Isolation and application of plant immune-activated dse fungi

Abstract

The present application relates to the field of microbial technology, in particular to a method for screening and application of plant immune activation DSE fungi, the present application is based on "directional trapping + mixed bacteria inhibition + symbiotic determination" separation technology, the isolation rate and new species discovery rate of DSE fungi are 28 times and 7 times higher than that of conventional direct isolation method respectively, the exploration of strain resources is greatly improved, and the inhibition ability to mixed bacteria is improved by improving the separation medium, which further improves the fungal isolation rate; In addition, the DSE strain with plant immune activation function is screened by using strain-cucumber symbiotic determination, whole plant dry weight evaluation method, combined with cucumber wilt in vivo plate determination and other comprehensive evaluation methods, to replace in vitro with in vivo, to replace direct inhibition of pathogenic bacteria with indirect activation of plant immunity, to break through the limitation of traditional antagonistic method only for specific pathogenic bacteria, to realize efficient identification of broad-spectrum eliciting strains, and to provide new ideas and methods for screening of biocontrol microorganisms.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the isolation and screening methods and applications of plant immune-activated DSE fungi. Background Technology

[0002] Dark septate endophytes (DSEs) are a type of soil fungi that can colonize plants. As important plant symbiotic microorganisms, they play a crucial role in enhancing plant growth, development, and innate immunity. Plants are home to a rich diversity of endophytic fungi, but many are potential or opportunistic pathogens or saprophytes. Beneficial DSEs belong to a "low abundance, high function" group, and most are difficult to culture. Therefore, conventional direct isolation methods for DSEs from plant tissues or soil often fail due to their low abundance, slow growth under artificial culture conditions, and weak niche competition. They are frequently overtaken and inhibited by rapidly growing contaminating microorganisms, resulting in low isolation rates and significant time, manpower, material, and financial costs. Therefore, there is an urgent need to establish an efficient DSE isolation technology.

[0003] Soil is the primary habitat and source of DSE fungi. Commonly, direct isolation methods are used to isolate DSE fungi from soil, such as diluting soil leachate and streaking or spreading it on an isolation medium, or directly isolating them from plant roots. In previous experiments, the isolation rate of DSE using the conventional direct root isolation method was only 0.26%. However, our trapping method—using bait plants to trap soil fungi around the rhizosphere of the target plant, and then isolating endophytic fungi from the roots of the bait plants—achieved an isolation rate of 7.7% without improving the culture medium or optimizing isolation conditions. This trapping method increased the isolation rate by approximately 28 times and the new species discovery rate by 7 times compared to the conventional direct isolation method. Both the DSE isolation rate and the fungal discovery efforts were significantly improved. However, bait plants have varying sensitivities to DSE fungi; different bait plants have different trapping abilities for DSE, leading to different DSE isolation rates when different plants are used. Furthermore, different plant species attract DSE fungi with different characteristics, and specific host plants may enrich certain types of DSE fungi. In addition to using DSE-sensitive bait plants, it is also necessary to adjust the nutrients and culture conditions of the isolation medium to provide suitable nutrition for DSE, inhibit the growth of highly abundant dominant bacteria, and buy time and space for DSE growth, thereby improving the isolation rate of this type of microorganism.

[0004] Currently, traditional screening of biocontrol bacteria is mostly based on the antagonistic effect of biocontrol strains on pathogens. This involves using an in vitro confrontation culture method, where pathogens and test strains are simultaneously inoculated onto culture plates to evaluate the inhibitory effect of the test strain on the pathogen. This method relies on the culturability of the pathogen, but it cannot screen for unculturable diseases such as viruses and citrus Huanglongbing (HLB), limiting its effectiveness to specific pathogens. Furthermore, due to the widespread use of antagonistic screening methods, most of the biocontrol strains obtained so far are concentrated in common species such as Trichoderma, Streptomyces, and Bacillus, leading to increasing homogenization of biocontrol microorganisms and limiting the discovery of novel biocontrol microbial resources. Our research group discovered that DSE biocontrol bacteria can activate plant immunity but have no direct inhibitory effect on pathogen growth. Using traditional antagonistic screening methods would miss these biocontrol microorganisms with unique disease control mechanisms. Therefore, it is urgent to establish a screening system based on plant immune activation as an indicator to fully explore and utilize these special biocontrol resources. Since plant immune-activating strains typically exhibit good and broad-spectrum control efficacy against a variety of plant diseases, the discovery and utilization of DSE fungi with this function is expected to significantly broaden their application scope and enhance their application potential. Summary of the Invention

[0005] In view of the above, it is necessary to develop a method and application for isolating and screening plant immune-activating DSE fungi, which can screen out DSE strains with broad-spectrum immune-activating activity against a variety of crops, thereby enabling DSE fungi to overcome the barriers of crop species and achieve broad-spectrum disease resistance against a variety of crop diseases.

[0006] A method for isolating and screening plant immune-activating DSE fungi, wherein the isolation and screening method is as follows: (1) Obtaining cucumber roots: After germinating cucumber seeds, they were sown in soil samples for cultivation; (2) Isolation and purification of strains: After cleaning and disinfecting the roots of cucumber seedlings, they were placed in 1 / 2 PSA medium or modified 1 / 2 CM medium for culture. After the colonies grew, they were promptly transferred to PDA medium for culture to obtain purified strains. (3) By re-inoculation and microscopic examination of roots, strains that coexist with cucumbers were screened. The purified strains obtained in step (2) were co-cultured with cucumber plants on petri dishes for 14 days to form the experimental group. The coexistence of the strains with cucumbers in the experimental group was investigated. Cucumber plants without inoculation were used as the control group. The roots of cucumber plants were washed and dried to determine the dry weight of the whole plant. (4) Compare the dry weight of the whole cucumber plant in the experimental group and the control group in step (3), and select the experimental group strains whose dry weight of the whole cucumber plant is not significantly different from that of the control group or significantly higher than that of the control group as candidate immune-activating DSE fungi. (5) The candidate immune-activated DSE fungi from step (4) are evaluated for immune activation in an indoor in vivo plate for cucumber wilt disease, thus obtaining the immune-activated DSE fungi.

[0007] Furthermore, the 1 / 2PSA medium consists of 100 g / L potato, 10 g / L sucrose, and 15 g / L agar; the modified 1 / 2CM medium consists of 8.5 g / L corn flour, 15 g / L agar powder, and 10 g / L monk fruit polysaccharide.

[0008] Furthermore, the monk fruit polysaccharide was obtained by water extraction and alcohol precipitation.

[0009] Furthermore, in step (2), the culture temperature of the 1 / 2 PSA medium or the modified 1 / 2 CM medium is 20°C.

[0010] Furthermore, the immune activation evaluation method is as follows: after co-culturing the candidate strain with cucumber for 7 days, the symbiont along with the culture medium is transferred to a water agar medium containing the pathogen. After the control group cucumber seedlings show obvious disease, the disease grade of the plants is investigated, and the disease index and control effect are calculated. The grading standards for the plant disease grade are: Grade 0: no symptoms; Grade 1: the area of ​​yellowing or wilting of true leaves and cotyledons does not exceed 50% of the total area; Grade 2: the area of ​​yellowing or wilting of true leaves and cotyledons exceeds 50% of the total area; Grade 3: the leaves wilt or die, only the growing point survives; Grade 4: the whole plant is severely wilted, to the point of death. The disease index is calculated as follows: Disease index = [∑(number of diseased plants at each level × corresponding grade) / (total number of plants investigated × highest representative value)] × 100; The control effect is calculated as follows: Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100%; strains with a control effect >0.0% were selected as immune-activated DSE fungi.

[0011] Furthermore, in step (4), the experimental group strains with a significantly higher whole-plant dry weight than the control group are preferred as candidate immune-activating DSE fungi; in step (5), strains with a control effect ≥50.0% are preferred as excellent immune-activating DSE fungi.

[0012] Furthermore, the plant immune-activating DSE fungi screened by the isolation and screening method are strains of the genus *Cladosporium* (…). Cladophialophora impure LC3 and / or *Cladosporium guilloché* strains ( Cladophialophora guangxiense HX2; the strain of *Cladosporium* ( Cladophialophora impureThe accession number for LC3 is CGMCC NO. 41540, the depositary institution is the China General Microbiological Culture Collection Center, the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is October 12, 2024; the described *Cladosporium guilloché* strain (…) Cladophialophora guangxiense HX2 has the accession number CGMCC NO. 41498, the depositary institution is the China General Microbiological Culture Collection Center, the depositary address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is September 5, 2024.

[0013] This invention also includes the application of plant immune-activated DSE fungi isolated by the isolation and screening method in enhancing the plant's resistance to powdery mildew of bitter gourd, downy mildew of cucumber, and / or wilt of cucumber.

[0014] The present invention has the following beneficial effects.

[0015] 1. This invention, based on the "directional trapping + contaminant inhibition + symbiotic assay" isolation technology, significantly improves the isolation rate of DSE fungi compared to traditional methods. Targeted trapping using host plants specifically enriches DSE fungi that infect plant roots, making it easier to enrich symbiotic strains colonizing plant roots. The trapping method increases the isolation rate by approximately 28 times compared to conventional methods. To further improve the isolation rate, we combined oligotrophic culture medium and low-temperature culture to effectively inhibit the growth of contaminants during isolation. Symbiotic assays confirmed the acquisition of DSE fungi that mutually benefit from plant symbiosis, solving the isolation problem caused by low abundance and contaminant interference of DSE fungi. In subsequent experiments, the applicant used this method to screen and obtain 26 broad-spectrum growth-promoting and antagonistic strains, achieving efficient identification of novel biocontrol strains.

[0016] 2. The research group innovatively proposed the concept of "plant immune activation" DSE fungi. These DSE fungi do not have a significant antagonistic effect on pathogens, but they can enhance plant immunity by activating plant immunity, thereby improving the plant's broad-spectrum disease resistance. This approach uses live fungi instead of in vitro fungi and indirectly activates plant immunity instead of directly inhibiting pathogens, overcoming the limitations of traditional antagonistic methods that only target specific pathogens. The screening and discovery of these DSE fungi was made after the research group solved the problem of the difficulty in isolating DSE fungi due to their low abundance. In different plant trapping experiments, the research group found that cucumber is a good host for these DSE fungi. Plant trapping methods using cucumber as bait can effectively capture these DSE fungi. After trapping with cucumber plants, a screening system for these DSE fungi was established by combining strain-cucumber symbiosis assays and whole-plant dry weight evaluation methods, successfully selecting DSE fungi that have a mutualistic symbiotic relationship with plants. In the screening of immune-activated strains, live organisms were used instead of in vitro ones, shifting the approach from directly inhibiting pathogen growth to activating the plant's own immunity. The assessment of the plant's live immune activation capacity replaced the traditional in vitro antagonistic screening, overcoming the limitations of traditional methods that only target specific pathogens and achieving efficient identification of broad-spectrum resistant strains. Subsequent validation through plate antagonism experiments and pot experiments revealed that while this type of DSE fungus itself and its metabolites had no significant inhibitory effect on pathogens, it exhibited significant resistance to multiple diseases in pot experiments. This indicates that this type of DSE fungus enhances the plant's own immune activity and thus increases its resistance to diseases. The establishment of this screening method and system provides a new approach and pathway for subsequent screening of plant immune-activated DSE fungi, and it is a reproducible strain screening method.

[0017] 3. Based on the technical approach of inhibiting contaminating bacteria in oligotrophic culture media with different sugar sources, the research group also improved the isolation rate of endophytic fungi by modifying the 1 / 2CM culture medium, providing a new and efficient screening culture medium for the isolation of endophytic fungi.

[0018] Information on the preservation of biological materials.

[0019] The strain preservation information for LC3 of this application is: Cladosporium (Cladophialophora immunda) LC3, its classification name is: Cladophialophora unclean LC3, Chinese classification name: Cladosporium LC3, accession number: CGMCC NO. 41540; depositary institution: China General Microbiological Culture Collection Center; deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: October 12, 2024.

[0020] The strain preservation information for HX2 in this application is: *Cladosporium guangxiense*. (Cladophialophora guangxiense) HX2, its classification name is: Cladophialophora guangxienseHX2, Chinese classification name: Guangxi Cladosporium HX2, accession number: CGMCC NO. 41498, depositary institution: China General Microbiological Culture Collection Center; deposit address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: September 5, 2024. Detailed Implementation

[0021] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0022] Unless otherwise stated, each feature disclosed in this specification is merely one example of a series of equivalent or similar features.

[0023] Example 1.

[0024] This embodiment studies the isolation rate of soil plant endophytic fungi using the "directed trapping + contaminant inhibition + symbiotic assay" separation technology, as detailed below.

[0025] In our previous isolation experiments, DSE fungi were usually obtained by direct isolation from the root system. This method had a very low isolation rate; in previous experiments, our direct root isolation method typically achieved a DSE isolation rate of only 0.26%. However, by using a trapping method—that is, using bait plants to trap the rhizosphere soil fungi of the plant to be isolated, and then isolating the DSE from the roots of the bait plants—the isolation rate reached 7.7% without improving the culture medium or optimizing the isolation conditions. The trapping method improved the isolation rate by approximately 28 times and the new species discovery rate by 7 times compared to the conventional method. Both the isolation rate and the fungal discovery efforts were significantly improved. To further improve the isolation rate of the trapping method, we considered optimizing the isolation culture medium and isolation environment to further enhance the isolation rate. The optimization scheme is as follows.

[0026] 1. Obtaining cucumber roots: After disinfecting the cucumber seeds, rinse them with sterile water, place them on sterile filter paper to dry, and then transfer them to 90 mm pure agar medium plates. After germination in a dark environment in a 28 ℃ incubator, sow them in culture cups containing soil samples and harvest them after 30 days of cultivation.

[0027] 2. Comparison of the isolation effect of endophytic fungi in different culture media of root system: The root system of cucumber seedlings was rinsed clean under running water and disinfected by shaking with 0.005% Tween 20 and sterile water for 1 minute each time. The root system was then placed on 8 different culture media in Table 1 and cultured at 28 ℃. After the colonies grew, they were promptly transferred to PDA medium to obtain purified strains.

[0028]

[0029] The endophytic fungi of cucumber roots were isolated and cultured using the eight culture media listed in Table 1. The isolation rate of endophytic fungi was calculated using the following formula: Isolation rate (IR) = (Number of tissue samples from which endophytic fungi were isolated / Total number of tissue blocks from which isolated samples were obtained) × 100%.

[0030] The results are shown in Table 2 below.

[0031]

[0032] As shown in Table 2, 1 / 2 PSA medium exhibited the best isolation effect for endophytic fungi, with an isolation rate of 100.00%, followed by 1 / 2 CM and 1 / 2 PDA mediums. The isolation effects of the other mediums were relatively poor. This indicates that oligotrophic media are more conducive to the isolation of endophytic fungi. In such oligotrophic media, it is difficult for other bacteria to grow, but endophytic fungi are more likely to accumulate.

[0033] Therefore, it can be seen that by enriching cucumber new root systems, using oligonutrient (1 / 2 PSA medium) and low temperature (20 ℃) ​​culture to inhibit contaminating bacteria, the isolation rate of endophytic fungi can reach 100%.

[0034] 3. Optimization of Culture Medium: In the above experiments, we found that oligotrophic media significantly improved the isolation rate of endophytic fungi. The only difference between 1 / 2 PSA and 1 / 2 PDA was the type of sugar used, yet their isolation rates differed greatly. To find more efficient isolation media, considering our experimental needs and costs, we considered modifying the 1 / 2 CM medium to obtain another highly efficient medium for isolating DSE fungi. Specifically, this involved adding sugars or polysaccharides to the 1 / 2 CM medium to improve the isolation rate of endophytic fungi. In our technical research, we found that plant polysaccharides have a certain inhibitory effect on microorganisms. If we can find a polysaccharide that can effectively inhibit the growth of miscellaneous bacteria without inhibiting DSE fungi, we can greatly improve the isolation rate of DSE fungi. Therefore, we selected glucose, sucrose, aloe polysaccharide, and monk fruit polysaccharide for our experiments. Glucose and sucrose were purchased. Aloe polysaccharide and monk fruit polysaccharide were obtained by water extraction and alcohol precipitation: fresh aloe vera was crushed and fresh monk fruit was pulverized. They were then mixed with water at a solid-liquid ratio of 1:10, boiled to 100 °C and held for 15 min, then cooled to 80 °C and extracted for 2 h. The filtrate was filtered and concentrated to 1 / 10 of the original solution. After removing protein, 3 times the volume of 95% ethanol was added to make the final ethanol concentration reach 70%. The mixture was left to stand overnight at 4 °C and the precipitate was collected by centrifugation to obtain aloe polysaccharide and monk fruit polysaccharide. The test culture medium was prepared according to the ratio in Table 3.

[0035]

[0036] The four culture media listed in Table 3 were used to isolate and culture DSE fungi from cucumber roots. The isolation rate was calculated as above, and the results are shown in Table 4 below.

[0037]

[0038] As shown in Table 4, among the modified 1 / 2CM media with different sugars, only the addition of monk fruit polysaccharide resulted in a 100% isolation rate of endophytic fungi. The isolation rates of modified media 1 (with glucose added), modified media 2 (with sucrose added), and modified media 3 (with aloe polysaccharide added) were all lower than that of the 1 / 2CM media in Table 2 (87.33%). Therefore, it can be concluded that in the 1 / 2CM media, only the addition of monk fruit polysaccharide can effectively inhibit the enrichment of DSE fungi by other bacteria (modified media 4). Based on the above experiments, we can consider prioritizing 1 / 2 PSA media or 1 / 2CM media modified with monk fruit polysaccharide for the isolation of endophytic fungi from cucumber roots.

[0039] 4. Isolation of endophytic fungi in cucumber roots: After disinfecting cucumber roots, place them on 1 / 2 PSA medium or modified medium 4 and culture at 20 ℃. After the colonies grow, transfer them to PDA medium in time to obtain purified strains.

[0040] 5. Screening and Validation of Cucumber Symbiotic DSE Strains: Strains symbiotic with cucumber were screened through re-inoculation and root microscopic examination. The tested strains were inoculated onto oat medium (2.5 g / L oat flour, 7.5 g / L agar), with 3 inoculation points per dish, and cultured at 28 ℃ for 7-10 days. Seeds with white sprouts were transferred to the surface of colonies (1 seed per colony) and placed in culture flasks (5 replicates per strain, with a control without inoculation). After 14 days of culture, the symbiotic relationship was investigated under a microscope. Plants symbiotic with cucumber were selected as candidate plants, while plants without endophytic bacteria were used as controls. After washing the roots of the culture medium, the roots were dried at 55 ℃ for 48 h, and the dry weight was measured. Statistical analysis of the dry weight results was used as the judgment criterion.

[0041] The specific screening results for this symbiotic strain are shown in Table 5.

[0042]

[0043] As shown in Table 5, among the 13 endophytic fungi, two strains, HX2 and LC3, had significantly higher dry weights than the control group; two strains, GH15 and X21, had slightly higher dry weights than the control group (without significant difference); one strain, LZ67, had slightly lower dry weights than the control group (but without significant difference); and two strains, LC12 and SS17, had significantly lower dry weights than the control group. Microscopic examination of the roots revealed dark-colored septate hyphae or microsclerotia colonizing the intercellular spaces or cells of the roots. Therefore, these strains are DSE strains capable of establishing a symbiotic relationship with cucumber.

[0044] Example 2.

[0045] This example illustrates the in vivo screening of plant immune-activating DSE fungi, as detailed below.

[0046] 1. Using a cucumber wilt disease model to screen for immunologically active strains, the specific method was as follows: Strains with detectable dry weight from Table 5 were inoculated onto oat medium (MgSO4·7H2O 1.0 g, KH2PO4 1.5 g, NaNO3 1.0 g, oat flour 10 g, agar powder 11 g, distilled water 1000 mL), with 3 bacterial blocks (Ф=5 mm) per plate. These were then incubated in a dark incubator at 28 ℃ for 7 days. Cucumber seeds were surface-sterilized (refer to 1), germinated on pure agar medium until bud break, and then transplanted onto the bacterial colonies. These were then co-cultured in tissue culture flasks under the following conditions: temperature 28 ℃, light intensity 180 μmol / m². -2 s -1 The photoperiod was 16 h / d, with no inoculation serving as the control group. The cucumber wilt pathogen (FOC) was inoculated onto water agar medium, three inoculated blocks per dish, and cultured at 28 ℃ for 4 days. After co-culturing cucumber seedlings with the strain for 7 days, the symbiont and culture medium were transferred to water agar medium containing the pathogen and cultured in a light incubator for another 10-14 days. Once the control group cucumber seedlings showed obvious disease, the disease severity was assessed, the disease index was calculated, and the control effect was determined. The roots of the cucumber seedlings were washed, dried in a 55 ℃ oven, and their dry weight was measured.

[0047] Disease severity grading criteria: Grade 0: No symptoms; Grade 1: Yellowing or wilting of true leaves and cotyledons does not exceed 50% of the total area; Grade 2: Yellowing or wilting of true leaves and cotyledons exceeds 50% of the total area; Grade 3: Leaves wilt or die, only the growing point survives; Grade 4: The entire plant is severely wilted, to the point of death.

[0048] Disease index = [∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level representative value)] × 100.

[0049] Prevention and control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100%, and the results are shown in Table 6.

[0050]

[0051] As shown in Table 6, only strains GH15, HX2, LC3, and X21 could activate cucumber immunity, with control efficacy ranging from 15.51% to 92.82%. Among them, LC3 showed the best control efficacy, reaching 92.82%, followed by strain HX2. Other strains, LZ67, LC12, and SS17, had no control ability against cucumber wilt and even slightly accelerated the course of the disease. Combining this with Example 2, we found that although strain LZ67 had a slightly lower dry weight than the control group (the difference was not significant), it had no control effect against cucumber wilt. Fungi with significantly higher dry weights than the control group (LC3 and HX2) showed significantly better control efficacy against cucumber wilt than fungi with similar dry weights to the control group (GH15 and X21).

[0052] To further investigate the mechanism of action of the above-mentioned disease-controlling strains GH15, HX2, LC3 and X21 against cucumber wilt, we mainly studied whether the strains had an antagonistic effect on the pathogen of cucumber wilt or whether the metabolites had an inhibitory effect on cucumber wilt. We conducted in vitro antagonism experiments and antibacterial experiments of fermentation filtrate.

[0053] 1. The specific method for the in vitro antagonism experiment is as follows: Agar blocks containing bacterial colonies were prepared using a 5 mm aperture sampler and inoculated into a region 2 cm from the center of PDA medium. After 7 days of cultivation, pathogenic bacterial discs were inoculated at the same symmetrical distance. The experiment included a single-strain culture control group, with the test strain and the pathogen inoculated separately. The culture was carried out at 28 ℃ until the mycelia of the pathogenic control group plates completely covered the plates. The inhibition rate of the test strain against the pathogen was then measured, and the formula for the inhibition rate is as follows.

[0054] Inhibition rate (%) = [(Control colony diameter - Experimental group colony diameter) / Control group colony diameter] × 100%.

[0055] The tested strain was inoculated into PDB liquid medium and cultured at 28 ℃ and 120 r / min for 10 days with shaking. After removing mycelia by filtration with sterile gauze, the culture broth was filtered through a sterile filter membrane to obtain sterile fermentation filtrate. The fermentation filtrate was mixed with PDA medium (melted at approximately 50 ℃) at a ratio of 1:3 (v / v) to prepare PDA culture plates containing the fermentation filtrate. Pathogen mycelial discs with a diameter of 0.5 mm were inoculated into the center of the plates, with 6 replicates for each treatment. The plates were cultured at 28 ℃ until the pathogen control colonies filled the plates. The inhibition rate of the DSE strain against the pathogen was measured and calculated. The results are shown in Table 7.

[0056]

[0057] As shown in Table 7, the inhibition rates of the disease-resistant strains GH15, HX2, LC3, and X21 against the pathogen of cucumber wilt were very low, close to 0. This indicates that the disease-resistant strains GH15, HX2, LC3, and X21 screened out by us have no direct inhibitory effect on the pathogen of cucumber wilt.

[0058] 2. Antibacterial experiment of fermentation filtrate: The pathogen of cucumber wilt was inoculated on PDA plates containing fermentation filtrate of the disease-resistant strains GH15, HX2, LC3 and X21 and cultured for 10 days. The colony size of the experimental group and the blank control group (CK) was measured. The inhibition rate of the fermentation filtrate against the pathogen was calculated according to the above formula. The results are shown in Table 8.

[0059]

[0060] As shown in Table 8, the fermentation broths of the disease-resistant strains GH15, HX2, LC3, and X21 showed very low inhibition rates against the pathogen of cucumber wilt, close to 0. This indicates that the metabolites of the disease-resistant strains GH15, HX2, LC3, and X21 that we screened out have no inhibitory effect on the pathogen of cucumber wilt.

[0061] Therefore, in this embodiment, we found that the disease-resistant strains GH15, HX2, LC3, and X21 themselves have no antagonistic effect on the pathogen of cucumber wilt, and the fermentation broth has no inhibitory effect on the pathogen of cucumber wilt. However, they have a significant control effect on susceptible plants of cucumber wilt. This indicates that the disease-resistant strains GH15, HX2, LC3, and X21 improve the disease control effect against cucumber wilt by activating the plant's own immune activity, rather than by inhibiting the pathogen.

[0062] Example 3.

[0063] To further verify the immune activation ability of DSE disease-resistant strains GH15, HX2, LC3, and X21 in controlling different plant diseases, we conducted a broad-spectrum verification of the DSE strains in pot experiments for different disease control. The specific plan was as follows: disease-resistant strains GH15, HX2, LC3, and X21 were selected and inoculated into different plants to conduct pot experiments for different diseases, and strains with symbiotic immune activation ability on different plants were screened out, as detailed below.

[0064] (1) The effect of immune-activated strains on the control of cucumber wilt in potted plants.

[0065] (2) Preparation of bacterial suspension: The bacterial strains were inoculated into PDB medium and cultured on a shaker at 28 ℃ and 120 r / min for 10 days. The mycelium was collected by filtration through double-layer sterile gauze, washed three times with sterile water, squeezed dry, and then homogenized for 1 min with an appropriate amount of sterile water to prepare a suspension with a concentration of 5×10⁻⁶. 5 CFU / mL mycelial suspension.

[0066] Cucumber seeds were germinated at 30℃, then soaked in a bacterial suspension for 0.5 h, and sown in 10×10 cm seedling cups filled with seedling substrate. Each cup was then watered with 30 mL of bacterial solution, and the cups were covered with a film to maintain humidity indoors. After 10 days of cultivation, the seeds were transplanted into 19×17×20 cm pots and placed in a greenhouse for further cultivation. The pathogen, Fusarium, was activated and prepared at a concentration of 1×10⁻⁶. 6 cfu·mL -1 Fusarium spores were used to inoculate bitter gourd seedlings with Fusarium spores through root injury. Maintaining suitable temperature and humidity, the disease severity of the plants was assessed, and the disease index and control efficacy were calculated after significant disease development was observed in the control group of cucumber seedlings.

[0067] Disease severity grading criteria: Grade 0: No symptoms; Grade 1: Yellowing or wilting of true leaves and cotyledons does not exceed 50% of the total area; Grade 2: Yellowing or wilting of true leaves and cotyledons exceeds 50% of the total area; Grade 3: Leaves wilt or die, only the growing point survives; Grade 4: The entire plant is severely wilted, to the point of death.

[0068] Disease index = [∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level representative value)] × 100.

[0069] Prevention and control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100%. The results are shown in Table 9.

[0070]

[0071] As shown in Table 9, the disease-resistant strains GH15, HX2, LC3, and X21 all showed control efficacy against cucumber wilt under potted conditions. Among them, strain LC3 showed the highest control efficacy, reaching 87.88%, followed by strain HX2 with a control efficacy of 70.39%. However, the control efficacy was lower than that in the plate experiment, indicating that the potted environment and the plate environment have different effects on plants, and the plants have relatively higher immunity under the plate environment.

[0072] (2) The effect of immune-activated strains on potted control of cucumber downy mildew.

[0073] Preparation of bacterial suspension: as shown above.

[0074] Cucumber seeds were germinated at 30 ℃, then soaked in a bacterial suspension for 30 min, and sown in 10×10 cm seedling cups filled with seedling substrate. Each cup was then watered with 30 mL of bacterial solution. The cups were covered with a film to maintain humidity indoors, and after emergence, the film was removed and the seedlings were moved outdoors. After 10 days of cultivation, the seedlings were transplanted into 19×17×20 cm pots and placed in a greenhouse for natural disease development. Once the control group of cucumber seedlings showed obvious signs of disease, the disease severity was assessed, and the disease index and control efficacy were calculated.

[0075] Disease severity grading standards: Grade 0: No disease symptoms on leaves; Grade 1: Lesions cover less than 5% of the total leaf area; Grade 3: Lesions cover 6% to 10% of the total leaf area; Grade 5: Lesions cover 11% to 25% of the total leaf area; Grade 7: Lesions cover 26% to 50% of the total leaf area; Grade 9: Lesions cover more than 50% of the total leaf area.

[0076] Disease index = [∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level representative value)] × 100.

[0077] Prevention and control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100%. The results are shown in Table 10.

[0078]

[0079] As shown in Table 10, strains GH15, HX2, LC3, and X21 all showed control effects against cucumber downy mildew in potted plants under potted conditions. Strain HX2 had the highest control efficacy, reaching 81.37%, followed by strain LC3 with a control efficacy of 70.67%. The lowest control efficacy was observed in strains GH15 and X21, which also showed some control effects against cucumber downy mildew in potted plants, but the control effect was very poor, below 10%, and their effects were not significant compared to strains LC3 and HX2.

[0080] (3) The effect of immune-activated strains on the prevention and control of powdery mildew in potted bitter gourd.

[0081] Preparation of bacterial suspension: as shown above.

[0082] Bitter gourd seeds were germinated at 30℃. After sprouting, they were soaked in mycelial suspension for 30 minutes and sown in 10 cm × 10 cm nutrient cups containing seedling substrate. Each cup was then watered with 30 mL of mycelial solution. Water was used as a control. One seedling was left per cup, with 4 cups per treatment. When 4–6 true leaves appeared, the seeds were transferred to 19 × 17 × 20 cm pots and placed in a greenhouse for natural cultivation. After 10 days, approximately 1 × 10⁻⁶ seeds were used as a pre-treatment. 5 Spray a suspension of powdery mildew fungus (cfu / mL) on both sides of the leaves until the leaves are moist but not dripping wet.

[0083] After the control group had fully developed the disease, the disease severity was assessed. Fifteen leaves were examined from bottom to top on each plant, and the disease index was calculated. The disease severity grading standards are as follows: Grade 0: No powdery mildew on the entire leaf; Grade 1: A small amount of powdery mildew, with the affected area less than 1 / 3 of the leaf area; Grade 2: The affected area covers 1 / 3-2 / 3 of the leaf area, with obvious powdery mildew; Grade 3: The affected areas gradually merge into patches, covering more than 2 / 3 of the leaf area; Grade 4: Powdery mildew covers the entire leaf, and the powdery mildew falls off when the leaf is touched; Grade 5: Powdery mildew covers the entire leaf, the leaf begins to yellow, and gradually withers. The calculation methods for the disease index and control effectiveness are as follows.

[0084] Disease index = [∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level representative value)] × 100.

[0085] Prevention and control effect (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100%. The results are shown in Table 11.

[0086]

[0087] As shown in Table 11, strains GH15, HX2, LC3, and X21 all showed control efficacy against powdery mildew in bitter gourd under pot cultivation conditions. Among them, strain HX2 had the highest control efficacy of 75.18%, followed by strain LC3 with 74.02%. The control efficacy of both strains was above 70%, and the difference in control efficacy between the two strains was not significant. However, the control efficacy of strains HX21 and GH15 was significantly higher than that of strains X21 and GH15. Although strains X21 and GH15 showed control efficacy against powdery mildew in bitter gourd, their effects were not as significant as those of strains LC3 and HX2.

[0088] (4) The effect of immune-activated strains on the prevention and control of bacterial wilt in tomato plants.

[0089] Preparation of bacterial suspension: as shown above.

[0090] After tomato seeds were germinated, plump seeds with uniform sprout length were selected. These seeds were soaked in a bacterial suspension for 30 minutes, with an equal volume of sterile water used as a control (CK). Seeds were sown in seedling trays, and each seedling was inoculated with 30 mL of bacterial suspension as root-setting water. The trays were then placed on light-controlled cultivation racks for 25 days. After 25 days, the seedlings were transplanted into soil containing two-thirds of the soil infected with tomato bacterial wilt and placed in a greenhouse (temperature 30±5℃, relative humidity 80±5%, natural light). Disease incidence was assessed on day 45 after transplanting, and the incidence rate, disease index, and control efficacy were calculated. The tomato bacterial wilt disease index grading standards are as follows: Grade 0: No wilting symptoms; Grade 1: 1%-25% leaf wilting; Grade 2: 26%-50% leaf wilting; Grade 3: 51%-75% leaf wilting; Grade 4: 76%-100% leaf wilting. The formulas for incidence rate, disease index, and control efficacy are as follows.

[0091] Disease index = [∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level representative value)] × 100.

[0092] Prevention and control effect (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100%. The results are shown in Table 12.

[0093]

[0094] As shown in Table 12, strains GH15, HX2, LC3, and X21 all showed control efficacy against bacterial wilt of tomato under pot conditions. The best control efficacy was observed in strain HX2 at 73.06%, followed by strain LC3 at 45.22%. Strains GH15 and X21 also showed control efficacy against bacterial wilt of tomato in pots, but the control effect was very poor, below 10%, and the effect was not significant compared to strains LC3 and HX2.

[0095] In summary, in pot experiments on various plant diseases, we found that DSE fungi that can coexist with cucumbers and have a higher dry weight than the control group can control powdery mildew of bitter gourd, bacterial wilt of tomato, downy mildew of cucumber, and wilt of cucumber through seed treatment. Compared with the control group, the strains (HX2 and LC3) that significantly increased the dry weight of cucumber (P<0.05) showed a significant control effect on powdery mildew of bitter gourd, bacterial wilt of tomato, downy mildew of cucumber, and wilt of cucumber (the control efficacy can reach about 70%), and have field promotion value. Although the strains (GH15 and X21) that did not significantly increase the dry weight of cucumber have a certain control effect on these diseases, the effect is significantly lower than that of strains HX2 and LC3.

[0096] Furthermore, the research group demonstrated through plate confrontation experiments that strains LC3 and HX2 are effective against the pathogen of bacterial wilt in tomato. (Ralstonia solanacearum) No antibacterial activity was observed. The five-point inoculation method using detached leaves was used to test the antibacterial activity of strains LC3 and HX2 against the powdery mildew pathogen of bitter gourd. (Podosphaera xanthii) and cucumber downy mildew pathogen (Pseudoperonospora cubensis) The absence of antagonistic effects further demonstrates that the control efficacy of strains LC3 and HX2 against cucumber wilt, bitter gourd powdery mildew, tomato bacterial wilt, and cucumber downy mildew is due to the significant enhancement of plant immune activity rather than inhibition of pathogens. Increased plant immunity enhances resistance to common diseases, thus broadening the range of disease resistance and better suiting plant growth characteristics. In terms of control efficacy against these diseases, this type of DSE fungus exhibits the highest efficacy against cucumber wilt. Therefore, we can use the cucumber wilt susceptibility model as a preliminary screening model for resistance to this type of DSE fungus to more accurately obtain "plant immune-activated" strains.

[0097] The results of this study indicate that DSE fungi screened through cucumber root trapping, isolation, screening, and immune activation evaluation, if they have significant symbiotic effects with plants, can achieve disease resistance and prevention by coexisting with the host plant and activating the plant's own immunity. Therefore, they have excellent broad-spectrum activity. However, these DSE fungi and their secondary derivatives do not have a direct antibacterial effect on pathogens.

[0098] Therefore, we can preliminarily summarize the evaluation method for isolating, screening, and isolating DSE fungi that activate plant immunity as follows:

[0099] (1) Obtaining cucumber roots: After disinfecting the surface of cucumber seeds, rinse them with sterile water, dry them on sterile filter paper, and then transfer them to a 90 mm pure agar medium plate. Germinate them in a dark environment at 28℃ for 60 h until they show white sprouts. Then, sow them in a culture cup containing soil samples and harvest them after 30 days of cultivation.

[0100] (2) Isolation and purification of endophytic fungal strains: The roots of cucumber seedlings were collected and rinsed clean under running water. They were then disinfected by shaking with 0.005% Tween 20 and sterile water for 1 minute each time. The roots were then placed on 1 / 2 PSA medium and cultured at 20°C. After the colonies grew, they were promptly transferred to PDA medium for further culture to obtain purified strains.

[0101] (3) DSE strains symbiotic with cucumber were screened by re-inoculation and root microscopic examination. The test strains were then inoculated on oat medium (2.5 g / L oat flour, 7.5 g / L agar), with 3 inoculations per plate, and cultured at 28℃ for 7-10 days. Seeds with white spots were transferred to the surface of colonies (1 seed per colony) and placed in culture bottles (5 replicates per strain, with a control without inoculation). After 14 days of culture, the symbiotic relationship between the strains and cucumbers in the experimental group was investigated. Cucumber plants without inoculation were used as the control group. The roots of the cucumber plants were washed and the dry weight of the whole plant was measured and the colonization structure of the roots was examined under a microscope.

[0102] (4) Compare the dry weight of the whole cucumber plant in the experimental group and the control group in step (3), and select the experimental group strains whose dry weight of the whole cucumber plant is not significantly different from that of the control group or significantly higher than that of the control group as candidate immune-activated DSE fungi.

[0103] (5) The candidate immune-activated DSE fungi in step (4) were evaluated for immune activation in indoor petri dishes for cucumber wilt disease, and strains with a control effect of >0.0% on cucumber wilt disease were selected as immune-activated DSE fungi.

[0104] A better option is: in step (4), the experimental group strains with a significantly higher whole plant dry weight than the control group are selected as candidate immune-activated DSE fungi; in step (5), the strains with a control effect of ≥50.0% are selected as excellent immune-activated DSE fungi.

[0105] Example 4.

[0106] Based on the research in Example 2, this embodiment uses the same method to verify whether DSE fungi isolated from different root systems can isolate endophytic fungi with immune activity. The specific method is as follows.

[0107] 1. Isolation of endophytic fungi from Chinese cabbage roots: Following the method in Example 2, Chinese cabbage seeds were sown in culture cups containing soil samples and harvested after 30 days of culture.

[0108] 2. Isolation and purification of bacterial strains: The roots of cabbage seedlings were collected and rinsed clean under running water to disinfect the surface of the roots. They were then placed on 1 / 2 PSA medium and cultured at 20°C. After the colonies grew, they were promptly transferred to PDA medium to obtain purified bacterial strains.

[0109] 3. Strains symbiotic with Chinese cabbage were screened through re-inoculation and root microscopic examination. The tested strains were then inoculated onto oat medium, with three inoculation points per dish, and cultured at 28℃ for 7-10 days. Seeds with white sprouts were transferred to the surface of colonies (one seed per colony) and placed in culture bottles (five replicates per strain, with a control without inoculation). After 14 days of culture, the symbiotic relationship was investigated under a microscope. Plants symbiotic with cucumber were selected as candidate plants, and plants without endophytic fungi served as controls. After washing the roots of the culture medium, the roots were dried at 55℃ for 48 hours, and the dry weight was measured. Statistical analysis of the dry weight results was used as the judgment criterion. The specific endophytic fungi screened are shown in Table 13.

[0110]

[0111] As shown in Table 13, strain XW05 was selected as the candidate endophytic fungus with immune activity according to the selection method of Example 3.

[0112] Referring to the verification method in Example 2, the immunogenicity of candidate strains XW05, DE54, DE32 and SL22 was verified using a plate experiment on cucumber wilt disease. The results are shown in Table 14.

[0113]

[0114] As shown in Table 14, except for strain XW05, strains DE54, DE32, and SL22 showed no control effect against cucumber wilt. To further verify the disease resistance of strain XW05 against other diseases, we conducted pot experiments using strain XW05 against cucumber wilt, cucumber downy mildew, bitter gourd powdery mildew, and tomato bacterial wilt. The specific methods and steps are as described in Example 3, and the results are shown in Table 15.

[0115]

[0116] As shown in Table 15, strain XW05 has a certain control effect on cucumber wilt, but only 7.10%; the control effect on cucumber downy mildew, bitter gourd powdery mildew and tomato bacterial wilt is not significant. Inoculation with certain pathogens may even aggravate the disease. This indicates that using Chinese cabbage as a trapping plant cannot effectively screen out DSE fungi with immune activity. It suggests that cucumber roots are a targeted trapping plant for this type of "plant immune activation" endophytic fungi.

[0117] In summary, this invention, based on the isolation technology of "directional trapping + inhibition of other bacteria + symbiosis assay," significantly improves the isolation rate of endophytic fungi compared to traditional methods. It also proposes the concept of "plant immune activation" endophytic fungi. Through a comprehensive evaluation method combining cucumber plant trapping, strain-cucumber symbiosis assay, whole-plant dry weight evaluation, and cucumber wilt disease plate model, this type of endophytic fungi was successfully screened. Verification showed that this type of endophytic fungi does not rely on the antagonistic effect of the strain against specific pathogens to achieve disease resistance, but rather enhances the plant's own immunity to achieve disease resistance. Pot experiments verified that this type of endophytic fungi overcomes the limitations of traditional antagonistic screening techniques and has a strong broad-spectrum disease resistance effect against plant diseases. Furthermore, the applicant has improved the 1 / 2 cm medium, providing a new medium for the efficient isolation of endophytic fungi and offering new ideas and methods for subsequent efficient isolation of endophytic fungi.

[0118] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Plant immune activation DSE fungi for Phialophora clavis Cladophialophora immunda ) LC3 and / or Phialophora guangxiensis Cladophialophora guangxiense ) HX2 in the resistance to bitter gourd powdery mildew, cucumber downy mildew and / or cucumber fusarium wilt. The said branch of bottle (Blastocaulis) (Blastocaulis) Cladophialophora immunda The accession number of LC3 is CGMCC NO. 41540, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is Beijing Chaoyang District North No. 3, Courtyard 1, Chenxi Road; Date of collection: October 12, 2024; The Guangxi Cladosporium ( Cladophialophora guangxiense The accession number of HX2 is CGMCCNO.41498, the depositary institution is the China General Microbiological Culture Collection Center, the depositary address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is September 5, 2024.

2. A method for isolating and screening plant immune-activating DSE fungi, characterized in that, The separation and screening method is as follows: (1) Obtaining cucumber roots: After germinating cucumber seeds, they were sown in soil samples for cultivation; (2) Isolation and purification of strains: The roots of cucumber seedlings were collected, cleaned and disinfected, and then placed in 1 / 2 PSA medium or modified 1 / 2 CM medium. The culture was carried out in medium, and after the colonies grew, it was promptly transferred to PDA medium for further culture to obtain purified strains. (3) By re-inoculation and microscopic examination of roots, strains that coexist with cucumbers were screened. The purified strains obtained in step (2) were co-cultured with cucumber plants on petri dishes for 14 days to form the experimental group. The coexistence of the strains with cucumbers in the experimental group was investigated. Cucumber plants without inoculation were used as the control group. The roots of cucumber plants were washed and dried to determine the dry weight of the whole plant. (4) Compare the dry weight of the whole cucumber plant in the experimental group and the control group in step (3), and select the strains in the experimental group whose dry weight of the whole cucumber plant is significantly higher than that in the control group as candidate immune-activating DSE fungi. (5) The candidate DSE fungi for immune activation in step (4) were evaluated for indoor in vivo plate immune activation in cucumber wilt disease. The price was determined by selecting strains with a control effect ≥50.0% as immune-activated DSE fungi; The 1 / 2PSA medium consists of 100 g / L potato, 10 g / L sucrose and 15 g / L agar; the modified 1 / 2CM medium consists of 8.5 g / L corn flour, 15 g / L agar powder and 10 g / L monk fruit polysaccharide. The culture temperature for step (2) of the 1 / 2PSA medium or the modified 1 / 2CM medium is 20℃.

3. The separation and screening method according to claim 2, characterized in that, The mogro fruit polysaccharide was obtained by water extraction and alcohol precipitation.

4. The separation and screening method according to claim 2, characterized in that, The evaluation method for immune activation in step (5) is as follows: after co-culturing the candidate strain with cucumber for 7 days, the symbiont along with the culture medium is transferred to a water agar medium containing pathogens. After the control group cucumber seedlings show obvious disease, the disease grade of the plants is investigated, the disease index is calculated, and the control effect is evaluated. The grading criteria for plant disease severity are as follows: Grade 0: No symptoms; Level 1: The area of ​​yellowing or wilting of true leaves and cotyledons does not exceed 50% of the total area; Level 2: The area of ​​yellowing or wilting of true leaves and cotyledons exceeds 50% of the total area; Level 3: Leaves are wilted or dead, only the growing point survives; Level 4: The entire plant is severely wilted, to the point of death. The disease index is calculated using the following formula: Disease Index = [Σ (Number of diseased plants at each level × Corresponding level) / (Total number of plants surveyed × ...] [Superior representative value] × 100; The formula for calculating the prevention and control effect is: Prevention and control effect (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100%.

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