A pink-like sporidiobolus and application thereof

Abstract

The present application relates to the field of microorganism and plant protection, and particularly relates to a kind of spores of pink lock (Sporidiobolus roseus) and its application in the prevention and control of plant root-knot nematode. Sporidiobolus pararoseus The strain is isolated from orchard soil, is easy to survive in soil, and through indoor and field control tests, it is shown that it has good control effect on root-knot nematode. Therefore, the present application provides an effective biocontrol strain for preventing and controlling the harm of root-knot nematode, helps the green prevention and control of root-knot nematode, and has great application value.

Description

Invention Field

[0001] This invention relates to the fields of microorganisms and plant protection, specifically to a species of yeast, *Sporidiobolus pararoseus*, and its application in the control of plant root-knot nematodes. Background Technology

[0002] Root-knot nematode disease is an important soil-borne disease of crops, caused by root-knot nematodes infecting the root system. Root-knot nematodes primarily infect the fibrous roots of crops. Early-stage larvae typically invade from the posterior part of the root tip, moving towards the tip and stimulating cells to form permanent feeding sites. At these feeding sites, the cell nuclei divide, but the cell walls do not, forming multinucleated giant cells, approximately 100 times the size of normal cells. Once these giant cells form, the nematode begins to develop, transforming from a thread-like shape to a sausage-shaped form. Generally, J2 nematodes transform into J3 nematodes 14 days after invasion, and then into J4 nematodes 4-6 days later, subsequently evolving into pear-shaped adults. Root-knot nematodes primarily reproduce parthenogenetically, with males only forming under unfavorable environmental conditions. Males generally do not feed. Parthenogenesis, compared to sexual reproduction, typically results in a shorter reproductive cycle and a larger reproductive output, which is beneficial for the nematode's reproduction and survival.

[0003] Root-knot nematodes are globally distributed plant parasitic nematodes. The typical symptom of their damage is the formation of root knots of varying sizes after infecting the root system. Root-knot nematode infection impairs the root system's absorption and upward transport of water and nutrients. Above-ground symptoms mainly include slow growth, wilting, yellowing leaves, and organ malformation. Directly related to agricultural production, this results in reduced yield and quality, causing significant economic losses. In my country, root-knot nematodes are one of the major soil-borne pathogens, capable of infecting over 3,000 host plant species belonging to 114 families, particularly the Cucurbitaceae, Solanaceae, and Brassicaceae families. The earliest report of root-knot nematodes in my country was in 1932 by Tu Zhi, who reported a severe outbreak of root-knot nematode disease in tomatoes in southern China. Globally, plant parasitic nematodes cause an estimated $157 billion in losses annually (Abad 2008). Root-knot nematode disease occurs in both greenhouse and open-field environments, especially in greenhouses. When crops are continuously planted in the same location, the disease occurs repeatedly, generally reducing yield by 30% to 70%. Root-knot nematode disease has become a serious obstacle in greenhouse vegetable production.

[0004] The strategy for controlling root-knot nematodes includes: first, selecting disease-resistant varieties and cultivating healthy seedlings; second, using high-temperature fumigation and sunlight sterilization in summer, and soil tilling and freezing treatment in winter; third, crop rotation with onions, garlic, and other field crops; and finally, using nematicides or biological agents for control. Biological control is a key research focus, characterized by its green and safe nature, and is closely related to the green control of agricultural pests and diseases, thus receiving increasing attention. Research on biocontrol bacteria isolated from root-knot nematodes involves various microorganisms, including fungi and bacteria. Among bacteria, Bacillus species have been extensively studied, with Bacillus subtilis, Bacillus sturdius, and Bacillus punctata being reported for their use in controlling root-knot nematode diseases. Among fungi, various Trichodermas, such as Trichoderma viride, Trichoderma hookeriana, and Trichoderma harzianum, have also been reported, while other filamentous fungi, such as Paecilomyces lilacinus, Verticillium, and Pinctonia, have also been applied to the control of root-knot nematode diseases. There is relatively little research on yeast. Yeast is a single-celled fungus that is easy to cultivate and preserve, can survive for a long time in soil, and can produce abundant secondary metabolites, making it a promising candidate for the development of biocontrol bacteria. Summary of the Invention

[0005] The inventors isolated and identified a strain of *Syngonium rosenbergii* from the rhizosphere soil of an orchard and studied its control effect on root-knot nematode disease, laying the foundation for its application in agricultural production to control root-knot nematode disease.

[0006] Therefore, the purpose of this invention is to provide a strain of *Cyclophorus rosenbergii* Sp-S06 for the control of root-knot nematode disease. This *Cyclophorus rosenbergii* strain is derived from orchard rhizosphere soil and exhibits good efficacy against root-knot nematodes. Experiments have demonstrated that the isolated *Cyclophorus rosenbergii* strain exhibits good repellent, lethal, and control effects against root-knot nematodes both indoors and in the field. This is of great significance for controlling the damage caused by root-knot nematodes and ensuring the sustainable development of the vegetable industry.

[0007] This strain was deposited on October 11, 2022, at the China General Microbiological Culture Collection Center (CGMCC), a Budapest Treaty International Collection Unit for Microorganisms. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The accession number is CGMCC NO. 25890, and the classification is *Sporidiobolus pararoseus*. It has been confirmed to be viable.

[0008] The strain was cultured in a shaker. Second-instar larvae of root-knot nematodes were treated with the culture medium and its supernatant, and its lethality against the nematodes was tested. The results showed that the culture medium achieved a 100% lethality rate against nematodes within 12 hours. The in-plate confrontation method was used to test the strain's repulsiveness to nematodes, confirming that the fermentation broth and supernatant of the strain could significantly repel root-knot nematodes. In indoor potted plants, the control effect against root-knot nematodes reached 75.5%. In the field, the fermentation broth was used to irrigate the soil before transplanting cucumber seedlings to control root-knot nematodes, confirming a 50.8% control efficacy against root-knot nematode disease. In the field, the strain promoted the growth of cucumber plants by 17.2%.

[0009] Therefore, a second aspect of the present invention is to provide the application of the *Syndrome rubrum* strain Sp-S06 in the control of root-knot nematodes in crops such as vegetables.

[0010] Furthermore, a third aspect of the present invention provides a method for controlling root-knot nematodes using the aforementioned *Candida citrinum* strain Sp-S06, which involves fermenting and culturing the *Candida citrinum*, inoculating the fermentation broth into soil seedling holes, and then planting plant seedlings in the seedling holes for normal management and cultivation.

[0011] Preferably, the fermentation culture involves inoculating the *Synthia spp.* strain Sp-S06 into a liquid culture medium and fermenting it for 2-4 days at 25-32°C and 150-200 rpm.

[0012] More preferably, the liquid culture medium is NYDB medium: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose, pH: 7.0-7.2, sterilized at 121°C for 20 min.

[0013] More preferably, the culture conditions are fermentation culture at 28°C and 180 rpm for 3 days.

[0014] In a specific implementation, the plant seedling is a cucumber seedling, but it is also applicable to other vegetable crops such as cucumbers, melons, watermelons, tomatoes, and peppers.

[0015] The fourth aspect of this invention is to provide a biocontrol agent for controlling root-knot nematodes, which is prepared by fermentation broth or concentrate obtained from the fermentation culture of the aforementioned *Syndrome affinis* strain Sp-S06.

[0016] The beneficial effects of this invention are as follows: the fermentation broth of *Cyperus rotundus* strain Sp-S06 achieves a 100% mortality rate against second-instar larvae of root-knot nematodes, a control effect of 75.5% in pot experiments, and a field control effect of 50.8%, promoting plant growth by 17.2%. Furthermore, the fermentation broth of this strain significantly repels root-knot nematode larvae, demonstrating high and stable control efficacy and significant application potential in agricultural production. Simultaneously, *Cyperus rotundus* strain Sp-S06 is a yeast isolated from rhizosphere soil, making it safe and harmless to crops, thus exhibiting safety characteristics in agricultural applications. This invention is the first to screen *Cyperus rotundus* and apply it to the control of root-knot nematodes, demonstrating significant application value in biocontrol. Attached Figure Description

[0017] Figure 1 Morphology of *Syndrome rosenbergii* strain Sp-S06.

[0018] Figure 2 Schematic diagram of nematode chemotaxis test. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to specific implementation examples. First, the *Saccharomyces rosenbergii* Sp-S06 strain was isolated, purified, and cultured, and then fermented. The nematicidal activity and nematode repellency activity of the fermentation broth and supernatant were tested. Simultaneously, under pot and field conditions, the fermentation broth of *Saccharomyces rosenbergii* Sp-S06 strain was inoculated onto cucumbers to control root-knot nematodes.

[0020] Example 1: Isolation, purification and identification of *Saccharomyces roxburghii* strain Sp-S06

[0021] Soil samples were evenly collected from the rhizosphere of apple trees in the orchard of Yizhihuiying Village, Langfang City, totaling 3 portions, each 200g, and mixed thoroughly. 50g of this mixture was added to 1000mL of distilled water, stirred well, and allowed to stand for 3 minutes. 1mL of the supernatant was taken and diluted 100 times with sterile water. 50μL of the diluted liquid was then spread onto lactic acid PDA plates (200g potato, 20g glucose, 5mL lactic acid, 15g / L agar powder, diluted to 1L, pH=5.0). For the preparation of the solution, 200g of peeled potatoes were weighed, cut into pieces, and boiled for 15 minutes. The potato pieces and residue were filtered out using gauze. The filtrate was then mixed with 20g glucose, 15g agar, and 5ml lactic acid, and autoclaved at 121℃ for 20 minutes. The resulting solution was then plated in petri dishes for the isolation and purification of yeast. Lactic acid medium was used to maintain an acidic pH of 5-6, allowing yeast to grow normally while inhibiting the growth of other microorganisms within this acidic range. The role of lactic acid is to suppress other contaminating bacteria, facilitating yeast isolation and purification. After the soil dilution was evenly spread, the plates were inverted and incubated at 28°C for 2 days. Once colonies appeared on the plates, single-colony purification was performed. Single colonies were picked, streaked onto lactic acid PDA agar plates for isolation and purification, yielding single colonies for classification and identification. Approximately 30 single colonies grew on each plate, randomly numbered starting from 1 (Sp-S01). These numbered single colonies were then propagated and identified, resulting in the sixth single-colony strain, Sp-S06. Morphological analysis of the strain was performed according to the *Handbook of Fungal Identification*.

[0022] See Figure 1 The Sp-S06 strain was obtained and cultured on lactic acid PDA plates at 30°C. The strain grew rapidly, with colonies reaching approximately 1.5-2.0 mm in diameter after 3 days. Colonies were orange-red or magenta, cheesy to mucilaginous, with raised, smooth or wrinkled surfaces, reflecting or not reflecting light, and smooth or etched edges. Cells were elliptical, unilaterally budding, and exhibited fungal buds, annulus, and sediment formation; spores were kidney-shaped. The Sp-S06 strain was acid-tolerant, growing normally at pH 5-6. Morphological analysis of the Sp-S06 strain according to the *Handbook of Fungal Identification* confirmed it as *Sporidiobolus pararoseus*.

[0023] Further molecular biological identification was performed. Single colonies were picked up with a sterile toothpick and gently tapped to the bottom of a PCR tube to adhere the bacterial cells, serving as a PCR template for bacterial PCR amplification. The 26S rRNA sequence D1 / D2 region was amplified using universal fungal identification primers NL1 (forward primer) and NL4 (reverse primer). The PCR amplification reaction system was 50 μL, containing 25 μL ExTaq enzyme, 1 μL forward primer, 1 μL reverse primer, 3 μL DNA template, and 20 μL sterile water. Amplification conditions: 94℃ for 5 min, 94℃ for 30 s, 50℃ for 30 s, 72℃ for 1 min, 30 cycles; amplification was terminated at 72℃ for 10 min. The amplified products were separated and identified by 1% agarose gel electrophoresis. The PCR products were directly subjected to bidirectional sequencing and compared with BLAST on the NCBI website.

[0024] The D1 / D2 region sequence of yeast 26S rDNA was amplified using primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3'). The PCR product was subjected to 1% agarose gel electrophoresis. Sequencing analysis showed that the amplified 26S rDNA sequence of the strain was 618 bp long (SEQ ID NO: 1). BLAST analysis of the strain's 26S rDNA sequence showed that strain Sp-S06 had 100% similarity to *Sporidiobolus pararoseus*. The strain was identified as *Sporidiobolus pararoseus*. The strain was deposited.

[0025] Example 2: Effects of *Syndrome rosenbergii* strain Sp-S06 on nematode attraction and repulsion.

[0026] Experimental culture medium and its formulation:

[0027] NYDA medium: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose, solid medium supplemented with 15-20 g / L agar, pH: 7.0-7.2, dispensed into Erlenmeyer flasks and sterilized at 121°C for 20 min.

[0028] NYDB medium: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose, pH: 7.0–7.2. Dispense into Erlenmeyer flasks and sterilize at 121°C for 20 min.

[0029] (1) Preparation of fermentation broth of Sp-S06 strain

[0030] The *Synthia spp.* Sp-S06 strain was purified and activated by streaking on NYDA agar plates and cultured at 28°C for 3 days. Activated colonies were scraped off with an inoculation loop and inoculated into 250 mL Erlenmeyer flasks containing 100 mL of NYDB liquid medium. The flasks were then cultured at 28°C with shaking (180 rpm) for 3 days. The OD value of the fermentation broth was measured... 600 When the concentration reached 1.0, the fermentation broth was used for experiments. Simultaneously, a portion of the fermentation broth was centrifuged at 10,000 rpm for 3 minutes to precipitate the cells. The supernatant was used for experiments to analyze the repulsion behavior of second-instar larvae of root-knot nematodes (Meloidogyne spp.).

[0031] (2) Preparation of root-knot nematodes for the experiment

[0032] Root-knot nematodes were collected from the greenhouse of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The roots of severely infested pepper plants were removed, gently rinsed with water, and egg masses were carefully removed from the root surface. These egg masses were then disinfected in 0.5% sodium hypochlorite for 3 minutes, followed by rinsing three times with sterile water to remove any remaining reagent. The disinfected egg masses were then placed in petri dishes containing a small amount of sterile water and incubated at 25°C for 24-48 hours. The hatched second-instar larvae of the root-knot nematodes were collected for testing.

[0033] (3) Test methods

[0034] Chemotaxis of root-knot nematodes was analyzed using petri dishes (9cm). The petri dishes were divided into 6 sections (1-6) from left to right and 6 sections from top to bottom, for a total of 6×6 small squares. Figure 2 Plates were prepared using 0.5% water agar. 20 μl of fermentation broth was added to the center of the left edge of the petri dish (position B). Approximately 100 second-instar root-knot nematode larvae (20 μl) were added to the center of the dish (position A). Nematode movement was observed at 25°C. In this study, nematode movement from the center towards the bacterial culture (parts 1-3) was defined as attraction, while movement from the center away from the culture (parts 4-6) was defined as repulsion. At 12 h, the number of nematodes in each part of the dish and the number of nematodes still remaining at the inoculation site were counted and compared. Each treatment consisted of 10 petri dishes, and the experiment was independently repeated 3 times. The experiment also included a treatment with 20 μl of SP-S06 strain supernatant and a control with 20 μl of sterile water.

[0035] The results showed that at 12 hours, second-instar larvae exhibited strong rejection of both the fermentation broth and supernatant treatments (Table 1). In both the bacterial culture and supernatant treatments, nematodes tended to move away from the fermentation broth and supernatant, with the number of nematodes in the rejection zone exceeding that in the attraction zone. In the fermentation broth treatment, the number of nematodes in the attraction and rejection zones were 17.2 and 80.5, respectively. A similar situation was observed in the supernatant treatment, with 22.1 and 79.4 nematodes in the attraction and rejection zones, respectively. In the water control, the number of nematodes in the attraction and rejection zones was not significantly different, at 49.5 and 51.0, respectively (Table 1).

[0036] Table 1. Analysis of nematode repulsion (unit: nematodes)

[0037] Fermentation liquid 17.2 4.7 80.5 Supernatant 22.1 5.4 79.4 Water comparison 49.5 5.3 51.0

[0038] Example 3: Nematicidal Activity Analysis of *Cyclophorus rosenbergii* Sp-S06 Strain

[0039] (1) Preparation of fermentation broth of Sp-S06 strain

[0040] The Sp-S06 strain was purified and activated by streaking on NYDA agar plates and cultured at 28°C for 3 days. Activated colonies were scraped off with an inoculation loop and inoculated into 250 mL Erlenmeyer flasks containing 100 mL of NYDB liquid medium. The flasks were then cultured at 28°C with shaking (180 rpm) for 3 days. The OD value of the fermentation broth was measured... 600 When the concentration reached 1.0, the fermentation broth and its 10-fold and 100-fold dilutions prepared with sterile water were used for the experiment. At the same time, a portion of the fermentation broth was centrifuged at 10,000 rpm for 3 min to precipitate the cells. The supernatant and the 10-fold and 100-fold dilutions prepared with sterile water were used for the experiment to treat second-instar larvae of root-knot nematodes (Meloidogyne spp.) and to determine the lethal effect of the fermentation broth of strain Sp-S06 on root-knot nematodes.

[0041] (2) Preparation of root-knot nematodes for the experiment

[0042] Southern root-knot nematodes were collected from the greenhouse of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Roots of severely infested pepper plants were removed, gently rinsed with water, and egg masses were carefully removed from the root surface. These egg masses were then disinfected in 0.5% sodium hypochlorite for 3 minutes, followed by rinsing three times with sterile water to remove any remaining reagent. The disinfected egg masses were then placed in petri dishes containing a small amount of sterile water and incubated at 25°C for 24-48 hours. The hatched second-instar larvae were collected for testing.

[0043] (3) Test methods

[0044] In a sterile 24-well cell culture plate, 1 mL of fermentation broth was added to each well, followed by 10 μL of nematode suspension containing approximately 100 nematodes. Each treatment was repeated in 10 wells (samples), with 3 replicates. Treatments were performed using the same method, including 10-fold and 100-fold dilutions of the fermentation broth, as well as supernatant treatments with 10-fold and 100-fold dilutions of the supernatant. Sterile water was used as a control. The plates were incubated at room temperature for 24 hours, and the mortality of root-knot nematodes was observed. The corrected mortality rate was calculated, representing the nematode-killing effect. The formula for calculating the corrected mortality rate is as follows:

[0045] Corrected mortality rate (%) = 100 × (mortality rate of treated nematodes - mortality rate of control nematodes) / (1 - mortality rate of control nematodes). (4) Experimental results:

[0046] The above experiments determined the lethality of fermentation broth and supernatant treatment on root-knot nematodes after 24 hours. The results showed that different fermentation broth dilutions had varying effects on nematodes. The corrected mortality rate of root-knot nematodes was 100% in both the original fermentation broth and the 10-fold dilution. The control effect decreased after a 100-fold dilution, but still reached 55.3% (Table 2). The nematode-killing effect of the supernatant of the Sp-S06 strain fermentation broth was similar to that of the fermentation broth. The corrected mortality rate of root-knot nematodes was 100% in both the supernatant and the 10-fold dilution, and the nematode-killing effect was 65.6% in the 100-fold dilution of the supernatant (Table 3). These experiments demonstrate that the Sp-S06 strain fermentation broth has a good control effect on root-knot nematodes.

[0047] Table 2. Nematode-killing effect of fermentation broth of Sp-S06 strain at different concentrations.

[0048] Repeat 1 100 100 57.1 1.3 Repeat 2 100 100 56.3 1.5 Repeat 3 100 100 54.5 1.7 Corrected mortality rate 100％ 100％ 55.3％ -

[0049] Table 3. Nematode-killing effects of supernatant at different concentrations

[0050] Repeat 1 100 100 67.3 2.1 Repeat 2 100 100 66.4 1.8 Repeat 3 100 100 65.2 1.7 Corrected mortality rate 100％ 100％ 65.6％ -

[0051] Example 4: Effect of Cyclospora pinkisiformis Sp-S06 strain on the control of root-knot nematode disease in potted plants

[0052] (1) Test methods

[0053] The *Syndrome rosenbergii* sp-S06 strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of NYDB liquid medium and cultured at 28 °C with shaking (180 rpm) for 3 days. The absorbance of the fermentation broth was measured when the OD value... 600=1.0 was used for control experiments. The cucumber variety "Zhongnong 16" was selected. After seed disinfection and germination, seedlings were raised in seedling trays. The seedlings were used for the experiment when they developed one true leaf. The cucumber seedlings with one true leaf were transplanted into seedling pots (10cm in diameter, 10cm in height) with a disinfected peat moss and vermiculite substrate (v / v = 2:1). Seven days after transplanting, when the second true leaf appeared, inoculation was performed. 15mL of Sp-S06 strain fermentation culture was inoculated into the rhizosphere soil of the cucumber seedlings. One day later, 500 second-instar root-knot nematode larvae were inoculated per plant. The preparation of the second-instar root-knot nematode larvae was the same as in Example 2. The plants were managed normally at room temperature, and the number of root knots was measured after 5 weeks to calculate the control effect of Sp-S06 strain against root-knot nematodes. The experiment included a water control and a pesticide treatment. The pesticide treatment used 10% thiazophos granules (Ishihara, Japan), with a dosage calculated at 2 kg per acre, approximately 0.03 g per seedling pot. To apply, gently make shallow grooves or small holes on the substrate surface around the seedling roots, evenly sprinkle the granules into the substrate, and then gently cover with the substrate. Normal management was followed after application. Each treatment consisted of 10 cucumber seedlings, replicated three times, with a water control as a baseline. The formula for calculating the control effect in potted plants is as follows:

[0054] Control efficacy (%) = (1 - number of root knots in treatment / number of root knots in control) × 100.

[0055] (2) Test Results

[0056] Thirty-five days after inoculation with root-knot nematodes, the soil around the cucumber seedling roots was gently washed away, and the number of root knots on the roots was measured to analyze the control effect (Table 4). The experiment showed that the number of root knots on cucumber roots was significantly reduced after treatment with *Cyperus rosenbergii* Sp-S06 strain, with an average of 23.8 root knots per plant, compared to an average of 97.0 root knots per plant in the control treatment. The control effect of *Cyperus rosenbergii* Sp-S06 strain against root-knot nematodes reached 75.5%. The control effect of thiazophos reached 93.5% in the thiazophos treatment. This indicates that *Cyperus rosenbergii* Sp-S06 strain can effectively control cucumber root-knot nematodes. Although its control effect is lower than that of the chemical agent thiazophos, it has potential and value in the control of root-knot nematode disease.

[0057] Table 4. Number of root knots and control effects of different treatments

[0058] Water comparison 100.3 95.5 95.2 97.0 - Thiazolphosphine 6.4 6.9 5.7 6.3 93.5％ Sp-S06 22.8 23.6 25.0 23.8 75.5％

[0059] Example 5: Field control efficacy of *Syndrome rosenbergii* strain Sp-S06 against root-knot nematode disease

[0060] (1) Test methods

[0061] Sp-S06 inoculum was inoculated into 1000mL Erlenmeyer flasks containing 500mL NYDB liquid medium and cultured at 28℃ with shaking (180rpm) for 3 days, yielding approximately 10L of *Syngonium rosenbergii* fermentation broth. The fermentation broth was used for control experiments when OD=1.5. The experiment was conducted in a greenhouse at the Yizhihuiying Vegetable Research Institute base in Langfang City, Hebei Province, an area with severe root-knot nematode infestation, suitable for field efficacy evaluation. The cucumber variety used was "Zhongnong 16," and field trials were conducted when the cucumber seedlings had two fully unfolded true leaves. Each plot consisted of one bed, 1.1 meters wide and 6 meters long, planted in double rows with a plant spacing of 40cm and a row spacing of 50cm, with approximately 30 seedlings per bed. The experiment was replicated in three plots (beds), randomly distributed, and conducted simultaneously in two different greenhouses.

[0062] Before transplanting, dig 15cm deep holes at the designated planting locations for the seedlings. Dilute 50ml of the Sp-S06 strain fermentation broth 10 times with water and pour it into the seedling holes. After the bacterial solution has completely penetrated into the soil and there is no standing liquid, transplant the cucumber seedlings into the holes. The experiment also included a water control and a chemical pesticide treatment. For the chemical pesticide treatment, 10% thiazophos granules (Fukido, Ishihara, Japan) were used at the recommended dosage (2kg / acre). After transplanting, normal production management was implemented. 45 days later, the occurrence of root-knot nematodes was monitored, and the disease severity and disease index were statistically analyzed to calculate the control effect. Simultaneously, the above-ground plant height was measured, and the growth-promoting effect of the strain on cucumber plant height was compared and analyzed.

[0063] The disease severity, disease index, and prevention efficacy are calculated as follows (Table 5):

[0064] Table 5. Disease Grading of Root Knot Severity in Individual Plants

[0065]

[0066]

[0067] The Disease Index (DI) is calculated using the following formula (1):

[0068] DI=(∑(s×n) / (N×S))×100…………(1)

[0069] In the formula:

[0070] ∑—The sum of the products of the numerical value of each disease level and the number of plants at each disease level; s—The numerical value of each disease level; n—The number of diseased plants at each disease level; N—The total number of plants surveyed; S—The numerical value of the highest disease level.

[0071] Formula for calculating prevention and control effect:

[0072] Prevention and control effect (%) = (1 - disease index of treatment / disease index of control) × 100……..........…(2)

[0073] Formula for calculating growth-promoting effect:

[0074] Growth promotion rate (%) = (Treatment plant height / Control plant height - 1) × 100 ………(3)

[0075] (2) Test Results

[0076] The experiment showed that treatment with *Cyclospora rosenbergii* Sp-S06 significantly reduced the incidence of root-knot nematode disease in cucumbers, and decreased the number of root knots (Table 6). In the water control, the disease index was 70.4, while the disease index after treatment with Sp-S06 was 34.7, indicating a control efficacy of 50.8% against root-knot nematodes. The disease index after treatment with thiazophos was 26.0, with an average control efficacy of 63.1%. These results demonstrate that although *Cyclospora rosenbergii* Sp-S06 does not compare to the chemical pesticide thiazophos, it is an effective biocontrol agent for cucumber root-knot nematode disease, exhibiting the advantages of green control. It has significant potential for application in agricultural production for the control of root-knot nematodes.

[0077] Meanwhile, the plant height was measured (Table 6). The results showed that the *Cyclophorus rosenbergii* Sp-S06 strain promoted growth. The average plant height of cucumber plants treated with this strain was 208.2 cm, while the height of the water control was 177.6 cm, resulting in a growth promotion rate of 17.2%. In contrast, the average plant height treated with thiazophos was 196.5 cm, with a growth promotion rate of only 10.6%. These results indicate that the *Cyclophorus rosenbergii* Sp-S06 strain significantly outperformed the thiazophos treatment in promoting growth, demonstrating a significant growth-promoting effect.

[0078] Table 6 Disease index and prevention and control effects of different treatments

[0079]

[0080]

Claims

1. A species of *Clostridium pinki* ( Sporidiobolus pararoseus ), characterized in that, Its accession number is CGMCC NO.25890.

2. The application of *Synthia spp.* as described in claim 1 in the control of root-knot nematodes.

3. The method for controlling root-knot nematodes using *Synthia spp.* as described in claim 1, characterized in that, After fermenting and culturing the *Syngonium rosenbergii*, the fermentation broth was inoculated into soil seedling holes, and then plant seedlings were planted in the seedling holes for normal management and cultivation.

4. The method as described in claim 3, characterized in that, The fermentation culture involves inoculating the *Syngonium rosenbergii* strain into a liquid culture medium and fermenting it for 2-4 days at 25-32°C and 150-200 rpm.

5. The method as described in claim 4, characterized in that, The liquid culture medium was NYDB medium: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose, pH: 7.0–7.2, sterilized at 121°C for 20 min.

6. The method as described in claim 4, characterized in that, The culture conditions were 28℃ and 180rpm for 3 days of fermentation.

7. The method according to any one of claims 4 to 6, characterized in that, The plant seedlings mentioned are vegetable seedlings.

8. The method according to any one of claims 4 to 6, characterized in that, The plant seedlings are cucumber, melon, watermelon, tomato, and pepper seedlings.

9. A biocontrol agent for controlling root-knot nematodes, characterized in that, It is prepared using the fermentation broth or concentrate obtained from the fermentation culture of *Syngonium oryzae* as described in claim 1.

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