Complex microbial inoculant and application thereof in promoting growth of zizania aquatica

Abstract

The invention belongs to the technical field of biopesticides, and provides a complex microbial inoculant and an application thereof in promoting growth of zizania aquatica, the complex microbial inoculant is composed of Ustilago esculenta G1 and Streptomyces hygroscopicus Sh-2, the strain preservation number of the Ustilago esculenta G1 is CGMCC (China General Microbiological Culture Collection Center) No.40037, the preservation number of the Streptomyces hygroscopicus Sh-2 is CGMCC No.33980, the collection number of the Streptomyces hygroscopicus Sh-2 is CGMCC No.33980, the collection number of the Streptomyces hygroscopicus Sh-2 is CGMCC No.33980, and the collection number of the Streptomyces hygroscopicus Sh-2 is CGMCC No.33980. When the complex microbial inoculant is applied to zizania aquatica cultivation, the biodiversity of the zizania aquatica growth environment can be enriched, the utilization rate of the fertilizer is increased, and then the yield of zizania aquatica is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological pesticides. Specifically, it relates to the application of a compound bacterial agent in promoting the growth of water bamboo Background Art

[0002] The diversity of the microbial population in the surrounding environment of plants plays an extremely important role in plant growth, and the growth process of water bamboo crops is a typical example of the interaction between microorganisms and plants. The scientific name of water bamboo is Zizania latifolia, which is a perennial rhizomatous herb of the Gramineae family. In the early stage, wild Zizania latifolia, like other ordinary herbaceous plants, would go through the processes of plant growth, flowering, and fruiting. The seeds it produced were called wild rice, which was one of the "six grains" in ancient China. When Zizania latifolia is infected by Ustilago esculenta P. Henn., they will stop germinating and flowering, thus hindering the reproduction of the plant. Instead, the reproduction of Zizania latifolia turns into the accumulation of nutrients, manifested as the stem of Zizania latifolia becoming extremely thick and forming a fleshy stem, and the fleshy stem part is the water bamboo that people often eat currently. Due to its rich nutrition and delicious taste, water bamboo has become a widely cultivated aquatic vegetable crop

[0003] The formation of the fleshy stem of water bamboo is closely related to the infection of Ustilago esculenta. At the same time, water bamboo has a relatively high demand for nutrients such as fertilizers during its growth process. Therefore, a large amount of chemical compound fertilizers are usually applied during the cultivation of water bamboo. In addition, in order to promote the early pregnancy of water bamboo and increase its yield, the chemical agent sodium敌磺钠 is commonly used as a pregnancy-promoting agent for water bamboo. However, the long-term and large-scale use of chemical fertilizers and pesticides has led to a significant reduction in the richness of microbial species in farmland soil, a decrease in beneficial microbial communities, and an increase in resistant pathogenic bacteria. The silt environment for water bamboo cultivation has a strong adsorption and fixation effect on most of the fertilizers applied, making them insoluble nitrogen, phosphorus, and potassium that are difficult to be directly absorbed and utilized by plants. Therefore, farmers often increase the amount of fertilizers and pesticides used to ensure the nutritional and growth-promoting needs of plants, while excessive chemical fertilizers and pesticides have an impact on and pose a threat to the organisms in the surrounding environment Summary of the Invention

[0004] In view of the above problems, the purpose of the present invention is to provide a compound bacterial agent composed of Ustilago esculenta G1 and Streptomyces hygroscopicus Sh-2 and its application in promoting the growth of water bamboo, which enriches the diversity of environmental microorganisms in the water bamboo field, improves the utilization efficiency of fertilizers, and thus promotes the growth of water bamboo

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

[0006] A compound bactericide for promoting the growth of water bamboo, which is composed of Ustilago esculenta G1 and Streptomyces hygroscopicus Sh-2. The preservation number of the strain Ustilago esculenta G1 is CGMCC No. 40037, and the preservation number of Streptomyces hygroscopicus Sh-2 is CGMCC NO. 33980.

[0007] The present invention provides the application of the above compound bactericide in enriching the biodiversity of the water bamboo growth environment; further, significantly increasing the species richness and species diversity in the soil.

[0008] The present invention provides the application of the above compound bactericide in improving the fertilizer utilization rate; further, improving the utilization rate of any one or more of nitrogen fertilizer, phosphorus fertilizer and potassium fertilizer; still further, increasing the content of nitrogen, phosphorus and potassium that can be effectively utilized in the soil, and / or increasing the content of total nitrogen, total phosphorus and total potassium in the plant.

[0009] The present invention provides the application of the above compound bactericide in increasing the yield of water bamboo.

[0010] The present invention is further configured that in the above application, Ustilago esculenta G1 and Streptomyces hygroscopicus Sh-2 in the compound bactericide are composed according to the viable bacteria amount ratio of (1~10):1.

[0011] The present invention is further configured that in the above application, Ustilago esculenta G1 and Streptomyces hygroscopicus Sh-2 in the compound bactericide are composed according to the viable bacteria amount ratio of (2~5):1.

[0012] The present invention provides a planting method using the above compound bactericide, which is to add the microbial bactericide containing the compound bactericide to the irrigation water or evenly spread it in the field or spray it on the leaf surface after dilution during the seedling stage of water bamboo; further, it is to perform the treatment of the microbial bactericide within 35~55 days after the water bamboo is planted.

[0013] The present invention is further configured that the planting method using the above compound bactericide includes:

[0014] Irrigation treatment: About 40 days after the water bamboo is planted, the microbial bactericide is directly applied to the inlet of the irrigation water at a dose of 200g~400g active ingredient per mu, so that the medicament is distributed throughout the whole field along with the irrigation water; or,

[0015] Broadcasting: Approximately 40 days after planting, the microbial inoculant is evenly broadcast into the water chestnut field at a rate of 1000g to 4000g of the formulation per acre; or,

[0016] For foliar spraying, dilute the microbial agent with 50g to 200g of active ingredients per acre and spray it. The water consumption per acre should be controlled at 50kg to 80kg.

[0017] The present invention is further configured such that the form of the microbial agent includes aqueous suspension, granules, and microcapsules;

[0018] In the aforementioned microbial agents, the amount of the active ingredient, by weight percentage, ranges from 5% to 20% for aqueous suspensions and granules, and from 10% to 30% for microcapsules.

[0019] The present invention is further configured such that the preparation method of the compound microbial agent includes the following steps: washing the solid culture medium filled with spores after cultivation with an appropriate amount of sterile water to obtain a suspension of Streptomyces hygroscopicus Sh-2 spores, and mixing it evenly with the culture of Smut fungus G1 cells or liquid semi-finished product after cultivation in the required proportion to obtain a compound microbial agent composed of two microorganisms.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention uses a compound microbial agent composed of smut fungus G1 and streptomyces hygroscopicus Sh-2 for the cultivation of water bamboo. The application of this compound microbial agent enriches the diversity of environmental microorganisms in water bamboo fields, improves fertilizer utilization efficiency, and thus promotes the growth of water bamboo.

[0022] (2) By spraying or irrigating the field, the two beneficial bacteria become the dominant bacteria and colonize and grow in the plants and environment, so that the insoluble fertilizers in the environment are transformed into effective phosphorus, water-soluble nitrogen and potassium that can be directly utilized by the plants, thereby promoting the growth and incubation of water chestnuts, and meeting the actual needs of water chestnut farmers and the green and environmentally friendly requirements of reducing the use of chemical fertilizers and pesticides. Attached Figure Description

[0023] Figure 1 Image showing water-absorbing spots on the surface of a colony of Streptomyces hygroscopicus Sh-2.

[0024] Figure 2 This is an electron micrograph of spores from the Streptomyces hygroscopicus Sh-2 strain.

[0025] Figure 3 Phylogenetic tree of 16S rRNA from Streptomyces hygroscopicus.

[0026] Preservation Matters

[0027] The *Ustilago maydis* G1 strain of this invention was deposited on January 4, 2022, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 40037, located at No. 3, Beichen West Road, Chaoyang District, Beijing. *Streptomyces hygroscopicus* Sh-2 was deposited on March 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC NO. 33980, located at No. 3, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0029] I. Identification of the strain

[0030] The compound microbial agent described in this invention is composed of Ustilago esculenta G1 and Streptomyces hygroscopicus. The Ustilago esculenta G1 has the accession number CGMCC No. 40037 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 4, 2022. The morphological characteristics, culture characteristics, and 18S rRNA sequence of this strain have been disclosed in the invention patent application document with publication number CN117187074A.

[0031] The Streptomyces hygroscopicus Sh-2 strain, with accession number CGMCC NO.33980, was deposited at the China General Microbiological Culture Collection Center on March 26, 2025. The morphological characteristics, culture characteristics, and 16S rRNA sequence of this strain are as follows:

[0032] (1) Morphological characteristics:

[0033] like Figure 1 and Figure 2As shown, strain Sh-2, after being cultured at 28°C for 3 days on ATCC 172 medium, exhibited white colonies, which turned grayish-white after approximately 10 days of further culture. Single colonies were pie-shaped, with raised outer edges and a central depression between the outer edge and the center. Initially, the white colonies had a dry, rough surface; later, after turning gray, the colony surface became smooth with water-absorbing spots.

[0034] (2) 16S rRNA of strain Sh-2

[0035] The 16S rRNA nucleotide sequence of strain Sh-2, as shown in SEQ ID No. 1, is 1463 bp in length. BLAST homology comparison and phylogenetic analysis indicate that this strain belongs to the genus *Streptomyces*. Figure 3 As shown in the phylogenetic tree based on the 16S rRNA sequence of the strain, the closest relative is *Streptomyces hygroscopicus* in the strain library. The 16S rRNA sequence similarity between the two is 100%, indicating that they are homologous.

[0036] Based on the above culture and morphological characteristics, combined with 16S rRNA sequence comparison, strain Sh-2 is considered to belong to Streptomyces hygroscopicus in the genus Streptomyces.

[0037] II. Methods for Microbial Culture and Preparation of Microbial Agents

[0038] The *Ustilago mays* G1 species was produced using the fungal culture method disclosed in CN117187074A.

[0039] The *Streptomyces* genus *Streptomyces* Sh-2 is cultured using solid-state methods. There are no specific requirements for the solid culture medium used; common actinomycete media such as Goethe's agar, oat broth, and ATCC 172 are acceptable. There are also no strict limitations on culture conditions such as temperature and time; for example, the culture temperature can be 26-30℃, and the culture time can be 10-15 days. These media composition, culture temperature, and culture time can be adjusted appropriately according to actual conditions to suit the growth of the fungus and spore production.

[0040] After the solid culture medium is filled with spores, it is washed with an appropriate amount of sterile water to obtain a spore suspension. This suspension is then mixed with the cultured *Ustilago maydis* G1 cells or liquid semi-finished product in the required ratio. The biomass mixing ratio of the two microorganisms is *Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2 at (1~10):1 to obtain a compound microbial agent composed of the two microorganisms.

[0041] The two microbial mixtures mentioned above, mixed in the specified proportions, can be further supplemented with various carriers such as solid-phase, liquid-phase, and emulsifying dispersants to formulate the desired dosage forms, such as aqueous suspensions, granules, and microcapsules. The proportions of the active ingredients in various formulations can be appropriately increased or decreased according to different intended uses.

[0042] By weight percentage, the effective ingredient content of aqueous suspensions and granules typically ranges from 5% to 20%, while the effective ingredient content of microcapsules typically ranges from 10% to 30%.

[0043] The proportions of active ingredients in various formulations can be adjusted appropriately according to different intended uses. Generally, the total viable count of the active ingredients ranges from 100 million CFU / mL to 10 billion CFU / mL in the aqueous suspension, and from 100 million CFU / g to 10 billion CFU / g in the granules and microcapsules.

[0044] In practical use, this microbial agent can be diluted to a suitable concentration before use, or it can be used directly.

[0045] III. Application Methods and Scope

[0046] 35-55 days after planting water chestnuts, the prepared microbial compound inoculant, with *Ustilago maydis* G1 and *Streptomyces hygroscopicus* Sh-2 as active ingredients, is added to irrigation water or applied evenly in the field by broadcasting or foliar spraying after dilution to promote water chestnut growth and earlier fruiting. Specifically:

[0047] For irrigation treatment, about 40 days after planting water chestnuts, apply 200g to 400g of active ingredient per acre directly to the inlet of the irrigation water so that the agent is distributed throughout the field with the irrigation water; apply the agent again two weeks later.

[0048] For broadcasting, about 40 days after planting water chestnuts, apply 1000 g to 4000 g of the formulation product (taking 5% granules as an example) per mu (approximately 667 square meters) evenly to the water chestnut field.

[0049] For foliar spraying, the bacterial agent described in this invention is diluted with 50 g to 200 g of active ingredients per acre and sprayed. The water consumption per acre can be 30 kg to 80 kg.

[0050] Example 1

[0051] (1) Cultivation of Ustilago maydis G1 and preparation of intermediate products from culture

[0052] The culture medium was prepared with the following proportions (by weight): 2% glucose, 1% soluble starch, 1% yeast extract, 0.2% potassium chloride, 0.5% magnesium sulfate, 0.2% ammonium nitrate, and 0.05% dipotassium hydrogen phosphate. The pH was adjusted to 7.2, and the medium was sterilized at 121°C for 25 minutes.

[0053] The *Ustilago maydis* G1 strain grown on PSA slant was inoculated into the aforementioned culture medium cooled to room temperature and cultured at 28°C for 16-24 hours. Using this culture as seed, it was inoculated into the fermentation broth composed of the above culture medium at a ratio of 5-20% and cultured at 28°C with aeration and stirring for 36-72 hours.

[0054] Take 1000 kg of the fermentation broth, concentrate it under reduced pressure with a membrane, control the vacuum degree at 0.1 MPa and the temperature at below 60℃, so that the volume of the culture is concentrated to about 100 kg, and obtain liquid intermediate product 1.

[0055] Intermediate product 1 was spray-dried to obtain approximately 20 kg of solid intermediate product 2. The inlet temperature of the spray dryer was controlled at 140℃~180℃, and the outlet temperature was controlled at 70℃~80℃.

[0056] The number of viable bacteria per unit weight of the obtained solid or liquid intermediate product was determined by the dilution plating method.

[0057] (2) Culture of Streptomyces hygroscopicus Sh-2

[0058] To prepare oatmeal culture medium, add 30g of rolled oats and 20g of agar to 1000mL of water and sterilize at 121℃ for 20min; or prepare Gao's No. 1 culture medium by adding 20g of soluble starch, 1g of KNO3, 0.5g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of NaCl, 0.01g of FeSO4·7H2O, and 20g of agar to 1000mL of water and sterilizing at 121℃ for 20min; or prepare ATCC172 culture medium. Dispense the sterilized culture media into Φ12cm petri dishes and allow them to cool and solidify before use.

[0059] Sterile water was added to the slant culture of *Streptomyces hygroscopicus* Sh-2 to prepare a seed-spore suspension. 1 mL of this suspension was evenly spread onto the prepared culture medium. After incubation at 28°C for 10-14 days, the surface of the medium was covered with spores, at which point the spores could be harvested. 5-10 mL of sterile water was added to the confluent culture dish, and the spores were scraped off with a sterilized cotton swab to prepare a spore suspension. The number of spores per milliliter of liquid was counted using a micro-counter, and the viable cell count per unit weight was determined using the dilution plating method.

[0060] Example 2

[0061] Preparation of water suspension

[0062] By weight, 2 parts of sizing agent LS, 1 part of xanthan gum, 1 part of urea, 1 part of coconut oil, and 0.1 parts of sodium salicylate were added to several parts of intermediate product 1 obtained in Example 1. Based on the viable cell count determined by the dilution plating method, the prepared *Streptomyces hygroscopicus* spore suspension was added at a ratio of 1:1 (*Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2), mixed, and stirred until homogeneous. By adjusting the ratio of the liquid intermediate to the additives, and adding water if necessary, aqueous suspensions with different contents were prepared.

[0063] Example 3

[0064] Preparation of water suspension

[0065] By weight, take several portions of intermediate product 2 obtained in Example 1. Based on the viable count determined by the dilution plating method, add the prepared *Streptomyces hygroscopicus* Sh-2 spore suspension at a ratio of *Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2 of 4:1. Then, add water as needed, along with 2 parts of wetting agent LS, 1 part of xanthan gum, 1 part of urea, 1 part of coconut oil, and 0.1 parts of sodium salicylate. Mix and stir until homogeneous. By adjusting the ratio of water to additives, aqueous suspensions with different contents can be obtained.

[0066] Example 4

[0067] Granule preparation

[0068] By weight, several portions of intermediate product 2 obtained in Example 1 were taken. Based on the viable bacterial count determined by the dilution plating method, *Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2 were added to the spore suspension prepared in Example 2 at a ratio of 9:1. Several portions of light calcium carbonate, 2 portions of wetting agent SXC, 2 portions of dispersant NNO, and 15 portions of corn cob powder were then taken, pulverized and mixed using a pulverizer. An appropriate amount of water was added, and the mixture was extruded and granulated. The granules were then dried in an oven at 54 ℃ for 1 h. Granules with different contents were obtained by adjusting the ratio of bacterial powder to light calcium carbonate.

[0069] Example 5

[0070] Microencapsulation preparation

[0071] 20g of poly(ethylene terephthalate) (PET) granules were dissolved in 200mL of dichloromethane. A certain volume of intermediate product 1 obtained in Example 1 (or intermediate product 2 mixed with water in the required proportion) was added, along with a suspension of *Streptomyces hygroscopicus* Sh-2 spores (prepared by adding *Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2 spores in a 1:1 ratio based on the viable cell count determined by the dilution plating method) and a gelatin solution. The mixture was stirred and dispersed for 30-45 minutes to obtain a water / oil emulsion. Then, 1600mL of 1% gelatin solution was added to this emulsion to obtain a water / oil / water emulsion. Finally, the mixture was distilled at 45°C to remove the dichloromethane solution, yielding water-soluble microcapsules. Different formulation contents were obtained by adjusting the ratio of intermediate product 1 and gelatin solution during the preparation of the water / oil emulsion.

[0072] Example 6

[0073] The effect of a 1 billion CFU / mL aqueous suspension of *Ustilago maydis* G1 and *Streptomyces hygroscopicus* Sh-2 on the microbial community around *Zanthoxylum bungeanum* was studied, with a viable count ratio of *Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2 of 4:1.

[0074] The effects of microbial agents on the periphery microbial community of water chestnut were tested using a field trial. Two treatments were set up: a compound microbial agent treatment and a conventional chemical agent, sodium dichlorophenoxylate treatment. Each treatment group had three replicates, and each replicate covered an area of ​​60 m². 2 Forty days after planting, water chestnuts were treated with a foliar spray using 400 mL of microbial inoculant diluted in 45 L of water per acre. A second spray was applied after a two-week interval. Four weeks (28 days) after the final application, a five-point sampling method was used to collect whole plant samples and soil samples from the roots of each plot. All surface debris and plant remains were removed from the soil. The plant and soil samples from each plot were mixed and brought back to the laboratory. The plant samples were cut into approximately 2 cm pieces, mixed thoroughly, and then quartered. Soil samples were air-dried overnight at 40°C, mixed thoroughly, and then quartered. Microbial diversity was assessed on the prepared samples. The results are shown in Tables 1-3.

[0075] Table 1. Comparison of the number of characteristic microbial sequences around water chestnut after treatment with microbial agents and conventional pesticides.

[0076]

[0077] Table 1 shows that after treatment with the microbial agent, the number of characteristic sequences representing 16S rRNA of prokaryotes in *Zizania latifolia* plants increased by 60.06% compared to the control group; the number of characteristic sequences representing 18S rRNA of eukaryotes increased by 90.91% compared to the control group. In the soil, the number of characteristic sequences of 16S rRNA and 18S rRNA in the microbial agent treatment increased by 1.77% and 32.18% respectively compared to the control.

[0078] Table 2. Comparison of microbial community composition at the taxonomic level of *Zizania latifolia* after treatment with microbial agents and conventional pesticides.

[0079]

[0080] The results in Table 2 show that after treatment with the inoculant, prokaryotes on plants increased at the phylum, class, order, family, genus, and species levels, while those in the soil increased at the genus and species levels. Eukaryotes in both plants and soil showed a significant increase at the phylum, class, order, family, genus, and species levels.

[0081] Further Alpha diversity analysis was conducted using indices such as Chao1, ACE, Shannon, Simpson, and PD-whole tree to compare the species richness and species diversity of the samples. The results are shown in Table 3.

[0082] Table 3. Comparison of Alpha diversity of microorganisms around water chestnut after treatment with microbial agents and conventional agents.

[0083]

[0084] As shown in Table 3, on plants, both prokaryotes and eukaryotes showed significant increases in the ACE and Chao1 indices (measuring species richness) after inoculant treatment compared to conventional treatment. The Shannon and Simpson indices (measuring species diversity) also increased to some extent. The PD-whole tree diversity index, calculated based on phylogenetic trees, showed a substantial increase, indicating higher species richness and community diversity on plants after inoculant treatment. In soil, compared to conventional treatment, the ACE and Chao1 indices for both prokaryotes and eukaryotes significantly increased after inoculant treatment, while the Shannon and Simpson indices showed no significant change. The PD-whole tree index increased to some extent, indicating a significant increase in species richness in the soil after inoculant treatment, although species diversity also increased slightly.

[0085] Example 7

[0086] Experiment on the promotion of fertilizer absorption by water chestnuts using a 1 billion CFU / mL aqueous suspension of *Ustilago maydis* G1 and *Streptomyces hygroscopicus* Sh-2, wherein the ratio of viable bacteria of *Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2 was 4:1.

[0087] The experiment included two treatments: a 200 mL / mu compound microbial agent treatment and a conventional chemical agent treatment. Each treatment had three replicate plots. The experimental plot area was 66 m². 2 Each ridge between plots was 1.0 m wide, and each plot had its own inlet and outlet water system. Forty days after the water chestnuts established themselves, all fields were treated with 50 kg / mu of 45% compound fertilizer (N (nitrogen) – P (phosphorus) – K (potassium): 15–15–15). The conventional group received foliar spraying with chemical agents, while the microbial agent treatment was also done via foliar spraying. Starting from day 2, soil samples (0-20 cm depth) and above-ground water chestnut plants were taken every 6 days. A total of 3 samplings were conducted. The experimental results are shown in Table 4.

[0088] Table 4. Comparison of nitrogen, phosphorus, and potassium content in soil and plants after inoculant treatment and conventional treatment.

[0089]

[0090] The results in Table 4 show that after one week of treatment with the microbial agent, the content of available nitrogen, phosphorus, and potassium in the soil was significantly higher than that in the conventional chemical treatment group. The content of total nitrogen, total phosphorus, and total potassium in the plants also increased to a certain extent, indicating that the microbial agent can significantly improve the utilization efficiency of fertilizers in the soil and promote the absorption of nitrogen, phosphorus, and potassium by plants.

[0091] Example 8

[0092] A study on the growth of summer water chestnuts using a 1 billion CFU / mL aqueous suspension of *Ustilago maydis* G1 and *Streptomyces hygroscopicus* Sh-2, wherein the viable cell ratio of *Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2 was 4:1.

[0093] The experiment included three treatments: a compound microbial agent treatment, a single agent treatment with *Ustilago maydis* (1 billion CFU / mL *Ustilago maydis* G1 aqueous suspension), and a conventional chemical treatment with sodium disulfide. Each treatment area was 66 m². 2 Three replicates were used. Fifty days after summer planting of water chestnuts in the field, compound microbial agents and single-agent smut fungi were applied at a dosage of 900 mL / mu, and chemical agents at 900 g / mu, using 30 kg of water per mu as foliar spray. A second treatment was applied two weeks later. The results are shown in Table 5.

[0094] Table 5 Comparison of the number of water bamboo shoots harvested after summer harvest (individual shoots) after treatment with microbial agent and conventional treatment.

[0095]

[0096] The results showed that after spraying the compound microbial agent, the total yield after four harvests increased by 10.9% compared with the single agent treatment of Ustilago maydis; and the total yield increased by 38.4% compared with the conventional agent treatment.

[0097] Example 9

[0098] An experiment was conducted to promote the growth of autumn water bamboo with 10 billion CFU / g of *Ustilago maydis* G1 and *Streptomyces hygroscopicus* Sh-2 granules, in which the live bacteria ratio of *Ustilago maydis* G1 to *Streptomyces hygroscopicus* Sh-2 was 9:1.

[0099] The experiment included two treatments: a compound microbial agent treatment and a single agent treatment with *Ustilago maydis* (10 billion CFU of *Ustilago maydis* G1 granules). Each treatment covered an area of ​​66 m². 2 Three replicates were used. Forty-five days after autumn planting of water chestnuts in the field, the fungicide was applied as a foliar spray at a rate of 100 g / mu (approximately 667 square meters) with 30 kg of water per mu (approximately 200 kg / mu). A second treatment was applied two weeks later. Harvesting was conducted every four days from the first harvest, for a total of seven harvests. The results are shown in Table 6.

[0100] Table 6. Comparison of the number of water chestnuts harvested in autumn after treatment with microbial agent and conventional treatment.

[0101]

[0102] The experimental results in Table 6 show that, after spraying the compound microbial agent, the total yield after 7 harvests increased by 15.22% compared with the single agent treatment of Ustilago maydis.

[0103] The experimental results show that the microbial agent described in this invention enriches the microbial diversity of the water chestnut field environment, improves fertilizer utilization efficiency, and thus promotes water chestnut growth and increases yield.

[0104] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A complex microbial agent for promoting growth of Zizania latifolia Turcz, characterized by, Composed of Ustilago esculenta G1 and Streptomyces hydrophila ( Streptomyces hygroscopicus The fungal composition is Sh-2, and the strain preservation number of Ustilagoesculenta G1 is CGMCC No. 40037. The Streptomyces hydrophila ( Streptomyces hygroscopicus The accession number for Sh-2 is CGMCC NO.33980.

2. The application of the complex microbial agent in claim 1 in the growth environment of Zizania latifolia to improve the biological diversity.

3. The application of the complex microbial agent in claim 1 to improve the utilization rate of fertilizer.

4. Use according to claim 3, characterized in that, The utilization rate of any one or more of nitrogen fertilizer, phosphorus fertilizer and potassium fertilizer is improved.

5. The application of the complex microbial agent in claim 1 to improve the yield of Zizania latifolia.

6. The use according to any one of claims 2 to 5, characterized in that, Ustilagoesculenta G1 and Streptomyces hydrophila ( Streptomyces hygroscopicus Sh-2 is composed of live bacteria in a ratio of (1~10):

1.

7. Use according to any one of claim 6, characterized in that, Ustilagoesculenta G1 and Streptomyces hydrophila ( Streptomyces hygroscopicus Sh-2 is composed of live bacteria in a ratio of (2~5):

1.

8. A planting method using the complex microbial inoculant of claim 1, characterized by, During the seedling stage of Zizania latifolia, the microbial agent containing the complex microbial agent is added to irrigation water or evenly spread in the field or diluted for foliar spraying treatment.

9. The method of claim 8, wherein the microbial inoculant is applied to the plant at a rate of about 1 x 10<6> to about 1 x 10<9> CFU per plant. The form of the microbial agent includes water suspension, granules and microcapsules.

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