Co-inoculant Response of Microbial Consortia on Physiology of Blackgram [Vigna mungo (L.) Hepper] Seed Germination

Pulses belonging to the Leguminoceae family are the chief source of protein in the Indian vegetarian diet. Among the pulses, blackgram contains 25-26% protein (Amuthaselvi et al., 2019). India is the largest producer as well as consumer of blackgram covering an area of 41.4 lakh ha with a production and productivity of about 22.3 lakh tonnes and 538 kg/ha respectively during 2020-21 (Indiastat, 2022). The reason for low productivity in pulses may be due to the fact that they are normally grown in marginal lands of low fertility status with inadequate soil moisture.

The use of quality seed alone could increase the productivity by 15-20%. Biological seed treatment with beneficial microbes is an environmentally sound approach for improving the vigour of seeds. Film coating is one of the methods of microbial inoculation in which a thin even coating of microbes with a binder material are coated onto the seeds.

The legume-Rhizobium seed inoculation has been known long back due to its role in nitrogen fixation in the root nodules. The majority of plant-microbe interactions research concentrate on a single plant-microbe association at a time and clear laboratory demonstration of co-inoculation of Rhizobium with other beneficial microbes are still very few.

The rhizobia-bean symbiosis, when in association with Arbuscular Mycorrhizal Fungi (AMF) is known for its benefit of better supply of phosphorus (Sanginga et al., 2000). Pink Pigmented Facultative Methylotrophic bacteria (PPFM) were known to influence seed germination and seedling establishment (Ivanova et al., 2001). Recently, a novel bacterial species named Bacillus velezensis, exhibits growth promotion and antagonistic activity against major plant fungal pathogens (Myo et al., 2019).

Positive influence of microbes on co-inoculation have been reported for Rhizobium - PPFM interaction in groundnut (Priya et al., 2019) and in pigeonpea (Raja et al., 2019a), Rhizobium - AMF in common bean (Tajini et al., 2011) and blackgram (Choudhury and Azad, 2004). Microbes interact with the plant system and alter the production of metabolites resulting in distinct root exudation pattern. In line with this, present work was hypothesized that a quadritrophic interaction is established between Rhizobium sp. BMBS, AMF, PPFM (Methylobacterium extorquens AM1) or B. velezensis VB7 and blackgram for seed germination and seedling vigour improvement.

The laboratory experiment was carried out at the Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore during 2021-22. Freshly harvested blackgram variety VBN 8 seeds were obtained from Krishi Vigyan Kendra, Vamban, Tamil Nadu. The bio inoculants viz., Rhizobium sp. BMBS, Methylobacterium extorquens AM1 (PPFM) used in the experiment were obtained from the Department of Microbiology and Bacillus velezensis VB7 was obtained from the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore. The bacterial cultures were maintained at a population of 108 cfu mL^-1 while the Arbuscular Mycorrhizal Fungi (AMF) in the form of liquid formulation was procured from Uyir Organic Farmers Market, Coimbatore with a spore count of 1x 103 spores ml^-1.

The surface sterilized seeds were first coated with 20 per cent gum arabic followed by the coating with bioinoculants as per the treatments viz., T₀ - Control (Dry seed), T₁ - Rhizobium sp.  BMBS + AMF + Methylobacterium extorquens AM1 and T₂ - Rhizobium sp. BMBS + AMF + Bacillus velezensis VB7. For co-inoculation of bioinoculants in the treatment T₁ and T₂, a cocktail of the above mentioned bioinoculants were taken in equal ratios. The seeds after shade drying were used for the physiological and biochemical analysis. The experiment was conducted by adopting completely randomized block design (CRD) with seven replications.
 
Determination of physiological parameters
 
The blackgram seeds were tested for different physiological parameters viz., germination, root length, shoot length, dry matter production, vigour index (ISTA 2015) and seed metabolic efficiency (Srivastava and Sareen, 1974).
 
Determination of biochemical parameters
 
The dehydrogenase activity was estimated according to Kittock and Law (1968) and α-amylase activity as per the procedure described by Paul et al., (1970). The protease activity was analysed according to the method described by Li et al., (2011) and total free amino acid according to Ching and Ching (1964).
 
Metabolite profiling
 
Treated and untreated seeds were kept for germination using paper medium (between paper). The seven days old seedlings of uniform size were transplanted into glass test tubes containing Hoagland’s nutrient solution (Hoagland and Arnon, 1950). Root exudates were collected on 15th day with ethyl acetate in equal volume (1:1, v/v) and then concentrated by natural evaporation. The elute was then dissolved in one ml of MS grade methanol and subjected to identification of metabolites through Gas Chromatography-Mass Spectroscopy using Shimadzu GCMS-TQ8040 NX. One µL aliquots of the reaction mixture was injected into the gas chromatograph in 1:10 split mode. The separation was performed in Rtx-5MS column, which has a 30 m length, 0.25 mm ID and 0.25 m film thickness and was made up of 5 percent diphenyl dimethyl polysiloxane. The quadrupole mass spectrometer was operated in an electron ionization mode at 70 eV. The scan range was set to 50-650 m/z. Mass-spectrum interpretation was done utilizing the NIST Standard Reference Database 1A in NIST V2 data version.
 
Statistical analysis
 
Statistical analysis was performed by subjecting the data to one way analysis of variance (ANOVA) and analysing them by Least Significant Difference test for statistical significance at pd”0.05 using AGRESS software (Gomez and Gomez, 1984).

The co-inoculation of microbes through film coating performed better than the control seeds. Highly significant difference in germination (97%) was found in seeds coated with combination of Rhizobium sp. BMBS + AMF + M. extorquens  AM1 followed by Rhizobium sp. BMBS + AMF + B. velezensis VB7 (95%) (Table 1).



Initially, inoculated seeds showed higher germination which might be due to the production of phytohormone, as phytohormone influences seed germination (Mia et al., 2012). Methylobacterium produce phytohormones such as cytokinins and auxins, which were known to stimulate seed germination (Lee et al., 2006).

Seeds coated with Rhizobium sp. BMBS + AMF + M. extorquens AM1 recorded the longer root (20.40 cm) and shoot length (21.52 cm) which was on par with Rhizobium sp. BMBS + AMF + B. velezensis VB7 (20.11 cm and 21.39 cm) respectively (Table 1). Similar findings of inoculation of Rhizobium or PPFM on germination and seedling vigour improvement was studied earlier in blackgram (Raja et al., 2019) and pigeonpea (Raja et al., 2019a) seeds. Induction of longer roots was a growth response that might be attributed due to the production of Indole Acetic Acid (IAA) by Rhizobium sp. (Mohite, 2013), PPFM (Pattnaik et al., 2017), B. velezensis (Meng et al., 2016). The increase in seed germination and seedling length were considered typical gibberellins-like responses. Microbes were known to modulates the level of ROS at the time of germination (Gomes and Garcia, 2013).

Dry matter production, vigour index and seed mobilisation efficiency were higher in Rhizobium sp. BMBS + AMF + M. extorquens AM1 (0.237 g 10 seedlings-1, 22.99, 1.11) followed by Rhizobium sp. BMBS + AMF + B. velezensis VB7 (0.224, 21.28, 1.06), respectively (Table 1). Microbes through phytohormone and hydrolysing enzymes production interact with the seedlings and facilitates the nutrient mobilization from endosperm to embryo, that could reflect in the dry matter production and seed mobilisation efficiency. Reactive oxygen species (ROS) roles have been recognised in weakening of endosperm and mobilisation of food reserve during seed germination (El-maarouf-bouteau and Bailly 2008).

No significant difference was found in dehydrogenase activity (Fig 1A) among the treatments.



The changes in enzymatic activities such as α-amylase activity (22.6 mg maltose min) (Fig 1B), protease activity (0.269 units/mg of protein) (Fig1C) and total free amino acids (0.005 μg 25 seeds-1 25ml^-1) (Fig 1D) were higher in Rhizobium sp. BMBS + AMF + M. extorquens AM1 followed by Rhizobium sp. BMBS + AMF + B. velezensis VB7.  Previous studies showed the ability of Rhizobium strains for the production of α-amylase (Oliveira et al., 2007) and protease enzymes (Dhole and Shelat, 2022). Besides Rhizobium, PPFM was also found to produce protease enzyme (Jayshree et al., 2014). It was clear from the physiological and biochemical studies, that co-inoculation of Rhizobium sp. BMBS + AMF + M. extorquens AM1 through film coating exhibited a profound effect on vigorous seedling production of blackgram.

GC-MS based untargeted metabolomic analysis was launched to compare the metabolic difference that occurred in primary and secondary metabolism of control and bio-inoculant coated seeds in the root exudates pattern of hydroponically grown blackgram seedlings. The compounds identified in root exudates of seedlings with their potential uses were shown in Table 2. 



This study revealed that, more number of compounds responsible for antioxidant and antimicrobial activity were released by seeds coated with Rhizobium sp. BMBS + AMF + M. extorquens AM1 and Rhizobium sp. BMBS + AMF + B. velezensis VB7 than the root exudates of control seedlings. In line with this, changes in root exudates compound upon Pseudomonas fluorescens inoculation in tomato was reported by Kamilova et al., (2006). Similarly, volatile organic compounds (VOC) produced by rhizobacteria were involved in their interaction with plant-pathogenic microorganisms and host plants by elucidating antimicrobial and plant-growth modulating activities (Vespermann et al., 2007).

During microbial interaction of Rhizobium sp. BMBS + AMF + M. extorquens AM1, it produced distinct metabolites such as phenyl (2-phenyl-1, 3-dioxolan-2-yl) methanol, sabinene, squalene. Phenyl (2-phenyl-1,3-dioxolan-2-yl)methanol had highest peak area percent of 6.21 possessing antifungal activity (Van gestel​ et al., 1980). Sabinene which is a monoterpene has been found to involve in starch and sucrose metabolism and plant growth regulation (Grulova et al., 2022). Squalene belonging to triterpene is an antioxidant (Huang et al., 2009) and scavenges the free radical damage (Micera et al., 2020).

In nutshell, interaction between seed and bioinoculants viz., Rhizobium sp. BMBS + AMF + Methylobacterium extorquens AM1 released plant growth promoting substance, which played a role in increasing the seed germination, seedling length and dry matter production ultimately resulted in increased seedling vigour.

This investigation evaluated the responses of seed germination, physiological, biochemical parameters and root exudates pattern to co-inoculation of Rhizobium sp. BMBS and AMF with Methylobacterium extorquens AM1 and Bacillus velezensis in blackgram.  We found that the response pattern to inoculation was highly influenced by Rhizobium sp. BMBS, AMF and Methylobacterium extorquens AM1 co-inoculation through coating enhanced seed germination, vigour, production of enzymes and useful metabolites.

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