Symbiotic Modulation as a Driver of Niche Expansion of Coastal Plants in the San Juan Archipelago of Washington State

Abstract

Modern evolutionary theory and population genetics posit that adaptation and habitat expansion of plants result from processes exclusive to their genomes. Here, we present studies showing that plants can grow across complex habitat gradients by modulating symbiotic associations with Class 2 fungal endophytes. Endophyte analysis of three native (Leymus mollis, Distichlis spicata, and Salicornia pacifica) and one invasive (Spartina anglica) plant growing across adjacent microhabitats in the San Juan Archipelago altered associations with Class 2 fungal endophytes in response to soil salinity levels. At the microhabitat interfaces where the gradation of salinity varied, the plants were colonized by endophytes from both microhabitats. A reciprocal transplant study along a salt gradient demonstrated that Leymus mollis (dunegrass) required endophytes indigenous to each microhabitat for optimal fitness and/or survival. In contrast, when dunegrass and Grindelia integrifolia (gumweed) were found growing in low salinity, but high drought habitats, these plant species had their own unique dominant endophyte association regardless of geographic proximity and conferred drought but not high salt stress tolerance. Modulation of endophyte abundance occurred in planta based on the ability of the symbiont to confer tolerance to the stress imposed on plants. The ability of an endophyte to confer appropriate stress tolerance resulted in a significant increase of in planta fungal abundance. Conversely, the inability of an endophyte to confer stress tolerance resulted in a decrease of in planta fungal abundance. Our studies indicate that Class 2 fungal endophytes can provide a symbiotic mechanism for niche expansion and phenotypic plasticity across environmental gradients.

Keywords: Class 2 endophytes; fungal endophytes; plant-fungal interactions; stress tolerance; symbiosis.

Figure 1

Endophyte distribution across a salt gradient. Endophytes were isolated from plants (N = 30/location) collected from beach, slope, and upper meadow habitats within a contiguous population of Leymus mollis (dunegrass). Fungal growth media plates (0.1x PDA) on the left represent the dominant endophyte (<70%) identified from plants in each location. Soil conductivity measurements were taken and the highest salinity levels found to occur on the beach, moderate levels on the slope, and low salinity levels in the upper meadow.

Figure 2

Laboratory studies were conducted in double-decker magenta boxes with four-week-old dunegrass plants (5 plants /magenta box, x10 reps of each treatment). Treatments were uninoculated controls, or symbiotically colonized F. culmorum, F. redolens, or Alternaria sp. plants. The average plant health score (*) and wet biomass (**) of five magenta boxes/treatment exposed to 0 mM or 300 mM NaCl after 2 weeks ± standard deviation values were recorded (table on top). The plant health score was assessed on a scale of 1–5 (1=dead, 2=severely wilted and chlorotic, 3=wilted +/- chlorosis, 4=slightly wilted, and 5=healthy w/o lesions or wilting). The highest salt tolerance was found with F. culmorum, and moderate and no salt tolerance were observed with F. redolens and Alternaria sp. plants, respectively, when compared to uninoculated controls. No differences in plant health or biomass were observed in treatments in the absence of salt stress. In the presence of 300 mM NaCl stress, differences in plant health and biomass were observed in F. culmorum and F. redolens treatments, and no plant health or biomass differences were observed in Alternaria sp. treatments when compared to uninoculated controls. A representative photograph showing health differences in dunegrass treatments (lower panel) exposed to salt stress (300 mM NaCl). Statistical analysis was performed using Student's t-test. P < 0.05 values were determined to be significant.

Figure 3

Drought stress studies in double-decker magenta boxes containing uninoculated or symbiotic (C. cladosporioides or A. alternata) treated ryegrass seeds (545 seeds/box) grown for 12 days when fluid was removed from the lower chamber of (N = 4 magenta boxes) to begin drought stress. Four additional magenta boxes remained as hydrated controls. After 9 days in the absence of fluids, 200 mls of fluid was added back to all the boxes and plants were allowed to recover for 7 days and the wet biomass (g) of ryegrass shoots were assessed. No significant differences in biomass were observed in the absence of stress (SD = 0.39–0.79; P = 0.209–0.394) and statistically significant differences were observed in both symbiotic plants (indicated with *; SD = 0.12–0.28; P = 0.014–0.016) when compared to uninoculated plants in the presence of drought stress. A representative photograph of ryegrass plants prior to shoot harvests after 9 days of drought stress (bottom panel). Statistical analysis was conducted with Student's t-test to determine significance between two groups (i.e., symbiotically treated vs. uninoculated controls). Values of P < 0.05 were considered to be statistically significant.

Figure 4

Modulation of endophytes in planta dunegrass (Leymus mollis). Laboratory studies were conducted in double-decker magenta boxes with four-week-old dunegrass plants (x5 plants/magenta box, x3 reps of each treatment) colonized with F. culmorum beach isolate that does impart salt tolerance or F. culmorum ATCC isolate that does not impart salt tolerance, exposed either to no (0 mM NaCl; green bars) or high salt stress (300 mM NaCl, blue bars) for 7 days. When visible stress symptoms were observed in the F. culmorum ATCC isolate, the lower stem and roots of five plants were pooled and processed for cfu analysis by grinding plant tissues in an osmotic solution and plating aliquots onto fungal growth media (0.1x PDA). The in planta cfu abundance of the F. culmorum beach isolate remained the same in the presence and absence of salt stress. In contrast, in the presence of salt stress, the abundance of F. culmorum ATCC isolate decreased significantly. In each graph, ± standard deviation bars are indicated and the different lowercase letters above the bars indicate significant differences (Duncan's Grouping; P < 0.001).