Pair Of Degenerate Primers Research Articles

Targeting polyketide synthase gene pool within actinomycetes: new degenerate primers

Natural products provide a unique element of molecular diversity and biological functionality and they are still indispensable for drug discovery. The polyketides, comprising a large and structurally diverse family of bioactive natural products, have been isolated from a group of mycelia-forming Gram-positive microorganisms, the actinomycetes. Relatively high amino acid sequence identity of the actinomycetes type I polyketide synthases (PKSs) was used to design three degenerate primer pairs for homology-based PCR detection of novel PKS genes, with particular interest into PKSs involved in biosynthesis of immunosuppressive-like metabolites. The stepdown PCR method, described here, enables fast insight into the PKS arsenal within actinomycetes. Designed primers and stepdown PCR were applied for the analysis of two natural isolates, Streptomyces sp. strains NP13 and MS405. Sequence analysis of chosen clones revealed the presence of two distinctive sequences in strain Streptomyces sp. NP13, but only one of these showed homology to PKS-related sequences. On analysing PCR amplicons derived from Streptomyces sp. strain MS405, three different PKS-related sequences were identified demonstrating a potential of designed primers to target PKS gene pool within single organism.

Cloning and characterization of resistance gene analogs from Avena species

Sequences analogous to plant resistance genes of the NBS-LRR class were cloned from the genomic DNA of 11 Avena species with different genomes and levels of ploidy. Three pairs of degenerate primers were used, based on conserved DNA sequence motifs belonging to the NBS domain, and 33 sequences were identified. These were subdivided into 7 classes depending on nucleotide sequence identity. Despite the high level of degeneracy, the primers behaved in a highly selective way; the majority of sequences from the different species obtained with every primer combination showed strong identity and were considered homologous. For most species, only one sequence of each class was identified in each genome, suggesting that duplicated sequences are fairly divergent. The strong identity among specific NBS sequences precludes any conclusions being made on the evolution of these species. The genomic organization of the RGA sequences was explored using those of A. strigosa as probes in Southern blots involving digested DNA from 15 Avena species. The hybridization patterns showed wide diversity both among sequences within a species and among species for each sequence. However, the dendrogram generated using the RFLPs showed relationships among species to be in good agreement with those previously established using other molecular markers.

First Report of a Lettuce-Infecting Sequivirus in Chile.

Sequiviruses are isometric aphidborne plant viruses. Dandelion yellow mosaic virus (DaYMV), genus Sequivirus, was isolated from dandelion and lettuce in Europe. Lettuce mottle virus (LeMoV), a putative sequivirus, is often found in mixed infections with Lettuce mosaic virus (LMV) in Brazil (3). DaYMV, LeMoV and LMV cause similar mosaics in field-grown lettuce. Differences in biology and sequence suggest that DaYMV and LeMoV are distinct species (2). Forty-two and 101 lettuce samples with mosaic symptoms collected from two locations near Santiago during a survey of lettuce viruses in Chile in 2002 and 2003, respectively, were analyzed for the presence of LeMoV using reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted (1) and used for RT-PCR with the specific LeMoV primers pairs Lmo3 (5' ACATGAGCACTAGTGAGG 3') and Lmo4 (5' AGATAGAGCCGTCT GGCG 3') (2). One of the 42 and three of the 101 samples produced the expected 300-bp fragment. Isometric particles of 30 nm diameter, typical of a sequivirus, were visualized by transmission electron microscopy. These samples were tested using RT-PCR for the presence of LMV and Cucumber mosaic virus (CMV), but no mixed infections were observed. One isolate, Ch36, was reamplified with the degenerate primer pairs DALE 1 (5' GARTTCAACATGCACGCCAG 3') and DALE 2 (5' TTTTTCTCCCCATYCGTCAT 3') which amplify part of the putative replicase gene (2) and produced a 563-bp fragment that was cloned on pGEM-T Easy (Promega, Madison, WI) and sequenced. The Ch36 product (EMBL Accession No. AM039965) showed 97% amino acid identity with LeMoV from Brazil, 79% with DaYMV, 72% with the sequivirus Parsnip yellow fleck virus, and 34% with the waikavirus Maize chlorotic dwarf virus. To our knowledge, this is the first report of a sequivirus in field lettuce in Chile, and although the virus was found at low incidence, this report extends the range of LeMoV to the western side of the Cordillera de Los Andes. The impact of LeMoV needs to be further analyzed in Chile, Brazil, and possibly other South American countries.

PriFi: using a multiple alignment of related sequences to find primers for amplification of homologs

Using a comparative approach, the web program PriFi () designs pairs of primers useful for PCR amplification of genomic DNA in species where prior sequence information is not available. The program works with an alignment of DNA sequences from phylogenetically related species and outputs a list of possibly degenerate primer pairs fulfilling a number of criteria, such that the primers have a maximal probability of amplifying orthologous sequences in other phylogenetically related species. Operating on a genome-wide scale, PriFi automates the first steps of a procedure for developing general markers serving as common anchor loci across species. To accommodate users with special preferences, configuration settings and criteria can be customized.

Development of Antigen-Capture Enzyme-Linked Immunosorbent Assay and RT-PCR for Detection of Turkey Astroviruses

Turkey astrovirus (TAstV) is an important agent of poult enteritis. The diagnosis of astroviruses has been dependent mainly on electron microscopy (EM) or immune EM (IEM). To develop other simple, rapid, and reliable diagnostic assays, two antigen-capture enzyme-linked immunosorbent assays (AC-ELISAs), polyclonal AC-ELISA and monoclonal AC-ELISA, were developed in this study. Monoplex and multiplex reverse transcription-polymerase chain reactions (RT-PCRs) were also developed using nondegenerate primer sets specific to the capsid region and degenerate primer pairs specific to the polymerase area of two TAstV. EM was included for comparison. Fecal or intestinal contents samples from naturally and experimentally infected poults with enteritis were examined using the developed assays. The polyclonal AC-ELISA had higher sensitivity and wider detection spectrum than the monoclonal AC-ELISA with group-specific monoclonal antibody (MAb), whereas the monoclonal AC-ELISA had very high specificity but lower sensitivity, which was estimated at 0.06 microg of viral proteins. Small round viruses (SRV) that could be astroviruses or other small viruses were detected in 34.4% of the samples examined by EM. The monoplex RT-PCR results amplified with primers SRV-1-3 and SRV-1-5 revealed that the positive rate of astroviruses was 45.3%, which was 10.9% higher than that of EM even if other SRVs were not excluded. Multiplex RT-PCR with SRV-1-3 and SRV-1-5 and AFCP-F1 and AFCP-R1 and the monoplex RT-PCR with degenerate primers verified that the positive rate of astroviruses was 59.4%, which was 25% higher than that of EM. Both RT-PCRs showed good specificity and wider detection spectrum compared with earlier published data.

Biochemical and genetic characterization of the β-lactamases of Y. aldovae, Y. bercovieri, Y. frederiksenii and ‘ Y. ruckeri’ strains

The β-lactamases of five strains each of Y. aldovae and ‘ Y. ruckeri’, and 10 strains each of Y. bercovieri and Y. frederiksenii were examined phenotypically and genetically. β-Lactamase activity and induction assays and SDS-PAGE were applied for phenotypic characterization of these enzymes. Genotypically, PCR experiments applying degenerated primer pairs for the detection of AmpC β-lactamase genes were performed. All yersiniae yielded specific amplification products for ampC and all these strains expressed β-lactamases. Each species produced its own, species-specific AmpC β-lactamase. Inducibility of these enzymes was shown for Y. bercovieri, but not for the low-level enzyme producing species Y. aldovae and ‘ Y. ruckeri’. In contrast to these species, induction tests for Y. frederiksenii revealed heterogenous results. Whereas the β-lactamases of 6 of 10 strains were inducible, the enzyme activities after induction in the remaining four were similar to those measured without an inducer. In addition to the AmpC enzyme, all Y. frederiksenii strains expressed a second β-lactamase belonging to Ambler class A. The present study enlarges the knowledge about the β-lactamases of four novel Yersinia species that are likely to be involved in human disease. β-Lactamases of Y. aldovae and ‘ Y. ruckeri’ have been characterized for the first time.

Rapid and Accurate Identification of Human Isolates of Pasteurella and Related Species by Sequencing the sodA Gene

The identification of Pasteurella and related bacteria remains a challenge. Here, a 449- to 473-bp fragment (sodA(int)) internal to the sodA gene, encoding the manganese-dependent superoxide dismutase, was amplified and sequenced with a single pair of degenerate primers from the type strains of Pasteurella (18 strains), Gallibacterium (1 strain), and Mannheimia (5 strains) species. The sodA(int)-based phylogenetic tree was in general agreement with that inferred from the analysis of the corresponding 16S rRNA gene sequences, with members of the Pasteurella sensu stricto cluster (Pasteurella multocida, Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis) forming a monophyletic group and Gallibacterium and Mannheimia being independent monophyletic genera. However, the sodA(int) sequences showed a markedly higher divergence than the corresponding 16S rRNA genes, confirming that sodA is a potent target to differentiate related species. Thirty-three independent human clinical isolates phenotypically assigned to 13 Pasteurella species by a reference laboratory were successfully identified by comparing their sodA(int) sequences to those of the type species. In the course of this work, we identified the first Gallibacterium anatis isolate ever reported from a human clinical specimen. The sodA(int) sequences of the clinical isolates displayed less than 2.5% divergence from those of the corresponding type strains, except for the Pasteurella pneumotropica isolates, which were closely related to each other (> 98% sodA(int) sequence identity) but shared only 92% sodA(int) identity with the type strain. The method described here provides a rapid and accurate tool for species identification of Pasteurella isolates when access to a sequencing facility is available.

A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods

Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in food and pharmaceutical products. As they form one of the most important groups in both human and animal feces, their use as fecal indicator organisms in raw milk products has recently been proposed. Bifidobacteria species isolated in humans are different from those isolated in animals. It should therefore be possible to determine contamination origin (human or animal). A method of detecting the Bifidobacterium genus was developed by PCR targeting the hsp60 gene. The genus Bifidobacterium was identified by PCR amplification of a 217-bp hsp60 gene fragment. The degenerated primer pair specific to the Bifidobacterium genus used was tested for it specificity on 127 strains. Sensitivity was measured on artificially contaminated samples. Food can however be a difficult matrix for PCR testing since it contains PCR inhibitors. So an internal PCR control was used. An artificially created DNA fragment of 315 bp was constructed. The PCR detection method was tested on raw milk and cheese samples and compared with three culture-based methods, which comprised enrichment and isolation steps. The enrichment step used Brain Heart Infusion medium with propionic acid, iron citrate, yeast extract, supplemented with mupirocin (BHMup) or not (BH) and the isolation step used Columbia blood agar medium, supplemented with mupirocin (CMup) or not (C). The method using mupirocin at both enrichment and isolation steps and the PCR method performed from the culture in BHMup enrichment medium were shown to be the most efficient. No significant difference was observed in raw milk samples between PCR from BHMup and the culture-based method BHMup/CMup, while a significant difference was noticed between the same methods in raw milk cheese samples, which would favor using PCR. The results suggested that PCR on the hsp60 gene was convenient for a rapid detection of bifidobacteria in raw milk and raw milk cheese samples and that bifidobacteria always present throughout raw milk cheese production could be efficiently used as fecal indicators.

The abundance of nahAc genes correlates with the 14C-naphthalene mineralization potential in petroleum hydrocarbon-contaminated oxic soil layers

In this study, we evaluated whether the abundance of the functional gene nahAc reflects aerobic naphthalene degradation potential in subsurface and surface samples taken from three petroleum hydrocarbon contaminated sites in southern Finland. The type of the contamination at the sites varied from lightweight diesel oil to high molecular weight residuals of crude oil. Samples were collected from both oxic and anoxic soil layers. The naphthalene dioxygenase gene nahAc was quantified using a replicate limiting dilution-polymerase chain reaction (RLD-PCR) method with a degenerate primer pair. In the non-contaminated samples nahAc genes were not detected. In the petroleum hydrocarbon-contaminated oxic soil samples nahAc gene abundance [range 3 × 10 1–9 × 10 4 copies (g dry wt soil) −1] was correlated (Kendall non-parametric correlation r 2 = 0.459, p < 0.01) with the aerobic 14C-naphthalene mineralization potential (range 1 × 10 −5–0.1 d −1) measured in microcosms at in situ temperatures (8 °C for subsurface and 20 °C for surface soil samples). In these samples nahAc gene abundance was also correlated with total microbial cell counts ( r 2 = 0.471, p < 0.01), respiration rate ( r 2 = 0.401, p < 0.01) and organic matter content ( r 2 = 0.341, p < 0.05). NahAc genes were amplified from anoxic soil layers indicating that, although involved in aerobic biodegradation of naphthalene, these genes or related sequences were also present in the anoxic subsurface. In the samples taken from the anoxic layers, the aerobic 14C-naphthalene mineralization rates were not correlated with nahAc gene abundance. In conclusion, current sequence information provides the basis for a robust tool to estimate the naphthalene degradation potential at oxic zones of different petroleum hydrocarbon-contaminated sites undergoing in situ bioremediation.

Isolation, characterization, and development of WRKY genes as useful genetic markers in Theobroma cacao

There is currently an international effort in improving disease resistance and crop yield in Theobroma cacao L., an economically important crop of the tropics, using marker-assisted selection for breeding. We are developing molecular genetic markers focusing upon gene families involved with disease resistance. One such family is the WRKY proteins, which are plant-specific transcriptional factors associated with regulating defense responses to both abiotic and biotic stresses. Degenerate PCR primers were designed to the highly conserved DNA-binding domain and other conserved motifs of group I and group II, subgroups a-c, WRKY genes. Sixteen individual WRKY fragments were isolated from a mixture of T. cacao DNA using one pair of primers. Of the 16 WRKY loci investigated, seven contained single nucleotide polymorphisms within the intron as detected by sequence comparison of the PCR products. Four of these were successfully converted into molecular markers and mapped in an F2 population by capillary electrophoresis-single strand conformation polymorphism analysis. This is the first report of a pair of degenerate primers amplifying WRKY loci directly from genomic DNA and demonstrates a simple method for developing useful genetic markers from members of a large gene family.

Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon.

The final steps in butyrate synthesis by anaerobic bacteria can occur via butyrate kinase and phosphotransbutyrylase or via butyryl-coenzyme A (CoA):acetate CoA-transferase. Degenerate PCR and enzymatic assays were used to assess the presence of butyrate kinase among 38 anaerobic butyrate-producing bacterial isolates from human feces that represent three different clostridial clusters (IV, XIVa, and XVI). Only four strains were found to possess detectable butyrate kinase activity. These were also the only strains to give PCR products (verifiable by sequencing) with degenerate primer pairs designed within the butyrate kinase gene or between the linked butyrate kinase/phosphotransbutyrylase genes. Further analysis of the butyrate kinase/phosphotransbutyrylase genes of one isolate, L2-50, revealed similar organization to that described previously from different groups of clostridia, along with differences in flanking sequences and phylogenetic relationships. Butyryl-CoA:acetate CoA-transferase activity was detected in all 38 strains examined, suggesting that it, rather than butyrate kinase, provides the dominant route for butyrate formation in the human colonic ecosystem that contains a constantly high concentration of acetate.

Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy ( Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin.

Antimicrobial susceptibility patterns, β-lactamases, and biochemical identification of Yokenella regensburgei strains

Yokenella regensburgei is an opportunistic human pathogen that phenotypically resembles Hafnia alvei. The susceptibility of 10 Y. regensburgei strains to 75 antimicrobial agents was examined, applying a microdilution procedure in cation-adjusted Mueller-Hinton broth (CAMHB) and IsoSensitest broth (ISB). β-Lactamases were characterized phenotypically with β-lactamase activity and induction assays. Genotypically, PCR experiments applying degenerated primer pairs for the detection of AmpC β-lactamase genes were performed. Examining the phenotypic properties of Yokenella and 76 H. alvei strains with commercial identification systems and conventional tests, a database for an accurate biochemical separation of Y. regensburgei from H. alvei was established. In CAMHB, all tested yokenellae were resistant or at least of intermediate susceptibility to penicillin G, oxacillin, amoxicillin, amoxicillin-clavulanate, cefaclor, cefazoline, loracarbef, cefoxitin, all tested macrolides, lincosamides, streptogramins, ketolides, fusidic acid, glycopeptides, linezolid, and rifampicin. All Yokenella strains were sensitive to several β-lactams, all tested aminoglycosides, chloramphenicol, folate-pathway inhibitors, fosfomycin, nitrofurantion, quinolones, and tetracyclines. In ISB, the minimum inhibitory concentration (MIC) values of several β-lactams were one to four MIC doubling dilution steps lower than those found in CAMHB (depending on the β-lactam). All yokenellae yielded specific amplification products for ampC, and all of these strains expressed β-lactamases that were strongly inducible. Hydroxyproline amidase, maltosidase, tri-peptidase, proline deaminase, catalase reaction, Voges-Proskauer test, and fermentation of glycerol, melibiose and myo-inositol were suitable parameters to separate Y. regensburgei from H. alvei.

Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris.

A pair of degenerate primers was used for amplification and cloning of a DNA fragment containing parts of the P. pastoris URA5 and SEC65 genes. Using additional information from a partial genomic sequence of P. pastoris, we cloned and sequenced a 1.9 kb chromosomal fragment containing the complete orotate-phosphoribosyltransferase-encoding URA5 gene. A disruption cassette was constructed by replacing a small part of the open reading frame with a kanamycin-resistance gene. The P. pastoris wild-type strain NRRL Y-11430 was transformed with the disruption cassette and an ura5 auxotrophic strain was identified. To generate marker constructs that can be reused in successive transformations of a single strain, we constructed two lacZ-PpURA3-lacZ and lacZ-PpURA5-lacZ cassettes and used them to disrupt PpOCH1. The PpURA3 and PpURA5 genes in the disruptants were then successfully recycled by selecting for resistance to 5'-fluoro-orotic acid. We also assembled a set of modular plasmids that can be used for the stable genetic modification of P. pastoris via a double cross-over event. The sequence presented here has been submitted to the EMBL data library under Accession No. AY303544.

Molecular markers linked to the blast resistance gene Pi-z in rice for use in marker-assisted selection.

Rice blast, caused by the fungal pathogen Pyricularia grisea, is a serious disease affecting rice-growing regions around the world. Current methods for identification of blast-resistant germplasm and progeny typically utilize phenotypic screening. However, phenotypic screens are influenced by environmental conditions and the presence of one resistance gene can sometimes phenotypically mask other genes conferring resistance to the same blast race. Pi-z is a dominant gene located on the short arm of chromosome 6 that confers complete resistance to five races of blast. Using sequence data found in public databases and degenerate primer pairs based on the P-loop, nucleotide binding sites and kinase domain motifs of previously cloned resistance genes, we have developed PCR-based DNA markers that cosegregate with the gene. These markers are polymorphic in a wide range of germplasm, including the narrow crosses characteristic of applied rice-breeding programs. They can now be used as a low cost, high-throughput alternative to conventional phenotypic screening for direct detection of blast resistance genes, allowing rapid introgression of genes into susceptible varieties as well as the incorporation of multiple genes into individual lines for more-durable blast resistance.

The partial sequence of RNA 1 of the ophiovirus Ranunculus white mottle virus indicates its relationship to rhabdoviruses and provides candidate primers for an ophiovirus-specific RT-PCR test.

A 4018 nucleotide sequence was obtained for RNA 1 of Ranunculus white mottle virus (RWMV), genus Ophiovirus, representing an incomplete ORF of 1339 aa. Amino acid sequence analysis revealed significant similarities with RNA polymerases of viruses in the family Rhabdoviridae and a conserved domain of 685 aa, corresponding to the RdRp domain of those in the order Mononegavirales. Phylogenetic analysis indicated that the genus Ophiovirus is not related to the genus Tenuivirus or the family Bunyaviridae, with which it has been linked, and probably deserves a special taxonomic position, within a new family. A pair of degenerate primers was designed from a consensus sequence obtained from a relatively conserved region in the RNA 1 of two members of the genus, Citrus psorosis virus (CPsV) and RWMV. The primers, used in RT-PCR experiments, amplified a 136 bp DNA fragment from all the three recognized members of the genus, i.e. CPsV, RWMV and Tulip mild mottle mosaic virus (TMMMV) and from two tentative ophioviruses from lettuce and freesia. The amplified DNAs were sequenced and compared with the corresponding sequences of CPsV and RWMV and phylogenetic relationships were evaluated. Assays using extracts from plants infected by viruses belonging to the genera Tospovirus, Tenuivirus, Rhabdovirus and Varicosavirus indicated that the primers are genus-specific.

Molecular Characterization of a New Tomato Begomovirus from India.

The Asian Vegetable Research and Development Center's (AVRDC) tomato breeding lines derived from Lycopersicon hirsutum f. glabratum B 6013 × L. esculentum H-24 and carrying the Ty-2 resistance gene located on chromosome 11 are tolerant to tomato leaf curl disease in Karnataka State, southern India (3), where several isolates of Tomato leaf curl Virus-Bangalore (GenBank Accession Nos. L11746, Z48182, and AF165098) and Tomato leaf curl virus-Karnataka (GenBank Accession No. U38239) are reported to infect tomatoes. The only area in south and southeast Asia where these AVRDC tomato breeding lines were found susceptible to begomovirus infection is Thailand, where several bipartite Tomato yellow leaf curl virus isolates (GenBank Accession Nos. X63015, X63016; AF141922, AF141897; and AF511529, AF511528) are reported to be prevalent. However, in field trials conducted in the fall of 1999 in Bodeli, Gujarat State, western India, the AVRDC breeding lines showed typical symptoms of begomovirus infection, such as leaf curling and vein clearing. The presence of a different tomato begomovirus was suspected. Viral DNA from a symptomatic plant from Bodeli was amplified by polymerase chain reaction (PCR) using the begomovirus-specific degenerate primer pair PAL1v1978/PAR1c715 (4) and the expected 1.4-kb PCR product was obtained. Based on the sequence of the 1.4-kb DNA product, specific primers were designed to complete the DNA-A sequence. The DNA-A of the virus associated with tomato leaf curl from Bodeli consists of 2,759 nucleotides (GenBank Accession No. AF413671) and contains six open reading frames (ORFs V1, V2, C1, C2, C3, and C4). The DNA-A sequence of the Bodeli isolate had highest sequence identities of 98 and 98.3%, respectively, with viruses causing tomato leaf curl from Varanasi, Uttar Pradesh State, northern India (GenBank Accession No. AF449999) collected in the fall of 1999 and Panchkhal, Nepal (GenBank Accession No. AY234383) collected in early 2000. There was no evidence for the presence of DNA-B in the Bodeli, Panchkhal, or Varanasi virus isolates using DNA-B specific primer pairs DNABLC1/DNABLV2 and DNABLC2/DNABLV2 (2). However, a 1.3-kb DNA-beta was detected in the Panchkhal and Varanasi isolates using the primer pair Beta01/Beta02 (1). Sequence comparisons with begomovirus sequences available in the GenBank database showed that these three virus isolates and GenBank Accession No. AY190290 collected in 2001 from Varanasi shared more than 97% sequence identity with each other and should be considered closely related strains of the same virus. These four virus isolates belong to a new distinct tomato geminivirus species because their sequences share less than 88% sequence identities with the next most closely related virus, Tomato leaf curl virus-Karnataka (GenBank Accession No. U38239). This new tomato leaf curl virus is prevalent in western India, northern India, and Nepal.

Molecular Characterization of a New Begomovirus Associated with Tomato Yellow Leaf Curl and Eggplant Yellow Mosaic Diseases in Thailand.

Leaf curl and yellowing symptoms on tomato, and yellow mosaic symptoms on eggplant, are frequently observed in Kanchanaburi Province, Thailand. DNA was extracted from leaves of 13 symptomatic tomato and six symptomatic eggplant samples by the method of Gilbertson et al (1). Viral DNA was amplified by polymerase chain reaction (PCR) using the begomovirus-specific degenerate primer pair PAL1v1978/PAR1c715 (3), which amplified a 1.4-kb fragment of DNA-A. All samples, except one eggplant sample, yielded the expected product. The 1.4-kb PCR products of one tomato and one eggplant sample were cloned and sequenced. Both begomoviruses from tomato and eggplant contained a DNA-B component, amplified using two degenerate primer pairs, DNABLC1/DNABLV2 and DNABLC2/DNABLV2 (2). Based on sequences of the DNA products amplified by the degenerate primer pairs, specific primers were designed to complete the DNA-A and DNA-B sequences. Computer-assisted sequence comparisons were performed with geminivirus sequences available in the laboratory at the Asian Vegetable Research and Development Center, Taiwan and in the GenBank sequence database. Both tomato (GenBank Accession Nos. AF511529 and AF511528) and eggplant (GenBank Accession Nos. AF511530 and AF511527) virus isolates contain the conserved geminivirus sequence-TAATATTAC on the DNA-A and B. Based on the high sequence identities of 99% DNA-A and 98% DNA-B, these two virus isolates are considered to be the same species. Although the genome organization of these two isolates was the same as Tomato yellow leaf curl virus Thailand (TYLCTHV; GenBank Accession Nos. X63015 and X63016), including six open reading frames (ORFs) on the DNA-A (AV1, AV2, AC1, AC2, AC3, and AC4) and two ORFs on the DNA-B (BV1 and BC1), sequence comparisons showed highest DNA-A sequence identity (73%) with Ageratum yellow vein virus from Singapore (GenBank Accession No. X74516) and highest DNA-B identity (77%) with the TYLCTHV (X63016). To our knowledge, these tomato- and eggplant-infecting viruses from Thailand constitute a distinctly novel bipartite Begomovirus species.

Screening for tolerance to bavistin, a benzimidazole fungicide containing methyl benzimidazol-2-yl carbamate (MBC), in Beauveria bassiana: Sequence analysis of the β-tubulin gene to identify mutations conferring tolerance

The entomopathogenic Beauveria bassiana is a potential biopesticide. Fungicide-tolerant isolates of this fungus would have an added advantage of being compatible with the conventional chemical methods of pest control. Therefore, 30 isolates of the fungus were screened for tolerance to bavistin a commonly used benzimidazole fungicide containing methyl benzimidazol-2-yl carbamate (MBC). Germination and growth bioassays in the presence of 0.05% bavistin were conducted for screening. Three tolerant isolates were identified, showing tolerance up to 2% bavistin. Mutation in the beta-tubulin gene is known to confer tolerance to MBC; the nine known mutation sites in the gene involved were sequenced in the tolerant strains. The beta-tubulin gene from codons 1-405 was amplified using two pairs of degenerate primers, designed for the conserved region of the beta-tubulin gene and sequenced. From the sequence suitable primers were designed for the regions flanking the nine known sites involved in mutations conferring MBC tolerance. The amplified products with these primers from the MBC-tolerant isolates were sequenced and in two (ARSEF 1315 and ARSEF 1316) a mutation was detected in the 198 codon resulting in replacement of glutamic acid with lysine. In the third tolerant isolate (ITCC 913) no mutation could be detected in any of the nine known sites conferring tolerance to MBC. To complete the sequencing of the beta-tubulin gene, the remaining part (from codon 405 onwards) of the gene was isolated by a three-prime gene walk. The gene sequence showed a close homology to fungal beta-tubulin genes with four introns.

Molecular Characterization of Tomato and Chili Leaf Curl Begomoviruses from Pakistan.

Leaf curl or yellowing symptoms, typical of those caused by begomovirus infection, are commonly observed in chili (Capsicum annuum) and tomato (Lycopersicon esculentum) plantings in Pakistan. One chili sample with leaf curl symptoms was collected in 1998 in Multan (Punjab Province), and two tomato samples with leaf curl and yellowing symptoms were collected from Islamabad and Dargai (North West Frontier Province) in 2000 and 2001, respectively. Virus DNA was first amplified by polymerase chain reaction using the degenerate primer pair PAL1v1978/PAR1c715 (3). The expected 1.4-kb PCR products were obtained from the three samples. Based on the sequences of the 1.4-kb DNA products, specific primers were designed to complete each of the DNA-A sequences. Two primer pairs, DNABLC1/DNABLV2 and DNABLC2/DNABLV2, were used for the detection of DNA-B (2). The genome of the tomato leaf curl isolate from Islamabad contained a DNA-A of 2,739 nucleotides (GenBank Accession No. AF448059), a DNA-B of 2,728 nucleotides (GenBank Accession No. AY150304), and had 94% nucleotide identity in the common region. The genome of the tomato leaf curl isolate from Dargai contained a DNA-A of 2,740 nucleotides (GenBank Accession No. AF448058), a DNA-B of 2,686 nucleotides (GenBank Accession No. AY150305), and had 96% nucleotide identity in the common region. Each of the tomato isolates contained eight predicted open-reading frames (ORFs) (AV1, AV2, AV3, AC1, AC2, AC3, AC4, and AC5) in the DNA-A and two predicted ORFs (BV1 and BC1) in the DNA-B. The DNA-A nucleotide sequence identity of the Islamabad isolate and Dargai tomato isolate is 96% and that of DNA-B is 88%. Sequence comparisons with begomovirus sequences available in the GenBank sequence database showed that these two tomato virus isolates had the highest sequence identity with Tomato leaf curl New Delhi virus-Severe (GenBank Accession No. U15015) from northern India (more than 95% for DNA-A and less than 90% for DNA-B). The DNA-A of the virus associated with chili leaf curl from Pakistan (GenBank Accession No. AF336806) consists of 2,754 nucleotides, containing six predicted ORFs (AV1, AV2, AC1, AC2, AC3, and AC4). The chili virus was unrelated to the two tomato begomovirus isolates from Pakistan, with which it shares less than 75% nucleotide identity. Sequence comparisons show highest sequence identity (87%) with Tomato leaf curl Bangladesh virus (GenBank Accession No. AF188481). DNA-beta of 1.3 kb was detected in the chili begomovirus isolate using Beta01/Beta02 primers (1). There was no evidence for the presence of a DNA-B in the chili begomovirus isolate when tested by the two DNA-B specific primer pairs. Based on DNA sequence comparisons, the chili leaf curl virus from Pakistan, to our knowledge, constitutes a distinct, new monopartite begomovirus.