CN111560337B - Bacillus for promoting recovery of submerged plants in high organic matter bottom mud - Google Patents
Bacillus for promoting recovery of submerged plants in high organic matter bottom mud Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
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- 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
Abstract
The invention belongs to the technical field of polluted water bioremediation and discloses a stratospheric bacillus (Bacillus subtilis)Bacillus stratosphericus) PC2 and its use in promoting the recovery of submerged plants in high organic matter load sediments. Aiming at the problem that the submerged plant is difficult to recover under the condition of high organic matter content of the water body sediment, the invention screens the rhizosphere sediment of the submerged plant to obtain a PGPR strain, and after the growth promotion performance of the strain is evaluated, a stratospheric bacillus PC2 which has obvious promotion effect on the growth of the submerged plant in the sediment with high organic matter load is obtained, the preservation number of the strain is CCTCC M2020188, and the strain is inoculated to the rhizosphere of the submerged plant under the stress of the high organic matter content sediment, so that the purpose of quickly recovering the submerged plant community can be realized.
Description
Technical Field
The invention belongs to the technical field of polluted water body bioremediation, and particularly relates to a rhizosphere growth-promoting bacterium, namely Bacillus stratosphericus (PC 2), separated from rhizosphere sediments of submerged plants, wherein the strain can promote the recovery of the submerged plants in high organic matter load sediments.
Background
The submerged plant is used as an important primary producer in a water body, plays a role in regulating and controlling the material circulation and energy flow of a water ecosystem, plays an important role in maintaining biological diversity and clear water stable state, and is mainly realized in the ecological effect through the modes of absorbing water body nutrient substances, preventing bottom mud from being resuspended, providing a biological shelter place, competitively inhibiting water bloom algae and the like. However, the sediment is one of the main factors influencing the growth of the submerged plants, and has a determining function in the aspects of the growth, the shape and the distribution characteristics of the submerged plants. The submerged plant recovery process is often disturbed by the problems of high load sediment stress and the like, and the excessive nutrient salt in the sediment can stress the seeds of the submerged plant to germinate, the seedlings to grow and even have toxic action, thereby influencing the whole water ecological recovery process.
Plant growth-promoting rhizobacteria (PGPR) is a generic term for a group of bacteria that colonize the Plant rhizosphere and can directly or indirectly promote Plant growth. At present, a plurality of PGPR strains are separated from crops such as rice, wheat and the like, medicinal plants and economic forest trees. A large number of researches show that the rhizosphere beneficial microorganisms have the capability of enhancing the abiotic stress tolerance of plants such as drought stress, saline-alkali stress, heavy metal stress, nutrient deficiency and the like. For example, after the PGPR strain Streptomyces thermocarbonylydus is inoculated to rice, the drought resistance of the rice is improved by enhancing the dissolved phosphorus and secreting proline, phytohormone and siderophore, and the physiological indexes of dry weight, fresh weight, chlorophyll and the like are obviously improved. Thus, inoculation of PGPR also has potential for restoration of submerged plants under abiotic stress. At present, related application of PGPR focuses on terrestrial plants, and no report on the use of the rhizosphere growth-promoting bacteria for submerged plant restoration exists. For the recovery of submerged plants in the water body with high organic matter sediments, the propagation bodies and plants are made to cope with the environmental stress by utilizing the mutual beneficial symbiotic relationship of plants and microorganisms, so that the biomass is expanded and stably grows, and the method is the key for successful ecological restoration of lakes with high organic matter sediments.
Disclosure of Invention
Aiming at the problems that the growth of submerged plants in a high organic matter lake is inhibited, so that the ecological restoration process of the lake is slow and the like, the invention provides a rhizosphere growth-promoting bacterium stratosphericus Bacillus (Bacillus stratosphericus) PC2, the submerged plants in high organic matter load sediments are recovered by using the strain, and the plant height, the root length, the overground fresh weight and the root fresh weight of the submerged plants under the condition of high organic matter bottom mud can be obviously improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
the applicant obtains rhizosphere growth-promoting bacteria from submerged plant rhizosphere sediments through screening, and a rhizosphere growth-promoting bacteria strain is screened through growth-promoting performance evaluation (determination of four growth-promoting indexes of P-dissolving, indoleacetic acid-producing, cytokinin-producing capability and 1-aminocyclopropane-1-carboxylic acid deaminase activity), has the highest P-dissolving capability and higher indoleacetic acid-producing and cytokinin-producing capabilities, is Bacillus stratosphericus (PC 2), and is sent to a Chinese typical culture collection center for preservation at 6-8 months in 2020, and is classified and named: bacillus stratosphericus (Bacillus stratosphericus) PC2, accession number: CCTCC M2020188.
In the specific embodiment of the invention, the applicant compares the influence of the stratosphericus strain PC2 and other 3 rhizosphere growth-promoting bacteria on the germination of the eel grass seeds and the seedling growth of the eel grass seeds in the high organic matter sediments, and the results show that compared with the blank treatment group, the plant height of the eel grass seeds in the stratosphericus strain PC2 treatment group after germination is increased by 95.7%, and the plant height, the root length, the overground fresh weight and the root fresh weight of the eel grass after 120 days of culture are respectively increased by 165.0%, 17.4%, 378.8% and 165.1%. Therefore, the stratosphericus strain PC2 has a remarkable growth promoting effect on plants (such as tape grass) under high organic matter stress.
Compared with the prior art, the method has the advantages and beneficial effects as follows:
1. the carbon source and the nitrogen source which can be utilized by the bacillus stratosphere PC2 have wide range and are easy to culture.
2. The stratospheric bacillus PC2 is applied to roots of high-organic-matter submerged plants, the effect is remarkable, the plant height can be increased by 165.0% compared with blank treatment, the root length can be increased by 17.4% compared with blank treatment, the overground fresh weight can be increased by 378.8% compared with blank treatment, and the root fresh weight can be increased by 165.1% compared with blank treatment.
3. Compared with other methods for restoring submerged plants, the method applies PGPR to restoring the submerged plants under the stress of high organic matters for the first time, only needs to supplement bacterial suspension, is simple to operate, and is green and environment-friendly.
Drawings
FIG. 1 is a photomicrograph (1000 Xmagnification) of Bacillus stratosphericus PC 2.
FIG. 2 shows the effect of PGPR inoculation on the plant height of germinated Sophora alopecuroides seeds.
FIG. 3 is a graph showing the effect of PGPR inoculation on the growth of seedlings of Sophora alopecuroides.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the content of the present invention, but the content of the present invention is not limited to the following examples.
Example 1 isolation and screening of submerged plant PGPR
(1) Sample collection
Two sediments with high organic matter content (OM ═ 17.35%) and low organic matter content (OM ═ 4.94%) in Hangzhou West lake are selected as two treatments (numbered as H and L), each treatment is carried out in parallel, and the tape grass is planted in a glass jar for 70 days. Collecting the root system of herba Swertiae Dilutae and the deposit around the root system of herba Swertiae Dilutae about 10g, and refrigerating at 4 deg.C. In addition, the locus where the eel grass, the curly pondweed, the watermifoil, the black alga and the microtooth eyeweed in good growth state are located is directly selected from the natural lake of the Hangzhou west lake with low organic matter substrate sludge as the rhizosphere soil collection point (the high organic matter demonstration area has no submerged plant growth), and the number is S. About 10g of plant roots and deposits attached to the periphery of the roots are collected at each site, and the roots and the deposits are refrigerated and stored in a refrigerator at 4 ℃.
(2) Preliminary screening
H, L and 10g of S-group tape grass root system and peripheral attached sediments are respectively weighed in a sterilized conical flask, 90mL of sterile water and sterilized glass beads are added, and shaking culture is carried out at the temperature of 28 ℃ and the speed of 170r/min for 30min to obtain bacterial suspension. Through gradient dilution, 10 is obtained-4、10-5、10-6And (3) respectively sucking 0.1mL of bacterial suspension liquid into a bacterial suspension liquid plate with the concentration, coating the bacterial suspension liquid plate on a root system secretion culture medium for primary screening of growth-promoting bacteria, treating three concentration gradients in each treatment, repeating three gradients in each treatment, and culturing for 5 days in a constant-temperature incubator at 28 ℃. Respectively selecting dominant strains for separation and purification, and storing at-80 ℃ for later use.
The culture medium is root secretion culture medium, a proper amount of tape grass in H, L groups of experimental devices is taken, roots are respectively soaked in ultrapure water, leaves are placed outside and periodically sprayed with water, illumination culture is carried out for 6 hours, root soak solution is freeze-dried to obtain root secretion, and the root secretion solution with the final concentration of 20mg/L is prepared. Sterilizing the root exudate by a 0.22 mu m filter membrane, placing the sterilized exudate into a water bath at 60 ℃ for preheating, sterilizing by 40g/L agar culture medium high-pressure steam, cooling the sterilized exudate into the water bath to 60 ℃, and then mixing the sterilized exudate and the cooled exudate with water according to the volume ratio of 1: 1 mixing to obtain the primary screening culture medium.
(3) Double sieve
And (3) further measuring four growth promotion indexes of P dissolution, indoleacetic acid (IAA) production capability, Cytokinin (CKs) production capability and 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase activity of the dominant strain obtained by primary screening, and finally screening to obtain three optimal strains (PC2, H19 and L3) which are stored in a refrigerator at the temperature of-80 ℃ for rhizosphere inoculation of submerged plants.
TABLE 1 Table of three PGPR basic information
PGPR is mainly used for promoting the growth and development of crops by synthesizing certain plant hormones, such as auxin analogs, cytokinin, enzyme substances for degrading ethylene and the like. Among the four growth promoting indexes, IAA and CKs can be used as plant hormones to accelerate cell division and growth, so that plant growth is promoted. ACC deaminase can help plants break down the synthetic precursor ACC of ethylene, metabolize it to alpha-ketobutyrate and ammonia, and use it as a carbon and nitrogen source, supplying the bacteria with their growth needs. Phosphorus is one of the essential nutrient elements for plant growth and development, and when the plant body is deficient in phosphorus, the synthesis of important biomolecules such as protein, nucleic acid, coenzyme and the like is affected, so that plant branches are reduced, and the plant is short and small. Phosphorus in combination with Fe in soil3+、Ca2+And Al3+And the like are combined to be water-insoluble and are difficult to be absorbed by plants. The phosphorus-dissolving bacteria can convert insoluble or insoluble phosphorus in plant rhizosphere and natural soil into soluble phosphorus which is easily absorbed and utilized by plants. The PC2 strain has the highest P-dissolving capacity, high IAA and CKs producing capacity and comprehensive capacity greater than H19 and L3, so that the strain has higher application value.
The 16S rRNAs of the three PGPR strains are respectively shown in SEQ ID NO.1-3, and the specific information is shown in Table 1. The PC2 strain has been sent to the China center for type culture Collection (CGMCC) for collection at 6/8/2020, under the classification name: bacillus stratosphericus (Bacillus stratosphericus) PC2, accession number: CCTCC M2020188.
Example 2 Effect of PGPR on Germination of Swertia amara seeds in high organic matter load sediments
Selecting high organic matter sediment (OM ═ 17.35%) and making high-temperature sterilization, then spreading it in culture dish with diameter of 12cm, uniformly placing 100 disinfected and sterilized bitter grass seeds, adding pure water to make them pass through the sediment, every treatment is parallel, at the same time setting no-bacteria as blank control, and making four treatment groups of CK, PC2, H19 and L3, and every culture dish is initially added with bacterial liquor (OD)6001)1mL, adding pure water once after 5 days, supplementing pure water once every 3 days, placing in a 2000lux light incubator for 10 days in total, and illuminating for 12 hours and darkness for 12 hoursAnd measuring the plant height after the experiment is finished.
The experimental result is shown in figure 1, the plant heights of the PC2, H19 and L3 treated groups are increased by 95.7%, 69.6% and 81.2% compared with the blank, the growth promotion effect on the germinated tape grass seeds under the stress of high organic matters is obvious, and the effect of the strain PC2 is optimal.
Example 3 Effect of PGPR on the growth of Sophora alopecuroides seedlings in high organic matter load sediments
Four treatment groups of CK, PC2, H19 and L3 are arranged, and the organic matter content of the selected high organic matter sediment is 17.4%. In each treatment, 15 planting cups (diameter 5cm and height 8cm) are uniformly placed in a box of 35X 50X 30cm, sediment of 5cm height is paved at the bottoms of the planting cups, and five sowthistle seedlings with the same size and biomass are planted in each planting cup. The experiment is carried out under outdoor natural illumination, the experiment time is 7 months to 11 months of 2019 years, the culture temperature range is 5-36 ℃, the experiment period is 120 days, the water is added every 3 days, and the plant rhizosphere is supplemented with a bacterial liquid (OD) by using a disposable dropper every 15 days 6001, 2mL), the blank treatment was replaced with ultrapure water. And (4) respectively collecting samples at 20d, 40d, 60d, 90d and 120d, randomly selecting three planting cups for each treatment in each sampling, collecting plant samples, and measuring the plant height, the root length, the overground fresh weight and the root fresh weight of the plant samples.
The experimental results are shown in fig. 2, and the plant height of the plants is obviously inhibited by blank treatment along with the experimental time. After the experiment is finished, the plant height, the root length, the overground fresh weight and the root fresh weight of the PC 2-treated group are respectively increased by 165.0 percent, 17.4 percent, 378.8 percent and 165.1 percent compared with the blank treatment; the plant height, the root length, the overground fresh weight and the root fresh weight of the H19 treatment group are respectively increased by 143.2 percent, 6.5 percent, 329.8 percent and 92.8 percent compared with the blank treatment; the plant height, the root length, the overground fresh weight and the root fresh weight of the L3 treated group are respectively increased by 89.3 percent, 6.5 percent, 189.0 percent and 72.3 percent compared with the blank treatment, and the comprehensive influence on the growth promotion of the tape grass is PC2> H19> L3.
Sequence listing
<110> institute of aquatic organisms of Chinese academy of sciences
<120> bacillus for promoting recovery of submerged plants in high organic matter bottom mud
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cttgacatcc tctgacaacc ctagagatag ggctttccct tcggggacag agtgacaggt 1020
ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080
aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa 1140
accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200
gtgctacaat ggacagaaca aagggctgcg agaccgcaag gtttagccaa tcccacaaat 1260
ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa 1320
tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380
ccacgagagt ttgcaacacc cgaagtcggt gaggtaacct ttatggagcc agccgccgaa 1440
gtgcagagtg g 1451
<210> 2
<211> 1452
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aggctgcggc gtgctataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt 60
tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120
gaaaccgggg ctaataccgg atggttgttt gaaccgcatg gttcaaacat aaaaggtggc 180
ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240
accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420
gaacaagtac cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540
cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg 600
agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag 660
cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720
actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780
gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840
cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960
gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag 1020
gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080
gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac 1140
aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200
acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa 1260
atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt 1320
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac cttttaggag ccagccgccg 1440
aagtgacaga tt 1452
<210> 3
<211> 1458
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggaatggcgg cgtgcctata catgcaagtc gagcgaatgg attaagagct tgctcttatg 60
aagttagcgg cggacgggtg agtaacacgt gggtaacctg cccataagac tgggataact 120
ccgggaaacc ggggctaata ccggataaca ttttgaaccg catggttcga aattgaaagg 180
cggcttcggc tgtcacttat ggatggaccc gcgtcgcatt agctagttgg tgaggtaacg 240
gctcaccaag gcaacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga 300
gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt 360
ctgacggagc aacgccgcgt gagtgatgaa ggctttcggg tcgtaaaact ctgttgttag 420
ggaagaacaa gtgctagttg aataagctgg caccttgacg gtacctaacc agaaagccac 480
ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttat ccggaattat 540
tgggcgtaaa gcgcgcgcag gtggtttctt aagtctgatg tgaaagccca cggctcaacc 600
gtggagggtc attggaaact gggagacttg agtgcagaag aggaaagtgg aattccatgt 660
gtagcggtga aatgcgtaga gatatggagg aacaccagtg gcgaaggcga ctttctggtc 720
tgtaactgac actgaggcgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780
ccacgccgta aacgatgagt gctaagtgtt agagggtttc cgccctttag tgctgaagtt 840
aacgcattaa gcactccgcc tggggagtac ggccgcaagg ctgaaactca aaggaattga 900
cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960
ccaggtcttg acatcctctg aaaaccctag agatagggct tctccttcgg gagcagagtg 1020
acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080
gagcgcaacc cttgatctta gttgccatca ttaagttggg cactctaagg tgactgccgg 1140
tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct 1200
acacacgtgc tacaatggac ggtacaaaga gctgcaagac cgcgaggtgg agctaatctc 1260
ataaaaccgt tctcagttcg gattgtaggc tgcaactcgc ctacatgaag ctggaatcgc 1320
tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380
gtcacaccac gagagtttgt aacacccgaa gtcggtgggg taaccttttt ggagcccagc 1440
cgcctaagtg acagagtt 1458
Claims (3)
1. Bacillus stratosphericus (Bacillus stratosphericus) PC2, characterized in that the strain has a accession number of CCTCC M2020188.
2. Use of the bacillus stratosphericus PC2 of claim 1 to promote the recovery of submerged plants in high organic matter load sediments.
3. The use according to claim 2, wherein the submerged plant is tape grass.
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Non-Patent Citations (5)
| Title |
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| Bacillus as Plant Growth Promoting Rhizobacteria (PGPR): A Promising Green Agriculture Technology;Kashyap B.K.等;《 Plant Health Under Biotic Stress》;20190509;219-236 * |
| Plant growth-promoting rhizobacteria isolation from rhizosphere of submerged macrophytes and their growth-promoting effect on Vallisneria natans under high sediment organic matter load;Wang C等;《Microb Biotechnol》;20210128;726-736 * |
| Vertical distribution of microbial communities in chromium-contaminated soil and isolation of Cr(Ⅵ)-Reducing strains;Liu B等;《Ecotoxicol Environ Saf.》;20190520;242-251 * |
| 根际促生菌对沉水植物的促生效应及其与沉积物氮磷赋存形态的关系;王会会 等;《水生生物学报》;20210315;299-307 * |
| 江苏沿海滩涂植物根际促生菌的筛选鉴定及其耐盐促生特性;王小兵等;《扬州大学学报(农业与生命科学版)》;20160630(第02期);84-89 * |
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Application publication date: 20200821 Assignee: WUHAN THOUGHT OF FORGING ANEW ENVIRONMENT ENGINEERING Co.,Ltd. Assignor: Institute of Hydrobiology, Chinese Academy of Sciences Contract record no.: X2023980045919 Denomination of invention: A Bacillus strain promoting the recovery of submerged plants in high organic matter sediment Granted publication date: 20211214 License type: Exclusive License Record date: 20231108 |