WO2023240910A1 - 一株耐盐芽孢杆菌及其应用 - Google Patents

一株耐盐芽孢杆菌及其应用 Download PDF

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WO2023240910A1
WO2023240910A1 PCT/CN2022/130479 CN2022130479W WO2023240910A1 WO 2023240910 A1 WO2023240910 A1 WO 2023240910A1 CN 2022130479 W CN2022130479 W CN 2022130479W WO 2023240910 A1 WO2023240910 A1 WO 2023240910A1
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coletotrichum
bacillus
grape
strain
diaporthe
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French (fr)
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燕继晔
李兴红
张玮
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北京市农林科学院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
    • A01G17/02—Cultivation of hops or vines
    • 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
    • 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
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • 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
    • 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
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Definitions

  • the present disclosure belongs to the field of microbial application, and specifically relates to a salt-tolerant Bacillus (Bacilus halotolerans) and its application in preventing and treating plant diseases such as grape branch diseases.
  • Bacillus Bacillus halotolerans
  • Grape (Vitis vinifera L.) is one of the important fruit trees in the world. In 2020, the global grape planting area was 6.9509 million hectares, and the output was 78.0343 million tons. my country's grape planting area is 767,500 hectares, ranking second in the world; its output is 14.8431 million tons, ranking first in the world (FAO, 2020). Grapes are widely planted in my country, but most of the country's ecological zones are sub-suite for grape growing. Summers are hot and rainy, and in the north, the soil is buried to prevent cold in winter. Grape diseases have always been an important hidden danger that limits the yield and quality of my country's grapes. They occur all year round and need to be prevented. Including grape downy mildew, grape powdery mildew, grape gray mold, grape white rot, grape anthracnose and grape branch diseases.
  • Grape branch disease is an important vascular disease that harms grapes. There are many types, including grape wilt (Esca complex disease), grape canker (Botryosphaeria dieback), grape dieback (Eutypa dieback), Five species including Diaporthe dieback and Black foot disease (Ye Qingtong, Li Yameng, Zhou Yueyan, et al. The occurrence and harm of grape branch diseases at home and abroad and the types of pathogenic bacteria. Journal of Fruit Trees, 2021, 38 (2):278-292.). Grape branch diseases not only cause a decrease in the yield and quality of grapes, but also shorten the life of the tree and cause more serious losses. According to statistics, the global cost of renewing dead plants caused by grape branch diseases reaches 1.5 billion US dollars every year.
  • Grape branch diseases are a new type of diseases discovered in my country in the past ten years, among which grape canker and vine blight are the main types of grape branch diseases in my country (Yan J Y, Xie Y, Zhang W, et al.Species of Botryosphaeriaceae involved in grapevine dieback in China.Fungal Diverisity.2013,61:221-236.Manawasinghe I S, Dissanayake A J, Li X, et al. High Genetic Diversity and Species Complexity of Diaporthe Associated With Grapevine Dieback in China.Frontier s in Microbiology,2019,10:1936.). Since the pathogenic bacteria of grape branch diseases have the characteristics of latent infection and systemic agents have poor targeting effect on them, there is still a lack of effective prevention and control measures in current production. Therefore, it is urgent to explore efficient prevention and control measures for grape branch diseases.
  • Bacillus is rich in resources and is currently the most widely developed and applied type of biocontrol bacteria. It can produce spores to resist harsh conditions and reproduce quickly, which is beneficial to the production, processing, transportation and storage of preparations.
  • the bacteriostatic range of Bacillus is also very wide. Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, etc. have been used to control grape gray mold, downy mildew and other diseases. Grape diseases.
  • Bacillus subtilis can inhibit the growth of Lasiodiplodia theobromae, Neofusicoccum parvum and other pathogens that cause grape canker and cause mycelial deformities.
  • Field treatment of grape pruning wounds can significantly Reduce the incidence of grape canker (Rusin C, Rossicavalcanti F, Lima P, et al. Control of the fungi Lasiodiplodia theobromae, the causal agent of dieback, in cv.syrah grapevines. Acta Scientiarum Agronomy, 2020, 43:e44785.) .
  • Bacillus halotolerans is a Bacillus with biocontrol potential.
  • halotolerant Bacillus also has the function of promoting plant growth (Eirini-Evangelia Thomloudi, and Panagiotis Katinakis. Genomic and metabolomic insights into secondary metabolites of the novel Bacillus halotolerans Hil4, an endophyte with promising antagonistic activity against gray mold and plant growth promote ing potential,Microorganisms,2021,9:2508). Therefore, screening Bacillus that can control grape branch diseases can open up new ways for the effective control of grape branch diseases. Since there are certain differences in the biocontrol potential between different strains of the same Bacillus, the development and application of Bacillus biocontrol preparations requires in-depth exploration and full development of the biocontrol potential of the target strains.
  • the present disclosure provides a strain of halotolerant Bacillus and its application.
  • halotolerant Bacillus of the present disclosure is classified and named as halotolerant Bacillus (Bacilus halotolerans). It is collected from Huailai County, Zhangjiakou, Hebei province. It is isolated from grape diseased branch tissue of grape branches and is named halotolerant Bacillus (Bacilus). halotolerans) BJ-3, has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing) on December 16, 2021, with the deposit number CGMCC No. 24115 .
  • the halotolerant Bacillus of the present disclosure does not produce pigment on the LB medium, and the colonies are milky white and opaque, round or nearly round, with rough edges and obvious wrinkles on the surface.
  • the physiological and biochemical characteristics of the halotolerant Bacillus BJ-3 are positive Gram staining, can produce cellulase, but does not produce protease or amylase.
  • the halotolerant Bacillus of the present disclosure has antibacterial activity against plant pathogenic fungi and has a broad antibacterial spectrum.
  • the plant pathogenic fungi are Lasiodiplodia theobromae, Botryosphaeria dothidea, Neofusicoccum parvum, Diaporthe sojae, and Diaporthe eres), Diaporthe honkonggensis, Botrytis cinerea, Coletotrichum viniferum, Coletotrichum aenigma, Coletotrichum gloeosporioides, Coletotrichum fructicola, Siam Colletotrichum siamense, Coletotrichum acutatum, Neopestalotiopsis sp., Neopestalotiopsis rosae, Coniela vitis, Dactylonectria macrodidyma, Fusarium oxysporum, dikaryotic Rhizoctonia fusion group A, dikaryotic Rhizoctonia Fusion group G, Alter
  • the halotolerant Bacillus BJ-3 strain of the present disclosure can inhibit the conidial germination of Lasiodiplodia theobromae, with an EC50 value of 3.35 ⁇ 10 6 cfu/mL.
  • the results of the indoor disease prevention test by the in vitro branch inoculation method and the field efficacy test show that the halotolerant Bacillus BJ-3 strain of the present disclosure has a good control effect on grape canker.
  • the disclosed halotolerant Bacillus BJ-3 strain has a significant inhibitory effect on grape canker on isolated green branches of grapes, and the fermentation broth of strain BJ-3 with a concentration of 1.08 ⁇ 10 8 CFU/mL has a protective effect on grape canker.
  • the therapeutic effect and prevention effect are 91.75% and 86.94% respectively;
  • the disclosed salt-tolerant Bacillus BJ-3 strain fermentation broth has a field control effect of 65.56% on grape branch diseases at a concentration of 5.6 ⁇ 10 7 CFU/mL, which is significantly better than the control biocontrol agents and chemical agents. It has biocontrol potential against grape canker. It can be used to prepare biocontrol agents or microbial fertilizers against grape branch disease fungi.
  • the pathogenic fungi of grape branch diseases are Lasiodiplodia theobromae, Neofusicoccum parvum, Botryosphaeria dothidea, Diaporthe eres, and Soybean Diaporthe sojae, Neopestalotiopsis sp., Dactylonectria macrodidyma.
  • halotolerant Bacillus described in the present disclosure in promoting the growth of grape seedlings also falls within the protection scope of the present disclosure.
  • the halotolerant Bacillus fermentation culture liquid, the dilution of the fermentation culture liquid, the fermentation culture liquid filtrate or the dilution of the fermentation culture liquid filtrate is used to perform root irrigation treatment on the grape seedlings.
  • the diluted solution of the fermentation culture liquid filtrate is used for root irrigation treatment.
  • the dilution of the fermentation culture liquid filtrate is obtained by diluting the fermentation culture liquid filtrate 10,000 times with clean water.
  • the dilution of the fermentation culture liquid filtrate is to pour the seed liquid into a fermentation culture medium containing 100mL (maltose 20g/L, peptone 10g/L, bran 10g/L, CaCO 3 10g/L, NaHPO 3 2g/L , KH 2 PO 3 1g/L; pH value 6.8) in a 500mL triangular flask, with an inoculation amount of 10%, and cultured with shaking at 28°C and 180rpm/min for 72h to obtain the fermentation broth; the fermentation broth was centrifuged in a high-speed refrigerated centrifuge at 4°C and 10000rpm for 15 -20 minutes, collect the supernatant and filter it through a bacterial filter, discard the precipitate, and obtain a sterile fermentation filtrate.
  • a fermentation culture medium containing 100mL (maltose 20g/L, peptone 10g/L, bran 10g/L, CaCO 3 10g/L, NaHPO 3 2g/L
  • the halotolerant Bacillus of the present disclosure has antibacterial activity against plant pathogenic fungi and has a broad antibacterial spectrum, and particularly has a good control effect on grape canker.
  • This strain has a good growth-promoting effect on grape seedlings.
  • This strain has simple culture conditions, rapid growth and easy preservation, and is suitable for industrial production.
  • Figure 1 shows the colony morphology and scanning electron micrograph of strain BJ-3.
  • Figure 2 shows the amylase detection results of strain BJ-3; among them, left: Escherichia coli JM109, right: strain BJ-3.
  • Figure 3 shows the detection results of strain BJ-3 protease; among them, left: Escherichia coli JM109, right: strain BJ-3.
  • Figure 4 shows the cellulase detection results of strain BJ-3; among them, left: Escherichia coli JM109, right: strain BJ-3.
  • Figure 5 shows a phylogenetic tree based on 16SrDNA.
  • Figure 6 shows a phylogenetic tree based on the gyrB gene.
  • Figure 7 shows the growth-promoting effect of different treatments of strain BJ-3 on grape seedlings (42d); where, a: CK, b: 10000-fold dilution of the fermentation medium for BJ-3 culture, c: BJ-3 fermentation Filtrate 10000 times dilution, d: 5.8 ⁇ 10 6 CFU/mL BJ-3 fermentation bacteria liquid.
  • Figure 8 shows the effects of different treatments of strain BJ-3 on the growth of new grape seedlings; among them, treatment 1 is the BJ-3 fermentation broth with a concentration of 5.8 ⁇ 10 6 CFU/mL, and treatment 2 is the fermentation culture for BJ-3 culture.
  • the base is a 10,000-fold dilution
  • treatment 3 is a 10,000-fold dilution of BJ-3 fermentation filtrate
  • treatment 4 is a clean water control.
  • Figure 9 shows the effects of different treatments of strain BJ-3 on the leaf area of grape seedlings; among them, treatment 1 is the BJ-3 fermentation broth with a concentration of 5.8 ⁇ 10 6 CFU/mL, and treatment 2 is the fermentation medium for BJ-3 culture. 10,000-fold dilution, treatment 3 is a 10,000-fold dilution of BJ-3 fermentation filtrate, and treatment 4 is a clean water control.
  • Figure 10 shows the effects of different treatments of strain BJ-3 on the chlorophyll content of grape seedlings; among them, treatment 1 is the BJ-3 fermentation broth with a concentration of 5.8 ⁇ 10 6 CFU/mL, and treatment 2 is the fermentation medium for BJ-3 culture. 10,000-fold dilution, treatment 3 is a 10,000-fold dilution of BJ-3 fermentation filtrate, and treatment 4 is a clean water control.
  • the tissue separation method was used to isolate pathogenic bacteria. The method is as follows: (1) Observe and record the symptoms of grape branch disease; ( 2) Cut 2cm branches at intervals of 10cm from the parts with different degrees of disease, remove the diseased bark of the grape branches, cut out 5mm2 tissue blocks from the diseased and healthy junction of the branch tissue, and use 2% sodium hypochlorite Disinfect for 2 minutes, disinfect with 70% ethanol for 30 seconds, wash with sterile water 3 times, put sterilized filter paper on the tissue block to dry, and after the water is absorbed, place the tissue block on the PDA plate.
  • Amylase selective medium (1L): 1g soluble starch, 5g peptone, 5g glucose, 5g NaCl, 5g beef extract, 15g agar.
  • Detection method After culturing the strain to be tested in LB solid medium for 3 days, pick a single bacterial colony and connect it to the amylase detection medium, incubate at a constant temperature of 28°C for 24 to 48 hours, and observe the transparent circle.
  • Test results As shown in Figure 2, there is no obvious transparent circle around the colony of the strain to be tested, indicating that strain BJ-3 does not produce amylase.
  • Detection plate Protease selection medium (1L): 10.0g peptone, 5.0g NaCl, 0.1g CaCl 2 , 100.0mL skim milk (added when the medium is 45°C after sterilization), 15g agar.
  • Detection method After culturing the strain to be tested in LB solid medium for 3 days, pick a single bacterial colony and connect it to the protease detection medium, incubate at a constant temperature of 28°C for 24 to 48 hours, and observe the transparent circle.
  • Detection plate Cellulase selection medium (1L): CMC-Na 10g, peptone 10g, yeast powder 10g, KH 2 PO 4 1g, NaCl 15g, agar 15g
  • Detection method After culturing the strain to be tested in LB solid medium for 3 days, pick a single bacterial colony and connect it to the cellulase detection medium, incubate at a constant temperature of 28°C for 24-48 hours, and observe the transparent circle.
  • Test results As shown in Figure 4, there is an obvious transparent circle around the colony of the strain to be tested, indicating that strain BJ-3 produces cellulase.
  • the genomic DNA of BJ-3 was extracted, and the bacterial genomic DNA was used as a template, and bacterial 16S rDNA universal primers 27f and 1492r were used for PCR amplification.
  • reaction system used for PCR amplification 2 ⁇ Taq PCR Mix 25 ⁇ L, DNA template (100 ng/ ⁇ L) 2 ⁇ L, upstream and downstream primers (10 ⁇ mol/L) 1 ⁇ L each, make up 50 ⁇ L system with sterile ddH 2 O.
  • PCR reaction program 94°C for 3min; 35 cycles of 94°C for 30s, 54°C for 30s, 72°C for 90s; 72°C for 10min; insulation: 4°C.
  • the PCR amplification product was detected by electrophoresis, and the fragment length was approximately 1500 bp (sequence 1 in the sequence listing).
  • the genomic DNA of bacteria BJ-3 was extracted, and the genomic DNA of this bacterial strain was used as a template, and the bacterial gyrB gene universal primers gyrB-F and gyrB-R were used for PCR amplification.
  • gyrB-F 5’-TTGRCGGHRGYGGHTATAAAGT-3’
  • reaction system used for PCR amplification 2 ⁇ Taq PCR Mix 25 ⁇ L, DNA template (100 ng/ ⁇ L) 2 ⁇ L, upstream and downstream primers (10 ⁇ mol/L) 1 ⁇ L each, make up 50 ⁇ L system with sterile ddH 2 O.
  • PCR reaction program 94°C for 4 min; 35 cycles of 94°C for 30 s, 55°C for 45 s, 72°C for 1 min; 72°C for 10 min; insulation: 4°C.
  • the PCR amplification product was detected by electrophoresis, and the fragment length was approximately 900 bp (sequence 2 in the sequence listing).
  • strain BJ-3 was different from halotolerant Bacillus strain CR-119 ( GenBank accession number: NR115283) and halotolerant Bacillus strain CECT 5687 (GenBank accession number: DQ903179) were aggregated together. Based on this, strain BJ-3 was identified as halotolerant Bacillus (Bacilus halotolerans). Strain BJ-3 is deposited in the China General Microbial Culture Collection and Management Center (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), with a deposit date of December 16, 2021, and a deposit number of CGMCC No.24115.
  • Cultivate BJ-3 on LB solid medium at 25°C for 2 to 3 days then pick a single colony into 1 mL of LB liquid medium, shake culture at 37°C and 200 rpm/min for 8 to 10 hours, and set aside.
  • Inhibition rate (%) (radius of control colony - radius of treated colony)/radius of control colony ⁇ 100%
  • strain BJ-3 has obvious inhibitory effects on different plant pathogenic fungi tested.
  • the inhibition rate on mycelial growth ranges from 54.74% to 91.62%.
  • Diaporthe sojae OSS1 has the best mycelial growth inhibition effect, with an inhibition rate of 91.62%.
  • Research results show that BJ-3 has a broad spectrum of inhibitory effects on common plant pathogenic bacteria.
  • the length of the conidial germ tube exceeds 1/2 of the spore diameter, it is recorded as a germinated spore.
  • the spore germination rate of the blank control reaches more than 90%, check the spore germination status of each treatment. Each treatment was repeated and randomly observed more than 3 visual fields, the total number of spores investigated was not less than 200, and the germination number and total number of spores were recorded respectively. Then convert the bacterial solution concentration-inhibition rate into the corresponding logarithm-probability value, and calculate the virulence regression equation, correlation coefficient (r) and EC 50 .
  • the indoor biological activity measurement results of strain BJ-3 against grape canker fungus GX-5-5 are shown in Table 2.
  • the results show that the BJ-3 bacterial liquid has a significant inhibitory effect on the germination of GX-5-5 spores, and the test concentration is 8 ⁇
  • the inhibitory rate of BJ-3 bacterial suspension of 10 5 to 4 ⁇ 10 7 CFU/mL on the conidial germination of grape canker ranges from 16.72% to 98.47%, in which the BJ-3 bacterial suspension concentration is 2 ⁇ 10 7 CFU/mL.
  • the inhibition rates of spore germination were 97.95% and 98.47% respectively, the EC 50 value was 3.35 ⁇ 10 6 cfu/mL, and the r value was above 0.9. Therefore, the effect of strain BJ-3 on grape Canker fungus has a strong inhibitory effect on spore germination, and may have the ability to control grape canker by inhibiting the infection of the host by the fungus.
  • Example 4 Determination of the preventive and therapeutic effect of strain BJ-3 on grape canker by in vitro branch inoculation method
  • the inoculum uses annual grape green branches. On the day of inoculation, collect healthy semi-lignified summer black grape green branches from the vineyard. After cutting off the leaves, clean the surface with clean water first, and then wipe and disinfect with 75% alcohol wipes before use. Rinse the surface with sterile water and dry it for later use.
  • the pathogenic bacteria tested was grape canker strain GX-5-5.
  • the pathogenic fungi tested were cultured on PDA medium at 28°C for 3 days and set aside for later use.
  • the spray method to treat the disinfected grape green branches with the fermentation broth of strain BJ-3.
  • the treatment concentrations are BJ-31.08 ⁇ 10 6 CFU/mL, 1.08 ⁇ 10 7 CFU/mL, and 1.08 ⁇ 10 8 CFU/mL, and then
  • the green branches were inserted into a 9 cm diameter plastic bowl filled with moist vermiculite substrate and cultured in an artificial climate chamber at 25°C and 60% relative humidity for 24 hours.
  • each treatment was inoculated with 7 branches and repeated three times. After inoculation, continue culturing for 48 hours in the inoculation room at 25°C with a relative humidity of 90%, and then adjust the relative humidity to 60%. After 7 to 10 days of inoculation, the incidence and lesion length of each treatment were measured using SPSS 18 software. Perform statistical analysis on the data. Calculated as follows:
  • Control effect (%) [(control lesion length - treated lesion length)/(control lesion length - bacterial cake diameter)] ⁇ 100%
  • the therapeutic effect was measured by inoculating the bacteria first, and then treating the inoculated green branches with different bacterial solutions and sterile water 48 hours after inoculation.
  • the methods and materials used were the same as those for the protective effect measurement.
  • control effect is 86.94%
  • control effect of the control agent Bacillus is 73.72%
  • control effect of BJ-3 fermentation broth with a concentration of 1.08 ⁇ 10 7 CFU/mL on grape canker is 51.03%
  • control effect of the control agent is 51.03%.
  • Fermentation medium maltose 20g/L; peptone 10g/L; CaCO 3 10g/L; NaHPO 3 2g/L; KH 2 PO 3 1g/L, bran 10g/L; the balance is water; adjust ⁇ 6.8.
  • Fermentation conditions temperature 32° ⁇ , stirring speed 200r/min, ventilation volume 1:0.5-1:1.2, culture time 22h. After culturing in a 120L fermentation tank, BJ-3 fermentation broth was obtained with a concentration of 5.6 ⁇ 10 9 CFU/mL.
  • the evaluation of the control effect of the fermentation broth of strain BJ-3 on grape branch diseases was carried out using the field plot comparative test method.
  • the test site was selected on varieties where grape branch diseases occur all year round and are susceptible to infection.
  • the location was COFCO, Huailai County, Zhangjiakou City, Hebei province.
  • Treatment 1 is BJ-3 fermentation broth with a concentration of 5.6 ⁇ 10 7 CFU/mL; Treatment 2 is 10 billion CFU/g Bacillus subtilis WP 1000 times liquid; Treatment 3 is 10% difenoconazole WG 1000 times liquid; Treatment 4 is a blank control with clean water; the application method is root irrigation, 1L per plant, more than 200 grape plants per treatment, 3 repetitions, apply once on July 9 and July 20, 2021, before application and 70 days after application (grape near-maturity period) to investigate the disease status of grape plants respectively, and calculate the diseased plant rate (%) and control effect (%).
  • the calculation formula is as follows:
  • Control effect (%) [1-(pre-drug diseased plant rate in the blank control area ⁇ post-drug diseased plant rate in the treatment area)/(post-drug diseased plant rate in the blank control area ⁇ pre-drug diseased plant rate in the treatment area)] ⁇ 100%
  • strain BJ-3 fermentation broth on grape branch diseases The field disease prevention effect of strain BJ-3 fermentation broth on grape branch diseases is shown (Table 4), 5.6 ⁇ 10 7 CFU/mL BJ-3 fermentation broth, 10 billion CFU/g Bacillus subtilis WP 1000 times solution and 10 % difenoconazole WG 1000 times, the average control effect after root irrigation application of three pesticides was 65.56%, 42.34% and 27.30% respectively.
  • the BJ-3 fermentation broth with a concentration of 5.6 ⁇ 10 7 CFU/mL had a negative impact on grapes.
  • the control effect of canker disease was extremely significantly higher than that of the other two chemical treatments, and there was no significant difference between the latter two. During the entire test process, the three chemical treatments showed no harm to the test crop grapes.
  • the field efficacy test results show that the average control effect of the two tested fungicides and BJ-3 fermentation bacteria on grape branch diseases is 27.30-65.56%, among which BJ-3 fermentation bacteria (5.6 ⁇ 10 7 CFU/mL), the highest efficacy was 65.56%, significantly compared with Bacillus subtilis and the chemical fungicide difenoconazole, which have biocontrol potential in preventing grape branch diseases.
  • the average disease rate in the table is the average of three replicates.
  • Preparation of seed liquid Use a 250 mL triangular flask to put 100 mL of sterilized LB liquid culture medium. Pick a single colony from BJ-3 and insert it into the LB liquid culture medium. Shake at 28°C and 180 rpm/min. Incubate for 12h.
  • Fermentation culture BJ-3: Pour the seed liquid into 100mL of fermentation medium (maltose 20g/L, peptone 10g/L, bran 10g/L, CaCO 3 10g/L, NaHPO 3 2g/L , KH 2 PO 3 1g/L; pH value 6.8) in a 500mL Erlenmeyer flask, with an inoculation amount of 10%, and cultured with shaking at 28°C and 180rpm/min for 72h to obtain a fermentation broth.
  • fermentation medium maltose 20g/L, peptone 10g/L, bran 10g/L, CaCO 3 10g/L, NaHPO 3 2g/L , KH 2 PO 3 1g/L; pH value 6.8
  • the grape variety tested was the 'Marseland' variety, and the material was cutting seedlings.
  • the cutting seedlings were planted in flowerpots with a diameter of 22.5cm and a height of 13.5cm.
  • the root irrigation method was used to determine the growth-promoting effect of strain BJ-3 fermentation broth on grape seedlings. When the grape seedlings grow to 5-7 leaves, root irrigation treatment is carried out. Each pot of grape seedlings is filled with 150mL of roots. There are 4 treatments in total. Treatment 1 is BJ-3 fermentation broth with a concentration of 5.8 ⁇ 10 6 CFU/mL, and treatment 2 is It is a 10,000-fold dilution of the fermentation medium for BJ-3 culture (the medium without cultivating BJ-3). Treatment 3 is a 10,000-fold dilution of BJ-3 fermentation filtrate. Treatment 4 is a clear water control. Each treatment has 10 grape seedlings. . The grape plants were placed in the greenhouse and cultured at 25 ⁇ 2°C.
  • the test results of the growth-promoting effects of different treatments of strain BJ-3 on grape seedlings are shown in Figures 7 to 10.
  • the results show that the BJ-3 fermentation broth (treatment 1) with a concentration of 5.8 ⁇ 10 6 CFU/mL has a positive effect on the new growth of grape seedlings. There was no obvious promotion effect.
  • the 10,000-fold dilution of BJ-3 fermentation filtrate (Treatment 3) significantly increased the leaf area and chlorophyll content of grape seedlings by 8.67% and 8.97% respectively compared with the clear water control (Treatment 4). This indicates that strain BJ-3 may have the ability to improve the photosynthetic performance of grape seedlings by increasing leaf area and chlorophyll content.

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Abstract

一株耐盐芽孢杆菌BJ-3及其应用，该耐盐芽孢杆菌菌株BJ-3的保藏编号为CGMCC No.24115。该耐盐芽孢杆菌菌株具有对植物病原菌的抑菌活性并且具有较宽的抑菌谱。室内和田间试验结果表明，BJ-3发酵液对葡萄枝干病害具有一定的防治效果。此外，该菌株发酵滤液对葡萄叶面积和叶绿素含量具有明显的促进作用。

Description

一株耐盐芽孢杆菌及其应用 技术领域
本公开属于微生物应用领域，具体涉及一株耐盐芽孢杆菌(Bacilus halotolerans)及其在防治葡萄枝干病害等植物病害上的应用。
背景技术
葡萄(Vitis vinifera L.)是世界上重要的果树之一，2020年，全球葡萄种植面积为695.09万公顷，产量为7803.43万吨。我国葡萄种植面积为76.75万公顷，位居世界第二；产量为1484.31万吨，位居世界首位(FAO,2020)。葡萄在我国种植广泛，但我国多数生态区为次适宜葡萄种植区，夏季炎热多雨，北方冬季埋土防寒，葡萄病害一直是限制我国葡萄的产量和品质的重要隐患，常年发生和需要防治的病害包括葡萄霜霉病、葡萄白粉病、葡萄灰霉病、葡萄白腐病、葡萄炭疽病和葡萄枝干病害等。
葡萄枝干病害是危害葡萄的一类重要的维管束病害，种类较多，主要包括葡萄衰枯病(Esca complex disease)、葡萄溃疡病(Botryosphaeria dieback)、葡萄顶枯病(Eutypa dieback)、葡萄蔓枯病(Diaporthe dieback)、葡萄黑根病(Black foot disease)等5种(叶清桐,李亚萌,周悦妍,等.国内外葡萄枝干病害的发生危害与病原菌种类.果树学报,2021,38(2):278-292.)。葡萄枝干病害不仅引起葡萄的产量、品质下降还可缩短树体寿命造成更严重的损失，据统计，全球每年仅用于更新种植葡萄枝干病害引起的死亡植株的费用达15亿美元，被认为是未来30年影响葡萄产业可持续健康发展的重要病害之一(Hofstetter V,Buyck B,Croll D,et al.What if esca disease of grapevine were not a fungal disease？.Fungal Diversity,2012,54(1):51-67.)。葡萄枝干病害是我国近十几年来新发现的一类病害，其中葡萄溃疡病和蔓枯病是我国主要的葡萄枝干病害类型(Yan J Y,Xie Y,Zhang W,et al.Species of Botryosphaeriaceae involved in grapevine dieback in China.Fungal Diverisity.2013,61:221-236.Manawasinghe I S,Dissanayake A J,Li X,et al.High Genetic Diversity and Species Complexity of Diaporthe Associated With Grapevine Dieback in China.Frontiers in Microbiology,2019,10:1936.)。由于葡萄枝干病害病原菌具有潜伏侵染的特点，同时内吸性药剂对其靶向效果差，当前生产中仍然缺乏有效的防控措施。因此，探索葡萄枝干病害的高效防控措施迫在眉睫。
生物防治具有无污染、不易产生抗性等优点，是防控作物病害的重要措施之一，近年来在农业绿色高质量发展的背景下，得到了高度重视和广泛应用。芽孢杆菌资源丰富，是目前开发应用最广泛的一类生防细菌。能产生芽孢抵抗恶劣的条件，且繁殖速度快，有利于制剂的生产、加工、运输和贮藏。芽孢杆菌的抑菌范围也很广，枯草芽孢杆菌(Bacilus subtilis)、解淀粉芽孢杆菌(Bacilus amyloliquefaciens)、短小芽孢杆菌(Bacilus pumilus)等已应用于防治葡萄灰霉病、霜霉病等多种葡萄病害。已有研究发现枯草芽孢杆菌能抑制引起葡萄溃疡病的可可毛色二孢(Lasiodiplodia theobromae)、小新壳梭孢(Neofusicoccum parvum)等病菌的生长并引起菌丝畸形，田间处理葡萄剪枝伤口可以显著降低葡萄溃疡病的发生率(Rusin C,Rossicavalcanti F,Lima P,et al.Control of the fungi Lasiodiplodia theobromae,the causal agent of dieback,in cv.syrah grapevines.Acta Scientiarum Agronomy,2020,43:e44785.)。耐盐芽孢杆菌(Bacillus halotolerans)是一种具有生防潜力的芽孢杆菌，近年来其对植物病害的控制作用得到了重视，研究表明耐盐芽孢杆菌对棉花黄萎病具有控制作用，对葡萄灰霉病、番茄灰霉病、番茄根结线虫病、小麦赤霉病和豌豆根腐病等也有一定的抑制作用(Eirini-Evangelia Thomloudi,and Panagiotis Katinakis.Genomic and metabolomic insights into secondary metabolites of the novel Bacillus halotolerans Hil4,an endophyte with promising antagonistic activity against gray mold and plant growth promoting potential,Microorganisms,2021,9:2508；Muhammad Nadeem Hassan，Vegetable associated Bacillus spp.suppress the pea(Pisum sativum L.)root rot caused by Fusarium solani Raheela Riaz 1,Biological Control 2021:104610；Bacillus halotolerans strain LYSX1-induced systemic resistance against the root-knot nematode Meloidogyne javanica in tomato，Ann Microbiol(2019)69:1227–1233)。另外，耐盐芽孢杆菌还具有促进植物生长的功能(Eirini-Evangelia Thomloudi,and Panagiotis Katinakis.Genomic and metabolomic insights into secondary metabolites of the novel Bacillus halotolerans Hil4,an endophyte with promising antagonistic activity against gray mold and plant growth promoting potential,Microorganisms,2021,9:2508)。因此，筛选具有对葡萄枝干病害有控制作用的芽孢杆菌，能够为葡萄枝干病害的有效控制开辟新途径。而由于同一种芽孢杆菌不同株系间的生防潜力具有一定差异，因此芽孢杆菌生防制剂的开发应用过程中，需要深入挖掘和充分开发目标菌株的生防潜力。
发明内容
为了解决上述问题，本公开提供了一株耐盐芽孢杆菌及其应用。
本公开的耐盐芽孢杆菌，分类命名为耐盐芽孢杆菌(Bacilus halotolerans)，采集自河北省张家口怀来县，分离自葡萄枝干病害的葡萄病枝干组织，命名为耐盐芽孢杆菌(Bacilus halotolerans)BJ-3，已于2021年12月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号)，保藏编号为CGMCC No.24115。
本公开的耐盐芽孢杆菌在LB培养基上不产色素，菌落为乳白色不透明，圆形或近圆形，边缘不光滑，表面有明显褶皱。所述耐盐芽孢杆菌BJ-3的生理生化特征为革兰氏染色阳性，可产纤维素酶，不产蛋白酶、淀粉酶。
本公开的耐盐芽孢杆菌具有对植物病原真菌的抑菌活性并且具有较宽的抑菌谱。所述植物病原真菌为可可毛色二孢(Lasiodiplodia theobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthe sojae)、甜樱间座壳(Diaporthe eres)、Diaporthe honkonggensis、灰葡萄孢(Botrytis cinerea)、葡萄炭疽菌(Coletotrichum viniferum)、隐秘炭疽菌(Coletotrichum aenigma)、胶孢炭疽菌(Coletotrichum gloeosporioides)、果生炭疽菌(Coletotrichum fructicola)、暹罗炭疽菌(Coletotrichum siamense)、尖孢炭疽菌(Coletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsis rosae、Coniela vitis、Dactylonectria macrodidyma、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)、核盘菌(Sclerotinia sclerotiorum)。对上述22种植物病原真菌45个菌株的菌丝生长抑制率在54.74％-91.62％之间。该菌株在植物真菌病害生物制剂开发方面具有非常好的应用前景。
本公开的耐盐芽孢杆菌BJ-3菌株可抑制可可毛色二孢(Lasiodiplodia theobromae)分生孢子萌发，EC50值为3.35×10 6cfu/mL。
室内离体枝条接种法防病试验和田间药效试验结果表明，本公开的耐盐芽孢杆菌BJ-3菌株对葡萄溃疡病具有较好的控制作用。本公开的耐盐芽孢杆菌BJ-3菌株对葡萄离体绿枝条上的葡萄溃疡病有明显抑制作用，浓度为1.08×10 8CFU/mL的菌株BJ-3发酵液对葡萄溃疡病的保护作用和治疗作用防治效果分别为91.75％和86.94％；
本公开的耐盐芽孢杆菌BJ-3菌株发酵液在浓度为5.6×10 7CFU/mL时对葡萄枝干病害 的田间防治效果可达65.56％，显著优于对照生防菌剂和化学药剂，具有防治葡萄溃疡病的生防潜力。可用于制备针对葡萄枝干病害真菌的生防菌剂或微生物菌肥。其中，葡萄枝干病害病原真菌为可可毛色二孢(Lasiodiplodia theobromae)、小新壳梭孢(Neofusicoccum parvum)、葡萄座腔菌(Botryosphaeria dothidea)、甜樱间座壳(Diaporthe eres)、大豆间座壳(Diaporthe sojae)、Neopestalotiopsis sp.、Dactylonectria macrodidyma。
本公开所述的耐盐芽孢杆菌在促进葡萄幼苗生长中的应用也属于本公开的保护范围。所述应用中，利用所述的耐盐芽孢杆菌发酵培养液、发酵培养液的稀释液、发酵培养液滤液或者发酵培养液滤液的稀释液，对葡萄幼苗进行灌根处理。
优选的，当葡萄幼苗长至5-7片叶，用发酵培养液滤液的稀释液进行灌根处理。
在本公开的一个优选的技术方案中，发酵培养液滤液的稀释液是将发酵培养液滤液用清水稀释10000倍得到的。
其中，发酵培养液滤液的稀释液是将种子液倒入装有100mL的发酵培养基(麦芽糖20g/L，蛋白胨10g/L，麸皮10g/L，CaCO 3 10g/L，NaHPO 3 2g/L，KH 2PO 3 1g/L；pH值为6.8)的500mL三角瓶中，接种量10％，28℃180rpm/min振荡培养72h，得到发酵液；发酵液经高速冷冻离心机4℃10000rpm离心15-20min，收集上清并经细菌过滤器过滤，丢弃沉淀，即得到无菌的发酵滤液。
本公开的耐盐芽孢杆菌具有对植物病原真菌的抑菌活性并且具有较宽的抑菌谱，特别对葡萄溃疡病具有较好的控制作用。该菌株对葡萄幼苗具有很好的促生作用。该菌株培养条件简单、生长迅速且容易保存，适合用于工业生产。
附图说明
图1示出了菌株BJ-3的菌落形态和扫描电镜照片。
图2示出了菌株BJ-3的淀粉酶检测结果；其中，左：大肠杆菌Escherichia coli JM109，右：菌株BJ-3。
图3示出了菌株BJ-3蛋白酶检测结果；其中，左：大肠杆菌Escherichia coli JM109，右：菌株BJ-3。
图4示出了菌株BJ-3纤维素酶检测结果；其中，左：大肠杆菌Escherichia coli JM109，右：菌株BJ-3。
图5示出了基于16SrDNA系统进化树。
图6示出了基于gyrB基因的系统进化树。
图7示出了菌株BJ-3不同处理对葡萄幼苗的促生效果(第42d)；其中，a：CK，b：BJ-3培养用发酵培养基10000倍稀释液，c：BJ-3发酵滤液10000倍稀释液，d：5.8×10 6CFU/mL的BJ-3发酵菌液。
图8示出了菌株BJ-3不同处理对葡萄幼苗新稍生长的影响；其中，处理1为浓度5.8×10 6CFU/mL的BJ-3发酵液，处理2为BJ-3培养用发酵培养基10000倍稀释液，处理3为BJ-3发酵滤液10000倍稀释液，处理4为清水对照。
图9示出了菌株BJ-3不同处理对葡萄幼苗叶面积的影响；其中，处理1为浓度5.8×10 6CFU/mL的BJ-3发酵液，处理2为BJ-3培养用发酵培养基10000倍稀释液，处理3为BJ-3发酵滤液10000倍稀释液，处理4为清水对照。
图10示出了菌株BJ-3不同处理对葡萄幼苗叶绿素含量的影响；其中，处理1为浓度5.8×10 6CFU/mL的BJ-3发酵液，处理2为BJ-3培养用发酵培养基10000倍稀释液，处理3为BJ-3发酵滤液10000倍稀释液，处理4为清水对照。
具体实施方式
实施例1.菌株BJ-3的获得和鉴定
采集自河北省张家口怀来县，分离自表现葡萄枝干病害症状的葡萄病枝干组织，采用组织分离法进行病原菌分离，方法如下：(1)观察并记录葡萄枝干病样的症状；(2)对不同发病程度的部分每间隔10cm各切取2cm的枝干，去掉葡萄枝干病样的树皮，从枝干组织的病健交界处切出5mm 2的组织块，用2％的次氯酸钠消毒2分钟，70％的乙醇消毒30s，无菌水洗3次，把组织块上放灭菌的滤纸晾干，待水分吸干之后把组织块放在PDA平板上，每个PDA平板放置4～5个组织块，封口膜封口；(3)于25℃，黑暗条件下培养3d，3d后观察、统计菌落生长情况并计算相应的分离比率；(4)挑取菌落边缘少量菌丝于新的PDA或其他易诱导产孢的培养基中，待产生分生孢子后进行单孢分离。在分离病原真菌的过程中，发现一株细菌对病原真菌具有很好的拮抗作用，将其在LB培养基平板划线纯化，获得细菌的纯培养物，经鉴定，获得了一株对病原菌具有拮抗作用的菌株，将其命名为BJ-3，菌株BJ-3的菌落形态如图1所示，菌落为圆形或近圆形，乳白色，边缘不光滑，表面有不明显褶皱。
1.菌株BJ-3的生理生化特征
1.1淀粉酶的检测
检测培养基：淀粉酶选择培养基(1L)：可溶性淀粉1g，蛋白胨5g，葡萄糖5g，NaCl 5g，牛肉膏5g，琼脂15g。
检测方法：将待测菌株在LB固体培养基中培养3d后，挑取单菌落点接在淀粉酶检测培养基上，28℃恒温培养24～48h，观察透明圈。
检测结果：如图2所示，待测菌株菌落外围没有明显透明圈产生，说明菌株BJ-3不产生淀粉酶。
1.2蛋白酶检测
检测平板：蛋白酶选择培养基(1L)：蛋白胨10.0g，NaCl 5.0g，CaCl 2 0.1g，脱脂牛奶100.0mL(灭菌之后待培养基45℃时加入)，琼脂15g。
检测方法：将待测菌株在LB固体培养基中培养3d后，挑取单菌落点接在蛋白酶检测培养基上，28℃恒温培养24～48h，观察透明圈。
检测结果：如图3所示，待测菌株菌落外围没有明显透明圈产生，说明菌株BJ-3不产生蛋白酶。
1.3纤维素酶检测
检测平板：纤维素酶选择培养基(1L)：CMC-Na 10g，蛋白胨10g，酵母粉10g，KH 2PO 41g，NaCl 15g，琼脂15g
检测方法：将待测菌株在LB固体培养基中培养3d后，挑取单菌落点接在纤维素酶检测培养基上，28℃恒温培养24-48h，观察透明圈。
检测结果：如图4所示，待测菌株菌落外围有明显透明圈产生，说明菌株BJ-3产生纤维素酶。
2菌株BJ-3的分子鉴定
2.1基因组DNA的提取：
将保存的菌液涂平皿后，25℃培养3d后，用接种环刮下菌体后使用LB液体培养基洗出，采用北京博迈德基因技术有限公司细菌基因组DNA快速提取试剂盒提取细菌的基因组DNA。
2.2 16S rDNA的PCR扩增：
对BJ-3进行基因组DNA的提取，并以这株细菌基因组DNA为模板，选用细菌16S rDNA通用引物27f和1492r进行PCR扩增。
PCR扩增所用引物：
27f：5’-AGAGTTTGATCCTGGCTCAG-3’
1492r：5’-TACCTTGTTACGACTT-3’
PCR扩增所用反应体系：2×Taq PCR Mix 25μL，DNA模板(100ng/μL)2μL，上下游引物(10μmol/L)各1μL，用无菌ddH 2O补足50μL体系。
PCR反应程序：94℃3min；94℃30s，54℃30s，72℃90s，35个循环；72℃10min；保温：4℃。
PCR扩增产物通过电泳检测，片段长度约为1500bp(序列表中序列1)。
2.3gyrB基因的PCR扩增
对细菌BJ-3进行基因组DNA的提取，并以这株细菌基因组DNA为模板，选用细菌gyrB基因通用引物gyrB-F和gyrB-R进行PCR扩增。
PCR扩增所用引物：
gyrB-F：5’-TTGRCGGHRGYGGHTATAAAGT-3’
gyrB-R：5’-TCCDCCSTCAGARTCWCCCTC-3’
PCR扩增所用反应体系：2×Taq PCR Mix 25μL，DNA模板(100ng/μL)2μL，上下游引物(10μmol/L)各1μL，用无菌ddH 2O补足50μL体系。
PCR反应程序：94℃4min；94℃30s，55℃45s，72℃1min，35个循环；72℃10min；保温：4℃。
PCR扩增产物通过电泳检测，片段长度约为900bp(序列表中序列2)。
2.4序列分析与分子鉴定
PCR产物经1％琼脂糖凝胶电泳验证后，送至北京博迈德基因技术有限公司测序。所获得序列与NCBI网站GenBank数据库中的序列进行比对，获得与测序16S rDNA序列、gyrA基因序列相似度最高的菌株种类。结果显示，BJ-3菌株的与耐盐芽孢杆菌(Bacilus halotolerans)菌株TRM85649的16S rDNA序列相似度为98.47％，与耐盐芽孢杆菌(Bacilus halotolerans)菌株LYSX1的gyrB基因序列相似度为99.77％。将其初步鉴定为耐盐芽孢杆菌近缘种群菌株。
BJ-3菌株的16S rDNA和gyrB基因PCR扩增片段测序结果分别如序列表中序列1和序列2所示。
根据基因片段序列，利用MEGA软件，分别基于16S rDNA和gyrB基因利用ML方法构建系统发育树，如图5和图6所示，结果表明菌株BJ-3分别与耐盐芽孢杆菌菌株CR-119(GenBank登录号：NR115283)和耐盐芽孢杆菌菌株CECT 5687(GenBank登录号：DQ903179)聚合到一起，据此，将菌株BJ-3鉴定为耐盐芽孢杆菌(Bacilus halotolerans)。将菌株BJ-3保 藏于中国普通微生物菌种保藏管理中心(地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所)，保藏日期2021年12月16日，保藏编号为CGMCC No.24115。
实施例2.菌株BJ-3CGMCC No.24115的抑菌活性测定
采用对峙培养法，以22种植物病原真菌的45个菌株(植物病原真菌菌株信息见表1，由北京市农林科学院植病综防研究室按照形态学及分子鉴定方法和柯赫氏法则进行鉴定)为靶标菌，测定菌株BJ-3的拮抗活性。
将BJ-3在LB固体培养基上25℃培养2～3d，后挑取单菌落于1mL的LB液体培养基内，37℃200rpm/min摇培8～10h，备用。将45个病原菌菌株在PDA平板上培养3～5d，用打孔器在菌落边缘区域打孔制成直径为5mm的病原菌菌饼，放置到PDA平板中央，同时吸取4μL培养好的菌悬液滴加在直径为6mm的滤纸片上，放置距平板边缘约1cm处，1个板放4个滤纸片，并以滤纸片上滴加同样剂量的清水作为对照，重复3次，28℃培养箱中培养5d，观察抑菌带的产生，测量相对内生拮抗细菌BJ-3方向病原菌菌丝生长的半径(D)和抑菌带宽度(d)，计算抑菌率。
抑制率(％)＝(对照菌落半径-处理菌落半径)/对照菌落半径×100％
表1.菌株BJ-3对45株植物病原真菌的抑菌活性
Figure PCTCN2022130479-appb-000001
Figure PCTCN2022130479-appb-000002
通过对峙培养法，测得菌株BJ-3对22种植物病原真菌45个菌株的抑菌活性，测定结果如表1所示。由实验结果可知，菌株BJ-3对供试的不同植物病原真菌都具有明显的抑制作用，对菌丝生长的抑制率在54.74％-91.62％之间，对引起葡萄蔓枯病的大豆间座壳(Diaporthe sojae)OSS①菌丝生长抑制效果最佳，抑制率可达91.62％。研究结果表明，BJ-3对常见植物病原菌的抑制作用有广谱性。
实施例3.菌株BJ-3对葡萄溃疡病菌Lasiodiplodia theobromae分生孢子萌发的抑制作 用测定
1实验方法
1.1葡萄溃疡病菌分生孢子悬浮液的制备
将保存的葡萄溃疡病菌Lasiodiplodia theobromae菌株GX-5-5接种于PDA平板，28℃培养2～3d，将活化的菌再转接至新的PDA平板上，28℃培养7～10d，产孢后用无菌水洗脱分生孢子，并用4层无菌纱布过滤，去除菌丝和杂质，最终用无菌水稀释分生孢子悬浮液浓度至1×10 5个孢子/mL，备用。
1.2菌株BJ-3菌悬液制备
将菌株BJ-3在LB固体培养基上28℃培养2～3d，后挑取单菌落于20mL的LB液体培养基内，28℃200rpm/min摇培8～10h，备用。
1.3菌株BJ-3菌悬液对葡萄溃疡病菌孢子萌发的抑制作用测定
用无菌水将制备的BJ-3菌悬液浓度分别调整至8×10 5、1.6×10 6、4×10 6、8×10 6、2×10 7、4×10 7CFU/mL，按体积比1∶1将孢子悬浮液分别与BJ-3的6种不同浓度菌悬液混匀，以体积比1∶1的孢子悬浮液和无菌水为对照，每个处理重复3次。分别吸取100μL混合液滴加在灭菌凹玻片上，将凹玻片放置铺有湿润滤纸的培养皿中，放入培养箱28℃培养，在10～12h时镜检，观察分生孢子萌发情况。
1.4数据统计与分析
以分生孢子芽管的长度超过孢子直径的1/2记为已萌发孢子，当空白对照孢子萌发率达到90％以上时，检查各处理孢子萌发情况。每处理各重复随机观察3个以上视野，调查孢子总数不少于200个，分别记录萌发数和孢子总数。然后将菌液浓度--抑制率转化为相应对数--机率值，求出毒力回归方程、相关系数(r)和EC 50。
计算公式：
R＝Ng/Nt×100
(R-孢子萌发率；Ng-孢子萌发数；Nt-调查的孢子总数)，
Re＝Rt/R 0×100
(Re-处理校正孢子萌发率；Rt-处理孢子萌发率；R 0-空白对照孢子萌发率)，
I＝(R 0-Re)/R 0×100(I-孢子萌发相对抑制率)。
2实验结果
菌株BJ-3对葡萄溃疡病菌GX-5-5的室内生物活性测定结果见表2，结果表明，BJ-3菌液对GX-5-5孢子萌发有明显的抑制作用，供试浓度8×10 5～4×10 7CFU/mL的BJ-3菌液对葡萄 溃疡病菌的分生孢子萌发抑制率在16.72％～98.47％，其中BJ-3菌悬液浓度在2×10 7CFU/mL和4×10 7CFU/mL时，对孢子萌发的抑制率分别为97.95％和98.47％，EC 50值为3.35×10 6cfu/mL，r值在0.9以上，因此，菌株BJ-3对葡萄溃疡病菌孢子萌发有较强的的抑制作用，可能具有通过抑制病菌对寄主的侵染实现控制葡萄溃疡病的能力。
表2菌株BJ-3对葡萄溃疡病菌GX-5-5的室内生物活性测定结果
Figure PCTCN2022130479-appb-000003
实施例4.离体枝条接种法测定菌株BJ-3对葡萄溃疡病的防治作用
1实验方法
1.1菌株BJ-3发酵液的制备
用灭菌的牙签挑取菌株BJ-3单菌落接种于装有10mL的LB液体培养基中，28℃180rpm振荡培养12h后转入装有100mL的发酵培养基的三角瓶中28℃180rpm振荡培养72h，得到菌株BJ-3的发酵液，用无菌水稀释制成10 6CFU/mL、10 7CFU/mL、10 8CFU/mL三种浓度的发酵液，4℃保存。
1.2接种体及供试病原菌的培养制备
接种体选用一年生葡萄绿枝条，接种当天从葡萄园采集健康当年生半木质化的夏黑葡萄绿枝条，剪去叶片后，先用清水清洗表面，后用75％的酒精湿巾擦拭消毒再用无菌水冲洗表面晾干，备用。
供试病原菌为葡萄溃疡病菌GX-5-5菌株，将供试病原真菌在PDA培养基上28℃条件下培养3d，备用。
1.3菌株BJ-3对葡萄溃疡病的防治作用
1.3.1菌株BJ-3对葡萄溃疡病的保护作用测定
采用喷雾方法用菌株BJ-3发酵液处理经消毒处理的葡萄绿枝条，处理浓度分别为BJ-31.08×10 6CFU/mL、1.08×10 7CFU/mL、1.08×10 8CFU/mL，然后将绿枝条插入装有湿润蛭石基质的直径为9cm的塑料钵中，在25℃、相对湿度60％的人工气候室培养24h。随后，用灭菌的直径为4.0mm的打孔器在枝条节间中部处打取直径大小为4mm，深1mm的伤口，剥掉 伤口部位的组织，将培养3d的葡萄溃疡病菌沿菌落边缘向内1cm处一圈，用4.0mm打孔器均匀打成菌饼，用灭菌的牙签将菌饼置于枝条伤口处，菌丝面接触葡萄组织，用封口膜将接种菌饼的位置包好，重新插入塑料钵中，分别以接种空白PDA菌块喷施内生拮抗细菌发酵液、接种葡萄溃疡病菌喷施无菌水和接种空白PDA菌块喷施无菌水的绿枝条作为对照，以5×10 6CFU/mL100亿CFU/mL枯草芽孢杆菌WP作为对照药剂。每个处理接种7根枝条，3次重复。接种后继续在25℃接种室中，相对湿度90％条件下培养48h，然后调整相对湿度至60％，在接种7～10d后对各处理的发病情况和病斑长度进行测量，用SPSS 18软件对数据进行统计分析。计算公式如下：
防治效果(％)＝[(对照病斑长度-处理病斑长度)/(对照病斑长度-菌饼直径)]×100％
1.3.2菌株BJ-3对葡萄溃疡病的治疗作用的测定
治疗作用的测定是先接种病菌，接种后48h再用不同的菌液及无菌水处理接种的绿枝条，其所用方法及材料与保护作用测定相同。
2实验结果
通过离体绿枝条人工接种，测得菌株BJ-3对葡萄溃疡病的防治效果，结果如表3所示。结果表明，三种浓度的菌株BJ-3发酵液与5×10 6CFU/mL100亿CFU/mL枯草芽孢杆菌WP对葡萄溃疡病的保护作用防治效果均无显著差异，其中1.08×10 8CFU/mL浓度的BJ-3发酵液防治效果最佳，为91.75％；1.08×10 8CFU/mL的BJ-3发酵液对葡萄溃疡病的治疗作用显著高于BJ-3其他浓度发酵液及对照药剂芽孢杆菌，防效为86.94％，对照药剂芽孢杆菌的防治效果为73.72％，浓度为1.08×10 7CFU/mL的BJ-3发酵液对葡萄溃疡病的防治效果为51.03％，与对照药剂的防治效果无显著差异，由此可见，菌株BJ-3对葡萄溃疡病的治疗作用好于对照药剂枯草芽孢杆菌的防治效果。
表3菌株BJ-3发酵菌液对葡萄溃疡病的防治效果(室内人工接种)
Figure PCTCN2022130479-appb-000004
Figure PCTCN2022130479-appb-000005
实施例5.菌株BJ-3发酵菌液对葡萄枝干病害的田间防治效果评价
1实验材料与方法
1菌株BJ-3发酵菌液的制备
发酵培养基：麦芽糖20g/L；蛋白胨10g/L；CaCO 3 10g/L；NaHPO 3 2g/L；KH 2PO 3 1g/L，麸皮10g/L；余量为水；рН6.8。
发酵条件：温度为32°С，搅拌速度200r/min，通气量1:0.5-1:1.2，培养时间22h。120L发酵罐培养，获得BJ-3发酵液，浓度为5.6×10 9CFU/mL。
2菌株BJ-3发酵菌液对葡萄溃疡病的田间防治
菌株BJ-3发酵液对葡萄枝干病害的控制作用效果评价采用田间小区对比试验方法进行，试验地选在葡萄枝干病害常年发生且易感染的品种，地点为河北省张家口市怀来县中粮长城桑干酒庄(怀来)酿酒葡萄种植基地，品种为‘马瑟兰’，试验共设4个处理。处理1为BJ-3发酵液，浓度为5.6×10 7CFU/mL；处理2为100亿CFU/克枯草芽孢杆菌WP 1000倍液，处理3为10％苯醚甲环唑WG 1000倍液；处理4为清水空白对照；施药方式为灌根，每株1L，每处理200株以上葡萄植株，3个重复，于2021年7月9日和7月20日分别施药一次，施药前和施药用70天后(葡萄近成熟期)分别调查葡萄植株发病情况，计算病株率(％)及防治效果(％)，计算公式如下：
病株率(％)＝病株数/调查总株数×100％
防治效果(％)＝[1-(空白对照区药前病株率×处理区药后病株率)/(空白对照区药后病株率×处理区药前病株率)]×100％
2.试验结果
菌株BJ-3发酵液对葡萄枝干病害的田间防病效果表明(表4)，5.6×10 7CFU/mL的BJ-3发酵液、100亿CFU/克枯草芽孢杆菌WP 1000倍液和10％苯醚甲环唑WG 1000倍3个药剂处理灌根施药后平均防治效果分别为65.56％、42.34％和27.30％，其中浓度为5.6×10 7CFU/mL的BJ-3发酵液对葡萄溃疡病的防治效果极显著高于其他两个药剂处理，后两者之间无显著差异，整个试验过程中，三种药剂处理对供试作物葡萄未见药害。
综上所述，田间药效试验结果表明，供试的2种杀菌剂和BJ-3发酵菌液对葡萄枝干病害的平均防治效果为27.30-65.56％，其中BJ-3发酵菌液(5.6×10 7CFU/mL)的药效最高为65.56％，显著对照的枯草芽孢杆菌和化学杀菌剂苯醚甲环唑，具有防治葡萄枝干病害的生防 潜力。
表4菌株BJ-3发酵菌液对葡萄枝干病害的田间防治效果
Figure PCTCN2022130479-appb-000006
注：表中平均病株率为三个重复的平均值。
实施例6.菌株BJ-3发酵液对葡萄幼苗的促生作用测定
1.材料与方法
1.1菌株BJ-3发酵液及发酵滤液的制备
1.1.1种子液制备：使用250mL容量的三角瓶装入100mL已灭菌的LB液体培养基，将BJ-3挑取单菌落接入LB液体培养基内，在28℃180rpm/min条件下，震荡培养12h。
1.1.2发酵培养：BJ-3：将种子液倒入装有100mL的发酵培养基(麦芽糖20g/L，蛋白胨10g/L，麸皮10g/L，CaCO 3 10g/L，NaHPO 3 2g/L，KH 2PO 3 1g/L；pH值为6.8)的500mL三角瓶中，接种量10％，28℃180rpm/min振荡培养72h，得到发酵液。
1.1.3发酵滤液的制备：发酵液经高速冷冻离心机4℃10000rpm离心15-20min，收集上清并经细菌过滤器过滤，丢弃沉淀，即得到无菌的发酵滤液，-20℃保存备用。
1.2供试植物
供试葡萄品种为‘马瑟兰’品种，材料为扦插苗，将扦插苗栽植在口径22.5cm，高13.5cm花盆中。
1.3菌株BJ-3发酵液对葡萄幼苗的促生作用
采用灌根法测定菌株BJ-3发酵液对葡萄幼苗的促生作用。当葡萄幼苗长至5-7片叶，进行灌根处理，每盆葡萄苗灌根150mL，共设4个处理，处理1为浓度5.8×10 6CFU/mL的BJ-3发酵液，处理2为BJ-3培养用发酵培养基(未培养BJ-3的培养基)10000倍稀释液，处理3为BJ-3发酵滤液10000倍稀释液，处理4为清水对照，每个处理10株葡萄苗。将葡萄植株置于温室中，25±2℃培养。
1.4数据统计与分析
于灌根7d、14d、21d、28d、35d和42d时，分别观察测量葡萄新稍长度，在第42d时同时对叶绿素含量、叶片面积进行测定，用SPSS 25软件分析对数据进行分析。
2.结果
菌株BJ-3不同处理对葡萄幼苗的促生作用的试验结果见图7～10，结果表明，浓度为5.8×10 6CFU/mL的BJ-3发酵液(处理1)对葡萄幼苗新稍生长无明显促进作用，BJ-3发酵滤液10000倍稀释液(处理3)对葡萄幼苗的叶面积和叶绿素含量显著高于其他处理，相比清水对照(处理4)分别提高了8.67％、8.97％。说明菌株BJ-3可能具有通过提高叶面积和叶绿素含量改善葡萄幼苗光合性能的能力。
以上所述实施例仅展示了本公开的几种实施方式，其描述较为具体，但对本公开而言只是说明性的，而非限制性的。对于本领域普通技术人员来说，在不脱离所附权利要求所限定的精神和范围的情况下，可做出许多修改、变化和改进，这些都属于本公开的保护范围。
Figure PCTCN2022130479-appb-000007
Figure PCTCN2022130479-appb-000008

Claims (12)

  1. 一株耐盐芽孢杆菌，名称为耐盐芽孢杆菌BJ-3，其特征在于，所述耐盐芽孢杆菌已保存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.24115。
  2. 根据权利要求1所述的耐盐芽孢杆菌，其特征在于，所述耐盐芽孢杆菌具有针对植物病原真菌的抑菌活性。
  3. 根据权利要求2所述的耐盐芽孢杆菌，其特征在于，所述植物病原真菌为可可毛色二孢(Lasiodiplodia theobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthe sojae)、甜樱间座壳(Diaporthe eres)、Diaporthe honkonggensis、灰葡萄孢(Botrytis cinerea)、Coletotrichum viniferum、隐秘炭疽菌(Coletotrichum aenigma)、胶孢炭疽菌(Coletotrichum gloeosporioides)、果生炭疽菌(Coletotrichum fructicola)、暹罗炭疽菌(Coletotrichum siamense)、尖孢炭疽菌(Coletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsis rosae、Coniela vitis、Dactylonectria macrodidyma、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)和核盘菌(Sclerotinia sclerotiorum)中的一种或多种。
  4. 权利要求1-3中任一项所述的耐盐芽孢杆菌在制备抑制植物病原真菌的生防菌剂或微生物菌肥中的应用。
  5. 根据权利要求4所述的应用，其特征在于，所述植物病原菌为可可毛色二孢(Lasiodiplodia theobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthe sojae)、甜樱间座壳(Diaporthe eres)、Diaporthe honkonggensis、灰葡萄孢(Botrytis cinerea)、Coletotrichum viniferum、隐秘炭疽菌(Coletotrichum aenigma)、胶孢炭疽菌(Coletotrichum gloeosporioides)、果生炭疽菌(Coletotrichum fructicola)、暹罗炭疽菌(Coletotrichum siamense)、尖孢炭疽菌(Coletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsis rosae、Coniela vitis、Dactylonectria macrodidyma、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)和核盘菌(Sclerotinia sclerotiorum)中的一种或多种。
  6. 一种针对植物病原真菌的生防菌剂，其特征在于，所述生防菌剂的活性成分为权利要求1-3中任一项所述的耐盐芽孢杆菌。
  7. 根据权利要求6所述的生防菌剂，其特征在于，所述植物病原菌为可可毛色二孢(Lasiodiplodia theobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthe sojae)、甜樱间座壳(Diaporthe eres)、Diaporthe honkonggensis、 灰葡萄孢(Botrytis cinerea)、Coletotrichum viniferum、隐秘炭疽菌(Coletotrichum aenigma)、胶孢炭疽菌(Coletotrichum gloeosporioides)、果生炭疽菌(Coletotrichum fructicola)、暹罗炭疽菌(Coletotrichum siamense)、尖孢炭疽菌(Coletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsis rosae、Coniela vitis、Dactylonectria macrodidyma、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)、核盘菌(Sclerotinia sclerotiorum)中的一种或多种。
  8. 一种针对植物病害的微生物菌肥，其特征在于，所述微生物菌肥的活性成分为权利要求1-3中任一项所述的耐盐芽孢杆菌。
  9. 根据权利要求8所述的微生物菌肥，其特征在于，导致所述植物病害的植物病原菌为可可毛色二孢(Lasiodiplodia theobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthe sojae)、甜樱间座壳(Diaporthe eres)、Diaporthe honkonggensis、灰葡萄孢(Botrytis cinerea)、Coletotrichum viniferum、隐秘炭疽菌(Coletotrichum aenigma)、胶孢炭疽菌(Coletotrichum gloeosporioides)、果生炭疽菌(Coletotrichum fructicola)、暹罗炭疽菌(Coletotrichum siamense)、尖孢炭疽菌(Coletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsis rosae、Coniela vitis、Dactylonectria macrodidyma、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)、核盘菌(Sclerotinia sclerotiorum)中的一种或多种。
  10. 权利要求1-3中任一项所述的耐盐芽孢杆菌在促进葡萄幼苗生长中的应用。
  11. 一种促进葡萄幼苗生长的方法，利用权利要求1-3中任一项所述的耐盐芽孢杆菌发酵培养液、发酵培养液的稀释液、发酵培养液滤液或者发酵培养液滤液的稀释液，对葡萄幼苗进行灌根处理。
  12. 根据权利要求11所述的方法，其特征在于，当葡萄幼苗长至5-7片叶，用权利要求1-3中任一项所述的耐盐芽孢杆菌发酵培养液滤液的稀释液进行灌根处理。
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