CN114752530B - Composite antibacterial agent for inhibiting powdery mildew of rubber tree - Google Patents

Composite antibacterial agent for inhibiting powdery mildew of rubber tree Download PDF

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CN114752530B
CN114752530B CN202210476849.0A CN202210476849A CN114752530B CN 114752530 B CN114752530 B CN 114752530B CN 202210476849 A CN202210476849 A CN 202210476849A CN 114752530 B CN114752530 B CN 114752530B
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bacillus subtilis
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lactobacillus plantarum
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CN114752530A (en
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刘文波
秦春秀
缪卫国
李潇
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Hainan University
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Abstract

The invention provides a compound bacteriostatic agent for inhibiting rubber tree powdery mildew, which consists of bacillus subtilis fermentation liquor A, bacillus subtilis fermentation liquor B, lactobacillus plantarum fermentation liquor and chitosan, wherein the total viable count of the bacillus subtilis and the lactobacillus plantarum is more than or equal to 3 multiplied by 10 7 cfu/ml, wherein the concentration of chitosan is 0.1-0.3wt%, the ratio of viable count of bacillus subtilis to lactobacillus plantarum is (5-15):1-10, wherein betel nut powder is added in the fermentation medium for preparing bacillus subtilis fermentation broth A by 0.1-0.5wt%, and semen Alpiniae powder is added in the fermentation medium for preparing bacillus subtilis fermentation broth B by 0.1-0.5wt%. The composite bacteriostatic agent disclosed by the invention belongs to active ingredients with different action mechanisms, has a better inhibition effect on powdery mildew of rubber trees under the synergistic effect, and can effectively prevent and treat the powdery mildew of the rubber trees.

Description

Composite antibacterial agent for inhibiting powdery mildew of rubber tree
Technical Field
The invention relates to a composite antibacterial agent, in particular to a composite antibacterial agent for inhibiting powdery mildew of rubber trees.
Background
Natural rubber is an important national strategic material, and the planting area of the natural rubber exceeds 113 ten thousand hm 2 Wherein, the planting area of Hainan and Yunnan accounts for more than 95 percent of the whole country. Powdery mildew caused by obligate parasitic rubber tree powdery mildew is the most serious disease of the rubber tree, and is also the biological disaster most concerned by the rubber farmers in Hainan and Yunnan. Pathogenic bacteria only infect tender leaves, buds and inflorescences of the rubber tree, so that diseases are mainly popular in spring of a large number of tender leaves of the rubber tree. The disease was found in Hainan in 1951 in China, and the disease was first pandemic in Hainan in 1959, and since then occurred annually and was severeThe occurrence frequency of the disease years is also higher and higher. When the disease is serious, the glue yield can be reduced by 30-45%. Therefore, how to prevent powdery mildew of rubber tree is a focus of attention.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a composite antibacterial agent for inhibiting powdery mildew of rubber trees, which can effectively prevent and treat the powdery mildew of the rubber trees.
The first aspect of the invention provides a composite bacteriostatic agent for inhibiting powdery mildew of rubber tree, which consists of bacillus subtilis fermentation liquor A, bacillus subtilis fermentation liquor B, lactobacillus plantarum fermentation liquor and chitosan, wherein the total viable count of bacillus subtilis and lactobacillus plantarum in the composite bacteriostatic agent is more than or equal to 3 multiplied by 10 7 cfu/ml, wherein the concentration of chitosan is 0.1-0.3wt%, the ratio of viable count of bacillus subtilis to lactobacillus plantarum is (5-15):1-10, wherein betel nut powder is added in the fermentation medium for preparing bacillus subtilis fermentation broth A by 0.1-0.5wt%, and semen Alpiniae powder is added in the fermentation medium for preparing bacillus subtilis fermentation broth B by 0.1-0.5wt%.
Preferably, 0.3wt% of betel nut powder is added to the fermentation medium for preparing the bacillus subtilis broth a.
Preferably, 0.25wt% of katsumade cardamom powder is added to the fermentation medium for preparing the bacillus subtilis broth B.
Preferably, the ratio of the number of living bacteria of the bacillus subtilis to the lactobacillus plantarum is (8-12):3-7.
The fermentation medium for preparing the lactobacillus plantarum fermentation broth is not particularly limited herein, and any medium may be used as long as it is a medium capable of fermenting and culturing lactobacillus plantarum, and those skilled in the art can reasonably select the medium according to the nature of the strain.
Wherein, the fermentation medium for preparing the bacillus subtilis fermentation broth A is a basic fermentation medium to which betel nut powder is added, and the invention is not particularly limited herein, and any medium can be used as long as it can ferment and culture bacillus subtilis, and those skilled in the art can perform the fermentation cultureSo as to reasonably select according to the nature of the strain. For example, the composition of the basal fermentation medium may be: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05 to 0.1wt%, peanut oil 0.1 to 0.5wt%, and balance water, initial pH7.5, the composition of the fermentation medium for preparing the bacillus subtilis broth A in the invention can be: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05-0.1wt%, peanut oil 0.1-0.5wt%, betel nut powder 0.1-0.5wt%, and water for the rest, the pH is 7.5.
In a specific embodiment of the invention, the fermentation medium for preparing the bacillus subtilis broth a comprises: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.3wt% and balance water, starting pH7.5.
The fermentation medium for preparing the bacillus subtilis fermentation broth B is a basic fermentation medium to which katsumadai powder is added, and the invention is not particularly limited herein, and any medium can be used as long as it can ferment and culture bacillus subtilis, and those skilled in the art can reasonably select according to the nature of the strain. For example, the composition of the basal fermentation medium may be: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05 to 0.1wt%, peanut oil 0.1 to 0.5wt%, and balance water, initial pH7.5, the composition of the fermentation medium for preparing the bacillus subtilis broth B in the invention can be: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05-0.1wt%, peanut oil 0.1-0.5wt%, semen Alpiniae powder 0.1-0.5wt%, and water for the rest, initial pH7.5。
In a specific embodiment of the invention, the fermentation medium for preparing the bacillus subtilis broth B comprises: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.25wt%, and the balance water, starting at pH7.5.
In a second aspect, the present invention provides a method for preparing a composite bacteriostatic agent according to the first aspect of the present invention, comprising the following steps: inoculating bacillus subtilis into a culture solution for liquid seed culture, and then respectively inoculating two parts of bacillus subtilis into a fermentation culture medium containing 0.1-0.5wt% of betel nut powder and a fermentation culture medium containing 0.1-0.5wt% of semen katsumadai powder for liquid fermentation culture to obtain bacillus subtilis fermentation liquor A and bacillus subtilis fermentation liquor B; inoculating lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain lactobacillus plantarum fermentation solution; then uniformly mixing the bacillus subtilis fermentation liquor A, the bacillus subtilis fermentation liquor B, the lactobacillus plantarum fermentation liquor and chitosan to obtain the compound feed; wherein the total viable count of the bacillus subtilis and the lactobacillus plantarum is more than or equal to 3 multiplied by 10 7 cfu/ml, chitosan concentration of 0.1-0.3wt%, and viable cell count ratio of Bacillus subtilis and Lactobacillus plantarum of (5-15):1-10.
Preferably, 0.3wt% of betel nut powder is added to the fermentation medium for preparing the bacillus subtilis broth a.
Preferably, 0.25wt% of katsumade cardamom powder is added to the fermentation medium for preparing the bacillus subtilis broth B.
Preferably, the ratio of the number of living bacteria of the bacillus subtilis to the lactobacillus plantarum is (8-12):3-7.
The culture medium for seed culture is not particularly limited, and any culture medium may be used as long as it is a culture medium capable of seed culture of Bacillus subtilis, and any culture medium may be used as long as it is a culture medium capable of seed culture of Lactobacillus plantarum, and those skilled in the art can reasonably select the culture medium according to the nature of the strain.
The fermentation medium for preparing the lactobacillus plantarum fermentation broth is not particularly limited herein, and any medium may be used as long as it is a medium capable of fermenting and culturing lactobacillus plantarum, and those skilled in the art can reasonably select the medium according to the nature of the strain.
The fermentation medium for preparing the bacillus subtilis fermentation broth A is a basic fermentation medium, betel nut powder is added into the basic fermentation medium, and the basic fermentation medium is not particularly limited herein, and any medium can be used for fermenting and culturing the bacillus subtilis, so that a person skilled in the art can reasonably select the medium according to the nature of the strain. For example, the composition of the basal fermentation medium may be: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05 to 0.1wt%, peanut oil 0.1 to 0.5wt%, and balance water, initial pH7.5, the composition of the fermentation medium for preparing the bacillus subtilis broth A in the invention can be: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05-0.1wt%, peanut oil 0.1-0.5wt%, betel nut powder 0.1-0.5wt%, and water for the rest, the pH is 7.5.
In a specific embodiment of the invention, the fermentation medium for preparing the bacillus subtilis broth a comprises: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.3wt% and balance water, starting pH7.5.
Wherein, the fermentation medium for preparing the bacillus subtilis fermentation broth B is a basic fermentation medium, and the invention is not particularly limited, and any fermentation medium can be used as long as the bacillus subtilis fermentation broth B can be used for fermenting and culturing bacillus subtilis, and the bacillus subtilis fermentation broth B can be reasonably selected by a person skilled in the art according to the nature of strains. For example, the basal fermentation mediumThe composition may be: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05 to 0.1wt%, peanut oil 0.1 to 0.5wt%, and balance water, initial pH7.5, the composition of the fermentation medium for preparing the bacillus subtilis broth B in the invention can be: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05-0.1wt%, peanut oil 0.1-0.5wt%, semen Alpiniae powder 0.1-0.5wt%, and water for the rest, with an initial pH of 7.5.
In a specific embodiment of the invention, the fermentation medium for preparing the bacillus subtilis broth B comprises: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.25wt%, and the balance water, starting at pH7.5.
The third aspect of the invention provides the application of the composite bacteriostatic agent in inhibiting powdery mildew of rubber trees.
The fourth aspect of the invention provides an application of the composite bacteriostatic agent in preventing and treating powdery mildew of rubber trees.
The active ingredients of the compound bacteriostatic agent comprise bacillus subtilis and fermentation metabolites thereof, lactobacillus plantarum and fermentation metabolites thereof, betel nuts, bacillus subtilis fermentation metabolites, katsumadai seed fermentation metabolites, and chitosan, and the active ingredients belong to active ingredients with different action mechanisms, have better inhibition effects on powdery mildew of rubber trees under synergistic effects, can effectively prevent and treat the powdery mildew of rubber trees, and all the ingredients belong to green, environment-friendly, non-toxic active ingredients with dual biological regulation functions of drug effect and fertilizer effect.
Detailed Description
The invention will be further described with reference to specific examples to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the specific embodiment of the present invention, the present invention is not particularly limited herein, and the present invention is applicable to any type of culture medium capable of seed-culturing bacillus subtilis, and any type of culture medium capable of seed-culturing lactobacillus plantarum, and those skilled in the art can reasonably select the culture medium according to the nature of the strain.
In the specific embodiment of the present invention, the present invention is not particularly limited herein, and any medium may be used as long as it is a medium capable of fermenting and culturing lactobacillus plantarum, and those skilled in the art can appropriately select the medium according to the nature of the strain.
In a specific embodiment of the present invention, the fermentation medium for preparing the bacillus subtilis fermentation broth a is a basic fermentation medium to which betel nut powder is added, and the present invention is not particularly limited herein, and any medium can be used as long as it can perform fermentation culture on bacillus subtilis, and those skilled in the art can reasonably select according to the nature of the strain.
In a specific embodiment of the present invention, the fermentation medium for preparing the bacillus subtilis fermentation broth B is a basic fermentation medium to which katsumadai seed powder is added, and the present invention is not particularly limited herein, and any medium may be used as long as it is a medium capable of fermenting and culturing bacillus subtilis, and those skilled in the art can reasonably select according to the nature of the strain.
Example 1
Inoculating Bacillus subtilis into culture solution for liquid seed culture, and inoculating two parts of the culture solution into fermentation medium containing Areca catechu powder and fermentation medium containing semen Alpiniae Katsumadai powder respectively for liquid fermentation culture to obtain Bacillus subtilis fermentation broth A and Bacillus subtilis fermentation brothB, a step of preparing a composite material; inoculating lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain lactobacillus plantarum fermentation solution; then uniformly mixing the bacillus subtilis fermentation liquor A, the bacillus subtilis fermentation liquor B, the lactobacillus plantarum fermentation liquor and chitosan to ensure that the viable count of the bacillus subtilis is 8 multiplied by 10 7 cfu/ml, the viable count of Lactobacillus plantarum is 7×10 7 cfu/ml, chitosan concentration was 0.2wt%.
The fermentation medium for preparing the bacillus subtilis fermentation broth A contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.3wt%, and balance water, with an initial pH of 7.5;
the fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.25wt%, and the balance water, starting at pH7.5.
Example 2
Inoculating bacillus subtilis into a culture solution for liquid seed culture, and then respectively inoculating two parts of bacillus subtilis into a fermentation culture medium containing betel nut powder and a fermentation culture medium containing semen katsumadai powder for liquid fermentation culture to obtain a bacillus subtilis fermentation solution A and a bacillus subtilis fermentation solution B; inoculating lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain lactobacillus plantarum fermentation solution; then uniformly mixing the bacillus subtilis fermentation liquor A, the bacillus subtilis fermentation liquor B, the lactobacillus plantarum fermentation liquor and chitosan to ensure that the viable count of the bacillus subtilis is 12 multiplied by 10 7 cfu/ml, the viable count of Lactobacillus plantarum is 3×10 7 cfu/ml, chitosan concentration was 0.1wt%.
The fermentation medium for preparing the bacillus subtilis fermentation broth A contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.3wt%, and balance water, with an initial pH of 7.5;
the fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.25wt%, and the balance water, starting at pH7.5.
Example 3
Inoculating bacillus subtilis into a culture solution for liquid seed culture, and then respectively inoculating two parts of bacillus subtilis into a fermentation culture medium containing betel nut powder and a fermentation culture medium containing semen katsumadai powder for liquid fermentation culture to obtain a bacillus subtilis fermentation solution A and a bacillus subtilis fermentation solution B; inoculating lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain lactobacillus plantarum fermentation solution; then uniformly mixing the bacillus subtilis fermentation liquor A, the bacillus subtilis fermentation liquor B, the lactobacillus plantarum fermentation liquor and chitosan to ensure that the viable count of the bacillus subtilis is 12 multiplied by 10 7 cfu/ml, the viable count of Lactobacillus plantarum is 3×10 7 cfu/ml, chitosan concentration was 0.3wt%.
The fermentation medium for preparing the bacillus subtilis fermentation broth A contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.5wt% and balance water, with an initial pH of 7.5;
the fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.1wt%, and the balance water, starting at pH7.5.
Example 4
Inoculating bacillus subtilis into the culture solution for liquid seed culture, and inoculating two parts of bacillus subtilis respectivelyPerforming liquid fermentation culture in a fermentation medium containing betel nut powder and a fermentation medium containing semen Alpiniae powder to obtain a bacillus subtilis fermentation broth A and a bacillus subtilis fermentation broth B; inoculating lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain lactobacillus plantarum fermentation solution; then uniformly mixing the bacillus subtilis fermentation liquor A, the bacillus subtilis fermentation liquor B, the lactobacillus plantarum fermentation liquor and chitosan to ensure that the viable count of the bacillus subtilis is 8 multiplied by 10 7 cfu/ml, the viable count of Lactobacillus plantarum is 7×10 7 cfu/ml, chitosan concentration was 0.3wt%.
The fermentation medium for preparing the bacillus subtilis fermentation broth A contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.1wt% and balance water, with an initial pH of 7.5;
the fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.5wt%, and the balance water, starting at pH7.5.
Example 5
Inoculating bacillus subtilis into a culture solution for liquid seed culture, and then respectively inoculating two parts of bacillus subtilis into a fermentation culture medium containing betel nut powder and a fermentation culture medium containing semen katsumadai powder for liquid fermentation culture to obtain a bacillus subtilis fermentation solution A and a bacillus subtilis fermentation solution B; inoculating lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain lactobacillus plantarum fermentation solution; then uniformly mixing the bacillus subtilis fermentation liquor A, the bacillus subtilis fermentation liquor B, the lactobacillus plantarum fermentation liquor and chitosan to ensure that the viable count of the bacillus subtilis is 15 multiplied by 10 7 cfu/ml, the viable count of Lactobacillus plantarum is 1×10 7 cfu/ml, chitosan concentration was 0.2wt%.
Preparation of Bacillus subtilisThe fermentation medium of the bacterial fermentation liquid A contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.3wt%, and balance water, with an initial pH of 7.5;
the fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.25wt%, and the balance water, starting at pH7.5.
Comparative example 1
Respectively inoculating bacillus subtilis and lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain a bacillus subtilis fermentation solution and a lactobacillus plantarum fermentation solution; then mixing the bacillus subtilis fermentation liquor and the lactobacillus plantarum fermentation liquor uniformly to ensure that the viable count of the bacillus subtilis is 8 multiplied by 10 7 cfu/ml, the viable count of Lactobacillus plantarum is 7×10 7 cfu/ml。
The fermentation medium for preparing the bacillus subtilis fermentation broth comprises the following components: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, and balance water, starting at pH7.5.
The fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.25wt%, and the balance water, starting at pH7.5.
Comparative example 2
Respectively inoculating bacillus subtilis and lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain a bacillus subtilis fermentation solution and a lactobacillus plantarum fermentation solution; then the bacillus subtilis fermentation liquor and the lactobacillus plantarum fermentation liquor are addedMixing to obtain viable count of Bacillus subtilis of 12×10 7 cfu/ml, the viable count of Lactobacillus plantarum is 3×10 7 cfu/ml。
The fermentation medium for preparing the bacillus subtilis fermentation broth comprises the following components: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.3wt% and balance water, starting pH7.5.
The fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, and balance water, starting at pH7.5.
Comparative example 3
Respectively inoculating bacillus subtilis and lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain a bacillus subtilis fermentation solution and a lactobacillus plantarum fermentation solution; then mixing the bacillus subtilis fermentation liquor and the lactobacillus plantarum fermentation liquor uniformly to ensure that the viable count of the bacillus subtilis is 15 multiplied by 10 7 cfu/ml, the viable count of Lactobacillus plantarum is 1×10 7 cfu/ml。
The fermentation medium for preparing the bacillus subtilis fermentation broth comprises the following components: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, and balance water, starting at pH7.5.
The fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, and balance water, starting at pH7.5.
Example 6: bacteriostasis experiment
The powdery mildew (O.heveae) of the rubber tree is obtained from the city of the Yan, hainan province, inoculated on the rubber tree seedlings of the susceptible variety cultivated in the incubator and stored in a living body.
Taking the biocontrol microbial preparation of examples 1-5 and the mixed fermentation broth of comparative examples 1-3, respectively, diluting the fermentation broth with sterile water until the total viable count is 5×10 5 cfu/ml of diluent. The conidium of the fresh rubber tree powder spore is swept down into sterile water by a writing brush, 100 mu L of diluent is respectively added, and the sterile water is used as a blank control. After culturing in a constant temperature incubator at 25 ℃ for 24 hours, 20 mu L of the culture medium is placed on a concave glass slide by a pipette, observation is carried out under a microscope, each treatment is repeated 3 times, and the germination rate and the spore germination inhibition rate are calculated.
Spore germination rate (%) = number of germination/total number of spores x 100%
Spore germination inhibition (%) = (control germination rate-treated germination rate)/control germination rate x 100%.
The results are shown in Table 1.
TABLE 1
Treatment group Total number of spores Germination count (number) Germination rate (%) Germination inhibition ratio (%)
Example 1 150 10 6.67 87.08
Example 2 98 6 6.12 88.13
Example 3 107 7 6.54 87.32
Example 4 116 5 4.31 91.64
Example 5 175 8 4.57 91.14
Comparative example 1 182 63 34.62 32.89
Comparative example 2 143 54 37.76 26.79
Comparative example 3 84 38 45.24 12.30
Blank control 95 49 51.58 /
The table shows that the invention has good inhibition effect on rubber powdery mildew, and the effect is better than other treatment groups.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims (8)

1. A composite antibacterial agent for inhibiting powdery mildew of rubber tree is characterized by comprising bacillus subtilis fermentation liquor A, bacillus subtilis fermentation liquor B, lactobacillus plantarum fermentation liquor and chitosan, wherein the total viable count of bacillus subtilis and lactobacillus plantarum in the composite antibacterial agent is more than or equal to 3 multiplied by 10 7 cfu/ml, wherein the concentration of chitosan is 0.1-0.3wt%, the ratio of viable count of bacillus subtilis to lactobacillus plantarum is (5-15):1-10, wherein betel nut powder is added in the fermentation medium for preparing bacillus subtilis fermentation broth A by 0.1-0.5wt%, and semen Alpiniae powder is added in the fermentation medium for preparing bacillus subtilis fermentation broth B by 0.1-0.5wt%.
2. The composite bacteriostat of claim 1, wherein 0.3wt% of betel nut powder is added to a fermentation medium for preparing bacillus subtilis fermentation broth a, and 0.25wt% of katsumade cardamom powder is added to a fermentation medium for preparing bacillus subtilis fermentation broth B.
3. The composite bacteriostat of claim 1, wherein the fermentation medium for preparing bacillus subtilis broth a comprises: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05-0.1wt%, peanut oil 0.1-0.5wt%, betel nut powder 0.1-0.5wt%, and rest water, initial pH7.5;
the fermentation medium for preparing the bacillus subtilis fermentation broth B contains: corn flour 2-4wt%, bean cake powder 2-4wt%, bran powder 2-4wt%, K 2 HPO 4 2-2.5wt％、KH 2 P0 4 0.5-1wt％、MgSO 4 0.05-0.1wt%, peanut oil 0.1-0.5wt%, semen Alpiniae powder 0.1-0.5wt%, and water for the rest, with an initial pH of 7.5.
4. A composite bacteriostatic agent according to claim 3, characterized in that the fermentation medium for preparing bacillus subtilis broth a contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, betel nut powder 0.3wt%, and balance water, with an initial pH of 7.5;
the fermentation medium for preparing the bacillus subtilis fermentation broth B contains: 3wt% of corn flour, 3wt% of bean cake powder, 3wt% of bran powder and K 2 HPO 4 2.15wt％、KH 2 P0 4 0.8wt％、MgSO 4 0.075wt%, peanut oil 0.3wt%, semen Alpiniae powder 0.25wt%, and the balance water, starting at pH7.5.
5. The composite bacteriostat of claim 1, wherein the ratio of viable count of bacillus subtilis to lactobacillus plantarum is (8-12):3-7.
6. A method of preparing a composite bacteriostatic agent according to any one of claims 1-5, comprising the steps of: inoculating bacillus subtilis into a culture solution for liquid seed culture, and then respectively inoculating two parts of bacillus subtilis into a fermentation culture medium containing 0.1-0.5wt% of betel nut powder and a fermentation culture medium containing 0.1-0.5wt% of semen katsumadai powder for liquid fermentation culture to obtain bacillus subtilis fermentation liquor A and bacillus subtilis fermentation liquor B; inoculating lactobacillus plantarum into a culture solution for liquid seed culture and liquid fermentation culture to obtain lactobacillus plantarum fermentation solution; then uniformly mixing the bacillus subtilis fermentation liquor A, the bacillus subtilis fermentation liquor B, the lactobacillus plantarum fermentation liquor and chitosan to obtain the compound feed; wherein the total viable count of the bacillus subtilis and the lactobacillus plantarum is more than or equal to 3 multiplied by 10 7 cfu/ml, chitosan concentration of 0.1-0.3wt%, and viable cell count ratio of Bacillus subtilis and Lactobacillus plantarum of (5-15):1-10.
7. Use of the composite antibacterial agent of any one of claims 1 to 5 for inhibiting powdery mildew of rubber tree.
8. Use of the compound bacteriostat of any one of claims 1-5 for controlling powdery mildew of rubber trees.
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