Keywords

1 Introduction

Since the onset of civilization, agriculture has been the most important source of income (Gouda et al. 2018). Food security has been one of society’s primary issues for a long time, and any element that threatens it has been one of society’s challenges. With an increasing population rate and an unsustainable traditional agricultural system, farmers and the government are struggling with how to produce enough food to fulfill global demand (Prajapati et al. 2022a, b; Khoshru et al. 2020). According to the FAO, agricultural land covers 38.47% of the world’s land area, and while 28.43% of that land is arable, only 3.13% is permanently used for crop production. The issue has deteriorated as 20–25% of land worldwide is degraded each year, with another 5–10 million hectares destroyed each year. The movement of nutrients, energy, and carbon between soil organic matter, the soil environment, the aquatic ecosystem, and the atmosphere has a significant impact on agricultural productivity, water quality, and climate change (Gouda et al. 2018).

Abiotic stresses are major constraints of plant growth and development, which in turn affects crop yield, food quality, and global food security. Under stress conditions, numerous parameters such as biochemistry, molecular biology, and physiology of plants are affected. The use of chemical pesticides and inorganic fertilizers causes environmental pollution and degradation of soil fertility. During the stress period, the plant releases certain exudates that can act as a signaling mechanism to alter or create a healthy rhizosphere soil community (Shaikh et al. 2022; Prajapati et al. 2022a, b).

A well-studied and sustainable alternative for improving plant growth and soil fertility is the application of plant growth-promoting bacteria (PGPB) as biofertilizers, which possess functional traits that regulate the growth, development, and productivity of crops. These growth-promoting effects are due to the improvement of the availability and biosynthesis of several limiting macro- and micronutrients, as well as crop protection against stressful environmental conditions. Plant growth-promoting microorganism (PGPM) is a term that applies to all microorganisms (e.g., bacteria, actinomycetes, fungi, and algae) that have a beneficial effect on plant growth through the action of either direct or indirect mechanisms (e.g., mineral nutrition, ethylene reduction, disease suppression). PGPMs have a significant role in sustainable agriculture. They increase the production of various crops, improve soil fertility, promote diversity and interaction with other beneficial microorganisms, inhibit the growth and infective action of potential pathogens, and generally maintain the sustainability of the systems (Prajapati et al. 2022a, b; Santoyo et al. 2021).

The application of microbial consortia to agricultural fields is an innovative natural approach, which can help plants tolerate different stress conditions and enhance plant growth as compost is made up of diverse microbial consortia that can function in different temperature segments (Sathiavelu 2021).

2 Strategies for the Development of Microbial Consortia/Rhizobacterial Consortia

2.1 What Are Microbial Consortia?

Rhizobacteria that stimulate plant growth are a symbiotic association between plants and microbes found in the rhizosphere that boost plant growth (Rochlani et al. 2022). The roots are referred to as rhizomes, and the surrounding environment is referred to as spheres. The rhizosphere is the zone of soil that surrounds a plant’s root system. The zone, which is around 1 mm wide, has no defined edges. Rhizobacteria are bacteria found in the rhizosphere that can create an environment for roots (Rochlani et al. 2022; Jha and Saraf 2015). The varied microbial communities of the rhizosphere enable the formation of microorganisms that can stimulate plant growth under abiotic conditions via direct and indirect mechanisms (Rochlani et al. 2022; Shaikh et al. 2022; Saraf et al. 2017).

Currently, agriculture is heavily dependent on mineral fertilizers and inorganic pesticides (inorganic), and the impact of their continuous application is reflected in deteriorating soil health and increased resistance to pests and pathogens (Prajapati et al. 2022a, b). In the past 40 years, usage of nitrogen fertilizers has increased by sevenfold and pesticide usage by threefold. In the future, these trends will continue unabated, as the application threefold of both inorganic fertilizers and pesticides is expected to increase by an additional threefold by 2050, which will cause unprecedented damage to the agroecosystem (Sekar et al. 2016). Engineering the plant rhizomicrobiome is an alternative approach to increasing soil health and enhancing plant productivity (Pindi and Satyanarayana 2012). Microbial interaction in the rhizosphere provides plants with multiple plant growth-promoting traits and different stress-tolerant traits apart from enhancing their own population and function (Sekar et al. 2016; Keswani et al. 2014). The inconsistency in the performance of a single microbial product in field application has emphasized the need for co-inoculation or consortia of the microbial products (Santoyo et al. 2021).

Although numerous studies have shown that single microorganisms can benefit plants, it is becoming increasingly clear that when a microbial consortium (mixed culture)—two or more interacting microorganisms—is involved, additive or synergistic results can be assumed. This is owing, in part, to the fact that multiple species can perform a range of activities in an ecosystem like the rhizosphere. The use of mixed cultures of beneficial microorganisms as soil inoculants is based on the principles of natural ecosystems, which are sustained by the quality and quantity of their inhabitants and specific ecological parameters, i.e., the greater the diversity and number of inhabitants, the higher the order of their interaction and the more stable the ecosystem (Higa 1994). The mixed culture technique is essentially an attempt to apply these ideas to natural systems such as agricultural soils in order to alter the microbial balance in favor of enhanced plant growth, productivity, and protection (Santoyo et al. 2021; Higa 1994;).

Beneficial plant growth stimulation mechanisms include increased nutrient availability, phytohormone modulation, biocontrol, and biotic and abiotic stress tolerance exerted by various rhizosphere microbial players such as plant growth-promoting bacteria (PGPB) and fungi such as Trichoderma and Mycorrhizae (Prajapati et al. 2022a, b; Santoyo et al. 2021).

The influence of different PGPR strains on plants has been thoroughly investigated in recent years, leading to the commercialization of a significant number of microbial inoculums (Santos Villalobos et al. 2018; Reed and Glick 2013). The construction of bacterial consortia has received interest as a feasible technique for sustainable food production to improve the beneficial capabilities exhibited by these bacteria. In rare circumstances, a consortium of several strains of the same species can display improved activity and be considered. Due to their coverage of a varied set of plant growth promotion and biological regulatory mechanisms, bacterial consortia have been shown to boost beneficial traits in plants as compared to individual strains (Ju et al. 2019). The adoption of these consortia is a viable technique for improving agricultural crops under drought (Joshi et al. 2020), salinity (Sharma et al. 2022c; Nawaz et al. 2020), heavy metal (Prajapati et al. 2022a, b), nutrient uptake (Rana et al. 2012), pests, and phytopathogenic diseases (Villa-Rodriguez et al. 2019). Furthermore, some bacterial consortiums can fix nitrogen, convert some inaccessible nutrients into assimilable forms, produce phytohormones, and chelate iron, all of which are important in maintaining soil quality and health; these can also mitigate the negative effects of some conventional nonsustainable farming techniques (Shaikh et al. 2022).

Rhizobacterial consortia are classified into two types: simple and complex. The fermentation method or protocol (generation of a large population of bacteria to be later made into an inoculant), in which strains are grown individually or in combination with other species/strains in a suitable medium for all PGPR species (Bashan and Prabhu 2020), is the difference. This is an essential stage because a greater number of species often results in a greater number of interactions between strains, resulting in changes in metabolite secretions. The effectiveness of bacterial consortia in field conditions, on the other hand, is reliant on the type and function of strains utilized, where some elements demand special consideration, such as tolerance to severe climatic conditions, survival, and persistence in the soil after inoculation (Gosal and Kaur 2017; Verbruggen et al. 2013;).

The source of the strain isolation influences the selection of these strains because consortium members must grow in the environmental conditions (soil type, host, and climate) where they will be applied. Additionally, when two or more strains form a rhizobacterial consortium, each strain not only competes with the others functionally for plant growth promotion but also complements the others for soil and/or plant establishment (Sharma et al. 2020; Ney et al. 2018; Morriën 2016; Pandey et al. 2012) (Fig.9.1).

Fig. 9.1
An illustration represents that the microbial consortia of P G P B + P G P R, Arbuscular mycorrhiza + P G P R, and Trichoderma + P G P R promote plant growth and fitness under abiotic stress. The synergistic beneficial effects are listed on the right side.

Combining different rhizosphere microorganisms to form microbial consortia can promote plant growth, development, and nutrient uptake while also improving the plant defense system against diseases and enhancing tolerance to various environmental stresses

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Though the concept of the consortium is theoretically feasible, developing a consortium is a challenge for researchers due to factors such as the mutual compatibility of microbes, their reliance on one another, and the task of maintaining inoculum potential while not depleting plant resources excessively during mutualism/symbiosis. There is currently no conventional or experimentally confirmed process for screening and choosing promising consortia among a vast number of theoretically feasible consortia. The traditional hit-and-trial strategy yields a vast number of combinations and time-consuming approaches (at least 4 months), which are applied at random, leaving room for scientific improvisation. To address this specific issue, we explored the various methodologies for evaluating rhizobacterial microbial consortia (Fig. 9.2), as outlined below.

Fig. 9.2
A flow diagram has 6 steps. The steps are as follows. 1. Check the plant growth stimulating properties of the selected rhizobacteria and classify into groups. 2. Pair every rhizobacteria member. 3. Examine P G P Bs' capacity. 4. Investigate the mitotic growth pattern and inoculum potential of individual P G P Bs. 5. Invitro rapid bioassay, and 6. Recheck and select the most promising consortia combination for greenhouse studies.

The “strategies” for selecting the most promoting consortia of plant growth-promoting bacteria

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2.1.1 Step 1: Analysis of Traits of Plant Growth-Promoting Rhizobacteria

Beneficial bacteria and fungi that act as plant growth-promoting microorganisms (PGPMs) can alleviate stress and stimulate plant growth in two ways: indirectly by inducing defense mechanisms against phytopathogens and/or directly by solubilizing mineral nutrients (nitrogen, phosphate, potassium, iron, and so on), producing plant growth-promoting substances (e.g., phytohormones), and secreting specific enzymes (e.g., 1-aminocyclopropane1-carboxylate deaminase).

To prepare possible rhizobacteria consortia, all selected rhizobacteria could evaluate for plant growth-enhancing properties. The following are plant growth-enhancing attributes:

  1. (a)

    Phosphate solubilization: The ability of rhizobacteria to solubilize insoluble phosphates has been investigated using Pikovskaya’s medium (Pikovskaya 1948). Each rhizobacterial culture spot could subsequently be inoculated in the center of Pikovskaya’s media agar plates with tricalcium phosphate [Ca3(PO4)2] as an insoluble phosphate source. Rhizobacteria that can dissolve insoluble phosphates will generate halos. Using the diameter of clearing halo zones, the P solubilization index (PSI) is determined using the formula below (Rathod and Saraf 2021a; Jain et al. 2020).

    $$ \mathrm{Phosphate}\ \mathrm{solubilization}\ \mathrm{index}\ \left(\mathrm{PSI}\right)=\frac{\mathrm{Colony}\ \mathrm{diameter}+\mathrm{Halozone}\ \mathrm{diameter}}{\mathrm{Colony}\ \mathrm{diameter}} $$
  2. (b)

    Siderophore production: Iron [Fe (III)] is required by all organisms as a cofactor for numerous critical metabolic activities. Siderophores are low-molecular-weight organic ligands secreted by soil microorganisms that bind to iron and release it for microbial absorption. Iron shortage in metal-stressed soils can be remedied using siderophores generated by various bacterial genera. Siderophores increase IAA production by chelating hazardous metal species, and IAA has been shown to benefit metal remediating plants. The CAS agar method is used to determine siderophore production (Schwyn and Neilands 1987). In the dark blue medium, the formation of a bright zone with yellowish (hydroxamate), pinkish (catecholate), and whitish (carboxylate) colors indicated the production of siderophore (Saraf et al. 2017).

  3. (c)

    Hydrogen cyanide and ammonia production: The production of HCN and ammonia is regarded as an indirect plant growth enhancer. A volatile chemical with antifungal qualities is HCN. In addition to helping the host plant meet its nitrogen needs, ammonia production can help prevent disease invasion. HCN’s strong toxicity against phytopathogens makes it a popular biocontrol agent in agricultural systems. However, HCN is also used to chelate metal ions and therefore indirectly contributes to the availability of phosphate (Mahmud et al. 2021). Alström and Burns (1989) found that the synthesis of HCN by rhizobacterial culture could be determined by the color change of filter paper. A change in the color of the filter paper from yellow to light brown, brown, or reddish-brown had been recorded as a weak (+), moderate (++), or strong (+++) production of HCN. NH3 production could be determined by the method described by Cappuccino and Sherman (1992). The formation of yellow to brown precipitate showed the presence of NH3 (Trivedi et al. 2018; Jha and Saraf 2011).

  4. (d)

    Indole acetic acid production (IAA): The synthesis of indole acetic acid was determined using the method described by Bric et al. (1991). The pink color that developed after adding Salkowsky’s reagent to cell-free supernatant was spectrophotometrically measured (Shah et al. 2020; Patel et al. 2012).

  5. (e)

    Biocontrol activity: The agar diffusion method can be used to assess antibacterial and antifungal activity (Sharma et al. 2022b; Thakkar and Saraf 2015).

2.1.2 Step 2: Compatibility Efficiency Studies

Individual plant growth-promoting rhizobacteria in the consortia must cultivate in synchrony to exert synergistic effects on plant growth. For that, a paired-wise growth performance study could be conducted in vitro to examine the presence of any antagonism among individual members of the two and more plant growth-boosting rhizobacteria groups. The compatibility efficiency assay has been constructed so that every PGPM member in group I received challenges with every other PGPM member in group II and group III and more. Overnight-grown broth cultures of the relevant PGPMs (one from each group I and II) could be streaked in two halves of nutrient agar. After incubation at optimum temperature, all plates were evaluated for the presence of any zones of inhibition at the colony borders where the two cultures intersected. If the counterpart did not show any zone of inhibition, it suggests the absence of any diffusible toxins or volatile substances that could cause antagonism against each other (Rathod et al. 2020; Rathod and Saraf 2021b; Prasad and Babu 2017). This study provides proof that the tested plant growth-boosting rhizobacterial consortia are growing in a mutually noninhibitory manner, paving the way for further research on microbial consortia.

2.1.3 Step 3: Sensitivity to Physical and Chemical Conditions

Temperature responses might have been measured in the 20–45 °C range, while pH can be studied at optimal temperatures in the 5–11 range (with unit interval). In both trials, an aliquot of overnight-developed plant growth-promoting rhizobacterial cultures has been employed as inoculum. Each experiment was carried out in triplicate to ensure that the results appeared reproducible. Rhizobacteria growth has been assessed spectrophotometrically after overnight incubation at various temperatures and pH levels (Sharma and Saraf 2022).

2.1.4 Step 4: PGPR Growth and Mitotic Behavior

Generation times of co-habiting rhizobacteria in microbial consortia would ensure a balance in the relative inoculum density of distinct isolates. If one microbial consortia member develops faster, it may deplete the medium’s nutrients and provide unsuitable growth circumstances for the other members of the consortia. The same mitotic growth behavior of PGPR consortia supports co-survival ability and their attractiveness as prospective candidates for consortia creation. At the most optimum temperature and pH conditions, the growth kinetics of each plant growth-promoting rhizobacteria can be studied. Overnight-developed plant growth-promoting rhizobacteria culture was inoculated in triplicate in nutrient broth and incubated in a BOD incubator at 150 rpm. Then, at regular intervals, culture broth aliquot could be collected and a growth curve could be produced with time (in hours) on the x-axis and absorbance (OD measured at 600–610 nm) on the y-axis (Sharma et al. 2021; Jha and Saraf 2012). The generation time can be calculated using standard methods. The generation time is the amount of time it takes for the cells (or population) to divide (Todar 2015).

$$ \mathrm{G}\ \left(\mathrm{generation}\ \mathrm{time}\right)=\kern0.5em \frac{t\left(\mathrm{time},\mathrm{in}\ \mathrm{minute}\ \mathrm{or}\ \mathrm{hours}\right)}{n\ \left(\mathrm{number}\ \mathrm{of}\ \mathrm{generations}\right)} $$

n = 3.3logb/B

$$ \mathrm{G}\ \left(\mathrm{generation}\ \mathrm{time}\right)=\kern0.5em \frac{t\left(\mathrm{time},\mathrm{in}\ \mathrm{minute}\ \mathrm{or}\ \mathrm{hours}\right)}{3.3\log b/B} $$

t = time interval in hours or minute.

B = number of bacteria at the beginning of a time interval.

b = number of bacteria at the end of the time interval.

n = number of generations (number of times the cell population doubles during the time interval).

2.1.5 Step 5: Design of Microbial Consortia

Microbial consortia combinations can be studied for their plant growth stimulating efficiency using a two- and three-factorial design approach, ensuring that each consortium comprises one member from group I (perhaps rhizobacteria) with two members from groups II and III (may be endophyte) and may be between different rhizobacteria. Under aseptic conditions, equal amounts of overnight-grown cultures of the various PGPMs (~108–109 cfu/mL) are mixed together to form consortia combinations, which would then be employed for application in agriculture crop and selecting consortia combinations that show significant increments in vegetative growth parameters as compared with noninoculated crop (Sharma et al. 2022c; Mishra and Sundari 2017; Jha and Saraf 2012).

2.1.6 Step 6a: Rapid Plant Bioassay

Mishra and Sundari (2017) created a quick plant bioassay technique that has been utilized to assess the microbial community. It is an in vivo plant growth study in which seeds are surface sterilized according to standard protocol, coated with the corresponding consortia combinations, and placed for germination. Mishra and Sundari (2017) established a novel experimental setup termed the “tube-in-tube” approach in their laboratory using germinated seeds with healthy radicals and plumules. The cap of the sterilized Eppendorf tube (1.5 mL) has been removed, and the bottom could be subsequently cut to create an opening. Germinated seeds were then transferred aseptically into this Eppendorf tube, and the assembly could then be placed in an autoclaved glass test tube (50 mL capacity), giving rise to the term “tube-in-tube” (Fig. 9.3).

Fig. 9.3
A flow diagram of the Rapid plant bioassay. The cap and bottom tip of the autoclaved Eppendorf tube are cut. A seedling is placed inside the Eppendorf tube and kept inside a test tube. The test tube is plugged and filled with a considerable amount of liquid medium.

Rapid plant bioassay (“tube-in-tube”) approach: (a) autoclave Eppendorf (1.5 mL); (b, c) Eppendorf cap, and bottom removed; (d, e) Eppendorf germinated seedling; f, Eppendorf in an autoclaved test tube with seedling; (g) a glass test tube containing medium; (h) plugged tube-in-tube system to preserve aseptic state for plant growth (Mishra and Sundari 2017)

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It is an in vivo plant growth study in which seeds can surface sterilized as per standard protocol and coated with the respective consortia combinations (detailed in seed germination paragraph) and placed for germination. Germinated seeds with healthy radicals and plumules could be selected and transferred to the novel experimental setup called the “tube-in-tube” method developed by Mishra and Sundari (2017) in their laboratory. The cap of the sterilized Eppendorf tube (1.5 mL) could be removed and bottom cut to make an aperture. Germinated seeds could be transferred aseptically into this Eppendorf, and the assembly could place in an autoclaved glass test tube (50 mL capacity), thus drawing its name “tube-in-tube” method. The test tube may hold 10 mL of half-strength modified Melin–Norkrans medium, free of glucose and malt. The entire “tube-in-tube” system could be then closed with sterile cotton to maintain aseptic conditions and incubated for 10 days at 30 2 °C with no sunshine regulation. To keep the root system in the dark, the bottom portion of the complete test tube rack carrying the setup was wrapped in a black sheet of paper (Fig. 9.2). SEM has been used to visually confirm bacterial attachment with plant roots after harvest. The influence of various PGPR consortia on plant growth may be assessed using four parameters: root length (RL), shoot length (SL), root dry weight (RDW), and shoot dry weight (SDW). The geometrical mean for the different combinations of RL and SL could be determined, and deviations among replicates could be reported as standard deviations (Mishra and Sundari 2017). This rapid plant bioassay technique (“tube-in-tube method”) proved to be effective for screening a large set of consortia combinations in a short span of time (Fig.9.3).

2.1.7 Step 6b: Pot Experiments

Rhizobacterial consortiums with promising plant growth-boosting properties are evaluated for seed germination. Seeds are surface sterilized with 0.2 percent HgCl2 for 2 min before being washed in sterile distilled water for 10 min. Seedlings for 7–8 h in YEMA/nutrient broth with a pre-screened rhizobacteria consortium combination in log phase containing approximately 108–109 CFU/mL are held at optimal temperature in a shaker. Control seeds are immersed in a sterile medium. The seeds are then dried aseptically in laminar air flow overnight before being employed in pot experiments. For pot experiments, only sterile soil should be used. Standard protocols could be used to analyze physicochemical parameters. Transfer the sterile soil to pots; the amount of soil used for pot studies is determined by the size of the pots. Standard germination (percentage) of seeds can be counted until no further germination occurs (Rathod et al. 2021; Jha and Saraf 2012). Seedling vigor indices could be determined using the formula proposed by Abdul-Baki and Anderson (1973) as follows: -

$$ \mathrm{Seedling}\ \mathrm{Vigour}\ \mathrm{Index}\ \left(\mathrm{SVI}\right)=\mathrm{Total}\ \mathrm{Seedling}\ \mathrm{Length}\ \left(\mathrm{cm}\right)\times \mathrm{Germination}\ \mathrm{Percentage}\ \left(\%\right) $$

After 1 month, the vegetative growth parameters are assessed to assess the influence of microbial consortia on plant growth in comparison to noninoculated pots. Confirm the presence of microbial consortia on the roots of plants using scanning electron microscopy (SEM) and transmission electron microscope (TEM) techniques.

By applying step-to-step strategies, the best and most manageable number of consortia are thus shortlisted for further field trials to improve productivity in a sustainable manner.

3 Microbial Consortia on Plant Roots: Scanning Electron Microscopy (SEM)/Transmission Electron Microscopy (TEM)

Basically, SEM is used in microbiology for analyzing the organism’s morphological structure and measurement of size. Nowadays, it is widely used for the observation of microorganisms adhering on plant parts (Root, Shoot, etc.). Trivedi and Saraf (2019) studied endophytes from the Ricinus communis plant’s stem, leaves, and root. Despite being an excellent tool for investigating ultrastructure, scanning electron microscopy (SEM) is less frequently used than transmission electron microscopy for microbes such as viruses or bacteria. SEM could be used for visual confirmation of bacterial association with plant roots. Olivares et al. (2017) observed the H. seropedicae strain HRC54 attached with humic acid plates on the sugarcane leaf surface. Kim and Krcmcr (2005) utilized SEM techniques to detect IAA-producing bacteria viz. Pseudomonas putida, Bacillus megaterium, Bradyrhizobium japonicum, etc., which had been isolated by the IAA screening method based on an in situ membrane assay (Bric et al. 1991) (Fig. 9.4).

Fig. 9.4
An SEM of P G P C in the roots of the Sorghum bicolor plant. The arrows point to the capsule-shaped P G P C on the roots.

SEM of PGPC association with Sorghum bicolor plant roots (Mishra and Sundari 2017)

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Mishra and Sundari (2017) developed the microbial consortia with Pseudomonas and diazotrophs applied to Sorghum bicolor plant as plant growth-promoting consortium (PGPC). Colonization pattern of primary tomato roots by Pseudomonas fluorescens SEM aids in evaluating consortium formation and activity on plants to identify specific microorganisms present in the plant when the consortium can measure multiple microorganisms, as well as their size and structure.

Transmission electron microscopy (TEM) is used for seen internal structure by sending an electron beam across a sample. As a result, an image of the sample’s internal structure is created up to 50 million times from its original size. TEM, on the other hand, produces a two-dimensional image. The organism having the ability to accumulate metal has been seen in TEM (Avendaño et al. 2016). Trivedi and Saraf (2019) examined selenium accumulation in endophytic selenobacteria using transmission electron microscopy (TEM) (Fig. 9.5). Sodium selenite had decreased and accumulated in the bacterial cell because of the accumulation of elemental selenium. The bacterial cell revealed red coloring because the reduced form of sodium selenite is red in color. In the presence of selenite, most of the selenium build-up occurred in the internal cell membrane of bacteria with wavelengths of 174.65 nm, 74.79 nm, and 76.32 nm, according to TEM imaging (Trivedi et al. 2020).

Fig. 9.5
A TEM of Parburkholderia megapolitana species M G T 9 in the scale of 500 nanometers. The species has a rod-shaped structure with measurements marked on it.

Transmission electron microscopy (TEM) of selenium particle accumulation around endophytic selenobacterial isolates Parburkholderia megapolitana sp. MGT9. (Trivedi and Saraf 2019)

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Transmission electron microscopy (TEM) micrographs showed the spherical NPs, which had an average diameter of 12 nm. By mixing it with an aqueous solution of AgNO3, Argemone mexicana leaf extract serves as a capping and reducing agent in the creation of AgNPs. Plant-based green synthesis of silver nanoparticles and its effective role in abiotic stress tolerance in crop plants were explained by Alabdallah and Hasan (2021). The use of TEM is beneficial in the field of microbiology because it can be used to identify and measure the structure and size of microorganisms that can bind or chelate metals, especially in contaminated sites and waste treatment facilities for dump yards. TEM images can also be used to resolve metal-contaminated fields or accumulate micro and macro metals in crops.

4 Role of Microbial Consortia as Efficient Biofertilizer

Microbial inoculants are mixtures that contain live algae, fungus, and bacteria, whether in alone or in a consortium, to boost plant growth and increase agricultural output. Beneficial microbes (algae, fungus, and bacteria alone or in a consortium) in biofertilizers improve soil chemical and biological attributes by fixing nitrogen, cellulolytic activity, iron, or phosphate (Mahmud et al. 2021; Seenivasagan and Babalola 2021). Microbes mostly as biofertilizers accomplish beneficial actions such as phosphorus solubilization, nitrogen fixation, siderophore formation, hydrogen cyanide, and ammonia synthesis, and the production of plant growth chemicals. Because of the presence of these bacteria, plants have antagonistic effects on a variety of phytopathogens (Rochlani et al. 2022; Jha and Saraf 2012). They inhabit the rhizosphere, whether applied to seed, plant surfaces, roots, or soil, and through their biological activity, they improve nutrient bioavailability, boost plant growth, and increase soil microflora. As a result, they are preparations that quickly restore soil fertility (Mahmud et al. 2021; Seenivasagan and Babalola 2021; Jha and Saraf 2015). They are critical elements of integrated nutrient management (INM) strategies for increasing soil productivity and sustainability while also preserving the environment because by being pollution-free, cost-effective, and a source of renewable nutrients to plants to replenish synthetic fertilizers in a sustainable production system (Yadav and Sarkar 2019). According to Panda (2011), the impact of bio-fertilizers on crop improvement ranges from 35% to 65% (Mahmud et al. 2021). The continual application of biofertilizer to the land for 3–4 years can retain fertility due to the efficacy of parental inoculums, which can successfully maintain plant growth and multiplication. They improve the texture, pH, and other characteristics of the soil. Biofertilizers are low-cost, sustainable sources of plant nutrients that are supplemental artificial fertilizers. In comparison to chemical fertilizers, biofertilizers are more environmentally friendly; they can be created from natural sources, are less likely to cause damage, and aid in the development of healthy soil. To some extent, plants are cleansed of chemical fertilizers that are precipitated (Seenivasagan and Babalola 2021). Depending on their capabilities, such as delivering nutrients to plants and acting as natural pest deterrents, a wide range of microorganisms can be used as biological fertilizers at the industrial level (Rochlani et al. 2022). When considering biofertilizer as a modern agricultural tool, its use is critical as a component of integrated nutrient management, a reduction in the use of hazardous chemicals, a cost-effective source of renewable energy for plants, and a source of renewable energy for plants in sustainable agriculture (Seenivasagan and Babalola 2021).

5 Mechanisms as Biofertilizer

Biofertilizers are classified into several categories based on their functional capabilities, such as nitrogen-fixing biofertilizers, phosphate biofertilizers, micronutrient biofertilizers, and plant growth-promoting rhizobacteria, among others. Nitrogen-fixing biofertilizers increase soil nitrogen levels by absorbing atmospheric nitrogen and releasing it to plants. Azotobacter, Nostoc, Rhizobium, and Azospirillum are a few examples (Itelima et al. 2018). Phosphate biofertilizers are divided into two types: phosphorous solubilizing biofertilizers (PSB) and phosphorus mobilizing biofertilizers (PMB). PSB dissolves insoluble phosphate from organic and inorganic sources. Bacillus, Pseudomonas, Penicillium, Aspergillus, and other bacteria are examples (Etesami et al. 2017). Phosphorus is transferred from the soil to the root cortex via PMB. Arbuscular Mycorrhiza is one example (AM fungi). Micronutrient biofertilizers include silicate and zinc solubilizer bacteria. In soil, these bacteria break down silicates and aluminum silicates. Bacillus sp. is one example. Plant growth-promoting rhizobacteria (PGPR) are bacteria that live in the rhizosphere (Upadhyay et al. 2019). The rhizosphere is a thin layer of soil surrounding the roots characterized by high levels of biochemical activities and composed of plants, bacteria, fungi, and soil constituents. They boost plant growth by functioning as bioprotectants, biostimulants, and nutrient enhancers (Fig. 9.6).

Fig. 9.6
A diagram of the plant's underground and aboveground areas. When biofertilizers are inoculated into the plant, they improve soil health, maintain microbial consortia in the soil, maintain a symbiotic relationship, eradicate soil pathogens, eliminate the need for chemical fertilizers, and provide a sufficient supply of nutrients.

The effect of biofertilizers on plant growth and soil health. [VAM vesicular-arbuscular mycorrhiza, ISA increased soil aggregation, ISP increased soil porosity] (Mahmud et al. 2021). https://www.researchgate.net/figure/Schematic-representation-Influence-of-biofertilizers-on-plant-growth-performance-and_fig1_353131148

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The mechanism of action refers to the biological and chemical process by which microorganisms contained in biofertilizers exert their effects on the plant’s rhizosphere. Plant growth rhizobacteria can execute a variety of mechanisms that increase plant growth and development, eventually leading to sustainable agriculture methods. Direct mechanisms of these rhizospheric bacteria can increase plant growth by increasing nutrient intake via nitrogen fixation, phosphate solubilization, phytohormone production, and exopolysaccharide production, resulting in sustainable and eco-friendly agri-science perspective. These microorganisms also have an indirect role in plant protection by producing antibiotics, hydrogen cyanide, siderophores, and other biocontrol chemicals (Rochlani et al. 2022; Prajapati et al. 2022a, b; Panchal et al. 2022). Surprisingly, these relationships between plant–root and microbial communities have been labeled as symbiosis. As the former decomposes unavailable nutrients into an available form, the latter benefit from root exudates such as carbohydrates, proteins, sugars, vitamins, mucilage, amino acids, and organic acids (Vives-Peris et al. 2020), which modify biochemical properties of the rhizosphere by acting as a messenger between the microbes and the plants (Shaikh et al. 2022; Vives-Peris et al. 2020).

6 Role of Microbial Consortia to Remediate Abiotic Stress

6.1 Abiotic Stress Affecting Crop

There are various types of abiotic stress that affect soil and ultimately crop productivity. These stresses are salinity stress (increase in salts concentration in soil), drought stress (insufficient water availability to plants), heavy metal stress (excessive harmful metals in soil), temperature stress (very high and very low temperature), and nutrients stress (insufficient nutrients in the soil) (Fig. 9.7). In this review, we describe three major soil stress: drought, salinity, and heavy metals. Drought and salt stress have a complex relationship that affects almost every element of a plant’s life. Both these stresses have the most detrimental effects on agriculture (de Oliveira et al. 2013). Stress causes disturbance in photosynthesis, resulting in leaf senescence, the formation of excessive reactive oxygen species (ROS), nutritional deprivation, and the breakdown of cellular organelles and metabolism, all of which result in diminished plant growth (de Oliveira et al. 2013). Another key soil stress is heavy metal stress, which is getting more intense because of a variety of anthropogenic influences (Glick 2010). Unchecked population growth and the industrial revolution are accumulating toxic metals and organic wastes in soil, rendering it unfit for agricultural techniques and detrimental to all living things (Glick 2010).

Fig. 9.7
A radial diagram of microbial consortia. Surrounding labels are salinity stress, drought stress, heavy metals stress, hydrocarbon stress, heat and p H stress, and nutrient deficient stress.

Microbial consortia as biofertilizer to remediate abiotic stress

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One of the most common abiotic factors impacting crop plants is water deprivation. Drought stress occurs when the amount of water available in the soil diminishes. Drought produces a variety of harmful consequences on plants that are multifaceted in their effects. From seed germination to maturity and senescence, the plants respond to drought stress at physiological, biochemical, and molecular levels (Tiwari et al. 2017). Because plants need to use groundwater, their root length increases under mild drought stress (Forni et al. 2017), while extremely dry conditions can slow root growth. However, PGPR under stress conditions modifies root architecture and boosts plant nutrient absorption and water drawing ability (Shaikh et al. 2022; Kasim et al. 2013). These rhizobacteria could be able to grow under stressful conditions and provide a beneficial effect on plants to cope with stressful environments (Jain et al. 2020; Jain and Saraf 2021; Bilal et al. 2018). ACC (1-aminocyclopropane-1-carboxylate) deaminase activity, production of exopolysaccharide (EPS) (Panchal et al. 2022) and volatile organic compounds (VOCs), osmolyte and antioxidant production, enhanced mineral nutrient uptake, phytohormone production, and modulation are among the mechanisms proposed by PGPM to overcome drought stress in plants. The PGPRs are bestowed through these pathways, either singly or jointly, to counteract drought stress in plants (Gontia-Mishra et al. 2020). Microbial consortia of Pseudomonas putida NBRIRA + Bacillus amyloliquefaciens NBRISN13 mitigates drought stress in chickpea by enhancing physiological parameters such as shoot length, root length, and fresh and dry weight of root and shoot, modulates defense enzymes such as superoxide dismutase, catalase, lipid peroxidase, and enhance soil enzymes activity and microbial diversity in the rhizosphere region under drought stress (Kumar et al. 2016).

Soil salinity is defined a salt level that exceeds the plant’s requirements. When the electrical conductivity (EC) in the soil surrounding the root zone reaches 4 dS/m (40 mM NaCl), the soil becomes saline (Egamberdieva et al. 2017). Excessive salt concentrations causing low water availability create drought-like conditions and result in altering the physicochemical features of soil and interfering with nutrient uptake, rendering nutrients unavailable to plants. Salt stress affects plant growth, photosynthetic capacity, CO2 assimilation, and nitrogen content and leads to ion toxicity, which results in oxidative stress (Liu et al. 2015). However, the combination of compatible yet dissimilar genera of microbes can greatly boost plant growth under saline circumstances and may aid in salinity amelioration (Kapadia et al. 2021). Microbial consortia provide a variety of essential tasks under salinity conditions, including promoting plant growth, acting as osmoprotectants, antioxidants, and biocontrol agents, and reducing stress in the soil. Microbial consortia of four rhizobacteria strains Bacillus sp. + Delftia sp. + Enterobacter sp. + Achromobacter sp. helps to overcome the salinity stress in tomato. Consortia alleviate salt stress in tomatoes by increasing plant growth parameters, chlorophyll content, mineral uptake, accumulation, and transportation to a different part of the plant (Kapadia et al. 2021).

Heavy metals (HMs) are described as elements with metallic characteristics and a wide molecular weight range, which includes transition metals. Metal concentrations in soil have risen considerably because of the industrial revolution and human activities (Dabhi et al. 2021; Sharma et al. 2021). Plant metabolism and growth are harmed by the abundant HM in soil, which is absorbed and translocated to numerous organs of plants (Cheng 2003). Excess metals in soil have an adverse effect on soil characteristics and fertility, making it unfit for agricultural uses (Khan et al. 2012). Due to well-known plant growth-enhancing mechanisms such as hormone production (IAA, GA), siderophore generation, nitrogen fixation, and phosphate solubilization, PGPR can be used to help phytoremediation contaminated sites (Ojuederie and Babalola 2017). Heavy metal tolerance and accumulation by plants may be significantly influenced by heavy metal-resistant bacteria living in the rhizosphere. Rhizobacteria isolated from the landfill site and mining areas are able to tolerate heavy metal stress (Sharma and Saraf 2022). Application of rhizobacteria consortia of Bacillus cereus MG257494.1, Alcaligenes faecalis MG966440.1, and Alcaligenes faecalis MG257493.1 shows tolerance against heavy metals (Cu, Pb, Cd, and Zn), and their application on sorghum mitigate heavy metal stress by increasing the dehydrogenase activity, decreasing metal accumulation in plant parts and soil, also regulating bioaccumulation factor (BAF) of heavy metals (Abou-Aly et al. 2021). Some of the applications of microbial consortia under abiotic stress are listed in Table 9.1.

Table 9.1 Abiotic stress alleviation by microbial consortia
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7 Conclusions

Microbial consortium is part of the plant microbiome that interacts synergistically to promote plant growth and health through the production of metabolites with antibiotic activity and by solubilizing nutrients and making them available to the plant, forming nodules to fix nitrogen, and producing plant-growth-stimulating phytohormones or enzymes that degrade ethylene precursors, such as ACC deaminase. This review presents a consortium screening protocol as a step-to-step strategy to develop microbial consortia to construct, evaluate, and shortlist the most potent microbial consortia. The review described a factorial design involving two and more representative groups and has many PGPRs to facilitate the selection of the most auspicious combinations for larger greenhouse trials before developing bio-inoculants. In vivo, rapid plant bioassays are obligatory to evaluate the performances of microbial consortia even when the isolates exhibit similar preference to physiological growth conditions, synergy in co-culture, and high mitotic activity. SEM and TEM help in the evaluation of the development of consortium and their activity on the plant to identify the microorganism, especially present in the plant where the consortium can measure multiple microorganisms, their size, and structure too. Biofertilizers have been used to boost crop production by augmenting the plant’s available nutrients through the organic matter decomposition process. Two main reasons necessitate the use of biofertilizers in today’s crop production. The first is to increase the use of biofertilizers, which results in the corresponding increase in crop yield, and the second is the long-term use of synthetic fertilizers degrading the soil besides other threats to our health and environment. The efficacy of biofertilizers can be enhanced by sound knowledge and long-time practical experience in a diverse soil type. Application of microbial inoculants, especially consortia, will be one of the solutions to alleviate plant abiotic stress, and enhanced plant growth and productivity under stress conditions have been reported. The directed use of microbial consortia will facilitate the production of plants in a more sustainable way that, eventually, will not depend on agrochemicals.