ACC deaminase positive Enterobacter-mediated mitigation of salinity stress, and plant growth promotion of Cajanus cajan: a lab to field study

Abstract

Salinity is a major abiotic stress that negatively impacts plant health and soil microbiota. ACC (1-aminocyclopropane carboxylic acid) deaminase producing microorganisms act as natural stress busters that protect plants from different kinds of stresses. The study focused on the isolation of potent, indigenous, multi-trait ACC deaminase producers. The shortlisted ACC deaminase producers were checked for their ability to promote growth of Cajanus cajan, and mitigate stress under laboratory conditions followed by validation of their potency in naturally saline field conditions. Physiological stress markers were assessed to evaluate the impact of salinity in plants treated with ACC deaminase producer, compared to controls. Further, the contribution of ACC deaminase in stress mitigation was demonstrated by using a chemical inhibitor for ethylene biosynthesis. This study presents a polyphasic approach, transitioning from the rhizospheric soil to the laboratory to validation in the field, and puts forth a promising eco-friendly alternative for sustainable agriculture.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-021-01031-0.

Introduction

The current agricultural practice of using chemicals like fertilizers and pesticides has significantly deteriorated soil health, and has eventually rendered a large fraction of soil unfit for agriculture. The prominent abiotic stresses, salinity, and drought, limit agricultural productivity worldwide. Salinity stress has a deleterious effect on the plant, particularly the roots (Schwarz and Grosch 2003) and plant-associated microbiome (Yan et al. 2015). It creates oxidative stress, leading to the production of reactive oxygen species (ROS), thereby limiting the absorption of several micro and macro-nutrients, and creating an osmotic and ionic imbalance. Poor agricultural practices, the use of chemicals, and irrigation with saline water have led to increased prevalence of areas inflicted by salt (Jamil et al. 2011).

A plant is no longer considered an individual entity but is viewed as a holobiont, i.e., a complex community of diverse microflora associated with specific plant species that have co-existed and co-evolved in response to numerous environmental disturbances (Bang et al. 2018). Environmental stressors, such as abiotic and biotic stress factors, shape the soil microbial community, which in turn affects plant physiology and vice-versa. Thus, the survival of a holobiont depends on a bidirectional flow of signals that help it adapt to and tolerate harsh environments. The plant-associated microbiome under stressed conditions has a rich pool of bacteria with diverse functions, which can be utilized for application as bioinoculants (Ullah et al. 2019).

ACC (1-aminocyclopropane carboxylic acid) deaminase activity of microorganisms in the soil microbial community plays a nodal role in stress mitigation, and functions as one of the most crucial plant growth-promoting (PGP) properties (Glick 2014). Environmental stresses lead to an increase in “stress ethylene levels” in plants, which are reduced explicitly by ACC deaminase-producing microbes. Thus, altered plant physiology favors these microbes, which in turn modify the plant’s physiology by reducing the deleterious levels of ethylene (Glick 2014). ACC deaminase producers play an important role in mitigating the effects of stresses, such as salinity (Singh and Jha 2016a), drought (Saikia et al. 2018), and floods on plant growth (Barnawal et al. 2012; Glick 2014).

The implementation of PGPR with ACC deaminase-producing ability for mitigation of stresses is an attractive technology. Some studies have highlighted the role of ACC deaminase producers in plant stress alleviation under laboratory conditions (Kruasuwan and Thamchaipenet 2018; Sarkar et al. 2018; Win et al. 2018). However, one of the main limitations of employing PGPR as bioinoculants is their reduced efficacy and survivability in environmental conditions. Limited studies have implemented ACC deaminase producers for stress mitigation in certain crops under field conditions (Nadeem et al. 2009; Aamir et al. 2013; Kiani et al. 2016). The target crop in the study was Cajanus cajan (pigeonpea), which is known as a sturdy and robust crop (Qiao et al. 2011). It was hypothesized to be an apt model system for isolation of multifarious ACC deaminase-producing strains that would be pre-programmed for the rhizosphere of C. cajan. Despite C. cajan being an economically important crop, there has been little enhancement in its productivity in the last five decades (Saxena and Nadarajan 2010). To the best of our knowledge, there exists no report demonstrating the efficacy of an indigenous ACC deaminase producer in salinity stress mitigation in C. cajan under field conditions. Hence, the objective of this study was the isolation of an indigenous multi-trait ACC deaminase producer for mitigation of salinity stress and enhancement of crop productivity. An indigenous strain would ascertain enhanced survival and efficacy in the rhizosphere. Consequently, the performance of the strains was first tested in the laboratory followed by evaluation in pots and farmer’s fields for plant growth promotion, and mitigation of salinity stress. Additionally, the direct contribution of ACC deaminase towards the dual role was demonstrated by inhibiting the ethylene biosynthetic pathway using a chemical inhibitor.

Materials and methods

Induction of salinity stress

A plant growth experiment was set up in the IIT Delhi Nursery in a randomized block design to simulate salinity stressed conditions in the rhizosphere of C. cajan UPAS 120 as described by Anand et al. (2020). Seeds of C. cajan, UPAS 120, which is an early maturing variety with a life-cycle of 120 days, were procured from National Seed Corporation (NSC), ICAR-Indian Agricultural Research Institute (IARI), New Delhi. The soil used for the experiment had a pH of 7.9; electrical conductivity of 0.6 dS m⁻¹; organic matter of 3.15%, with an organic carbon content of 1.79%; and respiratory activity of 649 µg CO₂ per day 50 g⁻¹ soil. Nitrogen, phosphorus, and potassium (available NPK content) content of soil were 371, 298, and 597 kg ha⁻¹, respectively. The treatments applied to C. cajan were medium salinity stress (MS, 150 mM NaCl, 6–7 dS m⁻¹ electrical conductivity) and high salinity stress (HS, 180 mM NaCl, 8–10 dS m⁻¹ electrical conductivity]. The stress was induced 30 days after sowing (DAS). For salinity treatments, the pots were watered with respective NaCl solutions at the water holding capacity of the pots. The electrical conductivity of soil was regularly monitored. The experiment was carried out during Kharif (May–July) season with mean daily maximum temperature ranging from 28 to 45 °C (with a decrease in temperature as the months progressed). The relative humidity was between 55 and 80% and the maximum rainfall was in July. Samplings were performed at 45, 60, 75, and 90 DAS.

Seed sterilization and seed bacterization

Seed sterilization and bacterization were done as described earlier (Gupta et al. 2012). Seeds of C. cajan UPAS 120 were surface sterilized with 70% ethanol for 30 s followed by sterilization with sodium hypochlorite (0.01%) for 2 min. The seeds were then washed thoroughly with 0.01 N HCl to remove sodium hypochlorite and then rinsed eight times with sterile water. Seeds of approximately similar size and shape were selected for seed bacterization (by visually observing seed size, and also by passing through a coarse sieve having mesh size of approximately 0.8 cm). Seed bacterization was done by using a fixed number of seeds and coating them with liquid culture of selected bacterial strains so as to have a count of ~ 10⁶ CFU/seed. The seeds for control (C) were used without such an amendment.

Isolation of indigenous ACC deaminase producers

Samples were collected from C. cajan rhizospheric soil from induced-salinity stress experiment set up in the IIT Delhi Nursery. Isolation of ACC deaminase producers was done using the protocol of Penrose and Glick (2003) with certain modifications. After sample collection, roots were shaken vigorously by hand for 10 min. to ensure removal of loosely-bound soil particles. Then, by using a brush, the rhizospheric soil fractions, tightly adhering to the roots of C. cajan, were collected in sterile falcons (Barillot et al. 2013). Initially, 1 g of rhizospheric soil sample was inoculated in Pseudomonas agar F (PAF) medium and was incubated at 28 °C with shaking at 200 rpm in an incubator shaker for 24 h. This was followed by sub-culturing in PAF medium, and incubation as mentioned above. Subsequently, inoculum from this suspension was added to Dworkin and Foster (D&F) minimal medium with ammonium sulfate as the sole nitrogen source followed by incubation under conditions as above. This step was repeated using D&F minimal medium supplemented with 3 mM ACC as the sole nitrogen source instead of ammonium sulfate. Further, the bacterial suspension grown in D&F with ACC, were serially diluted at appropriate dilutions, and plated on the D&F medium supplemented with 3 mM ACC using agarose, which has very low nitrogen content, as the solidifying agent. Plating of the serially diluted suspensions was also performed on nutrient agar (NA) for determining bacterial count. The plates were incubated for up to 72 h with periodic monitoring of the growth of colonies. Colony forming units per ml (CFU/ml) were recorded. Colonies that were morphologically distinct were selected and sub-cultured for purity; 20% (w/v) glycerol stocks were prepared and stored at − 80 °C. The isolates were then screened for ACC deaminase activity.

ACC deaminase activity

ACC deaminase activity was estimated for all isolates obtained according to the protocol mentioned by Penrose and Glick (2003) and expressed in µmoles α-ketobutyrate mg⁻¹ protein h⁻¹. The ACC deaminase activity of isolates was estimated prior to activation of the culture for the induction of ACC deaminase expression. The range for visible results in the gnotobiotic assay was 0.02–0.4 µmoles α-ketobutyrate mg⁻¹ protein h⁻¹ (Penrose and Glick 2003).

Gnotobiotic root elongation assay

Gnotobiotic root elongation assay was performed as per the protocol described earlier (Penrose and Glick 2003). Seeds of C. cajan UPAS 120 were sterilized and bacterized with the isolated bacterial strains (Gupta et al. 2012). They were grown in seed pouches in a plant growth chamber under 16 h daylight and 8 h dark, at 30 °C ± 2 °C with relative humidity between 60 and 70% during the experiment for eight days.

Characterization of the isolates for salt tolerance and other PGP properties

The salt tolerance ability of the chosen bacterial strains was tested by growing them in Tryptic soy broth (TSB) with varying concentrations of NaCl (1–15%). Other desirable PGP traits were assessed using standard protocols, viz., indole acetic acid (IAA) production (Gordon and Weber 1951), nitrogen fixation ability by culturing the bacteria in minimal media devoid of nitrogen source (Jensen’s and Burk media) (Dobereiner 1988), ammonia production (Bharucha et al. 2013) and phosphate solubilization ability by spot inoculating bacterial strains onto NBRIP (National Botanical Research Institute Phosphate solubilizing growth medium) and Pikovskaya agar plates (Nautiyal 1999). Siderophore production was assessed by spot inoculating the bacterial strains onto chroma azurole S (CAS) plates, followed by incubation at 28 °C for 4 days. The plates were checked for halo zone around bacterial colonies (Schwyn and Neilands 1987). The biocontrol ability of the strains was tested using the standard protocol as mentioned by Lorck (1948) for the production of hydrogen cyanide (HCN). Briefly, this involved observing the change in color of filter paper saturated with a picric acid solution (2.5 g l⁻¹ picric acid in 12.5 g l⁻¹ Na₂CO₃). Protease activity was checked by spot inoculation of bacterial strains on casein agar plates with skimmed milk (Bharucha et al. 2013). Nitrate reduction assay was performed as per the standard protocol (Cappuccino and Sherman 1992).

ACC deaminase activity, phosphate solubilization, and IAA production under salinity stress

ACC deaminase activity, phosphate solubilization, and IAA production assays were performed in the presence of salt to assess the ability of the bacterial strains to perform under salinity stress conditions. The salt concentration added to the medium for each bacterial strain for determining ACC deaminase activity assay was the maximum salt tolerance capability of the respective strain (10% for ACC deaminase producing bacterial strains G, L, and W). Salt in similar concentrations was added for assays carried out for IAA production and phosphate solubilization, according to standard protocols (Gordon and Weber 1951; Oliveira et al. 2009).

Phylogenetic affiliation of potent ACC deaminase-producing strain

DNA was extracted from the ACC deaminase-positive bacterial strain G using 10% SDS (sodium dodecyl sulphate), 1X TE (Tris–EDTA; buffer for lysis), and cetyltrimethylammonium bromide (CTAB)–NaCl buffer (Nishiguchi et al. 2002). The taxonomic affiliation of the bacteria was confirmed by sequence analysis of 16S rRNA gene. Primers, 27F and 1492R (Lane 1991), were used for amplification of the 16S rRNA gene using 2X PCR Master Mix (Bioline, London, UK) in a reaction volume of 50 µl; 10 pmol of each primer and 50 ng of DNA were subjected to PCR under the conditions described by Lane (1991). The PCR product was sequenced using an ABI3500 sequencer (Applied Biosystems, USA). The nucleotide sequence obtained was compared with the existing 16S rRNA sequences in the GenBank database using National Center for Biotechnology Information (NCBI) BLAST. The sequence was submitted to the NCBI database under the accession number MH988744. Gelatinase and coagulase tests were performed according to standard protocols (Hass and Defago 2005).

Plant growth experiment in a pot with the application of Enterobacter strain G

Plant growth experiment was set up in pots in the IIT Delhi Nursery to test the ability of the shortlisted Enterobacter strain G for plant growth promotion and alleviation of salinity stress. The soil used in this experiment had a pH of 8.10 (in water), organic matter content of 4.06%, an electrical conductivity of 0.653 dS m⁻¹, an organic carbon content of 1.83%, and a respiratory activity of 653 µg CO₂ per day 50 g⁻¹ soil, and no known history of pesticide application. The available nitrogen, phosphorus, and potassium content of soil were 363, 302, and 601 kg ha⁻¹, respectively. Seed sterilization and bacterization (CFU of 2 × 10⁶/seed) were performed as per the protocol of Gupta et al. (2012). C. cajan UPAS 120 seeds were sown in pots containing 8 kg of soil, and salinity treatment (HS, 180 mM NaCl) was administered at 15 DAS. The two control treatments for the experiment were non-inoculated sterilized seeds (i) without salt treatment (C), and (ii) with 180 mM NaCl (C₁₈₀). The experiment was set up including a total of 15 replicates each for control (unamended seeds) as well as salinity treatments. The experiment was carried out during Kharif (June–November) season with a mean daily maximum temperature ranging from 30 to 42 °C (with a decrease in temperature as the months progressed). The relative humidity was between 55 and 80%, and the maximum rainfall was in July. Sampling was performed at 120 DAS. Plant growth was analyzed with a set of biometric parameters, such as shoot length, dry weight, and root length.

Plant physiological parameters

Mitigation of salinity stress was assessed by estimation of various physiological parameters, such as proline, lipid peroxidation, Na⁺/K⁺ ion ratio, chlorophyll content, total soluble sugar (TSS), auxin content, and membrane stability index (MSI). Chlorophyll was estimated from the leaf sample (0.5 g) homogenized in 80% acetone (10 ml). The pigment extracted was quantified spectrophotometrically at 645 nm and 663 nm. The chlorophyll levels were calculated using the following formula (Singh and Jha 2016a):

$$\text{Chl}a=12.7{\text{A}}_{663}-\text{2}.{\text{59A}}_{645}$$

$$\text{Chl}b=22.9{\text{A}}_{645}-\text{4}.{\text{67A}}_{663}$$

Total protein (Bradford 1976), auxin (Andreae and Ysselstein 1959), and soluble sugars (Singh and Jha 2016a) were estimated using standard methods.

Leaf tissue was used for measuring the MSI as per the methodology of Singh and Jha (2016a). Leaf tissue samples (0.2 g) were placed in 10 ml of double-distilled water in two sets. One set was incubated in a boiling water bath for 10 min, and electrical conductivity (C1) was measured using a conductivity meter. The other set was heated at 40 °C for 30 min, and electrical conductivity (C2) was measured after incubation.

$$\text{MSI}=\left[1-\left(\frac{\text{C}2}{\text{C}1}\right)\right]\times 100$$

Estimation of proline and lipid peroxidation

Leaf tissue samples (0.5 g) were homogenized in 5% sulfosalicylic acid (w/v) and centrifuged at room temperature at 8000×g for 10 min. Proline concentrations were estimated by the standard protocol (Bates et al. 1973). Alcoholic extract was prepared by crushing 0.5 g of leaf tissue in 5 ml of 80% ethanol. Lipid peroxidation was assessed by estimating the malondialdehyde (MDA) concentration as per the protocol of Hodges et al. (1999).

Ion analysis

Shoot tissue was used for the estimation of the concentration of ions, viz., Na⁺, K⁺, Ca²⁺, and Mg²⁺. Shoot tissue was ground in liquid nitrogen, and 0.2 g of the ground material was digested with 2 ml of ultra-pure nitric acid (Sigma-Aldrich, St. Louis, MO, USA). The volume was adjusted to 50 ml using MQ water and centrifuged for 10 min at 8000×g at room temperature for removal of any residual debris. One ml of aliquot was used for ion analysis by Microwave plasma-atomic emission spectrometry (MP-AES) (4200 MP-AES, Agilent Technologies, Santa Clara, CA, USA).

Plant growth experiment with the application of Enterobacter strain G in the presence of an inhibitor of ethylene biosynthesis (pot study)

Plant growth experiment was set up under controlled conditions in a plant growth chamber to assess the performance of the Enterobacter strain G in the presence of an inhibitor of ethylene biosynthesis, i.e., cobalt chloride (CoCl₂). Seeds of C. cajan UPAS 120 were sterilized and bacterized with the Enterobacter strain G by the protocol of Gupta et al. (2012). The seeds were then placed in sterilized tubes with 0.7% water agar and kept in a plant growth chamber with 16-h daylight and 8-h dark format at 30 °C ± 2 °C with relative humidity between 60 and 70% during the experiment. The inhibitor CoCl₂ was added to specific tubes during the preparation of water agar, i.e., at a final concentration of 2 µM CoCl₂. Similarly, salt was added to specific tubes during the preparation of water agar, i.e., for high salinity treatment (180 mM NaCl). The plants were harvested after 21 DAS, and their root and shoot length, and fresh and dry weight were measured.

Plant growth experiment in the naturally saline agricultural field with the application of Enterobacter strain G

Plant growth experiment was set up in a randomized block design in an agricultural field of Phaphund, Uttar Pradesh, India (Coordinates: 26.5998°N, 79.4648°E), with naturally stressed saline conditions. The soil was loamy and had an electrical conductivity of 10.562 dS m⁻¹ and a pH of 8.2. The C. cajan UPAS 120 seeds were sterilized and subsequently bacterized (CFU per seed ~ 10⁶) as per the protocol of Gupta et al. (2012). The experiment in the saline field was set up by sowing seeds in 40 replicates each for control (unamended seeds) as well as treatment (Enterobacter strain G amended seeds). Sowing was done on furrows in the field with a dibbling method, whereby plant to plant distance and row to row distance was maintained at approximately 20 cm and 50 cm, respectively. Water from tube wells was used for irrigation, as per the farmers’ practice. The experiment was carried out during Kharif (June–November) season with mean daily maximum temperature ranging from 30 to 42 °C (with a decrease in temperature as the months progressed). The relative humidity was between 55 and 80% and the maximum rainfall was in July (171.25 mm), while October and November received no rainfall. Sampling was performed at 15, 45 and 180 DAS. Plant growth was only visually monitored at the first time point, while at the second (45 DAS) and third time points (180 DAS; harvest stage) plant growth was analysed with a set of biometric parameters such as shoot length, dry weight and root length. Further, plant physiological markers, viz. proline and lipid peroxidation, were measured for assessment of the extent of mitigation of salinity stress by the Enterobacter strain G.

Statistical analysis

The data generated from the gnotobiotic root elongation assay, biochemical assays for PGP properties, all plant growth experiments were subjected to one-way analysis of variance (ANOVA) using Duncan’s multiple range test (DMRT) at p < 0.05 in the SPSS statistical system (SPSS 16.0 for Windows). The dependent variables were root length, biochemical parameters, plant growth/stress parameters, and the independent variables were the various treatments.

Results

Isolation of ACC deaminase producers

Isolation of ACC deaminase producing isolates was performed using the spread plate method wherein appropriate dilutions were plated onto D&F medium after enrichment. The major perturbations in bacterial count were observed at the first and final time points (Table 1). At the final time-point, all treatments exhibited enriched bacterial count [64% and 134% for high salinity stress (HS, 180 mM) and medium salinity stress (MS, 150 mM), respectively] compared to the control. The bacterial abundance on D&F media representing putative ACC deaminase producers showed differences among the treatments; HS showed an increment of 46% compared to the control. Further, MS had a marginal increase in abundance with a value of 18.8%.

Table 1.

Percentage change in the abundance of bacterial count at different time-points along with the plant’s growth under salinity stress

Time-point	High salinity	Medium salinity
Bacterial count
45 DAS	− 17.5	37.2
60 DAS	63.2	53.7
75 DAS	72.2	92.7
90 DAS	64.7	134.7
Putative ACC deaminase producers
45 DAS	54.8	− 8.1
60 DAS	122.3	− 5.7
75 DAS	52.2	− 16.5
90 DAS	46.6	18.8

“−” signifies a decrease

Percentage values are from replicate data (n = 3) with respect to control of corresponding time-point

ACC deaminase activity and gnotobiotic root elongation assay

Morphologically distinct colonies (120) were picked from HS treatment and tested for ACC deaminase activity assay. Nine isolates were selected based on their ACC deaminase activity (in µmoles α-ketobuytrate mg⁻¹ protein h⁻¹), viz., B (0.638 ± 0.03), G (1.29 ± 0.04), H (0.483 ± 0.034), L (0.775 ± 0.060), N (0.849 ± 0.075), S (1.20 ± 0.019), T (0.407 ± 0.011), W (0.816 ± 0.101), and V (0.703 ± 0.006). The isolates were then tested by gnotobiotic root elongation assay for visual confirmation of plant growth promotion (Fig. 1). The bacterial isolates G, H, L, and S out-performed the other isolates with root length enhancement of 77%, 79%, 108%, and 83%, respectively, in comparison to the control plants.

Fig. 1.

Elongation of root by ACC deaminase producing bacterial isolates (B, G, H, L, N, S, T, W, and V) under gnotobiotic conditions. Significantly different values (p < 0.05) have been marked by lowercase letters. Error bars represent the standard deviation for n = 3

Salient PGP properties of ACC deaminase producing isolates and their salt tolerance

The ACC deaminase producing isolates demonstrated multi-trait PGP properties. All the isolates were positive for IAA production, ammonia production (except isolates H, L and S which were negative for ammonia production), phosphate solubilization, nitrate reduction, siderophore production, and nitrogen fixation, while they were negative for HCN production, and proteolytic activity. The isolates were further tested for salt tolerance, and among them, the isolates G, L, and W tolerated stress up to 10% NaCl, the isolates S and H exhibited tolerance up to 7.5% NaCl, and the isolates B, N, and T could tolerate up to 5% NaCl concentration. Further, the isolates G, L, and W were tested for ACC deaminase activity, IAA production, and phosphate solubilization under the maximum bearable salt concentration, i.e., 10% NaCl (Table S1).

Phylogenetic identification of ACC deaminase-producing bacterial strain

Based on ACC deaminase activity under salinity stress and multi-trait PGP ability, strain G was selected for further experimentation and validation for plant growth promotion and salinity stress mitigation. The closest match for 16S rRNA sequence of G strain (99.86%) was Enterobacter sp. (Query coverage-100%; Accession number-KY755351). The 16S rRNA sequence for the G strain was submitted to the NCBI database under the accession number MH988744. The Enterobacter strain G was found to be negative for gelatinase and coagulase assay.

Effect of Enterobacter strain G on C. cajan in salinity stress (pot experiment)

The effect of the Enterobacter strain G on the growth of C. cajan was first monitored in a pot experiment. Strain G significantly enhanced all plant physiological parameters (Table S2; Fig. S1). Moreover, strain G led to enhancements of 106%, 78%, 35% and 120% in root length, shoot length, dry weight, and fresh weight, respectively, at 180 mM NaCl (G₁₈₀) salt concentration, compared to the respective control salt treatments (C₁₈₀). With respect to the stress parameters, treatment with strain G in the presence of salt (G₁₈₀) led to a reduction of 94%, 48%, and 53% in proline, Na⁺/K⁺ ion ratio, and MDA levels, respectively, compared to the control salt treatment (C₁₈₀) (Fig. 2).

Fig. 2.

Effect of Enterobacter strain G on plant morphological parameters in growth experiment of Cajanus cajan in pots. The treatments are C₁₈₀—control with high salinity (180 mM NaCl), G₁₈₀—treatment of Enterobacter strain G with high salinity (180 mM NaCl), and C—control without salinity stress

Effect of Enterobacter strain G on C. cajan in the presence of a chemical inhibitor of ethylene biosynthesis

To assess the contribution of ACC deaminase in mitigation of stresses and promotion of plant growth, Enterobacter strain G was applied in a plant growth experiment in the presence of CoCl₂, which is a chemical inhibitor of ethylene biosynthesis. The application of chemical inhibitors enhanced the shoot length, root length and dry weight in CoCl₂-treated plants (C₁₈₀ − Co) as compared to the untreated plants (C₁₈₀) (Fig. 3). However, the increment was highest in the presence of the ACC deaminase producer, strain G without the chemical inhibitor (G₁₈₀) (shoot length 71%, root length 65% and dry weight 75%) as compared to the control plants with salinity stress (C₁₈₀). In fact, upon comparing the best performer, G₁₈₀, with G₁₈₀ − Co, root enhancement was found to be reduced by 18.5% in the latter. Similar trend of reduced enhancement of shoot length and dry weight was witnessed for plants amended with Enterobacter strain G in the presence of CoCl₂ (G₁₈₀ − Co) as compared to treatment with the Enterobacter strain G in absence of CoCl₂ (G₁₈₀).

Fig. 3.

Effect of inoculation of Enterobacter strain G, along with an inhibitor of ethylene biosynthesis (CoCl₂), on plant growth parameters (shoot length, root length, dry weight). Significantly different values (p < 0.05) have been marked by lowercase letters. Error bars represent the standard deviation for n = 3. C, control; C₁₈₀, control with high salinity (180 mM NaCl); C₁₈₀ + Co, control with high salinity (180 mM NaCl) with CoCl₂; G₁₈₀ + Co, treatment of Enterobacter strain G with high salinity (180 mM NaCl) with CoCl₂; G₁₈₀, treatment of Enterobacter strain G with high salinity (180 mM NaCl)

Effect of Enterobacter strain G on C. cajan in naturally saline farmer’s field

Performance of Enterobacter strain G was validated in an agricultural field under long-term naturally saline environment. Enterobacter strain G performed well in the naturally stressed saline environments. The marked impact of the amendment could be seen right from the first sampling time point, i.e., 15 DAS (Fig. S2a), where the non-treated plants had a severe mortality. The Enterobacter strain G treated plants had high germination and survival rate. Plant health was significantly improved by the application of this potent strain. At 45 DAS, marked enhancement in shoot length (183%), root length (105%) and dry weight (257%) were observed. Significantly enhanced plant growth parameters and grain yield of C. cajan were also observed at the harvest stage (180 DAS) for plants amended with Enterobacter strain G as compared to control plants without the amendment (Table 2). Application of strain G led to reductions in proline (50%) and MDA (55%) levels.

Table 2.

Effect of Enterobacter strain G on plant morphological parameters in growth experiment of Cajanus cajan in naturally stressed field

Treatment	No. of pods per plant	No. of branches per plant	Plant height (cm)	Dry weight (g)	Proline (mMol g−1 FW−1)	MDA (μMol g−1 FW−1)	Grain yield (kg ha−1)
Control	22 ± 3.6a	11.67 ± 0.57a	47 ± 4.03a	17.36 ± 2.18a	2.12 ± 0.14b	0.067 ± 0.002b	223 ± 27.6a
Strain G	65.3 ± 6.65b	22 ± 1b	132.1 ± 5.50b	95.3 ± 2.75b	0.98 ± 0.11a	0.031 ± 0.005a	642 ± 25.6b

Significantly different values (p < 0.05) have been marked by lowercase letters. Values are depicted with their standard deviation for n = 5

Discussion

Under stressed conditions, plants shape their rhizospheric microbiome in such a way that it ensures the survivability of the metaorganism, i.e., the plant and its associated microbiome (Bang et al. 2018). Therefore, stressed conditions can create an ideal reservoir for the isolation of beneficial bacteria producing ACC deaminase, which mitigates biotic and abiotic stresses. The application of bioinoculants is still under-utilized because of the reduced efficacy and survivability of the bacterial strains from the lab to the field. There is an enhanced possibility of isolating stress-tolerant bacteria upon inducing the stresses (Naylor and Coleman-Derr 2018; Ullah et al. 2019). Hence, such an approach has been proposed and/or adopted for isolation of potent stress-tolerant strains (Yaish et al. 2016; Nordstedt and Jones 2020). Thus, a plant growth experiment was set up with C. cajan for experimental induction of salinity stress to isolate indigenous ACC deaminase positive bacterial strains from the rhizosphere of the same crop. The isolated strain would be better suited to the rhizospheric environment when inoculated back in the rhizosphere and thus could enhance plant survivability and productivity under stress as demonstrated by Sood et al. (2018).

Penrose and Glick’s (2003) protocol for isolation of ACC deaminase producers has been employed as a standard procedure in many studies (Barnawal et al. 2012; Singh and Jha 2016a; Stromberger et al. 2017; Saikia et al. 2018; Chandra et al. 2019). The impact of two abiotic stressors on bacterial community dynamics in C. cajan rhizosphere was demonstrated earlier (Anand et al. 2020). Such a comparative estimation was performed by Stromberger et al. (2017), wherein ACC deaminase producers were enumerated for different genotypes of winter wheat grown under different irrigation regimes, including decreasing soil water availability. As in the study by Anand et al. (2020), the salinity treatments demonstrated significant shifts in response to the stress, isolation for potent ACC deaminase producers was performed from them, to specifically target the members enriched under the selection pressure. The total bacterial count and ACC deaminase producers were compared. It was observed that the first time point had the least bacterial count in the high salinity treatment. This could be indicative of the fact that at the first time point, the microbiome was still acclimatizing to the stress levels and did not achieve an equilibrium (Naylor and Coleman-Derr 2018). It is noteworthy to add that the condition of high salinity significantly enhanced the chances of isolating probable ACC deaminase producers. A greater abundance of ACC deaminase producers has been reported in an earlier study, where the presence of stress conditions (increased solar radiations) reportedly enhanced the population of ACC deaminase producers (Timmusk et al. 2011).

The nine isolates (B, G, H, L, N, S, T, V, and W) with potent ACC deaminase activity exhibited an enhancement in root length when assayed under gnotobiotic conditions. Comparable results have been observed in another study where an increase in root length was observed when canola seedlings were inoculated with ACC deaminase producers (Madhaiyan et al. 2006). Furthermore, the enhancement in root length was associated with the physiological effect of ACC deaminase activity of the strains. The nine shortlisted ACC deaminase producers exhibited multi-PGP traits. Other studies have also reported that ACC deaminase producers, exhibiting multi-PGP traits like production of IAA, phosphate solubilization, siderophore production etc., are capable of mediating plant growth promotion and stress mitigation in different plants (Saikia et al. 2018; Kang et al. 2019; Zarei et al. 2020). The bacterial strain G demonstrated maximum PGP properties and ACC deaminase activity under salinity stress. Therefore, it was selected for validation of plant growth promotion ability and mitigation of salinity stress in C. cajan. The strain G was phylogenetically related to the genus Enterobacter. In recent times, Enterobacter has gained prominence as a PGPR. It is found to be associated with roots as an endophyte and as free-living; in both forms, it has a beneficial impact on the plant growth, and is a promising microbial agent for agricultural amendment (Saikia et al. 2018).

The potential of the Enterobacter strain G was further validated by performing plant growth experiment in pots. Enhanced plant biometrics, such as shoot length, root length and biomass established the PGP potential of the Enterobacter strain G. Furthermore, the mechanistic action of mitigation of salinity stress by the strain was also evaluated by estimating specific plant physiological parameters. Similar observations with respect to specific plant physiological markers (proline, MDA, MSI, Na⁺/K⁺ ratio and TSS) for mitigation of stresses have been reported by other studies (Barnawal et al. 2012; Singh and Jha 2016a; Saikia et al. 2018). Proline acts as an osmolyte, and one of the mechanisms by which a bacterium protects the plant from salinity or oxidative stress is by stimulating proline levels (Claussen 2005), but with reduced yield and productivity (Taiz and Zeiger 2002). Studies have reported an increase (Saikia et al. 2018) as well as a decrease (Barnawal et al. 2012) in plant proline levels on treatment with a bioinoculant. The lowering of plant proline levels in the present study is suggestive of the fact that the plants were lesser impacted by the stress due to mechanisms different from proline accumulation (Barnawal et al. 2012; Palaniyandi et al. 2014; Singh and Jha 2016b). MDA is a direct measure of lipid peroxidation and lipid cell membrane damage in the case of oxidative stress conditions (Mittler 2002). Reduction in MDA levels demonstrates alleviation of stress conditions; the strain G caused a reduction in MDA levels under high salinity conditions. Salinity stress conditions stimulate the uptake of Na⁺ ion and reduce the uptake of K⁺ and Ca²⁺ ions (Hamdia et al. 2004). Therefore, for maintenance of ionic balance and regulating the levels of Na⁺ ions, restriction of excessive Na⁺ ions and decreased Na⁺/K⁺ ratio could be a possible mechanism for salinity tolerance by the plants (Hamdia et al. 2004). Treatment with the strain G also reduced Na⁺ levels in the plant and increased the uptake of K⁺ and Ca²⁺ ions, as depicted by a decline in Na⁺/K⁺ ratio. Chlorophyll content, one of the physiological parameters, is a marker for the photosynthetic ability of the plant and is adversely affected by salinity stress (Singh and Jha 2016a). Enhancement of the photosynthetic ability of the plant, as observed in our study, is a clear indication of stress alleviation. Auxin levels also increased, suggesting a healthier plant system. Like proline, soluble sugars also act as osmoregulants; lowering of TSS levels were observed in the treated set compared to the levels in the respective control plants. Similar observations have been reported by Goicoechea et al. (2005). Reports have also suggested that the increase in TSS levels can be one of the mechanisms of action for mitigation of salinity stress (Singh and Jha 2016a). Hence, the mechanism of action of alleviation of salinity stress by a microbe can include a set of factors, which play a cumulative role in the observed effect.

As Enterobacter strain G exhibited multiple PGP properties, to evaluate the contribution of ACC deaminase production in growth promotion and mitigation of salinity stress, plant growth experiment was conducted in the presence of a chemical inhibitor of ethylene biosynthesis, CoCl₂. The chemical inhibitor-treated plants exhibited increment in plant attributes compared to the control, however, the treatment of Enterobacter strain G without Co (G₁₈₀) yielded the best results. Similar results have been reported earlier using pea (Shaharoona et al. 2007) and rice seedlings (Etesami et al. 2014). Hence, the overall increment in plant growth promotion and mitigation of stresses can be attributed to the synergistic impact of the array of PGP properties exhibited by the strain together with the contribution of ACC deaminase production.

Subsequently, the potential of Enterobacter strain G was validated in a naturally stressed saline agricultural field for plant growth promotion and mitigation of stress in C. cajan. Plant growth was significantly enhanced, both in terms of plant height, biomass and productivity. The control plants without the inoculation displayed severe mortality, which was considerably improved with the application of the Enterobacter strain G (data not shown). Strain G led to a decrease in proline and MDA levels similar to observations in the experiments in plant growth chamber and pots. Though there are many studies advocating the impact of ACC deaminase producers on plant growth and mitigation of salinity stress for various crops in laboratory conditions (Kruasuwan and Thamchaipenet 2018; Sarkar et al. 2018; Win et al. 2018), there are few studies that have amended the isolates for mitigation of salinity stress in farmers’ fields. Studies have shown alleviation of salinity stress in field trials by ACC deaminase positive bacteria for crops such as maize (Nadeem et al. 2009), mung-bean (Aamir et al. 2013) and sunflower (Kiani et al. 2016), however no report is available for plant growth promotion and salinity stress mitigation in C. cajan by ACC deaminase producing Enterobacter sp. Thus, the novelty of this work is reflected by the successful application of an indigenous ACC deaminase producing strain, isolated from the rhizosphere of C. cajan, in mitigating salinity stress in C. cajan. The efficacy of the indigenous ACC deaminase producing strain was validated by estimating its target impacts on C. cajan under controlled conditions in laboratory as well as validation under naturally saline field condition. The application of an ethylene biosynthesis chemical inhibitor, CoCl₂, further delineated the role of ACC deaminase, being produced by indigenously isolated Enterobacter strain G, in mitigation of salinity stress in C. cajan.

The effect of the Enterobacter strain G on plant growth and mitigation of salinity stress also supports the fact that the indigenous strain isolated from the rhizosphere of C. cajan would have enhanced survivability and efficacy. Hence, the indigenous Enterobacter strain G demonstrates a promising potential for application in the field as it served the dual purpose of plant growth promotion and mitigation of salinity stress for C. cajan by functioning at multiple levels.

Conclusion

The study successfully isolated and tested the indigenous multi-trait ACC deaminase-producing Enterobacter strain G for its ability to act as a stress-buster and plant probiotic not just under controlled conditions but also in farmer’s fields. The performance of the applied strain was assessed by monitoring plant attributes including characteristic stress markers. The study also emphasized upon the contribution of ACC deaminase production by the potent strain in exhibiting plant beneficial attributes by employing a chemical inhibitor for ethylene biosynthesis. Therefore, the results of the study demonstrate the potential of Enterobacter strain G for mitigation of salt-stress in C. cajan, with an understanding of the impact of such an application on plant physiology.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

GA and SS conceptualized the study, SS acquired financial support, GA and SS designed the experiments, GA, VLS, SD and AB performed the experiments and compiled the data, SS supervised the work, GA wrote the original draft, AB and SS reviewed and edited the manuscript, all authors approved the manuscript.

Funding

The work was supported by Science and Engineering Research Board, Department of Science and Technology, Govt. of India (Grant No. YSS/2015/001437) granted to SS. GA wishes to acknowledge his fellowship from IIT Delhi. AB wishes to acknowledge her fellowship from Science and Engineering Research Board, Department of Science and Technology, Government of India (PDF/2018/001905).

Declaration

Conflict of interest

The authors declared that they have no conflict of interest.