CN114806993B - Bacterial enzyme composition for preventing and controlling root-knot nematode and preparation method thereof - Google Patents
Bacterial enzyme composition for preventing and controlling root-knot nematode and preparation method thereof Download PDFInfo
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- CN114806993B CN114806993B CN202210586649.0A CN202210586649A CN114806993B CN 114806993 B CN114806993 B CN 114806993B CN 202210586649 A CN202210586649 A CN 202210586649A CN 114806993 B CN114806993 B CN 114806993B
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- root
- alkyl
- cysteine sulfoxide
- lcc1
- bacillus subtilis
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Abstract
The invention relates to the technical field of biological medicines, in particular to a bacterial enzyme composition for preventing and controlling root knot nematodes and a preparation method thereof. Bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 was overexpressed and integrated in Bacillus subtilis. The bacillus subtilis over-expression strain is utilized, lactose is used for inducing to obtain high-enzyme-activity protein in the fermentation process, enzyme is secreted out of cells, and enzyme dry powder is obtained by spray drying; and (3) integrating the expression strain with bacillus subtilis, obtaining an enzyme product which is constantly and stably expressed through a fermentation process, and performing spray drying to obtain the microbial inoculum/enzyme dry powder. The substrate and the bacterial enzyme dry powder are compounded to form a product, so that the in-situ catalysis of the enzyme to produce the insecticidal product diallyl thiosulfinate is realized, and the rapid killing and long-term control effects of the root-knot nematode are realized. The plate inhibition experiment shows that the product has 100% lethal effect on soil root-knot nematodes, and has obvious control effect on greenhouse and field crop root-knot nematodes.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a bacterial enzyme composition for preventing and controlling root knot nematodes and a preparation method thereof.
Background
Root-knot nematodes severely restrict the sustainable development of agriculture in China, and are particularly shown in that the root-knot nematodes can infect the roots of crops to form root nodules, so that the crops are difficult to absorb nutrition, wither and die. The nematode disease has wide disease incidence range, serious disease and difficult control. Especially in greenhouse cultivation, root knot nematode diseases have developed into a destructive pest in many old sheds due to continuous cropping planting. There is therefore a need for products that are effective against root knot nematodes.
At present, the chemical control is quick and simple, and the harm of the root-knot nematode can be effectively controlled. Common chemical line killers include fosthiazate, 1.8% avermectin emulsifiable concentrate, 10% acephate granules, methyl bromide, dichloropropene and the like. However, chemical nematicides have the problems of high toxicity, serious environmental pollution, easy generation of drug resistance for nematodes and the like. Thus, with the increasing public awareness of environmental protection and increasing nematode resistance, the use of chemical nematicides is increasingly limited.
Biological control is increasingly gaining importance in the development of control products for root knot nematodes because of its outstanding advantages of green safety. The most studied fungi and bacteria are used, for example, the paecilomyces lilacinus and bacillus subtilis which are mentioned in patent 202110568678.X & lt- & gt, a microbial agent for preventing and treating tomato root-knot nematode and a preparation method thereof & gt, and 202110553160.9 & lt- & gt, a microbial agent for effectively preventing and treating root-knot nematode and a preparation method thereof & gt are used, and the microbial agent has an effect of preventing and treating the root-knot nematode, but the instability of the microbial agent itself makes the application effect of the product unstable.
Researchers find that biological control such as killing root knot nematodes by using some natural insecticidal active substances has the advantages of environmental protection, safety, effectiveness and the like. For example, diallyl thiosulfinate is naturally present in broken plant tissue, has a broad spectrum of bacteriostatic and insecticidal efficacy, and is almost impossible for bacteria and fungi to develop resistance. At present, most common is chemical synthesis, but the cost is high, chloropropene is remained, or the bulb of the lily is crushed and hydrolyzed and then is extracted, but the operation is complex, the cost is high, and the application of the bulb in agriculture is greatly limited.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a bacterial enzyme composition for preventing and controlling root-knot nematodes and a preparation method thereof.
The technical scheme of the invention is as follows:
a bacterial enzyme composition for root knot nematode control, characterized in that: the bacterial enzyme composition is a mixture of bacillus subtilis bacterial agent, S-alkyl-L-cysteine sulfoxide lyase LCC1, S-allyl-L-cysteine sulfoxide and an activator PLP.
The compound diallyl thiosulfinate for effectively preventing and treating the root-knot nematode is obtained by carrying out catalytic reaction on a substrate S-allyl-L-cysteine sulfoxide by S-alkyl-L-cysteine sulfoxide lyase LCC 1.
As a preferable technical scheme, the S-alkyl-L-cysteine sulfoxide lyase LCC1 overexpressed by the bacillus subtilis, the bacillus subtilis microbial inoculum for integrally expressing the S-alkyl-L-cysteine sulfoxide lyase LCC1, the substrate S-allyl-L-cysteine sulfoxide and the activator PLP are prepared according to the weight ratio (6-1): (6-1): (1-6): (0.001-0.003).
As a preferable technical scheme, a bacterial enzyme composition for preventing and treating root-knot nematodes reacts to generate an active substance diallyl thiosulfinate, and the medium is aqueous solution and reacts at normal temperature.
As a preferred technical scheme, the composition of S-alkyl-L-cysteine sulfoxide lyase LCC1, bacillus subtilis microbial inoculum, substrate S-allyl-L-cysteine sulfoxide and PLP is used for preventing and controlling root-knot nematodes of greenhouse crops and open-air crops in the modes of root dipping, root irrigation, flushing, fertilizer spreading, hole spreading or strip spreading.
The S-alkyl-L-cysteine sulfoxide lyase LCC1 is obtained by over-expressing and secreting an expression vector in a host cell; further, the host cell may be bacillus subtilis WB800N, WB, WB600, SCK6, etc.; further, the expression vector may be pHT43, pHT01, pHT08, pHT09 or pHT10, etc.
As a preferable technical scheme, the specific preparation method comprises the following steps:
(1) The bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 is constructed to a secretory expression plasmid pHT43, wherein the protein sequence of the S-alkyl-L-cysteine sulfoxide lyase LCC1 is shown as a sequence 1 in a sequence table.
(2) Transforming the over-expression plasmid constructed in the step (1) into a bacillus subtilis host WB 800N;
(3) Adding 10-20% corn starch into the fermentation liquid, spray drying to obtain enzyme powder, and carrying out catalytic reaction;
(4) The fermentation medium comprises the following components in percentage by mass and volume (g/l): 5-10 parts of lactose, 10-40 parts of bean pulp powder, 10-30 parts of glucose, 20-30 parts of corn flour, 0.1-0.5 part of manganese sulfate, 5-10 parts of yeast powder, 4-10 parts of corn steep liquor, 2-5 parts of sodium chloride, 3-5 parts of monopotassium phosphate, 0.5-1 part of magnesium sulfate, 0.3-0.5 part of ferric chloride and 7.2 parts of pH, and placing the materials into a 5L fermentation tank, and sterilizing the materials at 121 ℃ for 20 min.
The bacillus subtilis bacterial agent is obtained by recombining and integrating S-alkyl-L-cysteine sulfoxide lyase LCC1 into a host cell through an integrated vector, and further, the host cell can be bacillus subtilis WB800N, WB, WB600, SCK6 and the like; further, the integration vector may be pIEFBPR, p7Z6, p7S6, pTSC or the like.
As a preferable technical scheme, the specific preparation method comprises the following steps:
(1) The bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 is constructed into a secretion type integrative plasmid pIEFBPR, wherein the protein sequence of the S-alkyl-L-cysteine sulfoxide lyase LCC1 is shown as a sequence 1 in a sequence table.
(2) Transforming the recombinant plasmid in (1) into bacillus subtilis WB800N and integrating protease coding gene on WB800N chromosomebprSequence middle;
(3) The fermentation broth is not required to be centrifuged, 5-10% of dextrin, 5-15% of corn starch and 5-10% of glycerol are added, and catalytic reaction is carried out after enzyme powder containing microbial inoculum is obtained through spray drying;
(4) The formula of the fermentation medium according to the mass volume ratio (g/l) is as follows: 10-40 parts of bean pulp powder, 10-30 parts of glucose, 20-30 parts of corn flour, 0.1-0.5 part of manganese sulfate, 5-10 parts of yeast powder, 4-10 parts of corn steep liquor, 2-5 parts of sodium chloride, 3-5 parts of potassium dihydrogen phosphate, 0.5-1 part of magnesium sulfate, 0.3-0.5 part of ferric chloride and 7.2 parts of pH, and placing the materials into a 5L fermentation tank, and sterilizing at 121 ℃ for 20 min.
The biological fermentation method is used for producing S-alkyl-L-cysteine sulfoxide lyase LCC1, and catalyzing substrate S-allyl-L-cysteine sulfoxide to obtain the product diallyl thiosulfinate. Because the S-alkyl-L-cysteine sulfoxide lyase LCC1 and the S-allyl-L-cysteine sulfoxide are respectively very stable, the defect of poor storage stability of the product is overcome by utilizing the way of in-situ reaction of the enzyme and the substrate. This route is explained in detail below:
1. bacillus subtilis WB800N overexpressing S-alkyl-L-cysteine sulfoxide lyase LCC1 strain construction: cloning bacterial S-alkyl-L-cysteine sulfoxide lyase LCC1 gene sequence to over-expression vector pHT43, transforming recombinant plasmid into host bacterium WB800N, screening transformant with chloramphenicol (5 μg/ml), and sequencing to determine the correctness of the transformant.
2. Bacillus subtilis WB800N recombinant integration S-alkyl-L-cysteine sulfoxide lyase LCC1 strain construction: cloning bacterial S-alkyl-L-cysteine sulfoxide lyase LCC1 gene sequence to multiple cloning site between bpr front and DR sequences of homologous arm of integration vector pIEFRPHPR, transforming host bacterium WB800N with recombinant plasmid and integrating into protease encoding gene on WB800N chromosomebprIn the middle of the sequence, spectinomycin (100. Mu.g/ml) was screened for first crossover transformants. The transformants were grown in antibiotic-free LBG (LB+1% glucose) medium for 24 h, and the second crossover occurred spontaneously. The culture broth was plated with LBG plates containing IPTG, and single colonies were picked and spot-half-verified (spectinomycin resistant/non-resistant). And selecting a single colony without resistance for culture, and preserving after PCR verification and sequencing are correct.
3. And (3) strain fermentation:
(1) Bacillus subtilis WB800N fermentation over-expressing S-alkyl-L-cysteine sulfoxide lyase LCC 1: single colonies were picked from the plates and inoculated into 5 ml liquid LB medium, chloramphenicol (5. Mu.g/ml), cultured overnight at 37℃at 220 rpm; inoculating the first-stage seeds into a triangular flask filled with 150 ml liquid LB culture medium according to the inoculation amount of 1-3% by volume, and culturing at 37 ℃ and 220 rpm until OD 600 In the range of 38-40. At a volume ratio of 5%The second-level seeds are inoculated into a 5L fermentation tank containing 3L liquid fermentation medium for culture at 30 ℃ for 6-8 hours, and 5-10 g/l of inducer lactose is added. The fermentation regulation parameters are controlled as follows: dissolved oxygen control: 40%; and (3) temperature control: 30 ℃; pH control: and the pH value is 7.0 after 0-6 h.
(2) Recombinant integration of bacillus subtilis WB800N fermentation expressing S-alkyl-L-cysteine sulfoxide lyase LCC 1: single colony is picked from the flat plate and inoculated to 5 ml liquid LB culture medium, and the culture is carried out at 37 ℃ and 220 rpm overnight; inoculating the first-stage seeds into a triangular flask filled with 150 ml liquid LB culture medium according to the inoculation amount of 1-3% by volume, and culturing at 37 ℃ and 220 rpm until OD 600 In the range of 38-40. The secondary seeds were inoculated in an inoculum size of 5% by volume into a 5L fermenter containing 3L liquid fermentation medium for cultivation at 30 ℃. The fermentation regulation parameters are controlled as follows: dissolved oxygen control: 40%; and (3) temperature control: 30 ℃; pH control: and the pH value is 7.0 after 0-6 h.
(3) The formula of the fermentation medium according to the mass volume ratio (g/l) is as follows: 10-40 parts of bean pulp powder, 10-30 parts of glucose, 20-30 parts of corn flour, 0.1-0.5 part of manganese sulfate, 5-10 parts of yeast powder, 4-10 parts of corn steep liquor, 2-5 parts of sodium chloride, 3-5 parts of monopotassium phosphate, 0.5-1 part of magnesium sulfate, 0.3-0.5 part of ferric chloride and 7.2 parts of pH, and placing the materials into a 5L fermentation tank, and sterilizing the materials at 121 ℃ for 20 min.
4. Spray drying:
(1) And (3) performing spray drying on the fermentation liquor of the overexpression S-alkyl-L-cysteine sulfoxide lyase LCC1 to obtain an enzyme powder product. The operation flow is as follows: bacillus subtilis WB800N fermentation broth overexpressing S-alkyl-L-cysteine sulfoxide lyase LCC1 was added with a fluidizing agent (V/W): 10-20% of corn starch and stirring at 100 rpm for 20-30 min to enable the fluidizer to be fully dispersed in the fermentation liquor, so that the uniformity of feed liquid in a spray drying link is ensured. The temperature of the air inlet of the spray dryer is 150-160 ℃, the temperature of the air outlet is 70-80 ℃, and the rotating speed of the peristaltic pump is 2000 ml/h.
The corn starch is fully dispersed in the fermentation liquid, so that the fluidity of the material in the spray drying link can be improved. The corn starch has strong hygroscopicity and smaller granularity, can be coated on the surface of the bacteria to provide protection for the dried bacteria, can be used as a carbon source of bacillus subtilis, and is beneficial to improving the reviving rate of the spores in the application stage of the product. In addition, the addition amount is economical and reasonable, so that the fluidity of the feed liquid in the spray drying process can be effectively improved, and a certain protection can be provided for thalli in a final viable bacteria preparation finished product.
(2) Recombinant integration S-alkyl-L-cysteine sulfoxide lyase LCC1 fermentation broth spray drying to obtain bacterial enzyme mixture. The operation flow is as follows: recombinant integration of S-alkyl-L-cysteine sulfoxide lyase LCC1 Bacillus subtilis WB800N fermentation broth with addition of fluidizing agent (V/W): 5-10% of dextrin, 5-15% of corn starch and 5-10% of glycerol, and stirring at 100 rpm/min for 20-30 min to enable the fluidizer to be fully dispersed in the fermentation broth, so that the uniformity of feed liquid in a spray drying link is ensured. The temperature of the air inlet of the spray dryer is 150-160 ℃, the temperature of the air outlet is 70-80 ℃, and the rotating speed of the peristaltic pump is 2000 ml/h.
The corn starch is fully dispersed in the fermentation liquid, so that the fluidity of the material in the spray drying link can be improved. The corn starch has strong hygroscopicity and smaller granularity, can be coated on the surface of the bacteria to provide protection for the dried bacteria, can be used as a carbon source of bacillus subtilis, and is beneficial to improving the reviving rate of the spores in the application stage of the product. Maltodextrin can form an effective protective layer on the surface of the bacterial body, and glycerin can penetrate through the cell wall and the cell membrane, and can replace the original water molecules in the cell membrane to form hydrogen bonds with phospholipid and protein in the drying process so as to stabilize the integrity of the cell membrane. The three protective agents in the proportion can effectively improve the storage period and the revival rate of the product.
5. Substrate S-allyl-L-cysteine sulfoxide preparation:
weighing sodium hydroxide (or weighing 50-100 ml of ammonia water) according to the final concentration of 0.8-4.5 mol/l, and dissolving in 5: 1-4: 1, adding L-cysteine solid with the final concentration of 0.4-2.5 mol/L into the ethanol water solution, adding bromopropene with the same mol concentration as cysteine after the L-cysteine solid is completely dissolved, and reacting for 5-12 h while stirring. And regulating the pH value of the solution to 4-6 by using acetic acid, filtering, and drying in an oven to obtain the S-allyl-L-cysteine.
Taking the S-allyl-L-cysteine and hydrogen peroxide prepared in the first step according to the mass volume ratio of (1-3) g: (6~2) ml, for 5min. Adding precooled ethanol, and drying to obtain the S-allyl-L-cysteine sulfoxide.
6. Compounding the bacterial enzyme composition:
the bacterial agent for over-expressing the S-alkyl-L-cysteine sulfoxide lyase LCC1, the bacterial agent for recombining and integrating the S-alkyl-L-cysteine sulfoxide lyase LCC1, the substrate S-allyl-L-cysteine sulfoxide and the coenzyme PLP are prepared according to the weight ratio of (6-1): (6-1): (1-6): (0.001-0.003).
The over-expressed enzyme has high enzyme activity, can rapidly catalyze and synthesize the bactericidal substance thiosulfinate, and has rapid control effect on root-knot nematodes. The microbial inoculum of the integrated expression S-alkyl-L-cysteine sulfoxide lyase LCC1 can produce enzyme stably for a long time after being recovered, thereby achieving the purpose of preventing and controlling root-knot nematodes stably for a long time.
7. Biological enzyme method catalysis preparation of diallyl thiosulfinate:
and (3) reacting the bacterial enzyme composition with water as a medium at room temperature to obtain the product diallyl thiosulfinate for resisting the root-knot nematode. When the plant is applied, the root knot nematodes can be prevented and treated by dipping roots, root irrigation, flushing, fertilizer spreading, hole spreading or strip spreading.
The beneficial effects of the invention are as follows:
(1) According to the invention, the product diallyl thiosulfinate is obtained by an in-situ catalysis mode of the S-alkyl-L-cysteine sulfoxide lyase LCC1 and a substrate S-allyl-L-cysteine sulfoxide, so that the problem of reduced activity of the product in the storage period is avoided, and the defect that the existing diallyl thiosulfinate product is unstable and easy to degrade is effectively solved.
(2) Compared with the prior art, the novel S-alkyl-L-cysteine sulfoxide lyase LCC1 is secreted and expressed with high activity by selecting bacillus subtilis as an expression strain. Heterologous high expression can be obtained in bacillus subtilis using bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC 1. The secretory expression does not need wall breaking release enzyme, and can realize rapid catalytic synthesis of the product diallyl thiosulfinate in vitro.
(3) According to the invention, the bacterial enzyme composition of the enzyme preparation and the bacterial agent is obtained by combining the over-expression and integration of the S-alkyl-L-cysteine sulfoxide lyase LCC1 on a bacillus subtilis genome, after application, the quick-acting insecticidal effect can be realized by quickly generating insecticidal products, and the quick-acting and long-acting effect of preventing and controlling root-knot nematodes can be realized by continuously generating the S-alkyl-L-cysteine sulfoxide lyase LCC1 in the growth process of thalli. Breaks the limit of the application of the microbial agent in the field of preventing and controlling the root-knot nematodes caused by the defect of instability of the microbial agent.
Drawings
FIG. 1 is a diagram of the verification of transformants of an overexpressing strain;
FIG. 2 is a diagram of verification of recombinant integrative transformants;
FIG. 3 shows the result of a killing experiment of adult root knot nematodes;
FIG. 4 shows the results of an experiment for the hatching inhibition of eggs of the root knot nematode;
FIG. 5 shows the experimental results of greenhouse cucumber root-knot nematode control;
fig. 6 shows the experimental results of control of large Tian Fanjia root-knot nematodes.
Detailed Description
The present invention will be further described with reference to the following specific drawings, so that technical means, technical features, objects and technical effects of the present invention can be easily understood.
Example 1:
construction of bacillus subtilis over-expression S-alkyl-L-cysteine sulfoxide lyase LCC1 strain
(1) Designing a primer according to the gene sequence of the S-alkyl-L-cysteine sulfoxide lyase LCC1 and performing PCR amplification to obtain an S-alkyl-L-cysteine sulfoxide lyase LCC1 gene fragment; extraction of pHT43 expression vector plasmid extraction double enzyme cutting and glue recovery.
(2) The recovered S-alkyl-L-cysteine sulfoxide lyase LCC1 gene fragment and pHT43 plasmid fragment were ligated. Ligation product transformation competent cellsE. coliTransformants were picked up for DH 5. Alpha. And full-length sequencing of the gene fragments.
(3) The recombinant plasmid was transferred into bacillus subtilis WB800N competent cells, resuscitated, and plated on LB plates (100 μg/ml Amp). Transformants were picked and cultured in LB medium (100. Mu.g/ml Amp) at 37℃with shaking for 14h. The PCR and electrophoresis results are shown in FIG. 1, the correct transformant is preserved with 15% glycerol, and the transformant is kept at-20 ℃ for later use, and simultaneously delivered to a worker for full-length sequencing of the gene fragment.
The transformant strain with correct sequencing is the S-alkyl-L-cysteine sulfoxide lyase overexpression strain.
Example 2:
construction of bacillus subtilis recombinant integrated S-alkyl-L-cysteine sulfoxide lyase LCC1 strain
Constructing recombinant plasmid pIEFBPR-S-alkyl-L-cysteine sulfoxide lyase LCC1 gene, cloning bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 gene sequence to multiple cloning site between homologous arm bpr front and DR sequence of integrated vector pIEFBPR, transforming host strain WB800N with recombinant plasmid and integrating protease encoding gene on WB800N chromosomebprIntermediate the sequences. Spectinomycin (100. Mu.g/ml) was screened for first crossover transformants. The transformants were grown in antibiotic-free LBG (LB+1% glucose) medium for 24 h, and the second crossover occurred spontaneously. The culture bacterial liquid is transferred to LBG culture medium, induced by IPTG, cultured (or directly coated with LBG plate containing IPTG), induced by IPTGmazFLethal gene expression, if no second exchange occurs, the bacteria cannot grow. The LBG plates containing IPTG were coated and single colonies were picked and half-verified (spectinomycin resistant/non-resistant) at double plate spots. And selecting a single colony without resistance for culture, and preserving after PCR verification is correct, wherein as shown in figure 2, transformants No. 1, no. 2, no. 3 and No. 4 are constructed S-alkyl-L-cysteine sulfoxide lyase recombinant integrated expression strains.
Example 3:
bacillus subtilis over-expressing S-alkyl-L-cysteine sulfoxide lyase LCC1 and bacillus subtilis recombinant and integrated to express S-alkyl-L-cysteine sulfoxide lyase LCC1 are subjected to tank fermentation
(1) Bacillus subtilis WB800N fermentation over-expressing S-alkyl-L-cysteine sulfoxide lyase LCC 1:
the fermentation medium was formulated as follows:
the formula of the fermentation medium (g/l) according to the mass-volume ratio (g/l) is as follows: 20 parts of bean pulp powder, 20 parts of glucose, 20 parts of corn flour, 0.2 part of manganese sulfate, 5 parts of yeast powder, 4 parts of corn steep liquor, 2 parts of sodium chloride, 3 parts of monopotassium phosphate, 0.5 part of magnesium sulfate, 0.3 part of ferric chloride and 7.2 parts of pH, and placing the materials into a 5L fermentation tank for sterilization at 121 ℃ for 20 minutes.
Primary seed culture: single colonies were picked from the plates and inoculated into 5 ml liquid LB seed medium, chloramphenicol (5. Mu.g/ml), cultured overnight at 37℃at 220 rpm.
Secondary seed culture: inoculating the first seed into a triangular flask containing 150 ml liquid LB culture medium according to the inoculation amount of 3% by volume, culturing at 37deg.C and 220 rpm until OD 600 Up to 39.2.
Culturing on a tank to obtain an over-expressed S-alkyl-L-cysteine sulfoxide lyase LCC1 fermentation broth: the secondary seeds are inoculated into a 5L fermentation tank containing 3L liquid fermentation medium according to the inoculation amount of 5% by volume for culture at 30 ℃, 8g/l of inducer lactose is added into 6h, and the culture is continued until 36 hours. The fermentation regulation parameters are controlled as follows: dissolved oxygen control: 40%; and (3) temperature control: 30 ℃; pH control: and the pH value is 7.0 after 0-6 h.
(2) Recombinant integration of bacillus subtilis WB800N fermentation expressing S-alkyl-L-cysteine sulfoxide lyase LCC 1:
the fermentation medium was formulated as follows:
the mass volume ratio (g/l) formula of the fermentation medium is as follows: 30 parts of bean pulp powder, 20 parts of glucose, 20 parts of corn flour, 0.1 part of manganese sulfate, 5 parts of yeast powder, 4 parts of corn steep liquor, 5 parts of sodium chloride, 4 parts of monopotassium phosphate, 0.7 part of magnesium sulfate, 0.4 part of ferric chloride and 7.2 parts of pH, and placing the materials into a 5L fermentation tank for sterilization at 121 ℃ for 20 minutes.
Primary seed culture: single colonies were picked from the plates and inoculated into 5 ml liquid LB seed medium, 37℃at 220 rpm, and cultured overnight.
Secondary seed culture: inoculating the first seed into a triangular flask containing 150 ml liquid LB culture medium according to the inoculation amount of 3% by volume, culturing at 37deg.C and 220 rpm until OD 600 Up to 39.
Culturing on a tank to obtain a fermentation broth of the recombinant and integrally expressed S-alkyl-L-cysteine sulfoxide lyase LCC 1: the secondary seeds were inoculated in an inoculum size of 5% by volume into a 5L fermenter containing 3L liquid fermentation medium and cultured at 30℃for 36 hours. The fermentation regulation parameters are controlled as follows: dissolved oxygen control: 40%; and (3) temperature control: 30 ℃; pH control: and the pH value is 7.0 after 0-6 h.
Example 4:
preparation of bacterial enzyme composition
Preparing microbial inoculum and enzyme preparation dry powder: (1) Preparation of over-expressed S-alkyl-L-cysteine sulfoxide lyase LCC1 dry powder: 3L of Bacillus subtilis WB800N fermentation broth over-expressing S-alkyl-L-cysteine sulfoxide lyase LCC1 was added with fluidizing agent (V/W): 450 And g of corn starch is stirred at 100 rpm for 30 min, so that the fluidizer is fully dispersed in the fermentation broth, and the uniformity of feed liquid in a spray drying link is ensured. The temperature of the air inlet of the spray dryer is 150-160 ℃, the temperature of the air outlet is 70-80 ℃, and the rotating speed of the peristaltic pump is 2000 ml/h.
(2) The preparation flow of the recombinant integrated S-alkyl-L-cysteine sulfoxide lyase LCC1 bacterial agent dry powder comprises the following steps: 3L recombinant integration of S-alkyl-L-cysteine sulfoxide lyase LCC1 Bacillus subtilis WB800N fermentation broth added with fluidizing agent (V/W): 300 g dextrin, 450 g corn starch and 300 g glycerin, and stirring at 100 rpm/min for 25min to enable the fluidizer to be fully dispersed in the fermentation broth, so as to ensure the uniformity of the feed liquid in the spray drying link. The temperature of the air inlet of the spray dryer is 150-160 ℃, the temperature of the air outlet is 70-80 ℃, and the rotating speed of the peristaltic pump is 2000 ml/h.
Preparation of the bacterial enzyme composition product: 500g of over-expression S-alkyl-L-cysteine sulfoxide lyase LCC1 enzyme dry powder, 500g of recombinant integrated S-alkyl-L-cysteine sulfoxide lyase LCC1 microbial inoculum dry powder, 300 g of substrate S-allyl-L-cysteine sulfoxide and 0.5g of pyridoxal phosphate activating agent PLP are mixed together, fully stirred and uniformly mixed, and placed at room temperature in a drying place for standby, thus obtaining the product.
Example 5:
test for killing nematodes by products
The bacterial enzyme composition of example 4 was selected and reacted in situ to produce diallyl thiosulfinate for root knot nematode experiments.
(1) Adult root knot nematode killing experiment
After 7 days incubation, the nematode culture solution was observed under a split microscope, so that the nematode bodies were observed to be active, and then the nematode culture solution was uniformly divided into 6-well plates, and 1 control group and four experimental groups each contained 1ml nematode culture solution. After adding substrate S-allyl-L-cysteine sulfoxide, bacterial enzyme dry powder and activator PLP according to substrate concentration gradient, in-situ reaction is carried out to generate diallyl thiosulfinate, and incubation is continued at 28 ℃ for 12h, and then the activity of the diallyl thiosulfinate is observed under a split microscope. As a result, it was found that nematodes in the control group without any treatment could still flex and move, whereas nematodes in the experimental group treated with the substrate and the enzyme all had dead and inactive, and the mortality was 100%. Wherein the minimum substrate concentration is 20.125 mug/ml, the root-knot nematodes can be killed, and the concentration of the reduced product diallyl thiosulfinate is 10 mug/ml. As shown in fig. 3, the control group and the lowest substrate concentration group were microscopic photographs after incubation of 12h.
(2) Root knot nematode egg hatching inhibition experiment
Four days of incubation were taken from the root knot nematode solution, which was egg-based. In 1ml of nematode egg liquid, S-allyl-L-cysteine sulfoxide was set to the following gradient: 160. Mu.g/ml, 80.0. Mu.g/ml, 40.25. Mu.g/ml, 20.125. Mu.g/ml, and 10. Mu.l of enzyme solution was added to each group, and incubation of nematode eggs was observed after 12h. The results showed that most of the nematode solution was eggs before the experiment, and that eggs in the control group were largely hatched to root knot nematode larvae without any treatment, while eggs in the four experimental groups to which the substrate was added were still in the state of eggs. Wherein the hatching of the root-knot nematode eggs can be inhibited when the lowest substrate concentration is 20.125 mug/ml, and the concentration of the reduced product diallyl thiosulfinate is 10 mug/ml. Fig. 4 is a photomicrograph of the control group and the lowest substrate concentration group after incubation of 12h.
Example 6:
application effect experiment of the product of the invention on serious cucumber plants infected by root-knot nematodes
The bacterial enzyme composition of example 4 was selected and reacted in situ to produce diallyl thiosulfinate for greenhouse cucumber root knot nematode control experiments.
For root systems seriously infected with root-knot nematodes, the simple killing of the nematodes cannot achieve a good effect in a short time due to the lack of normal root system absorption nutrition. Therefore, the bacteria-enzyme composition compound hole fertilizer, the water-soluble fertilizer and the amino acid rooting fertilizer are applied in a matched manner, so that the effective combination of prevention and treatment is realized, and the action effect of synergistic reinforcement prevention and treatment is realized.
Cucumber plants severely infected with root-knot nematodes are selected for potting control experiments. In the experimental group, the bacterial enzyme composition is applied to the plant root by the compound hole fertilization and then is watered; meanwhile, 0.1-0.5 g of the composite water-soluble fertilizer product of the bacterial enzyme composition is weighed and dissolved in 1L tap water, and the amino acid liquid fertilizer 100ml is added and fully stirred until the bacterial enzyme composition is dissolved, and the liquid medicine is irrigated once every other week for 3 times. Recovery of affected plants was observed during this period, and root nodule remnants were observed. Meanwhile, cucumber without liquid medicine is used as a control group.
The invention achieves remarkable effect in the aspect of inhibiting the root-knot nematode by utilizing the reaction of enzyme and substrate. The results are shown in FIG. 5. The bacteria enzyme composition product generates bactericidal substances after application, and can kill root knot nematodes when the concentration of the substrate S-allyl-L-cysteine sulfoxide is 20.125 mug/ml, and the concentration of the reduced product diallyl thiosulfinate is 10 mug/ml. Similarly, incubation of the root knot nematode egg was completely inhibited at a substrate S-allyl-L-cysteine sulfoxide concentration of 20.125 μg/ml, giving a reduced product diallyl thiosulfinate concentration of 10 μg/ml. When the product is used for treating plants infected with the root-knot nematode, the plant wilting condition is observed to be obviously improved after 3 continuous application times. And observing the condition of the plant root system, wherein root knot nematode nodules continuously expand, and the plant also has new roots for germination, and the new roots are not provided with the nodules. The leaf wilting condition of the upper leaf of the plant is improved by observing the leaf, and as shown in figure 5, the product has remarkable control effect on cucumber plant root-knot nematodes.
Example 7:
the invention has the application effect on tomato plants with serious root-knot nematode infection
The bacterial enzyme composition of example 4 was selected and reacted in situ to produce diallyl thiosulfinate for greenhouse cucumber root knot nematode control experiments.
Tomatoes planted in open air in a field with serious root-knot nematode dip dyeing are selected as field efficacy comparison tests of the bacterial enzyme composition. Tomatoes with serious root-knot nematode infection are selected for transplanting, and two treatments are carried out: blank control group, direct normal transplanting; in the experimental group, the bacterial enzyme composition is applied to the root of tomatoes by a hole application method and watered during transplanting, and 20 repeated tests are adopted for each treatment. During the treatment, the bacterial enzyme complex is irrigated with water, and the treatment is carried out once every other week. After 30 days of treatment, the growth state of the tomato plants is observed, and the root knot nematode infection condition of the plant root systems is investigated.
The test results are shown in fig. 6, which demonstrate that: the tomato roots of the No. 1 blank control group are seriously infected by nematodes, the number of root knots is large, and the plants grow poorly; no. 2 test group root has no obvious root knot, the infection rate is obviously reduced, the plant growth is good, and the root knot nematode infection is effectively prevented and controlled under the action of the bacteria enzyme composition.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the present invention. Equivalent changes and modifications of the invention are intended to fall within the scope of the present invention.
Sequence listing
<110> university of Shandong
<120> a bacterial enzyme composition for controlling root-knot nematode and a method for preparing the same
<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
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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
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Ala Gly Leu His Cys Ala Pro Leu Ala His Glu Thr Ala Gly Thr Leu
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Ala Thr Gly Thr Leu Arg Phe Ser Phe Gly Trp Gln Thr Thr Pro Glu
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Glu Ile Thr Trp Thr Ile His Ala Leu Gln Glu Leu Leu Ile
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Claims (5)
1. A bacterial enzyme composition for root knot nematode control, characterized in that: the bacterial enzyme composition is a mixture of bacillus subtilis bacterial agent for integrally expressing S-alkyl-L-cysteine sulfoxide lyase LCC1, pyridoxal phosphate (PLP) serving as an activator and S-allyl-L-cysteine sulfoxide, and the protein sequence of the S-alkyl-L-cysteine sulfoxide lyase LCC1 is as follows:
MKDLVYLNYAATSYKKFPATIEALTAYLAENQFMNYGRNAPLLREGLPLLETRQLLADFFQAPSAAQITFTNNATTSLNLALAGILQPGDHVITTMLEHHAVARPLHLLEKERGISVTYVACQKTGLLDVEDIQRAWRTNTKALVMTHASNVLGTILPIEECFQWAQQKGLLTVLDAAQTAGFLPIKMTQMAIDVLAFTGHKSLYGLAGIGGLAFSERGAEAVKPLMAGGTGSHSNSFDQPSFLPDKFEAGTLNSLGILSLNSSIKELNKIGLAAIQKHERTLMQNFLNGLSGLPVTILGTKDVAQTVPVVSITLWNQEETVVAQQLAEQYGIMTRAGLHCAPLAHETAGTLATGTLRFSFGWQTTPEEITWTIHALQELLI。
2. a bacterial enzyme composition for root-knot nematode control according to claim 1, characterized in that: the weight ratio of the bacillus subtilis over-expressed and secreted S-alkyl-L-cysteine sulfoxide lyase LCC1 to the bacterial agent for the bacillus subtilis integrated and expressed S-alkyl-L-cysteine sulfoxide lyase LCC1 to the substrate S-allyl-L-cysteine sulfoxide to the activator PLP is (6-1): (6-1): (1-6): (0.001 to 0.003).
3. A bacterial enzyme composition for root knot nematode control according to any one of claims 1 to 2, characterized in that: the S-alkyl-L-cysteine sulfoxide lyase LCC1 is obtained by over-expressing and secreting an expression vector in a host cell; the host cell is one of bacillus subtilis WB800N, WB, WB600 or SCK 6; the expression vector is one of pHT43, pHT01, pHT08, pHT09 or pHT 10.
4. A bacterial enzyme composition for root knot nematode control according to any one of claims 1 to 2, characterized in that: the bacillus subtilis microbial inoculum is obtained by recombining and integrating S-alkyl-L-cysteine sulfoxide lyase LCC1 into a host cell through an integrated vector, wherein the host cell is one of bacillus subtilis WB800N, WB, WB600 or SCK 6; the integrating vector is one of pIEFBPR, p7Z6, p7S6 or pTSC.
5. Use of a bacterial enzyme composition for root-knot nematode control according to claim 1, characterized in that: the bacterial enzyme composition is used for preventing and controlling root-knot nematodes of greenhouse crops and open-air crops, and the mode of preventing and controlling the root-knot nematodes by the bacterial enzyme composition is root dipping, root irrigation, flushing, fertilizer spreading, hole spreading or strip spreading.
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