Development of a viral biopesticide for the control of the Guatemala potato tuber moth Tecia solanivora.

The Guatemala potato tuber moth Tecia solanivora (Povolny) (Lep. Gelechiidae) is an invasive species from Mesoamerica that has considerably extended its distribution area in recent decades. While this species is considered to be a major potato pest in Venezuela, Colombia, and Ecuador, currently no specific control methods are available for farmers. To address this issue we developed a biopesticide formulation to be used in integrated pest management of T. solanivora, following three steps. First, search for entomopathogenic viruses were carried out through extensive bioprospections in 12 countries worldwide. As a result, new Phthorimaea operculella granulovirus (PhopGV) isolates were found in T. solanivora and five other gelechid species. Second, twenty PhopGV isolates, including both previously known and newly found isolates, were genetically and/or biologically characterized in order to choose the best candidate for a biopesticide formulation. Sequence data were obtained for the ecdysteroid UDP-glucosyltransferase (egt) gene, a single copy gene known to play a role in pathogenicity. Three different sizes (1086, 1305 and 1353 bp) of egt were found among the virus isolates analyzed. Unexpectedly, no obvious correlation between egt size and pathogenicity was found. Bioassays on T. solanivora neonates showed a maximum of a 14-fold difference in pathogenicity among the eight PhopGV isolates tested. The most pathogenic PhopGV isolate, JLZ9f, had a medium lethal concentration (LC(50)) of 10 viral occlusion bodies per square mm of consumed tuber skin. Third, we tested biopesticide dust formulations by mixing a dry carrier (calcium carbonate) with different adjuvants (magnesium chloride or an optical brightener or soya lecithin) and different specific amounts of JLZ9f. During laboratory experiments, satisfactory control of the pest (>98% larva mortality compared to untreated control) was achieved with a formulation containing 10 macerated JLZ9f-dead T. solanivora larvae per kg of calcium carbonate mixed with 50 mL/kg of soya lecithin. The final product provides an interesting alternative to chemical pesticides for Andean farmers affected by this potato pest.

Experimental mixtures of Phthorimaea operculella granulovirus isolates provide high biological efficacy on both Phthorimaea operculella and Tecia solanivora (Lepidoptera: Gelechiidae).

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/mm(2) to 0.10 OB/mm(2)) more pathogenic than isolate Peru applied alone. In T. solanivora they were between 2.3 and 4.9-fold (from 12.29 OB/mm(2) to 1.25 OB/mm(2)) 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.

Genetic and biological analysis of Colombian Phthorimaea operculella granulovirus isolated from Tecia solanivora (Lepidoptera: Gelechiidae).

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.

Cydia pomonella granulovirus genotypes overcome virus resistance in the codling moth and improve virus efficiency by selection against resistant hosts.

Cydia pomonella granulovirus (CpGV) has been used for 15 years as a bioinsecticide in codling moth (Cydia pomonella) control. In 2004, some insect populations with low susceptibility to the virus were detected for the first time in southeast France. RGV, a laboratory colony of codling moths resistant to the CpGV-M isolate used in the field, was established with collection of resistant insects in the field followed by an introgression of the resistant trait into a susceptible colony (Sv). The resistance level (based on the 50% lethal concentrations [LC(50)s]) of the RGV colony to the CpGV-M isolate, the active ingredient in all commercial virus formulations in Europe, appeared to be over 60,000-fold compared to the Sv colony. The efficiency of CpGV isolates from various other regions was tested on RGV. Among them, two isolates (I12 and NPP-R1) presented an increased pathogenicity on RGV. I12 had already been identified as effective against a resistant C. pomonella colony in Germany and was observed to partially overcome the resistance in the RGV colony. The recently identified isolate NPP-R1 showed an even higher pathogenicity on RGV than other isolates, with an LC(50) of 166 occlusion bodies (OBs)/microl, compared to 1.36 x 10(6) OBs/microl for CpGV-M. Genetic characterization showed that NPP-R1 is a mixture of at least two genotypes, one of which is similar to CpGV-M. The 2016-r4 isolate obtained from four successive passages of NPP-R1 in RGV larvae had a sharply reduced proportion of the CpGV-M-like genotype and an increased pathogenicity against insects from the RGV colony.

An isometric virus of the potato tuber moth Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) has a tri-segmented RNA genome.

A small isometric virus has been isolated from larvae of the Guatemala potato tuber moth, Tecia solanivora (Povolny), collected in Ecuador. It was designated the Anchilibi virus (AnchV). The non-enveloped viral particles have an estimated diameter of 32+/-2 nm. Three major proteins were found in virions, with estimated sizes of 102.0+/-2.1, 95.8+/-2.0 and 92.4+/-1.5 kDa for AnchV as determined by polyacrylamide gel electrophoresis. After denaturing agarose gel electrophoresis, the genome of AnchV appeared to be a tri-segmented single-stranded RNA with fragment sizes of 4.1+/-0.2, 2.8+/-0.2 and 1.65+/-0.2 kb. In addition to a high virulence towards its original host, AnchV also caused high mortality in larvae of two other potato tuber moth species, Phthorimaea operculella (Zeller) and Symmetrischema (tangolias) plaesiosema (Turner). Electron microscopy confirmed that AnchV replication occurs in the cell cytoplasm, mainly in vesicles. Several important characteristics exhibited by this virus differ from those reported for known families of insect viruses. Thus, AnchV might be member of a new taxonomic group.

Characterization and authentication of insect cell lines using RAPD markers.

Random amplified polymorphic DNA (RAPD) analysis was used to characterize 11 insect cell lines, including six from lepidoptera (five species), one from diptera and four from coleoptera (one species: Leptinotarsa decemlineata). Whatever the order and even when comparing two closely related species from the same genus (Spodoptera), the DNA fingerprints are very different from one species or from one primer to the other. On the other hand, two independently isolated cell lines from the lepidopteran Phthorimaea operculella produce nearly identical profiles with only minor differences. Finally, a statistical analysis based on Nei's similarity coefficient was performed on the fingerprints of four independent cell lines from the Colorado potato beetle, L. decemlineata. Each possesses a common recognizable pattern also found in field-collected insects, while also showing a series of polymorphic markers which allow one to distinguish each cell line from the three others. RAPD fingerprinting, together with the use of appropriate statistics, thus constitutes a highly specific method both for the authentication of the species from which a cell line was developed and for the individual characterization of each cell line from a given species.