CN105886447A - Bacillus strain DR011 and plant drought-enduring inducting agent prepared from bacillus strain DR011 - Google Patents

Bacillus strain DR011 and plant drought-enduring inducting agent prepared from bacillus strain DR011 Download PDF

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CN105886447A
CN105886447A CN201610507771.9A CN201610507771A CN105886447A CN 105886447 A CN105886447 A CN 105886447A CN 201610507771 A CN201610507771 A CN 201610507771A CN 105886447 A CN105886447 A CN 105886447A
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bacillus
cgmcc
strain
drought
plants
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王云鹏
王晓莉
熊清平
石莹莹
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Huaiyin Institute of Technology
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • 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
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • 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
    • 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

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Abstract

The invention discloses a bacillus strain DR011 and a plant drought-enduring inducting agent prepared from the bacillus strain DR011. The strain is bacillus sp., and is collected in CGMCC (China General Microbiological Culture Collection Center), on the date of May 23, 2016; the collection number is CGMCC No. 12482. The strain is prepared through DR011 strain liquid fermentation culture; the plant drought-enduring performance is improved; when a bacterium agent of DR011 is used in a greenhouse, a good effect is achieved on improving the drought-enduring performance of tomatoes and cucumbers; the re-watering is performed after the 10 days of the drought of the cucumbers, and the survival rate reaches 80.1 percent; the re-watering is performed after 15 days of the drought of the tomatoes, and the survival rate reaches 90 percent.

Description

Bacillus strain DR011 and the drought tolerance in plants derivant prepared with it
Technical field
The invention belongs to field of agricultural microorganism, relate to Bacillus strain DR011 and the drought tolerance in plants prepared with it lures Lead agent.
Background technology
China's arid and semi-arid lands's area accounts for area 1/2, and arid has become limiting plant growth growth, gene Expressing and the important eco environment facto of yield, the harm to plant occupies first place in all abiotic harm.Along with the whole world The unusual weather conditions of property and the destruction of ecological balance, soil desertification increasingly and salinization of soil, Arid Problem will be the most serious.The whole nation is every The crop failure that year causes because of arid estimates to reach 20%, improves the important measures that the drought-resistant property of crop is disaster mitigation.Therefore, The drought-resistance ability of raising crop is one of hot issue in modern agriculture research work.Except cultivating the kind of drought resistance, The degeneration-resistant protective agent finding safety non-toxic in nature is also the important means improving Crop Drought Resistance.
Plant growth-promoting rhizobacteria (plant growth promoting rhizobacteria, PGPR) refers to free life Live and promote plant growing and to the absorption of mineral nutrition and utilization at soil or the class in root system of plant of growing nonparasitically upon another plant, and can suppress The useful mushroom of harmful organism.Plant is not had pathogenic by PGPR, and has prophylaxis effect and growth-promoting functions to plant.
Plant growth-promoting rhizobacteria PGPR improves plant rhizosphere soil ecological environment, especially has plant root growth aobvious The facilitation write.PGPR promotes plant growing by fixed nitrogen, molten phosphorus, secretion auxin and antibiotic agents, carries The high plant opposing respond to environment-stress such as arids.Therefore, it is used for improving plant by plant growth-promoting rhizobacteria PGPR Drought tolerance there is the biggest application potential.
Summary of the invention
It is an object of the invention to: provide a kind of Bacillus strain DR011 and with the drought tolerance in plants derivant that it is prepared, adopt The bacteria preparation prepared with microbial strains DR011, improves drought resistance in plants, increases plant survival rate under drought environment.
The technical solution of the present invention is: this Bacillus strain DR011, identified belongs to bacillus, classification NamedBacillus sp., it is preserved in China Committee for Culture Collection of Microorganisms on May 23rd, 2016 the most micro- Bio-Centers (CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research Institute, culture presevation number is CGMCC No. 12482.
Wherein, drought tolerance in plants derivant prepared by described bacterial strain DR011, the preparation method of this drought tolerance in plants derivant is: The seed bacterium solution of bacillus cereus DR011 is seeded to fermentation culture in LB fermentation culture with 1:100 volume ratio, 28-30 DEG C, 180-200rpm cultivates 40-48 h；Then 5000-6000 rpm is centrifuged 10-15 min, is diluted making bacterium with aquesterilisa Agent, in microbial inoculum finished product, viable bacteria total concentration is 1 × 109 ~ 1×10 10 CFU/mL。
Wherein, described preserving number is that the bacillus cereus DR011 seed bacterium solution preparation method of CGMCC No.12482 is: By bacillus cereus DR011 inoculation that preserving number is CGMCC No.12482 to 28 DEG C of cultivations in LB culture fluid, 180 rpm Shaken cultivation terminates when being 0.8 to the OD value of 600 nm to cultivate.
Wherein, described preserving number is that drought tolerance in plants prepared by the Bacillus strain DR011 of CGMCC No.12482 lures The application process leading agent is: described microbial inoculum dilutes 100 ~ 200 times, root irrigation after arid 10-15 days.
The invention has the beneficial effects as follows: this biological preparation a series of is asked entirely without bring because of the use of chemical agent Topic, the beneficially No-harmful apple orchard of crop, it is effectively improved the Drought resistance of plant.
Detailed description of the invention
Embodiment 1: the screening of Bacillus strain DR011
Bacillus cereus DR011(CGMCC No.12482) by harbour town, Huai'an, Jiangsu Province Fructus Lycopersici esculenti rhizosphere soil separates acquisition；Will Tomato Root System is cut into the small pieces of 1cm, soaks 5 min, the alcohol-pickled 1-2 of mass concentration 70% with the sodium hypochlorite of mass concentration 1% Min, sterile water wash 3 times；The solution 100 μ l taking last cleaning is applied to R2Cultivate on A solid medium, if 48 h Rear asepsis growth then thinks that surface sterilization is clean, aseptic；Take the sample that 3 g disinfect to be placed in bacterium mortar, add 27 ml aseptic Mass concentration 0.85% NaCl, macerate tissue, grind, with aseptic mass concentration 0.85% NaCl gradient dilution；Take 10-1、10 -2 、10 -3The each 100 μ l of diluent of three gradients are applied to R2On A, cultivate 48 h for 28 DEG C；Single bacterium colony that picking is maximum It is inoculated into fresh R2A solid medium, through repeatedly switching obtain purification bacterial strain, by bacterial strain after purification with 40% glycerol be stored in- In 70 DEG C of ultra cold storage freezers.
Authentication method: this bacterial strain is identified by morphological characteristic, bio-chemical characteristics and 16S rDNA sequence analysis.
Morphological feature: Gram-positive bacillus, produces spore, white, translucent, and smooth moistening bacterium colony slightly swells, circular Neat bacterium colony, without pod membrane, peritrichous.
16S rRNA gene amplification and sequence analysis: bacillus DR011 is at R2A culture medium is cultivated extremely for 28 DEG C Logarithmic (log) phase, is centrifuged 5 min with 12 000 r/min and collects thalline, use the genome of Shanghai SBS Genetech gene technology company limited DNA rapid extraction test kit, extract antibacterial genomic DNA, with extract DNA product as template, with bacterial 16 S rRNA expand Increase universal primer U8-27 (5 '-AGAGTTTGATC (AC) TGGCTCAG-3 '), L1494-1514 (5 '-CTACGG (AG) TACCTTGTTACGAC-3 ') from genomic DNA, amplify 16S rDNA genetic fragment；It is biological that PCR primer serves the raw work in sea Engineering Co., Ltd checks order；Tetraploid rice is carried out to measuring 16S rRNA gene order, knot by BLAST software Fruit be shown in Table 1, therefore, by this identification of strains be bacillus (Bacillus sp.).
Table 1 DR011 bacterial strain 16SRDNA checks order comparison result
Bacterial strain Most like bacterial strain and accession number Similarity (%)
DR011 Bacillus sp . NR_118950.1 99
The preparation of embodiment 2:DR011 microbial inoculum
By bacillus cereus DR011(CGMCC No.12482) inoculation is to 28 DEG C of 180 rpm shaken cultivation in LB culture fluid After 16 h, in superclean bench, sample its OD value at 600 nm of survey every 2 h, terminate when OD value is 0.8 to cultivate, this Bacterium solution is as seed bacterium solution；Seed bacterium solution is seeded to fermentation culture in LB fermentation culture with 1:100 volume ratio, 28 DEG C, 180 Rpm cultivates 48 h, and then 6000 rpm are centrifuged 10 min, takes to precipitate and i.e. obtains described microbial inoculum DR011, in gained biocontrol agent Viable bacteria total concentration is 1 × 108 ~ 1×10 9 CFU/mL。
The preparation of embodiment 3:DR011 microbial inoculum
By bacillus cereus DR011(CGMCC No.12482) inoculation is to 28 DEG C of 180 rpm shaken cultivation in LB culture fluid After 16 h, in superclean bench, sample its OD value at 600 nm of survey every 2 h, terminate when OD value is 0.8 to cultivate, this Bacterium solution is as seed bacterium solution；Seed bacterium solution is seeded to fermentation culture in LB fermentation culture with 1:100 volume ratio, 29 DEG C, 190 Rpm cultivates 44 h, and then 5500 rpm are centrifuged 12min, takes to precipitate and i.e. obtains described microbial inoculum DR011, lives in gained biocontrol agent Bacterium total concentration is 1 × 108 ~ 1×10 9 CFU/mL。
The preparation of embodiment 4:DR011 microbial inoculum
By bacillus cereus DR011(CGMCC No.12482) inoculation is to 28 DEG C of 180 rpm shaken cultivation in LB culture fluid After 16 h, in superclean bench, sample its OD value at 600 nm of survey every 2 h, terminate when OD value is 0.8 to cultivate, this Bacterium solution is as seed bacterium solution；Seed bacterium solution is seeded to fermentation culture in LB fermentation culture with 1:100 volume ratio, 30 DEG C, 200 Rpm cultivates 40 h, and then 5000 rpm are centrifuged 15 min, takes to precipitate and i.e. obtains described microbial inoculum DR011, in gained biocontrol agent Viable bacteria total concentration is 1 × 108 ~ 1×10 9 CFU/mL。
Certainly, described in utilization improve drought resistance in plants plant rhizosphere Promoting bacteria DR011 prepare microbial inoculum be not limited to Upper method, every can mass propgation DR011, and keep the method for its biological and ecological methods to prevent plant disease, pests, and erosion activity to be used equally to prepare the present invention being claimed Drought resistance in plants induction microbial inoculum.
Embodiment 5: greenhouse test measures DR011 microbial inoculum and improves Fructus Cucumidis sativi drought tolerance effect
Cucumber variety is excellent No. 1 of Tianjin of Tianjin Cucumber Inst.'s selection-breeding；The seed presoaking and germinating 48h (25 DEG C) of cucumber variety After be seeded in seedling culture hole plate, be placed in greenhouse, normal management is watered, transplant seedlings after 15d to volume be 450cm2Basin alms bowl in, every other day Water 25mL；Start after transplanting seedlings 15 days to process, if following 2 process:
(1) DR011 microbial inoculum processes: each Seedling pouring 20mL rhizosphere bacterium solution, after the 5d that normally waters, and simulation arid of cutting off the water supply Process；
(2) matched group: every Seedling pouring 20mL LB, after the 5d that normally waters, simulation Osmotic treatment of cutting off the water supply；
Simulating drought waters after processing 10d again, adds up survival rate after 24 h that again water；Drought resistance coefficient assay method: cut off the water supply After, follow the tracks of the appearance observing Fructus Cucumidis sativi arid symptom, the wilting degree shown according to cucumber plant, according to following grade scale pair Each repetition plant division carries out investigation statistics；0 grade: plant strain growth is normal, without wilting phenomenon；1 grade: plant strain growth is normal, only 1 The performance of sheet leaf is slight wilts；2 grades: plant is wilted and increases the weight of, wilting 3 grades occur in 2 leaves: plant is substantially wilted, and more than 3 leaves all withers Listless；Blade is wilted sagging, recovers difficulty.
Statistical result is calculated as follows drought resistance coefficient: drought resistance coefficient (%)=[1-∑ (strain number × corresponding stage at different levels Number)/(total strain number × 3)] × 100；After cutting off the water supply 3 days, overall in 50% plant generation moderate wilt, respectively to process drought resisting Coefficient is made comparisons, and the drought resistance coefficient of DR011 microbial inoculum process group increases by 43.90 % than matched group.
Experimental result: after drought stress processes 10 d, DR011 microbial inoculum process group and matched group Fructus Cucumidis sativi all show serious Wilting symptom, but after 24 h that again water, the fast quick-recovery of DR011 microbial inoculum process group Fructus Cucumidis sativi energy, and through statistics survival rate It is 80.1%, and matched group recovers slowly, and survival rate is only 36.32%；These results indicate that DR011 microbial inoculum can be notable Improve the drought tolerance of Fructus Cucumidis sativi, the recovery capability of Fructus Cucumidis sativi after enhancing drought stress.
Embodiment 6: greenhouse test measures DR011 microbial inoculum and improves Fructus Lycopersici esculenti drought tolerance effect
Tomato variety is Shanghai 903；Educating in dish by being seeded in after tomato seeds presoaking and germinating 48h, normal management is watered, after 15d Transplant seedlings to volume be 450cm2Basin alms bowl in, water 25mL tap water every other day；Start after transplanting seedlings 15 days to process, if following 2 process:
(1) rhizosphere bacterium solution processes: each Seedling pouring 20mL rhizosphere bacterium solution, after the 5d that normally waters, and simulation arid of cutting off the water supply Process；
(2) matched group: every Seedling pouring 20mL LB, after the 5d that normally waters, simulation Osmotic treatment of cutting off the water supply；
Simulating drought waters after processing 15d again, adds up survival rate after 24 h that again water；Experimental result: drought stress processes After 15 d, DR011 microbial inoculum process group and matched group Fructus Lycopersici esculenti all show serious wilting symptom, but after 24 h that again water, The fast quick-recovery of DR011 microbial inoculum process group Fructus Lycopersici esculenti energy, and be 90.00% through adding up survival rate, and matched group recovers slowly, and Survival rate is only 35.33%；These results indicate that DR011 microbial inoculum can significantly improve the drought tolerance of Fructus Lycopersici esculenti, after strengthening drought stress The recovery capability of Fructus Lycopersici esculenti.

Claims (4)

1. Bacillus strain DR011, is characterized in that: it is identified belongs to bacillus, and Classification And Nomenclature isBacillus sp., it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on May 23rd, 2016, Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, and culture presevation number is CGMCC No. 12482。
2. the drought tolerance in plants derivant that prepared by bacterial strain DR011, is characterized in that the preparation method of this drought tolerance in plants derivant is: by bud The seed bacterium solution of spore bacillus DR011 is seeded to fermentation culture in LB fermentation culture, 28-30 DEG C, 180-with 1:100 volume ratio 200rpm cultivates 40-48 h；Then 5000-6000 rpm is centrifuged 10-15 min, is diluted making microbial inoculum, bacterium with aquesterilisa In agent finished product, viable bacteria total concentration is 1 × 109 ~ 1×10 10 CFU/mL。
Drought tolerance in plants derivant prepared by bacterial strain DR011 the most according to claim 2, is characterized in that described preserving number is The bacillus cereus DR011 seed bacterium solution preparation method of CGMCC No.12482 is: be CGMCC No.12482 by preserving number Bacillus cereus DR011 inoculation is to 28 DEG C of cultivations in LB culture fluid, the OD at 180 rpm shaken cultivation to 600 nm Value is to terminate when 0.8 to cultivate.
Drought tolerance in plants derivant prepared by bacterial strain DR011 the most according to claim 2, is characterized in that described preserving number is The application process of drought tolerance in plants derivant prepared by the Bacillus strain DR011 of CGMCC No.12482 is: described microbial inoculum Dilute 100 ~ 200 times, root irrigation after arid 10-15 days.
CN201610507771.9A 2016-07-01 2016-07-01 Bacillus strain DR011 and plant drought-enduring inducting agent prepared from bacillus strain DR011 Pending CN105886447A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456922A (en) * 2018-11-30 2019-03-12 江苏大学 The beach the Halophilic Bacterium bacterial strain Hua Jin bacillus of one plant of raising alec fermentation quality
CN114409446A (en) * 2022-01-21 2022-04-29 领先生物农业股份有限公司 Low-water solid-state fermentation method of drought-tolerant bacteria and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
李晓舟: "枯草芽孢杆菌B504菌株形态学及生理生化性状研究", 《农民致富之友》 *
王春娟: "蜡质芽孢杆菌AR156诱导植物耐旱及抗病机理研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 *
王春娟等: "根围促生细菌(PGPR)蜡质芽孢杆菌AR156对番茄的诱导耐旱性研究", 《农业生物技术学报》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456922A (en) * 2018-11-30 2019-03-12 江苏大学 The beach the Halophilic Bacterium bacterial strain Hua Jin bacillus of one plant of raising alec fermentation quality
CN109456922B (en) * 2018-11-30 2021-09-10 江苏大学 Moderately halophilic bacteria strain Zhan beach bacillus for improving fermentation quality of fish paste
CN114409446A (en) * 2022-01-21 2022-04-29 领先生物农业股份有限公司 Low-water solid-state fermentation method of drought-tolerant bacteria and application thereof

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Application publication date: 20160824