FORINSEC

The general goal of the project was to design and produce new active ingredients based on baculovirus (OBs) and the Bt (Cry+Vip3A) insecticide protein, with a wide spectrum of hosts and better insecticide properties than the product currently on the market. Another parallel goal was to identify molecules with antioxidant, photoprotection or synergetic activity on the active ingredient. To promote those effects, the metabolites had to be formulated as microcapsules together with the new active ingredients (OBs) or (Cry+vip3A), in order to improve their stability during storage and their persistence and effectiveness as insecticide when used in the field.

In the first place, work was done on developing and obtaining new active baculovirus ingredients with a wider spectrum of hosts and improved insecticide properties. Work was done with several isolates and variants (and mixtures of them), and the best one was determined based on its all-around insecticide properties, and which one was the most pathogenic and productive. Subsequently, the physical characteristics and genetic structure of the OBs that made up the active ingredient were determined. On one hand, we saw that the four wild isolates analysed in this project (HearMNPV, AcMNPV, AnfaMNPV and MbMNPV), have OBs that are very similar in size and internal structure. The specific analysis of the structure of the ODVs was also similar in the four wild isolates analysed. Likewise, through qPCR it was additionally confirmed that the various genotypic variations were in the same relative proportion as in the artificial combination used to coinfect the S. exigua larvae. Lastly, in this first block of tasks the active ingredient (OBs) with the optimum genetic structure was produced.

Secondly, a new strain of Bacillus thuringiensis (Bt) with a wider spectrum of hosts and improved insecticide characteristics was characterised. To do that, first Bt strains were selected that produced a combination of proteins (Cry+Vip3A) with a wide spectrum of hosts and high insecticide power. Then the production process of the Cry and Vip3A proteins was optimised in laboratory fermenters, so it could be scaled to a semi-industrial level in fermenters of more than 1000 litres. Because laboratory conditions frequently cannot be extrapolated to large volume production, and it was necessary to test the scalability of production of the active substance.

In the third block of activity, bioactive compounds were identified that could be used as additives in the new formulation to increase the persistence of the insecticide effect during storage and after being used in the field. Thirty-eight extracts from different plant raw materials were analysed. In the first place, their extraction performance was analysed and, secondly, their synergetic capacity with the different active materials (both baculovirus (BV) and Bt) was analysed. After the first tests, six extracts were selected because of their enhancing capacity and extraction performance. The E37 extract was ultimately chosen as the most promising because it increased activity up to 14 times in comparison with the control. To optimise the extraction method three extracts were chosen, which will be included in the optimum formulation for the encapsulation trials.

Lastly, the formulation of the technical product through micro-encapsulation was tackled. The first phase was to analyse the polymers for encapsulating the active substances. Encapsulation using proteins was chosen, and then virion encapsulation was tested so the synergetic plant extracts studied previously could be included. After obtaining the microcapsules, their activity against susceptible insects was evaluated. Likewise, the photoprotection capacity of the covering and the viability of the OBs under determined temperature and pH conditions was analysed, and the results were highly satisfactory. Furthermore, a simplification of the preparation process of the microcapsules was designed in order to improve the potential for scalability and industrial scale implementation. Despite the results being promising, a slight increase in the particles was observed, which could be problematic when they are ingested by larvae in the early lagoon larval instars of the smaller phytophagous species.

Consequently, we propose continuing to work on the issue. And we expect to propose a project that continues to explore this work, because it is seen as a strategic line of research in bioinsecticides whose goal is to produce new generations of cheaper, more efficient and more sustainable microbial insecticides.