WO2023272996A1 - 一种防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂及其制作方法 - Google Patents

一种防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂及其制作方法 Download PDF

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WO2023272996A1
WO2023272996A1 PCT/CN2021/122000 CN2021122000W WO2023272996A1 WO 2023272996 A1 WO2023272996 A1 WO 2023272996A1 CN 2021122000 W CN2021122000 W CN 2021122000W WO 2023272996 A1 WO2023272996 A1 WO 2023272996A1
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nano
bacillus
fruit
silver composite
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French (fr)
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周游
黄俊生
杨腊英
汪军
郭立佳
梁昌聪
刘磊
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中国热带农业科学院环境与植物保护研究所
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/54—1,3-Diazines; Hydrogenated 1,3-diazines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the invention relates to the field of bacterial agents for preventing and treating Polychaetosporum fruit rot and anthracnose of fruits, in particular to a nano-silver composite Bacillus bacterial agent for preventing and treating Polychaetosporum fruit rot and anthracnose of lotus mist fruit and a preparation method thereof.
  • the fruit rot and anthracnose of Lianwu fruit are common and cause heavy losses in the production of Lianwu, both of which can cause fruit drop and fruit rot of Lianwu.
  • Pestalotiopsis versicolor fruit rot of lotus mist fruit is caused by Pestalotiopsis versicolor
  • anthracnose of lotus mist fruit is caused by Colletotrichum fructicola. Because the lotus mist fruit period coincides with the high temperature and rainy season, the two diseases spread and developed rapidly, causing farmers to use excessive chemical pesticides in the production process, which easily leads to environmental pollution and food poisoning.
  • the use of biological control of plant diseases is the general trend.
  • Biological control is an eco-friendly control method that can provide guarantee for the sustainable development of agriculture.
  • Bacillus has biocontrol mechanisms such as producing antagonistic substances, competing with pathogenic bacteria for nutrients and space, and inducing plant resistance. Successfully used to control bacterial and fungal diseases of a variety of plants. Studies have shown that using two or more biocontrol bacteria with synergistic effects to control diseases is more effective than using a single bacterium.
  • the synergistic mechanism between bacteria in disease control may be that bacteria with different biocontrol mechanisms (competition, production of antagonistic substances, parasitism/hyperparasitism) have complementary disease control mechanisms.
  • the antagonistic substances produced by different types of biocontrol bacteria complement each other, and different types of strains induce the production of new resistant substances under the condition of co-existence.
  • Nano-silver has a broad-spectrum antibacterial ability, can penetrate the cell wall of microorganisms and enter the body, has a good ability to kill many pathogenic bacteria and fungi, and has a high valence state and a huge specific surface area, and is not easily oxidized in the air And precipitation, has been used in antibacterial products, such as nano-silver antibacterial gauze, nano-silver wound stickers and nano-silver paint.
  • the physical and chemical synthesis of nano-silver is costly and produces toxic substances, while the biosynthesis of nano-silver is cheap, safe and environmentally friendly, and has the characteristics of sustainable development. It has gradually become a research hotspot in the field of nano-silver synthesis.
  • Microbial metabolites can be used as reducing agents and stabilizers to biosynthesize nano-silver, but due to the broad-spectrum antibacterial properties of nano-silver, the source of bacteria and production efficiency are limited.
  • Cyprodinol is a new type of plant growth regulator used to increase plant chlorophyll content and promote plant growth, but there is no report that it can improve plant disease resistance.
  • the patent finds that the compound bacillus fermentation broth added with cyprodinol can greatly improve fruit resistance-related enzymes and increase disease resistance.
  • CN104672006A a kind of lotus mist special medicine fertilizer, comprises following components: 18-22 parts of cow dung, 15-18 parts of chicken manure, 20-40 parts of rice straw , 10-20 parts of bean cake, 10-20 parts of bagasse, 40-50 parts of urea, 30-40 parts of superphosphate, 30-40 parts of potassium sulfate, 2-5 parts of trace elements, 3-5 parts of compound amino acids, 15- 25 parts of Chinese herbal medicine extract, 1-2 parts of Bacillus subtilis, 0.6-1 part of Chaetomium globosa.
  • the patent uses the interaction between Bacillus subtilis and Chaetomium globosa to prevent and control various diseases. However, the control effect of the special medicinal fertilizer after use is not recorded.
  • the present invention proposes a nano-silver composite bacillus bacterial agent and a preparation method thereof for preventing and treating P.
  • Bacillus amyloliquefaciens HW05 was cultured in LB liquid medium (containing: tryptone 10g, yeast extract 5g, NaCl 10g per liter) at 37°C and 180rpm. Scale culture for 24 hours to produce Bacillus amyloliquefaciens HW05 seed solution; Bacillus subtilis BLG010 in LB liquid medium (each liter contains: tryptone 10g, yeast extract 5g, NaCl 10g), at 37°C, 180rpm Small-scale culture for 24 hours to produce Bacillus subtilis BLG010 seed liquid;
  • nano-silver composite bacillus bacterial agent by weight, 94-96 parts of nano-silver composite bacillus suspension prepared in step (2), 2-2.5 parts of guar gum, fatty alcohol 0.8-1.5 parts of polyoxyethylene ether (JFC), 1-1.5 parts of polyvinylpyrrolidone, 0.2-0.3 parts of castor oil polyoxyethylene ether (EL-35), and 0.8-1.5 parts of cyprodinol are mixed evenly.
  • JFC polyoxyethylene ether
  • EL-35 castor oil polyoxyethylene ether
  • step (1) the Bacillus amyloliquefaciens is provided by the Institute of Environment and Plant Protection of the Chinese Academy of Tropical Agricultural Sciences, preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and the preservation number is CGMCC No.10273, the strain No. HW05, its properties refer to patent CN108690821A.
  • step (1) the Bacillus subtilis is provided by the Institute of Environment and Plant Protection of the Chinese Academy of Tropical Agricultural Sciences, and is preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures.
  • the preservation number is CGMCC No.5953, and the strain number is BLG010, its properties refer to patent CN102747020A.
  • step (2) the preparation of the nano-silver composite bacillus suspension: in parts by weight, add 35 parts of organic fermentation nutrient base, 15 parts of inorganic fermentation nutrient base, 1000 parts of distilled water After stirring evenly, sterilize by high-pressure steam at 121°C for 30 minutes and at a pressure of 100kPa.
  • the organic fermentation nutrient base is composed of the following raw materials in parts by weight: 6-8 parts of cassava flour, 5-7 parts of soybean meal, 4-5 parts of amino acids, 3-4.5 parts of yeast powder, seaweed 3-6 parts of essence, 5-8 parts of fish protein powder, 5-7.5 parts of brown sugar, mix well, crush and pass through 80-mesh sieve.
  • the organic fermentation nutrient base is composed of the following raw materials in parts by weight: 7 parts of cassava flour, 6 parts of soybean meal, 4.5 parts of amino acid, 3.75 parts of yeast powder, 4.5 parts of seaweed extract, 6.5 parts of fish protein powder part, 6.25 parts of brown sugar, mix evenly, smash through 80 mesh sieve.
  • the inorganic fermentation nutrient base is composed of the following raw materials in parts by weight: 10-15 parts of diammonium phosphate, 1-2 parts of potassium nitrate, 3-5 parts of zinc sulfate, 2-3 parts of magnesium sulfate 0.05-0.1 part ammonium molybdate, 0.5-1 part sodium chloride.
  • the inorganic fermentation nutrient base is composed of the following raw materials in parts by weight: 12.5 parts of diammonium phosphate, 1.5 parts of potassium nitrate, 4 parts of zinc sulfate, 2.5 parts of magnesium sulfate, 0.075 parts of ammonium molybdate, 0.75 parts of sodium chloride.
  • step (3) the preparation of the nano-silver composite bacillus bacterial agent: in parts by weight, 95 parts of the nano-silver composite bacillus suspension prepared in step (2), 2.25 parts of guar gum 1.15 parts of fatty alcohol polyoxyethylene ether (JFC), 1.25 parts of polyvinylpyrrolidone, 0.25 parts of castor oil polyoxyethylene ether (EL-35), and 1.15 parts of cyprodinol and mix well.
  • JFC fatty alcohol polyoxyethylene ether
  • EL-35 castor oil polyoxyethylene ether
  • step (3) the entire configuration process is carried out at a temperature of 60°C.
  • the nano-silver composite bacillus bacterial agent for the present invention to prevent and treat Polychaeta spp. fruit rot and anthracnose can not only effectively inhibit P.
  • the growth of lotus mist; the combined use of silver nitrate solution and cyprodinol can promote the growth of lotus mist and improve the antibacterial effect of bacteria; It can continuously provide nutrition for bacillus, improve the persistence ability of bacillus on the surface of fruit, prolong the antibacterial function, and then make the nano-silver composite bacillus bacterial agent of the present invention achieve the prevention and treatment of polychaete fruit rot and anthracnose of lotus mist fruit Effect.
  • nano-silver composite bacillus bacterial agent of the invention can solve the ills caused by the abuse of chemical pesticides, and enable farmers to produce pollution-free green food.
  • Fig. 2 embodiment 1 nano-silver composite bacillus inoculum affects fruit catalase (CAT) activity;
  • Fig. 3 embodiment 1 nano-silver composite bacillus inoculum influences on fruit malondialdehyde (MDA) content;
  • Bacillus amyloliquefaciens HW05 was cultured in LB liquid medium (containing: tryptone 10g, yeast extract 5g, NaCl 10g per liter) at 37°C and 180rpm. Scale culture for 24 hours to produce HW05 seed liquid; the production method of Bacillus subtilis BLG010 seed liquid is the same as the above-mentioned Bacillus amyloliquefaciens.
  • Organic fermentation nutrient base it is composed of the following raw materials in parts by weight: 7 parts of cassava flour, 6 parts of soybean meal, 4.5 parts of amino acids, 3.75 parts of yeast powder, 4.5 parts of seaweed extract, 6.5 parts of fish protein powder, 6.25 parts of brown sugar, Mix evenly, break through an 80-mesh sieve, and prepare.
  • Inorganic fermentation nutrient base by weight, it consists of the following raw materials: 12.5 parts of diammonium phosphate, 1.5 parts of potassium nitrate, 4 parts of zinc sulfate, 2.5 parts of magnesium sulfate, 0.075 parts of ammonium molybdate, and 0.75 parts of sodium chloride Mix well and make.
  • nano-silver composite bacillus bacteria agent by weight, 95 parts of nano-silver composite bacillus suspensions prepared in step (2), 2.25 parts of guar gum, fatty alcohol polyoxyethylene ether (JFC) 1.15 parts, polyvinylpyrrolidone 1.25 parts, castor oil polyoxyethylene ether (EL-35) 0.25 parts, cyproridinol 1.15 parts and mix well.
  • JFC fatty alcohol polyoxyethylene ether
  • EL-35 castor oil polyoxyethylene ether
  • Nano-silver compound bacillus agent to prevent fruit rot drop 0.1ml of Pestalotiopsis versicolor and Colletotrichum fructicola on the peduncle of the fruit (the most susceptible part) of the fruit Mixed spore liquid (the spore concentration of each pathogen is 1 ⁇ 10 3 /ml).
  • the control (CK) fruit is only inoculated with pathogenic bacteria, and after the fruit is inoculated with pathogenic bacteria, the whole fruit is sprayed with 500 times the nano-silver composite bacillus bacterial agent of the present invention.
  • Each replicate contained 4 fruits, 3 replicates each for the control (CK) and treatment.
  • the fruits used in the test are all picked from the same orchard in the same batch and have the same maturity and no damage on the surface.
  • the test fruits are placed in the culture conditions of 20°C, 85% humidity, 12 hours of light and 12 hours of darkness.
  • CAT can reduce hydrogen peroxide (H 2 O 2 ) to water (H 2 O) and oxygen (O 2 ), thereby Reduce the harm of active oxygen to plants.
  • the test fruits for the determination of catalase (CAT) were divided into CK and treatment.
  • the surface of the fruit was sprayed with mixed spore liquid of Pestalotiopsis versicolor and Colletotrichum fructicola (the spore concentration of each pathogen was 1 ⁇ 10 3 /ml).
  • the CK fruit is only inoculated with pathogenic bacteria, and the whole fruit is sprayed with 500 times the nano-silver composite Bacillus bacterial agent invented by this patent after the fruit is inoculated with pathogenic bacteria.
  • the fruits used in the test are all picked from the same orchard in the same batch and have the same maturity and no damage on the surface.
  • the test fruits are placed in the culture conditions of 20°C, 85% humidity, 12 hours of light and 12 hours of darkness. Both CK and treatment fruits had 50 fruits.
  • For each determination of CAT three samples were randomly selected for testing, and each sample was tested three times in parallel.
  • Malondialdehyde (MDA) is one of the products of cell membrane lipid peroxidation.
  • the content of MDA in plants increases under adversity, so its content can be used to indirectly reflect the damage degree of plant cells. Every time MDA is measured, three samples are randomly selected for testing, and each sample is subjected to three parallel tests, and the fruit processing method is the same as the detection method (2) in Example 1.
  • the fruits used in the test are all picked from the same orchard in the same batch and have the same maturity and no damage on the surface.
  • the test fruits are placed in the culture conditions of 20°C, 85% humidity, 12 hours of light and 12 hours of darkness.
  • Fig. 1 showed that CK fruit pedicles rotted 5 days after inoculation with pathogenic bacteria, and produced fruiting bodies 7 days later. However, the fruit sprayed with nano-silver compound Bacillus inoculum had no symptoms. The results showed that spraying nano-silver composite Bacillus inoculum could effectively prevent fruit rot caused by Pestalotiopsis versicolor and Colletotrichum fructicola.
  • FIG. 2 shows that the nano-silver composite Bacillus inoculum can significantly reduce the CAT enzyme activity of the fruit after the 6th day.
  • the content of H 2 O 2 in its body is less than that of CK fruit, which in turn regulates the decrease of CAT enzyme activity.
  • Figure 3 shows that the nano-silver composite Bacillus inoculum can significantly reduce the MDA content of the fruit. This may be due to the fact that the spraying of the inoculum prevents the fruit from being infected by pathogenic bacteria, thereby protecting the plasma membrane structure of the fruit cells and function.
  • Fig. 4 shows that the nano-silver composite Bacillus inoculum can weaken the decline of fruit anthocyanin content, thereby slowing down the darkening of the peel color.
  • Table 1 shows that after 10 days of spraying the nano-silver composite Bacillus bacterial agent of the present invention, only 3 types of fungi were isolated on the surface of the pericarp, while 10 types of fungi were isolated on the surface of the CK pericarp. This shows that spraying nano-silver compound Bacillus inoculum can significantly reduce the fungus on the surface of the fruit peel and reduce the probability of pathogenic bacteria infecting the fruit.
  • Table 2 shows that the CK fruit began to rot on a large scale on the 9th day, while the rotten rate of the treated fruit was only 8.33% on the 12th day.
  • the nano-silver composite bacillus bacterial agent of the present invention can reduce fruit rot.
  • Bacillus amyloliquefaciens HW05 was cultured in LB liquid medium (containing: tryptone 10g, yeast extract 5g, NaCl 10g per liter) at 37°C and 180rpm. Scale culture for 24 hours to produce HW05 seed liquid; the production method of Bacillus subtilis BLG010 seed liquid is the same as the above-mentioned Bacillus amyloliquefaciens.
  • Organic fermentation nutrient base by weight, it consists of the following raw materials: 6 parts of cassava flour, 5 parts of soybean meal, 4 parts of amino acid, 3 parts of yeast powder, 3 parts of seaweed essence, 5 parts of fish protein powder, and 5 parts of brown sugar , mixed evenly, crushed through a 80-mesh sieve, and prepared.
  • Inorganic fermentation nutrient base in parts by weight, it consists of the following raw materials: 10 parts of diammonium phosphate, 1 part of potassium nitrate, 3 parts of zinc sulfate, 2 parts of magnesium sulfate, 0.05 part of ammonium molybdate, and 0.5 part of sodium chloride , mixed evenly, prepared.
  • nano-silver composite bacillus suspension in parts by weight, add 30 parts by weight of organic fermentation nutrient base, 10 parts of inorganic fermentation nutrient base, and 950 parts of distilled water in a vertical fermenter and stir evenly.
  • the high temperature steam sterilization temperature is 121°C, the time is 30min, and the pressure is 100kPa.
  • nano-silver composite bacillus bacteria agent by weight, 94 parts of nano-silver composite bacillus bacteria suspensions prepared in step (2), 2 parts of guar gum, fatty alcohol polyoxyethylene ether (JFC) 0.8 part, polyvinylpyrrolidone 1 part, castor oil polyoxyethylene ether (EL-35) 0.2 part, cyprodinol 0.8 part and mix well.
  • JFC fatty alcohol polyoxyethylene ether
  • EL-35 castor oil polyoxyethylene ether
  • Table 3 shows that the rot rate of the treated fruit is only 9.02% on the 12th day, and the nano-silver composite bacillus bacterial agent of the present invention can reduce fruit rot.
  • microbial seed liquid small-scale culture of Bacillus amyloliquefaciens HW05 in LB liquid medium containing: tryptone 10g, yeast extract 5g, and NaCl 10g per liter at 37°C and 180rpm 24h, produce HW05 seed liquid; Bacillus subtilis BLG010 seed liquid production method is the same as above-mentioned Bacillus amyloliquefaciens.
  • Organic fermentation nutrient base by weight, it consists of the following raw materials: 8 parts of cassava flour, 7 parts of soybean meal, 5 parts of amino acids, 4.5 parts of yeast powder, 6 parts of seaweed essence, 8 parts of fish protein powder, and 7.5 parts of brown sugar , mixed evenly, crushed through a 80-mesh sieve, and prepared.
  • Inorganic fermentation nutrient base by weight, it consists of the following raw materials: 15 parts of diammonium phosphate, 2 parts of potassium nitrate, 5 parts of zinc sulfate, 3 parts of magnesium sulfate, 0.1 part of ammonium molybdate, and 1 part of sodium chloride , mixed evenly, prepared.
  • nano-silver composite bacillus suspension in parts by weight, add 40 parts by weight of organic fermentation nutrient base, 20 parts of inorganic fermentation nutrient base, and 1050 parts of distilled water in a vertical fermenter and stir evenly.
  • the high temperature steam sterilization temperature is 121°C
  • the time is 30min
  • the pressure is 100kPa.
  • After the temperature drops below 37°C add 1.5 parts of Bacillus amyloliquefaciens HW05 seed solution in parts by weight, and add 3 parts of Bacillus subtilis after 6 hours Bacillus BLG010 seed solution, fed with sterile air, the ventilation rate was 19L/min, the pressure was 0.04MPa, and the constant temperature was 38°C.
  • nano-silver composite bacillus bacteria agent by weight, 96 parts of nano-silver composite bacillus suspensions prepared in step (2), 2.5 parts of guar gum, fatty alcohol polyoxyethylene ether (JFC) 1.5 parts, polyvinylpyrrolidone 1.5 parts, castor oil polyoxyethylene ether (EL-35) 0.3 parts, cyprodinol 1.5 parts and mix well.
  • JFC fatty alcohol polyoxyethylene ether
  • EL-35 castor oil polyoxyethylene ether
  • Table 4 shows that the rot rate of the treated fruit was only 9.76% on the 12th day.
  • the nano-silver composite bacillus bacterial agent of the present invention can reduce fruit rot.
  • Comparative example 1 Effect of different inorganic fermentation nutrient bases and organic fermentation nutrient bases on sporulation rate and bacteriostatic rate
  • Example 1 reduce the use of a raw material in the inorganic fermentation nutrient base or organic fermentation nutrient base, and use a raw material with a similar effect to replace the raw material in the present invention: see the following table for specific ingredients
  • Bacteriostatic rate (%) [(control group pathogenic fungal colony radius-treatment group pathogenic fungal colony radius)/control group pathogenic fungal colony diameter] ⁇ 100%.
  • Table 7 shows that the sporulation yield of processing 9 and treatment 10 is higher than that of Example 1, and the bacteriostasis efficiency of Processing 7 is higher than that of Example 1, but according to the sporulation yield and bacteriostasis efficiency consideration, organic fermentation nutrient base and inorganic fermentation of the present invention The combination of nutrient bases has the best performance.
  • step (3) use different raw materials to prepare film-forming agent, replace nano-silver composite spore suspension with distilled water, investigate and make film water vapor transmission rate and integrity.
  • Treatment 1 2.5 parts of guar gum, 0.8 part of fatty alcohol polyoxyethylene ether (JFC), 1 part of polyvinylpyrrolidone, 0.3 part of castor oil polyoxyethylene ether (EL-35), 94 parts of distilled water, mix uniform.
  • JFC fatty alcohol polyoxyethylene ether
  • EL-35 castor oil polyoxyethylene ether
  • Treatment 2 3 parts of guar gum, 0.8 part of fatty alcohol polyoxyethylene ether (JFC), 0.2 part of castor oil polyoxyethylene ether (EL-35), 94 parts of distilled water, and stir evenly.
  • JFC fatty alcohol polyoxyethylene ether
  • EL-35 castor oil polyoxyethylene ether
  • Treatment 3 3 parts of polyvinylpyrrolidone, 0.8 part of fatty alcohol polyoxyethylene ether (JFC), 0.2 part of castor oil polyoxyethylene ether (EL-35), 94 parts of distilled water, and stir evenly.
  • JFC fatty alcohol polyoxyethylene ether
  • EL-35 castor oil polyoxyethylene ether
  • Integrity Spray the surface of lotus mist fruit, wait for the film to dry naturally, then place the fruit on the window sill in direct sunlight, avoid biological contact with ants, insects or birds during the day, and cover it with a breathable cover at night to prevent contact with foreign objects.
  • the damage of the film was observed with a magnifying glass after 5 days. The damage is assessed in 6 grades: Grade 1, broken in more than 20 places; Grade 2, broken in more than 15 places; Grade 3, broken in more than 10 places; Grade 4, broken in more than 5 places; Grade 5, broken in less than 2 places; Grade, no damage.
  • Water vapor permeability 24M/At, the unit is g/m 2 /day.
  • M is the mass loss of the assembled disc (g)
  • A is the exposed area of the membrane (m 2 )
  • t is the time (h)
  • Integrity evaluation index 6 grades to evaluate the damage: Grade 1, broken more than 20 places; Grade 2, broken more than 15 places; Grade 3, broken more than 10 places; Grade 4, broken more than 5 places; Grade 5, broken low In 2 places; Grade 6, no damage.
  • Comparative example 3 The inhibitory effect of different bacillus and silver nitrate combined fermentation broth on the growth of pathogenic bacteria
  • Bacillus subtilis BLG010 and Bacillus amyloliquefaciens HW05 grown in the dark on Luria-Bertani (LB) medium at 28°C for 3 days were diluted with sterile distilled water to a concentration of OD 600 0.1. The experiment was divided into 6 treatments.
  • Treatment 1 Add 2ml HW05 spore suspension into 200ml liquid medium as HW05 treatment.
  • Treatment 2 Add 2ml BLG010 spore suspension into 200ml liquid medium as BLG010 treatment.
  • Treatment 3 Add 2ml HW05 spore suspension and 2ml BLG010 bacterial suspension into 200ml liquid medium as HW05+BLG010 treatment.
  • Treatment 4 Add 2ml HW05 spore suspension and 0.068g AgNO 3 into 200ml liquid medium as HW05+AgNO3 treatment.
  • Treatment 5 Add 2ml BLG010 spore suspension and 0.068g AgNO 3 into 200ml liquid medium as BLG010+AgNO3 treatment.
  • Treatment 6 Add 2ml HW05 spore suspension, 2ml BLG010 bacterial suspension and 0.068g AgNO 3 into 200ml liquid medium as HW05+BLG010+AgNO 3 treatment
  • the above-mentioned liquid culture medium was prepared according to the organic fermentation nutrient base, inorganic fermentation nutrient base and distilled water in the weight ratio of 35:15:1000 as described in Example 1, and sterilized at 121° C. for 30 minutes at high temperature. After culturing for 74 hours, centrifuge the above-mentioned fermented broth at 25° C. at 1300 rpm for 15 minutes, discard the supernatant, add 30 times distilled water to the precipitate, and make the precipitate into a suspension.
  • Pestalotiopsis versicolor and Colletotrichum fructicola cultured on the PDA plate for 5 days were respectively punched with a 5mm diameter hole puncher to take the part of the outermost circle of the colony that grew uniformly.
  • Cakes were inoculated in the center of the PDA plate, and incubated in the dark at 28°C for 24 hours. After culturing for 24 hours, four holes were punched at the periphery of the pathogenic bacteria colony at equal distances with a puncher with a diameter of 5 mm, and 100 ⁇ l of fermentation broth from treatment 1 to treatment 6 were added to each hole (the bacterial suspension was at least 0.2 cm below the medium plane.
  • Bacteriostatic rate (%) [(control group pathogenic fungus colony radius-treatment group pathogenic fungus colony radius)/control group pathogenic fungus colony diameter] ⁇ 100%
  • the fermentation liquid obtained by co-fermentation of single or compound Bacillus plus silver nitrate has a higher growth inhibition rate of pathogenic bacteria than that of single or compound Bacillus fermentation liquid, which shows that nano-silver converted from silver nitrate can enhance the antibacterial ability of the whole fermentation liquid .

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Abstract

本发明提供一种防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂及其制作方法，该菌剂包括纳米银复合芽孢菌菌悬液，纳米银复合芽孢菌菌悬液包括以下重量份的原料：枯草芽孢杆菌1～3份、解淀粉芽孢杆菌0.5～1.5份；纳米银复合芽孢菌菌悬液使用硝酸银溶液调节浓度为2-3mmol/L。本发明菌剂同时有效抑制异色拟盘多毛孢菌和果生刺盘孢菌，该菌剂具有成膜能力，喷施后能在果实表面形成保护膜，该膜能保持菌剂中的复合芽孢杆菌及纳米银长期附着在果实表面。

Description

一种防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂及其制作方法 技术领域
本发明涉及防治果实拟盘多毛孢果腐病及炭疽病菌剂领域，特别涉及一种防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂及其制作方法。
背景技术
莲雾果实拟盘多毛孢果腐病及炭疽病是莲雾生产中常见且引起重大损失的病害，均能引起莲雾落果和果腐病。莲雾果实拟盘多毛孢果腐病由异色拟盘多毛孢(Pestalotiopsis versicolor)引起，莲雾果实炭疽病由果生刺盘孢菌(Colletotrichum fructicola)引起。由于莲雾果期适逢高温多雨季节，导致这两种病害蔓延发生发展迅速，造成农户在生产过程中过量使用化学农药，容易导致环境污染和造成食品中毒。目前，使用生物防治植物病害是大势所趋，生物防治是生态友好型防治手段，能为农业的可持续发展提供保障。
芽孢杆菌具有产生拮抗物质、和病原菌竞争营养和空间、诱导植物产生抗性等生防机制，是目前广泛应用的生防菌，具有优良的拮抗植物病原菌和诱导植物产生抗病性的能力，已经成功的用于防治多种植物的细菌和真菌病害。有研究表明，利用具有协同作用的两种或者多种生防菌防治病害比用单一菌的防治效果要高。在防治病害时菌与菌之间的协同作用机制可能是，具有不同生防机制(竞争、产生拮抗物质、寄生/重寄生)的菌防治病害机制互补。同时，不同种类的生防菌产生的拮抗物质互补，不同种类菌株在共同存在条件下诱导产生新的抗性物质。目前，利用两种或者多种生防菌复合防治植物病害已经成为生物防治领域的热点研究方向。然而，不是菌株间的随意组合就能产生协同作用，多数菌种之间具有相互拮抗，不能达到菌株间的协同增效效果，反而会导致微生物的生防功能降低甚至菌株失活。
纳米银具有广谱抗菌能力，能穿透微生物的细胞壁进入体内，对众多病原细菌和真菌有较好的杀灭能力，并且具有较高的价态和巨大的比表面积，在空气中不易被氧化和沉淀，已经用于抗菌产品中，如纳米银抗菌纱布，纳米银创伤贴和纳米银涂料。纳米银的物理和化学合成成本高和产生有毒物质，而生物合成纳米银价格低廉、安全和环境友好，具有可持续发展的特征，逐渐成为纳米银合成领域的研究热点。微生物代谢物可作为还原剂和稳定剂生物合成纳米银，但由于纳米银的广谱抗菌性，限制了菌种来源和生产效率。
环丙嘧啶醇是一类新型的用于提高植物叶绿素含量和促进植物生长的植物生长调节剂，但是未见报道其有提高植物抗病能力。本专利发现加入环丙嘧啶醇的复合芽孢杆菌发酵液能极大的提高果实抗性相关酶和增加抗病能力。
现有技术中也有利用枯草芽孢杆菌防治莲雾病害的报道，例如CN104672006A一种莲雾专用药肥，包括以下组分：18‐22份牛粪、15‐18份鸡粪、20‐40份稻草、10‐20份豆饼、10‐20份蔗渣、40‐50份尿素、30‐40份过磷酸钙、30‐40份硫酸钾、2‐5份微量元素、3‐5份复合氨基酸、15‐25份中草药提取液、1‐2份枯草芽孢杆菌、0.6‐1份球毛壳菌。该专利使用枯草芽孢杆菌和球毛壳菌相互作用进行防治各种病害。但是未记载该专用药肥使用后的防治效果。CN101748078B新颖液化淀粉芽孢杆菌菌株及其应用，液化淀粉芽孢杆菌具有抑制莲雾果腐菌的作用，但是未见记载使用液化淀粉芽孢杆菌进行抑制果腐病的技术水平。
发明内容
鉴以此，本发明提出一种防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂及其制作方法。
本发明的技术方案是这样实现的：
(1)微生物菌种子液的制备：将解淀粉芽孢杆菌HW05在LB液体培养基(每升含：胰蛋白胨10g，酵母提取物5g，NaCl 10g)中，于37℃，180rpm的条件下进行小规模培养24h，生产解淀粉芽孢杆菌HW05种子液；枯草芽孢杆菌BLG010在LB液体培养基(每升含：胰蛋白胨10g，酵母提取物5g，NaCl 10g)中，于37℃，180rpm的条件下进行小规模培养24h，生产枯草芽孢杆菌BLG010种子液；
(2)纳米银复合芽孢杆菌菌悬液的制备：按重量份计，在立式发酵罐中加入有机发酵营养基30～40份、无机发酵营养基10～20份、蒸馏水950～1050份，搅拌均匀后经高压蒸汽灭菌温度为121℃，时间为30min，压力为100kPa，在温度降至37℃以下后，按重量份计，加入0.5～1.5份解淀粉芽孢杆菌HW05种子液，6小时后加入1～3份枯草芽胞杆菌BLG010种子液，通入无菌空气，通气量为16～19L/min，保压0.02～0.04MPa，恒温34～38℃，转速170～200rpm，8小时后加入硝酸银溶液调节浓度为2-3mmol/L，每升纳米银复合芽孢杆菌菌悬液当中含复合芽孢杆菌2～3mmol，经过70～80h的发酵时间，当孢子浓度达到8.0×10 8个/mL～1×10 9个/mL且纳米银产率>80％时停止发酵。将温度提高至55-65℃，转速110～140rpm，保持4～6h，促 进芽孢杆菌形成芽胞，增强其抗逆性。在20～30℃进行离心，离心参数为1200-1400rpm，时间为10-12min，弃去上清液，按重量比计，加入蒸馏水和沉淀重量比为25～35:1，获得含纳米银的菌悬液。
(3)纳米银复合芽孢杆菌菌剂的制备：按重量份计，将步骤(2)制得的纳米银复合芽孢菌菌悬液94-96份，瓜尔豆胶2-2.5份，脂肪醇聚氧乙烯醚(JFC)0.8-1.5份，聚乙烯吡咯烷酮1-1.5份，蓖麻油聚氧乙烯醚(EL-35)0.2-0.3份，环丙嘧啶醇0.8-1.5份混合均匀。
进一步的，步骤(1)中，所述解淀粉芽孢杆菌由中国热带农业科学院环境与植物保护研究所提供，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.10273，菌株号HW05，其性状参见专利CN108690821A。
进一步的，步骤(1)中，所述枯草芽孢杆菌由中国热带农业科学院环境与植物保护研究所提供，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.5953，菌株号BLG010，其性状参见专利CN102747020A。
进一步的，步骤(2)中，所述纳米银复合芽孢杆菌菌悬液的制备：按重量份计，在立式发酵罐中加入有机发酵营养基35份、无机发酵营养基15份、蒸馏水1000份，搅拌均匀后经高压蒸汽灭菌温度为121℃，时间为30min，压力为100kPa，在温度降至37℃以下后，按重量份计，加入1份解淀粉芽孢杆菌HW05种子液，6小时后加入2份枯草芽胞杆菌BLG010种子液，通入无菌空气，通气量为18L/min，保压0.03MPa，恒温36℃，转速185rpm，8小时后加入硝酸银溶液至终浓度为2.5mmol/L，经过75h的发酵时间，当孢子浓度达到9.0×10 8个/mL且纳米银产率＝85％时停止发酵。将温度提高至60℃，转速125rpm，保持5h。在25℃进行离心，离心参数为1300rpm，时间为11min，弃去上清液，按重量比计，加入蒸馏水与沉淀的重量比为30:1。
进一步的，步骤(2)中，所述有机发酵营养基按重量份由以下原料组成：木薯粉6-8份，豆粕5-7份，氨基酸4-5份，酵母粉3-4.5份，海藻精3-6份，鱼蛋白粉5-8份，红糖5-7.5份，混合均匀，打碎过80目筛。
进一步的，步骤(2)中，所述有机发酵营养基按重量份由以下原料组成：木薯粉7份，豆粕6份，氨基酸4.5份，酵母粉3.75份，海藻精4.5份，鱼蛋白粉6.5份，红糖6.25份，混合均匀，打碎过80目筛。
进一步的，步骤(2)中，所述无机发酵营养基按重量份由以下原料组成：磷酸二铵10-15份、硝酸钾1-2份，硫酸锌3-5份、硫酸镁2-3份、钼酸铵0.05-0.1份，氯化钠0.5-1份。
进一步的，步骤(2)中，所述无机发酵营养基按重量份由以下原料组成：磷酸二铵12.5份、硝酸钾1.5份，硫酸锌4份、硫酸镁2.5份、钼酸铵0.075份，氯化钠0.75份。
进一步的，步骤(3)中，所述纳米银复合芽孢杆菌菌剂的制备：按重量份计，将步骤(2)制得的纳米银复合芽孢菌菌悬液95份，瓜尔豆胶2.25份，脂肪醇聚氧乙烯醚(JFC)1.15份，聚乙烯吡咯烷酮1.25份，蓖麻油聚氧乙烯醚(EL-35)0.25份，环丙嘧啶醇1.15份混合均匀。
进一步的，步骤(3)中，整个配置过程在温度60℃条件下进行。
与现有技术相比，本发明的有益效果是：
1、本发明防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂不仅能够有效同时抑制莲雾异色拟盘多毛孢菌和果生刺盘孢菌，而且促进莲雾生长；硝酸银溶液和环丙嘧啶醇联合使用能够促进莲雾生长和提高菌剂的抑菌效果；成膜剂不仅能有效阻止外界病原菌对果实的侵染，而且在果实表面形成膜后能持续为芽孢杆菌提供营养，提高芽孢杆菌在果实表面的宿存能力，延长抑菌功能，进而使本发明纳米银复合芽孢杆菌菌剂达到防治莲雾果实拟盘多毛孢果腐病及炭疽病的效果。
2、由于莲雾果实病害发生严重，导致生产中大量使用农药，造成生态环境恶化和食品安全等不良后果。使用本发明的纳米银复合芽孢杆菌菌剂能解决滥用化学农药造成的弊病，能让农户生产出无公害的绿色食品。
附图说明
图1实施例1纳米银复合芽孢杆菌菌剂防治果实腐烂；
图2实施例1纳米银复合芽孢杆菌菌剂对果实过氧化氢酶(CAT)活性影响；
图3实施例1纳米银复合芽孢杆菌菌剂对果实丙二醛(MDA)含量影响；
图4实施例1纳米银复合芽孢杆菌菌剂对果实花青苷含量影响。
具体实施方式
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。
本发明实施例所用的实验方法如无特殊说明，均为常规方法。
本发明实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
1、实验过程
(1)微生物菌种子液的制备：将解淀粉芽孢杆菌HW05在LB液体培养基(每升含：胰蛋白胨10g，酵母提取物5g，NaCl 10g)中，于37℃，180rpm的条件下进行小规模培养24h，生产HW05种子液；枯草芽孢杆菌BLG010种子液生产方式同上述解淀粉芽孢杆菌。
(2)有机发酵营养基：按重量份计由以下原料组成：木薯粉7份，豆粕6份，氨基酸4.5份，酵母粉3.75份，海藻精4.5份，鱼蛋白粉6.5份，红糖6.25份，混合均匀，打碎过80目筛，制得。
(3)无机发酵营养基：按重量份计，由以下原料组成：磷酸二铵12.5份、硝酸钾1.5份，硫酸锌4份、硫酸镁2.5份、钼酸铵0.075份，氯化钠0.75份混合均匀，制得。
(4)纳米银复合芽孢杆菌菌悬液的制备：按重量份计，在立式发酵罐中按重量份加入有机发酵营养基35份、无机发酵营养基15份、蒸馏水1000份搅拌均匀，经高压蒸汽灭菌温度为121℃，时间为30min，压力为100kPa，在温度降至37℃以下后，按重量份计，加入1份解淀粉芽孢杆菌HW05种子液，6小时后加入2份枯草芽胞杆菌BLG010种子液，通入无菌空气，通气量为17L/min保压0.03MPa，恒温36℃，转速185rpm，8小时后加入硝酸银溶液调节浓度为2.5mmol/L，经过75h的发酵时间，当孢子浓度达到9.0×10 8个/m且纳米银产率＝85％时停止发酵。将温度提高至60℃，转速125rpm，保持5h，在25℃进行离心，离心参数为1300rpm，时间为11min，弃去上清液，按重量比计，加入蒸馏水与沉淀的重量比为30：1，溶解沉淀。
(5)纳米银复合芽孢杆菌菌剂的制备：按重量份计，将步骤(2)制得的纳米银复合芽孢菌菌悬液95份，瓜尔豆胶2.25份，脂肪醇聚氧乙烯醚(JFC)1.15份，聚乙烯吡咯烷酮1.25份，蓖麻油聚氧乙烯醚(EL-35)0.25份，环丙嘧啶醇1.15份混合均匀。
2、检测方法
(1)纳米银复合芽孢杆菌菌剂防治果实腐烂：果实均在果实蒂部(最易发病部位)滴0.1ml异色拟盘多毛孢(Pestalotiopsis versicolor)和果生刺盘孢菌(Colletotrichum fructicola)混合孢子液(每种病原菌的孢子浓度均为1×10 3/ml)。对照(CK)果实仅接种病原菌，处理果实接种病原菌后用500倍本专利发明的纳米银复合芽孢杆菌菌剂喷施全果。每个重复含4个果实，对照(CK)和处理各3个重复。试验用果实均为同个果园同一批采摘且成熟度一致的表面无损伤果实，试验果实放置在20℃，湿度为85％，12小时光照12小时黑暗的培养条件中。
(2)纳米银复合芽孢杆菌菌剂对果实过氧化氢酶(CAT)活性影响：CAT可将过氧化氢(H 2O 2)还原为水(H 2O)和氧气(O 2)，从而减少活性氧对植物的危害。测定过氧化氢酶(CAT)的试验果实分为CK和处理。果实表面均用异色拟盘多毛孢(Pestalotiopsis versicolor)和果生刺盘孢菌(Colletotrichum fructicola)混合孢子液喷施(每种病原菌的孢子浓度均为1×10 3/ml)。CK果实仅接种病原菌，处理果实接种病原菌后用500倍本专利发明的纳米银复合芽孢杆菌菌剂喷施全果。试验用果实均为同个果园同一批采摘且成熟度一致的表面无损伤果实，试验果实放置在20℃，湿度为85％，12小时光照12小时黑暗的培养条件中。CK和处理果实均为50个果实。每次测定CAT均随机取三份样品进行试验，每份样品进行三次平行试验
(3)纳米银复合芽孢杆菌菌剂对果实丙二醛(MDA)含量影响：丙二醛(MDA)是细胞膜脂过氧化产物之一。逆境情况下植物MDA含量增高，所以利用它的含量间接地反映植物细胞受损程度。每次测定MDA均随机取三份样品进行试验，每份样品进行三次平行试验，果实处理方式同实施例1检测方式(2)。试验用果实均为同个果园同一批采摘且成熟度一致的表面无损伤果实，试验果实放置在20℃，湿度为85％，12小时光照12小时黑暗的培养条件中。
(4)纳米银复合芽孢杆菌菌剂对果实花青苷含量影响：测定花青苷均随机取三份样品进行试验，每份样品进行三次平行试验，果实处理方式同实施例1检测方式(2)。试验用果实均为同个果园同一批采摘且成熟度一致的表面无损伤果实，试验果实放置在20℃，湿度为85％，12小时光照12小时黑暗的培养条件中。
(5)纳米银复合芽孢杆菌菌剂对果实表皮真菌种类的影响：测定表皮真菌的果实均不接种病原菌，试验果实分为CK和处理。CK果实不做如何处理，处理果实用500倍本专利发明的纳米银复合芽孢杆菌菌剂喷施全果。检测表皮真菌的CK和处理果实均为9个。
(6)纳米银复合芽孢杆菌菌剂对果实腐烂率的影响：果实均不接种病原菌，试验果实分为CK和处理。CK果实不做如何处理，处理果实用500倍本专利发明的纳米银复合芽孢杆菌菌剂喷施全果。测定腐烂率的CK和处理果实均为60个。腐烂部位大于整果10％以上记为腐烂，腐烂率＝腐烂果实/60*100％。试验用果实均为同个果园同一批采摘且成熟度一致的表面无损伤果实，试验果实放置在20℃，湿度为85％，12小时光照12小时黑暗的培养条件中。
3、实验结果参见图1至图4及实验分析
(1)(图1)表明，接种病原菌5天后，CK果实蒂部出现腐烂，7天后产生子实体。而喷施纳米银复合芽孢杆菌菌剂的果实无发病症状。结果说明，喷施纳米银复合芽孢杆菌菌剂能有效避免由异色拟盘多毛孢(Pestalotiopsis versicolor)和果生刺盘孢菌(Colletotrichum fructicola)造成的果实腐烂。
(2)(图2)表明，纳米银复合芽孢杆菌菌剂在第6天后能明显降低果实的CAT酶活性，这可能的原因是，喷施菌剂造成果实避免遭受病原菌侵染，由此导致其体内的H 2O 2含量比CK果实的少，进而调控CAT酶活性下降。
(3)(图3)表明，纳米银复合芽孢杆菌菌剂能明显降低果实的MDA含量，这可能的原因是，喷施菌剂造成果实避免遭受病原菌侵染，从而保护果实细胞的质膜结构和功能。
(4)(图4)表明，纳米银复合芽孢杆菌菌剂能减弱果实花青苷含量下降，从而减缓果皮颜色变暗。
表1 莲雾果实存储10d后表皮真菌种类
处理果实 CK果实
Aspergillus spp. Alternaria spp.
Cladosporium spp. Aspergillus spp.
Penicillium spp. Cladosporium spp.
  Colletotrichum spp.
  Diaporthe spp.
  Fusarium spp.
  Lasiodiplodia spp.
  mucor spp.
  Penicillium spp.
  Pestalotiopsis spp.
(5)表1表明，喷施本专利发明的纳米银复合芽孢杆菌菌剂10天后，果皮表面仅分离出3类真菌，而CK果皮表面分离出10种真菌。由此说明，喷施纳米银复合芽孢杆菌菌剂可明显减少果皮表面真菌，减低病原菌对果实的侵染概率。
表2 纳米银复合芽孢杆菌菌剂对果实腐烂率的影响
  3d 6d 9d 12d
处理果实 0 0 3.33％ 8.33％
CK果实 0 0 41.67％ 68.33％
(6)表2表明，CK果实在第9天时开始大规模腐烂，而处理果实在第12天时腐烂率仅为8.33％。本发明纳米银复合芽孢杆菌菌剂能减少果实腐烂。
实施例2
1、实验过程
(1)微生物菌种子液的制备：将解淀粉芽孢杆菌HW05在LB液体培养基(每升含：胰蛋白胨10g，酵母提取物5g，NaCl 10g)中，于37℃，180rpm的条件下进行小规模培养24h，生产HW05种子液；枯草芽孢杆菌BLG010种子液生产方式同上述解淀粉芽孢杆菌。
(2)有机发酵营养基：按重量份计，由以下原料组成：木薯粉6份，豆粕5份，氨基酸4份，酵母粉3份，海藻精3份，鱼蛋白粉5份，红糖5份，混合均匀，打碎过80目筛，制得。
(3)无机发酵营养基：按重量份计，由以下原料组成：磷酸二铵10份、硝酸钾1份，硫酸锌3份、硫酸镁2份、钼酸铵0.05份，氯化钠0.5份，混合均匀，制得。
(4)纳米银复合芽孢杆菌菌悬液的制备：按重量份计，在立式发酵罐中按重量份加入有机发酵营养基30份、无机发酵营养基10份、蒸馏水950份搅拌均匀，经高温蒸汽灭菌温度为121℃，时间为30min，压力为100kPa，在温度降至37℃以下后，按重量份计，加入0.5份解淀粉芽孢杆菌HW05种子液，6小时后加入1份枯草芽胞杆菌BLG010种子液，通入无菌空气，通气量为16L/min保压0.02MPa，恒温34℃，转速为170rpm，8小时后加入硝酸银溶液调节浓度为2mmol/L，经过70h的发酵时间，当孢子浓度达到8.0×10 8个/m且纳米银产率＝85％时停止发酵。将温度提高至55℃，转速110rpm，保持4h，促进芽孢杆菌形成芽胞，增强其抗逆性。在25℃进行离心，离心参数为1200rpm，时间为10min，弃去上清液，按重量比计，加入蒸馏水与沉淀的重量比为25:1，溶解沉淀。
(5)纳米银复合芽孢杆菌菌剂的制备：按重量份计，将步骤(2)制得的纳米银复合芽孢菌菌悬液94份，瓜尔豆胶2份，脂肪醇聚氧乙烯醚(JFC)0.8份，聚乙烯吡咯烷酮1份，蓖麻油聚氧乙烯醚(EL-35)0.2份，环丙嘧啶醇0.8份混合均匀。
2、检测方法
同实施例1中检测方法(6)
3、实验结果
表3 纳米银复合芽孢杆菌菌剂对果实腐烂率的影响
  3d 6d 9d 12d
处理果实 0 0 4.05％ 9.02％
表3表明，处理果实在第12天时腐烂率仅为9.02％，本发明纳米银复合芽孢杆菌菌剂能减少果实腐烂。
实施例3
1、实验过程
(1)微生物菌种子液的制备：将解淀粉芽孢杆菌HW05在LB液体培养基每升含：胰蛋白胨10g，酵母提取物5g，NaCl 10g中，于37℃，180rpm的条件下进行小规模培养24h，生产HW05种子液；枯草芽孢杆菌BLG010种子液生产方式同上述解淀粉芽孢杆菌。
(2)有机发酵营养基：按重量份计，由以下原料组成：木薯粉8份，豆粕7份，氨基酸5份，酵母粉4.5份，海藻精6份，鱼蛋白粉8份，红糖7.5份，混合均匀，打碎过80目筛，制得。
(3)无机发酵营养基：按重量份计，由以下原料组成：磷酸二铵15份、硝酸钾2份，硫酸锌5份、硫酸镁3份、钼酸铵0.1份，氯化钠1份，混合均匀，制得。
(4)纳米银复合芽孢杆菌菌悬液的制备：按重量份计，在立式发酵罐中按重量份加入有机发酵营养基40份、无机发酵营养基20份、蒸馏水1050份搅拌均匀，经高温蒸汽灭菌温度为121℃，时间为30min，压力为100kPa，在温度降至37℃以下后，按重量份计，加入1.5份解淀粉芽孢杆菌HW05种子液，6小时后加入3份枯草芽胞杆菌BLG010种子液，通入无菌空气，通气量为19L/min保压0.04MPa，恒温38℃，8小时后加入硝酸银溶液调节浓度为3mmol/L，经过80h的发酵时间，当孢子浓度达到1×10 9个/m且纳米银产率＝85％时停止发酵。将温度提高至65℃，转速140rpm，保持6h，促进芽孢杆菌形成芽胞，增强其抗逆性。在30℃进行离心，离心参数为1400rpm，时间为12min，弃去上清液，按重量比计，加入蒸馏水与沉淀的重量比为35:1，溶解沉淀。
(5)纳米银复合芽孢杆菌菌剂的制备：按重量份计，将步骤(2)制得的纳米银复合芽孢菌菌悬液96份，瓜尔豆胶2.5份，脂肪醇聚氧乙烯醚(JFC)1.5份，聚乙烯吡咯烷酮1.5份，蓖麻油聚氧乙烯醚(EL-35)0.3份，环丙嘧啶醇1.5份混合均匀。
2、检测方法
同实施例1中检测方法(6)
3、实验结果
表4 纳米银复合芽孢杆菌菌剂对果实腐烂率的影响
  3d 6d 9d 12d
处理果实 0 0 4.09％ 9.76％
表4表明，处理果实在第12天时腐烂率仅为9.76％。本发明纳米银复合芽孢杆菌菌剂能减少果实腐烂。
对比例1 不同无机发酵营养基、有机发酵营养基对产孢率和抑菌率影响
1、实验过程
在实施例1基础上，减少无机发酵营养基或有机发酵营养基中一种原料的使用，使用作用相近的原料代替本发明中原料：具体配料见下表
表5 对无机发酵营养基进行调整
Figure PCTCN2021122000-appb-000001
表6 对有机发酵营养基进行调整
Figure PCTCN2021122000-appb-000002
2、检测方法
将在PDA平板上培养5天的果生刺盘孢菌用直径5mm打孔器取菌落最外圈生长一致的部分打制菌饼，接种在PDA平板中央，28℃暗培养1天。在培养1天后，病原菌菌落外围位置用直径5mm的打孔器等距离打4个孔，将处理1至处理16的100μl发酵液分别加入各个孔(菌悬液低于培养基平面至少0.2cm)，在第5天时测量各处理菌落半径，计算抑制率。抑菌率(％)＝[(对照组病原真菌菌落半径一处理组病原真菌菌落半径)/对照组病原真菌菌落直径]×100％。
3、实验结果
表7 不同处理产孢量与抑菌效果
Figure PCTCN2021122000-appb-000003
表7表明处理9和处理10的产孢量比实施例1高，处理7的抑菌效率比实施例1高，但是根据产孢量和抑菌效率考虑，本发明有机发酵营养基和无机发酵营养基搭配表现效果最优。
对比例2 使用不同原料制备成膜剂
在实施例2的基础上，步骤(3)中，使用不同原料制备成膜剂，将纳米银复合芽孢菌菌 悬液替换为蒸馏水，考察制得膜水蒸气透过率和完整性。
1、实验过程
(1)处理1：瓜尔豆胶2.5份，脂肪醇聚氧乙烯醚(JFC)0.8份，聚乙烯吡咯烷酮1份，蓖麻油聚氧乙烯醚(EL-35)0.3份、蒸馏水94份，混合均匀。
(2)处理2：瓜尔豆胶3份、脂肪醇聚氧乙烯醚(JFC)0.8份、蓖麻油聚氧乙烯醚(EL-35)0.2份、蒸馏水94份，搅拌均匀。
(3)处理3：聚乙烯吡咯烷酮3份、脂肪醇聚氧乙烯醚(JFC)0.8份、蓖麻油聚氧乙烯醚(EL-35)0.2份、蒸馏水94份，搅拌均匀。
2、检测方式
水蒸气透过率：在室温下将5mL的蒸馏水放入一个内径为32mm的玻璃培养皿中，然后用不同处理获得的薄膜覆盖培养皿。将处理好的培养皿称重，并固定在低转速平底摇床上，24h后，重新称重培养皿。根据以下等式计算水蒸气渗透率＝24M/At，单位为g/m 2/day。其中M为组装盘的质量损失(g)，A为膜的暴露面积(m 2)，t为时间(h)。
完整性：喷涂莲雾果实表面，等膜自然风干后，将果实放置于窗台日光直射处，白天避免蚂蚁、虫或鸟类等生物接触，夜晚用透气罩覆盖防治外物接触。考虑到常温下离体果实失水严重，故5天后用放大镜观察膜的破损情况。分6个等级评估破损情况：1级，破碎超过20处；2级，破碎超过15处；3级，破碎超过10处；4级，破碎超过5处；5级，破碎低于2处；6级，无破损。
3、实验结果
水蒸气渗透率＝24M/At，单位为g/m 2/day。其中M为组装盘的质量损失(g)，A为膜的暴露面积(m 2)，t为时间(h)
完整性评价指标：分6个等级评估破损情况：1级，破碎超过20处；2级，破碎超过15处；3级，破碎超过10处；4级，破碎超过5处；5级，破碎低于2处；6级，无破损。
表8 不同配方膜的水蒸气透过率和完整性
名称 水蒸气透过率 膜完整性
处理1 420.21(±20.34) 6级
处理2 510.8(±11.58) 2级
处理3 492.54(±10.29) 4级
实验结果表明(表8)，本发明制得的成膜剂水蒸气透过率低于450，这表明该处理的膜能有效阻止水分从果实分向环境的转移。并且，处理1在5天后仍无观察到破碎。
对比例3 不同芽孢杆菌及硝酸银组合发酵液对病原菌生长的抑制效果
将在28℃，Luria-Bertani(LB)培养基上黑暗培养3天的枯草芽孢杆菌BLG010和解淀粉芽孢杆菌HW05，用无菌蒸馏水稀释成浓度为OD 600＝0.1。试验分为6个处理。
1、实验过程：
处理1：将2ml HW05孢子悬浮液加入含200ml液体培养基中作为HW05处理。
处理2：将2ml BLG010孢子悬浮液加入含200ml液体培养基中作为BLG010处理。
处理3：将2ml HW05孢子悬浮液和2ml BLG010菌悬液加入含200ml液体培养基中作为HW05+BLG010处理。
处理4：将2ml HW05孢子悬浮液和0.068g AgNO 3加入含200ml液体培养基中作为HW05+AgNO3处理。
处理5：将2ml BLG010孢子悬浮液和0.068g AgNO 3加入含200ml液体培养基中作为BLG010+AgNO3处理。
处理6：将2ml HW05孢子悬浮液和2ml BLG010菌悬液和0.068g AgNO 3加入含200ml液体培养基中作为HW05+BLG010+AgNO 3处理
(2)上述液体培养基按实施例1中所述有机发酵营养基、无机发酵营养基、蒸馏水按重量份比35：15：1000配置，121℃，30分钟高温灭菌。培养74小时后，将上述处理发酵液在25℃1300rpm条件下离心15min，弃上清液，将沉淀加入30倍的蒸馏水，将沉淀制成悬浮液。
2、检测方式
将在PDA平板上培养5天的异色拟盘多毛孢菌(Pestalotiopsis versicolor)和果生刺盘孢菌(Colletotrichum fructicola)分别用直径5mm打孔器取菌落最外圈生长一致的部分打制菌饼，分别接种在PDA平板中央，28℃暗培养24小时。在培养24小时后，病原菌菌落外围位置用直径5mm的打孔器等距离打4个孔，将处理1至处理6的100μl发酵液分别加入各个孔(菌悬液低于培养基平面至少0.2cm)，第3和第5天时测量各处理菌落半径，计算抑制率。抑菌率(％)＝[(对照组病原真菌菌落半径一处理组病原真菌菌落半径)/对照组病原真菌菌落直径]×100％
3、实验结果
表9 不同芽孢杆菌及硝酸银组合发酵液对病原菌生长的抑制效果(％)
Figure PCTCN2021122000-appb-000004
结果表明(表9)，复合芽孢杆菌的发酵液，比单个芽孢杆菌的发酵液对病原菌的菌丝生长抑制率高。类似的，复合芽孢杆菌加硝酸银的发酵液对病原菌的生长抑制率，比单个芽孢杆菌的发酵液高。在6种处理中，HW05+BLG010+AgNO 3处理的发酵液抑制病原菌生长能力最高，在第5天时对异色拟盘多毛孢(Pestalotiopsisversicolor)的抑制率达到50.81％，对果生刺盘孢菌(Colletotrichumfructicola)的抑制率达到57.9％。单独或者复合芽孢杆菌加硝酸银共同发酵获得的发酵液，比单独或者复合芽孢杆菌发酵液对病原菌的生长抑制率高，这说明由硝酸银转化得到的纳米银可以增强整体发酵液的抑菌能力。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂，其特征在于，包括纳米银复合芽孢菌菌悬液，所述纳米银复合芽孢菌菌悬液包括以下重量份的原料：枯草芽孢杆菌1～3份、解淀粉芽孢杆菌0.5～1.5份；所述纳米银复合芽孢菌菌悬液使用硝酸银溶液调节浓度为2-3mmol/L。
  2. 如权利要求1所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂，其特征在于，包括以下重量份的原料：纳米银复合芽孢菌菌悬液94-96份，瓜尔豆胶2-2.5份，脂肪醇聚氧乙烯醚0.8-1.5份，聚乙烯吡咯烷酮1-1.5份，蓖麻油聚氧乙烯醚0.2-0.3份，环丙嘧啶醇0.8-1.5份。
  3. 如权利要求1所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂，其特征在于，所述纳米银复合芽孢菌菌悬液包括以下重量份的原料：有机发酵营养基30～40份、无机发酵营养基10～20份、蒸馏水950～1050份。
  4. 如权利要求1所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂，其特征在于，含有有机发酵营养基，所述有机发酵营养基按重量份包括以下原料组成：木薯粉6-8份、豆粕5-7份、氨基酸4-5份、酵母粉3-4.5份、海藻精3-6份、鱼蛋白粉5-8份、红糖5-7.5份。
  5. 如权利要求1所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂，其特征在于，含有无机发酵营养基，所述无机发酵营养基按重量份包括以下原料组成：磷酸二铵10-15份、硝酸钾1-2份、硫酸锌3-5份、硫酸镁2-3份、钼酸铵0.05-0.1份、氯化钠0.5-1份。
  6. 如权利要求1所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂，其特征在于，所述解淀粉芽孢杆菌(Bacillus amyloliquefaciens)由中国热带农业科学院环境与植物保护研究所提供，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.10273，菌株号HW05。
  7. 如权利要求1所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂，其特征在于，所述枯草芽孢杆菌(Bacillus subtilis)由中国热带农业科学院环境与植物保护研究所提供，保藏于中国微生物菌种保藏管理委 员会普通微生物中心，保藏编号为CGMCC No.5953，菌株号BLG010。
  8. 如权利要求1所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂的制备方法，其特征在于，包括以下步骤：
    (1)微生物菌种子液的制备：将解淀粉芽孢杆菌HW05在LB液体培养基中，于37℃，180rpm的条件下进行培养24h，生产解淀粉芽孢杆菌HW05种子液；将枯草芽孢杆菌BLG010在LB液体培养基中，于37℃，180rpm的条件下进行培养24h，生产枯草芽孢杆菌BLG010种子液；
    (2)纳米银复合芽孢杆菌菌悬液的制备：按重量份计，在立式发酵罐中加入有机发酵营养基、无机发酵营养基、蒸馏水，搅拌均匀后经高压蒸汽灭菌，在温度降至37℃以下后，加入解淀粉芽孢杆菌HW05种子液，6小时后加入枯草芽胞杆菌BLG010种子液，通入无菌空气，通气量为16～19L/min，保压0.02～0.04MPa，恒温34～38℃，转速170～200rpm，8小时后加入硝酸银溶液，经过70～80h的发酵时间，当孢子浓度达到8.0×10 8个/mL～1×10 9个/mL且纳米银产率>80％时停止发酵；将温度提高至55-65℃，转速110～140rpm，保持4～6h；在20～30℃进行离心，离心参数为1200-1400rpm，时间为10-12min，弃去上清液，按重量比计，加入蒸馏水和沉淀重量比为25～35:1，获得纳米银复合芽孢杆菌菌悬液；
    (3)纳米银复合芽孢杆菌菌剂的制备：按重量份计，将步骤(2)制得的纳米银复合芽孢菌菌悬液与瓜尔豆胶、脂肪醇聚氧乙烯醚、聚乙烯吡咯烷酮、蓖麻油聚氧乙烯醚、环丙嘧啶醇混合均匀，获得纳米银复合芽孢杆菌菌剂。
  9. 如权利要求8所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂的制备方法，其特征在于，步骤(3)整个配置过程在温度60℃条件下进行。
  10. 如权利要求1所述的防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂，其特征在于，纳米银复合芽孢杆菌菌剂用水稀释500-600倍后使用，在莲雾果实表面喷施使用。
PCT/CN2021/122000 2021-06-30 2021-09-30 一种防治莲雾果实拟盘多毛孢果腐病及炭疽病的纳米银复合芽孢杆菌菌剂及其制作方法 WO2023272996A1 (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117695428A (zh) * 2023-10-17 2024-03-15 山东威高新生医疗器械有限公司 一种可以接触创面的医疗级硅橡胶
CN117981757A (zh) * 2024-04-02 2024-05-07 中国农业科学院作物科学研究所 一种喷施处理提高小麦抗假禾谷镰刀菌引起的茎基腐病害的组合物及方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117397698A (zh) * 2023-12-15 2024-01-16 中国农业大学 一种含纳米银颗粒木醋液抑菌剂及其制备方法和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102747020A (zh) * 2012-07-18 2012-10-24 中国热带农业科学院环境与植物保护研究所 一株枯草芽孢杆菌及其应用
CN102888428A (zh) * 2011-07-21 2013-01-23 中国科学院过程工程研究所 一种利用解淀粉芽胞杆菌lsse-62合成纳米银的方法
CN103274825A (zh) * 2013-06-09 2013-09-04 苏州仁成生物科技有限公司 一种防治芹菜软腐病的多元微生物专用肥及其制备方法
CN107960103A (zh) * 2014-12-29 2018-04-24 Fmc有限公司 用于促进植物生长和治疗植物疾病的微生物组合物和使用方法
CN108690821A (zh) * 2017-12-07 2018-10-23 中国热带农业科学院环境与植物保护研究所 一株高效解淀粉芽孢杆菌及其菌剂和应用

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101254017A (zh) * 2007-03-02 2008-09-03 钟燿年 包含纳米银的保鲜用品及其制备方法与用途
EP2920273A1 (en) * 2012-11-15 2015-09-23 BASF Corporation Mulch and potting soil compositions containing microorganisms and related methods
CN106367367A (zh) * 2016-08-29 2017-02-01 上海诺同农业科技有限公司 一种生防性复合微生物菌液、复合微生物肥料及生产方法
CN107881129B (zh) * 2017-11-06 2020-11-10 安徽六国化工股份有限公司 一株解淀粉芽孢杆菌及其菌剂、菌剂制备方法与应用
CN108034611A (zh) * 2018-01-12 2018-05-15 中国科学院成都生物研究所 一株解淀粉芽孢杆菌及其应用
TWI729523B (zh) * 2019-10-05 2021-06-01 味丹企業股份有限公司 液化澱粉芽孢桿菌、液化澱粉芽孢桿菌用於製備抑制或殺死病原菌之組成物之用途及其代謝產物用於製備抑制或殺死病原菌之組成物之用途
CN111040976B (zh) * 2020-01-06 2021-04-13 安徽农业大学 一株解淀粉芽孢杆菌及其应用
CN111349590B (zh) * 2020-04-29 2022-04-08 福建省农业科学院植物保护研究所 一株解淀粉芽孢杆菌及其在防治马铃薯晚疫病中的应用
CN112940979B (zh) * 2021-03-18 2022-07-19 深圳大学 细菌菌株、提取物、纳米农药及其应用与制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102888428A (zh) * 2011-07-21 2013-01-23 中国科学院过程工程研究所 一种利用解淀粉芽胞杆菌lsse-62合成纳米银的方法
CN102747020A (zh) * 2012-07-18 2012-10-24 中国热带农业科学院环境与植物保护研究所 一株枯草芽孢杆菌及其应用
CN103274825A (zh) * 2013-06-09 2013-09-04 苏州仁成生物科技有限公司 一种防治芹菜软腐病的多元微生物专用肥及其制备方法
CN107960103A (zh) * 2014-12-29 2018-04-24 Fmc有限公司 用于促进植物生长和治疗植物疾病的微生物组合物和使用方法
CN108690821A (zh) * 2017-12-07 2018-10-23 中国热带农业科学院环境与植物保护研究所 一株高效解淀粉芽孢杆菌及其菌剂和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU WEI, SHI CHENG: "Research on Simulation Test Platform for Railway Train Control Safety Information Monitoring", CHINA RAILWAY, SIGNAL & COMMUNICATION RESEARCH INSTITUTE,CHINA ACADEMY OF RAILWAY SCIENCES CORPORATION LIMITED, CN, vol. 6, 15 June 2021 (2021-06-15), CN , pages 141 - 146, XP093018523, ISSN: 1001-683X, DOI: 10.19549/j.issn.1001-683x.2021.06.141 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117695428A (zh) * 2023-10-17 2024-03-15 山东威高新生医疗器械有限公司 一种可以接触创面的医疗级硅橡胶
CN117981757A (zh) * 2024-04-02 2024-05-07 中国农业科学院作物科学研究所 一种喷施处理提高小麦抗假禾谷镰刀菌引起的茎基腐病害的组合物及方法

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