Impact of Seed Coating with Prebiotics on the Physiological Performance and Soil Microbial Activity of Blackgram (Vigna mungo L.) during Storage

Blackgram (Vigna mungo L.) is the major pulse crop grown over 4.63 million hectares with a total (Agricultural Statistics Division, DES, MoAF and W, 2022) production of 2.78 million tonnes and a productivity rate of 600 kg per hectare. In Tamil Nadu, it is cultivated on 407,000 hectares, yielding a total production of 269,000 tonnes and a productivity of 660 kg per hectare (AICRP PC Report, 2023). Significant improvements in crop yields due to high-yielding crop varieties, innovative farming practices and the application of chemical fertilizers, has also sparked worries regarding its impact on environmental quality and soil health (Pramanik et al., 2023) after Green revolution era.
       
Soil microbial diversity and abundance are key indicators of soil fertility. Enhancing the population of beneficial microorganisms in soil can significantly support plant growth and development (Wei et al., 2024) through nutrients and carbon. Under biotic and abiotic stresses, microorganisms can stimulate the production of indole-3-acetic acid (IAA) and osmolytes such as glycine betaine and proline. Additionally, microorganisms’ play a major role  in stress tolerance by solubilizing mineral phosphates, fixing nitrogen, producing organic acids and synthesizing enzymes like ACC-deaminase, chitinase and glucanase, thereby enhancing plant growth (Nanda et al., 2019). Plant growth-promoting rhizobia (PGPR) enhance nutrient uptake from the rhizosphere and produce beneficial substances. They indirectly protect plants by suppressing soil-borne pathogens and directly stimulate plant growth by producing growth regulators like gibberellins, auxins and cytokinin’s (Nagargade et al., 2018).
       
In response, prebiotics are substances that enhance microbial diversity and soil health by fostering the growth of microorganisms that naturally exist within the soil-plant ecosystem. These are natural obtained from agro-industrial wastes such as sewage sludge, biochar, humus, animal manure and compost. These substances improve soil structure, enhance biochemical activity and boost microbial populations and diversity, particularly in degraded soils (Vassileva et al., 2020; Alahmad et al., 2023).
       
Seed coating is a process of seed enhancement technique which can deliver essential micro and macronutrients or bio stimulants, ultimately results in boosting germination, seedling vigour and establishment (Amirkhani et al., 2016; Shinde et al., 2017). Film coating is the process of directly applying a thin layer of a beneficial coating agent to the seed coat’s surface, often up to 5% of the seed weight (Pedrini et al., 2020). Film seed coating mediated by chitosan and polyethylene glycol (PEG) was successful in promoting castor germination and vigorous plant growth (Chandrika et al., 2024). Maize seeds coated with slow release nutrients led to better emergence potential and enhanced yield attributes (Dong et al., 2016).

With this background in mind, this research aims to evaluate the effects of prebiotic seed coating formulations on the physiological and soil microbiological properties of black gram over six months and to assess the stability of the formulation after storage.

The seed coating experiment was conducted on blackgram cultivar ‘VBN 11’ in the Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore during January 2024 - July 2024. The initial quality of blackgram seeds was with 92% germination and 8% seed moisture content.
 
Seed coating procedure
 
Blackgram seeds were coated with Rotary Seed Coater. The seed coating formulation was prepared using various nutrient substances combined at appropriate ratio. The nutrient used was Murashige and Skoog media (17%), chitosan (3%), glycerol (34%), Beef extract (34%), gelatin (7%), Carboxy methyl cellulose (2.5%), potassium dihydrogen phosphate (1.25%) and magnesium sulphate (1.25%). These substances were mixed with white polymer. The coating formulation obtained was self-sticky nature. The seeds were coated with formulation at 2 different doses such as T₁: 6g kg^-1 and T₂: 8g kg^-1. Uncoated seeds served as control (To). After coating, seeds were shade dried (25°C) for one h.
 
Experiment 1: Storage of coated seeds
 
Coated and uncoated seeds were stored for six months in cloth bags under storage conditions (temperature 30°C and 45% relative humidity). Then, stored seeds were sown under prescribed germination conditions (temperature 25°C and 65% relative humidity) recommended by ISTA to determine the potential of seeds at monthly intervals. The experiment was conducted in a completely randomized design with seven replications. During storage, seed germination, seedling vigour, spermosphere and rhizosphere microbial activity was recorded at monthly up to six months of storage. Germination percentage was determined by sand and soil method (1:1). Each treatment replicated four times. At the end of germination test, ten normal seedlings were randomly selected and the shoot and root length were measured in centimeters. Further, the seedlings were kept in an oven at 80±20°C for 72 h for measuring seedling dry weight. The seedling vigour index (SVI) was calculated as the product of the germination percentage and seedling length as suggested by Abdul-Baki and Anderson (1973).
       
One day after sowing, the seeds were carefully excavated from the germination tray and the soil attached to the seeds was carefully collected for spermosphere activity. Subsequently, at 7 days after sowing (DAS), the seedlings were removed from the trays, ensuring the roots remained intact. The soil adhered with the roots was then collected for rhizosphere activity. The bacterial and fungal population in both the spermosphere and rhizosphere were determined using serial dilution and plating methods (Parkinson et al., 1971). Additionally, the dehydrogenase enzyme activity in the rhizosphere soil was measured following the procedure outlined by Casida et al., (1964).
 
Experiment 2: Evaluation of formulation stability
 
In a parallel experiment, the same seed coating formulation was prepared and stored for six months at refrigerated conditions (5°C). After this storage period, a fresh batch of blackgram seeds was coated with the stored formulation, using the same doses (T₁: 6 g kg-¹ and T₂: 8 g kg-¹) as in the first experiment. Freshly prepared formulation was also coated on blackgram seeds at 2 doses (T₃: 6 g kg-¹ and T₄: 8 g kg-¹) and T0 serves as control (uncoated seeds). Observations on seed germination, seedling vigor and spermosphere and rhizosphere microbial activity and dehydrogenase activity were recorded with the same methodologies as described earlier.
 
Statistical analysis
 
The laboratory experiment was laid out in a Completely Randomized block design (CRD). All data were analyzed using GRAPES software. ANOVA was performed at P<0.05 and treatment means were compared with Duncan’s multiple range test (DMRT). Graphical analyses of seedling growth, soil microbial activity, enzyme levels were carried out using Origin software.

Coated seeds maintain their properties during long-term storage. Seed coatings deliver essential substances like microorganisms, nutrients and growth regulators, while also containing protective chemicals, reducing the need for spraying or fertilizing (Torre-Roche  et al., 2020). Seed coating with prebiotics produced significant effect evaluated in the laboratory in all physiological and microbial attributes irrespective of the treatment dosage. Over the 6-month storage period, the speed of germination showed a gradual decline in all treatments. The initial values at 0 month after storage (MAS) indicated 8 g kg^-1 had the highest germination speed (8.23) compared to control (7.40). After 6 MAS, 8 g kg^-1 maintained a relatively higher speed of germination (6.95) over control (6.20). Germination percentage also decreased over time, with 8 g kg^-1 maintaining higher germination rates (91%) at 6 MAS over control (85%). The prebiotic coating at 8 g kg^-1 dose helped in preserving a higher germination rate throughout the storage period (Fig 1). Over time, both root and shoot lengths declined across treatments, with 8g kg-1 consistently having higher root lengths than 6 g kg^-1 and control. At 6 MAS, 8 g kg-1 exhibited a root length of 12.33 cm, followed by 6 g kg^-1 (12.36 cm) and control (11.90 cm). The shoot length for 8 g kg^-1 remained slightly higher than the other treatments at each MAS, with a final value of 17.1 cm compared to 17.4 cm in 6g kg^-1 and 16.7 cm in control at 6 MAS (Fig 2). There was a decrease in dry matter production for all treatments over the storage period. Initially, 8 g kg^-1  showed the highest dry matter production (0.403 g/10 seedlings), while at 6 MAS, the values declined, with 8 g kg^-1 at 0.196 g/10 seedlings compared to 6 g kg-1 (0.193 g/10 seedlings) and control (0.194 g/10 seedlings). Vigour index values also followed a similar trend, with 8g kg^-1 achieving the highest values initially and maintaining a slight edge at the end of the storage period (Table 1). At 0 MAS, 8g  kg^-1 showed the highest bacterial population in the spermosphere (77.0´10u  CFU/g soil) and even after 6 MAS, the population remained relatively higher at 17.0 compared to T0 (11.0) (Fig 3). Similarly, the rhizosphere bacterial population was highest in 8 g kg^-1 at 6 MAS. Prebiotic seed coating formulation at 8 g kg^-1 had consistently resulted in fungal populations over control (Table 2). Increased microbial activity resulted in increased soil enzyme activity which has been proved in our study. Dehydrogenase activity, an indicator of soil microbial activity, was initially highest in 8 g kg^-1 (24.56 µg TPF/g soil/day) and remained significantly elevated across the storage period. At 6 MAS, 8 g kg^-1 recorded highest dehydrogenase activity (17.93 µg TPF/g soil/day) (Fig 4). Soil microorganisms play a vital role in improving seed germination by synthesizing plant growth-promoting hormones (Wu et al., 2016). They improve soil structure by reducing compaction, thereby improving porosity and aeration, water permeability, creating optimal conditions for root growth and for efficient uptake of water and nutrients. Indigenous microbial strains enhance germination rates, increasing plant biomass accumulation, seed yield, resilience to abiotic stress in various agricultural crops (Rocha et al., 2019). During seed germination, seeds emit exudates into the spermosphere, promoting microbial proliferation, which is crucial for the successful germination of various crop seeds (Nelson, 2018). Rhizosphere microor-ganisms are crucial in regulating and producing plant hormones, greatly influencing growth and development of plants. Bacterial IAA (Indole 3 acetic acid) specifically enhances root growth by promoting cell elongation and division. Gibberellin initiates stem elongation and further plant growth. Ethylene helps in root hair formation and seed germination (Glick, 2012).












       
In the parallel study on stability of formulation, at a higher dose of newly prepared formulation (8 g kg^-1) significantly enhanced several growth parameters compared to the control and stored formulations. Germination rate improved slightly, with newly prepared formulation (8 g kg^-1) achieving the highest germination (98 %) and the highest seedling vigour index (3464). Root and shoot lengths were also most pronounced in newly prepared formulation (8 g kg^-1) showing 15.96 cm and 19.13 cm, respectively, with corresponding improvements in dry matter production (0.403 g/10 seedlings) (Table 3).      

                           

Additionally, microbial analysis revealed that newly prepared formulation (8 g kg^-1) treatment had the highest bacterial and fungal populations in both the spermosphere and rhizosphere, especially at higher dilutions, along with a peak in dehydrogenase activity (25.10 µg TPF/g soil/day), suggesting enhanced microbial and biochemical activity in the soil (Fig 5). Prebiotics can reshape native soil microbial communities, enhancing bacterial and fungal diversity while, recruiting microorganisms with specialized ecological functions (Hellequin et al., 2020). Soil microbe’s increases plant disease resistance by adjusting soil pH, enriching soil organic carbon and competing with pathogens for essential resources. Additionally, certain rhizosphere microbes fix atmospheric nitrogen, mobilize soil phosphorus and potassium and supply vital nutrients to plants (Wei et al., 2024). These findings collectively demonstrate that the newly prepared formulation, particularly at 8g kg-1 is more effective in promoting plant growth and microbial activity than the old formulation and untreated control.


       
The study showed that prebiotic seed coating, particularly at the 8 g kg^-1 dose, in enhancing seed germination, growth parameters and microbial activity both initially and over a 6-month storage period. The sustained vigour, microbial diversity and soil enzyme activity suggest that this formulation can contribute significantly to improved plant health and productivity.

This study evaluates the impact of prebiotic seed coating on the physiological performance and soil microbial activity of blackgram (Vigna mungo L.), focusing on germination, vigor and microbial populations over six months. The results show that prebiotic-coated seeds, especially at higher doses (8 g kg-1), maintained superior growth parameters, microbial activity and enzyme levels compared to control seeds, supporting enhanced plant and soil health even after 6 months of storage.