CN114916561B

CN114916561B - Biopesticide for preventing and treating crop pathogenic bacteria

Info

Publication number: CN114916561B
Application number: CN202210592308.4A
Authority: CN
Inventors: 杨春玉; 刘远翔; 李春芳
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2022-11-18
Anticipated expiration: 2042-05-27
Also published as: CN114916561A

Abstract

The invention relates to the technical field of biological medicines, in particular to a biological pesticide for preventing and treating crop pathogenic bacteria. Based on a bacteria source S-alkyl-L-cysteine sulfoxide lyase LCC1, in an escherichia coli expression strain E, high expression is realized through codon optimization; and (3) after the fermentation liquor is centrifuged, carrying out spray drying on the supernatant fluid of the crushed thalli to obtain high-concentration enzyme dry powder, and further combining the enzyme dry powder with an activator PLP, a substrate S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide to prepare a composition dry powder product. When in use, the bactericide can be directly spread or applied in holes, or is uniformly mixed with water to be sprayed, flushed, irrigated to roots or drip-irrigated, and only a small amount of bactericide is needed to be applied, so that the bactericide has an obvious effect on preventing and treating pathogenic fungi and bacteria.

Description

Biopesticide for controlling crop pathogenic bacteria

Technical Field

The invention relates to the technical field of biological medicines, in particular to a biological pesticide for preventing and treating crop pathogenic bacteria.

Background

With the increasingly severe global desertification and desertification situations, the ploughable land is less and less, the contradiction that more people and less land are in China is more prominent, and the main method for solving the problems is to improve the multiple cropping index (continuous cropping cultivation) and increase the dosage of pesticide and fertilizer. However, the continuous cropping diseases such as soil-borne diseases and the like cause large-area yield reduction and failure of crops. Repeated over-planting and excessive application of a large amount of chemical fertilizers and pesticides cause serious soil hardening, ecological environment change, serious continuous cropping obstacles and breeding of plant diseases and insect pests. The common morbidity is 10-30%, the plants are usually withered to cause seedling shortage and ridge breaking, the serious morbidity can reach 80-90%, even death in the whole garden causes failure, and the disease is a destructive disease.

At present, the method for preventing and treating crop continuous cropping diseases is mainly a chemical pesticide method, but the effect of preventing and treating soil-borne diseases is not ideal when a large amount of pesticides are applied, pathogenic bacteria can generate drug resistance, the prevention difficulty is increased, and more importantly, environmental pollution and soil structure change are caused. Compared with chemical pesticides, biological control has the advantages of environmental protection, safety, effectiveness and the like if certain natural bacteriostatic active substances are utilized to kill pathogenic bacteria.

Dialkyl thiosulfinates belong to the class of two alkyl-substituted thiosulfinates, some of which occur naturally in disrupted plant tissue. The substances have broad-spectrum antibacterial performance, and have killing effect on gram-negative bacteria, gram-positive bacteria and fungi. It binds to sulfhydryl-containing enzymes required for key metabolic steps and inactivates such enzymes to kill pathogenic bacteria, and bacteria and fungi are less likely to develop resistance to them.

The prior synthesis method of dialkyl sulfo-sulfinate still mainly uses a chemical synthesis method, and researchers in the prior art use chemically synthesized dialkyl sulfo-sulfinate to inhibit the growth of mixed bacteria in the process of spirulina cultivation, such as patent CN112741090A. However, the dialkyl sulphosulfinate prepared by chemical synthesis has two disadvantages, namely, toxic substances such as chloropropene and the like are always remained in the method with high pollution, and the preparation process is relatively complex; secondly, the dialkyl sulphosulfinate is unstable and difficult to store, can be gradually converted into dialkyl thioether substances, and has some bacteriostatic activity after conversion, but is not good enough. This has led to the fact that dialkyl sulphosulfinates prepared by chemical synthesis have not been widely used.

In order to solve the stability problem, pesticides 401 and 402, namely diethyl thiosulfinate, are developed by chemical synthesis in China, the product has a strong sterilization effect, is known as a bacteriostatic king and is relatively stable when being stored in missible oil, but the diethyl thiosulfinate is not a naturally-occurring substance but a biological-control product, has certain toxicity to human bodies, and has strong irritation when being applied at high concentration.

In contrast, diallyl thiosulfinate and dipropyl thiosulfinate are natural substances generated after bulbs of liliaceae crops are crushed, and the pathogenic bacteria control by using the diallyl thiosulfinate and the dipropyl thiosulfinate accords with the concept of green environmental protection. Because the diethyl sulfinate (ethylicin) has stronger killing effect on plant pathogenic fungi, and meanwhile, the low-concentration diethyl sulfinate can be added to improve the bacteriostatic activity of the natural thiosulfinate, the low-concentration diethyl thiosulfinate can not cause stimulation to human bodies. The natural bacteriostatic substance diallyl thiosulfinate and dipropyl thiosulfinate can be quickly, simply and conveniently synthesized by a biocatalysis method, so that the pollution problem caused by a chemical synthesis method and the hazard problem of non-natural thiosulfinate to human bodies can be avoided.

However, the studies on the biocatalytic synthesis of disulfinic acid esters, diallylthiosulfinate and dipropylthiosulfinate, have been relatively rare. At present, the two products are most commonly extracted after the bulb of the liliaceae plant is broken and enzymolyzed, so the operation is complex and the cost is high, and the application of the natural thiosulfinate in agriculture is limited again.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a biopesticide for controlling crop pathogenic bacteria. The natural bacteriostatic active substances diallyl thiosulfinate and dipropyl thiosulfinate are quickly and simply synthesized by a biocatalysis method, so that the green biological control of pathogenic bacteria is realized.

The technical scheme of the invention is as follows:

the idea of the invention is to produce natural bactericidal substances of diallyl thiosulfinate, dipropyl thiosulfinate and diethyl thiosulfinate by a biological catalysis method. The composition is formed by combination, so that the biocatalysis of the bacteria source S-alkyl-L-cysteine sulfoxide lyase LCC1 is realized, and the large-scale in-situ preparation of three types of bactericidal substances is realized. The composition with safer bactericidal activity is formed by combination.

The preparation process of biological pesticide for preventing and controlling crop pathogenic bacteria can be divided into three parts. The method is characterized by comprising the steps of preparing a large amount of S-alkyl-L-cysteine sulfoxide lyase LCC1, simply synthesizing substrates S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide, and compounding the efficient sterilization composition. The S-alkyl-L-cysteine sulfoxide lyases are capable of cleaving cysteine sulfoxide-S-conjugates at the β -position C-S to produce thiosulfinates. The protein sequence of the bacteria source S-alkyl-L-cysteine sulfoxide lyase LCC1 is as follows:

MKDLVYLNYAATSYKKFPATIEALTAYLAENQFMNYGRNAPLLREGLPLLETRQLLADFFQAPSAAQITFTNNATTSLNLALAGILQPGDHVITTMLEHHAVARPLHLLEKERGISVTYVACQKTGLLDVEDIQRAWRTNTKALVMTHASNVLGTILPIEECFQWAQQKGLLTVLDAAQTAGFLPIKMTQMAIDVLAFTGHKSLYGLAGIGGLAFSERGAEAVKPLMAGGTGSHSNSFDQPSFLPDKFEAGTLNSLGILSLNSSIKELNKIGLAAIQKHERTLMQNFLNGLSGLPVTILGTKDVAQTVPVVSITLWNQEETVVAQQLAEQYGIMTRAGLHCAPLAHETAGTLATGTLRFSFGWQTTPEEITWTIHALQELLI。

firstly, producing S-alkyl-L-cysteine sulfoxide lyase LCC1, screening to obtain a bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 sequence due to the difficulty in purification of plant cysteine sulfoxide lyase and poor heterologous expression effect, performing codon optimization, then realizing heterologous expression of the bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 in escherichia coli, culturing a large amount of the engineering strain, adding lactose as an inducer to promote the strain to express a large amount of target enzyme, and then collecting and crushing thalli at high pressure to obtain a crude enzyme solution. Adding maltodextrin with the mass fraction of 10-15% into the crude enzyme solution, fully dissolving, and then carrying out spray drying to obtain dry enzyme powder.

Secondly, a large amount of substrates S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide are synthesized by a chemical method, the used reagents are low in price, pollution-free, mild in reaction conditions, free of complex equipment, simple and convenient to operate, high in conversion rate and high in purity. The preparation method comprises the following steps: the first step is a substitution reaction, which is based on the principle that cysteine and substituents such as bromopropene, bromopropane and bromoethane undergo a substitution reaction under alkaline conditions with ethanol as a medium to form an L-cysteine-S-conjugate. In the first step, the volume ratio of ethanol to water is (4-5): 1, taking the sodium hydroxide as a medium, dissolving a certain amount of sodium hydroxide particles in the medium to form a solution, wherein the final concentration of the sodium hydroxide is 0.8-4.5 mol/L, and then adding a certain amount of L-cysteine to stir until the sodium hydroxide is completely dissolved, wherein the final concentration is 0.4-2.5 mol/L. Then adding substituent groups with the same equivalent weight as that of the L-cysteine, such as bromopropylene, bromopropane and bromoethane, sealing the system by using a sealing film, continuously stirring until the solution is transparent and clear, and then stirring overnight for reaction for 8-12 hours. After the reaction is finished, acetic acid is added to adjust the pH value to 4-6 so as to neutralize sodium hydroxide and to ensure that the product S-alkyl-L-cysteine in the first step is crystallized and separated out in a large quantity. After a little cooling, the solid can be filtered out and dried in a 65 ℃ oven. The second reaction, which is an oxidation reaction in which the thioether bond in the S-alkyl-L-cysteine prepared in the first step can be oxidized to the sulfoxide form using a 30% hydrogen peroxide solution, takes only a few minutes and is largely exothermic. And ending the reaction when the S-alkyl-L-cysteine is completely dissolved in 30 percent hydrogen peroxide solution. At this time, cold ethanol is added to precipitate white solid, which is fixed and dried to obtain the target substrate S-alkyl-L-cysteine sulfoxide, such as S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide.

Then, mixing dry enzyme powder, a substrate and activator PLP powder according to the mass ratio of (1-10): (1-3): (0.001-0.005) in a certain proportion. Wherein the substrate comprises three components, namely S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide, and the mass ratio of the three components is (3-4): (3-4): (2-4). And performing bacteriostasis experiments on the prepared mixture of the two substrates for inhibiting fungi and bacteria, and comparing to obtain the optimal substrate combination formula.

If S-ethyl-L-cysteine sulfoxide is not added, the mass ratio of S-allyl-L-cysteine sulfoxide to S-propyl-L-cysteine sulfoxide is (1. And performing bacteriostasis experiments on the prepared mixture of the two substrates for inhibiting fungi and bacteria, and comparing to obtain the optimal substrate combination formula.

Two application methods of a biological pesticide for preventing and controlling crop pathogenic bacteria are provided. 0.1-1 g of the product can be taken and added with 1L of water, and after the product is fully stirred and dissolved, the product is sprayed, drip-irrigated or flushed; the composition may also be applied directly to the site or to the site.

The invention has the beneficial effects that:

(1) The invention avoids using chemical method to directly synthesize dialkyl sulphosulfinate, thereby avoiding pollution, and simultaneously, the product of the invention is natural and pollution-free because toxic substituent groups such as chloropropene and the like are not used.

(2) The invention utilizes a biocatalysis method to synthesize two natural substances of diallyl thiosulfinate and dipropyl thiosulfinate, and simultaneously utilizes a biocatalysis method to synthesize diethyl thiosulfinate and combines the diethyl thiosulfinate with a proper amount for application, and the combination of the two substances can obtain better sterilization effect.

(3) The invention uses a biological catalysis method to compound enzyme and substrate after drying to form a composition product, solves the problem of activity reduction caused by instability of the thiosulfinate synthesized in advance, and can fully exert the biological activity of the thiosulfinate.

(4) The substrate synthesis method adopted by the invention has the advantages of simple steps, rapid synthesis, no pollution, low reagent price, high purity of the synthesized substrate and low production cost.

Drawings

FIG. 1 shows the verification of E.coli BL21 transformant;

FIG. 2 is a graph of the inhibition of three phytopathogens by two products;

FIG. 3 is a graph showing the inhibition of plant soft rot pathogens by two products;

FIG. 4 is a graph of the stability of spray dried enzyme powder when left at different temperatures.

Detailed Description

In order to make the technical means, technical features, objects and technical effects of the present invention easily understandable, the present invention is further described below with reference to the specific drawings.

Example 1:

optimization of conditions for efficient expression of enzymes

Escherichia coli is used for expressing the bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1, and in order to obtain high expression quantity and low production cost of protein, the sequence is sent to the generation for sequence synthesis after codon optimization.

The optimized sequence is connected with a pET24a expression vector, the expression vector is transformed into escherichia coli BL21, a transformant is selected for PCR verification, and the size of a target fragment is 1000-1500bp and is shown in figure 1. Transformants which were confirmed to be correct were arbitrarily selected, and for example, transformant No. 4 was selected and inoculated into 1L of LB medium for fermentation culture.

(1) Induction temperature: the induction time is respectively set to 16 ℃ for 12h, 20 ℃ for 10h, 25 ℃ for 8h and 30 ℃ for 6h.

(2) IPTG concentration: IPTG addition was set at 0.1, 0.25, 0.5, 1.0mM, respectively.

(3) IPTG addition time: respectively at OD _600nm When the concentration reached 0.4, 0.6, 0.8 or 1.0, IPTG was added to induce the cells.

(4) Lactose induction:

through serial optimization, the optimal expression conditions are finally determined as follows: at OD _600nm The enzyme activity unit is defined as enzyme amount required for catalyzing and generating 1 mu g of pyruvic acid under the conditions of 37 ℃ and pH 7, when S-allyl-L-cysteine sulfoxide is taken as a substrate, the maximum enzyme yield of crude enzyme liquid is 2970U/ml, and the specific enzyme activity reaches 456U/mg, and the specific enzyme activity is shown in table 1.

Table 1: enzyme activity determination of crude enzyme solution

Example 2:

preparation of enzyme Dry powder

And (2) carrying out heterogeneous expression on the Escherichia coli liquid of the bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 to collect thalli, crushing cells under high pressure, and carrying out spray drying on the crushed liquid to form enzyme dry powder. Wherein, the spray drying condition is that the inlet temperature is 150-180 ℃, 10-15% of maltodextrin and 5-10% of soluble starch are added as protective agents, and the enzyme activity loss after drying is 42%; the enzyme powder is prepared by a freeze drying process, trehalose is used as a protective agent, the pre-freezing temperature is-40 ℃, the trehalose concentration is 1-2%, the thickness of an enzyme solution is 3-5mm, the pre-freezing time is 10 hours, and the enzyme activity loss of the prepared freeze-dried enzyme preparation is 13%. Comprehensive cost, and more economical and effective preparation of the enzyme by adopting a freeze drying mode.

Example 3:

stability of enzyme Dry powder

The enzyme dry powder was placed at 4 deg.C, 25 deg.C, 30 deg.C, 35 deg.C and 40 deg.C, respectively, and sampled once a month at intervals for 6 months. The enzyme activity was measured by DNPH method in Tris-HCl buffer at pH 7.0. The relative enzyme activity value was calculated with the initial enzyme activity as 100%, and a curve of enzyme activity stability was plotted as shown in FIG. 4. Thus, the dry enzyme powder has good stability and is suitable for long-term storage.

Example 4:

product preparation

According to the catalytic reaction characteristics of the enzyme, the composition proportion and the formula of the product are optimized to form soluble powder containing the enzyme stabilizer, the activator PLP and the substrate. The powder can be directly applied to the field or the root of crops, and can also be used in modes of spraying, drip irrigation, flushing application and the like after being dissolved in water.

Example 5:

experiment of product bacteriostasis

The first is the inhibition of phytopathogenic fungi. Taking two products with proper amount, namely, the ratio of the substrate to the S-allyl-L-cysteine sulfoxide to the substrate to the S-propyl-L-cysteine sulfoxide to the S-ethyl cysteine sulfoxide is 4:4:2 water is added to prepare a solution, and the ratio of S-allyl-L-cysteine sulfoxide to S-propyl-L-cysteine sulfoxide is 1:1 Water was added to prepare a solution, and after 30 minutes, the amounts of the products diallylthiosulfinate and dipropylthiosulfinate in the solution were measured by the DNPH method. Three phytopathogenic fungi, which can cause the rotting of sweet potatoes and the withering of tomatoes, respectively, were activated with solid PDA medium. After activation, three plant pathogenic fungi are respectively inoculated on a PDA (personal digital assistant) flat plate to serve as a control group, two reaction liquids of the product are respectively added into a PDA solid flat plate in an experimental group, the concentration of thiosulfinate is controlled to be 20ug/ml, then the three activated plant pathogenic fungi are inoculated on a flat plate in the experimental group, and the three activated plant pathogenic fungi are kept standing and cultured for about 3 days at the temperature of 30 ℃ to observe the growth conditions of the control group and the experimental group. As shown in FIG. 2, the plant pathogenic fungi in the control group grew vigorously, while none of the plant pathogenic fungi in the experimental group grew, indicating that the final effective ingredient of the product had excellent fungistatic properties.

The inhibitor also has good inhibition effect on the pathogenic bacteria of the soft rot of plants. The method is broth dilution (turbidimetry), and the principle is that a product with a certain concentration and a liquid culture medium containing the test bacteria are subjected to a series of multiple dilutions, whether the test bacteria grow visually or not is observed after the culture, and the lowest concentration of the product capable of inhibiting the visible growth of the test bacteria in the flesh and eyes is defined as the lowest (or Minimum) Inhibitory Concentration (MIC) of the product.

The test bacterial liquid is then adjusted to an absorbance value of 0.090-0.10 (measured with a UV-1700 UV-Vis spectrophotometer) at a wavelength of 625nm using sterilized normal saline, and then mixed with sterilized MH broth in a ratio of 1: and (5) diluting by 200. Adding tested bacterium liquid into a row of 1-11 holes of 12 holes in a 96-hole plate, adding a blank MH culture medium into the 12 th hole, adding 190 mu l of the bacterium liquid into the first hole, adding 100 mu l of the bacterium liquid into the other holes, adding 10 mu l of the product into the first hole, fully blowing, sucking and uniformly mixing, then adding 100 mu l of the product into the next hole, fully diluting according to a 2-time dilution method, removing 100 mu l of the product after blowing, sucking and uniformly mixing the product into the 10 th hole, and not performing any treatment on the 11 th hole. Two replicates were made and kanamycin was used as a control, and the cultures were incubated at 37 ℃ for 16-18 h.

The concentrations of diallyl thiosulfinate and dipropyl thiosulfinate were estimated by DNPH determination of pyruvic acid concentration according to the previous method, and adjusted to such a concentration that the concentration of the drug solution in the first well was 79.85. Mu.g/ml. As shown in FIG. 3, it is understood that the MIC value of the product with S-ethyl-L-cysteine sulfoxide against the pathogenic bacteria of soft rot is 9.9. Mu.g/ml, and the MIC value of the product without S-ethyl-L-cysteine sulfoxide against the pathogenic bacteria of soft rot is 19.8. Mu.g/ml, indicating that the addition of S-ethyl-L-cysteine sulfoxide can enhance the bacteriostatic effect of the product

Based on the two bacteriostasis experiments, the minimum bacteriostasis concentration of the product is determined to be 20 mu g/ml, and according to the MIC value, the dry enzyme powder, the substrate fine powder and the enzyme activator PLP powder are mixed according to the mass ratio of 4:1:1, the components are mixed evenly to form the product, and the product is prepared at present according to the amount of 0.6 g/L. Wherein the components of the substrate fine powder are S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide, and the mass ratio of the S-allyl-L-cysteine sulfoxide to the S-ethyl-L-cysteine sulfoxide is 4:4:2.

in summary, the embodiments of the present invention are merely exemplary and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made according to the content of the claims of the present invention should fall within the technical scope of the present invention.

Sequence listing

<110> Shandong university

<120> biopesticide for controlling crop pathogenic bacteria

<140> 2022105923084

<141> 2022-05-27

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 382

<212> PRT

<213> Bacillus cereus (Bacillus cereus)

<400> 1

Met Lys Asp Leu Val Tyr Leu Asn Tyr Ala Ala Thr Ser Tyr Lys Lys

1 5 10 15

Phe Pro Ala Thr Ile Glu Ala Leu Thr Ala Tyr Leu Ala Glu Asn Gln

20 25 30

Phe Met Asn Tyr Gly Arg Asn Ala Pro Leu Leu Arg Glu Gly Leu Pro

35 40 45

Leu Leu Glu Thr Arg Gln Leu Leu Ala Asp Phe Phe Gln Ala Pro Ser

50 55 60

Ala Ala Gln Ile Thr Phe Thr Asn Asn Ala Thr Thr Ser Leu Asn Leu

65 70 75 80

Ala Leu Ala Gly Ile Leu Gln Pro Gly Asp His Val Ile Thr Thr Met

85 90 95

Leu Glu His His Ala Val Ala Arg Pro Leu His Leu Leu Glu Lys Glu

100 105 110

Arg Gly Ile Ser Val Thr Tyr Val Ala Cys Gln Lys Thr Gly Leu Leu

115 120 125

Asp Val Glu Asp Ile Gln Arg Ala Trp Arg Thr Asn Thr Lys Ala Leu

130 135 140

Val Met Thr His Ala Ser Asn Val Leu Gly Thr Ile Leu Pro Ile Glu

145 150 155 160

Glu Cys Phe Gln Trp Ala Gln Gln Lys Gly Leu Leu Thr Val Leu Asp

165 170 175

Ala Ala Gln Thr Ala Gly Phe Leu Pro Ile Lys Met Thr Gln Met Ala

180 185 190

Ile Asp Val Leu Ala Phe Thr Gly His Lys Ser Leu Tyr Gly Leu Ala

195 200 205

Gly Ile Gly Gly Leu Ala Phe Ser Glu Arg Gly Ala Glu Ala Val Lys

210 215 220

Pro Leu Met Ala Gly Gly Thr Gly Ser His Ser Asn Ser Phe Asp Gln

225 230 235 240

Pro Ser Phe Leu Pro Asp Lys Phe Glu Ala Gly Thr Leu Asn Ser Leu

245 250 255

Gly Ile Leu Ser Leu Asn Ser Ser Ile Lys Glu Leu Asn Lys Ile Gly

260 265 270

Leu Ala Ala Ile Gln Lys His Glu Arg Thr Leu Met Gln Asn Phe Leu

275 280 285

Asn Gly Leu Ser Gly Leu Pro Val Thr Ile Leu Gly Thr Lys Asp Val

290 295 300

Ala Gln Thr Val Pro Val Val Ser Ile Thr Leu Trp Asn Gln Glu Glu

305 310 315 320

Thr Val Val Ala Gln Gln Leu Ala Glu Gln Tyr Gly Ile Met Thr Arg

325 330 335

Ala Gly Leu His Cys Ala Pro Leu Ala His Glu Thr Ala Gly Thr Leu

340 345 350

Ala Thr Gly Thr Leu Arg Phe Ser Phe Gly Trp Gln Thr Thr Pro Glu

355 360 365

Glu Ile Thr Trp Thr Ile His Ala Leu Gln Glu Leu Leu Ile

370 375 380

Claims

1. A composition for controlling crop pathogens, comprising: dry enzyme powder comprising a bacterially derived S-alkyl-L-cysteine sulfoxide lyase LCC1, an activator pyridoxal phosphate PLP, and a substrate; wherein the substrate is selected from one or more of S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide; substrate: bacteria source S-alkyl-L-cysteine sulfoxide lyase LCC1 dry enzyme powder: the activator is (1 to 10): (1 to 3): (0.001 to 0.005); the protein sequence of the bacteria source S-alkyl-L-cysteine sulfoxide lyase LCC1 is as follows: <xnotran> MKDLVYLNYAATSYKKFPATIEALTAYLAENQFMNYGRNAPLLREGLPLLETRQLLADFFQAPSAAQITFTNNATTSLNLALAGILQPGDHVITTMLEHHAVARPLHLLEKERGISVTYVACQKTGLLDVEDIQRAWRTNTKALVMTHASNVLGTILPIEECFQWAQQKGLLTVLDAAQTAGFLPIKMTQMAIDVLAFTGHKSLYGLAGIGGLAFSERGAEAVKPLMAGGTGSHSNSFDQPSFLPDKFEAGTLNSLGILSLNSSIKELNKIGLAAIQKHERTLMQNFLNGLSGLPVTILGTKDVAQTVPVVSITLWNQEETVVAQQLAEQYGIMTRAGLHCAPLAHETAGTLATGTLRFSFGWQTTPEEITWTIHALQELLI. </xnotran>

2. The composition for controlling crop pathogens according to claim 1, wherein: the substrate comprises S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide, and the corresponding proportion is (3 to 4): (3 to 4): (2 to 4).

3. The composition for controlling pathogenic bacteria in crops of claim 1, wherein: the substrate comprises S-allyl-L-cysteine sulfoxide and S-propyl-L-cysteine sulfoxide, and the corresponding proportion is (1 to 3): 1.

4. the composition for controlling crop pathogens according to claim 1, wherein: the S-alkyl-L-cysteine sulfoxide lyases are capable of cleaving cysteine sulfoxide-S-conjugates at the β -position C-S to produce thiosulfinates.

5. The composition for controlling pathogenic bacteria in crops of claim 1, wherein: the preparation method of the bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 comprises the following steps: expressing a bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 in a host cell, crushing the cell to obtain a crude enzyme solution, adding maltodextrin with the mass fraction of 10-15% into the crude enzyme solution, and performing spray drying to obtain dry enzyme powder.

6. The composition for controlling crop pathogens according to claim 2, wherein: the three substrates S-allyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide and S-ethyl-L-cysteine sulfoxide are S-alkyl-L-cysteine sulfoxide, and the preparation method of the S-alkyl-L-cysteine sulfoxide comprises two steps of substitution reaction and oxidation reaction.

7. The composition for controlling crop pathogens according to claim 6, wherein: the steps of the substitution reaction are as follows:

(1) The volume ratio of the ethanol to the water is (4 to 5): 1, and taking the mixture as a medium;

(2) Dissolving a certain amount of sodium hydroxide particles in the medium to form a solution, wherein the final concentration of the sodium hydroxide is 0.8-4.5 mol/l;

(3) Adding L-cysteine, stirring until the L-cysteine is completely dissolved, wherein the final concentration of the L-cysteine is 0.4 mol to 2.5 mol/L;

(4) Adding a substituent with the same equivalent weight as that of the L-cysteine, such as one or more of bromopropene, bromopropane and bromoethane, sealing a system, and stirring for reaction for 8 to 12 hours;

(5) After the reaction is finished, adding acetic acid to adjust the pH to 4 to 6;

(6) After a little cooling the solid is filtered off and dried in an oven to obtain S-alkyl-L-cysteine.

8. The composition for controlling pathogenic bacteria in crops of claim 6, wherein: the oxidation reaction comprises the following steps:

(1) Dissolving S-alkyl-L-cysteine prepared in the substitution reaction by 30 percent hydrogen peroxide solution;

(2) Adding cold ethanol to precipitate white solid, and drying to obtain the target substrate S-alkyl-L-cysteine sulfoxide.

9. Use of a composition according to claim 1 for controlling crop pathogens in any of the following applications:

1) The solid of the composition can be directly applied, scattered or hole-applied to the roots of crops to prevent and control continuous cropping cultivation diseases or bacterial and fungal diseases of various crops;

2) The solid of the composition can be dissolved in water before use, is uniformly mixed and then is sprayed on leaves of crops or disease-causing parts of plants to prevent and treat continuous cropping cultivation diseases or bacterial and fungal diseases of various crops;

3) The solid of the composition can be dissolved in water, and the mixture is uniformly mixed and used for root irrigation, drip irrigation or flushing application to prevent and treat continuous cropping cultivation diseases or bacterial and fungal diseases of various crops.