CA2470669A1 - Isoflavonoid compounds and use thereof - Google Patents

Abstract

The use of a one or more Isoflavonoid compound Signals which may be with an agriculturally acceptable carrier, applied prior to planting, up to 365 days or more, either directly to the seed or transplant of a non-legume crop or a legume crop, or applied to the soil that will be planted either to a non-legume crop or a legume crop, for the purpose of increasing yield and/or improving seed germination and/or improving earlier seed emergence and/or improving nodulation and/or increasing crop stand density and/or improving plant vigour and/or improving plant growth, and/or increasing biomass, and/or earlier fruiting, all including in circumstances of seedling and plant transplanting.

Description

r Isoflavonoid Compounds and Use Thereof THE INVENTION
The invention relates to the use of a one or more (soflavonoid compound Signals which may be with an agriculturally acceptable carrier, applied prior to planting, up to 270 days or more, either directly to the seed of a non-legume crop or a legume crop, or applied to the soil that will be planted either to a non-legume crop or a legume crop, for the purpose of increasing yield and/or improving seed germination and/or improving earlier seed emergence and/or improving nodulation andlor increasing .
crop stand density andlor improving plant vigour andlor improving plant growth, all including in circumstances of seedling and plant transplanting.
Background Agricultural Practices Agriculture in the developing world frequently utilizes a practice of intercropping plant species to maximize land productivity. That practice frequently involves a legume crop interspaced sow by row with another plant species of regional value. It has long been known that the non-leguminous crop generally benefits in yield for having been in intimate .contact at the root level with the legumes. This has traditionally been thought to be due to the legumes known benefit of returning fixed plant-utilizable nitrogen to the soil through the residual of its own nitrogen-fixing symbiosis with the rhizobia bacteria. This nitrogen, it was viewed, was utilizE:d by the intercrop growing better.
In the developed world intercropping legumes with othc;r agricultural crops, while known and understood, is simply not feasible. It is a practice requiring hand field maintenance for best results and in volume agriculture the equipment does not lend itself to the disparity in plant heights and size experienced i.e. soybeans (a legume) is physically much different from maize.

North American Cropping practices Intercropping practices from the developing world are, in developed countries, translated into crop rotation agricultural practices where a single crop is grown on the land one year and another crop is grown in the following year. These crops are so rotated as to best maintain the land and reduce its nutrient loss and may involve two, three or four crops in regular rotation year-by-year.
One of these crops will be a legume, the type depending on soil, markets, region etc and might involve soybeans, peas, beans, alfalfa, clover etc. - all legumes with their own symbiotic relationship with a particular rhizobia bacterial species - and each bacterial species producing a specific LCO structure for the particular plant species host.
It has long been held that the crop rotation must include a legume because of their ability to . leave a nitrogen residue available to the following crop - that residue generally recognized to: be a pound of Nitrogen for each bushel of legume seed harvested i.e. for soybeans 40-50 ibs N per acre available to the next, generally non-leguminous crop.
In North America the major crop rotations are (1) Corn-soybeans and used through the major production states of the USA - Illinois, Ohio, Iowa; Nebraska - and (2) Wheat-Peas, in western Canada.
Scientific Development The legume symbiosis with rhizobia is now much better understood while not yet fully explained. It involves and requires a series of plant and microbial signals to iriitiate the plant tissue changes, which will protect and support the rhizobia internally to the root where it can undertake nitrogen gas conversion to plant utilizabie nitrogen utilizing energy from the plant.
It is now known scientifically that the rhizobial signal sent to the legume plant to initiate root tissue changes is a Lipo-chito-oligosaccharide (LCO) and is termed Nod Factor in this application. Its production arose from the adjacent rhizobia bacteria receiving legume root exuded isaflavonoid chemicals - aiso termed signals -that switch on the genes for the production of these LCOs.
It is further appreciated here through present studies and patents that LCOs have a plant growth function not restricted to legumes. It has been demonstrated that non-legume seeds as well as legume seeds germinate earlier in the presence of minute (10-'to 10-'2 M) levels of LCO in solution.
It has been further demonstrated that foliar LCO applications to many plants (corn, soybeans, peas, tomatoes) leads to their earlier flowering and higher yield (not yet published). The mechanism for these phenomena continues to be under study.
The objects of the present invention include application and use of one or more Signals and compositions thereof to a non-legume including a seed, resulting in increased yield and/or improved seed germination, and/or increased stand density, andlor earlier emergence, and/or improved- plant vigour, and/or improved plant growth; including but not Limited to:
where the non-legume includes but is not limited to a seed, tuber, transplant, or vegetative cutting;
where the non-legume is grown for use in agriculture" horticulture, silviculture, or gardening;
where the non-legume is sown into land that had previously been sown to a legume crop, or which has an indigenous population of rhizobia;
where a Signal is applied to the non-legume crop up to 270 days in advance of planting;
where a Signal is applied with an agriculturally acceptable carrier such as, but not limited to, water, seed treatments, inoculants, herbicides, fungicides, insecticides, fertilizers, growth promoters, or horticultural media;

where the soil to be planted with the non-legume crop has been pre-treated with a specific symbiotic rhizobia or has an indigenous population of rhizobia;
where the seed has been treated with a specific symbiotic rhizobium or rhizobia;
One or more Signal and compositions thereof applied to the soil which will be planted with a non-legume crop, resulting in increased yield and/or improved seed germination, andlor increased stand density, and/or earlier emergence, andlor improved plant vigour, and/or improved plant growth; including but not limited to:
where the non-legume is grown for use in agriculture, horticulture, silviculture, or gardening;
where the non-legume is sown into land that had previously been sown to a legume crop or has an indigenous population of rhizobia;
where the non-legume is sown -into land that had not previously been sown to a legume crop;
where a Signal is applied with an agriculturally acceptable carrier such as, but not limited to, water, seed treatments, inoculants, herbicides, fungicides, insecticides, fertilizers, growth promoters, or horticultural media;
where the soil to be planted with the non-legume crop has been pre-treated with a specific symbiotic rhizobium or rhizobia or has an indigenous population of rhizobia;
where the seed has been treated with a specific symbiotic rhizobium or rhizobia;
One or more Signal and compositions thereof applied to~ a legume including a seed, resulting in increased yield and/or improved seed germination, andlor increased stand density, andlor earlier emergence, and/or improved plant vigour, andlor improved plant growth; including but not limited to:
where the legume includes but is not limited to a seed, tuber, transplant, or vegetative cutting;

where the legume is grown for use in agriculture, horticulture, silviculture, or gardening;
where the legume is sown into land that had previously been sown to a legume crop or has an indigenous population of rhizobia;
where the legume is sown into land that had not previously been sown to a legume crop;
where the Signal is applied to the legume crop up to 270 days in advance of planting;
where the Signal is applied with an agriculturally acceptable carrier such as, but not limited to, water, seed treatments, inoculants, herbicides, fungicides, insecticides;
fertilizers, growth promoters, or horticultural media;
where the soil to be planted with the legume crop has been pre-treated with a specific symbiotic rhizobium or rhizobia or has an indigenous population of rhizobia;
where the seed has been treated with a specific symbiotic rhizobia;
One or more Signal and compositions thereof applied to the soil, which will be planted with a legume crop, resulting in increased yield and/or improved seed germination, andlor increased stand density, and/or earlier emergence, and/or improved plant vigour, and/or improved plant growth, including but not limited to:
where the legume is grown for use in agriculture, horticulture, silviculture, or gardening;
where the legume is sown into land that had previously been sown to a legume crop or has an indigenous population of rhizobia;
where the legume is sown inta land that had not previously been sown to a legume crop;

where the Signal is applied with an agriculturally acceptable carrier such as, but not limited to, water, seed treatments, inoculants~, herbicides, fungicides, insecticides, fertilizers, growth promoter, or horticultural media;
where the soil to be planted with the legume crop has been pre-treated with a specific symbiotic rhizobium or rhizobia or has an indigenous population of rhizobia;
where the seed has been treated with a specific symbiotic rhizobium or rhizobia;
Seedling growth-stimulant A Seed application of soybean isoflavonoid signals In intercropping, the response of the non-legume crop rnay be explained as in fact due to the LCOs produced by the legume (bean) plants migrating to the roots of the non-legume and initiating a growth response in that crop. This is a reasonable assumption with present knowledge.
In view of knowledge of agriculture, of soybeans and soybean crop rotations with corn and of Peas in rotation with wheat, of legume isoflavonoid signals which induce iricreased nodufation through increased LCO production by the rhizobial cells, of manufacture and application of soybean and pea rhizobial seed-inoculants and the persistence of the rhizobia in soil, the present inventors have considered the application of soybean and other isoflavonoid signal in agriculturally effective and useful amounts to corn seed and other legume and non-legume species, including crop and horticultural. varieties, including for transplanting, to cause the inducement of LCO production by the indigenous rhizobial population in the soil and that this LCO might lead to increased growth at the seedling stage when the young plants are being established and the other desirable effects all as aforesaid.
This concept was then broadened to encompass horticultural crops where the seeding and potting mix could be seeded with a level of Bradyrhizobium (for Soybearis) or of other Rhizobium species provided they were capable of inducement by their appropriate isoflavonoid signals to produce meaningful levels of L_CO
at the seedling roots where it could act as a growth stimulant to non-leguminous bedding plants and horticultural crops.
This was tested.
(1 ) Corn growth stimulation.
Potting soil was seeded (inoculated) with sufficient Bradyrhizobium japonicum from a commercial soybean inoculant to attain 100,000 active cells per gram of soil, a level mid point to recognized rotational corn bean land where the soil population will be between 10,000 and 1,000,000 active bacterial cells per gram of soil.
Corn seed coated with various levels of the isoflavonoid inducer, genistein, were planted in this soil in pots in the greenhouse in such a way that germination could be determined as well as height differences from the untreated control over the first month of growth. The levels tested were 0, 50, 100, 200, 300 and 400 uM
genistein solution applied at the rate of 0.3 ml (300 u1) per 100 corn seeds, a normal application rate in agriculture.
Further batches of such treated seed were stored in a dry cool 22°C
room for a period of up to 6 months and representative samples withdrawn monthly for retesting for germination and growth, thus determining the capacity of the concept for pre-treati~nent of seed from the previous harvest at harvest time. These studies continue and are being improved in methodology as they progress.
(2) Bedding Plant growth stimulation Potting mix was seeded with Bradyrhizobium japonicum at 500,000 and 5,000,000 active cells per ml of mix. Seeds of 8 different bedding plant varieties were sown in the seeded mix and genistein isoflavonoid inducer was applied to the rhizobia in a number of ways from coating the seeds to watering with the signal inducer.
The seeds were then assessed for germination either- as increased amount or increased rate. The young seedlings were then assessed for growth as measured by height for a number of weeks while in the seedling trays.

Transplanted Growth Cherry tomato seedlings (5-week old) were transplanted into 5" pots seeded with rhizobia at 1 e6 cellslml (2 and 20 uM Seed Coater) or without rhizobia (control and LCO treatments). Seed Coater and LCO solutions were prepared with water and 50mllplant applied to plant after transplantation. Ripened fruit (orange or red) were collected 8 weeks after transplantation.
Summary: (1 ) Seed Coater soil applied to transplanted cherry tomato can enhance early fruit number. ~ (2) Seed Coater signals more effective than LCO signal when applied to soil around transplanted roots.

Data List Table/Fig Crop/parameter Lbcation/yearPlanted time ember Table 1 Soybean/nodule Greenhouse Immediately Table 2 Soybean/nodule Greenhouse One month 2002 later Table 3 Corn/dry weight Greenhouse Immediately Table 4 Cornldry weight Greenhouse One month 2002 later Fig.1 Soybeanlgermination Greenhouse Immediately Fig. 2 Soybean/germination Greenhouse One month 2002 later Fig:3 Soybeanlheight Greenhouse Immediately Fig.4 Corn/germination Greenhouse Immediately Fig.S Cornlheight Greenhouse Immediately Fig. fi Corn/height Greenhouse One month 2002 later Fi . 7 Tomato Trans lant Fruit Number Table 5 Corn/yield MAC Immediately fieldl2003 Table 6 Soybean/germinationlyield MAC 5 weeks fieid/2003 Table 7 Soylnodufe/biomass MAC 5 weeks field/2003 Table 8 PealgerminationlnodulelbiomasslyieldMA,C Immediately fieldl2003 Table 9 Soybeanlyield 5 sites in Immediately Table 10 Wheatlgermination MAC Immediately field12003 Table 11 Corn/germination 5 sites in Immediately Table 12 Soybeanlgerminationlyield 2 sites of Immediately UoG/2003 a Table 1. Effect of SeedCoater Dose on soybean nodulation when soybean seed treated and sown immediately, Cumulative weight and Number of nodules from16 plants at 24 days Signal Total NoduleNodule Nodule Nodule # vs number on weight control weight vs.
16 (g) of applied (uM) plants 16 plants control 0 253 0.119 0 0 50 315 0.147 24.50% 23.84%

100 260 0.135 2.70% 13.65%

200 281 0.121 11.20% 1.50%

300 306 0.127 20.94% 6.99%

400 313 0.125 23.70% 5.64%

Notes, Greenhouse study conducted in 4" pots inoculated with Apex at 105 cellslg of greenhouse soil before planting, 8 pots per treatment. 10U gram of soybean seed was treated with 0.3 ml of each solution in a plastic bag. Treated seed was planted into pot immediately.
Conclusion, 1. All strengths of Seed Coater treated seed and planted immediately increased nodule number and weight.
2. 50 uM strength proved the best dose for both nodule number and weight when applied and planted immediately.

Table 2. Effect of SeedCoater dose on soybean nodulation when soybean seed treated one month in advance of sowing.
Cumulative weight and Number of nodules from 16 plants at 23 days Signal Total NoduleNodule weightNodule Nodule weight ~# vs number on (g) of 16 control vs. control applied (uM) plants plants 0 336 0.18 0 0 50 373 0.19 11.01 % 7.22%

100 365 0.19 8.63% 3.33%

200 369 0.20 9.82a~ 11.67%

300 410 0.24 22.02% 33.89%

400 382 0.20 13.69% 13.33%

Notes, Greenhouse study conducted in 4" pots inoculated with inoculants at 105 cellslg of greenhouse soil before planting, 8 pots per treatment. 100 gram of soybean seed was treated with 0.3 ml of each solution in a plastic bag. Treated seed was stored at room temperature for 30 days.
Conclusions 1. All strengths of Seed Coater increased nodule number and nodule weight when applied 30 days in advance 2. 300 uM strength was the best dose for both nodule number and weight when applied 30 days in advance.

3. Application of Seed Coater 30 days in advance required a higher dose (300 uM) than when applied and sown immediately (50 uM - Table 1 ).

Table 3 Effect of Seed Coater dose on corn plant dry weight in greenhouse study (Planted immediately after treatment) Treatment Dry weight (gram)IplantIncreased over control 0.0 uM 0.8367 50 a M 0.9024 7.8 100 uM 0.8987 7.4%

200 uM 0.9501 13.5%

300 uM 0.9672 15.6%

400 uM 0.9299 11.1 Notes;
1. Inoculated Bradyrhizobium japonicum at 105 cfulml in soil 2. Plant at time zero (Table 3) or 1 month later (Table 4) 3. 2 plants/pot and 8 pots/treatment 4. Greenhouse temperature over 30C for a few days in April, which affected plant growth in the greenhouse (Table 4) so that plants got bigger compared to plants in Table 3 5: Plants were harvested for biomass 31 days (Table 3) and 32 days (Table 4) after sowing Gonclusion;

No difference in plant height was seen, but plant dry matter increased by all treatments (7-15.6% over control) by 31 days after sowing Table 4. Effect of SeedCoater dose on Corn plant dry weight in greenhouse study (planted 1 month after treatment) Treatment Dry weight (gram)IplantIncreased over control 0.0 a M 3.0056 50 uM 3.2844 8.5%

100 uM 3.0650 1.8%

200 uM 3.6975 21.1 300 aM 3.2456 7.3%

400 uM 3.3781 11.3%

Conclusion;
All Seed Coater treatments increased both plant heighfi and dry weight at 32 days after sowing, but dry weight increased up to 21 % at applied strength of 200 uM.

Table 5. Effect of SeedCoater dose on corn grain yield Treatments Harvested Wet Grain Yield Grain Yield Wet Grain Grain (kg/2 rows) (kglha) (kgl2 Yield rows) (kg/ha) 250uM 8.05 b 5963.0 6.53 b 4840.1 b b 400uM 9.63 a 7133.3 7.71 a 5713:6 a a 600uM 8.17 b 6051.9 6.50 b 4817.0 b b Untreated control7.63 b 5244.4 6.14 b 4546.4 b b Significant at Yes Yes Yes Yes 5%

Notes;
1. Treated seeds were stored at room temperature (20°C) for one month before planting 2. Soil was seeded with inoculants at 105 cellslgram soiF before planting 3. Seedling stand was examined 1 month after planting and data (not listed) showed that Seed Coater did not affect seed emergence when applied 1 month after treatment.
4. Corn grain was harvested from the two middle rows of each plot (13.5 M2) at MAC farm (Harvesting date: Oct. 30, 2003, Seeding: May 23, 2003) 5. Grain. yield corrected to dry weight by drying approx. 500 gramlplot at 60°C for days.
Conclusion:
1. All treatments of Seed Coater increased corn grain yield by 6%-25.6% over control 2. 400 uM significantly increased both wet and dry grain yield Table 6. Effect of Seed Coater dose on soybean seed germination and final grain yield Treatments Germination % Yield (Kglha) 200 NM one month 46.OOa 2102.19 a 300 ~M one month 37.75b 1970.114 b 400 ~M one month 42.00ab 2040.Fifi a Untreated Control 39.25b 1530.57 c Significance at 5% Yes Yes.

Table 7. Effect of Seed Coater dose on soybeari nodulation and biomass Treatments Growing Stages V3 , Bloomin Nodule Nodule Shoot Nodule Nodule Shoot Number Dry Weight Dry Number Dry WeightDry on Weight on Weight (g) from (g) of 5 plants (g) from5 plants (g) of 5 plants 5 plants 5 plants 5 plants 200 pM one 122:8 0.2281 7.9 184 0.4994 21.70 month 300 pM one 96.5 0.2629 7.4 186 0.4994 23.56 month 400 pM one 121.8 0.2689 6.77 161 0.4304 19.64 month Untreated 104.0 0.2012 5.21 164 0.4329 15.31 Control Significance No No No No No No at 5%
Notes:
1. Experiment was conducted on E. Lods farm of McGill University in 2003 2. Seeds pre-treated by Seed Coater on April 4, 2003 and stored at room temperature (20°C), and sown on May 30 (5 weeks).
3. Germination or stand % was examined on July 2, counting seedling in 2-meter long row from two middle rows of each plot.
4. Soil was seeded with rhizobia at 10~ cellslgram on May 30 just before planting 5. Soybean grain in whole plot was harvested by a combine on Oct.17, 2003 Conclusion:
There were:
1. Increased seed emergence by strength at 200 and 400 uM dosages, and statistically significant at 200 uM strength.
2. Significantly increased grain yield by all treatments 3. Increased nodulation and biomass by all treatments, however, not statistically.

Table 8. Effect of SeedCoater Dose on pea seed emergence, nodulation and yield under field conditions Treatments Stand Nodule Nodule Average Dry Bulacre Increase nodule Number Weight Weight in weight bu/ac on 5 (g) of (g) 5 of (mg) plants plants Shoot s Control 95 217.75 0.2227 1.04b 8.81 26.5b 0 50pM 93 265.75 0.2633 1.13ab 10.17 29.8a 3.3 100~,M 98 287.75 0.2991 1.24ab 8.61 28.Oab 1.5 200~.M 91.25 196.25 0.2931 1.52a 10.14 29.6a 3.1 400~M 87.5 216.5 0.2585 1.20ab 9.01 29.4a 2.9 600~M 93 245.75 0.2970 1.33ab 9.02 28.3ab 1.8 Significant NS NS NS Yes NS Yes 5%

Notes; -.
1. Experiment was conducted on E. Lods farm of McGill University in 2003 2. Make stock solution of Naringenin (70 mM) and Hesperetin (30 mM) with DMSO and dilute to the strengths needed for each seed treatment with water 3. Pea seed (cv. Delta) was treated and planed immediately in plots which was seeded with Rhizobia at 105 cellslgram of soil 4. Seed germination was examined on June 9, 2003 (sown on May 16, 2003) 5. Nodulation examined on June 27, 2003 by sampling 5 plants per plot 6. Pea was harvested on August 6, 2003 using a combine and grain was dried at 60°C for 3 days Conclusion;
1. There is no difference among treatments on extent of germination of pea.
2. Seeds treated with SeedCoater at 100p,M showed the maximum germination.
There was no significant difference when compared to control 3. SeedCoater increased nodulation and biomass, but not significantly.
However, nodule weight was significantly improved at 200 uM
4. Most treatments significantly increased pea grain yield, some up to 3 bu/ac.

Table 9. Response in soybean yield (Bulac) at 5 sites in USA 2003 Locations Treatments Untreated controlWarden RTA (W- W-RTA + Seed RTA) Coater Brookston, IN 32.97 31.50 40.03 Tolono, IL 36.43 33.80 37.73 Walbash, IN 43.78 44.85 45.14 Wolcott, IN 31.03 36.70 35.83 Mt. Hope, WI 32.90 34.39 38.13 Average yield 35.42 36.25 39.37 of 5 sites vs. control 0.00 2.34 11.15 vs. W-RTA -2.29 0.00 8.61 Significant at B B A
5%

Notes;
1. Seeds were treated at 300 uM and planted immediately in repeat soybean lands at 5 sites in USA
2. High quality soybean seed commercially treated with Fungicide (Warden RTA) was employed in this trial in USA

Conclusion;
1. Seed Coater significantly increased soybean grain yield over yields from untreated and Warden RTA seeds Table 10. Effect of SeedCoater Dose on spring wheat seed emergence (%) in field trial on E. Lods farm of McGill University 2003 Percent of treated seed emerged at 4 weeks F~eplicates Treatments 1 2 3 4 Average Control 56 64 ' 52 36 52b 100uM 68 72 60 64 66ab 200uM 60 ~ 72 80 64 69a 400uM 60 68 68 76 68a 600uM 80 60 80 48 67ab Notes;
1. 100 treated wheat seeds were planted in each plot of field immediately 2. Spring wheat seed was coated by chemicals 3. Emergence was examined at 4 weeks after sowing in field 4. The field trial was terminated because plots were damaged by animals. No yield data available from this trial.
Conclusion:

Seed Coater significantly improved wheat seed emergence at strength of 200-400 uM

Table 11. Effect of Seed Coater treatment on fungicide (Maxim XL) treated corn seed emergence at 5 sites in USA, 2003 (% Field Emergence) Treatments Indiana Illinois Iowa (1) Iowa Nebraska Average (2) MaximXL 86.88 86.07 76.79 58.21 84.29 78.448b MaximXL + 85.63 85 79:29 77.5 91.07 83.698a Seed Coater Notes;
1. 250 uM (liquid) of Seed Coater directly applied to corn seed (Hybrid) at 3mllkg seed before sowing.
2. Seeds treated with Seed Coater were sown immediately after treatment at 5 sites in USA
3. Chemical (fungicide) coated corn seed was used in this trial 4. The Contracted field trials failed and contractor did not submit any yield data Conclusions;
Seed Coater significantly improved corn seed emergence in USA 2003 Table 12. Effect of Seed Coater on soybean stand and grain yield in field trials 2003 (the University of Guelph) Treatment Plants/M Grain yield (kglha) Huron Park Ridgetown Huron Park Ridgetown Untreated 21.2 ab 56 1926 a 3177 ab control Seed Coater 22.23 a 49 2026 a 3227 a Inoculant 2 19.8 ab 53 1992 a 2967 c Inoculant 1 13.88 b 47 1842 b 3056 be Significant LSDo.os NS LSDo.~ LSDo.~

Notes;
1. Seed Coater treated seed immediately planted in repeat soybean lands 2. 'Soybean seed was treated with Seed Coater of 300 uM at 3ml/kg seeds Conclusion;
1: In general, Seed Coater did not negatively affect soybean seed emergence in the fields 2. Seed Coater increased soybean grain yield over other inoculant treatments and control. However, significance was only seen over control (at 0.1 alpha).
The increase was not significant over other inoculant treatments.

Claims (32)

1. The use of one or more Signal or an agricultural composition thereof to increase yield and/or improve seed germination, and/or increase stand density, and/or result in earlier emergence, and/or improve plant vigour, and/or improve plant growth, and/or increase biomass, and/or result in earlier fruiting in a non-legume plant or crop thereof, by applications to said plant or crop.

2. The use as in Claim 1 where the non-legume plant or crop thereof is a seed, tuber, transplant, or vegetative cutting.

3. The use as in Claim 1 or 2 where the non-legume is grown for use in agriculture, horticulture, silviculture, or gardening.

4. The use as in any one of Claim 1 to 3 above, where the non-legume is sown into land that had previously been sown to a legume crop, or which has an indigenous population of rhizobia.

5, The use as in any one of Claims 1 to 3 above, where the non-legume is sown into land not previously sown to a legume crop.

6. The use as in any one of Claims 1 to 5 above, where the Signal is applied up to 365 days in advance of planting.

7. The use as in any one of Claims 1 to 6 above, where the one or more Signal is applied with an agriculturally acceptable carrier, comprising one or more selected from the group of water, seed treatments, inoculants, herbicides, fungicides, insecticides, fertilizers, growth promoters, and horticultural media.

8. The use as in any one of Claims 1 to 7 above, where the soil to be planted to the non-legume crop has been pre-treated with a specific symbiotic rhizobia or has an indigenous population of rhizobia.

9. The use as in any one of Claims 1 to 8 where the non-legume has been treated with a specific symbiotic rhizobia.

10.The application of one or more Signal or agricultural composition thereof to soil which will be planted to a non-legume plant or crop, to increase yield and/or improve seed germination, and/or increase stand density, and/or increase biomass, and/or result in earlier emergence, and/or result in earlier fruiting, and/or improve plant vigour, and/or improve plant growth, in said plant or crop.

11.The application as in Claim 10 where the non-legume is grown for use in agriculture, horticulture, silviculture, or gardening

12.The application as in Claims 10 or 11 where the non-legume is sown into land that had previously been sown to a legume crop or has an indigenous population of rhizobia

13.The application as in any one of Claims 10 or 11 where the non-legume is sown into land that had not previously been sown to a legume crop.

14.The application as in any one of Claims 10 to 13, where the Signal is applied with an agriculturally acceptable carrier comprising any one or more selected from water, seed treatments, inoculants, herbicides, fungicides, insecticides, fertilizers, growth promoters, and horticultural media.

15.The application as in any one of Claims 10 to 14 where the soil to be planted to the non-legume crop has been pre-treated with one or more symbiotic rhizobia or has an indigenous population of rhizobia.

16.The application as in any one of Claims 10 to 15 where the plant or crop has been treated with one or more symbiotic rhizobia.

17.The use of one or more Signal or agricultural composition thereof to increase yield and/or improve seed germination, and/or increase stand density, and/or increase biomass, and/or result in earlier emergence, and/or result in earlier fruiting, and/or improve plant vigour, and/or improve plant growth, and/or increase nodule number, and/or increased nodule weight, in a legume plant or crop thereof, by application to said plant or crop.

18.The use as in Claim 17 where the legume is a seed, tuber, transplant, or vegetative cutting.

19.The use as in Claim 17 or 18 where the legume is grown for use in agriculture, horticulture, silviculture, or gardening

20.The use as in any one of Claims 17 to 19 where the legume is sown into land that had previously been sown to a legume crop or has an indigenous population of rhizobia.

21.The use as in any one of Claims 17 to 19 where the legume is sown into land that had not previously been sown to a legume crop.

22.The use as in any one of Claims 17 to 21 where the Signal is applied up to 365 days in advance of planting.

23.The use as in any one of Claims 17 to 22 where the Signal is applied with an agriculturally acceptable carrier comprising one or more selected from water, seed treatments, inoculants, herbicides, fungicides, insecticides, fertilizers, growth promoters, or horticultural media.

24.The use as in any one of Claims 17 to 23 where the soil to be planted to the legume crop has been pre-treated with one or more symbiotic rhizobia or has an indigenous population of rhizobia.

25.The use as in any one of Claims 17 to 24 where the plant or crop has been treated with one or more specific symbiotic rhizobia.

26.The application of one or more Signal or agricultural composition thereof to soil, which will be planted to a legume plant or crop, to increase yield and/or improve seed germination, and/or increase stand density, and/or earlier emergence, and/or improve plant vigour, and/or improve plant growth, and/or increase nodule number, and/or increase nodule weight, and/or increase biomass, and/or result in earlier fruiting in said plant or crop.

27.The application as in Claim 26 where the legume is grown for use in agriculture, horticulture, silviculture, or gardening.

28.The application as in Claims 26 or 27 where the legume is sown into land that had previously been sown to a legume crop or has an indigenous population of rhizobia.

29.The application as in Claims 26 or 27 where the legume is sown into land that had not previously been sown to a legume crop

30. The application as in any one of Claims 26 to 29 where the Signal is applied with an agriculturally acceptable carrier comprising one or more selected from water, seed treatments, inoculants, herbicides, fungicides, insecticides, fertilizers, growth promoter, or horticultural media.

31.The application as in any one of Claim 26 to 30 where the soil to be planted to the legume crop has been pre-treated with one or more symbiotic rhizobia or has an indigenous population of rhizobia.

32. The application as in any one of Claims 26 to 31 where the plant or crop has been treated with one or more symbiotic rhizobia.