Dual Inoculation with Rhizophagus irregularis and Bacillus megaterium Improves Maize Tolerance to Combined Drought and High Temperature Stress by Enhancing Root Hydraulics, Photosynthesis and Hormonal Responses

Abstract

Climate change is leading to combined drought and high temperature stress in many areas, drastically reducing crop production, especially for high-water-consuming crops such as maize. This study aimed to determine how the co-inoculation of an arbuscular mycorrhizal (AM) fungus (Rhizophagus irregularis) and the PGPR Bacillus megaterium (Bm) alters the radial water movement and physiology in maize plants in order to cope with combined drought and high temperature stress. Thus, maize plants were kept uninoculated or inoculated with R. irregularis (AM), with B. megaterium (Bm) or with both microorganisms (AM + Bm) and subjected or not to combined drought and high temperature stress (D + T). We measured plant physiological responses, root hydraulic parameters, aquaporin gene expression and protein abundances and sap hormonal content. The results showed that dual AM + Bm inoculation was more effective against combined D + T stress than single inoculation. This was related to a synergistic enhancement of efficiency of the phytosystem II, stomatal conductance and photosynthetic activity. Moreover, dually inoculated plants maintained higher root hydraulic conductivity, which was related to regulation of the aquaporins ZmPIP1;3, ZmTIP1.1, ZmPIP2;2 and GintAQPF1 and levels of plant sap hormones. This study demonstrates the usefulness of combining beneficial soil microorganisms to improve crop productivity under the current climate-change scenario.

Keywords: PGPR; aquaporin; arbuscular mycorrhiza; combined drought and heat stress; maize; root hydraulic conductivity.

Figure 1

(A) Shoot dry weight and (B) root dry weight in maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 15) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on Duncan’s test.

Figure 2

(A) Shoot water content and (B) relative electrolyte leakage in maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 15) (shoot water content) or (n = 6) (relative electrolyte leakage) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on Duncan’s test.

Figure 3

(A) Stomatal conductance (gs) and (B) photosystem II efficiency in the light-adapted state (ΔFv/Fm′) in maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 8) (gs) or (n = 10) (ΔFv/Fm’) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on Tukey–Kramer test.

Figure 4

(A) Net photosynthetic activity (An) and (B) instantaneous water-use efficiency (WUEi) in maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 8) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on the Tukey–Kramer test.

Figure 5

(A) Osmotic root hydraulic conductivity (Lo) and (B) hydrostatic root hydraulic conductivity (Lpr) in maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 8) (Lo) or (n = 7) (Lpr) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on Duncan’s test.

Figure 6

Expression of ZmPIP1;3 (A) and ZmPIP2;2 (B) in roots of maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 3) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on Duncan’s test.

Figure 7

Expression of ZmTIP1;1 (A) and GintAQPF1 (B) in roots of maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 3) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on Duncan’s test.

Figure 8

(A) ABA, (B) JA and (C) JA-Ile contents in sap of maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 8) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on Duncan’s test or Tukey–Kramer test in the case of JA-Ile.

Figure 9

(A) SA and (B) IAA contents in sap of maize plants inoculated or not (control) with a PGPR strain of Bacillus megaterium (Bm), with the arbuscular mycorrhizal fungus Rhizophagus irregularis (AM) or with both microorganisms (AM + Bm). Plants were cultivated under standard ambient temperature and well-watering conditions (well-watered) or subjected to a combined drought and high temperature stress (drought + T) for 15 days before harvest. Data represent the means (n = 8) ± S.E. Different letters indicate significant differences between treatments (p < 0.05) based on Duncan’s test.