Xavier Léry

Cydia pomonella granulovirus Genotypes Overcome Virus Resistance in the Codling Moth and Improve Virus Efficiency by Selection against Resistant Hosts

ABSTRACT 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 [LC50s]) 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 LC50 of 166 occlusion bodies (OBs)/μl, compared to 1.36 × 106 OBs/μl 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.

Genetic and Biological Analysis of Colombian Phthorimaea operculella Granulovirus Isolated from Tecia solanivora (Lepidoptera: Gelechiidae)

ABSTRACT 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.

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.

MULTIPLICATION OF A GRANULOSIS VIRUS ISOLATED FROM THE POTATO TUBER MOTH IN A NEW ESTABLISHED CELL LINE OF PHTHORIMAEA OPERCULELLA

SummaryA newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95.The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.

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.

Costa Rican soils contain highly insecticidal granulovirus strains against Phthorimaea operculella and Tecia solanivora

The aim of this work was to isolate and characterize novel Phthorimaea operculella granulovirus (PhopGV) strains from Costa Rican soils. Three novel strains, named PhopGV‐CR3, PhopGV‐CR4 and PhopGV‐CR5, were isolated from three locations in Costa Rica, Alvarado, Zarcero and Abangares, respectively, by means of soaking potato tubers with diluted soil samples. An additional strain, PhopGV‐CR2, was identified from diseased larvae from a Tecia solanivora laboratory culture. Restriction fragment length polymorphisms obtained for each isolate with six restriction endonucleases (RENs) allowed their identification as four distinct PhopGV strains. Both REN and Polymerase chain reaction analyses indicated the existence of an array of genotypic variants present in all isolates. Bioassays in P. operculella and T. solanivora showed that PhopGV‐CR3 was well adapted to the two coexisting hosts with high levels of pathogenicity against both pest species. The mean lethal dose values of this strain were 2.8 OBs/mm2 for P. operculella and less than 0.5 OBs/mm2 for T. solanivora. We conclude that PhopGV‐CR3 shows great promise for soil application against these pests in Costa Rican potato crops.

Establishment of a cell line derived from embryos of the potato tuber mothPhthorimaea operculella (Zeller)

SummaryA cell line from the main insect pest of potatoes in tropical and subtropical areas,Phthorimaea operculella (Zeller), was obtained from embryoculture. These cells were cultured in Grace’s modified medium. The cell line, designated ORS-Pop-93, had a heterogeneous population consisting of spherical and spindle cells with great capacity to adhere and a doubling time of 40 h. They were subcultured for more than 60 passages. Their polypeptidic profile was different from profiles of other lepidopteran cell lines. The cell line supports the multiplication of theAutographa californica nuclear polyhedrosis virus.

Responses of different geographic populations of two potato tuber moth species to genetic variants of Phthorimaea operculella granulovirus

Phthorimaea operculella granulovirus (PhopGV) belongs to the genus Betabaculovirus of the arthropod‐infecting Baculoviridae. PhopGV is able to infect several gelechiid species. Among them are the potato tuber moths Phthorimaea operculella Zeller and Tecia solanivora Povolny (both Lepidoptera: Gelechiidae). In various South American countries, PhopGV‐based biopesticides are used to control either P. operculella or T. solanivora. Many trials have indicated that a particular viral isolate can exhibit very distinct pathogenicity when infecting different host species or different populations of one host species. In this study, we compared host–pathogen interactions using various PhopGV isolates and various populations of P. operculella and T. solanivora. Virus isolates from P. operculella were more pathogenic against their original host species than against T. solanivora. A PhopGV isolated from T. solanivora was less efficient against P. operculella. In addition, virus isolates differed in pathogenicity toward their hosts (i.e., lethal concentrations of isolates ranged from low to high). Unexpectedly, we also found that host populations of one species from distinct geographic origins did not differ significantly in susceptibility to the same PhopGV isolate. This was the case for both host species and for five PhopGV isolates. Comparative restriction fragment length polymorphism (RFLP) analyses of 11 isolates including those used in bio‐assays indicated three main regions of variation in the genome of PhopGV, corresponding to the regions of open reading frame PhopGV046, gene PhopGV129 (egt), and repeat 9 (located between open reading frames PhopGV083 and PhopGV084). Comparison of the nucleotide sequences of the insertions/deletions present in these regions were carried out for the most variable isolate, JLZ9f. The results are discussed in the context of the production and use of PhopGV as a biological agent against these two pest species.

A new permissive cell culture obtained from Latoia viridissima (Lepidoptera, Limacodidae).

Two viruses were isolated on Latoia viridissima (Lepidoptera, Limacodidae), defoliator of oil palm in Côte d’Ivoire, a picornavirus (G. Fédière, R. Philippe? J. C. Veyrunes, and P. Monsarrat, Entomophaga 37, 354-357,1990) and a nuclear polyhedrosis baculovirus (N. K. Mouassi, Oléagineux, in press). No lines were known from this family and as no permissive cell culture existed to multiply these viruses, we have attempted to obtain a permissive cell line from hemocytes of this insect. Larvae from four different field samples were used for the culture of 75 C35 petri dishes. Hemolymph obtained by the “dropping” method (J. M. Quiot, Doctorat, Montpellier? France, 1975) was put directly into petri dishes with Grace medium containing 10% fetal bovine serum (FBS), modified form (X. Léry and G. Fédière, J. Invertebr. Pathol. 55,47-51,1990), with the addition of 5%0 NaN, and incubated at 28°C (all chemicals from Sigma). After 4 hr of culture, most of the hemocytes adhered to the flask walls. During the first days of the culture, the different types of hemocytes described by T. J. Kurtti and M. A. Brooks ( J . Inuertebr. Pathol. 15, 341-350, 1970) were observed. After 1 to 2 weeks, small fibroblast-like cells (prohemocytes) were the only cells remaining in the cultures. They occurred in two forms, fibroblast-like adherent cells and small rounded suspension cells representing mitotic prohemocytes (Fig. 1). During the first 10 days, in 40% of the cultures, no multiplication occurred and cells died. In a few cultures, characteristic polyhedra were found in the cells. After purification and analysis they appeared to be nuclear polyhedrosis viruses and represented endogenous infections. During the following weeks, a low rate of multiplication was observed in 50% of the remaining cultures. The vigorous multiplication was generally observed af-