U.S. patent application number 10/005412 was filed with the patent office on 2002-10-03 for bacterial inoculants for enhancing plant growth.
Invention is credited to Chelius, Marisa K., Kaeppler, Shawn M., Triplett, Eric W..
Application Number | 20020142917 10/005412 |
Document ID | / |
Family ID | 22950632 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142917 |
Kind Code |
A1 |
Triplett, Eric W. ; et
al. |
October 3, 2002 |
Bacterial inoculants for enhancing plant growth
Abstract
A biological inoculant for enhancing the growth of plants is
disclosed. The inoculant includes the bacterial strains
Herbaspirillum seropedicae 2A, Pantoea agglomerans P101, Pantoea
agglomerans P102, Klebsiella pneumoniae 342, Klebsiella pneumoniae
zmvsy, Herbaspirillum seropedicae Z152, Gluconacetobacter
diazotrophicus PA15, with or without a carrier. The inoculant also
includes strains of the bacterium Pantoea agglomerans and K.
pneumoniae which are able to enhance the growth of cereal grasses.
Also disclosed are the novel bacterial strains Herbaspirillum
seropedicae 2A, Pantoea agglomerans P101 and P102, and Klebsiella
pneumoniae 342 and zmvsy.
Inventors: |
Triplett, Eric W.;
(Middleton, WI) ; Kaeppler, Shawn M.; (Oregan,
WI) ; Chelius, Marisa K.; (Greeley, CO) |
Correspondence
Address: |
David M. Kettner
Quarles & Brady LLP
1 South Pinckney Street
P O Box 2113
Madison
WI
53701-2113
US
|
Family ID: |
22950632 |
Appl. No.: |
10/005412 |
Filed: |
December 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60251137 |
Dec 4, 2000 |
|
|
|
Current U.S.
Class: |
504/117 ;
435/252.1; 504/100 |
Current CPC
Class: |
C12R 2001/22 20210501;
C12N 1/00 20130101; A01N 65/44 20130101; C12R 2001/00 20210501;
A01N 63/20 20200101; C12N 1/205 20210501; A01N 43/40 20130101; A01N
65/20 20130101; A01N 43/40 20130101; A01N 2300/00 20130101 |
Class at
Publication: |
504/117 ;
504/100; 435/252.1 |
International
Class: |
A01N 063/00; A01N
025/26; C12N 001/12; C12N 001/20 |
Goverment Interests
[0002] The disclosed invention was made with United States
government support awarded under contract DE-FC05-92OR22072 by the
Unites States Department of Energy. The United States Government
has certain right to this invention.
Claims
We claim:
1. An inoculum for application to plants, said inoculum comprising
a carrier and an effective quantity of bacteria, the bacteria
selected from Herbaspirillum seropedicae 2A (ATCC No. PTA-2742),
Pantoea agglomerans P101 (ATCC No. PTA 2744), Pantoea agglomerans
P102 (ATCC No. PTA 2740), Klebsiella pneumoniae 342 (ATCC No.
PTA-2743), Klebsiella pneumoniae zmvsy (ATCC No. PTA-2741),
Herbaspirillum seropedicae Z152 (ATCC No. 35894), Gluconacetobacter
diazotrophicus PA15 (ATCC No. 49037) and mutant strains derived
therefrom, said mutant strains able to enhance the growth of
plants.
2. An inoculum for application to plants, the inoculum comprising a
carrier and an effective quantity of a Klebsiella pneumoniae
bacterial strain.
3. An inoculum for application to plants other than legume plants,
the inoculum comprising a carrier and an effective quantity of a
Pantoea agglomerans bacterial strain.
4. A biologically pure bacterial culture wherein the bacteria is
selected from Herbaspirillum seropedicae 2A (ATCC No. PTA-2742),
Pantoea agglomerans P101 (ATCC No. PTA 2744), Pantoea agglomerans
P102 (ATCC No. PTA 2740), Klebsiella pneumoniae 342 (ATCC No.
PTA-2743), and Klebsiella pneumoniae zmvsy (ATCC No. PTA-2741).
5. A biologically pure culture of a mutant strain, the mutant
strain derived from either Herbaspirillum seropedicae 2A (ATCC No.
PTA-2742), Pantoea agglomerans P101 (ATCC No. PTA 2744), Pantoea
agglomerans P102 (ATCC No. PTA 2740), Klebsiella pneumoniae 342
(ATCC No. PTA-2743), or Klebsiella pneumoniae zmvsy (ATCC No.
PTA-2741), wherein the mutant strain retains the ability to enhance
the growth of plants.
6. A method for enhancing the growth of a plant, the method
comprising the step of placing in the vicinity of the plant an
effective quantity of bacteria, the bacteria selected from
Herbaspirillum seropedicae 2A (ATCC No. PTA-2742), Pantoea
agglomerans P101 (ATCC No. PTA 2744), Pantoea agglomerans P102
(ATCC No. PTA 2740), Klebsiella pneumoniae 342 (ATCC No. PTA-2743),
Klebsiella pneumoniae zmvsy (ATCC No. PTA-2741), Herbaspirillum
seropedicae Z152 (ATCC No. 35894), Gluconacetobacter diazotrophicus
PA15 (ATCC No. 49037) and mutant strains derived therefrom, said
mutant strains able to enhance the growth of plants.
7. The method of claim 6 wherein the plant is either a cereal grass
plant or a legume plant.
8. A method for enhancing the growth of a plant, the method
comprising the step of placing in the vicinity of the plant an
effective quantity of a Klebsiella pneumoniae bacterial strain.
9. The method of claim 8 wherein the plant is either a cereal grass
plant or a legume plant.
10. A method for enhancing the growth of a plant other than a
legume plant, the method comprising the step of placing in the
vicinity of the plant an effective quantity of a Pantoea
agglomerans bacterial strain.
11. A seed from a cereal grass plant coated with an effective
quantity of bacteria to enhance growth, the bacteria selected from
Herbaspirillum seropedicae 2A (ATCC No. PTA-2742), Pantoea
agglomerans P101 (ATCC No. PTA 2744), Pantoea agglomerans P102
(ATCC No. PTA 2740), Klebsiella pneumoniae 342 (ATCC No. PTA-2743),
Klebsiella pneumoniae zmvsy (ATCC No. PTA-2741), Herbaspirillum
seropedicae Z152 (ATCC No. 35894), Gluconacetobacter diazotrophicus
PA15 (ATCC No. 49037) and mutant strains derived therefrom.
12. The seed of claim 11 wherein the coating also includes a
carrier for the bacteria.
13. A method for identifying Pantoea agglomerans and Klebsiella
pneumoniae bacterial strains having the ability to enhance the
growth of a cereal grass plant, said method comprising the steps
of: isolating a bacterial isolate wherein the isolate is either a
Pantoea agglomerans bacterial strain or a Klebsiella pneumoniae
bacterial strain; planting a cereal grass seed or a cereal grass
seedling with said test material in a planting medium; growing said
planted cereal grass seed or said cereal grass seedling for a time
sufficient to allow for a growing seedling to develop and be
evaluated for growth enhancement; and evaluating the growing
seedling for evidence of enhanced growth when compared to a growing
seedling grown in the absence of the test material.
14. An inoculum for application to plants, the inoculum comprising
a carrier and an effective quantity of bacteria wherein the
bacteria is identified according to the method of claim 13.
15. A method for enhancing the growth of a cereal grass plant, said
method comprising the steps of placing in the vicinity of the plant
an effective quantity of bacteria wherein the bacteria is
identified to enhance the growth of a cereal grass plant according
to the method of claim 13.
16. A seed from a cereal grass plant coated with an effective
quantity of the bacteria identified according to the method of
claim 13.
17. The seed of claim 16 wherein the coating also includes a
carrier for the bacteria.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional.
Application No. 60/251,137, filed Dec. 4, 2001.
BACKGROUND OF THE INVENTION
[0003] Significant research has been conducted in recent years on
the use of biological control agents to increase agricultural
productivity and efficiency. These studies have shown that various
microorganisms are able to suppress plant pathogens or supplement
plant growth, thus offering an attractive alternative to chemical
pesticides which are less favored because of their effect on human
health and environmental quality.
[0004] Several screening programs have been used to isolate
biological control agents effective in facilitating plant growth or
combating pests in the laboratory or in the field. An example of
one such biological control agent is Bacillus thuringiensis, which
has been shown to produce toxic proteins lethal to certain insects.
Another example is the bacterial strain Bacillus cereus UW85 (ATCC
No. 53522), which has been found to protect alfalfa seedlings from
damping off caused by Phytophthora medicaginis, tobacco seedlings
from Phytophthora nicotianae, cucumber fruits from rot caused by
Pythium aphanidermatum, and peanuts from Sclerotinia minor (See
U.S. Pat. No. 4,877,738). In addition, Bacillus cereus AS4-12 (ATCC
No. 55609) has been found to increase the efficacy in fostering the
growth and establishment of alfalfa plants in the upper mid-western
United States (See U.S. Pat. No. 5,552,138).
[0005] Earlier experiments have shown that strains of Pantoea
agglomerans may also be able to increase the yield of legumes and
inhibit the growth of phytopathogenic fungi. Hoflich and Ruppel,
"Growth stimulation of pea after inoculation with associative
bacteria," Microbiol. Res., 149:99-104 (1994). P. agglomerans
(formerly Enterobacter agglomerans) is a Gram-negative
Enterobacterium often found associated with plants, water, soil, or
foodstuffs. P. agglomerans is also a diazotroph, and able to fix
nitrogen in both pure culture and in association with wheat.
Merbach et al., "Dinitrogen fixation of microbe-plant associations
as affected by nitrate and ammonium supply," Isotopes Environ.
Health Stud., 34:67-73 (1998). It has also been reported to produce
two auxins and two cytokines in pure culture. Scholz et al.,
"Development of DAS-ELISA for some selected bacteria from the
rhizosphere," Zentralbl. Mikrobiol. 146:197-207 (1991);
Scholz-Seidel C. and Ruppel S., Nitrogenase and phytohormone
activities of Pantoea agglomerans in Culture and their reflection
in combination with wheat plants," Zentralbl. Mikrobiol.
147:319-328 (1992). Even with these studies, however, little is
known about the interaction between P. agglomerans and cereal
grasses, and whether P. agglomerans may serve as an effective
biocontrol agent.
[0006] Klebsiella pneumoniae is also a member of the family
Enterobacteriaceae and a known nitrogen fixing bacterium, i.e. able
to convert atmospheric nitrogen into ammonium. K. pneumoniae is a
free-living soil bacterium and unlike other nitrogen-fixing
bacteria, such as Rhizobium, K. pneumoniae does not participate in
symbiotic interactions with leguminous plants. K. pneumoniae has
also not yet been shown to be effective in enhancing the growth of
cereal grasses.
[0007] The mechanisms by which biological control agents are able
to increase agricultural productivity and efficiency are diverse,
and will vary depending upon the unique characteristics of each
particular agent. It is believed, for example, that certain
bacteria are able to control root rot in plants by competing with
fungi for space on the surface of the plant root. It is also
believed that competition between various bacterial strains in a
plant's native microflora may stimulate root growth and increase
the uptake of mineral nutrients and water to enhance plant yield.
Alternatively, toxins produced by certain bacterial species are
believed to facilitate plant growth by controlling bacterial
species pathogenic to the plant. Bacterially produced antibiotics
are an example of such toxins.
[0008] Some have suggested that bacterial strains other than those
presently identified may also prove to be beneficial to crop
plants. In particular, it is quite possible that some of these
bacterial strains may be particularly helpful in cultivating
various field crops as a result of relationships formed between
plant and bacteria. The present invention discloses several such
bacterial strains.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention is summarized as a method for
enhancing the growth of a plant using an inoculum comprising an
effective quantity of bacteria selected from the group consisting
of Klebsiella pneumoniae 342, Klebsiella pneumoniae zmvsy, Pantoea
agglomerans P101, Pantoea agglomerans P102, Herbaspirillum
seropedicae 2A, Herbaspirillum seropedicae Z 152, Gluconacetobacter
diazotrophicus PA15, and any mutations thereof which retain the
ability to enhance plant growth. The present invention also
includes the bacterial inoculant of the above method, and a plant
seed coated with the bacterial inoculant.
[0010] In addition, the present invention includes a method for
identifying strains of Pantoea agglomerans and Klebsiella
pneumoniae which have the ability to enhance the growth of cereal
grasses. Also included is an inoculum comprising the bacterial
strains identified by said method and a method for enhancing the
growth of cereal grasses using said bacterial strains. The present
invention also includes a plant seed coated with the inoculum.
[0011] The present invention is further characterized in that novel
bacterial strains capable of enhancing the growth of a plant have
been isolated from the environment. These strains include
Herbaspirillum seropedicae 2A (ATCC No. PTA-2742), Pantoea
agglomerans P101 (ATCC No. PTA 2744), Pantoea agglomerans P102
(ATCC No. PTA 2740), Klebsiella pneumoniae 342 (ATCC No. PTA-2743),
Klebsiella pneumoniae zmvsy (ATCC No. PTA-2741), and mutations
thereof which retain the ability to enhance the growth of
plants.
[0012] It is an object of the present invention to provide a
bacterial inoculant effective in facilitating the germination
and/or growth of plants.
[0013] It is another object of the present invention to provide a
biological agent capable of improving crop yield without additional
chemical agents.
[0014] Other objects, advantages and features of the present
invention will become apparent from the following specification
when taken in conjunction with the accompanying claims.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0015] None.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention is directed toward certain bacterial
strains and inoculants capable of enhancing the growth of plants.
The bacterial strains utilized in the present invention include
Herbaspirillum seropedicae 2A, Herbaspirillum seropedicae Z152,
Pantoea agglomerans P101, Pantoea agglomerans P102, Klebsiella
pneumoniae 342, Klebsiella pneumoniae zmvsy, Gluconacetobacter
diazotrophicus PA15, and any mutations thereof which retain the
ability to enhance the growth of plants. Also included are strains
of the bacterium P. agglomerans and K. pneumoniae which are able to
enhance the growth of cereal grasses as identified by the method
described below. As used herein, the above bacterial strains shall
sometimes be referred to collectively as "enhancing" bacteria.
[0017] Bacterial strains H. seropedicae 2A, P. agglomerans P101, P.
agglomerans P102, K. pneumoniae 342, and K. pneumoniae zmvsy are
believed to be new to the world, and were deposited with the
American Type Culture Collection ("ATCC"), Manassas, Va. 20110-2209
U.S.A., on Nov. 30, 2000. The bacterial strain H. seropedicae 2A
was isolated from a maize plant cultivated in Hancock, Wis. during
the summer of 1999, and has been given the ATCC Patent Deposit
Designation PTA-2742. P. agglomerans P101 and P. agglomerans P102
were isolated from switch grass plants collected from a remnant
prairie near Plover, Wis., and have been given the ATCC Patent
Deposit Designation PTA 2744 and PTA 2740, respectively. K.
pneumoniae 342 was isolated from a very nitrogen efficient line of
maize from Mexico cultured in a greenhouse in Madison, Wis., and
has been given the ATCC Patent Deposit Designation PTA-2743. K.
pneumoniae zmvsy was isolated in 1994 from a maize plant grown on a
farm in Madison, Wis., and has been given the ATCC Patent Deposit
Designation PTA-2741.
[0018] Bacterial strains H. seropedicae Z152 (ATCC No. 35894) and
G. diazotrophicus PA15 (ATCC No. 49037) were both previously known
and available from the ATCC, but never shown to enhance the growth
of cereal grasses. Both bacterial strains were isolated in the late
1980s from sugarcane in Brazil.
[0019] It is anticipated that certain mutants of the enhancing
bacteria may also enhance plant growth comparable to the
non-mutated forms set forth above. Mutants of the enhancing
bacteria may include both naturally occurring and artificially
induced mutants. Certain of these mutants will be found to enhance
cereal grasses or legumes using the plant enhancement assay
described below. Others mutants may be induced by subjecting the
enhancing bacteria to known mutagens, such as
N-methyl-nitrosoguanidine, using conventional methods. Similar
mutants have been made from useful Bacillus cereus strains such as
UW85 (ATCC No. 53522) and AS4-12 (ATCC No. 55609) as described in
U.S. Pat. Nos. 4,877,738 and 5,552,138, respectively, the
disclosure of which is hereby incorporated by reference.
[0020] The data set forth below in the Examples demonstrate that
other strains of the bacterium P. agglomerans and K. pneumoniae
will also be effective in enhancing the growth of cereal grasses
when used in accordance with the present invention. These strains
may be isolated using methods commonly known in the art for
isolating free-living organisms from the environment, and their
ability to enhance plant growth may be verified using any one of
many plant enhancement assays.
[0021] The following is a disclosure of one such plant enhancement
assay whereby a bacterial isolate, or the like, may be tested for
its ability to enhance the growth of a cereal grass next to which
it is placed. The seed or seedling of the cereal grass to be
enhanced is planted in a planting medium and watered with a
nutrient solution. The planting medium may be a damp soil,
vermiculite in water, an agar-based formulation, or any other
planting medium in which the seed or seedling will grow and
develop. The bacterial isolate is placed at least in the immediate
vicinity of the seed or seedling. Such placement shall be
understood to be in the "immediate vicinity" of the seed or
seedling if the bacterial isolate or any soluble exudate of a
bacterium being tested will be in actual contact with the
germinating seedling. After a time sufficient for seedling growth,
seedlings developing from the planted seed may be evaluated for
visual evidence of enhanced growth when compared to controls.
[0022] The bacterial inoculants of the present invention act
through an unknown mechanism to enhance plant growth. While the
mechanism by which these inoculants enhance plant growth is not
understood, it is possible that the mechanism involves an
antagonistic action by the enhancing bacterium on other organisms
which may inhibit and/or retard the germination and growth of the
plant seedling. The method of action may alternatively involve a
symbiotic relationship of some unknown type.
[0023] It is broadly intended within the scope of the present
invention that the bacterial inoculant of the present invention be
inoculated into the soil with plant seeds so that a culture of the
enhancing bacteria will develop in the root system of the plant as
it grows. To facilitate this co-culturing, it is preferred that the
inoculant, preferably diluted with a suitable extender or carrier,
either be applied to the seeds prior to planting or introduced into
the seed furrows when the seeds are planted. The bacterial
inoculant so delivered may be any viable bacteria culture capable
of successful propagation in the soil.
[0024] One advantageous technique is that the bacterial inoculant
be applied to the seeds through the use of a suitable coating
mechanism or binder prior to the seeds being sold into commerce for
planting. The process of coating seed with such an inoculum is
generally well known to those skilled in the art. For example, the
enhancing bacteria may be mixed with a porous, chemically inert
granular carrier as described by U.S. Pat. No. 4,875,921
(incorporated herein by reference).
[0025] Alternatively, the bacterial inoculant may be prepared with
or without a carrier and sold as a separate inoculant to be
inserted directly into the furrows into which the seed is planted.
The process for inserting such inoculants directly into the furrows
during seed planting is also generally well known in the art.
[0026] The enhancing bacteria may also be obtained in a
substantially pure culture. A "substantially pure" culture shall be
deemed to include a culture of bacteria containing no other
bacterial species in quantities sufficient to interfere with the
replication of the culture or be detected by normal bacteriological
techniques.
[0027] Whether the bacterial inoculants are coated directly on the
seed or inserted into the furrows, the enhancing bacteria is
preferably diluted with a suitable carrier or extender so as to
make the culture easier to handle and to provide a sufficient
quantity of material so as allow easy human handling. For example,
a peat based carrier may be used as described by Bosworth et al,
"Alfalfa yield response to inoculation with recombinant strains of
Rhizobium meliloti carrying an extra copy of dct and/or modified
nifA expression," Appl. Environ. Microbiol., 60:3815-3832 (1994),
incorporated herein by reference. In addition, it has been
discovered that perlite, vermiculite and charcoal materials are
suitable carrier substances. It is believed that many other
non-toxic and biologically inert substances of dried or granular
nature are also capable of serving as carriers for the enhancing
bacteria.
[0028] The density of inoculation of these bacterial cultures onto
seed or into the furrows should be sufficient to populate the
sub-soil region adjacent to the roots of the plant with viable
bacterial growth. An effective amount of bacterial inoculant should
be used. An effective amount is that amount sufficient to establish
sufficient bacterial growth so that the yield from the plant is
increased.
[0029] As stated above, the enhancing bacterial strains are
isolated from the roots of exceptionally vigorous plants grown
under conventional cultivation practices. Once isolation of the
strains was made, the bacterial culture had to be cultivated to
generate sufficient quantities of material for proper seed
treatment. It has been discovered here that the inoculation of
various cereal grasses with the enhancing bacterial strains results
in significantly improved growth of the cereal grass plants. As
will be appreciated by any person skilled in plant husbandry, the
rate of growth or improvement in growth of any given crop is
subject to many variables. It has been found here, however, that
the co-cultivation of the bacterial inoculant of the present
invention with cereal grasses is of significant advantage in at
least some typical field conditions. It is believed that this
co-cultivation technique will result generally in improved yield
and improved growth of cereal grasses in field applications.
[0030] It is also anticipated that the inoculation of various
legumes with the enhancing bacterial strains may result in
significantly improved growth of legume plants.
[0031] It will be appreciated by one skilled in the art that a
bacterial inoculant of the type described herein offers several
significant potential advantages over the chemical inoculants or
growth hormones or similar agents commonly used in agriculture
today. By the very nature of the bacterial inoculant, the enhancing
bacterial strains are self-sustaining in a continuous fashion once
they are introduced into the furrows with the plant seed.
Therefore, there is no need for retreatment of the plants during
the crop season. The bacterium grows in cultivation along with the
plants and should continue to exhibit its beneficial effect on the
plant throughout the agricultural season. This is in strong
contrast to chemical growth agents or fungicides which must be
retreated periodically to have a continuing effect on inhibition of
the fungus in question or to help improve the plant growth
throughout its life cycle. Since the bacterial inoculant of the
present invention can be inoculated onto the seeds using a dry or
wet formulation, the application of this technique is relatively
simple to the farmer since the seeds can be inoculated prior to
distribution. In this way, a significant economic advantage is
achievable.
[0032] The following non-limited examples are intended to
illustrate the present invention.
EXAMPLES
Example 1
[0033] The bacterial strains which make up the bacterial inoculants
of the present invention were isolated (or obtained from the ATCC
in the case of Gluconacetobacter diazotrophicus PA15 and
Herbaspirillum seropedicae Z152) and grown in culture on petri
dishes at 28.degree. C. to create crop inoculating propagules. The
culturing media used for Klebsiella pneumoniae 342, Klebsiella
pneumoniae zmvsy, Pantoea agglomerans P101 and Pantoea agglomerans
P102 was Luria-Bertani medium. The medium used for
Gluconacetobacter diazotrophicus PA15 was AcD medium, which
contains per liter: 0.64 g K.sub.2HPO.sub.4, 0.16 g
KH.sub.2PO.sub.4, 0.2 g MgSO.sub.4.7H.sub.2O, 0.2 g g
CaSO.sub.4.2H.sub.2O, 20 g sucrose, 2 mg NaMoO.sub.4, 3 mg
FeSO.sub.4, 2 g MES buffer, 1 g malic acid, and 0.1 g yeast
extract. This medium was adjusted to pH 6.5 prior to autoclaving.
Bacterial strains Herbaspirillum seropedicae 2A and Herbaspirillum
seropedicae Z152 were cultured on BSM medium as described by
Bergersen et al., "The Growth of Rhizobium in Synthetic Medium,"
Aust. J. Biol. Sci., 14:349-360 (1961). All media contained 15 g/L
of agar.
[0034] Wheat seeds from three cultivars (Trenton, Russ, and Stoa)
were inoculated with the bacterial propagules to determine each
bacterial strains' ability to affect plant growth. Seeds were first
surface sterilized using 70% ethanol for 30 seconds and 10% bleach
for 2 minutes, followed by 6 washes with sterile water. A cell
suspension of approximately 10.sup.8 cells/ml was then added to the
seeds for a few hours prior to planting to inoculate the wheat
seeds.
[0035] Inoculated and uninoculated wheat seeds were planted
approximately 1 cm deep in pots containing 2 liters of a 1:1
sand/vermicullite mixture. Plants were watered with a nutrient
solution described by Chelius et al., "Immunolocalization of
dinitrogenase reductase produced by Klebsiella pneumoniae in
association with Zea mays L.," Appl. Environ. Microbiol.,
66:783-787 (2000), containing all essential nutrients except
nitrogen. Four seeds were planted per pot which were thinned to two
plants per pot after two weeks.
[0036] After six weeks of growth, the above ground portions of the
plants were harvested, placed in a paper bag, and dried for one
week in driers at about 80.degree. C. After drying, the plants were
weighed. The values shown in Table 1 below were obtained by
comparing the average dry weight of the treated plants to the
average dry weight of untreated plants.
1TABLE 1 Changes in Dry-Shoot Weight of Wheat by Inoculation with
Bacterial Inoculants Wheat Ave. Dry Weight Dry Weight Cultivar
Bacterial Inoculant (mg/plant) Difference Trenton Uninoculated .257
-- K. pneumoniae 342 .789 .532 K. pneumoniae zmvsy .500 .243 H.
seropedicae 152 .455 .198 H. seropedicae 2A .440 .183 P.
agglomerans 101 .747 .490 P. agglomerans 102 .601 .344 G.
diazotrophicus PA15 .382 .125 Russ Uninoculated .402 -- K.
pneumoniae 342 .416 .014 K. pneumoniae zmvsy .467 .065 H.
seropedicae 152 .400 -.002 H. seropedicae 2A .454 .052 P.
agglomerans 101 .545 .143 P. agglomerans 102 .672 .270 G.
diazotrophicus PA15 .392 -.010 Stoa Uninoculated .269 -- K.
pneumoniae 342 .380 .111 K. pneumoniae zmvsy .253 -.016 H.
seropedicae 152 .336 .067 H. seropedicae 2A .213 -.056 P.
agglomerans 101 .429 .160 P. agglomerans 102 .472 .203 G.
diazotrophicus PA15 .299 .030
Example 2
[0037] Field tests of corn were performed at the University of
Wisconsin Hancock Agricultural Research Station (Hancock, Wis.) in
which maize seeds of different varieties were inoculated with
bacterial inoculants of the present invention.
[0038] The bacterial strains used were isolated (or obtained from
the ATCC in the case of Gluconacetobacter diazotrophicus PA15 and
Herbaspirillum seropedicae Z152) and grown in culture on petri
dishes at 28.degree. C. to create crop inoculating propagules. Most
strains were cultured on Bergersen's synthetic medium (Bergersen F.
J., "The growth of Rhizobium in synthetic media," Aust. J. Biol.
Sci., 14:349-360 (1961)). Bacterial strains Klebsiella pneumoniae
342 and Klebsiella pneumoniae zmvsy were cultured on Luria-Bertani
medium (Sambrook et al., "Molecular cloning: a laboratory manual,"
(Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.,
1989)). Gluconacetobacter diazotrophicus PA15 was cultured on AcD
medium (Burris R. H., "Comparative study of the response of
Azotobacter vinelandii and Acetobacter diazotrophicus to changes in
pH," Protoplasma, 183:62-66 (1994)).
[0039] Maize seeds were inoculated prior to planting by coating 1
kg of seeds with a 125 mL bacterial suspension in 8 grams of peat
(0.566 gCaCO.sub.3, 0.288 g charcoal, 10 mL 40% Vitalive (Research
Seeds Inc., St. Joseph, Mo., USA) in a plastic bag. The cell
suspension contained approximately 8.times.10.sup.8 cells per mL to
ensure an inoculum density of 10.sup.8 cells per seed. Cell number
per seed was verified after inoculation by suspending seeds in
water and plating various dilutions on the appropriate medium. The
seeds included seeds from maize inbred lines B73 and Mo17; the
hybrid B73.times.Mo17; two lines with a significant teosinte
background, 1/4 teosinte background, and 1/2 teosinte background;
and various hybrid lines from Pioneer.
[0040] Seeds were planted within 48 hours of inoculation in rows
thirty feet in length, with 30 seeds planted per row. Rows were
approximately 36 inches apart. Weeds were controlled chemically by
standard agricultural practices for the region. Fertilizer was
applied prior to planting at rates of 17.2 g m.sup.-2
P.sub.2O.sub.5 and 51.5 g m.sup.-2 K.sub.2O. Where nitrogen was
applied, it was applied at a rate of 224 kg NH.sub.4NO.sub.3
ha.sup.-1 prior to planting. An uninoculated control was included
with each maize line.
[0041] After the growing season, the treated corn was harvested and
grain weight and moisture measured for each plot. Yield was
determined as grain yield in t ha.sup.-1, standardized to 15.5%
moisture. Grain was harvested with a combine equipped with a
HarvestMaster data logger (HarvestMaster, Inc., Logan, Utah, USA)
for the determination of weight and moisture levels of the grain
from each plot. The values shown in Table 2 below illustrate the
increased yield per acre (or hectare), after correcting for
moisture levels. Grain yield is expressed in metric t ha.sup.-1.
Only those combinations where statistically significant increases
were obtained are listed.
2TABLE 2 Yield Increase of Corn by Inoculation with Bacterial
Inoculants N Yield % Yield Year Corn Variety Bacterial Inoculant
(+/-) (t ha.sup.-1) Increase 1998 Pioneer 3905 H. Seropedicae Z152
+ 10.6 12.0 Pioneer 3905 G. diazotrophicus PA15 + 9.96 5.4 Pioneer
3905 K. pneumoniae zmvsy + 10.8 13.9 B73 .times. Mo17 H.
Seropedicae Z152 + 8.67 14.4 B73 .times. Mo17 G. diazotrophicus
PA15 + 9.50 25.3 B73 .times. Mo17 K. pneumoniae zmvsy + 8.40 10.7
1/2 teosinte H. Seropedicae Z152 + 6.04 10.4 1/2 teosinte K.
pneumoniae zmvsy + 6.31 15.3 Pioneer 3921 G. diazotrophicus PA15 -
5.8 18.1 1999 Pioneer 3751 H. Seropedicae Z152 + 7.81 13.4 Pioneer
3751 K. pneumoniae zmvsy + 8.2 18.7 Pioneer 36H36 H. Seropedicae
Z152 + 7.17 8.0 Pioneer 36H36 K. pneumoniae zmvsy + 6.64 10.6
Pioneer 3921 H. Seropedicae Z152 - 2.88 18.2 Pioneer 3921 G.
diazotrophicus PA15 - 5.08 18.1 Pioneer 3921 P. agglomerans P101 -
2.6 13.1 Pioneer 3921 P. agglomerans P103 - 2.96 30.1 Pioneer 3905
K. pneumoniae 342 - 3.14 22.0 B73 .times. Mo17 P. agglomerans P102
- 1.53 19.9
Example 3
[0042] Additional field tests were performed at the University of
Wisconsin Arlington Agricultural Research Station (Arlington, Wis.)
in a manner similar to Example 2. After the growing season, the
treated corn was harvested and grain weight and moisture measured
for each plot. The values shown in Table 3 below illustrate the
increased yield per acre (or hectare), after correcting for
moisture levels. Grain yield is expressed in metric t ha.sup.-1.
Only those combinations where statistically significant increases
were obtained are listed.
3TABLE 3 Yield Increase of Corn by Inoculation with Bacterial
Inoculants N Yield % Yield Year Corn Variety Bacterial Inoculant
(+/-) (t ha.sup.-1) Increase 1998 Pioneer 3905 H. Seropedicae Z152
+ 8.96 19.5 Pioneer 3921 H. Seropedicae Z152 + 8.75 8.5 1/2
teosinte H. Seropedicae Z152 + 4.46 8.3 1999 Pioneer 36H36 H.
Seropedicae Z152 + 7.66 18.8 Pioneer 36H36 G. diazotrophicus PA15 +
7.88 23.4 Pioneer 36H36 K. pneumoniae zmvsy + 7.58 18.6 Pioneer
3921 H. Seropedicae Z152 + 6.77 13.2 Pioneer 3921 K. pneumoniae
zmvsy + 6.72 7.3 Pioneer 3905 H. Seropedicae Z152 + 6.78 12.6
Pioneer 3905 G. diazotrophicus PA15 + 6.92 14.4 Pioneer 3905 K.
pneumoniae 342 + 6.68 30.5 Pioneer 3905 P. agglomerans P103 + 6.04
18.0 Pioneer S1501 H. Seropedicae Z152 + 7.55 17.5 B73 .times. Mo17
P. agglomerans P103 + 6.27 9.4 2000 Pioneer 36H36 H. Seropedicae
Z152 + 15.51 7.3 Pioneer 36H36 P. agglomerans P103 + 13.56 17.8
Pioneer 3921 K. pneumoniae zmvsy + 15.9 20.0 Pioneer 3921 H.
Seropedicae Z152 + 14.86 12.1
Example 4
[0043] Additional field tests were performed at the University of
Wisconsin Lancaster Agricultural Research Station (Lancaster, Wis.)
in a manner similar to Examples 2 and 3. Plots in four other
states, Iowa, Indiana, Illinois and Nebraska were established with
the same design. All tests included the application of nitrogen as
described above. After the growing season, the treated corn was
harvested and grain weight and moisture measured for each plot. The
values shown in Table 4 below illustrate the increased yield per
acre (or hectare), after correcting for moisture levels. Grain
yield is expressed in metric t ha.sup.-1. Only those combinations
where statistically significant increases were obtained are
listed.
4TABLE 4 Yield Increase of Corn by Inoculation with Bacterial
Inoculants Yield % Yield Year Corn Variety Bacterial Inoculant
Location (t ha.sup.-1) Increase 2000 Pioneer 36H36 K. pneumoniae
342 Lancaster 16.39 25.8 Pioneer 36H36 H. Seropedicae Z152 Iowa
8.89 10.7 Pioneer 36H36 H. Seropedicae Z152 Nebraska 9.77 1.8
Pioneer 36H36 H. Seropedicae Z152 Illinois 11.07 6.4 Pioneer 36H36
G. diazotrophicus PA15 Iowa 9.25 15.2 Pioneer 36H36 G.
diazotrophicus PA15 Nebraska 9.83 2.4 Pioneer 36H36 G.
diazotrophicus PA15 Illinois 11.18 7.4 Pioneer 36H36 G.
diazotrophicus PA15 Indiana 8.4 8.1 Pioneer 36H36 P. agglomerans
P103 Iowa 8.94 11.3 Pioneer 36H36 P. agglomerans P103 Illinois
10.74 3.2 Pioneer 33A14 H. Seropedicae Z152 Iowa 10.77 1.6 Pioneer
33A14 H. Seropedicae Z152 Nebraska 10.24 4.7 Pioneer 33A14 H.
Seropedicae Z152 Illinois 12.82 19.6 Pioneer 33A14 H. Seropedicae
Z152 Indiana 10.03 8.1 Pioneer 33A14 G. diazotrophicus PA15 Iowa
11.09 1.6 Pioneer 33A14 G. diazotrophicus PA15 Nebraska 9.88 1.0
Pioneer 33A14 G. diazotrophicus PA15 Illinois 12.46 16.2 Pioneer
33A14 G. diazotrophicus PA15 Indiana 9.41 1.4 Pioneer 33A14 P.
agglomerans P103 Nebraska 10.17 4.0 Pioneer 33A14 P. agglomerans
P103 Illinois 12.63 17.8 Pioneer 33A14 P. agglomerans P103 Indiana
10.60 14.2
Example 5
[0044] Rice seeds were inoculated with the bacterial inoculants as
described in Example 1 above to determine each bacterial strains'
ability to affect the growth of rice plants. Seeds were first
surface sterilized using 70% ethanol for 30 seconds and 10% bleach
for 2 minutes, followed by 6 washes with sterile water. A cell
suspension of approximately 10.sup.8 cells/ml was then added to the
seeds to inoculate the seeds for a few hours prior to planting.
[0045] Inoculated and uninoculated rice seeds were planted
approximately 1 cm deep in pots containing 2 liters of a 1:1
sand/vermicullite mixture. Plants were watered with a nutrient
solution described by Chelius et al., "Immunolocalization of
dinitrogenase reductase produced by Klebsiella pneumoniae in
association with Zea mays L.," Appl. Environ. Microbiol.,
66:783-787 (2000), containing all essential nutrients except
nitrogen. Four seeds were planted per pot which were thinned to two
plants per pot after two weeks.
[0046] After six weeks of growth, the rice shoots of the plants
were harvested, placed in a paper bag, and dried for one week in
driers at about 80.degree. C. After drying, the plants were
weighed. The values shown in Table 5 below were obtained by
comparing the average dry weight of the treated plants to the
average dry weight of untreated plants.
5TABLE 5 Changes in Dry-Shoot Weight of Rice by Inoculation with
Bacterial Inoculants Ave. Dry Weight Dry Weight Bacterial Inoculant
(mg/plant) Difference Uninoculated 240.4 -- K. pneumoniae 342 247.4
7.0 P. agglomerans 101 376.5 136.1 P. agglomerans 102 273.3 32.9 G.
diazotrophicus PA15 248.2 7.8
* * * * *