Benzothiadiazole (BTH) induces resistance to Pepper golden mosaic virus (PepGMV) in pepper (Capsicum annuum L.).

Pepper is an economically important crop in many countries around the world but it is susceptible to many diseases. In Mexico, diseases caused by bipartite begomoviruses have emerged as important problems in pepper. Several control strategies have been explored wiht little success; most of them are based on the avoidance of virus transmission and the breeding for resistance. Abiotic inducers can act at various points in the signaling pathways involved in disease resistance, providing long-lasting, wide-spectrum resistance. Benzothiadiazole (BTH) shares the property of activating the systemic acquired resistance pathway downstream from the SA signaling. In this work, resistance to PepGMV infection was induced in pepper plants by activating the SA pathway using BTH treatment. The resistance was characterized by evaluating symptom appearance, virus accumulation and viral movement. Our results showed that BTH could be an attractive alternative to induce geminivirus resistance in pepper plants without a significant damage of the fruit quality and productivity.

Benzothiadiazole (BTH) induces resistance to Pepper golden mosaic virus (PepGMV) in pepper (Capsicum annuum L.)

Pepper is an economically important crop in many countries around the world but it is susceptible to many diseases. In Mexico, diseases caused by bipartite begomoviruses have emerged as important problems in pepper. Several control strategies have been explored wiht little success; most of them are based on the avoidance of virus transmission and the breeding for resistance. Abiotic inducers can act at various points in the signaling pathways involved in disease resistance, providing long-lasting, wide-spectrum resistance. Benzothiadiazole (BTH) shares the property of activating the systemic acquired resistance pathway downstream from the SA signaling. In this work, resistance to PepGMV infection was induced in pepper plants by activating the SA pathway using BTH treatment. The resistance was characterized by evaluating symptom appearance, virus accumulation and viral movement. Our results showed that BTH could be an attractive alternative to induce geminivirus resistance in pepper plants without a significant damage of the fruit quality and productivity.

Characterization of Rhynchosia yellow mosaic Yucatan virus, a new recombinant begomovirus associated with two fabaceous weeds in Yucatan, Mexico.

Rhynchosia minima (L.) DC. (Fabaceae) plants exhibiting bright golden mosaic symptoms were previously associated with begomovirus infection in Yucatan, México [1]. To characterize the begomovirus infecting these plants, the complete bipartite genome was cloned and sequenced. Sequence comparisons indicated that the virus was distinct from all other begomoviruses known to date, including those previously identified from symptomatic R. minima, and the name Rhynchosia yellow mosaic Yucatan virus (RhYMYuV) is proposed. Pairwise comparisons indicated that RhYMYuV DNA-A [2,597 nt, (EU021216)] and DNA-B [2,542 nt, (FJ792608)] components shared the highest nt sequence identity with Cabbage leaf curl virus (CaLCuV), 87% for component A and 71% for component B. Phylogenetic analysis indicated that both components of RhYMYuV are most closely related to other New World begomoviruses, having as closest relatives immediate outliers to the major Squash leaf curl virus (SLCV) clade. Recombination analysis of the RhYMYuV genome indicated that the DNA-A component has arisen through intermolecular recombination. R. minima plants inoculated with the monomeric clones developed a bright yellow mosaic similar to symptoms observed in naturally infected plants, confirming that the clones were infectious. Nicotiana benthamiana plants biolistically inoculated with monomeric clones developed curling and chlorosis in the newly emerging leaves. RhYMYuV was also detected in symptomatic Desmodium sect. Scorpiurus Benth. (Fabaceae) that were collected near the RhYMYuV-infected plants.

A New Strain of Tomato chino La Paz virus Associated with a Leaf Curl Disease of Tomato in Baja California Sur, Mexico.

Since 2001, geminivirus-like disease symptoms have been observed in tomato plants on the Baja California Peninsula of Mexico. These diseases have been associated with large populations of Bemisia tabaci (Genn.) in commercial fields and have caused dramatic decreases in expected yields. Leaf samples from tomato plants displaying symptoms of stunting and severe upward leaf curling were collected in March 2002 in fields located near the city of La Paz, Baja California Sur (BCS). Total DNA was extracted and tested for the presence of geminiviral DNA using polymerase chain reaction (PCR) with begomovirus-specific degenerate primer pairs PALIv1978/PARIc494 and PALIc1978/PARIv494 (4). PCR products of the expected size (~1.16 and ~1.45 kb) were obtained, cloned into pGEM-T Easy (Promega, Madison, WI), and sequenced. Restriction fragment length polymorphism analysis of the PCR fragments was performed using EcoRI, HindIII, PstI, and XbaI. Restriction fragment patterns were the same for all amplicons and no evidence of mixed infection was obtained. In addition, experimental transmission by whiteflies and inoculations by biolistics consistently induced severe leaf epinasty and stunted growth on tomato seedlings. The complete (2,606 nt) DNA-A sequence of the infecting virus was determined (GenBank Accession No. AY339618) and compared with viral sequences available at GenBank-EMBL databases using BLASTN and the CLUSTAL program (MegAlign, DNASTAR, Madison, WI). The highest nucleotide identity was obtained with the recently described Tomato chino Baja California virus, ToChBCV (90.2%, GenBank Accession No. AY339619), isolated from tomato plantings in El Carrizal, BCS, 100 km from La Paz (3). The second and third best scores were obtained with Tomato severe leaf curl virus from Nicaragua (ToSLCV-NI, 79.6%, GenBank Accession No. AJ508784) and Guatemala (ToSLCV-GT94, 73.8%, GenBank Accession No. AF130415), respectively. Overall, sequence similarity with other New World begomoviruses was rather low (less than 70% identity). Careful analysis of differences between the La Paz isolate and its closest relative, ToChBCV from El Carrizal, revealed that they display different Ori-associated iterons (i.e., replication (Rep)-binding sites) having GGAGTA and GGGTCY core sequences, respectively (1). Moreover, sequence comparisons of the Rep-binding domain (aa 1-120) showed that these domains are only 71% identical. Current taxonomic criteria for begomoviruses establishes that a virus DNA-A sequence identity below 89% with its closest relative is indicative of a separate species (2). Since the La Paz and El Carrizal isolates share 90.2% nt identity, they should be considered strains of a same virus species, recently renamed Tomato chino La Paz virus, ToChLPV (2). Nevertheless, the remarkable differences in their putative replication specificity determinants suggest that ToChLPV and ToChLPV-[BCS] could be incompatible in replication, an interesting issue that should be experimentally addressed. References: (1) G. R. Arguello-Astorga et al. Virology 203:90, 1994. (2) C. Fauquet and J. Stanley. Arch. Virol. 150:2151, 2005. (3) R. J. Holguín-Peña et al. Plant Dis. 89:341, 2005. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

A New Begomovirus Causes Tomato Leaf Curl Disease in Baja California Sur, Mexico.

More than 10,000 ha of tomatoes are grown in the field and greenhouses on the Baja California Peninsula of Mexico. Information about the etiology of geminivirus-like diseases affecting tomato crops in all horticultural regions in the area has been difficult to obtain and assess. From 2001 through 2003, stunting, foliar discoloration, reduced leaf size, and leaf crumpling symptoms were observed and analyzed in one large area of tomato plantings in El Carrizal (near the city of La Paz in Baja California Sur). This leaf curl disease resembled that caused by Chino del tomate virus and has been observed at levels of incidence ranging from 60 to 90%. DNA isolated from symptomatic plants was analyzed using DNA hybridizaton and polymerase chain reaction (PCR) amplification of the 5' regions of the replication and coat protein genes, including the intergenic region (3). Comparisons of the nucleotide sequence (GenBank Accession No. AY339619) with corresponding sequences in GenBank resulted in 84.2% identity with Tomato mild mottle virus and 61.7% with Tomato severe leaf curl virus; both isolates originate from Central America. The relatively low nucleotide sequence identities from its closest relatives suggested that the virus may be a new begomovirus species of unambiguous American ancestry. In a phylogenetic analysis using PAUP 4.0 software, the Baja California isolate clustered in a separate group from other Mexican sequences. Moreover, the iteron (iterative sequences motifs associated in virus replication) arrangements (1) are unique among known New World begomoviruses, but identical to analogous elements from a tobacco-infecting begomovirus from China. On the other hand, it is well known that there are interactions between geminiviruses in mixed infections in some horticultural areas of Mexico (2). To determine the identity of the putative geminivirus involved in the disease, we used selected restriction enzyme (EcoRI, HindIII and XbaI) analysis and PCR with specific primers. No evidence of mixed infections with other geminiviruses was obtained. DNA fragments of the expected size (1.1 kb) showed different digestion patterns compared with other well-characterized geminiviruses isolated from Mexico such as Chino del tomate virus, Pepper huasteco yellow vein virus, Tomato leaf curl Sinaloa virus, and Pepper golden mosaic virus. Epidemiological, experimental, and natural host range studies indicated that the Baja California isolate has a relatively narrow host range infecting tomatoes, peppers (Capsicum annuum L.), and Peruvian apple (Nicandra physalodes L.). Reproduction of characteristic leaf curling symptoms in tomato seedlings infected with viruliferous whiteflies (Bemisia tabaci Genn.) and inoculated biolistically using infectious DNA (0.5 µg/ml) as inoculum were obtained. Koch's postulates were completed using PCR and DNA hybridization to confirm virus identity. These results confirm that the Baja California isolate is different from other begomoviruses isolated from Mexico. The virus is tentatively named Tomato chino Baja California virus (ToChBCV), genus Begomovirus, family Geminiviridae. References: (1) G. R. Arguello-Astorga et al. Arch. Virol. 146:1465, 2001. (2) J. Mendez-Lozano et al. Phytopathology 93:270, 2003. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Identification of the minimal sequence required for vascular-specific activity of Tomato mottle Taino virus Replication-associated protein promoter in transgenic plants.

A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep(459::Rep)) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep(459::Rep) showed an expression activity 10-40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep(459::Rep) (pRep(459), pRep(324), pRep(203), pRep(145), pRep(132) and pRep(115)), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep(115) version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.

Pepper golden mosaic virus Affecting Tomato Crops in the Baja California Peninsula, Mexico.

In the state of Baja California Sur, tomatoes (Lycopersicon esculentum Mill.) are cultivated on approximately 1,000 ha. Occurrence of viral diseases is currently causing low yields and severe losses. Virus-like symptoms (severe leaf curling, stunting, reduced leaf size, and mosaic patterns) were observed on 99% of tomato plants in 2002 in La Paz, Baja California Sur. Whiteflies (Bemisia tabaci Gennadius) were present in affected fields and appeared to be associated with the disease. The virus was experimentally transmitted from infected plants to tomato and peppers seedlings by whiteflies and as infectious DNA (replicative form) by mechanical and biolistic inoculation. Symptoms similar to those found in the field were observed in experimental transmission assays. DNA from inoculated plants was extracted and analyzed by DNA hybridization and polymerase chain reaction (PCR) using degenerate (1) and specific (2) primers. The PCR products (1.1 kb) obtained from the common region (GenBank Accession No. AY368336) suggested the presence of a bipartite geminivirus. The nucleotide sequence of the PCR products showed a 98% identity to Pepper golden mosaic virus-Tamaulipas strain (PepGMV-Tam) in the intergenic region (IR). Similar identities (97%) were obtained by using the predicted amino acid sequences of the amino termini of the coat proteins. Identities in the replication proteins (92%) and IR iterative sequence analyses show that the PepGMV-La Paz isolate is a closely related strain of PepGMV-Tam. To our knowledge, this is the first report of PepGMV affecting tomato crops in Baja California Sur, Mexico. References: (1) M. R. Rojas et al. Plant Dis. 77:340. 1993. (2) I. Torres-Pacheco et al. Phytopathology 86:1186, 1996.

Tomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: implications for the delimitation of cis- and trans-acting replication specificity determinants.

Over the last decade, the tomato production in Cuba has been affected by new whitefly-associated diseases. In addition to the well-documented presence of Tomato yellow leaf curl virus (TYLCV) along the island, the occurrence of bipartite begomoviruses has also been reported. One of them, tentatively named Tomato mottle Taino virus (ToMoTV), has now been cloned and characterized at the molecular level. Its genomic organization is similar to other bipartite geminiviruses. Phylogenetic analyses placed ToMoTV in a subcluster with other geminiviruses isolated in the Caribbean Basin: Tomato mottle virus (ToMoV), Bean dwarf mosaic virus, Abutilon mosaic virus, Sida golden mosaic virus and Potato yellow mosaic virus (PYMV). Biolistic inoculation of tobacco and tomato plants with cloned viral DNA showed that ToMoTV pseudorecombines with PYMV-GP as predicted by the identity of their iterative elements, whereas it does not show the same ability with ToMoV, even when their replication-associated proteins (Rep and REn) show the highest percentage of similarity. A comparative analysis of Rep proteins from begomoviruses that are able to produce viable reassortants suggests that some key elements for virus replication specificity are located in the first ten amino acids of this protein.

First Report of a Geminivirus Associated with Leaf Curl in Baja California Peninsula Tomato Fields.

Since November 2001, geminivirus-like symptoms (stunting, reduced leaf size, and leaf curling "chino") have been observed in tomato (Lycopersicon esculentum Mill.) plantings in Baja California Sur, Mexico. Samples of symptomatic plants were collected from commercial fields and analyzed by traditional and molecular methods for the presence of geminiviruses. Inocula prepared from infected plants were experimentally transmitted to tomato seedlings and Datura stramonium by mechanical inoculation and whitefly transmission. Leaf curling and interveinal chlorosis symptoms similar to those found in the field were observed in inoculated tomato and D. stramonium. DNA from infected plants was extracted and analyzed by polymerase chain reaction (PCR) and electrophoresis using degenerate primers PALIv1978/PARIc494 (1). PCR fragments of the expected size (1.1 kb) for the common region (CR) were obtained from 28 of 64 plants, cloned and sequenced (GenBank Accession No. AY336088). Comparisons of CR sequences with the NCBI database by using BLAST and MegAlign (DNASTAR, London) indicated that the Baja Californian isolates were New World bipartite begomoviruses sharing the highest nucleotide sequence identity (93%) with a partially characterized geminivirus (Tomato severe leaf curl virus (ToSLCV); GenBank Accession No. AF130415) from Guatemala. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993.

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. References: (1) J. Garzon-Tiznado et al. Phytopathology 83:514, 1993. (2) M. Paxidamis et al. Arch. Virol. 144:703, 1999. (3) J. L. Potter et al. Plant Dis. 84:1045, 2000. (4) I. Torres-Pacheco et al. Phytopathology 86:1186, 1996.

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. References: (1) I. Torres-Pacheco et al. Phytopathology 86:1186, 1996. (2) P. Umaharan et al. Phytophatology 88:1262, 1998.

Molecular characterization of the RNA3 of asparagus virus 2.

Asparagus virus 2 (AV 2) has been detected in all major asparagus growing areas around the world and it has been suggested that, in association with other pathogens, it might be an important factor for the condition called asparagus decline. Although AV 2 was first reported in the 70's, only few of its biochemical characteristics have been actually studied. Some properties of an AV 2 isolate from Guanajuato, Mexico were characterized and its RNA3 cloned and sequenced. Electrophoretic analysis of AV 2 RNA showed 3 genomic RNAs (RNA1. 3.0; RNA2 2.7; and RNA3 2.3 kb) and a fourth, subgenomic, RNA4 of 1.1 kb. In vitro translation data confirmed that coat protein is produced by translation of the subgenomic RNA4, as reported for other bromoviruses. Sequence analysis of AV 2 RNA3 showed two open reading frames identified by sequence comparisons and immunological assays as coding the movement and coat proteins (MP and CP, respectively). AV 2 CP showed a high percentage of identity with the CPs from other members of the subgroup II of the ilarvirus genus. The percentage of identity with other ilarviruses decreased dramatically and in some cases was lower than the values obtained with members of different genera.

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. References: (1) J. E. Polston et al. Plant Dis. 81:1358, 1997. (2) J. E. Polston et al. Plant Dis. 83:984, 1999. (3) P. L. Ramos et al. Plant Dis. 80:1208, 1996.

Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication.

A phylogenetic and structural analysis of the intergenic region of 22 dicot-infecting and 8 monocot-infecting geminiviruses was carried out. The analysis allowed the identification of iterative sequence motifs 8-12 nucleotides in length, whose organization (number, orientation, and spacing) is highly conserved within each of the three major lineages of dicot-geminiviruses, according to the phylogeny derived from the amino acid sequences of the replication-associated protein (AL1). The iterated elements differ in sequence even between closely related viruses, and are found in the vicinity of the putative TATA box of the AL1 gene in all dicot-infecting geminiviruses. Analogous elements were identified also in monocot-infecting geminiviruses, but the arrangement was different, since one of the iterative sequences is part of the conserved hairpin structure essential for replication of all the members of this viral family. We propose here that the iterated sequences are the specific binding sites of the geminiviral replication-associated proteins and show that the hypothesis is in agreement with the experimental data available to date. Additionally, a model of geminivirus replication that involves the participation of host transcription factors in the process is presented.