Roles of Rhizobacteria in Cereals

Abstract

The rhizosphere is a dynamic ecosystem consisting of a plethora of microorganisms. The rhizosphere microbiome plays diverse roles in cereal plants. Among them, the bacterial population associated with roots including exophyte microbes and endophytes has a direct impact on plant development and health. In this chapter, we describe the rhizosphere bacterial microbiome in cereals, meta-genomics studies, isolation and identification of rhizobacterial endophytes and exophytes in different cereal plants, characterization of cereal rhizobacteria, and the potential roles of the rhizobacteria in cereal crops. The potential roles of these microbes will be pathogenic, parasitic, neutral, growth-promoting, stress-tolerant, biocontrol, etc. Overall, this chapter will explore the recent research advances and updates in rhizobacteria in cereal crops.

1. Introduction

The rhizosphere is the thin layer or narrow region of soil (approx. 1 mm) surrounding the roots of a plant, and it is directly influenced by root activities [1, 2]. The rhizosphere consists of three main zones, namely, endo-rhizosphere, rhizoplane, and ecto-rhizosphere [1]. The endo-rhizosphere is the innermost zone of the rhizosphere which includes plant root tissues such as the cortex and endodermis. Ecto-rhizosphere is the outer-most zone which comprises soil particles adjacent to the roots. The rhizoplane consists of the cortex, epidermis, and mucilage. It is the root surface that interacts with soil microbes and soil particles [1]. Bacteria inhabiting the rhizosphere, i.e., rhizobacteria, are found in all three zones of the rhizosphere. The endo-rhizosphere is inhabited by root endophytes (microbes living inside the roots) while the exophytes (microbes living outside of the roots) inhabit the ecto-rhizosphere [3]. Epiphytic (surface-dwelling) microbes colonize the rhizoplane (Figure 1) [4]. Rhizosphere is home to many organisms including bacteria, fungi, actinomycetes, protozoa, algae, nematodes, and arthropods [1, 5]. The rhizosphere microbiome plays different roles in cereal plants, however, bacterial microbes in them have sparked great importance for further studies. Rhizosphere microbial studies have been carried out in different cereal crops including rice, wheat, oat, barley, maize, rye, and sorghum [2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25].

Figure 1.

The structure and interactions of the cereal rhizosphere.

Root epidermal cells release root exudates which consist of an array of compounds, including organic acid ions, inorganic ions, siderophores, sugars, vitamins, amino acids, purines, nucleosides, and enzymes, to the soil [1]. Polysaccharide mucilage is produced by the root cap [17]. Soil microbes exploit these compounds as carbon and energy sources [2]. As a result, the microbial density in the rhizosphere increases up to 10 to 100 times than the bulk soil. The rhizosphere is therefore called the second genome [1, 26] or an extended genome [20] of plants. The majority of the rhizosphere community is bacteria, with approx. 10⁴ bacterial species/g of soil [1].

The rhizosphere microbial community is mainly considered substrate-driven, and it may vary with the plant genotype, plant developmental stage, and environmental factors [1, 17]. The root exudates influence plant-rhizobacteria and rhizobacteria-rhizobacteria interactions [2]. The plant-rhizobacteria interactions could be pathogenic, parasitic, neutral, growth-promoting, stress-tolerant, and antimicrobial [1]. Root exudates may attract beneficial bacteria. For example, plant growth-promoting rhizobacteria (PGPR) could be recruited through a process called chemotaxis. These PGPRs may boost plant growth and provide a protective function under abiotic and biotic stresses [2, 3]. On the other hand, the secretion of antimicrobial compounds by roots may inhibit microbial growth [1]. Some root exudates are involved in antibiosis (microbial inhibition due to antibiotics and toxins), parasitism (microbial inhibition due to cell wall-degrading enzymes), and systemic resistance (microbial inhibition by inducing plant defense mechanisms) [1].

Different mechanisms have been proposed to explain the exophytic and endophytic microbial profiles. The exophytic bacteria are prone to frequent fluctuations, thus highly dependent on environmental conditions, host genotype, and the soil microbial community. It is suggested that autolysis of root epidermal cells facilitates exo-bacterial invasion in internal plant tissues. Successful colonization of the plant root tissues could make them stable root endophytes [1, 3]. However, plant endogenic microflora that have co-evolved with the plant host could be present prior to exophytic invasions [3]. The endophytic community may influence the exophytic profile by modifying the root morphology [3].

In addition to plant-rhizobacteria interactions, communication among rhizobacteria is important for establishing a healthy rhizosphere. Signaling molecules called autoinducers produced by bacteria in response to a stimulation or at a particular stage of life, interact with receptors of other bacterial cells. A certain bacterial cell density is required to trigger bacterial gene expression that is responsible for cell-cell communication. This process is defined as quorum-sensing [1].

Rhizobacterial interactions with plants are categorized as positive/beneficial, negative/harmful, or neutral [2, 3, 17]. The positive mechanisms of plant growth-promoting rhizobacteria have been elucidated by previous researchers which could be classified broadly as direct and indirect [2, 17]. The production of plant growth regulators, enzymes and siderophores, solubilization of minerals, and symbiotic nitrogen fixation, directly influence plant growth, hence defined as direct mechanisms. Reports show that some PGPRs may use multiple mechanisms for accomplishing plant growth enhancement [17]. Mechanisms such as antagonistic activity against phytopathogens and induced systemic resistance have indirect effects on plant growth promotion [2, 17]. Negative interactions of rhizobacteria with plants include phytopathogenicity that adversely affects plant health [3].

Different species of rhizobacteria have been reported over the past few decades. Some examples include Azospirillum, Klebsiella, Pseudomonas, Azotobacter, Alcaligens, Enterobacter, Burkholderia, Arthobacter, Serratia, Bacillus and Stenotrophomonas [2]. Certain strains of PGPR belonging to Bacillus, Enterobacter, Burkholderia, Acinetobacter, Alcaligenes, Arthrobacter, Azospirillum, Azotobacter, Beijerinckia, Erwinia, Flavobacterium, Rhizobium and Serratia are utilized in a global scale to enhance crop productivity [13]. Diazotrophic soil bacteria such as Rhizobium are members of the PGPR [2]. Siderophore-producing bacteria are involved in iron sequestration for plants [6, 27]. Some of the PGPRs such as Rhizobium, Azospirillum, Pseudomonas, Flavobacterium, Arthrobacter, and Bacillus have been identified to enhance tolerance to abiotic stresses such as high salinity [27]. Some PGPR produces secondary metabolites such as hydrogen cyanide, 2,4-diacetylphloroglucinol, and antibiotics (e.g., phenazine) that play a role in antagonism [27]. Chromium stress in wheat is alleviated by Pseudomonas sp. whereas barley inoculated with Arthrobacter mysorens 7 and Flavobacterium sp. L-30 helps survival in lead-contaminated soil [8]. Certain rhizobacteria are available for commercial use as soil formulations, i.e., bioinoculants. Some examples are Arthrobacter mysorens 7, Flavobacterium sp. L30, and Klebsiella mobilis CIAM 880 [8].

This chapter will review the rhizobacteria present in different cereal crops, their roles, applications in agriculture, challenges, and future directions. In addition, this will further discuss the rhizosphere bacterial microbiome, identification and characterization studies of rhizobacteria, and the potential roles of the rhizobacteria in cereals such as maize, rice, wheat, barley, rye, and sorghum.

2. Identification and characterization of rhizobacteria

Rhizobacteria in the cereal rhizosphere are identified and characterized in numerous ways. They could be mainly categorized into culture-independent and culture-dependent approaches. Culture-dependent methods involve bacterial isolation, identification, and characterization. Culture-independent methods usually include meta-analyses such as meta-genomics, meta-transcriptomics, and meta-proteomics [4]. Bacterial identification and characterization in cereals can be carried out on isolated colonies or on root and/or soil samples as a whole [5, 11, 20]. Rhizobacteria are characterized using bioassays that target their biochemical properties and pathways. Figure 2 summarizes the methods used in the identification and characterization of rhizobacteria.

Figure 2.

The methods of identification and characterization of rhizobacteria.

2.1 Identification of rhizobacteria

2.1.1 Culture-dependent identification methods

In the studies of culture-based methods, a sample of the rhizosphere is suspended in a sterile solution (water/buffer) under aseptic conditions. The diluted rhizosphere suspension could then be plated on bacterial culture media such as tryptic soy agar [7, 21] and Luria-Bertani agar [19]. Single bacterial colonies are purified using subsequent culturing steps [5, 19]. Conventionally, culturable bacteria are identified mainly using phenotypic characters such as colony color, texture, size, form, margin, elevation, microscopic features, etc. [7, 12]. Microscopic techniques allow for determining the cell morphology, and more importantly the habitat and colonization patterns of bacteria [4]. Finally, bacteria are identified with reference to taxonomic keys. The disadvantages of conventional methods are the inability to capture large quantities of microorganisms, especially in the soil, and the reliance on culturable isolates. Many microorganisms would have remained unknown unless for the development of molecular identification techniques [4].

Purified cultivable bacterial colonies could further be isolated and studied using molecular identification. The first step of molecular identification is nucleic acid (DNA/RNA) extraction, followed by polymerase chain reactions (PCR) [4]. Bacteria are identified by amplifying the universal bacterial 16S rRNA gene [4]. Table 1 provides a summary of different target regions of the 16S rRNA gene used in previous studies with their respective primer pairs [5, 9, 11, 24, 25]. PCR techniques become useful for the functional identification of rhizobacteria as they allow to targeting of PGP traits with specific primers. For example, primers have been designed to target the gene encoding dinitrogenase reductases (nifH), and ammonia monooxygenase (amoA) [4].

Amplified region	Primers	Size of the amplified region	Reference
V3–V4 region of the 16S rRNA gene	341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′)	N/A	[19]
16S rRNA gene rrs	T7-PA and PH	1.5 kb	[11]
16S rRNA gene	fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rD1 (5′-AAGGAGGTGATCCAGCC-3′)	1.5 kb	[21]
16S rRNA gene	27F and 1492R	N/A	[19]
V4 region of the 16S rRNA gene	515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACVSGGGTATCTAAT-3′)	N/A	[25]
16S rRNA gene partial sequence	pA (5′-AGAGTTTGATCCTGGCTCAG-3′) and pH (5′-AAGGAGGTGATCCAGCCGCA-3′)	N/A	[28]
16S rRNA gene	27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1401r (5′-CGGTGTGTACAAGACCC-3′)	1.4 kb	[10]
16S rRNA gene	63F primer; (5′-CAGGCCTAACACATGCAA GTC-3′), 20F primer (5′-AGAGTTTGATCATGGCTCAG-; 3′) and 1500R primer (5′-GGTTACCTTGTTACGACTT-3′)	N/A	[16]

Table 1.

Regions of the 16S rRNA gene used for molecular identification of rhizobacteria.

N/A: Not available.

Next, PCR products are sequenced, mainly using the Sanger sequencing technique. Taxonomy is assigned with reference to 16S databases such as GreenGenes [25] and SILVA [20, 21]. One limitation of the 16S rDNA identification is the interference from chloroplast 16S rDNA and mitochondrial 18S rDNA, resulting in inaccurate molecular identification [29]. Therefore, generally, mitochondria, chloroplasts, archaea, and eukaryote-associated sequences are removed after classification [25]. Different pipelines are used for sequence analysis, for example, the Quantitative Insights Into Microbial Ecology (QIIME) analysis [20, 21]. DNA sequence identification is usually followed by a phylogenetic analysis. The evolutionary relatedness among bacterial isolates is determined by constructing phylogenetic trees according to various algorithms [19, 21, 28].

Some other molecular identification methods include hybridization/probing, fingerprinting, preparation of clone libraries, and microarrays [4]. Nucleic acid hybridization (DNA-DNA or DNA-RNA) is based on the affinity of DNA or RNA fragments to respective probes. DNA fingerprinting is another molecular technique used in bacterial identification where organisms are differentiated according to their unique DNA. Some of the fingerprinting methods successfully used are restriction fragment length polymorphism (RFLP), polymerase chain reaction-restriction fragment length (PCR-RFLP), denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), temperature gradient gel electrophoresis (TGGE), single-strand conformational polymorphisms (SSCP), ribosomal internal spacer analysis (RISA), length heterogeneity-PCR (LH- PCR), and amplified ribosomal DNA restriction analysis (ARDRA) [4, 22, 29]. In order to examine wheat rhizobacteria [9], conducted a DGGE analysis by amplifying the region from 8 to1492 bp and the V3 region of the 16S rRNA gene using the primer pairs, GM3f-GM4r and 338f-520r, respectively. The total DNA from the rhizosphere of barley was extracted by [10], and targeted for PCR amplification of 16S rRNA genes, followed by a T-RFLP analysis.

DNA microarrays, commonly known as DNA chips or bio-chips can be used to investigate specific genes of rhizobacteria or specific taxa within a community [4]. To identify bacterial taxa enriched in the course of barley monoculture [10], compared the rhizobacterial communities from three vegetation cycles, using a taxonomic 16S rRNA microarray containing 1033 probes. The data showed similar and distinct taxa in the three cycles [10]. Another study tested the effect of the evolutionary changes in eukaryotes on the composition of their associated microbiome using chloroplast sequences of Poaceae genotypes, namely, maize, sorghum, and wheat [11]. Their 16S rRNA taxonomic microarray analysis study revealed the correlation between rhizobacterial communities and Poaceae genotypes. They could also identify distinct bacterial taxa involved in cooperation (e.g., Rhodospirillales and Bacillales), symbiosis (e.g., Rhizobiales), and parasitism (e.g., Agrobacterium and Xanthomonas). In some studies, PCR products are transferred into vectors and cloned to prepare DNA clone libraries. Clone libraries can comprise complementary DNA (cDNA) or genomic DNA [4].

Another well-established method for identifying cultivable bacterial strains is the fatty acid analysis. It is a fast and cheap method, and a large number of conservative characteristics can be subjected to quantitative analysis [7]. The authors analyzed the fatty acid profiles of 1188 isolates of rhizobacteria in barley. The analysis separated the isolates into three distinct groups, with Pseudomonas, Cytophaga, and Gram-positive bacteria, respectively, as the predominant bacterial taxa. Bacterial communities such as rhizobacteria are further analyzed using metabolomics which measures the metabolic profile, including primary and secondary metabolites, at a specific time point. This approach is based on analytical techniques such as nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS) [4].

2.1.2 Culture-independent identification methods

Culture-independent identification techniques do not necessarily need culturable bacterial isolates and are often carried out on a community of bacteria (e.g. soil samples and plant tissues), based on meta-analyses. Meta-analyses provide information on bacterial communities and their interactions. Some of the approaches are, meta-genomics, meta-transcriptomics, meta-proteomics, and metabolomics. Meta-genomics allows the identification and diversity and abundance calculations of both culturable and unculturable microbiomes. The bacterial abundance is measured in relation to the copy number of the 16S rRNA gene [19], and the degree of gene expression is examined using transcriptomics [11]. Transcriptomics is useful in identifying active genes or pathways. The gene expression level can be quantified by bioinformatics software [25]. Meta-proteomics provides a protein analysis at the bacterial community level [4].

Several sophisticated high-throughput sequencing techniques, i.e., next-generation sequencing methods, have been developed in the recent past for fast, efficient, and simultaneous identification of multiple organisms. Table 2 provides some high-throughput sequencing techniques used in meta-genomics studies for the characterization of bacteria on a community scale.

Sequencing platform	PCR amplified region	PCR primers	Reference
Illumina MiSeq	V3-V4 hypervariable region of the 16S rRNA gene	341F (5′-CCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′)	[21]
Ion Torrent	N/A	N/A	[25]
Illumina MiSeq	16S rRNA gene	338F and 806R with specific primers synthesized using barcodes	[20]
PacBio RS II	Full length of 16S rRNA genes	27F and 1492R	[5]
Illumina Miseq and HiSeq	N/A	341F (5′-CCTACACGACGCTCGGCATCTTCGGATTTA-3′) and 805R (5′-GACTGGAGTTCCTTGGCACCCGAATTCCA-3′)	[24]

Table 2.

High-throughput sequencing techniques used for meta-genomics studies of bacterial communities.

N/A: Not available.

The analysis of sequence data includes various statistical and bioinformatics methods. In addition to taxonomical classification, the sequence data can be classified as core, shared, and unique taxa [25]. Statistical software such as R [25] and IBM SPSS [16] are used to analyze data using several statistical models [20]. Statistical models allow calculations of alpha diversity (within-sample richness) and beta diversity (between-sample dissimilarity) [19, 24, 25]. Bacterial correlation network analyses highlight the interactions among bacterial taxa, and determine the keystone taxa that are critical for maintaining community structure and function under different soil management practices [25].

2.2 Characterization of rhizobacteria

Rhizobacteria can be characterized according to their biochemical properties. Bacterial colonies isolated from cereal crops could be grown in selective or differential media, and identified based on their substrate-specificity [30]. Staining techniques, particularly Gram staining and endospore staining, aided with microscopic observations are also used for characterization [31]. A less time-consuming method of biochemical characterization is the use of the BIOLOG system. This method provides a phenotypic fingerprint of bacteria by assessing a microorganism up to the species level using carbon source utilization assays and chemical sensitivity assays in special microplates [28].

Assessing the functional roles of bacteria is not always possible since only the culturable rhizobacteria can be tested using bioassays [29]. However, several biochemical tests have been developed to qualitatively and/or quantitatively assess the biochemical properties of bacteria, for example, carbon sources and the production of enzymes. In an attempt to biochemical characterization of rhizobacteria [21], conducted several bioassays to determine plant growth-promoting traits. Rhizobacteria were tested for casein hydrolyzation, phosphate solubilization, potassium solubilization, zinc solubilization, production of siderophores, and salt tolerance. All the assays were conducted in agar plates incorporated with selective/inhibitory reagents. The number of positive isolates was counted for each functional assay [21]. The ability of rhizobacteria to produce IAA, fix nitrogen, and control Fusarium graminearum, F. proliferatum, F. verticillioides, and F. boothii were analyzed by [19].Table 3 provides a brief description of some bioassays used in bacterial characterization. Software packages such as PICRUSt provide predicting models for functional profiles of bacterial communities based on the 16S rRNA gene sequences [24], which are applicable for unculturable bacteria as well.

Bioassay	Special constituents of the medium	Observation of a positive isolate	Reference
Hydrolyzation of casein	5% skimmed milk powder, 0.5% pancreatic digest of casein, 0.25% yeast extract, 0.1% D-glucose	A clear halo in the medium surrounding the bacterial colony	[21]
Solubilization of phosphate	Tricalcium phosphate and Pikovskayas agar	A halo zone in the medium surrounding the bacterial colony	[21]
Solubilization of potassium	Potash feldspar and Pikovskayas agar	A halo zone in the medium surrounding the bacterial colony	[21]
Solubilization of zinc	Zinc oxide and Pikovskayas agar	A halo zone in the medium surrounding the bacterial colony	[21]
Production of Siderophores: iron solubilization	Chrome azurol S (CAS) and hexadecyltrimethylammonium bromide (HDTMA)	An orange halo in the medium surrounding the bacterial colony	[21, 28]
Salt tolerance	Different concentrations of sodium chloride (NaCl)	High growth/colony count compared to the control	[21]
Antagonistic activity - Dual culture	N/A	Pathogen growth inhibition	[19]
IAA production	Nutrient broth supplemented with tryptophan	Development of red color in the supernatant of the bacterial culture broth with the addition of orthophosphoric acid and Salkowaskis reagent	[28]
	LB broth supplemented; with L-tryptophan (200 mg/l)	Optical density of the cell-free supernatant after adding Salper reagent	[16]
Nitrogen fixation	Nitrogen-free malate (NFM/NFB) medium: malic acid, KH2PO4, MgSO4.7H2O, NaCl, CaCl2, Na2MoO4, MnSO4.H2O, EDTA, 0.5% bromothymol blue, biotin, KOH	Development of blue color in the NFB medium	[2]

Table 3.

Assays for biochemical characterization of rhizobacteria.

N/A: Not available.

3. Contribution of cereal rhizobacteria to face current challenges in agriculture

The global population is estimated to be nearly 10 billion over the next two decades, and a constant supply of food is essential to feed them [32]. It is challenging for researchers, breeders, and farmers to meet these demands within the existing arable lands while minimizing the use of agrochemicals [32]. Cereals are prone to many diseases by pests and pathogens, leading to yield loss. Climate change and human activities contribute to the development of undesirable environments with high salinity, high temperature, water stress, and less fertile soils. Cereals such as maize, rice, wheat, barley, rye, and sorghum, are staple foods of the majority of the world population, and their cultivation covers a large proportion of arable lands. Plant growth-promoting rhizobacteria from cereals have therefore been studied as a sustainable, eco-friendly approach in various agricultural aspects such as soil nutrient supplements and biocontrol agents [2, 6]. There is evidence for PGPR inoculations to enhance crop yield, and plant growth, and alter the microbial population in the soil (Table 4). However, their mechanisms are yet to be fully understood, and efficient application methods are yet to be developed [1]. This section highlights the importance of probing for beneficial rhizobacteria with examples from previous studies on plant hormone production, mineral solubilization, biocontrol properties, managing biotic and abiotic stress situations, and bioremediation.

Rhizobacteria	Inoculated cereal crop	Potential role/s	Reference
Brevibacterium frigoritolerans, Bacillus thuringiensis, and Bacillus velezensis	Wheat (China)	Increase plant height, root length, dry weight, and fresh weight	[23]
Pseudomonas sp.	Wheat	Tolerant to chromium (Cr)	[8]
Bacillus megaterium M3, Bacillus subtilis OSU142, Azospirillum brasilense Sp245, and Raoultella terrigena	Wheat and barley	Increase root and shoot dry weight under cold stress	[31]
Bacillus amyloliquefaciens and Microbacterium oleovorans	Maize	Protection against Fusarium verticillioides	[2]
Azospirillum brasilense + Bradyrhizobium japonicum	Maize	Increase seed germination and initial development of seedlings	[17]
Sinorhizobium sp. A15, Bacillus sp. A28, Sphingomonas sp. A55 and Enterobacter sp. P24	Maize	Increase IAA synthesis and early plant growth rate under conservation tillage	[19]
Pseudomonas fluorescens BRM-32111 + Burkholderia pyrrocinia BRM-32113	Upland rice (Brazil)	Increase biomass, leaf area, root length, root biomass, chlorophyll content, rate of carbon assimilation, rubisco carboxylation efficiency, water use efficiency, and tolerance to allelochemicals in seedlings under greenhouse conditions	[14]
Stenotrophomonas maltophilia, Enterobacter sp., Bacillus sp., Ochrobactrum haematophilum and Pseudomonas aeruginosa	Rice	Limit the mycelial growth of Rhizoctonia solani and Fusarium oxysporum; Increase germination percentage, seed vigor index, total dry biomass, and nitrogenase activity	[2]
Arthrobacter, Klebsiella, Pseudomonas, Bacillus, Proteus and Staphylococcus	Barley	Increase bioremediation of cadmium (Cd)-contaminated soils	[8]
Arthrobacter mysorens 7, Flavobacterium sp. L30, and Klebsiella mobilis CIAM 880	Barley	Solubilize Cd; Increase root elongation in Cd-contaminated soil; Nitrogen fixation	[8]
Arthrobacter mysorens 7 and Flavobacterium sp. L-30	Barley	Tolerant to lead (Pb)	[8]
Pseudomonas putida	Fall rye (Secale cereal)	Tolerant to petroleum hydrocarbon (PHC)	[12]
Bacillus and Sphingomonas	N/A	Mineralize heavy-metals; Degrade organic pollutants	[24]
Geobacter	N/A	Involved in iron and sulfate cycles; Degrade (anaerobic) polycyclic aromatic hydrocarbons (PAHs)	[24]

Table 4.

Potential PGP roles of rhizobacteria in cereal crops.

N/A: Not available.

3.1 Phytohormone production and mineral solubilization

Most PGPRs have the ability to produce indole-3-acetic acid (IAA), a type of auxin that stimulates root growth and the formation of lateral roots and root hairs [16]. Cytokinins, gibberellins, and inhibitors of ethylene may also be responsible for altered root morphology, facilitating nutrient uptake [15]. A mixture of Azospirillum brasilense with Bradyrhizobium japonicum could increase seed germination and initial development of maize [17]. Application of the rhizobacteria, Pseudomonas fluorescens BRM-32111 and Burkholderia pyrrocinia BRM-32113 on upland rice seeds in Brazil increased biomass, leaf area, root length, root biomass, chlorophyll content, rate of carbon assimilation, rubisco carboxylation efficiency and water use efficiency in seedlings under greenhouse conditions [14].

Crop-associated indigenous nitrogen-fixers may be agronomically important since they could supply a portion of the crop’s total nitrogen requirement [29]. Mineral solubilization by PGPR is another key tool for increasing nutrient uptake efficiency. The PGPR is adapted to fulfill the requirements of both nutrient-deficient and excessive soils. The effect of five nitrogen-fixing bacteria (Bacillus licheniformis RC02, Rhodobacter capsulatus RC04, Paenibacillus polymyxa RC05, Pseudomonas putida RC06, and Bacillus OSU-142), and two phosphate-solubilizing bacteria (Bacillus megaterium RC01 and Bacillus M-13) was tested in barley under greenhouse conditions by [33]. Bacillus M-13 and B. megaterium RC01 significantly increased the phosphate availability in soil. All the tested PGPR strains were diazotrophic. Among them, BacillusOSU-142 performed the best. Further experiments revealed that the inoculation of nitrogen-fixers in barley had a significant impact on the uptake of nitrogen (N), iron (Fe), manganese (Mn), and zinc (Zn) [33].

3.2 Biocontrol agents

Biotic agents such as phytopathogens and pests are a major threat to cereal production. Even though synthetic pesticides are efficient in disease management, continuous use may lead to negative impacts such as increasing disease resistance, soil and water toxicity, and accumulation of residues in food chains [18]. Biocontrol agents are organisms that significantly reduce pest and pathogen levels. They are assumed to be less harmful due to their biological origin and hence considered as an alternative to environmentally deleterious agrochemicals. Biocontrol agents can be applied by seed dipping, spraying, through irrigation, or as solid inoculants [30].

Several biocontrol agents have been experimented. Some PGPR such as Bacillus amyloliquefaciens and Microbacterium oleovorans can protect maize against Fusarium verticillioides when applied in the form of seed coatings. Based on that, [2] conducted a similar experiment using five selected rhizobacterial isolates as inoculations on rice seeds. The isolates: Stenotrophomonas maltophilia, Enterobacter sp., Bacillus sp., Ochrobactrum haematophilum, and Pseudomonas aeruginosa, exhibited multiple PGP attributes and successfully limited the mycelial growth of two fungal pathogens, Rhizoctonia solani and Fusarium oxysporum, in vitro. PGPR-inoculated plants had a substantially higher germination percentage, seed vigor index, and total dry biomass compared to the control [2]. Further, the nifH gene responsible for regulating nitrogen fixation was also detected in rhizobacterial isolates with significant nitrogenase activity [2].

3.3 Managing abiotic stress

Some of the major abiotic stresses on cereals are salinity, drought, low and high temperatures, and nutrient deficiencies [22, 26]. High saline content in the soil leads to osmotic stress, nutrient deficiency, ion toxicity, hormonal imbalance, and oxidative stress in plants. As a consequence, photosynthesis, protein synthesis, lipid metabolism, and stomatal closure are interfered, leading to lower crop yields [13]. Salt-tolerant rhizobacteria enhance root and shoot biomass, nutrient and water uptake, chlorophyll content, and disease resistance under saline conditions [13]. Some PGPRs form biofilms which provide plants protection from external stresses, while supporting their growth and increasing crop quality [13].

The rhizosphere of a halotolerant barley cultivar (Hordeum maritimum With.) was investigated by [16], in search for halotolerant rhizobacteria. Three halotolerant bacterial strains: Bacillus mojavensis S1, B. pumilus S2, and Pseudomonas fluorescens S3, were isolated and inoculated on a salt-sensitive barley cultivar. The bacteria showed an increase in IAA and proline production under salinity stress, which corresponds to plant adaptation in saline conditions. In a study in China by [23], wheat plants inoculated with three salt-tolerant plant growth-promoting rhizobacteria: Brevibacterium frigoritolerans, Bacillus thuringiensis, and Bacillus velezensis, increased in plant height, root length, dry weight, and fresh weight. Another group of scientists in China examined the rhizosphere bacterial diversity and soil metabolome of sea rice SR86 seedlings, over a range of salinity levels [5]. A rhizobacterial co-occurrence network of SR86 seedlings described keystone taxa involved in coping with salt stress. Further analysis on the network explained the potential contribution of keystone taxa, and specific metabolites in salt tolerance. Four rhizobacterial strains capable of alleviating salt stress and promoting seedling growth under salinity stress were isolated, characterized, and inoculated on SR86 plants. The inoculants were re-isolated and characterized for verification. Bacterial phyla, namely, Proteobacteria, Firmicutes, Desulfobacterota, and Verrucomicrobiota were detected in PGPR-applied soil [5].

In another study [31], investigated the performance of PGPR applications compared to a fertilizer treatment in wheat and barley plants under cold stress. The PGPR used were, Bacillus megaterium M3, Bacillus subtilis OSU142, Azospirillum brasilense Sp245, and Raoultella terrigena. The authors observed a statistically significant difference between PGPR inoculations and the fertilizer treatment with respect to root and shoot dry weight.

3.4 Plant acclimatization for toxicity/phytoremediation

Due to a lack of agricultural lands to meet the needs of the growing population, environments that are generally considered undesirable for cultivation may also be used for crop production. Heavy metals are common soil contaminants. Microbial activity under heavy-metal stress can change the pH of soil and consequently, its absorption and adsorption characteristics [8, 34]. However, high levels of heavy metals can cause significant changes in the microbial community structure [24, 34].

The effect of PGPR on barley plant growth in Cd-polluted soil was tested by [8] as a measure of increasing barley yield in Cd-polluted soils. Three commercially available PGPR: Arthrobacter mysorens 7, Flavobacterium sp. L30 and Klebsiella mobilis CIAM 880 were found to be successful Cd solubilizers, out of which K. mobilis CIAM 880 was the most effective. In addition, all three PGPRs were able to fix nitrogen and actively colonize the barley root system and rhizosphere which resulted in significant root elongation in Cd-contaminated soil. The authors used several measurements such as Cd accumulation in bacteria, gas chromatography analysis, IAA production, and a mathematical model simulation to propose the complex mechanism of bacterial Cd tolerance [8]. Moreover, rhizobacteria of the genera, Arthrobacter, Klebsiella, Pseudomonas, Bacillus, Proteus, and Staphylococcus were proposed as good candidates for accelerated bioremediation of soils heavily contaminated with Cd [8].

Research has been conducted to investigate the transformations of heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) in rhizosphere soils, and the adaptive responses of the rhizobacterial community in rice in China [24]. The dominant genera of rhizobacterial community in HM-PAH co-contaminated paddy rhizosphere soil were Bacillus, Massilia, Sphingomonas, and Geobacter, and their higher abundances appeared at the tillering stage and heading stage of rice. Bacillus and Sphingomonas are known to mineralize heavy metals and degrade organic pollutants whereas Geobacter is known for contributing to iron or sulfate cycles, and anaerobically degrading PAHs [24]. These phenomena explain their richness in HM-PAH co-contaminated paddy rhizosphere soil. In a similar study, fall rye (Secale cereal) grown in petroleum hydrocarbon (PHC)-impacted soil treated with two strains of Pseudomonas putida showed a gradual decrease in the detrimental effects of PHC over 3 years [12]. The two Pseudomonas strains were introduced to the rhizosphere by seed treatment, and field trials were conducted at an oil refinery land farm in Canada. Transcriptomics data revealed significant upregulation of two genes under stress conditions. P. putida is also known as a PGPR that produces auxin.

Another soil contaminant is allelochemicals which are the secondary metabolites of plants and microorganisms that accumulate due to long-term consecutive cropping systems. High concentrations of allelochemicals have detrimental effects on plant growth and development. During a study in Brazil, [14] observed allelochemicals in rice, maize, and sorghum plantations. The introduction of PGPR tolerant to allelochemicals was suggested as a method to overcome this challenge [14]. Phytoremediation is another strategy that uses rhizobacteria that have the ability to sequester, degrade, and transform contaminants [12, 29].

3.5 Cultural practices

Cultural practices, for example, the use of fertilizer and antimicrobials, tillage, crop rotations, intercropping, etc., can influence the rhizosphere composition, either enhancing or depleting beneficial bacteria [22]. Under conservation tillage, maize seeds inoculated with PGPR significantly promoted the early plant growth rate which promotes high grain yield [19]. The PGPR strains, Sinorhizobium sp. A15, Bacillus sp. A28, Sphingomonas sp. A55 and Enterobacter sp. P24 used for inoculations was previously isolated from the rhizosphere of maize grown in the same area [19]. The PGPR isolates were capable of IAA synthesis. These PGPR significantly increased the abundance and species richness of rhizobacteria which was supported by a 16S rRNA analysis. The molecular analysis further described changes in certain bacterial classes and genera in response to certain PGPR inoculations, indicating the role of PGPR in coordinating the ecological functions of the rhizosphere [19]. For example, the relative abundance of the class Alphaproteobacteria and the genera Sphingomonas, Candidatus solibacter, and Bryobacter were significantly reduced with all four PGPR inoculations while a significantly high relative abundance of the genus Streptomyces was observed in all inoculation except A15 [19]. A similar study in China by [20], confirmed that the crop growth stage, long-term tillage practices, and short implementation years of crop rotation significantly affected the phylogenetic diversity of root-associated bacteria in wheat. The work was supported by co-occurrence networks and a correlation analysis. Acidobacteria, Actinobacteria, Chloroflexi, and Proteobacteria were the four dominant phyla present in all samples [20]. The variations in the wheat rhizosphere composition under nitrogen-phosphorous-potassium fertilizer treatments were evaluated by [21]. According to the results of greenhouse experiments, fertilizer addition decreased the proportion of nutrient-solubilizing bacteria in wheat.

4. Challenges of rhizobacteria applications in agriculture

Once beneficial rhizobacteria with PGP traits are successfully identified, the next step is to implement methods for their application under field conditions. Attempts have been made to genetically engineer PGP traits to rhizobacteria. Beneficial traits such as nitrogen fixation, phosphorous solubilization, IAA biosynthesis, naringenin biosynthesis, biocontrol, and rhizoremediation, have become successful in the transfer and optimization within some rhizobacteria [32]. However, PGPR colonization and performance under real field conditions have not always been consistent [26]. As discussed in previous sections, the rhizobacterial population is highly dependent on environmental factors, host factors as well as cultural practices. Figure 3 outlines the challenges and future directions of PGPR in agriculture.

Figure 3.

Challenges and future directions of plant growth-promoting rhizobacteria in agriculture.

The performance of rhizobacteria in the natural environment may vary from that under in vitro conditions. Further, not all rhizosphere bacteria can successfully be cultured in synthetic media. For effective results, the PGPR should be able to survive in foreign soils while competing with the established microbial community [26]. Moreover, host specificity plays a role in successful rhizobacterial colonization. For example, attempts to modify non-host soils with Rhizobium have not always been successful. Biocontrol agent inoculations have met varying degrees of failure in disease control, therefore, their use in pest management is not well established [3]. Sometimes, undesirable genetic regulation might repress PGP traits that are genetically engineered to rhizobacteria. Therefore, bioinoculants should be prepared addressing the above issues.

So far, several approaches have been proposed. One strategy is to engineer and transfer PGP traits into selected efficacious rhizobacterial isolates [32]. Another suggestion is to modify the entire bacterial rhizosphere community during inoculation. By inoculating bacteria directly into root tissues as endophytes, it is expected that they will later spread to the soil or control root characteristics and eventually manipulate the rhizosphere community [3]. The application of a consortium of compatible PGPR rather than one bacterial isolate alone has a better influence on improving the adaptability of plants exposed to stress conditions [22, 26]. Crop rotations and intercropping, especially with leguminous crops can influence the development of favorable associations of exophytes and endophytes with plants [3, 26]. The cereal-legume cropping system exchanges PGPR, particularly the diazotrophs from legumes, and may promote rhizobacterial community diversity, soil health, and plant growth. This is a promising strategy to optimize resource-use efficiency and crop yield in less fertile agricultural lands [22]. Exploiting PGPR from extreme environments is another approach to address biotic and abiotic stresses in cereals. Further exploring and improving knowledge on plant-PGPR interactions, particularly at the molecular level altogether will help determining efficient ways of utilizing cereal rhizobacteria as eco-friendly, sustainable agricultural applications.

5. Conclusion

The cereal rhizosphere is an excellent source of bacteria possessing various functions. Rhizobacterial exophytes and endophytes have a direct impact on cereal plant health and nutrition. Several techniques are used to identify and characterize rhizobacteria, mainly characterized by culture-dependent and culture-independent approaches. Exploiting the potential roles of these microbes will lead to effective, sustainable, and eco-friendly applications in agriculture. Beneficial cereal rhizobacteria will be eco-friendly alternatives for sustainable agriculture.

Acknowledgments

Funding was provided by Agriculture and Agri-Food Canada.

Conflict of interest

The authors declare no conflict of interests.