Ustilago esculenta and application thereof
By using a microbial inoculant made from Ustilago esculenta, the problems of toxicity and high cost of existing water bamboo growth promoters have been solved, achieving green and environmentally friendly promotion of water bamboo growth and increased yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- EAST CHINA UNIV OF SCI & TECH
- Filing Date
- 2023-06-19
- Publication Date
- 2026-05-12
AI Technical Summary
Existing fertilization agents for water bamboo, such as sodium dichloroisocyanurate, have toxicity issues. Hormonal fertilization agents are costly and have unstable effects. The method of artificially inoculating water bamboo with Ustilago maydis is labor-intensive and ineffective, making it difficult to promote on a large scale.
Using Ustilago esculenta as the main active ingredient, microbial agents are produced through in vitro large-scale bio-fermentation, including aqueous suspensions, granules, and microcapsules. These agents are applied to the field by spraying or irrigation to promote colonization and growth of water chestnuts and encourage seed formation.
It achieves green and environmentally friendly promotion of water bamboo growth and early fruiting, significantly increasing yield, solving the toxicity and cost problems of existing fruiting agents, and improving plant survival rate and fruiting rate.
Smart Images
Figure SMS_1 
Figure SMS_3 
Figure HDA0004291968700000011
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biological pesticides. Specifically, it is a Ustilago esculenta and its application. Background Art
[0002] Zizania latifolia is a perennial rhizomatous herb of the Gramineae family. It is an important aquatic vegetable native to China and is widely cultivated in the Yangtze River basin and areas south of it. Currently, it has become the second largest aquatic vegetable in China after lotus root and has important economic value. A large number of studies at home and abroad have shown that the interaction between the formation of enlarged stems in Zizania latifolia and the infection of its endophytic fungus, Ustilago esculenta, is very close. After Ustilago esculenta infects Zizania latifolia, the flowering of Zizania latifolia is inhibited, and the stem of Zizania latifolia is stimulated to expand, forming an edible enlarged stem. Under natural conditions, Ustilago esculenta has two phenotypes, namely the spore type and the hypha type. In most cases, Ustilago esculenta is passed on in the spore type and exists in the Zizania latifolia plant. Therefore, there are three phenotypes of Zizania latifolia in the field: normal Zizania latifolia (Ustilago esculenta exists in the hyphal state and can be transformed into spores after being placed for a long time), grey Zizania latifolia (the stem of Zizania latifolia is full of Ustilago esculenta spores), and male Zizania latifolia (the stem does not expand).
[0003] Existing research has shown that spraying appropriate drugs for regulating the formation of enlarged stems during the cultivation of Zizania latifolia can regulate the growth of Zizania latifolia, regulate the time of formation of enlarged stems by regulating the growth of Zizania latifolia plants and Ustilago esculenta, and increase the field yield of Zizania latifolia. Among them, sodium thiram is a relatively commonly used promoter for the formation of enlarged stems in the current field management of Zizania latifolia. Sodium thiram is a systemic protective fungicide. Existing research has shown that sodium thiram has moderate toxicity to birds and the LD 50 for mice is 60 mg / kg body weight. The EU issued a ban on the use of sodium thiram in July 2002.
[0004] Some people also regulate the formation of enlarged stems in Zizania latifolia based on the vegetative growth of Zizania latifolia plants. Spraying a certain proportion of mixed plant growth hormones as a promoter for the formation of enlarged stems in Zizania latifolia during cultivation can promote the formation of enlarged stems in Zizania latifolia. However, due to the high cost of using hormones as promoters for the formation of enlarged stems at present, and the large influence of the proportion control between hormones on the local microhabitat, resulting in large differences in effects in different regions, it has not been widely used.
[0005] In addition to using promoters for the formation of enlarged stems, there are also literature reports on using an artificial inoculation system of Ustilago esculenta. In a greenhouse environment, wild Zizania latifolia seedlings were infected using an optimized artificial syringe inoculation technique, and plants that could produce normal Zizania latifolia were obtained. The method of inoculating Ustilago esculenta using an artificial syringe also has problems such as a large workload, difficulty in obtaining mutant strains, low survival rate and formation rate of enlarged stems of plants, and cannot be widely promoted and used. Summary of the Invention
[0006] The inventors of this application isolated *Ustilago maydis* from *Zizania latifolia* plants in the Shanghai area, and after purification, induced a mycelial type of *Ustilago maydis* (MT type). After large-scale in vitro bio-fermentation, the MT type *Ustilago maydis* was then applied in the field via spraying or irrigation to establish itself as the dominant fungus, promoting the growth of *Zizania latifolia* and avoiding adverse factors such as gray *Zizania latifolia*, thus meeting the practical needs of *Zizania latifolia* farmers and fulfilling the requirements of green and environmentally friendly practices.
[0007] Therefore, in a first aspect, the present invention provides a *Ustilago esculenta* fungus, with accession number CGMCC No. 40037.
[0008] In a second aspect, the invention provides the application of the aforementioned Ustilago esculenta for preparing a microbial agent to promote the growth and bud formation of water bamboo.
[0009] According to the present invention, the microbial inoculant is available in the form of an aqueous suspension, granules, and microcapsules.
[0010] A third aspect of the present invention provides a microbial agent for promoting the growth and seed formation of water bamboo, wherein the microbial agent uses the aforementioned Ustilago esculenta as the main active component.
[0011] According to the present invention, the Ustilago esculenta, which is the main active component, is a concentrated solution of its fermentation culture.
[0012] According to an alternative approach, the Ustilagoesculenta, which is the main active component, is a solid obtained by drying a concentrated broth of its fermentation culture.
[0013] According to the present invention, the microbial inoculant is available in the form of an aqueous suspension, granules, and microcapsules.
[0014] According to the present invention, 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.
[0015] A fourth aspect of the present invention provides a method for using the aforementioned microbial inoculant:
[0016] For irrigation treatment, about 40 days after planting water chestnuts, apply the microbial agent directly to the inlet of the irrigation water at a dosage of 200g to 400g of active ingredients per acre, so that the agent can be distributed throughout the field with the irrigation water.
[0017] For broadcasting, about 40 days after planting water chestnuts, the microbial inoculant is evenly broadcast into the water chestnut field at a dosage of 1000g to 4000g of the formulation product per acre.
[0018] For foliar spraying, dilute the microbial agent with 50g to 200g of active ingredients per acre before spraying, and control the water usage per acre to 50kg to 80kg.
[0019] The present invention has the following beneficial effects:
[0020] 1. Using the Ustilago esculenta fungus of the present invention as the main active ingredient to prepare a microbial inoculant for use in the field of water bamboo can promote the growth of water bamboo, enable water bamboo to form fruit earlier and significantly increase yield.
[0021] 2. After large-scale in vitro bio-fermentation, the Ustilago esculenta of this invention can be applied to the field by spraying or irrigating to make it the dominant microbial community and promote its colonization and growth in the plant, thereby promoting the growth of water chestnuts. At the same time, it does not produce adverse factors such as gray water chestnuts, thus meeting the actual needs of water chestnut farmers and the requirements of green environmental protection. Attached Figure Description
[0022] Figure 1 The colony morphology of strain G1 on solid medium is shown.
[0023] Figure 2 The image shows a photomicrograph of strain G1 magnified 1000 times.
[0024] Preservation Matters
[0025] The *Ustilago maydis* G1 strain of this invention was deposited on January 4, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40037. Detailed Implementation
[0026] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0027] I. Acquisition and Identification of Strains
[0028] In June 2020, the inventors of this application isolated and purified a strain G1 from water bamboo plants collected in the suburbs of Shanghai, and then induced mutagenesis to obtain a strain that promotes water bamboo growth and fruit development. This strain G1 is a microbial species belonging to the genus *Ustilago*, and is classified as *Ustilago esculenta*. This *Ustilago esculenta* G1 strain was deposited on January 4, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40037.
[0029] The morphological characteristics, culture characteristics, 18S rRNA sequence, and species classification results of Ustilago esculenta CGMCC No. 40037 (strain G1) are as follows:
[0030] 1. Morphological characteristics
[0031] like Figure 1 and Figure 2 As shown, after about 2 weeks of culture on PSA medium, white mycelia appeared around the colonies of strain G1. Under in vitro culture conditions, the strain mostly exists in yeast-like, short rod-like, budding, and branched mycelial forms, and it is difficult for it to form spores to complete its life cycle.
[0032] 2. 18S rRNA of strain G1
[0033] The 18S rRNA nucleotide sequence of strain G1 is shown in SEQ ID No.1, with a length of 1770 bp. BLAST homology comparison and evolutionary analysis showed that strain G1 belongs to the genus Ustilago, and its closest relative is Ustilago esculenta Zhe2H in the strain library. The 18S rRNA sequence similarity between the two is 100%, indicating that they are homologous.
[0034] Based on the above culture and morphological characteristics, combined with 18S rRNA sequence comparison, it is believed that strain G1 of the present invention belongs to Ustilagoesculenta in the genus Ustilagoesculenta.
[0035] II. Cultivation of strain G1 and preparation of biological cells
[0036] The *Ustilago mays* G1 strain of this invention can be cultured using conventional fungal production methods to obtain the corresponding culture. The cultivation method is submerged liquid culture. To facilitate industrial-scale cultivation, it is advisable to inoculate the bacterial suspension of the above-mentioned production strain onto the culture medium and then aerate and stir it.
[0037] There are no specific requirements for the nutrient sources in the culture medium. The medium can contain carbon sources, nitrogen sources, and other nutrient sources commonly used in microbial culture. The ideal carbon-to-nitrogen ratio in the culture medium is 3:1 to 5:1.
[0038] Carbon sources can be starch, dextrin, glucose, glycerol, sucrose, mannitol, etc.
[0039] Nitrogen sources can be peptone, soybean meal, peanut powder, yeast, meat extract, rice bran, corn steep liquor, wheat bran, ammonium salts, urea, nitrates, and other organic or inorganic nitrogen-containing compounds.
[0040] Other nutrient sources can be supplemented with appropriate amounts of inorganic salts, such as table salt, phosphates, and metallic salts such as potassium, calcium, manganese, and iron.
[0041] If necessary, some animal, vegetable, or mineral oils can be added as defoamers.
[0042] There are no strict limitations on culture conditions such as temperature, pH, and time; the most important factors are those suitable for the strain's production and those that maximize the yield of the active components. For example, the pH range of the culture medium can be 6–8, preferably close to neutral; the culture temperature should be 20–35°C; and the aeration ratio should be between 1:0.3 and 1:4 (aeration ratio: the ratio of the volume of air passing through the culture medium per minute to the volume of the culture medium). These components, pH, culture temperature, and aeration conditions should be adjusted appropriately according to the actual situation to obtain the best results.
[0043] Starting from the cultures obtained above, bacterial cell cultures can be prepared through appropriate methods. For example, the cultures can be concentrated into high-concentration liquid semi-finished products using vacuum membrane concentration, which can then be used to further prepare liquid target formulation products. Alternatively, solid semi-finished products can be obtained by methods such as ventilation drying or spray drying. The obtained solid semi-finished products can then be pulverized and used to prepare target formulation products.
[0044] III. Microbial inoculants
[0045] The microbial agent described in this invention is a microbial agent that promotes the growth and fruit development of water chestnuts, with *Ustilago maydis* G1 as the main active ingredient (effective component). In preparing the microbial agent of this invention with *Ustilago maydis* G1 as the effective component, various carriers commonly used in pesticides or fertilizers in this technical field, such as solid-phase, liquid-phase, and emulsifying dispersants, can be used, and it can be formulated into dosage forms such as aqueous suspensions, granules, and microcapsules. Simultaneously, some adjuvants can generally be appropriately added to various formulations, such as dispersants, wetting agents, penetrants, spreading agents, adhesives, defoamers, foaming agents, thickeners, and stabilizers. The carrier used in the formulation can be a solid carrier such as sawdust, talc, bentonite, clay, kaolin, diatomaceous earth, white carbon black, vermiculite, quicklime, silica sand, ammonium sulfate, or urea, or a liquid carrier such as isopropanol, xylene, cyclohexane, or methylnaphthalene. The dispersant described herein may be an alcohol sulfonate, an alkyl aryl sulfonate, a lignin sulfonate, a polyoxyethylene glycol ether, a polyoxyethylene alkyl aryl ether, or a polyoxyethylene sorbitan monoalkyl ester. The adjuvant may be carboxymethyl cellulose, polyethylene glycol, gum arabic, xanthan gum, etc.
[0046] The proportions of active ingredients in various formulations can be adjusted appropriately according to different intended uses. By weight percentage, the active ingredient content of aqueous suspensions and granules typically ranges from 5% to 20%, while that of microcapsules typically ranges from 10% to 30%.
[0047] In practical use, the microbial agent of the present invention can be diluted to a suitable concentration before use, or it can be used directly.
[0048] The microbial agent of the present invention can be used in combination with other pesticides and fertilizers, but cannot be mixed with antifungal pesticides.
[0049] IV. Application Methods and Scope
[0050] The microbial agent of the present invention, with *Ustilago maydis* G1 as the active ingredient, can be used to promote the growth and fruit formation of water bamboo. Specifically, about 40 days after planting water bamboo, the microbial agent can be added to irrigation water or applied evenly in the field by broadcasting or by foliar spraying after dilution to promote the growth of water bamboo and advance the fruit formation.
[0051] 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.
[0052] For broadcasting, about 40 days after planting water chestnuts, apply 1000g to 4000g of the formulation product (taking 5% granules as an example) per mu (approximately 667 square meters) evenly to the water chestnut field.
[0053] For foliar spraying, the microbial agent of the present invention is diluted with 50g to 200g of active ingredients per acre and sprayed. The water consumption per acre can be 50kg to 80kg.
[0054] Example 1: Cultivation of active bacterial strains and preparation of intermediate products from cultures
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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℃.
[0059] Example 2: Preparation of aqueous suspension
[0060] By weight, take 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, and add them to several parts of intermediate product 1 obtained in Example 1. Mix and stir evenly. By adjusting the ratio of liquid intermediate to additives, and adding water if necessary, water suspensions with different contents can be obtained.
[0061] Example 3: Preparation of Aqueous Suspension Agent
[0062] Take intermediate product 2 obtained in Example 1 by weight, add a certain amount of water, then add 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 evenly. By adjusting the ratio of water to additives, water suspensions with different contents can be obtained.
[0063] Example 4: Granule Preparation
[0064] By weight, intermediate product 2 obtained in Example 1, light calcium carbonate, 2 parts wetting agent SXC, 2 parts dispersant NNO, and 15 parts sawdust were pulverized and mixed in 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°C for 1 hour. Granules with different contents were obtained by adjusting the ratio of bacterial powder to light calcium carbonate.
[0065] Example 5: Preparation of Microcapsules
[0066] 20g of polyethylene terephthalate (PET) granules were dissolved in 200ml of dichloromethane. An appropriate volume of intermediate product 1 obtained in Example 1 (or intermediate product 2 mixed with water in the desired proportion) and gelatin solution were then added. The mixture was stirred and dispersed for 30–45 min to obtain a water / oil emulsion. Then, 1600ml of a 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.
[0067] The following field trials verify the effects of the microbial inoculant of the present invention on promoting the growth and conception of water bamboo.
[0068] Example 6: Trial of early spring planting of water chestnuts promoted by 5% water smut G1 aqueous suspension.
[0069] To evaluate the feasibility of the aqueous suspension of the present invention in promoting the formation of water bamboo shoots, a field trial was conducted to test the effects of the aqueous suspension on the growth and formation of water bamboo shoots.
[0070] The experiment included three treatments: a water suspension treatment, a conventional sodium dichloroisocyanurate fertility-inducing agent treatment, and a treatment without fertility stimulants. Each treatment had three replicates. Each replicate area was 250 m². 2 .
[0071] Forty-five days after planting water chestnuts in early spring, 1 liter of microbial inoculant was added to 60 kg of irrigation water and sprayed on the leaves. The results are shown in Table 1.
[0072] Table 1: Comparison of the number of water chestnuts harvested after water suspension treatment and conventional treatment
[0073]
[0074] The results in Table 1 show that, compared with the conventional sodium dichloroisocyanurate fertilization-promoting treatment group, the total yield of water chestnut increased by 30% to 40% after spraying the aqueous suspension of the present invention, and the early harvest was significantly improved.
[0075] Example 7: Trial of promoting fruit development in summer-planted water chestnuts using 10% Ustilago maydis G1 microcapsules.
[0076] The experiment included two treatments: microcapsule treatment and conventional sodium dichloroisocyanurate treatment. Each treatment covered an area of approximately 1200 square meters. There were no replicates and no control group without fertility-promoting agents.
[0077] Fifty days after summer planting of water chestnuts in the field, 1 liter of microcapsule was added to 60 kg of irrigation water and sprayed on the leaves. The results are shown in Table 2.
[0078] Table 2: Comparison of harvested water chestnut yield (kg) after microencapsulation treatment and conventional treatment
[0079] Planting / days Endophytic bacteria treatment (kg) Standard processing (kg) 76 11.86 7.70 79 28.96 14.78 81 70.84 55.82 84 103.14 67.70 87 134.58 88.32 90 141.26 107.10 93 138.98 102.50 96 130.64 104.66 99 116.70 100.64 total: 876.96 649.22
[0080] The results in Table 2 show that, compared with conventional treatment, spraying the microcapsule agent of the present invention results in a longer harvest period and a 30% to 40% increase in total yield.
[0081] The above results indicate that the novel microbial agent of the present invention, with *Ustilago maydis* G1 as the effective ingredient, can promote the growth of *Zizania latifolia*, induce early fruiting, and increase yield.
[0082] Sequence list information:
[0083] DTD Version: V1_3
[0084] Filename: 231073SEQ.xml
[0085] Software Name: WIPO Sequence
[0086] Software version: 2.1.1
[0087] Generation Date: 2023-06-15
[0088] Basic Information:
[0089] Current application / Intellectual Property Office: CN
[0090] Current application / applicant file name: 231073
[0091] Applicant's Name or Title: East China University of Science and Technology
[0092] Applicant's name or title / language:zh
[0093] Applicant's name or title / Latin name: ECUST
[0094] Invention Title: A type of smut fungus (Ustilago esculenta) and its application (zh)
[0095] Total sequence count: 1
[0096] sequence:
[0097] Serial Number (ID): 1
[0098] Length: 1770
[0099] Molecular type: RNA
[0100] Feature location / qualifier:
[0101] -source,1..1770
[0102] >mol_type,rRNA
[0103] >organism, Ustilago esculenta
[0104] residues:
[0105]
[0106]
Claims
1. A type of water chestnut smut fungus ( Ustilago esculenta ), characterized in that, The *Smutella ursodesifolia* ( Ustilago esculenta The accession number for this item is CGMCC No. 40037.
2. The *Ustilago maydis* fungus described in claim 1 (… Ustilago esculenta The application of ) is characterized by, Used to prepare microbial agents for promoting the growth and inoculation of water bamboo shoots.
3. The application according to claim 2, characterized in that, The microbial agents are available in the form of aqueous suspensions, granules, and microcapsules.
4. A microbial inoculant for promoting the growth and seed formation of water bamboo, characterized in that, The microbial agent is the *Smutweed* species described in claim 1 (…). Ustilago esculenta () is the main active component.
5. The microbial agent according to claim 4, characterized in that, Wild rice smut fungus (as the main active component) Ustilago esculenta () is a concentrated solution of its fermentation culture.
6. The microbial agent according to claim 4, characterized in that, Wild rice smut fungus (as the main active component) Ustilago esculenta It is the solid obtained by drying the concentrated liquid of its fermentation culture.
7. The microbial agent according to claim 4, characterized in that, The microbial agents are available in the form of aqueous suspensions, granules, and microcapsules.
8. The microbial agent according to claim 4, characterized in that, By weight percentage, the active ingredient ranges from 5% to 20% for aqueous suspensions and granules, and from 10% to 30% for microcapsules.
9. The method of using the microbial inoculant according to any one of claims 4 to 8: For irrigation treatment, 40 days after planting water chestnuts, apply microbial inoculants at a dosage of 200g to 400g of active ingredients per acre directly to the inlet of the irrigation water so that the inoculants can be distributed throughout the field with the irrigation water. For broadcasting, 40 days after planting water chestnuts, the microbial inoculant is evenly broadcast into the water chestnut field at a dosage of 1000g to 4000g of the formulation product per acre; For foliar spraying, dilute the microbial agent with 50g to 200g of active ingredients per acre before spraying, and control the water usage per acre to 50kg to 80kg.