Squash Blot Research Articles

First Report of Tomato yellow leaf curl virus Infecting Tomato, Tomatillo, and Peppers in Guatemala.

In Guatemala and other Central American countries, whitefly-transmitted geminiviruses (begomoviruses) cause economically important diseases of tomato (Solanum lycopersicum) and pepper (Capsicum annuum). Disease symptoms include stunted and distorted growth and leaf curling, crumpling, light green to yellow mosaic, purpling, and vein swelling. In Guatemala, at least eight bipartite begomovirus species infect tomato or peppers (1), but their role and relative importance is unclear. As part of an Integrated Pest Management strategy to manage these diseases, surveys for begomovirus symptoms in pepper and tomato have been conducted in the Salama Valley, Sanarate, and other locations since 2003, and begomoviruses were identified by squash blot hybridization, PCR and DNA sequencing. Beginning in 2006, a new type of symptom, stunted upright growth and upcurled leaves with yellowing of the margins and interveinal areas, was observed in tomato and tomatillo plants in the Salama Valley and Sanarate. These symptoms were similar to those induced by the exotic monopartite begomovirus Tomato yellow leaf curl virus (TYLCV). Evidence that TYLCV caused these symptoms came from positive results in high stringency squash blot hybridization tests with a TYLCV probe, and amplification of the expected size of ~0.3- and 2.8-kb fragments in PCR tests with TYLCV capsid protein (CP) gene and full-length component primer pairs, respectively (3). Sequence analyses of PCR-amplified CP fragments and portions of full-length fragments revealed 97 to 99% identity with isolates of TYLCV-Israel (TYLCV-IL). The complete nucleotide sequence of an isolate from the Salama Valley (GenBank Accession No. GU355941) was >99% identical to those of TYLCV-IL isolates from the Dominican Republic, Florida, and Cuba and ~97% identical to those of isolates from Mexico and California. Thus, this TYLCV-IL isolate (TYLCV-IL[GT:06]) was probably introduced from the Caribbean Region. To further characterize begomoviruses in the Salama Valley, leaf samples were collected from 44 and 118 tomato plants showing symptoms of begomovirus infection in March 2006 and 2007, respectively, and from 106 symptomatic pepper plants in March 2007. Begomovirus infection was confirmed in 42 of 44 and 93 of 118 of the tomato samples and 100 of 106 of the pepper samples based on PCR amplification of the expected size of ~0.6- and 1.1-kb DNA fragments with the begomovirus degenerate primers pairs AV494/AC1048 and PAL1v1978/PAR1c496, respectively (2,4). Sequence analyses of cloned PCR-amplified fragments revealed that 3 of the 44 and 16 of the 118 tomato samples collected in 2006 and 2007, respectively, and 9 of the 106 pepper samples were infected with TYLCV based on >97% identity with TYLCV-IL. In all samples, TYLCV was present in mixed infections with other begomoviruses. The introduction of TYLCV adds to the already high level of genetic complexity of bipartite begomovirus infection of tomatoes and peppers in Guatemala and will undoubtedly complicate disease management efforts.

Evidence of local evolution of tomato-infecting begomovirus species in West Africa: characterization of tomato leaf curl Mali virus and tomato yellow leaf crumple virus from Mali

Tomato yellow leaf curl (TYLC) and tomato leaf curl (ToLC) diseases are serious constraints to tomato production in Mali and other countries in West Africa. In 2003 and 2004, samples of tomato showing virus-like symptoms were collected during a survey of tomato virus diseases in Mali. Three predominant symptom phenotypes were observed: (1) TYLC/ToLC (stunted upright growth and upcurled leaves with interveinal yellowing and vein purpling), (2) yellow leaf crumple and (3) broccoli or bonsai (severe stunting and distorted growth). Squash blot (SB) hybridization with a general begomovirus probe and/or SB/PCR analyses revealed begomovirus infection in plants with each of these symptom phenotypes and no evidence of phytoplasma infection. Sequence analysis of PCR-amplified begomovirus fragments revealed two putative new begomovirus species associated with the TYLC/ToLC and yellow leaf crumple symptom phenotypes, respectively. Full-length clones of these begomoviruses were obtained using PCR and overlapping primers. When introduced into N. benthamiana and tomato plants, these clones induced upward leaf curling and crumpling (the TYLC/ToLC-associated begomovirus) or downward leaf curl/yellow mottle (yellow leaf crumple-associated begomovirus) symptoms. Thus, these begomoviruses were named tomato leaf curl Mali virus (ToLCMLV) and tomato yellow leaf crumple virus (ToYLCrV). The genome organization of both viruses was similar to those of other monopartite begomoviruses. ToLCMLV and ToYLCrV were most closely related to each other and to tobacco leaf curl Zimbabwe virus (TbLCZV-[ZW]) and tomato curly stunt virus from South Africa (ToCSV-ZA). Thus, these likely represent tomato-infecting begomoviruses that evolved from indigenous begomoviruses on the African continent. Mixed infections of ToLCMLV and ToYLCrV in N. benthamiana and tomato plants resulted in more severe symptoms than in plants infected with either virus alone, suggesting a synergistic interaction. Agroinoculation experiments indicated that both viruses induced symptomatic infections in tomato and tobacco, whereas neither virus induced disease symptoms in pepper, common bean, small sugar pumpkin, African eggplant, or Arabidopsis. Virus-specific PCR primers were developed for detection of ToLCMLV and ToYLCrV and will be used to further investigate the distribution and host range of these viruses.

Host range of an NPV and a GV isolated from the common cutworm, Agrotis segetum: pathogenicity within the cutworm complex

The term cutworm covers a range of species with a similar life history that can be very damaging pests on a wide range of crops. Attacks by cutworms are often made up of more than one species; thus, the most cost effective microbial control agent needs to be pathogenic for multiple species within this complex. In this study we investigate the host range of Agrotis segetum NPV and A. segetum GV for other cutworm species and closely related Noctuinae. Eight species, A. segetum, Agrotis ipsilon, Agrotis exclamationis, Agrotis puta, Noctua comes, Peridroma saucia, Xestia sexstrigata, and Xestia xanthographa, were clearly susceptible to AgseNPV, which was confirmed by DNA analysis. Aglais urticae, Diarsia rubi, Noctua pronuba, and Xestia c-nigrum were not susceptible to AgseNPV at the doses used. Noctua fimbriata, Noctua janthina, and Ochroplura plecta gave ambivalent results: larvae died of NPV infection when they were challenged with AgseNPV, but these individuals only produced weak positives in a squash blot analysis and there was insufficient DNA for confirmation by restriction endonuclease profiling. These ambivalent results could suggest either a weak infection by AgseNPV or partial homology between their own virus and AgseNPV. The untreated control insects of several species died of NPV infection, which indicates that these field-collected insects were probably carrying a vertically transmitted NPV. Fewer species were tested with AgseGV and only N. pronuba and N. comes were susceptible. N. fimbriata and Helicoverpa armigera were not susceptible to AgseGV.

Reservoir Weed Hosts of Tomato Yellow Leaf Curl Begomovirus from Tanzania

A survey was initiated to detect tomato yellow leaf curl virus (TYLCV) and identify its reservoir weed hosts in six regions (Arusha, Morogoro, Dodoma, Iringa, Kilimanjaro and Dar es Salaam) in Tanzania. Three farms were randomly selected in each region. Assessment of TYLCV incidence was done by relating the number of infected tomato plants to the total number of plants assessed along a diagonal in five quadrants measuring 4m ‐ 4m in size (one at each corner of the farm and one at the centre). Disease severity was scored on a scale of 0 to 4 (where 0 = no symptoms and 4 = very severe symptoms). Within and outside each farm, weeds showing TYLCV-like symptoms were collected and either squash-blotted, dot-blotted or both on nylon membranes. The membranes were hybridized with DIG-labelled probe synthesized for the detection of TYLCV from Sardinia (TYLCV-Sar) following standard protocols. Selected plant species were experimentally inoculated with screenhouse cultures of TYLCV representative isolates from the six regions using Bemisia tabaci to determine their host status. Results indicated that TYLCV incidence and severity were significantly higher (P = 0.05) in Dodoma region than the rest of the regions. In Iringa region, the incidence and severity of TYLCV were the lowest of all regions. TYLCV was detected in 12 of the 17 dot-blotted samples and in all the 21 squashed samples using the non-radioactively labelled riboprobes. Similarly, five plant species (Capsicum annuum, Datura stramonium, Lycopersicon esculentum, Nicotiana glutionsa and N. tabacum) tested in the screenhouse were infected by the six TYLCV isolates used. It is recommended that weeds within and outside tomato farms be removed to eliminate or reduce sources of virus inoculum. The dot and squash blot techniques are convenient for field detection of the virus, and are especially useful for the detection of early and latent infections so that management strategies can be initiated and implemented.

Spread of Tomato yellow leaf curl virus Sar from the Mediterranean Basin: Presence in the Canary Islands and Morocco.

Severe outbreaks of tomato yellow leaf curl disease occurred during summer and autumn 1999 in tomato (Lycopersicon esculentum Mill.) crops in the Vecindario Region of Gran Canaria (Canary Islands, Spain) and Agadir (southwestern Atlantic coast of Morocco). Symptoms of the disease included upward curling of leaflet margins, reduction of leaflet area, and yellowing of young leaves, as well as stunting and flower abortion. High populations of whiteflies, Bemisia tabaci Gen., were present on tomatoes in Agadir, and analysis of adult individuals by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) identified them as the biotype Q. Samples were collected from symptomatic tomato plants: 5 plants from Gran Canaria and 22 from three areas in Agadir, (7 from Agadir/1, 12 from Agadir/2, and 3 from Agadir/3) in the Koudya Region. Samples were analyzed for Tomato yellow leaf curl virus (TYLCV) Sar or Is (genus Begomovirus, family Geminiviridae) infection by squash blot hybridization under high stringency conditions with digoxigenin-labeled DNA probes specific to TYLCV-Sar or -Is, as described previously (1,3). The TYLCV-Sar probe hybridized to the five samples from Gran Canaria, and the TYLCV-Is probe hybridized to the 22 samples from Agadir. The TYLCV-Sar probe also hybridized to the three samples from Agadir/3. Primer pairs MA-14/MA-15 and MA-30/MA-31, designed for specific amplification of the intergenic region (IR) of TYLCV-Sar or -Is reported from Spain, respectively (1), were used in PCR to amplify one sample each from Gran Canaria, Agadir/1, and Agadir/3. A fragment of the expected size was obtained from the samples from Gran Canaria and Agadir/3 using MA14/MA15 (342 bp) and from the two samples from Agadir using MA30/MA31 (357 bp). PCR products were directly sequenced (GenBank Accession nos. AF215819 to AF215822). The nucleotide sequences of the IR fragments amplified from the Gran Canaria and Agadir/3 sample using MA-14/MA-15 indicated their closest relationship (99.0 and 96.7% identity, respectively) was to the corresponding region of a TYLCV-Sar isolate reported from Spain (GenBank Accession no. L27708). The nucleotide sequences of the IR fragments amplified from the Agadir/1 and Agadir/3 samples using MA-30/MA-31 indicated their closest relationship (98.1% identity) was to the corresponding region of the TYLCV-Is isolate reported from Spain (GenBank Accession no. AF071228). Based on the hybridization and sequence data, we conclude that the symptomatic plants from Gran Canaria were infected by TYLCV-Sar, those from Agadir/1 and Agadir/2 were infected by TYLCV-Is, and those from Agadir/3 had mixed infections with TYLCV-Is and TYLCV-Sar. The presence of TYLCV-Is in Morocco has been described recently (2). However, this is the first report of TYLCV-Sar in the Canary Islands and Morocco and extends its geographic range beyond the Iberian Peninsula and Italy.

A New Bipartite Geminivirus (Begomovirus) Causing Leaf Curl and Crumpling in Cucurbits in the Imperial Valley of California.

During fall 1998, volunteer watermelons (Citrullus lunatus L. (Thunb.) Matsum. & Nakai) showing leaf curl, crumpling, and yellowing symptoms were found in a commercial honeydew melon (Cucumis melo L. subsp. melo Inodorus group) field in the Imperial Valley of California. The plants were infected with a begomovirus (family Geminiviridae, genus Begomovirus) based on (i) a positive response in squash blots probed with a general begomovirus DNA probe (1) and (ii) amplification of DNA-A (≈1.2 kb) and DNA-B (≈1.4 kb) fragments by polymerase chain reaction (PCR) with degenerate DNA-A (PAL1v1978/PAR1c496) and DNA-B (PBL1v2040/PBR1c970) primers, respectively (3). The DNA-A and -B fragments were cloned and sequenced (GenBank accession nos. AF224760 [DNA-A] and AF224761 [DNA-B]). The DNA-A and -B fragments had a nearly identical (99.5%) common region (CR) of 186 (DNA-A) and 187 (DNA-B) nucleotides, indicating they were from the same begomovirus. Database searches conducted with these sequences revealed no high degree of sequence identity (i.e., >90%) with other begomoviruses, including Squash leaf curl virus (SqLCV [2]) from southern California. The partial AC1 sequence (669 nt) was most identical to Tomato severe leaf curl virus (ToSLCV) from Guatemala (83%) and SqLCV (81%), the partial AV1 sequence (135 nt) was most identical to Tomato golden mosaic virus from Brazil (84%) and SqLCV (81%), and the CR was most identical to Squash yellow mottle virus from Costa Rica (81%), ToSLCV (81%), and SqLCV (77%). The partial BV1 sequence (465 nt) was most identical to Bean calico mosaic virus and SqLCV (72%), and the partial BC1 sequence (158 nt) was most identical to SqLCV (75%). Watermelon seedlings bombarded with a DNA extract from infected watermelon volunteers developed crumpling and distortion symptoms, whereas seedlings bombarded with gold particles alone developed no symptoms. Geminivirus infection in symptomatic seedlings was confirmed by PCR. These results suggest a new begomovirus caused the disease symptoms in the watermelon volunteers. Leaf crumpling and curling symptoms were not observed in spring melons in the Imperial Valley in 1999, but on 2 July and 17 August 1999, cantaloupe (C. melo L. subsp. melo Cantalupensis group), muskmelon (C. melo L. subsp. melo Cantalupensis group), and watermelon plants with leaf crumpling and yellowing were found. These plants were infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments (97 to 98% identity for CR and partial AC1 sequence). A survey of fall melons, conducted 23 to 24 September 1999, revealed widespread symptoms of leaf curl and crumpling on new growth of muskmelon plants in all seven commercial fields examined (estimated incidence 25 to 50%) and on watermelon volunteers. No such symptoms were observed on leaves of honeydew melons. Symptomatic muskmelon and watermelon leaves, collected from eight locations throughout the Imperial Valley, were infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments. Thus, a new begomovirus has emerged in the Imperial Valley; the name Cucurbit leaf crumple virus (CuLCrV) is proposed.

Improved Diagnostic Techniques for Tomato Yellow Leaf Curl Virus in Tomato Breeding Programs

Breeding for tomatoes resistant to tomato yellow leaf curl geminivirus (TYLCV) is difficult, mainly due to the lack of an accurate system for selecting resistant plants. The variable nature of the genotypes used (wild or wild-derived genotypes and advanced breeding lines) makes the development of a reproducible selection procedure necessary during the breeding program. Serological and nucleic acid-based diagnostic techniques for detecting TYLCV were evaluated for their sensitivity, reliability, and possibility of quantification in order to select the combination of techniques that provides the most rapid and accurate characterization of the resistance level of each item. Squash blot followed by the more sensitive polymerase chain reaction (PCR) detection of inconclusive samples is recommended for the screening of new resistant sources among asymptomatic Lycopersicon spp., as they accumulate extremely low viral concentrations that cannot be detected by serological methods. A triple antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA), modified to reduce the background, can be used in large-scale field screening of advanced breeding lines if combined with the more sensitive squash blot, which is less affected by the age and state of the tissue. Hybridization methods were also more appropriate to assess viral distribution throughout the plant than TAS-ELISA or even PCR, which fails to reliably detect TYLCV in tissues like roots.

Immunological Detection of Helicoverpa armigera(Lepidoptera: Noctuidae) Ingested by Heteropteran Predators: Time-Related Decay and Effect of Meal Size on Detection Period

Indirect enzyme-linked immunosorbent assay (ELISA), dot blot, and squash blot were compared for sensitivity and ease of use for detecting Helicoverpa armigera (Hübner) egg antigen using an egg-specific monoclonal antibody. The best results were obtained by dot blot, although several indirect ELISA conditions were tried. Three of the main predator species in Mediterranean vegetable crops—Dicyphus tamaninii Wagner and Macrolophus caliginosus Wagner (Hemiptera: Miridae), and Orius majusculus (Reuter) (Hemiptera: Anthocoridae)—which had fed on H. armigera eggs, were assayed by dot blot to detect egg antigen in their gut. The prey was detected in all samples. Another study was conducted to determine the effect of meal size and time after feeding on the prey detection period in D. tamaninii. The proportion of prey remaining at various time intervals after feeding was measured. Curves were fitted to exponential and linear models and detection periods were estimated. The decline in prey detection over time was fitted with an exponential model for predators that ate 1 egg and a linear model for predators that ate 10 eggs. Therefore, meal size produces a considerable difference in the rate of decline and the estimated detection periods.

First Report of Tomato Mottle Geminivirus Infecting Tomatoes in Yucatan, Mexico

Whitefly-transmitted geminiviruses are a major constraint on tomato production in Mexico (3). In the Yucatan State, these viruses can cause serious losses in late season plantings. As part of an effort to characterize these viruses, leaf samples from four tomato plants showing symptoms of geminivirus infection, such as stunted growth and leaf mottling and deformation, were collected from a single field in the Yucatan State in February, 1996. Geminivirus nucleic acids were detected in leaf samples from all four plants by squash blot hybridization analysis with a general DNA probe for Western Hemisphere whitefly-transmitted geminiviruses (2). Nicotiana benthamiana plants inoculated with sap prepared with leaf tissue from one plant developed stunted growth and leaf mottling and deformation. When graft-transmitted from N. benthamiana to tomato, the geminivirus(es) induced leaf mottling and deformation, which were similar to symptoms in the field-collected tomato plants. The presence of geminivirus DNA in the sap- and graft-inoculated plants was confirmed with the polymerase chain reaction (PCR) and degenerate primers for the DNA-A (PAL1v1978 and PAR1c496) or DNA-B (PBL1v2040 and PCRc1) components of whitefly-transmitted geminiviruses (4). Using PCR and these degenerate primers, approximately 1.1-kb DNA-A and approximately 0.6-kb DNA-B fragments were amplified from DNA extracts prepared from leaves of each of the four Yucatan tomato plants. No DNA fragments were amplified from these extracts with primers for pepper huasteco geminivirus (pAL1c2329 and pAL1v1471, or pBR1c840 and pBL1v1830). To determine the identity of the geminivirus(es) infecting these tomato plants, the PCR-amplified DNA-A and DNA-B fragments from one of the samples were cloned and sequenced. Comparisons made with these sequences revealed two distinct types of DNA-A and DNA-B clones, indicating a mixed infection of at least two bipartite geminiviruses. DNA-A and DNA-B sequences of one set of clones were >97% identical to sequences of tomato mottle geminivirus (ToMoV) from Florida (1). The presence of ToMoV in all four tomato leaf samples was demonstrated by the PCR-mediated amplification of a 0.9-kb DNA-A fragment with ToMoV-specific primers (pAL1v2295 and pAR1c580). The identity of this 0.9-kb DNA fragment was further confirmed based upon its hybridization with a full-length clone of ToMoV DNA-A under high stringency conditions (2). A data base search made with the sequence of the other type of DNA-A clone revealed sequence identities of <70% with various bipartite geminiviruses (e.g., identities of 70% with tomato mottle, 69% with Sida golden mosaic, 67% with bean dwarf mosaic, and 66% with taino tomato mottle and with potato yellow mosaic), which confirmed that a second geminivirus was present in a mixed infection with ToMoV in this tomato leaf sample. To confirm the bipartite nature of this geminivirus, a DNA-B fragment that contained the common region (CR) sequence was amplified from the same sample with PCR and primers PBL1v2040 and PBR1c970 (a degenerate primer that anneals within the BV1 open reading frame; F. M. Zerbini and R. L. Gil-bertson, unpublished data), cloned, and sequenced. The CR sequence of this DNA-B fragment was 96% identical to that of the DNA-A fragment, which establishes the presence of another bipartite geminivirus in this sample. This is the first report of ToMoV in Mexico. These results also suggest that at least two bipartite geminiviruses may infect tomatoes in the Yucatan Peninsula.

Partial Characterization of Two Whitefly-Transmitted Geminiviruses Infecting Tomatoes in Venezuela.

Whitefly-transmitted geminiviruses can cause significant yield losses on tomatoes (Lycopersicon esculentum Mill.) in Venezuela. To identify the geminivirus(es) infecting tomatoes in Venezuela, 20 tomato samples from commercial tomato fields in four states and one weed (Euphorbia heterophylla L.) sample were examined for geminivirus infection. All samples showed symptoms generally associated with geminivirus infection, including golden or yellow mosaic, mottling, crumpling and/or distortion of leaves, and, in some cases, stunted and distorted growth. Through the use of squash blot hybridization analysis and a general probe for Western Hemisphere whitefly-transmitted geminiviruses (4), geminivirus nucleic acids were detected in 19 of 20 tomato samples and the weed sample. No samples were infected with tomato yellow leaf curl virus (TYLCV), based on squash blot hybridization analysis with a TYLCV-specific probe. With polymerase chain reaction (PCR) and degenerate primers for whitefly-transmitted geminiviruses (PAL1v1978 and PAR1c496) (4), an approximately 1.2-kb DNA-A fragment was amplified from the 19 squash blot-positive tomato samples and from the weed sample. No DNA fragment was amplified from any samples when TYLCV-specific primers (PTYC1v2406 and PTYIRc287) (3) were used. PCR-amplified DNA-A fragments from four samples representing four different states [Monagas (5L), Guarico (3M), Aragua (3R), and Portuguesa (2U)] were cloned and sequenced. Partial AC1, AV1, and complete common region (CR) sequences of the 5L, 3M, and 2U DNA-A fragments were 92 to 93, 93, and 95 to 97% identical, respectively, indicating that these were DNA-A clones of the same virus. Furthermore, these sequences were 91 to 92, 92 to 95, and 93 to 95% identical, respectively, to sequences of homologous regions of potato yellow mosaic virus (PYMV), indicating that these tomato-infecting geminiviruses are isolates or strains of PYMV. The partial AC1, AV1, and complete CR sequences of the 3R DNA-A fragment were 79, 95, and 77% identical to those of 5L, 3M, and 2U clones, respectively, suggesting that this is a different geminivirus. These sequences were 75 to 87, 82 to 88, and 73 to 81% identical, respectively, to sequences of homologous regions of other tomato geminiviruses, including tomato golden mosaic from Brazil, tomato mottle from Florida, and tomato leaf crumple from Mexico. The bipartite nature of the geminiviruses that were present in the 5L, 3M, 3R, and 2U samples was suggested by the amplification of a DNA-B fragment with degenerate DNA-B primers (PBL1v2040 and PCRc1) (4). These results suggest at least two distinct bipartite Western Hemisphere whitefly-transmitted geminiviruses are associated with tomato virus diseases in Venezuela, and that one of these (sample 3R) may be an undescribed geminivirus. The sequence of the DNA-A fragment from the weed sample was not closely related to the tomato-infecting geminiviruses and, therefore, this weed was not an alternate host of these viruses. Furthermore, because PYMV has been shown to infect tomatoes and cause yellow mosaic symptoms (1), it would be of interest to determine the relationship of PYMV and tomato yellow mosaic geminivirus (ToYMV), which has been reported infecting tomatoes in Venezuela (2), but has not been characterized on the molecular level.

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.

DNA probes for the Anopheles punctulatus complex.

Genomic DNA probes were made for two recently identified members of the Anopheles punctulatus complex; Anopheles sp. near punctulatus from Papua New Guinea and Anopheles farauti No. 7 from the Solomon Islands. The probes are species-specific and with the use of 32P labeling sensitive enough so that a squash blot of only a small segment of the mosquito is required for identification. The 119-basepair (bp) probe for An. sp. near punctulatus and the 1,106-bp probe for An. farauti No. 7 have been sequenced in full and the probes have been tested on field collected specimens. These probes now make it possible to distinguish An. sp. near punctulatus and An. farauti No. 7 from the other eight members of the An. punctulatus complex. A pan-species probe was also made from the 18S ribosomal DNA that binds to DNA from all members of the complex. These three probes complete the set required for distinguishing all known members of the An. punctulatus complex by DNA hybridization.

The mariner transposable element in the Drosophilidae family

The distribution of the mariner transposable element among Drosophilidae species was investigated using three different techniques, i.e. squash blots, Southern blots and PCR amplification, using two sets of primers (one corresponding to the Inverted Terminal Repeats and the other to two conserved regions of the putative transposase). Our results and those of others show that the distribution of mariner is not uniform and does not follow the phylogeny of the host species. An analysis of geographical distribution, based on endemic species, shows that mariner is mainly present in Asia and Africa. At least two hypotheses may be proposed to explain the specific and geographical distributions of this element. Firstly, they may be the results of several horizontal transmissions between Drosophila species and/or between Drosophila species and one or several donor species outside the Drosophilidae family. Secondly, these particular distributions may correspond to the evolution of the mariner element from an ancestral copy which was present in the ancestor of the Drosophilidae family.

DNA Probes Can Be Used To Discriminate between Tephritid Species at all Stages of the Life Cycle (Diptera: Tephritidae)

We isolated DNA sequences from the genomes of three Tephritid species. These can be used as probes to make rapid and reliable species identifications using material from any stage of the life cycle. The three Tephritid species are the Mediterranean fruit fly, Ceratitis capitata (Weidemann); the oriental fruit fly, Bactrocera dorsalis (Hendel), and the melon fly, Bactrocera cucurbitae (Coquillett). These isolated DNA sequences can be used to probe specimen material such as single eggs, larvae, or adult body parts on dot blots or squash blots to determine rapidly which species are present when infestations are found. The actual species identifications are made either on the basis of a direct positive result with a particular probe or by the process of elimination from multiple probings. The availability of species-specific DNA probes for use on minimal amounts of material from any stage of the life cycle or body part can facilitate greatly the identification of specimens which are otherwise indistinguishable or unsuitable for classification on the basis of morphological criteria. This technique also allows material to be analyzed immediately, which obviates the need for rearing and handling of material collected at immature stages. Although these probes have been developed for the three Tephritid species described here, the methods used are applicable to any group of organisms.

DNA Probes for Identifying the Members of the Anopheles punctulatus Complex in Papua New Guinea

Genomic DNA probes were made for five members of the Anopheles punctulatus complex of mosquitoes found in Papua New Guinea. Specific DNA probes were developed for An. punctulatus, An. koliensis, and three sibling species, An. farauti No. 4, An. farauti No. 5, and An. farauti No. 6, by differentially screening total genomic DNA libraries of individual species and sibling species with homologous DNA against heterologous DNA labeled with 32P. Probes ranged from 273 to 630 bp. Identification of species can be made from squash or dot blots using only a segment of the mosquito (i.e, head, thorax, abdomen, or even legs), allowing for concurrent analysis of the remainder of the mosquito for other epidemiologic characteristics.

DNA probes for the detection of Fasciola hepatica in snails

Fasciola hepatica, also called the large liver fluke, is a trematode which can infect most mammals. Monitoring the infection rate of snails, which function as intermediate hosts and harbour larval stages of F. hepatica, is an important component of epidemiological studies on fascioliasis. For this purpose, DNA probes were generated which can be used for the detection of F. hepatica larvae in snails. Four highly repetitive DNA fragments were cloned in a plasmid vector and tested by Southern blot hybridization to the DNA of various trematodes for specificity and sensitivity. The probes Fhr-I, Fhr-II and Fhr-III hybridized only to F. hepatica DNA. Fhr-IV contained ribosomal RNA gene sequences and cross-hybridize with the DNA from various other trematode species. Squash blot analysis showed that the different probes were able to detect the parasite larvae in trematode-infected snails even as isolated single larvae. No signals were obtained in squash blots of uninfected snails. Probes Fhr-I, Fhr-II and Fhr-III are thus useful specific tools for studying the epidemiology of fascioliasis. The probe Fhr-IV, because of its broader spectrum, can be used to detect the larvae of a wide range of trematode species of waterbirds, which are the causative agents of swimmer's itch.

Distribution and conservation of sequences homologous to the 1731 retrotransposon in Drosophila.

The distribution of 1731 retrotransposon-hybridizing sequences in the family Drosophilidae has been studied using a 1731 probe from Drosophila melanogaster. Squash blot and Southern blot analyses of 42 species reveal that the 1731 sequences are widespread within both the Sophophora and Drosophila subgenera and are also present in the genera Scaptomyza and Zaprionus. Hence the 1731 retrotransposon family appears to have a long evolutionary history in the Drosophilidae genome. Differences of hybridization signal intensity suggested that the 1731 sequence is well conserved only in the three species most closely related to D. melanogaster (D. simulans, D. mauritiana, and D. sechellia). A survey of insertion sites in numerous different populations of the previous four species by in situ hybridization to polytene chromosomes has shown in all cases both chromocentric hybridizations and a low number of sites (0-5) on the chromosomal arms. This number of sites is among the lowest observed in D. melanogaster and D. simulans when 1731 is compared with other retrotransposon families. In addition, we have observed species-specific patterns of the chromocentric hybridization signal, suggesting rapid modifications of the beta-heterochromatin components since the radiation of the melanogaster subgroup.

Vigorous growth of fusion products allows highly efficient selection of interspecific potato somatic hybrids: molecular proofs

An early identification of fusion products was based on the presumed vigorous growth of hybrid calluses after fusion between Solanum brevidens and S. tuberosum leaf protoplasts. The S. brevidens protoplasts were unable to form multicellular colonies under the applied culture conditions. Three size groups of calluses were separated and analyzed at two different early phases of culture period. "Squash blot" hybridization with a S. brevidens specific repetitive DNA probe showed that the group of the largest calluses consisted of putative somatic hybrids with a frequency of 80-100% in three independent experiments. Furthermore, approximately 80-95% of the middle sized calluses and 33-90% of the smallest ones were shown to be hybrid. The unexpectedly high percentage of fusion products, even in the case of the smallest calluses, may result from the suppression of the development of parental potato colonies in cultures with mixed cell population. Till this time 120 independent colonies selected as putative hybrids have been regenerated into plants. All of them exhibited hybrid phenotype, and their hybrid origin was proved by cytological and restriction fragment length polymorphism analyses.

A simple and rapid technique for identification of large numbers of individual mosquitoes using DNA hybridization

A general method for obtaining species-specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.