Prevalence and risk factors of bovine tuberculosis in dairy cattle in Eritrea.

BACKGROUND: The prevalence of bovine tuberculosis (BTB) in dairy cattle in the three major milk producing regions of Eritrea was assessed by subjecting 15,354 dairy cattle, 50 % of Eritrea's dairy cattle population, to the single intradermal comparative tuberculin test (SICTT). Skin test results were interpreted according to guidelines of the World Organization for Animal Health (OIE) with >4 mm as cutoff in skin thickness increase. In addition, we studied the relation between 'physiological' variables related to pregnancy and lactation, and the variable 'region' on the probability to be skin test positive. RESULTS: The BTB prevalences at animal and herd levels were: 21.5% and 40.9% in Maekel, 7.3% and 10% in Debub, and 0.2% and 1.6% in the Anseba region, respectively. Overall, in the regions included, prevalence was 11.3% (confidence interval (CI) 95% CI, 11.29 - 11.31%) and 17.3% (95% CI, 17.27-17.33%), at animal and herd level, respectively. Considering positive herds only, the animal BTB prevalence was 36.8%, 30.1%, and 1.8%, in Maekel, Debub and Anseba, respectively, and the overall animal prevalence within these herds was 32%. In adult dairy cattle the probability of positive reactivity in the SICTT test was highest in pregnant animals as compared to the other categories. CONCLUSION: This study reports persistent prevalence of BTB as defined by positive SICTT in the dairy sector of Eritrea, especially in the regions of Maekel and Debub that are located in the central highlands of the country. To our understanding this is the first report that has encompassed all the major dairy farms in Eritrea and it will be instrumental in advocating future BTB control programs in the dairy sector.

First Report of Watermelon chlorotic stunt virus Infecting Watermelon in Saudi Arabia.

In the Saudi Arabian deserts, watermelon [Citrullus lanatus (Thunb.)] is cultivated in the lowlands and wadis (washes) where water accumulates following rainfall, and where heat, salt, and drought stress are common constraints on production. During the spring of 2014, watermelon leaves exhibited yellowing and severe chlorotic mottling symptoms. The foliar symptoms were reminiscent of Watermelon chlorotic stunt virus (WmCSV), a bipartite begomovirus previously reported in several neighboring countries (1,3). Ten samples were collected from three farms in the Leith region, where 100% of the watermelon plants were symptomatic. Total nucleic acids were extracted from the symptomatic watermelon plants and were subjected to PCR using WmCSV DNA-A specific primers designed based on a complete genome sequence (GenBank Accession No. AJ012081), WmCSVF-3'-CGTGCTGTTGCCCCCACTGT-5' and WmCSVR-3'-CCTGCATATCTCGTGCCAGAATC-5' to obtain an expected size fragment of 1,111 bp located between the nucleotide (nt) coordinates 400-1510. The amplicons (one per sample) were cloned, and the DNA sequence was determined for each and used to search the NCBI database. The top hits for sequences obtained from all 10 samples were to WmSCV sequences, with shared nt identity values of 97 to 98%. To clone the full-length begomoviral DNA-A and DNA-B components, nucleic acids were subjected to rolling circle amplification (RCA) (2). The RCA products were cloned into the pGEM7 plasmid vector using the unique restriction sites, identified as HindIII for DNA-A, and as EcoRI for DNA-B, respectively. Ten DNA-A clones and two DNA-B component clones were sequenced. The DNA-A components ranged in size from 2,751 (KM066100) to 2,752 bp (KJ939448), whereas the DNA-B components were 2,757 bp in size (KJ939447). Analysis of the viral sequences from the DNA-A clones indicated they had the characteristics of a typical genome of a begomovirus DNA-A component that consist of a hairpin stem-loop structure in the intergenic region, two tandem copies of the iteron (TGGAGAC) located between the nt coordinates 2675 and 2680, 2682 and 2688 predicted to be involved in Rep binding, and six predicted genes encoding proteins with high sequence identity to those encoded by other WmCSV isolates. The 10 DNA-A component sequences were aligned with sequences for previously described WmCSV isolates available in GenBank using Muscle, followed by pairwise comparisons using SDT software (4). The analysis revealed that the cloned DNA-A components shared 99 to 100% nt sequence identity with each other, and 97 to 98% nt identity with WmCSV isolates reported from Yemen (AJ012081), Jordan (EU561237), Iran (AJ245652), and Sudan (AJ245650). Further, the WmCSV DNA-B from Saudi Arabia shared the highest nt identity with sequences from Yemen (AJ012082) at 96%, Iran (AJ245653) at 95%, and only 94% with DNA-B from both Sudan (AJ245651) and Jordan (EU561236). To our knowledge, this is the first report of WmCSV in Saudi Arabia. WmCSV poses a serious threat to the production of this highly valued crop in Saudi Arabia and considerably reduces crop yield (1). References: (1) I. D. Bedford et al. Eur. J. Plant Pathol. 100:243, 1994. (2) A. Idris et al. Plant Dis. 97:910, 2007. (3) A. Kheyr-Pour et al. Phytopathology 90:629, 2000. (4) B. Muhire et al. Arch. Virol. 158:1411, 2013.

Identification of Weed Hosts of Tomato yellow leaf curl virus in Cyprus.

An extensive study was conducted during 2007 and 2008 in three major tomato production areas of Cyprus, where Tomato yellow leaf curl virus (TYLCV) is commonly found, to assess the incidence and prevalence of naturally infected weed species that could serve as TYLCV reservoirs. Approximately 4,000 of the most common dicotyledonous plants belonging to 122 species from 25 families were collected, identified, and tested for TYLCV presence using serological and molecular methods. The tests included a previously reported conventional polymerase chain reaction (PCR) assay and a real-time TaqMan PCR assay developed and optimized in this study. Real-time PCR was found to be the most sensitive technique, and enabled the detection of TYLCV in 461 samples of 49 different species belonging to the families Amaranthaceae, Chenopodiaceae, Compositae, Convolvulaceae, Cruciferae, Euphorbiaceae, Geraniaceae, Leguminosae, Malvaceae, Orobanchaceae, Plantaginaceae, Primulaceae, Solanaceae, Umbelliferae, and Urticaceae. The results further indicated that the host range of TYLCV in Cyprus is far more extensive than previously documented and, therefore, new management strategies are required. These should focus on the control of alternative virus hosts during the growing season and in crop-free periods.

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.

First Report of "Candidatus Liberibacter psyllaurous" (synonym "Ca. L. solanacearum") Associated with 'Tomato Vein-Greening' and 'Tomato Psyllid Yellows' Diseases in Commercial Greenhouses in Arizona.

During the winter of 2006-2007, plants in commercial tomato greenhouses (GH-1 and GH-2; total 320 acres [129.5 ha]) in Arizona were infested with the potato psyllid Bactericera cockerelli (Sulc) and more than 60% and ~20% of the plants, respectively, exhibited leaf curling, chlorosis, and shortened internodes. In addition, some plants in GH-1 developed an unusual 'vein-greening' phenotype. Nucleic acids were isolated from 10 symptomatic and three asymptomatic plants from each greenhouse. PCR primers designed to amplify a phytoplasma-like 16S rDNA (850 bp) yielded the expected size product from GH-1 samples, whereas samples from GH-2 and the asymptomatic samples from both greenhouses did not. Several 16S rDNA PCR products (3 of 60) when cloned and sequenced, surprisingly shared 97% homology with 'Candidatus Liberibacter asiaticus' (GenBank No. GQ926917). PCR primers PSY680F 5'-GTTCGGAATAACTGGGCGTA-3' and PSY1R 5'-CCCATAAGGGCCATGAGGACT-3', based on the resultant 16S rDNA sequences, were used to amplify a 680-bp fragment from plant DNA extracts and psyllid lysates (1). A robust PCR product (~680 bp) was obtained from 10 of 10 GH-1 plant extracts (GQ926918) and from a GH-1-derived psyllid colony (28 of 35 adults) (GQ926919) and the tomato plants on which they were reared. In contrast, no 680-bp product was obtained from GH-1 asymptomatic plants (0 of 3), GH-2 plants (0 of 10 symptomatic; 0 of 3 asymptomatic), GH-2-derived psyllid colonies (0 of 35 adults), or psyllid colony tomato plants (data not shown). At least three 680-bp amplicons for each sample type were cloned and 8 to 10 inserts were sequenced for each. BLAST analysis revealed that all 680-bp sequences shared 99 to 100% nt identity with the analogous 16SrDNA from "Ca. Liberibacter psyllaurous" (2) and synonym "Ca. L. solanacearum" (3). A second molecular marker was obtained with the 1611F and 480R primers (2) to amplify the 16SrDNA-23S-ITS (980 bp) from >3 plant extracts and psyllid lysates that tested positive for liberibacter. Clustal W alignment of the 16S-23S-ITS sequences from GH-1 original tomato plants and psyllid colony plants (GQ926920) and psyllids (GQ926921) indicated they were 100% identical to each other and BLAST analysis indicated 99 to 100% shared identity with "Ca. L. psyllaurous" (EU812558) (synonym "Ca. L. solanacearum"). Transmission electron microscopy examination of GH-1 and GH-2 psyllids revealed rod and pleomorphic-shaped bacteria (0.5 to 2.0+ µm) at the brain-salivary gland interface in psyllids from the GH-1 liberibacter-positive colony. No such bacteria were observed in GH-2 liberibacter-negative psyllids. These results support an etiological role of a new liberibacter spp. in the development of the 'vein-greening' symptom phenotype. In contrast, the GH-2 'yellows' phenotype is reminiscent of 'psyllid toxicity' in tomato colonized by B. cockerelli (4). To our knowledge, this is the first report of distinct psyllid-associated diseases in greenhouse tomato in Arizona, one associated with a new 'Ca. Liberibacter' spp., manifest as 'vein-greening' disease, and the other associated with psyllid feeding, in which liberibacter is undetectable in plants and psyllids, and is manifest as the 'tomato psyllid yellows' disease. References: (1) D. R. Frohlich et al. Mol. Ecol. 8:1683, 1999. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) H. J. Pack. Utah Agric. Exp. Stn. Bull. 209, 1929.

First Report of "Candidatus Liberibacter psyllaurous" or "Ca. Liberibacter solanacearum" Associated with Severe Foliar Chlorosis, Curling, and Necrosis and Tuber Discoloration of Potato Plants in Honduras.

From 2006 to 2009, all commercial potato fields in Azacualpa F.M. Honduras were heavily infested with the potato psyllid Bactericera cockerelli (Sulc.). Plants exhibited interveinal chlorosis, vein-greening, downward curling, stunting, above ground tuber formation, and brownish flecks in some tubers. Disease incidence ranged from 50 to 95%. Leaf samples and psyllids were collected from seven fields in two potato-growing regions of Honduras. Total DNA was purified from the leaves of 30 symptomatic and three asymptomatic plants. DNA was extracted from 20 adult and 10 immature (4th to 5th instar) psyllids according to Frohlich et al (1). PCR primers, PSY680F 5'-GTTCGGAATAACTGGGCGTA-3' and PSY1R 5'-CCCATAAGGGCCATGAGGACT-3', were used to amplify a 680-bp fragment of the 16S rDNA for the recently described "Candidatus Liberibacter physallaurous" (2) and "Ca. L. solanacearum" (3). PSY1R/PSY680F primer design was based on the association of a previously undescribed liberibacter with vein-greening symptoms in greenhouse tomato plants in Arizona from 2006 to 2007 (GenBank Accession No. GQ926918) that lead to the hypothesis that a similar bacterium could be associated with symptomatic potato plants in Honduras. PCR amplification, cloning, and sequencing of the resultant 16S rDNA amplicons indicated that 17 of 30 potato plants, 8 of 20 adult and 7 of 10 third to fourth instar psyllids, respectively, were positive for liberibacter based on 99 to 100% shared nucleotide sequence (nt) identity with the analogous sequence from liberibacter (EU812558 [2]). To substantiate these results, a second molecular marker was targeted using the 1611F and 480R primers (~980 bp) that amplify the 16S-23S-ITSrDNA of liberibacter (2) for selected liberibacter-positive samples (above). Amplicons of the expected size were obtained from 12 of 17 potato and 7 of 10 immature psyllids. No PCR product of the expected size was obtained from asymptomatic potato samples or the PCR negative (water) control. The resultant PCR amplicons were cloned and 12 to 15 clones per amplicon were sequenced. The sequences were aligned and the percentage pair wise nt identity was calculated by Clustal W revealing that the 16S rDNA and 16S-23S-ITS sequences, respectively, shared 99 to 100% nt identity with each other. BLAST analysis against the NCBI database indicated that the 16S rRNA sequences from potato plants (GQ926922) and immature psyllids (GQ926923), and the 16S-ITS-23S sequence from potato plants (GQ926924) and immature psyllids (GQ926925), shared 98.5 to 100% nt identity with 'Ca. Liberibacter' reported from potato (EU812556; [2,4]) and tomato (EU812558, EU812559, EU935005; [2,3]). Evidence for the widespread presence of liberibacter and the potato psyllid in potato fields in Honduras, together with foliar and tuber symptoms that are reminiscent of those recently described in potato plants in the United States affected with 'zebra chip' disease (4), suggest that a similar or identical disease of the potato also occurs in Honduras. This emergent disease poses a serious threat to potato production in Honduras and elsewhere in Central America. References: (1) D. R. Frohlich et al., Mol. Ecol. 8:1683, 1999. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 78:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007.

Geminivirus-mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton.

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar 'Deltapine 5415' bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30 degrees C/26 degrees C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22 degrees C/18 degrees C. However, endogenous gene silencing decreased at 30 degrees C/26 degrees C. There was an approximately 7 d delay in the onset of gene silencing at 22 degrees C/18 degrees C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.

Melon chlorotic leaf curl virus: characterization and differential reassortment with closest relatives reveal adaptive virulence in the squash leaf curl virus clade and host shifting by the host-restricted bean calico mosaic virus.

The genome components of the Melon chlorotic leaf curl virus (MCLCuV) were cloned from symptomatic cantaloupe leaves collected in Guatemala during 2002. The MCLCuV DNA-A and DNA-B components shared their closest nucleotide identities among begomoviruses, at approximately 90 and 81%, respectively, with a papaya isolate of MCLCuV from Costa Rica. The closest relatives at the species level were other members of the Squash leaf curl virus (SLCV) clade, which is endemic in the southwestern United States and Mexico. Biolistic inoculation of cantaloupe seedlings with the MCLCuV DNA-A and -B components resulted in the development of characteristic disease symptoms, providing definitive evidence of causality. MCLCuV experimentally infected species within the Cucurbitaceae, Fabaceae, and Solanaceae. The potential for interspecific reassortment was examined for MCLCuV and its closest relatives, including the bean-restricted Bean calico mosaic virus (BCaMV), and three other cucurbit-infecting species, Cucurbit leaf crumple virus (CuLCrV), SLCV, and SMLCV. The cucurbit viruses have distinct but overlapping host ranges. All possible reassortants were established using heterologous combinations of the DNA-A or DNA-B components. Surprisingly, only certain reassortants arising from MCLCuV and BCaMV, or MCLCuV and CuLCrV, were viable in bean, even though it is a host of all of the "wild-type" (parent) viruses. The bean-restricted BCaMV was differentially assisted in systemically infecting the cucurbit test species by the components of the four cucurbit-adapted begomoviruses. In certain heterologous combinations, the BCaMV DNA-A or -B component was able to infect one or more cucurbit species. Generally, the reassortants were less virulent in the test hosts than the respective wild-type (parent) viruses, strongly implicating adaptive modulation of virulence. This is the first illustration of reassortment resulting in the host range expansion of a host-restricted begomovirus.

Two Distinct Isolates of Tomato yellow leaf curl virus Threaten Tomato Production in Arizona and Sonora, Mexico.

Severe yellow leaf curl and plant stunting symptoms were observed in tomato plants from two home gardens in central Arizona (Phoenix area) and a tomato field in Sonora, Mexico during the fall of 2006. Disease symptoms were reminiscent of those reported in Florida during 1994 (4) and more recently in tomato fields in the Pacific Coast state of Sinaloa, Mexico found to be infected with the exotic Tomato yellow leaf curl virus (TYLCV) (2). Total DNA was extracted from two symptomatic tomato plants from Arizona and Sonora and used as a template in PCR. PCR products of the core region of the begomovirus coat protein gene (Cp) were cloned (n = 3) and the DNA sequence was determined. BLAST analysis of the 579 bases with sequences available in the NCBI GenBank database indicated the closest match was to an isolate of the monopartite begomovirus TYLCV from Israel, which was known to have been introduced into the Caribbean region, including Puerto Rico, the southeastern United States, and Mexico from 1990 to 1996 (1,4). The full-length TYLCV genome (approximately 2,800 bases) was amplified for a field isolate from each location by rolling circle amplification (RCA) using TempliPhi (Amersham Biosciences, Piscataway, NJ). RCA products were cloned into the plasmid vector pGEM7 (Promega, Madison, WI) that had been previously digested with SacI endonuclease. The complete TYLCV genome sequence was determined for six clones from each RCA product. Nucleotide analysis indicated that the complete TYLCV genome sequences from Sonora and Arizona, respectively, shared 97.6 and 97.7% nt identity. The comparative sequence analysis indicated that TYLCV-Sonora (TYLCV-Son) (GenBank Accession No. EF210555) was 99.1% nt identical to TYLCV reported recently from Culiacan, Mexico (GenBank Accession No. DQ631892). In contrast, TYLCV-AZ (GenBank Accession No. EF210554) shared 99.3% identity with an isolate from Texas, TYLCV-TX (GenBank Accession No. EF110890) (3). Interestingly, the TX and AZ TYLCV isolates contained a unique 29-nt deletion in the intergenic region (IR) between the TATA-box and the nonanucleotide, initiating at nt coordinate 2696. Except for the deletion in the IR region of the AZ and TX isolates, these viruses shared 97.6 to 99.1% nt identity to other TYLCV isolates reported in the Western Hemisphere. The genome sequence for TYLCV-Son shares high nt identity with TYLCV isolates identified in the Yucatan Peninsula and Pacific Coast of Mexico (2), the Caribbean region, and the southeastern United States, suggesting that a single TYLCV species was introduced and has spread throughout North America and the Caribbean (4). The absence of other TYLCV isolates in the Western Hemisphere with the novel 29-nt deletion noted for the TX and AZ isolates suggests that the latter two isolates originated from the same U.S. source. In Mexico, TYLCV was first introduced in the east coast and Yucatan region approximately in 1996. From there, this isolate has spread to the western part of the country (Sinaloa and Sonora) from 2004 to 2006 (2). Similarly, in the United States, TYLCV was introduced and spread in the eastern U.S. states beginning in 1994 (4), where it had been confined until it was discovered in Texas (3) and now Arizona during 2006. References: (1) J. Bird et al. Plant Dis. 85:1028, 2001. (2) J. K. Brown and A. M. Idris. Plant Dis. 90:1360, 2006. (3) T. Isakeit et al. Plant Dis. 91:466, 2007. (4) J. E. Polston et al. Plant Dis. 78:831, 1994.

Widespread Outbreak of Cucurbit yellow stunting disorder virus in Melon, Squash, and Watermelon Crops in the Sonoran Desert of Arizona and Sonora, Mexico.

Bright yellow, interveinal chlorosis was observed for the first time on leaves of the older and mid-growth of cucurbit plants in southern Arizona and Sonora (Mexico) during September and October of 2006. Some cultivars exhibited substantial yield losses of 30 to 80%. In Arizona, symptoms were in Cucumis melo (muskmelon and honeydew melon) fields in the Yuma Valley and Hyder. In Sonora, honeydew and muskmelon, Cucurbita pepo (acorn, spaghetti, and summer [yellow and zucchini] squash), and Citrullus lanatus (watermelon) were symptomatic in Hermosillo, whereas, in Caborca, honeydew and cantaloupe developed similar symptoms. Interveinal chlorosis was observed in 60 to 100% of the plants in each field. Crops planted mid-to-late season were 100% infected, whereas, the early-season fields experienced approximately 60 to 80% incidence. All symptomatic fields in the Sonoran Desert and vicinity were infested by the whitefly Bemisia tabaci (Genn.), which was identified as the 'B biotype' on the basis of mitochondria COI sequence analysis (data not shown). Whitefly population levels were variable and ranged from 5 to 200 per plant. Total RNA was isolated from leaf samples collected from symptomatic plants using Tri Reagent (Molecular Research Center, Cincinnati, OH). Purified RNA was used in reverse transcriptase-PCR with primers specific to the Cucurbit yellow stunting disorder virus (CYSDV) coat protein (CP) gene (RNA2-deoxyribonucleotide coordinates 4927-4950 and 5657-5679) for the suspected whitefly-transmitted bipartite CYSDV (4). PCR yielded the CYSDV CP fragment, at 753 bp (GenBank Accession Nos. EF21058 and EF21059), which was cloned into pGEM T-Easy and sequenced in both directions using universal primers. The CYSDV CP nucleotide sequences (n = 16) obtained from acorn squash, honeydew melon, muskmelon, yellow squash, and watermelon had 99 to 100% identity. The Arizona (AZ) and Sonora (SON) CYSDV CP sequences shared 99 to 100% identity with previously described CYSDV isolates from the Eastern Hemisphere (GenBank Accession Nos. DQ903105 and DQ903108) and also with two isolates of CYSDV collected during 2004 from Zacapa Valley, Guatemala (GenBank Accession Nos. EF21060 and EF21061) (J. K. Brown, unpublished data). CYSDV is a member of the genus Crinivirus, family Closteroviridae. CYSDV was first identified in cucumber and melon crops in the Middle East approximately 15 years ago and 10 years ago in Spain (1). Most recently, this virus was introduced into Texas (2), Guatemala (J. K. Brown, unpublished data), and Arizona and California (3). CYSDV has therefore emerged as an important and potentially worldwide threat to the production of cultivated cucurbits (3). The threat appears to be significant in light of the introduction or establishment of the exotic B. tabaci biotypes B and Q vectors, which also originated in the Middle Eastern-North African-Mediterranean region. To our knowledge, this is the first report of CYSDV infecting field-grown C. pepo (four types) and watermelon, reported previously only as experimental laboratory hosts, and of CYSDV in two types of melon (C. melo) in Mexico. References: (1) A. Celix et al. Phytopathology 86:1370, 1996. (2) J. Kao et al. Plant Dis. 84:101, 2000. (3) Y.-W. Kuo et al. Plant Dis. 91:330, 2007. (4) L. Rubio et al. J. Gen. Virol. 82:929, 2001.

Tomato yellow leaf curl virus in Tomato in Texas, Originating from Transplant Facilities.

Tomato yellow leaf curl virus (TYLCV), a monopartite virus in the genus Begomovirus (family, Geminiviridae) from the Middle East, is one of the most damaging whitefly-transmitted viruses of tomato (Lycopersicon esculentum) worldwide. TYLCV was first identified in the United States in 1997 in Florida (4), and most recently, in the Pacific Coast states of Mexico where fresh market tomatoes are grown for the U.S. market (1). During September 2006, tomatoes grown from transplants in Waller County, TX exhibited shortened internodes, stunting and puckering of leaflets, green vein banding, and diffuse chlorosis. The disease incidence in two fields (4 ha total) was 95% and yield was substantially reduced. Many of the transplants were symptomatic at planting. The transplants originated from two facilities in Hidalgo County, TX. Both facilities had experienced heavy infestations of the whitefly, Bemisia tabaci (Genn.), during transplant production. At the same time, transplants produced in Uvalde and Bexar counties, TX, where whitefly infestations were also prevalent, had similar virus symptoms. Total DNA was extracted from the leaves of symptomatic tomato plants from 10 samples from these four counties and amplified by PCR (2). DNA samples from Waller, Hidalgo, and Uvalde counties were cloned, and a partial fragment of the viral coat protein gene (core Cp) was sequenced. BLAST analysis of the core Cp sequences of each sample confirmed the presence of TYLCV. No other begomovirus was detected, and all attempts to amplify a bipartite begomovirus by PCR using degenerate DNA-B specific primers (3) were unsuccessful. The full-length TYLCV DNA was amplified from three samples using the rolling circle amplification method as described (1), cloned, and the sequences were determined. The three sequences shared 99.6 to 100% nt identity and so only one sequence was deposited in the NCBI GenBank database (Accession No. EF110890) (1). Analysis of the complete genome nucleotide sequence corroborated TYLCV identity predicted by core Cp analysis that was 98.1% identical with TYLCV from Egypt (GenBank Accession No. AY594174) and Spain (GenBank Accession No. AJ489258), 97.6% with TYLCV from Mexico (GenBank Accession No. DQ631892), and 96.5% with TYLCV-Is (GenBank Accession No. X15656). Additionally, a Southern blot with TYLCV as the probe detected replicating (double-stranded) TYLCV DNA in all samples consisting of three plants from Uvalde County and 21 plants from Bexar County. To our knowledge, this is the first report of TYLCV in Texas that occurred in two transplant production areas approximately 400 km apart. Transplants produced in Uvalde and Bexar counties were planted there, while Hidalgo County transplants were shipped outside of the usual range of the whitefly. Hidalgo County has a subtropical climate, which can allow overwintering of TYLCV and the whitefly vector, allowing the establishment and spread of this virus in the future. References: (1) J. K. Brown and A. M. Idris. Plant Dis. 90:1360, 2006. (2) J. K. Brown et al. Arch. Virol. 146:1581, 2001. (3) A. M. Idris and J. K. Brown. Phytopathology 88:648, 1998. (4) J. E. Polston et al. Plant Dis. 83:984, 1999.

Introduction of the Exotic Monopartite Tomato yellow leaf curl virus into West Coast Mexico.

Leaf curl symptoms that are reminiscent of begomovirus (genus Begomovirus, family Geminiviridae) infection were observed widespread in the tomato crop during the early fall 2005 through the spring 2006 growing seasons in Sinaloa State, Mexico. Symptoms were widespread in three major valleys (Culiacan, Guasave, and Los Mochis) that are largely dedicated to fresh-market tomato production for the U.S. market from October to June. Symptoms included stunting of leaves, shortened internodes, distortion of leaf margins, and green vein banding. Fruit set was reduced significantly (as much as 90%) on the portion of the plant that developed above the point of symptom expression. Tomato fields were heavily infested with the B biotype of the whitefly Bemisia tabaci (Genn.) vector and no other insect vectors were noted in the fields. Total DNA was extracted from six symptomatic tomato plants (two from each valley) and used as template to amplify, clone, and sequence the core region of the begomovirus CP. BLAST analysis of begomovirus sequences available in the NCBI GenBank database indicated the closest match was the Old World monopartite begomovirus Tomato yellow leaf curl virus (TYLCV) from Israel (Accession No. X15656) at 97.8% shared nucleotide (nt) identity. The full-length genome was amplified for each of six isolates using TempliPhi (Amersham Biosciences, Piscataway, NJ) and cloned into the pGEM7 vector (Promega, Madison, WI). The complete DNA genome sequence was determined for eight clones by primer walking. Cloned TempliPhi products sequenced represented two to three isolates from each valley. Results indicated that the isolates (n = 8) were 98.9 to 100% identical (Accession No. DQ631892) to each other, and they shared 98% identity with TYLCV isolates reported from the Caribbean Region and Florida. This highly virulent begomovirus of tomato, originating in Israel, was first reported in Mexico from 1996 to 1997 when it was identified in tomato plants in the Yucatan Peninsula (1) (>1,500 miles from Sinaloa). The latter report followed the introduction of TYLCV in tomato seedlings from Florida into several eastern U.S. states (3,4) and then into Puerto Rico (2). The introduction of TYLCV into Sinaloa where tomato production is highly concentrated is significant because the region supplies the majority (as much as 93%) of fresh-market tomatoes to the western United States from October to June (>$750 million dollars). Of equal importance is the immediate proximity of the pandemic to California where more than 90% of the processing tomatoes in the United States are grown. References: (1) J. T. Ascencio-Ibáñez et al. Plant Dis. 83:1178, 1999. (2) J. Bird et al. Plant Dis. 85:1028, 2001. (3) M. T. Momol et al. Plant Dis 83:487, 1999. (4) J. E. Polston and P. K. Anderson, Plant Dis. 81:1358, 1997.

Introduction of the New World Squash leaf curl virus to Squash (Cucurbita pepo) in Egypt: A Potential Threat to Important Food Crops.

Squash plants showing leaf curling, yellow mottling, and reduced fruit set were observed in fields in Giza, Egypt in spring 2005. These particular symptoms had not been observed previously in zucchini squash plants in Egypt, but were reminiscent of those caused by begomoviruses (Geminiviridae) that are known to occur in the region, including Watermelon chlorotic stunt virus. Squash plants were heavily infested with the whitefly Bemisia tabaci (Genn.), the only known vector of begomoviruses. Total nucleic acids were isolated from symptomatic squash leaves using the cetyltrimethylammoniumbromide method, and extracts were subjected to polymerase chain reaction (PCR) analysis using two sets of PCR primers. One primer set (prAV2644 and prAC1154) was designed to amplify a fragment that contains the entire viral coat protein (Cp), while the second primer set (prBV1855 and prBC656) was designed to amplify the common region (CR) of DNA-B of begomoviruses (1). The expected size fragments were cloned and the sequence was determined for five clones each. Unexpectedly, the Cp and the CR-B fragments shared their highest nucleotide sequence (nt) identity among well-characterized begomoviruses to the bipartite Squash leaf curl virus (SLCV) native to the western United States. A third primer set (prAC344 and prAV1134) (1) was subsequently used to amplify the remainder of the putative SLCV DNA-A. The fragment was cloned and the DNA sequence was determined. Assembly of the overlapping DNA-A fragments resulted in a complete DNA-A component sequence of 2,636 nt, which is identical to the expected size of the SLCV DNA-A component (GenBank Accession No. DQ285019). Comparison with the latter sequence indicated that the Egyptian squash isolate shared 98% nt identity with SLCV. The sequence for the DNA-B fragment (1,162 nt) shared 94% nt identity with SLCV and was deposited in GenBank as Accession No. DQ285020. The high-shared nt identity with SLCV (2) from the United States suggests that this isolate, herein SLCV-EG, has been introduced into Egypt. The relatively low DNA-B nt sequence identity was a not a surprise since this component is normally less conserved even between strains of a single begomoviral species. Introduction of SLCV is not only potentially significant to the domestic production of crop species in the Cucurbitaceae but also for legume crops. SLCV has a broad host range that also includes members of the Fabaceae, which includes species that contribute significant sources of protein for much of Egypt's population. The virus thus far is thought to be present only in Lower Egypt, however, it could feasibly threaten legume and cucurbit crops if it spreads to Upper Egypt. To our knowledge, this is the first begomovirus of New World origin to become established in the Old World. References: (1) A. M. Idris and J. K. Brown. Phytopathology 88:648, 1998. (2) S. G. Lazarowitz. Virology 180:70, 1991.

Cotton leaf curl Gezira virus-satellite DNAs represent a divergent, geographically isolated Nile Basin lineage: predictive identification of a satDNA REP-binding motif.

Cotton leaf curl Gezira virus (CLCuGV), a species of the genus Begomovirus (family Geminiviridae), was recently cloned from cotton, okra, and Sida alba plants exhibiting leaf-curling and vein-thickening symptoms in Sudan. Here, we describe a previously unknown lineage of single-stranded DNA satellite (satDNA) molecules, which are associated with CLCuGV, and are required for development of characteristic disease symptoms. Co-inoculation of cotton and Nicotiana benthamiana plants with satDNAs cloned from cotton, okra, and S. alba, together with CLCuGV as the 'helper virus' resulted in the development of characteristic leaf-curling and vein-thickening symptoms in both hosts. An anatomical study of symptomatic, virus-infected cotton leaves revealed that spongy parenchyma cells had developed instead of collenchyma cells at the sites of vein thickening. Phylogenetically, the CLCuGV-associated satDNAs from Sudan, together with their closest relatives from Egypt, form a new satDNA lineage comprising only satDNAs from the Upper and Lower Nile Basins. Analysis of satellites and their helper virus sequences identified a predicted REP-binding site consisting of the directly repeated sequence, 'CGGTACTCA', and an inverted repeated sequence, 'TGAGTACCG', which occur in the context of a 17-nucleotide motif. The conserved REP-binding motif identified herein, together with strict geographic isolation, and apparent host-restriction, may be the collective hallmark of these new satDNA-begomovirus lineages, extant in the Nile Basin.

Evidence for interspecific-recombination for three monopartite begomoviral genomes associated with the tomato leaf curl disease from central Sudan.

Two distinct viral genotypes were identified in the same tomato plant collected from Gezira, Sudan and are provisionally designated Tomato leaf curl Sudan virus (ToLCSDV-Gez) and Tomato yellow leaf curl virus-Sudan (TYLCV-SD). A third genotype was identified in tomato samples collected in Shambat, Sudan (ToLCSDV-Sha). The ToLCSDV-Gez and ToLCSDV-Sha isolates were approximately 90% identical, TYLCV-SD from Gezira shared approximately 93% identity with TYLCV-Mld. Recombination analyses identified two fragments in the ToLCSDV-Gez and TYLCV-SD genomes, providing evidence that these two genomes had undergone intermolecular recombination. A half unit size (737 nt) single-stranded satellite DNA was associated with ToLCSDV-Gez and TYLCV-SD.

Sequence analysis and classification of apparent recombinant begomoviruses infecting tomato in the nile and mediterranean basins.

ABSTRACT Numerous whitefly-transmitted viral diseases of tomato have emerged in countries around the Nile and Mediterranean Basins the last 20 years. These diseases are caused by monopartite geminiviruses (family Gemini viridae) belonging to the genus Begomovirus that probably resulted from numerous recombination events. The molecular biodiversity of these viruses was investigated to better appreciate the role and importance of recombination and to better clarify the phylogenetic relationships and classification of these viruses. The analysis partitioned the tomato-infecting begomoviruses from this region into two major clades, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus. Phylogenetic and pairwise analyses together with an evaluation for gene conversion were performed from which taxonomic classification and virus biodiversity conclusions were drawn. Six recombination hotspots and three homogeneous zones within the genome were identified among the tomatoinfecting isolates and species examined here, suggesting that the recombination events identified were not random occurrences.

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.

Cotton leaf crumple virus Is a Distinct Western Hemisphere Begomovirus Species with Complex Evolutionary Relationships Indicative of Recombination and Reassortment.

ABSTRACT The bipartite DNA genome of Cotton leaf crumple virus (CLCrV), a whitefly-transmitted begomovirus from the Sonoran Desert, was cloned and completely sequenced. The cloned CLCrV genome was infectious when biolistically delivered to cotton or bean seedlings and progeny virus was whitefly-transmissible. Koch's postulates were completed by the reproduction of characteristic leaf crumple symptoms in cotton seedlings infected with cloned CLCrV DNA, thereby verifying the etiology of leaf crumple disease, which has been known in the southwestern United States since the 1950s. Sequence comparisons confirmed that CLCrV has a genome organization typical of yet sufficiently divergent from all other bipartite begomoviruses to justify recognition as a distinct species. Phylogenetic analyses indicated that CLCrV has a complex evolutionary history probably involving both recombination and reassortment. The relatively low nucleotide sequence identity (77%) of the common region shared by the CLCrV DNA-A and DNA-B components and the distinct phylogenetic relationships of each component are consistent with component reassortment. Sequence analyses indicated that the CLCrV DNA-A component was likely derived by recombination among ancestors of two divergent clades (e.g., the Squash leaf curl virus [SLCV] clade and the Abutilon mosaic virus clade) of Western Hemisphere begomoviruses. The CLCrV DNA-B component also may have originated by recombination among an ancestor of the SLCV clade and another distantly related but unknown Western Hemisphere begomovirus.

Two Newly Described Begomoviruses of Macroptilium lathyroides and Common Bean.

ABSTRACT Macroptilium lathyroides, a perennial weed in the Caribbean region and Central America, is a host of Macroptilium yellow mosaic Florida virus (MaYMFV) and Macroptilium mosaic Puerto Rico virus (MaMPRV). The genomes of MaYMFV and MaMPRV were cloned from M. lathyroides and/or field-infected bean and the DNA sequences were determined. Cloned A and B components for both viruses were infectious when inoculated to M. lathyroides and common bean. Comparison of the DNA sequences for cloned A and B components with well-studied begomovirus indicated that MaMPRV (bean and M. lathyroides) and MaYMFV (M. lathyroides) are unique, previously undescribed begomo-viruses from the Western Hemisphere. Phylogenetic analysis of viral A components indicated that the closest relative of MaYMFV are members of the Bean golden yellow mosaic virus (BGYMV) group, at 76 to 78% nucleotide identity, whereas the closest relative for the A component of MaMPRV was Rhynchosia golden mosaic virus at 78% nucleotide identity. In contrast, BGYMV is the closest relative for the B component of both MaYMFV and MaMPRV, with which they share approximately 68.0 and approximately 72% identity, respectively. The incongruent taxonomic placement for the bipartite components for MaMPRV indicates that they did not evolve entirely along a common path. MaYMFV and MaMPRV caused distinctive symptoms in bean and M. lathyroides and were transmissible by the whitefly vector and by grafting; however, only MaYMFV was mechanically transmissible. The experimental host range for the two viruses was similar and included species within the families Fabaceae and Malvaceae, but only MaYMFV infected Malva parviflora and soybean. These results collectively indicate that MaMPRV and MaYMFV are new, previously undescribed species of the BGYMV group, a clade previously known to contain only strains and isolates of BGYMV from the Caribbean region that infect Phaseolus spp. Both MaYMFV and MaMPRV may pose an economic threat to bean production in the region.

Emergence of a New Cucurbit-Infecting Begomovirus Species Capable of Forming Viable Reassortants with Related Viruses in the Squash leaf curl virus Cluster.

ABSTRACT Cucurbit leaf curl virus (CuLCV), a whitefly-transmitted geminivirus previously partially characterized from the southwestern United States and northern Mexico, was identified as a distinct bipartite begomovirus species. This virus has near sequence identity with the previously partially characterized Cucurbit leaf crumple virus from California. Experimental and natural host range studies indicated that CuLCV has a relatively broad host range within the family Cucurbitaceae and also infects bean and tobacco. The genome of an Arizona isolate, designated CuLCV-AZ, was cloned and completely sequenced. Cloned CuLCV-AZ DNA A and B components were infectious by biolistic inoculation to pumpkin and progeny virus was transmissible by the whitefly vector, Bemisia tabaci, thereby completing Koch's postulates. CuLCV-AZ DNA A shared highest nucleotide sequence identity with Squash leaf curl virus-R (SLCV-R), SLCV-E, and Bean calico mosaic virus (BCaMV) at 84, 83, and 80%, respectively. The CuLCV DNA B component shared highest nucleotide sequence identity with BCaMV, SLCV-R, and SLCV-E at 71, 70, and 68%, respectively. The cis-acting begomovirus replication specificity element, GGTGTCCTGGTG, in the CuLCV-AZ origin of replication is identical to that of SLCV-R, SLCV-E, and BCaMV, suggesting that reassortants among components of CuLCV-AZ and these begomoviruses may be possible. Reassortment experiments in pumpkin demonstrated that both reassortants of CuLCV-AZ and SLCV-E A and B components were viable. However, for CuLCV-AZ and SLCV-R, only one reassortant (SLCV-R DNA A/CuLCV-AZ DNA B) was viable on pumpkin, even though the cognate component pairs of both viruses infect pumpkin. These results demonstrate that reassortment among sympatric begomovirus species infecting cucurbits are possible, and that, if generated in nature, could result in begomoviruses bearing distinct biological properties.