Evaluation of Bacillus thuringiensis crystal (CRY6A) and carica papaya cystatin toxins against the banana weevil (Cosmopolites sordidus) using a novel diet and construction of a plant transformation vector with two stacked toxin genes

Abstract

The banana weevil (Cosmopolites sordidus) is a major pest of banana, plantain and Ensete. Despite the existence of control strategies that combine cultural, biological, use of botanical extracts and chemical control methods, banana weevil damage and the associated crop losses have persisted. Although resistance is available in wild banana, conventional breeding is not feasible. Genetic engineering as an alternative strategy to conventional breeding or in combination has been suggested for the provision of weevil resistant banana varieties to farmers, especially with maintained taste and cultural preferences. However, this has been hampered among other experimental factors, by the lack of an artificial diet to perform rapid bioassay experimentation to screen resistant banana cultivars and potential candidate proteins for a transgenic approach. This research modified an artificial diet in which banana weevil neonates were able to develop up to adult stage in 48 days compared to 36 days on natural banana stem diet. It also reports on the use of this diet to evaluate resistance in banana germplasm and the in-vitro efficacy of Bacillus thuringiensis Cry6A as well as Carica papaya cystatin (CpCYS) toxins against C. sordidus. The laboratory produced recombinant Cry6A and CpCYS proteins and their use in the diets is described. The modified artificial diet allowed preliminary evaluation of efficacy and potential of these two genes in the control of banana weevil. Fifty percent mortality (LD50) of neonate larvae was observed at 0.24 parts per million and 0.15parts per million for Cry6A and CpCYS respectively. Both genes were cloned together into a plant expression cassette to create a plant transformation binary vector. This research therefore modified an artificial diet that sustained 70% of banana weevil through all growth stages and using this diet established the efficacy of two recombinant proteins; a basis for developing transgenic bananas with improved resistance against banana weevils.