Diverse endophytic microbes colonize seeds forming some of the first bacterial associations in a plant's life cycle (López et al., 2018). These microbes include both bacteria and fungi (Nair and Padmavathy, 2014; Chimwamurombe et al., 2016). Seed endophytes have been observed to contribute to seed germination and cell elongation (Verma et al., 2017; Khalaf and Raizada, 2018). In addition, they form the initial microbial association for the promotion of the overall health of plants (Khalaf and Raizada, 2016). Seed endophytes can also remain quiescent in latent seeds. This means they only become active when germination begins. Furthermore, seed endophytes may be passed through to progeny with some changes occurring in the microbiome due to pathogenic infections, environmental changes or other stresses (López et al., 2018).

Seeds endophytic bacteria contribute positively to the general health of plants. Several species and genera have been identified as plant growth-promoting endophytic bacteria. Endophytic rice seedlings analysis revealed a diverse group of bacteria including Enterobacter asburiae, Pantoea dispersa and Pseudomonas putida. These were found to produce auxins, solubilize phosphates and inhibit pathogenic fungi (Verma et al., 2017). Through nitrogen fixation (Verma et al., 2017), hormone production (Chimwamurombe et al., 2016; Khalaf and Raizada, 2018) and antimicrobial activity (Nair and Padmavathy, 2014), endophytes improve abiotic stress tolerance and increase germination rates (Suman et al., 2016). Furthermore, they are also able to regulate hormone concentration thereby improving plant adaptation to environmental strains (Asaf et al., 2017).

Root nodules are small structures typically found on legume roots. These nodules are small ranging between 2 and 5 mm containing up to 10⁹ bacterial cells (Downie, 2014). Root nodule formation is triggered by simultaneous correlations between plants and their soil environment. The release of Nod factors into the soil by rhizobia temporarily activates plant genes that code for specific hormones (Spaink, 2000; Poehlman et al., 2019). Peptide hormones, for example, together with signal receptors and low levels of nitrogen in soil induce nodule formation with close association with nitrogen fixing bacteria (Taleski et al., 2018). However, nodule formation may be negatively affected by the absence of specific strains, low quorum and failure to colonize the rhizosphere (Prasanna et al., 2017). Though root nodules are mostly colonized by nitrogen fixing rhizobia, other microorganisms may also be found present in the nodules (Martínez-Hidalgo and Hirsch, 2017).

The formation of root nodules with the eventual colonization by bacteria is not fully understood however, it is known that nitrogen fixation is a result of this process. The process of nodulation is triggered by nitrogen levels in the soil with low levels initiating hormone signaling in the form of C-terminally encoded peptides (Verma et al., 2010; Taleski et al., 2018). Nod factors are produced by the bacteria as a response to signal molecules from the plant. These chemical signals include flavonoids which trigger the activation of Nod factor regulatory genes in bacteria (Spaink, 2000). This begins the process of infection with the rhizobial bacteria attached to root hairs. Once plant cell membranes detect the Nod factors, root hair deformation follows. A process that results in the nodule structure (Downie, 2014). Microbial interactions with roots tend to be location specific. Figure 1 below illustrates the specificity of different bacteria with the root system.

Bacteria associated with root nodules include Mesorhizobium, Rhizobium and Sinorhizobium (Verma et al., 2010). In addition, species from the Bacillus, Bradyrhizobium and Leifsonia genera have been isolated from legume nodules in semiarid regions. Microbacterium endophytic isolates have also been isolated from root nodules (Nunes et al., 2018; Muresu et al., 2019). The symbioses have the advantage of promoting plant growth by increasing nitrogen uptake and assisting in disease tolerance and resistance. The bacteria may also solubilize phosphate or produce plant hormones which increase plant growth (Busby et al., 2017; Muresu et al., 2019). Plants consequently take advantage of the symbiotic relationship with bacteria present in the soil facilitating the formation of root nodules (Lawless et al., 2018).

The rhizosphere is described as the soil region closest to the roots. It acts as a platform for close interaction within the biosphere around the roots of plants (Jha and Saraf, 2015) and is largely influenced by the plant roots themselves (Ai et al., 2011; Semenov et al., 2020). Therefore, bacteria that colonize the rhizosphere are known as rhizobacteria (Haiyambo et al., 2015).

Through the action of root exudates and essentially chemotaxis (Figure 2) the rhizosphere is a microbe-rich zone (Orozco-Mosqueda et al., 2018; Swarnalakshmi et al., 2020). Also referred to as inter-kingdom signaling, chemotaxis forms the basis for the initial colonization of the rhizosphere by microbes (Venturi and Keel, 2016). As a result, it is a site for biological functions including microbial activity (Fernández Lópeza et al., 2013) and water regulation (Zhang et al., 2020). Both fungal and bacterial organisms form the population of microbes that occupy the rhizosphere (Bui and Franken, 2018; Liu et al., 2019; Leontidou et al., 2020; Sharma et al., 2020).

Rhizobacteria possess the unique ability to influence plant systems both directly and indirectly (Enebe and Babalola, 2018). They offer positive support and influence the crops by performing or facilitating various biological processes. These include solubilisation of inorganic forms of essential compounds (Kaushal and Kaushal, 2015; Puri et al., 2020), biological nitrogen fixation (Tamagno et al., 2018) and antimicrobial activity (Qiu et al., 2012; Martínez-Hidalgo and Hirsch, 2017) among others. The microbial community of the rhizosphere, as such, is heavily influenced by microbes present in the general soil mass (Mendes et al., 2014).

The rhizosphere forms the primary stage for the exchange of nutrients and compounds between the plants and rhizobacteria. This is made possible by carbon rich root exudates that make the rhizosphere a nutrient rich region. This favors microbial growth (Orozco-Mosqueda et al., 2018; Semenov et al., 2020). The physical characteristics of the rhizosphere also create a suitable environment to accommodate both aerobic and anaerobic bacteria among others (Jha and Saraf, 2015; Chawngthu et al., 2020).

One important role played by the rhizosphere is the contribution it makes to water uptake from the bulk soil into plant roots. The uptake of water by plants from the bulk soil is a well understood process, however, the influence of the rhizosphere is often overlooked. Through an intricate interaction between the plant and rhizosphere, the water uptake is regulated (Carminati et al., 2010). This is initiated by plant roots that have been observed to produce a gel like substance (mucilage) that is held within the rhizosphere. Mucilage modifies rhizospheric soil properties resulting in improved water storage (Zeppenfeld et al., 2017; Zhang et al., 2020). Mucilage also has an additional function of inducing hydrophobicity in the event of reduced water availability. This allows for biophysical protection of the plant from drought (Kroener et al., 2016).

In addition, research strongly suggests that rhizospheric influence may differ depending on the age of the roots. This implies, therefore, that distal (younger) roots experience a greater mucilage occurrence to improve water uptake compared to proximal (older) roots (Carminati, 2013). Therefore, the hydraulic properties of the rhizosphere together with root exudates play a crucial regulatory role in water uptake by plants.

Root exudates are nutrient rich carbon sources ideal for microbial communities. They also offer a certain degree of influence on the microbiome (Semenov et al., 2020). Due to this influence and its physical properties, the rhizosphere creates an ideal environment for microbes. With this, the rhizosphere is able to house a wide variety of microbes whose composition is often influenced by plant roots (Essel et al., 2019). Distinct differences in microbiomes between the bulk soil and rhizosphere exist, however, the multiplicity decreases around the rhizosphere (Cui et al., 2019). In addition, the rhizospheric microbiome is more functionally structured compared to the bulk soil. This strongly points toward ecological stability within the rhizosphere (Zhang et al., 2017; Tian et al., 2022).

The rhizospheres of all plants are characterized by bacteria from several different genera. These include Bacillus, Enterobacter and Pseudomonas (Haiyambo et al., 2015). Some of the most abundant bacterial genera that have been identified within the rhizosphere are Lactococcus, Nocardioides, Pseudarthrobacter, Rhizobium and Streptomyces (Essel et al., 2019). The rhizosphere of legumes also includes a similar microbial profile. Rhizobacteria isolated from the chickpea rhizosphere include Azotobacter chroococcum, Bacillus pumilis, Bacillus subtilis and Pseudomonas aeruginosa (Pandey et al., 2019). Hence, other legumes like dolichos bean (L. purpureus) that have formed beneficial symbioses with bacteria become ideal candidates for sustainable intercropping practices.

Ethylene is a phytohormone with a regulatory role necessary for plant growth when in low concentrations. However, abiotic and biotic stresses trigger a different response (Ghosh et al., 2018). Stress events such as drought and higher temperatures induce the production of plant growth limiting compounds such as ethylene (Gupta and Pandey, 2019a). During drought stress, a frequent problem in arid and semi-arid regions, ethylene is produced as a stress signal. The increased water stress accelerates the oxidation of 1-aminocyclopropane-1-carboxylic acid from S-adenosyl methionine. A reaction that results in the production of ethylene (Danish and Zafar-Ul-Hye, 2020). An unregulated increase in “stress ethylene” results in the death of shoots and roots leading to the plant eventually failing to thrive (Singh et al., 2015). The presence of the enzyme ACC deaminase regulates the amount of ethylene in the plant. This is done by the hydrolysis of ACC to ammonium and α-ketobutyrate (Penrose and Glick, 2003). Studies have noted that ACC deaminase can effectively eliminate drought stress effects and this has been observed in pea crops (Ghosh et al., 2018).

ACC deaminase is also especially useful in increasing plant stress tolerance in events of high salinity and pathogenic infections (Bhattacharyya and Jha, 2012). Furthermore, the presence of ACC deaminase promotes nodule formation supporting plant growth. Some bacterial species produce ACC deaminase that actively breaks down ACC to ammonium and α-ketobutyrate (Belimov et al., 2001; Tsukanova, 2017). It has been noted, however, that ACC deaminase activity is higher in phosphorous deficient environments than in phosphorous abundance (Alemneh et al., 2020). Essentially ACC deaminase, by reducing the amount of ethylene, can influence an increase in root length (in the event of water stress) and improved nutrient uptake (in situations of nutrient deficiency) (Alemneh et al., 2020).

To determine the presence of ACC deaminase, bacterial isolates are tested for their ability to utilize ACC as the sole source of nitrogen (in the form of ammonium) (Penrose and Glick, 2003). This is achieved by inoculating the bacterial samples onto augmented Dworkin Foster minimal salt media with added ACC. Growth on these plates would indicate the presence of active ACC deaminase. An additional step measures the activity by determining the amount of α-ketobutyrate and ammonium produced (Ali et al., 2014). The process of the production of ammonia and α-ketobutyrate via ACC deaminase activity is shown in Figure 3 below. Molecular analysis of the isolates via 16S rRNA primers provides their identities. Some known bacteria species which are capable of hydrolyzing ACC include Pseudomonas putida strain Am2, P. brassicacearum strain Am3, Variovorax paradoxus strain Bm2, P. putida strain Bm3 (Belimov et al., 2001), P. fluorescens strain FPG3 (Ali et al., 2014), Paenibacillus sp. strain SG_AIOA2 and Aneurinibacillus aneurinilyticus (Gupta and Pandey, 2019a).

Minerals in insoluble forms cannot be taken up and utilized by plants, hence the need for chemical fertilizers. Phosphorous is one such mineral (Khandare et al., 2020). Phosphate solubilizing bacteria convert inorganic phosphate (Pi or PO43-) into more soluble forms (HPO42- or H₂PO₄) that can be taken up and utilized by the plant. Bacteria achieve this by secreting acids that facilitate solubilization. Succinic acid is one such acid produced by several strains of Bacillus megaterium (Suleman et al., 2018; Zheng et al., 2018).

Phosphorous is an essential nutrient required for the growth and development of plants. It is a crucial element in DNA and RNA, adenosine triphosphate (ATP) and phospholipids (Daneshgar et al., 2018). Thereby positively contributing to photosynthesis, root elongation and nitrogen fixation (Matse et al., 2020). The availability of phosphorous to plants is crucial in soils with low concentrations of biologically available phosphates (Khandare et al., 2020). Furthermore, by using phosphate solubilizing PGPR in agriculture the use of environmentally damaging phosphate fertilizers is avoided. These phosphate fertilizers are known to leach heavy metals into water sources (Bhattacharyya and Jha, 2012).

To characterize bacteria for phosphate solubilization, isolates are grown on Pikovaskya's agar plates with 2% inorganic Tricalcium phosphate (Ca₃(PO₄)₂ (Pandey et al., 2019) or a tris-minimal medium with added zinc phosphate (Shahab et al., 2009) and monitored. A molecular technique may also be employed in the identification and characterization of phosphate solubilising bacteria. This method entails the identification of phosphate solubilising genes in bacterial isolates. Using gene specific primers, genes may be identified (Zheng et al., 2018). This, however, is an inconclusive technique as it only indicates the ability of the bacteria to solubilise phosphates but does not reveal the level of expression of the genes.

Bacteria known to solubilize inorganic phosphate include P. fluorescens, P. putida, Xanthomonas maltophilia (Gupta et al., 2014), Enterobacter agglomerans and Rhizobium leguminosarum (Bhattacharyya and Jha, 2012). Some studies have identified bacterial strains in co-inoculation studies that improve phosphorus uptake. Improved phosphorous content was observed when Rhizobium spp strains (CHB1120 and CHB1121) were inoculated with Azotobacter vinelandii (strain G31) and Bacillus aryabhattai (strain Sb) (Matse et al., 2020).

Iron is one of the most crucial elements for plant growth and is essential for plants to maintain ion homeostasis. It is also an essential component as plants are the main source of iron for humans. Iron deficiency in plants, therefore, is a serious problem (Rai et al., 2021). Some PGPR can produce siderophores that improve the uptake of iron by plants. These siderophores, by forming chelating complexes, promote plant growth by improving the availability of iron to plants and microbes. Siderophores are low molecular weight compounds released by organisms that have a high chelating affinity for ferric iron (Dudeja and Giri, 2014). These compounds solubilise ferric iron into more soluble forms (Fe³⁺ complexes) that are more easily taken up by plant cells (Gamit and Tank, 2014).

The functions of the siderophores promote plant health. As previously mentioned, nitrogen is an essential nutrient required by all plants. For nitrogen to be fixed, bacteria require the enzyme nitrogenase which contains iron. Therefore, sufficient amounts of iron are required (Singh et al., 2018). Iron is also an essential mineral required by plants for growth and development. Using iron-chelating siderophores, PGPR improve the uptake of iron in iron-deprived soils (Dastager et al., 2011). Siderophores also play a secondary role in biocontrol. By chelating ferric iron, they reduce the availability of free living iron in the soil which is required by phytopathogenic microbes (Bhattacharyya and Jha, 2012; Majeed et al., 2015). This has been observed in the control of pathogenic fungi by reducing the availability of iron (Penrose and Glick, 2003; Goswami et al., 2014).

Ligands that chelate iron (III) are used to classify and identify siderophores, these include carboxylates, catecholates and hydroxamates (Louden et al., 2011). Chrome azurol S (CAS) agar, with a pH indicator, is often used as a universal identifier for siderophore production tests. Isolates are inoculated onto CAS agar and observed for color change. The presence of a yellow halo around inoculated isolates indicates siderophore production (Schwyn and Neilands, 1987; Batista et al., 2017). Siderophore producing rhizobacteria in the genera Azadirachta, Azotobacter, Bacillus, Pseudomonas and Rhizobium contribute positively to plant growth and improvement of chlorophyll content (Gamit and Tank, 2014; Gupta et al., 2015). Pseudomonas sp. strain GRP3 from V. radiata supports iron uptake because of efficient siderophore production (Glick, 2012). Some siderophore producing species include Bradyrhizobium japonicum, Rhizobium leguminosarum and Sinorhizobium meliloti (Bhattacharyya and Jha, 2012).

Indole-3- acetic acid (IAA), a growth-promoting auxin, stimulates root elongation and root hair growth. It is synthesized from tryptophan (Lu et al., 2018). However, previous studies have also identified bacteria that can produce IAA without the use of a tryptophan precursor (Kumari et al., 2016). It is an essential plant growth-promoting compound that offers positive support during drought stress, nutrient deficiency, and high salinity.

Extended periods without water (drought stress) mean the amount of water available to plants decreases continuously. However, IAA creates a metabolic reaction that improves water and nutrient uptake (Etesami, 2018). IAA stimulates root elongation and increases root hairs during drought stress. Furthermore, by increasing cell-water uptake efficiency and protein synthesis, IAA promotes embial activity. This in turn promotes increased nutrient uptake, (by longer roots) and induces flowering and fruiting (by delayed abscission) (Mohite, 2013).

The presence of IAA has also been attributed to increased salt tolerance by plants. By improving and maintaining the homeostasis of auxins and phytohormones, IAA supports salt tolerance. This is of importance as high salinity affects hormone production and balance (Saleem et al., 2021). Plants infected with IAA producing PGPB have been found to contain higher levels of antioxidant enzymes which increase salt tolerance (Viscardi et al., 2016). However, salt tolerance may also be enhanced with physical modifications induced by IAA. Khalid and Aftab (2020) observed salt tolerance samples with IAA. They attributed this tolerance to a possible increased salinity tolerance threshold made possible by the improved root length and cell extension.

IAA production may be assessed from bacterial isolates and quantified using different methods. Microbial analysis of IAA production often follows the growth of isolates in Luria-Bertani (LB) broth with tryptophan and incubated while shaking. Samples will thereafter be centrifuged and supernatant extracted for quantification using a spectrophotometer (Rajendran et al., 2012). Isolates can also be grown in yeast malt dextrose broth and quantification of IAA can be done using thin layer chromatography (Mohite, 2013). Some IAA producing genera include Azotobacter, Azospirillum, Bacillus, Kocuria, Pseudomonas, and Rhizobia (Bhattacharyya and Jha, 2012; Goswami et al., 2014).

Biotic stresses are major threats to crop production and yield and often results from fungal, bacterial, or viral infections. These infections cause great losses. Sub-Saharan Africa has recorded losses of more than 220, 000 tons due to fungal infections in common beans. The result of this on a global scale is approximately 800 million people being undernourished (Burke, 2010; Rajendran et al., 2012). Therefore, the antifungal activity of biofertilisers is an important characteristic.

One important fungal pathogen to legumes is Colletotrichum lindemuthianum. It affects L. purpureus (dolichos bean) and causes anthracnose disease which often results in yield loss. V. radiata (mung bean) is also susceptible to anthracnose infection with losses sometimes reaching up to 60% of planted crops (Bhutani et al., 2018). Other important fungal species are in the genus Fusarium. These include F. oxysporum and F. solani which are common pathogens that affect legumes (Burke, 2010; Eid and Fouda, 2021). Antifungal activity of plants by endophytic bacteria, therefore, is beneficial and contributes to plant growth-promoting activities (Haiyambo et al., 2015).

The antifungal activity of endophytic bacteria may be determined by molecular analysis or microbiological techniques. Molecular analysis of bacterial endophytes with primers allows for the detection of genes for antifungal compounds. Previous studies have identified the following genes phzC-phzD, prnD, pltc, phz, phlD and hcnAB to code for the production of antifungal compounds such as phenazine, phenazine-1-carboxylic acid and pyrrolnitrin (Bahroun et al., 2018). Metagenomics may also be used to detect antifungal clones in isolates, however, this method often results in low detection (Burke, 2010).

Antifungal compounds produced by endophytic bacteria actively inhibit the growth of pathogenic fungi. Microbial analysis of antifungal activity follows the concept of the inhibitory potential of isolates (Bhattacharyya and Jha, 2012). Isolates from V. radiata have been found to produce hydrogen cyanide which actively inhibits pathogenic fungi (Bhutani et al., 2018). To determine antifungal activity, fungal isolates are grown on potato dextrose agar (PDA) plates co-inoculated with bacterial isolates with antifungal abilities (Rajendran et al., 2012). Zones of inhibition indicate the degree of efficacy of antifungal compounds produced.

PGPB with antifungal activity can be isolated from different plants. An endophytic bacterium (Paenibacillus polymyxa SK1) isolated from bulbs of the Lilium lancifolium was found to possess significant antifungal activity. P. polymyxa SK1 was shown to actively inhibit Botrytis cinerea, Botryosphaeria dothidea, Fusarium fujikuroi and F. oxysporum, all detrimental fungal pathogens (Khan, et al., 2020b). Some Staphylococcus strains have been found to reduce drought stress but also inhibit fungal infections in plants (Eid and Fouda, 2021). Streptomyces murinus is a well-studied endophyte with antifungal activity. The most significant activity has been observed against Gibberella fujikuroi, Aspergillus niger and Aspergillus fumigatus all-important plant pathogens (Sun et al., 2013).