Collection of non-toxic endophyte strains adapted to cold and wet climate in northern Japan and development of new technology for its practical use

本研究项目的目的是寻找适合日本北部寒冷和潮湿气候的无毒内生菌菌株，并研究其实际应用。为此，我们系统地采集了感染内生新伤寒菌的多年生黑麦草和高羊茅。其中，采自青森县福仓市的一株多年生黑麦草麦角氨酸和糖素B含量较低，对家畜有毒害作用。福冈多年生黑麦草生态型内生真菌形态与野生型内生真菌相似，通过核糖体RNA基因的研究，鉴定为lolii新伤寒菌。将分离得到的福仓生态型内生菌接种到其他无内生菌基因型上。接种植株的Lolitrem B含量从低到极高。这被认为是内生菌株与植物基因型之间的相互作用。研究了低磷营养液培养条件下内生菌侵染（E+）植株的生长特性。在多年生黑麦草和高羊茅中，E+植株的次生根系都较大。此外，还澄清了内生菌对矿物质吸收的有益影响。E+高羊茅叶片中磷、硫含量高，E+多年生黑麦草叶片中磷含量高。在实际应用E+饲料植物时，需要检测内生菌产生的生物碱对动物的毒性。为了阐明家畜体内内生生物碱的毒性机制，研究人员在小鼠身上进行了实验。饲喂内生菌密集感染的种子粉时，小鼠没有出现踉跄现象，但体温升高，旋转性能下降。

The aim of this research project was to find non-toxic endophyte strains adapted to cold and wet climate in northern Japan and to study for the practical use. For this purpose, we systematically collected perennial ryegrass and tall fescue plants infected with Neotyphodium endophytes. Among them a perennial ryegrass plant collected from Fukaura, Aomori prefecture was low in ergovaline and lolitrem B contents which were toxic to livestock. Endophyte fungi of Fukaura perennial ryegrass ecotype were morphologically similar to wild type endophyte and identified as Neotyphodium lolii from the investigation of ribosome RNA genes. The endophyte isolated Fukaura ecotype was inoculated to other endophyte free genotypes. Lolitrem B contents of inoculated plants ranged from low to extremely high levels. This was thought to an interaction between the endophyte strain and plant genotype. Growth characteristics of endophyte-infected (E+) plants were investigated under low P nutrient solution culture condition. In both perennial ryegrass and tall fescue, E+ plants showed larger secondary root system. Moreover, it was clarified that an endophyte has a beneficial influence for the mineral absorption. Mineral compositions in leaf blade were high in phosphorus and sulfur of E+ tall fescue plants and in phosphorus of E+ perennial ryegrass plants. In case E+ forage plants were practically used, it is required to check toxicity of alkaloid produced by endophyte to animals. Mice have been used to clarify the mechanism underlying the toxicity of endophyte alkaloids in livestock. When seeds powder densely infected with endophyte was fed, no mice showed any signs like stagger, but showed increased body temperature and decreased rotarod performance.