CN110229764B - Bacillus belgii YC666 and application thereof - Google Patents
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Abstract
The invention provides a Bacillus velezensis YC666 strain and application of the Bacillus velezensis YC666 strain in aluminum toxicity soil remediation, aluminum toxicity stress alleviation and plant aluminum toxicity stress resistance improvement. Bacillus velezensis YC666 is significantly resistant to Al (III), Mn (II), Co (II) metal stress and acid stress; YC666 can adsorb aluminum ions through its biological membrane; YC666 can relieve the stress of plant root system aluminum toxicity, relieve the toxic action of aluminum toxicity to plant growth, and improve the resistance of plant to aluminum toxicity.
Description
Technical Field
The invention relates to the field of metal pollution bioremediation, in particular to a bacillus beiLeisi YC666 strain and application thereof.
Background
Acid soil aluminum toxicity has become the most prominent limiting factor for crop growth (Kochian et al, 2004; Liu et al, 2014). Excessive accumulation of aluminum entering crops in humans and animals through the food chain can lead to permeable encephalopathies, senile dementia, alzheimer's disease, etc. (Klotz et al, 2017; Martinez et al, 2017). In China, the acid soil is spread over 14 provinces, and the total area reaches 203 km 2 Approximately 21% of the total cultivated land area nationwide (li.e., queen and xylonite, 2013). More worrisingly, the problem of acid rain precipitation, which is becoming more serious, is further aggravating the acidification of the soil, the scope and strength of which is still increasing. In actual agricultural activities, the application of lime can relieve aluminum toxicity, but the cost is high, secondary pollution is easily caused, soil hardening and other problems are caused, and therefore, the development of an environment-friendly bioremediation technology is urgently needed.
The microorganism has the characteristics of fast growth, low cost, environmental protection and the like, and shows great advantages in the aspect of restoring aluminum toxicity. For example, Penicillium microphyllum F-13(Penicillium janthinoleur) secretes organic acids and raises the pH of the medium, thus having high aluminum tolerance, but its mycelium does not absorb aluminum (Zhang et al, 2002). The Cryptococcus 14-3-3(Cryptococcus podzolicus) adsorbs or absorbs aluminum through thalli, so that the content of active aluminum in a culture medium is reduced by 85.1%, and the aluminum resistance can reach 200mM (cuojin, 2016). The bacillus has the advantages of fast growth, suitability for industrial production, long shelf life and the like, and shows huge potential and market prospect in soil remediation, however, few reports are made on the aspect of alleviating aluminum toxicity by the bacillus.
Biofilms are another survival strategy for microorganisms in response to aluminum toxicity stress. For example, after culturing bacillus mucilaginosus HJ07 and bauxite for 12 days in a mixed manner, a significant biofilm is formed on the surface of the bauxite, thereby promoting the decomposition of the bauxite (xiao guo et al, 2013). Al (iii) promotes bacteria in the reclaimed water to form a biofilm, causing bacterial flocculation to clog reclaimed water porous media, and therefore it is desired to reduce aluminum content in reclaimed water to reduce biofilm formation (dawn spring, 2017). Jackson et Al (2009) reports that pseudomonads, Sphingomonas and bacilli (Pseudomonas sp., Sphingomonas sp., and Bacillus sp.) on the surface of a bioreactor form a mixed bacteria biofilm, and the adsorption rates of Al, Ni and Zn in sewage respectively reach 85%, 65% and 97%, so that the bacteria biofilm has important practical significance for eliminating metal pollution such as aluminum in sewage. However, the bacillus biofilm technology is adopted to eliminate root aluminum toxicity and promote plant growth, and the report on the aspect of eliminating root aluminum toxicity and promoting plant growth is not seen at home and abroad.
Therefore, aiming at the current situation of aluminum toxicity harm in acid soil, particularly the problem that the acid soil area in south China is continuously increased, a novel bioremediation way is provided for the acid soil aluminum toxicity by digging a bacillus resource which has high yield of biological film, slows down medium aluminum toxicity by the biological film absorbing aluminum, colonizes by the biological film adhering to a root system and promotes the growth of the plant root system.
Disclosure of Invention
The invention aims to provide a bacillus beleisi YC666 and application thereof.
The technical scheme adopted by the invention is as follows:
a strain of Bacillus velezensis YC666, deposited in Guangdong province collection of microorganisms, address: building 5 of the first 100 th courtyard college, building 59, zip code: 510075, accession number: GDMCC No. 60627.
An agent comprising Bacillus velezensis YC666 as described above.
The application of the Bacillus velezensis YC666 in the aluminum toxicity soil remediation is provided.
Further, the soil is an acid soil.
An aluminum-toxicity soil bioremediation method, which uses the Bacillus velezensis YC666 to adsorb aluminum ions in soil.
Further, the soil is an acid soil.
The application of the Bacillus velezensis YC666 in alleviating plant aluminum toxicity stress is provided.
Further, the aluminum toxicity stress is aluminum toxicity stress in an acidic environment.
A method for relieving plant aluminum toxicity stress comprises applying a microbial inoculum containing Bacillus velezensis YC666 to plant root system.
Further, the aluminum toxicity stress is aluminum toxicity stress in an acidic environment.
The invention has the beneficial effects that:
the invention provides a Bacillus belgii strain YC666, wherein YC666 has obvious resistance to Al (III), Mn (II), Co (II) metal stress and acid stress; YC666 can adsorb aluminum ions through its biological membrane; YC666 can relieve the stress of plant root system aluminum toxicity, relieve the toxic action of aluminum toxicity to plant growth, and improve the resistance of plant to aluminum toxicity.
Drawings
FIG. 1 is a YC666 phylogenetic tree diagram.
FIG. 2 is a photograph of YC666 showing significant resistance to Al (III), Mn (II), and Co (II) toxicity (2A plate, 2B bar).
FIG. 3 shows that YC666 is significantly resistant to acid (3A plate culture, 3B liquid culture).
FIG. 4 is a YC666 biofilm and pellicle configuration.
FIG. 5 is a graph of the capacity of YC666 biofilm to adsorb aluminum ions (5A liquid culture photograph, 5B biofilm weight histogram, 5C biofilm adsorbed aluminum content histogram).
FIG. 6 shows the colonization of YC666 by biofilm formation adhering to the root system of Arabidopsis.
FIG. 7 is the effect of YC666 on the growth of Arabidopsis plants under aluminum stress.
Detailed Description
Bacillus belgii YC666 as deposited in the Guangdong province culture Collection (GDMCC) in 2019 at 09.04.21 with accession number GDMCC No. 60627: hibernation No. 59, building No. 5, Hippocampus No. 100, Guangzhou, Guangdong province institute for microorganisms.
YC666 is obtained by separating and purifying soil in Guangzhou fire mountain forest park, belongs to gram-positive bacteria, has spores, rods, and is aerobic and motile; the colony formed after 24h of culture on LB solid medium is white circle with wrinkled surface.
The strain was identified by whole genome sequencing by collecting YC666 cultured in LB broth for 24h, and extracting YC666 DNA sample with genomic DNA Kit (genomic DNA Mini Kit, Qiagen, Germany). And obtaining a sequencing result of YC666 by adopting an Illumina high-throughput sequencing method. Then, the YC666 sequencing results were spliced and assembled using CLC bio. genomics work platform to obtain the whole genome sequence of YC666 with length 3,886,997bp, total 3917 genes (table 1). The YC666 whole genome was annotated with Rapid Annotation Subsystem Technology (RAST 2.0) software to preliminarily define the functional information of 2755 genes, 106 genes among which were used for stress response (table 2), thereby indicating that YC666 has a better repair of stress-related genes. In addition, the NCBI BLAST website is adopted to align with Bacillus model strains 168,3610, FZB42 and the like to obtain 16S rRNA, cheA and ropB sequences of YC666, the similarity of the sequences and the Bacillus belgii FZB42 is 100%, the YC666 is preliminarily identified as the Bacillus belgii and is named as the Bacillus belgii YC666, and the 16S rRNA, cheA and ropB gene sequences of the strains are determined as shown in SEQ ID NO:1, 2 and 3. A diagram of the YC666 phylogenetic tree is shown in FIG. 1.
TABLE 1 YC666 genomic sequence characterization
TABLE 2 distribution of YC666 Coding DNA Sequence (CDS)
Resistance of YC666 to Al (III), Mn (II), Co (II) metal stress
In order to evaluate the resistance of YC666 to metal ions, the invention adopts a flat filter paper diffusion method (disc diffusion assay) to determine, which is as follows: collecting LB culture solution (OD) of YC666 600 About 0.4) 100. mu.L was added to LB medium containing 4ml of 0.75% agar at 50 ℃ and mixed well, and then poured into a 1.5% LB solid plate containing 15 ml. After the medium was solidified (about 10 minutes), a filter paper sheet (diameter 6.5mm) to which each metal ion (5. mu.L) was added was placed in the center of the plate, and cultured overnight at 37 ℃. And (4) photographing and determining the diameter of the inhibition zone of the metal ions on the YC 666. The concentrations of the respective metal ions were as follows: 1M AlCl 3 ，1M MnCl 2 ，1M CuCl 2 ，100mM ZnCl 2 And 100mM CoCl 2 . The results (FIG. 2) show that this strain is significantly resistant to YC666 metal stresses Al (III), Mn (II), Co (II), compared to Bacillus subtilis model strain CU 1065.
Acid resistance of YC666
In order to evaluate the acid resistance potential of YC666, the invention adopts a flat filter paper diffusion method, 1M hydrochloric acid 10 mu L is added into filter paper sheets, and other experimental steps are shown in the metal stress resistance verification experiment. In addition, a 96-well microplate liquid culture method is adopted, which comprises the following specific steps: first, 200. mu.L of LB liquid medium at different pH concentrations was added to wells of a 96-well cell culture plate, and 2. mu.L of each strain broth (OD) was added to each well 600 About 0.4), culturing the strain in a shaking table at 37 ℃ and 100rpm for 18h, observing the growth condition of the strain under different pH concentrations, and finding out the lowest pH value of the growth of the strain.
The flat filter paper diffusion method result (figure 3) shows that YC666 has obvious acid resistance effect compared with CU1065 (the diameter of a inhibition zone is 2.97cm), and the diameter of the inhibition zone is 2.48 cm. The results of the microplate liquid culture show that: the CU1065 strain can grow above pH 5.0, while YC666 can grow above pH 4.3. Thus, YC666 has significant acid tolerance and resistance to hydrochloric acid stress as compared to control bacillus subtilis CU 1065.
YC666 biofilm producing ability
Biofilm is an important strategy for bacteria to cope with stress environment, and in order to evaluate the biofilm-producing ability of YC666, biofilm formation experiments using LBGM plates were performed, and the results showed that YC666 formed a biofilm morphology of light blue petals (about 2.6cm in diameter) compared to bacillus subtilis model strain NCIB3610 when cultured at 30 ℃ for 5 days. The results of the thin skin formation experiments using LBGM liquid medium (FIG. 4) showed that YC666 formed wrinkled yellow thin skin when cultured at 30 ℃ for 1 day, which had a morphology similar to that of the Bacillus subtilis model strain NCIB3610, but the thin skin yield was significantly higher than that of NCIB3610 (FIG. 4). Thus, YC666 has the ability to produce biofilm.
Ability of YC666 biomembrane to adsorb aluminum ion
To investigate the ability of YC666 biofilm to alleviate aluminum toxicity, CU1065 and YC 66624 h were grown in Msgg broth at 30 ℃ with different aluminum concentrations (0,0.5,1,5mM), respectively, and both strains were inhibited by Al (III) treatment and CU1065 was completely free of biofilm formation at 0.5mM Al (III) concentration compared to no aluminum. YC666 had biofilm formation at Al (III) concentrations of 0,0.5,1,5mM, 4.16, 3.48, 3.24, 2.28mg/ml, respectively. Further, the ICP-MS method is adopted to measure the aluminum concentration in the biological membrane, and the result (figure 5) shows that the aluminum content in the YC666 biological membrane is obviously higher than that in CU1065, which shows that the YC666 biological membrane reduces the aluminum content in the culture medium by adsorbing and enriching aluminum, thereby achieving the effect of relieving aluminum toxicity.
YC666 biofilm adhesion root system colonization ability
The glycerol freeze-dried tube YC666 with the temperature of-80 ℃ is selected and streaked on an LB culture medium, and is cultured overnight at the temperature of 37 ℃. Picking a single colony to an LB liquid culture medium, and performing shake culture at 150rpm and 37 ℃ for 14h to obtain a seed solution; inoculating the obtained seed liquid into a fermentation culture medium according to the inoculation amount of 1%, and performing shake culture at 37 ℃ for 10-14h at the rotation speed of 150r/min to obtain YC666 fermentation liquid.
To evaluate the formation of YC666 on the surface of the plant root systemThe biofilm colonization ability is determined by taking Arabidopsis thaliana plant as test material, placing sterile germinated 5-day Arabidopsis thaliana seedling in 96-well microplate, adding 300 μ L LBGM culture medium, and collecting YC666 fermentation liquid 3 μ L (OD 666 fermentation liquid) 600 About 0.4) was added to each well and cultured with shaking at 23 ℃ and 100 rpm. Samples were taken at 0h, 12h, 24h, 48h, and the roots were washed with PBS buffer and stained with SYTO13 solution (1500-fold diluted with PBS phosphate buffer) for 2 min, washed with PBS buffer, and imaged by scanning at 488nm excitation wavelength and 507nm emission wavelength on a Leica SP5 Confocal Scanning Laser Microscope (CSLM). The method successfully detects that YC666 forms a large number of biofilms on the surface of Arabidopsis root system (FIG. 6).
Ability of YC666 to alleviate plant aluminum toxicity stress
In order to investigate the effect of YC666 on the alleviation of the plant aluminum toxicity stress, after the Arabidopsis thaliana treated by NCIB3610 and YC666 strains is cultured for 7d at 22 ℃ in a liquid culture medium by a hydroponics method without adding aluminum, the root growth of the Arabidopsis thaliana treated by a control strain is obviously inhibited in an aluminum culture solution with 25 MuM, while the inhibition degree of the root growth of the Arabidopsis thaliana treated by the YC666 strain is obviously reduced (figure 7), which shows that YC666 can alleviate the toxic action of aluminum toxicity on the Arabidopsis thaliana growth and improve the resistance of the plant to the aluminum toxicity.
Claims (10)
1. A strain of Bacillus velezensis YC666, deposited in Guangdong province collection of microorganisms, address: building 5 of the first 100 th courtyard college, building 59, zip code: 510075, accession number: GDMCC No. 60627.
2. An agent comprising Bacillus velezensis (Bacillus velezensis) YC666 as claimed in claim 1.
3. The use of Bacillus velezensis YC666 as claimed in claim 1 in the remediation of aluminous soils.
4. Use according to claim 3, characterized in that: the soil is acid soil.
5. An aluminum toxicity soil bioremediation method is characterized in that: the use of Bacillus velezensis (Bacillus velezensis) YC666 as claimed in claim 1 for the adsorption of aluminium ions in soil.
6. The aluminous soil remediation method of claim 5, wherein: the soil is acid soil.
7. Use of Bacillus velezensis YC666 according to claim 1 for alleviating plant aluminium toxicity stress.
8. Use according to claim 7, characterized in that: the aluminum toxicity stress is aluminum toxicity stress in an acidic environment.
9. A method for relieving aluminum toxicity stress of plants, which is characterized by comprising the following steps: applying a microbial inoculum containing Bacillus velezensis YC666 as claimed in claim 1 to the root system of a plant.
10. The method of claim 9, wherein: the aluminum toxicity stress is aluminum toxicity stress in an acidic environment.
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