Preparation of trichoderma composite flora and application of trichoderma composite flora in prevention and control of crop fusarium wilt and soil improvement

Abstract

The invention discloses preparation of a trichoderma composite flora and application of the trichoderma composite flora in prevention and control of crop fusarium wilt and improvement of soil, and relates to the field of agricultural production, and the composite flora is prepared by mixing trichoderma asperellum (5 * 10 < 7 > CFU / L), streptomyces (108 CFU / L), Chinese monospora (108 CFU / L), ensifer adhesion (108 CFU / L), pseudomonas nitroreducens (108 CFU / L) and paenibacillus alvei (108 CFU / L) according to the volume ratio of 6: 1: 1: 1: 1: 1: 1: 1 (V: V). According to the composite flora in the scheme, the composite flora is used for promoting growth of cucumber plants, and compared with a clear water control group (Foc), the fresh weight of cucumbers is increased by 55.51%, the stem diameter is increased by 12.76%, and the plant height is increased by 34.25%. The composite flora can also effectively prevent and treat fusarium wilt of cucumbers, and the prevention and treatment effect reaches 71.43%. Compared with the Trichoderma asperellum FJ035 treated group, the Trichoderma asperellum FJ035 has the advantages that the Trichoderma asperellum FJ035 is better than the Trichoderma asperellum FJ035 which is independently applied in the aspects of promoting the growth of cucumber plants and preventing and treating cucumber fusarium wilt, and compared with the Trichoderma asperellum FJ035 treated group, the control effect is improved by 56.25%. Meanwhile, the composite flora can be used for improving soil and preventing and treating multi-crop fusarium wilt.

Description

1. Technical Field

[0001] The present invention relates to Trichoderma asperellum for agricultural production, and particularly relates to a preparation method and application of a composite microbial community mainly composed of Trichoderma asperellum FJ035 for controlling crop wilt disease and improving soil quality. 2. Background Art

[0002] Crop wilt disease caused by infection of Fusarium oxysporum is a worldwide soil-borne fungal disease, posing a serious threat to the main agricultural production areas in China. Long-term and extensive use of chemical fungicides to control wilt disease not only causes pesticide residues and environmental pollution, threatening human health, but also leads to a vicious cycle of increasing pesticide use doses and disease severity with the continuous increase of pathogen resistance. Although measures such as crop rotation have good effects, they are cumbersome to operate and costly. Fusarium oxysporum has multiple special forms and physiological races, and is prone to horizontal gene transfer, resulting in the loss of crop resistance during continuous planting. At present, the use of microbial agents to control wilt disease has been widely accepted, but most single microbial agents have unstable field effects and single functions. With the overplanting and fertilization of land in China's agricultural production, the soil quality is deteriorating continuously, and the continuous cropping obstacle is particularly prominent, such as soil nutrient element imbalance, frequent and severe occurrence of diseases, poor crop growth and development, etc., leading to a series of agricultural product safety problems related to soil health.

[0003] Trichoderma spp. is an important microbial resource for biological pesticides and biological fertilizers. Because of its simple fermentation and production and convenient use, it not only has good antagonistic effects on various soil-borne pathogenic fungi, but also can improve soil quality, and is widely used in agricultural production. However, in different field environments, single biocontrol strains may have a competitive relationship with native microbiota, with unstable colonization ability and single function, resulting in differences and instabilities in control effects. The control effects of currently known Trichoderma asperellum strains on plant soil-borne diseases are still not very satisfactory. Therefore, it is of great significance to reasonably combine native microorganisms that have a beneficial interaction with Trichoderma asperellum strains to construct a new type of microbial community for suppressing and preventing diseases and improving soil quality for solving crop wilt disease and improving soil quality. 3. Summary of the Invention

[0004] The present invention has first discovered a strain of Trichoderma asperellum FJ035, and the Latin taxonomic name of this strain is: Trichoderma asperellum. This strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 30, 2023. The abbreviation of the depositary institution is CGMCC, with the address: No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number: CGMCC No. 40942.

[0005] One object of the present invention is to provide a preparation method of a composite bacterial community for controlling crop fusarium wilt. This composite bacterial community takes Trichoderma asperellum FJ035 as the main member to promote crop growth and control fusarium wilt. Another object of the present invention is to provide an application for improving continuously cropped obstacle soil.

[0006] The technical solution adopted by the present invention is: to provide a preparation method and an application of a composite bacterial community for controlling crop fusarium wilt. The composite bacterial community is prepared by mixing Trichoderma asperellum FJ035, Streptomyces rubiginosus, Sinomonas atrogrisea, Ensifer adhaerens, Pseudomonas nitroreducens, and Paenibacillus alvei. The mixed bacterial community is prepared by mixing Streptomyces rubiginosus, Sinomonas atrogrisea, Ensifer adhaerens, Pseudomonas nitroreducens, and Paenibacillus alvei.

[0007] The Pseudomonas nitroreducens B1-22 selected in the present invention has a deposit number of CGMCC No. 29051, and this strain has been described in the patent "Pseudomonas nitroreducens B1-22 and Its Application" (CN 118620798 B). The Ensifer adhaerens B1-9 selected in the present invention has a deposit number of CGMCC No. 29052, and this strain has been described in the patent "Ensifer adhaerens B1-9 and Its Application" (CN118165895 B). The Sinomonas atrocyanea B1-1 selected in the present invention has a deposit number of CGMCC No. 29053, and this strain has been described in the patent "Sinomonas atrocyanea B1-1 and Its Application" (CN 118147022 B). The Paenibacillus alvei B1-35 selected in the present invention has a deposit number of CGMCC No. 29054, and this strain has been described in the patent "Paenibacillus alvei B1-35 and Its Application" (CN 118620797 B). The Paenibacillus alvei B1-33 selected in the present invention has a deposit number of CGMCC No. 29055, and this strain has been described in the patent "Paenibacillus alvei B1-33 and Its Application" (CN 118562685 B). The Streptomyces griseorubiginosus B1-3 selected in the present invention has a deposit number of CGMCC No. 29056, and this strain has been described in the patent "Streptomyces griseorubiginosus B1-3 and Its Application" (CN 118497090 B).

[0008] The above-mentioned complex microbial community is used to promote crop growth.

[0009] In the above-mentioned scheme, the complex microbial community is used to promote the growth of cucumber plants. Compared with the clear water control group (Foc), the fresh weight of cucumbers has increased by 55.51%, the stem diameter has increased by 12.76%, and the plant height has increased by 34.25%.

[0010] The effect of the above-mentioned complex microbial community in promoting crop growth is superior to that of Trichodema asperellum FJ035 and the mixed bacterial group respectively.

[0011] In the above - mentioned scheme, the composite microbial community is used to promote the growth of cucumber plants. Compared with the Trichodema asperellum FJ035 treatment group, the fresh weight of cucumbers increased by 54.69%, the stem diameter increased by 17.36%, and the plant height increased by 34.50%. Compared with the mixed - bacteria group treatment group, the fresh weight of cucumbers increased by 51.92%, the stem diameter increased by 19.90%, and the plant height increased by 15.22%.

[0012] The above - mentioned composite microbial community is superior to Trichodema asperellum FJ035 and the mixed - bacteria group in preventing and controlling plant diseases.

[0013] In the above - mentioned scheme, the control effect of the microbial community on cucumber fusarium wilt reaches 71.43%; compared with the Trichodema asperellum FJ035 treatment group, the control effect is increased by 56.25%. Compared with the mixed - bacteria group treatment group, the control effect is increased by 62.16%.

[0014] The above - mentioned mixed - bacteria group has no inhibitory effect on the growth of Trichodema asperellum FJ035.

[0015] In the above - mentioned scheme, there is no inhibitory effect on the growth of Trichoderma after the bacteria in the composite microbial community are mixed; Sinomonas sp. B1 - 1, Ensifer adhaerens B1 - 9, Pseudomonas nitroreducens B1 - 22 and Paenibacillus alvei B1 - 33 in the composite microbial community have a promoting effect on the growth of Trichodema asperellum FJ035 on insoluble phosphate medium and nitrogen - free medium.

[0016] The above - mentioned composite microbial community is used to improve the soil.

[0017] In the above - mentioned scheme, the composite microbial community significantly increases the contents of total nitrogen, organic matter, available phosphorus and available potassium in the soil, as well as the cation exchange capacity.

[0018] The above - mentioned composite microbial community can prevent and control fusarium wilt of multiple crops.

[0019] In the above - mentioned scheme, the greenhouse field control effect of the composite microbial community on cucumber fusarium wilt reaches 67.09%; the greenhouse field control effect on pepper fusarium wilt reaches 64.03%; the greenhouse field control effect on eggplant fusarium wilt reaches 66.99%; the greenhouse field control effect on tomato fusarium wilt reaches 66.79%.

[0020] Beneficial effects:

[0021] The composite microbial community provided by the present invention has a better ability to promote the growth of crops and prevent and control cucumber fusarium wilt than the application of single - strain Trichoderma. In production, it has a high control effect on fusarium wilt of various common melon crops such as cucumber fusarium wilt, pepper fusarium wilt, eggplant fusarium wilt, tomato fusarium wilt, etc., and the control effect is greater than 60%.

[0022] Depository description

[0023] Trichoderma asperellum FJ035, taxonomically named Trichoderma asperellum, was deposited at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was November 30, 2023, and the deposit number was CGMCC No. 40942.

[0024] Trichoderma asperellum FJ069, taxonomically named Trichoderma asperellum, was deposited by the inventor at the China General Microbiological Culture Collection Center (CGMCC) on June 19, 2019. The address of the depositary institution was No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), and the deposit number was CGMCC No. 17976.

[0025] Pseudomonas nitroreducens B1-22, taxonomically named Pseudomonas nitroreducens, was deposited by the inventor at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was November 20, 2023, and the deposit number was CGMCC No. 29051.

[0026] Ensifer adhaerens B1-9, taxonomically named Ensifer adhaerens, was deposited by the inventor at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was November 20, 2023, and the deposit number was CGMCC No. 29052.

[0027] Sinomonas atrocyanea B1-1, taxonomically named Sinomonas atrocyanea, was deposited by the inventor at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was November 20, 2023, and the deposit number was CGMCC No. 29053.

[0028] Paenibacillus alvei B1-35, taxonomically named Paenibacillus alvei, was deposited by the inventor at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was November 20, 2023, and the deposit number was CGMCC No. 29054.

[0029] Paenibacillus alvei B1-33, classified and named as Paenibacillus alvei, was deposited by the inventor at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC). The deposit address is No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is November 20, 2023, and the deposit number is CGMCC No. 29055.

[0030] Streptomyces griseorubiginosus B1-3, classified and named as Streptomyces griseorubiginosus, was deposited by the inventor at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC). The deposit address is No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is November 20, 2023, and the deposit number is CGMCC No. 29056. IV. Description of the Drawings

[0031] Figure 1 Cucumber plants treated with complex microbial community 1 for controlling cucumber Fusarium wilt in the pot experiment of the present invention.

[0032] Figure 2 Picture of the co-culture of the bacteria of complex microbial community 1 of the present invention and Trichoderma asperellum FJ035 in a nutrient-rich medium.

[0033] Figure 3 Picture of the co-culture of the bacteria of complex microbial community 1 of the present invention and Trichoderma asperellum FJ035 in a nutrient-deficient medium. V. Specific Embodiments

[0034] The present invention will be described in detail below in conjunction with specific embodiments. The embodiments are for understanding rather than limiting the present invention.

[0035] As introduced in the background art, the existing Trichoderma asperellum agents are still not very effective in controlling cucumber Fusarium wilt. The biological pesticides have unstable effects and are relatively demanding on conditions such as the temperature of the use environment and the soil community state. Based on this, the present invention provides a novel complex microbial community 1 for controlling cucumber Fusarium wilt.

[0036] Example 1: Isolation, preservation and determination of the ability to inhibit pathogenic bacteria of Trichoderma asperellum FJ035

[0037] 1. Trichoderma asperellum strain FJ035 was collected from a pumpkin field in Fuzhou, Fujian Province.

[0038] Soil sample collection: The five-point diagonal sampling method was used for soil collection. That is, 5 equal points were selected on the diagonal of the collection plot, and then a soil sampler with a depth of 25 cm was used to drill about 15 - 20 cm of soil samples at each point. Then the 5 soil samples were mixed evenly, and about 50 g of soil samples were taken by the quartering method and put into a self-sealing bag. They were sub-packed into 50 mL plastic bottles in the laboratory and stored at 4°C for later use.

[0039] Isolation of Trichoderma: Using the dilution plate method, take 10g of soil sample from the refrigerator and put it into a triangular flask containing 90mL of sterilized water and 5-10 glass beads with a diameter of 4mm, and then culture it on a shaker at 200r / min for 30min. Take 5mL and pour it into a triangular flask containing 45mL of sterilized water. After mixing, draw 5mL and transfer it to a triangular flask containing 45mL of sterilized water. After fully mixing, draw 0.2mL on a plate with a diameter of 9cm, spread it evenly with an applicator, and culture it in a constant temperature incubator at 28℃. After 48-72h, pick the colony that produces green conidia, transfer it to a PDA plate, and continue to culture it in a constant temperature incubator at 28℃. When the colony diameter grows to 4-5cm, transfer it to a PDA plate again for constant temperature culture at 28℃.

[0040] 2. Collection of Trichoderma spinulosa strain FJ035

[0041] The Trichoderma aspergillus strain FJ035 was vacuum freeze-dried and preserved at the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) on November 30, 2023, with the preservation number CGMCC No.40942 and a preservation period of 30 years.

[0042] 3. The identification result was Trichoderma aculeatus through morphological identification combined with molecular biology (gene sequencing). The TEF sequence is shown in Sequence Table 1.

[0043] 4. Trichoderma spinulosum FJ035 inhibits the growth of pathogenic fungi

[0044] The strain of Trichoderma spinulosa FJ035 and plant pathogenic fungi were inoculated on PDA plates and cultured at 28°C for 72-96h. The bacterial cakes were punched out from the edge of the colony with a 5mm puncher and then transferred to the PDA plates at the same time, with the two bacterial cakes 5cm apart. The plates were cultured at 28°C for 10 days and then the inhibition rate and reparasitism ability were calculated. The plates without Trichoderma and only with pathogens were used as controls. The experiment was repeated 3 times. The inhibition rate and reparasitism ability were calculated according to the following formula and standard:

[0045] Inhibition rate (%) = ((control pathogen radius - confrontation pathogen radius) / control pathogen radius) × 100%

[0046] Heavy parasitism ability classification standard:

[0047] +++++: The Trichoderma hyphae completely cover the pathogen.

[0048] ++++: Trichoderma hyphae have moderate coverage of pathogens.

[0049] +++: Trichoderma hyphae weakly cover the pathogen.

[0050] ++: The Trichoderma hyphae only weakly covered the pathogen.

[0051] +: Trichoderma hyphae and pathogenic hyphae do not cover each other.

[0052] Table 1 Antibacterial effect and hyperparasitic ability of Trichoderma asperellum FJ035 of the present invention

[0053]

[0054]

[0055] Trichoderma asperellum FJ035 of the present invention has a high antibacterial effect on common pathogenic bacteria of melon crops such as Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum f. sp. melonis, Fusarium oxysporum f. sp. luffae, and Fusarium oxysporum f. sp. niveum, and the antibacterial rate is greater than 70%.

[0056] The hyphae of Trichoderma asperellum FJ035 of the present invention have a certain hyperparasitic ability on common pathogenic bacteria of melon crops such as Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum f. sp. melonis, Fusarium oxysporum f. sp. luffae, and Fusarium oxysporum f. sp. niveum. Among them, the hyperparasitic ability on Fusarium oxysporum f. sp. cucumerinum is the strongest, and the hyphae of Trichoderma asperellum FJ035 completely cover the pathogenic bacteria.

[0057] Example 2: Preparation method of microbial community

[0058] 1. Activation treatment of required strains.

[0059] Streptomyces griseorubescens B1-3 with preservation number CGMCC NO.29056, Sinomonas atra B1-1 with preservation number CGMCC NO.29053, Ensifer adhaerens B1-9 with preservation number CGMCC NO.29052, Pseudomonas nitroreducens B1-22 with preservation number CGMCC NO.29051, Paenibacillus alvei B1-33 with preservation number CGMCC NO.29055, Paenibacillus alvei B1-35 with preservation number CGMCC NO.29054, Trichoderma asperellum FJ035 with preservation number CGMCC NO.40942, and Trichoderma asperellum FJ069 with preservation number CGMCC NO.17976 were subjected to activation treatment; the activation treatment method was: inoculating Trichoderma asperellum FJ035 and FJ069 on PDA plate medium and culturing at a constant temperature of 28 °C for 72 h; inoculating Streptomyces griseorubescens B1-3, Sinomonas atra B1-1, Ensifer adhaerens B1-9, Pseudomonas nitroreducens B1-22, Paenibacillus alvei B1-33, and Paenibacillus alvei B1-35 on solid TSB plate medium and culturing at a constant temperature of 30 °C for 72 h.

[0060] PDA medium: 200 g of potatoes, 20 g of glucose, 18 g of agar powder, 1 L of distilled water.

[0061] TSB solid medium: 17.0 g of tryptone, 3.0 g of soy peptone, 2.5 g of D(+)-glucose, 5 g of NaCl, 3.28 g of K2HPO4·3H2O, 18 g of agar, 1 L of distilled water, pH = 7.3.

[0062] 2. Preparation of spore suspension.

[0063] Preparation of Trichoderma spore suspension: The activated Trichoderma asperellum FJ035 and FJ069 strains were inoculated on PDA plates and cultured at 28 °C for 5 - 7 d. The spores on the PDA plates were scraped with a spreader to prepare a spore suspension with a concentration of 5×10 7 CFU / L and stored at 4 °C for later use.

[0064] Preparation of bacterial liquid: The activated single bacterial colonies were picked and inoculated into liquid TSB medium respectively, cultured at 30 °C and 210 r / min for 72 h, with a concentration of about 10 8 CFU / L and stored at 4 °C for later use.

[0065] TSB liquid medium: 17.0 g of tryptone, 3.0 g of soy peptone, 2.5 g of D(+)-glucose, 5 g of NaCl, 3.28 g of K2HPO4·3H2O, 1 L of distilled water, pH = 7.3, sterilized at 121 °C for 20 min for later use.

[0066] 3. Preparation of fermentation broth.

[0067] The bacterial liquid with a concentration of 10 8 CFU / L prepared in Example 2-2 was filtered through a 0.22 μm filter membrane to obtain bacterial fermentation broth; the bacterial fermentation broths were mixed in equal volumes, that is, Streptomyces griseorubiginosus B1-3: Sinomonas atrogrisea B1-1: Ensifer adhaerens B1-9: Pseudomonas nitroreducens B1-22: Paenibacillus alvei B1-33: Paenibacillus alvei B1-35 was 1:1:1:1:1:1 (V:V:V:V:V) to obtain a mixed bacterial fermentation broth.

[0068] 4. Preparation of composite flora.

[0069] The bacterial liquids with a concentration of 10 8 CFU / L were mixed in equal volumes, that is, Streptomyces griseorubiginosus B1-3: Sinomonas atrogrisea B1-1: Ensifer adhaerens B1-9: Pseudomonas nitroreducens B1-22: Paenibacillus alvei B1-33: Paenibacillus alvei B1-35 was 1:1:1:1:1:1 (V:V:V:V:V) to obtain a bacterial mixed liquid of the mixed bacterial group, and then the bacterial mixed liquid was mixed with a concentration of 5×10 7Equal volumes of the Trichoderma asperellum FJ035 spore suspension at 7 CFU / L were mixed to obtain a spore suspension of complex flora 1 with a bacteria:Trichoderma asperellum FJ035 ratio of 1:1 (V:V); the bacterial mixed liquid was mixed with an equal volume of the Trichoderma asperellum FJ069 spore suspension of the same species but different subspecies at a concentration of 5×10

[0070] Example 3: Growth promotion effect of the complex flora on cucumber seedlings and effect of preventing and controlling cucumber fusarium wilt

[0071] Table 1 Treatments of the pot experiment of the complex flora of the present invention

[0072]

[0073] The pot experiment of this study was carried out in the greenhouse of the Green Agro-biological Agent Creation Engineering Center of Hainan University, Hainan Province from August to October 2021. After cucumber seeds were disinfected with 75% ethanol and 1% (v / v) sodium hypochlorite for 10 min, they were rinsed 3 times with sterile water, placed in a sterile culture dish (for germination. After 48 h, cucumber seeds with consistent growth were selected and transplanted into pots (500 g of sterilized soil per pot, 4 seedlings were planted in each pot). Treatments were started 5 days after transplantation (Table 1), with 3 pots for each treatment and repeated three times. The plant height, stem diameter, fresh weight of 35-day-old cucumbers and the incidence of fusarium wilt were measured.

[0074] Grading standard for cucumber fusarium wilt:

[0075] Grade 0: The stem base and main root grow healthily without any rot or necrosis; Grade 1: There are a small number of rot lesions on the stem base and main root, and the cucumber shows no obvious wilting and is relatively healthy as a whole, with the rot or necrosis area less than 25%; Grade 2: The rot or necrosis area on the stem base and main root accounts for 25%-50% of the total area, and many fibrous roots are rotten or necrotic. The cucumber has mild wilting symptoms but can still grow; Grade 3: The rot or necrosis area on the stem base and main root accounts for 50%-75%, the wilting of the cucumber is obvious and difficult to recover, a large number of fibrous roots are necrotic, and the growth is significantly inhibited; Grade 4: 75%-100% of the roots are completely necrotic or the above-ground part completely wilts and dies.

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

[0077] Control effect (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100.

[0078] Table 2 Growth promotion of cucumber plants and disease inhibition ability of the complex flora of the present invention

[0079]

[0080] It can be seen that the effect of the composite microbial community 1 of the present invention in preventing and controlling plant diseases and its ability to promote plant growth are superior to those of the mixed bacteria, Trichodema asperellum FJ035, and the composite microbial community 2 constructed by Trichodema asperellum FJ069 of the same species but different subspecies.

[0081] Example 4: Interaction within the composite microbial community 1

[0082] 1. Effect of bacteria in the composite microbial community 1 on the growth of Trichodema asperellum FJ035 on a rich nutrient medium plate.

[0083] Colony antagonism: A mycelial disc with a diameter of 5 mm was punched from the edge of the Trichodema asperellum FJ035 colony and transferred to the center of a TSB plate. Then, two 5-cm parallel lines were drawn with a toothpick to pick single colonies of 6 bacteria at a distance of 2.5 cm from the center of the plate on both the left and right sides; after culturing the plate at 30 °C for 10 d, the inhibition rate was observed and calculated. Using only Trichodema asperellum FJ035 as the control, the experiment was repeated 3 times. The inhibition rate was calculated according to the following formula and standard:

[0084] Inhibition rate (%) = ((radius of the control Trichodema asperellum FJ035 - radius of the confronting Trichodema asperellum FJ035) / radius of the control Trichodema asperellum FJ035) × 100%

[0085] Bacterial liquid antagonism: A mycelial disc with a diameter of 5 mm was punched from the edge of the Trichodema asperellum FJ035 colony and transferred to the center of a TSB plate. Three 6-mm holes were punched on the TSB solid plate 2 cm away from the center, and the distance between adjacent two holes was equal. 100 μL of single bacterial liquid and mixed bacterial liquid were respectively dropped into the holes, and the experiment was repeated three times.

[0086] Fermentation broth antagonism: A mycelial disc with a diameter of 5 mm was punched from the edge of the Trichodema asperellum FJ035 colony and transferred to the center of a TSB plate. Three 6-mm holes were punched on the TSB solid plate 2 cm away from the center, and the distance between adjacent two holes was equal. 100 μL of single bacterial fermentation broth and mixed bacterial fermentation broth were respectively dropped into the holes, and the experiment was repeated three times.

[0087] Table 3 Effect of bacterial strains in the composite microbial community 1 of the present invention on the growth of Trichodema asperellum FJ035 on a rich nutrient medium plate

[0088]

[0089] It can be seen that the colonies and bacterial suspensions of Paenibacillus alvei B1-33 and B1-35 of the present invention have obvious inhibitory effects on the growth of Trichoderma asperellum FJ035, but their fermentation broths have no effect on the growth of Trichoderma asperellum FJ035; the mixed bacterial suspension and the mixed fermentation broth have no significant effect on the growth of Trichoderma.

[0090] 2. Effect of bacteria in composite flora 1 on the growth of Trichoderma asperellum FJ035 on a rich medium-deficient medium plate.

[0091] Antagonism of bacterial suspension: Take a mycelial cake with a 5-mm punch from the edge of the Trichoderma asperellum FJ035 colony and transfer it to the center of an insoluble phosphate, potassium feldspar, and nitrogen-free medium plate. Select three points on the solid plate 2 cm from the center, with the distance between adjacent two points. Spot inoculate 100 μL of a single bacterial suspension and a mixed bacterial suspension of bacteria. The experiment is repeated three times.

[0092] Table 4 Effect of bacterial strains in composite flora 1 of the present invention on the growth of Trichoderma asperellum FJ035 on a rich medium-deficient medium plate

[0093]

[0094]

[0095] It can be seen that on a rich medium-deficient medium plate, Streptomyces griseorubescens B1-3, Sinomonas atrocyanea B1-1, Ensifer adhaerens B1-9, Pseudomonas nitroreducens B1-22, and Paenibacillus alvei B1-33 and B1-35 of the present invention have no obvious inhibitory effect on the growth of Trichoderma asperellum FJ035; when the growth of Trichoderma asperellum FJ035 is inhibited on an insoluble phosphate and nitrogen-free medium, Sinomonas atrocyanea B1-1, Ensifer adhaerens B1-9, Pseudomonas nitroreducens B1-22, and Paenibacillus alvei B1-33 have a significant promoting effect on the growth of Trichoderma asperellum FJ035.

[0096] Example 5: Mitigation effect of composite flora 1 on the deterioration of physical and chemical properties of continuous cropping soil

[0097] The pot experiment of this study was carried out in the greenhouse of the Green Agro-biological Agent Creation Engineering Center of Sanya Nanfan Research Institute, Hainan University, Hainan Province from February to April 2024. After disinfecting cucumber seeds with 75% ethanol and 1% (v / v) sodium hypochlorite for 10 minutes, they were rinsed 3 times with sterile water and placed in a sterile petri dish for germination. After 48 hours, cucumber seeds with consistent growth were selected and transplanted into pots (the size of the pots was 60×40×16.5 cm, filled with 10 kg of cucumber continuous cropping soil, a total of 6 pots). Five days after transplantation, they were treated with the same volume of sterile water (Control) or complex microbial community 1 respectively, with 3 pots for each treatment and repeated three times. The plant height, stem diameter, fresh weight of 35-day-old cucumbers and the incidence of Fusarium wilt were measured. After 40 days, the soil was collected to measure the soil pH, total nitrogen, organic matter, available phosphorus, available potassium and cation exchange capacity, so as to understand the alleviating ability of the simplified functional microbial community application on the chemical properties of continuous cropping soil.

[0098] Table 5 Chemical properties of continuous cropping soil after the application of complex microbial community 1 of the present invention

[0099] It can be seen that after the application of complex microbial community 1 of the present invention, the contents of total nitrogen, organic matter, available phosphorus and available potassium in the soil, as well as the cation exchange capacity, can be significantly increased; there is no significant effect on the soil pH value.

[0100] Example 6: Control effect of complex microbial community 1 on Fusarium wilt of multiple crops

[0101] The greenhouse experiment of this study was carried out in the field greenhouse of the Green Agro-biological Agent Creation Engineering Center of Hainan University, Hainan Province from November 2023 to February 2024. Cucumber, pepper and tomato seeds were surface-disinfected and then rinsed 3 times with sterile water, placed in a sterile petri dish for germination. Cucumbers, peppers and tomatoes with consistent growth were selected and transplanted into seedling trays, and then transplanted into the field greenhouse when they grew to the two-leaf stage; eggplant seedlings at the two-leaf stage were directly purchased from the farmers' market in Danzhou City and transplanted. One week after transplantation, complex microbial community 1 was inoculated, and the pathogenic bacteria were inoculated again two days later. After 21-30 days, the incidence of Fusarium wilt of cucumbers, peppers, eggplants and tomatoes was measured.

[0102] Table 6 Control effect of complex microbial community 1 of the present invention on Fusarium wilt of multiple crops

[0103]

[0104] Complex microbial community 1 of the present invention has a high control effect on common melon crop Fusarium wilts such as cucumber Fusarium wilt, pepper Fusarium wilt, eggplant Fusarium wilt and tomato Fusarium wilt, and the control effect is greater than 60%.

[0105] Sequence Listing 1:

Claims

1. A Trichoderma complex population, characterized in that it is It consists of Streptomyces griseorubiginosus B1-3 with the preservation number of CGMCC NO.29056, Sinomonas atrogrisea B1-1 with the preservation number of CGMCC NO.29053, Ensifer adhaerens B1-9 with the preservation number of CGMCC NO.29052, Pseudomonas nitroreducens B1-22 with the preservation number of CGMCC NO.29051, Paenibacillus alvei B1-33 with the preservation number of CGMCC NO.29055, Paenibacillus alvei B1-35 with the preservation number of CGMCC NO.29054, and Trichoderma asperellum FJ035 with the preservation number of CGMCC NO.40942.

2. The composite flora according to claim 1, characterized in that: Streptomyces B1-3: Sinomonas atrogrisea B1-1: Ensifer adhaerens B1-9: Pseudomonas nitroreducens B1-22: Paenibacillus alvei B1-33: Paenibacillus alvei B1-35 were all mixed and configured into a mixed bacterial population at a volume ratio of 1:1:1:1:1:1 under the same concentration condition; Trichoderma asperellum FJ035 was mixed and configured into a composite bacterial population with the aforementioned mixed bacterial population at a volume ratio of 1:1 under the same concentration condition as each of the aforementioned individual bacteria.

3. Preparation of a Trichoderma composite bacterial population and its application in preventing and controlling crop fusarium wilt and improving soil; wherein the Trichoderma composite bacterial population consists of Streptomyces griseorubiginosus B1-3 with the preservation number of CGMCC NO.29056, Sinomonas atrogrisea B1-1 with the preservation number of CGMCC NO.29053, Ensifer adhaerens B1-9 with the preservation number of CGMCC NO.29052, Pseudomonas nitroreducens B1-22 with the preservation number of CGMCC NO.29051, Paenibacillus alvei B1-33 with the preservation number of CGMCC NO.29055, Paenibacillus alvei B1-35 with the preservation number of CGMCC NO.29054, and Trichoderma asperellum FJ035 with the preservation number of CGMCC NO.40942.

4. Preparation of a Trichoderma composite bacterial population and its application in promoting crop growth; wherein the Trichoderma composite bacterial population consists of Streptomyces griseorubiginosus B1-3 with the preservation number of CGMCC NO.29056, Sinomonas atrogrisea B1-1 with the preservation number of CGMCC NO.29053, Ensifer adhaerens B1-9 with the preservation number of CGMCC NO.29052, Pseudomonas nitroreducens B1-22 with the preservation number of CGMCC NO.29051, Paenibacillus alvei B1-33 with the preservation number of CGMCC NO.29055, Paenibacillus alvei B1-35 with the preservation number of CGMCC NO.29054, and Trichoderma asperellum FJ035 with the preservation number of CGMCC NO.40942.

5. Use of the composite flora according to claim 3 or 4, characterized in that: The growth of Trichoderma asperellum FJ035 is not inhibited by the mixed bacterial population.

6. The application of the composite flora according to claim 5, characterized in that: The mixing of bacteria in the complex microbial community has no inhibitory effect on the growth of Trichoderma; B1-1 of Sinomonas, B1-9 of Ensifer adhaerens, B1-22 of Pseudomonas nitroreducens, and B1-33 of Paenibacillus alvei in the complex microbial community have a promoting effect on the growth of Trichoderma asperellum FJ035 on the insoluble phosphate medium and nitrogen-free medium.

7. Use of the composite flora according to claim 3, characterized in that: The complex microbial community is used for soil improvement.

8. Use of the composite flora according to claim 7, characterized in that: The complex microbial community significantly increases the contents of total nitrogen, organic matter, available phosphorus, and available potassium in the soil, as well as the cation exchange capacity.

9. Use of the composite flora according to claim 3, characterized in that: The complex microbial community can prevent and control the wilt diseases of multiple crops.

10. The application of the composite flora according to claim 9, characterized in that: The wilt diseases of multiple crops are cucumber wilt disease, pepper wilt disease, eggplant wilt disease, and / or tomato wilt disease.

11. Use of the composite flora according to claim 4, characterized in that: The complex microbial community can promote the growth of cucumbers.

12. The application of the composite flora according to claim 11, wherein: The complex microbial community significantly increases the fresh weight, stem diameter, and plant height of cucumber plants.

13. Trichoderma asperellum FJ035 with antibacterial effect and hyperparasitic ability, characterized in that Its preservation number is CGMCC NO.40942.

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