Lysinibacillus elongate E and application thereof in prevention and treatment of root-knot nematode
By using Bacillus longiformis E to prepare a biological agent, the problems of soil pollution and drug resistance caused by chemical control of root-knot nematodes were solved, achieving a highly efficient and safe control effect against root-knot nematodes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-02
- Publication Date
- 2026-04-28
Smart Images
Figure CN121931003A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a strain of long-shaped lysine-containing Bacillus E and its application in the control of root-knot nematodes. Background Technology
[0002] Root-knot nematode disease is a soil-borne disease caused by pathogenic nematodes belonging to the genus *Meloidogyne* of the superfamily Heteroderidae in the class Nematoda of the class Nematoda. It is one of the most destructive diseases affecting crop production globally, severely restricting agricultural output. In recent years, with the rapid expansion of greenhouse agriculture in my country, especially the area under greenhouse vegetable cultivation, the occurrence and damage of root-knot nematode disease have shown a significant increasing trend in both northern and southern regions. Among them, the southern root-knot nematode (*Meloidogyne incognita*), due to its wide host range and strong environmental adaptability, has become the most prevalent and serious dominant species, posing a continuous threat to the yield and quality of high-value vegetables such as cucumbers, tomatoes, and peppers.
[0003] Currently, the control of root-knot nematode disease mainly relies on a comprehensive management strategy, primarily chemical control supplemented by agricultural and physical control methods. While chemical nematicides are rapidly effective, long-term reliance on them has led to a series of serious problems, including soil residue pollution from highly toxic pesticides, ecological damage, agricultural product safety risks, and the continuous emergence of pesticide resistance in nematode populations. Therefore, finding safe and efficient alternative control strategies has become an urgent need for the industry. Against this backdrop, biological control has attracted significant attention due to its environmentally friendly and sustainable characteristics. Current resources used for nematode biological control mainly focus on plant-derived extracts (such as matrine), antibiotic-producing actinomycetes (such as *Paecilomyces lilacinus*), predatory or parasitic biocontrol fungi (such as *Prconia chlamydosporium*), and nematicidal bacteria (such as *Pasteurella multocida* and *Bacillus*).
[0004] Existing research on *Bacillus longiformis* mainly focuses on its potential applications as a probiotic, particularly in aquaculture and food fermentation, while reports on its use as a nematicide are scarce. Furthermore, compared to the residues and toxicity of chemical pesticides in the environment, *Bacillus longiformis* is widely distributed in nature, has a relatively small impact on soil, and is relatively safe for the environment, aligning with the trend of sustainable agricultural development. Summary of the Invention
[0005] The purpose of this invention is to provide a strain of long-shaped lysine-containing Bacillus E and its application in the control of root-knot nematodes, so as to solve the problems existing in the prior art.
[0006] To achieve the above objectives, the present invention provides the following solution:
[0007] This invention provides a strain of *Lysinibacillus macroides* E, with the accession number CGMCC No. 33877.
[0008] The present invention provides a method for culturing the above-mentioned long-shaped lysine spores E, the method comprising the steps of inoculating the long-shaped lysine spores E into a culture medium and culturing it;
[0009] The culture medium is based on LB liquid medium and also includes 10 g / L glucose, 15 g / L yeast extract and 10 g / L magnesium sulfate.
[0010] Optionally, the culture temperature is 28°C and the rotation speed is 180 r / min.
[0011] This invention provides the application of the above-mentioned long-shaped lysine-containing Bacillus E in the preparation of biological agents.
[0012] The present invention provides a biological agent comprising the above-mentioned Bacillus longiformis E, the fermentation broth of Bacillus longiformis E, or the supernatant of the fermentation broth of Bacillus longiformis E.
[0013] Optionally, the method for preparing the fermentation broth includes the step of inoculating the long-shaped lysine-containing Bacillus E into a culture medium and culturing it to obtain the fermentation broth.
[0014] Optionally, the culture medium is LB liquid medium;
[0015] Alternatively, the culture medium is based on LB liquid medium and further includes 10 g / L glucose, 15 g / L yeast extract and 10 g / L magnesium sulfate.
[0016] Optionally, the culture temperature is 28°C and the rotation speed is 180 r / min.
[0017] This invention provides the application of the above-mentioned long-shaped lysine-containing Bacillus E or the above-mentioned biological agent in the control of root-knot nematodes.
[0018] The present invention provides a method for controlling root-knot nematodes, comprising the step of applying the above-mentioned long-shaped lysine-containing Bacillus E or the above-mentioned biological agent to the plants to be treated.
[0019] The present invention discloses the following technical effects:
[0020] This invention provides a microorganism, *Lysinibacillus macroides* E, which is effective in controlling root-knot nematodes. The strain's preservation number is CGMCC No. 33877. The efficacy of this strain was verified through indoor nematicidal activity assays and field trials. The results showed that in the indoor nematicidal activity assay, the fermentation broth of *Lysinibacillus macroides* E strain exhibited the best efficacy, with a 98.50% lethality rate against nematodes; 50% of the fermentation broth still maintained high activity (87.56%). In field trials, this strain also effectively controlled tomato root-knot nematode disease, with a control efficacy of 48.56%. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 The images show the colony morphology (A), Gram staining results (B), and 16S rDNA cluster analysis (C) of strain E.
[0023] Figure 2 This is a growth curve of strain E;
[0024] Figure 3 The control effects of different treatments on tomato root-knot nematode disease (partial); where A is the control (CK); B is abamectin; and C is the fermentation broth of Bacillus longiformis strain E.
[0025] Figure 4 The effects of different carbon sources (A), nitrogen sources (B), and inorganic salt sources (C) on the nematicidal efficacy of Bacillus longiformis strain E. Detailed Implementation
[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0031] Example 1: Isolation and identification of Lysinibacillus macroides E
[0032] 1. Isolation of strains
[0033] Healthy *Ageratina adenophora* plants were selected. The surface soil of the plants was rinsed with running tap water for 2 hours. The plants were then transferred to a clean bench and the roots were sterilized and removed. The roots were soaked in 75% (v / v) ethanol for 3 minutes, rinsed with sterile water, and then soaked in 2.5 wt% sodium hypochlorite for 7 minutes. They were then soaked in 75% (v / v) ethanol for 20 minutes. Finally, the roots were rinsed six times with sterile water to remove the disinfectant, and then ground. The resulting bacterial solution was inoculated into LB liquid medium and PDB liquid medium and cultured in a constant temperature shaker at 30°C and 100 r / min for 24 hours for enrichment. The sterile water from the final rinse served as a blank control.
[0034] The enriched bacterial suspensions were inoculated onto NA solid medium and incubated upside down in a dark incubator at 28°C. Colony growth was observed on the plates. Each treatment was repeated three times. After a clear colony growth was observed on the plates, a loopful was picked and inoculated onto a new blank plate using the streak plating method. This purification process was repeated multiple times until a single colony was obtained. The nematicidal activity of each bacterial strain was initially screened (100 μL of each strain's bacterial suspension at a concentration of 2 × 10⁻⁶). 8 (CFU / mL) was added to 20 μL of a suspension of approximately 50 southern root-knot nematodes, with sterile water as a control (CK). Each treatment was repeated three times. After incubation at 25°C for 24 h, the number of dead nematodes was observed and recorded under a stereomicroscope. The nematodes were stimulated with 1 mol / L NaOH; stiffness was considered a sign of death. The mortality rate and corrected mortality rate were calculated using the following formula: ; ), and strain E with better activity was obtained.
[0035] 2. Identification of strains
[0036] LB (Leydig-Bacillus thuringiensis) liquid medium: peptone (10.0 g / L), yeast extract (5.0 g / L), NaCl (10.0 g / L). LB solid medium is the LB liquid medium formula with 15.0 g / L agar powder added.
[0037] Strain E was activated on LB solid medium using the streak plate method. After incubation at 28°C for 2-3 days, the characteristics of single colonies, such as size, color, shape, elevation, texture, and edge, were observed, described, and recorded with reference to Bergey's Manual of Bacteriological Identification and Microbiology.
[0038] The colony morphology and Gram staining results of strain E are as follows: Figure 1 As shown in Figures A and B. The results show that strain E is round, pale yellow, smooth, opaque, with a flat and glossy surface and relatively neat edges. It is a Gram-positive bacterium.
[0039] The activated strain E was inoculated at a volume fraction of 1% into 100 mL of LB liquid medium and cultured at 28℃ with shaking at 180 r / min for 72 h. OD values at different time points were measured using sterile LB medium as a blank control. 600 Values were used to plot the growth curve of the strain, and the results are as follows: Figure 2 As shown in the figure. The results showed that the tests for VP, methyl red, indole, esterase, protease, citrate, and gelatin hydrolysis were negative, while the tests for catalase, urease, starch hydrolysis, cellulose hydrolysis, lead acetate reduction, and organic acid production were positive.
[0040] The physiological and biochemical characteristics of strain E were identified according to the "Handbook of Systematic Identification of Common Bacteria". The physiological and biochemical characteristics of strain E are shown in Table 1. The results showed that strain E was positive for catalase test, urease test, starch hydrolysis test, cellulose hydrolysis test and lead acetate reduction test, and negative for VP test, methyl red test, indole test, esterase test, protease test, citrate utilization test and gelatin hydrolysis test, indicating that it can produce organic acids.
[0041] Table 1. Partial physiological and biochemical characteristics of strain E
[0042]
[0043] Note: +: positive reaction; -: negative reaction.
[0044] Templates were prepared using a bacterial genomic DNA extraction kit. 16S rDNA was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.1) and 1492R (5′-ACGGCTACCTTGTTACGACT-3′, SEQ ID NO.2). A 50 μL reaction mixture contained 45 μL of 1×TSE102 Taq PCR Mix, 2 μL of each primer, and 1 μL of template. The program was as follows: pre-denaturation at 98℃ for 2 min, followed by 35 cycles of 98℃ for 10 s, 56℃ for 10 s, and 72℃ for 10 s, and then extension at 72℃ for 5 min. After passing 1.0% agarose gel electrophoresis, the PCR products were sent to Beijing Qingke Kunming Company for sequencing. The nucleotide sequence of the obtained 16S rDNA is shown in SEQ ID NO.3.
[0045]
[0046] The 16S sequence was uploaded to NCBI for BLAST alignment, yielding other sequences with extremely high sequence similarity (high homology sequences). After downloading the high homology sequences, a phylogenetic tree was constructed using MEGA 7.0 neighbor-joining to determine the strain's taxonomic position. The phylogenetic tree is shown below. Figure 1 As shown in Figure C, strain E clusters with Lysinibacillus macroides MK389440.1 and Lysinibacillus macroides NR 114920.1. Figure 1 The results from A and C indicate that strain E is *Lysinibacillus macroides*.
[0047] The present invention names strain E as Lysinibacillus macroides E, which was deposited on March 20, 2025 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33877.
[0048] Example 2: Indoor Nematode Activity Assay
[0049] Southern root-knot nematode (M. incognita) was collected from severely diseased cucumber roots in a greenhouse in Luxi County, Yunnan Province. It was propagated and cultured on susceptible tomato plants in a greenhouse at Yunnan Agricultural University, which provided the samples. Tomato roots with abundant egg sacs were selected, rinsed with clean water to remove soil and impurities, and plump egg sacs were placed in a 1wt% sodium hypochlorite solution for 3 minutes for sterilization. They were then rinsed three times with sterile water and placed in sterile petri dishes for incubation at 25°C. Newly hatched second-instar larvae were collected every 24 hours. This nematode was used for indoor contact nematode activity.
[0050] The *Bacillus longiformis* E strain was activated using the streak plating method. Single colonies with good growth were picked and inoculated into 50 mL of LB liquid medium, and cultured at 28℃ and 180 r / min for 48 h with shaking to obtain the fermentation broth of *Bacillus longiformis* E strain. The fermentation broth of *Bacillus longiformis* E strain was centrifuged at 12000 r / min for 5 min, and the supernatant was collected for later use. It was diluted with sterile water to prepare 50% fermentation broth (diluted 2 times) and 10% fermentation broth (diluted 10 times). The fermentation broth of *Bacillus longiformis* E strain was centrifuged at 12000 r / min for 5 min, and then filtered through a microporous membrane (0.22 µm) for sterilization to obtain sterile supernatant and bacterial cells. The centrifuged bacterial cells were then used to prepare a bacterial suspension (concentration 2 × 10⁻⁶) with sterile water. 8(CFU / mL). Take 20 mL of Bacillus longiformis strain E fermentation broth and supernatant, respectively, and autoclave at 121℃ for 30 min to obtain sterilized fermentation broth and sterilized supernatant for later use (to be used within 4 h). Nematodes were treated using the immersion method. 100 μL of sterilized fermentation broth and sterilized supernatant were added to 96-well plates, and 20 μL of Southern root-knot nematode suspension (approximately 50 nematodes) was added to each well. Sterile water was used as a control (CK). Each treatment was repeated three times. After incubation at 25℃ for 24 h, the number of dead nematodes was observed and recorded under a stereomicroscope. The nematodes were stimulated with 1 mol / L NaOH; stiffness was considered death. The mortality rate and corrected mortality rate were calculated using the following formula.
[0051] ;
[0052] .
[0053] The results are shown in Table 2. As can be seen from Table 2, the fermentation broth of *Bacillus longiformis* strain E showed the best lethality against nematodes at 98.50%. The 50% fermentation broth maintained high activity (87.56%), while the activity of the 10% fermentation broth decreased significantly (30.73%), indicating that the effective component decreased with increasing dilution factor. The effectiveness of the *Bacillus longiformis* strain E fermentation broth decreased significantly after sterilization, from 98.50% to 88.42%.
[0054] Table 2. Effects of various components of the fermentation broth from *Bacillus longiformis* strain E on nematode mortality.
[0055]
[0056] Example 3: Tomato Field Trial
[0057] The study was conducted in August 2025 at the experimental field behind Yunnan Agricultural University. The soil is red soil, where tobacco is grown year-round, and root-knot nematode disease has occurred every year, with a moderate degree of disease incidence.
[0058] Select plump tomato seeds, disinfect their surface with a 1% NaClO solution for 5 minutes, rinse repeatedly with sterile water 3-5 times, air dry, and sow in seedling trays. After the tomato seedlings reach the 4-leaf stage, transplant them to the field. Planting spacing is 0.3m, row spacing is 0.4m, with 11 tomato plants per row. Two days after transplanting, inoculate the tomato seedlings with 48-hour fermentation broth of *Bacillus longiformis* strain E using a root drenching method, at a volume of 150 mL per plant. A control treatment was prepared by root drenching with equal volumes of water, LB broth, and a 1500× dilution of 3.2% abamectin EC. Each treatment was replicated three times, with 15 seedlings per replicate. The plots were randomly distributed. After 30 days of cultivation, the incidence of root-knot nematode disease was classified according to the grading standard of GB / T17980.38—2000 (Ministry of Agriculture and Rural Affairs of the People's Republic of China. Field Efficacy Test Guidelines for Pesticides (I) Control of Root Nematode Diseases with Nematicides: GB / T 17980.38-2000 [S]. China Standards Press, 2000.). The nematode reduction rate, root-knot index, and control efficacy were calculated. The calculation formulas for the nematode reduction rate, root-knot index, and control efficacy are as follows:
[0059] ;
[0060] ;
[0061] .
[0062] Grading method for diseased plants: Grade 0: No galls on the roots; Grade 1: A small number of small galls on the roots; Grade 3: Two-thirds of the roots are covered with small galls; Grade 5: The roots are covered with small galls and have secondary galls; Grade 7: The roots have formed fibrous root clusters.
[0063] The results are shown in Table 3 and Figure 3 As shown in Table 3, after 30 days of treatment, the disease index of the water treatment group was 83.33, while the disease indices of tomatoes treated with abamectin and Bacillus longiformis E strain fermentation broth were 41.27 and 42.86, respectively. The nematode reduction rates in the soil were 62.32% and 52.95%, respectively, and the control efficacies were 50.47% and 48.56%, respectively. Root phenotypes for different treatments are shown in Table 3. Figure 3 Therefore, it can be seen that under field conditions, the fermentation broth of Bacillus longiformis strain E can effectively control tomato root-knot nematode disease.
[0064] Table 3. Control efficacy of different treatments against root-knot nematode disease in tomatoes under potted conditions.
[0065]
[0066] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (p<0.05).
[0067] Example 4: Optimization of Liquid Culture Medium Composition
[0068] LB liquid medium (LB) was used as the basal medium. Different carbon sources (starch, mannitol, maltose, glucose, lactose, sucrose, and potato starch as carbon sources to be screened) were added to a final concentration of 10 g / L. Other components remained unchanged. Each treatment was repeated three times. *Bacillus longiformis* strain E was inoculated into the medium at a 1% (v / v) inoculum. After incubation at 28°C and 180 r / min for 48 h with shaking, the nematicidal activity of the supernatant was measured (method as in Example 2). Water was used as the control group (CK). The results are as follows: Figure 4 As shown in A, there were no significant differences among the treatments except for the starch treatment. However, the mortality rate of nematodes in the glucose treatment was close to 100%. Therefore, glucose was finally determined to be the optimal carbon source.
[0069] Using LB liquid medium as the basal medium and glucose (final concentration 10 g / L) selected above as the carbon source, different nitrogen sources (peptone, yeast extract, tryptone, beef extract, ammonium nitrate, potassium nitrate, ammonium sulfate, ammonium chloride, urea, and nitrogen-free sources as the nitrogen sources to be screened) were added to a final concentration of 15 g / L. Each treatment was repeated three times. *Bacillus longiformis* E strain was inoculated into the medium at a 1% (v / v) inoculum and cultured at 28°C and 180 r / min for 48 h with shaking. The nematicidal activity of the supernatant was then measured (method as in Example 2). Water was used as the control group (CK). The results are as follows: Figure 4 As shown in B, the mortality rate of nematodes in the treatment with added yeast extract was close to 80%, which was significantly higher than other treatments. Therefore, yeast extract was finally determined to be the optimal nitrogen source.
[0070] The glucose (final concentration 10 g / L) and yeast extract (final concentration 15 g / L) selected in the above experiments were fixed. Different inorganic salts (sodium chloride, calcium chloride, copper sulfate, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, and manganese sulfate as the inorganic salts to be screened) were added to each treatment at a final concentration of 10 g / L. Other components remained unchanged. Each treatment was repeated three times. *Bacillus longiformis* E strain was inoculated into the culture medium at a 1% (v / v) inoculum. After incubation at 28℃ and 180 r / min for 48 h with shaking, the nematicidal activity of the supernatant was measured (method as in Example 2). Water was used as the control group (CK). The results are as follows: Figure 4 As shown in C, the mortality rate of nematodes treated with sodium chloride, magnesium sulfate, and potassium dihydrogen phosphate was close to 100%. However, the mortality rate of nematodes was the highest in the magnesium sulfate treatment. Therefore, magnesium sulfate was finally determined to be the most suitable inorganic salt.
[0071] The optimal culture medium for Bacillus longiformis strain E was determined to be: LB liquid medium as the base medium + 10 g / L glucose + 15 g / L yeast extract + 10 g / L magnesium sulfate.
[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A strain of long-shaped lysine-containing Bacillus macroides E, characterized in that, The preservation number of the long-shaped lysine spores E is CGMCC No. 33877.
2. The method for culturing *Bacillus longiformis* E according to claim 1, characterized in that, The cultivation method includes the step of inoculating the long-shaped lysine-containing Bacillus E into a culture medium and culturing it. The culture medium is based on LB liquid medium and also includes 10 g / L glucose, 15 g / L yeast extract and 10 g / L magnesium sulfate.
3. The cultivation method according to claim 2, characterized in that, The culture temperature was 28℃ and the rotation speed was 180 r / min.
4. The application of the long-shaped lysine-containing Bacillus E as described in claim 1 in the preparation of biological agents.
5. A biological agent, characterized in that, The biological agent includes the *Bacillus longiformis* E as described in claim 1, the fermentation broth of the *Bacillus longiformis* E, or the supernatant of the fermentation broth of the *Bacillus longiformis* E.
6. The biological agent according to claim 5, characterized in that, The method for preparing the fermentation broth includes the steps of inoculating the long-shaped lysine-containing Bacillus E into a culture medium, culturing it, and obtaining the fermentation broth.
7. The biological agent according to claim 6, characterized in that, The culture medium is LB liquid culture medium; Alternatively, the culture medium is based on LB liquid medium and further includes 10 g / L glucose, 15 g / L yeast extract and 10 g / L magnesium sulfate.
8. The biological agent according to claim 6, characterized in that, The culture temperature was 28℃ and the rotation speed was 180 r / min.
9. The application of the long-shaped lysine-containing Bacillus E as described in claim 1 or the biological agent as described in any one of claims 5-8 in the control of root-knot nematodes.
10. A method for controlling root-knot nematodes, characterized in that, The procedure includes applying the long-shaped lysine-containing Bacillus E as described in claim 1 or the biological agent as described in any one of claims 5-8 to the plants to be treated.