Trichoderma harzianum Tr21 antagonistic to corn stalk rot pathogen and its application

Abstract

The present invention relates to the field of biological control technology and discloses a Trichoderma harzianum strain Tr21 that antagonizes the Pythium stalk rot pathogen of corn and its uses. Trichoderma harzianum strain Tr21 is deposited with the General Microbiology Center of the China National Committee for the Collection of Microorganisms and has a deposit number of CGMCC No. 40325. Trichoderma harzianum strain Tr21 has a strong antagonistic effect against the Pythium stalk rot pathogen of corn and can be used to control Pythium stalk rot of corn. It also has broad antagonistic effects against plant pathogenic fungi.

Description

Technical Field

[0001] This invention belongs to the field of biological control, specifically relating to Trichoderma harzianum Tr21, an antagonistic fungus against Pythium stalk rot pathogen of corn, and its application. Background Technology

[0002] Maize stalk rot is a significant disease prevalent in maize-producing regions worldwide, and it is a serious problem in all maize-growing areas of my country. The pathogens causing maize stalk rot are complex, with over 20 reported species. These pathogens can cause stalk rot either alone or in combination. In my country, the main pathogens causing stalk rot are *Fusarium* spp. and *Pythium* spp.

[0003] Among various maize stalk rot diseases, Pythium stalk rot stands out due to its sudden onset and difficulty in control, making it a prevalent disease with significant impact on maize production worldwide. Pythium stalk rot is a soil-borne disease that occurs in the later stages of maize growth and is highly susceptible to environmental influences. Once it occurs, there are no effective field control measures. Therefore, the most practical measure to control Pythium stalk rot is to cultivate and promote disease-resistant varieties.

[0004] Currently, the main methods for controlling Pythium stalk rot in maize are planting resistant varieties, agricultural control, chemical control, and biological control. Planting resistant varieties is the most economical and effective method. However, there are significant differences in resistance to Pythium stalk rot among maize varieties, with highly resistant varieties being relatively few. In production, there are few effective chemical control agents for Pythium stalk rot; the main methods are chemical seed dressing agents containing metalaxyl and increased potassium fertilizer application, but fungicides are not very effective in controlling Pythium stalk rot. The long-term and large-scale use of chemical pesticides has led to increasingly prominent problems such as pesticide residues, environmental pollution, pesticide resistance in pests and diseases, disruption of the microecological balance in farmland, and threats to the health of organisms. Biological control uses microorganisms with disease-preventing and growth-promoting effects as biocontrol agents to control plant diseases. In recent years, due to its safety and high efficiency, it has gradually gained attention and become an important alternative to chemical control. However, current research on the biological control of Pythium stalk rot in maize is extremely limited, and there is a lack of microbial agents with good control effects against the pathogen of Pythium stalk rot in maize. Summary of the Invention

[0005] In view of this, the present invention provides a Trichoderma afroharzianum Tr21 strain that antagonizes the corn pyrolysis stem rot pathogen. The Trichoderma afroharzianum Tr21 strain of the present invention has the effect of preventing and controlling a variety of plant diseases, especially having a good control effect on the corn pyrolysis stem rot pathogen.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a Trichoderma afroharzianum Tr21, with accession number CGMCC NO.40325.

[0008] In this invention, the EF-1α gene sequence of Trichoderma harzianum Tr21 is shown in SEQ ID NO:1, and the RPB2 gene sequence is shown in SEQ ID NO:2.

[0009] In this invention, the morphological characteristics of *Trichoderma harzianum* Tr21 are as follows: the colonies are round, initially white, gradually turning green; the hyphae are filamentous, relatively long and thin, resembling cotton wool, growing radially from the center outwards; the conidiophores are flask-shaped to ampoule-shaped, swollen in the middle, with the thinnest tip capable of producing spores, and the flask cells are solitary or in whorls of 2 to 5; the conidia are spherical or ellipsoidal, single-celled, smooth, light green, turning dark green when mature.

[0010] The present invention also includes the above-mentioned Trichoderma harzianum culture, which is a substance obtained by culturing the above-mentioned Trichoderma harzianum Tr21 in a microbial culture medium.

[0011] As an optional implementation, the culture comprises a suspension of Trichoderma harzianum Tr21, a spore suspension, a fermentation broth, and / or its metabolites.

[0012] Another object of the present invention is to provide the use of the above-mentioned Trichoderma harzianum Tr21 and / or its cultures in any of the following:

[0013] 1) Inhibit plant pathogens; the plant pathogens are at least one of the following: *Pythium sacchariformis*, *Pythium sibiricum*, *Pythium sibiricum*, *Fusarium graminearum*, *Fusarium oxysporum*, *Fusarium verticillatum*, *Fusarium moniliforme*, *Curviflora zeylanica*, *Curviflora lunata*, *Curviflora zeylanica*, *Rhizoctonia graminearum*, *Fusarium graminearum*, *Fusarium solani*, *Phytophthora infestans*, and *Alternaria ginseng*.

[0014] 2) Preparation of inhibitors for the plant pathogens described in 1);

[0015] 3) Prevention and control of plant diseases caused by the plant pathogens mentioned in 1);

[0016] 4) Prepare inhibitors for the plant diseases described in 3).

[0017] Preferably, the plant diseases include at least one of the following: corn stalk rot, corn ear rot, corn root rot, corn sheath rot, corn leaf spot, Curvularia leaf spot, wheat root rot and leaf spot, wheat sheath blight, wheat root rot, wheat stem base rot, wheat Fusarium head blight, soybean root rot, ginseng root rot, ginseng black spot, ginseng blight, and tobacco root rot.

[0018] As one possible implementation, the concentration of *Trichoderma harzianum* Tr21 used is 10. 6 ~10 8 spores / mL.

[0019] As one possible implementation, the plant includes corn, and the plant disease includes corn pyroxene stem rot.

[0020] The present invention also provides a microbial inoculant or fertilizer containing the aforementioned Trichoderma harzianum Tr21 and / or culture.

[0021] Compared with the prior art, the present invention has the following advantages and effects:

[0022] The *Trichoderma harzianum* Tr21 of this invention exhibits good antagonistic activity against various *Pythium* stalk rot pathogens of corn and can be used for the control of *Pythium* stalk rot in corn. Simultaneously, *Trichoderma harzianum* Tr21 of this invention has a broad spectrum of antagonistic activity against plant pathogenic fungi and can be used for the control of various diseases in food crops, economic crops, and medicinal plants.

[0023] Preservation Instructions

[0024] Trichoderma afroharzianum Tr21 was deposited on September 23, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.40325; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0025] Figure 1 This is a phylogenetic tree of the Tr21 strain obtained based on the Ef1-α gene sequence.

[0026] Figure 2 This is a phylogenetic tree of the Tr21 strain obtained based on the RPB2 gene sequence.

[0027] Figure 3 The inhibitory effect of different concentrations of sterile fermentation broth of Tr21 on Pythium saccharidosis.

[0028] Figure 4 The inhibitory effect of different concentrations of sterile fermentation broth of Tr21 on Pythium sibiricum.

[0029] Figure 5 The inhibitory effect of different concentrations of sterile fermentation broth of Tr21 on Pythium spp. Detailed Implementation

[0030] This invention isolates and purifies Trichoderma afroharzianum Tr21 from the rhizosphere soil of maize stalk rot diseased plants in Shangshui County, Zhoukou City, Henan Province. This strain has good control efficacy against maize stalk rot and also has a broad spectrum of antagonistic plant pathogenic fungi.

[0031] Currently, this African Trichoderma afroharzianum has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.40325; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: September 23, 2022; and classified as Trichoderma afroharzianum Tr21.

[0032] The morphological characteristics of Trichoderma harzianum Tr21 of the present invention are as follows: the colonies are round, initially white, and gradually turn green; the hyphae are filamentous, relatively long and thin, and cotton-like, growing radially from the center to the periphery; the conidiophores are flask-shaped to ampoule-shaped, swollen in the middle, and the apex is the thinnest and can produce spores, and the flask cells are solitary or in whorls of 2 to 5; the conidia are spherical or ellipsoidal, single-celled, smooth, light green, and turn dark green when mature.

[0033] The EF-1α gene sequence of Trichoderma harzianum Tr21 of the present invention is shown in SEQ ID NO:1, and the RPB2 gene sequence is shown in SEQ ID NO:2.

[0034] Trichoderma africa Tr21 was identified as a fungus belonging to the genus Trichoderma (T. afriharzianum) of the subphylum Deuteromycetes, class Hyphomycetes, order Hyphomycetes.

[0035] In this invention, the aforementioned *Trichoderma harzianum* culture refers to the substance obtained by culturing *Trichoderma harzianum* Tr21 in a microbial culture medium, including but not limited to bacterial suspensions, spore suspensions, fermentation broths, and / or metabolites of *Trichoderma harzianum* Tr21. The microbial culture medium here refers to a medium capable of promoting the growth and propagation of *Trichoderma harzianum* Tr21, including but not limited to PDA medium, and improved media on PDA medium for better growth or metabolism of *Trichoderma harzianum* Tr21. Those skilled in the art can adjust or improve the culture medium and culture conditions according to actual culture circumstances.

[0036] The African Trichoderma Tr21 and its culture of the present invention have the function of inhibiting a variety of plant pathogens and can be used as the sole or partial active ingredient of plant pathogen inhibitors. Preferably, the African Trichoderma Tr21 and its cultures of the present invention have inhibitory effects on a variety of plant pathogenic fungi, including Pythium inflatum, Pythium arrhenomanes, Pythium aristosporum, Fusarium graminearum, F. oxysporum, F. verticillioides, F. proliferatum, Bipolaris maydis, Curvularia lunata, Bipolaris sorokiniana, Rhizoctonia cerealis, F. pseudoograminearum, F. solani, P. cactorum, and Alternaria panax.

[0037] The *Trichoderma harzianum* Tr21 and its cultures of the present invention achieve the effect of preventing and controlling plant diseases by inhibiting plant pathogens, and can be used as the sole or partial active ingredient of plant disease inhibitors. As one embodiment, the plant diseases of the present invention include at least one of the following: corn stalk rot, corn ear rot, corn root rot, corn sheath rot, corn short spot, *Curvularia* leaf spot, wheat root rot and leaf spot, wheat sheath blight, wheat root rot, wheat stem base rot, wheat scab, soybean root rot, ginseng root rot, ginseng black spot, ginseng blight, and tobacco root rot. Among them, *Trichoderma harzianum* Tr21 of the present invention has a particularly strong antagonistic effect on the corn stalk rot pathogens *Pythium spp.*, *Pythium spp.*, and *Pythium spp.*, with 10-fold and 20-fold dilutions achieving 100% inhibition rates against *Pythium spp.*, *Pythium spp.*, and *Pythium spp.* in plate cultures. An appropriate concentration is beneficial for *Trichoderma harzianum* Tr21 to exert a good control effect against plant pathogens; both high and low concentrations are detrimental to the antagonistic effect of *Trichoderma harzianum* Tr21 against plant pathogens. Experiments have shown that the *Trichoderma harzianum* Tr21 of this invention exhibits good control efficacy against plant pathogens at a concentration of 10... 6 ~10 8 At a concentration of 1 spore / mL, it can effectively control corn stalk rot caused by Pythium sclerotium, Pythium virgaureum, and Pythium spp.

[0038] Another aspect of the present invention provides a microbial inoculant or fertilizer containing *Trichoderma harzianum* Tr21 and / or its culture. The microbial inoculant or fertilizer of the present invention can be produced according to methods well known to those skilled in the art. The formulation type of the microbial inoculant or fertilizer can be in the form of solution, dispersant, suspension, granules, etc. It can be applied by spraying, broadcasting, furrow application, or irrigation.

[0039] In one embodiment, the microbial inoculant or fertilizer of the present invention further includes microbial strains different from the *Trichoderma harzianum* Tr21 of the present invention. The combined use of different microorganisms broadens the host spectrum of the microbial inoculant, improves the control effect against plant pathogens, and delays the development of drug resistance. The microbial fertilizer of the present invention may also include organic and inorganic fertilizers required for plant growth. By combining *Trichoderma harzianum* Tr21 or its culture with organic and inorganic fertilizers to prepare a microbial fertilizer, the growth-promoting effect of the microbial fertilizer is enhanced while controlling plant diseases.

[0040] In one embodiment, the present invention's *Trichoderma harzianum* Tr21 or its culture can be prepared as a microbial inoculant or fertilizer by freeze-drying or dissolving in a suitable solvent. Freeze-drying employs conventional methods in the art. The solvent may be water, a microbial culture medium, or the like.

[0041] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The plant pathogens used in the following embodiments are plant pathogen strains preserved in the laboratory and are known types of plant pathogens. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0042] Example 1

[0043] Isolation and purification of Trichoderma strains

[0044] During the corn maturity period, a five-point sampling method was used to collect soil samples from the top 10-20 cm layer around diseased corn plants in fields affected by corn stalk rot. Soil samples from the same plot were mixed evenly, numbered, and placed in plastic bags, then brought back to the laboratory and stored at 4℃. A total of 124 soil samples were collected.

[0045] The dilution plate method was used. 10g of soil sample was taken from the refrigerator and placed in an Erlenmeyer flask containing 90ml of sterile water and 5-10 glass beads (4mm diameter). The flask was then shaken at 200 rpm for 30 minutes. 5ml of the solution was poured into an Erlenmeyer flask containing 45ml of sterile water and mixed well. Another 5ml of this solution was transferred to the Erlenmeyer flask containing 45ml of sterile water and mixed thoroughly. 0.2ml of this solution was then transferred onto a 9cm diameter plate and spread evenly using a spreader. The plate was incubated at 28℃. After 48–72 hours, colonies producing green conidia were picked and transferred to PDA plates. The plates were then incubated at 28℃ until the colony diameter reached 4–5cm. A total of 109 *Trichoderma* strains were obtained.

[0046] The inhibitory effect of the above-mentioned *Trichoderma* strains on the pathogen of *Pythium stalk rot* in maize was tested using the mycelial growth rate method. *Pythium stalk rot* pathogens *Pythium spp.*, *Pythium spp.*, and *Pythium spp.*, which were preserved in the laboratory, were activated. The activated pathogens and *Trichoderma* were cultured separately on PDA plates for 5 days. After sufficient colony growth, 10 mycelial cakes (0.5 cm in diameter) were cut from the edge of the *Trichoderma* colonies and inoculated into liquid culture medium (100 mL PD / 250 mL Erlenmeyer flasks). The cultures were incubated at 28℃ and 180 rpm for 5 days with shaking. The cultured liquid was centrifuged at 4℃ and 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane to obtain a sterile fermentation broth for use. The obtained fermentation broth was added to PDA medium at a volume ratio of 10% to prepare plates. Fresh mycelial blocks (0.5 cm in diameter) of *Pythium spp.*, *Pythium spp.*, and *Pythium spp.* were inoculated into the center of each plate. PDA plates with an equal amount of PD were used as controls. Each treatment was repeated three times. The inoculated plates were placed in a dark incubator at 28°C. After the control plates were fully colonized, the colony diameter of each treatment was measured using the cross-sectional method, and the inhibition rate was calculated.

[0047] Inhibition rate = (colon diameter of control pathogen - colony diameter of treated pathogen) / (colon diameter of control pathogen - 0.5 cm) × 100%.

[0048] As shown in Table 1, the fermentation broth of Trichoderma strain Tr21 has a high inhibitory effect on the three Pythium species, with an inhibition rate of 100% for all of them.

[0049] Table 1. Determination of the inhibition rate of Tr21 against Pythium spp.

[0050]

[0051] Example 2

[0052] Morphological identification of Tr21 strain

[0053] Using a punch, collect 0.5 cm diameter mycelial discs from the edge of a Tr21 colony and place them on a PDA plate. Incubate at 28°C in the dark. Observe the growth rate and color changes of the colonies on the petri dish daily. After 3-5 days of colony incubation, pick mycelia to prepare slides and observe the morphology of mycelia, conidia, and conidiophores under an optical microscope.

[0054] Results: After activation on PDA medium, strain Tr21 exhibited rapid aerial mycelial growth. Colonies were round, initially white, gradually turning green. Hyphae were filamentous, relatively long, and cotton-like, radiating outwards from the center. Conidiophores were flask-shaped to ampoule-shaped, swollen in the middle, tapering to a spore-bearing apex; flask cells were solitary or arranged in whorls of 2–5. Conidia were spherical or ellipsoidal, single-celled, smooth, and pale green, turning dark green when mature. Based on these morphological characteristics, the strain was preliminarily identified as a species of the genus *Trichoderma*.

[0055] Example 3

[0056] Molecular identification of Tr21 strain

[0057] Trichoderma strain Tr21 was cultured on PDA medium for 5 days, and spores and mycelium were collected. Total DNA of the fungus was extracted using the CTAB method.

[0058] PCR amplification was performed using primers for the EF-1α gene and the RPB2 gene, respectively. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0059] The primers for the EF-1α gene are as follows:

[0060] EF1-728F: 5'-CATCGAGAAGTTCGAGAAGG-3';

[0061] TEF1LLErev: 5'-AACTTGCAGGCAATGTGG-3'.

[0062] The primers for the RPB2 gene are as follows:

[0063] 5F: GAYGAYMGWGATCAYTTYGG;

[0064] 7CR: CCCATRGCTTGYTTRCCCAT.

[0065] The PCR reaction system was 25 μL, consisting of 12.5 μL of 2×Es Taq MasterMix (Kangwei Century), 1 μL of template DNA, 0.5 μL each of forward and reverse primers (10 μmol / L), and ddH2O to a final volume of 25 μL.

[0066] PCR amplification reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; annealing temperature depending on the primers: 53℃ (primer EF1-728F and TEF1LLErev) and 51℃ (primer 5F and 7CR) for 30 s; 72℃ extension for 1 min; 35 cycles; final extension at 72℃ for 7 min.

[0067] PCR amplification products were directly sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared for homology in GenBank. Simultaneously, based on the obtained sequences and other pathogen-related sequence information obtained from GenBank, a phylogenetic tree was constructed using MEGA 5.2 software to determine the taxonomic position of the pathogen.

[0068] PCR amplification of EF-1α and RPB2 genes in strain Tr21 yielded products of 1280 bp and 1143 bp, respectively. Sequencing of these products is shown in SEQ ID NO:1 and SEQ ID NO:2. Homology comparison of the EF-1α and RPB2 gene sequences of strain Tr21 in GenBank revealed that strain Tr21 was most closely related to the EF-1α gene sequence of *Trichoderma africanum* strain Z19 (99.84% homology) and most closely related to the RPB2 gene sequence of *Trichoderma africanum* strain Tri-1 (99.74% homology). Phylogenetic tree construction using MEGA 5.2 software showed that strain Tr21 clustered with *Trichoderma africanum*, indicating that Tr21 belongs to the genus *Trichoderma* *Trichoderma africanum*.

[0069] Based on the culture characteristics, morphological features, EF-1α gene sequence, and RPB2 gene sequence analysis of strain Tr21, it was identified as a fungus belonging to the genus Trichoderma of the class Hyphomycetes, order Hyphomyales, and class Myxosporidioides, specifically *T. afroharzianum*.

[0070] Example 4

[0071] Antibacterial spectrum determination of strain Tr21

[0072] The plate confrontation method was used to confront strain Tr21 with various major pathogenic fungi of maize, including *Hymenocortis macrantha*, *Sclerotium macrantha*, *Curvularia macrantha*, *Root rot*, *Sheath rot*, *Root rot*, *Stem rot*, *Fusarium graminearum*, *Root rot*, *Root rot*, *Gynostemma pentaphyllum*, *Root rot*, *Gynostemma ginseng*, *Black spot*, *Phytophthora blight*, and *Root rot* of tobacco, on PDA medium. The cultures were incubated in the dark at 28°C. After the mycelia in the control group had fully colonized the medium, the colony diameter was measured using the cross-crossing method. PDA medium inoculated only with pathogens served as a control. The experiment was repeated three times to calculate the inhibition rate of strain Tr21 on the mycelial growth of the pathogens.

[0073] The experimental results (Table 2) show that the African Trichoderma harzianum strain Tr21 exhibits good inhibitory effects on the mycelial growth of all 13 tested plant pathogenic fungi, with inhibition rates exceeding 60%, demonstrating a broad antifungal spectrum. Specifically, it showed an inhibition rate of 80.71% against *Trichoderma harzianum*, the causal agent of ginseng blight, and over 70% against *Trichoderma root rot*, *Trichoderma small spot*, *Trichoderma ear rot*, *Trichoderma stem rot*, *Trichoderma black spot*, *Trichoderma scab*, and *Trichoderma sheath rot*, with inhibition rates of 77.33%, 73.60%, 73.37%, 73.37%, 73.35%, 70.89%, and 70.12%, respectively.

[0074] Table 2. Inhibitory effect of strain Tr21 on the colony growth of tested plant pathogenic fungi.

[0075]

[0076]

[0077] Example 5

[0078] Inhibitory effect of different concentrations of fermentation broth of strain Tr21 on *Pythium spp.*, the causal agent of corn stem rot.

[0079] Aseptic fermentation broth of *Trichoderma* was prepared according to the method in Example 1. The fermentation broth was added to PDA medium at different volume ratios to prepare plates, resulting in concentrations of 10-fold, 20-fold, 50-fold, and 100-fold dilutions. Fresh mycelial blocks (0.5 cm in diameter) of *Pythium spp.*, *Pythium spp.*, and *Pythium spp.* were inoculated at the center of each plate. PDA plates with an equal amount of PD were used as controls. Each treatment was repeated three times. The inoculated plates were placed in a 28°C incubator in the dark. After the control plates were fully colonized, the colony diameter for each treatment was measured using the cross-sectional method, and the inhibition rate was calculated.

[0080] Inhibition rate = (colon diameter of control pathogen - colony diameter of treated pathogen) / (colon diameter of control pathogen - 0.5 cm) × 100%.

[0081] The results are shown in Table 3. The 10-fold and 20-fold dilutions of the Tr21 strain fermentation broth all showed 100% inhibition rates against *Pythium spp.*, *Pythium spp.*, and *Pythium spp.* The 50-fold dilution also showed inhibition rates exceeding 50% against all three *Pythium* species, with the highest inhibition rate against *Pythium spp.* at 76.00%. The 100-fold dilution showed a decrease in inhibition rate, with the highest inhibition rate against *Pythium spp.* at 50.22%.

[0082] Table 3. Inhibition rate of different concentrations of Tr21 fermentation broth against Pythium blight

[0083]

[0084] Example 6

[0085] Control efficacy of strain Tr21 against stalk rot of maize Pythium spp.

[0086] Preparation of Pythium corn kernel culture: Pythium sacchariformis, Pythium virgaureum, and Pythium spp. were cultured on PDA plates for 5 days. Mycelia and spores were washed off each plate with 150 mL of sterile water. Pythium solution was inoculated into corn kernel culture medium at a ratio of 1% (v / w) and cultured in the dark at 28℃. When mycelia covered the corn kernels, the inoculum was obtained as Pythium corn kernel culture.

[0087] Tr21 was inoculated onto PDA plates and cultured in the dark at 28℃ for 4-7 days. After the colonies had fully grown, 10 mycelial discs (0.5 cm in diameter) were cut from the edge of each disc and inoculated into PD liquid medium (100 mL PD / 250 mL Erlenmeyer flask). The culture was then incubated at 28℃ with shaking at 180 rpm for 5 days. The mycelium was removed by filtration to obtain the fermentation broth. The spore concentrations of the fermentation broth were prepared to a concentration of 1×10⁻⁶. 8 cells / ml, 1×10 7 cells / ml and 1×10 6 Seeds were collected in a 7:2:1 volume ratio and sterilized at 121℃ for 2 hours to obtain a sterile substrate. Healthy Zhengdan 958 seeds of uniform size were selected, disinfected with 75% alcohol for 30 seconds, then with 2% sodium hypochlorite for 10 minutes, and washed three times with sterile water. Seeds disinfected by soaking in different concentrations of Tr21 fermentation broth for 3 hours were dried and sown in sterile soil, with Zhengdan 958 seeds soaked in sterile water serving as a control. 20-30g of each of the three Pythium maize kernel cultures were placed in each pot, covered with 3cm of sterile substrate, then 5 soaked corn kernels were sown, and finally covered with soil. The disease severity of each treatment was assessed after 20 days, and the control effect was calculated.

[0088] Grading standards for Pythium stalk rot in seedlings: Grade 0: Normal overall plant growth, disease-free; Grade 1: Basic normal growth of both above-ground and underground parts of corn, lesions covering less than 1 / 4 of the total root surface area, root color white with brown tinges; Grade 2: Significantly inhibited above-ground and underground growth, pale leaves, plant height only 3 / 4 of the control, few and short lateral roots, no fibrous roots, lesions confluent, lesion area covering 1 / 4 to 1 / 2 of the total root surface area, root color roughly white and brown; Grade 3: Extremely abnormal growth of both above-ground and underground parts, above-ground parts showing signs of wilting and yellowing, plant height only 1 / 2 of the control, very small lateral roots, lesions covering 1 / 2 to 3 / 4 of the total root surface area, root color brown with white tinges; Grade 4: Germination but failure to emerge, almost suffocating and dying, lesions covering more than 3 / 4 of the total root surface area, roots brown.

[0089] The calculation methods for the disease index and prevention efficacy are as follows:

[0090] Disease index = 100 × ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value);

[0091] Prevention and control effect (%) = (disease index of blank control - disease index of treatment) / disease index of blank control.

[0092] The experimental results (Table 4) show that different concentrations of Tr21 fermentation broth have certain control effects on Pythium stem rot caused by Pythium sclerotiorum, Pythium virgaureum, and Pythium spp., with the concentration of 1×10⁻⁶ showing the best control effect. 6 At a concentration of [number] cells / mL, the control efficacy against the three Pythium species stem rot was the best, with control efficacies of 74.48%, 80.76%, and 73.71%, respectively.

[0093] Table 4. Control efficacy of Tr21 against Pythium stalk rot in maize.

[0094]

[0095] The above embodiments are the best implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An African Trichoderma harzianum ( Trichoderma afroharzianum ) strain Tr21, the preservation number of the African Trichoderma harzianum strain Tr21 is CGMCC NO.40325.

2. The African Trichoderma harzianum strain Tr21 according to claim 1, characterized in that The EF-1α gene sequence of the Trichoderma harzianum Tr21 is shown in SEQ ID NO: 1, and the RPB2 gene sequence is shown in SEQ ID NO:

2.

3. The culture of the Trichoderma harzianum strain Tr21 according to any one of claims 1 to 2, characterized in that The culture comprises a bacterial suspension and / or fermentation liquid of the Trichoderma harzianum strain Tr21.

4. Use of the Trichoderma harzianum strain Tr21 according to any one of claims 1 to 2 and / or the culture according to claim 3 in any of the following: 1) Inhibiting plant pathogens; the plant pathogen is at least one of the following: Pythium spp., Pythium spp., Pythium spp., corn ear rot, corn leaf spot, Curvularia spp., corn root rot, corn sheath rot, wheat root rot, wheat stem base rot, wheat head blight, soybean root rot, ginseng root rot, ginseng black spot, ginseng blight, and tobacco root rot; 2) preparing the inhibitor of the plant pathogenic bacteria described in 1); 3) Prevent and control corn stalk rot caused by Pythium sphaeroides, Pythium spp. and Pythium iris; 4) preparing the plant disease inhibitor described in 3).

5. The use according to claim 4, characterized in that The concentration of the African Trichoderma harzianum strain Tr21 was 10 6 ~10 8 spores / mL.

6. A microbial agent or fertilizer, characterized in that: Contains the Trichoderma harzianum strain Tr21 according to any one of claims 1 to 2, and / or the culture according to claim 3.

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