Soybean protease inhibitors increase Bacillus thuringiensis subs. israelensis toxicity against Hypothenemus hampei

Abstract

Introduction. The coffee berry borer (Hypothenemus hampei Ferrari, CBB) is one of the most devastating pests on coffee plantations around the world. Although CBB is susceptible to the effect of δ–endotoxins of Bacillus thuringiensis subs. israelensis (Bti) at laboratory level, the efficacy of this control method is poor in the field, presumably due to the inactivation by digestive proteases different to those required for protoxin activation. Objective. To study whether the addition of a soybean flour extract enriched with protease inhibitors (PI), mixed with Bti crystals and spores (Bti-sc) in an artificial diet, could improve the toxicity of Bti against CBB. Materials and methods. This study was performed in San José, Costa Rica, between 2012 and 2013. A set of adult female CBB insects was exposed to a mixture containing different concentrations of a partially purified soybean meal extract with active PI and lyophilized Bti-sc, and were tested through a bioassay in artificial diet to estimate the sub-lethal concentration (LC50). The mortality results were validated by observing the dissected midgut, whose ultrastructure was analyzed by transmission electron microscopy. Results. The soybean extracts partially degraded the Bti-sc complex, it reduced its LC50 by almost four times (from 1.135 to 0.315 µg µl-1) and enhanced CBB mortality in a concentration-dependent manner. Histological analyses of the midgut confirmed this synergistic effect, since severe epithelial damage to the intestinal epithelium of CBB exposed to Bti-sc + PI was visualized compared to Bti-sc alone. Conclusions. The combination of a soybean extract enriched in PI and Bti-sc enhanced the mortality effect over CBB, which was confirmed by the midgut collapse. Soybean flour is a cost-effective supplement that could increase Bti effectiveness against CBB and delay the appearance of biological resistance.

Deficiencies in the Risk Assessment of Genetically Engineered Bt Cowpea Approved for Cultivation in Nigeria: A Critical Review

“…In addition, with cowpea, plant compounds, such as the protease inhibitors (PI) produced in the plants, can cause Bt toxins to degrade more slowly than in isolation. This can result in higher toxicity of the r-Bt toxins if taken up together with the plant tissue when compared to toxins in isolation [ 35 , 36 , 37 , 38 , 39 ]. Indeed, there is evidence that the toxicity of Bt toxins is increased if combined with PI [ 40 , 41 , 42 , 43 , 44 ].…”

“…These organisms should be subjected to specific tests in the laboratory, or a greenhouse, to provide the relevant data on the toxicity and impact of the plant material. The investigations should include synergistic effects of Bt toxins and PI as described and evidenced [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ]. As far as hazards are concerned, the effects on non-target organisms need to be fully investigated, as plant material from the Bt cowpea may have unexpected and severely adverse effects on ecosystem services, the food web and biodiversity of exposed insects or wild species, e.g., birds or other non-target organisms.…”

“…Therefore, empirical testing of the relevant microbial species, including the plant microbiome and long-term impact on the soil rhizosphere, is necessary before concluding on the environmental safety of Bt cowpea. The experiments should also take into account synergistic effects such as those made evident by several publications [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ].…”

“…In the case of cowpea, plant compounds, such as PI, can cause the r-Bt protein to show higher toxicity than it would in isolation. As shown, there are specific findings evidencing an increase in the toxicity of Bt proteins if combined with PI present in cowpea [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ]. Such synergetic effects not only enhance the effectivity (toxicity) of Bt toxins but may also impact their selectivity (for overview, see [ 32 ]).…”

“…However, such studies are of little value as these experimental conditions do not allow identification of synergistic effects, which are likely to occur in the plant material and which might cause higher overall toxicity in the diet. The effects caused by the PI described by [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ] that leads to higher toxicity of Bt toxins show that the approach chosen by the applicant is inadequate.…”