Elucidating the mystery of the tripartite symbiosis plant – mycorrhizal fungi – dark septate endophytes

This study provides information on the tripartite symbiotic relationships formed by arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) in crops growing in the semiarid region of the Canadian Prairie. We found the symbiotic root systems of wheat, pea, chickpea and lentil to be morphologically distinct. The relationship between DSE and AMF abundance in roots ranged from negative in lentil to positive in wheat. Introduction Class II fungal endophytes, commonly called dark septate endophytes (DSE), and arbuscular mycorrhizal fungi (AMF) have a worldwide distribution. AMF are well known to increase plant growth, productivity and stress tolerance (Lozano et al., 2008; Tripathi et al., 2008) and improve P and N use efficiency (Fernández et al., 2011; Harman & Mastouri, 2010). However relatively few studies on DSE in the context of crop production have been conducted despite of the potential of these fungi to improve the productivity of different plants by reducing the impact of biotic and abiotic stresses (Yuan et al., 2010). Possible interactions between DSE and arbuscular mycorrhizal fungi (AMF) colonizing the roots of crop plants still awaits research attention. The goal of this study was to document and compare the symbioses formed between AMF and DSE in the roots of important crops grown in the semiarid region of the Canadian Prairie: wheat and three pulse, pea, chickpea and lentil. Materials and methods The field experiment was conducted in 2010 at the Agriculture and Agri-Food Canada Semiarid Prairie Agricultural Research Centre (SPARC), Swift Current, SK, Canada (latitude: 50°17’N; longitude: 107°41’W, elevation 825 m). The symbioses involving AMF and DSE were tested in four different crops using a randomized complete block design with 4 replicates per treatment. Plots were 4 x 8 m. Crop plants were: hard red spring wheat (cv. Lillian), kabuli chickpea (cv. CDC Frontier), yellow pea (cv. CDC Meadow), and red lentil (cv. CDC Maxim CL). Root colonization was evaluated in root samples at mid-bloom stage after staining according to the protocol of Vierheilig et al. (1998). The percentage of root colonization by AMF and DSE structures was measured using the gridline intersect method (Giovannetti and Mosse, 1980) under the microscope, at 100 x magnifications. Root density and root length colonized by the fungi were measured by staining the whole root system in 0.8% Toluidine Blue solution for 5 min just before scanning, as described in Costa et al. (2001). Images were saved and analyzed using the program WinRhizo PRO V 2003 for Windows (Régent Instrument Inc., Québec). Root length colonized (RLC) was calculated as: colonization values X root length measurements. Plant harvest index (HI) was calculated as the ratio of seed weight to the total weight of the harvested material. Results were analyzed using one-way ANOVA and the significance between treatment means was assessed by protected LSD Student’s t tests, in JMP version 8 (SAS Institute Inc., Cary, NC, USA). Pairwise correlations were also conducted using JMP version 8. Transformations were applied to the percentage data before analyses to comply with the requirement of the tests (Scherrer, 1984). Results In roots, AMF and DSE have different morphologies and form different structures that make possible their identification by microscopic examination of stained roots (Fig. 1). AMF colonized the roots more abundantly than DSE (Fig. 2). Melanized hyphae and microsclerotia were formed by DSE in the roots of the different crops roots (Fig. 1 D, E, F) often very close to AMF colonized spots (Fig. 1F). DSE hyphae were septate and usually thicker than those of AMF (Fig.1 B, E). Typically, AMF formed intraradical spores, vesicles and arbuscules, and their hyphae did not have septa (Fig. A, B, C). These structures were more or less abundant depending on the plant-fungal association. Figure 1. AMF and DSE structures observed in the roots of wheat. A: AMF hyphae with spore; B: AMF hyphae; C: AMF hyphae and intracellular arbuscules; D: DSE melanized microsclerotia; E: DSE melanized hyphae; F: DSE melanized hyphae overlapping AMF spores in the background. A B C