*2.3.1. Accumulation of osmolytes*

**2.3. Production of plant hormones and other beneficial plant metabolite**

124 Wheat Improvement, Management and Utilization

There are five groups of plant hormones of well-known PGRs, namely auxins, gibberellins, cytokinins, ethylene and abscisic acid [60]. Direct plant growth promotion includes symbiotic and non-symbiotic PGPR, which functions through production of these plant hormones [11, 61–63]. Much attention has been given on the role of phytohormone auxin. Production of indole-3-ethanol or indole-3-acetic acid (IAA), the compounds belonging to auxins, which is known to stimulate in cell elongation, division and differentiation responses in plants, has been reported for several bacterial genera [12, 17, 64]. PGPR promote root growth by increasing root surface area, which, in turn, promotes nutrient uptake, thereby indirectly stimulating plant growth positively [52, 65]. Khalid et al. reported a correlation between *in vitro* auxin

production and increase in early growth parameters of inoculated wheat seeds [66].

reported [75–78].

such as temperature, pH and oxygen [79].

Inoculation with *A. brasilense* Cd and the application of pure IAA to the roots both increased root length, number of lateral roots and number of root hairs in wheat as observed by earlier workers [67, 68]. IAA-producing *Azospirillum* sp. also promoted alterations in the growth and development of wheat (*Triticum aestivum* L.) plants [69–72]. Bacteria of the *Azotobacter* genus synthesize auxins, cytokinins and GA-like substances, and these growth materials are the primary substances controlling the enhanced growth [73]. These hormonal substances, which originate from the rhizosphere or root surface, affect the growth of the closely associated higher plants. The highest concentration of IAA is produced by bacterial strain *P. fluorescens* and *Kocuria* varians [74]. Specifically for wheat, the positive effect of PGPR via IAA has been

When applied in optimum concentrations, bacterial indole-3-acetic acid (IAA), synthesized by gram-positive and -negative, photosynthetic, methylotrophic and cyanobacteria, is reported to stimulate root hair formation, at the same time increasing the length and the number of primary and lateral roots [66, 72, 79]. IAA synthesis by these bacteria is reported to be affected by tryptophan, vitamins, salt and oxygen levels, as well as pH, temperature, carbon and nitrogen source. For example, IAA from *Azospirillum brasilense* Sp245 stimulates early plant development and increases significantly the plants and roots yield (in dry weight) and the N-uptake efficiency of wheat [71, 80]. The ability to synthesize ABA, particularly under stressful conditions, for example, salinity, and to affect the ABA level in plants was detected in PGPB from the genera *Azospirillum, Bacillus, Pseudomonas, Brevibacterium* and *Lysinibacillus* [15, 81, 82]. Both plants and bacteria can be synthesized via several pathways, including the indole-3-pyruvic acid (IPA), indole-3-acetamide (IAM) and indole-3-acetonitrile (IAN) pathways, which are often regulated by tryptophan, carbon and nitrogen availability, a reduction in growth rate and abiotic factors

As a PGPR application to wheat seedlings, Sachdev et al. reported that IAA producing *Klebsiella* strains significantly increased the root length and shoot height, when compared with the control, in pot experiments [83, 84]. Similarly, Khalid et al. reported up to 28% higher grain yields in wheat grown in field as a result of seed inoculation in peats with high auxinproducing rhizobacteria [66]. The capability of auxin synthesis detected in many bacterial strains from the genera *Azospirillium, Pseudomonas, Bacillus*, etc., is thought to underlie the activation of plant root growth by these microorganisms [81]. Sadeghi et al. demonstrated Proline is a known osmoprotectant, promoting the protection of the plant from drought, salt and other stresses [94]. Alternative to proline accumulation, another defence strategy is to increase total soluble sugar level in plants under salinity stress. PGPRs have been demonstrated to enhance wheat stress tolerance via osmolyte accumulation as reported in Refs. [95–97]. Ali et al. used *P. putida* AKMP7 resulting in significant increase in proline levels in heat-stressed wheat plants [98].

Yegorenkova et al. suggested that lectin-carbohydrate interactions are involved in the initial stages of bacterial-plant root attachment [99]. Additionally, PGPR producing extracellular polymeric substance are reported to enhance greatly the soil volume macropores and the rhizosphere aggregation of soil, which results in increased water and fertiliser availability to plants [46].

### **2.4. Siderophore and exopolysaccharide production by PGPR**

With its unique physico-chemical properties, iron (Fe) has a key role in plant growth, taking part in several metabolic pathways including TCA cycle, nitrogen fixation, respiration and ETC, oxidative phosphorylation and photosynthesis, biosynthetic regulation (chlorophyll, toxin, vitamins, antibiotic, cytochrome and pigment) and as cofactor for numerous enzymes [100]. Following this, iron deficiency (typically caused by low iron bioavailability) is frequently seen at elevated pH, alkali soils in dry regions, as well as in case of excessive fertilizer and pesticides application.

Siderophores are small iron carriers, chemically high-affinity iron chelating compounds secreted by PGPRs and are among the strongest soluble Fe3+ binding agents known. Comprehensive information on the role of siderophores in increasing iron oxide solubility and promoting dissolution in soils requires the consideration of the rates of various processes such as siderophore exudation, the uptake, and the degradation rates [101]. In BNF, siderophores are expected to play significant role, since in its very essence, nitrogenase requires Fe [102], also supported by a high correlation between N and Fe uptake.

Siderophore productions promote the crop growth, or protect the plant against pathogens. Produced by microorganisms, these are found in soil solutions and influence Fe nutrition of plants [103]. The role of siderophores has been reported as signalling molecules and as such, their use points to avenues for novel agricultural applications [54].

The wheat seed inoculation was tested for their effect on wheat in terms of healthier germination and productivity. The organisms used were siderophoregenic pyoverdin-producing *Pseudomonas putida* and *Pseudomonas aeruginosa* strains from two diverse habitats. Inoculation with siderophoregenic PGPR increased percentage germination, shoot height, shoot and root length, weight of spikelets, chlorophyll content, grain yield and iron content [100, 104, 105]. Inoculated wheat plants showed increase in total iron uptake and physiologically available iron contents. *Acinetobacter calcoaceticus* obtained from wheat rhizosphere produces catechol type of siderophores during exponential phase, which is influenced by iron content of medium [106]. Ca, Cd and Mg ions and succinic acid stimulated the synthesis of the siderophore examined, whereas Zn and Pb ions partially decreased its level.

Some PGPR strains may also protect plants from salt and drought stress by producing exopolysaccharides (EPS), binding, in turn, Na+ or by biofilm formation [107]. Resultingly, reduced Na+ results in lower Na+ uptake and high K+/Na+ ratio, promoting survival in salt-stressed conditions [107, 108]. Another example is the wheat seedling inoculation by EPS producing strain of *Pantoea agglomerans* (NAS206) isolated from the wheat rhizosphere, growing in a Moroccan vertisol. It had a positive effect on aggregation and stabilization of root-adhering soil, by increased mean aggregate diameter and macroporosity [109].

#### **2.5. PGPR and plant nutrient uptake**

Seed inoculation with the bacterium has been found to improve the growth and nutrient uptake of wheat seedlings via promotion of the plant growth and increased root surface area or the general root architecture [110]. With enlarged root hairs, nutrient uptake is promoted [21, 71, 77, 111].

The PGPR effects also increase N and P uptake in field trials [112], presumably, by stimulating greater plant root growth. Both *A. chroococcum* and *P. agglomerans* were found to increase plant growth, plant dry matter, as well as N and P uptake [25, 113]. *Azospirillum*-inoculated plants under drought conditions had increased Mg, K and Ca contents compared to non-inoculated plants [62, 114–117]. The increase in nutrient accumulation/uptake due to biofertilizers/ PGPR was previously reported in wheat [118–120]. Sharma et al. reported that the majority of 13 tested *Pseudomonas* spp. strains increased the macro (N, P, K and S) and micronutrients uptake (Cu, Fe, Zn and Mn) in wheat [102, 121].

Inoculation of efficient plant-growth-promoting actinobacterial *Streptomyces* species significantly improved the Fe, Mn and P content of wheat plants when compared with an uninoculated control [43, 105]. Yasin et al. investigated the effects of selenate fertilization and bacterial inoculation on Se uptake and plant growth [122]. They found that *Bacillus pichinotyi* enhanced wheat growth, dry weight, shoot length and spike length, Se and Fe concentration in wheat kernels and stems. Selenium (Se) is an essential trace element for humans [123], and they reported that inoculation with rhizospheric microorganisms significantly enhanced wheat Se content.

### **2.6. Alleviation of abiotic stress in wheat by PGPR**

Abiotic stress is the major cause of decreasing crop productivity worldwide. The application of the combination of PGPR and mycorrhizal fungi alleviates the stress conditions, as reported by Nadeem et al., via the regulation of hormones, nutrition uptake and growth [124]. Similar outcomes have been reported by Cakmakci et al. for wheat and spinach plants [77]. Enzymatic activities in the leaves of these plants such as glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and glutathione S-transferase have been observed.

Additionally, numerous studies suggested that both IAA and ACC deaminase-producing bacteria protect plants most effectively, against a wide range of different stresses [125]. Notable reports among those are *Azospirillum* strains helping to cope with salt stress [126–128] and *Bacillus* and *Azospirillum* leading to improve heat tolerance in wheat [129].

#### *2.6.1. Drought*

Siderophore productions promote the crop growth, or protect the plant against pathogens. Produced by microorganisms, these are found in soil solutions and influence Fe nutrition of plants [103]. The role of siderophores has been reported as signalling molecules and as such,

The wheat seed inoculation was tested for their effect on wheat in terms of healthier germination and productivity. The organisms used were siderophoregenic pyoverdin-producing *Pseudomonas putida* and *Pseudomonas aeruginosa* strains from two diverse habitats. Inoculation with siderophoregenic PGPR increased percentage germination, shoot height, shoot and root length, weight of spikelets, chlorophyll content, grain yield and iron content [100, 104, 105]. Inoculated wheat plants showed increase in total iron uptake and physiologically available iron contents. *Acinetobacter calcoaceticus* obtained from wheat rhizosphere produces catechol type of siderophores during exponential phase, which is influenced by iron content of medium [106]. Ca, Cd and Mg ions and succinic acid stimulated the synthesis of the sidero-

Some PGPR strains may also protect plants from salt and drought stress by producing exopolysaccharides (EPS), binding, in turn, Na+ or by biofilm formation [107]. Resultingly, reduced Na+ results in lower Na+ uptake and high K+/Na+ ratio, promoting survival in salt-stressed conditions [107, 108]. Another example is the wheat seedling inoculation by EPS producing strain of *Pantoea agglomerans* (NAS206) isolated from the wheat rhizosphere, growing in a Moroccan vertisol. It had a positive effect on aggregation and stabilization of root-adhering

Seed inoculation with the bacterium has been found to improve the growth and nutrient uptake of wheat seedlings via promotion of the plant growth and increased root surface area or the general root architecture [110]. With enlarged root hairs, nutrient uptake is promoted

The PGPR effects also increase N and P uptake in field trials [112], presumably, by stimulating greater plant root growth. Both *A. chroococcum* and *P. agglomerans* were found to increase plant growth, plant dry matter, as well as N and P uptake [25, 113]. *Azospirillum*-inoculated plants under drought conditions had increased Mg, K and Ca contents compared to non-inoculated plants [62, 114–117]. The increase in nutrient accumulation/uptake due to biofertilizers/ PGPR was previously reported in wheat [118–120]. Sharma et al. reported that the majority of 13 tested *Pseudomonas* spp. strains increased the macro (N, P, K and S) and micronutrients

Inoculation of efficient plant-growth-promoting actinobacterial *Streptomyces* species significantly improved the Fe, Mn and P content of wheat plants when compared with an uninoculated control [43, 105]. Yasin et al. investigated the effects of selenate fertilization and bacterial inoculation on Se uptake and plant growth [122]. They found that *Bacillus pichinotyi* enhanced wheat growth, dry weight, shoot length and spike length, Se and Fe concentration in wheat kernels and stems. Selenium (Se) is an essential trace element for humans [123], and

their use points to avenues for novel agricultural applications [54].

phore examined, whereas Zn and Pb ions partially decreased its level.

soil, by increased mean aggregate diameter and macroporosity [109].

**2.5. PGPR and plant nutrient uptake**

126 Wheat Improvement, Management and Utilization

uptake (Cu, Fe, Zn and Mn) in wheat [102, 121].

[21, 71, 77, 111].

Drought stress, exhibited as limited water supply, usually causes a severe loss in plant yield, where the combination of severity and duration are critical factors for plant survival [130]. The application of PGPR can counteract damaging effects of moisture stress, and therefore boost crop yields. Creus et al. reported that growing *Azospirillum brasilense* Sp245-primed wheat under drought stress conditions resulted in large increase in water content and potential, and apoplastic water function in both shoots and roots compared to the non-primed plants [62].

Moreover, Pereyra et al. reported that *Azospirillum* inoculation provided a better water status in wheat seedlings under osmotic stress due to morphological modifications of the coleoptile xylem architecture [131]. *Azospirillum*-inoculated wheat seedlings subjected to osmotic stress developed significant higher coleoptiles, with higher fresh weight and better water status than non-inoculated seedlings [132]. In this regard, ABA-producing bacteria *Azospirillum* promoted resistance of *Arabidopsis*, maize and wheat plants to soil drought [81]. *Azospirillum brasilense* INTA Az-39-inoculated wheat plants under typical dry land farming conditions exhibited better growth and increased vegetative growth, shoot and root dry matter accumulation, grain number and grain yield [133]. According to Arzanesh et al. results, inoculation of wheat with *Azospirillum* spp. can alleviate drought stress on plant growth and yield through adjusting plant water characters [134].

Inoculation of wheat with *Burkholderia phytofirmans* PsJN significantly diluted the adverse effects of drought on relative water contents and CO<sup>2</sup> assimilation rate, thus improving the photosynthetic rate, water use efficiency and chlorophyll content over the uninoculated control [135]. In a similar study conducted on wheat under water stress environment showed that mycorrhizal inoculation enhanced the activities of antioxidant enzymes such as peroxidase and catalase compared to those in uninoculated control plants [136]. Several other studies report similar outcomes [137].

#### *2.6.2. Salinity*

Salinity decreases the yield of many crops because salt inhibits plant photosynthesis, protein synthesis and lipid metabolism. Nutrient contents decrease in the roots and shoots with increasing NaCl concentration in the growth medium. PGPR counteract osmotic stress and help plant growth. Investigations on interaction of PGPR with other microbes and their effect on the physiological response of crop plants under different soil salinity regimes are still in incipient stage.

Rhizobacteria that are residing within the rhizosphere of plants growing in saline habitats may have already been adapted to salt stress that may be a valuable resource to develop crop inoculants. Raheem and Ali isolated rhizobacteria that were producing beneficial plant growth-promoting metabolites such as IAA and ACC-deaminase activity [138]. The isolation of indigenous microorganisms from the stress-affected soils and screening on the basis of their stress tolerance and PGP traits may be useful in the rapid selection of efficient strains that could be used as bio-inoculants for stressed crops [139, 140]. For several durum cultivars, PGPR efficacy in mitigating salt stress in tetraploid wheat is salt level and bacterial strainspecific [128, 141, 142]. There are some instances of ameliorating salt-stricken cereal crops by PGPR's. Salinity stress in the wheat was alleviated by inoculations with four strains of PGPR, *Pseudomonas fluorescens* 153, 169, *Pseudomonas putida* 108 and 4 [143]. Upadhyay et al. considered the impact of PGPR inoculation on the growth and antioxidant of wheat under saline conditions [46]. In a follow-up study, Upadhyay et al. investigated the effects of two salt-tolerant PGPR (*B. subtilis* and *Arthobacter* sp.) on wheat plants under different salinity regimes and the results obtained demonstrated alleviation of the salinity stress effects on plants treated with bacteria [97]. Similar outcome has been reported by Nia et al. for *Azospirillum* strains on wheat plants [144]. Several PGPR of the genus *Pseudomonas* contain ACC-deaminase enzyme, and when inoculated into plant roots may sustain plant growth under salinity [125, 142].

#### *2.6.3. Mitigation of cold stress in wheat by PGPR*

The over-wintering ability of PGPR is fundamental when considering uses in colder climates. De Freitas and Germida reported that *Pseudomonas* species are able to over-winter in sufficient quantities on the roots of winter wheat [145]. It has also been argued that antifreeze protein activity of many bacterial species may contribute to their survival in colder climates [146–148].

The effect of inoculation with 12 psychrotolerant *Pseudomona* strains on cold alleviation and growth of wheat seedling at cold temperature was investigated in Ref. [105]. Psychrotolerant PGPR inoculation improved metabolite levels, such as chlorophyll, anthocyanin, free proline, total phenolics, starch content, physiologically available iron, proteins and amino acids that are sign of alleviation of cold stress in wheat plants.

Higher chlorophyll content in leaves of cold acclimated winter wheat over control plants was also reported [105]. Proline is a dominant amino acid that accumulates in many organisms upon exposure to environmental stress and plays multiple roles in plant adaptation to stress. Also increased proline content in wheat plant at low temperature with the bacterial inoculation is an indication to chilling tolerance [105].

Turan et al. conducted greenhouse experiments in wheat and barley under cold stress conditions to determine the growth, freezing injury, antioxidant enzyme activity effect of four different rhizobacteria and boron [149]. The authors showed that boron+PGPR treatments have positive effect on root and shoot growth, H<sup>2</sup> O2 , and SOD, POD and CAT antioxidant enzyme activities of wheat and barley plants under cold and control conditions. This suggests that the PGPB application can ameliorate the deleterious effects of cold stress by increasing chlorophyll content, photosynthetic activity and relative water content, altering mineral uptake, and decreasing membrane damage, increasing cold tolerance in wheat and barley.

#### *2.6.4. Metal stress tolerance in wheat*

*2.6.2. Salinity*

128 Wheat Improvement, Management and Utilization

incipient stage.

[146–148].

*2.6.3. Mitigation of cold stress in wheat by PGPR*

are sign of alleviation of cold stress in wheat plants.

tion is an indication to chilling tolerance [105].

Salinity decreases the yield of many crops because salt inhibits plant photosynthesis, protein synthesis and lipid metabolism. Nutrient contents decrease in the roots and shoots with increasing NaCl concentration in the growth medium. PGPR counteract osmotic stress and help plant growth. Investigations on interaction of PGPR with other microbes and their effect on the physiological response of crop plants under different soil salinity regimes are still in

Rhizobacteria that are residing within the rhizosphere of plants growing in saline habitats may have already been adapted to salt stress that may be a valuable resource to develop crop inoculants. Raheem and Ali isolated rhizobacteria that were producing beneficial plant growth-promoting metabolites such as IAA and ACC-deaminase activity [138]. The isolation of indigenous microorganisms from the stress-affected soils and screening on the basis of their stress tolerance and PGP traits may be useful in the rapid selection of efficient strains that could be used as bio-inoculants for stressed crops [139, 140]. For several durum cultivars, PGPR efficacy in mitigating salt stress in tetraploid wheat is salt level and bacterial strainspecific [128, 141, 142]. There are some instances of ameliorating salt-stricken cereal crops by PGPR's. Salinity stress in the wheat was alleviated by inoculations with four strains of PGPR, *Pseudomonas fluorescens* 153, 169, *Pseudomonas putida* 108 and 4 [143]. Upadhyay et al. considered the impact of PGPR inoculation on the growth and antioxidant of wheat under saline conditions [46]. In a follow-up study, Upadhyay et al. investigated the effects of two salt-tolerant PGPR (*B. subtilis* and *Arthobacter* sp.) on wheat plants under different salinity regimes and the results obtained demonstrated alleviation of the salinity stress effects on plants treated with bacteria [97]. Similar outcome has been reported by Nia et al. for *Azospirillum* strains on wheat plants [144]. Several PGPR of the genus *Pseudomonas* contain ACC-deaminase enzyme, and when inoculated into plant roots may sustain plant growth under salinity [125, 142].

The over-wintering ability of PGPR is fundamental when considering uses in colder climates. De Freitas and Germida reported that *Pseudomonas* species are able to over-winter in sufficient quantities on the roots of winter wheat [145]. It has also been argued that antifreeze protein activity of many bacterial species may contribute to their survival in colder climates

The effect of inoculation with 12 psychrotolerant *Pseudomona* strains on cold alleviation and growth of wheat seedling at cold temperature was investigated in Ref. [105]. Psychrotolerant PGPR inoculation improved metabolite levels, such as chlorophyll, anthocyanin, free proline, total phenolics, starch content, physiologically available iron, proteins and amino acids that

Higher chlorophyll content in leaves of cold acclimated winter wheat over control plants was also reported [105]. Proline is a dominant amino acid that accumulates in many organisms upon exposure to environmental stress and plays multiple roles in plant adaptation to stress. Also increased proline content in wheat plant at low temperature with the bacterial inoculaPlant growth-promoting bacteria are able to also grow in heavy metal-contaminated environment and protect plants against heavy metals toxicity in contaminated soils [150, 151]. Hasnain and Sabri reported that upon *Pseudomonas* sp. inoculation of wheat in Pakistan, growth was stimulated, less toxic ions were taken up and increased auxin content was observed [152].

Under Cr stress conditions, Shahzadi et al. reported root length, shoot length, root dry weight and shoot dry weight, respectively, as compared to uninoculated control plants upon inoculation of wheat seeds with *Pseudomonas fluorescens* Q14 and *Bacillus thuringiensis* KAP5 [153]. In this context, ACC-deaminase producing PGPR could play vital role in improving the plant growth under metal-stress condition and they may enhance bioremediation process in Cr-contaminated environment. Similarly, Jamali et al. studied the relationship of bacterial Cr mobilization in soil with total Cr accumulation in wheat [154]. Hassan et al. reported that inoculation with PGPR decreased the deleterious effects of cadmium pollution by chelating and influencing its bioavailability and increased the wheat growth [155]. Singh et al. found that PGPR having ACC-deaminase activity were resistant against Cd, Cr, Pb and Cu toxicity, and increased the wheat and pigeon pea growth [156]. Consequently, uses of rhizospheric microorganisms are generally considered as safe, cost effective and reliable technique for elimination of heavy metals from environmental compartments [150, 157, 158]. Govindasamy et al. observed that growth-promoting ability of rhizoacteria containing ACC deaminase in wheat seedlings through modulation of stress ethylene synthesis enhanced root elongation significantly and minimized ethylene synthesis in wheat seedlings under induced cadmium stress condition [159].

#### **2.7. Improve yield and quality of wheat**

Beneficial rhizobacteria associated with cereals has increased recently and several studies clearly demonstrated the positive and beneficial effects of PGPR on growth and yield of wheat at different environment under variable ecological conditions (Turan et al., 2010).

Zn solubilizing rhizobacteria significantly influenced the growth, yield and Zn concentration of wheat grain over uninoculated control and Zn fertilizer [160, 161]. Similarly, increased nutrient concentrations in wheat due to inoculation were reported in Refs. [5, 118, 162–165]. It is pointed out by Mäder et al. that microbial inoculants have been shown to be a valid option for sustainable high quality wheat production in low-input areas, promising to improve the nutritional status and health of the rural population [163]. In a survey of 20 years of experiments, Okon and Labandera-Gonzalez reported that 60–70% of the experiments showed yield increases due to inoculation, with statistically significant increases in yield from 5 to 30% [31].

*Pseudomonas* strains significantly increased grain yield of wheat [23, 49, 143, 166]. Similarly, Shaharoona et al. reported that N use efficiency increased in response to inoculation with *Pseudomonas fluorescens* at all fertilizer levels in wheat [167]. PGPR isolates significantly increased shoot and root length, shoot and root dry weight, grain weight per spike, shoot and root N content and also enhanced the N contents of inoculated wheat seedlings [168]. Barneix et al. reported that inoculation of wheat with *Bacillus simplex* and *Bacillus firmis* resulted in consistent increase in dry matter and wheat grain quality. A number of other *Bacillus* spp. isolated from wheat rhizosphere have also been investigated for their growth-promoting property in wheat having similar effects on dry weight [10, 40, 169], the latter focusing on isolating and characterizing PGPRs. Trials with rhizosphere-associated plant growth-promoting N2 -fixing and P-solubilising *Bacillus* and other species indicated yield increases in many crops such as wheat [43, 51, 170, 171]. In wheat, several rhizobacteria have been reported as improving grain yield, grain protein concentration or both [3, 135, 140, 164, 172].
