Douglas P. Maxwell

Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses

Geminiviruses are widely recognised as a serious threat to vegetable production in many tropical and subtropical regions. This has increased the need for accurate identification of these viruses. Geminiviruses are well suited to polymerase chain reaction (PCR) methods because they replicate via a double-stranded, circular DNA form. Degenerate PCR primers were designed to anneal to highly conserved nucleotide sequences identified in the genomes of 10 whitefly-transmitted geminiviruses. The PCR primers were tested for their effectiveness in the amplification of viral DNA fragments from the DNA-A and/or DNA-B components of 15 previously uncharacterized geminiviruses from the Americas, the Caribbean Basin, and Africa (.)

Tomato yellow leaf curl virus in the Dominican Republic: Characterization of an Infectious Clone, Virus Monitoring in Whiteflies, and Identification of Reservoir Hosts

ABSTRACT Epidemics of tomato yellow leaf curl disease (TYLCD) in the Dominican Republic in the early to mid-1990s resulted in catastrophic losses to processing tomato production. As part of an integrated management approach to TYLCD, the complete nucleotide sequence of a full-length infectious clone of an isolate of Tomato yellow leaf curl virus (TYLCV) from the Dominican Republic (TYLCV-[DO]) was determined. The TYLCV-[DO] genome was nearly identical in sequence (>97%) and genome organization to TYLCV isolates from Israel and Cuba. This established that TYLCV-[DO] is a bonafide TYLCV isolate (rather than a recombinant virus, such as isolates from Israel [Mild], Portugal, Japan, and Iran), and provided further evidence for the introduction of the virus from the eastern Mediterranean. A reduction in the incidence of TYLCV in the northern and southern processing tomato production areas of the Dominican Republic has been associated with the implementation of a mandatory 3-month whitefly host-free period (including tomato, common bean, cucurbits, eggplant, and pepper). Monitoring TYLCV levels in whiteflies, by polymerase chain reaction with TYLCV-specific primers, established that the incidence of TYLCV decreased markedly during the host-free period, and then gradually increased during the tomato-growing season. In contrast, TYLCV persisted in whiteflies and tomato plants in an area in which the host-free period was not implemented. Surveys for TYLCV reservoir hosts, conducted to identify where TYLCV persists during the host-free period, revealed symptomless infections in a number of weed species. The implications of these findings for TYLCV management in the Dominican Republic are discussed.

Pseudorecombination between infectious cloned DNA components of tomato mottle and bean dwarf mosaic geminiviruses

A newly described whitefly-transmitted geminivirus infecting tomato plants in Florida induces yellow mottling symptoms on leaves, and stunted and distorted growth. The DNA-A and DNA-B components were cloned from extracts of field-infected tomato tissue; excised monomers or uncut tandem dimers of these clones were infectious when co-inoculated on to Nicotiana benthamiana by rub-inoculation. Tomato plants inoculated directly with the DNA-A and DNA-B dimers, or indirectly by sap or graft transmission from N. benthamiana plants previously infected with the dimers, developed symptoms similar to those observed in field-infected plants. This tomato geminivirus is different from previously characterized geminiviruses, and has been named tomato mottle geminivirus (ToMoV). DNA sequence comparisons revealed that ToMoV is closely related to bean dwarf mosaic geminivirus (BDMV) and abutilon mosaic geminivirus. Infectious pseudorecombinants were made by exchanging the cloned infectious DNA components of ToMoV and BDMV and inoculating N. benthamiana plants. The presence of the inoculated DNA components in systemically infected plants was confirmed by characterization of DNA-A and DNA-B fragments amplified by the polymerase chain reaction. This is the first report of pseudorecombination between two distinct geminiviruses. The implications of this finding in geminivirus evolution are discussed.

Differentiation of bean-infecting geminiviruses by nucleic acid hybridization probes and aspects of bean golden mosaic in Brazil

A bean golden mosaic geminiviral isolate from Goiania, Goias, Brazil (BGMV-BZ), was determined to be transmitted by whiteflies and induced golden mosaic symptoms and diagnostic geminiviral inclusion bodies and ultrastructural abnormalities in infected bean leaves. In contrast to BGMV isolates from Central America and the Caribbean, BGMV-BZ could not be sap-transmitted to beans. Cloned geminiviral DNA components were used as DNA probes for the rapid and specific detection of BGMV-BZ and three sap-transmissible bean-infecting geminiviral isolates-BGMV from Guatemala (GBMV-GA) and the Dominican Republic (BGMV-DR) and bean dwarf mosaic geminivirus from Colombia (BDMV-CO) (.)

Use of the asymmetric polymerase chain reaction and DNA sequencing to determine genetic variability of bean golden mosaic geminivirus in the Dominican Republic.

A combination of the polymerase chain reaction (PCR), asymmetric PCR (A-PCR) and DNA sequencing was used to determine the nucleotide sequence of a hypervariable region of the bipartite genome of bean golden mosaic geminivirus (BGMV). This region, which was part of the intergenic region of the DNA-B component, was amplified using primers designed from the nucleotide sequence of a DNA-B component clone (pDRB1) of an isolate of BGMV from the Dominican Republic (BGMV-DR). pDRB1 is infectious on beans when coinoculated with the DNA-A component of BGMV-DR (pDRA1), and typical bean golden mosaic symptoms are observed on infected plants. Bean leaf tissue infected with BGMV was collected at five separate field locations in the Dominican Republic and the hypervariable region was amplified by PCR, ssDNA was produced using A-PCR, and partial nucleotide sequences were determined. The sequences of the hypervariable region from the field-collected samples ranged from 95% (one sample) to 98% (four samples) identical to the sequence of pDRB1. This contrasts with sequence identities of 86, 75 and 46% between the pDRB1 hypervariable region and the hypervariable regions of BGMV isolates from Guatemala, Puerto Rico and Brazil respectively, and 42% with bean dwarf mosaic geminivirus. These results indicate that Dominican Republic isolates of BGMV are very similar and should be considered isolates of the same virus (BGMV-DR), and that the infectious clones of BGMV-DR are representative of BGMV isolates in the Dominican Republic. The procedures described for DNA extraction from leaf tissue and for production of high quality ssDNA using PCR and A-PCR are rapid and efficient and could be applied to studies of variability and epidemiology of other viruses.

ESTIMATION OF RELATEDNESS BETWEEN PHYTOPHTHORA SPECIES BY ANALYSIS OF MITOCHONDRIAL DNA

The relatedness between four Phytophthora megasperma isolates [P. megasperma f. sp. glycinea (Pmg) from soybean, P. megasperma f. sp. medicaginis (Pmm) from alfalfa, and two broad host-range isolates from apple or alfalfa], P. cactorum, P. cryptogea, and P. parasitica var. nicotianae (Ppn) was investigated by a quantitative analysis of restriction fragment length polymorphisms of mitochondrial DNAs. Nucleotide sequence divergence values among P. megasperma isolates were within the same range as between the P. megasperma isolates and the three other Phytophthora species. An unrooted phylogenetic network based on nucleotide sequence divergence values indicated that several evolutionary lines exist for P. megasperma. These data are consistent with the observed variability of morphological, physiological and biochemical characteristics within this fungal group and support the contention that P. megasperma comprises several biological species which are the result of convergent evolution and which are not closely related. A consensus restriction map of the mtDNAs of Pmg, Pmm, and Ppn was constructed, and numerical analyses of conserved and variable restriction cleavage sites also support the separation of Pmg and Pmm into distinct species.

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

Summary.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.

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.

Microbodies and glyoxylate-cycle enzyme activities in filamentous fungi.

SummaryMicrobodies were observed in the hyphal tips of all 14 fungi investigated. Their morphology varied among the fungi and their numbers were influenced by the growth medium. Microbodies were closely associated with mitochondria in one fungus and with the endoplasmic reticulum in several fungi. Catalase was not detected in microbodies with the diaminobenzidine cytochemical procedure even though catalase activity was present in extracts of these fungi. The activities of the glyoxylate-cycle enzymes isocitrate lyase and malate synthase were affected by the growth medium and were particulate in the two fungi studied by differential centrifugation. Microbodies are abundant, and they are ubiquitous among the fungi and in some cases they may contain glyoxylate-cycle enzymes.

Genetic Diversity Among Geminiviruses Associated with the Weed Species Sida spp., Macroptilium lathyroides, and Wissadula amplissima from Jamaica

Genetic diversity among geminiviruses associated with three common weeds in Jamaica was studied using digoxigenin-labeled geminiviral DNA probes, polymerase chain reaction with degenerate primers for DNA-A and DNA-B, nucleic acid sequencing, and derived amino acid sequences. Geminiviruses with bipartite genomes were found in Sida spp., Macroptilium lathyroides, and Wissadula amplissima. The geminiviruses detected in Sida spp. and M. lathyroides were nearly identical and were both designated Sida golden mosaic geminivirus (SidGMV-JA), whereas the geminivirus in W. amplissima was sufficiently different to be designated Wissadula golden mosaic geminivirus (WGMV). Nucleotide sequence comparisons of the common regions and the N-terminal regions of the AC1 (rep) and AV1 ORFs, together with the derived amino acid sequence comparisons of the N-terminal parts of BC1 and BV1 ORFs were used to determine their similarities to other geminiviruses. SidGMV-JA was most similar to potato yellow mosaic geminivirus (PYMV). We propose that these two geminiviruses (SidGMV-JA and PYMV) define a new geminivirus cluster, the potato yellow mosaic virus (PYMV) cluster. WGMV was most similar to members of the Abutilon mosaic virus cluster but is not likely to be included in the Abutilon phylogenetic group because of the divergent sequence of the common region. These results indicate that geminiviruses infecting some weeds in Jamaica are distinct from crop-infecting geminiviruses in Jamaica and define a new geminivirus cluster.