Aspergillus submananiii from deep sea and application of Aspergillus submananiii in prevention and treatment of alien species invasion

Abstract

The invention discloses Aspergillus sp. Aspergillus sp. CHNSCLM-1229, which is derived from cold water coral in Mariinensis, and the preservation number of the Aspergillus sp. Aspergillus sp. CHNSCLM-1229 is CGMCC (China General Microbiological Culture Collection Center) No: 41021. It is found through experiments that the extract obtained through separation from an Aspergillus submananii CHNSCLM-1229 fermentation culture has the remarkable inhibitory activity on attachment of alien invasive species, such as seascia sachatinii, of fouling organisms. Therefore, the extract of the fungus Aspergillus submananii CHNSCLM-1229 derived from deep sea has a wide application and development prospect in the preparation of the product for inhibiting the attachment of alien invasive species of the fouling organisms.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an extract of Aspergillus subramanianii CHNSCLM-1229 (with a preservation number of CGMCC No: 41021) from a deep-sea cold-water coral symbiotic fungus and application thereof in inhibiting the attachment of alien invasive species such as Aspergillus subramanianii, Ciona indica, Aspergillus serrata, Aspergillus tannica, Aspergillus serrata, Aspergillus tannica, Aspergillus truncatus and Aspergillus chrysanthemi. Background Art

[0002] Alien invasive species can cause serious damage or adverse effects on ecosystems and human life (Xu Haigen, Qiang Sheng. Alien Invasive Species in China, 2018; Petr et al. Biol. Rev. Camb. Philos. Soc., 2020, 95(6): 1511-1534.). With the rapid development of the global economy, the risk of alien species invasion is increasing year by year. my country is one of the countries most affected by alien species invasion in the world (Wan Fanghao, et al. Invasion Biology, 2011.). As an important member of the marine fouling community, many species of ascidian are classified as alien invasive species.

[0003] Sea squirts are diverse in shape and adapt to a wide range of salinity. Typical invasive species include Ciona intestinalis, Ciona stalk, Ciona savarinata, Ciona dermatophyte, Ciona serrata and Ciona chrysanthemi, among which Ciona intestinalis belongs to Chordata, Ascidian, Ciona serrata, and is a protozoan widely found in the Yellow Sea and Bohai Sea. The main harm of Ciona intestinalis is to compete with sea cucumbers for nutrition, consume dissolved oxygen in the water body, seriously affect the algae balance of the water body, and cause water body hypoxia. In addition, Ciona intestinalis is easy to attach to hulls, rocks, aquaculture facilities or other materials, compete with aquatic animals for living space, and seriously affect other biological survival (Delsuc F, et al. Nature, 2006, 439 (7079): 965-968.). In 2010, Ma Hongming and others collected Ciona savignyi for the first time in Qingdao and Dalian, which was the first recorded species in my country (Ma Hongming, et al. Acta Hydrobiologica Sinica, 2010, 34(5):1056-1059.). As a highly invasive marine organism, Ciona savignyi can usually complete habitat conversion and expansion in different environments in a short period of time, which has a great impact on the global ecological environment, and also caused huge environmental damage and economic losses.

[0004] There are two conventional methods to kill sea squirts. One is to eliminate sea squirts through physical methods, i.e. filtration, and effectively reduce the harm caused by sea squirts by filtering or changing water. However, this method is not only labor-intensive, but also not applicable to aquaculture areas or sea areas. The other method is to kill sea squirts through drugs. This has also been the method used by seafarers and aquatic disease prevention and control personnel for many years. However, since sea squirts are protists and are highly resistant to general insecticides, no breakthrough has been made in the use of drugs to kill sea squirts.

[0005] The study of natural products is the basis for the development and utilization of biological resources. It can provide many lead compounds with novel structures and has far-reaching significance for the development of drugs. Compared with the secondary metabolites of terrestrial organisms, the special environment of the ocean (high salt, high pressure, lack of oxygen, low temperature and lack of light, etc.) determines the unique structural characteristics of the secondary metabolites of marine organisms, such as unique and complex structures, containing rare special groups, containing halogens, etc. The application of marine natural products to the invasion of alien species has important research value. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a solution for inhibiting the fouling and attachment of tunicates by alien invasive species.

[0007] The main purpose of the present invention is to provide a strain of Aspergillus subramanianii CHNSCLM-1229, a symbiotic fungus of cold-water corals in the Mariana Trench, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit date of December 29, 2023 and a deposit number of CGMCC No: 41021. The extract of the fungus can significantly inhibit the fouling of sea squirts by alien invasive species.

[0008] The outstanding advantage of the present invention is that the fungus Aspergillus subramanianii is isolated from the cold-water corals in the Mariana Trench for the first time, and is cultured by solid and liquid fermentation. Extracts are obtained by normal phase column chromatography elution, reverse phase column chromatography elution, and macroporous resin column chromatography elution. These extracts show significant inhibitory effects on the attachment of alien invasive species to fouling sea squirts.

[0009] The fungal extract obtained by the method of the present invention can effectively and conveniently inhibit the attachment of alien invasive species to fouling ascidian.

[0010] The following examples of the present invention illustrate examples of extract preparation and activity testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Attached Figure 1The inhibitory effect of different concentrations of crude extract KXHG-03-41 of Aspergillus subramanianii CHNSCLM-1229 on the attachment of Ascidian sabinoides.

[0012] Attached Figure 2 The inhibitory effects of the extract components KXHG-03-41-A, KXHG-03-41-B and KXHG-03-41-C separated by normal phase column chromatography from Aspergillus subramanianii CHNSCLM-1229 on the attachment of Ascidian sabinoides after two-fold dilution.

[0013] Attached Figure 3 IC of KXHG-03-41-B, a crude extract of Aspergillus subramanianii CHNSCLM-1229, inhibiting the adhesion of Ascidian sabinoides 50 . DETAILED DESCRIPTION

[0014] The present invention will be further described below in conjunction with specific embodiments.

[0015] Example 1 Isolation, purification and identification of Aspergillus subramanianii CHNSCLM-1229, a fungus associated with cold-water corals in the Mariana Trench

[0016] 1. Pretreatment of Mariana Trench Cold Water Coral Samples

[0017] Cold-water coral samples were collected from the Mariana Trench, rinsed with sterile water, dried with absorbent paper, soaked in 75% alcohol for 30 seconds, rinsed three times with sterile artificial seawater, and cut into 1 cm pieces with a sterile knife. 3 The organization is reserved.

[0018] 2. Isolation of symbiotic fungi from cold-water corals in the Mariana Trench

[0019] Tissue section method: in a sterile environment, take the pre-treated coral sample material and place it on a 3% salinity PDA medium plate; homogenization method: in a sterile environment, grind the coral sample with sterile artificial seawater to three concentrations, namely: 10 -1 , 10 -2 , 10 -3 (Four concentrations of homogenate were finally obtained), 100 μL was taken respectively and inoculated into 3% salinity PDA medium plates, and spread evenly with a spreader. The above plates were placed in a 28°C constant temperature incubator; the 3% salinity PDA medium composition is: 200g potato, 20g glucose, 30g sea salt, 1L water, 20g agar.

[0020] 3. Purification of fungi associated with cold-water corals in the Mariana Trench

[0021] After culturing for about 2 days, observe whether there are single colonies growing, and inoculate the grown single colonies into a newly prepared 3% salinity PDA medium plate. Repeat the separation and purification process until a pure strain is obtained.

[0022] 4. Identification of fungi associated with cold-water corals in the Mariana Trench

[0023] The fungus was molecularly identified using ITS-rRNA sequencing. The ITS sequence sequenced and fed back by Qingdao Qingke Zixi Biotechnology Co., Ltd. was compared with BLAST on NCBI to obtain similar strains with a homology of more than 99%, and the fungus was determined to be Aspergillus subramanianii.

[0024] Example 2 Solid fermentation production of Aspergillus subramanianii

[0025] Take out the cryovial containing Aspergillus subramanianii CHNSCLM-1229 from the -80℃ ultra-low temperature freezer, inoculate the strain on the PDA plate, and activate the strain in a 28℃ biochemical incubator for 3 to 5 days; in a sterile clean bench, pick the colony with an inoculation loop and inoculate it into the solid fermentation medium in a 1L conical flask, and the inoculated conical flask is placed at room temperature (25℃) for 60 days. The composition of the solid fermentation medium is: add 70g of rice to a 1L conical flask, and add 3% salinity PDB liquid medium to soak the rice 2mm. The components of the 3% salinity PDB liquid medium are: 200.0g / L potato, 20.0g / L glucose, 30.0g / L sea salt, and the pH of the medium is 7.

[0026] Example 3 Liquid static fermentation production of Aspergillus subramanianii

[0027] The colonies were picked from the plate and inoculated into the liquid fermentation medium in a 500 mL conical flask, and the inoculated conical flask was placed at room temperature (25°C) for 40 days. The liquid fermentation medium composition is as follows: 250 mL of 3% salinity PDB liquid medium was added to a 500 mL conical flask. The 3% salinity PDB liquid medium composition is: 200.0 g / L potato, 20.0 g / L glucose, 30.0 g / L sea salt, and the pH of the medium is 7.

[0028] Example 4 Liquid Shaking Fermentation Production of Aspergillus subramanianii

[0029] The colonies were picked from the plate and inoculated into the liquid fermentation medium in a 500 mL conical flask. The inoculated conical flask was placed in a shaker at 28°C, the speed was selected to be 160 rpm, and cultured for 15 days. The liquid fermentation medium composition is as follows: 250 mL of 3% salinity PDB liquid medium was added to a 500 mL conical flask. The components of 3% salinity PDB liquid medium are: 200.0 g / L potato, 20.0 g / L glucose, 30.0 g / L sea salt, and the pH of the medium is 7.

[0030] Example 5 Extraction of the fermentation broth

[0031] 5.1 Solid fermentation broth extraction

[0032] The solid culture medium was extracted with ethyl acetate solvent for 3 times, and the ethyl acetate solvent was removed by concentration under reduced pressure to obtain a crude extract, which was named KXHG-03-41.

[0033] 5.2 Liquid fermentation broth extraction

[0034] The liquid culture medium was extracted 3-4 times with ethyl acetate solvent, the organic phases were combined, and the organic solvent was removed by concentration under reduced pressure to obtain a crude extract. The crude extract obtained by liquid static fermentation was named KXHG-03-41-1, and the crude extract obtained by liquid shaking fermentation was named KXHG-03-41-2.

[0035] Example 6 Normal phase column chromatography separation of extract KXHG-03-41

[0036] After the obtained crude extract is fully dissolved with an organic solvent, 200-300 mesh normal phase silica gel is added, and the ratio of silica gel to sample is 1.2:1-1.5:1. The crude extract is gradient eluted with organic solvents ethyl acetate and petroleum ether in different proportions to obtain three polar segment component extracts, segment A is a 50% ethyl acetate / petroleum ether polar segment, segment B is a 100% ethyl acetate polar segment, and segment C is a 30% methanol / ethyl acetate polar segment. The three polar segments are named KXHG-03-41-A, KXHG-03-41-B and KXHG-03-41-C.

[0037] Example 7 Reverse Phase Column Chromatographic Separation of Extract KXHG-03-41

[0038] After the obtained crude extract is fully dissolved with an organic solvent, reverse phase silica gel is added, and the ratio of silica gel to sample is 1.2:1-1.5:1. After the blank silica gel is balanced with the corresponding solvent, the silica gel after adsorbing the sample is added, and the crude extract is gradient eluted using eluents of different polarities to obtain 3 polar segment component extracts, segment D is a 40% methanol / water polar segment, segment E is an 80% methanol / water polar segment, and segment F is a 100% methanol polar segment. The three polar segments are named KXHG-03-41-D, KXHG-03-41-E and KXHG-03-41-F.

[0039] Example 8 Macroporous resin column chromatography separation of extract KXHG-03-41

[0040] The macroporous resin chromatography column is first washed with pure water, and the required elution buffer is prepared at room temperature and poured into the column. The column is tilted so that the buffer flows freely to the bottom and thoroughly absorbs moisture from the cushion particles at the bottom.

[0041] Distribute the dissolved extract sample evenly on the top of the chromatography column, and then slowly inject the extract sample buffer. When all the extract samples are added, add appropriate buffer to allow the solid phase to gradually flow down until the extract sample reaches equilibrium in the solid phase. After the extract sample is successfully loaded into the column, perform gradient elution. Three polar segment components are obtained, segment G is a 10% ethanol / water polar segment, segment H is a 50% ethanol / water polar segment, and segment I is a 100% ethanol polar segment. The three polar segments are named KXHG-03-41-G, KXHG-03-41-H and KXHG-03-41-I.

[0042] Example 9 Test of inhibitory activity against ascidian adhesion

[0043] 9.1 Preparation of test compounds

[0044] All samples were dissolved in DMSO and sterilized by filtration using a 0.22 μm filter membrane.

[0045] 9.2 Experimental Methods

[0046] (1) Collection of experimental animals: Several adult Ciona savignyi from the laboratory marine aquaculture system were taken, the membranes were peeled off, and the egg and sperm threads were broken with tweezers. A sufficient amount of eggs and sperm were collected for in vitro artificial fertilization. 7 minutes after fertilization, the excess sperm was washed away with filtered seawater, and the culture dish containing the fertilized eggs was placed in an incubator at 18°C ​​and cultured in the dark. After 18-20 hours of culture, the fertilized eggs developed into swimming larvae, and the swimming larvae were collected with a pipette into a 24-well plate (30 animals were added to each well).

[0047] (2) Test on the inhibitory effect of crude extract on larval metamorphosis: The crude extract was dissolved in DMSO at a certain concentration to prepare a crude extract stock solution with a concentration of 50 μg / mL (for primary screening) or several concentration gradients below 50 μg / mL (for rescreening). Use a pipette to add the crude extract solution to the 24-well plate containing the ascidian larvae, adding 0.5 μL to each well, and the total seawater volume of each well was 500 μL; 0.5 μL of DMSO was added to the control group, and no additional treatment was performed on the seawater control group; three replicates were set for each crude extract treatment and control group. After adding the crude extract and DMSO, the 24-well plate was placed on a shaker and shaken evenly (10 min). The 24-well plate was then placed in an incubator and cultured at 18°C ​​in the dark. After about 18 hours, the metamorphosis of the ascidian larvae was observed under a stereomicroscope, and the number of larvae with fully retracted tails in each well was recorded. The inhibitory effect of the crude extract was judged by the difference between the proportion of larvae with complete tail retraction in each experimental group and that in the control group (“animals with complete tail retraction / total number of animals*100%”);

[0048] (3) Select the extract components of the present invention with excellent initial screening activity, and further reduce the concentration for gradient screening. The extract components KXHG-03-41-A, KXHG-03-41-B and KXHG-03-41-C separated by normal phase column chromatography were tested, and the final test concentrations were 0.78, 1.56, 3.125, 6.25, 12.5, and 25 μg / mL, respectively.

[0049] 9.3 Conclusion

[0050] (1) As attached to the instruction manual Figure 1 As shown, the crude extract of Aspergillus subramanianii KXHG-03-41 of the present invention has significant inhibitory activity on the attachment of Ascidian sabinoides, and still has a strong inhibitory effect at a concentration of 6.25 μg / mL.

[0051] (2) As attached to the instruction manual Figure 2 As shown, the extract components of the present invention (KXHG-03-41-A, KXHG-03-41-B and KXHG-03-41-C) can still maintain a strong inhibitory activity on the adhesion of Ascidiana saburoi after reducing the concentration. For example, at 0.78 μg / mL, the extract component KXHG-03-41-B still has a strong significant difference compared with the control group.

[0052] (3) As attached to the instruction manual Figure 3 As shown in the figure, GraphPad Prism 8 software was used to fit the experimental data with a nonlinear regression equation to determine the relationship between the inhibition rate and the test concentration, and the half-inhibitory concentration IC of the crude extract component KXHG-03-41-B for inhibiting the attachment of Ascidian sabinosum was calculated. 50It is 0.572μg / mL.

[0053] In summary, the extract of the present invention has an outstanding effect in inhibiting the attachment of the alien invasive species ascidian, and can be used to prevent the attachment of alien invasive species to foul the ascidian in bays and different sea areas, providing a new application product for the prevention and control of alien species invasion.

[0054] Finally, it should be noted that there are other ways to implement the present invention; accordingly, the embodiments of the present invention will be described as examples, but are not limited to the contents described in the present invention, and may also be modifications made within the scope of the present invention or equivalent contents added in the claims; all publications or patents cited in the present invention will be used as references of the present invention.

Claims

1. A fungus symbiotic with cold-water corals in the Mariana Trench, characterized in that: The symbiotic fungus is Aspergillus subramanianii CHNSCLM-1229, and its preservation number is CGMCC No:41021.

2. The fungus according to claim 1, characterized in that The fermentation method of the fungus Aspergillus subramanianii CHNSCLM-1229 is solid fermentation and liquid fermentation.

3. The solid fermentation according to claim 2, characterized in that The fermentation method comprises the following steps: taking out a cryopreserved tube containing Aspergillus subramanianii from an ultra-low temperature refrigerator at -80°C, inoculating the strain on a PDA plate, and activating the strain in a biochemical incubator at 28°C for 3 to 5 days; picking a colony with an inoculation loop and inoculating it in a solid fermentation medium in a 1L conical flask in a sterile clean bench, and placing the inoculated conical flask at 25°C for 60 days.

4. The solid fermentation according to claim 3, characterized in that The solid fermentation medium is composed of: adding 70g of rice into a 1L conical flask, adding 3% salinity PDB liquid medium to soak the rice by 2mm; The components of 3% salinity PDB liquid culture medium are: 200.0 g / L potato, 20.0 g / L glucose, 30.0 g / L sea salt, and the pH of the culture medium is 7.

5. The liquid fermentation according to claim 2, characterized in that: The fermentation method comprises the following steps: taking out a cryopreserved tube containing Aspergillus subramanianii from an ultra-low temperature refrigerator at -80°C, inoculating the strain on a PDA plate, activating the strain in a biochemical incubator at 28°C for 3 to 5 days; picking a colony with an inoculation loop in a sterile clean bench and inoculating the colony in a liquid fermentation medium in a 500 mL conical flask, and culturing the culture for 40 days or fermenting the culture on a shaking table for 15 days.

6. The liquid fermentation according to claim 5, characterized in that The liquid fermentation medium is composed of: adding 250 mL of 3% salinity PDB liquid medium into a 500 mL conical flask. The 3% salinity PDB liquid medium contains: 200.0 g / L potato, 20.0 g / L glucose, 30.0 g / L sea salt, and the pH of the medium is 7.

7. The liquid fermentation method according to claim 5, characterized in that: The static culture temperature was 25°C for 40 days, and the shaking fermentation conditions were 28°C, 160 rpm, for 15 days.

8. The fermentation method according to claim 2, characterized in that: The crude extracts obtained by different fermentation methods were extracted three times with ethyl acetate solvent, concentrated under reduced pressure to remove the ethyl acetate solvent, and obtained the crude extracts.

9. The crude extract according to claim 8, characterized in that The obtained crude extract is further eluted by normal phase column chromatography, reverse phase column chromatography and macroporous resin column chromatography to obtain extract components with different polarities.

10. The extraction method according to claim 9, characterized in that: The elution method of normal phase column chromatography is: gradient elution of the crude extract using eluents of different polarities to obtain three polar segment components, segment A is a 50% ethyl acetate / petroleum ether polar segment, segment B is a 100% ethyl acetate polar segment, and segment C is a 30% methanol / ethyl acetate polar segment.

11. The extraction method according to claim 9, characterized in that: The reverse phase column chromatography elution method is: using different proportions of methanol and water mixed solvents to gradient elute the crude extract to obtain three polar segment components, segment D is a 40% methanol / water polar segment, segment E is an 80% methanol / water polar segment, and segment F is a 100% methanol polar segment.

12. The extraction method according to claim 9, characterized in that: The elution method of macroporous resin column chromatography is as follows: using a mixed solvent of ethanol and water in different proportions to gradiently elute the crude extract to obtain three polar segment components, segment G being a 10% ethanol / water polar segment, segment H being a 50% ethanol / water polar segment, and segment I being a 100% ethanol polar segment.

13. Use of the Aspergillus subramanianii CHNSCLM-1229 extract of claim 1 in inhibiting the attachment of invasive alien species such as Ascidianus sabinoides, Ciona indica, Ascidianus sabinoides ... and Ascidianus sabinoides.

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