BIOLOGICAL SEED-COATING AGENT (SCA) INCLUDING FERMENTATION BROTH OBTAINED BASED ON CO-CULTIVATION OF TRICHODERMA AND BACILLUS SEQUENTIALLY INOCULATED, AND PREPARATION METHOD THEREOF

Abstract

A biological seed-coating agent (SCA) including a fermentation broth obtained based on co-cultivation of Trichoderma and Bacillus sequentially inoculated, and a preparation method thereof are provided. According to a principle of synthesis biology, Trichoderma that induces disease resistance and stress resistance and Bacillus that antagonizes pathogens and promotes crop growth are inoculated in stages for co-fermentation to prepare a microbial co-culture solution. The microbial co-culture solution is mixed with diatomaceous earth and brassinolide according to a specified ratio to prepare a biological SCA in a dosage form of a powder. The biological SCA includes high contents of spores and antagonistic and growth-promoting substances.

Claims

1. A fermentation broth obtained based on a co-cultivation of Trichoderma and Bacillus sequentially inoculated, wherein the Thrichoderma is first inoculated at an amount 1% v/v to 2% v/v of a total fermentation amount and cultivated at 25 C. to 28 C. for 35 h to 45 h, then the Bacillus is inoculated at an amount 2.0% v/v to 3.0% v/v of the total fermentation amount, and the Trichoderma and the Bacillus are co-cultivated for 48 h at 28 C. to 30 C.

2. A biological seed-coating agent (SCA), comprising the fermentation broth obtained based on the co-cultivation of the Trichoderma and the Bacillus sequentially inoculated according to claim 1.

3. The biological SCA according to claim 2, wherein in the fermentation broth obtained based on the co-cultivation of the Trichoderma and the Bacillus, a content of a marker alamethicin is 0.04 g/L to 0.05 g/L, a content of indoleacetic acid (IAA) is 900 g/L to 1,000 g/L, a content of a Trichoderma spore is 1.010.sup.8 cfu/L to 9.010.sup.8 cfu/L, and a content of a Bacillus spore is 1.010.sup.9 cfu/L to 9.010.sup.9 cfu/L.

4. The biological SCA according to claim 2, wherein the biological SCA is in a dosage form of a dry powder.

5. The biological SCA according to claim 2, comprising: 20% w/w to 30% w/w of microbial biomass obtained after the co-cultivation of the Trichoderma and the Bacillus, 40% w/w to 60% w/w of a diatomaceous earth, and 12% w/w to 20% w/w of brassinolide; wherein the brassinolide is in a form of a 0.1% solution.

6. A preparation method of the biological SCA according to claim 2, comprising the following steps: S1: preparing the fermentation broth obtained based on the co-cultivation of the Trichoderma and the Bacillus; S2: mixing the fermentation broth obtained based on the co-cultivation of the Trichoderma and the Bacillus with a diatomaceous earth and brassinolide; and S3: detecting a quality of the biological SCA in a dosage form of a powder.

7. The preparation method of the biological SCA according to claim 6, wherein the Trichoderma is Trichoderma aureoviride (T. aureoviride) SG3403, and the Bacillus is Bacillus subtilis (B. subtilis) B22.

8. The preparation method of the biological SCA according to claim 6, wherein S1 specifically comprises: inoculating the Trichoderma into a potato dextrose agar (PDA) medium, cultivating the PDA medium inoculated with the Trichoderma at 28 C. for 5 d, punching the PDA medium inoculated with the Trichoderma with a 5 mm puncher to obtain a resulting Trichoderma disc, inoculating the resulting Trichoderma disc into a potato dextrose (PD) medium, and cultivating the resulting Trichoderma disc in the PD medium at 28 C. for 3 d to obtain a resulting Trichoderma seed culture; inoculating the resulting Trichoderma seed culture into a co-culture medium in a fermentation tank, and conducting A fermentation for 35 h to 45 h; inoculating the Bacillus into a lysogeny broth agar (LBA) medium, cultivating the LBA medium inoculated with the Bacillus at 30 C. for 2 d, punching the LBA medium inoculated with the Bacillus with the 5 mm puncher to obtain a resulting Bacillus disc, inoculating the resulting Bacillus disc into a lysogeny broth (LB) medium, and cultivating the resulting Bacillus disc in the LB medium for 2 d to obtain a resulting Bacillus seed culture; and inoculating the resulting Bacillus seed culture into the co-culture medium with a Trichoderma fermentation broth, and further conducting the fermentation for 45 h to 50 h.

9. The preparation method of the biological SCA according to claim 8, wherein the co-culture medium in the fermentation tank comprises: 20 g/L of molasses, 20 g/L of a yeast extract powder, and 20 g/L of a corn flour, and a pH of the co-culture medium is adjusted with sodium hydroxide to 5.8 to 6.0.

10. The preparation method of the biological SCA according to claim 8, wherein the resulting Trichoderma seed culture is inoculated at an amount 1.0% to 2.0% of a total fermentation amount, and the resulting Bacillus seed culture is inoculated at an amount 2.0% to 3.0% of the total fermentation amount; at an end of the fermentation, a dissolved oxygen (DO) level rebounds to 100% of a DO level at a start of the fermentation, a pH is 7.3, a rotational speed is 120 rpm, a co-fermentation time is 45 h to 50 h, a Bacillus spore formation rate is 85%, and a Trichoderma chlamydospore formation rate is 90%; and indexes for evaluating an antagonism and a growth promotion of the biological SCA obtained through a co-fermentation mode of the Trichoderma and the Bacillus are as follows: a content of the Trichoderma chlamydospore is 1.010.sup.8 cfu/g to 2.010.sup.8 cfu/g, a content of the Bacillus spore is 3.0-10.sup.9 cfu/g to 5.010.sup.9 cfu/g, a content of antimicrobial peptaibol alamethicin is 0.1 g/g to 0.5 g/g, and a content of IAA is 2,000 g/g to 2,500 g/g.

11. The preparation method of the biological SCA according to claim 6, wherein in the fermentation broth obtained based on the co-cultivation of the Trichoderma and the Bacillus, a content of a marker alamethicin is 0.04 g/L to 0.05 g/L, a content of indoleacetic acid (IAA) is 900 g/L to 1,000 g/L, a content of a Trichoderma spore is 1.010.sup.8 cfu/L to 9.010.sup.8 cfu/L, and a content of a Bacillus spore is 1.010.sup.9 cfu/L to 9.010.sup.9 cfu/L.

12. The preparation method of the biological SCA according to claim 6, wherein the biological SCA is in a dosage form of a dry powder.

13. The preparation method of the biological SCA according to claim 6, wherein the biological SCA comprises: 20% w/w to 30% w/w of microbes obtained after the co-cultivation of the Trichoderma and the Bacillus, 40% w/w to 60% w/w of a diatomaceous earth, and 12% w/w to 20% w/w of brassinolide; wherein the brassinolide is in a form of a 0.1% solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows the influence of SCAs on a wheat emergence rate; and

[0019] FIG. 2 shows the control effects of SCAs obtained based on co-cultivation of Trichoderma and Bacillus for Fusarium head blight (FHB).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The present disclosure is described in detail below with reference to specific examples. The following examples will help those skilled in the art to further understand the present disclosure, but do not limit the present disclosure in any way. It should be noted that those of ordinary skill in the art can further make several variations and improvements without departing from the idea of the present disclosure. These all fall within the protection scope of the present disclosure.

Example 1

[0021] A biological SCA obtained based on co-fermentation of Trichoderma and Bacillus was prepared, and specific steps were as follows:

[0022] 1. Plate cultivation of Trichoderma and Bacillus: Trichoderma was inoculated into a PDA medium and cultivated at 28 C. for 3 d. Bacillus was inoculated into an LBA medium and cultivated at 30 C. for 2 d.

[0023] PDA medium: 200 g of a potato was taken, peeled, diced, and then steamed; a resulting supernatant was taken, 20 g of glucose and 20 g of an agar powder were added, and a resulting mixture was diluted with deionized water to 1 L, dispensed into 250 mL erlenmeyer flasks, and autoclaved at 121 C. for 30 min.

[0024] PD medium: 200 g of a potato was taken, peeled, diced, and then steamed; a resulting supernatant was taken, 20 g of glucose was added, and a resulting mixture was diluted with deionized water to 1 L, dispensed into 250 mL erlenmeyer flasks, and autoclaved at 121 C. for 30 min.

[0025] LBA medium: 5 g of a yeast extract, 10 g of sodium chloride, 10 g of tryptone, and 20 g of an agar powder were mixed, a resulting mixture was diluted with deionized water to 1 L and dispensed into 250 mL erlenmeyer flasks, a pH was adjusted with NaOH to 7.0, and a resulting medium was autoclaved at 121 C. for 20 min.

[0026] LB medium: 5 g of a yeast extract, 10 g of sodium chloride, and 10 g of tryptone were mixed, a resulting mixture was diluted with deionized water to 1 L and dispensed into 250 mL erlenmeyer flasks, a pH was adjusted with NaOH to 7.0, and a resulting medium was autoclaved at 121 C. for 20 min.

[0027] 2. Preparation of a Trichoderma-Bacillus co-fermentation medium: 20 g/L corn flour, 20 g/L yeast extract powder, and 20 g/L molasses were mixed, and a pH of a resulting medium was adjusted with sodium hydroxide to 6.0 to 6.5.

[0028] 3. Control of a sequential inoculation and co-fermentation process: In a 300 L fermentation tank, a fermentation medium was sterilized at 121 C. for 30 min; after the sterilization was completed, a Trichoderma seed culture was inoculated into a co-culture medium (1% v/v), and fermentation was conducted for 40 h with a ventilation of 1 vvm, a rotational speed of 180 rpm, a temperature of 28 C., and a pH adjusted by ammonia water to 4.5 to 5.5, a Bacillus seed culture was inoculated into a resulting fermentation broth (2.0% v/v), and co-fermentation was conducted for 48 h; and when a Bacillus spore formation rate reached 85% and a Trichoderma chlamydospore formation rate reached 90%, a resulting fermentation broth was discharged.

[0029] 4. Detection of a quality of the co-fermentation: Contents of IAA and alamethicin in each of a Trichoderma-Bacillus co-fermentation supernatant and a Trichoderma fermentation supernatant were detected. In the co-fermentation supernatant, an alamethicin content was 0.1039 g/L and an IAA content was 2159.53 g/L, which were significantly higher than those in a supernatant obtained after fermentation of Trichoderma alone and a supernatant obtained after fermentation of Bacillus alone; and contents of the antimicrobial peptaibol (alamethicin) and IAA in a co-fermentation broth were significantly higher than those in a single-strain fermentation broth (as shown in Table 1).

[0030] 5. Preparation of a dry-powder SCA based on co-cultivation of Trichoderma and Bacillus After the co-fermentation was completed, enriched mixed spores were separated through SLS by a centrifuge, the enriched mixed spores, diatomaceous earth, and 0.1% brassinolide were thoroughly mixed according to a mass ratio of 3:6:1, oven-dried in a spore dryer (at 45 C.) until a moisture content was 5%, and sieved through an 80-mesh sieve to obtain a Trichoderma-Bacillus-brassinolide dry-powder biological SCA.

TABLE-US-00001 TABLE 1 Analysis of contents of alamethicin and IAA in a supernatant obtained after co-fermentation of Trichoderma and Bacillus and a supernatant obtained after fermentation of Trichoderma alone Component Component content, Fermentation type type g/L Co-fermentation of Trichoderma and IAA 2159.53 Bacillus Alamethicin 0.1039 Fermentation of Trichoderma alone IAA 23.82 Alamethicin 0.0865 Fermentation of Bacillus alone IAA 620.45 Alamethicin 0.0120

Example 2

[0031] A growth-promoting effect of the biological SCA obtained based on co-cultivation of Trichoderma and Bacillus was detected in the field.

[0032] Test site: Henan Academy of Agricultural Sciences, Zhumadian

[0033] Test Steps:

[0034] 1. Preparation of field experimental materials

[0035] Variety: Jimai 22

[0036] Biological SCA: A dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus

[0037] Chemical SCAs: 90% thiamethoxam, 8.5% tebuconazole, and a 1.5% film-forming agent (control)

[0038] 2. The chemical SCAs (agent-to-seed ratio: 1:200) and the dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus (agent-to-seed ratio: 1:100) each was used for seed dressing.

[0039] 3. 20 d after the seeds were sown, the influence of each treatment on wheat emergence was investigated.

[0040] 4. There was a very significant difference in emergence rate between the biological SCA and the blank control; and the influence of the biological SCA on a wheat emergence rate was similar to or better than the influence of the chemical SCAs on a wheat emergence rate (as shown in FIG. 1, it should be noted that data in FIG. 1 were an average of 5 replicates and a standard error thereof. **: one-way analysis of variance (ANOVA) indicated a very significant difference, 0.01<P<0.001).

Example 3

[0041] A control effect of the dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus for soil-borne diseases of corn was verified in the field.

[0042] Test site. A farm of Shenyang Agricultural University

[0043] Test Steps:

[0044] 1. Field experimental materials:

[0045] 2. Variety: Zhengdan 958

[0046] 3. Biological SCA: A dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus, which was used with an agent-to-seed ratio of 1:100.

[0047] 4. Chemical SCAs: 90% thiamethoxam, 8.5% tebuconazole, and a 1.5% film-forming agent, which each was used with an agent-to-seed ratio of 1:200.

[0048] 5. Sowing mode: Dibbling, 4,000 plants/mu.

[0049] 6. Pathogen inoculation: When sowing, Fusarium graminearum (F graminearum) and Rhizoctonia solani (R. solani) each was inoculated into the soil at an inoculum size of 5%, in mid-to-late July, a lower leaf sheath was inoculated by the wheat grain-cultured R solani; and in early August, a fruit ear was inoculated with Fusarium verticilloides (F. verticilloides) by a toothpick method.

[0050] Control Effect Investigation and Production Test.

[0051] 1. At a milk ripening stage, 5 diagonal points were investigated for each treatment, 4 rows were randomly selected for each point, and 50 plants were randomly investigated for each row. A disease incidence (including a lodging rate) of each treatment was investigated, and a field control effect of stalk rot was statistically analyzed.

[0052] 2. At a milk ripening stage, 5 diagonal points were investigated for each treatment, 4 rows were randomly selected for each point, and 50 plants were randomly investigated for each row. A disease incidence and a disease level per plant for each treatment were investigated, and a disease index and a field control effect for corn sheath blight were statistically analyzed.

[0053] Disease indexes of corn sheath blight were shown in Table 2.

TABLE-US-00002 TABLE 2 Grading criteria for com sheath blight Disease rank Symptom description 1 The 4th leaf sheath under a fruit ear and lower leaf sheaths are diseased. 3 The 3rd leaf sheath under a fruit ear and lower leaf sheaths are diseased. 5 The 2nd leaf sheath under a fruit ear and lower leaf sheaths are diseased. 7 The 1st leaf sheath under a fruit ear and lower leaf sheaths are diseased 9 A fruit ear and upper leaf sheaths are diseased.

[0054] 3. At a milk ripening stage, 4 rows of each treatment were randomly selected, and 10 ears were randomly selected from each row. Bracts were peeled off, a disease level of each ear was investigated and recorded, and a disease index and a control effect for the ear were statistically analyzed. Grading criteria for disease incidence of corn ears were shown in Table 3.

TABLE-US-00003 TABLE 3 Grading criteria for corn ear rot Disease rank Symptom description 1 A diseased area is 0% to 1% of a total area of a fruit ear. 3 A diseased area is 2% to 10% of a total area of a fruit ear. 5 A diseased area is 11% to 25% of a total area of a fruit ear. 7 A diseased area is 26% to 50% of a total area of a fruit ear. 9 A diseased area is 51% to 100% of a total area of a fruit ear.

[0055] 4. Yield Measurement Method

[0056] At a harvest stage, 5 points were selected for each treatment to investigate 10 m.sup.2 of plants. 10 to 20 plants were selected from each point and investigated, and a number of corn ears, a thousand-grain weight, and a moisture content in grains at harvest each was measured. A yield of each plot was recorded, a yield per acre was calculated, and a yield increase rate in contrast to a blank control (in the blank control, neither the dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus or the chemical SCA was added) was calculated.

[0057] 5. Field test results showed that the dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus could effectively reduce the occurrence of stalk rot and significantly increase the yield (as shown in Table 4).

TABLE-US-00004 TABLE 4 Corn stalk rot control effect and yield increase effect of the dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus Number Control of Yield Incidence effect ears/10 Thousand- Yield increase Treatment (%) (%) m.sup.2 grain weight/g kg/mu rate (%) Dry-powder 0.93 0.46a 74.86 53.67a 372.80 15.55a 736.80 9.72a 19.28 SCA obtained based on co- cultivation of Trichoderma and Bacillus Chemical 1.39 0.80a 62.43 54.33a 350.10 31.65a 712.95 3.11a 15.42 SCA Blank 3.70 0.46b 53.33a 317.10 31.65a 617.70 9.95b control

[0058] The dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus exhibited an excellent control effect for corn sheath blight and an excellent yield increase effect (as shown in Table 5 and Table 6).

TABLE-US-00005 TABLE 5 Corn sheath blight control effect and yield increase effect of the dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus (plot 1) Number Control of Yield Disease effect ears/10 Thousand- Yield increase Treatment index (%) m.sup.2 grain weight/g kg/mu rate (%) Dry-powder 33.81 5.67ab 48.80 59.33a 352.67 7.68a 746.50 11.33a 10.31 SCA obtained based on co- cultivation of Trichoderma and Bacillus Chemical 42.01 1.60b 36.38 58.33a 349.23 19.83a 720.15 3.11a 6.43 SCA Blank 66.03 3.06c 56.33a 317.03 16.78a 676.73 10.51a control

[0059] The SCA obtained based on co-cultivation of Trichodermna and Bacillus could improve the ear rot control effect and yield increase rate (Table 6).

TABLE-US-00006 TABLE 6 Corn sheath blight control effect and yield increase effect of the dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus (plot 2) Number Control of Yield Disease effect ears/10 Thousand- Yield increase Treatment index (%) m.sup.2 grain weight/g KG/667 m.sup.2 rate (%) Dry-powder 23.81 0.58a 36.86 59.37a 363.67 9.14a 757.94 9.40a 16.93 SCA obtained based on co- cultivation of Trichoderma and Bacillus Chemical 26.06 1.65a 30.89 57.67a 352.67 14.50a 726.67 8.39a 12.10 SCA Blank 37.71 0.58b 57.67a 328.83 8.20a 648.21 7.67b control

Example 4

[0060] Control effects of SCAs obtained based on co-cultivation of Trichoderma and Bacillus for FHB

[0061] 1. Test site: A farm of Shanghai Jiao Tong University (Minhang Campus)

[0062] Test materials: Bacillus subtilis (B. subtilis) BS-22, Trichoderma aureoviride (T. aureoviride) SG3403, F. graminearum, Rhizoctonia solani, and wheat variety Ningmai 13.

[0063] 2. Preparation of dry-powder and liquid SCAs: A microbial co-culture solution, a film-forming agent, and water were mixed in a ratio of 8:1:1 to obtain a liquid SCA. A microbial co-culture solution and talcum powder were mixed in a ratio of 1:5 (ml:g) and then oven-dried in a 40 C. oven for 5 h to 6 h to obtain a dry-powder SCA.

[0064] 3. F. graminearum was cultivated in a shaking flask for about 7 d (210.sup.8 cfu/L).

[0065] 4. Pot soil was prepared with an organic seedling medium and vermiculite in a ratio of 3:1 (V/V). The prepared pot soil was filled into flower pots (highdiameter: 27 cm23 cm). Then coated wheat seeds (Ningmai 13) were sown, with 10 seeds per pot and a covered soil thickness of 1 cm.

[0066] 5. 14 d to 21 d after sowing, three wheat plants were randomly selected from each pot and then tested for the plant height and fresh weight. At a wheat flowering stage, an F graminearum spore suspension inoculum was diluted 50-fold (110.sup.7 cfu/L) and then sprayed onto wheat plants, and the wheat plants were highly moisturized with a humidifier. 20 d later, the incidence of FHB was observed, and the disease index and control effect were investigated. FIG. 1 shows the influence of treatment of different SCAs on a wheat emergence rate.

[0067] 6. FIG. 2 shows the control effects of SCAs obtained based on co-cultivation of Trichoderma and Bacillus for FHB. It can be seen from FIG. 2 that control effects of treatments with a microbial agent obtained after fermentation of Bacillus alone, a microbial agent obtained after fermentation of Trichoderma alone, and the dry-powder SCA obtained after co-fermentation of Trichoderma and Bacillus reach 28.4%, 13.4%, and 66.2%, respectively (namely, the bars d, cd, and b for the dry-powder SCA in the figure), indicating that a control effect of the treatment with the dry-powder SCA for FHB is significantly higher than that of other treatments; and

[0068] control effects of treatments with a microbial agent obtained after fermentation of Bacillus alone, a microbial agent obtained after fermentation of Trichoderma alone, and the liquid SCA obtained after co-fermentation of Trichoderma and Bacillus reach 10.9%, 20.4%, and 45.2%, respectively (namely, the bars c, d, and a for the liquid SCA in the figure), indicating that a control effect of the treatment with the liquid SCA for FHB is significantly higher than that of other treatments.

[0069] It should be noted that B in FIG. 2 represents a microbial agent obtained after fermentation of Bacillus alone; T represents a microbial agent obtained after fermentation of Trichoderma alone; and BT represents an SCA obtained after co-fermentation of Trichoderma and Bacillus. For significance differences of the above control effects: P<0.05.

[0070] Compared with the prior art, one or more embodiments of the present disclosure have the following beneficial effects.

[0071] 1. A fermentation broth obtained after co-fermentation of Trichoderma and Bacillus has higher contents of alamethicin and IAA than a fermentation broth obtained after fermentation of Trichoderma alone. It should be noted that, unlike the co-cultivation used in the prior art, co-fermentation is adopted in the present disclosure, which can overcome an antagonism or competition effect between microorganisms during co-cultivation to cause growth of a specified microorganism.

[0072] 2. The dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus has a more stable spore content than the liquid SCA.

[0073] 3. The dry-powder SCA obtained based on co-cultivation of Trichoderma and Bacillus has more excellent functions of controlling diseases and promoting crop growth than a microbial agent obtained based on fermentation of Trichoderma or Bacillus alone and a simple combination of microbial agents obtained based on separate fermentation of the two.

[0074] The present disclosure is not limited to the above specific implementations, and a person skilled in the art can make various variations or modifications within the scope of the claims without affecting the essence of the present disclosure.

[0075] The specific examples of the present disclosure are described above. It should be understood that the present disclosure is not limited to the above specific implementations, and a person skilled in the art can make various variations or modifications within the scope of the claims without affecting the essence of the present disclosure. The examples of the present disclosure and features in the examples may be arbitrarily combined with each other in a non-conflicting situation.