Drought-Stress Tolerance in Wheat Seedlings Conferred by Phenazine-Producing Rhizobacteria

Abstract

The specific role of phenazines produced by rhizosphere-colonizing Pseudomonas in mediating wheat seedling drought-stress tolerance and recovery from water deficit was investigated using Pseudomonas chlororaphis 30-84 and isogenic derivatives deficient or enhanced in phenazine production compared to wild type. Following a 7-day water deficit, seedlings that received no-inoculum or were colonized by the phenazine mutant wilted to collapse, whereas seedlings colonized by phenazine producers displayed less severe symptoms. After a 7-day recovery period, survival of seedlings colonized by phenazine-producing strains exceeded 80%, but was less than 60% for no-inoculum controls. A second 7-day water deficit reduced overall survival rates to less than 10% for no-inoculum control seedlings, whereas survival was ∼50% for seedlings colonized by phenazine-producers. The relative water content of seedlings colonized by phenazine-producers was 10-20% greater than for the no-inoculum controls at every stage of water deficit and recovery, resulting in higher recovery indices than observed for the no-inoculum controls. For 10-day water deficits causing the collapse of all seedlings, survival rates remained high for plants colonized by phenazine-producers, especially the enhanced phenazine producer (∼74%), relative to the no-inoculum control (∼25%). These observations indicate that seedlings colonized by the phenazine-producing strains suffered less from dehydration during water deficit and recovered better, potentially contributing to better resilience from a second drought/recovery cycle. Seedlings colonized by phenazine-producing strains invested more in root systems and produced 1.5 to 2 fold more root tips than seedlings colonized by the phenazine mutant or the no-inoculum controls when grown with or without water deficit. The results suggest that the presence of phenazine-producing bacteria in the rhizosphere provides wheat seedlings with a longer adjustment period resulting in greater drought-stress avoidance and resilience.

Keywords: Pseudomonas; drought-stress tolerance; phenazine; plant–microbe interactions; wheat.

FIGURE 1

Colonization by phenazine-producing strains enhanced seedling investment in roots and altered root system architecture under non-stressed conditions. Wheat seedlings were grown in 1-gallon pots containing autoclaved soil inoculated either with 30-84Enh, 30-84WT, or 30-84ZN, or no inoculum (control). Seedlings were grown 21 days. (A) Representative images of root scans for each treatment. Plant growth characteristics including: (B) whole plant dry weight, (C) root dry weight and (D) root/shoot investment (based on dry weights), and root system characteristics obtained from WinRhizo software including, (E) total root length, (F) root surface area, and (G) number of root tips per plant. Data are the mean and standard error (N = 18 plants). Values with the same letter do not differ statistically as determined by a Fishers protected Tukey test (P > 0.05).

FIGURE 2

Colonization by phenazine-producing strains reduced plant drought stress symptoms and enhanced whole plant relative water content. Wheat seedlings were grown unstressed for 21 days in cones containing autoclaved soil inoculated either with 30-84Enh, 30-84WT, or 30-84ZN, or no inoculum (control). Following this establishment period, plants were not watered for 7 days. All plants were then re-watered for 7 days at 3-day intervals. (A) Pictures of seedlings on Day 7 of the water deficit in the moderate water-deficit experiment. (B) Relative water content of above ground plant tissue was measured: before water deficit (Day 0), during water deficit (Days 3 and 7), and 7 days after re-watering (Day 14). Data are the mean and standard errors (N = 6 plants).

FIGURE 3

Colonization by phenazine-producing strains improved seedling survival and recovery from drought stress. Wheat seedlings were grown unstressed for 21 days in cones containing autoclaved soil inoculated either with 30-84Enh, 30-84WT, or 30-84ZN, or no inoculum (control). After this establishment period, plants were not watered for 7 days (A, moderate water-deficit experiment) or 10 days (B, severe water-deficit experiment). All plants were then watered and survival and recovery index were determined after 7 days of well-watered conditions. Survival was expressed as the percentage of plants surviving the Moderate or Severe water-deficit after 7 days of recovery. Recovery Index was based on the amount of above ground tissue recuperated (RI-0 = no recovery, RI-1 = slight new growth, RI-2 = recovery of partial leaf, RI-3 = recovery of one or more entire leaves). Data are the means and standard errors (Blocks = 4, Reps = 20). Values with the same letter do not differ statistically as determined by a Fishers protected Least Significantly Difference (LSD) test (P > 0.05).

FIGURE 4

Severe water-deficit experiment: effect of phenazine-producing strains on root system development. (A) Non-vernalized seedlings and (B) vernalized plants were grown in autoclaved soil inoculated either with 30-84Enh, 30-84WT, or 30-84ZN, or no inoculum (control). Non-vernalized seedlings were grown 21 days under well-watered conditions and then exposed to two water-stressed cycles (10 and 7 days); Vernalized seedlings were grown to the jointing stage under well-watered conditions and then exposed to a 15-day water deficit. Root system metrics were obtained from WinRhizo software including total root length, root surface area, and number of root tips per plant. These experiments were repeated once. Data are the mean and standard errors. Values with the same letter do not differ statistically as determined by a Fishers protected Tukey test (P > 0.05), N = 5 plants (two replicate plants per scan).