Increased fitness of rice plants to abiotic stress via habitat adapted symbiosis: a strategy for mitigating impacts of climate change

Abstract

Climate change and catastrophic events have contributed to rice shortages in several regions due to decreased water availability and soil salinization. Although not adapted to salt or drought stress, two commercial rice varieties achieved tolerance to these stresses by colonizing them with Class 2 fungal endophytes isolated from plants growing across moisture and salinity gradients.Plant growth and development, water usage, ROS sensitivity and osmolytes were measured with and without stress under controlled conditions.The endophytes conferred salt, drought and cold tolerance to growth chamber and greenhouse grown plants. Endophytes reduced water consumption by 20-30% and increased growth rate, reproductive yield, and biomass of greenhouse grown plants. In the absence of stress, there was no apparent cost of the endophytes to plants, however, endophyte colonization decreased from 100% at planting to 65% compared to greenhouse plants grown under continual stress (maintained 100% colonization).These findings indicate that rice plants can exhibit enhanced stress tolerance via symbiosis with Class 2 endophytes, and suggest that symbiotic technology may be useful in mitigating impacts of climate change on other crops and expanding agricultural production onto marginal lands.

Figure 1. Effects of symbiosis on growth response on young seedlings in the absence of stress.

The number of plants/treatment are indicated (N = XX) below. Statistical analysis was performed using Duncan's multiple-range test. Values with the same letters are not significantly different. A) Representative photo of three day old rice seedlings (N = 30 total) that were nonsymbiotic (NS) or symbiotic (S) with SaltSym. B) Growth of rice seedlings were measured by assessing the dry biomass (mg) of (N = 10/rep) three day old rice seedling that were NS or S with the SaltSym or TempSym1. Each assay was repeated three times. Statistical analysis indicated that S plants were statistically larger (SD≤0.06; P≤0.0002) than NS plants.

Figure 2. Representative time-lapse photos of rice growth and development.

NS (left panel) and S (SaltSym; right panel) rice seedlings (N = 3) were photographed every 20 min over a ten-day period. Sterilized, imbibed rice seeds were inoculated with fungal spore solutions (S) or mock-inoculated (NS) for 48 hours on water agar plates until seed germination occurred. Three S and NS seedlings were randomly chosen and imbedded into the silica sand layer on top of the vertical plant growth apparatus, and the time-lapse photography initiated (identified as time point t = 0). Zero, three, six and ten day time points are represented by t = 0, t = d3, t = d6, and t = d10, respectively.

Figure 3. Effects of salt, cold and drought stress and water usage in S and NS rice plants under laboratory conditions.

The number of plants or seedlings/treatment are indicated by (N = XX), and the % survival and health of surviving plants is indicated in parentheses after each treatment (Fig. 3A &C). Plant health was based on comparison to NS controls and rated from 1 to 5 (1 = dead, 2 = severely wilted and chlorotic, 3 = wilted +/− chlorosis, 4 = slightly wilted, 5 = healthy w/o lesions or wilting). All assays described from left to right and images are representative of all plants/treatment. A) Rice plants (N = 60), no stress controls (labeled "C") representative of both S and NS plants (100%, 5), S with SaltSym (100%, 5), or NS (0%, 1) exposed to 300 mM NaCl for 21 days. While all plants bent over with age, unstressed controls and salt exposed S plants remained hydrated while the NS plants wilted. B) The % rice seedling development at 5–20°C of NS (black bars) and TempSym1 colonized (grey bars) treatments were assessed. After the initial sterilization and imbibing process (see Materials and Methods), seeds (N = 20) were placed on agar water media plates, and +/− inoculated with the fungal symbiont, and % seedling development assessed after ten days. Statistical analysis (Duncan's multiple-range test; SE≤4.48; P≤0.001) indicated the % seedling development was significantly higher at 5°C and 10°C in S treatments but not at 15°C and 20°C. C) Representative photo of five week old rice plants (N = 140–210) that are NS or S with TempSym1 (TS1). From Left to right: control plants (C) were kept hydrated and healthy (100%, 5) for both treatments. The remaining plants were not watered for 15,12, 9,7, 5, and 3 days. Post drought stress, plants were re-hydrated for 2 days and viability assessed. After 3 days, NS plants began to show the effects of drought (70%, 2; 25%, 1) and after>5 days, NS plants succumbed to the stress (>90%, 1). In contrast, S plants did not show the effects of drought until after 12 days (70%, 1; 30%, 2). Moreover, S plants remained green and robust in the absence of water for 9 days (≥90%, 5) as visualized by a general thicker, green canopy. D) Fluid usage of 5 week old NS or S (SaltSym, TempSym1, and TempSym2) rice plants (N = 60) as ml of fluid used over a ten day period. Statistical analysis (Duncan's multiple-range test) indicated significant differences in fluid usage (SD≤7.51; P<0.05) with all three symbionts using less fluid compared to NS plants.

Figure 4. The effect of stress on root, shoot and seed yields of mature plants under greenhouse conditions.

Two month old NS and S (SaltSym) rice plants (N = 30) were +/− exposed to a gradual increase in NaCl concentrations (0–300 mM) over an additional three-month period. Non-stressed NS and S (SaltSym) plants were hydrated with 1x Hoagland's solution supplemented with 5 mM CaCl₂ throughout the length of the study (5 months total). Stress treatments began after 2 months and plants were exposed to 100 mM NaCl for 3 weeks, then increased 200 mM NaCl for an additional 3 weeks, and then increased and maintained at 300 mM NaCl until the completion of the study (5 months total). A) Root and shoot biomass (N = 5) of NS and S (SaltSym) plants in the absence (-; grey bar graphs) and presence (+; white bar graphs) of salt stress. Statistical analysis (Duncan's multiple-test range) indicated S plants had significantly larger shoot biomass in the presence and absence of stress (SE<2.88; P<0.0001). Significant differences between S and NS treatments in root size were observed (SE<2.81; P<0.0031) in +/− salt stress treatments. No significant differences in root biomass were observed when comparing NS+ versus NS-, or S+ versus S- (Values with the same letters are not significantly different). B) Seed yields of plants (N = 25) of NS and S (SaltSym) plants in the absence (-) and presence (+) of salt stress. Statistical analysis (Duncan's multiple-test range) indicated S+ plants had higher yields than NS+ plants, and S- plants had higher yields than NS- plants (SD≤0.17;P<0.01). C) Representative photo of NS and S (SaltSym) plants grown in the absence (-) and presence (+) of a gradual increase in salt stress (0–300 mM NaCl, see above).

Figure 5. Effect of symbiosis on plant osmolyte concentrations and paraquat-induced photobleaching (ROS) under laboratory conditions.

A) Five week old rice plants (N = 30) that were NS or colonized with SaltSym or TempSym1 exposed for ten days in the absence (-) and presence (+) of salt stress (300 mM NaCl), at which point, the effects of stress began to show in NS plants treatments (≥70% wilted +/−chlorosis). SaltSym imparts salt tolerance and TempSym1 does not. Osmolyte concentrations (milliosmoles per kg wet weight) of roots and shoots were assessed and statistical analysis (Duncan's multiple-range test; SE≤9.98 &<23.73 for root, and shoot, respectively; P<0.0001 for root and shoot) indicated significantly higher levels in the shoots of S plants compared to NS plants in the absence of salt stress, and no statistical differences between treatments in the presence of salt stress. No significant differences were observed in roots in the absence of salt stress. In the presence of stress, Tempsym1+ showed significantly lower level of osmolytes than SaltSym+ and NS+ treatments. Values with the same letters are not significantly different. B) NS and S (SaltSym and TempSym1 & 2) plants exposed to salt (300 mM NaCl, 10 days) and drought stress (3 days) were tested for paraquat-induced photobleaching (ROS activity). Time points were chosen when symptoms began to appear (wilting and chlorosis) in NS stressed plants. Leaf disks (N = 9) from 9 independent plants were used for ROS assays. Leaf disks were sampled from leaf tissues of similar size, developmental age, and location for optimal side-by-side comparisons. Values indicate the number of leaf disks out of a total of nine that bleached white after exposure to paraquat indicating ROS generation. Statistical analysis (Duncan's multiple-range test) indicated that in the absence of stress, little to no (0–11%) photo beaching occurred in all the treatments. In contrast, significant differences occurred with 100% of the NS plant disks for both salt and drought stress bleaching white compared to only 11–22% of the S plant disks (P<0.0001). ND =  not determined.