Nodule Synthetic Bacterial Community as Legume Biofertilizer under Abiotic Stress in Estuarine Soils

Abstract

Estuaries are ecologically important ecosystems particularly affected by climate change and human activities. Our interest is focused on the use of legumes to fight against the degradation of estuarine soils and loss of fertility under adverse conditions. This work was aimed to determine the potential of a nodule synthetic bacterial community (SynCom), including two Ensifer sp. and two Pseudomonas sp. strains isolated from Medicago spp. nodules, to promote M. sativa growth and nodulation in degraded estuarine soils under several abiotic stresses, including high metal contamination, salinity, drought and high temperature. These plant growth promoting (PGP) endophytes were able to maintain and even increase their PGP properties in the presence of metals. Inoculation with the SynCom in pots containing soil enhanced plant growth parameters (from 3- to 12-fold increase in dry weight), nodulation (from 1.5- to 3-fold increase in nodules number), photosynthesis and nitrogen content (up to 4-fold under metal stress) under all the controlled conditions tested. The increase in plant antioxidant enzymatic activities seems to be a common and important mechanism of plant protection induced by the SynCom under abiotic stress conditions. The SynCom increased M. sativa metals accumulation in roots, with low levels of metals translocation to shoots. Results indicated that the SynCom used in this work is an appropriate ecological and safe tool to improve Medicago growth and adaptation to degraded estuarine soils under climate change conditions.

Keywords: SynCom; abiotic stress; degraded estuarine soils; legumes nodulation; plant growth promoting endophytes.

Figure 1

Growth parameters and nodulation. (A) Dry weight of shoot (top graphs) and roots (bottom graphs), (B) length of shoot and roots, (C) number of nodules, and (D) nitrogen content in non-inoculated and inoculated M. sativa plants after 60 days in pots under greenhouse conditions using different substrates. Values are means ± S.D. (n = 16). Different letters and symbols indicate means that are significantly different from each other (One-way ANOVA, LSD test, p < 0.0001). C-: non inoculation; N10: inoculation with Ensifer sp. N10; CSN: inoculation with the SynCom formed by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensifer sp. N10, and Ensifer sp. N12; heavy metal treatment: soil from the middle marshes of the Odiel river as substrate; salt treatment: sterilized soil from the upper marshes of the Odiel river supplemented with 60 mM of NaCl as substrate; drought treatment: sterilized soil from the upper marshes of the Odiel river as substrate and plants exposed to drought stress; high temperature treatment: sterilized soil from the upper marshes of the Odiel river as substrate and plants exposed to 40 °C.

Figure 2

Photosynthetic parameters after the exposition of different stressful conditions. (A) Total chlorophyll content, (B) net photosynthetic rate (A_N), (C) maximum quantum efficiency of PSII photochemistry (Fv/Fm), (D) quantum efficiency of PSII (Φ_PSII), (E) intrinsic water use efficiency (iWUE), and (F) electron transport efficiency (ETR) in non-inoculated and inoculated M. sativa plants after 60 days in pots under greenhouse conditions in the case of heavy metal and salt treatments, and during the stress in the case of drought and high temperature treatments. Values are means ± S.D. (n = 16). Different letters and symbols indicate means that are significantly different from each other. (One-way ANOVA, LSD test, p < 0.0001). C-: non inoculation; N10: inoculation with Ensifer sp. N10; CSN: inoculation with the SynCom formed by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensifer sp. N10, and Ensifer sp. N12; heavy metal treatment: soil from the middle marshes of the Odiel river as substrate; salt treatment: sterilized soil from the upper marshes of the Odiel river supplemented with 60 mM of NaCl as substrate; drought treatment: sterilized soil from the upper marshes of the Odiel river as substrate and plants exposed to drought stress; high temperature substrates: sterilized soil from the upper marshes of the Odiel river as substrate and plants exposed to 40 °C.

Figure 3

Antioxidant enzymes activities after the exposition of different stressful conditions. (A) Guaiacol peroxidase (GPx), (B) superoxide dismutase (SOD), (C) catalase (CAT), and (D) ascorbate peroxidase (GPx) in shoots (top graphs) and roots (bottom graphs) of non-inoculated and inoculated M. sativa plants after 60 days in pots under greenhouse conditions in the case of heavy metal and salt treatments, and during the stress in the case of drought and high temperature treatments. Values are means ± S.D. (n = 16). Different letters and symbols indicate means that are significantly different from each other. (One-way ANOVA, LSD test, p < 0.0001). C-: non inoculation; N10: inoculation with Ensifer sp. N10; CSN: inoculation with the SynCom formed by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensifer sp. N10, and Ensifer sp. N12; heavy metal treatment: soil from the middle marshes of the Odiel river as substrate; salt treatment: sterilized soil from the upper marshes of the Odiel river supplemented with 60 mM of NaCl as substrate; drought treatment: sterilized soil from the upper marshes of the Odiel river as substrate and plants exposed to drought stress; high temperature substrate: sterilized soil from the upper marshes of the Odiel river as substrate and plants exposed to 40 °C.

Figure 4

Metal/loids concentration after the exposition of heavy metal stress. (A) arsenic (As), (B) cadmium (Cd), (C) copper (Cu), and (D) zinc (Zn) concentrations in shoots and roots of M. sativa plants inoculated with different inoculums after 60 days in pots under greenhouse conditions using soil from the middle marshes of the Odiel river as substrate. Values are means ± S.D. (n = 16). Different letters indicate means that are significantly different from each other (One-way ANOVA, LSD test, p < 0.0001). C-: non inoculation; N10: inoculation with Ensifer sp. N10; CSN: inoculation with the SynCom formed by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensifer sp. N10, and Ensifer sp. N12.