CN115010548A - Special biological compound fertilizer applied to pear fire germ antagonistic biocontrol - Google Patents
Special biological compound fertilizer applied to pear fire germ antagonistic biocontrol Download PDFInfo
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- CN115010548A CN115010548A CN202210371235.6A CN202210371235A CN115010548A CN 115010548 A CN115010548 A CN 115010548A CN 202210371235 A CN202210371235 A CN 202210371235A CN 115010548 A CN115010548 A CN 115010548A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Soil Sciences (AREA)
- Fertilizers (AREA)
Abstract
The invention discloses a biological compound fertilizer specially applied to pyricularia agrestis antagonistic biocontrol, which comprises a compound microbial inoculum and a compound material, wherein the compound material comprises cow dung, sheep dung, oil residue and mineral humic acid, the compound microbial inoculum is inoculated according to the mass percent of 0.3-0.5% of the compound material to obtain the pyricularia agrestis antagonistic biocontrol biological compound fertilizer, the invention creatively provides the compound microbial fertilizer which can specially aim at preventing and controlling pyricularia agrestis, stable microecology is formed by compounding strains in a specific proportion, higher viable count is ensured, normal growth of plants can be ensured, and the compound microbial fertilizer has obvious pyricularia agrestis prevention and control effect, the yield is increased by about 16.56% when the compound microbial fertilizer is applied to planting of the pyricularia agrestis, organic matter content in soil can be increased, physical and chemical properties of the soil are obviously improved, soil pesticide pollution, heavy metal pollution, germ pollution and the like can be effectively improved and degraded, thereby improving the cultivated land quality, improving the soil productivity and having wide practical value for preventing and controlling the pear fire blight.
Description
Technical Field
The invention relates to the technical field of research and development of microbial fertilizers. Specifically, the invention relates to a biological compound fertilizer specially applied to pear fire pathogen antagonistic biocontrol, and the technical fields of preparation and application thereof.
Background
The pear fire blight (fire blue of pear) is a destructive bacterial disease caused by Erwinia amylovora, and pathogenic bacteria are harmful organisms for entry quarantine in China. The disease causes that flowers, leaves and branches infected by the erwinia amylovora show the symptoms of blackening and withering and look like fire. Erwinia amyloliquefaciens can infect more than 220 plants of more than 40 genera, and is mainly harmful to pome fruit trees. The pear fire blight causes massive death of fruit trees, is easy to spread and difficult to prevent and treat, and often causes huge economic loss. The disease is determined to be caused by bacteria in 1883, so the pear fire blight is also the first bacterial plant disease in the world. The toxicity and the transmission of the amylovora are very strong, and one orchard can be destroyed in a single growing season.
The erwinia amylovora is a straight-rod bacterium, the outer surface of which is coated with capsules and the outer surface of which is coated with periphytic flagella, the size of which is 0.9-1.8 multiplied by 0.6-1.5 mu m, and most of which are single-grown. The optimum growth temperature is 28 ℃, the humidity is 70 percent, and the pH value is 6. It can propagate at 5-37 deg.c and propagate one generation at 25 deg.c for about 75 min. The erwinia amylovora can be carried to adjacent plants by birds, insects, wind, rain, dew and the like, and can infect parts such as flowers, fruits, leaves, stems, actively growing twigs, rootstocks and the like of host plants. Because the existence and the migration capacity of the erwinia amylovora are strong, and the diversity of susceptible varieties and tissues is high, a unified and effective radical target is difficult to find, so that the treatment of the erwinia amylovora is very difficult. At present, the prevention and control means for the pear fire blight mainly comprise isolation quarantine, pesticide application, resistance variety cultivation, pruning and tree digging and the like. In the aspect of control, in addition to continuous improvement and strict execution of existing control measures, development of new chemical agents to cope with drug resistance is still required, development of biological agents at a price suitable for orchard application is also required, and cultivation and screening of resistant varieties with strong resistance and stable resistance are also required. In view of the factors such as the easy generation of drug resistance, the control of customs on reagent residues, and the consumption preference of people on organic agricultural products, the importance of biological prevention and control measures is increasingly prominent.
Chinese patent (patent publication No. CN112358366A) discloses a composite bacterial fertilizer for preventing and treating the pear fire blight, which is prepared by Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens and Candida guilliermondii according to a certain proportion, can promote plant growth and has obvious pear fire blight prevention and treatment effect, is applicable to saline-alkali soil, and can reduce the risk of pear tree infection to the pear fire blight. However, in the practical application process, the series of composite bacterial fertilizers have the problems of complex preparation process, high production cost, short fertilizer effect duration, poor nitrogen-phosphorus-potassium fertilizer effect and further improved yield of bergamot pears when applied to planting of Korla bergamot pears.
Disclosure of Invention
Aiming at the technical current situations that the preparation process of the existing bio-control fertilizer for the fire blight of pears is complex, the cost is high, the fertilizer efficiency time is short, and the yield of the Korla bergamot pears needs to be further improved. The invention provides a biological compound fertilizer specially applied to pyricularia agrestis antagonistic biocontrol, which comprises a compound microbial inoculum and a compound material, wherein the compound material comprises cow dung, sheep dung, oil residue and mineral humic acid, the compound microbial inoculum is inoculated according to the mass percent of 0.3-0.5% of the compound material to obtain the pyricularia agrestis antagonistic biocontrol biological compound fertilizer, the invention creatively provides the compound microbial fertilizer which can specially prevent and control pyricularia agrestis, stable microecology is formed by compounding strains in a specific proportion, higher viable count is ensured, the coordination and compounding among the strains and safety are verified, the normal growth of the plants of the pyricularia agrestis can be ensured, the pyricularia agrestis preventive effect is obvious, the application of the compound fertilizer to the plantation of the pyricularia agrestis, the yield is increased by about 16.56% on average, the organic matter content in soil can be increased, the physicochemical property of the soil can be obviously improved, and soil pollutants such as pesticide pollution, heavy metal pollution, germ pollution and the like in soil are effectively degraded, so that the cultivated land quality is improved, the soil production capacity is improved, and the method has wide practical value for preventing and treating the pear fire blight.
The invention provides a biological compound fertilizer specially applied to pear fire germ antagonism biocontrol, which comprises a compound microbial inoculum and a compound material, wherein the compound material comprises 15-25 parts of cow dung, 15-25 parts of sheep dung, 30-50 parts of oil residue and 20-40 parts of mineral source humic acid in parts by weight; the composite microbial inoculum is inoculated according to the mass percent of 0.3-0.5% of the composite material.
Furthermore, the invention provides a biological compound fertilizer specially applied to pear fire pathogen antagonistic biocontrol, which comprises a compound microbial inoculum and a compound material, wherein the compound material comprises 20 parts of cow dung, 20 parts of sheep dung, 40 parts of oil residue and 30 parts of mineral humic acid in parts by weight; the composite microbial inoculum is inoculated according to the mass percent of 0.4 percent of the composite material.
The invention provides a biological compound fertilizer specially applied to pyrus pyricularis antagonism biocontrol, wherein the compound microbial agent is obtained by mixing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (CGMCC NO. 1640) and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor according to the mass ratio of 1:1: 1.
Meanwhile, the invention provides a preparation method of the biological compound fertilizer specially applied to pear fire pathogen antagonistic biocontrol, which is specifically obtained by the following steps:
(1) selecting low-temperature stored Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (CGMCC NO. 1640) and Bacillus mucilaginosus (Bacillus mucosus) CGMCC NO.1641, respectively inoculating to an NA culture medium plate, culturing at 30 ℃ for about 12h, selecting single colonies, respectively inoculating to 200m LNB culture solution, and performing activation culture in a 30-DEG C160 rpm constant temperature shaking table for 12h or 24h to serve as seed liquid.
(2) Respectively inoculating the seed liquid obtained in step (1) into fermentation tanks filled with 7L of corresponding liquid culture medium, and performing fermentation culture for 10-15h until viable count reaches 5 × 10 9 cfu/mL。
(3) And (3) compounding the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the Bacillus megaterium (CGMCC NO. 1640) and the Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor obtained in the step (2) according to a specific mass ratio to prepare the composite microbial inoculum.
(4) Collecting cow dung, sheep dung with water content below 85% and no other impurity, frying (1: 1: 2), stacking to height of 1-1.5 m, width of 1.5-2 m, and length of 4m, and fermenting for more than 40 days until the material is completely decomposed; controlling the water content to be 60-65% in the fermentation process, and judging the water content: the hand grips a handle of material, and the fingers can be sewed with water but do not drip water, and the materials are scattered when falling to the ground.
(5) And (4) mixing the compound microbial inoculum prepared in the step (3) with the compound material prepared from the completely decomposed cattle and sheep manure and oil residue material prepared in the step (4) and mineral humic acid according to a proportion, uniformly stirring and packaging to obtain the biological compound fertilizer specially applied to the pear fire germ antagonistic biocontrol.
The invention provides application of a biological compound fertilizer applied to biocontrol of erwinia amylovora in preparation of a fertilizer for preventing and treating erwinia amylovora.
Through the technical scheme, the invention has the following technical effects:
(1) the invention provides three biocontrol bacteria applied to a biological compound fertilizer specially applied to pyricularia agrestis antagonistic biocontrol to carry out antagonistic determination on pyricularia agrestis, the diameter of a bacteriostatic spot of bacillus amyloliquefaciens is (9.03mm) > bacillus megatherium (4.38mm) > bacillus mucilaginosus (0.78mm), and the number of viable bacteria of the biological compound fertilizer obtained by inoculation according to a certain proportion reaches 3.03 multiplied by 10 8 CFU/mL is close to the initial spore number of the compound microbial inoculum, when the biological fertilizer is diluted by 10 times, the antibacterial effect is the highest, and the antibacterial diameter is 5.71 +/-0.51 mm.
(2) Although reports about the application of Bacillus Subtilis and Bacillus amyloliquefaciens or partial Bacillus in the antagonistic biocontrol of erwinia amylovora have been disclosed in the prior art, the biggest problem of microbial strains is to explore and study clearly to find out the safe, effective, coordinated and efficient combination of suitable strains, which is a known problem in the industry and also a technical problem restricting the application of the microbial strains to meet the most key problem, and the problem that the strains can not be randomly selected and can be combined and applied to the production industry. The invention selects and uses Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (CGMCC NO. 1640) and Bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC NO.1641, wherein two strains of the Bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and the Bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC NO.1641 are new strains which are automatically screened by an applicant at the early stage, the invention is applied to the early stage research, the compatibility and safety compounding of the three strains are verified by scientific research and production, the invention further obtains the biological compound fertilizer by searching for a suitable compound material and developing the biological compound fertilizer specially aiming at the application of pyrus pyricularia antagonism biological control, not only can ensure the normal growth of the plant of the bergamot pear and has obvious pyrus pyricularia pyrifolia linn prevention and control effect, the biological compound fertilizer is applied to the planting of the bergamot pear, the average content of the bergamot pear is about 16.56%, and the organic matter content in the soil is increased, the soil physical and chemical properties are obviously improved, and soil pollutants such as pesticide pollution, heavy metal pollution, germ pollution and the like in soil can be effectively degraded, so that the farmland quality is improved, the soil production capacity is improved, and the method has wide practical value for preventing and treating the pear fire blight.
(3) The invention provides a biological compound fertilizer specially applied to biocontrol of erwinia amylovora, and soil pollutants such as pesticide pollution, heavy metal pollution, germ pollution and the like in soil are improved and degraded by applying biotechnology, so that the quality of cultivated land is effectively improved, and the soil production capacity is improved. Wherein the content of heavy metal mercury is reduced by 9.09%, the content of lead is reduced by 1.00%, the content of chromium is reduced by 80.77%, the content of hexachloro cyclohexane is reduced by 77.78%, and the content of dichlorodiphenyl trichloroethane is reduced by 81.25%.
(4) The invention provides a biological compound fertilizer specially applied to pear fire germ antagonistic biocontrol and a biological organic fertilizer with similar effective components sold in the market, wherein the biological compound fertilizer lasts for 25 days in a device with simulated fertilization conditions, the quick-acting nitrogen, phosphorus and potassium of the biological compound fertilizer at a 15cm position of a simulated plough layer are obviously higher than those of a contrast group, and the biological compound fertilizer has the condition of obviously improving the utilization rate of the quick-acting nitrogen, phosphorus and potassium of the fertilizer.
(5) The biological compound fertilizer specially applied to the pear fire pathogen antagonism biocontrol provided by the invention has an obvious yield increase effect on the Korla pear, the yield is increased by about 16.56% on average, and the sensory, quality, nutrition and sanitary safety of the Korla pear are further evaluated, and the results all meet the requirements.
Drawings
FIG. 1 is a plate antagonism test chart of three biocontrol bacteria provided by the present invention; in the figure, A is a plate antagonism figure, B is a bacteriostatic diameter statistic, 1 represents bacteriostatic plaque of bacillus amyloliquefaciens, 2 represents bacteriostatic plaque of bacillus megaterium, and 3 represents bacteriostatic plaque of bacillus mucilaginosus.
FIG. 2 is a schematic diagram of a test soil column device.
FIG. 3 is a graph showing the change of the 15cm quick-acting nitrogen of the soil after simulated fertilization of different fertilizers along with the culture time.
FIG. 4 is a graph showing the variation of 15cm fast-acting phosphorus in soil with culture time after simulated fertilization of different fertilizers.
FIG. 5 is a graph showing the variation of 15cm quick-acting potassium in soil with culture time after simulated fertilization of different fertilizers.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples.
Materials and equipment involved in the present invention:
the invention adopts the following materials: nutrient Agar (NA) culture medium, Nutrient Broth (NB) culture medium, MRS culture medium, agar powder and the like are all domestic analytical pure reagents. The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ATCC NO.1978, the Bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and the Bacillus mucilaginosus (Bacillus mucosus) CGMCC NO.1641 which are selected by the invention are taken as early-stage known strains, can be called or purchased by a person with ordinary skill in the art through a microorganism strain preservation center of the Chinese academy of sciences (China microorganism strain preservation management Committee CGMCC), and are taken as known materials for scientific research and production in the field.
All materials, reagents and equipment selected for use in the present invention are well known in the art and are not intended to limit the practice of the present invention.
Example 1: biological compound fertilizer specially applied to pear fire germ antagonism biocontrol
The biological compound fertilizer specially applied to pear fire germ antagonism biocontrol comprises a compound microbial inoculum and a compound material, wherein the compound material comprises 15-25 parts of cow dung, 15-25 parts of sheep dung, 30-50 parts of oil residue and 20-40 parts of mineral humic acid in parts by weight; inoculating the composite microbial agent according to the mass percent of 0.3-0.5% of the composite material, wherein the composite microbial agent is obtained by mixing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor according to the mass ratio of 1:1: 1.
Example 2: biological compound fertilizer specially applied to pear fire germ antagonism biocontrol
The embodiment provides a biological compound fertilizer specially applied to pyretobacter sphaeroides antagonistic biocontrol on the basis of embodiment 1, and the biological compound fertilizer comprises a compound microbial inoculum and a compound material, wherein the compound material comprises 20kg of cow dung, 20kg of sheep dung, 40kg of oil residue and 30kg of mineral humic acid; the composite microbial agent is inoculated according to the mass percent of 0.4 percent of composite materials, wherein the composite microbial agent is obtained by mixing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor according to the mass ratio of 1:1: 1.
Example 3: special biological compound fertilizer applied to pear fire germ antagonistic biocontrol
The embodiment provides a biological compound fertilizer specially applied to pyretobacter sphaeroides antagonistic biocontrol on the basis of embodiment 1, and the biological compound fertilizer comprises a compound microbial inoculum and a compound material, wherein the compound material comprises 15kg of cow dung, 15kg of sheep dung, 30kg of oil residue and 20kg of mineral humic acid; inoculating the composite microbial agent according to the mass percent of 0.3 percent of the composite material, wherein the composite microbial agent is obtained by mixing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor according to the mass ratio of 1:1: 1.
Example 4: biological compound fertilizer specially applied to pear fire germ antagonism biocontrol
The embodiment provides a biological compound fertilizer specially applied to biocontrol of erwinia amylovora antagonism on the basis of embodiment 1, and the biological compound fertilizer comprises a compound microbial inoculum and a compound material, wherein the compound material comprises 25kg of cow dung, 15kg of sheep dung, 50kg of oil residue and 40kg of mineral humic acid; inoculating the composite microbial agent according to the mass percent of 0.5 percent of the composite material, wherein the composite microbial agent is obtained by mixing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor according to the mass ratio of 1:1: 1.
Example 5: biological compound fertilizer specially applied to pear fire germ antagonism biocontrol
The embodiment provides a biological compound fertilizer specially applied to pyretobacter sphaeroides antagonistic biocontrol on the basis of embodiment 1, and the biological compound fertilizer comprises a compound microbial inoculum and compound materials, wherein the compound materials comprise 20kg of cow dung, 22kg of sheep dung, 45kg of oil residue and 35kg of mineral humic acid; inoculating the composite microbial agent according to the mass percent of 0.4% of the composite material, wherein the composite microbial agent is obtained by mixing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor according to the mass ratio of 1:1: 1.
Example 6: preparation of biological compound fertilizer specially applied to pear fire pathogen antagonism biocontrol
The embodiment provides a preparation method of a biological compound fertilizer specially applied to pyricularia pyricularis antagonistic biocontrol on the basis of the embodiments 1-5, and the preparation method specifically comprises the following steps:
(1) selecting low-temperature stored Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (CGMCC NO. 1640) and Bacillus mucilaginosus (Bacillus mucosus) CGMCC NO.1641, respectively inoculating to an NA culture medium plate, culturing at 30 ℃ for about 12h, selecting single colonies, respectively inoculating to 200mLNB culture solution, and performing activation culture in a 30-DEG C160 rpm constant temperature shaking table for 12h or 24h to serve as seed liquid.
(2) Respectively inoculating the seed liquid obtained in step (1) into fermentation tanks filled with 7L of corresponding liquid culture medium, and performing fermentation culture for 10-15h until viable count reaches 5 × 10 9 cfu/mL, obtaining fermentation liquor of each bacterium.
(3) And (3) compounding the Bacillus amyloliquefaciens, Bacillus megaterium (CGMCC NO. 1640) and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor obtained in the step (2) according to a specific mass ratio to prepare the composite microbial agent.
(4) Collecting cow dung, sheep dung with water content below 85% and no other impurity, frying (1: 1: 2), stacking to height of 1-1.5 m, width of 1.5-2 m, and length of 4m, and fermenting for more than 40 days until the material is completely decomposed; controlling the water content to be 60-65% in the fermentation process, and judging the water content: the hand grips a handle of material, and the fingers can be sewed with water but do not drip water, and the materials are scattered when falling to the ground.
(5) And (4) mixing the compound microbial inoculum prepared in the step (3) with the compound material prepared from the completely decomposed cattle and sheep manure and oil residue material prepared in the step (4) and mineral humic acid according to a proportion, uniformly stirring and packaging to obtain the biological compound fertilizer specially applied to the pear fire germ antagonistic biocontrol.
Example 7: biological safety evaluation of biological compound fertilizer specially aiming at pear fire pathogen antagonistic biocontrol
According to the general technical criteria for biological safety of microbial fertilizers (NY 1109-2006), the principles, procedures, test methods and result evaluation methods of microbial fertilizer strain safety classification catalogues, different strains and product selection toxicology tests are specified according to the national standard. The principle of selecting toxicological tests required by the state is to carry out different safety evaluations according to production strains, the strains for producing the microbial fertilizer are divided into four levels of management, the safety classification of the strains has a catalogue, the strains not listed in the appendix except rhizobium and Lactobacillus (Lactobacillus) need to be subjected to toxicological tests.
The first level is the strain that is exempt from toxicology testing.
The second level is the strain that needs to be tested for acute toxicity.
The third level is the strain to be tested for pathogenicity.
The fourth stage is forbidden strains.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (CGMCC NO. 1640) and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 which are selected and used in the invention all belong to strains which are not used for toxicological tests in '1.4 decomposed phosphorus potassium compound bacteria' in the first level, but do not belong to strains which are required to be used for acute toxicity (LD50) tests in the second level; the strain does not belong to the third-level strain which needs pathogenicity test; is not the fourth forbidden strain.
On the basis of the above examples 1 to 5, acute toxicity tests of the prepared biocomposite fertilizer are further provided as follows.
Preparation of (I) toxigenic liquid culture medium
1. Potato-yeast extract-sucrose culture medium
Taking 200-300g of peeled potatoes, cutting into small pieces, adding 1000mL of water, boiling for 20min, filtering by using gauze to prepare potato juice, supplementing water to 1000mL, adding 10g of yeast extract and 100g of cane sugar, subpackaging, and carrying out autoclaving at 121 ℃ for 15 min.
2. Wort-yeast extract culture medium
Adding 10g yeast extract into 1000mL of wort, subpackaging, and autoclaving at 121 ℃ for 15 min.
Malt extract-peptone medium
1000mL of wort was added with 1g of peptone, and subpackaged and autoclaved at 121 ℃ for 15 min.
3. Glucose aspargine culture medium
Adjusting pH to 7.2-7.4, subpackaging, and autoclaving at 121 deg.C for 15 min.
4. Gao's synthetic No.1 culture medium
Adjusting pH to 7.2-7.4, subpackaging, and autoclaving at 121 deg.C for 15 min.
5. Wort culture medium
200mL of wort was dispensed and autoclaved at 121 ℃ for 15 min.
0.5% peptone Medium
2.0g of glucose, 0.6g of yeast extract, 1.0g of peptone and 4.0g of agar are taken, distilled water is added to the mixture to be 200mL, and the mixture is subpackaged and autoclaved at 121 ℃ for 15 min.
6. Culture preparation
After the pure culture (suitable culture medium slant, culture at 28 +/-1 ℃ for 5-7d) is confirmed to be the pure culture, respectively inoculating the pure culture into a suitable toxigenic culture medium, respectively inoculating general strains into three toxigenic culture media of wort-yeast extract, wort-peptone and potato-yeast extract-sucrose, inoculating actinomycetes into a glucose asparagines culture medium and a Gao's synthetic No.1 culture medium, inoculating Phaffia rhodozyma into a malt extract culture medium and a 0.5% peptone culture medium, and culturing for 14d at 28 +/-1 ℃. The culture was steamed for 1h and filtered, and the filtrate was partially concentrated at 80 ℃ for 2.5 times for use, the remainder being used directly for the experiment.
(II) mouse virulence test
Each toxigenic liquid culture medium needs 80 mice, each half of the mice is male and female, and the mice are randomly divided into 4 groups, and each group contains 10 mice. The dose group is set as follows: a culture medium blank control group, a culture medium 2.5-time concentrated blank control group, a culture stock solution group and a culture 2.5-time concentrated group. The test substance is administered to the mice at the dosage of 20.0mL/kg BW for one-time gavage, and then the mice are observed for 14d, and the toxic manifestation and death condition of the mice in the experimental process are recorded.
(III) statistics of test data
Carrying out homogeneity of variance test on initial weight and final weight of the mouse, and carrying out statistical treatment on data meeting the requirement of homogeneity of variance by using a t test for comparing mean values of two samples; and carrying out statistical treatment on the data with uneven variance by using a t' test.
(IV) determination of results
The initial body weight of the mouse is balanced among groups, the tested object has no adverse effect on the final body weight, the mouse does not have toxic reaction or death in the experimental process, and the safety of the tested strain can be judged.
The tests prove that the tested strains of the Bacillus amyloliquefaciens, the Bacillus megaterium CGMCC NO.1640 and the Bacillus mucilaginosus CGMCC NO.1641 selected from the biological compound fertilizers provided by the embodiments 1 to 6 are safe, the compatibility of the strains meets the biological safety standard, the application of the antagonistic biocontrol of the erwinia amylovora is safe, and the key of the safety of the selected strains is that whether the selected strains are safe or not, whether the selected strains are subjected to safety evaluation and test verification and whether the corresponding biological fertilizer is safe or not is prepared by using the selected strains.
Example 8: biological safety detection test of biological compound fertilizer special for pear fire pathogen antagonistic biocontrol
The safety results of the finished bio-organic fertilizer are shown in table 1. As can be seen from the results in Table 1, the bio-compound fertilizer meets the actual non-toxicity level in the requirements of acute oral toxicity test and evaluation of fertilizer registration (NY 1980-2010).
Table 1: the result of the acute oral toxicity test of the biological compound fertilizer
The tests prove that the biological compound fertilizer provided by the embodiments 1 to 6 meets the biological safety, is safe in the application of the pyricularia oryzae antagonistic biocontrol, and is also the key for solving the biological safety commonly existing in the current microbial fertilizer.
Example 9: performance test of biological compound fertilizer specially aiming at pear fire germ antagonistic biocontrol
In this example, on the basis of examples 1 to 6, the inhibition effect of the screened strains and the prepared biological compound fertilizer after compounding on erwinia amylovora is examined.
(1) Test protocol
Bacillus megaterium, Bacillus amyloliquefaciens and Bacillus mucilaginosus are provided by Xinjiang Heisen biotechnology, Inc., a pear fire blight strain NCPPB1665 from the plant quarantine laboratory of Nanjing agriculture university, LB culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl and 1000mL of pure water. Adjusting pH to 7.0, sterilizing at 121 deg.C for 20min, adding agar to obtain solid culture medium: 0.8-0.9g/50 mL.
(2) Test method
Dipping a small amount of pear fire blight by using an inoculating loop, streaking on an LB solid culture medium, and then putting the solid culture medium in a constant-temperature incubator at 28 ℃ for 24 hours; selecting single colony, inoculating into culture flask containing 25mL LB liquid culture medium, shake culturing in constant temperature shaking table at 28 deg.C and 220r/min for 24 hr, and adjusting the shaking bacteria liquid to OD 600 0.3 (about 1 × 10) 8 cfu/mL) for use; inoculating the biocontrol bacteria to be tested into LB liquid culture medium, performing shake culture in a constant temperature shaking table at 28 ℃ and 220r/min, and adjusting the shaking bacteria liquid to OD 600 0.3 (about 1 × 10) 8 cfu/mL) for use; weighing 10g of biological compound fertilizer, dissolving in 100mL of sterile water to obtain bacterial suspension diluted by 10 times, and diluting the bacterial suspension to 10 times, 100 times and 500 times in a multiple ratio for activity determination; the test uses a plate coating method and dilutedAbsorbing 100 mu L of the bacterial suspension, coating the bacterial suspension on an LB solid culture medium, repeating the dilution times for 3 times, observing the result after constant-temperature culture at 28 ℃ for 24 hours, counting the number of bacterial colonies grown by coating plates at each dilution time, calculating the number of the viable bacteria of the composite microbial inoculum, and comparing the number with the initial spore number, wherein the viable bacteria number (CFU/mL) of the composite microbial inoculum is (bacterial colony number multiplied by dilution times)/0.1 mL; dripping 10 mu L of antagonistic bacteria suspension on a bacteria-containing flat plate, setting blank control, repeating each treatment for 3 times, culturing at constant temperature of 28 ℃ for 48h, observing the antibacterial effect, measuring the diameter of an antibacterial ring and the diameter of a bacterial colony by a cross method, and measuring the diameter (mm) of the antagonistic bacteria, namely the diameter of the antibacterial ring and the diameter of the bacterial colony. The specific test results are shown in figure 1 and tables 2-3.
(3) Test results
As can be seen from the data in the attached figure 1, the antagonism effect of 3 strains of biocontrol bacteria is measured by a plate confrontation method, and the antagonism effect has the sequence of bacillus amyloliquefaciens (9.03mm) > bacillus megaterium (4.38mm) > bacillus mucilaginosus (0.78 mm); as can be seen from the data in tables 2 to 3, the number of viable bacteria in the biofertilizer was 3.03X 10 8 The CFU/g is very close to the initial spore number of the preparation, so that the biocontrol bacteria in the preparation can keep activity for a long time, the biological fertilizer has good inhibition effect on the erwinia amylovora bacteria under the concentration of 10, 100 and 500 times of dilution, wherein the effect is the best when the biocontrol bacteria is diluted by 10 times, and the bacteriostasis diameter is 5.71 +/-0.51 mm.
Table 2: viable count of composite microbial inoculum with different dilution times
| Dilution factor | Colony count (number/mL) of |
| 10 7 | 4、3、1 |
| 10 6 | 30、35、27 |
| 10 5 | 351、341、337 |
| Viable count (CFU/mL) | 3.03×10 8 |
Table 3: inhibition effect of biological fertilizer on erwinia amylovora
| Concentration (multiple) of biofertilizer | Bacteriostatic diameter (mm) |
| 10 | 5.71±0.51 |
| 100 | 3.83±0.31 |
| 500 | 0.81±0.14 |
Example 10: application of biological compound fertilizer specially applied to pear fire pathogen antagonism biocontrol
In this example, on the basis of examples 1 to 7, the formula of example 2 and the preparation method of example 6 of the present invention are applied to the planting of the Xinjiang Korla bergamot pear, wherein the biological compound fertilizer is specially applied to the antagonistic biocontrol against Pyricularia oryzae. Comparing the influence of different treatments on the change of pesticides, heavy metals and germs in the soil, the influence of the biological compound fertilizer on improving the cultivated land quality and the soil production capacity is investigated.
(1) Test protocol
Test site: von Nanedy pear garden of sixteen cents in gardening field, Kuerle Shaoydong, Xinjiang
Test time: month 10 of 2018 to month 9 of 2019
Control product: huisen commercial organic fertilizer (control 1), commercial biological organic fertilizer (control 2) of a certain manufacturer, commercial organic fertilizer (control 3) of a certain manufacturer
The soil condition of the test field is as follows: the soil type of the test is desert soil (sampled in 2018, 10 and 21 days), and the basic soil conditions before the test are as follows: 11.6g/kg of organic matter, 58.3mg/kg of hydrolysis nitrogen, 11.8mg/kg of quick-acting phosphorus, 142.1mg/kg of quick-acting potassium and 7.42 of pH value; before the test, the conditions of heavy metals and pesticide residues in the test soil are as follows: 15.237 mg/kg of lead, 0.011mg/kg of mercury, 0.056mg/kg of arsenic, 0.043mg/kg of cadmium, 46.377mg/kg of chromium, 0.26mg/kg of organic phosphorus pesticide residue, 0.18mg/kg of hexachloro cyclohexane and 0.16mg/kg of dichlorodiphenyl trichloroethane.
The trial article species are as follows: korla bergamot pear. Planting in 2002 for 3 months, planting holes with diameter of 0.8m, depth of 0.6 m and plant row spacing of 4.5 x 3.5 m, and entering full bearing age when the tree crown has a diameter of about 3.2 m and has a age of 16 years in 2018. And (3) performing four treatments in the test, repeating the three treatments for three times, setting 12 cells, randomly arranging the cells in groups, wherein each test cell is 1 mu, 10 trees are randomly divided in each cell, the length of each cell is 22.5 meters, the width of each cell is 7 meters, the area of each cell is 157.5 square meters for comparison, 30cm ridges are arranged between the cells, and the field management is consistent.
Table 4: effect of different treatments on Pear yield
As can be seen from the data in Table 4, the yield of the bergamot pears is the highest by applying the biological compound fertilizer provided by the invention, the average yield per mu reaches 923.6kg, and the yield is increased by 67.7kg and 7.91% compared with the 'Huisen' organic fertilizer; compared with the biological organic fertilizer of a certain factory sold in the market, the yield of each mu is increased by 117.2kg and 14.53%; compared with the biological commercial fertilizer of a certain factory on the market, the yield of each mu is increased by 131.2kg averagely and increased by 16.56 percent.
Table 5: analysis of variance
Analysis of variance for the different treatment yields in table 5 shows that treatment F is 48.64954, F > F0.01(3,6), indicating that the difference between the different treatments reaches a very significant level of 1%.
Table 6: multiple comparisons
As can be seen from the multiple comparison results in Table 6, the difference of the yield of the biological compound fertilizer reaches an obvious level compared with other treatment products, which shows that the biological compound fertilizer provided by the invention has an obvious yield increase effect, and compared with the Heisen commercial organic fertilizer, the microbial live bacteria has obvious difference of the yield indexes of the bergamot pears.
(2) Test of influence of different fertilizers on soil nutrient change
Table 7: effect of different Fertilizer treatments on soil nutrient changes
As can be seen from the data in Table 7, after the biological compound fertilizer provided by the invention is treated, the organic matter of soil is improved by 2.3g/kg, the hydrolyzed nitrogen is improved by 2.1mg/kg, the organic phosphorus is improved by 0.8mg/kg, the quick-acting potassium is improved by 3.1mg/kg, and the viable count is improved by 0.8 million/g.
(3) Effect of different fertilizers on changes of soil heavy metals and persistent organic pollutants
Table 8: influence of different fertilizer treatments on main physical and chemical properties of soil
As can be seen from the data in Table 8, by applying the biological compound fertilizer provided by the invention, the mercury content in soil is reduced by 0.001mg/kg and 9.09%, the arsenic content is reduced by 0.004mg/kg and 7.14%, and the cadmium content is reduced by 0.010mg/kg and 26.26%; the lead content is reduced by 0.152mg/kg and 1.00 percent; the chromium content is reduced by 0.928mg/kg and reduced by 2.00 percent; the total content of organophosphorus pesticide residues is reduced by 0.21mg/kg, which is reduced by 80.77%; the content of the hexachloro-cyclohexane soprocide is reduced by 0.14mg/kg and 77.78 percent, the content of the dichlorodiphenyl trichloroethane is reduced by 0.13mg/kg and 81.25 percent, and the biological compound fertilizer provided by the invention is proved to have obvious improvement on heavy metal and pesticide residue in soil.
(4) Test of residence time of different fertilizers in plough layer
In the experiment, the distribution relation of effective agriculture in the plough layer as a root layer is judged by comparing the residence time distribution of the effective components of the biological compound fertilizer and the commercially available organic fertilizer in the plough layer.
Test protocol: the test adopts a device shown in the attached figure 2, and the device is formed by connecting an upper part, a middle part and a lower part of organic glass cylinders with the same specification (phi 10cm multiplied by 11cm), wherein: the upper section glass cylinder and the middle section glass cylinder are not provided with ground and are separated by gauze, soil is prevented from leaking from the culture column during sampling, the bottom of the lowest section glass cylinder is provided with uniform small holes so as to facilitate smooth outflow of runoff water, and specific test results are shown in attached figures 3-5.
As can be seen from the data in the attached figure 3, in the test period from the time of applying to the land to 25 days under the simulated fertilization condition, the content of the quick-acting nitrogen of the other groups B3 and B4 (which are 15cm under the table) (simulated plough layer plant root system layers) is 25-400% higher than that of the quick-acting nitrogen of the comparison groups B0, B1 and B2 along with the change of the culture time.
As can be seen from the data in the attached figure 4, in the test period from the time of applying to the land to 25 days under the simulated fertilization condition, the content of the quick-acting phosphorus in the groups B3 and B4 which are 15cm (simulated plough layer plant root system layers) below the other tables is 12-210% higher than that of the quick-acting phosphorus in the comparative groups B0, B1 and B2 along with the change of the culture time.
As can be seen from the data in the attached figure 5, in the test period from the time of applying to the land to 25 days under the simulated fertilization condition, the content of the quick-acting potassium in the groups B3 and B4 which are 15cm (simulated plough layer plant root system layer) below the other tables is 6-170% higher than that of the quick-acting potassium in the comparison groups B0, B1 and B2 along with the change of the culture time.
The data show that the biological compound fertilizer provided by the invention has equivalent effective components with the commercial biological organic fertilizer and lasts for 25 days in equipment under the simulated fertilization condition, and three repeated reading test data show that the quick-acting nitrogen, phosphorus and potassium of the biological compound fertilizer provided by the invention at a position 15cm (simulated plant root system layer) of a simulated plough layer are obviously higher than those of a comparison group. The method has the advantage of remarkably improving the utilization rate of the quick-acting nitrogen, phosphorus and potassium of the fertilizer.
(5) Influence of applying the biological compound fertilizer on the quality of Korla bergamot pears
In the test, on the basis of tests 1 to 4, the nutritional quality of the korla pears produced by applying the formula in the embodiment 2 and the biological compound fertilizer prepared by the preparation method in the embodiment 6 is identified, and the sensory, nutritional quality and hygienic indexes of the korla pears are evaluated according to the detection standards such as CAQS/MTYX2004-2019, GB/T21488-2008, GB/T12947-2008, GB/T5009.268-2016, GB2762-2017 and GB2763-2019, and the specific results are shown in table 9.
Table 9: influence of different fertilizer treatments on the quality of Korla bergamot pears
According to the test data in the table 9, the test tests are performed on sensory, nutritional quality and health safety indexes of the Korla bergamot pears, the biological compound fertilizer provided by the invention is applied to planting of the Korla bergamot pears, the bergamot pears are regular in shape and uniform in size, and the green and glossy peel of the bergamot pears, compared with the reference of similar products, the soluble solid content of the Korla bergamot pears obtained by the invention is increased by 11.1%, the solid-acid ratio is increased by 45%, the sugar-acid ratio is increased by 47.9%, the total acid content is reduced by 23.1%, VC (is increased by 7.1%), potassium (is increased by 14.9%), calcium (is increased by 18.2%), iron (is increased by 33.5%), and zinc (is increased by 60.0%), so that the product of the invention is better in taste, richer in nutrition and better in flavor; the content of harmful heavy metals and pesticide residues is further determined, the determination results are below the limit, the edible safety is realized in 14 days 4 months in 2020, the obtained Korla bergamot pear product is detected by a national famous special-quality new agricultural product nutrition quality evaluation and identification mechanism of a quality alkalinity inspection test center for microbial fertilizers and edible fungus strains in agricultural rural areas, and the comprehensive evaluation of the product is finally given as follows: the Korla bergamot pears produced by applying the biological compound fertilizer have the characteristics of good sensory quality, excellent nutritional quality, safety and health, and are comprehensively evaluated to be of superior grade.
In conclusion, according to the technical scheme of the biological compound fertilizer for preventing and treating the pear fire blight provided by the invention creatively, stable microecology is formed and higher viable count is ensured by compounding the strains in a specific proportion, so that not only can the normal growth of plants be ensured, but also the remarkable pear fire blight prevention and treatment effect is achieved, the yield is increased by about 16.56% on average when the biological compound fertilizer is applied to the planting of Korla bergamot pears, the organic matter content in soil can be increased, the physical and chemical properties of the soil are obviously improved, the capability of effectively improving and degrading soil pollutants such as soil pesticide pollution, heavy metal pollution, germ pollution and the like can be improved, the cultivated land quality is improved, the soil production capability is improved, and the biological compound fertilizer has wide practical value for preventing and treating the pear fire blight.
The present invention is not limited to the above embodiments, and those skilled in the art can implement the present invention in other various embodiments according to the present disclosure. Therefore, the automatic fast search technical scheme and thought of the invention can be simply changed or modified and fall into the protection scope of the invention.
Claims (5)
1. The biological compound fertilizer is characterized by comprising 15-25 parts by weight of cow dung, 15-25 parts by weight of sheep dung, 30-50 parts by weight of oil residue and 20-40 parts by weight of mineral humic acid; the composite microbial inoculum is inoculated according to the mass percent of 0.3-0.5% of the composite material.
2. The biological compound fertilizer special for the pyricularia agrestis antagonistic biocontrol as claimed in claim 1, wherein the compound material comprises 20 parts of cow dung, 20 parts of sheep dung, 40 parts of oil residue and 30 parts of mineral humic acid in parts by weight; the composite microbial inoculum is inoculated according to the mass percent of 0.4 percent of the composite material.
3. The biological compound fertilizer special for the pyricularia agrestis antagonistic biocontrol as claimed in claim 1, wherein the compound microbial inoculum is obtained by mixing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (CGMCC No. 1640) and Bacillus mucilaginosus (Bacillus mucopolysaccharides) CGMCC No.1641 fermentation broth according to a mass ratio of 1:1: 1.
4. The preparation method of the biological compound fertilizer specially applied to pyricularia agricola antagonistic biocontrol as claimed in claim 1, characterized by specifically adopting the following steps:
(1) selecting Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus megaterium (CGMCC NO. 1640) and Bacillus mucilaginosus (Bacillus mucosus) CGMCC NO.1641 which are stored at low temperature to be respectively inoculated to an NA culture medium plate, culturing for about 12 hours at 30 ℃, selecting single colonies to be respectively inoculated to 200mLNB culture solution after the single colonies grow out, and performing activation culture for 12 hours or 24 hours in a constant temperature shaking table at 160rpm at 30 ℃ to serve as seed liquid;
(2) respectively inoculating the seed liquid obtained in step (1) into fermentation tanks filled with 7L of corresponding liquid culture medium, and performing fermentation culture for 10-15h until viable count reaches 5 × 10 9 cfu/mL, obtaining fermentation liquor of each bacterium;
(3) compounding the Bacillus amyloliquefaciens, Bacillus megaterium (CGMCC NO. 1640) and Bacillus mucilaginosus (Bacillus mucoginosus) CGMCC NO.1641 fermentation liquor obtained in the step (2) according to a specific mass ratio to prepare a composite microbial inoculum;
(4) collecting cow dung, sheep dung and oil (1: 1: 2) with water content below 85% and no other obvious impurities, piling to height of 1-1.5 m, width of 1.5-2 m and length of 4m, and fermenting for more than 40 days until the materials are completely decomposed; controlling the water content to be 60-65% in the fermentation process, and judging the water content: the material is gripped by hands, and the material is scattered when falling to the ground after being sewed with water but not dripping;
(5) and (4) mixing the compound microbial inoculum prepared in the step (3) with the compound material prepared from the completely decomposed cattle and sheep manure and oil residue material prepared in the step (4) and mineral source humic acid according to a proportion, uniformly stirring and packaging to obtain the biological compound fertilizer specially applied to the pear fire pathogen antagonistic biocontrol.
5. Use of the biological compound fertilizer of any one of claims 1 to 4 in the preparation of a fertilizer specifically directed to the control of fire blight of pears.
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