阅读说明：本技术 一种刺激树干上捕食线虫真菌生长的营养基质及其制备方法和用法 (Nutrient medium for stimulating growth of nematode-trapping fungi on trunk and preparation method and application thereof ) 是由 杨晓燕 肖文 佘容 李飞腾 于 2021-07-29 设计创作，主要内容包括：本发明公开了一种刺激树干上捕食线虫真菌生长的营养基质及其制备方法和用法,包括营养基质S或营养基质E包括,其中：营养基质S包括：灭菌土壤0.001-100重量份；以及培养基1-1000重量份；营养基质E包括：灭菌土壤浸出液0.001-100重量份；以及培养基1-1000重量份。本发明营养基质具有刺激树干上原有捕食线虫真菌生长的功能。(The invention discloses a nutrient substrate for stimulating the growth of nematode-trapping fungi on a trunk, a preparation method and a use method thereof, wherein the nutrient substrate comprises a nutrient substrate S or a nutrient substrate E, wherein: the nutrient medium S comprises: 0.001-100 parts by weight of sterilized soil; and 1-1000 parts by weight of a culture medium; the nutrient substrate E comprises: 0.001-100 parts by weight of sterilized soil extract; and 1-1000 parts by weight of culture medium. The nutrient medium has the function of stimulating the growth of the original nematode-trapping fungi on the trunk.)

1. A nutrient medium for stimulating the growth of nematode-trapping fungi on a tree trunk, the nutrient medium comprising nutrient medium S or nutrient medium E, wherein:

the nutrient medium S comprises: 0.001-100 parts of sterilized soil and 1-1000 parts of culture medium;

the nutrient substrate E comprises: 0.001-100 parts of sterilized soil extract and 1-1000 parts of culture medium.

2. The nutrient medium for stimulating the growth of nematode-trapping fungi on a trunk of a tree according to claim 1, wherein the culture medium is a liquid culture medium; preferably, the liquid medium is a potato dextrose medium.

3. The nutrient medium for stimulating the growth of nematode-trapping fungi on a trunk of a tree according to claim 1, wherein the sterilized soil is obtained by autoclaving soil from the bottom of Yunnan pine trees.

4. The nutrient medium for stimulating the growth of nematode-trapping fungi on trunks according to any one of claims 1 to 3, wherein the sterilized soil is 1 to 50 parts by weight and the culture medium is 100 to 200 parts by weight.

5. The nutrient medium for stimulating the growth of nematode-trapping fungi on trunks according to any one of claims 1 to 3, wherein the sterilized soil extract is 1 to 50 parts by weight, and the culture medium is 100 to 200 parts by weight.

6. A nutrient medium for stimulating the growth of nematode-trapping fungi on tree trunks according to any one of claims 1 to 3 or 5, wherein the sterile soil leachate is prepared from sterile soil by a method comprising:

adding sterilized soil into water, stirring, mixing, and standing;

and filtering the supernatant after standing, wherein the filtrate is the sterilized soil leaching solution.

7. A method of preparing a nutrient medium as claimed in any one of claims 1 to 6 for stimulating the growth of nematode-trapping fungi on a trunk, comprising the steps of:

putting the culture medium into a container, and adding sterilized soil or sterilized soil leachate;

sterilizing under high pressure, and cooling;

obtaining the nutrient substrate for stimulating the growth of the nematode-trapping fungi on the trunk.

8. The method of making a nutrient medium for stimulating growth of nematode-trapping fungi on a tree trunk of claim 7, wherein the autoclaving is performed at 121 ℃ for 30 min.

9. Use of a nutrient medium as claimed in any one of claims 1 to 6 for stimulating the growth of nematode-trapping fungi on a tree trunk, wherein: the nutrient medium S is uniformly applied to the trunk by using a brush; the nutrient medium E is sprayed on the tree trunk by using a spraying method.

10. The amount of the nutrient medium according to claim 9 for stimulating the growth of nematode-trapping fungi on a tree trunk, wherein: the application amount of the nutrient medium for stimulating the growth of the nematode-trapping fungi on the trunk is 200mL per tree.

Technical Field

The invention relates to a nutrient medium for stimulating the growth of nematode-trapping fungi on a trunk, a preparation method of the nutrient medium for stimulating the growth of the nematode-trapping fungi on the trunk and a use method of the nutrient medium for stimulating the growth of the nematode-trapping fungi on the trunk.

Background

Nematode disease causes serious economic losses to the agriculture, forestry and animal husbandry industries worldwide, and particularly, pine wood nematodes cause destructive pine wilt. The existing methods for preventing and treating nematode parasitic diseases mainly comprise the following steps: physical control, chemical control, agricultural control and biological control, and no effective drug aiming at the pine wilt is found at present. The physical prevention method for the nematode disease has low efficiency, the chemical prevention method has better effect but has larger negative effect on the environment and human body, the agricultural prevention and control effect is not ideal, the breeding time period is longer, and more scholars focus on green biological prevention and control along with the discovery of more and more biological prevention strains.

The biological control means widely used at present mainly comprise: preparing biocontrol preparation by using natural enemies (fungi, bacteria, actinomycetes and the like) of nematodes; extracting secondary metabolites of specific strains for biocontrol; biological control by means of genetic engineering, etc. The biological control means of the pine wilt disease mainly aims at the intermediate host of monochamus alternatus hope, but not the source of pine nematode. Although a great deal of biological control research is available, more biological control research still stays in a laboratory stage and is not expanded to field tests or field tests, the effect of the commercialized biological control agent is not ideal, and the existing control means are important to control.

Disclosure of Invention

In view of the above problems, the present invention provides, in one aspect, a nutrient medium for stimulating the growth of nematode-trapping fungi on a trunk, the nutrient medium for stimulating the growth of nematode-trapping fungi on a trunk having a function of stimulating the growth of the original nematode-trapping fungi on the trunk.

The technical scheme is as follows: a nutrient medium for stimulating the growth of nematode-trapping fungi on a tree trunk, the nutrient medium comprising nutrient medium S or nutrient medium E, wherein:

the nutrient medium S comprises: 0.001-100 parts of sterilized soil and 1-1000 parts of culture medium;

the nutrient substrate E comprises: 0.001-100 parts of sterilized soil extract and 1-1000 parts of culture medium. .

Further, the culture medium is a liquid culture medium; preferably, the liquid medium is potato dextrose agar medium.

Further, the sterilized soil is obtained by subjecting soil under Yunnan pine forest to high pressure steam sterilization.

Further, the sterilized soil is 1-50 parts by weight, and the culture medium is 100-200 parts by weight.

Further, the sterilized soil leachate is 1-50 parts by weight, and the culture medium is 100-200 parts by weight.

Further, the sterilized soil leachate is prepared by the following method of sterilized soil:

adding sterilized soil into water, stirring, mixing, and standing;

and filtering the supernatant after standing, wherein the filtrate is the sterilized soil leaching solution.

The invention also provides a preparation method of the nutrient medium for stimulating the growth of nematode-trapping fungi on the trunk.

A preparation method of the nutrient medium for stimulating the growth of nematode-trapping fungi on the trunk comprises the following steps:

putting the culture medium into a container, and adding sterilized soil or sterilized soil leachate;

sterilizing under high pressure, and cooling;

obtaining the nutrient substrate for stimulating the growth of the nematode-trapping fungi on the trunk.

Further, the autoclaving was 121 ℃ for 30 min.

The invention also provides a use method of the nutrient medium for stimulating the growth of nematode-trapping fungi on the trunk.

The use of the nutrient medium for stimulating the growth of nematode-trapping fungi on a trunk is characterized in that the nutrient medium for stimulating the growth of nematode-trapping fungi on the trunk is applied by the following method: the nutrient medium S is uniformly applied to the trunk by using a brush; the nutrient medium E is sprayed on the tree trunk by using a spraying method.

Further, the application amount of the nutrient medium for stimulating the growth of the nematode-trapping fungi on the trunk of the tree is 200 mL/tree.

The invention principle and the beneficial effects are as follows:

most of the research on biocontrol has been focused on finding new biocontrol strains, neglecting the few or unculturable microorganisms that are inherently present in the habitat, which may be the key species for biocontrol, but only in small numbers and cannot function. Researches show that ecological factors play a very important role in the performance and activity of microorganisms with active biological control capacity, and from the ecological perspective, the mode of adding strains into the environment leads the competitiveness of biocontrol strains to be not as good as that of native species due to environmental differences, so that the strains cannot exert the potential of the biocontrol strains and finally cannot achieve the ideal biological control effect; on the other hand, the artificially added living things change the original ecosystem and may damage the balance of the original ecosystem, thereby causing adverse effects. The application is based on the ecological theory, the eye light is transferred from the cultured biocontrol bacteria to the key species stimulating the original habitat to grow, the large environment-soil for microbial growth is used for preparing the preparation, and the microbial growth which is in the environment but has few quantity and can not play the function is stimulated.

Nematode-trapping fungi, as a class of vegetative hyphae, specialize to form trapping organs to trap nematodes, have the potential to serve as biocontrol strains of nematode disease. Therefore, the nematode-trapping fungi which widely live in soil or on sapropel and are not separated from healthy trunks are selected as research objects, and the Yunnan pine is adopted as a research environment to research whether the aim of preventing the pine wood nematodes can be achieved by stimulating the growth of the nematode-trapping fungi on the trunk of the Yunnan pine.

Drawings

FIG. 1 shows the time-dependent change trend of the total detection rate of nematode-trapping fungi in group S and group E of the nutrient medium of the present invention;

FIG. 2 is a time-space variation law of the detection rate of nematode-trapping fungi in group E of the nutrient substrate of the invention;

FIG. 3 is the space-time variation law of nematode-trapping fungi of nutrient medium group S of the present invention;

FIG. 4 shows the vertical distribution pattern of nematode-trapping fungi on Pinus yunnanensis trunks in a blank control experiment of the present invention.

Detailed Description

The present invention will be further explained below.

The examples provided herein are merely to further illustrate the invention and should not be construed as limiting the invention in any way.

It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well known in the art, unless otherwise specified, in the following.

In the invention, the detection rate calculation formula is as follows:

in the data analysis, Excel2019 software is used for sorting and primarily analyzing data, SPSS20.0 software is used, and the Pearson chi-square test is adopted for statistically analyzing the data under the condition that the test level is 0.05.

In the invention, the sample collection method comprises the following steps:

collecting bark of Yunnan pine tree with sterile knife (the trunk is 0-5cm, 5-10cm, 10-20cm, 20-40cm, 40-80cm, 80-120cm, and 120-200cm away from ground). The harvested bark was collected in 1 sample bag No. 5 (100mm x 150mm) and the number and height of the tree was recorded on the sample bag. And after each subsection is collected, fully burning the knife, slightly cooling the knife, and collecting the next subsection sample. Finally, 7 segmented samples of the same tree are collectively placed in 1 No. 7 sampling bag (200mm multiplied by 140mm), the sampling time and the tree number are recorded, and then the next Yunnan pine bark is collected.

In the invention, the separation method of the nematode-trapping fungi comprises the following steps:

the samples were processed using the bait plate method. Before sample scattering, the barks are broken into small pieces with proper sizes through a sampling bag, and the samples are scattered in the CMA with the diameter of 90mm by adopting a five-point sample scattering method. When the sample is scattered, a certain gap needs to be left between every two points, and the sample needs to be spaced by about 1cm from the outer edge of the culture dish so as to be convenient for observation and purification. One petri dish per bag of sample at each height. About 5000 total-dentate revival nematodes were added as baits to each well-sown dish and incubated at room temperature for 4 weeks. And then purifying the nematode-trapping fungi by adopting a monospore picking method.

The method for identifying the nematode-trapping fungi comprises the following steps:

(ii) morphological identification

Digging an observation room with the length of 2cm multiplied by 2cm in the center of a corn culture medium by using an aseptic scalpel, picking a pure culture block of the nematode-trapping fungi by using an aseptic toothpick, placing the pure culture block in the corner of the observation room and contacting with the corn culture medium, placing the pure culture block in a constant-temperature incubator at the temperature of 26.5 ℃ for culturing for a week, and taking a picture under a differential interference microscope by using a sticky sheet method after the observation room is full of hyphae; and (3) adding 1-2 drops of holodentate revival nematode larva suspension (about 20-30 holodentate revival nematodes) into an observation chamber after photographing, observing under a stereoscope after 24h, determining the type of a predatory organ, and carrying out morphological identification on the nematode-predatory fungi by combining the shape of conidia, the growing mode and the type of the predatory organ.

② molecular identification

For the strain which can not be identified by morphology, extracting DNA of the nematode-trapping fungi by a kit method, carrying out electrophoresis detection, carrying out multi-gene fragment sequencing on the extracted DNA, and carrying out molecular identification on the DNA.

Example 1

A10 x 10m Yunnan pine plot is selected from a mountain with few people walking, 2kg of soil under the Yunnan pine forest is collected in the experimental plot, and the soil is sieved by a 10-mesh sieve to remove rotten leaves and dead branches, and then the soil is taken back to the laboratory. The soil was autoclaved 2 times for use (121 ℃, 30min, 2 times sterilization at intervals of at least 8 h).

Example 2 preparation of soil leachate

10g of the soil which had been autoclaved 2 times in example 1 was weighed into a beaker, 10mL of distilled water was added, and the mixture was mixed with a glass rod and allowed to stand for 1 hour. Folding the filter paper in half for 2 times in the funnel, placing the funnel on a 20mL measuring cylinder, pouring the supernatant after standing into the funnel by adopting a glass rod drainage mode until the liquid level in the measuring cylinder rises to 10 mL.

Example 3

The soil extract from the graduated cylinder of example 2 was poured into erlenmeyer flasks filled with Potato Dextrose Broth (PDB) (i.e., 5mL of soil extract, PDB100mL) at the ratio of nutrient media type E in Table 1 and autoclaved 1 time (121 ℃ C., 30 min). And cooling to obtain a nutrient medium E based on the ecological theory, and filling the nutrient medium E into a prepared 400mL sterile spray kettle for later use.

Example 4

The soil which had been sterilized 2 times in example 1 was placed in an Erlenmeyer flask filled with Potato Dextrose Broth (PDB) (i.e., soil 5g, PDB100mL) in the proportions of nutrient media type S in Table 1 and autoclaved 1 time (121 ℃, 30 min). And cooling to obtain the nutrient medium S based on the ecological theory, and filling the nutrient medium S into a 400mL sterile spray kettle prepared in advance for later use.

TABLE 1

Example 5

6 Pinus yunnanensis in the experimental sample plot are selected as experimental objects for effect monitoring, the nutrient medium E filled in the sterile spray pot in example 3 and the nutrient medium S filled in the sterile spray pot in example 4 are respectively taken, and the 6 Pinus yunnanensis are divided into 2 groups which are respectively a nutrient medium E group and a nutrient medium S group.

Spraying 2 corresponding nutrient substrates (the nutrient substrate E is sprayed on the nutrient substrate E group, and the nutrient substrate S is brushed on the nutrient substrate S group) on the 0-200cm distance from the ground of the main trunk of the Yunnan pine to ensure that the main trunk of the Yunnan pine in the whole range is completely wet, and the application amount of the nutrient substrate of each tree is about 200 mL.

1 week, 2 weeks, 3 weeks and 4 weeks after the nutrient substrates E and S are applied, a Yunnan pine bark sample is collected according to the sample collection method of the invention, and the Yunnan pine bark sample is separated, purified and identified according to the separation method and the identification method of the nematode-trapping fungi of the invention, and the results are as follows:

(ii) Total check out of conditions

After application of the nutrient media E and S, the nematode-trapping fungi are distributed in the range of 0-200cm on the trunk of the Yunnan pine and the detected species are increased. The detected species belong to Dactylellina, and are respectively 4 kinds as follows: monochoria drechsleri (Dactylella drechsleri), monochoria ellipsoidea (Dactylella ellipspora), monochoria stenocephalata (Dactylella parvicolla) and monochoria laxa (Dactylella lyopaca).

Variation rule of nematode-trapping fungi on time scale

The total detection rate of nematode-trapping fungi on the trunk of Yunnan pine of the group E of the nutrient substrate shows a trend that the total detection rate is firstly reduced and then increased along with the time lapse, and finally the total detection rate shows an increasing trend (as shown in figure 1). The total detection rate of nematode-trapping fungi at week 4 was 44.76%, which is significantly higher than at week 1 (29.52%). The total detection rate of the nematode-trapping fungi on the trunk of the Yunnan pine of the nutrient medium S group shows the same rule as that of the nematode-trapping fungi E along with the time (as shown in figure 1). The total detection rate of nematode-trapping fungi at week 4 was 62.86%, which is significantly higher than that at week 1 (48.57%).

After 2 different nutrient mediums were applied, the total detection rate of nematode-trapping fungi in the nutrient medium group S was consistently higher than that in the nutrient medium group E (fig. 2). The results of the Pearson chi-square test of the total detection rate of the nematode-trapping fungi in the 2 groups at week 1, week 2, week 3 and week 4 are respectively as follows: chi shape²＝83.886，P<0.001；χ²＝41.429，P<0.001；χ²＝119.573，P<0.001；χ²＝32.640，P<0.001. The total detection rates of nematode-trapping fungi on the trunks of Yunnan pine of the nutrient medium group E and the nutrient medium group S are both statistically different (as shown in the following table 2).

TABLE 2 Total Perweek nematode-trapping fungus detection Rate Difference between nutrient substrate group E and nutrient substrate group S

③ change rule of nematode-trapping fungi on spatial scale

The Yunnan pine trunk is subdivided into an upper section, a middle section and a lower section. The lower section is 0-20cm, the middle section is 20-80cm, and the upper section is 80-200 cm.

The detection rates of the nematode-trapping fungi on different sections of the Yunnan pine trunks of the nutrient medium E group are different along with the change trend of time. In the 1 st week, the nematode-trapping fungi at the lower section (0-20cm) of the trunk of the Yunnan pine has the most rapid response to the nutrient medium E group, the detection rate is 35.56%, the nematode-trapping fungi at the upper section (80-200cm) have the detection rate of 33.33%, the nematode-trapping fungi at the middle section (20-80cm) have the slower response, and the detection rate is 16.67%. With the lapse of time, the detection rates of the nematode-trapping fungi at the lower section (0-20cm) and the upper section (80-200cm) of the trunk of the Yunnan pine are reduced and increased, and the overall trend is increased. The detection rate of nematode-trapping fungi in the middle section (20-80cm) of Yunnan pine trunk shows a fluctuation rising trend. The detection rate of the nematode-trapping fungi at the 4 th week of the lower section (0-20cm) of the Yunnan pine is increased to 44.44 percent, and the detection rate of the nematode-trapping fungi (66.67 percent) is only second to the detection rate of the nematode-trapping fungi at the middle section (20-80 cm). The detection rate of the nematode-trapping fungi at the upper section (80-200cm) is only 23.33 percent and does not exceed the detection rate (33.33 percent) at the 1 st week (as shown in figure 3).

The Pearson chi-square test results of 3 segmented detection rates of the upper section (80-200cm), the middle section (20-80cm) and the lower section (0-20cm) of the nematode-trapping fungi of the nutrient medium E group are respectively as follows: chi shape² _{General assembly}＝115.569，P<0.001；χ² _{In the upper middle}＝80.597，P<0.001；χ² _{Up and down}＝39.868，P<0.001；χ² _{Middle lower part}＝62.592，P<0.001. The detection rates of 3 segments of the nematode-trapping fungi of group E of the nutrient substrate were statistically different with time (Table 3 below).

TABLE 3 differential rates of 3 subsections of nematode-trapping fungi in different time periods for nutrient substrate E group

The detection rates of the nematode-trapping fungi of different sections on the trunk of the Yunnan pine of the nutrient medium S group have different trends along with the change of time. In the 1 st week, the response of the nematode-trapping fungi at the lower section (0-20cm) of the trunk of the Yunnan pine tree to the nutrient substrate S is the most rapid, the detection rate is 55.56%, the nematode-trapping fungi at the middle section (20-80cm) is 50%, and the nematode-trapping fungi at the upper section (80-200cm) is 36.67%. With the increase of the Yunnan pine tree trunk sections, the detection rate of nematode-trapping fungi shows a gradually-decreasing trend. With the lapse of time, the detection rates of nematode-trapping fungi at the lower section (0-20cm), the middle section (20-80cm) and the upper section (80-200cm) of the Yunnan pine trunk show the trend of decreasing and increasing. The detection rate of nematode-trapping fungi at the 4 th week of the lower section (0-20cm) is 53.33 percent and is not greatly different from the detection rate at the 1 st week; the detection rate of nematode-trapping fungi in the middle section (20-80cm) is 80.00 percent, is obviously higher than that in the 1 st week and is higher than that of nematode-trapping fungi in the upper section (80-200cm) and the lower section (0-20cm) of the same section; the detection rate of the nematode-trapping fungi at the upper section (80-200cm) is 60.00 percent, which is obviously higher than that at the 1 st week and is slightly higher than that at the lower section (0-20cm) of Yunnan pine at the 4 th week (as shown in figure 4).

The Pearson chi-square test results of 3 segmental detection rates of the upper section (80-200cm), the middle section (20-80cm) and the lower section (0-20cm) of the nematode-trapping fungi of the nutrient medium S group are respectively as follows: chi shape² _{General assembly}＝39.349，P<0.001；χ² _{In the upper middle}＝0.455，P＝0.929；χ² _{Up and down}＝25.522，P<0.001；χ² _{Middle lower part}＝30.511，P<0.001. With the change of time, the detection rates of the upper section (80-200cm) and the lower section (0-20cm), the middle section (20-80cm) and the lower section (0-20cm) of the nematode-trapping fungi in the S group are all statistically different, but the detection rates of the upper section (80-200cm) and the middle section (0-20cm) are not statistically different (the following table 4).

TABLE 4 differential rates of 3 subsections of nematode-trapping fungi in different time periods for nutrient substrate S group

The results of Pearson chi-square test of the segmented detection rate of nematode-trapping fungi in the nutrient medium E group and the nutrient medium S group at 1 week, 2 weeks, 3 weeks and 4 weeks are respectively as follows: chi shape²＝15.740，P<0.001；χ²＝29.454，P<0.001；χ²＝6.259，P＝0.044；χ²＝15.919，P<0.001. The fractional detection rates of nematode-trapping fungi on the trunk of Yunnan pine of group E and group S in each week are statistically different (Table 5 below).

TABLE 5 differential detection rates of nematode-trapping fungi at different time periods for nutrient substrate group E and nutrient substrate group S

COMPARATIVE EXAMPLE 1 (blank control with EXAMPLE 5)

In the experimental plot, 3 pinus yunnanensis were selected as blank control and numbered as k1, k2, k 3.

According to the sample collection method, a Yunnan pine bark sample is collected, and the nematode-trapping fungi separation method and the nematode-trapping fungi identification method are used for separating, purifying and identifying the nematode-trapping fungi from the Yunnan pine bark sample.

This example does show the presence of nematode-trapping fungi on the trunk of Yunnan pine without the application of a nutrient medium, but only on 1 trunk of pine (0-5cm) near the soil part (Monacrasporium. sp), which fails to obtain pure cultures due to late contamination and the species could not be identified (see FIG. 4).

From the results of example 5 and the results of comparative example 1, it can be seen that: the nutrient medium E and the nutrient medium S both have the function of stimulating the growth of the original nematode-trapping fungi on the trunk, and the stimulation effect of the nutrient medium E and the nutrient medium S can be continuously existed within 1 month. The stimulation effect of nutrient medium S was better compared to nutrient medium E. The spraying intensity of the upper section is weaker than that of the other sections. The biological control effect of the nutrient medium which is developed from the ecological perspective and can stimulate the growth of the biological control strain in the primary environment is better.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.