Chaetomium f8 strain and use thereof

Abstract

The present invention belongs to the technical field of microorganisms, and specifically relates to Chaetomium F8 and the use thereof. The Chaetomium F8 is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC No.40973. The provided Chaetomium F8 can be used to effectively promote the growth of wheat, maize and rice. In addition, the provided Chaetomium F8 has the functions of promoting straw degradation and phosphorus solubilization, remarkably promotes soil agglomeration under the field condition, can provide strain resources for agricultural straw degradation, and provides reference for further development and utilization of Chaetomium.

Description

A Chaetomium F8 strain and its application

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on February 4, 2024, with application number CN202410154770.5 and invention name “A Chaetomium truncatulum F8 strain and its application”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present application belongs to the field of microbial technology, and specifically relates to a Chaetomium F8 strain and its application. Background Art

[0003] Chaetomium fungi are common endophytic fungi in plants and have biocontrol effects on a variety of plant diseases. However, little research has been conducted on Chaetomium truncatulum in the industry. The morphological characteristics of this species were described in detail by Asgari and Zare in 2011, but no functional reports have been found (Asgari, B., Zare, R., 2011. The genus Chaetomium in Iran, a phylogenetic study including six new species. MYCOLOGIA 103, 863-882.). Subsequently, Chaetomium truncatulum was reported to be a pathogenic fungus of agricultural pests (Mantzoukas, et al., 2020. Stored product pests as models for trapping entomopathogenic fungi from olive tree orchards in Western Greece. Journal of Stored Products Research 87, 101584.). Existing Chaetomium strains primarily control plant diseases and insect pests, but few are known to promote straw degradation or soil improvement. For example, patent CN105524841A discloses a broad-spectrum plant disease biocontrol Chaetomium strain that has beneficial effects against several common plant pathogens in production, such as Fusarium oxysporum, Botrytis cinerea, and root rot. However, it does not promote straw degradation or soil improvement.

[0004] Summary of the Invention

[0005] The purpose of this application is to make up for the deficiencies of the existing technology and provide a Chaetomium F8 strain and its application to promote wheat growth and have the functions of promoting straw degradation and solubilizing phosphorus.

[0006] The present application provides a Chaetomium truncatulum F8 strain, which is deposited in the China Center for Microorganism Collection with a deposit number of CGMCC No.40973.

[0007] The present application also provides the use of Chaetomium F8 and / or its metabolites described in the above technical solution in any one or more of the following:

[0008] (1) Promote straw degradation;

[0009] (2) Promote the dissolution of ineffective phosphorus and the degradation of sugars in the soil;

[0010] (3) Improve soil structure and promote plant growth.

[0011] Preferably, the plants include wheat, corn and rice.

[0012] The present application also provides a composite bacterial agent, which comprises Chaetomium F8 and Mortierella strain;

[0013] The Chaetomium sp. F8 is deposited in the China Center for Microbiological Collection with the deposit number CGMCC No. 40973;

[0014] The mortierella fungus system includes Mortierella elongata, Mortierella alpina, Mortierella sp. F34 and Mortierella capitata.

[0015] Preferably, the concentration of Chaetomium F8 in the composite bacterial agent is 3×10 7 ~8×10 7 cfu / g, the concentrations of Mortierella longi, Mortierella alpina, Mortierella unnamed F34 and Mortierella cephalosporium were 2×10 6 ~10×10 6 cfu / g.

[0016] The present application also provides a method for preparing the composite bacterial agent described in the above technical solution, comprising the following steps:

[0017] Mixing Chaetomium F8 and Mortierella strains for expansion culture to obtain a mixed bacterial solution; inoculating the mixed bacterial solution into a fermentation substrate for fermentation culture to obtain a composite bacterial agent;

[0018] The ratio of the number of live bacteria inoculated with Chaetomium F8, Mortierella longi, Mortierella capitis, Mortierella unnamed F34 and Mortierella alpina is 2-8:1:1:1:1;

[0019] Calculated by mass, the fermentation matrix includes 1000 parts of wheat straw, 80-120 parts of woody peat, 180-220 parts of soybean meal, 1.67-2.00 parts of potassium chloride, 0.96-1.10 parts of potassium dihydrogen phosphate and 6-12 parts of humic acid; the water content of the fermentation matrix is ​​35%-45%.

[0020] Preferably, the expanded culture comprises mixing Chaetomium F8 and Mortierella strains and then inoculating the mixture onto a culture medium, wherein the ratio of Chaetomium F8 to culture medium is 0.1-1 cfu:1 mL.

[0021] Preferably, the ratio of the mixed bacterial solution to the fermentation substrate is 100 mL: 100 g; the concentration of the mixed bacterial solution is 10 8 ~10 9 cfu / mL.

[0022] Preferably, the temperature of the expanded culture is 25° C., the rotation speed is 180 rpm, and the culture time is 5 days; the temperature of the fermentation culture is 20-28° C., and the culture time is 7 days.

[0023] The present application also provides the use of the composite bacterial agent described in the above technical solution or the composite bacterial agent prepared by the preparation method described in the above technical solution in any one or more of the following:

[0024] (1) Promote plant growth and increase crop yield;

[0025] (2) Improve plant disease resistance and stress resistance;

[0026] (3) Increase the nitrogen content and available phosphorus content in the soil;

[0027] (4) Promote the formation of soil aggregates. Beneficial effects

[0028] This application provides a Chaetomium truncatulum F8 strain, deposited with the China National Microorganism Collection Center under the accession number CGMCC No. 40973. Examples demonstrate that Chaetomium F8 can effectively promote wheat growth, promote straw degradation, and solubilize phosphate. This strain provides a bacterial resource for agricultural straw degradation and offers a reference for the further development and utilization of Chaetomium.

[0029] The present application compounds the Chaetomium F8 and the Mortierella fungus system to obtain a composite bacterial agent that can effectively promote the degradation of lignin, enhance the disease resistance of plants, and effectively promote plant growth, increase crop yields, and also has certain advantages in promoting the degradation of sugar substances.

[0030] Biological deposit information

[0031] Chaetomium F8, biologically classified as Chaetomium truncatulum, was deposited in the China Center for Microorganism Collection on November 7, 2023, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 40973. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments.

[0033] Figure 1 shows the colony morphology of the F8 strain;

[0034] Figure 2 is the identification evolutionary tree of the F8 strain;

[0035] FIG3 is a dry matter statistical result of the combined strains of Chaetomium F8 and Mortierella in liquid co-culture;

[0036] Figure 4 shows the combined culture of Chaetomium F8 and Mortierella composite inoculum;

[0037] FIG5 shows the corn field growth conditions of the control group and the experimental group in Test Example 5;

[0038] FIG6 shows the corn field growth conditions of the control group and the experimental group in Test Example 5;

[0039] Figure 7 shows the relative abundance of soil bacterial communities in the maize rhizosphere at the phylum level;

[0040] Figure 8 shows the relative abundance of soil bacterial communities in the maize rhizosphere at the family level;

[0041] Figure 9 shows the relative abundance of soil bacterial communities in the maize rhizosphere at the genus level;

[0042] Figure 10 shows the relative abundance of soil fungal communities in the maize rhizosphere at the phylum level;

[0043] Figure 11 shows the relative abundance of soil fungal communities in the maize rhizosphere at the family level;

[0044] Figure 12 shows the relative abundance of soil fungal communities in the maize rhizosphere at the genus level;

[0045] Figure 13 shows the growth of rice in saline-alkali soil;

[0046] Figure 14 shows the relative abundance of rice rhizosphere soil bacterial communities at the phylum level;

[0047] Figure 15 shows the relative abundance of rice rhizosphere soil bacterial communities at the family level;

[0048] Figure 16 shows the relative abundance of rice rhizosphere soil bacterial communities at the genus level;

[0049] Figure 17 shows the relative abundance of fungal communities in rice rhizosphere soil at the phylum level;

[0050] Figure 18 shows the relative abundance of fungal communities in rice rhizosphere soil at the family level;

[0051] Figure 19 shows the relative abundance of fungal communities in rice rhizosphere soil at the genus level. DETAILED DESCRIPTION

[0052] The present application provides a Chaetomium truncatulum F8 strain, with a deposit number of CGMCC No. 40973. The present application isolated and screened Chaetomium F8 strain from moderately saline-alkali soil in the Songnen Plain, and it has been shown to increase the straw degradation rate, the content of available phosphorus, and the concentration of reducing sugars in the culture medium.

[0053] Based on the effects of Chaetomium F8 provided herein, the use of Chaetomium F8 and / or its metabolites in promoting one or more of straw degradation, promoting the dissolution of unavailable phosphorus in soil, improving soil structure, and promoting plant growth also falls within the scope of protection of this application. In this application, the plants preferably include wheat, corn, and rice.

[0054] The present application also provides a composite bacterial agent, which comprises Chaetomium F8 and Mortierella strain;

[0055] The deposit number of Chaetomium F8 is CGMCC No.40973;

[0056] The mortierella fungus system includes Mortierella elongata, Mortierella alpina, Mortierella sp. F34 and Mortierella capitata.

[0057] In this application, the concentration of Chaetomium F8 in the composite bacterial agent is preferably 3×10 7 ~8×10 7 cfu / g; the concentrations of the long spore mold, alpine spore mold, unnamed spore mold F34 and cephalosporium are preferably 2×10 6 ~10×10 6cfu / g. The long spore mold described in this application is preferably the long spore mold with a deposit number of CGMCC No. 14151 in Patent No. ZL201710907142.X; the alpine spore mold is preferably the alpine spore mold with a deposit number of CGMCC No. 17077 in Patent No. ZL201911182423.9; the unnamed spore mold F34 is disclosed in Patent No. ZL202211478572.1 and has a deposit number of CGMCC No. 40122; the head spore mold is preferably the head spore mold with a deposit number of CGMCC No. 18560 in Patent No. ZL201911127227.1.

[0058] The present application also preferably provides a method for preparing the composite bacterial agent described in the above technical solution, comprising the following steps:

[0059] The Chaetomium F8 strain and the Mortierella strain are mixed and cultured to obtain a mixed bacterial solution; the mixed bacterial solution is inoculated into a fermentation substrate for fermentation culture to obtain a composite bacterial agent;

[0060] The ratio of the number of live bacteria inoculated with Chaetomium F8, Mortierella longi, Mortierella capitis, Mortierella unnamed F34 and Mortierella alpina is 2-8:1:1:1:1;

[0061] Calculated by mass, the fermentation matrix includes 1000 parts of wheat straw, 80-120 parts of woody peat, 180-220 parts of soybean meal, 1.67-2.00 parts of potassium chloride, 0.96-1.10 parts of potassium dihydrogen phosphate and 6-12 parts of humic acid; the water content of the fermentation matrix is ​​35%-45%.

[0062] In the present application, the Chaetomium F8 strain and the Mortierella strain are mixed and cultured to obtain a mixed bacterial liquid.

[0063] In the present application, the culture medium used for the expanded culture is preferably PD culture medium. The present application also preferably adds 4.76g of ammonium sulfate per liter of PD culture medium. The temperature of the expanded culture is preferably 25°C, the rotation speed is preferably 180rpm, and the culture time is preferably 5d; the expanded culture preferably includes mixing Chaetomium F8 and Mortierella system and inoculating them onto the culture medium, and the inoculation ratio of the Chaetomium F8 strain and the culture medium is preferably 0.1-1cfu / mL culture medium, and more preferably 0.5cfu / mL culture medium; the inoculated live bacteria count of the Chaetomium F8 strain, Mortierella longi, Mortierella capitis, Mortierella unnamed F34 and Mortierella alpina is preferably 2-8:1:1:1:1, more preferably 2:1:1:1:1:1.

[0064] After obtaining the mixed bacterial solution, the present application inoculated the mixed bacterial solution into a solid fermentation matrix for solid fermentation culture, with a ratio of 10 mL of the mixed bacterial solution inoculated per 100 g of the solid matrix to obtain a composite bacterial agent.

[0065] In the present application, the fermentation substrate comprises, by weight, 1000 parts of wheat straw, 80-120 parts of woody peat, 180-220 parts of soybean meal, 1.67-2.00 parts of potassium chloride, 0.96-1.10 parts of potassium dihydrogen phosphate, and 6-12 parts of humic acid, more preferably 1000 parts of wheat straw, 100 parts of woody peat, 200 parts of soybean meal, 1.67 parts of potassium chloride, 0.96 parts of potassium dihydrogen phosphate, and 10 parts of humic acid; the moisture content of the fermentation substrate is 35-45%, more preferably 40%; the fermentation culture temperature is preferably 20-28°C, and the culture time is preferably 7 days. In the present application, it is preferred that after culturing in a clean bench for 3 days, the culture is transferred to an indoor environment and continued for 4 days.

[0066] In this application, the ratio of the mixed bacterial solution to the fermentation substrate is preferably 100 mL: 100 g; the concentration of the mixed bacterial solution is preferably 10 8 ~10 9 cfu / mL, more preferably 10 9 cfu / mL.

[0067] The present application also provides the use of the composite bacterial agent described in the above technical solution or the composite bacterial agent prepared by the preparation method described in the above technical solution in any of the following items:

[0068] (1) Promote plant growth and increase crop yield;

[0069] (2) Improve plant disease resistance and stress resistance;

[0070] (3) Increase the nitrogen content and available phosphorus content in the soil;

[0071] (4) Promote the formation of soil aggregates.

[0072] In order to further illustrate the present application, a Chaetomium F8 strain and its application provided by the present application are described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0073] Example 1

[0074] A strain F8 was isolated from the moderately saline-alkali soil of the Songnen Plain (soil pH 9.0, alkalinity 15%) using the dilution plating method. The culture medium used for isolation was Bengal rose agar (Sigma-Aldrich), and the culture medium used for purification was PDA medium (200 g potato, 20 g sucrose, 20 g agar, and 1000 mL deionized water).

[0075] The F8 strain was identified with reference to Chinese patent CN202211477950.4. The colony morphology is shown in Figure 1, where a represents the colony morphology of the F8 strain on the front of a PDA plate, b represents the colony morphology of the F8 strain on the back of a PDA plate, and c represents the colony morphology of the F8 strain on soil. The ITS sequence of the F8 strain is as follows: 5'--3' (SEQ ID No. 1). After comparison with the NCBI website, representative sequences were selected and a phylogenetic tree was constructed using Mega 7.0.0.26, as shown in Figure 2.

[0076] As shown in Figures 1 and 2, the colony morphology of the F8 strain is white colonies with vigorous aerial hyphae. The strain F8 is Chaetomium truncatulum, and was named Chaetomium F8 and biologically preserved.

[0077] Example 2

[0078] Prepare 3 replicates as follows:

[0079] (1) In a clean bench, the mycelium of Chaetomium F8 was picked and placed on a PDA plate medium (200 g potato, 20 g sucrose, 20 g agar and 1000 mL deionized water) for activation. The plate was cultured in the dark at 25 °C for 5 days. The Chaetomium F8 agar truffles were punched with a hole puncher with a diameter of 1 cm and 8 mm, respectively. The thickness of the Chaetomium F8 agar truffles was 5 mm.

[0080] (2) Prepare enough clean and uniform straw and cut it into 1cm thick pieces with scissors. 3 Weigh 0.50 g of yeast extract, 20.0 g of straw, 5.0 g of tricalcium phosphate, 0.50 g of ammonium sulfate, 0.20 g of potassium chloride, 0.10 g of magnesium sulfate heptahydrate, 0.0001 g of manganese sulfate monohydrate, and 0.0001 g of ferrous sulfate heptahydrate, add to deionized water, adjust the pH to 7.0, dilute to 1 L, sterilize at 121°C for 20 min, and let cool for later use to obtain straw culture medium.

[0081] (3) Inoculating two pieces of Chaetomium F8 agar truffles with a diameter of 1 cm prepared in step (1) into the straw culture medium prepared in step (2), culturing on a shaking table at 25° C. and a rotation speed of 170 rpm for 5 to 7 days to obtain a culture solution.

[0082] Comparative Example 1

[0083] Prepare 3 replicates as follows:

[0084] (1) Pour the prepared PDA culture medium (200 g potato, 20 g sucrose, 20 g agar, and 1000 mL deionized water, natural pH) into a plate culture dish and condense to obtain an agar block with a thickness of 0.3 cm. The PDA plate culture dish was incubated upright at 25°C in the dark for 5 days, and holes were punched with a hole puncher with a diameter of 1 cm and 8 mm, respectively, to obtain a blank agar block with a thickness of 3 mm.

[0085] (2) Prepare enough clean and uniform straw and cut it into 1cm thick pieces with scissors. 3 Weigh 0.50 g of yeast extract, 20.0 g of straw, 5.0 g of tricalcium phosphate, 0.50 g of ammonium sulfate, 0.20 g of potassium chloride, 0.10 g of magnesium sulfate heptahydrate, 0.0001 g of manganese sulfate monohydrate, and 0.0001 g of ferrous sulfate heptahydrate, add to deionized water, adjust the pH to 7.0, dilute to 1 L, sterilize at 121°C for 20 min, and let cool for later use to obtain straw culture medium.

[0086] (3) The blank agar block with a diameter of 1 cm in step (1) was inoculated onto the straw culture medium, and cultured on a shaking table at 25° C. and a rotation speed of 170 rpm for 5 to 7 days to obtain a culture solution.

[0087] Test Example 1

[0088] Determination of soluble phosphorus

[0089] Molybdenum antimony reagent: Weigh 10.0g of ammonium molybdate and dissolve it in 250mL of 60°C water, cool and dilute to 300mL. Slowly inject 181mL of concentrated H2SO4 (analytical grade) into 800mL of water, cool, then inject dilute sulfuric acid into the molybdic acid solution and stir thoroughly. Weigh 0.3g of potassium antimony tartrate and add it to the ammonium molybdate dilute sulfuric acid solution. Dilute to 2L with water to obtain the molybdenum antimony reagent. Store in a brown bottle for long-term storage. When using, add 1.5g of L-ascorbic acid per 100mL of molybdenum antimony reagent to obtain the molybdenum antimony solution.

[0090] Accurately pipette 1 mL of the culture medium of the three groups of repetitions in Example 2 and the three groups of repetitions in Comparative Example 1 into a 25 mL volumetric flask, accurately add 12.5 mL of distilled water with a burette, and then add 2.5 mL of molybdenum antimony antimony reagent with a pipette, shake well, let it stand for 30 minutes, adjust the volume, shake well, and perform colorimetry at a wavelength of 700 nm.

[0091] The standard curve used in the determination of soluble phosphorus is y = 1.3935x, R 2 =0.9959, x is the absorbance value, and y is the concentration of orthophosphate in the system.

[0092] The straw degradation rate in the culture medium of Example 2 and Comparative Example 1 was calculated using the drying difference subtraction method: the straw in the culture bottles of three replicates in Example 2 and three replicates in Comparative Example 1 was screened and washed, and a small amount of hyphae on the surface was removed with tweezers. The straw in the culture bottles was dried at 50°C to constant weight for 12 to 24 hours, and the dry weight of the straw after degradation was recorded. The calculation formula (1) is as follows:

[0093] Straw degradation rate = (straw dry weight - straw dry weight after degradation) / straw dry weight × 100% (1);

[0094] SPSS 20.0 statistical software was used for analysis. The experimental data were expressed as “mean ± standard deviation” and Tukey sb (K) test was used to test the homogeneity of the data. The results are shown in Table 1.

[0095] Table 1 Results of determination of phosphate solubilization effect and straw degradation rate of Chaetomium F8 in Comparative Example 1 and Example 2

[0096] Note: “**” in the table means there is significance at the P=0.01 level.

[0097] According to Table 1, the inoculation of Chaetomium F8 increased the straw degradation rate and the content of available phosphorus in the culture solution, indicating that Chaetomium F8 promoted the dissolution of tricalcium phosphate.

[0098] Example 3

[0099] Weigh 80g of nutrient soil (purchased from Henan Fengwo Agricultural Materials Co., Ltd.) and place it in a 1L triangular flask, adjust the water content to 30%, seal it with a breathable sealing film, and sterilize it at 121°C for 20min. After the nutrient soil cools, wheat seeds are sown into the nutrient soil under aseptic conditions to a sowing depth of 1cm. At the same time, inoculate 3 pieces of Chaetomium F8 agar truffles prepared in Example 2 to a depth of 1cm. After sealing, culture in a biochemical incubator. After 2d of emergence, remove the seedlings under aseptic conditions, leaving 1 seedling per bottle, and culture them at 25°C for 16h (light intensity of 30,000lm) every day, then transfer to 15°C for dark culture for 8h, and cultivate them for 15d to obtain seedlings.

[0100] Comparative Example 2

[0101] The same as Example 3, except that the Chaetomium F8 agar block was replaced with the blank agar block prepared in Comparative Example 1.

[0102] Test Example 2

[0103] Experiment on promoting root growth of wheat at seedling stage by Chaetomium sp. F8

[0104] The cultured seedlings of Example 3 and Comparative Example 2 were respectively taken, and the wheat root parameters were measured using a root scanner. The results are shown in Table 2.

[0105] Table 2 Root parameters of wheat at seedling stage in Example 3 and Comparative Example 2

[0106] Note: “**” represents significance at the P=0.01 level, and “*” represents significance at the P=0.05 level.

[0107] As shown in Table 2, inoculation with Chaetomium F8 significantly increased the total root length and total root area of ​​wheat, and reduced the number of root crowns, number of intersections, and total root volume of the root system. This suggests that Chaetomium F8 mainly promotes the elongation of the taproot of wheat at the seedling stage.

[0108] Example 4

[0109] 1. Four species of Mortierella fungi (Mortierella longifolia, Mortierella alpina, Mortierella sp. F34, and Mortierella cephalosporin) were inoculated into PDA medium and incubated at 25°C in the dark for 5 days for activation. After activation, agar tubes of each Mortierella strain were punched with an 8 mm diameter cork punch to obtain agar tubes with a diameter of 8 mm and a thickness of 5 mm. Each activated Mortierella strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of potato dextrose liquid medium, with an inoculum size of one mycelial tube of each fungus. The culture was shaken at 25°C in the dark for 5 days at 180 rpm to obtain a mixed culture of Mortierella.

[0110] 2. Disinfection and germination of corn

[0111] Inbred B73 maize seeds (available from the China Crop Germplasm Information Network) were mutagenized in 0.4% EMS (CAS No. 62-50-0) at 20°C for 12 hours, then soaked in 5% sodium thiosulfate solution for 10 minutes and rinsed with water for 2 hours to obtain mutant material. After planting, individuals without root hairs were screened to obtain the rtl6 mutant. Experiments were conducted using both the inbred B73 maize material (parent) and the rtl6 mutant, which exhibited a root hairless phenotype after EMS mutagenesis.

[0112] Fifty parental corn seeds and 50 mutant corn seeds were sterilized in a 10% hydrogen peroxide solution for 5 minutes, then washed five times with distilled water to completely remove any residual hydrogen peroxide. After soaking in distilled water for 10 hours, the seeds were primed. Specifically, 30 seeds were placed in a 90mm glass Petri dish with a 90mm clean filter paper, 5-10mL of distilled water, and the endosperm facing upward. The dish was then covered to maintain a moist environment. The seeds were incubated at 28°C for three days to obtain primed corn.

[0113] 3. Planting

[0114] The soil from Fengzhuang Village, Gongyi City, Henan Province was selected as potting soil. The soil type was sandy damp soil with a pH of 7.7, an organic matter content of 12.71 g / kg, a total nitrogen content of 0.62 g / kg, and a moisture content of 23%. After passing through a 2 mm sieve, the soil was potted for later use. Each pot was filled with 300 g of soil, watered thoroughly, and soaked for 2 hours before the corn prepared in step 2 was placed, root-side down, and lightly covered with 30 g of dry soil. The planted corn was placed in a constant temperature incubator and cultured for 16 hours at 28 ° C light intensity (light intensity of 30,000 lm) every day, followed by 8 hours of dark culture at 20 ° C.

[0115] 4. When the corn plants grow to 3-4 leaves, remove the seedlings and leave one seedling per pot. Divide 50 pots of parent corn plants into a parent control group and a parent plus bacteria group, and 50 pots of mutant corn plants into a mutant control group and a mutant plus bacteria group, and inoculate them according to the following method:

[0116] Parent control group: 4 blank agar blocks with a diameter of 8 mm and a thickness of 5 mm prepared in Comparative Example 1 were inoculated per pot 15 mm below the topsoil layer;

[0117] Parent plus bacteria group: Each pot was inoculated with 4 different Mortierella agar tuber strains 15 mm below the surface soil layer, with one agar tuber strain of each Mortierella strain inoculated;

[0118] Mutant control group: consistent with the parental control group;

[0119] Mutant-added bacteria group: consistent with the parent-added bacteria group.

[0120] After inoculation, cover the soil with moist, loose, sieved soil without compacting it to facilitate observation of mycelial growth. After inoculation, water the soil surface with a watering can to keep it moist. After 14 days of incubation, collect corn plant and soil samples for measurement of relevant parameters.

[0121] Test Example 3

[0122] Evaluation of the Effects of Mortierella Composite Strain Used in Composite Microbial Agents on Corn Growth

[0123] Method for determining available phosphorus: The soil sample in Example 4 was air-dried to constant weight, 5 g of the air-dried soil sample was weighed and poured into a 250 mL plastic bottle. One spoonful of phosphorus-free activated carbon and 100 mL of 0.5 mol / L sodium bicarbonate solution were added, the pH was adjusted to 8.5, the bottle stopper was tightly plugged, and after shaking on a shaker for 30 min, the solution was immediately filtered with phosphorus-free filter paper and the filtrate was collected in a 100 mL conical flask. 1 mL of the filtrate was drawn into a 25 mL volumetric flask, 15 mL of distilled water was accurately added with a burette, and then 2.5 mL of molybdenum antimony antimony reagent was added with a pipette. The volume was adjusted to 50 mL after shaking well. After standing for 30 min, the solution was shaken well again and colorimetry was performed at a wavelength of 800 nm.

[0124] The biomass of each group of corn samples in Example 4 was measured, and the phosphatase activity was measured using a soil neutral phosphatase activity assay kit (Suzhou Mengxi Biotechnology Co., Ltd.). The results are shown in Tables 3 and 4.

[0125] Table 3 Determination of biomass and soil-related indicators of corn under different treatments

[0126] Note: Different letters indicate significant differences at the P = 0.05 level. Soil neutral phosphatase activity (U) is defined as: 1 μmol of phenol released per gram of soil per day at 37°C is one unit of enzyme activity.

[0127] Table 4 Results of corn root growth parameters

[0128] Note: Different letters represent significant differences at the P=0.05 level.

[0129] As shown in Tables 3 and 4, the Mortierella strain significantly increased the root dry weight and total dry weight of the parent corn, and significantly increased the content of phosphatase in the rhizosphere soil of corn. The root dry weight, aboveground dry weight, and total weight of the mutant were significantly lower than those of the parent. Inoculation of the Mortierella strain significantly increased the number of root tips and root forks of the parent corn, and reduced the average root diameter and root density. This indicates that the Mortierella strain increased the bifurcation and elongation of the normal corn root system. The results of the root hair-less mutant showed that the Mortierella strain significantly increased the average root diameter, and the root length, number of root tips, and root forks were reduced, but not to a significant level. This indicates that the promotion of root growth by the Mortierella strain is strongly dependent on root hairs.

[0130] Test Example 4

[0131] Effects of different fertilizers on composite bacterial systems

[0132] Under liquid and solid culture medium conditions, different types of fertilizers were selected and added to the culture medium to explore the effect of fertilizer type on the compound bacterial agent.

[0133] The screening medium was prepared by adding chemical fertilizers to the PDA / PD medium in the equivalent ratio of N-P2O5-K2O of the topsoil to 150:75:150 kg / ha. The specific ingredients are shown in Table 6.

[0134] Table 6 Ingredients of culture medium with different fertilizer types

[0135] Two solid culture medium treatments were set up: treatment 1 was inoculated with F8 alone, and treatment 2 was inoculated with both F8 and Mortierella strains. Three culture mediums were inoculated for each treatment. In treatment 1, a single 8 mm diameter bacterial block was inoculated in the center of the plate. In treatment 2, an F8 bacterial block of the same size was inoculated in the center of the plate, with Mortierella bacteria blocks of equal size inoculated around the F8 (Figure 4a). Colony diameters were measured after 28 h, and hyphal growth rates were calculated.

[0136] SPSS 20.0 statistical software was used for analysis. The experimental data were expressed as “mean ± standard deviation” and Tukey sb (K) test was used to test the homogeneity of the data. The results are shown in Table 7.

[0137] Table 7 Growth rate of Chaetomium F8 under different conditions

[0138] Note: Different letters represent significant differences at the P=0.05 level.

[0139] During the solid culture stage, consider the growth promotion and balanced ratio of the target strain to the Mortierella colony, ensuring that the strain ratio difference is within one order of magnitude. A 1:1 ratio is the most effective. Table 7 shows that potassium chloride has the most balanced growth promotion effect on Chaetomium F8 in the presence of Mortierella colonies. Based on the results of previous experiments, ammonium sulfate and potassium chloride are the preferred fertilizers.

[0140] Under liquid culture conditions, a cellophane chamber was placed in a 250 mL Erlenmeyer flask. During inoculation, 20 mL of culture medium was added to the sterile cellophane chamber. Four 8 mm diameter F8 agar tubes were then inoculated into the cellophane chamber. Eighty mL of culture medium was added to the Erlenmeyer flask, and four Mortierella species were inoculated, with two 8 mm diameter agar tubes for each species. Cultures were incubated in the dark at 28°C for five days at 180 rpm. After incubation, the mycelium in the cellophane chamber and the Erlenmeyer flask were oven-dried at 50°C, and the dry matter weight was recorded. The results are shown in Figure 3. The analysis of variance in Figure 3 was performed using Tukey's sigmoid curves with a significance level of 0.05. As shown in Figure 3, ammonium sulfate had the greatest effect on the growth of Chaetomium F8 in liquid culture, followed by potassium dihydrogen phosphate, and then by urea. For the Mortierella consortium, urea was the most effective, followed by monocalcium phosphate, and then by ammonium sulfate. After weighing the combined strains, ammonium sulfate was found to be the most effective.

[0141] Example 5

[0142] The culture conditions were the same as in Example 4, except that 2.38 g of ammonium sulfate was added to 500 mL of PDA medium to prepare a liquid culture medium. The cellophane chamber was removed, and four species of Mortierella fungi (Mortierella longifolia, Mortierella alpina, Mortierella sp. F34, and Mortierella cephalosporium) were cultured separately according to the method of step 1 of Example 4 to obtain a mixed bacterial solution (Figure 4b).

[0143] The preparation method of solid fermentation matrix is ​​as follows: cut wheat straw to a length of about 2 cm or less, weigh 1000 g of wheat straw, 100 g of woody peat, 200 g of soybean meal, 1.67 g of potassium chloride, 0.96 g of potassium dihydrogen phosphate and 10 g of humic acid, mix them thoroughly, add water until they are moist but do not drip when squeezed by hand (water content is about 40%), put them into polypropylene bags, each bag containing about 100 g of sample, seal them, and sterilize them at 121°C for 2 hours to obtain solid fermentation matrix.

[0144] Each bag of solid fermentation matrix was inoculated with 100 mL of mixed bacterial solution and grown statically in a clean bench for two days at a temperature maintained between 20 and 28°C. It was then placed in a room temperature environment at 20 to 28°C and statically cultured for three days to allow the mycelium to cover the matrix. A composite bacterial agent was obtained (c in Figure 4). After mixing, it was applied to the field according to the amount.

[0145] Test Example 5

[0146] Effects of Chaetomium F8-Mortierella Composite on Corn Growth

[0147] A field experiment was conducted in June 2023 at the Ministry of Agriculture's North China Wheat-Corn Rotation Nutrition and Fertilization Scientific Observation Experimental Station. Corn seeds of the Yudan 132 variety were selected. Six pools were set up in the field, each with five rows of approximately 30 holes, alternating between odd and even. The pools were divided into a control group and a test group, with three pools in each group. The following treatments were applied:

[0148] Experimental Group (M+F8): Corn was inoculated with the composite inoculant prepared in Example 5 at the five-leaf stage. Approximately 10 g of the composite inoculant was inoculated into the rhizosphere of the corn. Each hole was covered with old straw after inoculation. Three parallel pools were used for this experiment.

[0149] Control group (CK): No composite bacterial agent was inoculated, but an equal amount of solid fermentation substrate was added. Other conditions were the same as those of the experimental group. This experiment set up three parallel groups of three pools.

[0150] (1) The effects of the combined strain of F8 and Mortierella on soil chemical properties, plant traits, and plant root hormones were tested during the tasseling stage of corn in the field; the growth status of corn plants, soil physical properties, and yield were tested during the maturity stage. The results are shown in Tables 8 to 12 and Figures 5 to 6.

[0151] Table 8 Effects of F8 and Mortierella combined strains on soil chemical properties

[0152] Table 9 Effects of microbial inoculation on soil physical properties

[0153] Table 10 Effects of combined strains of F8 and Mortierella on plant traits

[0154] Table 11 Effects of combined strains of Chaetomium F8 and Mortierella on root hormone content

[0155] Table 12 Effect of F8 and Mortierella sp. combined strains on yield indicators

[0156] As shown in Figures 5-6 and Tables 8-12, inoculation with the inoculum significantly increased soil nitrate nitrogen, with an increase of up to 52.40%. Furthermore, inoculation with the composite inoculum significantly increased the proportion of macroaggregates and mesoaggregates in the soil, while reducing the proportion of microaggregates and clay particles. Observation of plant phenotypic traits revealed that inoculation with the inoculum significantly increased corn stem fresh weight, leaf fresh weight, total fresh weight, plant height, and the ratio of stem diameter by 47.08%, 59.28%, 59.28%, 24.18%, and 17.41%, respectively. This directly demonstrates that inoculation with the inoculum is beneficial to corn plant growth. Measurements of corn root hormone levels revealed increases in root abscisic acid (ABA), growth hormone (IAA), jasmonic acid (JA), and zeatin riboside (ZR), with the auxin content increasing significantly, indicating that the combined effects of inoculation with the inoculum contribute to corn drought resistance and growth through endogenous root assistance. The inoculation of combined bacteria significantly increased the average number of ears per plant by 16.87%, and significantly improved the 100-grain weight, number of grains per ear, and fresh weight of corn ears, indicating that the inoculation of combined bacteria can increase corn yield by increasing the 100-grain weight and the number of ears, thereby increasing the number of grains per ear.

[0157] (2) The abundance of fungal bacteria in the corn rhizosphere was detected by high-throughput sequencing, and the results are shown in Figures 7 to 12. As shown in Figures 7 to 12, for the bacterial component, the abundance of the most dominant bacteria at each classification level in the embodiment was reduced, and there was no significant difference in the remaining bacteria. In the fungal component, compared with the control group, the abundance of Ascomycota, Chaetomiaceae, Botryotrichum, Acrophialophora and Aspergillus in the experimental group was significantly increased, which increased by 12%, 1.6 times, 1 times, 5 times and 1.5 times, respectively. At the same time, the abundance of Basidiomycota and Talaromyces was significantly reduced, which decreased by 53% and 83%, respectively. Among them, the Chaetomiaceae family is the inoculant, and Botryotrichum has the function of degrading lignin (Sun et al., 2023. Continuous wheat / soybean cropping influences soybean yield and rhizosphere microbial community structure and function, Agronomy.). The genus Acrophialophora has a strong antagonistic effect with seven pathogens, including Cylindrospermum destructum, and has a certain preventive and control effect on windproof root rot (Han Zhongming et al. A strain of Acrophialophora MR-57 and its application, p. 22.). This shows that the composite bacterial agent prepared in this application can promote the degradation of lignin and enhance the disease resistance of plants.

[0158] Test Example 6

[0159] Effects of Chaetomium-Mortierella Composite Fungus on Rice Growth

[0160] Thirty rice seeds of the Songliao 186 variety (purchased from Inner Mongolia Hengzheng Group Baoanzhao Agricultural Industry and Trade Co., Ltd.) were selected and placed in a disposable culture dish. Distilled water was added and the culture dish lid was covered to keep the environment moist. The seeds were cultured in an incubator for 3 days to accelerate germination.

[0161] The potting soil used was saline-alkali soil from Northeast China, with a total nitrogen content of 0.47 g / kg, an alkaline nitrogen content of 46.50 mg / kg, an available phosphorus content of 17.50 mg / kg, an available potassium content of 153 mg / kg, an organic matter content of 10.58 g / kg, a pH of 9.16, and a soil salt content of 1.08 g / kg. The soil was passed through a 2 mm sieve, 200 g of which was weighed and placed in a pot, with each pot containing approximately 300 g of soil. After being watered thoroughly, rice plants with relatively consistent growth after germination were selected and sown in the soil at a sowing depth of 5 mm. The sown rice was divided into a control group and a test group. After growing in an incubator for one week, the rice was processed according to the following method.

[0162] Experimental group: The composite bacterial agent prepared in Example 5 was inoculated into the rice rhizosphere, with each hole inoculated with about 5 g of the composite bacterial agent. After inoculation, the rice was covered with moist soil to a thickness of about 1 cm.

[0163] Control group: No composite bacterial strain was inoculated, and 5 g of solid fermentation matrix was added.

[0164] Three parallels were set up in each control group and experimental group, with one pot of rice in each parallel. Fifteen rice plants were inoculated in each pot at a depth of 1 cm. The plants were cultured under natural conditions. After 3 days, the seedlings were removed, leaving one seedling in each bottle. The plants were cultured for a total of 40 days. The growth status of the rice is shown in Figure 13, with the rice in the control group on the left and the rice in the experimental group on the right.

[0165] After the incubation, the root systems of the experimental and control rice plants were scanned and their plant height, aboveground biomass, root proline content, root malondialdehyde content, root catalase content, and root superoxide dismutase content were measured. At the same time, the ammonium nitrogen content in the non-rhizosphere soil was measured.

[0166] Scan the root system using a root scanner.

[0167] The aboveground biomass was measured by oven-drying weighing method.

[0168] Rice root proline, malondialdehyde, catalase, and superoxide dismutase levels were measured using proline, malondialdehyde, catalase, and superoxide dismutase assay kits (Suzhou Keming Biotechnology Co., Ltd.), respectively. Fresh samples were homogenized in an ice bath and centrifuged at 8000 g at 4°C for 10 min. The supernatant was collected and assayed according to the kit procedures. The supernatant was allowed to stand at room temperature for 30 min, and colorimetry was performed at 560 nm.

[0169] Superoxide dismutase activity U = [inhibition percentage ÷ (1-inhibition percentage) × V total] ÷ (W × V sample ÷ V total) = 11.11 × inhibition percentage ÷ (1-inhibition percentage) ÷ W.

[0170] Here, inhibition percentage = (A control tube - A assay tube) ÷ A control tube × 100%.

[0171] Where: Vreaction: total volume of the reaction system, 0.2 mL (volume of the reaction in the well plate);

[0172] Sample V: the sample volume added to the reaction system, 0.018 mL (tissue fluid after grinding and centrifugation);

[0173] V total sample: volume of extract added, 1 mL;

[0174] W is the sample mass, g.

[0175] SPSS statistical software was used for analysis, and the experimental data were expressed as "mean ± standard deviation". The results are shown in Tables 13 to 16.

[0176] Table 13 Results of rice root system in control group and experimental group

[0177] Note: “**” represents significance at the P=0.01 level, and “*” represents significance at the P=0.05 level.

[0178] Table 14 Rice biomass parameters of the control group and the experimental group

[0179] Note: “**” indicates significant difference at the P=0.01 level.

[0180] Table 15 Hormone content of rice roots in the control group and the experimental group

[0181] Note: "**" indicates significance at the P = 0.01 level, and "*" indicates significance at the P = 0.05 level. Definition of peroxidase: 1 unit of enzyme activity is defined as the degradation of 1 μmol of hydrogen peroxide per gram of tissue per minute.

[0182] Table 16 Determination of relevant indicators in paddy soil of control group and experimental group

[0183] Tables 13-16 show that the root length, root surface area, root volume, number of root tips, and number of forks in the experimental group were significantly higher than those in the control group, increasing by 4.6-fold, 4-fold, 4-fold, 3.5-fold, and 6-fold, respectively, indicating that the composite bacterial system strongly promoted rice root growth. Furthermore, the fresh weight of roots and aboveground parts increased by 2-fold, and plant height increased by 46%. Superoxide dismutase, malondialdehyde, and catalase activities in rice roots were higher than those in the control group, indicating that Chaetomium enhanced the antioxidant capacity of rice roots and improved lipid oxidation and reactive oxygen species scavenging. There were no significant differences in ammonium nitrogen, total nitrogen, organic matter, and organic carbon content in the soil between the experimental group and the control group, suggesting that the composite bacterial system may promote rice growth through biostimulation.

[0184] The microbial community structure of the root zone of the above-mentioned rice was determined using high-throughput sequencing methods (Li et al., 2020), and the results are shown in Figures 14 to 19.

[0185] Figures 14 to 19 show that the relative abundance of Proteobacteria, Rhodospirillaceae, Thiobacillus, Phenylobacterium, Azospirillum, and Enhydrobacter increased in the experimental group compared to the control group. Proteobacteria accounted for the largest proportion, with an abundance increase of 9.3%. Azospirillum showed the largest increase, with a 15-fold increase. Azospirillum was the second-largest increase, with a 12-fold increase. Among the fungi, the abundance of Pseudeurotium increased tenfold. Among them, Azospirillum can associate with the rhizosphere of plants to fix nitrogen, which has the functions of increasing plant height, promoting plant development, enhancing the salt-alkali resistance of crops, and increasing yield (Cassán et al., 2020. Everything you must know about Azospirillum and its impact on agriculture and beyond. Biology and Fertility of Soils 56, 461-479.). Aquatic bacteria can produce cellulases and chitinases, promoting the degradation of carbohydrates (Hao Zhikui, Wu Hangui, Xi Limin, 2013. Screening and identification of chitinase-producing Enhydrobacter sp. MBRH2 strains. The 8th National Conference on Medical Biochemistry and Molecular Biology, the 5th National Conference on Clinical Applied Biochemistry and Molecular Biology, 2013 Joint Academic Symposium on Biochemistry and Molecular Biology of Six Provinces and One City in East China, Qingdao, Shandong, China, p. 1.; Premalatha et al., 2015. Optimization of cellulase production by Enhydrobacter sp. ACCA2 and its application in biomass saccharification. Frontiers in Microbiogy 6.).Pseudeurotium is salt- and alkali-tolerant and can utilize lignin and humus with extremely low oxygen content (Ran et al., 2016. Isolation of exceedingly low oxygen consuming fungal strains able to utilize lignin as carbon source. Cellulose Chemistry and Technology 50, 811-817.; Georgieva et al., 2023. Haloalkalitolerant fungi from sediments of the Big Tambukan Saline Lake (Northern Caucasus): Diversity and Antimicrobial Potential, Microorganisms.). Thus, the composite fungus prepared in this application has an advantage in promoting plant growth compared to the control group.

[0186] Based on the above content, it can be seen that the Chaetomium F8 provided in this application has the function of promoting straw degradation and phosphorus solubilization, significantly promotes soil agglomeration under field conditions, can provide bacterial resources for agricultural straw degradation, and provide a reference for the further development and utilization of Chaetomium.

[0187] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. A Chaetomium truncatulum F8 strain, deposited with CGMCC No. 40973.

2. A Chaetomium mushroom block, characterized in that: The mycelia of Chaetomium sp. F8 according to claim 1 are activated by placing them in a PDA plate culture medium and cultured in the dark at 25° C. for 5 days to obtain the product.

3. The Chaetomium mushroom block according to claim 2, characterized in that The thickness of the Chaetomium mushroom block is 5 mm, and the diameter is 1 cm or 8 mm.

4. A Chaetomium culture solution, characterized in that The Chaetomium fungus block according to claim 2 or 3 is inoculated into a straw culture medium, and cultured on a shaking table at 25° C. and 170 rpm for 5 to 7 days; The straw culture medium comprises 0.50 g / L yeast extract, 20.0 g / L straw, 5.0 g / L tricalcium phosphate, 0.50 g / L ammonium sulfate, 0.20 g / L potassium chloride, 0.10 g / L magnesium sulfate heptahydrate, 0.0001 g / L manganese sulfate monohydrate, 0.0001 g / L ferrous sulfate heptahydrate, and the balance, with a pH value of 7.

0.

5. Use of the Chaetomium F8 and / or its metabolites according to claim 1, the Chaetomium block according to claim 2 or 3, or the Chaetomium culture solution according to claim 4 in one or more of the following: (1) Promote straw degradation; (2) Promote the dissolution of ineffective phosphorus and the degradation of sugars in the soil; (3) Increase the soluble phosphorus content in the soil; (4) Improve soil structure; (5) Promote plant growth.

6. The use according to claim 5, characterized in that The plants include one or more of wheat, corn and rice.

7. A composite bacterial agent, characterized in that: The composite bacterial agent includes Chaetomium F8 and Mortierella strain; The deposit number of the Chaetomium sp. F8 is CGMCC No. 40973; The mortierella fungus system includes Mortierella elongata, Mortierella alpina, Mortierella sp. F34 and Mortierella capitata.

8. The composite bacterial agent according to claim 7, characterized in that The concentration of Chaetomium F8 in the composite bacterial agent is 3×10 7 ~8×10 7 cfu / g; The concentrations of the long spore mold, the alpine spore mold, the unnamed spore mold F34 and the cephalospore mold were 2×10 6 ~10×10 6 cfu / g.

9. The method for preparing the composite bacterial agent according to claim 7 or 8, characterized in that: The following steps are involved: Mixing Chaetomium F8 and Mortierella strains for expansion culture to obtain a mixed bacterial solution; inoculating the mixed bacterial solution into a fermentation substrate for fermentation culture to obtain a composite bacterial agent; The ratio of the number of live bacteria inoculated with Chaetomium F8, Mortierella longi, Mortierella capitis, Mortierella unnamed F34 and Mortierella alpina is 2-8:1:1:1:1; Calculated by mass, the fermentation matrix includes 1000 parts of wheat straw, 80-120 parts of woody peat, 180-220 parts of soybean meal, 1.67-2.00 parts of potassium chloride, 0.96-1.10 parts of potassium dihydrogen phosphate and 6-12 parts of humic acid; the water content of the fermentation matrix is 35%-45%.

10. The preparation method according to claim 9, characterized in that The expanded culture comprises mixing Chaetomium F8 and Mortierella strains and then inoculating the mixture onto a culture medium, wherein the ratio of Chaetomium F8 to culture medium is 0.1-1 cfu:1 mL.

11. The preparation method according to claim 9, characterized in that The ratio of the mixed bacterial solution to the fermentation substrate is 100 mL: 100 g; the concentration of the mixed bacterial solution is 10 8 ~10 9 cfu / mL.

12. The preparation method according to claim 9 or 10, characterized in that: The temperature of the expanded culture is 25° C., the rotation speed is 180 rpm, and the culture time is 5 days; the temperature of the fermentation culture is 20-28° C., and the culture time is 7 days.

13. Use of the composite bacterial agent according to claim 7 or 8 or the composite bacterial agent prepared by the preparation method according to any one of claims 9 to 12 in one or more of the following: (1) Promote plant growth and increase plant yield; (2) Improve plant disease resistance and stress resistance; (3) Increase nitrogen and soluble phosphorus content in the soil; (4) Promote the formation of soil aggregates.

14. The use according to claim 13, characterized in that The plants include one or more of wheat, corn and rice.

15. A method for promoting corn growth, characterized in that: include: At the five-leaf stage of corn, the composite microbial agent according to claim 7 or 8 or the composite microbial agent prepared by the preparation method according to any one of claims 9 to 12 is inoculated into the rhizosphere of corn, and straw is covered after inoculation; the inoculation method of the composite microbial agent is hole application, and 10 g of the composite microbial agent is inoculated into each hole.

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