Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real-time qPCR assay to detect the genus Dickeya and Dickeya dianthicola.

AIMS: Dickeya species are high consequence plant pathogenic bacteria; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, Dickeya dianthicola. METHODS AND RESULTS: A signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome-based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed a detection limit of 10 fg DNA. CONCLUSION: The developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding programme. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.

Draft Genome Sequences of Two Dickeya dianthicola Isolates from Potato.

Here, we report two annotated draft genome sequences of Dickeya dianthicola isolates from potatoes collected in Delaware and West Virginia. The genomes of strains DE440 and WV516 show 99% similarity to each other and 96% and 95% similarity to the European strains IPO 980 and RNS04.9, respectively.

Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.

The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.

Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.

Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.

A novel virus of the genus Cilevirus causing symptoms similar to citrus leprosis.

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

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.

Post-transcriptional gene silencing in controlling viruses of the Tomato yellow leaf curl virus complex.

Tomato yellow leaf curl disease (TYLCD) is caused by a group of geminiviruses that belong to the Tomato yellow leaf curl virus (TYLCV) complex and are transmitted by the whitefly (Bemisia tabaci Genn.). The disease causes great yield losses in many countries throughout the Mediterranean region and the Middle East. In this study, the efficacy of post-transcriptional gene silencing (PTGS) to control the disease caused by TYLCV complex was investigated. Non-coding conserved regions from the genome of TYLCV, Tomato yellow leaf curl virus-mild, tomato yellow leaf curl Sardinia virus, tomato yellow leaf curl Malaga virus, and tomato yellow leaf curl Sardinia virus-Spain [2] were selected and used to design a construct that can trigger broad resistance against different viruses that cause tomato yellow leaf curl disease. The silencing construct was cloned into an Agrobacterium-binary vector in sense and antisense orientation and used in transient assay to infiltrate tomato and Nicotiana benthamiana plants. A high level of resistance was obtained when plants were agro-infiltrated with an infectious clone of the Egyptian isolate of TYLCV (TYLCV-[EG]) or challenge inoculated with TYLCV, TYLCV-Mld, and TYLCSV-ES[2] using whitefly-mediated transmission 16-20 days post infiltration with the silencing construct. Results of the polymerase chain reaction showed that the resistance was effective against all three viruses. Furthermore, dot blot hybridization and PCR failed to detect viral DNA in symptomless, silenced plants. A positive correlation between resistance and the accumulation of TYLCV-specific siRNAs was observed in silenced plants. Together, these data provide compelling evidence that PTGS can be used to engineer geminivirus-resistant plants.

Development of a real-time PCR for Tomato yellow leaf curl Sardinia virus.

Recently, tomato yellow leaf curl disease has become important for the tomato grown both in greenhouse and field conditions in Tunisia. Here, we describe a rapid, specific, reliable, and sensitive real-time PCR, based on TaqMan chemistry, for Tomato yellow leaf curl Sardinia virus (TYLCSV). This method proved suitable for the detection and quantification of this virus in tomato, pepper and bean plants. It detected the virus even in the samples that were negative by conventional assays.

First Report of Tomato spotted wilt virus on Tomatoes in Lebanon.

During the spring and summer of 2004, an epidemic of Tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) was observed in an isolated tomato field at an elevation of 1,000 m in Lebanon. Symptoms were characteristic of TSWV (2). Seedlings came from a nursery in the coastal area of Byblos. In the spring of 2005, TSWV-like symptoms (2) appeared on tomato in the same mountainous area, as well as on tomato, pepper, and lettuce crops in the Byblos coastal area. Initial diagnosis using TSWV ImmunoStrip Tests (Agdia, IN) gave positive results on tomato and lettuce samples. When these samples were analyzed using reverse transcription-polymerase chain reaction, a specific band (619 nt) was observed in symptomatic samples but not in healthy controls (1). Amplicons were cloned into the pGEM-T easy vector (Promega, Madison, WI) and three clones were sequenced in both directions (GenBank Accession No. DQ131804). Sequence analysis revealed more than 99% nucleotide identity (GenBank Accession Nos. AY744476, AJ297611, and AJ418781) and 99% amino acid identity and 100% amino acid similarity (GenBank Accession Nos. AAU95409, CAA85356, and CAD11452) to the nucleocapsid protein of several TSWV isolates. To our knowledge, this is the first report of TSWV in Lebanon. To prevent rapid spread, farmers were informed about the disease, its vector, and appropriate preventive control measures. References: (1) S. Adkins and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) G. Marchoux et al. Plant Pathol. 40:347, 1991.

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.

A New Tomato-Infecting Begomovirus in Barbados.

In September 1998, tomato plants in Barbados exhibited symptoms of severe leaf curling without marginal chlorosis. These symptoms were often associated with an increase in whitefly (Bemisia tabaci) populations. DNA was extracted from leaf tissue from symptomatic tomato plants. Polymerase chain reaction (PCR) was performed with DNA-A degenerate primer pair PAC1v1978/PAV1c715, which amplifies part of the rep gene, the cp gene, and the common region (CR), and with DNA-B primer pair PBC1v2039/PBV1c800, which amplifies part of the bc1 and bv1 genes and the CR (2). The amplified PCR fragments of DNA-A and DNA-B were 1.3 and 1.4 kb, respectively, which are the expected sizes from bipartite, whitefly-transmitted geminiviruses of the Western Hemisphere (2). DNA sequence of the cloned fragments of DNA-A and DNA-B are available as GenBank No. AF213013 and AF213014, respectively. The 181 nucleotides of the CR of DNA-A had a nucleotide identity of 96% with the CR of DNA-B, which indicates that this is a bipartite begomovirus. Pairwise comparisons using DNASTAR (DNASTAR, Madison, WI) of the sequenced part of DNA-A was most similar to Cabbage leaf curl virus (CaLCuV, 69%, U65529) and Squash leaf curl virus extended host range isolate (SqLCV-E, 64%, M38183), and <59% to 13 other bipartite Western Hemisphere geminiviruses and Tomato yellow leaf curl virus from Israel (X15656). Pairwise comparisons of the DNA-B fragment sequence was 59 and 55% similar to CaLCuV (U65530) and SqLCV-E (M38182), respectively. Phylogenetic analysis of DNA-A of the major groups of Western Hemisphere begomoviruses placed the Barbados tomato-infecting geminivirus in the cluster with CaLCuV and SqLCV-E (1), while DNA-B analysis placed it with CaLCuV. The DNA-A amplified fragment was used as a probe at high stringency with the dot blot hybridization assay using the Genius II labeling and detection kit (Boeringer Mannheim) to detect this geminivirus in tomato and several other plant species, which had typical geminiviral symptoms. Strong hybridization signals were obtained for all 23 tomato plants with symptoms, weak signals were observed for two of three muskmelon and two of seven watermelon plants, all with leaf curling symptoms. No hybridization signals were observed for peppers with leaf curling symptoms and two weed species, Macroptilium lathyroides and Rhynchosia minima, with golden mosaic symptoms or with the symptomless plant species used as negative controls. The weak signals observed from watermelon and muskmelon samples indicated the presence of low virus titer or geminiviruses distinct from this tomato virus. The presence of viral DNA in these two plant species was confirmed by PCR with degenerate primers described above. Resulting database searches of sequences in the GenBank revealed that the Barbados tomato virus appears to be a previously unreported virus. This new virus is given the provisional name Tomato leaf curl Barbados virus (ToLCBBV). References: (1) J. C. Faria et al. Phytopathology 84:321, 1994. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

First Report and Characterization of Rhynchosia golden mosaic virus in Honduras.

Rhynchosia minima was suspected to be a weed host of Bean golden yellow mosaic virus (BGYMV, previously designated Bean golden mosaic virus type II). Leaf tissue that exhibited yellow mosaic foliar symptoms characteristic of a geminivirus infection was collected in the Comayagua Valley in Honduras in July 1999. Extraction of viral DNA from the symptomatic leaves was accomplished with the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Subsequent viral DNA amplification was accomplished with degenerative primers for the cp gene (AV494/AC1048) (4). The 570-bp fragment was cloned into the pGEM T-Easy vector (Promega Corp., Madison, WI) producing the recombinant plasmid pRhyb5. The viral insert was sequenced, and from this sequence, specific primers (RHc549 and RHv29) were designed to amplify the remaining part of DNA-A. The 2.1-kb-amplified polymerase chain reaction (PCR) fragment was cloned into the pGEM T-Easy vector producing the recombinant plasmid (pRhya-sp), and the viral insert was sequenced. Nucleotide sequence comparison (GAP program, Wisconsin Package Version 10.0, Genetics Computer Group, Madison, WI) of the complete 2,624-bp DNA-A (GenBank accession no. AF239671) to geminiviruses representing the major phylogenetic clusters (1) showed nucleotide identities ranging from 63 to 82%. Sequence comparisons for the common region and rep, trap, ren, and cp genes with the most closely related geminivirus, Pepper hausteco virus (PHV, X70418), gave 76, 82, 79, 81, and 82% nucleotide identities, respectively. There is a direct repeat (TATCGGT) of 7 nt 5' (viral sense polarity) of the conserved TATA box, and this repeat is most analogous to that in PHV (1). Specific primers were designed in the complementary sense (RGBc2414, BGBc2553) from the common region DNA-A sequence and used with a degenerative viral sense primer for the DNA-B (PBC1v2039) (3) to amplify a 647-bp fragment. Sequence comparison for the common region (134 nt from the rep gene start codon toward the 3' end) from the DNA-B sequence had 88% nt identity to the DNA-A sequence, thus indicating that this geminivirus is bipartite. These sequence analyses indicated that this geminivirus isolated from R. minima is distinct from previously described geminiviruses, and we propose the name Rhynchosia golden mosaic virus (RGMV). From rep gene sequence alignments, RGMV has an apparent genome recombination between Old and New World geminiviruses (Tomato yellow leaf curl virus and Bean dwarf mosaic virus) as previously noted for PHV (2). Our results indicate that RGMV is a distinct geminivirus from BGYMV, and, thus, additional studies are needed to establish the importance of R. minima as a reservoir for vegetable-infecting geminiviruses. This study is the first report of another virus in the PHV phylogenetic cluster and is thus of importance in the understanding of recombinant viruses and their phylogenetic relationship to other characterized geminiviruses. References: (1) J. C. Faria et al. Phytopathology 84:321, 1994. (2) M. Padidam et al. Virology 265:218, 1999. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) S. Wyatt and J. K. Brown. Phytopathology 86:1288, 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.

Polymerase chain reaction detection of viruliferous Bemisia tabaci (Homoptera: Aleyrodidae) with two tomato-infecting geminiviruses.

The sweetpotato whitefly, Bemisia tabaci (Gennadius), is an important pest worldwide. A new biotype of sweetpotato whitefly, biotype B, causes damage by direct feeding and by the transmission of plant viruses, such as geminiviruses. In the Mediterranean area, tomato yellow leaf curl geminivirus (TYLCV) is the most serious disease of tomatoes. Another whitefly-transmitted geminivirus, tomato mottle geminivirus (ToMoV), is presently a serious problem in tomato production in west-central and southwestern Florida. Because of the increasing incidence of whitefly-transmitted geminiviruses, it is necessary to develop rapid and simple diagnostic methods for the detection of viruliferous whiteflies. The polymerase chain reaction is a sensitive and specific technique for the detection and identification of plant pathogens. Polymerase chain reaction methods were used successfully to amplify 1.1-kb DNA fragments from individual viruliferous B. tabaci carrying either TYLCV or ToMoV, and no amplified DNA fragments were obtained when nonviruliferous B. tabaci adults were processed similarly. Southern hybridization analysis proved that fragments amplified from viruliferous B. tabaci adults were viral DNA. This polymerase chain reaction-based detection method is sensitive enough to detect TYLCV and ToMoV in individual viruliferous B. tabaci in mixed samples of up to 25 (1 viruliferous: 24 nonviruliferous) and 10 (1 viruliferous: 9 nonviruliferous) individuals, respectively. The potential uses of this polymerase chain reaction-based detection method in epidermiological studies of geminiviruses are discussed.