Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
PEA AND LENTIL PRODUCTION-ENHANCING
COMPOSITIONS AND METHOD USING SAME
FIELD OF THE INVENTION
The present invention relates to agriculture. More
particularly, the present invention relates to the promotion of growth of
peas and/or lentils. More particularly, the invention relates to the
development of pea and/or lentil production-enhancing compositions
comprising a signal molecule and to methods using same. The invention
also relates to compositions and methods for increasing nodulation,
nitrogen-fixation and grain yield of peas and/or lentils. In particular, the
invention relates to an increase in nodulation, nitrogen fixation and grain
yield of peas and/or lentils grown under field conditions which inhibit or
reduce nitrogen fixation and/or yield.
BACKGROUND OF THE INVENTION
The knowledge that elements in the soil influence root
nodulation has long been recognized. Indeed, the Romans transferred
soil from successful legume fields to unsuccessful ones in order to
improve the quality of the latter. It has since then been demonstrated that
one important soil element responsible for nodulation is soil bacteria. The
family Rhizobiaceae consists of a heterogeneous group of gram-negative,
aerobic, non-spore-forming rods that can invade and induce a highly
differentiated structure, the nodule (on the roots, and in some instances,
stems of leguminous plants), within which atmospheric nitrogen is
reduced to ammonia by the bacteriod. The family Rhizobiaceae contains
four genera, Rhizobium, Bradyrhizobium, Sinorhizobium and
Azorhizobium. The host plant is most often of the family Leguminosae.
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The element nitrogen (N) is essential to all living
organisms because it is a component of many biologically important
molecules. The most important of these include nucleic acids, amino
acids, proteins, and porphyrins, which occur in large amounts in all living
cells. To be able to multiply, grow, or just survive, organisms require a
source of N. The ability to reduce atmospheric dinitrogen (N2) is limited
to prokaryotes. Legumes and a few other plant species have the ability
to fix atmospheric N through symbiotic relationships. In the case of
legumes, N2 fixation is carried out by prokaryotes, Rhizobium or
Bradyrhizobium in nodules located on the plant root (Sprent and Sprent,
1990). Symbiotic association takes place in highly specialized root
organs known as nodules that result from the association between the
host plant and endosymbiotic Rhizobia. Inside these nodules, the
bacteriod provide reduced nitrogen to the plant while in return the plant
provides carbon and energy to the rhizobia. Symbiotic nitrogen fixation is
dependent on the genotypes or both the host plant and the Rhizobium
strain and the interaction of these symbionts with the pedoclimatic factors
and the environmental conditions. In this process, bacterium-plant
interactions and communications are highly specific. For instance, R.
leguminosarum nodulates pea (Pisum) while R. meliloti nodulates alfalfa
(Medicago). Nodulation compatibility between a particular legume cultivar
and a rhizobial strain is determined by the presence of appropriate genes
carried by both the plant and the microbe. The molecular mechanisms of
recognition between Rhizobium and legumes can be considered as a
form of cell-to-cell interorganismal communication. A precise exchange
of molecular signals between the host plant and rhizobia over space and
time is essential to the development of effective root nodules. The first
apparent exchange of signals involves the secretion of phenolic
compounds, flavonoids and isoflavonoids, by host plants. These signal
compounds are often excreted by the portion of the root with emerging
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root hairs, a region that is most susceptible to infection by rhizobia
(Verma, 1992). These compounds have been shown to activate the
expression of nod genes in rhizobia, stimulating production of the
bacterial Nod factor (Kondorosi, 1992). This Nod factor has been
identified as a lipo-chitooligosaccharide (Carlson et al., 1993), able to
induce many of the early events in nodule development, including
deformation and curling of plant root hairs, the initiation of cortical cell
division, and induction of root nodule meristems. In pea for example,
naringenin, hesperetin and luteolin are the major components of plant
root exudates which induce the nod genes of R. leguminosarum. Other
such substances active at very low concentrations (10-6 to 10-' M) have
been shown to stimulate bacterial nod gene expression within minutes.
However, the effectiveness of isoflavonoids is found to vary between
cultivars.
The "common" nod genes, designated nodA, 8 and C,
which are associated with the early stages of infection and modulation, are
structurally conserved among Rhizobium strains. In R. meliloti, R.
leguminosarum, and R. trifolii, the nodA, 8 and C genes are organized in
a similar manner and are believed to be coordinately transcribed as a
single genetic operon. The DNA region adjacent and 5' to nodA has been
found to contain a fourth modulation gene, designated nodD, which is
transcribed divergently from the nodABC operon. nodD has been found
to function in the regulation of expression of nodABC and other
modulation genes. nodD has been shown to interact with the flavonoid
from a host plant and to initiate the coordinated expression of nod genes
for the production of lipo-chitooligosaccharide return signal molecules.
Comparisons of the DNA sequences and the deduced
amino acid sequences of the encoded nodD product confirm the
presence of significant sequence conservation of these genes among
strains of Rhizobium. nodD mutants in the various species of Rhizobium
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do not, however, display the same nodulation phenotypes. It now
appears that many species of Rhizobium carry multiple nodD-like genes,
on their Sym plasmids.
Another similarity in the nod regions) of Rhizobium
strains is the presence of conserved sequence elements within the
promoter regions of certain inducible nod genes. These conserved
sequences, first identified in the nodABC promoter region, are termed the
nod-box and are believed to function in induced nod gene expression,
possibly as regulatory protein binding sites.
The structure of the lipo-chitooligosaccharide signal
molecules are species-dependent and determined by the host specific
genes which encode enzymes that modify the basic lipo-
chitooligosaccharide molecules, thereby contributing to host specificity.
The chemical nature of the flavonoid as well as the NodD sequence is
partly responsible for the host specificity of the legume-Rhizobium
interaction. The capacity of a flavonoid to interact with a nodD gene
product is strongly affected by its molecular structure. The structure of the
nod gene inducers of R. leguminosarum, R. trifolii and R. meliloti was
found to be difFerent (Spaink et al., 1987). Numerous flavonoid structures
have been reported as natural nod gene inducers from various legumes,
including flavanone, flavones, flavonols and isoflavones (Verma, 1992).
Individual species can release numerous aglycone nod gene inducers.
Thus, nod gene-inducing flavonoids have usually been identified by using
bacterial strains containing a suitable nod gene and an inducible nod
promoter fused to the Escherichia coli IacZ reporter gene. With these
constructs, nod gene expression can be monitored as ~3-galactosidase
activity (Van Brussel et al., 1990) in in vitro experiments.
The specific components of legume exudate that act to
induce nodulation genes in several species of Rhizobium and
Bradyrhizobium have been identified as flavonoids. Luteolin was reported
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to be the component of alfalfa exudates that induces nodABC expression
in R. meliloti. Three clover exudate constituents: 4',7-dihydroxyflavone,
geraldone and 4'-hydroxy-7-methoxyflavone were reported to induce the
nodulation genes of R. trifolii. Two pea exudate components: eriodictyol,
5 and apigenin-7-O-glucoside were reported to induce the nodulation genes
of R. leguminosarum. In addition, molecules having structures related to
those of the inducers found in exudates, were assessed for their ability to
induce. Inducers of Rhizobium nodulation genes appear in general to be
limited to certain substituted flavonoids, and the range of compounds to
which a Rhizobium responds is species specific. Since host range is
used to classify Rhizobium strains into different species, this suggests
that differential response to inducer molecules is involved in the
mechanism of determination of host range.
In view of the above, it is clear that the exchange of
signals between legume and bacterial strain and intricacies thereof, while
very complex, are shared between different legumes and the Rhizobium
and Bradyrhizobium genera. The manner in which nodulation genes are
regulated is also conserved among Rhizobium and Bradyrhizobium
strains.
While combinations of rhizobia and plants can be
compatible, nodulation failure can still occur in the field (Robson and
Bottomley, 1991). Poor nodulation which can lead to substantial loss of
yield, has been attributed to a range of environmental conditions,
including unfavorable soil pH, high salinity, presence of ions such as
nitrate and deficiencies in essential elements, including calcium (Zahran,
1999). Furthermore, soil temperature is an important environmental
variable, which affects legume nodulation and nitrogen fixation. In
addition to the reduction in the nodulation at temperature extremes, there
are also specific temperature-sensitive legume-Rhizobium combinations.
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The inability of a legume to nodulate strongly is often
attributed to a breakdown of the early events of nodulation such as
stimulation of root hair curling and formation of infection threads.
Unfavorable environmental conditions are often the culprit. Factors that
have been proposed to restrict these early events of nodulation (and are
often referred to as stress conditions) include salinity (Zahran and Sprent,
1986), low levels of calcium or phosphorus (Hicks and Loynachan, 1987)
and temperature (Zhang et al., 1996). Molecular techniques have shown
how changes in environmental conditions can affect the production of
signal molecules by legumes in vitro. For example, the exudation from
subclover roots of flavonoid compounds required for nod gene induction
in R. leguminosarum bv. trifolii was reduced when the plants were grown
at a pH <5 (Richardson et al., 1988).
The presence of combined nitrogen also limits the
nodulation of legumes while nitrogen (as ammonia) has been shown to
limit the induction of the nodA8C genes (Dusha et al., 1989).
Temperature affects legumes non-specifically and
through plant metabolic processes such as respiration, photosynthesis
and transpiration. The temperature range for the symbiotic system is
narrower than that of the plant supplied with fertilizer nitrogen. Symbiosis
ceases when it is exposed to extreme temperatures. Low temperatures
delay root hair infection, and decrease nodulation and nitrogenase
activity. It has been noted that all stages of nodule formation and
functioning are affected by suboptimal root zone temperatures (RZTs)
and experiments have generally indicated that early nodule development
processes are the most sensitive. Initiation and establishment of nitrogen
fixation in alfalfa is subject to severe constrains at low temperatures: in
controlled environment studies with this legume, growth is reduced to
24% and 75% at root temperatures of 13°and 8°C, respectively
when
compared to growth at 21 °C (Cralle and Heichel, 1982). It has also
been
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reported that in the case of soybean, the time between inoculation and
onset of dinitrogen fixation is delayed by 2 to 3 days for each degree
decrease in temperature from the optimum 25°C (Zhang et al., 1995).
Nodulation is completely inhibited when plants are grown under
10°C.
Finally, low temperature was found to decrease both the biosynthesis of
isoflavonoids and the excretion of those signal compounds from plant root
cells to soil rhizosphere (Zhang et al., 1995).
Grain legume crops play an important role in agricultural
production, primarily through their role in protein and fat production for
animal and human nutrition. Although numerous legume species are
cultivated, few legumes are suitable for growing in cool season areas
including Canada.
Pea and lentil, which have been adapted in temperate
to subtropical regions, are some of the most important legume crops in
Canada. They are cultivated on a total area of 2.6 and 1.0 million acres,
respectively, and are used for grain as well as for soil improvement in
crop rotations. In order to maximize the yield potential of a pulse crop, a
number of production factors, including fertility of the field, must be taken
into account. A sustainable alternative to nitrogen fertilizer for legume
crop is the atmospheric nitrogen fixation in symbiotic association with
microbes.
Production of N fertilizer, in Canada as elsewhere, is
economically ($1 billion per year in Canada), energetically (equivalent to
million barrels of oil per year) and environmentally (produce 15 million
25 tones of C02 per year, ground water-polluting N03 and ozone-destroying
NOX) expensive. In eastern Canada the farm community spends
approximately $150 x 106 per year for N fertilizer. Nitrogen fixation is the
sustainable alternative to N fertilizer. Therefore, an understanding of the
mechanism of suboptimal RZT effects on pea and lentil nodulation and
30 N2 fixation and finding methods to reduce this restriction by low RZT
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would allow an increased use of this NZ fixing cash crop, and a decreased
reliance on potentially polluting N fertilizers in cool season areas. The
ability to overcome the negative effects of suboptimal RZTs could also be
applied to other conditions that negatively affect nitrogen fixation (water
stress, high pH, temperatures etc.).
Due to the number of benefits which can result from the
establishment of rhizobia:legume symbiosis, a number of strategies have
been devised to promote nodulation of legumes.
US patent 4,878,936 to Handelsman et al., teaches a
method for enhancing nodulation of legumes which includes inoculation
in the immediate vicinity of the roots thereof, an effective quantity of
bacteria which enhance nodulation. However, the results are based on
controlled laboratory conditions, not on field studies. Moreover, the
laboratory conditions used, involved temperatures above 25°C which are
not expected to be limiting for nodulation.
US patent 5,141,745 to Rolfe et al., discloses flavones,
some of which are leguminous plant exudates, which induce expression
of certain nod genes in Rhizobium strains. Rolfe et al., however, do not
assess whether their results, all obtained under laboratory conditions,
translate into increase nodulation and growth of the leguminous plant
under field conditions.
The art is replete with examples demonstrating that
results obtained under the laboratory setting are not predictive of the field
situation. Typically, a good controlled environment provides optimal
levels of soil nutrients, soil pH, soil moisture, air humidity, temperature
and light. The plants are usually widely spaced so that they do not
compete for light. In some cases environmental factors such as carbon
dioxide may even be optimized. The field environment is vastly more
complicated than that of the controlled environment setting. The soil
varies in its chemistry and texture in a fractal pattern, such that, while the
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soil of a research site can be characterized in general, it is variable at
every level within the confines of the experimental area. In a controlled
environment setting plants are usually produced in sterilized rooting
media (pasteurized soil, sterile sand, or some form of artificial rooting
media) and there is no soil micro flora or fauna. Field soil, on the other
hand, is an ecosystem; it contains an enormous number of bacteria,
fungi, protista, algae, and soil insects. The climate and related
atmospheric factors (light intensity, relative humidity, temperature,
rainfall,
carbon dioxide concentration of the air, presence of pollutants etc.) vary
constantly under unpredictable field conditions. Thus, a researcher may
impose a nutrient limitation in the field, but if the conditions are dry and
water is more limiting to plant growth than the nutrient in question, there
will be no discernable effect due to nutrient treatments.
The inability to extrapolate from a laboratory to a field
setting is illustrated by work conducted in the 1970's and early 1980's on
soybean with strains of 8. japonicum which were hypothesized to be more
energy efficient when fixing nitrogen. Because of the extreme stability of
the triple bond in the dinitrogen molecule nitrogen fixation was known to
be a very energy expensive process. In addition, it was discovered that
the enzyme which fixed dinitrogen into biologically useful ammonia
(nitrogenase) leaked high energy electrons to protons, so that every time
one dinitrogen molecule was fixed into two ammonia molecules, one
dihydrogen (the product of two protons plus two electrons) was produced.
This constituted a waste of energy by the plant-bacterium symbiotic
system. Shortly afterward it was discovered that some strains of 8.
japonicum contained an enzyme that took up the hydrogen formed and
took the high energy electrons back off the protons, hence recovering
much of the energy that would have been lost (Schubert et al. 1978).
This lead to speculation that strains containing these "uptake
hydrogenases", referred to as Hup+ strains, would be more efficient and
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lead to improved plant growth, as the plant would have to supply less
energy (as organic acids) to the bacteria for each ammonia molecule
received from them. Albrecht et al. (1979) compared soybean plants
inoculated with Hup+ and Hup- strains of B. japonicum under greenhouse
5 conditions (laboratory conditions). Average total nitrogen contents and
total dry weights of Hup+ inoculated plants were shown to be larger than
those of plants inoculated with Hup- strains. However, under field
conditions, Albrecht et al. (1979) were unable to detect an increase in dry
matter production or yield between Hup+ and Hup- strains. These results
10 were confirmed by numerous field condition studies. During the course of
these confirmations however, a superior strain of B. japonicum (532C),
which is now included in almost all soybean inoculants used to produce
soybean in Canada, was identified (Hume et al., 1990). Strikingly, this
strain is Hup-.
This example provides a blatant proof involving
soybean, that results obtained in a controlled milieu are a priori not
predictive of the field situation.
US Patent 5,175,149 of Stacey et al., teaches that the
mere coating of the leguminous seeds or sowing of the soil with the
desired bacterial strains does not necessarily lead to the desired
inoculation of the plant. Therefore, they provide a means for inducing
nodulation on the roots of leguminous plants that is independent of the
presence of rhizobial bacteria, by using the bacterial signal molecule
directly (lipo-chitooligosaccharide), thereby by-passing the plant signal
molecule (flavonoids).
US Patent 5,229,113 ('113) issued to Kosslak et al.,
relates to nodulation-inducing compositions and methods of selectively
activating nod genes under the control of a soybean exudate inducible
promoter responsive to inducer molecules. Similarly to US Patent
5,141,745, '113 does not teach or suggest that their compositions and
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methods are operational under field conditions and/or under conditions
that inhibit or delay modulation.
PCT patent application WO 94/25568, which was
published November 10, 1994 in the name of Rice et al., discloses cold
tolerant strains of Rhizobium which are useful for improving modulation,
nitrogen fixation and overall crop size under field conditions. However,
it is unclear whether the cold-selected strains indeed provided an
advantage to plant growth, final grain yield and protein yield, since in
certain experiments plants receiving the temperate strains performed
better than those plants having the cold-temperature selected strain. This
results corroborates the findings of Lynch and Smith, 1994 which
suggested that inoculation with 8. japonicum strains from cold
environments is unlikely to enhance soybean N2-fixation under cool soil
conditions. Lynch et al., 1994 also suggested that, indeed, the host plant,
and not the bacterial strain, mediates at least a significant portion of the
sensitivity of N2-fixation under low RZT. Further WO 94/25568 (see
below) teaches that commercial rhizobial inoculants are not consistent in
their efficacy and performance, and modulation failures after use of
commercial inoculants are common. This is explained by the inability of
inoculant strains to out-compete indigenous rhizobial bacteria for root-
infection sites, once again demonstrating the non-predictability of lab
results to the field conditions. In any event WO 94/25568 fails to provide
any teaching or suggestion as to the involvement of the signal molecules
in the initiation of the modulation event and their effect under field
conditions.
US Patent 5,432,079 to Johansen et al., relates to the
isolation of Rhizobium strains having improved symbiotic properties.
Once again this Patent fails to teach an enhancement of growth and/or
yield of a legume under field conditions. Moreover, this document is
silent on the use of flavonoids or the like to achieve that goal. It teaches
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however that a higher expression of the nod genes does not necessarily
provide an advantage, but can be detrimental to the competitive ability of
the Rhizobium strains.
In Brazil, with an established population of 104
Bradyrhizobium cells/gm soil and 106 Rhizobium cells/gm soil, genistein
treated (40 pM) bean or soybean seeds showed a 15 and 20% increase
in nodule numbers, respectively (Hungria and Stacy, 1997).
An understanding of the mechanism of suboptimal RZT
effects on pea and/or lentil nodulation and N2 fixation and the
identification of methods to reduce this growth restriction by low RZT
would allow an increased use of this NZ fixing cash crop, and a decreased
reliance on potentially polluting N fertilizers in cool season areas. Thus,
there remains a need to elucidate the mechanism which explains the
inhibitory activity of suboptimal RZTs on nodulation and nodule formation
in pea and/or lentil and to determine how to reduce the negative effect of
suboptimal RZTs on the pea and/or lentil NZ fixation symbiosis under cool
spring conditions or other conditions which inhibit or delay this symbiosis.
Such elucidation or determination would provide a significant advantage
to the production of legumes. For example, it would be advantageous to
understand whether the poor nodulation of pea and/or lentil at suboptimal
RZTs are related to the plant's ability to synthesize and/or excrete plant-
to-bacterial signal molecules.
There thus remains a need to reduce the negative
efFects of environmental factors on nodulation and nodule formation and
to provide compositions and methods enabling the enhancement of grain
yield and protein yield of peas and/or lentils grown under environmental
conditions that inhibit or delay nodulation thereof.
It would also be desirable to provide formulations that
could affect the plant-microbe interaction, so as to overcome problems of
nodulation and nitrogen fixation. Whereas a nodulation booster
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enhancing composition which acts to exploit the signalling mechanism,
and methods of use thereof have been successfully developed for
soybean (Smith and Zhang, USP 5,922,316), no such compositions have
been developed for peas and/or lentils.
There therefore remains a need to provide a pea and/or
lentil inoculant/booster composition which could improve yield in the field,
in view of the fact that such crops are planted in the soils in Canada and
elsewhere and therefore face nodulation inhibiting conditions.
Recent reviews on nodulation factors and Rhizobium
symbiosis are available: Spaink, 1995, "Molecular basis of infection and
nodulation by Rhizobium - the ins- and outs of sympathogenesis", Ann.
Rev. Phytopathol. 33:345-368.
The present invention seeks to meet these and other
needs.
The present description refers to a number of
documents, the content of which is herein incorporated by reference in
their entirety.
SUMMARY OF THE INVENTION
The invention relates to the demonstration that low root
zone temperature (RZT)-induced delays in nitrogen fixation and
nodulation in peas and lentils can be compensated using a composition
comprising signal molecules.
Compositions and methods are provided to overcome
the inhibitory effects of low RZTs on pea and/or lentil, comprising an
application of an agriculturally effective amount of a nodulation gene
inducing compound in admixture with an agriculturally suitable carrier
medium.
In accordance with one embodiment of the present
invention, there is provided a method for enhancing nodulation and/or
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nitrogen fixation and/or grain yield of pea and/or lentil grown in the field
under conditions which inhibit nodulation of the pea and/or lentil,
comprising a supply in the vicinity of one of a seed and root of the pea
and/or lentil with a nodulation enhancing amount of a nodulation gene-
s inducing compound. In one embodiment, the supply of the nodulation
gene-inducing compound is exogenous (e.g. a composition comprising
same and an agriculturally suitable carrier medium is provided). In
another embodiment, the supply is endogenous (i.e. the pea or lentil plant
used is engineered to express a chosen nodulation gene-inducing
compound in accordance with the present invention, at a higher level, or
to express additional nodulation gene-inducing compounds not usually
expressed by the pea or lentil plant, or to express a combination of
nodulation gene-inducing compounds or a different level thereof).
In accordance with another feature of the present
invention, there is provided a method to improve compositions of the
present invention, comprising a use of the strains and assaying methods
of the present invention (e.g. ~i-galactosidase assays) to test other
nodulation gene-inducing compounds, derivatives thereof or combinations
of same.
Thus in a first aspect, the present invention features
compositions for enhancing grain yield of pea and/or lentil grown under
environmental condition which inhibit or delay the nodulation thereof.
In a related aspect, the invention features methods for
enhancing grain yield of pea and/or lentil grown under environmental
condition which inhibit or delay nodulation thereof.
In one preferred embodiment, the present invention
features compositions and methods for enhancing grain yield of pea
and/or lentil grown under low root zone temperature conditions.
Further broad aspects of the instant invention include a
method of increasing the growth and/or seed yield of pea and/or lentil
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crops grown under environmental conditions which inhibit or delay
nodulation thereof with an agriculturally effective amount of a composition
comprising a Rhizobial strain in admixture with a flavonoid nodulation
gene inducing compound and an inoculant carrier medium.
5 In accordance with the present invention, there is
provided a composition for enhancing grain yield of pea and/or lentil
grown under environmental conditions that inhibit or delay nodulation
thereof, the composition comprising an agriculturally effective amount of
a nodulation gene-inducing compound in admixture with a suitable carrier
10 medium.
In accordance with the present invention, there is also
provided a method for enhancing grain yield of pea and/or lentil grown in
the field comprising: a) incubating a rhizobial strain which nodulates the
pea and/or lentil with an agriculturally effective amount of a nodulation
15 gene-inducing compound in admixture wth an agriculturally suitable
carrier medium; and
b) inoculating in the vicinity of one of a seed and root of
the pea and/or lentil with the rhizobial strain of a).
While the instant invention is demonstrated by
experiments performed using in particular hesperetin, naringenin and
luteolin as preferred nodulation inducing compounds, the invention is not
so limited. U.S. Patent 5,141,745 teaches the molecular structural
features that are associated with nodulation inducing activity of plant
exudates. Therein, a number of flavonoids, isoflavonoids, flavones
including flavanones, flavanols and dihydroflavanols, isoflavones,
coumarins and related molecules were assayed for nodulation inducing
activity. Nodulation inducing activity was found to reside in a structurally
identifiable group of compounds not limited to those flavones associated
in particular with legumes which include specifically substituted flavones,
flavonones (dihydroflavones), flavanols (3-hydroxyflavones) and
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dihydroflavanols. The basic flavone ring structure common to flavones,
flavonones, flavanols and dihydroflavanols is requisite for activity. Within
the group of flavones, it is clear that substitution at the 7th position with
a hydroxyl group leads to a strong stimulatory activity.
However, flavones or dihydroflavones substituted with
either hydroxyl or methoxyl at both the 3' and 4' positions require in
addition to 7-hydroxylation a hydroxyl group at the 5 position for activity.
The fact that taxifolin and naringein, both flavanones, have stimulatory
activity indicates that the double bond in the flavone fused ring (between
positions 2 and 3) is not necessary for modulation gene-inducing activity.
This implies that all flavones and dihydroflavanols having substitution
patterns as described above have potential modulation inducing activity.
As exemplified in the present application, synthetic as
well as natural modulation gene-inducing compounds are encompassed
by the scope of the present invention. Thus, the present invention
provides the means and the methods to screen and select nod gene
inducing compounds which could be used in the compositions and
methods of the present invention.
Direct or indirect methods of pea or lentil inoculation can
be employed. During direct inoculation the bacterium is applied directly
to the seed prior to sowing. This can most simply be accomplished by
spraying the seed with or dipping the seed into a liquid culture containing
a desired Rhizobium strain and a modulation gene inducer (or combination
thereof). A preferred method of direct inoculation is pelleting of the seed
with an inoculating composition containing a Rhizobium strain and a
modulation gene-inducing factor. Generally, the bacterium is applied to
a carrier material and a pellet is formed with the carrier surrounding the
seed. Many diverse carriers are known in the art and include, among
others, peat, soil, calcium carbonate, dolomite, gypsum, clay minerals,
phosphates, titanium dioxide, humus and activated charcoal. Any
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agriculturally suitable material can be employed. An adhesive material is
often included in such a pellet to insure that the carrier remains in contact
with the seed. Again, many acceptable adhesives are known including,
among others, synthetic glues, vegetable glues, gelatin and sugars. In
general, the carrier and any adhesive used are chosen to insure viability
of the inoculant strain and retention of activity of nodulation gene-inducing
factor. Pelleted inoculated seed containing an inducing factor can be
directly sown into the field. Alternatively, a conventionally prepared
inoculated seed or seed pellet containing the desired strain can be
contacted with an inducing composition containing an effective amount
of a nodulation gene inducer before, with or after sowing of the inoculated
seed .
The concentration of nod gene inducing compound will
be adapted to the particular situation at hand by the skilled artisan. For
example, the skilled artisan will take into account the level of severity of
inhibition or delay of the environmental conditions on nodulation, the
responsiveness of the nod genes of the rhizobial strain to the nod gene
inducing compound, the method of application of the composition, etc.
The upper limit of the effective concentration is determined by toxicity of
the nod gene inducing compound toward the rhizobial strain or, if
applicable, by the solubility limit of the inducer in the carrier chosen.
During indirect inoculation, an inoculating composition
of the present invention containing an inoculant strain with an effective
concentration of a nodulation gene inducer is introduced in the vicinity of
the seed at the time of sowing.
Having now demonstrated that nodulation gene-inducing
factors are effective under field conditions, another use of the present
invention is for the selective induction in bacterial genes containing a
legume nodulation gene-inducing promoter and a structural gene under
its control. Expression of this structural gene under the control of a nod
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gene-inducing promoter can be activated by the addition of the activator
therefor. Having demonstrated that these promoters are affected by
environmental factors such as temperature, the present invention
provides a means to somehow regulate, through the field conditions, the
level of expression of the structural gene under the control of the above-
mentioned promoter. Construction of such chimeras can be adapted
using conventional methods by the skilled artisan.
It should also be understood that pea and lentil could be
engineered to express the nod gene-inducing compounds of the present
invention at higher levels. Similarly, such engineered plants could
express a combination of such nod gene-inducing compounds. It will be
clear to the skilled artisan to which the present invention pertains that
engineering of the pea and lentil plants can be through a qualitative
and/or quantitative expression of the nod gene-inducing compounds of
the present invention.
In a particular embodiment, the engineered plants are
transgenic peas and/or transgenic lentil plants. Method to engineer and
obtain transgenic plants are known in the art.
The term "environmental conditions which inhibit or delay
nodulation" should be interpreted herein as designating environmental
conditions which postpone or inhibit nodulation and nitrogen fixation and
include, without being limited thereto: temperature stress, water stress,
salinity, pH stress as well as inhibitory soil nitrogen concentrations or
fixed nitrogen.
As used herein, the term "enhancing grain yield" refers
to an enhancement of grain yield of pea and/or lentil of treated plants in
accordance with the present invention or adaptations thereof as
compared to control plants.
"An agriculturally effective amount of a composition" for
increasing the growth of legume crops in accordance with the present
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invention refers to a quantity which is sufficient to result in a
statistically
significant enhancement of growth and/or of protein yield and/or of grain
yield of such a legume crop as compared to the growth and grain yield of
a control crop (e.g. not treated).
The term "immediate vicinity of a seed or roots" refers
to any location of a seed or roots wherein if any soluble material or
composition is so placed, any exhibit of the plant or of the bacteria, or
bacterial cells will be in actual contact with the seed as it germinates or
the roots as they grow and develop.
By "nodulation gene-inducing" or "nod gene-inducing" is
meant bacterial genes involved in nodule establishment and function.
The term "recombinant DNA" as known in the art refers
to a DNA molecule resulting from the joining of DNA segments. This is
often referred to as genetic engineering.
As used herein, the term "gene" is well known in the art
and relates to a nucleic acid sequence defining a single protein or
polypeptide. A "structural gene" defines a DNA sequence which is
transcribed into RNA and translated into a protein having a specific amino
acid sequence thereby giving rise the a specific polypeptide or protein.
A "heterologous" (i.e. a heterologous gene) region of a
DNA molecule is a subsegment segment of DNA within a larger segment
that is not found in association therewith in nature. The term
"heterologous" can be similarly used to define two polypeptidic segments
not joined together in nature. Non-limiting examples of heterologous
genes include reporter genes such as luciferase, chloramphenicol acetyl
transferase, ~i-galactosidase, and the like which can be juxtaposed or
joined to heterologous control regions (i.e. a nod gene promoter region)
or to heterologous polypeptides.
The term "vector" is commonly known in the art and
defines a plasmid DNA, phage DNA, viral DNA and the like, which can
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serve as a DNA vehicle into which DNA of the present invention can be
cloned. Numerous types of vectors exist and are well known in the art.
The term "expression" defines the process by which a
gene is transcribed into mRNA (transcription), the mRNA is then being
5 translated (translation) into one polypeptide (or protein) or more.
The terminology "expression vector" defines a vector or
vehicle as described above but designed to enable the expression of an
inserted sequence following transformation into a host. The cloned gene
(inserted sequence) is usually placed under the control of control element
10 sequences such as promoter sequences. The placing of a cloned gene
under such control sequences is often referred to as being operably
linked to control elements or sequences.
Operably linked sequences may also include two
segments that are transcribed onto the same RNA transcript. Thus, two
15 sequences, such as a promoter and a "reporter sequence" are operably
linked if transcription commencing in the promoter will produce an RNA
transcript of the reporter sequence. In order to be "operably linked" it is
not necessary that two sequences be immediately adjacent to one
another.
20 Expression control sequences will vary depending on
whether the vector is designed to express the operably linked gene in a
prokaryotic or eukaryotic host or both (shuttle vectors) and can
additionally contain transcriptional elements such as enhancer elements,
termination sequences, tissue-specificity elements, and/or translational
initiation and termination sites.
As used herein, the terms "molecule", "compound" or
"agent" are used interchangeably and broadly to refer to natural, synthetic
or semi-synthetic nod gene-inducing compounds of the present invention.
The agents can be selected and screened by a variety of means including
random screening, rational selection and by rational design using for
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example protein or ligand modelling methods such as computer
modelling. Of course, these agents/compounds can be tested in the
assays and field trials of the present invention. The terms "rationally
selected" or "rationally designed" are meant to define compounds which
have been chosen based on the configuration of the herein shown active
compounds of the present invention.
As exemplified herein, the level of gene expression of
the reporter gene (e.g. the level of luciferase, or ~i-gal, produced) within
the treated cells can be compared to that of the reporter gene in the
absence of the compound(s). The difference between the levels of gene
expression indicates whether the molecules) of interest synergizes
another nod gene-inducing compound, antagonizes its activity, increases
nod promoter activity when used alone, and the like. The magnitude of
the level of reporter gene product expressed (treated vs. untreated cells)
provides a relative indication of the strength of that molecules) as an
agonist, antagonist or inducer.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following non
restrictive description of preferred embodiments thereof, given by way of
example only with reference to the accompanying drawings.
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 time course of growth and ~i-
galactosidase activity by strains 1477 and 5280 in the presence of 1 pM
naringenin;
Figure 2 shows the effect of different signal molecules
on nod gene expression of R. Leguminosarum strains 1477 and 5280;
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Figure 3 shows the induction of promoter nodC-IacZ as
a function of the inducer concentration with strain R. Leguminosarum
1477 for 24 hours;
Figure 4 shows the effect of combined inducer at
different ratios on nod gene activity with strain R. Leguminosarum 1477
in TY medium for 24 hours induction;
Figure 5 shows the effect of time of addition of inducer
hesperetin (A) and naringenin (B) in the growth culture on nod gene
activity in the R. Leguminosarum strain 1477;
Figure 6 shows the effect of temperature on nod gene
induction by different inducer compounds;
Figure 7 shows the growth and (3-galactosidase activity
in R. Leguminosarum grown at 15°C and 28°C in the presence of
hesperetin;
Figure 8 shows the effect of inducer concentration on
nod gene activity of R. Leguminosarum 1477 grown at 28° and
15°C;
Figure 9 shows the time course of ~i-galactosidase
activity displayed by strain R. Leguminosarum 1477 grown at 15°C in the
presence of the inducer hesperetin;
Figure 10 shows the effect of preinduced cells of
Rhizobium sp. on nodule number, nodule biomass and shoot biomass of
the pea variety Celeste harvested at 60 days after inoculation; and
Figure 11 shows the effect of inoculation with preinduced
cells of Rhizobium sp. on nodulation and biomass production of lentil.
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
Laboratory, growth chamber and greenhouse studies
were carried out to develop a technology for sustainable nodulation and
nitrogen fixing capabilities of field pea and lentil by incorporating signal
molecules to a system capable of enhancing nodulation and fixing
nitrogen at low root zone temperatures (RZTs) below 10°C. To overcome
the low temperature effect on induction of nodulation in pea and lentil,
appropriate signal molecules for nod gene induction were identified using
reporter gene-containing R. Leguminosarum strains. Out of a number of
signal compounds including apigenin, daidzein, genistein, hesperetin,
kaempferol, luteolin, naringenin and rutin; hesperetin was found to be the
most effective inducer. Moreover, it was shown to be heat stable. It was
also shown to be an effective inducer in the presence of other inducer
molecules. In one preferred embodiment, a composition comprising
hesperetin and another inducer was shown to be especially effective. In
one particular preferred embodiment, a composition comprising
hesperetin and naringenin (at a ratio of 7:3) was shown to be a potent
inducer of pea nod gene activity.
Controlled environment experiments in growth chamber
and greenhouse were conducted on pea and lentil to determine whether
the preinduced Rhizobium with hesperetin at a concentration of 10pM
increase nodulation at suboptimal temperature of 17°C. Under such
conditions, a significant increase in nodulation and greater biomass
production was observed with preinduced cell inoculation to both pea and
lentil as compared to uninduced cell inoculation. Indeed, a 120% increase
in nodulation and 48% increase in biomass production resulted from an
inoculation of field pea with preinduced (with hesperetin) Rhizobium sp.
Similarly, inoculation of lentil with luteolin induced Rhizobium, lead to a
55% increase in nodulation and a 30% increase in biomass production.
In order to validate these laboratory condition studies, trials were carried
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out under experimental field conditions. These field experiments showed
that preinduced Rhizobium with pea signal molecules significantly
improved the plant nodulation process and final grain yield. Plants which
received peat-based preinduced Rhizobium cells showed a 32% increase
in nodule numbers and 57% of pod number per plant, as compared to
plants inoculated with uninduced cells. The grain yield of plants
inoculated with preinduced cells also increased 10.9%, as compared to
that of the corresponding control plants. Moreover, a direct application
of the signal molecules to the seed surface also significantly increased
nodule number and final grain yield by up to 64 and 12%, respectively
over control.
Taken together, commercial inoculant preparation for
pea and lentil would greatly benefit from the inclusion of exogenous gene
inducing compounds (or from inducing compounds producing organisms).
Since such inducers are active at low concentrations, their addition to
such inoculant preparations should be possible at a low cost.
It should also be recognized by the skilled artisan that
transgenic plants expressing a combination of the signal molecules of the
present invention, or expressing at least one such signal molecule at
higher levels (or more potent forms thereof) could also be used in
accordance with the present invention.
The present invention is illustrated in further detail by the
following non-limiting examples.
EXAMPLE 1
Compositions enhancing Rhizobium nod gene
expression in vitro
Bacterial strains and girowth conditions
Bacterial strains used in this study were R.
Leguminosarum 840/pRL1-pJl J 1477 with nodC-IacZ gene fusion reporter
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plasmid, R. Leguminosarum 840/pRLIJI 1478 with nodD-IacZ gene fusion
reporter plasmid (Rossen et al., 1985), R. Leguminosarum by trifolii
LPR5045 lacking Sym plasmid and with cloned nodD1 gene from R.
Leguminosarum bv. vices (5280), nodD1 gene from R. Leguminosarum
5 bv. trifolii (5283) and nodD1 from R. meliloti (5284) respectively (Spanik
et al., 1987). The strains contain a complete nodD gene including their
own constitutively expressed promoter (Spanik et al., 1987). Besides
these, one rhizobial strain Rhizobium sp. (collected from the Department
of Plant Science at McGill University) was used for plant nodulation
10 experiments.
Cells to be used for induction experiments were
pregrown at 28°C on solid YEM medium containing yeast extract and
mannitol (Hooykaas et al., 1979). For stable maintenance of the
recombinant plasmids and the strains, the medium was supplemented
15 accordingly with streptomycin (400 pg/ml) and chloramphenicol (10
pg/ml) and tetracycline (2 Ng/ml). After growth for 48 hours the plates
were stored for a period of 7 days at 4°C. Experiments were carried out
in test tubes containing 5 ml of TY medium. Tubes were inoculated with
5% inoculum pregrown overnight in TY medium. Unless otherwise stated,
20 cultures were induced at the beginning of inoculation. Final concentration
of inducers varied with the experiment. Cultures were incubated at 28°C
on a shaker at 180 rpm and induction was monitored at different time
intervals.
Bioassay for nod gene-inducing activi~
25 Fresh cells grown in induction medium were used for ~3-
galactosidase assay. Unless otherwise indicated, ~3-galactosidase
activities were calculated as described by Miller (1972). The f3-
galactosidase activities of the bacteria that had not been exposed to
flavonoid were used as background reading. The origin of the signal
molecules naringenin, hesperetin, apigenin, luteolin, rutin, kaemferol,
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genistein and daidzein that were tested for the nod gene inducing ability
were obtained from Sigma Chemical Co.
Plant nodulation test
Plant nodulation tests were performed both under
controlled atmosphere plant growth chamber and greenhouse conditions.
Seeds of pea and lentil were surface sterilized by immersion in 95%
ethanol for five minutes followed by running in sterile water and then
immersion in 5% commercial bleach for 20 minutes. Then at least five
washes with sterile water were carried out. The seeds were allowed to
imbibe water by incubating for four hours prior to sowing in 1:1 (v/v) sand
and turface.
Growth chamber experiment
Experiments with lentil were executed by growing lentil
plants in test tubes (200x25mm) on modified Hoagland's agar (Hoagland
and Arnon, 1950). Surface-sterilized seeds were germinated on petri
dishes containing 1.5% agar at room temperatures. Upon germination,
two seedlings were transferred to each tube. After another two days of
growth, plants were inoculated with test strains.
Pot experiments for both pea and lentil, in growth
chambers, were carried out in five-inch pots on sterile sand and turface
in 1:1 (v/v) ratio. Six surface sterilized seed were sown in each pot and
upon germination at 22°C, seedlings were thinned to two plants per pot.
Plants were supplied with Hoagland's plant nutrient solutions once a
week. The experiment was carried out in four replicates. The growth
chambers were set up at a temperature of 22°C and a relative humidity
of 75%. The photon flux density was approx. 300 ~cmol m-Z sec' (Philips
TLF 60W/33 fluorescent tubes), and the day-length was set at 16 hours.
Greenhouse experiment
Greenhouse experiments were carried out in an
environmentally controlled research greenhouse located at McGill
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University, MacDonald campus. Light levels were maintained at an
irradiance of 300 ,umol m~2 s-' for a 16:8 hour (day: night) photo period and
a constant air temperature of 17°C.
Non sterilized Turface (Applied Industrial Materials
Corp., Deerfield, IL):sand in 1:1 (v/v) mixture was used as the plant
rooting medium. During the experiment, plants were watered with a
modified Hoagland's solution (Hoagland and Arnon, 1950) in which the
CaN03 and KN03 were replaced with 1 mM C~CI , and 1 mM
K2HP04 plus 1 mM K~i P~J respectively, to provide a nitrogen-free
solution.
Two pregerminated seedlings were transplanted into
each five-inch pot. Plants were watered every alternate day and provided
with Hoagland's nutrient medium once a week.
Inoculation
Inoculum was prepared by growing selected rhizobial
strains in tryptone yeast extract (TY) medium containing appropriate
antibiotics and inducers. Overnight grown cultures were pelleted by
centrifuging at 8000 rpm, and resuspended into 0.5% saline solution at
a concentration of 1.0 x 109 CFU per ml. For each plant in the pot, one
ml of culture suspension was applied in the root rhizosphere of each
plant.
Data collection
Growth chamber and greenhouse plants were harvested
for nodule count and dry matter measurement at 6 and 8 weeks after
transplanting, respectively. Dry matter of biomass was determined by
drying them at 80°C for 48 hours.
Results
Selection of reporter gene containing Rhizobium strains
To develop an efficient inoculant and seed treatment
process, a selection of appropriate signal compounds for optimum
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expression of nod genes in Rhizobium was utilized. Further, a suitable
reporterireference strain was helpful to assess the level of stimulation of
Nod factor producing genes by signal compounds.
Two rhizobial strains containing reporter nod genes were
obtained from Jhon Innes Centre, Netherlands. Of them are, Rhizobium
leguminosarum pIJ1477 with a plasmid carrying Rhizobium nodC gene
fused with E. coli IacZ and R. leguminosarum pIJ1478 carrying nodD-IacZ
fusion. Both strains have the nitrogen fixing plasmid pRL1Jl. Three other
isogenic strains of R. Leguminosarum bv. trifolii 5045, containing a IacZ
fusion with nodD gene from three different origin, were obtained from the
Institute of Molecular Plant Science, Leiden University, Netherlands.
Strain RBL5280 carrying a Lac-Z fusion with nodD1 gene from R.
Leguminosarum bv. viceae, RBL5283 carrying a Lac-Zfusion with nodD1
gene from R. leguminosarum bv. trifolii, and strain RBL5284 carrying a
lac-Z fusion with nodD1 gene from R. meliloti. In this case the host strain
is devoid of nitrogen fixing Sym plasmid. These strains were able to grow
on yeast extract manitol (YEM) and tryptone yeast extract agar (TY)
medium. Strains were tested for their growth and nod gene induction in
the presence of signal compound naringenin (1 NM) at 28°C to select a
suitable strain for this study. Strain 1477 showed a [3-galactosidase
activity of approximately 2000 Miller units at 24 hours of growth while
strain 5280 and 5283 showed 101 and 112 Miller units at 42 hours of
incubation in the presence of 1 pM naringenin, respectively. Strain 1478
did not show any ~3-galactosidase activity. Similarly, strain RBL5284
harboring the nodD1 gene of R. meliloti was not induced by naringenin in
comparison with other strains, hence it cannot be used as an indicator
strain (Spaink et al., 1987). Since strains 1477 and 5280 showed a
significant ~i-galactosidase activity, they were selected as indicator strains
for the detailed study presented hereinbelow. Figure 1 represents the
growth and ~i-galactosidase activity units shown by the strain 1477 and
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5280, respectively. Strain 1477 showed approximately 2500 Miller units
at 24 hours of growth while strain 5280 showed 800 Miller units at 48
hours of incubation in the presence of 1 pM naringenin (Figure1).
Determination of more efficacious plant-to-microbe siginalling compounds
for inducting of R. leguminosarum nod genes
Determination of appropriate signal compounds for
optimum expression of nod genes in Rhizobium is preferred for the
development of an effective inoculant and seed treatment process for
legume crops and compositions thereof. Hence the effect of different nod
gene inducers on nod gene induction was investigated. A number of
commercially available flavones, flavanones, isoflavanones and related
flavonoids were tested for their ability to induce the nod gene promoter
using indicator strains 1477 and 5280. To select the most potent inducer,
both strains were grown in the presence of eight different signalling
compounds including apigenin, daidzein genistein, hesperetin,
kaempferol, luteolin, naringenin and rutin at a concentration of 5 pM.
Inducers were added at the beginning of the inoculation. The flavanones,
hesperetin and naringenin and the flavones apigenin and luteolin
appeared to be the most active inducers among the compounds tested
and the maximal induction level varied with the particular signalling
compound (Figure 2). Flavanol and kaempferol were found to be poor
inducers for strain 5280 which only showed an induced response that was
double that of background in the absence of inducer. All other
compounds tested were found inactive for both strains. Among the
signalling compounds, maximal induction was shown by strain 1477 in the
presence of inducer hesperetin (9,560 Miller units of 13-galactosidase
activity). The next most effective inducer for strain 1477 was naringenin,
with a corresponding 13-galactosidase activity of 4,369 Miller units. For
strain 5280, apigenin showed the strongest activity at 4,369 Miller units.
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The second strongest inducer for strain 5280 was luteolin, with a
corresponding 13-galactosidase activity of 4,092 units (Figure 2).
Taken together, these results indicate that the selection
of the best signalling compound is strain dependent. Since hesperetin
5 appeared to be the most active inducer overall, this flavanone was used
to study induction in more detail.
Determination of the optimum concentration of the signal compounds
To determine the effect of signal molecules on growth
and the optimum concentration of inducers for maximum nod gene
10 expression, strain 1477 was grown in the presence of all the above-
selected inducer compounds at 5 different concentrations ranging from
0 to 20 ,uM. Cells grown in the same medium without signal molecule
were used as controls. The experiment was conducted at 28°C.
Samples from the cultures were collected at different intervals to
15 determine cell growth by measuring optical density at 600 nm and nod
gene induction by determining ~i-galactosidase activity.
The growth of Rhizobium cultures incubated with 20 ,uM
of the different signal compounds was monitored after a growth period of
48 hours to determine whether the compounds affected the growth
20 thereof. Although many of the isoflavones and flavonols were not strong
nod gene inducers, they showed no negative effects on cell growth
(Table 1 ), as the optical densities of all the cultures were close to 2Ø
(Furthermore, no significant difference in growth was observed between
the growth of the strains in the absence and presence of signalling
25 compounds at the highest concentration level (20,uM) of inducers added
at the beginning of inoculation).
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TABLE 1
Effect of signal molecules on the growth of
R. leguminosarum 1477
Signal compoundConcentration O.D.
(pm) at 600
nm
16h 24h
48h
None 0.0 1.94 1.96 2.14
Apigenin 20 1.72 1.90 1.92
Hesperetin 20 1.82 1.86 1.96
Luteolin 20 1.76 1.94 1.94
Naringenin 20 . 1.90 2.08 1.96
Kaempferol 20 1.63 1.95 1.82
Rutin 20 1.52 1.66 1.73
Genistein 20 1.59 1.66 1.84
Daidzein 20 1.70 1.98 1.78
The response of the nod gene activity to increasing
concentrations of the inducers hesperetin, apigenin, and naringenin was
linear and reached its maximum level at concentrations of 10 and 15uM
respectively (Figure 3). With luteolin, maximum induction was obtained
at 20pM level. Results also indicated that increase in concentration of
isoflavone and flavonol above these levels did not enhance nod gene
induction under the conditions tested (data not shown).
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Induction of nod gene by mixtures of hesperetin and naringenin at
different ratios
Individual legume sp. can release numerous nod-gene
inducers. For example, alfalfa, vetch and common bean release between
five to nine different flavonoid nod-gene inducers. The presence of more
than one nodD genes in the respective rhizobial strains suggests that
various flavonoids released from their host plants, may bind to different
NodD proteins. In addition, it underlines the complexity of the
establishment of the plant to bacteria symbiosis mechanisms. As the
nod-gene inducing activity was shown by four different flavonoid
structures in the strain 1477, specific interaction between the inducers at
different concentrations might have increase the induction capability.
Hence, an experiment was carried out to identify improved combination
ratios of the inducers. In this particular embodiment, different ratios of the
two most potent inducers, hesperetin and naringenin, were tested to
identify combinations enabling a maximal expression of nod genes.
Hesperetin and naringenin were thus mixed at six different ratios and
added to the TY medium during inoculation with strain 1477. Individual
inducers at a concentration of 10NM were also used to provide a better
control for the combination effects. Induction levels corresponding to the
~i-galactosidase activities are represented in Figure 4.
The results obtained revealed that the hesperetin and
naringenin, in a 7:3 mixture, increased ~i-galactosidase activity
significantly, compared to individual or equimolar applications of inducers.
The effectiveness of naringenin was found to increase in the combination
where a lower amount of hesperetin (1:9) was present, as compared to
an equimolar or higher level of hesperetin in the medium (Figure 4).
Induction in this condition was also found higher than with naringenin
alone at a final concentration of 10NM.
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Taken together, these results indicate that nod gene
induction can be effectively enhanced by a combination of inducers. In
addition, they indicate that induction of the nod gene by particular ratios
of the two inducers are shown to be more effective than with a single
inducer.
Effect of time of addition of inducers
Assays were designed to test whether induction of nod
genes is affected by the time of addition of inducers to the growth
medium. In this experiment, both hesperetin and naringenin were tested
individually with strain 1477. Inducers at a concentration of 10NM were
added at 0, 7, and 16 hours of incubation and associated ~i-galactosidase
activities were determined.
A similar pattern of nod gene induction was observed
with hesperetin and naringenin added at different incubation period.
Higher ~i-galactosidase activity was obtained when inducers were added
at 7 hours of growth as compared to an addition at 0 and 16 hours of
growth. ~i-galactosidase activity at 7 hours was found to be approximately
double that of ~i-galactosidase when the inducer was added at 16 hours
of growth (Figure 5). Maximum ~3-galactosidase activity for all of the
additions were obtained at 24 hours of growth. After 24 hours, a
decrease in ~i-galactosidase activity was observed, except for a 16 hour
addition, for which activities remained constant until the end of
experiment.
Effect of growth temperature on nod gene induction
The optimal temperatures range for symbiotic nitrogen
fixation by Rhizobium ranges between 22 to 28°C. Temperatures outside
of this range are inhibitory. Infection and early nodule development are
the most sensitive steps in the nodulation process. It has been observed
that by lowering the incubation temperature from 28° to 18°C,
the number
and relative concentrations of the Nod metabolites produced by R.
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Leguminosarum bv. trifolli is affected. Further, when more inducer is
required for maximum induction, the temperature is out of the optimum
temperature range, as was observed in the case of soybean symbiosis
with 8. japonicum (Zhang et al., 1996). Hence, to establish nodulation at
low temperature, the effect of growth temperature on inducibility of nod
gene expression by signalling compounds needs to be determined.
The temperature effect was determined by growing
Rhizobium strain 1477 at two different temperatures in the presence of
four different inducer compounds apigenin, luteolin, naringenin and
hesperetin at 10 NM concentration. Growth was monitored by measuring
optical density of the culture at 600 nm and inducibility was determined
by measuring ~i-galactosidase activity at different time intervals.
The results showed that the nod gene induction by
hesperetin was significantly affected by temperatures. Lower levels of
gene expression were observed at lower temperatures than at higher
temperature (28°C) (i.e. suboptimal versus optimal). The effect was
much more pronounced with the inducer hesperetin compared to the
other inducers used in the experiment. Induction of ~3-galactosidase
activity at 15°C was found to be almost half of that observed at
28°C.
However, the level of induction was still comparable to that obtained in
the presence of other inducers at 28°C (Figure 6).
Growth measurement results from hesperetin-induced
culture (Figure 7) showed a lower growth rate at low temperature of
15°C
compared to the growth at 28°C. Maximum growth at low temperature
(15°C) was found at 60 hours of incubation, while the highest cell
density
at 28°C was at 24 hours (Figure 7). This suggests that lower expression
obtained at low temperature is probably due to lower cell growth.
Therefore a longer induction period was needed to reach maximal
expression. However, the maximal gene expression level was still 75%
lower than that obtained from 24 hours incubation at 28°C.
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To improve nod gene activity at suboptimal
temperatures, an experiment was carried out by increasing hesperetin
concentration up to 40 ~cM and growing R. Leguminosarum 1477 cells at
15° and 28°C. The results (Figure 8) showed that with an
increase in
5 hesperetin concentration in the medium, expression level in terms of ~i-
galactosidase activity, was decreased. The highest level of (3-
galactosidase activity (9,000 unit) was obtained at a hesperetin
concentration of 10 pM in the medium when cells were grown at 28°C. At
15°C, the maximum activity (6,000) also occurred at 10 NM hesperetin.
10 Thus, the level of expression was about 70% lower than that obtained at
28°C (Figure 8). These results suggest that the lower level of activity
at
low temperature is associated with the growth of the strains. Figure 9
shows that maximum f3-galactosidase activity was obtained at 120 hours
of growth at 15°C in the presence of 5 to 30 ~cM hesperetin (Figure 9)
15 while at 28°C incubation, only 24 hours are required to reach
maximum
activity (Figure 7).
Determination of stability of siginal molecules to the heat
Chemical and biological structures of signal molecules
are very specific for induction of specific rhizobial strains. Thus, any
20 process that could have an effect on the molecular structure of the
inducers, are likely to render the inducer less effective in achieving nod
gene expression. In the process of formulating an effective inoculant and
seed treatment composition, it is possible that the signal compound would
be exposed to high temperatures that could affect the induction efficiency
25 of the signalling compounds. Therefore, heat stability of the proper signal
compound is preferable to ensure maximum nod gene expression.
Heat stability of signal compounds was determined by
measuring the nod gene activity of Rhizobium by using heat-treated
signal compounds - hesperetin and naringenin. Both compounds (10 ,uM)
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36
were heat-treated by autoclaving at 121 °C for 15 minutesl. The same
signal compounds without heat-treatment, at the same concentration
were used as controls. ~3-galactosidase activity was measured at 24 hours
of incubation. Heat-treatment hesperetin did not significantly decrease
nod gene induction capacity. However, heat-treatment of naringenin
decreased the induction capacity of nod gene by about 25% (Table 2).
TABLE 2
(3-galactosidase activity in presence of autoclaved
and unautoclaved hesperetin and naringenin grown
in TY medium for 24 hours.
Inducer Heating Concentration ODsoo ~-gal unit
condition of
inducer NM
None ------- 0 1.52 243.42
HesperetinNot autoclaved10 1.07 8281.31
autoclaved 10 1.32 8005.30
NaringeninNot autoclaved10 1.45 4800.00
Autoclaved 10 1.47 3656.46
Test of plant nodulation using induced and uninduced rhizobial cells
Growth chamber experiment
With an aim at overcoming the negative effect of
temperature, plant nodulation tests on pea and lentil were carried out
initially with hesperetin-induced Rhizobium cells. Two different Rhizobium
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37
strains, R. leguminosarum 1477, and a commercial strain Rhizobium sp.
were used. Induced inoculants were prepared by growth in n medium
at 28°C in the presence of 10 pM hesperetin. Uninduced cells were
prepared without adding signalling compound to the medium. Plants were
inoculated at an inoculation rate of 1 x 109 cells/plant. Pea plants were
grown in pots with sand and turface at a 1:1 ratio, and lentils were
maintained in test tubes on agar slants. One set of plants was incubated
at 17°C and the other at 24°C. One experiment with lentil was
carried
out in pots at 24°C containing sand and turface as root medium. Data
were taken after six weeks of growth and plant growth, and determined
nodule and shoot biomass (Tables 3 and 4).
CA 02439421 2003-08-29
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38
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CA 02439421 2003-08-29
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CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
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CA 02439421 2003-08-29
WO 01/72126 41 PCT/CA01/00075
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42
TABLE 6
Effect of preinduced rhizobial cells on plant nodule number and
plant biomass production of lentil in the greenhouse at 17°C
Nodule Shoot
number DM
Strains Inducer #/2 plantst% g/2 plantst%
Control 194 552
Apigenen 300 55 545 -1
Rhizobium sp Luteolin 300 55 719 30
Nagingenin220 13 470 -15
Hesperetin278 43 643 16
Control 187 714
Apigenen 178 -5 650 -3
R.leguminosarum Luteolin 304 63 765 7
1477 Nagingenin222 19 589 -18
Hesperetin245 31 757 6
TABLE 7
The percentage increases of plant nodule number and shoot
biomass production with different pea and rhizobial genotypes
17C 24C
Strains # of Nodule Shoot # of Nodule Shoot
nodule weight weightnodule weight weight
Var. Bohatyr
Rhizobium 51 25 42 28 23 16
sp
8.1.1477 29 -4 11 20 5 3
Celeste
Rhizobium 120 75 47 42 30 21
sp
8.1.1477 38 -2 -11 35 16 17
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A significant difference in nodule number was found
between pea plants having received preinduced or uninduced cells and
these differences were especially notable on plant shoot dry weight. Pea
plants inoculated with induced cells at 17°C showed about 46 to 74%
increase in modulation and 9 to 18% increase in shoot biomass
production, as compared to plants inoculated with uninduced cells (Table
3). Similarly, plant grown at 24°C showed a 28 to 35% increase in
modulation and 3 to 16% increase in shoot biomass production. Thus, the
results indicated that the responses in modulation and biomass production
by inoculating pre-induced cells were higher for plants grown at low
temperature as compared to plants grown at optimum temperature
(Table 3).
Like pea, lentil plants grown in the test tube revealed a
temperature-dependent increase in modulation and biomass production
upon inoculating thereof with pre-induced cells. Increases of 51 to 66%
in nodule number and 4 to 7% in biomass production were observed
when plants were grown at 17°C. On the other hand, plants grown at
24°C, showed increases of 41 to 73% in modulation and 23 to 43% in
biomass production. Depending on the rhizobial strains used,
experiments carried out in pots at 24°C also demonstrated a 53 to 73%
increase in nodule number and 14 to 53% increase in shoot biomass
production (Table 4). The results presented in Tables 3 and 4
demonstrated that under low root zone temperature conditions, induced
cells more effectively affected plant modulation and dry matter
accumulation of pea plants, while the better response of lentil plants to
the preinduced Rhizobium cells was more pronounced at normal growth
temperature (24°C). For both crops, modulation responses varied as a
function of the rhizobial strain used. The increases of nodule number and
shoot biomass were more pronounced in every case in which the plants
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44
received the pre-induced commercial Rhizobium sp. compared to the R.
Leguminosarum 1477 strain.
Taken together, the overall results thus suggest that
induced cells have positive effects on plant nodulation with both pea and
lentil and different rhizobial strains at different growth temperatures.
Greenhouse experiment
Based on the results from growth chamber experiment,
plant inoculation tests were conducted under greenhouse environmental
conditions. The combination of two pea cultivars and a lentil cultivar with
two rhizobial strains were completely randomized into this experiment
(with 4 replications) at 17°C. Both strains were preinduced separately
by
apigenin, luteolin, naringenin and hesperetin. Cells were induced for 16
hours at 28°C. Plants were harvested for nodule count and dry matter
measurements eight weeks after inoculation. The results obtained are
presented in Tables 5, 6 and 7 and Figures 10 and 11.
Generally, a significant increase in nodule number,
nodule dry mass and shoot dry weight with plants receiving preinduced
cell was obtained over those plants receiving uninduced cells on both
lentil and pea. However, plant responses varied significantly with the
strains, signal molecules and the cultivars. The preinduced commercial
strain Rhizobium sp. had a greater nodulation as compared to preinduced
R. Leguminosarum 1477 on both pea varieties - Bohatyr and Celeste
(Table 5). Maximum increase in nodule number, nodule biomass and
shoot biomass was obtained with hesperetin, followed by naringenin
(Figure 10 and Table 5). Increased nodule number was reflected on
nodule dry mass, shoot biomass and the number of pod. A 120%
increase in nodule number, 48% increase in shoot biomass and 46%
increase in pod number was observed with the Celeste cultivar
inoculation with hesperetin induced Rhizobium sp. cells. In the case of
varietal comparison, only a 51 % increased nodulation and 42% shoot dry
matter was observed on the pea variety Bohatyr by inoculating hesperetin
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induced Rhizobium sp. cells (Table 5 and 7). However, increases in pod
number increase was found to be higher with the Bohatyr than the
Celeste. This may be due to the early podding characteristics of the
Bohatyr variety.
5 In contrast to pea, a greater nodulation of lentil and
therefore nitrogen fixation was obtained with luteolin-induced Rhizobium
cells rather than with hesperetin-induced cells. Depending on which
strains were induced, a 55 to 63 % increase in nodulation and an 18 to
30% increase in biomass production was observed with luteolin-induced
10 cells. Cells induced with hesperetin also increased nodulation and plant
biomass of lentils, although these effects were not as strong as luteolin-
induced cells (Table 6 and Figure 11 ).
Discussion
Symbiotic nodule formation by Rhizobium bacteria and
15 plant hosts is a complex process, which involves the expression of
nodulation genes, the expression of which is triggered by signalling
compounds in the respective host plants. Specific environmental
conditions such as low RZTs has been shown to affect the initial steps in
the nodulation process, as for example the production and excretion of
20 both the plant-to-microbe and microbe-to-plant signalling molecules
(Smith and Zhang, 1999). A few studies have shown that the nodulation
of economically important legume crops such as soybean and bean, can
be enhanced by the exogenous application of nodulation gene inducing
compounds (Smith and Zhang, 1999, U.S.P. 5,922,316; Abdalla, 1994).
25 It was unknown, until the present invention, whether
signal molecules could overcome an environmental-stress-induced
reduction in nodulation, and/or nitrogen fixation and/or mass of pea
and/or lentil. The data provided herein show that the negative effects of
low RZT on pea and lentil nodulation and nitrogen fixation can be reduced
30 or completely overcome by using nodulation gene-inducing compounds
to preinduce rhizobial cells. In addition, greenhouse and field
observations showed that the application of signal molecular compounds
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46
can significantly enhance nodulation and grain yield of pea and lentil
plants.
Of the large number of commercially available
flavanones, isoflavones and other related compounds that were tested,
naringenin and hesperetin and to a lesser extent apigenin and luteolin
were found to be very powerful inducers of the nodC promoter of R.
Leguminosarum (Figure 2). R. Leguminosarum 1477 showed specificity
towards flavonoids and isoflavonoids since genistein and daidzein were
not shown to be active inducers of nod gene in R. Leguminosarum. While
specificity is shown to be dependent on the actual flavonoid or
isoflavonoid compound used, having now shown that such compounds
can compensate for a stress-induced inhibition of nodulation, the present
invention enables the skilled artisan to use assays such as the ~i-
galactosidase assay used herein to identify other flavones or isoflavones
which could be used in accordance with the present invention (introduced
into the compositions of the present invention).
The potency of nod gene induction displayed by the
different compounds tested may be related to structural differences
thereof and to their capacity to inhibit nod gene expression in different
rhizobia. For example, genistein inhibits nodF induction in R.
leguminosarum. bvs. viceae and trifolii (Firmin et al., 1986); naringenin
inhibits nod gene induction in R. meliloti (Peters and Long, 1988) and
some 8. japonicum strains (Kosslak et al., 1990). Some isoflavones have
even been reported to inhibit the induction of R. leguminosarum by pea
root exudate (Firman et al., 1986).
Furthermore, flavonoid levels have been shown to affect
legume nodulation and N2 fixation directly (Appelbaum, 1990). Kapulnik
et al. (1987) have, for example, reported that the superior nodulation and
Nz fixation of HP32 alfalfa, as compared to HP alfalfa, was associated
with a 77% increase in the amount of luteolin in plant tissue. It has been
shown herein that an optimum concentration of inducer is required for
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maximum induction of nod genes based on ~i-galactosidase activity
(Figure 6).
It is known that different legume species secrete a
number of different inducer compounds. For example, the alfalfa plant
secretes more than 400 natural nod gene-inducing compounds. To date
there is no clear understanding as to how legumes profit by releasing
more than one nod gene-inducing flavonoid. The presence of more than
one nodD gene in R. meliloti or R. Leguminosarum suggests that various
flavonoids released from the host plant may activate different nodD
genes. Common bean release natural nod gene inducers belonging to
four different classes of flavonoids (Hungria et al., 1991). Thus, R.
Leguminosarum nod gene products may be activated by a number of
different flavonoids in a concentration-dependent manner. This concept
is supported by the data presented herein with R. Leguminosarum 1477
which showed that higher ~3-galactosidase activity was observed with a
7:3 combination of hesperetin to naringenin (Figure 4).
The magnitude of the effect of the inoculation with
preinduced strains is also strain dependent. The strains used in this study
have shown clear differences in their abilities to produce and excrete Nod
metabolites at a low temperature (Figure 6). In contrast to strain 1477,
strain 5280 showed reduced activity when this strain was grown at
suboptimal temperature of 17°C in the presence of the same amount of
inducer compounds (Figure 6). The difference in activity between the
strains could be due to differences in excretion rather than in production
of Nod metabolites, which is more sensitive to environmental stresses
(Mckay and Djordjevic, 1993). Moreover, it is apparent from Figures 6 and
7, that although extensive nod gene activity, based on f3-galactosidase
activity, is evident at 28°C, lowering the temperature to 17°C
markedly
reduces the f3-galactosidase activity at the maximum growth level of the
cell cultures .
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Studies on subtropical legumes have shown that RZTs
lower than 25°C decrease both nodulation and nodule function. All
stages of the establishment of the symbiotic relationship in soybean
investigated to date have been shown to be inhibited by suboptimal RZT
(Zhang and Smith, 1995). Suboptimal RZT retard root hair infection more
than nodule initiation, nodule development or nitrogen assimilation
(Gibson, 1971 ) which may be associated with production Nod factors, the
return signal associated with Rhizobium.
Nodule formation and biomass production under
controlled environment conditions at suboptimal temperature both in
growth chamber and in greenhouse studies were greater when
preinduced rhizobial cells were used as inoculant. Similar results were
observed for both pea and lentil. These observations suggested that
applying preinduced cells can be used to overcome an environmental
condition which inhibits nodulation and especially which inhibits early
stages of nodulation. As exemplified herein, preinduced rhizobial cells,
according to the present invention, were shown to overcome low
temperature inhibition on plant nodulation and nitrogen fixation. However
the extent of increase in nodulation and biomass production over
uninduced cell inoculation varies with the plant cultivars and the rhizobial
strains used. Although overall nodule formation was higher with the strain
R. Leguminosarum 1477, the % increase in nodulation and biomass
production was higher when using preinduced cells from the commercial
Rhizobium strain. In addition, among the four different signal molecules,
preinduced cells with hesperetin showed better nodulation on pea plants,
which corresponds with the results obtained in laboratory experiments
where maximum nod gene activity in terms of f3-galactosidase activity was
obtained from hesperetin induced culture (Figure 2). However, the
efficacy of the inducer varied with the legume tested. Whereas
hesperetin induced cells performed better on pea plants, luteolin induced
cells performed better on lentil plants (Tables 5 and 6) indicating that
specificity in legume-rhizobia combinations are determined, at least in
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part, by the secretion of specific signal molecules. In any event, it is clear
from the present invention that there is a qualitative and a quantitative
difference in the ability of the nodulation regulons to operate in the same
background.
In term of temperatures, nodulation responses (i.e
difference between nodule and biomass production by inoculating
preinduced and uninduced cells) were more than double at a lower
temperature of 17°C than at optimal temperature of 24°C (Table
7).
Mckay and Djordjevic (1993) demonstrated that nodule occupancy by
different rhizobial strains, in some cases, is determined by certain
environmental changes. In addition to the reduction of nodulation at
temperature extremes, there are also specific temperature-sensitive
legume-rhizobium combinations as was found for R. Leguminosarum bv.
trifolii; strain TA1 forms nodule with Trifolium subterraneum cv.
woogenellup at above 25°C but not below 22°C, although it
nodulates a
range of other cultivars at the lower temperatures (Lews-Henderson and
Djordjevic, 1991). The ability of different strains to produce and release
Nod metabolites is likely to be a major determinant of nodule occupancy.
Hence screening of strains suitable for low temperature rhizobium-legume
combinations should be determined for specific environments.
Taken together from the laboratory and greenhouse
experiments presented above, better nodulation and nitrogen fixation
performances in field pea and lentil were demonstrated using preinduced
strains under low soil temperature conditions. The present invention
therefore provides a cost-effective method to enhance the efficacy of
inoculants.
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EXAMPLE 2
Application of pea symbiotic signal molecules in Rhizobium
leguminosarum increases pea symbiotic nitrogen fixation,
plant dry matter and final grain yield under field conditions
5 Three field experiments were conducted at the E. Lods
Agronomy Research Centre of McGill University, Mcdonald campus, in
Ste-Anne-De-Bellevue, Quebec, to address this objective. In the first
experiment, different pea inoculant formulations and the pea signal
compounds (PeaSignal) were tested on two varieties. In the second
experiment, the interaction of the PeaSignal and other commercial pea
inoculants was tested. In the third experiment, the best rate of application
of PeaSignal was evaluated. In all experiments, the size of the
experiment plots were 7.65 m2, with 6 rows spaced 30 cm apart. The
experimental site had previously grown peas, and therefore the R.
leguminosarum population in this land amounted to 1.0 x 106 cells per
gram of soil. Treatments were randomized in four replicate blocks. The
seeds of all pea cultivars used in the experiments were obtained from
commercial seed sources. To avoid the cross contamination of
treatments, all three experiments were hand planted.
EXPERIMENT 1
This experiment was arranged as split-plot design with
4 blocks. Two pea varieties, Bohatyr and Celeste, were treated as the
main-plot factors. The inoculant treatments and the PeaSignal were the
sub-plot factors. The treatments were as follows: pre-induced R.
leguminosarum cells on dry peat, untreated cells on dry peat, pre-induced
cells in a liquid culture, uninduced cells in a liquid culture, the PeaSignal
applied to the seed and applied to the furrow and an untreated control.
The first two inoculant treatments were cultured and then injected into
sterile peat, which would be a typical formulation for a peat-based pea
inoculant.
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Preinduced cell inoculation
Peat based application
The commercial strain Rhizobium sp. was used for all
three field experiments. Preinduced Rhizobium culture was prepared by
growing up the culture in the presence of 10 NM hesperetin for 24 hours.
One set of cultures was grown in the absence of signal compounds and
therefore is considered as the uninduced treatment. Both the induced and
uninduced cells were collected by centrifugation and mixed with peat to
produce a cell density of 2.0 x 10g cells/gram, and a final moisture
content of 39%. The induced cell preparation was termed as "AffixP+"
and uninduced cell preparation was termed as "AffixP-". Seeds were
treated with the inoculants at the rate of 3.0 grams of peat inoculant per
kilogram of seed and were planted within two hours of treatment.
Preinduced cells in a liguid culture
To follow the protocol of the growth chamber and
greenhouse experiments previously done, an experiment was carried out
with induced and uninduced cells suspended in 0.5% saline or in water.
Twenty ml of the cell suspension containing 1.0x10' cells/ml was applied
into the furrow by syringe, on top of the sown seeds.
PeaSiginal-liquid nodulation booster
Seed treatments
PeaSignal was prepared by incorporating signal
molecules hesperetin and naringenin in a 7:3 ratio mixed with biofactors
of Rhizobium sp. The final concentration of the signal compounds in
PeaSignal was 208 ~M. One part of PeaSignal was diluted with 3 parts
of water and 3.0 ml of this solution was used to treat 1 kilogram of seed.
In furrow application
PeaSignal was applied at the rate of 74 ml per hectare.
This rate of application of active product was achieved by taking 36 ml of
distilled water and adding 0.315 ml of PeaSignal. This solution was then
applied to the seeds in the furrows of the plot by syringe.
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52
EXPERIMENT 2
The interaction of PeaSignal with a commercial liquid
pea inoculant was tested in this experiment. The liquid inoculant used
was Liqui-Prep, supplied by Urbana Laboratories of St. Joseph Mo.. First,
18,5 ml of PeaSignal was mixed with 60 ml of Liqui-Prep, and incubated
at room temperature for about one hour. After this period, 2.86 ml of the
PeaSignal inoculant mixture was applied onto 1 kg of pea seeds.
EXPERIMENT 3
This experiment was also arranged as a split-plot design
with four blocks. The main plot factor was the pea variety and the
different application rates were the sub-plot factor. The PeaSignal was
applied as a seed treatment. The activated compound of PeaSignal at
0, 30, 60, 120, 300 and 600 ~M were tested in this experiment to
determine the most effective concentration of signal molecules in
PeaSignal.
Data collection
Plant nodule number, nodule biomass, shoot biomass
accumulation and pod number were measured from a five plant
subsample, which was collected randomly from each plot at 8 weeks after
planting. At maturity, the plots were harvested by a plot combine to
measure grain yield.
Statistical analyrsis
The data were analysed statistically by using the
Statistical Analysis System (SAS) computer package(SAS Institute Inc,
1998). When analysis of variance showed a significant treatment effect
(p<0.05), the LSD tests were applied to make comparisons among the
means (p<0.05) (Steel and Torrie, 1980).
RESU LTS
Preincubation of Rhizobium leguminosarum with pea
symbiotic signal molecules significantly improved pea nodulation
processes and plant dry matter accumulation and grain yield under field
conditions. Compared to the uninduced control, induced cells
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
53
dramatically increased plant nodule number, nodule dry weight and final
grain yield. The grain yield of peas receiving the preincubated cells was
10.94% higher than the uninoculated control (Table 8).
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
54
d
N ~ Z
a~ N N
.C O
fn ~ . (6
L ~ +~'-'- +
~
a
L
O O *
1 C o
V m C Q N ONO
G ~
+
y d
C
V C
a
O
~ r
C O O
(a O 00 N
C _t0 ~t ~
~
ao y
c
J
+ .-.
Q tL0 ~ ~
+, c EO W i
co co a yp
,~ ~ Q O O N C
N O
G7 ~ ~ p .
O O
Z + U
.Q
a
O ~
C 'a L C
N ~
O ~ ~ ca
C C 0 \
C f6 O
C
_ f~ N O
y
Q T M ~ L
~ \ N ~- ~ j
.Q C C *k C+ON
.a ~ Z E
O
.Q cG tn U E
ni O _fnU C (B
C U
U ~ ~ (B
N '~ U
J C 'D
H C ~ W
~ ~ I
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
Pea nodulation, shoot dry matter, and final grain yield
responded differently to peat-based products containing either preinduced
R. leguminosarum by pea symbiotic signal molecules or uninduced cells.
Plants having received preincubated R. leguminosarium had 32.51,
5 144.56, and 57.85% more nodule number, nodule dry weight and pod
number, respectively, than those having received uninduced cells under
field conditions (Table 9). The data presented in Tables 8 and 9
demonstrate that pea nodulation and grain yield are dramatically
increased by applying preincubated R, leguminosarum cells, either using
10 a peat based product or in liquid format.
CA 02439421 2003-08-29
WO 01/72126 5 6 PCT/CAOI/00075
d
c O
c a_~ _
c
Q ~ ~ Z
O +~ ~ MO
V ~ (~ ~ N N
V ~C (a O
_ C
O O
w
Q. U
c
L #
~
_
~
U7 ~ M O O
L~
d
N
N
'C _
N = (n
~ D O ~ Z
c+rC~ ~ o
d T C _
d M O
Q. L ti cB p~ h
I G7 d ~ r
N
J
t.-~.
~ a ~ a ~ ~ o
O d o
0 ~ U
C . D ~ O O
L ~ + p_
OQ O Z
L
O C M *
~
' ,~ ca M ~ o ..
r
O r r ~ ~L
s " r N >
Z + N
i~ O
"a N
ceiO N ~ U
N U ~ U
7 Q U ~ ~_ (B
~ ~ .'<tN
O d
~ C C ~S U
N ~ ~ o
d H
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
57
Direct application of the pea symbiotic signal molecules,
as PeaSignal, either into the furrow and onto the seed surface
significantly increased plant nodule number, and numerically increased
plant dry matter and final grain yield (Table 10). Application of PeaSignal
into the furrow and onto the seed surface significantly increased nodule
number by 64.72, and 69.33%, respectively, compared to untreated
plants under the same field conditions. The nodule mass was also
greatly increased by the PeaSignal application (42.44 % for the furrow
application, and 42.22% for the seed treatment). However, as there was
a high level of experimental variablity in this field, this parameter was not
statistically significant. Yield was also increased by in-furrow application
of PeaSignal by 11.70%.
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
58
a~ ~ ~
Z
~ Z
~ fl '-'
co o co 0 0
*~- N N N
~j r
V (a
C ~. +
~
01
_ _
d
C Z Z
Q d 0 0 n O
(B M M \ o
Q- N M ~ O
M M N N N
M M
O
U
O ~
z _
0 ' O o
N f~ OO o
- M f~ O ~ M
(B ~ ~t ~ O 00
p Sricfl
+
+
o
_
m . ~ ~ ~ cn
U ~ .~ ~ Z
J t~71 _ n ~ Z
N 00 07 o O O
- ~t 0O ~. N
N ~ w ~ e- O ~ N
~C ~ ~ O O O N N O
d O ~ d'
c Z a + +
V
~
_ _ ~
N .. O O
C E C 0 \ o
H7 p C Q N
N M I~ M n
N ~
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f!! ~ .~ U
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. ~ _ ~ -a
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' c~ *''c~ Y c
~ v U U
o ...o
~ c ~ (n
O .' o 0
- ~ a~ c y y
cn ~ +i +I
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
59
To determine whether a commercial liquid pea
inoculant's performance could be improved by incorporation of PeaSignal,
the latter was premixed with a liquid product (from Urbana, USA), and
then the seeds were treated before planting. Generally speaking,
incubation of the liquid inoculant with the PeaSignal improved nodulation
and increased grain yield and plant dry weight (Table 11).
CA 02439421 2003-08-29
WO 01/72126 6 ~ PCT/CA01/00075
L
c ~ ~n cfl
_ ~ N N o
d (~ N N
~
>' O
C ~ t
L
~_. Z
Q
C d C 00 O
ca ~ Z ca M ~ o
Q. O d- N
O a ~ O N N
N
'a >
O p
C .-.
d L7
d m ~ ~ M M Z
Q- d C
C ~ ~ ~ ~ ~ o
O O C Q ~ ~- O
r = (B d' ~ O
t4 Q ~ r
~ ~
_ t
LiJ p
r .-.
cC O .~
C O O
_c~f O O o
Q 00 O N n
CO M ~ C
U ~ ~ o ~ o
O L p t
c d Z U_
N
L
C1 ._ 'U * 'U
~ ~ a
O C o o ca
O ~ ~ O M M N
L- _ ~ ~ O
a7 O ~ '/ t cB
.~ Z
N
d
w O
C O
C~ U E
C
O ~ t N U
7
r (B
o
d _ U
_f
I- J J ti
W
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
61
In experiment 3, the best rate of application of PeaSignal
was determined by this field trial. The results (Table 12) showed that the
best application rate of the PeaSignal product was at 300 ,uM of active
compound, which was twice that of the application rate in experiment 1,
or 1.23 ml of PeaSignal for each kg of pea seed.
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
62
ti
a~
v ~ ~ Z
_= a ~ , N N
y_ ~; y
(~ "~'
> ~ 0
V O ~ N
C
O
L
Q G7
C_ d
a ~ Z
_G> O C M O
V (/~ Z _(aCO ~
... ~ Q..O tf~n
C ~h M
3 G -
O
t4
U
o
V L
d
~ ~
N C O O o
N V C ~_ fB O ~
+.' W . ~ ~ ~ O (Op
W O C G~ ~ v~ ~ d' cfl
J
t.
O +,
V ~ #
. C ~
d O - o O
O
L ~ ~ O T U
~ O O ~ O
O
Z +
V
_
G. __ E C O M
Q. o
of .ca ~ (a ~ ~ .
+~ a7 Q- O O M 'N
N ~ O O to
d ~ ~ z ~ ~ aj >
0 '. f N
d
a z o
w C
O c3 ~ N
C C X ~
O_ O_ N C U
a"' '~' N N N
E (B
O ~ O ot5
p C (a ~ C
d H
Q. JJ
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
63
DISCUSSION
Thus, the field experiments herein show that a
preincubation of R. leguminosarium with at least one pea symbiotic signal
compound increases pea nodulation, plant dry matter and pod number.
However, the final grain yield is not as positively enhanced as expected
from the field nodule count. Even though some of the treatment
numerically increased final grain yield by up to 11 %, it was not a
statistically significant improvement as compared to control. Without
being limited by particular hypotheses, a number of reasons might explain
this result: (1 ) the Montreal area is not a pea production area. The hot
summer conditions might have negatively affected seed filling. (2) The
experimental variability was relatively large, which might have influenced
the statistical analysis. (3) Field harvesting was late. As application of at
least one pea symbiotic signal compound improved nodulation and
possible nitrogen accumulation as well, it should have increased plant
growth and development. All of these led plants to mature earlier than
the untreated control plant. The late harvest resulted in severe seed
shattering from the pods, especially for plants receiving the PeaSignal
treatments. This may have also reduced the treatment effect as well.
Nevertheless, the present invention shows that signal
molecules (i.e. flavonoid compounds) could be used to release the
inhibition of nodulation and nitrogen fixation of pea and/or lentil grown in
the field. More particularly, the present invention provides strong
evidence that such signal molecules should significantly improve the yield
of pea and/or lentil grown in the field under condtiions which inhibit and/or
delay N fixation and nodulation and especially low RZTs.
The demonstration that the addition of signal molecules
increases nodulation and yield of pea and lentil grown in the field, and
relieves the nodulation-inhibiting conditions caused by environmental
factors, serves to validate the laboratory and greenhouse methods used
to identify and select agents which could be used in the field as
production enhancing compositions and methods for pea and lentil.
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WO 01/72126 6 4 PCT/CA01/00075
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.
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
REFERENCES
Abdalla, M. H. 1994. Some phenolic compounds enhance modulation and
nitrogen fixation in a soybean Bradyrhizobium japonicum system. Phyton-
Annales. Rei. Botanicae. 33: 249-256.
5
Appelbaum, E. 1990. The Rhizobium/Bradyrhizobium-legume symbiosis.
In: Gresshoff PM, ed. Molecular biology of symbiotic nitrogen fixation.
Boca Raton: CRC Press, 131-158.
10 Carlson, R.W. et al., 1993, The structures and biological activities of the
lipo-oligosaccharide modulation signals procedure by type I and II strains
of Bradyrhizobium japonicum, J.B.C. 268:18372-18381.
Coventry, D. R. and Evans, J. 1989. Symbiotic nitrogen fixation and soil
15 activity. P. 103-137. In A. D. Robson (ed.), Soil acidity and plant growth.
Academic Press, New York.
Cralle, H. T. and Heeichel, G. H. 1982. Temperature and chilling
sensitivity of nodule nitrogenase activity of unhardened alfalfa. Crop. Sci.
20 21:300-304.
Dusha, I, Bakos, A., Kondorosi, A., de Bruijn, F. J. and Schell, J. 1989.
The Rhizobium meliloti early modulation genes (nodABC) are nitrogen
regulated: isolation of a mutant strain with efficient modulation capacity on
25 alfalfa in the presence of ammonium. Mol. Gem. Genet. 219: 89-97.
Firmin, J. L. Wilson, K. E., Rossen, Land Johnston, A. W. B. 1986.
Flavonouid activation of modulation genes in Rhizobium reversed by other
compounds present in plants. Nature. 324: 90-92.
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
66
Gibson, A. H. 1971. Factors in the physical and biological environment
affecting nodulation and nitrogen fixation by legumes. Plant and Soil.
(Special Volume): 139-152.
Hicks, P.M. and Loynachan, T. E. 1987. Phosphorus fertilisation reduces
vesicular-arbuscular myccorrhizal infection and changes nodule
occupancy of field-grown soybeans. Agronomy. 79: 841-844.
Handelsman, J.E. and Halverson, L.J., 1989, Method to enhance root
nodulation in legumes and inoculant preparation therefor,
U.S.P. 4,878,936.
Hoagland, D. R. and Arnon, D. I. 1950. A water culture method for
growing plants without soil. California Agricultural Experimental Station,
Circular.347:25-32.
Hooykaas, P. J. J., klapwijk, P. M., Nuti, M. P., Schilperoort, R. A. and
Rorsch, A. 1979. Transfer of the agrobacterium tumefaciens Ti plasmid
to a virulant agrobacteria and to Rhizobium ex plants. J. Gen. Microbiol.
98 477-484.
Hungria, M., Joshep, M. and Philips, D. A. 1991. Anthocyanidins and
flavonols, major nod-gene inducers from seeds of a black-seeded
common bean (Phaseolus vulgaris L.). Plant Physiol. 97: 751-758.
Hungria, M. and Stacey, G. 1997. Molecular signal exchanged between
host plants and Rhizobia: Basis aspects and potential application in
agriculture. Soil Biol. Biochem. 29: 819-830.
Ian and Djordjevic, 1993.
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
67
Johansen, E. and Appelbaum, E.R., 1995, Method for selecting mutants
of legume-nodulating bacteria enhanced in competition,
U.S.P. 5,432,079.
Kapulnik, Y., Joseph, C. M. and Philips, D. A. 1987. Flavone limitations
to root nodulation and symbiotic nitrogen fixation in alfalfa. Plant Physiol.
84: 1193-1196.
Kondorosi A., 1992, Regulation of nodulation genes in rhizobia. In
Verme, D.D.S. (ed), Molecular Signals in plant-microbe communication,
pp. 325-340, CRC. Press Boca. Raton. FL.
Kosslak, R., Bookland, R. and Appelbaum, G.R., 1993, Bradyrhizobium
japonicum nodulation inducing factor, U.S.P. 5,229,113
Kosslak, R. M., Joshi, R.S., Bowen, B. A., Paaren H. E. and Appelbaum.
E. R. 1990. Strain-specific inhibition of nod gene indution in
Bradyrhizobium japonicum by flavonoid compounds. Appl. Environ.
Microbiol. 56: 1333-1341.
Lews-Henderson, W. R. and Djordjevic. 1991. A cultivar-specific
interaction between Rhizobium Leguminosarum bv. trifolli and
subterranean clover is conditioned by nodM, other bacterial cultiver
specificity genes, and a single recessive host gene. J. Bacteriol. 173:
2791-2799.
Lynch, D.H. and Smith, D.L., 1994, The effect of low root zone temperature
stress on two soybean (glycine max) genotypes when combined with
Bradyrhizobium strains of varying geographic origin, Physiol. Plant 90:105-
113.
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
68
Mckay, I. A. and Djordjevic, M. A. 1993. Production and excretion of Nod
metabolites by Rhizobium Leguminosarum bv. trifolii are disrupted by the
same environmental factors that reduce nodulation in the field. Appl. Environ.
Microbiol. 59 : 3385-3392.
Miller, J. H. 1972. Experiments in molecular genetics. P. 352-355. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N. Y.
Peters, N. K. and Long, S. R. 1988. Alfalfa root exudates and compounds
which promote or inhibit induction of Rhizobium meliloti nodulation genes.
Plant Physiol. 88: 396-400.
Psom, 1996, Nodulation factors in plant microbe interactions, In Stacey et al.
(eds.), Pub. Chapman and Hall.
Rice et al., WO 94/25568.
Richardson, A. E., Simpson, R. Djordjevic and Rolfe, B. G. 1988. Expression
and induction of nodulation genes in Rhizobium trifolii at low pH and in the
presence of Ca and AI. Appl. Environ. Microbiol. 54: 2541-2548.
Robson, A. D. and Bottomley, P. J. 1991. Limitations in the use of legumes in
agriculture and forestry, P. 320-349. In M. J. Dilworth and A. R. Glenn (ed),
Biology and Biochemistry of nitrogen fixation. Elsevier, Amsterdam.
Rolfe, B.G., Redmond, S.W., Batley, M. and Djordjevic, M.A., 1992,
Nodulation inducing factors, U.S.P. 5,141,745.
Rossen, L., Shearman, C. A., Johnston, A. W. B. and Downie, J. A. 1985. The
nodD gene of Rhizobium Leguminosarum is autoregulatory and in the
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
69
presence of plant exudate induces the nod A, B, C genes. The EMBO J. 4:
3369-3373.
Schubert et al. 1978.
Smith, D. L and. Zhang, F. 1999. Composition for enhancing grain yield and
protein yield of legumes grown under environmental condition that inhibit or
delay modulation thereof. U. S. patent 5,922,316.
Spaink, H.P., 1995, Molecular basis of infection and modulation by Rhizobium
and oats of sympathogenesis, Ann. Rev. Phytopathol. 33:345-368.
Spaink, H. P., Wijffelman, C. A., Pees, E., Okker, R. J. H. and Lugtenberg, B.
J. J. 1987. Rhizobium modulation gene nodD as a determinant of host
specificity. Nature. 328: 337-340.
Sprent, J.L. and Sprent P., 1990, Nitrogen fixing organisms, Chapman and
Hall, New York, 249 p.
Stacey, G., Carlson, R.W. and Spaink, H., 1992, Pentasaccharide
phytohormones and methods for their use, U.S. P. 5,175,149.
Van Brussel, A. A. N., Recourt, K., Pees, E., Spaink, H., Tak, T., Wiffelman,
C. A. T., Kijne, J. and Lutenberg, B. J. J. 1990. A bio-var-specific signal of
R.
Leguminosarum bv. viciae induces increased modulation gene-inducing activity
in root exudate of Vicia sativa subsp. Nigra. J. Bacteriol. 172 : 5394 -5401.
Verma, DPS, 1992, Signals in cool nodule organogenesis and endocytosis of
Rhizobium plant soil, 4:373-382.
CA 02439421 2003-08-29
WO 01/72126 PCT/CA01/00075
Zahran, H. H. and J. I. Sprent. 1986. Effect of sodium chloride and
polyethylene glycol on root hair infection and nodulation of Vicia faba L.
plants
by Rhizobium Leguminosarum. Planta (Berlin) 167: 303-309.
5 Zahran, H.H., 1989, Rhizobium legumes symbiosis and nitrogen fixation under
conditions and in an arid climate, Microbiology and Molecular Biology Reviews
63:963-989.
Zhang, F., Lynch, D. H. and Smith, D. L. 1995, Impact of low root zone
10 temperatures in soybean (Glycine max) on nodulation and nitrogen fixation,
Env. Exp. Bot. 35:279-285.
Zhang, F. and Smith, D. L. 1996 , Preincubation of Bradyrhizobium japonicum
with genistein accelerates nodule development of soybean (glycine max) at
15 suboptimal root zone temperature, PIantPhysol. 108:961- 986.
Zhang, F., Charles T.C., Pan B. and Smith D., 1996, Inhibition of the
expression of Bradyrhizobium japonicum nod genes at low temperatures, Soil
Biology and Biochemistry 28:1579-1583.
