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.
Table 1: Survival of Rhizobium and AM fungi in powder formulation under room temperature storage.
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 12th
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 12th
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
seed both in single as well as combined formulations and at 12th
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 12th
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.
Table 2: Survival of Rhizobium and AM fungi in coated seeds of greengram and blackgram under storage.
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).
Table 3: Seed germination and vigour index of greengram coated with Rhizobium and AM fungi under storage condition.
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).
Table 4: Seed germination and vigour index of blackgram coated with Rhizobium and AM fungi under storage condition.
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.