Abstract:

Background and Aims The fungal endophytes in grasses grow intercellularly and systemically in aboveground plant tissues. Vertically transmitted asexual endophytes (Neotyphodium) forming asymptomatic infections of cool-season grasses have been repeatedly derived from sexual species (Epichloı) that abort host inflorescences. The phylogenetic distribution of seed-transmitted endophytes is strongly suggestive of cocladogenesis with their hosts. Endophytes are considered plant mutualists: they receive nutrition and protection from the host plant while the host plant may benefit from enhanced competitive abilities and increased resistance to herbivores, pathogens, and various abiotic stresses. Most studies of grass-endophyte associations have been undertaken with two economically important plant species, Lolium perenne and Festuca arundinacea. The interactions between endophytes and other grasses in natural plant communities, however, are seldom concerned. Much less attention has been paid to native grasses in Inner Mongolia Steppe, which lies in the north of China and is an important part of Steppe in Eurasia, and only limited published symbiosis studies are available in this natural ecosystem. Thereby, a more comprehensive view of the ecology, diversity of endophytes and their interactions with host plants becomes essential.
Methods Our research of grass (Achnatherum sibiricum) associated with Neotyphodium endophyte (a new grass-endophyte association) was carried out in the Inner Mongolia Steppe, China. Twenty-seven endophyte strains were isolated from four A. sibiricum populations including Hulingol (H), Xi Ujimqin Qi (X), Inner Mongolia Grassland Ecosystem Research Station of the Chinese Academy of Sciences (IMGERS-CAS) (I), and the Leymus chinensis plot of the IMGERS-CAS (L), respectively. Colony morphology, growth rate on potato dextrose agar (PDA) and conidial measurements of isolates were used to characterize diversity of endophytes hosted by A. sibiricum. The genetic polymorphisms of Neotyphodium species from the four A. sibiricum populations were also analyzed using random amplified polymorphic DNA (RAPD) markers.
Key Results Four morphological groups of Neotyphodium species and five individual morphological types were described based on the colony morphology, growth rate, and conidial shape and size. A total of 463 bands were obtained using 20 RAPD primers, of which 461 bands were polymorphic. A total of 93 unique loci were obtained, accounting for 20.1% of the total amplified loci. Nei's diversity index (h) and Shannon's diversity index (I) were 0.238 and 0.387, respectively. In the four populations (H , X , I, and L), the value of h was 0.122 0, 0.180 4, 0.146 3 and 0.210 7, respectively. The four populations may be ranked in a descending order as L > X > I > H, and the I of the four populations had a similar trend. The fixation index (F_ST), the coefficient of gene differentiation (G_ST), and the genetic differentiation (Hsp-Hpop)/Hsp (estimated by Shannon's diversity index) were 0.205 1, 0.300 4, and 0.355 0, respectively. The genetic variation of Neotyphodium species mainly existed within populations. Genetic similarities within each population were found to be in the range of 0.647 9-0.943 8 in similarity, and the average genetic distance among the four populations was 0.285 8, varying from 0.057 8 to 0.433 9. Clustering analysis based on Nei's unbiased genetic distance and UPGMA method showed that there were seven distinct RAPD genetic groups.
Conclusions The high incidence of endophytic infection in A. sibiricum was indicative that the presence of the endophyte gave the grass a competitive advantage over non-infected grasses. The study also indicated that there were higher genetic diversity based on morphological and RAPD results, and that there were relatively high genetic variations among the four populations and higher genetic differentiation of Neotyphodium sp. within a geographical region. Most of the genetic groups were related to geographical origins of the strains. The dendrogram of genetic distance matched rather well with the morphological data, as reflected by the four morphotypes.

Key words: Endophyte, Achnatherum sibiricum, Neotyphodium, Morphotypes, RAPD, Genetic diversity