Biocontrol bacterium preparation for preventing and treating anthracnose of rubber trees and preparation of biocontrol bacterium preparation

Abstract

The invention belongs to the field of rubber tree cultivation, and particularly relates to a biocontrol bacterium preparation for preventing and treating anthracnose of rubber trees and preparation of the biocontrol bacterium preparation. The biocontrol microbial agent is prepared from the following components in parts by weight: 0.5 to 1 part of bacillus velezensis HN-2, 1 to 1.5 parts of bacillus laterosporus, 5 to 10 parts of mango polysaccharide, 20 to 40 parts of herba king grass, 10 to 20 parts of clausena lansium peel, 15 to 25 parts of pitaya stem and 8 to 12 parts of rubber leaf. According to the biocontrol microbial inoculum, waste pitaya stems in the pitaya cultivation process, waste rubber leaves in the rubber tree cultivation process and wampee peel are used for preparing an extracting solution for strain fermentation, and the biocontrol microbial inoculum has good inhibitory activity on colletotrichum gloeosporioides of the rubber trees and has good application prospects in the aspect of preventing and treating the colletotrichum gloeosporioides of the rubber trees.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of rubber tree cultivation, and particularly relates to a biocontrol agent preparation for preventing and treating rubber tree anthracnose and preparation thereof. BACKGROUND

[0002] Rubber tree anthracnose fungus (Colletotrichum gloeosporioides) is one of the main diseases of rubber tree leaves, and the pathogenic bacteria can invade the leaves of rubber trees at all stages. The pathogen invades from the leaf tip or leaf edge, and after the lesion expands, a circular, oval or irregular patch can be formed. The lesion is dark brown to gray white with a ring-shaped patch, the edge is black brown, and slightly raised. Severe rubber trees will cause leaf drop, and the disease will affect the health of rubber trees and the normal secretion of rubber latex, thereby reducing the yield and quality of rubber latex, and directly affecting the yield and application of rubber. Therefore, it is crucial to develop an efficient and environmentally friendly preparation for preventing and treating rubber tree anthracnose for rubber tree cultivation. SUMMARY

[0003] The present application provides a biocontrol agent preparation for preventing and treating rubber tree anthracnose and preparation thereof, which has good antagonistic effect on rubber tree anthracnose fungus.

[0004] The technical solution of the present application is as follows:

[0005] A biocontrol agent preparation comprises the following components in parts by weight: Bacillus velezensis HN-20.5-1 part, Bacillus laterosporus 1-1.5 parts, mango polysaccharide 5-10 parts, king grass 20-40 parts, yellow skin 10-20 parts, dragon fruit stem 15-25 parts, and rubber leaf 8-12 parts. The Bacillus velezensis HN-2 has a preservation number of CCTCC NO: M 2018382.

[0006] Further, the biocontrol agent preparation comprises the following components in parts by weight: Bacillus velezensis HN-20.8 parts, Bacillus laterosporus 1 part, mango polysaccharide 7 parts, king grass 30 parts, yellow skin 15 parts, dragon fruit stem 20 parts, and rubber leaf 10 parts.

[0007] Further, the preparation method of the mango polysaccharide is as follows: mango peels are dried, crushed, and then extracted with distilled water by ultrasonic wave. The supernatant is obtained by filtration, and then 80% of the supernatant volume of anhydrous ethanol is added for precipitation. After centrifugation, the precipitate is obtained, and then washed with anhydrous ethanol, acetone and diethyl ether for 3 times, and dried to obtain the mango polysaccharide.

[0008] Further, the concentration of the Bacillus velezensis is greater than or equal to 10 8 cfu / mL, and the concentration of the Bacillus laterosporus is greater than or equal to 10 8cfu / mL.

[0009] Further, the said pitaya peel is fresh pitaya peel; the said dragon fruit stem is fresh dragon fruit stem; the said king grass and rubber leaf blade are dry king grass and rubber leaf blade.

[0010] The preparation method of the above-mentioned biocontrol agent preparation comprises the following steps:

[0011] (1) the king grass and rubber leaf blade are mixed and crushed, then water is added for decoction, filtration, and the extract is concentrated to 1 / 6-1 / 5 of the volume; the pitaya peel and dragon fruit stem are ground and the filtrate is taken;

[0012] (2) the extract after concentration is added with bacillus velezensis HN-2 and bacillus laterosporus for fermentation for 2-3 days to obtain a fermentation liquor;

[0013] (3) the fermentation liquor is added with mango polysaccharide for further fermentation for 1 day.

[0014] Further, in the step (1), the water is added in an amount of 5-8 times the total weight of the king grass and rubber leaf blade for decoction for 20-30 min.

[0015] Further, in the step (1), the pitaya peel and dragon fruit stem are ground by a colloid mill, then water is added in an amount of the weight of the pitaya peel and dragon fruit stem, stirred uniformly, and the filtrate is taken.

[0016] Further, in the steps (2) and (3), the fermentation temperature is 25-28 DEG C, and the bacteria are shaken at 200 r / min.

[0017] The biocontrol agent preparation has the application in the preparation of the prevention and treatment of the rubber tree anthracnose fungus.

[0018] The biocontrol agent preparation has the application in the preparation of the prevention and treatment of the rubber tree anthracnose fungus.

[0019] The biocontrol agent preparation has the application in the preparation of the prevention and treatment of the rubber tree anthracnose fungus. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0021] In the following embodiments, the fresh mangosteen peel is used, the fresh dragon fruit stem is used, and the dry aboveground parts of the king grass and the dry rubber leaves are used.

[0022] The preparation method of the mango polysaccharide is as follows: the mango peel is taken, dried, crushed, added with distilled water for ultrasonic extraction, filtered to obtain supernatant, added with 80% of the volume of the supernatant of anhydrous ethanol for precipitation and standing, then centrifuged to obtain a precipitate, and the precipitate is washed with anhydrous ethanol, acetone and diethyl ether in sequence for three times, and dried to obtain the mango polysaccharide.

[0023] Embodiment 1

[0024] The biocontrol agent preparation comprises the following components in parts by weight: 5 parts of Bacillus velezensis HN-20, 1 part of Bacillus laterosporus, 5 parts of mango polysaccharide, 20 parts of king grass, 10 parts of mangosteen peel, 15 parts of dragon fruit stem and 8 parts of rubber leaves. The preservation number of the Bacillus velezensis HN-2 is CCTCC NO: M 2018382. The concentration of the Bacillus velezensis is 10 8 cfu / mL, and the concentration of the Bacillus laterosporus is 10 8 cfu / mL.

[0025] The preparation method of the biocontrol agent preparation comprises the following steps:

[0026] (1) The king grass and the rubber leaves are mixed and crushed, then water with a weight of 5 times the total weight of the king grass and the rubber leaves is added for decoction for 20-30 min, filtration is performed, and the extract is concentrated to 1 / 5 of the volume; the mangosteen peel and the dragon fruit stem are ground by using a colloid mill, then water with a weight of the mangosteen peel and the dragon fruit is added for stirring to be uniform, filtration is performed to obtain filtrate; and the extract and the filtrate are combined to obtain a mixed solution;

[0027] (2) The mixed solution is added with the Bacillus velezensis HN-2 and the Bacillus laterosporus for fermentation for 2 days to obtain a fermentation liquor; the fermentation process is performed at a temperature of 25℃ and a shaking speed of 200 rpm;

[0028] (3) The fermentation liquor is added with the mango polysaccharide for further fermentation for 1 day, and the fermentation process is performed at a temperature of 25℃ and a shaking speed of 200 rpm.

[0029] Embodiment 2

[0030] A biocontrol agent preparation, comprising the following components in parts by weight: Bacillus velezensis HN-20.8 parts, Bacillus laterosporus 1 part, mango polysaccharide 7 parts, king grass 30 parts, yellow skin 15 parts, dragon fruit stem 20 parts, rubber leaf 10 parts. The concentration of the Bacillus velezensis is 10 9 cfu / mL, and the concentration of the Bacillus laterosporus is 10 9 cfu / mL.

[0031] The preparation method of the above-mentioned biocontrol agent preparation, comprising the following steps:

[0032] (1) Mix and crush the king grass and rubber leaves, then add 5 times the weight of the total king grass and rubber leaves in water and decoct for 25 min, filter, and concentrate the extract to 1 / 6 of the volume; grind the yellow skin and dragon fruit stem with a colloid mill, then add water to the weight of the yellow skin and dragon fruit, stir evenly, filter, and take the filtrate; combine the extract and the filtrate to obtain a mixed solution;

[0033] (2) Add Bacillus velezensis HN-2 and Bacillus laterosporus to the mixed solution and ferment for 93 days to obtain a fermentation liquor; the fermentation temperature is 27℃, and the bacteria are shaken at 200 rpm;

[0034] (3) Add mango polysaccharide to the fermentation liquor and continue to ferment for 1 day, and the fermentation temperature is 27℃, and the bacteria are shaken at 200 rpm.

[0035] Example 3

[0036] A biocontrol agent preparation, comprising the following components in parts by weight: Bacillus velezensis HN-21 parts, Bacillus laterosporus 1.5 parts, mango polysaccharide 10 parts, king grass 40 parts, yellow skin 20 parts, dragon fruit stem 25 parts, and rubber leaf 12 parts; the preservation number of the Bacillus velezensis HN-2 is CCTCC NO: M 2018382. The concentration of the Bacillus velezensis is 10 8 cfu / mL, and the concentration of the Bacillus laterosporus is 10 8 cfu / mL.

[0037] The preparation method of the above-mentioned biocontrol agent preparation, comprising the following steps:

[0038] (1) Mix and crush the king grass and rubber leaves, then add 8 times the weight of the total king grass and rubber leaves in water and decoct for 30 min, filter, and concentrate the extract to 1 / 5 of the volume; grind the yellow skin and dragon fruit stem with a colloid mill, then add water to the weight of the yellow skin and dragon fruit, stir evenly, filter, and take the filtrate; combine the extract and the filtrate to obtain a mixed solution;

[0039] (2) mixed liquid is added with Bacillus velezensis HN-2 and Bacillus laterosporus to ferment for 3 days to obtain a fermentation liquor; the temperature is 28 DEG C during the fermentation process, and the bacteria are shaken at 200 r / min;

[0040] (3) the fermentation liquor is added with mango polysaccharide to continue to ferment for 1 day, and the temperature is 28 DEG C during the fermentation process, and the bacteria are shaken at 200 r / min.

[0041] The bacteriostatic effect of the biocontrol agent preparation is investigated

[0042] Rubber tree anthracnose fungus (Colletotrichum gloeosporioides) is used as the test pathogenic fungus to investigate the bacteriostatic effect. The biocontrol agent preparations prepared in the three examples are used to investigate the bacteriostatic effect. The comparative examples are prepared according to the following proportions and preparation methods.

[0043] Comparative example 1: 1 part of Bacillus laterosporus, 7 parts of mango polysaccharide, 30 parts of king grass, 15 parts of yellow skin peel, 20 parts of dragon fruit stem, and 10 parts of rubber leaf. The concentration of the Bacillus laterosporus is 10 9 cfu / mL. The preparation method is referred to example 2.

[0044] Comparative example 2: 0.8 parts of Bacillus velezensis HN-20, 7 parts of mango polysaccharide, 30 parts of king grass, 15 parts of yellow skin peel, 20 parts of dragon fruit stem, and 10 parts of rubber leaf. The concentration of the Bacillus velezensis is 10 9 cfu / mL.

[0045] Comparative example 3: the proportions and preparation methods of the materials are all referred to example 2, and the difference is that the yellow skin peel and the dragon fruit stem are ground, and then the extraction liquid is obtained after the king grass and the rubber leaf are boiled with water.

[0046] Comparative example 4: the proportions and preparation methods of the materials are all referred to example 2, and the difference is that the mango polysaccharide is directly added to the mixed liquid, and then the Bacillus velezensis HN-2 and the Bacillus laterosporus are added for fermentation.

[0047] Comparative example 5: the proportions and preparation methods of the materials are all referred to example 2, and the difference is that the king grass and the rubber leaf are soaked in an equal amount of water during the preparation process, and then the yellow skin peel and the dragon fruit stem are ground together, and then the filter liquid is obtained after filtration.

[0048] The biocontrol agents prepared in each example group and the comparative example group are diluted with sterile water to a dilution liquid with a concentration of 5x10 5 cfu / mL for standby.

[0049] The antibacterial activity of each sample was determined by the flat plate confrontation method. The rubber tree anthracnose fungus (Colletotrichum gloeosporioides) cake was taken on the PDA medium, and after the mycelium grew throughout the plate, the cake was taken, the mycelium was downward, and was inoculated into the center of a new PDA plate. The cake was punched symmetrically on the upper, lower, left and right sides at a distance of 20 mm with a 5 mm puncher, and 30 ul of the diluent of the example group and the comparative example group was added to the hole. At the same time, the plate of the rubber tree anthracnose fungus cake was used as a control (CK). It was placed in a 28℃ incubator and cultured for 7 days, and then the antibacterial results were observed and recorded. The relative inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - 5) x 100%. In the formula, each colony diameter unit is "mm"; "5" is the initial diameter of the treated colony. The antibacterial rates of each treatment group are shown in Table 1.

[0050] Table 1 Comparison of antibacterial activity of different samples

[0051] No. Sample added Relative inhibition rate (%) 1 Example 1 84.51 2 Example 2 86.84 3 Example 3 83.75 4 Comparative Example 1 46.70 5 Comparative Example 2 54.39 6 Comparative Example 3 55.26 7 Comparative Example 4 61.04 8 Comparative Example 5 38.77

[0052] As can be seen from the table, the biocontrol agent prepared in the three examples of the present application has significant inhibitory activity on rubber tree anthracnose, and the relative inhibition rates are 84.51%, 86.84% and 83.75%, respectively. In Comparative Example 1 and Comparative Example 2, Bacillus velezensis HN-2 and Bacillus laterosporus were removed, and the rest of the operation was the same as Example 2, but the inhibition rate was significantly reduced. It shows that the biocontrol agent described in the present application can significantly improve the antibacterial effect of the biocontrol agent by adding Bacillus velezensis HN-2 and Bacillus laterosporus for fermentation treatment.

[0053] In Comparative Examples 3-5, although the composition of the components is the same as the examples, the preparation method is different from the example group, and the antibacterial activity of the rubber tree anthracnose fungus is reduced to varying degrees. The above experiment shows that although the composition of the biocontrol agent in the three comparative examples is the same as the examples, the preparation method is different from the three examples, and the antibacterial activity of the biocontrol agent prepared finally is reduced to varying degrees relative to the three examples. The above experimental results show that different preparation methods have a significant impact on the antibacterial activity of the biocontrol agent. In the present application, the mixed liquid prepared by decocting Wangcao and rubber leaves with water and then adding pitaya stem and yellow peel filtrate is used for fermentation of Bacillus velezensis HN-2 and Bacillus laterosporus, and then adding mango polysaccharide for secondary fermentation, which can significantly improve the antibacterial activity of the prepared biocontrol agent.

[0054] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A biocontrol agent preparation, characterized in that, The biocontrol agent preparation comprises the following components in parts by weight: Bacillus velezensis HN-20.5-1 parts, Bacillus laterosporus 1-1.5 parts, mango polysaccharide 5-10 parts, king grass 20-40 parts, yellow skin 10-20 parts, dragon fruit stem 15-25 parts, and rubber leaf 8-12 parts; the Bacillus velezensis HN-2 has a preservation number of CCTCC NO: M 2018382.

2. The biocontrol agent as described in claim 1, characterized in that, The biocontrol agent preparation comprises the following components in parts by weight: Bacillus velezensis HN-20.5-1 parts, Bacillus laterosporus 1-1.5 parts, mango polysaccharide 5-10 parts, king grass 20-40 parts, yellow skin 10-20 parts, dragon fruit stem 15-25 parts, and rubber leaf 8-12 parts; the Bacillus velezensis HN-2 has a preservation number of CCTCC NO: M 2018382.

3. The biocontrol agent as described in claim 1, characterized in that, The preparation method of the mango polysaccharide comprises the following steps: taking mango peel, drying, crushing, adding distilled water, ultrasonic extraction, filtering, taking supernatant, adding 80% of the volume of the supernatant of anhydrous ethanol, precipitating and standing, then centrifuging to take the precipitate, and washing the precipitate with anhydrous ethanol, acetone and diethyl ether in sequence, and drying to obtain the mango polysaccharide.

4. The biocontrol agent as described in claim 1, characterized in that, The concentration of the said Bacillus velezensis is ≥ 10 8 cfu / mL, and the concentration of the Paenibacillus latericius is ≥ 10 8 cfu / mL.

5. The biocontrol agent as described in claim 1, characterized in that, The yellow skin is fresh yellow skin, the dragon fruit stem is fresh dragon fruit stem, and the king grass and rubber leaf are dry above-ground parts of king grass and rubber leaf.

6. The method of preparing the biocontrol bacterial preparation according to any one of claims 1 to 5, wherein, The method comprises the following steps: (1) taking king grass and rubber leaf, mixing and crushing, then adding water to decoct, filtering, and concentrating the extract to 1 / 6-1 / 5 of the volume; grinding the yellow skin and dragon fruit stem, and taking the filtrate; (2) adding Bacillus velezensis HN-2 and Bacillus laterosporus to the concentrated extract to ferment for 2-3 days to obtain a fermentation liquor; (3) adding mango polysaccharide to the fermentation liquor to continue fermenting for 1 day.

7. The production method according to claim 1, wherein: In step (1), the amount of water added is 5-8 times the total weight of the king grass and rubber leaf, and the decoction is performed for 20-30 min.

8. The production method according to claim 1, wherein: In step (1), the yellow skin and dragon fruit stem are ground by a colloid mill, then water is added to the weight of the yellow skin and dragon fruit, stirred uniformly, and the filtrate is taken.

9. The production method according to claim 1, wherein: In steps (2) and (3), the fermentation temperature is 25-28℃, and the bacteria are shaken at 200 r / min.

10. Use of the biocontrol agent preparation according to any one of claims 1-5 in the preparation of a preparation for preventing and treating the rubber tree anthracnose fungus.