Development of a new bioinsecticde base on a Chrysodeixis chalcites nucleopolyhedrovirus from the Canary Islands

Abstract

Chrysodeixis chalcites (Lepidoptera: Noctuidae) causes valuable economic damage in banana crops in the Canary Islands. Effective control of this pest with chemical insecticides requires many applications that increase production costs and render serious environmental hazards and chemical residues that hamper banana commercialization. In these situations, microorganism-based bioinsecticides, particularly baculoviruses, constitute one of the most realistic alternatives for efficient pest control programs. Under natural conditions, C. chalcites populations are affected by the C. chalcites nucleopolyhedrovirus (ChchSNPV) (Fam. Baculoviridae, Alphabaculovirus). The aim of this thesis has been to address seme of the biotechnological developments necessary to obtain a new biopesticide based on a ChchSNPV strain indigenous of the Canary Islands. Firstly, the genetic diversity of ChchSNPV in the Canary Islands was evaluated. During the course of a large field sampling, 97 larvae died with the typical signs and symptoms of NPV disease. These NPV isolates were grouped into five different strains as indicated by restriction endonuclease analysis of their viral DNA genome. The most widespread and prevalent isolate, ChchSNPV-TFI (ChchTFI), was also the most pathogenic and virulent against a local insect population. Genotypic characterization of this isolate revealed that ChchTF1 is composed of at least eight different genotypes, being ChchTF1-A the most isolated one and also the most prevalent in the wild-type population (36%). None of the pure genotypes was as pathogenic as the wild-type population, suggesting the occurrence of interactions among genotypes that increase the pathogenicity of the wild-type mixture. However, ChchTF1 was less virulent than single genotypes which was correlated with a higter occlusion body (OB) yield. ChchTF1 is genotypically structured to maximize its transmissibility in nature. Experimental testing mixtures of genotypes revealed that the co-occluded mixture composed by the three most prevalent genotypes, ChchTF1-ABC, in a proportion similar to that found in the wild-type population, was even more pathogenic than the wild-type population. Unexpectedly, the virulence of the co-occluded mixture was improved by 33 h in relation to the wild-type isolate.

Therefore, the ChchTF1-ABC co-occluded mixture was selected as the active ingredient for a bioinsecticide to control C. chalcites in the Canary Islands. Sequencing analysis of the most frequent genotypes, ChchTF1-A, -B and ¿C, and of the least frequent ones, ChchTF1-G and ¿H, revealed a high similarity (around 99%) between them. Two regions of genome variability, located in the hoar and bro-d genes, were identified that could account for differences in the phenotype of these genotypes. One of the greatest limitations in developing baculoviruses as biocontrol agents is the mass production system, which to date, is performed in vivo. To optimize ChchTF1 OB production, variables such as larval stage, inoculation time, viral suspension concentration and larval density were evaluated. Lots of 150 larvae 24 h post L6 molting infected with virus concentrations that killed 90% of inoculated larvae (LC90) yielded greater OB productions (8.07x1013Obs/container), than lower densities (1, 25, 50 and 100) and similar to those yielded in the highest larval density (200). This represented a 59,645-fold increase over the inoculated concentration. Thereafter, the susceptibility of different C. chalcites instars to ChchTF1 was evaluated. Susceptibility to viral infection decreased as larvae aged and second instars (L2) were 104-fold more susceptible than sixth instars (L6). The virus speed of kill was 42 h faster in L2 than in L6 and OB production increased in late instars and L6 larvae were 23-fold more productive than L4. Formulation of ChchTF1 with optical brighteners increased the virus insecticidal properties. For instance addition of 1% of Tinopal UNPA-GX increased the pathogenicity between 4.43 to 397-fold depending of the larval instar. The virulence was also improved by 14 to 26 h. However, OB yield was reduced by 8.5-fold. The sinergistic activity of Leucophor UO was significantly lower. In sum, formulation of ChchTF1 with optical brighteners may improve the efficiency of the product as they allow to achieve control levels at lower virus concentrations. These formulations seem particularly useful in field situations where it is necessary to control different larval stages simultaneously with a single dose. Finally, the efficacy of the ChchTF1 in controlling larval infestations of C. chalcites in tomato and banana crops was compared with a chemical (indoxicarb) and a biological inseticide (Bacillus thurigiensis var kurstaki), commonly used in these crops. ChchTF1 treatments (1x109Obs/l) were 3 to 4-fold more effective in reducing larval infestations than the chemical or Bt treatments. Considering thatpesticides are usually applied to banana crops at volumes fo 1600-2000 l/ha, for an application rate of 1x109Obs/l, the amount of virus inoculum for an hectare would be 1.6-2.0x102 Obs. Given that our system yields as much as 8.1x1013OBs from 150 NPV-killed larvae, 40 to 50 ha could be protected efficiently with this inoculum. All this information has been used to make a patent application and is the basis for development of a new biopesticide. This new biopesticide, besides being the most effective biocontrol agent against C. chalcites to this day, is a very useful tool for the implementation of Integrated Pest Management programs and the establishment of a sustainable agriculture.