CN112592869B - Bacillus indians with efficient coral larva attachment and metamorphosis inducing effect and application thereof - Google Patents
Bacillus indians with efficient coral larva attachment and metamorphosis inducing effect and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
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- 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
- Y02A10/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE at coastal zones; at river basins
- Y02A10/26—Artificial reefs or seaweed; Restoration or protection of coral reefs
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- 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
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses a strain of Bacillus indians with an effect of efficiently inducing attachment and metamorphosis of coral larvae and application thereof. Bacillus indiana (Bacillus indicus) SCSIO 43739, accession number: CCTCC NO: m2020835. The invention separates and obtains the Bacillus indicus (Bacillus indicus) SCSIO 43739 from the clustered helmet-shaped coral in the reindeer coral reef area of the Sanlu in Hainan province of China, has the function of efficiently inducing the attachment and metamorphosis of coral larvae, can be used for promoting the attachment and metamorphosis of the coral larvae, and can be used for ecological restoration of coral reefs.
Description
The technical field is as follows:
the invention belongs to the technical field of microorganisms, and particularly relates to Indian bacillus with an effect of efficiently inducing attachment and metamorphosis of coral larvae and application of Indian bacillus.
Background art:
coral reefs are one of the most diverse and complex marine ecosystems in the world, distributed in tropical and subtropical shallow sea areas between 30 degrees south latitude and 30 degrees north latitude (stabbler, 2011). The area of the coral reef around the world is about 30 ten thousand square kilometers, and is mainly distributed in india-pacific and atlantic-caribbean regions (stabbler, 2011). Coral reefs provide considerable economic, social, and ecological value to mankind through coastal protection, marine foods, tourist sightseeing, and marine medicine (Costanza et al, 2014). However, global coral reefs are facing severe degradation due to global warming, rising surface temperatures, ocean acidification, and interference with human activities (Hughes et al, 2003). Over the past 20 years, the global coral albinism event has caused the death of many thousands of square kilometers of coral and coral reef organisms (Hughes et al, 2017). Coral reefs in China are mainly distributed in the waters of the south China sea, the Hainan island, the Taiwan island, hong Kong and the coast of south China (such as Fujian, Guangdong and Guangxi coasts) (Zhang et al, 2001). Since the 80 s of the 20 th century, the same face a severe decline situation under the influence of human activities such as coastal engineering construction, destructive fishing, aquaculture, etc. (caiyulin et al, 2018). Coral is used as the main reef-building organism of the coral reef ecosystem, and the dynamic change of the population quantity directly influences the stability of the coral reef ecosystem. In the life history of coral, the attachment and metamorphosis of its larva are the bottleneck of new coral individual supplement, and the success or failure is directly related to the change of coral population. Therefore, the improvement of the attachment and metamorphosis rate of the coral larvae has important ecological significance for the supplement and construction of coral reef groups.
Coral is a symbiont composed of hydranth of coral and a variety of microorganisms including bacteria, fungi, archaea, viruses and protists. Abundant microorganisms not only provide a source of nutrition for corals, but also participate in energy metabolism and immune system formation of corals (Foo et al, 2017). In addition, there have been studies showing that microorganisms play an important role in settlement metamorphosis of coral larvae, which may be effected by forming biofilms or culture solutions of single bacteria. As in 2001, Negri et al isolated from calcified coral algae for the first time to obtain a pseudoalteromonas strain could significantly induce metamorphosis of larvae of Cervus elaphus (Acropora willisae) and Cervus porophus (Acropora millefora) (Negri et al, 2001). Subsequent isolation of bacteria from different species from different environments can also induce attachment or metamorphosis of different coral larvae, including bacteria of the genera Roseivivax, Thalassomonas, Marinobacter, and Cytophaga (ritchai et al, 2012; Shikuma et al, 2014; Sharp et al, 2015). These findings indicate that there may still be a large number of inducible strains to be exploited in the marine environment. Although the coral reef protection and repair work has been carried out for many years, the effect is very little, and the existing mature repair technology for establishing a nursery by using coral asexual propagules has high cost and large workload, and cannot be cultivated on a large scale due to the limitation of the coral asexual propagules (Small et al, 2019; Randall et al, 2020). Therefore, a strain which can efficiently induce the attachment and metamorphosis of coral larvae and is non-toxic is obtained by screening, so that the replenishment rate of new coral individuals is improved, not only is germplasm resources provided for the cultivation of a coral nursery, but also the genetic diversity of the coral is protected, and the recovery and the stability of coral reef groups are facilitated.
The invention content is as follows:
the first purpose of the invention is to provide a strain of Bacillus indicus (Bacillus indicus) SCSIO 43739 which is separated from Sanlu's coral reef area clustered helmet coral (Galaxea fascicularis) in China Hainan and has the function of efficiently inducing attachment and metamorphosis of coral larvae, wherein the strain is preserved in China Center for Type Culture Collection (CCTCC) at 12 and 3 days of 2020, and the address is as follows: wuhan university, the preservation number: CCTCC NO: m2020835.
The result of the measurement of the growth curve of the indian bacillus SCSIO 43739 shows that the growth rate of the indian bacillus SCSIO 43739 is fast, the growth cycle is short, and the indian bacillus SCSIO 43739 gradually enters the plateau stage at 16 h.
The induction effect of different concentrations of Bacillus Indian SCSIO 43739 was verified, and the result shows that the final concentration of the strain is 3.2 × 107cfu/ml, the induction effect is best, the co-cultured coral larvae are metamorphosed within 24 hours, and the metamorphosis rate is 43%; and the adhesion is carried out within 48h, and the adhesion rate reaches 70 percent.
Therefore, the second object of the present invention is to provide the use of the above-mentioned Bacillus indian SCSIO 43739 or a microbial preparation containing Bacillus indian SCSIO 43739 for ecological restoration of coral reefs.
Preferably, the application in coral reef ecological restoration is inducing attachment and metamorphosis of coral larvae.
Preferably, the coral larvae are goblet antler shaped coral (Pocillopora damicornis) larvae.
Further preferably, the larva of the goblet of deer antler is put in a concentration of 3.2X 107cfu/ml of Bacillus indiae SCSIO 43739.
It is a third object of the present invention to provide a microbial preparation for inducing attachment metamorphosis of coral larvae, which comprises Bacillus indiani SCSIO 43739 as an active ingredient.
The invention separates and obtains the Bacillus indicus (Bacillus indicus) SCSIO 43739 from the clustered helmet-shaped coral in the reindeer coral reef area of the Sanlu in Hainan province of China, has the function of efficiently inducing the attachment and metamorphosis of coral larvae, can be used for promoting the metamorphosis of the coral larvae, and can be used for ecological restoration of coral reef.
Bacillus indiana (Bacillus indicus) SCSIO 43739 was deposited at the chinese culture collection center (CCTCC) at 12 months and 3 days 2020, address: wuhan university, the preservation number: CCTCC NO: m2020835.
Description of the drawings:
FIG. 1 is a phylogenetic tree of Bacillus indiae SCSIO 43739;
FIG. 2 is a growth curve of Bacillus indiae SCSIO 43739;
FIG. 3 is a graph showing the effect of different concentrations of Bacillus indiae SCSIO 43739 on settlement and metamorphosis of Callitha cervi larvae.
The specific implementation mode is as follows:
the present invention will be described more specifically with reference to the following examples, but is not limited thereto.
Example 1: isolation, purification and characterization of Bacillus indiae SCSIO 43739
Separation of the strains: bacillus Indigofera SCSIO 43739 was isolated and purified from Corallium japonicum Kishinouye collected from the coral reef area of the red deer in Hainan province of China in 11 months of 2017, and stored in-80 deg.C with 50% glycerol.
Identification of the 16s sequence of bacillus indiana SCSIO 43739: extracting Bacillus India SCSIO 43739 strain genome DNA by using a bacterial DNA extraction kit (Takara, Japan), measuring the purity and concentration of the DNA by using Nannodrop1000, and amplifying qualified DNA by 16s rRNA genes, wherein amplification primers are respectively as follows: the forward primer was 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), the reverse primer was 1492R (5'-CGGTTACCTTGTTACGACTT-3'); the PCR amplification system is as follows: 12.5. mu.l of TaqPCR mix, 1. mu.l of DNA template, 0.5. mu.l of each of primers 27F/1492R, ddH2Make up to 25. mu.l of O. The PCR amplification conditions were: pre-denaturation at 95 deg.C for 10min, denaturation at 9 deg.C for 1min, annealing at 54 deg.C for 1min, extension at 72 deg.C for 2min, 35 cycles, and final extension at 72 deg.C for 10 min. The band of interest (about 1500bp) was confirmed by running 1.2% agarose gel electrophoresis under a 260nm gel imager, and the PCR product containing the band of interest was sequenced by the company (Tianyihui Biotech, Inc., Guangzhou) and the sequence thereof was shown as SEQ ID NO. 1. Uploading the obtained genetic sequence to an Ezbiocloud platform to obtain a kindred sequence, performing multi-sequence alignment by using Align in MEG6.0 software, and constructing a phylogenetic tree by using a Neighbor-join method. The results are shown in FIG. 1: the closely related sequence of Bacillus coli SCSIO 43739 is Bacillus indicus LMG 22858 with a similarity of 99.93%, which belongs to the phylum Firmicutes, class Bacillaceae (Bacillus), order Bacillales (Bacillus), family Bacillaceae (Bacillus), genus Metabacillus (Metabacillus). Thus, indian bacillus SCSIO 43739 was named: bacillus indifferentus (Bacillus indicus) SCSIO 43739, which was administered 12 months and 3 days 2020Deposited in China Center for Type Culture Collection (CCTCC) with the address: wuhan university, the preservation number: CCTCC NO: m2020835.
Example 2
Activation of Bacillus indiae SCSIO 43739
Taking out Bacillus India SCSIO 43739 stored at-80 deg.C, thawing at room temperature, mixing, spreading 200ul of the bacterial liquid on 2216E solid culture medium, culturing in 28 deg.C incubator, selecting single colony, and streaking.
The 2216E solid culture medium comprises the following components:
2216E composition of solid Medium: tryptone 5g, Yeast extract 1g, FePO4·2H2O0.1 g, NaCl 25 g, agar 18g, ddH2O1000 mL; mixing the above culture medium components, adjusting pH to 7.0-8.0, and sterilizing.
Second, preparation of Bacillus India SCSIO 43739 thallus
Preparation of the thallus: a single colony of Bacillus India SCSIO 43739 was picked from the plate, inoculated into MA liquid medium, and cultured on a shaker (32 ℃ C., 180 rpm). And (5) centrifuging at 8000rpm for 5min to remove supernatant after the bacterial liquid is cultured to be in a turbid state, and obtaining the experimental thallus.
Wherein the MA liquid culture medium comprises the following components:
composition of MA liquid Medium: difco Marine Broth 18g, NaCl 9g, ddH2O1000 mL; mixing the above culture medium components, adjusting pH to 7.0-8.0, and sterilizing.
Thirdly, drawing of the growth curve of the Indian bacillus SCSIO 43739
Drawing a strain growth curve: a single colony of Bacillus India SCSIO 43739 was inoculated into 250ml MA broth and cultured in a shaker at 180rpm at 28 ℃. Taking the first sample after 4h, sampling every 2h, measuring the OD value at 600nm by using a spectrophotometer, and carrying out zero setting calibration by using an MA liquid culture medium for 3 times each time. As shown in FIG. 2, the number of Bacillus Indian SCSIO 43739 increased gradually with the increase of the culture time, and the strain entered the logarithmic expression after 4 hoursGrowing until the number of the thallus is maximum at 16h gradually entering a plateau stage, and culturing until the thallus number is maximum at 18h, wherein the OD is the maximum600At 1.058, the number of cells began to decrease.
Fourth, screening of the optimum Induction concentration of Bacillus India SCSIO 43739
Screening of optimal induction concentration: a single colony of Bacillus India SCSIO 43739 was inoculated into MA liquid medium and cultured on a shaker (32 ℃ C., 180 rpm). When the bacterial liquid is cultured to a platform stage, centrifuging at 8000rpm for 5min to remove supernatant, resuspending by using seawater filtered by a 0.2-micron industrial filter bag, diluting to 6 concentration gradients, and respectively adding into a 6-hole culture plate, wherein 6 final concentrations of the bacterial strain are respectively as follows: 3.2X 108cfu/ml、1×108cfu/ml、3.2×107cfu/ml、1×107cfu/ml、 3.6×106cfu/ml、1×106cfu/mL, containing 10 larvae and 10mL of inoculum per well, for a total of 3 replicates. In the control group, the bacteria solution was replaced with filtered seawater of equal volume. Placing 6-well culture plate in a place with illumination intensity of 250 phototns.m-2·s-1The culture was carried out at room temperature controlled at 28 ℃. + -. 1 ℃. The metamorphosis number and the attachment number of the coral larvae are recorded by naked eyes and anatomical lens observation at intervals of 24h for 72 h. The results are shown in FIG. 3, the concentration is 3.2X 107cfu/ml and 1X 107The bacterial liquid of cfu/ml can induce coral larva to metamorphose within 24h, the metamorphosis rate is 43% and 17%, wherein the concentration is 1 × 107The metamorphosis rate of larvae in the cfu/ml bacterial liquid group is the highest in 48 hours and reaches 63 percent; furthermore, the concentration was 1X 107The bacterial liquid of cfu/ml can induce coral larva to attach within 24h, the attachment rate is 10%, and the attachment rate of 48h larva reaches the highest and is 33%. And the concentration is 3.2X 107The bacterial liquid of cfu/ml can induce coral larva to attach in 48h, and the attachment rate of the larva is 70 percent, which is higher than that of the bacterial liquid group and the control group with other concentrations. Further, the concentration was 3.6X 106The cfu/ml bacterial solution can induce coral larva to attach in 72 hours, but the attachment rate is lower and is 6%. The bacteria liquid groups and the control group with the rest concentration do not generate attachment and metamorphosis during the experiment period, and are both the larvas in the wave state. In summary, the concentration is 3.2X 107Bacterial liquid of cfu/ml Bacillus indiae SCSIO 43739The coral larva settlement and metamorphosis inducing effect is optimal, the coral larva settlement and metamorphosis inducing effect can be achieved within 24 hours, the metamorphosis rate can reach 43%, the coral larva settlement inducing effect can be achieved within 48 hours, and the settlement rate can reach 70%.
The above are only preferred embodiments of the present invention, and it should be noted that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Sequence listing
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<120> bacillus indians with high-efficiency coral larva attachment and metamorphosis inducing effect and application thereof
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Claims (6)
1. Bacillus indiana (Bacillus indicus) SCSIO 43739, accession number: CCTCC NO: m2020835.
2. Use of the indian bacillus SCSIO 43739 or a microbial preparation containing the indian bacillus SCSIO 43739 according to claim 1 for ecological restoration of coral reefs.
3. The use as claimed in claim 2, wherein the coral reef ecology restoration is inducing settlement and metamorphosis of coral larvae.
4. The use as claimed in claim 3, wherein the coral larvae are goblet-shaped coral (Pocillopora damicornis) larvae.
5. The use as claimed in claim 4, wherein the larvae of the coral antler cup are introduced into the aqueous medium at a concentration of 3.2X 107cfu/ml of Bacillus indiae SCSIO 43739.
6. A microbial agent for inducing settlement and metamorphosis of coral larvae, which comprises the Bacillus indiani SCSIO 43739 of claim 1 as an active ingredient.
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| CN202110003695.9A CN112592869B (en) | 2021-01-04 | 2021-01-04 | Bacillus indians with efficient coral larva attachment and metamorphosis inducing effect and application thereof |
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| CN112592869B true CN112592869B (en) | 2022-02-22 |
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| JP2005160316A (en) * | 2003-11-28 | 2005-06-23 | Tetra Co Ltd | Method for marine culturing of coral and apparatus therefor |
| CN105779367A (en) * | 2016-05-23 | 2016-07-20 | 广东海洋大学 | Coral-associated marine bacillus amyloliquefaciens strain CoMb-9 and application thereof |
| WO2019029394A1 (en) * | 2017-09-07 | 2019-02-14 | 中国科学院南海海洋研究所 | Marine microbial agent and preparation method therefor |
| CN109618983A (en) * | 2018-12-03 | 2019-04-16 | 中国科学院南海海洋研究所 | A method of improving coral larva settlement and metamorphosis |
| CN110036947A (en) * | 2019-04-23 | 2019-07-23 | 深圳信息职业技术学院 | A method of induction coral Metamorphore improves adhesive ability |
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| JP2005160316A (en) * | 2003-11-28 | 2005-06-23 | Tetra Co Ltd | Method for marine culturing of coral and apparatus therefor |
| CN105779367A (en) * | 2016-05-23 | 2016-07-20 | 广东海洋大学 | Coral-associated marine bacillus amyloliquefaciens strain CoMb-9 and application thereof |
| WO2019029394A1 (en) * | 2017-09-07 | 2019-02-14 | 中国科学院南海海洋研究所 | Marine microbial agent and preparation method therefor |
| CN109618983A (en) * | 2018-12-03 | 2019-04-16 | 中国科学院南海海洋研究所 | A method of improving coral larva settlement and metamorphosis |
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| 珊瑚藻对黄癣蜂巢珊瑚浮浪幼虫生长和附着的影响;聂磊等;《海南热带海洋学院学报》;20190430;第26卷(第2期);21-26 * |
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