CN114451425B - Application of fructus forsythiae extract in preventing and treating kiwifruit canker - Google Patents

Abstract

The invention discloses application of a forsythia suspense extract in preventing and treating kiwifruit canker caused by pseudomonas syringae kiwifruit pathogenic variety, wherein the forsythia suspense extract is an ethanol extract of forsythia suspense or an n-butanol re-extraction extract of the ethanol extract of forsythia suspense. The forsythia suspense extract provided by the invention has an inhibitory activity effect on kiwi canker pathogenic bacteria, and is expected to be developed into a pollution-free plant-derived pathogenic bacteria inhibiting and killing agent due to the characteristics that the forsythia suspense raw material has wide sources, is non-toxic to human bodies, is harmless to crops and has easy degradation when being used as a plant-derived pathogenic bacteria inhibiting and killing agent.

Description

Application of fructus forsythiae extract in preventing and treating kiwifruit canker

Technical Field

The invention belongs to the field of plant pesticides, and particularly relates to application of a forsythia suspense extract to prevention and treatment of kiwifruit canker.

Background

Plant diseases are an important factor affecting agricultural production, and different pathogenic bacteria can cause diseases of various crops, which causes great loss to agricultural economy. The kiwifruit canker is a destructive bacterial disease seriously threatening the production of kiwifruit and is listed as a national forest plant quarantine object. The disease is fierce in coming, causes damage to the whole garden due to epidemic years, causes great economic loss, reduces yield, causes fruit peel thickening, turns sour fruit taste, reduces fruit size, varies fruit shape, reduces quality and reduces commodity value. The commercialization of agricultural chemicals began in the early days, but the agricultural chemicals were chemical ones at that time, and the rapid development of agricultural chemicals brought a great benefit in the yield and quality of agricultural products. However, with the continuous popularization and application of chemical pesticides, the harm caused by the chemical pesticides is not negligible. The use of a large amount of chemical pesticides can cause serious pollution to the environment, can cause damage to non-target organisms, and can cause harm to the health of human beings due to pesticide residues. With the improvement of the life quality of people, more attention is paid to food safety. Therefore, in order to enable agriculture to develop sustainable health, the development of new pesticides which do not harm the environment and human health becomes an important task for pesticide research, biological pesticides becomes a research hotspot of modern agriculture development, and plant-derived pesticides are an important field in biological pesticides. Because the plant source pesticide has the following characteristics: the earth has rich plant resources and various varieties; different from chemical pesticides, the biological pesticide of plant source has stronger pertinence and cannot cause damage to non-target organisms; the plant source extract has diversified components, the active components have synergistic effect, and the action targets of different active components on diseases are different, so compared with chemical pesticides, when the plant source biological pesticide is adopted, pathogenic bacteria are not easy to generate drug resistance on the pesticide; since the botanical pesticide is a natural product derived from nature, the botanical pesticide can be naturally digested in nature, and substances destructive to the environment can not be generated, so that the development of the efficient biopesticide has important significance for protecting ecological environment, human and animal health.

Since 2008 pseudomonas syringae kiwifruit pathogenic variety (Psa) has been threatening the world kiwifruit industry, destroying orchards in europe, south america, and new zealand. The "kiwifruit canker disease" caused by pseudomonas syringae kiwifruit pathovar cata (Psa) was first reported in italy and it has resulted in economic losses of tens of millions of dollars in the next four years. The disease is quick in outbreak and causes serious loss to the kiwi fruit producing areas all around the world. At present, chemical agents are mainly applied to control kiwifruit canker in production, and researches show that Psa has generated resistance to partial pesticides.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to provide a new plant-source antibacterial drug source for preventing and treating kiwifruit canker caused by pseudomonas syringae kiwifruit pathogenic variety.

The technical scheme of the invention is as follows: use of a forsythia suspense extract for the control of kiwifruit canker disease caused by pseudomonas syringae kiwifruit pathogenic varieties, the forsythia suspense extract being any one of the following:

a. an ethanol extract of the fruit of weeping forsythia,

b. n-butanol re-extraction of ethanol extract of fructus forsythiae.

The raw material of the forsythia is the fruit or the whole plant of the forsythia.

Further, the ethanol extract adopts ethanol with volume fraction of not less than 75%. Further 90-98% ethanol, preferably 95% ethanol.

Further, the n-butanol re-extract of the ethanol extract of forsythia is prepared by the following method:

(1) leaching the dried and crushed fructus forsythiae with 95% ethanol for 12-24 hours, then ultrasonically extracting at 45-50 ℃ for 4-6 hours to fully leach active ingredients in the fructus forsythiae, and collecting leaching liquor;

(2) concentrating the collected leaching solution to obtain ethanol extract of fructus forsythiae, and recovering ethanol as solvent;

(3) extracting the ethanol extract of fructus forsythiae with n-butanol, and concentrating to obtain n-butanol extract.

Further, the n-butanol extract was concentrated using vacuum at 35 ℃.

The forsythia extract for inhibiting and killing kiwifruit canker disclosed by the invention can be used for preparing a preparation for inhibiting and killing kiwifruit canker pathogenic bacteria. Wherein the ethanol extract of fructus forsythiae or N-butanol re-extract of the ethanol extract of fructus forsythiae can be directly used for preparing a preparation for inhibiting and killing pathogenic bacteria of kiwifruit ulcer after dissolving in N, N-dimethylformamide.

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

1. the fructus forsythiae active component for inhibiting and killing the pathogenic bacteria of the kiwifruit ulcer disclosed by the invention can be prepared into the inhibiting and killing preparation of the pathogenic bacteria of the kiwifruit ulcer in the dosage forms of aqueous solution, soluble powder, wettable powder, concentrated suspension, granules and the like. The preparation method of various dosage forms is a conventional method in the field, and the used auxiliary agent in the preparation process of various dosage forms is a common auxiliary agent in the field of pesticides. The concentration of fructus forsythiae extract in the solution prepared by the dosage forms during use is in the range of 0.1mg/mL-1000 mg/mL.

2. The forsythia suspense extract provided by the invention has an inhibitory activity effect on kiwi canker pathogenic bacteria, and is expected to be developed into a pollution-free plant-derived pathogenic bacteria inhibiting and killing agent due to the characteristics that the forsythia suspense raw material has wide sources, is non-toxic to human bodies, is harmless to crops and has easy degradation when being used as a plant-derived pathogenic bacteria inhibiting and killing agent.

Drawings

FIG. 1 anti-PSA activity of different extracts of Forsythia suspensa; 1 is n-butanol extract, 2 is ethanol extract, 3 is ethyl acetate extract, and 4 is petroleum ether extract

FIG. 2 comparison of anti-PSA activity of alcoholic forsythia extract with other plant extracts;

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

Example 1 preparation of Total ethanol extract of Forsythia suspense and re-extract of petroleum ether, re-extract of ethyl acetate, re-extract of n-butanol

1. Pulverizing dried fructus forsythiae, and sieving with 40 mesh sieve to obtain fructus forsythiae powder.

2. 95% ethanol (not less than 75%, typically 90-98%) in an amount corresponding to about 7 times (typically controlled in the range of 5-10 times) the mass of the feedstock was extracted at room temperature (typically at a temperature in the range of 18-24 ℃) for about 20 hours (typically controlled in the range of 12-24 hours).

3. Ultrasonic waves are carried out for 6h (generally controlled in the range of 4-8h) at about 50 ℃. Filtering with Buchner funnel under negative pressure, and removing residue to obtain filtrate.

4. Further extracting the residue with 95% ethanol with volume of about 6 times (generally 5-10 times) of the raw material for 6 hr (generally 4-8 hr) to extract active components from fructus forsythiae, filtering with Buchner funnel under negative pressure, and removing residue to obtain filtrate.

5. Combining the filtrates, concentrating the filtrate with rotary evaporator in water bath at about 50 deg.C (generally controlled at 45-55 deg.C) to obtain ethanol extract, and storing at 4 deg.C.

6. Taking 20g of fructus forsythiae extract, adding 10 times of n-butanol for extraction, filtering by using filter paper to obtain filtrate, adding n-butanol with proper volume into the residual solid for repeated extraction, and filtering by using filter paper to obtain filtrate. Until the extract is colorless, and vacuum concentrating the filtrate at 35 deg.C with rotary evaporator to obtain n-butanol re-extract of fructus forsythiae alcohol extract.

7. And (3) taking 20g of forsythia ethanol extract, adding 10 times of ethyl acetate for extraction, filtering by using filter paper to obtain filtrate, adding the ethyl acetate with proper volume into the residual solid again for repeated extraction, and filtering by using the filter paper to obtain the filtrate. Until the extract is colorless, concentrating all the obtained filtrate at 35 deg.C under vacuum with rotary evaporator to obtain ethyl acetate extract of fructus forsythiae alcohol extract.

8. And (3) taking 20g of forsythia ethanol extract, adding petroleum ether with the volume 10 times that of the forsythia ethanol extract for extraction, filtering by using filter paper to obtain filtrate, adding petroleum ether with the proper volume into the residual solid again for repeated extraction, and filtering by using the filter paper to obtain the filtrate. Until the extract is colorless, concentrating all the obtained filtrate at 35 deg.C under vacuum with rotary evaporator to obtain petroleum ether re-extract of fructus forsythiae alcoholic extract.

EXAMPLE 2 determination of biological Activity

Under the condition of separation, the antibacterial activity of each extract on pseudomonas syringae kiwi fruit pathogenic variant (Psa) is compared by a filter paper sheet method. Firstly, selecting common chemical qualitative filter paper, preparing the filter paper into a circular filter paper sheet with the diameter of 6mm by using a puncher, and putting the circular filter paper sheet into a centrifugal tubeAnd after high-pressure steam sterilization, drying for later use. Transferring the Psa strain to an LB solid plate for activation, inoculating a single colony into an LB liquid culture medium by using an aseptic inoculating loop after 2-3 times of activation, carrying out shake culture at 25 ℃ and 120rpm/min for 24h, sucking bacterial liquid, and diluting the bacterial liquid with the LB liquid culture medium to the concentration of 1 × 10⁷CFU/mL. Under the aseptic condition, sucking 100 mul of diluted bacterial suspension, uniformly coating the diluted bacterial suspension on an LB solid plate, standing for 2-3 minutes, clamping sterilized filter paper sheets by using sterile forceps, and pasting the filter paper sheets on the surface of the LB solid plate, wherein 3 filter paper sheets are uniformly placed on each plate and are arranged in a regular triangle. A small amount of the four forsythia extractum prepared in example 1 is prepared into a solution with the concentration of 100mg/mL by using N, N-dimethylformamide to serve as a sample for standby experiment.

Respectively sucking 5 mu L of each liquid medicine (the medicine content is 0.5mg), dripping the liquid medicine on a filter paper sheet, marking the liquid medicine, taking the treatment of dripping 5 mu L of dimethyl sulfoxide as a blank control and the treatment of dripping 5 mu L of streptomycin sulfate with the concentration of 50mg/mL as a positive control, and repeating each treatment for three times. The flat plate after adding the medicine is kept stand for 1h at room temperature, and then is put into a constant temperature incubator with the temperature of 25 ℃ for 24 h. And taking out the filter paper, observing whether an obvious bacteriostatic zone exists around the filter paper, and measuring the diameter of the bacteriostatic zone. Results are expressed as mean and standard deviation (mean ± SD). The judgment criteria are shown in Table 1

TABLE 1 criteria

TABLE 2 bacteriostatic Activity of Forsythia suspensa extract on Psa

Note: the diameter of the inhibition zone is expressed by the mean value plus or minus standard deviation; lower case letters indicate significance p < 0.05.

As shown in FIG. 1 and Table 2, it is understood that the inhibitory effect of the re-extract of n-butanol from forsythia on Psa is the best, and at a concentration of 100mg/mL, the zone diameter is 16.53. + -. 0.12mm, the MIC is 0.125mg/mL, and the inhibitory activity exceeds that of ethanol extract of forsythia (MIC is 0.5 mg/mL). Secondly, the extract with stronger inhibitory action is the re-extracted extract of forsythia ethyl acetate, the diameter of the inhibition zone is 14.47 +/-0.21 mm, the effect is slightly lower than that of the ethanol extract, and the extract has significant difference (p is less than 0.05) with the re-extracted extract of n-butyl alcohol. The fructus forsythiae petroleum ether re-extract has poor bacteriostatic action, the diameter of a bacteriostatic zone is 12.67 +/-0.17 mm, and the bacteriostatic activity of the fructus forsythiae petroleum ether re-extract is remarkably different from that of an n-butyl alcohol re-extract (p is less than 0.05). Therefore, the forsythia n-butyl alcohol re-extracted substance has the best bacteriostatic effect on Psa. This indicates that the polarity of the bacteriostatic active substance in forsythia is relatively high and is mainly concentrated in the n-butanol re-extract.

Example 3 MIC determination of minimum inhibitory concentration

The minimum inhibitory concentrations MIC of the ethanol extract and n-butanol re-extract were determined with partial modification to the method of Yang et al [ International Journal of Antimicrobial Agents,2006,27(4):325-330 ]. The concentration of fructus forsythiae extract and re-extract is set to 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6 and 4.0mg/mL, and corresponding volume of medicated culture medium (without Psa) is set as blank control and DMSO is set as negative control. Incubated at 28 ℃ for 24h and OD600nm was measured using a microplate reader. The concentration at which the absorbance of the test group was not significantly different from that of the control group was defined as the minimum inhibitory concentration. mu.L of the above culture medium was applied, and cultured at 28 ℃ for 48 hours to observe colony formation. The concentration corresponding to plates with no longer growing colonies was defined as the minimum inhibitory concentration (Lovato et al, Frontiers in microbiology,2019(10): 2362). The results showed that the MIC of the ethanol extract was 0.5mg/mL and the MIC of the n-butanol re-extract was 0.125 mg/mL.

Example 4

The minimum inhibitory concentration of the 4 plant ethanol extracts on Psa is further determined by using a two-fold dilution method through the inhibitory activity of the ethanol crude extracts of 4 medicinal plants such as fructus forsythiae, liquorice, scutellaria baicalensis and selfheal on Psa. The minimum inhibitory concentration of the ethanol extract of forsythia was found to be 0.5mg/mL for Psa, the minimum inhibitory concentration of licorice and scutellaria was found to be 6.25mg/mL, 2.4mg/mL, and the minimum inhibitory concentration of selfheal was found to be 12.5 mg/mL. The minimum bacteriostatic concentration of the selfheal to Psa is 2 times that of the liquorice and the radix scutellariae, and 4 times that of the fructus forsythiae, so that the inhibition effect of the fructus forsythiae to Psa is stronger than that of the liquorice, the radix scutellariae and the selfheal.

Example 5 field test

The field experiment effect shows that the kiwi fruit tree coated with the forsythia suspense sterilization preparation has better growth vigor, the original branches which do not sprout start to grow new leaves, the disease spots start to be reduced, and no infectious bacterium liquid is seen; the Chinese gooseberry treated by infusion has better growth vigor than Chinese gooseberry trees treated by scraping disease spots and applying medicine. The worst effect is the kiwi fruit tree without scraping the lesion and applying the medicine. In the field experiment process, the infusion treatment is easier to be absorbed by plants, the lesion tissues can be quickly reached, and the loss of the liquid medicine in the treatment process is less, so the treatment effect is most obvious. For the kiwi fruit trees which are scraped and treated by the medicine, the medicine can directly act on the bacterial plaque infection part under the plant epidermis and can be easily absorbed by the plant, so the effect is better. For the kiwi fruit trees which are not scraped with disease spots and are directly coated with the medicine, the medicine is slowly absorbed due to the obstruction of plant epidermis, and the medicine is easily washed by rainwater in rainy days to cause medicine loss, so the effect is obviously poor.

Through field tests, the prevention and control effects of basal application of the bio-organic fertilizer and infusion of the composite bactericide containing the forsythia suspensa extract on the kiwi fruit trees in the winter dormancy stage are preliminarily confirmed.

Claims (6)

1. The fructus forsythiae extract is used for preventing and treating kiwifruit canker caused by pseudomonas syringae kiwifruit pathogenic variety, and is ethanol extract of fructus forsythiae or n-butanol re-extract of ethanol extract of fructus forsythiae.

2. The use according to claim 1, wherein the ethanol extract is obtained by using ethanol with a volume fraction of not less than 75%.

3. Use according to claim 2, characterized in that the ethanol extract uses an extraction solvent of 90-98% ethanol by volume fraction.

4. Use according to claim 3, characterized in that the ethanol extract uses 95% by volume of ethanol as extraction solvent.

5. The use as claimed in claim 1, wherein the n-butanol re-extract of the ethanol extract of forsythia suspensa is prepared by the following method:

(1) leaching the dried and crushed fructus forsythiae with 95% ethanol for 12-24 hours, then ultrasonically extracting at 45-50 ℃ for 4-6 hours to fully leach active ingredients in the fructus forsythiae, and collecting leaching liquor;

(2) concentrating the collected leaching solution to obtain ethanol extract of fructus forsythiae, and recovering ethanol as solvent;

(3) extracting the ethanol extract of fructus forsythiae with n-butanol, and concentrating to obtain n-butanol extract.

6. Use according to claim 5, characterized in that the concentration of the n-butanol extract is carried out by vacuum concentration at 35 ℃.

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