Predatory tubulomyces sp. And application of predatory tubulomyces sp. In biological prevention and control of plant diseases

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to predatory tubulomyces sp. And application of the predatory tubulomyces sp. In biological prevention and control of plant diseases. The predatory tubulomyces sp. Strain NSE202 is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 3, 2023, and the preservation number is CGMCC NO.26375. The invention further discloses a preparation method of the predatory tubulomyces sp. Strain NSE202. According to the present invention, the growth of a variety of plant pathogens such as Erwinia amyloliquefaciens, pectobacterium carotovorum subsp. Carotovora, pear rust pathogen and the like is inhibited through the predation manner, and the broad-spectrum pathogen resistance of the tubulomyces sp. NSE202 is represented; meanwhile, the tubulomyces sp. Strain NSE202 can effectively inhibit the infection of pear fire blight bacteria and cotton verticillium wilt bacteria on pyrus betulaefolia and cotton, reduces the morbidity of pear fire blight and cotton verticillium wilt, and can be used for biological control of plant diseases caused by phytopathogens.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to predatory tube creeping bacteria and application thereof in biological control of plant diseases. Background Art

[0002] Plant diseases are one of the natural disasters that seriously endanger agricultural production. They have a great impact on the growth, development and yield of crops, causing huge economic losses. According to statistics, the disease losses of major crops in the world account for about 20% to 40% of the total crop output, and the direct economic losses are as high as billions of dollars each year, of which 70% to 80% of the diseases are caused by pathogenic fungi.

[0003] At present, plant diseases in agricultural production are mainly controlled by chemical means, while over-reliance on and abuse of chemical pesticides have led to a series of problems such as pest resistance, excessive pesticide residues, and environmental pollution, which seriously affect the green and sustainable development of agriculture. Relevant research and practice have shown that the use of living microorganisms and their active substances to develop biological agents for the prevention and control of plant diseases can achieve good prevention effects that are better than or equivalent to chemical pesticides, have strong selectivity, are not prone to resistance, are safe and efficient, and are environmentally friendly. They meet the requirements of sustainable agricultural development and have gradually become an important means of plant disease prevention and control.

[0004] In recent years, the biocontrol microorganisms that have received more attention mainly come from Pseudomonas, Bacillus, Burkholderia, Lysobacterium and Trichoderma, and the antimicrobial effects involved mainly include secretion of antimicrobial secondary metabolites, niche or resource competition, secretion of antimicrobial proteins, etc. However, the entry of biocontrol microorganisms into the environment is affected by multiple factors such as the variability of environmental factors, immune recognition during plant-microorganism interactions, and plant rhizosphere regulation, making it difficult for biocontrol microorganisms to colonize in open environments and unstable control effects, resulting in a certain degree of limitation in the practical application of biological agents.

[0005] In fact, in the biological control of insect pests or animal diseases, using their natural enemies and predator-prey relationships for biological control can achieve better control effects. There are also some predators among microorganisms. Including Haemococcus, Bdellovibrio, Vibrio phagocytophilus, Micavibrio, Daptobacter, Bacteroidetes and Myxobacteria. Among them, Bdellovibrio can directly invade the periplasmic space of cells to prey on Gram-negative bacteria, and is widely used in the aquaculture industry to improve water quality and treat bacterial diseases of aquatic animals. Myxobacteria are a type of Gram-negative bacteria with broad-spectrum predation capabilities. They often sense prey in a group sliding manner, and then secrete a large amount of hydrolases or secondary metabolites to prey on a variety of bacteria and fungi, showing potential application value in the biological control of plant diseases.

[0006] Herpetosiphon is the only predatory group in the Chloroflexi phylum and is a deeply branched bacterial group in evolution. It is widely distributed in soil, fresh water and decaying organic matter. It can glide on solid surfaces, capture prey through wolf pack attack mode, and secrete a large number of hydrolases to prey on a variety of bacteria and fungi. In addition, Herpetosiphon can also produce abundant new secondary metabolites and has multiple antibacterial mechanisms.

[0007] At present, the genus Herpetosiphon only includes five species of microbial groups: Herpetosiphon aurantiacus, Herpetosiphongeysericola, Herpetosiphon giganteus, Herpetosiphon.gulosus and Herpetosiphonllansteffanensis. Studies have found that all discovered species of this genus have predatory functions, and the prey objects are quite extensive. However, the preyed microorganisms reported so far mostly involve human and animal pathogens, and their applications are limited to the field of public health. For example, the Chinese patent with patent number 202110820711.3 discloses a predatory Herpetosiphon and its application in the preparation of a drug to inhibit methicillin-resistant Staphylococcus aureus. However, there have been no reports on the antibacterial activity of Herpetosiphon microorganisms against different plant pathogenic microorganisms and their application in the biological control of plant diseases. Therefore, it is of great significance for the biological control of plant diseases to carry out research on the antibacterial activity of Herpetosiphon microorganisms against different plant pathogenic microorganisms and develop the application of Herpetosiphon microorganisms in the biological control of plant diseases. Summary of the invention

[0008] In order to solve the problem that there is no application of microorganisms of the genus Sphaeroides in the biological control of plant diseases in the prior art, the present invention proposes a predatory Sphaeroides genus and its application in the biological control of plant diseases. To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] The present invention provides a predatory tube mushroom strain NSE202, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on January 3, 2023, with a deposit number of CGMCC NO.26375.

[0010] The present invention also provides a culture comprising the predatory tube mushroom strain NSE202.

[0011] The present invention also provides a plant disease biological agent, which includes strain NSE202, a culture of strain NSE202, or a mixture of one or more of the cultures.

[0012] Preferably, the culture comprises a bacterial strain broth, a bacterial strain fermentation broth or a sterile filtrate of a fermentation broth.

[0013] The present invention also provides a biological fertilizer, which comprises the biological microbial agent and plant growth nutrient elements.

[0014] Preferably, the biocontrol agent or biofertilizer is processed into an agriculturally acceptable formulation.

[0015] The present invention also provides the use of the predatory tube mushroom strain NSE202 or the culture in preventing and controlling plant pathogens.

[0016] Preferably, the plant pathogenic bacteria include plant pathogenic bacteria, plant pathogenic fungi and plant pathogenic oomycetes.

[0017] Preferably, the plant pathogenic bacteria include any one of Erwinia amylovora, Pectobacterterium carotovorum subsp. Carotovorum, Dickeya fangzhongdai, Acidovorax citrulli, and Pseudomonas syringae pv. Syringae;

[0018] The plant pathogenic fungi include any one of Verticillium dahliae, Fusarium oxysporum f.sp.vasinfectum, Fusarium verticillioides, Rhizoctonia solani, Fusarium culmorum, Alternaria tenuissima, and Valsa mali var.pyri;

[0019] The plant pathogenic oomycetes include Phytopahthora capsici.

[0020] The present invention also provides a method for biological control of plant diseases, wherein the strain NSE202, the culture, the biological agent for plant diseases or the biological fertilizer is applied to plants to prevent the plants from being infected by plant pathogens.

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

[0022] The present invention provides a predatory tubercle fungus and its application in biological control of plant diseases. The present invention studies the antibacterial activity of tubercle fungus microorganisms against different plant pathogenic microorganisms, and provides a tubercle fungus microorganism strain NSE202 and its new application in biological control of plant diseases. The research results of the present invention prove that the tubercle fungus microorganisms have the potential to become a new type of microbial agent, which is of great significance for solving the prevention and control problems of existing biocontrol agents such as narrow antibacterial spectrum and poor combined prevention effect.

[0023] The present invention proves through research results that the strain NSE202 found by the present invention shows good control effects on plant bacterial diseases such as fire blight and cotton fungal diseases such as Verticillium wilt, and can be applied to plant disease biological control. The present invention uses a preyed fungus induction method to separate and purify a strain NSE202 from soil samples collected from Tianshan Grand Canyon National Forest Park in Urumqi, Xinjiang, and is identified as the Herpetosiphonp strain NSE202. The strain can prey on a variety of plant pathogenic fungi and plant pathogenic bacteria during its growth process to meet its own nutritional needs and show a broad spectrum of antibacterial activity. Based on indoor potted plant experiments, it is shown that the strain NSE202 involved in the present invention shows good control effects on plant bacterial diseases such as fire blight and cotton fungal diseases such as Verticillium wilt, and is a biocontrol strain with good application prospects, which can be used for the biological control of plant diseases caused by plant pathogens, and shows great application potential in the biological control of plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The morphology of strain NSE202, wherein Figure a is a morphological diagram of the colony of strain NSE202; Figure b is a scanning electron microscope image of strain NSE202;

[0025] Figure 2 This is a phylogenetic tree constructed based on the 16S rDNA gene;

[0026] Figure 3 The antagonistic effect of strain NSE202 on seven plant pathogenic fungi;

[0027] Figure 4 is the antagonistic rate of strain NSE202 against seven plant pathogenic fungi;

[0028] Figure 5 is the antibacterial ability of strain NSE202 against Phytophthora capsici;

[0029] Figure 6 The results of the predation test of strain NSE202 on 5 plant pathogenic bacteria on the lawn;

[0030] Figure 7It is the number of residual living cells of five plant pathogenic bacteria after 5 days of the fungus moss predation experiment;

[0031] Figure 8 The results of confrontation culture between strain NSE202 and five plant pathogenic bacteria;

[0032] Fig. 9 It is the number of residual viable cells of five plant pathogenic bacteria after 5 days of confrontation culture;

[0033] Fig.10 The antibacterial activity of the extracellular secondary metabolites secreted by strain NSE202 against Erwinia amylovora. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0035] The culture medium and its specific formula used in the following examples are as follows:

[0036] LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L, pH 7.0.

[0037] LBS liquid culture medium: soluble starch 7.0 g / L, tryptone 1.0 g / L, yeast extract 5.0 g / L, MgSO4·7H2O 1.0 g / L, pH 7.2-7.4.

[0038] VY / 4 medium: Angel yeast 2.5g / L, CaCl2 1.0g / L, agar powder 15g / L, pH 7.2-7.4.

[0039] WCX medium: CaCl2 1.0 g / L, agar powder 15 g / L, pH 7.2. After sterilization, add cycloheximide to a final concentration of 25 μg / mL.

[0040] TPM medium: 10.0mmol / L Tris-HCl, 1.0mmol / L KH2PO4, 1.97g / L MgSO4·7H2O, 15g / L agar powder.

[0041] Czapek medium: NaNO3 3.0g / L, K2HPO4 1.0g / L, MgSO4·7H2O 0.5g / L, KCl 0.5g / L, FeSO4 0.01g / L.

[0042] V8 medium: V8 vegetable juice 100 mL, CaCO3 1.0 g, deionized water 900 mL, agar 15 g / L.

[0043] Example 1

[0044] 1. Isolation and purification of strains

[0045] Soil samples with a depth of 5 cm were collected from the Tianshan Grand Canyon National Forest Park in Urumqi, Xinjiang. Small stones and rotten wood were removed with a 40-mesh sieve and then naturally air-dried at room temperature. 20 g of air-dried soil samples were weighed and placed in a sterile culture dish, placed in an oven at 65°C for 30 min, and after the soil samples were cooled, actinomycin solution (final concentration of 25 μg / mL) was added to soak, and the residual liquid was discarded after soaking overnight (time ≥ 12 h) for later use.

[0046] Erwinia amylovora was inoculated into LB liquid medium, cultured overnight at 30°C with shaking (time ≥ 12h), 1mL of bacterial suspension was drawn into a centrifuge tube, centrifuged at 12000rpm for 1min, the bacteria were collected, rinsed 3 times with sterile water, and 100μL was left as bacterial suspension. The bacterial suspension was dipped into a sterile inoculation loop and a "field" line was drawn on the surface of WCX medium containing actinomycin (final concentration of 25μg / mL), and 0.5g of the above-treated soil sample was placed in the center of four squares after drying, and the WCX medium was cultured at 30°C. After 48h, the granular bacterial mass induced in the WCX medium was picked up with a sterilized fine needle tip under a stereomicroscope, and inoculated on the surface of VY / 4 medium, and the VY / 4 medium was cultured at 30°C. When a transparent bacterial film grows on the VY / 4 medium, promptly pick up the outer edge of the transparent bacterial film and transfer it to a fresh VY / 4 plate for further purification until no foreign bacteria grow, and obtain strain NSE202. Inoculate the isolated and purified strain NSE202 into LB culture medium, and culture it overnight (for ≥12 hours) at 30°C and 180rpm. Observe the state of the culture medium. If the culture medium is clear and transparent, it indicates that the strain is a pure strain (because the creeping tube bacteria do not grow in the LB culture medium); if the culture medium is turbid, it proves that other foreign bacteria are mixed in the colony. Transfer the pure strain NSE202 into 20% glycerol and store it in a -80°C refrigerator.

[0047] 2. Identification of strain NSE202

[0048] 2.1 Observation of the morphology and culture characteristics of strain NSE202

[0049] The surface of the colony of strain NSE202 inoculated on VY / 4 plate was moist, orange-yellow, and slid and expanded. After about 5 days, hill-like aggregates and a network structure formed by aggregation were observed, and the edges of the colonies were curled ( Figure 1a); Vegetative cells are non-branching filaments and multicellular. As the culture time increases, the filaments break and form single cells ( Figure 1 b).

[0050] 2.2 Sequencing and analysis of the 16S rRNA gene of strain NSE202

[0051] Fresh cells of strain NSE202 were taken and total genomic DNA was extracted using a bacterial genome extraction kit (TIANamp Bacteria DNA Kit, TIANGEN). The 16S r RNA gene was amplified using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID No.2) and 1492R (5'-TACGGCTACCTTGTTACGACTT-3', as shown in SEQ ID No.3). After the amplified product was detected by 10g / L agarose gel electrophoresis, the PCR product was cloned and sequenced by Shanghai Sangon Biotechnology Co., Ltd. The sequencing results are shown in SEQ ID No.1:

[0052]

[0053] The obtained sequences were compared by BLAST in the GenBank database, DNAstar software was used for homology comparison, and MEGA7.0 software was used to construct the 16S rRNA gene phylogenetic tree by the neighbor-joining method. Figure 2 The results show that strain NSE202 has the highest similarity (99.4%) with the model strain of Herpetosiphon giganteus Hp a2, and they are clustered on the same phylogenetic branch in the phylogenetic tree, while the 16S rRNA gene sequence similarity with the strain Herpetosiphon gulosus 2-13 published in the invention patent (patent number: 202110820711.3) is only 98.8%, and they are not clustered in the same cluster on the phylogenetic tree, indicating that the above strain NSE202 and the strain Herpetosiphon gulosus 2-13 disclosed in the invention patent (patent number: 202110820711.3) are not clustered on the same evolutionary branch, indicating that the two are not the same strain. Based on the morphological characteristics and molecular identification results, strain NSE202 was preliminarily identified as Herpetosiphonp spp., which has been deposited in the Budapest Treaty International Microbiological Depository: China General Microbiological Culture Collection Center (CGMCC). The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 3, 2023, and the deposit number is CGMCC NO.26375. The name of strain NSE202 is changed to Herpetosiphonp NSE202.

[0054] 3. Determination of the predatory activity of NSE202 against various plant pathogenic fungi

[0055] 3.1 Activation of plant pathogenic fungi: Pick the mycelium blocks of Verticillium dahliae, Fusarium oxysporum f.sp.vasinfectum, Fusarium verticillioides, Rhizoctonia solani, Fusarium culmorum, Alternaria tenuissima, and Valsa mali var.pyri from the test tube slope, transfer them to a fresh PDA solid plate, and culture and activate them in a 26°C incubator. When the mycelium expands about 1 cm, use a sterilized blade to cut a 2×2 mm agar block along the edge of the mycelium expansion, transfer it to another fresh PDA plate again, and use it for subsequent experiments after the mycelium expands about 2 cm.

[0056] 3.2 Preparation of NSE202: Inoculate NSE202 into 3 mL of LBS test tube culture medium, shake at 160 rpm and 30°C for 2 days; collect the bacteria by centrifugation at 10,000 rpm for 10 min, wash them 3 times with TPM liquid culture medium, and finally resuspend them with 500 μL of TPM liquid culture medium to obtain a bacterial suspension of NSE202.

[0057] 3.3 Plate confrontation test: Use a sterilized blade to cut a 2×2mm agar block along the edge of the hyphae expansion of the above-mentioned activated pathogenic fungi and transfer it to the middle of the VY / 4 plate and culture it in a 26℃ incubator. When the hyphae expands about 0.5cm, inoculate the NSE202 bacterial suspension symmetrically along a straight line about 1.5cm away from the edge of the hyphae. The straight-line distance is about 3cm, and 20μL is inoculated in each straight line. After natural drying, place the VY / 4 plate in a 28℃ incubator for culture. The solid plate inoculated with the above-mentioned activated plant pathogenic fungi alone was used as a control, and each group of experiments was repeated 3 times. After 5 days of confrontation, use a fully automatic colony counter to take pictures and measure the colony area of ​​the plant pathogenic fungi, and calculate the antagonism rate. The experimental results are shown in Figure 3 and Figure 4 .

[0058] The calculation formula is: antagonistic rate (%) = (colony area of ​​control group - colony area of ​​treatment group) / (colony area of ​​control group) × 100%.

[0059] Depend on Figure 3 and Figure 4It can be seen that the creeping tube fungus NSE202 has a good predatory effect on the above selected plant pathogenic fungi. The creeping tube fungus biofilm expands into the plant pathogenic fungal colony, most of the hyphae collapse, and the growth of plant pathogenic fungi is significantly restricted. The antagonistic efficiency of creeping tube fungus NSE202 against Alternaria pseudoverticillium and Fusarium oxysporum wilt specialized type is as high as more than 90.0%, and it also has a high antagonistic efficiency against black rot pear variety of apple, Rhizoctonia solani, Verticillium dahliae, Alternaria tenuissima and Fusarium oxysporum, which are 79.6%, 80.6%, 72.0%, 80.6% and 65.6% respectively.

[0060] The above results indicate that the creeping tube fungus NSE202 exhibits good predation ability against a variety of plant pathogenic fungi, demonstrating its good broad-spectrum anti-pathogenic fungi ability.

[0061] 4. Determination of the predatory activity of NSE202 against plant pathogenic oomycetes

[0062] Phytopahthora capsici was activated and cultured using V8 medium, and then the activated Phytophthora was transferred to VY / 4 medium. When the colony diameter grew to about 1-2 cm, the suspension of NSE202 was inoculated symmetrically along a straight line about 1.5 cm from the edge of the hyphae. The straight-line distance was about 3 cm, and 20 μL was inoculated in each straight line. After natural drying, the VY / 4 plate was placed at 28 ° C for 3 days to observe the collapse of the colony. The solid plate inoculated with the activated plant pathogenic oomycete alone was used as a control, and 3 replicates were set for each group of experiments.

[0063] The co-culture results showed (see Figure 5 ), the bacterial film of NSE202 expanded into the colony of Phytophthora capsici, the hyphae of the contact part collapsed, and the growth of Phytophthora capsici was obviously restricted. The results showed that NSE202 also showed good predation ability on Phytophthora capsici.

[0064] 5. Determination of the ability of NSE202 to prey on plant pathogenic bacteria

[0065] 5.1 Preparation of pathogenic bacteria and NSE202

[0066] Erwinia amylovora, Pectobacterterium carotovorum subsp. Carotovorum, Dickeyafangzhongdai, Acidovorax citrulli, and Pseudomonas syringae pv. Syringae were activated respectively, and single colonies were picked and inoculated into LB culture medium, and cultured in a constant temperature shaker at 30°C and 160 rpm for 24 h. The cells were centrifuged at 12000 rpm for 1 min, and the cells were collected. After rinsing with sterile water for 3 times, they were resuspended with TPM buffer to obtain a pathogenic bacterial suspension for later use.

[0067] After the pure culture of NSE202 was activated on a VY / 4 plate, an appropriate amount of colonies were scraped and transferred to LBS culture medium, and cultured at 30°C and 160rpm for 2 days. The prepared NSE202 bacterial suspension was centrifuged at 10,000rpm for 10 minutes, the supernatant was removed and the cells were collected, then washed 3 times with TPM liquid culture medium, and finally resuspended with 500μL TPM liquid culture medium to obtain the strain NSE202 bacterial suspension for use.

[0068] 5.2 Evaluation of the ability of NSE202 to prey on plant pathogenic bacteria

[0069] (1) Predation test on bacterial lawn: 50 μL of pathogenic bacterial suspension was vertically inoculated on TPM nutrient-free solid culture medium. After natural air drying, 3 μL of strain NSE202 bacterial suspension was inoculated in the center of pathogenic bacterial lawn and natural air drying was performed. The control group was inoculated with 3 μL of TPM liquid culture medium. The culture was cultured at 30°C for 5 days, and the expansion of the tube-climbing bacteria was observed on days 1, 3, and 5. On the 5th day, the bacterial lawn was scraped off with a sterile capillary tube and mixed with 1 mL of sterile water. The dilution coating method was used to take 100 μL of each gradient and evenly coated on the LB plate. The plate was placed in a 30°C constant temperature incubator and cultured until a single colony grew. The number of pathogenic bacterial colonies was counted, the number of residual living cells was calculated, and the predation ability was evaluated.

[0070] The results of the fungus moss predation experiment showed that ( Figure 6 , Figure 7 ), after one day of culture, it can be observed that the creeping tube fungus NSE202 can diffusely grow using plant pathogenic bacteria as nutrients. After 5 days of culture, the creeping tube fungus NSE202 can completely spread and cover the entire pathogenic bacterial lawn. At this time, the entire colony was scraped and the residual viable cells of pathogenic bacteria were counted on the LB solid plate by the dilution coating method. It was found that the biomass of the square-headed Dixodactylum had the largest decrease, which decreased by 5 orders of magnitude, and the number of viable cells increased from 1.3×109 cfu / mL decreased to 7.9×10 4 cfu / mL; followed by Pectobacterium carotovora subspecies, which decreased by 4 orders of magnitude, with the number of viable cells from 2.7×10 9 cfu / mL decreased to 5.9×10 5 cfu / mL; the next is Erwinia amylovora, which dropped by 3 orders of magnitude, with the number of viable cells from 9.7×10 8 cfu / mL decreased to 2.1×10 5 cfu / mL; while Acidobacterium citrullum and Pseudomonas syringae pv. syringae decreased by 2 and 1 orders of magnitude, respectively.

[0071] (2) Confrontation culture test: In order to further count and evaluate the movement and predation ability of strain NSE202, NSE202 was cultured in confrontation with five plant pathogenic bacteria, and the movement direction and expansion speed of NSE202 were observed regularly. Specific methods: 50 μL of pathogenic bacterial suspension was vertically suspended on TPM solid culture medium, and air-dried naturally. 3 μL of strain NSE202 suspension was inoculated near the edge of the pathogenic bacterial lawn. The control group was inoculated with 3 μL of TPM liquid culture medium. After air-drying, it was placed in a 30℃ constant temperature incubator for 5 days. During this period, the movement direction of NSE202 was observed regularly. After 5 days, the lawn was scraped off with a sterile capillary tube and mixed with 1 mL of sterile water. Using the dilution coating method, 100 μL of each gradient was evenly coated on the LB plate. The plate was placed in a 30℃ constant temperature incubator for culture until a single colony grew. The number of pathogenic bacterial colonies was counted, the number of residual living cells was calculated, and the predation ability was evaluated.

[0072] Depend on Figure 8 and Fig. 9 It can be seen that the creeping tube fungus NSE202 has obvious tropism for the five plant pathogenic bacteria and can cover and prey on the plant pathogenic bacterial lawn. After 5 days, the lawn was scraped and diluted and spread, and the number of residual living cells of pathogenic bacteria was calculated. The results showed that the creeping tube fungus NSE202 had the strongest ability to expand its prey outward, and the biomass of Dadickia decreased by 3 orders of magnitude; the biomass of Erwinia amylovora, Pectobacterium carrotum subsp. carrotum, and Acidophilus citrullus decreased by 2 orders of magnitude, and the biomass of Pseudomonas syringae pv. syringae decreased by 1 order of magnitude.

[0073] The above results indicate that the creeping tube fungus NSE202 has good predation ability on a variety of plant pathogenic bacteria, showing its good broad-spectrum anti-pathogenic bacteria ability.

[0074] 6. Antibacterial effect of fermentation supernatant of S. cerevisiae NSE202 on plant pathogens

[0075] The LBS liquid medium was used to ferment the tube fungus NSE202, and the culture was shaken at 30°C and 180rpm for 4 days. The fermentation supernatant was collected by centrifugation at 4°C and 10000rpmn for 10min. Ammonium sulfate crystals were added to the obtained 1.0L fermentation supernatant until 80% saturation, and the crude protein was allowed to stand at 4°C for 12h to fully precipitate. The protein suspension was centrifuged at 4°C and 12000rpmn for 20min, the supernatant was discarded, the precipitate was collected, and resuspended with 1.5mL50mmol / L pH 7.0 Tris-HCl buffer, and then dialyzed with a 0.5KDa dialysis bag and 50mmol / L pH 7.0 Tris-HCl buffer as the dialysis fluid to remove ammonium sulfate. The liquid in the dialysis bag was collected and sterilized by filtration with a 0.22μm bacterial filter. Take 500μL of the crude protein extract and 500μL of the Erwinia amylovora suspension (OD 600 =1.0) co-cultured for 3 times, 30°C, 180rpm shaking culture for 24h, and then diluted and coated to count the number of residual viable cells of Erwinia amylovora, with Tris-HCl buffer as control. The results showed that the prepared protein crude extract had a strong antibacterial activity against Erwinia amylovora, and the number of residual viable cells increased from 3.52×10 10 cfu / mL decreased to 1.66×10 6 cfu / mL, indicating that the creeping tube fungus NSE202 can secrete some protein substances to inhibit the growth of Erwinia amylovora

[0076] Referring to the preparation method in the invention patent (patent number: 202110820711.3), the fermentation medium is LBS liquid, and a 2% volume ratio of macroporous resin XAD-16 is added before inoculation. The creeping tube bacteria NSE202 is inoculated at a volume ratio of 2%, and the culture is shaken at 30°C and 180rpm for 4 days. The resin is collected by filtering with gauze, and 2 times the volume of methanol is added and placed at 30°C for shaking and extraction for 1h, and the methanol is filtered and collected; the extraction process is repeated 3-4 times until the methanol is colorless. The collected methanol is combined and evaporated to dryness under reduced pressure at 35°C and 40rpm on a rotary evaporator. Fermentation of 1.0L of the strain obtained a total of 1.05g of crude extract of the fermentation broth. The above crude extract was dissolved with 4mL of dimethyl sulfoxide (DSMO): water (v / v 1:1) and filtered and sterilized with a 0.22μm bacterial filter to obtain a prepared crude extract solution. The prepared fermentation broth crude extract solution was diluted with sterile deionized water to concentrations of 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL and 1.5625 mg / mL of fermentation broth crude extract solution. 500 μL of each concentration of fermentation broth crude extract solution was co-cultured with 500 μL of Erwinia amylovora suspension (OD600=1.0), repeated 3 times, 30°C, 180 rpm shaking culture for 24 hours, diluted and coated to count the number of residual viable cells of Erwinia amylovora, with DSMO aqueous solution as control.

[0077] The results are as follows Fig.10 As shown in Figure 2, when the concentration of the fermentation broth crude extract was 100 mg / mL, 50 mg / mL, and 25 mg / mL, the number of residual viable cells of Erwinia amylovora increased from 2.93×10 10 cfu / mL decreased to 8.22×10 8 cfu / mL, 2.54×10 9 cfu / mL and 3.27×10 9 cfu / mL, showing a certain antibacterial activity. When the concentration of the fermentation broth crude extract was lower than 25 mg / mL, it had no inhibitory effect on the growth of Erwinia amylovora.

[0078] The above results show that there are obvious differences between the Herpetosiphon gulosus 2-13 involved in the present invention and the invention patent (patent number: 202110820711.3), that is, the secondary metabolites in Herpetosiphon gulosus 2-13 have good antibacterial effects and still have antibacterial activity at a concentration of 6.25 mg / mL, while in the fermentation supernatant of the climbing tube fungus NSE202 involved in the present invention, both protein substances and secondary metabolites have certain antibacterial activity, but the antibacterial activity of protein substances is stronger.

[0079] 7. Evaluation of the application of NSE202 in biological control of plant diseases

[0080] In order to study the biocontrol effect of the creeping tube fungus NSE202 involved in the present invention on plant pathogens in actual application, Erwinia amylovora, the pathogen of fire blight of amylopectin, and Verticillium dahliae, the pathogen of cotton wilt, were selected as model pathogen materials, and the disease prevention and control ability of the creeping tube fungus NSE202 in the aboveground part of the plant and in the soil environment was verified through potted experiments on pear and cotton.

[0081] 7.1 Potted experiment on Pyrus betulaeformis to determine the control effect of NSE202 on fire blight

[0082] (1) Preparation of NSE202 and pathogen inoculum

[0083] The activated NSE202 was inoculated into 3 mL of LBS test tube culture medium, and then inoculated into 200 mL of LBS shake flask culture medium, and then inoculated into 30 ° C, 160 rpm shake flask culture medium for 3-4 days to obtain the fermentation liquid of NSE202. The pathogen of amylovora amylovora was activated, and a single colony was selected and inoculated into LB culture medium, and then shaken and cultured in a constant temperature shaker at 28 ° C, 160 rpm for 24 hours until the OD value of the bacterial liquid reached 0. 600 =1.0, dilute to 10 with sterile water 7 cfu / mL, and obtain the pathogen suspension for later use.

[0084] (2) Protective effect of NSE202 against fire blight

[0085] This part of the experiment was conducted in a greenhouse, and two-year-old potted pear seedlings were selected as inoculation materials. The fermentation liquid of the creeping tube fungus NSE202 was sprayed with a handheld pressure sprayer until the leaves and branches were completely wet. After 48 hours, the above pathogen suspension was sprayed and inoculated. Three pots (about 15 branches per pot) were sprayed for each treatment, and repeated 3 times. At the same time, a 4000-fold liquid control of agricultural streptomycin (North China Pharmaceutical Factory, 72% effective ingredient) and a sterile water blank control (i.e., sterile water was sprayed 48 hours later and then inoculated with apricot fire blight bacteria, CK) were set up. The disease situation was observed every day, the number of diseased branches was recorded, the length of branch dead, the proportion of branch dead length to the length of inoculated branches, and the disease level were measured, the incidence rate and disease index were calculated, and the prevention effect was statistically calculated. After the experiment, the diseased plant materials were sterilized by dry heat and destroyed.

[0086] The results of the protective test showed (as shown in Table 1) that spraying the fermented liquid of NSE202 with the seedlings of Pyrus betulaeformis beforehand could significantly reduce the incidence and disease index of Pyrus betulaeformis seedlings, with an average protective effect of 77.46% for 10 to 20 days. The protective effect of NSE202 fermented liquid was the highest (87.28%) on the 10th day after spraying, and it could still be maintained at 71.87% on the 20th day, which was slightly lower than the protective effect of agricultural streptomycin (84.71%).

[0087] Table 1 The protective effect of NSE202 on fire blight of Pyrus betula

[0088]

[0089]

[0090] (3) Therapeutic efficacy of NSE202 against fire blight

[0091] The inoculation order of pathogens and NSE202 in the therapeutic test was opposite to that in the protective test, that is, the amylopectin pathogen solution was first sprayed on the pear seedlings and then the fermentation liquid of NSE202 was sprayed 48 hours later. Other test materials, culture conditions, and prevention efficacy investigation methods were consistent with those in the protective test.

[0092] The disease grading standards for potted Pyrus betula seedlings are as follows: Grade 0 (no lesions on branches); Grade I (the length of lesions on branches is 1 / 3 of the length of inoculated branches); Grade III (the length of lesions on branches is 1 / 3-2 / 3 of the length of inoculated branches); Grade V (the length of lesions on branches is more than 2 / 3 of the length of inoculated branches).

[0093] Incidence rate (%) = (number of diseased branches / total number of inoculated branches) × 100%; Disease index = ∑ (number of diseased branches at each level × representative value of disease level) / (total number of inoculated branches × highest level value) × 100; Branch blight prevention effect (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0094] The results of the therapeutic test showed (as shown in Table 2) that spraying the fermented liquid of the creeping tube fungus NSE202 had a significant therapeutic effect on the branch blight of Pyrus betulaeformis seedlings. The control effect reached 71.89% on the 10th day of spraying, and the control effect decreased from 15 to 20 days, and the average control effect reached 66.10% on the 10th to 20th day.

[0095] Table 2 The therapeutic efficacy of NSE202 on fire blight of Pyrus betula

[0096]

[0097] 7.2 Cotton pot experiment to determine the control effect of NSE202 on cotton Verticillium wilt

[0098] (1) Preparation of spore suspensions of NSE202 and Verticillium dahliae

[0099] The activated creeping tube fungus NSE202 was inoculated into 3 mL of LBS test tube culture medium, and cultured at 30°C and 160 rpm for 24 h. Then, all of the culture medium was inoculated into 200 mL of LBS shake flask culture medium, and cultured at 30°C and 160 rpm for 3 to 4 days for later use.

[0100] Using a sterile cork puncher, three 5 mm diameter cakes were taken from the edge of the activated Verticillium dahliae colony and inoculated into 200 mL of Czapek medium. The culture was shaken at 26 °C and 180 rpm for 7 days. The spore suspension was filtered through sterile gauze and the concentration was calculated using a hemocytometer and diluted to 1.0 × 10 6 cfu / mL, for future use.

[0101] (2) Potted plant control effect of creeping tube fungus NSE202 on cotton Verticillium wilt

[0102] Vermiculite and nutrient soil were mixed evenly in a volume ratio of 3:7 and then placed in a flower pot. At the same time, cotton seeds disinfected with 75% ethanol were soaked for 12 hours, and planted in flower pots after they turned white. When they grew to the 1-leaf stage, 10 seedlings were selected for each pot as one treatment. The cotton was cultivated in a solar greenhouse with a temperature of 23-30°C and a relative humidity of more than 60%. When the cotton grew to the mature 2-3 leaf stage, 10 mL of the fermentation culture solution of the creeping tube fungus NSE202 was inoculated into the roots of each cotton plant. After 7 days, the cotton roots were wounded, and the prepared spore suspension of Verticillium dahliae (1.0×10 6 cfu / mL) and 5mL Czapek medium was used as the control. Each treatment was repeated 3 times. 60 days after inoculation of Verticillium dahliae, the stems were cut open to investigate the disease situation, and the disease index and potted control efficacy were calculated.

[0103] The cotton verticillium wilt disease grading standard is graded from 0 to 4, with the following criteria: Grade 0: normal, no brown spots on the cross section; Grade 1: sporadic brown spots on the cross section, occupying less than 25% of the area; Grade 2: the brown area occupies 25-50% of the cross section; Grade 3: the brown area occupies 50-75% of the cross section; Grade 4: the brown area occupies more than 75% of the cross section, and the cross section has almost completely turned brown.

[0104] Incidence rate (%) = (number of diseased plants / total number of plants) × 100%;

[0105] Disease index = 100 × ∑ (number of plants at each severity level × representative value of each level) / (number of plants surveyed × highest representative value)

[0106] Control effect (%) = [(control group disease index - treatment group disease index) / control group disease index] × 100%

[0107] The test results showed (as shown in Table 3) that the incidence rate and disease index of cotton plants treated with root irrigation with the fermented culture solution of Sphaerotheca spp. NSE202 were significantly lower than those of the control, and the control efficiency reached 63.64%.

[0108] Table 3 Biocontrol effect of creeping tube fungus NSE202 on cotton Verticillium wilt in potted plant experiment

[0109] deal with Incidence / % Disease index Prevention effect / % NSE202 40.00 26.67±1.18 63.64±1.61 Comparison 86.67 73.33±5.14 —

[0110] Based on indoor pot plant experiments, it was shown that the creeping tubercle NSE202 involved in the present invention exhibited good control effects on plant bacterial diseases such as fire blight and plant fungal diseases such as cotton wilt, and is a biocontrol strain with good application prospects, which can be used for the biological control of plant diseases caused by plant pathogens. The creeping tubercle microorganisms and their new applications in the biological control of plant diseases have been developed.

[0111] The present invention provides a predatory tubercle fungus and its application in biological control of plant diseases. The present invention studies the antibacterial activity of the tubercle microorganism strain NSE202 against different plant pathogenic microorganisms, and provides a tubercle microorganism (tubercle fungus NSE202) and its new application in biological control of plant diseases. The research results of the present invention prove that the tubercle microorganism has the potential to become a new type of microbial agent, which is of great significance for solving the prevention and control problems of existing biocontrol agents such as narrow antibacterial spectrum and poor combined prevention effect.

[0112] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0113] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0114] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Predatory tube mushroom strain NSE202, characterized in that The strain NSE202 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 3, 2023, with the deposit number CGMCCNO.26375.

2. A culture comprising the predatory tube mushroom strain NSE202 according to claim 1.

3. A plant disease biological agent, characterized in that: The biological agent includes strain NSE202, a culture of strain NSE202, or a mixture of one or more of the culture of strain NSE202.

4. A plant disease biological agent according to claim 3, characterized in that: The culture includes bacterial liquid of the strain, fermentation liquid of the strain or sterile filtrate of the fermentation liquid.

5. A biological fertilizer, characterized in that: The biological fertilizer comprises the biological agent as claimed in claim 3 and plant growth nutrient elements.

6. A plant disease biological agent according to claim 3 or a biological fertilizer according to claim 5, characterized in that: The biocontrol agent or biofertilizer is processed into an agriculturally acceptable preparation.

7. Use of the predatory tube mushroom strain NSE202 according to claim 1 or the culture according to claim 2 in controlling plant pathogens.

8. The use according to claim 7, characterized in that: The plant pathogens include plant pathogenic bacteria, plant pathogenic fungi and plant pathogenic oomycetes.

9. The use according to claim 8, characterized in that: The plant pathogenic bacteria include any one of Erwinia amylovora, Pectobacterterium carotovorum subsp. Carotovorum, Dickeya fangzhongdai, Acidovorax citrulli, and Pseudomonas syringaepv. Syringae; The plant pathogenic fungi include any one of Verticillium dahliae, Fusarium oxysporum f.sp. vasinfectum, Fusarium verticillioides, Rhizoctonia solani, Fusarium culmorum, Alternaria tenuissima, and Valsamali var. pyri; The plant pathogenic oomycetes include Phytopahthora capsici.

10. A method for biological control of plant diseases, characterized in that: The predatory tube mushroom strain NSE202 according to claim 1, the culture according to claim 2, the plant disease biological agent according to claim 3 or the biological fertilizer according to claim 5 is applied to plants to prevent and control the plants from being infected by plant pathogens.

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