Isolation and characterisation of novel fungal root endophytes from Elymus repens (Poaceae) for resistance to Fusarium, Gaeummanomyces and Pyrenophora

Abstract

Barley diseases are predominantly controlled using genetic resistance and fungicides. However, pathogen populations are becoming increasingly tolerant to the active compounds in fungicides and the European Union is endorsing integrated pest management, including biocontrol, in order to promote sustainable agriculture. In this thesis the root endophyte community of Elymus repens was studied using direct amplicon sequencing as well as a culture dependent method combined with standard DNA barcoding. The aim was to identify how different the total fungal community, determined by amplicon sequencing, was between five sites, and also among individual plants, in order to investigate the importance of these factors in endophyte discovery. Direct amplicon sequencing revealed that the five sites had distinct fungal endophyte communities and on average a root system contained 151 OTUs. In addition, the individual plants showed a large variation in species richness ranging from 96 to 239 OTUs out of in total 715 OTUs. The community of fungi that could be cultured on three of the most frequently used isolation media, PDA, MEA and 2 % MEA, were compared and the community was characterised using three DNA barcoding regions including ITS, LSU and TEF1α. The ITS barcoding region identified a higher number of OTUs than the other two regions and the OTU richness was influenced by the culture medium. PDA retrieved the largest number of OTUs but did not include approximately 50 % of the total unique OTUs. Combinations of media are therefore highly recommended for endophyte isolation purposes. Amplicon sequencing identified 349 OTUs from site III and the culturing method identified 66 OTUs. Between these two estimates of community richness there was an overlap of ten shared OTUs and there was no correlation between the widely distributed OTUs identified by direct amplicon sequencing and the widely isolated OTUs. A total of 24 fungal isolates from the Elymus collection were screened against Fusarium culmorum and a subset was furthermore tested against Gaeumannomyces graminis and Pyrenophora teres. Treatment with Periconia macrospinosa strain E1 significantly (P ≤ 0.05) reduced disease symptoms in two out of four experiments. Seed treatment with Lasiosphaeriaceae sp. isolate E10 reduced net blotch symptoms significantly but only in one out of three experiments. The ecological role of endophytes is believed to fall in the spectrum between latent pathogen, pathogen facilitator, neutral and antagonistic. However, here endophytic strains from E. repens seemed to remain neutral and in a few cases antagonistic when changing the setting to a barley host attacked by fungal pathogens in a controlled environment.