WO2022038625A1 - Wickerhamomyces anomalus -a polyfunctional isolate, formulated as biological input for agricultural & horticultural use - Google Patents

Abstract

A method of growing environmentally friendly and benign marine yeast strain, Wickerhamomyces anomalus, MSD1, for use as a biological input with multiple activities such as solubilizing insoluble nutrients for the uptake of nutrients, resulting in enhanced growth and yield in subjects, protective activities against plant fungal pathogens, inducing resistance under biotic and abiotic stress and inherent capability to be viable under adverse environments and compatibility to chemical fertilizers & pesticides. The invention also includes a method, composition, and a kit comprising processed MSD1 in a suitable carrier, which can be delivered as a product, 5MIN, for application in agriculture and horticulture use. The invention also covers a pre-formulation medium during processing step to retain viability and enhanced shelf life of MSD1 before delivering to a subject and thereby retain its inherent multifunctional attributes.

Description

Wickerhamomyces anonuuus -A polyfunctional isolate, formulated as biological input for Agricultural & Horticultural use

Field of the Invention

The present invention relates to a biostimulant product with multiple plant growth promoting properties and plant protective activities against fungal phyto pathogens. It includes a method of producing the same and its use in agriculture and horticulture for improved plant growth and yield.

Background of the Invention

Agriculture is the backbone of country’s economy and farmers have to be provided with adequate resources to not only protect his farmland but also to provide sufficient supply of crops to the community. The adequate resources include sufficient supply of seeds, fertile soil, water, fertilizers and pesticides. In agriculture and horticulture, fertilizers include chemical fertilizers, which are meant to nourish the soil but ends up as a toxic agent, which gets drained into ground water and thereby affecting aquatic life and essentially ends up in food chain itself. The use of pesticides to curb the bacterial and fungal infections may also lead to leaching of these chemicals from soil to ground water and become toxic to the entire ecosystem. Continuous and unbalanced use of chemical fertilizers results in reduced nutrient uptake efficiency of plants; decreased crop yield; deterioration of soil, groundwater and atmosphere, accompanied by losses of the applied fertilizers.

In contrary to the use of chemical fertilizers, application of bio-stimulants/ bio fertilizers/PGPR is benign and environmentally friendly to the soil and the entire ecosystem. Soil is the natural source for supplying both macro and micro nutrients to the plants, which undergo a complex dynamic equilibrium of solubilization and insolubilization, and largely dependent on the pH of soil, micro-flora, which affects their accessibility to plant roots for efficient absorption. In situations when soil is not be amenable to provide necessary nutrients or less fertile and for plants with stunted growth, there is a need for supplemental source to aid the soil for nutrients uptake.

Nitrogen, phosphorus and potassium are primary plant nutrients, whereas calcium, magnesium and sulphur are secondary nutrients; and zinc, iron, manganese, copper, boron, molybdenum and chlorine form the micro-nutrients.

These form the critical nutrients in plant nutrition, which possess specific important plant functions but differ in their requirement levels. Of all the above mentioned macro & micro nutrients, Nitrogen, phosphorus and potassium plays a vital role in a plants growth along with critical minor nutrients including sulphur, zinc & iron, and their effect of deficiency are shown below in a tabular form.

There are various types of biostimulant composition known in the prior art, which supplements the soil or growth medium in nourishing the necessary nutrients for plant growth in agriculture and horticulture.

In US 2019/0021339 Al, disclosed the method of producing a biostimulant, with a diverse group of beneficial microorganisms for treatment in the soil. The aqueous biostimulant produced photosynthetic bacteria, Lactobacillus sp., fermenting fungi/yeast (a range of organisms including Wickerhamomyces anomalus as one of them). This invention included plurality of microorganism in the composition and most importantly there was considerable down time on mixing with the manure before the bio-stimulant can find its application as biopesticide, probiotics and animal healthcare etc.,

In WO 2018/094075, disclosed the production of a microbes-based composition with the growth by products such as biosurfactants, soporolipid (SLP) and/or mannosylerythrithol lipid (MEL) along with other pesticidal components. Different embodiments of the invention disclosed the use of wide range of microbes including a group of gram positive, gram negative bacteria (Pseudomonas syringae, Bacillus subtilis), and combination of yeast species including Wickerhamomyces sp., as one of the isolates but not limited to other species of Pichia including Pichia anomala, Pichia guielliermondii, Pichia kudrizvzevii one or combinations of solid state and submerged fermentation. The microbial composition with biosurfactants was recommended for the control of pests, especially nematodes, when applied on the field. This study clearly focused on the use of surfactants produced by a group of microbes for its antinematicidal activity added along with a range of oils in the composition and their coincidental effect on plant growth. There was no mention or data on some of the essential nutrients in the plant growth.

WO 2017/174503, disclosed the use of microorganism free compositions with inactivated microbes along with carriers for its biostimulant activity and plant growth promoting functions. The microbes were selected from a range of bacterial, fungal and yeast species with Wickerhamomyces anomalus as one of the isolates in the composition, with Alternaria alternata being the main component of the composition prepared. Also, the invention disclosed the plant growth promoting capability of volatile organic compounds (VOC’s) produced by the microbes (a consort used for preparing the compositions). Microbial free compositions along with the metabolites produced by these organisms in the culture medium, which was either separated, or as inactivated microbes was prepared as a product for its application in the field. Though this invention provided an instant effect on the plants, sustained effect is questionable, since the basic source is metabolites.

WO 2019/023034, disclosed the microbe based composition using Pichia clade of yeasts (P.anomala, P.guilliemondii, P.kudriavzevii in combinations) and/or growth by products such as biosurfactants (Sophorolipids & Mannosyl erythritol lipids), enzymes and metabolites for improved production in agriculture, horticulture and livestock’s. This application covered treating or preventing nutrient deficiency in plant, especially phosphorous deficiency. However, other essential nutrient elements were not covered and more importantly this application covered cocktail of microorganisms along with other growth byproducts in the composition.

US 2015/0232804 Al, disclosed the use of complex microbial flora with Wickerhamomyces anomalus either alone or in combination with a set of microbial isolates including species of Bacillus, Pseudomonas, Acinetobacter, Rheinheimera. This invention explored the method of making biological pulping solution for paper making, and producing fibers (textile), cellulose (for use as an additive).

IN218260 B, disclosed a method for producing a biological fertilizer composition which displayed its ability to fix atmospheric nitrogen, solubilizing phosphorous, potassium while utilizing other carbon sources but replacing the use of chemical fertilizers. The composition included at least one yeast cell component and combination of six strains. This invention mainly focused on production of a biofertilizer composition with combination of one or more of yeasts species preferably from Sachharomyces sp., for nitrogen fixation, decomposition of Phosphorous and Potassium compounds. The effect of this biofertilizer composition under in vivo condition and further field studies to understand nutrients uptake or plant growth promotion were not reported.

WO 2019/162913 Al, disclosed preparation of an agricultural biostimulant for stimulating the growth of plants, seeds and to enrich the soil. The composition contained a biofertilizer (bacteria/fungi/virus), seaweed extract powder, plant elicitor, natural plant extracts with pesticide activity and wax as fluency agent which altogether enhanced the soil quality and in turn plants growth & yield. This invention failed to cover the effects of this composition on the plants, and changes observed in the treated plants.

Srinivasan et al. (Seaweed Res. Utiln., 39(2): 39 - 46, 2017) reported screening of microorganism from seaweeds for different plant growth promoting activities. But this study did not cover understanding the potency of the isolate towards plants for multiple activities including protective activities.

Khunnamwong et al, 2019 (Khunnamwong, P., Lertwattanasakul, N., Jindamorakot, S., Suwannarach, N., Matsui, K., & Limtong, S. (2019). Evaluation of antagonistic activity and mechanisms of endophytic yeasts against pathogenic fungi causing economic crop diseases. Folia Microbiologica, 1-18) has reported the isolation of endophytic Wicker hamomyces anomalus from the leaf tissues of rice, sugarcane and com. This article studied the antifungal activity of these W.anomalus strains against most of the foliar pathogens of rice, corn and sugarcane It has to be noted that reported properties with the isolates of Wickerhamomyces anomalus were tested only in vitro and the application of these outside the lab conditions as a product were not explored..

Limtong et al., 2020 (Limtong, S., Into, P., & Attarat, P. (2020). Biocontrol of Rice Seedling Rot Disease Caused by Curvularia lunata and Helminthosporium oryzae by Epiphytic Yeasts from Plant Leaves. Microorganisms, 8(5), 647) reported the isolation of Wickerhamomyces anomalus from the surface of rice, sugarcane and corn. This study focused on understanding the antifungal activity against two pathogens viz., Curvularia lunata and Helminthosporium oryzae causing rice seedling rot disease. This article focused on studying phosphate and zinc oxide solubilization efficiency of the isolate, quantification of solubilized minerals were not reported and the nutrient solubilization efficiency of the isolate under in vivo and field conditions were not explored.

Kumla et al., 2020 (Kumla, J., Nundaeng, S., Suwannarach, N., & Lumyong, S. (2020). Evaluation of Multifarious Plant Growth Promoting Trials of Yeast Isolated from the Soil of Assam Tea (Camellia sinensis var. assamica) Plantations in Northern Thailand. Microorganisms, S(8), 1168) reported the isolation of Wickerhamomyces sp., from the soil of Assam tea plantations. The properties of Indole Acetic Acid (IAA) and Siderophore production by W.xylosivorus and W.mori were reported. Further, the degree of solubilization of Phosphate and Zinc were 1-2 in the solubilization index, which are considered as activity with only medium solubilization potential. This article studied the reported properties using active culture as such and failed to test the same as a product, which poses several challenges.

Few of the above published articles reported only select properties of yeast strain Wickerhamomyces anomalus. Some of these properties were explored only in the lab conditions, but the biggest challenge remains in exploring this outside of the lab conditions, i.e. field study, which comes with issues such as mode of delivery, packaging this without losing the activity of microorganism to achieve a desired outcome. In essence, the search is on for an effective plant growth promoting microbes, which works in all conditions, that is economical, and an effective alternative for increasing crop productivity.

The end user, a farmer, needs access to products that are easy to use, without worrying about the mixing proportions of different supplements, associated toxicity and side effects, if any due to cross contamination. Thus there is a need for a simpler stimulant which would pack all necessary ingredients and deliver all the desired results in one pack and thereby improve the productivity in agriculture and horticulture.

Objectives of the Invention

It is a general objective of the invention to provide a method of growing microorganism, bio-stimulant composition and a kit for use in agriculture and horticulture, which avoids aforementioned drawbacks of the known prior art.

It is the main objective of the invention, the isolate from the culture medium with no more than Wickerhamomyces anomalus (MSD1), resulting in plethora of growth promoting and protective activities against fungal phytopathogens.

It is also an objective of the invention, the uptake of nutrients resulting in conversion of insoluble nutrients from a subject into a soluble form and thereby readily absorbed by products resulting in growth.

It is another objective of the invention, MSD1 produces siderophore.

Yet another objective of the invention is the use of a biostimulant composition comprising not more than one microorganism, Wickerhamomyces anomalus (MSD1) and a carrier, as a biostimulant for manifesting all of plant growth promoting and protective activities against fungal phytopathogens.

In yet another objective of the invention, the invention includes a kit for plant growth promoting and protective activities against fungal phytopathogens. Summary of the Invention

This invention broadly solves the problem of related art by a method of growing, separating cell-biomass, Wickerhamomyces anomalus (MSD1), from a culture medium and mixing the isolate with a pre-formulation medium to retain viability of MSD1 and preserve the shelf life of MSD1, after processing of the culture medium, but before mixing with a carrier for delivery to a subject.

This marine based yeast strain, MSD1, with no other microorganisms shows plethora of growth promoting activity in product by effectively converting insoluble nutrients from subjects to soluble nutrients, in a readily absorbable form in products.

In addition, MSD1 also shows protective activities against fungal phytopathogens. MSD1 is formulated with a carrier for use as commercial plant biostimulants product in agriculture and horticulture, which is easy to handle and can be stored at room temperature. Additionally, a kit comprising this biostimulatory composition also exhibits all of the above properties.

The first aspect of the invention relates to a method of growing a marine yeast strain for use in agriculture and horticulture comprising: a) Growing said marine yeast strain in a culture medium; b) Assessing the culture medium by collecting samples at different time points until it reaches stationary phase; c) Processing the culture medium to retrieve concentrated wet biomass from step b); d) Mixing said wet biomass in a pre-formulation medium; and e) Mixing with a carrier for delivery to a subject

Wherein said wet biomass contains no more than one microorganism;

Wherein the microorganism is Wickerhamomyces anomalus, MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical-elongate cells with multilateral budding;

Wherein said MSD1 shows plethora of growth promoting activities in a subject; and protective activities against fungal phytopathogens; Wherein said pre-formulation medium enables MSD1 to be dormant, retaining its viability during post processing, and thereby prolongs shelf life of MSD1 before delivery to the subject.

The second aspect of the invention covers a biostimulant composition for agriculture and horticulture comprising: a) A wet biomass of MSD1 from a culture medium; b) Mixing MSD1, after processing from step a) with a carrier to obtain 5MIN; and c) Selecting a formulation to deliver said 5MIN to a subject;

Wherein the wet biomass contains only MSD1;

Wherein said MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical -elongate cells with multilateral budding; wherein said MSD1 shows plethora of growth promoting activities in a subject; and protective activities against fungal phytopathogens;

Wherein a pre-formulation medium is added after the processing in step b) but before mixing with a carrier, whereby said pre-formulation medium enables MSD1 to be dormant, while retaining its viability and prolongs shelf life of MSD1 before delivery to the subject.

This aspect of the invention also covers growth protective activities in a subject and protective activities from fungal phytopathogens.

The third aspect of the invention covers a kit for use in agriculture and horticulture comprising: a) MSD1 obtained from a culture medium; b) Mixing MSD1, after processing from step a) with a carrier to obtain 5MIN; and c) Selecting a suitable formulation to deliver said 5MIN to a subject;

Wherein the culture medium contains only MSD1 and no other microorganism;

Wherein MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical-elongate cells with multilateral budding; Wherein said MSD1 shows plethora of growth promoting activities in a subject; and protective activities against fungal phytopathogens;

Wherein a pre-formulation medium is added after the processing in step b) but before mixing with a carrier, whereby said pre-formulation enables MSD1 to be dormant, while retaining its viability and prolongs shelf life of MSD1 before delivery to the subject .

This aspect also covers growth promoting activities in a subject and protective activities from fungal phytopathogens.

The broader scope of applicability of the present invention will be apparent from the detailed description below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, should not be construed as the limitations to the invention, and it is within the scope of those skilled in the art to make various changes and modifications, such as mutation of the microorganism, other nutrients, sources, and subjects, to the spirit and scope of the invention from this detailed description.

Brief Description of the Drawings

Figure 1 shows Colony morphology of Wickerhamomyces anomalus (MSD1) in Pikovskaya’s agar medium observed from front and back of the agar plates.

Figure 2 shows the Gram’s reaction of Wickerhamomyces anomalus (MSD1)

Figure 3 shows the Agarose gel electrophoresis image, showing the DNA isolated from MSD1 and the amplified ITS region.

Figure 4 shows the phylogenetic tree of Wickerhamomyces anomalus (MSD1)

Figure 5 shows the method of counting viable cells and dead cells using Haemocytometer cell counting chamber.

Figure 6 Iron transformation efficacy of 5MIN in tomato plants in shade house condition in comparison to control Figure 7 shows the fresh weight of Radish cotyledons on treatment with MSD1 against water control and BAP control treatments under laboratory conditions.

Figure 8 shows the effect of 5MIN on Coleoptile elongation rate and growth of maize seeds in comparison to untreated water control.

Figure 9 shows the photographs showing variations in elongation of maize coleoptiles after 72hrs of treatment with 5MIN in comparison to water and IAA control.

Figure 10 shows the effect of 5MIN on seed germination, growth and vigor index of green gram by seed treatment method.

Figure 11 shows the effect of 5MIN on seed germination, growth and vigor index of radish by seed treatment method.

Figure 12 shows the orange halo zone displayed by 5MIN product against uninoculated control in Pikovskaya’ s agar medium with CAS reagent.

Figure 13 shows the orange halo zone displayed by Active ingredient of 5MIN product (MSD1) against uninoculated control in Pikovskaya’ s agar medium with CAS reagent.

Figure 14 shows the orange halo zone displayed by Pseudomonas fluorescens (positive control) against uninoculated control in Pikovskaya’ s agar medium with CAS reagent.

Figure 15 shows the growth data of 5MIN treated groundnut plants in comparison to control at in-house field.

Figure 16 shows the growth & yield data of 5MIN treated rice plants (Ponni variety) in comparison to control at Vadakumelur village, Panruti Taluk, Cuddalore district, Tamil Nadu. Figure 17 shows the growth& yield data of 5MIN treated rice plants (BPT variety) in comparison to control at Vadakumelur village, Panruti Taluk, Cuddalore district, Tamil Nadu.

Figure 18 shows the effect of 5MIN on the growth & yield of paddy at Mullakolathur village, Kanchipuram, Tamil Nadu in comparison to control

Figure 19 shows the effect of 5MIN on the growth & yield of rice plants in comparison to positive control.

Detailed Description:

Selected Definitions

All the terms used in this application carry ordinary meaning as known in the prior art unless otherwise specified. Few other specific definitions used in this invention are explained below, which applies throughout this specification. Claims provide broader definition unless and otherwise specified.

The culture medium refers to both solid and liquid medium, which supports the growth of MSD1.

The term “sample” refers to a small quantity or an aliquot taken for analysis which is representative of the whole population.

The term “assessing” refers to evaluating parameters such as colony forming units (CFU)/ml, pH, microbial contamination in the samples tested.

The term “processing” refers to steps involved in mass multiplication of MSD1, and separation to get the isolate ready for further characterization and studies involved in this invention.

The term “isolation” with respect to the current invention refers to the removal of a microbial isolate from a native flora of microorganism as present in the natural environment.

The term “separation” refers to the removal of MSD1 cells from the culture medium after the incubation period of 48-72hrs. The term “Inoculum” or a “seed inoculum” refers to the small quantity of cells required to initiate the mass multiplication of MSD1 in the said culture medium.

The wet biomass refers to isolate obtained after processing, which is filtration and centrifugation after reaching a desired level (colony forming units) from the culture medium. The isolate is not further purified and the concentrate obtained is considered wet biomass, which was further characterized and studied.

A pre-formulation medium is a medium added to concentrated wet biomass before the post processing step such as dehydration, to preserve the activity of microorganism used in this invention and thereby enhance the shelf life of microorganism.

The colony forming units (CFU) refers to the number of viable (ability to grow and multiple) yeast cells in the sample tested.

The marine yeast strain or microorganism in the specification refers to the isolated microorganism, W ickerhamomyces anomalus (MSD1), and whose strain is shown to exhibit all the properties explained here in this description and which is deposited in patent deposit bank of MTCC, Chandigarh, India [Ref. No. Patent deposit MTCC 25284 dated 01.07.2019], It is possible for those skilled in the art to come up with mutants or other variations of the strain associated with this invention or combinations involving this strain to exhibit all or some of the properties covered in this invention.

The agriculture and horticulture covered in this invention refers to plants, saplings, seed, soil, seedlings, potting mixture, and hydroponics.

The growth promoting activity exhibited by this strain, mainly refers to positive modulation or stimulatory or growth promoting activities in subjects and its features namely: Seed germination, Coleoptile elongation rate, root length, shoot length, dry weight, vigor index, early flowering, fruit setting, number of cobs, number of pods, number of branches, enlarged leaf surface area.

Similarly protective activities refer to inhibiting the growth of few fungal plant pathogens in soil, viz., Sclerotium rolfsii, Rhizoctonia solani, Botrytis cinerea, Colletotrichum gloeosporioides, and eliciting resistance and enhancing defense potential in plants.

The kit in this invention refers to a commercial product including the product information sheet with instructions to use or a kit with premixed ingredients for improving plant health or crop care.

The use of word “effective amount” in this invention refers to anything that brings about a positive or a modulatory effect in products covered under this invention, by adding a specified amount listed under this invention to the subjects covered under this invention.

The invention provides a method of growing, separating, processing of the culture medium to yield a wet biomass, Wicker hamomyces anomalus (MSD1). This is further mixed with a pre-formulation medium, which retains its viability and thereby prolongs the shelf life of MSD1 before delivery to a subject. This marine based yeast strain, MSD1, with no other microorganism, shows plethora of growth promoting activity in plants by effectively converting insoluble nutrients from subjects to soluble nutrients, which can be readily absorbed in products. In addition, MSD1 also showed protective activities against fungal phytopathogens. MSD1 is mixed with a pre-formulation medium, “Stanes formulation medium”, before dehydration and further mixed with a carrier, to yield 5MIN, for delivery to variety of subjects as a biostimulatory composition for use in agriculture and horticulture. Additionally, kit comprising this biostimulatory composition also exhibits all of the above properties.

The first embodiment of the invention covers a method of growing a marine yeast strain for use in agriculture and horticulture comprising: a) Growing said marine yeast strain in a culture medium; b) Assessing the culture medium by collecting samples at different time points until it reaches the stationary phase; c) Processing the culture medium to retrieve concentrated wet biomass from step b); d) Mixing wet biomass in a pre-formulation medium; and e) Mixing with a carrier for delivery to a subject; Wherein said wet biomass contains no more than one microorganism;

Wherein the microorganism is Wickerhamomyces anomahis. MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical-elongate cells with multilateral budding; wherein said MSD1 shows plethora of growth promoting activities in a subject; and protective activities against fungal phytopathogens;

Wherein said pre-formulation medium enables MSD1 to be dormant, retaining its viability during post processing step, and thereby prolongs shelf life of MSD1 before delivery to the subject.

Isolation and Characterization of MSD1

The marine yeast strain used in this and other embodiments of the invention was isolated from the marine macroalgae sample (Sargassum sp.,) collected from Mandapam Beach Park, Rameswaram, Tamil Nadu, India. After washing twice, sufficient quantity of the sample with sterilized RO water, ground and mixed in 90 ml of saline (0.85% NaCl) solution, kept in shaker for 30 minutes. Then the samples are serially diluted up to IO'¹⁰ dilution and plated on Zobell marine agar (ZMA), Potato Dextrose agar (PDA), Starch Casein agar (SCA) plates and incubated at 28°C ±2°C for 5-7 days (Singh, R.P., V.A. Mantri, C.R.K. Reddy and B. Jha 2011. Isolation of seaweed-associated bacteria and their morphogenesis inducing capability in axenic cultures of the green alga Ulva fasciata. Aquat. Biol., 12: 13-21).

The bacterial population from the collected marine macroalgae was 4.2 x 10⁵ CFU/g and fungal population was 1.0 x 10³ CFU/g.

The marine yeast strain Wickerhamomyces anomalus referred to as MSD1 is identified microscopically, morphologically, biochemically & through molecular analysis by ITS gene amplification (using universal primer pair ITS1 and ITS2). The amplified product is sequenced and subjected to identification using Basic Local Alignment Search Tool (BLAST) and Ribosomal database project (RDP) homology search. The phylogenetic tree is constructed with related sequences retrieved from the NCBLNucleotide databases using the maximum likelihood algorithm with bootstrap values based on 1000 replications in MEGA version 5.1.

The characterization of MSD1 include biochemical tests with 21 different sugars (i.e., melibiose, fructose, inositol, inulin, maltose, Arabinose, xylose, Mannitol, trehalose, adonistol, dextrose, lactose, rhamnose, sorbitol, galactose, dulcitol and sucrose), Gram’s reaction, formation of germ tube test, positive/negative reactions for IMVIC, gelatin hydrolysis, nitrate reduction and H2S production (Table 1). MSD1 is found to be Gram positive budding yeast, and showed positive for citrate utilization test. MSD1 is capable of utilizing various carbon sources such as dextrose, fructose, maltose, mannose, Salicin & sucrose. Formation of germ tube is not observed with this MSD1 isolate.

The colony morphology of the isolate, MSD1, on Pikovskaya’s agar medium is creamy white color, circular shape and size ranged from 1-6 mm (Figure.1). The cells are Gram positive; spherical-elongate, with multilateral budding (Figure.2). Based on the microscopical, morphological and biochemical analysis (Table 1), the isolate (MSD1) is identified and further confirmed through molecular analysis by ITS gene amplification (Figure.3).

Based on the BLAST homology search, the similarity between the ITS gene sequences of MSD1 showed 100% resemblance to Wickerhamomyces anomalus with an e-value of 0.0. Phylogenetically, MSD1 is clustered within the clade of Wickerhamomyces anomalus (Figure. 4) confirming it as Wickerhamomyces anomalus and it is submitted to NCBI with an accession number MF 174856 (https://www.ncbi.nlm.nih.gov/nuccore/MF174856) dated 29.05.2017. Whole genome sequence (WGS) of MSD1 was carried out by sequence analysis; the isolate was confirmed as Wickerhamomyces anomalus. The WGS of MSD1 was deposited at GenBank/NCBI under the accession number SRR10092046 and BioProject number

PRJNA556347(https://datavi ew.ncbi.nlm.nih.gov/object/PRJNA5563477revi ewer =3dn0qimfp435s37na6fr53oke8). The associated Illumina HiSeq 4000 subreads are available under the SRA accession number SRR9822044.

Table 1: Biochemical characterization of Wickerhamomyces anomalus (MSD1) Optimization of Growth Conditions of MSD1

In this embodiment and other embodiments of the invention, the culture medium for growing MSD1 is preferably selected from, Stanes growth medium, Yeast Peptone Dextrose (YPD) Broth, Potato Dextrose (PDB) Broth, Sabouraud Dextrose broth, Pikovskaya’s medium, Nitrogen free malic acid medium, Starkey mineral salt (SMS) medium supplemented with 1% Sodium thiosulphate, LM Broth, Zinc oxide (ZO agar), modified Chrome Azurol S (CAS) agar medium, nutrient broth medium supplemented with tryptophan (0.1g/l), DF medium, Bushnell hass medium supplemented with 10% coconut oil, G1 Medium (Yeast extract -lOg/L, Peptone - 20g/L, Dextrose 20g/L, K2HPO4- 2g/L, KH2PO4 - Ig/L) and combination thereof. And more preferably the culture medium is Stanes growth medium. MSD1 is grown in lab scale, pilot scale and large-scale fermentors and not limited to continuous, batch, fed batch with an optimized experimental condition.

Similarly, the effect of temperature, initial pH and incubation time is optimized for growing MSD1 in laboratory condition. Effect of temperature (25°C, 30°C, 37°C, 40°C, 42°C), different pH (3, 5, 7, 9) and incubation period (0^th hour, 8^th hour, 24^th hour, 32^nd hour, 48^th hour, 56^th hour, 72^nd hour) on the growth of MSD1 in G1 media to obtain maximum cell viability (Table 2). The optimized parameters achieved in each step i.e. temperature 30°C, pH 7.0 and incubation period of 72 hrs, was fixed as a optimized parameters for all the optimization studies in this invention.

MSD1 isolate shows good growth in terms of cell viability preferably at 30°C (2.0 x 10⁷ CFU/ml) compared to 25°C and 37°C but above 40°C, observed growth was nil (Table 2a). The growth of the isolate MSD1 shows higher cell viability preferably in the range of pH 5 to 9 (Table 2b), indicating its survivability at a wide range of pH. And more preferably the optimum pH for the growth of MSD1 is in the range of pH 5 - 7. The optimum incubation period that supports maximum growth of MSD1 is in the range of 60 to 72 hrs and more preferably 72 hrs (1.8xlO⁸CFU/ml) (Table 2c).

Table 2a: Effect of temperature (at constant pH of 7.0 and incubation period of 72 hrs) on the growth of Wickerhamomyces anomalus (MSD1) under static condition

Table 2b: Effect of pH (at constant temperature of 30°C and incubation period of 72hrs) on the growth of Wickerhamomyces anomalus (MSD1) under static condition

Table 2c: Effect of incubation period (at constant temperature of 30°C and pH of pH 7.0) on the growth of Wickerhamomyces anomalus (MSD1) under static condition.

The above optimized conditions of pH, temperature and incubation time for mass multiplication of the isolate was used in the culture medium. Mass multiplication and Formulation of MSD1

Further, in this and other embodiments of the invention, processing the culture medium to retrieve concentrated wet biomass is not limited to filtration, centrifugation, and combinations thereof. It may also include sedimentation, flocculation and others that are known to those skilled in the art.

The amount of wet biomass of Wicker hamomyces anomalus, MSD1, after fermentation in pilot scale fermentor can vary from 2% to 7%, preferably from 3% to 6% by wet weight of the culture medium and cell viability could vary from IxlO⁸ CFU/mL to 5xl0⁹ CFU/mL, preferably between 5xl0⁸ CFU/mL to lxlO⁹CFU/mL. It can be achieved with 0.5 to 3% of active seed inoculum in the presence of nutrients such as carbon preferably ranging from 2% to 5%, organic nitrogen preferably of 0.2-1%, phosphates preferably between 0.1 to 0.4%, chlorides of about 0.1 to 0.4% and sulfates of 0.2 to 0.6%. Aforementioned optimized fermentation conditions include maintaining temperature between 28°C to 32°C, and pH within the range of pH5.0 to pH7.5 and incubation periods ranging from 48-72 hours. The process time in pilot scale fermentor preferably could be from 48 to 72 hrs with agitation preferably ranging from 100 to 200 rpm, aeration ranging between 0.4 to 1.5 VVM (Vessel Volume per Minute). The wet biomass of Wickerhamomyces anomalus, MSD1, is then separated by conventional separation process, such as filtration or continuous centrifugation.

In accordance with various embodiments of the invention, after processing of the culture medium to yield wet biomass, followed by continuously mixing the concentrated form of wet biomass with a pre-formulation medium, referred as Stanes formulation medium, which is eco- friendly water encapsulatable soluble material in the liquid form such as Polyphosphate preferably from 0.2 to 1%, nonreducing sugar i.e. trehalose, sucrose preferably 2 to 10%, polyols i.e. polyethylene glycol, polypropylene glycol, glycerol preferably 0.1 to 1% and skim milk preferably 10 to 20%,. The water may be removed after the encapsulation treatment with the above ingredients, such as by vacuum drying, spray-drying or freeze-drying. Since post processing involves various steps including dehydration of MSD1 after separation of wet biomass. It is essential that MSD1 has to be dormant, retain its viability with prolonged shelf life before delivery to the subjects, and Stanes formulation medium ensures MSD1 is stable, viable with prolonged shelf life.

This pre-formulation more preferably may comprise water-soluble carriers such as sucrose, glucose, lactose, water soluble starch, cellulose etc. preferably in the range 50 to 95% with MSD1 for immediate revival of microbes. The choice of carrier includes but not limited to sucrose, glucose, lactose, water soluble starch and the combination thereof. The ratio of MSD1 to said carrier preferably be in the range 10-40%, or 15-45%, 20-40%, or 30-50% or more preferably 5- 50%. This mixture is 5MIN.

Further, the choice of formulation for delivery of 5MIN includes but not limited to water soluble powder, particulate solid, liquid, dispersions, suspensions, emulsions and combinations thereof. And these formulations of 5MIN are delivered to subjects, and choice of subjects include but not limited to soil, seeds, compost, manure, culture tubes, petri plates, culture flasks, hydroponics, potting mixture and any combinations thereof.

In another embodiment of the invention, a biostimulant composition for agriculture and horticulture comprising: a) A wet biomass of MSD1 from a culture medium; b) Mixing MSD1, after processing from step a), with a carrier to obtain 5MIN; and c) Selecting a formulation to deliver said 5MIN to a subject;

Wherein the wet biomass contains only MSD1;

Wherein said MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical -elongate cells with multilateral budding;

Wherein said MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical -elongate cells with multilateral budding;

Wherein said MSD1 shows plethora of growth promoting activities in a subject; and protective activities against fungal phytopathogens; Wherein a pre-formulation medium is added after the processing in step b) but before mixing with a carrier, whereby said pre-formulation enables MSD1 to be dormant, while retaining its viability and prolongs shelf life of MSD1 before delivery to the subject.

All the protocol involved in isolation, growth, processing and formulation of MSD1 from the culture medium are similar to the previous embodiment. The choice of culture medium, formulation and subjects are discussed in previous embodiment, which extends to this embodiment as well.

In yet another embodiment of the invention, a kit for use in agriculture and horticulture comprising: a) MSD1 obtained from a culture medium; b) Mixing MSD1, after processing from step a), with a carrier to obtain 5MIN; and c) Selecting a suitable formulation to deliver said 5MIN to a subject;

Wherein the culture medium contains only MSD1 and no other microorganism;

Wherein said MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical -elongate cells with multilateral budding;

Wherein said MSD1 shows plethora of growth promoting activities in a subject; and protective activities against fungal phytopathogens;

Wherein a pre-formulation medium is added after the processing in step b) but before mixing with a carrier, whereby said pre-formulation enables MSD1 to be dormant, while retaining its viability and prolongs shelf life of MSD1 before delivery to the subject.

All the protocol involved in isolation, growth, processing and formulation of MSD1 from the culture medium are similar to the previous embodiment. The choice of culture medium, formulation and subjects are discussed in previous embodiment, which extends to this embodiment as well. In yet another embodiment of the invention, a siderophore producing Wickerhamomyces anomalus, MSD1 in growth medium incorporated with CAS + HDTMA reagent, is characterized to produce 15-20pg/ml.

Properties of MSD1:

In all the embodiments of the invention described above, MSD1 exhibits stimulatory and growth enhancing activities with the protocols explained in the examples section (see Table 3 and associated examples thereof). The choice of subjects in all the embodiments include but not limited to soil, seeds, compost, manure, culture tubes, petri plates, culture flasks, hydroponics, potting mixture and combinations thereof.

The growth promoting activities of MSD1 is exhibited by 5MIN, which converts insoluble nutrients to soluble nutrients, whereby said soluble nutrients resulting in plethora of growth promoting activities in the subjects covered under various embodiments of the invention.

The growth promoting activities in various embodiments of the invention include but not limited to Nitrogen fixation (Example 1); phosphate solubilization (Example 2); Sulphate oxidation (Example 3); Iron transformation (Example 4a, 4b); Zinc solubilization (Example 5a, 5b, 5c); Cytokinin like activity (Example 6); Siderophore production (Example 9); IAA production (Example 10); ACC Deaminase production (Example 11); Biosurfactant and emulsification activity (Example 15).

The insoluble nutrients covered under various embodiments of the invention include but not limited to tricalcium phosphate, Zinc oxide, Ferric citrate, sodium thiosulphate and combinations thereof. And more specifically, the insoluble nutrients are either intrinsically present or externally added to the subjects. The concentration and proportion of insoluble nutrients are mentioned in the examples section.

The soluble nutrients under various embodiments of the invention include but not limited to Nitrogen; Zinc; Ferrous; and Sulphur; the uptake of soluble nutrients by the product result in plant growth promoting activities in the respective products.

The products include plants but not limited to application of plants alone, and the plant growth promoting activities is measured preferably based on increase in growth or yield of the product in the range of 1-30%, or 5-25%, 10- 20% and more preferably 15-30% of the product.

The other properties of MSD1 include protective activities against fungal phytopathogens. These include, but not limited to, antifungal activity (Example 12), Volatile Organic Compounds (VOC) (Example 13), biofilm formation (Example 14), biosurfactant and emulsification activities (Example 15).

MSD1 produced siderophore in the range of 1-20 pg/ml, 5-10 pg/ml, 10-15, and more specifically between 15-20 pg/ml of siderophore in the growth medium, see Example 9.

Vitro and In Vivo conditions

Examples Microscopy: Microscopic methods were used to identify the culture purity as well as total viable and dead cells within 15 minutes. The adopted methods and protocols are described below.

1. Wet mount technique: A drop of the sample including but not limited to the seed inoculum, intermediate samples collected during mass multiplication process, concentrated slurry before formulation was placed on to a clean glass slide, covered with a cover slip and observed under 40x and lOOx light microscope to visualize yeast cells morphology or presence of any other contaminants.

2. Gram staining technique: A thin smear was prepared with a loopful of sample i.e., slant culture, seed inoculum, intermediate samples collected during mass multiplication process, concentrated slurry before formulation in a clean grease free slide, air dried for a minute and heat fixed by passing the slide over the flame for few seconds. Gram staining was done as per prescribed protocol and the slide was observed under light microscope (lOOx oil immersion) to visualize Gram positive budding/oval yeast cells or gram negative broken/damaged cells or presence of any other contaminants.

3. Haemocytometer cell count: Serially diluted the samples i.e., seed inoculum, intermediate samples collected during mass multiplication process, concentration, before formulation up to 10'⁵ dilution. For each diluted sample, a drop of Methylene Blue was added and mixed properly using vortex. The sample mixed with dye (20pl) was transferred into the counting chamber and observed under light microscope (lOOx oil immersion). Number of cells present in the outer 4 squares (Figure 5) was counted and the cell concentration was calculated based on Total cell count in 4 squares x 2500 x dilution factor.

Culturing Method

4. Pure culture technique - Quadrant Streaking

Quadrant streaking was done in selective media (Pikovskaya’ s agar) and common media (Nutrient agar/ TSA agar) to verify the purity of slant culture, seed inoculum, intermediate samples collected during mass multiplication process, concentrated slurry before and after formulation, dehydrated active ingredients & final product. And the quadrant streaked plates were incubated at 30°C for 48-72 hrs. After incubation MSD1 colonies were observed as creamy white color, circular shape and size ranged from l-6mm.

5. Total Viable cell count - Spread plate technique

Spread plating was done in selective media (Pikovskaya’ s agar) and common media (Nutrient agar/ TSA agar) to verify the purity as well as total viable count of seed inoculum, intermediate samples collected during mass multiplication process, concentrated slurry before and after formulation, dehydrated active ingredients & final product as 5MIN. Serial dilution of the sample was performed in sterile saline (0.85% NaCl) and spread plating was done on Pikovskaya’s agar & Nutrient agar medium, incubated at 30°C for 48-72 hrs. Number of colony forming units were counted as CFU/ml or CFU/g of the sample and presence of contaminants were cross verified against the colony morphology of MSDl. I. Studies on Plant Growth Promoting Activities and Solubilization of Minerals

Example 1: In vitro Nitrogen fixation assay:

5MIN was inoculated in nitrogen free malic acid broth medium containing bromothymol blue indicator and incubated at 28 ± 2°C for 7 days and uninoculated sterile broth served as control. The development of blue color in the medium indicated its ability to fix nitrogen was quantified at 595nm in UV spectrophotometer by a modified (Indian Standard, 14806:2000. Azospirillum inoculants. Bureau of Indian Standards, New Delhi, India, 1-13 pp) lab method (Table 4).

Table: 4 Plant growth promotion properties displayed by 5MIN Example 2. In vitro Phosphate solubilization assay:

Qualitative analysis of Phosphate solubilization by 5MIN was analyzed with Pikovskaya’s agar medium (Pikovskaya, R.I. 1948. Mobilization of phosphates in soil in connection with the vital activities of some microbial species. Mikrobiologiya, 17: 362-370) and incubated at 28 ± 2°C for 7-14 days. After incubation, the zone of solubilization was measured and the phosphate solubilization activity was estimated as per the reported method(Indian Standard, 14807:2000. Phosphate solubilizing bacterial inoculant (PSBI) - Specification. Bureau of Indian Standards, New Delhi, India, 1-4 pp) .Uninoculated broth/agar plate served as control and as per literature Bacillus megaterium was used as positive control [Thant S, Aung NN, Aye OM, et al. Phosphate solubilization of Bacillus megaterium isolated from non-saline soils under salt stressed conditions. J Bacteriol Mycol Open Access. 2018;6(6):335-341. DOI: 10.15406/jbmoa.2018.06.00230 ].

Quantitative estimation of soluble phosphorus was assessed by inoculating 5MIN in 50 ml of Pikovskaya’s broth and incubated at 28 ± 2°C on a rotary shaker for 7-14 days. After incubation, the soluble phosphorus was estimated as per the method (Indian Standard, 14807:2000. Phosphate solubilizing bacterial inoculant (PSBI) - Specification. Bureau of Indian Standards, New Delhi, India, 1-4 pp) (Table 4).

Example 3. In vitro Sulphate oxidation assay:

The sulphate oxidation potential of 5MIN was evaluated by the method described (Starkey, RL, Collins VG. 1923. Autotrophs. In: Methods in Microbiology, J. R. Norris, D. W. Ribbons (Eds), New York: Academic press, 38, 55-73; El-Tarabily, KA, Soaud, AA, Saleh, ME, Matsumoto, S, 2006. Isolation and charaterisation of sulfur-oxidising bacteria, including strains of Rhizobium, from calcareous sandy soils and their effects on nutrient uptake and growth of maize (Zea mays L.). Australian Journal of Agricultural Research, 57, 101-111; Nunthaphan V, Siriom B, Nipon P, 2015. Optimizing sulfur oxidizing performance of Paracoccuspantotrophus isolated from leather industry wastewater. Energy Procedia, 79, 629-633). The qualitative analysis of oxidized sulphate was carried out on Starkey Mineral Salt (SMS) agar medium supplemented with 1% Sodium thiosulphate and incubated at 28 ± 2°C for 7-10 days. After incubation, presence of yellow colour halo zone around & beneath colonies indicated positive for sulphate oxidation.

Quantitative estimation of sulphate was assessed by inoculating 5MIN in 50ml of SMS broth supplemented with 1% sodium thio sulphate medium and incubated at 28 ± 2°C for 7-10 days under shaking at 120rpm. After incubation, the culture broth was centrifuged at 10000 rpm for 10 minutes. The supernatant was collected and sulphate was estimated by Barium chloride method.

5MIN showed considerable oxidation of sulphate in SMS agar medium supplemented with sodium thiosulfate, visualized by the presence of yellow colour zone around and beneath the colonies. The reduction in pH of the inoculated broth to pH 4.5, when compared to uninoculated control with a pH of 7.0 confirms the oxidation potential of 5MIN. The reduction of pH indicates that the organic acids produced by 5MINaidedtherelease of sulfate from sodium thiosulfate and increased the availability of sulfate. A significant reduction in the pH and a concurrent significant increase in the sulfate concentration (121.69 pg/ml) was detected in the cell free culture filtrate of 5MIN (Table 4).

Example4a. Iron transformation efficiency of 5MIN under invitro conditions:

5MIN was inoculated* into LM broth (0.02 % yeast extract, 0.01 % peptone, 0.6 % NaCl, 10 mM sodium bicarbonate, 10 mM HEPES) supplemented with carbon substrates (5 mM lactate, 5 mM succinate, 5 mM glycerol, 1 mM acetate), 50 mM ferric citrate, 5 mM sodium molybdate to a final pH 7.2. The samples were incubated anaerobically at room temperature until a visual turbidity/change in color appeared (after 7d ) against the control product.

Analytical technique:

Fe (II) was quantified using a spectrophotometric assay with ferrozine modified method from Stookey (1970). The visible absorption spectrum of the ferrous complex of ferrozine exhibited single sharp peaks with maximum absorbance at 562 nm with Fe (Il)-grown cultures (From LM broth) taken under sterile conditions. About 0.1 to 0.5 mL of suspended sample was withdrawn after vigorous shaking and transferred into the assay test tube. The reaction mixture composed of 1 mol L'¹ HC1 and ferrozine solution, where ferrozine reacts for 10 min with the Fe (II) and the absorbance at 562 nm was measured using a UV spectrophotometer.

Standard curve

Ferrous sulfate was used as a standard to determine the unknown concentration in the test sample. The standard curve was plotted against concentrations ranging from 1 to 5mM. Higher transformation of ferric form to ferrous form was observed with 5MIN in medium supplemented with sodium succinate.

Example 4b. Study on iron transformation efficacy of 5MIN in tomato plants under shade house condition:

The experiment was conducted at shade house facility with Ferric citrate (800ppm) by soil application. Four weeks old, uniform and healthy tomato saplings were used for this study. Soil samples were analyzed before and after treatment with Img of 5MIN product against the control product (Figure 6).

Fe (II) was quantified spectrophotometrically with ferrozine (Stookey,1970). It was ensured that the sampling was done from different regions of the pot for testing of ferrous ion.100 pL of sample was transferred to assay test tube to check the ferric to ferrous conversion. The reaction mixture composed of Test sample (200 to 400 pL), water (Volume made up to 2 mL), 1 mol L'¹ HC1 (1 mL) and ferrozine solution (200 pL). The reaction mixture was allowed to react for 10 mins with Fe (II) and the absorbance at 562 nm was measured using a UV spectrophotometer. Ferric citrate was used as a standard to determine the unknown concentration in the test sample.

Results

5MIN treated soil showed 185.71% increase in Fe transformation after 30days of treatment (Table 5)

Table 5: Quantification of Fe (II) in treated samples Example 5a. In vitro Zinc Solubilization assay: i. Qualitative analysis: 5MIN was evaluated for its Zinc solubilization activity qualitatively by streaking on mineral salt agar medium supplement with 0.1% Zinc oxide (ZO agar) (Shaikh SS, Saraf MS (2017). Optimization of growth conditions for zinc Solubilizing Plant Growth associated Bacteria and Fungi. J Adv Res Biotech 2(1): 1-9; Saravanan VS, Subramoniam SR, Raj SA. 2003. Assessing in vitro solubilization potential of different zinc solubilizing Bacterial (ZSB) isolates. Brazilian Journal of Microbiology (2003) 34: 121-125) solubilization (mm) is measured and recorded (Table 4). The product (5MIN) showed potential zinc solubilization zone in zinc oxide medium, producing a clear zone of 45mm. ii. Quantitative estimation of Zn by AAS:

Quantitative estimation of the solubilization of Zinc by 5MIN was assessed in 50 ml of mineral salt broth medium supplemented with 0.1% Zinc oxide and incubated 28 ± 2°C on a rotary shaker for 7-15 days. After incubation, the culture broth was filtered through Whatman No.1 filter paper and 10 ml of this solution was nebulized in an Atomic Absorption Spectrophotometer (AAS) to determine the available zinc content as described.

Culture filtrate was filtered through Whatman Nol filter paper and the response of the instrument was optimized by adjusting the burner height and flame. Aspirated water to get zero absorption and when the stable response was observed, standards (at least 4) were aspirated and noted down the absorption. The test sample was then aspirated and absorption as noted down. Calibration curve was plotted with net absorption value of the standard against concentration in pg/ml of metals. Absorption value of the sample was located on the calibration curve and the concentration of Zinc in samples were calculated using the formula,

Zinc (mg/1) = Observed zinc cone. (mg/l)/sample volume X Final diluted volume

It was noted that of Zinc was released by 5MIN (509.2ppm) through solubilization of zinc oxide supplemented in the minimal salt broth medium (Table 4). A shift in pH to acidic (pH4.5) after growth of MSD1 (in 5MIN product) in the broth was observed, from the initial pH of 7.0. The release of Zinc from ZnO was enabled through the production of organic acids (drop in pH).

Example 5b: Effect of 5MIN on Zinc uptake in tomato plants by Dutch bucket based hydroponic system (In Vivo studies)'.

Zinc plays a vital role in metal component of different enzymes (Marschner, H., 1993. Zinc uptake from soils, In. Robson, A.D. (ed.), Zinc in Soils and Plants, pp: 59-77. Kluwer, Dordrecht, The Netherlands; Marschner, H., 1995. Mineral Nutrition of Higher Plants, 2nd edition. Academic Press, London ) and an essential trace element in various functions in plant like increasing the chlorophyll content and antioxidant enzymes (Sbartai, H., M.R. Djebar, R. Rouabhi, I. Sbartai and H. Berrebbah, 2011. Anti oxidative response in tomato plants Lycopersicon esculentum L. roots and leaves to Zinc. American-Eurasian J. Toxicol. Sci., 3: 41-46; Martin, R., J. Broadley, W. Philip, P.H. John, Z. Ivan and L. Alexander, 2007. Zinc in Plants. New Phytol., 173: 677-702).

To study the effect of 5MIN on Zn uptake in plants, four weeks old uniform and healthy tomato saplings were used in this experiment. The trial was conducted in green house condition (95% RH with 25±2°C and 16h/8h day/night photoperiod). Plants in Hoagland medium (Tl) served as positive control and plants in Hoagland medium treated with ZnO without 5MIN and ZnSo4 (T3) along with plants treated with water alone (T4) were considered as negative control group (Table 6).

Table 6: Details of treatment groups for studying Zinc uptake in tomato plants Results:

Table 7: Effect of 5MIN on zinc uptake in tomato plants

The dry matter product (DMP) of plant consists of all its constituents excluding water. In this example, Zinc mineral deficiency was observed to be directly proportional to DMP. Correspondingly, DMP ratios were lower in Zn- deficient than in Zn sufficient plants (Cakmak, I., Yilmaz, A., Ekiz, H., Torun, B., Erenoglu, B. & Braun, H. J.1996. Zinc deficiency as a critical nutritional problem in wheat production in Central Anatolia. Plant and Soil, 180, 165 - 172.& Narwal, R.P., Malik, R.S., 2011. Interaction of Zn with other nutrients. Indian J. Fert. 7 (10), 140-150)

5MIN solubilizes ZnO and release Zn (in available form) for plant uptake/absorption.5MIN treated plants showed higher Zn concentration in plant tissue due to the solubilization and mobilization potential of MSD1 (Active ingredient in 5MIN) in soil. A higher DMP was noted in the 5MIN treated plantlets than Hoagland and plants without 5MIN treatment. The plants treated with regular zinc & water control were healthy and with increased height than the plants given water control (T4). Increased concentration of Zinc in 5MIN treated plants indicates that Zn was made available by MSD1 present in the 5MIN formulation for plant uptake. The presence of 5MIN in the solution played a catalytic role for Zn uptake (51.51%) with a higher DMP (97.3 %) than Hoagland control (Table 7).

Example 5c. Effect of 5MIN on Zinc uptake in Green gram at Green house In Vivo Studies)'.

Zinc is an essential micronutrient for microorganisms and plants and present in the enzyme system as co-factor and metal activator for many enzymes (Parisi, B.; Vallee, B.L. Metal enzyme complexes activated by zinc. J. Biol. Chem., 179: 803-807, 1969). Since zinc is vital in the nutrition and physiology of both eukaryotic and prokaryotic organisms, the addition of microbial based zinc solubilizers in soil enables its uptake in plants. Zinc uptake study for 5MIN in green gram plants was conducted at green house, T-Stanes and Company, Ltd. The treatment details are depicted in Table 8.

Table 8: Details of treatment groups studied for Zn uptake studies in green gram

The application of 5MIN enabled the conversion of insoluble form of zinc oxide in the soil medium to soluble form and its availability to plants. The treatments (T4 & T5) with 5MIN product showed significant release of Zn (426.63ppm & 1042ppm) & absorption by the plants (Table 9). Even under high concentration of zinc oxide the active ingredient present in the product 5MIN was able to thrive, colonize in the soil by utilizing the soil nutrients & thereby producing organic acids to solubilize ZnO to Zn. The survival & growth of green gram treated plants showed significant difference in 5MIN treated (T4&T5) at high & low concentration of Zinc oxide, when compared to 5MIN untreated groups (T2&T3).

Table 9: Effect of 5MIN on Zinc uptake in Green gram plants

II. Studies on the plant growth promoting and anti-pathogenic activities of the product 5MIN

Example 6: Cytokinin like activity displayed by MSD1 on radish cotyledons:

Cytokinins are plant hormones that enhance cell division by stimulating the process of mitosis. They are made naturally by plants but have been synthesized by numerous plant growth promoting microbes. Increased mitosis results in plant growth and the formation of shoot apical meristems and floral buds, as well as the development of seeds and fruits. The cytokinin like activity of MSD1 was tested following a reported method (Letham, D.S., 1971. Regulators of cell division in plant tissues. XII. A cytokinin bioassay using excised radish cotyledons. Physiol. Plant. 25:391-396). MSD1 was grown in nutrient broth for 7 days and centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected and adjusted to pH 2.8 with IN HC1. The pH adjusted culture filtrate was mixed with equal volume of ice cold diethyl ether and allowed to stand for 4 hrs at 4° C with an intermittent shaking. The organic phase was evaporated to dryness in the dark and the residue was dissolved in 2.0 ml of absolute methanol and used for radish cotyledon bioassay (Letham, D.S., 1971. Regulators of cell division in plant tissues. XII. A cytokinin bioassay using excised radish cotyledons. Physiol. Plant. 25:391-396). Radish seeds were washed with distilled water and allowed to germinate on blotter paper (in dark) at 24-25°C for 3 days. Cotyledons of uniform weight were selected, placed on filter paper in petri plates and 10 mL of 2mM potassium phosphate buffer of pH 5.9 was added, followed by addition of 1 mL of cell free culture extract. The different treatments were, 1) water control (Tl), 2) Nutrient Broth served as Broth control (T2), 3) Methanol as solvent control (T3), 4) BAP +ve as standard chemical control (T4), 5) Pseudomonas fluorescens +ve organism control (T5), 6) MSD1 as cell free extract (T6) and 7) MSD1-R in the media optimized with specific components (T7).

Cytokinin like activity exhibited by MSD1 was studied on radish cotyledons. Increase in shoot length with earlier appearance of shoot apical meristems (SAMs) was observed in MSD1 (T6) treatment (within 2 days) than the positive chemical (T4) and microbial (T5) control. The cotyledony growth was earlier (within 2 days) in MSD1-R (T7) when compared to all other treatments. Comparatively, a slow growth and SAM appearance was observed in PF (T5).

Based on the results obtained, percentage increase in the fresh weight of cotyledon was calculated. BAP served as a standard to calculate the percentage increase among different treatments (Table 10 & Figure 7).

different treatment groups

MSD1 showed a higher percentage (14.59) of increase in fresh weight when compared to BAP (standard synthetic chemical). Early greening with more shoot apical meristems (SAM) were observed in both MSD1 and MSD1-R treated cotyledons. The positive control PF (Pseudomonas fhiorescens) did not promote cotyledony growth. BAP had no deleterious effect even after 72hrs (Figure 7, Table 10). Hence the present study clearly establishes the beneficial effect of MSD1 metabolites on cell division enhancing activity.

Example 7: Cell division enhancement potential of 5MIN product on maize coleoptiles under laboratory conditions:

The growth potentiating effect of 5MIN was tested In vitro on maize seeds germinated in perlite: peat (1 : 10). The seedlings were checked for coleoptile elongation after 72 hrs. The coleoptiles were dissected from the embryo and subjected to the following treatments (Park, C. J., Kim, K. J., Shin, R., Park, J. M., Shin, Y. C. and Paek, K. H. 2004. Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J. 37: 186- 198). Water treated plants served as control group and IAA treated plants as positive control group and 5MIN treated plants as test group here for this experiment (Table 11).10 coleoptiles per treatment were used in each assay.

Table ll:Treatment details for Audus test of the product 5MIN

The coleoptiles treated with water showed lower elongation rate compared to 5MIN treated coleoptiles. 5MIN treated seed samples showed a significant increase in length (104.41%) of the coleoptile and a higher fresh weight 39.57% (139.58 mg) (Table 12, Figures 8 &9). 5MIN treated samples showed a significant elongation of the coleoptile compared to standard (IAA). Enhanced cell division activity that was observed in seeds treated with 5MIN could be due to the production of cytokinins like growth substances.

Table 12: Variation in elongation rate & fresh weight of maize coleoptiles treated with 5MIN in comparison to control

Example 8: Effect of 5MIN on Dry Matter Production & Vigor Index of seedlings of Greengram and Radish:

The influence of 5 MIN in enhancing the germination potential of seeds was evaluated as per the procedure prescribed by ISTA. Substrate mixture containing perlite/peat (1 : 10) was filled in jiffy trays, moistened with sterile MilliQ water (20ml/well). The 5MIN treated and untreated (water control) seeds were sown and kept under 16h light/8h dark photo period, at 24°C, 80%RH. After 14 days of seedling establishment, data on the seed germination (%), root length (cm), shoot length (cm), dry matter production & vigor index I & II was recorded. The trays were transferred to greenhouse condition after the emergence of the seedling.

Germination % = Total no. of Normal Seedlings produced/Total no. of Seeds Sown x 100. The seedlings used for growth measurement were placed in a paper towel and dried in shade for 24 h and then transferred to a hot air oven maintained at 85 ± 2°C for 24 h. The dried seedlings were removed from the hot air oven and cooled in the desiccators over silica gel. Dry weight was recorded and the mean values were expressed in dry weight of seedlings.

Vigor index values are calculated using the following formula for each of the treatments and replications and the mean values were expressed in whole number (Abdul-Baki, A. A. and J.D. Anderson, 1973. Vigor determination in soybean seed by multiple criteria. Crop Sci., 13: 630-633 & Reddy, Y.T.N. and Khan, M.M. (2001) Effect of osmopriming on germination, seedling growth and vigour of khirni (Mimusopshexandra) seeds. Seed Res. 29(1): 24-27).

Vigor index I = Germination (%) x Total Seedling Length (cm)

Vigor index II = Germination (%) x Dry matter production (g seedlings'¹⁰)

The germination enhancing potential of 5MIN was studied in green gram & radish seeds.5MIN (0.1%) treated green gram (seeds soaked for 24hrs) showed significant increase on dry matter production (27.54%), vigor index 1 (19.52%) & vigor index 2 (30.18%) when compared to water treated control, whereas, for 10 minutes soaking period, the results were similar to water control (FigurelO). Treatment of radish seeds with 5MIN (0.1%) product showed significant results (10 minutes & 24hrs soaking) when compared to water control. The seeds soaked for 24hrs exhibited higher germination percentage (7.14%), dry matter production (13.7%), vigor index 1(18.3%) & vigor index 2 (21.82%) when compared to water treated control (Figurel l). The above results imply that 5MIN product has a profound effect on enhancing the cell division and dry matter production in plants.

Example 9: Siderophore Production by 5MIN:

Qualitatively, the 5MIN product was assayed for siderophore production in the growth medium supplemented with Chrome Azurol S (CAS) and HDTMA (hexa decyl trimethyl ammonium bromide) solution (Alexander, D.B., and Zuberer, D.A. 1991. Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil soils, 12, 39). After inoculation, the plates were incubated at 28 ± 2°C for 48-72 hours. Development of orange halos around colonies, qualitatively determined the siderophore production. Pseudomonas fluorescens served as positive control with uninoculated medium as media control for this experiment.

In quantitative estimation, 5MIN product was inoculated into the growth medium supplemented with CAS and HDTMA solution and incubated at 28 ± 2°C for 7-10 days. After incubation, the culture broth was centrifuged at 3000rpm for 15 minutes. The pH of supernatant was adjusted to 2.0 with HC1 and equal quantity of ethyl acetate was added in separating funnel, mixed well and ethyl acetate fraction was collected. This process was repeated for three times to bring the entire quantity of siderophore from the supernatant. The ethyl acetate fractions were pooled, air dried and dissolved in 5ml of 50% ethanol. Five ml of ethyl acetate fraction was mixed with 5ml of Hathway’s reagent (1 ml of 0. IM FeC13 in 0.1N HC1 to 100ml of distilled water containing 1 ml of potassium ferric cyanide). The absorbance for dihydroxy phenol was read at 700 nm. Dihydroxy benzoic acid was used as standard. The quantity of siderophore synthesized was expressed as pg/ml of culture filtrate.

Result: Siderophore production by 5MIN was observed through formation of orange halo zones around the colonies in the agar medium (Figure 12, 13) against the positive control (Pseudomonas fluor e see ns (Figure 14). 5MIN product was quantitatively evaluated for the siderophore production which was found to produce about 15-20pg/ml of siderophore (Table 4).

Example 10: Estimation of Indole Acetic Acid (IAA) Production:

Indole Acetic Acid production was determined by In vitro method (Holt, JG., NR Krieg and Sneath, PAP. 1994. Bergy’s Manual of Determinative Bacteriology. 9^th Ed, Williams and Wilkins Pub, Baltimore). 5MIN was grown in nutrient broth medium supplemented with tryptophan (0.1g/l) and incubated at 28 ± 2°C for 24-72 hours. After incubation, culture was centrifuged at 3000 rpm for 30 min. 1ml of the culture supernatant was mixed with 2ml of Salkowski reagent (35% perchloric acid; 1 ml 0.5 FeCh) and incubated for 30 minutes at room temperature in dark condition. Development of pink colour indicates IAA production and optical density (OD) valve was measured at 530nm in UV spectrophotometer. IAA concentration was measured from the standard graph of IAA obtained in the range of 10-100 pg/ml (Table 4).

Example 11: Production of ACC Deaminase:

ACC deaminase producing microorganisms utilize amino cyclopropane- 1 carboxylic acid (ACC) as a nitrogen source. Three culture conditions were tested viz., DF minimal medium alone as negative control, DF medium plus (NH4)2 SO4 (2 g/1) as positive control and DF medium plus 3 mM/L ACC as the selective medium. 50 pl of the 5MIN was inoculated and incubated at 30 °C for 48 hours under shaking at 120 rpm. After incubation, optical density of the suspensions was measured spectrophotometrically at 405 nm (Table 3). The formation of a- ketoglutarate in presence of ACC was observed both in test (5MIN inoculated) and positive control (Pseudomonas sp.) after 48 hours of incubation. Activity of ACC in 5MIN inoculated broth (4.19 pg/ml) was similar to positive control (4.17 pg/ml)

III. Antagonistic activity displayed by 5MIN:

Example 12. Antifungal Activity of 5MIN studied by Dual Culture Method:

The antagonistic activity of 5MIN against fungal phyto-pathogens (Macrophomina sp., Fusarium oxysporum & Sclerotium rolfsii) was analyzed by agar streak method & dual culture method.

The antifungal activity of 5MIN was analyzed by agar streak method by a straight line streak on potato dextrose agar (PDA) medium, with simultaneous streaking of the fungal phyto-pathogens (Macrophomina sp., Fusarium oxysporum & Sclerotium rolfsii) at right angle and incubated at 28 ± 2°C for 3-4 days (Dhanasekaran, D., N Thajuddin and A. Panneerselvam 2011. Applications of Actinobacterial Fungicides in Agriculture and Medicine. In Tech Open Access Publisher. ISBN: 978-953-307-670-6, 151-174 pp). The zone of inhibition against the fungal phyto-pathogens was noted.

Based on the positive results obtained from agar streak method, the antifungal activity of 5MIN was tested by dual culture technique using PDA medium. A mycelial disc of the fungal pathogen (5mm dia) was placed at one end of the petri plate and 5MIN was streaked one centimeter away from the periphery of the petri plate just opposite to the mycelial disc of the pathogen. PDA plate simultaneously inoculated with fungal pathogen served as control. Visual observation on the inhibition of pathogenic fungal growth was recorded after 4- 5days of incubation in comparison to control. The radial growth of mycelium (in mm) was measured and percent inhibition (PI) was calculated.

PI = C - T/C x 100 Where, C is the growth of test pathogen (mm) in the absence of the antagonistic isolate;

T is the growth of test pathogen (mm) in the presence of the antagonistic isolate.

Result: Antifungal activity of 5MIN tested against Fusarium oxysporum, Macrophomina spp., Sclerotium rolfsii by agar streak & dual culture method, showed significant antagonistic activity against Sclerotium rolfsii with a zone of inhibition (12 mm) and mycelial growth inhibition (42%) through dual culture technique (Table 18). 5MIN did not exhibit antagonistic activity against Fusarium oxysporum & Macrophomina sp. in the In vitro assays.

Table 13: Antagonistic activity displayed by 5MIN against fungal phytopathogens

** Note: Antifungal activity was not observed against pathogens Fusarium oxysporum & Macrophomina sp., by agar streak method & dual culture method.

Example 13. Studies on Production of Volatile Organic Compound by 5MIN Detection of volatile compounds generation by 5MIN and its anti-phyto pathogenic effect against Sclerotium rolfsii, Rhizoctonia solani, Botrytis cinerea, Colletotrichum gloeosporioides& Fusarium oxysporum by sealed plate technique

The potential role of volatile compounds of 5MIN produced against the above mentioned fungal pathogens was evaluated by measuring the growth of the fungal mycelium through sealed plate technique. Volatile compounds were detected using a reported method (Jayaswal RK, Fernandez M, Upadhyay RS, Visintin L, Kurz M, Webb J, el al. Antagonism of Pseudomonas cepacia&yy ay. phytopathogenic fungi. Curr Microbiol 1993; 26: 17-22; PMID:7679303)). 5MIN and fungal pathogens were cultivated in PDA medium in separate plates and plate inoculated with 5MIN was placed over fungal pathogen plate, avoiding direct contact between the two, sharing only the air. Both plates were sealed from the bottom with parafilm and plates were incubated at 28°C for 5 to 6 days. All experiments were performed in triplicate and represented as mean values. The radial growth of mycelium (in mm) was measured and percent inhibition (PI) was calculated.

PI = C - T/C x 100

Where, C is the growth of test pathogens (mm) in the absence of the 5MIN;T is the growth of test pathogens (mm) in the presence of 5MIN.

Results of the growth inhibition of fungal mycelium were visible after the fourth day of incubation. The strongest effect on growth inhibition (87.8%) was observed against Sclerotium rolfsii. followed by Rhizoctonia solani (75%), B. cinerea (64.6%), C. gloeosporioides (57.5%) & lowest activity (22%) was observed against F. oxysporum (Table 14).

The inhibition of fungal mycelial growth was evident by the morphological and microscopic analysis of fungi. B. cinerea in the presence of 5MIN changed its hyphal morphology, which included hyphal swelling & distortion, whereas the pathogen, C. gloeosporioides showed degradation of fungal cell walls, cell breakage & leakage of intracellular substances, alterations in hyphal morphology, and large amounts of balloon-shaped cells & ruptured mycelia. The growth inhibition (22%) of F. oxysporum was low & this result was more evident by the mycelial colour changing from pink to white in the presence of 5MIN & the cotton like texture in the absence of 5MIN changing to flattened, slender hyphae in the presence of 5MIN. Microscopic analysis of fungi co-cultivated with 5MIN showed evidence of the morphological changes resulting from production of volatile compounds synthesized by MSD1.

Table 14: Growth inhibition effects against fungal phytopathogens by volatile compounds produced by MSD1

Example 14.Biofilm formation:

The probable mechanisms by which MSD1 displays its ability to thrive in harsh environments was studied through the following experiments of biofilm formation, surfactant & emulsification properties exhibited by the isolate.

The ability to form a biofilm was assessed using adherence assay (Ruzicka F, Hol a V, Votava M &Tekkalov_a R (2007). Importance of biofilm in Candida parapsilosis and evaluation of its susceptibility to antifungal agents by colorimetric method. Folia Microbiol 52: 209-214). Wells of a 96-well micro-titer plates containing sterile YPD broth (180pl) were inoculated with 5MIN suspension (20pl) and incubated for 48 h at 25 °C. Controls containing only sterile YPD broth were included. After incubation, the wells were emptied, rinsed with water and air-dried at room temperature. The adherent bio-film layer was stained with an aqueous solution of violet crystal 1% (w/v) for 20 min, rinsed with water, and air-dried. The bound dye was eluted from each well with 200 pl of ethanol. The absorbance of each well was measured at 655 nm. Biofilm formation was considered positive in those wells where absorbance was higher than the mean of the negative control. Uninoculated sterile YPD broth served as control. The results presented in table 15 below confirm the biofilm formation by this MSD1 isolate.

Table 15: Determination of Biofilm formation by 5MIN

Note: Increasing levels of absorbance reflect the ability of the active ingredient in the 5MIN to form a biofilm. Each value represents a mean standard deviation (SD). Example 15.Studies on the Biosurfactant & Emulsification Activity:

5MIN was inoculated in Bushnell horse medium (50 ml) supplemented with coconut oil (10%) and incubated at 30°C for 72 hours under shaking at 120 rpm. After incubation, total viable count (CFU/ml) was analyzed by serial dilution technique followed by spread plating. Biosurfactant activity was determined using oil spreading technique. Distilled water was added to a large Petri dish (15 cm diameter) followed by the addition of 20 pl of crude oil to the surface of water with 10 pl of supernatant of culture broth. The diameter of clear zones of triplicate assays from the same sample was determined (Morikawa, M., Hirata, Y., and Imanaka, T. 2000. A study on the structure-function relationship of lipopeptide biosurfactants. Biochimica et B iophy si ca Acta (BBA) - Molecular and Cell Biology of Lipids, 1488 (3), 211-218)) (Table 16).

Emulsification activity was determined by the addition of 2ml of n-hexadecane to equal volume of purified culture supernatant of 5MIN in a test tube which was mixed vigorously with vortex mixer for 2 minutes. The tubes were incubated at 25°C and the emulsification index (El) was determined after a given time (t) according to equation given below.

Elt = (He/Ht) X 100

Where He and Ht are the height of emulsion and total height of the liquid in the tube, respectively. All emulsification indexes were performed in duplicate (Table 17). Supernatant collected from the sterile uninoculated Bushnell hass broth medium +10% coconut oil without 5MIN served as control group. Samples with 0.5% Tween 20 served as positive control.

The biosurfactant activity for 5MIN determined by oil spreading test method showed a significant oil clearance zone of 38.5 mm. The emulsification index (El) of the biosurfactant produced by 5MIN was 64.28% & with standard (0.5% Tween 20) it was 76.7% (Table 17).

oil spreading technique

Table 17: Emulsification index of 5MIN after 24hrs of incubation

IV. Compatibility Studies of 5MIN Example 16. Compatibility of 5MIN with Agrochemicals (DAP & Urea):

Agrochemicals are a part of the cultivation practices in Agriculture farming by farmers. Hence the compatibility of microbial based product 5MIN with commonly used agrochemicals was studied. The survival of the active ingredients of 5MIN at different concentration of DAP & urea was studied for period of one month and the results represented as total viable cell count (CFU/ml).

Different concentrations (1%, 2.5% and 5%) of DAP & urea solution were prepared and inoculated with 5MIN. The above mixture was kept at room temperature for a month & total viable count (CFU/ml) was enumerated periodically (Initial, 7^th day, 14^th day and 30^th day) by using standard technique. Briefly, one milliliter of sample was taken &serially diluted in sterile saline (0.85% NaCl) up to 10'¹⁰dilutions. Samples (O. lml)from the serially diluted tubes were streaked on Pikovskaya’s agar plates and incubated at 28 ± 2°C for 3 days & the colony forming unit was expressed as CFU/ml (Table 18 & Table 19). Positive control (in the absence of DAP & urea) was included for comparing the growth of the culture in the presence of agrochemicals.

The viable count initially ranged from 2.0 x 10⁸ to 5.6 x 10⁸ CFU/ml at all concentrations of DAP, in comparison to control (without DAP). After 7th day, 14th day & 30th day, the total viable count (CFU/ml) in the DAP, showed a decline (Table 18). The concentration of DAP hampered to an extent though adaptability of the Al of 5MIN was observed till 7^th day. Similar results were observed with urea and the reduction was less on 7^th day compared to 30^th day. (Table 19).

Table 18: Effect of DAP on the growth & survival of the isolate MSD1

Table 19: Effect of the Urea on the growth & survival of the isolate MSD1

Example 17: Compatibility of 5MIN with fungicide and insecticide

The compatibility of 5MIN with commonly used fungicide/insecticides was checked to integrate the use of the product with the regular package of practices. Three different concentrations (1500ppm, 2000ppm & 2500ppm) of fungicide (Carbendazim with Brand name of Bavistin) and insecticide (Dimethyl (E)-l-methyl-2-(m ethylcarbamoyl) vinyl phosphate) with Brand name of Monocrotophos was added into YPD broth medium and sterilized. The sterilized media was inoculated with 5MIN at 2.8 X 10⁸ CFU/ml and incubated at 28 ± 2°C for 72hrs and total viable count was analyzed by using standard plate count method. Media inoculated with 5 MIN and without inclusion of Bavistin and Monocrotophos served as control group.

Result: The compatability of the product was confirmed by enumerating the viability of the Al of 5MIN (CFU/ml) and the results (Table 20 & 21) showed that the cells were tolerant to the fungicide and insecticide. The viability of the cells indicated that the Al of 5MIN was able to tolerate, thrive & survive even at high concentration of the Bavistin (2500ppm) and Monocrotophos (2500ppm).

Table 20: Compatibility of 5MIN with fungicide Bavistin

Table 21: Compatibility of 5MIN with insecticide Monocrotophos

Example 18: Compatibility of 5MIN with different concentration of chlorinated water

The effect of chlorinated water on the activity of 5MIN was tested with two different concentrations (150ppm & 200ppm) of sodium hypochlorite and total viable count was estimated (after 24^th hour, 96^th hour) by standard plate method. Sample (1ml) was taken & serially diluted in sterile saline (0.85% NaCl) up to IO'¹⁰ dilutions and spread plating done on Pikovskaya’s agar plates and incubated at 28 ± 2°C for 3 days &the number of colonies were expressed as CFU/ml (Table 22). Sterile water (with 5MIN product) but without chlorine served as control group. It was observed that 5MIN was tolerant to chlorine at all concentrations for prolonged time of contact (Table 22).

Table 22: Compatibility of 5MIN at different concentration of chlorinated water

The results infer that the product can be dissolved in chlorinated waters (150ppm & 200ppm) that is often found used for preparing the agri inputs.

Example 19.Effect of NaCl and KC1 on the growth and Phosphate solubilizing ability of MSD1:

The ability of the marine yeast (MSD1) to grow and solubilize tri-calcium phosphate (TCP) in the presence of different concentrations of NaCl & KC1 were assessed by measuring the concentration of soluble phosphorous (ppm) using ascorbic acid method (Indian Standard, 14807:2000. Phosphate solubilizing bacterial inoculant (PSBI) - Specification. Bureau of Indian Standards, New Delhi, India, 1-4 pp). MSD1 culture was inoculated into Pikovskaya’s medium supplemented with different concentration of NaCl & KC1 (0.5%, 1%, 2.5%, 5%, 7.5%, 10% & 12.5%) and incubated at 28 ± 2°C on a rotary shaker for 7-14 days. After incubation, the soluble phosphorus was estimated by using ascorbic acid method (Indian Standard, 14807:2000. Phosphate solubilizing bacterial inoculant (PSBI) - Specification. Bureau of Indian Standards, New Delhi, India, 1-4 pp).

The total viable count (6.7 x 10⁸ CFU/ml) was stable at 0.5% NaCl concentration when compared to control, uninoculated sterile broth, and other treatments (Table 23). The amount of soluble Phosphorus released, in general, with increase in NaCl concentration up to 2.5% and declined from 5% NaCl. Phosphorus solubilization of the inorganic insoluble phosphate was observed to increase at 0.5% NaCl concentration (16 mg/lOOml) and also at 2.5% (15.5 mg/lOOml) & 1% (15 mg/lOOml). The solubilization (P) of the isolate was not affected at high concentration of NaCl except at 7.5%.

The total viable count (3.7 x 10⁸ CFU/ml) was observed to be maximum with 0.5% KC1 concentration when compared to other concentration of KC1 (Table 24). The isolate MSDlwas also tested for its ability to solubilize tricalcium phosphate in the presence of different concentrations of KC1 in Pikovskaya’s broth. The amount of Phosphorous released was found to increase with 2.5% KC1 concentration (14.8 mg/lOOml) and declined at 5% & 7.5%.

There was a reduction in pH (6.5) from near neutral to acidic (3.9) on the 7^th day indicating the activity of the microbe at high salt concentration.

Table 23: Influence of NaCl on growth and P-solubilizing ability of Wickerhamomyces anomalus (MSD1)

Table 24: Influence of KC1 on growth and P solubilizing ability of Wickerhamomyces ano ma! us (MSD1) Example 20. Effect of NaCl & KC1 on the viability of the isolate MSD1:

MSDl’s ability to survive in different concentrations of NaCl was tested in Yeast Peptone Dextrose broth medium supplemented with various concentrations (0.5%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15% and 25%) of NaCl and

KC1. After incubating the culture at 28 ± 2°C on a rotary shaker for 72hrs, the cell viability was enumerated in Pikovskaya’s agar medium and results were expressed as CFU/ml.

The study on increasing concentration of sodium chloride (NaCl) and potassium chloride (KC1) on the growth of MSD1 was essential to understand whether growth of MSD1 was inhibited under high EC of soils up to high NaCl and KC1 (25%). The total viable count of 5MIN was maximum in 7.5% of NaCl (1.7 x 10⁹ CFU/ml) and KC1 (1.2 x 10⁹ CFU/ml) when compared to control and other concentrations of NaCl and KC1 (Table 25). The maximum tolerance of 5MIN was observed up to 12.5% of NaCl (2.4 x 10⁵ CFU/ml) and 20% of KC1

(1.0 x 10⁵ CFU/ml).

Table 25: Survivability of 5MIN in different concentrations of NaCl and KC1

V. Field efficacy trials of 5MIN product tested in different crops at different fields:

Example 21. In vivo efficacy study of 5MIN on the growth and yield of groundnut plants at in house field:

The in vivo efficacy of 5MIN on the growth & yield of groundnut, cluster beans, cauliflower and tomato plants were evaluated at in-house field & the experimental protocol is depicted in Table 26. Surface sterilization of Groundnut (Arachis hypogoea L.) seeds was carried out by dipping in 0.1 % HgCF for 5 minutes and thoroughly washing with sterile distilled water thrice. After air drying, the sterilized seeds were soaked in 5MIN product suspensions (2.0 X 10⁹ CFU/g) for 30 minutes, followed by drying in shade for 30 minutes. Thirty seeds/plots were sowed at equal distance with a spacing of 45 cm between the rows. The plots were watered everyday till germination and then at the interval of two days. During the experiment & after harvesting, the following parameters including biometric data viz., plant height, fresh weight & dry weight, pods & fodder yield (Kg), physico-chemical properties, total bacterial count, total fungal count and total viable count of active ingredient in 5MIN were analyzed by standard methods (Table 27 & Figure 15). Plants not treated with 5MIN served as untreated control (Tl). Plants treated with a commercial product (“CONSORT NPK”) and plants treated with standard chemical fertilizer served as positive controls. (T3,T4,T5).

Table 26: Trial protocol adopted for evaluating the effect of 5MIN on the growth & yield of groundnut

*Note - T3 - CONSORT NPK

T4&T5 treatment groups with standard chemical fertilizer tested with the recommended dosage. Results:

The data presented in the Tables 31,32,33 reveals that 5MIN showed a marked effect on plant growth (fresh weight and dry weight), proliferation of Al present in the product 5MIN (CFU/g in the soil) & yield (g), over the uninoculated control. 5MIN (lOOg/acre) treated groundnut plants with the recommended agronomical package and practices showed significant increase in plant height (23.49%), no. of branches/plant (21.73%), fresh fodder weight (21.25%), no. of pods/plant (28.77%), pods weight/plants (22.06%), dry fodder weight (17.55%), 100 pods weight (15.70%), 100 kernel weight (4.65%) & pod yield/acre (15.56%) when compared to untreated control (Table 27, 28 and Figure 15). The soil analysis data after harvest showed significant results in plots treated with 5MIN (Table 28).Parakhia, A.M and Rajani, V.V. 2009. Phosphate solubilizing microorganisms increase yield in groundnut (Arachis hypogaea). JMycol Pl Pathol., 39(2), 294-296.) reported that soil yeast phosphate solubilizer (Torulospora globosa) treatment showed maximum pod & fodder yield in groundnut, when compared to other treatments i.e. Bacillus circulans, B. coagulans, B. brevis & Aspergillussp., . The results of the present study, is in concurrence to the findings of the report, as 5MIN was similarly found to increase the pod & fodder yield in groundnut.

Table 27: Effect of 5MIN on the growth & yield of Groundnut at in-house field in comparison to control

Table 28: Total bacterial & fungal count enumerated before commencement& after completion of field trials from different treatment groups

Note: Tl = Control; T2=5MIN; T3= CONSORT NPK (Positive control); T4= Recommended Dose P2O5 (50kg/ha); T5= Recommended Dose P2O5 (25kg/ha)

Example 22. In vivo Efficacy study of 5MIN on the growth and yield of tomato, cluster beans, cauliflower & Sunflower at inhouse field.

Table 29: Cultural practices followed during the period of efficacy trial on selected crops at In-house field

Result:

The 5MIN treated tomato, cluster beans, cauliflower &sunflower showed significant increase in the yield of about 28.05%, 23.16%, 4.95% & 37.92%, respectively, when compared to untreated control (Table 30,31,32,33).

Table 30: Effect of 5MIN on the growth & yield of tomato plants in field condition

Table 31: Effect of 5MIN on the growth & yield of cluster beans plants in field condition

Table 32: Effect of 5MIN on the growth & yield of cauliflower at in-house field

Table 33: Effect of 5MIN on the growth & yield of sunflower at inhouse field

Example 23: In vivo efficacy study of 5MIN on the growth and yield of cowpea at external fields: The in vivo efficacy of 5MIN was assessed in cowpea plants at external fields & trial details are presented in Table 34.

Tab e 34: Cultural practices followed during the period of efficacy trial at external field

The effect of 5MIN product on cowpea was assessed in external field at Arumugagoundanur, Coimbatore. The results showed significant effect on the growth & yield of the treated cowpea plants, when compared to control. 5MIN @ lOOg/acre significantly increased the yield of cowpea (20.94%) when compared to control (Table 35).

Table 35: Effect of 5MIN on the growth & yield of cowpea at external farm (Arumugagoundanur, Coimbatore)

Example 24: In vivo efficacy study of 5MIN on the growth and yield of rice at Vadakumelur village, Panruti Taluk, Cuddaloredist, Tamil Nadu

The in vivo efficacy of product 5MIN was assessed in Rice plants at external fields & the trial details are presented in Table 36. The plants were analyzed for the growth parameters such as no. of tillers per plant, Shoot length (cm) of per tillers, No. of leaves per tillers, Length of the panicle (cm), No. of grains per panicle, 100 grains weight (g), Fresh weight (g) of per tillers, Dry weight (g) of per tillers, total bacterial count, total fungal count & total viable count of active ingredients in 5MIN were analyzed by standard methods.

Table 36: Trial protocol adopted for trials in rice plants at external fields Results: The external field trial on paddy was conducted at Vadakumellur village near Neyveli, Cuddalore dist. Tamil Nadu. The data presented in Figure 16 & 17reveals that 5MIN showed significant effect on the growth & yield in paddy. The growth parameters tested as listed above & yield data with 2 varieties of rice (BPT &Ponni), showed an increase in No. of tillers (43.40%), shoot length of tillers (5.27%), fresh weight of tillers (21.61%), dry weight of tillers (24.67%), no. of grains per panicle (33.44%) and 100 grains weight (5.19%). The BPT rice variety showed moderate response to the application of the product 5MIN in terms of tillers (34%), shoot length of tillers (1.44%), dry weight of tillers (8.16%), no. of grains per panicle (17.03%) and 100 grains weight (3%) (Fig. 19), when compared to untreated control. Periodic monitoring of the inoculants in soil showed its colonization in the rhizosphere in the midst of the native micro-flora (Table 37).

Table 37: Total bacterial & funga count enumerated before & after completing the experiment at paddy field (external field)

Example 25: In vivo efficacy study of 5MIN on the growth and yield of rice at Mullakolathur village, Kanchipuram, Tamil Nadu

The in vivo efficacy of product 5MIN was assessed in Rice plants at external farmer’s field at Mullakolathur village, Kanchipuram, Tamil Nadu & the trial details are presented in table 41. The plants were analyzed for the growth parameters such as plant height (cm), no. of tillers per plant, panicle length (cm), No. of grains per panicle, flag leaf length (cm), 1000 grains weight (g) and yield (t/acre) were analyzed by standard method.

Table 38: Trial protocol adopted for conducting external field trials in rice plants at Mullakolathur village, Kanchipuram, Tamil Nadu Result:

The field efficacy trial results of 5MIN product (lOOg/acre) showed significant effect on the vegetative growth and yield of paddy crop when compared to control (farmer practices) in external field at Mullakolathur village, Kanchipuram, Tamil Nadu (Table 39). The 5MIN treated paddy plants showed significant increase in plant height (9.03%), no. of tillers per plant (20.04%), panicle length (20.5%), no. of grains per panicle (10.15%), flag leaf length (23.31%), 1000 grain weight (11.66%) and grain yield (25.22%) when compared to control (farmer practice) (Figure 18).

Example 26: In Vivo efficacy study of 5MIN in rice plants conducted by Agriculture University

The product 5MIN was tested by the Department of Agronomy, Faculty of Agriculture, Annamalai University, Annamalai Nagar, Chidambaram-608002, Tamil Nadu. The Field Experiment was conducted in farmer’s field located at ThittuKattur village, Cuddalore district, Tamil Nadu. The Farm field is geographically situated at 11° 29' North latitude and 79° 44' East longitude at an altitude of + 5.79 m above mean sea level during Samba season of 2018 in paddy (variety BPT 5204) under saline conditions.

The experiment was laid out in Randomized Block Design with three replications and the treatments groups as mentioned in Table 40. Rice seedlings were raised in dry nursery beds. The fertilizers were applied to the experimental field as per the recommended schedule of 120:40:40 kg of N, P2O5 and K2O ha'¹, 100 per cent and 50 per cent of NPK (Commercial product) and 5MIN @ 300 &100 g ha'¹, Consort NPK@ 6 kg ha'¹ was applied uniformly at basal to the respective treatment plots as per the treatment schedule. Untreated plants served as control group and plants treated with the product “Consort NPK”, containing consortium of Azotobacter, Bacillus megaterium &Frateuria aurantia served as positive control group.

vegetative and flowering stage

Table 40: Details of treatments groups for experiments on rice plants

*RDF - Recommended Dose of Fertilizer

Ten hills in rice was chosen at random within each net plot and tagged for recording biometric observations viz., plant height, leaf area, DMP, number of tillers m'², panicle length, number of grains panicle'¹’ thousand grain weight and grain yield.

Results:

The results of the university trials demonstrate that rice plants treated with 5MIN exhibited significant improvement in growth and yield attributes of rice. 100 % RDF (recommended fertilizer) + soil application of 5MIN @ 300 g/acre at basal before transplantation & one application at vegetative and flowering stage significantly registered the highest plant height, leaf area and DMP of 90.15 cm, 37.90 cm'² and 8450 kg ha'¹ respectively at harvest stages (Table 41). The yield and yield attributes of the plants treated with 100 % RDF + soil application of 5 MIN @ 300 g/acre at basal before transplantation & one application at vegetative and flowering stage significantly registered the highest number of tillers nr² ( 410.50), panicle length (26.65 cm), number of grains panicle'¹ (218.60) and grain yield (5620 kg ha'¹). The results showed significant increase on the plant height (15.16%), leaf area (48.39%), no. of tillers nr² (15.02%), panicle length (18.66%), no. of grains panicle'¹ (27%), dry matter production (15.82%) & grain yield (15.82%) when compared to standard product. (Consort NPK as positive control) (Figure 19).

plants

Note:

T1 = Control (Farmers practice)- 100 % RDF alone (NPK @ 120: 40: 40 kg ha'¹) without application of 5MIN;

T2 = 50% RDF + soil application of 5MIN @ 300 g ha'¹ at basal before transplantation, vegetative & flowering stage;

T3 = 50% RDF + soil application of 5MIN @ 100 g ha'¹ at basal before transplantation, vegetative & flowering stage;

T4 = 100% RDF + soil application of 5MIN @ 300 g ha'¹ at basal before transplantation, vegetative & flowering stage;

T5 = 100% RDF + soil application of 5MIN @ 100 g ha'¹ at basal before transplantation, vegetative & flowering stage;

T6 = 50% RDF + soil application of Consort @ 6 kg ha'¹;

T7 = Soil application of Consort @ 6 kg ha'¹ alone (without chemical fertilizers)

Example 27. Pathogenesis related (PR) Gene elicitation studies in tomato & Rice plants under Polyhouse conditions Crop productivity is strongly affected by the biotic and abiotic stresses in surrounding environments. The plant PR proteins have been studied to combat numerous biotic and abiotic stress that are classified into 17 classes based on their amino acid sequence, serological relationship, and biological activities (Van Loon, L. C., Pierpoint, W. S., Boiler, T. and Conejero, V. 1994. Recommendations for naming plant pathogenesis related proteins. Plant Mol. Biol. Report. 12:245-264; Van Loon, L. C. and van Strien, E. A. 1999. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol. Mol. Plant Pathol. 55:85-97). Adaptation to biotic and abiotic stress with minimum effect on the growth and improvement in yield of crops can be mainly attributed due to the overexpression of stress responsive genes in plants (Li, J. B., Luan, Y. S. and Yin, Y. L. 2014. SpMYB overexpression in tobacco plants leads to altered abiotic and biotic stress responses. Gene 547: 145-151; Shi, W., Hao, L., Li, J., Liu, D., Guo, X. and Li, H. 2014. The Gossypium hirsutum WRKY gene GhWRKY39-l promotes pathogen infection defense responses and mediates salt stress tolerance in transgenic Nicotiana benthamiana. Plant Cell Rep. 33:483-498).

Three of the PR gene families, PR-1 (Antifungal), PR-2 (P-1, 3- glucanases), and PR-5 (Osmotins), have been reported to encode proteins that can confer increased resistance to phyto-pathogenic fungi, when over expressed in plants (Broglie K, Chet I, Holliday M, Cressman R, Riddle P, Knowlton S, Mauvais CJ, Broglie R. Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science.PR-1 family members have 14 to 17 kDa molecular weight, are mostly basic in nature, show induction with salicylic acid (SA) or pathogens and are commonly used as a marker for SA dependent SAR pathway (Mitsuhara I, Iwai T, S. Seo Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121/180) Mol. Genet. Gen., 279 (2008), pp. 415-427). PR2 encoding for P-1, 3-glucanases are pathogenesis-related (PR) proteins, was reported to play an important role in plant defense responses to pathogen infection. PR5 gene encodes osmotin-like proteins play important role in both biotic stress (antifungal) as well as abiotic stress (osmotic & cold stress).

Reports shows PR10 family proteins are involved in anti -biotic stresses, such as antifungal (Chen, Z. Y., Brown, R. L., Damann, K. E. and Cleveland, T. E. 2007. Identification of maize kernel endosperm proteins associated with resistance to aflatoxin contamination by Aspergillus flavus. Phytopathology 97:1094-1103; Xie, Y. R., Chen, Z. Y., Brown, R. L. and Bhatnagar, D. 2010. Expression and functional characterization of two pathogenesis- related protein 10 genes from Zea mays. J. Plant Physiol. 167: 121-130, anti-bacteria (Flores et al., 2002; Xie et al., 2010), anti-viral (Park et al., 2004), and anti-nematicidal ( Andrade, L. B., Oliveira, A. S., Ribeiro, J. K., Kiyota, S., Vasconcelos, I. M., de Oliveira, J. T. and de Sales, M. P. 2010. Effects of a novel pathogenesis-related class 10 (PR- 10) protein from Crotalaria pallida roots with papain inhibitory activity against rootknot nematode Meloidogyne incognita. J. Agric. Food Chem. 58:4145-4152).

Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthegrisea. Mol. Plant-Microbe Interact. 14:877-886), jasmonic acid inducible pathogenesis related class 10 (JIOsPRIO) (Jwa, N. S., Agrawal, G. K., Rakwal, R., Park, C. H. and Agrawal, V. P. 2001. Molecular cloning and characterization of a novel Jasmonate inducible pathogenesis-related class 10 protein gene, JIOsPRIO, from rice (Oryza sativa L.) seedling leaves. Biochem. Biophys. Res. Commun. 286:973-983), and RSOsPRIO (Hashimoto, M., Kisseleva, L., Sawa, S., Furukawa, T., Komatsu, S. and Koshiba, T. 2004. A novel rice PR10 protein, RSOsPRIO, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway. Plant Cell Physiol. 45:550-559). All those PR10 family genes are induced by Magnaporthe oryzae infection and jasmonic acid treatment (Hashimoto, M., Kisseleva, L., Sawa, S., Furukawa, T., Komatsu, S. and Koshiba, T. 2004. A novel rice PR10 protein, RSOsPRIO, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway. Plant Cell Physiol. 45:550-559; Jwa, N. S., Agrawal, G. K., Rakwal, R., Park, C. H. and Agrawal, V. P. 2001. Molecular cloning and characterization of a novel Jasmonate inducible pathogenesis-related class 10 protein gene, JIOsPRIO, from rice (Oryza sativa L.) seedling leaves. Biochem. Biophys. Res. Commun. 286:973-983 & McGee, J. D., Hamer, J. E. and Hodges, T. K. 2001. Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthegrisea. Mol. Plant-Microbe Interact. 14:877-886), suggesting that those PR10 family genes may be functional redundant in rice.

PR10 family proteins are involved in multiple anti -pathogen processes, and are generally localized in the intracellular spaces (Van Loon and van Strien, 1999). The PR10 family proteins consist of three a-helices and seven antiparallel P-strands. Those structure elements enclose a large hydrophobic cavity which is most likely related with their functional relevance (Fernandes, H., Michalska, K., Sikorski, M. and Jaskolski, M. 2013. Structural and functional aspects of PR10 proteins. FEBS J. 280: 1169-1199). The above reports implied that PR1, 2, 5 and 10 plays a key role in multiple stress tolerance. Based on the significance of the pathogen related proteins, the expression/elicitation of PR genes in Rice and Tomato treated with 5MIN, without any pathogen induction was evaluated.

Differential expression of PR genes in tomato and rice leaves treated with 5MIN:

Induced resistance is associated with the expression of disease resistance marker genes. To determine whether the stimulatory effect of 5MIN on defense related genes in tomato and rice plants by quantitative real time polymerase chain reaction (qRT-PCR) was studied. To our knowledge, this is the first report on PR gene elicitation studies in plant using MSD1 (Al of 5MIN product).

Example27a: Studies on the elicitation effect of 5MIN on pathogenesis related genes in tomato leaves:

. The elicitation effect of 5MIN in tomato plants was studied at vegetative stage, after 120 hrs of spraying. Plants treated with water only served as control (Table 42).

Methods: An experiment was conducted in poly-house facility at T. Stanes & Co. Ltd at the developmental stage of (25 days old) tomato plants. The details of the treatments for assessing the effect on PR gene (PR1, PR2, and PR5) expression are as follows:

Table 42: Treatment and sampling details in tomato plants (Exp. No. 27a)

Expression analysis (qPCR studies)

RNA Extraction

The leaves from all the treatment group plants were collected in duplicates at 0^th and 4^th day after treatment (For Exp. No. 26a) and transported to the laboratory in ice cold condition. The RNA was extracted using HiMedia RNA express reagent according to the manufacturer’s protocol. The extracted RNA was quantified on Nanodrop lite. The quality of the RNA was checked using the agarose gel electrophoresis. cDNA Synthesis

One pg of total RNA was used to synthesize cDNA with cDNA synthesis kit (Hi-cDNA Synthesis Kit) according to manufacturer’s instructions. cDNA synthesized was quantified by Nanodrop quantification method.

Primer Used for analysis

Based on the literature, the primers for tomato (PR1, 2 and 5) were validated using real time PCR from the synthesized cDNA. Amplification curves were monitored for 30 cycles. Melting curves were analyzed for single product formation in qPCR runs. The sequences of the primers used are depicted in the Table 43.

Table 43: Details of primers used PR Gene elicitation studies in tomato plants

RT-PCR:

Real Time (RT-PCR) was carried out by using Bio-Rad SYBR Green master mix; lOpl reaction volume for each sample was prepared in 96-well PCR plate (BioRad) according to the manufacturer’s instruction. After preparing the reaction mixture RT PCR was carried out in BioRad Real-Time PCR. qPCR thermal cycling steps consisted of initial denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 s, 58.0 for 30 s, 72°C for 1 min. The melting temperatures for all genes were analyzed using Melt Curve analysis from 65.0°C to 95.0°C with an increment of 0.5°C for 0:05-MIN. The sigmoid curves, relative normalized gene expression and melt curve were obtained through qPCR. Samples from water treated control served as the base line of normalized expression.

Results

The expression pattern of PR1, 2 and 5 genes were studied from the transcripts obtained from the leaves before and after treatment of all treated groups of tomato plants. Normalized expression was derived from the real time PCR data. The expression pattern of pathogenesis-related genes PR-1, PR-2 and PR-5, considered as markers for salicylic acid dependent systemic acquired resistance (SAR), was examined in the leaves of tomato plants treated with salicylic acid.

Uninduced expression of both PR2 & 5 genes were observed similarly in all groups initially indicating the time induced expression. On day 4, both salicylic acid and 5MIN treated samples showed relatively enhanced expression of PR genes (PR 1, 2 & 5). 5MIN treated samples showed an enhanced PR5 expression (2-folds). Thus a sustained PR5 gene elicitation was observed on tomato plants treated with 5MIN by soil & foliar application.

Example 27b: Studies on the elicitation effect of 5MIN in leaves of tomato and rice plants:

Methods:

25days old germinated seedlings of tomato & 2 weeks old rice seedlings were used for this experiment. Uniform and evenly grown plantlets were selected for transplantation and further utilized for the studies on Pathogenesis-related (PR) gene elicitation (Table 44) in plants. Plants treated with water alone served as control and “Consort NPK” served as positive control.

Table 44: Treatment and sampling details in tomato and rice plants Primer Used for analysis Based on the literature, the primers for rice (PRla and PR10) were validated using real time PCR from the synthesized cDNA.

Table 45: Detai s of primers used PR Gene elicitation studies in tomato and rice plants

The leaves from all the treatment group plants were collected in duplicates at at 0^th, 2^nd and 7^th day after treatment (for Exp. No. 2) and transported to the laboratory in ice cold condition. The RNA isolation, cDNA synthesis, nanodrop quantification, and real time PCR for the primers were implemented using the procedure mentioned in Exp.No. 26a.

Results

Gene expression in tomato:

The expression pattern of PR1, 2 and 5 genes were studied from the transcripts obtained from the leaves (tomato)at 0^th, 2^nd and 7^th day after treatment. Normalized expression with respect to water control was derived from the real time PCR data.

In this study, the expression of three PR genes (PR1, PR2, and PR5) serving as markers for SA signaling (Fu and Dong, 2013) were analyzed. The 5MIN product showed marked increase in stimulating the PR genes of tomato leaves compared to the control [Consort NPK treated plants]. The product 5 MIN showed an increase in elicitation effect of PR1 (45%) and PR 2 (20%) compared to control (leaves treated with water). The elicitation levels of the expressed gene sustained for a longer period (7d) in tomato plants.

The P-1, 3-glucanase gene (PR2) showed a 15.5fold higher expression in plants treated with 5MIN compared to control and the elicitation effect sustained till 7^th day after treatment.

Gene expression in Rice Pathogenesis-related (PR10) proteins play multiple roles in plant development, biotic and abiotic stress tolerance in rice and overexpression of a PR Protein 10 enhances the tolerance of rice to stress (Wu et al 2016). Rice PR10 genes are also induced by various abiotic stresses, such as cold, salt and drought (Hashimoto et al., 2004; Kim et al., 2008b), suggesting that PR10 protein may have multiple function in tolerance to both biotic and abiotic stresses.

The expression pattern of PR1 and 10 genes was studied from the transcripts obtained from the leaves of all the treated plants (on 0^th, 2^nd and 7^th day after treatment). The up-regulation of the expression of PR10 gene was persistent till 7^thday in plants treated with 5MIN product and also immediately after treatment (27 %) compared to positive control plants (Consort NPK treated).

Thus, it is evident from the above studies, that the increased expression of the genes PR5 and PR10 in tomato and rice plants respectively, that the application of 5MIN to crops can enhance the defense potential in plants.

Claims

We claim:

1. A method of growing a marine yeast strain for use in agriculture and horticulture comprising: a) Growing said marine yeast strain in a culture medium; b) Assessing the culture medium by collecting samples at different time points until it reaches stationary phase; c) Processing the culture medium to retrieve concentrated wet biomass from step b); d) Mixing said wet biomass in a pre-formulation medium; and e) Mixing with a carrier for delivery to a subject;

Wherein said wet biomass contains no more than one microorganism;

Wherein the microorganism is Wicker hamomyces anomalus (MSD1) characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical-elongate cells with multilateral budding;

Wherein said MSD1 shows plethora of growth promoting activities in a subject; and protective activities against fungal phytopathogens;

Wherein said pre-formulation medium enables MSD1 to be dormant, retaining its viability during post processing step, and thereby prolongs shelf life of MSD1 before delivery to the subject.

2. The method of claim 1, wherein the culture medium is selected from Stanes growth medium, Yeast peptone Dextrose (YPD) Broth, Potato Dextrose Broth (PDB), Potato Dextrose Agar (PDA), Sabouraud’s Dextrose agar (SDA), Pikovskaya’s medium, Nitrogen free malic acid medium, Starkey mineral salt (SMS) medium supplemented with 1% Sodium thiosulphate, LM Broth, Zinc oxide (ZO) agar, modified Chrome azurol S (CAS) agar medium, Pikovskaya’s broth with CAS & HDTMA reagents, nutrient broth medium supplemented with tryptophan (0.1g/l), DF medium, Bushnell hass medium supplemented with 10% coconut oil,Gl Medium(Yeast extract -10g/l, peptone-20g/l, dextrose- 20g/l, K2HPO4-2g/l and KFbPC -lg/l), and combination thereof.

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3. The method of claim 2, wherein the culture medium further comprising of carbon, nitrogen and other ion source.

4. The method of claim 1, wherein the culture medium is Stanes growth medium, further comprising of: a) Carbon source is selected from one or more of glucose and sucrose in the range of 2-5% in water; b) Nitrogen source is selected from one or more of yeast extract, peptone, malt extract, ammonium sulphate and urea in the range 0.2-1% in water; and c) Ion source is selected from one or more of Potassium, Magnesium, and Sodium chloride in the range of 0.1 - 1.0% in water.

5. The method of claim 1, wherein the culture medium is maintained at 28°C to 32°C and incubated for 48-72 hrs.

6. The method of claim 1, wherein the culture medium is agitated between 100 to 200 RPM.

7. The method of claim 1, wherein cell count before processing the culture medium is at least IxlO⁹ CFU/ml.

8. The method of claim 1, wherein the processing of the culture medium to yield MSD1 is selected from filtration, centrifugation, and combinations thereof.

9. The method of claim 1, wherein MSD1 is mixed with the pre-formulation medium, said pre-formulation medium is Stanes formulation medium.

10. The method of claim 9, wherein the Stanes formulation medium comprising: a) Trehalose or sucrose in the range of 2 to 10%, or combinations thereof; b) Sodium Tripolyphosphate or Sodium metaphosphate in the range of 0.2 to 1% or combinations thereof; c) polyethylene glycol, polypropylene glycol and glycerol in the range of 0.1 to 1% or combinations thereof; and d) skim milk in the range of 10 to 20%.

11. The methods of claim 1 or claim 9, wherein the post processing of MSD1 is dehydration, selected from vacuum drying, spray drying, freeze drying and combinations thereof, to yield dehydrated MSD1.

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12. The methods of claim 1 or claim 11, wherein the post processed MSD1 is mixed with the carrier, said carrier is selected from the group consisting of sucrose, glucose, lactose, water soluble starch and a combination thereof.

13. The method of claim 12, wherein ratio of MSD1 to said carrier is 5% to 50%, whereby said mixture is 5MIN.

14. The method of claim 13, wherein formulation for delivery of 5MIN is selected from the group consisting of water-soluble powder, particulate solid, liquid, dispersions, suspensions, emulsions and combinations thereof.

15. The methods of claim 1 or claim 13, wherein 5MIN is delivered to the subject, wherein the subject is selected from soil, seeds, compost, manure, culture tubes, petri plates, culture flasks, hydroponics, potting mixture and combinations thereof.

16. The method of claim 15, wherein 5MIN converts insoluble nutrients to soluble nutrients, whereby said soluble nutrients resulting in plethora of growth promoting activities in the said subject.

17. The methods of claim 1 or claim 16, wherein the plethora of growth promoting activities in the subject comprising: a) Nitrogen fixation; b) Phosphate solubilization; c) Zinc solubilization; d) Sulphate oxidation; e) Iron transformation; f) IAA production; g) ACC Deaminase production; h) Siderophore production; i) Enhancing the biotic, abiotic stress tolerance and resistance; j) Cytokinin like activity; k) Biosurfactant; and l) Emulsification activity.

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18. The method of claim 16, wherein said insoluble nutrients is selected from the group consisting of tricalcium phosphate, Zinc oxide, Ferric citrate, sodium thiosulphate and combinations thereof.

19. The methods of claim 16 or claim 18, wherein said insoluble nutrients are intrinsically or externally added to the subjects.

20. The method of claim 16, wherein the soluble nutrients are utilized by a product, whereby said product is plants.

21. The method of claim 16, wherein uptake of the soluble nutrients comprising: a) Nitrogen; b) Phosphorous; c) Zinc; d) Ferrous; and e) Sulphur;

Whereby said soluble nutrients result in an increase of at least 15-30% in growth and yield of the product.

22. The method of claim 1, wherein said protective activities against fungal phytopathogens, is selected from antagonistic activities of biofilm formation, volatile organic compounds (VOC), and upregulation of pathogenesis related proteins and combinations thereof.

23. The methods of claim 1 or claim 15, wherein an effective amount of 5MIN delivered to the subject is preferably lOOg/acre.

24. A siderophore producing Wicker hamomyces anomalus (MSD1) characterized to produce 15-20pg/ml of siderophore in a growth medium incorporated with CAS and HDTMA reagent.

25. A bio-stimulant composition for agriculture and horticulture comprising: a) A wet biomass of MSD1 from a culture medium; b) Mixing MSD1, after processing from step a), with a carrier to obtain 5MIN; and c) Selecting a formulation to deliver said 5MIN to a subject;

Wherein the wet biomass contains only MSD1;

80 Wherein said MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical -elongate cells with multilateral budding;

Wherein said MSD1 shows plethora of growth promoting activities in a subject and protective activities against fungal phytopathogens;

Wherein a pre-formulation medium is added after the processing in step b) but before mixing with a carrier, whereby said pre-formulation medium enables MSD1 to be dormant, while retaining its viability and prolongs shelf life of MSD1 before delivery to the subject.

26. The bio-stimulant composition of claim 25, wherein MSD1 growth and processing from the culture medium are as claimed in claims 2 to 11.

27. The bio-stimulant composition of claim 25, wherein said carrier is selected from the group consisting of sucrose, glucose, lactose, water soluble starch and a combination thereof.

28. The bio-stimulant composition of claim 25, where in the ratio of MSD1 to said carrier is 5% to 50%.

29. The bio-stimulant composition of claim 25, wherein the formulation for delivery to the subject is selected from the group consisting of water-soluble powder, particulate solid, liquid, dispersions, suspensions, emulsions and combinations thereof.

30. The bio-stimulant composition of claim 25, wherein said subject is selected from soil, seeds, compost, manure, culture tubes, petri plates, culture flasks, hydroponics, potting mixture and combinations thereof.

31. The bio-stimulant composition of claim 25, wherein 5MIN converts insoluble nutrients to soluble nutrients, whereby said soluble nutrients resulting in plethora of growth promoting activities in the subject.

32. The bio-stimulant composition of claim 25, wherein the plethora of growth promoting activities as claimed in claims 16-21.

33. The bio-stimulant composition of claim 25, wherein the composition showed protective activities in said subject from fungal phytopathogens as in claim 22.

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34. The bio-stimulant composition of claim 25, wherein an effective amount of said 5MIN delivered to the subject is preferably lOOg/acre.

35. A kit for use in agriculture and horticulture comprising: a) MSD1 obtained from a culture medium; b) Mixing MSD1, after processing from step a) with a carrier to obtain 5MIN; and c) Selecting a suitable formulation to deliver said 5MIN to a subject;

Wherein the culture medium contains only MSD1 and no other microorganism;

Wherein said MSD1, characterized as circular shaped creamy white colored, colony size ranging from l-6mm, spherical -elongate cells with multilateral budding; wherein said MSD1 shows plethora of growth promoting activities in a subject; and protective activities against fungal phytopathogens;

Wherein a pre-formulation medium is added after the processing in step b) but before mixing with a carrier, whereby said pre-formulation enables MSD1 to be dormant, while retaining its viability and prolongs shelf life of MSD1 before delivery to the subject.

36. The kit of claim 35, wherein MSD1 growth and processing from the culture medium under conditions as claimed in claims 2 to 11.

37. The kit of claim 35, wherein said carrier is selected from the group consisting of sucrose, glucose, lactose, water soluble starch and a combination thereof.

38. The kit of claim 35, wherein the ratio of MSD1 to said carrier is 5% to 50%.

39. The kit of claim 35, wherein the formulation for delivery is selected from the group consisting of water-soluble powder, particulate solid, liquid, dispersions, suspensions, emulsions and combinations thereof.

40. The kit of claim 35, wherein said subject is selected from soil, seeds, compost, manure, culture tubes, petri plates, culture flasks, hydroponics, potting mixture and combinations thereof.

82 The kit of claim 35, wherein 5MIN converts insoluble nutrients to soluble nutrients, whereby said soluble nutrients resulting in plethora of growth promoting activities in said subject. The kit of claim 35, wherein the plethora of growth promoting activities as claimed in claims 16-21 in said subject. The kit of claim 35, wherein the kit showed protective activities in said subject from fungal phytopathogens as in claim 22. The kit of claim 35, wherein an effective amount of 5MIN delivered to the subject is preferably lOOg/acre.

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