CN110551666A - Bacillus amyloliquefaciens YB-130, microbial preparation and application thereof - Google Patents
Bacillus amyloliquefaciens YB-130, microbial preparation and application thereof Download PDFInfo
- Publication number
- CN110551666A CN110551666A CN201910954686.0A CN201910954686A CN110551666A CN 110551666 A CN110551666 A CN 110551666A CN 201910954686 A CN201910954686 A CN 201910954686A CN 110551666 A CN110551666 A CN 110551666A
- Authority
- CN
- China
- Prior art keywords
- bacillus amyloliquefaciens
- wheat
- microbial preparation
- culture medium
- microbial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
the invention discloses a bacillus amyloliquefaciens YB-130, a microbial preparation thereof and application thereof, aiming at solving the technical problems of poor quality, low yield and heavy diseases of wheat, wherein the bacillus amyloliquefaciens YB-130 is obtained by screening, is classified and named as Bacillus amyloliquefaciens, and has the preservation number of CGMCC NO.16229, and the microbial preparation is provided and is prepared from the following raw materials of, by mass, 60-80% of bacillus amyloliquefaciens CGMCC NO.16229 bacterial liquid, 8-10% of adhesive, 1-2% of stabilizer and 100% of filler, and has good application effects in the aspects of improving the quality and the yield of wheat, inhibiting the synthesis of wheat gibberella DON toxin, preventing and controlling the diseases of crops and the like.
Description
Technical Field
The invention relates to the technical field of microbial preparations, in particular to bacillus amyloliquefaciens YB-130, a microbial preparation thereof and application thereof.
Background
wheat is one of the most important grain crops in the world, and in northern areas of China, wheat is the crop with the largest planting area and plays a decisive role in agricultural production.
With the improvement of the quality of life, people have higher and higher requirements on the quality of wheat. At present, the total wheat production in China basically meets the requirements of people, the supply is changed from long-term shortage into basic balance, and the wheat is redundant in abundance. People have higher requirements on the quality of wheat grains, the requirements of the people are changed from the new stage of solving the problem of satiety to high quality, safety and health, and the main contradiction of wheat is changed from the total quantity deficiency to the structural contradiction of insufficient quality.
Wheat flour is classified into three types of strong gluten, medium gluten and weak gluten according to the use of wheat. According to the national standards of high-quality strong gluten wheat (GB/T17892-1999 and GB/T17983-1999), the main indexes of the first-grade strong gluten wheat are as follows: the protein content (dry basis) is more than or equal to 15.0 percent, the wet gluten content is more than or equal to 35.0 percent, the dough stabilization time is more than or equal to 10.0 min, the cutin rate is more than or equal to 70 percent, the falling number is more than or equal to 300 s, the baking quality score is more than or equal to 80, the bulk density is less than or equal to 770 g/L, the water content is less than or equal to 12.5 percent, the imperfect grains are less than or equal to 6.0, the total impurity content is less than or equal to 1; the main indexes of the secondary strong gluten wheat are as follows: the protein content (dry basis) is more than or equal to 14.0 percent, the wet gluten content is more than or equal to 32.0 percent, the dough stabilization time is more than or equal to 7.0 min, the cutin rate is more than or equal to 70 percent, the falling number is more than or equal to 300 s, the baking quality score is more than or equal to 80, the bulk density is less than or equal to 770 g/L, the water content is less than or equal to 12.5 percent, the imperfect grains are less than or equal to 6.0, the total impurity content is less than or equal to 1; the main indexes of weak gluten wheat are as follows: the protein content is less than or equal to 11.5 percent, the wet gluten is less than or equal to 22.0 percent, the dough stabilizing time is more than or equal to 2.5 min, the wheat cutin rate is less than or equal to 30 percent, and the falling number is less than or equal to 300 s; the medium gluten wheat is a type between the secondary gluten wheat and the weak gluten wheat, and the wheat flour is mainly used for processing foods such as steamed bread, noodles and the like.
The wheat grains and the flour quality thereof have direct relation with variety breeding and improvement; therefore, breeding new high-quality wheat varieties is one of the important ways for improving the quality of wheat; however, the breeding cycle is long, the cost is high, the practical problems in the prior art are difficult to solve, and the currently popular main cultivated variety resources cannot be utilized or excavated.
Disclosure of Invention
The invention aims to solve the technical problems of screening out the bacillus amyloliquefaciens YB-130 with new functions and a microbial preparation thereof, and applying the bacillus amyloliquefaciens YB-130 to improving the quality and the yield of wheat and biological control so as to solve the technical problems of poor quality, low yield and serious diseases of the wheat.
In order to solve the technical problems, the invention adopts the following technical scheme:
A bacillus amyloliquefaciens YB-130 with excellent functions is obtained by screening from a plurality of strains, is classified and named as Bacillus amyloliquefaciens, and is preserved in China general microbiological culture Collection center (CGMCC) (the address: No. 3 of Xilu No.1 of Beijing Kogyo district facing Yang, Beijing city; zip code: 100101) in 8 months and 7 days in 2018, and the preservation number is CGMCC NO. 16229.
the microbial preparation is prepared from the following raw materials in percentage by mass: 60-80% of bacillus amyloliquefaciens CGMCC number 16229 bacterial liquid, 8-10% of adhesive, 1-2% of stabilizer and 100% of filler.
Preferably, the adhesive is at least one of maltodextrin, soybean meal and gelatin, so that the adhesive property of the medicament on germs can be improved, and the prevention and treatment effect can be better exerted; the stabilizer is bentonite or carboxymethyl cellulose, and can prevent the decomposition of effective components or the deterioration of physical properties in the storage process of the microbial preparation; the filler is at least one of talcum powder, attapulgite, clay and white carbon black, and can be used as a carrier attached to flora and also can increase the dispersibility of the preparation.
Preferably, the effective viable count of the bacillus amyloliquefaciens CGMCC number 16229 bacterial liquid is 10 10 -10 12/g.
preferably, the preparation method of the bacillus amyloliquefaciens CGMCC number 16229 bacterial liquid comprises the following steps:
(1) Inoculating bacillus amyloliquefaciens CGMCC number 16229 into a solid culture medium, and culturing at 28-32 ℃ for 32-48 hours;
(2) inoculating the strain cultured in the step (1) into a primary seed culture medium, wherein the initial pH value is 7.0-7.2, and culturing at 24-37 ℃ for 20-24 hours to obtain primary seeds;
(3) Inoculating the primary seeds into a secondary seed culture medium according to the inoculation amount of 1-6% of the volume ratio, aerating, and culturing for 28-40 hours to obtain secondary seeds;
(4) And (3) inoculating the second-level seeds into a fermentation tank according to the inoculation amount of 1-6% of the volume ratio, and performing high-density fermentation culture to obtain the compound microbial inoculum.
preferably, the formula of the primary seed culture medium comprises 10g of corn flour, 5g of glucose, 15g of bean cake powder, 5g of fish meal, 5g of CaCO 3 5, (NH 4) 2 SO 4 1g, 0.3g of K 2 HPO 4 0.3, 0.2g of MgSO 4 & 7H 2 O and 1000ml of water.
Preferably, the formula of the secondary seed culture medium comprises 12.98g of corn flour, 5g of glucose, 20.11g of bean cake powder, 5g of fish meal, 6.90g of CaCO 3 6.90, (NH 4) 2 SO 4 1g, 0.3g of K 2 HPO 4 0.3, 0.2g of MgSO 4.7H 2 O, 0.2g of MnSO 4. H 2 O and 1000ml of water.
The bacillus amyloliquefaciens or the microbial preparation can be effectively applied to the aspects of improving the quality of wheat, improving the yield of the wheat, inhibiting the synthesis of wheat gibberellic disease DON toxin, biologically preventing and treating plant diseases caused by fusarium and the like.
Compared with the prior art, the invention has the main beneficial technical effects that:
1. The invention screens out a bacillus amyloliquefaciens YB-130 with new functions or functions, which is classified and named as Bacillus amyloliquefaciens, and the preservation number is CGMCC NO. 16229.
2. the bacillus amyloliquefaciens or the microbial preparation thereof can effectively improve the yield and the quality of field wheat, the yield of the treated wheat is averagely more than 16 percent, the protein content is averagely as high as about 15 percent, and the yield is obviously higher than that of the conventional treatment.
3. the bacillus amyloliquefaciens or the microbial preparation thereof can prolong the functional period of wheat leaves and delay the mature period, thereby being beneficial to the later filling and thousand seed weight improvement of wheat and further improving the yield and the quality of the wheat.
4. the bacillus amyloliquefaciens or the microbial preparation thereof is environment-friendly, has low production cost, can realize green prevention and control of crop diseases, and is beneficial to pollution-free production of crops.
Drawings
FIG. 1 is a photograph of the experiment of the opposition in the rescreened dish for inhibiting Fusarium graminearum.
FIG. 2 is a photograph of the experiment of inhibiting the strain YB-130 from the confronting test in a fusarium graminearum dish.
FIG. 3 is a map of the phylogenetic tree of strain YB-130.
FIG. 4 is a statistical plot of the effect of co-cultivation of strain YB-130 with PH-1 on the synthesis of PH-1 toxin.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.
The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the reagents and culture media are all conventional reagents and culture media which are sold on the market if not specified; the test methods involved are conventional methods unless otherwise specified.
example 1: isolation, screening and identification of Bacillus amyloliquefaciens YB-130
(1) sample collection
wheat ear diseases are collected in wheat fields of Xihua county, late Yingxiang village, Xihua county, Yangrong, 5, 15 days in 2016, and are brought back by a fresh-keeping bag for later use.
(2) Separation and purification
And taking the collected ear tissues of the wheat plants as the separation objects of the endophytic bacteria.
Taking ear grains at different parts of the ear in the filling period, respectively taking 1g of sample, washing the sample with running water for multiple times, naturally drying the sample, soaking the sample in 75% ethanol for 1 min, soaking in 2% NaClO for 3 min, soaking in 75% ethanol for 30 s, washing the sample with sterile water for 2 times, and finally washing the sample with a water-coated NA culture medium plate for checking the sterilization effect. And (3) placing the sample with the sterilized surface on a flat plate, picking different single colonies according to the characteristics of the form, color, size and the like of the grown colonies, recording the serial numbers of the single colonies, repeatedly purifying, and then connecting an NA inclined plane for storage.
Through the separation, 56 bacillus-like strains are separated out altogether, then 7 strains with antagonistic action on fusarium graminearum are obtained through primary screening by a plate mutual opposition method, 5 strains with strong antagonistic action, namely YB-130, XH-16, XH-43, XH-25 and XH-22 (see table 1 and figure 1) are obtained through secondary screening by a cup butterfly method, wherein the antagonistic effect of the YB-130 strains is the best (see figure 2).
TABLE 1 inhibitory Effect of six antagonistic bacteria on wheat take-all pathogen
。
(3) Bacteriostatic detection
Through further experimental study, YB-130 is determined to have bacteriostatic effect on the following 10 pathogenic bacteria, and the results are shown in Table 2.
TABLE 2 bacteriostatic profile of YB-130 strain
。
note: "+ +": the antibacterial belt is more than 10 mm; "+": 5-10 mm of antibacterial band;
"+": the antibacterial zone is less than 5 mm; "-": the antibacterial zone is less than 3 mm.
Therefore, YB-130 shows a broad-spectrum bacteriostatic effect, wherein the bacteriostatic effect on wheat scab, verticillium dahliae of cotton, maize bacterial wilt and the like is better.
(4) Identification of Strain YB-130
1) Morphological characterization
The YB-130 strain with better rescreening effect is cultured in an LB culture plate for 1d at 28 ℃, the colony morphology and the color are observed, the YB-130 grows well on an LB culture medium, the colony is milk white, no pigment is generated, the colony morphology is irregular, the surface is provided with a bulge and forms an irregular fold, slight fishy smell is generated after 24d of culture, the bacteria fall into a cluster when picked up and have mucus, and gram staining is positive (G +).
2) Physiological and biochemical identification
A part of physiological and biochemical indexes of Bergey's Manual of bacteria identification (eighth edition) and general Manual of bacteria identification are measured and observed by referring to the methods of Bergey's Manual of bacteria identification (eighth edition) and general Manual of bacteria identification (JJ), and the results are shown in Table 3.
TABLE 3 physiological and biochemical characteristics of Strain YB-130
。
note: "+" indicates a positive reaction, and "-" indicates a negative reaction.
As can be seen from Table 3, strain YB-130 is capable of liquefying gelatin, hydrolyzing starch, reducing nitrate, peptonizing milk, producing catalase, not hydrolyzing cellulose, producing glucose, utilizing glucose, fructose, galactose, xylose, maltose, raffinose and mannitol, strain YB-130 is resistant to NaCl concentration of 7%, and does not grow when pH is less than 5 or pH is greater than 9, according to Bergey bacteria identification Manual (eighth edition) and common bacteria identification Manual, morphological characteristics and physiological and biochemical characteristics of YB-130 are in accordance with the classification standards of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
3) Molecular biological identification
BLAST analysis of the YB-130 sequence and related sequences in a GenBank database shows that 128 homologous sequences matched with the sequence are provided, 16S rRNA sequences with higher homology and representative strains are selected to construct a phylogenetic tree, and as shown in FIG. 3, the strain YB-130 and Bacillus amyloliquefaciens (FZB 42) are in the same branch of the evolutionary tree and have homology of 99.6%.
And (3) identifying the YB-130 strain as the bacillus amyloliquefaciens by combining the morphological, physiological and biochemical characteristics and sequence homology analysis results of the YB-130 and related strains 16S-23S rRNA ISR (Bacillus amyloliquefaciens) (Bacillus amyloliquefaciens).
example 2: culture of Bacillus amyloliquefaciens YB-130
The method comprises the following specific steps:
(1) Culture in a culture dish: inoculating the bacillus amyloliquefaciens YB-130 on a solid culture medium LB under the aseptic condition, and culturing for 32-48 hours at the temperature of 28-32 ℃, preferably 30 ℃;
(2) First-order seed culture: inoculating the strain cultured in the step (1) into a primary seed culture medium under an aseptic condition, wherein the initial pH value is 7.0-7.2, and culturing at 24-37 ℃ for 20-24 hours to prepare a primary seed;
The formula of the primary seed culture medium comprises 10g of corn flour, 5g of glucose, 15g of bean cake powder, 5g of fish meal, 5g of CaCO 3 5, (NH 4) 2 SO 4 1g, 0.3g of K 2 HPO 4 0.3, 0.2g of MgSO 4 & 7H 2 O and 1000ml of water;
(3) Secondary seed culture: inoculating the primary seeds into a fermentation culture medium according to the inoculation amount of 1-6% by volume percentage, aerating, and culturing for 28-40 hours to obtain secondary seeds;
the secondary seed culture medium comprises 12.98g of corn flour, 5g of glucose, 20.11g of bean cake powder, 5g of fish meal, 6.90g of CaCO 3 6.90, (NH 4) 2 SO 4 1g, 0.3g of K 2 HPO 4 0.3, 0.2g of MgSO 4.7H 2 O, 0.2g of MnSO 4.H 2 O and 1000ml of water;
(4) Fermentation culture: inoculating the second-level seeds into a fermentation tank according to the inoculation amount of 1-6% by volume percentage, and performing high-density fermentation culture to obtain a bacterial suspension of the strain.
Example 3: preparation of bacillus amyloliquefaciens YB-130 microbial preparation
(1) mixing the concentrated bacterial liquid prepared in the example 2 with talcum powder and attapulgite, and spray-drying to prepare mother powder;
(2) Adding maltodextrin into the mother powder to prepare a microbial preparation;
Wherein the mass percentages of the bacterial liquid, the maltodextrin and the bentonite are 60%, 8% and 1%, and the total mass percentage of the talcum powder and the attapulgite is 100%. The effective viable count of the bacillus amyloliquefaciens YB-130 is 1000 hundred million/gram.
example 4: preparation of bacillus amyloliquefaciens YB-130 microbial preparation
(1) Mixing the concentrated bacterial liquid obtained in the embodiment 2 with argil and clay, and performing spray drying to prepare mother powder;
(2) adding soybean powder and bentonite into the mother powder to obtain the microbial preparation.
Wherein, the mass percentages of the bacteria liquid, the soybean meal and the bentonite are 70 percent, 9 percent and 1.5 percent, and the clay are complemented to 100 percent.
Example 5: preparation of bacillus amyloliquefaciens YB-130 microbial preparation
(1) mixing the concentrated bacterial liquid prepared in the example 2 with white carbon black, and performing spray drying to prepare mother powder;
(2) Adding gelatin and carboxymethyl cellulose into the mother powder to obtain the microbial preparation.
Wherein, the mass percentages of the bacteria liquid, the gelatin and the carboxymethyl cellulose are 80%, 10% and 2%, and the balance of the white clay and the clay is 100%.
Example 6: verification test for influence of microbial preparation on field wheat yield and quality
(1) design of experiments
The experimental site: test demonstration base of agricultural academy of sciences of Henan province
wheat variety: zheng wheat 366, sowing 25 jin/mu, applying base fertilizer and topdressing according to the conventional method.
Pre-sowing treatment: normally soil preparation, shallow ploughing, uniform mechanical sowing and average row spacing of 21 cm.
Experiment time: 10 months-2018 months in 2017.
Experimental treatments as shown in table 5, the trial was set up with 4 agent treatments and 1 blank control treatment, repeated 3 times per treatment, randomized block arrangement, with 20 x 30m 2 per treatment cell area, the trial treatments are as in table 4.
TABLE 4 Experimental treatment
。
The seed dressing method comprises the following steps: seed water adding proportion 3: 100, fully mixing the medicament with water, uniformly stirring seeds, and airing for later use.
The spraying method comprises the following steps: the application dosage of each test agent is shown in table 4, and the application is carried out for the 1 st time in the 4 months of 2016 and 20-25 days of the spike filling period of wheat; in 2016, the 5 months and the 1-5 days, the 2 nd pesticide application is carried out in the wheat flowering period, a knapsack electric sprayer is adopted for spraying, and the pesticide is prepared by a secondary dilution method, wherein the water consumption per mu is 30 kg.
(2) Effect of microbial Agents on yield
Sampling five points in each cell before harvesting, investigating two lines of 1m at each point, measuring the average line spacing (21 cm), and investigating the number of wheat ears which is equivalent to the number of ears per mu; selecting representative 20 ears continuously to calculate the average ear grain number, weighing thousand grain weight after drying, and calculating the theoretical yield by 85 folds, wherein the result is shown in table 5.
TABLE 5 comparison of wheat yield test results
。
note: the mean values for each treatment were shown to differ by a different lower case letter to a 5% significance level. The same goes for
Therefore, the following steps are carried out: the evaluation of the yield of the wheat in the field shows that the microbial preparation in the example 3 has the best treatment effect, the yield is increased by 24.3 percent, and the yield is obviously higher than that of other treatments. 32% tebuconazole was applied once with a yield increase of 19.7%.
(2) Detecting the effect of a microbial preparation on quality
The quality detection is finished in wheat research institute of agricultural science institute of Henan province, five points are sampled in each district before harvesting, about 1Kg of panicle part of 2 is taken at each point, 5 points are mixed uniformly, the mixture is dried and prepared into powder, the powder is placed for about 15 days and is then cooked, the detection is carried out, and the steps are repeated for 3 times.
the detection method comprises the following steps:
1) Milling: milling wheat according to the hardness of wheat grains by referring to the AACC26-20 method;
2) Moisture content of flour: according to the method of GB/T5497-1985, the method adopts a constant weight method at 105 ℃;
3) Powder quality: according to the method of GB/T14614-2006, a dough kneading bowl of 50g is adopted, the dough consistency is controlled to be 480-520 FU, and the powder curve result is automatically analyzed by using system software;
4) Gluten index: the content of the wet gluten and the gluten index are determined according to the method of GB/T5506.2-2008, the dried gluten index utilizes 2020 type dried gluten to determine a baking oven, and the weight of the baked dried gluten is multiplied by 10 to obtain the dried gluten value of the sample.
The results are shown in Table 6.
TABLE 6 wheat yield test results
。
Therefore, the following steps are carried out: by detecting the quality of the field wheat, the microbial preparation in the example 3 has the best treatment effect, the protein content is 15.0%, and the settling time is 16.8 minutes, which is obviously higher than that of other treatments. Bacillus amyloliquefaciens B7900 (commercially available) was administered twice.
It was also observed in the experiment that the microbial preparation treatment of example 3 extended the functional stage of wheat leaves and delayed the maturation stage by 3 days, which is very beneficial for the late filling and thousand kernel weight improvement of wheat.
Example 7: test for verifying influence of YB-130 strain on synthesis of wheat scab germ DON toxin
Deoxynivalenol (DON) is a metabolite of fusarium, has the highest toxicity, can cause poisoning of people and livestock, and has a toxic effect on germination of wheat cells, tissues and seeds.
in order to explore a mechanism that microbial agents affect yield and quality of wheat in fields, a YB-130 strain and a fusarium graminearum PH-1 strain are co-cultured, and the influence of synthesis of DON toxin of the PH-1 strain is detected.
The method comprises the following specific steps:
(1) Inoculating fusarium graminearum PH-1 strain to a flat plate made of a PDA culture medium for culturing for 5d, picking PDA bacterial blocks with the diameter of 1cm by using toothpicks respectively to a sterile triangular flask filled with 50ml of CMC culture medium, and shaking at 25 ℃, 220rpm for 5 d;
(2) respectively inoculating the YB-130 strain and the XH-25 strain separated in the example 1 to a solid LB culture medium for 2 days, selecting a single colony, inoculating the single colony to a liquid LB culture medium, and keeping for 36 hours for later use;
(3) Filtering the culture medium after the PH-1 strain is shaken by using a single-layer Miracloth, transferring the culture medium into a sterile 50ml centrifuge tube, centrifuging at 3000rpm for 10min to collect conidia, washing the conidia cleanly by using sterile water, transferring the conidia into a sterile 50ml triangular flask, and adjusting the concentration of the conidia to 10 4/ml by using a TBI culture medium;
(4) 2ml of TBI-spore suspension is put into a 24-well plate, 10 6 YB-130 and XH-25 bacteria are respectively added at the same time, only PH-1 bacteria are cultured as positive control, and the incubation is carried out for 7d in a dark place at 25 ℃, and each is repeated for 3 times.
(5) DON toxin assay kits were used and operated as described.
As shown in FIG. 4, it can be seen that YB-130 has a significant effect on the synthesis of PH-1 toxin by co-culturing the strain YB-130 with PH-1, and the content of PH-1 toxin is only 28.5 ppm; after the XH-25 bacteria and the PH-1 are co-cultured, the toxin content is obviously higher than YB-130 and reaches 92.3 ppm; the toxicity of the control culture alone with PH-1 can reach 129.6 ppm. It is shown that YB-130 can effectively reduce the content of fusarium graminearum toxin, thereby improving the quality of wheat.
Example 8: verification test for preventing and treating stem base rot by microbial preparation
The indoor pot culture experiment is carried out in a greenhouse of agricultural academy of sciences in Henan province, and the used matrix material is formed by mixing turfy soil and vermiculite. The tested wheat variety is Zheng wheat 366, and the treatment method comprises the following steps:
(1) Inoculating the fungus cake: the method comprises the steps of taking fusarium graminearum (stored in a molecular biology laboratory of plant protection research institute of agricultural academy of sciences of Henan province) as a pathogen to be tested, transferring the fusarium graminearum to a PDA flat plate, punching the fusarium graminearum into stipes with the diameter of 1cm for later use by using a puncher, filling a substrate material into a nutrition pot, flattening the surface of the substrate material, placing the stipes on the substrate with the stipe facing upwards, sowing seeds with different treatments, placing one seed on each stipe, and covering a sowing layer with the substrate;
(2) seed dressing treatment of the microbial preparation of example 3 (see table 4): sterilizing wheat seeds for 10min by 1% sodium hypochlorite, washing with sterile water for 3 times, accelerating germination, soaking seeds for 1h by using a microbial preparation after exposure to white, airing the surfaces of the seeds, and sowing for later use;
(3) The microbial preparation of example 3 was applied to root irrigation (final concentration 10 6/cfu). wheat seeds were sterilized with 1% sodium hypochlorite for 10min, washed 3 times with sterile water, pregerminated, white-exposed and then sowed, 1ml of the preparation was applied to the root of each wheat plant with wheat open-bud, and applied again by the day 10 of sowing;
(4) medicament control: seed dressing and seed soaking treatment are carried out on the wheat by a difenoconazole file;
(5) Comparison: the wheat seeds are sterilized by 1 percent of sodium hypochlorite for 10min, washed by sterile water for 3 times, germinated, exposed and sowed.
Each treatment was repeated 5 times, 15 pellets per pot, cultured at 25 ℃ for 28 days, and then root-washed for investigation. And (5) calculating the investigation result after the contrast is completely diseased.
Grading standard:
0: no symptoms;
1: browning blastoderm nodes;
2: blastoderm nodes, mediterranean stems or leaves\37559;
3: the hypocotyl nodes, the underground stems or the leaf pins are all browned;
4: browning the underground stem, leaf pin and lower stem;
5: the stems have the symptoms of dry rot and rot;
6: plant death due to stalk rot;
7: seedling withering or seed decay.
The results of the control experiments by potting are shown in table 7.
TABLE 7 prevention and cure effect of microbial preparation on wheat stem basal rot pot culture
。
Therefore, the following steps are carried out: the microbial preparation can prevent and treat wheat stem basal rot, has the best effect of accompanying soil prevention and treatment, is higher than difenoconazole and reaches more than 77.5 percent, and the hole application effect is lower than the effect of accompanying soil.
Example 9: application verification test of microbial preparation in wheat scab prevention and treatment
The field test design is the same as example 6, and the investigation method, time and times are as follows: and (3) sampling at 5 points on the diagonal of each cell in the early stage of wheat maturity at about 24 days of 5 months in 2016, investigating 100 spikes at each point, grading by the percentage of the area of the withered spikes in the whole spike area, and recording the number of diseased spikes at each level and the total number of investigated spikes. The specific classification criteria are as follows:
Level 0: disease-free of whole ear
Level 1: the area of the susceptible panicle occupies less than one fourth of the whole panicle area.
And 3, level: the area of the susceptible ear accounts for one fourth to one half of the total ear area.
and 5, stage: the area of the susceptible ear accounts for one half to three quarters of the total ear area.
and 7, stage: the area of the susceptible panicle occupies more than three-fourths of the area of the whole panicle.
The drug effect calculation method comprises the following steps:
。
the field test results are shown in table 8.
TABLE 8 wheat scab drug effect test results
。
it can be seen that the control effect of the different bactericides on wheat scab after being applied for 2 times is that the control effect of 2% tebuconazole is the best (75.6%), the control effect of the microbial preparation in example 3 is the second best, and is equivalent to the control effect of 2% tebuconazole, and is obviously higher than 57.3% of that of the commercial biocontrol preparation, which indicates that the microbial preparation has longer lasting period and good control effect in field. Has the potential of further developing into biological pesticides.
While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.
Claims (10)
1. the bacillus amyloliquefaciens YB-130 has the preservation number of CGMCC NO. 16229.
2. The microbial preparation is characterized by being prepared from the following raw materials in percentage by mass: 60-80% of bacillus amyloliquefaciens CGMCC number 16229 bacterial liquid, 8-10% of adhesive, 1-2% of stabilizer and 100% of filler.
3. The microbial formulation of claim 2, wherein the adhesive is at least one of maltodextrin, soy flour, gelatin; the stabilizer is at least one of bentonite and carboxymethyl cellulose; the filler is at least one of talcum powder, attapulgite, clay and white carbon black.
4. The microbial preparation according to claim 2, wherein the effective viable count of the bacillus amyloliquefaciens CGMCC number 16229 is 10 10 -10 12/g.
5. the microbial preparation according to claim 4, wherein the preparation method of the bacterial liquid of Bacillus amyloliquefaciens CGMCC number 16229 comprises the following steps:
(1) Inoculating bacillus amyloliquefaciens CGMCC number 16229 into a solid culture medium, and culturing at 28-32 ℃ for 32-48 hours;
(2) Inoculating the strain cultured in the step (1) into a primary seed culture medium, wherein the initial pH value is 7.0-7.2, and culturing at 24-37 ℃ for 20-24 hours to obtain primary seeds;
(3) inoculating the primary seeds into a secondary seed culture medium according to the inoculation amount of 1-6% of the volume ratio, aerating, and culturing for 28-40 hours to obtain secondary seeds;
(4) And (3) inoculating the second-level seeds into a fermentation tank according to the inoculation amount of 1-6% of the volume ratio, and performing high-density fermentation culture to obtain the compound microbial inoculum.
6. The microbial preparation according to claim 5, wherein the primary seed culture medium is prepared from corn flour 10g, glucose 5g, bean cake powder 15g, fish meal 5g, CaCO 3 5g, (NH 4) 2 SO 4 1g, K 2 HPO 4 0.3.3 g, MgSO 4 & 7H 2 O0.2 g, and water 1000 ml.
7. The microbial preparation according to claim 5, wherein the secondary seed culture medium is prepared from corn flour 12.98g, glucose 5g, bean cake powder 20.11g, fish meal 5g, CaCO 3 6.90g, (NH 4) 2 SO 4 1g, K 2 HPO 4 0.3.3 g, MgSO 4.7H 2 O0.2 g, MnSO 4.H 2 O0.2 g, and water 1000 ml.
8. use of a bacillus amyloliquefaciens according to claim 1 or a microbial preparation according to claim 2 for improving the quality and/or yield of wheat.
9. Use of a bacillus amyloliquefaciens according to claim 1 or a microbial preparation according to claim 2 for inhibiting synthesis of a DON toxin of fusarium graminearum.
10. Use of a bacillus amyloliquefaciens according to claim 1 or a microbial preparation according to claim 2 for the biological control of a plant disease caused by fusarium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910954686.0A CN110551666B (en) | 2019-10-09 | 2019-10-09 | Bacillus amyloliquefaciens YB-130, microbial preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910954686.0A CN110551666B (en) | 2019-10-09 | 2019-10-09 | Bacillus amyloliquefaciens YB-130, microbial preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110551666A true CN110551666A (en) | 2019-12-10 |
| CN110551666B CN110551666B (en) | 2022-07-19 |
Family
ID=68742381
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910954686.0A Active CN110551666B (en) | 2019-10-09 | 2019-10-09 | Bacillus amyloliquefaciens YB-130, microbial preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110551666B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029693A (en) * | 2020-09-30 | 2020-12-04 | 河南省农业科学院植物保护研究所 | Bacillus amyloliquefaciens with growth promoting effect and capable of preventing and treating sesame stem blight and fusarium wilt and application thereof |
| CN112608860A (en) * | 2020-12-17 | 2021-04-06 | 河南省农业科学院植物保护研究所 | Bacillus amyloliquefaciens YB130 and application thereof |
| CN116172015A (en) * | 2022-11-01 | 2023-05-30 | 邹城市农业农村局 | Seed dressing agent for preventing and treating wheat stem-based rot, seed coating agent and comprehensive prevention and treatment method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433351A (en) * | 2011-12-21 | 2012-05-02 | 河南省农业科学院 | Trichoderma viride chitinase gene Tvchi and expression product and application thereof |
| CN104498386A (en) * | 2014-11-25 | 2015-04-08 | 山西农业大学 | Preparation method and applications of wild jujube endophytic bacillus amyloliquefaciens new strain SZ23 and fermentation broth |
| WO2017105050A1 (en) * | 2015-12-15 | 2017-06-22 | 김진철 | Composition for preventing plant diseases, containing, as active ingredient, bacillus amyloliquefaciens strain producing iturin, and use thereof |
| CN109182197A (en) * | 2018-09-29 | 2019-01-11 | 淮阴工学院 | A kind of bacillus amyloliquefaciens CPLK1314 and its application in feed storage |
| US20190133136A1 (en) * | 2016-06-06 | 2019-05-09 | Establissements J. Soufflet | Microbial Strains for Biologically Controlling Fusarium Head Blight |
| CN110129238A (en) * | 2019-05-31 | 2019-08-16 | 河南科技大学 | Bacillus amyloliquefaciens and its application, biocontrol agent with a variety of prophylaxis effects |
-
2019
- 2019-10-09 CN CN201910954686.0A patent/CN110551666B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433351A (en) * | 2011-12-21 | 2012-05-02 | 河南省农业科学院 | Trichoderma viride chitinase gene Tvchi and expression product and application thereof |
| CN104498386A (en) * | 2014-11-25 | 2015-04-08 | 山西农业大学 | Preparation method and applications of wild jujube endophytic bacillus amyloliquefaciens new strain SZ23 and fermentation broth |
| WO2017105050A1 (en) * | 2015-12-15 | 2017-06-22 | 김진철 | Composition for preventing plant diseases, containing, as active ingredient, bacillus amyloliquefaciens strain producing iturin, and use thereof |
| US20190133136A1 (en) * | 2016-06-06 | 2019-05-09 | Establissements J. Soufflet | Microbial Strains for Biologically Controlling Fusarium Head Blight |
| CN109182197A (en) * | 2018-09-29 | 2019-01-11 | 淮阴工学院 | A kind of bacillus amyloliquefaciens CPLK1314 and its application in feed storage |
| CN110129238A (en) * | 2019-05-31 | 2019-08-16 | 河南科技大学 | Bacillus amyloliquefaciens and its application, biocontrol agent with a variety of prophylaxis effects |
Non-Patent Citations (3)
| Title |
|---|
| WEN XU ET AL.: "Isolation, Identification, and Complete Genome Assembly of an Endophytic Bacillus velezensis YB-130, Potential Biocontrol Agent Against Fusarium graminearum", 《FRONT. MICROBIOL.》 * |
| 张臻: "两株生防菌对小麦赤霉病和茎基腐病防治效果的评价", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
| 林琪童等: "小麦茎基腐病生防菌株YB-161的分离鉴定及防效测定", 《植物保护学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029693A (en) * | 2020-09-30 | 2020-12-04 | 河南省农业科学院植物保护研究所 | Bacillus amyloliquefaciens with growth promoting effect and capable of preventing and treating sesame stem blight and fusarium wilt and application thereof |
| CN112029693B (en) * | 2020-09-30 | 2022-06-07 | 河南省农业科学院植物保护研究所 | Bacillus amyloliquefaciens with growth promoting effect and capable of preventing and treating sesame stem blight and fusarium wilt and application thereof |
| CN112608860A (en) * | 2020-12-17 | 2021-04-06 | 河南省农业科学院植物保护研究所 | Bacillus amyloliquefaciens YB130 and application thereof |
| CN112608860B (en) * | 2020-12-17 | 2022-06-28 | 河南省农业科学院植物保护研究所 | Bacillus amyloliquefaciens YB130 and application thereof |
| CN116172015A (en) * | 2022-11-01 | 2023-05-30 | 邹城市农业农村局 | Seed dressing agent for preventing and treating wheat stem-based rot, seed coating agent and comprehensive prevention and treatment method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110551666B (en) | 2022-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106591185B (en) | Bacillus amyloliquefaciens plant subspecies and preparation and application of microbial inoculum thereof | |
| CN106350470B (en) | The production method and its application of bacillus amyloliquefaciens and microbial inoculum and the microbial inoculum | |
| CN106591193B (en) | One plant of bacillus amyloliquefaciens with the degeneration-resistant effect of wide spectrum growth-promoting | |
| CN105420156A (en) | Bacillus amyloliquefaciens with phosphate solubilizing, disease preventing and growth promoting functions and application thereof | |
| CN110551666B (en) | Bacillus amyloliquefaciens YB-130, microbial preparation and application thereof | |
| CN109749961B (en) | Microbial agent for repairing garlic continuous cropping soil and preparation method and application thereof | |
| CN107509744B (en) | Compound biopesticide for preventing and treating wheat stem basal rot and preparation method and application thereof | |
| CN105505836A (en) | Bacillus subtilis and methods for preparing and using bacillus subtilis microbial agent for controlling potato scab | |
| CN111944716B (en) | Special compound microbial agent for tobacco seedling culture and preparation method and application thereof | |
| CN109053253A (en) | A kind of biological organic fertilizer and preparation method thereof of solid root lodging-prevention | |
| US20060018883A1 (en) | Microbial preparation & method for preventing and curing the bacterial wilt the plant and its use | |
| CN103131641A (en) | T.hariamum multivariate fermentation method and application | |
| CN110218678B (en) | Anti-continuous cropping soil remediation agent and preparation method thereof | |
| CN113234602B (en) | Chaetomium globosum, microbial inoculum, seed soaking liquid and application | |
| CN101619293B (en) | Streptomyces vinaceusdrappus, filtering method and application | |
| CN110240527A (en) | A kind of bacteria agent and preparation method thereof and biological bacterium organic fertilizer and preparation method thereof | |
| CN114107070B (en) | Pityrosporum indicum preparation and preparation method thereof | |
| CN113243398B (en) | Biocontrol preparation for preventing and treating edible fungi and slugs as well as preparation method and application of biocontrol preparation | |
| CN112501028B (en) | Efficient microbial agent suitable for dendrobium wilt and preparation method thereof | |
| CN111172064B (en) | Bacillus laterosporus and application thereof | |
| CN103340107B (en) | Celest coated bacterium soil nutrition pot method for authenticating cotton fusarium wilt resistance | |
| CN107236689B (en) | One fluorescent pseudomonads pf27 and its application in plant growth-promoting | |
| CN114436698B (en) | Liquid compound microbial fertilizer and application thereof in agricultural production | |
| CN115005216B (en) | Application of nano chitin in promoting melon fruit and vegetable production | |
| CN114058555B (en) | Preparation and application methods of biological seed coating agent for preventing and treating corn stem rot |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |