Shelf Life Studies of Seed Coat Formulation of Rhizobium and Arbuscular Mycorrhizal Fungus (AMF) for Pulses - A New Perspective in Biofertilizer Technology

Pulses remain as one of the most important vegetarian diet in India. India is one of the leading countries in blackgram and greengram production. India shares 70 per cent of the total world blackgram and greengram production. Rhizobium, a root nodulating bacterium fixes the atmospheric nitrogen in the nodules of leguminous crops and satisfies nitrogen budget to a significant level and also contributes for the subsequent crop. The existence of arbuscular mycorrhizal fungi (AMF) in association with plant root system is known for decades and this fungal partner satisfies the crop nutritional (N, P, Fe, Zn, etc.,) and water requirements and protects plants from biotic/ a biotic stresses. In case of Rhizobium and AMF, plants reward their microbial partners with photosynthetically assimilated carbon (Puschel et al., 2017).

Biofertilizers maintain the soil health by various mechanisms viz., N2 fixation, PO4 solubilization, K solubilization, production of plant growth regulators, etc., (Sinha et al., 2014). Major portion of chemical fertilizers are lost into the environment when applied to soil and these biofertilizer organisms through any of the above said processes makes it available to the plants (Adesemoye and Kloepper, 2009). Hence biofertilizers remains as one of the major components of integrated nutrient management.

Advances in biofertilizer technology have led to production of liquid formulations from carrier based formulations. With an attempt to progress further in biofertilizer technology, a seed coat formulation of Rhizobium and spore formulation of AMF was made by the Department of Agricultural Microbiology, TNAU, Coimbatore -3 and evaluated for its shelf life under storage and survival in coated seeds.

The most reported types of seed coating are seed dressing, film coating and pelleting. Tested in more than 50 plant species with seeds of different dimensions, forms, textures and germination types (e.g., cereals, vegetables, fruits, pulses and other legumes). Seed coating has been studied using various species of plant growth-promoting bacteria, rhizobia, Trichoderma and to a lesser extent mycorrhizal fungi. Most of the studies regarding PBM applied via seed coating are aimed at promoting crop growth, yield and crop protection against pathogens. Studies have shown that coating seeds with PBM can assist crops in improving seedling establishment and germination or achieving high yields and food quality, under reduced chemical fertilization.

All these works were carried in the Dept. of Agricultural Microbiology, AC and RI, Madurai as well as in TNAU, Coimbatore during 2017-2019
 
Survival of Rhizobium on seed coat formulation under storage
 
Powder formulation of Rhizobium (COC 10) was prepared with the cell load of 1012 cells g^-1 and stored in polythene pouches at room temperature. Rhizobium was grown in yeast extract mannitol broth on large volume for 5 days till the population reaches 108 cfu mL^-1. The cultures were concentrated using TFF (tangential flow filtration) system and freeze dried. The freeze dried powder was blended with water soluble carrier dextrin and the population was standardized to 1012 cfu g^-1 and the packing was done in Polythene pouches and sealed and stored under room temperature. Destructive sampling was done at monthly intervals and population was assessed up to 12 months of storage. Serial dilution and plating method with Congored Yeast Extract Mannitol Agar (YEMA) was used for assessing the survival of Rhizobium. Plates were incubated for 4-5 days at 28±2^oC and the population was enumerated.
 
Survival of AM fungi on seed coat formulation under storage
 
AM fungi (Rhizopagus intraradices)was multiplied under Root organ culture in Petriplates  using MSR medium and spores were harvested after dissoluting the phytagel in the medium. Harvesting was done through centrifugation. Harvested spores were blended with water soluble carrier Dextrin and the product was formulated with the spore load of 1040 g^-1  of the product. The formulated product is packed in polyethylene pouches and stored at room temperature. Destructive sampling was done at monthly intervals and spore load was assessed up to 12 months of storage using wet sieving and decanting method as described by Gerdeman and Nicolson (1963) followed by microscopic count under stereozoom microscope.
 
Survival of Rhizobium and AM fungi in coated seeds under storage
 
Blackgram (var. co 6) and greengram (var. co 8) seeds were surface sterilized and treated with seed coat formulation of Rhizobium containing 1012  CFU g^-1 and AM fungi containing spore load of 1040 g^-1 and the seeds were packed in polythene pouches , sealed and stored at room temperature. The following are the treatments used.
 
Treatments
 
1. Single inoculation of Rhizobium @ 25g ha^-1 seed (1012 CFU/g).
2. Single inoculation of AM fungi @ 250 g ha^-1 seed (2.6 lakh spores).
3. Combined inoculation of Rhizobium and AM fungi.
4. Uninoculated control.

For each treatment 10 replications were maintained. Population of Rhizobium and spores of AM fungi were assessed using standard protocol at monthly intervals.
 
Testing the viability of coated seeds under storage
 
Greengram and blackgram seeds coated with Rhizobium and AM fungi individually as well as in combinations were evaluated for germination and vigour index.
 
Germination (%)
 
Four replicates of 100 seeds were uniformly placed on standard germination paper roll-towel medium (ROLL towel medium, ISTA, 1993) and kept in germination room maintained at 25 ± 2oC and 90 ± 2 per cent relative humidity. After the test period of 14 days, the seedlings were evaluated  for germination percentage.
 
Vigour index (VI)
 
Vigour Index (VI) was calculated (Abdul-Baki and Anderson, 1973) by adopting the following formula after estimating shoot length and root length and expressed as whole number.

VI = Germination (%) x Mean total length of seedling in cm 

On fourteenth day, ten normal seedlings per replication from roll towel medium were carefully removed at random from each treatment and the root length was measured from the base to the top of the primary root and the mean value was calculated and expressed in cm. Same way shoot length was measured from the base of the shoot to tip of primary leaf on the 14^th day and the mean value was expressed in cm. For every plant total length was calculated and the mean value is derived.

Survival of Rhizobium and AM fungi in formulation under storage
 
Shelf life is a very important criterion, that should be given due importance during the development of a new formulation. The success of a new product depends solely on its shelf life. The survival of Rhizobium and AM fungi in the newly developed formulation was assessed at monthly intervals. Initial Rhizobium population in the formulation was 12.6 log CFU g^-1. Only a slight reduction in population was observed and no significant reduction was noticed till 12 months of study and it was 11.3 log CFU g^-1  (Table 1). The above results suggest that the newer powder formulation of Rhizobium can be stored up to 12 months at room temperature without any significant loss in the population. According to FCO (Fertilizer Control Order, 1985) standards the biofertilizer Rhizobium should contain the cell load of 5x107 Cells g^-1 of a product. The product tested here had the population of i.e. 1011 cells g^-1 and hence satisfy the FCO standards.



Many researchers have developed the formulation of Rhizobium with silica gel, chitosan, vermiculite, perlite etc., Mixture of the polymers can also be investigated as carriers (Bashan, 1998; John et al., 2011). Denardin and Freire (2000) reported that mixture of polymers of natural or synthetic gave the best result of maintaining the cell population for a period of 6 months. Not only the polymers are playing a role in maintaining the cell load, the form of cells what we are using for the preparation of the formulation is much important than using the fresh vegetative cells for increasing the shelf life of a product. Freeze dried cells gave the higher cell count than the recommendation so that the population loss during processing can be managed.

AM spore load in the formulation decreased  from 1040 spores g-1 to 1000 spores g^-1 at 6 months of storage and at the end of 12 months storage period the spore load in the formulation was 925 g^-1 (Table 1). Though reduction was noticed it is sufficient to colonize the seedlings upon seed coating. With this count at least each seed should contain one propagule, so that it makes colonization in the roots successfully. Hence, the results suggest that the new AM formulation can be stored up to one year with the required spore load at room temperature. According to FCO, the AM inoculant should contain the minimum of 100 viable propagules g^-1 of the product at the time of delivery. The product tested in our study recorded higher than the standard and also the population maintained during storage is also up to the standard. Formulation plays a major role on the survival of the organisms under storage and application as well as its efficiency on the crop plants (Herrmann and Lesueur, 2013). Hence it is utmost important to have better shelf life for the biofertilizers to get popularized and to have better effect on crop plants.

Efforts are made nowadays by the researchers to formulate a range of biofertilizers to ensure maximum viability (Brar et al., 2012). Shelf life of inoculants is a very major factor for their efficacy which mainly depends on several factors (production technology, carrier and packaging material used, transport activity) to sustain the quality of inoculants (Arora et al., 2010).

Our results showed the better cell viability of Rhizobium and AM fungi with  good shelf life of one year and  which are suitable for seed coating.
 
Survival of Rhizobium and AM fungi on coated seeds under storage
 
According to Bashan et al., (2014), only a few research attempts have been made on delivery mechanisms of biofertilizers. The success of a new formulation is decided by the successful delivery of the product under field conditions. With this aim, the newly developed seed coat formulation of Rhizobium and AMF was coated on greengram and blackgram seeds and evaluated for their survival in the coated seeds. Initial population of Rhizobium in greengram seed was 9.0 log CFU/g and significant reduction in population was noticed after 6 months of storage and the population at 12th month was 2.0 log CFU g^-1 of seed. In combined inoculation treatment also initial population was 9.0 log CFU g^-1 and here also significant reduction was noticed after 6 months of storage and the final population at 12^th month was 2.5 log CFU g^-1  of seed. With regard to AM spore load, initial count was 12.4spores g^-1 seed in single inoculation of AM as well as combined inoculation of Rhizobium and AM fungi. Though the spore count was reduced during storage it was found significant only after 6th month of storage, at 12^th month the spore count was 2.2 and 2.4 g^-1 seed respectively for single and combined inoculation.

In blackgram initial population of Rhizobium was 9.2g^-1  seed both in single as well as combined formulations and at 12^th month of storage the population was 3.0 and 3.5 log CFU g^-1 seed respectively. AM spore count was 13.2 and 12.8 g^-1 seed in single and combined formulations. The spore count was reduced slowly under storage and at 12^th month the spore count was 1.6 and 2.0 g^-1 seed respectively in single and combined formulation (Table 2). The results indicated that population of Rhizobium as well as AMF was not significantly reduced till 6 months of storage at room temperature in single as well as combined inoculations in blackgram and greengram. Sufficient population of Rhizobium was maintained up to 9 months of storage in coated seeds of greengram and upto 11 months of storage in coated seeds of blackgram. AM spore count was also found sufficient upto 9 months of storage in blackgram as well as greengram.



Survival of the inoculant bacterial strains on the seed are mainly affected by desiccation, the toxic seed coat exudates as well as the higher the temperatures (Deaker et al., 2004). Bacterial survival on seed in turn directly affects the total yield of legume crops (Brockwell and Bottomley 1995). Muller and Berg, (2008) studied the survival of S. plymuthica cells on the seeds of oilseed rape applied through seed pelleting, film coating and bio-priming. They reported the survival in seed pelleting and film coating were found improved when the seeds were stored at 4°C when compared to 20°C. The survival in bioprimed seeds was unaffected irrespective of the temperature. Improper inoculant formulation or having limited survival on seed surface can influence different kinds of seed coating (O’Callaghan, 2016).The results of the present study confirmed the significant survival of the Rhizobial cells and AMF spores on the coated seeds of greengram and blackgram when stored at room temperature, which showed the suitability of the formulation for seed coating. Bacterial survival on seed directly affects the total legume yield (Brockwell and Bottomley 1995). However, emphases should also be given on techniques for increasing population density and survival of rhizobial strains in inoculants
 
Viability of coated seeds under storage
 
Greengram and blackgram seeds coated with Rhizobium and AM fungi individually as well as in combinations were evaluated for germination. In greengram, seed germination was not affected due to the coating of seed with newer formulation of Rhizobium and AM individually as well as in combination. It was observed as 100% in all the treatments during the initial stage of storage. No inhibition of germination was observed. Seedling vigour was significantly enhanced due to seed coating with AM and Rhizobium and it was found reduced significantly after 3-4 months of storage. At 12th month in control the seedling vigour was 2571 and it was enhanced to 2910 in combined inoculation of Rhizobium and AM fungi. Inoculation of Rhizobium registered an increase of 5.0% over control, AM recorded 6.4% increase over control and the combined inoculation recorded 13.2 % increase in seedling vigour over uninoculated control (Table 3) at 12 months of storage. Several studies showed the positive influence of AM and Rhizobium on plant growth. Inoculated plant performed superior in terms of plant growth and quality over control. All plant growth and yield parameters were observed higher in plant samples inoculated with VAM and Rhizobium in combination as compared to single inoculation of VAM or Rhizobium. Dual inoculation resulted 10, 24, 17, 21 and 14% increase in seed protein. leaf chlorophyll seed fiber and ash content and number of seed pod-1 over single application when averaged over two years. Combined application of Rhizobium + VAM enhanced seed yield by 45% over control and 24% and 28% over single inoculation of VAM and Rhizobium respectively in lentils (Yaseen et al., 2016).



In blackgram, the seed germination was not affected due to coating of seeds with Rhizobium and AM fungi under storage upto 12 months at room temperature. It was 100% in all the treatments at 0 day of storage. A slight reduction in seed germination was noticed in all the treatments. Generally combined inoculation registered higher germination than other treatments.  No significant reduction in vigour index was observed in all the treatments up to 4 months of storage. At 12th month combined inoculation had a significant increase in vigour index over all the other treatments with an increase of 13.3 to 17.7 percent (Table 4). The results indicated that seed germination was not affected due to the coating of seeds with Rhizobium and AM fungi and the vigour index was significantly enhanced in inoculated plants compared to uninoculated control. Sharon et al., (2015) reported that the germination of seeds is more sensitive to quality changes and is known to be affected due to storage conditions. Germination and vigour index are essential factors, which should be considered for a successful crop. Seed coating with microbial inoculants should not alter these properties of a seed. The results of the present study confirmed that the seed coating with the newer formulations of Rhizobium and AMF, does not affect the seed stability during the storage period. Seed coating technology have been evaluated with different crop plants such as cereals, vegetables, fruits and legumes with the use of plant growth promoting bacteria and mycorrhizal fungi and  have shown that coating seeds with beneficial micro organisms can assist crops in germination, improving seedling establishment and achieving high yields and food quality, under reduced chemical fertilization. (Rocha et al., 2019).



Seed coating with plant beneficial microorganisms is a promising technology which allows a precise application of minor amounts of inocula at the seed- soil interface (Scott, 1989) and ensuring that the inoculated organisms are readily accessible at germination as well as early developmental stages of the plant, stimulating healthy and rapid establishment and consequently maximizing the crop production (Colla et al., 2015). Further seed coating significantly reduce the amount of inoculum needed as well as avoiding the wastage of inoculum through non targetted delivery. Also left over after seed treatment can be reduced through this new formulation since it coats the seed very thin.

Shelf life and stability are the two perspectives that should be considered during the development of a new biofertilizer formulation. From the present study it was evident that the newer freeze dried powder formulation of Rhizobium and AMF can be stored up to 12 months at room temperature without any significant loss in the population and spore load respectively. The outcome of the current research concludes that, this new seed coat formulation of Rhizobium and AMF may serve as forward milestone in the biofertilizer technology. In future many more newer formulations which ensure the reachability of the seed and the rhizosphere, as well as the fortification of the formulation with stimulating compounds such as flavanoids, precursors and plant hormones are need to be worked out.