NZ614079B2 - Microbial inoculants and fertilizer compositions comprising the same - Google Patents
Microbial inoculants and fertilizer compositions comprising the same Download PDFInfo
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- NZ614079B2 NZ614079B2 NZ614079A NZ61407912A NZ614079B2 NZ 614079 B2 NZ614079 B2 NZ 614079B2 NZ 614079 A NZ614079 A NZ 614079A NZ 61407912 A NZ61407912 A NZ 61407912A NZ 614079 B2 NZ614079 B2 NZ 614079B2
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- lactobacillus
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- 230000001743 silencing Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000004856 soil analysis Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 235000020354 squash Nutrition 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000001131 transforming Effects 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
- 230000003442 weekly Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G2/00—Vegetative propagation
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/02—Other organic fertilisers from peat, brown coal, and similar vegetable deposits
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/90—Apparatus therefor
- C05F17/921—Devices in which the material is conveyed essentially horizontally between inlet and discharge means
- C05F17/939—Means for mixing or moving with predetermined or fixed paths, e.g. rails or cables
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/02—Acetobacter
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/72—Candida
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/141—Feedstock
- Y02P20/145—Feedstock the feedstock being materials of biological origin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
Disclosed herein are microbial inoculants for use in increasing plant growth, plant productivity and/or soil quality, comprising strains of one or more bacterial species selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapi and Lactobacillus zeae. Optionally the microbial inoculants also comprise a strain of Acetobacter fabarum and/or a strain of Candida ethanolica. Also provided are fertilizer compositions comprising said microbial inoculants. obial inoculants also comprise a strain of Acetobacter fabarum and/or a strain of Candida ethanolica. Also provided are fertilizer compositions comprising said microbial inoculants.
Description
Microbial inoculants and fertilizer compositions comprising the
same
Field of the Art
The present sure relates lly to microbial inoculants, particularly for use
as izers, comprising one or more microbial species or strains as described herein, a d
to fertilizer compositions comprising such sms. The disclosure also relates to
methods of promoting plant growth, sing availability of nutrients in the soil and
remediating degraded soils and pastures using ial inoculants and fertilizer
compositions of the present disclosure.
Background
The use of fertilizers to enhance plant and crop production and overcome poor soil
quality is widespread. Most commonly employed commercially available fertilizers are
inorganic chemical fertilizers. Such chemical fertilizers can be expensive to produce, can
be hazardous to use and are often associated with environmentally damaging
consequences, such as nitrate contamination in run off and ground water. Environmental
sustainability can be promoted by limiting the use of al fertilizers.
Fertilizer compositions comprising microorganisms (so-called rtilizers") are
increasingly considered as atives to tional al fertilizers. The y of
specific bacterial species to promote plant growth has long been recognised. For example,
nitrogen-fixing bacteria such as Rhizobium species provide plants with essential
nitrogenous compounds. Species of Azotobacter and Azospirillum have also been shown
to promote plant growth and increase crop yield, promoting the accumulation of nutrients
in . However bacteria of these genera are often unable to compete effectively with
native soil and plant flora, thereby requiring the application of impractically large volumes
of inoculum. Various Bacillus and Pseudomonas species have also found application in
microbial-based fertilizers.
To date, biofertilizers have typically met with limited success, often not proving to
be efficacious under real farming conditions. There remains a need for improved
microbial-based fertilizers that are effective in providing nts for plant growth and are
environmentally safe and nonhazardous.
Summary of the Disclosure
A first aspect of the t disclosure provides a microbial inoculant for use in
increasing plant growth, plant productivity and/or soil quality, comprising s of one or
more bacterial species selected from Lactobacillus parafarraginis, Lactobacillus buchneri,
Lactobacillus rapi and acillus zeae.
In a particular ments the inoculant comprises two of said acillus
species, three of said Lactobacillus species or all of said Lactobacillus species. The
ant may represent a symbiotic combination of two or more or three or more of said
Lactobacillus species.
The Lactobacillus parafarraginis strain may be Lactobacillus parafarraginis Lpl8.
In a particular embodiment the Lactobacillus parafarraginis strain is Lactobacillus
parafarraginis Lpl8 deposited with National Measurement Institute, Australia on 27
October 20 1 under Accession Number V1 /022945 .
The Lactobacillus buchneri strain may be Lactobacillus buchneri Lb23. In a
particular embodiment the Lactobacillus buchneri strain is Lactobacillus buchneri Lb23
deposited with National Measurement Institute, lia on 27 October 201 1 under
Accession Number VI 1/022946.
The Lactobacillus rapi strain may be Lactobacillus rapi Lr24. In a particular
embodiment the Lactobacillus rapi strain is Lactobacillus rapi Lr24 ted with
National Measurement Institute, Australia on 27 October 201 1 under Accession Number
VI 1/022947.
The Lactobacillus zeae strain may be Lactobacillus zeae Lz26. In a particular
ment the Lactobacillus zeae strain is Lactobacillus zeae Lz26 deposited with
National ement ute, Australia on 27 October 201 under Accession Number
V 1/022948.
[001 ] An inoculant of the first aspect may further comprise a strain of Acetobacter
fabarum. The acter fabarum strain may be Acetobacter fabarum Afl5. In a
particular embodiment the Acetobacter fabarum strain is Acetobacter fabarum Afl5
deposited with the National Measurement Institute, lia on 27 October 201 1 under
ion Number VI 1/022943.
An inoculant of the first aspect may further comprise a yeast. The yeast may be a
strain of Candida ethanolica. The Candida ethanolica strain may be Candida ethanolica
Ce31. In a particular embodiment the Candida ethanolica strain is Candida ethanolica
Ce31 deposited with the National ement Institute, Australia on 27 r 2011
under Accession Number V I 44.
One or more of the strains in the inoculant may be encapsulated. Where multiple
strains are encapsulated, the strains may be individually ' encapsulated or combined in a
single ulation.
A second aspect of the present disclosure provides a microbial inoculant
comprising at least one Lactobacillus species, at least one Acetobacter species and at least
one Candida species.
In a particular embodiment the at least one Lactobacillus species is selected from
Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapt and Lactobacillus
zeae. In a further particular embodiment, the microbial ant comprises at least one
strain of each of said Lactobacillus species. In a further particular embodiment, the
Lactobacillus parafarraginis is strain Lpl8 (deposited under Accession Number
V I 1/022945), Lactobacillus buchneri is strain Lb23 (deposited under Accession Number
V 1/022946), Lactobacillus rapi is strain Lr24 (deposited under Accession Number
VI 1/022947) and Lactobacillus zeae is strain Lz26 (deposited under Accession Number
V I 1/022948).
In a particular embodiment the at least one Acetobacter s is Acetobacter
fabarum. In a further ular embodiment the Acetobacter fabarum is Afl5 (deposited
under Accession Number V I 1/022943).
In a particular embodiment the at least one Candida species is Candida ethanolica.
In a further particular embodiment the Candida ethanolica is Ce31 (deposited under
Accession Number VI 1/022944).
A third aspect of the present disclosure provides a microbial inoculant comprising
at least one bacterial strain ed from Lactobacillus rraginis Lpl 8, Lactobacillus
buchneri Lb23, Lactobacillus rapi Lr24 and Lactobacillus zeae Lz26.
An inoculant of the third aspect optionally further comprises Acetobacter fabarum
Afl5 and/or Candida ethanolica Ce31.
An inoculant of the first, second or third aspect may be used as a fertilizer.
A fourth aspect of the present disclosure provides a fertilizer composition
comprising a microbial inoculant of the first, second or third aspect. The fertilizer
ition may optionally se one or more additional components such as organic
material, humic nces, penetrants, utrients, micronutrients and other soil
and/or plant additives.
A fifth aspect of the present disclosure provides a method for increasing plant
growth and/or productivity, the method comprising applying to the plant, plant seeds or to
the soil in which the plant or plant seeds are grown an effective amount of a microbial
inoculant of the first, second or third aspect or a fertilizer composition of the fourth aspect.
A sixth aspect of the present disclosure provides a method for improving soil
quality, the method comprising applying to soil or to the plants or plant seeds in said soil
an effective amount of a microbial inoculant of the first, second or third aspect or a
izer ition of the fourth aspect.
In accordance with the above aspects the plant may be, for example, a pasture
plant, crop plant (including fruit and vegetable ) or ornamental plant. The crop may
be, for e, any human or animal food crop or crop for use as fuel or for
pharmaceutical production. The food crop may be, for example, a fruit, vegetable, nut,
seed or grain.
A seventh aspect of the present disclosure provides a method for remediating
degraded soil or pasture, the method comprising applying to the soil or pasture an effective
amount of a microbial inoculant of the first, second or third aspect or a fertilizer
composition of the fourth aspect.
Brief Description of the Drawings
Aspects and embodiments of the present disclosure are described , by way of
non-limiting example only, with reference to the following gs.
Figure 1. Root pment in tick bean plants, treated as described in Example 5.
A, control group; B, T40 treatment group; C, SGL40 treatment group; D, T25%GL40
ent group; E, GL40 treatment group.
Figure 2. Average rate of change of growth (height) of tomato plants over a 20
day treatment period in three different soils (A-C), treated as described in Example 6.
Squares ent IMP Bio treated seedlings, diamonds represent FlowPhos treated
ngs, triangles ent IMP Bio plus FlowPhos treated seedlings, crosses ('c')
represent ted (water only) seedlings.
Figure 3. Comparison of plant height, foliage size and root development in tomato
seedlings, treated as described in Example 6. GreatLand = IMP Bio treated seedling.
Figure 4. Comparison of vegetative growth (and density of growth) of strawberry
plants, treated as described in Example 8. A, conventional fertilizer treated plants after 3
months. B, IMP Bio treated plants after 3 months.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by those of ry skill in the art to which the
disclosure belongs. Although any methods and materials similar or lent to those
described herein can be used in the practice or testing of the present disclosure, typical
methods and materials are described.
The articles "a" and "an" are used herein to refer to one or to more than one (i.e. ~ o
at least one) of the grammatical object of the article. By way of example, "an element"
means one element or more than one element.
In the context of this specification, the term "about," is understood to refer to
range of numbers that a person of skill in the art would consider lent to the recited
value in the context of achieving the same function or result.
Throughout this specification and the claims which follow, unless the context
requires otherwise, the word "comprise", and variations such as "comprises" or
"comprising", will be understood to imply the inclusion of a stated integer or step or group
of integers or steps but not the exclusion of any other integer or step or group of integers or
steps.
The term "plant productivity" as used herein refers to any aspect of growth or
development of a plant, that is a reason for which the plant is grown. Thus, for purposes of
the present sure, improved or increased plant tivity refers broadly to
improvements in biomass or yield of leaves, stems, grain, fruit, vegetables, s, or
other plant parts harvested or used for various purposes, and improvements in growth of
plant parts, including stems, leaves and roots. For example, when referring to food crops,
such as grains, fruits or vegetables, plant tivity may refer to the yield of grain, fruit,
bles or seeds harvested from a particular crop. For crops such as e, plant
productivity may refer to growth rate, plant density or the extent of groundcover. "Plant
growth" refers to the growth of any plant part, including stems, leaves and roots. Growth
may refer to the rate of growth of any one of these plant parts.
The term "yield" refers to the amount of produced biological material and may be
used interchangeably with "biomass". For crop plants, "yield" may also mean the amount
of harvested al per unit of production or per area (e.g. hectare). Yield may be
defined in terms of quantity or quality. The harvested material may vary from crop to
crop, for e, it may be seeds, above-ground s, below-ground s (e.g.
potatoes), roots, fruits, or any other part of the plant which is of economic value. "Yield"
also encompasses yield ity of the . "Yield" also asses yield potential,
which is the m obtainable yield under optimal growth conditions. Yield may be
dependent on a number of yield components, which may be monitored by certain
parameters. These parameters are well known to persons d in the art and vary from
crop to crop. For example, breeders are well aware of the specific yield components and
the corresponding parameters for the crop they are aiming to improve. For e, key
yield parameters for potato include tuber weight, number of tubers, and number of stems
per plant.
By "improving soil quality" is meant increasing the amount and/or bility of
nutrients required by, or beneficial to plants, for growth. By way of example only, such
nutrients include en, phosphorous, potassium, copper, zinc, boron and molybdenum.
Also encompassed by the term "improving soil quality" is reducing or minimising the
amount of an element that may be detrimental to plant growth or development such as, for
example iron and manganese. Thus, improving soil quality by use of microbial inoculants
and fertilizer compositions of the present disclosure thereby assists and promotes the
growth of plants in the soil.
The term "remediating" as used herein in relation to degraded pasture or soil refers
to the improvement in plant nutrient content in the soil to facilitate improved plant growth
and/or yield. Degraded pasture includes overgrazed pasture.
As used herein, the term "effective amount" refers to an amount of microbial
inoculant or fertilizer composition applied to a given area of soil or vegetation that is
ient to effect one or more beneficial or desired outcomes, for example, in terms of
plant growth rates, crop yields, or nutrient availability in the soil. An "effective amount"
can be provided in one or more administrations. The exact amount required will vary
depending on factors such as the identity and number of individual strains employed, the
plant species being treated, the nature and condition of the soil to be treated, the exact
nature of the ial inoculant or fertilizer composition to be applied, the form in which
the inoculant or fertilizer is applied and the means by which it is applied, and the stage of
the plant growing season during which application takes place. Thus, it is not le to
specify an exact "effective amount". r, for any given case, an appropriate
"effective " may be determined by one of ordinary skill in the art using only routine
experimentation.
The term "crop" as used herein refers to any plant grown to be harvested or used
for any economic purpose, including for example human foods, livestock fodder, fuel or
pharmaceutical production (e.g. poppies).
The term "optionally" is used herein to mean that the uently described
feature may or may not be present or that the subsequently bed event or circumstance
may or may not occur. Hence the specification will be understood to include and
encompass embodiments in which the feature is present and embodiments in which the
feature is not present, and embodiments in which the event or circumstance occurs as well
as embodiments in which it does not.
In accordance with the present disclosure, novel microbial ants and microbial
fertilizer compositions are presented which find application in increasing plant
productivity and improving soil quality. In particular embodiments the microbial species
present in the microbial inoculant or fertilizer composition provide a symbiotic
combination of sms.
In the broadest embodiments, a microbial ant of the present disclosure
comprises strains of one or more bacterial Lactobacillus species. The Lactobacillus
species may be selected from Lactobacillus parafarraginis, Lactobacillus buchneri,
Lactobacillus rapt and Lactobacillus zeae. The inoculant may further comprise at least
one Acetobacter s and at least one a species.
The Lactobacillus parafarraginis strain may be Lactobacillus parafarraginis Lpl8.
In a particular embodiment the Lactobacillus parafarraginis strain is acillus
parafarraginis Lpl8 deposited with National Measurement Institute, Australia on 27
r 201 1 under Accession Number V 1/022945. The Lactobacillus buchneri strain
may be Lactobacillus buchneri Lb23. In a particular embodiment Lactobacillus
buchneri strain is Lactobacillus buchneri Lb23 deposited with National Measurement
Institute, Australia on 27 October 2011 under Accession Number V I 1/022946. The
Lactobacillus rapi strain may be Lactobacillus rapi Lr24. In a particular embodiment the
acillus rapi strain is Lactobacillus rapi Lr24 deposited with National Measurement
ute, Australia on 27 October 201 under ion Number V 47. The
Lactobacillus zeae strain may be acillus zeae Lz26. In a particular embodiment the
Lactobacillus zeae strain is Lactobacillus zeae Lz26 deposited with National Measurement
Institute, Australia on 27 October 201 1 under Accession Number V 1/022948.
The inoculant may further comprise a strain of Acetobacter fabarum. The
Acetobacterfabarum strain may be Acetobacterfabarum Afl5. In a particular ment
the Acetobacter fabarum strain is Acetobacter fabarum Afl5 deposited with the National
Measurement Institute, Australia on 27 October 201 1 under Accession Number
VI 1/022943.
The inoculant may further comprise a yeast. The yeast may be a strain of Candida
ethanolica. The Candida lica strain may be Candida ethanolica Ce31. In a
particular embodiment the Candida ethanolica strain is Candida ethanolica Ce31
deposited with the National Measurement Institute, Australia on 27 October 2011 under
Accession Number VI 1/022944.
The concentrations of each microbial strain to be added to ial inoculants and
fertilizer compositions as disclosed herein will depend on a variety of factors including the
identity and number of individual strains ed, the plant species being treated, the
nature and condition of the soil to be treated, the exact nature of the microbial inoculant or
fertilizer composition to be applied, the form in which the inoculant or fertilizer is applied
and the means by which it is applied, and the stage of the plant growing season during
which application takes place. For any given case, appropriate concentrations may be
determined by one of ordinary skill in. the art using only routine experimentation. By way
of example only, the concentration of each strain present in the ant or fertilizer
composition may be from about 1 x 102 cfu/ml to about 1 x 10 10 cfu/ml, and may be about
1 x 103 cfu ml, about 2.5 x 103 , about 5 x 103 cfu/ml, 1 x 104 cfu/ml, about 2.5 x 104
cfu/ml, about 5 x 104 cfu/ml, 1 x 10 cfu/ml, about 2.5 x 10 , about 5 x 10 cfu/ml, 1
x 106 cfu/ml, about 2.5 x 106 cfu/ml, about 5 x 106 , 1 x 107 cfu/ml, about 2.5 x 107
cfu/ml, about 5 x 107 cfu/ml, 1 x 10 cfu/ml, about 2.5 x 08 cfu/ml, about 5 x 108 cfu/ml, 1
x 109 cfu/ml, about 2.5 x 109 cfu/ml, or about 5 x 109 cfu/ml. In particular exemplary
ments the final concentration of the Lactobacillus strains is about 2.5 x 10s cfu/ml,
the final concentration of Acetobacter fabarum may be about 1 x 106 cfu/ml and the final
concentration of Candida ethanolica may be about 1 x 10 cfu/ml.
Also plated by the present disclosure are variants of the microbial strains
described herein. As used herein, the term "variant" refers to both naturally occurring and
specifically developed variants or s of the microbial s disclosed and
exemplified herein. Variants may or may not have the same identifying biological
characteristics of the specific strains exemplified herein, provided they share similar
advantageous properties in terms of promoting plant growth and providing nutrients for
plant growth in the soil. Illustrative examples of suitable methods for preparing variants of
the microbial strains exemplified herein include, but are not d to, gene ation
techniques such as those ed by inseftional elements or transposons or by
homologous recombination, other recombinant DNA techniques for modifying, inserting,
deleting, activating or silencing genes, intraspecific protoplast , mutagenesis by
irradiation with ultraviolet light or X-rays, or by treatment with a chemical mutagen such
as nitrosoguanidine, methylmethane sulfonate, nitrogen mustard and the like, and
iophage-mediated transduction. Suitable and applicable methods are well known in
the art and are described, for example, in J . H . Miller, Experiments in lar Genetics,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A
Short Course in Bacterial Genetics, Cold Spring Harbor tory Press, Cold Spring
Harbor, N.Y. (1992); and J. Sambrook, D. Russell, Molecular Cloning: A Laboratory
Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001),
inter alia.
Also assed by the term "variant" as used herein are microbial strains
phylogenetically closely related to strains disclosed herein and strains possessing
substantial sequence identity with the strains disclosed herein at one or more
phylogenetically informative markers such as rRNA genes, elongation and initiation factor
genes, RNA polymerase subunit genes, DNA gyrase genes, heat shock protein genes and
recA genes. For example, the 16S rRNA genes of a "variant" strain as contemplated herein
may share about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% sequence identity with a strain disclosed herein.
Microbial inoculants and fertilizer compositions of the present disclosure may
optionally further comprise one or more additional microbial organisms, for example,
additional agronomically beneficial microorganisms. Such agronomically beneficial
microorganisms may act in synergy or t with, or otherwise cooperate with the
organisms of the t disclosure in the inoculant or fertilizer. Examples of
agronomically beneficial microorganisms include Bacillus sp., Pseudomonas sp.,
Rhizobium sp., Azospirillum sp., Azotobacter sp., phototrophic and cellulose degrading
ia, Clostridium sp., Trichoderma sp. and the like. Those skilled in the art will
appreciate that this list is merely exemplary only, and is not limited by reference to the
specific examples here provided.
In the soil nment, inoculated bacteria can find survival ult among
naturally occurring itor and predator organisms. To aid in survival of
microorganisms present in microbial inoculants and fertilizer itions of the present
disclosure upon application in the environment, one or more of the s may be
encapsulated in, for example, a suitable polymeric matrix. In one example, encapsulation
may se alginate beads such as has been described by Young et al, 2006,
Encapsulation of plant growth-promoting bacteria in alginate beads enriched with humic
acid, Biotechnology and Bioengineering 95:76-83, the disclosure of which is incorporated
herein by reference in its ty. Those skilled in the art will appreciate that any suitable
encapsulation material or matrix may be used. Encapsulation may be achieved using
methods and techniques known to those skilled in the art. Encapsulated rganisms
can include nutrients or other components of the inoculant or fertilizer composition in
addition to the microorganisms.
Those d in the art will appreciate that any plant may benefit from the
application of microbial inoculants and fertilizer compositions of the present disclosure to
soil, seeds and/or vegetation. Particular embodiments are employed to aid the growth,
development, yield or productivity of crops and pastures or other plants of economic value,
including ntals and plants grown for oils or biofuel. The crop plant may be, for
example, a food crop (for humans or other animals) such as any fruit, vegetable, nut, seed
or grain producing plant. Exemplary crop plants include, but are not limited to, tubers and
other below-ground vegetables (such as potatoes, beetroots, radishes, carrots, onions, etc.),
ground-growing or vine vegetables (such as pumpkin and other members of the squash
family, beans, peas, asparagus, etc.), leaf vegetables (such as lettuces, chard, spinach,
alfalfa, etc.), other vegetables (such as tomatoes, brassica including broccoli, avocadoes,
etc.), fruits (such as berries, , stone fruits including nectarines and peaches, tropical
fruits including mangoes and s, apples, pears, mandarins, oranges, mandarins, kiwi
fruit, coconut, etc.), cereals (such as rice, maize, wheat, barley, millet, oats, rye etc.), nuts
(such as macadamia nuts, peanuts, brazil nuts, hazel nuts, walnuts, almonds, etc.), and
other economically valuable crops and plants (such as sugar cane, soybeans, sunflower,
, sorghum, pastures, turf grass, etc).
Microbial inoculants and fertilizer compositions of the present disclosure may be
applied directly to plants, plant parts (such as foliage) or seeds, or alternatively may be
applied to soil in which the plants are growing or to be grown or in which seeds have been
or are to be sown. Application may be by any suitable means and may be on any suitable
scale. For example, application may comprise g, spreading or spraying, including
broad scale or bulk spreading or ng, g of seeds before planting, and/or
drenching of seeds after planting or seedlings. Those skilled in the art will appreciate that
le means of application may be used in combination (for example soaking of seeds
prior to ng followed by drenching of planted seeds and/or application to seedlings or
mature plants). Seeds, ngs or mature plants may be treated as many times as
appropriate. The number of applications ed can readily be determined by those
skilled in the art depending on, for example, the plant in question, the stage of
development of the plant at which treatment is initiated, the state of health of the plant, the
growth, environmental and/or climatic conditions in which the plant is grown and the
purpose for which the plant is grown. For example, in the case of flowering crops such as
tomatoes, it may be desirable to apply the microbial inoculant or fertilizer composition
once or more than once during the flowering .
Thus, in accordance with the present disclosure, microbial inoculants and fertilizer
/ ts as disclosed herein may be prepared in any suitable form depending on the means
by which the inoculant or izer composition is to be applied to the soil or to plant seeds
or vegetation. Suitable forms can include, for example, slurries, liquids, and solid forms.
Solid forms include powders, granules, larger particulate forms and pellets. Solid form
fertilizer particles can be encapsulated in water soluble coatings (for example dyed or
undyed gelatin spheres or capsules), extended release coatings, or by micro-encapsulation
to a free flowing powder using one or more of, for example, gelatin, polyvinyl alcohol,
ellulose, cellulose acetate phthalate, or styrene maleic anhydride. Liquids may
e aqueous ons and aqueous suspensions, and emulsifiable concentrates.
In order to achieve effective dispersion, on and/or conservation or stability
within the environment of inoculants and izer compositions disclosed herein, it may
be advantageous to formulate the inoculants and compositions with suitable carrier
components that aid sion, adhesion and conservation/stability. Suitable carriers will
be known to those skilled in the art and include, for example, chitosan, vermiculite,
compost, talc, milk powder, gels and the like.
Additional components may be incorporated into ants and fertilizer
compositions of the present disclosure, such as humic substances, trace elements, organic
material, penetrants, utrients, micronutrients and other soil and or plant additives.
Humus or humic substances that may be incorporated may include, but are not
limited to, humic acid derived from, for example oxidised lignite or leonardite, fulvic acid
and humates such as potassium humate.
Organic material added may include, but is not limited to, ids, animal
manure, compost or composted organic byproducts, activated sludge or processed animal-
or ble byproducts (including blood meal, feather meal, cottonseed meal, ocean kelp
meal, seaweed extract, fish emulsions and fish meal).
Penetrants include, but are not limited to, non-ionic wetting agents, detergent based
tants, silicones, and/or organo-silicones. le penetrants will be known to those
skilled in the art, non-limiting examples including polymeric polyoxyalkylenes, allinol,
nonoxynol, nol, oxycastrol, TRITON, TWEEN, Sylgard 309, Silwet L-77, and
Herbex (silicone/surfactant blend).
ary trace elements for inclusion in microbial inoculants and fertilizer
compositions are provided in Example 1. However those skilled in the art will recognise
that suitable trace elements are not limited thereto, and that any trace elements (natural or
synthetic) amy be employed.
Optional further soil and/or plant additives that can be added to inoculants and
fertilizer compositions of the t disclosure include, for example, water ng
agents such as zeolites, enzymes, plant growth hormones such as ellins, and pest
control agents such as acaracides, insecticides, fungicides and cides.
The reference in this ication to any prior publication (or information derived
from it), or to any matter which is known, is not, and should not be taken as an
acknowledgment or admission or any form of suggestion that that prior publication (or
information derived from it) or known matter forms part of the common l
knowledge in the field of endeavour to which this specification relates.
The present disclosure will now be described with reference to the following
ic examples, which should not be construed as in any way limiting the scope of the
invention.
Examples
The following examples are illustrative of the invention and should not be
construed as limiting in any way the general nature of the disclosure of the description
throughout this specification.
Example 1- Microbial strains
The following microbial strains were used in the production of a tilizer.
Lactobacillus parafarraginis Lpl8 was isolated from an environmental source.
Partial 16S rRNA sequencing indicated 100% similarity to acillus parafarraginis
AB 262735 which has a risk group of 1 (TRBA). When cultured on MRS media for 3 days
at 34°C, anaerobically, Lpl8 produces cream, round, slight sheen, convex, colony diameter
l-2mm (facultative be). Its microscopic appearance is Gram positive, non-motile,
short rods rectangular, mainly diploid. Lactobacillus parafarraginis Lpl8 was deposited
with the National Measurement Institute, Australia on 27 October 201 1 under Accession
Number V I 1/022945.
Lactobacillus buchneri Lb23 was isolated from an environmental . l
16S rRNA sequencing indicated 99% similarity to Lactobacillus buchneri AB 429368
which has a risk group of 1 . When cultured on MRS media for 4 days at 34°C,
anaerobically, Lb23 produces cream, shiny, , colony diameter l-2mm (facultative
anaerobe). Its microscopic appearance is Gram positive, non-motile, rods in chains.
Lactobacillus buchneri Lb23 was deposited with the National Measurement Institute,
Australia on 27 October 201 1 under Accession Number V I 1/022946.
Lactobacillus rapi Lr24 was isolated from an environmental source. Partial 16S
rRNA sequencing indicated 99% similarity to Lactobacillus rapi AB 366389 which has a
risk group of 1 (DSMZ). When cultured on MRS media for 4 days at 34°C, anaerobically,
Lr24 es cream, round, shiny colonies with a diameter of 0.5mm (facultative
anaerobe). Its microscopic appearance is Gram positive, non-motile, short rods single or
d. Lactobacillus rapi Lr24 was deposited with the National Measurement Institute,
Australia on 27 October 201 1under Accession Number VI 1/022947.
Lactobacillus zeae Lz26 was isolated from an nmental source. Partial 16S
rRNA cing indicated 99% similarity to Lactobacillus zeae AB .1 which has
a risk group of 1 (TRBA). When cultured on MRS media for 48 hours at 34°C,
anaerobically, Lz26 produces white, round, shiny, convex, colonies with a er of
lmm (facultative anaerobe). Its microscopic appearance is Gram positive, non-motile,
short rods almost coccoid, diploid and some chains. Lactobacillus zeae 2 6 was
deposited with the National Measurement ute, Australia on 27 October 201 1 under
Accession Number V I 1/022948.
Acetobacter fabarum Afl5 was isolated from an environmental source. Partial 16S
rRNA sequencing indicated 100% similarity to Acetobacter fabarum AM 905849 which
has a risk group of 1 (DSMZ). When ed on Malt extract media for 3 days at 34°C,
AF15 produces opaque, round, shiny, convex, colony diameter lmm (aerobic). Its
copic appearance is Gram negative, rods single or diploid. Acetobacter fabarum
Afl5 was ted with the National Measurement Institute, Australia on 27 October
201 1 under Accession Number V I 1/022943.
Candida ethanolica Ce31 was isolated from an nmental source. l 16S
rRNA cing indicated 89% similarity to Candida ethanolica AB534618. When
cultured on Malt extract media for 2 days at 34°C, Ce31 produces cream, flat, dull,
roundish, colony diameter 2-3mm (aerobic). Its microscopic appearance is budding, ovoid
yeast. Candida ethanolica Ce3 1 was deposited with the National Measurement Institute,
Australia on 27 r 201 1 under Accession Number V 1/022944.
Maintenance of cultures
30% glycerol stocks were made of each isolate and maintained at -80°C for longterm
culture storage. Short-term storage of the cultures are maintained at 4°C on agar
slopes (3 month storage) and on agar plates which are subcultured monthly. To maintain
the isolates original traits, a fresh plate is made from the -80°C stock following three plate
subcultures.
Inoculum and growth media
The Lactobacillus s were grown with or without air (I. rapi prefers
anaerobic) either in MRS broth (Difco) or on MRS agar plates depending on application.
The cultures were routinely grown for 2 days at a mesophilic temperature of 30-34°C. The
Acetobacter and Ethanolica strains are grown aerobically either in Malt extract broth
(Oxoid) or on Malt extract agar plates depending on application. The cultures are routinely
grown for 2 days at a mesophilic temperature of 30-34°C.
Fermenter 'seed'preparation
For individual strains, using a sterile nichrome wire a single colony is removed
from a fresh culture plate and transferred to a universal bottle containing 15mL of sterile
media. The bottle is securely placed in a g tor set at 30°C, 140rpm for 48hrs
(L.rapi is not shaken). After incubation a cloudy bacterial growth should be visible. 'Seed'
inoculation bottles are stored at 4°C until required (maximum 1 week).
Typically a 5% bacterial inoculation is required for a fermenter run. The stored
15ml culture seed is added to a Schott bottle containing a volume of e media which is
% of the total fermenter working volume. The culture is incubated and shaken in the same
way as the 15ml seed. Large scale automatic ters are used to grow pure es of
each isolate. There is an automatic feed of , antifoam and glucose. Typically the
temperature is ined at 30-34°C, pH 5.5 but the oxygen and agitation varies
depending on the microorganism.
Sample analysis
After each large scale culturing of an isolate a sample is aseptically withdrawn and
a viability count undertaken using 0 fold serial dilutions, performed in a laminar flow
hood. A wet slide is also prepared and purity observed using a phase contrast microscope
to double check for contaminants that may be present but unable to grow on the culture
media. After 48 hours the viability plates are d for a pure culture (same colony
morphology) and the colonies counted to produce a colony forming unit per ml (cfu/ml)
value. A Grams stain is also performed.
Example 2- Pasture trials
Field trials on pasture were conducted using a tlizer as sed herein, in
comparison to untreated pasture and pasture treated with conventional inorganic fertilizers.
The biofertilizer (hereinafter "IMP Bio") comprised the six microbial strains listed
in Example 1, at final concentrations of 2.5 x 10 c u ml for each of the Lactobacillus
strains, 1.0 x 10 cfu/ml for Candida ethanolica and 1.0 x 106 cfu/ml for Acetobacter
m. The strains were grown as described in Example 1 and mixed with 2% trace
elements, 0.3% humate (Soluble Humate, LawrieCo), 3% molasses and 0.1-0.2%
phosphoric acid. Phosphoric acid was added to the point where pH was in the range 3.8 to
4.0. The trace elements ent typically comprised the following (per 1,000L):
Table 1 Trace ts component of biofertilizer
Conventional inorganic fertilizers used as comparators were Spray Gro Liquid
Urea, DAP (diammonium phosphate), and :16:11 commercial NPK mix.
Sites for the pasture trials were ed based on ll levels, soil type, pasture
composition and past izing practices. The following locations in Tasmania were used:
Nabageena (high rainfall; rye grass, cocksfoot, Yorkshire fog and other grasses), Cuprona
(high rainfall; rye grass), West lle/Upper Burnie (high rainfall; rye ,
Connorville (dryland pasture; degraded) and Connorville (irrigated pasture; rye grass).
At each location, multiple 4 x 10m strips of pasture were prepared by mowing to a
height of 45mm (and removal of clipped plant material prior to fertilizing). At West
Moorville/Upper Burnie and Nabageena, IMP Bio was applied to replicate plots at 20L/ha,
30L/ha or 50L/ha, and 14:16:11 NPK mi was applied to replicate plots at 250kg/ha. At
West Moorville, DAP was also applied to replicate plots at 125kg/ha. At Cuprona, IMP
Bio was applied to replicate plots at 20L/ha, 30L/ha or 50L/ha, and Spray Gro Liquid Urea
was applied at 50L/ha. At Connorville, IMP Bio was d to replicate plots at 20L/ha,
30L/ha or 50L/ha, and DAP was applied to replicate plots at 125kg/ha. IMP Bio and
SprayGro Urea were applied as large ts through 2m backpack boom sprays in a
single pass. 14:16:11 NPK mix and DAP were applied by hand distribution. At each
location, ate control (unfertilized) plots were set aside.
Plant yield and leaf nutrient content were analysed 6-8 weeks after treatment.
Results for plant yield are shown in Table 2 below. These s indicate that the
IMP Bio fertilizer produced yields at least similar to, and in some cases superior to,
conventional inorganic fertilizers.
Table 2. Yield (kg/ha/day)
Plant material nutrient analysis was conducted, as shown in Table 3 below. Key
elements ed by, or beneficial to, the pasture for growth (such as nitrogen,
phosphorous, postassium, calcium, copper, zinc, boron, molybdenum) were present in
plant material from the IMP Bio treated plots at levels equivalent to or higher than those
plots d with the comparator conventional inorganic fertilizer, despite these nutrients
not being added in the IMP Bio fertilizer.
Table 3able 3
Nutrient Control . Conventional IMP Bio
' fertilizer. ‘ 30L/ha
Connorville irriated
Nigerian (8/22 .1. VVVVV _____
Phoghgrus (%) '
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Calcium (%)
Ma ; esium %
sodium..(?_/.9L_.W.......-.
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Nitrogen : horus ratio
fiitmrogen ssium ratio
...£3.11.rb.<11.1_=..1:11.t.£<_>.$sn ratio
Crude rotein %N x 6.25
21221.5.qu,,,,,,,,,,,,,,,,,,, 0.34 0-31
.......__ W--.9121_..........
s_s._1.n</.2.....................__. 2.26 ngzWW 211‘
341.91%)W, 0-25 024 4 0-24
............................ -W
CarEnLQ._.._..__ 419._._.WW44_1 43 9
Ca1cium_(%) 078
- WWWWWWWWWW95.9..............<1117W..-
Ma 411151124)...............................................11.1.2.4......._ 0120 ,
.. .: ......... 1111.8 __
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w........................ ......
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-.......... fl. '_‘‘‘‘‘‘
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Eflmem)_-w-._.,.w - 172 114 120
......................... ______
_Boronggp_m2 7.7 8.3
_____m ____1.9_mmmmm -.-,_ _.
M01 b69291 m 1-3 1.0
..........._ _ ....... ........;........111
Cobaltgmemwm ._.n.~.__9;1.................................. <0-‘1 <0-1
~~~~~~
Silicon $2221.) .m,~._‘__._._... 316 244 '
W_-__.. 35,8 .
Nitrossy Sumhugflio 7 0
, .,,__§.2.4.___M “WA?_____________ W.
gnihepgheflls ratio 5 3
...............ESMMMAJ......... W..-
M§9.i9£a_8§i1m§.tj9.m 41.7. ._._w_,_‘2_-§_M Vania!..............m
Carbonflsmiflitmgsyfiig..........................................Z82___;8_1.................-............26.43.........a
Crude n %Nx6.25 9.8 9.8 10.2
.Nitrogeqm 3 68
......... _ M. ‘ W W_MMWW...................,
- mmwgw.4239
__ _
Potassigm (%) _} __, 3:13"
Sul h95_§‘122_.~.._..,wwm “WEE?
Carboewgfyo) 436
- ‘ a........................ .............
Calcium (%) W,WW*051
Mamgigrfl’élmn..flm._~,._#______-.-m_024__._
Sodium,_,(%) __, , -026..........
00231922911.....................................1.3J..-”
2m ‘3me M25: -
Mm_mm,... ' -106 -m.........
.m)._m_____________ H.491...............
.,,.§9322.,:92m.2.......... l 6.2
................ ...........
M‘Mgbzbmmgpm) 0-3
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ML...“........... <0-1
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@3313 Phggshorpmggtio 4 9.5
_ “““““
Nitrogen : Potassium ratio 1.1 1.1 1.3
Carbon : Nitrogen ratio 11.8 12.3 11.9
Crude protein (%N x 6.25) 23.0 22.5 23.0
Example 3- Soil quality
To determine the effect of a biofertilizer as disclosed herein on soil quality, 2 x
150g of soil from a farm in Tasmania were each weighed into 2 x clean 150ml Schott
bottle. lOmls of a 1:10 dilution of IMP Bio fertilizer (see Examples 1 and 2) was dripped
over the top of the soil in one bottle and the lid replaced and incubated at 34°C for one
week. The second bottle had no biofertilizer added was incubated 34°C. The soil from
both bottles was analysed by nmental Analytical Laboratories (EAL, rn Cross
University Lismore, NSW) using standard soil testing methods.
The results for the one week treatment of soil with IMP Bio are summarised in
Table 4. Soil tests on the untreated ted sample are not shown as these were
substantially the same as the initial untreated soil test. It is clear from the soil tests on the
two treated samples that there is a marked difference in the soil after incubation with IMP
Bio. The second sample analysed, shows a general trend of increasing the levels of
available cations um, magnesium, potassium, sodium and all trace elements - zinc,
manganese, iron and copper) and ammonium nitrogen, while the total levels under the acid
extractions were slightly lower across all nutrients. Organic matter increased by 1%
(14.6% to 15.5%) between the samples dates. The overall decrease in total nts does
not appear to be significant.
There was a greater than three-fold se in ammonium nitrogen, although no
increase in nitrates. This indicates an increase in mineralisation of en from the
organic en pool, and may be linked to the transformation of organic material, the
level of which in this soil is particularly high. This could also indicate nitrogen fixation.
Table 4
Example 4 - Potato trials
A field trial was conducted in which Bondi y potatoes were treated with the
IMP Bio biofertilizer (see Example 2) at planting. The trial was conducted at ouse,
Tasmania. IMP Bio was applied in furrows to rows 30m long at a rate of 50L ha, either
alone, or together with the conventional chemical fertilizer 516 at either 650kg/ha
(delivering 32kg/ha en, 63kg/ha phosphorus and lOOkg/ha potassium) or 1250kg/ha
(delivering 63kg/ha nitrogen, 125kg/ha phosphorus and 200kg/ha potassium). In a fourth
replicate, 516 was applied at /ha together with the fungicide Amistar. Four
plots of 4m length were dug from each treatment and tubers assessed for size and yield.
The results are shown in Table 5.
Table 5. Potato yield
There was an increase in stem s per plant in the IMP Bio treatment, which
is desirable r stem numbers typically correlating with higher tuber numbers). The
reduction in large (>350g) tubers observed with IMP Bio treatment is also significant as
larger tubers have lower commercial value than seed sized tubers (45-350g). onally,
the 14% se (5 tonnes/ha) in seed weight in the IMP Bio compared to the 516 +
Amistar treatment is also of significant economic value. The IMP Bio treated potato plants
were also observed to be approximately three weeks more developed (in terms of maturity)
than those treated with 516.
Example 5 - Tick bean trials
A greenhouse experiment was ted to establish the effect of IMP Bio
biofertilizer (see Example 2) on tick bean plant growth, compared to the commercial
fertilizer Baileys TriStar (8.3%N, 0% P, 16% K, 14% S, 1% Fe, 2% Mg).
The treatment groups and regimes employed for seedlings post-germination were
as follows:
Control: 300 mΐ water
"T40": 300 m TriStar at 40L/ha
"SGL40": 300 m IMP Bio at 40L/ha
"T25%GL40": 300 m TriStar 25% plus IMP Bio at 40L/ha
"GL40": 300 mΐ IMP Bio at 40L/ha
Seeds in the T40, SGL40 and 40 groups were soaked for 1 hour in 100ml
of a 1:10 dilution of IMP Bio on prior to planting. Control and GL40 seeds remained
dry prior to planting. Three replicates of each treatment group (and two replicates of the
control group) were used. Seeds were planted 5mm deep in the middle of each pot and the
pots placed in a temperature-controlled greenhouse at 16-18°C under hydroponic lights.
After germination, all seedlings were d every two weeks (for a total of four weeks)
using the ents described above. Seedlings were watered once a day.
At the conclusion of the experiment it was observed that the tallest plants, and the
plants with the strongest main stem were those of the T25%GL40 treatment group.
Overall, the best growth was observed in the T25%GL40 and SGL40 groups (data not
shown). However the most noticeable ences observed were in root development (see
Figure 1). Roots of the control plants were the least dense and the shortest (Figure 1A).
Roots of the T40 plants had good root density and length (Figure IB), however
pment was not as extensive as in the plants treated with IMP Bio. In the SGL40
plants the root system shown good desnity and length (Figure 1C). Root s were
present as were black nodule-like growths. In the T25%GL40 plants the root system was
more dense and longer than in other treatment groups (Figure ID). Root nodules were
present but black nodule-like growth was not seen. In the GL40 plants the root system was
similarly dense, long and well developed (Figure IE). Root nodules were present as were
black nodule-like growths.
Example 6 - Tomato trials
A ouse experiment was conducted to investigate the effect of IMP Bio
biofertilizer (see Example 2) on the growth rate of tomato plants over a 20 day period.
Tomato seedlings were provided by Cedenco. Water only was used as a control, and the
commercial izer FlowPhos (Yara Nipro) used as a comparator. Seedlings were potted
into 50 mm pots in one of three different soils obtained from different locations (Cedenco)
and drenched once with either: (i) 10 ml water; (ii) 0 ml of IMP Bio (100 ml in 900 ml
water); (iii) 0 ml of FlowPhos (7.5 ml in 900 ml water); or (iv) 10 ml of FlowPhos plus
IMP Bio (7.5 ml FlowPhos and 100 ml IMP Bio made to a total volume of 1000ml with
water). Three replicates of the control (water) group and eight replicates of each of the
ent groups. Plants were d twice a day with 30 ml water. Plant height was
measured every third day over the 20 day period of the ment.
The e rate of change of growth (height) of tomato seedlings over the 20 day
period for all treatment groups, in each of the three soils, is shown in Figure 2. As can be
seen, the IMP Bio treated plants were the only plants that tently showed increases in
growth over the course of the experiment, resulting in taller plants. Figure 3 shows an
exemplary comparison of difference in plant height, foliage and root system development
in control plants, FlowPhos treated plants and IMP Bio (GreatLand) treated plants, in
which the advantages of IMP Bio treatment are clearly evident.
A field trial was then conducted at Timmering, Victoria in which tomato plants
were treated with IMP Bio by foliar application during flowering, either at a rate of 80L/ha
or 40L ha during early flowering followed by 40L ha during mid flowering. Yield of
tomato fruit was determined and compared to the yield from the same number of untreated
plants. For the plants that received 80L ha IMP Bio, total fruit yield was 149.87 tonnes/ha,
compared to 128.87 tonnes/ha for the untreated plants. For the plants that received two
applications of 40L/ha IMP Bio, total fruit yield was 130.15 /ha, compared to 103.05
tonnes/ha for the untreated plants.
Example 7 - Macadamia trials
A field trial was conducted in which macadamia trees in a 100 ha farm in Lismore,
NSW were treated with the IMP Bio biofertilizer (see Example 2) by spraying at the rate of
40L/ha, every 2-3 months for a period of 12 . IMP Bio was applied in conjunction
with chemical izer (Easy N Fertilizer), the same fertilizer used for at least the previous
four years. The yield of macadamia nuts following the 12 month treatment was
approximately 70 tonnes, compared to an average yield of 35 tonnes per year over the
previous four years. The benefits offered by the IMP Bio biofertilizer d for a
significant reduction in the application of al fertilizer.
Leaf and soil analysis was also conducted at four sites across the farm after 45 days
of IMP Bio use. Significant increases were observed in levels of zinc, manganese, iron
and boron in macadamia leaves, and in ammonium nitrogen, nitrate nitrogen,' phosphorus,
ium, calcium, copper and boron in the soil.
Example 8 · Strawberry trials
A field trial was conducted in Beerwah, Qld to establish the effect of IMP Bio
biofertilizer (see Example 2) on strawberry plant growth and fruit yield over an 8 ha plot.
The IMP Bio was applied at a rate of 40L ha to the soil pre-planting, again at the same rate
at planting, and weekly during the vegetative growth and flowering stage (weeks 2-4),
during the fruiting stage (weeks 5-8) and during the g stage (weeks 9-16). In
comparison to conventional fertilizer (NitroPhoska(blue) applied preplanting at 1000
kg ha), plant growth rate was icantly increased and plants showed increased
vegetative growth and leaf area (Figure 4). Fruit yield was also significantly increased
(38,000 kg as compared to 20,000 kg).
e 9 - Other trials
Preliminary trials have also been conducted on sugar cane, lettuce,
raspberries, roses, wheat, basil and turf grass (golf course green). In each case IMP Bio
biofertilizer (see e 2) was observed to result in increased rate of growth of plants
compared to untreated plants (data not shown).
Claims (6)
1. A microbial fertilizer, comprising strains of three or more bacterial species selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapi and Lactobacillus zeae.
2. A microbial fertilizer according to claim 1 n the inoculant comprises three of said acillus species.
3. A microbial fertilizer according to claim 1 wherein the inoculant comprises all of said Lactobacillus species.
4. A microbial fertilizer according to any one of claims 1 to 3 sing a symbiotic combination of said three or more acillus species.
5. A microbial fertilizer according to any one of claims 1 to 4 wherein the Lactobacillus rraginis strain is Lactobacillus parafarraginis Lp18.
6. A microbial fertilizer according to claim 5 wherein the Lactobacillus parafarraginis strain is Lactobacillus parafarraginis Lp18 deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number V
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161555535P | 2011-11-04 | 2011-11-04 | |
US61/555,535 | 2011-11-04 | ||
PCT/AU2012/001355 WO2013063658A1 (en) | 2011-11-04 | 2012-11-05 | Microbial inoculants and fertilizer compositions comprising the same |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ614079A NZ614079A (en) | 2016-07-29 |
NZ614079B2 true NZ614079B2 (en) | 2016-11-01 |
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