WO2021185021A1 - 一种有利于盐碱地玉米生长的微生物菌剂yf及其应用 - Google Patents

一种有利于盐碱地玉米生长的微生物菌剂yf及其应用 Download PDF

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WO2021185021A1
WO2021185021A1 PCT/CN2021/077128 CN2021077128W WO2021185021A1 WO 2021185021 A1 WO2021185021 A1 WO 2021185021A1 CN 2021077128 W CN2021077128 W CN 2021077128W WO 2021185021 A1 WO2021185021 A1 WO 2021185021A1
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bacillus
microbial
soil
saline
corn
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沙月霞
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宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室）
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Priority to US17/624,209 priority Critical patent/US11700856B2/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the present invention relates to the technical field of microorganisms, in particular to the application of a microbial inoculum YF in promoting the growth of corn in saline soil and the prevention and treatment of stalk rot.
  • saline-alkali land accounts for 10% of the world's total area.
  • the total area of saline-alkali land in China that affects arable land exceeds 500 million mu.
  • the area of saline-alkali land ranks third among all countries in the world. Among them, the area with agricultural development potential accounts for China's total arable land. 10% or more.
  • the pH value of saline soil is generally high.
  • Microbial agents can not only improve the quality of soil, but also repair saline-alkali soil in combination with plants. At the same time, they can prevent diseases, insects and promote growth of corn, tomato, chickpeas and other plants, and increase their protein content and solubility. Sugar content, etc., and increase its absorption of minerals and nitrogen.
  • Corn is an important food crop in the world, and its planting scale is only behind wheat. With changes in global climate conditions, continuous cropping, high-density cultivation, etc., the ecological environment of corn fields has also changed, and the structure of the dominant bacterial community of pathogens in the soil has changed, and the accumulation of diseased bodies is increasing, and the community structure of beneficial microorganisms And the number has also undergone major changes. These factors lead to the imbalance of the soil microbial community structure in the corn field, and the occurrence of corn stalk base rot is becoming more and more serious.
  • Corn stalk basal rot is a typical soil-borne disease caused by multiple pathogenic bacteria, and it is one of the main diseases that endanger the world's corn production. Under normal circumstances, the output loss is 10-25%, and it can reach 75% in severe years. Resistant varieties, chemical seed coating agents and increased application of potassium fertilizer are the main production measures at present, but the selection of varieties takes a long time, and the residues of chemical seed coating agents can easily pollute the ecological environment, threaten human health, and increase soil salinity. The pathogenic bacteria are prone to drug resistance. Reducing the number of pathogenic bacteria in the soil and the soil micro-ecological environment that interfere with their growth are the key means to prevent and control corn stem rot. Therefore, the development of microbial agents for corn stalk basal rot has important scientific significance and application value for food security and the sustainable development of the corn industry.
  • Chen et al. used Trichoderma asperellum granules to be mixed with chemical fertilizers and applied to the soil before sowing, which has a significant control effect on corn stalk rot.
  • microbial agents that can be used for corn growth and disease control in saline-alkali soils. It is necessary to develop microbial agents that are beneficial to the growth of corn in saline-alkali soils and the prevention and control of stalk rot.
  • the present invention provides a compound microbial agent that is beneficial to the growth of corn in saline-alkali land and has a good control effect on corn stalk rot in saline-alkali land.
  • the present invention adopts the following technical solutions.
  • a microbial inoculum YF is provided.
  • the microbial inoculum YF includes four strains; the four strains are Bacillus sonorensis B-2 and Bacillus paralicheniformis.
  • B-26, Bacillus sonorensis N-16, Bacillus pumilus N-17, all four strains are deposited in the General Microbiology Center (CGMCC) of the China Microbial Species Collection Management Committee (CGMCC).
  • CGMCC General Microbiology Center
  • CGMCC General Microbiology Center
  • CGMCC General Microbiology Center
  • the address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is March 5, 2020.
  • the preservation number of Sonora Desert Bacillus B-2 is CGMCC NO.19454, Bacillus paralicheniformis B
  • the deposit number of -26 is CGMCC NO.19449
  • the deposit number of Sonora Desert Bacillus N-16 is CGMCC NO.19448,
  • the deposit number of Bacillus pumilus N-17 is CGMCC NO.19450; the four strains are suitable for growth
  • the conditions are temperature 30-37°C, pH>8.0, using Horikoshi I, modified LB-I or modified LB-II medium.
  • the mass ratio of Sonora Desert Bacillus B-2, Bacillus paralicheniformis B-26, Sonora Desert Bacillus N-16 and Bacillus pumilus N-17 is 1:1:1:1; microbial agent YF The number of viable Bacillus spp.>1 billion CFU/g.
  • a microbial inoculum YF which contains the inoculum powder prepared by the above four strains, and also includes a carrier, humic acid, regulators and trace elements.
  • the content of each component in the microbial inoculum YF is :Bacterial powder 48-58%; carrier 35-45%; humic acid 3-8%; regulator 0.4-1.2%; trace element 0.6-1.8%.
  • the carrier is sheep dung
  • the regulator is calcium sulfate (CaSO 4 ⁇ 2H 2 O)
  • trace elements include zinc sulfate (ZnSO 4 ), ferrous sulfate (FeSO 4 ⁇ 7H 2 O) and potassium sulfate (K 2 SO 4 ).
  • each component in the microbial inoculum YF is: 48-58% of the inoculum powder; 35-45% of the carrier; 3-8% of humic acid; 0.4-1.2% of calcium sulfate; 0.2-0.6% of zinc sulfate; Iron 0.2-0.6%; potassium sulfate 0.2-0.6%.
  • the carrier is decomposed sheep dung.
  • each component in the microbial agent YF is: 54% of the bacterial powder; 40% of the carrier; 5% of humic acid; 0.4% of calcium sulfate; 0.2% of zinc sulfate; 0.2% of ferrous sulfate; 0.2% of potassium sulfate.
  • a microbial agent YF in the preparation of a medicine for preventing and treating corn stalk rot in saline-alkali land is provided.
  • the present invention provides a microbial agent YF, comprising Bacillus Sonora Desert B-2, Bacillus paralicheniformis B-26, Bacillus Sonora Desert Bacillus N-16, Bacillus pumilus N-17
  • the mass ratio of Sonora Desert Bacillus B-2, Bacillus paralicheniformis B-26, Sonora Desert Bacillus N-16 and Bacillus pumilus N-17 is 1:1:1:1; microbial inoculants
  • the number of viable bacteria of Bacillus in YF is more than 1 billion CFU/g, and the effect is better.
  • the functional bacteria of the microbial agent YF is isolated from the saline-alkali soil, and has the characteristics of high salt-tolerance, lowering soil pH, improving soil nutritional status and better disease prevention and growth effects; humic acid can increase fertilizer efficiency, improve soil and promote The effect of plant growth is often used as an auxiliary preparation of microbial fertilizer; the carrier is sheep dung, which has a wide range of sources and low cost.
  • Calcium sulfate is a soil amendment for saline-alkali soil (Wang Minghua. Effects of amendments on physiological characteristics of soda saline-alkali soil and corn.
  • the microbial agent YF provided by the present invention has a good control effect on corn stalk rot in saline-alkali land, and has significant antibacterial effect on a variety of soil-borne plant pathogens; it has obvious micro-ecological regulation and control effect on corn field soil in saline-alkali land and improves soil Diversity and structure of fungal and bacterial communities in the soil, reducing the abundance of pathogenic bacteria in the soil; it has a good effect on improving the soil of the saline-alkali corn farmland, effectively reducing the soil pH, and increasing the content of soil organic carbon, nitrate nitrogen, total nitrogen and available potassium ; It has an obvious growth-promoting effect on corn, increasing the corn emergence rate by 107.75%, increasing the corn plant height by 46.68%, and increasing the corn stem thickness by 48.54%; significantly increasing the effective ear number, corn ear thickness, ear length, ear weight and reducing baldness rate .
  • the microbial inoculum YF of the present invention does not contain pathogenic bacteria, heavy metals and toxic chemical substances, and is suitable for soil treatment for planting corn without causing additional burden on the soil. It is an environmentally friendly green microbial inoculum. The sustainable development of the corn industry and the effective utilization of saline-alkali land resources have important scientific significance and application value.
  • Figure 1 is a phylogenetic tree of strain B-2 based on 16S rDNA sequencing.
  • Figure 2 is a phylogenetic tree of strain N-16 based on 16S rDNA sequencing.
  • Figure 3 is a phylogenetic tree of strain N-17 based on 16S rDNA sequencing.
  • Figure 4 is a phylogenetic tree of strain B-26 based on 16S rDNA sequencing.
  • Figure 5 is a picture of corn ears after harvest.
  • Figure 6 shows the effect of microbial inoculants on soil bacterial community structure in corn fields.
  • Figure 7 Effects of microbial inoculants on soil fungal community structure in corn fields.
  • Figure 8 is the principal coordinate (PCoA) analysis (OTU level) of the soil bacterial community in the corn field after the application of microbial inoculants.
  • the present invention provides a microbial inoculum YF.
  • the microbial inoculum YF contains four strains; the four strains are Bacillus sonorensis B-2 and Bacillus paralicheniformis B-26. , Bacillus sonorensis N-16, Bacillus pumilus N-17, all four strains are deposited in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, the preservation date is 2020 March 5, 2010; among them, the deposit number of Bacillus Sonora Desert B-2 is CGMCC NO.19454, the deposit number of Bacillus paralicheniformis B-26 is CGMCC NO.19449, and Bacillus Sonora Desert N
  • the deposit number of -16 is CGMCC NO.19448, and the deposit number of Bacillus pumilus N-17 is CGMCC NO.19450; the suitable growth conditions for the four strains are temperature 30-37°C, pH>8.0, Horikoshi I, modified LB -I or modified
  • the mass ratio of Sonora Desert Bacillus B-2, Bacillus paralicheniformis B-26, Sonora Desert Bacillus N-16 and Bacillus pumilus N-17 is 1:1:1:1; microbial agent YF The number of viable Bacillus spp.>1 billion CFU/g.
  • the microbial inoculum YF contains the inoculum powder prepared by the above-mentioned four strains, as well as carrier, humic acid, regulator and trace elements.
  • the content of each component is: inoculum powder 48-58%; carrier 35-45 %; Humic acid 3-8%; Regulator 0.4-1.2%; Trace element 0.6-1.8%.
  • the carrier is sheep dung; preferably, the carrier is decomposed sheep dung.
  • the regulator is calcium sulfate (CaSO 4 ⁇ 2H 2 O), and trace elements include zinc sulfate (ZnSO 4 ), ferrous sulfate (FeSO 4 ⁇ 7H 2 O) and potassium sulfate (K 2 SO 4 ).
  • each component in the microbial inoculum YF is: 48-58% of the inoculum powder; 35-45% of the carrier; 3-8% of humic acid; 0.4-1.2% of calcium sulfate; 0.2-0.6% of zinc sulfate; Iron 0.2-0.6%; potassium sulfate 0.2-0.6%.
  • the content of each component in the microbial inoculum YF is: 54% of the inoculum powder; 40% of the carrier; 5% of humic acid; 0.4% of calcium sulfate; 0.2% of zinc sulfate; 0.2% of ferrous sulfate; 0.2% of potassium sulfate %.
  • Isolation of Bacillus halophilis Collect the soil from the saline-alkali cornfield in Pingluo County, Ningxia Hui Autonomous Region, add 10g of the sample to 100mL of sterile water, shake well for about 15min to disperse the soil particles, and then dilute the sample with sterile water.
  • the coated plate is wrapped with a sealing film and placed upside down in an incubator at 37°C.
  • Isolation of alkalophilic bacillus Collect the soil from the saline-alkali corn field in Guyuan City, Ningxia Hui Autonomous Region, and put the soil sample in a water bath at 80°C for 10 minutes, and then apply the dilution plate coating method on Horikoshi I, modified LB-I or modified LB-II medium Carry out separation. Observe the morphology of the colonies on each plate, and distinguish and number different colonies according to the characteristics of colony size, color, dryness and wetness, shape, edge, and transparency. Single colonies with different colony morphologies were selected for continuous streaking culture until pure culture was obtained, and they were preserved by freezing with glycerol at -80°C.
  • Horikoshi I medium (g/L): glucose 10.0g, peptone 5.0g, yeast extract 5.0g, K 2 HPO 4 1.0g, MgSO 4 0.2g, Na 2 CO 3 10.0g (sterilized separately), agar 15.0 ⁇ 20.0g, pH 9, sterilized at 121°C for 20 minutes.
  • LB-I medium (75.0g/L NaCl): tryptone 10.0g, yeast extract 5.0g, NaCl 75.0g, agar 15-20g, water 1,000mL, pH 9.0.
  • LB-II medium (150.0g/L NaCl): tryptone 10.0g, yeast extract 5.0g, NaCl 150.0g, agar 15-20g, water 1,000mL, pH 9.0.
  • the above culture medium was placed in a high-pressure steam sterilization pot and sterilized at 121°C for 20 minutes.
  • Detection of amylase inoculate a newly activated single colony on an LB plate containing 0.2% soluble starch and culture for 48 hours to form a significant bacterial lag. Add Lugo's iodine solution to the plate for 10 minutes and wash the plate with 70% ethanol. , A strain that can produce amylase, on a black background, a colorless transparent circle can be formed around the colony growth area. If there is a transparent circle, it indicates that the strain can produce amylase. 3 replicates for each treatment. The results are shown in Table 2.
  • Protease detection puncture and inoculate the activated strain to be tested on a 1% skimmed milk agar plate. After incubating at 30°C for 24, 48, 72 hours, observe the production of the outer transparent circle. The appearance of the transparent circle indicates the production of protease. 3 for each treatment The results are shown in Table 2.
  • Glucanase detection The test bacteria are inoculated on a plate containing ABP medium, and after culturing at 30°C for 48 or 72 hours, observe whether there is a digestion circle in the plate. If there is a digestion circle, it indicates that there is production of glucanase. There are 3 replicates for each treatment, and the results are shown in Table 2.
  • Test of siderophore use CAS medium to detect siderophore. Inoculate the activated test bacteria on the CAS detection medium plate. After incubating at 30°C for 72 hours, observe whether there is an orange halo in the plate. If it appears orange, then Indicates the production of siderophore, and each treatment was repeated 3 times. The results are shown in Table 2.
  • Antagonistic B-26 strain 1.7 0.4 0.4 0.7 +
  • the number indicates the radius of the transparent circle, that is, the ability to produce enzymes; "-" means that no such enzymes are produced
  • Morphological identification is based on the "Manual for Identification of Common Bacterial Systems", combined with molecular biology technology 16 SrDNA sequencing to identify the species of the selected antagonistic bacteria, strain B-2 was identified as Bacillus sonorensis, the results are shown in the figure 1; N-16 is identified as Bacillus sonorensis, the results are shown in Figure 2; N-17 is identified as Bacillus pumilus, the results are shown in Figure 3; B-26 is identified as Bacillus paralicheniformis (Bacillus paralicheniformis), the results are shown in Figure 4.
  • halophilic antagonistic bacteria refer to Barbosa's determination method, streak one functional strain on half of the LB solid medium plate, and inoculate another functional strain on the other half vertically streak at the same time , Each treatment was repeated 3 times. The plate was placed in a biochemical incubator at 37°C, and the compatibility reaction between the two strains was observed and recorded after 24 hours. The two strains that do not produce antibacterial bands are compatible, and vice versa. Compatibility results showed that the four strains did not inhibit each other.
  • LB liquid medium yeast extract 5g, peptone 10g, sodium chloride 5g, water 1000mL, pH7.4 ⁇ 7.6, 121°C sterilized for 30min.
  • LB solid medium 15g agar powder is added to the liquid medium.
  • Microbial agent YF (100kg) is mainly composed of the following components: Sonora Desert Bacillus B-2, N-16, Bacillus pumilus N-17 and Bacillus paralicheniformis B-26 as a functional bacteria preparation of microbial powder 54kg ( Each strain powder is mixed according to the mass ratio 1:1:1:1), 5kg of humic acid, 400g (0.4%) of calcium sulfate, 200g (0.2%) of zinc sulfate, 200g (0.2%) of ferrous sulfate, 200g of potassium sulfate ( 0.2%), the carrier is 40 kg of decomposed sheep dung, the above components are evenly mixed to prepare the microbial inoculum YF, the number of viable bacteria of Bacillus reaches more than 1 billion CFU/g, and it is stored at low temperature.
  • test examples all use the ingredient formula of the best embodiment 1.
  • the glutinous corn variety Hannuozao 60 was planted in saline-alkali land according to different treatments for 14 days, and the seedling emergence rate was investigated. Plant height and stem thickness (the plant circumference of the first node) were measured after 30 days of planting to calculate the growth-promoting effect of microbial agent YF on corn in saline-alkali land. , The results are shown in Table 6.
  • Table 7 Yield-promoting effect of microbial agent YF on corn in saline-alkali land (1)
  • Maize stalk base rot grading standard (individual plant damage): Level 1: The whole plant grows normally, the middle and lower leaves appear with wilt/green wilt symptoms, the stem base grows normally, and the ears grow normally; Level 3: The whole plant leaves appear The symptoms of bacterial wilt, the stem base grows normally, and the ears grow normally; Grade 5: The whole plant leaves have typical symptoms of bacterial wilt, the stem base is discolored and slightly waterlogged, and the ears are basically normal; Grade 7: Plant leaves have typical symptoms of bacterial wilt.
  • the base of the stem is obviously soft but not toppling, the ears are drooping, and the grains are not full; Grade 9: The whole plant is dead and toppling, the vascular bundles at the base of the stem are broken, and the grains are shriveled. See Table 9 for the control effect of microbial agent YF on corn stalk rot in saline soil.
  • Bacterial amplification primers are 338F: 5'-barcode-ACTCCTACGGGAGGCAGCA-3', 806R: 5'-GGACTACHVGGGTWTCTAAT-3', PCR amplification of the variable region of bacterial 16S rRNA gene V3-V4, the primers are from Shanghai Meiji Biomedical Technology Co., Ltd.
  • the formal PCR test uses a 20 ⁇ L reaction system: 10 ⁇ PCR Buffer 2 ⁇ L, 2.5mmol/L dNTPs 2 ⁇ L, 5 ⁇ mol/L forward primer 0.8 ⁇ L, 5 ⁇ mol/L reverse primer 0.8 ⁇ L, rTaq Polymerase 0.2 ⁇ L, BSA 0.2 ⁇ L, Template DNA10ng , Add ddH 2 O to 20 ⁇ L.
  • PCR amplification conditions 95°C 3min; cycle number ⁇ (95°C 30s, 55°C 30s, 72°C 45s); 72°C 10min, 10°C until the end of the reaction.
  • the second round of PCR amplification products were detected by 2% agarose gel electrophoresis, the PCR products were recovered by cutting the gel, and eluted with Tris-HCl; 2% agarose electrophoresis was used for detection.
  • the PCR products were detected and quantified with QuantiFluor TM -ST blue fluorescence quantification system (Promega), and then mixed in corresponding proportions.
  • the mixed products were sequenced on the Miseq 2 ⁇ 300 platform of Illumina.
  • the effect of microbial agent YF on soil bacterial community structure in saline-alkali corn fields is shown in Figure 6.
  • the fungal amplification primers are ITS1F: 5'-TCCGTAGGTGAACCTGCGC-3', ITS2R 5'-CTCGGACGAGGATCCTCGCC-3', and ITS1-ITS2 region for PCR amplification.
  • ITS amplified polymerase chain reaction (PCR) system 25 ⁇ L: ddH 2 O 17.25 ⁇ L, Bufferl 2.5 ⁇ L, dNTPs 2 ⁇ L, primer ITS1F 1 ⁇ L, primer ITS2R 1 ⁇ L, HiFi enzyme 0.25 ⁇ L, template DNA 1 ⁇ L .
  • the raw data obtained by high-throughput sequencing is subjected to quality control and software splicing to filter out low-quality sequences.
  • the effective sequence similarity is greater than or equal to 97% sequence clustering into taxonomic units (Operational Taxonomic Units).
  • QIIME software uses the QIIME software to analyze the composition of a single sample, and calculate the Coverage, Chao, Shannon index of the sample and so on.
  • the larger the Shannoneven value the lower the community uniformity.
  • the larger the Shannon value the higher the community diversity.
  • Coverage is the coverage index, which characterizes the detection probability of the sequence of the microbial sample. The higher the value, the higher the probability of the detected sequence, which can reflect the true situation of the microorganisms in the sample.
  • Sobs refers to the richness index, the greater the value, the higher the richness.
  • the effects of microbial agent YF on soil bacterial community diversity in saline-alkali corn fields are shown in Table 10 and Figure 8.
  • the effects of microbial agent YF on the diversity of soil microbial fungi community in saline-alkali corn fields are shown in Table 11 and Figure 9.
  • the microbial agent YF provided by the invention has obvious growth-promoting effects on corn, significantly promotes the emergence rate, increases plant height and plant stem thickness, and increases ear thickness, ear length, ear weight, number of ears, and number of effective ears of waxy corn in saline-alkali land
  • the number of rows and kernels can significantly reduce the length of corn bald tips; it has a good control effect on corn stalk rot in saline-alkali land, and has a significant antibacterial effect on a variety of soil-borne pathogens; it has obvious micro-ecological regulation on the soil of saline-alkali corn field , Improve the diversity and structure of fungal and bacterial communities in the soil, reduce the abundance of pathogens in the soil; have a better effect on improving the soil of the saline-alkali corn farmland, effectively reducing the soil pH, and increasing the soil organic carbon, nitrate nitrogen, total nitrogen and Available potassium content.
  • the microbial inoculum YF of the present invention does not contain pathogenic bacteria, heavy metals and toxic chemical substances, and is suitable for soil treatment for planting corn without causing additional burden on the soil. It is an environmentally friendly green microbial inoculum.
  • the sustainable development of the corn industry and the effective utilization of saline-alkali land resources have important scientific significance and application value. Therefore, the present invention has good industrial applicability.

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Abstract

提供了一种微生物菌剂YF，该菌剂包含保藏编号为CGMCC NO.19454的索诺拉沙漠芽孢杆菌(Bacillus sonorensis) B-2、保藏编号为CGMCC NO.19449的副地衣芽孢杆菌(Bacillus paralicheniformis) B-26、保藏编号为CGMCC NO.19448的索诺拉沙漠芽孢杆菌(Bacillus sonorensis) N-16和保藏编号为CGMCC NO.19450的短小芽孢杆菌(Bacillus pumilus) N-17。还提供了微生物菌剂YF在防治盐碱地玉米茎基腐病的药物制备中的应用。

Description

一种有利于盐碱地玉米生长的微生物菌剂YF及其应用
本申请要求于2020年03月19日提交中国专利局、申请号为202010198243.6、发明名称为“一种有利于盐碱地玉米生长的微生物菌剂YF及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及微生物技术领域，尤其涉及一种微生物菌剂YF在促进盐碱地玉米生长及茎基腐病的防治中的应用。
背景技术
盐碱地作为一种重要的土地资源，其土质改良和利用一直是农业研究领域的重点和热点，也是我国耕地后备资源开发与粮食增产潜在保障的基础。全球盐碱地面积占全球总面积的10％，在中国对耕地有影响的盐碱地总面积超过5亿亩，盐碱地面积在全世界所有国家中位居第三，其中具有农业发展潜力的占中国耕地总面积10％以上。盐碱地的pH值普遍偏高，过量的盐浓度会导致土壤退化，改变土壤渗透和基质潜力，并降低土壤微生物活性，对粮食产量形成了较大威胁。现阶段主要采取的是物理、化学、生物这三类方法来进行盐碱地修复，这几种方法耗费的成本较高，且很难从根本上解决土壤盐渍化问题，而解决这个问题就需从土质上入手，学者认为微生物肥料能从根本上改良土质。随着人们对生态环境的关注且土地资源日益短缺，以天然土壤微生物为主要成分的微生物菌剂应用在盐碱农业中越来越受到重视。微生物菌剂不仅能够改善土壤的质量，还能够与植物联合修复盐碱土壤，同时对玉米、番茄、鹰嘴豆等植物起到良好的防病、防虫和促生作用，提升其蛋白质含量、可溶性糖含量等，并增加其矿物质和氮的吸收。
玉米是世界上重要的粮食作物，种植规模仅排在小麦之后。随着全球气候条件的变化、重茬连作、高密度栽培等，玉米田生态环境随之改变，土壤中病原菌的优势菌群结构发生改变，病残体的积累数量越来越大，有益微生物群落结构和数量也发生较大变化。这些因素导致玉米田土壤微生物群落结构失衡，玉米茎基腐病的发生日趋严重。
玉米茎基腐病是由多种病原菌复合侵染引起的典型土传病害，是危害世界玉米生产的主要病害之一。一般情况下产量损失在10～25％，严重年份可以达到75％。抗性品种、 化学种衣剂和增施钾肥是目前生产上的主要措施，但是品种选育时间较长，化学种衣剂的残留易污染生态环境，也会威胁人类健康，加重土壤的盐渍化，病原菌易产生抗药性。降低土壤中的病原菌数量与干扰其生长的土壤微生态环境是防治玉米茎基腐病的关键手段。因此，研发用于玉米茎基腐病的微生物菌剂对于粮食安全和玉米产业可持续发展具有重要的科学意义和应用价值。
众所周知，土壤中存活着多种病原菌，其数量与活性影响着土壤微生物群落结构的稳定，土壤微生物生态环境的不平衡会加重农作物土传病害的危害。研究表明，农艺措施和生物制剂对土壤微生物群落多样性以及种群组成的影响显著。微生物菌剂广泛用于改善土壤微生物的生态环境，达到防病、防虫、促生和增产效果。吕宁等研究发现枯草芽孢杆菌(Bacillus subtilis)可湿性粉剂滴施后对棉花黄萎病防治效果明显，土壤真菌、细菌和放线菌的数量与物种丰度随着施药量的增加而显著增加。Chen等采用棘孢木霉菌(Trichoderma asperellum)颗粒剂在播种前与化肥混合施入土壤，对玉米茎腐病防效显著。但是目前可以用于盐碱地玉米生长及病害防治的微生物菌剂较少，有必要研发有利于盐碱地玉米生长及茎基腐病防治的微生物菌剂。
发明内容
为了解决上述农业生产存在的技术问题及现有技术的不足，本发明提供了一种有利于盐碱地玉米生长、对盐碱地玉米茎基腐病有良好防治效果的复配微生物菌剂。
为达此目的，本发明采用以下技术方案。
[根据细则26改正12.03.2021]　
在第一方面，提供一种微生物菌剂YF，微生物菌剂YF中包含四种菌株；四种菌株分别为索诺拉沙漠芽孢杆菌(Bacillus sonorensis)B-2、副地衣芽孢杆菌(Bacillus paralicheniformis)B-26、索诺拉沙漠芽孢杆菌(Bacillus sonorensis)N-16、短小芽孢杆菌(Bacillus pumilus)N-17，四种菌株均保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号，保藏日期为2020年3月5日；其中，索诺拉沙漠芽孢杆菌B-2的保藏编号为CGMCC NO.19454，副地衣芽孢杆菌B-26的保藏编号为CGMCC NO.19449，索诺拉沙漠芽孢杆菌N-16的保藏编号为CGMCC NO.19448，短小芽孢杆菌N-17的保藏编号为CGMCC NO.19450；四种菌株适宜的生长条件为温度30-37℃，pH>8.0，采用Horikoshi I、改良LB-I或者改良LB-II培养基。
索诺拉沙漠芽孢杆菌B-2、副地衣芽孢杆菌B-26、索诺拉沙漠芽孢杆菌N-16和短 小芽孢杆菌N-17的质量比为1:1:1:1；微生物菌剂YF中芽孢杆菌的活菌数＞10亿CFU/g。
在第二方面，提供一种微生物菌剂YF，包含上述的四种菌株制备的菌剂粉，还包括载体、腐殖酸、调节剂和微量元素，微生物菌剂YF中各组分的含量为：菌剂粉48-58％；载体35-45％；腐殖酸3-8％；调节剂0.4-1.2％；微量元素0.6-1.8％。
载体为羊粪，调节剂为硫酸钙(CaSO 4·2H 2O)，微量元素包括硫酸锌(ZnSO 4)、硫酸亚铁(FeSO 4·7H 2O)和硫酸钾(K 2SO 4)。
微生物菌剂YF中各组分的含量为：菌剂粉48-58％；载体35-45％；腐殖酸3-8％；硫酸钙0.4-1.2％；硫酸锌0.2-0.6％；硫酸亚铁0.2-0.6％；硫酸钾0.2-0.6％。
载体为腐熟羊粪。
微生物菌剂YF中各组分的含量为：菌剂粉54％；载体40％；腐殖酸5％；硫酸钙0.4％；硫酸锌0.2％；硫酸亚铁0.2％；硫酸钾0.2％。
在第三方面，提供一种微生物菌剂YF在防治盐碱地玉米茎基腐病的药物制备中的应用。
为了达到上述目的，本发明提供一种微生物菌剂YF，包含索诺拉沙漠芽孢杆菌B-2、副地衣芽孢杆菌B-26、索诺拉沙漠芽孢杆菌N-16、短小芽孢杆菌N-17，索诺拉沙漠芽孢杆菌B-2、副地衣芽孢杆菌B-26、索诺拉沙漠芽孢杆菌N-16和短小芽孢杆菌N-17的质量比为1:1:1:1；微生物菌剂YF中芽孢杆菌的活菌数＞10亿CFU/g，效果较好。
微生物菌剂YF的功能菌分离于盐碱地土壤，具有高度耐盐碱性、降低土壤pH、改良土壤营养状况和较好防病促生效果的特性；腐殖酸具有肥料增效、改良土壤和促进植物生长的效果，常常用作微生物肥料的辅助制剂；载体选择羊粪，来源广泛，成本较低。硫酸钙为盐碱地土壤改良剂(王明华.改良剂对苏打盐碱土及玉米生理特性的影响.哈尔滨：东北农业大学,2016.)；微量元素均为增强玉米植株抗玉米茎基腐病的营养元素(李保军,张兰松,朱静,等.氮磷钾锌对玉米茎基腐病及产量的影响.河北农业科学，2016，20(4)：41-44.；王晓鸣,吴全安,张培坤.硫酸锌防治玉米茎基腐病的研究.植物保护,1999.(2):23-25.)
本发明的有益效果如下：
本发明提供的微生物菌剂YF对盐碱地玉米茎基腐病有良好防效，并且对多种土传植物病原菌有显著抑菌作用；对盐碱地玉米田土壤具有较明显的微生态调控作用，改善 土壤中真菌和细菌群落多样性和结构，降低土壤中的病原菌丰度；对盐碱地玉米农田土壤有较好的改良作用，有效降低土壤pH，增加土壤有机碳、硝态氮、全氮和速效钾含量；对玉米有明显的促生作用，提高玉米出苗率107.75％，增加玉米株高46.68％、提高玉米茎粗48.54％；显著增加有效穗数、玉米穗粗、穗长、穗重和降低秃头率。本发明的微生物菌剂YF不含致病菌，不含重金属和有毒害化学物质，适合用于种植玉米的土壤处理，且不会对土壤造成额外负担，属于环保型的绿色微生物菌剂，对玉米产业可持续发展和盐碱地资源有效利用具有重要的科学意义和应用价值。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中:
图1是菌株B-2基于16S rDNA测序的***发育进化树。
图2是菌株N-16基于16S rDNA测序的***发育进化树。
图3是菌株N-17基于16S rDNA测序的***发育进化树。
图4是菌株B-26基于16S rDNA测序的***发育进化树。
图5是收获后的玉米穗图片。
图6是微生物菌剂施用后玉米田土壤细菌群落结构的影响。
图7微生物菌剂施用后玉米田土壤真菌群落结构的影响。
图8是微生物菌剂施用后玉米田土壤细菌群落的主坐标(PCoA)分析(OTU水平)。
图9微生物菌剂YF施用后玉米田土壤真菌群落的主坐标(PCoA)分析(OTU水平)。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互任意组合。
本发明提供一种微生物菌剂YF，微生物菌剂YF中包含四种菌株；四种菌株分别为索诺拉沙漠芽孢杆菌(Bacillus sonorensis)B-2、副地衣芽孢杆菌(Bacillus paralicheniformis)B-26、索诺拉沙漠芽孢杆菌(Bacillus sonorensis)N-16、短小芽孢杆菌(Bacillus pumilus)N-17，四种菌株均保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏日期为2020年3月5日；其中，索诺拉沙漠芽孢杆菌B-2的保藏编号为CGMCC NO.19454，副地衣芽孢杆菌B-26的的保藏编号为CGMCC NO.19449，索诺拉沙漠芽孢杆菌N-16的保藏编号为CGMCC NO.19448，短小芽孢杆菌N-17的保藏编号为CGMCC NO.19450；四种菌株适宜的生长条件为温度30-37℃，pH>8.0，采用Horikoshi I、改良LB-I或者改良LB-II培养基。
索诺拉沙漠芽孢杆菌B-2、副地衣芽孢杆菌B-26、索诺拉沙漠芽孢杆菌N-16和短小芽孢杆菌N-17的质量比为1:1:1:1；微生物菌剂YF中芽孢杆菌的活菌数＞10亿CFU/g。
微生物菌剂YF中包含上述的四种菌株制备的菌剂粉，还包括载体、腐殖酸、调节剂和微量元素，各组分的含量为：菌剂粉48-58％；载体35-45％；腐殖酸3-8％；调节剂0.4-1.2％；微量元素0.6-1.8％。
载体为羊粪；优选的，载体为腐熟羊粪。
调节剂为硫酸钙(CaSO 4·2H 2O)，微量元素包括硫酸锌(ZnSO 4)、硫酸亚铁(FeSO 4·7H 2O)和硫酸钾(K 2SO 4)。
微生物菌剂YF中各组分的含量为：菌剂粉48-58％；载体35-45％；腐殖酸3-8％；硫酸钙0.4-1.2％；硫酸锌0.2-0.6％；硫酸亚铁0.2-0.6％；硫酸钾0.2-0.6％。优选的，微生物菌剂YF中各组分的含量为：菌剂粉54％；载体40％；腐殖酸5％；硫酸钙0.4％；硫酸锌0.2％；硫酸亚铁0.2％；硫酸钾0.2％。
实施例1四种菌株的获得
(1)嗜盐碱菌的分离
喜盐芽孢杆菌的分离：采集宁夏回族自治区平罗县盐碱地玉米田土壤，将10g样品加入到100mL无菌水中，充分振荡约15min分散土壤颗粒，然后用无菌水将样品进行梯度稀释。将稀释好的10 -2、10 -3、10 -4、10 -5和10 -6浓度的样品各0.1mL涂布在含不同NaCl浓度的：Horikoshi I、改良LB-I或者改良LB-II培养基上，涂布后的平板用封口膜包好，倒置于37℃培养箱内培养，3～7d后从平板上挑取不同表型特征的单菌落，梯度划线分离获得纯菌株，挑取纯化菌株的单菌落，转移至40％甘油中，在-80℃超低 温冰箱中保存备用。
嗜碱芽孢杆菌的分离：采集宁夏回族自治区固原市盐碱地玉米田土壤，将土壤样品进行80℃水浴10min后，采用稀释平板涂布法在Horikoshi I、改良LB-I或者改良LB-II培养基上进行分离。观察各平板上的菌落形态，根据菌落大小、颜色、干湿、形状、边缘、透明度等特征区分不同的菌落并进行编号。挑取菌落形态不同的单菌落进行连续划线培养，直至获得纯培养，采用-80℃甘油冷冻法保藏。
Horikoshi I培养基(g/L)：葡萄糖10.0g，蛋白胨5.0g，酵母浸出物5.0g，K 2HPO 4 1.0g，MgSO 4 0.2g，Na 2CO 3 10.0g(单独灭菌)，琼脂15.0～20.0g，pH 9，121℃灭菌20min。
改良LB-I培养基(75.0g/L NaCl)：胰蛋白胨10.0g，酵母浸出物5.0g，NaCl 75.0g，琼脂15～20g，水1 000mL，pH值为9.0。
改良LB-II培养基(150.0g/L NaCl):胰蛋白胨10.0g，酵母浸出物5.0g，NaCl 150.0g，琼脂15～20g，水1 000mL，pH值为9.0。以上培养基均置于高压蒸汽灭菌锅中，121℃灭菌20min。
(2)嗜盐碱型拮抗菌的筛选
①嗜盐碱型拮抗菌的筛选
在PDA培养基平板***放置尖孢镰刀菌(Fusarium oxysporum)、串珠镰刀菌(Fusarium moniliforme)、西瓜枯萎病菌专性致病型(Fusarium oxysporum f.sp.niveum)、立枯丝核菌(Rhizoctonia solani)和烟草黑胫病菌(Phytophthora parasitica var.nicotianae)菌饼，待病原菌菌落生长2天后，分别在菌落边缘两侧相距2cm处接种供试菌株菌饼(5mm)，立枯丝核菌与供试菌株同时对峙培养，置于培养箱28℃黑暗培养，4次重复。2天后测量病原菌菌落直径和抑菌带，结果见表1。
表1嗜盐碱菌对多种土传病原菌的拮抗作用
Figure PCTCN2021077128-appb-000001
Figure PCTCN2021077128-appb-000002
②嗜盐碱型拮抗菌的产酶能力测试
淀粉酶的检测：取新活化的单菌落接种于含有0.2％可溶性淀粉的LB平板上，培养48h，形成明显菌落后，在平板上滴加卢哥式碘液染色10min，用70％乙醇洗板，能产生淀粉酶的菌株，在黑色的背景下，其菌落生长处周围可形成无色的透明圈，如果有透明圈表明菌株可以产生淀粉酶，每个处理3个重复，结果见表2。
蛋白酶的检测：将活化的待测菌株穿刺接种于1％脱脂牛奶琼脂平板上，30℃培养24、48、72h后观察***透明圈的产生，出现透明圈表明有蛋白酶的产生，每个处理3个重复，结果见表2。
葡聚糖酶检测：待测菌接种于含有ABP培养基的平板上，30℃培养48、72h后，观察平板中是否出现消解圈，若有消解圈产生则表明有葡聚糖酶的产生，每个处理3个重复，结果见表2。
嗜铁素检测：嗜铁素用CAS培养基检测，将活化的待测菌接种于CAS检测培养基平板上，30℃培养72h后，观察平板中是否产生橘黄色晕圈，若出现橘黄色则表明有嗜铁素的产生，每个处理重复3次，结果见表2。
纤维素酶的检测：将分离到的菌株活化后接种于纤维素筛选培养基平板上，28℃恒温倒置培养2d，用0.1％的刚果红染色液浸染10min，再用1mol/L的NaCl溶液脱色5min。若菌株产纤维素酶，则会在菌落周围出现清晰的透明圈，每个处理重复3次，结果见表2。
表2嗜盐碱型拮抗菌的产酶能力
                                     单位：cm
菌株名称 淀粉酶 蛋白酶 葡聚糖酶 纤维素酶 嗜铁素
拮抗菌B-2菌株 2.10 0.52 0.54 0.67 -
拮抗菌N-16菌株 1.93 0.82 - 0.55 -
拮抗菌N-17菌株 - 0.7 - 0.73 -
拮抗菌B-26菌株 1.7 0.4 0.4 0.7 +
数字表示透明圈半径，即产酶能力；“-”表示不产生该类酶
(3)嗜盐碱型拮抗菌的鉴定
形态学鉴定依据《常见细菌***鉴定手册》，结合分子生物学技术16 SrDNA测序对筛选出的拮抗菌进行种类鉴定，菌株B-2鉴定为索诺拉沙漠芽孢杆菌(Bacillus sonorensis)，结果见图1；N-16鉴定为索诺拉沙漠芽孢杆菌(Bacillus sonorensis)，结果见图2；N-17鉴定为短小芽孢杆菌(Bacillus pumilus)，结果见图3；B-26鉴定为副地衣芽孢杆菌(Bacillus paralicheniformis)，结果见图4。
实施例2微生物菌剂YF的配制
①嗜盐碱型拮抗菌之间的相容性：参考Barbosa的测定方法，在LB固体培养基平板上一半划线接种一种功能菌株，同时在其另一半垂直划线接种另一种功能菌株,每个处理重复3次。将平板放置在37℃的生化培养箱中培养，24h后观察和记录两菌株之间的相容性反应。两菌株之间不产生抑菌条带的为相容，反之为不相容。相容性结果表明4株菌株之间不相互抑制。
LB液体培养基：酵母提取物5g，蛋白胨10g，氯化钠5g，水1000mL，pH7.4～7.6，121℃灭菌30min。LB固体培养基：在液体培养基基础上加入15g琼脂粉。
②微生物菌剂YF的配制
超低温保藏的索诺拉沙漠芽孢杆菌B-2、N-16、短小芽孢杆菌N-17和副地衣芽孢杆菌B-26活化24h后，分别接种单菌落制备种子发酵液，将种子发酵液分别按照6％比例转移到发酵罐中，30℃条件下，180rpm/min振荡培养48h后获得发酵液。培养好的4种菌液按照1:1:1:1的比例混合均匀，将混合好的菌液用草炭吸附制成粉剂。
实施例1
微生物菌剂YF(100kg)主要由以下成分组成：索诺拉沙漠芽孢杆菌B-2、N-16、短小芽孢杆菌N-17和副地衣芽孢杆菌B-26作为功能菌制备的微生物粉剂54kg(各个菌株粉剂按照质量比1:1:1:1混合)、腐殖酸5kg、硫酸钙400g(0.4％)、硫酸锌200g(0.2％)、硫酸亚铁200g(0.2％)、硫酸钾200g(0.2％)，载体为腐熟羊粪40kg，以上组分混合均匀制成微生物菌剂YF，芽孢杆菌的活菌数达到10亿CFU/g以上，低温保存。
本发明的微生物菌剂YF的实施例的各组分含量如表3所示。
表3实施例中各组分含量
Figure PCTCN2021077128-appb-000003
测试例1
测试例均采用最优实施例1的成分配方。
1)微生物菌剂YF生物活性的测定
(1)微生物菌剂YF在盐碱地玉米农田土壤的拌土处理
在宁夏回族自治区平罗县黄渠桥盐碱荒地种植糯玉米-旱糯早60，种植前将微生物菌剂YF粉剂均匀撒施在农田土壤表面(40kg/亩)，然后用旋耕机旋耕(深度为20cm左右)，将微生物菌剂与土壤均匀混合。设计2个处理：微生物菌剂YF；空白对照CK：不施用任何农药和肥料，试验小区面积为60m 2，每个处理4个重复小区。以下研究均在此实验田进行。
(2)微生物菌剂YF对盐碱地玉米农田土壤的改良作用：
玉米种植45d(乳熟期)采集农田土壤(距离植株15cm，深度为0-20cm)，土壤样本采用冰盒带回实验室，检测土壤pH值、有机碳、有机质、全氮、全磷、硝态氮、有效磷、速效钾等理化指标，结果见表4和表5。
表4微生物菌剂YF对盐碱地玉米农田土壤的改良作用(1)
Figure PCTCN2021077128-appb-000004
注：每组数据为4个重复的平均值，小写字母是显著性差异(最小极差法，P＜0.05)
表5微生物菌剂YF对盐碱地玉米农田土壤的改良作用(2)
Figure PCTCN2021077128-appb-000005
注：每组数据为4个重复的平均值，小写字母是显著性差异(最小极差法，P＜0.05)
(3)微生物菌剂YF对盐碱地玉米的促生作用
糯玉米品种旱糯早60在盐碱地根据不同处理种植14d后调查出苗率，种植30d后测量株高和茎粗(第一节部位植株周长)，计算微生物菌剂YF对盐碱地玉米的促生效果，结果见表6。
表6微生物菌剂YF对盐碱地玉米的促生效果
Figure PCTCN2021077128-appb-000006
注：每组数据为4个重复的平均值，小写字母是显著性差异(最小极差法，P＜0.05)
(4)微生物菌剂YF对盐碱地玉米的促产作用
玉米成熟期(种植约80d)调查结穗率和有效穗数，采集成熟玉米穗，测量糯玉米穗长、鲜重、秃头、穗粒数、行粒数、穗粗(穗中部位置的周长)，评价微生物菌剂YF对盐碱地玉米的促产效果，结果见表7和表8。
表7微生物菌剂YF对盐碱地玉米的促产效果(1)
Figure PCTCN2021077128-appb-000007
注：每组数据为4个重复的平均值，小写字母是显著性差异(最小极差法，P＜0.05)
表8微生物菌剂YF对盐碱地玉米的促产效果(2)
Figure PCTCN2021077128-appb-000008
注：每组数据为4个重复的平均值，小写字母是显著性差异(最小极差法，P＜0.05)
(5)微生物菌剂YF对盐碱地玉米茎基腐病的预防效果
在玉米乳熟期调查盐碱地玉米茎基腐病的发病率和病情指数，发病率以有无病斑为主。玉米茎基腐病分级标准(植株个体受害程度)：1级：全株生长正常，中下部叶片出现青枯/青黄枯症状，茎基生长正常，果穗生长正常；3级：全株叶片出现青枯症状，茎基生长正常，果穗生长正常；5级：全株叶片出现典型青枯症状，茎基部变色且稍有水浸状，果穗基本正常；7级：植株叶片出现典型青枯症状，茎基部明显***但不倒状，果穗下垂，籽粒不饱满；9级：全株枯死且倒伏，茎基部维管束破裂，籽粒干瘪。微生 物菌剂YF对盐碱地玉米茎基腐病的防治效果见表9。
表9微生物菌剂YF对盐碱地玉米茎基腐病的防治效果
Figure PCTCN2021077128-appb-000009
注：每组数据为4个重复的平均值，小写字母是显著性差异(最小极差法，P＜0.05)
测试例2
微生物菌剂YF对盐碱地玉米田土壤的微生态调控作用
(1)微生物菌剂YF对盐碱地玉米田土壤微生物群落结构的影响
玉米乳熟期采集农田土壤(距离植株15cm，深度为0～20cm)，土壤样本采用冰盒带回实验室。提取各个处理组的土壤样本基因组总DNA，然后利用1％琼脂糖凝胶电泳检测抽提的基因组DNA。细菌扩增引物为338F：5′-barcode-ACTCCTACGGGAGGCAGCA-3′，806R：5′-GGACTACHVGGGTWTCTAAT-3′，对细菌16S rRNA基因V3–V4可变区进行PCR扩增，引物由上海美吉生物医药科技有限公司设计合成，测序区域合成带有barcode的特异引物。PCR正式试验采用20μL反应体系：10×PCR Buffer 2μL，2.5mmol/L dNTPs 2μL，5μmol/L正向引物0.8μL，5μmol/L反向引物0.8μL，rTaq Polymerase 0.2μL，BSA 0.2μL，Template DNA10ng，补ddH 2O至20μL。PCR扩增条件：95℃ 3min；循环数×(95℃ 30s，55℃ 30s，72℃ 45s)；72℃ 10min，10℃直到反应结束。对第二轮PCR扩增产物用2％琼脂糖凝胶电泳检测，切胶回收PCR产物，Tris-HCl洗脱；2％琼脂糖电泳检测。将PCR产物用QuantiFluor TM-ST蓝色荧光定量***(Promega公司)进行检测定量，之后进行相应比例的混合。混合后的产物用Illumina公司的Miseq 2×300平台测序。微生物菌剂YF对盐碱地玉米田土壤细菌群落结构的影响见图6。
真菌扩增引物为ITS1F：5'-TCCGTAGGTGAACCTGCGC-3'，ITS2R 5′-CTCGGACGAGGATCCTCGCC-3′，ITS1-ITS2区进行PCR扩增。ITS扩增的聚合酶链式反应(Polymerase chain reaction，PCR)体系(25μL)：ddH 2O 17.25μL，Bufferl 2.5μL，dNTPs 2μL，引物ITS1F 1μL，引物ITS2R 1μL，HiFi酶0.25μL，模板DNA 1μL。 PCR反应条件：93℃预变性3min，93℃变性45s，57℃复性45s，72℃延伸90s，35个循环。扩增产物回收后，由上海美吉生物医药科技有限公司完成测序。微生物菌剂YF对盐碱地玉米田土壤微生物真菌群落结构的影响见图7。
(2)微生物菌剂YF对盐碱地玉米田土壤微生物群落多样性的影响
高通量测序得到的原始数据进行质量控制和软件拼接，过滤掉低质量的序列。将有效序列相似性在≥97％序列聚类成为分类单元(Operational Taxonomic Units)。再用QIIME软件进行单样品组成分析，计算样品的Coverage、Chao、Shannon指数等。Shannoneven值数值越大，说明群落均匀度越低。Shannon值越大，说明群落多样性越高。Coverage是覆盖度指数，表征微生物样本序列的检测概率，数值越高表明检测出的序列概率越高，能够反映样本中微生物的真实情况。Sobs指丰富度指数，数值越大丰富度越高。微生物菌剂YF对盐碱地玉米田土壤细菌群落多样性的影响见表10和图8。微生物菌剂YF对盐碱地玉米田土壤微生物真菌群落多样性的影响见表11和图9。
表10微生物菌剂YF对盐碱地玉米田土壤微生物细菌群落多样性的影响
Figure PCTCN2021077128-appb-000010
表11微生物菌剂YF对盐碱地玉米田土壤微生物真菌群落多样性的影响
Figure PCTCN2021077128-appb-000011
需要说明的是：在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包含一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备 所固有的要素。在没有更多限制的情况下，由语句“包括一个…”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上实施例仅用以说明本发明的技术方案，而非对其限制。尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
工业实用性
本发明提供的微生物菌剂YF对玉米有明显的促生作用，显著促进出苗率、增加株高和植株茎粗，增加盐碱地糯玉米穗粗、穗长、穗重、穗粒数、有效穗数、行粒数，明显降低玉米秃尖长度；对盐碱地玉米茎基腐病有良好防效，并且对多种土传病原菌有显著抑菌作用；对盐碱地玉米田土壤具有较明显的微生态调控作用，改善土壤中真菌和细菌群落多样性和结构，降低土壤中的病原菌丰度；对盐碱地玉米农田土壤有较好的改良作用，有效降低土壤pH，增加土壤有机碳、硝态氮、全氮和速效钾含量。本发明的微生物菌剂YF不含致病菌，不含重金属和有毒害化学物质，适合用于种植玉米的土壤处理，且不会对土壤造成额外负担，属于环保型的绿色微生物菌剂，对玉米产业可持续发展和盐碱地资源有效利用具有重要的科学意义和应用价值。因此，本发明具有良好的工业实用性。

Claims (8)

  1. 一种微生物菌剂YF，其特征在于，所述微生物菌剂YF中包含四种菌株；
    所述四种菌株分别为索诺拉沙漠芽孢杆菌(Bacillus sonorensis)B-2、副地衣芽孢杆菌(Bacillus paralicheniformis)B-26、索诺拉沙漠芽孢杆菌(Bacillus sonorensis)N-16、短小芽孢杆菌(Bacillus pumilus)N-17，所述四种菌株均保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏日期为2020年3月5日；
    其中，所述索诺拉沙漠芽孢杆菌B-2的保藏编号为CGMCC NO.19454，所述副地衣芽孢杆菌B-26的的保藏编号为CGMCC NO.19449，所述索诺拉沙漠芽孢杆菌N-16的保藏编号为CGMCC NO.19448，所述短小芽孢杆菌N-17的保藏编号为CGMCC NO.19450；
    所述四种菌株适宜的生长条件为温度30-37℃，pH>8.0，采用Horikoshi I、改良LB-I或者改良LB-II培养基。
  2. 如权利要求1所述的微生物菌剂YF，其特征在于，所述索诺拉沙漠芽孢杆菌B-2、所述副地衣芽孢杆菌B-26、所述索诺拉沙漠芽孢杆菌N-16和所述短小芽孢杆菌N-17的质量比为1:1:1:1；所述微生物菌剂YF中芽孢杆菌的活菌数＞10亿CFU/g。
  3. 一种微生物菌剂YF，其特征在于，所述微生物菌剂YF中包含如权利要求2所述的四种菌株制备的菌剂粉，还包括载体、腐殖酸、调节剂和微量元素，所述微生物菌剂YF中各组分的含量为：
    Figure PCTCN2021077128-appb-100001
  4. 如权利要求3所述的微生物菌剂YF，其特征在于，所述载体为羊粪，所述调节剂为硫酸钙(CaSO 4·2H 2O)，所述微量元素包括硫酸锌(ZnSO 4)、硫酸亚铁(FeSO 4·7H 2O)和硫酸钾(K 2SO 4)。
  5. 如权利要求4所述的微生物菌剂YF，其特征在于，所述微生物菌剂YF中各组分的含量为：
    Figure PCTCN2021077128-appb-100002
  6. 如权利要求4所述的微生物菌剂YF，其特征在于，所述载体为腐熟羊粪。
  7. 如权利要求5所述的微生物菌剂YF，其特征在于，所述微生物菌剂YF中各组分的含量为：
    Figure PCTCN2021077128-appb-100003
  8. 如权利要求1-7任一项所述的微生物菌剂YF在防治盐碱地玉米茎基腐病的药物制备中的应用。
PCT/CN2021/077128 2020-03-19 2021-02-20 一种有利于盐碱地玉米生长的微生物菌剂yf及其应用 WO2021185021A1 (zh)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111647527A (zh) * 2020-05-21 2020-09-11 浙江工业大学 一种利用枯草芽孢杆菌改良盐碱土和保肥的方法
CN113832060A (zh) * 2021-09-26 2021-12-24 青岛力力惠生物科技股份有限公司 一种抗重茬微生物菌剂及其在农业生产中的应用
CN114107130A (zh) * 2021-12-16 2022-03-01 天津科技大学 一种复合微生物菌肥及其制备方法和应用
CN114287195A (zh) * 2021-12-28 2022-04-08 滨州学院 一种提高滨海盐碱地防护林建造成活率的方法

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN113186132B (zh) * 2021-05-07 2023-05-23 广东丽豪生物农业有限公司 一种适于玉米种植的微生物菌剂和应用
CN113621532B (zh) * 2021-07-08 2024-01-16 东营市华科农业科技有限公司 含耐盐碱副地衣芽孢杆菌的复合微生物菌剂及其制备方法
CN114107096A (zh) * 2021-11-10 2022-03-01 甘肃省科学院生物研究所 玉米种植地生物法改善连作障碍微生物菌剂及其制备方法
CN114582439B (zh) * 2022-03-10 2022-11-04 新疆楚强生物科技有限公司 一种基于应用场景的土壤盐碱地改良剂筛选方法及***
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CN117431195B (zh) * 2023-12-21 2024-05-17 北京绿氮生物科技有限公司 一种耐盐碱的促生菌、促生菌菌剂及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015118516A1 (en) * 2014-02-10 2015-08-13 Biofil Mikrobiológiai, Géntechnológiai És Biokémiai Kft. Soil bacteria for inoculating stress soils
CN106947725A (zh) * 2017-05-24 2017-07-14 北京本农科技发展有限公司 微生物菌剂及其在盐碱地土壤改良中的应用
CN111304132A (zh) * 2020-03-19 2020-06-19 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） 一种有利于盐碱地玉米生长的微生物菌剂yf及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102039229B1 (ko) * 2017-12-07 2019-10-31 재단법인 포항테크노파크 비만, 당뇨와 지방간의 예방과 치료 효과를 갖는 바실러스 혼합 조성물 및 이의 용도
CN109082398B (zh) * 2018-10-19 2020-04-07 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） 一株防治稻瘟病的枯草芽孢杆菌及其应用
CN110373207A (zh) * 2019-07-29 2019-10-25 苏州沣懿生物科技有限公司 一种盐碱地改良的微生物菌剂及其制备工艺

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015118516A1 (en) * 2014-02-10 2015-08-13 Biofil Mikrobiológiai, Géntechnológiai És Biokémiai Kft. Soil bacteria for inoculating stress soils
CN106947725A (zh) * 2017-05-24 2017-07-14 北京本农科技发展有限公司 微生物菌剂及其在盐碱地土壤改良中的应用
CN111304132A (zh) * 2020-03-19 2020-06-19 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） 一种有利于盐碱地玉米生长的微生物菌剂yf及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SHA, YUEXIA ET AL.: "Preventive and Promoting Efficacy of Microbial Agents Mixed with Soil Against the Maize Stem Basal Rot in Ningxia", CHINESE AGRICULTURAL SCIENCE BULLETIN, vol. 49, no. 4, 15 February 2021 (2021-02-15), pages 141 - 144,154, XP055851417, ISSN: 1000-6850 *
WANG CQ ET AL.: "Effects of Bacillus velezensis FKM10 for Promoting the Growth of Malus hupehensis Rehd. and Inhibiting Fusarium verticillioides", FRONT MICROBIO, vol. 10, no. 1, 10 January 2020 (2020-01-10), pages 1 - 16, XP055851448, ISSN: 1664-302X *
WU. XIAOWEI ET AL.: "Breeding and compounding of saline-alkali-tolerant microbial compound and its improvement effect on saline-alkali soil", JIANGSU AGRICULTURAL SCIENCES, vol. 43, no. 6, 25 June 2015 (2015-06-25), pages 346 - 349, XP055851422, ISSN: 1002-1302 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111647527A (zh) * 2020-05-21 2020-09-11 浙江工业大学 一种利用枯草芽孢杆菌改良盐碱土和保肥的方法
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CN113832060B (zh) * 2021-09-26 2023-02-10 青岛力力惠生物科技股份有限公司 一种抗重茬微生物菌剂及其在农业生产中的应用
CN114107130A (zh) * 2021-12-16 2022-03-01 天津科技大学 一种复合微生物菌肥及其制备方法和应用
CN114107130B (zh) * 2021-12-16 2024-03-15 天津北洋百川生物技术有限公司 一种复合微生物菌肥及其制备方法和应用
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