Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
USE OF LIPO-CHITOOLIGOSACCHARIDES FOR INCREASING PHOTOSYNTHESIS IN PLANTS AND
CORRESPONDING METHODS AND COMPOSITIONS
FIELD OF THE INVENTION
The present invention relates to agriculture. More particularly,
the invention relates to a method of increasing photosynthesis of a plant and
more particularly of a crop plant. In addition, the invention relates to a
method of
increasing photosynthesis and/or growth and/or yield in crop plants,
comprising
an exposure thereof to lipo-chitooligosaccharides, and compositions therefor.
BACKGROUND OF THE INVENTION
Bacteria of the genera Rhizobium, Bradyrhizobium,
Sinorhizobium and Azorhizobium, collectively known as the rhizobia, form
specialized organs called nodules on the roots, and sometimes stems, of
legumes and fix atmospheric nitrogen within these structures. Nodule formation
is a highly specialized process that is modulated by signal molecules. In
general,
this phase of the interaction is a two step process. Initially, plant-to-
bacteria
signal molecules, usually specific fiavonoids or isoflavonoids, are released
by
roots of the host plants. In response to the plant-to-bacteria signals the
microsymbiont releases bacteria-to-plant signal molecules, which are lipo-
chitooligosaccharides (LCOs), so called nod factors (also nol and noe) genes
very rapidly (only a few minutes after exposure) and at very low
concentrations
(10-7 to 1078 M) (Peters et at., 1986). Generally this is through an
interaction with
nodD, which activates the common nod genes, although the situation may be
more complex, as is the case in B. japonicum where nodD,, nodD2 and nodVW
are involved (Gillette & Elkan 1996; Stacey 1995). Nod genes have been
identified in the rhizobia that form nitrogen fixing relationships with
numbers of
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the Fabiaceae family (see 5,549,718 and references therein). Recently, the
plant-to-bacteria signal molecules have been shown to promote soybean
nodulation and nitrogen fixation under cool soil temperatures (CA 2,179,879)
and
increase the final soybean grain yield on average of 10% in the field and up
to
40% under certain conditions. (Long, 1989; Kondorosi, 1991; Schenes et al.,
1990; Boone et al., 1999). Among the products of thenod genes induced by the
plant phenolic signal molecules are various enzymes involved in the synthesis
of a series of lipo chitooligosaccharides (LCOs) (Spaink, 1995; Stacey, 1995).
These newly synthesized LCOs act as bacterium-to-plant signals, inducing
expression of many of the early nodulin genes (Long, 1989).
LCO signal molecules are composed of three to five 1-4 R
linked acetylglucosamine residues with the N-acetyl group of the terminal non-
reducing sugar replaced by an acyl chain. However, various modifications of
the
basic structure are possible and these, at least in part, determine the host
specificity of rhizobia (Spaink et al., 1991; Schultze et al., 1992).
Lipo-chitiooligosaccharides are known to affect a number
of host plant physiological processes. For example, they induce: root hair
deformation (Spaink et a/., 1991), ontogeny of compete nodule structures
(Fisher
and Long, 1992; Denarie and Cullimore, 1993), cortical cell division (Sanjuan
et
a/., 1992; Schlaman et al., 1997) and the expression of host nodulin genes
essential for infection thread formation (Horvath et a!., 1993; Pichon eta!.,
1993,
Minami et al., 1996). LCOs have also been shown to activate defense-related
enzymes (Inui et al., 1997). These bacterium-to-plant signals exert a powerful
influence over the plant genome and, when added in the absence of the
bacteria,
can induce the formation of root nodules (Truchet et al., 1991). Thus, the
bacteria-to-plant signals can, without the bacteria, induce all the gene
activity for
nodule organogenesis (Denarie et al., 1996; Heidstra & Bisseling, 1996).
Moreover, the above-mentioned activities induced by LCOs can be produced by
concentrations as low as 1014 M (Stokkermans et al. 1995). The mutual
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exchange of signals between the bacteria and the plant are essential for the
symbiotic interaction. Rhizobia mutants unable to synthesize LCOs will not
form
nodules. Analysis of the B. japonicum nod genes indicates that ability to
induce
soybean nodulation requires at least 1) a basic tetrameric Nod factor
requiring
only nodABC genes or 2) a pentameric LCO (C18:1, C16:0 or C16: fatty acid and
a methyl-fucose at the reducing end, sometimes acetylated) requiring nodABCZ
genes (Stokkermans et al. 1995).
When added to the appropriate legume, LCOs can cause the
induction of nodule meristems (Denarie et al., 1996), and therefore cell
division
activity. LCOs have also been shown to induce cell cycle activities in an in
vitro
system: (a carrot embryogenesis system) at levels as low as 10-14 M (De Jong
et al. 1993).
A chemical structure of lipo chitooIigosaccharides, also
termed "symbiotic Nod signals" or "Nod factor", has been described in U.S.
Patents 5,549,718 and 5,175,149. These Nod factors have the properties of a
lectin ligand or lipo-oligosaccharide substances which can be purified from
bacteria or synthesized or produced by genetic engineering.
The process of N2 fixation is energy intensive requiring about
10-20% of the carbon fixed by the plant. It has been estimated that an average
of about 6 mg of carbon is required per mg of nitrogen fixed (Vance and
Heichel,
1991). Enhanced photosynthesis, due to the Bradyrhizobium-soybean
association has been previously reported. Imsande (1989a,b) reported enhanced
net photosynthesis and grain yield in soybean inoculated with Bradyrhizobium
japonicum compared with plants that were not inoculated but adequately
supplemented with N fertilizer. Recently, Phillips et al., (1999) showed that
lumichrome might act as a signal molecule in the rhizosphere of alfalfa
plants,
leading to increased respiration and net carbon assimilation during early
stages
of the Sinorhizobium meliloti-alfalfa symbiosis.
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Methods to increase plant dry matter accumulation and yield
are essential as world population is projected to increase by 4 billion (66%)
during the next fifty years (United Nations, Population Division, 1998). In
the last
fifty years world crop output increased by 2.5 fold, with little increase the
area of
land cropped (Hoisington et al., 1999). Given the projected increase in world
population we must provide another 2.5 fold increase during the next 50 years
if everyone is to have reasonably reliable access to food (James, 1997).
However, the primary causes of increased food production during the last 50
years (increases in harvest index, the amount of land under irrigation and the
use
of fertilizers, particularly N fertilizer) are largely exhausted. A century of
plant
breeding has resulted in little or no increase in the photosynthetic rates of
most
crop plants (Moss and Musgrave, 1971; Evans 1975, 1980). There thus remains
a tremendous need to increase the photosynthetic rates and growth of crop
plants. There also remains a need to increase production of crop plants.
There have been considerable efforts to enhance
photosynthesis in crop plants with a view to increase plant productivity.
Makela
et al., (1999) reported enhanced photosynthesis under drought and salinity
stress in tomato and turnip rape following foliar application of
glycinebetanine at
very low concentrations. Foliar application of methanol also increased
photosynthesis in a number of plants (Noumora and Benson, 1991). Johnson
and Stelizer (1991) reported increased photosynthesis in loblolly pine by
application of sub-lethal doses hexazinone.
While the effects of plant-to-bacteria signal molecules (i.e.
isoflavones) on nodulation, nitrogen fixation, growth and protein yield of
legumes,
such as soybean, and on bacteria-to-plant signal molecules (LCOs) on
nodulation and nitrogen fixation in legumes have been described under certain
conditions, the effect of the bacteria-to-plant signal molecules on the growth
of
non-legumes is unknown. In fact, the role of such bacteria-to-plant signal
molecules on non-legumes has yet to be reported. In addition, the effect
ofLCOs
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on processes other than nodulation of legumes has yet to be described.
Moreover, while LCOs have been associated with a growth-promoting effect in
the early stages of the initiation of the symbiotic relationship between plant
and
bacteria, it remains to be determined whether LCOs can have an effect on
plants
5 at later stages of their life cycle.
There thus remains a need to assess the effect of LCOs on
plant growth and especially on later stages thereof. Moreover, there remains a
need to assess whether LCO comprising compositions can have an effect on the
synthetic rate and/or growth of plants in general and especially of non-legume
plants.
There also remains a need to better understand the workings
of the complex homeostatic system which is involved in the regulation of
photosynthesis. Moreover, there remains a need to assess the role of LCOs on
photosynthesis of plants.
The present invention seeks to meet these and other needs.
SUMMARY OF THE INVENTION
The invention concerns a composition for enhancing the
photosynthetic rate, and/or growth, and/or yield of a plant and especially of
a crop
plant. More specifically, the present invention relates to a composition
comprising
an LCO which can increase the photosynthetic rate, and/or growth, and/or yield
of a legume, in addition to acting as a trigger to initiate legume symbiotic
nitrogen
fixation. More particularly, the invention relates to methods and compositions
to
enhance the photosynthetic rate, and/or growth, and/or yield of a plant and
especially of a crop plant grown under field conditions. In certain
embodiments,
the plant is a non-legume. In further embodiments, the invention relates to
methods and compositions to increase the photosynthetic rate, and/or
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growth, and/or yield of a legume, more particularly soybean, and especially to
a
legume grown under field conditions.
Surprisingly, the compositions of the present invention act not
only on a legume such as soybean, but on plants in general, as exemplified
with
a number of non-legume crops. More specifically, these non-legume crops are
exemplified with diversified and evolutionary divergent crops such as corn,
rice
(Poaceae); melon (Cucurbitaceae); canola (Brassicaceae); apple (Rosaceae);
and grape (Vitaceae). The present invention thus also refers to compositions
for
enhancing photosynthetic rate, and/or growth, and/or yield of non-legumes.
More
particularly, the invention relates to compositions comprising an LCO for
enhancing photosynthetic rate, and/or growth, and/or yield of non-legumes. Non-
limiting examples of such non-legumes include cotton, corn, rice, canola,
potato,
cucumber, cantaloupe, melon, lettuce, apple, grape and beet.
Broadly therefore, the present invention relates to
compositions comprising an LCO for promoting growth of a crop. Non-limiting
examples of plant crops include monocot, dicot, members of the grass family
(containing the cereals), and legumes.
More specifically, therefore, the present invention concerns
the demonstration that an administration of LCOs to a plant significantly
increases the photosynthetic rate thereof. More particularly, the present
invention
demonstrates that spraying LCOs on the leaves of a plant (e.g. a foliar
application) significantly increases the photosynthetic rate thereof. The
present
invention therefore relates to compositions to increase the photosynthetic
rate
of plants in general. In addition, the present invention relates to methods of
increasing the photosynthetic rate of evolutionary divergent plants,
comprising
an application of an agriculturally effective dose of LCOs. In a particularly
preferred embodiment, the invention relates to an acute application of LCOs by
a spraying of the leaves of the plants and to its effect on the growth and/or
yield
of plants and especially of field grown plants.
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In a particular set of experiments, a composition of the
present invention, comprising an LCO, was shown to significantly enhance the
photosynthetic rate of evolutionary divergent plants such as soybean
(Fabaceae), corn, rice (Poaceae), melon (Cucurbitaceae), canola
(Brassicaceae), apple (Rosaceae) and grape (Vitaceae), under greenhouse
conditions.
In another set of experiments in the field, a composition of the
present invention comprising an LCO was shown to significantly enhance the
photosynthetic rate of soybean, corn, apple and grape.
While the present invention has been demonstrated using
evolutionary divergent plants, the invention should not be so limited. Indeed,
it
will be clear to a person skilled in the art to which the present invention
pertains,
that based on the evolutionary distance between the types of plants tested and
their similar response to an application of LCOs, that it is expected that
other
types of plants should respond similarly to the LCO application, by displaying
an
increase in the photosynthetic rate and/or yield thereof. Of note, the group
of
Smith et al. (the group from which the instant invention stems) has also shown
that LCOs can significantly enhance seed germination and/or seedling
emergence and/or growth, and/or break the dormancy of numerous types of non-
legume plant families, including Poaceae, Cucurbitaceae, Malvaceae,
Asteraceae, Chenopodiaceae and Solonaceae. More specifically, the non-
legume crops used included corn, cotton, cantaloupe, lettuce, potato and beet.
Thus, the biological activity of LCOs on early stages of plants in general has
also
been demonstrated.
Based on (1) the evolutionary divergence of the tested crops,
which display an increased photosynthetic rate after LCO treatment; and (2)
the
effect of LCO on germination, and seedling emergence (and of the breaking of
dormancy of potato tubers) of evolutionary divergent plants, it is expected
that
the photosynthetic rates and yield-increasing effects demonstrated by the
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methods and compositions of the present invention can be applied to plants in
general. More particularly, it relates to compositions and methods for
different
plant families including but not limited to Poaceae, Cucurbitaceae, Malvaceae,
Asteraceae, Chenopodiaceae, Brassicaceae, Rosaceae, Vitaceae, Fabaceae
and Solonaceae. More specifically, crops within the scope of the present
invention include without limitation corn, cotton, cantaloupe, melon,
cucumber,
canola, lettuce, potato, apple, grape and beet. Non-limiting examples of crop
plants also include monocot, dicot, members of the grass family (containing
the
cereals), and legumes.
Thus, the present invention relates to agricultural
compositions comprising at least one LCO (and methods of using same) for
promoting photosynthetic rate increases and/or increase in yield of a crop. It
should be clear to a person skilled in the art that other photosynthetic rate
increasing-, and/or yield-increasing compounds could be added to the
compositions of the present invention.
The Applicant is the first to show that a composition
comprising an LCO can have a significant effect on the photosynthetic rate of
legumes. Moreover, the Applicant is the first to show the surprising effect of
signal molecules involved in the bacteria-legume signalling on the
photosynthetic
rate and growth of non-legume plants.
It should also be understood that conventional plants and
genetically engineered plants can be used in accordance with the present
invention. In one particular and preferred embodiment of the present
invention,
non genetically-engineered plants are treated with the composition and/or
method of the present invention.
While the photosynthetic rate and/or yield enhancing
capabilities of the compositions of the instant invention are demonstrated
under
field conditions with corn, apple, grape and soybean, it is expected that
other
crops should also show the same type of response to LCOs treatment. These
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plants include without limitation significantly divergent plants in ten
distinct
families: (1) corn, the only monocot tested herein, in the family of grasses
(Poaceae), which also contains the cereals; (2) cucumber and cantaloupe, the
latter being a plant used horticulturally, and being slow to germinate at low
temperature [its base temperature is about 14 C] (Cucurbitaceae); (3) cotton,
one of the most important fibre crops on the planet (Malvaceae); (4) lettuce
(Asteraceae); (5) beet (Chenopodiaceae); (6) potato, a very important crop
(Solonaceae, which also includes tobacco, peppers and tomato); and two
families of legumes (7) canola, representing the mustard group (Brassicaceae)
and (8) soybean (representative of oil seed crop), bean (representative of a
crop
for human consumption) and red clover and alfalfa (forage legumes) (all of the
Fabaceae family); (9) apple, representing Rosaceae; and (10) grape,
representing Vitaceae.
In view of the evolutionary distance between the above-listed
plants, and of their similar response to LCO treatment under greenhouse
conditions or field conditions, it can be predicted that such results will
apply to
crop plants in general. It follows that a person skilled in the art can adapt
the
teachings of the present invention to other crops. Non-limiting examples
thereof
include tobacco, tomato, wheat, barley, rice, sunflower and plants grown for
flower production (daisy, carnation, pansy, gladiola, lilies and the like). It
will be
understood that the compositions can be adapted to specific crops, to meet
particular needs.
In accordance with one embodiment of the present invention,
there is thus provided an agricultural composition for enhancing a plant crop
photosynthetic rate and/or growth thereof comprising a photosynthetic rate-
promoting amount of at least one lipo-chitooligosaccharide (LCO) together with
an agriculturally suitable carrier.
In accordance with another embodiment of the present
invention, there is therefore provided a use of an agricultural composition
for
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enhancing a plant crop photosynthetic rate and/or growth thereof comprising a
photosynthetic rate-promoting amount of at least one lipo-chitooligosaccharide
(LCO) together with an agriculturally suitable carrier.
In accordance with another embodiment of the present
5 invention, there is provided a use of an agricultural composition for
enhancing the
photosynthetic rate and/or growth of a plant crop comprising treatment of a
leaf of
the plant crop with a photosynthetic rate-promoting amount of at least one
lipochitooligosaccharide (LCO) together with an agriculturally suitable
carrier.
In accordance with yet another embodiment of the present
10 invention, there is provided a method for increasing the photosynthetic
rate
and/or growth of a plant, comprising a treatment of a leaf of a plant with a
composition comprising an agriculturally effective amount of a lipo
chitooligosaccharide (LCO) in admixture with an agriculturally suitable
carrier
medium, wherein the effective amount enhances the photosynthetic rate and/or
growth of the plant in comparison to an untreated plant.
In accordance with yet another embodiment of the present
invention, there is provided a method for enhancing the photosynthetic rate
and/or growth of a plant, comprising a treatment of a leaf of a plant crop
with a
composition comprising an agriculturally effective amount of at least one
lipochitooligosaccharide (LCO) in admixture with an agriculturally suitable
carrier
medium, wherein the effective amount enhances the photosynthetic rate and/or
growth of the plant crop in comparison to an untreated plant.
In addition, in accordance with another embodiment of the
present invention, there is therefore provided a method for enhancing the
photosynthetic rate and/or growth of a plant crop comprising incubating a
rhizobial strain which expresses a lipo chitooligosaccharide (LCO) in the
vicinity
of a leaf of the plant such that the LCO enhances the photosynthetic rate
and/or
growth of the plant crop as compared to an untreated plant.
In addition, in accordance with another embodiment of the
present invention, there is therefore provided a method for enhancing the
photosynthetic rate and/or growth of a plant crop comprising incubating a
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10a
rhizobial strain which expresses at least one lipo chitooligosaccharide (LCO)
on a
leaf of the plant crop such that the LCO enhances the photosynthetic rate
and/or
growth of the plant crop, as compared to an untreated plant.
In addition, in accordance with another embodiment of the
present invention, there is provided a use of an agricultural composition
comprising an agriculturally effective amount of at least one
lipochitooligosaccharide (LCO) in admixture with an agriculturally suitable
carrier
medium, for enhancing the photosynthetic rate and growth of a plant crop
comprising treatment of a leaf of said plant crop with said composition,
wherein
said effective amount enhances the photosynthetic rate and growth of said
plant
crop in comparison to an untreated plant.
The terms "lipochitin oligosaccharide" and "lipo-
chitooligosaccharide" are used herein interchangeably.
The terminology "grown under field conditions" will be
understood to cover the conditions to which a plant is subjected when grown in
the field, as opposed to when grown under more controlled conditions, such as
greenhouse conditions.
As used herein, the term "LCO" refers broadly to a Nod factor
which is under the control of at least one nodulation gene (nod gene), common
to
rhizobia. LCO therefore relates to a bacteria-to-plant signal molecule which
induces the formation of nodules in legumes and enables the symbiotic bacteria
to colonize same. Broadly, LCOs are lipo chitooligosaccharide signal
molecules,
acting as phytohormones, comprising an oligosaccharide moiety having a fatty
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acid condensed at one of its end. An example of an LCO is presented below as
formula
CizORI
O CHIOR,
ORS O
01 R2 ORi O G
4 0
NH-R- õ
NH-00-Rs
in which:
G is a hexosamine which can be substituted, for example, by an acetyl group on
the nitrogen, a sulfate group, an acetyl group and/or an ether group on an
oxygen,
R,, R2, R3, R5, R6 and R7, which may be identical or different, represent H,
CH3CO-, CXHYCO- where x is an integer between 0 and 17, and y is an integer
between 1 and 35, or any other acyl group such as for example a carbamyl,
R4 represents a mono-, di- or triunsaturated aliphatic chain containing at
least 12
carbon atoms, and
n is an integer between 1 and 4.
More specific LCOs from R. meliloti have also been described
in 5,549,718 as having the formula II
35
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OR
/ OSO3H
CH2
CHOH CH2
HO 0 4 O
O
NH 0
NH
HO I HO I OH
I CO . HO NH
C-H CO
H-C 013
(k I -nz)s
CH
II
CH
I
ccH2)s
CHI
in which R represents H or CH3CO- and n is equal to 2 or 3.
Even more specific LCOs include NodRM, NodRM-1,
NodRM-3. When acetylated (the R = CH3CO-), they become AcNodRM-1, and
AcNodRM-3, respectively (U.S. 5,545,718).
LCOs from B. japonicum have also been characterized in
U.S. 5,175,149 and 5,321,011. Broadly, they are pentasaccharide
phytohormones comprising methylfucose. A number of these B. japonicum-
derived LCOs are described : BjNod-V (C18:1); BjNod-V (Ac, C18:1), BjNod-V
(C16:1); and BjNod-V (Ac, C16:0), with "V" indicating the presence of five N-
acetylgiucosamines; "Ac" an acetylation; the number following the "C"
indicating
the number of carbons in the fatty acid side chain; and the number following
the
":" the number of double bonds.
It shall also be understood that compositions comprising
different LCOs, are encompassed within the scope of the present invention.
Indeed, while the present invention is exemplified with LCOs obtained from B.
japonicum, R. leguminosarum and S. meliloti, and in particular NodBj-V(C18:1,
MeFeu), any LCO produced by a rhizobia which is capable of entering into a
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nitrogen fixation relationship with a legume (i.e. a member of the Fabiaceae
family) is expected to show the same properties as that of theLCOs exemplified
herein. It will be clear to the person of ordinary skill that the selection of
a
rhizobia known to be expressing LCOs at high levels, or known to express an
LCO having an effect on a broader spectrum of legumes (such as NGR234)
could be advantageous.
It will also be clear that the LCO compositions of the present
invention could also comprise more than one signal molecule. Non-limiting
examples of such compositions include agricultural compositions comprising in
addition to one LCO: (1) at least one additional LCO; (2) at least one plant-
to-
bacteria signal molecule; (3) gibberellic acid or other agents or compounds
known to promote growth or fitness of plants; and mixtures of such
compositions
(1), (2) or( 3).
It shall be clear that having identified new uses for LCO,
bacteria could be genetically engineered to express nod genes and used for
producing LCOs or for direct administration to the plants and/or seeds.
Thus, while the instant invention is demonstrated in particular
with LCOs from Bradyrhizobium japonicum, and a selected legume and non-
legume crops, the invention is not so limited. Other legume crops, non-legume
crops and rhizobial strains may be used using the same principles taught
herein.
Preferred matching of rhizobia with legume crop groups include, for example:
rhizobial species Legume crop group
R. meliloti alfalfa, sweet clover
R. leguminosarum peas, lentils
R. phaesolii beans
Bradyrhizobium japonicum soybeans
R. trifolii red clover
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As will be apparent to the person of ordinary skill to which the
present invention is directed, the growth-stimulating compositions of the
present
invention can be applied to other crop plants and especially to other warm
climate adapted crop plants (plants or crops having evolved under warm
conditions [i.e. tropical, subtropical or warm temperature zones] and whose
metabolism is optimized for such climates). It should be understood that the
photosynthesis-enhancing compositions of the present invention should find
utility whenever a particular crop is grown in a condition which limits its
growth.
For example, whenever a particular plant crop is grown at a temperature (or
under environmental parameters) which is below its optimum temperature for
photosynthesis and/or growth. Such temperatures are known in the art. For
example, optimum temperatures for germination of corn, soybean, rice and
cotton are 30 C, 34-36 C, 30-32 C, and 34 C, respectively. The minimum
germination temperatures (or base temperatures) for these crops are 9 C, 4C,
8 to 10 C, and 14 C, respectively, while the maximum germination temperatures
are 40 C, 42-44 C, 44 C and 37 C, respectively. The compositions of the
present
invention therefore find utility, among other things, in enhancing
photosynthesis
of warm climate adapted crops when grown at temperatures between their base
temperature for photosynthesis and/or growth. The compositions of the present
invention find utility in general in enhancing the photosynthesis rate and/or
growth of crop plants when grown under conditions which delay or inhibit the
photosynthesis and/or growth thereof. Non-limiting examples of such inhibiting
conditions (as known from their signalling inhibition in bacteria-legume
interactions, their inhibition or delay of the bacteria-plant symbiotic
relationship)
include pH stress, heat-stress, and water stress.
It will be nevertheless recognized that the compositions and
methods of the present invention also can enhance growth of plants grown under
optimal conditions.
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Thus, the compositions and methods of the present invention
should not be limited to plants growing under sub-optimal conditions.
The term "environmental conditions which inhibit or delay the
bacterial-plant symbiotic relationship" should be interpreted herein as
designating
5 environmental conditions which postpone or inhibit the production and
exchange
of signal molecules between same and include, without being limited thereto:
conditions that stress the plant, such as temperature stress, water stress, pH
stress as well as inhibitory soil nitrogen concentrations or fixed nitrogen.
"An agriculturally effective amount of a composition" for
10 increasing the growth of crop plants in accordance with the present
invention
refers to a quantity which is sufficient to result in a statistically
significant
enhancement of the photosynthetic rate, growth and/or yield (e.g. protein or
grain
yield) of the plant crop as compared to the photosynthetic rate and/or growth,
and/or yield of the control-treated plant crop. As will be seen below, the
15 photosynthetic and/or yield-promoting activity of the LCOs are observable
over a
broad range of concentrations. Indeed, LCO photosynthetic rate-promoting
activities can be observed at an applied concentration of about 10-14 to about
10-5 M, preferably about 10-12 to about 10-6 M and more preferably about 10-10
to
about 10-6 M and even more preferably about 10-10 to about 10-7 M. As shown
herein, however, the best of photosynthetic rate-promoting concentration of
LCO
depends on the growth conditions (e.g. controlled vs environmental) and on the
treated plant. A person skilled in the art will be able to adapt the range or
actual
concentration of LCO in the composition to satisfy his or her need.
While a direct method of inoculation with the composition of
the present invention is preferred, an indirect method can also be employed.
During direct inoculation the composition is applied directly onto the plant
and
preferably by foliar application. This can be accomplished, for example, by
spraying the leaves. The indirect method of inoculation would be based on an
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16
application of a rhizobia expressing an LCO of the present invention onto the
plant.
Foliar applications such as spray treatments of leaves are
well-known in the art. Of course, the method of administration of a
composition
of the present invention to the leaves can be adapted by a skilled artisan to
meet
particular needs.
The time at which the compositions and methods of the
present invention are effective in enhancing a plant's photosynthetic, and/or
growht, and/or yield thereof, in accordance with the present invention is from
as
soon as a leaf is present until physiological maturity of the plant. More
particularly, the administration of the composition should occur between the
seedling stage and the late pod filing stages. Thus, the administration can
occur
during the seedling, flowering and pod filing stages.
The recitation "short season condition" refers herein broadly
to temperatures of the middle and temperate zones and shorter. Typically, the
active growing season is around 1/2 to 2/3 of the year. Short season
conditions
broadly refers to a frost-free period of less than half the year, often on the
order
of 100 frost-free days.
By "nodulation gene-inducing" or "nod gene-inducing" is
meant bacterial genes involved in nodule establishment and function.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the invention, reference will
now be made to the accompanying drawings, showing by way of illustration a
preferred embodiment thereof, and in which:
Figure 1 shows the effect of lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu) over time on percent increase in photosynthetic rate of
soybean (cv Bayfield) under greenhouse conditions;
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Figure 2 shows the effect of lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu) on photosynthetic rate of corn (cv Pioneer 3921) (at the time
of maximum effect, 2 days after treatment)
Figure 3 shows the effect of lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu) on photosynthetic rate of rice (cv Cypress) (at the time of
maximum effect, three days after treatment);
Figure 4 shows the effect of lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu) on photosynthetic rate of canola (cv Springfield) (at the time
of maximum effect, two days after treatment);
Figure 5 shows the effect of lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu) on photosynthetic rate of melon (cv Nova) (at the time of
maximum effect, three days after treatment);
Figure 6 shows the effect of Lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu) on photosynthetic rate of apple (cv Empire) under field
conditions (at the time of maximum effect, five days after treatment)
Figure 7 shows the effect of lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu) on photosynthetic rate of grape (cv DuChaunac) under field
conditions (at the time of maximum effect, three days after treatment);
Figure 8 shows the effect of Lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu), over time, on photosynthetic rate of soybean (cvBayfield)
under field conditions; and
Figure 9 shows the effect of Lipo-chitooligosaccharide Nod Bj
V(C18:1, MeFeu) on photosynthesis of corn under field conditions (at the time
of maximum effect, two days after treatment).
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following non-
restrictive
description of preferred embodiments with reference to the accompanying
drawing which is exemplary and should not be interpreted as limiting the scope
of the present invention.
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DESCRIPTION OF THE PREFERRED EMBODIMENT
The research reported herein was conducted to study the
effects of foliar applications of LCO on the photosynthetic rates of a host
plant
(soybean) and non-host plants (rice, melon, canola, and corn) under green
house conditions. Also, field experiments were conducted to study the effect
of
LCO application on photosynthesis by corn, grape, apple and soybean. Field
experiments were also carried through to the examination of yield and yield
components.
During the course of work on the ability of LCOs to stimulate
seed germination of plants, it was observed that seedlings left exposed to a
composition comprising LCOs, following germination, continued to grow faster.
The possibility that an application of LCO to leaves of seedlings would
increase
their photosynthetic rates, leading to faster growth rates, was thus formerly
tested. It was thereby shown that LCOs increase the photosynthetic rates
and/or
yield of plants in general, as exemplified both under greenhouse conditions
and
under field conditions with a number of evolutionary divergent plants.
Lipochitin oligosaccharide (LCO) nod Bj V (C18:1, MeFeu)
isolated from Bradyrhizobium japonicum 532C was evaluated for its effect on
the
photosynthetic rates of a number of crop plants belonging to diverse botanical
families: soybean (Fabaceae) corn, rice (Poaceae), melon (Cucurbitaceae),
canola (Brassicaceae) apple (Rosaceae) and grape (Vitaceae). LCO enhanced
photosynthesis of all the plants tested. However, theextent of the responses
are
dependent on the plant species and the concentration LCO used. Under green
house conditions soybean (cv Bayfield) showed the largest increase in
photosynthesis due to LCO spray; on an average there was a 50% increase in
photosynthetic rate. As LCO application resulted in increased stomatal
aperture
without any increase in leaf internal C02 concentration, the data indicate
that
there was an increase in C02 uptake by chloroplasts, which lead to increased
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stomatal opening. LCO sprayed plants had more leaf area and dry weight than
water sprayed controls. Under field conditions LCO spray was tested on
soybean, corn, apple and grape plants. In the case of soybean the spray
applied
at the seedling, flowering and podfilling stages, resulted in increased branch
number, leaf area, pod number, plant dry matter and grain yield. LCO
application
enhanced grain yield by 33-44%. The data illustrate that LCOs can be used to
increase the productivity of a wide range of crops.
The present invention is illustrated in further detail by the
following non-limiting examples.
EXAMPLE 1
Production extraction and purification of lipo-chitooligosaccharides
(LCOs)
Bacterial culture
Bradyrhizobiumjaponicum (strain 532C) was grown at 28 C
in yeast mannitol medium (YEM) (Mannitol 1 Og, K2HPO4 0.5g, MgSO4 7H20 0.2g,
NaCl 0.1g, yeast extract 0.4g and distilled water 1000 mL), pH 6.8, shaken at
150 rpm until the OD620 reached 0.4-0.6 (4-6 days) in the dark. Thereafter, 2
L
of bacterial subculture was started by inoculating with material from the
first
culture (5 mL of the first culture per 250 mL of YEM media), for 5-7 days
(OD620
- 0.8-1.0), as above. At this stage, 0.25mL of50 pM genistein (in methanol)
was
added to each 250 mL of bacterial subculture (genistein concentration of 5 pM)
and the culture was incubated for 48-96 hours.
Extraction of LCOs
Two liters of bacterial subculture were phase-partitioned against 0.8
L of HPLC-grade 1-butanol by shaking overnight. The upper butanol layer was
transferred to a 1 L evaporation flask and concentrated to 2-3mL of light
brown,
viscose material with a rotary evaporator operated at 80 C (Yamota RE500,
Yamato, USA). This extract was resuspended in 4mL of 18% acetonitrile and
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kept in the dark at 4 C in a sealed glass vial until use. HPLC analysis
(Waters,
MA, USA) was conducted with a Vydac C18 reversed-phase column (Vydac, CA,
USA; catalogue # 218TP54) with a flow rate of 1.OmL min' and a Vydac guard
column (catalogue # 218GK54). As a baseline 18% acetonitrile (AcN/ H2O; w/w)
5 was run through the system for at least 10 min prior to injection. The
sample was
loaded and isocratic elution was conducted with 18% AcN for 45 min to remove
all non-polar light fractions. Thereafter, gradient elution was conducted for
90
min. with 18-82% AcN. The LCO was eluted at 94-96 min of HPLC run time.
The chemical identity of the LCO was confirmed by mass
10 spectrometer (MS-MS) analysis to be Nod Bj V (C18:1 MeFeu) (R. Carlson ,
Complex Carbohydrate Research Centre, University of Georgia, Athens, USA)
and by root hair deformation assay (Prithiviraj et al., 2000).
Plant Material
Briefly, seeds of soybean (cv AC Bravor) were surface
15 sterilized with 2% sodium hypochlorite for 2 min and washed with at least
four
changes of sterile distilled water. The seeds were then placed on 1.5% water
agar (20 mL) in 9 cm diameter Petri dishes (two seeds per plate). The Petri
dishes were incubated in the dark at 25 C for 7-8 days; during this time the
seeds germinated and developed tap and lateral roots on the agar surface.
20 Lateral roots with abundant root hairs, which could be easily distinguished
by the
fluffy appearance they imparted to the lateral roots, were excised with a
sterile
scalpel. These lateral roots were placed on sterile grease free glass slides
containing 40-60 pL of LCO solution. The slides were then placed in a moist
chamber and incubated for 24 h at 25C in the dark. At the end of the
incubation
time the slides were removed and the roots were fixed in a staining solution
[methylene blue (0.02% w/v) + glycerol (20% v/v) + phenol (10%w/v)]. Slides
were observed under a light microscope for root hair deformation.
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EXAMPLE 2
LCO treatment and data collection for
the greenhouse experiments
Plant treatment
Soybean (cv Bayfield) seeds were surface sterilized with 2%
sodium hypochlorite for 3-4 minutes, washed with several changes of sterile
distilled water and germinated in plastic trays containing sterile
vermiculite.
Seedlings at the two-leaf stage, about seven days of planting, were
transplanted
into 15 cm plastic pots containing promix (Premier Brands Inc., New Rochelle,
NY, USA). Pots were placed in a greenhouse maintained at 25 2 C with a
day/night cycle of 16/8h. Plants were watered as required.
Seeds of rice (Oryza sativa cv Cypress), canola (Brassica
napus cv Springfield), corn (Zea mays cv Pioneer 3921) and melon (Cucumis
melo cv Nova) were surface sterilized with 2% sodium hypochlorite for 3-4 min,
washed with several changes of sterile distilled water and planted in plastic
pots
(15 cm dia) containing promix (Premier Brands Inc., New Rochelle, NY, USA).
LCO treatment
Concentrations of LCO (10"6 M - 10-12 M) were made with
distilled water containing 0.02% Tween 20TM. A control treatment, containing
0.02% Tween 20, but no LCO was also applied. Since the rates of growth and
development differed among the plant species used in the experiments, spray
treatment was conducted at different times after planting In general, the
spray
was applied when the plants were big enough to allow easy measurements of
leaf photosynthetic rates. The following are the ages of the plants when the
sprays were conducted: soybean 21 days after planting (DAP), corn 25 DAP, rice
45 DAP, melon (35 DAP) and canola 30 DAP. The plants were sprayed with LCO
solutions until dripping. The sprays were applied with an atomizer (Nalgene,
USA). Each plant required 2-3 mL of spray solution. Each treatment was
replicated at least five times and organized on the green house bench in a
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randomized complete block design. Each experiment (with each crop species)
was repeated at least twice.
Data collection
Photosynthesis was recorded every 24h using a Li-Cor 6400
portable photosynthesis system (Li-Cor Inc., Lincoln, Nebraska, USA) for 6
days.
In the case of soybean the photosynthesis in the second nodal leaf from the
top
was recorded while in the other species used in the photosynthetic rate was
measured for the top-most fully expanded leaf. Soybean plants were harvested
after seven days of LCO treatment and dried at 80 C for 48 h. Data were
analyzed with the Statistical Analysis System (SAS Inc., NC, USA). Percent
increase in photosynthesis over the control was calculated. Multiple means
comparisons were conducted with an ANOVA protected LSD test, thus, the LSD
test was not performed if the ANOVA test did not indicate the presence of
differences due to treatment.
EXAMPLE 3
Field Experiments (year 1999)
Soybean
The soybean experiment was conducted at the Lods
Agronomy Research Centre, McGill University, Macdonald Campus, Ste-Anne-
de-Bellevue, Quebec, Canada during the period June to September, 1999. A
randomized complete block design with three blocks was followed. The plot size
was 2 x 4 m with a row to row spacing of 25cm and 10 cm between plants within
a row. Seeds of soybean (cv OAC Bayfield), treated with commercial
Bradyrhizobiumjaponicum inoculant (Bios Agriculture Inc., Quebec, Canada) at
the rate of 3g per kilogram of seed, were hand planted.
At 25 days after planting twenty plants in each plot were
randomly marked and sprayed until dripping with LCO solutions (10-6, 10.8 and
10-10M) containing 0.02% Tween 20 with a hand sprayer. The plants on either
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side, within the row, of the marked plants were also sprayed. A second spray
was carried out at flowering stage and a third spray at pod filling.
Apple, Grape and Corn
These experiments were conducted at the horticultural
research facility of McGill University, Ste-Anne-de-Bellevue, Quebec, Canada
during July 2000. LCO of different concentrations (10-8, and 10-10M) were
prepared as described above. Branches of apple (cv Empire) and Grapes (cv
De Chaunac) were sprayed with LCO and the photosynthesis was observed
every 24h for five days with a Li-Cor 6400 portable photosynthesis system (Li-
Cor Inc., USA). Each treatment was applied to three branches from the same
plant. Care was taken to ensure that the branches were on the same level and
orientation. Part of each branch was sprayed with LCO and the remaining part
served as a control. The control portion of the branch was sprayed with
distilled
water containing the same amount of Tween 20 as the LCO treatment solution.
Observations were taken on 15 leaves per replicate for each treatment. For
both
apple and grape the entire procedure was repeated twice on two different
plants.
Single row corn plots (Pioneer 3921) were established during
the 1999 and 2000 cropping seasons. The rows were 75 cm apart andtheir was
an average of 20 cm between plants. The plants were sprayed at 40 DAP.
Photosynthetic rates were recorded each day for 5 days after spray
application.
However, multiple sprays of LCO on corn were not possible due to limitations
of LCO supplies, and because only single row plots were used yields were not
recorded.
Field Data collection
As with the indoor experiments, photosynthetic readings were
taken every day for five days after the application of LCO. For soybean
additional
developmental and agronomic data were collected. The first harvest was
conducted at 25 days after the first spray treatment. Five plants were
harvested
from each plot and the following growth variables were analyzed: plant height,
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number of branches, number of leaves, leaf area, number of flower clusters,
number of pods, number of nodules, dry weights of leaves, stem and roots. The
final harvest was conducted after physiological maturity of the plants Fehr et
al.,
1971); at this time the remaining fifteen treated plants from each plot were
harvested and data on number of branches, number of pods, number of seeds
and grain yield per plant was collected.
EXAMPLE 4
Effect of LCOs on the photosynthetic rate of soybean and non-legumes
under greenhouse conditions
LCO spray increased the photosynthetic rate of soybean
even at very low concentrations (Table 1).
Table 1. Effect of lipo-chitin oligosaccharide (Nod Bj V (C18:1, MeFeu))
on photosynthesis ( mol m_2 sec-1) of soybean under greenhouse
conditions.
Treatment Days after treatment
1 2 4 5 6
Control 11.2 d@ 8.1 c 10.1 d 12.1 c 10.4 a
10-6 14.9 ab 12.1 a 16.2 a 16.7 ab 13.1 a
10-' 12.1 cd 9.1 be 12.9 bcd 14.1 be 11.1 a
10-8 13.8 b 8.1 be 12.3 cd 16.4 ab 10.3 a
10-9 15.8 a 8.4 bc 15.7 ab 17.5 ab 11.2 a
10-10 13.6 be 8.7 be 14.6 abc 16.9 ab 11.2 a
10-11 14.0 b 8.7 bc 17.4 a 17.9 a 12.5 a
10-12 15.0 ab 10.3 ab 16.9 a 17.0 ab 12.0 a
LSD (p<0.05) 1.67 2.24 3.15 3.78 2.87
@ means with in the same column, followed by the same letter are not
significantly different
(p < 0.05) by ANOVA protected LSD test.
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The photosynthesis rate increased from day 1 up to day 4
after which it decreased and by day 5 it generally reached levels not
different
from the control plants. However, the maximum increase in photosynthesis was
observed on day four in most treatments. Percent increase in photosynthesis
5 over the control varied with the concentration of LCO spray (Fig. 1). LCO at
10-
"M caused the greatest increase in photosynthetic rate followed by 1012 M,
with
these maxima occurring at four days after treatment, while other
concentrations
caused more sustained increases in photosynthesis, that remained higher than
the control for more extended periods of time. LCO treatments caused an
10 increase in the leaf area and dry weight of soybean at seven days after
treatment
(Figs. 2 and 3). shoot dry weights of treated plants were statically (p <
0.05)
higher than those of the control plants, while leaf areas were only increased
numerically (p = 0.09).
LCO treatment also enhanced the photosynthetic rates of
15 non-legumes: corn (Fig. 4), rice (fig. 5), canola (fig.6) and melon (fig.
7). It was
evident that the days for maximum increase and the most effective
concentration
of LCO differed among the species. In general a 10-20% increase in
photosynthesis was common. For the C3 plants (rice, melon, canola) the
increased in photosynthetic rates were always accompanied by a concomitant
20 increases in stomatal conductance and transpiration while the intercellular
CO2
concentration was unaffected by the treatments. For corn (a C4 plant) LCO
application increased photosynthetic rate, decreased leaf internal CO2
concentration and did not significantly alterstomatal aperture, These data
argue
that the increase in photosynthetic rate was due to an increase in
photosynthetic
25 uptake of C02 inside the leaf, which, in the case of C3 plants, triggered
an
increase in stomatal aperture. Had it been the case that increased stomatal
aperture was the primary cause of the increased photosynthetic rates one would
have expected increases in the internal CO2 concentration of the leaf
(Morison,
1998).
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EXAMPLE 5
Effect of LCOs on the photosynthetic rate, growth
and yield of soybean and non-legumes grown
under field conditions (year 1999)
Grape, Apple and Corn
LCO spray also caused increases in the photosynthetic rates
of field-grown apple and grape (Figs 8 and 9). In case of apple,
photosynthesis
increase peaked at five days after treatment; the 10-$ M LCO treatment
resulted
in a photosynthetic rate of 14.1 mol CO2 m-2s-1, while the rate was 10.8 mol
C02 m_2 s' for the control. As with the other crops there were increases in
stomatal conductance without any effect on theCi. LCO treatment also increased
transpiration (Fig. 6). In grapes, the greatest increase in photosynthetic
rate
occured three days after treatment with the 10-'OM LCO treatment, and this
resulted in a concomitant increase in stomatal conductance. LCO application
increased the photosynthetic rate of field grown corn by a maximum of
approximately 10% (Fig. 10) at two days after treatment application. While LCO
application did cause reduced Ci levels in the greenhouse (p = 0.05) there was
no such effect on Ci in field grown plants.
Soybean
In general, the photosynthetic responses of soybean in the
field were similar to those observed under greenhouse conditions. LCO
treatment resulted in increases in the photosynthetic rates from day one to
day
four after application. The most effective concentration was 106 M, which
resulted in a photosynthetic rate of 24 mmol M-2 sec' on day three as compared
to 20 mmol m-2sec' for the control (Fig. 10). The increase in photosynthetic
rate
was accompanied by increases in stomata) conductance; again the 106 M LCO
treatment resulted in the highest stomatal conductance values. However, the
effect of LCO in the field grown plants were less pronounced than for green
house gown plants and required higher concentration for better effects. The
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27
requirement for higher concentrations may have been due to leaf anatomical
differences; field grown plants usually have thicker cuticles than green house
grown plants. It might also have been the case that epiphytic microorganisms,
or the leaves themselves, may have produced chtinases that degraded the LCO.
Given the likelihood of lower levels of microbial activity under greenhouse
conditions, both of these could have contributed to the need for higher LCO
concentrations in the field than the greenhouse. The lower degree of response
under field conditions may have been due the greater environmental
variability,
and increased likelihood of at least some other stresses imposing limitations,
at
least some of the time, under field conditions. Raschke et al., (1979)
observed
differences in stomatal sensitivity to C02 level between green house and field
grown maize. Similarly, Talbott et al. (1996) showed differences in stomatal
sensitivity to C02 between growth cabinet and greenhouse plants. LCO
treatment resulted in increased transpiration, probably due to increased
stomatal
aperture.
LCO spray resulted in increased growth of soybean plants.
There were increases in the following growth variables: number of branches,
number of leaves and leaf area. However, plant height was not affected by LCO
treatment. There also increases in the yield variables number of pod clusters
per
plant, number of pods and total number of seeds per plant. The latter resulted
in increases in seed yield that ranged from 33.7 to 44.8% (Table 2).
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Table 2. Effect of lipo-chitin oligosaccharide
(Nod Bj V (C18:1, MeFeu)) leaf area and shoot
dry weight of soybean under greenhouse conditions
Treatment Leaf area (cm2) Shoot dry weight
(mg)
Control 188.0 ab@ 951.6 b
10-6 223.6 a 1065.8 ab
10-7 193.6ab 1135.2a
10_$ 217.6 ab 1095.7 ab
10-9 194.3 ab 999.9 ab
10-10 195.3 b 1012.3 ab
1011 202.6 ab 931.6 b
10-12 230.3 a 1060.8 ab
LSD (p<0.05) 36.2 168.3
@ means with in the same column, followed by the same letter are not
significantly different
(p < 0.05) by ANOVA protected LSD test.
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Cu
r` (0
N N 6) ln N N
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L O N CCU: (ci
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The results presented demonstrate that foliar application of
LCO Nod Bj (C1:18 MeFeu) causes enhanced photosynthesis in both host and
non-host plants. For C3 plants the increase in photosynthesis was always
accompanied with increases in stomatal conductance, although without change
5 in Ci values, while for corn (a C4 plant) the stomata) aperture did not
increase
and the Ci values delined under green house conditions. In both cases the data
indicate that increases in photosynthesis due to LCO treatment is due to more
efficient C02 uptake inside the leaf. For the C3 plants this lead to increased
stomatal aperture. Because the stomata of the C3 plants were more opened
10 there were concomitant increases in transpiration for the leaves of LCO
treated
plants. These results were similar to those observed for glycinebetanine
application (Rajasekaran et al., 1997; Makela et al., 1999). Foliar
application of
glycinebetanine enhanced net photosynthesis and water use efficiency and
mitigated drought and salinity stress. Increased stomatal conductance have
been
15 positively correlated with the yield in a number of crops and it has been
suggested that selection for increased stomata] conductivity will result in
enhanced yields (Lu et at., 1998; Morrison et at., 1999). The link between
stomatal aperture and photosynthetic rate would seem to apply in the case of
the
C3 plants tested here, although, it is clear that, the case of LCO
application, the
20 more open stomata were the result of greater photosynthetic CCU uptake by
the
chloroplasts, and not the primary cause of increased photosynthetic rates.
Dinitrogen fixation is energy intensive process. About 10-20%
of the photosynthates of a nitrogen-fixing legume are consumed in N2 fixation.
If this were not compensated by an increase in net photosynthesis it would
lead
25 to reduction in the crop yield as compared to plants receiving nitrogen
fertilizer,
and such photosynthetic compensation has been demonstrated (Imsade, 1983).
However, mechanisms by which plants compensate for the increased demand
during this, and other, plant-microbe interactions are unknown. Our work
suggests that this might be controlled by the LCO bacteria-to-plant signal
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31
molecules. Several lines of evidence suggest that nodulated soybean plants
have higher net photosynthetic rates than those acquiring their nitrogen from
mineral forms available in the rooting medium (lmsande, 1989a,b). This might
be
brought about either by increase in photosynthesis due to improved efficiency
in
the dark reactions or by enhanced efficiency of the photosystems as reported
by
Maury et al. (1993), or both.
Recently, Phillips et al. (1999) isolated lumichrome, a
breakdown product of riboflavin, in the rhizosphere of alfalfa plants during
early
nodulation and showed that it caused increased respiration and photosynthetic
carbon fixation. In an earlier experiment we observed enhanced germination and
early growth of diverse crop plants due to LCO treatment (unpublished results)
and this led us to hypothesize that LCO improves early growth through
increased
photosynthesis. The results of the present experiment support the above
hypothesis. Identification of specific high affinity receptors for LCOs
remains
elusive. However, two class of receptors for LCO have been characterized
recently (Stacey et al., 2000; Bono et al., 1995; Gressent et al., 1999). This
led
us to hypothesize that one of these receptors is associated with the
nodulation
process and the other with a more generalized process that triggers the growth
machinery of plants when exposed to chitin and related compounds, such as
LCOs. The observation that this stimulation occurred in such a wide variety of
angiosperms (the work reported here shows effects in five plant families, all
angiosperms: Poaceae, Fabaceae, Brassicaceae, Rosaceae, Vitaceae)
suggests that this LCO response mechanism is at least as old as the
angiosperms. There are several reports of the presence of nod factor
responsive genes in non-legumes such as rice (Kouchi et al. 1999; Reddy et al
1998). These may play a role in the detection of, and response to, plant
pathogens, many of which contain chitin in their cell walls. Presumably, more
vigorous growth is a response to the presence of a detected pathogen. There
are several reports of enhanced photosynthesis due to fungal pathogens (Ayers,
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1979; 1981) this might be due to the stress responses of the plant and could
be
mediated by cell wall fragments that are chitin oligomers.
The phenomenon of enhanced photosynthesis and yield due
to application of LCO, as observed in this study, might explain, at least in
part,
the increased productivity of legume-non legume intercropping systems and crop
rotations. Hungria and Stacey (1997) reported enhanced growth and yield of
intercropped corn and bean as compared to the monocrops and postulated that
this increase might be due to the reciprocal stimulation ofA. lipoferum and R.
tropici in the soil by the root exudates of corn and bean. To our knowledge
this
is the first report of LCO enhancement of photosynthesis in legumes and non-
legumes. LCOs, besides mediating the early events of nodulation, also act as
signals for enhanced photosynthesis in a number of plants and this opens the
possibility of harnessing these signal molecules for improving crop
production,
and ultimately, world food production.
EXAMPLE 6
Effect of LCOs on the photosynthetic rate of
Soybean and corn grown under field conditions
(year 2000)
Rhizobium Leguminosarum (127K105) and Sinorhizobium
meliloti (RCR 2011) were cultured in modified Bergerson minimal media (Spaink
et al., 1992) for four days, when the OD (620) of the culture had reached 0.37
for
S. meliloti and 0.28 for R. leguminosarum, isoflavonoid nariginin was added to
R. leguminosarum to final concentration of 5 M and luteolin at 5 M was added
to S. meliloti. The cultures were further incubated for five days and they
were
extracted using the method as described for Bradyrhizobium japonicum. LCO of
R. leguminosarum eluted at 27-31 min of HPLC run while that of S. meliloti
eluted
at 35-38min.
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LCO of R. leguminosarum enhanced photosynthesis of
soybean and was more effective as compared to the LCO of S. meliloti. LCO
from S. meliloti enhanced the photosynthesis of corn (Tables 4 & 5).
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Table 4. Effect of LCOs of Rhizobium leguminosarum (127K105) and
Sinorhizobium meliloti (RCR 2011) on photosynthetic rates of soybean
(cv Bayfield) two days after treatment
Treatment Photosynthe Conductance Ci (mmol Transpiration
sis ( mol (mol H2O m-2 s') C02 mol-') (mmol H2O m-
C02 m-2s') 2s')
LCO from
Rhizobium 14.5a 0.26a 277.Oa 3.42ab
Leguminosarum
(127K105) 10-
61
LCO from
Rhizobium 16.Oa 0.29a 272.6a 4.2a
Leguminasarum
(127K105)
10-8M
LCO from
Sinorhizobium 12.36b 0.15bc 241.0b 2.89b
meliloti (RCR
2011)
10-6M
LCO from
Sinorhizobium 14.5a 0.26ab 272.3 a 2.89b
meliloti (RCR
2011)
10-8M
Control 12.03b
0.13c 224.3b 2.72
LSD (p<0.05) 2.12 0.10 28.9 0.96
In column numbers followed by same letters are not significantly different
(p<0.05) by ANOVA protected LSD test.
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Table 5. Effect of LCOs of Rhizobium leguminosarum (127K105) and
Sinorhizobium meliloti (RCR 2011) on photosynthetic rates of corn (cv
Pioneer 3921) two days after treatment
5
Treatment Photosynth Conductance Ci (mmol Transpiratio
esis ( mol (mol H2O m-2 s') CO2 mol-') n (mmol
CO2 m-2s') H2O m 2s 1)
LCO from
Rhizobium 25.9ab 0.13b 63.8a 3.3bc
Leguminasarum
(127K105)
10-6M
LCO from
Rhizobium 30.5ab 0.17ab 78.3ab 4.1 ab
Leguminasarum
(127K105)
10-8M
LCO from
Sinorhizobium 26.9b 0.14b 57.4ab 3.4bc
meliloti (RCR
2011)
10-6M
LCO from 35.1 a 0.21a 88.5ab 4.9a
Sinorhizobium
meliloti (RCR
2011) 10-8M
Control 23.1 b 0.11 42.6b 2.7
LSD (p<0.05) 7.9 0.07 42.9 1.3
In column numbers followed by same letters are not significantly different
(p<0.05) by ANOVA protected LSD test.
Table 6 shows the effect of foliar spray of LCO on yield
of soybean during the year 2000. LCO enhanced all the yield
components, LCO at 10-6 M and 10-10 M showed the maximum effects.
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LCO 10-6 M improved the yield by about 60%. The increase in yield was
due to the increase in the number of pods/ plant. The 100-seed weight
was not increased by LCO spray during the 2000 field season.
Table 6. Effect of LCO on yield of soybean (2000 cropping season)
Treatment Pods/ Pod weight/ Seeds/ 100-seed Seed Seed
plant plant (g) plant weight yield/pl yield
(g) ant (t/ha)
(g)
LCO10-6M 46.8 a 31.4 a 118.0 a 17.4 a 21.0 a 10.5 a
LCO 10-8M 39.1 b 24.1 b 96.4 b 17.8 a 16.2 b 8.1 b
LC010-10M 47.7 a 29.1 ab 117.7 a 18.1a 21.8 a 10.9 a
Control 28.3 c 18.7 c 70.4 c 17.7 a 13.2 b 6.6 b
LSD 7.6 5.2 18.4 2.7 3.8 1.9
In columns numbers followed by same letters are not significantly different
(p<0.05) by
an ANOVA protected LSD test.
Yields are at 0% seed moisture.
Yields were calculated by sampling 10 randomly selected plants per plot,
determining
the yield per plant and assuming an average stand of 500,000 plants per ha.
Taken together, the results of Tables 4 and 5 show that the
photosynthetic rate-promoting effects observed with the B. japonicum LCO
NodBj-V(C18:1, MeFeu) during the 1999 experiments are also observable with
LCOs obtained from other rhizobia. Thus, addition of the promiscuous rhizobial
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strain NGR234, known to promote the nodulation of a wide range of legumes or
others, are also expected to enhance the photosynthetic rate of plants
similarly
to the data presented herein.
Data on the effect offoliar spray of LCO on yield of soybean
during the year 2000 also shows a yield-increasing effect, similar to that
shown
in the year 1999. More specifically, in 2000, LCO application enhanced
pods/plant and seed yield. Data suggests that (1) LCO at 10-6M showed the
maximum effect; (2) LCO at 10-6M improved the yield by more than 100%; and
(3) the increase in yield was due to the increase in the number of pods/
plant.
It is noteworthy that the 1999 and 2000 cropping seasons
were very different. As compared to an average cropping season, 1999 was a
hot-dry year while 2000 was a cold-wet year.
Taken together with the 1999 results of the effects of LCO
application on the photosynthetic rate and on yield, those of 2000 show that
the
LCO effect thereon is robust over a wide range of environmental conditions.
Also of note, the LCO application in the field experiments in
year 2000 were by spraying whole plots, as opposed to individual plants
(1999).
Thus, the LCO effects described in the present invention are also observable
when using large production application methods.
CONCLUSION
The present invention demonstrates that LCO composition
can significantly enhance the photosynthetic rate of legumes and non-legumes
grown under laboratory conditions (e.g. greenhouse conditions). Furthermore,
these greenhouse condition results are validated in the field using soybean,
grape, corn and apple. The LCO effect is further shown to be observable with
different LCOs, thereby validating the photosynthetic rate-enhancing activity
of
LCOs in general. In addition, the present invention shows that the
photosynthetic
rate-enhancing effect of LCOs on plants is robust across the environment field
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conditions. The similar increases in photosynthetic rates and yield for the
tested
crop (e.g. soybean) imply that yield increases are to be expected from LCO
application on a wide range of crops. The present invention thus provides
agricultural compositions and methods by which LCO can be used to enhance
the photosynthetic rate, growth and yield of a crop under controlled and
diversified field conditions.
Although the present invention has been described herein
above by way of preferred embodiments thereof, it can be modified, without
departing from the spirit and nature of the subject invention as defined in
the
appended claims.
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