U.S. patent application number 13/037880 was filed with the patent office on 2011-09-01 for compositions and methods for increasing biomass, iron concentration, and tolerance to pathogens in plants.
This patent application is currently assigned to University of Delaware. Invention is credited to Harsh Bais, Venkatachalam Lakshmannan, Darla Janine Sherrier.
Application Number | 20110212835 13/037880 |
Document ID | / |
Family ID | 44505584 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212835 |
Kind Code |
A1 |
Bais; Harsh ; et
al. |
September 1, 2011 |
COMPOSITIONS AND METHODS FOR INCREASING BIOMASS, IRON
CONCENTRATION, AND TOLERANCE TO PATHOGENS IN PLANTS
Abstract
Methods for producing greater biomass in a plant, increasing the
drought tolerance of a plant, producing a decreased lignin
concentration in a plant, producing a greater iron concentration in
a plant, or inhibiting fungal infection in a plant comprise
administering Bacillus subtilis FB17 to the plant, the seed of the
plant, or soil surrounding the plant or the seed in an amount
effective to produce greater biomass, increase the drought
tolerance, produce a decreased lignin concentration, produce a
greater iron concentration, or inhibit fungal infection in the
plant compared to an untreated plant, respectively. Agricultural
carriers and seed coatings comprising Bacillus subtilis FB17 are
provided. The biomass of a plant which has been administered
Bacillus subtilis FB17 can be converted to a biofuel or can be used
as a food crop or in other uses.
Inventors: |
Bais; Harsh; (Newark,
DE) ; Sherrier; Darla Janine; (Hockessin, DE)
; Lakshmannan; Venkatachalam; (Newark, DE) |
Assignee: |
University of Delaware
Newark
DE
|
Family ID: |
44505584 |
Appl. No.: |
13/037880 |
Filed: |
March 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61309134 |
Mar 1, 2010 |
|
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61414108 |
Nov 16, 2010 |
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61416039 |
Nov 22, 2010 |
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Current U.S.
Class: |
504/100 ;
424/93.462; 44/307; 504/117 |
Current CPC
Class: |
A01N 63/00 20130101;
A01N 63/10 20200101; A01N 63/00 20130101; A01N 25/00 20130101; A01N
2300/00 20130101 |
Class at
Publication: |
504/100 ;
504/117; 44/307; 424/93.462 |
International
Class: |
A01N 25/26 20060101
A01N025/26; A01N 63/00 20060101 A01N063/00; C10L 1/00 20060101
C10L001/00; A01P 3/00 20060101 A01P003/00 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] Research leading to the disclosed inventions was funded, in
part, by National Science Foundation (NSF) Grant No. 0923806 and
NSF Grant No. IOS-0814477, with technical support from the USDA
Experimental Field Station in Georgetown, Del. at the University of
Delaware. Accordingly, the United States Government may have
certain rights in this invention.
Claims
1. A method for producing a greater biomass in a plant comprising
administering Bacillus subtilis FB17 to the plant, the seed of the
plant, or soil surrounding the plant or the seed in an amount
effective to produce a greater biomass in the plant compared to an
untreated plant.
2. The method of claim 1, comprising administering the Bacillus
subtilis FB17 to the seed of the plant.
3. The method of claim 1, wherein the plant is selected from the
group consisting of a corn plant, a soybean plant, a rice plant,
and a tomato plant.
4. The method of claim 1, wherein the plant is a bioenergy crop
plant.
5. The method of claim 4, wherein the plant is Brachypodium
distachyon.
6. The method of claim 1, comprising administering the Bacillus
subtilis FB17 in an amount effective to produce a greater biomass
in the plant by about 5% to about 100% compared to an untreated
plant.
7. The method of claim 2, comprising administering the Bacillus
subtilis FB17 to the seed in an amount of about 1.times.10.sup.6
CFU/seed to about 1.times.10.sup.8 CFU/seed.
8. A method for producing a greater drought tolerance in a plant
comprising administering Bacillus subtilis FB17 to the plant, the
seed of the plant, or soil surrounding the plant or the seed in an
amount effective to produce a greater drought tolerance in the
plant compared to an untreated plant.
9. The method of claim 8, comprising administering the Bacillus
subtilis FB17 to the seed of the plant.
10. The method of claim 8, wherein the plant is selected from the
group consisting of a corn plant, a soybean plant, a rice plant,
and a tomato plant
11. A method for producing a decreased lignin concentration in a
plant comprising administering Bacillus subtilis FB17 to the plant,
the seed of the plant, or soil surrounding the plant or the seed in
an amount effective to produce a decreased lignin concentration in
the plant compared to an untreated plant.
12. The method of claim 11, comprising administering the Bacillus
subtilis FB17 to the seed of the plant.
13. The method of claim 11, wherein the plant is selected from the
group consisting of a corn plant, a soybean plant, a rice plant,
and a tomato plant.
14. The method of claim 12, comprising administering the Bacillus
subtilis FB17 to the seed in an amount of about 1.times.10.sup.6
CFU/seed to about 1.times.10.sup.8 CFU/seed.
15. A method for producing a biofuel, said method comprising
converting the biomass of the plant of claim 11 which has been
administered Bacillus subtilis FB17 to said biofuel.
16. A biofuel produced according to claim 15.
17. A method for producing a greater iron concentration in a plant
comprising administering Bacillus subtilis FB17 to the plant, the
seed of the plant, or soil surrounding the plant or the seed in an
amount effective to produce a greater iron concentration in the
plant compared to an untreated plant.
18. The method of claim 17, wherein the plant is a rice plant.
19. The method of claim 18, comprising administering the Bacillus
subtilis FB17 to the seed of the rice plant prior to planting.
20. The method of claim 18, comprising administering the Bacillus
subtilis FB17 in an amount effective to produce a greater iron
concentration in the rice plant by at least about 25% compared to
an untreated rice plant.
21. The method of claim 19, comprising administering the Bacillus
subtilis FB17 to the seed in an amount of about 1.times.10.sup.6
CFU/seed to about 1.times.10.sup.8 CFU/seed.
22. A method for inhibiting infection of a plant by a fungal
pathogen comprising administering Bacillus subtilis FB17 to the
plant, the seed of the plant, or soil surrounding the plant or the
seed in an amount effective to inhibit infection of the plant by
the fungal pathogen compared to an untreated plant.
23. The method of claim 22, wherein the plant is a rice plant and
the fungal pathogen is rice blast.
24. The method of claim 23, comprising administering the Bacillus
subtilis FB17 to the seed of the rice plant prior to planting.
25. The method of claim 24, comprising administering the Bacillus
subtilis FB17 to the seed of the rice plant in an amount of about
1.times.10.sup.6 CFU/seed to about 1.times.10.sup.8 CFU/seed.
26. The method of claim 23, wherein symptoms of the rice blast are
reduced by about 5% to about 100% compared to an untreated
plant.
27. An agricultural carrier comprising Bacillus subtilis FB17.
28. A plant seed coating comprising Bacillus subtilis FB17.
29. A plant seed comprising the coating of claim 28.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to application Ser. No.
61/309,134, filed Mar. 1, 2010, application Ser. No. 61/414,108,
filed Nov. 16, 2010, and application Ser. No. 61/416,039, filed
Nov. 22, 2010, which are incorporated herein by reference in their
entirety and for all purposes.
FIELD OF THE INVENTION
[0003] This invention relates generally to the use of plant growth
promoting rhizobacteria to enhance various characteristics of plant
growth, including increasing biomass, increasing drought tolerance,
decreasing lignin content, increasing seed germination, increasing
iron concentration, and increasing tolerance to pathogens. In
particular, embodiments of the present invention relate to the
administration of Bacillus subtilis FB17 to plants. The resulting
plants can be used in the production of biofuels, food, or for
other purposes.
BACKGROUND OF THE INVENTION
[0004] Food security has always been a top priority throughout the
world, and an escalating concern for the environmental impact of
crop production necessitates the development and use of novel
methods to enhance productivity while protecting the environment
Plant biologists develop and implement strategies for efficient
production of crop plants with the aim of ensuring the availability
of essential raw materials to the world's growing population.
However, the development of biofuel and renewable technologies adds
to this challenge since they have also become an increasingly
important priority. Therefore, there is an increased need for
improved approaches to enhance crop yield in diverse field
conditions.
[0005] The multitude of different geographic environments and
climates throughout the world present different types of challenges
in generating increased biomass and yield potential in crop plants.
Drought is a major factor which limits crop production globally.
Long-term drought or short-term drought in the growing season can
severely limit or even eliminate crop production. Changes in global
weather patterns have affected the frequency and intensity of
drought, even in prime cropping regions of the world.
[0006] Nutrient availability also limits crop production. Soil
augmentation with nutrients is costly and energy intensive, and
even when nutrients are available in sufficient quantities, crop
plants are sometimes inefficient at nutrient uptake. Poor uptake of
essential nutrients results in lower yields and food crops with
lower nutritional values. For example, rice (the seed of the
monocot plants Oryza sativa or Oryza glaberrima) is the most
important staple food for over two-thirds the world's population,
providing a significant proportion of the calories consumed. Since
rice is the main staple food for much of the global population,
producing rice with higher levels of iron can have a major impact
on reducing micronutrient malnutrition throughout the world, as
iron deficiency is one of the most widespread micronutrient
deficiencies in humans worldwide.
[0007] Pathogen stress also limits productivity. Plants must invest
energy to survive pathogen attack, and this diversion of energy
results in lower yields. Plants also modify their composition to
restrict disease progression, and these changes often make crop
processing more difficult. Further, some crop pathogens cannot be
limited effectively by genetic diversity, nor chemical control, and
have significant impact on crop production globally.
[0008] Rice blast (Magnaporthe grisea or Magnaporthe oryzae) is a
plant-pathogenic fungus that causes a serious disease affecting
rice. It causes economically significant crop losses annually,
contributing to an estimated 40% in crop yield. Rice blast destroys
enough rice to feed millions of people throughout the world every
growth season. Since rice is an important food staple for much of
the world, the effects of rice blast have a broad impact on human
health and the environment. Rice shortfalls contribute directly to
human starvation. The rice blast further contributes to crop loss
and requires the use of additional resources to compensate for
reduced yield. There continues to be a great need for strategies
that enhance various characteristics of plant growth in diverse
growing conditions, such as tolerance to drought stress, tolerance
to pathogen pressure, nutrient availability, and ultimately crop
yield, so that greater amounts of food with increased nutrition can
be available to the global population, and for other important
benefits, such as biofuel production.
SUMMARY OF THE INVENTION
[0009] An embodiment of the present invention provides a method for
producing a greater biomass in a plant comprising administering
Bacillus subtilis FB17 to the plant, plant seed, or soil
surrounding the plant or plant seed in an amount effective to
produce a greater biomass in the plant compared to an untreated
plant.
[0010] Another embodiment provides a method for increasing the
drought tolerance of a plant comprising administering Bacillus
subtilis FB17 to the plant, plant seed, or soil surrounding the
plant or plant seed in an amount effective to increase the drought
tolerance of the plant compared to an untreated plant.
[0011] Another embodiment provides a method for producing a
decreased lignin concentration in a plant comprising administering
Bacillus subtilis FB17 to the plant, plant seed, or soil
surrounding the plant or plant seed in an amount effective to
produce a decreased lignin concentration in the plant compared to
an untreated plant.
[0012] Another embodiment provides a method for increasing the seed
germination in plants comprising administering Bacillus subtilis
FB17 to the plants, plant seeds, or soil surrounding the plants or
plant seeds in an amount effective to increase the seed germination
of the plants compared to untreated plants.
[0013] Another embodiment provides a method for producing a greater
iron concentration in a plant, particularly a rice plant,
comprising administering Bacillus subtilis FB17 to the plant, the
seed of the plant, or soil surrounding the plant or the seed in an
amount effective to produce a greater iron concentration in the
plant compared to an untreated plant.
[0014] Another embodiment provides a method for inhibiting growth
of a plant fungal pathogen and infection of a plant, particularly a
rice plant, by a fungal pathogen, particularly rice blast,
comprising administering Bacillus subtilis FB17 to the plant, the
seed of the plant, or soil surrounding the plant or the seed in an
amount effective to inhibit infection of the plant by the fungal
pathogen.
[0015] Additional embodiments provide agricultural carriers and
seed coatings comprising Bacillus subtilis FB17. The biomass of a
plant which has been administered Bacillus subtilis FB17 can be
converted to a biofuel, and the crop produced can be used safely
for human or animal foodstock, or for other purposes.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1: Morphometric analysis (number of branches, number of
leaves, shoot height, shoot weight, root length, root weight) of
Brachypodium distachyon (Bd2-1) plants treated with B. subtilis
FB17 compared to controls. This shows that inoculation with B.
subtilis FB17 enhances plant morphology.
[0017] FIG. 2: Biochemical analysis of Brachypodium distachyon
plants treated with B. subtilis FB17 compared to controls, as
measured by total chlorophyll and carotenoids. This shows that B.
subtilis FB17 inoculation positively impacts the ability of plants
to collect light energy.
[0018] FIG. 3: Amounts of B. subtilis FB17 and controls recovered
from soil and Brachypodium distachyon roots. This demonstrates
persistence of association with the root of B. subtilis FB17
inoculated plants.
[0019] FIG. 4: Total biomass gain in different plant species
treated with B. subtilis FB17. Significant increase (.about.28%) in
both aerial and root biomass was observed with Z. mays (M017).
[0020] FIG. 5: Quantitative data showing the increased root and
shoot biomass in the B. subtilis FB17 seed treated Z. mays Mo-17
plants.
[0021] FIG. 6: Quantitative data showing the increased leaf numbers
in the B. subtilis FB17 seed treated bioenergy crop Brachypodium
distachyon (genotype Bd2-1) plants.
[0022] FIG. 7: Quantitative data showing the increased root and
shoot biomass in the B. subtilis FB17 seed treated bioenergy crop
Brachypodium distachyon (genotype Bd2-1) plants.
[0023] FIG. 8: Quantitative data showing the increased root and
shoot biomass in the B. subtilis FB17 seed treated Zinnia sp. `Red
Spider`.
[0024] FIG. 9: Quantitative data showing the increased root and
shoot biomass in the B. subtilis FB17 seed treated Zinnia sp. `Red
Spider`.
[0025] FIG. 10: Total chlorophyll content in plants treated with B.
subtilis FB17. Significant increase in total chlorophyll content
was observed in tomato (14%), Z. mays CML10 (72%) and CML258 (87%)
post FB17 treatment.
[0026] FIG. 11: Total carotenoid content in plants treated with B.
subtilis FB17. Significant increase in total carotenoid content was
observed in soybean (31%) and Z. mays M017 (82%) post FB17
treatment.
[0027] FIG. 12: Quantitative data showing the increased
photosynthetic efficiency in the B. subtilis FB17 seed treated
Mo-17 plants.
[0028] FIG. 13: Quantitative data showing the increased
photosynthetic efficiency in the B. subtilis FB17 seed treated
bioenergy crop Brachypodium distachyon (genotype Bd2-1) plants.
[0029] FIG. 14: Quantitative data showing the increased
photosynthetic efficiency in the B. subtilis FB17 seed treated
Zinnia sp. `Red Spider`.
[0030] FIG. 15: Quantitative data showing the increased
photosynthetic efficiency in the B. subtilis FB17 seed treated
Exotic Corn CML 10 and CML 258.
[0031] FIG. 16: Percentage germination enhancement in seeds treated
with B. subtilis FB17. Significant increase in total germination
percentage content was observed in tomato (6%), Z. mays M017 (2.1%)
and CML258 (14%) post FB17 treatment. Notably, exotic corn line
CML258 germination increased dramatically.
[0032] FIG. 17: Growth Rate in Zea mays treated with B. subtilis
FB17.
[0033] FIG. 18: Water holding capacity in plants treated with B.
subtilis FB17. Significant increase in total water holding capacity
and retention was observed in tomato (2.1%) and Z. mays M017 (3.5%)
post FB17 treatment.
[0034] FIG. 19: Drought tolerance in plants treated with B.
subtilis FB17. Significant increase in growth rate under drought
treatments (no water) was observed in M017, i.e., 37.5% increase
over drought stressed (no water) uninoculated treatment control
post FB17 treatment
[0035] FIG. 20: B. subtilis FB17 seed treatment reduces lignin
content in corn. Significant reduction of total lignin content was
observed in Z. mays (M017=46%; CML10=64% and CML58=49%) post FB17
treatment under no stress conditions.
[0036] FIG. 21: Aerial & root biomass increase in Oryza sativa
(Nipponbare) treated with B. subtilis FB17. Significant increase in
total biomass was observed in O. sativa (rice; cultivar Nipponbare)
(over 200%) post FB17 treatment.
[0037] FIG. 22: Iron concentration observed in Bacillus subtilis
FB17 treated rice plants compared to untreated rice plants. This
data shows that inoculation with FB17 results in greater crop yield
and higher concentrations of iron in the rice grain.
[0038] FIG. 23: Summary of the effects of B. subtilis FB17 on
different traits in multiple plant species.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The applicants have discovered that a strain of plant growth
promoting rhizobacteria (PGPR), Bacillus subtilis FB17, exhibits
surprising effects when administered to plants. B. subtilis strain
FB17 was originally isolated from red beet roots in North America
(see Fall et al. 2004 System Appli. Microbiol. 27: 372-379,
incorporated herein by reference). This strain was isolated from
beet root on the basis of its ability to form surface biofilm and
dendritic growth.
[0040] In particular, Bacillus subtilis FB17 has provided a
surprising enhancement of biomass in phylogenetically diverse
plants, as well as increased photosynthetic efficiency and enhanced
growth rates in drought conditions. Administration of Bacillus
subtilis FB17 to plants has also resulted in decreased
concentrations of lignin in plants, which can provide important
benefits in the field of bioenergy, as lignin is one of the chief
barriers in converting plant biomass to biofuel. With respect to
rice plants, Bacillus subtilis FB17 has provided a surprising
enhancement in the iron concentration in rice and has also been
shown to attenuate the growth of rice blast, a fungal pathogen that
destroys rice crops around the world. The present invention
provides methods for increasing biomass, increasing drought
tolerance, decreasing lignin content, increasing seed germination,
increasing iron concentration, and increasing tolerance to
pathogens in various plants, particularly crop plants such as corn,
soybean, and rice plants. The present invention also provides
agricultural carriers and seed coatings comprising Bacillus
subtilis FB17.
[0041] An embodiment of the present invention provides a method for
producing a greater biomass in a plant comprising administering
Bacillus subtilis FB17 to the plant, the seed of the plant, or soil
surrounding the plant or the seed in an amount effective to produce
a greater biomass in the plant compared to an untreated plant. As
used herein, the biomass in a plant refers to the total mass of the
plant's matter. Unless specified otherwise, biomass comprises both
aboveground biomass (i.e., aerial biomass, including but not
limited to stem, leaves, and/or grain) and belowground biomass
(i.e., roots). The biomass of a plant that has been administered
Bacillus subtilis FB17 can be measured according to known methods.
In one embodiment, the biomass of the plant is measured according
to the dry weight (DW) of the plant in grams.
[0042] The biomass of a plant that has been administered Bacillus
subtilis FB17 can be measured at a timepoint that is between about
7 days to about 100 days, about 10 days to about 75 days, or about
15 days to about 35 days following administration of Bacillus
subtilis FB17 to the plant. Alternatively, the biomass of a crop
plant that has been administered Bacillus subtilis FB17 can be
measured at the time that the plant is harvested to collect its
grain or produce, i.e., at the time that the mature crop plant such
as a corn, soybean, or tomato plant, is gathered from a field. As
an example, a crop plant that has been administered Bacillus
subtilis FB17 according to a method of the present invention
produces a greater amount of the total aboveground and belowground
biomass as measured in grams of dry weight, in an amount of at
least about 1%, between about 5% and about 200%, between about 5%
and about 100%, between about 7.5% and about 75%, between about
15.degree. A) and about 60%, or between about 30% and about 55%
greater than an untreated plant. In one embodiment, a method
comprises administering Bacillus subtilis FB17 to the plant seed
prior to planting the seed in soil in an amount effective to
produce a greater biomass in the plant in an amount of between
about 5% to about 100% greater than an untreated plant, following
the administration of Bacillus subtilis FB17. For example, as
illustrated in FIG. 4, an increase of about 28% in aerial and root
biomass was observed in Bacillus subtilis FB17 treated corn,
compared to untreated corn 15 days post treatment.
[0043] Another embodiment of the present invention provides a
method for producing a greater drought tolerance in a plant
comprising administering Bacillus subtilis FB17 to the plant, the
seed of the plant, or soil surrounding the plant or the seed in an
amount effective to produce a greater drought tolerance in the
plant compared to an untreated plant. A drought is the absence of
rainfall or irrigation for a period of time sufficient to deplete
soil moisture and injure plants. Drought stress results when water
loss from the plant exceeds the ability of the plant's roots to
absorb water and when the plant's water content is reduced enough
to interfere with normal plant processes. A plant responds to a
lack of water by halting growth and reducing photosynthesis and
other plant processes in order to reduce water use. As used herein,
drought tolerance refers to a plant's growth rate per day in the
absence of water, for example, grams per day of biomass increase in
a Bacillus subtilis FB17 inoculated plant compared to an untreated
plant. For example, as illustrated in FIG. 19, corn plants seed
treated with Bacillus subtilis FB17 in the absence of water
produced about 37.5% greater growth rate per day compared to
untreated plants 15 days post-treatment. In one embodiment, a
method comprises administering Bacillus subtilis FB17 to the plant,
soil surrounding the plant, or the plant seed prior to planting the
seed in soil in an amount effective to produce a greater drought
tolerance in the plant in an amount of at least about 10% greater
than an untreated plant, following the administration of said
Bacillus subtilis FB17.
[0044] Another embodiment of the present invention provides a
method for producing a decreased lignin concentration in a plant
comprising administering Bacillus subtilis FB17 to the plant, the
seed of the plant, or soil surrounding the plant or the seed in an
amount effective to produce a decreased lignin concentration in the
plant compared to an untreated plant. The lignin concentration can
be measured according to known methods. For example, as illustrated
in FIG. 20, plants treated with Bacillus subtilis FB17 exhibited
between about 46% and about 64% decreases in the number of
lignified cells observed in untreated plants. Lignin is an integral
component of plants and is found within plant cell walls, as well
as between plant cells. Lignin is one of the chief barriers to
converting plant biomass to biofuel. Cellulose, another plant
component, is currently the main source for biofuels. While
cellulose is easily fermented to alcohol, lignin does not convert
using existing fermentation processes and renders extraction of
fermentable sugars difficult. It is therefore beneficial to produce
plants that have decreased concentrations of lignin. The present
invention provides biofuels that are produced by converting any of
the biomass of a plant (e.g., the entire biomass of the plant or
any part of the biomass of the plant) that has been administered
Bacillus subtilis FB17 according to any of the methods of the
present invention to a biofuel. The biomass of a plant that has
been administered Bacillus subtilis FB17 can be converted to
biofuel by any known method, such as by fermentation of the sugar
components of the plant.
[0045] Another embodiment of the present invention provides a
method for increasing the rate of seed germination in plants
comprising administering Bacillus subtilis FB17 to the plants,
plant seeds, or soil surrounding the plants or plant seeds in an
amount effective to increase the seed germination of the plants
compared to untreated plants. For example, as illustrated in FIG.
16, increases in total germination percentages were observed in
tomato and corn plants following administration with Bacillus
subtilis FB17.
[0046] Another embodiment of the present invention provides a
method for producing a greater iron concentration in a plant,
particularly a rice plant, comprising administering Bacillus
subtilis FB17 to the plant, the seed of the plant, or soil
surrounding the plant or the seed in an amount effective to produce
a greater iron concentration in the plant compared to an untreated
plant. As iron deficiency is one of the most widespread
micronutrient deficiencies in humans, and rice is the most
important staple food for a large part of the world's population,
rice plants produced according to methods of the present invention
can provide important nutritional benefits throughout the world.
The iron concentration of a plant that has been administered
Bacillus subtilis FB17 can be measured according to known methods,
including inductively coupled plasma-atomic emission spectroscopy
(ICP-AES), inductively coupled plasma mass spectroscopy (ICP-MS),
or other standard methods. In one embodiment, the iron
concentration of the plant is measured according to the milligrams
of iron per kilogram of the dry weight of the plant. As illustrated
in FIG. 22, an approximately 81% increase in iron content was
observed in FB17-treated rice plants compared to untreated plants,
as measured by mg of iron per kg of dry weight of the plant
[0047] Suitable rice plants for use in the invention include Oryza
sativa, Oryza glaberrima, and all subspecies and cultivars thereof.
The iron concentration of a rice plant that has been administered
Bacillus subtilis FB17 can be measured at the time that the rice is
harvested to collect its grain or produce, i.e., at the time that
the mature rice grains are gathered from a field. Alternatively,
the iron concentration of a rice plant that has been administered
Bacillus subtilis FB17 can be measured at a time-point that is
between, for example, about one week to about five months,
preferably about three months following administration of Bacillus
subtilis FB17 to the rice plant. A rice plant that has been
administered Bacillus subtilis FB17 according to a method of the
present invention produces a greater amount of iron, as measured,
for example, in grams of iron per gram of dry weight of aboveground
and belowground biomass of the rice plant.
[0048] As an example, a rice plant that has been administered
Bacillus subtilis FB17 according to a method of the present
invention produces a greater amount of iron per aboveground and
belowground biomass dry weight of the rice plant, in an amount that
is at least about 5%, between about 10% and about 200%, between
about 25% and about 150%, between about 50% and about 100%, between
about 70% and about 90%, between about 75% and about 85%, or about
80% greater than an untreated plant. For example, in one
embodiment, a method comprises administering Bacillus subtilis FB17
to a rice seed prior to planting the rice seed in soil in an amount
effective to produce a greater iron concentration in the rice plant
in an amount of at least about 25% greater than an untreated plant,
following the administration of said Bacillus subtilis FB17.
[0049] Another embodiment of the present invention provides a
method for inhibiting infection of a plant by a fungal pathogen
comprising administering Bacillus subtilis FB17 to the plant, the
seed of the plant, or soil surrounding the plant or the seed in an
amount effective to inhibit infection of the plant by the fungal
pathogen compared to an untreated plant. Examples of plants include
rice and barley plants, such as the rice cultivar Nipponbare. In a
particular embodiment, the present invention provides methods for
inhibiting infection of a rice plant by a fungal pathogen,
particularly rice blast, comprising administering Bacillus subtilis
FB17 to the rice plant, the seed of the rice plant, or soil
surrounding the rice plant or the seed in an amount effective to
inhibit infection of the rice plant by the fungal pathogen compared
to an untreated rice plant. As used herein, "rice blast" refers to
the plant-pathogenic fungus Magnaporthe grisea or Magnaporthe
oryzae.
[0050] Symptoms of rice blast include lesions or spots (which may
be, for example, white or gray) produced on any part of the plant,
particularly on aerial or above-ground parts of the plant, such as
the leaves. As used herein, "inhibiting infection" refers to the
production of a reduced fungal infection in the rice plant, as
measured by a reduction in the symptoms of the fungal infection,
for example, by a reduced number of lesions on the aerial portions
of the rice plant compared to an untreated plant, or a reduced size
of some or all of the lesions. For example, in particular
embodiments, Bacillus subtilis FB17 is administered to a rice
plant, the seed of the rice plant, or soil surrounding the rice
plant or the seed in an amount effective to reduce the number of
lesions on the rice plant caused by rice blast by about 5% to about
100%, about 10% to about 80%, about 20% to about 60%, or about 25%
to about 45%, compared to an untreated rice plant. Without being
bound to any theory, it is believed that B. subtilis FB17 produces
an antifungal volatile compound which attenuates or inhibits M.
oryzae's growth. In particular embodiments, in order to inhibit the
growth of rice blast and infection of a rice plant, Bacillus
subtilis FB17 is administered to a rice seed in an amount of
between about 1.times.10.sup.7 CFU/seed to about 1.times.10.sup.9
CFU/seed, more preferably about 1.times.10.sup.8 CFU/seed, and the
seed is subsequently planted in soil.
[0051] As used herein, an "untreated plant" refers to a plant of
the same species and grown under substantially the same conditions
(e.g., for the same amount of time, in the same climate, and
cultivated according to the same methods using the same materials,
with biomass, drought tolerance, lignin concentration, iron
concentration, fungal infection, and other characteristics being
measured according to the same methods) as a plant which has been
administered Bacillus subtilis FB17 according to a method of the
present invention, except that the untreated plant has not been
administered Bacillus subtilis FB17. As used herein, a
characteristic of a plant that has been administered Bacillus
subtilis FB17, such as a greater biomass, greater drought
tolerance, decreased lignin concentration, greater iron
concentration, or decreased fungal infection, compared to an
untreated plant, refers to a greater biomass, greater drought
tolerance, decreased lignin concentration, greater iron
concentration, or decreased fungal infection as measured at the
same timepoint, respectively.
[0052] In certain embodiments of the methods described herein,
Bacillus subtilis FB17 is administered to a seed in an amount of
between about 1 ml/kg of a Bacillus subtilis FB17 inoculum (i.e., 1
ml/kg of 0.5 Optical Density (OD) at wavelength 600 nm as measured
using a SmartSpec Bio Rad spectrophotometer of Bacillus subtilis
FB17 grown overnight in LB medium) to about 50 ml/kg, preferably
between about 5 ml/kg to about 25 ml/kg, more preferably between
about 10 ml/kg to about 15 ml/kg, most preferably about 12.5 ml/kg.
In alternative embodiments, the Bacillus subtilis FB17 is
administered to a seed in an amount of between about
1.times.10.sup.6 CFU/seed to about 1.times.10.sup.9 CFU/seed, more
preferably between about 1.times.10.sup.7 CFU/seed to about
1.times.10.sup.8 CFU/seed.
[0053] The methods of the present invention can be used to treat
many types of plants (as well as their seeds or surrounding soil)
to increase biomass, increase drought tolerance, decrease lignin
content, increase seed germination, increase iron concentration,
and increase tolerance to pathogens. The plants may include
monocots or dicots. In particular, the plants may include crops
such as corn, soybean, tomato, rice, or barley. Additional examples
of plants that can be treated according to methods of the present
invention include Arabidopsis thaliana and Zinnia, as well as
bioenergy crop plants, i.e., plants that are currently used or have
the potential to be used as sources of bioenergy (e.g., plants
which are useful in producing biofuels), such as Brachypodium
distachyon.
[0054] According to the invention, Bacillus subtilis FB17 may be
administered to a plant by any known method wherein all or part of
the plant is treated, such as by root, seed, or foliar inoculation.
For example, Bacillus subtilis FB17 can be administered to the
aerial portions of a plant, such as the leaves and stem, to the
roots of the plant, to the seed of the plant prior to planting the
seed in soil, or to the soil surrounding the plant or plant seed.
Methods of administration include drenching, spraying, coating,
injection, or other methods known to those of ordinary skill in the
art. As used herein, administering Bacillus subtilis FB17 refers to
either one-time administration, repeated administration (i.e.,
administering Bacillus subtilis FB17 more than one time), or
continuous administration. The Bacillus subtilis FB17 can be
administered at any point in the life cycle of the plant (e.g.,
before or after germination). For example, Bacillus subtilis FB17
can be administered to a plant's seed prior to planting the seed in
soil and prior to germination. Alternatively, Bacillus subtilis
FB17 can be administered to the plant, the seed of the plant, or
the soil surrounding the plant after germination has occurred. Once
treated with Bacillus subtilis FB17, seeds can be planted in soil
and cultivated using conventional methods for generating plant
growth.
[0055] According to embodiments of the present invention, Bacillus
subtilis FB17 can be administered to a plant, plant seed, or soil
either alone or in a mixture with other materials. For example,
Bacillus subtilis FB17 can be administered in a composition that
consists essentially of Bacillus subtilis FB17 in a growth medium
without any additional additives or materials. Alternatively,
Bacillus subtilis FB17 can be administered in a composition that
comprises Bacillus subtilis FB17 in a growth medium, a carrier,
such as water, an aqueous solution, or a powder. The growth medium,
carrier, aqueous solution, or powder may contain additional
additives, such as an insecticide or fungicide. Alternatively,
Bacillus subtilis FB17 can be administered separately with other
additives or materials being applied at different times. In certain
embodiments, Bacillus subtilis FB17 is administered in a
composition that comprises Bacillus subtilis FB17 in an amount of
between about 1 ml/kg (i.e., 1 ml/kg of 0.5 Optical Density (OD) at
wavelength 600 nm as measured using a SmartSpec Bio Rad
spectrophotometer of Bacillus subtilis FB17 grown overnight in LB
medium) to about 50 ml/kg, preferably between about 5 ml/kg to
about 25 ml/kg, more preferably between about 10 ml/kg to about 15
ml/kg, most preferably about 12.5 ml/kg. In alternative
embodiments, Bacillus subtilis FB17 is administered in a
composition that comprises Bacillus subtilis FB17 in an amount of
between about 1.times.10.sup.6 CFU/seed to about 1.times.10.sup.9
CFU/seed, more preferably between about 1.times.10.sup.7 CFU/seed
to about 1.times.10.sup.8 CFU/seed.
[0056] The present invention further provides agricultural carriers
comprising Bacillus subtilis FB17, which can be applied to plants
(e.g., roots), to soil surrounding the plants, or to seeds prior to
planting, as well as seed coatings comprising Bacillus subtilis
FB17 which can be applied to plant seeds. The present invention
also provides a plant seed, preferably a crop plant seed (e.g., the
seed of a corn plant, a soybean plant, a rice plant, a tomato
plant, or a bioenergy crop plant such as Brachypodium distachyon),
that is coated with Bacillus subtilis FB17, such that all or part
of the seed has a coating or film comprising Bacillus subtilis
FB17. The agricultural carrier may comprise Bacillus subtilis FB17
in an amount of between about 1 ml/kg of a Bacillus subtilis FB17
inoculum (i.e., 1 ml/kg of 0.5 Optical Density (OD) at wavelength
600 nm as measured using a SmartSpec Bio Rad spectrophotometer of
Bacillus subtilis FB17 grown overnight in LB medium) to about 50
ml/kg, between about 5 ml/kg to about 25 ml/kg, between about 10
ml/kg to about 15 ml/kg, or about 12.5 ml/kg. The seed coating may
comprise Bacillus subtilis FB17 in an amount of between about
1.times.10.sup.6 CFU/seed to about 1.times.10.sup.6 CFU/seed, more
preferably about 1.times.10.sup.7 CFU/seed. The agricultural
carrier and seed coating may each consist essentially of Bacillus
subtilis FB17 in a growth medium without any additional additives
or materials. Alternatively, the agricultural carrier and seed
coating may each comprise Bacillus subtilis FB17 in a growth
medium, such as water, an aqueous solution, or a powder. The growth
medium, aqueous solution, or powder may contain additional
additives, such as an insecticide or fungicide.
[0057] The present invention has both basic and applied
applications. In a broad sense one could use the methods described
herein to increase biomass (e.g., in alternative plant species used
for biofuel or to impact yield potential of crop plants) and to
confer enhanced drought tolerance. Compared to transgenic
approaches, these methods are immediately applicable to any plant,
without the time required for gene identification, generation and
characterization of transgenic lines, and is free of the regulatory
and social issues related to the use of trans genes. Compared to
the use of traditional agronomic practices (applications of
chemical fertilizers and water), the methods described herein are
less resource and labor intensive for the grower and are more
environmentally friendly. In addition, application of chemical
fertilizers is known to enhance crop disease susceptibility by the
induction of rapid weak growth, whereas plants grown with this
method do not demonstrate enhanced disease susceptibility. Compared
to other rhizobacteria that are used for seed treatment, FB17
requires low inoculums to confer beneficial results. Finally, these
methods are compatible with organic farming practices, whereas
other methods described above (e.g., the application of chemical
fertilizers) are not.
[0058] A deposit of B. subtilis strain FB17 has been available
since prior to Mar. 1, 2010, at the Delaware Biotechnology
Institute, 15 Innovation way, Room # 145, Newark, Del. 19711. A
deposit of B. subtilis strain FB17 will also be made with the
American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209 USA. Access to this ATCC
deposit will be available during the pendency of the application to
the Commissioner of Patents and Trademarks and persons determined
by the Commissioner to be entitled thereto upon request. The
deposit will be maintained in the ATCC Depository, which is a
public depository, for a period of 30 years, or 5 years after the
most recent request, or for the enforceable life of the patent,
whichever is longer, and will be replaced if it becomes nonviable
during that period. The deposit will be available as required by
foreign patent laws in countries wherein counterparts of the
subject application, or its progeny are filed.
[0059] Further, the subject deposit will be stored and made
available to the public in accord with the provisions of the
Budapest Treaty for the Deposit of Microorganisms, i.e., it will be
stored with all the care necessary to keep it viable and
uncontaminated for a period of at least five years after the most
recent request for the furnishing of a sample of the deposit, and
in any case, for a period of at least thirty (30) years after the
date of deposit or for the enforceable life of any patent which may
issue disclosing the culture.
[0060] The following examples are provided to describe the
invention in greater detail and are intended to illustrate, not
limit, the invention. "UD10-22," as stated in some of the figures
described below, refers to Bacillus subtilis FB17.
EXAMPLES
Example 1
[0061] Brachypodium distachyon and corn plants were germinated and
grown for 21 days. Once in 5 days (3 times), 5 ml of 0.5 OD B.
subtilis FB17 per pot was added. For control, 5 ml of 0.5 OD of E.
coli OP50 per pot was added. FB17 and OP50 had been grown overnight
in LB medium and optical density (OD) at wavelength (600 nm) was
taken using SmartSpec (Bio Rad) spectrophotometer. Ten days after
the final treatment, plants were analyzed. The controls described
in all the experiments herein refer to plants that were not treated
with bacteria or that were treated with E. coli OP50.
[0062] Brachypodium distachyon (Bd2-1) and corn plants treated with
B. subtilis FB17, bacterial control E. coli, or mock treatment were
grown in 4.times.4 inch pots in standard conditions (22-25.degree.
C., 60% humidity, 16 h light-8 h dark photoperiod) for 30 days post
treatment. Aerial and root biomass of the energy crop B. distachyon
increased with FB17 treatment. FIG. 1 shows that the biomass of B.
distachyon treated with FB17 was enhanced at a statistical level.
FIG. 2 depicts an increase in photosynthetic efficiency observed in
B. distachyon treated with FB17. The B. distachyon treated with
FB17 contained more chlorophyll and total caroetenoids than
controls, demonstrating robust plant health. FIG. 3 shows amounts
of FB17 recovered from soil surrounding B. distachyon roots. The
figure reveals that FB17 is associated much more strongly with B.
distachyon roots compared to the E. coli, suggesting the true
rhizobacterial nature of FB17.
[0063] Corn plants also exhibited an increase in aerial and root
biomass after growing for 30 days following treatment with B.
subtilis FB17, bacterial control E. coli OP50, or mock
treatment.
Example 2
[0064] Arabidopsis thaliana seeds were germinated and grown for 21
days. Once in 5 days (3 times), 5 ml of 0.5 OD B. subtilis FB17 per
pot was added. For control, 5 ml of 0.5 OD of E. coli OP50 per pot
was added. FB17 and OP50 had been grown overnight in LB medium and
optical density (OD) at wavelength (600 nm) was taken using
SmartSpec (Bio Rad) spectrophotometer. Ten days after the final
treatment, plants were subjected to drought (i.e., no water was
added) at 25.degree. C. with 40% humidity for 4 weeks. Thirty days
post-treatment, drought was assessed through loss of stay green
phenotype in the untreated plants compared to the FB17 treated
plants, indicating that FB17 confers enhanced drought tolerance in
Arabidopsis.
Example 3
[0065] Seed treatment of B. subtilis FB17 promotes biomass
enhancement in Corn Mo17, CML258, CML10, Zinnia, and Brachypodium
distachyon.
[0066] To test the effect of B. subtilis FB17 on biomass
enhancement in corn (Mo17, CML258, CML10), soybean (Will-82),
tomato (Solanum lycopersicum), Zinnia, and Brachypodium distachyon
(an energy crop model), 50 seeds (n=50) per plant species were seed
treated with B. subtilis FB17 (about 1.times.10.sup.7 cfu/seed or
12.5 ml/kg of 0.5 Optical Density (OD) Bacillus subtilis FB17 grown
overnight in LB medium, at wavelength 600 nm as measured using a
SmartSpec Bio Rad spectrophotometer). Post seed treatment seeds
were individually sown in pots (4.times.4 inches) with a soil mix
for germination and biomass studies. Interestingly, seed treatment
of B. subtilis FB17 promoted root and shoot growth for all the
tested crop species. Measurements were taken 15 days post
treatment.
[0067] Seed treated plants promoted increased root biomass
resulting in denser root systems rather than increased root length.
A denser root system results from increased lateral roots and root
hairs providing more available uptake of water and nutrients.
[0068] Zea mays var. CML258 resulted in about 16% increase in
aerial biomass (g DW) over control. Zea mays var. CML10 resulted in
about 9% increase in aerial biomass (g DW) over control. Zea mays
var. Mo-17 resulted in about 38% increase in aerial biomass (g DW)
over control. Brachypodium resulted in about 40% increase in aerial
biomass (g DW) over control. A significant increase of about 28% in
total aerial and root biomass was observed with Z. mays (M017) over
control. FIG. 4 illustrates the total biomass gain in plants
treated with B. subtilis FB17.
[0069] FIG. 5 illustrates quantitative data showing the increased
root and shoot biomass in the B. subtilis FB17 seed treated Mo-17
plants. FIG. 6 illustrates quantitative data showing the increased
leaf numbers in the B. subtilis FB17 seed treated bioenergy crop
Brachypodium distachyon (genotype Bd2-1) plants. FIG. 7 illustrates
quantitative data showing the increased root and shoot biomass in
the B. subtilis FB17 seed treated bioenergy crop Brachypodium
distachyon (genotype Bd2-1) plants. FIGS. 8 and 9 illustrate
quantitative data showing the increased root and shoot biomass in
the B. subtilis FB17 seed treated Zinnia sp. `Red Spider`.
Example 4
[0070] Seed treatment of B. subtilis FB17 promotes photosynthetic
efficiency in corn and tomato.
[0071] To test the effect of B. subtilis FB17 on photosynthetic
efficiency in corn (Mo17, CML258, CML10), soybean (Will-82), tomato
(Solanum lycopersicum), Zinnia, and Brachypodium (an energy crop
model), 50 seeds (n=50) per plant species were seed treated with B.
subtilis FB17 (12.5 ml/kg or 1e7 cfu/seed). Leaves post 15-32 days
of treatment were harvested and analyzed for total chlorophyll
content. Results showed that B. subtilis FB17 inoculated corn and
tomato plants (tomato and exotic lines of Corn CML258 and CML10)
showed enhanced chlorophyll and carotenoid content compared to the
untreated samples, as depicted in FIGS. 10 and 11.
[0072] The increased total chlorophyll values have the potential to
promote increased vigor and biomass as seen with CML258 and CML10.
The total chlorophyll content of B. subtilis FB17 seed inoculated
tomatoes resulted in an increase of about 14%. Even more
significant are exotic corn lines CML258 and CML10 with an increase
of about 87% and about 72% increases, respectively.
[0073] Although there are increases in total chlorophyll content,
that does not signify that the total carotenoid content will also
correspond with an increased value. Tomato and Zinnia had
significantly reduced total carotenoid percent when inoculated with
B. subtilis FB17 and compared with untreated seeds. Corn CML258 and
CML10 had significantly increased total carotenoid percents, while
soybean, corn Mo17, and Brachypodium did not show statistically
significant differences between treated and untreated seeds.
[0074] FIG. 12 illustrates quantitative data showing the increased
photosynthetic efficiency in the B. subtilis FB17 seed treated
Mo-17 plants. FIG. 13 illustrates quantitative data showing the
increased photosynthetic efficiency in the B. subtilis FB17 seed
treated bioenergy crop Brachypodium distachyon (genotype Bd2-1)
plants. FIG. 14 illustrates quantitative data showing the increased
photosynthetic efficiency in the B. subtilis FB17 seed treated
Zinnia sp. `Red Spider`. FIG. 15 illustrates quantitative data
showing the increased photosynthetic efficiency in the B. subtilis
FB17 seed treated Exotic Corn CML 10 and CML 258.
Example 5
[0075] Seed treatment of B. subtilis FB17 promotes germination in
corn and tomato plants.
[0076] To test the effect of B. subtilis FB17 on percentage
germination increase in corn (Mo17, CML258, CML10), soybean
(Will-82), tomato (Solanum lycopersicum), Zinnia, and Brachypodium
(an energy crop model), 50 seeds (n=50) per plant species were seed
treated with B. subtilis FB17 (12.5 ml/kg or 1e7 cfu/seed). Final
germination percents were scored 8 days post date sown. Results
showed that B. subtilis FB17 treatment promoted statistically
significant germination response in tomato and corn, as depicted in
FIG. 16.
[0077] B. subtilis FB17 treated tomato seeds and exotic corn line
CML 258 had a 5.9% and 14% increase in germination percent,
respectively.
[0078] The B. subtilis FB17 seed treatment had neutral and positive
effects for all of the crop species tested. There was not a
statistically negative response to germination percent when seed
treatments were applied.
Example 6
[0079] To test the effect of B. subtilis FB17 in corn (Mo17,
CML258, CML10), soybean (Will-82), tomato (Solanum lycopersicum),
Zinnia, and Brachypodium (an energy crop model), 50 seeds (n=50)
per plant species were seed treated with B. subtilis FB17 (1e7
cfu/seed or 12.5 ml/kg of 0.5 Optical Density (OD) Bacillus
subtilis FB17 grown overnight in LB medium, at wavelength 600 nm as
measured using a SmartSpec Bio Rad spectrophotometer). Post seed
treatment seeds were individually sown in pots (4.times.4 inches).
Measurements were made post 15 days of treatment. FIG. 17
illustrates the growth rate in Zea mays following treatment with B.
subtilis FB17. FIG. 18 illustrates water holding capacity in plants
treated with B. subtilis FB17. Significant increase in total water
holding capacity and retention was observed in tomato (2.1%) and Z.
mays M017 (3.5%) post FB17 treatment. FIG. 19 illustrates drought
tolerance in plants treated with B. subtilis FB17. Significant
increase in growth rate under drought treatments was observed in
M017 (37.5% increase over no water treatment control) post FB17
treatment. FIG. 20 illustrates that B. subtilis FB17 seed treatment
reduces lignin content in corn. Significant reduction of total
lignin content was observed in Z. mays (about 46% reduction in
M017; about 64% reduction in CML10, and about 49% reduction in
CML58) post FB17 treatment.
Example 7
[0080] FIG. 21 illustrates aerial & root biomass increase in
rice plants, Oryza sativa (Nipponbare), treated with B. subtilis
FB17 60 days after inoculation. Overnight cultures of FB17 grown in
LB were used to generate an inoculum of 10.sup.8 cells per ml. Four
week old hydroponically grown rice plants (cultivar Nipponbare)
were used for FB17 supplementation. The rice plants that were
administered B. subtilis FB17 exhibited an increase of about 200%
biomass compared to untreated rice plants.
Example 8
[0081] To evaluate if Bacillus subtilis FB17 colonizes rice roots,
rice plants (cultivar Nipponbare) were inoculated with Bacillus
subtilis FB17 and the roots of the rice plants were observed 96 hrs
post-inoculation by confocal scanning laser microscopy.
Observations confirmed that the beneficial rhizobacteria (Bacillus
subtilis FB17) form biofilm in planta. In particular, the data
suggest that Bacillus subtilis FB17 efficiently colonizes the rice
roots post 96 hrs of treatment, indicating that rice roots support
the colonization of beneficial microbes.
[0082] To evaluate if rhizobacterial treatment of rice plants
inflicts any changes in stomatal aperture, applicants analyzed rice
plants treated with rhizobacteria. Results showed that
rhizobacterial treatment of rice with Bacillus subtilis FB17
greatly reduced the stomatal aperture in treated rice plants
(cultivar Nipponbare). In the case of Bacillus subtilis FB17
treatment, guard cells were observed 1 week after addition of
Bacillus subtilis FB17. These results suggest that B. subtilis FB17
(Bacillus subtilis FB17) inflicts a general stomatal closure
response in both monocots and dicot plants as evidenced with both
A. thaliana and rice.
[0083] To evaluate if Bacillus subtilis FB17 attenuates the growth
of rice blast, applicants exposed Magnaporthe oryzae to Bacillus
subtilis FB17 cultures. Qualitative compartment plates and
quantitative data showed that Bacillus subtilis FB17 attenuated the
growth of M. oryzae as shown by reduced radial growth in the
Bacillus subtilis FB17 exposed fungal cultures. Comparison with the
controls (TY & LB) demonstrates the extent to which the
pathogen would grow with no treatment. As shown in Table 1,
Bacillus subtilis FB17 restricted M. oryzae's growth by about 25%
in vitro. These results suggest that B. subtilis FB17 produces an
antifungal volatile compound which may attenuate or inhibit M.
oryzae's growth.
TABLE-US-00001 TABLE 1 Percent fungal growth Fungal colony avg.
relative to control Treatment diameter (cm) treatment TY control
3.175 100 LB control 3.098 100 Bacillus subtilis FB17 2.342
75.59
[0084] Bacillus subtilis FB17 induced systemic resistance in rice
and barley to Magnaporthe oryzae. Both rice and barley plants that
were exposed to M. oryzae had reduced lesion formation on rice
leaves and barley cotyledons, respectively, in FB17-treated plants
compared to controls, as shown in Table 2 ("infected" was defined
as a leaf having as least one typical diamond-shaped Blast lesion
on it).
TABLE-US-00002 TABLE 2 # infected leaves Total # lesions None/Mo 3
23 FB17/Mo 5 13
Example 9
[0085] To evaluate if Bacillus subtilis FB17 increases iron
fortification in rice, the applicants analyzed the overall iron
content in rice leaves, roots, and grains in plants supplemented
with Bacillus subtilis FB17, using inductively coupled
plasma-atomic emission spectroscopy (ICP-AES). Results showed that
Bacillus subtilis FB17 supplementation to rice helps mobilize iron
in planta, i.e., the essential element iron is actively taken up by
the plant where it is utilized for plant growth and development. As
illustrated in FIG. 22, an 81% increase in iron content was
observed in FB17-treated rice plants compared to untreated control,
as measured by mg of iron per kg of dry weight of the plant
("UD1022," as stated in FIG. 1 refers to Bacillus subtilis FB17).
Thus, administration of Bacillus subtilis FB17 to plants,
particularly rice plants, can greatly enhance the nutritional value
of food by increasing iron concentrations in the food.
[0086] FIG. 23 summarizes the effects of B. subtilis FB17 on
different traits in multiple plant species described above.
[0087] Although the present invention has been described in
connection with specific embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications and variations
of the described compositions and methods of the invention will be
apparent to those of ordinary skill in the art and are intended to
be within the scope of the appended claims.
* * * * *