USOO9249061B2

(12) United States Patent (10) Patent No.: US 9.249,061 B2

Harman et al. (45) Date of Patent: Feb. 2, 2016

(54) HIGHLY EFFICIENT ORGANIC FERTILIZER (56) References Cited

AND COMPONENTS THEREOF
U.S. PATENT DOCUMENTS
(71) Applicant: CORNELL UNIVERSITY, Ithaca, NY
(US) 4,908,220 A 3, 1990 Shih et al.
5,071.462 A 12, 1991 Kimura ............................... 71/7
(72) Inventors: Gary E. Harman, Geneva, NY (US);
Xingen Lei, Ithaca, NY (US) (Continued)
(73) Assignee: Cornell University, Ithaca, NY (US) FOREIGN PATENT DOCUMENTS
(*) Notice: Subject to any disclaimer, the term of this CN 101117300 A 2, 2008
patent is extended or adjusted under 35 CN 1.01619006 A 1, 2010
U.S.C. 154(b) by 0 days.
(21) Appl. No.: 14/360,520
(Continued)
OTHER PUBLICATIONS
(22) PCT Filed: Nov. 21, 2012
(86). PCT No.: PCT/US2O12/066329 Eman F. Sharaf et al., “Keratinolytic activity of purified alkaline
keratinase produced by Scopulariopsis brevicaulis (Sacc.) and its
S371 (c)(1), amino acids profile”, Saudi Jornal of Biological Sciences (2011018,
(2) Date: May 23, 2014 117-121.*
(87) PCT Pub. No.: WO2013/078365 (Continued)
PCT Pub. Date: May 30, 2013 Primary Examiner — Wayne Langel
(65) Prior Publication Data (74) Attorney, Agent, or Firm — LeClairRyan, a
US 2014/O323297 A1 Oct. 30, 2014 Professional Corporation
(57) ABSTRACT
Related U.S. Application Data The present invention relates to a fertilizer comprising at least
(60) Provisional application No. 61/563,355, filed on Nov. one microbe selected from the group consisting of Tricho
23, 2011. derma viride (NRRL B-50520), Scopulariopsis brevicaulis
(51) Int. Cl. (NRRL B-50521), Scopulariopsis brevicaulis (NRRL
C05C3/00 (2006.01) B-50522), and combinations thereof, and a substrate which is
COSC II/00 (2006.01) acted upon by the at least one microbe to release nitrogen. The
invention also relates to a fertilizer comprising at least one
(Continued) microbe selected from a keratin degrading microorganism
(52) U.S. Cl. and a keratin Substrate which is acted upon by the at least one
CPC. C05B 7700 (2013.01); C05B 1700 (2013.01); microbe to release nitrogen. The invention further relates to a
C05C3/00 (2013.01); C05C5/00 (2013.01); method for enhancing growth of plants with the fertilizer, and
(Continued) methods of making a fertilizer. The invention further relates
to an isolated Trichoderma viride strain (NRRL B-50520)
(58) Field of Classification Search and two isolated Scopulariopsis brevicaulis strains (NRRL
CPC ............ C05F 11/08: C05C3/00; C05C 11/00 B-50521, NRRL B-50522).
USPC ........................................... 71/6-10, 54, 64.1
See application file for complete search history. 22 Claims, 4 Drawing Sheets
 US 9.249,061 B2
Page 2

(51) Int. Cl. 2010, 0028.303 A1 2/2010 Martin, Jr. et al.

2012fO255334 A1* 10/2012 Gans ................................... T1/6
C05B 700 (2006.01) 2013,00556.35 A1* 3, 2013 Harman .. 47.58.1 R
C05E II/08 (2006.01) 2014/034.9847 A1* 11/2014 Schrader ....................... 504,100
C05E I/00 (2006.01) 2014/0351998 A1* 1 1/2014 Gupta .................. CO7K 14,415
CI2R L/885 (2006.01) 800,278
CI2R L/645 (2006.01) FOREIGN PATENT DOCUMENTS
CI2P2/06 (2006.01)
C05B (7/00 (2006.01) CN 102.159084 A 8, 2011
C05C5/00 (2006.01) EP O295968 B1 5, 1994
C05C 9/00 (2006.01) EP 1150934 A1 11, 2001
C05D I/02 (2006.01) WO
WO
9929.177
2010119032
A1
A2
6, 1999
10, 2010
C05D3/00 (2006.01)
C05D 5/00 (2006.01) OTHER PUBLICATIONS
C05D 9/02 (2006.01)
C05E II/02 (2006.01) International Search Report and Written Opinion for corresponding
(52) U.S. C. Application No. PCT/US2012/066329 (mailed Feb. 8, 2013) (12
CPC. C05C 9/00 (2013.01); C05C II/00 (2013.01); pages).
C05D I/02 (2013.01); C05D3/00 (2013.01); ATCC Deposit No. 20900. “Trichoderma viride Persoon: Fries,
anamorph.” ATCC: The Essentials of Life Science Research (depos
C05D5/00 (2013.01); C05D 9/02 (2013.01); ited 1991) (1 page).
C05F I/005 (2013.01); C05F II/02 (2013.01); ATCC Deposit No. 36840, "Scopulariopsis brevicaulis (Saccardo)
C05F II/08 (2013.01); C12P21/06 (2013.01); Bainier, anamorph.” ATCC: The Essentials of Life Science Research
CI2R I/645 (2013.01); CI2R 1/885 (2013.01) (deposited 1997) (2 pages).
“Microbial inoculant.” Wikipedia (last modified Sep. 25, 2012) (3
(56) References Cited pages).
“PAR4 13-0-0: Granulated Feather Meal.” Bridgewell Resources
U.S. PATENT DOCUMENTS (dated 2011) (2 pages).
“Natural Fertilizers: Feather Meal.” North Country Organics (dated
5,366,532 11/1994 Fages et al. Mar. 3, 2011) (1 page).
5,976,210 1 1/1999 Sensibaugh Natural Fertilizers: Cheep Cheep 4-3-3, OMRI Listed, North Coun
6,228,806 5, 2001 Mehta try Organics (dated Nov. 1, 2011) (3 pages).
6,440,692 8/2002 Koyama et al. "Container Gardening Tips: Biofertilizer' (dated 2010) (2 pages).
6,596,272 7/2003 Cheung "Endophyte.” Wikipedia (last modified Oct. 15, 2012) (3 pages).
8,163,672 4/2012 Birthisel ....................... 504,101
2002.0053229 5/2002 Varshovi ............................. T1/6 “Biofertilizer.” Wikipedia (last modified Oct. 29, 2012) (2 pages).
2005.0020449 1, 2005 Blais First Office Action and English Translation corresponding to Chinese
2007/013 1009 6/2007 Westbrook et al. ................. T1/6 Patent Application No. 20128.0062108.2 (mailed Sep. 1, 2015).
2008.0318777 12/2008 Lin .......................... COSD 9.02
504,117 * cited by examiner
U.S. Patent Feb. 2, 2016 Sheet 1 of 4 US 9.249,061 B2

Figure 1
U.S. Patent Feb. 2, 2016 Sheet 2 of 4 US 9.249,061 B2

Figures 2A-2B
U.S. Patent US 9.249,061 B2

3eis,
U.S. Patent US 9.249,061 B2
 US 9.249,061 B2
1. 2
HIGHLY EFFICIENT ORGANIC FERTILIZER tional 8 percent—in spite of a projected population
AND COMPONENTS THEREOF increase of 30 percent by the year 2030. Reductions of
that magnitude will only be possible if governments
This application is a national stage application under 35 target all the watershed's sources of nutrient pollution.
U.S.C. S371 of PCT Application No. PCT/US2012/066329, 5 Excess nitrogen and phosphorus, along with sediment, is a
filed Nov. 21, 2012, which claims the benefit of U.S. Provi leading cause of recurring poor water quality in the Bay
sional Patent Application Ser. No. 61/563,355, filed Nov. 23, and the waters that feed it. About 30 percent of the Bay's
2011, which is hereby incorporated by reference in its phosphorus load comes from urbanand Suburban runoff.
entirety. Those same developed lands account for 10 percent of
The Subject matter of this application was made with Sup 10 the nitrogen-tainted runoff. Yet not nearly enough has
port from the National Science Foundation, Grant No. been done to reduce nutrient runoff from developed
0.945724. The government has certain rights in the invention. lands.
While Maryland regulators are requiring farmers to do
FIELD OF THE INVENTION better at controlling nutrient-laden runoff from their
15 fields, the state is mostly ignoring the watersheds domi
This invention relates to highly efficient organic fertilizer nant crop: grass. Throughout the Bay watershed, nearly
and components thereof. 3.8 million acres are now planted in turfgrass, and the
acreage is growing as residential development expands
BACKGROUND OF THE INVENTION and replaces farm fields. Turfgrass is Maryland's big
gest crop by far, with as much as 1.3 million acres
Fertilizers for lawns and other purposes frequently consist planted in grass statewide. That compares with 1.5 mil
of an inorganic mixture of salts that provide nitrogen, phos lion acres planted for all other crops in Maryland in
phorus, and potassium. Fertilizers for different purposes may 2009. Yet it is the least regulated of the state's major
be liquids or Solids, and contain a wide range of nitrogen: CropS.
phosphorus:potassium (“N:P:K)as well as other plant nutri 25 Turfgrass becomes a pollution problem when it is covered
ents depending on the purpose for which the products are with too much fertilizer, which contains nitrogen and
intended. Typically, fertilizer products are primarily com phosphorus. The nutrients in fertilizer can help maintain
posted of salts of inorganic compounds, and so do not qualify healthy lawns, but in excess they can wash into nearby
for organic status. waters when it rains or snows. Excess fertilizer nutrients
Many traditional fertilizer products function very well. 30 can also seep directly into groundwater. Whether the
However, there is increasing concern, because Such fertilizers fertilizer is organic or chemical, its nutrients can harm
pollute water due to their release of high levels of nitrates into the Bay and local waterways.
ground water. One fertilizer product has a formulation of Tracking fertilizer use on developed land is such a low
32:2:8 (% N:PO: KOs). Of that 32%, the analysis on the priority that the state doesn't keep statistics on it, but
packaging indicates that 3.8% is ammonium compounds, 35 Maryland Department of Agriculture records show
53% is urea, 39% is water soluble N, and 3.8% other N. The “nonfarm use fertilizer sales are quickly catching up to
nitrate and urea are immediately available for leaching into farm fertilizer sales. The best estimates suggest that
ground water if these compounds are not taken up by plants. Maryland landowners apply at least 86 million pounds
Unfortunately, plants typically use only about 33% of the of nitrogen fertilizer to state lawns every year.
total nitrogen fertilizer applied (Arnall et al., “Relationship 40 This fertilizer makes its way into rivers and the Bay. In one
Between Nitrogen Use Efficiency and Response Index in Suburban Baltimore watershed, researchers found 56
Winter Wheat,” J. Plant Nutr. 32:502-515 (2009)), while the percent of the nutrients in a local stream came from lawn
remainder may be metabolized to nitrous oxides that are fertilizer. Scientists in Texas, Wisconsin, Minnesota,
potent greenhouse gases or leach into Soil and Surface water Connecticut and Canada have also confirmed that pol
as nitrates and nitrites, where they may be toxic (EPA limit for 45 lutants in lawn fertilizer can significantly harm Surface
drinking water 10 and 1 ppm, respectively) and where they water quality.
may result in areas where decay of algae and other microbes Id. at 1-2.
create anaerobic Zones where plants, fish, and other inhabit Several ordinances on Long Island request "since the
ants cannot survive. Fertilizers that do not have such adverse health of the children and citizens of Township, their water,
environmental impacts are needed. 50 the environment and Long Island Sound will all benefit from
Fertilizers for lawn uses are being modified due to regula the decreased use of chemical fertilizers and lawn pesticides,
tory concerns about eutrophication of waters. Lawn fertilizers the Board of Selectmen urges all citizens to voluntarily
containing phosphorus are banned by Some localities for that refrain from the use of chemical fertilizers and lawn pesti
reason. Nitrate is at least as serious a pollutant as phosphorus, cides and urges the use of organic lawn care.” Pesticides/
and many locations, such as Long Island for example, would 55 Heribicides Model Municipal Ordinances and Regulations,
like to limit water pollution from this source. The seriousness Rivers Alliance of Connecticut. There is a need and potential
of the problem can be illustrated from the following quote market for organic lawn fertilizers with low likelihood to
regarding Chesapeake Bay (Dewar et al., “Urban Fertilizers pollute water. Regulations such as the above stop short of
and the Chesapeake Bay: An Opportunity for Major Pollution requiring that only organic fertilizers be used, primarily
Reduction.” Executive Summary, Environment Maryland, 60 because there are no non-polluting, organic, and cost-effec
Research and Policy Center (2011)): tive products that perform adequately.
For more than 26 years, states in the Chesapeake Bay Synthetic fertilizers that are primarily used are quite effec
region have attempted to clean up the Bay, but it contin tive in providing quality lawns, but they are damaging to the
ues to choke on a lethal overdose of pollution. In order to environment. Unfortunately, as noted Supra, organic fertiliz
achieve a clean, Sustainable Bay, states in the Bay water 65 ers themselves are no panacea. They may also pollute waters
shed will have to reduce nitrogen levels in Bay waters since they still require the same amount of nitrogen to provide
another 30 percent and reduce phosphorus by an addi strong, lush lawns. Typically, current organic fertilizers have
 US 9.249,061 B2
3 4
serious shortcomings. These include the failure to provide The present invention also relates to an isolated Tricho
adequate green-up of lawns shortly after application, and they derma viride strain deposited with Agricultural Research Ser
typically contain only 7 to 9% N of any sort. This means, that vice Culture Collection under number NRRL B-50520.
on a pound-for-pound basis, they cannot provide sufficient N Another aspect of the present invention relates to an iso
for lush, green lawns unless high rates are used. In addition, to lated Scopulariopsis brevicaulis Strain deposited with Agri
obtain adequate performance, they must be applied at 2-6 cultural Research Service Culture Collection under number
NRRL B-50521.
times the rate that Scotts(R) is applied. Thus, a bag of Scotts(R)
fertilizer that weighs about 371b will fertilize 15,000 square Another aspect of the present invention relates to an iso
feet and last for 1-2 months (the company advocates four lated Scopulariopsis brevicaulis Strain deposited with Agri
10 cultural Research Service Culture Collection under number
applications per growing season); equivalent results with a NRRL B-50522.
low Norganic fertilizer will require about 3 bags to deliver the This invention relates to the development of highly effec
same amount of N. These factors make traditional organic tive organic fertilizers that can be used for many purposes and
fertilizers inconvenient to use—customers typically don't can minimize or eliminate groundwater pollution from
want to carry around and spread 100-125 pounds offertilizer 15 nitrates. It includes the identification of slow-release nitrogen
when 30-35 pounds of standard synthetic fertilizers would forms and methods of their use. It also includes microbial
suffice. agents that can degrade complex forms of nitrogen, especially
The fact that so much more needs to be applied makes proteins produced from agricultural waste products, and
traditional organic fertilizers uneconomical. Even if a 25 release plant-available nitrogen in the Soil or in fermenters. It
pound bag of organic fertilizer is slightly less expensive than describes the use of microbes that are endophytic plant sym
inorganic fertilizer, the total price for more than 100 pounds is bionts that enhance the activity of the fertilizers described
much higher. Commercial inorganic fertilizer typically retails herein and also enhance plant growth and performance. The
for about $55 per bag, while the same amount of N in most fertilizers are efficient methods of delivery of these organ
organic fertilizers will cost more than S100. isms.
The present invention is directed to overcoming these and 25 The requirements for user-acceptable and nonpolluting
other deficiencies in the art, and provides an answer to many fertilizers are several. First, there is a need for a universally
of the problems of traditional fertilizer while providing sev applicable mechanism whereby plants can take up N fertilizer
eral distinct paths to new and unique products that will meet more efficiently. One method to accomplish this is through
the needs of both users and limit environmental problems. the use of endophytic plant symbiotic fungi or bacteria that
30 increase plants’ abilities to use nitrogen fertilizer more effi
SUMMARY OF THE INVENTION ciently (“nitrogen use efficiency” or “NUE'). NUE can be
induced by increasing plant root growth and by physiological
The present invention relates to a fertilizer including at changes in plants. A number of microorganisms are poten
least one microbe selected from the group consisting of Tri tially useful in this regard. For example, these include bacte
choderma viride (NRRL B-50520), Scopulariopsis brevicau 35 ria designated as Plant Growth Promoting Rhizobacteria
lis (NRRL B-50521), Scopulariopsis brevicaulis (NRRL (“PGPR), the Basidiomycete Piriformospora indica, and
B-50522), and combinations thereof. The fertilizer also mycorhizzal fungi such as those in the genus Glomus. Other
includes a Substrate which is acted upon by the at least one examples include T. harzianum strain RR17Bc (ATCC acces
microbe to release nitrogen. sion PTA9708), T. harzianum F11 Bab (ATCC accession PTA
Another aspect of the present invention relates to a method 40 9709), T. atroviride strain WW10TC4 (ATCC PTA accession
of enhancing growth of plants. The method includes provid 9707), and T. vixens strain 41 (ATCC accession 20476)).
ing a fertilizer comprising at least one microbe selected from These same fungi also induce numerous other beneficial
the group consisting of Trichoderma viride (NRRL changes in plants such as resistance to disease, resistance to
B-50520), Scopulariopsis brevicaulis (NRRL B-50521), abiotic stresses like drought and salt, and increased abilities
Scopulariopsis brevicaulis (NRRL B-50522), and combina 45 of plants to photosynthesize, thereby providing additional
tions thereof; and a substrate which is acted upon by the at benefits to plants.
least one microbe to release nitrogen. The method further For lawns, the present invention provides an organic fertil
includes contacting the fertilizer with plants or plant seeds izer that has an analysis of 12-16:0-10:5-15 that is function
under conditions effective to enhance the growth of the plants ally equivalent to a widely sold inorganic lawn fertilizer that
or plant seeds compared to plants or plant seeds to which the 50 has an analysis of 30:2:8 (N:PO: KO). Also described is a
fertilizer was not applied. prototype process of producing a granular fertilizer Suitable
The present invention also relates to a fertilizer that for dispersal instandard drop or broadcast fertilizer spreaders
includes at least one microbe selected from a keratin degrad that contains the organisms that are claimed in this invention.
ing microorganism and a keratin Substrate which is acted Also described is a method for producing a powdered formu
upon by the at least one microbe to release nitrogen. 55 lation that can be incorporated into potting media, applied to
Another aspect of the present invention relates to a method furrows and the like, and also a liquid organic fertilizer for
of making a fertilizer comprising amino acids and ammonia. mulation process. The result is a new fertilizer, which may be
This method includes providing at least one microbe selected classified as organic.
from the group consisting of Trichoderma viride (NRRL Another useful attribute that can be provided by compo
B-50520), Scopulariopsis brevicaulis (NRRL B-50521), 60 nents offertilizers is the slow release of nutrients. Fertilizers
Scopulariopsis brevicaulis (NRRL B-50522), and combina that are composed simply of soluble salts release their nutri
tions thereofand providing a substrate which is acted upon by ents rapidly and provide a flush of nutrients into ground water,
the at least one microbe to release nitrogen. The method and they rapidly become pollutants. As described herein,
further includes contacting the at least one microbe and the microorganisms, particularly selected Trichoderma Strains,
substrate under conditions effective to cause the at least one 65 can markedly enhance plant growth, improve resistance to
microbe to act on the Substrate and produce a liquid Suspen abiotic stresses, increase plant nitrogen use efficiency,
sion comprising amino acids and ammonia. increase root growth and development, and markedly
 US 9.249,061 B2
5 6
enhance general plant performance. It is highly advantageous and, therefore, there will be little or no release of nitrates into
to incorporate such beneficial microbes directly onto or into ground water. This environmental advantage will be even
fertilizers so that the benefits of both the fertilizers and ben greater if the microbial release of N is combined with other
eficial microbes can be conveniently applied at the same time microbes that increase root growth and improve plant nitro
and in the same product. However, the high salt content of 5 gen use efficiency. Further, the same invention, with minor
most synthetic fertilizers will severely damage or kill applied modifications, can be used to produce a soluble source or
microbes contained within or on the fertilizers. This invention amino acids and ammonia that are highly beneficial for use as
describes fertilizers and methods of making fertilizer gran a plant fertilizer.
ules that are composed primarily of proteins that are not The present invention satisfies the needs created by con
injurious to microbial agents. Pollution is particularly likely if 10 ventional fertilizers, by providing a system or systems com
fertilizers are overapplied, if there is excessive water from prising fertilizers with specific compositions and functions
rain or irrigation, or when they are applied to soils that are combined with microorganisms that degrade complex pro
sandy and that leach nutrients. Since it cannot be anticipated teinaceous Substrates to provide nutrients, especially nutri
where or how fertilizers are applied, nitrogen may be in a ents to growing plants. It also includes microorganisms (i.e.,
form that is released slowly, so that plants can take up more of 15 endophytic plant symbionts) that enhance plant growth and
the applied nutrients as it is released slowly rather than all at performance and provide other advantages to plant growth.
once. There are two basic methods of approach. First, a stan The fertilizers of the present invention are excellent delivery
dard process in the industry is to produce nitrogen in various agents for these organisms.
forms that release nitrogen slowly. There are a number of slow The present invention is expected to provide revolutionary
release forms of nitrogen in commercial trade today. These benefits to the fertilizer industry. First, the fertilizer of the
include urea-formaldehyde, methylene urea, Sulfur coated present invention provides, for the first time, a totally organic
urea, and others. These are well known in the field, see for or biopreferred fertilizer that performs as well as standard
example U.S. Pat. No. 8,182.572 to King et al., which is widely-sold inorganic fertilizers. It also serves as a fertilizer
hereby incorporated by reference in it entirety. Second, there that is as convenient and easy-to-use as conventional inor
are various complex forms of nitrogen, usually in the form of 25 ganic fertilizers and is useful as a lawn fertilizer that has
proteins, manures or composts. These materials release their greatly reduced pollution of ground and Surface waters with
nitrogen slowly, as a consequence of microbial activity. nitrates. The fertilizer of the present invention also provides a
“Organic' materials may be certified, and the rules pertain method to produce a new class of organic Soluble fertilizers
ing to them are agency dependent. The Organic Materials that include immediately available nitrogen for plant growth
Research Institute (“OMRI) certified products according to 30 in the form of amino acids and ammonia. The only product
their own specifications. OMRI is a private agency with well available to organic (OMRI certified) growers that contains
established certification. Many, but not all agricultural waste immediately available nitrogen is Chilean nitrate, and its use
products such as composts, manures, and the like, can be is limited by OMRI rules, and it may be banned from organic
certified, but the requirements by which they are made are use in the near future. A mixture of amino acids and ammonia
relatively stringent. A few well-defined chemicals can be 35 would be a very useful new product that can be used either as
used, but their uses are strictly governed. For example sodium a component of other fertilizers or else as a liquid stand-alone
nitrate is acceptable only if it is a mined form (called Chilean fertilizer. The present invention describes, for the first time, a
nitrate) and not a manufactured version, and even with this method of deriving an OMRI registerable mixture of amino
material its use is limited. Another, near-organic certification acids and ammonia and the composition of Such as product.
is the USDA BioPreferred product listing. Products with this 40 The focus is on lawn fertilizers, which are probably the most
designation are required to be derived from bio-based farm demanding, and perhaps the materials used in the greatest
products to a certain percentage of the total. The minimum amounts, but the principles and developments are equally
content of farm-derived products for fertilizers is 71%. This applicable to fertilizers for other applications. Products with
can include many of the same materials that are listed by these capabilities do not currently exist.
OMRI, but there are exceptions. For example, peat moss is 45
eligible for certification by OMRI, but not by the BioPre BRIEF DESCRIPTION OF THE DRAWINGS
ferred program. Composts, manures, sources of protein from
both farm animals and plants are acceptable for both. One FIG. 1 shows dry granules prepared using a feather pow
significant difference is that the OMRI registered products der-gelatin mixture coated with feature degrading microbes,
have to be 100% of their certified products, but for BioPre 50 before and after moistening for two days. The fungi that were
ferred, up to 29% can be other than products on the BioPre coated on the outside of the granules grew moderately. The
ferred list. Thus, it is possible to use some of the synthetic granules were modified by the addition of media that would
slow-release synthetic nitrogen forms with a BioPreferred Support the growth of Trichoderma rapidly and abundantly.
product, but not with an OMRI-labeled product. The dry granules are approximately 2 mm in diameter.
The present invention describes microbes (i.e., fungi) that 55 FIGS. 2A-2B illustrate fertilizer comparisons using prepa
effectively degrade keratin in feathers/feather meal. These rations described in the present invention or commercially
microbes rapidly release amino acids and ammonia from obtained. In FIG. 2A, the treatments used were PT (organic
feather meal in semi-solid media. For example, inoculum of fertilizer, NRRL B-50521, and K1-K4, which included T.
the fungi has many uses and can be prepared as a dry spore harzianum strain RR17Bc (ATCC accession PTA 9708), T.
preparation or as a liquid formulation. The present invention 60 harzianum F11 Bab (ATCC accession PTA 9709), T. atro
also describes a method for producing a granule that can be viride strain WW10TC4 (ATCC PTA accession 9707), and T.
broadcast, with or without a coating of microbes, or with or virens strain 41 (ATCC accession 20476)); STB (Scotts(R)
without having the microbes incorporated within the granule, Turf Builder(R); P (organic fertilizer with NRRL B-50521):
that provide a slow-release microbially-activated release of and C (untreated control). In FIG. 2B, the treatments used
nitrogen for plant growth, especially when combined with 65 were PT (organic fertilizer, NRRL B-50521, and K1-K4, as
other beneficial fungi. The combination of microbially-acti described supra); OFC (organic fertilizer granules without
vated nitrogen systems release almost no nitrates into Soils any fungi); and P (organic fertilizer with NRRL B-50521).
 US 9.249,061 B2
7 8
FIGS. 3A-3D show harvest weights of grass over time with Trichoderma harzianum T-22, Plant Dis. 84:377-393
using different fertilizer treatments. (2000), which is hereby incorporated by reference in its
FIG. 4 shows root development of turfgrown in a sand:peat entirety). Equipment and procedures for Such applications are
mix for about 6 weeks and fertilized with the granules well known and used in various agricultural industries. In one
described in this example with and without the beneficial embodiment of the present invention, the fertilizer further
microbial mix T. virens stain NRRL B-50521 and Tricho comprises a Supplemental microbe in the form of a Tricho
derma strains K1-K4 (T. harzianum strain RR17Bc, T. har derma species. The Trichoderma species can be selected from
zianum F11 Bab, T. atroviride strain WW10TC4, and T. virens the group consisting of Trichoderma vixens, Trichoderma
strain 41). The petridish labeled 11 shows root growth on turf harzianum, Trichoderma atroviride, and combinations
fertilized with the granules without the beneficial microbes. 10 thereof.
The dish labeled 12 contained the organisms incorporated Trichoderma grows intercellularly in the root epidermis
into the granules at about 10 cfu/g, while the dish labeled 13 and cortex and induces the Surrounding plant cells to deposit
was fertilized with granules coated with the beneficial fungi cell wall material and produce phenolic compounds. This
in a dextran mixture (10 colony forming units (cfus)/ml in plant reaction limits the Trichoderma growth inside the root
2% tapioca dextran to form a thin continuous coat). 15 (see Yedidia et al., “Induction and Accumulation of PR Pro
teins Activity During Early Stages of Root Colonization by
DETAILED DESCRIPTION OF THE INVENTION the Mycoparasite Trichoderma harzianum Strain T-203.”
Plant Physiol. Biochem. 38:863-873 (1999), which is hereby
The present invention relates to a fertilizer including at incorporated by reference in its entirety). Endophytic plant
least one microbe selected from the group consisting of Tri symbionts have much longer periods of efficacy since they
choderma viride (NRRL B-50520), Scopulariopsis brevicau have the ability to grow with plants and in the environment;
lis (NRRL B-50521), Scopulariopsis brevicaulis (NRRL therefore if conditions are favorable for them, they may have
B-50522), and combinations thereof. The fertilizer also effects for weeks or months. These organisms may develop on
includes a Substrate which is acted upon by the at least one or in plant roots and provide benefits to plants for at least the
microbe to release nitrogen. 25 life of an annual crop (Harman, G. E., “Myths and Dogmas of
The present invention can be carried out with all types of biocontrol. Changes in Perceptions Derived from Research
Substrate that is acted upon by a microbe to release nitrogen, on Trichoderma harzianum T-22. Plant Dis. 84:377-393
including hair, hooves, and feather. In one embodiment, the (2000) and Harman et al., “Changing Paradigms on the Mode
Substrate is any protein selected from the group consisting of of Action and Uses of Trichoderma spp. for Biocontrol.”
feather, feather meal, and a derivative of urea. Any type of 30 Outlooks Pest Manag. 19:24-29 (2008), which are hereby
feather may be employed, including but not limited to incorporated by reference in their entirety). The establish
chicken, turkey, and duck feather. The present invention is ment of living hyphae of the beneficial organisms in the root
applicable to the degradation and utilization of all Substrate cortex results in chemical communication with the plant. In
materials that release nitrogen. Some embodiments, the fertilizer contains other Supplemen
Any of a number of organisms or beneficial microbes can 35 tal microbes, such as those selected from the group consisting
be added to the fertilizer of the present invention. These of Piriformospora indica, a plant growth promoting rhizobac
include those Strains described in WO 2010/091337 to Har teria, mycorrhizal fungi, and combinations thereof.
man; Harman, “Multifunctional Fungal Plant Symbionts: As a consequence, reprogramming of plant gene expres
New Tools to Enhance Plant Growth and Productivity.” New sion occurs, and numerous benefits to the plant result. This
Phytol. 189:647-49 (2011); Lorito et al., “Translational 40 capability of growing on, or conidial germination on, root
Research on Trichoderma: From 'Omics to the Field, Annu. Surfaces makes possible many kinds of application methods.
Rev. Phytopathol. 48:395-417 (2010); Shoresh et al., These include, but are not limited to, seed treatments, appli
“Induced Systemic Resistance and Plant Responses to Fungal cation to soils or planting mixes as drenches that penetrate the
Biocontrol Agents.” Annu. Rev. Phytopathol. 48:21-43 soil Volume and in furrow application at the time of planting,
(2010), which are hereby incorporated by reference in their 45 broadcast or spray applications to Soil Surfaces containing
entireties. Other beneficial organisms include mycorrhizal roots. It also permits the use of very Small amounts of inocu
fungi, plant growth promoting rhizobacteria, aZospirillum, lum (10 S of g/ha) applied as a seed treatment, but that then
and nitrogen fixing rhizobia. These organisms have very results in Subsequent proliferation of the organism on roots,
many useful attributes. The fungi change gene expression and causing season-long effects, including plant protection,
cause plants to exhibit increased resistance to disease-causing 50 greater root proliferation and enhanced exploration of the soil
pathogens (“resistance to biotic stresses'), exhibit increased by roots (Adams et al., “Trichoderma harzianum Rifai 1295
resistance to environmental stresses such as resistance to 22 Mediates Growth Promotion of Crack Willow (Salix fra
drought, salt or temperature (“resistance to abiotic stresses'), gilis) Saplings in Both Clean and Metal-contaminated Soil.”
and increase the efficiency of uptake of nitrogen (“improved Microbial. Ecol. 54:306–313 (2007); Harman, G. E., “Myths
nitrogen use efficiency INUEI”). Beneficial microbes and 55 and Dogmas of Biocontrol. Changes in Perceptions Derived
endophytic plant microbes of the present invention may pro from Research on Trichoderma harzianum T-22. Plant Dis.
vide improved nitrogen use efficiency. Beneficial microbes 84:377-393 (2000); Harman et al., “Changing Paradigms on
may be formulated or mixed to prepare granules or liquid the Mode of Action and Uses of Trichoderma spp. for Bio
Suspensions. These can be mixed directly into Soils or potting control. Outlooks Pest Manag. 19:24-29 (2008); and Har
mixes. The term soil in this specification is used to include 60 man et al., “Trichoderma Species—Opportunistic, Avirulent
any medium capable of Supporting the growth of plants and, Plant Symbionts.” Nature Rev. Microbiol. 2:43-56 (2004),
therefore, includes common soil, humus, manure, compose, which are hereby incorporated by reference in their entirety).
sand, and the like, and also artificially created plant growth Trichoderma Strains Suitable for the present invention (e.g.,
media. The preparations are then mixed into the Soil or plant Trichoderma viride, Trichoderma vixens, Trichoderma har
ing mix volume for greenhouse applications or into the upper 65 zianum, and Trichoderma atroviride) are strains with strong
volume of field soil (Harman, G. E., “The Dogmas and Myths abilities to colonize roots. This ability is known as rhizo
of Biocontrol. Changes in Perceptions Based on Research sphere competence, which is used herein to describe those
 US 9.249,061 B2
10
organisms capable of colonizing the root surface or the Sur The substrate of the present invention can be blended with
face plus Surrounding soil volume (rhizoplane and rhizo other sources of plant nutrients, including potassium, phos
sphere, respectively), when applied as a seed or other point phorus, iron or minor nutrients.
source at the time of planting in absence of bulk flow of water. In one embodiment, the fertilizer further comprises a non
Thus, the organisms of the present invention have the physi proteinaceous binding agent selected from the group consist
ological and genetic ability to proliferate in and on the root as ing of dextrans, starches, polyvinylchloride, and combina
it develops. Rhizosphere competence is not an absolute term, tions thereof.
and degrees of this ability may occur among strains (Harman, In another embodiment, the fertilizer may include a source
G. E., “The Development and Benefits of Rhizosphere Com 10
of phosphorus selected from the group consisting of rock
petent Fungi for Biological Control of Plant Pathogens. J. phosphate, sodium phosphate, potassium phosphate, bone
Plant Nutrition 15:835-843 (1992); U.S. Pat. Nos. 4,996,157 meal, and combinations thereof.
and 5,165,928 to Smith, which are hereby incorporated by In yet another embodiment, the fertilizer may include a
reference in their entirety). Procedures for measuring rhizo Source of potassium selected from the group consisting of
sphere competence are known to those skilled in the art (Har 15 potassium chloride, potassium phosphate, potassium sulfate,
man et al., “Combining Effective Strains of Trichoderma Jersey green sand, organic sources of potassium, animal
harzianum and Solid Matrix Priming to Improve Biological manure, and combinations thereof. For organic uses, sources
Seed Treatments.” Plant Disease 73:631-637 (1989); Har of potassium include Jersey green sand, and organically listed
man, G. E., “The Myths and Dogmas of Biocontrol. Changes potassium sulfate and chloride.
in Perceptions Based on Research with Trichoderma har The fertilizer may, alternatively, include minor nutrients
zianum T-22, Plant Disease 84:377-393 (2000): Kloepperet selected from the group consisting of salts, Substances that
al., “A Review of Issues Related to Measuring Colonization contain iron, cobalt, manganese, magnesium, copper, cal
of Plant Roots by Bacteria.” Can. J. Microbiol. 38:1219-1232 cium, boron, Zinc, and combinations thereof. These are
(1992), which are hereby incorporated by reference in their readily available from a variety of sources.
entirety). 25 Sulfur is already present in the amino acids contained in the
The fertilizer of the present invention may be in granule, substrate, including, for example, feather meal. Useful fertil
pellet, dust, powder, slurry, film, and/or liquid Suspension izers may be prepared with a variety of N:P:K ratios with or
form. In one embodiment, the fertilizer is in the form of a without other nutrients by blending or addition of other
granule or pellet which contains gelatinas a Solidifying agent. Sources of organic Substrates other than N. In one embodi
The gelatin may be 5-20 wt % of the granule or pellet, or more 30 ment, the fertilizer has a nitrogen content between 10-20
wt %.
preferably, 7-8 wt % of the granule or pellet. In another In one embodiment, the fertilizer contains a growing
embodiment, the fertilizer includes at least 104 colony form media. Growing media of the present invention may include,
ing units/g (“cfu') of the at least one microbe in the substrate. but is not limited to, soil, sand compost, peat, rice hulls, coir,
In yet another embodiment, the fertilizer is in the form of a 35 cocopeat, soilless growing media containing organic and/or
liquid Suspension including amino acids and ammonia. The inorganic ingredients, artificial plant-growth substrates, poly
microbes of the present invention grow, and so by weight their mer-based growth matrices, hydrophonic nutrient and growth
quantities are vanishingly small. 104 cfu is equivalent to a Solutions, organic soil amendments, water, and mixtures
proportion of the microbial formulation as little as one part in thereof.
107 of the substrate. However, the formulation can be made 40 The fertilizer may include a carrier selected from the group
in a number of different ways and, therefore, can easily be a consisting of water, aqueous solutions, slurries, and powders.
100x variation in the concentration of the Trichoderma or Alternatively, the fertilizer may include an additive such as,
other microbe in the granule or pellet. but not limited to, insecticide, fungicide, nematicide, addi
The fertilizer of the present invention also contemplates the tional organic fertilizer, bioinsecticide, biofungicide, bion
addition of a Supplemental source of nutrients. These include, 45 ematicide, agricultural or horticultural adjuvants, stickers,
for example, Soil, water, urea, ammonium nitrate, sources spreaders, Surfactants, dispersants, humectants, UV pro
providing nitrogen, phosphorous, potassium, calcium, mag tectants, and mixtures thereof. The fertilizer can also contain
nesium, Sulfur, and other micronutrients. There are at least a source of organic carbon Such as, but not limited to, compost
sixteen chemical elements known to be useful in a plants and biochar.
growth and Survival. The sixteen chemical elements are 50 Another aspect of the present invention relates to a fertil
divided into two main groups: non-mineral and mineral. The izer that includes at least one microbe selected from a keratin
non-mineral nutrients includehydrogen (H), oxygen (O), and degrading microorganism and a keratin Substrate which is
carbon (C) and are found in the air and water. The remaining acted upon by the at least one microbe to release nitrogen.
13 nutrients are minerals, which come from the soil, and are The methods of the present aspectare carried out in accor
dissolved in water and absorbed through a plant's roots. The 55 dance with the previous aspect.
mineral nutrients are further divided into two groups: macro In one embodiment, the fertilizer includes at least one
nutrients and micronutrients. Macronutrients include but are microbe selected from the group consisting of Trichoderma
not limited to N. P. S. K., Ca,Mg,Na, and Si. Micronutrients viride (NRRL B-50520), Scopulariopsis brevicaulis (NRRL
include, but are not limited to, Fe, Mn, Cu, Zn, Mo, B, and Cl. B-50521), Scopulariopsis brevicaulis (NRRL B-50522), and
Micronutrients are needed in only very small (i.e., micro) 60 combinations thereof.
quantities. These nutrients and their benefits to plants would Keratin degrading microorganisms of the present aspect
be well known to a person of skill in the art. The fertilizer of can include microorganisms of the genus Streptomyces,
the present invention may also contain a soluble nitrogen including but not limited to, Streptomyces avermitilis, Strep
Source selected from the group consisting of nitrate, ammo tomyces coelicolor, and Streptomyces violaceOruber.
nia, ammonium salts, amino acids, urea, fish meal or extract, 65 This aspect of the present invention can be carried out using
compost extract, kelp extract, shrimp extract, shellfish any of the additives and any modes of application described
extract, and combinations thereof. above on any of the above noted plants.
 US 9.249,061 B2
11 12
Another aspect of the present invention relates to a method house applications or into the upper Volume of field soil
of enhancing growth of plants. The method includes provid (Harman, G., “The Myths and Dogmas of Biocontrol.
ing a fertilizer comprising at least one microbe selected from Changes in Perceptions Based on Research with Trichoderma
the group consisting of Trichoderma viride (NRRL harzianum T-22.” Plant Disease 84:377-393 (2000), which is
B-50520), Scopulariopsis brevicaulis (NRRL B-50521), hereby incorporated by reference in its entirety). Equipment
Scopulariopsis brevicaulis (NRRL B-50522) and combina and procedures for Such contacting are well known and used
tions thereof and a substrate which is acted upon by the at in various agricultural industries. Typical rates are 0.2 to 10
least one microbe to release nitrogen. The method further kg of product containing 107 to 10 colony forming units (cfu)
includes contacting the fertilizer with plants or plant seeds per cubic meter of planting mix or soil.
under conditions effective to enhance the growth of the plants 10 Contacting can also be done by liquid application
or plant seeds compared to plants or plant seeds to which the (drenches) for greenhouse or nursery soils and soil mixes.
fertilizer was not applied. Liquid Suspensions of the beneficial microorganisms can be
The methods of the present aspectare carried out in accor prepared by mixing dry powder formulations into water or
dance with the previous aspect. another carrier, including fertilizer Solutions, or by diluting a
In one embodiment of the present aspect of the invention, 15 liquid formulation containing the organism in water or other
the fertilizer contains gelatin which makes up 5-20 wt % of aqueous solutions, including those containing fertilizers. In
the granule or pellet. either case, the formulation may include other organic or
In practicing all aspects of the present invention, the fertil non-organic additives to aid in dissolving or applying the
izer may be prepared in a formulation containing organic or mixture. Solutions can then be used to water planting mixes
inorganic materials that aid in the delivery or contacting of the either prior to planting or else when plants are actively grow
organism to the recipient plant or plant seed. Furthermore, in ing, such as by field irrigation. Typically, 10 to 400 ml of
all aspects of the present invention described herein, contact product (usually 150/inn or smaller in particle size) contain
ing of the fertilizer with plants or plant seeds, or other plant ing 107 to 10 cfu are mixed with 100 L of water for such
material, may be carried out either simultaneously with, applications.
before, or after the introduction of the plant, plant seed, or 25 Seeds are commonly treated using slurry, film-coating or
other plant propagative material into a growing media or area. pelleting by processes well known in the trade (Harman et al.,
The plant, plant seed, or other plant material can be estab “Factors Affecting Trichoderma hamatum Applied to Seeds
lished (propagated) in any Suitable planting media, as As a Biocontrol Agent.” Phytopathology 71:569-572 (1981);
described Supra, without limitation, as well as in any Suitable Taylor et al., “Concepts and Technologies of Selected Seed
environment, for example, agreenhouse or field environment. 30 Treatments.” Ann. Rev. Phytopathol. 28:321-339 (1990), both
A person of skill in the art would readily be able to establish of which are hereby incorporated by reference in their
the requirements suitable for sustaining and/or propagating a entirety). The microbes of the present invention can effec
plant. tively be added to any such treatment, provided that the for
Regardless of the orderin which contacting the organism to mulations do not contain materials injurious to the beneficial
plant, seed, or other plant material is carried out, the following 35 organism. Depending on the organism in question, this may
are all suitable methods in accord with the present invention include chemical fungicides. Typically, powder or liquid for
for bringing the fertilizer and plant material of choice in mulations (107 to 10" cfu/g) of the organism are suspended in
contact. Non limiting examples of these methods include aqueous Suspensions to give a bioactive level of the organism.
broadcast application, drop application, rotary application, The liquid typically contains adhesives and other materials to
liquid or dry in-furrow application, direct incorporation into 40 provide a good level of coverage of the seeds and may also
soils or greenhouse planting mixes, spray application, irriga improve its shape for planting or its cosmetic appeal.
tion, injection, dusting, pelleting, or coating of the plant or the Contacting can also be accomplished by dry powders con
plant seed or the planting medium with the fertilizer. It also is taining beneficial organisms which are applied as a dust to
possible to produce a granular formulation Suitable for drop roots, bulbs, or seeds. Generally, fine powders (usually 250/in
or broadcast spreading, a powdered formulation for addition 45 or smaller) are dusted onto seeds, bulbs or roots to the maxi
to potting mixes or directly in field applications, or a liquid mum carrying powder (i.e., until no more powder will adhere
fertilizer using the systems described herein. to the treated surface). Such powders typically contain 107 to
The fertilizer of the present invention may be applied in the 10 cfu/g.
same manner as conventional fertilizers. As known to those Liquid Suspensions of the beneficial microorganisms can
skilled in the relevant art, many methods and appliances may 50 be prepared by mixing dry powerformulations into water or
be used. In one embodiment, a mixture of microbes of the other aqueous carriers, including fertilizer Solutions, or by
present invention and Substrate are applied directly to Soil or diluting a liquid formulation containing the microbe in water
plants. In another embodiment, dried powders of the or otheraqueous solutions. Such solutions can then be used to
microbes of the present invention and Substrate are applied to water planting mixes either prior to planting or else when
soil or plants. The fertilizer may be applied to soil, by spread 55 plants are actively growing. Liquid Suspensions of products
ers, sprayers, and other mechanized means which may be may be injected under pressure into the root Zone of appro
automated. The fertilizer may be applied directly to plants, for priate plants through a hollow tube located at the depth
example, by soaking seeds and/or roots, or spraying onto desired by the application. Equipment for Such application is
leaves. Such application may be made periodically, such as well known in the horticulture industry. Alternatively, sus
once per year, or per growing season, or more frequently as 60 pensions or powders containing appropriate organisms can be
desired. Although not necessary, the fertilizer of the invention applied into wells or other aqueous environments in the soil.
can also be used in conjunction or in rotation with other types Liquid Suspensions of products may be prepared as described
of fertilizers. above for preparing drenches. Such materials may be added
Beneficial microbes may be formulated or mixed to pre to the furrow into which seeds are planted or small plants are
pare granules, dusts, or liquid Suspensions. These can be 65 transplanted. Equipment for Such applications is widely used
mixed directly into Soils or planting mixes. The preparations in the agricultural industry. Typical rates of application are
are then mixed into the Soil or planting mix volume for green 0.5 to 10 kg of product (107 to 10 cfu/g) per hectare of field.
 US 9.249,061 B2
13 14
Granules can be broadcast onto soil surfaces that contain pears and quince, crops that include beets, Sugarbeets, red
growing plants, to soil at time of planting, or onto soils into beets, carrots, celeriac, chicory, horseradish, parsnip, radish
which seeds or plants will be planted. Typical rates of appli rutabaga, Salsify, turnips; shadehouse and other nursery crops
cation range from 1 to 500kg of product containing 10 to 10 that include deciduous trees (maple, oak), ornamentals,
cfu/g depending on the plants to be treated and the goals of the grapes, citrus, pine; Small grains that include rye, wheat,
treatment. Alternatively, spray Solutions can be prepared and Sorghum, and millet; Stone fruits that include apricots, cher
applied to give similar rates (Harman, G. E., "The Dogmas ries, nectarines, peaches, plums, prunes, tree nuts: almonds,
and Myths of Biocontrol. Changes in Perceptions Based on beech nuts, Brazil nuts, butternuts, cashews, chestnuts, fil
Research with Trichoderma harzianum T-22. Plant Dis. berts, hickory nuts, macadamia nuts, pecans, pistachios, and
80:736-741 (1996); Lo etal, “Biological Control of Turfgrass 10 walnuts; tuber crops that include potatoes, Sweet potatoes,
Diseases with a Rhizosphere Competent Strain of Tricho yams, artichoke, cassava, and ginger. Other examples include
derma harzianum," Plant Disease 80:736-741 (1996); Lo et those grasses associated with turfgrass, turf sports fields,
al., “Improved Biocontrol Efficacy of Trichoderma har parks, established and new preparation of golf course tees,
zianum 1295-22 for Foliar Phases of Turf Diseases by Use of greens, fairways and roughs, seed production and sod pro
Spray Applications.” Plant Dis. 81:1132-1138 (1997), which 15 duction. Plants that may be treated also include petunia, pel
are hereby incorporated by reference in their entirety). argonium, poinsettia, chrysanthemum, carnation, and Zinnia.
For the purposes of the present invention, all methods Plant growth enhancement of the present invention may be
which describe application are designed to accomplish the in the form of greater root mass, greater depth of rooting,
same purpose, i.e., to provide a means of application that will greater shoot mass, greater length of shoots, increased leaf
result in effective colonization of the root by the beneficial greenness, increased yields, and improved Stand and vigor.
organism (Harman et al., “Potential and Existing Uses of Plant growth can be established and ascertained by other
Trichoderma and Gliocladium For Plant Disease Control and means besides the extrinsic properties listed above. A person
Plant Growth Enhancement. In Trichoderma and Gliocla of skill in the art would readily be able to establish physical,
dium, Harman et al., eds. Vol. 2, London: Taylor and Francis biochemical or genetic assays to identify and/or quantify
(1998), which is hereby incorporated by reference in its 25 plant growth or viability.
entirety). Plants derive numerous advantages from root colonization
The present invention can be used to treat a wide variety of by the fertilizer disclosed herein. One advantage is protection
plants of their seeds. As used herein, the fertilizer of the of plants against diseases by direct action of the microbe
present invention Supports or enhances plant growth, if in the strains on pathogenic microbes (see Chet, L., “Trichoderma—
presence of the fertilizer in the soil, or applied to the roots, 30 Application, Mode of Action, and Potential as a Biocontrol
stems, leaves or other parts of the plant, the plant or a part of Agent of Soilborne Plant Pathogenic Fungi. In Innovative
the plant gains viability, size, weight, rate of germination, rate Approaches to Plant Disease Control, pp. 137-160, 1. Chet,
of growth, or rate of maturation. Thus, the fertilizer has utility ed., J. Wiley and Sons: New York (1987), which is hereby
in any kind of agricultural, horticultural, and forestry prac incorporated by reference in its entirety) or other deleterious
tice. The fertilizer can be used for large scale commercial 35 soil microflora (Bakker et al., “Microbial Cyanide Production
farming, in open fields or in greenhouse, or even in interiors in the Rhizosphere in Relation to Potato Yield Reduction and
for decorative plants. Preferably, the fertilizer is used to Pseudomonas spp-Mediated Plant Growth-Stimulation.” Soil
enhance the growth of crop plants, such as, but not limited to, Biol. Bio-Chem. 19:451-457 (1987), which is hereby incor
cereal crops, vegetable crops, fruit crops, flower crops, and porated by reference in its entirety). Microbes offer protec
grass crops. For example, the fertilizer compositions may be 40 tion against plant pathogens due to systemic induction of
used with dicots and monocots. More particularly, plants resistance. This permits plants to be protected at a point
treated in accordance with the present invention include any widely separated (temporally or spatially) from application of
plant Susceptible to fungal or plant pathogen. For example, microbes (see Harman et al., “Trichoderma Species—Oppor
plants treated in accordance with the present invention tunistic, Avirulent Plant Symbionts.” Nature Microbiol. Rev.
include, but are not limited to, agronomic row or other field 45 2:43-56, (2004), which is hereby incorporated by reference in
crops that include buckwheat, beans (soybean, Snap, dry), its entirety). For example, through induced resistance, the
corn (grain, seed, Sweet corn, silage, popcorn, high oil), cot microbes disclosed herein can control foliar pathogens even
ton, canola, peas (dry, succulent), peanuts, safflower, and when it is present only on the roots.
Sunflower; alfalfa hay and forage crops that include alfalfa, Another advantage is that the fertilizer of the present inven
clover, vetch, and trefoil; berries and small fruits that include 50 tion can provide protection against abiotic stress due to
blackberries, blueberries, currants, elderberries, gooseber drought (water deficit), disease or other unfavorable plant
ries, huckleberries, loganberries, raspberries, strawberries, growth conditions. Often times, plants may be cultivated in
grapes, bulb crops: garlic, leeks, onions, shallots, and orna climates where the crop is exposed to many biotic and abiotic
mental bulbs; citrus fruits that include citrus hybrids, grape stresses such as plant diseases and drought. Drought condi
fruit, kumquat, limes, oranges, and pummelos, cucurbit veg 55 tions affect gene expression, amino acid profiles, and photo
etables that include cucumbers, melons, gourds, pumpkins, synthesis in plants thereby inducing stress. The majority of
squash, and flowers; bedding plants and ornamentals; fruiting these responses may be delayed in plants treated with the
Vegetables that include eggplant, Sweet and hot peppers, fertilizer of the present invention. It may be possible to
tomatillos, tomatoes, herbs, spices, and mints; hydroponic improve the tolerance of plants to drought by treating plants
crops that include cucumbers, tomatoes, and lettuce; herbs 60 with microbes of the present invention in the field. Plants with
and spices; leafy vegetables and cole crops that include aru improved tolerance to drought, disease, and stress would be
gula, celery, chervil, endive, fennel, lettuce (head and leaf), of benefit to the farmers by stabilizing crop yields and prof
parsley, radicchio, rhubarb, spinach, Swiss chard, broccoli, itability.
Brussels sprouts, cabbage, cauliflower, collards, kale, kohl The microbes used in the fertilizer disclosed in the present
rabi, mustard greens, and asparagus; legume vegetable and 65 invention may result in more and deeper roots and reduce the
field crops that include Snap and dry beans, lentils, succulent nitrogen requirement for plant growth presumably by
and dry peas, peanuts, and Soybeans; pome fruit that include enhancing nitrogen uptake. This capability can also be used to
 US 9.249,061 B2
15 16
reduce nitrogen requirements for plant producers. These Research Service Culture Collection under number NRRL
strains can also increase tolerance of plants to drought. B-50520. The isolated Trichoderma viride strain may be in a
To understand the relevance of the present invention, a biologically pure form.
consideration of the plant yield plateau is of importance. Another aspect of the present invention relates to an iso
Plants generally respond to increasing nitrogen fertilizer lev lated Scopulariopsis brevicaulis Strain deposited with Agri
els with increased yield and growth up to a point and then the cultural Research Service Culture Collection under number
yield increase levels off; this is the yield plateau above which NRRL B-50521. The isolated Scopulariopsis brevicaulis
use of nitrogen fertilizer no longer increases yields. Planting strain may be in a biologically pure form.
seeds treated with the microbes (i.e., Trichoderma viride Another aspect of the present invention relates to an iso
(NRRL B-50520), Scopulariopsis brevicaulis (NRRL 10 lated Scopulariopsis brevicaulis Strain deposited with Agri
B-50521), and Scopulariopsis brevicaulis (NRRL B-50522)) cultural Research Service Culture Collection under number
of the present invention have been shown to increase plant NRRL B-50522. The isolated Scopulariopsis brevicaulis
growth and productivity even under conditions of substantial strain may be in a biologically pure form.
nitrogen deficiency (see Harman, G., “Myths and Dogmas of
Biocontrol. Changes in Perceptions Derived from Research 15 EXAMPLES
on Trichoderma harzianum T-22, Plant Dis. 84:377-393
(2000); Harmanetal, “Enhancing Crop Performance and Pest The following examples are provided to illustrate embodi
Resistance with Genes from Biocontrol Fungi. In M. Vurro, ments of the present invention but are by no means intended
J. Gressel, T. Butt, G. E. Harman, A. Pilgeram, R.J. St. Ledger to limit its scope.
and D. L. Nuss (eds.), Enhancing Biocontrol Agents and
Handling Risks pp. 114-125. IOS Press, Amsterdam (2001); Example 1
Harman et al., “Trichoderma Species—Opportunistic, Aviru
lent Plant Symbionts.” Nature Rev. Microbiol. 2:43-56 Isolation of Effective Microorganisms that Degrade
(2004), which are hereby incorporated by reference in their Waste Proteins
entirety). Plants grown in the presence of the symbiotic bio 25
control fungus frequently are greener and more vigorous One of the most widespread proteins present in agricultural
(Harman, G., “Myths and Dogmas of Biocontrol. Changes in wastes is keratin. This is the structural component of hair,
Perceptions Derived from Research on Trichoderma har feathers and other similar substances. While keratin is gener
zianum T-22, Plant Dis. 84:377-393 (2000), which is hereby ally degraded slowly in soil or elsewhere, there are microbes
incorporated by reference in its entirety). Previous research 30 that degrade this substrate relatively rapidly.
has found that in the presence of T. harzianum, yield plateaus The organisms of the present invention can be grown in
were reached with 40-50% less nitrogen fertilizer than in its liquid fermentation and ammonia and amino acids will be
absence (Harman et al., “Enhancing Crop Performance and released. For use with the NRRL strains described herein, an
Pest Resistance with Genes from Biocontrol Fungi. In M. additional nutrient may be used. Such as tripticase Soy broth
Vurro, J. Gressel, T. Butt, G. E. Harman, A. Pilgeram, R.J. St. 35 (Difco Products). Feather meal at 1 to 10% w/v is added to the
Ledger and D. L. Nuss (eds.), Enhancing Biocontrol Agents tripticase soy broth in water and the mixture is sterilized by
and Handling Risks pp. 114-125. IOS Press, Amsterdam autoclaving. When the organisms are added to this sterile
(2001), which are hereby incorporated by reference in their medium, feather meal is degraded. The release of ammonia
entirety). This means that nitrogen fertilizer rates could be from feather meal creates an alkaline environment. Since
reduced by this amount without a yield decrease. This has 40 ammonia at a high pH is strongly volatile, the presence of this
great potential both for decreasing evolution of NO from material is easily detected by smell. Systems for harvesting
soils, since less total fertilizer is applied, and since a greater and continuous removal of the ammonia are readily available
percentage of the applied nitrogen must be taken up by the and known to those skilled in the art. One method is simply to
plant, the requirement for nitrogen in plant metabolism is not have a closed sterile closed loop outside the fermentation
expected to be altered, and, therefore, the only way to obtain 45 vessel that includes a sterile air sparging device. As air is
the added N in the plant is via enhanced N use efficiency. introduced through the fermentation liquid, ammonia will be
Another aspect of the present invention relates to a method removed and can be trapped by passage into an acidic solu
of making a fertilizer comprising amino acids and ammonia. tion, vinegar would be satisfactory. This will result in the
This method includes providing at least one microbe selected formation of ammonium acetate, which can be readily used as
from the group consisting of Trichoderma viride (NRRL 50 a fertilizer, and this sparging step will result in concentration
B-50520), Scopulariopsis brevicaulis (NRRL B-50521), of the ammonia for further use. If it is desired also to remove
Scopulariopsis brevicaulis (NRRL B-50522), and combina the amino acids, this step can be accomplished by passage of
tions thereofand providing a substrate which is acted upon by the fermentation liquid through an appropriate ion exchange
the at least one microbe to release nitrogen. The method resin, from which the amino acids can be eluted and thereby
further includes contacting the at least one microbe and the 55 concentrated. At the same time, this sparging process will
substrate under conditions effective to cause the at least one remove the excess ammonia that drives up the pH and thereby
microbe to act on the Substrate and produce a liquid Suspen allow the fermentation to continue. This can be developed
sion comprising amino acids and ammonia. into a continuous flow system for efficient production ammo
This aspect of the present invention can be carried out using nia.
any of the additives and any modes of application described 60 The present invention sought to determine effective
above on any of the above noted plants. microbes that degrade proteins. To accomplish this, feather
In this aspect of the present aspect, the fertilizer may be in meal was mixed with peat moss and moistened the mixture
a liquefied form of all or a part of the substrate (i.e., keratin) (composition: 1 L peat moss, 1 L feather meal and 400 ml of
as a result of fermentation. Such fermentation will produce water), autoclaved the mixture on two Successive days, and
amino acids, peptides, and/or ammonia. 65 inoculated the mixture with 1 g composted chicken manure.
Another aspect of the present invention relates to an iso The peat moss was used as bulking/aeration component and
lated Trichoderma viride strain deposited with Agricultural the composted chicken manure was considered a good source
 US 9.249,061 B2
17 18
of keratin (i.e., feather)-degrading microorganisms. Within (April 2007), which is hereby incorporated by reference in its
six days of inoculation, a strong odor of ammonia was entirety). The same is true for most other fungi in the genus
present, indicating degradation of the feathers to ammonia, Trichoderma.
which is an excellent natural nutrient for plants. The organ A recent review lists organisms that produce keratinases
isms present in the ammonia-scented media were plated using (Brandelli et al., “Biochemical Features of Microbial Kerati
dilution techniques on both potato dextrose agar ("PDA") nases and Their Production and Applications. Applied
(“Difico') and on tripticase soyagar (“TSA) (“Difco') made Microbiology and Biotechnology 85:1735-1750 (2010),
up according to the manufacturer's directions. Microbial which is hereby incorporated by reference in its entirety). S.
growth on the PDA, which is a general mycological medium, brevacaulis is described as a producer of keratinase, but T.
was generally poor, but on TSA, which is a hydrolyzed Soy 10 viride is not. However, there is a single report of production of
protein, numerous fungi grew. These, Surprisingly, consisted keritanase by a strain of T. viride from India (Jyoti et al.,
of only three different microorganisms that apparently were “Keratinolytic Enzymes From Trichoderma viride and
able to grow well on feather meal while most other of the Graphium cuneiferum Isolated From Poultry Farms at
abundant microbes that occur in compost could not. These Jabalpur, India.” Cryptogamic Botany 4:30-33 (1993), which
fungi were isolated, obtained in pure form by single sporing 15 is hereby incorporated by reference in its entirety).
and stored on silica gel at -20°C., which is a very good way
of preserving fungi. Example 3
The fungi isolated from the chicken manure compost were
inoculated into tripticase soy broth (“TSB) and grown for Methods of Production of Useful Formulations
three days. Good growth of all microorganisms was obtained
in the TSB and 50 ml of the actively growing cultures was Feather meal is a coarse brown powder. Dry fertilizers for
added to the feather meal:peat mixture contained insterilized use in many applications need to be applied as granules
plastic boxes with flanged lids that provide good aeration of through drop or rotary dispersal systems. Therefore, a method
the contents of the boxes. After six days of growth, Samples was developed for making a useful granule. A method for
were removed and extracted and the levels of soluble nitrogen 25 Solidifying and production of granules with dimensions of
compounds were determined. The extracts were analyzed for 2-5 mm in diameter was required. The initial granule was
amino acids (ninhydrin assay) or ammonia (Nessler's prepared by adding sufficient water to the feather powder to
reagent; LaMotte Testing kit). produce a slurry and then dissolving in this slurry gelatin to a
Promising results were obtained with the fungi obtained. final concentration of about 7% relative to feather powder.
The ammonia level, with lysine as the standard, for the con 30 This material was spread in a sheet and dried and the result
trol sample was about 19 ug/ml, while the values were 600, was a hard brown sheet. This was then ground and sieved to
3240, and 4900 ug/g, respectively for the three strains. The provide particles of the desired size. It will be appreciated that
amino acid levels for the same samples were 2800, 3003, and a similar composition can be extruded and dried to give
5600 ug/g for the same three strains, while the control values appropriately sized particles. Similarly, these mixtures can
were about 3 ug/ml. These assays were repeated several times 35 also be used in pelleting equipment, followed by appropriate
with similar results. Several strains in particular degraded sizing.
keratin more efficiently than any other tested microbes. The granules thus produced do not contain any added
microorganisms. Some systems for producing granules or
Example 2 pellets involve heat and pressure, so a coating procedure was
40 developed for the pellets. Conidia of the Trichoderma strains
Identification of the Keratin-Degrading Fungi and known to provide benefits, including enhanced nitrogen use
Bacteria efficiency were added to 0.1% Crystal Tex (tapioca dextran,
National Starch) to give about 10 colony forming units per
The fungi identified in Example 1, supra, were sent to the ml of Suspension. This mixture was applied to the Surface of
USDA type culture collection for deposit in their patent col 45 the dried pellets to give a Smooth and continuous coat that did
lection. The strains were designated as NRRL B-50520, not appreciably increase the size of the granules. Thus, the
NRRL B-50521, and NRRL B-50522 for the green-, brown living fungi were applied as a dry coating. From this location,
and white-spored strains, respectively. These fungi were they grew immediately when the pellet was moistened (FIG.
identified as Trichoderma viride (NRRL B-50520), and 1).
Scopulariopsis brevicaulis (NRRL B-50521 and NRRL 50 In later experiments, direct addition of the strains to the
B-50522 are both isolates of this fungi). Both species have mix while moist and before it was extruded to form the pellets
penicillate conidiophores, but subtle differences in the was examined. This method works very well and the organ
arrangements of the conidiophores clearly place them in their isms remain stable so long as drying temperatures remained
respective genera. The ITS regions of each of the isolates at or below 38°C. Selection of the appropriate microorgan
were also sequenced and assessed by GenBank BLAST que 55 isms (i.e., microbes) is helpful in obtaining the best results.
ries. As a result, their identity is confirmed as follows: These microbes serve two functions: (1) some microbes have
NRRL B-50520 Trichoderina viride the ability to degrade feather meal or other recalcitrant pro
NRRL B-50521—Scopulariopsis brevicaulis teins, and (2) Some microbes act as endophytic plant sym
NRRL B-50522—Scopulariopsis brevicaulis bionts. In the experiments of the present invention, in the
Trichoderma viride has a very low risk profile. The US 60 absence of microbes, N, as judged by turf plant greenup only
Environmental Protection Agency states, relative to use of a occurs 4-6 weeks after application. By this time, in the pres
strain within this genus as a biocontrol agent against fungal ence of the degrading microbes, most of the N is already
pathogens, “the Agency anticipates that no additional health released. The endophytic plant microorganisms have many
effects data are required for the currently registered sites for functions, one of which is increased plant nitrogen use effi
Trichoderma polysporum and T. viride' (Trichoderma spe 65 ciency (“NUE'). If this microbial component is included,
cies Summary Document, Registration Review: Initial then the effective N value of the fertilizer is increased. Thus,
Docket, Unites States Environmental Protection Agency the amount of N in a 14% N fertilizer effectively becomes
 US 9.249,061 B2
19 20
28% when the NUE contribution is included. This is a useful that were produced were tested against Scotts(R Turf
aspect of the present invention if this invention is to be com Builder.R, a widely used home fertilizer which has an analysis
petitive with, and as effective, as standard synthetic fertilizer of 30:2:8 N:P:K (as N. P.O.s and KO). By comparison, it was
with 30% N. Plants still require the same level of N to grow anticipated that the analysis of the fertilizer that was prepared
and thrive, so the only way that a fertilizer in the present would 15-16:0:0.3, so it would not be a complete fertilizer. P
invention becomes as effective at the same application rate was not included, since this is not allowed in many jurisdic
(useful for economic competitiveness) is through the use of tions because of the danger of pollution of Pinto water which
NUE enhancing organisms. results in eutrophication. The N level was less than 50% as
Standard synthetic fertilizers contain high levels of salts, high as that of the Scotts(R fertilizer, and the Klevel is sub
and the osmotic potential and direct effects of these compo 10 optimal. Thus, if the performance of the fertilizer prepara
nents is lethal to microbes. However, in this invention, syn tions of the present invention approaches that of Scotts(R), it
thetic salts are almost completely eliminated, and thus, a demonstrates that (a) a significant amount of N is being
granule containing a microbe is safe for the microbe. Most of released from the fertilizer, (b) there is increased efficacy of
the granule is protein, which is not toxic to the microbes. uptake of N induced in the turf plants by the fungi added, and
Thus, either the coating or direct mixing of the microbial 15 (c) there is much less availability of N to leach into ground
agents is one component of this invention. water. After all, plants still need the same amount of N to grow
The microbial component and the nonliving components and, if only half as much nitrogen is being added, there must
can be formulated into a single granule or other mixture. be greater uptake of Nand there can be only small amounts of
There are many cases in the literature where, for example, N that leach into groundwater.
microbes added to or living in the Soil increase plant growth The tests were conducted in an annual rye grass planted
including NUE. However, users will be unwilling to apply a into a 1:1 sandy loam:peat mix that contains almost no free
number of different organisms and other products to accom nitrogen. Annual rye grass was planted in flats and after
plish a valuable result. The inclusion of all the living and seedlings were well established, about two weeks after plant
nonliving components into a single product or granule is one ing, the various fertilizers were applied. Granules were
component of this invention. 25 applied at the 1x rate, which is the level offertilizer applied at
This embodiment of the invention provides a granule com the recommended rate for Scotts R. Turf Builder(R). In addi
posed almost entirely of protein. Proteins are 12-14% N, so tion, every treatment was applied at two times this rate. The
since gelatin and feather meal are both animal proteins the first experiments contained a number of treatments as fol
necessary value is obtained. lows:
For some applications, a granular product is not necessary 30 No treatment control
for some applications. Powders are frequently used, for Granules applied without any microorganisms
example as components of plant growth media and as mate Granules applied with B-50520
rials to be added directly to fields or gardens, typical nitrogen Granules coated with B-50521
values for a granule such as this is 12-14%. Admixtures of Granules coated with B-50522
feather meal and microorganisms described herein can be 35 Granules applied with B-50520--Trichoderma strains
used directly. K1-K4 (described in the next example)
In addition, any number of additions can be made to this Granules coated with B-50521+Trichoderma strains
basic granule or powder. An example is the liquid ammonia K1-K4
and amino acid mixture extracted from the growth medium as Granules coated with B-50522+Trichoderma strains
in Example 2, supra. This can be used directly in the dry 40 K1-K4.
powder or granule or concentrated to give an even higher level ScottS(R) Turf Builder(R) fertilizer
of N. Since feather powder or gelatin will provide very little All were applied at the 1x and 2x rate. In this example, K1-K4
immediately available N. such additions are useful for pro consisted of the following strains: T. harzianum strain
viding immediate green up. Other Suitable nitrogen sources RR17Bc (ATCC accession PTA9708); T. harzianum F11 Bab
are fish meals or extracts, compost extracts, kelp extracts, 45 (ATCC accession PTA9709), T. atroviride strain WW10TC4
extracts of sea animals such as shrimp or shellfish. Inorganic (ATCC PTA accession 9707) and T. virens strain 41 (ATCC
Sources include nitrates, inorganic ammonia Sources and the accession 20476).
like. In addition, organically-listed products Such as K from The results of these trials were successful and are summa
greensand or organically listed potassium sulfate, and other rized in FIGS 2A-2B and FIGS 3A-3D.
sources of K that are not listed can be added. Similarly, P can 50 In FIG. 2A, the treatments used were PT (organic fertilizer,
be supplied as bone meal (organic) or from various inorganic NRRL B-50521, and K1-K4, which included T. harzianum
salts. For lawn applications, P is being banned and a compo strain RR17Bc (ATCC accession PTA 9708), T. harzianum
nent of use of feather meal or gelatin is that P is not present. F11 Bab (ATCC accession PTA 9709), T. atroviride strain
WW10TC4 (ATCC PTA accession 9707), and T. virens strain
Example 4 55 41 (ATCC accession 20476)); STB-Scotts(R Turf BuilderR);
P-organic fertilizer with NRRL B-50521; and C=untreated
Fertilizer for Grass control. In FIG. 2B, the treatments used were PT (organic
fertilizer and NRRL B-50521); OFC organic fertilizer gran
A very demanding use of fertilizers is for application to ules without any fungi; P (organic fertilizer with NRRL
grass in lawns, sports arenas, parks, golf courses and other 60 B-50521). FIGS. 2A-2B show growth about 10 days after
similar uses. The fertilizers must provide a highly attractive fertilization by Scattering the granules across the Surface of
surface and last for a considerable period of time. The granule the Soil in which the grass was growing. Increases in growth
was prepared to contain 73.8% feather meal, 13% Chilean are primarily a function of available soluble nitrogen. As can
nitrate, 7.3% gelatin, and 5.7% Jersey green sand (source of be seen, the growth of grass was substantially greater with the
K). The Chilean nitrate is currently organically certified and 65 organic fertilizer in the presence of NRRL B-50521 than with
contains 16% N, so its nitrogen content is just a bit above that any treatment without this organism. Comparable results
of the protein components. As a comparison, the preparations were seen using NRRL B-50520 or NRRL B-50522. Growth
 US 9.249,061 B2
21 22
of the grass was also greater than with the commercial lawn free nitrogen in any form is likely to be smaller in soils with
fertilizer at this time period. The organic fertilizer control also the samples of the present invention than with the standard
grew better than the control with no granules; in part this can chemical fertilizer. Data from soil sampled nine days after
be attributed to the small amount of organic Chilean nitrate fertilization is given in Table 1 that follows. 1 g of soil was
(NaNO primarily) that was added to the granules as a starter extracted per 3.6 ml of extraction buffer and the colorimetric
fertilizer. This illustrates the utility of the invention disclosed tests described supra were used to measure the soluble nitro
here, which includes microbially-mediated slow release gen in the extracts. For the amino acid levels, data was based
nitrogen. on a dilution curve with lysine as the standard. All of these
FIGS. 3A-3D contain graphs showing growth over time. To assays were done at the 1x the rate of fertilizer application.
obtain this data, grass was clipped from the flats, dried, and 10
weighed to simulate a mowing event (in FIGS. 2A-2B, the TABLE 1
grass was ready for cutting) and this was continued over the
course of the experiment. All of the cuttings were done the Amounts of Different Forms of Soluble Nitrogen in Soil
same day, so the graphs represent the amount of grass that was Nitrate
harvested between the last cutting and the next and days after 15
Treatment (Igg) Ammonia (ugg) Amino acids (Lugg)
harvest represents the day after fertilization at which the
cutting was made. Untreated
Scotts (R)
>5
75
21
2O
43
68
The untreated grass had, at its highest level of cuttings, 800 Turf Builder (R)
mg/flat at 7 days after planting, and thereafter the cutting K1-K4 on granules 5 11 86
weights declined to very low levels (FIG. 3A). The organic B-50520 on granules 5 15 90
fertilizer at the 1x rate was similar to the untreated control, but B-50521 on granules 7.5 15 107
K1-K4 -- B-50520 5 22 68
at the 2x rate, the weights were substantially larger. The K1-K4 -- B-50521 5 22 75
organic fertilizer at the first cutting was high, which no doubt
represents the nitrogen supplied by the Chilean nitrate that
was provided in the granule. After another week, the grass 25 The values for nitrate in soil were exactly as predicted.
weights dropped and then increased again (FIG. 3A). This With the chemical standard fertilizer, the level of nitrate is
second increase is no doubt due to hydrolysis of the feather more than an order of magnitude greater than with the
meal by the native soil microflora. untreated control soil of with any of the feather-meal-based
The standard, Scotts(R Turf Builder(R), initially supported granular products. Ammonia levels change little across treat
modest growth at the 1x level, but this increased at the second 30
ments, probably because any ammonia released is immedi
cutting. Growth decreased again at the third cutting and ately taken up by the turf which was already growing and
increased again finally at the last cutting (FIG. 3B). This established. However, the levels of amino acids did change.
likely reflects changes in availability of different nitrogen The greatest level of amino acids was with the granules with
Sources. However, at double time rate, grass yields were NRRL B-50521. This correlates well with the rapid increase
extremely low at the second and third cutting times (FIG.3B). 35
in growth of turf early on when fertilized with granules con
This was caused by burning of the grass at this level of taining this organism (FIG. 3D). Interestingly, the addition of
application, which never occurred with any of the feather K1-K4 to the granules containing either B-50520 or B-50521
meal based systems. reduced the levels of amino acids. This may be a consequence
At the 1x rate, strain NRRL B-50520 gave grass cutting of the apparent conversion of reduced forms of nitrogen
yield results that were substantially better than the control 40
(amino acids or ammonia) to nitrate. If the concept of
organic fertilizer (FIGS. 3A and 3C). Strain NRRL B-50521 enhanced turfgrowth by in vivo hydrolysis of the feather meal
gave rapid release of nitrogenas evidenced by very high grass is correct, then the pool sizes of nitrogenous compounds in
yields at the first cutting, but then grass yields in most cases the turf plants themselves ought to change both quantitatively
declined dramatically by the second cutting. This is due to and qualitatively. Table 2 gives the concentrations in plant
very high release of soluble nitrogen and then the substrate 45
tissue of nitrate, ammonia and amino acids (as lysine equiva
was depleted. lents) that could be extracted from grass seedlings 14 days
In these experiments, it should be noted that the Scotts(R) after planting. In these experiments, grass was dried and then
fertilizer is a relatively complete fertilizer while, as noted extracted and the levels of the various soluble N forms mea
earlier, the test materials used contained no phosphorus and sured by procedures already described.
inadequate levels of potassium. 50

TABLE 2
Example 5
Concentration of Nitrate. Ammonia, and Amino Acids in Plant Tissue
Pool Sizes of Soluble Nitrogen Compounds in Soil Nitrate
and Grass Fertilized with Different Materials 55
Treatment (Igg) Ammonia (ugg) Amino acids (Lugg)
It is indicated in the above examples that the fertilizer Untreated control
Scotts (R)
10
700
936
962
2600
3380
prepared by other methods will have low levels of leaching of Turf Builder (R)
nitrates into ground water. If this is true, then levels of nitrate K1-K4 on granules 15 686 421.2
should be lower than in soils fertilized with standard chemical 60 B-50520 on granules 15 1477 3380
fertilizers. At the same time, other forms of soluble nitrogen B-50521 on granules 15 1352 3900
K1-K4 -- B-50520 150 988 3224
should increase since degradation of feather meal results in K1-K4 -- B-50521 150 910 3224
release of amino acids and ammonia. These compounds
would only be converted to nitrate by activity of soil micro
flora over time. However, amino acids and ammonia are likely 65 This data confirms the expectations that the source of nitro
to be rapidly taken up by plants (or microflora) and so pool gen makes a difference in the forms of Soluble nitrogen in
sizes will not accumulate to large levels. Thus, the amount of plant tissues. It must be emphasized that these values are Snap
 US 9.249,061 B2
23 24
shots of the size of the active nitrogen metabolic pool in provide an extended life of the product. The granules so
plants, and that there is a constant rebalancing between produced were coated or contained Trichoderma strains
uptake from the environment and metabolism of the nitrogen K1-K4 plus Trichoderma viride NRRL B-50520 to provide
on to proteins and other nitrogenous compounds in the turf. the NUE benefit to make the product perform in a manner
The untreated control has low levels of all forms of N, which 5 equivalent to Scotts(R Turf Builder(R) with 32% N.
explains the poor growth noted above. With the exception of In addition, another product was prepared. This one con
B-50520 and B-50521 on granules, ammonia levels vary tained 46% organic protein sources, primarily feather meal,
within a small range, but in the case of these two fungi on and 26% garden compost. It also included methylene urea to
granules, the levels are considerably higher. The levels of a level of 23% of the total and the remainder was composed of
ammonia appeared to be reduced if K1-K4 was added to 10 Chilean nitrate and potassium Sulfate. This product had an
B-50520 or B-50521. This may be explained by the conver N:P:K ratio of 16:0:3 and contained 71% BioPreferred com
sion of ammonia to nitrate by these fungi, as Suggested by the ponents. This product was expected to have similar capabili
increase in nitrate in seedlings grown in the presence of both ties as the one described in the previous paragraph; however,
fungi. it was expected to be about 20% cheaper because of the
Seedlings grown in the presence of the commercial fertil 15 reduction in the amount of the organic protein. It is also an
izer have very high levels of nitrate, again as would be advantage to have a greater amount of organic matter in the
expected. A principal nitrogenous component of this fertilizer preparation from the compost.
is nitrate and this clearly is reflected in the values obtained. The granules also can contain nonproteinaceous binders
High levels of nitrates are pollutants of water, and these data (e.g., gelatin) Such as dextrans, starches, polyvinyl chloride or
reflect the point that the amount of nitrate saturates the plants other similar materials.
abilities to absorb this nutrient and much of this will end up as Granules produced using the technologies and systems
water and air pollutants. Seedlings grown in the presence of described in this herein perform well in actual tests. However,
feather meal granules and microbial mixture K1-K4, and they provide their best performance when combined (either
NRRL B-50520 or B-50521, all had only about 2% as much through coating of granules or internal incorporation, with
nitrates in their tissue as did those grown in the presence of the 25 beneficial microorganisms). FIG. 4 shows one example of
inorganic lawn fertilizer. As seen in the previous example, this, that of increased turf root development. Strains K1-K4
they grew much better than the levels of nitrate in their tissues have been repeatedly shown to increase plant root growth
would predict. However, the pool sizes of amino acids were from seed treatments or other methods of application in
enhanced, as was the level of ammonia. Amino acids and wheat, maize, tomatoes, and other crops. This attribute of
ammonia are downstream of nitrate in plant assimilation and 30 increasing plant root development and deeper colonization of
nitrogen transformation in plants, nitrate needs to be trans soil is a component of the abilities of K1-K4.
formed to ammonia and then on to amino acids before they FIG. 4 shows root development of turfgrown in a sand: peat
can become assimilated and converted to a structural or active mix for about 6 weeks and fertilized with the granules
component of plant metabolism. So, this higher level of described in this example with and without the beneficial
readily available N without the energy-expensive step of 35 microbial mix T. vixens stain NRRL B-50521 and Tricho
reduction to ammonia will improve plant growth and nitrogen derma strains K1-K4. Root growth on turf fertilized with the
use efficiency. Also demonstrated is the potential for greatly granules without the beneficial microbes is show in the petri
reduced nitrate pollution of ground and Surface water. Sur dish labeled 11. The petridish labeled 12 contained the organ
prisingly, the level of nitrate in plants when B-50520 or isms incorporated into the granules at about 10 cfu/g, while
B-50521 were added together with K1-K4, the level of nitrate 40 the dish labeled 13 was fertilized with granules coated with
in plant tissue increased. This may be because there are inter the beneficial fungi in a dextran mixture (10 colony forming
actions between these groups of fungi that result in greater units (cfus)/ml in 2% tapioca dextran to form a thin continu
oxidation of amino acids or ammonia to nitrate. ous coat). These are variations on the methods of adding
beneficial microbes. The greater root growth (turf roots are
Example 6 45 quite small and fine) are evident in the lower half of the petri
dishes with either method of application of the beneficial
Production of BioPreferred Fertilizer Products microbes but absent in the turf fertilized with the granules in
the absence of the beneficial microbes.
As shown in FIG. 1, the formulation of the products used to There are two microbial components to this invention.
generate the data in that figure Supported growth of grass 50 First, microorganisms that increase degradation of natural
about as well as Scotts(R fertilizer, and at only a slightly recalcitrant sources of N such as feather meal to convert them
elevated application rate for about 5 weeks. However, there into simple compounds that can be used for plant nutrition.
after, results were less effective, while Scotts(R synthetic fer Second, microorganisms. Such as Kl-K4 that have the ability
tilizer was effective for about 8 weeks. to increase plant growth and productivity, including enhanc
In addition, it was discovered that the price of feather meal 55 ing root growth, NUE, improving resistance to abiotic
increased 2-3 fold over the Summer of 2012, which over stresses, and improving plant photosynthetic capabilities.
priced products containing it. The answer seemed to be to Although preferred embodiments have been depicted and
produce a BioPreferred, rather than a product which could be described in detail herein, it will be apparent to those skilled
OMRI certified. As noted earlier, BioPreferred products are in the relevant art that various modifications, additions, Sub
required to contain, at a minimum, 71% of the total as organic 60 stitutions, and the like can be made without departing from
farm materials. Thus, a synthetic slow release nitrogen Source the spirit of the invention and these are therefore considered to
with a high N level could be used. A granule was prepared that be within the scope of the invention as defined in the claims
contained, on a dry weight basis, 71% organic protein (pri which follow.
marily feather meal), 20% methylene urea (a slow release What is claimed:
synthetic nitrogen Source), with the remainder divided 65 1. A fertilizer comprising:
between potassium sulfate and Chilean nitrate. The N:P:K at least one microbe selected from the group consisting of
ratio was about 17:0:9 and the slow release N was expected to Trichoderma viride (NRRL B-50520), Scopulariopsis
 US 9.249,061 B2
25 26
brevicaulis (NRRL B-50521), Scopulariopsis brevicau tural or horticultural adjuvants, stickers, spreaders, sur
lis (NRRL B-50522), and combinations thereof and factants, dispersants, humectants, UV protectants, and
a substrate which is acted upon by the at least one microbe mixtures thereof.
to release nitrogen. 14. The fertilizer of claim 1, wherein the fertilizer further
2. The fertilizer of claim 1, wherein the substrate is a 5 comprises:
protein selected from the group consisting of feather, feather a source of organic carbon selected from the group con
sisting of compost and biochar.
meal, and a derivative of urea. 15. A fertilizer comprising:
3. The fertilizer of claim 1 further comprising: at least one microbe selected from the group of Tricho
a Supplemental microbe in the form of a Trichoderma 10 derma viride (NRRL B-50520), Scopulariopsis brev
species. icaulis (NRRL B-50521), Scopulariopsis brevicaulis
4. The fertilizer of claim 1 further comprising: (NRRL B-505822), and combinations thereof and
a Supplemental microbe selected from the group consisting a keratin substrate which is acted upon by the at least one
of Piriformospora indica, a plant growth promoting microbe to release nitrogen.
rhizobacteria, mycorrhizal fungi, and combinations 15
16. The fertilizer of claim 15 further comprising:
thereof. at least one microbe selected from the group consisting of
5. The fertilizer of claim 1, wherein the fertilizer is in Streptomyces avermitilis, Streptomyces coelicolor,
granule, pellet, dust, powder, slurry, film, and/or liquid sus Streptomyces violaceOruber, and combinations thereof.
pension form. 17. The fertilizer of claim 15 further comprising:
6. The fertilizer of claim 1, wherein the fertilizer further a Supplemental microbe in the form of a Trichoderma
comprises: species.
a nonproteinaceous binding agent selected from the group 18. The fertilizer of claim 15 further comprising:
consisting of dextrans, starches, polyvinylchloride, and a Supplemental microbe selected from the group consisting
combinations thereof. of Piriformospora indica, a plant growth promoting
7. The fertilizer of claim 1, wherein the fertilizer further 25
rhizobacteria, mycorrhizal fungi, and combinations
comprises: thereof.
a soluble nitrogen source selected from the group consist 19. The fertilizer of claim 15, wherein the fertilizer is in
ing of nitrate, ammonia, ammonium salts, amino acids, granule, pellet, dust, powder, slurry, film, and/or liquid sus
urea, fish meal or extract, compost extract, kelp extract, pension form.
shrimp extract, shellfish extract, and combinations 30
20. The fertilizer of claim 15, wherein the fertilizer is in the
thereof. form of a liquid suspension comprising amino acids and
ammonia.
8. The fertilizer of claim 1, wherein the fertilizer further 21. A method of enhancing growth of plants, the method
comprises:
a source of phosphorus selected from the group consisting comprising:
of rock phosphate, sodium phosphate, potassium phos 35
providing a fertilizer comprising:
phate, bone meal, and combinations thereof. at least one microbe selected from the group consisting
9. The fertilizer of claim 1, wherein the fertilizer further of Trichoderma viride (NRRL B-50520), Scopulari
comprises: opsis brevicaulis (NRRL B-50521), Scopulariopsis
a source of potassium selected from the group consisting of brevicaulis (NRRL B-50522), and combinations
potassium chloride, potassium phosphate, potassium 40 thereof and
Sulfate, Jersey green sand, organic sources of potassium, a substrate which is acted upon by the at least one
and combinations thereof. microbe to release nitrogen; and
10. The fertilizer of claim 1, wherein the fertilizer further contacting the fertilizer with plants or plant seeds under
comprises: conditions effective to enhance the growth of the plants
minor nutrients selected from the group consisting of salts, 45
or plant seeds compared to plants or plant seeds to which
Substances that contain iron, cobalt, manganese, magne the fertilizer was not applied.
sium, copper, calcium, boron, zinc, and combinations 22. A method of making a fertilizer comprising amino
thereof. acids and ammonia, the method comprising:
11. The fertilizer of claim 1, wherein the fertilizer further providing at least one microbe selected from the group
comprises: 50
consisting of Trichoderma viride (NRRL B-50520),
a growing media. Scopulariopsis brevicaulis (NRRL B-50521), Scopu
12. The fertilizer of claim 1, wherein the fertilizer further lariopsis brevicaulis (NRRL B-50522), and combina
comprises: tions thereof;
a carrier selected from the group consisting of water, aque providing a substrate which is acted upon by the at least one
ous solutions, slurries, and powders. 55
microbe to release nitrogen; and
13. The fertilizer of claim 1, wherein the fertilizer further contacting the at least one microbe and the substrate under
comprises: conditions effective to cause the at least one microbe to
an additive selected from the group consisting of insecti act on the substrate and produce a liquid suspension
cide, fungicide, nematicide, additional organic fertilizer, comprising amino acids and ammonia.
bioinsecticide, biofungicide, bionematicide, agricul