CN110982764A - Bacillus tequilensis S12 for preventing and treating rice blast and application thereof - Google Patents
Bacillus tequilensis S12 for preventing and treating rice blast and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to a Bacillus tequilensis S12 for preventing and treating rice blast and application thereof, and particularly discloses a Bacillus tequilensis strain S12 and application of a S12 strain in preventing and treating rice blast, wherein the strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 18347. The bacillus tequilensis S12 strain and the metabolite thereof have strong antagonism on rice blast, can obviously inhibit the pathogenicity of the rice blast, and further effectively prevent and control the occurrence of rice blast, so the bacillus tequilensis S12 strain and the metabolite thereof obtained by the invention can be used for biological prevention and control of the rice blast. In addition, the biocontrol microbial inoculum is safe to people and livestock, does not have the problems of environmental pollution and the like, and has the advantages of simple preparation method, low cost and simple use.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to bacillus tequilensis S12 for preventing and treating rice blast and application thereof.
Background
Rice is one of the three cereal crops that produce over five million tons per year in the world, and is the major source of basic food for 30 billions of people per day in the world (particularly asia). Rice blast (rice blast disease) is one of the most serious rice diseases, and all rice production areas in the world occur, so that the yield of rice can be reduced by 10-30% every year. Diseases can occur in all tissue parts of rice, for example, leaf plague caused by leaf diseases is the most common disease, and leaf death can be caused in severe cases; besides leaves, the stem can also be attacked to form node plague, and plants are easy to fall and wither after the part is diseased; when the disease occurs in heading stage, panicle blast and stem blast are easy to form, and when the disease rate is high, the filling can be seriously influenced, so that the quality and the yield of rice are reduced, and in addition, the rice panicle blast can also occur.
The occurrence of rice blast has strict requirements on temperature and humidity, particularly the latter. Generally speaking, in rainy seasons of rice planting areas, the daily average temperature is 24-32 ℃, the deep water irrigation and ventilation of rice fields are poor, and in addition, the relative humidity often exceeds 90% due to field shading, so that the temperature and humidity are particularly suitable for the occurrence and prevalence of rice blast. The rice can be infected by diseases in each growth and development stage, and has two susceptible stages in comparison, namely a seedling stage, because the tissue is tender, the disease resistance is weak, and the infection is easy to form the leaf plague or the stem plague; secondly, in the heading stage, rain is often generated in the development stage, water drops are accumulated at the heading part, conidia are easy to adsorb, and spike-stalk blast is induced.
The rice blast fungus (Magnaporthe oryzae or Pyricularia oryzae) is a pathogenic bacterium of rice blast, which is a filamentous fungus belonging to ascomycetes in sexual generations. The conidia of the rice blast fungus are differentiated from the end of a conidiophore, are mostly formed by three cells after being matured, are pear-shaped, fall on the surface of rice tissue through wind blowing or rain washing, and are induced to form a specialized infected cell-attachment cell (apressorium), and the cell generates huge turgor pressure by precipitating a layer of thick melanin on the inner side of a cell wall and synthesizing a large amount of glycerol in the cell, thereby invading the rice tissue to grow and expand in the rice tissue, and causing scabs. The infected hypha can spread from the lesion to the air to form conidiophores, and
conidia were differentiated at the ends and subjected to another round of infection. In addition to infecting rice, the filamentous fungi may also infect other plants such as: barley, wheat, and some weeds of the grass family.
Similar to the control of other diseases, the rice blast germs follow the principle of 'prevention is the main and comprehensive control'. For example, several rice blast resistance genes have been cloned so far in breeding disease-resistant varieties; at present, chemical control is still a conventional control method for controlling rice blast, common chemical pesticides comprise tricyclazole, cyanamide and the like, and the generation of the rice blast is mainly inhibited by inhibiting a melanin biosynthesis pathway. Since chemical control may cause pesticide residue, there is a risk of endangering human health and ecological environment, and in recent years, green control technologies mainly for biological control have been widely recognized, and vigorous development is urgently needed.
The main microbial pesticide includes bacteria, actinomycetes and fungi. Zhaixia etc. separates 403 actinomycetes from 48 soil samples collected all over the country, and 30 actinomycetes have inhibition effect on rice blast, wherein the inhibition effect of fresh streptomycin flavacin WM2-4 on rice blast is the best, and the inhibition rate reaches 89.35%. The research of the Roxburgh orchid and the like also shows that the fungi of the trichoderma have good antibiotic effect on the rice blast. In recent years, researchers have begun to study the application of fungal viruses in the prevention and control of rice blast and have obtained preliminary results. The microbial pesticide for preventing and controlling rice blast is mainly prepared from bacteria, wherein bacillus is abundant. The bacillus subtilis and the bacillus pumilus are screened in the spring equal way, and the germination of the meristematic daughter of the rice blast germ and the formation of an attachment cell can be obviously inhibited. Penhuaxian and the like are separated from the roots of rice to obtain bacillus cereus and bacillus subtilis with better preventing and treating effects on rice blast germs. In addition, scientific research workers find that the field control effect of the fermentation liquor of the paenibacillus brevis on the leaf blast reaches 57-64 percent, and the fermentation liquor has good commercial application potential.
The research and application of adopting biological pesticide to prevent and control rice blast is the requirement of sustainable development of rice production, and can meet the requirement of people on the safe production of main grain crops of rice. Although plant protection workers have separated a plurality of biocontrol strains which can obviously inhibit the growth and development of rice blast fungi at present, the field still has a large gap from effectively preventing and controlling rice blast, and the field also lacks of the biocontrol strains which have high-efficiency rice blast preventing and controlling capability and are easy to colonize in rice fields, so that the search for new high-efficiency biocontrol strains is helpful for green prevention and control of rice blast.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the bacillus tequilensis S12 for preventing and treating the rice blast and the application thereof, wherein the bacillus tequilensis S12 has strong antagonism on the rice blast, can obviously inhibit the pathogenicity of the rice blast, and further effectively prevent and treat the rice blast.
Biological sample preservation information:
the Bacillus tequilensis S12 is classified as Bacillus tequilensis (Bacillus tequilensis) and is preserved in China general microbiological culture Collection center (CGMCC) in 7-29 th of 2019 at the address of No. 3 of West Lu 1 of the Korean area of Beijing, and the microbial research institute of the Chinese academy of sciences at the strain accession number of CGMCC No. 18347.
The technical scheme adopted by the invention is as follows:
a Bacillus tequilensis (Bacillus tequilensis) strain is preserved in China general microbiological culture Collection center in 29 months in 2019, with the preservation number of CGMCC No.18347, and is named as S12; the proposed classification is named: bacillus tequilensis; latin has the chemical name: bacillus tequilenesis S12.
The application of the Bacillus tequilensis strain S12 in preventing and controlling rice blast.
The application of the Bacillus tequilensis strain S12 in preparing a medicament for preventing and treating rice blast.
The biocontrol microbial inoculum produced by the Bacillus tequilensis strain S12 is utilized.
Furthermore, the dosage form of the biocontrol microbial inoculum is a liquid preparation.
The preparation method of the biocontrol microbial inoculum comprises the following steps:
(1) activating the Bacillus tequilensis S12 strain on a PDA plate culture medium, selecting a single colony to culture on a PDA slant culture medium, and obtaining a cultured strain;
(2) inoculating the cultured strain into a PDB culture solution for culture to obtain a seed bacterial solution of the Bacillus tequilensis S12 strain;
(3) and carrying out amplification fermentation culture on the seed bacterial liquid of the Bacillus tequilensis S12 strain in a PDB culture solution to obtain the biocontrol microbial inoculum.
Further, in the step (1), the pH value of the PDA culture solution is 7.0-7.2, and the PDA culture solution comprises the following components: 20% of potato boiled filtrate, 2% of glucose, 2% of agar and 76% of purified water; the activation temperature is 28-34 ℃, and the activation time is 20-24 h; the culture time is 12-18h, and the culture temperature is 25-35 ℃.
Further, in the steps (2) and (3), the pH value of the PDB culture solution is 7.0-7.2, and the PDB culture solution comprises the following components: 20% of potato boiled filtrate, 2% of glucose and 78% of purified water.
Further, the preparation method of the cooked potato filtrate comprises the following steps: cutting potato into pieces, adding 4-5 times of water, cooking for 20-30min, and filtering to obtain cooked potato filtrate.
Further, in the step (2), the cultivation is shaking table shaking cultivation, the shaking table shaking speed is 180-200 rpm, the cultivation temperature is 28-30 ℃, and the cultivation time is 20-28 hours.
Further, in the step (3), the volume ratio of the seed bacterium liquid of the Bacillus tequilensis S12 strain to the PDB culture liquid is 1 (90-110); the culture is shaking table oscillation culture, the shaking table oscillation speed is 180-200 rpm, the culture temperature is 28-30 ℃, and the culture time is 40-56 hours.
Further, the active ingredient of the biocontrol microbial inoculum is bacillus tequilensis S12 thallus and/or metabolite thereof.
Further, the concentration of the viable bacteria of the Bacillus tequilensis S12 in the biocontrol microbial inoculum is 0.5-1 multiplied by 109CFU/mL。
The biocontrol microbial inoculum produced by the Bacillus tequilensis strain S12 is applied to preventing and controlling rice blast.
The invention has the beneficial effects that:
(1) the bacillus tequilensis S12 is separated from soil of a paddy field of Jilin province, can be colonized in the paddy field and has no adverse effect on the growth of rice. The solid medium confronting culture shows that the S12 strain has strong antagonism to rice blast germs, and the diameter of the inhibition zone can reach 46 mm; in-vitro inoculation experiments show that the S12 strain can obviously inhibit the pathogenicity of rice blast bacteria; greenhouse inoculation and field experiments show that S12 can effectively prevent and control rice blast and has excellent prevention and control effects on leaf blast and panicle neck blast, so that the Bacillus tequilensis S12 strain can be used for biological prevention and control of rice blast.
(2) The Bacillus tequilensis obtained by the invention has the advantages of easily obtained culture conditions, great commercial potential and good development and application prospects.
(3) The bacillus tequilensis S12 strain is a biological control agent developed specially for rice blast. Because the biological agent is a biological agent, a series of problems caused by the use of chemical pesticides are completely avoided, so that the method is favorable for pollution-free production of rice, and farmers can reduce or eliminate the use amount of other chemical pesticides on the premise of ensuring the yield and quality of rice, thereby not only saving expenses for farmers, but also being favorable for producing green products and improving benefits. In addition, the S12 strain is separated from the paddy field, so that the strain can coexist with the paddy field for a long time and can be symbiotic with the paddy field mutually, and the strain is sprayed on paddy plants for disease control, does not change the nutritional quality of the rice, and is safe and reliable for the eating of the rice and the processed products of the rice.
(4) The preparation method of the biocontrol microbial inoculum prepared by utilizing the Bacillus tequilensis S12 strain is characterized in that the biocontrol microbial inoculum is prepared from the Bacillus tequilensis S12 culture solution, and the biocontrol microbial inoculum is simple in preparation method, low in cost, high in biological safety and suitable for industrial production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a colony morphology of Bacillus tequilensis S12 on LB medium plates;
FIG. 2 is an ITS sequence amplification electropherogram of 16S rDNA of Bacillus tequilensis S12;
FIG. 3 is a photograph showing the culture of Bacillus tequilensis S12 against Pyricularia oryzae;
FIG. 4 is a microscopic view of Bacillus tequilensis S12 biocontrol agent for inhibiting germination of conidia of Pyricularia oryzae;
FIG. 5 is a graph showing the effect of a Bacillus tequilensis S12 biocontrol agent on the pathogenicity of Pyricularia oryzae at different time points;
FIG. 6 is a greenhouse test picture showing the effect of Bacillus tequilensis S12 biocontrol agents on rice blast control.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1
The embodiment provides a preparation method of a cooked potato filtrate, which comprises the following steps: taking 200g of potatoes, cleaning, peeling, cutting into pieces, adding 800g of water, cooking for 20min, and filtering to obtain cooked potato filtrate;
also provides a PDA culture solution with a pH value of 7.0: the components comprise 20% of potato boiled filtrate, 2% of glucose, 2% of agar and 78% of purified water;
also provided is a PDB broth with a pH of 7.0: the components comprise 20% of potato boiled filtrate, 2% of glucose and 78% of purified water.
The embodiment also provides a preparation method of the biocontrol microbial inoculum for effectively preventing and treating rice blast, which comprises the following specific steps:
(1) activating the Bacillus tequilensis S12 strain on a PDA plate culture medium at 28 ℃ for 24h, selecting a single colony on a PDA slant culture medium, and culturing at 25 ℃ for 18h to obtain a cultured strain;
(2) inoculating the cultured strain into a PDB culture solution, performing shaking table shake culture at the speed of 180rpm for 20 hours at the temperature of 28 ℃, and obtaining a seed bacterial solution of the Bacillus tequilensis S12 strain;
(3) inoculating the seed bacterial liquid of the Bacillus tequilensis S12 strain into a PDB culture solution at a volume ratio of 1:90, performing shaking table shaking amplification fermentation culture at a speed of 180rpm at 28 ℃, and culturing for 40h to obtain the biocontrol microbial inoculum.
Example 2
The embodiment provides a preparation method of a cooked potato filtrate, which comprises the following steps: taking 200g of potatoes, cleaning, peeling, cutting into pieces, adding 1000g of water, cooking for 30min, and filtering to obtain cooked potato filtrate;
also provides a PDA culture solution with a pH value of 7.2: the components comprise 20% of potato boiled filtrate, 2% of glucose, 2% of agar and 78% of purified water;
also provided is a PDB broth with pH 7.2: the components comprise 20% of potato boiled filtrate, 2% of glucose and 78% of purified water.
The embodiment also provides a preparation method of the biocontrol microbial inoculum for effectively preventing and treating rice blast, which comprises the following specific steps:
(1) activating the Bacillus tequilensis S12 strain on a PDA plate culture medium at 34 ℃ for 20h, selecting a single colony on a PDA slant culture medium, and culturing at 35 ℃ for 12h to obtain a cultured strain;
(2) inoculating the cultured strain into a PDB culture solution, performing shaking culture at the speed of 200rpm for 28h at the temperature of 30 ℃, and obtaining a seed bacterial solution of the Bacillus tequilensis S12 strain;
(3) inoculating the seed bacterial liquid of the Bacillus tequilensis S12 strain into a PDB culture solution at a volume ratio of 1:110, performing shaking table shaking amplification fermentation culture at a speed of 200rpm at 30 ℃, and culturing for 56h to obtain the biocontrol microbial inoculum.
Example 3
The embodiment provides a preparation method of a cooked potato filtrate, which comprises the following steps: taking 200g of potatoes, cleaning, peeling, cutting into pieces, adding 900g of water, cooking for 25min, and filtering to obtain cooked potato filtrate;
also provides a PDA culture solution with a pH value of 7.1: the components comprise 20% of potato boiled filtrate, 2% of glucose, 2% of agar and 78% of purified water;
also provided is a PDB broth with pH 7.1: the components comprise 20% of potato boiled filtrate, 2% of glucose and 78% of purified water.
The embodiment also provides a preparation method of the biocontrol microbial inoculum for effectively preventing and treating rice blast, which comprises the following specific steps:
(1) activating the Bacillus tequilensis S12 strain on a PDA plate culture medium at 31 ℃ for 22h, selecting a single colony on a PDA slant culture medium, and culturing at 30 ℃ for 15h to obtain a cultured strain;
(2) inoculating the cultured strain into a PDB culture solution, performing shaking culture at the speed of 190rpm for 24 hours at the temperature of 29 ℃, and obtaining a seed bacterial solution of the Bacillus tequilensis S12 strain;
(3) inoculating the seed bacterial liquid of the Bacillus tequilensis S12 strain into a PDB culture solution in a volume ratio of 1:100, performing shaking table shaking amplification fermentation culture at a speed of 190rpm at 29 ℃, and culturing for 48h to obtain the biocontrol microbial inoculum.
Examples of the experiments
Screening and identification of Bacillus tequilensis S12 strain
(1) Collecting samples: collecting a sample, namely paddy field soil in a dormant season in the early winter, wherein the place is the pine city in Jilin province; and (5) taking frozen soil blocks with the surface layer of 5-20 cm, packaging the frozen soil blocks with polyethylene plastic bags, and taking the frozen soil blocks back to the laboratory for separation.
(2) Strain separation: bottling 50mL sterile water in 100mL triangular flask, weighing 5g soil sample (removing residual branches and broken leaves), shaking on shaking table for 30min, taking out, standing for 10min, sucking supernatant, and diluting with sterile water in gradient concentration of 10%-3、10-4、10-5And 10-6Then, 100. mu.L of each of the solutions was pipetted into LB medium (each liter containing tryptone 10g, yeast extract 5g, NaCl 5g, agar powder 15g) and applied uniformly in triplicate for each concentration. Culturing at 28 ℃ for 24-48 h, selecting a single colony according to different forms, colors and the like, streaking and purifying on an LB culture medium, and placing the purified strain on a slant culture medium for later use. The S12 strain is one of the bacteria obtained by the above method. The inventor has preserved the strain in China general microbiological culture Collection center with the preservation date of 2019, 7 months and 29 days and the preservation number of CGMCC No. 18347.
(3) Colony characteristics of biocontrol strain S12: streaking on an LB solid plate, culturing at 28 ℃ for 48h, and observing that colonies of the S12 strain are rough and irregular circles under a microscope, the outer edge of the colonies presents radial ripples, white, opaque and non-wetting degree (shown in figure 1), the S12 strain is a gram-positive bacterium, the bacterial body is rod-shaped, the spore ellipse is columnar, and the spore is not capsule and can move.
(4) Molecular identification of biocontrol strain S12: genomic DNA of S12 strain was extracted and 16SrDNA fragment of S12 strain was amplified using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1429R (5'-GCTACCTTGTTACGACTT-3'). The reaction system is as follows: 10mmol/LdNTP mix, 0.5. mu.L; 10 XPCR buffer, 2.5. mu.L; 1. mu.L (10. mu. mol/mL) of each of the upstream and downstream primers; template DNA, 1. mu.L; Ex-Taq, 0.2. mu.L (5U); ddH2O, 18.8 μ L; the amplification procedure was: pre-denaturation at 94 ℃ for 3 min; then carrying out denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extending at 72 ℃ for 60s, and circulating for 30 times; extension at 72 ℃ for 6 min. The amplified fragments were separated by agarose gel electrophoresis (as shown in FIG. 2, M (DNA marker) was DL 2000). After purification, the sequencing is carried out by the corporation of Venezetian Biotechnology engineering (Shanghai) and the sequence to be tested is compared at NCBI website (https:// blast. NCBI. nlm. nih. gov/blast. cgi) to obtain the identification information. The strain S12 was identified as Bacillus tequilensis, Bacillus tertalyticus (shown in Table 1), both of which are a bacterium, by sequence analysis of the 16SrDNA gene fragment.
TABLE 1 alignment of 16S rDNA sequences of biocontrol strain S12
Second, the inhibitory Activity of Bacillus tequilensis S12 against Pyricularia oryzae
The test was carried out by plate-faced culture, and the solid plate was a PDA medium (each liter contains 200g of boiled filtrate of potato, 20g of glucose, 15g of agar powder, and pH 7.0-7.2). The method comprises inoculating rice blast fungus JJ88 on one side of PDA culture medium, inoculating prepared Bacillus tequilensis S12 strain on the other side by streaking, culturing at 28 deg.C, and measuring the inhibition ability of S12 strain on the growth of rice blast fungus with sterile water as Control (CK). Each treatment was repeated three times, and the growth of rice blast germs was observed and measured after 7 days. The results of the confrontation culture showed that the colonies of Pyricularia oryzae spread very slowly toward the inoculated side of the S12 strain, were completely inhibited in the later stage, and could not continue to spread, and even if the observation period was prolonged to 14 days, the colonies did not spread any more, while the colonies of the control spread well (as shown in FIG. 3). The measurement shows that the diameter of the zone of inhibition of rice blast germs can reach 46 +/-2.4 mm. The test shows that the Bacillus tequilensis S12 strain can inhibit the growth of rice blast germ hypha obviously.
Preparation of bio-control bacterium agent of bacillus tequilensis S12 and detection of bacteriostatic activity of bio-control bacterium agent
(1) Preparation of bio-control bacterium agent of Bacillus tequilensis S12
And (3) culturing S12 seed bacterial liquid: the above-mentioned confrontation culture test has shown that the S12 strain cultured on PDA produces a compound that inhibits the growth of Pyricularia oryzae. Therefore, the invention further adopts PDB liquid culture medium (each liter contains 200g of potato boiled filtrate and 20g of glucose, and the pH value is 7.0-7.2) with similar formula to carry out fermentation culture on the S12 biocontrol strain. And (3) selecting an S12 fresh colony subjected to streak culture, inoculating the colony into a PDB culture solution, and culturing for about 24 hours at the temperature of 28-30 ℃ and the shaking speed of a shaking table of 180-200 rpm. Sampling and measuring the OD value at the position of 600nm every 3h in an ultraclean workbench after 16h, and zeroing with a culture solution without inoculation in the measuring process until the OD value of the seed bacterial solution reaches 0.5-0.8, and finishing the culture to obtain the seed bacterial solution of S12.
S12 preparation of the biocontrol microbial inoculum: the S12 seed bacterial liquid is subjected to amplification fermentation culture in PDB according to the volume ratio of 1: 100. Culturing for 48 hours at the temperature of 28-30 ℃ and the shaking speed of a shaking table of 180-200 rpm to obtain the biocontrol microbial inoculum, wherein the concentration of viable bacteria is 0.5-1 multiplied by 109CFU/mL。
(2) Antibacterial activity determination method of bacillus tequilensis S12 biocontrol microbial inoculum
And testing the bacteriostatic activity of the S12 biocontrol microbial inoculum by adopting an Oxford cup method. Specifically, the S12 biocontrol microbial inoculum is obtained according to the method, and is filtered by a 0.22 mu m filter membrane to obtain sterile fermentation liquor for later use. The periphery of the PDA culture medium is inoculated with rice blast fungus JJ88, 100 mu l of aseptic fermentation liquor of the Bacillus tequilensis S12 strain is inoculated into an aseptic oxford cup which is inserted in advance in the center, the mixture is placed at 28 ℃ for opposite culture, and the inhibition capacity of the S12 fermentation liquor on the growth of the rice blast fungus is measured. PDB was used as a control and fermentation broth containing S12 cells was used as a parallel treatment. Each treatment was repeated three times, and the growth of rice blast germs was observed and measured after 7 days.
TABLE 2 determination of inhibitory Activity of Bacillus tequilensis S12 biocontrol agent against Magnaporthe grisea growth
| S12 biocontrol microbial inoculum | S12 biocontrol microbial inoculum filtrate | Control | |
| Diameter of bacteriostatic circle (mm) | 47±3.1a | 39±1.9ab | 0c |
Note: values-mean ± standard error, different lower case letters indicate significance of difference (p < 0.05); a. ab and c are both expressed by standard errors in the labeling method of "heart method".
As shown in Table 2, the results of the confrontation culture showed that the colony expansion of the rice blast germs toward the side of the S12 fermentation broth was significantly inhibited, and the diameter of the zone of inhibition was measured to be 39. + -. 1.9 mm. The bacteriostatic effect of the fermentation liquor containing the S12 cells is consistent with that of the determination of the inhibitory activity of the Bacillus thuringiensis S12 on rice blast germs, and the diameter of a bacteriostatic zone can reach 47 +/-3.1 mm. Statistical analysis (t test) shows that S12 biocontrol microbial inoculum and filtrate thereof have extremely obvious bacteriostatic effects (ab and c) compared with control, in addition, the microbial inoculum containing S12 cells has higher bacteriostatic activity and obvious difference (a and b) than the sterile filtrate thereof, namely in the PDB fermentation biocontrol microbial inoculum of the Bacillus tequilensis S12 strain, not only live cells have the effect of inhibiting rice blast bacteria, but also fermentation liquor from which the live cells are removed can also extremely obviously inhibit the growth of rice blast bacteria hyphae.
Fourth, the inhibitory activity of the bio-control agent of the Bacillus tequilensis S12 on the germination of conidia of Magnaporthe grisea is determined
The rice blast germs are transmitted as pests through conidia, and after the conidia are attached to the surfaces of rice, the rice blast germs germinate and develop attachment cells to invade the rice, so that diseases are caused. Inhibiting the germination of conidia can block the progress of infection from the beginning.
The section determines the inhibitory activity of the S12 biocontrol microbial inoculum on the germination of conidia of magnaporthe grisea. Taking 10 μ l of Magnaporthe grisea JJ88 conidium solution (concentration 5X 10)4CFU/mL) is dripped on a glass slide, and 2 mu l of PDB fermentation biocontrol microbial inoculum (0.5-1 multiplied by 10) of the Bacillus tequilensis S12 strain is added9CFU/mL), 2. mu.l of PDB was added as a Control (CK), incubated at 28 ℃ for 12 hours under moisture retention, and then S12 was analyzed for the inhibition of conidium germination of Pyricularia oryzae. Microscopic observation shows that the S12 biocontrol agent can remarkably inhibit the germination of conidia of the rice blast fungus, wherein 81% of the conidia cannot germinate and have incomplete morphology, and occasionally germinated few spores grow to form short and multi-bent germ tubes and lose the capacity of continuous growth, while the conidia of the rice blast fungus of the control germinate well (as shown in figure 4). The test shows that the bio-control bacterium agent of the bacillus tequilensis S12 has a destructive effect on the conidia of the rice blast fungus and can remarkably inhibit the germination and the extension of a germ tube.
Five, the activity of the bio-control bacterium agent of the Bacillus tequilensis S12 for inhibiting the pathogenicity of rice blast bacterium is determined
The inhibitory activity of the bio-control agent of the Bacillus tequilensis S12 on the pathogenicity of rice blast fungus is determined by taking a barley variety 'Qianjin-Nuo' which is very susceptible as a host and inoculating experiments on in vitro leaves. The experiment sets a plurality of treatments around the time point of inoculation of rice blast germs, and applies the S12 biocontrol microbial inoculum at different time points respectively to test the preventing and treating effects of S12 on the rice blast. 8 mul of Magnaporthe grisea JJ88 spore solution (concentration 1X 10) is added dropwise to each inoculation point5CFU/mL), 2. mu.l of PDB biocontrol bacterium agent (0.5-1 × 10) of Bacillus tequilensis S12 strain was added at different time points9CFU/mL), 2. mu.l of PDB was added as a Control (CK), and the mixture was incubated at 25 to 28 ℃ for 7 days with moisture retention, and then S12 was analyzed for the inhibition of rice blast pathogenic bacteria pathogenicity. Experimental results show that the application of the S12 biocontrol microbial inoculum 12 hours before and at the same time as the inoculation of the Magnaporthe grisea or the application of the S12 biocontrol microbial inoculum 4-48 hours after the inoculation of the Magnaporthe grisea can obviously inhibit the occurrence of the Magnaporthe grisea, only pinpoint-sized scabs can be formed at the inoculation part and cannot be expanded, and the control treatment causes the disease normally(as shown in fig. 5).
TABLE 3 determination of inhibitory Activity of Bacillus tequilensis S12 biocontrol agent against pathogenic force of Pyricularia oryzae
| Point of application S12 | 12HBI | 0 | 4HPI | 12HPI | 24HPI | 48HPI |
| Pathogenic power of Pyricularia oryzae% | 4±2.1 | 1.6±0.8 | 5.1±2.5 | 8.3±2.9 | 8.9±3.1 | 12.5±3.7 |
| Control effect% | 96±3.1 | 98.4±1.4 | 94.9±3.9 | 91.7±4.1 | 91.1±3.5 | 87.5±3.9 |
Note: mean ± standard error; the pathogenicity of rice blast germs is reflected by the lesion area of the inoculation part, and a relative value is calculated by comparing the pathogenicity with the pathogenicity of control treatment at the same time point; HBI represents the number of hours of inoculation of the biocontrol microbial inoculum before inoculation of Magnaporthe grisea; HPI represents the number of hours of inoculation of the biocontrol microbial inoculum after inoculation of Magnaporthe grisea; 0 represents that the rice blast germs and the biocontrol microbial inoculum are inoculated simultaneously.
As shown in Table 3, the test shows that the pathogenic activity of the rice blast germs can be effectively prevented by applying the Bacillus tequilensis S12 biocontrol microbial inoculum in advance, the control effect is over 90 percent, even if the S12 biocontrol microbial inoculum is applied after the rice blast germs infect hosts for 48 hours, the further infection of the rice blast germs can be effectively controlled, and the control effect reaches 87.5 percent. Therefore, the bacillus tequilensis S12 biocontrol microbial inoculum has both prevention and treatment effects on rice blast.
Greenhouse test of biocontrol microbial inoculum of bacillus thuringiensis S12 on rice blast control effect
The invention adopts a greenhouse inoculation experiment to test the control effect of the bacillus tequilensis S12 biocontrol microbial inoculum on rice blast. Sowing a rice cultivar Jijing 88 in a greenhouse seedling raising tray, spraying a PDB fermentation biocontrol microbial inoculum (1-2 multiplied by 10) of a Bacillus tequilensis S12 strain when the rice cultivar Jijing 88 grows to a three-leaf stage (about 18 days)8CFU/mL), PDB medium as Control (CK); spraying after 36h to inoculate rice blast fungus JJ88 spore liquid (with concentration of 5 × 10)5CFU/mL), incubation and moisture preservation, and analyzing the disease after 7 d.
Grading standard of leaf blast disease:
grade 0, no disease;
grade 1, the disease spots are small and few, and the area of the disease spots accounts for less than 1% of the area of the leaves;
2, the disease spots are small and much, or large and little, and the area of the disease spots accounts for 1 to 5 percent of the area of the leaves;
grade 3, the disease spots are large and more, and the area of the disease spots accounts for 5 to 10 percent of the area of the leaves;
grade 4, large and much scabs, wherein the area of the scabs accounts for 10 to 50 percent of the area of the leaves;
and 5, the lesion area accounts for more than 50% of the leaf area, and the whole leaf is withered.
Calculating the formula: incidence rate = number of leaves affected/total number of leaves investigated × 100; the disease index ∑ (number of onset of disease at each stage × representative value at each stage)/(total number of examined leaves × highest representative value) × 100.
TABLE 4 greenhouse test of biocontrol microbial inoculum of Bacillus tequilensis S12 for controlling rice blast
| Treatment of | The incidence of disease% | Index of disease condition | Control effect% |
| S12 | 9±2.1b | 4.8±1.4b | 87.9±3.5a |
| CK | 100a | 39.8±1.9a | 0b |
Note: mean ± standard error, different lower case letters indicate significance of difference (p <0.05)
The experimental result shows that the early spraying of the S12 biocontrol microbial inoculum has an efficient protection effect on rice, can remarkably inhibit the occurrence of rice blast, has few scabs, has the morbidity of only 9% of the control, occasionally has tiny scabs and is difficult to expand, has the control effect of the S12 biocontrol microbial inoculum of 87.9%, and has the control group with normal morbidity and scab expansion (as shown in figure 6 and table 4). The experiment shows that the bio-control strain S12 of the Bacillus tequilensis can effectively prevent and control rice blast.
Field test of biocontrol bacterium agent of bacillus hepta-tequilensis S12 on rice blast control effect
The invention finally adopts field rice field test to test the control effect of the bacillus tequilensis S12 biocontrol microbial inoculum on rice blast. Performing field efficacy test of Bacillus tequilensis S12 biocontrol microbial inoculum in Yilazi town of Yongji county of Jilin province, spraying PDB fermentation biocontrol microbial inoculum (1-2 × 10) of S12 strain on a cultivated variety of long-grain incense8CFU/mL); the contrast agent is tricyclazole and iprobenfos wettable powder (produced by Baoqing crop science and technology limited in Hebei), the application amount is 100-150 g/mu, and 40-50 kg of water is added. The average area per test cell is 50m2No control was performed as a negative control. 3 replicates were set per treatment. The control effect of the bacillus tequilensis S12 biocontrol microbial inoculum on leaf blast and panicle blast is investigated 3-4 weeks before harvest.
The method for calculating the grading standard, incidence rate and disease index of the leaf blast disease is shown in 'greenhouse test of the biocontrol microbial inoculum of bacillus tequilensis S12 on the control effect of the rice blast'.
Grading standard of ear blast disease condition:
grade 0, no disease;
grade 1, attack of individual branches and stalks;
grade 2, about 1/3 branch and peduncle diseases;
grade 3, the neck or main axis of the ear is attacked;
grade 4, panicle neck attack, most blighted grains;
grade 5, the development of the neck of the ear causes the white ear. Calculating the formula: incidence rate = number of diseased ears/total number of investigated ears × 100;
the disease index ∑ (number of onset at each stage × representative value at each stage)/(total number of examined ears × highest representative value) × 100.
TABLE 5 field test of biocontrol bacterial agent of Bacillus tequilensis S12 for preventing and controlling rice blast
Description of the drawings: mean ± standard error, different lower case letters indicate significance of difference (p <0.05)
As shown in Table 5, the field test results show that spraying of the bio-control fungicide of Bacillus tequilensis S12 has an efficient protection effect on rice, can remarkably inhibit the occurrence of rice blast, can effectively prevent and control leaf blast and panicle blast, has the prevention effect of 83.8 percent and 82.8 percent respectively, has few scabs, and has small and difficult expansion of the occasionally occurring scabs; compared with a control medicament, the control effect of the S12 biocontrol microbial inoculum has no significant difference. The test shows that the bacillus tequilensis S12 biocontrol microbial inoculum can effectively prevent and control the occurrence of rice blast in the field, can replace chemical pesticides, and is used for green and efficient control of the rice blast in the rice field.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. A Bacillus tequilensis (Bacillus tequilensis) strain is preserved in China general microbiological culture Collection center in 29 months in 2019, with the preservation number of CGMCC No.18347, and is named as S12.
2. Use of the Bacillus tequilensis strain of claim 1 for controlling rice blast.
3. The use of the Bacillus tequilensis strain of claim 1 in the preparation of a medicament for the control of rice blast.
4. A biocontrol microbial agent produced by the Bacillus tequilensis strain of claim 1.
5. The biocontrol microbial inoculant according to claim 4 wherein said dosage form is a liquid formulation.
6. A method for preparing the biocontrol microbial inoculum of claim 5, comprising the steps of:
(1) activating the Bacillus tequilensis S12 strain on a PDA plate culture medium, selecting a single colony to culture on a PDA slant culture medium, and obtaining a cultured strain;
(2) inoculating the cultured strain into a PDB culture solution for culture to obtain a seed bacterial solution of the Bacillus tequilensis S12 strain;
(3) and carrying out amplification fermentation culture on the seed bacterial liquid of the Bacillus tequilensis S12 strain in a PDB culture solution to obtain the biocontrol microbial inoculum.
7. The preparation method of the biocontrol microbial inoculum according to claim 6, wherein in the step (1), the pH value of the PDA culture solution is 7.0-7.2, and the PDA culture solution comprises the following components: 20% of potato boiled filtrate, 2% of glucose, 2% of agar and 76% of purified water; the activation temperature is 28-34 ℃, and the activation time is 20-24 h; the culture time is 12-18h, and the culture temperature is 25-35 ℃;
in the steps (2) and (3), the pH value of the PDB culture solution is 7.0-7.2, and the PDB culture solution comprises the following components: 20% of potato boiled filtrate, 2% of glucose and 78% of purified water;
the preparation method of the cooked potato filtrate comprises the following steps: cutting potato into pieces, adding 4-5 times of water, cooking for 20-30min, and filtering to obtain cooked potato filtrate.
8. The preparation method of the biocontrol microbial inoculum according to claim 6, wherein in the step (2), the culture is shaking table shaking culture, the shaking table shaking speed is 180-200 rpm, the culture temperature is 28-30 ℃, and the culture time is 20-28 h;
in the step (3), the volume ratio of the seed bacterial liquid of the Bacillus tequilensis S12 strain to the PDB culture solution is 1 (90-110); the culture is shaking table oscillation culture, the shaking table oscillation speed is 180-200 rpm, the culture temperature is 28-30 ℃, and the culture time is 40-56 hours.
9. The method for producing a biocontrol microbial inoculum according to claim 6, wherein the biocontrol microbial inoculum comprises the effective component of Bacillus tequilensis S12 cells and/or metabolites thereof, and the concentration of viable bacteria of Bacillus tequilensis S12 in the biocontrol microbial inoculum is 0.5 to 1 x 109CFU/mL。
10. The use of the biocontrol microbial inoculum of claim 4 or 5 in the control of rice blast.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107849518A (en) * | 2014-05-28 | 2018-03-27 | 株式会社第格林产业 | The preparation for natural plant protecting and plant enhancing is developed from the raw bacillus of new plant endogenetic bacteria rice of rice rhizosphere separation and using it |
| CN108998394A (en) * | 2018-08-16 | 2018-12-14 | 浙江农林大学 | A kind of bacillus and its application |
-
2020
- 2020-01-06 CN CN202010008665.2A patent/CN110982764A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107849518A (en) * | 2014-05-28 | 2018-03-27 | 株式会社第格林产业 | The preparation for natural plant protecting and plant enhancing is developed from the raw bacillus of new plant endogenetic bacteria rice of rice rhizosphere separation and using it |
| CN108998394A (en) * | 2018-08-16 | 2018-12-14 | 浙江农林大学 | A kind of bacillus and its application |
Non-Patent Citations (1)
| Title |
|---|
| LI HUI等: "Isolation and evaluation of endophytic Bacillus tequilensis GYLH001 with potential application for biological control of Magnaporthe oryzae", 《PLOS ONE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553085A (en) * | 2020-11-27 | 2021-03-26 | 云南农业大学 | Non-toxic rice blast strain for preventing and controlling rice blast and application thereof |
| CN112553085B (en) * | 2020-11-27 | 2022-11-01 | 云南农业大学 | Nontoxic rice blast strain for preventing and controlling rice blast and application thereof |
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