Trichoderma koningii LCTK248 strain, biocontrol agent and application thereof

Abstract

The invention belongs to the technical field of microbial prevention and control of plant diseases and insect pests, and particularly discloses a Trichoderma koningii strain LCTK248, which is preserved in the General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms of Institute of Microbiology of Chinese Academy of Sciences, and has the preservation number of CGMCC No: 41747. The Trichoderma koningii strain LCTK248 provided by the invention is rapid in growth, large in sporulation quantity, high in yield and high in yield, and can be used for preparing a Trichoderma koningii strain LCTK248. The trichoderma koningii LCTK248 has a very remarkable inhibiting effect on various plant pathogenic bacteria, the bacteriostasis rate reaches 95.1%-99.8%, and the disease control rate reaches 85%-91%, which indicates that the trichoderma koningii LCTK248 has broad-spectrum plant pathogenic fungus resisting activity and has huge biocontrol potential and development and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial control of plant diseases and insect pests, and specifically relates to a biocontrol Trichoderma koningii strain LCTK248 having a highly effective inhibitory effect on plant pathogens such as tea anthracnose bacteria and a biocontrol preparation prepared therefrom. Background Art

[0002] tea tree( Camellia sinensis ),coffee( Arabic coffee ), macadamia nuts( Macadamia three-leaved ),rubber( Brazilian rubber tree ) and rice balls ( White-tailed deer ) are representatives of the Yunnan-Guizhou Plateau's specialty crops, which are widely planted in the area and have important economic value. Colletotrichum Anthracnose, caused by infection with the fungus (Aglaonema spp.), is a major foliar disease that is widespread and severely damaging to all of these crops. Currently, the main preventive measures for anthracnose are strengthening crop cultivation management and chemical control with fungicides. However, the use of chemical fungicides is known to cause problems such as crop residue, ecological pollution, and the development of pesticide resistance in pathogens. Biopesticides offer a promising approach to overcoming the shortcomings of chemical pesticides. While several biocontrol agents have been reported, the long-term use of traditional chemical agents has led to the development of resistance in pathogens, resulting in a decline in control effectiveness. Furthermore, with adjustments in crop structure and environmental changes, new diseases continue to emerge, necessitating the development of new biocontrol agents to address these challenges. These new biocontrol agents often possess stronger antimicrobial activity and a broader antimicrobial spectrum. This research and development can improve disease control effectiveness and reduce pesticide costs. Summary of the Invention

[0003] The present invention has obtained a biocontrol strain LCTK248 with strong inhibitory activity against important crop pathogens such as anthrax, which has a good preventive and control effect on various diseases. It was identified as Trichoderma koningii ( Trichoderma koningii ) LCTK248; it is deposited at the General Microbiology Center of the China General Microbiology Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, under the CGMCC No. 41747. Its pathogenic mechanism, which inhibits the growth and spore formation of pathogens by producing toxins, was discovered. Experimental analysis of its key biological characteristics enabled optimization of its propagation conditions. These findings provide promising strains for the further development of biofungicides for plant disease control and lay the technical foundation for the creation of biopharmaceuticals.

[0004] 1) Discovery and identification of LCTK248: This strain was isolated from a moss growing on tea stems; antagonism assays showed an inhibition rate of 97.36±1.68% (n=5) against Colletotrichum truncatum; morphological and molecular identification revealed that it was a Trichoderma koningii strain. Trichoderma kongingii ).

[0005] 2) Morphological characteristics of LCTK248: The colony grows rapidly on potato dextrose agar plate. o After culturing in the dark at 47°C for 3 days, the colony diameter was 86.5 ± 1.6 mm (n=5), with a growth rate of 27.9 mm / d. The colony was initially round or nearly round, with sparse and transparent hyphae in the vigorous growth area around it; the hyphae in the central area were more developed and white. After 2 days, a large amount of spores were produced, and the color turned blue-green and gradually deepened. Microscopically, the hyphae are light green, septate, and branched at nearly right angles, with a diameter of 1.2-3.1µm (n=30). Conidiophores differentiate from the hyphae, with a diameter of 2.1-4.4µm (n=30). These solitary, septate conidiophores further form bottle-shaped branches (spore-bearing cells), which are light green, 11.6-19.1µm long (n=30), and slightly swollen at the ends to bear conidia. Conidia are mostly elliptical, with a few being round, green, and measuring 4.8-6.2×2.3-4.0µm (n=30).

[0006] 3) The antibacterial effect of LCTK248 on different important crop pathogens: LCTK248 has a strong inhibitory effect on plant pathogens, especially on corn white spot pathogen ( Epicoccus latusicollum ), tobacco wilt pathogen ( Fusarium oxysporum )、Southwestern Polygala leaf spot ( Pestalotiopsis microspora ), Colletotrichum sinensis ( Colletotrichum camelliae ), Macadamia Anthracnose ( C. fructicola ) and Colletotrichum oryzae ( C. Florin ), etc., the inhibition rate of LCTK248 reached 95.1%~99.8%, indicating that LCTK248 has a very strong antibacterial or bactericidal effect on a variety of crop pathogens.

[0007] 4) Safety of LCTK248 in Different Crops: Leaf inoculation results showed that LCTK248 did not infect corn, tobacco, grapes, tea, coffee, or rice. This demonstrates that LCTK248 is safe for disease control in these crops and does not cause phytotoxicity.

[0008] 5) Bactericidal mechanism of LCTK248: The volatile substances of LCTK248 had an inhibition rate of 73.1% and 57.6% against Colletotrichum oleraceus and Fusarium oxysporum, respectively.

[0009] 6) Optimized culture conditions of LCTK248: The optimum temperature range for LCTK248 growth and sporulation is 10-30 o C, the optimum temperature is 26 o C and 28 o C, when 0 o C and above 37 o C, the fungus stopped growing and producing spores; it could grow at pH 4-13, with the optimal pH for growth and spore production being pH 7-8; LCTK248 grew fastest and most vigorously in complete darkness and under 4 h / d of light, but grew very slowly when the light intensity ranged from 8 to 24 h / d; LCTK248 was best suited for growth and spore production on media containing glucose and maltose, but grew more slowly on media containing other carbon sources; yeast powder and peptone were suitable for the growth of LVTK248, while the other two nitrogen sources were less suitable; LCTK248 could grow on all nine culture media tested, but grew fastest and produced spores most vigorously on PDA and PSA.

[0010] The present invention provides a strain of Trichoderma koningii LCTK248, which is classified as: Trichoderma koningii Trichoderma koningii, The accession number is CGMCC NO.41747, deposited in the General Microbiology Center of China Culture Collection Administration.

[0011] The use of the Trichoderma koningii LCTK248 or its conidia liquid in inhibiting the growth of plant pathogens or in preparing a preparation for inhibiting the growth of plant pathogens, wherein the pathogen is E. Epicoccus latusicollum ), Fusarium oxysporum ( Fusarium oxysporum ), Microsporum pseudodiscoideum ( Pestalotiopsis microspora )、Tea Sporangium ( Colletotrichum camelliae ), Colletotrichum spp. ( Colletotrichum. Fructicola ) or Colletotrichum philinii ( Colletotrichum fioriniae ) one or more.

[0012] The use of the Trichoderma koningii LCTK248 or its conidia liquid in preventing and controlling crop diseases or in preparing a preparation for preventing and controlling crop diseases, wherein the pathogen of the crop disease is corn white spot pathogen ( Epicoccus latusicollum ), tobacco wilt pathogen ( Fusarium oxysporum ), Southwestern Polygala leaf spot pathogen ( Pestalotiopsis microspora ), tea tree anthracnose ( Colletotrichum camelliae ), grape and macadamia anthracnose ( Colletotrichum fructicola ) or Colletotrichum oryzae ( Colletotrichum Florin )

[0013] A biocontrol preparation comprises the Trichoderma koningii LCTK248 or its conidia liquid.

[0014] A method for preventing and controlling crop diseases, wherein the pathogen of the crop diseases is corn white spot pathogen ( Epicoccus broad-necked ), tobacco wilt pathogen ( Fusarium oxysporum ), Southwestern Polygala leaf spot pathogen ( Pestalotiopsis microspora ), tea tree anthracnose ( Colletotrichum camelliae ), grape and macadamia anthracnose ( Colletotrichum fructicola ) or Colletotrichum oryzae ( Colletotrichum fioriniae ), the method comprises applying the conidia liquid of Trichoderma koningii LCTK248 to crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 .Trichoderma koningii ( C. koningii ) Morphological characteristics of LCTK248 strain; Note: A.&B. Potato dextrose agar (PDA) medium 25 o C. Front and back of the colony after 48 hours of dark culture; C. Mycelium; D. Conidiophores differentiated from the mycelium; E. Bottle-shaped branches formed at the end of the conidiophores, with conidia forming at the tip of the 'phialopile'; F. LCTK248 produces a large number of conidia under culture conditions.

[0016] Figure 2 PCR-amplified sequences of the ITS1 (top) and TEF1a (bottom) genes of the LCTK248 strain. Figure 3 .Neighbor-joining phylogenetic tree of LCTK248 strain and related fungi; Note: Based on ITS sequences from related strains and TEF1α Gene splicing sequence, neighbor-joining phylogenetic tree constructed using MEGA6.0 software. The scale shows the difference between the splicing sequences of paired species, and the number at each node is the bootstrap support rate (%) that the paired species are the same species.

[0017] Figure 4 .Inhibitory effect of Trichoderma koningii LCTK248 on six plant pathogens; Note: 1) A1, B1, C1, D1, E1 and F1 show the effects of LCTK248 on corn white spot pathogen (Esphaeroides spp. Epicoccus latusicollum ), tobacco wilt pathogen (Fusarium oxysporum Fusarium oxysporum ), Southwestern Polygala leaf spot pathogen (Microsporum polysporum Pestalotiopsis microspora), tea anthracnose fungus (Tea thorn Colletotrichum Colletotrichum camelliae ), grape and macadamia anthracnose (Colletotrichum oleraceus Colletotrichum. fruit-growing ) and Colletotrichum oryzae anthracnose fungi (Colletotrichum philiniae Colletotrichum fioriniae ) of the antibacterial effect; A2, B2, C2, D2, E2 and F2 were the controls for each treatment (the pathogen was cultured opposite to the PDA medium round cake). 2) The inoculation method was to place a 5mm diameter pathogen cake in the center of the plate culture medium, and place a 5mm diameter Corning Trichoderma cake at 20mm symmetrical positions on all four sides of the pathogen cake. 3) After treatment, o C in the dark for 7 days and then observe and photograph.

[0018] Figure 5 Results of a safety (pathogenicity) test of Trichoderma koningii LCTK248 on leaves of different crops; Note: 1) A, B, C, D, E and F are the images of LCTK248 bacterial cake (Φ5 mm) after puncture inoculation at 25 o C) Leaves of corn, tobacco, grapes, tea, coffee, and glutinous rice plants placed under RH>90% and 10 h / d light for 21 days (no infection or disease). 2) Tea plants inoculated with brown spot pathogen (CK1) and glutinous rice plants inoculated with anthracnose pathogen (CK2) served as positive controls. CK1 and CK2 are lesions photographed 7 days after pathogen inoculation. DETAILED DESCRIPTION

[0019] Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention. In the examples, where specific techniques or conditions are not specified, the techniques or conditions described in the literature within the art or the product specifications are used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased. The present invention will be further described in detail below with reference to examples.

[0020] The present invention provides a strain of Trichoderma koningii LCTK248, which is classified as: Trichoderma koningii Trichoderma king's, Deposit number: CGMCC NO.41747, deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit date: January 7, 2025. The biological material was received and registered by the deposit center on January 7, 2025. The viability of the biological material was tested by the deposit center on January 7, 2025, and the result was viable.

[0021] Example 1 Acquisition and Identification of Trichoderma koningii LCTK248 1) Acquisition of LCDK248: Samples of tea rhizosphere soil, tea leaves, and epiphytic mosses were collected from tea gardens in Lincang, Yunnan. These samples were isolated and purified using PDA and NA agar plates, yielding 16 pure culture isolates of fungi and 31 pure culture isolates of bacteria, respectively. Antagonistic activity against Colletotrichum spp., a pathogen causing tea anthracnose, was identified through screening. The screening results showed that LCDK248 exhibited the strongest antagonistic activity against Colletotrichum spp., achieving a rate of 97.36 ± 1.68% (n = 5), significantly higher than that of other antagonistic bacteria and fungi. Further investigation of this fungus was therefore conducted.

[0022] ) Identification of LCTK248: Its species identification was carried out by combining morphology and molecular biology.

[0023] (1) Morphological identification: The strain was inoculated on a 90 mm diameter PDA plate medium and o After culturing in the dark at 47°C for 3 days, the colony morphology was observed, the colony diameter was measured five times, and the colony growth rate (mm / d) was calculated. The shape, color, size, spore production and other characteristics of the colony were described. The characteristics of the fungus were observed and photographed under a microscope. The diameters of 30 hyphae and conidiophores and the sizes of 30 conidia were measured. According to the observed and measured morphological characteristics ( Figure 1 ).

[0024] (2) Morphological characteristics of LCTK248: The colony grew rapidly on potato dextrose agar plate culture medium. After culturing at 25°C in the dark for 3 days, the colony diameter was 86.5 ± 1.6 mm (n=5), and the growth rate was 27.9 mm / d. The colony was initially round or nearly round, with sparse and transparent hyphae in the vigorous growth area around it. The hyphae in the middle area were more developed and white. After 2 days, a large amount of spores were produced, and the color turned blue-green and gradually deepened. Microscopically, the hyphae are light green, septate, and branched at nearly right angles, with a diameter of 1.2-3.1 µm (n=30). Conidiophores differentiate from the hyphae, with a diameter of 2.1-4.4 µm (n=30). These solitary, septate conidiophores further form bottle-shaped branches (spore-bearing cells), which are light green, 11.6-19.1 µm long (n=30), and slightly swollen at the ends to bear conidia. Conidia are mostly elliptical, with a few being round, green, and measuring 4.8-6.2 × 2.3-4.0 µm (n=30).

[0025] Based on the literature of authoritative fungal classification, LCTK248 was initially determined to be a Trichoderma genus ( Trichoderma sp.) fungi.

[0026] Molecular biological identification: Mycelia were scraped from LCTK248 colonies cultured for 5 days on PDA medium, and genomic DNA was extracted from the mycelia using the cetyltrimethylammonium bromide (CTAB) method. ITS and ITS4 (5'-TCCGTAGGTGAACCTGCGG-3' / 5'-TCCTCCGC TTATTGATATGC-3') and ef1 / ef2 (5'-ATG GGTAAGGAGGACAAGAC-3' / 5'- GGAG GTACCAGTGATCATGTT-3') primers were used to amplify the ITS and TEF1-1α The gene sequence fragments were recovered and purified, and the amplified products were sent to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the two sequences were 556 bp long and 609 bp long respectively. Figure 2 ). ITS and TEF1-1α The gene sequences were analyzed by BLAST-n online (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi); the ITS and TEF1-1α Gene sequence, and the phylogenetic tree was constructed using the spliced ​​sequences of the two ( Figure 3 ). The results of molecular biological identification showed that LCTK248 was Trichoderma koningii ( Trichoderma koningii ).

[0027] Example 2 Determination of the antibacterial and disease control effects of LCTK248 Trichoderma koningii and its safety on crops 1) Determination of the antibacterial effect of LCTK248 on different important crop pathogens: The tested plant pathogens include corn white spot pathogen ( Epicoccus latusicollum ), tobacco wilt pathogen ( Fusarium oxysporum ), Southwestern Polygala leaf spot pathogen ( Pestalotiopsis microspora ), tea tree anthracnose ( Colletotrichum camelliae ), Macadamia Anthracnose ( Colletotrichum fructicola )、Anthracnose of rice flower ( Colletotrichum Florin ) and Corning Trichoderma LCTK248. These fungi were inoculated onto PDA culture plates and o After culturing in the dark for 5 days in a constant temperature incubator at 37°C, a 5 mm diameter bacterial cake was punched out using a hole punch. The bacterial cake of each pathogen was placed in the center of a 90 mm diameter culture medium plate, and the bacterial cake of Trichoderma koningii LCTK248 was placed at a distance of 25 mm from the bacterial cake on all four sides ( Figure 4 ); PDA solid culture medium cakes were placed around the pathogen cakes for control, and each treatment was repeated 5 times. oAfter culturing in the dark at 40°C for 7 days, the diameter of the pathogen colony was measured by the cross-cross method, and the colony area was calculated. The inhibition rate of Trichoderma koningii LCTK248 against various pathogens was calculated [(%) = (control colony area - treated colony area) / control colony area × 100%].

[0028] From the test results (Table 1), it can be seen that Corning Trichoderma LCTK248 has a strong inhibitory effect on the growth of 6 plant pathogens, with an inhibition rate of 95.1%~99.8%, which shows that Corning Trichoderma LCTK248 has a very strong antibacterial or bactericidal effect on a variety of crop pathogens.

[0029] Table 1. Antibacterial effect of Trichoderma koningii LCTK248 on 6 crop pathogens Note: Colony diameter and area are the mean ± standard error of five replicates. The inhibitory effects of Trichoderma koningii LCTK248 on the six pathogens reached extremely significant levels (P<0.001).

[0030] ) Disease control effect of Trichoderma corningii LCTK248: Prepare healthy potted tea seedlings, macadamia seedlings and rice ball seedlings in the greenhouse; use LCTK248 cultured on PDA flat medium for 14 days to prepare a concentration of about 10 8 spores / mL of Trichoderma liquid, use the liquid to spray the leaves of three kinds of plants evenly. 24 hours after the spraying of Trichoderma leaves, use the spore suspension of tea thorn spore, fruit thorn spore and Ferrinia thorn spore (about 10 6 Tea, macadamia, and rice spore seedlings were sprayed foliarly with the pathogen spore solution (spores / mL). Negative controls were sprayed with the pathogen spore solution alone, while positive controls were sprayed with difenoconazole solution 24 hours later. Ten pots of plants were replicated for each treatment. After treatment, the seedlings were covered with black cloth to block light and maintain moisture for 24 hours. Thereafter, regular watering and moisturizing were performed. After 21 days, the disease severity (%) was recorded and the disease control rate (%) was calculated.

[0031] The test results (Table 2) showed that the control efficacy of LCTK248 Trichoderma liquid against anthracnose of tea trees, macadamia nuts and rice flowers was 85.2%~88.5%, which was close to the control efficacy of 40% difenoconazole suspension concentrate (SA) (91.7%~95.3%).

[0032] Table 2. The control effect of LCTK248 Trichoderma on anthracnose of three crops 3) Safety test of Trichoderma koningii LCTK248 on different crops: The tested crops include corn ( Corn ), ordinary cigarettes ( Nicotiana tabacum ),Grape( Wine grapevine ),Tea( Camellia sinensis ), Arabica coffee( Coffee bean ) and rice balls ( White-tailed deer Healthy plant leaves from the field were collected and inoculated with Trichoderma koningii LCTK248 using the in vitro graft inoculation method. The collected fresh leaves were placed on moist filter paper on a porcelain plate, with five leaves from each plant placed. Several micropores were pierced at four locations on both sides of the vein of each leaf using a flame-sterilized sewing needle, with a Trichoderma koningii LCTK248 cake placed at each location. For the positive control, tea leaves were inoculated with brown spot pathogen ( Epicoccum sorghum ) mushroom cakes and inoculation of anthracnose fungi on rice flower leaves ( Colletotrichum Florin ) bacterial cake, cover the inoculation tray with plastic wrap to keep it moist. After inoculation, place the leaves in saturated humidity and dark conditions for 24 hours, and then o C, RH>90% and 10 h / d light in a growth chamber, with appropriate watering daily to maintain moisture. After 7 days of cultivation, the positive control was photographed, and after 21 days, the leaves treated with Trichoderma koningii LCTK248 were observed and photographed.

[0033] Test results ( Figure 5 ) showed that typical lesions formed on the leaves of two plants inoculated with the pathogen (positive control) 7 days after inoculation; negative control leaves treated with PDA culture medium cake (not shown) and leaves of various plants treated with T. koningii LCTK248 remained healthy 21 days after treatment, with no infection or disease observed. This indicates that T. koningii LCTK248 is safe for these crops and will not cause "phytotoxicity" in future applications.

[0034] Example 3 Analysis of the antibacterial mechanism of Trichoderma koningii LCTK248 Colletotrichum oleraceus and Fusarium oxysporum were selected as pathogens for testing. The inhibitory effects of volatile metabolites of LCTK248 on the pathogens were evaluated using a double-plate interleaving method. Colonies cultured at 27°C in the dark for 5 days were obtained. A cake (d = 8 mm) from the edge of the colony was inoculated in the center of a polydimethylsiloxane (PDA) plate. A similar cake of pathogens of the same size was inoculated in the center of a new PDA plate. The two plates were interleaved and sealed with parafilm. An uninoculated PDA plate was placed on the inoculated PDA plate as a control. Each treatment was replicated five times. After 7 days of incubation at 28°C in the dark, the diameter of the pathogen colonies was measured, and the colony area was calculated from this. The inhibitory rate of the volatile compounds against the pathogens was then calculated [(%) = (control colony area - treated colony area) / control colony area × 100%]. The test results showed that the volatile substances of Trichoderma corningii LCTK248 had an antibacterial rate of 73.1% and 57.6% against Colletotrichum oleraceus and Fusarium oxysporum, respectively. This preliminarily indicates that the production of volatile substances with antibacterial effects is one of the important mechanisms of Trichoderma corningii LCTK248 to inhibit the growth and spore production of pathogens.

[0035] Example 4 Biological characteristics of Trichoderma koningii LCTK248 and optimization of culture conditions The effects of varying temperature, pH, photoperiod, culture medium, and carbon and nitrogen sources on the growth of Trichoderma koningii LCTK248 were tested. For each biological test, the required culture medium was prepared according to the recipe, sterilized at 121°C (0.10-0.12 MPa) for 30 minutes, and then poured into 9 cm Petri dishes to prepare culture plates. LCTK248 Trichoderma strains stored at 4°C were removed from a refrigerator, left at room temperature for 24 hours, and then inoculated onto PDA plates. After incubation at 25°C for 7 days, the plates were punched out using a sterile 5 mm borer. Each experimental treatment was repeated five times.

[0036] ① Temperature test. Using PDA culture plates, set 13 temperature treatments: 0°C, 4°C, 10°C, 15°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 34°C, 37°C, 40°C, and 45°C. After incubation in the dark for 4 days, colony diameters were measured using the cross-hatch method. The average colony diameter and growth rate for each treatment were calculated. Colony growth and sporulation were also observed and recorded.

[0037] ②Acidity test. Use a PDA culture medium plate and set 10 pH values: pH 4, 5, 6, 7, 8, 9, 10, 11, 12, and pH 13. Adjust the pH of the culture medium to the desired value using 1.0 M HCl solution and 1.0 M NaOH solution.

[0038] ③ The light experiment used PDA culture medium plates, and set the light duration treatments of 0, 4, 8, 12, 18, and 24 h / d during the culture period.

[0039] ④ Carbon source test. Eight different carbon source media were prepared using NA as the basal medium (control) and added with appropriate amounts of potato starch, corn starch, soluble starch, maltose, glucose, sucrose, lactose, or galactose. Each carbon source content was 30 g / L. Each treatment was repeated five times.

[0040] ⑤ Nitrogen source test. CDA was used as the basal culture medium. Yeast powder, peptone, ammonium sulfate, or ammonium chloride were added to prepare a culture medium with a nitrogen source content of 2.0 g / L.

[0041] ⑥ Culture medium test. Test the growth of pathogens on 9 culture media including PDA, PSA, CDA, OA, CMA, NA, PCA, WA and MEA.

[0042] Except for experiments ① and ③, all other experiments were conducted by observing the growth of colonies after culturing in the dark at 25°C for 4 days. The colony diameters were measured using the cross method, and the mean colony diameter and growth rate of each treatment were calculated. The Duncan's new multiple range method was used to test the significance of differences between different treatments in the same experiment.

[0043] Analysis of the results showed that the optimum temperature range for the growth and sporulation of T. koningii LCTK248 was 10–30°C, with the optimum temperatures being 26°C and 28°C. Growth and sporulation ceased at temperatures above 0°C and above 37°C. LCTK248 grew at a pH range of 4–13, with an optimum pH of 7.0–8.0. LCTK248 grew fastest and most vigorously in complete darkness and was also able to grow at 4 h / d, but growth slowed at light levels of 8–24 h / d. LCTK248 was best suited for growth and sporulation on media containing glucose and maltose, but grew more slowly on other carbon sources tested (Table 3). Yeast extract and peptone were most suitable for LCTK248 growth, while the other six nitrogen sources were less suitable (Table 4). LCTK248 grew on all nine culture media tested, but grew fastest and produced the most sporulation on PDA and PSA.

[0044] Table 3. Effects of temperature, pH and photoperiod on the growth of LCTK248 Note: Each observation value of each experiment is the mean ± standard error of 5 replicates; different capital letters after the values ​​in the same experiment indicate that the differences between treatments are extremely significant (P < 0.01, Duncan's new multiple range test).

[0045] Table 4. Effects of different carbon and nitrogen sources on the growth of LCTK248 Note: The colony diameter of LCTK248 was measured after culturing for 7 days. Each value is the mean ± standard deviation of 5 replicates. Different lowercase letters indicate significant differences among the treatments with different carbon or nitrogen sources (P<0.05).

Claims

1. A Trichoderma koningii Trichoderma koningii ) strain LCTK248, which is deposited in the General Microbiology Center of China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, with strain deposit number CGMCC No.: 41747.

2. The Konningia spp. LCTK248 strain and its extracts in claim 1 are effective against corn white spot pathogen ( Epicoccus latusicollum ), tobacco wilt pathogen ( Fusarium oxysporium )、Southwestern Polygala leaf spot ( Pestalotiopsis microspora ), tea tree anthracnose ( Colletotrichum camelliae ), Macadamia Anthracnose ( Colletotrichum fructicola ) or Colletotrichum oryzae ( Colletotrichum fioriniae ) has a very high inhibitory effect. Under inoculation conditions, it has a very good control effect on anthracnose of tea trees, macadamia nuts and rice flowers.

3. The optimal culture scheme of the Konningia spp. LCTK248 strain in claim 1 is: using potato dextrose agar (PDA) or potato sucrose agar (PSA) with a pH of pH 7.0 to pH 8.0, at 26 o C~28 o C in the dark, with glucose and maltose as the optimal carbon sources and peptone and yeast powder as the optimal nitrogen sources. Under these conditions, the LCTK248 strain grew rapidly and produced spores vigorously.

4. The volatile substances of the K. corningii LCTK248 strain in claim 1 have an inhibition rate of 73.1% and 57.6% against Colletotrichum oleraceus and Fusarium oxysporum, respectively. This preliminarily indicates that the production of volatile substances with antibacterial effects is one of the important mechanisms of K. corningii LCTK248 to inhibit the growth and spore production of pathogens.

5. The conidia liquid of Trichoderma corningii LCTK248 in claim 1 has a control effect of 85.2% to 88.5% on anthracnose of tea trees, macadamia nuts and rice flowers, which is approximately lower than the control effect of 40% difenoconazole suspension concentrate of 91.7% to 95.3%.

6. Using the corn dermatophyte LCTK248 strain or its effective volatile substance biological preparation in claim 1 as a foliar spray can effectively control corn leaf spot, tobacco wilt, southwestern polygala leaf spot and various plant anthracnose diseases. This is a green prevention and control technology for crop fungal diseases.

7. Use of the Trichoderma koningii LCTK248 or its conidia liquid according to claim 1 in inhibiting the growth of plant pathogens or in preparing a preparation for inhibiting the growth of plant pathogens, characterized in that: The pathogen is E. Epicoccus latusicollum ), Fusarium oxysporum ( Fusarium oxysporium ), Microsporum pseudodiscoideum ( Pestalotiopsis microspora )、Tea Sporangium ( Colletotrichum camelliae ), Colletotrichum spp. ( Colletotrichum. Fructicola ) or Colletotrichum philinii ( Colletotrichum fioriniae ) one or more.

8. Use of Trichoderma koningii LCTK248 or its conidia liquid according to claim 1 in preventing and controlling crop diseases or in preparing a preparation for preventing and controlling crop diseases, characterized in that: The pathogen of the crop disease is corn white spot pathogen ( Epicoccus latusicollum ), tobacco wilt pathogen ( Fusarium oxysporium ), Southwestern Polygala leaf spot pathogen ( Pestalotiopsis microspora ), tea tree anthracnose ( Colletotrichum camelliae ), grape and macadamia anthracnose ( Colletotrichum. fructicola ) or Colletotrichum oryzae ( Colletotrichum fioriniae ) 9. A biocontrol agent, characterized in that The invention comprises the Trichoderma koningii LCTK248 or its conidia liquid according to claim 1.

10. A method for preventing and controlling crop diseases, characterized in that: The pathogen of the crop disease is corn white spot pathogen ( Epicoccus latusicollum ), tobacco wilt pathogen ( Fusarium oxysporium ), Southwestern Polygala leaf spot pathogen ( Pestalotiopsis microspora ), tea tree anthracnose ( Colletotrichum camelliae ), grape and macadamia anthracnose ( Colletotrichum. fructicola ) or Colletotrichum oryzae ( Colletotrichum fioriniae ), the method comprises applying the conidia liquid of Trichoderma koningii LCTK248 according to claim 1 to crops.

Images