Microbial fertilizer, preparation method and application thereof

Abstract

The present application relates to the technical field of sugarcane smut prevention and treatment, and particularly relates to a microbial fertilizer, a preparation method thereof and application thereof in prevention and treatment of smut. The microbial fertilizer comprises, by weight, 40-60 parts of cow dung, 2-4 parts of corn straw biochar, 4-6 parts of mulberry branch and 8-14 parts of mixed bacterial agent. The mixed bacterial agent is composed of Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1 and Trichoderma longibrachiatum Z2. The microbial fertilizer has a positive promoting effect on sugarcane and can significantly stimulate the self-defense response of sugarcane.

Description

Technical Field

[0001] This invention relates to the field of sugarcane smut control technology, specifically to a microbial fertilizer, its preparation method, and its application in controlling smut. Background Technology

[0002] Sugarcane smut is a common and serious sugarcane disease that poses a significant threat to the healthy growth of sugarcane. After an outbreak, it typically causes losses of 10% to 30%, and in severe cases, losses can reach 50% to 70%. Sugarcane smut is a cumulative disease, and its severity increases with the number of years of cultivation, especially on sugarcane grown in dryland areas.

[0003] In addition, there are also studies reporting on the biological control of sugarcane smut. Liao Yongmei and Xiong Guoru both discovered bacterial strains that have antagonistic effects against sugarcane smut fungus, such as Pseudomonas, Acinetobacter, and Bacillus subtilis (Liao Yongmei, Zhang Guiying, Huang Dingan, et al. Screening and identification of antagonistic bacteria against sugarcane smut fungus [J]. Guangxi Agricultural Biological Science, 2004, (03): 197-201 and Xiong Guoru, Zhao Gengfeng, Wu Suran, et al. Screening and identification of antagonistic bacteria HAS against sugarcane smut fungus [J]. Journal of Tropical Crops, 2013, 34 (06): 1149-1154). Sinha discovered that the sugarcane endophytic fungus Pseudomonas aeruginosa B18 can inhibit the activity of sugarcane smut fungus and enhance the stress resistance of sugarcane by regulating the content of hydrolytic enzymes 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 binding of *Ustilago maydis* (Xi Pinggen. Study on the inhibition of sexual binding of *Ustilago maydis* 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 Sexual Mating and Hyphal Growth Using a Bacterial Isolate [J]. Frontiers In Microbiology, 2017, (8): 778.).Trichoderma, Bacillus pumilus, and Burkholderia gladioli have all been shown to inhibit 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): 1872-853). 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 purpose of this invention is to provide a microbial fertilizer for controlling smut, its preparation method, and its application in the control of smut.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A microbial fertilizer, by weight, comprises: 40-60 parts animal manure, 2-4 parts straw biochar, 4-6 parts mulberry branches, and 8-14 parts mixed microbial agent; said mixed microbial agent is composed 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, and the preservation number of Bacillus altitudinis... The accession number for X2 is GDMCC NO:65099, and the accession number for 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 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on September 4, 2024.

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

[0009] In one preferred embodiment, the microbial fertilizer, by weight, comprises: 50 parts animal manure, 3.3 parts straw biochar, 5 parts mulberry branches, and 11 parts mixed microbial agent.

[0010] In one preferred embodiment, the carbonization temperature of the straw biochar is 450-550℃, and the specific surface area is 14-17m². 2 / g, with an average pore size of 8-10nm and a pore capacity of 25-29m³. 3 / kg.

[0011] In one preferred embodiment, the animal feces contain 40-43 wt% total carbon, 1-4 wt% total nitrogen, a carbon-to-nitrogen ratio 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-to-nitrogen ratio of 70-80, and a moisture content of 55-70%.

[0013] In one preferred embodiment, the animal feces are cow dung.

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

[0015] In one preferred embodiment, the volume ratio of Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1, and Trichoderma longibrachiatum Z2 in the mixed microbial agent 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 microbial agent is 5 × 10⁻⁶. 6 -1×10 8 cfu / mL.

[0017] After multiple experiments, it was verified that the microbial fertilizer containing Bacillus subtilis X1, Bacillus altitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1, and Trichoderma longibrachiatum Z2, when added at the same concentration and volume, exhibited the best growth-promoting effect on plants, but poor effect on smut. The absence of any one of these microorganisms, or an excessively high or low proportion of any one microorganism, resulted in a decrease in effectiveness.

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

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

[0020] S1. Mix the raw materials other than the mixed microbial agent in a certain proportion and then compost them to obtain organic fertilizer;

[0021] S2. Add mixed microbial agents to the organic fertilizer for secondary fermentation;

[0022] S3. Air dry to obtain the microbial fertilizer. Natural air drying is preferred.

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

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

[0025] In one preferred embodiment, during composting fermentation, the fermentation temperature of the organic fertilizer is controlled at 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℃, dark conditions, and humidity is 50-60%.

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

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

[0029] Based on the same inventive concept, the present invention also claims protection for the application of the microbial fertilizer in the prevention and control of plant smut.

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

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

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

[0033] (1) The physicochemical properties and nutrient composition of the organic fertilizer were optimized through secondary fermentation by functional microorganisms. The secondary fermentation process played a crucial role in promoting the decomposition of lignocellulose components in the fertilizer. Through this process, the humic acid content in the fertilizer was increased, while the microbial toxicity of the fertilizer was reduced. The combined effect of these factors significantly enhanced the compostability of the fertilizer and improved its quality.

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

[0035] (3) The microbial fertilizer described above is significantly more effective than other bio-organic fertilizers in controlling sugarcane smut. In addition, this fertilizer also has a positive promoting effect on sugarcane growth, effectively increasing the sugar content and biomass of sugarcane, thereby achieving the goal of increasing sugarcane yield.

[0036] (4) When sugarcane is invaded by pathogens, it triggers a sophisticated defense mechanism. In response, sugarcane produces chitinase and β-1,3-glucanase, both of which effectively resist pathogen invasion. In addition, sugarcane adjusts the activity of its reactive oxygen species scavenging enzymes, thereby increasing the accumulation of reactive oxygen species in the body to kill pathogens.

[0037] (5) By applying the microbial fertilizer, the activities of chitinase, β-1,3-glucanase, catalase, and peroxidase in sugarcane can be effectively increased, stimulating the sugarcane's own defense mechanisms and thus enhancing its ability to resist pathogen invasion. In addition, the microbial fertilizer can also alleviate oxidative stress in sugarcane to a certain extent, providing a guarantee for the healthy growth of sugarcane.

[0038] In summary, the microbial fertilizer described above has a significant ability to stimulate the sugarcane's own defense response, effectively adjust the physiological changes of sugarcane, promote its growth, and control diseases. Therefore, the microbial fertilizer described above has broad application prospects in ensuring normal sugarcane growth and increasing sugarcane yield. Attached Figure Description

[0039] Figure 1 Growth photos of the fungus that causes sugarcane smut after 10 days of culture;

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

[0041] Figure 3 Phylogenetic tree of 5 antagonistic strains;

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

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

[0044] Figure 6 This describes the changes in the composition of lignocellulose.

[0045] Figure 7 The changes in humic acid composition in the fertilizer before and after fermentation;

[0046] Figure 8 The diseased plant and its black rhizome are shown in Figure a; Figure a shows the diseased plant (left) and healthy plant (right) in pots; Figure b shows the diseased plant and its black rhizome in the field.

[0047] Figure 9 Growth indicators for sugarcane under different treatments;

[0048] Figure 10 The effects of different treatments on sugarcane sucrose content;

[0049] Figure 11 To evaluate the β-1,3-glucanase activity of sugarcane under different treatments;

[0050] Figure 12 To measure the chitinase activity of sugarcane under different treatments;

[0051] Figure 13 To measure the peroxidase activity of sugarcane under different treatments;

[0052] Figure 14 The catalase activity of sugarcane under different treatments was measured. Detailed Implementation

[0053] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0054] The data processing and statistical analysis methods of this invention are as follows: Barcode sequence identification is used, combined with the FLASH online tool to achieve accurate splicing of sequence pairing segments. Simultaneously, FASTP software is used for data quality control to remove low-quality sequences. Subsequently, the sequence is compared with a reference database to eliminate chimeric contamination. Using the QIIME2 software platform, deep sequence denoising, species annotation, and phylogenetic tree construction are performed to generate a set of amplicon sequence variants (ASVs) and their corresponding feature abundance matrices.

[0055] SPSS 24 software was used to assess the significance of differences between the two groups of data based on the independent sample t-test. To further reveal the heterogeneity of microbial community structure, non-metric multidimensional scaling analysis (NMDS) was applied to visually display the distance and similarity between communities. For the analysis of the association between soil chemical factors and protozoan communities, redundancy analysis (RDA) and correlation heatmaps were implemented using the Lianchuan Bio Cloud Platform. LEfSe analysis and correlation coefficient plots were completed using the Novogene Cloud Platform. Mantel analysis was implemented using the vegan and MASS packages in R software. The completion of other charts was also achieved using the bioinformatics cloud platform, Excel 2021, Origin 2022 software, and Adobe Illustrator.

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

[0057] Corn stalk biochar powder: purchased from Xingyuan Environmental Protection Co., Ltd., and its specific properties are shown in Table 1. Calcium carbonate powder was purchased directly from a calcium carbonate processing plant near the market in Dacai Township, Huanjiang County, Hechi City, Guangxi Zhuang Autonomous Region. Cow manure came from a cattle farm near the experimental field 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). The properties of all materials are shown in Table 2.

[0058] Table 1 Physicochemical properties of corn straw biochar

[0059]

[0060] Table 2 Properties of Initial Compost Materials

[0061]

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

[0063] 1. Experimental materials and instruments

[0064] The screening material for the pathogen causing sugarcane smut was fresh sugarcane smut rhizomes (variety Guitang 42, a susceptible variety of smut) collected from a sugarcane plantation at the Guangxi Academy of Agricultural Sciences. The functional verification of the inoculant also used sugarcane rhizomes of this variety.

[0065] Soil for screening antagonistic strains: rhizosphere soils of healthy and smut-infected sugarcane in sugarcane plantations of the Guangxi Academy of Agricultural Sciences, and disease-suppressing soils that have been fertilized with organic fertilizer for a long time.

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

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

[0068] Main instruments: DNA extraction kit (QIAprep Spinminiprep Kit250, USA); spectrophotometer (model 722, Shanghai Yidian Analytical Instrument Co., Ltd.); pure water system (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, isolation, and culture of the pathogen of sugarcane smut.

[0070] Fresh, newly formed sugarcane smut rhizomes, not yet broken through the leaf sheath, were collected from sugarcane plantations at the Guangxi Zhuang Autonomous Region Academy of Agricultural Sciences and laid out at 16-20℃ and 10-20% relative humidity for 8-12 hours. This step aims to maximally inhibit the growth of contaminating fungi (especially bacteria) on the black rhizomes, significantly reducing contamination of the sugarcane smut fungus's teliospores and ensuring a high germination rate (90%-98%). After thoroughly drying the black rhizomes, the surface was disinfected by wiping with 75% alcohol in a clean bench. The leaf sheaths were then peeled back, and the teliospores were scraped from the black rhizomes using sterile tweezers, removing impurities. The purified teliospores were aliquoted into 50mL centrifuge tubes, sealed with sterile sealing film, and stored at 4℃.

[0071] Weigh 1.5g of streptomycin sulfate and mix it with sterile water to prepare a streptomycin sulfate solution with a concentration of 1500mg / L (used to inhibit bacterial growth). Pick about 3g of treated sugarcane smut teliospores and put them into a beaker containing 250mL of streptomycin sulfate solution. Stir and let stand for 1 minute, then perform serial dilutions with streptomycin sulfate solution. Since streptomycin sulfate is not heat-resistant, after the PDA medium has been sterilized at high temperature, add another 1.5g of streptomycin sulfate when the temperature drops to about 60℃. Take 50μL of spore solution and spread it on PDA medium. Incubate at 28℃ for 3 days, then pick 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 isolated and purified using a dilution-spreading method. 5g of rhizosphere soil sample was weighed and placed in a 250mL Erlenmeyer flask containing 45mL of deionized water. The mixture was stirred on a constant-temperature shaker for 30min at 30℃ and 180rpm. The soil suspension was serially diluted with deionized water, and 50μL of each dilution was added to NA and PDA agar plates and spread evenly. The plates were incubated at 37℃ and 28℃ respectively. Single colonies with distinct characteristics were picked and purified until single colonies appeared on the plates. The purified bacteria and fungi were inoculated onto NA and PDA agar plates respectively and stored at 4℃ for short-term use, activating them every 3 months.

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

[0075] (1) Preliminary screening of antagonistic strains

[0076] Activation culture of Sporisorium scitamineum, the causal agent of sugarcane smut: Collect newly formed, yet unexposed, diseased rhizomes, disinfect the surface, peel off the leaf sheath, pick up a small number of black teliospores with sterile tweezers, dilute them in sterile water, spread the diluted Sporisorium scitamineum spore solution on PDA plates, and incubate them in a constant temperature incubator at 28℃ for 2 days. Then, transfer the plate to a clean bench, ignite the inoculation loop, cool it, and pick up the newly germinated pathogens to inoculate them onto a new PDA medium plate.

[0077] Preliminary screening of antagonistic strains of sugarcane smut fungus: When the smut fungal colonies on PDA plates to be individually inoculated reached approximately 2 mm in diameter, the plate confrontation method was used. Four isolated and purified single bacteria were inoculated at 20 mm intervals between the centers of each plate. Bacteria were inoculated using an inoculation loop, and fungi were inoculated using a 5 mm diameter perforator. Each treatment was repeated in triplicate, and a control group was also included. The plates were cultured for 10-15 days. The degree of antagonism was determined by the distance from the bacterial and fungal inoculation points to the outer edge of the fungus. Colonies exhibiting significant inhibition of sugarcane smut growth were selected, numbered, and recorded.

[0078] More than 100 bacterial strains and more than 60 fungal strains were isolated and purified from the fine roots and rhizosphere soil of healthy / disease-prone sugarcane and from soil that had been fertilized with organic fertilizer for a long time. They were stored in a 4C refrigerator for subsequent plate confrontation experiments. In the initial screening, 14 bacterial strains and 26 fungal strains with antagonistic ability were identified.

[0079] (2) Rescreening of antagonistic strains

[0080] The pathogen of sugarcane smut was inoculated in the center of PDA medium. When the diameter of the sugarcane smut colony was about 2 mm, the strains obtained from the initial screening were inoculated 1 cm from the edge of the plate. Only one functional strain was inoculated per plate, and each colony was set up in triplicate. 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 preserved. The inhibition rate (the ratio of the diameter of the control smut colony minus the diameter of the treated group colony to the diameter of the control colony) was used to compare the inhibitory ability of the antagonistic bacteria against sugarcane smut.

[0081] Further screening of the strains obtained from the initial screening revealed a group of bacteria and fungi with high inhibition rates against pathogens. Some of the data are shown in Table 3. Some strains also had high inhibition rates, but these are not listed.

[0082] Table 3. Inhibition rate of the rescreened strains against *Ustilago maydis*.

[0083]

[0084] 5. Identification of pathogens and antagonistic strains

[0085] Single colonies selected from the rescreening were transferred to the corresponding liquid culture media (LB for bacteria, PDA for fungi). Genomic DNA was extracted from each strain using bacterial and fungal genomic DNA extraction kits, respectively. PCR amplification was performed using universal primers 27F and 1492R for bacterial 16S rDNA and ITS1 and ITS4 for fungal ITS. The PCR reaction volume was 50 μL: 2 μL template DNA, 25 μL PCR premix, 1 μL each of the two primers, and dd H2O to a final volume of 50 μL. Conditions: 95℃ for 5 min; 95℃ for 30 s; 57℃ for 30 s; 72℃ for 5 min; 30 cycles; 72℃ for 5 min. The products were examined by electrophoresis, and the target bands were obtained. The products were then purified by gel extraction and sent to Beijing Qingke Company for sequencing.

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

[0087] Spores scraped from *Scutellaria baicalensis* were diluted with pure water and spread on plates, resulting in the growth of numerous white fungi. The number of colonies showed a significant negative correlation with the dilution factor. Based on literature, this white fungus is presumed to be *S. scritamineum*. The hyphae are pure white, densely packed, smooth, and round. Figure 1 Compared to other fungi, this bacterium grows relatively slowly on PDA medium. It takes 3 days to observe obvious colonies, the colony diameter is about 10 mm at 5 days, the average colony diameter is 22 mm at 10 days, and the average colony diameter is 30 mm at 15 days.

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

[0089] The ITS sequence obtained from sequencing the single strain B1 isolated from the smut virus was compared for homology using the BLAST website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Seven strains with greater than 99% homology to B1 were selected, and a phylogenetic tree was further constructed using MEGA 11 software, such as... Figure 2 As shown, the analysis results indicate that strain Bl belongs to the genus Sporisorium, specifically Sporisorium scitamineum.

[0090] The sequences obtained from the sequencing of the five rescreened strains were BLAST-aligned on the NCBI website. For each strain, 10 other strains with 99% similarity were selected. A preliminary phylogenetic tree was constructed using MEGA11 software to compare the sequences of antagonistic bacteria of *Smutella smut* and its similar strains. The tree structure was then further refined on the Itol website. Based on the alignment results, phylogenetic trees were constructed using 10 similar strains from each strain. The results are as follows: Figure 3As shown. Through comparison and phylogenetic tree analysis, the five strains obtained belong to two genera: Bacillus and Trichoderma. Three strains belong to the genus Bacillus: Bacillus subtilis, Bacillus altitudinis, and Bacillus cereus; the other two strains belong to the genus Trichoderma: Trichoderma longibrachiatum and Trichoderma harzianum.

[0091] 6. Interactions among antagonistic strains of sugarcane smut fungus

[0092] The absence of mutual inhibition of growth and reproduction among microorganisms is a prerequisite for the construction of synthetic microbial communities and a guarantee for the better functioning of these communities in biocontrol. To verify whether there is antagonistic interaction among the obtained dominant bacteria, the screened strains were inoculated on PDA plates. Bacteria were streaked in pairs, and bacterial-fungal antagonism was achieved by inoculating two bacteria 1 cm from the edge of the culture medium. Fungi were paired using a perforator. Three parallel experiments were set up. The plates were incubated at 30℃, and the growth of the strains was observed for phenomena such as failure to produce spores, colonies concave to one side, or small diameter. Strains with no growth inhibition or weak antagonistic interaction were selected for the next step of preparing the synthetic microbial community for controlling sugarcane smut.

[0093] Interactions among highly effective antagonistic strains of sugarcane smut can be found in Figure 4 The results showed that the two fungal strains, Z1 and Z2, grew relatively quickly. The study indicated that *Trichoderma* and *Bacillus* species exhibit similar mechanisms for controlling sugarcane smut, primarily involving competition for living space and nutrients, secretion of substances antagonistic to pathogens, promotion of sugarcane growth, and induction of immune responses. *Trichoderma harzianum*, in particular, demonstrates remarkable resilience, reproduces at a much faster rate than typical functional fungi, is adaptable to various soil types, and once it establishes a symbiotic relationship with its host plant, it is largely unaffected by adverse external environments. None of the five fungi exhibited significant growth inhibition or antagonistic effects.

[0094] Some of the bacteria showed significant antagonistic and growth-inhibiting effects with the two fungi Z1 and Z2, and were therefore excluded.

[0095] Example 2: Fertilizer Preparation

[0096] 1. Preparation of organic fertilizer

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

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

[0099] (2) PMC: Cow manure (50kg) plus mulberry branches (5kg) are the main composting materials, with 6% biochar added. The carbon-nitrogen ratio of the compost is about 25-30. No calcium carbonate is added to experimental group 1 (experimental group 1: organic fertilizer containing only biochar is used as the control group).

[0100] (3) CaPMC: Based on experimental group 1, 6% calcium carbonate powder was added at the beginning of composting and mixed evenly with cow manure and mulberry branch raw materials (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 bio-organic fertilizer

[0102] The spore concentrations of three bacteria (Bacillus subtilis, Bacillus altitudinis, and Bacillus cereus) and two fungi (Trichoderma harzianum and Trichoderma longibrachiatum) were adjusted to the same order of magnitude, i.e., 10⁻⁶, using the dilution plating method and hemocytometer. 8 The bacterial solutions were inoculated at the same volume ratio into (1) cow dung and mulberry branch compost (PM), (2) cow dung and mulberry branch biochar compost (PMC), and (3) calcareous cow dung and mulberry branch biochar compost (CaPMC). The total inoculation volume of the bacterial solutions was 20% (V / W) of the dry matter of the cow dung and mulberry branch compost product. Fermentation was carried out for 2 weeks (secondary fermentation process) to prepare bio-organic fertilizer PMA, carbon-based bio-organic fertilizer PMCA, and calcareous carbon-based bio-organic fertilizer CaPMCA. During fermentation, the compost was turned over every 2 days and naturally air-dried until the moisture content was about 25% to obtain different microbial organic fertilizers. During the initial antagonistic experiments, it was found that the control effect of the five bacteria alone on sugarcane smut was weak, and the effects of other combinations of two or three were also not ideal. After further exploration, it was found that the best effect was achieved by combining the five bacteria in a ratio of 1:1:1:1:1. Therefore, this ratio was used to make bio-organic fertilizer, and its impact on plants and its actual disease resistance in the field and in pots were investigated.

[0103] 3. Sample collection

[0104] Samples were collected at 0 and 14 days after inoculation. The collection method was as follows: 250g of samples were collected from four points (front, back, left, and right) within a 10cm radius of the center of the pile. After mixing, the samples were divided into three portions and packaged into self-sealing bags. The bags were stored at 4℃ for the determination of basic properties, at -80℃ for the determination of microorganisms, and air-dried for the determination of 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 (day 0) and after secondary fermentation (day 14). Fresh samples (samples stored at 4°C) were mixed with pure water at a ratio of 1:10 (w / w) and shaken at 170 rpm for 2 hours. After standing for 30 minutes, the supernatant was collected and the pH was measured using a pH meter.

[0109] Total carbon and total nitrogen: The air-dried sample was ground and passed through an 85-mesh sieve. 20 mg of the sample was weighed and the total nitrogen and total carbon content was determined using an elemental analyzer.

[0110] The results are shown in Table 4.

[0111] Table 4 Physicochemical properties of fertilizer 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). In the table, PMA, PMC and CaPMCA represent bio-organic fertilizer (control group), carbon-based bio-organic fertilizer with only biochar added, and calcium-based carbon-based bio-organic fertilizer with calcium carbonate and biochar added, respectively.

[0114] At 25℃, microbial activity can be effectively stimulated without causing microbial death or reduced activity due to excessively high temperatures. However, in actual secondary fermentation processes, the fermentation temperature rises due to heat production from microbial metabolism and heat exchange with the external environment. Experimental data show that maintaining a stable temperature of around 27℃ throughout the fermentation process ensures microbial activity while avoiding the adverse effects of excessively high temperatures on the fermentation process.

[0115] In the initial composting trials (single fermentation), special attention was paid to the high-temperature fermentation stage. This stage is crucial in composting because high temperatures effectively kill or inhibit harmful microorganisms in the feces, thus ensuring the safety and harmlessness of the compost product. Experimental results showed that the high-temperature (50℃) fermentation stage in all treatments was maintained for more than 15 days, fully meeting the composting temperature requirements of the "Hygienic 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 secondary solid-state fermentation. pH indicates the acidity or alkalinity of the fertilizer, which directly affects plant growth; a suitable pH range facilitates nutrient absorption by plants. Total nitrogen is one of the main nutrient elements that influence plant growth. Total carbon has a significant impact on soil structure and microbial activity. The carbon-nitrogen ratio reflects the carbon-nitrogen balance of the fertilizer and has a significant impact on the decomposition of organic matter by soil microorganisms.

[0117] After adding the bacterial solution, the moisture content of the organic fertilizer was around 65%, which is relatively high. After the secondary fermentation process, the moisture content of all fertilizers was reduced to below 30% as stipulated in the agricultural industry standard NY884-2012. This change indicates that the fermentation process effectively reduced the moisture content of the fertilizer and improved its stability.

[0118] Before and after fermentation, the pH values ​​of all three fertilizers decreased by 0.01, with no significant difference among them. This small change is significant because it indicates that the fertilizers did not undergo anaerobic acid production during fermentation, meaning no obvious acidification occurred. This further demonstrates that the entire fermentation process took place under aerobic conditions, ensuring its smooth progress and the stability of the fertilizer quality.

[0119] All three fertilizers showed an increase in total carbon and total nitrogen content before and after fermentation. The increase in total nitrogen was particularly significant for the calcium-based carbon-based bio-organic fertilizer (CaPMA), rising from 2.07% to 2.34%, an increase of 0.27%. The carbon and nitrogen-containing substances introduced during the addition of biochar and bacterial solution are the main reasons for this phenomenon. Simultaneously, these substances effectively promote the decomposition of organic matter during composting.

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

[0121] Calcium carbonate was added during the initial composting process of calcium-based carbon-based bio-organic fertilizer treatment. This measure effectively removes a large number of harmful microorganisms from the fertilizer and affects the total amount of microorganisms during secondary fermentation. Due to the reduction in the total number of microorganisms, their consumption of nitrogen and carbon in the environment is correspondingly reduced. Therefore, the addition of calcium carbonate demonstrates that it increases the nitrogen and carbon content of the compost pile.

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

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

[0124] In summary, the increase in total carbon content of the three fertilizers before and after fermentation indicates significant chemical changes during the composting process. Among them, the calcium-based bio-organic fertilizer (CaPMA) showed the most significant increase in total nitrogen, which may be related to the addition of biochar and bacterial solution. Simultaneously, the addition of calcium carbonate also contributed to increasing the nitrogen and carbon content of the fertilizer. Furthermore, the carbon-to-nitrogen ratio of all three fertilizers was below 20, indicating that they were fully composted and met favorable application conditions.

[0125] 4.2 Seed Germination Index (GI)

[0126] For each treatment group, 5g of fresh samples from 0 and 14 days of secondary fermentation were added to 50mL of pure water and shaken on a shaker at 170rpm for 30min, followed by filtration. 5mL of the filtrate was added to a petri dish lined with filter paper, and 50 uniformly sized cucumber seeds were selected, spread evenly on the petri dish, and placed in a 25℃ constant temperature incubator for 2 days in the dark.

[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 As shown.

[0129] The germination index (GI) is an important indicator frequently used to evaluate the toxicity of fertilizers before and after composting and to assess the degree of fertilizer maturity. Analyzing this index provides insights into the biotransformation and degradation processes during composting, enabling the production of safer and more efficient fertilizers for agriculture.

[0130] Figure 5 The changes in seed germination rate in the fertilizer before and after fermentation are shown in the figure. As can be seen from the figure, the seed germination rate of the fertilizer before fermentation was generally above 75%, while after secondary fermentation, the seed germination rate of the fertilizer was all above 80%. This result indicates that the secondary fermentation process further promotes the decomposition of the compost pile and reduces the biotoxicity of cow manure compost.

[0131] In the composting process, besides fermentation, the use of additives is also an important means to improve fertilizer quality. Studies have found that the addition of calcium carbonate and biochar can both increase the seed germination rate of the compost and reduce its phytotoxicity. When both are added simultaneously, it is even more beneficial for the recovery of the compost and the reduction of biotoxicity.

[0132] Generally, a seed germination rate exceeding 50% indicates that the compost pile is fully decomposed. A germination rate exceeding 80% indicates complete decomposition. The results show that the combined use of biochar and calcium carbonate not only significantly improves the degree of manure composting but also reduces the biotoxicity of the products.

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

[0134] Neutral detergent: Accurately weigh 18.6 g of analytical grade disodium ethylenediaminetetraacetate (EDTA) and 6.8 g of analytical grade sodium borate into a beaker, dissolve them by heating, then add 30 g of analytical grade sodium dodecyl sulfate and 10 mL of analytical grade glycol ether. Weigh 4.56 g of analytical grade anhydrous sodium phosphate, dissolve it by heating, mix it with the neutral detergent, and bring the volume to 1 L. Acidic detergent: Weigh 20 g of analytical grade hexadecyltrimethylammonium bromide into a beaker and bring the volume to 1 L with 0.5 mol / L sulfuric acid.

[0135] Lignocellulose content: determined using the Van der Waals washing method.

[75] The method for determining the lignocellulose content in fertilizer after the fixed decomposition period and secondary fermentation is as follows: 0.5g of dried and weighed air-dried sample was transferred to a feed fiber analysis filter bag (three replicates per group), a condenser was attached, and the sample was placed in an automatic fiber analyzer. Neutral detergent was added (1L of neutral detergent was added to each batch of 12 samples) and boiled for 1 hour. After boiling, the filter bag was removed, rinsed with hot water and spun dry more than 5 times until there was no foam in the washing liquid. The bag was then rinsed twice with 20mL of acetone. Finally, the filter bag was dried in a 105℃ oven to obtain sample a1, which was then weighed to obtain A1.

[0136] After drying and weighing, sample a1 was fitted with a condenser and placed in a fully automatic fiber tester. Acidic detergent was added (1 L of acidic detergent was added to each batch of 12 samples) and boiled for 1 hour. After boiling, the filter bag was removed, rinsed with hot water and spun dry, and then rinsed with a small amount of acetone. Finally, the filter bag was placed in a 105℃ oven to dry sample a2, and the acidic detergent lignin content A2 was obtained by weighing.

[0137] Acid-washed lignin was added to 5 mL of sulfuric acid for digestion at room temperature for 3 hours, rinsed, and dried to obtain sample a3. The sample a3 was then weighed to obtain sample A4. Sample a3 was then ignited in a muffle furnace at a temperature above 500 degrees Celsius and weighed to obtain sample A4.

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

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

[0140] In fertilizer production, the content of lignocellulose components affects the overall quality of the fertilizer and also reflects the degree of compost stabilization. The lignocellulose content showed a significant decreasing trend during fermentation. For example... Figure 6 As shown, after fermentation, the lignocellulosic content in PMA, PMCA, and CaPMCA was 14.95%, 18.69%, and 14.08%, respectively, which decreased by 21.89%, 19.26%, and 16.54% compared with before fermentation. This indicates that the lignocellulosic components were effectively degraded during composting.

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

[0142] Trichoderma microorganisms can produce lignin-degrading enzymes, thereby enhancing the degradation of lignin in straw. In this study, the degradation rate of hemicellulose in CaPMCA fertilizer was higher than in other treatments. This may be related to the fact that, compared to the hard "outer 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, making it more easily degraded by microorganisms.

[0143] Furthermore, this invention also found that the addition of calcium carbonate is beneficial for promoting the degradation of hemicellulose and cellulose during composting. Calcium carbonate can regulate the pH of the compost, providing a more suitable growth environment for microorganisms, thereby promoting the degradation of lignocellulose components. In contrast, the degradation rate of lignocellulose components decreased in the treatment with only biochar added. This may be related to the lignin content at the start of fermentation and the loss of moisture and reduction in the total volume of the compost pile during fermentation.

[0144] In conclusion, the addition of exogenous functional microorganisms, calcium carbonate, and other exogenous substances during composting helps promote the degradation of lignocellulose components, thereby improving fertilizer quality.

[0145] 4.4 Determination of humic acid components

[0146] Preparation of test solution: Potassium dichromate was extracted using sodium pyrophosphate method.

[79] The specific method is as follows: Accurately weigh 5.00g of the air-dried sample that has passed through an 85-mesh sieve. Add 100mL of a mixed extraction solution of sodium pyrophosphate and sodium hydroxide (44.6g of analytical grade sodium pyrophosphate and 4g of analytical grade sodium hydroxide, diluted to 1L with deionized water, pH approximately 13), shake for 5min, let stand for 14h, then shake well and filter. Collect the filtrate in a 100mL Erlenmeyer flask for analysis.

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

[0148] Determination of carbon content in humic acid: Take 10 mL of the test solution, adjust the pH to 3 with 0.5 mol / L sulfuric acid, incubate at 80℃ in a water bath for 30 min, let stand for 14 h, centrifuge and filter. Dissolve the precipitate in small amounts several times with 0.05 mol / L sodium hydroxide, and dilute to 50 mL. Take 10 mL of the diluted liquid, adjust the pH to approximately 7 with 0.5 mol / L sulfuric acid. After precipitation occurs, evaporate to dryness in a water bath and determine the carbon content using the potassium dichromate fusion method.

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

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

[0151] During composting, microorganisms decompose the organic matter in waste into 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 degree of compost maturity.

[0152] Figure 7 The changes in humic acid composition before and after composting are shown. As can be seen from the figure, the humic acid content of each fertilizer showed an increasing trend after fermentation. This indicates that during fermentation, the decomposition of organic matter by microorganisms promoted the humification process, resulting in an increase in the humic acid content of the fertilizers.

[0153] Besides 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, and has an important impact on soil structure and soil fertility.

[0154] During fermentation, a significant increase in humic acid content was observed in the fertilizers. At the end of fermentation, the humic acid content in the calcium-based charcoal bio-fertilizer, charcoal-based bio-organic fertilizer, and bio-organic fertilizer increased by 0.917 g / kg, 1.32 kg, and 1.24 g / kg, respectively. Simultaneously, the humic acid content in the organic fertilizer 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 compost pile, while the presence of biochar facilitated the conversion of organic matter into humic acid by microorganisms.

[0155] also, Figure 7 It also indicates that the content of fulvic acid changed before and after fermentation. However, this change was not significant, which may be because fulvic acid, as a component with a high degree of oxidation in humic matter, is affected by a variety of factors in terms of its content and changes.

[0156] Humulus-Fullness ratio (DP) is another important indicator reflecting the degree of compost maturity. A higher DP value indicates higher fertilizer quality. The DP values ​​before and after secondary fermentation for CaPMCA, PMCA, and PMA treatments were 2.22 and 3.14; 1.83 and 3.15; and 2.22 and 3.14, respectively. After secondary fermentation, the DP of the fertilizer increased. During fermentation, microorganisms utilize the organic matter in the fertilizer to synthesize new humic acids, while the fulvic acid content in the fertilizer did not increase significantly. Therefore, at the end of the experiment, the DP showed an increasing trend.

[0157] When calcium carbonate and biochar are present simultaneously, the fertilizer's DP value reaches its highest level after fermentation. This demonstrates that calcium carbonate and biochar significantly improve fertilizer quality.

[0158] In summary, secondary fermentation deepens the humification process of fertilizers, effectively improving their quality. Furthermore, the addition of functional microorganisms and biochar can further promote the humification process, increasing the humic acid and humic acid content in the fertilizer, thereby improving soil structure and fertility.

[0159] Example 3

[0160] 1. Experimental materials and instruments

[0161] Experimental Locations: Both field and pot experiments 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 field experiment was conducted in Xiadong Village, Dacai Township, while the pot experiment was conducted in an open-air greenhouse within the Karst Ecosystem Observation Station. The research subject was the most widely planted variety in Huanjiang area, "Guitang 44" (cultivated from sugarcane stalks collected from the field). Soil for the pot experiment was taken from the field experiment plot. The soil was dried, ground, and sieved to remove stones and branches before use. The soil properties were as follows: pH 5.37, organic matter content 14.64 g / kg, total nitrogen content 1.89 g / kg, total phosphorus content 1 g / kg, and total potassium content 5.72 g / kg.

[0162] Main instruments and equipment: constant temperature shaker; flow analyzer (AutoAnalyzer3-AA3, SealAnalytic, USA), multi-functional 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 11.55 mL of glacial acetic acid and dilute with distilled water to 1000 mL. Solution B: Weigh 16.4 g of anhydrous sodium acetate, dissolve in distilled water and bring the volume to 1000 mL. Mix 105 mL of Solution A and 395 mL of Solution B, adjust the pH to 5.2, and dilute to 1000 mL to obtain a 100 mmol / L, pH 5.2 acetate buffer), etc.

[0164] 2. Preparation of pathogenic spore solution

[0165] Collection of pathogenic spores: Collect fresh black whip-like samples of sugarcane, thoroughly dry them at approximately 15℃ and 15% relative humidity, disinfect the sugarcane surface with alcohol, and collect the teliospore powder from the black whip-like spikes into 50mL centrifuge tubes using sterilized tweezers for storage. When using, take 25g of spore powder and dilute it in 10kg of water (concentration approximately 5×10⁻⁶). 6 (Spores / mL), dilute and spread on a plate before use to ensure spore activity.

[0166] 3. Pot and field experiment design

[0167] 3.1 Pot Experiment Design

[0168] Six treatments were set up for potted plants, with 10 replicates in each treatment group. Each pot contained 20 kg of soil and 1.5 kg of fertilizer (pre-mixed evenly with the soil, the fertilizer used was the fertilizer prepared in Example 1). 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 containers were foam boxes (30cm×20cm×10cm). The sugarcane seed stalks were buried 10cm 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 the base fertilizer and no treatment was performed. It served as the control group. The organic fertilizer was mainly composed of cow manure (50 kg) and mulberry branches (5 kg). The carbon-nitrogen ratio of the compost was about 25-30. No microorganisms were added for 2 weeks of fermentation (secondary fermentation process). During fermentation, the pile was turned over every 2 days and naturally air-dried until the moisture content was about 25%.

[0170] (2) Pathogen treatment group (SS): Based on the blank control group, the group was treated with 10 kg of pathogen spore liquid and the pathogen was drenched in the roots once a month (April-August);

[0171] (3) Bio-organic fertilizer treatment group (PMA): Bio-organic fertilizer made from fermented organic fertilizer and compound antagonistic bacteria agent was used as base fertilizer. 10 kg of pathogen spore liquid was used for treatment. Pathogen root irrigation was carried out once a month (April-August) and compared with treatment (2). The compound antagonistic bacteria agent was prepared by dilution coating method and hemocytometer to adjust the spore liquid concentrations of 3 bacteria (Bacillus subtilis, Bacillus altitudinis, and Bacillus cereus) and 2 fungi (T. Harzianum and T. longibrachiatum) to the same order of magnitude, i.e., 10. 8 CFU / mL. The total inoculation volume of the bacterial solution was 20% (V / W) of the dry matter weight of the pig manure and mulberry branch compost product.

[0172] (4) Carbon-based bio-organic fertilizer treatment group (PMCA): Based on the bio-organic fertilizer treatment group (PMA), 6% biochar was added as base fertilizer and treated with 10kg pathogen spore liquid. Pathogen root irrigation treatment was carried out once a month (April-August) as a control group and compared with treatment (3).

[0173] (5) Calcium-based 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 liquid was used for treatment. Pathogen root irrigation treatment was carried out once a month (April-August). 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 liquid was used for treatment. Pathogen root irrigation was carried out once a month (April-August) as a control group and compared with treatment (3).

[0175] (7) Commercial microbial fertilizer treatment group (CB): Commercial microbial fertilizer was used as the base fertilizer, and 10 kg of pathogen spore liquid was used for treatment. Pathogen root irrigation was carried out once a month (April-August). The purpose was to compare the biocontrol effect of commercial microbial fertilizer and self-made microbial fertilizer.

[0176] Except for the control group, all other treatments were soaked in pathogen spore solution for 1 hour before planting. After the sugarcane emerged, the roots were drenched again with pathogen spores. Subsequently, the CaPMCA, PMCA, CaPMA and PMA treatment groups were drenched once a month with a compound antagonistic bacterial agent, while other treatment groups were given an equal amount of blank liquid culture medium.

[0177] 3.2 Field Trial Design

[0178] Consistent with the treatments in the pot experiment, there were 7 treatment groups: (1) Blank control (C): only fertilization treatment without the addition of pathogens; (2) Organic fertilizer treatment (SS): organic fertilizer was applied and pathogens were treated at the same time; (3) Bio-organic fertilizer (PMA): bio-organic fertilizer was applied and pathogens were treated at the same time; (4) Carbon-based bio-organic fertilizer treatment group (PMCA): carbon-based bio-organic fertilizer was applied and pathogens were treated at the same time; (5) Calcium carbon-based bio-organic fertilizer treatment (CaPMCA): calcium carbon-based bio-organic fertilizer was applied and pathogens were treated at the same time; (6) Calcium bio-organic fertilizer treatment group (CaPMA); and (7) Commercial microbial fertilizer treatment (CB): commercial microbial fertilizer was applied and pathogens were treated at the same time.

[0179] The field trial employed a randomized block design with four blocks, each containing seven plots (3m × 6m) randomly assigned to six treatments. To minimize interference between treatments, each plot was separated by 5mm thick PP boards extending 20cm above ground and 30cm below the soil surface. Each plot contained three rows of sugarcane, planted with double buds, with a row spacing of 1m. To prevent the spread of pathogens between plots, different plots were separated using plastic film and steel frames. The sugarcane variety was Guitang 44 ratoon sugarcane (45 plants per plot). In addition to the required organic and bio-fertilizers, compound fertilizer (15% N, P, K), urea, superphosphate, and potassium chloride were used to ensure nutrient consistency across treatments. Fertilization was performed twice, on March 27th and June 27th.

[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 streaks in the center of the plant are considered diseased) is counted weekly. The disease index is calculated when the disease stabilizes (no new diseased plants appear within two weeks). Disease incidence and control rate calculations:

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

[0184] Prevention and control rate = (Incidence rate in control group - Incidence rate in treatment group) / Incidence rate in control group × 100%

[0185] Sugarcane leaves were collected in July and August and stored at -4°C (for measuring changes in sugarcane enzyme activity) and -80°C (for measuring microbial community and quantity).

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

[0187] Table 5. Control effect of sugarcane smut in pot experiments

[0188] Processing group C SS PMA PMCA CaPMCA CaPMA CB Incidence rate % 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 rate of sugarcane smut under different treatments.

[0190] Table 6. Field experiment results on the control effect of sugarcane smut.

[0191] Processing group C SS PMA PMCA CaPMCA CaPMA CB Incidence rate % 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 rate of sugarcane smut under different treatments.

[0193] Sugarcane, as an important economic crop, has its yield and quality affected by various factors, among which smut is a common and destructive disease. To simulate the pathogen infection process under natural conditions and evaluate the control effect, spores of *Ustilago maydis* were injected near the roots of sugarcane. Two months after inoculation, some sugarcane showed typical symptoms of smut infection, such as thin, elongated stalks and increased tillering. The disease worsened over time. Three months later, black filaments formed by numerous *Ustilago maydis* spores appeared in the center of some sugarcane plants.

[0194] Six months after inoculation with the pathogen, the number of diseased sugarcane plants in the field stabilized, with no new diseased plants appearing. In pot experiments, sugarcane treated with *Ustilago maydis* showed a significantly increased incidence of disease, approximately five times higher than in the field trials. Compared to the field environment, the soil volume in pots is smaller, and the ecological environment is relatively simpler, making the balance of microorganisms more susceptible to interference and disruption by pathogens. Within the limited soil space, pathogens can more easily multiply and spread rapidly, thus more easily infecting sugarcane plants and causing disease.

[0195] In the control group (C), which only received PDA culture solution and basal fertilizer, no diseased plants were found in either the pot experiment or the field experiment. 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 disease incidence rate was 33.33% in the pot experiment and 6.70% in the field experiment. Although the disease incidence rate did not exceed 50% in either environment, the incidence rate in the pot experiment was significantly higher than that in the field experiment. Guitang 44 is a moderately resistant variety to sugarcane smut, exhibiting some resistance to infection by *Ustilago maydis*. Due to the smaller soil volume and simpler ecological environment in the pot environment, Guitang 44 was more susceptible to pathogen attack, leading to a higher disease incidence rate. This phenomenon also demonstrates that microorganisms are key to the effectiveness of disease-suppressing soil.

[0197] Compared with ordinary bio-organic fertilizer (PMA), compound bio-organic fertilizers (PMCA, CaPMCA) with added functional materials showed superior control effects. According to data in Tables 5 and 6, compound bio-organic fertilizers with added functional materials significantly reduced the incidence of sugarcane smut. In pot experiments, the incidence of sugarcane smut after treatment with ordinary bio-organic fertilizer was 11.42%. In field experiments, the control rate of ordinary bio-organic fertilizer was 67.73%.

[0198] In pot experiments, carbon-based bio-organic fertilizer achieved a control rate of 76.92% against smut, significantly higher than that of ordinary bio-organic fertilizer (p<0.05). Meanwhile, calcium-based carbon-based bio-organic fertilizer showed control effects of 88.00% and 100% in pot and field experiments, respectively, also significantly higher than that of ordinary bio-organic fertilizer (p<0.05).

[0199] The introduction of functional materials can remove harmful substances from fertilizers and promote the production of metabolites that inhibit pathogens. These metabolites play a significant role in the soil, effectively suppressing the growth and reproduction of sugarcane smut fungus. Simultaneously, the functional materials also have a positive impact on the secondary fermentation process, further optimizing fertilizer properties and providing more favorable conditions for the growth of beneficial microorganisms.

[0200] The above results indicate that the impact of sugarcane smut fungus on sugarcane varies significantly under different environmental conditions. Furthermore, compound bio-organic fertilizers with added functional materials exhibit a more significant effect in controlling sugarcane smut.

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

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

[0203] The application of bio-organic fertilizer significantly promotes the growth of sugarcane. Sugarcane plants treated with bio-organic fertilizer showed significantly higher growth indicators, including plant height, stem diameter, and number of leaves, compared to the control treatment. In pot experiments, the plant heights of sugarcane treated with carbon-based bio-organic fertilizer (PMCA), bio-organic fertilizer (PMA), and commercial microbial fertilizer (CB) were 1.35 cm, 1.24 cm, and 1.22 cm, respectively, significantly higher than the control treatment (C, mean plant height 1.14 cm) and the pathogen treatment (SS, mean plant height 0.93 cm). The mean plant height of sugarcane in the calcareous carbon-based agricultural waste material treatment group (CaPMCCA) was 1.55 cm, significantly higher than other treatment groups. Figure 9 a).

[0204] Sugarcane smut is a live, nutritional parasitic fungus that competes with sugarcane for nutrients and forms chlamydospores at the central tip of the sugarcane, causing the sugarcane plant to stop growing in later stages. Figure 9(b) Therefore, the sugarcane plants treated with pathogens had a 18.4% lower plant height compared to the control group. However, when sugarcane was treated with calcium-based carbonaceous agricultural waste materials, the beneficial microorganisms within them effectively inhibited the growth of sugarcane smut and improved the form of soil nutrients, promoting nutrient absorption by plants. Therefore, the sugarcane treated with CaPMCCA had a 6.14% higher plant height compared to the control group. Simultaneously, the biomass of sugarcane was also increased after treatment with calcium-based bio-organic fertilizer; the dry weight of sugarcane treated with calcium-based bio-organic fertilizer was 316.67 g, 46% higher than the control group. The dry weight of sugarcane in the pathogen-treated group was 133.33 g, a 38.46% decrease compared to the healthy treatment. This indicates that the application of calcium-based carbonaceous agricultural waste materials not only promotes the growth of sugarcane plants but also effectively resists the invasion of sugarcane smut, increasing sugarcane biomass and yield.

[0205] Infection with smut fungus has a significant negative impact on sugarcane growth, specifically manifested as leaf wilting, reduced photosynthetic capacity, and decreased sugar content. According to... Figure 10 The data shown indicate that in pot experiments, sugarcane samples infected with the pathogen (SS) had a sugar content reduced to 9.00%, a 40% decrease compared to the control group (C, sucrose content 15.03%). Sugarcane samples treated with bio-organic fertilizer (PMA), carbon-based bio-organic fertilizer (PMCA), and calcareous carbon-based agricultural waste material (CaPMCA) achieved sucrose contents of 14.04%, 15.23%, and 15.70%, respectively. Calcium-based carbon-based agricultural waste material exhibited a more significant promoting effect during treatment, increasing sugar synthesis in sugarcane by approximately 4%.

[0206] In a field setting, the sucrose content in sugarcane decreased by approximately 8% in the experimental group treated only with pathogens. Compared to the potted environment, calcium-based biochar agricultural waste materials showed a more significant promoting effect on sugar synthesis in crops under field conditions, increasing it by approximately 12%. This may be due to the synergistic effect of biochar, calcium carbonate, and bio-organic fertilizer, which increased the content of mineral nitrogen and available potassium in the soil, thereby improving soil properties and fertility.

[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, and add 35 μL β-mercaptoethanol to a 100 mmol / L, pH 5.2 acetate buffer solution. Adjust the volume to 100 mL and store at 4°C for later use. Extraction buffer for chitinase: Weigh 29 mg EDTA, add 35 μL β-mercaptoethanol to a 100 mmol / L, pH 5.2 acetate buffer solution. Adjust the volume to 100 mL, shake to dissolve, and store at 4°C for later use.

[0210] Preparation of crude chitinase and β-1,3-glucanase enzyme solutions: 2.0 g of sugarcane leaves were weighed and homogenized in 10.0 mL of extraction buffer. The homogenate was centrifuged at 12000 g for 30 min, and the supernatant was collected. 2 mL of the supernatant was mixed with 10 mL of acetone and kept at -20℃ for 4 h. The mixture was then centrifuged at 12000 g for 20 min, and the precipitate was collected. The precipitate was washed with acetone and dried under nitrogen. The precipitate was dissolved in 50 mmol / L, pH 5.2 acetate buffer, centrifuged at 12000 g for 20 min, and the supernatant was collected and stored at low temperature for later use. All reactions were carried out at 4℃, and all samples were placed in a 4℃ refrigerator beforehand.

[0211] Enzyme activity 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] In the formula, W is the weight of the sample, in grams; t is the reaction time, in 4 hours; A 标 Based on the fluorescence value of the standard, A was obtained. 净 This indicates the sample fluorescence value.

[0214] Chitinase and β-1,3-glucanase belong to the PR-3 and PR-2 families of pathogenesis-related proteins (PRs), respectively, and 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 broad resistance to a variety of fungal diseases. They are one of the key products of the plant's defense response

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

[0215] In pot experiments, there were significant differences in the activity of β-1,3-glucan in sugarcane among different treatments (p<0.05), see [link to relevant documentation]. Figure 11In the SS group (inoculated only with *Ustilago maydis*), the activity of β-1,3-glucanase in the leaves was 90.83% higher than that in the control group (C). Compared with the SS treatment, the application of bio-organic fertilizer further increased the activity of β-1,3-glucanase in sugarcane leaves. The activities of β-1,3-glucanase in sugarcane leaves after PMA, PMCA, and CaPMCA treatments were 29.61%, 215.37%, and 365.31% higher than those after the SS treatment, respectively. Figure 12 ).

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

[0217] After applying bio-organic fertilizers (PMA, PMCA, CaPMCA) during sugarcane cultivation, the chitinase activity in sugarcane leaves was increased. Compared with the SS treatment, the chitinase activities in sugarcane leaves treated with PMA, PMCA, and CaPMCA increased by 394.56%, 597.67%, and 608.53%, respectively. 4.4 CAT and POD Activity Determination

[0218] POD activity is expressed as the ability of a 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] In the formula V 反总 Total volume of the reaction system: 0.2 mL; V 样 The volume of crude enzyme solution added to the reaction system is 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 start of the reaction and the absorbance value measured 90 s after the start of the reaction.

[0221] CAT activity was determined using the ammonium molybdate colorimetric method. One unit of enzyme activity is defined as the rate at which one gram of tissue catalyzes the degradation of 1 μmol of H₂O₂ per minute.

[0222]

[0223] In the formula V 样 The volume of crude enzyme solution added to the reaction system is 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 messenger role in plants, which is crucial for the development of plant resistance to antibiotics and can effectively regulate the broad-spectrum resistance of plants to a variety of pathogens

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

[0225] In the pathogen-treated group (SS), the POD activity in sugarcane leaves was significantly lower than that in the healthy control (C) (p<0.05). In the field, the POD activity in the pathogen-treated group was 443.68 U / g, a decrease of 27.25% compared to the healthy control; in the pot environment, the POD activity in the pathogen-treated group was 253.28 U / g, a decrease of 19.99% compared to the healthy plant treatment. Figure 13 This result indicates that the activity of POD enzyme in sugarcane leaves was inhibited to some extent under the influence of pathogens. The invasion of pathogens leads to the production of large amounts of reactive oxygen species (ROS) within the sugarcane to combat the invading pathogens, thereby inhibiting the activity of POD enzymes.

[0226] After treatment with PMA, PMCA, and CaPMCA, the POD activity in sugarcane leaves all showed an increasing trend. Compared with treatment group C, in pot experiments and field experiments, the POD activity of sugarcane increased by 0.15% and 28.53%, 2.25% and 62.90%, and 2.94% and 61.89%, respectively. Figure 13 Although these improvements did not reach a statistically significant level, a positive effect of bio-organic fertilizer on POD activity could still be observed. Bio-organic fertilizer provides nutrients and microbial agents, enhancing the disease resistance of sugarcane and thus maintaining the stability of POD enzyme activity in sugarcane.

[0227] The above results indicate that the POD activity of sugarcane leaves exhibits a consistent trend under different environmental treatments. POD activity is inhibited under the influence of pathogens, while it increases under bio-organic fertilizer treatment.

[0228] Catalase (CAT) is also an important member of the plant's reactive oxygen species (ROS) scavenging system, and together with peroxidase (POD), it constitutes a key enzyme in the plant's defense system. Under both field and pot conditions, CAT activity in sugarcane leaves showed a consistent trend. In the treatments affected only by the pathogen, the CAT activities in sugarcane leaves were 14.41 U / g and 22.05 U / g, respectively, representing decreases of 36.45% and 17.36% compared to the healthy treatment. Figure 14 ).

[0229] In PMA, PMCA, and CaPMCA treatments, CAT activity in sugarcane leaves increased. Compared with healthy plants, under different pot and field conditions, CAT activity in PMA, PMCA, and CaPMCA treatments increased by 28.40% and 31.82%; 74.52% and 100.94%; and 153.77% and 121.21%, respectively. Unlike its effect on POD activity, calcium-based carbon-based agricultural waste materials significantly increased CAT activity in sugarcane leaves (p<0.05).

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

[0231] 3.1 Experimental Materials and Instruments

[0232] The main experimental equipment includes: DNA extraction kit (EZNA) MicroElute Genomic DNA Kit (Omega, USA); Roche Real-Time PCR System; Spectrophotometer; Flow Analyzer (AutoAnalyzer 3-AA3, Seal Analytic, USA); Flame Atomic Absorption Spectrometer; Alkali Dispersion Dispersion Plate, etc.

[0233] 3.2 Experimental Design

[0234] Pot and field trial design (same as Example 2):

[0235] 3.3 Performance Testing

[0236] 3.3.1 Determination of soil physicochemical properties

[0237] pH: The air-dried soil sample was mixed with pure water at a ratio of 1:2.5 (w / w) and shaken at 170 rpm for 2 hours. After standing for 30 minutes, the supernatant was collected, and the pH was measured using a pH meter. Total carbon, total nitrogen, total phosphorus, and total potassium: The air-dried sample was ground and passed through an 85-mesh sieve. 20 mg of the sample was weighed and the total carbon content was determined using the potassium dichromate fusion method. Total nitrogen was determined using a digestion method combined with an AA3 continuous flow analyzer. 50 mg of the air-dried sample, passed through an 85-mesh sieve, was digested using a three-acid method, and the total phosphorus and total potassium contents were determined using an AA3 continuous flow analyzer and a flame atomic absorption spectrometer, respectively.

[0238] Determination of readily available nutrients (AN, AP, AK): AN: Alkaline diffusion method. Weigh 1.50g (accurate to 0.01g) of air-dried soil sample passed through an 85-mesh sieve and spread it evenly in 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 evenly on the soil sample. Perform a reagent blank test. Add 3.0mL of boric acid-indicator solution to the inner chamber of the diffusion dish. Apply alkaline adhesive to the edge of the outer chamber of the diffusion dish, cover it with a frosted glass slide, and rotate it several times to ensure the frosted glass slide adheres completely to the edge of the diffusion dish. Rotate the frosted glass slide to expose a narrow slit, add 10.0mL of sodium hydroxide solution to the outer chamber of the diffusion dish, and immediately cover it tightly with the frosted glass slide. Gently rotate the diffusion dish horizontally to fully mix the solution in the outer chamber with the soil sample. Then carefully secure the frosted glass slide by crossing two rubber bands in a cross shape. Place it in a constant temperature incubator and keep it at 40℃ for 24 hours. Titrate the amount of ammonia absorbed in the boric acid in the inner chamber with a 0.01 mol / L hydrochloric acid standard solution. The endpoint is reached when the color changes from blue to purple-red. Record the volume of hydrochloric acid standard solution used and calculate the AN content.

[0239] AP: The solution was extracted with reagent and then determined using AA3. Weigh 5.00 g of air-dried soil that had passed through an 85-mesh sieve, add 25 mL of hydrochloric acid-sulfuric acid extractant, and vortex at 180 r / min for 5 min at a constant temperature of 25℃. Filter the solution using double-layer slow quantitative filter paper. Perform a reagent blank test on the filtrate before testing, and complete the determination within 48 h.

[0240] AK: After extraction with ammonium acetate, the determination was performed using a flame atomic absorption spectrometer. 5.00 g of air-dried soil sample that had passed through an 85-mesh sieve was weighed into a 100 mL extraction bottle, 50.0 mL of 1 mol / L ammonium acetate solution was added, the bottle was tightly sealed, and the mixture was shaken at 180 r / min for 30 min at 20℃-25℃. The mixture was then filtered immediately, and a reagent blank test was performed simultaneously. The analysis was completed within 48 h.

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

[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 (p<0.05) between different treatments for the same indicator.

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

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

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

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

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

[0251] In summary, this invention involves adding Bacillus subtilis, Bacillus cereus, Bacillus alpineus, Trichoderma harzianum, and Trichoderma longicornis, all of which have highly antagonistic effects against the sugarcane smut pathogen Sporisorium scitanmineum, to organic fertilizer at 20% of the fertilizer's dry matter content, along with 6% biochar and calcium carbonate, to prepare a compound bio-organic fertilizer for targeted control of sugarcane smut. The effectiveness of this microbial fertilizer in controlling sugarcane smut was demonstrated through indoor pot experiments and field trials. The microbial fertilizer of this invention has the following performance advantages:

[0252] (1) The combined effect of calcium carbonate and biochar improves the quality and maturity of agricultural waste composting and enhances the utilization of agricultural straw.

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

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

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

[0256] The embodiments described above are merely illustrative of implementation methods of the present invention and should not be construed as limiting the scope of the invention, nor are they intended to impose any limitation on the structure of the invention. It should be noted that those skilled in the art can make various changes and modifications without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A microbial fertilizer, characterized in that, The product, by weight, comprises: 40-60 parts animal manure, 2-4 parts straw biochar, 4-6 parts mulberry branches, 8-14 parts mixed microbial agent, and 2-4 parts calcium carbonate; the mixed microbial 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, and the preservation number of Bacillus altitudinis... The accession number for X2 is GDMCC NO: 65099, and the accession number for Bacillus subtilis X1 is GDMCC NO: 65098. In the mixed bacterial agent, the volume ratio of Bacillus subtilis X1, Bacillus saltitudinis X2, Bacillus cereus X3, Trichoderma harzianum Z1, and Trichoderma longibrachiatum Z2 is 1:1:1:1:

1.

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

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 14-17 m². 2 / g, with an average pore size of 8-10 nm and a pore capacity of 25-29 m³ / g. 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, 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 was 5 × 10⁻⁶. 6 -1×10 8 cfu / mL.

5. A method for preparing the microbial fertilizer according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Mix the raw materials other than the mixed microbial agent in a certain proportion and then compost them to obtain organic fertilizer; S2. Add mixed microbial agents to the organic fertilizer for secondary fermentation; S3. Air dry to obtain the microbial fertilizer.

6. The method according to claim 5, characterized in that, During composting and fermentation, the fermentation temperature of the organic fertilizer should be controlled at 60-70℃ and the humidity at 60-75%; the secondary fermentation temperature should be 35-38℃, under dark conditions, and the humidity at 50-60%.

7. The method according to claim 5, characterized in that, The secondary fermentation takes 12-16 days, and the pile is turned over every 2-3 days.

8. The application of the microbial fertilizer according to any one of claims 1-4 in the prevention and control of plant smut.

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