Neotyphodium endophytes in the native perennial grass Hordeum comosum from Patagonia (Argentina).

IANNONE, L.J.; IRIZARRI, J.G.N.; PÉREZ, L.I. & P.E. GUNDEL.

Lanzhou

Congreso; The 8th International Symposium on Fungal Endophyte of Grasses; 2012

In Argentina, Neotyphodium endophytes were isolated from 20 host species and other 16 grass species were reported to be associated with Neotyphodium-like endophytes (Iannone et al. 2012). In this country, the hybrid Epichloë festucae x Epichloë typhina species, Neotyphodium tembladerae, seems to be the most common endophyte infecting at least 19 grass species included in six genera, but other Neotyphodium species were also described. Hordeum comosum J. Presl., has been reported as a host grass (Vila Aiub et al. 2001) but their endophytes have not been isolated and characterized. In this work we study the endophytes of Hordeum comosum performing a preliminary characterization of their incidence in natural populations and their phylogenetic relationships with other endophytes from Argentina. Plants and seeds of Hordeum comosum were collected in 9 different collection sites in western-central Patagonia among 42º00S and 46º00?W, in Chubut Province (Fig.1a). Seeds of at least 20 plants were collected in each population and endophyte incidence was evaluated by the inspection of thirty seeds under microscope as described by (Iannone et al. 2010). Endophytes were isolated from superficially sterilized seeds and leaf sheaths accordingly to Iannone et al. (2009). Isolates were single spore isolated (Iannone et al., 2009) and grown on PDA in darkness at 24°C for morphological characterization. The phylogenetic relationships of the endophytes of H. comosum with other endophytes from Argentina and other Hordeum species were studied. One isolate of each population was selected for DNA sequencing. Regions of the genes encoding β-tubulin (tubB) and translation elongation factor 1-α (tefA) were amplified and sequenced. DNA isolation and PCR reactions were performed as described by Moon et al. (2004) and Iannone et al. (2009). Both genes were firstly amplified using non selective primers to establish the possibility of a hybrid origin, by the presence of two dye-terminator peaks at any single nucleotide position, result of two different alleles of the gene (Moon et al., 2004). Three isolates were chosen for phylogenetic analyses using tefA gene sequences and each allele was selectively amplified accordingly to Iannone et al. 2012. Maximum Parsimony analyses were performed using Winclada (Nixon, 1999) and Bayesian analyses were conducted with MrBayes 3.1 (Ronquist and Huelsenbeck 2003). The evolution model for MrBayes was K2+G (gamma= 0.59), established with Mega 5.05 (Tamura et al., 2011). Reference sequences of Epichloë species and of Neotyphodium isolates from other Hordeum species (Moon et al. 2004) were included in the analyses. The incidence of endophytes in seeds of H. comosum populations ranged from 0 to 100%. Whereas endophyte incidence ranged from 74% to 100% in E+ populations, three populations were endophyte free. Two isolates were obtained from each population. All of the isolates presented white cottony to felted mycelium and colony diameter reached 10 to 15 mm after 30 days on PDA. Conidia were 6 to 8 µm in length, variable in shape, mostly allantoids. Moon shaped and lemmon shaped conidia were also observed and some isolates also presented abundant fusoid conidia up to 11 µm long (Fig1 b, c, d). All the isolates presented two alleles of each tubB and tefA genes indicating a hybrid origin. The phylogeny of tefA gene (Fig. 1e) of the three isolates studied indicated that two isolates (2701 and 2703) were close related to N. tembladerae. The allele 1 of the isolate (2702) was close related with E. amarillans and with the allele 1 of the endophyte of Hordeum bogdanii whereas the other allele was derived from the same E. typhina ancestor that those from other endophytes from Argentina. These preliminary results suggest a wider host range (20 species) for N. tembladerae, and suggest the existence of a new hybrid endophyte in Argentina. A thorough analysis is required to determine the endophyte incidence pattern in relation to environmental variables, establishing whether the hybrids associate differentially to environmental variables, establishing whether the hybrids associate differentially to populations.