CA2439421A1 - Flavonoid compositions for improving pea and lentil production - Google Patents

Abstract

Compositions for enhancing grain yield and protein yield of a pea and/or lentil grown under environmental conditions that inhibit or delay nodulation thereof are provided. The compositions comprise a nodulation gene-inducing compound such as flavones. Moreover, methods for enhancing grain yield and protein yield of a pea and/or lentil grown under environmental conditions th at inhibit or delay nodulation thereof are provided. In one embodiment, the methods comprise an addition of an agriculturally effective amount of a nodulation gene-inducing compound such as flavones, in the vicinity of the seed or root of the pea and/or lentil. In particular, a condition which inhibits root nodulation is a root zone temperature below 25 ~C.

Description

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.

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 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 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 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.

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 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

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 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 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 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 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 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 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 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 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 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 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 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 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 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;

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.

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 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.

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 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, 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 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 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 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.

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).

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).

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.

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.

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.

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) 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).

(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 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).

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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 The percentage increases of plant nodule number and shoot biomass production with different pea and rhizobial genotypes 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 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 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 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 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 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 .

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 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.

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.

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.

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.

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.

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 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).

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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.

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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%.

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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.

Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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Claims (23)

WHAT IS CLAIMED IS:

1. A method for enhancing grain yield of pea and/or lentil grown in the field under conditions which inhibit nodulation thereof, comprising a treatment in the vicinity of one of a seed and root of said pea and/or lentil with a composition comprising an agriculturally effective amount of a nodulation gene-inducing compound in admixture with an agriculturally suitable carrier medium, wherein said effective amount enhances grain yield of said pea and/or lentil grown in the field under said condition which inhibit nodulation, in comparison to an untreated pea and/or lentil grown under the same conditions.

2. The method of claim 1, wherein said composition comprises at least one flavonoid compound.

3. The method of claim 2, wherein said flavonoid compound is selected from at least one of hesperetin, naringenin and luteolin.

4. The method of claim 3, wherein a rhizobial strain which nodulates said pea and/or lentil is exposed to a concentration of said flavonoid compound ranging from about 0.5 µM to about 20 µM.

5. The method of claim 4, wherein said flavonoid concentration ranges from about 1 µM to about 10 µM.

6. The method of claim 1, 2, 3 or 4, wherein said condition which inhibits nodulation is low root zone temperatures (LRZT).

7. The method of claim 6, wherein said LRZT is below about 25°C to about 17°C.

8. The method of claim 6, wherein said LRZT is from about 17°C to about 10°C.

9. A method for enhancing grain yield of pea and/or lentil grown in the field under a condition which inhibits the nodulation thereof, comprising:
a) incubating a rhizobial strain which nodulates said pea and/or lentil with an agriculturally effective amount of a nodulation gene-inducing compound, in admixture with an agriculturally suitable carrier medium, wherein said effective amount enhances grain yield of said pea and/or lentil grown in the field under said condition which inhibits the nodulation thereof in comparison to an untreated pea and/or lentil grown under the same conditions; and b) inoculating in the vicinity of one of a seed and root of said pea and/or lentil said rhizobial strain of a).

10. The method of claim 9, wherein said composition comprises a flavonoid compound.

11. The method of claim 9 or 10, wherein said condition which inhibits the nodulation thereof is low root zone temperature.

12. A method for enhancing nodulation of pea and/or lentil, comprising a treatment in the vicinity of one of a seed and root of said pea and/or lentil with a composition comprising an agriculturally effective amount of a nodulation gene-inducing compound in admixture with an agriculturally suitable carrier medium, wherein said effective amount enhances nodulation of said pea and/or lentil.

13. The method of claim 12, wherein said pea and/or lentil is grown in the field under a condition which inhibits the nodulation thereof.

14. The method of claim 13, wherein said condition which inhibits the nodulation is low root zone temperature.

15. The method of claim 14, wherein said composition comprises a flavonoid compound.

16. A method for enhancing nodulation of pea and/or lentil, comprising:
a) incubating a rhizobial strain which nodulates said pea and/or lentil with a composition comprising an agriculturally effective amount of a nodulation gene-inducing compound, in admixture with an agriculturally suitable carrier medium, wherein said effective amount enhances nodulation of said pea and/or lentil in comparison to an untreated soybean grown under the same conditions; and b) inoculating in the vicinity of one of a seed and root of said pea and/or lentil said rhizobial strain of a).

17. The method of claim 16, wherein said pea and/or lentil is grown in the field under a condition which inhibits nodulation thereof.

18. A composition for enhancing grain yield and protein 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 medium.

19. The composition of claim 18, further comprising a rhizobial strain, wherein said rhizobial strain nodulates said pea and/or lentil and wherein said nodulation (nod) gene-inducing compound is effective in inducing the nod genes of said rhizobial strain.

20. The composition of claim 19, wherein said nodulation gene-inducing compound is selected from hesperetin, naringenin and luteolin.

21. The composition of claim 18, wherein said nod gene-inducing compound is effective in inducing the nod genes of native soil rhizobial strain and wherein said composition is effective in enhancing grain yield and protein yield of pea and/or lentil.

22. A method for enhancing nodulation and/or 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-inducing compound.

23. The method of claim 22, wherein said supply is an endogenous supply.