Sporosarcina sp. strain and application thereof

Abstract

The application discloses a strain of Sporosarcina and application thereof, and belongs to the technical field of microorganisms. The strain of Sporosarcina QD264 provided by the application is identified as a new species of Sporosarcina through phenotype characteristics, chemical classification characteristics and 16S rRNA gene phylogenetic analysis, 16S rRNA sequence comparison with similar species in EzbioCloud, and the results of whole genome ANI and dDDH calculation, and the numerical values are less than 95% and 70% respectively. Volatile components in the fermentation liquor of the strain QD264 can rapidly spread in the soil space, increase the contact opportunity with nematodes, thereby improving the nematode killing efficiency, and overcoming the disadvantages of traditional nematode control agents, i.e. the contact probability with nematodes is reduced due to the existence in fixed space, and the control effect is not obvious. In addition, the waste of traditional pesticide spraying in a large range can be avoided, the cost is reduced, and the environmental pollution is reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus occulta and its application. Background Technology

[0002] Plant root-knot nematodes are a type of sessile, obligate endoparasitic nematode. During infection, they obtain nutrients from the host plant by forming feeding sites, causing root swelling and the formation of root knots, thereby impairing the plant's ability to absorb water and nutrients. Affected plants typically exhibit weakened growth, wilting leaves, yellowing leaves, root deformities, reduced yield, and lower fruit quality. Root-knot nematodes have a very wide host range, covering vegetable crops such as cucumbers, tomatoes, peppers, and beans, as well as grain and oilseed crops such as corn, potatoes, and soybeans. These nematodes are distributed in most parts of southern my country and some warmer regions in the north, and occur on almost every landmass globally except Antarctica. It is estimated that parasitic nematodes cause annual economic losses of up to US$358.24 billion to 37 important crops worldwide, including approximately US$215.77 billion to 20 basic food crops and approximately US$142.47 billion to 17 important economic crops. In addition, the proportion of major crop-growing areas around the world affected by plant nematodes is generally high. For example, 90% of banana-growing areas worldwide are affected by plant nematode diseases.

[0003] In crop production, root-knot nematode disease often occurs in combination with other diseases. Root-knot nematodes play a pioneering role: they create numerous wounds in the roots, providing entry points for other pathogens and thus triggering complex infections. The synergistic effect of multiple pathogens often leads to more complex symptoms in the host, creating a cumulative damage effect where "1+1>2". For example, the pathogen of tobacco bacterial wilt—Ralstonia solanacearum (Ralstonia solanacearum)—is a prime example. Ralstonia solanacearum The interaction between the nematode and the southern root-knot nematode significantly exacerbates the disease. In recent years, with global warming, the expansion of protected cultivation areas, continuous cropping, and increasingly complex soil environments, root-related complex diseases have become commonplace, seriously restricting agricultural production in my country.

[0004] Bacillus spp. ( SporosarcinaBelonging to the domain Bacteria, phylum Firmicutes, class Bacillus, order Bacillusales, family Bacillusaceae, this genus of bacteria are aerobic or facultative anaerobic, Gram-positive rod-shaped bacteria, typically 0.5–1.2 μm wide and 1.3–4.0 μm long. Widely distributed in nature, they are commonly found in soil, water bodies, and plant rhizospheres, adaptable to various ecological conditions, and are among the microbial groups with the highest urease activity. Since the 20th century, *Bacillus* has been proven to be a highly efficient biocementing microorganism, used for microbially induced calcium carbonate (MICP) precipitation in soil reinforcement, building self-repair, wind erosion control, and restoration of stone artifacts. These bacteria possess a unique spore-forming ability, capable of entering a dormant state under extreme conditions such as nutrient deficiency, ultraviolet radiation, dryness, high temperature, freezing, or chemical disinfectants, and resuming activity once the environment becomes suitable. This characteristic allows them to maintain their metabolic potential even in harsh habitats.

[0005] Bacillus subtilis possesses multiple mechanisms, including antagonistic effects, promotion of plant growth, production of various enzyme types, and induction of plant defense responses, playing a crucial role in tolerance to biotic and abiotic stresses. Besides directly acting on pathogens, it can also induce systemic resistance and promote plant growth, thereby enhancing the plant's disease resistance.

[0006] The present invention aims to provide a new strain of *Dystrophus sporeans* with excellent nematicidal effects. Summary of the Invention

[0007] The first objective of this invention is to provide a strain of *Spore-forming Micrococcus* (Spore-forming Micrococcus). Sporosarcina The second object of the present invention is to provide the application of the Bacillus sp. strain QD264.

[0008] The first objective of this invention is achieved as follows: a Bacillus subtilis strain QD264, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with the number GDMCC No: 68269, on May 20, 2026, at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0009] The second objective of this invention is achieved by applying the Bacillus spores strain QD264 in the control of plant root-knot nematodes.

[0010] The beneficial effects of this invention are as follows: 1. The Bacillus spores strain QD264 provided by this invention was isolated from forest soil in Gaoligong Mountain, Longling County, Baoshan City, Yunnan Province. Phenotypic characteristics, chemical taxonomic characteristics, and phylogenetic analysis of the 16S rRNA gene showed a similarity of less than 99.72% with similar species in EzbioCloud. Further analysis using whole-genome ANI and dDDH showed similarities of less than 95% and 70%, respectively, thus identifying it as a new species of Bacillus spores.

[0011] 2. The fermentation broth of Bacillus spp. strain QD264 provided by this invention contains both non-volatile and volatile nematicidal components. The volatile components can rapidly diffuse and distribute within the soil space, increasing the chance of contact with nematodes and thus improving nematicidal efficiency. This characteristic overcomes the drawback of traditional liquid, solid, oil-based, and live bacterial nematicides, which have reduced contact with nematodes due to their fixed spatial location, resulting in less effective control. Furthermore, it avoids the waste of large-scale spraying of traditional pesticides, reduces pesticide usage, lowers costs, and reduces environmental pollution.

[0012] 3. The fermentation broth of the new Bacillus spp. strain QD264 provided by this invention has a mortality rate of over 90% against root-knot nematodes, and the mortality rate of the fermentation broth diluted 50 times against root-knot nematodes exceeds 83%, with an action time of no more than 12 hours. It rapidly kills nematodes in the soil through diffusion in the soil space, and has good application prospects in the control of plant root-knot nematodes.

[0013] 4. The volatile nematicidal component provided by this invention originates from a novel strain of Bacillus spp., QD264. This strain can survive in the soil environment, thereby continuously producing volatile active ingredients within the soil. Unlike other nematicides that use non-active cells (whose activity diminishes over time), strain QD264 of this invention can act as a live-cell agent, providing long-lasting control. Attached Figure Description

[0014] Figure 1 The morphological characteristics of the Bacillus subtilis strain QD264 of this invention are shown in Figure A, where A is a colony morphology diagram of strain QD264 on ISP1 medium; and B is a Gram-stained microscopic morphology diagram of strain QD264. Figure 2 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of the Bacillus spores strain QD264 of the present invention; Figure 3The image shows the microscopic morphology of the volatile components in the fermentation broth of Bacillus spp. strain QD264 in Experimental Example 1 of this invention, as well as the lethality of Southern Root-knot Nematodes. In this image, D represents the microscopic morphology of Southern Root-knot Nematodes in the control group on blank LB medium, and E represents the microscopic morphology of Southern Root-knot Nematodes in the experimental group (nematode death).

[0015] Figure 4 The images show the microscopic morphology of nematodes killed by direct action of the fermentation broth of Bacillus cereus strain QD264 of this invention; where A represents the morphology of naturally dead nematodes in the negative control group (M9 buffer), and B represents the intact morphology of nematodes killed by the fermentation broth. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0017] This invention relates to a strain of *Bacillus sporeans*, wherein the strain is *Bacillus sporeans* (… Sporosarcina strain QD264 (sp.) ， It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with the number GDMCC No: 68269, the deposit date is May 20, 2026, and the deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0018] The 16S rRNA gene sequence of the *Bacillus occulta* strain is shown in SEQ ID NO. 1.

[0019] The present invention also provides a fungal agent based on the aforementioned Bacillus occulta strain.

[0020] The microbial agent composition includes at least one of the live cells of the *Bacillus occulta* strain or its fermentation broth.

[0021] The present invention further provides a fermentation broth, the preparation method of which is carried out according to the following steps: 1) After activating the Bacillus spores, the strain was inoculated into a seed culture medium and cultured on a shaker at 36℃±1℃ at a speed of 130~160r / min for 24~36h to obtain the seed liquid; 2) The fermentation seed liquid obtained in step 1) is inoculated into the fermentation medium and cultured on a shaker at 36℃±1℃ at a speed of 140~180 r / min for 72~160 hours. The fermentation product is centrifuged or filtered to remove the cells, and the supernatant is collected to obtain the fermentation broth.

[0022] The seed culture medium and fermentation culture medium mentioned in step 2) are both LB liquid culture medium.

[0023] The present invention further provides a method for controlling root-knot nematodes, which involves bringing the root-knot nematodes into contact with the volatile components released from the fermentation liquid.

[0024] The fermentation liquid is applied to the rhizosphere soil of the crop, covered with soil and sealed with mulch film, and the volatile components in the fermentation liquid are used to fumigate and kill root-knot nematodes in the root system.

[0025] The present invention further provides the application of the strain, the inoculant, or the fermentation broth in the control of plant root-knot nematodes.

[0026] The root-knot nematodes include Southern root-knot nematodes ( Meloidogyne incognita Javan root-knot nematodes ( Meloidogyne javanica ) or Northern root-knot nematodes ( Meloidogyne hapla ).

[0027] Example 1: Isolation, Purification, and Identification of Strains 1. Strains Isolation and Purification 10g of soil sample collected from the Gaoligong Mountain forest in Longling County, Baoshan City, Yunnan Province was placed in an Erlenmeyer flask containing 90 mL of sterile water and shaken at 180 rpm for 30 min to prepare 10 -1 Soil suspension. Take 10 -1 Add 1 mL of suspension to 9 mL of sterile water, and then serially dilute 10-fold to 10⁻⁶. -5 Take 10 samples respectively. -4 10 -5 50 μL of diluted bacterial suspension was spread onto LB solid medium (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15–20 g agar, diluted to 1 L with water; pH adjusted to 7.0–7.2; autoclaved at 121°C for 20 min) plates. The medium was spread evenly using a sterile spreader, sealed with Parafilm, and incubated upside down in a 37°C incubator for 3–4 days.

[0028] After colonies have grown, single colonies are selected based on their morphology, size, color, and other characteristics and transferred to fresh LB solid medium plates. The streak purification is repeated 2-3 times to obtain pure culture strains, which are originally numbered QD264 in the "Southwest Germplasm Resources-Microbial Bank" of Yunnan University (managed and operated by the State Key Laboratory of Biological Resources Conservation and Utilization of Yunnan University).

[0029] 2. Phenotypic characteristics of the strain 2.1 Strain morphology and culture characteristics Microscopic morphological observation and culture characteristics analysis of the purified strain QD264 showed that strain QD264 is a Gram-positive rod-shaped bacterium. Figure 1(B) can produce spores and is motile. It can grow normally on various conventional bacterial culture media such as ISP1, ISP2, TSA, LB, R2A, and NA, with the best growth performance in ISP1 medium, where the colonies are round, moist, and glossy. Figure 1 (A). Aeration experiments confirmed that strain QD264 is a strictly aerobic microorganism.

[0030] 2.2 Physiological and biochemical characteristics of the strain The growth environment adaptability and substrate degradation ability of strain QD264 were determined to clarify its basic physiological and biochemical characteristics.

[0031] The results showed that the growth temperature range of strain QD264 was 15~40℃, with the optimal growth temperature being 35~40℃; the NaCl tolerance concentration range was 0~8% (w / v), with the best growth under the condition of no exogenous NaCl (0%); and the growth pH tolerance range was 6.0~9.0.

[0032] 3. Chemical classification characteristics of bacterial strains The cellular characteristic components of strain QD264 were detected and analyzed to clarify its chemical taxonomic indicators.

[0033] The results showed that the main respiratory quinone of strain QD264 was MK-7; the main polar lipid components included phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine; and the main cellular fatty acid components were anteiso-C15:0 and iso-C15:0.

[0034] 4. Molecular identification of the 16S rRNA gene of the strain Total genomic DNA was extracted from strain QD264 using a bacterial genomic DNA extraction kit. PCR amplification was performed using universal primers 27F and 1492R for the bacterial 16S rRNA gene to obtain the target gene fragment. The amplified product was purified and sequenced to obtain the complete 16S rRNA gene sequence of strain QD264, as shown in SEQ ID NO:1.

[0035] BLAST homology comparison of SEQ ID NO:1 sequence with NCBI GenBank database showed that strain QD264 is similar to *Saccharomyces cerevisiae*. Sporosarcina luteola Y1 T The 16S rRNA gene sequence similarity was 99.72%. Further calculations using whole-genome ANI and dDDH showed similarities below 95% and 70%, respectively. This, combined with the phenotypic characteristics, chemotaxonomic features, and phylogenetic analysis of the 16S rRNA gene of strain QD264, further supports this finding. Figure 2 Strain QD264 was identified as a new species of the genus *Bacillus*. Sporosarcina sp.)

[0036] Example 2: Preparation of fermentation broth from strain QD264 1. Seed culture: The pure culture QD264 obtained from the plate purification in Example 1 was transferred to multiple 250 mL Erlenmeyer flasks containing 100 mL of LB liquid medium (formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0-7.2, autoclaved at 121℃ for 20 min). The Erlenmeyer flasks were then placed on a reciprocating shaking shaker and cultured at 36℃±1℃ and 145 r / min for 20 h to obtain the fermentation seed culture.

[0037] 2. Fermentation Culture: The prepared fermentation seed culture was inoculated into LB liquid medium at a rate of 5% (v / v). Erlenmeyer flasks were used, with a volume of 150 mL of culture, and the culture was incubated at 37°C and 150 rpm for 72 hours with shaking. The fermentation culture was then centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was collected, filtered through a 0.22 μm microporous membrane, and the filtrate was collected to obtain the cell-free fermentation broth.

[0038] Experimental Example 1: Detection of nematicidal activity of fermentation broth from strain QD264 The fermentation broth prepared in Example 2 was subjected to a nematicide test for its volatile components. 1. Preparation of M9 buffer solution Weigh out 6.0 g of anhydrous disodium hydrogen phosphate, 3.0 g of potassium dihydrogen phosphate, 5.0 g of sodium chloride, and 0.25 g of magnesium sulfate heptahydrate. Dissolve in 800 ml of distilled water and bring the volume to 1 L. Autoclave at 121°C for 30 min, then cool before use. 2. Test methods Volatile nematicidal effects were tested on three root-knot nematodes using double-compartment petri dishes (divided plates, 9 cm in diameter). The root-knot nematodes were physically isolated from the fermentation broth and came into contact only through volatile components.

[0039] In one compartment of a double-compartment culture dish, inject 0.5 mL of sterile M9 buffer and add 100–150 second-instar larvae of *Symplocos sulphureus*, *Javanese nematode*, or *Northern nematode* (each nematode was tested separately, not mixed). Add 50 μL of the fermentation broth prepared in Example 2 to the center of the other compartment. Immediately seal the plate with Parafilm and incubate at 22°C. Observe the survival of root-knot nematodes in the nematode compartment under a stereomicroscope every 30 minutes. If the nematodes are found to be stiff and unresponsive to needle stimulation, they are considered dead. Observe continuously for 24 hours. Set up a blank LB medium group and a negative control group. Calculate the nematode mortality rate and corrected mortality rate at 8 h, 16 h, 20 h, and 24 h according to Zhang et al. (2016). The experiment was repeated 5 times.

[0040] Mortality rate (%) = (Number of dead nematodes / Total number of nematodes used in the experiment) × 100 Nematode corrected mortality rate (%) = (Nematode mortality rate in experimental group - Nematode mortality rate in control group on blank LB medium) / (1 - Nematode mortality rate in control group on blank LB medium) × 100.

[0041] Negative control group: 0.5 mL of sterile M9 buffer and 100-150 root-knot nematodes were added to the nematode compartment, and 50 μL of sterile M9 buffer (instead of fermentation broth) was added to the center of another compartment.

[0042] Blank LB medium control group: Add 0.5 mL of sterile M9 buffer and 100-150 root-knot nematodes to the nematode compartment, and add 50 μL of sterile LB liquid medium (instead of fermentation broth) of uninoculated strain QD264 to the center of another compartment. All other procedures are the same as those for the experimental group.

[0043] Table 1. Results of lethality tests on nematodes by volatile components of the fermentation broth prepared in Example 2.

[0044] Note: The data in the table represent the lowest mortality rates of the three root-knot nematodes (Southern Root-Knot Nematode, Javan Root-Knot Nematode, and Northern Root-Knot Nematode) at the same treatment time after the volatile components were applied to them respectively.

[0045] The results in Table 1 show that the volatile components in the fermentation broth prepared in Example 2 have a 16-hour lethality of over 89% against Southern Root-Knot Nematode, Javan Root-Knot Nematode, and Northern Root-Knot Nematode.

[0046] Experimental Example 2: Lethality test of the fermentation broth prepared in Example 2 against nematodes. 1. Lethality test of the fermentation broth prepared in Example 2 on nematodes. Experimental Method: 700 µL of the fermentation broth prepared in Example 2 was added to a cell culture dish, followed by a 20 µL suspension of root-knot nematodes (80–100 second-instar Southern root-knot nematodes, Javanese root-knot nematodes, or Northern root-knot nematodes; each nematode was tested separately and not mixed). The mixture was then placed in a 22°C incubator. The number of live and dead nematodes was recorded under a stereomicroscope every 30 minutes. Nematodes were considered dead if they exhibited stiffness and no response to needle stimulation. A blank LB medium group (using 700 µL of sterile M9 buffer instead of the fermentation broth) and a negative control group (using 700 µL of sterile LB liquid medium (blank LB medium) instead of the fermentation broth) were set up. Nematode mortality and corrected mortality were calculated according to Zhang et al. (2016). The experiment was repeated 5 times.

[0047] Mortality rate (%) = (Number of dead nematodes / Total number of nematodes used in the experiment) × 100 Nematode corrected mortality rate (%) = (Nematode mortality rate in experimental group - Nematode mortality rate in control group on blank LB medium) / (1 - Nematode mortality rate in control group on blank LB medium) × 100.

[0048] The results are shown in Table 2.

[0049] Table 2. Lethality test results of the fermentation broth stock solution (cell-free) prepared in Example 2 against plant root-knot nematodes.

[0050] Note: The data in the table are the lowest values ​​of the three root-knot nematodes (Southern Root-knot Nematode, Javan Root-knot Nematode, and Northern Root-knot Nematode) after the fermentation broth prepared in Example 2 was applied to them at the same treatment time.

[0051] As shown in Table 2, the fermentation broth prepared in Example 2 had a 16-hour lethality of over 95% against Southern Root-knot Nematode, Javan Root-knot Nematode, and Northern Root-knot Nematode.

[0052] 2. Lethality test of nematodes after the fermentation broth prepared in Example 2 was diluted 50 times. The fermentation broth (cell-free) prepared in Example 2 was diluted 50 times and then subjected to a nematode lethality test according to the above method. The test results are shown in Table 3.

[0053] Table 3. Lethality test results of the fermentation broth (cell-free) diluted solution (×50) prepared in Example 2 against plant root-knot nematodes.

[0054] Note: The data in the table are the lowest values ​​of the mortality rates of the three root-knot nematodes after the fermentation broth (cell-free) diluted solution (×50) was applied to Southern root-knot nematode, Javan root-knot nematode, and Northern root-knot nematode.

[0055] As shown in Table 3, the original fermentation broth prepared in Example 2, after being diluted 50 times, had a mortality rate of over 85% on second-instar larvae of Southern Root-knot Nematode, Javan Root-knot Nematode, and Northern Root-knot Nematode after 16 hours of exposure.

Claims

1. A strain of Bacillus subtilis, characterized in that, The strain is *Bacillus occulta* (Bacillus). Sporosarcina strain QD264 (sp.) ， It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with the number GDMCC No: 68269, the deposit date is May 20, 2026, and the deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

2. The *Saccharomyces occulta* strain according to claim 1, characterized in that, The 16S rRNA gene sequence of the *Bacillus occulta* strain is shown in SEQ ID NO.

1.

3. A fungal agent prepared based on the *Sclerotium occulta* strain of claim 1.

4. The inoculum preparation made from the *Sclerotium occulta* strain according to claim 3, characterized in that, The microbial agent composition includes at least one of the live cells of the *Bacillus occulta* strain or its fermentation broth.

5. A fermentation broth, characterized in that, The fermentation broth is prepared according to the following steps: 1) The *Bacillus spp.* strain described in claim 1 is activated and inoculated into a seed culture medium, and cultured on a shaker at 36℃±1℃ at a speed of 130~160r / min for 24~36h to obtain a seed liquid; 2) The fermentation seed liquid obtained in step 1) is inoculated into the fermentation medium and cultured on a shaker at 36℃±1℃ at a speed of 140~180r / min for 72~160 hours. The fermentation product is centrifuged or filtered to remove the cells, and the supernatant is collected to obtain the fermentation broth.

6. The fermentation broth according to claim 5, characterized in that, The seed culture medium and fermentation culture medium mentioned in step 2) are both LB liquid culture medium.

7. A method for controlling root-knot nematodes, characterized in that, The root-knot nematodes are brought into contact with the volatile components released from the fermentation broth of claim 5.

8. The method for controlling root-knot nematodes according to claim 7, characterized in that, The fermentation liquid is applied to the rhizosphere soil of the crop, covered with soil and sealed with mulch film, and the volatile components in the fermentation liquid are used to fumigate and kill root-knot nematodes in the root system.

9. The application of the strain of claim 1, the inoculum of claim 3, or the fermentation broth of claim 5 in the control of plant root-knot nematodes.

10. The application according to claim 9, characterized in that, The root-knot nematodes include Southern root-knot nematodes ( Meloidogyne incognita Javan root-knot nematodes ( Meloidogyne javanica ) or Northern root-knot nematodes ( Meloidogyne hapla ).

Images