Microbial fertilizer as well as preparation method and application thereof

Abstract

The invention relates to the technical field of sugarcane smut prevention and treatment, in particular to a microbial fertilizer, a preparation method thereof and application of the microbial fertilizer in smut prevention and treatment. The microbial fertilizer comprises the following components in parts by weight: 40-60 parts of cow dung, 2-4 parts of corn straw charcoal, 4-6 parts of ramulus mori and 8-14 parts of a mixed microbial agent, the mixed bacterial agent is prepared from bacillus subtilis X1, bacillus altitudinis X2, bacillus cereus X3, Trichoderma harzianum Z1 and Trichoderma longibrachiatum Z2, and the mixed bacterial agent is prepared from bacillus subtilis X1, bacillus altitudinis X2, bacillus cereus X3, Trichoderma harzianum Z1, Trichoderma longibrachiatum Z2, bacillus altitudinis X2, bacillus cereus X3, Trichoderma harzianum Z1, Trichoderma longibrachiatum Z2, Trichoderma harzianum Z2, Trichoderma harzianum Z2, Trichoderma harzianum Z2, Trichoderma harzianum Z2, Trichoderma harzianum Z2, Trichoderma harzianum Z3, Trichoderma The microbial fertilizer disclosed by the invention has a positive promoting effect on the sugarcane, and meanwhile, the self defense response capability of the sugarcane can be obviously stimulated.

Description

Technical Field

[0001] The invention relates to the technical field of sugarcane smut prevention and control, and in particular to a microbial fertilizer and a preparation method thereof and application of the microbial fertilizer in preventing and controlling sugarcane smut. Background Art

[0002] Sugarcane smut is a common and serious sugarcane disease, which poses a great threat to the healthy growth of sugarcane. After the disease occurs, it generally causes a loss of 10% to 30%, and in severe cases, the loss can even be as high as 50% to 70%. Sugarcane smut is a cumulative disease, and its incidence increases year by year with the increase of planting years, especially in dryland sugarcane.

[0003] In addition, there are also studies reporting on the biological control of sugarcane smut. Liao Yongmei and Xiong Guoru both found bacterial strains that have antagonistic effects on sugarcane smut, such as Pseudomonas, Acinetobacter, Bacillus subtilis, etc. (Liao Yongmei, Zhang Guiying, Huang Dingan, et al. Screening and identification of antagonistic bacteria against sugarcane smut [J]. Guangxi Agricultural Biological Sciences, 2004, (03): 197-201 and Xiong Guoru, Zhao Gengfeng, Wu Suran, et al. Screening and identification of antagonistic bacteria HAS against sugarcane smut [J]. Journal of Tropical Crops, 2013, 34 (06): 1149-1154). Sinha found that sugarcane endophyte Pseudomonas aeruginosa B18 can inhibit the activity of sugarcane whip smut fungus and enhance the stress resistance of sugarcane by regulating the content of hydrolases and antioxidant enzymes (Singh P, Singh RK, Guo DJ, et al. Whole Genome Analysis of Sugarcane Root-Associated Endophyte Pseudomonas aeruginosa B18-A Plant Growth-Promoting Bacterium With Antagonistic Potential Against Sporisorium scitamineum[J]. Frontiers In Microbiology, 2021, (12): 628376). Xi Pinggen and Liu Shiyin reported that Pseudomonas fluorescens YJHM58 and Pseudomonas ST4 can inhibit the sexual mating of sugarcane smut fungus respectively (Xi Pinggen. Research on the inhibition of sexual mating of sugarcane smut fungus by Pseudomonas fluorescens YJHM58 [D]. Doctoral dissertation. Guangdong: South China Agricultural University, 2010. and Liu S, Lin N, Chen Y, et al. Biocontrol of Sugarcane Smut Disease by Interference of Fungal SexualMating and Hyphal Growth Using a Bacterial Isolate [J]. Frontiers In Microbiology, 2017, (8): 778.).Trichoderma, Bacillus pumilus, and Burkholderia gladiolus have all been shown to have inhibitory effects on sugarcane smut (Jayakumar V, Ramesh Sundar A, Viswanathan R. Biological Suppression of Sugarcane Smut with Endophytic Bacteria [J]. Sugar Tech, 2018, 21 (4): 653-660 and Tegene S, Dejene M, Terefe H, et al. Evaluation of native Trichoderma isolates for the management of sugarcane smut (Ustilagoscitaminea) in sugar plantations of Ethiopia [J]. Cogent Food And Agriculture, 2021, 7 (1): 1872853). However, the ability of a single strain to regulate soil microorganisms is limited, which is not conducive to maintaining the stability of the soil ecosystem. Summary of the invention

[0004] The present invention aims to provide a microbial fertilizer for preventing and controlling smut, a preparation method thereof and application thereof in preventing and controlling smut.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] A microbial fertilizer comprises, by weight, 40-60 parts of animal manure, 2-4 parts of straw biochar, 4-6 parts of mulberry branches, and 8-14 parts of a mixed bacterial agent; the mixed bacterial agent consists of Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1, and Trichoderma longibrachiatum Z2; the preservation number of Bacillus cereus X3 is GDMCC NO: 65100, the preservation number of Trichoderma harzianum Z1 is GDMCC NO: 65101, the preservation number of Trichoderma longibrachiatum Z2 is GDMCC NO: 65102, the preservation number of Bacillus altitudinis X2 is GDMCC NO: 65103, the preservation number of The deposit number of X2 is GDMCC NO:65099, and the deposit number of Bacillus subtilis X1 is GDMCC NO:65098.

[0007] Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1 and Trichoderma longibrachiatum Z2 are deposited in Guangdong Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit date is September 4, 2024.

[0008] Among them, animal manure, straw biochar and mulberry branches are calculated based on dry weight.

[0009] In one of the preferred embodiments, the microbial fertilizer includes, by weight: 50 parts of animal manure, 3.3 parts of straw biochar, 5 parts of mulberry branches, and 11 parts of mixed bacterial agents.

[0010] In one preferred embodiment, the carbonization temperature of the straw biochar is 450-550°C and the specific surface area is 14-17m 2 / g, average pore size is 8-10nm, pore capacity is 25-29m 3 / kg.

[0011] In one preferred embodiment, the animal manure has a total carbon content of 40-43 wt%, a total nitrogen content of 1-4 wt%, a carbon-nitrogen ratio C:N of 25-27, and a water content of 60-75%.

[0012] In one preferred embodiment, the mulberry branches have a total carbon content of 60-70 wt%, a total nitrogen content of 0.5-1.5 wt%, a carbon-nitrogen ratio C:N of 70-80, and a water content of 55-70%.

[0013] In a preferred embodiment, the animal manure is cow manure.

[0014] In one preferred embodiment, the straw biochar is corn straw biochar, wherein the cow dung, corn straw biochar and mulberry branches are calculated by dry weight.

[0015] In a preferred embodiment, in the mixed bacterial agent, the volume ratio of Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1 and Trichoderma longibrachiatum Z2 is 1:1:1:1:1.

[0016] In one preferred embodiment, the spore concentration of Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1 and Trichoderma longibrachiatum Z2 in the mixed bacterial agent is 5×10 6 -1×10 8 cfu / mL.

[0017] After multiple tests and verifications, in the microbial fertilizer, Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1 and Trichoderma longibrachiatum Z2 have the best growth-promoting effect on plants when added at the same concentration and volume, but have a poor effect on smut. If any of the microorganisms is missing, or the proportion of any of the microorganisms is too large or too small, the effect will be reduced.

[0018] In one of the preferred embodiments, the microbial fertilizer further comprises 2-4 parts of calcium carbonate by weight.

[0019] Based on the same inventive concept, the present invention also claims a method for preparing the microbial fertilizer, comprising the following steps:

[0020] S1, mixing the raw materials other than the mixed bacterial agent in proportion and then stacking and fermenting to obtain organic fertilizer;

[0021] S2, adding mixed bacterial agents to the organic fertilizer for secondary fermentation;

[0022] S3, air drying to obtain the microbial fertilizer, preferably natural air drying.

[0023] In one preferred embodiment, during the pile fermentation and the secondary fermentation, a temperature sensor is used to detect the temperature of the cow dung, and a humidity sensor is used to detect the humidity of the cow dung.

[0024] In one preferred embodiment, the method for regulating temperature and moisture during pile fermentation and secondary fermentation includes: regulating the temperature and humidity of organic fertilizer by turning the pile at high temperature and adding water.

[0025] In one preferred embodiment, during the pile fermentation, the fermentation temperature of the organic fertilizer is controlled to be 60-70° C., and the humidity is 60-75%.

[0026] In one preferred embodiment, the secondary fermentation process is as follows: fermentation temperature is 35-38°C, dark conditions, and humidity is 50-60%.

[0027] In one preferred embodiment, the secondary fermentation time is 12-16 days, and the compost is turned every 2-3 days.

[0028] In one preferred embodiment, the moisture content of the microbial fertilizer after air drying is 23-27%.

[0029] Based on the same inventive concept, the present invention also claims to protect the use of the microbial fertilizer in preventing and controlling plant smut.

[0030] In a preferred embodiment, the amount of microbial fertilizer added is 0.5-1 kg fertilizer / 10 kg soil.

[0031] In a preferred embodiment, the plant is sugarcane.

[0032] The microbial fertilizer of the present invention has the following beneficial effects:

[0033] (1) After secondary fermentation by functional microorganisms, the physical and chemical properties and nutritional components of organic fertilizers are optimized. However, the secondary fermentation process plays an important role in promoting the decomposition of lignocellulose components in fertilizers. Through this process, the content of humic acid in fertilizers is increased, and the microbial toxicity of fertilizers is also reduced. The combined effect of these factors makes the secondary fermentation process significantly enhance the maturity of fertilizers and improve the quality of fertilizers.

[0034] (2) The combined effect of calcium carbonate and biochar also contributes to the improvement of fertilizer quality. They not only increase the nutritional components in the fertilizer, but also accelerate the degradation of lignocellulose during composting and fermentation. The addition of these two materials increases the content of humic acid in agricultural waste compost products, promotes the resource utilization and harmlessness of agricultural waste, and at the same time, is conducive to maintaining the stability of the soil ecosystem.

[0035] (3) The microbial fertilizer is significantly better than other biological organic fertilizers in preventing and controlling sugarcane smut. In addition, the fertilizer also shows a positive promoting effect on sugarcane growth, can effectively increase the sugar content in sugarcane, increase its biomass, and thus achieve the purpose of increasing sugarcane yield.

[0036] (4) When sugarcane is invaded by pathogens, a strict defense mechanism is triggered in its body. In response, sugarcane will autonomously produce chitinase and β-1,3-glucanase, which can effectively resist the invasion of pathogens. In addition, sugarcane will adjust the activity of its active oxygen scavenging enzymes to increase the accumulation of active oxygen in the body, thereby achieving the purpose of killing pathogens.

[0037] (5) By applying the microbial fertilizer, the activities of chitinase, β-1,3-glucanase, catalase and peroxidase in sugarcane can be effectively improved, and the defense mechanism of sugarcane itself can be stimulated, thereby enhancing its ability to resist the invasion of pathogens. In addition, the microbial fertilizer can also alleviate the oxidative stress reaction in sugarcane to a certain extent, providing a guarantee for the healthy growth of sugarcane.

[0038] In summary, the microbial fertilizer has a significant ability to stimulate the self-defense response of sugarcane, can effectively adjust the physiological changes of sugarcane, promote the growth of sugarcane and prevent and control diseases. Therefore, the microbial fertilizer has broad application prospects in ensuring the normal growth of sugarcane and increasing sugarcane yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a photo of the growth of the pathogen of sugarcane smut whip after 10 days of cultivation;

[0040] Figure 2 Phylogenetic tree of single strain B1 isolated from sugarcane smut whip

[0041] Figure 3 is the phylogenetic tree of the five antagonistic strains;

[0042] Figure 4 To antagonize the interaction between fungi and bacteria;

[0043] Figure 5 is the change of seed germination rate in fertilizer before and after fermentation;

[0044] Figure 6 The changes of lignocellulose components;

[0045] Figure 7 The changes of humic acid components in fertilizers before and after fermentation;

[0046] Figure 8 Figure 1 shows diseased plants and their black stigma; Figure a shows diseased plants (left) and healthy plants (right) in pots, and Figure b shows diseased plants and their black stigma in the field;

[0047] Fig. 9 It is the growth index of sugarcane with different treatments;

[0048] Fig.10 The effects of different treatments on sugarcane sugar content;

[0049] Fig.11 β-1.3 glucanase activity of sugarcane with different treatments;

[0050] Fig.12 Chitinase activity of sugarcane treated with different methods;

[0051] Fig.13 Peroxidase activity of sugarcane with different treatments;

[0052] Fig.14 Catalase activity of sugarcane with different treatments. DETAILED DESCRIPTION

[0053] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0054] All data processing and statistical analysis methods of the present invention are as follows: Barcode sequence identification is used, and the FLASH online tool is combined to realize the accurate splicing of the sequence paired end read segments, and fastp software is used to implement data quality control and eliminate low-quality sequences. Subsequently, it is compared with the reference database to exclude chimera contamination. With the help of the QIIME2 software platform, deep sequence denoising, species annotation and phylogenetic tree construction are performed to generate amplicon sequence variants (ASVs) sets and corresponding feature abundance matrices.

[0055] The significant difference between the two groups of data was evaluated based on the independent sample t-test using SPSS24 software. To further reveal the heterogeneity of microbial community structure, non-metric multidimensional scaling analysis (NMDS) was used to intuitively display the distance and similarity between communities. In order to analyze the correlation between soil chemical factors and protist communities, redundancy analysis (RDA) and correlation heatmap were implemented using the Lianchuan Biological Cloud Platform. LEfSe analysis and correlation coefficient plot were completed using the Novo Cloud Platform. Mantel analysis was implemented using the vegan package and MASS package of R software. The completion of the remaining charts was also completed relying on the bioinformatics cloud platform, Excel 2021, Origin 2022 software and AI (Adobe Illustrator).

[0056] Experimental instruments: carbon and nitrogen element analyzer elementar (Vario EL); pH meter; fully automatic fiber tester; constant temperature incubator, etc., are all commonly used instruments.

[0057] Corn straw biochar powder: purchased from Xingyuan Environmental Protection Co., Ltd., and its specific properties are shown in Table 1. Calcium carbonate powder was directly purchased from a calcium carbonate processing plant near the market in Dacai Township, Huanjiang County, Hechi City, Guangxi Zhuang Autonomous Region. Cow dung came from a cattle farm near the field test plot, and mulberry branches came from local farmers. The composting process (primary fermentation) was carried out at the Huanjiang Karst Ecosystem Observation and Research Station (Hechi, Guangxi), and the secondary fermentation process was carried out at the Institute of Subtropical Agricultural Ecosystems, Chinese Academy of Sciences (Changsha, Hunan). All material properties are shown in Table 2.

[0058] Table 1 Physical and chemical properties of corn straw biochar

[0059]

[0060] Table 2 Properties of initial composting materials

[0061]

[0062] Example 1: Screening and identification of antagonistic strains

[0063] 1. Experimental materials and instruments

[0064] The screening material for the pathogenic bacteria of sugarcane smut was fresh sugarcane smut whips (the variety was Guitang 42, which is susceptible to smut) collected from the sugarcane plantation of Guangxi Academy of Agricultural Sciences. The functional verification of the fungicide was also carried out using the seed stems of the sugarcane variety.

[0065] Soil for screening antagonistic strains: rhizosphere soil of healthy and smut-infected sugarcane in the sugarcane plantation of Guangxi Academy of Agricultural Sciences and disease-suppressing soil with long-term application of organic fertilizer.

[0066] Potting soil: Taken from the Institute of Subtropical Agriculture Ecology, Chinese Academy of Sciences. Remove large stones, roots, dead branches, etc. from the soil before use.

[0067] Main culture media: beef extract peptone (NA) and potato dextrose agar (PDA) medium.

[0068] Main instruments: DNA extraction kit (QIAprep Spin mini prep Kit 250, USA); spectrophotometer (model 722, Shanghai Yidian Analytical Instrument Co., Ltd.); water purifier (Synthesis A10, MILLIPORE, USA); clean bench (SW-CJ-1FD, Suzhou Jingmei Co., Ltd.); constant temperature shaker (SPH-103B, Shanghai Changping Co., Ltd.); constant temperature incubator (XT5116IN, Hangzhou Xuetan Co., Ltd.).

[0069] 2. Collection and Isolation and Cultivation of Sugarcane Smut Pathogen

[0070] Freshly formed sugarcane smut whips that have not broken out of the leaf sheath were collected from the sugarcane plantation of the Guangxi Academy of Agricultural Sciences and spread in an environment of 16-20°C and a relative humidity of 10-20% for 8-12 hours. The purpose of this step is to maximally inhibit the reproduction of miscellaneous bacteria (especially bacteria) on the black whip-shaped spikes, greatly reduce the contamination of miscellaneous bacteria on the winter spores of sugarcane smut fungi, and ensure that the winter spores maintain a high germination rate (90%-98%). After the black whip-shaped spikes are fully dried, the surface is wiped and disinfected with 75% alcohol in a clean bench, and then the leaf sheaths are peeled off, and the winter spores on the black whip-shaped spikes are scraped with sterilized tweezers to remove impurities. The winter spores after the impurities are removed are divided into 50mL centrifuge tubes, sealed with sterilizable sealing film, and stored in a refrigerator at 4°C.

[0071] Weigh 1.5g of streptomycin sulfate and stir and mix in sterile water to prepare a streptomycin sulfate solution with a concentration of 1500mg / L (for inhibiting bacterial growth). Pick about 3g of the treated winter spores of sugarcane whip smut fungus, put them into a beaker containing 250mL of streptomycin sulfate solution, stir and let stand for 1min, and dilute them in a gradient with streptomycin sulfate solution. Since streptomycin sulfate is not resistant to high temperatures, add 1.5g of streptomycin sulfate to the PDA medium after high temperature sterilization when the temperature drops to about 60℃, draw 50μL of spore liquid and apply it to the PDA medium, place it in a 28℃ incubator for 3d, and then pick out a single bacteria for purification.

[0072] 3. Isolation and purification of total bacteria and fungi in soil

[0073] All culturable bacteria and fungi in the soil were separated and purified using the dilution coating method. Weigh 5g of rhizosphere soil sample, put it into a 250mL triangular flask containing 45mL of deionized water, and mix and shake it on a constant temperature shaker for 30min, with the parameters set to 30℃ and 180rpm. The mixed soil suspension was aspirated and gradiently diluted with deionized water, and 50pL of different gradient solutions were aspirated and added to NA and PDA culture medium plates, and spread evenly. The two coated culture medium plates were placed in constant temperature incubators at 37℃ and 28℃, respectively, and single colonies with different colony characteristics were picked and cultured and purified until single colonies appeared on the plate. The purified bacteria and fungi were inoculated on NA and PDA culture media, respectively, and placed in a 4C refrigerator for short-term storage and standby, and activated every 3 months.

[0074] 4. Screening of antagonistic strains against sugarcane smut

[0075] (1) Preliminary screening of antagonistic strains

[0076] Activation culture of sugarcane smut fungus: Collect diseased whips that have just formed and have not yet exposed, disinfect the surface and peel off the leaf sheath, pick up a few black winter spores with sterilized tweezers, stir and dilute in sterile water, dilute the spore liquid of sugarcane whip smut fungus (Sporisoriumscitamineum) and spread it on the PDA plate and place it in a constant temperature incubator at 28℃ for 2 days, move it to the clean bench for operation, burn the inoculation loop and cool it, pick up the newly germinated pathogens and inoculate them on a new PDA culture medium plate.

[0077] Initial screening of antagonistic strains of sugarcane smut: When the smut colonies on the PDA plate to be inoculated with the pathogen grow to a diameter of about 2 mm, four isolated and purified single bacteria are inoculated at a distance of 20 mm from the center of each plate using the plate confrontation method. Bacteria are inoculated with an inoculation loop, and fungi are inoculated with a 5 mm diameter puncher. Three replicates are set for each treatment, and a control group is set at the same time. The culture is continued for 10-15 days. The antagonistic degree is determined by the distance from the bacterial and fungal inoculation points to the outer edge of the pathogen, and the colonies that have a significant inhibitory effect on the growth of sugarcane whip smut are selected and numbered and recorded.

[0078] More than 100 bacterial strains and 60 fungal strains were isolated and purified from healthy / disease fine roots and rhizosphere soil of sugarcane and soil with long-term application of organic fertilizer, and stored in a 4C refrigerator for subsequent plate confrontation tests. A total of 14 bacterial strains and 26 fungal strains with antagonistic ability were screened out in the initial screening.

[0079] (2) Rescreening of antagonistic strains

[0080] The sugarcane smut pathogen was inoculated in the center of the PDA medium. When the diameter of the sugarcane smut colony was about 2 mm, the strain obtained by the initial screening was inoculated at a position 1 cm away from the edge of the plate. Each plate was inoculated with only one functional bacteria, and each colony was set up with 3 replicates. A control group was also set up. The culture was continued for 10-15 days, and the bacteria and fungi with strong antagonistic ability were recorded and saved. The inhibition rate (the ratio of the diameter of the control smut colony minus the diameter of the treated group to the diameter of the control group) was used to compare the degree of inhibition of the antagonistic bacteria on sugarcane whip smut fungus.

[0081] The strains obtained in the initial screening were further screened to select a group of bacteria and fungi with high inhibition rates against pathogens. Some of the data are shown in Table 3. The inhibition rates of some strains were also high but were not listed.

[0082] Table 3 Antibacterial rate of rescreened strains against sugarcane whip smut fungus

[0083]

[0084] 5. Identification of pathogens and antagonistic strains

[0085] Pick the rescreened single colonies to the corresponding liquid culture medium (LB for bacteria and PDA for fungi), and use the bacterial genome extraction kit and the fungal genome extraction kit to extract the genomic DNA of each strain. PCR amplification was performed using the bacterial 16S rDNA universal primers 27F and 1492R, and the fungal ITS universal primers ITS1 and ITS4. PCR reaction system 50μL: template DNA 2μL, PCR premix 25μL, two primers 1μL each, dd H2O supplemented to 50μL. Conditions: 95℃5min; 95℃30s; 57℃30s; 72℃5min; 30 cycles; 72℃5min. The product was tested by electrophoresis, and the target band was obtained. The gel was cut and purified and recovered, and sent to Beijing Qingke Company for sequencing.

[0086] The morphological identification results of the pathogen of sugarcane smut are as follows:

[0087] The spores scraped from the black smut whip were diluted with pure water and spread on the plate. A large number of white fungi grew out. The number of colonies was significantly negatively correlated with the dilution factor. Combined with the literature, it was speculated that the white fungi were S. scitamineum. The hyphae were pure white, densely grown, smooth and round. Figure 1 Compared with other fungi, this fungus grows relatively slowly on PDA culture medium. Obvious colonies can only be observed after 3 days of culture. The colony diameter is about 10 mm at 5 days, the average colony diameter is 22 mm after 10 days of culture, and the average colony diameter is 30 mm after 15 days of culture.

[0088] The results of molecular biological identification of the pathogen of sugarcane smut are as follows:

[0089] The ITS sequences obtained by sequencing the single strain B1 isolated from the whip of smut were compared with each other using the BLAST website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and 7 strains with homology greater than 99% with B1 were selected. The phylogenetic tree was further constructed using the MEGA 11 software, as shown in Figure 2 . Figure 2 As shown, the analysis results showed that strain B1 was Sporisorium scitamineum of the genus Sporisorium.

[0090] The sequences of the five re-screened strains were compared by BLAST on the NCBI website. Ten strains with a similarity of 99% were selected for each strain. A preliminary phylogenetic tree was constructed for the sequences of the antagonistic bacteria of sugarcane smut and its similar strains using the MEGA11 software. The tree was then further beautified on the Itol official website. Based on the comparison results, the phylogenetic tree was constructed by combining the 10 similar strains. The results are shown in the figure below. Figure 3As shown. Through comparison and phylogenetic tree, it can be seen that the 5 strains obtained belong to two genera, Bacillus and Trichoderma. Among them, 3 strains belong to Bacillus, namely Bacillus subtilis, Bacillus altitudinis and Bacillus cereus; the other 2 strains belong to Trichoderma, namely Trichoderma longibrachiatum and Trichoderma Harzianum.

[0091] 6. Interactions between antagonistic strains of sugarcane smut pathogen

[0092] The absence of mutual inhibition of growth and reproduction between microorganisms is a prerequisite for the construction of synthetic flora and a guarantee for the flora to better perform its biological control function. In order to verify whether there is antagonism between the dominant bacteria, the rescreened strains were inoculated on PDA plates, and cross-streaking was performed between the bacteria. The antagonism between bacteria and fungi was inoculated at 1 cm from the edge of the culture medium, and the fungi were confronted with each other using a puncher. Three parallel tests were set up. The strains were placed in a 30°C constant temperature incubator for culture, and the strains were observed to see whether they did not produce spores, whether the colonies were concave to one side or had a smaller diameter during their growth. The strains with no mutual growth inhibition or weaker antagonism were selected for the preparation of synthetic flora for the next step of controlling sugarcane whip smut fungus.

[0093] The interaction relationship between the highly antagonistic strains of sugarcane smut is shown in Figure 4 , the results showed that the two fungi Z1 and Z2 grew faster. Studies have shown that Trichoderma and Bacillus have similar ways of preventing and controlling sugarcane smut, mainly including competition for living space and nutrients, secreting substances that have antagonistic effects on pathogens, promoting the growth of sugarcane and inducing its immune response. Among them, Trichoderma harzianum has tenacious vitality and its reproduction speed is much faster than that of general functional bacteria. It is suitable for various soil types. Once it establishes a symbiotic relationship with the host plant, it is basically difficult to be affected by adverse external environments. There is no obvious growth inhibition or antagonism between these five fungi.

[0094] However, some bacteria had obvious antagonism and growth inhibition effects with the two fungal strains Z1 and Z2, so they were excluded.

[0095] Example 2: Preparation of fertilizer

[0096] 1. Preparation of organic fertilizer

[0097] Two experimental groups were set up according to whether or not exogenous calcium carbonate was added (a small-scale fermentation test was conducted using a turnover box (50 cm × 40 cm × 30 cm)):

[0098] (1) PM: Cow dung (50 kg) and mulberry branches (5 kg) were used as the main composting materials, and the carbon-nitrogen ratio of the compost was about 25-30. No biochar and calcium carbonate were added to the control group (control group: excluding the influence of background values ​​such as composting materials and composting process on the experiment);

[0099] (2) PMC: Cow dung (50 kg) and mulberry branches (5 kg) were used as the main composting materials, 6% biochar was added, and the carbon-nitrogen ratio of the compost was about 25-30. No calcium carbonate was added in experimental group 1 (experimental group 1: organic fertilizer containing only biochar was used as the control group);

[0100] (3) CaPMC: Based on Experimental Group 1, 6% calcium carbonate powder was added at the initial stage of composting and mixed evenly with the cow dung and mulberry branches (Experimental Group 2: calcium carbonate was added to organic carbon fertilizer to verify the effect of calcium carbonate on the fermentation quality of organic fertilizer).

[0101] 2. Preparation of biological organic fertilizer

[0102] The spore concentrations of three bacterial strains (Bacillus subtilis, Bacillus altitudinis and Bacillus cereus) and two fungal strains (T. Harzianum and T. longibrachiatum) were adjusted to the same order of magnitude by dilution coating method and hemocytometer counting. 8 cfu / mL, each bacterial solution was inoculated into (1) cow dung and mulberry branch compost (PM), (2) cow dung and mulberry branch biochar compost (PMC), and (3) calcium cow dung and mulberry branch biochar compost (CaPMC) at the same volume ratio, and the total volume of bacterial solution inoculation was 20% (V / W) of the dry matter mass of pig dung and mulberry branch compost products, and fermented for 2 weeks (secondary fermentation process) to prepare bio-organic fertilizer PMA, charcoal-based bio-organic fertilizer PMCA and calcium charcoal-based bio-organic fertilizer CaPMCA. During the fermentation, the compost was turned once every 2 days, and the compost was naturally air-dried to a moisture content of about 25% to obtain different microbial organic fertilizers. In the early stage of bacterial antagonism experiments, it was found that the prevention and control ability of the five bacteria alone against sugarcane smut was weak, and the effects of other combinations of two or three were not ideal. After exploration, it was found that the best combination of the five bacteria was 1:1:1:1:1. Therefore, this ratio was used to make biological organic fertilizer, and its effects on the plants and its actual disease resistance in the field and potted plants were examined.

[0103] 3. Sample collection

[0104] Samples were collected 0 and 14 days after inoculation of the bacterial solution. The collection method was as follows: taking the center of the pile as the origin, 250 g of samples were collected from the front, back, left and right sides within a radius of 10 cm. After mixing, they were divided into three portions and packaged into self-sealing bags. They were stored in a refrigerator at 4°C for determining basic properties, in a refrigerator at -80°C for determining microorganisms, and in an air-dried state for determining nutrient content.

[0105] 4. Performance Testing

[0106] The performance test results are as follows:

[0107] 4.1 Basic physical and chemical properties

[0108] pH: Samples were collected before inoculation (0 day) and after secondary fermentation (14 days). Fresh samples (samples stored in a 4°C refrigerator) were mixed with pure water at a ratio of 1:10 (w / w), shaken at 170 rpm for 2 h, and allowed to stand for 30 min. The supernatant was collected and the pH was measured using a pH meter.

[0109] Total carbon and total nitrogen: Grind the air-dried sample and pass it through an 85-mesh sieve. Weigh 20 mg and use an elemental analyzer to determine the content of total nitrogen and total carbon.

[0110] The results are shown in Table 4.

[0111] Table 4 Physicochemical properties of fertilizers before and after fermentation

[0112]

[0113] Note: The results in the table are mean ± standard deviation. Different lowercase letters indicate that there are significant differences in the same treatment before and after fermentation (p<0.05). PMA, PMC and CaPMCA in the table represent bio-organic fertilizer (control group), charcoal-based bio-organic fertilizer with only biochar added, and calcium charcoal-based bio-organic fertilizer with calcium carbonate and biochar added, respectively.

[0114] Under 25℃, the activity of microorganisms can be effectively stimulated, and at the same time, the microorganisms will not die or their activity will not be reduced due to excessively high temperatures. However, in the actual secondary fermentation process, the fermentation temperature will rise due to the heat generated by the metabolism of the microorganisms and the heat exchange with the external environment. Experimental data show that during the entire fermentation process, the temperature is stable at around 27℃, which not only ensures the activity of the microorganisms, but also avoids the adverse effects of excessively high temperatures on the fermentation process.

[0115] In the early stage of composting test (primary fermentation), special attention was paid to the high temperature fermentation stage. This stage is the key in composting, because high temperature can effectively kill or inhibit harmful microorganisms in feces, thereby ensuring the safety and harmlessness of compost products. The experimental results show that the high temperature (50°C) fermentation stage of all treatments was maintained for more than 15 days, which fully meets the requirements for composting temperature in the "Hygiene Standard for Harmless Treatment of Feces" (GB7959-2012).

[0116] Table 4 shows the pH, total nitrogen, total carbon and carbon-nitrogen ratio of the three fertilizers before and after the secondary fermentation of solids. The pH value indicates the acidity and alkalinity of the fertilizer, which has a direct impact on the growth of plants. The appropriate pH range helps plants absorb nutrients. Total nitrogen is one of the main nutrients in fertilizers that affects plant growth. Total carbon has an important influence on soil structure and microbial activity. The carbon-nitrogen ratio reflects the carbon-nitrogen balance of the fertilizer and has an important influence on the decomposition of organic matter by soil microorganisms.

[0117] After adding bacterial liquid, the moisture content of organic fertilizer is about 65%, which is a relatively high moisture content. After the secondary fermentation process, the moisture content of all fertilizers is reduced to below 30% stipulated in the agricultural industry standard "NY884-2012". This change shows that the fermentation process effectively reduces the moisture content of the fertilizer and improves its stability.

[0118] Before and after fermentation, the pH values ​​of the three fertilizers all decreased by 0.01, and there was no significant difference between the three. This change is small but significant. It shows that during the fermentation process, the fertilizer did not undergo anaerobic acid production, that is, no obvious acidification occurred during the fermentation process. This further shows that the entire fermentation process was carried out under aerobic conditions, ensuring the smooth progress of the fermentation process and the stability of the fertilizer quality.

[0119] The total carbon and total nitrogen content of the three fertilizers increased before and after fermentation. The total nitrogen content of calcium charcoal-based bio-organic fertilizer (CaPMA) increased particularly significantly, from 2.07% to 2.34%, an increase of 0.27%. The carbon- and nitrogen-containing substances introduced when adding biochar and bacterial liquid are the main reasons for this phenomenon. At the same time, these substances can effectively promote the decomposition of organic matter during the composting process.

[0120] The total carbon content of fertilizer in the CaPMA treatment group showed a significant increase. The addition of biochar may be the main reason for this change. Biochar itself is rich in carbon, so it significantly increased the total carbon content in the fertilizer.

[0121] Calcium carbonate is added during the early composting process of calcium carbon-based bio-organic fertilizer. This measure can remove a large number of harmful microorganisms in the fertilizer and affect the total amount of microorganisms in the secondary fermentation process. Due to the reduction in the total amount of microorganisms, their consumption of nitrogen and carbon in the environment is also reduced accordingly. Therefore, it is shown that the addition of calcium carbonate will increase the nitrogen and carbon content of the pile.

[0122] However, although the total nitrogen and carbon contents of the three fertilizers increased before and after fermentation, there was no significant difference, which indicates that the loss of nitrogen and carbon during the fertilizer fermentation process was relatively small and the fertilizer had a strong nutrient retention capacity.

[0123] In addition, the carbon-nitrogen ratio is an important indicator for evaluating the maturity of fertilizers. At the beginning and end of fermentation, the carbon-nitrogen ratios of the three fertilizers were all below 20. According to relevant research, fertilizers with a carbon-nitrogen ratio below 20 can be considered fully mature. Therefore, these three fertilizers have completed a full maturity process during the fermentation process.

[0124] In summary, the total carbon content of the three fertilizers increased before and after fermentation, indicating that significant chemical changes occurred during the fertilizer maturation process. Among them, the total nitrogen content of calcium charcoal-based bio-organic fertilizer (CaPMA) increased most significantly, which may be related to the addition of biochar and bacterial liquid. At the same time, the addition of calcium carbonate also helps to increase the nitrogen and carbon content of the fertilizer. In addition, the carbon-nitrogen ratio of the three fertilizers is less than 20, indicating that they have been fully decomposed and have good application conditions.

[0125] 4.2. Seed germination index (GI)

[0126] For each treatment group, 5 g of fresh samples from 0 and 14 days of secondary fermentation were added to 50 mL of pure water, shaken in a shaker set at 170 rpm for 30 min, and then filtered. 5 mL of filtrate was added to a culture dish covered with filter paper, 50 cucumber seeds of uniform size were selected, spread on the culture dish, and placed in a 25°C constant temperature incubator in the dark for 2 days.

[0127] GI (%) = (seed germination rate of compost extract × root length) / (seed germination rate of control group × root length) × 100.

[0128] The results are as follows Figure 5 shown.

[0129] The germination index (GI) is an important indicator that is often used to evaluate the toxicity of fertilizers before and after composting and to detect the maturity of fertilizers. By analyzing this index, we can gain a deeper understanding of the biological transformation and material degradation during the composting process and provide safer and more efficient fertilizers for agricultural production.

[0130] Figure 5 The figure shows the changes in the germination rate of seeds in the fertilizer before and after fermentation. It can be seen from the figure that the germination rate of seeds in the fertilizer before fermentation is generally above 75%, and after the secondary fermentation treatment, the germination rate of seeds in the fertilizer is greater than 80%. This result shows that the secondary fermentation process further promotes the maturity of the pile and reduces the biological toxicity of cow dung compost.

[0131] In the composting process, in addition to fermentation, the use of additives is also an important means to improve the quality of fertilizers. Studies have found that the addition of calcium carbonate and biochar can increase the germination rate of seeds in the compost and reduce the phytotoxicity of the compost. When both are added at the same time, it is more beneficial to the recovery of the compost and the reduction of biological toxicity.

[0132] Generally speaking, when the germination rate of seeds exceeds 50%, the pile is considered mature. When the germination rate exceeds 80%, it means that the pile is fully mature. The results show that the combined use of biochar and calcium carbonate can not only significantly improve the maturity of manure compost, but also reduce the biological toxicity of the product.

[0133] 4.3 Determination of cellulose, hemicellulose and lignin content

[0134] Neutral detergent: Accurately weigh 18.6g analytically pure disodium ethylenediaminetetraacetate (EDTA) and 6.8g analytically pure sodium borate in a beaker, heat to dissolve, then add 30g analytically pure sodium dodecyl sulfate and 10mL analytically pure glycol ether. Weigh 4.56g analytically pure anhydrous sodium phosphate, heat to dissolve, mix with the former and adjust the volume to 1L. Acidic detergent: Weigh 20g analytically pure hexadecyltrimethylammonium bromide in a beaker and adjust the volume to 1L with 0.5mol / L sulfuric acid.

[0135] Lignocellulose content: measured by Fan's washing method

[75] The content of lignocellulose in the fertilizer after the maturity period and secondary fermentation is determined as follows: 0.5 g of the air-dried sample that has been dried and weighed is transferred to a feed fiber analysis filter bag (three replicates are performed in each group), and after being covered with a condensing device, it is placed in a fully automatic fiber tester, and neutral detergent is added (one pot for every 12 samples, and 1L of neutral detergent is added to each pot) and boiled for 1 hour. After boiling, the filter bag is taken out, rinsed with hot water and dried for more than 5 times until there is no foam in the washing liquid, rinsed with 20 mL of acetone twice, and finally the filter bag is placed in a 105°C oven to dry to obtain sample a1, which is weighed to obtain A1.

[0136] The dried and weighed a1 sample is covered with a condensing device and placed in a fully automatic fiber tester. Acid detergent is added (1L of acid detergent is added to each pot for every 12 samples) and boiled for 1 hour. After boiling, the filter bag is taken out, rinsed with hot water, dried, and rinsed with a small amount of acetone. Finally, the filter bag is placed in a 105°C oven to dry to obtain sample a2, which is then weighed to obtain the acid detergent lignin content A2.

[0137] The acid washed lignin was added to 5 mL of sulfuric acid and digested for 3 h at room temperature, rinsed and dried to obtain sample a3, which was weighed to obtain A3. Sample a3 was placed in a muffle furnace and burned at a temperature above 500 degrees, and then weighed to obtain A4.

[0138] The hemicellulose content (%) is (A1-A2) / 0.5, the cellulose content (%) is (A2-A3) / 0.5, and the lignin content (%) is (A3-A4) / 0.5.

[0139] The results are as follows Figure 6 shown.

[0140] In fertilizer production, the content of lignocellulose components is related to the overall quality of the fertilizer and also reflects the degree of compost stabilization. The lignocellulose components show a clear downward trend during the fermentation process. Figure 6 As shown in the figure, after the fermentation, the wood contents in PMA, PMCA and CaPMCA were 14.95%, 18.69% and 14.08%, respectively, which were reduced by 21.89%, 19.26% and 16.54% compared with those before fermentation, respectively. This indicates that the lignocellulose components were effectively degraded during the composting process.

[0141] The cellulose content in the CaPMCA treatment was 7.96%, and the degradation rate was as high as 22.34%, which was significantly higher than the other two piles. The hemicellulose content in the PMA, PMCA, and CaPMCA piles was 9.41%, 11.43%, and 9.07% respectively after the fermentation, and the degradation rates were 30.79%, 15.72%, and 33.31% respectively. This shows that in the composting process, exogenous functional microorganisms played a positive role in promoting the degradation of lignocellulose components.

[0142] Trichoderma microorganisms can produce lignin-degrading enzymes, thereby improving the degradation of lignin in straw. In this study, the degradation rate of hemicellulose in CaPMCA fertilizer was higher than that of other treatments, which may be related to the fact that compared with the hard "shell" of lignin and the crystal structure of cellulose forming a network, hemicellulose is composed of shorter and highly branched polysaccharide chains with a lower degree of polymerization and is more easily degraded by microorganisms.

[0143] In addition, the present invention also found that the addition of calcium carbonate is beneficial to promote the degradation of hemicellulose and cellulose during the composting process. Calcium carbonate can adjust the pH of the compost, provide a more suitable growth environment for microorganisms, and thus promote the degradation of lignocellulose components. In contrast, the degradation rate of lignocellulose components in the treatment where only biochar was added actually decreased. This may be related to the lignin content at the beginning of fermentation and the loss of water and the reduction of the total amount of the compost during the fermentation process.

[0144] In summary, during the composting process, the addition of exogenous substances such as exogenous functional microorganisms and calcium carbonate can help promote the degradation of lignocellulose components, thereby improving the quality of fertilizer.

[0145] 4.4 Determination of humic acid components

[0146] Preparation of test solution: Sodium pyrophosphate extraction and potassium dichromate method

[79] The specific method is as follows. Accurately weigh 5.00g of the sample that has been air-dried and passed through an 85-mesh sieve. Add 100mL of a mixed extract of sodium pyrophosphate and sodium hydroxide (44.6g of analytically pure sodium pyrophosphate and 4g of analytically pure sodium hydroxide, dilute to 1L with deionized water, pH about 13) and shake for 5min. After standing for 14h, shake and filter, and collect the filtrate in a 100mL conical flask for testing.

[0147] Determination of total carbon content of humic acid and fulvic acid: Take 10mL of the test solution and adjust the pH to about 7 with 0.5mol / L sulfuric acid. After the solution precipitates, heat it in a water bath and evaporate it to dryness, and determine the total carbon content using the potassium dichromate fusion method.

[0148] Determination of humic acid carbon content: Take 10mL of the test solution, adjust the pH to 3 with 0.5mol / L sulfuric acid, keep it in a water bath at 80℃ for 30min, let it stand for 14h and centrifuge, dissolve the precipitate with 0.05mol / L sodium hydroxide in small amounts and multiple times, and adjust the volume to 50mL. Take 10mL of the liquid after the volume is taken and adjust the pH to about 7 with 0.5mol / L sulfuric acid. After the solution precipitates, heat it in a water bath and evaporate it to dryness, and determine the carbon content using the potassium dichromate fusion method.

[0149] The carbon content percentage of fulvic acid can be obtained by subtracting the carbon content percentage of humic acid from the total carbon content percentage of humic acid and fulvic acid.

[0150] The results are as follows Figure 7 shown.

[0151] During the composting process, microorganisms decompose organic matter in waste to produce humus, thereby stabilizing and rendering organic waste harmless. Humic acid in humus has a profound impact on soil structure, soil ecology and soil fertility, and is one of the key indicators for judging the maturity of compost.

[0152] Figure 7 The figure shows the changes in humic acid components before and after the fermentation of the pile. It can be seen from the figure that after the fermentation treatment, the humic acid content of each fertilizer showed an increasing trend. This shows that during the fermentation process, the decomposition of organic matter by microorganisms promoted the humification process, which increased the humic acid content of the fertilizer.

[0153] In addition to humic acid, humic substances in fertilizers also include humic acid (HA), fulvic acid (FA) and humin (HS). Among them, humic acid has the characteristics of high molecular weight and high stability, which has an important impact on soil structure and soil fertility.

[0154] During the fermentation process, a significant increase in the content of humic acid in the fertilizer was observed. At the end of fermentation, the humic acid in calcium charcoal-based biofertilizer, charcoal-based bio-organic fertilizer, and bio-organic fertilizer increased by 0.917 g / kg, 1.32 kg, and 1.24 g / kg, respectively. At the same time, the content of humic acid in organic fertilizers with added biochar was higher than that in the control group. These results indicate that the newly added microorganisms promoted the formation of humic acid in the pile, and the presence of biochar helped microorganisms convert organic matter into humic acid.

[0155] also, Figure 7 It also shows that the content of fulvic acid changes before and after fermentation. However, this change is not obvious, which may be because fulvic acid, as a component with a higher degree of oxidation in humus, its content and changes are affected by many factors.

[0156] Hufu ratio (DP) is another important indicator that reflects the maturity of compost. The larger the DP value, the higher the quality of the fertilizer. The DP values ​​before and after the secondary fermentation of CaPMCA, PMCA, and PMA treatments were 2.22 and 3.14; 1.83 and 3.15; 2.22 and 3.14, respectively. After completing the secondary fermentation, the Hufu ratio of the fertilizer was improved. During the fermentation process, microorganisms use the organic matter in the fertilizer to synthesize new humic acid, while the content of fulvic acid in the fertilizer did not increase significantly. Therefore, at the end of the experiment, the Hufu ratio showed an increasing trend.

[0157] When calcium carbonate and biochar are present at the same time, the DP value of the fertilizer reaches the highest after fermentation is completed. This proves that calcium carbonate and biochar have a significant effect on improving the quality of fertilizer.

[0158] In summary, secondary fermentation deepens the humification effect of fertilizers and can effectively improve the quality of fertilizers. At the same time, by adding functional microorganisms and biochar, the humification process of compost can be further promoted, and the humic acid and humic acid content in fertilizers can be increased, thereby improving soil structure and soil fertility.

[0159] Example 3

[0160] 1. Test materials and instruments

[0161] Experimental location: Both the field experiment and the pot experiment were conducted in Dacai Township, Huanjiang Maonan Autonomous County, Hechi City, Guangxi Zhuang Autonomous Region (107°50′~108°42′E, 24°43′~25°32′N). The specific location of the field experiment was Xiadong Village, Dacai Township, and the pot experiment was conducted in an open-air greenhouse in the Karst Ecosystem Observation Station. The research object was "Guitang No. 44", which is the most planted in Huanjiang area (cultivated by taking sugarcane stems from the field). The soil for the pot experiment was taken from the field experiment plot. The soil was dried, ground, sieved to remove stones and branches and leaves, and the soil properties were as follows: pH was 5.37, organic matter content was 14.64g / kg, total nitrogen content was 1.89g / kg, total phosphorus content was 1g / kg, and total potassium content was 5.72g / kg.

[0162] Main instruments and equipment: constant temperature shaker; flow analyzer (AutoAnalyzer3-AA3, SealAnalytic, USA), multifunctional microplate reader (Infinite M200 PRO), 96-well plate, superoxide dismutase (SOD) activity kit (Catalog Number AKAO001M), peroxidase (POD) activity kit (Catalog Number AKAO005M), catalase (CAT) activity kit (Catalog Number AKAO003-2M), etc.

[0163] Main reagents and solutions: disodium ethylenediaminetetraacetate (EDTA, analytical grade), ascorbic acid (analytical grade), β-mercaptoethanol (analytical grade), pH 5.2 acetate buffer (Solution A: take 11.55mL of glacial acetic acid and dilute it to 1000mL with distilled water. Solution B: weigh 16.4g of anhydrous sodium acetate, dissolve it in distilled water and make up to 1000mL. Take 105mL of solution A and 395mL of solution B, mix them, adjust the pH to 5.2, and dilute to 1000mL, which is 100mmol / L, pH 5.2 acetate buffer), etc.

[0164] 2. Preparation of pathogen spore solution

[0165] Collect pathogen spores: Collect fresh black stigma samples of sugarcane, fully dry them in an environment with a temperature of about 15°C and a relative humidity of about 15%, disinfect the surface of the sugarcane with alcohol, and use sterilized tweezers to collect the winter spore powder on the black whip-like stigma into a 50mL centrifuge tube for storage. When using, take 25g of spore powder and add 10kg of water (concentration is about 5×10 6 Spores / mL), diluted and spread on plates before use to ensure the activity of spores.

[0166] 3. Potted and field experiment design

[0167] 3.1 Potted experiment design

[0168] There were 6 potted plants in total, and each treatment group had 10 replicates; each pot contained 20 kg of soil and 1.5 kg of fertilizer (evenly mixed with the soil in advance, and the fertilizer used was the fertilizer prepared in Example 1), and the NPK nutrients in each pot were adjusted to be consistent using compound fertilizer (N:P:K=15:15:15), urea, superphosphate, and potassium chloride. The container was a foam box (30 cm×20 cm×10 cm), and the sugarcane seed stems were buried 10 cm below the soil surface. Planting began on April 27. The specific treatment group design is shown below.

[0169] (1) Blank treatment group (C): organic fertilizer was used as base fertilizer without any treatment, serving as the control group; the organic fertilizer was cow dung (50 kg) and mulberry branches (5 kg) as the main compost materials, the carbon-nitrogen ratio of the compost was about 25-30, and no microorganisms were added for fermentation for 2 weeks (secondary fermentation process), during which the compost was turned over every 2 days and naturally dried to a moisture content of about 25%.

[0170] (2) Pathogen treatment group (SS): Based on the blank control group, the plants were treated with 10 kg of pathogen spore solution and the roots were irrigated with pathogens once a month (April to August);

[0171] (3) Bio-organic fertilizer treatment group (PMA): The bio-organic fertilizer made by fermenting organic fertilizer and composite antagonistic bacteria was used as base fertilizer, treated with 10 kg of pathogen spore liquid, and the pathogen spore liquid was irrigated once a month (April to August) for comparison with treatment (2); the preparation method of the composite antagonistic bacteria was to adjust the spore liquid concentrations of three bacteria (Bacillus subtilis, Bacillus altitudinis and Bacillus cereus) and two fungi (T. Harzianum and T. longibrachiatum) to the same order of magnitude, i.e., 10 8 cfu / mL. The total volume of bacterial solution inoculation is 20% (V / W) of the dry matter of pig manure and mulberry twig compost product.

[0172] (4) Carbon-based bio-organic fertilizer treatment group (PMCA): 6% bio-char was added as base fertilizer to the bio-organic fertilizer treatment group (PMA), and 10 kg of pathogen spore solution was used for treatment. Root irrigation with pathogens was performed once a month (April to August) as a control group for comparison with treatment (3);

[0173] (5) Calcium carbon-based bio-organic fertilizer treatment (CaPMCA): 6% calcium carbonate powder was added as base fertilizer to the carbon-based bio-organic fertilizer treatment group (PMCA), and 10 kg of pathogen spore solution was used for treatment. Pathogen root irrigation was performed once a month (April to August), and the effect of calcium carbonate was verified by comparison with treatment (5);

[0174] (6) Calcium bio-organic fertilizer treatment group (CaPMA): 6% calcium carbonate powder was added as base fertilizer to the bio-organic fertilizer treatment group (PMA), and 10 kg of pathogen spore solution was used for treatment. Root irrigation with pathogens was performed once a month (April to August) as a control group for comparison with treatment (3);

[0175] (7) Commercial fertilizer treatment group (CB): Commercial fertilizer was used as base fertilizer and treated with 10 kg of pathogen spore solution. Root irrigation with pathogens was performed once a month (April to August). The purpose was to compare the biocontrol effects of commercial fertilizers with those of homemade fertilizers.

[0176] Except for the control group, the sugarcane seed stems of the other treatments were soaked in pathogen spore liquid for 1 hour before planting. After the sugarcane seedlings emerged, the pathogen spores were used for root irrigation again. After that, the CaPMCA, PMCA, CaPMA and PMA treatment groups were treated with compound antagonistic bacteria once a month, and the other treatment groups were added with the same amount of blank liquid culture medium.

[0177] 3.2 Field trial design

[0178] The treatments were consistent with those in the potted plant experiment, including (1) blank control (C): only fertilization without adding pathogens; (2) organic fertilizer treatment (SS): application of organic fertilizer and pathogen treatment at the same time; (3) bio-organic fertilizer (PMA): application of bio-organic fertilizer and pathogen treatment at the same time; (4) charcoal-based bio-organic fertilizer treatment group (PMCA): application of charcoal-based bio-organic fertilizer and pathogen treatment at the same time; (5) calcium charcoal-based bio-organic fertilizer treatment (CaPMCA): application of calcium charcoal-based bio-organic fertilizer and pathogen treatment at the same time; (6) calcium bio-organic fertilizer treatment group (CaPMA); and (7) commercial bacterial fertilizer treatment (CB): application of commercial bacterial fertilizer and pathogen treatment at the same time. There were a total of 7 treatment groups.

[0179] The field experiment adopted a randomized block design, with 4 blocks, each block had 7 plots (3m×6m) randomly assigned to 6 treatments. To reduce the interference between treatments, each plot was separated by a 5mm PP board, which extended to 20cm above the ground and 30cm below the soil surface. Each plot had 3 rows of sugarcane, planted in double bud segments, with a row spacing of 1m. At the same time, to prevent the spread of pathogens between plots, plastic film and steel frames were used to isolate different plots. The sugarcane variety was Guitang No. 44 perennial root sugarcane (45 plants in each plot). In addition to applying the required organic fertilizer and biological fertilizer, compound fertilizer (N, P, K content were all 15%), urea, superphosphate, and potassium chloride were used to adjust the nutrients of each treatment group. Fertilization was carried out twice on March 27 and June 27.

[0180] 4. Performance Testing

[0181] 4.1 Disease statistics and sample collection

[0182] Two months after planting, the number of diseased plants (those with obvious black whips in the center of the plant) was counted once a week, and the disease index was calculated when the disease was stable (no new diseased plants within two weeks).

[0183] Incidence rate = diseased plants / total number of plants × 100%.

[0184] Control rate = (incidence rate of control group - incidence rate of treatment group) / incidence rate of control group × 100%

[0185] Sugarcane leaves were collected from July to August and stored in a -4°C refrigerator (for measuring changes in sugarcane enzyme activity) and -80°C (for measuring microbial communities and quantities).

[0186] The results are shown in Tables 5, 6 and Figure 8 shown.

[0187] Table 5 Effect of controlling sugarcane smut in potted plant experiment

[0188] Treatment Group C SS PMA PMCA CaPMCA CaPMA CB Incidence % 0.00f 33.33a 11.42b 7.69c 4.00d 7.54 12.50b Prevention and control rate% -- -- 65.73c 76.92b 88.00a 77.38 62.50c

[0189] Note: Different lowercase letters in the table indicate significant differences in the incidence and control rates of sugarcane smut in different treatments

[0190] Table 6 Effect of field experiment on prevention and control of sugarcane smut

[0191] Treatment Group C SS PMA PMCA CaPMCA CaPMA CB Incidence % 0.00d 6.70a 2.20b 2.20b 0.00d 2.42 2.20b Prevention and control rate% -- -- 67.16b 67.16b 100.00a 63.88 67.16b

[0192] Note: Different lowercase letters in the table indicate significant differences in the incidence and control rates of sugarcane smut in different treatments.

[0193] As an important economic crop, the yield and quality of sugarcane are affected by many factors, among which smut is a common and destructive disease. In order to simulate the infection process of pathogens under natural conditions and evaluate the control effect, the spores of sugarcane whip smut were injected near the roots of sugarcane. Two months after the inoculation of the pathogen, some sugarcanes showed typical symptoms of smut infection, such as slender stems and increased tillers. Over time, the disease worsened. Three months later, black tassels formed by a large number of sugarcane whip smut spores appeared in the center of some sugarcanes.

[0194] Six months after the pathogen was inoculated, the number of diseased plants in the sugarcane field stabilized and no new diseased plants appeared. In the pot experiment, the disease situation of sugarcane was significantly aggravated after the sugarcane whip smut fungus was treated, and the incidence rate was about 5 times that of the field experiment. Compared with the field environment, the total amount of soil in the pot is less and the ecological environment is relatively simple, so the balance between microorganisms is more susceptible to interference and destruction by pathogens. In the limited soil space, pathogens are more likely to reproduce and spread rapidly, making it easier to infect sugarcane plants and make them sick.

[0195] In the control group (C) where only PDA culture medium and base fertilizer were added, no diseased plants were found in either pot tests or field tests. This indicates that sugarcane plants can grow healthily in the soil in the absence of pathogens.

[0196] In the pathogen treatment group (SS) inoculated with pathogen spores, the incidence rate in the pot test was 33.33%, while the incidence rate in the field test was 6.70%. Although the incidence rate in both environments did not exceed 50%, the incidence rate in the pot test was significantly higher than that in the field test. Guitang 44 is a medium-resistant variety to sugarcane smut, and this variety has a certain resistance to infection with sugarcane whip smut. Due to the small amount of soil in the pot environment and the relatively simple ecological environment, Guitang 44 is more susceptible to pathogens, resulting in a higher incidence rate. At the same time, this phenomenon also proves that microorganisms are the key to the role of disease-suppressing soil.

[0197] Compared with ordinary bio-organic fertilizer (PMA), composite bio-organic fertilizer (PMCA, CaPMCA) with added functional materials showed a more superior control effect. According to the data in Tables 5 and 6, composite bio-organic fertilizer with added functional materials can significantly reduce the incidence of sugarcane smut. In the pot test, the incidence of sugarcane smut after ordinary bio-organic fertilizer treatment was 11.42%. In the field experiment, the control rate of ordinary bio-organic fertilizer was 67.73%.

[0198] The control rate of carbon-based bio-organic fertilizer against smut in pot experiments reached 76.92%, significantly higher than that of ordinary bio-organic fertilizer (p<0.05). The control effect of calcium carbon-based bio-organic fertilizer in pot and field experiments was 88.00% and 100%, respectively, also significantly higher than that of ordinary bio-organic fertilizer (p<0.05).

[0199] The introduction of functional materials can remove harmful substances in fertilizers and promote the production of metabolites with pathogen-inhibiting effects. These metabolites play a huge role in the soil, effectively inhibiting the growth and reproduction of sugarcane whip smut fungus. At the same time, functional materials also have a positive impact on the secondary fermentation process, further optimizing the properties of fertilizers and providing more favorable conditions for the growth of beneficial microorganisms.

[0200] The above results show that the effects of sugarcane whip smut on sugarcane vary significantly under different environments. Secondly, the composite bio-organic fertilizer with added functional materials has a more significant effect in preventing and controlling sugarcane smut.

[0201] 4.2 Determination of plant height, stem diameter and number of leaves of sugarcane

[0202] Two months after planting, the plant height, plant fresh weight, dry weight, and sugarcane sugar content of each group were counted and the results are shown below.

[0203] The application of bio-organic fertilizer has a significant promoting effect on the growth of sugarcane. The growth indicators of sugarcane plants treated with bio-organic fertilizer, such as plant height, stem diameter, and number of leaves, were significantly higher than those of the control treatment. In the potted experiment, the plant heights of sugarcane plants treated with charcoal-based bio-organic fertilizer (PMCA), bio-organic fertilizer (PMA), and commercial bacterial fertilizer (CB) were 1.35 cm, 1.24 cm, and 1.22 cm, respectively, which were significantly higher than those of the control treatment (C, the average plant height was 1.14 cm) and the pathogen treatment (SS, the average plant height was 0.93 cm). The average plant height of sugarcane in the calcium charcoal-based agricultural waste material treatment group (CaPMCCA) was 1.55 cm, which was significantly higher than that of the other treatment groups ( Fig. 9 a).

[0204] Sugarcane smut is a parasitic fungus that competes for nutrients in the parasitic sugarcane and forms thick spores in the center of the sugarcane, causing the sugarcane plant to stop growing in the later stage ( Fig. 9b). Therefore, the height of sugarcane plants treated with pathogens decreased by 18.4% compared with the control group. However, when sugarcane was treated with calcium carbon-based agricultural waste materials, the beneficial microorganisms in them could effectively inhibit the growth of sugarcane whip smut, improve the existence form of soil nutrients, and promote the absorption of nutrients by plants. Therefore, the height of sugarcane treated with CaPMCCA group increased by 6.14% compared with the control group. At the same time, the biomass of sugarcane was also improved after the treatment of calcium bio-organic fertilizer. The dry weight of sugarcane after the treatment of calcium bio-organic fertilizer was 316.67g, which was 46% higher than that of the control group. The dry weight of sugarcane in the pathogen treatment group was 133.33g, which was 38.46% lower than that of the healthy treatment. This shows that the application of calcium carbon-based agricultural waste materials can not only promote the growth of sugarcane plants, but also effectively resist the invasion of sugarcane smut, and increase the biomass and yield of sugarcane.

[0205] Ustilago infection has a significant negative impact on sugarcane growth, manifested in leaf wilting, decreased photosynthesis capacity, and reduced sugar content. Fig.10 The data shown in the pot experiment showed that the sugar content of the sugarcane samples infected with pathogens (SS) was reduced to 9.00%, which was 40% less than that of the control group (C, sucrose content was 15.03%). The sucrose content of the sugarcane samples treated with bio-organic fertilizer (PMA), carbon-based bio-organic fertilizer (PMCA) and calcium carbon-based agricultural waste materials (CaPMCA) reached 14.04%, 15.23% and 15.70% respectively. The calcium carbon-based agricultural waste materials showed a more prominent promoting effect during the treatment process, which increased the synthesis of sugar in the sugarcane by about 4%.

[0206] In the field environment, the sucrose content in sugarcane decreased by about 8% in the experimental group treated with pathogens only. Compared with the potted environment, calcium carbon-based agricultural waste materials have a more obvious promoting effect on the synthesis of sugar in crops under field conditions, with an increase of about 12%. This may be due to the synergistic effect of biochar, calcium carbonate, and biological organic fertilizer, which increases the content of mineral nitrogen and the level of available potassium in the soil, thereby improving the properties and fertility of the soil.

[0207] 4.3 Determination of chitinase and β-1,3-glucanase activities

[0208] Using MUB fluorescence spectrophotometry

[99] The activities of chitinase and β-1,3-glucanase were determined, and the preparation method of crude enzyme solution was optimized. The specific steps are as follows:

[0209] Extraction buffer for β-1,3-glucanase: weigh 29 mg EDTA, 0.1 g ascorbic acid, absorb 35 μL β-mercaptoethanol, add to 100 mmol / L, pH 5.2 acetate buffer, dilute to 100 mL, store at low temperature (4°C) for later use. Extraction buffer for chitinase: weigh 29 mg EDTA, absorb 35 μL β-mercaptoethanol, add to 100 mmol / L, pH 5.2 acetate buffer, dilute to 100 mL, shake to dissolve, store at low temperature (4°C) for later use.

[0210] Preparation of crude chitinase and β-1,3-glucanase enzyme solutions: Weigh 2.0 g of sugarcane leaves respectively, add 10.0 mL of extraction buffer and grind into a homogenate, centrifuge at 12000g for 30 min, and collect the supernatant. Take 2 mL of supernatant and mix with 10 mL of acetone, keep at -20°C for 4 hours, centrifuge at 12000g for 20 min, and collect the precipitate. Wash the precipitate with acetone and blow dry the precipitate with nitrogen, dissolve the precipitate in 50 mmol / L, pH 5.2 acetate buffer, collect the supernatant after centrifugation at 12000g for 20 min, and store at low temperature for later use. The above reaction processes were all carried out at 4°C, and all were placed in a 4°C refrigerator in advance.

[0211] The activity of the enzyme is expressed as fluorescence activity and the calculation method is as follows:

[0212] Fluorescence activity (μmol / (h×g)) = (A 净 / A 标 ) / (W×t)

[0213] Where W is the weight of the sample, g; t is the reaction time, 4h; A 标 According to the fluorescence value of the standard; 净 Indicates the fluorescence value of the sample.

[0214] Chitinase and β-1,3-glucanase are respectively classified into the PR-3 and PR-2 families of pathogenesis-related proteins (PR), and both are widely present in plants

[106] . Chitinase and β-1,3-glucanase have the ability to decompose chitin and β-1,3-glucan, the main components of fungal cell walls, respectively, and have a wide range of resistance to a variety of fungal diseases. They are one of the key products of the defense response in plants

[107] . β-1,3-glucanase and chitinase also play an important role in the process of sugarcane resistance to smut. By measuring the activity of chitinase and β-1,3-glucanase, the occurrence of defense response in sugarcane can be reflected.

[0215] In the pot experiment, there were significant differences in the activity of β-1,3-glucan in sugarcane between different treatments (p<0.05). Fig.11The activity of β-1,3-glucan in the leaves of the group SS inoculated with only sugarcane whip smut fungus was 90.83% higher than that of the control group C. Compared with the SS treatment, the activity of β-1,3-glucanase in the sugarcane leaves was further increased after the application of bio-organic fertilizer. The activity of β-1,3-glucanase in the sugarcane leaves after PMA, PMCA, and CaPMCA treatments was 29.61%, 215.37%, and 365.31% higher than that of the SS treatment, respectively. Fig.12 ).

[0216] In the potted plant experiment, the chitinase activity in the sugarcane leaves of the group inoculated with sugarcane whip smut fungus (SS) was not significantly different from that of the healthy control (C). This result indicates that when sugarcane is infected with smut fungus, β-1,3-glucanase in its body is the main substance for sugarcane to resist smut fungus infection.

[0217] After applying bio-organic fertilizers (PMA, PMCA, CaPMCA) during sugarcane planting, the chitinase activity in sugarcane leaves was improved. Compared with SS treatment, the chitinase activity in sugarcane leaves after PMA, PMCA, and CaPMCA treatments increased by 394.56%, 597.67%, and 608.53%, respectively. 4.4 CAT and POD activity determination

[0218] POD activity is expressed by the ability of the sample to catalyze the oxidation of guaiacol by H2O2. One unit of enzyme activity is defined as: A per gram of tissue per milliliter of oxidation system per minute 470 Change 0.005.

[0219]

[0220] Where V 反总 : Total volume of reaction system, 0.2mL; V 样 : The volume of crude enzyme solution added to the reaction system, 10 μL = 0.01 mL; V 样总 : total volume of crude enzyme solution, 1 mL; T: reaction time, 60 s = 1 min; W: sample mass, g; ΔA is the difference between the absorbance value measured 30 s after the reaction started and the absorbance value measured 90 s after the reaction started.

[0221] CAT activity was determined using the ammonium molybdate colorimetric method. Definition of 1 enzyme activity unit: 1 μmol H2O2 degradation catalyzed per gram of tissue per minute is defined as one enzyme activity unit.

[0222]

[0223] Where V 样 : The volume of crude enzyme solution added to the reaction system, 0.02 mL; V 样总 : Total volume of crude enzyme solution, 1 mL; VS2 : Final volume of the reaction system, 0.02 mL; W: Sample mass, g; x: H2O2 concentration in the sample calculated from the standard curve, μmol / ml.

[0224] Reactive oxygen species (ROS) play a key role as messengers in plants. They play a decisive role in the development of plant resistance and can effectively regulate the broad-spectrum resistance of plants to a variety of pathogens

[108] . When plants detect pathogen invasion, they accumulate ROS to trigger pathogen poisoning reactions. However, excessive ROS can also cause damage to the plants themselves. POD is an enzyme that is ubiquitous in plants. It can use H2O2 as an oxidant to remove ROS in plants and participate in a variety of plant physiological processes. Therefore, changes in POD activity can reflect the dynamic process of stress resistance in plants as the environment changes.

[0225] In the pathogen treatment group (SS), the POD activity of sugarcane leaves decreased significantly compared with the healthy control (C) (p<0.05). In the field environment, the POD activity of sugarcane in the pathogen treatment group was 443.68U / g, which was 27.25% lower than that of the healthy treatment; in the potted environment, the POD activity of sugarcane in the pathogen treatment group was 253.28U / g, which was 19.99% lower than that of the healthy plant treatment ( Fig.13 ). This result shows that under the influence of pathogens, the POD activity of sugarcane leaves was inhibited to a certain extent. The invasion of pathogens caused a large amount of reactive oxygen species (ROS) to be produced in the sugarcane body to deal with the invading pathogens, thereby inhibiting the activity of the POD enzyme.

[0226] After PMA, PMCA, CaPMCA and other treatment groups, the POD activity in sugarcane leaves showed an upward trend. Compared with the C treatment group, the POD activity of sugarcane in the pot experiment and field experiment increased by 0.15% and 28.53%, 2.25% and 62.90%, and 2.94% and 61.89%, respectively. Fig.13 ). Although these improvements did not reach statistically significant levels, the positive effects of bio-organic fertilizer on POD activity were still observed. Bio-organic fertilizer can provide nutrients and microbial agents to enhance the disease resistance of sugarcane, thereby maintaining the stability of POD enzyme activity in sugarcane.

[0227] The above results show that the POD activity of sugarcane leaves showed a consistent trend under different environmental treatments. Under the influence of pathogens, POD activity was inhibited; under the treatment of biological organic fertilizer, POD activity increased.

[0228] Catalase (CAT) is also an important member of the plant's body to remove reactive oxygen species (ROS), and together with peroxidase (POD), it constitutes a key enzyme in the plant's defense system. Under field and potted conditions, the CAT activity in sugarcane leaves showed a consistent trend of change. In the treatments affected only by pathogens, the CAT activity in sugarcane leaves was 14.41U / g and 22.05U / g, respectively, which was 36.45% and 17.36% lower than that in the healthy treatment ( Fig.14 ).

[0229] The CAT activity of sugarcane leaves increased in the PMA, PMCA and CaPMCA treatments. Compared with the healthy treated plants, the CAT activity in the PMA, PMCA and CaPMCA treatments increased by 28.40%, 31.82%, 74.52%, 100.94%, 153.77%, 121.21% respectively in potted plants and fields. Different from the effect on POD activity, calcium carbon-based agricultural waste materials can significantly increase the CAT activity in sugarcane leaves (p<0.05).

[0230] Example 4: Effects of calcium carbon-based bio-organic fertilizer on sugarcane rhizosphere microorganisms

[0231] 3.1 Test materials and instruments

[0232] The main experimental equipment includes: DNA extraction kit (EZNA MicroElute Genomic DNA Kit, Omega, USA); Roche real-time fluorescence quantitative PCR instrument; spectrophotometer; flow analyzer (AutoAnalyzer 3-AA3, Seal Analytic, USA); flame atomic absorption spectrometer; alkaline diffusion dish, etc.

[0233] 3.2 Experimental design

[0234] Potted plant and field test design (same as Example 2):

[0235] 3.3 Performance Testing

[0236] 3.3.1 Determination of soil physical and chemical properties

[0237] pH: Mix the air-dried soil sample with pure water at a ratio of 1:2.5 (w / w) and shake at 170 rpm for 2 hours. After standing for 30 minutes, take the supernatant and measure the pH using a pH meter. Total carbon, total nitrogen, total phosphorus, and total potassium: Grind the air-dried sample through an 85-mesh sieve, weigh 20 mg, and use the potassium dichromate fusion method to determine the total carbon content; total nitrogen is determined by digestion method with AA3 continuous flow analyzer; weigh 50 mg of the air-dried sample that has passed an 85-mesh sieve and digest it with the three-acid method, and then use AA3 continuous flow analyzer and flame atomic absorption spectrometer to determine the total phosphorus and total potassium content.

[0238] Determination of available nutrients (AN, AP, AK): AN: Alkaline diffusion method, weigh 1.50g (accurate to 0.01g) of air-dried soil sample that has passed through an 85-mesh sieve and evenly spread it on the outer chamber of the diffusion dish, add 1.00g of zinc-ferrous sulfate reducing agent to the outer chamber of the soil, and spread it on the soil sample, and perform a reagent blank test. Add 3.0mL of boric acid-indicator solution to the inner chamber of the diffusion dish. Apply alkaline glue above the edge of the outer chamber of the diffusion dish, cover the frosted glass and rotate it several times to make the frosted glass completely adhere to the edge of the diffusion dish. Rotate the frosted glass to reveal a slit, add 10.0mL of sodium hydroxide solution to the outer chamber of the diffusion dish, and immediately cover it with a frosted glass. Gently rotate the diffusion dish horizontally to fully mix the outer chamber solution with the soil sample, and then carefully use two rubber bands to cross and tighten them in a cross shape to fix the frosted glass. Place it in a constant temperature box and keep it warm at 40℃ for 24h. Use 0.01 mol / L hydrochloric acid standard solution to titrate the amount of ammonia absorbed in the boric acid in the inner chamber. When the color changes from blue to purple-red, the end point is reached. Record the volume of the hydrochloric acid standard solution used and calculate the AN content.

[0239] AP: After extraction with reagent, use AA3 for determination. Weigh 5.00g of air-dried soil that has passed through an 85-mesh sieve, add 25mL of hydrochloric acid-sulfuric acid extractant, vortex at 180r / min for 5min at a constant temperature of 25℃, filter with double-layer slow quantitative filter paper, and perform a reagent blank test while the filtrate is being tested, and the determination is completed within 48h.

[0240] AK: After ammonium acetate extraction, use flame atomic absorption spectrometer to determine. Weigh 5.00 g of air-dried soil sample that has passed through an 85-mesh sieve into a 100-mL extraction bottle, add 50.0 mL of 1 mol / L ammonium acetate solution, cover the bottle stopper, shake well, oscillate at 180 r / min for 30 min at 20°C-25°C, filter immediately, perform a reagent blank test at the same time, and complete the analysis within 48 h.

[0241] The results are shown in the following table.

[0242] Table 7 Effects of different fertilizers on soil nutrients

[0243]

[0244]

[0245] Note: The data in the table are mean ± standard deviation. Different lowercase letters indicate significant differences between different treatments for the same index (p<0.05).

[0246] In the field test, the TOC content of soil in different treatment groups ranged from 13.59 g / kg to 16.90 g / kg, and there was no significant difference between the treatments. The biochar in the fertilizer slightly increased the organic carbon content of the soil, resulting in higher TOC content in the CaPMCA and PMCA treatment groups.

[0247] The soil TN content of different treatment groups ranged from 1.32g / kg to 1.74g / kg, and the AN content ranged from 0.13g / kg to 0.18g / kg. The TN content of the pathogen treatment group was 11% to 30% lower than that of other treatments (p<0.05), but there was no significant change in the soil AN content between different treatment groups. The composition of soil microorganisms is related to the absorption of N by sugarcane.

[0248] Compared with the control group, the TP and AP contents in the soil of the other treatment groups increased significantly, with the TP content increasing by 0.46 g / kg to 0.69 g / kg and the AP content increasing by 0.09 g / kg to 0.15 g / kg. Changes in the microbial community will change the form of phosphorus in the soil, leading to an increase in total phosphorus and available phosphorus in the soil.

[0249] The TK content in the soil of all treatment groups was between 1.43g / kg and 20.43g / kg, and the AK content was between 0.54g / kg and 1.04g / kg. The AK content in the soil of the calcium carbon-based bio-organic fertilizer and carbon-based bio-organic fertilizer treatment groups was significantly higher than that of the other fertilizer treatment groups and the control group (p<0.05), and there was no significant difference in AK content in other treatment groups (Table 7).

[0250] In the pot experiment, the TOC content in the soil of different treatment groups was between 14.19g / kg and 21.64g / kg. Unlike the field experiment, CaPMCA and PMCA significantly increased the TOC content in the soil. The TN content in the soil of different treatment groups was between 1.69g / kg and 2.18g / kg, and the AN content was between 0.13g / kg and 0.20g / kg. There was no significant difference in the TN and AN content in the soil of all treatment groups. The TP content in the soil of all treatment groups was between 2.18g / kg and 2.62g / kg, and the AP content was between 0.02g / kg and 0.12g / kg. The TK content in the soil of all treatment groups was between 19.27g / kg and 22.53g / kg, and the AK content was between 0.52g / kg and 2.88g / kg. There was no significant difference in the TK content in the soil of all treatment groups.

[0251] In summary, the present invention adds Bacillus subtilis, Bacillus cereus, Bacillus alpinus, Trichoderma harzianum and Trichoderma longibrachiatum, which have highly antagonistic effects on sugarcane smut pathogens (Sporisorium scitanmineum), to organic fertilizer in an amount of 20% of the dry matter of the fertilizer, and simultaneously adds 6% of biochar and calcium carbonate to prepare a composite bio-organic fertilizer for targeted prevention and control of sugarcane smut. The prevention and control benefits of the microbial fertilizer are verified through indoor potted plant tests and field tests. The microbial fertilizer of the present invention has the following performance advantages:

[0252] (1) The combined effects of calcium carbonate and biochar improve the quality and maturity of agricultural waste compost and improve the utilization effect of agricultural straw.

[0253] (2) Calcium carbon-based agricultural waste materials alleviate the pollution of soil caused by the long-term use of chemical fertilizers, improve soil fertility, and increase crop yields.

[0254] (3) The application of calcareous carbon-based agricultural waste materials increased microbial activity and recruited Bacillus, Trichoderma, and Pseudomonas.

[0255] (4) Calcium carbon-based agricultural waste materials use biological means to prevent and control the occurrence of diseases, reducing the use of chemical pesticides.

[0256] The above-mentioned embodiments are only embodiments of the present invention and cannot be understood as limiting the scope of the present invention, nor are they limiting the structure of the present invention in any form. It should be pointed out that, for those of ordinary skill in the art, several changes and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A microbial fertilizer, characterized in that: The invention comprises, by weight, 40-60 parts of animal manure, 2-4 parts of straw biochar, 4-6 parts of mulberry branches, and 8-14 parts of a mixed bacterial agent; the mixed bacterial agent consists of Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1, and Trichoderma longibrachiatum Z2; the accession number of Bacillus cereus X3 is GDMCC NO: 65100, the accession number of Trichoderma harzianum Z1 is GDMCC NO: 65101, the accession number of Trichoderma longibrachiatum Z2 is GDMCC NO: 65102, the accession number of Bacillus altitudinis X2 is GDMCC NO: 65103, the accession number of The deposit number of X2 is GDMCC NO:65099, and the deposit number of Bacillus subtilis X1 is GDMCC NO:65098.

2. The microbial fertilizer according to claim 1, characterized in that The microbial fertilizer includes, by weight: 50 parts of animal manure, 3.3 parts of straw biochar, 5 parts of mulberry branches, and 11 parts of mixed bacterial agents.

3. The microbial fertilizer according to claim 1, characterized in that: The carbonization temperature of straw biochar is 450-550℃ and the specific surface area is 14-17m 2 / g, average pore size is 8-10nm, pore capacity is 25-29m 3 / kg; in animal feces, the total carbon content is 40-43wt%, the total nitrogen content is 1-4wt%, the carbon-nitrogen ratio C:N is 25-27, and the water content is 60-75%; in mulberry branches, the total carbon content is 60-70wt%, the total nitrogen content is 0.5-1.5wt%, the carbon-nitrogen ratio C:N is 70-80, and the water content is 55-70%.

4. The microbial fertilizer according to claim 1, characterized in that: In the mixed bacterial agent, the volume ratio of Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1 and Trichoderma longibrachiatum Z2 is 1:1:1:1:

1.

5. The microbial fertilizer according to claim 1, characterized in that: In the mixed bacterial agent, the spore concentration of Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1 and Trichoderma longibrachiatum Z2 is 5×10 6 -1×10 8 cfu / mL.

6. The microbial fertilizer according to any one of claims 1 to 5, characterized in that: The microbial fertilizer further comprises, by weight, 2-4 parts of calcium carbonate.

7. A method for preparing the microbial fertilizer according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, mixing the raw materials other than the mixed bacterial agent in proportion and then stacking and fermenting to obtain organic fertilizer; S2, adding mixed bacterial agents to the organic fertilizer for secondary fermentation; S3, air drying to obtain the microbial fertilizer.

8. The method according to claim 7, characterized in that During the pile fermentation, the fermentation temperature of the organic fertilizer is controlled to be 60-70° C., and the humidity is 60-75%; the fermentation temperature is 35-38° C., in the dark condition, and the humidity is 50-60%.

9. The method according to claim 7, characterized in that: The secondary fermentation time is 12-16 days, and the pile is turned every 2-3 days.

10. Use of the microbial fertilizer according to any one of claims 1 to 6 in preventing and controlling plant smut.

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