CN113481108B

CN113481108B - Nutritional matrix for stimulating growth of nematode-trapping fungi on trunk, and preparation method and application method thereof

Info

Publication number: CN113481108B
Application number: CN202110865908.9A
Authority: CN
Inventors: 杨晓燕; 肖文; 佘容; 李飞腾
Original assignee: Dali University
Current assignee: Dali University
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2023-09-12
Anticipated expiration: 2041-07-29
Also published as: CN113481108A

Abstract

The application discloses a nutrition substrate for stimulating growth of nematode-trapping fungi on trunks, and a preparation method and a use method thereof, wherein the nutrition substrate comprises a nutrition substrate S or a nutrition substrate E, and the nutrition substrate comprises the following components: the nutrient substrate S comprises: 0.001-100 parts by weight of sterilized soil; 1-1000 parts by weight of a culture medium; the nutrient substrate E comprises: 0.001-100 parts by weight of sterilized soil leaching solution; 1-1000 parts by weight of a culture medium. The nutrient medium has the function of stimulating the growth of the original nematode-trapping fungi on the trunk.

Description

Nutritional matrix for stimulating growth of nematode-trapping fungi on trunk, and preparation method and application method thereof

Technical Field

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

Background

Nematode disease causes serious economic losses to the agroforestry and livestock industries worldwide, especially pine nematodes, and can cause devastating pine wilting disease. The current control method for nematode parasitic diseases mainly comprises the following steps: physical control, chemical control, agricultural control and biological control, no effective medicine for pine wilting is found at present. While the physical prevention and control method for nematode disease has low efficiency, the chemical prevention and control method has better effect but larger negative influence on environment and human body, and the agricultural prevention and control effect is not ideal, the breeding time period is longer, and along with the discovery of more biocontrol strains, more students gather eyes on green biological prevention and control.

The biological control means widely used at present mainly comprise: preparing a biocontrol preparation by utilizing natural enemies (fungi, bacteria, actinomycetes and the like) of nematodes; extracting secondary metabolites of specific strains for biological control; biological control is performed by means of genetic engineering. The biological prevention and treatment means of pine wilt mainly aims at the intermediate host, namely Monochamus alternatus, but not at the source, namely pine wood nematodes. Although a great deal of biological control research exists, more biological control research remains in laboratory stage, and is not expanded to field test or field test, but the effect of commercial biological control agent is not ideal, and the current control means are heavy "control" and not heavy "control".

Disclosure of Invention

Based on the above problems, in one aspect, the present application provides a nutrient medium for stimulating growth of nematode-trapping fungi on a trunk, which has a function of stimulating growth of original nematode-trapping fungi on the trunk.

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

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

the nutrient substrate E comprises: 0.001-100 parts by weight of sterilized soil leaching solution and 1-1000 parts by weight 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 sterilized by high-pressure steam from the soil under the Yunnan pine forest.

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

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

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

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

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

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

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

filling the culture medium into a container, and adding sterilized soil or sterilized soil leaching solution;

sterilizing under high pressure and cooling;

the nutrient medium for stimulating the growth of the nematode-trapping fungi on the trunk is obtained.

Further, the autoclave was at 121℃for 30min.

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

The method for using the nutrient medium for stimulating the growth of the nematode-trapping fungi on the trunk, which comprises the following steps: the nutrition substrate S is applied to the trunk with a brush; the nutrient substrate E is applied by spraying to trunk.

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

The application has the following principle and beneficial effects:

most of the research on biocontrol has focused on finding new biocontrol strains, and neglecting the naturally occurring small or non-viable number of microorganisms in the habitat, they may be key species for biocontrol, simply because of their low numbers, which cannot function. Researches show that ecological factors play a very important role in the performance and activity of microorganisms with active biological control capability, and from the ecological perspective, the mode of adding strains into the environment can lead to the fact that the competition of biocontrol strains is not as good as that of native species due to environmental differences on one hand, so that the strains cannot exert the potential of the strains, and finally the ideal biological control effect cannot be achieved; on the other hand, artificially added organisms change the original ecological system, and the balance of the original ecological system may be destroyed, thereby causing adverse effects. Based on ecological theory, the application moves eye light from culturing biocontrol bacteria to stimulating the growth of key species in original habitat, uses the large environment of microorganism growth-soil to prepare the preparation, stimulates the growth of microorganisms which are existing in the environment but have small quantity and cannot function.

Nematode-trapping fungi have the potential to act as biocontrol strains for nematode disease as a class of fungi that specialize in forming predators to trap nematodes. Therefore, the nematode-trapping fungi which are widely living in soil or on saprophytes are selected as research objects, and the Yunnan pine is adopted as a research environment to explore whether the purpose of preventing 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 is a graph showing the overall rate of detection of nematode-trapping fungi of the present application over time for a group S and a group E of nutrient substrates;

FIG. 2 shows the time-space variation law of the detection rate of the nematode-trapping fungi in the nutrition matrix group E;

FIG. 3 shows the time-space variation law of the group S nematode-trapping fungi of the nutrient matrix of the application;

fig. 4 is a vertical distribution pattern of nematode-trapping fungi on the trunk of pinus yunnanensis in a blank control experiment according to the present application.

Detailed Description

The present application will be further described below.

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

It will be clear to a person skilled in the art that hereinafter, unless otherwise indicated, the materials and methods of operation used in the present application are well known in the art.

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

in the data analysis of the application, excel2019 software is used for sorting and preliminary analysis, SPSS20.0 software is used for statistical analysis of the data by using Pearson chi-square test under the condition that the test level is 0.05.

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

the bark of each Pinus yunnanensis (trunk 0-5cm,5-10cm,10-20cm,20-40cm,40-80cm,80-120cm,120-200cm from the ground) was harvested in sections using a sterile knife. The collected bark was collected in 1 number 5 sampling bags (100 mm. Times.150 mm) and the number and height of the tree were recorded on the sampling bags. After each section is collected, the knife needs to be fully burnt, and after the knife is cooled slightly, the next section sample is collected. Finally, the 7 segmented samples of the same tree are put into a No. 7 sampling bag (200 mm multiplied by 140 mm) in a concentrated mode, sampling time and tree numbers are recorded, and then the next Yunnan pine bark is collected.

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

samples were processed using the bait plate method. Before sample spreading, breaking bark into small blocks with proper size through a sampling bag, and spreading samples in CMA with the diameter of 90mm by adopting a five-point sample spreading method. When the sample is spread, a certain gap needs to be reserved between every two points, and the sample needs to be spaced about 1cm away from the outer edge of the culture dish for observation and purification. One bag of sample at each level was spread out over one petri dish. About 5000 full-tooth reviving nematodes are added to each petri dish after sample spreading as baits, and the petri dishes are cultured for 4 weeks at room temperature. And then purifying the nematode-trapping fungi by adopting a single spore isolation method.

In the application, the identification method of the nematode-trapping fungi comprises the following steps:

(1) morphological identification

Digging a 2cm multiplied by 2cm observation chamber in the center of a corn culture medium by using a sterile scalpel, picking up pure culture fungus blocks of the nematode-trapping fungi by using a sterile toothpick, placing the pure culture fungus blocks at corners of the observation chamber and contacting the corn culture medium, placing the pure culture fungus blocks in a constant temperature incubator at 26.5 ℃ for culturing for one week, and taking a picture under a differential interference microscope by adopting a sticking method after the mycelia grow up in the observation chamber; after photographing, 1-2 drops of the full-tooth reviving nematode larva suspension (about 20-30 full-tooth reviving nematodes) are added into an observation chamber, and after 24 hours, the type of predatory organs is determined by observation under a stereoscopic vision, and morphological identification is carried out on the predatory nematode fungi by combining the conidium form, the breeding mode and the type of the predatory organs.

(2) Molecular characterization

Extracting DNA of nematode-trapping fungi from strains which cannot be identified in morphology by adopting a kit method, performing electrophoresis detection, sequencing the extracted DNA by using a polygene fragment, and carrying out molecular identification.

Example 1

Selecting a 10X 10m Yunnan pine sample plot on a mountain with less personnel walking, collecting 2kg of soil under the Yunnan pine tree forest in the experimental sample plot, sieving with a 10-mesh sieve, removing rotten leaves and dried branches, and carrying the rotten leaves and dried branches back to a laboratory. The soil was autoclaved 2 times (121 ℃,30min,2 sterilization intervals of at least 8 h) for use.

EXAMPLE 2 preparation of soil leachate

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

Example 3

The soil extract from the measuring cylinder of example 2 was poured into conical flasks (i.e., 5mL of soil extract, 100mL of PDB) containing Potato Dextrose Broth (PDB) in the proportions of nutrient base type E in Table 1, and autoclaved 1 time (121 ℃ C., 30 min). After cooling, a nutrient substrate E based on ecological theory is obtained, and the nutrient substrate E is put into a prepared 400mL sterile spray pot for standby.

Example 4

The soil of example 1, which had been sterilized 2 times, was placed in an Erlenmeyer flask (i.e., 5g of soil, 100mL of PDB) filled with Potato Dextrose Broth (PDB) in the proportions of nutrient substrate type S in Table 1, and autoclaved 1 time (121 ℃ C., 30 min). After cooling, a nutrient substrate S based on ecological theory is obtained, and the nutrient substrate S is filled into a prepared 400mL sterile spray pot for standby.

TABLE 1

Example 5

6 Yunnan pine pieces are selected as experimental objects for effect monitoring in the experimental plot, and the nutrient substrate E filled in the sterile spray pot in the example 3 and the nutrient substrate S filled in the sterile spray pot in the example 4 are respectively taken, wherein the 6 Yunnan pine pieces are divided into 2 groups, namely a nutrient substrate E group and a nutrient substrate S group.

The corresponding 2 nutrient matrixes are sprayed by using a spray can at 0-200cm of the main trunk of the Yunnan pine from the ground (the nutrient matrix E group is sprayed with the nutrient matrix E and the nutrient matrix S group is brushed with the nutrient matrix S), so that the main trunk of the Yunnan pine is completely wetted within the whole range, and the nutrient matrix application amount of each tree is about 200mL.

The sample collection method according to the application collects samples of Yunnan pine bark 1 week, 2 weeks, 3 weeks and 4 weeks after the application of the nutrient medium E and S, and the separation method of the nematode-trapping fungi and the identification method of the nematode-trapping fungi according to the application, the separation, purification and identification of the nematode-trapping fungi are carried out on the samples of Yunnan pine bark, and the results are as follows:

(1) total detection situation

After application of the nutrient substrates E and S, there was a distribution of nematode-trapping fungi in the range of 0-200cm on the trunk of Pinus yunnanensis and an increase in detected species. The species detected were all of the genus Dactylellina, 4 of which were: acremonium terrestris (Dactylellina drechsleri), acremonium ellipsoidea (Dactylellina ellipspspora), acremonium leptospora (Dactylellina parvicolla) and Acremonium acutum (Dactylellina lysipaga).

(2) Law of change of nematode-trapping fungi on time scale

The total detection rate of the nematode-trapping fungi on the trunks of the Yunnan pine group E of the nutrient matrixes is reduced and then increased with the lapse of time, and finally the rising trend is shown (shown in figure 1). The total detection rate of the nematode-trapping fungi at week 4 is 44.76%, which is obviously higher than that at week 1 (29.52%). The total detection rate of the nematode-trapping fungi on the trunks of the nutrient matrix S group of Yunnan pine showed the same law with the lapse of time as E (as shown in figure 1). The total detection rate of the nematode-trapping fungi at week 4 is 62.86%, which is obviously higher than that at week 1 (48.57%).

After application of 2 different nutrient substrates, the total rate of detection of nematode-trapping fungi in nutrient substrate group S was consistently higher than that in nutrient substrate group E (fig. 2). The Pearson chi-square test results for the total detection rate of 2 groups of nematode-trapping fungi at weeks 1, 2, 3 and 4, respectively, were: x-shaped articles ² ＝83.886，P<0.001；χ ² ＝41.429，P<0.001；χ ² ＝119.573，P<0.001；χ ² ＝32.640，P<0.001. There were statistical differences in the total weekly detection rate of nematode-trapping fungi on the trunks of both nutrient matrix group E and nutrient matrix group S pinus yunnanensis (table 2 below).

TABLE 2 total weekly differences in detection rate of nematode-trapping fungi in nutrient matrix group E and nutrient matrix group S

(3) Law of change 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 80-200cm.

The detection rate of the nematode-trapping fungi on the trunks of the Yunnan pine group of the nutrient matrix E is different from different sections, and the trend of the fungus-trapping fungi changes with time. At week 1, the response of the nematode-trapping fungi at the lower section (0-20 cm) of the trunk of Yunnan pine to the nutrient matrix E group is the most rapid, the detection rate is 35.56%, the detection rate is 33.33% for the nematode-trapping fungi at the upper section (80-200 cm), and the detection rate is 16.67% for the nematode-trapping fungi at the middle section (20-80 cm) when the response is slower. Over time, the detection rate of the nematode-trapping fungi at the lower section (0-20 cm) and the upper section (80-200 cm) of the trunk of the Yunnan pine tree shows a decreasing and increasing trend, and the total trend is shown. The detection rate of the nematode-trapping fungi at the middle section (20-80 cm) of the trunk of the Yunnan pine shows a fluctuation type rising trend. The detection rate of the nematode-trapping fungi at the 4 th week of the lower section (0-20 cm) of the Yunnan pine is increased to 44.44 percent, which is next to the detection rate of the nematode-trapping fungi at the middle section (20-80 cm) (66.67 percent). The detection rate of the upper section (80-200 cm) of the nematode-trapping fungi is only 23.33%, and does not exceed the detection rate of week 1 (33.33%) (as shown in FIG. 3).

The Pearson chi-square test results of the upper section (80-200 cm), the middle section (20-80 cm) and the lower section (0-20 cm) of the nutrient matrix E group of the nematode-trapping fungi are respectively as follows: x-shaped articles ² _{Total (S)} ＝115.569，P<0.001；χ ² _{Upper middle part} ＝80.597，P<0.001；χ ² _{Up and down} ＝39.868，P<0.001；χ ² _{Middle lower part} ＝62.592，P<0.001. There were statistical differences in the detection rate of 3 segments of the nutrient matrix group E nematode-trapping fungi over time (Table 3 below).

TABLE 3 detection rate differences of 3 segments of nematode-trapping fungi in different time periods of nutrient matrix group E

The trend of the detection rate of the nematode-trapping fungi of different segments on the trunks of the nutrient substrate S group Yunnan pine is not the same with time. At week 1, the response of the nematode-trapping fungi at the lower section (0-20 cm) of the trunk of Yunnan pine to the nutrient substrate S is the most rapid, the detection rate is 55.56%, the second is the nematode-trapping fungi at the middle section (20-80 cm), the detection rate is 50%, and the last is the nematode-trapping fungi at the upper section (80-200 cm), the detection rate is 36.67%. With the increase of the stem segments of the Yunnan pine, the detection rate of the nematode-trapping fungi shows a gradually decreasing trend. Over time, the detection rate of the nematode-trapping fungi at the lower section (0-20 cm), the middle section (20-80 cm) and the upper section (80-200 cm) of the trunk of the Yunnan pine is gradually reduced and gradually increased. The detection rate of the nematode-trapping fungi at the 4 th week of the lower section (0-20 cm) is 53.33%, which is not much different from that at the 1 st week; the detection rate of the nematode-trapping fungi in the middle section (20-80 cm) is 80.00%, which is obviously higher than that in week 1, and is higher than that in the upper section (80-200 cm) and the lower section (0-20 cm) of the same period; the detection rate of the upper section (80-200 cm) of the nematode-trapping fungi is 60.00%, which is obviously higher than that of the lower section (0-20 cm) of the Yunnan pine at week 1, and is slightly higher than that of the lower section (0-20 cm) of the Yunnan pine at week 4 (as shown in figure 4).

The Pearson chi-square test results of the upper section (80-200 cm), the middle section (20-80 cm) and the lower section (0-20 cm) of the nutrient substrate S group of the nematode-trapping fungi are respectively as follows: x-shaped articles ² _{Total (S)} ＝39.349，P<0.001；χ ² _{Upper middle part} ＝0.455，P＝0.929；χ ² _{Up and down} ＝25.522，P<0.001；χ ² _{Middle lower part} ＝30.511，P<0.001. Over time, there was a statistical difference in the detection rates of the upper (80-200 cm) and lower (0-20 cm), middle (20-80 cm) and lower (0-20 cm) sections of the S group of nematode-trapping fungi, but there was no statistical difference in the detection rates of the upper (80-200 cm) and middle (0-20 cm) (Table 4 below).

TABLE 4 detection rate differences of 3 segments of nematode-trapping fungi in different time periods of nutrient matrix group S

The Pearson chi-square test results of the segmented detection rates of nematode-trapping fungi in nutrient substrate group E and nutrient substrate group S were respectively: x-shaped articles ² ＝15.740，P<0.001；χ ² ＝29.454，P<0.001；χ ² ＝6.259，P＝0.044；χ ² ＝15.919，P<0.001. There were statistical differences in the fractional detection rates of weekly predatory nematode fungi on trunks of both group E and group S of yunnan pine (table 5 below).

TABLE 5 differences in the segmented detection rates of nematode-trapping fungi for different time periods for nutrient substrate group E and nutrient substrate group S

Comparative example 1 (comparison with the blank of example 5)

3 Yunnan pine subjects were selected as blank control in the experimental plots and numbered as k1, k2, k3.

According to the sample collection method, a Yunnan pine bark sample, a separation method of nematode-trapping fungi and an identification method of nematode-trapping fungi are collected, and separation, purification and identification of the nematode-trapping fungi are carried out on the Yunnan pine bark sample.

In this example, the nematode-trapping fungi were indeed present on the trunk of Pinus yunnanensis without the application of a nutrient medium, but were detected only on the trunks (0-5 cm) of the soil-proximal parts of 1 pine tree (Monascosporium. Sp), and no pure culture could be obtained due to late contamination, and no species could be identified (see FIG. 4).

From the results of example 5 and comparative example 1, it can be seen that: the nutrient substrate E and the nutrient substrate 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 substrate E and the nutrient substrate S can be continuously existed within 1 month. Compared with the nutrient substrate E, the stimulation effect of the nutrient substrate S is better. The spray intensity of the upper section is weaker than that of the other sections. The nutrition matrix which is developed from the ecological perspective and can stimulate the growth of the biocontrol strain in the primary environment has better biocontrol effect.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A use of a nutritional matrix for stimulating growth of nematode-trapping fungi on a tree trunk, comprising:

the nutrient substrate for stimulating the growth of the nematode-trapping fungi on the trunk comprises a nutrient substrate S or a nutrient substrate E, wherein the nutrient substrate S is used by uniformly coating the nutrient substrate S on the trunk by using a brush; the nutrition matrix E is applied by spraying to trunk; wherein:

the nutrient substrate S comprises: 1-50 parts of sterilized soil and 100-200 parts of culture medium;

the nutrient substrate E comprises: 1-50 parts by weight of sterilized soil leaching solution and 100-200 parts by weight of culture medium;

the culture medium is a liquid culture medium;

the liquid culture medium is a potato glucose culture medium;

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

2. The method of claim 1, wherein the sterilized soil is produced by autoclaving soil under the pine forest of yunnan pine.

3. Use of a nutrient substrate for stimulating the growth of nematode-trapping fungi on trunks according to any of claims 1-2, wherein the sterile soil leachate is made of sterile soil by the following method:

adding sterilized soil into water, stirring, mixing, and standing; filtering the supernatant after standing, wherein the filtrate is sterilized soil leaching liquid.