Elimination Of Fungal Endophytes From Roegneria Kamoji

Epichloae endophytes, including Epichloe spp. and their asexual relatives Neotyphodium spp. live within cool season grasses. Neotyphodium spp. have been documented to enhance resistance to insect herbivory, and tolerance to drought and to grasses. In this study, I focus on application techniques and basic biological characters of a new symbiont, Roegneria kamoji/endophyte.First, we used two different methods, rose bengal staning and tissue clearing, to detect endophyte distributing in sheath of Roegneria plant.20tillers collected from mature plants in wild and20seedlings rosed in lab were used. Fungal hyphae were mostly parallel to longitudinal leaf axis in seedlings, while winding in tillers. In the seedlings, I can hardly detected hyphae by actic acid clearing method as a result of the morphological character. While the detection rate of lactic acid clearing method,90%was higher than the75%of staining method in tillers. So the lactic acid clearing method is adapted to detect endophyte in tillers.Second, more than3000seeds were collected from a single Roegneria plant in Zijin Mountain, Nanjing in May2010. Through rose bengal staining50randomly selected dry seeds, the seed endophyte-infected rate turned out to be100%. Then, every50seeds were placed in a test rube as a group, experimental groups were treated for12,14,16,18,20,22or24hours in40℃,50℃and60℃temperature water bath respectively, control groups were put in the room temperature. All the treated seeds were put in vermiculite for germinating. After the third true leaf grow out, the sheath epidermis of first true leaf was stained to detect the endophytic fungi. The results indicated that,22h and24h of treatment on50℃and14h to24h treatment on60℃can make the endophyte mycelium within seeds loss vertical transmission ability. We isolated and identified this endophyte fungi strains Rnj0171as E. yangzii based on morphological characteristics and phylogenetic relationships.Third, we collected all mature seeds from two different endophyte infected Roegneria plants, and constructed two EI/EF populations. Agronomic properties including tiller number, plant height, width and so on were investigated in the next3springs. Endophyte infection of each plant was detected after the seeds matured in the early summer. Results shows that, Roegneria plants infected with stain Rnj0146grow better and develop more tillers (about38%) comparing with endophyte free in the first spring, however, plant height, width of the pseudostems and leaves were not significantly affected by the endophyte infection. In the second and third year, all difference between El and EF plants become undetectable. The1000grain weight of EF seeds was5.15±0.68g in2010and5±0.41g in2011, both of them were significant higher than those of EI seeds. However, Roegneria plants infected with stain Rnj0171have fewer tillers and reproductive tillers. Morphological and phylogenetic evidences indicated the fungal isolates Rnj0146and Rnj0171should be N. sinicum and E. yangzii respectively.At last,8strains and Roegneria tillers were used to explain why endophyte dead in seeds after temperature treatment. Results showed that,6of8strains could not survive after50℃treatment for24h, and all of the8strains dead after60℃treatment, whlie30℃and40℃have no influence to the endophyte fungi. Roegneria tillers infected with endophyte showed the similar conclusion. So the high temperature may lead to the endophyte death in seeds.Combinedly, in this study, we constructed two Roengeria EI and EF systems using a dry-high temperature meathod, and then detected their characters. Results indicated that, N. sinicum could improve the growth of Roegneria, while E. yangzii have opposite effects.