NZ625835B2 - Plant growth-promoting microbes and uses therefor - Google Patents
Plant growth-promoting microbes and uses therefor Download PDFInfo
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- NZ625835B2 NZ625835B2 NZ625835A NZ62583512A NZ625835B2 NZ 625835 B2 NZ625835 B2 NZ 625835B2 NZ 625835 A NZ625835 A NZ 625835A NZ 62583512 A NZ62583512 A NZ 62583512A NZ 625835 B2 NZ625835 B2 NZ 625835B2
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
-
- 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
- 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
-
- 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
- C12R2001/07—Bacillus
Abstract
Disclosed is an isolated microbial strain SGI-003-H11 (Pantoea agglomerans) deposited as NRRL B-50483. Further, disclosed is an isolated microbial strain comprising a DNA sequence exhibiting at least 99% sequence identity to the nucleotide sequences of SEQ ID NO:1 in the Sequence Listing; and further wherein said microbial strain has a plant growth-promoting activity. r wherein said microbial strain has a plant growth-promoting activity.
Description
PLANT GROWTH-PROMOTING MICROBES AND USES THEREFOR
This application claims the benefit of US. provisional application 61/570,237, filed
December 13, 2011 and which is incorporated by reference herein in its entirely, including all
, , and claims.
FIELD OF THE INVENTION
The present invention relates to the field of sustainable agriculture. Specifically,
the disclosure provides microbial itions and methods useful for the production of crop
plants. In particular, the compositions and methods disclosed herein are useful for enhancing
plant growth and/or suppressing the development of plant pathogens and pathogenic diseases.
INCORPORATION OF SEQUENCE LISTING
The material in the accompanying Sequence Listings is hereby incorporated by
reference into this application. The accompanying file, named
“SGIl540_1WO_CRF_OF_SL_ST25.txt” was created on December 13, 2012 and is 20 KB.
The files can be ed using Microsoft Word on a computer that uses Windows OS.
BACKGROUND OF THE INVENTION
The microflora surrounding plants is very diverse, ing bacteria, fungi, yeast,
algae. Some of these microorganisms may be deleterious to plants, and are often referred to
as pathogens, while others may be beneficial to plants by promoting plant growth and crop
productivity. Recent advances in soil iology and plant biotechnology have resulted in
an increased interest in the use of microbial agents in agriculture, horticulture, forestry and
nmental management. In particular, a number of microorganisms known to be present
in soil ecological niche, generally known as rhizosphere and rhizoplane, have received
considerable attention with respect to their ability to promote plant . Indeed, the
rhizosphere soil ents a good reservoir of microbes for the potential ion of
beneficial microbes. Plant rhizosphere can n ns of microorganisms in one gram of
soil. In theory, microbial inoculants, without human intervention, have a low survival rate
and efficacy in their natural soil environment because of the insufficient colony forming units
per gram of soil. Therefore, since the 1960’s, a number of biofertilizers that have an increased
2012/069579
colony um potential concentration have been developed and commercialized in an
attempt to reduce the need for chemical fertilizers.
In addition, research ted in recent years has shown that microorganisms
can be used as biological l agents to increase agricultural tivity and efficiency.
These studies have shown that various rganisms are able to suppress plant pathogens
and/or supplement plant growth, thus offering an attractive alternative to chemical pesticides
with are less favored because of their ially negative impact on human health and
environment quality.
Microorganisms which can colonize plant roots and stimulate plant growth are
generally known as plant growth-promoting microbes (PGPM). In the past two decades,
many PGPM species having positive influence on the grth of a wide variety of crop plants
have been reported. PGPM are often universal symbionts of higher plants, and are able to
enhance the adaptive potential of their hosts through a number of mechanisms, such as the
fixation of molecular nitrogen, the mobilization of recalcitrant soil nutrients (e.g., iron,
phosphorous, sulfur etc), the synthesis of phytohormones and vitamins, and the
decomposition of plant materials in soils which often increases soil organic matter. Also,
certain microbes can tate plant growth by controlling microbial species pathogenic to the
plant (i.e., phytopathogens). For example, some beneficial microbes can control root rot in
plants by competing with filngi for space on the e of the plant root. In other instances,
competition among various microbial strains in a s native microflora can stimulate root
growth and increase the uptake of mineral nutrients and water to enhance plant yield.
Therefore, biofertilizers can be developed as products based on microorganisms that naturally
live in the soil. By increasing the population of ial microorganisms in the soil through
artificial inoculation, these soil rganisms can boost their biological activity and, thus,
supply the plants with important nutrients and beneficial factors that enhance their growth.
The inoculation of cultivated plants with PGPM is generally seen as a promising
agricultural approach, for it allows pests to be controlled without using ides in large
amounts. As environmental concerns about groundwater quality with excess fertilizer and
pesticide exposure in foods grow, biological alternatives are becoming necessary. Thus,
developing biological treatment compatible with fertilizers and ides or even reducing
the amount of these chemical compounds could be a significant advancement in the
agricultural industry. It has been ished that stimulation of plant growth by PGPM is
2012/069579
often closely related to the ability of the PGPM to colonize plant roots. However, vely
little attention has been given to the development of efficient selection procedures for
obtaining microbial s with high root-colonizing ability. The lack of such selection
procedures slows down the study of plant-bacterial symbioses, and the ment of PGPM
in agriculture.
Therefore, there is a continuing need for the identification of new PGPM and/or
testing of their compatibility with existing commercially available crop management
products. Moreover, additional investigation is also needed to compare pure culture strains
versus complementary mixed strains of microorganisms that form synergistic consortia. Such
mixed consortia might have greater potential for consistent performance with better
itive ability under different environmental and growth conditions.
SUMMARY OF THE INVENTION
Microbial s and cultures are provided herein. Microbial itions and
methods of use thereof to enhance the growth and/or yield of a plant are also ed. Also
provided are methods for the treatment of plant seeds by using the microbial compositions
disclosed herein. Further provided are methods for preventing, inhibiting, or treating the
development of plant pathogens or the development of phytopathogenic diseases. The
disclosure also provides non-naturally occurring plant varieties that are varieties artificially
infected with a microbial endophyte of the invention. Seed, uctive tissue, vegetative
tissue, regenerative tissues, plant parts, or progeny of the non-naturally occurring plant
varieties are also provided. The disclosure further provides a method for preparing
agricultural compositions.
In one aspect, the present disclosure provides isolated microbial strains, isolated
cultures thereof, biologically pure cultures thereof, and enriched es thereof. In n
preferred embodiments of this aspect, the microbial strain can be 3-Hll ited as
NRRL B-50483); SGI-OZO-AOl ited as NRRL B-50484); SGIG06 (deposited as
NRRL B-50485); 6-G07 (deposited as NRRL B-50486), or a strain derived from any
one of said strains. In some other preferred embodiments, the microbial strain can comprise a
nucleotide or amino acidsequence that exhibits at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or at least 99.5% sequence identity to any one of the 168 ribosomal and/or recA
nucleotide sequences and/or amino acid sequences in the ce Listing. In some
embodiment the microbial strain also has a plant growth-promoting activity as described
herein.
Also provided are microbial itions that include a microbial strain of the
invention or a culture thereof. Such microbial compositions according to some red
embodiments may comprise an agriculturally effective amount of an additional compound or
composition, in which the additional compound or composition may be a fertilizer, an
acaricide, a bactericide, a fungicide, an insecticide, a microbicide, a nematicide, or a
pesticide. In some other preferred embodiments, the microbial compositions may fiarther
include a carrier. In yet other preferred embodiments, the carrier may be a plant seed. In
certain embodiments of this aspect, the microbial ition is prepared as a formulation
that can be an emulsion, a colloid, a dust, a granule, a pellet, a powder, a spray, an on,
or a solution. In some other preferred embodiments, the microbial compositions may be seed
coating formulations. In yet another aspect, plant seeds that are coated with a microbial
composition in accordance with the present invention are also ed.
In another aspect, there are provided methods for ng plant seeds. Such
methods include exposing or contacting the plant seeds with a microbial strain according to
the present invention or a culture f.
In another aspect of the invention, provided herein are methods for enhancing the
growth and/or yield of a plant. In some embodiments, such method involves applying an
ive amount of a microbial strain in accordance with the present invention or a culture
f to the plant, or to the plant's surroundings. In some other embodiments, the method
involves growing a microbial strain in accordance with the t invention or a e
f in a grth medium or soil of a host plant prior to or concurrent with host plant
growth in said grth medium or soil. In preferred embodiments, the plant may be a corn
plant or a wheat plant. In some other embodiments, the microbial strain or culture thereof
may be established as an endophyte on the plant.
In another aspect of the present invention, there are provided s for
preventing, inhibiting or treating the development of a plant pathogen. Such methods include
growing a microbial strain according to the invention or a culture thereof in a growth medium
or soil of a host plant prior to or concurrent with host plant grth in said grth medium or
soil. In some preferred embodiments, the plant pathogen may be a microorganism of the
genus Colletotrz'chum, Fusarz'um, Gibberella, Monographella, Penicillium, or Stagnospora.
In some particularly preferred embodiments, the plant pathogen may be Colletotrz'chum
graml'nz'cola, Fusarz'um graml'nearum, Gibberella zeae, Monographella nivall's, Penicillium
sp., or Stagnospora nodurum.
Another fiarther aspect of the invention provides methods for preventing,
inhibiting or treating the development of plant pathogenic e of a plant. Such s
e applying to the plant, or to the plant's surroundings, an effective amount of a
microbial strain according to the invention or a culture thereof. In some preferred
embodiments, the microbial strain or a culture f may be applied to soil, a seed, a root, a
flower, a leaf, a portion of the plant, or the whole plant.
Another further aspect of the invention provides non-naturally occurring plants.
The turally occurring plants are artificially infected with a microbial strain of the
invention or a culture thereof Further provided in some embodiments of this aspect are seed,
reproductive , tive , regenerative tissues, plant parts, and progeny of the
non-naturally occurring plants.
Another aspect of the invention provides methods for preparing an agricultural
composition. Such methods involve ating the microbial strain according to the present
ion or a culture f into or onto a substratum and allowing it to grow.
In another aspect the invention provides an isolated strain, an isolated culture
thereof, a biologically pure culture thereof, and an enriched culture of a microorganism of the
genus Pantoea. In one embodiment the rganism comprises a DNA sequence or amino
acid sequence coding for a 16S rRNA gene or a recA protein having at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% or at least 99.5% sequence identity to a sequence coding
for 16S rRNA gene or recA n disclosed in the Sequence Listing. In another
embodiment the invention provides a genus of microorganisms comprising any of the DNA
sequences or amino acid sequences described above and which enhances the grth and/or
yield of a plant, as described herein.
These and other objects and features of the invention will become more fully
apparent from the following detailed description of the ion and the claims.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise , all terms of art, notations and other scientific terms or
terminology used herein are intended to have the meanings commonly understood by those of
skill in the art to which this invention pertains. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready reference, and the
inclusion of such ions herein should not necessarily be ued to ent a
substantial difference over what is generally understood in the art. Many of the techniques
and ures described or referenced herein are well understood and commonly employed
using conventional methodology by those d in the art.
The singular form “a”, “an”, and “the” include plural references unless the context
clearly dictates ise. For example, the term “a cell” includes one or more cells,
including mixtures thereof
Bactericidal: the term “bactericidal”, as used herein, refers to the ability of a
ition or substance to increase mortality or inhibit the growth rate of bacteria.
Biological control: the term “biological control” and its iated form
“biocontrol”, as used herein, is defined as control of a pathogen or insect or any other
undesirable organism by the use of at least a second organism other than man. An example of
known mechanisms of biological control is the use of microorganisms that control root rot by
mpeting filngi for space on the surface of the root, or microorganisms that either inhibit
the growth of or kill the pathogen. The “host plant” in the context of biological control is the
plant that is susceptible to disease caused by the pathogen. In the context of isolation of an
organism, such as a bacterium or fungal species, from its natural environment, the “host
plant” is a plant that supports the growth of the bacterium or filngus, for example, a plant of a
species the bacterium or fiangus is an endophyte of.
An "effective amount", as used herein, is an amount sufficient to effect beneficial
or desired results. An effective amount can be administered in one or more administrations.
In terms of treatment, inhibition or protection, an effective amount is that amount sufficient to
rate, stabilize, reverse, slow or delay progression of the target infection or disease
. The expression "effective microorganism" used herein in nce to a microorganism
is intended to mean that the subject strain exhibits a degree of promotion of plant grth
and/or yield or a degree of inhibition of a pathogenic disease that exceeds, at a statistically
significant level, that of an untreated control. In some instances, the expression "an effective
amount" is used herein in reference to that ty of microbial treatment which is necessary
to obtain a beneficial or desired result relative to that ing in an untreated control under
suitable conditions of treatment as described herein. For the purpose of the t disclosure,
the actual rate of application of a liquid formulation will usually vary from a minimum of
about l X 103 to about l X 1010 viable cells/mL and preferably from about l X 106 to about
X 109 viable cells/mL. Under most conditions, the strains of the invention described in the
es below would be optimally effective at application rates in the range of about l X
106 to l X 109 viable cells/mL, assuming a mode of application which would achieve
substantially uniform t of at least about 50% of the plant tissues. If the microorganisms
are applied as a solid formulation, the rate of application should be controlled to result in a
comparable number of viable cells per unit area of plant tissue surface as obtained by the
entioned rates of liquid treatment. Typically, the microbial compositions of the present
invention are biologically effective when delivered at a concentration in excess of 106 CFU/g
(colony g units per gram), preferably in excess of 107 CFU/g, more preferably 108
CFU/g, and most preferably at 109 CFU/g.
Composition: A “composition” is intended to mean a ation of active agent
and at least another compound, carrier, or composition, which can be inert (for example, a
detectable agent or label or liquid carrier) or active, such as a fertilizer.
A "control plant", as used in the present disclosure, provides a nce point for
measuring changes in phenotype of the subject plant, may be any suitable plant cell, seed,
plant component, plant tissue, plant organ or whole plant. A control plant may comprise, for
example, (a) a wild-type plant or cell, z'.e., of the same genotype as the starting material for
the c alteration which resulted in the subject plant or cell; (b) a plant or cell of the
genotype as the starting material but which has been transformed with a null construct (i.e., a
construct which has no known effect on the trait of st, such as a construct comprising a
reporter gene); (c) a plant or cell which is a non-transformed segregant among progeny of a
subject plant or cell; (d) a plant or cell which is genetically identical to the t plant or
cell but which is not exposed to the same treatment (e.g., fertilizer treatment) as the t
plant or cell; (e) the subject plant or cell itself, under conditions in which the gene of interest
is not sed; or (f) the subject plant or cell itself, under conditions in which it has not
WO 90628
been exposed to a particular treatment such as, for example, a fertilizer or combination of
fertilizers and/or other chemicals.
Culture, isolated e, biologically pure culture, and enriched e: As used
herein, an isolated strain of a microbe is a strain that has been removed from its natural
milieu. As such, the term ted" does not necessarily reflect the extent to which the
microbe has been purified. But in different embodiments an “isolated” culture has been
purified at least 2x or 5x or 10x or 50x or 100x from the raw material from which it is
isolated. As a non-limiting example, if a culture is isolated from soil as raw material, the
organism can be isolated to an extent that its concentration in a given quantity of purified or
partially purified material (e.g., soil) is at least 2x or 5x or 10x or 50x or 100x that in the
original raw material. A "substantially pure culture" of the strain of e refers to a
culture which contains substantially no other microbes than the d strain or s of
microbe. In other words, a substantially pure e of a strain of microbe is substantially
free of other inants, which can include microbial contaminants as well as undesirable
al contaminants. Further, as used herein, a "biologically pure" strain is intended to
mean the strain separated from materials with which it is normally associated in nature. Note
that a strain associated with other s, or with compounds or materials that it is not
normally found with in nature, is still defined as "biologically pure." A monoculture of a
particular strain is, of course, "biologically pure." In different embodiments a “biologically
pure” culture has been purified at least 2x or 5x or 10x or 50x or 100x from the material with
which it is normally associated in nature. As a non-limiting example, if a culture is normally
associated with soil in nature, the organism can be biologically pure to an extent that its
concentration in a given quantity of purified or partially purified material with which it is
normally associated in nature (e.g. soil) is at least 2x or 5x or 10x or 50x or 100x that in the
al unpurified material. As used herein, the term "enriched culture" of an isolated
ial strain refers to a microbial culture wherein the total microbial population of the
culture contains more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain .
Culturing: The term ‘culturing’, as used herein, refers to the propagation of
organisms on or in media of various kinds.
As used herein, an hyte" is an endosymbiont that lives within a plant for at
least part of its life without causing apparent disease. Endophytes may be transmitted either
ally (directly from parent to offspring) or horizontally (from individual to unrelated
2012/069579
individual). Vertically-transmitted fiangal endophytes are typically asexual and transmit from
the maternal plant to offspring Via fiangal hyphae penetrating the host's seeds. Bacterial
endophytes can also be transferred vertically from seeds to seedlings (Ferreira et al., FEMS
Microbiol. Lett. 287:8-14, 2008). Conversely, horizontally-transmitted endophytes are
typically sexual, and transmit Via spores that can be spread by wind and/or insect vectors.
Microbial endophytes of crop plants have received erable attention with respect to their
ability to control disease and insect infestation, as well as their potential to promoting plant
growth.
Fungal en: For purposes of this invention it is understood that the use of
term fungal pathogen or fungus is intended to include both the sexual (teleomorphic) stage of
this organism and also the asexual (anamorphic) stage, also referred to as the perfect and
imperfect fungal stages, respectively. For example, the anamorphic stage of Fusarium
gramz'nearum is ella zeae.
Fungicidal: As used herein, “fiangicidal” refers to the ability of a ition or
nce to decrease the rate of grth of fiangi or to increase the mortality of fungi.
Mutant: As used herein, the term “mutant” or “variant” in nce to a
microorganism refers to a modification of the parental strain in which the desired biological
actiVity is similar to that expressed by the parental strain. For example, in the case of
lderia the “parental strain” is defined herein as the original Burkholderz’a strain before
mutagenesis. s or variants may occur in nature without the intervention of man. They
also are obtainable by treatment with or by a variety of methods and compositions known to
those of skill in the art. For example, a parental strain may be treated with a chemical such as
N—methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma,
x-ray, or adiation, or by other means well known to those practiced in the art.
Nematicidal: The term icidal”, as used herein, refers to the ability of a
substance or composition to increase mortality or inhibit the growth rate of nematodes.
Pathogen: The term "pathogen" as used herein refers to an organism such as an
alga, an arachnid, a bacterium, a fungus, an insect, a nematode, a parasitic plant, a protozoan,
a yeast, or a virus capable of producing a disease in a plant or animal. The term
"phytopathogen" as used herein refers to a enic organism that infects a plant.
Percentage of sequence identity: “percentage of sequence identity”, as used
, is determined by comparing two optimally locally aligned ces over a
comparison window defined by the length of the local alignment between the two sequences.
The amino acid sequence in the comparison window may comprise ons or deletions (e.
g., gaps or overhangs) as compared to the reference ce (which does not comprise
additions or deletions) for l alignment of the two ces. Local alignment n
two sequences only includes segments of each ce that are deemed to be ently
similar according to a criterion that depends on the algorithm used to perform the alignment
(e. g. BLAST). The percentage of sequence identity is calculated by determining the number
of positions at which the identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the number of matched
positions by the total number of positions in the window of comparison and multiplying the
result by 100. Optimal alignment of sequences for comparison may be conducted by the local
homology algorithm of Smith and an (1981) Add. APL. Math. 2:482, by the global
homology alignment algorithm of Needleman and Wunsch (J M01. Biol. 48:443, 1970), by
the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:
2444, 1988), by heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST,
BLAT, SIM, BLASTZ), or by inspection. Given that two sequences have been identified for
comparison, GAP and BESTFIT are preferably employed to determine their optimal
alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length
are used. The term “substantial sequence identity” between polynucleotide or polypeptide
ces refers to polynucleotide or polypeptide comprising a sequence that has at least
50% ce identity, preferably at least 70%, preferably at least 80%, more preferably at
least 85%, more preferably at least 90%, even more preferably at least 95%, and most
preferably at least 96%, 97%, 98% or 99% sequence identity compared to a reference
ce using the programs. In addition, pairwise sequence homology or sequence
similarity, as used refers to the percentage of residues that are similar between two sequences
aligned. Families of amino acid residues having similar side chains have been well defined in
the art. These families include amino acids with basic side chains (e.g., , arginine,
histidine), acidic side chains (e.g., aspartic acid, ic acid), uncharged polar side chains
(e.g., glycine, gine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), ranched side chains (e.g., ine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Query nucleic acid and amino acid sequences can be searched against subject
nucleic acid or amino acid sequences residing in public or proprietary databases. Such
searches can be done using the National Center for Biotechnology Information Basic Local
Alignment Search Tool (NCBI BLAST V 2.18) program. The NCBI BLAST program is
available on the intemet from the National Center for Biotechnology Information
(blast.ncbi.nlm.nih.gov/Blast.cgi). Typically the ing parameters for NCBI BLAST can
be used: Filter options set to "default”, the Comparison Matrix set to M62”, the Gap
Costs set to “Existence: 11, Extension: 1”, the Word Size set to 3, the Expect (E threshold)
set to le-3, and the minimum length of the local alignment set to 50% of the query ce
length. Sequence identity and similarity may also be determined using GenomeQuestTM
re (Gene-IT, Worcester Mass. USA).
The term “pest” as used herein refers to an undesired organism that may include,
but not limited to, bacteria, fungi, plants (e.g., weeds), nematodes, insects, and other
pathogenic animals. “Pesticidal”, as used , refers to the ability of a substance or
composition to decrease the rate of growth of a pest, z'.e., an undesired organism, or to
increase the mortality of a pest.
y: As used herein, "progeny" includes descendants of a particular plant or
plant line. Progeny of an instant plant include seeds formed on F1, F2, F3, F4, F5, F6 and
uent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation
plants, or seeds formed on F1BC1, F1BC2, F1BC3, and subsequent generation plants. The
designation F1 refers to the y of a cross between two parents that are genetically
distinct. The designations F2, F3, F4, F5 and F6 refer to subsequent generations of self— or sib-
pollinated progeny of an F1 plant.
Variant: as used herein in reference to a c acid and ptide, the term
“variant” is used herein to denote a polypeptide, protein or polynucleotide molecule with
some differences, generated synthetically or naturally, in their amino acid or nucleic acid
sequences as ed to a reference polypeptide or polynucleotide, respectively. For
example, these differences include substitutions, insertions, deletions or any desired
combinations of such changes in a reference polypeptide or polypeptide. Polypeptide and
protein variants can further consist of changes in charge and/or post-translational
modifications (such as glycosylation, methylation. phosphorylation, etc.)
The term “variant”, when used herein in reference to a microorganism, is a
microbial strain having fying characteristics of the species to which it belongs, while
having at least one nucleotide sequence variation or identifiably different trait with respect to
the parental strain, where the trait is genetically based able). For example, for a Bacillus
thuringiensis 020_AOl strain having a plant growth-promoting activity, identifiable traits
include 1) the ability to suppress the development of fungal phytopathogens, including
Fusarium earum, Gibberella zeae, spora nodurum, Colletotrichum
graminicola; 2) the ability to enhance seed yield in wheat; and 3) having a 168 rRNA gene
with nucleotide ce with greater than 95%, greater than 96%, greater than 97%, greater
than 98%, or greater than 99% sequence identity to the 168 rRNA gene of Bacillus
thuringiensis 020_A01; can be used to confirm a variant as Bacillus thuringiensis 020_A01.
Yield: As used herein, the term "yield" refers to the amount of harvestable plant
material or plant-derived product, and is normally defined as the measurable produce of
economic value of a crop. For crop plants, "yield" also means the amount of harvested
material per acre or unit of production. Yield may be defined in terms of quantity or quality.
The harvested material may vary from crop to crop, for example, it may be seeds, above
ground biomass, roots, fruits, cotton fibers, any other part of the plant, or any plant-derived
product which is of economic value. The term "yield" also encompasses yield potential,
which is the m obtainable yield. Yield may be dependent on a number of yield
components, which may be red by certain ters. These parameters are well
known to persons skilled in the art and vary from crop to crop. The term "yield" also
encompasses harvest index, which is the ratio between the harvested biomass over the total
amount of s.
All publications and patent applications mentioned in this specification are herein
incorporated by reference to the same extent as if each individual publication or patent
ation was cally and individually indicated to be incorporated by reference.
No admission is made that any reference constitutes prior art. The discussion of
the references states what their authors assert, and the applicants e the right to
challenge the accuracy and ence of the cited documents. It will be clearly understood
that, although a number of prior art publications are referred to herein, this reference does not
constitute an admission that any of these documents forms part of the common general
knowledge in the art.
The sion of the general methods given herein is ed for illustrative
purposes only. Other alternative methods and embodiments will be apparent to those of skill
in the art upon review of this disclosure.
Plant Growth-Promoting Microorganisms
Diverse plant-associated microorganisms can positively impact plant health and
physiology in a variety of ways. These beneficial microbes are generally referred to as plant
growth-promoting microorganisms (PGPMs). The term “plant growth-promoting activity”, as
used herein, asses a wide range of improved plant properties, including, for example
without limitation, improved en fixation, improved root development, increased leaf
area, increased plant yield, increased seed germination, increased photosynthesis, or an
increased in accumulated biomass of the plant. In various embodiments the improvement is
an at least 10% increase or at least 25% increase or at least 50% se or at least 75%
increase or at least a 100% increase in the property being measured. Thus, as non-limiting
examples, the microbes may produce an above stated percentage increase in nitrogen fixation,
or an above stated increase in total root , or in leaf area or in plant product yield (e. g.,
an above stated percentage increase in plant product weight), or an increased percentage of
seeds that germinate within 10 days or 14 days or 30 days, or rate of photosynthesis (e.g.,
determined by C02 consumption) or accumulated biomass of the plant (e.g., determined by
weight of the plant). The plant product is the item — usually but not arily — a food item
produced by the plant. The yield can be determined using any convenient method, for
example, bushels or pounds of plant product ed per acre of planting. To date, isolated
strains of over two dozen genera of microorganisms have been reported to have plant growth-
promoting ty and/or biocontrol activity, and new genera and species with similar
activities are still being discovered. Additionally, within some bacterial genera, multiple
species and subspecies of biocontrol agents have been identified and can be found across
multiple spatial , from the global level to farm level, and even on single .
rmore, it has been reported that some individual microbial isolates may display
trol and/or plant growth-promoting ty not only on the plants or crops from which
they were obtained but also on other crops. This indicates the generalist nature of some
WO 90628
genotypes, especially those with a wide geographic distribution. As discussed above, if
introduced in sufficient numbers and active for a sufficient duration, a single microbial
population can have a significant impact on plant health.
Several mechanisms have been postulated to provide an explanation for the
positive impact of PGPMs on plant growth enhancement. The beneficial effects of the
microorganisms on plant growth can be direct or indirect.
The term “direct plant -promoting microorganism”, for the purpose of this
disclosure, refers to a microorganism that can enhance plant grth in the absence of
pathogens. As discussed in more detail below, examples of direct plant grth promotion
include (a) biofertilization, (b) stimulation of root growth, (c) rhizoremediation, and (d) plant
stress control. In addition, several PGPMs have been reported to promote plant growth
indirectly via isms of biological control, z'.e., by reducing the level of disease, for
example antibiosis, induction of systemic resistance, and competition with pathogens for
nutrients and niches.
Biofertilizers: Microbial fertilizers supply the plant with nutrients and thereby can
promote plant growth in the absence of pathogen re. Non-limiting es of
microbial isolates that can directly promote plant grth and/yield include Ng-fixing bacteria
Rhizobz'um and hl'zobium species that, through symbiotic nitrogen fixation, can form
s on roots of leguminous plants, in which they convert atmospheric N2 into a
which, in contrast to heric N2, can be used by the plant as a nitrogen source. Other
examples include Azospz'rz'llum species, which are free-living N2-fixers that can ize and
increase yield of cereal crops such as wheat, sorghum, and maize. Despite 'rz'llum ’s N2-
fixing capacity, the yield increase caused by inoculation by Azospz'rillum is often attributed to
increased root development and thus to increased rates of water and mineral . In this
respect, several rhizobacteria like Azotobacter spp. have been reported to be capable of
producing a wide array of ormones (e.g., auxins, cytokinins) and enzymes (e.g.,
pectinase). Many of these phytohormones and enzymes have been shown to be intimately
involved in the infection process of symbiotic bacteria-plant associations which have a
regulatory influence on nodulation by Rhizobz'um.
In many ces, PGPMs also can affect the plant growth and development by
modifying nutrient uptake. They may alter nt uptake rates, for example, by direct
effects on roots, by effects on the environment which in turn modify root behavior, and by
competing directly for nutrients (Gaskin et al., Agricult. Ecosyst. n. 12: 99-116, 1985).
Some factors by which PGPM may play a role in modifying the nutrient use efficiency in
soils include, for example, root geometry, nutrient solubility, nutrient availability by
producing plant ial ion form, ioning of the nutrients in plant and ation
efficiency. For example, a low level of e phosphate can limit the growth of plants.
Some plant growth-promoting microbes are capable of solubilizing phosphate from either
organic or inorganic bound phosphates, thereby facilitating plant growth. Several enzymes of
microbial origin, such as nonspecific phosphatases, phytases, phosphonatases, and C-P
lyases, release soluble phosphorus from organic compounds in soil. For example, an
increased solubilization of inorganic phosphorous in soil has been found to enhance
phosphorus uptake in canola seedling using Pseudomonas puticla as well as increased sulfur-
oxidation and sulfur uptake ton and Germida, Can. J. Microbiol. 37: 521-529, 1991;
Banerjee, Phytochemicals and Health, vol. 15, May 18, 1995).
Phytostimulators: Some microorganisms can produce substances that ate the
growth of plant in the e of pathogens. For example, the production of plant hormones
is a characteristic of many plant-associated rganisms. For all five classical
phytohormones, i. e., auxin, ethylene, abscisic acid, nin, and gibberellin, synthesis as a
secondary metabolite has been demonstrated for at least one bacterial and/or fungal species
(for review, see, e.g., Kim et al., Appl. n. Microbiol, Vol. 77, 5:1548—1555, 2011).
Some microorganisms can also produce secondary lites that affect phytohormone
production in plants. ly, the best-known example is hormone auxin, which can
promote root growth. Other examples include pseudomonads which have been reported to
produce indole acetic acid (IAA) and to enhance the amounts of IAA in plants, thus having a
profound impact on plant biomass production (Brown, Annual Rev. Phytopathology, 68: 181-
197, 1974). For example, Tien et al. (Applied Environmental Microbiol, 37:1016-1024,
1979) ed that inoculation of nutrient solutions around roots of pearl millet with
Azospirillum brasiliense resulted in increased shoot and root weight, an increased number of
lateral roots, and all lateral roots were densely covered with root hairs. Plants supplied with
combinations of IAA, gibberellins and kinetin showed an increase in the production of lateral
roots similar to that caused by Azospirilla. Although the biological cance of these
ormones and plant-hormone-like materials are not fillly understood, the growth
2012/069579
stimulating activity of these microorganisms is commonly attributed to their production of
these materials.
In addition, other hormones as well as certain volatile c compounds (VOCs)
and the cofactor pyrrolquinoline quinone (PQQ) also stimulate plant growth. For example,
some rhizobacteria, such as strains of the bacterial species B. sabtilis, B. amyloliqaefaciens,
and Enterobacter cloacae, promote plant growth by ing VOCs. The highest level of
growth promotion has been observed with 2,3-butanediol and 3-hydroxybutanone (also
referred to as n) as elicitors of induced systemic resistance. The cofactor PQQ has been
described as a plant growth promoter, which acts as an antioxidant in plants. Some reports
suggests that effect may be indirect because PQQ is a cofactor of several enzymes, e. g.,
involved in ngal activity and induction of systemic resistance.
Stress controllers: Plant -promoting microorganisms that contain the
enzyme l-aminocyclopropane-l-carboxylic acid (ACC) deaminase facilitate plant growth and
development by decreasing plant ethylene . Such microorganisms take up the ethylene
precursor ACC and convert it into 2-oxobutanoate and NH3. Several types of stress have been
reported to be relieved by ACC deaminase producers, such as, for example, stress from the
effects of athogenic bacteria, stress from polyaromatic hydrocarbons, stress from
heavy metal such as Ca2+ and Ni2+, and stress from salt and drought.
In addition, several PGPM strains that induced yield increases of potato have been
ed to produce extracellular siderophores that bind Fe“, making it less available to
certain member of natural microflora (Kloepper et al., Nature 286: 885-886, 1980). These
rhizobacteria excrete low molecular weight, high affinity -chelating microbial cofactors
that specifically enhance their acquisition of iron by binding to membrane bound siderophore
receptors. One of the siderophores produced by some pseudomonad PGPMs is known as
pseudobactin that inhibits the growth of Erwinia cartovora (causal organism for soft-rot of
) (see, e.g., Kloepper et al., Current Microbiol. 4: 317-320, 1980). Additions of
pseudobactin to the grth medium inhibited soft-rot infection and also d the number
of pathogenic fiangi in the potato plant along with a significant increase in potato yield. Most
evidence to support the siderophore theory of biological control by PGPM comes from work
with the dines, one class of sideophores that comprises the fluorescent pigments of
fluorescent pseudomonads [Demange et al., in Iron Transport in es, Plants and
Animals eman et al., eds.), pp 167-187, 1987]. According to the siderophore theory,
pyoverdines demonstrate certain onal strain specificity which is due to selective
recognition of outer membrane phore receptors r et al., Soil Biology and
Biochemistry 19: 443-450, 1989).
Isolated cultures of the invention
As described in more detail in the Examples section of the present disclosure,
Applicants have discovered several novel microorganisms that are effective promoters of
plant growth and plant yield. In many cases, the isolated rganisms are also effective in
suppressing the development of several plant enic diseases. The microbial es
were selected from a pool of approximately 5,000 microbial strains obtained from
environmental samples collected from several ons throughout the United States. Initial
selection of the microorganisms was based on the ability of the microorganisms to colonize
plant roots and to produce chemical compounds and s that are considered to be
important for their ction with plants. The microorganisms were also bio-assayed for
their y to suppress the development of various fungal phytopathogens in an in vitro
antagonism assay. Selected microbial microorganisms were then bio-assayed in greenhouse
studies on commercial wheat and corn ies for the ability of the microbial strains to
promote plant growth and for their ability to preserve seed yield potential.
Taxonomic analysis further determined that representative microorganisms
bed in the present disclosure are closely related to the ial genera Bacillus,
lderia, Herbaspirillum, Pantoea, and Pedobacter.
Deposit of Biological Material
Purified cultures of microbial strains described in the present disclosure were
deposited in the Agricultural Research Service Culture Collection located at 1815 N.
sity Street, Peoria, IL 61604, USA (NRRL) in accordance with the Budapest Treaty
for the purpose of patent procedure and the regulations thereunder (Budapest Treaty).
Accession numbers for these deposits are as follows:
Table 1: Microbial isolates and corresponding accession numbers
Strain ID Provisional Taxonomy
SGIH11 NRRL B-50483 Pantoea agglomerans 003_H11
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SGI-OZO-AOl NRRL B-50484 Bacillus thuringiensis 020_AOl
SGTG06 NRRL B-50485 Burkholderz'a metallica 026_G06
SGTG07 NRRL B-50486 Burkholderz'a Vietnamz'ensz's 026_G07
The microbial strains have been deposited under conditions that ensure that access
to the culture will be available during the cy of this patent application to one
determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37
C.F.R. §l.l4 and 35 U.S.C. §122. The deposits represent ntially pure cultures of the
deposited strains. The deposits are available as required by foreign patent laws in countries
wherein counterparts of the subject application or its progeny are filed. r, it should be
understood that the availability of a deposit does not constitute a license to practice the
subject invention in tion of patent rights granted by mental action.
Preferred microorganisms of the present invention have all of the fying
characteristics of the deposited strains and, in particular, the fying characteristics of
being able to e plant growth and/or yield as described herein, and the identifying
characteristics as being able to suppress the development of fungal phytopathogen as
described herein. In particular, the preferred microorganisms of the present invention refer to
the deposited microorganisms as bed above, and strains derived therefrom.
The microbiological compositions of the present invention that comprise isolated
microbial strains or cultures f can be in a variety of forms, including, but not d to,
still cultures, whole cultures, stored stocks of cells, mycelium and/or hyphae (particularly
glycerol stocks), agar strips, stored agar plugs in glycerol/water, freeze dried stocks, and
dried stocks such as lyophilisate or mycelia dried onto filter paper or grain seeds. As defined
elsewhere , “isolated culture” or grammatical equivalents as used in this disclosure and
in the art is understood to mean that the referred to culture is a culture fluid, pellet, scraping,
dried sample, lyophilisate, or section (for example, hyphae or mycelia); or a support,
ner, or medium such as a plate, paper, filter, matrix, straw, pipette or pipette tip, fiber,
needle, gel, swab, tube, vial, particle, etc. that contains a single type of organism. In the
present invention, an isolated culture of a microbial antagonist is a culture fluid or a scraping,
pellet, dried preparation, lyophilisate, or section of the microorganism, or a support,
container, or medium that contains the microorganism, in the absence of other organisms.
The present disclosure further provides compositions that contain at least one
ed ial strains or cultures thereof of the present invention and a r. The carrier
may be any one or more of a number of carriers that confer a variety of ties, such as
sed stability, wettability, dispersability, etc. g agents such as natural or synthetic
tants, which can be nonionic or ionic surfactants, or a combination thereof can be
included in a composition of the invention. Water-in-oil emulsions can also be used to
formulate a composition that includes at least one isolated microorganism of the present
invention (see, for example, US. Patent No. 7,485,451, incorporated by reference herein).
Suitable formulations that may be prepared include wettable powders, granules, gels, agar
strips or pellets, thickeners, and the like, microencapsulated particles, and the like, liquids
such as aqueous flowables, aqueous suspensions, water-in-oil emulsions, etc. The formulation
may include grain or legume products (e.g., ground grain or beans, broth or flour derived
from grain or beans), starch, sugar, or oil. The carrier may be an agricultural carrier. In
certain preferred embodiments, the carrier is a seed, and the composition may be applied or
coated onto the seed or allowed to saturate the seed.
In some embodiments, the agricultural carrier may be soil or plant growth
medium. Other agricultural carriers that may be used include water, izers, plant-based
oils, humectants, or combinations thereof Alternatively, the agricultural carrier may be a
solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, ulite, seed
cases, other plant and animal products, or combinations, including granules, pellets, or
suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as
carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in
loam, sand, or clay, etc. Formulations may include food sources for the ed organisms,
such as barley, rice, or other biological materials such as seed, plant parts, sugar cane
bagasse, hulls or stalks from grain processing, ground plant al (“yard ) or wood
from ng site refuse, t or small fibers from recycling of paper, fabric, or wood.
Other suitable formulations will be known to those skilled in the art.
In the liquid form, e.g., ons or suspensions, the microorganisms of the
present invention may be mixed or suspended in water or in aqueous solutions. Suitable
WO 90628
liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other
liquid carriers.
Solid compositions can be prepared by dispersing the microorganisms of the
invention in and on an appropriately divided solid carrier, such as peat, wheat, bran,
vermiculite, clay, talc, ite, diatomaceous earth, fuller's earth, pasteurized soil, and the
like. When such ations are used as wettable powders, biologically compatible
dispersing agents such as non-ionic, anionic, amphoteric, or cationic dispersing and
emulsifying agents can be used.
In a preferred embodiment, the compositions contemplated herein enhance the
growth and yield of crop plants, such as wheat, barley, oat, and corn and, when used in
sufficient amounts, to act as microbial izer. These compositions, similarly to other
biofertilizer agents, can have a high margin of safety because they typically do not burn or
injury the plant.
As described in great detail throughout the present disclosure, ing plant
growth and plant yield may be ed by application of one or more of the microbiological
compositions of the present invention to a host plant or parts of the host plant. The
compositions can be applied in an amount ive to enhance plant growth or yield relative
to that in an untreated l. The active constituents are used in a concentration sufficient to
enhance the grth of the target plant when applied to the plant. As will be apparent to a
skilled person in the art, ive concentrations may vary depending upon various factors
such as, for example, (a) the type of the plant or agricultural commodity; (b) the
physiological condition of the plant or agricultural commodity; (c) the concentration of
pathogens affecting the plant or agricultural commodity; (d) the type of e injury on the
plant or agricultural commodity; (e) weather conditions (e.g., temperature, humidity); and (f)
the stage of plant disease. According to the t invention, typical concentrations are those
higher than l X 102 CFU/mL of carrier. Preferred concentrations range from about l X 104
to about 1 X 109 CFU/mL, such as the concentrations g from l X 106 to l X 108
CFU/mL. More preferred concentrations are those of from about 37.5 to about 150 mg dry
bacterial mass per milliliter of carrier (liquid composition) or per gram of carrier (dry
formulation).
In some ments, the amount of one or more of the microorganisms in the
compositions of the present invention can vary depending on the final formulation as well as
size or type of the plant or seed utilized. ably, the one or more microorganisms in the
compositions are present in about 2% w/w/ to about 80% w/w of the entire formulation. More
preferable, the one or more microorganisms employed in the compositions is about 5% w/w
to about 65% w/w and most ably about 10% w/w to about 60% w/w by weight of the
entire formulation.
As it will be appreciated by those skilled in the art, the microbiological
compositions of the invention may be applied to the target plant using a variety of
conventional methods such as dusting, coating, injecting, rubbing, rolling, dipping, spraying,
or brushing, or any other appropriate technique which does not significantly injure the target
plant to be treated. Particularly preferred methods include the inoculation of growth medium
or soil with suspensions of microbial cells and the coating of plant seeds with ial cells
and/or spores.
Typically, the compositions of the invention are chemically inert; hence they are
compatible with substantially any other constituents of the application schedule. They may
also be used in ation with plant growth affecting substances, such as fertilizers, plant
growth regulators, and the like, provided that such compounds or substances are biologically
compatible. They can also be used in combination with biologically compatible pesticidal
active agents as for example, ides, nematocides, fiangicides, insecticides, and the like.
When used as biofertilizers in their commercially available formulations and in
the use forms, prepared from these formulations, the active microbial strains and
compositions according to the present invention can furthermore be present in the form of a
e with synergists. Synergists are compounds by which the ty of the active
compositions is increased without it being necessary for the synergist added to be active
itself.
When used as biofertilizers in their commercially available formulations and in
the use forms, prepared from these formulations, the active microbial strains and
itions according to the invention can furthermore be present in the form of a mixture
with inhibitors which reduce the degradation of the active compositions after application in
the t of the plant, on the surface of parts of plants or in plant tissues.
The active microbial strains and compositions according to the invention, as such
or in their ations, can also be used as a mixture with known izers, acaricides,
icides, fiangicides, insecticides, microbicides, nematicides, pesticides, or combinations
of any thereof, for example in order to widen the spectrum of action or to t the
development of resistances to pesticides in this way. In many cases, synergistic effects result,
z'.e., the activity of the mixture can exceed the activity of the individual components. A
e with other known active compounds, such as growth tors, safeners and/or
semiochemicals is also contemplated.
In a preferred embodiment of the present invention, the compositions may further
include at least one chemical or biological fertilizer. The amount of at least one chemical or
ical fertilizer employed in the compositions can vary depending on the final
formulation as well as the size of the plant and seed to be treated. Preferably, the at least one
al or biological fertilizer employed is about 0.1% w/w to about 80% w/w based on the
entire formulation. More preferably, the at least one chemical or biological fertilizer is
present in an amount of about 1% w/w to about 60% w/w and most preferably about 10%
w/w to about 50% w/w.
The microbiological compositions of the present invention preferably include at
least one biological fertilizer. ary biological fertilizers that are suitable for use herein
and can be included in a microbiological ition according to the present invention for
promoting plant growth and/yield include microbes, s, bacteria, fiangi, genetic
material, plant, and natural products of living organisms. In these compositions, the
microorganism of the present invention is isolated prior to formulation with an additional
sm. For example, microbes such as but not limited to species of Achromobacter,
Ampelomyces, Aareobasidiam, Azospl'rillam, Azotobacter, Bacillus, Beaaveria,
Bradyrhizobiam, Candida, Chaetomz'am, Cordyceps, Cryptococcas, Dabaryomyces, 'a,
Erwinia, Exophz'lz'a, Gliocladz'am, Herbaspz'rz'llam, Lactobacz'llas, Mariannaea, Microecocas,
Paecz'lomyces, Paenibacillas, Pantoea, , Pseudomonas, Rhizobz'am, Saccharomyces,
Sporobolomyces, Stenotrophomonas, Streptomyces, Talaromyces, and Trichoderma can be
provided in a composition with the microorganisms of the present invention. Use of the
microbiological compositions according to the present invention in ation with the
microbial microorganisms disclosed in US. Patent Appl. Nos. USZOO30172588A1,
US20030211119A1; US. Pat. Nos. 7,084,331; 7,097,830; 7,842,494; PCT Appl. No.
WO2010109436A1 is also particularly preferred.
In a preferred embodiment of the present ion, the compositions may further
include at least one chemical or biological pesticide. The amount of at least one chemical or
ical pesticide employed in the compositions can vary depending on the final
formulation as well as the size of the plant and seed to be treated. Preferably, the at least one
chemical or biological pesticide employed is about 0.1% w/w to about 80% w/w based on the
entire formulation. More preferably, the at least one chemical or biological pesticide is
present in an amount of about 1% w/w to about 60% w/w and most preferably about 10%
w/w to about 50% w/w.
A y of chemical pesticides is apparent to one of skill in the art and may be
used. Exemplary chemical pesticides include those in the carbamate, organophosphate,
organochlorine, and prethroid classes. Also included are chemical control agents such as, but
not limited to, benomyl, borax, captafol, captan, chorothalonil, formulations containing
copper; ations containing dichlone, an, iodine, zinc; fiangicides that inhibit
ergosterol biosynthesis such as but not limited to blastididin, cymoxanil, fenarimol,
flusilazole, folpet, imazalil, one, maneb, manocozeb, metalaxyl, oxycarboxin,
myclobutanil, oxytetracycline, PCNB, pentachlorophenol, prochloraz, onazole,
quinomethionate, sodium ite, sodium DNOC, sodium lorite, sodium
phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazole, thiabendazolel, thiophanate-
methyl, triadimefon, tricyclazole, triforine, validimycin, vinclozolin, zineb, and ziram.
The microbiological compositions of the t invention preferably include at
least one biological pesticide. Exemplary biological pesticides that are suitable for use herein
and can be included in a microbiological ition according to the present ion for
preventing a plant pathogenic disease include microbes, animals, bacteria, fungi, genetic
material, plant, and natural products of living organisms. In these compositions, the
microorganism of the present invention is isolated prior to formulation with an additional
organism. For example, es such as but not limited to species of Ampelomyces,
Aareobasidiam, Bacillus, Beaaveria, a, Chaetoml'am, Cordyceps, Cryptococcas,
Dabaryomyces, Erwinia, Exophz'lz'a, Gliocladz'am, Mariannaea, Paecilomyces, Paenibacillas,
Pantoea, Pichia, Pseudomonas, Sporobolomyces, Talaromyces, and Trichoderma can be
ed in a composition with the rganisms of the present invention, with fungal
strains of the Muscodor genus being particularly preferred. Use of the microbiological
compositions according to the present invention in combination with the microbial
nists disclosed in US Patent No. 7,518,040; US Patent No. 346; US Patent No.
6,312,940 is also ularly preferred.
Examples of fiangi that can be ed with microbial s and compositions
of the t invention in a composition include, without limitation, Muscodor species,
Aschersom'a aleyrodz's, Beauverl'a bassiana ("white muscarine"), Beauveria brongm'artz'z',
Chladosporl'um herbarum, Cordyceps clavulata, Cordyceps entomorrhl'za, Cordyceps facis,
eps gracilz's, Cordyceps melolanthae, Cordyceps militaris, Cordyceps myrmecophila,
Cordyceps ravenell'l', Cordyceps sinensis, Cordyceps sphecocephala, Cordyceps subsessz'lz's,
Cordyceps unilateralz's, Cordyceps variabilis, Cordyceps washingtonensis, Culicinomyces
clavosporus, phaga gryllz', Entomophaga maimaiga, Entomophaga muscae,
phaga bullz', Entomophthora plutellae, Fusarz'um laterz'tz'um, Hirsutella
citrz'formz's, Hirsutella thompsom', Metarhz'zz'um anisoplz'ae ("green muscarine"), Metarhz'zz'um
flaviride, Muscodor albus, Neozygz'tesflorz'dana, Nomuraea rileyi, Paecilomyces farinosus,
Paecz'lomyces roseus, Pandora neoaphidis, Tolypocladz'um cylindrosporum,
Verticz'llz'um lecam’z’, Zoophthora radicans, and mycorrhizal species such as Laccarz’a bicolor.
Other mycopesticidal species will be apparent to those skilled in the art.
The present invention also provides methods of treating a plant by application of
any of a variety of customary formulations in an effective amount to either the soil (i.e., in-
furrow), a n of the plant (i.e., drench) or on the seed before planting (126., seed coating
or dressing). Customary formulations include solutions, emulsifiable concentrate, wettable
powders, sion concentrate, soluble powders, granules, suspension-emulsion
concentrate, natural and synthetic materials nated with active compound, and very fine
control release capsules in polymeric substances. In certain embodiments of the present
invention, the microbial itions are formulated in s that are available in either a
ready-to-use formulation or are mixed er at the time of use. In either embodiment, the
powder may be admixed with the soil prior to or at the time of planting. In an alternative
embodiment, one or both of either the plant growth-promoting agent or biocontrol agent is a
liquid formulation that is mixed together at the time of treating. One of ordinary skill in the
art tands that an effective amount of the inventive compositions depends on the final
formulation of the composition as well as the size of the plant or the size of the seed to be
treated.
Depending on the final formulation and method of application, one or more
suitable additives can also be introduced to the compositions of the present ion.
ves such as carboxymethylcellulose and natural and synthetic polymers in the form of
powders, granules or latexes, such as gum arabic, chitin, polyvinyl alcohol and polyvinyl
e, as well as natural phospholipids, such as cephalins and lecithins, and synthetic
olipids, can be added to the present compositions.
In a preferred embodiment, the microbiological compositions are formulated in a
single, stable solution, or emulsion, or suspension. For solutions, the active chemical
compounds are typically dissolved in solvents before the ical agent is added. Suitable
liquid solvents include petroleum based aromatics, such as xylene, toluene or
alkylnaphthalenes, aliphatic arbons, such as cyclohexane or ns, for example
petroleum fractions, mineral and vegetable oils, alcohols, such as butanol or glycol as well as
their ethers and esters, ketones, such as methyl ethyl ketone, methyl isobutyl ketone or
cyclohexanone, strongly polar ts, such as dimethylformamide and dimethyl sulphoxide.
For on or suspension, the liquid medium is water. In one embodiment, the chemical
agent and biological agent are suspended in separate liquids and mixed at the time of
application. In a preferred embodiment of suspension, the chemical agent and biological
agent are combined in a ready-to-use formulation that exhibits a reasonably long shelf-life. In
use, the liquid can be d or can be applied foliarly as an ed spray or in-fiarrow at
the time of planting the crop. The liquid composition can be introduced in an effective
amount on the seed (i.e., seed coating or ng) or to the soil (226., III-filI'I'OW) before
germination of the seed or directly to the soil in contact with the roots by utilizing a variety of
techniques known in the art including, but not limited to, drip irrigation, sprinklers, soil
injection or soil drenching.
Optionally, stabilizers and buffers can be added, ing alkaline and alkaline
earth metal salts and organic acids, such as citric acid and ic acid, inorganic acids, such
as hydrochloric acid or sulfuric acid. Biocides can also be added and can include
formaldehydes or formaldehyde- releasing agents and derivatives of benzoic acid, such as phydroxybenzoic
acid.
Pathogens
The skilled artisan in the art will recognize that the methods and compositions
according to the t invention in principle can be applied to suppress the development of
any plant pathogens or any phytopathogenic diseases. It is not intended that the invention be
limited to a particular culture types or cell types. For example, microbial cells that undergo
complex forms of differentiation, filamentation, sporulation, etc. can also be used for the
methods and compositions of the present invention.
Examples of phytopathogenic diseases that are suitable for applications of the
s and materials of the present ions include, but are not limited to, diseases
caused by a broad range of pathogenic fungi. The methods of the present ion are
preferably applied against pathogenic fungi that are important or interesting for agriculture,
horticulture, plant biomass for the production of biofuel molecules and other chemicals,
and/or ry. Of particular interest are pathogenic Pseudomonas species (e.g.,
Pseudomonas solanacearum), Xylella fastidiosa; Ralstonia solanacearum, monas
campestris, Erwim'a ora, Fusarz'um species, Phytophthora species (e.g., P. infestans),
Botrytz's species, Leptosphaerz’a species, y mildews (Ascomycota) and rusts
iomycota), etc.
miting examples of plant pathogens of interest include, for instance,
Acremonium strictum, Agrobacterium tumefaciens, Alternarz'a ata, Alternarz'a solanz',
Aphanomyces euteiches, Aspergillus fumigatus, Athelz'a 'z', Aureobasidium pullulans,
Bz'polarz's zeicola, Botrytis cinerea, Calonectrl'a kyotensz's, Cephalosporz'um maydz's,
Cercospora medicagz'nz's, Cercospora sojina, Colletotrichum coccodes, Colletotrichum
fragarl'ae, Colletotrichum m'cola, Coniella dl'plodz'ella, opsz's psychromorbida,
Corynespora cassz'z'cola, Curvularz'a pallescens, Cylindrocladl'um crotalarz'ae, Dz'plocarpon
earlianum, Diplodl'a gossyz'na, Dz'plodl'a spp., Epicoccum , Erysz'phe cichoracearum,
Fusarz'um gramz'nearum, Fusarz'um oxysporum, Fusarz'um oxysporumfsp. tuberosz', Fusarz'um
prolz'feratum var. prolz'feratum, Fusarz'um solanz', Fusarz'um verticillz'oides, Ganoderma
boninense, 'chum candidum, Glomerella tucumanensis, Guignardz'a bidwellz'z',
Kabatz'ella zeae, Leptosphaerulina briosz'ana, Leptotrochila medicagz'nz's, Macrophomina,
Macrophomina lina, Magnaporthe , Magnaporthe oryzae, Microsphaera
manshurica, Monilz'm'a fructz’cola, haerella fijiensis, Mycosphaerella fragarz'ae ,
Nigrospora oryzae, Ophz'ostoma ulmz', Pectobacterz'um carotovorum, Pellz'cularz'a sasakl'z'
(Rhizoctom'a salami), Peronospora manshurica, Phakopsora pachyrhizz', Phoma faveata,
Phoma medicagz'nz's, Phomopsz's longicolla, Phytophthora cinnamomi, Phytophthora
erythroseptz'ca, Phytophthora l'ae, Phytophthora infestans, Phytophthora medicagz'nz's,
Phytophthora megasperma, Phytophthora palmz'vora, haera leucotrz'cha,
Pseudopezz'za medicagz'm's, Puccinia graml'm's subsp. Triticz' (UG99), Puccinia sorghz',
Pyricularz'a grisea, Pyricularz'a oryzae, Pythz'um ultimum, Rhizoctom'a ', Rhizoctom'a
zeae, Rosellz'nz'a sp., Sclerotz'm'a tiorum, Sclerotinina trl'folz'orum, Sclerotl'um 'z',
Septorz'a glycines, Septorz'a lycopersz'cz', Setomelanomma turcica, Sphaerotheca macularz's,
Spongospora subterranea, Stemphylz'um Sp, Synchytrl'um 'otl'cum, Thecaphora
(Angiosorus), Thielavz'opsz's, Tilletz'a indica, Trichoderma viride, Ustz'lago maydz's,
Verticz'llz'um albo-atrum, Verticz'llz'um ae, Verticz'llz'um dahlz'ae, Xanthomonas
axonopodz's, Xanthomonas oryzae pv. oryzae.
In a preferred embodiment of the present invention, the methods and materials of
the invention are useful in suppressing the development the pathogens Aspergillusfumz’gatus,
Botrytis cinerea, Cerpospora betae, Colletotrz'chum sp., Curvularl'a spp., Fusarz'um sp.,
Ganoderma boninense, Geotrz'chum candidum, Gibberella sp., Monographella sp.,
haerella fijiensis, Phytophthora palmz'vora, hthora ramorum, Penicillium sp.,
Pythz'um ultimum, Rhizoctom'a solam', Rhizopus spp., phyllum spp., Sclerotz'm'a
sclerotiorum, Stagnospora sp., Verticz'llz'um dahlz'ae, or Xanthomonas axonopodz's. In a
particularly red ment, the inventive methods and materials may be used to
suppress the development of several plant pathogens of cial importance, including
'um graminearum NRRL-5883, Monographella nivalz's ATCC MYA-3968, Gibberella
zeae ATCC-l6lO6, Stagnospora nodurum ATCC-26369, Colletotrz'chum graminz'cola
ATCC-34l67, and Penicillium sp. pathogens.
Seed coating formulation
In a particularly preferred embodiment, the microbial itions of the present
invention are formulated as a seed treatment. It is contemplated that the seeds can be
substantially mly coated with one or more layers of the microbial compositions
disclosed herein using tional methods of mixing, spraying or a combination thereof
through the use of treatment application equipment that is specifically ed and
manufactured to accurately, safely, and efficiently apply seed treatment products to seeds.
Such equipment uses various types of coating technology such as rotary coaters, drum
coaters, fluidized bed ques, spouted beds, rotary mists or a combination f. Liquid
seed treatments such as those of the present invention can be d via either a spinning
"atomizer" disk or a spray nozzle which evenly distributes the seed treatment onto the seed as
it moves though the spray pattern. Preferably, the seed is then mixed or tumbled for an
additional period of time to achieve additional treatment distribution and drying. The seeds
can be primed or ed before coating with the inventive compositions to increase the
uniformity of germination and emergence. In an ative embodiment, a dry powder
ation can be metered onto the moving seed and allowed to mix until completely
distributed.
Another aspect of the invention provides seeds treated with the subject microbial
compositions. One embodiment provides seeds having at least part of the surface area coated
with a microbiological composition according to the present invention. In a specific
embodiment, the microorganism-treated seeds have a microbial spore concentration or
microbial cell concentration from about 106 to about 109 per seed. The seeds may also have
more spores or microbial cells per seed, such as, for example 1010, 1011 or 1012 spores per
seed. The microbial spores and/or cells can be coated freely onto the seeds or, preferably,
they can be formulated in a liquid or solid composition before being coated onto the seeds.
For example, a solid ition comprising the microorganisms can be prepared by mixing
a solid carrier with a sion of the spores until the solid carriers are impregnated with the
spore or cell suspension. This mixture can then be dried to obtain the desired particles.
In some other embodiments, it is contemplated that the solid or liquid microbial
compositions of the t invention further contain functional agents capable of protecting
seeds from the harmful effects of selective herbicides such as ted carbon, nutrients
(fertilizers), and other agents capable of ing the germination and quality of the
products or a combination thereof.
Seed g methods and compositions that are known in the art can be
particularly useful when they are modified by the addition of one of the embodiments of the
present invention. Such coating methods and apparatus for their application are disclosed in,
for example, US. Pat. Nos. 5,918,413; 5,554,445; 5,389,399; 4,759,945; and 4,465,017. Seed
coating compositions are disclosed, for example, in US. Pat. Appl. No. US20100154299,
US. Pat. Nos. 5,939,356; 5,876,739, 5,849,320; 5,791,084, 5,661,103; 5,580,544, 5,328,942;
4,735,015; 587; 080, 4,339,456; and 4,245,432, among others.
WO 90628 2012/069579
A variety of additives can be added to the seed treatment formulations comprising
the inventive compositions. Binders can be added and include those composed preferably of
an adhesive polymer that can be natural or synthetic Without phytotoxic effect on the seed to
be . The binder may be selected from polyvinyl es; polyvinyl acetate copolymers;
ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; nyl alcohol copolymers;
celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses,
hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones;
ccharides, including starch, modified starch, dextrins, maltodextrins, alginate and
chitosans; fats; oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene
chloride and vinylidene chloride copolymers; m lignosulfonates; acrylic copolymers;
polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers;
polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.
Any of a variety of colorants may be employed, including organic chromophores
classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine,
anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine,
phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that can be
added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt,
molybdenum and zinc. A polymer or other dust control agent can be applied to retain the
ent on the seed surface.
In some specific ments, in on to the microbial cells or spores, the
coating can further comprise a layer of adherent. The adherent should be non-toxic,
biodegradable, and adhesive. Examples of such materials include, but are not limited to,
nyl acetates; polyvinyl acetate mers; polyvinyl alcohols; polyvinyl alcohol
copolymers; celluloses, such as methyl celluloses, hydroxymethyl celluloses, and
hydroxymethyl propyl celluloses; dextrins; alginates; sugars; molasses; polyvinyl
pyrrolidones; polysaccharides; proteins; fats; oils; gum arabics; gelatins; ; and starches.
More examples can be found in, for example, US. Pat. No. 7,213,367 and US. Pat. Appln.
OlOOl$9693.
Various additives, such as adherents, dispersants, surfactants, and nutrient and
buffer ingredients, can also be included in the seed treatment formulation. Other conventional
seed treatment additives include, but are not d to, coating agents, wetting agents,
buffering agents, and polysaccharides. At least one agriculturally acceptable carrier can be
added to the seed treatment formulation such as water, solids or dry powders. The dry
powders can be derived from a variety of materials such as calcium carbonate, gypsum,
vermiculite, talc, humus, activated charcoal, and various phosphorous compounds.
In some embodiment, the seed coating composition can comprise at least one filler
which is an organic or inorganic, natural or synthetic component with which the active
components are combined to facilitate its application onto the seed. Preferably, the filler is an
inert solid such as clays, natural or synthetic silicates, silica, , waxes, solid fertilizers
(for example ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz,
attapulgite, montmorillonite, bentonite or diatomaceous , or synthetic minerals, such as
silica, alumina or silicates, in particular ium or magnesium silicates.
The seed treatment formulation may fiarther include one or more of the following
ingredients: other pesticides, including compounds that act only below the ;
fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and s of each of
those materials, and the like; herbicides, including compounds ed from glyphosate,
carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, ol ethers,
pyridazinones, uracils, phenoxys, ureas, and benzoic acids; herbicidal safeners such as
benzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide, various dihaloacyl,
idinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and
oxime derivatives; chemical fertilizers; ical fertilizers; and trol agents such as
other naturally-occurring or recombinant bacteria and fungi from the genera Rhizobz'um,
Bacillus, monas, Serratz'a, Trichoderma, Glomus, Gliocladz'um and mycorrhizal fungi.
These ingredients may be added as a separate layer on the seed or alternatively may be added
as part of the seed g composition of the invention.
Preferably, the amount of the novel composition or other ingredients used in the
seed treatment should not inhibit germination of the seed, or cause phytotoxic damage to the
seed.
The ation that is used to treat the seed in the present invention can be in the
form of a suspension; emulsion; slurry of particles in an aqueous medium (e.g., ;
wettable powder; wettable granules (dry flowable); and dry granules. If ated as a
suspension or slurry, the concentration of the active ingredient in the formulation is
preferably about 0.5% to about 99% by weight (w/w), preferably 5-40% or as otherwise
formulated by those skilled in the art.
As mentioned above, other conventional inactive or inert ingredients can be
orated into the formulation. Such inert ingredients include but are not limited to:
conventional sticking agents; dispersing agents such as methylcellulose, for example, serve as
combined dispersant/sticking agents for use in seed treatments; polyvinyl alcohol; lecithin,
polymeric dispersants (e. g., polyvinylpyrrolidone/vinyl acetate); thickeners (e.g, clay
thickeners to improve viscosity and reduce settling of particle sions); emulsion
stabilizers; surfactants; antifreeze compounds (e.g, urea), dyes, colorants, and the like.
Further inert ingredients useful in the present invention can be found in McCutcheon's, vol. 1,
"Emulsifiers and Detergents," MC hing Company, Glen Rock, N.J., U.S.A., 1996.
Additional inert ingredients useful in the present invention can be found in McCutcheon's,
vol. 2, "Functional Materials," MC Publishing Company, Glen Rock, N.J., , 1996.
The coating formulations of the present invention can be applied to seeds by a
variety of methods, including, but not limited to, mixing in a container (e.g., a bottle or bag),
mechanical application, tumbling, ng, and immersion. A variety of active or inert
material can be used for contacting seeds with microbial compositions according to the
present invention, such as conventional film-coating materials including but not limited to
water-based film coating materials such as SEPIRETTM (Seppic, Inc., NJ.) and
OPACOATTM (Berwind Pharm. Services, PA.)
The amount of a composition according to the present invention that is used for
the treatment of the seed will vary depending upon the type of seed and the type of active
ingredients, but the treatment will comprise contacting the seeds with an agriculturally
ive amount of the inventive composition. As discussed above, an ive amount
means that amount of the ive ition that is sufficient to affect beneficial or
desired results. An effective amount can be stered in one or more administrations.
] In addition to the coating layer, the seed may be treated with one or more of the
following ingredients: other pesticides including fungicides and herbicides; herbicidal
safeners; fertilizers and/or biocontrol agents. These ients may be added as a separate
layer or alternatively may be added in the coating layer.
] The seed coating formulations of the present invention may be applied to the
seeds using a variety of ques and machines, such as fluidized bed techniques, the roller
mill method, rotostatic seed treaters, and drum coaters. Other methods, such as spouted beds
may also be useful. The seeds may be pre-sized before coating. After coating, the seeds are
typically dried and then transferred to a sizing machine for sizing. Such procedures are
known in the art.
The microorganism-treated seeds may also be enveloped with a film overcoating
to t the coating. Such overcoatings are known in the art and may be applied using
fluidized bed and drum film coating techniques.
In another embodiment of the present invention, compositions according to the
present ion can be uced onto a seed by use of solid matrix priming. For example,
a ty of an inventive composition can be mixed with a solid matrix material and then the
seed can be placed into contact with the solid matrix material for a period to allow the
composition to be introduced to the seed. The seed can then optionally be separated from the
solid matrix material and stored or used, or the mixture of solid matrix material plus seed can
be stored or planted directly. Solid matrix materials which are useful in the present invention
include polyacrylamide, starch, clay, silica, alumina, soil, sand, ea, rylate, or any
other material capable of absorbing or adsorbing the inventive composition for a time and
releasing that composition into or onto the seed. It is useful to make sure that the inventive
composition and the solid matrix material are compatible with each other. For example, the
solid matrix material should be chosen so that it can e the composition at a reasonable
rate, for example over a period of minutes, hours, or days.
In principle, any plant seed capable of germinating to form a plant can be treated
in accordance with the invention. Suitable seeds include those of cereals, coffee, cole crops,
f1ber crops, flowers, , legume, oil crops, trees, tuber crops, vegetables, as well as other
plants of the monocotyledonous, and ledonous s. Preferably, crop seeds are
coated include, but are not limited to, bean, carrot, corn, cotton, grasses, lettuce, peanut,
pepper, potato, rapeseed, rice, rye, sorghum, soybean, sugarbeet, sunflower, tobacco, and
tomato seeds. Most preferably, barley or wheat (spring wheat or winter wheat) seeds are
coated with the t compositions.
Preparing the microbial compositions according to the present invention
Cultures of the microorganisms may be prepared for use in the microbial
compositions of the invention using rd static drying and liquid fermentation techniques
known in the art. Growth is commonly ed in a bioreactor.
A bioreactor refers to any device or system that supports a biologically active
environment. As described herein a bioreactor is a vessel in which microorganisms including
the microorganism of the invention can be grown. A bioreactor may be any riate shape
or size for growing the microorganisms. A bioreactor may range in size and scale from 10
mL to liter’s to cubic meters and may be made of stainless steel or any other appropriate
material as known and used in the art. The bioreactor may be a batch type bioreactor, a fed
batch type or a continuous-type bioreactor (e.g., a continuous stirred r). For e, a
bioreactor may be a chemostat as known and used in the art of iology for growing and
harvesting microorganisms. A ctor may be obtained from any cial supplier (See
also Bioreactor System Design, Asenjo & Merchuk, CRC Press, 1995).
For small scale operations, a batch bioreactor may be used, for example, to test
and develop new processes, and for ses that cannot be converted to continuous
operations.
rganisms grown in a ctor may be suspended or immobilized. Growth
in the bioreactor is generally under aerobic conditions at suitable temperatures and pH for
growth. For the organisms of the invention, cell growth can be achieved at temperatures
between 5 and 37°C, with the preferred temperature being in the range of 15 to 30°C, 15 to
28°C, 20 to 30°C, or 15 to 25°C. The pH of the nutrient medium can vary n 4.0 and
9.0, but the preferred operating range is usually slightly acidic to neutral at pH 4.0 to 7.0, or
4.5 to 6.5, or pH 5.0 to 6.0. Typically, maximal cell yield is obtained in 20-72 hours after
inoculation.
Optimal conditions for the cultivation of the microorganisms of this invention
will, of course, depend upon the particular strain. However, by virtue of the conditions
applied in the selection process and general requirements of most microorganisms, a person
of ry skill in the art would be able to determine essential nutrients and conditions. The
microorganisms would typically be grown in aerobic liquid cultures on media which contain
sources of carbon, nitrogen, and inorganic salts that can be assimilated by the microorganism
and supportive of efficient cell growth. Preferred carbon sources are hexoses such as glucose,
but other sources that are readily lated such as amino acids, may be substituted. Many
inorganic and proteinaceous als may be used as nitrogen sources in the growth s.
Preferred en sources are amino acids and urea but others include gaseous ammonia,
inorganic salts of nitrate and ammonium, vitamins, purines, pyrimidines, yeast extract, beef
extract, proteose peptone, soybean meal, hydrolysates of casein, distiller's solubles, and the
like. Among the inorganic ls that can be incorporated into the nutrient medium are the
customary salts e of yielding calcium, zinc, iron, ese, ium, copper,
cobalt, potassium, sodium, molybdate, phosphate, e, chloride, borate, and like ions.
Without being limited thereto, use of potato dextrose liquid medium for fungal strains and
R2A broth premix for bacterial strains is preferred.
Novel p_lant varieties
] Also ed, in another aspect of the present invention, is a novel plant created
by artificially introducing a microbial endophyte of the invention into a plant that is fiee of
endophytic microorganisms. In some ments of this aspect, the microbial endophyte
introduced into the plant may be an endophytic microorganism having a plant growth-
promoting activity, a biological control activity, or a combination of both activities. A variety
of methods previously found effective for the introduction of a ial yte into
cereal grass species are known in the art. Examples of such methods include those described
in US. Pat. Appl. No. 20030195117Al, US. Pat. Appl. No. 20010032343Al, and US. Pat.
No. 7,084,331, among others. It will become nt to those skilled in the art that many of
the aforementioned methods can be useful for the making of a novel plant of the invention.
After artificial infection, it is preferred that a DNA sequence of the isolated
endophytic microorganism is amplified by PCR and the endophyte is confirmed by carrying
out a homology search for the DNA sequence amplified. Further, it is preferred that a foreign
gene that expresses an identifiable means is introduced into the above-mentioned endophytic
microorganism, and the presence of the colonization of the above-mentioned endophytic
microorganism infecting the plant is confirmed by the above-identifiable means using the
foreign gene.
Plants suitable for the methods of the invention
In principle, the methods and compositions according to the t invention can
be ed for any plant species. Monocotyledonous as well as dicotyledonous plant s
are particularly suitable. The methods and compositions are preferably used with plants that
are important or interesting for agriculture, horticulture, for the production of biomass used in
producing liquid fuel molecules and other als, and/or forestry.
Thus, the invention has use over a broad range of plants, preferably higher plants
pertaining to the classes ofAngiospermae and Gymnospermae. Plants of the subclasses of the
Dicotylodenae and the Monocotyledonae are particularly suitable. Dicotyledonous plants
belong to the orders of the Aristochiales, Asterales, s, Campanulales, Capparales,
Caryophyllales, Casuarinales, Celastrales, Cornales, sales, Dilleniales, Dipsacales,
Ebenales, Ericales, Eucomiales, biales, Fabales, Fagales, Gentianales, Geraniales,
Haloragales, lidales, Illiciales, Juglandales, Lamiales, Laurales, Lecythidales,
Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales,
Piperales, Plantaginales, Plumbaginales, Podostemales, Polemoniales, Polygalales,
Polygonales, ales, les, Rafilesiales, Ranunculales, Rhamnales, Rosales,
Rubiales, Salicales, es, Sapindales, Sarraceniaceae, ulariales, s,
Trochodendrales, Umbellales, Urticales, and Violales. Monocotyledonous plants belong to
the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales,
Cyperales, Eriocaulales, Hydrocharitales, es, Lilliales, Najadales, Orchidales,
Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plants belonging
to the class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales.
Suitable species may include members of the genus Abelmoschus, Abies, Acer,
Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo,
Atropa, Berberis, Beta, Bixa, ca, Calendula, Camellia, Camptotheca, Cannabis,
Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus,
Coflea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,
Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria,
Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha,
Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha,
thus, Musa, Nicotiana, Oryza, m, Papaver, Parthenium, Pennisetum, Petunia,
Phalaris, , Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum,
Salix, naria, Scopolia, Secale, Solanum, Sorghum, Spartina, ea, tum,
Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.
The methods and compositions of the present invention are preferably used in
plants that are important or interesting for agriculture, horticulture, biomass for the
production of biofuel molecules and other chemicals, and/or forestry. Non-limiting examples
include, for instance, Panicum virgatum (switchgrass), Sorghum r (sorghum,
sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. ycane), Populus
balsamifera (poplar), Zea mays (corn), Glycine max (soybean), ca napus (canola),
Triticum aestivum (wheat), Gossypium hirsutum n), Oryza sativa , Helianthus
annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), Pennisetum
glaucum (pearl millet), Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp.,
hus spp., Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum
(elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon clactylon
(bermudagrass), a arundinacea (tall fescue), Spartina pectinata (prairie cord-grass),
Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.
(eucalyptus), osecale spp. (triticum--wheat X rye), Bamboo, Carthamus tinctorius
(safflower), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis (oil
palm), Phoenix clactylifera (date palm), Archontophoenix cunninghamiana (king palm),
Syagrus romanzofiana (queen palm), Linum usitatissimum (flax), Brassica juncea, Manihot
esculenta (cassaya), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa
paradisiaca (banana), Solanum tuberosum (potato), ca ea (broccoli, cauliflower,
brusselsprouts), ia sinensis (tea), Fragaria ananassa berry), Theobroma cacao
(cocoa), Cofi’ea arabica e), Vitis vinifera ), Ananas comosus (pineapple),
Capsicum annum (hot & sweet pepper), Allium cepa (onion), s melo (melon),
Cucumis sativus (cucumber), Cucurbita maxima h), Cucurbita moschata (squash),
Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra),
Solanum melongena (eggplant), Papaver somniferum (opium poppy), Papaver orientale,
Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate,
Catharanthus roseus, Vinca rosea, Cinchona ofiicinalis, Coichicum autumnale, Veratrum
californica, Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographis paniculata,
Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica,
Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum
(Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, fia spp., Sanguinaria
canadensis, Hyoscyamus spp., Calendula ofiicinalis, Chrysanthemum parthenium, Coleus
forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber),
Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp.
, Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima
(poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats),
bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (f1r),
Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp.
(ryegrass), Phleum pratense (timothy), and conifers. Of interest are plants grown for energy
production, so called energy crops, such as cellulose-based energy crops like Panicum
virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus
(miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Andropogon
gerardii (big bluestem), Pennisetum purpureum (elephant , Phalaris arundinacea (reed
canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina
pectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giant reed), Secale
cereale (rye), Salix spp. w), Eucalyptus spp. (eucalyptus), osecale spp. (triticum—
wheat X rye), and ; and starch-based energy crops like Zea mays (corn) and Manihot
esculenta (cassava); and sugar-based energy crops like Saccharum sp. (sugarcane), Beta
vulgaris beet), and Sorghum bicolor (L) Moench (sweet sorghum); and biofiJel-
producing energy crops like Glycine max (soybean), Brassica napus (canola), Helianthus
annuus wer), Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinus
communis (castor), Elaeis guineensis an oil palm), Elaeis oleifera (American oil palm),
Cocos nucifera (coconut), Camelina sativa (wild flax), Pongamia pinnata (Pongam), Olea
europaea ), Linum usitatissimum (flax), Crambe abyssinica (Abyssinian-kale), and
Brassicajuncea.
The discussion of the general methods given herein is intended for rative
purposes only. Other alternative methods and embodiments will be apparent to those of skill
in the art upon review of this sure, and are to be included within the spirit and w
of this ation.
It should also be tood that the following examples are offered to illustrate,
but not limit, the invention.
EXAMPLES
EXAMPLE 1: Microorganism isolation from environmental samples
Identification of spore-forming rhizobacteria using a sonicated roots and serial
dilutions method. The ing microorganisms were isolated using a “sonicated roots,
serial dilutions” method as described below: the 6-G06 and SGIG07 isolates,
which were isolated from a needle-like grass sample; the SGI-04l-B03 isolate, which was
isolated from a wild rye sample; and the O-AOl isolate, which was isolated from a
wheat root tissues grown in a composite soil sample.
An enrichment procedure was developed to specifically fy spore-forming
rhizobacteria. Briefly, sonicated root extracts were heat treated to kill vegetative cells and
then plated onto a rich medium. Microorganisms that survived the heat treatment and formed
colonies were considered to be spore-formers. This method was found to be particularly
effective for selection of Gram-positive bacteria. Freshly sampled roots were used as starting
material for these enrichments. Fine sections found at the tip of roots are the youngest, can
have a high root hair density, and typically have high densities of rhizobacteria. A sterile
blade was used to section these areas of the roots into 5 - 10 cm ts, which were then
washed under sterile milliQ water to remove large soil particles. When needed, a more
rigorous wash was accomplished by g the roots into a 50 mL Falcon tube with 25 mL 1
X sterile phosphate buffered saline (1.6. PBS buffer) and vortexing for 1 minute. Each root
sample was subsequently suspended in 20 mL sterile PBS buffer and sonicated on ice for two
l-minute intervals at 8 watts using a Fisher Scientific Sonic Dismembrator. For heat
treatment, typically 1 mL of the sonicated root cell sion was transferred into a e
Eppendorf tube incubated in an 80°C water bath for 20 minutes. The heat treated cell
suspensions were allowed to cool to room temperature before ly diluted to
concentrations of 101, 102, 10'3, 10“, 10's, 106, and 107. 100 uL of each 10-fold dilution
was spread onto culture plates containing a microbiological medium fied with agar and
100 mg/L cycloheximide to inhibit fungal growth. In some cases, it was necessary to perform
a 1/10 or 1/100 dilution prior to plating in order to obtain the proper CFU density for colony
picking. Isolated es were picked using sterile pipette tips, arrayed into 96-well
microtiter plates each containing 150 uL 2 X YT liquid medium per well. The microtiter
plates were incubated for 1-2 days at 30°C in order to obtain a high cell y for r
characterization and archiving.
2012/069579
ion of biofilm-forming bacteria. The following microbial isolates were
isolated using a “biofilm former” method as described below: the 3-Hll isolate,
which was ed from a Yucca plant root sample; the SGIC09 isolate, which was
isolated from a grass root sample; and the 4-ElO isolate, which was from a Queen
Anne’s Lace plant sample.
Biofilm former method: In this procedure, biofilm-forming bacteria were ed
from sonicated root segments, as described by Fall et al. (Syst. Appl. Microbiol. 27,372-379,
2004). As described above, bacteria that form biofilms the e of a root are typically very
good root colonizing bacteria, In general, when such bacteria are present at high densities,
they can have a significant influence on plant health and can competitively exclude invading
pathogens. Briefly, sonication was used to remove bacterial and fungal cells that are loosely
attached to the root, leaving behind only those microbes that were strongly adhered to the
root surface. Both Gram-positive and Gram-negative biofilm-forming ia were selected
using this method.
y sampled roots were used as starting material for these enrichments. Fine
sections found at the tip of roots were the youngest tissues, had a high root hair density and
typically had high densities of rhizobacteria. A sterile blade was used to section these areas of
the roots into 5 - 10 cm segments, which were then washed by placing them into a 50 mL
Falcon tube with 25 mL l X PBS and vortexed for 1 minute. The debris from the wash was
allowed to settle, and then a sterile forceps was used to transfer the washed root segments to
50 mL Falcon tubes filled with 25 mL l X PBS, and sonicated on ice using a Fisher Scientific
Sonic Dismembrator for two 30 second intervals with a 30 second pause between . The
sonicated root samples were transferred to sterile plastic Petri dishes and allowed to dry
completely without lids inside a biosafety cabinet. Each root segment was then placed onto a
separate CMA plate containing 1% agar (10 g/L Casein digest, 10 g/L mannitol, 10 g/L agar).
Sometimes, a sterile forceps was used to push the root t into the agar media. The
plates were subsequently ted at 37°C and monitored for microbial growth. Typically
after 1-2 days, multiple microbial growths d from the root and onto the CMA media.
A sterile pipette tip was used to pick growths with unique morphologies along the segment
and each of these growths was transferred to the center of a CMA plate containing 0.3%
agarose. The CMA plates were uently incubated for 1-2 days at 37°C and monitored
for growth. Typically, biofilm-forming isolates displayed dendritic growth on this medium.
] A sterile loop was used to transfer biomass and streak-purify each isolate from the
CMA plates onto CMKA plates (2% agar, 1.2 g/L K2HPO4). The CMKA medium restricts
biofilm growth and allows for the picking of individual colonies for archiving.
EXAMPLE 2: Growth and storage of the ial isolates
The isolated bacteria were stored as a pure e. A bacterial colony was
transferred to a vial containing R2A broth liquid medium (Tecknova) and d to grow at
°C with shaking at 250 rpm for two days. The e was then transferred into vials
containing 15% glycerol and stored at -80°C.
EXAMPLE 3: DNA extraction, Sequencing and Taxonomy
A 20 ul aliquot of bacterial cell suspension was transferred to a 96-well PCR plate
containing 20 ul of a 2x lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1%
SDS, 400 ug/mL Proteinase K). Lysis conditions were as follows: 55°C incubation for 30
minutes, followed by 94°C incubation for 4 minutes. An aliquot of the lysis product was used
as the source of template DNA for PCR amplification.
For amplification of 16S rRNA region, each PCR mixture was prepared in a 20 ul
final volume reaction containing 4 ul of the bacterial lysis reaction, 2 uM of each PCR
primer, 6% Tween-20, and 10 ul of 2x ImmoMix (Bioline USA Inc, Taunton, MA). The
primers used for PCR amplification were M13-27F (5 ’ -
TGTAAAACGACGGCCAGTTAGAGTTTGATCCTGGCTCAG-3’ SEQ ID NO: 8) and
1492R M13-tailed (5’-CAGGAAACAGCTATGACCGGTTACCTTGTTACGACTT-3’;
SEQ ID NO: 9). The PCR was carried out in a PTC-200 personal thermocycler (MJ-
Research, MA, USA) as follows: 94°C for 10 minutes; 94°C for 30 seconds, 52°C for 30
seconds, 72°C for 75 s for 30 cycles; 72°C for 10 minutes. A 2 ul t of each PCR
product was run on a 1.0% e gel to confirm a single band of the expected size. Positive
bands were isolated, purified, and submitted for PCR cing. Sequencing was performed
in the forward and reverse priming directions by the J. Craig Venter Institute in San Diego,
Calif. using 454 technologies.
Homology search for the determined nucleotide sequence was conducted using the
DDBJ/GenBank/EMBL database. uently, the phylogenetic relationship of the
nucleotide sequence of the 16 rRNA genes was analyzed among the isolated ial strains
described herein, bacteria of the genera and species that exhibit high sequence homologies to
2012/069579
the isolated ial strains, and other wide varieties of bacterial genera and species, using
the ClustalW phylogenetic tree building program. Sequence identity and similarity were also
determined using GenomeQuestTM software (Gene-IT, Worcester Mass. USA). The sequence
analysis result revealed that the ial isolates SGI-003_Hl l, SGI-020_A01, SGI-
026_G06, SGI—026_G07, SGI—034_C09, SGI-034_E10, SGI—04l_B03 can be considered to
be related to the species of a agglomerans, Bacillus thuringiensis, lderz’a
metallica, Burkholderia vietnamz'ensz's, Bacillus pumilus, Herbaspirillum sp., Pedobacter sp.,
respectively, based upon >98% sequence homologies of each of the 16 rRNA sequences to
the respective microorganisms.
EXAMPLE 4: Biochemical Characteristics of the Bacterial Isolates
The isolated bacteria were further studied for properties important in their
interaction with plants. The studied properties included nitrogen fixation, siderophore
secretion, lization of inorganic phosphorus, production of ocyclopropane-l-
carboxylic acid (ACC) deaminase, tion of 2,3 diol, and the production of plant
growth hormone auxin. The s of in vitro mical assays are shown in Table 2.
Nitrogen fixation:
Bacterial cell suspensions were streaked on a solid medium of the following
composition which did not include a nitrogen source: KOH 4.0 g/L; K2HPO4 0.5 g/L;
MgSO4'7H20 0.2 g/L; NaCl 0.1 g/L; CaClz 0.02 g/L; FeSO4'7H20 0.005g/L;
NaMoO4'2H20 0.002 g/L; MnSO4'7H20 0.01 g/L; Malic Acid 5.0 g/L; Gellan Gum 0.l —
1.0 g/L; and optionally 0.5% v/v Bromothymol blue, pH 7.0. Gellan gum or agar
concentrations may be varied as necessary to achieve desired medium thickness; typically 0.5
g/L was used. Streaks were incubated at 30°C for 2 — 5 days. These plates were monitored
daily and colonies were ed as they appeared. In some cases, longer growth periods (up
to two weeks or greater) allowed for the capture of slower growing es. These streak
plates were typically colony-picked using 20 or 200 uL aerosol barrier pipette tips into 96-
well cell e plates filled with 150 uL/well of 2YT medium. Alternatively, isolates were
colony-picked from plates directly into N—free medium to confirm their N—free growth
abilities. The results, as summarized in Table 2, indicated that only the isolate SGIG07
showed nitrogen fixing activity at a detectable level.
Siderophore secretion:
This assay was used to identify bacterial isolates that were producing
siderophores, which are high-affinity Fe3+-chelating compounds, in vitro. Typically, the
microbial isolates were cultured on a minimal medium which was essentially free of Fe. All
glassware used throughout this assay was acid-washed and rinsed three times with milliQ
water to remove residual Fe which may alter assay results. The composition of the MM9
medium was as s: K2HPO4 0.5g/L; NH4Cl l.0g/L; MgSO4'H20 ; NaCl 05ng
PIPES Buffer 7.55g/L; Glucose 10.0g/L; Gluconic Acid 2.5g/L; Malic Acid 2.5g/L;
Casamino Acids 0.5 g/L. The medium was ed to pH 7.0 with 5N KOH, and sterilized
using a 0.2 uM filter (Corning).
This assay was typically run in a high-throughput format using a Beckman FX
liquid handling station and l cell culture plates with 150 uL MM9 growth medium per
well. Cultures and media were distributed and transferred aseptically using an autoclavable
pin-tool under a laminar flow hood. Following transfer, cultures were incubated at 30°C for 5
days. After tion, the culture supematants were ted via centrifiJgation using a 96-
well 0.22 uM filter plate. Ten microliters of filtered supernatant was transferred from each
well to a Falcon assay plate. A standard curve was prepared using desferrioxamine (DFO)
diluted in MM9 medium. Two-hundred microliters of the CAS assay solution [10 mM
HDTMA, Fe(III)-Solution: 1 mM F6C13.6H20, 10 mM HCl, 2 mM CAS] was added to each
of the supematants and standard wells, followed by incubation at room temperature for 20 —
minutes. The absorbance of the blue CAS assay on at 630 nm (SpectroMax M2) is
ely proportional to the siderophore concentration in each well (i.e., the assay solution
should change to an intense orange with greater quantities of siderophores).
Solubilization of inorganic phosphorus:
The ability of the ial isolates to lize l phosphate in vitro was
assessed as follows. Bacteria to be tested were streaked on an agar phosphate growth medium
[Hydroxylapatite — Ca10(PO4)5(OH)2 5.0g/L; NH4Cl l.0g/L; MgSO4'H20 0.2g/L; NaCl
05ng 7H20 0.01g/L; NazMoO4'7H20 0.01g/L; MnSO4'7H20 0.01g/L; Glucose
; Gluconic Acid 2.5g/L; Malic Acid 2.5 g/L; Casamino Acids 0.5g/L; Gellan Gum
.0g/L; pH 7.2)], and their growth was monitored daily. The culture medium had an opaque
appearance due the present of calcium phosphate. Bacterial growth and loss of the color of
the medium would be observed if the bacteria have dissolving ability of calcium phosphate.
Isolates having the y to solubilize the mineral phase phosphate would produce a clear
halo on the opaque medium surrounding the colony. As summarized in Table 2, the ability to
solubilize mineral phosphate was not able in any of the tested microorganisms as
determined by the in vitro assay described herein.
ACC deaminase p_roduction:
One of the major isms utilized by plant growth-promoting acteria
(PGPM) to facilitate plant grth and development is the lowering of ethylene levels by
deamination of l-aminocyclopropane-l-carboxylic acid (ACC), the immediate sor of
ethylene in plants. ACC deaminase catalyzes the hydrolysis of l-aminocyclopropane-l-
carboxylic acid (ACC) into (x-ketobutyrate and ammonia. The presence of the obutyrate
product can then be determined indirectly via a reaction with 2, 4-dinitrophenylhydrazine in
HCl to form a phenylhydrazone derivative. After an addition of NaOH, the amount of
ydrazone in solution can be determined spectrophotometrically by measuring its
absorbance at 540 nm (Penrose and Glick, Physiol Plant. May;ll8:lO-l, 2003). This assay
was typically run in a high-throughput format using 96-well cell culture plates. Each well
contained 150 uL DF salts growth medium supplemented with 2.0 g/L (NH4)2SO4. es
and media were distributed and transferred aseptically using an autoclavable pin-tool under a
laminar-flow hood. Following transfer, cultures were ted at 30°C for 2 days. After
ng turbidity, the cultures were transferred a second time using a sterile pin-tool under a
laminar-flow hood into 96-well plates containing 150 uL per well of DF salts grth medium
supplemented with 5 mM ACC as the sole nitrogen source, followed by a 4 day incubation at
°C. Absorbance of each culture at 600 nm was measured using a spectrophotometer.
es that displayed robust growth under these conditions (OD >0.2) were taken forward
for fiarther assay for ACC deaminase activity as described in Penrose and Glick, 2003, supra.
The test results, as summarized in Table 2, indicated that the following isolates
produced significant amounts of ACC deaminase: SGIHll, SGIG06, SGI
G07, and SGI-04l-B03.
2,3-butanediol production:
The ability of the bacterial isolates to synthesize tanediol in vitro was
assessed as follows using capillary gas chromatography mass oscopy as described by
Ryu et al. (Proc. Natl. Acad. Sci. USA. 100:4927-4932, 2003). This assay was typically run
in a hroughput format using 96-well cell culture plates with 150 uL DF salts growth
medium per well. A titer-tek may also be used when preparing a large number of plates for
primary screens of large isolate collections. Cultures and media were distributed and
erred aseptically using an autoclavable pin-tool under a laminar-flow hood. Following
transfer, cultures were incubated at 30°C for 5 days. After incubation, the culture supematants
were harvested via fiagation using a 96-well 0.22 uM filter plate. Fifty microliters of
filtered atant from each well was transferred to corresponding wells of a deep 96-well
plate containing 450 uL 50% methanol per well using a L200 multichannel pipette and sealed
with an adhesive plate seal, ed by 2,3-butanediol quantification assay using the
protocol described by Ryu et al. (2003, supra). The test results, as summarized in Table 2,
indicated that the following isolates ed significant s of 2, 3-butanediol: SGI-
003-Hl l, SGIC09, and SGI-04l-B03.
Production of auxin:
Auxins are es that can directly affect plant growth. This assay was
performed to determine if the bacterial isolates produced auxins, since many rhizosphere and
endophytic bacterial isolates are known to possess biochemical pathways that synthesize the
auxin indoleacetic acid (IAA) and its derivatives. Tryptophan is often a precursor in this
synthesis; and therefore, this assay quantified 1AA (auxin) production from bacterial es
grown on a medium supplemented with a low concentration of the amino acid phan.
This assay was typically run in a high-throughput format using 96-well cell
culture plates with 150 uL YT growth medium per well. When preparing a large number of
plates for primary screens of large isolate collections, a titer-tek was used. Cultures and
media were distributed and transferred aseptically using an autoclavable pin-tool under a
laminar-flow hood. Following transfer, cultures were incubated at 30°C for 5 days. After
incubation, the culture supematants were ted via filgation using a l 0.22
uM filter plate. Ten microliters of filtered supernatant from each well was transferred to a
Falcon assay plate. Two hundred microliters of the sky’s assay solution (Gordon and
Weber, Plant Physiol. 26:192-195, 1951) was added to each of the supernatant and standard
wells, followed by incubation at room temperature for 15 — 20 minutes. The reaction was
monitored by absorbance of the plate on the SpectroMax M2 at 535 nm as color change from
yellow to purple/pink of the Salkowsky’s assay solution was proportional to the concentration
of auxin (1AA) in each well. The test results, as summarized in Table 2, indicated that the
WO 90628
following isolates produced significant amounts of the phytohormone auxin: SGI—003-Hl l,
SGIA01, SGIC09, SGI—034-C09, and SGI—04l-B03.
Table 2: mical characteristics of the bacterial isolates (ND: not detectable).
Bacterial Isolates Biochemical Activity
Isolate ID Provisional Auxin ACC- 2,3— N-fixation Phosphoms-
Taxonomy production deaminase butanediol solubilization
003_Hll Pantoea
Yes Yes Yes ND ND
agglomerans
020_AOl Bacillus
Yes ND ND ND ND
thuringiensis
026_G06 Burklzolderz'a
ND Yes ND ND ND
metallica
026_G07 Burklzolderz'a
ND Yes ND Yes ND
vietnamiensis
034_C09 Bacillus pumilus ND Yes ND ND
034_ElO Herbaspz'rz'llum
ND ND ND ND ND
04 l_B03 Pedobacter Sp. Yes Yes Yes ND ND
EXAMPLE 5: Biocontrol ty of the bacterial isolates t fungal phytopathogens
An in vitro antagonism assay was used to assess the ability of the ed
bacterial s to suppress the pment of several plant fiangal pathogens, including
Fusarz'um graminearum NRRL-5883, aphella nivalz's ATCC MYA-3968, Gibberella
zeae ATCC-l6lO6, Stagnospora nodurum 6369, Colletotrichum graminz'cola
ATCC-34l67, and a Penicillium sp. pathogen. The assay was performed on potato dextrose
agar (PDA) medium. Isolated strains of bacteria were grown on one-fifth th Tryptic soy
broth agar (TSBA/S) for 24 h prior to use.
For each fungal pathogen, a conidial inoculum was ed by hyphal tipping an
actively growing colony of the fungus and transferring the hyphal strands to FDA agar
medium. After incubating the plates for 7 days at 25°C using a 12 h/day photoperiod, fiangal
conidia were washed from FDA plates using a weak phosphate buffer (0.004% phosphate
buffer, pH 7.2, with 0.019% MgClz). A suspension of fungal conidia in the weak phosphate
buffer ximately l X 105 conidia/mL) was then immediately sprayed over the agar
surface, and the sprayed plates were then ted at 25°C for 48-72 h prior to use in
antagonism tests.
To initiate the antagonism tests, cells of isolated ial strains were pointinoculated
at equal distances inside the perimeter of the plate. After five days, the bacterial
strains were scored as antibiosis positive when a visibly clear area (z'.e., growth inhibition
zone) that lacked mycelial grth existed around the perimeter of the ial colonies. The
results of antagonism assays, as summarized in Table 3, trated that each of the
microorganisms disclosed herein inhibited the development of several fungal
phytopathogens, including Fusarium graminearum, Monographella nivalis, Gibberella zeae,
Stagnospora m, Colletotrichum graminicola, Penicillium sp.
Table 3: Biocontrol actiVity of the bacterial isolates t fitngal
phytopathogens.
Bacterial Isolates Growth ssion of fungal pathogen (inhibition zone scored after 5 days of incubation)
Isolate Provisional Fusarium Monographella Gibberella Stagnospora Colletom'chum Penicillium
ID Taxonomy gramz'nearum nivalz's zeae nodurum gramz'm'cola sp.
003_H1 1 Pantoea
No Yes No No No No
agglomerans
020_A01 Bacillus
Yes No Yes Yes Yes No
thuringz'ensz's
026_G06 lderia
Yes Yes Yes Yes Yes
metallica
026_G07 lderia
Yes No No No No
viemamiensis
034_C09 Bacillus
No No Yes No No
pumilus
034_E10 Herbaspz'rillum
No No Yes No No
041_B03 Pedobacter sp. \0 Yes No Yes Yes Yes
EXAMPLE 6: Enhancement ofwheat yield potential
Effects of bacterial inoculation on plant growth and yield were studied in a
greenhouse with the isolate 0-A01. Microbial cell suspensions were prepared as
follows. 2YT medium, or similar growth media, broth cultures were inoculated from the
isolate’s glycerol stocks or streak plates. Typically, prior to use in the growth chamber,
greenhouse, or field, bacterial cultures were initiated 48 — 72 hours to allow the cultures to
reach late exponential phase. Isolates that have longer doubling times were initiated fiarther in
advance. Cultures were incubated at 30°C on a rotary shaker at 200 rpm. After growth, the
cells were pelleted at 10,000 X g for 15 min at 4°C and resuspended in 10 mM MgSO4 buffer
(pH 7.0). Cell densities were normalized for each isolate on a CFU/mL basis. Typically, ~109
CFU/mL suspensions were prepared for each isolate and transported on ice to the inoculation
site. Inoculations were performed by diluting these cell suspensions 1/20 in irrigation water to
a final density 5 X 107 CFU/mL. For 1 liter pot trials, 20 mL of this dilute cell suspension
was distributed evenly over the surface of each replicate pot.
] Greenhouse trial was conducted with a nutrient deficient field soil. After ng
large rocks and debris, f1eld soil was mixed thoroughly to ensure homogeneity. After filling,
soil in each of the pots was pressed down ~2 cm for a firm sowing layer. Seeds of a
commercial wheat cultivar (hard red spring wheat; Howe Seeds, Inc.) were sown in 1 liter
pots containing field soil medium (10.5 cm X 12.5 cm d er plastic pots). Two
grams of spring wheat seeds (approximately 70 seeds) were distributed evenly in each pot
and 50 mL of field soil were applied and spread evenly over seed layer. ing uniform
emergence of wheat coleoptile and uent emergence of first leaf, the plant population
was inoculated with 20 mL of 109 CFU/mL of SGI—020-A01. Plants of negative ls
received 20 mL of inoculum buffer only. Each ion was performed in 8 replicate flats,
each containing four 1 liter pots (n=4 per flat). The flats were randomly distributed over four
experimental blocks. The seeds and plants were then maintained in a greenhouse for 60 days
at ambient temperature (ranging from about 8°C to about 22°C) with l light cycles of
approximately 11.5 hours sunlight/ 12 hours dark throughout the trial. Plants were mly
bottom watered to appropriate hydration level depending on the temperature and stage of
growth. At approximately 30 days post , approximately 70 individuals per pot were
staked and loosely tied together to prevent cross contamination and to minimize positional
effects due to variation in plants g into other pots. At approximately 60 days post
sowing, plants were allowed to dry out in preparation for harvest. Wheat heads were
harvested at approximately 80 days post . Each wheat head was removed by g
just below the head. Wheat heads within each pot replicate were pooled, weighted, and
subsequently used as an estimate of yield potential. All plants in the population were
harvested on the same day and ents were harvested in a randomized order to eliminate
large differences in time in between harvesting between ents. As a result, wheat plants
treated with the isolate SGIA01 showed a 40% increase in yield potential compared to
control eated plants (2.95gram/pot vs. 2.10gram/pot). Averages and standard deviations
were documented across all 8 replicates and an ANOVA (Analysis of Variance) was
med. Efficacy of the microbial e SGIA01 in enhancing wheat yield potential
was fied by analyzing the wheat head yield in weight for each pot replicate. P-values of
<05 were considered significant.
E 7: Enhancement ofbiomass production in maize
] Effects of bacterial inoculation on plant grth and yield were studied in
greenhouse experiments with each of the following bacterial isolates: SGI—034-C09, SGI-
034-E10, SGIH11, SGIB03, SGIG06, and SGIG07. The greenhouse
trials were conducted with a nutrient deficient field soil. After removing large rocks and
debris, field soil was mixed thoroughly with potting soil (70:30) to ensure homogeneity.
After filling, soil in each of the pots was pressed down ~2 cm for a firm sowing layer. Seeds
of a commercial maize cultivar (Dow AgroSciences) were sown in 1 liter pots (10.5 cm X
12.5 cm tapered pots) each containing soil medium. Two maize s were distributed
evenly in each pot in embryo-up orientation, followed by application of 50 mL of field soil,
which was spread evenly over the seed layer. After germination, culling of one ng per
pot was performed if necessary so that each pot contained only one plant.
Following uniform emergence of maize coleoptile and subsequent emergence of
first leaf, the plant population was inoculated with ~20 mL of 109 CFU/ml of a microbial
isolate selected from the group of SGIC09, SGIE10, 3-H11, SGIB03,
SGIG06, and SGIG07. Microbial cell suspensions were prepared as described in
Example 6 above. Plants of negative controls received 20 mL of inoculum buffer only.
Each condition was performed in 8 replicate flats, each containing two 1 liter pots
(n=2 per flat). The flats were ly distributed over four experimental blocks. The seeds
and plants were then maintained in a greenhouse for 60 days at ambient temperature (ranging
from about 8°C to about 22°C) with diurnal light cycles of approximately 11.5 hours sunlight/
12 hours dark throughout the trial. Plants were uniformly bottom d to appropriate
hydration level depending on the temperature and stage of growth. Maize ground
biomass was harvested at approximately 60 days post sowing.
] All plants in the population were harvested on the same day and ents were
harvested in a randomized order to eliminate large differences in time in between harvesting
n treatments. Maize plants were analyzed for difference in total biomass. As
documented in Table 4, maize plants treated with each of the microbial isolates showed a
significant increase in total biomass as compared to control non-treated plants. Averages and
standard deviations were documented across all 8 replicates and an ANOVA (Analysis of
Variance) was performed.
Table 4: Efficacy of the microbial isolates in enhancing total plant biomass.
Treatment Plant biomass (g) p-Value Biomass Increase (%)
Non-treated 58.6 N/A N/A
SGIC09 106.3 <.0001 181%
SGIE10 103.6 <.0001 177%
SGIH11 100.7 <.0001 172%
1-B03 99.5 0.0001 170%
SGIG06 98.3 0.0002 168%
SGIG07 97.3 0.0003 166%
EXAMPLE 8: Seed coating treatment of wheat seeds and corn seeds
] Small scale seed treatment experiments were conducted by ing a procedure
described in Sudisha et al. (Phytoparasitz’ca, 37:161-169, 2009) with minor modifications.
Typically, a biopolymer stock solution was made by adding 1 gram of gum arabic powder
(MP Biomedical) to 9 mL water and mixing to homogeneity. Turbid cultures of actively
g ial cells or microbial spore preparations were washed with PBS and adjusted
to an OD600 of ~5.0. Three mL of the adjusted cell suspension was pelleted via
filgation in a 50 mL Falcon tube. The resulting supernatant was decanted, replaced with
3 mL biopolymer stock solution and the resulting suspension was mixed thoroughly.
Typically, approximately 25 g of seeds were added to the Falcon tube and usly shaken
or vortexed to ensure a uniform distribution of the gum/cell suspension. Coated seeds were
spread across plastic weigh boats to dry in a laminar flow hood until no longer tacky,
generally 3 hours with periodic mixing. The coated seeds were then stored at 4°C and
periodically tested for stability. A variety of wheat seeds and corn seeds were coated and
tested in the manner described above, including common hard red spring wheat varieties
Briggs, Faller, Glenn, Hank, RB07, ; hard red winter wheat varieties Jerry, McGill,
Overland; and maize seed variety DKC62-6l as well as a cial maize cultivar (Dow
AgroSciences).
Viability testing on the microbes used in seed coating formulation was performed
using a standard plate count method. Typically, a pre-determined amount of coated seeds was
tested for the presence of viable microbes by washing the seeds in an aliquot of appropriate
buffer and plating equivalent amounts of buffer on nutrient agar media. Viable colony-
forming-units were ined after 1-4 days incubation at 30°C. Viability test showed that
between l X 104 and 4 X 107 viable colony-forming-units per seed were present after
approximately five weeks of storage at 4°C. When seeds were coated with microbial spores,
the viability of the ty of tested microbes remained stable for at least four months,
including multiple interstate shipments across the United States, in and out of refrigerated
containers. When stored under refrigeration (4°C) the microbes survived on the seed coat
with little loss in viability over the test s. The results indicated that seeds coated with
compositions disclosed herein could be stored for extended periods under refrigeration and
ted that microbes would survive during periods of higher temperatures for distribution.
In on, germination rate of the coated seeds was tested and determined to be essentially
identical to l seeds, which were either seeds coated with gum arabic only or uncoated
seeds.
EXAMPLE 9: Solid State Formulation of the Microbial Comp_ositions
This section describes an ary ation of a microbial fertilizer where
the bacteria in accordance with the present invention are encapsulated and the fertilizer is in
solid form. Alginate beads are prepared as follows:
One milliliter of 30% glycerol is added to l, 1.5 or 2% sodium alginate solution,
depending on the alginate properties (M/G ratio) to obtain a final volume of 25 mL. Bacterial
cells from a 250 mL culture obtained from one of the bacterial isolates of the invention or
from a combination of two or more isolates is pelleted via centrifuged, then washed with a
saline solution (0.85% NaCl, w/v), suspended in 25 mL of alginate mixture, and mixed
thoroughly. This cell suspension is then added drop wise into a pre-cooled sterile 1.5 or 2%
(w/v) aqueous on of CaClz under mild agitation to obtain the bacterial-alginate beads.
These beads are allowed to harden for 2-4 h at room ature. Beads are collected by
g and are washed several times with sterile water and stored at 4°C. In order to preserve
the formulation, the fresh wet beads can be frozen at about -80°C prior to lyophilization at
about -45°C for 15 h. The lyophilized dry beads can be stored in appropriate ners, such
as sterile glass bottles.
To estimate the viable counts, the encapsulated bacteria can be ed from the
beads by resuspending 100 mg of beads in ate buffered saline (pH 7.0) for 30 min
followed by homogenization. The total number of released bacteria is determined by standard
plate count method after incubating at 30°C for 48 h. At one month intervals the cell densities
in the beads are enumerated using similar method.
E 10: Compatibility of the ial compositions with commercial filngicides
As environmental concerns are increasing about using pesticides in agriculture,
biological alternatives are increasingly perceived as inevitable. However, new biological
formulations must also allow organisms to survive and express their c beneficial
. Chemical ides are lly toxic not only towards deleterious microorganisms
but also to the beneficial ones. However, chance of survivability of these microbial agents
might have been enhanced when applied at reduced rates.
In the present study, peat-based carrier material is used for inoculation of both the
fungicide treated as well as bare crop seed. ial tolerability of filngicide is generally
evaluated in the following manner: a) bacteria inoculated bare seeds grown on an appropriate
bacterial growth medium such as trypticase soy agar (TSA; Tryptone 15g/L; Soytone 5 g/L,
sodium chloride 5g/L, and agar l5g/L) plates, b) fungicide-treated bacteria inoculated seeds
grown on common TSA plates, and, c) fungicide-treated bacteria inoculated seeds grown in
the sterile growth pouches. Typically, three trations of fiangicide are used in each of
the experiments: manufacturer’s recommended dose and two lower doses (at 75% and 50%
of the ended dose). Fungicide-treated bacteria inoculated seeds are stored after
inoculation and used at different time intervals (2 hrs, 4 hrs and 6 hrs) to examine the impact
on seed germination. Both seed germination and ial presence are monitored in petri-
plates. For the grth pouch study, fungicide-treated (recommended dose) seeds are used,
and root and hypocotyl lengths were measured at 7 days of seedling growth.
Some rhizobacterial isolates of the invention are compatible with several
commonly used fiangicides as ined by ial growth on fungicide-enriched TSA
plates. In general, both bare and fungicide-treated seeds, coated with inoculated peat show no
cant variation in germination compared to non-inoculated control. Moreover, growth-
promoting effects on root and total seedling lengths are observed in all rhizobacterial
treatments compared to non-inoculated control.
EXAMPLE 11: Development of non-naturally occurring cultivars and breeding program
Endophytic bacteria of the present ion are introduced into crop plants,
including cereals, of varying genotypes and geographic origin, lacking such endophytic fungi,
to create plant-endophyte combinations with improved agronomic characteristics, using
procedures analogous to those known in the art, including those described in US. Pat. Appl.
No. 20030195ll7Al; US. Pat. Appl. No. 20010032343Al; and US. Pat. No. 331,
among others. Thus, synthetic plant-endophyte combinations may be created and selected in a
breeding/cultivar pment program based on their ability to form and maintain a
mutualistic combination that results in an agronomic benefit. Rating of agronomic
characteristics of the combination may also be utilized in such a breeding m. These
characteristics may include, without limitation, drought tolerance, biomass accumulation,
ance to insect infestation, palatability to livestock (e.g., herbivores), ease of
reproduction, and seed yield, among others. Such combinations may differ in levels of
accumulation of microbial metabolites that are toxic to pests and weeds, including ergot
id levels, loline levels, peramine levels, or lolitrem levels, while displaying desired
agronomic teristics of crop plants, including resistance to insect feeding or ation,
ance to abiotic stress, bility to livestock, biomass accumulation, ease of
reproduction, and seed yield, among other traits.
E 12: Yield Study
Corn seeds (Zea mays) were coated with different ial treatments and sown
in a ed field. Each ent was replicated 5 times in random complete block design.
A single replicate consisted of four 30 feet long beds (rows), 60 seeds were sown (6 inches
apart) in each bed. For the observation purpose data was taken from the middle two rows
only.
Plant emergence was ed twice as shown in Table 5 below as the percentage
of plants in the replicate that had sprouted. Ten plants in the middle two rows of each plot
were tagged with a plastic ribbon to record the vital statistics such as the plant height,
chlorophyll measurement, plant weight etc. of the plants.
The plant heights (measuring the tip of the tallest/longest leaf) recorded 31 and 56
days after planting indicated that the plant height among the treatments was not significantly
different. Most of the plants were dry and leaves had shrunk by day 110 post planting,
therefore, in some of the cases, plants looked (measured) shorter than in previous
measurement, but overall, the plant height did not differ among the treatments. On the 5th
week of planting, chlorophyll content was measured (in SPAD units) from the lower leaves
(ca. 60 cm above the ground) and upper leaves (second fiJlly expanded leaf from the top) of
ten tagged plants of each plot. The chlorophyll content among the treatments did not
significantly differ. On the 110th and 111th day of ng, the crop was harvested. Ten
tagged plants from each plot were cut at the soil level and above the ground parts of the
plants were weighed (whole plant weight or WPtWt), corn ears were removed and the length
of the cob was measured (ear length with kernels) (region filled with marketable kernels
only), then the kernels were removed from the cob and their weight was measured for the
kernel weight per ear ear).
Shortly after the manual t of 10 plants per plot was over, a mechanical
harvester, Gleaner® K2 (Allis-Chalmers Mfrg, Milwaukee, WI) was brought in. This
machine mechanically removed the remaining plants from two middle rows of each plot,
removed the kernels from the cobs, and took the measurement of kernel moisture and weight
(10 ears + machine harvest). The ted total yield at 15.5 % moisture content (pounds of
corn kernel per acre) based on the weight (lb.) of kernels (include kernels from machine
harvested cobs+manual harvested cobs) was 10368.14 pounds per acre or 185.15 bushels per
acre for SGIH11 (Pantoea erans). This was the highest yield among all
organism treatments and cantly different from the treatments for us
amyloliquefaciens SGI-015_F03. The other high producer was the treatment with Bacillus
thuringiensis SGI-020_A01.
In conclusion, all the plants in the different treatments emerged and grew similarly
in the field conditions provided with the same amount of fertilizer, ergent herbicide
(with manual weed pulling later in the season), and weekly irrigation during the growing and
warm season, and pest control of ally the corn earworm. With all conditions equal,
treatment with SGIH11 ea agglomerans) produced the highest yield over the
non-microbe, control treatment. Thus, 003_H11 produced a yield of approximately 17%
higher than the control group. Thus, in various embodiments of the invention application of
an effective amount of the organism according to any of the methods described herein
produces at least 10% or at least 12.5% or at least 15% or about 17% higher yield than a
control group, which in some embodiments can be ined by pounds of plant product
(e. g., corn ears) per acre or bushels of plant product per acre. Organisms listed in Table 5 are
SGIH11 (Pantoea agglomerans); SGIF03 lus amyloliquefaciens); and SGI-
020-A01 (Bacillus thuringiensis).
Table 5
Organism Emergence Emergence Height Height Height
Date 6/20 6/24 7/11 8/5 9/28
Control 97.00 94.50 103.64 277.22 267.82
SGIH11 89.50 93.34 103.32 273.58 272.04
SGIF03 95.67 96.67 105.02 282.02 276.08
SGIA01 94.83 95.33 102.04 268.94 264.80
Organism Upper Leaf Lower Leaf WPtWt Ear Length w/ knls
Date 7/18 7/18 9/28 9/28
Control 43.24 60.85 493.24 14.98
3-H11 44.17 59.43 494.92 14.29
SGIF03 44.90 63.96 473.40 14.45
SGIA01 46.45 63.84 488.30 14.20
Organism Knl wt/ear(g) Knl wt/ear(lb) 10 ears+machine 10 ears+machine
Date 9/28 9/28 9/29 9/29
Control 168.62 0.37 157.81 8837.63
3-H11 171.96 0.38 185.15 10368.14
SGIF03 156.24 0.34 161.17 9025.62
SGIA01 158.68 0.35 163.09 9133.16
A number of embodiments of the invention have been described. Nevertheless, it
will be understood that elements of the ments described herein can be combined to
make additional embodiments and various modifications may be made t departing
from the spirit and scope of the invention. Accordingly, other embodiments, alternatives and
lents are Within the scope of the invention as described and claimed herein.
Headings Within the application are solely for the convenience of the reader, and
do not limit in any way the scope of the invention or its embodiments.
All publications and patent applications mentioned in this specification are herein
incorporated by reference to the same extent as if each individual publication or patent
application was specifically can individually ted to be incorporated by reference.
[Annotation] tboyc_piz
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE T OF MICROORGANISMS
FOR THE E OF PATENT URES
INTERNATIONAL FORM
TO RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Patent Dept. issued nt to Rule 7.1 by the
Synthetic Genomics, Inc. INTERNATIONAL DEPOSITARY AUTHORITY
11149 North Torrey Pines Rd. ‘ identified at the bottom of this page
La jolla, CA 92037
NANLE AND S OF DEPOSITOR
I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the DEPOSITOR: Accession number given by the
Pantoea agglomeram ATIONAL DEPOSITARY AUTHORITY:
003_H11 NRRL 13—50483
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I. above was accompanied by:
[I 1a scientific ption
SJa proposed mic designation
III. RECEIPT AND ACCEPTANCE
This ational Depositary Authority accepts the microorganism identified under 1. above, which was received by it on
March 28, ate of the original deposit)2
IV. RECEIPT OF RECUEST FOR CONVERSION
The microorganism identified under I. above, was received by this International Depositary Authority on
___________________
(date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received
by it on (date of receipt of request for conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: Agricultural Research Culture Signature(s) of person(s) having the power to represent the
Collection (NRRL) International Deposita A thority or of authorized official(s):
International Depositary Authority
Address: 1815 N. University Street
Peoria, Illinois 61604 U.S.A. Date:
Hflrfl
1 Mark with
a cross the applicable box.
2 Where Rule 6.4(d) applies, such date is the date
on which the status of international depositary authority was acquired.
5656
[Annotation] tboyc_piz
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSE OF PATENT URES
INTERNATIONAL FORM
TO RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Patent Dept. issued pursuant to Rule 7.1 by the
’ Synthetic Genomics, Inc. INTERNATIONAL DEPOSITARY AUTHORITY
11149 North Torrey Pines Rd identified at the bottom of this page
La Jolla, CA 92037
NAME AND ADDRESS OF DEPOSITOR
I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the DEPOSITOR: Accession number given by the
m tburiflgiemir INTERNATIONAL DEPOSITARY AUTHORITY.
020_A01 NRRL 13-50484
II. SCIENTIFIC PTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The rganism identified under I. above was accompanied by:
[I 1a scientific description
E121 proposed taxonomic designation
III. RECEIPT AND ACCEPTANCE
This International Depositary Authority accepts the microorganism identified under 1. above, which was ed by it on
March 28, 2011(date of the original deposit)2
IV. RECEIPT OF RE I UEST FOR CONVERSION
The microorganism identified under I. above, was received by this International Depositary Authority on_______________
(date of the original deposit) and a request to convert the original deposit to a deposit under the BudapestmTreaty was received
by it on (date of receipt of request for conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: Agricultural Research Culture Signature(s) of (s) having the power to represent the
Collection (NRRL) International Depositary Authoty or of authorized official(s):
International tary ity
Address: 1815 N. University Street i.
~ Peoria, Illinois 61604 USA. Date: Ll... p-“
1 Mark with
a cross the applicable box.
2 Where Rule 6.4(d) applies, such date is the date
on which the status of international depositary authority was acquired.
5757
[Annotation] tboyc_piz
[Annotation] piz
ST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSE OF PATENT PROCEDURES
INTERNATIONAL FORM
TO RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Patent Dept. issued pursuant to Rule 7.1 by the
Synthetic cs, Inc. INTERNATIONAL DEPOSITARY AUTHORITY
11149 North Torrey Pines Rd. identified at the bottom of this page
La Jolla, CA 92037
NAME AND ADDRESS OF DEPOSITOR
I. FICATION OF THE MICROORGANISM
Identification reference given by the DEPOSITOR: Accession number given by the
Burkba/dcria metal/1m INTERNATIONAL TARY ITY:
026_G06 NRRL B—50485
II. SCIENTIFIC PTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under 1. above was accompanied by:
E] 1a scientific description
Qa proposed taxonomic designation
III. RECEIPT AND ACCEPTANCE
This International Depositary Authority s the microorganism identified under I. above, which was received by it on
March 28, 201 1 (date of the original deposit)2
IV. RECEIPT OF REQUEST FOR CONVERSION '
The microorganism identified under I. above, was received by this ational Depositary Authority on
(date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received
by it on (date of receipt of request for conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Agricultural ch Culture Signature(s) of person(s) having the power to represent the
Collection (NRRL) International Depositary Authori or of authorized official(s):
International Depositary Authority ¢
Address: 1815 N. University Street
Peoria, Illinois 61604 U.S.A. Date: 1,, fl 3"”
1 Mark with
a cross the applicable box.
2 Where Rule 6.4(d) applies, such date is the date
on which the status of international depositary authority was acquired.
5858
[Annotation] tboyc_piz
BUDAPEST TREATY ON THE ATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSE OF PATENT PROCEDURES
ATIONAL FORM
TO RECEIPT IN THE CASE OF AN ORIGINAL T
Patent Dept. issued pursuant to Rule 7.1 by the
tic Genomics, Inc. INTERNATIONAL DEPOSITARY AUTHORITY
11149 North Torrey Pines Rd. identified at the bottom of this page
La Jolla, CA 92037
NAME AND ADDRESS OF DEPOSITOR
I. IDENTIFICATION OF THE MICROORGANISM
fication reference given by the TOR: Accession number given by the
Barbe/dank vietnamz'emzk INTERNATIONAL DEPOSITARY AUTHORITY:
026_G07 NRRL B—50486
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The rganism identified under I. above was accompanied by:
D 1a ific description
% proposed taxonomic designation
III. RECEIPT AND ACCEPTANCE
This International Depositary ity accepts the microorganism identified under I. above, which was received by it on
March 28, 2011(date of the original deposit)2
IV. RECEIPT OF RE 0 UEST FOR CONVERSION
The microorganism identified under 1. above, was received by this International Depositary Authority on ________________
(date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received
by it on (date of receipt of request for conversion).
V. INTERNATIONAL DEPOSITARY ITY
Name: Agricultural Research Culture Signature(s) of person(s) having the power to represent the
Collection (NRRL) International Depositary Authori or of authorized official(s):
International Depositary Authority
\ 2
Address: 1815 N. University Street
Peoria, Illinois 61604 USA. Date: '
“/4 3/1]
1 Mark with a cross the applicable box.
2 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.
5959
ation] tboyc_piz
GRDS003WO sequence listing
SEQUENCE LISTING
<110> SYNTHETIC GENOMICS, INC.
BULLIS, DAVID T.
GRANDLIC, CHRISTOPHER J.
MCCANN, RYAN
KEROVUO, JANNE S.
<120> PLANT GROWTH-PROMOTING MICROBES AND USES THEREFOR
<130> SGI1540-1WO
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 1436
<212> DNA
<213> Pantoea agglomerans
<220>
<221> misc_feature
<223> SGI bacterial isolate SGI-003_H11
<220>
<221> misc_feature
<223> encodes a 16S ribosomal RNA
<220>
<221> misc_feature
<222> (63)
<223> n is a, c, g, or t
<400> 1
ggca ggcctaacac gtcg agcggtagca cagagagctt gggt
gnngagcggc ggacgggtga gtaatgtctg ggaaactgcc tgatggaggg ggataactac
tggaaacggt agctaatacc gcataacgtc gcaagaccaa agtgggggac cttcgggcct
atca gatgtgccca gatgggatta gctagtaggt gaggtaatgg ctcacctagg
cgacgatccc tagctggtct gagaggatga ccagccacac tggaactgag acacggtcca
gactcctacg ggaggcagca gtggggaata aatg ggcgcaagcc tgatgcagcc
atgccgcgtg tatgaagaag gccttcgggt tgtaaagtac tttcagcgag gaggaaggcg
ataaggttaa taaccttgtc gattgacgtt actcgcagaa gaagcaccgg ctaactccgt
gccagcagcc atac ggagggtgca agcgttaatc ggaattactg ggcgtaaagc
ation] tboyc_piz
GRDS003WO sequence listing
gcacgcaggc ggtctgtcaa gtcggatgtg aaatccccgg gctcaacctg ggaactgcat
ccgaaactgg caggctagag tcttgtagag gggggtagaa ttccaggtgt agcggtgaaa
tgcgtagaga tctggaggaa taccggtggc ggcc ccctggacaa acgc
tcaggtgcga gggg agcaaacagg attagatacc ctggtagtcc acgctgtaaa
cgatgtcgac ttggaggttg ttcccttgag gagtggcttc cggagctaac gcgttaagtc
gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat gaattgacgg gggcccgcac
aagcggtgga gcatgtggtt taattcgatg gaag aaccttacct actcttgaca
tccagagaac ttagcagaga tgctttggtg ccttcgggaa ctctgagaca ggtgctgcat
1020
ggctgtcgtc agctcgtgtt gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt
1080
tgtt gccagcggtt cggccgggaa ctcaaaggag actgccagtg ataaactgga
1140
ggaaggtggg gatgacgtca agtcatcatg gcccttacga gtagggctac acacgtgcta
1200
caatggcata tacaaagaga ctcg cgagagcaag cggacctcat aaagtatgtc
1260
gtagtccgga tcggagtctg caactcgact ccgtgaagtc ggaatcgcta gtaatcgtgg
1320
atcagaatgc cacggtgaat acgttcccgg gccttgtaca ccgt cacaccatgg
1380
gagtgggttg aagt aggtagctta accttcggga gggcgcttac cacttt
1436
<210> 2
<211> 1440
<212> DNA
<213> Bacillus thuringiensis
<220>
<221> misc_feature
<223> SGI bacterial isolate 0_A01
<220>
<221> misc_feature
<223> encodes a 16S ribosomal RNA
<400> 2
cgtgcctaat acatgcaagt cgagcgaatg gattaagagc ttgctcttat gaagttagcg
[Annotation] piz
GRDS003WO sequence listing
gggt gagtaacacg tgggtaacct aaga taac tccgggaaac
cggggctaat accggataat attttgaact ttcg aaattgaaag gcggcttcgg
ctgtcactta gacc cgcgtcgcat tagctagttg gtgaggtaac ggctcaccaa
ggcaacgatg cgac ctgagagggt gatcggccac actgggactg ggcc
cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag
caacgccgcg tgagtgatga aggctttcgg aaac tctgttgtta gggaagaaca
agtgctagtt gaataagctg gcaccttgac ggtacctaac cagaaagcca cggctaacta
cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa
agcgcgcgca ggtggtttct taagtctgat gtgaaagccc acggctcaac cgtggagggt
cattggaaac tgggagactt agaa gaggaaagtg gaattccatg tgtagcggtg
aaatgcgtag agatatggag gaacaccagt ggcgaaggcg actttctggt ctgtaactga
cactgaggcg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt
aaacgatgag tgctaagtgt tagagggttt ccgcccttta gtgctgaagt taacgcatta
agcactccgc agta cggccgcaag gctgaaactc aaaggaattg acgggggccc
gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt
gacatcctct ccta gagatagggc ttctccttcg ggagcagagt gacaggtggt
1020
gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac
1080
tctt agttgccatc attaagttgg gcactctaag gtgactgccg gtgacaaacc
1140
ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg
1200
ctacaatgga cggtacaaag agctgcaaga ccgcgaggtg gagctaatct cataaaaccg
1260
ttctcagttc ggattgtagg ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg
ation] piz
GRDS003WO sequence listing
1320
cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca
1380
cgagagtttg ccga agtcggtggg gtaacctttt tggagccagc cgcctaaggt
1440
<210> 3
<211> 1414
<212> DNA
<213> lderia metallica
<220>
<221> misc_feature
<223> SGI bacterial isolate SGI-026_G06
<220>
<221> misc_feature
<223> encodes a 16S ribosomal RNA
<400> 3
catgcaagtc cagc acgggtgctt gcacctggtg gcgagtggcg aacgggtgag
taatacatcg gaacatgtcc tgtagtgggg gatagcccgg cgaaagccgg attaataccg
atct acggatgaaa gcgggggacc ttcgggcctc gcgctatagg gttggccgat
ttag ctagttggtg gggtaaaggc ctaccaaggc gacgatcagt agctggtctg
agaggacgac cagccacact gggactgaga cacggcccag actcctacgg gaggcagcag
tggggaattt tggacaatgg gcgaaagcct gatccagcaa tgccgcgtgt gtgaagaagg
ccttcgggtt gtaaagcact tttgtccgga aagaaatcct tgattctaat acagtcgggg
gatgacggta ccggaagaat cggc taactacgtg ccagcagccg cggtaatacg
gcga gcgttaatcg gaattactgg gcgtaaagcg tgcgcaggcg gtttgctaag
accgatgtga cggg ctcaacctgg gaactgcatt ggtgactggc aggctagagt
atggcagagg ggggtagaat tccacgtgta gcagtgaaat gcgtagagat gtggaggaat
accgatggcg aaggcagccc cctgggccaa tactgacgct catgcacgaa agcgtgggga
gcaaacagga ttagataccc tggtagtcca cgccctaaac gatgtcaact agttgttggg
[Annotation] tboyc_piz
GRDS003WO sequence listing
gattcatttc cttagtaacg tagctaacgc gtgaagttga ccgcctgggg agtacggtcg
caagattaaa agga attgacgggg acaa gcggtggatg atgtggatta
attcgatgca acgcgaaaaa ccttacctac ccttgacatg gtcggaatcc tgctgagagg
cgggagtgct cgaaagagaa ccggcgcaca ggtgctgcat ggctgtcgtc agctcgtgtc
1020
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtccttagtt gctacgcaag
1080
ctaa ggagactgcc ggtgacaaac cggaggaagg tgac gtcaagtcct
1140
catggccctt atgggtaggg cttcacacgt catacaatgg tcggaacaga gggttgccaa
1200
cccgcgaggg ggagctaatc ccagaaaacc gatcgtagtc gcac tctgcaactc
1260
gagtgcatga agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc
1320
ccgggtcttg tacacaccgc ccgtcacacc gtgg gttttaccag aagtggctag
1380
tctaaccgca aggaggacgg cggt agga
1414
<210> 4
<211> 1423
<212> DNA
<213> Burkholderia vietnamiensis
<220>
<221> misc_feature
<223> SGI ial isolate SGI-026_G07
<220>
<221> misc_feature
<223> encodes a 16S ribosomal RNA
<400> 4
tgccttacac atgcaagtcg aacggcagca cgggtgcttg cacctggtgg cgagtggcga
acgggtgagt aatacatcgg aacatgtcct gtagtggggg atagcccggc gaaagccgga
ttaataccgc atacgatcta aggatgaaag cgggggacct tcgggcctcg cgctataggg
ttggccgatg gctgattagc tagttggtgg ggcc taccaaggcg acgatcagta
gctggtctga gacc agccacactg ggactgagac acggcccaga ctcctacggg
[Annotation] tboyc_piz
3WO sequence listing
aggcagcagt ggggaatttt ggacaatggg cgaaagcctg atccagcaat gccgcgtgtg
tgaagaaggc cttcgggttg taaagcactt ttgtccggaa agaaatcctt ggctctaata
cagtcggggg gtac aata agcaccggct aactacgtgc cagcagccgc
ggtaatacgt agggtgcaag cgttaatcgg aattactggg cgtaaagcgt gcgcaggcgg
tttgctaaga ccgatgtgaa gggc tcaacctggg aactgcattg gtgactggca
ggctagagta tggcagaggg gggtagaatt ccacgtgtag cagtgaaatg cgtagagatg
tggaggaata ccgatggcga aggcagcccc ctgggccaat actgacgctc atgcacgaaa
gcgtggggag caaacaggat tagataccct ggtagtccac gccctaaacg atgtcaacta
gttgttgggg attcatttcc ttagtaacgt agctaacgcg tgaagttgac cgcctgggga
gtacggtcgc aagattaaaa ctcaaaggaa ttgacgggga cccgcacaag cggtggatga
ttaa ttcgatgcaa cgcgaaaaac cttacctacc cttgacatgg tcggaagccc
gatgagagtt gggcgtgctc gaaagagaac cggcgcacag gtgctgcatg gctgtcgtca
1020
gctcgtgtcg tgagatgttg ggttaagtcc gagc gcaacccttg tccttagttg
1080
ctacgcaaga gcactctaag gagactgccg gtgacaaacc aggt ggggatgacg
1140
tcaagtcctc atggccctta tgggtagggc ttcacacgtc atacaatggt cggaacagag
1200
ggttgccaac ccgcgagggg gagctaatcc cagaaaaccg atcgtagtcc cact
1260
ctgcaactcg agtgcatgaa gctggaatcg ctagtaatcg agca tgccgcggtg
1320
aatacgttcc cgggtcttgt acacaccgcc cgtcacacca tgggagtggg ttttaccaga
1380
agtggctagt gcaa ggaggacggt caccacggta gga
1423
<210> 5
<211> 1441
<212> DNA
[Annotation] tboyc_piz
GRDS003WO sequence listing
<213> Bacillus pumilus
<220>
<221> misc_feature
<223> SGI bacterial isolate SGI-034_C09
<220>
<221> misc_feature
<223> encodes a 16S ribosomal RNA
<220>
<221> misc_feature
<222> .(605)
<223> n is a, c, g, or t
<400> 5
gcggcgtgcc taatacatgc aagtcgagcg gacagaaggg agcttgctcc cggatgttag
cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa
accggagcta ataccggata gttccttgaa ccgcatggtt caaggatgaa agacggtttc
cact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc
acga tgcgtagccg gagg gtgatcggcc acactgggac tgagacacgg
cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg
agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa
caagtgcaag agtaactgct tgcaccttga cggtacctaa ccagaaagcc acggctaact
acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta
aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg
tcatnggaaa ctgggaaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt
cgta gagatgtgga ccag tggcgaaggc gactctctgg tctgtaactg
agga gcgaaagcgt ggggagcgaa taga taccctggta gtccacgccg
taaacgatga gtgctaagtg ttagggggtt tccgcccctt gcag ctaacgcatt
tccg cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc
cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct
[Annotation] tboyc_piz
GRDS003WO ce listing
tgacatcctc tgacaaccct agagataggg ctttcccttc ggggacagag tgacaggtgg
1020
tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa
1080
cccttgatct tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac
1140
cggaggaagg tgac gtcaaatcat catgcccctt atgacctggg ctacacacgt
1200
gctacaatgg acagaacaaa gggctgcgag accgcaaggt ttagccaatc ccacaaatct
1260
gttctcagtt cggatcgcag tctgcaactc gactgcgtga aatc gctagtaatc
1320
gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc
1380
acgagagttt gcaacacccg aagtcggtga ggtaaccttt ccag ccgccgaagg
1440
1441
<210> 6
<211> 1410
<212> DNA
<213> Herbaspirillum sp.
<220>
<221> misc_feature
<223> SGI ial isolate SGI-034_E10
<220>
<221> misc_feature
<223> encodes a 16S mal RNA
<400> 6
tgcaagtcga aacggcagca taggagcttg ctcctgatgg cgagtggcga acgggtgagt
aatatatcgg aacgtgccct agagtggggg ataactagtc gaaagactag ctaataccgc
atacgatcta cggatgaaag tgggggatcg caagacctca tgctcctgga gcggccgata
tctgattagc tagttggtgg ggtaaaagcc taccaaggca acgatcagta gctggtctga
gacc actg ggactgagac acggcccaga ctcctacggg aggcagcagt
tttt ggacaatggg ggcaaccctg atccagcaat gccgcgtgag tgaagaaggc
[Annotation] tboyc_piz
GRDS003WO sequence listing
cttcgggttg tctt ttgtcaggga agaaacggta gtagcgaata actattacta
atgacggtac ctgaagaata ggct aactacgtgc cagcagccgc ggtaatacgt
agggtgcaag tcgg aattactggg gcgt gcgcaggcgg aagt
cagatgtgaa atccccgggc tcaacctggg aattgcattt gagactgcac ggctagagtg
tgtcagaggg gggtagaatt ccacgtgtag aatg cgtagatatg tggaggaata
ccgatggcga aaggcagccc cctgggataa cgct catgcacgaa agcgtgggga
gcaaacagga ttagataccc tggtagtcca cgccctaaac gatgtctact cggg
tcttaattga cttggtaacg cagctaacgc gtgaagtaga ccgcctgggg agtacggtcg
caagattaaa actcaaagga attgacgggg acccgcacaa gatg atgtggatta
attcgatgca acgcgaaaaa ccttacctac ccttgacatg gatggaatcc tgaagagatt
tgggagtgct cgaaagagaa ccatcacaca ggtgctgcat ggctgtcgtc tgtc
1020
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtcattagtt gctacgaaag
1080
ggcactctaa tgagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcct
1140
catggccctt atgggtaggg cttcacacgt catacaatgg tacatacaga ccaa
1200
cccgcgaggg ggagctaatc ccagaaagtg tatcgtagtc cggattggag tctgcaactc
1260
gactccatga agttggaatc gctagtaatc gcggatcagc atgtcgcggt gaatacgttc
1320
ccgggtcttg tacacaccgc ccgtcacacc atgggagcgg gttttaccag aagtgggtag
1380
cctaaccgca aggagggcgc tcaccacggt
1410
<210> 7
<211> 1368
<212> DNA
<213> Pedobacter sp.
<220>
<221> misc_feature
[Annotation] piz
GRDS003WO sequence listing
<223> SGI bacterial isolate SGI-041_B03
<220>
<221> misc_feature
<223> encodes a 16S ribosomal RNA
<220>
<221> misc_feature
<222> .(529)
<223> n is a, c, g, or t
<400> 7
gaaagtggcg cacgggtgcg taacgcgtat gcaacctacc tggg ggatagcccg
gagaaatccg gattaatacc gcataaaatc acagtactgc atagtgcaat gatcaaacat
ttatgggaag aagatgggca tgcgtgtcat tagctagttg gcggggtaac ggcccaccaa
ggcgacgatg ggat ggat ggccccccac actggtactg agacacggac
cagactccta cgggaggcag cagtaaggaa tattggtcaa tggaggcaac tctgaaccag
ccatgccgcg aaga ctgccctatg aaac tgcttttatc cgggaataaa
cctgagtacg tgtacttagc tgaatgtacc ggaagaataa ggatcggcta actccgtgcc
agcagccgcg gtaatacgga ggatccaagc gttatccgga tttattgggt ttaaagggtg
cgtaggcggc ctgttaagtc aggggtgaaa gacggtagct caactatcng cagtgccctt
gatactgatg ggcttgaatg gactagaggt aggcggaatg agacaagtag cggtgaaatg
catagatatg tctcagaaca ccgattgcga aggcagctta ctatggtctt attgacgctg
aggcacgaaa ggat caaacaggat tagataccct ggtagtccac gccctaaacg
atgaacactc ggcg atacacagtc agcggctaag cgaaagcgtt aagtgttcca
cctggggagt acgctcgcaa gagtgaaact aatt gacgggggcc cgcacaagcg
gaggagcatg tggtttaatt cgatgatacg cgaggaacct tacccgggct tgaaagttag
tgaatcattt agagataaat gagtgagcaa tcacacgaaa gctg catggctgtc
gtcagctcgt gccgtgaggt gttgggttaa gtcccgcaac gagcgcaacc cctatgttta
1020
[Annotation] tboyc_piz
GRDS003WO sequence listing
gttgccagca cgttatggtg gggactctaa acagactgcc tgtgcaaaca gagaggaagg
1080
aggggacgac gtcaagtcat catggccctt acgtccgggg acgt atgg
1140
atggtacaga ctac atagcaatat gatgcgaatc tcacaaagcc attcacagtt
1200
cggattgggg tctgcaactc gaccccatga agttggattc gctagtaatc cagc
1260
aatgacgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcaagc catggaagtt
1320
gggggtacct aaagtatgta accgcaagga gcgtcctagg gtaaaacc
1368
<210> 8
<211> 39
<212> DNA
<213> Artificial sequence
<220>
<223> PCR primer M13-27F
<400> 8
tgtaaaacga cggccagtta gagtttgatc ctggctcag
<210> 9
<211> 37
<212> DNA
<213> Artificial sequence
<220>
<223> PCR primer 1492R M13-tailed
<400> 9
caggaaacag ctatgaccgg ttaccttgtt acgactt
<210> 10
<211> 1059
<212> DNA
<213> Pantoea agglomerans
<400> 10
atggctatcg acgaaaacaa acagaaagcg ttggcggcag cactgggcca gatcgaaaaa
ggta aaggctccat catgcgcctg ggtgaagacc gttccatgga cgtggaaact
accg tttc cctggatatc gccctcggcg ctggcggtct gccaatgggc
gtcg aaatctacgg gcctgaatct tccggtaaaa cgacgctgac cctgcaggtt
[Annotation] tboyc_piz
GRDS003WO sequence listing
atcgccgccg cgcagcgtga aggtaaaacc ttta tcgatgcaga gcacgcgctg
gtat atgcccgcaa gctgggcgtc gatatcgaca acctgctgtg ctctcagccg
gacaccggcg aacaggcgct ctgt gacgcgctgg cgcgctccgg tgcggtagac
gtgctggtgg tcgactccgt tgcggcgctg acgccgaaag cggaaatcga aggcgagatc
tctc acatgggcct cgcggcgcgt atgatgagcc aggcaatgcg ggcc
ggtaacctga agcagtccaa cacgctgctg atcttcatca accagatccg tatgaaaatt
ggcgtgatgt tcggtaaccc ggaaaccacc accggcggta acgcgctgaa attctacgcc
cgtc tggatatccg ccgtatcggt gcggtaaaag atggcgataa cgtcattggt
agcgaaaccc gcgtgaaggt cgtgaagaac aaaatcgccg cgccgttcaa gcaggcggag
ttccagatcc tctacggcga aggcatcaac ttcttcggcg agctggtcga tctgggcgtg
aaagagaagc tgattgaaaa agcgggcgcc tggtatagct acaacggcga caaaattggt
cagggtaaag cgaacgctat ctcctggctg aaagagaacc cggctgcggc gaaagagatc
gagaagaaag ttcgtgaact gctgctgaac aaccaggatg ccacgccgga cttcgcggtt
1020
gatggtaaaa gcgaagaagc aagcgaacag tga
1059
<210> 11
<211> 352
<212> PRT
<213> a agglomerans
<400> 11
Met Ala Ile Asp Glu Asn Lys Gln Lys Ala Leu Ala Ala Ala Leu Gly
1 5 10 15
Gln Ile Glu Lys Gln Phe Gly Lys Gly Ser Ile Met Arg Leu Gly Glu
25 30
Asp Arg Ser Met Asp Val Glu Thr Ile Ser Thr Gly Ser Leu Ser Leu
40 45
ation] tboyc_piz
GRDS003WO sequence listing
Asp Ile Ala Leu Gly Ala Gly Gly Leu Pro Met Gly Arg Ile Val Glu
50 55 60
Ile Tyr Gly Pro Glu Ser Ser Gly Lys Thr Thr Leu Thr Leu Gln Val
65 70 75 80
Ile Ala Ala Ala Gln Arg Glu Gly Lys Thr Cys Ala Phe Ile Asp Ala
85 90 95
Glu His Ala Leu Asp Pro Val Tyr Ala Arg Lys Leu Gly Val Asp Ile
100 105 110
Asp Asn Leu Leu Cys Ser Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu
115 120 125
Ile Cys Asp Ala Leu Ala Arg Ser Gly Ala Val Asp Val Leu Val Val
130 135 140
Asp Ser Val Ala Ala Leu Thr Pro Lys Ala Glu Ile Glu Gly Glu Ile
145 150 155 160
Gly Asp Ser His Met Gly Leu Ala Ala Arg Met Met Ser Gln Ala Met
165 170 175
Arg Lys Leu Ala Gly Asn Leu Lys Gln Ser Asn Thr Leu Leu Ile Phe
180 185 190
Ile Asn Gln Ile Arg Met Lys Ile Gly Val Met Phe Gly Asn Pro Glu
195 200 205
Thr Thr Thr Gly Gly Asn Ala Leu Lys Phe Tyr Ala Ser Val Arg Leu
210 215 220
Asp Ile Arg Arg Ile Gly Ala Val Lys Asp Gly Asp Asn Val Ile Gly
225 230 235 240
Ser Glu Thr Arg Val Lys Val Val Lys Asn Lys Ile Ala Ala Pro Phe
245 250 255
Lys Gln Ala Glu Phe Gln Ile Leu Tyr Gly Glu Gly Ile Asn Phe Phe
260 265 270
Gly Glu Leu Val Asp Leu Gly Val Lys Glu Lys Leu Ile Glu Lys Ala
275 280 285
[Annotation] tboyc_piz
GRDS003WO ce listing
Gly Ala Trp Tyr Ser Tyr Asn Gly Asp Lys Ile Gly Gln Gly Lys Ala
290 295 300
Asn Ala Ile Ser Trp Leu Lys Glu Asn Pro Ala Ala Ala Lys Glu Ile
305 310 315 320
Glu Lys Lys Val Arg Glu Leu Leu Leu Asn Asn Gln Asp Ala Thr Pro
325 330 335
Asp Phe Ala Val Asp Gly Lys Ser Glu Glu Ala Ser Glu Gln Asp Phe
340 345 350
Claims (24)
1. An enriched culture of a microbial SGIH11 strain deposited as NRRL B-50483.
2. An isolated culture of a ial SGIH11 strain ted as NRRL B-50483.
3. A biologically pure culture of a microbial SGIH11 strain deposited as NRRL B- 50483.
4. An isolated microbial strain ted as NRRL B-50483 or a strain derived therefrom;
5. An isolated microbial strain comprising a DNA ce exhibiting at least 99% sequence identity to SEQ ID NO:1; and further wherein said microbial strain has a plant growth-promoting activity.
6. A composition comprising a microbial strain or culture according to any one of claims 1-5, and an agriculturally effective amount of a compound or composition selected from the group consisting of a fertilizer, an acaricide, a bactericide, a fungicide, an insecticide, a microbicide, a nematicide, and a pesticide.
7. A composition comprising a microbial strain or culture according to any one of claims 1-5, and a carrier.
8. A composition ing to claim 7, wherein said carrier is a plant seed.
9. A composition according to claim 7, wherein said composition is a seed coating formulation.
10. A ition according to claim 7, wherein said composition is prepared as a formulation selected from the group consisting of an emulsion, a d, a dust, a e, a pellet, a powder, a spray, an emulsion, and a solution.
11. A plant seed having a coating comprising a ition according to claim 9.
12. A method for treating a plant seed, said method comprising a step of exposing or contacting said plant seed with a microbial strain or culture according to any one of claims 1-5.
13. A method for enhancing the growth and/or yield of a plant, said method comprising applying an effective amount of a microbial strain or culture according to any one of claims 1-5 to the plant, or to the s surroundings.
14. A method according to claim 13, wherein said microbial strain or e is grown in a growth medium or soil of a host plant prior to or concurrent with host plant growth in said growth medium or soil.
15. A method according to claim 13, wherein said plant is a corn plant or a wheat plant.
16. A method according to claim 13, wherein said microbial strain or culture is established as an endophyte on said plant.
17. A method for preventing, inhibiting or ng the development of a plant pathogen, said method sing growing a microbial strain or culture according to any one of claims 1-5 in a growth medium or soil of a host plant prior to or concurrent with host plant growth in said growth medium or soil.
18. A method ing to claim 17, wherein said plant pathogen is selected from the group consisting of Colletotrichum, Fusarium, Gibberella, Monographella, Penicillium, and Stagnospora sms.
19. A method according to claim 17, wherein said plant pathogen is selected from the group consisting of Colletotrichum graminicola, um graminearum, Gibberella zeae, Monographella nivalis, Penicillium sp., or Stagnospora nodurum.
20. A method for preventing, inhibiting or treating the pment of a pathogenic disease of a plant, said method comprising applying an effective amount of a microbial strain or culture according to any one of claims 1-5 to the plant, or to the plant's surroundings.
21. A method according to claim 20, wherein said microbial strain or culture is applied to soil, a seed, a root, a flower, a leaf, a portion of the plant, or the whole plant.
22. A non-naturally occurring plant that is a plant artificially infected with a microbial strain or culture according to any one of claims 1-5.
23. Seed, reproductive tissue, vegetative tissue, regenerative tissues, plant parts, or progeny of a turally occurring plant according to claim 22.
24. A method for ing an agricultural composition, said method comprising inoculating a microbial strain or culture according to any one of claims 1-5 into or onto a substratum and allowing said microbial strain or culture to grow at a temperature of 1- 37°C until obtaining a number of cells or spores of at least 102-103 per milliliter or per gram.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161570237P | 2011-12-13 | 2011-12-13 | |
US61/570,237 | 2011-12-13 | ||
PCT/US2012/069579 WO2013090628A1 (en) | 2011-12-13 | 2012-12-13 | Plant growth-promoting microbes and uses therefor |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ625835A NZ625835A (en) | 2016-02-26 |
NZ625835B2 true NZ625835B2 (en) | 2016-05-27 |
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