FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
“A MYCORRHIZAL COMBINATION, COMPOSITION AND USE THEREOF”
Applicant name and Address:
NATURAL PLANT PROTECTION LIMITED
Upl Ltd, Uniphos House, Madhu Park, C.D.Marg, Khar W, Mumbai 400052, India Nationality: IN
The following specification particularly describes the invention and the manner in which it is to be performed.

Field of Invention:
The present disclosure relates to a combination of mycorrhiza. The present disclosure also relates to compositions comprising said combination, and uses thereof. More particularly, the present disclosure relates to a biofertilizer comprising the combinations and compositions described herein.
Background:
Mycorrhizae are a collection of many species of symbiotic or beneficial soil-borne fungi that help nourish a host plant. The mycorrhizae, such as endomycorrhizae or ectomycorrhizae, can support the host plant physically and chemically. Physically, the mass of mycorrhizae hyphae increases the physical surface area that is available for water and nutrient absorption. The hyphae are smaller in diameter compared to root hairs and can grow through soil pores that are inaccessible to plant roots. The expanded reach of mycorrhizae hyphae can help reduce crop stress during drought by finding water at greater soil depths. Chemically, mycorrhizae cells excrete various organic acids that dissolve minerals in the soil rhizosphere making them available to the plant. The mycorrhizae hyphae also help break down rock, which increases the availability of nutrients such as potassium, calcium, zinc, and magnesium. Additionally, some mycorrhizae chemical excretions are toxic to soil pathogens, such as nematodes, and others can help provide protection to plants grown in soils with high concentrations of salt and toxic metals.
Mycorrhizae have been externally applied to the soil or the roots as a biofertilizer to increase nutrient absorption and help stimulate plant growth and yield. Research has shown that different mycorrhizal species promote different beneficial characteristics to plants. However, there is still an unquenched need in the art for mycorrhizal combinations which promote maximum yield increase, nutrient uptake, increase root and soil enzyme activity, promote root health, promote drought tolerance, improves plant establishment, promotes fruiting and flowering, improves tolerance to salinity and soil toxicities, and improves resilience of plants to nutrient and water stress. There is an unsatisfied need in the art for effective mycorrhizal combinations comprising the favorable mycorrhizal species and in optimum ratios to produce maximum impact on yield, stress tolerance and growth of the plant.

Thus, it is an object of the present disclosure to provide a mycorrhizal combination comprising mycorrhiza which increases plant growth and mycorrhizal colonization. It is also an object of the present disclosure to provide an efficacious mycorrhizal combination to aid plant growth and mycorrhizal colonization. It is also an object of the present disclosure to provide a composition comprising said mycorrhizal combination. It is also an object of the present disclosure to provide a method of growth stimulation in a plant or a plant part comprising applying said mycorrhizal combination.
Summary:
An aspect of the present disclosure provides a mycorrhizal combination comprising component I: Rhizophagus irregularis, or spores or propagules thereof; and component II: one or more other endomycorrhiza, or spores or propagules thereof; wherein the amount of component I is not less than 50% of the total amount of mycorrhiza.
In an embodiment, the components I and II are not identical.
In an embodiment, the proportion of component I in the combination is at least 50% of the total amount of mycorrhiza.
Another aspect of the present disclosure provides a composition comprising said mycorrhizal combination. Said composition further comprises atleast one agriculturally acceptable excipient.
Another aspect of the present disclosure provides a method of growth stimulation in a plant or a part thereof, wherein the method comprises applying to the plant, a part thereof, a plant propagation material, or a locus a mycorrhizal combination comprising:
component I: Rhizophagus irregularis, or spores or propagules thereof; and
component II: One or more other endomycorrhiza, or spores or propagules thereof;
wherein the amount of component I is not less than 50% of the total amount of mycorrhiza.
An aspect of the present disclosure provides a method of growth stimulation in a plant or a part thereof, wherein the method comprises applying to the plant, a part thereof, or a locus a

composition comprising a mycorrhizal combination and atleast one agriculturally acceptable excipient, wherein said mycorrhizal combination comprises:
component I: Rhizophagus irregularis, or spores or propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
and wherein the amount of component I is not less than 50% of the total amount of mycorrhiza.
The present disclosure provides new mycorrhizae-based biofertilizer compositions and biofertilizer formulations capable of serving as effective soil fertilization agents and growth promoters as well as enhancers.
An aspect also provides use of a mycorrhizal combination comprising
component I: Rhizophagus irregularis, or spores or propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
wherein the amount of component I is not less than 50% of the total amount of mycorrhiza;
or a composition comprising said combination for growth stimulation in a plant or a part thereof. In an embodiment said use is as a biofertilizer.
Detailed Description:
The following is a detailed description of embodiments of the present invention. The embodiments are in such detail as to clearly communicate the invention. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or

more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability.
Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises”, “comprising”, “includes” and “including” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances.
As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All provided ranges of values are intended to include the end points of the ranges, as well as values between the end points, and are independently combinable. As used herein, all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The headings and abstract of the disclosure provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
The following discussion provides many exemplary embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
Various terms as used herein are described below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
The term “about” as used herein encompasses variations of +/-10% and more preferably +/-5%, as such variations are appropriate for practicing the present invention.

The term “Rhizophagus irregularis” as used herein also refers to Glomus intraradices and subspecies and strains thereof.
An embodiment of the present disclosure provides a mycorrhizal combination comprising:
component I: Rhizophagus irregularis, or spores, propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
wherein the proportion of component I is not less than 50% of the total amount of mycorrhiza.
In an embodiment, the components I and II are not identical.
In an embodiment, the proportion of component I in the combination is at least 50% of the total amount of mycorrhiza.
In an embodiment, the proportion of component I in the combination is about 50% to about 99% of the total amount of mycorrhiza.
In an embodiment, the proportion of component II in the combination is about 1% to about 50% of the total amount of mycorrhiza.
In an embodiment, the ratio of component I to component II ranges from about 50:50 to about 99:1.
In an embodiment, the one or more other endomycorrhiza are selected from arbuscular mycorrhiza, ericoid mycorrhiza, arbutoid mycorrhiza, monotropoid mycorrhiza, orchid mycorrhiza, and combinations thereof.
In an embodiment, the one or more other endomycorrhiza are selected from the class Glomeromycota (glomeromycetes). In an embodiment the one or more other endomycorrhiza selected from the class Glomeromycota are selected from the order Archaeosporales, Diversisporales, Glomerales, Paraglomerales or combinations thereof. In an embodiment, the one or more other endomycorrhiza are selected from Glomaceae (Glomeraceae), Acaulosporaceae, Archaeosporaceae, Diversisporaceae, Pacisporaceae, Paraglomaceae, Gigasporaceae, Ambisporaceae, Geosiphonaceae, Claroideoglomeraceae, or combinations thereof. In an embodiment, the one or more other endomycorrhiza are selected from the genera Glomus, Entrophospora, Funneliformis, Rhizophagus, Septoglomus, Acaulospora,

Archaeospora, Paraglomus, Gigaspora, Pacispora, Sclerocystis, Dentiscutata, Scutellospora, or combinations thereof.
In an embodiment, the one or more other endomycorrhiza as provided herein the embodiments of the present disclosure does not comprise Rhizophagus irregularis.
5 In an embodiment, the one or more other endomycorrhiza are selected from Rhizophagus
clarus, Gigaspora margarita, Scutellospora heterogama, Glomus mossae, Septoglomus viscosum, Funneliformis mossae, and combinations thereof.
The term “Scutellospora heterogama” as used herein also refers to Dentiscutata heterogama and subspecies and strains thereof.
10 In an embodiment, the component II comprises one or more endomycorrhiza, or spores or
propagules thereof, selected from the group consisting of Rhizophagus clarus, Gigaspora margarita, Scutellospora heterogama, and combinations thereof. In a preferred embodiment, the component II comprises a combination of Rhizophagus clarus or spores or propagules thereof, Gigaspora margarita or spores or propagules thereof, and Scutellospora heterogama
15 or spores or propagules thereof.
In some embodiments, the component II of the combination of the present disclosure comprises
a combination of endomycorrhizal spores or propagules selected from: (i) spores or propagules
of Gigaspora margarita and Rhizophagus clarus, (ii) spores or propagules of Gigaspora
margarita and Scutellospora heterogama, (iii) spores or propagules of Rhizophagus clarus and
20 Scutellospora heterogama, or (iv) spores or propagules of Rhizophagus clarus, Gigaspora
margarita, and Scutellospora heterogama.
Thus, an embodiment of the present disclosure provides a mycorrhizal combination comprising
component I: Rhizophagus irregularis, or spores, propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof selected
25 from Rhizophagus clarus, Gigaspora margarita, Scutellospora heterogama, Glomus
mossae, Septoglomus viscosum, Funneliformis mossae, and combinations thereof;
wherein the proportion of component I is not less than 50% of the total amount of mycorrhiza.
8

In an embodiment, the combinations of the present disclosure may be further combined with ectomycorrhizae or spores or propagules thereof, helper bacteria, or combinations thereof.
Thus, an embodiment of the present invention provides a mycorrhizal combination comprising:
an endomycorrhizal component comprising
5 component I: Rhizophagus irregularis, or spores, propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
wherein the proportion of component I is not less than 50% of the total amount of mycorrhiza; and
an ectomycorrhiza, or spores or propagules thereof.
10 Thus, an embodiment of the present invention provides a mycorrhizal combination comprising:
an endomycorrhizal component comprising
component I: Rhizophagus irregularis, or spores, propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
wherein the proportion of component I is not less than 50% of the total amount of
15 mycorrhiza; and
a helper bacteria, or spores or propagules thereof.
Thus, an embodiment of the present invention provides a mycorrhizal combination comprising:
an endomycorrhizal component comprising
component I: Rhizophagus irregularis, or spores, propagules thereof; and
20 component II: one or more other endomycorrhiza, or spores or propagules thereof;
wherein the proportion of component I is not less than 50% of the total amount of mycorrhiza;
an ectomycorrhiza, or spores or propagules thereof; and
a helper bacteria.
9

In an embodiment, the ectomycorrhiza, spores or propagules thereof are selected from Laccaria, Pisolithus, Piriformospora, Scleroderma, Rhizopogon, and combinations thereof.
In an embodiment, the ectomycorrhiza, spores or propagules thereof are selected from
Piriformospora indica, Laccaria bicolor, Laccaria lacata, Rhizopogon roseolus, Laccaria
5 proxima, Pisolithus arhizus and Pisolithus tinctorius.
In an embodiment, the ectomycorrhiza, spores or propagules thereof includes a combination of Piriformospora indica, Laccaria bicolor and Pisolithus tinctorius. In an embodiment, the ectomycorrhiza, spores or propagules thereof includes a combination of Piriformospora indica, Laccaria bicolor, Laccaria lacata and Pisolithus tinctorius.
10 In an embodiment, the ectomycorrhiza or spores or propagules thereof and mycorrhizal
combinations described in the present disclosure may be combined in a ratio of 1:10 to 10:1. In a preferred embodiment, the ectomycorrhiza or spores or propagules thereof and mycorrhizal combinations described in the present disclosure may be combined in a ratio of 3:7 to 7:3.
In an embodiment, the helper bacteria are selected from Enterobacter, Pseudomonas,
15 Stenotrophomonas, Azospirillum, Azorhizobium, Azovibrio, Polyangium, Ramlibacter,
Rubrivivax, Sphingomonas, Rhizobium, Streptomyces, Amycolatopsis, Fraturia,
Pseudonocardia, Sphingomonas, Massilia, Methylobacterium, Bradyrhizobium, Bosea,
Bacillus, Paenibacillus, Azotobacter, Glucanoacetobacter or combinations thereof. In an
embodiment, the helper bacteria are selected from the group consisting of Pseudomonas
20 fluorescens, Bacillus subtilis, Bacillus aryabhattai, Rhizobium leguminosarum, Azorhizobium
caulidans, Fraturia aurentia, Bacillus megaterium, Bacillus amyloliquefaciens, Azotobacter chrococcum, Azospirilum lipoferum, Glucanoacetobacter diazotrophicus, Streptomyces viridochromogenes, Streptomyces hygroscopicus, Pseudomonas putida, Streptomyces koyangenesis, Streptomyces iarkyus, and combinations thereof.
25 In an embodiment, the helper bacteria and the mycorrhizal combination of the present
disclosure are admixed in a ratio of 1:100 to 100:1. In an embodiment, the helper bacteria and the mycorrhizal combination of the present disclosure are admixed in a ratio of 1:75 to 75:1. In an embodiment, the helper bacteria and the mycorrhizal combination of the present disclosure are admixed in a ratio of 1:50 to 50:1. In an embodiment, the helper bacteria and the
30 mycorrhizal combination of the present disclosure are admixed in a ratio of 1:30 to 30:1. In an
10

embodiment, the helper bacteria and the mycorrhizal combination of the present disclosure are admixed in a ratio of 1:20 to 20:1.
In an embodiment, the helper bacteria and the mycorrhizal combination of the present disclosure are admixed in a ratio of 1:17 to 2:1.
5 In an embodiment, the propagules of endomycorrhiza or ectomycorrhiza are in a size range of
60 – 150 microns, more preferably 70 – 80 microns. In a preferred embodiment, the mycorrhizal propagules are of 75 microns in size.
Another embodiment of the present disclosure provides a composition comprising the
mycorrhizal combinations described in the present disclosure. Said composition further
10 comprises atleast one agriculturally acceptable excipient.
In an embodiment, the agriculturally acceptable excipients are selected from any or a
combination of wetting agent, dispersing agent, binding agent, surfactant/emulsifier, anti-
freezing agent, mineral, filler, stabilizer, and preservative. However, it should be appreciated
that any other agriculturally acceptable excipients, as known to a person skilled in the art, may
15 be used to serve its intended purpose without departing from the scope of the present disclosure.
In an embodiment, the agriculturally acceptable excipients are present in an amount ranging from 0.3% to 99% by weight of the composition.
In an embodiment, the composition comprises a stabilizer. In an embodiment, the stabilizer is
selected from any or a combination of: humic acid, methyl cellulose, Xanthan gum,
20 polyvinylpyrrolidone (PVP), Tween and Gum Arabica.
In an embodiment, the agriculturally acceptable excipient is selected from a group comprising, but not limited to, a wetting agent, gelling agent, dispersing agent, binding agent, surfactant/emulsifier, anti-freezing agent, mineral, filler, preservative, and combinations thereof.
25 In an embodiment, the composition is a gel comprising a gelling agent as an agriculturally
acceptable excipient. An extract of Dodonaea viscosa may also be used as a binding agent.
In an embodiment, the mycorrhizal compositions may be composed and prepared in a way as embodied in the applications PCT/IN2023/050622 and/or PCT/IB2024/052717.
11

The wetting agent is selected from a group comprising, but not limited to, sulfosuccinates, naphthalene sulfonates, sulfated esters, phosphate esters, sulfated alcohol, alkyl benzene sulfonates, and combinations thereof.
The dispersing agent is selected from a group comprising, but not limited to, polycarboxylates,
5 naphthalene sulfonate condensates, phenol sulfonic acid condensates, lignosulfonates, methyl
oleyl taurates, polyvinyl alcohols, and combinations thereof.
The binding agent is selected from a group comprising, but not limited to, polyvinyl alcohols,
phenyl naphthalene sulphonates, lignin derivatives, polyvinyl pyrrolidone,
polyalkylpyrrolidone, carboxymethylcellulose, xanthan gum, polyethoxylated fatty acids,
10 polyethoxylated fatty alcohols, ethylene oxide copolymer, propylene oxide copolymer,
polyethylene glycols, polyethylene oxides, and combinations thereof. An extract of Dodonaea viscosa may also be used as a binding agent.
The surfactant is selected from a group comprising ionic surfactants, non-ionic surfactants, and combinations thereof. Non-limiting examples of ionic surfactants include sulfonic acids,
15 sulfuric acid esters, carboxylic acids, and salts thereof. Non-limiting examples of water soluble
anionic surfactants include alkyl sulfates, alkyl ether sulfates, alkyl amido ether sulfates, alkyl aryl polyether sulfates, alkyl aryl sulfates, alkyl aryl sulfonates, monoglyceride sulfates, alkyl sulfonates, alkyl amide sulfonates, alkyl aryl sulfonates, benzene sulfonates, toluene sulfonates, xylene sulfonates, cumene sulfonates, alkyl benzene sulfonates, alkyl
20 diphenyloxide sulfonate, alpha-olefin sulfonates, alkyl naphthalene sulfonates, paraffin
sulfonates, lignin sulfonates, alkyl sulfosuccinates, ethoxylated sulfosuccinates, alkyl ether sulfosuccinates, alkylamide sulfosuccinates, alkyl sulfosuccinamate, alkyl sulfoacetates, alkyl phosphates, phosphate ester, alkyl ether phosphates, acyl sarconsinates, acyl isethionates, N-acyl taurates, N-acyl-N-alkyltaurates, and alkyl carboxylates.
25 Non-limiting examples of the non-ionic surfactants include glycerol ethers, glycol ethers,
ethanolamides, sulfoanylamides, alcohols, amides, alcohol ethoxylates, glycerol esters, glycol esters, ethoxylates of glycerol ester and glycol esters, sugar-based alkyl polyglycosides, polyoxyethylenated fatty acids, alkanolamine condensates, alkanolamides, tertiary acetylenic glycols, polyoxyethylenated mercaptans, carboxylic acid esters, polyoxyethylenated
30 polyoxyproylene glycols, sorbitan fatty esters, or combinations thereof. Also included are
EO/PO block copolymers (EO is ethylene oxide, PO is propylene oxide), EO polymers and
12

copolymers, polyamines, and polyvinylpynolidones, sorbitan fatty acid alcohol ethoxylates and sorbitan fatty acid ester ethoxylates.
The anti-freezing agents is selected from a group comprising, but not limited to, ethylene glycol, propylene glycol, urea, glycerin, anti-freeze proteins, and combinations thereof.
5 The minerals are selected from a group comprising, but not limited to, kaolin, silica, titanium
(IV) oxide, rutile, anatase, aluminum oxides, aluminum hydroxides, iron oxide, iron sulfide,
magnetite, pyrite, hematite, ferrite, gregite, calcium carbonate, calcite, aragonite, quartz,
zircon, olivine, orthopyroxene, tourmaline, kyanite, albite, anorthite, clinopyroxene,
orthoclase, gypsum, andalusite, talc, fluorite, apatite, orthoclase, topaz, corundum, diamond,
10 tin, tin oxides, antimony, antimony oxides, beryllium, cobalt, copper, feldspar, gallium,
indium, lead, lithium, manganese, mica, molybdenum, nickel, perlite, platinum group metals, phosphorus and phosphate rock, potash, rare earth elements, tantalum, tungsten, vanadium, zeolites, zinc and zinc oxide, indium tin oxide, and combinations thereof.
The fillers are selected from a group comprising, but not limited to, diatomaceous earth, kaolin,
15 bentonite, precipitated silica, attapulgite, perlite, and combinations thereof.
In an embodiment, the combinations or compositions of the present disclosure may be
formulated as any of gel, aerosol, emulsifiable concentrate, wettable powder, soluble
concentrate, soluble powder, suspension concentrate, spray concentrate, capsule suspension,
water dispersible granule, granules, dusts, microgranule seed treatment formulation and the
20 likes.
Thus, in an embodiment, there is provided a composition comprising:
A. a mycorrhizal combination comprising
component I: Rhizophagus irregularis, or spores or propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
25 wherein the amount of component I is not less than 50% of the total amount of
mycorrhiza; and
B. atleast one agriculturally acceptable excipient.
13

An embodiment provides a biofertilizer comprising the combinations or compositions of the present invention.
In an embodiment, the mycorrhizal combination is present in the composition in an amount ranging from about 0.1% to about 90% by weight of the composition.
5 In an embodiment, the composition further comprises ectomycorrhiza or spores or propagules
thereof, helper bacteria, or combinations thereof.
In an embodiment, the composition of the present disclosure may be further combined with a
composition comprising ectomycorrhiza or spores or propagules thereof, helper bacteria, or
combinations thereof. In an embodiment, the composition of the present disclosure may be
10 combined with a composition comprising ectomycorrhiza or spores or propagules thereof, a
composition comprising helper bacteria, or combinations thereof.
In an embodiment, the composition comprising ectomycorrhiza or spores or propagules thereof
and composition comprising mycorrhizal combinations described in the present disclosure may
be combined in a ratio of 1:10 to 10:1. In a preferred embodiment, the composition comprising
15 ectomycorrhiza or spores or propagules thereof and composition comprising mycorrhizal
combinations described in the present disclosure may be combined in a ratio of 3:7 to 7:3.
In an embodiment, the ectomycorrhiza or spores or propagules thereof is present in the composition in an amount ranging from about 0.1% to about 90% by weight of the composition.
In an embodiment, the composition comprising helper bacteria and the composition comprising
20 mycorrhizal combination of the present disclosure are admixed in a ratio of 1:100 to 100:1.
In an embodiment, the helper bacteria are present in the composition in an amount ranging from about 0.1% to about 20% by weight of the composition.
Another embodiment of the present disclosure provides a method of growth stimulation in a
plant or a part thereof, wherein the method comprises applying to the plant, a part thereof, or a
25 locus a mycorrhizal combination comprising:
component I: Rhizophagus irregularis, or spores or propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
14

wherein the amount of component I is not less than 50% of the total amount of mycorrhiza.
An embodiment of the present disclosure provides a method of growth stimulation in a plant
or a part thereof, wherein the method comprises applying to the plant, a part thereof, a plant
propagation material, or a locus a composition comprising a mycorrhizal combination and
5 atleast one agriculturally acceptable excipient, wherein said mycorrhizal combination
comprises:
component I: Rhizophagus irregularis, or spores or propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
and wherein the amount of component I is not less than 50% of the total amount of
10 mycorrhiza.
“Growth stimulation” herein refers to initiation or acceleration of growth of the plant or a part thereof. Non-limiting examples of changes or improvement effected by the combinations and compositions of the present invention include: Improved plant nutrient use efficiency; Enhanced root and shoot growth; Improved reproductive heat stress tolerance; Improved
15 drought tolerance; Improved pollen tube growth; Enhanced pollen viability; Increased
fertilization and fruit set; Increased floral inflorescence primordia; Increased number of buds, pods and yield; Improved and strong root mass and architecture; Improved/increased bud development; Accelerated shoot or bud emergence; Enhanced vigor/uniformity of emergence; Improved and increased branching; Improved/increased diameter and strength;
20 Improved/increased inter-node length; Increase in number of yield structures (ears, fruits etc);
Increase in leaf area; Increased amount of chlorophyll, greening; Increased photosynthesis activity; Increase in CO2 fixation; Accelerated and improved flowering; Improvement in pollination; Enhanced fruit set & retention; Improvement in cell division for size and quality potential; Improvement in fruit finish; Freedom from pest such as fungal pests, viruses,
25 bacteria, weeds, insects, nematodes; Enhanced resistance against pest such as fungal pests,
viruses, bacteria, weeds, insects, nematodes; Increased plant weight; Increased plant height; Increased biomass such as higher overall fresh weight; Higher grain yield; More tillers; Larger leaves; Increased shoot growth; Increased protein content; Increased oil content; Increased starch content; Increased pigment content; Increased plant vigor; Improved vitality of the plant;
30 Improved plant growth; Improved plant development; Improved visual appearance of the plant
or a part thereof; Improved plant stand (less plant verse/lodging); Improved emergence;
15

Enhanced nodulation, in particular rhizobial nodulation; Bigger leaf blade; Increased yield
when grown on poor soils or unfavorable climate; Enhanced pigment content (e.g. Chlorophyll
content); Earlier flowering; Earlier fruiting; Earlier and improved germination; Earlier grain
maturity; Improved self-defence mechanisms; Improved stress tolerance and resistance of the
5 plants against biotic and abiotic stress factors such as fungi, bacteria, viruses, insects, heat
stress, cold stress, drought stress, UV stress and/or salt stress; Less non-productive tillers; Less dead basal leaves; Less input needed (such as fertilizers or water); Greener leaves; Complete maturation under shortened vegetation periods; Less fertilizers needed; Less seeds needed; Easier harvesting; Faster and more uniform ripening; Longer shelf-life; Longer panicles; Delay
10 of senescence; Stronger and/or more productive tillers; Better extractability of ingredients;
Improved quality of seeds (for being seeded in the following seasons for seed production); Reduced production of ethylene and/or the inhibition of its reception by the plant; Increased nutrient content; Increased content of fatty acids; Increased metabolite content; Increased carotenoid content; Increased sugar content; Increased amount of essential amino acids
15 Improved nutrient composition; Improved protein composition; Improved composition of fatty
acids; Improved metabolite composition; Improved carotenoid composition; Improved sugar composition; Improved amino acids composition; Improved or optimal fruit color; Improved leaf color; Higher storage capacity; and/or Higher processability of the harvested products.
In an embodiment, the combinations or the compositions of the present disclosure increase
20 shoot growth in a plant or a part thereof.
In an embodiment, the combinations or the compositions of the present disclosure increase root growth in a plant or a part thereof.
In an embodiment, the combinations or the compositions of the present disclosure cause or increase mycorrhizal colonization in a plant or a part thereof.
25 In an embodiment, the combinations or the compositions of the present disclosure may be
applied to seeds or any plant propagation material for seed treatment.
In an embodiment, the present disclosure provides a method of treating a seed of a plant comprising applying to the seed a mycorrhizal combination, wherein said mycorrhizal combination comprises:
30 component I: Rhizophagus irregularis, or spores or propagules thereof; and
16

component II: one or more other endomycorrhiza, or spores or propagules thereof;
and wherein the amount of component I is not less than 50% of the total amount of mycorrhiza.
In an embodiment, the present disclosure provides a method of treating a seed of a plant
5 comprising applying to the seed a composition comprising a mycorrhizal combination and
atleast one agriculturally acceptable excipient, wherein said mycorrhizal combination comprises:
component I: Rhizophagus irregularis, or spores, propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
10 and wherein the amount of component I is not less than 50% of the total amount of
mycorrhiza.
The term "plant propagation material" is to be understood to denote all the generative parts of
the plant such as seeds and vegetative plant material such as cuttings and tubers (e. g. potatoes),
which can be used for the multiplication of the plant. This includes seeds, roots, fruits, tubers,
15 bulbs, rhizomes, shoots, sprouts and other parts of plants. Seedlings and young plants, which
are to be transplanted after germination or after emergence from soil, are also included. These plant propagation materials may be treated prophylactically with a plant protection compound either at or before planting or transplanting. In an embodiment, the term includes agronomically useful plants, for example for example vegetable, fruit and cereal crops, and ornamental plants.
20 In an embodiment, the combinations or the compositions of the present disclosure may be
applied to crops or seeds. Examples of the crops on which the combinations and compositions of the present disclosure may be used include, but are not limited to, corn, rice, wheat, barley, rye, oat, sorghum, cotton, soybean, peanut, buckwheat, beet, rapeseed, sunflower, sugar cane, tobacco, etc.; solanaceous vegetables such as eggplant, tomato, pimento, pepper, potato, etc.;
25 cucurbit vegetables such as cucumber, pumpkin, zucchini, water melon, melon, squash, etc.;
cruciferous vegetables such as radish, white turnip, horseradish, kohlrabi, Chinese cabbage, cabbage, leaf mustard, broccoli, cauliflower, etc.; asteraceous vegetables such as burdock, crown daisy, artichoke, lettuce, etc; liliaceous vegetables such as green onion, onion, garlic, and asparagus; ammiaceous vegetables such as carrot, parsley, celery, parsnip, etc.;
17

chenopodiaceous vegetables such as spinach, Swiss chard, etc.; lamiaceous vegetables such as
Perilla frutescens, mint, basil, etc, strawberry, sweet potato, Dioscorea japonica, colocasia, etc.;
flowers, foliage plants, turf grasses; pome fruits such apple, pear, quince, etc.; stone fleshy
fruits such as peach, plum, nectarine, Prunus mume, cherry fruit, apricot, prune, etc.; citrus
5 fruits such as orange, lemon, rime, grapefruit, etc.; nuts such as chestnuts, walnuts, hazelnuts,
almond, pistachio, cashew nuts, macadamia nuts, etc.; berries such as blueberry, cranberry,
blackberry, raspberry, etc.; grape, kaki fruit, olive, plum, banana, coffee, date palm, coconuts,
etc. ; trees other than fruit trees; tea, mulberry, flowering plant, trees such as ash, birch,
dogwood, Eucalyptus, Ginkgo biloba, lilac, maple, Quercus, poplar, Judas tree, Liquidambar
10 formosana, plane tree, zelkova, Japanese arborvitae, fir wood, hemlock, juniper, Pinus, Picea,
and Taxus cuspidate, and the like.
In a preferred embodiment, the crops on which the combinations and compositions of the
present disclosure may be used include paddy, wheat, barley, maize, millet, fiber crops,
vegetable crops, fruit crops, spices, flowers, fodder and plantations. Preferably, the
15 combinations and compositions of the present invention may be used on tomato, brinjal, chili
and cucurbits such as cucumber and ridge gourd.
The combinations and the compositions of the present disclosure can be applied to a plant or a
part thereof by the use of conventional ground sprayers, granule applicators, watering
(drenching), drip irrigation, spraying, atomizing, broadcasting, dusting, foaming, spreading-
20 on, aerial methods of spraying, aerial methods of application, methods utilizing application
using modern technologies such as, but not limited to, drones, robots, and by other conventional
means known to those skilled in the art.
An embodiment also provides use of a mycorrhizal combination comprising
component I: Rhizophagus irregularis, or spores or propagules thereof; and
25 component II: one or more other endomycorrhiza, or spores or propagules thereof;
wherein the amount of component I is not less than 50% of the total amount of mycorrhiza;
or a composition comprising said combination for growth stimulation in a plant or a part thereof.
An embodiment also provides use of a mycorrhizal combination comprising
18

component I: Rhizophagus irregularis, or spores or propagules thereof; and
component II: one or more other endomycorrhiza, or spores or propagules thereof;
wherein the amount of component I is not less than 50% of the total amount of mycorrhiza;
or a composition comprising said combination as a biofertilizer.
5 In an embodiment, the combinations or the compositions of the present disclosure may be
mixed or applied in conjunction with other agrochemically active ingredients. The other agrochemically active ingredients may be selected from fertilizers, micronutrients, acaricides, algicides, antifeedants, avicides, bactericides, bird repellents, chemosterilants, fungicides, herbicide safeners, herbicides, insect attractants, insect repellents, insecticides, mammal
10 repellents, mating disruptors, molluscicides, nematicides, plant activators, plant-growth
regulators, rodenticides, synergists, virucides, derivatives thereof, biological control agents or mixtures thereof. The said mixtures may be premixed compositions or may be tank mixed at the time of application. The combinations or the compositions of the present disclosure may be applied with the other agrochemically active ingredients jointly or separately in a sequential
15 manner.
In an embodiment, the other agrochemically active ingredient is selected from the group consisting of insecticides, fungicides, biostimulants, and combinations thereof.
The mycorrhizae-based biofertilizer compositions and/or formulations in accordance with the
technology contained in the present invention is a multipurpose and multi-faceted product-it is
20 a soil conditioner, bio-remediator, and bio-control agent and has wide applications in
agriculture, plantations, horticulture, forestry. It offers sustainable and environment-friendly solutions to almost all cultivated plants and crops by enhancing nutrition and yields up to 5%-25% and curtailing chemical fertilizer inputs by 50%.
All headings and sub-headings are used herein for convenience only and should not be
25 construed as limiting the invention in any way.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
19

Examples
Example 1: Selection of mycorrhizal combination and efficacy of the combination on plant growth and mycorrhizal colonization
Seven treatments comprising solo mycorrhizal species and combination of mycorrhizal species
5 were applied to Tomato, Chili and Brinjal. Efficacy was checked with respect to increase in
root length, shoot length, and % root colonization by the mycorrhizal species. A control treatment was included wherein the treatment did not include any mycorrhizal species.
Table 1: Treatments comprising individual mycorrhizal species and combinations thereof.

Combination Mycorrhizal species
Combination 1 Rhizophagus irregularis
Combination 2 Rhizophagus clarus
Combination 3 Gigaspora margarita
Combination 4 Scutellospora heterogama
Combination 5 Rhizophagus irregularis, Rhizophagus clarus, Rhizophagus sp. in equal parts
Combination 6 Rhizophagus clarus, Gigaspora margarita (50:50 i.e. in equal parts)
Combination 7 Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita, Scutellospora heterogama (50:30:15:05)
10
Table 2: Efficacy of mycorrhizal combinations of Table 1 on plant growth and mycorrhizal colonization

Combination Root length (cm) Shoot length (cm) Endomycorrhizal root colonization (%)
20

Tomato Chilli Brinjal Tomato Chilli Brinjal Tomato Chilli Brinjal
Combination 1 34.95 32.74 24.35 72.92 57.75 69.05 84 80 85
Combination 2 31.25 31.85 23.05 70.35 59.95 69.39 85 82 81
Combination 3 35.15 31.45 22.95 70.63 58.76 68.32 82 86 89
Combination 4 35.95 29.56 23.27 72.56 57.54 71.43 85 83 86
Combination 5 31.05 30.14 24.35 71.03 56.34 73.85 82 81 89
Combination 6 30.23 29.34 23.24 72.85 57.82 72.92 80 88 83
Combination 7 38.45 34.35 28.45 78.35 60.16 75.34 92 94 95
Control 20.35 19.54 17.56 60.02 50.21 59.32 0 0 0
From Table 2, it is evident that the combination 7 provided the best results and more growth in all the three types of plants.
Example 2: Efficacy of mycorrhizal combination comprising varying proportions of mycorrhizal species.
5 6 gel formulations comprising combinations of varying proportions of endomycorrhizal
propagules of Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita and
Scutellospora heterogama were prepared and applied to tomato, chili and brinjal. The efficacy
of these combinations was checked with respect to root length, shoot length and root
colonization by the endomycorrhiza. A control treatment was included wherein the treatment
10 did not include any mycorrhizal species.
21

Table 3: Treatments comprising individual mycorrhizal species and combinations thereof.

Combination 1 Combination 2 Combination 3
Endomycorrhizal Percentage in Endomycorrhizal Percentage in Endomycorrhizal Percentage in
species the species the species the
combination combination combination
Rhizophagus 25 Rhizophagus 50 Rhizophagus 05
irregularis irregularis irregularis
Rhizophagus 25 Rhizophagus 30 Rhizophagus 15
clarus clarus clarus
Gigaspora 25 Gigaspora 15 Gigaspora 30
margarita margarita margarita
Scutellospora 25 Scutellospora 05 Scutellospora 50
heterogama heterogama heterogama
Combination 4 Combination 5 Combination 6
Endomycorrhizal Percentage in Endomycorrhizal Percentage in Endomycorrhizal Percentage in
species the species the species the
combination combination combination
Rhizophagus 15 Rhizophagus 30 Rhizophagus 20
irregularis irregularis irregularis
Rhizophagus 05 Rhizophagus 30 Rhizophagus 20
clarus clarus clarus
22

Gigaspora 50 Gigaspora 20 Gigaspora 30
margarita margarita margarita
Scutellospora 30 Scutellospora 20 Scutellospora 30
heterogama heterogama heterogama
Note: Combination 1-6 of example 2 differ from combination 1-7 of example 1
Table 4: Efficacy of mycorrhizal combinations of Table 3 on plant growth and mycorrhizal colonization

Combination Root length (cm) Shoot length (cm) Endomycorrhizal root colonization (%)

Tomato Chili Brinjal Tomato Chili Brinjal Tomato Chili Brinjal
1 34.65 33.45 23.85 73.05 58.15 68.35 85 82 85
2 38.65 35.15 29.25 79.15 60.35 75.65 95 97 95
3 29.85 30.25 24.65 72.95 57.92 67.95 85 85 86
4 34.85 30.95 23.45 70.54 57.65 67.85 83 85 87
5 34.14 32.55 24.30 70.15 58.25 70.35 84 88 83
6 34.95 30.35 24.25 71.85 56.75 70.75 86 84 85
Control 21.12 20.01 16.98 59.45 49.95 60.05 0 0 0
From the above table 4, it is evident that the combination wherein Rhizophagus irregularis
5 constituted 50% of the total mycorrhiza provided the best results in terms of plant growth.
23

Example 3: Efficacy of different formulations of Combination 2 of Table 3 on Cucurbits
The combination 2 of Table 3 was formulated as a gel and wettable powder and applied
to cucumber seeds and ridge gourd seeds before germination in a petri plate. The
5 germinated seeds were then transplanted into sterilized soil in pots.

Gel formulation of combination 2 of table 3 Wettable powder (WP) formulation of combination 2 of table 3
Humic acid – 25% Dextrose – 37%
Cellulose – 0.5% Lactose – 37%
Dodonaea viscosa extract –1% Cellulose – 1%
Endomycorrhiza – 20% (combination 2 of table 3) Humic acid – 5%
Water – 53.5% Endomycorrhiza – 20% (combination 2 of table 3)
A control was set up in both experiments which was devoid of endomycorrhiza.
Table 5: Efficacy of gel and WP formulations of combination 2 of table 3 on cucumber

Treatment Endomycorrhiza combination Days
required for germination Shoot length (cm) Root
length
(cm) Infectivity
potential
(%)
Control None 2 14 5.9 0.0
WP formulation combination 2 of table 3 1 20 9.5 95.0
Gel formulation combination 2 of table 3 1 20 9.8 95.0
Shoot and root length was calculated at 2 leaf stage
10 Table 6: Efficacy of gel and WP formulations of combination 2 of table 3 on ridge
gourd

Treatment Endomycorrhiza combination Days
required for germination Shoot length (cm) Root
length
(cm) Infectivity
potential
(%)
Control None 3 11.5 7.0 0.0
WP formulation combination 2 of table 3 2 19.5 8.5 94.0
Gel formulation combination 2 of table 3 2 20 8.8 95.0
Shoot and root length was calculated at 2 leaf stage
From the above table 5 and 6, it can be concluded that both formulations present similar effects in terms of germination period, shoot and root growth and demonstrate equivalent
24

infectivity potential, thus, concluding that the type of formulation does not affect the efficacy of the endomycorrhizal combination.
The infectivity potential as demonstrated in the examples of the specification is calculated by following procedure:
5 Subject roots (infected or uninfected) were removed from planting medium (such as soil)
and washed and rinsed with water. The roots were soaked in 10% KOH at 900C for 1 hour
or at 1200C for 15 mins. The roots were then rinsed in 1% HCl solution for 5 mins. The roots
were removed from HCl solution and stained in acidic glycerol solution containing Trypan
blue at 900C for 1 hour or at 1200C for 5 mins. The root specimens were placed in glass
10 petridish for destaining in destaining solution (50% glycerol) and observed under stereo
binocular / trinocular microscope.
Colonization is observed by infective points at the cortical cells and arbuscules, vesicles inside the cortical cells.
Percent Mycorrhiza colonization or Infectivity potential = (Total number of root
15 fragments infected / Total number of root fragments tested) X 100
25

We Claim:
1. A mycorrhizal combination comprising:
component I: Rhizophagus irregularis, or spores, propagules thereof; and component II: one or more other endomycorrhiza, or spores or propagules thereof; wherein the proportion of component I is not less than 50% of the total amount of mycorrhiza.
2. The mycorrhizal combination as claimed in claim 1, wherein the components I and II are not identical.
3. The mycorrhizal combination as claimed in claim 1, wherein the proportion of component I in the combination is about 50% to about 99% of the total amount of mycorrhiza.
4. The mycorrhizal combination as claimed in claim 1, wherein the proportion of component II in the combination is about 1% to about 50% of the total amount of mycorrhiza.
5. The mycorrhizal combination as claimed in any one of claims 1 to 4, wherein the ratio of component I to component II ranges from about 50:50 to about 99:1.
6. The mycorrhizal combination as claimed in claim 1, wherein the one or more other endomycorrhiza are selected from the genera Glomus, Entrophospora, Funneliformis, Rhizophagus, Septoglomus, Acaulospora, Archaeospora, Paraglomus, Gigaspora, Pacispora, Sclerocystis, Dentiscutata, Scutellospora, or combinations thereof.
7. The mycorrhizal combination as claimed in claim 6, wherein the one or more other endomycorrhiza are selected from Rhizophagus clarus, Gigaspora margarita, Scutellospora heterogama, Glomus mossae, Septoglomus viscosum, Funneliformis mossae, and combinations thereof.
8. The mycorrhizal combination as claimed in claim 1, wherein said combination is further combined with ectomycorrhizae or spores or propagules thereof, helper bacteria, or combinations thereof.
9. The mycorrhizal combination as claimed in claim 8, wherein said combination and the ectomycorrhizae or spores or propagules thereof are combined in a ratio of 1:10 to 10:1.

10. The mycorrhizal combination as claimed in claim 8, wherein said combination and the helper bacteria are admixed in a ratio of 1:100 to 100:1.
11. A composition comprising the mycorrhizal combination of claim 1 and atleast one agriculturally acceptable excipient.
12. The composition as claimed in claim 11, wherein the composition further comprises ectomycorrhiza or spores or propagules thereof, helper bacteria, or combinations thereof.
13. A biofertilizer comprising the mycorrhizal combination as claimed in claim 1 or a composition comprising said combination.
14. A method of growth stimulation in a plant or a part thereof, wherein the method comprises applying to the plant, a part thereof, or a locus a mycorrhizal combination as claimed in claim 1 or a composition comprising said combination.
15. Use of a mycorrhizal combination as claimed in claim 1 or a composition comprising said combination for growth stimulation in a plant or a part thereof or as a biofertilizer.