Distribution and rDNA-ITS sequence analysis of Epichloё endophyte symbiosis with Achnatherum sibiricum in mid-and eastern Inner Mongolia Steppe

The complex functions of fungal endophytes have received intensive study during the past three decades because of their roles in production loss of livestock and enhancement of biotic and abiotic stress resistances in hosts. Most of these studies focused on cultivated grasses, but little is known about the distribution and mechanisms of endophytes in native grass populations. Surveys of grass species associated with Neotyphodium and Epichloё fungal endophytes made in native grasslands of northern China suggested that endophytes are widely distributed in natural grass populations. Among them, Achnatherum sibiricum was found to associate with fungal endophytes in all sites investigated. Ach. sibiricum is a perennial, sparse bunch grass that inhabits temperate steppe in China. This grass can be found in different habitats such as meadows, typical steppes and forests. Its distribution may be largely determined by precipitation, while it is usually a companion species in communities and it sometimes can become the dominant species. Untill now, no toxicosis problems in grazing animals have been reported in pastures containing Ach. sibiricum.Six natural populations (IMGERS-CAS, Leymus chinensis plot of the IMGERS-CAS, Xi Ujimqin Qi, Hulingol, Arshan, Hailar) of Ach. sibiricum in mid- and eastern Inner Mongolia Steppe of varying distances from each other were chosen. Locations and descriptions of sampling sites were recorded. Infection frequency of fungal endophytes was detected and seeds from 30 Ach. sibiricum individuals were collected. Fungal endophytes of different morphologies were isolated from seeds and at each site, strains of different morphologies were used as internal transcribed spacers of the nuclear ribosomal DNA (rDNA-ITS) sequence analysis. Regions of rDNA-ITS were amplified, cloned, sequenced and submitted to GenBank. Furthermore, phylogenetic analysis was conducted with additional sequences conserved in GenBank. Fungal endophytes were found at high frequencies in all investigated local populations regardless of their location, elevation, annual precipitation, mean temperature and vegetation types, implying a systemic association between native grasses and fungal endophytes. In plants which have seed-transmitted endophytes, high infection frequency suggests a selective advantage compared with uninfected plants. This may be due to increased resistance to pathogens or herbivores. Another possible factor is that endophyte infection enhanced plant′s resistance to an abiotic stress, such as drought or fire. Nineteen strains from six populations were used in rDNA-ITS sequence analysis. They were IB from IMGERS-CAS; LB, LC, LL from Leymus chinensis plot of the IMGERS-CAS; XA, XB, XD, XE, XF, XG from Xi Ujimqin Qi; HA, HE from Hulingol; AA, AB, AC, AD from Arshan and EA, EB, EC from Hailar. Besides LC, LL and XE, which were pathogens belonged to Ascomycota but not Epichlo, there were only five informative sites in the alignment of 16 rDNA-ITS sequences and there were no correlation between differences of sequences and geographical distances. Among them IB, LB, AC and EB have the same sequences, which is consistent with the results of morphological observation. Results of Maximum parsimony tree and N-J tree based on ITS5.8S sequences of 16 fungal strains showed that all Epichlo symboints with Ach. sibiricum were clustered into one taxonomic group with 97% bootstrap support compared to other reference endophyte isolates used in this analysis. Therefore we propose that all Epichlo symboints share the same evolutionary origin. Their diverse morphologies and high genetic variations among different populations are attributed to broad distribution of Ach. sibiricum and differences in climate and plant community.