Department of Agricultural and Forestry Sciences, ETSIAM, University of Cordoba, C4 Building, Campus of Rabanales, 14071 Cordoba, Spain.
Department of Agronomy, ETSIAM, University of Cordoba, C4 Building, Campus of Rabanales, 14071 Cordoba, Spain.
J Invertebr Pathol. 2017 Oct;149:29-35. doi: 10.1016/j.jip.2017.06.007. Epub 2017 Jun 16.
Destruxin A is among the major secondary metabolites produced by the entomopathogenic ascomycete Metarhizium sp., and the lack of studies concerning production of destruxin A by the fungus is most likely the biggest obstacle for the registration of new fungal strains. Although several studies focus on the production of destruxin A in culture media, few studies examine destruxin A in vivo during host infection. In the current work, Galleria mellonella was used as an insect model to develop for the first time in vivo real-time PCR- and HPLC-MS-based quantification of fungal growth and metabolite production, respectively, during infection by two strains of M. brunneum. Total mortality of sixth instar G. mellonella larvae that were immersed in a suspension of 1.0×10conidiamL of M. brunneum EAMa 01/58-Su or BIPESCO5 strains reached 85.5% and 78.8%, respectively, and the percentage of cadavers with fungal outgrowth was low at 12.2% and 4.4%, respectively. The average survival time of treated larvae was 5.5days for both fungal strains. Using EAMa 01/58-Su and BIPESCO5 specific primer set, real-time PCR showed that the patterns of fungal growth were different for the two strains, whereas no significant differences were detected in the number of fungal sequence copies recovered from the infected larvae. EAMa 01/58-Su and BIPESCO5 strains secreted destruxin A from days 2 to 6 and from days 2 to 5 post treatment, respectively. For EAMa 01/58-Su and BIPESCO5, the maximum titer of destruxin A in the host was on day 4 at 0.369 and 0.06µg/larva, respectively, and throughout the pathogenic process, the total production was 0.6 and 0.09µg/larva, respectively. These results demonstrated that the strains pose a low hazard, if any, to humans and the environment. The methods used in this study to quantify fungal growth and metabolite production provided valuable data to better understand the role of destruxin A during the growth of M. brunneum in the host larvae and to monitor the fate of destruxin A in food chains.
destruxin A 是一种由昆虫病原子囊菌 Metarhizium sp. 产生的主要次生代谢物之一，而缺乏对该真菌产生 destruxin A 的研究很可能是新真菌菌株注册的最大障碍。尽管有几项研究集中在培养基中 destruxin A 的生产上，但很少有研究在宿主感染过程中研究体内的 destruxin A。在本研究中，首次使用家蚕作为昆虫模型，开发了体内实时 PCR 和 HPLC-MS 定量分析方法，分别用于感染两种 M. brunneum 菌株时真菌生长和代谢产物的产生。浸入 1.0×10 个分生孢子/mL M. brunneum EAMa 01/58-Su 或 BIPESCO5 菌株悬浮液的第六龄家蚕幼虫的总死亡率分别为 85.5%和 78.8%，真菌生长的尸体百分比分别为 12.2%和 4.4%。处理幼虫的平均存活时间均为 5.5 天。使用 EAMa 01/58-Su 和 BIPESCO5 特异性引物对，实时 PCR 显示两种菌株的真菌生长模式不同，而从感染幼虫中回收的真菌序列拷贝数没有差异。EAMa 01/58-Su 和 BIPESCO5 菌株分别在处理后第 2 至 6 天和第 2 至 5 天分泌 destruxin A。对于 EAMa 01/58-Su 和 BIPESCO5，宿主中 destruxin A 的最大浓度分别为第 4 天的 0.369 和 0.06μg/幼虫，整个致病过程中，总产量分别为 0.6 和 0.09μg/幼虫。这些结果表明，这些菌株对人类和环境的危害很小，如果有的话。本研究中用于定量真菌生长和代谢产物产生的方法提供了有价值的数据，有助于更好地了解 destruxin A 在宿主幼虫中 M. brunneum 生长过程中的作用，并监测 destruxin A 在食物链中的命运。
