Mérida, Yucatan) produced the larger PCR amplification product (750 bp) expected for monopartite begomoviruses. PCR products were cloned and sequenced to confirm their identity. The sequence was deposited in the GenBank Database (Accession no. AF168709) and compared with all geminivirus sequences deposited in the database. Analysis showed that the amplified fragment from the TYLCV strain present in the Yucatán is 99% identical to the isolate reported in the Dominican Republic and later found in Cuba (2). As previously noted, the isolate is almost identical to TYLCV-Isr (2). In addition to the PCR product, a full-length TYLCV clone was obtained directly from DNA extracts of an infected tomato plant. Further characterization of the full-length clone is underway. The fact that TYLCV was detected in two counties and in samples collected during two seasons confirms the presence of TYLCV in the Yucatán. Interestingly, although the first positive sample for TYLCV was collected during the winter of 1996 and 1997, current incidence is rather low-only two other positive samples have been detected in more recently collected samples. Perhaps the characteristics of the agriculture system in the Yucatán (small, disperse plots) or the presence of other geminiviruses have contributed to a slow spread of the virus. More comprehensive surveys are required to confirm the actual distribution of the pathogen in the area.

Complementation of Coat Protein Mutants of Pepper Huasteco Geminivirus in Transgenic Tobacco Plants

ABSTRACT The role of the pepper huasteco virus (PHV) coat protein (CP) gene during the infection was investigated in three different hosts by using mutations that produced truncated proteins and by complementation assays in transgenic plants. The infectivity analysis revealed that mutants that express truncated CP (CP7 and CP191) behave like the wild-type virus when inoculated onto pepper and Nicotiana benthamiana plants in terms of symptom expression and viral DNA movement. On the contrary, the CP7 mutant was unable to systemically infect tobacco plants, whereas only 10% of the plants inoculated with the CP191 mutant became infected. The CP7 mutant was complemented by coinoculating it with another geminivirus (taino tomato mottle virus). No complementation was observed in plants from nine transgenic tobacco lines expressing CP under the control of the cauliflower mosaic virus (CaMV) 35S promoter. However, 3 out of 10 lines expressing CP under the control of its own promoter (693 nucleotides) were able to complement the CP7 mutant. Interestingly, upon infection, the levels of CP mRNA in 693CP plants increased dramatically, probably due to transactivation of the CP promoter by the viral protein AC2.

Identification of a Sequence Element Involved in AC2-Mediated Transactivation of the Pepper Huasteco Virus Coat Protein Gene

The geminivirusAC2gene product transactivates the expression of the coat and movement protein (CPandBV1) genes, and this effect seems to be mediated by specific although hitherto unknowncis-acting elements. In this work we examined regions from theCPandBV1gene promoters of pepper huasteco virus (PHV) to define the sequence elements involved in regulation by AC2. Results from transient gene expression and transgenic plant assays suggest that a truncated 115-nt CP promoter is still responsive to the viral transactivator. This promoter contains three elements similar to a sequence motif termed conserved late element (CLE), which is found in the regulatory regions of many geminiviruses and that was previously suggested, on a theoretical basis, to be a potential functional target for AC2 (Argüello-Astorgaet al.(1994),Virology203, 90–100). To confirm these results, an oligonucleotide containing two CLE motifs was synthesized and characterized in gain-of-function experiments. Transient expression assays showed that this 29-nt sequence is able to confer AC2 responsiveness to heterologous promoters. A smaller oligonucleotide (16 nt) containing a single CLE also conferred this activity. In addition, when the CLE motifs were mutated in their original context (truncated 115-nt promoter), this modified promoter lost its ability to be transactivated by AC2. All these results support the involvement, at least in the case of PHV, of CLE sequences in the process of transactivation.

Distribution and Diversity of Geminiviruses in Trinidad and Tobago

ABSTRACT Seven crop and eight weed species from 12 agricultural locations in Trinidad and Tobago were assayed for the presence of whitefly-transmitted geminiviruses (WTGs) by using dot blot hybridization and polymerase chain reaction (PCR) amplification of the N-terminal coat protein sequence with degenerate primers. The amplified fragments were cloned and analyzed by restriction enzyme digestion to determine fragment length polymorphism among the cloned fragments. Representative clones were then sequenced and subjected to phylogenetic analysis to determine the sequence similarity to known WTGs. WTGs were found in every location sampled and in 10 of the 15 species investigated: Lycopersicon esculentum(tomato), Capsicum annuum (pepper), Capsicum frutescens (sweet pepper), Abelmoschus esculentus (okra), Phaseolus vulgaris (beans), Alternanthera tenella, Desmodium frutescens, Euphorbia heterophylla, Malva alceifolia, and Sida acuta. The geminiviruses infecting these plants were closely related to potato yellow mosaic virus from Venezuela (PYMV-VE) and tomato leaf curl virus from Panama (ToLCV-PA). However, in pepper, sweet pepper, okra, Alternanthera tenella, Euphorbia heterophylla, Des-modium frutescens, and in one sample of tomato, a PYMV-VE-related virus was found in mixed infections with a virus related to pepper huasteco virus. Full-length infectious DNA-A and DNA-B of a tomato-infecting geminivirus from Trinidad and Tobago were cloned and sequenced. DNA-A appears to be a recombinant derived from PYMV-VE or ToLCV-PA, and Sida golden mosaic from Honduras. The implications of these findings in the control of WTGs are discussed.

Los parientes silvestres del chile ( Capsicum spp.) como recursos genéticos

Se presentan resultados sobre la variabilidad genética, morfológica, en la capacidad de germinación y de resistencia al geminivirus PHV (virus huasteco del chile) de poblaciones silvestres de Capsicum annuum colectadas en el estado de Sinaloa para ilustrar la importancia de estudiar los parientes silvestres de las plantas cultivadas. El estudio de resistencia al geminivirus PHV realizado con el método biobalístico, mostró una elevada variabilidad entre las poblaciones con respecto a este carácter. Algunas poblaciones mostraron niveles muy bajos de síntomas de la enfermedad y son muy prometedoras para estudios posteriores sobre resistencia a virosis. El estudio de polimorfismo enzimático mostró que las poblaciones silvestres de C. annuum mantienen una elevada variabilidad genética (intervalo de heterocigosis de 0.255 a 0.325) . Todas las poblaciones silvestres de C. annuum mostraron una variabilidad elevada en diez de once caracteres morfológicos estudiados. En la mayoría de las pruebas de germinación hubo una gran variabilidad de respuesta entre las poblaciones estudiadas. Todas las poblaciones silvestres de C. annuum no germinaron en condiciones de oscuridad, lo cual sugiere que poseen mecanismos de latencia. No se encontró una relación entre la capacidad de germinación de las poblaciones y sus factores climáticos. En conclusión, los parientes silvestres del chile mantienen elevados niveles de variabilidad genética, morfológica, en su capacidad de germinación, y poseen genes potencialmente útiles, que los hace un recurso genético valioso que hay que estudiar y conservar.

Analysis of the infectivity of monomeric clones of pepper huasteco virus.

The infectivity of several monomeric clones of pepper huasteco virus was investigated. All clones were infectious when inoculated excised from the plasmid DNA. However, only certain clones were infectious when inoculated in the non-excised form. Constructs in which the cloning site lies inside regions or genes involved in replication (e.g. Rep-binding site, rep and AC2-AC3 genes) were not infectious, whereas constructs in which the site was located inside the CP or BC1 genes were infectious. A clone that interrupts the BV1 gene was not infectious suggesting an early of BV1 during the establishment of the infection. Linear viral clones containing different DNA fragments at both extremes were also infectious although with a lower efficiency. Analysis of the progeny suggested a precise excision mechanism since in most cases only wild type virus was recovered. The results suggest that excision could be linked to replication through a very specific recombination process.

Detection and Distribution of Geminiviruses in Mexico and the Southern United States

Plant samples from important horticultural areas in Mexico and the southern United States were collected during several seasons and analyzed for the presence of geminiviruses by a combination of agarose gel electrophoresis, molecular hybridization, and polymerase chain reaction amplification techniques. A general detection strategy confirmed the presence of geminiviruses in all horticultural areas of Mexico in pepper, tomato, tomatillo (Physalis ixocarpa), cucurbits, and tobacco. Specific detection procedures showed that pepper huasteco virus is widely distributed in Mexico; it was found in pepper and tomato samples in both coastal areas, as well as in central Mexico. It was also found in pepper samples from the Rio Grande Valley in southern Texas. Pepper jalapeno virus (PJV) and chino del tomate virus (CdTV) showed a more restricted distribution, although, in all cases, the viruses appeared to become more widely distributed over time. Partial DNA sequences of PJV and CdTV were also obtained. Comparative sequence analysis showed that PJV and the previously described Texas pepper geminivirus are probably strains of the same virus. The name pepper jalapeno virus is, thus, withdrawn to avoid further confusion. Similarly, CdTV showed a very high level of sequence identity with the recently described tomato leaf crumple virus (TLCrV), also suggesting that they both are strains of the same virus.

Complete nucleotide sequence of pepper huasteco virus: analysis and comparison with bipartite geminiviruses

The complete nucleotide sequence of pepper huasteco geminivirus (PHV) isolated in Northern Mexico was determined. The PHV genome consists of two circular ssDNA molecules of 2631 bases (PHV A) and 2589 bases (PHV B). PHV has a genome organization typical of a bipartite geminivirus with four open reading frames (ORFs) (AR1, AL1, AL2 and AL3) in component A and two (BR1 and BL1) in component B. An unexpected ORF was found in the complementary sense strand of PHV A. This ORF, termed AL5, is found entirely inside, but in the opposite orientation to AR1 (encoding the coat protein). AL5 shows some homology to equivalent but smaller ORFs predicted in other geminiviruses. Phylogeny trees based on pairwise comparisons of AR1, AL2, AL3, BL1 and BR1 predicted proteins placed PHV among the western hemisphere geminiviruses. A phylogeny tree based on AL1 (replicase-encoding ORF), on the other hand, placed PHV with eastern hemisphere geminiviruses, i.e. African cassava mosaic virus and the Sardinia and Israel isolates of tomato yellow leaf curl virus. Possible mechanisms for the 'hybrid or transition nature' of PHV are discussed.

Inoculation of Peppers with Infectious Clones of a New Geminivirus by a Biolistic Procedure

Several new viral diseases have appeared in almost all horticultural areas in Mexico. Transmission of their causal agents by whiteflies and host-range data suggest the involvement of geminiviruses in most cases, One example of a severe case is the «rizado amarillo» disease affecting peppers in northern Mexico (Tamaulipas State). The relationship of this disease to the tigre disease previously reported by Brown et al (7) is as yet unclear. Here we report the cloning and partial molecular characterization of the genome of a bipartite geminivirus associated with pepper rizado amarillo disease. The virus is tentatively named pepper huasteco virus (PHV). The cloned viral DNAs were infectious when inoculated by bombardment into pepper plants [...]