In vitro testing of fungal endophytes

In vitro tests were performed with representatives of each clade obtained by the analyses of internal transcribed spacer (ITS) sequences of the fungal isolates (Figures 1, 2) using leek (Allium porrum), a generally used host plant in DSE resynthesis experiments (see Mandyam and Jumpponen, 2008 (link); Knapp et al., 2012 (link)) to test the basic symbiotic nature of the fungi. Five replicates for each fungal isolate and five control plants were incubated in each series according to Knapp et al. (2012) (link). The fungus and isolates of its clade were considered a root endophyte if it colonized the roots without symptoms.
Root samples from the field and in vitro experiments were studied microscopically. The cleared roots were stained using the fluorescence labeled lectin, WGA-AlexaFluor488 (Wheat Germ Agglutinin, Alexa Fluor 488 conjugate, Molecular Probes W11261, Thermo Fisher Scientific, Lithuania), a cell-wall-specific dye used for in planta visualization of fungal endophytes (e.g., Andrade-Linares et al., 2011 (link)). Root samples were examined using a light microscope with Nomarski (differential interference contrast, DIC) optics and a Nikon Eclipse 80i microscope equipped with a Spot 7.4 Slider camera (Diagnostic Instruments, Inc.), differential interference contrast (DIC), and a filter wheel with excitation and emission filters for visualization of Alexa Fluor 488 probe.