Next Article in Journal
Food Autonomy within Food Sovereignty: Evidence from a Structural Model
Next Article in Special Issue
Delineating the Crop-Land Dynamic due to Extreme Environment Using Landsat Datasets: A Case Study
Previous Article in Journal
Evaluation of the Activity of Estragole and 2-Isopropylphenol, Phenolic Compounds Present in Cistus ladanifer
Previous Article in Special Issue
Melatonin: A Vital Pro-Tectant for Crops against Heat Stress: Mechanisms and Prospects
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 12
Issue 5
10.3390/agronomy12051140
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Inducing Drought Tolerance in Wheat through Exopolysaccharide-Producing Rhizobacteria

by
Muhammad Latif
1,2,
Syed Asad Hussain Bukhari
1,*,
Abdullah A. Alrajhi
3,*,
Fahad S. Alotaibi
3,
Maqshoof Ahmad
4,
Ahmad Naeem Shahzad
1,
Ahmed Z. Dewidar
5,6 and
Mohamed A. Mattar
5,6,7
1
Department of Agronomy, Bahauddin Zakariya University, Multan 60800, Pakistan
2
Department of Agronomy, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
3
King Abdulaziz City for Science and Technology (KACST), King Abdullah Road, Riyadh 11442, Saudi Arabia
4
Department of Soil Science, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
5
Prince Sultan Bin Abdulaziz International Prize for Water Chair, Prince Sultan Institute for Environmental, Water and Desert Research, King Saud University, Riyadh 11451, Saudi Arabia
6
Department of Agricultural Engineering, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia
7
Agricultural Engineering Research Institute (AEnRI), Agricultural Research Centre, Giza 12618, Egypt
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(5), 1140; https://doi.org/10.3390/agronomy12051140
Submission received: 19 April 2022 / Revised: 30 April 2022 / Accepted: 4 May 2022 / Published: 9 May 2022
(This article belongs to the Special Issue Strategizing Agricultural Management for Climate Change Adaptation and Mitigation-Series II)
Browse Figures
Versions Notes

Abstract

:
Wheat is the main staple food in the world, so it is the backbone of food security. Drought not only affects growth and development but also ultimately has a severe impact on the overall productivity of crop plants. Some bacteria are capable of producing exopolysaccharides (EPS) as a survival mechanism, along with other metabolites, which help them survive in stressful conditions. The present study was conducted with the aim of inducing drought stress tolerance in wheat through EPS-producing plant growth-promoting rhizobacteria (PGPR). In this regard, a series of laboratory bioassays were conducted with the aim to isolating, characterizing, and screening the EPS-producing PGPR capable of improving wheat growth under limited water conditions. Thirty rhizobacterial strains (LEW1–LEW30) were isolated from the rhizosphere of wheat. Ten isolates with EPS-producing ability were quantitatively tested for EPS production and IAA production ability. Four of the most efficient EPS-producing strains (LEW3, LEW9, LEW16, and LEW28) were evaluated for their drought tolerance ability along with quantitative production of EPS and IAA under polyethylene glycol (PEG-6000)-induced drought stress. The jar experiment was conducted under gnotobiotic conditions to examine the drought-tolerant wheat genotypes, and two wheat varieties (Johar-16, and Gold-16) were selected for further experiments. The selected varieties were inoculated with EPS-producing rhizobacterial strains and grown under control conditions at different stress levels (0, 2, 4, and 6% PEG-6000). The strain LEW16 showed better results for improving root morphology and seedling growth in both varieties. The maximum increase in germination, growth parameters, percentage, root diameter, root surface area, and root colonization was recorded in Johar-16 by inoculating LEW16 at 6% PEG-6000. Plant growth-promoting traits were tested on the top-performing strains (LEW3, LEW9, and LEW16). Through 16S rRNA sequencing, these strains were identified as Chryseobacterium sp. (LEW3), Acinetobacter sp. (LEW9), and Klebsiella sp. (LEW16), and they showed positive results for phosphorous and zinc solubilization as well as hydrogen cyanide (HCN) production. The partial sequencing results were submitted to the National Center for Biotechnology Information (NCBI) under the accession numbers MW829776, MW829777, and MW829778. These strains are recommended for their evaluation as potential bioinoculants for inducing drought stress tolerance in wheat.

1. Introduction

Water scarcity is the major issue under the current climate change scenario. Limited water availability for crop production compels farmers and scientists to find alternative means and management strategies to feed the increasing world population. As the water supply for agriculture is decreasing with every passing day, the food required to fulfill the demand of the rapidly growing population is on the rise; hence, further aggravating the overall deleterious impact of drought [1,2]. Moreover, the problem will gain more importance in the near future due to the expansion of agricultural activities to less fertile soils to meet the food requirements of a burgeoning population [3]. Therefore, improving crop growth and survival under water-limited conditions is emerging as a major challenge for scientists in modern-day agriculture.
Drought not only affects the growth and development but ultimately goes on to severely impact the overall productivity of crop plants [1,4]. Successful crop production is a major challenge in the presence of drought stress [5]. Losses incurred due to drought stress overwhelm losses from all the other causes as both aspects of stress, i.e., duration and severity, are very critical [4]. Researchers have already predicted that the severity and frequency of drought will increase under the current global climate change [6].
Many strategies are being used to improve the drought tolerance of crops. These strategies include traditional methods to select efficient crop varieties that have better a physiological performance under limited water availability and genetic engineering approaches to induce drought-tolerant genes in more productive crop varieties [7]. Water use efficiency and crop survival under drought is a complex phenomenon that is controlled by a number of genes. A large number of genes that respond to plant responses under drought stress have been discovered, but large gaps remain to be explored [8,9]. Selection of suitable crop variety is again a complicated and laborious process that needs to expose plants to a multitude of environmental under-field conditions. The separation of plant physiological responses under different types of stresses can be difficult as the response of one type of stress may overlap certain other stresses [10,11]. Moreover, plants at different growth stages respond differently to drought stress [12].
Under these circumstances, the use of plant growth-promoting rhizobacteria (PGPR) is a relatively simple and cheap alternative strategy for dealing with abiotic stresses [13]. A number of possible mechanisms by which these PGPR induce drought tolerance in crop plants have been reported, although the exact mechanism remains largely speculative. Under stress, the formation of a bacterial biofilm can lead to the development of an extracellular matrix providing a range of macromolecules essential for the growth and development of plants [14,15]. These biofilms are formed by the production of extra-cellular polymeric substances that are critical in plant–microbe interactions under stress environments. These exopolysaccharides have several folds higher water retention capacity than their mass [16].
Bacteria produce exopolysaccharides as a survival mechanism [17], along with other metabolites, which help them survive in stressful conditions [18]. The EPS produced by bacteria are mixtures of higher molecular weight polymers that stick to the surfaces and help in soil aggregation. This adherence with the soil particles retards their degradation as they can be very easily degraded if present in their innate state. Aggregation affects different aspects of soil properties such as infiltration, root penetration, aeration, and control of runoff. As exopolysaccharides are hygroscopic in nature [19], the production of these compounds enables these bacteria to maintain a higher water content in the microenvironment of the colonies and help them survive as the water potential recedes [20]. A wide range of exopolysaccharides are produced by these bacteria [21] depending on bacterial group, substrate, growth state, and cultivation time [19].
Wheat is main staple food of Pakistan, and it is the backbone of food security all over the globe. Although wheat can adapt to a range of moisture conditions with precipitation under different climates [22], its production is very prone to drought stress. Scientists are working on a range of technologies to improve wheat productivity under water-limited conditions. We hypothesized that the EPS production ability of rhizobacterial strains can help bacteria and plants to survive under limited water availability. Thus, the present study was conducted with the aim of isolating, characterizing, and examining drought-tolerant EPS-producing PGPR to induce drought stress tolerance in wheat.

2. Materials and Methods

Drought-tolerant, EPS- and IAA-producing rhizobacterial strains were isolated and characterized in this study. A series of studies were carried out to determine the various plant growth-promoting attributes of drought-tolerant rhizobacterial strains for this purpose. Ten wheat varieties were screened against various levels of PEG-6000 in growth room trial. Following that, selected EPS-producing bacterial strains were tested for their ability to improve wheat variety growth (Johar-16 and Gold-16) under PEG-induced drought-stressed gnotobiotic conditions. The best performing strains were identified using a commercial service for 16S rRNA sequencing.

2.1. Soil Samples Collection and Handling

Rhizosphere soil samples were collected from wheat fields in Bahawalpur, Pakistan (29°25′5.0448″ N, 71°40′14.4660″ E). Drought-tolerant rhizobacterial strains were isolated by transporting samples in sterile polythene bags to the laboratory and storing them in dry freeze conditions (−20 °C).

2.2. Isolation of Drought-Tolerant Rhizobacteria

One gram of soil samples was suspended in sterile distilled water and serially diluted up to 10−8. Then, 1 mL from the last three dilutions was poured into an agar plate containing Luria Bertani (LB) media having the composition: trypton (10 g), NaCl (10 g), yeast extract (5 g), and agar (20 g) adjusted pH 7.4 amended with polyethylene glycol (PEG-6000) at 4 g L−1. The plates were incubated at 32 ± 1 °C in an incubator (Model; LI15 Shel Lab, Sheldon Manufacturing, Inc., Cornelius, OR, USA). After 48 h of incubation, different colonies were selected based on color and morphology and streaked on new agar plates. Selected strains were purified by streaking, coded, and stored at −20 °C in a 50 percent glycerol stock for future experiments [23]. The isolates were grown in LB broth amended with 4% PEG-6000. The flask was cover with aluminum foil, sealed with Para film, and incubated in a rotary shaker (Model; SI9R-2, Shel Lab, Sheldon Manufacturing, Inc., USA) at 32 ± 1 °C. The incubation of the tubes was done under shaking at 30 ± 1 °C for 72 h. A UV-visible spectrophotometer (Carry 60; Agilent, Santa Clara, CA, USA) was used to measure bacterial growth in terms of optical density (OD600). Thirty bacterial isolates with the highest growth in PEG-amended media were chosen, coded (LEW1 to LEW30), and stored for future testing [24].

2.3. Exopolysaccharide Production

Exopolysaccharide production (EPS) bioassay was performed on the rhizobacterial isolates. Selected strains were inoculated on RCV agar plates and incubated for 48 h at 30 ± 1 °C. Mucoid formation around colonies was observed as an indicator of rhizobacterial isolates’ ability to produce EPS. The positive isolates were chosen for further testing [25]. The strains capable of producing EPS were tested for quantitative EPS production in liquid culture [26]. For this purpose, 100 mL of LB broth medium was inoculated with 100 µL of bacterial culture (OD600 0.6) and incubated in shaking incubator at 30 °C at 100 rpm. After 96 h, bacterial culture was centrifuged supernatant was separated in sterilized flask. Exopolysaccharide was precipitated by the addition of pre-chilled acetone in the supernatant (3:1 v/v). Freshly prepared exopolysaccharide was dried through lyophilization, and weight was measured [27]. The phenol-sulfuric acid method was used to quantify exopolysaccharides by comparing the standard curve developed with glucose standards [28]. The whole experiment was replicated three times.

2.4. Indole Acetic Acid (IAA) Production

The EPS-producing strains were then tested for their ability to produce Indole 3 acetic acid (IAA) in the presence and absence of L-tryptophan (L-tryp). The bioassay was carried out in vitro according to the standard protocol described by Bric et al. [29]. Overnight grown bacterial cultures were inoculated in LB liquid media supplemented with 4% PEG and 5% L-tryptophan, and tubes were incubated for 72 h in a rotary shaker (100 rpm) at 32 ± 1 °C. After incubation, tubes were removed from the rotary shaker and centrifuged for 20 min at 4500 rpm. After that, 3 mL of supernatant was combined with 2 mL of Salkowski reagent (2 mL of 0.5 M FeCl3·6H2O and 98 mL of 35 percent H2SO4) and left to develop for 20 min. Color was developed after the IAA standards (0, 10, 20, 30, 40, 50, and 100 mg kg−1) were prepared. A UV-Vis spectrophotometer (Carry 60; Agilent, USA) was used to measure absorbance at 535 nm. The concentration was determined by comparing the absorbance reading with the standard plotted curve. The parallel experiment was run to observe the IAA production by drought-tolerant rhizobacteria without addition of L-tryptophan. The whole experiment was replicated three times. Based on EPS production and IAA production ability, the four most efficient strains, i.e., LEW3, LEW9, LEW16, and LEW28, were selected for further experimentation.

2.5. Growth Potential of EPS-Producing Rhizobacterial Strains under PEG-6000-Induced Drought Stress

The goal of the bioassay was to see how different levels of PEG-6000 (0, 2, 4, 6, and 8%) affected the growth of EPS-producing strains in liquid culture [26]. The broth culture tubes were inoculated with 48 h old culture and incubated in rotary incubator (100 rpm) for 72 h at 28 ± 1 °C. The growth was observed in terms of absorbance in UV-Vis spectrophotometer at 600 nm wavelength.

2.6. The Effect of PEG-Induced Drought Stress on Selected Strains’ Growth, EPS Production, and IAA Production Ability

The effect of PEG-induced drought stress on selected strains’ growth, EPS-production, and IAA production ability was investigated. For this purpose, the selected strains were grown at different drought levels, i.e., 0, 2, 4, 6, and 8% of PEG-6000. The experiment was replicated three times. The broth culture tubes were inoculated with 48-hour-old culture and incubated for 72 h at 28 ± 1 °C in a rotary shaker at 100 rpm. After 24, 48, and 72 h, absorbance was measured using a UV-Vis spectrophotometer at 600 nm wavelength. Under PEG-induced drought stress, the amount of IAA and EPS produced by rhizobacterial strains was measured using a UV-Visible spectrophotometer, as defined by Bric et al. [29] and Dubois et al. [28], respectively.

2.7. Rhizobacterial Strains with Plant Growth-Promoting Properties

The plant growth-promoting characteristics of the selected EPS-producing strains with the ability to perform under drought stress were evaluated. The standard method described by Simons et al. [30] was used to test the root colonization of the selected rhizobacterial strains. For the determination of zinc (Zn) solubilization ability, the method of Fasim et al. [31] was used, whereas for the determination of qualitative phosphate solubilization, the strains were grown on Pikovskaya’s agar plates according to Pikovskaya [32]. The strains were also tested for their ability to produce hydrogen cyanide (HCN) using the standard protocol as described by Lorck [33].

2.8. Screening of Wheat Varieties against Drought Stress

Seeds of different wheat varieties were collected from the local market and research institutes. In the growth room, a jar experiment was carried out to screen wheat varieties for their ability to withstand drought stress. The growth room conditions were adjusted as 12 h dark with 15 ± 2 °C and 12 h light (1000 flux) with 15 ± 2 °C. The humidity was maintained at 70%. The plastic jars were filled with 600 g of river sand, irrigated with distilled water, and autoclaved. A completely randomized design (CRD) with three replications was used to arrange the jars. Twenty wheat varieties (Uqab-2000; Glaxy-13; Anaj-17; Punjab-11; AAS-11; Faisalabad-08; Bhakar-02; Lasani-08; Fareed-06; Johar-16; Gold-16; AS-02; NARC-11; Millat-11; AARI-11; Ujala-16; Shahkar-13; Seher-06; Shafaq-06; and SH-02) were screened under drought stress. PEG-6000 was used to develop different levels of drought (0, 3, 6, 9, and 12% (w/w)). Six seeds were sown in each jar and plant population was maintained at 3 plants jar−1 after germination. To meet the water and nutrient requirements, a half-strength Hoagland solution was used. Germination percentage was recorded, and germination speed was calculated using the formula described by Gairola et al. [34]. After 25 days, seedlings were harvested, and growth parameters recorded.

2.9. In Vitro Jar Trial for Plant Growth Promotion of Wheat Using EPS-Producing Rhizobacterial Strains

Based on previous results, selected EPS-producing, drought-tolerant PGPR strains were tested in a jar trial to improve the growth of wheat seedlings under drought stressed axenic conditions. Wheat seeds (Johar-16 and Gold-16) were disinfected by keeping them in 2% sodium hypochlorite for 10 min. Surface-sterilized wheat seeds were soaked in sterile filter paper before being inoculated with broth from the appropriate bacterial strains (48-hour-old culture). Wheat seeds were dipped in sterile distilled water for 30 min to serve as an uninoculated control. Drought levels were developed by applying PEG-6000 at 0, 2, 4, and 6% (w/w). Eight inoculated seeds were sown in a sterilized jar filled with 600 g river sand. For comparison, uninoculated control seeds were sown. A completely randomized design (CRD) with six replicates was used to arrange the jars. The germination percentage was observed, and germination speed was calculated using the formula described by Gairola et al. [34]. After 8 days of sowing, seedlings were uprooted, and root colonization was determined through serial dilution and pour plate technique as described by Iqbal et al. [35]. Seedlings from the remaining three replicates were harvested after 25 days to determine growth parameters such as root length, shoot length, root and shoot dry weight, and nutrient use efficiency.

2.10. Identification of EPS-Producing Rhizobacterial Strains

The phenotypes of the selected EPS-producing strains with plant growth-promoting properties (LEW9 and LEW16) were studied [36]. The 16S rRNA sequencing of the selected strains was also carried out in order to confirm the phylogeny of PGPR strains [37]. The data obtained through the partial sequencing of 16S rRNA of selected strains were analyzed on NCBI site using Blastn analysis. MEGA7 software was used to align the blast results of the closely related 16 nucleotide sequences by muscle alignment of the codons [38]. The microbial history was evaluated by constructing a neighbor-joining tree using the method described by Saitou and Nei [39], followed by analysis using the method described by Tamura et al. [40].

2.11. Statistical Analysis

Statistical analysis was performed on the data collected from various experiments. The CRD design in the factorial arrangement was used to construct ANOVA for experiments [41]. The data were compared by using LSD and Tukey’s tests, where applicable. To calculate standard error, mean, and draw graphs, Excel (MS Office 2010 and 365) was used.

3. Results

3.1. Isolation of Drought-Tolerant, EPS-Producing Rhizobacteria

Using LB growth media amended with 4% PEG-6000, thirty rhizobacterial colonies were isolated from soil samples collected from the rhizosphere of wheat in different fields in the Bahawalpur district. The Bahawalpur district is located at 29.35° N and 71.69° E, where annual rainfall is less than 150 mm, and more than 80% of rainfall occurs in monsoon season (June and July). These rhizobacterial isolates were coded as LEW1–LEW30.
The isolates’ drought tolerance was confirmed by growing them in liquid culture amended with PEG-6000 at 4% w/v. The bacterial growth was confirmed in terms of optical density (OD600) using UV-Visible Spectrophotometer (Model; Cary60; Agilent, USA). All the isolates showed growth in liquid culture amended with PEG-6000; however, these isolates showed different growth behavior in PEG-6000-amended liquid medium. The isolates LEW3, LEW9, LEW16, and LEW28 showed better growth as compared to other isolates (Table 1).
In agar plates containing RCV medium, these isolates were tested for their ability to produce EPS. Ten isolates (LEW3, LEW4, LEW9, LEW11, LEW16, LEW19, LEW21, LEW23, LEW26, and LEW28) were found to be capable of producing exopolysaccharides, as evidenced by mucoid growth on RCV agar plats (Table 1).

3.2. Quantification of Exopolysaccharides and IAA Production by Rhizobacterial Isolates

These isolates with the ability to show mucoid in PEG-amended RCV media were tested for EPS production in broth culture as well as quantitative IAA production in the presence and absence of L-tryptophan. All the tested strains produced EPS but with variable degrees (Table 2). Results revealed that the maximum EPS production of 86.2 µg mL−1 was recorded for the isolate LEW28 followed by LEW16 (81.3 µg mL−1), LEW3 (72.4 µg mL−1), and LEW9 (63.5 µg mL−1), whereas the minimum EPS production of 10.8 µg mL−1 was observed for the isolate LEW23. The IAA production of EPS-producing rhizobacterial isolates was tested in the presence and absence of L-tryptophan (Table 2).
In the presence and absence of L-tryptophan, the bioassay revealed that the majority of the isolates produced IAA. The isolates LEW4 and LEW21, however, did not produce IAA in both the cases. The isolates produced more IAA in the media amended with L-tryptophan. The isolate LEW28 has the maximum (13.17 µg mL−1) IAA production ability followed by LEW9, LEW3, and LEW16. Based on EPS production and IAA production ability, the four most efficient strains, i.e., LEW3, LEW9, LEW16, and LEW28, were selected for further experimentation.

3.3. Growth Potential of EPS-Producing Rhizobacterial Strains under PEG-6000-Induced Drought Stress

The results of an in vitro bioassay with various drought levels (0, 3, 6, and 9%; PEG-6000) revealed that bacterial growth decreased as the level of PEG-6000 increased (Figure 1). The isolate LEW9 showed the maximum growth in LB media where observed optical density was 1.22 at 600 nm wavelength. At 3% concentration of PEG-6000, the isolates LEW9 and LEW28 showed higher growth (1.00 OD600) followed by LEW16 (0.91 OD600) and LEW3 (0.85 OD600). The isolate LEW9 performed better under higher concentration of PEG-6000 and showed 0.81 and 0.66 OD600 at 6 and 9% concentration of PEG-6000, respectively. At 9% concentration of PEG-6000, the isolates LEW3, LEW16, and LEW28 showed 0.63, 0.60, and 0.61 OD600, respectively.

3.4. Effect of PEG-Induced Drought Stress on Growth, EPS Production, and IAA Production Ability of Selected Strains

The drought-tolerant rhizobacterial strains that produce EPS and IAA were grown at different levels of PEG-induced drought stress, and their growth, IAA production, and EPS production ability were monitored at 24 h intervals up to 72 h. According to the results (Table 3), increasing the level of PEG inhibited the growth of rhizobacterial strains. Furthermore, as the level of PEG increased, the length of the log phase decreased, resulting in an early stationary phase.
The isolates behaved differentially under different levels of drought. At increasing drought stress, only the isolate LEW9 showed significantly better growth at 9% concentration of PEG. The ability of the strains to produce IAA under normal and drought-stressed conditions also differed. The maximum IAA production (53.7 µg mL−1) was observed for isolate LEW9 after 72 h under normal conditions. Moreover, the minimum IAA production (4.40 µg mL−1) was observed for the isolate LEW3 at 9% concentration of PEG.
The tested isolates produced a rapid increase in IAA and EPS up to 48 h, after which the production of IAA and EPS was non-significant at a PEG concentration of 9%. The data revealed that the isolates have significantly different EPS production ability. After 72 h of growth under normal conditions, the strain LEW9 produced the highest quantity of EPS (122.0 µg mL−1).
The isolates LEW9 and LEW28 produced similar amounts of EPS at lower levels of drought (PEG concentration of 3%) when compared to each other, but they were significantly different from LEW3 and LEW16. The isolate LEW9 produced the highest quantity of EPS (60.3 µg mL−1) at a PEG concentration of 9%. Higher levels of PEG resulted in a significant decrease in EPS production when compared to lower levels.

3.5. Plant Growth-Promoting Traits of Selected Rhizobacterial Strains

Selected isolates were then tested for various plant growth-promoting properties (Table 4). All the tested isolates were potentially colonized with wheat roots. However, the isolates LEW3 and LEW9 were more efficient in colonizing wheat roots. These isolates showed 3.52 × 106 and 4.16 × 106 bacterial colonies cm of wheat root, respectively. The tested isolates also performed well in the agar plate assay for P solubilization. LEW3 and LEW9 isolates were more effective at solubilizing the insoluble source of P and had a larger halo zone around colonies. Similarly, the isolates LEW3, LEW9, and LEW16 efficiently grew in insoluble zinc and were able to solubilize Zn, as evidenced by the clearing zone around colonies. Three isolates, i.e., LEW3, LEW9, and LEW28, were positive for HCN production in the agar plate assay, which was evident from the change in filter paper color from yellow to pink.

3.6. Screening of Wheat Varieties against Drought Stress

Under controlled conditions, twenty wheat varieties were screened for drought tolerance at various levels of PEG. The varieties were grown under water-stressed conditions (PEG-6000 at 0, 2, 4, and 6%) and seedling growth was determined. The collected data were analyzed through principal component analyses (PCA) and better performing wheat varieties were selected for further experimentations (Figure 2). The wheat variety Johar-16 performed better for germination percentage, seedling dry biomass, shoot length, and root length at all drought levels. The next variety was Gold-16 which performed better at 6% of PEG-6000 (Figure 2a). Wheat variety AS-02 performed better at lower stressed levels such as 2% (Figure 2b) and 4% (Figure 2c) PEG-6000; however, its performance declined at 6% PEG-6000 (Figure 2d). Furthermore, the principal component analyses (PCA) revealed that wheat variety Aas-11 did not perform effectively at all drought levels. The wheat variety Lasani-08 and SH-02 showed better results under normal conditions; however, their performance was declined under drought stress. The wheat variety Johar-16 and Gold-16 performed better under higher drought level (at 6% PEG-6000) and therefore were selected for further experimentations.

3.7. In Vitro Jar Trial for Plant Growth Promotion of Wheat Using EPS-Producing Rhizobacterial Strains

The selected wheat varieties (Johar-16 and Gold-16) were inoculated with selected rhizobacterial strains (LEW3, LEW9, LEW16, and LEW28) and tested for plant growth promotion under controlled conditions at different drought levels, i.e., 0, 2, 4, and 6%, of PEG-6000. The results regarding seed germination and root morphology are presented in Figure 3. Under different levels of drought stress, the bacterial strains significantly increased the germination of wheat varieties Johar-16 and Gold-16 (Figure 3). Under normal conditions, wheat variety Johar-16 showed a maximum of 98 and 97% germination percentage when inoculated with strains LEW16 and LEW9, respectively. Under different levels of drought, however, inoculation with strain LEW16 produced better seed germination results in both varieties Johar-16 and Gold-16. The inoculation of strain LEW16 in wheat variety Gold-16 showed 87, 75, and 63% germination at 2, 4, and 6% PEG-6000, respectively. The wheat variety Johar-16 showed 83, 68, and 52% germination when inoculated with strain LEW16 at 2, 4, and 6% of PEG-6000, respectively, whereas the control treatment showed only 31% germination for both wheat varieties at 4% PEG-6000.
Wheat root diameter was increased by exopolysaccharide-producing plant growth-promoting rhizobacteria under drought-stressed conditions (Figure 3). Under normal conditions, the maximum root diameter (0.95 mm) was observed in variety Gold-16 by the inoculation of strain LEW16. The uninoculated control treatment had the smallest root diameter at all levels of drought stress, whereas the inoculation of strain LEW16 had the largest root diameter at all levels of PEG. At 6% PEG-6000, the inoculation of LEW16 showed the maximum root diameter, i.e., 0.49, and 0.52 mm in wheat variety Johar-16 and Gold-16, respectively.
The exopolysaccharide-producing, drought-tolerant rhizobacteria increased root surface area of wheat under water scarcity (Figure 3). The inoculation of strain LEW16 performed better under different levels of drought stress. At 6% PEG-6000, the maximum 23 and 16% increase in root surface area was observed in wheat variety Johar-16 and Gold-16, respectively, by the inoculation of strain LEW16. Potential exopolysaccharide-producing, drought-tolerant rhizobacteria improved root colonization in wheat under different levels of drought stress (Figure 3). Under different levels of drought, inoculation of exopolysaccharide-producing, drought-tolerant rhizobacterial strains increased root colonization in both wheat varieties Johar-16 and Gold-16. Under normal conditions, the maximum 8.1 × 105 colony forming units (CFU) per gram root was recorded in wheat variety Gold-16 by the inoculation of LEW3. However, under drought stress, the strain LEW16 and LEW28 performed better for root colonization. The maximum 3.1 × 105 CFU g−1 root was recorded in wheat variety Johar-16 whereas 3.6 × 105 CFU g−1 root was observed in wheat variety Gold-16 by the inoculation of strain LEW16 at 6% PEG-6000.
The capability of rhizobacteria to increase root length under water-stressed conditions by exopolysaccharide-producing rhizobacteria is presented in Figure 4. The inoculation of strain LEW28 showed better results at lower drought levels (2 and 4% of PEG-6000) where 27 and 53% increases were recorded in wheat variety Johar-16. Inoculation of LEW16 resulted in a maximum 92 percent increase in root length when compared to control at 6 percent PEG-6000. Figure 4 shows the results of a study on the ability of drought-tolerant rhizobacterial strains to increase shoot length in water-stressed conditions. Under different drought levels, the shoot length decreased, but this was improved by inoculating EPS-producing, drought-tolerant rhizobacterial strains. At 6% of PEG-6000, the inoculation of LEW16 showed the maximum, i.e., 27 and 28%, increase in root length in wheat varieties Johar-16 and Gold-16, respectively.
The exopolysaccharide-producing, drought-tolerant rhizobacteria improved seedling vigor index under different levels of drought stress. With increasing drought stress the seedling vigor index was decreased (Figure 4). Inoculation with exopolysaccharide-producing rhizobacteria, on the other hand, significantly improved seedling vigor index when compared to the control. Wheat variety Gold-16 showed better results compared with Johar-16. The inoculation of strain LEW16 showed the maximum, i.e., 7.9, 4.7, and 3.1, seedling vigor index in wheat variety Gold-16 at 2, 4, and 6% PEG-6000, respectively.
The results (Figure 4) describe the effectiveness of exopolysaccharide-producing, drought-tolerant rhizobacteria to improve seedling total dry biomass under different levels of drought stress. The minimum (0.1 g seedling−1) total dry biomass was recorded in wheat variety Johar-16 in uninoculated control treatment at 6% of PEG-6000. However, bacterial strain inoculation significantly increased the total dry biomass of both wheat varieties, Johar-16 and Gold-16, under different levels of drought stress when compared to the control. Due to inoculation with EPS-producing rhizobacterial strain LEW16, the maximum, i.e., 0.25 g, seedling−1 of total dry biomass was recorded in wheat variety Gold-16 at 6% PEG-6000, which is 44 percent more than control. The next best strain was LEW16, which showed a 41% increase in total dry biomass when compared to the control treatment.

3.8. Identification of Selected Rhizobacterial Isolates

Three strains were chosen for identification based on their performance in the pot and in the field. For this purpose, cell pellet was collected from 4 mL of overnight grown bacterial culture having 0.6 OD600. Then, DNA was extracted through the CTAB method. The extracted DNA was sent to Macrogen, Korea, for identification through the 16S rRNA gene sequencing technique. The DNA sequences obtained from Macrogen were blasted on the NCBI server. Using the software MEGA 7.0, a neighbor-joining phylogenetic tree was constructed by closely related taxa (Figure 5). The strains were identified as Chryseobacterium sp. (LEW3), Acinetobacter sp. (LEW9) and Klebsiella sp. (LEW16) and were submitted to NCBI gene bank database with accession numbers MW829776, MW829777, and MW829778, respectively.

4. Discussion

In the majority of countries, water scarcity is a major problem for crop production. Wheat is the staple food of Pakistan and among the major cereal crops over the globe. Irrigation is crucial for wheat productivity at various growth stages, and drought negatively affects its growth and germination. Drought-stressed crops have been shown to grow faster when exposed to EPS-producing PGPR [13,42]. They induce drought tolerance by colonizing the rhizosphere through the production of complex extra-cellular polysaccharides (EPS) along with other mechanisms such as modification in root architecture, production of growth regulators, and phytohormones such as IAA, siderophores, etc., reducing the production of ROS by regulating antioxidant enzyme systems, nutrient solubilization, and many more. These bacteria produce EPS, which is a mixture of higher molecular weight polymers that stick to surfaces and aid in soil aggregation and water potential maintenance.

4.1. Screening of Wheat Varieties under Drought Stress

Twenty wheat varieties were screened for drought tolerance. Under controlled conditions, all tested varieties showed more or less similar results. Drought stress, on the other hand, significantly reduced seed germination and seedling growth. Under PEG-induced drought stress, two wheat varieties, Johar-16 and Gold-16, showed significantly higher seed germination and growth than other varieties. This increase in germination and seedling growth could be due to these varieties’ adaptive responses. The reduction in germination and poor seedlings establishment are the early signs of water scarcity [43]. It has been reported that biochemical changes in seed occur shortly after imbibition and that these changes are influenced by soil water availability and potential [44]. Drought stress has also been linked to a delay in imbibition and, as a result, a lower germination rate. As a result, germination percentage and seedling vigor is negatively affected under drought stress [45].
Seedling growth may be slowed as a result of decreased cell division and elongation. Drought affects the morphological, physiological, and biochemical parameters of plants, thus decreasing the photosynthetic efficiency of crop plants. For example, Smolikova et al. [46] reported that delayed imbibition led the desiccation and hence both the seed viability and seedling vigor. These two traits are related to various biochemical changes predominantly related to the generation of reactive oxygen species (ROS) damaging and altering the genetic structure. Drought stress has a negative impact on germination because it causes desiccation, which affects seed viability and vigor.

4.2. Screening of EPS-Producing Plant Growth-Promoting Rhizobacteria under Drought Stress

Under drought stressed conditions, the EPS-producing, drought-tolerant rhizobacterial strains were evaluated for growth, IAA production, and EPS production. The strains exhibited variable drought stress growth as well as varying ability to produce IAA and EPS under drought-stressed conditions. Exopolysaccharides are complex compounds having water in their hydrated structure. These EPS help bacteria survive in stressful situations, and soil microbiologists use the same trait to induce drought tolerance in crop plants [13]. Under stressful conditions, a number of bacterial strains have been reported to produce EPS [47]. It has been reported that bacteria produce EPS as a survival mechanism [48] under stressful conditions [21], which are mixtures of higher molecular weight polymers including carbohydrates, proteins, and other organic compounds [49]. These EPS stick to the surfaces and help in the binding of soil particles leading to better soil aggregation.
Tested rhizobacterial strains produced significantly increased concentration of EPS under osmotic stress as compared to unstressed conditions, which indicates that these strains responded to stress [50]. The EPS molecule retain large quantity of water, which helps bacteria and plant to survive under drought stress [51]. The higher carbon concentration in the EPS molecule has been shown to improve water retention [52].
Under drought stress, the rhizobacterial strains produced higher concentrations of IAA than under normal conditions, according to the current study. Literature reported that IAA helps plants in cell division, and tissue differentiation [53]. Bacterial strains that can produce these compounds help crop plants cope with stress [54,55]. Furthermore, these bacterial strains have been reported to help plants cope with the negative effects of drought stress by engineering their physiology [13]. Plant growth is aided by the production of IAA in stressed situations [56].
Furthermore, in an agar plate assay, the tested strains showed phosphorous and zinc solubilization. Similarly, Intorne et al. [57] and Mumtaz et al. [58] described that solubilization of phosphorous and zinc by rhizobacteria improves their uptake and promotes plant growth and yield. The tested rhizobacteria possess hydrogen cyanide (HCN) production ability. The tested strains produced HCN that may help in balanced antioxidant activities. The results are similar to those of Agbodjato et al. [59] and Nazli et al. [47], who both reported HCN production under stressful conditions. Bacterial production of HCN acts as a biocontrol agent against a variety of pathogens, reducing disease spread [60].
The strains that were able to produce EPS, IAA, and other plant growth-promoting characters colonized the plant roots heavily in this study. The biochemical and enzymatic activities of bacteria help them in root colonization through modification in the secretion of root exudates [61]. As a result, bacterial strains that produce these compounds confer drought tolerance in plants [55].

4.3. Impact of EPS-Producing PGPR on Wheat Growth under Drought Stress

The selected wheat varieties Johar-16 and Gold-16 were inoculated with selected EPS-producing, drought-tolerant rhizobacterial strains and tested for the ameliorative effects on germination percentage, root development, and seedling growth. Germination percent was decreased under drought stress; however, inoculation of exopolysaccharide-producing rhizobacteria improve germination as compared to control. The EPS-producing bacteria retained water, which created a microenvironment that decreased osmotic stress [62]. The inoculation of rhizobacterial strains improved root colonization, root diameter, root surface area, root/shoot length, and seedling dry biomass in the current study, even under drought-stressed conditions [63]. In addition, inoculation of EPS-producing rhizobacteria has been shown to promote plant growth in drought-stressed conditions by stabilizing soil aggregates and retaining water [64].
The EPS-producing, drought-tolerant strains capable of improving wheat growth under limited water conditions were isolated from the rhizosphere of wheat. These strains showed different responses to PEG-induced drought stress and the maximum growth was shown by the strain LEW9. These strains have also been shown to be effective bioinoculants for reducing the negative effects of drought on wheat. The strain LEW9 provided the greatest improvement in wheat growth, yield, and physiological parameters, followed by LEW16 and LEW3. Based on the results of 16S rRNA sequencing these strains were identified as Acinetobacter sp. (LEW9), Klebsiella sp. (LEW16), and Chryseobacterium sp. (LEW3). These strains were rod-shaped with smooth colonies and Gram-negative in reaction. For Chryseobacterium sp. (LEW3), Acinetobacter sp. (LEW9), and Klebsiella sp. (LEW16), the partial sequences were submitted to GenBank under accession numbers MW829776, MW829777, and MW829778, respectively. Similarly, previous studies reported Chryseobacterium sp., Acinetobacter sp. and Klebsiella sp. as rod-shaped, spore-forming bacteria with Gram-negative in reaction [65,66].
Rhizosphere bacteria from these genera have been shown to help crop plants grow better when they are drought stressed. For example, Sharath et al. [67] reported that Acinetobacter sp. has a strong EPS-producing ability along with other multifarious traits. They regarded these bacteria as potential inoculants for improving the seed germination, seedling vigor, root length, and bolls weight of cotton under drought conditions. Leontidou et al. [68] described Chryseobacterium sp. as having multiple plant growth-promoting traits, including IAA production, ACC deaminase activity, and phosphate solubilization. They discovered that these strains contain a number of genes that may be involved in plant growth promotion and stress regulation, and that they can be used to boost crop yields under abiotic stress. Similarly, Liu et al. [69] discovered a gene in Klebsiella sp. that promotes plant growth by producing siderophore, IAA, phosphate solubilization, and N2 fixation. They claimed that these bacteria can grow in a variety of environments and that they can be used as bioinoculants in drought-stricken areas. The improved crop growth potential of selected strains LEW9, LEW16, and LEW3 in this study can be linked to their ability to solubilize zinc and phosphate, as well as their ability to produce EPS and IAA.

5. Conclusions

Drought stress tolerance in crop plants is induced by bacterial exopolysaccharides. Wheat growth was significantly improved when it was inoculated with EPS-producing rhizobacterial strains. In addition to the ability to produce EPS, these strains have different mechanisms such as nutrient solubilization, phytohormones (IAA), and HCN production. Drought tolerance in wheat plants can be induced by a variety of traits. The bacterial strain Acinetobacter sp. (LEW9) was found to be more effective in improving wheat growth physiology, biochemical parameters, and yield under drought stress, and is recommended for use in the development of a potential biofertilizer after a field experiment under natural conditions for inducing drought stress tolerance in wheat under arid to semi-arid agro-ecological conditions.
Future research should be focused on the characterization of more beneficial bacterial strains having the potential to produce exopolysaccharides along with multifarious growth-promoting traits for inducing drought stress tolerance in specific crop plants. Moreover, research should be focused on soil–plant–microbe interactions with the objective of evaluating EPS-producing bacteria under natural conditions and using these microbes in soils with variable physico-chemical properties and different agro-ecological zones.

Author Contributions

Conceptualization, data curation, methodology, formal analysis, writing—original draft, M.L. and S.A.H.B.; project administration, supervision, formal analysis, writing—review and editing, M.A. and A.N.S.; funding acquisition, writing—review and editing, A.A.A., F.S.A., A.Z.D. and M.A.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Vice Deanship of Research Chairs at King Saud University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

This work was financially supported by the Vice Deanship of Research Chairs at King Saud University.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Fahad, S.; Bajwa, A.; Nazir, U.; Anjum, S.A.; Farooq, A.; Zohaib, A.; Sadia, S.; Nasim, W.; Adkins, S.; Saud, S.; et al. Crop Production under Drought and Heat Stress: Plant Responses and Management Options. Front. Plant Sci. 2017, 8, 1147. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Islam, S.M.F.; Karim, Z. World’s Demand for Food and Water: The Consequences of Climate Change. In Desalination—Challenges and Opportunities; IntechOpen: London, UK, 2020. [Google Scholar]
  3. Foley, J.A.; Ramankutty, N.; Brauman, K.A.; Cassidy, E.S.; Gerber, J.S.; Johnston, M.; Mueller, N.D.; O’Connell, C.; Ray, D.K.; West, P.C.; et al. Solutions for a cultivated planet. Nature 2011, 478, 337–342. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Seleiman, M.F.; Al-Suhaibani, N.; Ali, N.; Akmal, M.; Alotaibi, M.; Refay, Y.; Dindaroglu, T.; Abdul-Wajid, H.H.; Battaglia, M.L. Drought Stress Impacts on Plants and Different Approaches to Alleviate Its Adverse Effects. Plants 2021, 10, 259. [Google Scholar] [CrossRef] [PubMed]
  5. Fathi, A.; Tari, D.B. Effect of Drought Stress and its Mechanism in Plants. Int. J. Life Sci. 2016, 10, 1–6. [Google Scholar] [CrossRef] [Green Version]
  6. Chapman, A.; Davies, W.; Downey, C. ADB Climate Risk Country Profile: Azerbaijan. Asian Development Bank. 2021. Available online: http://hdl.handle.net/11540/13785 (accessed on 3 May 2022).
  7. Fleury, D.; Jefferies, S.; Kuchel, H.; Langridge, P. Genetic and genomic tools to improve drought tolerance in wheat. J. Exp. Bot. 2010, 61, 3211–3222. [Google Scholar] [CrossRef] [Green Version]
  8. Panda, D.; Mishra, S.S.; Behera, P.K. Drought Tolerance in Rice: Focus on Recent Mechanisms and Approaches. Rice Sci. 2021, 28, 119–132. [Google Scholar] [CrossRef]
  9. Zargar, S.M.; Mir, R.A.; Ebinezer, L.B.; Masi, A.; Hami, A.; Manzoor, M.; Salgotra, R.K.; Sofi, N.R.; Mushtaq, R.; Rohila, J.S.; et al. Physiological and Multi-Omics Approaches for Explaining Drought Stress Tolerance and Supporting Sustainable Production of Rice. Front. Plant Sci. 2022, 12, 3603. [Google Scholar] [CrossRef]
  10. Lamers, J.; van der Meer, T.; Testerink, C. How Plants Sense and Respond to Stressful Environments. Plant. Physiol. 2020, 182, 1624–1635. [Google Scholar] [CrossRef] [Green Version]
  11. Zia, R.; Nawaz, M.S.; Siddique, M.J.; Hakim, S.; Imran, A. Plant survival under drought stress: Implications, adaptive responses, and integrated rhizosphere management strategy for stress mitigation. Microbiol. Res. 2020, 242, 126626. [Google Scholar] [CrossRef]
  12. Yang, F.; Li, H.; Wang, S.; Zhao, F.; Fang, F.; Guo, J.; Long, M.; Shen, Y. Differences in exopolysaccharides of three microbial aggregates. Environ. Technol. 2021, 1–13. [Google Scholar] [CrossRef]
  13. Nadeem, S.M.; Ahmad, M.; Tufail, M.A.; Asghar, H.N.; Nazli, F.; Zahir, Z.A. Appraising the potential of EPS-producing rhizobacteria with ACC-deaminase activity to improve growth and physiology of maize under drought stress. Physiol. Plant. 2020, 172, 463–476. [Google Scholar] [CrossRef]
  14. Pham, J.V.; Yilma, M.A.; Feliz, A.; Majid, M.T.; Maffetone, N.; Walker, J.R.; Kim, E.; Cho, H.J.; Reynolds, J.M.; Song, M.C.; et al. A Review of the Microbial Production of Bioactive Natural Products and Biologics. Front. Microbiol. 2019, 10, 1404. [Google Scholar] [CrossRef] [Green Version]
  15. Molina-Santiago, C.; de Vicente, A.; Romero, D. Bacterial extracellular matrix as a natural source of biotechnologically multivalent materials. Comput. Struct. Biotechnol. J. 2021, 19, 2796–2805. [Google Scholar] [CrossRef]
  16. Costa, O.Y.A.; Raaijmakers, J.M.; Kuramae, E.E. Microbial Extracellular Polymeric Substances: Ecological Function and Impact on Soil Aggregation. Front. Microbiol. 2018, 9, 1636. [Google Scholar] [CrossRef] [Green Version]
  17. Angelin, J.; Kavitha, M. Exopolysaccharides from probiotic bacteria and their health potential. Int. J. Biol. Macromol. 2020, 162, 853–865. [Google Scholar] [CrossRef]
  18. Meena, K.K.; Sorty, A.M.; Bitla, U.M.; Choudhary, K.; Gupta, P.; Pareek, A.; Singh, D.P.; Prabha, R.; Sahu, P.K.; Gupta, V.K.; et al. Abiotic Stress Responses and Microbe-Mediated Mitigation in Plants: The Omics Strategies. Front. Plant Sci. 2017, 8, 172. [Google Scholar] [CrossRef]
  19. Bibi, A.; Xiong, Y.; Rajoka, M.S.R.; Mehwish, H.M.; Radicetti, E.; Umair, M.; Shoukat, M.; Khan, M.K.I.; Aadil, R.M. Recent Advances in the Production of Exopolysaccharide (EPS) from Lactobacillus spp. and Its Application in the Food Industry: A Review. Sustainability 2021, 13, 12429. [Google Scholar] [CrossRef]
  20. Roberson, E.B.; Firestone, M.K. Relationship between Desiccation and Exopolysaccharide Production in a Soil Pseudomonas sp. Appl. Environ. Microbiol. 1992, 58, 1284–1291. [Google Scholar] [CrossRef] [Green Version]
  21. Nguyen, P.-T.; Nguyen, T.-T.; Bui, D.-C.; Hong, P.-T.; Hoang, Q.-K.; Nguyen, H.-T. Exopolysaccharide production by lactic acid bacteria: The manipulation of environmental stresses for industrial applications. AIMS Microbiol. 2020, 6, 451–469. [Google Scholar] [CrossRef]
  22. Fatima, Z.; Ahmed, M.; Hussain, M.; Abbas, G.; Ul-Allah, S.; Ahmad, S.; Ahmed, N.; Ali, M.A.; Sarwar, G.; Haque, E.U.; et al. The fingerprints of climate warming on cereal crops phenology and adaptation options. Sci. Rep. 2020, 10, 18013. [Google Scholar] [CrossRef]
  23. Marzan, L.W.; Hossain, M.; Mina, S.A.; Akter, Y.; Chowdhury, A.M.M.A. Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint. Egypt J. Aquat. Res. 2017, 43, 65–74. [Google Scholar] [CrossRef]
  24. Ansari, R.A.; Qureshi, A.A.; Ramteke, D.S. Isolation and characterization of heavy-metal resistant microbes from Industrial soil. Int. J. Environ. Sci. 2016, 6, 100–110. [Google Scholar]
  25. Tallgren, A.H.; Airaksinen, U.; von Weissenberg, R.; Ojamo, H.; Kuusisto, J.; Leisola, M. Exopolysaccharide-Producing Bacteria from Sugar Beets. Appl. Environ. Microbiol. 1999, 65, 862–864. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Verhoef, R.; de Waard, P.; Schols, H.A.; Siika-Aho, M.; Voragen, A.G. Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide. Carbohydr. Res. 2003, 338, 1851–1859. [Google Scholar] [CrossRef]
  27. De Vuyst, L.; Vanderveken, F.; van de Ven, S.; Degeest, B. Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in milk medium and evidence for their growth associated biosynthesis. J. Appl. Microbiol. 1998, 84, 1059–1068. [Google Scholar] [CrossRef]
  28. DuBois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 28, 350–356. [Google Scholar] [CrossRef]
  29. Bric, J.M.; Bostock, R.M.; Silverstone, S.E. Rapid In Situ Assay for Indoleacetic Acid Production by Bacteria Immobilized on a Nitrocellulose Membrane. Appl. Environ. Microbiol. 1991, 57, 535–538. [Google Scholar] [CrossRef] [Green Version]
  30. Simons, M.; van der Bij, A.J.; Brand, I.; de Weger, L.A.; Wijffelman, A.C.; Lugtenberg, B.J. Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 1996, 9, 600–607. [Google Scholar] [CrossRef]
  31. Fasim, F.; Ahmed, N.; Parsons, R.; Gadd, G.M. Solubilization of zinc salts by a bacterium isolated from the air environment of a tannery. FEMS Microbiol. Lett. 2002, 213, 1–6. [Google Scholar] [CrossRef]
  32. Pikovskaya, R.I. Mobilization of phosphorus in soil connection with the vital activity of some microbial species. Microbiologiya 1948, 17, 362–370. [Google Scholar]
  33. Lorck, H. Production of Hydrocyanic Acid by Bacteria. Physiol. Plant. 1948, 1, 142–146. [Google Scholar] [CrossRef]
  34. Gairola, K.C.; Nautiyal, A.R.; Dwivedi, A.K. Effect of temperatures and germination media on seed germination of Jatropha Curcas Linn. Adv. Biores. 2011, 2, 66–71. [Google Scholar]
  35. Iqbal, Z.; Ahmad, M.; Jamil, M.; Akhtar, M.F. Appraising the potential of integrated use of Bacillus strains for improving wheat growth. Int. J. Agric. Bot. 2020, 24, 1439–1448. [Google Scholar]
  36. Benson, H.J. Microbiological Application—A Lab Manual Ingeneral Microbiology, 5th ed.; W.C. Brown Publishers: Dubuque, IA, USA, 1990. [Google Scholar]
  37. Hussain, S.; Devers-Lamrani, M.; El Azhari, N.; Martin-Laurent, F. Isolation and characterization of an isoproturon mineralizing Sphingomonas sp. strain SH from a French agricultural soil. Biodegradation 2010, 22, 637–650. [Google Scholar] [CrossRef]
  38. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [Green Version]
  39. Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4, 406–425. [Google Scholar] [CrossRef]
  40. Tamura, K.; Nei, M.; Kumar, S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 2004, 101, 11030–11035. [Google Scholar] [CrossRef] [Green Version]
  41. Steel, R.G.D.; Torrie, J.H.; Dicky, D.A. Principles and Procedures of Statistics: A Biometrical Approach, 3rd ed.; McGraw Hill Book International Co.: Singapore, 1997; pp. 204–227. [Google Scholar]
  42. Khan, A.; Singh, A.V. Multifarious effect of ACC deaminase and EPS producing Pseudomonas sp. and Serratia marcescens to augment drought stress tolerance and nutrient status of wheat. World J. Microbiol. Biotechnol. 2021, 37, 198. [Google Scholar] [CrossRef]
  43. Harris, D.; Tripathi, R.S.; Joshi, A. On-farm seed priming to improve crop establishment and yield in dry direct-seeded rice. In Direct Seeding: Research Strategies and Opportunities; Pandey, S.M., Mortimer, L., Wade, T.P., Tuong, K., Lopes, K., Hardy, B., Eds.; International Research Institute: Manila, Philippines, 2002; pp. 231–240. [Google Scholar]
  44. Blunk, S.; Hoffer, J.; Brosda, S.; De Heer, M.I.; Sturrock, C.J.; Mooney, S.J. Impact of fruit orientation and pelleting material on water uptake and germination performance in artificial substrate for sugar beet. PLoS ONE 2020, 15, e0232875. [Google Scholar] [CrossRef]
  45. Liu, H.; Able, A.J.; Able, J.A. Transgenerational Effects of Water-Deficit and Heat Stress on Germination and Seedling Vigour—New Insights from Durum Wheat microRNAs. Plants 2020, 9, 189. [Google Scholar] [CrossRef] [Green Version]
  46. Smolikova, G.; Leonova, T.; Vashurina, N.; Frolov, A.; Medvedev, S. Desiccation Tolerance as the Basis of Long-Term Seed Viability. Int. J. Mol. Sci. 2020, 22, 101. [Google Scholar] [CrossRef]
  47. Nazli, F.; Wang, X.; Ahmad, M.; Hussain, A.; Bushra; Dar, A.; Nasim, M.; Jamil, M.; Panpluem, N.; Mustafa, A. Efficacy of Indole Acetic Acid and Exopolysaccharides-Producing Bacillus safensis Strain FN13 for Inducing Cd-Stress Tolerance and Plant Growth Promotion in Brassica juncea (L.). Appl. Sci. 2021, 11, 4160. [Google Scholar] [CrossRef]
  48. Primo, E.; Bogino, P.; Cossovich, S.; Foresto, E.; Nievas, F.; Giordano, W. Exopolysaccharide II Is Relevant for the Survival of Sinorhizobium meliloti under Water Deficiency and Salinity Stress. Molecules 2020, 25, 4876. [Google Scholar] [CrossRef]
  49. Morcillo, R.; Manzanera, M. The Effects of Plant-Associated Bacterial Exopolysaccharides on Plant Abiotic Stress Tolerance. Metabolites 2021, 11, 337. [Google Scholar] [CrossRef]
  50. Ali, S.Z.; Sandhya, V.; Rao, L.V. Isolation and characterization of drought-tolerant ACC deaminase and exopolysaccharide-producing fluorescent Pseudomonas sp. Ann. Microbiol. 2013, 64, 493–502. [Google Scholar] [CrossRef] [Green Version]
  51. Etesami, H. Plant–microbe interactions in plants and stress tolerance. In Plant Life Under Changing Environment; Academic Press: Cambridge, CA, USA, 2020; pp. 355–396. [Google Scholar]
  52. Yasmin, H.; Naeem, S.; Bakhtawar, M.; Jabeen, Z.; Nosheen, A.; Naz, R.; Keyani, R.; Mumtaz, S.; Hassan, M.N. Halotolerant rhizobacteria Pseudomonas pseudoalcaligenes and Bacillus subtilis mediate systemic tolerance in hydroponically grown soybean (Glycine max L.) against salinity stress. PLoS ONE 2020, 15, e0231348. [Google Scholar] [CrossRef]
  53. Vijay, R.; Ravichandran, V.; Boominathan, P. Screening of rice apoplast associated endophytic bacterial isolates for moisture stress tolerance and plant growth promoting traits. Madras Agric. J. 2019, 106, 5–11. [Google Scholar]
  54. Ahmad, M.; Zahir, Z.A.; Nazli, F.; Akram, F.; Arshad, M.; Khalid, M. Effectiveness of halo-tolerant, auxin producing Pseudomonas and Rhizobium strains to improve osmotic stress tolerance in mung bean (Vigna radiata L.). Braz. J. Microbiol. 2013, 44, 1341–1348. [Google Scholar] [CrossRef] [Green Version]
  55. Ghosh, D.; Gupta, A.; Mohapatra, S. A comparative analysis of exopolysaccharide and phytohormone secretions by four drought-tolerant rhizobacterial strains and their impact on osmotic-stress mitigation in Arabidopsis thaliana. World J. Microbiol. Biotechnol. 2019, 35, 90. [Google Scholar] [CrossRef]
  56. Abaid-Ullah, M.; Nadeem, M.; Hassan, M.; Ganter, J.; Muhammad, B.; Nawaz, K.; Shah, A.S.; Hafeez, F.Y. Plant growth promoting rhizobacteria: An alternate way to improve yield and quality of wheat (Triticum aestivum). Int. J. Agric. Biol. 2015, 17, 51–60. [Google Scholar]
  57. Intorne, A.C.; de Oliveira, M.V.V.; Lima, M.L.; da Silva, J.F.; Olivares, F.L.; Filho, G.A.D.S. Identification and characterization of Gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc. Arch. Microbiol. 2009, 191, 477–483. [Google Scholar] [CrossRef] [PubMed]
  58. Mumtaz, Z.M.; Ahmad, M.; Jamil, M.; Asad, S.A.; Hafeez, F. Bacillus strains as potential alternate for zinc biofortification of maize grains. Int. J. Agric. Biol. 2020, 20, 1779–1786. [Google Scholar]
  59. Agbodjato, N.A.; Noumavo, P.A.; Baba-Moussa, F.; Salami, H.A.; Sina, H.; Sèzan, A.; Bankolé, H.; Adjanohoun, A.; Baba-Moussa, L. Characterization of Potential Plant Growth Promoting Rhizobacteria Isolated from Maize (Zea mays L.) in Central and Northern Benin (West Africa). Appl. Environ. Soil Sci. 2015, 2015, 901656. [Google Scholar] [CrossRef] [Green Version]
  60. Crowley, D.E. Microbial siderophores in the plant rhizospheric. In Iron Nutrition in Plants and Rhizospheric Microorganisms; Barton, L.L., Abadía, J., Eds.; Springer: Dordrecht, The Netherlands, 2006; pp. 169–198. [Google Scholar]
  61. Hassan, M.; McInroy, J.; Kloepper, J. The Interactions of Rhizodeposits with Plant Growth-Promoting Rhizobacteria in the Rhizosphere: A Review. Agriculture 2019, 9, 142. [Google Scholar] [CrossRef] [Green Version]
  62. Ilyas, N.; Mumtaz, K.; Akhtar, N.; Yasmin, H.; Sayyed, R.; Khan, W.; Enshasy, H.; Dailin, D.; Elsayed, E.; Ali, Z. Exopolysaccharides Producing Bacteria for the Amelioration of Drought Stress in Wheat. Sustainability 2020, 12, 8876. [Google Scholar] [CrossRef]
  63. Lin, Y.; Watts, D.B.; Kloepper, J.W.; Feng, Y.; Torbert, H.A. Influence of Plant Growth-Promoting Rhizobacteria on Corn Growth under Drought Stress. Commun. Soil Sci. Plant. Anal. 2019, 51, 250–264. [Google Scholar] [CrossRef]
  64. Asghari, B.; Khademian, R.; Sedaghati, B. Plant growth promoting rhizobacteria (PGPR) confer drought resistance and stimulate biosynthesis of secondary metabolites in pennyroyal (Mentha pulegium L.) under water shortage condition. Sci. Hortic. 2019, 263, 109132. [Google Scholar] [CrossRef]
  65. Rani, F.M.; Rahman, N.I.A.; Ismail, S.; Alattraqchi, A.G.; Cleary, D.; Clarke, S.C.; Yeo, C.C. Acinetobacter spp. Infections in Malaysia: A Review of Antimicrobial Resistance Trends, Mechanisms and Epidemiology. Front. Microbiol. 2017, 8, 2479. [Google Scholar] [CrossRef] [Green Version]
  66. Kim, H.; Yu, S.M. Complete Genome Sequence of Chryseobacterium sp. Strain NBC 122, a Plant Growth-Promoting Bacterium Isolated from Freshwater. Microbiol. Resour. Announc. 2020, 9. [Google Scholar] [CrossRef] [Green Version]
  67. Sharath, S.; Triveni, S.; Nagaraju, Y.; Latha, P.C.; Vidyasagar, B. The Role of Phyllosphere Bacteria in Improving Cotton Growth and Yield Under Drought Conditions. Front. Agron. 2021, 3, 466. [Google Scholar] [CrossRef]
  68. Leontidou, K.; Genitsaris, S.; Papadopoulou, A.; Kamou, N.; Bosmali, I.; Matsi, T.; Madesis, P.; Vokou, D.; Karamanoli, K.; Mellidou, I. Plant growth promoting rhizobacteria isolated from halophytes and drought-tolerant plants: Genomic characterisation and exploration of phyto-beneficial traits. Sci. Rep. 2020, 10, 14857. [Google Scholar] [CrossRef] [PubMed]
  69. Liu, W.; Wang, Q.; Hou, J.; Tu, C.; Luo, Y.; Christie, P. Whole genome analysis of halotolerant and alkalotolerant plant growth-promoting rhizobacterium Klebsiella sp. D5A. Sci. Rep. 2016, 6, 26710. [Google Scholar] [CrossRef] [PubMed]
Agronomy 12 01140 g001 550
Figure 1. Effect of different levels of PEG-6000 on growth of EPS- and IAA-producing drought-tolerant rhizobacterial isolates in liquid culture after 72 h of incubation. Bars sharing same letter(s) do not differ from one another (p ≤ 0.05).
Figure 1. Effect of different levels of PEG-6000 on growth of EPS- and IAA-producing drought-tolerant rhizobacterial isolates in liquid culture after 72 h of incubation. Bars sharing same letter(s) do not differ from one another (p ≤ 0.05).
Agronomy 12 01140 g001
Agronomy 12 01140 g002 550
Figure 2. Screening of twenty wheat varieties grown at different drought levels. Germination percentage and seedling growth parameters were measured and plotted in Principal Component Analyses (PCA). (a) 0% PEG-6000; (b) 2% PEG-6000; (c) 4% PEG-6000; and (d) 6% PEG-6000.
Figure 2. Screening of twenty wheat varieties grown at different drought levels. Germination percentage and seedling growth parameters were measured and plotted in Principal Component Analyses (PCA). (a) 0% PEG-6000; (b) 2% PEG-6000; (c) 4% PEG-6000; and (d) 6% PEG-6000.
Agronomy 12 01140 g002
Agronomy 12 01140 g003 550
Figure 3. Efficiency of exopolysaccharide-producing, drought-tolerant rhizobacterial strains to improve seed germination, root development, root diameter; root surface area and root colonization; V1: Johar-16; V2: Gold-16.
Figure 3. Efficiency of exopolysaccharide-producing, drought-tolerant rhizobacterial strains to improve seed germination, root development, root diameter; root surface area and root colonization; V1: Johar-16; V2: Gold-16.
Agronomy 12 01140 g003
Agronomy 12 01140 g004 550
Figure 4. Efficiency of exopolysaccharide-producing, drought-tolerant rhizobacterial strains to improve root length, shoot length, seedling vigor index and total dry biomass; V1: Johar-16; V2: Gold-16.
Figure 4. Efficiency of exopolysaccharide-producing, drought-tolerant rhizobacterial strains to improve root length, shoot length, seedling vigor index and total dry biomass; V1: Johar-16; V2: Gold-16.
Agronomy 12 01140 g004
Agronomy 12 01140 g005 550
Figure 5. Neighbor-joining phylogenetic tree of Chryseobacterium sp. (LEW3), Acinetobacter sp. (LEW3), and Klebsiella sp. (LEW16) constructed by closely related taxa.
Figure 5. Neighbor-joining phylogenetic tree of Chryseobacterium sp. (LEW3), Acinetobacter sp. (LEW3), and Klebsiella sp. (LEW16) constructed by closely related taxa.
Agronomy 12 01140 g005
Table
Table 1. Growth in terms of optical density (OD600) and EPS production ability of rhizobacterial isolates in RCV medium.
Table 1. Growth in terms of optical density (OD600) and EPS production ability of rhizobacterial isolates in RCV medium.
Rhizobacterial IsolateProduction of EPSGrowth (OD600)Rhizobacterial IsolateProduction of EPSGrowth (OD600)
LEW1−0.95 ± 0.02LEW16 *+1.72 ± 0.04
LEW2−0.89 ± 0.02LEW17−0.89 ± 0.03
LEW3 *+1.62 ± 0.02LEW18−0.96 ± 0.03
LEW4 *+0.69 ± 0.02LEW19 *+0.81 ± 0.03
LEW5−0.76 ± 0.02LEW20−0.76 ± 0.02
LEW6−0.83 ± 0.04LEW21 *+0.74 ± 0.02
LEW7−0.94 ± 0.04LEW22−0.86 ± 0.04
LEW8−0.97 ± 0.03LEW23 *+0.94 ± 0.03
LEW9 *+1.36 ± 0.03LEW24−0.87 ± 0.03
LEW10−0.78 ± 0.02LEW25−0.77 ± 0.02
LEW11 *+0.86 ± 0.04LEW26 *+0.85 ± 0.03
LEW12−1.05 ± 0.03LEW27−0.74 ± 0.03
LEW13−0.67 ± 0.02LEW28 *+1.27 ± 0.06
LEW14−0.75 ± 0.04LEW29−0.89 ± 0.02
LEW15−0.93 ± 0.03LEW30−0.73 ± 0.04
* Isolates that have more growth in terms of OD600 and produced more EPS were selected for further experimentation (Results for production of EPS and growth behavior were confirmed by repeating the bioassays in three replications). The sign (+) expresses the occurrence of the tested attributes and the sign (−) denotes the lack of the tested trait.
Table
Table 2. Quantitative EPS production and IAA production in the presence and absence of L-tryptophan by rhizobacterial isolates.
Table 2. Quantitative EPS production and IAA production in the presence and absence of L-tryptophan by rhizobacterial isolates.
Rhizobacterial IsolateEPS Production
(µg mL−1)		IAA Production
Without L-TrypWith L-Tryp
LEW3 *72.4 ± 0.373.82 ± 0.11511.57 ± 1.213
LEW445.4 ± 0.48NDND
LEW9 *63.5 ± 0.562.76 ± 0.09512.71 ± 1.271
LEW1157.8 ± 0.382.71 ± 0.0626.15 ± 0.215
LEW16 *81.3 ± 0.848.36 ± 0.13710.29 ± 1.138
LEW1929.8 ± 0.651.84 ± 0.1254.17 ± 0.218
LEW2132.4 ± 0.61NDND
LEW2310.8 ± 0.571.79 ± 0.1363.56 ± 0.219
LEW2642.3 ± 0.731.52 ± 0.0735.14 ± 0.251
LEW28 *86.2 ± 0.984.74 ± 0.10613.17 ± 1.236
Values are mean of three replications ± SE. ND—production of IAA was not detected. * Isolates produced higher amount of IAA and were selected for further experimentation.
Table
Table 3. Effect of different levels of PEG-6000 on growth, IAA, and EPS production by the rhizobacterial strains.
Table 3. Effect of different levels of PEG-6000 on growth, IAA, and EPS production by the rhizobacterial strains.
Strains OD600 for Growth
24 h 48 h 72 h
03%6%9%03%6%9%03%6%9%
LEW30.66 bc0.55 de0.44 f0.42 f0.88 ab
0.95 a		0.73 de0.67 e0.53 g0.98 b–d0.85 e–g0.76 gh0.63 i
LEW90.74 ab0.65 dc0.51 ef0.46 ef0.79 b–d0.72 de0.54 g1.22 a1.03 bc0.81 fg0.66 hi
LEW160.62 cd0.51 ef0.46 ef0.42 f0.84 a–c0.70 de0.66 ef0.56 fg1.07 b0.91 d–f0.76 gh0.60 i
LEW280.78 a0.65 bc0.42 f0.42 f0.93 a0.76 c–e0.67 ef0.55 g0.94 c–e1.00 b–d0.79 g0.61 i
HSD
(p ≤ 0.05)		0.0951 0.1056 0.1067
EPS Production
Strains24 h 48 h 72 h
03%6%9%03%6%9%03%6%9%
LEW368.3 b–d64.3 c–e49.3 gh34.0 j85.7 ab73.0 c–e55.7 gh43.3 i101.7 c81.3 ef65.7 hi50.7 k
LEW977.7 a71.7 a–c54.7 fg44.3 hi94.0 a78.3 b–d64.7 e–g52.3 hi122.0 a107.3 bc79.7 fg60.3 ij
LEW1666.0 b–e59.3 ef49.7 gh36.3 j82.0 bc71.7 d–f61.7 g46.0 i99.0 cd90.7 de71.3 gh52.3 jk
LEW2872.0 ab61.3 d–f51.3 gh41.3 ij86.7 ab72.3 d–f63.3 fg49.7 hi112.0 b102.7 bc75.3 fg56.3 i–k
HSD
(p ≤ 0.05)		7.65 9.15 9.51
IAA Production
Strains24 h 48 h 72 h
03%6%9%03%6%9%03%6%9%
LEW317.3 c15.7 cd7.67 f1.43 g36.3 bc34.0 cd19.3 fg2.37 h47.7 a–d46.7 b–d24.0 f4.40 g
LEW924.7 a22.3 ab13.3 de2.33 g44.3 a40.3 ab23.3 f3.33 h53.7 a52.7 ab31.3 e6.97 g
LEW1619.3 bc18.7 bc10.0 ef1.60 g34.3 cd28.3 e17.7 g2.17 h45.3 cd44.7 d25.3 ef4.23 g
LEW2822.0 ab21.3 ab13.3 de1.80 g39.7 ab31.3 de20.3 fg2.87 h51.3 a–c49.3 a–d29.7 ef5.93 g
HSD
(p ≤ 0.05)		3.73 4.81 6.15
Data present as mean of three replicates. Means followed by same letter(s) do not differ from one another (p ≤ 0.05).
Table
Table 4. Plant growth-promoting attributes and MIC of PEG-6000 for selected rhizobacterial strains.
Table 4. Plant growth-promoting attributes and MIC of PEG-6000 for selected rhizobacterial strains.
CharacteristicLEW3LEW9LEW16LEW28
Root colonization (CFU cm−2)3.52 × 1064.16 × 1062.91 × 1062.34 × 106
Phosphate solubilization++++++
Zinc solubilization+++++++
Hydrogen cyanide production++−+
The symbols (+) and (++) represent the presence of traits, and the symbol (−) represents the absence of traits.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Latif, M.; Bukhari, S.A.H.; Alrajhi, A.A.; Alotaibi, F.S.; Ahmad, M.; Shahzad, A.N.; Dewidar, A.Z.; Mattar, M.A. Inducing Drought Tolerance in Wheat through Exopolysaccharide-Producing Rhizobacteria. Agronomy 2022, 12, 1140. https://doi.org/10.3390/agronomy12051140

AMA Style

Latif M, Bukhari SAH, Alrajhi AA, Alotaibi FS, Ahmad M, Shahzad AN, Dewidar AZ, Mattar MA. Inducing Drought Tolerance in Wheat through Exopolysaccharide-Producing Rhizobacteria. Agronomy. 2022; 12(5):1140. https://doi.org/10.3390/agronomy12051140

Chicago/Turabian Style

Latif, Muhammad, Syed Asad Hussain Bukhari, Abdullah A. Alrajhi, Fahad S. Alotaibi, Maqshoof Ahmad, Ahmad Naeem Shahzad, Ahmed Z. Dewidar, and Mohamed A. Mattar. 2022. "Inducing Drought Tolerance in Wheat through Exopolysaccharide-Producing Rhizobacteria" Agronomy 12, no. 5: 1140. https://doi.org/10.3390/agronomy12051140

APA Style

Latif, M., Bukhari, S. A. H., Alrajhi, A. A., Alotaibi, F. S., Ahmad, M., Shahzad, A. N., Dewidar, A. Z., & Mattar, M. A. (2022). Inducing Drought Tolerance in Wheat through Exopolysaccharide-Producing Rhizobacteria. Agronomy, 12(5), 1140. https://doi.org/10.3390/agronomy12051140

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop