A method for preparing high-yield sporangium of purple sporidiobolus rupestris

Abstract

The application belongs to the field of agricultural microorganism technology, and provides a preparation method of high-yield chlamydospores of Paecilomyces lilacinus. Purpureocillium lavendulum The conidium suspension of Paecilomyces lilacinus is washed out with sterile water, and then inoculated into a liquid induction medium, and high-yield chlamydospores can be obtained after 12-30h of shaking flask culture at 28 DEG C; the germination rate of the prepared chlamydospores can reach 100% after 48h of culture at 25 DEG C; compared with the control, the total number of root nodules is reduced by 89.50% in the pot experiment of Paecilomyces lilacinus chlamydospore treatment. The application only needs 18h from inoculation to induction of high-yield chlamydospores, has the advantages of short production cycle, high chlamydospore yield, simple culture condition and industrial production, and can be used for developing new preparations for preventing and treating root-knot nematode diseases, and has certain application value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a high-yielding purple purpureus (… Purple lavender Preparation method of thick-walled spores. Technical Background Plant parasitic nematodes are a significant disease affecting agricultural production, typically causing yield reductions of 20%–30% or even total crop failure. Currently, control methods for plant parasitic nematodes include: disease-resistant breeding, agricultural control, physical control, chemical control, and biological control. Among these, biological control primarily utilizes microorganisms and their metabolites to develop biological nematicides, which offer advantages such as long-lasting effectiveness, stability, relative safety, and environmental protection.

[0002] As a nematode biocontrol fungus, *Porphyromonas violaceum* ( P. lavender The biological control mechanism of *Porphyromonas violaceus* has a relatively good research foundation. Previous studies have reported on this mechanism. P. lavender When YMF1.00683 interacts with southern root-knot nematodes, purple purple purpureus (Porphyromonas violaceus) can be observed. P. lavender The process by which YMF1.00683 infects and decomposes southern root-knot nematodes ( Pathogens , 2022, 11(7): 795, and from purple purpureus ( P. lavender Several compounds with nematicidal activity were isolated and identified from YMF1.00683. Microorganisms, 2022, 10(7): 1343), indicating that *Porphyromonas violaceum* ( P. lavender YMF1.00683 has the potential to control plant pathogenic nematodes.

[0003] Chlamydospores are a special structure with a thick cell wall, derived from fungal hyphae or conidia. They are typically produced under unfavorable environmental conditions, such as low temperatures, unsuitable pH levels, and nutrient deficiencies, thus exhibiting greater resistance to adverse conditions. As a unique reproductive form, chlamydospores are characterized by their thick cell walls, strong resistance to adverse conditions, and long lifespan. Furthermore, biocontrol agents made from chlamydospores offer advantages such as good storage resistance, high stability, and long shelf life, which can compensate for the shortcomings of existing nematode biocontrol agents.

[0004] Repeated experiments have shown that *Porphyromonas violaceus* (… P. lavender It cannot grow at 35℃, and compared with the related species *Paecilomyces lilacinus*, it is more controllable, making it safer to develop into a live biocontrol agent. Summary of the Invention

[0005] This invention provides a new formulation for the control of root-knot nematode diseases, offering a high-yield purple purpureus fungus (…). Purple lavender The preparation method of chlamydospores provides a new formulation for the development of control agents against root-knot nematode diseases.

[0006] The purple purple purpureus used in this invention ( P. lavender The strain is the same as that described in the literature "Identification of Nematicidal Metabolites from Purpureocillium lavendulum" (Rui Liu, Zheng-XueBao, Guo-Hong Li, Chun-Qiang Li, Shao-Lin Wang, Xue-Rong Pan, Ke-Qin Zhang and Pei-Ji Zhao, Microorganisms, 2022-07-02), and is deposited in the Seed Resource Bank of the State Key Laboratory of Yunnan Biological Resources Conservation and Utilization, jointly constructed by Yunnan Province and the Ministry of Science and Technology, with accession number YMF1.00683.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a high-yield purple purple spore fungus ( Purple lavender The method for preparing chlamydospores, wherein the purple purple spores ( P. lavender Conidia produced on PDA solid medium are washed out with sterile water to prepare a conidia suspension, which is then inoculated into liquid induction medium and cultured in shake flasks at 28°C for 12-30 hours to produce high yields of thick-walled spores. The liquid induction medium is any one of the following five culture medium formulations: A. Potatoes 200 g / L, sucrose 20 g / L; B. Potatoes 200g / L, sucrose 20g / L, Ca(NO3)2 3g / L, KCl 1g / L; C. Potatoes 200g / L, sucrose 20g / L, (NH4)2SO4 3g / L, KCl 1g / L; D. Sucrose 20g / L, (NH4)2SO4 3g / L, KCl 1g / L; E, sucrose 20 g / L, Ca(NO3)2 3 g / L, KCl 1 g / L.

[0008] Preferably, the liquid induction culture medium is: C, potato 200g / L, sucrose 20g / L, (NH4)2SO4 3g / L, KCl 1g / L.

[0009] The specific preparation method of the conidial suspension is as follows: *Porphyromonas violaceus* (… P. lavender The inoculum was inoculated onto PDA solid medium and incubated at 28°C for 5 days. The conidia in the mycelial blocks were then washed and collected with sterile water to prepare a conidial suspension. The conidial suspension contained 6.042 × 10⁻⁶ spores. 6cfu / mL.

[0010] The inoculation amount of conidial suspension in the liquid induction medium was 1 mL containing 6.042 × 10⁶ spores per 250 mL of liquid induction medium. 6 Conidial suspension of cfu / mL conidia; culture conditions in shake flasks: 28℃, 120rpm for 18h.

[0011] The obtained purple purple purpureus ( P. lavender Chlamydospores were cultured at 25℃, and their germination was observed and calculated at 12h, 24h, 36h, and 48h. The obtained *Porphyromonas purpureus* (purple spores) P. lavender Thick-walled spores were used in a pot experiment to investigate root-knot nematode disease in tomatoes. After 40 days, the roots were pulled up and the number of root knots on the roots of each plant was recorded, and the root knot control efficacy was calculated.

[0012] In this invention, *Porphyromonas violaceus* (… P. lavender This method can rapidly produce large quantities of chlamydospores in liquid induction medium. From inoculation to the induction of large-scale chlamydospore production takes only 18 hours. This method is effective for preparing *Porphyromonas violaceum* (…). P. lavender Thick-walled spores have advantages such as short production cycle, high yield, short time consumption, low cost, simple culture conditions, and suitability for industrial production. They can also be used to control plant parasitic nematodes and can be used to develop new preparations for the control of root-knot nematode diseases, thus having great application value. Attached Figure Description

[0013] Figure 1 The purple purpureus in Example 1 ( P. lavender Microscopic image of thick-walled spores; Figure 2 The purple purpureus in Example 2 ( P. lavender Microscopic image of thick-walled spores; Figure 3 The purple purpureus in Example 3 ( P. lavender Microscopic image of thick-walled spores; Figure 4 The purple purpureus in Example 4 ( P. lavender Microscopic image of thick-walled spores; Figure 5 The purple purpureus in Example 5 ( P. lavender Microscopic image of thick-walled spores; Figure 6 The purple purpureus in Example 6 ( P. lavender Results of germination rate determination of thallus spores; Figure 7 The purple purpureus in Example 7 ( P. lavender Results of the efficacy of chlamydospores in controlling root knot disease in tomato root-knot nematode pot experiment. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited by them are incorporated herein by reference.

[0016] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0018] The following examples use *Porphyromonas violaceus* (purple purpureus) P. lavender It is deposited in the Seed Resource Bank of the State Key Laboratory of Yunnan Biological Resources Conservation and Utilization, jointly established by the Ministry of Science and Technology and Yunnan Province, with accession number YMF1.00683.

[0019] The culture medium formulations used in the following examples are as follows: PDA medium formula: Weigh 200 g of fresh potatoes, slice them and boil for 30 min. Filter the liquid through four layers of gauze, collect the filtrate and bring the volume to 1 L (the potato treatment method in the following liquid induction medium formula is the same). Add 20 g of glucose and 20 g of agar, and sterilize in a 121℃ moist heat autoclave for 30 min for later use.

[0020] The liquid induction medium is any one of the following five culture medium formulations: A. Potatoes 200 g / L, sucrose 20 g / L; B. Potatoes 200g / L, sucrose 20g / L, Ca(NO3)2 3g / L, KCl 1g / L; C. Potatoes 200g / L, sucrose 20g / L, (NH4)2SO4 3g / L, KCl 1g / L; D. Sucrose 20g / L, (NH4)2SO4 3g / L, KCl 1g / L; E, sucrose 20 g / L, Ca(NO3)2 3 g / L, KCl 1 g / L.

[0021] Example 1: A high-yielding purple purpureus ( Purple lavender The preparation method of chlamydospores, with specific steps as follows: (1) Activation and culture of strain: Pick one loopful of *Porphyromonas purpureus* using a sterile inoculation loop ( P. lavender For the bacterial culture, quickly insert an inoculation loop containing the culture into a 90mm PDA solid plate. Streak the culture medium on the surface of the plate in a "Z" pattern, starting from the edge and covering approximately one-fifth to one-quarter of the plate's area. Close the plate lid, rotate the plate approximately 120°, and flammate the inoculation loop. Cool the loop in air or against the inside of the plate lid, then streak from the end of the first streak to the next area. Repeat this process three times. Then, incubate the inoculated plate at 28°C for 5 days. After colonies have grown, use a sterile scalpel to cut single colonies into small pieces and transfer them to new PDA plates for further incubation for 5 days.

[0022] (2) Preparation of conidial suspension: Using a sterile scalpel, the colonies cultured on a new PDA plate for 5 days were cut into small pieces and transferred to an Erlenmeyer flask containing sterile water. The conidia were washed out by vortexing for 5 minutes. The filtrate was then filtered through sterile four-layer filter paper under sterile conditions to obtain the conidial suspension. Finally, the conidia were counted under a microscope, and the conidial suspension was diluted to 6.042 × 10⁻⁶. 6 CFU / mL is prepared for later use.

[0023] (3) Induction culture of chlamydospores: Prepare liquid induction medium A: potato 200 g / L, sucrose 20 g / L, sterilize in a humid heat sterilizer at 121℃ for 30 min. Under aseptic conditions, take 1 mL of the conidial suspension prepared in step (2) and inoculate it into 250 mL of liquid induction medium A, and culture in a shaker at 28℃ and 120 rpm. Set up three replicate samples for each group.

[0024] Observation of chlamydospore production: The production of chlamydospores was observed under a microscope at four time points: 12h, 18h, 24h, and 30h.

[0025] Chlamydospore staining and counting: Among the observations at the four time points mentioned above, the number of chlamydospores produced at 18 hours was the highest, and the counting was most suitable. Therefore, 18 hours was chosen as the time point for chlamydospore staining and counting. The counting results showed that under these conditions, *Porphyromonas violaceum* (*Porphyromonas violaceum*) produced at 18 hours of induction... P. lavender The number of chlamydospores produced was 8.4 × 10⁻⁶.4 cfu / mL. Optical microscope image of chlamydospores as shown below. Figure 1 As shown.

[0026] Example 2: A high-yielding purple purpureus ( Purple lavender The preparation method of chlamydospores involved preparing liquid induction medium B: potato 200 g / L, sucrose 20 g / L, Ca(NO3)2 3 g / L, KCl 1 g / L, with the remaining steps following the method described in Example 1. Counting results showed that under these conditions, after 18 h of induction, *Porphyromonas violaceus* (…)… P. lavender The number of chlamydospores produced was 3.68 × 10⁻⁶. 5 cfu / mL. Optical microscope image of chlamydospores as shown below. Figure 2 As shown.

[0027] Example 3: A high-yielding purple purpureus ( Purple lavender The preparation method of chlamydospores involved preparing liquid induction medium C: potato 200 g / L, sucrose 20 g / L, (NH4)2SO4 3 g / L, KCl 1 g / L, with the remaining methods being the same as described in Example 1. Counting results showed that under these conditions, after 18 h of induction, *Porphyromonas violaceus* (… P. lavender The number of chlamydospores produced was 1.04 × 10⁻⁶. 6 cfu / mL. Optical microscope image of chlamydospores as shown below. Figure 3 As shown.

[0028] Example 4: A high-yielding purple purpureus ( Purple lavender The preparation method of chlamydospores involved preparing liquid induction medium D: 20 g / L sucrose, 3 g / L (NH4)2SO4, and 1 g / L KCl. The remaining steps were the same as described in Example 1. Counting results showed that under these conditions, after 18 h of induction, *Porphyromonas violaceus* (…)… P. lavender The number of chlamydospores produced was 7.2 × 10⁻⁶. 5 cfu / mL. Optical microscope image of chlamydospores as shown below. Figure 4 As shown.

[0029] Example 5: A high-yielding purple purpureus ( Purple lavender The preparation method of chlamydospores involved preparing liquid induction medium E: 20 g / L sucrose, 3 g / L Ca(NO3)2, and 1 g / L KCl. The remaining steps were the same as described in Example 1. Counting results showed that under these conditions, after 18 h of induction, *Porphyromonas violaceus* (…)… P. lavender The number of chlamydospores produced was 1.6 × 10⁻⁶. 5 cfu / mL. Optical microscope image of chlamydospores as shown below. Figure 5 As shown.

[0030] Different induction media in Examples 1-5 for the effects of *Porphyromonas violaceus* (… P. lavender The production of chlamydospores is shown in Table 1: As shown in Table 1, the comparison between Examples 2 and 5, and Examples 3 and 4, shows that adding potato as a component of the induction medium increases the yield of chlamydospores. According to Examples 1-5, the chlamydospore yields in Examples 2 and 3, i.e., the induction media containing potato, (NH4)2SO4, or Ca(NO3)2, are higher than those in Examples 5 and 4, respectively, and both are significantly higher than in Example 1, which does not contain (NH4)2SO4, Ca(NO3)2, or KCl. The induction medium used in Example 3, with components of 200 g / L potato, 20 g / L sucrose, 3 g / L (NH4)2SO4, and 1 g / L KCl, produced 1.04 × 10⁻⁶ chlamydospores. 6 The highest yield was achieved at cfu / mL within the experimental range. As can be seen from the above examples, the addition of (NH4)2SO4, Ca(NO3)2, and potato can significantly increase the yield of *Porphyromonas violaceus* under these culture conditions. P. lavender The number of thick-walled spores produced.

[0031] This invention provides a purple purple purpureus ( P. lavender The method for efficient preparation of chlamydospores requires only 18 hours from inoculation to induction of chlamydospore production. It has the advantages of short production cycle, high chlamydospore yield, simple culture conditions, and is conducive to industrial production. It can be used to develop new preparations for the prevention and control of root-knot nematode diseases.

[0032] Example 6: The chlamydospore solution induced by any of the above methods was centrifuged at 9000 rpm and 25°C for 5 min. The precipitate was collected, washed repeatedly with sterile water, centrifuged 5 times, and then the chlamydospores were resuspended. They were then incubated at 25°C on 1.5% WA medium using the dilution-spreading method. The germination rate of the chlamydospores was observed and counted every 12 h. The results showed that the germination rate of the chlamydospores increased continuously with the increase of incubation time within the 48 h observation period, and the germination rate reached 100% after 48 h of incubation on nutrient-deficient WA medium. *Porphyromonas violaceus* (… P. lavender The germination rate of chlamydospores was measured as follows: Figure 6 As shown.

[0033] Example 7: Purple purpureus ( P. lavender The method for collecting chlamydospores is the same as in Example 6. The collected chlamydospores are diluted with an appropriate amount of sterile water and then counted under a microscope, so that each 200 mL of liquid contains approximately 5.4 × 10⁻⁶ spores. 7Thick-walled spores. The control group (CK) used no fungal inoculation with insects, and the experimental group used induced thick-walled spores. Each treatment was repeated 6 times. Tomato seedlings cultured for 3 days were irrigated, with each pot receiving approximately 5.4 × 10⁶ spores. 7 Thick-walled spores were used, and root-knot nematodes were added 10 days after seedling cultivation. Approximately 2000 second-instar larvae (J2) of root-knot nematodes were added to each pot. Watering was carried out intermittently according to weather conditions. After 40 days, the roots were pulled up and the number of root knots on the roots of each plant was recorded to calculate the root knot control efficacy. The results of the root knot control experiment on tomato root-knot nematode disease after 40 days showed that, compared with the control CK, *Porphyromonas violaceus* (… P. lavender Thick-walled spores showed good control effects against tomato root-knot nematode disease, reducing the total number of root knots by up to 89.50%, with a significant difference (P<0.01) as determined by the T-test. *Porphyromonas violaceus* (… P. lavender The results of the root-knot control efficacy test of chlamydospores against tomato root-knot nematode disease are as follows: Figure 7 As shown.

[0034] The liquid induction medium described in this invention can produce a large number of chlamydospores after 18 hours of induction culture, and the germination rate can reach 100% after 48 hours of culture on nutrient-deficient WA medium. The production cycle is short, the yield is high, the time consumption is short, the cost is low, and the culture conditions are simple, making it perfectly suitable for large-scale industrial production. Pot experiments on tomato root-knot nematode disease using the obtained chlamydospores showed good control effects after 40 days, reducing the total number of root knots by 89.50%. The difference was statistically significant (P<0.01) according to the T-test, indicating that this method can be used to develop new formulations for the control of root-knot nematode diseases and has certain application value.

[0035] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-yielding purple purpureus ( Purpureocillium lavendulum The method for preparing thallus spores is characterized by: The purple purpureus ( P. lavendulum YMF1.00683, the conidia produced on PDA solid medium were washed out with sterile water to prepare a conidia suspension, and then inoculated into liquid induction medium. High production of thick-walled spores can be achieved by shaking the culture in a flask at 28°C for 12-30 hours. The liquid induction medium is any one of the following five culture medium formulations: A. Potatoes 200 g / L, sucrose 20 g / L; B. Potatoes 200g / L, sucrose 20g / L, Ca(NO3)2 3g / L, KCl 1g / L; C. Potatoes 200g / L, sucrose 20g / L, (NH4)2SO4 3g / L, KCl 1g / L; D. Sucrose 20g / L, (NH4)2SO4 3g / L, KCl 1g / L; E, sucrose 20g / L, Ca(NO3)2 3 g / L, KCl 1g / L.

2. A high-yielding purple purpureus according to claim 1 ( Purpureocillium lavendulum The method for preparing thallus spores is characterized by: The liquid induction culture medium consisted of: C, potato 200 g / L, sucrose 20 g / L, (NH4)2SO4 3 g / L, and KCl 1 g / L.

3. A high-yielding purple purpureus according to claim 1 ( Purpureocillium lavendulum The method for preparing thallus spores is characterized by: The specific preparation method of the conidial suspension is as follows: *Porphyromonas violaceus* (… P. lavendulum The inoculum was inoculated onto PDA solid medium and incubated at 28°C for 5 days. The conidia in the mycelial blocks were then washed and collected with sterile water to prepare a conidial suspension. The conidial suspension contained 6.042 × 10⁻⁶ spores. 6 cfu / mL.

4. A high-yielding purple purple purpureus according to claim 1 ( Purpureocillium lavendulum The method for preparing thallus spores is characterized by: The inoculation amount of conidial suspension in the liquid induction medium was 1 mL containing 6.042 × 10⁶ spores per 250 mL of liquid induction medium. 6 Conidial suspension of cfu / mL conidia; culture conditions in shake flasks: 28℃, 120rpm for 18h.

5. A high-yielding purple purple purpureus according to claim 1 ( Purpureocillium lavendulum The method for preparing thallus spores is characterized by: The obtained purple purple purpureus ( P. lavendulum Thick-walled spores were cultured at 25°C on 1.5% WA medium. Germination of thalassiospores was observed and germination rate was calculated at 12h, 24h, 36h and 48h.

6. A high-yielding purple purple purpureus according to claim 1 ( Purpureocillium lavendulum The method for preparing thallus spores is characterized by: The obtained purple purple purpureus ( P. lavendulum A pot experiment was conducted on tomato root-knot nematode disease using chlamydospores. Each pot was irrigated with 5.4 × 10⁻⁶ spores. 7 One thick-walled spore was collected, and the roots were pulled up and surveyed 40 days later. The number of root knots on the roots of each plant was recorded, and the root knot control efficacy was calculated.

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