FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
“A BIOFERTILIZER”
Applicant name and Address:
NATURAL PLANT PROTECTION LIMITED of 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.
1

Field of invention
The present disclosure relates to the field of a biofertilizer. Particularly, the present disclosure relates to biofertilizer combinations and compositions comprising endophytic bacteria and mycorrhiza, and methods of uses thereof.
5 Background:
In agricultural practices, use of fertilizers is a regular practice for soil conditioning and boosting
plant health and yield. However, overuse of chemical fertilizers has produced negative side-
effects on soil health, soil microbiome, and environment along with causing toxicity in plants,
flora and fauna. As a result, use of biofertilizers is gaining momentum to overcome the negative
10 side effects of chemical supplements.
Biofertilizers are living microbes that enhance plant nutrition by either by mobilizing or increasing nutrient availability in soils. Various microbial taxa including beneficial bacteria and fungi are currently used as biofertilizers, as they successfully colonize the rhizosphere, rhizoplane or root interior and promote growth by increasing the supply or availability of
15 primary nutrients to the host plant. Arbuscular mycorrhizae (or “mycorrhiza”) are beneficial
fungi which create a conducive environment for nutrient uptake in the root zone of the plants. Mycorrhizae are a symbiotic association between plant roots and fungi. Their major role is to enhance nutrient and water uptake by the host plant by exploiting a larger volume of soil than roots alone can do. Mycorrhizae come in a number of forms, dependent upon both host plant
20 and fungal taxonomy. Mycorrhizae play important roles in plant nutrition, soil biology, and
soil chemistry. The current times have been witnessing several mycorrhizae based biofertilizers to improve soil health, plant health and yield.
Despite the availability of umpteen biofertilizers, there is still a dearth of research into optimal
mycorrhizal combinations with different properties which can impact soil health and plant
25 health at a maximum level. There still is a need for biofertilizer compositions involving
mycorrhizae which have enhanced beneficial functions exerted by mycorrhiza. There is a need for compositions which enhance and optimize mycorrhizal functions, thus, maximally benefiting the soil and crops. There is a need for biofertilizer compositions which promote and rejuvenate the soil health and soil microbiome.
2

In accordance with the need repurposed here, it is an object of the present disclosure to provide
a combination or a composition comprising mycorrhizae with enhanced mycorrhizal functions.
It is an object of the present disclosure to add an agent to the composition comprising
mycorrhizae which enhances the properties of mycorrhizae. It is an object of the present
5 disclosure to provide a combination or a composition comprising mycorrhizae and endophytic
bacteria. It is also an object of the present disclosure to improve plant health, yield and soil properties using a combination or a composition comprising mycorrhizae and endophytic bacteria.
Summary:
10 An aspect of the present disclosure provides a biofertilizer combination comprising an
endophytic bacteria selected from Pseudomonas fluorescens, Bacillus, Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture thereof; and one or more mycorrhiza.
An aspect of the present disclosure provides a biofertilizer combination comprising an
15 endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519,
Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza.
In an embodiment, the mycorrhiza may be selected from endomycorrhiza, ectomycorrhiza, or
20 combinations thereof. In another embodiment, the mycorrhiza may be in form of propagules
selected from endomycorrhiza propagules, ectomycorrhiza propagules, or combinations thereof.
Another aspect of the present disclosure provides a biofertilizer composition comprising an
endophytic bacteria selected from Pseudomonas fluorescens, Bacillus, Bacillus
25 amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture thereof; and
one or more mycorrhiza.
Another aspect of the present disclosure provides a biofertilizer composition comprising an endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC
3

25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza.
An aspect of the present disclosure provides a method of preparing a biofertilizer composition
comprising an endophytic bacteria selected from Pseudomonas fluorescens, Bacillus subtilis,
5 Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture
thereof; and one or more mycorrhiza.
An aspect of the present disclosure provides a method of preparing a biofertilizer composition
comprising an endophytic bacteria selected from Pseudomonas fluorescens deposited as
MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens
10 deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus
polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza.
An aspect of the present disclosure provides a method of improving plant health, yield and
vigor, wherein said method comprises contacting a plant or a part thereof, or a locus with a
biofertilizer combination comprising an endophytic bacteria selected from Pseudomonas
15 fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus
polymyxa, or a mixture thereof; and one or more mycorrhiza.
An aspect of the present disclosure provides a method of improving plant health, yield and
vigor, wherein said method comprises contacting a plant or a part thereof, or a locus with a
biofertilizer combination comprising an endophytic bacteria selected from Pseudomonas
20 fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus
amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza.
Another aspect of the present disclosure provides a use of a biofertilizer combination
25 comprising an endophytic bacteria selected from Pseudomonas fluorescens, Bacillus subtilis,
Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture thereof; and one or more mycorrhiza for improving plant health, yield, and vigor.
Another aspect of the present disclosure provides a use of a biofertilizer combination
comprising an endophytic bacteria selected from Pseudomonas fluorescens deposited as
30 MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens
4

deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza for improving plant health, yield, and vigor.
An aspect of the present disclosure also provides a kit comprising a component comprising an
5 endophytic bacteria selected from Pseudomonas fluorescens, Bacillus subtilis, Bacillus
amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture thereof; and another component comprising one or more mycorrhiza for improving plant health, yield, and vigor.
An aspect of the present disclosure also provides a kit comprising a component comprising an
10 endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519,
Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and another component comprising one or more mycorrhiza for improving plant health, yield, and vigor.
15 In an embodiment, the kit may be split into multiple components of endophytic bacteria and
one or more mycorrhiza to be mixed together or applied together, jointly, or sequentially or simultaneously.
Detailed Description:
For the purposes of the following detailed description, it is to be understood that the invention
20 may assume various alternative variations and step sequences, except where expressly
specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of materials/ingredients used in the specification are to be understood as being modified in all instances by the term "about".
Thus, before describing the present invention in detail, it is to be understood that this invention
25 is not limited to particularly exemplified process parameters or composition that may of course,
vary. It is also to be understood that the terminology used herein is for the purpose of describing
embodiments of the invention only and is not intended to limit the scope of the invention in
any manner. The use of examples anywhere in this specification including examples of any
terms discussed herein is illustrative only, and in no way limits the scope and meaning of the
30 invention or of any exemplified term. Likewise, the invention is not limited to various
embodiments given in this specification. Unless otherwise defined, all technical and scientific
5

terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
It must be noted that, as used in this specification, the singular forms “a,” “an” and “the” include
5 plural referents unless the content clearly dictates otherwise. The terms “preferred” and
“preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances.
As used herein, the terms “comprising” “including,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.
10 Unless otherwise stated, all exact values provided herein are representative of corresponding
approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate). As used herein, the term "about" refers to a measurable value such as a parameter, an amount, a temporal duration, and the like and is
15 meant to include variations of +/-15% or less, specifically variations of +/-10% or less, more
specifically variations of +/-5% or less, even more specifically variations of +/-1% or less, and still more specifically variations of +/-0.1% or less of and from the particularly recited value, in so far as such variations are appropriate to perform in the disclosure described herein.
Recitation of ranges of values are merely intended to serve as a shorthand method of referring
20 individually to each separate value falling within the range, unless otherwise indicated herein,
and each separate 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
25 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.
30 Keeping the objectives in mind, the inventors of the present disclosure surprisingly found that
combining endophytic bacteria with the mycorrhiza enhanced mycorrhizal functions and
6

impacted soil and plant health at a greater level. More particularly, the inventors found that
bacteria such as Pseudomonas, Bacillus, Paenibacillus greatly enhanced mycorrhizal functions
and worked symbiotically to produce pronounced effects in the plants where it was applied.
Moreover, the inventors observed, to their surprise, that the presence of endophytic bacteria in
5 combination with mycorrhiza increased colonization of the mycorrhiza.
Thus, an embodiment of the present disclosure provides a biofertilizer combination comprising an endophytic bacteria selected from Pseudomonas fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture thereof; and one or more mycorrhiza.
10 In an embodiment, the Pseudomonas fluorescens is deposited as MTCC 25519.
In an embodiment, the Bacillus subtilis is deposited as MTCC 25520.
In an embodiment, the Bacillus amyloliquefaciens is deposited as MTCC 25518.
In an embodiment, the Pseudomonas putida is deposited as MTCC 25607.
In an embodiment, the Paenibacillus polymyxa is deposited as MTCC 25609.
15 Thus, an embodiment of the present disclosure provides a biofertilizer combination comprising
an endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza.
20 An embodiment of the present disclosure provides a biofertilizer composition comprising the
biofertilizer combinations of the present disclosure.
Thus, an embodiment of the present disclosure provides a biofertilizer composition comprising
an endophytic bacteria selected from Pseudomonas fluorescens, Bacillus, Bacillus
amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture thereof; and
25 one or more mycorrhiza.
Therefore, an embodiment of the present disclosure provides a biofertilizer composition comprising an endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens
7

deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza.
In an embodiment, said combination comprises at least one endophytic bacteria and one or
more mycorrhiza. In an embodiment, said combination comprises at least two endophytic
5 bacteria and one or more mycorrhiza. In an embodiment, said combination comprises at least
three endophytic bacteria and one or more mycorrhiza. In an embodiment, said combination comprises at least four endophytic bacteria and one or more mycorrhiza. In an embodiment, said combination comprises five endophytic bacteria and one or more mycorrhiza.
In a preferred embodiment, the endophytic bacteria are a mixture of Pseudomonas fluorescens
10 deposited as MTCC 25519 and Bacillus subtilis deposited as MTCC 25520.
Another embodiment of the present disclosure provides a biofertilizer combination comprising Pseudomonas fluorescens deposited as MTCC 25519 and one or more mycorrhiza.
Another embodiment of the present disclosure provides a biofertilizer combination comprising Bacillus subtilis deposited as MTCC 25520 and one or more mycorrhiza.
15 Another embodiment of the present disclosure provides a biofertilizer combination comprising
Bacillus amyloliquefaciens deposited as MTCC 25518 and one or more mycorrhiza.
Another embodiment of the present disclosure provides a biofertilizer combination comprising Pseudomonas putida deposited as MTCC 25607 and one or more mycorrhiza.
Another embodiment of the present disclosure provides a biofertilizer combination comprising
20 Paenibacillus polymyxa deposited as MTCC 25609 and one or more mycorrhiza.
In an embodiment, the mycorrhiza is endomycorrhiza, ectomycorrhiza or combinations
thereof. Endomycorrhiza and ectomycorrhiza are biologically symbiotic soil fungi, which
colonize plant root systems and act as a living extension of the roots, increasing the absorptive
surface area and improving the plant nutrient and water efficiency. Through this symbiosis,
25 endomycorrhiza and/or ectomycorrhiza offers direct and indirect benefits to host plants such
as, but not limited to, stress resilience, drought resistance, and reduction in the fertilizer and irrigation requirements.
8

In an embodiment, the mycorrhiza comprises mycorrhizal propagules. In an embodiment, the mycorrhizal propagules are selected from propagules of endomycorrhiza, propagules of ectomycorrhiza or combinations thereof.
In an embodiment, the mycorrhizal propagules comprise propagules of endomycorrhiza
5 selected from arbuscular mycorrhiza, ericoid mycorrhiza, arbutoid mycorrhiza, monotropoid
mycorrhiza, orchid mycorrhiza or combinations thereof.
In an embodiment, the mycorrhizal propagules comprise propagules of endomycorrhiza
selected from the class Glomeromycota (glomeromycetes). In an embodiment, the mycorrhizal
propagules of the class Glomeromycota are selected from the order Archaeosporales,
10 Diversisporales, Glomerales, Paraglomerales or combinations thereof.
In an embodiment, the mycorrhizal propagules comprise of endomycorrhiza propagules,
wherein the endomycorrhiza propagules comprise propagules of Glomaceae (Glomeraceae),
Acaulosporaceae, Archaeosporaceae, Diversisporaceae, Pacisporaceae, Paraglomaceae,
Gigasporaceae, Ambisporaceae, Geosiphonaceae, Claroideoglomeraceae or combinations
15 thereof. In an embodiment, the mycorrhizal propagules comprise of endomycorrhiza
propagules, wherein the endomycorrhiza propagules comprise propagules of Glomus genera, Entrophospora, Funneliformis, Rhizophagus, Septoglomus, Acaulospora, Archaeospora, Paraglomus, Gigaspora, Pacispora, Sclerocystis, Dentiscutata, Scutellospora, and combinations thereof.
20 In an embodiment, the mycorrhizal propagules comprise propagules of endomycorrhiza
selected from a group consisting of Rhizophagus irregularis, 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
25 and subspecies and strains thereof.
In an embodiment, the mycorrhizal propagules comprise propagules of endomycorrhiza selected from a group consisting of Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita, Scutellospora heterogama, and combinations thereof.
9

In an embodiment, the mycorrhizal propagules comprise a combination of propagules of
endomycorrhiza. In some embodiments, the combination of the present disclosure comprises a
combination of endomycorrhizal propagules selected from: (i) propagules of Rhizophagus
irregularis and Rhizophagus clarus, (ii) propagules of Rhizophagus irregularis and Gigaspora
5 margarita, (iii) propagules of Rhizophagus irregularis and Scutellospora heterogama, (iv)
propagules of Rhizophagus clarus and Gigaspora margarita, (v) propagules of Rhizophagus
clarus and Scutellospora heterogama, (vi) propagules of Gigaspora margarita and
Scutellospora heterogama, (vii) propagules of Rhizophagus irregularis, Rhizophagus clarus,
and Gigaspora margarita, (viii) propagules of Rhizophagus irregularis, Rhizophagus clarus,
10 and Scutellospora heterogama, (ix) propagules of Rhizophagus irregularis, Gigaspora
margarita and Scutellospora heterogama, and (x) propagules of Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita and Scutellospora heterogama.
In a preferred embodiment, the combination of mycorrhizal propagules comprises a
combination of Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita and
15 Scutellospora heterogama.
In an embodiment, the mycorrhizal propagules comprise propagules of ectomycorrhiza selected from Laccaria, Pisolithus, Piriformospora, Scleroderma, Rhizopogon, and combinations thereof.
In an embodiment, the mycorrhizal propagules comprise, but are not limited to, propagules of
20 ectomycorrhiza selected from Piriformospora indica, Laccaria bicolor, Laccaria lacata,
Rhizopogon roseolus, Laccaria proxima, Pisolithus arhizus, Pisolithus tinctorius and combinations thereof.
In an embodiment, the mycorrhizal propagules include a combination of propagules of
ectomycorrhiza. In an embodiment, the combination of mycorrhizal propagules includes a
25 combination of Piriformospora indica, Laccaria bicolor and Pisolithus tinctorius. In an
embodiment, the combination of mycorrhizal propagules includes a combination of Piriformospora indica, Laccaria bicolor, Laccaria lacata and Pisolithus tinctorius.
In some embodiments, the composition of the present disclosure comprises a combination of
ectomycorrhizal propagules selected from: (i) propagules of Piriformospora indica and
30 propagules of Laccaria bicolor, (ii) propagules of Piriformospora indica and propagules of
Pisolithus tinctorius, (iii) propagules of Laccaria bicolor and propagules of Pisolithus
10

tinctorius, (iv) propagules of Piriformospora indica, propagules of Laccaria bicolor, and
propagules of Pisolithus tinctorius, (v) propagules of Piriformospora indica and propagules of
Laccaria lacata, (vi) propagules of Laccaria lacata and propagules of Pisolithus tinctorius,
(vii) propagules of Laccaria lacata and propagules of Laccaria bicolor, (viii) propagules of
5 Piriformospora indica, propagules of Laccaria lacata, and propagules of Pisolithus tinctorius,
(ix) propagules of Piriformospora indica, propagules of Laccaria bicolor and propagules of Laccaria lacata, (x) propagules of Laccaria bicolor, propagules of Laccaria lacata, and propagules of Pisolithus tinctorius and (xi) propagules of Piriformospora indica, propagules of Laccaria bicolor, propagules of Laccaria lacata, and propagules of Pisolithus tinctorius.
10 In an embodiment, the mycorrhizal propagules comprise a combination of endomycorrhiza and
ectomycorrhiza. The endomycorrhiza and ectomycorrhiza may be selected from the species described above.
In an embodiment, the endophytic bacteria and the mycorrhiza are admixed in a ratio of 1:100
to 100:1. In an embodiment, the endophytic bacteria and the mycorrhiza are admixed in a ratio
15 of 1:75 to 75:1. In an embodiment, the endophytic bacteria and the mycorrhiza are admixed in
a ratio of 1:50 to 50:1. In an embodiment, the endophytic bacteria and the mycorrhiza are admixed in a ratio of 1:30 to 30:1. In an embodiment, the endophytic bacteria and the mycorrhiza are admixed in a ratio of 1:20 to 20:1. In a preferred embodiment, the endophytic bacteria and the mycorrhiza are admixed in a ratio of 1:17 to 2:1.
20 The term “admixed” also refers to synonymous terms “combined”, “mixed” in the context of
the embodiments provided herein.
In an embodiment, wherein the endophytic bacteria are a mixture of Pseudomonas fluorescens
deposited as MTCC 25519, and Bacillus subtilis deposited as MTCC 25520, the Pseudomonas
fluorescens deposited as MTCC 25519, and the Bacillus subtilis deposited as MTCC 25520 are
25 admixed in a ratio ranging from 1:100 to 100:1, preferably 1:75 to 75:1, more preferably 1:50
to 50:1, even more preferably 1:30 to 30:1. In an embodiment, the preferred ratio is 1:20 to 20:1.
In an embodiment, the endophytic bacteria are present in an amount ranging from about 0.1%
to about 50% by weight of the total composition. In an embodiment, the endophytic bacteria
30 are present in an amount ranging from about 0.1% to about 30% by weight of the total
composition. In an embodiment, the endophytic bacteria are present in an amount ranging from
11

about 0.1% to about 20% by weight of the total composition. In an embodiment, the endophytic bacteria are present in an amount ranging from about 0.5% to about 10% by weight of the total composition. In an embodiment, the endophytic bacteria are present in an amount ranging from about 2% to about 10% by weight of the total composition.
5 The biofertilizer composition comprising said combination comprises endomycorrhiza,
ectomycorrhiza or a combination thereof in an amount ranging from about 0.1% to about 90% by weight of the composition.
The composition comprises endomycorrhiza in an amount ranging from about 0.1% to about 90% by weight of the composition. In an embodiment, the mycorrhizal propagules include
10 propagules of endomycorrhiza selected from one or more of Rhizophagus irregularis,
Rhizophagus clarus, Gigaspora margarita and Scutellospora heterogama. In an embodiment, the mycorrhizal propagules comprise a combination of propagules of endomycorrhiza. The combination of mycorrhizal propagules includes various combinations of Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita and Scutellospora heterogama as
15 described above.
In an embodiment, the biofertilizer combination or a composition comprising said combination comprises a combination of endomycorrhiza and ectomycorrhiza in weight ratios ranging from 1:10 to 10:1. In an embodiment, the weight ratio of endomycorrhiza to ectomycorrhiza in the biofertilizer combination or a composition comprising said combination ranges from 1:9 to 9:1.
20 In an embodiment, the weight ratio of endomycorrhiza to ectomycorrhiza in the biofertilizer
combination or a composition comprising said combination ranges from 3:7 to 7:3. In an embodiment, the weight ratio of endomycorrhiza to ectomycorrhiza in the biofertilizer combination or a composition comprising said combination ranges from 2:3 to 3:2. In an embodiment, the weight ratio of endomycorrhiza to ectomycorrhiza in the biofertilizer
25 combination or a composition comprising said combination ranges from 1:5 to 5:1, preferably
1:4 to 4:1.
In an embodiment, wherein the mycorrhizal propagules comprise a combination of endomycorrhiza and ectomycorrhiza, the propagules of endomycorrhiza are present in an amount ranging from about 10% to about 80% by weight of the mycorrhizal propagules.
12

In an embodiment, wherein the mycorrhizal propagules comprise a combination of endomycorrhiza and ectomycorrhiza, the propagules of ectomycorrhiza are present in an amount ranging from about 20% to about 90% by weight of the mycorrhizal propagules.
In an embodiment, mycorrhizal propagules comprise: (a) propagules of endomycorrhiza
5 selected from Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita,
Scutellospora heterogama and combinations thereof in an amount ranging from about 10% to
about 80% by weight of the mycorrhizal propagules; and (b) propagules of ectomycorrhiza
selected from Piriformospora indica, Laccaria bicolor, Laccaria lacata, Pisolithus tinctorius
and combinations thereof in an amount ranging from about 20% to about 90% by weight of the
10 mycorrhizal propagules.
In an embodiment, mycorrhizal propagules comprise: (a) propagules of endomycorrhiza
selected from Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita,
Scutellospora heterogama, and combinations thereof in an amount of about 70% by weight of
the mycorrhizal propagules; and (b) propagules of ectomycorrhiza selected from
15 Piriformospora indica, Laccaria bicolor, Laccaria lacata, Pisolithus tinctorius, and
combinations thereof in an amount of about 30% by weight of the mycorrhizal propagules.
In an embodiment, mycorrhizal propagules comprise: (a) propagules of endomycorrhiza
selected from Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita,
Scutellospora heterogama, and combinations thereof in an amount of about 30% by weight of
20 the mycorrhizal propagules; and (b) propagules of ectomycorrhiza selected from
Piriformospora indica, Laccaria bicolor, Laccaria, lacata, Pisolithus tinctorius, and combinations thereof in an amount of about 70% by weight of the mycorrhizal propagules.
In an embodiment, the number of propagules in the combinations or the compositions of the
present disclosure range from about 1X102 propagules/g to about 9X106 propagules/g. In an
25 embodiment, the combinations or the compositions of the present disclosure comprise about
2X102 to 2X105 propagules/g. In an embodiment, the combinations or the compositions of the present disclosure comprise about 1X105 propagules/g. In an embodiment, the combinations or the compositions of the present disclosure comprise about 2X102 propagules/g.
In an embodiment, the composition of the present disclosure further comprises one or more
30 agriculturally acceptable excipient. In an embodiment, the agriculturally acceptable excipients
are selected from any or a combination of wetting agent, dispersing agent, binding agent,
13

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 be used to serve its intended purpose without departing from the
scope of the present disclosure. In an embodiment, the agriculturally acceptable excipients are
5 present in an amount ranging from 0.3% to 99% by weight of the composition.
An embodiment of the present disclosure therefore provides a biofertilizer composition comprising the biofertilizer combinations of the present disclosure, and an agriculturally acceptable excipient.
Thus, an embodiment of the present disclosure provides a biofertilizer composition comprising
10 an endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519,
Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; one or more mycorrhiza; and an agriculturally acceptable excipient.
15 In an embodiment, the agriculturally acceptable excipient may be a stabilizer selected from any
or a combination of humic acid, cellulose, dextrose, lactose, methyl cellulose, Xanthan gum, polyvinylpyrrolidone (PVP), Tween and Gum Arabica. In an embodiment, the stabilizer is present in the composition in an amount ranging from about 0.2% to about 80% by weight of the composition.
20 An embodiment provides a biofertilizer composition comprising an endophytic bacteria
selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; one or more mycorrhiza; a stabilizer; and an agriculturally acceptable excipient. In an
25 embodiment, the agriculturally acceptable excipient is selected from a group comprising, but
not limited to, a wetting agent, dispersing agent, binding agent, surfactant/emulsifier, anti-freezing agent, mineral, filler, preservative, and combinations thereof.
The wetting agent is selected from a group comprising, but not limited to, sulfosuccinates,
naphthalene sulfonates, sulfated esters, phosphate esters, sulfated alcohol, alkyl benzene
30 sulfonates, and combinations thereof.
14

The dispersing agent is selected from a group comprising, but not limited to, polycarboxylates, 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,
5 phenyl naphthalene sulphonates, lignin derivatives, polyvinyl pyrrolidone,
polyalkylpyrrolidone, carboxymethylcellulose, xanthan gum, polyethoxylated fatty acids, polyethoxylated fatty alcohols, ethylene oxide copolymer, propylene oxide copolymer, polyethylene glycols, polyethylene oxides, and combinations thereof.
The surfactant is selected from a group comprising ionic surfactants, non-ionic surfactants, and
10 combinations thereof. Non-limiting examples of ionic surfactants include sulfonic acids,
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
15 sulfonates, xylene sulfonates, cumene sulfonates, alkyl benzene sulfonates, alkyl
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-
20 acyl taurates, N-acyl-N-alkyltaurates, and alkyl carboxylates.
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
25 glycols, polyoxyethylenated mercaptans, carboxylic acid esters, polyoxyethylenated
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 copolymers, polyamines, and polyvinylpynolidones, sorbitan fatty acid alcohol ethoxylates and sorbitan fatty acid ester ethoxylates.
30 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.
15

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,
5 orthoclase, gypsum, andalusite, talc, fluorite, apatite, orthoclase, topaz, corundum, diamond,
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.
10 The fillers are selected from a group comprising, but not limited to, diatomaceous earth, kaolin,
bentonite, precipitated silica, attapulgite, perlite, and combinations thereof.
An embodiment of the present disclosure provides a biofertilizer composition comprising the biofertilizer combinations of the present disclosure, and a plant extract.
An embodiment provides a biofertilizer composition comprising an endophytic bacteria
15 selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited
as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; one or more mycorrhiza; and a plant extract.
In an embodiment, the plant extract acts as a gelling agent or a binder. In an embodiment, the
20 plant extract belongs to a plant belonging to the genus Dodonaea. In an embodiment, the plant
extract belongs to a plant belonging to the genus Dodonaea viscosa.
In an embodiment, the extract of the plant belonging to Dodonaea genus is an aqueous extract.
In an embodiment, the extract of the plant belonging to Dodonaea genus is a solvent.
In an embodiment, the composition comprises the extract of the plant belonging to Dodonaea
25 genus in an amount ranging from about 0.1% to about 40% by weight of the composition. In
an embodiment, the composition includes the extract of the plant belonging to Dodonaea genus in an amount ranging from about 0.3% to about 30% by weight of the composition. In an embodiment, the composition includes the extract of the plant belonging to Dodonaea genus in an amount ranging from about 0.5% to about 20% by weight of the composition. In an
16

embodiment, the composition includes the extract of the plant belonging to Dodonaea genus
in an amount ranging from about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to
about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.3% to about 25%,
about 0.3% to about 20%, about 0.3% to about 15%, about 0.3% to about 10%, about 0.5% to
5 about 40%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 15%,
about 0.5% to about 10%, about 1% to about 40%, about 1% to about 30%, about 1% to about
25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, bout 2% to
about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about
2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 5% to about 40%,
10 about 5% to about 35%, about 5% to 30%, about 5% to about 20%, about 5% to about 15%,
about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 15% to about 30%, or about 20% to about 40%, by weight of the composition.
An embodiment provides a biofertilizer composition comprising an endophytic bacteria
selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited
15 as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida
deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; one or more mycorrhiza; a plant extract; and an agriculturally acceptable excipient.
An embodiment provides a biofertilizer composition comprising an endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited
20 as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida
deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; one or more mycorrhiza; a plant extract; a stabilizer; and an agriculturally acceptable excipient. In an embodiment, the agriculturally acceptable excipient may be a stabilizer selected from any or a combination of humic acid, cellulose, dextrose, lactose, methyl
25 cellulose, Xanthan gum, 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, dispersing agent, binding agent, surfactant/emulsifier, anti-freezing agent, mineral, filler, preservative, and combinations thereof.
The advantageous agricultural composition of the present disclosure is formulated in any of
30 formulations such as 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 likes.
17

In an embodiment, the composition is formulated into a granule, water soluble powder or a wettable powder.
Another embodiment of the present disclosure provides a granular biofertilizer composition,
the composition comprising: an endophytic bacteria selected from Pseudomonas fluorescens
5 deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus
amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; one or more mycorrhiza. In an embodiment, the mycorrhizal propagules comprise propagules of endomycorrhiza. In another embodiment, the mycorrhizal propagules comprise propagules of
10 ectomycorrhiza. In an embodiment, the mycorrhizal propagules comprise a combination of
endomycorrhizal and ectomycorrhizal propagules. In an embodiment, the composition further comprises a plant extract, a stabilizer and an agriculturally acceptable excipient. In an embodiment, the plant extract is an extract of a plant belonging to genus Dodonaea. In an embodiment, the stabilizer serves as a source of carbon aiding in improving survivability of
15 endophytic bacteria and mycorrhizal propagules and improving shelf life of the composition.
In some embodiments, the plant extract of Dodonaea viscosa also exerts stabilizing effect on endophytic bacteria and mycorrhizal propagules, aiding in improving shelf life of the composition.
In an embodiment, the composition comprises: (a) an endophytic bacteria selected from
20 Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC
25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited
as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof in
an amount ranging from about 0.1% to about 30%; (b) mycorrhizal propagules in an amount
ranging from about 5% to about 40% by weight of the composition; (c) an extract of Dodonaea
25 viscosa in an amount ranging from about 0.2% to about 20% by weight of the composition; (d)
a stabilizer in an amount ranging from about 0.2% to about 80% by weight of the composition;
and (e) an agriculturally acceptable excipient. In an embodiment, the stabilizer is selected from
a group comprising: humic acid, cellulose, dextrose, lactose, methyl cellulose, Xanthan gum,
polyvinylpyrrolidone (PVP), Tween (polysorbates), Gum Arabica, or combinations thereof. In
30 an embodiment, the stabilizer comprises humic acid and methyl cellulose. In an embodiment,
the stabilizer comprises humic acid and cellulose.
18

In an embodiment, the composition comprises: (a) an endophytic bacteria selected from
Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC
25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited
as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof in
5 an amount ranging from about 0.1% to about 30%; (b) mycorrhizal propagules in an amount
ranging from about 5% to about 40% by weight of the composition; (c) an extract of Dodonaea
viscosa in an amount ranging from about 0.2% to about 20% by weight of the composition; (d)
humic acid in an amount ranging from about 1% to about 50% by weight of the composition;
(e) cellulose or methyl cellulose in an amount ranging from about 0.1% to about 5% by weight
10 of the composition; and (f) an agriculturally acceptable excipient in an amount sufficient to
prepare said composition.
In an embodiment, the present disclosure provides a wettable powder of a biofertilizer composition comprising: an endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus
15 amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC
25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; one or more mycorrhiza. In an embodiment, the mycorrhizal propagules comprise propagules of endomycorrhiza. In another embodiment, the mycorrhizal propagules comprise propagules of ectomycorrhiza. In an embodiment, the mycorrhizal propagules comprise a combination of
20 endomycorrhizal and ectomycorrhizal propagules. In an embodiment, the composition further
comprises a stabilizer and an agriculturally acceptable excipient. a stabilizer selected from any or a combination of humic acid, cellulose, methyl cellulose, Xanthan gum, polyvinylpyrrolidone (PVP), Tween and Gum Arabica.
In an embodiment, the composition comprises: (a) an endophytic bacteria selected from
25 Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC
25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited
as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof in
an amount ranging from about 0.1% to about 30%; (b) mycorrhizal propagules in an amount
ranging from about 5% to about 40% by weight of the composition; (c) a stabilizer in an amount
30 ranging from about 0.2% to about 80% by weight of the composition; and (d) an agriculturally
acceptable excipient. In an embodiment, the stabilizer is selected from a group comprising: humic acid, cellulose, methyl cellulose, Xanthan gum, polyvinylpyrrolidone (PVP), Tween
19

(polysorbates), Gum Arabica, or combinations thereof. In an embodiment, the stabilizer comprises humic acid and methyl cellulose. In an embodiment, the stabilizer comprises humic acid and cellulose.
In an embodiment, the composition comprises: (a) an endophytic bacteria selected from
5 Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC
25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited
as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof in
an amount ranging from about 0.1% to about 30%; (b) mycorrhizal propagules in an amount
ranging from about 5% to about 40% by weight of the composition; (c) humic acid in an amount
10 ranging from about 1% to about 50% by weight of the composition; (d) cellulose or methyl
cellulose in an amount ranging from about 0.1% to about 5% by weight of the composition; and (e) an agriculturally acceptable excipient in an amount sufficient to prepare said composition.
In some embodiments, the amount of humic acid in the composition ranges from about 5% to
15 about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about
5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, or about 25% to about 50%, by weight of the composition.
In some embodiments, the amount of cellulose or methyl cellulose in the composition ranges
20 from about 0.1% to about 5%, about 0.1% to about 4.5%, about 0.1% to about 4%, about 0.1%
to about 3.5%, about 0.1% to about 3%, about 0.1% to about 2.5%, about 0.1% to about 2%, about 0.1% to about 1.5%, about 0.1% to about 1%, by weight of the composition.
An embodiment of the present disclosure provides a method of preparing a biofertilizer
composition comprising an endophytic bacteria selected from Pseudomonas fluorescens,
25 Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or
a mixture thereof; and one or more mycorrhiza.
An embodiment of the present disclosure provides a method of preparing a biofertilizer
composition comprising an endophytic bacteria selected from Pseudomonas fluorescens
deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus
30 amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC
20

25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza.
An embodiment provides a method of improving plant health, yield and vigor, wherein said
method comprises contacting a plant or a part thereof, a plant propagation material, a site or
5 medium of plant growth, or a locus with a biofertilizer combination or a composition
comprising said combination, wherein the combination comprises an endophytic bacteria selected from Pseudomonas fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture thereof; and one or more mycorrhiza.
An embodiment provides a method of improving plant health, yield and vigor, wherein said method comprises contacting a plant or a part thereof, a plant propagation material, a site or medium of plant growth, or a locus with a biofertilizer combination or a composition comprising said combination, wherein the combination comprises an endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza.
The agricultural benefit may be conferred by treating a seed, a seedling, a plant, a plant part, a
soil, or a combination thereof, with a composition or formulation of the present disclosure
20 under conditions leading to association between the composition and the seed, plant, plant part,
soil, or combinations thereof. Application of a formulation to a target is accomplished using any delivery method known in the art including, but not limited to dusting, fumigation, granule application, injection, misting, seed treatment, spraying, dipping, or coating.
In an embodiment, the combinations or the compositions of the present disclosure may be
25 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 a biofertilizer combination or a composition comprising said
combination, wherein the combination comprises an endophytic bacteria selected from
Pseudomonas fluorescens deposited as MTCC 25519, Bacillus subtilis deposited as MTCC
30 25520, Bacillus amyloliquefaciens deposited as MTCC 25518, Pseudomonas putida deposited
as MTCC 25607, Paenibacillus polymyxa deposited as MTCC 25609, or a mixture thereof; and
21

one or more mycorrhiza. In an embodiment, the method comprises applying a biofertilizer
combination or a composition comprising said combination, wherein the combination
comprises an endophytic bacteria selected from Pseudomonas fluorescens deposited as MTCC
25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens deposited as
5 MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus polymyxa
deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza propagules.
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,
10 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.
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,
15 rye, oat, sorghum, cotton, soybean, peanut, buckwheat, beet, rapeseed, sunflower, sugar cane,
tobacco, etc.; solanaceous vegetables such as eggplant, tomato, pimento, pepper, potato, etc.; 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,
20 crown daisy, artichoke, lettuce, etc; liliaceous vegetables such as green onion, onion, garlic,
and asparagus; ammiaceous vegetables such as carrot, parsley, celery, parsnip, etc.; 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
25 fruits such as peach, plum, nectarine, Prunus mume, cherry fruit, apricot, prune, etc.; citrus
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,
30 dogwood, Eucalyptus, Ginkgo biloba, lilac, maple, Quercus, poplar, Judas tree, Liquidambar
formosana, plane tree, zelkova, Japanese arborvitae, fir wood, hemlock, juniper, Pinus, Picea, and Taxus cuspidate, and the like.
22

In an 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.
The combinations and the composition of the present disclosure may be used at the time of
5 sowing or transplanting.
The combinations and the compositions of the present disclosure can be applied to a locus by
the use of conventional ground sprayers, granule applicators, watering (drenching), drip
irrigation, spraying, atomizing, broadcasting, dusting, foaming, spreading-on, aerial methods
of spraying, aerial methods of application, methods utilizing application using modern
10 technologies such as, but not limited to, drones, robots, and by other conventional means
known to those skilled in the art.
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,
15 algicides, antifeedants, avicides, bactericides, bird repellents, chemosterilants, fungicides,
herbicide safeners, herbicides, insect attractants, insect repellents, insecticides, mammal 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
20 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 manner.
In a preferred embodiment, the other agrochemically active ingredient is selected from the
group consisting of acephate + cypermethrin, copper sulphate + mancozeb, propineb,
25 thiamethoxam, folcisteine, Foltron®, Wuxal®, imidacloprid, carbendazim + mancozeb,
spinetoram, and orthosilicic acid (OSA).
Another embodiment of the present disclosure provides a use of a biofertilizer combination
comprising an endophytic bacteria selected from Pseudomonas fluorescens, Bacillus subtilis,
Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture
30 thereof; and one or more mycorrhiza for improving plant health, yield, and vigor.
23

Another embodiment of the present disclosure provides a use of a biofertilizer combination
comprising an endophytic bacteria selected from Pseudomonas fluorescens deposited as
MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens
deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus
5 polymyxa deposited as MTCC 25609, or a mixture thereof; and one or more mycorrhiza for
improving plant health, yield, and vigor.
An embodiment of the present disclosure also provides a kit comprising a component
comprising an endophytic bacteria selected from Pseudomonas fluorescens, Bacillus subtilis,
Bacillus amyloliquefaciens, Pseudomonas putida, Paenibacillus polymyxa, or a mixture
10 thereof; and another component comprising one or more mycorrhiza for improving plant
health, yield, and vigor.
An embodiment of the present disclosure also provides a kit comprising a component
comprising an endophytic bacteria selected from Pseudomonas fluorescens deposited as
MTCC 25519, Bacillus subtilis deposited as MTCC 25520, Bacillus amyloliquefaciens
15 deposited as MTCC 25518, Pseudomonas putida deposited as MTCC 25607, Paenibacillus
polymyxa deposited as MTCC 25609, or a mixture thereof; and another component comprising one or more mycorrhiza for improving plant health, yield, and vigor.
In an embodiment, the kit may be split into multiple components of endophytic bacteria and
one or more mycorrhiza to be mixed together or applied together, jointly, or sequentially or
20 simultaneously.
Examples:
Example 1: Endophyte + mycorrhiza WP compositions and their efficacy on root length, shoot length and mycorrhizal colonization.
Nine WP compositions (Composition 1 – 9) comprising endophytes; endomycorrhizal species
25 Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita, Dentiscutata heterogama
and ectomycorrhizal species Pisolithus tinctorius and Piriformospora indica were prepared to be applied to tomato and red gram. A control WP composition (Composition 10) comprising only endomycorrhizal species Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita, Dentiscutata heterogama and ectomycorrhizal species Pisolithus tinctorius and
24

Piriformospora indica was prepared for comparison. Compositions 1 – 10 are provided in table 1.
Application rate of the composition – 100g/acre
Application time – at the time of transplanting
5 No. of applications – one at the time of transplanting
Efficacy of the compositions on root length, shoot length and mycorrhizal colonization was evaluated 45 days after application.
Table 1: WP compositions comprising endophytes, endomycorrhiza and ectomycorrhiza.

Composition 1 Composition 2 Composition 3
Dextrose – 32% Dextrose – 32% Dextrose – 32%
Lactose – 32% Lactose – 32% Lactose – 32%
Cellulose – 1% Cellulose – 1% Cellulose – 1%
Humic acid – 5% Humic acid – 5% Humic acid – 5%
Endomycorrhiza – 20% Endomycorrhiza – 20% Endomycorrhiza – 20%
Ectomycorrhiza – 5% Ectomycorrhiza – 5% Ectomycorrhiza – 5%
Pseudomonas fluorescens – 2.5% Pseudomonas fluorescens – Pseudomonas fluorescens – 10% 5%
Bacillus subtilis – 2.5% Bacillus amyloliquifaciens – 5%
Composition 4 Composition 5 Composition 6
Dextrose – 32% Dextrose – 32% Dextrose – 32%
Lactose – 32% Lactose – 32% Lactose – 32%
Cellulose – 1% Cellulose – 1% Cellulose – 1%
Humic acid – 5% Humic acid – 5% Humic acid – 5%
Endomycorrhiza – 20% Endomycorrhiza – 20% Endomycorrhiza – 20%
Ectomycorrhiza – 5% Ectomycorrhiza – 5% Ectomycorrhiza – 5%
Pseudomonas fluorescens – 10% Pseudomonas fluorescens – 5% Pseudomonas fluorescens – 5%
Bacillus subtilis – 5% Pseudomonas putida – 5%
Composition 7 Composition 8 Composition 9
Dextrose – 32% Dextrose – 32% Dextrose – 32%
Lactose – 32% Lactose – 32% Lactose – 32%
Cellulose – 1% Cellulose – 1% Cellulose – 1%
Humic acid – 5% Humic acid – 5% Humic acid – 5%
Endomycorrhiza – 20% Endomycorrhiza – 20% Endomycorrhiza – 20%
Ectomycorrhiza – 5% Ectomycorrhiza – 5% Ectomycorrhiza – 5%
Pseudomonas putida – 5% Pseudomonas putida – 10% Pseudomonas fluorescens – 5%

Bacillus amyloliquifaciens – 5% Bacillus subtilis – 5%
Composition 10 (without endophytes)
Dextrose – 32%
Lactose – 32%
Cellulose – 1%
Humic acid – 5%
Endomycorrhiza – 20%
Ectomycorrhiza – 5%
Table 2: Efficacy of compositions of Table 1 on root length, shoot length and mycorrhizal colonization in tomato and red gram.

Compositions Root length (cm) Shoot length (cm) Mycorrhizal colonization (%)

Tomato Red gram Tomato Red gram Tomato Red gram
Composition 1 37.82 79.82 80.62 120.56 94 95
Composition 2 36.99 78.37 80.57 129.48 93 94
Composition 3 37.32 78.82 79.34 123.57 92 95
Composition 4 37.52 78.05 79.99 126.46 93 95
Composition 5 37.63 79.90 79.45 120.35 91 93
Composition 6 37.15 78.17 79.35 120.05 91 91
Composition 7 38.45 78.09 79.10 122.05 95 95
Composition 8 36.97 78.42 80.25 120.35 95 95
Composition 9 39.26 80.35 82.32 130.95 98 99
Composition 10 36.23 77.46 78.35 119.25 89 90.5
5 The above trial demonstrates a significant improvement in mycorrhizal colonization and shoot
and root length in red gram and tomato when endophytes are combined with mycorrhiza in a composition.
Example 2: Endophyte + mycorrhiza granular compositions and their efficacy on root length, shoot length and mycorrhizal colonization.
10 Nine granule compositions (Composition 11 – 19) comprising endophytes; endomycorrhizal
species Rhizophagus irregularis, Rhizophagus clarus, Gigaspora margarita, Dentiscutata heterogama and ectomycorrhizal species Pisolithus tinctorius and Piriformospora indica were prepared to be applied to tomato, chili and brinjal. A control granule composition (Composition 20) comprising only endomycorrhizal species Rhizophagus irregularis, Rhizophagus clarus,
15 Gigaspora margarita, Dentiscutata heterogama and ectomycorrhizal species Pisolithus

tinctorius and Piriformospora indica was prepared for comparison. Compositions 11 – 20 are provided in table 3.
Application rate of the composition – 100g/acre
Application time – at the time of transplanting
5 No. of applications – one at the time of transplanting
Efficacy of the compositions on root length, shoot length and mycorrhizal colonization was evaluated 45 days after application.
Table 3: Granular compositions comprising endophytes, endomycorrhiza and ectomycorrhiza.

Composition 11 Composition 12 Composition 13
Bentonite granules – 86% Bentonite granules – 86% Bentonite granules – 86%
Dodonaea viscosa extract – 1.0% Dodonaea viscosa extract – 1.0% Dodonaea viscosa extract – 1.0%
Amino acid – 2.5% Amino acid – 2.5% Amino acid – 2.5%
Humic acid – 3.5% Humic acid – 3.5% Humic acid – 3.5%
Endomycorrhiza – 4.0% Endomycorrhiza – 4.0% Endomycorrhiza – 4.0%
Ectomycorrhiza – 1.5% Ectomycorrhiza – 1.5% Ectomycorrhiza – 1.5%
Bacillus amyloliquifacies – 0.75%. Pseudomonas fluorescens – 1.5% Pseudomonas fluorescens – 0.75%
Bacillus subtilis – 0.75% Bacillus amyloliquifaciens – 0.75%

Composition 14 Composition 15 Composition 16
Bentonite granules – 86% Bentonite granules – 86% Bentonite granules – 86%
Dodonaea viscosa extract – 1.0% Dodonaea viscosa extract – 1.0% Dodonaea viscosa extract – 1.0%
Amino acid – 2.5% Amino acid – 2.5% Amino acid – 2.5%
Humic acid – 3.5% Humic acid – 3.5% Humic acid – 3.5%
Endomycorrhiza – 4.0% Endomycorrhiza – 4.0% Endomycorrhiza – 4.0%
Ectomycorrhiza – 1.5% Ectomycorrhiza – 1.5% Ectomycorrhiza – 1.5%
Pseudomonas fluorescens – 0.75% Pseudomonas putida – 0.75% Pseudomonas putida – 0.75%
Pseudomonas putida – 0.75% Bacillus amyloliquifaciens – 0.75% Peanibacillus sp. – 0.75%
Composition 17 Composition 18 Composition 19
Bentonite granules – 86% Bentonite granules – 86% Bentonite granules – 86%
Dodonaea viscosa extract – 1.0% Dodonaea viscosa extract – 1.0% Dodonaea viscosa extract – 1.0%
Amino acid – 2.5% Amino acid – 2.5% Amino acid – 2.5%
Humic acid – 3.5% Humic acid – 3.5% Humic acid – 3.5%

Endomycorrhiza – 4.0% Endomycorrhiza – 4.0% Endomycorrhiza – 4.0%
Ectomycorrhiza – 1.5% Ectomycorrhiza – 1.5% Ectomycorrhiza – 1.5%
Pseudomonas fluorescens – Paenibacillus sp. – 0.75% 0.75% Paenibacillus sp. – 1.5%
Bacillus subtilis – 0.75% Bacillus amyloliquifaciens – 0.75%
Composition 20 (without endophytes)
Bentonite granules – 87.5%
Dodonaea viscosa extract – 1.0%
Amino acid – 2.5%
Humic acid – 3.5%
Endomycorrhiza – 4.0%
Ectomycorrhiza – 1.5%
Table 4: Efficacy of compositions of Table 3 on root length, shoot length and mycorrhizal colonization in tomato, chili and brinjal.

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

Tomato Chilli Brinjal Tomato Chilli Brinjal Tomato Chilli Brinjal
Composition 11 34.29 32.57 26.98 77.67 60.32 77.34 93 94 92.5
Composition 12 33.76 31.95 25.01 77.15 59.95 75.15 94 95 94
Composition 13 33.29 31.67 25.16 75.55 60.59 74.25 91 93 93.5
Composition 14 35.35 33.32 29.34 79.00 60.52 75.32 92 94.5 94
Composition 15 36.52 33.95 27.98 76.28 62.15 73.26 91.5 93 95
Composition 16 34.83 32.10 25.33 75.95 61.56 76.95 92.5 94 94
Composition 17 38.56 34.68 31.23 80.98 63.99 80.16 95.5 96 96.5
Composition 18 35.26 31.56 27.44 78.55 60.32 75.98 91.5 93 93
Composition 19 34.75 32.37 24.98 77.95 60.95 77.95 92.5 94 93
Composition 20 33.09 31.17 24.32 75.45 60.05 73.15 89.5 90.5 89
5 The above trial demonstrates a significant improvement in mycorrhizal colonization and shoot
and root length in chili, brinjal and tomato when endophytes are combined with mycorrhiza in a composition.

We Claim,
1. A biofertilizer combination comprising an endophytic bacteria selected
from Pseudomonas fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens,
Pseudomonas putida, Paenibacillus polymyxa, or a mixture thereof; and one or more
5 mycorrhiza.
2. The biofertilizer combination of claim 1, wherein the Pseudomonas fluorescens is
deposited as MTCC 25519, Bacillus subtilis is deposited as MTCC 25520, Bacillus
amyloliquefaciens is deposited as MTCC 25518, Pseudomonas putida is deposited as
MTCC 25607, Paenibacillus polymyxa is deposited as MTCC 25609.
10 3. The biofertilizer combination of claim 1, wherein the combination comprises atleast two
endophytic bacteria.
4. The biofertilizer combination of claim 1, wherein the mycorrhiza is endomycorrhiza,
ectomycorrhiza or combinations thereof.
5. The biofertilizer combination of claim 4, wherein the mycorrhiza comprises
15 mycorrhizal propagules selected from propagules of endomycorrhiza, propagules of
ectomycorrhiza or combinations thereof.
6. The biofertilizer combination of claim 5, wherein the mycorrhizal propagules comprise
propagules of endomycorrhiza selected from a group consisting of Glomus genera,
Entrophospora, Funneliformis, Rhizophagus, Septoglomus, Acaulospora, Archaeospora,
20 Paraglomus, Gigaspora, Pacispora, Sclerocystis, Dentiscutata, Scutellospora, and
combinations thereof.
7. The biofertilizer combination of claim 6, wherein the mycorrhizal propagules comprise
propagules of endomycorrhiza selected from a group consisting of Rhizophagus
irregularis, Rhizophagus clarus, Gigaspora margarita, Scutellospora
25 heterogama, Glomus mossae, Septoglomus viscosum, Funneliformis mossae, and
combinations thereof.
8. The biofertilizer combination of claim 5, wherein the mycorrhizal propagules comprise
propagules of ectomycorrhiza selected from Laccaria, Pisolithus, Piriformospora,
Scleroderma, Rhizopogon, and combinations thereof.

9. The biofertilizer combination of claim 8, wherein the mycorrhizal propagules comprise propagules of ectomycorrhiza selected from Piriformospora indica, Laccaria bicolor, Laccaria lacata, Rhizopogon roseolus, Laccaria proxima, Pisolithus arhizus, Pisolithus tinctorius and combinations thereof.
5 10. The biofertilizer combination of claim 1, the endophytic bacteria and the mycorrhiza
are admixed in a ratio of 1:100 to 100:1.
11. A biofertilizer composition comprising the combination of claim 1.
12. A biofertilizer composition of claim 11, wherein the endophytic bacteria are present in an amount ranging from about 0.1% to about 50% by weight of the total composition.
10 13. A biofertilizer composition of claim 11, wherein the composition comprises
endomycorrhiza, ectomycorrhiza or a combination thereof in an amount ranging from about 0.1% to about 90% by weight of the composition.
14. A biofertilizer composition of claim 11, wherein the composition further comprises one or more agriculturally acceptable excipient.
15 15. A biofertilizer composition of claim 11, wherein the composition further comprises a
plant extract.
16. A biofertilizer composition of claim 11, wherein the composition is formulated into a
granule, water soluble powder or a wettable powder.
17. A method of improving plant health, yield and vigor, wherein said method comprises
20 contacting a plant or a part thereof, a plant propagation material, a site or medium of plant
growth, or a locus with a biofertilizer combination of claim 1, or a biofertilizer composition comprising said combination.
18. The biofertilizer combination of claim 1, wherein said combinations or a composition
comprising said combination is mixed or applied in conjunction with other agrochemically
25 active ingredients selected from fertilizers, micronutrients, acaricides, algicides,
antifeedants, avicides, bactericides, bird repellents, chemosterilants, fungicides, herbicide safeners, herbicides, insect attractants, insect repellents, insecticides, mammal repellents, mating disruptors, molluscicides, nematicides, plant activators, plant-growth regulators,

rodenticides, synergists, virucides, derivatives thereof, biological control agents or mixtures thereof.