Patent Application
20210068400
The present invention relates to compositions and methods for enhancing nitrogen fixation in plants. More specifically, the invention provides combinations of non-pathogenic, atmospheric nitrogen fixing, bacteria with one or more additional agents, and the use of said combinations in the fixation of nitrogen in several different species including graminaceous plants.
Nitrogen fixation is a process by which nitrogen in the Earth's atmosphere is converted into ammonia or other nitrogen-containing molecules which are then made available to living organisms for their metabolic and biosynthetic needs. In the case of plants, supply of nitrogen is needed from the early stage following germination until the plant has matured and developed its full crop yield potential.
The Gramineae family includes maize wheat and rice, which are the three main crops used world-wide for feeding the human population.
Unlike the Leguminosae plants that can fix atmospheric nitrogen by symbiosis with certain bacterial species, including those of the Rhizobium genus, the Gramineae family is not able to fix atmospheric nitrogen and growers need to use chemical fertilizers to supply the plants with the required amount of nitrogen, in order to improve crop yields.
This method of chemical fertilization, however, is not without significant problems, not least of which is massive contamination of the fresh water resources on the planet, leading to severe ecological damage. This may occur, for example, when nitrogen-containing fertilizers are washed out from the root zone of the plants and leak into the deeper aquifers and the fresh water reservoirs.
An urgent need therefore exists for alternative methods and compositions for enabling and/or enhancing the fixation of nitrogen in many plant species, in particular those of the Gramineae family. The present invention provides a solution for this need.
The present inventors have unexpectedly found that when certain bacteria, such as Rhizobium species are administered to plants in combination with certain other substances (as will be disclosed and described in detail hereinbelow), said combinations are capable of fixing atmospheric nitrogen and thereby supply the plant's nitrogen needs. This effect is particularly unexpected when the plants so treated are those of the Gramineae family, which, as explained hereinabove, are normally unable to obtain their nitrogen requirements by means of nitrogen-fixation mediated by bacteria present in the soil.
The present invention is primarily directed to a method for completely or partially supplying the nitrogen requirements of a plant, by means of administering to said plant a combination of non-pathogenic atmospheric nitrogen fixing bacteria together with one or more activating agents. In some cases, one or more fertilizers is also supplied together with said bacteria and activating agents. Thus, the present invention is primarily directed to a method for enabling fixation of atmospheric nitrogen in plant species that are normally unable to obtain their nitrogen intake in this manner.
In another aspect, the present invention provides a composition comprising a mixture of non-pathogenic nitrogen-fixing bacteria and one or more activating agents (as defined hereinabove and described hereinbelow).
In a further aspect, the present invention also provides a method for increasing the yield of a plant of agricultural or horticultural importance by means of:
In a still further aspect, the present invention further provides a method for increasing the yield of a plant of agricultural or horticultural importance by means of:
a) providing separately:
b) separately administering each of compositions (i) and (ii) to said host species.
In the above-disclosed methods and compositions, the nitrogen fixing non-pathogenic bacteria are, in one embodiment, members of the Rhizobium genus. In one preferred embodiment, the bacteria are of the species Rhizobium leguminosarum. Further examples of suitable bacteria will be disclosed hereinbelow.
In the above-disclosed methods the plant of agricultural or horticultural importance is, in one embodiment, a member of a species which is normally unable to obtain its nitrogen requirements by bacterial fixation of atmospheric nitrogen. In one embodiment, said plant species is a member of the Graminaea family. In one preferred embodiment, the plant species is maize. In another preferred embodiment, the plant species is wheat. In a still further preferred embodiment, the plant species is rice.
The present inventor have found, as disclosed hereinabove, that certain combinations of non-pathogenic nitrogen fixing bacteria and activating factors (whose properties will be described in detail hereinbelow) are capable of permitting nitrogen fixation in plant species (such as cereals) which are normally unable to obtain their nitrogen requirements in this way.
It has also been found by the inventors that the same combinations of nitrogen-fixing bacteria and activating factors also possess both anti-inflammatory and anti-microbial properties (directed against several different bacterial and fungal species, including those known to be plant pathogens).
The reason for this correlation between the ability of these combinations to permit nitrogen fixation in species that are normally unable to obtain nitrogen in this way and their anti-inflammatory and anti-microbial properties is not entirely clear.
Without wishing to be bound by theory, it is believed that by means of administering bacteria of the Rhizobium genus with the additional substances and agents set out in this disclosure, symbiosis develops between said bacteria and the root systems of plants of species such as those of the Gramineae family, thereby enabling fixation of atmospheric nitrogen within the plant. Again, without being bound by theory, it is possible that the reason that this symbiosis does not occur in the absence of said additional agents may be rejection of the Rhizobium bacteria by the Gramineae plants. It is therefore possible that the additional substances and agents which permit the aforementioned symbiosis to take place do so by means of preventing the development of this rejection mechanism.
Thus, by these means, plants of the Gramineae family—and of other species which are similarly unable to obtain their nitrogen needs via nitrogen-fixing bacteria alone—are able to satisfy their nitrogen requirements.
In one preferred embodiment, the plant is a species which is normally unable to obtain its nitrogen requirements by bacterial fixation of atmospheric nitrogen. In one particularly preferred embodiment, the plant species is a member of the Gramineae family. One example of such a species is maize (Zea mays)
In one preferred embodiment, the non-pathogenic atmospheric nitrogen fixing bacteria are bacteria belonging to the general class known as Rhizobia. The bacteria of this class are distributed among several different genii, and have the common feature of being able to fix nitrogen in certain plant species (such as legumes), after having been established within the root nodules of said plants.
Thus, in one embodiment of the present invention, the non-pathogenic atmospheric nitrogen fixing bacteria are Rhizobia, belonging to one or more genii selected from the group consisting of Bosea, Ochrobactrum, Devosia, Methylobacterium, Phyllobacterium, Rhizobium, Shinella, Sinorhizobium/Ensifer, Azorhizobium, Burkholderia and Cupriavidus.
In one particularly preferred embodiment, the Rhizobial bacteria are of the Rhizobium genus. Many different species of Rhizobium may be used in the combinations of the present invention, including R. alamii, R. alkalisoli, R. cauense, R. cellulosilyticum, R. daejeonense, R. etli, R. fabae, R. galegae, R. gallicum, R. giardinii, R. grahamii, R. hainanense, R. halophytocola, R. helanshanense, R. herbae, R. huautlense, R. indigoferae, R. leguminosarum, R. leucaenae, R. loessense, R. lupini, R. lusitanum, R. mesoamericanum, R. mesosinicum, R. miluonense, R. mongolense, R. multihospitium, R. nepotum, R. oryzae, R. petrolearium, R. phaseoli, R. pisi, R. pusense, R. qilianshanense, R. sphaerophysae, R. sullae, R. taibaishanense, R. tibeticum, R. tropici R. tubonense, R. undicola, R. vallis, R. vignae and R. yanglingense.
In some other embodiments, the nitrogen-fixing bacteria used to work the present invention may be of the Bradyrhizobim genus, for example, a species such as Bradyrhizobium japonicum.
In some cases, the Rhizobium species selected may be one which is already in commercial use for providing nitrogen requirements of leguminous species such as peanuts (groundnuts) and soya.
However, in one particularly preferred embodiment, the species used is Rhizobium leguminosarum. Although several different biovars of this species exist, in one preferred embodiment of the present invention, the biovar used is R. leguminosarum biovar viceae.
In another preferred embodiment, the non-pathogenic atmospheric nitrogen fixing bacteria are bacteria of the Clostridium genus. These anaerobic bacteria are particularly preferred when the combinations of the present invention are administered to crops such as rice which are grown under water-logged conditions. In one preferred embodiment of this aspect of the invention, the nitrogen-fixing Clostridium are selected from the group consisting of C. pasteurianum, C. acetobutylicum, C. beijerinckii, C. butyicum, C. hungatei and C. acidisoli.
It is to be noted that the term “nitrogen fixing bacteria” is used to indicate that these bacteria are generally capable of fixing atmospheric nitrogen in a large variety of vegetable and legume species, many of which (such as soya and peanuts) are of great economic value. However, as noted hereinabove, these bacteria, by themselves, are incapable of causing nitrogen fixation in cereal crops and rice.
In the context of the present invention, the term “activating agent” is used to denote a substance which when present in a mixture together with the non-pathogenic nitrogen fixing bacteria or when delivered separately therefrom, enables fixation of atmospheric nitrogen when administered to growing plant species that are normally unable to obtain their nitrogen requirements by this route. In some cases, this effect may be seen to be the result of a synergistic interaction between the non-pathogenic nitrogen fixing bacteria and the activating agents.
The present inventors have unexpectedly found that many of the activating agents suitable for use in the method of the present invention share a common feature, namely their ability to inhibit inflammatory mediators that are more generally associated with higher animal species (such as Tumor Necrosis Factor alpha [TNF-α]) rather than with plant species. Thus, in one preferred embodiment of the present invention, the one or more activating agents are substances having anti-inflammatory activity.
In one embodiment of the method of the invention, activating agents each have an IC50 for the inhibition of NO production of less than 1.6 mg/ml and/or an IC50 for the inhibition of TNF-α production of less than 2.4 mg/ml.
In another preferred embodiment of the method, each individual activating agents (whether used alone or in combination with other such agents) has an IC50 for the inhibition of NO production of less than 0.4 mg/ml and/or an IC50 for the inhibition of TNF-α production of less than 2.4 mg/ml.
In another preferred embodiment of the method, each individual activating agents (whether used alone or in combination with other such agents) has an IC50 for the inhibition of NO production of equal to or less than 0.15 mg/ml and/or an IC50 for the inhibition of TNF-α production of equal to or less than 2.4 mg/ml.
In another preferred embodiment of the method, each individual activating agents (whether used alone or in combination with other such agents) has an IC50 for the inhibition of NO production of equal to or less than 0.1 mg/ml and/or an IC50 for the inhibition of TNF-α production of equal to or less than 0.2 mg/ml.
In a still further preferred embodiment of the method, each individual activating agents (whether used alone or in combination with other such agents) has an IC50 for the inhibition of NO production of equal to or less than 0.05 mg/ml and/or an IC50 for the inhibition of TNF-α production equal to or less than 0.1 mg/ml.
In another preferred embodiment of the method, the activating agents are selected from the group consisting of Sclareol, Naringin, Nootkatone, Steviol glycoside and cannabidiol (CBD) and combinations thereof.
In one particularly preferred embodiment of the method, the one or more activating agents comprises cannabidiol (CBD). In this embodiment, the activating agents used in the method may further comprise agents or substances each having an IC50 for the inhibition of NO production of less than 1.6 mg/ml and/or an IC50 for the inhibition of TNF-α production of less than 2.4 mg/ml.
Said CBD may be obtained from many different sources, but in one preferred embodiment is supplied in the form of hemp oil.
In a yet further preferred embodiment of the method, the activating agents (including those having the qualitative and quantitative anti-inflammatory properties disclosed above) are derived from plant material (such as crude plant extracts, such as whole plant aqueous extracts, partially purified or fractionated extracts, purified extracts and synthetic analogues of active molecules present in said extracts).
In one preferred embodiment of this aspect of the invention, the plant-derived activating agents are herbal extracts selected from the group consisting of Aster tataricus, Cyperus rotundus and combinations thereof.
While the method of the present invention may be employed to promote nitrogen fixation in almost any vegetable or legume plant of commercial importance, in one preferred embodiment, the plant treated in the present method is a member of a species which is normally unable to obtain its nitrogen requirements by bacterial fixation of atmospheric nitrogen. In one preferred embodiment, the plant species is a member of the Graminaea family. Preferred (but non-limiting) examples of such species include maize, wheat and rice. In one particularly preferred embodiment, the plant species is maize. In another, it is wheat.
In some embodiments, the method of the present invention may further comprise the administration phosphorous-containing fertilizers. In one preferred embodiment, the fertilizer is Calirus.
In some embodiments of the presently-disclosed method, the combination of non-pathogenic, atmospheric nitrogen-fixing bacteria and one or more activating agents are administered by means selected from the group consisting of: application of slow-release granules to the soil in which the plants are being grown, seed coating and spraying the sowing trench or furrow. In some cases, the non-pathogenic, atmospheric nitrogen-fixing bacteria and the one or more activating agents are administered together in a single composition. In other embodiments, however, the non-pathogenic, atmospheric nitrogen-fixing bacteria and the one or more activating agents are administered in separate compositions.
In another aspect, the present invention provides a composition comprising a mixture of non-pathogenic nitrogen-fixing bacteria and one or more activating agents (as defined hereinabove and described hereinbelow).
Many different species and strains of non-pathogenic nitrogen-fixing bacteria may be used in combination with the activating agents described herein (i.e. in a single composition), or alternatively, may be administered in separate compositions. In the latter case, the two or more compositions may be administered either simultaneously or sequentially. The term ‘non-pathogenic’ is used in this context to indicate that the selected species have no, or very few, toxic or other deleterious effects on the host species to which the composition of the invention containing the bacteria are being administered.
In one preferred embodiment of the methods and compositions defined herein, the non-pathogenic bacteria are of the Rhizobia class. Suitable genii and species are disclosed herein.
In another preferred embodiment, the non-pathogenic nitrogen-fixing bacteria are of the Clostridium genus, in particular those species that are disclosed herein.
In some preferred embodiments the composition of the present invention further comprises (in addition to the non-pathogenic nitrogen fixing bacteria and the one or more activating agents) one or more phosphorous-containing fertilizers. Suitable fertilizers for this purpose include (but are not limited to) commercially-available preparations such as Calirus.
In one preferred embodiment, the combination of non-pathogenic bacteria, activating agents and fertilizers (when present) may be administered as a single composition. In other embodiments, some of these components may be administered separately.
Routes of administration of the combinations of the present invention include (but are not limited to) application of slow-release granules to the soil in which the plants are being grown, seed coating and spraying the sowing trench or furrow.
As mentioned hereinabove, it has been found by the present inventors that in some embodiments, the activating agents of the present invention may be characterized by their ability to inhibit one or more key inflammatory mediators such as TNF-α and/or nitric oxide (NO). Consequently, in one preferred embodiment of the present invention, the one or more activating agents used in the aforementioned method are substances capable of inhibiting the production of NO and/or TNF-α.
In one further preferred embodiment of the present invention, the activating agents each have an IC50 for the inhibition of NO production of less than 1.6 mg/ml and/or an IC50 for the inhibition of TNF-α production of less than 2.4 mg/ml.
In another preferred embodiment, each individual activating agents (whether used alone or in combination with other such agents) has an IC50 for the inhibition of NO production of less than 0.4 mg/ml and/or an IC50 for the inhibition of TNF-α production of less than 2.4 mg/ml.
In another preferred embodiment, each individual activating agents (whether used alone or in combination with other such agents) has an IC50 for the inhibition of NO production of equal to or less than 0.15 mg/ml and/or an IC50 for the inhibition of TNF-α production of equal to or less than 2.4 mg/ml.
In another preferred embodiment, each individual activating agents (whether used alone or in combination with other such agents) has an IC50 for the inhibition of NO production of equal to or less than 0.1 mg/ml and/or an IC50 for the inhibition of TNF-α production of equal to or less than 0.2 mg/ml.
In a still further preferred embodiment, each individual activating agents (whether used alone or in combination with other such agents) has an IC50 for the inhibition of NO production of equal to or less than 0.05 mg/ml and/or an IC50 for the inhibition of TNF-α production equal to or less than 0.1 mg/ml.
It is to be noted that the use of the IC50 value (i.e. the concentration of an agent which causes 50% of the maximal inhibition of a mediator, agonist or other biologically active molecule) as a means for comparing the potency of antagonists and other biologically- and pharmacologically-active molecules, is well-known to all skilled-artisans in this field. Briefly, the IC50 values may be obtained by plotting dose-response curves for a parameter such as inhibition of a particular inflammatory mediator, and extracting said values from said curves.
In another preferred embodiment, the activating agents are selected from the group consisting of Sclareol, Naringin, Nootkatone, Steviol glycoside and cannabidiol (CBD) and combinations thereof.
In one particularly preferred embodiment, the one or more activating agents comprises cannabidiol (CBD). In this embodiment, the activating agents used in the method may further comprise agents or substances each having an IC50 for the inhibition of NO production of less than 1.6 mg/ml and/or an IC50 for the inhibition of TNF-α production of less than 2.4 mg/ml.
Said CBD may be obtained from many different sources, but in one preferred embodiment is supplied in the form of hemp oil.
In a yet further preferred embodiment, the activating agents (including those having the qualitative and quantitative anti-inflammatory properties disclosed above) are derived from plant material (such as crude plant extracts, such as whole plant aqueous extracts, partially purified or fractionated extracts, purified extracts and synthetic analogues of active molecules present in said extracts).
In one preferred embodiment of this aspect of the invention, the plant-derived activating agents are herbal extracts selected from the group consisting of Aster tataricus, Cyperus rotundus and combinations thereof. Further suitable plant extracts are disclosed elsewhere herein.
In another aspect, the present invention also provides a method for increasing the yield of a plant of agricultural or horticultural importance by means of:
The present invention further provides a method for increasing the yield of a plant of agricultural or horticultural importance by means of:
a) providing separately:
b) separately administering each of compositions (i) and (ii) to said host species.
In the above-disclosed methods, the nitrogen fixing non-pathogenic bacteria are, in one embodiment, members of the Rhizobium genus. In one preferred embodiment, the bacteria are of the species Rhizobium leguminosarum.
In the above-disclosed methods the plant of agricultural or horticultural importance is, in one embodiment, a member of a species which is normally unable to obtain its nitrogen requirements by bacterial fixation of atmospheric nitrogen. In one embodiment, said plant species is a member of the Graminaea family. In one preferred embodiment, the plant species is maize. In another preferred embodiment, the plant species is wheat. In a still further preferred embodiment, the plant species is rice.
The advantages and benefits of the present invention will now be described in more detail in the following working Examples and accompanying drawings.
In this study, the following activating agents were mixed together and used in combination with the nitrogen fixing bacteria:
Sclareol, nootkatone, cannabidiol (CBD), naringin, steviol.
Since naringin and steviol are water soluble, while the other three activating agents are lipid soluble, two separate solutions—an oil phase and an aqueous phase—were prepared, as summarized in the following table, and then mixed using a high-shear mixer. As will be seen from this table, the oil phase contained (in addition to three of the activating agents) medium chain triglycerides (MCT) and a hydrolyzed sunflower lecithin (Giralec HE-60; E-322), while the aqueous phase also comprises water, glycerol and the non-ionic surfactant, sucrose palmitate (Sisterna PS750):
The drop size of the emulsion following mixing in the high-shear mixer was 214 nm.
In some of the treatments (as explained hereinbelow), some or all of the active substances were added in the form of granules to the furrow in which the plants had been seeded. The granules were prepared by soaking 1 kg of Perlite granules (having a mean diameter greater than 2 mm) in the following solution:
Generally, the nitrogen content of the growing plant in the field study was measured using a spectrophotometric method based on the standard method “4500-NO3_I. Cadmium Reduction Flow Injection Method” published by the Standard Methods Organization (<https://www.standardmethods.org/doi/full/10.2105/SMWW.2882.089). This method is based on the conversion of nitrates in an aqueous plant material extract to nitrites by passing the extract through a copperized cadmium column. Subsequent processing steps convert the nitrites to a magenta-colored dye having an absorbance peak at 540 nm.
The trial was sown on the format date 29 of Aug. 2017 using the pioneer maize silage variety number 32-W-68. The field was washed from possible nitrogen using sprinklers irrigation. The combination treatments administered to the plants which are of relevance for the present study were:
The amount of the solution containing the Rhibozium 1%, the activating agent emulsion and the fertilizer (Calirus) (treatments F and H) was calculated such that 2 liters per 1000 m row were added to the sowing trench.
In the case of treatment H, the granule quantity was adjusted to 4 Kg granules per 1000 m2.
The following parameters were monitored at either one timepoint (Oct. 29, 2017) or two timepoints (October 23 and Oct. 29, 2017) during growth of the maize plants:
As may be seen from the table and from the graph shown in
The height of the growing maize plants was measured at two timepoints: Oct. 23, 2017 and Oct. 29, 2017. As may be seen from the above table and from
As may be seen from these tabulated results obtained on Oct. 23, 2017 and Oct. 29, 2017, and from
As shown in the upper portion of the above table and the accompanying
As shown in this table, and summarized graphically in
Using a nominal scale of 1-10, the green color of the foliage in the maize plants was assessed on both Oct. 23, 2017 (upper part of table and
As in the case of the field study reported in Example 1, above, this trial was sown on the 29 of Aug. 2017 using the pioneer maize silage variety number 32-W-68. The field was washed from possible nitrogen using sprinklers irrigation. The combination treatments administered to the plants which are of relevance for the present study were:
The amount of the solution containing either the Rhibozium 1% (treatment 1) or Rhizobium 1%, and activating agent (treatment 2) was calculated such that 2 liters per 1000 m row were added to the sowing trench.
In both treatment regimes, the granule quantity was adjusted to 4 Kg granules per 1000m2.
The following parameters were monitored at either one timepoint (Oct. 29, 2017) or two timepoints (October 23 and Oct. 29, 2017) during growth of the maize plants:
As may be seen from the table and from the graph shown in
As may be seen from the above table and from
As may be seen from the above table and from
As shown in the upper portion of the above table and the accompanying
As shown in this table, and summarized graphically in
Using a nominal scale of 1-10, the green color of the foliage in the maize plants was assessed on both Oct. 23, 2017 (upper part of table and
This difference in green coloration and general vitality of the plants between the two treatment groups and the untreated control is also clear in the comparative photograph shown in
This trial was sown during the Israeli growing season of 2018 using the pioneer maize silage variety number W86. The field was washed from possible nitrogen using sprinklers irrigation. The combination treatments administered to the plants which are of relevance for the present study were:
The following parameters were monitored at one timepoint during growth of the maize plants, 3 months after they were sown:
The statistical significance of the difference between the various treatment groups was determined using the Tukey-Kramer HSD test.
The mean caliber for each of the 5 treatments (A-E) listed above was recorded, and the results obtained are shown below and in
These results indicate that each of the three treatments that contained the composition of the present invention (C-E) permitted the growing maize plants to achieve approximately the same plant thickness as that seen with the full nitrogen positive control (A). Each of the treatment regimes produced a mean plant thickness significantly greater than seen with the negative control plants (B).
The mean leaf width for each of the 5 treatments (A-E) listed above was recorded, and the results obtained are shown below and in
These results indicate that each of the three treatments that contained the composition of the present invention (C-E) permitted the growing maize plants to achieve approximately the same mean leaf width as that seen with the full nitrogen positive control (A). Each of the treatment regimes produced a mean leaf width significantly greater than seen with the negative control plants (B).
The mean cob weight from the main stems of 10 plants for each of the 5 treatments (A-E) listed above was recorded, and the results obtained are shown below and in
These results indicate that each of the three treatments that contained the composition of the present invention (C-E) resulted in approximatley the same mean cob weight as that seen with the full nitrogen positive control (A). Each of the treatment regimes produced a mean cob weight significantly greater than seen with the negative control plants (B).
The mean total plant weight of 10 plants for each of the 5 treatments (A-E) listed above was recorded, and the results obtained are shown below and in
These results indicate that each of the three treatments that contained the composition of the present invention (C-E) resulted in approximately the same mean total plant weight as that seen with the full nitrogen positive control (A). Each of the treatment regimes produced a mean total plant weight that was significantly greater than seen with the negative control plants (B).
The mean plant height of 10 plants seen with each of the 5 treatments (A-E) listed above was recorded, and the results obtained are shown below and in
These results indicate that each of the three treatments that contained the composition of the present invention (C-E) resulted in approximately the same mean total plant weight as that seen with the full nitrogen positive control (A). Although each of the treatment regimes produced a mean plant height that was slightly greater than seen with the negative control plants (B), this difference did not reach statistical significance.
This trial was sown during the Israeli growing season of 2018 using the pioneer maize silage variety number W86. The field was washed from possible nitrogen using sprinkler irrigation. The combination treatments administered to the plants which are of relevance for the present study were:
The average total leaf nitrogen content was measured at one timepoint during growth of the maize plants, 3 months after sowing.
The average total nitrogen content of the leaves was measured, and the results shown in the following table and in
These results indicate that each of the various treatments with the composition of the present invention resulted in nitrogen levels within the maize plants that were comparable with those obtained with the positive control (PC). Each of these treatments resulted in significantly higher leaf nitrogen levels than those seen in the untreated control group (NC). It may thus be concluded that treatment with the composition of the present invention allows Rhizobium bacteria to cause nitrogen fixation in growing maize plants.
Two different agricultural sites in Israel were selected for field trials in which the effects of compositions of the present invention on wheat crops were investigated. The various compositions were administered to the growing wheat (Galil variety) as described in Examples 3 and 4, hereinabove. The treatments used in this study are as follows:
B. Negative control (no nitrogen source)
A. Granules prepared according to Example 3, applied at a density of 4 kg granules per 1000 m2.
C. Granules prepared according to Example 3, applied at a density of 2 kg granules per 1000 m2.
F. Positive control—full commercial nitrogen. The plants were treated with 30 units of nitrogen per 1000 m2 by means of applying to this area 60kg urea containing 46% urea.
In the absence of treatment with granules containing a composition of the present invention, the roots of the growing wheat plants did not show any evidence of root nodule formation. This is seen in
Nodule development in these samples indicates the possible site of a symbiotic relationship between the administered Rhizobium bacteria and the plant root system, which has developed as part of the nitrogen fixation process induced by the treatment with the composition of the present invention.
The following parameters were measured in the wheat, in order to assess the effect of the treatment compositions on plant growth:
a) Number of side shoots;
b) Flag leaf width;
C) Main shoot diameter.
The results of these measurements are presented in the table, below:
These data are also presented graphically in
It may be seen from these results that all of the measured growth parameters are increased following treatment with either treatment A or treatment C, in relation to the negative control. In addition, said treatments also provide growth results either comparable with, or greater than, those obtained with the positive control.
The results for flag leaf nitrogen fixation are presented in the following table:
These results are also summarized graphically in
It may be seen from these results that both treatments A and C (compositions of the present invention) and the positive control caused an increase in flag leaf nitrogen content, as compared with the negative control. Both of these treatment regimens resulted in increases in nitrogen content similar to those caused by the positive control.
The following table summarizes the results for the effect of the treatments and controls on average wheat grain yield from each of 6 2 m plots:
These data are also presented in the form of a graph in
It may be seen from these results that both of the treatments containing a composition of the present invention and the positive control caused a significant increase in wheat grain yield in this field study (i.e. compared with negative control). The increase due to the two treatment regimens was numerically similar to that caused seen in the positive control group.
In all of the field trials reported hereinabove (Examples 1-5), the treatment regimens comprising combinations of both Rhizobium and an emulsion of the mixture of activating agents resulted in increased fixation of atmospheric nitrogen, as witnessed by the direct measurement of foliar nitrogen levels, the development of root nodules and the various growth-related parameters measured in these trials. This positive effect was seen regardless of the way in which the treatment combinations were administered.
Cucumber (Cucumis sativus L) seedlings are highly susceptible to fungal and bacterial pathogens attacking the seedling during the germination process and were therefore selected as a model plant to screen and calibrate the Rhizobium species and the phytochemicals that can cause activation thereof.
The potential phytochemicals were added to a mixture of 30 cc glucose 50% V/V substrate, 10 cc cocktail of fungal pathogens and 10 cc cocktail of bacterial pathogens in a Petri dish. The fungal cocktail contained: Botrytis cinerea, Rhizoctonia solani, Pythium spp. and non-pathogenic fungi used for the fermentation of tomatoes. The bacterial cocktail contained: Clavibacter michiganensis, Xanthomonas campestris, Pseudomonas syringae and non-pathogenic bacteria used for the fermentation of tomatoes.
Approximately 1000 potential phytochemicals were screened for their ability to activate, Rhizobium species by means of calculating a colony forming index for each test (0=no colony; 5=maximal colony size). The five phytochemicals listed above in the introduction to the Examples section were selected from the approximately 1000 phytochemicals tested on the basis of their superior performance as activating agents for Rhizobium species.
The optimal combination and concentrations of the five selected activating agents listed above were determined for each of the host organisms used in the studies reported below. The selected combinations were those found in preliminary studies to have the lowest possible concentrate that was capable of producing the desired protective effect. In this way, possible side effects and environmental pollution during the administration of these agents to the host organisms were avoided.
At the same time the phytochemicals were screened for their ability to eliminate a cocktail of bacterial and fungal pathogens. For the purposes of comparison between the various treatments, fungal and bacterial elimination indices were calculated (0=maximal elimination, 5=no elimination).
The test mixtures, containing the glucose substrate and fungal and bacterial cocktails mentioned above together with all five of the activating phytochemicals and a penetrator (MCT) and two type of surface active agent sugar ester and iso lecithin were used at four different concentrations: concentrations 1, 2, 3 and 4. In each case, the same amount of glucose substrate and fungal and bacterial cocktails—30 ml—was added to the mixture. Similarly, the concentration of the MCT and surface active agent were correlated to the concentration of the actives if Sclareol content at concentration 2 doubled then MCT and surface active agent concentrations were also doubled, and so on. However, the concentrations of the Rhizobium species and each of the five activating agents (given in %) were 3%. at concentration 1, 3 and 5% at concentration 2,4 as described in the Table I:
Rhizobium
5%
Stevia
Various different test mixtures containing different combinations of some or all of the five activating agents were used in this study, in accordance with the list of treatments given in Table II, below. In each case, the activating agents, Rhizobium species and substrate were used at the concentrations indicated in Table I. For example, when tested at Concentration 1, the concentration of sclareol in test mixtures containing that activating agent was 0.04%, while when tested at Concentration 2, sclareol was present at a concentration of 0.08%, and so on.
Rhizobium
Stevia
Preliminary results indicated that the optimal anti-fungal and anti-bacterial activity was obtained using test mixtures with concentration 2 and concentration 3 (see table above). Since Rhizobium species colony development was optimal using concentration 3, this was the concentration that was selected for use in the remainder of the study. The results obtained for fungal elimination, bacterial elimination and Rhizobium species activation (colony size) for the concentration 3 tests are summarized graphically in
As explained above, the three semi-quantitative indices used to assess the anti-fungal, anti-bacterial and activation properties are as follows:
Fungal index: 0 (no development) to 5 (maximum development)
Bacterial index: 0 (no development) to 5 (maximum development)
Rhizobium index (colony forming index): 0 (no development) to 5 (maximum development)
It may be seen from
The identification numbers of the actives:
Rhizobium complex
stevia
A second group of studies was aimed at investigating the effect of either eliminating one phytochemical from the full 5-component combination or of selectively altering the concentration of one or two components in the mixture.
As for Example 1.
The various test mixtures were used at either concentration 3 or concentration 4 (as defined in Example 1, above). The composition of each of these test mixtures is summarized in the following two tables:
Rhizobium
Stevia
Rhizobium
Stevia
As may be seen in
Similarly, as shown in
It may also be observed in
Furthermore,
These data indicate that mixtures containing less than the maximum five activating agents may be used to protect host organisms from fungal or bacterial attack. In addition, these results also indicate that optimization of the mixtures may be obtained by manipulating the concentration of one or more individual activating agents in the mixture.
In this study, the experiments performed in Example 7, above, were repeated using a different Rhizobium species preparation, namely a Rhizobium composition produced and sold by Bio-Lab Ltd., Jerusalem, Israel labeled as “Culture for growing groundnuts”.
As for Example 6.
The various test mixtures were used at either concentration 3 or concentration 4 (as defined in Example 6, above). The composition of each of these test mixtures is as summarized in Tables III and IV in Example 7, hereinabove.
This study confirms the results obtained in Example 7. Thus, as seen in
Of particular note is the fact that at concentration 3, the five-component activating agent mixture in which the Nootkatone and Stevia components are both at an elevated concentration (i.e. concentration 4, while all other components are at concentration 3; i.e. test mixture 8) has the greatest activity on all three indices (
These results, obtained with the Rhizobium species formulation confirm the findings obtained with the formulation (Example 7, hereinabove), indicating that the effects observed are not specific to any one particular Rhizobium preparation.
Following the results obtained with combinations of Rhizobium species and some or all of the five activating agents reported in Examples 6-8, hereinabove, said agents were investigated in order to look for common functional properties, in addition to their bactericidal, fungicidal and Rhizobium species—activating abilities.
Following a series of preliminary investigations, the present inventors unexpectedly found that each of the five activating agents tested in the studies presented hereinabove, also share a highly potent anti-inflammatory activity.
In order to investigate this further, three of the activating agents used in the previous Examples—both separately, in combination with each other and in combination with Rhizobium species—, were tested for their ability to inhibit the in vitro production in a cultured macrophage line of two key inflammatory inhibitors: nitric oxide (NO) and TNF-α. In addition, the viability of the macrophages was measured at appropriate IC50 values corresponding to the inhibition of NO and TNF-α, at the time that the anti-inflammatory assays were performed.
RAW 264.7 macrophages were grown in flat-bottomed flasks using a standard growth medium (DMEM supplemented with 5% FBS, antibiotics and glutamine. The cells were maintained in accordance with standard procedures well known in the art. After the cells reached confluence, they were removed from the flasks using mechanical means and then concentrated by centrifuging and resuspended in a small volume of fresh culture medium. The cell concentration was adjusted with growth medium in order that about 75,000 cells could be added to each well of a 96-well plate. A combination of 25 μg/mL LPS and 10 U/ml IFN-γ DMEM, was used for activation of the macrophages. The various test agents were added to the wells one hour prior to activation. The cells were then incubated for a further 24 hours, prior to assaying the inflammatory mediator production and cell viability.
The Alamar Blue assay of viability was performed by adding 100 μl of a 10% Alamar Blue solution to each well and incubating at 37° C. for 1-2 hr. Fluorescence was measured (excitation at 545 nm and emission at 595 nm) and expressed as a percentage of the values in untreated control cells.
The production of NO by the macrophages subjected to the various treatments was assayed using the Griess reagent (equal volumes of 1% sulphanilamide and 0.1% napthyethylene-diamine in 5% HCl). 70 μl of supernatant from each test well was transferred to a fresh 96-well plate and mixed with 70 μL of Griess reagent and the violet color produced was measured at 540 nm.
A sandwich ELISA was used to determine TNF-α concentration. The primary antibody was used at a concentration of 0.5 μg/mL in PBS. Serial dilutions of TNF-α standard from 0 to 1000 pg/mL in diluent (0.05% Tween-20, 0.1% BSA in PBS) were used as internal standard. TNF-α was detected with a biotinylated second antibody and an avidin peroxidase conjugate with TMB as detection reagent. The color development was monitored at 655 nm, taking readings after every 5 minutes. After 25 minutes, the reaction was stopped using 0.5 M sulphuric acid and the absorbance was measured at 450 nm.
The methods described above were used to determine the effects of Sclareol, Naringin and Steviol, and their combinations with each other and with Rhizobium species—, on NO and TNF-α production, and on cell viability. The results for the anti-inflammatory activities are presented as IC50 values for the inhibition o NO and TNF-α production in Table V, below, together with the cell viability results. In addition, comparable results obtained from the scientific literature (A. S. Ravipati et al. (2012) BMC Complementary and Alternative Medicine, 12:173 “Antioxidant and anti-inflammatory activities of selected Chinese medicinal plants and their relation with antioxidant content”) for two additional plant species—aqueous extracts of Aster tataricus and Cyperus rotundus—are presented at the end of the table. Extracts of these two species were investigated by the present inventors with regard to their fungicidal and bactericidal effects in combination with B. subtilis. The results of these investigations are presented in Example 5, hereinbelow.
The results obtained for the anti-inflammatory and viability assays of the cultured macrophages treated with the various agents are presented in Table V, below.
Rhizobium species
Aster tataricus
Cyperus rotundus
It may be seen that none of the treatment agents tested had any significant adverse effect on the viability of the macrophages. Consequently, any inhibition of the production of the two inflammatory mediators caused by these agents was not a result of a general cytotoxic effect.
It is to be noted from the table that when taken separately, the IC50 for NO inhibition of the three agents Sclareol, Naringin and Steviol are 0.04, 0.04 and 0.02, respectively. Furthermore, when used in combination with each other, said combination is even more potent, with an IC50 for NO inhibition of 0.004 in the absence of Rhizobium species—, and 0.001 in the presence of Rhizobium species—. If these results are compared with the comparable IC50 values for NO inhibition published for 44 selected plant extracts in the aforementioned paper by A. S. Ravipati et al. (2012), it will be seen that the values for Sclareol, Naringin and Steviol are at the lower extremity of the range of values in said paper (0.03-1.49), and in one case (Steviol) even beyond the lowest extent of that range. Similarly, if the mean value for Sclareol, Naringin and Steviol is compared with that for the 44 plants reported in the paper, it may be noted that the former (0.03) is much lower than the mean extracted from said published values (0.26).
A similar conclusion may also be drawn with regard to the inhibition of TNF-α by Sclareol, Naringin and Steviol when tested separately, with IC50 values of 0.08, 0.09 and 0.08, respectively (range=0.08-0.09; mean=0.083), compared with the published results for the 44 plant extracts in A. S. Ravipati et al. (2012) (range=0.07-2.5; mean—1.04).
It may thus be concluded that the three agents selected and tested in Examples 1-3, hereinabove, all have anti-inflammatory activity, and are more potent (i.e. have a lower IC50) than most of a set of 44 herbal extracts, commonly used in Chinese medicine (A. S. Ravipati et al. (2012)), with respect to NO and TNF-α inhibition.
Furthermore, it is of interest to note from Table V that even in the case of less potent anti-inflammatory plant extracts (Aster tataricus and Cyperus rotundus), said extracts are also effective as activating agents for Rhizobium species with regard to anti-fungal and anti-bacterial activity (as will be shown in Example 5, hereinbelow).
Tomato seedlings were inoculated with 10 cc of each test mixtures containing Rhizobium species and various combinations of activating phytochemicals. (including the bacterial and fungal cocktails described below) 10 hours after sowing.
The health of each plant was assessed 5 days following treatment, using a semi-quantitative inoculation index (0=healthy, 5=dead).
The composition and concentration of the various test mixtures, as well as the number of different activating agents used in combination are summarized in the following two tables (all concentrations are given as % v/v):
Rhizobium complex
stevia
Aster tataricus
Cyperus rotundus
Platycodon grandiflorus
Pleione bulbocadioides
The results of this inoculation study are summarized graphically in
These results indicate that the common ingredient found in the most active treatment mixtures is Cannabidiol (CBD; hemp oil), which was highly active even when present as the sole activating agent.
10 cc of each mixture of activating agents, Rhizobium species and additional components (as described in Tables I and II of Example 6, hereinabove) was sampled from the relevant petri dish and injected into 4 replicates of germinating cucumber seeds 10 hour after sowing.
The health of each plant was assessed 5 days following treatment, using a semi-quantitative inoculation index (0=healthy, 5=dead).
The results of this study are shown graphically in
As may be seen from the first (upper) graph in
The second graph in
At the highest concentration (concentration 4; last graph in
In summary: all of the multiple-component activating agent mixtures, as well as some of the mixtures containing only one activating agent, were effective at protecting cucumber plants in vivo, when used at concentrations 2 to 4. The semi-quantitative data obtained in this study correlate very well with the appearance of the plants that were subjected to the various treatments.
In this study, the effect of various combinations of Rhizobium species with activating agents on the survival of the pathogenic bacteria Clavibacter michiganensis sp. Michiganensis (Cmm) was investigated in vitro.
Various combinations of a 3% Rhizobium sp. preparation together with an emulsion containing 5 activating agents (E-91) or one of the components of said emulsion (naringin) and a culture of the plant pathogen Cmm (105-106 CFU/ml final concentration) were incubated in test tubes for up to 3 days (4 replicates per combination). At the end of the 3 day incubation period the contents of the test tubes containing all these components were plated on to growth medium and the numbers of colonies of Cmm and Rhizobium species for each test condition (CFU/ml) were measured.
The emulsion containing the 5 activating agents (Sclaerol, Naringin, Nootkatone, Stevia and CBD; referred to in the results table hereinbelow as 5% plant emulsion E-91) was prepared as described in Example 6, hereinabove.
The results obtained (CFU/ml) are presented in the following table:
Rhizobium sp.
5 × 105
Rhizobium sp.
2 × 102
3 × 102
9 × 101
5 × 104
1 × 105
3 × 105
2 × 105
3 × 105
It may be seen from these results that the only test mixture which was capable of reducing the Cmm count was the combination of 5% plant emulsion E-91 and 3% Rhizobium sp. This treatment caused a massive reduction in the Cmm count, from a control value of 5.25×105 to a final count of 172.5.
The combination of Naringin (as the sole activating agent) and 3% Rhizobium had no effect on the Cmm count (3.63×105). It may therefore be concluded that a combination of Rhizobium and naringin alone (i.e. in the absence of any other activating or anti-inflammatory agents) is unable to kill the Cmm pathogens.
This study was conducted in essentially the same manner as the study presented in Example 12. In the present study, however, the effect of the 5-component activating agent emulsion (5% plant emulsion E91) is compared with the following combinations of activating agents:
The results of these comparisons are set out in the following table:
Rhizobium sp.
7 × 105
8 × 106
It may be seen from these results that the combinations of 2, 3 or 4 activating agents together with Rhizobium (in each case, in the absence of CBD) had, in some cases, a minor inhibitory effect on the Cmm count. However, all of said partial combinations were far less effective than the complete 5-component activating agent emulsion when used in combination with Rhizobium.
In this study, the effect of a combination of the 5-component activating agent mixture E91 with 3% Rhizobium on the survival of two other plant pathogens—fungal species of the genus Alternaria and the gram negative bacteria Xanthomonas euvesicatoria was investigated. All materials and methods are as described hereinabove in Examples 12 and 13, except for the co-incubation time, which in this study was 2 days.
The results of this study are shown in the following table:
Rhizobium/XV
Rhizobium sp.
9 × 106
6 × 107
1 × 103
8 × 102
It may be seen from these results that the combination of the activating agents with Rhizobium caused a moderate reduction in the Xanthomonas euvesicatoria count after 2 days, as compared with the samples treated with the activating agents alone.
In the case of the Alternaria species, the reduction in the microbial count caused by the combination of the activating agents and Rhizobium as compared with the activating agents alone was much more significant.
It may be concluded that the compositions of the present invention have antimicrobial activity on a range of different bacterial and fungal species, including those species which are important plant pathogens.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2019/050082 | 1/21/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62619848 | Jan 2018 | US |
