Method for increasing the vigor and/or crop yield of agricultural plants under essentially non-existent pathogen pressure

Abstract

A method for increasing the vigor and/or crop yield of agricultural plants under essentially non-existent pathogen pressure, wherein the plants, the plant propagules, the seed of the plants and/or the locus where the plants are growing or are intended to grow are treated with an effective amount of a composition comprising a) the Bacillus subtilis strain with NRRL Accession No. B-21661 or a cell-free extract thereof, and/or a mutant of this strain or extract having all the identifying characteristics of the respective strain or extract as component (I), and b) optionally at least one chemical compound as component (II), selected from the active compound groups A) to J): A) strobilurins; B) carboxamides; C) azoles; D) heterocyclic compounds; E) carbamates; F) other active substances; G) growth regulators; H) herbicides; J) insecticides.

Claims

1. A method for increasing the vigor and/or crop yield of agricultural plants, wherein the plants, the plant propagules, the seed of the plants and/or the locus where the plants are growing or are intended to grow are treated with an effective amount of a composition comprising the Bacillus subtilis strain with NRRL Accession No. B-21661, a mutant of this strain having all the identifying characteristics of the strain, or a cell-free extract thereof; and wherein pathogen pressure is non-existent.

2. The method as claimed in claim 1, wherein the increased yield is characterized by increased plant weight, increased plant height, increased biomass, higher grain yield, more tillers, larger leaves, increased shoot growth, increased protein content, increased oil content, increased starch content, and/or increased pigment content.

3. The method as claimed in claim 1, wherein the plants, the plant propagules, the seed of the plants and/or the locus where the plants are growing or are intended to grow are treated with an effective amount of a composition comprising the Bacillus subtilis strain with NRRL Accession No. B-21661.

4. The method as claimed in claim 1, wherein treatment with the Bacillus subtilis strain with NRRL Accession No. B-21661, mutant of this strain having all the identifying characteristics of the strain or cell-free extract thereof results in an increase in vigor and/or crop yield in the agricultural plants; wherein the yield of a product of the plants is increased by at least 5% over the yield of the same product of the plants without application of the composition.

5. The method as claimed in claim 1, wherein the composition comprising the Bacillus subtilis strain with NRRL Accession No. B-21661, mutant of this strain having all the identifying characteristics of the strain or cell-free extract thereof increases the vigor and/or crop yield of agricultural plants independently of the pesticidal action of the composition.

6. The method as claimed in claim 1, wherein the seed is treated.

7. The method as claimed in claim 1, wherein the application is carried out as in-furrow and/or foliar treatment.

8. The method as claimed in claim 1, wherein a repeated application is carried out.

9. The method as claimed in claim 1, wherein the Bacillus subtilis strain with NRRL Accession No. B-21661, mutant of this strain having all the identifying characteristics of the strain or cell-free extract thereof is applied during the Pre-harvest interval.

10. The method as claimed in claim 1, wherein the agricultural plant is selected from soybean, corn, wheat, triticale, barley, oat, rye, rape, millet, rice, sunflower, cotton, sugar beet, pome fruit, stone fruit, citrus, banana, strawberry, blueberry, almond, grape, mango, papaya, peanut, potato, tomato, pepper, cucurbit, cucumber, melon, watermelon, garlic, onion, broccoli, carrot, cabbage, bean, dry bean, canola, pea, lentil, alfalfa, trefoil, clover, flax, elephant grass, grass, lettuce, sugarcane, tea, tobacco and coffee; each in its natural or genetically modified form.

11. The method as claimed in claim 4, wherein the yield of the product of the plants is increased by at least 10% over the yield of the same product of the plants without application of the composition.

12. The method as claimed in claim 1, wherein a commercially available formulation of the Bacillus subtilis strain is used.

13. The method as claimed in claim 2, wherein the increased yield is characterized by higher grain yield.

14. The method as claimed in claim 2, wherein the increased yield is characterized by increased plant height.

15. The method as claimed in claim 2, wherein the increased yield is characterized by increased plant weight.

16. The method as claimed in claim 2, wherein the increased yield is characterized by increased plant biomass.

Description

EXAMPLE 1: TOMATO

(1) Bacillus subtilis QST 713 was used to treat tomato seeds via a quasi-soil drench in the greenhouse. Specifically, tomato seeds were planted in steam-sterilized trays containing sterile media and grown in the greenhouse using standard techniques. Serenade ASO, which is a liquid formulation of Bacillus subtilis QST 713 containing 110.sup.9 CFU (Colony Forming Units)/g was applied to media at rates of 4 oz, 8 oz, and 16 oz per acre once, at the time of planting. The Serenade ASO product was applied as a spray application and not as a true drench, as the spray application did not provide enough water to cause germination. Other seeds not treated with the Serenade ASO product were used as negative controls.

(2) At the time of transplanting to the field, plants grown in media treated with Serenade ASO showed a higher vigor than those in the untreated control group based on grower observations of plant height, size of root mass as well as color and spindliness of leaves. In addition, a higher percentage of plants treated with the Serenade ASO product were able to be used in the field than those in the untreated control group (table 1a).

(3) TABLE-US-00001 TABLE 1a Plants that were able to Treatment be used in the field (%) Control 80.3 Serenade ASO 4 oz 83.0 Serenade ASO 8 oz 88.3 Serenade ASO 16 oz 87.7

(4) As can be seen in table 1a, Serenade ASO has a positive effect on plant health by increasing the plant's vigor resulting in an increased number of plants that could be used for planting in the field.

(5) Useable transplants treated with Serenade ASO and controls were subsequently planted in the field and grown under the same standard conditions (with all receiving the same watering, pesticide applications, and the like) until harvest. Due to the pesticide application, the plants could grow under essentially non-existent pathogen pressure. At harvest, plants treated with Serenade ASO at the time of planting in the greenhouse yielded more total weight of tomatoes and more marketable tomatoes than the untreated control (table 1b).

(6) TABLE-US-00002 TABLE 1b Yield (Total weight of Marketable tomatoes in 12 plots, tomatoes Treatment each containing 2 plants) (%) Control 359 46 Serenade ASO 4 oz/ac 366 78 Serenade ASO 8 oz/ac 397 71 Serenade ASO 16 oz/ac 368 77

(7) As can be seen in table 1 b, Serenade ASO has also a positive effect on plant health by increasing the plant's yield (total weight of tomatoes). In addition, the treatment with Serenade ASO results in increased vigor of the plants and cosequently in more marketable tomatoes compared to the untreated control plants.

EXAMPLE 2: PEPPER

(8) Bacillus subtilis QST 713 was used to treat pepper seeds via a quasi-soil drench in the greenhouse. Specifically, pepper seeds were planted in steam-sterilized trays containing sterile media and grown in the greenhouse using standard techniques. Serenade ASO, which is a liquid formulation of Bacillus subtilis QST 713 containing 1109 CFU/g, was applied to media at rates of 4 oz, 8 oz, and 16 oz per acre once, at the time of planting. The Serenade ASO product was applied as a spray application and not as a true drench, as the spray application did not provide enough water to cause germination. Other seeds not treated with the Serenade ASO product were used as negative controls.

(9) At the time of transplanting to the field, plants grown in media treated with Serenade ASO showed a higher vigor than those in the untreated control group based on grower observations of plant height, size of root mass as well as color and spindliness of leaves. In addition, a higher percentage of plants treated with the Serenade ASO product were able to be used in the field compared to those in the untreated control group (table 2).

(10) TABLE-US-00003 TABLE 2 Vigor (0 = no vigor; 10 = Plants that were able to Treatment optimal vigor) be used in the field (%) Control 3.7 95.3 Serenade ASO 4 oz/ac 4.0 96.0 Serenade ASO 8 oz/ac 5.3 97.0 Serenade ASO 16 oz/ac 6.7 97.0

(11) As can be seen in table 2, Serenade ASO has a positive effect on plant health by increasing the plant's vigor. In addition, the treatment with Serenade ASO results in more plants that were able to be used in the field compared to the untreated control plants which in turn will result in an increased overall yield.

EXAMPLE 3: BROCCOLI

(12) Bacillus subtilis QST 713 was used to treat broccoli seeds via a quasi-soil drench in the greenhouse. Specifically, broccoli seeds were planted in steam-sterilized trays containing sterile vermiculite and grown in the greenhouse using standard techniques. Serenade ASO, which is a liquid formulation of Bacillus subtilis QST 713 containing 110.sup.9 CFU/g, was applied to media at rates of 4 oz, 8 oz, and 16 oz per acre once, at the time of planting. The Serenade ASO product was applied as a spray application and not as a true drench, as the spray application did not provide enough water to cause germination. Other seeds not treated with the Serenade ASO product were used as negative controls.

(13) At the time of transplanting to the field, plants grown in media treated with Serenade ASO showed a higher vigor than those in the untreated control group based on grower observations of plant height, size of root mass, as well as color and spindliness of leaves. In addition, a higher percentage of plants treated with the Serenade ASO product were able to be used in the field compared to those in the untreated control group (table 3).

(14) As can be seen in table 3, Serenade ASO has a positive effect on plant health by increasing the plant's vigor. In addition, the treatment with Serenade ASO results in more plants that were able to be used in the field compared to the untreated control plants which in turn will result in an increased overall yield.

(15) TABLE-US-00004 TABLE 3 Vigor (0 = no vigor; 10 = Plants that were able to Treatment optimal vigor) be used in the field (%) Control 4.7 91.7 Serenade ASO 4 oz/ac 6.0 92.0 Serenade ASO 8 oz/ac 7.3 93.0 Serenade ASO 16 oz/ac 5.3 93.0

EXAMPLE 4: WHEAT

(16) Wheat seed was treated with Bacillus subtilis QST 713 by applying to the seeds a slurry of the Serenade ASO product at a rate of 4 oz, 8 oz, 12 oz or 16 oz per 100 lb seed. The slurry was prepared by mixing Serenade ASO with water. Seeds remained in the slurry for various periods of time, ranging from overnight to two weeks. Fields were seeded at a rate of 80-100 lb per acre. Seeds were applied to fields in which disease pressure was essentially non-existent. Consequently, growers would typically not engage in seed treatment for disease control.

(17) TABLE-US-00005 TABLE 4 Yield (bushels/ Treatment acre) Control 49.9 Difenoconazole + mefenoxam 65.7 Serenade ASO 4 oz + difenoconazole + mefenoxam 79.5 Serenade ASO 4 oz/100 lb seed 100.4 Serenade ASO 8 oz/100 lb seed 90.8 Serenade ASO 12 oz/100 lb seed 49.3 Serenade ASO 16 oz/100 lb seed 34.6

(18) As can be seen in table 4, Serenade ASO has a very positive effect on plant health by increasing the yield when applied below 10 oz/100 lb seed. When applied at higher amounts with this particular formulation, the yield may remain unaffected or may even decline. However, it is unknown whether this decline is due to a formulation inert in this particular formulation or due to the rate of active ingredient. One of ordinary skill in the art would be able to determine the optimal rate of application of component (I) with routine experimentation.

EXAMPLE 5: WHEAT

(19) The Serenade ASO product, which contains 1109 CFU/g Bacillus subtilis QST 713 was applied in furrow at the time of wheat seed planting along with the following starter fertilizer: 10-34-0 (10% nitrogen, 34% phosphate and 0% potassium) and/or Power Up (6% nitrogen, 18% phosphate and 6% potassium) at the rates/per acre shown below. Disease pressure was essentially non-existent, such that disease rates were not reported for this trial. This was a situation in which a grower would not typically apply the Serenade ASO product, as the cost would not be justified from a disease control perspective.

(20) TABLE-US-00006 TABLE 5 Yield (bushels/ Treatment acre) Control 21.7 3 gallons per acre (gpa) of 10-34-0 25.9 2 gpa of 10-34-0 + 1 gpa Power Up 28.3 2 gpa of 10-34-0 + 1 gpa Power Up + 31.0 Serenade ASO 8 oz/ac 2 gpa of 10-34-0 + 1 gpa Power Up + 30.1 Serenade ASO 16 oz/ac

(21) As can be seen in table 5, the application of Serenade ASO with the fertilizers resulted in an increased yield. In addition, in the case of in-furrow application of Serenade ASO, wheat heads displayed advanced maturity compared to the untreated control plants in which only 75% of the wheat heads had formed at the same time point.

EXAMPLE 6: LETTUCE

(22) The active compounds were used applying commercially available formulations and diluted according to the concentrations/dose rates as stated in table 6. Commercially available lettuce seedlings (Eichblatt) were used for the described greenhouse trial. 4 replications (pots with 1 plant each) were used per treatment. Plants were grown in commercially available substrate (Floradur A) at approx. 20 C. in the greenhouse. Drench applications using a volume of 25 ml of product solution or water (Control) were made on 16 consecutive days. On the last day, fresh weight was determined using all plant parts above ground.

(23) TABLE-US-00007 TABLE 6 Yield Fresh Treatment weight (g) Control 51.7 Serenade MAX 312 ppm 59.5 Serenade MAX 625 ppm 62.4 Serenade MAX 1250 ppm 74.1 Serenade MAX 2500 ppm 68.9

(24) As can be seen from table 6, Serenade MAX strongly increases the fresh weight of lettuce plants which is an essential parameter for vegetables.

EXAMPLE 7: SOYBEANS

(25) Soybeans were planted in December 2008 at the BASF experimental station in Campinas, San Antonio de Posse, Sao Paulo, Brazil. The variety Emprapa 48 was planted at a seeding rate of 300,000 plants per ha. Row spacing was 45 cm. Two trials were setup as a randomized bloc design with 6 replications. Plot size was 20 m.sup.2.

(26) Bacillus subtilis QST 713 was applied by foliar application to the vegetative parts of the soybean plants at developmental stage 23/29 (BBCH) followed by the foliar application of either Bacillus subtilis QST 713 alone or in tank mix with pyraclostrobin (applied as COMET at the beginning of flowering at the developmental stages 60/63 (BBCH). The active ingredients were applied using the commercial formulations Serenade (10%, WP with 5109 cfu/g) and Comet (250 g/L, EC). The formulations were used in the dose rates given in table 7. Total spray volume for foliar applications was 150 l/ha. Serenade was applied with 3 kg product per ha and Comet applied with a product rate of 0.4 l/ha. At maturity the crop was harvested and grain yield was measured in t/ha. Green leaf area was assessed 31 days after the last treatment (table 7) by estimating the green leaf area in 10 randomly chosen plants per plot.

(27) The efficacy (E) was calculated as % increase of green leaf area in the treatments compared to the untreated control according to the following formula:
E=a/b1.Math.100 E efficacy a corresponds to the green leaf area (%) of the treated plants and b corresponds to the green leaf area (%) of the untreated (control) plants

(28) An efficacy (E) of 0 means the green leaf area of the treated plants corresponds to that of the untreated control plants; an efficacy of 100 means the treated plants showed an increase in the green leaf area of 100%.

(29) TABLE-US-00008 TABLE 7 Grain GLA GLAE Yield Treatment PR FC FT AT (%) (%) (t/ha) Untreated 7.5 1.97 Serenade 3.0 kg/ha 10% WP 23/29 12.5 66.6 2.30 3.0 kg/ha 10% WP 60/63 Serenade 3.0 kg/ha 10% WP 23/29 20.0 166.6 3.08 3.0 kg/ha 10% WP 60/63 Pyra- 0.3 l/ha 250 g/l EC 60/63 clostrobin PR = Product rate; FC = Formulation concentration; FT = Formulation type; AT = Application time (BBCH); GLA = Green Leaf Area; Green Leaf Area (Efficacy)

(30) As can be seen in table 7, Serenade clearly increases the green leaf area duration (maintanance of green leaves) and the grain yield in soybeans compared to the untreated control. In addition, the results shown in table 7 demonstrate that the efficacy of the combination of Serenade and pyraclostrobin is even higher than for Serenade alone. An increase of the green leaf area is a visible sign of the enhanced plant vigor. Based on a prolonged maintanance of green leaves which in turn results in a prolonged photosynethic activity of the leaves as well as an overall srengthening of the plant, the plant is able to produce a higher yield.

EXAMPLE 8: SOYBEANS

(31) Soybeans were planted in 2009 at 9 locations across the soybean growing area of Midwest of the USA (IN, IL, IA, MO, NE, and SD). Planting dates ranched from May 7th at York, Neb., to June 22nd at Clarence, Mo. Bacillus subtilis QST 713 was applied to the vegetative parts of the soybean plants at developmental stage 23/29 (BBCH). The B. subtilis QST 713 was applied using the commercial formulation Serenade Max (14.3%, WP with 7.3109 cfu/g). Serenade Max was used in the dose rates given in Table 8. Total spray volume for foliar application ranched from 140 to 200 l/ha. Serenade Max was applied at 3 kg product per ha. At maturity the crop was harvested and grain yield was measured as t/ha (table 8). Green leaf area was assessed 36 to 66 days after the last treatment on seven of the trial locations (table 8) by estimating the green leaf area in 10 randomly chosen plants per plot. The efficacy was calculated as indicated above.

(32) TABLE-US-00009 TABLE 8 Grain GLA GLAE Yield Treatment PR FC FT AT (%) (%) (t/ha) Untreated 64.36 34.87 Serenade 3.0 kg/ha 14.3% WP 23/29 67.04 4.2 36.42 MAX PR = Product rate; FC = Formulation concentration; FT = Formulation type; AT = Application time (BBCH); GLA = Green Leaf Area; Green Leaf Area (Efficacy)

(33) As can be seen in table 8, Serenade Max increases the green leaf area duration and therefore improve photosynthetic activity of soybeans. In addition, Serenade Max strongly increases the grain yield; in this case by 1.55 t/ha in soybeans compared to the untreated control by improving the vigor of the soybean plants.

EXAMPLE 9: WINTER WHEAT

(34) Winter wheat was grown in the 2008/2009 growing season at 4 locations across Germany (Thuringia, Baden-Wuerttemberg, and Rhineland-Palatinate). Seeding of the crop ranged from September 21st to October 26th. The trials were setup in a randomized bloc design with 6 replications. Bacillus subtilis QST 713 was applied to the winter wheat plants at beginning of shooting (growth stage 31/32, BBCH). The fungicide spray sequence consisted of an application of epoxiconazole at beginning of shooting followed by an application of epoxiconazole in combination with pyraclostrobin at flag leaf stage (growth stage 37/39). The B. subtilis QST 713 was applied using the commercial formulation Serenade Max (14.3%, WP with 7.3109 cfu/g). Epoxiconazole was applied alone at beginning of shooting as the commercially available formulation Opus (125 g/l, SC). The combination of epoxiconazole and pyraclostrobin was applied as a ready to use developmental Opera formulation (SE) containing 62.5 g/l epoxiconazole and 85 g/l pyraclostrobin. Product rates are given in table 9. Total spray volume for the foliar applications were 300 l/ha. At maturity the crop was harvested and grain yield was measured as t/ha (table 9).

(35) TABLE-US-00010 TABLE 9 Observed Grain yield Yield increase Treatment PR FC FT AT (t/ha) (t/ha) Untreated 6.73 Epoxiconazole 0.8 l/ha 125 g/l SC 31/32 7.78 1.05 Epoxiconazole + 2.0 l/ha 147.5 SE 37/39 Pyraclostrobin Serenade 3.0 kg/ha 14.3% WP 31/32 6.87 0.14 Max Serenade 3.0 kg/ha 14.3% WP 31/32 7.93 1.20 Max Epoxiconazole 0.8 l/ha 125 g/l SC 31/32 Epoxiconazole + 2.0 l/ha 147.5 SE 37/39 Pyraclostrobin PR = Product rate; FC = Formulation concentration; FT = Formulation type; AT = Application time (BBCH);

(36) As can be seen in table 9, the Serenade Max treatment increased the yield of the wheat crop by 140 kg/ha. The joint application with an application sequence of Serenade Max together with the fungicide spray of epoxiconazole and epoxiconazole plus pyraclostrobin was even superior compared to the application of the fungicides (epoxiconazole plus pyraclostrobin) alone (1.2 vs. 1.05 t/ha). These findings demonstrate the improvement of the vigor of the wheat plants by Bacillus subtilis QST 713 and the superior effect of the combination of Bacillus subtilis QST 713 with fungicides compared to the effect of the solo application of Bacillus subtilis QST 713 or an application of a composition comprising as active ingredients only fungicides.

EXAMPLE 10: MAIZE (CORN)

(37) Maize was planted in 2009 at Carlyle, Ill., USA. The variety Burrus 616XLR was planted at a conventional seeding rate and a row spacing of 76 cm. The trial was setup as a randomized bloc design with 6 replications. Plot size was 18 m.sup.2. Pyraclostrobin was applied at developmental stage 34/37 (BBCH). Bacillus subtilis QST 713 (Serenade Max) was applied to the maize plants at developmental stage 34/37 (BBCH) followed by a second application at developmental stage 55/57 (BBCH). Bacillus subtilis QST 713 (Serenade Max) in combination with pyraclostrobin were applied as a tank mix at the developmental stage 34/37 (BBCH). The active ingredients were applied using commercial formulations Serenade Max (14.3%, WP with 7.3109 cfu/g) and Headline (250 g/L, EC). The formulations were used in the dose rates given in table 10. Total spray volume for foliar applications was 200 l/ha. Serenade Max was applied with 2.1 kg product per ha and Headline with a product rate of 0.44 l/ha. At maturity the crop was harvested and grain yield was measured as t/ha (table 10).

(38) The expected yield increase by the combination of the active compounds was estimated using Colby's formula (Colby, S. R., Calculating synergistic and antagonistic responses of herbicide combinations, Weeds, 15, pp. 20-22, 1967) and compared with the observed yield increase.
E=x+yx.Math.y/100Colby's formula: E expected efficacy, expressed as the numerical difference of the yield in t/ha to the untreated control, when using the mixture of the active compounds A and B at the concentrations a and b x efficacy, expressed as the numerical difference of the yield in t/ha to the untreated control, when using the active ingredient A at the concentration a y efficacy, expressed as the numerical difference of the yield in t/ha to the untreated control, when using the active ingredient B at the concentration b

(39) TABLE-US-00011 TABLE 10 Observed Expected Grain yield yield Yield increase increase Treatment PR FC FT AT (t/ha) (t/ha) (t/ha) Untreated 8.27 Pyraclostrobin 0.44 l/ha 250 g/l EC 34/37 8.32 0.05 Serenade Max 3.0 kg/ha 14.3% WP 34/37 8.42 0.15 3.0 kg/ha 55/57 Serenade Max 3.0 kg/ha 14.3% WP 34/37 8.66 0.39 0.19 Pyraclostrobin 0.3 l/ha 250 g/l EC PR = Product rate; FC = Formulation concentration; FT = Formulation type; AT = Application time (BBCH);

(40) Application of Serenade Max alone and the combination of Serenade Max and pyraclostrobin results in a clear yield increase. Compared to the yield increase of the solo application of Serenade Max or pyraclostrobin solo, the yield increase when the combination of Serenade Max and pyraclostrobin were applied togehter is even higher than could have been expected according to Colby's formula. This yield increase, which is about double as high as expected, clearly demonstrates the synergistic effect of the compositions according to the invention on the plant's vigor and the plant's yield.

EXAMPLE 11: WINTER WHEAT

(41) Winter wheat was grown in the 2008/2009 growing season at Cagnicourt in France. The variety Premio was sown November 1st at a seeding rate of 125 kg/ha. The trial was setup in a randomized bloc design with 6 replications and a plot size of 22.5 m.sup.2. Bacillus subtilis QST 713 was applied to the winter wheat plants at beginning of shooting (growth stage 31/32, BBCH). The fungicide spray sequence consisted of an application of epoxiconazole at beginning of shooting followed by an application of epoxiconazole in combination with pyraclostrobin at flag leaf stage (growth stage 37/39). The B. subtilis QST 713 was applied using the commercial formulation Serenade Max (14.3%, WP with 7.3109 cfu/g). Epoxiconazole was applied alone at beginning of shooting as the commercially available formulation Opus (125 g/l, SC). The combination of epoxiconazole and pyraclostrobin was applied as a ready to use developmental Opera formulation (SE) containing 62.5 g/l epoxiconazole and 85 g/l pyraclostrobin. Product rates are given in table 11. Total spray volume for the foliar applications were 300 l/ha. At end of heading and beginning of flowering, respectively, the number ears per m.sup.2 was counted (table 11). The expected increase in number of ears per m.sup.2 by the combination of the active compounds was estimated using Colby's formula (Colby, S. R., Calculating synergistic and antagonistic responses of herbicide combinations, Weeds, 15, pp. 20-22, 1967) and compared with the observed increase as described above.

(42) TABLE-US-00012 TABLE 11 No. of Observed Expected Treatment PR FC FT AT Ears/m.sup.2 increase increase Untreated 375 Epoxiconazole 0.8 l/ha 125 g/l SC 31/32 394 19 Epoxiconazole + 2.0 l/ha 147.5 SE 37/39 Pyraclostrobin Serenade Max 3.0 kg/ha 14.3% WP 31/32 385 10 Serenade Max 3.0 kg/ha 14.3% WP 31/32 406 31 27 Epoxiconazole 0.8 l/ha 125 g/l SC 31/32 Epoxiconazole + 2.0 l/ha 147.5 SE 37/39 Pyraclostrobin PR = Product rate; FC = Formulation concentration; FT = Formulation type; AT = Application time (BBCH);

(43) In this example, again, Serenade Max improved the health of the wheat plants leading to increased number of ears per m.sup.2. An increased number of ears per m.sup.2 was also observed from the fungicide spray sequence. The increase observed from the combined application of Serenade Max and the fungicides was higher than expected according to Colby's formula, as is shown in table 11. This result clearly illustrates the synergistic effect of the combination of Bacillus subtilis QST 713 with azoles and strobilurins on the vigor and yield of the wheat plants when applied together in a tank mix or a spray sequence.

EXAMPLE 12: PEAS

(44) The active compounds were used applying commercially available formulations and diluted according to the concentrations/dose rates as stated in the respective data tables.

(45) Seed treatment was done to seeds of peas using the Hege Seed Treatment equipment in a sequential approach. Pyraclostrobin was applied with a volume of 850 ml slurry per 100 kg seeds. Afterwards the stated amount of Serenade MAX was dissolved in a total volume of 8.3 L water (amount for 100 kg seeds) and applied sequentially in ten steps with drying of the seeds inbetween. The compounds were used as commercial finished formulations and diluted with water to the stated concentration of the active compound.

(46) Seeds of peas were sown in soil (10 seeds/pot, 10 replications/treatment) and incubated in the greenhouse at 20 C. for 12 days. Plants were harvested and pooled per treatment and the plant fresh weight was determined.

(47) The expected plant fresh weights of active compound mixtures were determined using Colby's formula as defined above [R. S. Colby, Calculating synergistic and antagonistic responses of herbicide combinations, Weeds 15, 20-22 (1967)] and compared with the observed plant fresh weights.

(48) As can be seen in table 12, the measured fresh weight when Serenade Max is applied strongly increases compared to the untreated control plants. In case a mixture according to the present invention is applied such as the combination of Serenade Max and pyraclostrobin, the fresh weight as an indicator for the plant's vigor and yield is increased even synergistically.

(49) TABLE-US-00013 TABLE 12 Dose rate Fresh Calculated (g/100 weight efficacy according Treatment kg seed) (g) to Colby (%) Untreated 55 Pyraclostrobin 5 47 (200 g/L, FS) Serenade Max 173 64 (14.3% B. subtilis, WP) Pyraclostrobin + 5 92 81 Serenade Max 173