Biofertilizer-biostimulant potential of endophyte and rhizospheric microorganisms for alfalfa tolerance to low phosphorus availability: Agrophysiological and biochemical aspects

Abstract

This PhD thesis is based on a combination of research approaches in laboratory and greenhouse mesocosms to field experiments. It aims at evaluating the biofertilizer-biostimulant potential of endophyte and rhizospheric bacteria on alfalfa (Medicago staiva L.) – Sinorhizobium (Ensifer) meliloti symbiosis under low phosphorus (P) availability. The emphasis was put on the agro-physiological and biochemical traits associated with low P availability tolerance. Hence, as a first step, we focused on the screening of four Moroccan alfalfa cultivars for their tolerance to P deficiency with a focus on acid phosphatase, oxidative stress, enzymatic and non-enzymatic antioxidant systems. The experiment was conducted under controlled conditions with two P forms, insoluble P using Ca3HPO4 versus a soluble P form (KH2PO4) at a final concentration of 250 μmol P. plant−1. week−1. The P stress was applied for two months. The obtained results indicated that the P-starvation significantly (p < 0.001) reduced the agronomic traits evaluated, such as plant dry biomass and leaf area. The root and shoot P contents were found (p < 0.001) decreased by low-P availability in the rooting medium. Significant (p < 0.001) increases in lipid peroxidation and oxidative damage to cells, evaluated through malondialdehyde (MDA) and hydrogen peroxide contents, were noted because of P stress. The results showed also, that low P availability significantly (p < 0.001) increased the enzymatic antioxidant responses reflected by the activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), and catalase (CAT). The non-enzymatic antioxidant molecules such as proline and total polyphenols significantly increased in alfalfa stressed plants. The behavior of the alfalfa cultivars tested was significantly different (p > 0.05). The cultivar Oued Lmaleh (OL) was found to be the least affected and the Demnate (DEM) was the most sensitive one, whereas the cultivars TATA and RICH showed a moderate tolerance. The second part of our work aims at the isolation and physiological characterization of rhizobacteria isolates from the Beni-Mella region. The isolated collection was tested for its biofertilizer-biostimulant potential through a set of traits such as tolerance to osmotic stress, qualitative and quantitative P solubilization, indole acetic acid (IAA) production, and exopolysaccharide production. The candidate isolates were preliminary identified using MALDI-TOF mass spectrometry, and then using housekeeping genes (gyrA, gyrB & rpoD). The selected plant growth-promoting rhizobacteria (PGPR) strains were used in the third part of our work as biofertilizer-biostimulant rhizobacteria to evaluate their synergistic effect on alfalfa growth under low-P availability. Hence, the synergistic effect of Pseudomonas alkylphenolica PF9, as phosphate solubilizing bacteria (PSB), and Sinorhizobium meliloti Rm41, as symbiotic nitrogen-fixing bacteria, on the Moroccan alfalfa cultivar OL under symbiotic nitrogen fixation and limited phosphorus (P) availability was focused. The results of this part indicated that the inoculation of alfalfa plants with Sinorhizobium strain alone or combined with Pseudomonas strain significantly (p < 0.001) improved the plant growth, the physiological and the biochemical traits focused in comparison to the uninoculated and P-stressed plants. For most sets of parameters, the improvement was more obvious in plants co-inoculated with both strains than in those inoculated with Sinorhizobium meliloti Rm41 alone. In fact, under limited P-availability, the co-inoculation with two strains significantly (p < 0.01) enhanced the growth of alfalfa plants evaluated by fresh and dry biomasses, plant height, and leaf area. The results indicated also that the enhancement noted in plant growth was positively correlated with the shoot and root P contents. Furthermore, the incensement in plant P contents in response to bacterial inoculation improved cell membrane stability, reflected by MDA and electrolyte leakage (EL) contents, and photosynthetic-related parameters such as chlorophyll contents, the maximum quantum yield of PS II (Fv /Fm) and stomatal conductance (gs). The last part of our PhD work was a field experiment during 2021 year in order to confirm the results obtained under controlled conditions. For this reason, a field trial was installed and evaluated during four consecutive cuttings, corresponding to growth and flowering period of alfalfa. The field was evaluated through some parameters related to forage biomass and forage quality. The results indicated that the rhizobacterial strains have a great potential on alfalfa production and its adaptation to low-P availability. Our findings suggest that rhizobacterial strains tested have a biofertilize-biostimulant potential for promoting alfalfa growth under low-P availability in field conditions. They are in concordance with those obtained under controlled conditions. Hence, the seed biopriming with the PSB tested constitutes a promising alternative to minimize the P problem in agricultural soils.