Bacillus tropicus HN44-2-F8 from the root of common wild rice and its biological preparation and application

Abstract

The application belongs to the field of biological pesticides, and specifically discloses a common wild rice root system bacillus tropicus HN44-2-F8, a biological preparation thereof and application. The bacillus tropicus in the application is named bacillus tropicus HN44-2-F8, which was preserved in the Guangdong Microbial Culture Collection Center on May 27, 2026, and the preservation number is GDMCC No: 68351. The strain of the bacillus tropicus HN44-2-F8 is derived from the root system of common wild rice and can well colonize in the root system of wild rice. The bacillus tropicus HN44-2-F8 has a significant inhibitory effect on rice blast fungus and an excellent biocontrol effect on rice blast, is environmentally friendly and non-toxic, is applied to the prevention and treatment of diseases by being applied to the soil for planting rice, has a small influence on the ecological environment, is safer and more reliable for the consumption of rice and the processing products of rice, has low culture condition requirements and has a good development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticide technology, and specifically relates to a common wild rice root-borne tropical Bacillus strain HN44-2-F8 and its biological agents and applications. Background Technology

[0002] Rice is the staple food for nearly half the world's population, and its safe production is crucial to the global food supply system. Rice blast is the most destructive fungal disease affecting rice production. This disease not only leads to a sharp reduction in yield but also severely impacts rice quality, threatening regional food security. Due to frequent physiological race variations of the rice blast fungus, chemical control faces the dual challenges of increasing drug resistance and ecological risks. Although breeding rice varieties with disease resistance is the most economical and effective method, the long breeding cycle and the linkage of many disease resistance genes with other undesirable agronomic traits mean that varietal resistance cannot keep up with the variation of the rice blast fungus, or that disease resistance and high yield and quality are not easily achieved simultaneously. Therefore, finding a rapid, efficient, economical, and safe method for controlling rice blast is of paramount importance. Biological control, as a long-lasting, broad-spectrum, and environmentally friendly method for disease control, has attracted increasing attention from researchers. Among these methods, the exploration of efficient and safe biocontrol bacteria resources has become a key breakthrough in the green control of diseases. Bacillus sp., with its strong resistance, broad antibacterial spectrum, and diverse metabolites, occupies an important position in the biological control of crop diseases. In recent years, scholars at home and abroad have conducted extensive research on the control of food crop diseases by Bacillus.

[0003] Bacillus, as an important plant growth-promoting bacterium, has become a hot resource for the development of biopesticides due to its ability to form stress-resistant spores, abundant metabolites, broad antibacterial spectrum, and environmental friendliness. Liu Youzhou et al. screened a strain of Bacillus amyloliquefaciens L-11 from rice rhizosphere soil. The fermentation broth showed an inhibition zone diameter of 32.5 mm against rice bakanae disease pathogens and an inhibition rate of 91.3% against rice false spore germination. The pot experiment showed a control efficacy of 78.6% against rice false spores. Ying Yufeng et al. isolated Bacillus velezensis DY-6-6 from the rhizosphere of tobacco. Treatment with B. velezensis DY-6-6 increased the malondialdehyde content of *Phytophthora parasitica*, decreased the activities of catalase and superoxide dismutase, and reduced protein content, indicating high biofilm activity. Furthermore, B. velezensis DY-6-6 promoted germination and growth in tobacco, significantly increasing seed germination rate, leaf area, plant height, and biomass. Its fermentation broth showed a relative preventive effect of 70.35% and a relative curative effect of 59.20% against tobacco black shank in indoor potted plants, consistent with fungicides. Cui Ruirui's team screened and obtained a strain of Bacillus amyloliquefaciens HX-23 that effectively controlled the occurrence of wilt disease in peppers, with a relative control effect of 23.16%. The HX-23 strain significantly promoted the growth of chili seedlings, increasing the fresh weight and dry weight of the whole plant by 17.70% and 22.35%, respectively. The HX-23 strain also exhibited good inhibitory effects against Fusarium oxysporum and promoted plant growth, providing strain resources and theoretical basis for the biological control of Fusarium oxysporum.

[0004] Numerous studies have shown that Bacillus can synergistically promote healthy rice growth through multiple mechanisms: First, it produces lipopeptide antibiotics such as surfactants, iturobrine, and fentanyl, which directly act on the cell membrane of pathogens, disrupting their structure and function; second, it secretes cell wall degrading enzymes such as chitinase and protease, hydrolyzing pathogen cell wall components; third, it limits pathogen colonization and infection by competing for nutrients and ecological niches in the rhizosphere and periplasm; fourth, it induces induced systemic resistance (ISR) in rice, activating the jasmonic acid / ethylene signaling pathway, increasing the activity of defense enzymes and phytoalexin content in the plant, and enhancing its systemic defense against rice blast; fifth, it can synthesize plant hormones such as indoleacetic acid and gibberellin, promoting root development and nutrient absorption in rice, and improving plant vigor and stress resistance. Bacillus also possesses advantages such as strong stress resistance, long-lasting colonization ability, and good formulation stability; its spore preparations can be stored at room temperature for more than a year, facilitating industrial production and field application. In summary, Bacillus, as a biocontrol microbial resource with great development value, shows promising prospects in the green control of rice diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Bacillus tropicalis* HN44-2-F8 from the roots of common wild rice, its biological agents, and applications. Its main application is in the control of rice blast, addressing the toxicity, residues, and environmental risks associated with existing chemical pesticides. The *Bacillus tropicalis* HN44-2-F8 described in this invention is isolated from the roots of healthy common wild rice. This strain can stably accumulate in wild rice roots, establishing a harmonious symbiotic relationship with the host. It exhibits a significant inhibitory effect on the growth of rice blast fungus, demonstrating excellent biocontrol potential. Its natural origin and good environmental compatibility not only effectively inhibit rice blast fungus but also ensure the safety of rice and its processed products for consumption. Furthermore, this strain has simple cultivation conditions and low production costs, possessing promising prospects for industrial development and application.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A strain of Bacillus tropicus HN44-2-F8, originating from common wild rice roots, is deposited at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The accession number of Bacillus tropicus HN44-2-F8 is GDMCC No: 68351, and the deposit date is May 27, 2026.

[0008] Furthermore, the rice germplasm used for the isolation and purification of Bacillus tropicalis HN44-2-F8 is the root system of common wild rice. More preferably, when Bacillus tropicalis HN44-2-F8 is cultured on LB medium at 28°C for 16 hours, the colonies are round with irregular edges, wrinkled surfaces, and a medium diameter of 2-5 mm. The colony texture is viscous or creamy.

[0009] Furthermore, the aforementioned Bacillus tropicalis HN44-2-F8 was applied in the prevention and control of rice blast.

[0010] Another object of the present invention is to provide a biological agent prepared based on the above-mentioned common wild rice root Bacillus tropicalis HN44-2-F8.

[0011] Furthermore, the biological agent is at least one of Bacillus tropicalis HN44-2-F8 fermentation broth, Bacillus tropicalis HN44-2-F8 supernatant, and Bacillus tropicalis HN44-2-F8 supernatant filtrate.

[0012] Furthermore, the preparation method of the Bacillus tropicalis HN44-2-F8 fermentation broth specifically includes the following steps: inoculating Bacillus tropicalis HN44-2-F8 into liquid LB medium and shaking culture for 16 hours, and then collecting the Bacillus tropicalis HN44-2-F8 fermentation broth.

[0013] Furthermore, the method for preparing the supernatant of Bacillus tropicalis HN44-2-F8 specifically includes the following steps: Bacillus tropicalis HN44-2-F8 is inoculated into liquid LB medium and cultured with shaking for 16 hours, and then the supernatant is collected by centrifugation at 10000-12000 rpm for 5-10 minutes to obtain the supernatant of Bacillus tropicalis HN44-2-F8;

[0014] Furthermore, the preparation method of the supernatant filtrate of Bacillus tropicalis HN44-2-F8 specifically includes the following steps: filtering the supernatant of Bacillus tropicalis HN44-2-F8 through a 0.45μm bacterial filter to remove bacteria, thereby obtaining the supernatant filtrate of Bacillus tropicalis HN44-2-F8.

[0015] Furthermore, the pH value of the liquid LB medium is 7.0.

[0016] Furthermore, the specific conditions for the 16-hour shaking culture are as follows: liquid LB medium is cultured at 28°C at a shaking rate of 180 rpm for 16 hours.

[0017] This invention also provides the application of the above-mentioned biological agent in the control of rice blast. Specifically, it provides the application of the above-mentioned biological agent in the preparation of a biopesticide for the control of rice blast.

[0018] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0019] First, the tropical Bacillus HN44-2-F8 obtained in this invention originates from the roots of common wild rice and can accumulate well in the roots. In addition, the bacteria screened from the roots of common wild rice in this invention, when used for the prevention and control of rice blast, only show an increase or decrease in the number of a certain microorganism among specific populations, without changing the category of microbial populations on rice and thus affecting the stability of the population. Furthermore, due to its long-term coexistence and mutualistic symbiosis with wild rice, it has formed a series of survival mechanisms similar to wild rice. Spraying it on the surface of rice tissues for disease prevention and control is safer and more reliable for the consumption of rice and its processed products.

[0020] Secondly, the tropical Bacillus HN44-2-F8 of this invention has a strong inhibitory effect on rice blast fungus, demonstrating excellent potential biocontrol efficacy against rice blast. Furthermore, its biological source is environmentally friendly, with minimal impact on the ecological environment. The Bacillus obtained in this invention has low requirements for culture conditions and shows great promise for development and application.

[0021] Third, the expected benefits and commercial value of the technical solution of this invention after transformation are as follows: Based on the fact that the microbial resources to be protected by this invention have a significant inhibitory effect on the growth of rice blast pathogen, researching and developing biopesticides based on this bacterium for the prevention and control of rice blast can effectively reduce the use of chemical pesticides, be environmentally friendly, ensure food security, and have good ecological benefits. Promoting the development of inoculants based on this bacterium can obtain economic benefits and commercial value.

[0022] Fourth, this invention further enriches the microbial resources related to biological control research both domestically and internationally. By employing different treatment methods, this invention can produce different microbial agents, solving the technical problem of the limited application of microbial pesticides. Through different treatments, this invention can be used not only to control rice blast but also in combination with other microorganisms to stabilize the microbial community in the rice growing environment, promoting healthy rice growth and improving its disease resistance.

[0023] Fifth, as a wild rice root fungus, derived from isolated strains of rice itself, this invention overcomes the technical bias of general biological pesticides having only a single effect. This invention can not only serve as an important means of controlling rice blast, but also stabilize the rice's own microbial community, contributing to rice growth and development and creating a more suitable microbial environment for rice survival. Attached Figure Description

[0024] Figure 1 This is a morphological diagram of the strain of Bacillus tropicalis HN44-2-F8 of the present invention on LB medium.

[0025] Figure 2 The figures show the inhibitory effects of different treatments of Bacillus tropicalis HN44-2-F8 on rice blast fungus (Guy11) and the control group. In the figure, A is the result of the confrontation culture between HN44-2-F8 cells (stretched around the edges) and rice blast fungus Guy11 (center), and B is the control result of normal growth of rice blast fungus Guy11 in LB liquid medium.

[0026] Figure 3The figures show the inhibitory effects of different treatments of the Bacillus tropicalis HN44-2-F8 biological agent of the present invention on rice blast fungus (Guy11) and the control group. Among them, A is the result of confrontation culture of HN44-2-F8 fermentation broth + rice blast fungus (Guy11), B is the result of confrontation culture of HN44-2-F8 fermentation supernatant + rice blast fungus (Guy11), C is the result of confrontation culture of HN44-2-F8 fermentation supernatant filtrate + rice blast fungus (Guy11), and D is the result of control treatment confrontation culture of LB liquid medium + rice blast fungus (Guy11).

[0027] Figure 4 The images show the microscopic results of the effect of Bacillus tropicalis HN44-2-F8 on the mycelium of rice blast fungus (Guy11) of the present invention. In the images, A is a microscopic result of the normal mycelium of rice blast fungus (Guy11), and B is a microscopic result of the abnormal mycelium of rice blast fungus (Guy11) after confrontation with the biocontrol fungus HN44-2-F8. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0029] Example 1: Isolation, purification and preservation of Bacillus tropicalis from the roots of common wild rice

[0030] (1) Preparation of LB medium: Weigh 10g of tryptone (Oxoid LTD LP0042, England), 5g of yeast extract (Oxoid LTD LP0021, England), and 10g of sodium chloride (NaCl, Sinopharm Chemical Reagent Co., Ltd., 10019318). Add pure water to make up to 1000 mL, stir evenly, and add 15g of agar when preparing solid medium. After heating and dissolving thoroughly, dispense into containers, sterilize at 121℃ for 20 min, and store for later use.

[0031] (2) Isolation and purification of tropical Bacillus:

[0032] The roots of common wild rice used for microbial isolation were collected from the Zengcheng Experimental Base of the Guangdong Academy of Agricultural Sciences.

[0033] The sample used for isolation was common wild rice. Healthy root tissue and the soil attached to it were taken from farmland planted in Zengcheng. The conventional tissue isolation method was followed with slight modifications: the roots and the soil attached to them were placed in an Erlenmeyer flask, sterile water was added for soaking, the mixture was shaken twice and allowed to stand for 10 minutes. After the supernatant appeared, 100 μL was taken and spread on LB solid medium plates. The plates were incubated at 28℃ for 24-48 hours. Single colonies that grew were picked and purified to obtain Bacillus tropicalis HN44-2-F8.

[0034] The Bacillus tropicus HN44-2-F8 was isolated from common wild rice at the Zengcheng Experimental Base of the Guangdong Academy of Agricultural Sciences. Its sequence is shown below: it has 99.39% homology with the 16S rRNA sequence of Bacillus tropicus JMT105-2 with accession number CP115303.1 in GenBank (the alignment results are shown in Table 1), confirming that it is classified as Bacillus tropicus and named Bacillus tropicus HN44-2-F8.

[0035] Table 1 shows the identification results of Bacillus tropicus based on whole-genome sequencing. Analysis of HN44-2-F8 was performed using the 16s and Nt databases. The identification results were obtained through ANI analysis by comparing the genome sequences of 13 Bacillus tropicus strains downloaded from NCBI.

[0036] Table 1. Species identification based on whole-genome sequencing

[0037] HN_44_2_F8.seq CP115303.1 Bacillus tropicus JMT105-2 99.3879

[0038] The 16S rRNA sequence fragment of Bacillus tropicalis HN44-2-F8 is as follows (Seq ID NO.1):

[0039]

[0040] Bacillus tropicalis HN44-2-F8 was cultured on LB medium at 28°C for 16 h. Colonies were typically round with irregular edges, wrinkled surfaces, and medium diameter, usually between 2-5 mm. The colony texture was viscous or creamy. (See...) Figure 1 ).

[0041] (3) Preservation of Bacillus tropicalis HN44-2-F8: The strain was deposited at Guangdong Provincial Microbial Culture Collection Center on May 27, 2026, with accession number GDMCC No: 68351.

[0042] Example 2: Determination of the activity of Bacillus tropicalis using the confrontation culture method

[0043] (1) The preparation of LB medium is the same as in Example 1; the preparation of LB liquid medium is the same as that of LB solid medium except that agar is not added. After the weighed reagent is fully dissolved, it is dispensed into conical flasks (100 mL of culture medium per flask), capped and wrapped, sterilized at 121 °C for 20 min, cooled and stored for later use.

[0044] (2) Preparation of single colonies of Bacillus tropicalis HN44-2-F8: Bacillus tropicalis was streaked onto LB medium plates and activated for 24 h. Single colonies were then picked and streaked for preservation.

[0045] (3) Preparation of fermentation broth, supernatant and supernatant filtrate of Bacillus tropicalis HN44-2-F8: Take the single colony of Bacillus tropicalis prepared in step (2) and inoculate it into the LB culture medium prepared in step (1) into an Erlenmeyer flask. Place it at 28℃ and shake at 180 rpm for 16 h to obtain fermentation broth of Bacillus tropicalis HN44-2-F8. Centrifuge the obtained fermentation broth at 12000 rpm for 10 min to obtain supernatant of Bacillus tropicalis HN44-2-F8. Then filter out the bacterial cells with a 0.45 m bacterial filter to obtain supernatant filtrate of Bacillus tropicalis HN44-2-F8.

[0046] (4) Activity assay:

[0047] ① Determination of the inhibitory effect of Bacillus tropicalis HN44-2-F8 on the growth of rice blast fungus: A circular filter paper with a diameter of 9cm was folded in half once, rotated 90° and folded in half a second time, and then folded in half again, forming a total of four creases. Four symmetrical points were marked on the bottom of the petri dish based on these creases. Figure 2 A). The rice blast fungus was inoculated into the center of a petri dish and incubated at 28°C for 4 days. The Bacillus tropicalis fermentation broth obtained in step (3) was then streaked and inoculated into four symmetrical spots. LB liquid medium was used as a control. Figure 2B), each with 3 replicates, were incubated at 28℃. After 7 days, the colony radius was measured, and the mycelial morphology of the inhibition zone edge and the control *Strombus rice* was observed. The results are as follows: Figure 3 As shown in Figure A. The Bacillus tropicalis fermentation broth obtained in step (3) was streaked onto four symmetrical points. The experiment was conducted using LB liquid medium ( Figure 2 B) served as a control, with three replicates for each control group, and was incubated at 28°C. After 7 days, the colony radius was measured, and the mycelial morphology of the inhibition zone edge and the control group was observed. The results are as follows: Figure 2 As shown in Figure A.

[0048] ② Determination of the inhibitory effect of Bacillus tropicalis HN44-2-F8 fermentation broth on the growth of rice blast fungus: The Bacillus tropicalis cells in step ① were replaced with 150 μL of Bacillus tropicalis fermentation broth in Oxford cups. The Oxford cups were placed vertically on the surface of the culture medium containing the pathogen, with the remaining steps unchanged. The results are as follows: Figure 3 As shown in A. Preparation of fermentation broth: Bacillus tropicalis was inoculated onto LB medium and cultured in a constant temperature incubator at 28°C for one day. The bacterial cells were then transferred to centrifuge tubes containing LB liquid medium and cultured in a shaker at 28°C at 180 rpm for 16 hours.

[0049] ③ Determination of the inhibitory effect of Bacillus tropicalis HN44-2-F8 fermentation supernatant and supernatant filtrate on the growth of rice blast fungus: The Bacillus tropicalis cells in step ① were replaced with 150 μL of Bacillus tropicalis fermentation supernatant and supernatant filtrate in Oxford cups, with the remaining steps unchanged. Preparation of fermentation supernatant and supernatant filtrate: The bacterial culture was centrifuged at 10000 rpm for 5 min to obtain the supernatant; the supernatant was drawn up with a 10 mL syringe, connected to a bacterial filter, and the filtered liquid was the fermentation supernatant filtrate. The results are as follows: Figure 3 B and Figure 3 As shown in C. The experiment was conducted using LB liquid medium ( Figure 3 D) As a control, each was set up in 3 replicates and incubated at 28℃.

[0050] ④ Take normally growing rice blast mycelia and rice blast mycelia cultured in confrontation with biocontrol bacterium HN44-2-F8, prepare slides separately, observe and photograph them under a microscope with a 40x objective lens, and the results are as follows: Figure 4 A and Figure 4 B.

[0051] Results Analysis: Figure 2 , Figure 3 and Figure 4It is known that the rhizobium tropicalis HN44-2-F8 of common wild rice has a significant inhibitory effect on the growth of rice blast fungus and causes malformation of the fungal hyphae. The aforementioned Bacillus tropicalis HN44-2-F8 can be used in the control of rice blast. Furthermore, biological agents based on Bacillus tropicalis HN44-2-F8 can be used in the control of rice blast; specifically, these biological agents can be used in the preparation of biopesticides for the control of rice blast.

[0052] The *Bacillus tropicalis* HN44-2-F8 strain described in this invention was isolated from the roots of healthy wild rice. This strain can stably accumulate in wild rice roots, establishing a harmonious symbiotic relationship with the host. It exhibits a significant inhibitory effect on the growth of rice blast fungus, demonstrating excellent biocontrol potential. Its natural origin and good environmental compatibility not only effectively inhibit rice blast fungus but also ensure the safety of rice and its processed products for consumption, making it environmentally friendly. Furthermore, this strain has simple cultivation conditions and low production costs, showing promising prospects for industrial development and application.

[0053] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of common wild rice rhizobium tropicalis HN44-2-F8, characterized by: The sample is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The accession number of the described Bacillus tropicalis HN44-2-F8 is GDMCC No: 68351, and the deposit date is May 27, 2026.

2. The common wild rice rhizobacterium tropicalis HN44-2-F8 according to claim 1, characterized in that: The rice germplasm used for the isolation and purification of the tropical Bacillus HN44-2-F8 was the root system of common wild rice.

3. The common wild rice rhizobacterium tropicalis HN44-2-F8 according to claim 1, characterized in that: The tropical Bacillus HN44-2-F8 was cultured on LB medium at 28°C for 16 hours. The colonies were round with irregular edges, wrinkled surfaces, and medium diameter, ranging from 2 to 5 mm. The colony texture was viscous or creamy.

4. The application of the tropical Bacillus HN44-2-F8 according to any one of claims 1-3 in the prevention and control of rice blast.

5. A biological agent, characterized in that: The biological agent was prepared based on the common wild rice root tropical Bacillus HN44-2-F8 as described in any one of claims 1-3.

6. The biological agent according to claim 5, characterized in that: It is at least one of the following: Bacillus tropicalis HN44-2-F8 fermentation broth, Bacillus tropicalis HN44-2-F8 supernatant, and Bacillus tropicalis HN44-2-F8 supernatant filtrate.

7. The biological agent according to claim 6, characterized in that: The preparation method of the fermentation broth of Bacillus tropicalis HN44-2-F8 specifically includes the following steps: Bacillus tropicalis HN44-2-F8 is inoculated into liquid LB medium and cultured with shaking for 16 hours, and then the fermentation broth of Bacillus tropicalis HN44-2-F8 is collected. The method for preparing the supernatant of Bacillus tropicalis HN44-2-F8 specifically includes the following steps: Bacillus tropicalis HN44-2-F8 is inoculated into liquid LB medium and cultured with shaking for 16 hours, and then the supernatant is collected by centrifugation at 10000-12000 rpm for 5-10 minutes to obtain the supernatant of Bacillus tropicalis HN44-2-F8. The preparation method of the supernatant filtrate of Bacillus tropicalis HN44-2-F8 specifically includes the following steps: filtering the supernatant of Bacillus tropicalis HN44-2-F8 through a 0.45μm bacterial filter to remove bacteria, thereby obtaining the supernatant filtrate of Bacillus tropicalis HN44-2-F8.

8. The biological agent according to claim 7, characterized in that: The pH of the liquid LB medium is 7.0; The specific conditions for the 16-hour shaking culture were as follows: liquid LB medium was cultured at 28°C at a shaking rate of 180 rpm for 16 hours.

9. The use of the biological agent according to any one of claims 5-8 in the prevention and control of rice blast.

10. The application of the biological agent according to claim 9 in the control of rice blast, characterized in that: The biological agent is used in the preparation of biopesticides for the prevention and control of rice blast.

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