Begomoviruses Associated with Bean Golden Mosaic Disease in Nicaragua.

Begomovirus spp. cause substantial losses in bean crops in tropical and subtropical regions of the Americas. The predominant Begomovirus sp. in Central America associated with golden mosaic symptoms in bean is Bean golden yellow mosaic virus (BGYMV). However, Calopogonium golden mosaic virus was previously found to infect bean crops in the northern region of Costa Rica. The objective of this research was to identify Begomovirus spp. that infect bean plants in different geographical regions of Nicaragua. In all, 126 samples of young bean leaves with symptoms of golden mosaic were collected from eight different regions of Nicaragua. Using DNA hybridization with specific probes, 120 samples tested positive for BGYMV, 14 samples tested positive for Squash yellow mild mottle virus, and 7 samples tested positive for Calopogonium golden mosaic virus. Sequence analysis of polymerase chain reaction-amplified products from three samples (MA-9 Managua, BE-8 Rivas, and SO-9 Granada) also indicated that the symptoms of golden mosaic in bean are associated with viral sequences from three different Begomovirus spp. Management of bean golden mosaic disease must take into account that BGYMV is the predominant virus (95% of the samples) and that 12% of the samples exhibited possible mixed infections or recombination events in the south and central geographical regions of Nicaragua.

Use of Tomato leaf curl virus (TYLCV) truncated Rep gene sequence to engineer TYLCV resistance in tomato plants.

Tomato yellow leaf curl disease causes severe losses in tomato production throughout Mediterranean countries including Tunisia. In order to generate engineered resistance to this disease, an intron-hairpin RNA construct harboring a Tomato yellow leaf curl Sardinia virus (TYLCSV) truncated replication-associated protein (Rep) gene was used to transform genotype of tomato plants. Prepared transgenic plants were agro-inoculated with Tunisian infectious strain of TYLCSV and screened for the resistance to infection. The infected transgenic plants were divided into 3 different groups according to their specific symptoms. Only one of them contained transgenic plants fully resistant to the tomato yellow leaf curl disease.

Evaluation of two gene-silencing constructs for resistance to tomato yellow leaf curl viruses in Nicotiana benthamiana plants.

Infiltration of Agrobacterium tumefaciens into intact plant leaves of N. benthamiana was used to test the efficiency of two virus-based silencing constructs conferring resistance to the closely related begomoviruses. The constructs contained the most conserved sequences of the coat protein (CP) gene and replication-associated protein (Rep) gene of Tomato yellow leaf curl Sardinia virus (Sicily strain) (TYLCSV-[Sic]). Both constructs formed a hairpin structure that enhanced the post-transcriptional gene-silencing mechanism. When agro-infiltrated plants were challenged separately with infectious viruses TYLCSV-[Sic] and Tomato yellow leaf curl virus (TYLCV), the plants showed resistance to TYLCSV-[Sic], but not to the related TYLCV.

Photosystem II Excitation Pressure and Photosynthetic Carbon Metabolism in Chlorella vulgaris.

Chlorella vulgaris grown at 5[deg]C/150 [mu]mol m-2 s-1 mimics cells grown under high irradiance (27[deg]C/2200 [mu]mol m-2 s-1). This has been rationalized through the suggestion that both populations of cells were exposed to comparable photosystem II (PSII) excitation pressures measured as the chlorophyll a fluorescence quenching parameter, 1 - qP (D.P. Maxwell, S. Falk, N.P.A. Huner [1995] Plant Physiol 107: 687-694). To assess the possible role(s) of feed-back mechanisms on PSII excitation pressure, stromal and cytosolic carbon metabolism were examined. Sucrose phosphate synthase and fructose-1,6-bisphosphatase activities as well as the ratios of fructose-1,6-bisphosphate/fructose-6-phosphate and sucrose/starch indicated that cells grown at 27[deg]C/2200 [mu]mol m-2 s-1 appeared to exhibit a restriction in starch metabolism. In contrast, cells grown at 5[deg]C/150 [mu]mol m-2 s-1 appeared to exhibit a restriction in the sucrose metabolism based on decreased cytosolic fructose-1,6- bisphosphatase and sucrose phosphate synthase activities as well as a low sucrose/starch ratio. These metabolic restrictions may feed-back on photosynthetic electron transport and, thus, contribute to the observed PSII excitation pressure. We conclude that, although PSII excitation pressure may reflect redox regulation of photosynthetic acclimation to light and temperature in C. vulgaris, it cannot be considered the primary redox signal. Alternative metabolic sensing/signaling mechanisms are discussed.

Redox Regulation of Light-Harvesting Complex II and cab mRNA Abundance in Dunaliella salina.

We demonstrate that photosynthetic adjustment at the level of the light-harvesting complex associated with photosystem II (LCHII) in Dunaliella salina is a response to changes in the redox state of intersystem electron transport as estimated by photosystem II (PSII) excitation pressure. To elucidate the molecular basis of this phenomenon, LHCII apoprotein accumulation and cab mRNA abundance were examined. Growth regimes that induced low, but equivalent, excitation pressures (either 13[deg]C/20 [mu]mol m-2 s-1 or 30[deg]C/150 ([mu]mol m-2 s-1) resulted in increased LHCII apoprotein and cab mRNA accumulation relative to algal cultures grown under high excitation pressures (either 13[deg]C/150 [mu]mol m-2 s-1 or 30[deg]C/2500 [mu]mol m-2 s-1). Thermodynamic relaxation of high excitation pressures, accomplished by shifting cultures from a 13 to a 30[deg]C growth regime at constant irradiance for 12 h, resulted in a 6- and 8-fold increase in LHCII apoprotein and cab mRNA abundance, respectively. Similarly, photodynamic relaxation of high excitation pressure, accomplished by a shift from a light to a dark growth regime at constant temperature, resulted in a 2.4- to 4-fold increase in LHCII apoprotein and cab mRNA levels, respectively. We conclude that photosynthetic adjustment to temperature mimics adjustment to high irradiance through a common redox sensing/signaling mechanism. Both temperature and light modulate the redox state of the first, stable quinone electron acceptor of PSII, which reflects the redox poise of intersystem electron transport. Changes in redox poise signal the nucleus to regulate cab mRNA abundance, which, in turn, determines the accumulation of light-harvesting apoprotein. This redox mechanism may represent a general acclimation mechanism for photosynthetic adjustment to environmental stimuli.

Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (I. Light-Harvesting Complex II Abundance and Zeaxanthin Content in Chlorella vulgaris).

The basis of the increased resistance to photoinhibition upon growth at low temperature was investigated. Photosystem II (PSII) excitation pressure was estimated in vivo as 1 - qp (photochemical quenching). We established that Chlorella vulgaris exposed to either 5[deg]C/150 [mu]mol m-2 s-1 or 27[deg]C/2200 [mu]mol m-2 s-1 experienced a high PSII excitation pressure of 0.70 to 0.75. In contrast, Chlorella exposed to either 27[deg]C/150 [mu]mol m-2 s-1 or 5[deg]C/20 [mu]mol m-2 s-1 experienced a low PSII excitation pressure of 0.10 to 0.20. Chlorella grown under either regime at high PSII excitation pressure exhibited: (a) 3-fold higher light-saturated rates of O2 evolution; (b) the complete conversion of PSII[alpha] centers to PSII[beta] centers; (c) a 3-fold lower epoxidation state of the xanthophyll cycle intermediates; (d) a 2.4-fold higher ratio of chlorophyll a/b; and (e) a lower abundance of light-harvesting polypeptides than Chlorella grown at either regime at low PSII excitation pressure. In addition, cells grown at 5[deg]C/150 [mu]mol m-2 s-1 exhibited resistance to photoinhibition comparable to that of cells grown at 27[deg]C/2200 [mu]mol m-2 s-1 and were 3- to 4-fold more resistant to photoinhibition than cells grown at either regime at low excitation pressure. We conclude that increased resistance to photoinhibition upon growth at low temperature reflects photosynthetic adjustment to high excitation pressure, which results in an increased capacity for nonradiative dissipation of excess light through zeaxanthin coupled with a lower probability of light absorption due to reduced chlorophyll per cell and decreased abundance of light-harvesting polypeptides.

Growth at Low Temperature Mimics High-Light Acclimation in Chlorella vulgaris.

Structural and functional alterations to the photosynthetic apparatus after growth at low temperature (5[deg]C) were investigated in the green alga Chlorella vulgaris Beijer. Cells grown at 5[deg]C had a 2-fold higher ratio of chlorophyll a/b, 5-fold lower chlorophyll content, and an increased xanthophyll content compared to cells grown at 27[deg]C even though growth irradiance was kept constant at 150 [mu]mol m-2 s-1. Concomitant with the increase in the chlorophyll a/b ratio was a lower abundance of light-harvesting polypeptides in 5[deg]C-grown cells as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by western blotting.The differences in pigment composition were found to be alleviated within 12 h of transferring 5[deg]C-grown cells to 27[deg]C. Furthermore, exposure of 5[deg]C-grown cells to a 30-fold lower growth irradiance (5 [mu]mol m-2 s-1) resulted in pigment content and composition similar to that in cells grown at 27[deg]C and 150 [mu]mol m-2 s-1. Although both cell types exhibited similar measuring-temperature effects on CO2-saturated O2 evolution, 5[deg]C-grown cells exhibited light-saturated rates of O2 evolution that were 2.8-and 3.9-fold higher than 27[deg]C-grown cells measured at 27[deg]C and 5[deg]C, respectively. Steady-state chlorophyll a fluorescence indicated that the yield of photosystem II electron transport of 5[deg]C-grown cells was less temperature sensitive than that of 27[deg]C-grown cells. This appears to be due to an increased capacity to keep the primary, stable quinone electron acceptor of photosystem II (QA) oxidized at low temperature in 5[deg]C- compared with 27[deg]C-grown cells regardless of irradiance. We conclude that Chlorella acclimated to low temperature adjusts its photosynthetic apparatus in response to the excitation pressure on photosystem II and not to the absolute external irradiance. We suggest that the redox state of QA may act as a signal for this photosynthetic acclimation to low temperature in Chlorella.

Mutational analysis of the putative nicking motif in the replication-associated protein (AC1) of bean golden mosaic geminivirus.

Geminiviruses are circular single-stranded DNA viruses that replicate by a rolling circle mechanism involving the viral-encoded AC1 protein. DNA nicking is necessary both for initiating replication of the covalently closed double-stranded DNA templates and for releasing unit-length monomers. The effects of mutations in a putative nicking motif (K101 A Y I D K106; E. V. Koonin and T. V. Ilyina, J. Gen. Virol. 73:2763-2766, 1992) of the AC1-derived protein for bean golden mosaic geminivirus isolate GA (BGMV-GA) were studied. The amino acids equivalent to Y103 and K106 of BGMV-GA are invariant in all whitefly-transmitted geminiviruses. Phaseolus vulgaris plant infectivity assays showed that the mutants K101-->H, K101-->A, and D105-->T produced symptoms, but mutants Y103-->A, Y103-->F, K106-->R, and K106-->H did not. A mutant with a stop codon in the N terminus of the AC4 open reading frame (ORF) produced the same symptoms as the wild-type BGMV-GA. Only those that were infectious replicated in NT-1 tobacco suspension cells. These results indicate that the Y103 and K106 residues are essential for replication, and that this putative DNA-nicking motif of the AC1 ORF may be functional in the rolling circle mechanism of replication for geminiviruses. The potential role of these mutants in the design of antiviral strategies is discussed.

The complete nucleotide sequence and genome organization of bean common mosaic virus (NL3 strain).

The nucleotide sequences of 3 cDNA clones corresponding to entire RNA genome of bean common mosaic virus NL3 strain have been determined. The RNA is 9612 nucleotides long, excluding a 3'-terminal poly(A) tail. A putative start codon located at nucleotide positions 170-172 initiates one large open reading frame that is terminated with a UAA codon at position 9368-9370. The predicted polyprotein has 3066 amino acids and an M(r) of 340.3 kDa. The positions of putative protein cleavage sites have been determined by analogy to consensus sequences in other potyviruses. The nucleotide sequences of the non-translated regions and the predicted amino acid sequences of BCMV NL3, were compared with those of other potyviruses. Comparison of the BCMV NL3 proteins with those of other potyviruses indicated a similar genomic organization, and high percentage of amino acid sequence identity in the cylindrical inclusion protein, nuclear inclusion 'b' protein and coat protein. BCMV NL3 displays the highest amino acid sequence identity with soybean mosaic virus.

An eye for an eye. A simplified model for teaching.

We developed an easily constructed, inexpensive eye model that consists of a Styrofoam head carved out to house a slide holder, a wide-angle, clinical photographic fundus slide, a plastic face, and an anatomically correct eye. The model can be used by students and residents to learn direct and indirect ophthalmoscopy, fundus photography, and laser photocoagulation. This model gives students and residents a realistic learning experience without inconveniencing or endangering patients.

trans-Dominant Inhibition of Geminiviral DNA Replication by Bean Golden Mosaic Geminivirus rep Gene Mutants.

ABSTRACT Geminiviruses are a group of single-stranded DNA viruses that cause major losses on a number of important crops throughout the world. Bean golden mosaic virus (BGMV) is a typical bipartite, whitefly-transmitted geminivirus that causes a severe disease on beans (Phaseolus vulgaris) in the Western Hemisphere. The lack of natural resistance to geminiviruses has led to attempts to engineer resistance, particularly through the use of pathogen-derived resistance strategies. The rep gene contains several conserved domains including nucleoside triphosphate (NTP)-binding and DNA-nicking domains and is the only geminiviral gene necessary for replication. Previous analysis by our group and others has demonstrated that the NTP-binding and DNA-nicking domains are necessary for geminiviral DNA replication. The ability of the rep gene and rep gene mutants to interfere with geminiviral DNA replication, when expressed in trans, was examined using a transient assay in a tobacco suspension cell culture system. Wild-type (wt) and mutant rep genes were cloned into plasmids under the control of the cauliflower mosaic virus 35S promoter for in planta expression and coinoculated into tobacco cells with infectious clones of various geminiviruses. The wt rep gene from BGMV-GA was able to support replication of BGMV-GA DNA-B. Several different rep gene mutants, with function-abolishing mutations in the NTP-binding or DNA-nicking domains, were potent trans-dominant inhibitors of geminiviral DNA replication.

Variability in Geminivirus Isolates Associated with Phaseolus spp. in Brazil.

ABSTRACT Bean golden mosaic geminivirus (BGMV) is the single most devastating virus of common beans in the tropical and subtropical Americas and the Caribbean Basin. The BGMV from Brazil, named BGMV-BZ, is considered distinct from BGMV-PR isolates from Puerto Rico, Guatemala, and the Dominican Republic because of DNA sequence data, the ability to form pseudorecombinants, and mechanical transmissibility properties. In bean-growing areas of Brazil, samples were collected from beans, lima beans, and the weed Leonurus sibiricus displaying typical symptoms of infection by geminiviruses. Viral DNA fragments comprising part of the rep gene, the common region, and part of the cp gene were amplified by polymerase chain reaction, cloned, and sequenced. The bean samples had geminivirus with sequences nearly identical to that of BGMV-BZ collected in Goiânia, state of Goiás, in 1986. The sample from lima bean contained a new species of geminivirus that induces symptoms similar to those induced by BGMV-BZ and was named lima bean golden mosaic virus (LBGMV-BR). While all sequences from bean samples clustered with BGMV-BZ, the sequence from the lima bean isolate stood alone. A mixed infection with abutilon mosaic geminivirus was also found in a single sample from the state of São Paulo. DNA sequence comparisons indicate that the virus isolate from L. sibiricus represents a new geminivirus species, designated here as leonurus mosaic virus.

A natural history study of experimental Staphylococcus epidermidis endophthalmitis.

The purpose of this study was to find a "threshold" quantity of organisms (i.e. inoculum) to produce clinical endophthalmitis and determine the natural course of intravitreal bacterial counts following inoculation in a rabbit model of Staphylococcus epidermidis endophthalmitis. S. epidermidis endophthalmitis was induced experimentally in 18 New Zealand white rabbits. Eyes were injected with 2.0 x 10(3) (Group I: n = 3), 2.0 x 10(4) (Group II: n = 3), 3.0 x 10(5) (Group III: n = 3), 3.0 x 10(6) (Group IV: n = 3), 3.0 x 10(7) (Group V: n = 3), or 3.0 x 10(8) (Group VI: n = 3) organisms. Serial quantitative bacterial cultures (colony counts) were performed on the vitreous every eight hours for 9 days. All eyes in Groups I and II became culture negative by 24-64 hours post-inoculation (PI). All eyes in Groups III-VI remained culture positive [approximately 600-4000 colony forming units (CFU) per cm3] at 48 to 72 hours PI and were stable for the remainder of the nine day study period. Previous work suggests that the host's inflammatory response is more important than had been recognized. Previous rabbit models of infectious endophthalmitis are known to become culture negative ("autosterilized") despite continued intraocular inflammation. This rabbit model demonstrates a "threshold" of infection where the host's immune response is overwhelmed and "autosterilization" does not occur. When inoculated with 3.0 x 10(5) or greater S. epidermidis organisms of this strain, continued active bacterial replication can now be studied in the rabbit.

First Report and Partial Molecular Characterization of a New Begomovirus Associated with Pigeon Pea in Puerto Rico.

Pigeon pea (Cajanus cajan (L.) Millsp.) is an important edible legume crop in Puerto Rico. In late fall 1997, symptoms of a golden mosaic virus were observed in a pigeon pea planting in the municipality of Villalba. The symptoms resembled those incited by the Rhynchosia mosaic virus, a whitefly-transmitted virus, in pigeon pea (1). Tests with the 3F7 antigeminivirus antibody from Agdia (Elkhart, IN) confirmed the presence of a begomovirus in these symptomatic pigeon peas. Extraction of DNA from dried symptomatic foliar tissue was accomplished by the methods described in Rojas et al. (2), and subsequent viral DNA amplification was accomplished with the coat protein (CP) gene polymerase chain reaction primer pair AV494-AC1048 (3). A 550-bp fragment was cloned and sequenced (GenBank Accession No. AY028308). For sequence analysis, the BLAST program at the National Institutes of Health was used. The first match was 91% with the CP gene sequence (Accession No. AF070924) of a begomovirus from Clitoria falcata from Puerto Rico. The next three matches were approximately 86% with a begomovirus (accession no. AF058024) from Macroptilium lathyroides from Puerto Rico, Dicliptera yellow mottle virus (Accession No. AF139168) from Florida, and Tobacco apical stunt virus (Accession No. AF076855) from Mexico. On the basis of sequence analysis of this conserved region of the CP gene, it is concluded that the virus described in this report is a new virus, and thus named Pigeon pea golden mosaic virus. The only sequence for a begomovirus from a Rhynchosia sp. in GenBank is Rhynchosia golden mosaic virus from Honduras (accession no. AF239671). On the basis of sequence comparisons, the virus from pigeon pea is not an isolate of the Honduran Rhynchosia golden mosaic virus. References: (1) J. Bird et al. 1975. Pages 3-25 in: Tropical Diseases of Legumes. J. Bird and K. Maramorosch, eds. Academic Press, NY. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) S. D. Wyatt and J. K. Brown. Phytopathology. 86:1288, 1996.

Calopogonium golden mosaic virus Identified in Phaseolus vulgaris from Western and Northern Regions of Costa Rica.

Bean golden yellow mosaic virus (BGYMV, GenBank Accession No. M91604) is reported to be the main bean-infecting (Phaseolus vulgaris) begomovirus in Central America (1,2). Another begomovirus, Bean calico mosaic virus (BCaMV, GenBank Accession No. AF110189), is known to infect P. vulgaris in central Mexico. Bean samples with the viral symptom of foliar chlorosis were collected in different regions of Costa Rica from 1994 to 2001. Total nucleic acids were extracted and tested using polymerase chain reaction (PCR) with degenerate primer pair PBL1v2039/PCRc2 for DNA-B, which can give a different fragment size for some begomoviruses (4). PCR fragments were obtained from 119 bean samples. Fifty samples from the central valley produced 600-bp PCR fragments, which is typical for BGYMV (4), and one sample produced two fragments (500 and 600 bp). One sample from western Costa Rica had a 550-bp fragment, and 69 samples from the western and northern regions produced 500-bp fragments. One of the latter samples (No. 01-1152) was selected for PCR with degenerate primer pairs PAL1v1978/PAR1c715 for DNA-A and PBL1v2039/PBR1v800 for DNA-B (4). The amplified PCR fragments were 1.35 and 1.4 kb for DNA-A and DNA-B, respectively. These are expected sizes for a Western Hemisphere begomovirus. The DNA-A fragment was cloned and sequenced (GenBank Accession No. AF439402). Sequence comparisons indicated that this uncharacterized begomovirus (No. 01-1152) had 98, 97, and 93% nucleotide identities with the rep gene, common region, and coat protein gene promoter of Calopogonium golden mosaic virus (CalGMV, GenBank Accession Nos. AF439748 and AF439749, respectively) (3). CalGMV had been isolated from a weed, Calopogonium sp., collected in 1991 near Quepos in the west and Tilaran in the north of Costa Rica (3). Phylogenetic analysis of the viral sequence of the begomovirus associated with No. 01-1152 placed it in the Squash leaf curl virus clade (1). BCaMV is also in this phylogenetic clade, but sequence comparisons between CalGMV and BCaMV had <90% nucleotide identity, and so are distinct species. CalGMV is the begomovirus associated with No. 01-1152, and this is an example of the movement of a weed-infecting begomovirus into an agricultural crop. Since this Calopogonium sp. is widespread in Central America, it is important to determine if CalGMV is present in beans in other regions. The data also indicate that BGYMV and CalGMV infect beans in two geographically distinct regions of Costa Rica. References: (1) J. C. Faria et al. Phytopathology 84:321, 1994. (2) W. McLaughlin et al. Plant Dis. 78:1220, 1994. (3) M. R. Rojas et al. Annu. Rep. Bean Improv. Coop. 35:150, 1992. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

PCR and DNA Hybridization Methods for Specific Detection of Bean-Infecting Begomoviruses in the Americas and Caribbean.

Begomoviruses are a major problem for common bean production in the tropics and subtropics of the Americas and the Caribbean. Multiplex polymerase chain reaction (PCR) primer pairs and nucleic acid hybridization probes have been developed to differentiate five bean-infecting begomoviruses and were used to assay reference and field-collected bean samples from Florida, Mexico, Central America, the Caribbean, and Brazil. Bean golden mosaic virus was found in Brazil, Bean calico mosaic virus in Mexico, and Bean golden yellow mosaic virus in Central America, the Caribbean, and Florida. Bean dwarf mosaic virus was not detected in any of the field samples. Tomato yellow leaf curl virus was found only in tomato samples from the Caribbean. These detection methods will provide tools to assist in the understanding of the epidemiology and diversity of geminiviruses as well as to facilitate resistance breeding, cultivar selection, and development of strategies for control.

First Report of Tomato yellow leaf curl virus Associated with Beans, Phaseolus vulgaris, in Cuba.

Beans with yellow mosaic and/or leaf crumple symptoms were collected in three fields in the southern area of the province of Havana, Cuba in December 2001 and February 2002. DNA was extracted from the fresh bean leaves of 25 samples (1). Dot blot hybridization was performed at high stringency with a specific probe for Tomato yellow leaf curl virus (TYLCV). The specific probe was prepared by alkaline phosphatase labeling of the polymerase chain reaction (PCR) fragment amplified with primer pair, PTYIRv21/PTYIRc287, containing the intergenic region (IR) of TYLCV, and chemiluminescent hybridization was completed as described by the manufacturer (AlkPhos Direct Labeling and Detection Systems, Amersham Pharmacia Biotech Inc., Piscataway, NJ). Four of the samples had positive hybridization signals. PCR was performed with overlapping primers for TYLCV (2) with the DNA extract from sample 01-44, which gave a positive hybridization signal with the TYLCV probe, and a 2.8-kb fragment was obtained. This fragment was cloned in pGem T-Easy (pBeTY44) and partially sequenced. Greater than 96% nt identity was obtained for the 591 nt of the IR and 504 nt of the N-terminus of the Rep gene with TYLCV (GenBank Accession No. AF260331). Also, PCR was completed on 11 of the 25 samples with the degenerate primer pair PAL1v1978/PAR1c715 for DNA-A (3). Eight samples gave fragment sizes of 1.4 kb and one sample gave a fragment of 1.3 kb. The 1.3-kb fragment from sample number 01-50 was cloned in pGem T-Easy (pBeBG50) and partially sequenced. Pairwise nucleotide comparisons with Bean golden yellow mosaic virus (BGYMV, GenBank Accession No. M91604) were 95% for 719 nt of the N-terminus of the Rep gene. These results are consistent with the association of both TYLCV and BGYMV in beans and have important implications for future disease management strategies. References: (1) G. P. Accotto et al. Eur. J. Plant. Pathol. 106:179, 2000. (2) M. K. Nakhla et al. Plant Dis. 78:926, 1994. (3) M. Rojas et al. Plant Dis. 77:340, 1993.

A New Geminivirus Associated with Tomato in the State of São Paulo, Brazil.

The apical growth of about 20% of young tomato plants in observed fields near Campinas, State of São Paulo, Brazil, had yellow streaking of veins. Leaf symptoms developed into patches of yellow mosaic and the leaves became wavy. The whitefly Bemisia tabaci Genn. transmitted a pathogen from the infected tomato plants to healthy tomato and potato plants, reproducing the original symptoms in tomato. The apical leaves of infected potatoes showed yellow or green mottle that developed into leaf distortion with yellow blotches, symptoms indistinguishable from potato-deforming mosaic disease (2). DNA was extracted from these tomato and potato plants (1). Using DNA from the infected tomato plant, polymerase chain reaction (PCR) with the degenerate primer pair PAC1v1978/ PAV1c715 (1), which amplifies part of the rep gene (AC1 ORF), the common region (CR), and part of the cp gene (AV1 ORF), and with the primer pair PBC1v2039/PBV1c800, which amplifies part of BC1 ORF, CR, and part of BV1 ORF, gave virus-specific DNA fragments of the sizes expected from a whitefly-transmitted geminivirus. These were cloned and the complete nucleotide (sequences for DNA-A (pToYA, GenBank accession no. U79998) and DNA-B (pToYB, GenBank accession no. U80042) fragments obtained. Nucleotide identity between the CRs (184 nucleotides) was 90%, strongly indicating that those fragments correspond to a bipartite subgroup III geminivirus. PCR with the DNA from infected potato gave the expected size fragment for DNA-A. The partial sequence of the rep gene was 100% identical to the homologous sequence from the PCR fragment from the infected tomato. A search in the GenBank, EMBL, DDBJ, and PDB databases, using the BLAST program, found no identical geminivirus. The highest identity for the CR was 75% to tomato mottle geminivirus-Florida (ToMoV) and 74% to bean golden mosaic virus-Brazil. For the rep gene, the highest identity was 73% to tomato yellow leaf curl virus-Israel, an Old World geminivi-rus, followed by 71% to tomato golden mosaic virus-Brazil (TGMV) and ToMoV. For the cp gene, the highest identity was 86% to TGMV, followed by 83% to squash leaf curl geminivirus. Therefore, we propose the name tomato yellow vein streak geminivirus (ToYVSV) for this distinct virus (2). References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) J. A. C. Souza-Dias et al. Summa Phytopathol. 22:57, 1996.

A New Geminivirus Associated with a Leaf Curl Disease of Tomato in Tanzania.

Leaf samples of tomato exhibiting yellow mottle, severe leaf curl, stunting, and upright stems were collected from Makutupora, Tanzania, in October 1994 by L. L. Black (AVRDC). Leaf tissue squashes on nylon membranes did not hybridize with DNA-A probes from tomato yellow leaf curl geminiviruses (TYLCVs) from Thailand (Thai) or Egypt (EG), an isolate of TYLCV-Isr (Israel). Polymerase chain reaction (PCR) with primer pair PAC1v1978/PAV1c715 (2), which specifically amplifies part of the rep (AC1) open reading frame (ORF), the intergenic region, and the cp (AV1) ORF of whitefly-transmitted geminiviruses, yielded a 1.5-kb fragment from a DNA extract of symptomatic tomato leaves. No virus-specific fragments were amplified from symptomless tomato leaves. The nucleotide sequence (GenBank accession no. U73478) of the PCR fragment (recombinant plasmid pAF1) was compared with the sequences of seven distinct subgroup III geminiviruses that infect tomato (1)-TYLCV-Isr, TYLCV-Sar (Sardinia), TYLCV-Thai, tomato leaf curl virus (TLCV)-;Aus (Australia), IndTLCV (India), TLCV-Ind (Bangalore I), and TLCV-Tai (Taiwan)-as well as three other subgroup III geminiviruses from the Old World: Indian cassava mosaic virus, African cassava mosaic virus, and mung bean yellow mosaic virus. Nucleotide sequence identities for the pairwise comparisons of the rep ORF (692 nucleotides) and the intergenic region (169 nucleotides) of this Tanzanian geminivirus with those of the 10 Old World geminiviruses showed low nucleotide identities, which were <79% for the rep ORF and <67% for the intergenic region. Since isolates of the same geminivirus usually have nucleotide sequence identities >90% (1), this Tanzanian geminivirus is considered to be different from all previously characterized Old World geminiviruses and is given the name TLCV-Tan. Further, tissue squash blots of samples previously collected from other areas in Tanzania gave strong positive reactions with the TYLCV-EG probe, so yet another geminivirus closely related to TYLCV-Isr may be present. References: (1) M. Padidam et al. J. Gen. Virol. 76:249, 1995. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.