The global population is predicted to reach 9.2 billion by 2050, with a projected increase in food demand. It seems necessary to increase agricultural production to feed the growing population. Current agricultural practices are aimed at maximizing the production of crops with scant care for the quality of produce, food safety, human health, and preservation of natural resources. Intensive reliance on agrochemicals has posed a challenge to the sustainability of the traditional farming system, as well as food security, protection of human health, and productivity of natural resources (Abbas et al., 2018). The most pressing concern is the loss of soil fertility, which reduces soil productivity. Therefore, employing natural organic and biological inputs, reversal of the decline in soil health may be possible. Legumes are well-known for developing and restoring soil fertility owing to their mutualistic relationship with symbiotic nitrogen-fixing bacteria. Chemical fertilizers are utilized to fulfill the nitrogen requirements of plants and attain higher yields of crops, but they cause negative ecological impacts (Tariq et al., 2020). Nitrogen-fixing rhizobia are inoculated in legume crops to gear up the natural process of biological nitrogen fixation to fulfill nitrogen requirements of crops and restore soil fertility (Naseem et al., 2018; Saeed et al., 2021). Effective nitrogen fixation symbiosis with rhizobia is key to the success of symbiosis relationships with legumes. However, various agricultural soils lack sufficient rhizobial populations. Also, naturally existing populations may be poor. It reduces their efficiency to fix nitrogen successfully, resulting in lower agricultural yield. Therefore, the use of rhizobial inoculants is an important means to combine its known significance in improving crop yield and biological control of plant diseases and pests (Yadav et al., 2021). These situations provide an opportunity to outline the use of symbiotic microorganisms to enrich the rhizosphere of legumes to promote effective nodules, nitrogen fixation, and the next prosperity of food production (Ali et al., 2017; Wolde-meskel et al., 2018; Matse et al., 2020). For example, rhizobial inoculants in legumes have been improving agricultural productivity and forming a familiar method since ancient times (Abdullahi et al., 2013), which provides an effective and appropriate method for legume rhizosphere and soil (Ahmad et al., 2019). This is vital for increasing crop yield in farmlands, especially where the supply of nitrogen fertilizers restricts crop production and productivity. The practice increases the infection establishment, nodulation, biomass, yield component, yield, and nutrient uptake of the legume crops (Khan et al., 2018).

The N is a critical element for legume crops because it is an integral component of chloroplast, phospholipids, and proteins (Nawaz et al., 2018). Uptake of nitrogen, its assimilation, and usage shows an essential part in improving the productivity of plants (Yuan et al., 2012). To offset the chemical-based agricultural industry, sustainable farming approaches are needed. From this perspective, exogenous use of amino acids is also considered to improve the productivity of crops. These are a type of phytostimulators (substances that enhance the growth of plants) that supply plant nutrients, which results in improved plant growth, yield, and commercial output (du Jardin, 2015; Rouphael et al., 2018). They are not only getting familiar with alleviating injuries of abiotic stresses to plants (Kowalczyk et al., 2008) but also serve as phytohormone precursors, i.e., tryptophan and methionine (Maeda and Dudareva, 2012; Calvo et al., 2014; Tatjana et al., 2015; Khan et al., 2019). Phytostimulators also regulate nitrogen uptake in plants (Miller et al., 2007), growth of roots (Walch-Liu and Forde, 2007; Calvo et al., 2014; Halpern et al., 2015; Weiland et al., 2016), and metabolism of antioxidants (Teixeira et al., 2017). Exogenous application of amino acids can promote root development, thereby increasing the ability of nitrogen fixation owing to an increase in root surface area for absorption of nutrients (Hildebrandt et al., 2015; Weiland et al., 2016). Direct use of amino acids or their products can change the absorption and assimilation of nitrogen, and various enzymes are involved in mediating this mechanism (Calvo et al., 2014; du Jardin, 2015). The latest research (Teixeira et al., 2017) found that the application of amino acids or their products through seeds or foliar treatment had different effects on legume crops. The use of amino acids alone as signal components improves antioxidant enzymes’ activities and results in nutrient absorption (Giose et al., 2012). Earlier studies have shown that L-methionine is used as an amino acid for the synthesis of growth regulators in plants (e.g., auxins, brassinosteroides, and cytokinins). Additionally, L-methionine is a precursor of multiple essential biomolecules such as vitamins, polyamines, cofactors (Paciorek et al., 2005; Yong et al., 2014), and antioxidants. However, the impact of single amino acids, particularly in seed applications, is poorly understood. In addition, most studies have used amino acid mixtures as well as other application strategies, such as soil treatment and foliar application (Sadak et al., 2015; Abd El-Aal, 2018; Khan et al., 2019). This study is based on the theory that the use of L-methionine alone and together with rhizobia can increase nitrogen absorption and other plant growth factors, and leads to increased productivity of chickpea. Therefore, the purpose of this research is to evaluate the effects of L-methionine alone and in combination with Rhizobium on the growth, physiology, and yield of chickpea crops.

Yearly biological nitrogen fixation generates 20–200 kg nitrogen per hectare in plant’s available forms in farming areas (Herridge et al., 2008) and maintains global food security. Among crops, most legumes are recognized because of their symbionts in root nodules with biological nitrogen fixation capabilities. Rhizobium-legume symbiosis has been investigated for decades as a reciprocal association. In this study, different rhizobial isolates were screened for plant growth promotion traits (Table 1). According to reports, the growth of rhizobium in the YEMA medium confirmed the morphological characteristics of Rhizobium (Somasegaran and Hoben, 1994). During culture preparation, Gram-negative and Congo red dye with non-absorbing behavior are typical characteristics of Rhizobium vulgaris (Abere et al., 2009). Studies have shown that a change in the color of the YEMA-BTB medium from blue to yellow indicates acid production and fast growth (Jida and Assefa, 2011). The diameter of colonies with oval and round edges is between 2 and 3.3 mm, which is consistent with other studies (Odee et al., 1997; Alemayehu, 2006). The biofilms and extracellular polysaccharides (EPSs) produced by the isolates may be universal components that protect bacteria from different biological and abiotic infestations in the soil (Khan et al., 2021). Rhizobia capable of tolerating environmental pressures may be more suitable for commercial inoculants (Saeed et al., 2021). These preliminary tests have previously been conducted to distinguish impurities in pure isolates of Rhizobia (Vincent, 1970). Rhizobium isolates were tested for IAA production in this study (Table 2). It is reported that the IAA produced by rhizobia plays a vital role in plant growth and interaction between Rhizobia and legumes. Production of plant hormones in nodules and their transport to the host through nodule symbionts was reported by Hunter (1989). Production of IAA from root nodule tryptophan by Rhizobia bacteria and its effect on nodule formation has been well-reported (Datta and Basu, 2000; Ghosh and Basu, 2006; Ghosh et al., 2013).

Rhizobium isolates were cultured in yeast extract mannitol (YEM) to test the potential for pH tolerance; they were grown in YEMA (Table 1). pH was maintained at 4–8, and 6–7 pH was found optimum for rhizobial growth. Nominal growth of the isolates was exhibited at pH 8 (Table 1). Deora and Singal (2010) reported that minimal variation in the pH of the medium might have a huge effect on the growth of nodule bacteria. Sensitivity of the Rhizobial isolates to high and low pH is observed commonly. It might be due to some strains of Rhizobium that have acid sensitivity, which has an important role in the regulation of internal pH and possibly accounts for their poor growth in cultures that have acidic pH (Riccillo et al., 2000).

All the isolates in this study were grown at different salt concentrations (Table 1), but it was found that Rhizobium has less ability to grow with the increase in salt concentration. It has been reported that extreme saline conditions suppress the proliferation and growth of Rhizobium. It has been speculated that some strains can stand and survive high salinity (Zahran, 1999; Keneni et al., 2010).

The most traditional microbes used as inoculants are “rhizobia.” They colonize the rhizosphere and form a symbiotic relationship with beans. They are employed to promote the productivity of plants by biological fixation of nitrogen; higher availability of plant nutrients by mobilization and dissolution, siderophores production, and release of plant growth hormones (Makoi et al., 2013; Koskey et al., 2021; Yadav et al., 2021). The present study showed a substantial role of Rhizobium inoculation on seedling emergence, as well as root and shoot elongation in pot study, as compared to untreated control (Table 3). This may be due to the inoculation by rhizobia of chickpea seeds by symbiotic nitrogen fixation to increase nitrogen absorption, which ultimately led to increased seedling height and root length. This result is similar to that of Farfour (2013). It reflects that rhizobial stain can improve the plant height of faba beans. Similarly, an increase in shoot and root length by bacterial strains was reported by El-Azeem et al. (2007). The results of the symbiosis effect (Table 3) show that the application of all 10 chickpea rhizobia in the study area is very effective. This result is similar to that of Gebremariam and Assefa (2018), which confirms that the symbiotic effect of Rhizobium strains isolated from faba beans is very effective.

A more multifarious amino acid cycle is crucial for symbiotic nitrogen fixation by Rhizobium in legumes. The use of amino acids in exogenous applications is getting more popular owing to their role in sustainable agriculture (Teixeira et al., 2017). By optimization of amino acid concentration, changes can be brought among various morphogenetic traits of crops; as higher concentrations mostly inhibitory role for growth in Cicer arietinum (John and Guha-Mukherjee, 1997). Various studies have shown that the optimal level of different amino acids depends on genotypes or species-specific characteristics. It should be determined before the recommended application (El-sharabasy et al., 2015). Gradually, plant ecologists are more interested in the role of amino acids in the nutritional life and productivity of plants (Rosati et al., 2000). We also found in field studies that when L-methionine was applied at a low concentration, the growth and yield attributes of chickpeas were higher compared with high concentrations (Table 4). Since L-methionine is also a precursor of ethylene, it is designated as a multifunctional plant hormone regulating plant growth and senescence. It has been observed that it is more beneficial at lower doses and has an inhibitory effect at higher doses (Khan, 2005; Khan et al., 2008).

In this research, it was found that inoculation with rhizobia could significantly promote the growth of chickpea sprouts and increase the number of pods and biomass. At the same time, inoculation with rhizobia can significantly affect the reproductive growth of chickpeas and increase the yield of pods and grains. This may be because inoculation with rhizobia can increase nitrogen content, promote the vegetative growth of plants, mainly root growth. Similar findings have also been reported in other studies in which chickpeas inoculated with rhizobia promoted plant growth, dry matter yield, pod number, grain yield, and nitrogen fixation under various climatic conditions. This is because of the extensive root system, nodulation ability, and strong and tall stems (Singh et al., 2003; Kasole et al., 2005). Our research shows that inoculation with rhizobia and L-methionine (5 mg L^–1) has a greater significant effect on pod number, plant height, biomass, and grain yield. It can be anticipated that L-met, L-tryptophan, and L-glycine not only provide plants with nutrients but also transmit signals (Teixeira et al., 2017). Smaller doses are enough to improve plants, and these molecules can be used as signals for better physiology of plants. Previous studies have shown that spraying amino acids on plants in the form of foliar spray is an encouraging technique (Sadak et al., 2014). In this way, L-methionine attracts plants to absorb sulfur and nitrogen more significantly depending on application rate (Price, 2012; Vincill et al., 2012; Forde and Roberts, 2014; Santi et al., 2017).

This study has noted that Rhizobium inoculation + L- met has a greater positive impact on seed emergence, plant height, and earliest harvest than the untreated control (Tables 4, 5). The reason behind this is that increase in seed germination percentage and crop maturity is considered typical a phytohormonal response. Initially, the inoculated plants confirmed better emergence, which is probably due to the production of plant growth hormones, as phytohormones affect seed germination and, eventually, crop maturity. The potential of microorganisms in promoting plant germination and improving development has been adjusted to the in vitro and in vivo conditions of some crops and ornamental plants. It has been recognized that the promotion of seedling growth with rhizobia can produce biologically active compounds. It has been reported that plant growth stimulants can be used for better germination and production of plants (Tsavkelova et al., 2007; Nazli et al., 2020; Rafique et al., 2021).

Inoculation with rhizobia under field conditions can cause significant differences in nodule count, dry weight, and volume (p ≤ 0.05) (Table 6). Changes in the number of root nodule plants-1 and dry weight of nodules plants^–1 indicate that the response of chickpea crops varies with rhizobia strains. Hence, this study agrees with a previous study. Jida and Assefa (2012) reported that faba bean produced a higher yield when a suitable symbiotic partner was provided. It has been found that rhizobia inoculation resulted in significant improvement in the dry weight of chickpea nodules (Table 5). Voisin et al. (2003) showed that an increase in the dry weight of tuberculosis is related to the higher efficiency of symbiotic relationships during tuberculosis development. Graham et al. (2004) reported that the dry weight of tuberculosis was a significant indicator of symbiosis efficiency. Efficient rhizobia isolates stimulate nodule formation. They give a higher fixation of N and biomass to crops. Rhizobium inoculation can improve nodulation and nitrogen fixation, thereby promoting plant growth (Thilakarathna et al., 2019; Umar et al., 2020). Nodule volume, count, and weight are increased in our study (Table 5).

Due to the incorporation of C₂H₄ and increase of root growth in the formation of nodules, the application of L-methionine at low to moderate concentrations had a positive effect on the nodules (nodule number and dry weight), and these positive effects weakened at higher doses. Some studies have also described the direct application of C₂H₄ exogenously released compounds (ethephon) to inhibit the formation of nodules (Grobbelaar et al., 1971; Drennan and Norton, 1972; Goodlass and Smith, 1979). This inhibitory effect is due to the high concentration of C₂H₄ used. We reported that ethylene gradually but continuously released L-met as a microbial metabolite to the rhizosphere. In addition, the plants’ response to L-met may be due to the interaction of ethylene released by microorganisms using some endogenous sources.

It was found that inoculation with rhizobia affects the physiological characteristics of chickpea (Tables 6, 7). An increase in leghemoglobin content has been recorded in this study, which may be due to better nodulation of chickpeas that results in higher leghemoglobin content in the nodular tissue. Previous studies have shown that because of better root and nodule development, leghemoglobin content in rhizobia is higher (Sidhu et al., 1967). Likewise, L-methionine has a positive effect on leghemoglobin content because it affects nodule formation. These results are similar to our conclusions.

It can be seen from our research that rhizobium and L-methionine have positive effects on plant nutrients, seed proteins, and photosynthetic pigments. Seeds inoculated with rhizobia can increase nodulation, nitrogen absorption, and seed protein because L-methionine has the function of maintaining protein structure for appropriate division, differentiation, and growth of cells. It gives enough nitrogen and sulfur to fulfill plants’ needs, which may be transformed. It is a polyamine and expands with modification of the molecular structure of hormones, allowing for nitrogen movement between cells and organs (Kakkar et al., 2000; Rudresh et al., 2005). It also acts as a buffer and carbon source. Plants treated with rhizobia and trace elements have high chlorophyll content.

Gwata et al. (2003) that chlorotic plants were not similar to plants with dark green leaves when grown in an N-free environment. It may be because the N in dark green leaves originated from biological fixation. Nitrogen-containing compounds produced because of N₂ fixation are transferred from nodules as ureides (allantoic acid and allantoin) and transferred to leaves for catabolism (Hildebrandt et al., 2015). It improved chlorophyll synthesis in plants, also indicated by better photosynthesis. The amount of chlorophyll dye indicates the physiological health of plants (Miller et al., 2007). By adding amino acids, better root development can enhance nitrogen fixation, thereby inducing the root surface to rise to absorb nutrients (Hildebrandt et al., 2015; Weiland et al., 2016). L-methionine can also be used as a growth regulator of cytokinins, brassinosteroids, and auxins to promote root germination and help plants absorb more nutrients (Davies, 2004). It ameliorates the homeostasis of endogenous hormones (Colla et al., 2017; Rouphael et al., 2017) and is the optimal compound for growing tips of roots (Davies, 2004). Elevated levels of L-methionine can affect plant hormones giving higher chlorophyll and chloroplasts and cytokinins (Bahari et al., 2013; Anne and Thomas, 2015). The expected requirement to promote the use of L-methionine may be the proximity of plant hormones (for example auxin and cytokinin). Plant hormones and signal compounds can increase photosynthetic activity, thereby increasing yield. Another possible mechanism may be the faster growth of roots treated with L-met. It gives a higher surface area for nutrients and water absorption (Colla et al., 2017; Rouphael et al., 2017). Its effects are then altered to better the cell growth and biomass of plants (Fawzy et al., 2012). It modifies normal growth behavior for good (Bahari et al., 2013; Romero et al., 2014).

In this study, the combined use of Rhizobium and 3 levels of L-methionine substantially enhanced the attributes of growth, physiological, chemical contents, and productivity of chickpea in two successive years of field trials. It could meet our interest in validating the benefits of biological inputs and reducing reliance on inorganic fertilizers and destructive farming practices. However, the combined use of Rhizobium and L-methionine, specifically a lower dose of L-methionine (5 mg L^–1) was the most effective in improving the productivity of chickpea. Hence, combined use of L-methionine and Rhizobium inoculation may be promoted to produce food using sustainable and safer inputs and preserve the most precious natural resources. Regardless of the various benefits of plant microbiome inoculation and amino acid application, there is a variation in the implementation of such types of practices by the farmer community. Therefore, researchers may now focus on reducing constraints of farmers by developing alternate technologies including integrated use of L-met and rhizobia in farming practices.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

MR and AA contributed to conceptualization. MR contributed to methodology and writing (original draft preparation). MR, MB, AAA-H, and AN contributed to software. MN, AAA-H, and AA contributed to validation. MR, AAA-H, MHS, and TA contributed to formal analysis. JH, AAA-H, and MHS contributed to resources. MR and MN contributed to data curation. MN, TA, AAA-H, MHS, and JK contributed to writing (review and editing). MN, AK, JK, MB, and AA contributed to supervision. MN and AM contributed to project administration. AAA-H and AM contributed to funding acquisition. All authors have read and agreed to the published version of the manuscript.

AK was employed by the company Agricultural Research, Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.