Compound microbial agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms and particularly relates to a compound microbial agent as well as a preparation method and application thereof. The invention provides a compound microbial agent, the microbial agent comprises a paenibacillus polymyxa w-115 fermentation liquid and a trichoderma asperellum W1 spore liquid, the preservation number of the paenibacillus polymyxa w-115 is CGMCC (China General Microbiological Culture Collection Center) NO.30027, and the preservation number of the trichoderma asperellum W1 is CGMCC NO.40312. The invention further provides a preparation method of the compound microbial agent. The embodiment of the invention shows that the compound microbial agent can effectively promote vegetative growth of plants, improve fruit quality and improve plant root systems and soil microflora.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a compound microbial inoculant, a preparation method thereof, and an application thereof. Background Art

[0002] Since the 21st century, with the rapid growth of the population, the demand for agricultural products has increased sharply. To address this challenge and respond to the national policy call for green and environment-friendly agriculture, scientific research personnel have actively explored new microbial inoculants to replace or partially replace chemical pesticides and chemical fertilizers.

[0003] Due to its strong stress resistance, heat resistance, acid and alkali resistance, etc., Bacillus has high biomass and spore yield in industrial production, which is beneficial to the processing and storage of products, and has become an ideal object for screening microbial inoculants. Bacillus can secrete hormones to promote the growth of plant roots, enhance the absorption of mineral nutrients and water by roots, so as to achieve the growth promotion effect; at the same time, it can colonize around the plant roots, induce the plant to produce systemic resistance, and improve the plant's ability to resist pathogenic bacteria.

[0004] However, in terms of promoting plant growth and controlling plant diseases, the existing technologies mostly rely on the use of single microbial strains. Although the combined use of different strains is expected to achieve complementary advantages, the commonly used compound microbial inoculants on the market are the combination of different Bacillus species. For example, the compound microbial inoculant of Brevibacillus brevis and Bacillus velezensis can significantly promote the early ripening of grapes, improve the fruit quality, and also significantly increase the soil enzyme activity (Che Jianmei et al., Biotechnology Bulletin). It is worth noting that although the application of Bacillus microbial inoculants in the agricultural field has achieved certain results, the specific effects of the compound microbial inoculant of Paenibacillus polymyxa and Trichoderma asperellum on plant growth promotion and quality improvement have not been reported in the existing technologies. Summary of the Invention

[0005] The purpose of the present invention is to provide a compound microbial inoculant, which includes the fermentation broth of Paenibacillus polymyxa w-115 and the spore liquid of Trichoderma asperellum W1. The embodiments of the present invention show that the compound microbial inoculant can effectively promote the vegetative growth of plants, improve the fruit quality, and improve the plant roots and soil microbial flora.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] First, the present invention provides a Paenibacillus polymyxa w-115, and the preservation number of the Paenibacillus polymyxa w-115 is CGMCC NO.30027.

[0008] The present invention provides a Trichoderma asperellum W1, and the preservation number of the Trichoderma asperellum W1 is CGMCC NO. 40312.

[0009] Second, the present invention provides a compound microbial inoculum, which includes the fermentation broth of Paenibacillus polymyxa w-115 and the spore liquid of Trichoderma asperellum W1 as described above.

[0010] Preferably, the viable count of the Paenibacillus polymyxa w-115 fermentation broth in the compound microbial inoculum is ≥1.0×10 8 CFU / mL, and the effective spore count of the Trichoderma asperellum W1 spore liquid is ≥8.0×10 8 CFU / mL.

[0011] Third, the present invention provides a compound microbial preparation, and the active ingredients of the compound microbial preparation include Paenibacillus polymyxa w-115 and Trichoderma asperellum W1 as described above and / or the metabolites containing the strains.

[0012] Fourth, the present invention provides a preparation method of a compound microbial inoculum, and the preparation method includes the following steps:

[0013] S1: Culturing the Paenibacillus polymyxa w-115 strain in a liquid medium to obtain a Paenibacillus polymyxa w-115 seed liquid; activating the Trichoderma asperellum W1 strain and inoculating it into a second fermentation medium for scale-up culture to obtain a Trichoderma asperellum W1 mixture, and then performing shallow pan culture and air-drying to obtain a Trichoderma asperellum W1 spore inoculum;

[0014] S2: Inoculating the Paenibacillus polymyxa w-115 seed liquid obtained in step S1 into a first fermentation medium for the first fermentation culture to obtain a Paenibacillus polymyxa w-115 fermentation broth; aseptically diluting the Trichoderma asperellum W1 spore inoculum obtained in step S1 to obtain a Trichoderma asperellum spore liquid;

[0015] S3: Mixing the Paenibacillus polymyxa w-115 fermentation broth and the Trichoderma asperellum spore liquid obtained in step S2 according to a certain volume ratio to obtain a compound microbial inoculum.

[0016] Preferably, the culture temperature for preparing the Paenibacillus polymyxa w-115 seed liquid in step S1 is 28°C - 30°C, and the rotation speed is 160 - 200 rpm.

[0017] Preferably, the inoculation amount of the Paenibacillus polymyxa w-115 seed liquid in step S2 is 8% - 10% of the total volume of the first fermentation medium, the first fermentation culture time is 19 - 24 h, the temperature is 30 - 32 °C, the rotation speed is 160 - 200 rpm, and the pH is 7.0 - 7.4.

[0018] Preferably, the time for the enlarged culture of Trichoderma asperellum W1 in step S1 is 3 - 4 d, and the temperature is 25 °C - 28 °C; the inoculation amount of Trichoderma asperellum W1 is 1 - 3 pieces of bacterial colonies.

[0019] Preferably, the volume ratio of the Paenibacillus polymyxa w-115 fermentation liquid to the Trichoderma asperellum W1 spore liquid in step S3 is 1:1 - 1:10.

[0020] Fifth, the present invention provides an application of the above-mentioned composite microbial inoculant or the above-mentioned composite microbial preparation or the microbial inoculant prepared by the above-mentioned preparation method in promoting one or more of plant vegetative growth, improving fruit quality, improving plant roots and soil microbial flora.

[0021] Sixth, the present invention provides an application method as described above. The application method is: diluting the above-mentioned composite microbial inoculant 10 - 80 times and then irrigating the roots of plants; the application frequency is once every 7 days in the first month, and once every 21 days starting from the second month until harvest; when applying, the irrigation amount of the diluted microbial preparation each time is 150 mL / plant.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The composite microbial inoculant provided by the present invention includes the Paenibacillus polymyxa w-115 fermentation liquid and the Trichoderma asperellum W1 spore liquid. The viable count of Paenibacillus polymyxa w-115 in the composite microbial inoculant is ≥ 1.0×10 8 CFU / mL, and the effective spore count of Trichoderma asperellum W1 in the composite microbial inoculant is ≥ 8.0×10 8 CFU / mL.

[0024] (2) The composite microbial inoculant obtained by compounding the Paenibacillus polymyxa w-115 fermentation liquid and the Trichoderma asperellum W1 spore liquid of the present invention can promote plant vegetative growth, improve fruit quality, and can also increase the yield, effectively improving the root system and the structure of the soil microbial flora.

[0025] (3) The results of the examples show that: 1) The composite microbial inoculant has a promoting effect on the growth of strawberry roots, increasing by 3.2% compared with the control, and increasing by 1.2% and 1.6% respectively compared with the single inoculants W1 and w-115. The composite microbial inoculant treatment shows the best performance in terms of plant height and chlorophyll index;

[0026] 2) For the treatment with the compound microbial inoculant, the average single fruit weight, sugar content, and Vc content increased by 1.45 - 2.94%, 2.5 - 3.93%, and 2.97 - 4.41% respectively compared to CK;

[0027] 3) The compound microbial inoculant can significantly increase the bacterial diversity in the strawberry root - zone soil. Its Shannon and Simpson indices are the highest, being 9.55 and 0.997 respectively, and increasing by 5.29% and 1.53% respectively compared to the control. It can be seen that the compound microbial inoculant of the present invention has good application prospects in plant cultivation.

[0028] Biological deposit certificate

[0029] Trichoderma asperellum W1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 5, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC NO. 40312.

[0030] Paenibacillus polymyxa w - 115 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 14, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC NO. 30027. Brief description of the drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 The left figure is a schematic diagram of the photo of Paenibacillus polymyxa w - 115 cultured on NA medium at 30°C for 24 h; the right figure is a schematic diagram of the photo of Trichoderma asperellum W1 cultured on PDA medium at 28°C for 3 days.

[0033] Figure 2 It is a picture of the strawberry root system for Example 3, which was taken by the Canadian Regent WinRHIZO root system analysis system; among them, from left to right are the pictures of strawberry root systems treated differently.

[0034] Figure 3 It is a schematic diagram of the soil bacterial community composition at the phylum classification level for different treatments in Example 5. Detailed implementation mode

[0035] The present invention provides a Paenibacillus polymyxa w-115, and the preservation number of the Paenibacillus polymyxa w-115 is CGMCC NO. 30027.

[0036] Paenibacillus polymyxa belongs to the phylum Firmicutes, the family Bacillaceae, and the genus Paenibacillus. It is a spore-forming Gram-positive bacterium that lives aerobically or facultatively anaerobically. Its cells are straight rods, sized (0.5-2.5 μm) × (1.2-10 μm), with a G+C content of 40%-50%; its colony characteristics are mostly light yellow or white and viscous, with a moist and smooth surface; it can move using peritrichous flagella, and produces elliptical spores in swollen sporangia; the optimal growth pH is 7.0, and the optimal temperature is 28-35 °C, as Figure 1 shown in the left figure.

[0037] The present invention provides a Trichoderma asperellum W1, and the preservation number of the Trichoderma asperellum W1 is CGMCC NO. 40312. The mycelium of Trichoderma asperellum W1 grows fast, is creeping, flat, the colony is relatively large and nearly circular, green, the surface is closely like a blanket, and the edge is white and flocculent, producing a large number of spores, as Figure 1 shown in the right figure.

[0038] The present invention provides a compound microbial inoculum, and the compound microbial inoculum includes the above-mentioned Paenibacillus polymyxa w-115 and Trichoderma asperellum W1.

[0039] The present invention also provides a compound microbial preparation, and the active ingredients of the compound microbial preparation include the above-mentioned Paenibacillus polymyxa w-115 and Trichoderma asperellum W1 and / or the metabolites containing the strains.

[0040] The present invention also provides a preparation method of a compound microbial inoculum, and the preparation method includes the following steps:

[0041] S1: Culturing the above-mentioned Paenibacillus polymyxa w-115 strain in a liquid medium to obtain a Paenibacillus polymyxa w-115 seed liquid; activating the above-mentioned Trichoderma asperellum W1 strain and inoculating it into a second fermentation medium for scale-up culture to obtain a Trichoderma asperellum W1 mixture, and then performing shallow pan culture and air-drying to obtain a Trichoderma asperellum W1 spore inoculum;

[0042] S2: Inoculating the Paenibacillus polymyxa w-115 seed liquid obtained in step S1 into a first fermentation medium for the first fermentation culture to obtain a Paenibacillus polymyxa w-115 fermentation liquid; aseptically diluting the Trichoderma asperellum W1 spore inoculum obtained in step S1 to obtain a Trichoderma asperellum spore liquid;

[0043] S3: Mix the Paenibacillus polymyxa w-115 fermentation broth and Trichoderma asperellum spore liquid obtained in step S2 in a certain volume ratio to obtain a composite microbial inoculant.

[0044] For the preparation of Paenibacillus polymyxa in step S1, the composition of the liquid medium is preferably 15 g of glucose, 8 g of peptone and 3 g of beef extract in 1000 mL of sterile distilled water.

[0045] When preparing the seed liquid of Paenibacillus polymyxa w-115, the culture temperature is 28 - 30 °C, preferably 30 °C, and the rotation speed is 160 - 200 rpm, preferably 160 rpm.

[0046] In the present invention, the inoculation amount of the seed liquid of Paenibacillus polymyxa w-115 in step S2 is 8% - 10% of the total volume of the first fermentation medium.

[0047] The first fermentation medium uses sterile distilled water as a solvent and includes the following components in mass concentration: 18 g / L of soybean cake powder, 12 g / L of corn flour, 33.3 g / L of yeast extract, 5 g / L of glucose, 0.3 g / L of dipotassium hydrogen phosphate, 4 g / L of disodium hydrogen phosphate, 0.3 g / L of manganese sulfate and 0.3 g / L of magnesium sulfate.

[0048] The pH of the fermentation medium is 7.0 - 7.4, preferably 7.1 - 7.2, and more preferably 7.2. The fermentation medium is preferably sterilized at high temperature. The high-temperature sterilization temperature is 115 - 123 °C, and the high-temperature sterilization time is preferably 0.6 h.

[0049] The time of the first fermentation culture is 19 - 24 h, preferably 20 - 22 h, and further preferably 20 h. The first fermentation temperature is 30 - 32 °C, preferably 31 - 32 °C, and further preferably 32 °C. The first fermentation rotation speed is 160 - 200 rpm, preferably 170 - 180 rpm, and further preferably 180 rpm.

[0050] During the first fermentation process, sampling starts after 16 h of fermentation, and sampling is carried out every 3 h. Colony plate counting and microscopic examination are performed. Fermentation stops when there is no obvious change in the viable bacteria count and spore ratio for 2 times, and Paenibacillus polymyxa w-115 fermentation broth is obtained. After fermentation is completed, the viable bacteria in the fermentation broth of w-115 ≥ 2.0×10 9 CFU / mL, and the proportion of spore quantity is ≥ 80%.

[0051] Inoculate Trichoderma asperellum W1 into the wheat grain mixture medium for subculture to obtain a Trichoderma asperellum W1 mixture; perform shallow pan culture on the Trichoderma asperellum W1 mixture to obtain a Trichoderma asperellum microbial inoculant;

[0052] The time for the enlarged culture is 3 - 4 days; after culturing for 3 - 4 days in the present invention, the surface of the wheat grain mixture medium begins to be covered with white mycelium, and dark green spores appear at some bag mouths. The temperature for the enlarged culture is 25 - 28 °C, preferably a constant temperature of 26 °C. The inoculation amount of Trichoderma asperellum W1 is 1 - 3 pieces of bacterial lawns, preferably 2 pieces of bacterial lawns; the diameter of the bacterial lawn is preferably 0.6 cm.

[0053] After the shallow pan culture, the present invention air-dries and sieves the culture obtained from the shallow pan culture to obtain a compound microbial inoculant. The air-drying in the present invention is preferably natural air-drying; the time for air-drying is preferably 48 h; the sieving is preferably through a 400-mesh sieve. After fermentation, the spore content of Trichoderma asperellum W1 ≥ 1.6×10 10 CFU / mL, and after diluting 10 times with water, a Trichoderma asperellum spore solution is obtained.

[0054] In the present invention, the culture of the wheat grain mixture medium preferably includes the following components in mass percentage: wheat grains 82 - 90%, wheat bran 2 - 8%, rice husk 6 - 9%, and calcium carbonate 1 - 2%. The wheat grain mixture medium in the present invention is preferably a wheat grain mixture medium sterilized at 115 - 121 °C for 20 - 30 min; the wheat grain mixture medium is also called the second fermentation medium.

[0055] Mix the fermentation broth of Paenibacillus polymyxa w-115 and the Trichoderma asperellum W1 spore solution according to a certain volume ratio, preferably a volume ratio of 1:1 - 1:10, more preferably 1:10. The effective viable count content of Paenibacillus polymyxa w-115 in the compound microbial inoculant ≥ 1.0×10 8 CFU / mL, and the effective spore count content of Trichoderma asperellum W1 ≥ 8.0×10 8 CFU / mL.

[0056] The present invention provides an application of the above-mentioned compound microbial inoculant or the microbial inoculant prepared by the above-mentioned preparation method in one or more of promoting plant vegetative growth, improving fruit quality, improving plant roots and soil microflora.

[0057] In the present invention, the plant is preferably tomato, cauliflower, cabbage, strawberry, more preferably strawberry; the strawberry is preferably Yuexiu.

[0058] In the present invention, the application method is to dilute the compound microbial preparation 10 - 80 times and then pour it into the rhizosphere of the plant by irrigation. The dilution multiple is preferably 10 - 40 times, and further preferably 10 - 20 times; the application frequency in the present invention is preferably once every 7 days in the first month, and once every 21 days starting from the second month until harvest; when applying, the irrigation amount of the diluted microbial preparation per plant is preferably 150 mL / plant.

[0059] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] In the embodiments of the present invention, the production processes, experimental methods or detection methods involved, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the art, and are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the name and apply the corresponding equipment, and implement them under conventional conditions or conditions recommended by the manufacturer.

[0061] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0062] Example 1 Preparation of Trichoderma asperellum W1 Spore Liquid

[0063] 1. Culture Medium

[0064] PDA culture medium: 200 g of potatoes, 20 g of glucose, 20 g of agar, 1000 mL of distilled water

[0065] The second fermentation culture medium is a wheat grain mixture culture medium: 88 wt.% of wheat grains, 5 wt.% of wheat bran, 6 wt.% of rice husks, 1 wt.% of calcium carbonate, by mass: 8.8 kg of wheat grains, 0.5 kg of wheat bran, 0.6 kg of rice husks, 0.1 kg of calcium carbonate.

[0066] 2. Preparation method of wheat grain mixture culture medium:

[0067] (1) Select wheat, wheat bran and rice husks without pests and diseases and place them in a container of clear water until the wheat grain mixture absorbs enough water;

[0068] (2) Lift the mixture in (1) and put it into boiling water and boil for 30 min;

[0069] (3) Filter the excess water from the mixture in (2), spread it out, naturally air-dry the surface water and then bag it. Each edible mushroom bag is filled with about 400 g of air-dried mixture, and 1% of calcium carbonate is added to the air-dried mixture. Tie the cotton plug tightly with a rubber band to seal the edible mushroom bag, and wrap the bag mouth with newspaper.

[0070] (4) Put the bagged cooked wheat grain mixture culture medium into a high-pressure steam sterilizer and sterilize it at a temperature of 121 °C for 30 min, and cool it for standby.

[0071] (5) After Trichoderma asperellum W1 was activated on a PDA plate, a puncher was used to cut it into agar discs, which were then inoculated into the wheat grain mixture medium in the edible mushroom bags, 2 discs per bag. Then, the bag mouths were tightly tied with cotton plugs. After slightly turning the spawn bags, they were placed in an incubator at a constant temperature of 26 °C for cultivation.

[0072] (6) After 3 days of cultivation, the surface of the wheat grain mixture began to be covered with white mycelia, and spores appeared at the mouths of some bags. After turning the wheat grain mixture medium, the bag mouths were opened and transferred to shallow trays for shallow tray fermentation. A layer of plastic wrap was covered on the shallow trays, and a 12 h light / 12 h dark treatment was carried out to induce the production of Trichoderma spores.

[0073] Every 3 days, the water droplets accumulated on the plastic wrap were shaken off, and the agglomerated culture was loosened with a wooden board. 6 days after inoculation, the spores could cover the surface of the wheat grain mixture in piles. At this time, the plastic wrap was removed, and the culture substrate was air-dried under natural conditions for 48 h. After sieving through a 400-mesh metal sieve, a large amount of Trichoderma asperellum W1 microbial inoculum could be obtained. The finally prepared Trichoderma asperellum W1 microbial inoculum (effective viable bacteria content ≥ 1.6×10 10 CFU / g) was diluted 10 times with sterile water to obtain Trichoderma asperellum W1 spore liquid.

[0074] Example 2 Preparation of Paenibacillus polymyxa w-115 Fermentation Broth

[0075] 1. Medium

[0076] NA medium: peptone 10 g, yeast extract 0.5 g, beef extract 3 g, distilled water 1000 mL

[0077] 2. Preparation method

[0078] A single colony of w-115 was picked and inoculated into NA liquid medium, and cultured at 30 °C and 160 rpm for 48 h to obtain a seed liquid. The effective viable bacteria count of the obtained seed liquid was 1×10 9 CFU / mL.

[0079] The seed liquid of w-115 was inoculated into a fermenter at an inoculation amount of 8% by volume. The fermentation medium was the first fermentation medium. The fermentation temperature was 32 °C, the fermentation rotation speed was 180 rpm, and the fermentation pH was 7.2. Sampling began after 16 h of fermentation, and sampling was carried out every 3 h for colony plate counting and microscopic examination. When the fermentation reached 23 h, the fermentation was stopped because there was no obvious change in the viable bacteria count and spore ratio for 2 times. After fermentation was completed, the viable bacteria count of the fermentation broth of w-115 ≥ 2.0×10 9 CFU / mL, and the proportion of spore quantity was 82%.

[0080] Example 3 Effect of Compound Microbial Inoculum on Vegetative Growth of Continuous Cropping Strawberries

[0081] The test crop was: strawberry (cultivar Yuexiu)

[0082] The compound microbial inoculant is prepared by mixing the Trichoderma asperellum W1 spore liquid and the Paenibacillus polymyxa w-115 fermentation liquid obtained in Examples 1 and 2 in a volume ratio of 1:10.

[0083] The experiment was carried out at A Huan Strawberry Horticultural Farm in Zhenhai District, Ningbo on September 5, 2021. Four treatments were set up. Among them, Treatment 1 was a 10-fold dilution of the Paenibacillus polymyxa w-115 fermentation liquid with an effective viable bacteria content ≥ 2.0×10 8 CFU·ml -1 , Treatment 2 was a 10-fold dilution of the Trichoderma asperellum W1 spore liquid (effective viable bacteria content ≥ 1.6×10 8 CFU·ml -1 ); Treatment 3 was a 10-fold dilution of the compound microbial inoculant; Treatment 4 was water, used as the control group.

[0084] On the 7th day after strawberry transplanting, the first application was made; then it was applied once a week. After 4 times (one month), it was changed to once every 21 days, and a total of 10 applications were made. The application amount per strawberry plant was 150 mL.

[0085] (1) During the budding stage, initial flowering stage, early fruit stage, and harvesting stage of strawberry growth, the plant height and chlorophyll SPAD value of strawberries were measured respectively; 20 consecutive strawberry plants were selected near the center of each plot. The distance from the highest point at the center of the strawberry to the ground surface at the root was measured with a ruler as the strawberry plant height. The SPAD (Soil and plant analyzer development) value was measured using a SPAD meter (chlorophyllmeter SPAD-502plus, Janpan) on the third functional leaf that unfolds outward from the strawberry heart leaf, and the average value was taken to reflect the chlorophyll level of strawberries. The results are shown in Table 1.

[0086] (2) After the strawberry fruit harvesting was completed, 10 consecutive strawberry plants were selected at the center of each plot. After carefully digging them out and gently washing the soil from the roots, the surface moisture was dried, and the fresh weight of the underground part of the strawberry roots was weighed; then it was blanched at 105°C for 30 min and dried to a constant weight at 70°C, and the dry matter weight of the underground part was weighed, as shown in Table 2. The strawberry root system pictures were taken by the Canadian Regent WinRHIZO root system analysis system, as Figure 2 shown

[0087] Table 1 Effects of compound microbial inoculant on the vegetative growth of strawberries

[0088]

[0089] As can be seen from Table 1: Compared with the single microbial agent, the combined use of w-115 and W1 (Treatment 3) has a promoting effect on the vegetative growth of strawberries. Among the four growth stages, Treatment 3 showed the best performance in terms of plant height and chlorophyll index, while the treatment with the lowest plant height was CK (Treatment 4).

[0090] The above results indicate that the fermentation broth of Paenibacillus polymyxa, the spore suspension of Trichoderma asperellum W1, and the compound microbial agent can all promote the growth of strawberry plant height and accelerate the synthesis of chlorophyll in strawberry leaves. Among them, the compound microbial agent of W1 and w-115 (Treatment 3) has a more obvious promoting effect than the single microbial agent (Treatment 1, 2).

[0091] Table 2 Effects of compound microbial agent on the root growth of strawberries

[0092]

[0093] As can be seen from Table 2, the application of the compound microbial agent (Treatment 3) can improve the root growth of strawberry plants and effectively increase the dry matter weight of the roots of strawberry plants. The single microbial agents (Treatment 1, 2) increased by 1.6% - 2.0% respectively compared with the control (Treatment 4). The compound microbial agent showed the best performance, increasing by 3.2% compared with the control, and increasing by 1.2% and 1.6% respectively compared with the single microbial agents of the spore suspension of Trichoderma asperellum W1 and the fermentation broth of Paenibacillus polymyxa w-115.

[0094] Example 4 Effects of compound microbial agent on the fruit quality of continuous cropping strawberries

[0095] The test method, test crop, and test treatment were the same as in Example 3.

[0096] Investigation of strawberry yield and fruit quality indicators:

[0097] After the strawberry fruits were harvested, the average single fruit weight, sugar content, and vitamin C content of the 1st - 4th generation fruits were measured respectively.

[0098] Twenty strawberry fruits with intact appearance and uniform fruit shape were selected for the determination of the average single fruit weight of strawberries. The sugar content of strawberries was measured using a WYH - type handheld refractometer (ATAQQ co.td.Janpan); the titratable acid content was determined by sodium hydroxide titration and converted to the percentage content of citric acid monohydrate; the vitamin C content of strawberries was determined using an ascorbic acid assay kit (Nanjing Jiancheng Bioengineering Institute).

[0099] Yield investigation: When the 1st generation fruit, 2nd generation fruit, 3rd generation fruit, and last generation fruit of strawberries were ripe, all the ripe strawberry fruits from each plant in each plot were picked from the beginning to the end of the harvest period, and the quality was counted. After conversion, the strawberry yield was obtained.

[0100] Table 3 Effects of compound microbial agent on the fruit quality of strawberries

[0101]

[0102] From the data in Table 3, it can be obtained that the treatments of applying the fermentation broth of Paenibacillus polymyxa w-115 and the spore liquid of Trichoderma asperellum W1 had higher average single fruit weight, sugar content, and Vc content in the fruits of batches 1 to 4 compared with the clear water control, indicating that applying microbial inoculants can improve the fruit quality of strawberries.

[0103] The treatment of applying the compound microbial inoculant of W1+w-115 showed the best performance among the above indicators. The average single fruit weight, sugar content, and Vc content increased by 1.45-2.94%, 2.5-3.93%, and 2.97-4.41% respectively compared with CK. Therefore, the compound microbial inoculant prepared by compounding W1 and w-115 can effectively improve the fruit quality of strawberries.

[0104] Example 5 Effect of compound microbial inoculant on the soil microbial flora of continuous cropping strawberries

[0105] The test method, test crop, and test treatment were the same as those in Example 3.

[0106] After the test, sampling points were selected by the five-point sampling method in each plot. Rhizosphere soil samples were collected by the shaking root method, mixed evenly and put into a sterile self-sealing bag, taken back to the laboratory with an ice box, and stored in a 4°C refrigerator.

[0107] Weigh 0.5 g of the well-mixed soil, extract and purify the soil DNA according to the DNA extraction kit. After the extracted DNA was detected by gel electrophoresis, the concentration of soil DNA was measured using a NanoDrop 2000c spectrophotometer.

[0108] Use the V3V4 forward primer: 343F TACGGRAGGCAGCAG (SEQ ID NO.1); reverse primer: 798R AGGGTATCTAATCCT (SEQ ID NO.2) to amplify the 16s rRNA gene for inspection.

[0109] Where the R group represents A or G;

[0110] Note: In the XML format file of the sequence list, r represents a or g.

[0111] Reaction system (30 μL): DNA ≥ 1 μL (50 ng), 2×Gflex PCR Buffer 15 μL, 1 μL each of 343F and 798R primers, 0.6 μL of Tks Gflex DNA Polymerase, and H2O was added to make up to 30 μL.

[0112] Reaction conditions: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 20 s, 26 cycles; 72°C for 5 min. After the amplified fragments were detected by gel electrophoresis without error, the soil DNA was sent to Shanghai OE Biotech Co., Ltd. for high-throughput sequencing and analysis.

[0113] Result analysis:

[0114] The soil bacterial community composition at the phylum classification level for different treatments is as Figure 3 shown. The relative abundance of Proteobacteria is the highest, accounting for 44.9% - 53.0%. The dominant bacteria at the phylum level under different treatments are Proteobacteria, Gemmatimonadetes, Bacteroidetes, Actinobacteria, and Firmicutes.

[0115] The relative abundances of Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes in T1 (Treatment 1), T2 (Treatment 2), and T3 (Treatment 3) are higher than those in the T4 (Treatment 4) control, indicating that the application of microbial inoculants can increase the relative abundances of Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes in the rhizosphere microbial flora of strawberry soil. Among them, the relative abundances of Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes in the T1 treatment increased by 4.14%, 16.5%, 47.2%, and 121% respectively compared with the control; the relative abundances of Proteobacteria, Actinobacteria, and Firmicutes in the T2 treatment increased by 20.2%, 14.9%, 26.0%, and 0.569% respectively compared with the control; the relative abundances of Proteobacteria, Actinobacteria, and Firmicutes in the T3 treatment increased by 11.7%, 15.0%, 33.2%, and 30.6% respectively compared with the control.

[0116] Since Paenibacillus polymyxa belongs to Firmicutes, the application of Bacillus can increase the proportion of Firmicutes in the soil microbial flora; Trichoderma asperellum is a beneficial fungus of the genus Trichoderma. Some studies have shown that Trichoderma can significantly improve soil physical and chemical properties, increase soil enzyme activity, increase the number of bacteria in the soil, and Trichoderma can also drive soil fungal interactions and enhance soil microbial diversity, thereby further optimizing the soil microbial community structure.

[0117] Therefore, the soil microbial diversity of different treatments was measured, and the results are shown in Table 4.

[0118] Table 4 Effects of different treatments on soil microbial diversity indices

[0119] Processing Chao1 Shannon Simpson Processing 1 1224.1 9.45 0.994 Processing 2 1236.2 9.42 0.994 Processing 3 1236.3 9.55 0.997 Processing 4 1222.8 9.07 0.982

[0120] Note: Chao1 reflects the richness of the community, and Shannon and Simpson reflect the species diversity of the community.

[0121] Result analysis: There was no significant difference in Chao1 between treatment 3 with compound microbial inoculant and control treatment 4; however, the Shannon and Simpson indices were significantly higher than those of control 4: The Shannon and Simpson indices of T1 were 9.45 and 0.994 respectively, increasing by 4.19% and 1.22% compared to the control; The Shannon and Simpson indices of T2 were 9.42 and 0.994 respectively, increasing by 3.86% and 1.22% compared to control 4; The Shannon and Simpson indices of T3 treatment were the highest, being 9.55 and 0.997 respectively, increasing by 5.29% and 1.53% compared to the control.

[0122] The improvement effect of T3 was the most obvious, indicating that applying the compound microbial inoculant was the best for improving the bacterial diversity in the soil of strawberry roots.

[0123] Example 5 Effects of different application frequencies of compound microbial inoculant on fruits of continuously cropped strawberries

[0124] The test crop was: strawberry (cultivar Yuexiu)

[0125] The microbial inoculant was Trichoderma asperellum W1 and Paenibacillus polymyxa w - 115 prepared in Examples 1 and 2;

[0126] A total of 4 treatments were set up in the test, namely: Treatment 1: Root irrigation application, once every 21 days; Treatment 2: Root irrigation application, once every 14 days; Treatment 3: Root irrigation application, once every 7 days; Treatment 4: Control, root irrigation with clear water, once every 7 days. Apply according to the above treatments in the month after transplanting, and apply every 21 days after one month until the strawberries are harvested.

[0127] After harvesting the strawberry fruits with different application frequencies of the compound microbial inoculant, when the first-generation, second-generation, third-generation, and last-generation fruits of strawberries were mature, pick all the mature strawberry fruits of each plant in each plot from the beginning to the end of the harvest period, and measure the average single fruit weight, sugar content, and vitamin C content of the 1st - 4th generation fruits respectively, as shown in Table 5.

[0128] Table 5 Effects of compound microbial inoculant on strawberry fruits

[0129]

[0130] Result analysis: Different application frequencies of the compound microbial inoculant can all improve the fruit quality of strawberries. Among them, treatment 3, that is, applying once every 7 days in the first month, had the most obvious improvement effect on the fruit quality of strawberries. The average single fruit weight of treatment 3 increased by 0.74 - 3.27% compared to the control, the sugar content increased by 0.73 - 1.84% compared to the control, and Vc increased by 0.04 - 3.26% compared to the control.

[0131] In summary, as can be seen from Examples 1 to 5, the compound microbial inoculant obtained by compounding the fermentation broth of Paenibacillus polymyxa w-115 and the spore liquid of Trichoderma asperellum W1 can promote the vegetative growth of plants, improve the fruit quality, increase the strawberry yield, and effectively improve the structure of the strawberry root system and soil microbial flora.

[0132] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite microbial agent, characterized in that: The composite microbial agent includes a fermentation liquid of Paenibacillus polymyxa W-115 and a spore liquid of Trichoderma aspergillus W1; The deposit number of the polymyxa bacillus w-115 is CGMCC NO.30027; The deposit number of the Trichoderma aspergillus W1 is CGMCC NO.40312.

2. The composite microbial agent according to claim 1, characterized in that: The number of viable bacteria of the fermentation liquid of Paenibacillus polymyxa W-115 in the composite microbial agent is ≥ 1.0×10 8 CFU / mL, the effective spore count of the Trichoderma spinulosa W1 spore solution is ≥8.0×10 8 CFU / mL.

3. A composite microbial preparation, characterized in that: The active ingredients of the composite microbial preparation include the polymyxa bacillus w-115 and Trichoderma aspergillus W1 described in claim 1 and / or metabolites containing the strains.

4. A method for preparing a composite microbial agent, characterized in that: The preparation method comprises the following steps: S1: Cultivating the Paenibacillus polymyxa W-115 strain according to claim 1 in a liquid culture medium to obtain a seed solution of Paenibacillus polymyxa W-115; Activating the Trichoderma aspergillus W1 strain according to claim 1 and inoculating it into a second fermentation medium for expansion culture to obtain a Trichoderma aspergillus W1 mixture, and then culturing it in a shallow dish and air-drying it to obtain a Trichoderma aspergillus W1 spore agent; S2: inoculating the polymyxa bacillus w-115 seed liquid obtained in step S1 into the first fermentation medium for the first fermentation culture to obtain polymyxa bacillus w-115 fermentation liquid; aseptically diluting the Trichoderma aspergillus W1 spore inoculum obtained in step S1 to obtain Trichoderma aspergillus spore liquid; S3: The fermentation liquid of Paenibacillus polymyxa W-115 obtained in step S2 and the spore liquid of Trichoderma aspergillus are mixed in a certain volume ratio to obtain a composite microbial agent.

5. The preparation method according to claim 4, characterized in that: The culture temperature for preparing the seed solution of Paenibacillus polymyxa W-115 described in step S1 is 28°C-30°C, and the rotation speed is 160-200 rpm.

6. The preparation method according to claim 4, characterized in that: In step S2, the inoculation amount of the polymyxa bacillus w-115 seed liquid is 8% to 10% of the total volume of the first fermentation medium, the first fermentation culture time is 19 to 24 hours, the temperature is 30 to 32° C., the rotation speed is 160 to 200 rpm, and the pH is 7.0 to 7.

4.

7. The preparation method according to claim 4, characterized in that: The time for expanding the culture of Trichoderma aspergillus W1 in step S1 is 3 to 4 days, and the temperature is 25° C. to 28° C.; the inoculation amount of Trichoderma aspergillus W1 is 1 to 3 mushroom lawns.

8. The preparation method according to claim 4, characterized in that: In step S3, the mixing volume ratio of the Bacillus polymyxa W-115 fermentation liquid and the Trichoderma aspergillus W1 spore liquid is 1:1 to 1:

10.

9. Use of the composite microbial agent according to any one of claims 1 to 2, the composite microbial preparation according to claim 3, or the composite microbial agent prepared by the preparation method according to any one of claims 4 to 8 in promoting plant nutritional growth, improving fruit quality, and improving plant root system and soil microbial flora.

10. The use according to claim 9, characterized in that: The application method is: dilute the above-mentioned composite microbial agent 10 to 80 times and then irrigate the roots of the plants; the application frequency is once every 7 days in the first month, and once every 21 days starting from the next month until harvest; when applying, the root irrigation volume of the diluted microbial agent is 150 mL / plant each time.

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