CN114410692A

CN114410692A - Preparation method of n-butanol extract of biocontrol strain HN-2 and application of n-butanol extract in preventing and treating powdery mildew of rubber

Info

Publication number: CN114410692A
Application number: CN202210047237.XA
Authority: CN
Inventors: 靳鹏飞; 褚凌龙; 缪卫国; 刘文波
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2022-01-04
Filing date: 2022-01-17
Publication date: 2022-04-29
Anticipated expiration: 2042-01-17
Also published as: CN114410692B

Abstract

The invention provides a preparation method of a n-butyl alcohol extract of a biocontrol strain HN-2 and application of the n-butyl alcohol extract in controlling rubber powdery mildew, wherein the invention deeply researches the action mechanism of a bacterium B velezensis HN-2 n-butyl alcohol extract in controlling rubber powdery mildew by separating and purifying active ingredients in a bacterial strain HN-2 metabolite, and the HN-2 fermentation active n-butyl alcohol extract is obtained by performing low-temperature extraction on HN-2 fermentation supernatant by taking an n-butyl alcohol organic solvent as an extracting agent, can be used for inhibiting the growth of rubber powdery mildew and inducing the resistance of rubber trees to the rubber powdery mildew, and provides an important basis for the development and utilization of the following Bacillus belgii and the control of the rubber powdery mildew.

Description

Preparation method of n-butanol extract of biocontrol strain HN-2 and application of n-butanol extract in preventing and treating powdery mildew of rubber

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a preparation method of a n-butanol extract of a biocontrol strain HN-2 and application of the n-butanol extract in preventing and treating rubber powdery mildew.

Background

Rubber tree powdery mildew (Erysiphe quercicola) belongs to fungi imperfecti, is a living obligate parasitic fungus and cannot leave a plant living tissue for culture, and the rubber tree powdery mildew caused by the Erysiphe quercicola is one of the most economically destructive diseases on rubber trees. In the chemical control of rubber tree powdery mildew, the types of common chemical agents are roughly divided into: chemical agents such as inorganic compounds, triazole compounds, methoxyacrylate derivatives (Strobilurins), and the like. For the prevention and treatment of rubber powdery mildew, the adopted inorganic compounds such as sulfur and the like can cause human poisoning and pollute the environment when being used in a large area, the use of sulfur powder is often influenced by weather, and meanwhile, the phenomenon of improving the drug resistance level is easy to occur when chemical agents such as tridemorph, triadimefon, sulfur powder, triadimefon and the like are adopted.

Biological control is the control of the occurrence and development of plant diseases by using plant roots and endogenous beneficial microorganisms called biocontrol bacteria. The biocontrol bacteria can affect the plant itself by secreting some compounds, or directly affect the pathogen itself, or compete with the pathogen for survival sites, reduce the number of the pathogen and the like in various regulation and control modes, so as to achieve different disease control effects. For biological control of powdery mildew, the control difficulty of the powdery mildew at present is high and the pathogenic mechanism is complex due to the influence of large difference of pathogenic bacteria species of powdery mildew of different crops. At present, the content of the application of the related extracts of the metabolites of the biocontrol strain to the biological control of the Erysiphe cichoracearum is not reported.

Disclosure of Invention

Therefore, the invention provides a preparation method of a n-butanol extract of a biocontrol strain HN-2 and application of the n-butanol extract in controlling powdery mildew of rubber.

The technical scheme of the invention is realized as follows:

the biocontrol bacterium B.velezensis HN-2 adopted by the invention is named as Bacillus belgii HN-2, and the preservation numbers are as follows: CCTCC NO: M2018382, which is disclosed in patent No. 201910239698.5, a patent of application of biocontrol bacterium HN-2 and its extract in preparation of preparation for inhibiting pathogenic fungi. The invention further deeply researches the action mechanism of the bacterial B.velezensis HN-2 n-butanol extract in the control of Erysiphe cichoracearum by separating and purifying the active ingredients in the bacterial strain HN-2 metabolite, and provides a theoretical basis for further expanding the development of B.velezensis biocontrol preparations.

A preparation method of n-butanol extract of biocontrol strain HN-2 comprises the following steps:

(1) performing propagation culture by using biocontrol bacteria B.velezensis HN-2, and centrifuging to obtain fermentation supernatant;

(2) mixing an n-butanol organic solvent and HN-2 fermentation supernatant according to a volume ratio of 0.5-1.5:1, and standing and extracting at a low temperature of 10-15 ℃ to obtain an upper n-butanol extract;

(3) performing rotary evaporation on the n-butanol extract, and freeze drying to obtain HN-2 fermentation active n-butanol extract.

Further explaining, in the step (1), the biocontrol bacterium B.velezensis HN-2 is subjected to propagation culture, namely, the strain HN-2 is inoculated into an LB liquid culture medium and cultured at the temperature of 27-28 ℃ and the rpm of 180-200 for 48 hours; the centrifugation speed was 10000 rpm.

Further explaining, in the step (2), the volume ratio of the n-butanol organic solvent to the HN-2 fermentation supernatant is 1: 1; the extraction time is more than or equal to 12 h.

Further, in the step (3), the rotary evaporation temperature is 50 ℃.

An active n-butanol extract of HN-2 prepared by the above method is provided.

An application of HN-2 fermentation active n-butanol extract in preventing and treating rubber powdery mildew can be used for inhibiting the growth of rubber powdery mildew.

A HN-2 fermentation active n-butanol extract antagonizes the growth of Blumeria necator by cell lysis and cytoplasmic extravasation of Blumeria necator spores, or by inhibiting cell wall or cell membrane synthesis.

An application of HN-2 fermentation active n-butanol extract in preventing and treating rubber powdery mildew can be used for inducing rubber tree resistance to rubber powdery mildew.

An HN-2 fermentation activity n-butanol extract is used for inducing rubber tree resistance to rubber powdery mildew by regulating and controlling rubber defense enzyme activity and expression level of defense genes.

Compared with the prior art, the invention has the beneficial effects that:

the invention adopts n-butyl alcohol as an extraction solvent, and performs low-temperature extraction on active ingredients in metabolites of biocontrol bacteria B.velezensis HN-2 to obtain an HN-2 fermentation active n-butyl alcohol extract, wherein the active ingredients and lipopeptide have very strong antibiotic activity and are used for inhibiting the growth of rubber powdery mildew, and experiments show that the extract can effectively regulate and control the activity of rubber defensive enzyme and the expression quantity of defensive genes, induce the resistance of rubber trees to rubber powdery mildew, realize that the biocontrol bacteria B.velezensis HN-2 n-butyl alcohol extract is widely applied to the control of rubber powdery mildew, and provide an important basis for the development and utilization of subsequent Bacillus belgii and the control of rubber powdery mildew.

Drawings

FIG. 1 is a bar graph of B.velezensis HN-2 n-butanol extract and propiconazole inhibition of E.quericola spore germination in an example of the present invention;

FIG. 2 is a graph showing the effect of B.velezensis HN-2 extract and propiconazole on the morphology of E.quericola spores sprayed on leaves according to the example of the present invention;

wherein group A, B, C was inoculated with E.quericola after foliar spray application of water, HN-2 extract (28. mu.g/mL) and propiconazole (28. mu.g/mL) for 12 hours, respectively; group D, E, F was prepared by foliar spray application of water, HN-2 extract (28. mu.g/mL), and propiconazole (28. mu.g/mL) 12 hours after E.quericola inoculation, respectively;

group D, E, F: after inoculating E.quericola for 12 hours, leaf surface was sprayed with water, HN-2 extract (28. mu.g/mL) or propiconazole (28. mu.g/mL), respectively

FIG. 3 is a bar graph of SOD enzyme activity of pre-treated and post-treated rubber leaves of an example of the present invention;

FIG. 4 is a bar graph of POD enzyme activity of rubber leaves of an embodiment of the present invention after and before treatment;

FIG. 5 is a bar graph of CAT enzyme activity of pre-treated and post-treated rubber leaves of an example of the present invention;

FIG. 6 is a bar graph of APX enzyme activity of pre-treated and post-treated rubber leaves according to an embodiment of the present invention;

FIG. 7 is a histogram of the changes in gene expression of HbLFG1, HbNPR1, HbCAT, and HbPOD after different treatments in the rubber leaf of the example of the invention.

Wherein in FIGS. 3-7, Benzyl-propiconazol with E.quericola is: after spraying propiconazole (28. mu.g/mL) to the leaf surface for 12 hours, E.quericola was inoculated. HN-2 with e.quericola is: after spraying HN-2 extract (28. mu.g/mL) to the leaf surface for 12 hours, E.quericola was inoculated. Water with e.quericola is: e.quericola was inoculated after spraying water on the leaf surfaces for 12 hours, respectively.

Querciclola with Benzyl-propiconazol is: after foliar inoculation with E.quericola for 12 hours, propiconazole (28. mu.g/mL) was sprayed. Quericola with HN-2: after 12 hours of leaf surface inoculation with E.quericola, HN-2 extract (28. mu.g/mL) was sprayed. Quericola with Water is: the leaf surface was inoculated with e.quericola for 12 hours before water was sprayed.

Benzyl-propiconazole is: only benzyl propiconazole (28. mu.g/mL) was sprayed. HN-2: only HN-2 extract (28. mu.g/mL) was sprayed. Water: only water was sprayed.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

Example 1 extraction of B.velezensis HN-2 fermentation broth active Material

Separating and storing B.velezensis HN-2 from soil, and inoculating the B.velezensis HN-2 on an LB plate for culture at 28 ℃; blumeria rubber powdery mildew (Oidium heveae) is collected from delirium, Hainan province, and inoculated on leaves of rubber trees in the ancient copper stage for culture.

Extracting active substances of Bacillus belgii HN-2 fermentation liquor:

inoculating the strain B.velezensis HN-2 into 50mL LB liquid culture medium, culturing at 28 deg.C and 180rpm for 48h, centrifuging at 10000rpm for 10min, and removing thallus to obtain strain HN-2 fermentation supernatant. The same amount of n-butanol was added to the resulting fermentation supernatant to mix the two solutions thoroughly, and the mixture was left to stand at 11 ℃ overnight for extraction (12 hours). And (3) introducing the mixed solution into a separating funnel to obtain an upper organic phase, performing rotary evaporation at 50 ℃ to obtain a precipitate, washing out the precipitate, and performing freeze drying to obtain the Bacillus beilesiensis HN-2 fermentation activity n-butanol extract.

Example 2 bacteriostasis test of Blumeria rubber powdery mildew spore suspension

The Erysiphe graminis spores cultured in example 1 are selected, and the spores are swept into a conical flask by a writing brush and the volume is adjusted to 50 ml. And subpackaging the spore solution into 1.5ml centrifuge tubes, and continuously and uniformly mixing during subpackaging. Individual tubes were labeled, with three replicates per treatment. Centrifuging at 10000rpm for 5min, slightly sucking the supernatant equal to the amount of the drug to be loaded, adding the test drug, mixing well, culturing at 28 deg.C and 180rpm, and counting with a blood counting plate for 12 hr, 24 hr and 48 hr respectively.

The test concentrations of the agents were:

fermentation of active n-butanol extract by Bacillus belgii HN-2 prepared in example 1: 500, 250, 125, 62.5, 31.25 mug/ml;

benzyl propiconazole: 500, 250, 125, 62.5, 31.25 mug/ml;

in FIG. 1A, the inhibition of E.quercinola spore germination by B.velezensis HN-2 n-butanol extracts at different concentrations (31.25, 62.5, 125, 250 and 500. mu.g/mL) can be seen, with different letters indicating that the same drug at the same concentration was statistically significant at different time points (P <0.05)

In FIG. 1B, the inhibitory effect of propiconazole on germination of E.quericola spores at various concentrations (31.25, 62.5, 125, 250 and 500. mu.g/mL) can be seen. Different letters indicate that at the same concentration, the same drug was statistically significant at different time points (P < 0.05).

The result shows that Bacillus velezensis HN-2 n-butyl alcohol extract can effectively inhibit in-vitro Erysiphe quericola spore germination. 50% Effective Concentration (EC) of velezensis HN-2 n-butanol extract on E.quericola spore suspension₅₀) It was 22.5. mu.g/mL. EC of propiconazole on e.quericola spore suspension₅₀It was 28.0. mu.g/mL.

Example 3 leaf surface inoculation bacteriostasis experiment of rubber powdery mildew

Selecting leaves of the rubber tree in the bronze stage with similar sizes, and respectively carrying out the following treatment:

a part of the leaves was selected and inoculated with Erysiphe necator, and a part of the leaves was selected and sprayed with propiconazole benzoate (28.0. mu.g/ml), N-butanol extract (28.0. mu.g/ml) of Bacillus belezii HN-2 prepared in example 1 and water. 12h after the first inoculation, leaves inoculated with Erysiphe graminis were sprayed with the same amounts of propiconazole benzoate (28.0. mu.g/ml), Bacillus belgii HN-2 n-butanol extract (28.0. mu.g/ml) and water. And (4) inoculating the rubber tree powdery mildew to the leaves which are sprayed with the pesticide.

Selecting leaves, and spraying benzyl propiconazole (28.0 μ g/ml), Bacillus belgii HN-2 n-butanol extract (28.0 μ g/ml) and water with the same concentration as the leaves to obtain a blank control group;

after the second inoculation, the time was started and the leaves were observed microscopically for powdery mildew at 0, 3, 6, 8, 12, 24 and 48h, respectively.

In fig. 2, in a to F, the spore morphology of e.quericola on leaves treated with b.velezensis HN-2 n-butanol extract and propiconazole is changed, the surface of spores is rough, even transparent holes are generated, and the size of spores is reduced to some extent compared with normal spores; the spore morphology did not change significantly in the blank control group, showing a smooth and intact appearance, thus indicating that b.velezensis HN-2 n-butanol extract was effective in inhibiting e.quericola spore germination on rubber leaves.

Example 4-B. influence of velezensis HN-2 extract on the Activity of rubber leaf defense-related enzymes

The enzyme activity assay was performed on 12, 24, 48, 72h treated leaves inoculated with Erysiphe graminis as described in example 3

Enzyme activity determination method-determination of antioxidant enzyme activity (SOD, POD, CAT, APX)

(1) Enzyme liquid extraction

Extracting solution: 50mM Tris-HCl buffer, pH7.0, containing 20% glycerol, 1mM EDTA, 1mM ASA, 1mM DTT, 1mM GSH, 5mM MgCl₂

② accurately weighing 0.10g of sample plant, crushing and grinding by using a grinder after quick freezing, then adding 2ml of precooled extracting solution, and centrifuging for 20min at 4 ℃ and 12000 rpm. The pellet was removed and only the supernatant was left for enzyme activity assay.

(2) Enzyme Activity assay

Firstly, SOD Activity measurement

Adding reaction reagent according to table 1, mixing, performing dark treatment on a control tube, placing a measuring tube in 4000lx sunlight for reaction for 40min, adjusting the control tube to zero after the reaction is finished, and respectively measuring A₅₆₀. SOD activity was calculated.

A_CKAbsorbance A of control tube_EDetermination of the absorbance of the tube

V-total volume of sample solution, mL Vt-amount of sample used in measurement, mL

FW-fresh weight of sample, g U_SODSOD enzyme activity, unit. mg^-1FW

TABLE 1 name and amount of reagents

Measurement of POD Activity

Adding reaction reagent according to Table 2, standing at 25 deg.C for 15min, adding enzyme solution, and rapidly mixing to determine A₄₇₀. Taking the time of 0.5-3.5min every 30s, and measuring A/min₄₇₀The change 0.01 was 1 enzyme activity unit (u), and the POD activity was calculated according to the formula.

t-reaction time

FW-fresh weight of sample, g U_PODPOD enzyme Activity, unit. mg^-1FW

TABLE 2 name and amount of reagents

(iii) determination of CAT Activity

The reagents were added as in Table 3, left at 25 ℃ for 15min, the enzyme solution was added, followed by 0.05ml of 750mM H₂O₂The reaction was started. Take 0.Reading A every 30s for 5-3.5min₂₄₀At a per minute₂₄₀The reduction of 0.1 is 1 enzyme activity unit (u), and the CAT activity is calculated according to the formula.

t-reaction time

FW-fresh weight of sample, g U_CATCAT enzyme Activity, unit. mg^-1FW

TABLE 3 name and amount of reagents

Determination of APX Activity

The reaction was started by adding the reagents according to Table 4, standing at 25 ℃ for 15min, adding the enzyme solution, and then adding 0.05ml of 30mM AsA. Taking 0.5-3.5min time period, and reading A every 30s₂₉₀At a per minute₂₉₀The reduction of 0.1 is 1 enzyme activity unit (u), and the APX activity is calculated according to the formula.

t-reaction time

FW-fresh weight of sample, g U_APXAPX enzyme Activity, unit. mg^-1FW

TABLE 4 name and amount of reagents

The results of measurement of antioxidant enzyme (SOD, POD, CAT, APX) activity of the present invention are shown in FIGS. 3 to 6, which include measurement of enzyme activity of the enzyme involved in defense of each rubber leaf in the prior treatment A and the subsequent treatment B (the prior treatment is treatment with a drug followed by inoculation, and the subsequent treatment is treatment with a drug followed by inoculation). As can be seen from the figure, in the previously treated experimental group, the enzymatic activities of SOD, CAT and APX were improved to some extent in the pre-experimental stage, while the enzymatic activity of POD was kept stable. In the post-treatment group, the enzymatic activity of SOD was slightly increased, while the enzymatic activity of POD reached a higher level. Meanwhile, in HN-2 group, the enzyme activity of CAT reached a maximum at 12 hours, then declined and remained stable again, while the enzyme activity of APX reached a maximum at 24 hours. Thus, the results indicate that spraying of n-butanol extract of b.velezensis HN-2 induces systemic resistance in rubber trees and increases the activity of enzymes involved in ROS clearance.

Example 5-B.quantitative (qRT-PCR) analysis of the Effect of velezensis HN-2 extract on defense-related Gene expression in rubber leaves

Rubber leaves at specific time points (12, 24, 48 and 72h) in example 3 were selected, RNA was extracted and reverse-transcribed into cDNA, and the cDNA concentration was adjusted to 100 ng/. mu.l. qRT-PCR detection was performed using RealUniversal Color PreMix (SYBR Green) kit. HbActin is used as an internal reference gene, the expression level of the gene is detected, and the primers for detecting the gene are shown in Table 5.

DNA primers used in Table 5

TABLE 6 qRT-PCR System

The quantitative analysis (qRT-PCR) of the expression of defense related genes in the rubber leaves comprises the following steps: expression of HbLFG1, HbPR1, HbHRS1 and HbCAT were analyzed.

HbLFG1 is a gene associated with e.quericola infestation, and expression of HbLFG1 was low in both experimental groups treated with propiconazole. In the group treated with HN-2 extract after inoculation with E.quericola, the expression level of HbLFG1 was highest at 24 hours, which was 1.3 times (12 hours) higher than the group treated with HN-2 extract first. The increase in the expression level of HbNPR1 occurred mainly in the E.quericola-preceded experimental group, reaching maximum values at 24 hours (HN-2 extract group) and 12 hours (propiconazole), the former being 1.7 times higher than the latter, respectively. In addition, in the experimental group treated with HN-2 extract, the activities of four defense-related antioxidant enzymes were relatively increased at each stage. This result indicates that control of e.quericola by HN-2 extract is moderately sustained, not temporarily inhibited.

In this experiment, the expression levels of HbPR1 and HbHRS1 remained stable, while the expression level of HbCAT increased significantly at 72h in the propiconazole group. In other experimental groups, there was no significant change in HbCAT expression. Also, the expression of HbPOD gene was increased in the group treated with benzylpropiconazole after inoculation with Corynebacterium diphtheriae, and was higher at 12 hours.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A preparation method of a n-butanol extract of biocontrol strain HN-2 is characterized by comprising the following steps: the method comprises the following steps:

(2) mixing an n-butanol organic solvent and HN-2 fermentation supernatant according to a volume ratio of 0.5-1.5:1, and standing and extracting at a low temperature of 10-12 ℃ to obtain an upper n-butanol extract;

2. The method for preparing n-butanol extract of biocontrol strain HN-2 as claimed in claim 1, wherein: in the step (1), the biocontrol bacterium B.velezensis HN-2 is subjected to propagation culture, namely, the strain HN-2 is inoculated into an LB liquid culture medium and cultured at 27-28 ℃ and 180-200 rpm for 48 hours; the centrifugation speed was 10000 rpm.

3. The method for preparing n-butanol extract of biocontrol strain HN-2 as claimed in claim 1, wherein: in the step (2), the volume ratio of the n-butanol organic solvent to the HN-2 fermentation supernatant is 1: 1; the extraction time is more than or equal to 12 h.

4. The method for preparing n-butanol extract of biocontrol strain HN-2 as claimed in claim 1, wherein: in the step (3), the rotary evaporation temperature is 50 ℃.

5. An HN-2 fermentation activity n-butanol extract obtained by the method for preparing the n-butanol extract of the biocontrol strain HN-2 as described in any one of claims 1 to 4.

6. Use of HN-2 fermentation active n-butanol extract as claimed in claim 5 for the control of powdery mildew of rubber: the HN-2 fermentation activity n-butanol extract is used for inhibiting the growth of Erysiphe cichoracearum.

7. Use of HN-2 fermentation active n-butanol extract as claimed in claim 6 for the control of powdery mildew of rubber: the HN-2 fermentation activity n-butanol extract antagonizes the growth of the powdery mildew by cell lysis and cytoplasm extravasation of the spores of the powdery mildew, or by inhibiting cell wall or cell membrane synthesis.

8. Use of HN-2 fermentation active n-butanol extract as claimed in claim 5 for the control of powdery mildew of rubber: the HN-2 fermentation activity n-butanol extract is used for inducing the resistance of the rubber trees to the powdery mildew of rubber.

9. Use of HN-2 fermentation active n-butanol extract according to claim 8 for the control of powdery mildew of rubber: the HN-2 fermentation activity n-butyl alcohol extract induces the resistance of the rubber trees to the rubber powdery mildew by regulating the activity of the rubber defense enzyme and the expression level of the defense genes.