Stenotrophomonas sp., microbial inoculant, and preparation method and application thereof

Abstract

This invention belongs to the field of agricultural microbial technology and discloses a strain of oligotrophomonad ( Stenotrophomonas sp.), named Oligotrophomonas ( Stenotrophomonas Oligotrophic monotypic bacteria (s.) R5 were deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231758. This invention also discloses microbial agents containing this strain and their application in controlling rice planthoppers. The Oligotrophic monotypic bacteria (s.) R5 of this invention... Stenotrophomonas The control effect of sp. R5 and microbial agents containing this strain on rice planthoppers can reach more than 90%, which significantly improves the control effect compared with conventional control methods and greatly improves the survival rate of rice.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, and in particular relates to an oligotrophic monoclonal bacterium, a microbial agent and its preparation method, and its application in the control of rice planthoppers. Background Technology

[0002] Rice is frequently attacked by various pests during its growth process, among which rice planthoppers, mainly including brown planthoppers, white-backed planthoppers, and gray planthoppers, are one of the most serious pest groups. Rice planthoppers use their piercing-sucking mouthparts to suck the sap from the phloem of rice plants, leading to malnutrition, stunted growth, yellowing and even death of leaves (commonly known as "planthopper burn" or "planthopper puncture"). More seriously, rice planthoppers are the main vectors for many devastating viral diseases of rice (such as rice serrated leaf dwarf virus, rice straw dwarf virus, and rice black-streaked dwarf virus).

[0003] Currently, the control of rice planthoppers mainly relies on the following methods:

[0004] Chemical pesticide control: Chemical insecticides (such as neonicotinoids, imidacloprid, thiamethoxam, thiamethoxam, and acetamiprid) remain the primary means of controlling rice planthoppers due to their rapid action and ease of use. However, the long-term, excessive, and irrational use of chemical pesticides has led to a series of serious problems. For example: Rapid development of resistance: Rice planthoppers, especially brown planthoppers, have developed high levels of resistance to many commonly used insecticides, significantly reducing control effectiveness and forcing farmers to increase the dosage and frequency of application. Environmental pollution and ecological damage: Pesticide residues pollute soil and water sources, damage the farmland ecosystem, threaten biodiversity, and in particular, kill beneficial organisms in rice paddies (such as spiders, black-shouldered green mirid bugs, and tassel wasps), disrupting the natural ecological balance control mechanism. Pesticide residues and food safety: Pesticide residues in rice have raised public concerns about food safety. Increased control costs: Resistance leads to increased pesticide dosage and frequency of treatment, significantly increasing production costs.

[0005] Agricultural and physical control methods include selecting insect-resistant varieties, scientific fertilizer and water management, and light trapping. While these methods are environmentally friendly, their effectiveness is often limited or unstable, making them difficult to use alone to effectively control pest outbreaks.

[0006] Biological control, including the protection and utilization of natural enemies: Protecting and utilizing natural enemies such as rice paddy spiders, black-shouldered green mirid bugs, and tassel wasps is an important means of ecological regulation. However, its effectiveness is easily affected by climate and agricultural practices (especially the use of chemical pesticides), and its ability to control pests is insufficient during large-scale outbreaks.

[0007] In conclusion, developing new control technologies for rice planthoppers that are efficient, safe, environmentally friendly, sustainable, and unlikely to induce resistance in pests is of great significance for ensuring safe rice production, reducing environmental pollution, and promoting green and sustainable agricultural development. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide an oligotrophic monotypic bacterium, a microbial agent, a method for preparing the same, and its application in the control of rice planthoppers.

[0009] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0010] A strain of oligotrophomonas ( Stenotrophomonas sp.), the oligotrophomonas ( Stenotrophomonas sp.) was named Oligotrophomonas ( Stenotrophomonas sp.)R5 was deposited on September 20, 2023 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231758.

[0011] As a general inventive concept, the present invention also provides a microbial inoculant, comprising the aforementioned oligotrophic monoclonal bacteria (…). Stenotrophomonas sp.)R5.

[0012] As a general inventive concept, the present invention also provides a method for preparing the above-mentioned microbial inoculant, wherein the oligotrophomonas ( Stenotrophomonas sp.) R5 was inoculated into LB medium and cultured to obtain bacterial suspension. The bacterial cells were collected, resuspended, and the bacterial agent was obtained.

[0013] In the above preparation method, preferably, the culture conditions are: culture on a shaker at 180-220 r / min at 25-30℃ for 10-14 h.

[0014] In the above preparation method, preferably, the OD600 value of the resuspended bacterial agent is 1.0-1.2.

[0015] In the above preparation method, preferably, the viable bacteria concentration of the microbial agent is 0.8 × 10⁻⁶. 8 -1.2×10 8 CFU / mL.

[0016] As a general inventive concept, the present invention also provides the application of the microbial agent as described above or the microbial agent prepared by the preparation method described above in the control of rice planthoppers.

[0017] In the above-described application, preferably, the microbial agent is sprayed onto the stems and / or leaves of rice.

[0018] In the above application, preferably, the amount of microbial agent used is 0.5-3 mL / strain, and the OD600 value of the microbial agent is 1.0-1.2.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The oligotrophomonas of the present invention ( Stenotrophomonas The strain R5 and microbial agents containing this strain can achieve a control effect of over 90% against rice planthoppers, significantly improving the control efficacy compared to conventional control methods and greatly increasing the survival rate of rice; furthermore, the oligotrophic monotypic bacteria (sp.) R5 and microbial agents containing this strain can achieve a control effect of over 90% against rice planthoppers, significantly improving the control efficacy and greatly increasing the survival rate of rice; Stenotrophomonas The R5 strain (sp.) is derived from rice, making it more environmentally friendly and unlikely to cause ecological harm or rice residue.

[0021] Biological Preservation Instructions

[0022] The oligotrophomonas involved in this invention is named Stenotrophomonas sp. R5 was deposited at the China Center for Type Culture Collection on September 20, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231758. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 It is the oligotrophomonad strain screened by this invention ( Stenotrophomonas Colony morphology diagram of sp.)R5.

[0025] Figure 2 It uses the oligotrophomonas bacteria of this invention ( Stenotrophomonas The survival rate of rice plants infected with rice planthoppers was improved by treating the microbial agent R5 (sp.)R5.

[0026] Figure 3 It uses the oligotrophomonas bacteria of this invention ( Stenotrophomonas The mortality rate of rice planthoppers infected with the microbial agent R5 (sp.) was reduced. Detailed Implementation

[0027] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] Example 1: Screening and Identification of Strains

[0031] 1. Screening of bacteria

[0032] Preparation of culture medium:

[0033] 1 / 2LB liquid culture medium: Weigh 5.0 g sodium chloride, 2.5 g yeast extract and 5.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, autoclave at 121°C for 30 minutes, and use after cooling.

[0034] NB liquid culture medium: Weigh 10.0 g of glucose, 3.0 g of beef extract and 5.0 g of peptone, and make up to 1000 mL with ultrapure water. Adjust the pH to 7.0, dispense into Erlenmeyer flasks, seal and sterilize by autoclaving at 121°C for 30 minutes. Use after cooling.

[0035] 1 / 2LB solid medium: Weigh 5.0 g sodium chloride, 2.5 g yeast extract and 5.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0036] NB solid medium: Weigh 10.0 g glucose, 3.0 g beef extract and 5.0 g peptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0037] Plump and healthy Huanghuazhan rice seeds from Zhouxi Town, Kaili City, Guizhou Province were selected. First, the seeds were soaked in 75% anhydrous ethanol for 10 minutes, then the ethanol was poured off, and the seeds were rinsed five times with sterile water. Next, they were soaked in 5% NaClO for 10 minutes, and then rinsed five times with sterile water. 100 μL of the sterile water from the final rinse was spread onto 1 / 2 LB solid culture medium and incubated in a constant temperature incubator for 5 days. The presence of bacterial growth on the medium was observed to ensure thorough sterilization of the rice seed surface.

[0038] Endophytic bacteria were isolated from rice seeds using 1 / 2 LB and NB liquid media. Surface-sterilized rice seeds were ground into powder in a sterile mortar with a small amount of liquid nitrogen. The rice seed powder was then inoculated into 250 mL of 1 / 2 LB and NB liquid media using a sterile weighing spoon. After 36 hours of incubation in a shaker (28 ℃, 180 rpm / min), 1 mL of the bacterial suspension was diluted to 1×10⁻⁶. 4 1×10 5 and 1×10 6 After dilution, 100 μL of the bacterial suspension was spread onto the corresponding solid culture medium and incubated upside down in a constant temperature incubator (28 ℃). The growth of the strains on the plates was observed periodically. After 5 days of growth, strains with different morphological characteristics were selected from the plates using an inoculation loop and streaked onto the corresponding solid culture medium to obtain purified single bacteria. After activation, the single bacteria were mixed with sterile glycerol at a ratio of 1:1, and 1.5 mL was aliquoted into 2 mL centrifuge tubes and stored at -80 ℃ for later use.

[0039] The selected strains with different morphological characteristics (numbered R1, R2, R3, R4, R5, and R6) were inoculated into LB liquid medium (pH 7.0) and cultured in a constant temperature shaker (28℃, 200 rpm) for 12 hours. The bacterial culture was then centrifuged at 6000 rpm for 3 min, the supernatant LB layer was discarded, and the bacterial cells were collected. Sterile water was added to the bacterial cells to adjust the OD600 value to 1.0 (viable cell count approximately 1 × 10⁻⁶). 8 (CFU / mL) was used to prepare a bacterial culture.

[0040] Rice planthoppers of the same age (3rd instar nymphs), healthy and disease-free, and reared for one generation in advance on pathogen-free rice seedlings were selected. Different morphological strains were prepared into single-strain bacterial suspensions (labeled R1, R2, R3, R4, R5, and R6, respectively) using the previously established method. 5 mL of each bacterial suspension was sprayed. A blank control group sprayed with an equal volume of 5 mL of sterile water and a negative control group sprayed with a suspension of a pathogen-free strain (Escherichia coli DH5α) were also set up. All treatments were placed in an artificial climate chamber at 28℃, 70%–80% relative humidity, and a photoperiod of 16 h light / 8 h darkness, using uniformly growing 3-leaf-1-heart stage rice seedlings as rearing media.

[0041] Standardized processing and observation were then carried out: three replicates were set up, and 20 rice planthoppers were placed in sterile petri dishes in each replicate. 5 mL of the corresponding bacterial solution (or control solution) was sprayed evenly with a calibrated micro-sprayer. After standing for 5 min, the plantshoppers were transferred to rice seedlings and covered with a breathable net. The cumulative mortality rate of rice planthoppers in each replicate group (number of dead planthoppers / total number of planthoppers × 100%) was recorded at 96 h. The results are shown in Table 1. The strain with the highest cumulative mortality rate at 96 h was selected as the strain R5 with the strongest pathogenicity to rice planthoppers.

[0042] Table 1. Cumulative mortality rate of rice planthoppers

[0043]

[0044] 2. Identification of fungal strains

[0045] The colony morphology of strain R5, which is most virulent against rice planthoppers, on agar plates is as follows: Figure 1 As shown, single colonies are 1 mm in diameter, round, with neat edges, hemispherical elevation, plump texture, smooth and moist surface, milky white, mucous-like, and stringy when picked up. The identification results of key physiological and biochemical characteristics are shown in Table 2 below.

[0046] Table 2 Key physiological and biochemical characteristics of Oligotrophomonas R5

[0047]

[0048] (+++: Diameter > 10mm, strong protease secretion capacity)

[0049] The most virulent strain R5 against rice planthoppers was selected and inoculated into LB broth for activation. After centrifugation using a high-speed refrigerated centrifuge, the bacterial cells were collected, and bacterial DNA was extracted using a bacterial DNA extraction kit according to the manufacturer's instructions. The amplified DNA products were sent to Hunan Saisiwei Biotechnology Co., Ltd. for sequencing and identification. The obtained endophytic bacterial gene sequences were then compared online with the NCBI database to identify the species of the endophytic bacteria.

[0050] Identification result: This strain is Stenotrophomonas sp. (Oligotrophomonas), named Oligotrophomonas ( Stenotrophomonas sp.)R5 was deposited at the China Center for Type Culture Collection on September 20, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231758.

[0051] Example 2: Preparation of microbial inoculants

[0052] Take the oligotrophic monoclonal bacteria isolated and screened in Example 1 ( Stenotrophomonas sp.) R5 was inoculated into LB medium (pH 7.0) and cultured in a constant temperature shaker (28℃, 200 rpm) for 12 hours. The bacterial culture was then centrifuged at 6000 rpm for 3 min, the supernatant LB was discarded, and the bacterial cells were collected. Sterile water was added to the bacterial cells to adjust the OD600 value to 1.0 (viable count of 1 × 10⁻⁶). 8 CFU / mL), shake well to obtain a sample containing oligotrophomonas (CFU / mL). Stenotrophomonas (sp.)R5 microbial inoculant.

[0053] Example 3: Application of microbial agents in the control of rice planthoppers

[0054] Select plump Huanghuazhan rice seeds, disinfect them with sodium hypochlorite, and then wash them with distilled water. Soak the seeds in a 28℃ constant temperature incubator for 4 days to promote germination. Then, use hydroponics to sow the seeds in plastic pots for cultivation. Once the rice seedlings have grown to three leaves and one bud, transplant them into soil-grown pots, with 10 seedlings per pot.

[0055] One hundred rice planthoppers (3rd instar nymphs, healthy and disease-free, and reared for one generation in advance on pathogen-free rice seedlings) were introduced into each rice pot. The microbial agent containing oligotrophomonas R5 prepared in Example 2 was sprayed onto the rice stems and leaves at doses of 5, 10, 15, 20, 25, and 30 mL / pot, and 30 mL of sterile water per pot (as a control) was sprayed onto the rice stems and leaves. Each treatment was replicated in triplicate.

[0056] One week after introducing a microbial agent containing *Oligotrophomonas* R5 into the rice planthopper population, the rice phenotype was observed, and survival was determined according to survival criteria (see Table 3). The number of dead planthoppers was calculated, and the survival rate of rice and the mortality rate of planthoppers were statistically analyzed. The results are shown in [Table 3]. Figure 2 , Figure 3 As shown.

[0057] Rice survival rate = (Total number of rice plants - Number of dead rice plants) / Total number of rice plants;

[0058] Rice planthopper mortality rate = (total number of rice planthoppers - number of dead rice planthoppers) / total number of rice planthoppers.

[0059] Table 3 Criteria for judging rice survival

[0060]

[0061] like Figure 3 As shown, the mortality rate of rice planthoppers increased significantly with increasing inoculant dosage. The treatment group sprayed with 20 mL of a microbial inoculant containing *Oligotrophomonas R5* showed the highest mortality rate, reaching 92%, with a statistically significant advantage. Correspondingly, as... Figure 2 As shown, the survival rate of rice was significantly protected after application of the microbial agent containing Oligotrophozoites R5. The rice survival rate reached a peak of 93% after spraying with 20 mL of the microbial agent containing Oligotrophozoites R5. These results indicate that the application of the microbial agent prepared in Example 2 can effectively kill rice planthoppers and protect rice, with the optimal dosage of 20 mL / pot (i.e., 2 mL / plant).

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a microbial inoculant in the control of rice planthoppers, wherein, The microbial agent includes Oligotrophomonas (… Stenotrophomonas sp.) R5, Oligotrophomonas ( Stenotrophomonas sp.)R5 was deposited on September 20, 2023 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231758.

2. The application as described in claim 1, characterized in that, The microbial agent is sprayed onto the stems and / or leaves of rice.

3. The application as described in claim 1, characterized in that, The amount of the microbial agent used is 0.5-3 mL / strain, and the OD600 value of the microbial agent is 1.0-1.2.

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