Synergism Among Nematicidal Crystal Proteins Cry6Aa, Cry5Ba And Cry55Aa From Bacillus Thuringiensis Against Root-Knot Nematode, Meloidogyne Incognita

Root-knot nematode is an important plant-parasitic pathogens, which causes drastic damage to crop production. Root-knot nematodes usually live underground so that it is very difficult to cope with them using traditional insecticides. In previous studies, researchers have reported that Cry6A and Cry5B were toxic to M. incognita and exhibited good control of M. incognita by constructing transgenic plants. However, a few nematicide cry genes were just documented, and thus to discover new cry genes or to explore new ways to control root-knot nematodes has great practical significance.1. Root-knot nematodes almost live together in the soil. In this study, M. incognita was isolated and identified from the infected root of tomato by molecular diagnostic key. Therefore, it is a good material to carry out further study on related root-knot nematodes.2. This study was carried out to improve the laboratory bioassay method established of Cry protein against root-knot nematode. In the absence of protein purified Cry5Ba, the result showed that the combination of 1.0μg/mL M-dihydroxybenzene and 2.5μg/mL Nystatin against M. incognita was not distinctively different compared with water agaist M. incognita. In the present of protein purified Cry5Ba and 20μg/mL BSA as negative control, no significant difference was showed in the three duplicates, which suggested the method improved was feasible.3. Based on bioinformatics analysis of Cry6Aa, Cry5Ba and Cry55Aa, the three proteins exhibited obvious differences. Furthermore, the interaction between total proteins of M. incognita and Cry6Aa, Cry5Ba and Cry55Aa was carried out by Ligand blot. The results showed that hybridization signals had distinctive differences.4. The three purified proteins Cry6Aa, Cry5Ba and Cry55Aa were combined at different ratios to assay the toxicities against M. incognita, and synergism between them has been assessed by synergistic factor (SF). The results showed that proteins Cry6Aa and Cry5Ba, as well as Cry6Aa and Cry55Aa, exhibited synergistic toxicity against M. incognita; at 1:1:1 ratio of Cry6Aa and Cry5Ba and Cry55Aa against M. incognita exhibited synergistic toxicity, while the interaction of Cry5Ba and Cry55Aa, Cry6Aa and Cry5Ba and Cry55Aa, against M. incognita exhibited additive toxicity. Additionally, the results also showed that the highest toxicity could be observed at 1:1 ratio of Cry6Aa and Cry5Ba, as well as of Cry6Aa and Cry55Aa, and that Cry6Aa and Cry55Aa exhibited higher toxicity against M. incognita than that of Cry6Aa and Cry5Ba.This study demonstrated that the combination of Cry6Aa/Cry5Ba and Cry6Aa/Cry55Aa against M. incognita exhibited obvious synergism. Therefore, the study could not only provide theoretical support for constructing transgenic plants with nematicidal genes cry6Aa/cry5Ba and cry6Aa/cry55Aa, and but also provide new strategy for delaying emergence of the root-knot nematode resistance.