A Trichoderma atroviride and its application

Abstract

The present invention discloses a Trichoderma atroviride and its application, belonging to the field of biological control of plant diseases. The Trichoderma atroviride was deposited at the China Center for Type Culture Collection (CCTCC) on July 19, 2022, with the deposit number of CCTCC No. M 20221135. The Trichoderma atroviride CJCCSJ-F-KY10039 was isolated from the forest system in Xishuangbanna. The fermentation supernatant of this Trichoderma atroviride strain can inhibit the growth of the hyphae of Fusarium graminearum and Fusarium verticillioides. Through the plate confrontation test, it was found that its strain has an inhibitory effect on the growth of Fusarium verticillioides and Fusarium graminearum. This strain has good application prospects in the biological control of Fusarium graminearum and Fusarium verticillioides in maize.

Description

Technical Field

[0001] The present invention relates to the field of biological control of plant diseases, and particularly to a Trichoderma atroviride and its application. Background Art

[0002] The genus Trichoderma belongs to Deuteromycetes and Hypocreaceae. It is a soil-inhabiting fungus widely distributed and also an efficient antagonistic and growth-promoting bacterium. Trichoderma is a kind of microorganism that grows in the soil without causing any symptoms. During the long-term evolution process, Trichoderma and plants have adapted to and interacted with each other. Since Weindling discovered its antagonism against pathogenic microorganisms in 1932, multiple Trichoderma strains have been isolated and applied to disease control.

[0003] Fusarium graminearum can cause diseases of various major crops, such as wheat scab, corn stalk rot, etc. Fusarium graminearum will metabolize different kinds of toxins in wheat grains, including nivalenol (NIV), deoxynivalenol (DON), and zearalenone (ZENralenol, ZEN), etc. DON toxin can cause vomiting, miscarriage in humans and livestock, etc.

[0004] Fusarium verticillioides is a pathogen that infects corn plants and causes a decline in world production. The toxin produced by Fusarium verticillioides exists in the form of fumonisin, which can accumulate in the grains and endanger the health of humans and livestock. The accumulation of fumonisin in the body can inhibit sphingolipid metabolism and cell cycle regulation, thus causing various effects such as esophageal cancer and the risk of neural tube defects in children. Based on this, it is necessary to control this disease to reduce yield losses. The control means usually control stalk rot by spraying fungicides on plants.

[0005] Resistant varieties and chemical pesticide control are usually common methods in the integrated control of plant diseases. However, chemical agents are not the best choice for controlling plant diseases due to reasons such as environmental residues and harm to non-target organisms. Biological control has gradually been widely applied in recent years due to its green and safe advantages. Isolating and screening Trichoderma strains with antagonistic effects against Fusarium graminearum and Fusarium verticillioides meets the needs of green production and has good application prospects. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a Trichoderma atroviride, and the fermentation supernatant of this Trichoderma atroviride strain can inhibit the growth of Fusarium graminearum and Fusarium verticillioides, and its strain has an antagonistic effect on Fusarium graminearum and Fusarium verticillioides.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a strain of Trichoderma atroviride, named Trichoderma atroviride CJCCSJ-F-KY10039, with a preservation number of CCTCC No. M20221135, preserved in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, and the preservation date is July 19, 2022.

[0009] The Trichoderma atroviride CJCCSJ-F-KY10039 provided by the present invention is isolated from the forest system in Xishuangbanna. The suitable growth environment is PDA medium, and it is cultured in a 28°C light incubator. It fills the culture dish in 72 hours. The colony color is green. Initially, it is white and then produces green spores as it grows. After culturing for 3 days, the conidia are white and aggregated in the center. Radial patterns are formed on the colony surface. After culturing for 5 days, green granular sporulation clusters are produced, with more sporulation clusters at the edge, showing a cushion shape, and the back is white. The hyphae are white, branched, smooth, and septate. The sporogenous cells are solitary, opposite, or whorled on the conidiophores. The sporogenous cells are flask-shaped, narrow at the base, wide in the middle, tapering upwards, and colorless. The conidia are solitary, nearly spherical to ovoid, unicellular, green, with smooth edges, and multiple spores aggregate to form a head shape. It has an aromatic smell. Chlamydospores are not seen.

[0010] To identify the phylogenetic status of this strain, the ITS DNA sequence of the isolated strain is sequenced. 20 reference strain sequences are obtained from the NCBI (GenBank) database, and the software BioEdit and MEGA11 are used to analyze the ITS DNA sequences of the isolated strain and the reference strains, and a phylogenetic tree of the isolated strain and the reference strains is constructed. Thus, it is determined that the Trichoderma atroviride strain system is the strain system of Trichoderma atroviride (attached Figure 3 ), and it is named CJCCSJ-F-KY10039.

[0011] The present invention finds that the inhibitory rate of the fermentation supernatant of Trichoderma atroviride CJCCSJ-F-KY10039 against the mycelia of Fusarium graminearum is 50-75%, and the inhibitory rate against the mycelia of Fusarium verticillioides is 15-45%. Through the plate confrontation test, it is found that it has an antagonistic effect on Fusarium graminearum and Fusarium verticillioides, and the inhibitory rates are 70-85% and 30-65% respectively.

[0012] The preparation method of the above Trichoderma atroviride CJCCSJ-F-KY10039 fermentation supernatant comprises the following steps: inoculate the Trichoderma atroviride strain into a PDA medium, culture it in a light incubator at 28 °C for 4 days, prepare a spore suspension, inoculate the spore suspension into a PD medium, and the inoculation amount is 10 5～8 cells / ml, culture at 28 °C and 200 rpm for 5 days, filter the spores and hyphae to obtain the supernatant, use a 0.22 μm aqueous microporous filter membrane for suction filtration to completely remove the bacteria, and collect the sterile fermentation supernatant for standby. Mix the obtained sterile fermentation supernatant with PDA, pour it into a 90 mm petri dish for standby.

[0013] Furthermore, the PDA medium contains: 20 g of glucose, 200 g of potato, 20 g of agar, and 1 L of deionized water.

[0014] Furthermore, the PD medium contains: 6 g of potato leaching powder, 20 g of glucose, and 1 L of deionized water.

[0015] Furthermore, the concentration of the spore suspension is 1×10 5～8 cells / ml.

[0016] Furthermore, the volume ratio of the sterile fermentation supernatant to PDA is 1:2 to 2.5.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The Trichoderma atroviride provided by the present invention comes from the tropical forest system in Xishuangbanna. Compared with other similar Trichoderma, except for higher temperature requirements, the culture conditions are extremely simple. Its fermentation supernatant has obvious antagonistic effects on Fusarium graminearum and Fusarium verticillioides. Its strain and metabolite solution can inhibit the mycelial growth of Fusarium graminearum and Fusarium verticillioides. This strain can be applied to green production, is environmentally friendly, safe, harmless, and pollution-free, and has good application prospects especially in the biological control of Fusarium graminearum and Fusarium verticillioides in corn.

[0019] Biological preservation description of Trichoderma atroviride CJCCSJ-F-KY10039:

[0020] Preservation institution: China Center for Type Culture Collection (CCTCC)

[0021] Preservation number: CCTCC No. M 20221135

[0022] Preservation date: July 19, 2022

[0023] Preservation address: Wuhan University, Wuhan, China

[0024] Taxonomic naming: Trichoderma atroviride Description of the Drawings

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings:

[0026] Figure 1 is the conidial morphology of Trichoderma atroviride CJCCSJ-F-KY10039;

[0027] Figure 2 is the colony morphology of Trichoderma atroviride CJCCSJ-F-KY10039 on PDA medium;

[0028] Figure 3 is the phylogenetic tree constructed based on the ITS rDNA sequence;

[0029] Figure 4 is the inhibitory effect diagram of the fermentation supernatant of Trichoderma atroviride strain CJCCSJ-F-KY10039 on the mycelial growth of Fusarium graminearum;

[0030] Figure 5 is the mycelial plate growth effect diagram of Fusarium graminearum with a blank control of 30% PD;

[0031] Figure 6 is the inhibitory effect diagram of the fermentation supernatant of Trichoderma atroviride strain CJCCSJ-F-KY10039 on the mycelial growth of Fusarium verticillioides;

[0032] Figure 7 is the mycelial plate growth effect diagram of Fusarium verticillioides with a blank control of 30% PD;

[0033] Figure 8 is the confrontation test effect diagram of Trichoderma atroviride CJCCSJ-F-KY10039 against Fusarium graminearum on a plate;

[0034] Figure 9 is the blank control of the confrontation test of Fusarium graminearum on a plate;

[0035] Figure 10 is the confrontation test effect diagram of Trichoderma atroviride CJCCSJ-F-KY10039 against Fusarium verticillioides on a plate;

[0036] Figure 11 is the blank control of the confrontation test of Fusarium verticillioides on a plate. Specific Embodiments

[0037] The present invention will be further described in detail below in conjunction with embodiments.

[0038] Example 1 Identification and Preservation of Trichoderma atroviride

[0039] Molecular biology identification: The DNA extraction method of Trichoderma atroviride uses a kit to extract fungal genomes (Vazyme Cat. DC104-01). The ITS4 and ITS5 sequences of DNA are amplified by PCR to obtain amplification products. After purification, the amplification products are directly subjected to bidirectional sequencing. The amplified ITS gene sequence is submitted to the BLAST program analysis on NCBI and identified as Trichoderma atroviride, named Trichoderma atroviride CJCCSJ-F-KY10039 (test number D1), and its DNA sequence is shown in SEQ ID No. 1. See the relevant figure in Figure 1 、 Figure 2 、 Figure 3 。

[0040] The PCR amplification primer sequences are ITS4 primer and ITS5 primer. The nucleotide sequence of the ITS4 primer (shown in SEQ ID No. 2) is 5'-TCCTCCGCTTATTGATATGC-3' and the nucleotide sequence of the ITS5 primer (shown in SEQ ID No. 3) is 5'-GGAAGTAAAAGTCGTAACAAGG-3'.

[0041] The reaction system for PCR amplification is: 10 μL of 2×PCR reaction premix, 1 μL of primer ITS4 primer with a concentration of 10 μmol / L, 1 μL of ITS5 primer with a concentration of 10 μmol / L, 2.5 μL of DNA template, and the volume is supplemented to 25 μL with double-distilled deionized water.

[0042] The reaction program for PCR amplification is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 50°C for 20 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 6 min, and termination at 4°C.

[0043] Preservation of Trichoderma atroviride CJCCSJ-F-KY10039: Trichoderma atroviride CJCCSJ-F-KY10039 is preserved in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The preservation date is July 19, 2022.

[0044] Example 2: Test on the inhibitory effect of the fermentation supernatant of Trichoderma atroviride strain CJCCSJ-F-KY10039 on the mycelial growth of Fusarium graminearum and Fusarium verticillioides

[0045] Preparation method of the fermentation supernatant:

[0046] Inoculate Trichoderma atroviride strain CJCCSJ-F-KY10039 (D1) in the middle of a 9-cm Petri dish containing PDA medium, and culture it in a 28°C light incubator for 4 days to prepare a spore suspension. Inoculate the PD medium with a Trichoderma spore suspension at a concentration of 1×10 6 cells / ml, culture it at 28°C and 200 rpm for 5 days, filter the fermentation broth to obtain the supernatant, and use a 0.22-μm aqueous microporous filter membrane for suction filtration to completely remove spores and mycelia, and then collect the sterile fermentation supernatant for standby. Mix the obtained sterile fermentation supernatant with PDA according to a volume ratio of 1:2, and pour it into a 90-mm Petri dish for standby.

[0047] Diameter 5-mm fungal discs of Fusarium graminearum and Fusarium verticillioides obtained by punching are respectively inoculated onto the plate containing the D1 fermentation supernatant (treatment) and the plate containing 30% PD (blank control), with 3 replicates. After culturing in a 28°C light incubator for 4 days, measure the colony areas of Fusarium graminearum and Fusarium verticillioides.

[0048] Inhibitory rate (%) = (control colony area - treatment colony area) / control colony area.

[0049] Calculated from the above formula, the inhibitory rates of the fermentation supernatant containing D1 on Fusarium graminearum and Fusarium verticillioides are 53.2% and 17.89% respectively. For specific results, see Table 2, Table 3 and Figures 4 - 7 .

[0050] Table 1 Comparison table of sample treatments

[0051] Treatment Sample Name D1 Fermentation Broth of Trichoderma atroviride Strain CJCCSJ - F - KY10039 CK 30% PD

[0052] Table 2 Inhibitory effect of the fermentation supernatant of Trichoderma atroviride strain CJCCSJ-F-KY10039 on the mycelial growth of Fusarium graminearum

[0053] Treatment Inhibitory Rate of Replicate 1 (%) Inhibitory Rate of Replicate 2 (%) Inhibitory Rate of Replicate 3 (%) Average Inhibitory Rate (%) D1 50.32 57.03 52.25 53.20±0.0345a CK 0 0 0 0

[0054] Table 3 Inhibitory effect of the fermentation supernatant of Trichoderma atroviride strain CJCCSJ-F-KY10039 on the mycelial growth of Fusarium verticillioides

[0055] Treatment Inhibitory Rate of Replicate 1 (%) Inhibitory Rate of Replicate 2 (%) Inhibitory Rate of Replicate 3 (%) Average Inhibitory Rate (%) D1 18.45 17.79 17.44 17.89±0.0294a CK 0 0 0 0

[0056] Example 3: Plate confrontation test of Trichoderma atroviride strain CJCCSJ-F-KY10039 against Fusarium graminearum and Verticillium dahliae

[0057] Mycelial cakes with a diameter of 5 mm were punched out from the mycelia of Trichoderma atroviride strain CJCCSJ-F-KY10039, Fusarium graminearum, and Verticillium dahliae. The mycelial cakes of Trichoderma and Fusarium graminearum, and Trichoderma and Verticillium dahliae were respectively inoculated at equal distances on both sides of the center point of a 9-cm Petri dish PDA medium (such as Figure 8 , 10). Only inoculating the mycelial cakes of Fusarium graminearum and Verticillium dahliae at the same position without inoculating the Trichoderma mycelial cake was used as a control (such as Figure 9 , 11). The colony radius of Fusarium graminearum and Verticillium dahliae towards the center point was measured respectively to calculate the inhibition rate. The inhibition rate of Trichoderma atroviride strain CJCCSJ-F-KY10039 against Fusarium graminearum was 52.15%, and the inhibition rate against Verticillium dahliae was 39.47%.

[0058] Inhibition rate (%) = (control colony radius - treated colony radius) / control colony radius.

[0059] From the above experimental results, we found that the Trichoderma atroviride strain of the present invention has a certain inhibitory effect on the mycelial growth of Verticillium dahliae and Fusarium graminearum. The results are shown in Table 4, Table 5 and Figures 8 - 11 .

[0060] Table 4 Plate confrontation effect of Trichoderma atroviride strain CJCCSJ-F-KY10039 against Fusarium graminearum

[0061] Treatment Inhibitory Rate of Replicate 1 (%) Inhibitory Rate of Replicate 2 (%) Inhibitory Rate of Replicate 3 (%) Average Inhibitory Rate (%) D1 55.00 50.23 51.21 52.15±2.519a CK 0 0 0 0

[0062] Table 5 Plate confrontation effect of Trichoderma atroviride strain CJCCSJ-F-KY10039 against Verticillium dahliae

[0063] Treatment Inhibitory Rate of Replicate 1 (%) Inhibitory Rate of Replicate 2 (%) Inhibitory Rate of Replicate 3 (%) Average Inhibitory Rate (%) D1 47.37 39.47 31.58 39.47±7.895a CK 0 0 0 0

[0064] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermentation supernatant of Trichoderma atroviride, characterized in that, The method for preparing the fermentation supernatant comprises the following steps: 1) The dark green Trichoderma strain was inoculated into PDA medium, and after culturing in a light incubator at 28° C. for 4 days, a spore suspension was prepared; 2) Inoculate the spore suspension into PD medium at an inoculum size of 10 5～8 cells / ml. After culturing at 28°C and 200 rpm for 5 days, filter the spores and mycelia and collect the supernatant. Use a 0.22-μm aqueous microporous membrane for suction filtration to completely remove the cells, and then collect the sterile fermentation supernatant for standby; 3) Mix the obtained sterile fermentation supernatant with PDA and pour into a 90 mm culture dish for later use; The Trichoderma atroviride ( Trichoderma atroviride ) was deposited at the China Center for Type Culture Collection (CCTCC) on July 19, 2022, with the deposit number of CCTCC No. M20221135.

2. The fermentation supernatant according to claim 1, characterized in that, The concentration of the spore suspension is 1×10 5～8 cells / ml.

3. The fermentation supernatant according to claim 1, characterized in that, The volume ratio of the sterile fermentation supernatant to PDA is 1:2-2.

5.

4. Use of the fermentation supernatant of Trichoderma atroviride according to claim 1 in antagonizing the growth of Fusarium graminearum ( Fursarium graminearum )

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