Background

Plant –root interface is considered as a unique niche for plethora of microorganism that act as key players in plant development and its resilience to various abiotic and biotic factors. Rhizosphere is estimated to host approximately 1010–1012 CFU/g soil of bacteria from 10,000 different species (McNear 2013). Plants attract the bacteria by secreting various molecules which are collectively termed as rhizodeposits. Plants spend upto 17% of its total photo assimilates as rhizodeposits. Rhizodeposits includes root exudates, sloughed off plant cells, mucilage, diffusates, organic acids, proteins, amino acids, sugar and phenolic compounds to be used for microbial growth. In exchange, these bacteria regulate the growth of host plant by supplying soluble nutrient, enhancing stress tolerance and disease resistance (Zheng et al. 2018). The rhizospheric plant–microbe interactions have attracted much attention. The studies objectified towards comprehensive sustainable agriculture have reported that rhizospheric bacteria have a crucial role in crop health and yield. The application of rhizobacteria in agricultural field is associated with increasing crop yield, soil health and plant’s tolerance (Simarmata et al. 2019). Rhizobacteria augment the plant development by various direct and indirect pathways. Direct pathways includes nutrient management by phosphate solubilisation, potassium solubilisation, biological nitrogen fixation, micronutrient supply, secretion of phytohormones like auxins, gibberallic acid, cytokinins and disease resistance by secretion of antibacterial, antifungal and anti-nematode factors (Kaur and Kaur 2018). On the other hand, indirect pathways include siderophore production, abiotic stress tolerance, ACC deaminase production, ammonia production. It has been reported that the climatic conditions, abiotic stress, plant host genetics, soil composition and anthropogenic activities significantly affects the availability and functioning of rhizobacteria in soil (Kaur and Kaur 2020; Igiehon and Babalola 2018).

The multifaceted advantages of plant growth promoting rhizobacteria (PGPR) can serve the agricultural benefits as a single entity towards replacing synthetic agrochemicals and chemicals fertilizers (Backer et al. 2018). Introduction of PGPR strains can help restore the soil health and prevent the ill effects of fertilizers. Living consortia of microbes are applied successfully to increase yield of wheat, canola, lettuce, tomato, banana, maize, chickpea and brinjal (Sneha et al. 2018). However, there is no single one fit biofertilizer that works for all crops and climatic conditions or that provides complete nutrient supply to plants. There is still a large gap between PGPR potential and its application as biofertilizer in fields for crop production (Vejan et al. 2016). These bacteria have shown high crop specificity and low success rate in varied climatic conditions. The inconsistency of rhizospheric bacteria to perform under different field conditions, limits its widespread usage for plant development. Differences in soil conditions and plant type effects the performance of rhizobacteria (Banerjee et al. 2006).The validation of application of rhizobacteria for field use is possible if their PGPR activity is enhanced through gene manipulations (Geetha and Joshi 2013).With the advancements in the molecular biology, synthetic biology and hand on genome editing techniques, there are vast opportunities to design a microbe that can provide all the three essential macronutrients (nitrogen, phosphorous, potassium) to the plants regardless of the crop. The knowledge of the mechanisms associated with the expression of plant disease resistance in PGPR has facilitated their better application by development of strains with enhanced activity or combination of different PGPR traits. This can also provide impetus to optimization of metabolic processes in PGPR to develop strains adapted to wide host range and environments. In this review, we summarize the recent advances in engineering plant growth promoting bacteria as living biofertilizer for sustainable agriculture (Tables 1 and 2).

Table 1 Engineered PGPR for enhanced macronutrient availability
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Table 2 Engineered PGPR for enhanced IAA production and biocontrol potential
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Genetic manipulations in rhizobacteria to augment plant growth promoting potential

  1. 1.

    Nitrogen fixation

In sustainable agricultural system, yield of crop relies upon the soil’s inherent fertility and microorganisms that regulate the transportability in addition to mineralization of essential nutrients for development of plants. Nutrient availability is appraised as the restricting factor of plant productivity (Choudhary et al. 2011). Nitrogen represents as one of the eminent nutrient principally required for the development of living beings specifically for the synthesis of amino acids. Bacteria belonging to genus Azorhizobium, Sinorhizobium, Bradyrhizobium and Mesorhizobium are reported as natural nitrogen fixers in leguminous plants while Frankia and Cyanobacteria are often found associated with non-leguminous plants (Kaur and Kaur 2018). PGPR strains like Pseudomonas sp. have shown the presence of nif genes required for nitrogen fixation. However, these microbe-plant associations are very specific and this symbiosis is observed in commercially important crops like rice, maize and wheat (James 2000). There is a need for the creation of potential recombinant strains exhibiting nitrogen fixating potential that can work for different crops. Earlier researchers have reported that transfer of functional nif (nitrogen-fixing) genes from K. pneumoniae to P. putida and E.coli have resulted in the mutant strains that were able to demonstrate nitrogenase activity by the express nif genes (Postgate and Kent 1987; Dixon and Postgate 1971). These studies confirmed that nitrogen fixation genes can be relocated from symbiotic to non-symbiotic bacteria through mobile genetic elements. Researchers are constantly working on the engineering of PGPR strains to boost plant growth under low nitrogen conditions.

In one such initiative, Setten et al. (2013) successfully transferred the nitrogenase encoding nifgenes from P. stutzeri A1501 to PGPR strain P. protegens Pf-5. X940 cosmid-mediated gene transfer induced constitutive expression of nitrogenase enzyme activity by the mutant strain in a nitrogen free medium. Similar nitrogen fixation ability was shown by P. putida, P. veronii and P. taetrolens when transformed by X940 cosmid integrated with nif genes. The results of this study unambiguously confirm that this technology of transfer of nif genes can be used for genetic manipulation in different bacterial strains. P. protegens Pf-5, a well-known biological control agent was found to work effectively to enhance the growth of both dicots (Arabidopsis and alfalfa) and monocots (maize and fescue) via increasing the availability of ammonium ions and nitrates in soil in response to nif genes. Therefore, it can be speculated that genetically modified P. protegens can be functionally used in many commercial crops (Setten et al. 2013).

Wang et al. (2013a, b) tried to express the nif genes in the heterogenous prokaryotic model by integrating nine genes from nif gene cluster of Paenibacillus sp. WLY78 to E.coli JM109 under the control of its native σ70-dependent promoter. The engineered E. coli JM109 synthesised active nitrogenase but its specific activity was only nearly 10% compared to the Paenibacillus sp. WLY78. The study raises an important ambiguity that the transfer of only nif genes to the heterogenous host may not yield the desired results and synthetic biology tools are necessary to increase the nitrogenase activity (Li et al. 2016). Promotor replacement is an artful synthetic biology approach to ease expression of nitrogen fixation which requires cluster of multiple genes organised into different operons. Transcription of these genes working in concert is managed by a complex cascade system of different promoter. These complex native regulatory elements and promoters can be replaced with simple expression systems. Wang et al. (2013a, b) cloned the entire nif gene cluster of 7 operons from Klebsiella pneumoniae to E.coli by replacing the native σ54 dependent promoters with T7 RNA polymerase–LacI expression system that yielded 100% nitrogenase activity. The newly constructed T7-dependent nif biological system cloned in E.coli when challenged with physiological and genetic conditions, bypassed the original complex regulatory circuits. However, only 42% nitrogenase activity could be seen by mimicking the same expression levels with variable-strength T7- dependent promoters. Therefore, the promoter choice can be vital in deciding the expression levels of a gene. Also, promoter replacement can be an important tool to switch the complex regulatory pathways into a more universal system and enhance the expression of nif gene in host (Dubeau et al. 2011).

Li et al. (2016) provided valuable insights towards enhancing the nitrogenase activity of the heterogenous host through genetic engineering. They cloned twenty eight selected genes from Klebsiella oxytoca and Paenibacillus sp. WLY78 and integrated them into two different vectors under the control of Paenibacillus nif promoter, followed by simultaneous transfer to the E. coli 78–7. These cloned genes included Fe–S cluster assembly (suf operon) & electron transport genes fldA, pfoAB and fer from Paenibacillus and Fe–S cluster assembly (nifSU gene) & electron transport specific for nitrogenase (nifFJ gene) from K. oxytoca. By this combined transfer of genes, the specific enzyme activity of nitrogenase from recombinant E. coli 78-7 was 50.1% of wild Paenibacillus (Fig. 1).

Fig. 1
figure 1

Combined assembly of Paenibacillus suf operon and electron transporter genes with Fe–S cluster assembly (nifSU gene) of K. oxytoca in the recombinant E. coli 78-7. A total of 28 selected genes from Paenibacillus and K. oxytoca were placed under the control of nif promoter in two cloning and expression vectors (pBC & Pck) and then are transferred to E. coli 78-7. The combined assembly of the potential K. oxytoca nifSU genes with Paenibacillus electron transporter genes (pfoABfldA) leads to enhanced nitrogenase activity

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Another invasive strategy involves transfer of the entire nitrogenase island with promoter genes from the native to PGPR strain. This is done to rule out the sensitivities related to oxygen supply raised by the transfer of only nif genes. Also, PGPR strain can be engineered for two or more characters simultaneously to enhance its PGPR potential. Such an initiative was carried out by Jing et al. (2018) in which they constructed a P. protegens Pf-5 strain to incorporate nitrogen fixation ability and enhance its innate antifungal traits. The nitrogenase island along with native promoter isolated from P. stutzeri DSM4166 was incorporated into a retS mutant strain. The engineered strain illustrated high nitrogenase activity as well as significant ammonium production as by acetylene reduction in L-medium. The mutant strains showed significant increase in fresh weight in dicots (cucumber) and monocot (wheat) and demonstrated high potential against R. solani infection (Jing et al. 2018).

  1. 2.

    Inorganic and organic phosphate solubilisation

After nitrogen, phosphorus is the second most substantial plant growth limiting nutrient. Even in the most fertile soils, the availability of phosphorous to plants is not more than 10 Μm (Aeron et al. 2019). Certain rhizospheric bacteria have the ability to convert inorganic phosphorus reserves to soluble form that can be assimilated by plants for their use. The phenotype for phosphate solubilisation is related to the synthesis of enzymes like phytase and phosphatase and secretion of low molecular weight organic acids. The presence of pyrroloquinoline quinone PQQ synthase gene discovered in Erwinia herbicola was correlated to mineral phosphate solubilisation (Goldstein and Liu 1987). They showed that the cloning of these genes to E.coli HB101 resulted into recombinant strain with comparable mineral solubilisation potential. Rodrı´guez et al. (2000) engineered Pseudomonas sp. PSS and Burkholderia cepacia IS-16 by transferring PQQ synthase gene from Erwinia herbicola. Enhanced mineral phosphate solubilisation (MPS) was observed in both the strains adding to their PGPR potential. Babu-Khan et al. (1995) and Liu et al. (1992) reported that expression of MPS trait is the result of indirect oxidation regulated by glucose dehydrogenase (GDH) which needs cofactor PQQ for formation of haloenzyme. E. coli K-12 synthesize apo-GDH but the cofactor (PQQ) is absent that results in lack of MPS phenotype. Babu-Khan et al. (1995) analysed that 369 bp single open reading frame that encodes the protein gabY, when cloned from P.cepacia (active solubilizer of inorganic phosphate) to E. coli K-12 resulted in the secretion of gluconic acid and induce MPS. It is rational here to suggest that gabY also play a key role in controlling expression of MPS and it can be considered as an option for genetic manipulation experiments in future. Farhat et al. (2013) cloned gdh gene and pqqABCDE gene encoding the apo- enzyme glucose dehydrogenase and its cofactor pqq from phosphate-solubilizing Serratia marcescens CTM 50650. Both the genes were subcloned and co-expressed in E.coli. The recombined strain was competent enough to solubilize wide range of inorganic phosphorous substrate including tricalcium phosphate, Gafsa rock phosphate and hydroxyapatite. These studies give a reason to support the fact that non-PGPR strains like E.coli offers potential for sustainable agricultural processes. However, field studies are needed, some of which are underway, to fully establish the potential of genetic manipulated strains.

Schulze et al. (2006) described that the process of nitrogen fixation by bacteria in field conditions is limited by the presence of insoluble phosphorous. Co-inoculation of PGPR strain with nitrogen-fixing ability and phosphorous mineralization has shown preferable nitrogen fixation and better plant growth in plants (Kumar and Chandra 2008). Instead of co-inoculation with two different PGPR strains, a single strain can also be engineered to express both these traits. For instance, Herbaspirillum seropedicae Z67, is a well characterized nitrogen-fixing endophytic bacteria known to niche roots, leaves and stem of agronomically important crops such as sugarcane, wheat, rice (Monteiro et al. 2008) but it lacks MPS phenotype. Unlike phosphate solubilising bacteria, its genome includes encodes GDH apoprotein but lacks the genes that encodes for the biosynthesis of its cofactor PQQ. pqqE gene (pyrroloquinoline quinine) of Erwinia herbicola, pqq gene cluster of Pseudomonas fluorescens B16 and pqq gene cluster of Acinetobacter calcoaceticus (pSS2) was subcloned and over expresses in H. seropedicae Z67 to construct mutant strains designated as Hs pJNK1, Hs pOK53 and Hs pSS2, respectively. Engineered Hs pOK53 and Hs pSS2 reported significant rock phosphate solubilisation due to the activity of GDH enzyme (Fig. 2). The mutant strains exhibit enhanced growth rate in N- free media showing the PGPR attributes of the bacteria were improved by the genetic modifications (Wagh et al. 2014). All these studies have involved cloned the desired genes using plasmid which is very unstable. However, to avoid horizontal gene transfer and enhance stability of engineered traits, future studies should be focussed on the chromosomal insertion of PQQ synthase genes in bacteria. The genomic integration of artificial citrate operon was carried out by Adhikary et al. (2014) in six-PGPR P. fluorescens strains using MiniTn7 transposon gene delivery system using method of electroporation. The NADH insensitive gltA1 (citrate synthase) and citC (citrate transporter) genes delivered to the genome of P. fluorescens conferred MSP due to enhanced gluconic acid and citric acid secretion. Although, this study provides interesting insights into the integration of phosphate solubilization genes but the efficiency of recombinant strains was not tested on the plants.

Fig. 2
figure 2

Engineering of Herbaspirillum seropedicae to overexpress gluconic acid production. pqq gene cluster of P. fluorescens B16 was subcloned and overexpresses in H. seropedicae Z67 to construct mutant strains with significant rock phosphate solubilisation. Single or multiple PQQ genes can be cloned for constructing different mutant strains

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It is important to note that soil also contains organic reserves of phosphorous in chelated form which can be solubilised by bacteria that secrets enzymes like acid phosphatase and phytases (Rodríguez and Fraga 1999). With the objective of developing an effective phosphate biofertilizer, Fraga et al. (2001) studied the organic phosphate solubilisation genes (Nap A) encoding for acid phosphatase in Morganella morganii. Nap A gene was sub cloned from the host bacterium and transferred to P. cepacia IS-16 strain which is a known inorganic phosphate solubilizer. The transformed clones reported significant enhancement in extracellular acid phosphatase which helped in organic phosphate solubilisation. Phytate is another, biological phosphate reserve present in soil that can be solubilised through enzyme phytases. To explore the possibility of engineering of PGPR for phytate solubilisation for agronomical use, Shulse et al. (2019), used synthetic biology approach to integrate the phytases encoding genes directly into the chromosome of three bacterial hosts which were characterized inorganic phosphate solubilizers. The gene sequence of 82 phylogenetically diverse phytase genes encoding three different class of enzyme comprising BPP (propeller phytase), HAP (histidine acid phosphatase) and CP (Cysteine phosphatase) was refactored and then synthesized their optimal expression in Proteobacteria host. The refactored genes were engineered Ralstonia sp. strain UNC404CL21Col, Pseudomonas putida KT2440 and P. simiae WCS417r. High levels of phytate hydrolysis was seen in 41 mutant strains whereas 12 engineered strains conferred the high growth rates on Arabidopsis thaliana test plant when phytate is supplied as the sole phosphate source. This study paves the road to chromosomal based integration of novel genes through genetic engineering techniques to generate PGPR strains.

Potassium (K) is third macronutrient decisive in resistance of plants towards pest and abiotic conditions. There are close to 80 different enzymes involved in plant processes like starch synthesis, sugar metabolism, nitrate reduction and photosynthesis that require K as cofactor (Hussain et al. 2016; Gallegos-Cedillo et al. 2016; Almeida et al. 2015). K-solubilizing microbial community effectively release insoluble inorganic pools of total soil K through solubilisation (Saha et al. 2016; Meena et al. 2014). Bacillus spp., Enterobacter spp., Burkholderia spp., Pseudomonas spp. are reported to be involved in K- solubilisation (Dong et al. 2019; Etesami et al. 2017). The mechanism of K-solubilization by these bacteria is similar to phosphate solubilisation (Meena et al. 2014; Uroz et al. 2009; Parmar and Sindhu 2013) and involves the production of low molecular weight organic acids which help in K solubilisation from its parent minerals (Saiyad et al. 2015; Keshavarz Zarjani et al. 2013; Prajapati et al. 2013). Very few studies are available that highlights the mechanism of K solubilisation in bacteria with almost no studies on genetic basis of K solubilisation. Biofertilizer industry lacks interest in potassium solubilising bacteria (KSB) due to low efficiency and rapid loss of K solubilisation phenotype. The gene manipulation technology can be used to enhance the production of organic acids similar to phosphate solubilising bacteria. However, there is no literature available supporting this hypothesis.

  1. 3.

    Chitinases activity

Plant pathogens are the major cause of decrease in crop yield and quality (Savary et al. 2012).Fungi are the chief causative agent of numerous diseases in plants. Chemical fungicides constitute the major strategic therapy to treat fungal infections in plants. But as these chemical agents have high persistence in soil. They possess great environment risks by contaminating water, targeting beneficial soil microorganisms and declining beneficial non target pollinators. Engineering PGPR to enhance their activity against fungal phytopathogens has gained much interest in recent decades. It has been suggested that PGPR can inhibit phytopathogens by different mechanism, including production of defence-related compounds, antimicrobial substances, siderophore production, nutrition improvement and activation of plant defence mechanism (Kaur and Kaur 2018). However, the natural occurring rhizobacteria exhibit weak antifungal potential while most of them demonstrate high plant or pathogen specificity (Huang et al. 2006).

Various studies have reported to inhibit phytopathogenic fungi by inoculating plants with chitinase-producing microbes. Chitinase is an active enzyme that disrupts the β-1, 4-linkages of N-acetyl-D-glucosamine of chitin, which is a vital biomolecule present in fungal cell wall (Gomes et al. 2001). Disruption of the N-acetyl-D-glucosamine bonds causes the weakening of the cell wall and sequentially causing cell lysis. Engineering PGPR for production of chitinase, serves as one of the important step towards disease suppression in plants. In a study, an engineered PGPR, B. vietnamiensis P418 was constructed by introducing a chitinase-encoding gene chi113 into its chromosome. The gene was isolated and cloned from Bacillus subtilis Ap113. The transformed cells had upgraded disease resistance potential against phytopathogenic fungi R. solani and Verticillium dahliae (Huang et al. 2006). Following this, chi113 gene was integrated into the chromosome of Burkholderia vietnamiensis P418 using transposon delivery vector pUTkm1. The transformed strain showed no reduced fitness and no loss of inherent potassium and phosphate solubilising activity. P418-37 showed enhanced in-vitro growth inhibition of R. solani, Fusarium oxysporum, Gaeumannomyces graminis, Rhizoctonia cerealis, Bipolaris sorokiniana and Verticillium dahliae (Zhang et al. 2012). Transformed strain, P418-37 showed significant repression of cotton wheat sheath blight Fusarium wilt, and tomato grey mould.

There are ample opportunities for chitinase gene from plant origin to be cloned into bacteria with novel qualities of plant growth promotion. In a study, Jayaraj et al. (2004) rice chitinase cDNA (RC 7) chitinase gene encoding 35 kDa class I chitinase was cloned into plasmid vector, pDSK519, and integrated into Azospirillum brasilense SP51eFL1 by biparental mating. The protein expression was demonstrated through western blotting which confirmed the presence of 35 kDa cell lysate which hydrolyzed various chitin substrates and was immuno-reactive to the barley chitinase antibody. The fusion proteins from cell lysates of transconjugated strain exhibited strong antifungal activity, however, inherent nitrogen-fixing ability were slightly diminished.

Other studies show that non-PGPR strains can also be engineered for introduction of chitinase expressing genes against various fungal pathogens. Liu et al. (2013) isolated chitinase genes (LbCHI31 and LbCHI32) from Limonium bicolor, and expressed it successfully in E. coli BL21 strain. The recombinant E.coli produced intracellular and extracellular chitinase with potential hydrolytic ability on cell walls of A. alternate (Fig. 1, Supplementary). In another study, PtChi19 gene encoding chitinase, isolated from Pseudoalteromonas tunicata CCUG 44952 T was integrated to the genome of E.coli BL21. The transformed strain harbouring PtChi19 exhibited prominent hyphal inhibition against fungal pathogens including F. oxysporum, A. niger, and Armillaria mellea (García-Fraga et al. 2015). Chitinase encoding gene ChiKJ406136 was isolated from the complete genome of Streptomyces sampsonii KJ40, which was then cloned into E. coli to express recombinant chitinase protein. The mutant E. coli produced chitinase which inhibited the mycelium growth in Cylindrocladium scoparium, Neofusicoccum parvum Crous, Cryphonectria parasitica and F.oxysporum Schl. This study shows E.coli can be used as an expression vector for the production of commercial grade chitinase for agricultural use (Li et al. 2018).

  1. 4.

    Indole acetic acid production

Indole acetic acid (IAA) is the functional auxin that is predominantly associated with the development of apical meristem and growth of adventitious and lateral roots (Olatunji et al. 2017). Plant produces an endogenous pool of IAA depending upon the prevailing environmental conditions, tissue type and availability of tryptophan precursor, concentration of IAA produced being monitored by rate of synthesis, its sequestering in different plant regions, conjugation and degeneration. This endogenous pool of IAA is usually suboptimal or optimal to support plant growth. Most of the rhizobacteria secretes IAA in the close vicinity to roots where plants can receive the hormone through roots. Acquisition of additional IAA produced by rhizobacteria enhances its concentration to optimal or supraoptimal levels, resulting in plant growth promotion. Miscellaneous strain of bacteria belonging to genus Azospirillum, Enterobacter, Pseudomonas, Myroides, Alcaligenes, Xanthomonas and Acetobacter have been reported to secret IAA and augment plant growth (Duca et al. 2018; Kaur and Kaur 2021).

The plant–microbe interaction is affected severely in stress conditions, suggesting that there is a need to engineer the natural IAA producing bacteria for over production of IAA. For this, Pseudomonas sp. UW4, an efficient PGPR strain acting efficiently in biotic as well as abiotic stress conditions was engineered for IAA-overproduction. Second copy of target IAA biosynthesis genes (ami, nit, nthA, nthB, phe) was introduced into the Pseudomonas sp. UW4 to construct transformant strain. Pseudomonas sp. UW4 transformants were reported to produce upto 2–3 times higher IAA (Duca et al. 2018). With the similar aim of constructing a superior bioinoculant that produce higher IAA, Malhotra and Srivastava (2006), worked with a natural isolate of Azosprillium brasilense SM. Azosprillium synthesize IAA via indole pyruvic acid (IPyA) pathway, which is subjected to extremely tight regulation. Constitutive indole acetamide (IAM) pathway genes, iaaM and iaaH from a heterologous host (P. syringae) were introduced to A. brasilense SM for the upregulation of IAA. With the IAM genes, bacteria were efficiently able to utilize indole acetamide as a substrate and produce 3 times higher levels of IAA than wild strain. Lateral branch development, root proliferation and dry weight of the sorghum plant were improved with the bioinoculation of engineered A. brasilense SM strain.

Deletion mutations can also be vital tool for the engineering of PGPR for higher production of IAA. Deletion mutations of structural gene acdS encoding 1-aminocyclopropane-1-carboxylate (ACC) deaminase resulted in mutant strain (B. phytofirmansYS2) that produced6 fold higher IAA than wild type, however, complete loss of ACC deaminase activity was also observed (Sun et al. 2009). Complementation of the ACC deaminase gene in the mutant YS2 (strain YS3) restores the ACC deaminase activity and high plant root elongation effect compared to the wild strain (Sun et al. 2009).

Non-rhizospheric bacteria offer better advantage over rhizospheric bacteria in terms of root colonization through metabolization of various plant root exudates (Ledger et al. 2012). Mechanism of quorum sensing (QS) offer them competitive advantage and better survival rates in particular niche. This encourages the quest to engineer these neutral bacteria to contribute towards the plant growth. C. pinatubonensis JMP134 is one such non-PGPR bacterium which is amenable to genetic manipulations. Zúñiga et al. (2018), constructed an QS dependent IAA biosynthesis device in strain C. pinatubonensis JMP134. A genetic circuit was created using Plux promoter and luxI, luxR genes from bacterium V. fischeri to C. pinatubonensis. A positive feedback QS circuit was capable of producing IAA and responding to N-(3-Oxohexanoyl)-L-homoserine lactone, 3-oxo-C6- HSL. iaaM gene (2-tryptophan monooxygenase) and iaaH gene (indole-3-acetamide hydrolase) from P. savastanoi 48 were assembled downstream the Plux-luxI sequence in the plasmid pSEVA-Plux-luxI. In-vivo and in-vitro test showed the self-induction of the device at higher concentration of bacterial cells leading to significant enhancement in lateral root expansion, primary root length, fresh weight and rosette area of Arabidopsis thaliana plants. This study emphasis the need that non-rhizospheric bacteria can be better utilized as a vehicle for agriculture applications.

  1. 5.

    Disease resistance

The pathogenic attack on plants produces an induced systematic resistance (ISR) initiated by the release of plant hormones. The PGPR residing in vicinity of effected plants release certain volatile compounds like 2,3-butanediol and 3-hydroxy-2-butanone (acetoin) that trigger ISR in plants and help control the disease progression. One of the simplest ways to combat the disease menace is to elicitate the defence pathway in plants as mediated by bacterial volatile organic compounds. Plants subjected to PGPR such as Pseudomonas, Bacillus and Serratia sp. have shown development of ISR and lower rates of pathogen attack (Kloepper et al. 2004). Biocontrol properties of PGPR may be improved by manipulating their genetic material to overexpress one or more traits (Glick and Bashan 1997). Recently, many researchers have worked towards enhancing the antibiotic secretion in PGPR, production of defence-related volatile compounds and overexpression of gene regulating secretion of broad spectrum antifungal compounds (Klemsdal and Tronsmo 1999).

Acetoin is a plant growth promoting volatile compound secreted by numerous PGPR strains including B. subtilis, B. velezensis and B. amyloliquefaciens IN937a. Acetoin production is critical for the elicitation of defence pathway in plants as the B. subtilis mutant strains (BSIP1174 and BSIP1173) fail to protect the plant against infection (Rudrappa et al. 2010). Acetoin production is also related to the binding of the PGPR strain to plant roots and enhanced plant growth promoting activity (Rudrappa et al. 2007). Higher acetoin levels are linked with the induction of defence enzymes such as polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL). Chung et al. (2016) have reported that B. velezensis is endowed with the innate ability to produce acetoin. However, the concentration of acetoin is generally lower than required functional levels. Peng et al. (2019), constructed the knockout strains of B. velezensis by deleting bdh, gdh and alsD genes by the double crossover homologous recombination method. The recombinant strain showed similar growth pattern and a significant increase in acetoin production compared to wild strain. A mild but effective immune response was elicited in plants in a ‘priming’ state for triggering defence response against pathogen. Hydrogen peroxide accumulation and callose deposition was enhanced significantly helping plants to fight against infection. The concentrations of POD, PPO and PAL were found to be significantly higher in plant leaves inoculated with transformed bacterial strains (Peng et al 2019).

Gene manipulations techniques involving downregulation of certain genes controlling the production of antibiotics and related antimicrobial compounds are widely used as a futuristic approach towards sustainable methods of biotic stress tolerance. Various Pseudomonas sp. like P. aeruginosa, P. fluorescens FD6, P. protegens CHA0, P. fuscovaginae, P. brassicacearum LBUM300 are involved in secretion of diacetypholoroglucinol (DAPG), pyoluteorin (Plt), pyochelin, hydrocyanic acid, phenazine-1-carboxylic acid (PCA) and pyoverdine antimicrobial compound with antifungal properties (Huang et al. 2017). RetS, a regulatory protein is believed to negatively control the phenotype of antibiotic production in PGPR strains by acting as a contender of the GacS/GacA system. Inactivation of RetS a sensory kinase downregulate the production of related antibiotics in P. fluorescens FD6 (Zhang et al. 2015; Reimmann et al. 2005). Jing et al. (2018) knockout retS genes to improve the antibiotics biosynthesis in strain P. protegens Pf-5. The production of DAPG was increased by 20–30 folds with pronounced activity against R. solani.

Similarly, P. protegens H78 a PGPR well known to produce DAPG and Plt was engineered using multiple gene knockout strategies to enhance the production of Plt by 14.3 times compared to wild strain (Shi et al. 2019). Four potential negative regulators controlling Plt production including an inhibitory sequence lying within the operator of the pltR (transcriptional activator gene), translational repressor gene (rsmE) in the Gac/Rsm-RsmE pathway, negative regulatory gene pltZ transporter operon pltIJKNOP and ATP-dependent protease gene lon encoding enzyme that downregulates the positive regulators were deleted. Plt ABC-type pltIJKNOP transporter operon was overexpressed in the engineered strain. Although, there was a significant enhancement in Plt production, the study revealed a declined cell growth rate in the later growth cycle. Another important observation was the fact that simultaneous deletion of multiple genes from the same regulatory pathway did not have additive effects on Plt production. Therefore, engineering studies should focus on the bracing the metabolic flux into the Plt synthesis pathway by splitting its competitive pathways. This explains the urgency for the studies differentiating different regulatory pathways underlying Plt biosynthesis to enable the researchers to precisely engineer regulatory pathway for enhance Plt production without effecting cell growth.

Phenazine-1-carboxylic acid (PCA) is another important broad spectrum antifungal compound found effective against Phytophthora capsici and Rhizoctonia solani AG1-IA, F. oxysporum that affects pepper, rice, tomato respectively (Upadhyay and Srivastava 2011). It has reported that P. chlororaphis GP72 have the potential to produce commercial grade PCA from glycerol (Liu et al. 2016), but the rate of production is much low (Poblete-Castro et al. 2019). Solaiman et al. (2016) reported that metabolic utilization of glycerol in P. chlororaphis NRRL B-30761 can be enhanced by co-expression of genes glpF and glpK encoding glycerol uptake facilitator and glycerol kinase. Along the same line, Song et al. (2020) constructed P. chlororaphis GP72 mutant by co-expressing genes glpF and glpK. GlpR gene which negatively regulates the glycerol utilization and mgsA gene regulating the conversion of glycerol to pyruvaldehyde was knocked out using DNA manipulation techniques (Fig. 3). The PCA production after 36 h was enhanced to 993.4 mg/L in mutant strain compared to 729.4 mg/L in wild strain. This study provides future insights for better utilization of genetic manipulation techniques for engineering PGPR strains for the synthesis of value added antifungal compounds.

Fig. 3
figure 3

Engineering of PGPR for enhanced production of Phenazine-1- carboxylic acid (PCA). GlpR gene which negatively regulates the glycerol utilization and mgsA gene regulating the conversion of glycerol to pyruvaldehyde was knocked out. P. chlororaphis GP72 mutant co-expressed glpF and glpK genes to overproduce PCA

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Limitations in current findings and future prospective

Figure 4 shows different tools that are presently in use for carrying out gene manipulations in PGPR. Generous amount of work has been done to fabricate novel PGPR biological units with desired functions or to engineer existing biological systems to augment their endowed potentials. Towards this approach, synthetic biology tools such as indexing biological parts, genome sequencing, synthetic genome synthesis and genome editing have played a key role. In context of PGPR, ligation of desired genes/set of genes and desired biological parts including promoters, terminators have been employed to enhance the PGPR potential in rhizobacteria. Desired functional genes are integrated successfully into host’s endogenous genome and overexpressed via transposon mediated modifications. Non-rhizospheric bacteria have also been engineered to show PGPR activity by the creation of synthetic genetic circuits. However, there are certain technical barriers in this approach that are being addressed by next generation sequencing methods. Biological parts that include promoters, terminators, operons are host specific and may not work in recombinant bacteria as expected. This calls out for need to build host-specific libraries of biological parts and the well-designed regulatory circuits. For the formation of new gene clusters biological parts should be simultaneously assembled (Lee et al. 2013).

Fig. 4
figure 4

Tools used for synthetic genome designing in PGPR. Genome sequencing, transcriptome analysis, genome synthesis and RNAi technologies are presently used for gene manipulation of PGPR. Construction and redesigning of PGPR in future requires the use of Zinc Finger Nuclease, Transcription Activator-Like Effector Nuclease and CRISPR/Cas9 nuclease

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Researchers have successfully knockout several downregulating genes through RNA interference (RNAi). Genome reduction approach by deletion or mutational inactivation of several genes is key tools in gene manipulation for enhancing PGPR activity in rhizobacteria. Off-target or unintended effects can often affects the RNAi-induced phenotypes (Martin and Caplen 2007). Fortunately, genome sequencing, transcriptome analysis, genome synthesis and RNAi technologies have helped designing of a predictable biological system. The current studies have focussed mainly on the isolation of desired gene and cloning it in host rhizobacteria or genome reduction approach to downregulate certain genes. Present studies are aimed at focussing single PGPR attribute of rhizobacteria and lack the characteristics of a so called ‘single fit biofertilizer’. Most of these current studies have not yet advanced into field trails. Despite these limitations, there is still a need to pay attention on engineering of PGPR for agricultural use. Creation of robust biological systems requires precise gene manipulations techniques that make changes to specific gene loci. Development of engineered endonucleases with versatile DNA binding domains such as CRISPR/Cas9 nuclease (Clustered Regularly Interspaced Short Palindromic Repeats), Zinc Finger Nuclease (ZFN) and Transcription Activator-Like Effector Nuclease (TALEN) have provided a major breakthrough in gene targeting technology (Zhang et al. 2018). However, there is a paucity of studies depicting the success of ZFN, TALEN and CRISPR/Cas9 nuclease for engineering of rhizobacteria for agriculture application.

Conclusion

Although underutilized, microbial consortia are expected to play crucial role in soil fertilization to create nutritional synergy with ecosystem. Commercial success of PGPR has been restrained due to inconsistency to perform in varied soil types, environment conditions, plant types and ecological zones. With wide agro-climatic zones, diverse cropping systems and different soil types, it is mandatory to involve genetic manipulation techniques for improving the survival rates and enhancing the efficiency of PGPR strains. The advances in next generation sequencing techniques, high specificity of gene editing and bioinformatics tools have allowed the deep insights into translation and expression of different genes of interest (Von Bergen et al. 2013; Melcher et al. 2014). The next generation agriculture revolution is possible only if ‘microbiome-driven cropping systems’ becomes a reality. Kumar and Dubey (2020) have described that the use of synthetic biology systems to purposely revamp the plant–microbe association can also enhance the overall sustainability and efficiency of PGPR. Apart from discussed microbe-based approach, plant based and the meta-organism based approach can also act as key players in enhancing the role of PGPR for plant use. CRISPR-CAS9 approach can be use to reshape the plant genome and root architecture to host PGPR and enhance their survival. In upcoming years, we can expect the use of metagenomics, other omic technologies, and genetic manipulation tools to be used in synergy for the selection of beneficial microbes. Although, voluminous amount of work has been done to engineer bacteria to enhance their beneficial characteristics; there is a need to redesign the strategies to evolve strains that can completely replace chemical fertilizers/pesticides.

Author contribution statement

The research proposal was designed by Dr. SK. Ms. RK, performed literature review and paper writing. Figures and tables were made by Ms. GK. Final paper editing and improvement was followed by Dr. SK.