CN104726361B - The method for assisting host plant to alleviate the bacterial strain 1JN2 of cadmium ion stress and prepare microbial inoculum using it - Google Patents
The method for assisting host plant to alleviate the bacterial strain 1JN2 of cadmium ion stress and prepare microbial inoculum using it Download PDFInfo
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Abstract
The invention discloses the method for assisting host plant to alleviate the degeneration-resistant bacterial strain 1JN2 of cadmium ion stress and prepare microbial inoculum using it, the degeneration-resistant bacterial strain 1JN2 is bacillus subtilis（Bacillus subtilis）, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 13rd, 2014, culture presevation number is CGMCC NO.9759；Degeneration-resistant bacterial strain 1JN2 is centrifuged into 10 min in LB nutrient solutions 28 DEG C of 180 rpm shaken cultivations 16 h, 6000 rpm, it is 1 × 10 to dilute to obtain viable bacteria total concentration in microbial inoculum, microbial inoculum finished product with aqua sterilisa resuspension9 ‑1×1010CFU/mL；Under greenhouse experiment, when capsicum and cucumber are transplanted, with 109CFU/mL microbial inoculum root irrigation, 20mL/ plants；Transplant after one week, with 30mg/L cadmium sulfate root irrigations, 20mL/ plants.The bacterial strain 1JN2 of the present invention can strengthen the tolerance of host plant under cadmium ion treatment conditions, 24h proceeds by ego resilience and exocellular polysaccharide solidification phenomenon occurs in 48h after cadmium ion processing, the adverse circumstance for helping host plant reply to be brought due to environmental pollution, ensures the safety in production of vegetables to a certain extent.
Description
Technical field
The invention belongs to technical field of plant protection, it is related to the degeneration-resistant bacterial strain for assisting host plant to alleviate cadmium ion stress
1JN2 and the method for preparing microbial inoculum using it.
Background technology
With the quickening and the sustainable growth of population of process of industrialization, heavy metal pollution turns into global problem.Agriculture
It is the class special soil that is influenceed strongly by mankind's activity with soil, its environmental quality and people's is healthy closely related.
Cure of Soil pollution is used essentially from Industrial " three Waste " and agricultural chemicals, a large amount of of chemical fertilizer, and pollutant can enter soil by irrigation water,
Also soil pollution can be caused by atmosphere pollution, aerial particulate matter (containing heavy metal and carcinogen etc.) dried wet deposition, with
The passage of time, the content of beary metal such as farmland topsoil cadmium, lead, copper, zinc has increased trend.Because the absorption of crops is made
With, heavy metal element, Transport And Transformation is into Crop Root cauline leaf and fruit from soil, so that the related weight for causing crops
Metallic pollution, jeopardizes the general level of the health of people.Wherein cadmium has become the arch-criminal for threatening agricultural production.Global annual release
Cadmium into environment is up to 3.9 × 104t.And among several heavy metal species that ring Hongchehu Lake area is polluted, the pollution of cadmium
It is several that degree is also higher than other.Cadmium pollution is very serious to the loss that planting industry is brought, it is often more important that these are by cadmium pollution
Agricultural product can not be weighed for the influence of human health.Cadmium poisoning can make muscle atrophy dysarthrasis, and skeleton pain is difficult
Bear, it is impossible to fall asleep, occur pathologic fracture, so that dead.Therefore, how to repair these by cadmium pollution soil and water body
Through being the problem of we are badly in need of facing.
At present, include for the reparation means that heavy metal pollution is used：Engineering reparation, electro reclamation, electric heating reparation, soil
Earth is eluted and chemical remediation, although these methods can alleviate heavy metal pollution to a certain extent, but exists one
A little drawbacks：Implement quantities big, investment cost is high, destroy soil structures, cause soil fertility to decline, and will also be to swapping out
Dirty soil is stacked or handled；Wherein chemical remediation is the existing forms for changing heavy metal, can not thoroughly eradicate, also exist
The possibility of reactivation pollution.In addition, by some specified plants to the extraction of metal ion, volatilization and the characteristic such as stable also
Heavy metal pollution reparation can be carried out using plant.This biological prosthetic mode due to effect it is good, it is easy to operate the features such as received
To increasing attention.But the reparation of uncultivated soil earth or water body is only intended to, for heavy metals in farmland pollution still
Helpless.
The heavy metal pollution of farmland and water body is repaired using beneficial microbe isolated in natural environment just can be with
Overcome this difficult.By the soil of heavy metal pollution, the fungi and bacterium of a variety of resistance to heavy metals are often enriched with, microorganism can be with
The toxicity of heavy metal-polluted soil is influenceed by a variety of modes of action.Microorganism remediation technology has small investment, efficiency high, in-situ treatment
The characteristic of low concentration unwanted pollutant, shows great potentiality in environmental improvement, can also coordinate economic development and environment
Relation between protection, realizes the sustainable development of society.
In summary, the plant rhizosphere growth-promoting of specific pollutants in fixing soil can be absorbed by screening and developing some
Bacterium coerces the pressure brought to assist host plant to alleviate cadmium ion, with good meaning and DEVELOPMENT PROSPECT.
The content of the invention
The purpose of the present invention is：A kind of degeneration-resistant bacterial strain 1JN2 for assisting host plant to alleviate cadmium ion stress and application are provided
It prepares the method for microbial inoculum, is provided among the production of vegetables and can assist in host plant and effectively alleviate cadmium ion stress pressure
Degeneration-resistant bacterial strain 1JN2, so that the adverse circumstance for helping host plant reply to be brought due to environmental pollution, ensures vegetables to a certain extent
Safety in production.
The present invention technical solution be：The degeneration-resistant bacterial strain 1JN2 that the assistance host plant alleviates cadmium ion stress is withered
Careless bacillus（Bacillus subtilis）, with being preserved in Chinese microorganism strain preservation management committee on October 13rd, 2014
Member can common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, culture presevation
Number be CGMCC NO.9759.
Wherein, the degeneration-resistant bacterial strain 1JN2 is for the method for preparing microbial inoculum：By degeneration-resistant bacterial strain 1JN2 in 28 DEG C of LB nutrient solutions
The h of 180 rpm shaken cultivations 16, then 6000 rpm centrifugations, 10 min, microbial inoculum, microbial inoculum finished product are diluted to obtain with aqua sterilisa resuspension
Middle viable bacteria total concentration is 1 × 109 -1×1010 CFU/mL。
Wherein, the microbial inoculum is in the application process for assisting host plant to alleviate cadmium ion stress：Under greenhouse experiment, in capsicum
When being transplanted with cucumber, with 109CFU/mL microbial inoculum root irrigation, 20mL/ plants；Transplant after one week, with 30mg/L cadmium sulfate pouring roots
Processing, 20mL/ plants.
Beneficial effects of the present invention are shown：Host plant is assisted to alleviate the degeneration-resistant bacterial strain 1JN2 and microbial inoculum of cadmium ion stress
The cadmium ion pollution for being specific to run into plant growth is developed, and it can be adsorbed and solid by the exocellular polysaccharide of itself
Determine cadmium ion, so as to reduce absorption and accumulation of the host plant for cadmium ion in environment, have compared to chemical treatment method
Cost is low, nontoxic, to environment and friendly people and animals the advantages of, can assist in plant and spend cadmium ion pollution adverse circumstance, and subtract
Lack absorption and accumulation of the plant for cadmium ion, so as to reduce food safety risk on source.
Brief description of the drawings
Fig. 1 is bacterial strain 1JN2 different time electromicroscopic photographs after cadmium ion processing.
In figure：First row A1-A4 is respectively control group and treatment group 12h colonies and unicellular form；B1-B4 points of second row
Wei not control group and treatment group 24h colonies and unicellular form；3rd row C1-C4 is respectively control group and treatment group 36h colonies
With unicellular form；4th row D1-D4 is respectively control group and treatment group 48h colonies and unicellular form；5th row E1-E4 is
Treatment group 48h exocellular polysaccharide forms.
Embodiment
With reference to specific embodiment further illustrate the present invention technical solution, these embodiments it is not intended that
It is the limitation to technical scheme.
Bacterium source：It is isolated from using dilution coated plate method in the ginger plant in Shandong Province Laiwu City Laixi town, strain number
For 1JN2, degeneration-resistant bacterial strain 1JN2 is bacillus subtilis（Bacillus subtilis）, in being preserved in on October 13rd, 2014
State's Microbiological Culture Collection administration committee common micro-organisms center, address：The Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Chinese Academy of Sciences
Institute of microbiology, culture presevation number is CGMCC NO.9759.
Bacterial strain is identified：By 16S rDNA sequence alignments, bacterial strain 1JN2 and bacillus subtilis（Genebank accession number
AB383135.1）Sequence similarity 99%；Other bacterial strain 1JN2 gram positives, 1-3mm is formed on LB culture mediums
Bacterium colony, edge is irregular, and dirty white, rough surface is opaque, with《Conventional bacteriological surveillance handbook》Middle bacillus subtilis
Characteristic is consistent, therefore is accredited as bacillus subtilisBacillus subtilis。
Embodiment 1：By degeneration-resistant bacterial strain 1JN2 in 28 DEG C of 180 h of rpm shaken cultivations 16 of LB nutrient solutions, then 6000 rpm
10 min are centrifuged, it is 1 × 10 to dilute to obtain viable bacteria total concentration in microbial inoculum, microbial inoculum finished product with aqua sterilisa resuspension9 -1×1010 CFU/
mL。
Embodiment 2：Degeneration-resistant bacterial strain 1JN2 is inoculated into the LB nutrient solutions containing final concentration of 15mg/L cadmium sulfates（Culture
Liquid preparation is deionized water with water）, 28 DEG C, 180 rpm shaking table cultures 24h；2mL is drawn from the nutrient solution after culture respectively
Its light absorption value is detected under 600nm, separately takes 1mL nutrient solutions to be digested to determine wherein concentration of cadmium ions.
The screening foundation of bacterial strain：By the inoculation after activation to the LB nutrient solutions containing final concentration of 15mg/L cadmium sulfates
In（Nutrient solution preparation is deionized water with water）, 28 DEG C, 180 rpm shaking table cultures 24h；Inhaled respectively from the nutrient solution after culture
Take 2mL to detect its light absorption value under 600nm, strain growth situation is judged with this；It is another to take the progress digestion of 1mL nutrient solutions to be used to
Wherein concentration of cadmium ions is determined, digestion method is as follows：9mL deionized waters are separately added into 1mL nutrient solutions, 50mL is transferred to
In small beaker, concentrated nitric acid 1mL is added, 80 DEG C of water-baths are evaporated to 1mL and add concentrated nitric acid 1mL, perchloric acid 0.5mL, mix follow-up
Continuous water-bath is evaporated to 1mL, cooling, adds 4mL deionized waters, and atomic emission spectrometry is utilized after mixing（ICP）Determine wherein cadmium
Ion concentration.
Table 1：Greenhouse experiments bacterial strain information
| Strain number | Light absorption value1 | Cd2+Concentration（mg/L） | Cd2+Concentration rate of descent（%） |
| 1JN2 | 1.71 | 8.66 | 42.3 |
Note：Inoculation is to containing 15mg/L Cd2+LB nutrient solutions in determine light absorption value under 600nm after culture 24h, with
Nonvaccinated nutrient solution is control test light absorption value, and numerical value is the average value of three repetition experiments in table.
Greenhouse experiments：The cucumber and capsicum variety selected in greenhouse experiments are respectively " excellent No. 1 of Xinjin " and " great Yu ox horns
King ", matrix used is purchased from Huai'an rice and fuel river agricultural science and technology Co., Ltd；Hole plate seedling growth is used first, treats that seedling grows 3-4 piece true leaves
After be transplanted in basin alms bowl, greenhouse experiment be 30 DEG C, light application time 16h, interlunation 8h；
3 repetitions of each treatment group, each repeat 24 plants of plant；The not inoculating strain of control group 1, is used after transplanting one week
30mg/L cadmium sulfate root irrigations, 20mL/ basins；The not inoculating strain of control group 2, normal culture；5 treatment group processing modes are such as
Under：During transplanting, with 109CFU/mL bacteria suspension root irrigation, 20mL/ basins；Transplant after one week, with 30mg/L cadmium sulfate pouring roots
Processing, 20mL/ basins；After greenhouse experiments plantlet of transplant, each treatment group root length, fresh weight, chlorophyll content, root are detected within the 21st day respectively
It is vigor and blade Relative electro-conductivity gesture, detection method reference《Plant physiology experiment is instructed》；Cadmium ion is dense in plant tissue
Degree is adopted to be detected with the following method：Each treatment group randomly selects 3 plants of plant, accurately weighs 1g axis, using dry method ash
Change（600 DEG C of ashing 4h）, 1% hydrochloric acid elution ash content, deionized water is settled to 5mL, utilizes atomic emission spectrometry（ICP）Determine
Wherein concentration of cadmium ions.
Table 2：Greenhouse experiments result
Resistance to cadmium mechanism：The microbial inoculum of embodiment 1 is diluted, it is 10 to obtain concentration7CFU/mL 1JN2 bacteria suspensions, by 1% inoculum concentration
Access contains Cd2+In final concentration of 0.015mol/L LB nutrient solutions（Nutrient solution preparation is deionized water with water）, 28 DEG C, 180
Rpm shaking table cultures, sample in 12h, 24h, 36h and 48h, thalline are collected by centrifugation respectively.
Fixed after ultrapure water elution using 2.5% glutaraldehyde, scanning electron microscopic observation（Agricultural University Of Nanjing's biological experiment religion
Center）, to be not added with the treatment group of cadmium ion as control, the results detailed in Fig. 1.
Test result indicates that：Relative to control group, bacterial strain 1JN2 can strengthen host plant under cadmium ion treatment conditions
Tolerance, with blank control phase specific root length increase 21-28%, fresh weight increase 19-32%, chlorophyll content increase 11-20%, root
It is vigor increase 363-526%, and reduces concentration of cadmium ions 23-40% in plant tissue；The bacterial strain is found by ESEM
24h starts that ego resilience can be carried out and exocellular polysaccharide solidification phenomenon occurs in 48h after cadmium ion processing.
Claims (3)
1. assist host plant to alleviate the degeneration-resistant bacterial strain 1JN2 of cadmium ion stress, it is characterized in that：The degeneration-resistant bacterial strain 1JN2 is withered grass
Bacillus（Bacillus subtilis）, it is preserved in China Committee for Culture Collection of Microorganisms on October 13rd, 2014
Common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, culture presevation number is
CGMCC NO.9759。
2. the degeneration-resistant bacterial strain 1JN2 described in claim 1 is used for the method for preparing microbial inoculum, it is characterized in that the preparation method of the microbial inoculum
It is：By degeneration-resistant bacterial strain 1JN2 in 28 DEG C of 180 h of rpm shaken cultivations 16 of LB nutrient solutions, then 6000 rpm centrifugations, 10 min, are used
It is 1 × 10 that aqua sterilisa resuspension, which dilutes to obtain viable bacteria total concentration in microbial inoculum, microbial inoculum finished product,9 -1×1010 CFU/mL。
3. the degeneration-resistant bacterial strain 1JN2 described in claim 2 is used for the method for preparing microbial inoculum, it is characterized in that the microbial inoculum is assisting host
Plant alleviate cadmium ion stress application process be：Under greenhouse experiment, when capsicum and cucumber are transplanted, with 109CFU/mL bacterium
Agent root irrigation, 20mL/ plants；Transplant after one week, with 30mg/L cadmium sulfate root irrigations, 20mL/ plants.
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| CN116836898B (en) * | 2023-09-04 | 2023-11-21 | 山东迈科珍生物科技有限公司 | Microbial modifier suitable for rice planting and application thereof |
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