CN117327591B

CN117327591B - Trichoderma atroviride and application thereof

Info

Publication number: CN117327591B
Application number: CN202311273281.3A
Authority: CN
Inventors: 祁金玉; 刘志华; 李昱霄; 汪莹; 唐世芬
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2024-06-21
Anticipated expiration: 2043-09-28
Also published as: CN117327591A

Abstract

The invention belongs to the technical field of biocontrol microorganisms, and particularly relates to trichoderma atroviride and application thereof. The classification of the trichoderma atroviride is Trichoderma paratroviride, and the preservation number is CGMCC No.40737. The trichoderma atroviride disclosed by the invention has high inhibition rate on the pathogenic bacteria of the pine tip disease, namely, the pine cone aschersonia (Sphaeropsis sapinea) and the red spot pathogenic bacteria, namely, the trichoderma pseudolariciresii (Pestalotiopsis funerea), and has remarkable growth promoting effect on the pinus sylvestris. The biological microbial inoculum taking the trichoderma atroviride as the active ingredient is pollution-free, does not produce environmental pollution, can reduce or even avoid other corresponding chemical pesticides, saves cost and improves the quality of the pinus sylvestris.

Description

Trichoderma atroviride and application thereof

Technical Field

The invention belongs to the technical field of biocontrol microorganisms, and particularly relates to trichoderma atroviride and application thereof.

Background

Pine is used as evergreen arbor and is a common tree species for greening and forestation. During the growth and development of pine, the pine is often affected by bacteria, which affects the growth of pine and even causes the death of the pine. The pine needle rot and the red pine needle rot are common diseases of pine trees, pathogenic bacteria of the pine needle rot are aschersonia pinella (Sphaeropsis sapinea), the host range is wide, a plurality of trees in the genus of pinaceae can be damaged, especially the damage of the pinus sylvestris is heaviest, the new tip of the pinus sylvestris is withered after the damage, the growth of plant height is blocked, the volume of materials is reduced, and even the whole plant withers. The pathogenic bacteria of the red spot blight is trichoderma reesei (Pestalotiopsis funerea), the red spot blight mainly damages new leaves of pine, the 2-year-old needle leaves are damaged even the red spot is caused when the disease is serious, the pinus sylvestris and the pinus koraiensis which are excellent forestation tree species are often damaged by the red spot blight and the red spot blight, and the chemical control can generate better control effect in a short period.

However, chemical control causes pathogenic bacteria to generate drug resistance, disease resistance of hosts is reduced, and microbial balance is destroyed after long-term application of chemical pesticides. However, the use of biocontrol bacteria is in many ways more advantageous than chemical control, and therefore, finding more and more efficient biocontrol strains is a technical problem to be solved by the person skilled in the art.

Disclosure of Invention

In view of the above technical problems, the present invention provides Trichoderma atroviride and its application.

In a first aspect of the present invention, there is provided a trichoderma atroviride (Trichoderma paratroviride) which was deposited at the China general microbiological culture collection center (CGMCC) at about 21 months of 2023, at the deposit address: the collection number of the microbial institute of China academy of sciences is CGMCC No.40737 in the Korean area North Star, west Lu No. 1, no. 3 of Beijing city.

In a second aspect of the invention, there is provided a culture of said Trichoderma atroviride, said culture being a fermentation broth.

In one embodiment of the invention, the fermentation is performed in PDA liquid medium.

In one embodiment of the invention, the fermentation temperature is 26-30℃and preferably 28 ℃.

In a third aspect of the present invention, a biological agent is provided, where the biological agent uses the trichoderma atroviride or the trichoderma atroviride fermentation broth that is propagated by industrial production as an active ingredient, and is diluted to a specified concentration to prepare a liquid biological agent, and porous substances such as turf and vermiculite can be used as an adsorbent to adsorb the trichoderma atroviride fermentation broth to prepare powder or granule.

In an embodiment of the invention, the biological agent is a liquid preparation.

In a fourth aspect, the invention provides application of the biological agent in preventing and treating pine wilt.

Further, the pathogenic bacteria of the pine needle rot disease is aschersonia pinensis (Sphaeropsis sapinea).

In one embodiment of the invention, trichoderma atroviride inhibits the growth of Saccharomycetes pinnatifida by a heavy parasitic mechanism.

In one embodiment of the invention, 20% of the fermentation broth of Trichoderma atroviride has significantly better inhibitory effect on the aschersonia aleyrodis than 10% and 30% of the fermentation broth.

Still further, the pest plants of pine needle disease include pinus sylvestris.

In a fifth aspect, the invention provides application of the biological agent in preventing and treating fusarium wilt.

Further, the pathogenic bacteria of red spot disease is trichoderma reesei (Pestalotiopsis funerea).

In one embodiment of the invention, the inhibition of trichoderma atroviride on trichoderma reesei is proportional to time, and the inhibition rate of trichoderma atroviride on trichoderma atroviride is 70.27% at 144 hours.

Further, the harmful plants of the red spot disease include Chinese pine.

In a sixth aspect of the invention, there is provided the use of the biological agent in promoting the growth of pinus sylvestris.

In one embodiment of the invention, the method of punching, root irrigation and inoculation is adopted, and the method of inoculating the near-dark green trichoderma after the pinus sylvestris is sown for 30d can remarkably improve the seedling height, fresh weight and dry weight of the pinus sylvestris.

The invention has the following beneficial effects:

the trichoderma atroviride disclosed by the invention can wind pathogenic bacteria and grow in parallel with pathogenic bacteria hyphae. The growth of pathogenic bacteria is inhibited through a heavy parasitic mechanism, the host cells are firstly identified, after the host cells are determined, the trichoderma atroviride is contacted and then surrounds or penetrates into mycelia of the host to grow, a series of complex processes such as parasitic processes are started by forming spiral mycelia, the death of the pathogenic bacteria is further caused, the inhibition rate of the pathogenic bacteria on the pine needle blight can reach 64.74%, the inhibition rate of the pathogenic bacteria on the pine needle blight can reach 70.27%, and the growth promoting effect on the pinus sylvestris is remarkable.

The biological microbial inoculum taking the trichoderma atroviride as the active ingredient is pollution-free, does not produce environmental pollution, can reduce or even avoid other corresponding chemical pesticides, saves cost and improves the quality of the pinus sylvestris.

Drawings

FIG. 1 shows the results of a plate-facing experiment of Trichoderma atroviride and Trichoderma pini, wherein FIG. a and FIG. b show the results of a plate-facing culture experiment of Trichoderma atroviride and Trichoderma pini, and FIG. a shows the parallel growth of Trichoderma atroviride mycelium and Trichoderma pini; FIG. b is a trichoderma atroviride hyphae and conidium entwining; FIG. c shows the cultivation of Trichoderma atroviride and Trichoderma reesei on the surface and FIG. d shows the cultivation of Trichoderma reesei alone.

FIG. 2 shows the inhibitory effect of Trichoderma atroviride broth on the germination of pathogenic bacteria spores of Rhizoctonia solani, wherein, FIG. A is a control and FIG. B is a treatment of Trichoderma atroviride.

Fig. 3 shows the prevention and treatment of the bacterial infection of the pine needle disease of the camphor tree by the trichoderma atroviride, wherein fig. A is a control group and fig. B is an experimental group.

FIG. 4 shows the results of a plate-stand experiment of Trichoderma atroviride and Pediopsis cumingii, FIGS. a and b show the results of a plate-stand culture experiment of Trichoderma atroviride and Pediopsis cumingii, and FIG. a shows the parallel growth of Trichoderma atroviride hyphae and Pediopsis cumingii; FIG. b is a trichoderma atroviride hyphae and trichoderma reesei intertwining; FIG. c shows the cultivation of Trichoderma atroviride and Trichoderma reesei on the surface and FIG. d shows the cultivation of Trichoderma atroviride alone.

FIG. 5 shows the growth of Trichoderma atroviride-promoted pinus sylvestris, wherein a is a control group and b is an experimental group.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1: isolation and identification of Trichoderma atroviride

(1) The separation of trichoderma adopts a conventional fungus separation method;

(2) Separation matrix: separating from soil in natural protection area of Yulong stream in Fu Shun city of Liaoning province;

(3) Separation time: 2021 month 12;

(4) And (3) identification: morphology identification and ITS, TEF, RPB molecule identification are respectively carried out, and the identification result is Trichoderma atroviride (Trichoderma paratroviride).

The Trichoderma atroviride (Trichoderma paratroviride) is preserved in China general microbiological culture Collection center (CGMCC) at the time of 2023, 7 and 21, and the preservation address is: the collection number of the microbial institute of China academy of sciences is CGMCC No.40737 in the Korean area North Star, west Lu No. 1, no. 3 of Beijing city.

Example 2: plate confrontation verifies the inhibition effect of Trichoderma atroviride on Sphaerocarpus gracilis pathogenic bacteria of Rhizoctonia cerealis

Trichoderma atroviride and Trichoderma pini (Sphaeropsis sapinea) isolated from the dried tip of Pinus sylvestris were first cultured on PDA plates, respectively. Taking Trichoderma atroviride and Pacific Saccharum sinensis Roxb cakes with diameters of 5mm by using a sterilized puncher, inoculating the cakes on two sides of the same PDA plate at a distance of about 4cm, and culturing in a 28 deg.C incubator. After the Trichoderma atroviride and the conidium matsutake hyphae are contacted, the interaction condition of the two fungi hyphae is observed by an optical microscope.

The results of the counter culture are shown in FIG. 1 and Table 1:

TABLE 1 counter cultivation of Trichoderma atroviride and Cryptosporidium Pini

Trichoderma atroviride is able to wind up pathogenic bacteria and grow in parallel with the mycelium of the Cellulars pinnata (FIGS. 1a,1 b). Trichoderma atroviride inhibits the growth of pathogenic bacteria by a heavy parasitic mechanism, which is in line with the heavy parasitic mechanism of Trichoderma atroviride. In this mechanism, first, the trichoderma recognizes the host cell, after the host cell is determined, the trichoderma grows around or penetrates the hypha of the host after contacting, and forms a series of complex processes such as the onset of parasitic action of the spiral hypha, and further causes death of pathogenic bacteria.

In the counter culture of Trichoderma atroviride and Trichoderma pini (FIGS. 1c,1 d), the inhibition of Trichoderma atroviride on the conidium pini was proportional to time. The inhibition rate of Trichoderma atroviride on the aschersonia aleyrodis is 64.74% at 96 h. The trichoderma atroviride has high growth speed and rapidly occupies the growth space to cover pathogenic bacteria.

Example 3: inhibition of Trichoderma atroviride fermentation broth on Pacific Saccharum sinensis Roxb

And (3) filtering the liquid culture solution of the trichoderma atroviride which is cultured for 15 days by using sterile gauze to remove hyphae, namely, obtaining fermentation liquor, and filtering and sterilizing the fermentation liquor under the sterile condition by using a Millex-HV Filter Unit sterilization Filter membrane (with the pore diameter of 0.22 mu m) for later use.

(1) Effect on the growth of the mycelium of the Pachylomyces Sonchus

The test adopts a mixed culture medium method, and a pathogen bacterial sheet with the diameter of 5mm is inoculated in the center of a PDA flat-plate culture medium containing 10%, 20% and 30% (V: V) fermentation liquor, and the culture is carried out in a constant temperature and humidity incubator at the temperature of 25 ℃ to measure the colony diameter at regular time. PDA plates containing 10%, 20%, 30% sterile water were used as controls, with 5 replicates per treatment.

The results are shown in Table 2:

TABLE 2 inhibition of Trichoderma fermentation broth on the growth of the mycelium of Pacific Saccharum sinensis Roxb

At 10% concentration, the inhibition rate of Trichoderma atroviride on the conidium Pini is increased and then decreased, and the inhibition rate of Trichoderma atroviride on the conidium Pini in 20% and 30% fermentation liquor is decreased with the increase of time. The maximum inhibition rate of the 20% fermentation liquor of the trichoderma atroviride is 33.88% at 24 hours, and the minimum inhibition rate of the 10% fermentation liquor is 3.31% at 24 hours. The inhibition effect of the fermentation liquor of the near-dark green trichoderma 20% on the conidium pine is obviously better than that of the fermentation liquor of 10% and 30%.

(2) Influence on germination of conidium pine

Mixing the trichoderma fermentation liquor and the conidium liquor according to the proportion of 1: mixing at a ratio of 1, and observing germination of the conidium by taking the spore liquid mixed by the culture medium as a control.

The Trichoderma atroviride fermentation liquid has an inhibiting effect on the germination of the conidium spores (figure 2), and the inhibiting effect of the Trichoderma atroviride on the germination of the conidium spores can reach 89.54% in 7 hours.

Example 4: prevention and treatment effect of trichoderma atroviride strain on pinus sylvestris

(1) Preparation of conidium suspensions: trichoderma atroviride was inoculated into a test tube of PDA liquid medium, cultured in an incubator at 28℃for 7 days, washed off with sterilized water, and after counting by a hemocytometer, the concentration of conidia was adjusted to 1X 10 ⁷/mL with sterilized water.

(2) Pathogenic bacteria spore of pinus sylvestris disease: soaking the needle leaves of the camphorwood pine needle disease collected in the field with sterile water to obtain the pathogen aschersonia aleyrodis.

(3) Inoculating pathogenic bacteria:

Selecting the camphor seed pine seedlings with 40-50 cm of consistent growth state, selecting 3 tips for each plant, and removing needle leaves in the middle of the tips. Then the following treatments are respectively carried out on the pinus sylvestris: A. control group: adding 50 mu L of the pathogenic bacteria spore suspension of the pinus sylvestris at the position of removing needle leaves at the tip of the pinus sylvestris; B. experimental group: spraying Shi Mumei conidium suspension to selected pinus sylvestris for 1 day, and adding 50 mu L of pinus sylvestris pathogenic bacteria spore suspension at the position of pinus sylvestris tip needle leaf removal; the inoculation site is then moisturized. 5 pine seedlings were treated each. After 50d, the pinus sylvestris is photographed and the morbidity is counted.

The results are shown in Table 3 and FIG. 3, the tip of 3-day camphor seed pine after inoculation with pathogenic bacteria showed a victim shape, the tip began to bend and droop, and the later onset tip died. Both A and B were inoculated with 15 shoots, of which 10 shoots died from A. 3 shoots died from B, 66.7% of A and 20% of B. Shows that the trichoderma atroviride has a certain control effect on the occurrence of the pinus sylvestris.

TABLE 3 statistics of incidence of pine cone tip disease in Cinnamomum camphora

Example 5: plate facing verification of inhibition of Trichoderma atroviride pathogenic bacterial plaque Mucor pulmonale by Trichoderma atroviride

Trichoderma atroviride and Pelargonium subtilis isolated from the pathogenic pinus koraiensis were first cultured on PDA plates, respectively. And (3) respectively taking Trichoderma atroviride and Mucor bescens cakes with diameters of 5mm by using a sterilized puncher, inoculating the cakes on two sides of the same PDA flat plate at a distance of about 4cm, and culturing in a 28 ℃ incubator. After the trichoderma atroviride and the trichoderma cumingii mycelium are contacted, the interaction condition of the two fungi mycelium is observed by an optical microscope.

The results of the counter culture are shown in FIG. 4 and Table 4: the inhibition of trichoderma atroviride by trichoderma atroviride is directly proportional to time. The inhibition rate of the trichoderma atroviride on the trichoderma cumingii is 70.27 percent at 144 hours. The trichoderma atroviride has high growth speed and rapidly occupies the growth space to cover pathogenic bacteria.

TABLE 4 counter cultivation of Trichoderma atroviride and Mucor subtilis

Example 6: protoxigenic effect of Trichoderma atroviride strain on pinus sylvestris

(1) Sowing seeds

The test pinus sylvestris seeds are provided by Liaoning province sand fixation forestation research institute. The seeds were sterilized with 0.5% KMnO4 for 0.5h, rinsed several times with clear water, and placed in a 25℃incubator for germination acceleration. Sowing the germinated pinus sylvestris seeds into a nutrition pot filled with sterilized soil, covering 30 seeds in each pot with 2cm thick sterilized soil, and watering thoroughly. After emergence of seedlings, the seedlings are fixed to 15 plants in each pot.

(2) Preparation of microbial inoculum

A sterile punch with a diameter of 0.5cm was used to cut out the Trichoderma atroviride cake cultured on PDA plate medium and inoculated into triangular flasks containing PDA medium. Shaking culture at 25deg.C on 160r/min shaker for 6d to obtain liquid microbial inoculum.

(3) Application of Trichoderma

And (3) inoculating the trichoderma atroviride by adopting a punching root-irrigation inoculation mode after 30d of sowing. 50ml of each pot was inoculated, 10 pots were inoculated altogether, and no Trichoderma treatment was applied to the control.

After 3 months of growth, 10 seedlings are randomly extracted from the pinus sylvestris by contrast and application of near-dark green trichoderma treatment, surface soil is gently shaken off, root attachment soil is washed off by running water, and the seedling height and the ground diameter of the plants are measured by a tape measure and a vernier caliper; the fresh weight is weighed by an electronic balance which absorbs water by using water absorption paper, and the dry weight is weighed after drying at 80 ℃.

The growth conditions of the different treated pinus sylvestris are shown in Table 5 and figure 5, and the result shows that the application of the near-dark green trichoderma can obviously improve the seedling height, fresh weight and dry weight of the pinus sylvestris.

TABLE 5 growth promoting effect of Trichoderma atroviride on Pinus sylvestris seedlings

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. Trichoderma atroviride (Trichoderma paratroviride), characterized in that the Trichoderma atroviride is preserved in China general microbiological culture Collection center (CGMCC) under the preservation number of CGMCC No.40737 at 7-21 of 2023.

2. The culture of trichoderma atroviride of claim 1, wherein said culture is a fermentation broth.

3. A biological agent comprising the trichoderma atroviride of claim 1 or the culture of claim 2 as an active ingredient.

4. Use of a biological agent according to claim 3 for controlling pine needle diseases, characterized in that the pathogenic bacteria of pine needle diseases are aschersonia pinensis (Sphaeropsis sapinea).

5. The use according to claim 4, wherein the harmful plant of pinocembria comprises pinocembria.

6. Use of the biological agent according to claim 3 for controlling fusarium wilt.

7. The use according to claim 6, characterized in that the pathogenic bacteria of the fusarium songaricum is trichoderma reesei (Pestalotiopsis funerea).

8. The use according to claim 7, wherein the harmful plants of the red spot disease comprise pinus koraiensis.

9. Use of the biological agent of claim 3 for promoting the growth of pinus sylvestris.