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Analysis of several Colombian Phthorimaea operculella granuloviruses isolated from Tecia solanivora: Detection of a new variable region in the PhopGV genome

Insect Pathogens and Insect Parasitic Nematodes
IOBClwprs Bulletin Vol.31, 2008
p. 83
Phthorimaea operculella
Tecia solanivora: Detection
a new
region in
VillamizarzCarlos Espineli,
-Louis Zeddarrr',
Cotesi, Miguel Lopez-Ferber"
IIRD. Centre de Recherche. 30380 St Christol-les-Ates, France; 2CORPOICA, Km 14 via
Mosquera. Bogota. Colombia; 3IRD, PUCE. Quito. Ecuador; ./LGEI. Ecole des Mines, Ales,
act: Five isolates of Phthorimaea opercu/ella granulovirus (PhopGV). obtained from Tecia
solanivora larvae sampled from three different regions of Colombia were studied. One isolate was
obtained from an infected larva collected in the north-east near the frontier with Venezuela. one from
the south-west near the frontier of Ecuador and the three others from the center of Colombia. They
were amplified on larvae of T. so/anivora reared in laboratory conditions. Isolates were characterized
using 12 specific restriction endonucleases and the amplification by PCR of the four variable regions
already mentionned for PhopGV. using specific set of primers. The results indicated differences
between the strains. The three isolates from the center part of Colombia present the same profiles, and
the Iwo others present submolar bands either with REN or with PCR analysis. The Colombian isolates
were compared with several PhopGVs originated from Peru. Ecuador and Tunisia and isolated from P.
operculella. T. so/anivora and S. tangolias. Using the 4 set of primers. the three isolates from the
center part of Colombia present a profile specific to T. solanivora isolates. In the two other isolates,
the submolar bands are the same than the ones found inP. apercu/ella profiles. indicating the presence
in the genome of at least 2 different profiles. A new variable region was detected in the 90-91 gene
region using another set of primers. With this set of primers. a 630 bp band appears. different from the
789 bp band usually found with the other PhopGV strains. The sequence of this part of the genome
indicates a deletion in the repetitive part of the 90-91 genes. This deletion appears to be a specific
modification only found in viral strains obtained from T. so/anivora in Colombia.
lnseet.Pathogens and
Parasitic Nematodes
o/r *
Itamar Glazer,
Lopez-Ferber &Cezary Tkaczuk
wprs BuJletin
srop Vol. 31, 2008
Intemational Organization for Biological
Noxious Animals and Plants
Organisation Intemationale de Lutte Biologique et Integree contre les Animaux et les Plantes Nuisibles
West Palaearctic Regional Section /Section Regionale Ouest Palearctique
... DNA fragments were stained with SYBR ® gold (Invitrogen) and visualized on a UV transilluminator (Chemi-Doc, Bio-Rad Laboratories, CA, USA), and the fragment sizes were estimated using 1 Kb Plus DNA ladder (Invitrogen). Restriction patterns of the viruses were analysed for the presence or absence of specific bands, comparing with the restriction pattern of a P. operculella granulovirus from Peru, coded as VG006(Léry et al., 2008). ...
The tomato leafminer Tuta absoluta (Meyrick 1917) (Lepidoptera:Gelechiidae) is a devastating pest, causing losses of up to 100%. An interesting tool for its control is the use of the insect specific viruses of Baculoviridae family. Despite its high potential, its use on T. absoluta larvae has been poorly studied. In this work, Colombian granuloviruses VG013 and VG003 isolated from T. absoluta and Tecia solanivora (Lepidoptera:Gelechiidae) larvae sampled in tomato and potato crops, respectively, were morphologically, biologically and molecularly characterized. Occlusion bodies showed ovoid shape containing one nucleocapsid. Restriction endonuclease analysis revealed a pattern similar to Phthorimaea operculella granulovirus and bioinformatics studies showed that both isolates are variants of that baculovirus specie. Similar mean lethal concentrations (LC50) on T. absoluta larvae were estimated for both viruses, although VG013 exhibited shorter mean time to death than VG003. The latter reached a higher OBs yield in comparison with VG013. These results demonstrated an interesting potential of evaluated betabaculoviruses to control T. absoluta populations and pointed key features to its use under field conditions.
... It is well established that different PhopGV isolates vary in their activity against different populations of P. operculella and alternate hosts [95][96][97][98]. PhopGV is able to infect other species of Gelechiidae, such as Tuta (Scrobipalpuloides) absoluta (Meyrick) and T. solanivora [99][100][101][102][103], and with the aim to find isolates also effective against these species, there is a renewed interest in characterization and evaluation of new isolates in several countries, in order to develop new biopesticidal products. The Colombian Corporation for Agricultural Research (CORPOICA) conducted samplings of T. solanivora larvae in Colombia with the purpose of finding local virus isolates. ...
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Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries) will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway.
Insects being the most diverse group, host a broad range of microorganisms. These insect–microbe associations not only account for basic nutritional deficiencies and biochemical needs but also play an essential role in sexual compatibility among mates. Besides these, over a hundred and thousands of bacteria, fungi, and entomopathogenic viruses have been isolated and identified from different insect orders, which is known to produce natural toxins. From diverse insect order, around one percent of insect pests are known to cause severe economic damage to humans and their domesticated animals and plants. Generations of chemical pesticides have been developed and given up owing to their broad range of toxicity, bioaccumulation, and development of resistance. With increased resistance of pest population to chemical pesticides, and negative impact on both environment and human health, there is an urgent need of a more natural and eco-friendly substitute. In this chapter, we have discussed the major microbial sources, i.e. fungi, bacteria, and viruses which are known to produce insect-toxic proteins that can be utilized for the production of biopesticides that are safer, cost-effective, and eco-friendly alternative to chemical pesticides to control pest population.
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Tecia solanivora es una de las plagas más limitantes del cultivo de la papa, para cuyo control el uso de granulovirus constituye una alternativa promisoria. Para el control de la plaga en condiciones de almacenamiento, en Colombia se produce un bioplaguicida en polvo a base de un granulovirus aislado en Perú a partir de larvas de Phthorimaea operculella. En un trabajo previo se aislaron tres granulovirus nativos provenientes de larvas de T. solanivora de los municipios de Chocontá, Mosquera y Carmen de Carupa en el departamento de Cundinamarca, los cuales podrían estar mejor adaptados al insecto y a las condiciones ambientales del país. En el presente trabajo, los tres aislamientos de granulovirus se evaluaron mediante un bioensayo en laboratorio utilizándolos formulados y sin formular y teniendo como patrón de comparación la cepa peruana. Los virus nativos formulados presentaron eficacias entre 88% y 100%, mientras que para el aislamiento peruano se obtuvo 88%. Estos resultados fueron significativamente diferentes de los obtenidos con los aislamientos sin formular, para los cuales la eficacia estuvo entre 36% y 86% para los aislamientos nativos y, 59% para el aislamiento peruano. También se comparó la cantidad de cuerpos de inclusión (CI) producidos por miligramo de larva con cada uno de los aislamientos, entre los que no se encontraron diferencias significativas. El rendimiento promedio fue de 4,4 x 107 CI/mg de larva. Los resultados permitieron seleccionar el aislamiento nativo proveniente de Mosquera (Cundinamarca) para el futuro desarrollo de un bioplaguicida por presentar los mayores niveles de virulencia.
Biological invasions constitute an important economical burden when they affect key resources for human alimentation, health or agronomic productions. Potato pests are important as this tuber is a key food source in Andean countries. The recent dispersion of the Guatemalan potato tuber moth, T. solanivora in South America can be traced back to the introduction in Venezuela, with progressive dispersion towards the South. Recent invasions provide, in addition, a unique model to analyse the process of adaptation of the whole receiving ecosystem to the new comers. This introduction of T. solanivora and its coexistence with the endemic potato tuber moth, Phthorimaea operculella offer us the possibility of studying the adaptation to T. solanivora of virus populations infeodated to the later. A survey has been carried out in the potato-producing regions of Colombia. From the T. solanivora larvae collected, granulovirus infections were detected in five different locations. All virus isolates are related to the previously described Phthorimaea operculella granulovirus (PhopGV). Differences in the pathogenicity against the two hosts were observed. Variability was detected in some isolates at two genetic markers. Genetically diverse populations appear to be more pathogenic for both hosts than genetically homogeneous populations. They provide a possible solution for the biological control of these insect pests. Artificial populations were constructed to mimic the mixed natural populations. They behave similarly from a biological point of view, but the evolution of the markers frequencies is not related to the biological efficacy, suggesting that undetected differences in the genomes could be responsible of this host adaptation. The productivity of the infections in both hosts has been studied as it constitutes a key point for the development of a biocontrol agent. The productivity in P. operculella (1.36 to 2.69 × 108 OBs/ mg) and T. solanivora (0.48 to 3.64 × 108 OBs/mg) are not very different. Genotypically mixed populations cannot be differentiated from homogeneous populations by their total production in one or the other host, however, the yields (virus output/doses to infect) show clear differences, mixed populations (natural or artificial) perform better in both hosts. No reduction in the pathogenicity for one host was observed after few cycles of replication of the virus population in the second host. Virus populations originally adapted to P. operculella had evolved to infect T. solanivora. In regions where both host are present, the populations developed a strategy to be efficient on both hosts.
The tomato leafminer Tuta absoluta is an important insect pest of tomato crops worldwide. Biological control using entomopathogenic viruses such as isolates of Phthorimaea operculella granulovirus (PhopGV) can be a promising tool for suitably controlling this pest. However to date, few viral isolates from T. absoluta have been reported. Therefore, the aim of this study was to develop an economic, fast and accurate immunological technique for granulovirus detection and to evaluate viral presence in T. absoluta larvae from field samples collected in Colombia. Polyclonal antibodies against PhopGV were produced by using hens and rabbits. The production system in hen was selected as the most efficient and simple method. Then, a dot-blot test was developed, demonstrating the ability of antibodies to recognize granulovirus. Finally, granulovirus presence in 1186 T. absoluta larvae collected in the most important tomato production areas in Colombia was evaluated, and eight isolates were found in Boyac�a and Cundinamarca (0.7% of occurrence). These results constitute the first report of a granulovirus isolated from T. absoluta larvae which had been naturally infected in Colombia’s tomato producing-fields, and thereby validates the use of the immunological technique to detect granulovirus in field conditions.
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During 2004 and 2005, native strains of granulovirus obtained from Tecia solanivora larvae found in Cundinamarca, Nariño and Norte de Santander were isolated, identified and characterized. The effects of these infectious agents were studied on development of the potato moths T. solanivora and Phthorimaea operculella, evaluating variables such as width of the cephalic capsule, signs of viral infection and mortality. The effect of formulation was also determined on the efficiency of the different isolates formulated as a powder for potato seed protection. Additionally, the viral productivity of each isolate was studied using spectrophotometry. The highest viral production yields were obtained in P. operculella larvae, which were significantly higher than those obtained in T. solanivora larvae for all native isolates. The granulovirus infection in T. solanivora and P. operculella larvae caused a greater duration and overlap of the larval instars. The process of formulation did not significantly affect the efficacy of the granulovirus. Through a technique of weighted indicators, the isolate from Norte de Santander was selected as most promising due to its high efficiency and productivity.
A granulovirus isolate collected from diseased Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) larvae in Costa Rican potato [Solanum tuberosum L. (Solanaceae)] crops was characterized at the molecular and biological level. Restriction endonuclease analysis identified this isolate as a novel P. operculella granulovirus (PhopGV) (Baculoviridae: Betabaculovirus) strain and was designated as PhopGV-CR1. In addition, PCR amplification of four specific variable genomic regions yielded multiple amplicons for two open reading frames, revealing the presence of different genotypic variants within the virus population. Biologically, PhopGV-CR1 was highly pathogenic for its two indigenous hosts, although significant differences of up to four-fold were detected against P. operculella [LD50 = 17.9 occlusion bodies (OBs) mm−2] and Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) (LD50 = 69.1 OBs mm−2). The two P. operculella colonies, from Costa Rica and France, were equally susceptible to PhopGV-CR1. Serial passage of PhopGV-CR1 over four generations in T. solanivora increased its pathogenicity by five-fold in three generations, suggesting an ongoing adaptation to its alternate host.
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The Guatemalan potato moth Tecia solanivora (Povolny) recently invaded part of South America, colonizing zones where Phthorimaea operculella (Zeller), another potato moth species belonging to the same group, was previously established. T. solanivora is now the major insect pest of potato in this area encompassing Venezuela, Colombia and Ecuador. P. operculella granulovirus (PhopGV) (Betabaculovirus) is a biocontrol agent to be considered for the simultaneous management of these two potato pests, instead of classical chemical insecticides. In a previous work, five PhopGV isolates were isolated in Colombia from T. solanivora and were tested against larvae of the same species showing variable efficacies. Infections with mixtures of different genotypes of Baculoviruses had been carried out in a wide range of species and several showed interesting results. In the present study, the effect of sequential passages of PhopGV in P. operculella and T. solanivora larvae was analyzed through biological assays. Three different mixtures containing a Peruvian PhopGV isolate (Peru) adapted to P. operculella and a Colombian PhopGV isolate (VG003) adapted to T. solanivora were tested. A preliminary analysis of the correlation between the genotypic marker egt gene and the level of pathogenicity after a variable number of replication cycles was made. Mixtures of virus isolates showed a higher efficacy in both hosts compared to individual PhopGV isolates. This higher pathogenicity was maintained through passages. In P. operculella the mixtures were between 2.8 and 23.6-fold (from 7.15 OB/mm2 to 0.10 OB/mm2) more pathogenic than isolate Peru applied alone. In T. solanivora they were between 2.3 and 4.9-fold (from 12.29 OB/mm2 to 1.25 OB/mm2) more pathogenic than isolate VG003 alone. Viral biopesticide containing a mixture of selected genotypes active against each hosts seemed suitable for the development of a biopesticide aimed to simultaneously control P. operculella and T. solanivora.
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Tecia solanivora (Lepidoptera: Gelechiidae) is an invasive potato pest of the north of South America that recently colonized zones where Phthorimaea operculella (Lepidoptera: Gelechiidae), a taxonomically related insect, was established. Nowadays, both species can be found in most areas in different proportions. The Phthorimaea operculella granulovirus (PhopGV) was found to efficiently control P. operculella and was used as a biopesticide in storage conditions. However, no appropriate biological control methods exist for T. solanivora, and the use of granulovirus isolates would provide a solution. The Colombian Corporation for Agricultural Research (CORPOICA) carried out several T. solanivora larva samplings in Colombia with the aim of finding potential isolates. Five geographical granulovirus isolates from T. solanivora (VG001, VG002, VG003, VG004, and VG005) were found, and molecular analysis by REN profiles shows three different genotypic variants in Colombia. Analysis of their genomes revealed their relatedness to PhopGV. Two isolates exhibited submolar bands in their REN patterns, suggesting a mixture of viral genotypes. These data were confirmed by PCR amplification and sequencing of particular regions of the viral genomes. Their biological activity was assayed on both hosts, T. solanivora and P. operculella. A significantly higher pathogenicity in both hosts was observed with isolates VG001 and VG005 than with isolate VG003 or a Peruvian isolate (from P. operculella) used as a reference in the bioassay. Based on their molecular and biological activity characteristics, VG001 and VG005 isolates should be selected for further analysis in order to establish their potential as biological control agents.
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