Corning Trichoderma LCTK248 strain, biocontrol agents and their applications

Abstract

This invention belongs to the field of plant disease and pest microbial control technology, specifically disclosing a type of Trichoderma cornuta ( Trichoderma koningii The strain LCTK248, deposited at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, with strain preservation number CGMCC No. 41747, is a *Trichoderma koningii* strain. This invention provides *Trichoderma koningii* LCTK248, which grows rapidly, produces a large number of spores, and exhibits significant inhibitory effects against various plant pathogens, with an inhibition rate of 95.1%–99.8% and a disease control rate of 85%–91%. This demonstrates that *Trichoderma koningii* LCTK248 possesses broad-spectrum antifungal activity against plant pathogens and has enormous biocontrol potential and promising prospects for development and application.

Description

Technical Field

[0001] This invention belongs to the field of plant disease and pest microbial control technology, specifically relating to a biocontrol Trichoderma tumefaciens strain LCTK248 that has a highly effective inhibitory effect on plant pathogens such as tea anthracnose fungus and its prepared biocontrol formulation. Background Technology

[0002] tea tree( Camellia sinensis ),coffee( Arabic coffee ), macadamia nuts ( Macadamia three-leaved ),rubber( Brazilian rubber tree ) and rice balls ( White-tailed deer These are representative of the characteristic crops of the Yunnan-Guizhou Plateau in my country, widely cultivated locally and possessing significant economic value. They are composed of different types of *Colletotrichum* (*Colletotrichum*). Colletotrichum Anthracnose (spp.), caused by fungal infection, is a significant foliar disease that is widespread and severely damaging to all these crops. Currently, prevention of crop anthracnose mainly relies on strengthening crop cultivation management and chemical control using fungicides. However, it is well known that the use of chemical fungicides can lead to problems such as crop residues, environmental pollution, and the development of drug resistance in pathogens. Biopesticides, on the other hand, are a good solution to overcome the shortcomings of chemical pesticides. Although some biocontrol strains have been reported, with the long-term use of traditional chemical agents, pathogens have gradually developed resistance, leading to a decline in control efficacy. Furthermore, with adjustments in planting structures and environmental changes, new diseases are constantly emerging. Therefore, it is necessary to develop new biocontrol bacteria to address these challenges. New biocontrol bacteria often have stronger antibacterial capabilities and a broader antibacterial spectrum. By developing new biocontrol bacteria, the control effect of diseases can be improved, and the cost of pesticides can be reduced. Summary of the Invention

[0003] This invention yielded a biocontrol strain, LCTK248, with strong inhibitory activity against important crop pathogens such as anthracnose fungus. It exhibits excellent preventative and control effects against various diseases. Based on morphological and molecular biological methods, it was identified as *Trichoderma cornigrum*. Trichoderma koningii LCTK248, deposited at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, strain accession number CGMCC No.: 41747, was discovered to inhibit pathogen growth and sporulation through toxin production. Its main biological characteristics were analyzed and determined experimentally, leading to optimization of its propagation conditions. These findings provide a valuable strain for further research and development of biological fungicides for plant diseases and lay the technological foundation for the creation of biological agents.

[0004] 1) Discovery and identification of LCTK248: This strain was isolated from moss epiphytic on tea plant stems; antagonistic tests showed that its inhibition rate against tea anthracnose fungus was 97.36±1.68% (n=5); morphological and molecular biological identification identified it as *Trichoderma cornuta*. Trichoderma kongingii ).

[0005] 2) Morphological characteristics of LCTK248: Colonies grow rapidly on potato dextrose agar plates, reaching 25... o After 3 days of incubation in the dark at C, the colony diameter was 86.5 ± 1.6 mm (n=5), and the growth rate was 27.9 mm / d. Initially, the colonies were round or nearly round, with sparse and transparent hyphae in the vigorous growth area at the periphery; the hyphae in the central area were more developed and white. After 2 days, a large number of spores were observed, turning blue-green and gradually deepening in color. Under a microscope, the hyphae are light green, septate, and branch at nearly right angles, with a diameter of 1.2–3.1 µm (n=30). Conidiophores differentiate on the hyphae, with a diameter of 2.1–4.4 µm (n=30). These conidiophores are solitary, septate, and then form bottle-shaped branches (sporogenesis cells). The conidiophores are light green, 11.6–19.1 µm long (n=30), and have slightly swollen ends bearing conidia. The conidia are mostly elliptical, with a few being round, green, and measuring 4.8–6.2 × 2.3–4.0 µm (n=30).

[0006] 3) Inhibitory effects of LCTK248 against various important crop pathogens: LCTK248 has a strong inhibitory effect on plant pathogens, particularly against the white spot disease pathogen of corn (… Epicoccus latusicollum ), Tobacco wilt pathogen ( Fusarium oxysporum ), Southwestern Polygala leaf spot disease ( Pestalotiopsis microspora ), Colletotrichum sinensis ( Colletotrichum camelliae macadamia nut anthracnose ( C. fructicola ) and rice flower anthracnose fungus ( C. Florin The inhibition rate of LCTK248 against six plant pathogens reached 95.1% to 99.8%, indicating that LCTK248 has a very strong inhibitory or bactericidal effect against a variety of crop pathogens.

[0007] 4) Safety of LCTK248 on different crops: Leaf inoculation results showed that LCTK248 did not infect corn, common tobacco, grapes, tea, coffee, and rice. This indicates that using LCTK248 to control diseases on these crops is safe and will not cause "phytotoxicity".

[0008] 5) Mechanism of action of LCTK248: The volatile substances of LCTK248 have antibacterial rates of 73.1% and 57.6% against Colchicum aestivum and Fusarium oxysporum, respectively.

[0009] 6) Optimized culture conditions for LCTK248: The suitable temperature range for the growth and sporulation of LCTK248 is 10~30℃. o C, the optimal temperature is 26°C. o C and 28 o C, when 0 o C and above 37 o At pH C, the fungus ceases to grow and sporulate; it can grow at pH 4–13, with the optimal pH for growth and sporulation being pH 7–8; it grows fastest and most vigorously under complete darkness with LCTK248, and can also grow under 4 h / d light, but growth is very slow when the light intensity is in the range of 8–24 h / d; LCTK248 is best suited for growth and sporulation on media containing glucose and maltose, and grows more slowly on other tested carbon source media; yeast extract and peptone are suitable for the growth of LCTK248, while the other two nitrogen sources are not very suitable; LCTK248 can grow on all 9 tested media plates, but grows fastest and sporulates most vigorously on PDA and PSA.

[0010] This invention provides the Corning Trichoderma LCTK248 strain, classified and named as: Corning Trichoderma Trichoderma koningii, Accession number: CGMCC NO.41747, deposited at the China General Microbiological Culture Collection Center.

[0011] The application of Corning Trichoderma LCTK248 or its conidial liquid in inhibiting the growth of plant pathogens or in the preparation of agents to inhibit the growth of plant pathogens, wherein the pathogen is *Plasmodium styracifolium* (…). Epicoccus latusicollum Fusarium oxysporum ( Fusarium oxysporum ), Microsporum simulans ( Pestalotiopsis microspora ), Tea Thorn Discs ( Colletotrichum camelliae ), fruit-borne thorny discs ( Colletotrichum. Fructicola ) or Philinia spinosa ( Colletotrichum fioriniae One or more of the following.

[0012] The application of Corning Trichoderma LCTK248 or its conidial liquid in the control of crop diseases or in the preparation of crop disease control agents, wherein the pathogen of the crop disease is *Trichoderma cornuta* (…). Epicoccus latusicollum ), Tobacco wilt pathogen ( Fusarium oxysporum ), the leaf spot pathogen of Polygala tenuifolia ( Pestalotiopsis microspora ), tea tree anthracnose fungus ( Colletotrichum camelliae ), anthracnose fungus of grape and macadamia trees ( Colletotrichum fructicola ) or the anthracnose fungus of the medicinal plant *Rhizophora stylosa* ( Colletotrichum Florin One or more of the following.

[0013] A biocontrol agent comprising the aforementioned Trichoderma Corning LCTK248 or its conidial solution.

[0014] A method for preventing and controlling crop diseases, wherein the pathogen of the crop disease is *Hymenopsula solani* (corn white spot pathogen). Epicoccus broad-necked ), Tobacco wilt pathogen ( Fusarium oxysporum ), the leaf spot pathogen of Polygala tenuifolia ( Pestalotiopsis microspora ), tea tree anthracnose fungus ( Colletotrichum camelliae ), anthracnose fungus of grape and macadamia trees ( Colletotrichum fructicola ) or the anthracnose fungus of the medicinal plant *Rhizophora stylosa* ( Colletotrichum fioriniae The method involves applying the conidial solution of Corning Trichoderma LCTK248 to the crop. Attached Figure Description

[0015] Figure 1 Corning Trichoderma ( C. koningii Morphological characteristics of strain LCTK248;

[0016] Note: A. & B. Potato dextrose agar (PDA) medium at 25°C o C. Colonies after 48 hours of incubation in the dark, front and back sides; C. Hyphae; D. Conidiophores differentiated on the hyphae; E. Bottle-shaped branches formed at the ends of the conidiophores, with conidia formed at the tips of the 'bottlenecks'; F. LCTK248 produces a large number of conidia under culture conditions.

[0017] Figure 2 PCR amplification yielded the ITS1 (top) and TEF1a gene (bottom) sequences of strain LCTK248;

[0018] Figure 3 .Neighbor-joining phylogenetic tree diagram of LCTK248 strain and related fungi;

[0019] Note: Based on ITS sequences from relevant strains and TEF1α Gene splicing sequences, neighbor-joining phylogenetic tree constructed using MEGA 6.0 software, scale bar showing the degree of difference between splicing sequences of paired species, and the number at each node representing the bootstrap support rate %) of paired species being the same species.

[0020] Figure 4 Inhibitory effect of Corning Trichoderma LCTK248 on 6 plant pathogens;

[0021] Note: 1) A1, B1, C1, D1, E1, and F1 respectively show the resistance of LCTK248 strain to the white spot disease pathogen of maize (Plasmodium styracifolium). Epicoccus latusicollum Fusarium oxysporum, the pathogen that causes tobacco wilt Fusarium oxysporum ), the leaf spot pathogen of *Polygala tenuifolia* (Microsporum simulans) Pestalotiopsis microspora Anthracnose fungus of tea trees (Tea thorn scab) Colletotrichum camelliae ), anthracnose fungus of grape and macadamia trees (Colletotrichum oryzae) Colletotrichum. fruit-growing ) and the anthracnose fungus of the medicinal plant *Colletotrichum philinense* Colletotrichum fioriniae The antibacterial effect of the bacteria was observed; A2, B2, C2, D2, E2, and F2 were the controls for each treatment (the pathogen was cultured in opposition to PDA culture medium discs). 2) The inoculation method was to place a 5 mm diameter pathogen disc in the center of the plate culture medium, and place 5 mm diameter Trichoderma Corning mycelium discs at symmetrical positions 20 mm apart on all four sides of the pathogen disc. 3) After treatment, the bacteria were incubated at 25°C. o After culturing in the dark at C for 7 days, observe and photograph.

[0022] Figure 5 . Results of safety (pathogenicity) testing of Corning Trichoderma LCTK248 on leaves of different crops;

[0023] Note: 1) A, B, C, D, E, and F are the inoculation results of LCTK248 mycelial cakes (Φ5 mm) after puncture wounding, at 25°C. o C. Leaves of corn, tobacco, grape, tea, coffee and rice flowers placed under RH>90% and 10 h / d light for 21 days (no infection or disease was induced); 2) Tea trees were inoculated with brown spot fungus (CK1) and rice flowers were inoculated with anthracnose fungus (CK2) as positive controls. CK1 and CK2 are lesions photographed 7 days after inoculation with pathogens. Detailed Implementation

[0024] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased. The invention will be further described in detail below with reference to examples.

[0025] The Corning Trichoderma strain LCTK248 provided by this invention is classified and named as: Corning Trichoderma. Trichoderma king's, Accession number: CGMCC NO.41747, deposited at the China General Microbiological Culture Collection Center, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit date: January 7, 2025. The biological material was received by the collection center on January 7, 2025 and registered. The viability of the biological material was tested by the collection center on January 7, 2025, and the result was that it was viable.

[0026] Example 1: Acquisition and identification of Corning Trichoderma LCTK248

[0027] 1) Acquisition of LCDK248: Samples of rhizosphere soil, tea tree branches and leaves, and epiphytic moss were collected from tea gardens in different locations in Lincang, Yunnan Province. These samples were brought back to the laboratory and purified using PDA and NA agar plates, yielding 16 fungal isolates and 31 bacterial isolates. Antagonistic bacteria with strong antagonistic activity were screened through antagonistic assays against *Colletotrichum spp.*, the anthracnose pathogen of tea. The screening results showed that the fungus LCDK248 exhibited the strongest antagonistic effect against *Colletotrichum spp.*, at 97.36 ± 1.68% (n=5), significantly higher than other antagonistic bacteria and fungi. Therefore, further in-depth research on this bacterium is warranted.

[0028] Identification of LCTK248: Its species was identified by combining morphological and molecular biological methods.

[0029] (1) Morphological identification: The strain was inoculated on PDA plates with a diameter of 90 mm and incubated at 25°C. o After incubation at C under darkness for 3 days, colony morphology was observed. Colony diameters were measured in five replicates, and colony growth rate (mm / d) was calculated. The shape, color, size, and sporulation characteristics of the colonies were described. The characteristics of the fungal cells were observed and photographed under a microscope. The diameters of 30 hyphae and conidiophores, and the size of 30 conidia were measured. Based on the observed and measured morphological characteristics... Figure 1 ).

[0030] (2) Morphological characteristics of LCTK248: The colonies grew rapidly on potato dextrose agar plates. After 3 days of incubation at 25°C in the dark, the colony diameter was 86.5 ± 1.6 mm (n=5) and the growth rate was 27.9 mm / d. The colonies were initially round or nearly round. The mycelium in the vigorous growth area around the periphery was sparse and transparent. The mycelium in the middle area was more developed and white. After 2 days, a large number of spores were observed, which turned blue-green and gradually deepened. Under a microscope, the hyphae are light green, septate, and branch at nearly right angles, with a diameter of 1.2–3.1 µm (n=30). Conidiophores differentiate on the hyphae, with a diameter of 2.1–4.4 µm (n=30). These conidiophores are solitary, septate, and then form bottle-shaped branches (sporogenesis cells). The conidiophores are light green, 11.6–19.1 µm long (n=30), and have slightly swollen ends bearing conidia. The conidia are mostly elliptical, with a few being round, green, and measuring 4.8–6.2 × 2.3–4.0 µm (n=30).

[0031] Based on authoritative fungal classification literature, LCTK248 has been preliminarily identified as belonging to the genus *Trichoderma*. Trichoderma sp.) fungi.

[0032] Molecular biological identification: Mycelia were scraped from LCTK248 colonies cultured for 5 days on PDA medium. Genomic DNA was extracted from the mycelia using the cetyltrimethylammonium bromide (CTAB) method. PCR amplification was performed using primer pairs ITS1 / ITS4 (5'-TCCGTAGGTGAACCTGCGG-3' / 5'-TCCTCCGCTTATTGATATGC-3') and ef1 / ef2 (5'-ATG GGTAAGGAGGACAAGAC-3' / 5'-GGAG GTACCAGTGATCATGTT-3') to amplify the ITS and ef1 genes from the DNA. TEF1-1α Gene sequence fragments were recovered, amplified, and sent to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing. Sequencing results showed two sequences, each 556 bp in length. What is meant by 609 bp? Figure 2 ). ITS and TEF1-1α Gene sequences were analyzed online (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) using BLAST-n comparison; ITS data for related Trichoderma species were downloaded from the NCBI GenBank database. TEF1-1α Gene sequences, and a phylogenetic tree constructed using the spliced ​​sequences of the two ( Figure 3 Molecular biological identification results showed that LCTK248 is *Trichoderma corniglita* (…). Trichoderma koningii ).

[0033] Example 2: Determination of the antibacterial and disease-controlling effects of LCTK248 Corning Trichoderma and its safety to crops.

[0034] 1) Determination of the antibacterial activity of LCTK248 against different important crop pathogens: The tested plant pathogens included *Hymenococcus solani*, the causal agent of white spot disease in maize. Epicoccus latusicollum ), Tobacco wilt pathogen ( Fusarium oxysporum ), the leaf spot pathogen of Polygala tenuifolia ( Pestalotiopsis microspora ), tea tree anthracnose fungus ( Colletotrichum camelliae macadamia nut anthracnose ( Colletotrichum fructicola ), Anthracnose fungus ( Colletotrichum Florin ) and Corning Trichoderma LCTK248. These fungi were inoculated onto PDA agar plates and incubated at 25°C. o After 5 days of dark incubation in a constant temperature incubator, mycelial discs with a diameter of 5 mm were punched out. Mycelial discs of each pathogen were placed in the center of a 90 mm diameter agar plate, and mycelial discs of *Trichoderma koningii* LCTK248 were placed 25 mm away from the mycelial discs at four different locations. Figure 4 The control group had PDA solid culture medium cakes placed around the bacterial cakes, with five replicates for each treatment. After inoculation, the plates were incubated at 25°C.o After culturing in the dark at C for 7 days, the diameter of the bacterial colonies was measured using the cross-cross method, and the colony area was calculated. The inhibition rate of Trichoderma koningii LCTK248 against various pathogens was then calculated [(%) = (control colony area - treated colony area) / control colony area × 100%].

[0035] The experimental results (Table 1) show that Corning Trichoderma LCTK248 has a strong inhibitory effect on the growth of six plant pathogens, with an inhibition rate of 95.1% to 99.8%. This indicates that Corning Trichoderma LCTK248 has a very strong inhibitory or bactericidal effect on a variety of crop pathogens.

[0036] Table 1. Antifungal effect of Corning Trichoderma LCTK248 against six crop pathogens

[0037]

[0038] Note: Colony diameter and area are the mean ± standard error of five replicates. The inhibitory effect of Corning Trichoderma LCTK248 treatment on all six pathogens was highly significant (P<0.001).

[0039] Disease control effect of Corning Trichoderma LCTK248: Healthy potted tea seedlings, macadamia seedlings, and rice flower seedlings were prepared in a greenhouse; LCTK248 cultured on PDA plates for 14 days was prepared to a concentration of approximately 10. 8 A Trichoderma solution with spores / mL was used to evenly spray the leaves of three plant seedlings. 24 hours after the Trichoderma foliar spray, a spore suspension of *Colletotrichum chamaejasminoides*, *Colletotrichum foetida*, and *Colletotrichum ferniensis* (approximately 10 mL) was then applied. 6 Foliar spraying was performed on tea seedlings, macadamia seedlings, and rice flower seedlings using spores ( / mL). The negative control was sprayed only with pathogen spore solution, while the positive control was sprayed with pathogen spore solution 24 hours after application of difenoconazole solution. Each treatment was replicated with 10 pots of plants. After treatment, seedlings were covered with black cotton cloth for 24 hours to maintain humidity, followed by regular and appropriate watering. Disease severity (%) was recorded and the disease control rate (%) was calculated after 21 days.

[0040] The experimental results (Table 2) show that the control efficacy of LCTK248 Trichoderma solution against anthracnose of tea trees, macadamia nuts and rice flowers is 85.2%~88.5%, which is close to the control efficacy of 40% difenoconazole suspension (SA) (91.7%~95.3%).

[0041] Table 2. Disease control effect of Trichoderma LCTK248 on anthracnose in three crops

[0042]

[0043] 3) Safety testing of Corning Trichoderma LCTK248 on different crops: The tested crops included maize (… Corn ), ordinary cigarettes ( Nicotiana tabacum ),Grape( Wine grapevine ),Tea( Camellia sinensis Small-bean coffee Coffee bean ) and rice balls ( White-tailed deer Healthy leaves were collected from the field and inoculated with *Trichoderma koningii* LCTK248 using an in vitro grafting method. The fresh leaves were placed on moist filter paper in a porcelain dish, with five leaves per plant species. Using a flame-sterilized sewing needle, several micro-holes were made at four points on both sides of the veins of each leaf, and a *Trichoderma koningii* LCTK248 mycelial cake was placed at each point. A positive control was prepared by inoculating tea leaves with *Trichoderma koningii* (also known as brown spot pathogen). Epicoccum sorghum ) Fungal cakes and inoculation of anthracnose fungus on rice flower leaves ( Colletotrichum Florin Prepare the mycelium cake by covering the inoculation tray with plastic wrap to maintain moisture. After inoculation, place the leaves in saturated humidity and darkness for 24 hours, then at 25°C... o The plants were grown in an incubator with a temperature of C, RH > 90%, and 10 h / d light, and were watered appropriately daily to maintain humidity. After 7 days of cultivation, a positive control was photographed, and after 21 days, the leaves of the Trichoderma Corning LCTK248-treated plants were observed and photographed.

[0044] Test results ( Figure 5 The results showed that leaves of two plant species inoculated with the pathogen (positive control) developed typical lesions 7 days after inoculation; negative control leaves treated with PDA medium cake (not shown in the figure) and leaves of various plants treated with Corning Trichoderma LCTK248 remained healthy 21 days after treatment, with no infection or disease observed. This indicates that Corning Trichoderma LCTK248 is safe for these crops and will not cause "phytotoxicity" in future applications.

[0045] Example 3: Analysis of the antibacterial mechanism of Trichoderma Corning LCTK248

[0046] *Colletotrichum spp.* and *Fusarium oxysporum* were selected as the pathogens to be tested. The inhibitory effect of volatile metabolites of LCTK248 on the pathogens was investigated using the double-plate inoculation method. Colonies cultured at 27°C in the dark for 5 days were collected, and mycelial discs (d=8mm) were inoculated from the edge of the colonies into the center of a PDA plate. Similarly, pathogenic mycelial discs of the same size were inoculated into the center of a new PDA plate. The two plates were then inoculated together and sealed with sealing film. A PDA plate inoculated with pathogens and an uninoculated pure PDA plate was used as a control. Each treatment was repeated 5 times. After culturing at 28°C in the dark for 7 days, the diameter of the pathogen colonies was observed and measured. The colony area was then calculated, and the inhibition rate of the antagonistic fungal volatile substances against the pathogens was calculated [(%) = (control colony area - treated colony area) / control colony area × 100%]. The test results showed that the volatile substances of Corning Trichoderma LCTK248 had inhibition rates of 73.1% and 57.6% against Colletotrichum spp. and Fusarium oxysporum, respectively. This preliminarily indicates that the production of volatile substances with antibacterial effects is one of the important mechanisms by which Corning Trichoderma LCTK248 inhibits the growth and sporulation of pathogens.

[0047] Example 4: Biological characteristics and culture condition optimization experiment of Trichoderma Corning LCTK248

[0048] The effects of different temperatures, pH levels, light exposure times, culture media, and C and N sources on the growth of *Trichoderma koningii* LCTK248 were tested. For each biological test, the required culture media were prepared in advance according to the formula, sterilized at 121℃ (0.10~0.12 MPa) for 30 min, and then poured into Φ9cm Petri dishes to prepare agar plates. Simultaneously, *Trichoderma koningii* strain LCTK248 stored at 4℃ was taken out, placed at room temperature for 24 h, and then inoculated onto PDA agar plates. After incubation at 25℃ for 7 days, mycelial cakes were collected from the colonies using a sterilized Φ5mm punch. Each test was performed five times.

[0049] ① Temperature test. Thirteen temperature treatments were set up using PDA agar plates: 0℃, 4℃, 10℃, 15℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 34℃, 37℃, 40℃, and 45℃. After treatment, the cells were incubated in the dark for 4 days. The colony diameter was measured using the cross-sectional method, and the average colony size and growth rate for each treatment were calculated. Colony growth and sporulation were observed and recorded.

[0050] ② pH test. Using PDA culture medium plates, set 10 pH values ​​(pH 4, 5, 6, 7, 8, 9, 10, 11, 12 and pH 13) for treatment. Adjust the pH of the culture medium to the required pH value using 1.0 mol HCl solution and 1.0 mol NaOH solution.

[0051] ③ PDA culture medium plates were used for the light exposure test, and light exposure durations of 0, 4, 8, 12, 18, and 24 h / d were set during the culture period.

[0052] ④ Carbon source experiment. Using NA as the basal medium (control), appropriate amounts of potato starch, corn starch, soluble starch, maltose, glucose, sucrose, lactose, or galactose were added to prepare eight different carbon source media with a carbon source content of 30 g / L. Each treatment was repeated five times.

[0053] ⑤ Nitrogen source test. Using CDA as the basal medium, yeast powder, peptone, ammonium sulfate or ammonium chloride were added to prepare a medium with a nitrogen source content of 2.0 g / L.

[0054] ⑥ Culture medium test. The growth of pathogens on nine culture media, including PDA, PSA, CDA, OA, CMA, NA, PCA, WA and MEA, was tested.

[0055] Except for experiments ① and ③, all other experiments were conducted by observing colony growth after incubation at 25°C in the dark for 4 days. The colony diameter was measured using the cross-cross method, and the average colony diameter and growth rate of each treatment were calculated. The significance of differences between different treatments in the same experiment was tested using Duncan's new multiple range test.

[0056] The results showed that the optimal temperature range for the growth and sporulation of *Trichoderma koningii* LCTK248 was 10–30 °C, with the optimum temperatures being 26 °C and 28 °C. The fungus ceased growth and sporulation at 0 °C and above 37 °C. LCTK248 could grow in the pH range of 4–13, with the optimum pH being 7.0–8.0. LCTK248 grew fastest and most vigorously under complete darkness, and could also grow at 4 h / d, but growth slowed down under 8–24 h / d light. LCTK248 was most suitable for growth and sporulation on media containing glucose and maltose, but grew more slowly on other tested carbon source media (Table 3). Yeast extract and peptone were most suitable for the growth of LCTK248, while the other six nitrogen sources were less suitable (Table 4). LCTK248 could grow on all nine tested media plates, but grew fastest and sporulated most vigorously on PDA and PSA media.

[0057] Table 3. Effects of temperature, pH, and light duration on the growth of LCTK248

[0058]

[0059] Note: Each observation in each experiment is the mean ± standard error of 5 replicates; different capital letters after the value in the same experiment indicate that the difference between treatments is highly significant (P<0.01, Duncan's new multiple range test).

[0060] Table 4. Effects of different carbon and nitrogen sources on the growth of LCTK248

[0061]

[0062] Note: LCTK248 colony diameter was measured after 7 days of culture. Each value is the mean ± standard deviation of 5 replicates. Different lowercase letters indicate significant differences between different carbon or nitrogen source treatments (P<0.05).

Claims

1. A Corning Trichoderma ( Trichoderma koningii The strain LCTK248 is deposited at the General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, with the strain accession number CGMCC No. 41747.

2. The use of the Corning Trichoderma LCTK248 or its conidial liquid as described in claim 1 in inhibiting the growth of plant pathogens or in the preparation of agents to inhibit the growth of plant pathogens, characterized in that, The pathogen is Epicoccus latusicollum , Fusarium oxysporium , Pestalotiopsis microspora , Colletotrichum camelliae , Colletotrichum Fructicola or Colletotrichum fioriniae One or more of them.

3. The use of Corning Trichoderma LCTK248 or its conidial liquid as described in claim 1 in the control of crop diseases or in the preparation of agents for the control of crop diseases, characterized in that, The crop diseases mentioned are one or more of the following: white spot disease of corn, wilt of tobacco, leaf spot of Polygala tenuifolia, anthracnose of tea, anthracnose of grape and macadamia trees, or anthracnose of the medicinal plant Rhizophora micrantha.

4. A biocontrol agent, characterized in that, It contains Corning Trichoderma LCTK248 or its conidial solution as described in claim 1.

5. A method for preventing and controlling crop diseases, characterized in that, The crop diseases mentioned are white spot disease of corn, wilt of tobacco, leaf spot of Polygala tenuifolia, anthracnose of tea, anthracnose of grape and macadamia trees, or anthracnose of medicinal plants such as Trichoderma gramineus. The method mentioned is to apply the conidial solution of Trichoderma gramineus LCTK248 as described in claim 1 to the crop.

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