A method for producing chlamydospores by liquid fermentation of Subseptate Digitaria tenuisporium

Abstract

The invention discloses a method for producing chlamydospores by liquid fermentation of subseptate spores of tenuisporium, and belongs to the field of agricultural biotechnology. Dactylellina haptotyla ) is activated on a PDA medium, and the D. haptotyla fungus blocks on the PDA medium or the conidia produced by the washed D. haptotyla on the PDA medium are inoculated into a liquid induction medium prepared with an amino-free yeast nitrogen source and glucose or sucrose for shake flask culture, so that a large number of chlamydospores can be produced. The liquid culture medium induces D. haptotyla to produce a large number of chlamydospores. The liquid induction medium is simple to prepare and does not require harsh culture conditions. It is easy to use the liquid induction medium for fermentation expansion of various scales, thereby expanding the application, which is beneficial to the research and development of plant parasitic nematode biocontrol agents and has good application prospects.

Description

Technical Field

[0001] The invention relates to a method for producing chlamydospores by liquid fermentation of subseptate spores of tenuisporium, and belongs to the field of agricultural biotechnology. Background Art

[0002] Crop diseases caused by plant parasitic nematodes are the second largest category of diseases after fungal diseases, especially root-knot nematodes, which are particularly harmful. Plant parasitic nematodes can directly damage their hosts or act as virus carriers. In addition, the wounds they cause are also an important cause of soil-borne diseases such as root rot, causing even more serious indirect losses. Therefore, the prevention and control of plant parasitic nematode diseases needs to be addressed urgently. The application of chemical nematicides is still the main measure for the prevention and control of plant parasitic nematodes; however, highly toxic and highly residual nematicides have obvious negative effects on the environment and human health; therefore, biological control has attracted much attention as an environmentally friendly and sustainable prevention and control strategy. Nematode-trapping fungi, as the natural enemy of parasitic nematodes, are one of the main sources for the development of nematode biocontrol agents.

[0003] Subseptidyl spores of tenuisporium Dactylellina haptotyla ) is a typical nematode-catching fungus that can produce sticky balls to capture nematodes, and enter the host through enzymatic degradation and mechanical pressure to obtain nutrition. In addition, studies have shown that D. haptotyla specifically produces 2-furoic acid, which is toxic to nematodes, during the process of catching nematodes; pot experiments have also shown that 2-furoic acid can effectively reduce the number of root knots in tomato roots. D. haptotyla is a nematode biocontrol fungus with development prospects.

[0004] At present, most of the nematode biocontrol agents developed by fungi are mycelium or conidia preparations, which have poor stability, short shelf life, and need to be produced on order, which seriously restricts their widespread use. Chlamydospores are thick-walled asexual spores transformed from fungal mycelium or conidia, and have strong resistance to adverse environmental conditions. As a special reproductive body, chlamydospores have the characteristics of strong stress resistance and long shelf life. They are insensitive to soil antibacterial effects and have greater biocontrol potential. They are good materials for the research and development of biocontrol agents. The preparations developed by them can make up for the shortcomings of existing nematode biocontrol agents.

[0005] At present, there is no report on inducing D. haptotyla to produce chlamydospores. Therefore, the present invention provides a method for producing a large amount of chlamydospores by liquid fermentation of D. haptotyla, providing a new preparation for developing prevention and treatment of root-knot nematode diseases. Summary of the invention

[0006] In order to enable Dactylocystis spp. to produce a large number of chlamydospores to develop new nematode biocontrol agents, the present invention provides a method for producing chlamydospores by liquid fermentation of Dactylocystis spp., and the specific steps are as follows:

[0007] (1) Activation and culture of D. haptotyla: D. haptotyla was activated and cultured on PDA medium. Plates with conidia or full hyphae were selected by observation under a microscope.

[0008] (2) Chlamydospore induction culture: This step is achieved by the following two methods:

[0009] Method 1: Rinse the plate producing conidia in step (1) with a liquid induction culture medium, collect the liquid to obtain a conidia suspension, and inoculate the conidia suspension into a liquid culture medium for induction culture to obtain a culture solution containing thick-walled spores.

[0010] Method 2: In step (1), the mycelium blocks that have grown all over the plate (6 cm) are directly inoculated into a liquid induction culture medium for induction culture to obtain a culture solution containing thick-walled spores.

[0011] The liquid induction medium is prepared from an amino-free yeast nitrogen source, glucose or sucrose, and water, wherein the water is purified water or tap water, the added amount of the amino-free yeast nitrogen source is 1.7-4.8 g / L, and the added amount of the glucose or sucrose is 20-40 g / L.

[0012] Preferably, the subseptate spore of the tenuisporium was deposited in the General Microbiological Center of the China Microorganism Collection Administration on May 27, 2024, with a deposit number of CGMCC No. 41325, and a classification name of: Dactylellina Happtotyla .

[0013] Preferably, the activation condition in step (1) is 28°C.

[0014] Preferably, in method 1 of step (2), the inoculation amount of the conidia suspension is: 8-12 mL of the conidia suspension is inoculated into every 100 mL of liquid induction medium, and the conidia suspension contains 4.8×10 5 ~8.4×10 5 Conidia.

[0015] Preferably, in the second method of step (2): one bacterial block (6 cm) with hyphae growing all over the plate is inoculated into every 100 mL of liquid induction culture medium.

[0016] Preferably, the conditions for the chlamydospore induction culture in step (2) method 1 and method 2 are: in a shaking incubator at 28°C and a rotation speed of 90-180 rpm for 6-7 days.

[0017] Beneficial effects of the present invention

[0018] The present invention realizes for the first time that D. haptotyla produces a large number of chlamydospores in a liquid induction culture medium. The liquid induction culture medium is simple to prepare and has no stringent requirements on culture conditions. The liquid induction culture medium is easy to use for fermentation expansion of various scales, thereby expanding the application, which is beneficial to the research and development of nematode biocontrol agents and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an optical microscopic image of the chlamydospores produced by D. haptotyla in Method 1 of Example 1.

[0020] Figure 2 This is an optical microscope image of the chlamydospores produced by D. haptotyla in Method 2 of Example 1.

[0021] Figure 3 This is an optical microscopic image of the thick-walled spores produced by D. haptotyla in Example 2.

[0022] Figure 4 This is an optical microscopic image of the thick-walled spores produced by D. haptotyla in Example 3.

[0023] Figure 5 This is an optical microscopic image of the thick-walled spores produced by D. haptotyla in Example 4.

[0024] Figure 6 This is an optical microscopic image of the thick-walled spores produced by D. haptotyla in Example 5.

[0025] Figure 7 This is an optical microscopic image of the thick-walled spores produced by D. haptotyla in Example 6.

[0026] Figure 8 This is an optical microscope picture of the thick-walled spores not produced by D. haptotyla in Comparative Example 1.

[0027] Fig. 9 This is an optical microscope picture of the thick-walled spores not produced by D. haptotyla in Comparative Example 2. DETAILED DESCRIPTION

[0028] In order to make the purpose, 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. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Unless defined otherwise, 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 belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0030] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0031] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0032] The D. haptotyla used in the following examples was deposited in the General Microbiological Center of the China Microbiological Collection Administration on May 27, 2024, with a deposit number of CGMCC No. 41325 and a classification name of: Dactylellina haptotyla .

[0033] The culture medium formula used in the following examples is as follows:

[0034] PDA culture medium formula: 200g fresh potato slices were boiled for 30min and filtered. 20g glucose and 15g agar were added to the filtrate. After complete dissolution, the volume was adjusted to 1L. Sterilized in a 121℃ wet heat autoclave for 30min and set aside.

[0035] Liquid induction medium formula: Liquid induction medium No. 1: YNB (amino-free yeast nitrogen source) medium 1.7g / L, glucose 20g / L, purified water 1L; Liquid induction medium No. 2: YNB (amino-free yeast nitrogen source) medium 4g / L, glucose 20g / L, tap water 1L; Liquid induction medium No. 3: YNB (amino-free yeast nitrogen source) medium 2g / L, glucose 40g / L, tap water 1L. Liquid induction medium No. 4: YNB (amino-free yeast nitrogen source) medium 4.8g / L, glucose 40g / L, tap water 1L; Liquid induction medium No. 5: YNB (amino-free yeast nitrogen source) medium 3g / L, glucose 30g / L, tap water 1L; Liquid induction medium No. 6: YNB (amino-free yeast nitrogen source) medium 3g / L, sucrose 30g / L, tap water 1L. All culture media were sterilized in a 121℃ wet heat sterilizer for 30min for later use.

[0036] Example 1

[0037] A method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium, the specific steps are as follows:

[0038] (1) Activation and culture of D. haptotyla: Use a sterile scalpel to cut D. haptotyla from PDA into pieces and inoculate them into new PDA solid culture medium plates (6 cm) and culture them at 28°C until conidia are produced or the plates are full of hyphae.

[0039] (2) Chlamydospore induction culture: This step can be achieved by the following two methods:

[0040] Method 1: Rinse the plate produced with conidia in step (1) with liquid induction medium No. 1, collect the liquid to obtain a conidia suspension, and count the conidia suspension under a microscope. The conidia content in the conidia suspension is about 6×10 4 ~7×10 4 8, 10 and 12 mL of the conidia suspension were inoculated into 100 mL of No. 1 liquid induction medium and cultured at 180 rpm and 28°C for 2 days. The culture was then transferred to 90 rpm and 28°C for further 5 days to obtain a culture solution containing thick-walled spores.

[0041] Method 2: Inoculate the mycelium blocks that cover the plate in step (1) directly into 100 mL of liquid induction medium No. 1 and culture at 180 rpm and 28°C for 2 days, then transfer to 90 rpm and continue culturing at 28°C for 5 days to obtain a culture solution containing thick-walled spores.

[0042] The production of chlamydospores was observed and counted every day from the second day. The results showed that the No. 1 liquid induction medium inoculated with bacterial blocks produced the most chlamydospores on the sixth day, with the number of chlamydospores produced being 2.02×10 8 / mL, optical microscope image of thick walled spores Figure 1 As shown in Figure 2, the liquid induction medium No. 1 inoculated with 10 mL of conidia suspension produced the most chlamydospores on the 6th day, with a number of 2.34×10 8 / mL, optical microscope image of thick walled spores Figure 2 shown.

[0043] Example 2

[0044] A method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium, the specific steps are as follows:

[0045] (1) Activation and culture of D. haptotyla: Use a sterile scalpel to cut D. haptotyla from PDA into pieces and inoculate them into new PDA solid culture medium plates (6 cm) and culture them at 28°C until the mycelium fills the plate.

[0046] (2) Chlamydospore induction culture: The mycelium blocks that covered the plate in step (1) were directly inoculated into 100 mL of liquid induction medium No. 2 and cultured at 180 rpm and 28°C for 2 days. The culture medium was then transferred to 90 rpm and cultured at 28°C for 5 days to obtain a culture solution containing chlamydospores.

[0047] The production of chlamydospores was observed every day from the second day. The results showed that chlamydospores could be induced on the fourth day, and the maximum number of chlamydospores was produced on the sixth day, with a total number of 2.20×10 8 / mL, optical microscope image of thick walled spores Figure 3 shown.

[0048] Example 3

[0049] A method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium, the specific steps are as follows:

[0050] (1) Activation and culture of D. haptotyla: Use a sterile scalpel to cut D. haptotyla from PDA into pieces and inoculate them into new PDA solid culture medium plates (6 cm) and culture them at 28°C until the mycelium fills the plate.

[0051] (2) Chlamydospore induction culture: The mycelium blocks that covered the plate in step (1) were directly inoculated into 100 mL of liquid induction medium No. 3 and cultured at 180 rpm and 28°C for 2 days. The culture medium was then transferred to 90 rpm and cultured at 28°C for 5 days to obtain a culture solution containing chlamydospores.

[0052] The production of chlamydospores was observed every day from the second day. The results showed that chlamydospores could be induced on the fourth day, and the maximum number of chlamydospores was produced on the sixth day, with a total number of 9.33×10 7 / mL. Optical microscope image of thick-walled spores Figure 4 shown.

[0053] Example 4

[0054] A method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium, the specific steps are as follows:

[0055] (1) Activation and culture of D. haptotyla: Use a sterile scalpel to cut D. haptotyla from PDA into pieces and inoculate them into new PDA solid culture medium plates (6 cm) and culture them at 28°C until the mycelium fills the plate.

[0056] (2) Thick-walled spore induction culture: The mycelium blocks that covered the plate in step (1) were directly inoculated into 100 mL of liquid induction medium No. 4 and cultured at 180 rpm and 28°C for 2 days. The culture medium was then transferred to 90 rpm and cultured at 28°C for 5 days to obtain a culture solution containing thick-walled spores.

[0057] The production of chlamydospores was observed every day from the second day. The results showed that chlamydospores could be induced on the fourth day, and the maximum number of chlamydospores was produced on the sixth day, with a total number of 1.42×10 8 / mL. Optical microscope image of thick-walled spores Figure 5 shown.

[0058] Example 5

[0059] A method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium, the specific steps are as follows:

[0060] (1) Activation and culture of D. haptotyla: Use a sterile scalpel to cut D. haptotyla from PDA into pieces and inoculate them into new PDA solid culture medium plates (6 cm) and culture them at 28°C until the mycelium fills the plate.

[0061] (2) Thick-walled spore induction culture: The mycelium blocks that covered the plate in step (1) were directly inoculated into 100 mL of liquid induction medium No. 5 and cultured at 180 rpm and 28°C for 2 days. The culture medium was then transferred to 90 rpm and cultured at 28°C for another 5 days to obtain a culture solution containing thick-walled spores.

[0062] The production of chlamydospores was observed every day from the second day. The results showed that chlamydospores could be induced on the fourth day, and the maximum number of chlamydospores was produced on the sixth day, with a total number of 1.04×10 8 / mL. Optical microscope image of thick-walled spores Figure 6 shown.

[0063] Example 6

[0064] A method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium, the specific steps are as follows:

[0065] (1) Activation and culture of D. haptotyla: Use a sterile scalpel to cut D. haptotyla from PDA into pieces and inoculate them into new PDA solid culture medium plates (6 cm) and culture them at 28°C until the mycelium fills the plate.

[0066] (2) Chlamydospore induction culture: The mycelium blocks that covered the plate in step (1) were directly inoculated into 100 mL of liquid induction medium No. 6 and cultured at 180 rpm and 28°C for 2 days. The culture medium was then transferred to 90 rpm and cultured at 28°C for 5 days to obtain a culture solution containing chlamydospores.

[0067] The production of chlamydospores was observed every day from the second day. The results showed that chlamydospores could be induced on the fourth day, and the maximum number of chlamydospores was produced on the sixth day, with a total number of 1.93×10 8 / mL. Optical microscope image of thick-walled spores Figure 7 shown.

[0068] Table 1 shows the effects of different induction culture media in Examples 1 to 6 on the production of chlamydospores by D. haptotyla.

[0069] Table 1 Effects of different induction media on the production of chlamydospores by D. haptotyla

[0070]

[0071] Comparative Example 1

[0072] For comparison, the only difference between this comparative example and Example 1 is that the liquid induction culture medium used is different, and the remaining method steps are the same as Example 1.

[0073] The liquid induction culture medium used in this comparative example is the whip-shaped spore thick-walled spore induction liquid culture medium reported in Application No. 202310462862.5: glucose (10 g / L), NaNO3 (3 g / L), MgSO4 (0.5 g / L), FeSO4 (0.01 g / L), KCl (0.5 g / L), K2HPO4 (1.0 g / L), and purified water (1 L).

[0074] The production of chlamydospores was observed every day from the second day. No chlamydospores were produced on the seventh day. The results were as follows: Figure 8 (Mycelial status on the 7th day) showed that Comparative Example 1 could not induce thick-walled spores.

[0075] Comparative Example 2

[0076] For comparison, the only difference between this comparative example and Example 1 is that the liquid induction culture medium used is different, and the remaining method steps are the same as Example 1.

[0077] The liquid induction culture medium used in this comparative example is the purple chlamydospore induction liquid culture medium reported in Application No. 202311574473.8: sucrose (20 g / L), (NH4)2SO4 (3 g / L), KCl (1 g / L), and purified water (1 L).

[0078] The production of chlamydospores was observed every day from the second day. No chlamydospores were produced on the seventh day. The results were as follows: Fig. 9 (Mycelial status on the 7th day) showed that Comparative Example 2 could not induce thick-walled spores.

[0079] By comparing the examples with the comparative examples, it was found that D. haptotyla could produce a large number of chlamydospores only when the liquid induction culture medium of the present invention was used for induction culture, while D. haptotyla did not produce chlamydospores when the liquid induction culture medium was used for induction culture.

[0080] In summary, the present invention provides a method for efficiently preparing chlamydospores of D. haptotyla. The method uses liquid culture to induce D. haptotyla to produce a large number of chlamydospores. The preparation process is simple to operate, the culture conditions are not harsh, and the culture medium is easy to use for various scales of fermentation, thereby expanding the application, which is beneficial to the research and development of plant parasitic nematode biocontrol agents and has good application prospects.

[0081] 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 aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A subseptate spore of the myxosporea Dactylellina haptotyla ) A method for producing chlamydospores by liquid fermentation, characterized in that: The specific steps are as follows: (1) Activation and culture of Dactylocystis spp.: Dactylocystis spp. was activated and cultured on PDA medium, and the plates with conidia or full hyphae were selected; (2) Chlamydospore induction culture: This step is achieved by one of the following two methods: Method 1: Rinse the plate producing conidia in step (1) with a liquid induction medium, collect the liquid to obtain a conidia suspension, inoculate the conidia suspension into a liquid induction medium for culturing to obtain a culture solution containing thick-walled spores; Method 2: Inoculate the bacterial mass with hyphae growing all over the plate in step (1) directly into a liquid induction culture medium for induction culture to obtain a culture solution containing thick-walled spores; The liquid induction medium is prepared from an amino-free yeast nitrogen source, glucose or sucrose, and water, wherein the water is purified water or tap water, the added amount of the amino-free yeast nitrogen source is 1.7-4.8 g / L, and the added amount of the glucose or sucrose is 20-40 g / L.

2. The method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium according to claim 1, characterized in that: The subseptate spore of the tenuisporium was deposited in the General Microbiological Center of the China Microorganism Collection Administration on May 27, 2024, with the deposit number: CGMCC No. 41325, and the classification name: Dactylellina haptotyla .

3. The method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium according to claim 1, characterized in that: The activation condition in step (1) is 28°C.

4. The method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium according to claim 1, characterized in that: In method 1 of step (2), the inoculation amount of the conidia suspension is: 8-12 mL of the conidia suspension is inoculated into every 100 mL of liquid induction medium, and the conidia suspension contains 4.8×10 5 ~8.4×10 5 Conidia.

5. The method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium according to claim 1, characterized in that: In the second method of step (2): inoculate 1 bacterial block with hyphae that covers the entire plate into every 100 mL of liquid induction culture medium.

6. The method for producing chlamydospores by liquid fermentation of Dactylotrichum tenuisporium according to claim 1, characterized in that: The conditions for the chlamydospore induction culture in step (2) method 1 and method 2 are 28° C. and cultured in a shaking incubator at a rotation speed of 90-180 rpm for 6-7 days.

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