A method for inducing trichoderma to produce chlamydospores

Abstract

The application discloses a method for inducing differentiation of Trichoderma to produce chlamydospores, and relates to the field of agricultural biotechnology. The method comprises the following steps: adding an inducing substance to a culture medium during fermentation culture to induce differentiation of Trichoderma to produce chlamydospores; and the inducing substance comprises at least one of ammonium persulfate, ammonium bisulfate, ammonium chloride or ammonium bromide. By adding the inducing substance at the beginning of fermentation or in the middle of fermentation, the spore type of Trichoderma can be changed from conidium to chlamydospore, so that the proportion of chlamydospores in the Trichoderma biocontrol preparation can be improved. Therefore, the method has important significance for solving the problem of short shelf life of Trichoderma. The method has a significant inducing effect in various culture media and various specifications of bioreactors, the inducing substance is low in price, simple in operation during use, suitable for large-scale production of various scales, and has a wide market prospect.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a method for inducing Trichoderma to differentiate and produce chlamydospores. Background Technology

[0002] With the long-term and extensive use of chemical agents, problems such as environmental pollution and drug resistance have become increasingly apparent. Therefore, the use of microorganisms or their metabolites to control plant diseases has attracted widespread attention. Trichoderma, as an important biocontrol fungus for plant diseases, has a significant growth-promoting effect on plants. In 1934, research first discovered that Trichoderma has antagonistic effects on several soil fungi, and subsequently, its role in controlling soil-borne diseases was gradually recognized. In recent years, Trichoderma has been widely considered the most likely biocontrol agent to replace many chemical fungicides. As a class of antagonistic microorganisms that can promote crop growth and are abundant in resources, they play an increasingly important role in the development of sustainable agriculture. Currently, Trichoderma is widely used to control soil-borne diseases such as potato dry rot, tobacco root rot, cucurbit wilt, tomato gray mold, cotton verticillium wilt, and crop gray mold. In horticultural plants, Trichoderma has a good inhibitory effect on various pathogens such as apple rot, poplar leaf blight, and poplar bark rot. It also has a good effect on the prevention and control of diseases of medicinal plants such as ginseng and chuanxiong.

[0003] Due to the broad application prospects of Trichoderma, large-scale industrial production of commercial formulations has become an inevitable trend. Currently, commercial Trichoderma agents mainly consist of conidia. Although Trichoderma conidia produce a large number of spores, their resistance to adverse conditions is weak, resulting in a high mortality rate during the spray drying process and subsequent transportation and storage, which makes them unsuitable for use as biological pesticides. Chlamydospores, on the other hand, have the characteristics of long survival time and strong resistance to adverse conditions. Therefore, chlamydospore formulations can extend the shelf life of fungal agents and increase the utilization value of Trichoderma. However, Trichoderma chlamydospores are dormant spores formed when individual cells swell, protoplasts condense, cell walls thicken, and the mycelium develops to a certain stage and encounters adverse external environments. Therefore, large-scale production of chlamydospores is difficult to achieve, making large-scale production of fungal chlamydospores one of the main technical bottlenecks in the industrialization of biocontrol fungi.

[0004] Therefore, finding a method that can effectively induce Trichoderma to differentiate and produce chlamydospores is key to overcoming this technical bottleneck. Summary of the Invention

[0005] The purpose of this invention is to provide a method for inducing Trichoderma to differentiate and produce chlamydospores, thereby solving the problems existing in the prior art. The method provided by this invention can induce Trichoderma to change its sporulation type from conidia to chlamydospores, which can greatly increase the proportion of chlamydospores in Trichoderma biocontrol agents and help extend the shelf life of Trichoderma.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for inducing Trichoderma to differentiate and produce chlamydospores, comprising the step of adding an inducing substance to the fermentation system during fermentation culture to induce Trichoderma to differentiate and produce chlamydospores;

[0008] The inducing substance includes at least one of ammonium persulfate, ammonium bisulfate, ammonium chloride, or ammonium bromide.

[0009] Furthermore, the amount of the inducing substance added relative to the culture medium in the fermentation system is 0.5-6 g / L.

[0010] Preferably, the amount of the inducing substance added relative to the culture medium in the fermentation system is 2 g / L.

[0011] Furthermore, the inducing substance is added 0-96 hours after the start of fermentation.

[0012] Preferably, the inducing substance is added 48 hours after the start of fermentation.

[0013] Furthermore, the fermentation culture temperature is 30°C and the rotation speed is 200 rpm.

[0014] The present invention also provides the application of an inducing substance in inducing Trichoderma differentiation to produce chlamydospores, wherein the inducing substance includes at least one of ammonium persulfate, ammonium bisulfate, ammonium chloride, or ammonium bromide.

[0015] The present invention discloses the following technical effects:

[0016] This invention, by adding an inducing substance at the initial or mid-stage of Trichoderma fermentation, can transform the sporulation type of Trichoderma from conidia to chlamydospores, thereby significantly increasing the proportion of chlamydospores in Trichoderma biocontrol agents. Therefore, this method is of great significance in solving the problem of short shelf life of Trichoderma. This induction method has significant induction effects in various culture media and bioreactors of various sizes. Furthermore, the inducing substance used is inexpensive, the operation is simple, and it is suitable for large-scale production of various sizes, with broad market prospects.

[0017] Chlamydospores, as a type of stress-resistant spore, differentiate to help the fungus survive adverse environmental conditions. Previous work screened and identified inducing substances that induce hyphal differentiation and chlamydospore production. Transcriptome analysis was performed to investigate the mechanism by which these inducing substances regulate sporulation, resulting in the construction of 12 cDNA libraries. After quality control, all samples achieved a Clean Data greater than 6.77 Gb, Q20 greater than 97%, and Q30 greater than 92%. GC content ranged from 53.46% to 54.17%. Analysis identified 1273 differentially expressed genes (DEGs) closely related to chlamydospore production. Further annotation analysis of these DEGs revealed the intrinsic mechanism by which the addition of inducing substances alters sporulation type and increases chlamydospore yield. The study found that the cell wall integrity (CWI) pathway plays a crucial role in cellular responses to external stress, and Wsc1, as a cell wall baroreceptor, mediates CWI pathway activation. This invention found that the addition of inducing substances significantly upregulated the expression of cell wall integrity and stress response components (WSCs), indicating that the addition of inducing substances altered the sporulation mode and increased chlamydospore production by introducing environmental stress into the cells. Furthermore, analysis revealed that when faced with environmental stress from the inducing substances, the cells also contracted carbohydrate metabolism, leading to significant changes in the expression levels of glycogen and lipid metabolism genes such as α-amylase (AMY), glucosylamylase (SGA1), phosphoglucose mutase (pgm), glycerol 3-kinase (GLYK), fatty acid synthase β subunit (Fas1), and fatty acid synthase α subunit (Fas2). This resulted in the accumulation of energy storage substances and changes in fatty acid types, providing a material basis for chlamydospore production. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] 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. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] In the following examples, *Trichoderma harzianum* T4 was used. Its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 17197, the accession date is March 25, 2019, and the accession location is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. *Trichoderma virens* T22 and *Trichoderma asperellum* Th have been disclosed in the literature “Jiang Xianquan et al., Postharvest stem spot rot of melon and its biological control, Journal of Plant Protection, 2007, 34(2): 129-135”.

[0024] The culture medium formulations used in the following examples are as follows:

[0025] The PDA medium formula is as follows: Cut 200g of potatoes into pieces, boil for 10 minutes, filter with gauze, take the extract and make up to 1L, add 20g of glucose and 20g of agar.

[0026] The shake flask liquid culture medium formula is as follows: soluble starch 23.85 g / L, wheat bran 4.83 g / L, ammonium sulfate 0.5 g / L, calcium carbonate 1 g / L, potassium dihydrogen phosphate 1 g / L and Tween 5 mL / L.

[0027] Example 1

[0028] (1) Trichoderma harzianum T4, Trichoderma viride T22, and Trichoderma th were inoculated onto PDA medium and cultured in a constant temperature incubator at 28℃ for 72 h. Mycelia were scraped off with a toothpick and placed in sterile water, vortexed thoroughly, and then filtered through gauze. The filtrate was used to count the conidia yield under a microscope and diluted to 1×10⁻⁶. 7 CFU / mL was used as the seed solution.

[0029] (2) Add 0.2g of the inducing substance ammonium persulfate to 100mL of shake flask liquid culture medium to obtain the induction-type shake flask liquid culture medium.

[0030] (3) The seed culture was inoculated at a ratio of 1% (v / v) into a 500 mL Erlenmeyer flask containing 100 mL of induction-type shake flask liquid medium, and fermented at 30 °C and 200 rpm for 168 h. After fermentation, the yield of chlamydospores was counted under a microscope. Three replicates were set up for each group.

[0031] Example 2

[0032] Same as Example 1, except that ammonium persulfate is replaced with ammonium bisulfate, while the amount added in step (2) remains the same.

[0033] Example 3

[0034] Same as Example 1, except that ammonium persulfate is replaced with ammonium chloride, while the amount added in step (2) remains the same.

[0035] Example 4

[0036] Same as Example 1, except that ammonium persulfate is replaced with ammonium bromide, while the amount added in step (2) remains the same.

[0037] Comparative Example 1

[0038] Same as Example 1, except that the original shake flask liquid culture medium (i.e. without the addition of ammonium persulfate) was used for fermentation culture.

[0039] Comparative Example 2

[0040] Same as Example 1, except that ammonium persulfate is replaced with ammonium oxalate, while the amount added in step (2) remains the same.

[0041] Comparative Example 3

[0042] Same as Example 1, except that ammonium persulfate is replaced with ammonium carbonate, while the amount added in step (2) remains the same.

[0043] Comparative Example 4

[0044] Same as Example 1, except that ammonium persulfate is replaced with ammonium bicarbonate, while the amount added in step (2) remains the same.

[0045] The sporulation of Trichoderma in Examples 1-4 and Comparative Examples 1-4 is shown in Table 1-3:

[0046] Table 1. Effects of different inducing substances on sporulation of Trichoderma harzianum T4 (cfu / mL)

[0047]

[0048] As shown in Table 1, compared to Comparative Example 1 (without any inducing agent), adding ammonium persulfate, ammonium bisulfate, ammonium chloride, or ammonium bromide as inducing agents can change the sporulation mode and significantly increase the yield of chlamydospores. Among them, ammonium persulfate (Example 1) showed the best effect, with a chlamydospore yield of 1.70 × 10⁻⁶. 9 cfu / L. In Comparative Examples 2, 3, and 4, ammonium salts such as ammonium oxalate, ammonium carbonate, and ammonium bicarbonate were added as inducing agents, respectively. The results showed that they were not conducive to the production of chlamydospores. Compared with Comparative Example 1 (without any inducing agents), they actually reduced the yield of chlamydospores.

[0049] Table 2. Effects of different inducing substances on sporulation of Trichoderma viride T22 (cfu / mL)

[0050]

[0051] Table 3. Effects of different inducing substances on sporulation of Trichoderma hydatids (Th) (cfu / mL)

[0052]

[0053] As shown in Tables 2 and 3, among *Trichoderma viride* T22 and *Trichoderma hydathodes* Th, the sporulation rate of each strain differs slightly due to the characteristics of the strains themselves. However, the effects of each inducing substance are basically the same. Ammonium sulfate, ammonium bisulfate, ammonium chloride, or ammonium bromide can still efficiently induce the production of chlamydospores as inducing substances.

[0054] Experimental Example 1

[0055] The final concentration of ammonium persulfate in step (2) of Example 1 was adjusted to 0.5, 1, 3, 4, 5, 6, and 7 g / L, respectively, while other steps were the same as in Example 1, and fermentation was carried out. After fermentation, the yield of chlamydospores was counted under a microscope, and the results are shown in Table 4. Three replicates were set up for each group.

[0056] Table 4. Effects of different ammonium persulfate concentrations on sporulation of Trichoderma harzianum T4 (cfu / mL)

[0057]

[0058]

[0059] As shown in Table 4, the addition concentration of ammonium persulfate in the range of 0.5-6 g / L can increase the yield of chlamydospores, with 2 g / L being the optimal concentration, yielding the highest yield of chlamydospores.

[0060] Experiment Example 2

[0061] (1) After inoculating Trichoderma harzianum T4 onto PDA medium, the mixture was cultured in a constant temperature incubator at 28℃ for 72 h. Mycelia were scraped off with a toothpick and placed in sterile water. After thorough vortexing, the mixture was filtered through gauze. The conidia yield of the filtrate was counted under a microscope and diluted to 1×10⁻⁶. 7 CFU / mL was used as the seed solution.

[0062] (2) The seed culture was inoculated at a ratio of 1% (v / v) into 500 mL Erlenmeyer flasks containing 100 mL of shake-flask liquid medium, as shown in Table 3. After fermentation at 30℃ and 200 rpm for 24, 48, 72 and 96 h respectively, 0.2 g of inducing substance ammonium persulfate was added per 100 mL of shake-flask liquid medium. Fermentation was then continued under the same conditions until the total fermentation time was 168 h. After the fermentation was completed, the yield of chlamydospores was counted under a microscope, and the results are shown in Table 5. Three replicates were set up for each group.

[0063] Table 5. Effects of different inducing substance addition times on sporulation of Trichoderma (cfu / mL)

[0064]

[0065] As shown in Table 5, when the inducing agent is added at different fermentation times, the yield of chlamydospores can be significantly increased regardless of whether the inducing agent is added at 24, 48, 72 and 96 hours of fermentation. The optimal time to add the inducing agent is 48 hours after the start of fermentation, when the yield of chlamydospores is the highest.

[0066] Example 5

[0067] (1) After inoculating Trichoderma harzianum T4 onto PDA medium, the mixture was cultured in a constant temperature incubator at 28℃ for 72 h. Mycelia were scraped off with a toothpick and placed in sterile water. After thorough vortexing, the mixture was filtered through gauze. The conidia yield of the filtrate was counted under a microscope and diluted to 1×10⁻⁶. 7 CFU / mL was used as the seed solution.

[0068] (2) The seed culture was inoculated into a 5L fermenter containing 3L of fermentation medium at an inoculation ratio of 1% (v / v). 30mL of silicone polyether defoamer was added, and fermentation was carried out at 30℃ and 200rpm for 48h. Then, 6g of ammonium persulfate was added to the fermenter, and the fermentation was continued under the same conditions for 120h. After the fermentation was completed, the yield of chlamydospores was counted under a microscope. Three replicates were set up for each group.

[0069] The fermentation medium formula used in this embodiment is: glucose 25g / L, corn steep liquor 5g / L, sodium chloride 25g / L, and molasses 50g / L.

[0070] Comparative Example 5

[0071] (1) After inoculating Trichoderma harzianum T4 onto PDA medium, the mixture was cultured in a constant temperature incubator at 28℃ for 72 h. Mycelia were scraped off with a toothpick and placed in sterile water. After thorough vortexing, the mixture was filtered through gauze. The conidia yield of the filtrate was counted under a microscope and diluted to 1×10⁻⁶. 7 CFU / mL was used as the seed solution.

[0072] (2) The seed culture was inoculated into a 5L fermenter containing 3L of fermentation medium at an inoculation ratio of 1% (v / v), and 30mL of silicone polyether defoamer was added. The fermentation was carried out at 30℃ and 200rpm for 168h. After the fermentation was completed, the yield of chlamydospores was counted under a microscope. Three replicates were set up for each group.

[0073] The fermentation medium formulation used in this comparative example is the same as that in Example 5.

[0074] The experimental results of Example 5 and Comparative Example 5 show that Comparative Example 5, without the addition of the inducing agent—ammonium persulfate, produced 1.42 × 10⁻⁶ chlamydospores in a 5L fermenter. 7 cfu / mL; while the chlamydospore yield of Example 5, with the addition of 2 g / L ammonium persulfate, was 8.46 × 10⁻⁶. 8 cfu / mL. This demonstrates that the addition of inducing substances can significantly increase the yield of chlamydospores under this culture method.

[0075] Example 6

[0076] (1) After inoculating Trichoderma harzianum T4 onto PDA medium, the mixture was cultured in a constant temperature incubator at 28℃ for 72 h. Mycelia were scraped off with a toothpick and placed in sterile water. After thorough vortexing, the mixture was filtered through gauze. The conidia yield of the filtrate was counted under a microscope and diluted to 1×10⁻⁶. 7 CFU / mL was used as the seed solution.

[0077] (2) The seed culture was inoculated into a 10L fermenter containing 7L of fermentation medium at an inoculation ratio of 1% (v / v). 60mL of silicone polyether defoamer was added, and fermentation was carried out at 30℃ and 200rpm for 48h. Then, 14g of ammonium persulfate was added to the fermenter, and the fermentation was continued under the same conditions for 120h. After the fermentation was completed, the yield of chlamydospores was counted under a microscope. Three replicates were set up for each group.

[0078] The fermentation medium used in this embodiment has the following formulation: corn flour 62.86 g / L, glycerol 7.54 mL / L, pH 4.17.

[0079] Comparative Example 6

[0080] (1) After inoculating Trichoderma harzianum T4 onto PDA medium, the mixture was cultured in a constant temperature incubator at 28℃ for 72 h. Mycelia were scraped off with a toothpick and placed in sterile water. After thorough vortexing, the mixture was filtered through gauze. The conidia yield of the filtrate was counted under a microscope and diluted to 1×10⁻⁶. 7 CFU / mL was used as the seed solution.

[0081] (2) The seed culture was inoculated into a 10L fermenter containing 7L of fermentation medium at an inoculation ratio of 1% (v / v), and 60mL of silicone polyether defoamer was added. The fermentation was carried out at 30℃ and 200rpm for 168h. After the fermentation was completed, the yield of chlamydospores was counted under a microscope. Three replicates were set up for each group.

[0082] The fermentation medium formulation used in this comparative example is the same as that in Example 6.

[0083] The experimental results of Example 6 and Comparative Example 6 show that, in a 10L fermenter, Comparative Example 6 produced 3.6 × 10⁻⁶ chlamydospores. 7 The cfu / mL yield was 2.87 × 10⁻⁶ cfu / mL, while the yield of spores in Example 6 with the addition of 2 g / L ammonium persulfate was 2.87 × 10⁻⁶ cfu / mL. 9 cfu / mL. This demonstrates that the addition of inducing substances can significantly increase the yield of chlamydospores under this culture method.

[0084] Example 7

[0085] (1) After inoculating Trichoderma harzianum T4 onto PDA medium, the mixture was cultured in a constant temperature incubator at 28℃ for 72 h. Mycelia were scraped off with a toothpick and placed in sterile water. After thorough vortexing, the mixture was filtered through gauze. The conidia yield of the filtrate was counted under a microscope and diluted to 1×10⁻⁶. 7 CFU / mL was used as the seed solution.

[0086] (2) The seed culture was inoculated at a ratio of 1% (v / v) into a 300L fermenter containing 210L of fermentation medium. 2000mL of silicone polyether defoamer was added. After fermentation at 30℃ and 200rpm for 48h, 420g of ammonium persulfate was added to the fermenter, and the fermentation was continued under the same conditions for another 120h. After the fermentation was completed, the yield of chlamydospores was counted under a microscope. Three replicates were set up for each group.

[0087] The fermentation medium used in this embodiment has the following formula: glucose 8g / L, corn flour 40g / L, molasses 50g / L, wheat bran 5g / L, soybean flour 32g / L, pH 6.5-7.0.

[0088] Comparative Example 7

[0089] (1) After inoculating Trichoderma harzianum T4 onto PDA medium, the mixture was cultured in a constant temperature incubator at 28℃ for 72 h. Mycelia were scraped off with a toothpick and placed in sterile water. After thorough vortexing, the mixture was filtered through gauze. The conidia yield of the filtrate was counted under a microscope and diluted to 1×10⁻⁶. 7 CFU / mL was used as the seed solution.

[0090] (2) The seed culture was inoculated into a 300L fermenter containing 210L of fermentation medium at an inoculation ratio of 1% (v / v), and 2000mL of silicone polyether defoamer was added. The fermentation was carried out at 30℃ and 200rpm for 168h. After the fermentation was completed, the yield of chlamydospores was counted under a microscope. Three replicates were set up for each group.

[0091] The fermentation medium formulation used in this comparative example is the same as that in Example 7.

[0092] The experimental results of Example 7 and Comparative Example 7 show that, in a 300L fermenter, the yield of chlamydospores produced in Comparative Example 7 was 4.78 × 10⁻⁶. 8 The cfu / mL yield was higher than that of Example 7, which had 2 g / L ammonium persulfate added, resulting in a chlamydospore yield of 3.93 × 10⁻⁶ cfu / mL. 9 cfu / mL. This demonstrates that the addition of inducing substances can significantly increase the yield of chlamydospores under this culture method.

[0093] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for inducing the differentiation of Trichoderma to produce chlamydospores, characterized in that, This includes the step of adding an inducing substance to the fermentation system during the fermentation process to induce the Trichoderma to differentiate and produce chlamydospores; The inducing substance is ammonium persulfate; The amount of the inducing substance added relative to the culture medium in the fermentation system is 2-6 g / L; The inducing substance is added 0-72 h after the start of fermentation.

2. The method according to claim 1, characterized in that, The amount of the inducing substance added relative to the culture medium in the fermentation system is 2 g / L.

3. The method according to claim 1, characterized in that, The inducing substance was added 48 hours after the start of fermentation.

4. The method according to claim 1, characterized in that, The fermentation culture was carried out at a temperature of 30°C and a rotation speed of 200 rpm.

5. The application of an inducing substance in inducing the differentiation of Trichoderma fungi to produce chlamydospores, characterized in that, The Trichoderma was induced to differentiate and produce chlamydospores by adding an inducing substance to the fermentation system. The inducing substance is ammonium persulfate; The amount of the inducing substance added relative to the culture medium in the fermentation system is 2-6 g / L; The inducing substance is added 0-72 h after the start of fermentation.

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