Microbiology Group, Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, USTHB, El Alia, BP 32, 16111, Bab Ezzouar, Algiers, Algeria.
ERI de Biotecnología y Biomedicina (BIOTECMED), Department of Genetics, Universitat de València, 46100, Burjassot, Spain.
Curr Microbiol. 2020 Oct;77(10):2904-2914. doi: 10.1007/s00284-020-02110-3. Epub 2020 Jul 10.
Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect's resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore-crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of "functional mortality" was used to discriminate between "nontoxic" and "toxic" isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore-crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130 kDa whereas the Bt isolate Bl5NA expresses a protein of 65-70 kDa. The LC-MS/MS results indicate that the major peptides in the 130 kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70 kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce β-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.
苏云金芽孢杆菌（Bt）是目前世界范围内应用最广泛的昆虫病原生物防治技术。为了筛选具有挑战性的新候选菌株，以应对昆虫抗药性的出现，我们对一个本土采集品系进行了大规模的生物测定和分子筛选。使用孢子-晶体混合物和 49 个含有至少一个编码鳞翅目特异性杀虫蛋白基因的 Bt 分离株的上清液培养物对三种鳞翅目幼虫（菜蛾、桃小食心虫和斜纹夜蛾）的新生幼虫进行了毒性测定。使用 30%的“功能死亡率”作为区分“非毒性”和“毒性”分离株的阈值。许多 Bt 分离株的毒性与 Btk-HD1 的毒性相当。然而，只有三个分离株（Bl4NA、Bl5NA 和 Bl9NA）在孢子-晶体混合物和上清液培养物中对三种鳞翅目物种均表现出高毒性。Bt 分离株 Bl4NA 和 Bl9NA 表达 130 kDa 的蛋白，而 Bt 分离株 Bl5NA 表达 65-70 kDa 的蛋白。LC-MS/MS 结果表明，Bl9NA 中 130 kDa 带的主要肽段为 Cry1Da、Cry1Ca、Cry1Ab 和 Cry1Aa，而 Bl5NA 中 70 kDa 带的主要肽段为 Cry1Aa 和 Cry1Ca。通过与 Btk-HD1 的上清液蛋白含量比较，表明这些菌株中过量表达了 Vip3 蛋白（很可能在 Bl4NA 和 Bl9NA 中是 Vip3Aa，而在 Bl5NA 中是 Vip3Ca）。此外，这三种 Bt 菌株不产生β-外毒素。基于我们的结果，这三个选定的菌株可能被认为是有前途的候选菌株，可用于害虫防治。
