Enhanced Salinity Tolerance of Medicago sativa, Roots AM Colonization and Soil Enzyme Activities by PGPR ^†

Abstract

Abiotic stresses such as salt are typical negative factors that have a considerable impact on agricultural output around the world. The goal of this study was to investigate the effect of halotolerant plant-growth-promoting rhizobacteria (PGPR) on plant growth and soil function under salinity stress. The consortium of four PGPR (Pseudomonas putida, Alcaligenes sp., Klebsiella sp., and Pseudomonas cedrina) was tested for its effect on growth, chlorophyll content, oxidative stress, and root arbuscular mycorrhizal (AM) colonization of Medicago sativa in pots experiment under salt stress. The bacteria’s impact on soil enzyme activity was also investigated. Overall, in comparison to the non-inoculated control, inoculating M. sativa plants with the bacterial consortium allowed us to overcome the unfavorable effects of NaCl stress and enhanced plant growth, root AM colonization, and leaf chlorophyll content. It also reduced the levels of oxidative damage indicators such as malondialdehyde, hydrogen peroxide, and proline. Furthermore, the consortium had a beneficial effect on the activities of soil phosphatase, β-galactosidase, and arylamidase. The bacterial consortium has the potential to be employed as bio-inoculants for plants growing under salt stress.

1. Introduction

Salinity is a problem altering large land area and damages soils [1]. Salt stress reduces plant nutrient uptake, affect many morphological, physiological, and biochemical parameters in different parts of plants [2].

Plant growth promoting rhizobacteria (PGPR) could be an important technology to enhance plants tolerance against abiotic stresses including salinity [3]. Many studies show that various genera of PGPR can promote plants growth and enhance crops productivity at many plants species [4]. The cooperation between PGPRs and arbuscular mycorrhizal fungi (AMF) in soil and plant tissues can be beneficial for plants through optimizing nutrition and protection against biotic and abiotic stresses [5]. Positive interactions between PGPR and AMF in stimulating plant growth and salinity tolerance are well improved [6]. Otherwise, PGPRs can increase many enzymes activity of soil under saline conditions which promote plants growth [7,8].

Thus, in this study we aimed to analyze the effect of the interaction of a consortium of four potential PGPR with M. sativa to alleviate salt stress on plants growth and oxidative stress, as well as on plant roots mycorrhizal colonization and soil enzyme activities.

2. Material and Methods

The bacteria, Pseudomonas putida, Alcaligenes sp., Klebsiella sp., and Pseudomonas cedrina, used in this study were previously isolated from southern Morocco and characterized for their PGP traits and their resistance to salinity [9]. Pot experiments were designed in order to study the effect of the bacterial consortium on M. sativa (Alfalfa) under salt exposure. Experiments were conducted in plastic pots containing soil amended with 2% NaCl. AMF inoculum was represented by the indigenous population of soil AMF.

The plantlets were inoculated with 2 mL of a suspension of the consortium of bacterial culture (10⁸ CFU mL⁻¹). For uninoculated plants, 2 mL of saline solution were added. Pots were placed in a greenhouse (about 16 h photoperiod, 26–30 °C day and 18–22 °C night) and watered daily. Plants were harvested after 30 days, subdivided in roots and shoots and washed with deionized water.

For plants morphological parameters, root and shoot dry weights were measured, after oven dying at 65 °C for 72 h. Chlorophyll content was quantified according to [10]. The proline content of the leaves was determined according to [11]. Hydrogen peroxide (H₂O₂) content of the leaves was determined according to [12]. The lipid peroxidation (malondialdehyde (MDA) content) in plant leaves tissues was measured according to [13].

Detection of AM colonization of roots was performed according to [14]. Percentage colonization of roots was recorded according to [15].

Determination of soil enzymes phosphatase (PHOS), β-galactosidase (GALA), arylamidase (AMID) and arylsulfatase (SULF) activities in the rhizospheric soils of M. sativa were performed, according to [16,17,18,19], respectively.

Data were submitted to ANOVA analysis for each data to determine significant differences between the means. A multiple range test at the 95% confidence level was performed using Tukey’s method.

3. Results and Discussion

3.1. Effet of PGPR on Plant Tolerance and AM Colonization under NaCL Stress

Bacterial inoculation of M. sativa plants induced a positive impact on plant growth (

Table 1. Effect of bacterial inoculation on plant growth and mycorrhizal colonization (mycorrhizal frequency (F%), mycorrhizal intensity (M%) and richness of arbuscules (A%)) of M. sativa roots under NaCl stress. Values with different letters are significantly different (p = 0.05). Means that do not share a letter are significantly different.

Treatment	Plant Growth		Mycorrhizal Colonization
	Shoot Dry Weight (mg)	Root Dry Weight (mg)	F%	M%	A%
Control	17.315 b	15.11 b	64.19 b	21.45 b	13.76 b
Consortium	26.611 a	18.050 a	74.55 a	37.06 a	21.58 a

). These results are in line with many studies improving a positive effect of PGPR inoculation in various plants cultures submitted to salt stress [4], and might be primarily linked to the PGP traits of the bacteria.

A significant (p < 0.05) increase of roots mycorrhizal colonization was recorded in plants inoculated with the bacterial consortium (F%, M%, and A%) of salt stressed plants (Table 1). This result suggests that these bacteria might act as mycorrhizal helper bacteria (MHB). The ability of AMF to improve plants growth and support tolerance to biotic and abiotic stresses is well documented in literature [20]. The positive impacts of bacteria on enhancing plants tolerance against salinity could also be due to their effect on mycorrhization.

The results show a significant (p < 0.05) increase of total chlorophyll content and a significant reduction of the indicators of oxidative damage levels (malondialdehyde and hydrogen peroxide), and proline content under salt stress, compared to non-inoculated plants (

Table 2. Effect of bacterial inoculation on chlorophyll, proline, MDA and H₂O₂ content of M. sativa leaves under NaCl stress. Values with different letters are significantly different (p = 0.05). Means that do not share a letter are significantly different.

Treatment	Chlorophyll (mg/g)	H2O2 (nmol/g)	MDA (µmol/g)	Proline (µmol/g)
Control	0.95 b	695.85 a	81.16 a	228.26 a
Consortium	1.32 a	499.60 b	61.55 b	109.21 b

). The reduction of MDA and H₂O₂ contents might be due to the activation of the antioxidant enzyme defensive pathway [21]. On the other hand, proline is linked with various functions (e.g., osmoregulation, scavenging free radicals, activation of detoxification pathways), and many plants accumulate this amino acid under stresses as a kind of adaptation against this condition [2]. The increase of proline content in inoculated plants might be due to high tolerance of inoculated M. sativa plant under salt stress.

3.2. Effet of PGPR on Soil Enzyme Activity

Enzymes activity of soil can be used as criteria to describe soil quality and fertility [22]. Our results showed that in NaCl treatment, the activities of phosphatase, galactosidase, and arylamidase were significantly (p < 0.05) increased in soils inoculated with bacteria (

Table 3. Effect of bacterial inoculation on soil enzymatic activities: galactosidase (GALA), phosphatase (PHOS), arylsulfatase (SULF) and arylamidase (AMID) of the rhizospheric soil of M. sativa plants under NaCl stress. Values with different letters are significantly different (p = 0.05). Means that do not share a letter are significantly different.

Treatment	Soil Enzyme Activities mU/g Dry Soil
	GALA	PHOS	SULF	AMID
Control	1.04 b	15.19 b	74.92 a	36.99 b
Consortium	1.800 a	20.33 a	73.43 a	47.04 a

). This positive effect might be through direct enzymes production by the microbial biomass of PGPRs or result indirectly from their interactions with rhizosphere microorganisms, AMF in particular. Indeed, the ability of AMF to improve soil enzymatic activities is recently documented in the meta-analysis of [23].

4. Conclusions

The use of PGPRs, tolerant under against salt stress, with effective PGP traits, able to cooperate positively with AM fungi, and to improve soil enzymes activities could optimize our ability to cultivate crops in saline environments.