A fungicide for controlling stem rot nematodes, its preparation and application

Abstract

This invention discloses a microbial agent for controlling stem rot nematodes, its preparation, and its application. The agent comprises *Bacillus mogarfianus* XCSF-04 and *Bacillus licheniformis* J117 or their fermentation products. The agent can be prepared by fermenting the two strains separately and then mixing them, or by co-fermentation. This microbial agent exhibits both rapid and high efficacy against stem rot nematodes, achieving a 24-hour corrected mortality rate of 85.00% and a 48-hour corrected mortality rate of 97.53%. Furthermore, its control effect in the field is significantly superior to that of the chemical pesticide thiazophos. This invention reduces the use of chemical pesticides through the combination of strains, avoiding pesticide resistance and environmental pollution, and is of great significance for ensuring the safe production of crops such as potatoes.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a fungicide for controlling stem rot nematodes, its preparation, and its application. Background Technology

[0002] Potato (Solanum tuberosum L.), as one of the world's most important food crops, is characterized by high yield, strong adaptability, and rich nutrition, serving as a major food source for residents in many countries and regions. Potato stem rot nematode disease is a highly destructive disease caused by the stem nematode (Ditylenchus destructor Thorne) during potato storage. This disease spreads rapidly and is difficult to control, posing a serious threat to the healthy development of my country's potato industry. Besides potatoes, the stem nematode can also infest various other crops, such as sweet potatoes, garlic, angelica, ginseng, and mint.

[0003] Currently, the control of stem rot nematodes mainly relies on chemical agents, such as thiazophos. However, the long-term and large-scale use of chemical agents not only induces nematodes to develop resistance, reducing control effectiveness, but also pollutes the soil environment, disrupts the balance of the ecosystem, and leads to harmful residues in agricultural products, posing a threat to human health. In addition, the control of stem rot nematodes also includes biological control, which mainly uses beneficial microorganisms or their metabolites to inhibit or kill stem rot nematodes. Compared with chemical agents, microorganisms can colonize in the soil and continuously exert their effects, achieving long-term control of nematode populations, reducing disease occurrence, and without causing environmental pollution and agricultural residue problems like chemical agents.

[0004] Currently, microbial control methods, represented by Bacillus, are highly anticipated, but the limitations of existing technologies in practical applications restrict their widespread adoption. For example, as described in patent CN117603878A, even with a viable count as high as 3.32 × 10⁻⁶, a type of Bacillus belychnophorus... 10 Under conditions of CFU / g, the incidence rate in field trials still exceeded 29%, and the disease index exceeded 15%, indicating limited control efficacy. As described in patent document CN120787974A, its compound microbial agent, at an application concentration as high as 1%, showed an inhibition rate of only 75.6% against stem nematodes after 48 hours, reaching 92.6% only after 72 hours, indicating severely insufficient rapid-acting effect. For example, the Bacillus licheniformis agent used by Liu Qiang et al. (2023) in the non-patent literature "Application Effect of Biological Agents in the Control of Sweet Potato Stem Nematode Disease" had a field application rate as high as 1.5 kg / 667m². 2 However, its insect control efficacy was only 24.95%, which reflects the serious insufficiency of the strain's own nematicidal potency.

[0005] As can be seen from the above, existing technologies lack a microbial solution capable of achieving efficient, rapid, and stable control of stem rot nematodes. Therefore, developing a highly specialized microbial strain with high nematicidal activity and suitability for complex field environments is of great significance in filling this technological gap. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, the present invention aims to provide a novel fungal agent for controlling stem nematodes that is both highly effective and fast-acting, as well as its preparation method and application.

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a microbial agent for controlling stem nematodes, comprising microorganisms and / or fermentation products;

[0009] The microorganisms include Bacillus mogavus XCSF-04 and Bacillus licheniformis J117;

[0010] Among them, the Bacillus mogarbhae XCSF-04 was identified as Bacillus mogarbhae. Bacillus mojavensis The *Bacillus licheniformis* J117 was deposited on October 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36379. The *Bacillus licheniformis* J117 described therein was identified as *Bacillus licheniformis*. Bacillus licheniformis It was deposited on October 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36435.

[0011] Preferably, the fermentation product is a mixture of fermentation broth obtained by fermenting Bacillus mogavus XCSF-04 and Bacillus licheniformis J117 separately and then mixing the resulting fermentation broths.

[0012] Alternatively, the fermentation product is a mixture of fermentation broth obtained by mixed fermentation of Bacillus mogavus XCSF-04 and Bacillus licheniformis J117.

[0013] Preferably, the bacterial concentration ratio of Bacillus mogavus XCSF-04 to Bacillus licheniformis J117 is (0.8-1.2):(0.8-1.2).

[0014] Preferably, the bacterial concentration ratio of Bacillus mogavus XCSF-04 to Bacillus licheniformis J117 is 1:1.

[0015] Preferably, when the fermentation product is a mixture of fermentation broth obtained by mixing Bacillus mogavus XCSF-04 and Bacillus licheniformis J117 after fermentation, the volume ratio of the fermentation broth of Bacillus mogavus XCSF-04 to the fermentation broth of Bacillus licheniformis J117 is (1-3):(1-3).

[0016] Preferably, the fermentation process includes inoculating the strain into a liquid culture medium for fermentation, centrifuging, and then collecting the supernatant to obtain the fermentation broth.

[0017] Secondly, the present invention provides a product for controlling stem nematodes, comprising the above-mentioned stem nematode control fungal agent and stem nematode control chemical agent.

[0018] Preferably, the chemical agent for controlling stem nematodes is thiazophos.

[0019] Preferably, the total volume ratio of the fermentation product of Bacillus mogavus XCSF-04 and the fermentation product of Bacillus licheniformis J117 to the volume of the thiazophosphonate is (1-3):(7-9).

[0020] Thirdly, the present invention provides an application of the above-mentioned fungicide for controlling stem rot nematodes, the application comprising:

[0021] A) The application of the aforementioned fungicide for controlling stem rot nematodes in the control of stem rot nematodes;

[0022] B) The application of the aforementioned stem rot nematode control agent in the preparation of products for controlling stem rot nematodes.

[0023] The present invention has the following beneficial effects:

[0024] The Bacillus licheniformis J117 and Bacillus mogarbhae XCSF-04 provided by this invention have excellent compatibility. No inhibition zone appears when they are co-cultured, and their growth does not inhibit each other. When combined, they can achieve synergistic prevention and control effects.

[0025] In terms of rapid efficacy, the 24-hour corrected mortality rate of stem nematodes after fermentation of the two strains individually and then mixed in a 1:1 ratio was 85.00%, significantly higher than that of the single strains (e.g., 85.54% for J117 alone and 81.89% for XCSF-04 alone). Regarding high efficacy, the 48-hour corrected mortality rate reached 97.53% (after fermentation and mixing), significantly higher than that of the single strains (90.61% for J117 and 90.38% for XCSF-04) and the mixed fermentation treatment (90.64%). Furthermore, the 48-hour corrected mortality rate was highest at the 1:1 ratio (93.61%).

[0026] Under the optimal spraying method, the compound microbial agent (XCSF-04+J117) treatment resulted in an incidence rate of 11.68% for stem rot nematodes, a disease index of 4.59, and a control efficacy of 77.53%, significantly higher than that of single microbial agents (XCSF-04: 57.55%, J117: 69.46%) and the chemical agent thiazophos (60.40%). Seed treatment showed similar efficacy to chemical agents. Specifically, the compound microbial agent seed treatment achieved a control efficacy of 57.76%, not significantly different from thiazophos (60.40%), and the incidence rate (16.19%) was significantly lower than that of single microbial agents.

[0027] In summary, this invention, through the formulation of biological agents, can reduce the amount of chemical agents (such as thiazophos), avoiding problems such as drug resistance, soil pollution, and agricultural product residues caused by long-term use of chemical agents, thus meeting the requirements of green agriculture development. This invention achieves highly efficient and rapid control of stem rot nematodes through the formulation of bacterial strains, demonstrating excellent field application results and high environmental safety, and is of great significance for ensuring the safe production of crops such as potatoes. Attached Figure Description

[0028] Figure 1 This is a diagram showing the results of the strain compatibility test in Example 1 of the present invention;

[0029] Figure 2 The results of the compatibility test for this invention are shown, where a is the blank control (CK) of strain XCSF-04-thiazophosphine; b is the concentration of strain XCSF-04-thiazophosphine at 25 μg·mL. -1 Treatment group; c was strain XCSF-04 with a thiazophosphonate concentration of 50 μg·mL. -1 Treatment group; d represents strain XCSF-04 with a thiazophosphonate concentration of 100 μg·mL. -1 Treatment group; e is the blank control (CK) of strain J117-thiazophosphine; f is the concentration of strain J117-thiazophosphine at 25 μg·mL. -1 Treatment group; g represents strain J117 with a thiazophosphonate concentration of 50 μg·mL. -1Treatment group; h represents strain J117 with a thiazophosphonate concentration of 100 μg·mL. -1 Processing group;

[0030] Figure 3 This invention provides a grading standard for potato stem rot nematode disease, wherein diseased potatoes a and b are grade 0; diseased potatoes c and d are grade 1; diseased potatoes e and f are grade 2; diseased potatoes g and h are grade 3; and diseased potatoes i and j are grade 4. Detailed Implementation

[0031] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0032] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0033] Example 1

[0034] 1. Strains compatibility test

[0035] In this embodiment, the test chemical reagent was 92% thiazophosphonate technical grade; the test strains were *Bacillus mogarf.* XCSF-04 and *Bacillus licheniformis* J117. *Bacillus mogarf.* XCSF-04 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 28, 2025, with accession number CGMCC No. 36379; *Bacillus licheniformis* J117 was deposited at the CGMCC on October 31, 2025, with accession number CGMCC No. 36435.

[0036] The compatibility of the bacterial strains was tested using the filter paper disc method. First, the fermentation broth was prepared. The fermentation broth was prepared by picking a single colony and inoculating it into a 250 mL Erlenmeyer flask containing 100 mL of sterile NB liquid medium. The flask was then placed in a shaker at 26±1℃ and fermented at 180 r / min for 72 h. The fermentation broth was then centrifuged at 6000 r / min for 5 min, and the supernatant was collected and stored at 4℃ for later use.

[0037] Spread 150 μL of J117 fermentation broth onto NA medium. Place double-layered sterile filter paper discs (1 cm diameter × 8 layers) symmetrically in the center of the medium. Add 150 μL of XCSF-04 bacterial suspension to each sterile filter paper disc. Perform three replicates. After incubation at 28°C in the dark for 48 hours, observe for the presence or absence of inhibition zones. The presence of inhibition zones indicates that the two strains cannot co-grow, while the absence of inhibition zones indicates that they can co-grow.

[0038] The test results are as follows Figure 1 As shown, no inhibition zone was produced when strain J117 and XCSF-04 were co-cultured, indicating that the two strains do not inhibit each other's growth, and biocontrol bacteria co-culture experiments can be carried out.

[0039] 2. Research on the compounding methods of biocontrol bacteria

[0040] The experiment was set up with five treatment groups:

[0041] (1) Water as control; (2) J117 fermentation broth stock solution; (3) XCSF-04 fermentation broth stock solution; (4) Fermentation treatment after mixing: J117 and XCSF-04 strains were mixed in equal mass ratio and inoculated into NB medium at an inoculation rate of 1%. The mixture was shaken and cultured at 150 rpm and 28℃ for 24 hours to obtain a mixed fermentation broth; (5) Mixing treatment after fermentation: The fermentation broth stock solutions obtained after 24 h of fermentation of the two strains were mixed in equal mass ratio to obtain a fermentation mixture.

[0042] The fermentation process described above was as follows: the strain was purified and cultured on NA medium for 48 h. After confirming the absence of contamination, a single colony was picked and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterile NB liquid medium. The flask was then placed on a shaker at 26±1℃ and fermented at 180 r / min for 72 h. The fermentation broth was then centrifuged at 6000 r / min for 5 min, and the supernatant was collected to obtain the fermentation broth.

[0043] A suspension of potato stem nematodes (20 μL per tube, containing approximately 150 nematodes) was added to 1.5 mL of fermentation broth for each treatment strain. After vortexing to mix, the mixture was incubated at 25°C in the dark for 24 hours. Each treatment was replicated in triplicate. After incubation at 25°C in the dark for 24 hours, the total number of nematodes and the mortality rate were determined by microscopy. The experimental results are shown in Table 1.

[0044] Table 1. Nematode mortality results

[0045]

[0046] Table 1 shows that after 24 and 48 hours of treatment with stem nematodes, the combined treatment with fermentation of both strains exhibited the highest inhibitory effect on stem nematodes, with corrected mortality rates of 89.91% and 97.53%, respectively. The corrected mortality rates at 24 and 48 hours were significantly higher than those when the two strains were applied alone. The inhibition rate of the combined strains after fermentation was roughly the same as the corrected mortality rate when the two strains were applied alone. Therefore, the method of fermenting the strains individually followed by mixing was chosen for subsequent strain combination experiments.

[0047] 3. Screening of biocontrol bacteria compounding methods and ratios

[0048] Eight treatment groups were established, with fermentation broths from two biocontrol bacteria fermented individually mixed at ratios of 1:1, 1:2, 1:3, 2:3, 3:2, 2:1, and 3:1, with water as a control. Each treatment was replicated three times, and the fermentation process was the same as above. After 24 hours of cultivation, the total number and mortality of potato stem rot nematodes were observed and recorded to determine the inhibitory effect of different biocontrol bacteria ratios on potato stem rot nematodes. The inhibitory effects of different biocontrol bacteria ratios on potato stem rot nematodes are shown in Table 2.

[0049] Table 2. Inhibitory effect of different compound ratios on potato stem rot nematode.

[0050]

[0051] Table 2 shows that the seven groups of biocontrol bacteria, treated with different compound ratios for 24 h and 48 h, exhibited varying nematode-killing effects against *S. rotten stem nematodes*, but the corrected mortality rates were significantly higher than the control group. Among them, the nematode-killing effect was strongest when the biocontrol bacteria XCSF-04 and J117 were mixed in a 1:1 ratio, with corrected mortality rates of 85.00% and 93.61% at 24 h and 48 h, respectively, showing significant differences from other treatment groups and confirming a significant synergistic effect between the two strains. The nematode-killing rate was second highest at a 3:1 ratio, reaching 75.22% and 80.46%, respectively. When the two strains were mixed in a 3:2 ratio, the inhibitory effect on *S. rotten stem nematodes* was poor, with corrected mortality rates of only 50.43% and 57.50% at 24 h and 48 h, respectively. Therefore, when strains XCSF-04 and J117 are used together, it is best to ferment them separately and then mix them in a 1:1 ratio before application. At this ratio, the contact killing effect on stem nematodes is most significant.

[0052] 4. Screening of the ratio of chemical agents to biocontrol agents for reduced application

[0053] 1) Compatibility test between biocontrol bacteria and chemical agents

[0054] Bacillus XCSF-04 and J117 bacterial suspensions were fermented separately (using the same method as above), and then mixed in a 1:1 mass ratio to obtain a mixed bacterial suspension. 200 μL of the mixed bacterial suspension was evenly spread onto a prepared NA solid medium plate. After the suspension was absorbed by the plate, a hole was punched in the center of the plate, and then 50 μL of prepared thiazophosphonate solutions of different concentrations were inoculated. The blank control group did not undergo any hole punching or drug addition treatment.

[0055] The preparation method of thiazophosphine stock solution is as follows: Mix 0.2 mL of 92% thiazophosphine crude oil with 0.8 mL of Tween 80 and 9 mL of dimethyl sulfoxide (DMSO), then dilute 10 times with sterile water to prepare a final concentration of 1918.28 μg·mL. -1 The thiazophosphatidylcholine stock solution was prepared. The thiazophosphatidylcholine stock solution was serially diluted with sterile water. 50 μL of the diluted solution was added to the prepared wells, resulting in concentrations of thiazophosphatidylcholine of 0, 25, 50, and 100 μg / mL in the wells. -1 Each treatment was repeated three times. Once the control colonies had covered the entire plate, observe whether an inhibition zone appeared around the wells. If no inhibition zone appeared, it indicates that the chemical agent had no inhibitory effect on biocontrol bacteria and could be used in combination.

[0056] The results of the compatibility test are as follows Figure 2 As shown in the figure. Compatibility test results showed that after perforating the plates and adding the chemical agent, no inhibition zones appeared on the plate surface, indicating that the thiazophos concentration was below 100 μg·mL. -1 At this concentration, the growth of the biocontrol bacteria was not significantly different from that of the control, and it had no inhibitory effect on either biocontrol bacteria. Therefore, it can be concluded that thiazophos can be used in combination with biocontrol bacteria XCSF-04 and J117 at this concentration.

[0057] 2) Screening test of the ratio of reduced chemical agent application to biocontrol bacteria

[0058] Bacillus suspensions XCSF-04 and J117 were fermented separately, then mixed in a 1:1 ratio, and subsequently combined with a 10-fold dilution of 92% thiazophos in a specific proportion. This mixture was used to conduct an indoor contact nematode test against stem rot nematodes, aiming to screen for the optimal combination of biological control bacteria and chemical agents. The experiment consisted of 5 treatment groups, with 3 replicates for each group. The fermentation method and the thiazophos preparation method were the same as described above.

[0059] Twenty-four hours after treatment, the total number of stem rot nematodes and the number of deaths were observed and recorded, and the corrected mortality rate was calculated. Simultaneously, the optimal ratio of reduced chemical pesticide application to biological control bacteria was determined to inhibit potato stem rot nematodes. The five treatment groups are as follows:

[0060] CK group: Water was used as a control.

[0061] Group A: The treatment group with a 10% reduction in thiazophos and the addition of 10% compound microbial agent (XCSF-04+J117);

[0062] Group B: The treatment group with a 20% reduction in thiazophos and the addition of 20% compound microbial agent (XCSF-04+J117);

[0063] Group C: The treatment group with a 30% reduction in thiazophos and the addition of 30% compound microbial agent (XCSF-04+J117);

[0064] Group D: Solution treatment group of 92% thiazophosphine diluted 10 times;

[0065] Group E: Treatment group with compound microbial agent (XCSF-04+J117).

[0066] The dosage of thiazophos combined with biological control agents in each treatment group is shown in Table 3. The lethality of chemical agents combined with biological control agents on stem rot nematodes is shown in Table 4.

[0067] Table 3. Dosage of thiazophos with reduced dosage combined with biological control agents in each treatment group.

[0068]

[0069] Table 4. Lethal effect of reduced chemical pesticide dosage combined with biocontrol agents on stem rot nematodes.

[0070]

[0071] Table 4 shows that among the treatments involving reduced chemical pesticide dosage combined with biocontrol agents, the treatment with 30% reduced thiazophos combined with 30% biocontrol agent (C) resulted in the highest corrected mortality rates for stem nematodes at 24h and 48h, at 75.70% and 92.58%, respectively. Compared with the thiazophos single-agent treatment (D), the corrected mortality rate at 48h increased by 0.59%, indicating a certain synergistic effect. Compared with the compound biocontrol agent treatment (E), the corrected mortality rates at 24h and 48h decreased by only 9.83% and 1.49%, respectively, and the differences were not significant. This indicates that the lethal effect of 30% reduced thiazophos combined with 30% biocontrol agent on stem nematodes is comparable to that of applying only chemical pesticides or the compound biocontrol agent, without a significant decreasing trend, and can effectively reduce the amount of chemical pesticides used. Compared to the treatment with a 30% reduction in thiazophos combined with 10% biocontrol agent (C), the corrected mortality rates of treatments with a 10% reduction in thiazophos combined with 10% biocontrol agent (A) and a 20% reduction in thiazophos combined with 20% biocontrol agent (B) were significantly lower. Among these, the treatment with a 10% reduction in thiazophos combined with 10% biocontrol agent (A) showed the worst lethality against stem nematodes, with corrected mortality rates of 52.72% and 74.49% at 24 and 48 hours, respectively. Compared to the thiazophos monotherapy treatment (D), the control efficacy was reduced by 36.27% and 17.5%, respectively; and compared to the combined biocontrol agent treatment (E), the reductions were 32.81% and 19.58%, respectively.

[0072] 5. Field efficacy study

[0073] 1) Field efficacy trials of biocontrol bacteria

[0074] The field trial was conducted on May 6, 2024, in a potato field in Etuoke Banner, Ordos City, where potato stem rot nematode infestation was severe. The potato variety planted was Xisen 6. The experimental plot had a row spacing of 0.9m and a length of 9m, arranged in a 2-row, 4-row layout, with a plot area of ​​1.8 × 9 = 16.2m². 2 The experiment included two application methods: seed dressing and spraying, with a total of 14 treatments, covering single-agent treatment groups, mixed-agent treatment groups, blank control groups, thiazophos group (10% thiazophos granules), and fungicide-agent mixture groups. Details are shown in Table 5.

[0075] The seed treatment process includes: using 20 kg of seeds per treatment plot; cutting the seed potatoes into pieces, drying and spreading them out before sowing; spraying the prepared bacterial fermentation liquid evenly with a sprayer; turning them over 3 times; and drying them for 4 hours to allow the fermentation liquid to fully absorb onto the seed surface before sowing.

[0076] The spraying steps include: after crop sowing, the inoculant is evenly sprayed onto the plants during the seedling stage.

[0077] The concentration of a single bacterial agent is 1×10 7 CFU / mL Bacillus mogavus XCSF-04 and a concentration of 1×107 Fermentation broth of Bacillus licheniformis J117 at cfu / mL; mixed inoculum at a concentration of 1×10 7 CFU / mL Bacillus mogavus XCSF-04 and a concentration of 1×10 7 The fermentation broth was prepared by fermenting Bacillus licheniformis J117 at a mass ratio of 1:1.

[0078] The thiazophosphine used in this experiment was all 10% thiazophosphine granules.

[0079] Table 5. Explanation of treatment groups in field efficacy trials of biocontrol bacteria.

[0080]

[0081] Each treatment was replicated in triplicate using a completely randomized block design. During the experiment, water and fertilizer management and cultivation practices remained consistent across all treatments. Potatoes were harvested in September of the same year (four months after planting), and a survey was conducted.

[0082] A three-point sampling method was used, with each point selecting a 3-meter double-row sampling survey. Based on... Figure 3 ( Figure 3 In the table, potatoes a and b are classified as level 0; potatoes c and d as level 1; potatoes e and f as level 2; potatoes g and h as level 3; and potatoes i and j as level 4. Following the grading standards for potato stem rot nematode disease (Table 6), each potato was cross-sectioned to grade the disease severity, and the disease rate was statistically analyzed. The disease index and relative control efficacy were then calculated. The calculation formula is as follows:

[0083] Incidence rate (%) = (Number of diseased potatoes / Number of potatoes surveyed) × 100;

[0084] Disease index = [∑(number of diseased potatoes × corresponding level representative value) / (total number of potatoes surveyed × highest level representative value)] × 100;

[0085] Prevention and control effect (%) = [(disease index of control area - disease index of treatment area) / disease index of control area] × 100.

[0086] Table 6 Grading Standards for Potato Stem Rot Nematode Disease

[0087]

[0088] The field control effects of single-agent and mixed-agent treatments on stem rot nematodes are shown in Table 7.

[0089] Table 7. Field control efficacy of single and mixed bacterial agent treatments against stem rot nematodes.

[0090]

[0091] As shown in Table 7, the control effects of different application methods and biocontrol agents on stem rot nematode disease were significantly better than the control (CK). Regarding application methods, field spraying showed better control, with lower incidence and disease index of stem rot nematodes compared to seed treatment. Specifically, the control effect of single-agent spraying of J117 (69.46%) was significantly higher than that of seed treatment (45.30%) by 24.16%. The control effect of compound microbial spraying (77.53%) was also significantly higher than that of seed treatment (57.76%).

[0092] In terms of control efficacy, under spraying conditions, strain J117 showed higher control efficacy than XCSF-04, with incidence rates of 14.24% and 21.01%, and control efficacy rates of 69.46% and 57.55%, respectively, which was 9.06% higher than that of 10% thiazophos granules. Under seed dressing treatment, the control efficacy of the two strains was not significantly different, with XCSF-04 showing slightly higher efficacy than J117, with incidence rates of 25.23% and 25.73%, and control efficacy rates of 49.02% and 45.30%, respectively. Both XCSF-04 and J117 showed lower control efficacy than those of 10% thiazophos granules applied by spraying, but the differences were not statistically significant. These results indicate that both biocontrol bacteria possess a certain ability to control stem rot nematodes, with spraying showing superior efficacy.

[0093] Table 7 shows the control effects of the combined biocontrol agents. The combined agents exhibited different control effects under different application methods. In seed treatment, the incidence of stem rot nematodes was only 16.19% after applying the combined agent of XCSF-04 and J117, significantly lower than the incidence rates of XCSF-04 (25.23%) and J117 (25.73%) alone. The disease index of the combined agent under this application method was also 1.79% and 2.55% lower than that of the single biocontrol agents, respectively. The control efficacy of the combined biocontrol agent was 57.76%, which was 8.74% and 12.46% higher than that of the single biocontrol agents XCSF-04 and J117, respectively. Furthermore, the control efficacy of the combined biocontrol agent was comparable to that of thiazophos (60.40%), with no significant difference. When sprayed, the incidence of stem nematode was only 11.68% after applying the compound microbial agent of XCSF-04 and J117, significantly lower than the incidence rates of XCSF-04 (21.01%) and J117 (14.24%) single microbial agents. The disease index of the compound microbial agent in this treatment was also 4.08% and 1.65% lower than that of the single biocontrol agents, respectively. The control efficacy of the compound biocontrol agent was 77.53%, which was 19.98% and 8.07% higher than that of the single biocontrol agents XCSF-04 and J117, respectively, demonstrating significant control efficacy. Furthermore, the control efficacy of the compound biocontrol agent was 17.13% higher than that of thiazophos (60.40%).

[0094] In summary, biocontrol agents XCSF-04 and J117 can effectively control stem rot nematode disease. Their combined application has a stronger inhibitory effect on stem rot nematode disease than their individual application, and spraying can achieve better control results.

[0095] The field control efficacy of the fungal-drug mixture against stem nematode is shown in Table 8.

[0096] Table 8. Field control efficacy of fungicide-fungicide mixtures against stem rot nematodes.

[0097]

[0098] Under the biocontrol seed treatment, the control effect of the mixed strain of biocontrol Bacillus XCSF-04+J117 with a 30% reduction in thiazophos (bacterial-drug mixture combination 3) was the most significant, at 61.74%, which was 3.98% higher than the control effect of the compound strain (XCSF-04+J117). The control effect was not significantly different from that of thiazophos alone (60.40%). Compared with the 10% and 20% reduction in thiazophos, it had a significant synergistic effect, with control effects increased by 35.08% and 19.5%, respectively.

[0099] Under biocontrol spraying treatments, compared with the control, both single application and reduced-dose combined application of the agent significantly reduced the incidence and disease index of potato stem rot nematode disease. The most significant reduction was achieved with a 1:1 mixture of Bacillus XCSF-04 and J117 combined with a 30% reduction in 10% thiazophos granules (combination 3). The incidence and disease index were only 6.48% and 4.78, respectively, representing a 4.71% reduction in incidence and a 15.65 reduction in disease index compared to the control. This treatment achieved a control efficacy of 76.60% against stem rot nematodes, a 16.20% increase compared to thiazophos alone (60.40%), showing a significant difference between treatments. This was second only to the control efficacy of the combined agent (XCSF-04+J117) (77.53%), although the difference was not significant. The control effects of 10% reduction in thiazophos (fungus-drug mixture 1) and 20% reduction in thiazophos (fungus-drug mixture 2) were not significantly different, at 54.53% and 59.21% respectively. However, compared with 30% reduction in thiazophos (fungus-drug mixture 3), the effects were reduced by 22.07% and 17.39% respectively, which was significantly different.

[0100] The above results indicate that when thiazophos is reduced by 30% and combined with biocontrol agents, the incidence of stem rot nematodes can be significantly reduced, the occurrence of the disease can be controlled, and the control effect is comparable to, or even better than, the application of chemical agents or biocontrol agents alone.

[0101] 3) Verification experiment on the effects of biocontrol bacteria on potato growth in the field.

[0102] While conducting field efficacy trials of biocontrol bacteria, the growth status of potatoes in the field was statistically analyzed, raw data were recorded, and the mean ± standard deviation was calculated. Analysis of variance (ANOVA) combined with multiple comparison methods (such as Duncan's method) was used to test the significance of differences between treatment groups (P < 0.05 level). The experimental results are shown in Tables 9 and 10. Table 9 shows the verification results of the effects of single-agent and mixed-agent treatments on growth status, while Table 10 shows the verification results of the effects of the combined bacterial and pesticide treatments on growth status.

[0103] The growth status includes potato plant height and stem diameter. Plant height refers to the vertical distance from the base of the plant to the top (unit: cm); stem diameter refers to the diameter of the stem at the base or a fixed point (unit: mm).

[0104] Table 9. Results of the validation experiment on the effects of single-agent and mixed-agent treatments on growth status.

[0105]

[0106] As shown in Table 9, there were significant differences in the effects of different treatment groups on potato plant height and stem diameter. Regarding plant height, at 60 days, the XCSF-04+J117 spray treatment group had the highest plant height, reaching 55.6±2.99 cm, significantly higher than most other treatment groups; while the CK group had the lowest plant height, only 42.15±3.76 cm. At 90 days, both the thiazophos application treatment group and the XCSF-04+J117 spray treatment group had relatively high plant heights, at 95.24±2.44 cm and 93.6±1.91 cm respectively, significantly higher than the CK group's 69.15±1.36 cm. This indicates that the combination of biocontrol bacteria and the use of chemical agents promoted potato plant height growth to some extent.

[0107] Regarding stem diameter, at 60 days, the XCSF-04+J117 spraying treatment group had the largest stem diameter, at 11.6±2.35 mm, significantly higher than the CK group's 6.93±1.15 mm. At 90 days, the XCSF-04+J117 spraying treatment group still had the largest stem diameter, reaching 12.27±2.55 mm, also significantly higher than the CK group's 7.93±1.35 mm. Overall, the biocontrol bacteria compound treatment groups showed outstanding performance in promoting potato stem diameter growth, especially the XCSF-04 and J117 compound treatment group, which had good effects on plant height and stem diameter growth whether applied as seed dressing or by spraying.

[0108] Table 10 Results of the validation experiment on the effect of the fungal drug mixture on growth status.

[0109]

[0110] As shown in Table 10, from the perspective of the difference in application methods, the promotion effect of spraying the microbial-fertilizer mixture on crop growth is significantly more prominent: the microbial-fertilizer mixture 3 (thiazophosphorus reduced by 30% and combined with 30% compound microbial agent) under this method reached a plant height of 58.53cm at 60 days, which is more significant than the plant height under the seed dressing method; by 90 days, the plant height of the sprayed treatment all exceeded 95cm, and the stem diameter was also stable above 11mm, while the plant height of the seed dressing method was generally below 90cm, and the stem diameter was around 11mm. The growth gap between the two methods gradually widened over time.

[0111] From different treatments, the combined effect of microbial-pesticide-fertilizer combination is better than that of thiazophos alone. The plant height and stem diameter of microbial-pesticide mixture 3 under the spraying method are significantly higher than those of other treatment groups at 60 days and 90 days. In contrast, the plant height and stem diameter of the blank control (CK) are much lower than those of other treatment groups at both 60 days and 90 days. This indicates that the microbial-pesticide mixture treatment has a strong promoting effect on crop growth and can also reduce the amount of chemical pesticides used.

[0112] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A fungicide for controlling stem rot nematodes, characterized in that, A fermentation product comprising microorganisms; The microorganisms include Bacillus moghaves (Bacillus) Bacillus mojavensis XCSF-04 and Bacillus licheniformis ( Bacillus licheniformis J117; The Bacillus mojavensis XCSF-04 has been preserved in the China General Microbiological Culture Collection Center on October 28, 2025, and the preservation number is CGMCC No. 36379; the Bacillus licheniformis J117 has been preserved in the China General Microbiological Culture Collection Center on October 31, 2025, and the preservation number is CGMCC No. 36435. The fermentation product is a mixture obtained by mixing the fermentation broth of Bacillus mojavensis XCSF-04 and the fermentation broth of Bacillus licheniformis J117, and the mass ratio of the fermentation broth of Bacillus mojavensis XCSF-04 and the fermentation broth of Bacillus licheniformis J117 is 1:

1. The preparation process of the fermentation broth comprises inoculating the two strains into liquid culture medium respectively, then performing fermentation, and obtaining the fermentation broth by centrifuging and taking the supernatant.

2. A product for controlling stem decay nematodes, characterized by, The application further discloses a chemical agent for controlling the stem decay nematode and a preparation method of the chemical agent.

3. The product for controlling the decay stem nematode according to claim 2, characterized by, The chemical agent for controlling the stem decay nematode is thiazolidine phosphine.

4. The product for controlling stem decay nematodes according to claim 3, characterized by, The volume ratio of the total volume of the fermentation broth of Bacillus mojavensis XCSF-04 and the fermentation broth of Bacillus licheniformis J117 to the volume of the thiazolidine phosphine is (1-3):(7-9).

5. The use of the decay stem nematode control agent according to claim 1, characterized in that, The application further discloses an application of the chemical agent for controlling the stem decay nematode. A) the application of the chemical agent for controlling the stem decay nematode in controlling the stem decay nematode; B) the application of the chemical agent for controlling the stem decay nematode in preparing a product for controlling the stem decay nematode.

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