WO1987002659A1

WO1987002659A1 - Microbial plant growth promoter and yield enhancer

Info

Publication number: WO1987002659A1
Application number: PCT/US1985/002159
Authority: WO
Inventors: Abraham I. Tenzer
Original assignee: Bio Organics Inc
Current assignee: Bio Organics Inc
Priority date: 1985-11-04
Filing date: 1985-11-04
Publication date: 1987-05-07
Anticipated expiration: 1988-05-04
Also published as: FI872921L; EP0246223A1; FI872921A7; EP0246223A4; FI872921A0; DK338487A; BR8507306A; DK338487D0; KR880700783A; NO872753D0; JPS63501286A; NO872753L

Abstract

A novel microbial plant growth promoting composition comprising a mixture of a bacteria and algae, and a method of promoting plant growth therewith.

Description

/ MICROBIAL PLANT GROWTH PROMOTER AND YIELD ENHANCER

Background of the Invention Field of the Invention The present invention concerns compositions and methods for plant growth promotion. More particularly the invention concerns microbial plant growth promoting compositions and methods for ther use.

Description of the Background Art Since man first began cultivating plant life, there has been an interest in promoting plant growth and increasing yields. This is particularly important in northern climates having limited growing seasons.

Various avenues have been pursued in attempts to promote plant growth. The use of microbes to achieve this result has been of great interest, particularly the use of nitrogen-fixing bacteria. Various means have been devised for contacting the roots of plants with nitrogen-fixing bacteria including inoculation of soil with such bacteria and treating seeds with bacteria-containing compositions. In addition to nitrogen-fixing bacteria, it is believed that other microorganisms found in the soil benefit plant growth. Although the exact mechanism of this beneficial action is not known, it has been proposed that such microorganisms break down complex soil components to nutrients that are assimilable by the plants. The conditions favoring microbial growth in tropical jungle soils are thought to partially account for the lush plant growth in those regions. Algae have also been employed for improving soil conditions and hence plant growth. For example, U.S. Patent 4,336,051 to Marguez describes soil-improving compositions containing gelidum type marine algae. Among the stated purposes of these compositions are to produce beneficial enzymes and to competitively inhibit the growth of pathogenic bacteria. In light of current and foreseeable demands on the world food supply, there is a continuing interest in developing methods and means of improving plant productivity.

Summary of the Invention

Iir accordance with the present invention a microbial plant.; growth-promoting composition comprises a mixture of a bacte_- a. and. an algae. Further disclosed are methods of promoting* plant growth by applying to plants or their environment plant growth-promoting amounts of a composition comprising a mixture of a bacteria and an algae.

Detailed Description of the Invention It has surprisingly been discovered that bacteria and algae, or their by-product, when combined and used according to the present invention, have a growth-promoting effect when applied to plants or their environmments.

The compositions of the present invention have been shown to have significant positive effects on early plant vigor and plant yields. Furthermore, use of the compositions of the present invention to promote growth in fruiting plants results in more fruit reaching marketable size faster than such plants grown without use of the composition. This is particularly beneficial to farming in northern climates, where an earlier harvest is important. Preferred bacteria for use according to the present invention are of the genera Escherichia and Bacillus, with B.subtilis being particularly preferred because of its non- pathogenicity. Algal components of the compositions are advantageously of the division Chlorophyta (green algae), with algae of the order Chlorococcales being preferred. Algae of the genus Chlorella are particularly preferred, with Chlorella saccharophilia being most preferred.

The bacterial and algal components of the composition of the present invention are advantageously separately prepared in appropriate nutrient mediums under growth conditions and then combined and incubated in a nutrient medium under growth conditions to produce a final product. Growth conditions include a physiologically acceptable pH and temperature. Nutrient mediums contain assimilable sources of carbon, nitrogen and essential trace minerals, vitamins and growth factors. Various sugars, such as lactose, glucose, or crude carbohydrates such as molasses, and the like may be used as suitable sources of carbon. Inorganic ammonium salts, such as ammonium nitrate, ammonium sulfate, ammonium phosphate and the like and protein extracts, such as meat broths, tryptose broth, soybean meal and the like can provide sources of nitrogen. Mineral salts, such as sodium, potassium, calcium, magnesium, and molybdenum salts of chloride, sulfate, phosphate and the like can be used to provide trace mineral requirements. In addition, essential growth factors, such as vitamins can be advantageously supplied as commercially available preparations, such as yeast extracts and the like. Particularly preferred nutrient media for preparing the bacterial and algal components are described below in Example I.

After the bacterial and algal cultures have been grown to suitable cell densities, e.g. from about 2xl0⁶ to lOxlO⁶, preferably about 4xl0⁶ to about 6xl0⁶ cells/ml for the bacterial culture and from but 10x10° to about

20xl0⁶, preferably about 14xl0⁶ to about lβxlO⁶ cells/ml for the algal culture, the two cultures are mixed together. To the resulting mixture is added additional nutrients, and the mixture is incubated under growth conditions. Whey may advantageously be used as a nutrient source, and those skilled in the art will recognize that other sources of carbon, nitrogen, minerals and growth factors, as described above, may be employed. The medium also advantageously includes a stabilizing amount of soresepinium (an extract from Yucca, obtained from Ritter Brothers, International, Los Angeles, California, USA). The concentration of soresepinium is preferably from about 0.5% to about 2%, preferably between about 1% and about 1.5% by volume, with a concentration of about 1.25% by volume being particularly preferred. A lipase is also preferably added to the mixture during the incubation in an amount sufficient to stabilize the mixture against decomposition. The concentr.ation of lipase is preferably from about 0.5% to about.2-%:,. preferably from about 1% to about 1.5% by volume, with a^~ concentration of about 1.25% by volume being particularly preferred. Generally, the mixture is incubated for about 1-4 weeks, preferably 2-3 weeks at a temperature of from about 15°C to about 40°C, preferably of from about 20°C to about 25°C. The resulting bacteria-algae compositions can be stabilized by terminating the growth of the cells by acidifying with a non-toxic acid, such as lactic acid or acetic acid. The pH is preferably lowered to about 3-5, preferably about 4.0. A preserving-amount of a preservative, such as sodium benzoate is also added.

Preferred compositions further contain vitamin B-12 in a stabilizing amount. It is particularly preferred that the vitamin B-12 be added to the composition in an amount within the range of from about 1.5 gm/gallon to about 5 gm/gallon of the composition, with an amount of about 2 gm/gallon of the composition being most preferred.

The present invention further relates to a method of enhancing plant growth using the microbial plant growth regulating composition. The preferred methods of plant treatment are irrigation or spraying. Alternatively, seeds may be treated with the composition by soaking the seeds preferably for from about 2 to about 24 hours in the composition prior to planting. The composition is applied in a growth-promoting amount. This amount will vary, depending on the application means, climatic and soil conditions, and the particular crop involved. Generally, from about 1 lb. to about 3 lbs. per acre is employed, preferably from about 1.5 lbs. to about 2 lbs. per acre. In hydroponic applications, the composition is added directly to the nutrient solution. The ratio of microbial composition to nutrient solution is advantageously approximately 0.5 to 3.0 ml microbial composition/80 gallon nutrient: medium.

The microbial plant growth regulating composition of this invention can be sprayed directly on plant foliage (foliar application) to increase plant growth and yield. For foliar application, generally from about 0.2 lb. to about 1 lb. per acre is employed, preferably from about 1/4 lb. to about 1/3 lb. per acre. Advantageously, the composition is mixed with a suitable carrier, such as water, to dilute the composition to a suitable concentra¬ tion to facilitate uniform application of the composition to the foliage. Absorption of the composition by foliage is promoted by the addition of small amounts (e.g., up to about 5% by weight) of one or more non-toxic foliar surfactants commonly used in the agriculture industry for application of materials to plant foliage, or other non-toxic foliar penetrants or wetting agents. Suitable absorption-promoting additives may include non-toxic anionic, cationic and nonionic surfactants, humic acid or a derivative and polyethylene glycol. Advantageously, a composition according to this embodiment includes additional plant nutrients such as assimilable nitrogen (N) , phosporous (P) and potassium (K) in, for example, an N-P-K ratio of 14:7:3. The .composition also advantageously includes small amounts (e.g., 0.1-0.5% by weight) of trace elements, such as molybdenum, selenium and boron, which are preferably chelates formed with, for example, ethylenediaminetetraacetic acid (EDTA). Additional small amounts of soresepinum (e.g., 0.01-0.1% by weight) can also be added to the composition for added stability. The invention is further illustrated by the following examples which are not intended to be limiting.

Example 1 Cultures of Bacillus subtilis and Chlorella sacchorophila were separately prepared and then combined and- treated to produce a final product according to the following^ procedure. Preparation: of: the Bacterial Culture Component

Samples: o£ Bacillus subtilis (obtained from the American Culture Collection of Rockville, Maryland as

ATCC #6461) were added to an enrichment medium consisting of lauryl tryptose broth (Difco #0241-02-7, Difco, Detroit, Michigan) containing about 3.5% by volume lactose at a pH of 6.8 to 7.0. The mixture was incubated at a temperature of 35°C plus or minus 1°C for 24 hours to complete replacement of lactose by CO2. The pH of the mixture was adjusted to between 6.5 and 6.8 with the aid of a pH sensitive dye (Bright Green'Laboratory Bile, Difco #007-01- 2). The mixture was then incubated at a temperature of 35°C plus or minus 1°C for 24 hours.

Isolation of the culture was accomplished using a nutrient agar medium (Difco #0001-02) composed of 3 g/L beef extract, 5 g/L peptone (Difco) and 15 g/L agar at a pH of 6.8 to 7.0. The mixture was then incubated at a temperature of 44.5°C plus or minus 0.1°C in a waterbath for 24 to 48 hours until a count of 5,000,000 cells/ml was achieved.

Preparation of the Algal Culture Component

Samples of Chlorella saccharophila (obtained from the American Type Culture Collection of Rockville, Maryland,

U.S.A. as ATCC #30408) were added to an algal protose agar medium consisting of 15 to 20 g/L agar, 0.2 to 0.3 g/L sodium nitrate, 2 g/L calcium chloride, 0.5 g/L magnesium sulfate, 15 g/L potassium phosphate, 3.0 g/L sodium chloride, 7.0 to 8.0 g/L protose peptone, 0.2 g/L molybdenum sulfate, and balance to one liter de-ionized water at a pH of 6.5 to 6.8. The mixture was incubated at a temperature of 22°C to 25°C for 2 to 3 weeks in sunlight. The mixture was transferred to a specific medium consisting of the algal protose agar medium described above but. without molybdenum sulfate for 2 to 3 days to isolate the culture and achieve a count of 15,000,000/ml. The count, was determined by a microscopic count procedure. The culture, was then centrifuged to separate the aqueous portion and the aqueous portion was discarded.

Preparation of the Microbial Plant Growth Regulating Composition (Bioagent)

The bacterial and algal cultures as prepared above were mixed together for a total count of 20,000,000/ml

(15,000,000/ml algae and 5,000,000/ml bacteria). One gallon of the mixture was added to 9 gallons of whey plus one pint of soresepinum, and incubated at a temperature of

20°C to 22°C for two to three weeks. The culture was mixed and checked for count four times daily. After 10 days

(approximately mid-cycle) one pint lipase (Difco

#0431-63-3) was added. At the end of the incubation cycle, the pH was adjusted to 4 plus or minus 0.2 using lactic acid titration and sodium benzoate was added to achieve a concentration of 5 ppm. Finally, 2 gram/gallon of vitamin

B-12 assay (Difco #6300-15-7) was added to produce the final product. The final product was stored at 55°F to

65°F away from sunlight.

Example II The following experiments were conducted to test the ability of the microbial composition (bioagent) described in Example I to enhance tomato plant growth. These experiments were conducted on two levels. One test was run in a commercial greenhouse using a bag system where a mixture of peatmoss and vermiculite was used in the bags. The other test made use of a growth chamber in which plants were grown in water for short periods of time under constant environmental conditions. In the growth chamber system the bioagents were added to the nutrient solution on a daily basis; while in the bag culture, the bioagents were added twice a week.

I. Greenhouse

Seeds were treated with bioagent and sown. Fifty percent of the seedlings had germinated by the end of the - first week. With the appearance of the first true leaf 1/4 ounce of fertilizer concentrate per gallon of H2O was used to water the seedlings. This was maintained for one week when 1/2 oz of fertilizer concentrate per gallon of water was used. After one month, the seedlings were placed into a bag culture system. During the first two weeks of growth in bags the nutrient solution conductivity was raised from 1.0 to 1.8. The nutrient solution. pH was maintained at about 6.5. The plants treated with bioagent received 1.5 ml of bioagent per 50 gallons of nutrient solution twice weekly.

The plants grown in bags containing a peatmoss: ver iculite mix showed a positive response to the addition of bioagents. In Figure 1 an enhanced growth rate as measured by plant height is evident in the bioagent treated plants. Table 1 shows increases in leaf and stem fresh weights of plants grown in the bioagents. Table 2 shows that the average number of fruit which set in the first cluster was higher in the bioagent treated plants and the weight of each fruit was also greater. The data suggests more fruit in the bioagent treated plants which may reach marketable size faster than in plants grown without bioagent.

II. Growth Chamber

In these experiments tomato seeds were germinated in vermiculite under constant misting. When the seedlings had the first true leaf 2 cm in length, they were removed from the vermiculite and their roots were washed. The seedlings were placed in 1 liter jars containing 750 ml of nutrient solution (conductivity 1.8, pH 6.5). The root zone was constantly aerated by forcing air into the solution with a small pump. Half of the containers received bioagent daily and half did not. The nutrient solution level was maintained between 700-750 ml by adding new solution as needed. The nutrient solution was replaced each week. These seedlings, after being placed in the nutrient solution, were grown for approximately 21 days in the growth chamber.

Two experiments were conducted. Table 3 contains the data from the first run. The data from the second run is contained in Tables 4 and 5. Again, as was the case with the first set, the results show a positive bioagent effect. The dry weight of leaves, stem, and roots is enhanced from 10-20% by bioagent application. Photosynthetic rates are up 29% and leaf areas 12.5%.

Table 1. A comparison of the effects of bioagent on stem and leaf fresh weight. These are final measurements and represent the average of 10 plants.

Leaf Stem Treatment Fresh Wt. (g) Fresh Wt. (g)

Bag Culture (+) Bioagent 227.2 102.3

Bag Culture (-) Bioagent 223.5 93.3

Table 2. The effect of bioagent on plant yield. These are final measurements and represent the average 10 plants.

Total Number of Fresh Weight/ Treatment Fruit/Cluster Wt. (g) Fruit

Bag Culture (+) Bioagent 3.0 (+25%) 654 21.8 (+53.8%) (23.1%)

Bag Culture (-) Bioagent 2.4 425 17.7 Table 3. The effect of bioagent on tomato seedlings grown in a growth chamber, (average 4 plants)

Photosynthetic Avg. Plant Rate Leaf Area

Treatment (mg Cθ2/hr) cm²)

Bioagent 510 (+51.7%) 242.19 (+14%)

Control 336 209.45

F R E S H E I G H T

Leaves ROOtS Stem

Bioagent 3.6(+0%) .46(+35%) 3 .70(+15%)

Control 3.6 .3 3 .17

Table 4. Growth Chamber Experiment. The effect of bioagent on dry weight accumulation.

Treatment Leaf Stem Root

Bioagent .28g .09g .06g (+27.2%) (+12.5%) (+20%)

Control .22g .08g .05g

Table 5. Growth Chamber Experiment. The effect of bioagent on photosynthesis and leaf area. Photosynthetic Avg. Plant

Treatment Rate (mg Cθ2/hr) Leaf Area

Bioagent 880 (+41.9%) 274.1 cm²

(+12.5%)

Control 620 240.3 cm²

Example 3 The following experiments were conducted to elucidate the effects of the microbial composition (bioagent) prepared as described in Example I on tomato plant growth in a nutrient film technique (NFT) hydroponic growing system. Procedure:

In these experiments the amount of the microbial composition (volume) added to the nutrient reservoirs and the number of times the bioagent was added each week were altered. The following treatment regime was used:

Treatment 1 - 2.4 ml microbial composition/80 gal. nutrient solution added daily for the duration of the experiment. Treatment 2 — 2.4 ml microbial composition/80 gal. nutrient solution added daily for 2 weeks followed by the addition of 1.2 ml microbial composition/80 gal. of nutrient solution added twice a week for the duration of the experiment. Treatment 3 - 2.4 ml microbial composition/80 gal. nutrient solution added daily for 2 weeks followed by the addition of 0.6 ml microbial comρosition/80 gal. of nutrient solution added twice a week for the duration of the experiment.

Treatment 4 - 0.6 ml microbial composition/80 gal. of nutrient solution added daily for 2 weeks followed by the addition of 1.2 ml microbial composition/80 gal. of nutrient solution added twice a week for an additional 2 weeks; this was followed by adding 2.4 ml microbial composition/80 gal. of nutrient solution twice a week for the duration of the experiment. Treatment 5 - Control; no microbial composition added.

Each treatment consisted of four NFT troughs 6 inches wide and an 80 gallon nutrient solution reservoir. The cultural practices employed were similar for each treatment and followed normal operating procedures for growing a commercial tomato crop in NFT. The 'Jumbo' cultivar was used throughout this study. Seeds were planted and allowed to grow for 48 days when the seedlings were transplanted into the troughs. The additions of the microbial composition began two weeks later.

Results:

The effects of the various treatments on the vegetative growth of tomatoes can be seen in Figures 2, 3,.and 4 as well as Table A. Figure 2 shows that when the addition of the microbial composition began there were little or no differences in height among the treatments. However, after only 2 weeks under the various treatment regimes, the bioagent treated plants were significantly taller than the controls. This response is also seen in Table A, where in every case the fresh weight of the leaves, roots, and stems are higher in the bioagent treated plants than in the control plants. Root length also appears to be somewhat greater in the bioagent treated plants when compared to those of the control.

Figure 3 also points out that in every case the average leaf area is significantly greater in the bioagent treated plants than in the control plants. This figure also demonstrates that treatment number 4 stimulated leaf growth more so than the other bioagent treatments. This is also demonstrated in Figure 4 where the stem diameter is much larger in treatments 4 and 1 than in the other experimental treatments.

Figure 5 (A and B) shows that treatments 1, 2, and 3 stimulate both an increase in total fruit number and total fruit weight when compared to the controls. Figure 6 (A and B) presents data on total yield approximately 12 weeks after the first addition of the microbial composition. It can be seen that plants in treatments 1, 2, and 3 and, treatment 2, in particular, developed reproductively sooner and therefore started producing marketable fruit earlier. Tables B and C also contain data concerning the effects of the microbial composition on growth. Treatments tended to increase the distance between clusters (Table B) . The microbial composition apparently stimulates earlier development of fruit and has a dramatic effect on yield particularly of the first cluster (Figure 7A). Treatments 1 and- 2 greatly enhanced first cluster yield of marketable fruit- (Figure 7A) . However, in the later clusters (2-6) treatments 3. and 4 are stimulatory (Figure 7B) . It appears that treatments 1 and 2 with the larger amounts of bioagent stimulate productivity early but the lower applications provided by treatments 3 and 4 may promote increased total marketable yield throughout the growing cycle.

Table A. The effect of the microbial composition on vegetative growth of tomato (cv. 'Jumbo'). The data were collected 2 weeks after the start of the treatment. Each data point is the average of 4 plants.

r e s h W e i g h t (Grams)

Treatment Root Number Leaf Stem Root Length (cm)

1 26.3 9.6 15.2 62.8

(+54.67%)

23.6 8.4 12.2 43.8

(+7.88%)

28.8 9.9 13.0 44.2

(+8.86%)

31.5 11.2 12.2 52.7

(+29.80%)

5 (control) 17.4 5.7 7.6 40.6 Table B, The effect of the microbial plant growth composition on the average distance between flower clusters.

Distance (cm) Between Clusters

T r e a t m e n t N u m e r

Cluster 1 2 3 4 5 (Control

0-1 25.7 24.2 28.2 28.7 22.2 1-2 21.8 21.4 22.8 18.9 18.0 2-3 17.9 19.9 22.2 18.3 19.3 3-4 22.5 22.2 21.5 18.0 18.3 4-5 15.5 16.6 20.0 16.8 17.2 5-6 13.3 11.4 17.2 18.0 16.9

Table C. The average number of flowers per cluster per plant as influenced by treatments with the microbial composition.

Average Flower Number/Cluster/Plant

T r e a t m e n t N u m b e r

Cluster 1 2 3 4 5 (Control

1 5.5 5.3 5.4 4.4 5.2

2 9.9 5.4 7.5 7.2 7.0

3 8.0 7.2 7.9 6.2 6.9

4 6.6 6.1 6.1 6.9 5.4

5 4.9 3.0 5.0 5.4 5.8

6 2.8 4.8 4.4 5.7 4.4

7 5.5 2.0 2.4 4.0 3.3

Total 43.2 33.8 38.7 39.8 38.0

Example 4 The microbial composition (bioagent) produced according to Example 1 (supra) was mixed with conventional plant fertilizer having an N-P-K ratio of 14:7:3, a carboxylate derivative of humic acid, trace element chelates (obtained from W.R. Grace and Co., Organic Chemicals Div. , Lexington, Massachusetts, U.S.A.), soresepinum and water to give the following mixture for foliar application in weight percent:

Mixture for Foliar Application

Bioagent 50% Total nitrogen (60%) 14%

Available phosphoric acid (32%) 7%

Soluble potash (45%) 3% Humic acid (carboxylate derivative, 13%) 5% - Trace element chelate (30%) 0.5%

Soresepinum 0.05%

Balance inert material (water) 20.45%

Total 100%

The carboxylate derivative of humic acid was obtained by mixing two parts natural crude humic acid powder composition (Agro-Lig™, American Colloid Company, Skokie, Illinois, U.S.A.) made from Leonardite shale, an organic substance mined in South Dakota, with one part of a mixture comprising 85% by weight technical grade HC1 and 15% by weight 2-p-methoxy phenyl ethylene bromide, to form a slurry. The slurry was allowed to react for about five weeks, during which a solvolysis reaction takes place wherein the 2-p-methoxy phenyl ethylene bromide is converted to 2-p-methoxy phenyl ethylene chloride, and a by-product of the humic acid powder composition is formed as a precipitate. The sedimented product (carboxylate derivative) was separated from the aqueous portion and mixed with Bioagent as set forth above.

The effects of the mixture for foliar application was demonstrated on the growth of the following plant species: Viburnum odoratissimum , Pittosporum tobria and Ilex cornuta "Burfordii." Foliage was sprayed with the foliar mixture (2 ml/3 gal. water) or control (0 ml/3 gal. water), once a week during the test to substantially cover the foliage. The results over a 4-week period are shown in Table I, and the results over a 12-week period are shown in Table II. Table I

Effect of Treatments on the Growth of Woody Ornamentals Over Four Weeks

Characteristics

Root

Height Plant Length

Name of Plant- (Inches) Weight (g) (Inches)

Viburnum", Foliar mixture 6.8 1.5 36.8 1.1 4.9 0.5 cdoratissimum- Control 3.0 0.3 15.5 2.8 1.8 0.5

Pittosporum Foliar mixture 8.1 1.9 41.5 1.7 6.1 0.2 tobria Control 3.5 0.3 13.5 2.5 2.5 0.6

Ilex cornuta Foliar mixture 9.5 2.1 67.5 1.8 6.6 0.3 Control 4.1 0.5 31.3 1.6 2.7 0.5

Table II

Effect of Treatments on the Growth of Woody Ornamentals Over 12 Weeks

Characteristics

Root

Height Plant Length

Name of Plant (Inches) Weight (g) (Inches)

Viburnum Foliar mixture 19.5 0.3 126.6 1.3 9.5 0.8 odoratissimum Control 6.2 0.5 21.5 1.5 2.5 0.5

Pittosporum Foliar mixture 24.5 0.5 128.3 1.2 11.8 0.3 tobria Control 6.5 0.3 21.5 0.8 3.5 0.5

Ilex cornuta Foliar mixture 18.5 2.5 125.5 1.2 12.5 0.5 Control 7.5 0.5 40.8 1.1 4.2 0.3

The data demonstrates that the foliar mixture, when sprayed on foliage , results in substantial increases in plant height, plant weight and root length over control .

Claims

WHAT IS CLAIMED IS:

1. A method for preparing a microbial plant growth promoting composition which comprises growing a bacterial culture in a first nutrient medium under growth conditions to a cell density of from about 2 x 10⁶ to about 10 x 10⁶ cells/ml; growing an algal culture in a second nutrient medium under growth conditions to a cell density of from about 10 x 10⁶ to about 20 x 10⁶ cells/ml, and then mixing the bacterial and algal cultures together and incubating the mixture in a third nutrient medium under growth conditions at a temperature of from about 10°C to about

40°C to produce the final product; said growth conditions including a physiologically acceptable pH and temperature.

2. The method of claim 1, wherein the third nutrient medium is incubated for a period of from about 1 to about 4 weeks.

3. A microbial plant growth-promoting composition produced by the method of claim 1.

4. The composition of claim 3 wherein said algae is of the Division Chlorophyta.

5. The composition of claim 4 wherein said bacteria is of the genus Bacillus.

6. The composition of claim 5 wherein said algae is of the order Chlorococcales.

7. The composition of claim 6 wherein said algae is of the genus Chlorella.

8. The composition of claim 7 wherein said bacteria is of the species B. subtilis.

9. The composition of claim 8 wherein said algae is of the species C. sacchorophilia.

10. The composition of claim 3, 4, 5, 6, 7, 8 or 9 wherein the bacteria are present at a concentration of from about 4 x 10⁶ to about 6 x 10⁶ cells per ml. and the algae are present at a concentration of from about 14 x 10⁶ to about 16 x 10⁶ cells per ml.

11. The composition of claim 10, which further comprises stabilizing amounts of soresepinium, lipase and vitamin B-12.

12. The composition of claim 11, wherein the soresepinium is present at a concentration of from about 0.5% to about 2% by volume, the lipase is present at a concentration of from about 0.5% to about 2% by volume and the vitamin B-12 is present at a concentration of from about 1.5 g to about 5 g per gallon.

13. The composition of claim 10 for foliar application by spraying, further including up to about 5% by weight of a material non-toxic to plants, the non-toxic material being selected from the group consisting of foliar surfactants, penetrants and wetting agents.

14. The composition of claim 11 for foliar application by spraying, further including up to about 5% by weight of a material non-toxic to plants, the non-toxic material being selected from the group consisting of foliar surfactants, penetrants and wetting agents.

15. The composition of claim 12 for foliar application by spraying, further including up to about 5% by weight of a material non-toxic to plants, the non-toxic material being selected from the group consisting of foliar surfactants, penetrants and wetting agents.

16. The composition of claim 13 wherein the non-toxic material is humic acid or a derivative thereof.

17. The composition of claim 14 wherein the non-toxic material is humic acid or a derivative thereof.

18. The composition of claim 15 wherein the non-toxic material is humic acid or a derivative thereof.

19. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 3 or 9.

20. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 10.

21. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 11.

22. A method of promoting the growth of plants comprising applying -to said plants a growth-promoting amount of the composition of claim 12.

23. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 13.

24. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 14.

25. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 15.

26. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 16.

27. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 17.

28. A method of promoting the growth of plants comprising applying to said plants a growth-promoting amount of the composition of claim 18.

29. The method of claim 19 wherein said composition is applied to said plants in an amount of from about 1 to about 3 pounds/acre by irrigation or spraying.

30. The method of claim 20 wherein said composition is applied to said plants in an amount of from about 1.5 to about 2 pounds/acre.

31. The method of claim 26 wherein said composition is applied to said plants in an amount of from about 0.2 to about 1 pound/acre by spraying.

32. The method of claim 26 wherein said composition is applied to said plants in an amount of from about 1/4-1/3 pound/ cre by spraying.