U.S. patent application number 14/609566 was filed with the patent office on 2016-08-04 for pseudomonas fluorescens inhibit annual bluegrass and rough bluegrass root growth and germination.
The applicant listed for this patent is The United States of America, as represented by the Secretary of Agriculture, The United States of America, as represented by the Secretary of Agriculture. Invention is credited to Ann C. Kennedy.
Application Number | 20160219886 14/609566 |
Document ID | / |
Family ID | 56544264 |
Filed Date | 2016-08-04 |
United States Patent
Application |
20160219886 |
Kind Code |
A1 |
Kennedy; Ann C. |
August 4, 2016 |
PSEUDOMONAS FLUORESCENS INHIBIT ANNUAL BLUEGRASS AND ROUGH
BLUEGRASS ROOT GROWTH AND GERMINATION
Abstract
A biocontrol agent containing one or more novel and isolated
Pseudomonas fluorescens strains (P. fluorescens biovar B strain XJ3
(NRRL B-50851), P. fluorescens biovar B strain XS18 (NRRL B-50852),
and P. fluorescens biovar A strain LRS12 (NRRL B-50853)) and a
biocontrol agent containing one, two or three of these bacteria
with an agriculturally acceptable carrier are useful for the
control of annual bluegrass and rough bluegrass root growth and
germination. The biocontrol agents of this invention may be applied
to the soil and/or seeds in the fall with inhibition occurring in
subsequent years. The biocontrol agent can also be used in
combination with herbicides that can inhibit the growth and seed
production of any standing annual bluegrass and/or fertilizer to
stimulate growth of the desirable plant that will compete with the
annual bluegrass weed and/or rough bluegrass. Methods for use of
these biocontrol agents to control to growth of annual bluegrass
and rough bluegrass are also provided.
Inventors: |
Kennedy; Ann C.; (Pullman,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary of
Agriculture |
Washington |
DC |
US |
|
|
Family ID: |
56544264 |
Appl. No.: |
14/609566 |
Filed: |
January 30, 2015 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C05G 3/60 20200201; A01N
25/08 20130101; A01N 63/00 20130101; A01N 63/00 20130101; C12R 1/39
20130101 |
International
Class: |
A01N 63/00 20060101
A01N063/00; C12R 1/39 20060101 C12R001/39; C05G 3/02 20060101
C05G003/02 |
Claims
1. A biocontrol agent effective for controlling annual bluegrass
and rough bluegrass root growth and germination comprising one or
more Pseudomonas fluorescens strains that inhibit annual bluegrass
and/or rough bluegrass root growth and seed germination, wherein
said one or more P. fluorescens strains are selected from the group
consisting of P. fluorescens biovar B strain XJ3 (NRRL B-50851), P.
fluorescens biovar B strain XS18 (NRRL B-50852), P. fluorescens
biovar A strain LRS12 (NRRL B-50853), and combinations thereof.
2. The biocontrol agent of claim 1 wherein the concentration of
said P. fluorescens applied to the area being treated is between
approximately 10.sup.5 to approximately 10.sup.11 cfu bacteria per
square meter of treated land.
3. The biocontrol agent of claim 1 further comprising a
herbicide.
4. The biocontrol agent of claim 1 further comprising a
fertilizer.
5. The biocontrol agent of claim 1 further comprising an
agriculturally acceptable carrier, optionally a herbicide, and
optionally a fertilizer.
6. The composition of claim 5 wherein said agriculturally
acceptable carrier is selected from the group consisting of seed,
rice, talc, carbohydrate, polysaccharide, polymeric porous
material, milk, water, medium, and pellet made from a cereal grain
flour or meal.
7. The composition of claim 5 wherein the concentration of said P.
fluorescens applied to the area being treated is between
approximately 10.sup.5 to approximately 10.sup.11 cfu bacteria per
square meter of treated land.
8. A method for controlling the growth of annual bluegrass and/or
rough bluegrass in an area in need of treatment comprising applying
the biocontrol agent of claim 1 in an amount effective to control
the growth of annual bluegrass and/or rough bluegrass to said area
in need of treatment.
9. The method of claim 8 wherein said amount of said P. fluorescens
is between approximately 10.sup.5 to approximately 10.sup.11 cfu
bacteria per square meter of said area in need of treatment.
10. The method of claim 8 wherein application of said biocontrol
agent occurs after rain or irrigation of the area in need of
treatment, prior to irrigating said area in need of treatment, or
when rain is anticipated.
11. The method of claim 8 wherein the air temperature in said area
in need of treatment is between approximately 32.degree. F. and
approximately 70.degree. F.
12. The method of claim 11 wherein said air temperature is between
approximately 32.degree. F. and approximately 50.degree. F.
13. The method of claim 8 wherein said biocontrol agent is applied
to bare soil or plants.
14. The method of claim 8 wherein said biocontrol agent is applied
via spraying, broadcasting, or injecting into the soil.
15. A method for controlling the growth of annual bluegrass and/or
rough bluegrass in an area in need of treatment comprising applying
an agriculturally acceptable carrier, a biocontrol agent,
optionally a herbicide, and optionally a fertilizer, in an amount
effective to control the growth of annual bluegrass and/or rough
bluegrass to said area in need of treatment; wherein said
biocontrol agent comprises one or more Pseudomonas fluorescens
strains selected from the group consisting of P. fluorescens biovar
B strain XJ3 (NRRL B-50851), P. fluorescens biovar B strain XS18
(NRRL B-50852), and P. fluorescens biovar A strain LRS12 (NRRL
B-50853).
16. The method of claim 15, wherein said agriculturally acceptable
carrier is selected from the group consisting of seed, rice, talc,
carbohydrate, polysaccharide, polymeric porous material, milk,
water, medium, and pellet made from a cereal grain flour or
meal.
17. The method of claim 15, wherein said amount of said P.
fluorescens is between approximately 10.sup.5 to approximately
10.sup.11 cfu bacteria per square meter of said area in need of
treatment.
18. The method of claim 15, wherein application of said biocontrol
agent occurs after rain or irrigation of the area in need of
treatment, prior to irrigating said area in need of treatment, or
when rain is anticipated.
19. The method of claim 15, wherein the air temperature in said
area in need of treatment is between approximately 32.degree. F.
and approximately 70.degree. F.
20. The method of claim 19, wherein said air temperature is between
approximately 32.degree. F. and approximately 50.degree. F.
21. The method of claim 15, wherein said biocontrol agent is
applied to bare soil or plants.
22. The method of claim 15, wherein said biocontrol agent is
applied via spraying, broadcasting, or injecting into the soil.
23. A kit for controlling of growth of annual bluegrass and rough
bluegrass comprising one or more containers containing a biocontrol
agent effective for controlling annual bluegrass and rough
bluegrass root growth or germination; optionally a container
containing a herbicide; optionally a container containing a
fertilizer; and instructions for applying said biocontrol agent to
the area in need of treatment; wherein said biocontrol agent
comprises Pseudomonas fluorescens selected from the group
consisting of P. fluorescens biovar B strain XJ3 (NRRL B-50851), P.
fluorescens biovar B strain XS18 (NRRL B-50852), P. fluorescens
biovar A strain LRS12 (NRRL B-50853), and combinations thereof.
24. A kit for controlling of growth of annual bluegrass and rough
bluegrass comprising one or more containers containing a biocontrol
agent effective in controlling annual bluegrass and/or rough
bluegrass root growth or germination; optionally a container
containing a herbicide; optionally a container containing a
fertilizer; and instructions for applying said biocontrol agent to
the area in need of treatment; wherein said biocontrol agent
comprises an agriculturally acceptable carrier and one or more
Pseudomonas fluorescens strains, wherein said one or more
Pseudomonas fluorescens strains are selected from the group
consisting of P. fluorescens biovar B strain XJ3 (NRRL B-50851), P.
fluorescens biovar B strain XS18 (NRRL B-50852), and P. fluorescens
biovar A strain LRS12 (NRRL B-50853).
25. The kit of claim 24, wherein said agriculturally acceptable
carrier is selected from the group consisting of seed, rice, talc,
carbohydrate, polysaccharide, polymeric porous material, milk,
water, medium, and pellet made from a cereal grain flour or meal.
Description
BACKGROUND OF INVENTION
[0001] 1. Field of Invention
[0002] This invention relates to novel isolated Pseudomonas
fluorescens strains that have been isolated, selected, and
characterized from the naturally occurring soil bacterial
populations because they inhibit the growth of annual bluegrass
(Poa annua L.) or inhibit the growth of both annual bluegrass (Poa
annua L.) and rough bluegrass (Poa trivialis), but do not inhibit
the growth of desired grasses, such as turfgrasses, cereal crops,
and native plants. This invention also relates to novel
compositions that contain the novel Pseudomonas fluorescens strains
described herein. In addition this invention relates to methods of
using the novel, isolated P. fluorescens strains and the novel
compositions, alone or in combination with other P. fluorescens
strains, herbicides, and/or fertilizers.
[0003] 2. Description of the Prior Art
[0004] Annual bluegrass (Poa annua L.) is a cool-season annual
grass that is a major weed species in turf, turfgrass seed
production, and golf courses of the western United States (Webster
and Nichols, 2012 Weed Science 60(2):145-157). Annual bluegrass
germinates in late summer or fall as soil temperatures fall below
70.degree. F. It continues to germinate throughout winter, allowing
several flushes of germination at any one site throughout the
season. The life cycles of annual bluegrass and turfgrass are very
similar, and annual bluegrass is often more competitive than most
turfgrasses. Because annual bluegrass is a grass weed growing with
turfgrass, selective control is difficult. Pre-emergent herbicides,
such as benefin, bensulide, dithiopyr, oryzalin, oxadiazon,
pendimethalin, prodiamine, and benefin/oryzalin, are successful in
limiting germination of annual bluegrass. A few post-emergent
herbicides reduce Poa annua L. growth, but they could also kill
desirable turfgrasses. As such, usage of these post-emergent
herbicides are limited. For example, foramsulfuron, sulfosulfuron,
and trifloxysulfuron can be used only on warm-season turfgrass.
Ethofumesate can be used in dormant bermudagrass, creeping
bentgrass, Kentucky bluegrass, tall fescue, perennial ryegrass, and
St. Augustine to reduce annual bluegrass infestations. Pronamide
can be used in warm-season turfgrass for established annual
bluegrass, but it is slow acting. Golf-course managers have few
tools to combat Poa annua L. and the invasion of this weed often
means that greens must be ripped out and replaced every ten
years.
[0005] Rough bluegrass (Poa trivialis) is also a cool-season annual
grass that is a major weed species. Similar to annual bluegrass,
rough bluegrass can outcompete turfgrasses and native plants.
[0006] Use of antagonistic microorganisms as bioherbicides against
some weeds has been previously reported (Kennedy, et al., 1991,
Soil Sci. Soc. Amer. J. 55:722-727; Kennedy, et al., 2001, Weed
Sci. 49:792-797; Makowski and Mortensen, 1998, Mycol. Res.
102(12):1545-1552). Xanthomonas campestris pv. poannua is sprayed
on plant leaves during mowing and reduces annual bluegrass in
bermudagrass. The bacterium enters the plant through the cut leaf
and causes systemic wilt, which kills the plant (Johnson, et al.,
1996, Weed Technology, 10(3):621-624). A germination arrest factor
(GAF) produced by Pseudomonas fluorescens strain WH6 and other
related species produce a compound that in-vitro reduced
germination of grassy weed species including annual bluegrass
(Banowetz, et al., 2008, Biological Control 46:380-390; Banowetz,
et al., 2009, Biological Control 50:103-110.) However, P.
fluorescens strain WH6 and other related species failed to reduce
germination of annual bluegrass in the field. GAF failed to inhibit
germination of grassy weed species in the field and the selectivity
of GAF is not known.
[0007] The physiological characteristics required for a bacterial
strain to suppress annual weeds are specific as to (1) the weed
growth to be controlled; (2) the specificity of the inhibition is
limited to that weed or similar weeds and lack activity against
crops or economically important plants; (3) the mode of action of
weed control; (4) the activity and ecological niche of the
microorganism; and (5) cultural practices and soil and climatic
conditions must be favorable for suppressive. Thus, information
about microbial treatments for control of weeds other than annual
bluegrass cannot be used to predict strains of microorganisms that
would reduce annual bluegrass under field conditions or predict
criteria for selecting such strains.
BRIEF DESCRIPTION OF THE INVENTION
[0008] It is an object of this invention to have three isolated and
novel Pseudomonas fluorescens strains that are effective in
controlling the root growth or the germination of annual bluegrass
and/or rough bluegrass. These three strains of P. fluorescens are
P. fluorescens biovar B strain XJ3 (NRRL B-50851), P. fluorescens
biovar B strain XS18 (NRRL B-50852), and P. fluorescens biovar A
strain LRS12 (NRRL B-50853), where P. fluorescens strain LRS12
(NRRL B-50853) controls the root growth or germination of both P.
annua L. and P. trivialis.
[0009] It is another object of this invention to have a biocontrol
agent that can control and/or inhibit annual bluegrass or can
control and/or inhibit both annual bluegrass (Poa annua L.) and
rough bluegrass root growth and seed germination and that the
biocontrol agent contains P. fluorescens biovar B strain XJ3 (NRRL
B-50851), P. fluorescens biovar B strain XS18 (NRRL B-50852), P.
fluorescens biovar A strain LRS12 (NRRL B-50853), combinations
thereof, and compositions containing one or more of these bacteria,
P. fluorescens strain LRS12 (NRRL B-50853) controls and/or inhibits
root growth and seed germination of both P. annua L. and P.
trivialis.
[0010] It is an object of this invention to have a biocontrol agent
that can control and/or inhibit annual bluegrass or can control
and/or inhibit both annual bluegrass and rough bluegrass root
growth and seed germination. The biocontrol agent contains P.
fluorescens biovar B strain XJ3 (NRRL B-50851), P. fluorescens
biovar B strain XS18 (NRRL B-50852), or P. fluorescens biovar A
strain LRS12 (NRRL B-50853), or combinations thereof, where P.
fluorescens strain LRS12 (NRRL B-50853) controls and/or inhibits
root growth and seed germination of both P. annua L. and P.
trivialis. The concentration of the bacteria applied to the treated
land ranges between approximately 10.sup.5 to approximately
10.sup.11 cfu bacteria per square meter of treated land. For one
alternative embodiment, if one single bacterial strain is present
in the biocontrol agent, the bacteria's concentration is
approximately 10.sup.8 cfu bacteria per square meter of treated
land. For another alternative embodiment, if two or all three
strains of bacteria are present in the biocontrol agent, the
concentration for each strain of bacteria ranges from approximately
10.sup.7 to approximately 10.sup.8 cfu bacteria per square meter of
treated land. It is another object of this invention that the
biocontrol agent contains a herbicide and/or a fertilizer.
[0011] It is an object of this invention to have a biocontrol agent
that can control and/or inhibit annual bluegrass or can control
and/or inhibit both annual bluegrass and rough bluegrass root
growth and seed germination and that the biocontrol agent contains
an agriculturally acceptable carrier and either P. fluorescens
biovar B strain XJ3 (NRRL B-50851), P. fluorescens biovar B strain
XS18 (NRRL B-50852), or P. fluorescens biovar A strain LRS12 (NRRL
B-50853), or combinations thereof, where P. fluorescens strain
LRS12 (NRRL B-50853) controls and/or inhibits root growth and seed
germination of both P. annua L. and P. trivialis. It is further
object of this invention that the biocontrol agent can optionally
contain a herbicide and/or a fertilizer. It is another object of
this invention that the agriculturally acceptable carrier can be a
seed from one or more desired plants, rice, talc, one or more
carbohydrates, one or more polysaccharides, one or more polymeric
porous materials, milk, water, medium, and pellets made from a
cereal grain flour or meal. The concentration of the bacteria
applied to the treated land ranges between approximately 10.sup.5
to approximately 10.sup.11 cfu bacteria per square meter of treated
land. For one alternative embodiment, if one single bacterial
strain is present in the biocontrol agent, the bacteria's
concentration is approximately 10.sup.8 cfu bacteria per square
meter of treated land. For another alternative embodiment, if two
or all three strains of bacteria are present in the biocontrol
agent, the concentration for each strain of bacteria ranges from
approximately 10.sup.7 to approximately 10.sup.8 cfu bacteria per
square meter of treated land.
[0012] It is another object of this invention to have a method for
controlling the growth of annual bluegrass and/or rough bluegrass
in an area in need of treatment by applying a biocontrol agent in
an amount effective to control the growth of annual bluegrass
and/or rough bluegrass to the area. It is a further object of this
invention that the biocontrol agent contains P. fluorescens biovar
B strain XJ3 (NRRL B-50851), P. fluorescens biovar B strain XS18
(NRRL B-50852), or P. fluorescens biovar A strain LRS12 (NRRL
B-50853), or combinations thereof; and optionally a herbicide
and/or a fertilizer, where P. fluorescens strain LRS12 (NRRL
B-50853) controls the growth of both P. annua L. and P. trivialis.
The concentration of the bacteria applied to the treated land
ranges between approximately 10.sup.5 to approximately 10.sup.11
cfu bacteria per area in need of treatment. For one alternative
embodiment, if one single bacterial strain is present in the
biocontrol agent, the bacteria's concentration is approximately
10.sup.8 cfu bacteria per area in need of treatment. For another
alternative embodiment, if two or all three strains of bacteria are
present in the biocontrol agent, the concentration for each strain
of bacteria ranges from approximately 10.sup.7 to approximately
10.sup.8 cfu bacteria per area in need of treatment. It is a
further object of this invention that the application of the
biocontrol agent occurs when the soil is moist (i.e., after rain or
irrigation) or prior to irrigating the area in need of treatment or
when rain is anticipated to occur shortly after application of the
biocontrol agent. It is another object of this invention that the
air temperature in the area in need of treatment should be between
approximately 32.degree. F. and approximately 70.degree. F. in one
embodiment, or between approximately 32.degree. F. and
approximately 50.degree. F. in another embodiment, when the
biocontrol agent is applied. One can apply the biocontrol agent to
bare soil and/or on plants present in the area being treated. One
can apply the biocontrol agent in any manner, including spraying,
broadcasting, or injecting into the soil.
[0013] It is another object of this invention to have a method for
controlling the growth of annual bluegrass and/or rough bluegrass
in an area in need of treatment by applying a biocontrol agent in
an amount effective to control the growth of annual bluegrass
and/or rough bluegrass to the area. It is a further object of this
invention that the biocontrol agent contains an agriculturally
acceptable carrier, and P. fluorescens biovar B strain XJ3 (NRRL
B-50851), P. fluorescens biovar B strain XS18 (NRRL B-50852), or P.
fluorescens biovar A strain LRS12 (NRRL B-50853), or combinations
thereof; and optionally a herbicide and/or a fertilizer, where P.
fluorescens strain LRS12 (NRRL B-50853) controls the growth of both
P. annua L. and P. trivialis. It is another object of this
invention that the agriculturally acceptable carrier can be a seed
from one or more desired plants, rice, talc, one or more
carbohydrates, one or more polysaccharides, one or more polymeric
porous materials, milk, water, medium, and pellets made from a
cereal grain flour or meal. The concentration of the bacteria
applied to the treated land ranges between approximately 10.sup.5
to approximately 10.sup.11 cfu bacteria per area in need of
treatment. For one alternative embodiment, if one single bacterial
strain is present in the biocontrol agent, the bacteria's
concentration is approximately 10.sup.8 cfu bacteria per area in
need of treatment. For another alternative embodiment, if two or
all three strains of bacteria are present in the biocontrol agent,
the concentration for each strain of bacteria ranges from
approximately 10.sup.7 to approximately 10.sup.8 cfu bacteria per
area in need of treatment. It is a further object of this invention
that the application of the biocontrol agent occurs when the soil
is moist (i.e., after rain or irrigation) or prior to irrigating
the area in need of treatment or when rain is anticipated to occur
shortly after application of the biocontrol agent. It is another
object of this invention that the air temperature in the area in
need of treatment should be between approximately 32.degree. F. and
approximately 70.degree. F. in one embodiment, or between
approximately 32.degree. F. and approximately 50.degree. F. in
another embodiment, when the biocontrol agent is applied. One can
apply the biocontrol agent to bare soil and/or on plants present in
the area being treated. One can apply the biocontrol agent in any
manner, including spraying, broadcasting, or injecting into the
soil.
[0014] It is another object of this invention to have a kit for the
control the growth of annual bluegrass and/or rough bluegrass
growth, which has one or more containers containing a biocontrol
agent effective in controlling annual bluegrass and/or rough
bluegrass root growth or germination; optionally a container
containing a herbicide; optionally a container containing a
fertilizer; and instructions for applying the biocontrol agent to
the area in need of treatment. The biocontrol agent contains an
agriculturally acceptable carrier and one or more of the following
Pseudomonas fluorescens strains: P. fluorescens biovar B strain XJ3
(NRRL B-50851), P. fluorescens biovar B strain XS18 (NRRL B-50852),
and P. fluorescens biovar A strain LRS12 (NRRL B-50853), where P.
fluorescens strain LRS12 (NRRL B-50853) controls root growth or
germination of both P. annua L. and P. trivialis. It is another
object of this invention that the agriculturally acceptable carrier
can be a seed from one or more desired plants, rice, talc, one or
more carbohydrates, one or more polysaccharides, one or more
polymeric porous materials, milk, water, medium, and pellets made
from a cereal grain flour or meal. The amount of P. fluorescens in
its container is such that between approximately 10.sup.5 to
approximately 10.sup.11 cfu P. fluorescens can be applied to the
area to be treated. For one alternative embodiment, if one single
P. fluorescens strain is present in the biocontrol agent, it is
present in an amount such that approximately 10.sup.8 cfu P.
fluorescens can be applied to the area to be treated. For another
alternative embodiment, if two or all three strains of P.
fluorescens are present in the biocontrol agent, each bacterial
strain is present in an amount such that approximately 10.sup.7 to
approximately 10.sup.8 cfu per bacterial strain can be applied to
the area to be treated.
[0015] It is another object of this invention to have a kit for the
control the growth of annual bluegrass and/or rough bluegrass
growth which has one or more containers containing a biocontrol
agent effective in controlling annual bluegrass and/or rough
bluegrass root growth or germination; optionally a container
containing a herbicide; optionally a container containing a
fertilizer; and instructions for applying the biocontrol agent to
the area in need of treatment. The biocontrol agent contains one or
more of the following Pseudomonas fluorescens strains: P.
fluorescens biovar B strain XJ3 (NRRL B-50851), P. fluorescens
biovar B strain XS18 (NRRL B-50852), and P. fluorescens biovar A
strain LRS12 (NRRL B-50853), where P. fluorescens strain LRS12
(NRRL B-50853) controls the root growth or germination of both P.
annua L. and P. trivialis. The amount of P. fluorescens in each
container is such that between approximately 10.sup.5 to
approximately 10.sup.11 cfu P. fluorescens can be applied to the
area to be treated. For one alternative embodiment, if one single
P. fluorescens strain is present in the biocontrol agent, the
amount in its container is such that approximately 10.sup.8 cfu P.
fluorescens can be applied to the area to be treated. For another
alternative embodiment, if two or all three strains of P.
fluorescens are present in the biocontrol agent, then each
bacterial strain is present in an amount such that approximately
10.sup.7 to approximately 10.sup.8 cfu per bacterial strain can be
applied to the area to be treated.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 illustrates the screening method of soil bacteria for
weed-suppressive bacteria used to identify bacteria that
selectively suppress Poa annua L. and Poa trivialis.
DEPOSIT OF BIOLOGICAL MATERIAL UNDER TERMS OF BUDAPEST TREATY
[0017] On Aug. 14, 2013, the inventor deposited samples of the
biological materials listed in Table 1 infra and that are described
and claimed herein with the U.S.D.A., Agricultural Research
Service, Patent Culture Collection located at the National Center
for Agricultural Utilization Research, 1815 N. University Street,
Peoria, Ill. 61604, in a manner affording permanence of the deposit
and ready accessibility thereto by the public if a patent is
granted. These deposits have been made under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure and the
regulations thereunder.
TABLE-US-00001 TABLE 1 Three strains of Pseudomonas fluorescens and
ARS Accession Number that suppress Poa annua L. Genus
species/strain designation ARS Accession Number Pseudomonas
fluorescens biovar B strain XJ3 NRRL B-50851 Pseudomonas
fluorescens biovar B strain XS18 NRRL B-50852 Pseudomonas
fluorescens biovar A strain NRRL B-50853 LRS12
[0018] All restriction on the availability to the public of these
biological materials identified herein and deposited as described
herein will be irrevocably removed upon the granting of a
patent.
[0019] The biological materials identified herein have been
deposited under conditions such that access to the microorganisms
are available during the pendency of the patent application to one
determined by the Commissioner to be entitled thereto under 37
C.F.R. .sctn.1.14 and 35 U.S.C .sctn.122.
[0020] The deposited biological materials will be maintained with
all the care necessary to keep them viable and uncontaminated for a
period of at least five years after the most recent request for the
furnishing of a sample of the deposited microorganism, and in any
case, for a period of at least thirty (30) years after the date of
deposit for the enforceable life of the patent, whichever period is
longer.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Annual bluegrass and rough bluegrass negatively affect many
turfgrasses (see Table 2 for a non-limiting list of common
turfgrasses) and reduce turfgrass and crop yield resulting in
economic loss. Annual bluegrass and rough bluegrass out-compete
desired turfgrasses and native plants by actively growing roots and
germinating seeds in cold temperatures, such as between
approximately 32.degree. F. and approximately 50.degree. F., or
between approximately 32.degree. F. and approximately 60.degree.
F., or 32.degree. F. and approximately 70.degree. F. Thus, during
these temperature ranges a biocontrol agent for these weeds would
extremely effective. After all, if one can inhibit root production
by annual bluegrass and/or rough bluegrass when they are most
actively growing and out-competing desired plants (e.g., turfgrass,
crops, and native plants), then one can help prevent these weeds
from establishing a strong root system that helps the weeds
continue to out-compete the desired plants at other
temperatures.
[0022] The biocontrol agent of this invention (P. fluorescens NRRL
B-50851, P. fluorescens NRRL B-50852, and/or P. fluorescens NRRL
B-50853; and/or compositions containing one or more of these
bacteria) is a slow acting biocontrol agent when compared to
herbicides. After application of the biocontrol agent of this
invention to the soil, it may take time for the bacteria of this
invention to multiply and inhibit the soil in close proximity to
the weeds' roots, or reside within the intracellular spaces of the
weeds' roots. While one may observe some reduction in the weeds'
growth during one growing season in the treated area, it may take
several growing seasons (three to four years) to observe
substantial reduction in the amount of annual bluegrass and/or
rough bluegrass growing in the treated area. Furthermore, one may
need to reapply this biocontrol agent after five or more years for
continual significant reduction in germination of the weeds that
may reenter the area.
[0023] For the purposes of this invention, turfgrass is used as
exemplary of a desired plant to be protected; however, the methods
and compositions described herein are applicable to other grasses,
crops, and native plants. Turfgrass species are present in golf
courses, public parks, residential lawns, sod production
facilities, sports fields, etc. in North and South America, Europe,
British Isles, North Africa, North Asia, Australia, and Antarctica.
Annual bluegrass (P. annua L.) and rough bluegrass (P. trivialis)
negatively impact the growth, health, and viability of turfgrass.
As such, it would be useful to have compositions and methods that
inhibit the growth of Poa annua L and P. trivialis.
TABLE-US-00002 TABLE 2 Common turfgrasses in the United States.
Common Name Scientific Name Alkaligrass Puccinellia distans
Bahiagrass Paspalum notatum Bentgrass - Colonial Agrostis
capillaris Bentgrass - Creeping Agrostis palustris Bentgrass -
Spike Agrostis exerata Bermudagrass Cynodon dactylon Blue grama
grass Bouteloua gracilis Bluegrass - Canada Poa compressa Bluegrass
- Kentucky Poa pratensis Bluegrass - Rough Poa trivialis
Buffalograss Buchloe dactyloides Carpetgrass Axonopus fissifolius
Centipedegrass Eremochloa ophiuroides Dichrondra Dichondra
micrantha Eastern Gamagrass Tripsacum dactyloides Fescue - Chewings
Festuca rubra var. commutate Fescue - Creeping Red Festuca rubra
var. rubra Fescue - Sheeps Fescue ovina var. ovina Fescue - Tall
Festuca arundinacea Fescue - Hard Festuca longifolia Grama Grass -
Blue Bouteloua gracilis Kikuyugrass Pennisetum clandestinum
Orchardgrass Dactylis glomerata Ryegrass - Perennial Lolium perenne
Saint Augustine grass Stenotaphrum secundatum Seashore Paspalum
Paspalum vaginatum Smooth bromegrass Bromus inermis Wheatgrass -
Bluebunch Agropyron spicatum Wheatgrass - Crested Agropyron
cristatum Zoysiagrass - Japonica Zoysia japonica Zoysiagrass -
Matrella Zoysia matrella Zoysiagrass - Tenuifolia Zoysia
tenuifolia
[0024] Chemical herbicides are well-known in the art field (see
Table 3 for a non-limiting example of herbicides). However, none of
the herbicides currently on the market can selectively control the
growth of P. annua L. (annual bluegrass) without harming the
desirable turfgrass. As such, the need exists for a
substance/composition that can control the growth of P. annua L. As
described herein, P. fluorescens NRRL B-50851, P. fluorescens NRRL
B-50852, and P. fluorescens NRRL B-50853 are three bacterial
strains that have been isolated from soil and selected because they
control the growth and/or germination of P. annua L. and/or P.
trivialis without harming the desirable turfgrass. It is noted that
P. fluorescens strain LRS12 (NRRL B-50853) inhibits or controls the
growth and/or germination of both P. annua L. and P. trivialis. The
other two bacteria do not inhibit or control the growth and/or
germination of P. trivialis.
[0025] For the purposes of this invention, the phrases "control the
growth" and "control the germination" can mean inhibit the growth
and/or reduce the growth of the indicated weeds and inhibit the
germination and/or reduce the germination of the indicated weeds,
respectively. The bacteria of the present invention exert this
"control" over the weeds by inhibiting the elongation of the root
cells, and thereby inhibiting or reducing root growth both during
the germination process and when the seedling or plant is growing.
When root growth is inhibited or reduced, the plant embryo,
seedling, and plant have a more difficult time absorbing the water
and nutrients needed from the soil. In turn, the growth of aerial
parts of the plant is reduced. A weed with a well-established root
system can usually compensate for the reduced or inhibited growth
of some of its roots. As such, this invention has maximum benefits
on seedlings or germinating seeds of the weeds, but can still help
slow the growth of established weeds with larger root systems. Not
wishing to be bound to any particular hypothesis, this invention
appears to cause the weed seeds to fail to germinate because the
inhibition or reduction of root cell growth and/or elongation of
the embryo plant's root cells result in the embryo plant dying
prior to appearance of aerial parts of the seedling.
[0026] The expressions "inhibit the growth", "inhibit the
germination", and "inhibit development" of annual bluegrass (P.
annua L.) and any similar expressions, refer to inhibition or
suppression of the germination, growth, and/or development of P.
annua L. When the annual bluegrass seed or seedlings are treated
with the biocontrol agents of this invention (P. fluorescens
strains described herein or compositions containing the P.
fluorescens strains described herein) at least approximately 20%
reduction in root growth (as measured by root dry weight) occurs
compared to untreated weed seedlings and/or seeds. In other
embodiments, inhibition of annual bluegrass occurs when weed
seedlings and/or seeds treated with the biocontrol agents of this
invention have at least approximately 25% reduction in root growth
(as measured by root dry weight) compared to untreated weed
seedlings and/or seeds. In another embodiment, inhibition of annual
bluegrass occurs when weed seedlings and/or seeds treated with the
biocontrol agents of this invention have at least approximately 30%
reduction in root growth (as measured by root dry weight) compared
to untreated weed seedlings and/or seeds.
[0027] In an alternative embodiment, inhibition of annual bluegrass
or rough bluegrass occurs when the weed seedlings and/or seeds
treated with the biocontrol agents of this invention (P.
fluorescens strains described herein or the compositions containing
the P. fluorescens strains described herein) have at least
approximately 20% reduction in shoot growth (as measured by shoot
dry weight) compared to untreated weed seedlings and/or seeds. In
yet another embodiment, inhibition of annual bluegrass occurs when
weed seedlings and/or seeds treated with the biocontrol agents of
this invention have at least approximately 25% reduction in shoot
growth (as measured by shoot dry weight) or stand (numbers of plant
m.sup.-2) compared to untreated weed seedlings and/or seeds. In yet
still another embodiment, inhibition of annual bluegrass occurs
when weed seedlings and/or seeds treated with the biocontrol agents
of this invention or the compositions of this invention have at
least approximately 30% reduction in shoot growth (as measured by
shoot dry weight) or stand (numbers of plant m.sup.-2) compared to
untreated weed seedlings and/or seeds.
[0028] The biocontrol agents of the present invention are
considered safe (that is, not having an undue adverse effect) on
desirable turfgrass, crop plants or desirable native plant species
when a crop or turfgrass or desirable native plant seedling and/or
seed, after exposure to a biocontrol agent of the present
invention, has no significant reduction in root growth (as measured
by root dry weight) and/or shoot growth (as measured by shoot dry
weight) or stand (as measured by numbers of plant m.sup.-2)
compared to a control crop plant seedling and/or seed. In another
embodiment, the biocontrol agents of the present invention are
considered safe (that is, not having an undue adverse effect) on a
crop plant when a crop plant seedling and/or seed, after exposure
to the biocontrol agent of the present invention, has less than
approximately 10% reduction in root growth (as measured by root dry
weight) or shoot growth (as measured by shoot dry weight) or stand
(as measured by numbers of plant m.sup.2) compared to a control
crop plant seedling and/or seed. In another embodiment, the
biocontrol agents of the present invention are considered safe
(that is, not having an undue adverse effect) on a crop plant when
a crop plant seedling and/or seed, after exposure to a biocontrol
agent of the present invention, has less than approximately 15%
reduction in root growth (as measured by root dry weight) or shoot
growth (as measured by shoot dry weight) or stand (as measured by
numbers of plant m.sup.-2) compared to a control crop plant
seedling and/or seed. The biocontrol agents of the present
invention are considered safe (that is, not having an undue adverse
effect) on a crop plant when a crop plant seedling and/or seed,
after exposure to a biocontrol agent of the present invention, has
less than approximately 20% reduction in root growth (as measured
by root dry weight) or shoot growth (as measured by shoot dry
weight) or stand (as measured by numbers of plant m.sup.-2)
compared to a control crop plant seedling and/or seed.
TABLE-US-00003 TABLE 3 Common herbicides and fertilizers that can
be applied with the Pseudomonas fluorescens strains to surface of
soil or injected into soil. Herbicide Preemergent Benefin Herbicide
Preemergent Bensulide Herbicide Preemergent Dithiopyr Herbicide
Preemergent Oryzalin Herbicide Preemergent Oxadiazon Herbicide
Preemergent Pronam Herbicide Preemergent Prodiamine Herbicide
Preemergent Pendimethalin Herbicide Preemergent Benefin/Oryzalin
Herbicide Post emergent Ethofumesate Herbicide Post emergent
Pronamide Fertilizer Macronutrient Nitrogen Fertilizer
Macronutrient Phosphate Fertilizer Macronutrient Potassium
Fertilizer Macronutrient Calcium Fertilizer Macronutrient Magnesium
Fertilizer Macronutrient Sulfur Fertilizer Micronutrient Iron
Fertilizer Micronutrient Zinc Fertilizer Micronutrient Molybdenum
Fertilizer Micronutrient Boron Fertilizer Micronutrient Copper
Fertilizer Macronutrient Lime Fertilizer Organic Amendment Bone
Meal
[0029] The biocontrol agents of this invention contain one or more
of P. fluorescens NRRL B-50851, P. fluorescens NRRL B-50852, and P.
fluorescens NRRL B-50853, alone or with an agriculturally
acceptable carrier. Further, the biocontrol agents of this
invention can contain one or more of the bacteria, an
agriculturally acceptable carrier, and at least one herbicide
and/or at least one fertilizer (see Table 3 for a non-limiting list
of herbicides and fertilizers that can be in the compositions of
this invention).
[0030] The biocontrol agents of this invention contain sufficient
amounts of one or more of P. fluorescens NRRL B-50851, P.
fluorescens NRRL B-50852, and P. fluorescens NRRL B-50853 described
herein to control the growth of P. annua L. and/or P. trivialis.
The concentration of each P. fluorescens in the biocontrol agents
of this invention is at least 10.sup.4 colony forming units (cfu)
per unit of composition. In another embodiment, the concentration
of each P. fluorescens in the biocontrol agents can range from
approximately 10.sup.5 to approximately 10.sup.11 cfu per unit of
composition. In another embodiment, the concentration of each P.
fluorescens in the biocontrol agents can range from approximately
10.sup.6 to approximately 10.sup.10 cfu per unit of composition. In
yet another embodiment, the concentration of each P. fluorescens in
the biocontrol agents can range from approximately 10.sup.7 to
approximately 10.sup.9 cfu per unit of composition. In another
embodiment, the concentration of each P. fluorescens in the
biocontrol agents of this invention is 10.sup.8 cfu per unit of
composition.
[0031] When two or more of the bacterial strains of this invention
are combined in a biocontrol agent, or applied sequentially or
together, the results are often better than when a single bacterial
strain is used alone. This result is surprising and unexpected.
Thus, one is able to apply a smaller amount of each bacterial
strain per unit area when two or more strains are used, than when
only one bacterial strain is applied. So, when any two of P.
fluorescens NRRL B-50851, P. fluorescens NRRL B-50852, and P.
fluorescens NRRL B-50853, or when all three bacterium are combined
in a biocontrol agent or applied sequentially or together, the
concentration of each bacterial strain can range from approximately
10.sup.6 to approximately 10.sup.9 cfu per unit of composition. In
another embodiment, the concentration of each P. fluorescens in a
biocontrol agent containing two or three of these bacteria can be
approximately 10.sup.7 cfu per unit of composition. When applying
two or three strains of these bacteria to a unit area, the
concentration of each bacterial strains applied can range from
approximately 10.sup.6 to approximately 10.sup.9 cfu per unit area,
or from approximately 10.sup.7 to approximately 10.sup.8 cfu per
unit area, or approximately 10.sup.7 cfu per unit area.
[0032] As used herein, the term "agriculturally acceptable carrier"
includes any liquid or solid carrier to which the bacteria of the
present invention can be added and that is not harmful to the
bacteria of the invention or the plants to which it is being
applied. An agriculturally acceptable carrier is any liquid or
solid that can be combined with the P. fluorescens of this
invention and that assists in the application of the P. fluorescens
strains of this invention to the soil so that the P. fluorescens
strains of this invention grow and colonize the soil and roots of
Poa annua L and/or Poa trivialis. Non-limiting examples of
agriculturally acceptable carriers include talc, starch, sucrose,
lactose, and other carbohydrates, polysaccharides, milk/skim milk,
cellulose, water, a buffered solution, methylcellulose, clay, sand,
peat, vermiculite, diatomaceous earth, a cereal grain flour or
meal, cotton meal, rice, seeds, and liquid media. A carrier can
also be a polymeric porous material, such as polyvinyl alcohol
(PVA), polyethylene glycol (PEG), polyacrylic acid (PAA),
poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymer of
poly(lactic-co-glycolic acid) (PLGA), poly(.epsilon.-caprolactic
acid) (PCLA), poly(.beta.-hydroxybutyric acid),
poly(.beta.-hydroxyvaleric acid), polydioxanone, poly(ethylene
oxide), poly(malic acid), poly(tartronic acid), polyphosphazene,
polyethylene (PE), polystyrene (PS), agar (alginate) or other
polysaccharides, gelatin, or combinations thereof. The carrier
should not kill the bacteria of the invention. Also the carrier
should release the bacteria of the invention into the soil after
application to the soil or onto plants after application onto the
plants. Such release of the bacteria may occur after a period of
rain or irrigation or other type of application of water to the
soil and/or plant. Any carrier that permits the bacteria of the
present invention to be delivered to the soil and/or target plant
in a manner such that the bacteria remains viable may be employed
in the composition so long as the carrier does not harm
turfgrasses, native plants, and crops.
[0033] A dry formulation of the bacteria of the present invention
can be produced by several drying methods including freeze drying,
air drying, spray drying, or fluidized bed drying. Skim milk,
sucrose, lactose, dextran, etc., can be added to the bacteria to
act as both protectants during the drying process and as
carriers.
[0034] In one embodiment, one or more of P. fluorescens NRRL
B-50851, P. fluorescens NRRL B-50852, and P. fluorescens NRRL
B-50853 can be combined with a cereal grain flour or meal and
formed into pellets. For example, liquid media containing
approximately 10.sup.8 of P. fluorescens NRRL B-50851, P.
fluorescens NRRL B-50852, or P. fluorescens NRRL B-50853 is
freeze-dried using known in the art methods. The freeze-dried media
is then mixed with a cereal grain flour and water and passed
through an extruder so that spherical pellets or cylindrical
pellets are made, and allowed to dry. One can then spread the
pellets onto the soil and/or plants in the area to be treated when
the ground is moist, or prior to rain being expected to occur, or
prior to irrigating the area. When wet, the a cereal grain flour
pellets decay and provide food for the P. fluorescens NRRL B-50851,
P. fluorescens NRRL B-50852, or P. fluorescens NRRL B-50853. In an
alternative embodiment, if one intends to use two or three of P.
fluorescens NRRL B-50851, P. fluorescens NRRL B-50852, and/or P.
fluorescens NRRL B-50853, then liquid media containing
approximately 10.sup.7 of each bacteria are sprayed-dried, and then
are mixed with the a cereal grain flour and water, are extruded
into the desired shape, and are allowed to dried. Optional pellet
shapes can include, but are not limited to conical, cube, cuboid,
prism (hexagonal or other shape), tetrahedron, octahedron, and
dodecahedron. The pellets can range in size from approximately 1
mm.sup.3 to approximately 10 cm.sup.3; or from approximately 10
mm.sup.3 to approximately 5 cm.sup.3, or from approximately 20
mm.sup.3 to approximately 1 cm.sup.3. Depending on the pellet's
shape and the mode of spreading, one may want to smooth the
pellet's edges to improve the spreading of the pellets. One can
broadcast spread pellets or aerial spray pellets.
[0035] In an alternative embodiment, one can freeze-dry the media
containing approximately 10.sup.8 of P. fluorescens NRRL B-50851,
P. fluorescens NRRL B-50852, or P. fluorescens NRRL B-50853, or a
combination thereof at a concentration of approximately 10.sup.7 of
bacteria with talc. Then one can mix the talc containing the
bacteria with seeds of desired plants. The talc/bacteria
combination sticks to the seeds. Then one can spread the
bacteria-coated seed of the desired plant.
[0036] Alternatively, one can mix a herbicide or fertilizer or both
with one or more of the bacteria (in liquid media or previously
dried via spray-drying or freeze-drying; optionally previously
combined with a carrier), optionally combine with a carrier, and
apply the mixture onto the soil or plants in the area desired to be
treated.
[0037] Not wishing to be bound to any particular theory, it is
believed that during colonization of P. annua L. and P. trivialis
roots, the P. fluorescens strains of this invention occupy the
intracellular spaces of the weeds' roots and produce one or more
inhibitory compounds that suppress root elongation of the weeds.
Not wishing to be bound to any particular hypothesis, it is
believed that the bacteria inhibit lipopolysaccharide production in
the cell wall and cell membrane in the root leading to the lack of
root elongation especially during the cool seasons from fall to
early spring. A similar mechanism of action occurs for the bacteria
of this invention with P. trivialis. The basis for this hypothesis
is that P. fluorescens strain D7 demonstrates this mechanism of
action with downy brome. See Gurusiddaiah, et al., 1994 Weed
Science 42(3): 492-501. Thus, to be effective, P. fluorescens NRRL
B-50851, P. fluorescens NRRL B-50852, and/or P. fluorescens NRRL
B-50853 should reside in close proximity to the root cells of
annual bluegrass or rough bluegrass. The lack of root growth of Poa
annua L. and P. trivialis during the fall, winter, and spring
(during time the plants actively grow) reduces the competitive
ability of these weeds and allows the desirable turfgrasses to
flourish. The bacteria can be successful in all environments where
Poa annua L. or P. trivialis grows, especially those areas with
cool or cold winters. Thus, application of the biocontrol agents of
this invention (the bacteria of this invention and compositions
containing the bacteria) are useful to control root growth of the
embryo or seedling of annual bluegrass and/or rough bluegrass.
[0038] P. fluorescens NRRL B-50851, P. fluorescens NRRL B-50852,
and P. fluorescens NRRL B-50853 grow and are metabolically active
during conditions that P. annua L. and P. trivialis are actively
growing roots, at cooler air temperatures (e.g., between
approximately 32.degree. F. and approximately 60.degree. F.) and
when the soil has high moisture content. When the air temperature
is too warm and/or when the soil is too dry, both the bacteria of
this invention and P. annua L. and P. trivialis go dormant. When
dormant, the polysaccharide coating of P. fluorescens NRRL B-50851,
P. fluorescens NRRL B-50852, and P. fluorescens NRRL B-50853
protect the bacteria from the harsh environmental conditions. In
one embodiment, application of the biocontrol agents described
herein (the bacteria and/or compositions containing the bacteria)
to the soil occurs in fall when air temperatures are below
50.degree. F. and rain is predicted or irrigation is intended
shortly after application. In another embodiment, application of
the biocontrol agents described herein to the soil when air
temperatures range from approximately 32.degree. F. to 80.degree.
F.; or approximately 35.degree. F. to approximately 60.degree. F.;
or approximately 35.degree. F. to approximately 55.degree. F.
Application occurs when the ground has sufficient moisture or when
rain is predicted or prior to irrigating the land. Alternatively,
one could have irrigated the land prior to application of the
biocontrol agents of this invention. Alternatively, one applies an
inhibitory amount of the biocontrol agents described herein with or
to turf and/or turfgrass seed. Application can occur to soil that
is bare of plants, to field of dormant weeds, to a field of
actively growing weeds, to a field with dormant turfgrass, or to a
field of actively growing turfgrasses. In general, one applies an
effective inhibitory amount of the biocontrol agents to the soil
surface or soil subsurface in an area where one wants to or is
growing turfgrass or small grain crop area.
[0039] The types of desirable crops and/or turfgrasses that the P.
fluorescens strains do not inhibit are listed in Table 4, infra.
Testing included all economically important plant species of the
area, and those plants known to be involved in ecosystem
maintenance. In agronomic ecosystems, the major crop species are of
interest. The U.S. Environmental Protection Agency (EPA) published
a list of the top 25 major agricultural crops. These plants were
placed on this list because of their economic importance, ecosystem
activity or total production values (EPA, 2011).
[0040] One can spray the biocontrol agents onto the bare soil or
onto a field of turfgrass. In one embodiment, spraying is performed
after a rain or irrigation (when the soil is moist). In another
embodiment, spraying is performed shortly before rain is
anticipated and/or during periods of high rain probability, and/or
prior to irrigating the area of application. Application of the
bacteria in liquid or suspension form and/or the compositions of
this invention may be accomplished by ground or aerial spraying
using equipment routine to one of skill in the art. The nozzle of
the sprayer may be adjusted for size by one of skill in the art to
accommodate the size of the area being treated and any plants in
that area, as well as the type and size of presently disclosed
carriers.
[0041] One can apply the bacteria of this invention or compositions
of this invention onto seed as a seed coating and then drill the
seed into the soil or broadcast the seed. In one embodiment, the
seed coated with the bacterium of this invention or compositions of
this invention can be drilled or broadcast in the fall of the year
into moisture, and the desired seed will spread the bacterium
throughout the soil. In another embodiment, the bacteria-coated
seed can be drilled or broadcast at any time prior to rain or
application of water (irrigate) to the area where the coated seed
are drilled or broadcasted. For example, to make a biocontrol agent
containing a seed coated with one or more of the bacteria described
herein, one grows one or more of the species of bacteria to
approximately 10.sup.8 cells/L, and then one can apply the bacteria
to the seed by making a slurry of approximately 10 oz. media per
approximately 60 pounds seed and rotate the slurry in a drum. After
the seeds are dry, one can direct drill or broadcast the biocontrol
agent (bacterial-coated seed) at recommended seeding rates, which
for wheat seed is approximately 60 pounds per acre. If one has
freeze-dried media containing approximately 10.sup.8 cells/L, then
one can apply the bacteria to the seed by making a slurry of
approximately 2 g freeze-dried bacteria with approximately 60
pounds per acre of seed and rotate the slurry in a drum. After the
seeds are dry, one can direct drill or broadcast seed at
recommended seeding rates, which for wheat seed is approximately 60
pounds per acre. For liquid spraying, one can use approximately 4
gallons of liquid media containing 10.sup.8 cells bacteria per L
per acre of land being treated or 2 g of freeze-dried media/cells
per acre. For 20 acres, apply approximately 80 gallons of liquid
per acre or approximately 40 g in 50 to 1000 gallons. Final minimum
concentration can be approximately 10 million cells per square foot
soil surface. The liquid spray can be applied by back pack sprayer,
ground sprayer or aerially applied.
[0042] Individual strains of bacteria can be cultured by standard
methods using well-known in the art techniques for a sufficient
amount of time (in one embodiment, approximately 32 hours) such
that sufficient bacteria grow.
[0043] The "root system" of a plant includes the rhizoplane (root
surface) and the rhizosphere (soil immediately surrounding the
roots).
[0044] One can have a kit containing one or more of the P.
fluorescens described herein for controlling the growth of annual
bluegrass and/or rough bluegrass. A kit may contain one or more
containers, each having one or more of the P. fluorescens described
herein, in a liquid or dry formulation, and written instructions
for using the kit to control the growth of annual bluegrass and/or
rough bluegrass. Alternatively, the kit may contain seeds or other
agriculturally acceptable carriers that contain the P. fluorescens
described herein. The kit may optionally contain one or more
herbicides and/or fertilizer that can be applied with the bacteria.
As discussed supra, the concentration of the P. fluorescens within
the kit can depend on the number of the bacteria contained within
the kit. For example if only one of the P. fluorescens described
herein is contained within the kit, the concentration of the
bacterium may be approximately 10.sup.8 per area for treatment.
Alternatively, if two of the P. fluorescens described herein are
within the kit, the concentration for each bacterium may be only
approximately 10.sup.7 per area for treatment. Further, if all
three of the bacteria described herein are contained in the kit,
then the concentration for each bacterium may be only approximately
10.sup.6 per area for treatment.
[0045] The terms "approximately" and "about" refers to a quantity,
level, value or amount that varies by as much as 30%, or in another
embodiment by as much as 20%, and in a third embodiment by as much
as 10% to a reference quantity, level, value or amount. As used
herein, the singular form "a", "an", and "the" include plural
references unless the context clearly dictates otherwise. For
example, the term "a bacterium" includes both a single bacterium
and a plurality of bacteria.
Example 1
Isolating Bacteria with Inhibitory Growth Activity
[0046] To reduce the growth of annual bluegrass, a bacterial strain
has the ability to establish and grow in the microhabitat where it
is to be used. The bacteria has the ability to colonize the root
system of annual bluegrass. Therefore, bacteria are selected by
isolating bacteria from the rhizoplane, the rhizosphere, or both
rhizoplane and rhizosphere of annual bluegrass and turfgrasses. In
addition the bacteria are able to selectively inhibit Poa annua L.
and not hurt the desirable plants, which are desirable turfgrass,
crops, and native plants.
[0047] The method for isolating the bacteria that can control of P.
annua L. and P. trivialis is illustrated in FIG. 1. A general
description of this isolation method follows. Turfgrass or annual
bluegrass are dug from the field and adhering soil is gently washed
from the roots with flowing tap water. The roots are excised from
the shoots. The bacteria are collected and isolated by placing the
excised roots in sterile water and shaking the mixture vigorously
to remove organisms clinging to the root surface. Then serial
dilutions are prepared and spread plated on SR agar (see Sands and
Rovira, 1970. Applied Microbiology 20:513-514). The plates are then
incubated 20.degree. C. Each isolate growing on the plates after 32
hours is then removed and streaked for isolation onto a new Petri
plate containing SR agar. Single colony isolates are then placed
into long-term storage until further testing is performed.
[0048] For long-term storage during the screening procedure,
inoculum from individual colonies is grown in SR broth, centrifuged
at room temperature (4,000 rpm for 10 minutes). The supernatant is
decanted, and the pellet is resuspended in 1 mL of fresh sterile SR
broth. This solution is mixed with sterile glycerol (1:1) in a two
mL cryovial (VWR International, Radnor, Pa.) and stored in
-80.degree. C. ultra-low freezer (Labconco, Kansas City, Mo.).
Example 2
In-Vitro Screening Isolated Bacterial Strains
[0049] Bacteria colonies isolated in Example 1 are screened to
select for bacteria strains that inhibit annual bluegrass growth
in-vitro as exhibited by reduction in root growth or germination as
compared to control plants. The bacteria strains inhibitory to
annual bluegrass are then tested against various turfgrass species
and other economically important plants. The bacteria strains that
do not deleteriously affect turfgrass growth in-vitro are selected
for further study.
TABLE-US-00004 TABLE 4 Suppression of root growth of various plant
species in agar bioassays investigating Pseudomonas fluorescens
strains XJ3, XS18, or LRS12. XJ3 XS18 LRS12 Root Growth Bioassay
Common Name Latin Name % Suppression MONCOTYLEDONS Annual bluegrass
Poa annua 100 98 94 Bahiagrass Paspalum notatum 0 10 0 Barley
Hordeum vulgare L. 0 0 0 Basin wildrye Elymus linareus 0 0 0
Bentgrass - Creeping Agrostis palustris 0 0 0 Bermudagrass Cynodon
dactylon 0 0 0 Birdseye bluegrama Bouteloua gracilis 0 0 0
Bluebunch wheatgrass Agropyron spicatum 20 0 0 Bluegrass - Kentucky
Poa pratensis 0 0 0 Bottlebrush squirreltail Elymus elymoides 0 0 0
Buffalograss Buchloe dactyloides 0 0 0 Carpetgrass Axonopus
fissifolius 0 0 0 Cody buffalograss Buchloe dactyloides 0 0 0
Columbia needlegrass Stipa columbian 0 0 0 Corn Zea mays L. 0 0 0
Dichrondra Dichondra micrantha 0 0 0 Downy brome Bromus tectorum L.
0 30 10 Eastern Gamagrass Tripsacum dactyloides 0 0 0 Fescue -
Creeping Red Festuca rubra var. rubra 10 0 0 Fescue - Tall Festuca
arundinacea 10 0 0 Idaho fescue Festuca idahoensis 0 0 0
Kikuyugrass Pennisetum clandestinum 0 0 0 Orchardgrass Dactylis
glomerata 0 0 0 Ryegrass - Perennial Lolium perenne 0 0 0 Saint
Augustine grass Stenotaphrum 0 0 0 secundatum Smooth bromegrass
Bromus inermis 20 0 0 Wheatgrass - Agropyron spicatum 10 10 20
Bluebunch Wheatgrass - Crested Agropyron cristatum 10 0 0
Zoysiagrass - Japonica Zoysia japonica 10 20 0 DICOTYLEDONS Alfalfa
Medicago sativa L. 0 0 0 Apple Malus Mill. 0 0 0 Beans Phaseolus L.
0 0 0 Big sagebrush Artemisia tridentata 0 0 0 Camelina Camelina
sativa L. 0 0 0 Canola Brassica napus L. 0 0 0 Celery Apium spp. L.
0 0 0 Chick peas Cicer arietinum 0 0 0 Clover Trifolium L. 0 0 0
Common vetch Vicia sativa L. 0 0 0 Cucumber Cucumis sativus L. 0 0
0 Faba bean Vicia faba 0 0 0 Flax Linum narbonense L. 0 0 0
Jointvetch Aeschynomene sp. L. 0 0 0 Lentil Lens culinaris Medik. 0
0 0 Lettuce Lactuca sativa L. 0 0 0 Mint Mentha L. 0 0 0 Northern
bedstraw Galium boreale L. 0 0 0 Pea Pisum sativum L. 0 0 0 Peanuts
Arachis L. 0 0 0 Pepper Capsicum L. 0 0 0 Phlox Phlox L. 0 0 0
Potato Solanum 0 0 0 Rannucula Ranunculus 0 0 0 Rapeseed Brassica
rapa L. 0 0 0 Safflower Carthamus tinctorius L. 0 0 0 Soybeans
Glycine max L. Merr. 0 0 0 Squash Cucurbita spp. 0 0 0 Sugar beets
Beta vulgaris 0 0 0 Sunflower Helianthus L. 0 0 0 Tomato Solanum 0
0 0 Vetch Vicia spp. 0 0 0
[0050] Individual bacteria colonies are inoculated into Pseudomonas
minimal salts medium (PMS) described by Gasson, Applied and
Environmental Microbiology 39:25-29 (1980), and grown at 20.degree.
C. (68.degree. F.) for 32 hours until late logarithmic growth
(about 10.sup.9 to 10.sup.11 cfu/mL culture medium).
[0051] Annual bluegrass growth inhibition is assessed by a seed
germination and root elongation assay. Briefly, one mL of test
bacterial cultures is added to water agar plates. The water agar
plates consist of 18 mL of 0.9% sterile, molten agar (50.degree.
C.) added to a Petri plate and allowed to solidify. Twenty annual
blue grass seed are added to the bacterial dishes, as well as to
control dishes with no bacteria, and allowed to grow at 15.degree.
C. After five days, plant root length is measured. The test
bacteria inhibit the annual bluegrass if root lengths of the
seedling treated with the bacteria are more than 20 percent less
than the control root lengths. This procedure is the first step in
determining those isolates to study further. Two plates are
prepared for each organism tested. Control plates are prepared by
using the non-inoculated growth medium instead of the bacteria cell
culture. The plates are slanted slightly so the roots grow down and
through the agar. Before root growth from the seedlings interferes
with each other, in about 5 days, the seedlings are pulled from the
agar, and root length and germination are recorded. The bacteria
showing significant inhibition of annual bluegrass root length or
lack of germination when compared to the control seeds are then
tested against desirable turfgrass, crop and native seedling growth
(infra). Significant inhibition of annual bluegrass in the in-vitro
test is defined as at least a 50% reduction in root length when
compared to the control or at least a 20% reduction in germination
when compared to the control.
[0052] Over 20,000 bacteria were isolated from soil in Example 1.
Half of these or 10,000 bacteria have some inhibitory effect on
annual bluegrass and desirable plants in water agar seed root
length inhibition and germination assays as tested in Example 2.
Yet, approximately 200 bacterial isolates had some inhibitory
effect to annual bluegrass seedlings, but did not inhibit grow of
turfgrass seedlings. These approximately 200 bacterial isolates are
then tested for sustained and sufficient inhibitory effect against
annual bluegrass when applied to soil in pots grown in the growth
chamber. Table 4, above, provides the results for Pseudomonas
fluorescens strains XJ3, XS18, and LRS12.
Example 3
Screening Bacterial Strains in the Growth Chamber
[0053] Bacterial strains showing significant inhibition of annual
bluegrass root length or germination in Example 2 (supra) are
subjected to a first screening in soil by separately growing annual
bluegrass plants and turfgrass species in a growth chamber or
greenhouse in the presence of the bacterial strain. Those bacteria
strains are selected that inhibit annual bluegrass without
deleteriously affecting desirable turfgrass.
[0054] Ritzville silt loam is obtained from a site in Lind, Wash.
and passed through a 4 mm screen and then a 2 mm screen to obtain a
uniform soil mixture. The screened silt loam is amended with 20%
sand by weight and is used as soil in the pots. Pots are filled
with soil and seeded with annual bluegrass or with turfgrass seeds.
6.4 cm diameter by 7.6 cm deep plastic pots seeded with 6-9 annual
bluegrass seeds and 7.6 cm diameter by 15.2 cm deep plastic pots
seeded with 5 turfgrass seeds are convenient sizes for growth
chamber or greenhouse studies.
[0055] The amount of bacteria per annual bluegrass seed in the test
pots is selected to optimize the selection of field-effective
strains and to minimize the selection of field-ineffective strains.
In particular, 10.sup.7 cfu of the test strain per annual bluegrass
test pot is applied to the soil surface. A rate of 10.sup.7 cfu
bacteria are applied per pot in 5 mL of water by dropping the
material onto the surface of the soil. Four turfgrass seeds are
planted on the surface of the pot and covered with sand to assess
the effect of the bacteria on turfgrass seeds and seedlings in
natural, non-autoclaved soil. Separate annual bluegrass and
turfgrass seed controls are prepared identical to the test samples
except without bacterial treatment as controls.
[0056] The soil is wetted to provide good plant growth, and the
pots incubated in the growth chamber with 14-hour day at 18.degree.
C. and a 10-hour night at 13.degree. C. After approximately 3 weeks
to approximately 4 weeks, the plants and soil are removed from the
pots; annual bluegrass and turfgrass seedlings and their roots are
washed with water until free of soil. The roots and shoots of the
annual bluegrass and turfgrass seedlings are excised, and are dried
at 60.degree. C. for 48 hours to remove water so that a comparison
to the control plants can be made.
[0057] To evaluate the bacterial treatment, root and shoot dry
weight of the annual bluegrass plants and turfgrass seedlings are
compared with the root and shoot dry weight of the control annual
bluegrass and turfgrass seedlings. Bacterial strains that cause the
treated annual bluegrass seedlings to have reduced root growth
(root dry weight), as compared to the control group root growth
(root dry weight), of at least 30% or that cause the treated annual
bluegrass seedlings to have reduced shoot growth (shoot dry
weight), as compared to the control group shoot growth (shoot dry
weight), of at least 30% are considered inhibitory to annual
bluegrass.
[0058] Of the less than 200 bacterial strains that may inhibit root
growth of annual bluegrass, but not inhibit root growth of
economically important crops or turfgrasses in the water agar
bioassay, ten bacterial strains are found to inhibit growth of
annual bluegrass but not turfgrass when applied to natural soils in
pots and grown in the greenhouse. Upon further testing of the 10
isolates, it is discovered that four isolates are inconsistent in
producing the compound(s) that inhibit root elongation as evident
by inconsistent inhibition of root elongation. The production of
anti-microbial activity disqualified another three strains from
further study. The results for Pseudomonas fluorescens strains XJ3,
XS18, or LRS12 are presented in Table 5 below. Only three bacterial
isolates are chosen for field studies; Pseudomonas fluorescens
strains XJ3, XS18, and LRS12.
TABLE-US-00005 TABLE 5 Suppression of various plant species grown
in natural, non-autoclaved soil in growth chambers after
application of three Pseudomonas fluorescens strains: either XJ3,
XS18, or LRS12. XJ3 XS18 LRS12 Plant Growth in Soil/Growth Chamber
Common Name Latin Name % Suppression MONCOTYLEDONS Annual bluegrass
Poa annua 96 97 91 Bahiagrass Paspalum notatum 0 0 0 Barley Hordeum
vulgare L. 0 0 0 Basin wildrye Elymus linareus 0 0 0 Bentgrass -
Creeping Agrostis palustris 0 0 0 Bermudagrass Cynodon dactylon 0 0
0 Birdseye bluegrama Bouteloua gracilis 0 0 0 Bluebunch wheatgrass
Agropyron spicatum 0 0 0 Bluegrass - Kentucky Poa pratensis 0 0 0
Bottlebrush squirreltail Elymus elymoides 0 0 0 Buffalograss
Buchloe dactyloides 0 0 0 Carpetgrass Axonopus fissifolius 0 0 0
Cody buffalograss Buchloe dactyloides 0 0 0 Columbia needlegrass
Stipa columbian 0 0 0 Corn Zea mays L. 0 0 0 Dichrondra Dichondra
micrantha 0 0 0 Downy brome Bromus tectorum L. 0 10 0 Eastern
Gamagrass Tripsacum dactyloides 0 0 0 Fescue - Creeping Red Festuca
rubra var. rubra 0 0 0 Fescue - Tall Festuca arundinacea 0 0 0
Idaho fescue Festuca idahoensis 0 0 0 Kikuyugrass Pennisetum
clandestinum 0 0 0 Orchardgrass Dactylis glomerata 0 0 0 Ryegrass -
Perennial Lolium perenne 0 0 0 Saint Augustine grass Stenotaphrum 0
0 0 secundatum Smooth bromegrass Bromus inermis 0 0 0 Wheatgrass -
Agropyron spicatum 0 0 0 Bluebunch Wheatgrass - Crested Agropyron
cristatum 0 0 0 Zoysiagrass - Japonica Zoysia japonica 0 0 0
DICOTYLEDONS Alfalfa Medicago sativa L. 0 0 0 Apple Malus Mill. 0 0
0 Beans Phaseolus L. 0 0 0 Big sagebrush Artemisia tridentata 0 0 0
Camelina Camelina sativa L. 0 0 0 Canola Brassica napus L. 0 0 0
Celery Apium spp. L. 0 0 0 Chick peas Cicer arietinum 0 0 0 Clover
Trifolium L. 0 0 0 Common vetch Vicia sativa L. 0 0 0 Cucumber
Cucumis sativus L. 0 0 0 Faba bean Vicia faba 0 0 0 Flax Linum
narbonense L. 0 0 0 Jointvetch Aeschynomene sp. L. 0 0 0 Lentil
Lens culinaris Medik. 0 0 0 Lettuce Lactuca sativa L. 0 0 0 Mint
Mentha L. 0 0 0 Northern bedstraw Galium boreale L. 0 0 0 Pea Pisum
sativum L. 0 0 0 Peanuts Arachis L. 0 0 0 Pepper Capsicum L. 0 0 0
Phlox Phlox L. 0 0 0 Potato Solanum 0 0 0 Rannucula Ranunculus 0 0
0 Rapeseed Brassica rapa L. 0 0 0 Safflower Carthamus tinctorius L.
0 0 0 Soybeans Glycine max L. Merr. 0 0 0 Squash Cucurbita spp. 0 0
0 Sugar beets Beta vulgaris 0 0 0 Sunflower Helianthus L. 0 0 0
Tomato Solanum 0 0 0 Vetch Vicia spp. 0 0 0
[0059] To evaluate the bacterial treatment on economically
important turfgrass species, on economically important crop plants
and on desirable native plant species, root and shoot dry weight of
the plants treated with the bacteria are compared with root and
shoot dry weight of the control plants with no bacterial treatment.
Any bacterium that did not deleteriously affect the treated
turfgrass or for which the turfgrass plants had less than
approximately 10% reduction in root growth (root dry weight) or
shoot growth (shoot dry weight) compared to the control plants, or
that caused at least approximately 30% reduction in annual
bluegrass shoot dry weight and/or root dry weight, are considered
being acceptable for testing in the next stage of testing in field
plots. See Table 5 for the results for Pseudomonas fluorescens
strains XJ3, XS18, or LRS12.
Example 4
Screening Bacterial Strains in the Test Plots
[0060] For the field test, annual bluegrass plants are grown in
plots that are substantially free of other weeds so that
variability resulting from the presence of other weeds is reduced.
Field test plots (treatment plots) are approximately 2 meters to
approximately 3 meters wide by approximately 9 meters long.
Negative control plots are within about 6 meters of the treatment
plot so that the treatment plot and non-treatment plot (negative
control plot) have similar soil conditions. A plot can range in
size from approximately 10 m.sup.2 to approximately 50 m.sup.2.
[0061] The plots seeded with annual bluegrass have about 50 seeds
m.sup.-2 to about 75 seeds m.sup.-2. Individual bacterial strains
(identified in Example 3 supra) are applied once to the soil as a
spray treatment. The spray treatment can be applied at any time
between seeding to 4 weeks after seeding. The bacteria are applied
to moist soil to help insure survival of the microorganisms. For
the spray treatment, each test bacteria, contained in distilled
water, are sprayed on to the soil to provide a concentration of
about 10.sup.7 cfu to about 10.sup.8 cfu of the bacteria m.sup.-2.
Negative control plots of annual bluegrass are treated identical to
the test plot of annual bluegrass except that no bacteria are
applied. To have statistical significance, each microorganism
treatment is replicated a minimum of three times.
[0062] Plots seeded with turfgrass (about 100 seeds m.sup.-2 to
about 150 seeds m.sup.-2) are seeded beside or with the annual
bluegrass plots. The bacteria mixed with distilled water are
sprayed on the test plots' soil to provide a concentration of about
10.sup.8 cfu of the bacteria m.sup.-2. The treatment is applied to
moist soil at any time between seeding to 4 weeks after seeding.
The negative control plots of turfgrass seed are treated identical
to the test plot of turfgrass seed except that no bacteria are
applied. To have statistical significance, each microorganism
treatment is replicated a minimum of three times.
[0063] To assess the effect of the bacterial treatment on the
inhibition of annual bluegrass in the field, after adequate growth
has occurred (e.g., about the 3- to 5-leaf-stage), at least one of
the following is obtained: root dry weight, shoot dry weight, or
stand (numbers of plant m.sup.-2). Root dry weight and shoot dry
weight are obtained as described in Example 3 (supra). Bacterial
strains that cause the treated annual bluegrass plants to average a
reduction in root growth (root dry weight), reduction in shoot
growth (shoot dry weight), or reduction in stand (numbers of plant
m.sup.-2) of at least 20% when compared to negative control annual
bluegrass plants are considered inhibitory to annual bluegrass in
the field test. To assess the effect of the bacterial treatment on
turfgrass, at least one of the following is obtained: root dry
weight, shoot dry weight, or stand. Root dry weight and shoot dry
weight are obtained as described in Example 3 (supra). Those
bacterial strains that cause turfgrass to average less than 10%
reduction in root growth (root dry weight) or shoot growth (shoot
dry weight) or stand (numbers of plant m.sup.-2) compared to
negative control crop plant plots are defined as not deleteriously
affecting the crop plants.
[0064] Of the approximately 200 bacteria isolates tested for
inhibitory effect against annual bluegrass in the test field in
this Example 4, only 20 bacteria isolates (0.1% of the original
number of bacteria isolated) have inhibitory effect on annual
bluegrass and no deleterious effect on turfgrass.
Example 5
Field Testing
[0065] The twenty most promising bacteria strains from the growth
chamber test and the field test (Examples 3, 4, respectively) are
then tested in the field near Pullman, Wash. Poa annua L. seeds
collected from Pullman are planted by hand in furrows. Because Poa
annua L. seeds are small, the seeds are mixed with sterile sand and
then scattered in the row to obtain a seeding of approximately one
seed per inch. Winter wheat cultivar `Madsen` is also planted in
rows at approximately one seed per inch. Each bacterial inoculum is
applied directly to seeds in the furrow at 20 mL/m to obtain a
concentration of approximately 10.sup.8 cfu per m.sup.2 that is
sufficient to obtain root colonization and plant inhibition.
(Kennedy, A. C., et al. 1991. Rhizobacteria suppressive to the weed
downy brome. Soil Sci. Soc. Amer. J. 55:722-727.) Control plots
having the same concentration of annual bluegrass seeds or winter
wheat cultivar `Madsen` seeds are treated with 20 mL of sterile
deionized water. The average annual precipitation at Pullman is
450-600 mm, and the soil is a Palouse silt loam. Rows are 1 m long
with 0.30 m between each row. Of the twenty strains of bacteria
that had favorable results in Example 4 above, eleven strains had
unfavorable results in this field test.
[0066] Of the nine bacterial strains with favorable results, three
bacterial strains, P. fluorescens XS18 (NRRL B-50852), P.
fluorescens XJ3 (NRRL B-50851), and P. fluorescens LRS12 (NRRL
B-50853) (2013 only) are subjected to additional testing. These
three strains are randomly applied to half of the rows in the fall
of each year. In early April 2012 or August 2013, Poa annua L.
plants in 7 rows are counted to obtain plot stands. The
above-ground shoot mass and below ground root mass of Poa annua L.
and winter wheat cultivar `Madsen` are determined using the methods
described supra. Bacterial strains are considered inhibitory to Poa
annua L. in this field test if Poa annua L. in the plots treated
with the bacteria averaged at least a 20% reduction in stand
(numbers of plant m.sup.-2); root growth (root dry weight of 0.25
m.sup.3 of soil); and shoot growth (shoot dry weight per 0.25
m.sup.2) when compared to control plots. Bacterial strains that
caused less than a 10% reduction in Kentucky bluegrass stand
(numbers of plant m.sup.-2); root growth (root dry weight of 0.25
m.sup.3 of soil); and shoot growth (shoot dry weight per 0.25
m.sup.2) compared to control winter wheat are considered as not
injurious to the crop.
[0067] All three bacteria strains tested in the field inhibit Poa
annua L. root growth more than 39% (Table 6). P. fluorescens strain
XS18, applied to the furrow in which Poa annua L. seed was planted
in the fall of 2011, inhibit shoot mass by 44% and root mass by 74%
at harvest in June of 2012. P. fluorescens strain XS18 also inhibit
Poa annua L. shoot mass by 78% and root mass by 79% when the
bacteria are applied to seed in the fall of 2012 and harvested in
August of 2013. When P. fluorescens strain XJ3 is applied to Poa
annua L. seed planted in the fall of 2011 shoot mass is inhibited
by 51% and root mass by 50%. P. fluorescens strain XJ3 inhibit Poa
annua L. shoot mass by 69% and root mass by 75%. P. fluorescens
strain LRS12 is applied to the furrow containing Poa annua L. seed
only in the fall of 2012. P. fluorescens strain LRS12 inhibit Poa
annua L. the least amount; shoot mass is inhibited by 42%, and root
mass is inhibited by 39%. These three bacteria isolates tested in
the field do not inhibit the growth of Kentucky bluegrass cultivar
`NuGlade" and winter wheat cultivar `Madsen` in the field trials
(data not shown).
TABLE-US-00006 TABLE 6 Field test data from application of
weed-suppressive bacteria to annual blue grass seeded in meter
rows. Shoot Shoot Shoot % Root Root Root % Control Bacteria
Inhibition Control Bacteria Inhibition Strain Year g m.sup.-1 g
m.sup.-1 % g m.sup.-1 g m.sup.-1 % P.f. strain XJ3 2011-2012 3.23
1.58 51 4.27 2.13 50 NRRL B-50851 2012-2013 5.24 1.65 69 7.92 1.98
75 P.f. strain XS18 2011-2012 3.81 2.12 44 5.24 1.34 74 NRRL
B-50852 2012-2013 4.6 1.01 78 6.77 1.40 79 P.f. strain LRS12
2011-2012 nd nd nd nd NRRL B-50853 2012-2013 2.32 1.34 42 4.42 2.71
39
Example 6
Rice as an Agriculturally Acceptable Carrier
[0068] One can coat rice (an agriculturally acceptable carrier)
with the one or more bacteria described herein to form the
biocontrol agent of this invention. One can broadcast the
bacteria-coated rice in the fall of the year into moisture, and the
rice will distribute the bacterium throughout the surface of the
soil. Alternatively, the bacteria-coated rice can be broadcast at
any time prior to rain or application of water (irrigate) to the
area where the coated rice are broadcasted.
[0069] To generate this bacteria-coated rice biocontrol agent, P.
fluorescens strain XJ3 (NRRL B-50851) is grown in PMS broth to
10.sup.8 cells/L. Then, 10 oz. media per 60 pounds of rice are
mixed together to form a slurry and are rotated in a drum at
25.degree. C. until the rice absorbs the media. After the rice is
dry, the bacteria-coated rice is broadcast distributed onto the
land to be treated at 30 pounds per acre. When the P. fluorescens
strain XJ3 (NRRL B-50851)-coated rice is applied to a field test
per Example 5, above, similar results are expected as described in
Table 6.
[0070] Alternatively, after culturing P. fluorescens strain XJ3
(NRRL B-50851) in PMS media to a density of 10.sup.8 cells/L, the
media is centrifuged to pellet the bacteria and the liquid is
decanted. The bacteria are freeze-dried to obtain a density of
2.times.10.sup.8 cells/g. Then, 2 g freeze-dried bacteria are mixed
with 30 pounds per acre of rice and water to form a slurry. This
slurry is rotated in a drum until all liquid is absorbed by the
rice. After the bacteria-coated rice are dry, one can direct drill
or broadcast the bacteria-coated rice at 30 pounds per acre.
Example 7
Pellets of Wheat Flour and P. fluorescens Strain XJ3 (NRRL
B-50851)
[0071] One can mix wheat flour (an agriculturally acceptable
carrier) with finely ground plant residue and add one or more
bacteria described herein, extrude the mixture of various
diameters, and cut the strings of bacteria into different lengths
to form the biocontrol agent of this invention. One can broadcast
the bacteria-embedded pellets or prills in the fall of the year
into moisture, and the pellets or prills will distribute the
bacterium throughout the surface of the soil. Alternatively, the
bacteria-embedded pellets or prills can be broadcast at any time
prior to rain or application of water (irrigate) to the area where
the pellets or prills are broadcasted.
[0072] To generate these pellets or prills embedded with the
biocontrol agent, P. fluorescens strain XJ3 (NRRL B-50851) is grown
in PMS broth to 10.sup.8 cells/mL. One can mix 454 g of a matrix,
such as wheat or plant flour together with 200 to 300 g of a
coarser matrix of finely ground plant residue or other materials.
To the flour/residue mixture, 250 to 350 mL of bacterial broth is
added to bring the material into a stiff ball. The amount of broth
needed will vary with the absorptive capacity of the materials. The
flour, residue, bacteria mixture is extruded through holes of
various diameter and cut to the width needed for the given
application. After the pellets are dry, the bacterial pellets are
broadcast distributed onto the land so that 10.sup.14 cells are
distributed per acre.
[0073] Alternately, one gram of freeze-dried bacteria can be added
to the dry mixture and 250 to 350 mL of water can be added to the
flour, residue, bacteria mixture. After culturing P. fluorescens
strain XJ3 (NRRL B-50851) in KB media to a density of 10.sup.8
cells/L, the media is centrifuged to pellet the bacteria and the
liquid is decanted. The bacteria are freeze-dried to obtain a
density of 4.times.10.sup.13 cells/g. Then, 2 g freeze-dried
bacteria are mixed with 454 g of flour/residue mixture and 250 to
350 mL of water is added to the flour, residue, and bacteria
mixture. The material is mixed to bring the material into a stiff
ball. The flour, residue, and bacteria mixture is extruded through
holes of various diameter and cut to the width needed for the given
application. After the bacterial embedded pellets are dry, one can
broadcast the pellets to obtain 10.sup.14 cells per acre.
[0074] The foregoing detailed description and certain
representative embodiments and details of the invention have been
presented for purposes of illustration and description of the
invention. It is not intended to be exhaustive or to limit the
invention to the precise forms disclosed. It will be apparent to
practitioners skilled in the art that modifications and variations
may be made therein without departing from the scope of the
invention. All references cited herein are incorporated by
reference.
* * * * *