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

本研究的目的是寻找适合日本北方寒冷潮湿气候的无毒内生菌菌株，并进行实际应用研究。为此，我们系统地收集了感染Neotyphodium内生菌的多年生黑麦草和高羊茅植物。其中，从青森县浦市采集的多年生黑麦草植物中麦角缬氨酸和lolitrem B含量低，这对家畜是有毒的。从形态上看，多年生黑麦草生态类型内生真菌与野生型内生真菌相似，经核糖体RNA基因分析，鉴定为Neotyphodium lolii。将内生菌分离的紫茎泽兰生态型接种到其他无内生菌的基因型中。Lolitrem B的含量从低到极高的接种植物。这被认为是内生菌菌株和植物基因型之间的相互作用。在低磷营养液培养条件下，研究了内生菌侵染（E+）植株的生长特性。在多年生黑麦草和高羊茅中，E+植物表现出较大的次生根系统。此外，阐明了内生菌对矿物质吸收具有有益影响。E+高羊茅植株叶片矿质元素含量以磷和硫含量高，E+多年生黑麦草植株以磷含量高。如果实际使用E+饲料植物，则需要检查内生菌产生的生物碱对动物的毒性。小鼠已被用于阐明家畜内生菌生物碱毒性的潜在机制。当喂食被内生菌密集感染的种子粉末时，没有小鼠表现出任何像蹒跚的迹象，但表现出体温升高和旋转棒性能下降。

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.