A long-branch Trichoderma CM5J-9 strain antagonistic to strawberry diseases and its screening method and application

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to a strain of Trichoderma longibrachiatum CM5J-9 that is highly effective in antagonizing strawberry diseases, and its screening method and application. The Trichoderma longibrachiatum CM5J-9 is deposited with the CGMCC NO.41989 on May 30, 2025, at the General Microbiology Center of the China Culture Collection Administration, and is classified as: Trichoderma longibrachiatum Trichoderma longibrachiatum . The long-branch Trichoderma CM5J-9 screened by the present invention has a significant antagonistic effect on the main pathogens in the soil of continuous strawberry planting, and has a significant preventive and control effect on strawberry-related diseases caused by them. The long-branch Trichoderma CM5J-9 provided by the present invention is isolated from the soil of continuous strawberry planting, has good environmental adaptability and disease prevention effect, and has a large spore production amount, and has great application potential.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a Trichoderma longibrachiatum CM5J-9 strain antagonistic to strawberry diseases, a screening method and an application thereof. Background Art

[0002] Strawberry (Fragaria × ananassa Duch.) is a popular fruit variety for its attractive color, aroma, and nutritional value. However, continuous strawberry cropping is subject to a complex diversity of soil pathogens, which are prone to recurring outbreaks under high-humidity, high-density planting conditions. To prevent disease outbreaks, regular application of multiple chemical pesticides is often necessary. This can lead to long-term food safety and environmental pollution issues, and can also cause pathogens to develop varying degrees of drug resistance. There is an urgent need to isolate biocontrol bacteria with broad-spectrum resistance to different strawberry pathogens to achieve green control of strawberry diseases.

[0003] Trichoderma, an important biocontrol microorganism, possesses multiple mechanisms of action, including pathogen antagonism and plant resistance induction, and is a major registered fungal biopesticide species. Currently, limited research has been conducted on the screening of in situ biocontrol agents for key pathogens in strawberry-growing areas, and the effectiveness of Trichoderma agents in controlling these diseases remains to be improved. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention provides a long-branch Trichoderma CM5J-9 that is highly effective in antagonizing strawberry diseases.

[0005] The present invention also provides a method for screening the Trichoderma longibrachiata CM5J-9.

[0006] Another object of the present invention is to provide the use of the above-mentioned Trichoderma longibrachiatum CM5J-9 in preventing and controlling strawberry diseases.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows:

[0008] The present invention provides a long-branch Trichoderma CM5J-9 that is highly effective in antagonizing strawberry diseases. The long-branch Trichoderma CM5J-9 has a deposit number of CGMCC NO.41989 and was deposited in the General Microbiology Center of the China Culture Collection Administration on May 30, 2025. It is classified and named as: long-branch Trichoderma Trichoderma longibrachiatum .

[0009] Preferably, the sequence of ITS amplification and sequencing of the Trichoderma longibrachiata CM5J-9 is shown as SEQ ID NO.1.

[0010] The present invention also provides a method for screening the above-mentioned Trichoderma longibrachiatum CM5J-9, comprising the following steps:

[0011] (1) The soil samples were diluted in sterile water, and then spread on Bengal red medium. The spread medium plates were cultured, and hyphae at the edge of the colonies of different morphologies were picked and inoculated into PDA medium for purification;

[0012] (2) The purified strain was subjected to a plate confrontation experiment with Siamese anthrax to obtain the target strain.

[0013] Preferably, in step (1), the soil sample is collected from a continuously cropped strawberry greenhouse, and the loosely attached soil within 2-3 cm from the strawberry roots is collected.

[0014] Preferably, in step (1), the specific dilution multiple of the gradient dilution is: 10 2 , 10 3 , 10 4 .

[0015] Preferably, in step (1), the culture is carried out in a constant temperature incubator at 28°C for 3 days.

[0016] The present invention also provides the use of the long-branch Trichoderma CM5J-9 in preventing and controlling diseases of continuously cropped strawberries.

[0017] The beneficial effects of the present invention are:

[0018] (1) The long-branch Trichoderma CM5J-9 obtained by the present invention has a significant antagonistic effect on the main pathogens in the soil of continuous strawberry planting, and has a significant preventive and control effect on strawberry-related diseases caused by them; the results of relevant tests show that the control rate of the strain provided by the present invention for related diseases can reach 100%.

[0019] (2) The strain Trichoderma longibrachiatum CM5J-9 provided by the present invention is isolated from strawberry continuous cropping soil, can adapt well to the soil microbial environment, and colonize in large quantities in the strawberry rhizosphere; it can reduce the amount of chemical pesticides used, which is beneficial to the pollution-free production of strawberries.

[0020] (3) The strain provided by the present invention has a large spore production capacity and has great application potential.

[0021] Preservation Information

[0022] Deposit date: May 30, 2025;

[0023] Depository: General Microbiology Center, China Culture Collection Administration;

[0024] Deposit number: CGMCC NO. 41989;

[0025] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0026] Postal code: 100101;

[0027] Classification and nomenclature: Trichoderma longifolia Trichoderma longibrachiatum . BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Plate confrontation test between CM5J-9 and Anthracnose bacteria (back of the plate); A. Anthracnose bacteria; b. CMCM5J-9 vs. Anthracnose bacteria confrontation image;

[0029] Figure 2 The colony morphology of CM5J-9 cultured on a PDA plate for 5 days; A is the front side of the colony; B is the back side of the colony;

[0030] Figure 3 is the microscopic structure of the CM5J-9 strain;

[0031] Figure 4 This is a plate confrontation experiment between the CM5J-9 strain and Aspergillus fumigatus (reverse side of the plate); A. Aspergillus fumigatus; B. CMCM5J-9 vs. Aspergillus fumigatus confrontation diagram;

[0032] Figure 5 Plate confrontation test between CM5J-9 and Mortierella (reverse side of plate); A. Mortierella; B. Confrontation picture of CM5J-9 vs. Mortierella;

[0033] Figure 6 This is a plate confrontation test between CM5J-9 and Fusarium fujikura (reverse side of the plate); A. Fusarium fujikura; B. CMCM5J-9 vs. Fusarium fujikura confrontation picture;

[0034] Figure 7 The control effect of CM5J-9 strain on Siamese anthracnose disease is shown in Figure 1. A is the treatment group inoculated with Siamese anthracnose alone; B is the group inoculated with Trichoderma longifolia CM5J-9; and C is the water control group.

[0035] Figure 8 The control effect of CM5J-9 strain on Aspergillus fumigatus disease; A is the treatment group inoculated with Aspergillus fumigatus alone; B is the group inoculated with Trichoderma longibrachiatus CM5J-9; C is the water control group;

[0036] Figure 9 Figure 1 is a graph showing the control effect of CM5J-9 strain on Mortierella disease; A is the treatment group inoculated with Mortierella alone; B is the group inoculated with Trichoderma longibrachiatum CM5J-9; C is the water control group;

[0037] Figure 10This is a diagram showing the control effect of the CM5J-9 strain on Fujikura Fusarium disease; among them, A is the treatment group inoculated only with Fujikura Fusarium; B is the group inoculated with long-branch Trichoderma CM5J-9; C is the clear water control group. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0039] Unless otherwise specified, the culture medium used in the present invention is a conventional culture medium in the art.

[0040] Example 1

[0041] 1. Screening of antagonistic bacteria

[0042] In a strawberry greenhouse that has been planted for 7 consecutive years, the complete strawberry root system and attached soil were collected, loose large pieces of soil were removed, and loose attached soil within 2-3 cm from the roots was collected by gently shaking the roots. 1 g of soil sample was diluted in sterile water to a dilution factor of 10. 2 , 10 3 , 10 4 , then 100 µl of each strain was spread onto Bengal Red Medium. The coated plates were incubated at 28°C. Starting on the third day, hyphae from the edges of the colonies of varying morphology were selected and inoculated onto PDA medium for purification. The purification process for each strain was repeated three times to obtain pure strains, which served as candidate strains for screening antagonistic bacteria.

[0043] The composition of the bengal culture medium (g / L) is shown in Table 1.

[0044] Table 1

[0045]

[0046] In the greenhouse under the same conditions as above, diseased strawberry roots were collected, and soil samples were collected, graded diluted, plated, and cultured in the same manner as above. The dominant colonies were selected and inoculated into PDA medium. Purification was performed three times to obtain strains for pathogen identification. Each test strain was inoculated into the roots of potted strawberries using the root wound method. The main pathogen strain was identified according to Koch's postulates. Combining morphological characteristics and ITS sequence alignment, it was identified as Siamese anthracnose ( Colletotrichum siamense ).

[0047] Antagonistic bacteria were screened from a candidate bacterial library using a plate standoff assay with Colletotrichum siamese. After activating the candidate antagonist and pathogen, 5 mm bacterial cakes were placed on either side of a PDA plate, 1 cm from the edge. A control plate containing only the pathogen served as the pathogen cake. Three replicates were set up for each antagonistic group. Both the standoff and control plates were incubated at 28°C until the control plate was about to touch the edge. The radius of each pathogen was measured, and the growth inhibition rate was calculated using the following formula:

[0048]

[0049] The strains with antagonistic effects on anthrax were screened and the CM5J-9 strain had the highest plate inhibition rate against the strawberry pathogen Colletotrichum siamese, which was 74.98±0.33% ( Figure 1 (A, B in Chinese).

[0050] 2. Identification of CM5J-9 strain

[0051] Morphologically, the fungus can produce a large number of conidia evenly distributed throughout the colony when cultured on PDA medium at 28℃ for 5 days. The mycelial layer is thin and there are few aerial hyphae. The conidia are dark green and velvety ( Figure 2 A), the reverse side of the colony is light yellow due to the yellow diffusible pigment produced ( Figure 2 The conidiophores of this fungus have a strongly developed central axis, from which solitary phialides arise towards the apex. As they extend further from the apex, secondary branches develop. Phialides arise directly from these secondary branches and are rarely arranged in a whorl. Phialides are often slender, slightly curved, and sometimes ampullate or columnar. Conidia are green, single-celled, and mostly oval ( Figure 3 ).

[0052] The ITS4 and ITS5 primers were used to amplify the ITS region of the bacterial genome. The primer pair sequences are:

[0053] ITS4 (SEQ ID NO.2): 5'-TCCTCCGCTTATTGATATGC-3';

[0054] ITS5 (SEQ ID NO. 3): 5'-GGAAGTAAAAGTCGTAACAAGG-3'.

[0055] The PCR amplification system consisted of 25 μl of 2× HiFi PCR Master Mix, 2 μl of ITS4 primer, 2 μl of ITS5 primer, 1 μl of gDNA, and ddH2O added to 50 μl. The PCR cycle parameters were: initial denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min (35 cycles total); extension at 72°C for 10 min; and storage at 4°C. A 1.0% agarose gel was then prepared and electrophoresed at 18 V / cm for 20 min. The agarose gel was stained with a nucleic acid dye and photographed using a UV gel imaging system. The target fragment was recovered from the agarose gel using a standard agarose gel DNA recovery kit, and the recovered product was sequenced. The sequence of the ITS amplified product from the CM5J-9 strain is shown in SEQ ID NO. 1. Based on the morphological characteristics and ITS sequence comparison results in Genbank, the strain CM5J-9 was identified as Trichoderma longibrachiatum. Trichoderma longibrachiatum This fungus is deposited in the General Microbiology Center of China Culture Collection Committee under the number CGMCC NO.41989. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Postal Code: 100101. Taxonomic name: Trichoderma longibrachiatum Trichoderma longibrachiatum .

[0056] The ITS amplified sequencing sequence of the CM5J-9 strain (as shown in SEQ ID NO.1) (635 bp) is:.

[0057] Example 2 Antagonistic Effects of Trichoderma longibrachiatum CM5J-9 on Different Strawberry Pathogens

[0058] Different pathogenic pure strains were inoculated into strawberries using the root wound method, and their pathogenicity was verified according to Koch's postulates, including Aspergillus fumigatus ( Aspergillus fumigatus )、Mortierella Actinomortierella ambigua ) and Fusarium fujikura ( Fusarium fujikuroi ).

[0059] After all strains were activated by plate culture, a 5 mm bacterial cake was placed at the edge of the colony. The CM5J-9 strain and each pathogen were placed on either side of a PDA plate, 1 cm from the edge, and incubated at 28°C. A plate containing only the pathogen cake served as a control. Three replicates were set for each treatment. After the control plate was incubated until it almost touched the edge, the pathogen radius of the control and opposite treatment groups was measured, and the pathogen growth inhibition rate was calculated. The calculation formula is as follows:

[0060]

[0061] The results showed that the long-branch Trichoderma CM5J-9 strain had a significant inhibitory effect on Aspergillus fumigatus, inhibiting its growth and having a heavy parasitic effect, with an inhibition rate of 75.41±0.61% ( Figure 4 (A, B in Chinese).

[0062] The results showed that the long-branch Trichoderma CM5J-9 strain had a significant inhibitory effect on Mortierella, producing an obvious transparent zone with an inhibition rate of 78.42±0.37% ( Figure 5 (A, B in Chinese).

[0063] The results showed that the long-branch Trichoderma CM5J-9 strain had a significant inhibitory effect on Fusarium fujikura, inhibiting its growth and having a heavy parasitic effect, with an inhibition rate of 68.64±0.52% ( Figure 6 (A, B in Chinese).

[0064] Example 3 Control effect of Trichoderma longibrachiatum CM5J-9 on different strawberry diseases

[0065] The Tianbao (also known as Zhangji) strawberry variety, which has a large planting area, was selected as the test plant. Plants with consistent growth were selected for potted plant experiments on the control effect. The soil used in the potted plants was the soil used in strawberry greenhouses. First, a conidia suspension of the CM5J-9 strain was prepared. The strain was cultured on PDA medium for 7 to 10 days. The conidia were blown off with sterile water supplemented with 0.05% Tween 80. After filtering to remove the hyphae, a 1×10 suspension was prepared by cell counting. 7 CFU / mL of conidia suspension. Prepare 1×10 6 CFU / mL of spore suspension of the above pathogens.

[0066] The bacterial suspension was inoculated using the root wound inoculation method. Prior to inoculation, the roots were slightly wounded with a sterilized razor blade. The bacterial suspension was then evenly inoculated at the base of the strawberry stem. Moistened sterile cotton wool was used to absorb the bacterial suspension and maintain moisture. Three treatment groups were set up, each with six replicates. Pathogen treatment (T1): 5 mL of pathogen spore suspension alone was inoculated. CM5J-9 + pathogen treatment (T2): 5 mL of CM5J-9 conidia suspension was inoculated 3 days later, followed by 5 mL of pathogen spore suspension. The water control (CK) was inoculated with 5 mL of sterilized water containing 0.05% Tween 80. Plants in each treatment group were placed in separate seedling boxes, maintained at high temperature and high humidity, and cultured under a 13.5-hour photoperiod and 10.5-hour dark cycle. The disease status of the plants was observed every day. After all bacterial solutions were inoculated and cultured for 14 days, the disease index of the strawberry plants under each treatment was calculated according to the "Guidelines for Field Efficacy Tests of Pesticides" GB / T 17980.119-2004, and the disease control effect was calculated.

[0067] The calculation formula is control effect (%) = (T1-T2) / (T1-CK)×100.

[0068] (1) The control effect of Trichoderma longifolia CM5J-9 on diseases caused by anthracnose

[0069] The results show that if Figure 7 As shown in Figure C, all strawberry plants in the water control group (CK) showed no disease symptoms. Figure 7 As shown in Figure A, the treatment group (T1) inoculated only with Siamese anthracnose developed black spots at the base of the stem within 8 days, and all leaves wilted or rotted within 10-12 days. Figure 7 As shown in Figure B, after inoculation with Trichoderma longibrachiatum CM5J-9 (T2), the pathological symptoms at the base of the stem were significantly alleviated, and the leaves showed no rot or significant wilting symptoms, with a disease control efficacy of 55.49%.

[0070] (2) The control effect of Trichoderma longifolia CM5J-9 on diseases caused by Aspergillus fumigatus

[0071] The results show that if Figure 8 As shown in Figure C, all strawberry plants in the water control (CK) showed no disease symptoms. Figure 8 As shown in middle A, the treatment group (T1) inoculated only with Aspergillus fumigatus developed large areas of rust on the leaves within 10-14 days, and flaky brown-red dot-like particles appeared on the cross section of the stem base. Figure 8 As shown in Figure B, after inoculation with Trichoderma longibrachiatum CM5J-9 (T2), the lesion symptoms at the stem base were significantly alleviated, the symptoms on the leaves were reduced, and the disease control efficacy was 57.15%.

[0072] (3) The control effect of Trichoderma longifolia CM5J-9 on diseases caused by Mortierella

[0073] The results show that if Figure 9 As shown in Figure C, all strawberry plants in the water control (CK) showed no disease symptoms. Figure 9 As shown in Figure A, in the treatment group (T1) inoculated only with Mortierella, the vascular bundles at the base of the stem turned yellow, dense brown spots appeared on the cut surface, and most of the leaves of the plant dried up on the 14th day. Figure 9 As shown in Figure B, after inoculation with Trichoderma longibrachiatum CM5J-9 (T2), the lesion symptoms at the base of the stem were significantly alleviated, and the leaves showed no rot or significant wilting symptoms, with a disease control efficacy of 76.40%.

[0074] (4) The control effect of Trichoderma longibrachiatum CM5J-9 on diseases caused by Fusarium fujikura

[0075] The results show that if Figure 10 As shown in C, all strawberry plants in the water control group showed no disease symptoms. Figure 10 As shown in Figure A, the treatment group (T1) inoculated only with Fusarium fujikura developed disease symptoms within 7 days, and obvious dark brown spots appeared on the stem section 14 days after inoculation. Figure 10 As shown in B, after inoculation with Trichoderma longibrachiatum CM5J-9 (T2), there were no lesions on the stem section and the plants were in a healthy state, with a disease control effect of 100%.

Claims

1. A strain of Trichoderma longifolia that is antagonistic to strawberry diseases ( Trichoderma longibrachiatum )CM5J-9, characterized in that, The long branch Trichoderma CM5J-9 has a deposit number of CGMCC NO.41989, and was deposited in the General Microbiology Center of China Culture Collection Administration on May 30, 2025; The sequence of the ITS amplified and sequenced Trichoderma longibrachiata CM5J-9 is shown in SEQ ID NO.

1.

2. An application of the long-branch Trichoderma CM5J-9 according to claim 1 in preventing and treating diseases of continuously cropped strawberries, characterized in that: The strawberry diseases are related diseases caused by Siamese anthracnose, Aspergillus fumigatus or Fusarium fujikura.

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