CN115010548B - Biological compound fertilizer specially applied to antagonism and prevention of pear fire germ - Google Patents
Biological compound fertilizer specially applied to antagonism and prevention of pear fire germ Download PDFInfo
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- CN115010548B CN115010548B CN202210371235.6A CN202210371235A CN115010548B CN 115010548 B CN115010548 B CN 115010548B CN 202210371235 A CN202210371235 A CN 202210371235A CN 115010548 B CN115010548 B CN 115010548B
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- 230000002265 prevention Effects 0.000 title claims abstract description 26
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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
Abstract
The invention discloses a biological compound fertilizer specially applied to pear fire germ antagonism prevention, which comprises a compound microbial inoculum and a compound material, wherein the compound material comprises cow dung, sheep dung, oil residue and mineral source humic acid, and the compound microbial inoculum is inoculated according to the mass percentage of 0.3-0.5% of the compound material to obtain the pear fire germ antagonism prevention biological compound fertilizer.
Description
Technical Field
The invention relates to the technical field of research and development of microbial fertilizers. In particular, the invention relates to a biological compound fertilizer specially aiming at application in piroxicam antagonism and the technical field of preparation and application thereof.
Background
The pear fire epidemic disease (fire blight of pear) is a destructive bacterial disease caused by erwinia amylovora (Erwinia amylovora), and pathogenic bacteria are entry quarantine pests in China. Flowers, leaves, shoots infested with erwinia amylovora show a darkened, dry-up condition, which looks like a fire, the etiology of which is known. The erwinia amylovora can infect more than 40 plants of genus 220, and is mainly harmful to kernel fruit type fruit trees. The pear fire epidemic disease causes a large number of deaths of fruit trees, is easy to spread and difficult to control, and often causes huge economic loss. The disease was determined to be caused by bacteria in 1883, and therefore, the pear fire disease is also the first bacterial plant disease in the world. The amyoviora has strong virulence and transmissibility, and can destroy an orchard in a single growing season.
The erwinia amylovora is straight-bar bacteria, the outer surface of the straight-bar bacteria is provided with a capsule, and the size of the periphyton flagella is 0.9-1.8x0.6-1.5 mu m, and most of the plagues are single growth. The optimal growth temperature is 28 ℃, the humidity is 70% and the pH is 6. It can reproduce at 5-37 deg.C and at 25 deg.C for 75 min. The erwinia amylovora can be carried to adjacent plants by birds, insects, wind, rain, dew and the like, and can infect flowers, fruits, leaves, stems, actively growing twigs, stock and other parts of host plants. As the existence and migration capability of the erwinia amylovora is strong, the diversity of susceptible varieties and tissues is high, and a unified and effective radical treatment target is difficult to find, the management of the erwinia amylovora is very difficult. At present, prevention and control means for pear fire epidemic diseases mainly comprise isolation quarantine, pesticide application, cultivation of resistant varieties, pruning, tree digging and the like. In terms of control, in addition to the continuous improvement and strict execution of existing control measures, there is still a need to develop new chemical agents to cope with resistance, and also to develop biological agents suitable for orchard applications at a price, and to cultivate and screen resistant varieties that are strong in resistance and stable in resistance. In view of the factors of easy generation of drug resistance, regulation of reagent residues by customs, consumption preference of people on organic agricultural products and the like, the importance of biological prevention and control means is increasingly prominent.
Chinese patent (patent publication No. CN 112358366A) discloses a compound bacterial fertilizer prepared from bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), pseudomonas fluorescens (Pseudomonas fluorescens) and candida mongolica (Candida guilliermondii) according to a certain proportion for preventing and treating pear fire blight, which 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 infection of pear fire blight. However, in the practical application process, the series of compound bacterial fertilizers have the problems of complex preparation process, high production cost, short fertilizer efficiency duration, poor nitrogen, phosphorus and potassium fertilizer efficiency and further improvement on the yield of bergamot pears when being applied to the planting of the Korla bergamot pears.
Disclosure of Invention
Aiming at the technical situations that the existing pear fire blight biocontrol fertilizer has complex preparation process, high cost, short fertilizer efficiency time and the yield of Korla bergamot pears needs to be further improved. The invention provides a biological compound fertilizer specially aiming at pear fire germ antagonism prevention, which comprises a compound microbial inoculum and a compound material, wherein the compound material comprises cow dung, sheep manure, oil residue and mineral source humic acid, and the compound microbial inoculum is inoculated according to the mass percentage of 0.3-0.5% of the compound material to obtain the pear fire germ antagonism prevention biological compound fertilizer.
The invention provides a biological compound fertilizer specially applied to antagonism of pear fire germ, which comprises a compound microbial inoculum and a compound material, wherein the compound material comprises, by weight, 15-25 parts of cow dung, 15-25 parts of sheep dung, 30-50 parts of oil residues and 20-40 parts of mineral source humic acid; the composite microbial inoculum is inoculated according to the mass percentage of 0.3-0.5% of the composite material.
Furthermore, the invention provides a biological compound fertilizer specially applied to antagonism and prevention of pear fire germ, which comprises a compound microbial inoculum and a compound material, wherein the compound material comprises, by weight, 20 parts of cow dung, 20 parts of sheep dung, 40 parts of oil residue and 30 parts of mineral source humic acid; the composite microbial inoculum is inoculated according to the mass percentage of 0.4 percent of the composite material.
The invention provides a biological compound fertilizer specially applied to antagonism of piroxicam, wherein the compound microbial inoculum is bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megatherium (Bacillus megaterium) CGMCC NO.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC NO.1641 fermentation liquor are mixed according to a mass ratio of 1:1:1.
Meanwhile, the invention provides a preparation method of the biological compound fertilizer specially aiming at application in antagonism of pear fire germ, which is prepared by the following steps:
(1) Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) preserved at low temperature, bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 are respectively inoculated to NA culture medium plates, cultured for about 12 hours at 30 ℃, single colonies are grown, picked and respectively inoculated to 200m LNB culture solution, and activated and cultured for 12 hours or 24 hours in a constant-temperature shaking table at 160rpm at 30 ℃ to be used as seed solution.
(2) Inoculating the seed solution obtained in step (1) into fermentation tank filled with 7L of corresponding liquid culture medium, fermenting and culturing for 10-15 hr until viable count reaches 5×10 9 cfu/mL。
(3) And (3) compounding the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) obtained in the step (2), bacillus megatherium (Bacillus megaterium) CGMCC NO.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC NO.1641 fermentation liquor according to a specific mass ratio to prepare the composite microbial inoculum.
(4) Collecting cow dung, sheep dung and frying (1:1:2) with water content below 85% and without obvious other impurities, and piling up to 1-1.5 m high, 1.5-2 m wide and 4m long, wherein the fermentation time is more than 40 days until the materials are thoroughly decomposed; controlling the moisture content to be 60-65% in the fermentation process, and judging the moisture: the hand grasps a piece of material, and the finger joints are water-permeable but not water-dripping, and the material can be scattered immediately after falling to the ground.
(5) And (3) mixing the composite microbial inoculum prepared in the step (3) with the composite material prepared from the completely decomposed cow and sheep manure, the oil residue material and the mineral source humic acid prepared in the step (4) according to a proportion, stirring uniformly, and packaging to obtain the biological composite fertilizer specially applied to antagonism and prevention of pear fire pathogens.
The invention provides an application of a biological compound fertilizer applied to antagonism and prevention of pear fire germ in preparing a fertilizer for preventing and treating pear fire blight.
Through the technical scheme, the invention has the following technical effects:
(1) The invention provides a method for antagonizing and measuring Pyricularia, which is specially used for three biocontrol bacteria applied in biological compound fertilizer applied in Pyricularia antagonism, wherein the diameter of bacterial plaque of Bacillus amyloliquefaciens is (9.03 mm) > that of bacillus megaterium (4.38 mm) > that of bacillus mucilaginosus (0.78 mm), and the number of viable bacteria of the biological compound fertilizer obtained by inoculating according to a certain proportion reaches 3.03x10 8 CFU/mL is close to the initial spore number of the composite microbial inoculum, and when the biological fertilizer is diluted by 10 times, the antibacterial effect is highest, and the antibacterial diameter is 5.71+/-0.51 mm.
(2) Although the prior art has disclosed reports about application of bacillus subtilis (Bacillus Subtilis) and bacillus amyloliquefaciens (Bacillus amyloliquefaciens) or part of bacillus in antagonism and prevention of piroxicam, the biggest problem of microbial strains is that research and study clearly shows how to find a suitable safe and effective synergistic efficient compatibility of strains, which is a known problem in the industry, and is a most critical technical problem restricting microbial strain application, and the microbial strains can not be applied in compatibility of any randomly selected strains, so that the microbial strains are applied in the production industry. The bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC NO.1641 are selected, two strains of bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC NO.1641 are new strains which are autonomously screened in the earlier stage of the applicant, and have been applied in the earlier stage research, compatibility and safety compounding of compatibility among the three strains are verified through scientific research production.
(3) The invention provides a biological compound fertilizer which is specially applied to antagonism and prevention of pear fire germ, and soil pesticide pollution, heavy metal pollution, germ pollution and other soil pollutants are improved and degraded by applying biotechnology, so that the cultivated land quality is effectively improved, and the soil productivity 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 hexakis is reduced by 77.78, and the content of the dribbling is reduced by 81.25%.
(4) The biological compound fertilizer specially applied to antagonism of piroxicam and the biological organic fertilizer with similar effective components on the market are continuously used for 25 days in equipment for simulating fertilization conditions, and quick-acting nitrogen, phosphorus and potassium of the biological compound fertilizer provided by the invention are obviously higher than those of a comparison group at 15cm of a simulated cultivation layer, so that the utilization rate of the quick-acting nitrogen, phosphorus and potassium of the fertilizer is obviously improved.
(5) The biological compound fertilizer specially applied to antagonism and prevention of pear fire germ has obvious yield increasing effect on Korla pear, the average yield is increased by about 16.56%, and the sensory, quality nutrition and sanitation and safety of Korla pear are further evaluated, so that the results all meet the requirements.
Drawings
FIG. 1 is a graph showing a panel antagonism test of three biocontrol bacteria provided by the invention; in the graph, A is a plate antagonism graph, B is antibacterial diameter statistics, 1 represents antibacterial plaques of bacillus amyloliquefaciens, 2 represents antibacterial plaques of bacillus megatherium, and 3 represents antibacterial plaques of bacillus mucilaginosus.
FIG. 2 is a schematic view of a column apparatus for testing.
FIG. 3 is a graph showing the change of 15cm quick-acting nitrogen in soil with time of cultivation after simulated fertilization of different fertilizers.
FIG. 4 is a graph showing the change of 15cm quick-acting phosphorus in soil with cultivation time after simulated fertilization of different fertilizers.
FIG. 5 is a graph showing the change of 15cm quick-acting potassium in soil with time of cultivation after simulated fertilization of different fertilizers.
Detailed Description
The present invention will be described with reference to the following examples, but the present invention is not limited to the examples.
The invention relates to materials and devices:
the materials adopted by the invention are as follows: nutrient Agar (NA) medium, nutrient Broth (NB) medium, MRS medium, agar powder and the like are all domestic analytical pure reagents. The three strains of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ATCC No.1978, bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 are all known as early-stage strains, and can be called or purchased by a person of ordinary skill in the art through the China center for type culture collection (China general microbiological culture Collection center) as a known material for scientific research production in the art.
All materials, reagents and equipment selected for use in the present invention are well known in the art and are not limiting to the practice of the invention.
Example 1: biological compound fertilizer specially applied to antagonism of pear fire germ
The biological compound fertilizer specially applied to antagonism and prevention of pear fire germ comprises a compound microbial inoculum and a compound material, wherein the compound material comprises, by weight, 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; the composite microbial inoculum is inoculated according to the mass percentage of 0.3-0.5% of the composite material, wherein the composite microbial inoculum is prepared by mixing bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megaterium (Bacillus megaterium) CGMCC NO.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC NO.1641 fermentation liquor according to the mass ratio of 1:1:1.
Example 2: biological compound fertilizer specially applied to antagonism of pear fire germ
The embodiment provides a biological compound fertilizer specially applied to antagonism of piroxicam on the basis of embodiment 1, wherein the biological compound fertilizer comprises a compound microbial inoculum and a compound material, and the compound material comprises 20kg of cow dung, 20kg of sheep dung, 40kg of oil residue and 30kg of mineral source humic acid; the composite microbial inoculum is inoculated according to the mass percentage of 0.4% of the composite material, wherein the composite microbial inoculum is obtained by mixing bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 fermentation liquor according to the mass ratio of 1:1:1.
Example 3: biological compound fertilizer specially applied to antagonism of pear fire germ
The embodiment provides a biological compound fertilizer specially applied to antagonism of piroxicam on the basis of embodiment 1, wherein the biological compound fertilizer comprises a compound microbial inoculum and a compound material, and the compound material comprises 15kg of cow dung, 15kg of sheep dung, 30kg of oil residue and 20kg of mineral source humic acid; the composite microbial inoculum is inoculated according to the mass percentage of 0.3% of the composite material, wherein the composite microbial inoculum is obtained by mixing bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 fermentation liquor according to the mass ratio of 1:1:1.
Example 4: biological compound fertilizer specially applied to antagonism of pear fire germ
The embodiment provides a biological compound fertilizer specially applied to antagonism of piroxicam on the basis of embodiment 1, wherein the biological compound fertilizer comprises a compound microbial inoculum and a compound material, and the compound material comprises 25kg of cow dung, 15kg of sheep dung, 50kg of oil residue and 40kg of mineral source humic acid; the composite microbial inoculum is inoculated according to the mass percentage of 0.5% of the composite material, wherein the composite microbial inoculum is obtained by mixing bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 fermentation liquor according to the mass ratio of 1:1:1.
Example 5: biological compound fertilizer specially applied to antagonism of pear fire germ
The embodiment provides a biological compound fertilizer specially applied to antagonism of piroxicam on the basis of embodiment 1, wherein the biological compound fertilizer comprises a compound microbial inoculum and a compound material, and the compound material comprises 20kg of cow dung, 22kg of sheep dung, 45kg of oil residue and 35kg of mineral source humic acid; the composite microbial inoculum is inoculated according to the mass percentage of 0.4% of the composite material, wherein the composite microbial inoculum is obtained by mixing bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 fermentation liquor according to the mass ratio of 1:1:1.
Example 6: preparation of biological compound fertilizer specially applied to antagonism of pear fire germ
The embodiment provides a preparation method of a biological compound fertilizer specially applied to antagonism and prevention of piroxicam on the basis of embodiments 1-5, which comprises the following steps:
(1) Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) preserved at low temperature, bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 are respectively inoculated to NA culture medium plates, cultured for about 12 hours at 30 ℃, single colonies are grown, picked and respectively inoculated to 200mLNB culture solution, and activated and cultured for 12 hours or 24 hours in a constant-temperature shaking table at 160rpm at 30 ℃ to be used as seed solution.
(2) Inoculating the seed solutions obtained in step (1) respectivelyFermenting in a fermentation tank containing 7L of corresponding liquid culture medium for 10-15 hr until the viable count reaches 5×10 9 cfu/mL, the fermentation liquor of each bacterium is obtained.
(3) And (3) compounding the bacillus amyloliquefaciens, bacillus megatherium (Bacillus megaterium) CGMCC NO.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) 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 and frying (1:1:2) with water content below 85% and without obvious other impurities, and piling up to 1-1.5 m high, 1.5-2 m wide and 4m long, wherein the fermentation time is more than 40 days until the materials are thoroughly decomposed; controlling the moisture content to be 60-65% in the fermentation process, and judging the moisture: the hand grasps a piece of material, and the finger joints are water-permeable but not water-dripping, and the material can be scattered immediately after falling to the ground.
(5) And (3) mixing the composite microbial inoculum prepared in the step (3) with the composite material prepared from the completely decomposed cow and sheep manure, the oil residue material and the mineral source humic acid prepared in the step (4) according to a proportion, stirring uniformly, and packaging to obtain the biological composite fertilizer specially applied to antagonism and prevention of pear fire pathogens.
Example 7: biological safety evaluation of biological compound fertilizer specially aiming at antagonism and prevention of pear fire germ
According to national general technical guidelines for microbial fertilizer biosafety (NY 1109-2006), rules of microbial fertilizer use strain safety classification catalogue, different strain and product selection toxicology test, procedures, test methods and result evaluation methods are specified according to national standard. The principle of national requirement selection toxicology test is to carry out different safety evaluations according to the strains for production, the strains for microbial fertilizer production are classified into four levels of management, the safety grades are catalogued, the strains which are not listed in the annex are all required to be subjected to toxicology tests except rhizoma bacteria and Lactobacillus.
The first stage is a strain free of toxicology testing.
The second stage is the strain for acute toxicity test.
And the third stage is a strain which needs to be subjected to a pathogenicity test.
The fourth stage is to disable the strain.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 belong to bacterial strain which is used for decomposing phosphorus and potassium compounds in the first stage and is not used for carrying out acute toxicity (LD 50) test in the second stage, wherein the bacterial strain is used for carrying out no toxicology test; the strain which does not belong to the third stage and needs to be subjected to pathogenicity test; not belonging to the fourth-stage forbidden strain.
On the basis of the above examples 1 to 5, the prepared biological compound fertilizer was further provided for acute toxicity test as follows.
Preparation of a liquid culture Medium for producing fluids
1. Potato-yeast extract-sucrose culture medium
Taking 200-300g of peeled potato, cutting into small pieces, adding 1000mL of water, boiling for 20min, filtering with gauze, preparing potato juice, supplementing water to 1000mL, adding 10g of yeast extract and 100g of sucrose, subpackaging, and sterilizing at 121 ℃ for 15min.
2. Wort-yeast extract culture medium
10g yeast extract was added to 1000mL wort, and the mixture was autoclaved at 121℃for 15min after sub-packaging.
Wort-peptone medium
To 1000mL of malt extract, 1g of peptone was added, and the mixture was autoclaved at 121℃for 15 minutes after packaging.
3. Glucose asparaginic acid culture medium
Adjusting pH to 7.2-7.4, packaging, and sterilizing at 121deg.C for 15min.
4. Gaoshi synthetic culture medium No.1
Adjusting pH to 7.2-7.4, packaging, and sterilizing at 121deg.C for 15min.
5. Wort medium
200mL wort was packaged and autoclaved at 121℃for 15min.
0.5% peptone Medium
Taking 2.0g of glucose, 0.6g of yeast extract, 1.0g of peptone and 4.0g of agar, adding distilled water to 200mL, subpackaging, and autoclaving at 121 ℃ for 15min.
6. Preparation of cultures
Pure cultures (suitable medium inclined plane, culture 5-7d at 28+ -1deg.C) of the strain to be examined or transferred are inoculated into suitable toxigenic culture medium respectively after being confirmed, general strains are inoculated into three toxigenic culture media of malt extract-yeast extract, malt extract-peptone and potato-yeast extract-sucrose, actinomycetes are inoculated into glucose asparaginic acid culture medium and Gao's synthetic No.1 culture medium, rhodotorula is inoculated into malt extract culture medium and 0.5% peptone culture medium, and cultured for 14d at 28+ -1deg.C. After flowing steam for 1h, the culture is filtered, and the obtained filtrate is partially concentrated for 2.5 times at the constant temperature of 80 ℃ for standby, and the rest is directly used for experiments.
(II) mouse toxicity test
80 mice are needed for each liquid culture medium, and the male and female halves are randomly divided into 4 groups of 10 mice each. The dose groups were set as follows: a culture medium blank group, a culture medium 2.5-time concentrated blank group, a culture stock solution group and a culture 2.5-time concentrated group. The test object is subjected to one-time gastric lavage on the mice at a dosage of 20.0mL/kg BW for 14d, and poisoning performance and death condition of the mice during the experiment are recorded.
(III) statistics of test data
Carrying out variance alignment test on initial weight and final weight of the mice, and carrying out statistical treatment on data meeting the requirement of variance alignment by using t test of average comparison of two samples; statistical processing is performed on the data with irregular variance by t' test.
(IV) result determination
The initial weight of the mice is balanced among groups, the test object has no adverse effect on the final weight, the mice do not have toxic reaction or die in the experimental process, and the safety of the test strain can be judged.
The tests prove that three strains of bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus megatherium (Bacillus megaterium) CGMCC No.1640 and bacillus mucilaginosus (Bacillus mucilaginosus) CGMCC No.1641 selected from the biological compound fertilizers provided in the embodiments 1 to 6 are safe as tested strains, the compatibility of the strains meets the biological safety standard, and the application of the strains in antagonism and prevention of pear fire bacteria is safe, so that the biological compound fertilizer is also a key point for solving the problem that whether the selected strains are safe or not in general existence in the existing microbial fertilizers, whether the selected strains are subjected to safety evaluation and test verification, and whether the selected strains are safe or not to prepare the corresponding biological fertilizer.
Example 8: biological compound fertilizer biosafety detection test specially aiming at antagonism and prevention of pear fire germ
The safety results of the finished bio-organic fertilizer are shown in Table 1. As shown by the results in Table 1, the biological compound fertilizer meets the actual non-toxic grade in the national standard for fertilizer registration acute oral toxicity test and evaluation requirement of the people's republic of China (NY 1980-2010).
Table 1: the acute oral toxicity test result of the biological compound fertilizer
The tests prove that the biological compound fertilizer provided in the embodiments 1 to 6 accords with biological safety, is safe in application of antagonism and prevention of pyriform bacteria, and is in fact a key for solving the problem of the existing biological safety of the microbial fertilizer.
Example 9: performance test of biological compound fertilizer specially aiming at antagonism and prevention of pear fire germ
The inhibition effect of the strain screened by the invention and the biological compound fertilizer prepared after compounding on the erwinia amylovora is examined on the basis of examples 1-6.
(1) Test protocol
Bacillus megaterium, bacillus amyloliquefaciens and Bacillus mucilaginosus are supplied by Xinjiang Huison biotechnology Co., ltd., pyricularia strain NCPPB1665 from Nanjing university plant quarantine laboratory, LB medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl and 1000mL of pure water. Adjusting pH to 7.0, sterilizing at 121deg.C for 20min, and adding agar to obtain solid culture medium: 0.8-0.9g/50mL.
(2) Test method
Dipping a small amount of pear fire epidemic disease by an inoculating loop, streaking on an LB solid culture medium, and then placing the solid culture medium in a constant temperature incubator at 28 ℃ for culturing for 24 hours; picking single colony, inoculating into culture flask containing 25mL LB liquid medium, shake culturing at 28deg.C in 220r/min constant temperature shaking table for 24 hr, and adjusting shaking bacterial liquid to OD 600 =0.3 (about 1×10) 8 cfu/mL) for standby; inoculating biocontrol bacteria to be detected into LB liquid culture medium, shake culturing in a constant temperature shaking table at 28 ℃ and 220r/min, and regulating the shaking bacterial liquid to OD 600 =0.3 (about 1×10) 8 cfu/mL) for standby; 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 by the ratio for activity measurement; the test adopts a flat plate coating method, 100 mu L of diluted bacterial suspension is coated on LB solid culture medium, each dilution multiple is repeated 3 times, the observation result is carried out after the constant temperature culture for 24 hours at 28 ℃, the colony number of each dilution multiple coating plate is counted, the viable count of the composite bacterial agent is calculated, and compared with the initial spore number, the viable count (CFU/mL) = (colony number multiplied by dilution multiple)/0.1 mL of the composite bacterial agent is obtained; and (3) dropwise adding 10 mu L of antagonistic bacteria suspension to a bacteria-containing flat plate, setting a blank control, repeating each treatment for 3 times, culturing at a constant temperature of 28 ℃ for 48 hours, observing the antibacterial effect, measuring the diameter of a bacteriostasis zone and the diameter of a colony by using a crisscross method, and measuring the diameter of the bacteriostasis (mm) of antagonistic bacteria=the diameter of the bacteriostasis zone-the diameter of the colony. The specific test results are shown in FIG. 1 and tables 2-3.
(3) Test results
As can be seen from the data of FIG. 1, the antagonism of 3 strains of biocontrol bacteria was measured by a plate counter method, and the order of antagonism was that the order of the antagonism was that of Bacillus amyloliquefaciens (9.03 mm) > Bacillus megaterium (4.38 mm) > Bacillus mucilaginosus (0.78 mm); as is clear from the data in tables 2 to 3, the viable count of the biofertilizer was 3.03X10 8 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, and the biological fertilizer has good inhibition effect on erwinia amylovora at 10 times of dilution, 100 times of dilution and 500 times of dilution, wherein the best effect is achieved at 10 times of dilution, and the inhibition diameter is 5.71+/-0.51 mm.
Table 2: viable count of composite microbial inoculum with different dilution factors
| Dilution factor | Colony count (number/mL) of composite bacterial preparation |
| 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 of bio-fertilizer on erwinia amylovora
| Concentration (times) of biofertilizer | Antibacterial 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 aiming at application in antagonism of pear fire germ
The application of the biological compound fertilizer specially aiming at the application in antagonism of pear fire germ provided by the formula of the embodiment 2 and the preparation method of the embodiment 6 in planting of Xinjiang Korla bergamot pears is provided on the basis of the embodiments 1-7. The influence of different treatments on the change of pesticides, heavy metals and germs in the soil is compared, and the influence of the biological compound fertilizer on improving the quality of cultivated land and improving the productivity of the soil is examined.
(1) Test protocol
Test site: sixteen-division Feng Naide pear garden in Xinjiang Korla Sha-eastern gardening field
Test time: 10 in 2018 to 9 in 2019
Control product: huison' commodity organic fertilizer (control 1), commercial biological organic fertilizer of certain manufacturer (control 2), commercial commodity organic fertilizer of certain manufacturer (control 3)
Soil condition of test field: the soil type of the test is desert soil (sampling is carried out on the day 21 of 10 months in 2018), and the basic condition of the soil before the test is as follows: 11.6g/kg of organic matters, 58.3mg/kg of hydrolyzed nitrogen, 11.8mg/kg of quick-acting phosphorus, 142.1mg/kg of quick-acting potassium and pH value of 7.42; soil heavy metal and pesticide residue conditions of test land before test: 15.237mg/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 organophosphorus pesticide residues and 0.16mg/kg of drop nasal discharge.
Test crop variety: korla bergamot pear. Planting in 2002 in 3 months, planting holes with diameters of 0.8m, depths of 0.6 m and plant row spacing of 4.5X3.5 m, and tree ages of 16 years in 2018 and tree crowns with diameters of about 3.2 m, and entering full bearing period. Four treatments are tested, three repetitions are carried out, 12 cells are arranged, random block arrangement is carried out, each test cell is 1 mu, 10 trees are randomly defined in each cell, the length is 22.5 meters, the width is 7 meters, the area is 157.5 square meters and is used as a comparison, 30cm ridge is arranged between the cells, and field management is consistent.
Table 4: influence of different treatments on bergamot pear yield
As can be seen from the data in Table 4, the yield of bergamot pears is highest by applying the biological compound fertilizer provided by the invention, the average acre yield reaches 923.6kg, and the yield is increased by 67.7kg and 7.91% compared with the average acre yield of the organic fertilizer 'Huison' of the company; compared with the bio-organic fertilizer sold in a certain manufacturer, the yield per mu is increased by 117.2kg and the yield is increased by 14.53 percent; compared with the commercial bio-commodity fertilizer of a certain manufacturer, the yield per mu is increased by 131.2kg and 16.56%.
Table 5: analysis of variance
From table 5, analysis of variance of the yields of the different treatments showed that treatment f=48.64954, F > F0.01 (3, 6), indicating that the difference between the different treatments reached a very significant level of 1%.
Table 6: multiple comparisons
As shown by the multiple comparison results in the table 6, the difference number of the yield of the biological compound fertilizer reaches a remarkable level compared with other treatment yields, and the biological compound fertilizer provided by the invention has remarkable yield increasing effect, and compared with the 'Huison' commodity organic fertilizer, the biological compound fertilizer provided by the invention has remarkable difference of microbial viable bacteria on the yield index of bergamot pears.
(2) Test of the Effect of different fertilizers on soil nutrient Change
Table 7: effects 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 matters in soil are increased by 2.3g/kg, the hydrolytic nitrogen is increased by 2.1mg/kg, the organic phosphorus is increased by 0.8mg/kg, the quick-acting potassium is increased by 3.1mg/kg, and the viable count is increased by 0.8 million/g.
(3) Influence of different fertilizers on soil heavy metal and persistent organic pollutant changes
Table 8: influence of different fertilizer treatments on main physicochemical properties of soil
As can be seen from the data in Table 8, the application of the biological compound fertilizer provided by the invention reduces the mercury content in soil by 0.001mg/kg, reduces by 9.09%, reduces the arsenic content by 0.004mg/kg, reduces by 7.14%, reduces the cadmium content by 0.010mg/kg, and reduces by 26.26%; the lead content is reduced by 0.152mg/kg and 1.00%; the chromium content is reduced by 0.928mg/kg by 2.00%; the total content of organophosphorus pesticide residues is reduced by 0.21mg/kg and 80.77%; the content of hexakis is reduced by 0.14mg/kg, the content of driping is reduced by 77.78%, the content of driping is reduced by 0.13mg/kg, and the content of driping is reduced by 81.25%, which shows that the biological compound fertilizer provided by the invention has remarkable improvement on heavy metal and pesticide residues in soil.
(4) Test of residence time of different fertilizers in cultivated layer
The test judges the distribution relation of the effective agriculture serving as the root system layer in the cultivation layer by comparing the residence time distribution of the effective ingredients of the biological compound fertilizer provided by the invention and the effective ingredients of the commercial organic fertilizer in the cultivation layer.
Test protocol: the test uses the device shown in figure 2, which 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 11 cm), wherein: the upper and middle section glass cylinders are free of ground, gauze is used for separation, soil leaks from the culture cylinder during prevention and control sampling, and uniform small holes are formed in the bottom of the lowest section glass cylinder so as to facilitate smooth outflow of runoff water, and specific test results are shown in figures 3-5.
As can be seen from the data of FIG. 3, in the test section from the application of the soil to 25 days under the simulated fertilization condition, the other groups B3 and B4 which are 15cm below the table (simulated cultivated layer plant root system layer) are 25-400% higher than the quick-acting nitrogen-containing bacteria of the comparative groups B0, B1 and B2 along with the change of the culture time.
As can be seen from the data of FIG. 4, in the test section from the application of the soil to 25 days under the simulated fertilization condition, the other groups B3 and B4 which are 15cm below the table (simulated cultivated layer plant root system layer) are 12-210% higher than the quick-acting phosphorus content bacteria of the comparative groups B0, B1 and B2 along with the change of the culture time.
As can be seen from the data of FIG. 5, in the test section from the application of the soil to 25 days under the simulated fertilization condition, the other groups B3 and B4 which are 15cm below the table (simulated cultivated layer plant root system layer) are 6-170% higher than the groups B0, B1 and B2 of quick-acting potassium with the change of the culture time.
The data show that the biological compound fertilizer provided by the invention and the biological organic fertilizer with the equivalent effective components sold in the market last for 25 days in equipment under the simulated fertilization condition, and three repeated-reading test data show that the biological compound fertilizer provided by the invention has obviously higher quick-acting nitrogen, phosphorus and potassium than a comparison group at 15cm (simulated plant root system layer) of a simulated cultivation layer. Has the obvious condition of improving the utilization rate of quick-acting nitrogen, phosphorus and potassium of the fertilizer.
(5) Effect of application of the biological Compound fertilizer of the invention on the quality of Korla bergamot pears
The experiment is based on experiments 1-4, the nutritional quality of the Korla pears produced by applying the biological compound fertilizer provided by the formula of the embodiment 2 and the preparation method of the embodiment 6 is identified, and the sensory, nutritional quality and health indexes of the Korla pears are evaluated according to the detection standards of CAQS/MTYX2004-2019, GB/T21488-2008, GB/T12947-2008, GB/T5009.268-2016, GB2762-2017, GB2763-2019 and the like, and the specific results are shown in the table 9.
Table 9: influence of different fertilizer treatments on the quality of Korla bergamot pears
As can be seen from the test data in Table 9, the biological compound fertilizer provided by the invention is applied to the planting of the Korla pears by testing the sensory, nutritional quality and sanitary safety indexes of the Korla pears, the fruits of the pears are regular and uniform in size, the peel is greenish and glossy, compared with the reference of similar products, the soluble solid content of the Korla pears is improved by 11.1%, the solid acid ratio is improved by 45%, the sugar-acid ratio is improved by 47.9%, the total acid content is reduced by 23.1%, the VC (improved by 7.1%), the potassium (improved by 14.9%), the calcium (improved by 18.2%), the iron (improved by 33.5%) and the zinc (improved by 60.0%), so that the product provided by the invention has better taste, richer nutrition and better flavor; the method further determines the content of harmful heavy metals and pesticide residues, the determination results are below a limit, the eating is safe, and the product is obtained in the invention for 14 days in 2020, and the product is detected by a national brand-new agricultural product nutrition quality evaluation and identification mechanism of an agricultural rural microbial fertilizer and edible fungus strain quality alkalinity test center, and finally gives out comprehensive evaluation of the product as follows: the Korla bergamot pear produced by applying the biological compound fertilizer has the characteristics of good sensory quality, excellent nutrition quality, safety and health, and comprehensive evaluation of the quality.
In summary, the technical scheme of the biological compound fertilizer capable of being used for preventing and treating the pear fire blight provided by the invention is that the strain with a specific proportion is compounded to form stable microecology and ensure a higher viable count, so that the normal growth of plants can be ensured, the pear fire blight prevention and treatment effect is obvious, the average yield is increased by about 16.56% when the biological compound fertilizer is applied to the planting of Korla 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 and germ pollution can be improved, the cultivated land quality is improved, the soil production capacity 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 using other various embodiments according to the present disclosure. Therefore, the design of some simple changes or modifications by adopting the automatic quick searching technical scheme and thought of the invention falls into the protection scope of the invention.
Claims (2)
1. The biological compound fertilizer is characterized by comprising a compound microbial inoculum and a compound material, wherein the compound material comprises, by weight, 20 parts of cow dung, 20 parts of sheep dung, 40 parts of oil residues and 30 parts of mineral source humic acid; inoculating the composite microbial inoculum according to the mass percentage of 0.4% of the composite material; the composite microbial inoculum is bacillus amyloliquefaciensBacillus amyloliquefaciens) ATCC No.1978, bacillus megateriumBacillus megaterium) CGMCC No.1640 and Bacillus mucilaginosus @ and its preparing processBacillus mucilaginosus) Mixing CGMCC NO.1641 fermentation liquor according to the mass ratio of 1:1:1 to obtain;
the compound fertilizer is prepared by the following steps:
(1) Selecting bacillus amyloliquefaciens preserved at low temperatureBacillus amyloliquefaciens) ATCC No.1978, bacillus megateriumBacillus megaterium) CGMCC No.1640 and Bacillus mucilaginosus @ and its preparing processBacillus mucilaginosus) Inoculating CGMCC NO.1641 to NA culture medium plates respectively, culturing at 30 ℃ for about 12 hours, picking single colony, inoculating to 200m LNB culture solution respectively, and activating and culturing in a constant temperature shaking table at 160rpm at 30 ℃ for 12 hours or 24 hours to obtain seed solution;
(2) Inoculating the seed solution obtained in step (1) into fermentation tanks containing 7L of corresponding liquid culture medium, fermenting and culturing for 10-15 hr until viable count reaches 5×10 9 cfu/m L, obtaining fermentation liquor of each bacterium;
(3) The bacillus amyloliquefaciens obtained in the step (2) is treatedBacillus amyloliquefaciens) ATCC No.1978, bacillus megaterium ]Bacillus megaterium) CGMCC No.1640 and Bacillus mucilaginosus @ and its preparing processBacillus mucilaginosus) The CGMCC NO.1641 fermentation liquor is compounded according to the mass ratio to prepare the composite microbial inoculum;
(4) Collecting cow dung, sheep dung and oil residue (1:1:2) with water content below 85% and without obvious other impurities, and piling up to 1-1.5 m high, 1.5-2 m wide and 4m long, wherein the fermentation time is more than 40 days until the materials are thoroughly decomposed; controlling the moisture content to be 60-65% in the fermentation process, and judging the moisture: a hand grasps a material, the finger joint is water-permeable but not water-dripping, and the material is immediately scattered after falling to the ground;
(5) And (3) mixing the composite microbial inoculum prepared in the step (3) with the composite material prepared from the completely decomposed cow and sheep manure, the oil residue material and the mineral source humic acid prepared in the step (4) according to a proportion, stirring uniformly, and packaging to obtain the biological composite fertilizer specially applied to antagonism and prevention of pear fire pathogens.
2. The use of a biocomposite fertilizer according to claim 1 for the preparation of a fertilizer specifically directed against pear fire blight.
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