Bacillus having effect of inducing corn to resist northern corn leaf blight (<i>Exserohilum turcicum</i>) of corn and application

Abstract

The disclosure relates to bacillus having an effect of inducing corn to resist Northern leaf blight and applications of the bacillus. The bacillus having an effect of inducing corn to resist Northern corn leaf blight is Bacillus megaterium which was deposited on Apr. 16, 2019 with a deposit number of CGMCC No. 17573. The Bacillus megaterium of the disclosure can induce corn to generate resistance to Northern corn leaf blight during seed germination and corn growth to activate the immune response of corns and stimulate immune control of Northern leaf blight of corn. The method described herein is long in coming does not pollute environment and soil, is a green, simplified and new control method of Northern leaf blight of corn, and has wide application and popularization prospect.

Claims

1. A fermentation broth comprising Bacillus megaterium and liquid fermentation culture medium, wherein the Bacillus (B.) megaterium is deposited with the General Microbiology Center of China Microbial Species Conservation Management Committee (CGMCC), Beijing, China, and accorded Deposit No. 17573, and wherein the fermentation broth induces resistance of corn to Northern leaf blight.

2. The fermentation broth of claim 1, wherein the fermentation broth further comprises beef extract, peptone, sucrose, and/or water.

3. The fermentation broth of claim 1, wherein the fermentation broth comprises B. megaterium at a concentration of at least about 1×10.sup.7 CFU/ml.

4. The fermentation broth of claim 3, wherein the fermentation broth comprises B. megaterium at a concentration of about 1×10.sup.7 CFU/ml, about 1×10.sup.8 CFU/ml, about 1×10.sup.9 CFU/ml, or about 1×10.sup.10 CFU/ml.

5. A method of preparing the fermentation broth of claim 1, wherein the method comprises culturing a single strain of B. megaterium to obtain an activated B. megaterium, inoculating a liquid fermentation culture medium with the activated B. megaterium, and growing the activated B. megaterium in the liquid fermentation culture medium to a concentration of at least about 1×10.sup.7 CFU/ml thereby obtaining the fermentation broth of claim 1.

6. The method of claim 5, wherein culturing the single strain of B. megaterium to obtain activated B. megaterium comprises growing B. megaterium on a solid bacterium medium.

7. The method of claim 5, wherein the liquid fermentation culture medium comprises beef extract, peptone, sucrose, and/or water.

8. The method of claim 7, wherein growing the activated B. megaterium comprises growing at a temperature of about 25 to 28° C. for about two or three days in the liquid fermentation culture medium.

9. The method of claim 8, wherein growing the activated B. megaterium comprises shaking the in the liquid fermentation culture medium with a rotating speed of 150 rpm.

10. The method of claim 5, wherein the method comprises growing the activated B. megaterium to a concentration of about 1×10.sup.7 CFU/ml, about 1×10.sup.8 CFU/ml, about 1×10.sup.9 CFU/ml, or about 1×10.sup.10 CFU/ml.

11. A method of inducing resistance of corn to Northern leaf blight comprising treating corn seeds with the fermentation broth of claim 1 and planting the treated corn seeds.

12. The method of claim 11, wherein treating the corn seeds comprises coating the corn seeds with the fermentation broth with kneading and drying the coated seed in the shade prior to planting.

13. The method of claim 12, wherein the coating is carried out at a mass ratio of 40 grams of the corn seeds to 1 gram of the fermentation broth.

Description

DESCRIPTION OF THE EMBODIMENTS

(1) In order to further describe but not limit the disclosure in detail, the following examples are given. The terms “a,” “an,” “the” and similar referents used in the context of describing the claimed subject matter (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

(2) As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of” The transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. In particular embodiments, lack of a material effect is evidenced by lack of a statistically-significant reduction in the embodiment's ability to induce resistance of corn to Northern leaf blight.

(3) When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±15% of the stated value; ±10% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; ±1% of the stated value; or ±any percentage between 1% and 20% of the stated value.

(4) The disclosure describes fermentation broth comprising isolated Bacillus megaterium and liquid fermentation culture medium, wherein the Bacillus (B.) megaterium is deposited with the General Microbiology Center of China Microbial Species Conservation Management Committee (CGMCC), Beijing, China, on Apr. 16, 2019, and accorded Deposit No. 173573, and wherein the fermentation broth induces resistance of corn to Northern leaf blight. In embodiments, the fermentation broth also includes beef extract, peptone, sucrose, and/or water. The concentration of B. megaterium in the fermentation broth includes at least 1×10.sup.7 CFU/ml. In embodiments, the concentration of B. megaterium in the fermentation broth is about 1×10.sup.7 CFU/ml, about 1×10.sup.8 CFU/ml, about 1×10.sup.9 CFU/ml, or about 1×10.sup.10 CFU/ml. In embodiments, the disclosure describes a composition including the fermentation broth.

(5) The disclosure also describes a method of preparing the fermentation broth described herein, wherein the method includes culturing a single strain of B. megaterium to obtain an activated B. megaterium, inoculating a liquid fermentation culture medium with the activated B. megaterium, and growing the activated B. megaterium in the liquid fermentation culture medium to a concentration of at least about 1×10.sup.7 CFU/ml thereby obtaining the fermentation broth. In embodiments, the liquid fermentation culture medium includes beef extract, peptone, sucrose, and/or water. In embodiments, culturing the single strain of B. megaterium to obtain activated B. megaterium includes growing B. megaterium on a solid bacterium medium. In embodiments, the method described herein includes growing the activated B. megaterium at a temperature of 25 to 28° C. for about two or three days in the liquid fermentation culture medium using a rotary shaker at a speed of 150 rpm (revolutions per minute). In embodiments, the method includes growing the activated B. megaterium to a concentration of about 1×10.sup.7 CFU/ml, about 1×10.sup.8 CFU/ml, about 1×10.sup.9 CFU/ml, or about 1×10.sup.10 CFU/ml.

(6) The disclosure also describes a method of using the fermentation broth described herein or a composition comprising the fermentation broth described herein to induce resistance of corn to Northern leaf blight. The method of inducing resistance of corn to Northern leaf blight includes treating corn seeds with the fermentation broth and sowing the treated corn seeds or planting the treated corn seeds in the ground. In embodiment, treating the corn seeds includes coating the corn seeds with the fermentation broth with kneading followed by drying the coated seed in the shade. In embodiments, coating the corn seeds is carried out at a mass ratio of 40 grams of corn seeds to 1 gram of fermentation broth.

EXAMPLES

Example 1

(7) Preparation of Bacillus Fermentation Broth Having an Effect of Inducing Corn to Resist Northern Leaf Blight.

(8) The bacillus having an effect of inducing corn to resist Northern leaf blight is Bacillus megaterium. The Bacillus megaterium is classified as Bacillus megaterium Sneb85 and was deposited on Apr. 16, 2019 with deposit number CGMCC No. 17573, at the General Microbiology Center of China Microbial Species Conservation Management Committee, and conservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 courtyard, Beichen West Road, Chaoyang District, Beijing.

(9) The method of culturing and activating the bacillus having an effect of inducing corn to resist Northern leaf blight of corn may include the following steps:

(10) (1) Activation and Culture of Strains

(11) Single strain was obtained by plate culture of Bacillus megaterium for 3 days (d) at 28° C. using a solid bacterium medium.

(12) The formula of the bacterial culture medium was as follows: beef extract peptone agar culture medium (NA): beef extract 3 g, peptone 10 g, sucrose 10 g, agar 17-20 g, distilled water 1000 mL, and pH 7.0.

(13) (2) Liquid Fermentation Culture

(14) The strains obtained after activation culture were inoculated into a liquid fermentation medium, the fermentation temperature was 25-28° C., the rotating speed of a shaking table was 150 r.Math.min.sup.−1 and the fermentation time was 2-3 d.

(15) The formula of the liquid fermentation medium was as follows: beef extract 3 g, peptone 10 g, sucrose 10 g, distilled water 1000 mL, and pH 7.0.

(16) The concentration of the fermentation broth of the obtained Bacillus megaterium reached 1×10.sup.7 CFU.Math.ml.sup.−1 or more.

Example 2

(17) Field Effect Test for Controlling Northern Leaf Blight of Corn with Bacillus megaterium Sneb85 Fermentation Broth

(18) Test site: Corn experimental field, Kangping County, Shenyang City, Liaoning Province. The Northern leaf blight of corn was serious in this experimental field last year;

(19) Corn variety: Zhengdan958, and seeds were purchased on the market.

(20) Treatment:

(21) Treatment 1 was seed coating treatment with Bacillus megaterium Sneb85 fermentation broth;

(22) Treatment 2 was uncoated (CK1) corn seeds;

(23) Treatment 3 was coating with beef extract peptone liquid culture medium (CK2);

(24) Coating treatment method: corn seeds were coated with the prepared Bacillus megaterium fermentation broth having adjusted concentration in example 1; the surfaces of corn seeds were first disinfected with 5.0% sodium hypochlorite solution for 5 min, then washed with sterile water for 3-5 times, and dried. Coating treatment was carried out in a mass ratio of liquid to seeds of 1:40 (namely, 40 g of seeds corresponds to 1 g of fermentation broth). In order to make the coating uniform, the seeds were kneaded in the coating process, then dried in the shade, and then seeded.

(25) Field Control Effect:

(26) At the mature stage of corn plants, the disease progression of Northern leaf blight of corn was investigated and the control effect was calculated. The results showed that the disease index of Northern leaf blight of corn treated with an Sneb85 fermentation product was less than that for the control, and the field control effect was 58.94% in Kangping City, Shenyang City, Liaoning Province. There was significant difference between the coated corn and the untreated corn. It can be seen that corns coated with Sneb85 fermentation broth before sowing can produce resistance to Northern leaf blight of corn at the maturity stage, and the disease resistance effect of corns is induced to be relatively stable, showing a good control effect.

(27) TABLE-US-00001 TABLE 1 field control effect of corn seeds coated with Sneb85 fermentation broth product Treatment Disease index Control effect Sneb85 35.6 58.94% CK1 (uncoated) 93.3 — CK2 (beef extract peptone 86.7 — liquid culture medium)

(28) All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entireties as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

(29) The subject matter described above is provided by way of illustration only and should not be construed as limiting. Various modifications and changes may be made to the subject matter described herein without following the example embodiments and applications illustrated and described, and without departing from the true spirit and scope of the present disclosure, which is set forth in the following claims.