阅读说明：本技术 一种食线虫真菌厚垣孢子舔块的制备方法与应用 (Preparation method and application of chlamydospore licking block of nematophagous fungus ) 是由 王波波 杨明彩 吴倩倩 蔡葵蒸 王孝伟 周世良 王奕彤 杨明娟 徐斌 于 2020-07-08 设计创作，主要内容包括：本发明提供一种食线虫真菌厚垣孢子舔块的制备方法,其特征在于：所述的制备方法采用的舔块组分,以重量份计,包括：尿素5~10份,糖蜜15~20份,食盐10~25份,钠型膨润土5~10份,羧甲基纤维素0~2份,磷酸二氢钙0~5份,复合维生素1-1.5份,麸皮20~30份,花生油1~2份,<I>D.flagrans</I>厚垣孢子粉1-2份；所述舔块含0.5~2×104/g个D.flagrans厚垣孢子。将舔块用于饲喂牛羊,对胃肠线虫的杀虫率达到83%以上。(The invention provides a preparation method of a chlamydospore licking block of nematophagous fungi, which is characterized by comprising the following steps: the licking block component adopted by the preparation method comprises the following components in parts by weight: 5-10 parts of urea, 15-20 parts of molasses, 10-25 parts of salt, 5-10 parts of sodium bentonite, 0-2 parts of carboxymethyl cellulose, 0-5 parts of monocalcium phosphate, 1-1.5 parts of compound vitamin, 20-30 parts of bran and 1-2 parts of peanut oil, D.flagrans 1-2 parts of chlamydospore powder, wherein the licking block contains 0.5-2 × 104/g of D.flagrans chlamydospores, and the licking block is used for feeding cattle and sheep and has the insecticidal rate of more than 83% on gastrointestinal nematodes.)

1. A preparation method of a chlamydospore licking block of nematophagous fungi is characterized in that: the licking block component adopted by the preparation method comprises the following components in parts by weight: 5-10 parts of urea, 15-20 parts of molasses, 10-25 parts of salt, 5-10 parts of sodium bentonite, 0-2 parts of carboxymethyl cellulose and monocalcium phosphate0 to 5 parts of vitamin complex, 1 to 1.5 parts of composite vitamin, 20 to 30 parts of bran, 1 to 2 parts of peanut oil,D. flagrans1-2 parts of chlamydospore powder, wherein the licking block contains 0.5-2 × 10⁴Per g isD. flagransChlamydospores.

2. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the preparation method comprises the following steps: the adopted strain is flagellum type ArthrobotrysArthrobotrysflagransIsolate SDH035 with preservation number CGMCC NO. 11210.

3. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the preparation method comprises the following steps: comprises thatD. flagransPreparation of chlamydospore powder: flagellate type arthrobotrys of nematode-trapping fungiD. flagransAnd carrying out batch culture on the SDH035 isolate on a corn-wheat combined grain culture medium, wherein the culture temperature is 25-30 ℃, the relative humidity is 80-100%, and the culture time is 15-25 d, and then drying the grain containing spores until the moisture content in the grain is below 15%.

4. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 3, which is characterized in that: the above-mentionedD. flagransPreparation of chlamydospore powder, wherein the spore content of the prepared chlamydospore powder is 1-2 × 10⁶Per gram.

5. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the preparation method comprises the following steps: the preparation method comprises the following steps: firstly, 30% of bran powder by mass is weighed, then the spore powder is added into the bran powder, and the two are fully and uniformly mixed; the bran powder containing the spore powder is then thoroughly mixed with the remaining bran powder and the remaining components of the formulation.

6. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the licking block comprises the following components in parts by weight: 25 parts of urea, 75 parts of molasses, 105 parts of NaCl, 25 parts of sodium bentonite, 15 parts of carboxymethyl cellulose, 10 parts of monocalcium phosphate, 5 parts of composite vitamin, 225 parts of bran powder, 5 parts of spore powder and 10 parts of edible peanut oil.

7. The method for preparing the licking block of chlamydospore of nematophagous fungus according to claim 1, which is characterized in that: the lick block prepared: the spore germination rate reaches over 27.32-38.65%.

8. The application of the chlamydospore lick block of the nematophagous fungus is characterized in that: the licking block is used for feeding cattle and sheep, and the insecticidal rate of gastrointestinal nematodes reaches more than 83%.

Technical Field

The invention relates to the technical field of preparation of licking blocks for feeding domestic ruminants, in particular to a preparation method and application of a chlamydospore licking block of nematophagous fungus.

Background

The gut nematodes of domestic ruminants are a major parasitic disease that occurs worldwide and cause severe economic losses in animal husbandry. The main harm of the disease to cattle and sheep is chronic nutrition consumption, production performance reduction and growth retardation, and severe people die from cachexia finally along with the development of disease conditions. For a long time, such epidemic diseases have relied primarily on chemical drugs to prevent and treat insect repellents. However, with the widespread use of anthelmintics, resistance of current animal endoparasites to the three general classes of anthelmintics (benzimidazoles, avermectins/ivermectins, levamisole) frequently occurs and is prevalent and prevalent in some countries or regions. To prevent the continued spread of parasite resistance, researchers recommend rotation, elimination of pasture feces, use of plants that naturally contain tannin, use of vaccines that harbor antigens, and use of natural enemies of nematodes for biological control; the nematode-eating fungi are used for killing infectious larvae (L3) in the environment to control infection sources, so that the method not only saves manpower, material resources and application cost, but also is one of the most direct and effective control strategies with application prospects at present.

Nematophagous fungiArthrobotrys(Duddingtonia)flagrans(synonyms of the same thingsTrichotheciumflagrans) Has been extensively studied and valued in the biological control of ruminant parasites, and in most of the literature the habit of this bacterium is known asDuddingtoniaflagrans。D. flagransDuring culture, a large number of double-walled chlamydospores are produced, which are resistant to the digestive enzymes in the animal's digestive tract without being inactivated. Therefore, spores of the strain can be added into animal feed and discharged out of the body along with fecal ova after passing through the digestive tract, so that a large number of parasitic nematode larvae hatched in feces can be killed, the quantity of L3 is reduced, and the aim of biological prevention and control is fulfilled.

However, since the domestic lack of excellent candidate strainsD. flagransThis severely limits the research progress of animal parasitic nematode biocontrol, and the introduction of foreign species carries the risk of genetic recombination and species invasion. Therefore, the nematophagous fungi are widely separated and identified at 87 sites in 20 provinces (autonomous region/direct prefecture city) and 48 counties in China since 2011, and are separated and obtained for the first time in ChinaD. flagrans13 strains, and the morphology, molecular genetics, predation mechanism, in vitro insecticidal test, in vivo animal digestive tract test and other biological characteristics of local isolates are comprehensively researched, and the results show that the isolated strains in ChinaD. flagransThe strain is cultured in morphologyThe strain is different from foreign strains in sex, is proved to be a new isolate adapted to the geographical characteristics of China, and has great potential as a candidate strain for developing a biocontrol agent.

To achieve the biological control of parasitic nematodes in livestock, the biological control is not only maintainedD. flagransThe spores have stable activity and can be transferred into animal excrement to effectively kill the L3, and the spores need to be put into feed for feeding, storage and transportation. A good dosage form is the key of commercialization of the biocontrol agent, and has been adopted in ChinaD. flagransThe vacuum freeze-drying preparation of the isolate chlamydospore needs large-scale equipment, has larger power consumption and high product cost, and is very difficult to effectively feed a trace amount of freeze-drying preparation to animals; the chlamydospore survival rate is greatly reduced in this process, the germination rate is very low and is not sufficient to effectively kill L3.

In addition, patent CN201610578955.4 discloses a powdery preparation of nematode-trapping fungi and its application; inoculating nematophagous fungus isolate on culture medium (preferably corn-wheat combined grain culture medium), culturing chlamydospore in batch at 25-28 deg.C and relative humidity above 80%, terminating culture for 15-25 days, and collecting culture containing spores. Collecting culture containing spore and grains, spreading in a container, and air drying at 35 deg.C for 3-5 days; or vacuum drying at 35-40 deg.C under vacuum degree of 0.082-0.085 MPa; or naturally drying, and finally harvesting the dried substance for standby preparation and crushing. And mixing the dried culture with bran according to the mass ratio of 1:5 ‒ 1:10, crushing the mixture by a crusher, and uniformly stirring to obtain the powdery preparation of the nematode-trapping fungi. The obtained subpackaged preparation is culture of fungi. The cultures cannot be stored for a long period of time. The bacterial powder stored for several months has obviously reduced insecticidal rate, and can not effectively kill L3, thereby failing to achieve the purpose of biological prevention and control of gastrointestinal parasitic nematodes.

In summary, the prior art has the following disadvantages: the chlamydospore preparation of nematophagous fungi in the prior art is difficult to be effectively eaten by animals; the chlamydospore preparation of nematophagous fungi in the prior art is not storable, and the insecticidal rate is obviously reduced after the chlamydospore preparation is stored under common indoor conditions.

Disclosure of Invention

O.11210, preservation date 2015, 8 months and 31 days; the preservation unit: china general microbiological culture Collection center (CGMCC), which is abbreviated as CGMCC; and (4) storage address: western road No.1, north chen west city of township, 3, institute for microbiology, china academy of sciences, common microbiology center of the committee for preservation and management of microbial cultures, china, and zip code 100101. The SDH035 isolate was assigned the sequence accession number KP2575930 at NCBI (GenBank).

Chlamydospore production: recovering SDH035 strain preserved on 2% corn flour agar (CMA) slant culture medium, selecting partial hypha or spore with inoculating loop to 0.4% CMA, culturing in 30 deg.C constant temperature incubator for 5-7 d, taking 4 pieces of agar block with length of 4mm × 4mm from colony growth edge, inoculating to 50 mL potato glucose (PD) liquid culture medium, shaking at 180 r/min and 30 deg.C for 6d, enlarging and culturing the liquid cultured seed by PD broth according to 10% inoculum size for 1 time in the same way as above, inoculating to sterilized grain culture medium according to 5-10% seed size of grain culture medium (corn + wheat =2: 1), culturing in 25 deg.C constant temperature incubator for 21d in dark, taking grain with mycelium, adding appropriate amount of 0.01% Tween-80 solution to elute chlamydospore, filtering the bacterial solution with 4 layers of gauze to remove mycelium, counting with a blood cell counting plate, and calculating chlamydospore per ml.

A preparation method of a chlamydospore lick block of nematophagous fungi comprises the following steps:

a preparation method of a chlamydospore licking block of nematophagous fungi is characterized in that: the licking block component adopted by the preparation method comprises the following components in parts by weight: 5-10 parts of urea, 15-20 parts of molasses, 10-25 parts of salt, 5-10 parts of sodium bentonite, 0-2 parts of carboxymethyl cellulose, 0-5 parts of monocalcium phosphate, 1-1.5 parts of compound vitamin, 20-30 parts of bran and 1-2 parts of peanut oil,D. flagrans1-2 parts of chlamydospore powder, wherein the licking block contains 0.5-2 × 10⁴Per g isD. flagransChlamydospores. The lick block prepared: the spore germination rate reaches 27.32-38.65%The above.

The preparation method comprises the following steps: the adopted strain is flagellum type ArthrobotrysArthrobotrysflagransIsolate SDH035 with preservation number CGMCC NO. 11210.

The preparation method comprises the following steps: comprises thatD. flagransPreparation of chlamydospore powder: flagellate type arthrobotrys of nematode-trapping fungiD. flagransAnd carrying out batch culture on the SDH035 isolate on a corn-wheat combined grain culture medium, wherein the culture temperature is 25-30 ℃, the relative humidity is 80-100%, and the culture time is 15-25 d, and then drying the grain containing spores until the moisture content in the grain is below 15%.

The content of spores in the prepared spore powder is 1-2 × 10⁶Per gram.

The preparation method comprises the following steps: the preparation method comprises the following steps: firstly, 30% of bran powder by mass is weighed, then the spore powder is added into the bran powder, and the two are fully and uniformly mixed; the bran powder containing the spore powder is then thoroughly mixed with the remaining bran powder and the remaining components of the formulation.

Further, the licking block component comprises the following components in parts by weight: 25 parts of urea, 75 parts of molasses, 105 parts of NaCl, 25 parts of sodium bentonite, 15 parts of carboxymethyl cellulose, 10 parts of monocalcium phosphate, 5 parts of composite vitamin, 225 parts of bran powder, 5 parts of spore powder and 10 parts of edible peanut oil.

The application of the chlamydospore lick block of the nematophagous fungus is characterized in that: the licking block is used for feeding cattle and sheep, and the insecticidal rate of gastrointestinal nematodes reaches more than 83%.

Compared with the prior art, the invention has the beneficial effects that:

(1) the chlamydospore licking block of the nematophagous fungus prepared by the invention has high spore germination rate, and the spore germination rate in the licking block reaches over 27.32-38.65%.

(2) The biological control method has obvious effect on the biological control of the gastrointestinal nematode diseases of the cattle and sheep; the chlamydospore lick block of the nematophagous fungus prepared by the invention is used for feeding sheep, and the insecticidal rate reaches more than 83 percent.

(3) The chlamydospore lick block of the nematophagous fungus prepared by the invention is storage-resistant and does not depend on a protective agent; under the condition of no addition of a protective agent, the licking block still keeps the insecticidal rate of more than 90 percent when stored for 7 months under the daily indoor condition.

(4) The drug resistance gene of animal parasitic nematodes to anthelmintics is widely spread in the breeding industry all over the world, and has posed a great threat to animal husbandry production. The use of nematophagous fungi for biological control and the like is recommended. Application of nematophagous fungiD.flagransChlamydospore licks can be used to reduce free life stages of parasites in the environment, such as eggs, larvae at stage 1-3, by feeding domestic animals. Compared with vaccine development, the preparation has the advantages of small technical difficulty, low cost and long validity period, and directly controls source infection, so the preparation is a chemical substitution or supplement method with the most application prospect, and is a control method conforming to natural ecological balance. The preparation method is simple, the operation flow is less, complex equipment is not needed, the cost is reduced, and the preparation method is more beneficial to large-scale production, storage and transportation. In addition, the licking block can effectively prevent and control parasitic nematodes of animals while supplementing daily required nutrition for the animals, and the prevention and control strategy belongs to the field of using nematode-eating fungi contained in the licking blockD. flagransAnd the third-stage larvae in the excrement are killed, so that the nematode infection of the animals is reduced from the source. The product of the invention has the characteristics of easy storage, transportation, simple production process, easy large-scale production, convenient production practice and application and low cost.

Drawings

FIG. 1 is a diagram of the appearance of a licking block of chlamydospores of the nematophagous fungus;

FIG. 2 is a graph showing the results of the measurement of spore germination rates of chlamydospore lick blocks in example 4;

FIG. 3 shows the results of in vitro insecticidal effect of chlamydospore lick blocks stored under 6 different conditions for 1-7 months in example 6.

Detailed Description