CN110468086B

CN110468086B - Streptomyces griseus HL-50 and composite microbial inoculum and application thereof

Info

Publication number: CN110468086B
Application number: CN201910896534.XA
Authority: CN
Inventors: 高志强; 籍立民; 娄倩倩
Original assignee: Shandong Longchang Animal Health Product Co ltd
Current assignee: Shandong Longchang Animal Health Products Co ltd
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2020-05-15
Anticipated expiration: 2039-09-20
Also published as: CN110468086A

Abstract

The invention discloses a streptomyces griseus HL-50 strain, a composite microbial inoculum and application thereof, wherein the strain is classified as streptomyces griseusStreptomyces griseusThe microbial inoculum is preserved in the China general microbiological culture Collection center, and the preservation number is as follows: CGMCC No. 18424. The bacterial colony of the strain is round, light earthy yellow, small in area and provided with a bulge in the middle, the surface of the bacterial colony is slightly velvet and opaque, aerial hyphae grow well and are milky white, the suitable growth temperature is 24-35 ℃, and the pH value is 6-8. The invention also provides a compound microbial inoculum prepared by using the streptomyces griseus HL-50 and the eucalyptus globulus oil, the compound microbial inoculum can effectively drive off various parasites infected by freshwater fish, has natural components and excellent quality, can promote the balance of the flora in the body of the freshwater fish and keep the health, further reduces the culture cost, and has wide application prospect.

Description

Streptomyces griseus HL-50 and composite microbial inoculum and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a streptomyces griseus HL-50 strain, a composite microbial inoculum thereof and application thereof.

Background

When the aquatic animals are cultured, various parasites and bacteria can be caused due to incomplete pond cleaning, poor water quality management, improper prevention and the like, so that huge loss is directly or indirectly caused to the whole aquaculture industry. In order to maintain the culture benefit, antibacterial pesticides become the just-needed products for aquaculture. However, in order to simply pursue the instant effect, the traditional medicine has the situations of heavy metal exceeding, unstable medicine dosage and the like, so that the stability of water quality is damaged, the health of aquatic animals is damaged, the immunity of the aquatic animals is reduced, the death events can be directly caused seriously, the production benefit is finally reduced, the potential safety hazard is also brought to consumers, and the drug resistance of the aquatic animals can be generated by excessively using the traditional insecticide, so that the drug failure is caused. In this situation, probiotics are used as an important means to maintain the health and stable development of aquaculture. The microbial preparation not only has good insect expelling and sterilizing effects, but also can decompose and consume feed residues, aquatic animal carcasses, excrement and the like, purify water quality, increase dissolved oxygen in water and reduce the accumulation of harmful substances. Therefore, it is very important to screen out the strains which have good sterilizing and insect expelling effects from the environment and can prepare natural, healthy and safe sterilizing and insect expelling medicines for aquatic animals.

The essential oil is volatile aromatic substance extracted from flower, leaf, stem, root or fruit of plant by steam distillation, extrusion, cold soaking or solvent extraction. The essential oil is sensitive to heat and light, so the essential oil needs to be stored in a dark container and a shade place, and the essential oil is mainly used for maintenance products and aromatherapy at present due to the characteristics of special aroma and organic solvent solubility of the essential oil, but the plant essential oil has various functions, and has the effects of resisting bacteria, preventing spasm, promoting growth, promoting cell metabolism and cell regeneration, enhancing immunity, resisting oxidation and the like besides the traditional functions of flavoring, relieving and refreshing. The plant essential oil is gradually becoming a high-quality and safe aquatic feed additive or aquatic medicine due to the functions and the characteristics of safety, high efficiency and stability. The essential oil is eucalyptus essential oil which is light in color, not thick in sense and lasting in smell, and finally influences the using effect of the essential oil. However, the eucalyptus lobular (eucalyptus globulus) is distributed in Australia, the eucalyptus lobular (eucalyptus globulus) grows in a very arid sandy land environment, more eucalyptol components are contained than general eucalyptus globulus, the extraction rate is high, essential oil produced by the eucalyptus globulus is high in content and good in quality, is a precious medical raw material, and can be widely used in the fields of antibiosis and insect repelling.

Therefore, it is necessary to develop a natural and efficient compound microbial inoculum which has good antibacterial and insect-expelling effects on aquatic animals and does not generate drug resistance and harm to animals.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention provides a streptomyces griseus HL-50 strain, a composite microbial inoculum thereof and application thereof. The screened Streptomyces griseus HL-50 can achieve the effects of sterilization and insect repelling, does not influence the health of aquatic animals, and can be matched with eucalyptus multiceps essential oil to prepare a compound microbial inoculum with better insect repelling effect.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides a streptomyces griseus HL-50, which is classified as streptomyces griseus and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 18424.

Furthermore, the colony of the streptomyces griseus HL-50 is round, light earthy yellow, small in area, provided with a protrusion in the middle, and slightly velvet and opaque on the surface of the colony; aerial hyphae grow vigorously and are milky white.

Furthermore, the proper growth temperature of the streptomyces griseus HL-50 is 24-35 ℃, and the proper growth pH value is 6-8.

The invention also provides a composite microbial inoculum containing the streptomyces griseus HL-50.

Further, the complex microbial inoculum comprises: the bacteria content is not less than 5 × 10⁸CFU/g of Streptomyces griseus HL-50 and Eucalyptus multicastus essential oil.

Further, the eucalyptus multicladus essential oil is prepared from eucalyptus multicladus leaves and comprises 1, 8-cineole, B-cymene, limonene, α -pinene, terpinen-4-ol, allojunene, α -terpineol, P-cymene, diacetone alcohol, β -pinene, β -myrcene, α -phellandrene, α -terpene, gamma-terpene, mucoaldehyde, carvone, phellandrene, 4-chirp alkene, eucalyptene and B-eucalyptol.

Further, the eucalyptus globulus oil is prepared by the following preparation method: cleaning, drying and crushing leaves of eucalyptus globulus; obtaining crude oil of eucalyptus multicuput essential oil through a steam distillation kettle, a condenser and an oil-water demixer; and removing impurity oil in a reduced pressure rectifying tower to obtain the eucalyptus multicuput essential oil.

The invention also provides application of the compound microbial inoculum containing the streptomyces griseus HL-50 in preparing a pesticide for freshwater fishes.

Further, the application method of the complex microbial inoculum comprises the following steps: the application method of the complex microbial inoculum comprises the following steps: the compound microbial inoculum is uniformly mixed in the edible feed for freshwater fishes according to the dosage of 0.5-1.5 kg/ton feed, and the feed is continuously fed for 4-7 days.

Further, the freshwater fish comprises: grass carp, crucian carp, silver carp, Japanese sea bass, minnow, catfish, trout, tilapia, herring and bighead carp.

Further, the insect repellent is capable of killing the following parasites: sporozoon, Trichodina, dactylogyrus, lernaea, Gyrodactyla, Torulopsis molesta, Clinodinella, Taenia and Cryptotrichum.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the Streptomyces griseus HL-50 has good parasite expelling and sterilizing effects on freshwater fishes, and has the advantages of stable character, simple culture, mild culture condition, wide application condition and no environmental pollution.

(2) The raw material of the eucalyptus multicasts essential oil in the composite microbial inoculum is the Australian eucalyptus multicasts leaves, the ingredients are natural, no toxic or side effect is caused, the ingestion and the growth of freshwater fishes are not influenced, the culture water quality of the freshwater fishes is not influenced, and the prepared eucalyptus multicasts essential oil has good quality and has good insect expelling effect.

(3) The streptomyces griseus HL-50 and eucalyptus globulus essential oil are prepared into the compound microbial inoculum, the compound microbial inoculum can effectively repel various parasites such as dactylogyrus, trichiorrhiza, trichodina, siphonostegia, sporulaspongopus, ichthyophthirius multiformis, ciliate and the like infected by freshwater fishes, and can be taken orally to adjust the balance and health of flora in the fishes; the composite microbial inoculum can effectively inhibit the propagation of harmful floras in the freshwater fish culture water body and reduce bacterial infection while expelling insects, further improves the growth of fishes, is safe to use, does not generate any toxic or side effect, is simple to prepare, is environment-friendly, has a wide application range and a good insect expelling effect, can save the culture cost, and has a wide application prospect.

Drawings

FIG. 1 is a photograph of a colony of Streptomyces griseus HL-50 isolated according to the present invention on a Gao's synthetic agar medium No. one.

FIG. 2 is a flow chart of a preparation process of eucalyptus multicuput essential oil according to the present invention.

FIG. 3 is a table of specific components of eucalyptus globulus oil according to the present invention.

Fig. 4 is an experimental result of the compound microbial inoculum containing streptomyces griseus HL-50 of the invention used for sporular worm, trichodina and dactylogyrus in freshwater fish, fig. 4A shows that parasites are observed on gill parts of crucian carps, and fig. 4B shows that the gill parts of crucian carps are recovered to be normal and the parasites disappear after 7 days of using the compound microbial inoculum of the invention.

FIG. 5 is an experimental result of the composite bacterial agent containing Streptomyces griseus HL-50 for lernaea in freshwater fish, FIG. 5A is the lernaea observed on the gill of the grass carp, and FIG. 5B is the restoration of the gill of the grass carp to normal after 7 days of using the composite bacterial agent.

FIG. 6 shows the experimental results of the Streptomyces griseus HL-50-containing complex microbial inoculum for the trichiorrhiza in freshwater fish, FIG. 6A shows the trichiorrhiza observed by a microscope, and FIG. 6B shows the disappearance of the trichiorrhiza after 7 days of using the complex microbial inoculum.

Fig. 7 is an experimental result of the compound microbial agent containing streptomyces griseus HL-50 for the polyculture parvus in the pelteobagrus fulvidraco, fig. 7A shows that white spots are formed on the surface of the pelteobagrus fulvidraco infected with the polyculture parvus, and fig. 7B shows that the polyculture parvus is detected by microscopic examination; FIG. 7C shows the disappearance of the white spots in the body after the complex microbial inoculum of the invention is used.

FIG. 8 shows the results of the application of the composite bacterial agent containing Streptomyces griseus HL-50 in grass carp, and FIG. 8A shows that the branchia part infected with the pipe worms is inflamed and has much mucus; FIG. 8B is a microscopic picture of a tubular worm; FIG. 8C shows the disappearance of the Pieris obliquus after using the complex microbial inoculum of the present invention.

FIG. 9 shows the results of the application of the composite bacterial agent containing Streptomyces griseus HL-50 in grass carp to Chinese , and FIG. 9A shows the rotten gill of Chinese infected by grass carp; FIG. 9B shows the microscopic Chinese fish gill ; FIG. 9C shows that the gill part of the grass carp returns to be normal after the composite microbial inoculum of the invention is used.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific examples.

Example 1

1. Screening of Streptomyces griseus HL-50

Taking soil as a sample, and uniformly mixing the soil and sterile water to prepare the soil 10^-1Diluting the solution, and respectively preparing 10^-2、10^-3、10^-4、10^-5And (4) diluting the solution. Respectively take 10^-3、10^-4、10^-5Coating the diluent on Gao's synthetic first agar culture medium, culturing at 30 deg.C for 7d, selecting obvious colony, purifying for multiple times to obtain single colony, named HL-50, and storing in Gao's synthetic first agar slant culture medium. The formula of the Gao's synthetic agar medium I is as follows: KNO₃1g of soluble starch, 20g of soluble starch, K₂HPO₄0.5g，MgSO₄·7H₂O 0.5g，NaCl 0.5g，FeSO₄0.01g, agar 20g, distilled water 1000mL, pH 7.2.

As shown in FIG. 1, the colony biological characteristics of the strain HL-50 in the Gao's synthetic agar-No. I culture medium are as follows: the colony is round, small in area, slightly protuberant in the middle, light earthy yellow, slightly velvet and opaque on the surface, and aerial hyphae grow vigorously and are milky white. The growth temperature of the strain is 16-40 ℃, the suitable growth temperature is 24-35 ℃, the growth pH value is 4-9, and the suitable pH value is 6-8.

2. 16S rRNA sequencing of Streptomyces griseus HL-50

Extracting genome DNA of the strain HL-50, using the genome DNA as a template, performing PCR amplification by using a 16S rRNA universal primer to obtain a 16S rRNA amplification sequence, and sequencing the 16S rRNA amplification sequence to obtain a nucleotide sequence shown as SEQ ID No: 1, performing Blast comparison on the sequence, and judging that the strain HL-50 is the Streptomyces griseus with the highest homology with the Streptomyces griseus in a GenBank gene library as a result.

And (3) performing strain preservation on the screened streptomyces griseus HL-50, wherein the preservation unit comprises the following steps: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 8 month 27 year 2019; the preservation number of the Streptomyces griseus is CGMCC No. 18424.

Example 2

1. Preparation method of eucalyptus globulus oil

The preparation process flow of the eucalyptus multibladybutton essential oil in the composite microbial inoculum is shown in figure 2, firstly, leaves of the eucalyptus multibladybutton are placed in a washing tower to be washed, then the leaves are placed in a dryer to be dried, then the leaves are crushed in a crusher and then respectively enter a steam distillation kettle, a condenser and an oil-water separator to separate an oil phase and a water phase, wherein the water phase enters an ether extractor to separate waste water, liquid containing ether enters an ether recovery tower to recover the ether, the recovered ether and oil phase liquid are added into a solid bed adsorption dryer to further remove moisture in the oil phase, and finally the oil phase liquid is added into a reduced pressure rectification tower to be rectified so as to remove impurity oil, so that the eucalyptus multibladybutton essential oil is obtained.

The eucalyptus globulus oil prepared in the embodiment is stored in a sealed and lightproof container and is placed in the dark, then, the eucalyptus globulus oil is subjected to chromatographic analysis, and the detected specific components are shown in fig. 3, from the two graphs, the components with the largest relative content in the eucalyptus globulus oil are 1, 8-cineole (83.8%), which is also the most main effective component in the eucalyptus globulus oil, and have obvious antibacterial and insect-repellent effects, and other components comprise β -cymene (3.43%), limonene (2.5%), α -pinene (18%), isomyrcene (1.08%), terpinene-4-ol (0.88%), α -terpineol (0.58%), eucalyptol (0.54%), P-cymene (0.51%) and the like.

2. Preparation of complex microbial inoculum

The prepared eucalyptus globulus oil has bacteria content of not less than 5 × 10⁸And uniformly mixing CFU/g streptomyces griseus HL-50 to prepare the compound microbial inoculum. When the compound microbial inoculum is used, the compound microbial inoculum is mixed in the edible feed of freshwater fish according to the dosage of 0.5-1.5 kg/ton feed, and the feed is fed according to normal feeding management for 4-7 days continuously.

Example 3

The composite microbial inoculum prepared in the example 2 is subjected to an insect expelling experiment, the test site is selected from Jiangsu blackberry lily, the breeding area is 80 mu, and the breeding mode is shown in the following table:

breeding mode

After parasites are detected at the gill part of the fish, 1kg of the compound microbial inoculum is mixed with 1 ton of feed according to normal feeding management, and the feed is continuously fed for 7 days and is fed for 3 times every day. After 7 days, the gill parasites were again examined. As shown in fig. 4, in the initial stage of feeding, the gills of the crucian are infected with sporozoon, trichodina and dactylogyrus; after feeding for 7 days, the gills of the crucian carps are not infected by the sporozoon, and the trichodina and dactylogyrus are not existed under a microscope, which shows that the compound microbial inoculum has obvious insect expelling effect on the freshwater fishes.

Example 4

The test site is selected from Jiangsu Belamcanda chinensis, the cultivation area is 100 mu, and the cultivation mode is shown in the following table:

breeding mode

After the fish gill part is checked and found to be lernaea, 1kg of the compound microbial inoculum is mixed with 1 ton of feed according to normal feeding management, and the feed is continuously fed for 7 days and is fed for 3 times every day. After 7 days, the gill parasites were again examined. As shown in fig. 5, in the early feeding period, the gill part of the grass carps is infected with lernaea; after feeding for 7 days, the lernaea falling off of the gill part of the grass carps is detected, which can show that the composite microbial inoculum has obvious insect expelling effect on freshwater fishes.

Example 5

The test site is selected from the yellow plum in Hubei, the culture area is 25 mu, and the culture mode is shown in the following table:

breeding mode

After the gill part of the fish is checked and found to be the third generation worms, 1kg of the compound microbial inoculum is mixed with 1 ton of feed according to normal feeding management, and the feed is continuously fed for 7 days and is fed for 3 times every day. After 7 days, the gill parasites were again examined. As shown in fig. 6, during the initial feeding period, the gills of the grass carps are infected with the trichinella; after feeding for 7 days, the third generation insects on the gill parts of the grass carps are detected to shed, which can show that the compound microbial inoculum has obvious insect expelling effect on the freshwater fishes.

Example 6

The pelteobagrus fulvidraco is bred in lake of Zhejiang, and white point-shaped objects are formed on the body surface of the pelteobagrus fulvidraco in 5 months, and the pelteobagrus fulvidraco is detected by a microscope to be a majority of small melon worms. 1kg of the compound microbial inoculum prepared in the example 2 is mixed with 1 ton of feed according to normal feeding management, and the feed is continuously fed for 7 days and is fed for 3 times per day. After 7 days, the condition of the ectoparasites was checked again. As shown in fig. 7, in the early feeding stage, the body surface of the pelteobagrus fulvidraco is infected with the majority of ichthyophthirius multifiliis; after feeding for 7 days, the multocarpus fasciatus falls off, and white spots disappear, which indicates that the compound microbial inoculum has obvious parasite expelling effect on the pelteobagrus fulvidraco.

Example 7

The test site is selected in Yueyang in Hunan, weever in California is cultured, the tail of the weever is found to have a wound, and the weever is detected by microscopic examination. 1kg of the compound microbial inoculum prepared in the example 2 is mixed with 1 ton of feed, and the feed is continuously fed for 7 days and is fed for 3 times per day according to normal feeding management. After 7 days, the fish tails were again examined for parasites. In the initial feeding stage, the tail of the Micropterus salmoides is infected with the Trichodina; after feeding for 7 days, the weever on the tail part of the weever is detected to fall off, which can show that the weever has obvious insect expelling effect on the Micropterus salmoides.

Example 8

The test site selects the Jiangsu Kyara, the grass carps are bred, the grass carps die sporadically, the gill part is found to be red and swollen, the mucus is increased, and a large amount of the squints are detected from the wound. 1kg of the compound microbial inoculum prepared in the example 2 is mixed with 1 ton of feed, and the feed is continuously fed for 7 days and is fed for 3 times per day according to normal feeding management. After 7 days, the fish gill parasites were again examined. As shown in fig. 8, in the initial feeding period, the branchia part of the grass carps is infected with the pipe worms; after feeding for 7 days, the falling of the pipe worms is detected, which can show that the compound microbial inoculum has obvious insect expelling effect on the freshwater fishes.

Example 9

The test site is selected from the Shanyang in Anxiang county of Hunan province, the culture area is 20 mu, and the culture mode is shown in the following table:

breeding mode

When the fish gill part was found to be festered, the test revealed a Chinese , which was fed continuously for 7 days 3 times a day with 1 ton of the feed mixed with 1kg of the complex microbial inoculum prepared in example 2, according to normal feeding management. After 7 days, the gill parasites were again examined. As shown in fig. 9, in the initial feeding period, the gills of the grass carps are infected with Chinese ; after feeding for 7 days, the pellicles of the grass carps are detected to fall off , and the gills return to be normal, which can show that the grass carps have obvious insect expelling effect on freshwater fishes.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

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<120> Streptomyces griseus HL-50 strain, and composite microbial inoculum and application thereof

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gtagggcgca agcgttgtcc ggaattattg ggcgtaaaga gctcgtaggc ggcttgtcac 540

gtcggatgtg aaagcccggg gcttaacccc gggtctgcat tcgatacggg ctagctagag 600

tgtggtaggg gagatcggaa ttcctggtgt agcggtgaaa tgcgcagata tcaggaggaa 660

caccggtggc gaaggcggat ctctgggcca ttactgacgc tgaggagcga aagcgtgggg 720

agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgttgggaac taggtgttgg 780

cgacattcca cgtcgtcggt gccgcagcta acgcattaag ttccccgcct ggggagtacg 840

gccgcaaggc taaaactcaa aggaattgac gggggcccgc acaagcagcg gagcatgtgg 900

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Claims

1. A Streptomyces griseus HL-50 is characterized in that the classification name is Streptomyces griseusStreptomyces griseusThe microbial inoculum is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 18424; the colony of the streptomyces griseus HL-50 is round, light earthy yellow, small in area, provided with a bulge in the middle, and slightly villous and opaque on the surface of the colony; aerial hyphae grow vigorously and are milky white; the proper growth temperature of the streptomyces griseus HL-50 is 24-35 ℃, and the proper growth pH value is 6-8.

2. The compound bacterial agent containing the streptomyces griseus HL-50 as claimed in claim 1, wherein the compound bacterial agent comprises: the bacteria content is not less than 5 × 10⁸CFU/g of Streptomyces griseus HL-50 and Eucalyptus multicastus essential oil.

3. The composite microbial inoculum of claim 2, wherein the eucalyptus multiclada essential oil is prepared from eucalyptus multiclada leaves and comprises 1, 8-cineole, β -cymene, limonene, α -pinene, terpinen-4-ol, allomyrcene, α -terpineol, P-cymene, diacetone alcohol, β -pinene, β -myrcene, α -phellandrene, α -terpene, gamma-terpene, mucoaldehyde, carvone, phellandral, 4-chirp alkene, eudesmene and β -eucalyptol.

4. The use of the complex microbial inoculum containing streptomyces griseus HL-50 as claimed in claim 2 in the preparation of an anthelmintic for freshwater fish; the method is characterized in that: the application method of the complex microbial inoculum comprises the following steps: the compound microbial inoculum is uniformly mixed in the edible feed for freshwater fishes according to the dosage of 0.5-1.5 kg/ton feed, and the feed is continuously fed for 4-7 days.

5. Use according to claim 4, characterized in that: the freshwater fish comprises grass carp, crucian carp, silver carp, California perch, minnow, catfish, trout, tilapia, herring and bighead carp.

6. Use according to claim 4, characterized in that: the insect repellent is capable of killing the following parasites: sporozoan, trichodina, lernaea and dipterus.