A method for producing chlamydospores of the dark-septate endophytic fungus phialophora richardsiae by liquid fermentation

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a method for producing chlamydospores of dark septate endophytic fungus Phialophora fastigiata by liquid fermentation. The present application finds out the culture medium and culture conditions suitable for the growth of Guangxi Phialophora fastigiata HX2 through single factor experiment, response surface optimization experiment and the like. After optimization, the method greatly improves the yield of chlamydospores produced by Phialophora fastigiata, effectively reduces the production cost of chlamydospores, and prolongs the shelf life of the microbial agent. According to the calculation, the yield of chlamydospores is increased by 139.8% compared with the traditional PDB culture medium, the cost is reduced by 47%, and the shelf life is prolonged by about 8 months. The utilization of the growth-promoting and immunity-inducing strain HX2 is effectively improved. The culture medium is simple to prepare, the fermentation process is stable, and the method is suitable for large-scale production. The prepared chlamydospore preparation has strong stress resistance and long shelf life, and provides a new effective way for the industrialization of the Phialophora fastigiata strain in the later stage.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for producing dark-colored septate endophytic fungi, Cladosporium chlamydospores, by liquid fermentation. Background Technology

[0002] Thick-walled spores ( Chlamydospore Thick-walled spores (CFS) are formed from mycelial cells through fragmentation or cell wall thickening. Their thick walls are composed of chitin, glucan, and polysaccharides, with some outer layers containing melanin-like substances that enhance their resistance to adverse conditions. They are typically round, spindle-shaped, rectangular, or barrel-shaped, often growing in chains or clusters. Upon maturity, they may divide into single cells and germinate independently. They are a special dormant structure formed by fungi under specific conditions, primarily used to resist adverse environments and maintain survival. In existing technologies, we know that chlamydospore production is quite difficult and has significant specific requirements for fungal strains. This is mainly because: most fungi reproduce primarily through conidial growth, while chlamydospore production requires extreme environments for induction, exhibiting strong dependence on stress. Most chlamydospores require nutrient deprivation (e.g., carbon / nitrogen imbalance), high temperatures (e.g., 35-40℃ for *Trichoderma*), extreme pH (e.g., pH 3 for *Cladosporium spp.*), or oxidative stress (H2O2 treatment) to form. The conditions require high precision control. The core difficulty in inducing production for different fungal strains lies in the strong strain specificity, complex induction conditions, and unstable large-scale processes. Furthermore, the strain specificity is significant: for example, *Trichoderma* chlamydospores require… The induction logic of *Metarhizium anisopliae* differs significantly between species, requiring a phased regulation process of "acidification followed by alkali supplementation." Furthermore, the sporulation mechanism of *Thylakoidea* is unclear: the molecular regulatory network is complex, with multiple signaling pathways, including the TOR signaling pathway, ROS (reactive oxygen species) signaling, and MAPK pathway, jointly regulating sporulation; the functions of key genes (such as *Trichoderma*'s Tvhyd1 and Tvstp1) are not fully understood; and cell differentiation is irreversible: once formed, *Thylakoidea* spores may enter a dormant state, requiring additional steps to break dormancy (such as specific temperature or enzyme treatment). Bottlenecks exist for large-scale production, such as unstable fermentation processes; traditional solid-state fermentation (e.g., wheat bran substrate) is susceptible to contamination by other microorganisms, while liquid submerged fermentation is prone to shear stress that damages mycelial structure. These conditions limit the production and formation of *Thylakoidea* spores.

[0003] Because chlamydospores have cell walls rich in chitin and melanin, they typically form under stress conditions such as nutrient deficiency, extreme temperatures, high salt concentrations, or abnormal pH. Chlamydospores can withstand adverse environments such as drought, high temperatures, and chemical substances for a long time, and can survive even after the mycelium dies. When conditions are suitable, they will germinate into new mycelium. The thick-walled structure allows them to be spread by wind, water, or animals, thus expanding the distribution range of fungi. Therefore, developing fungal biocontrol agents with chlamydospores as the core is considered a key direction to overcome existing technological bottlenecks.

[0004] Cladosporium ( Cladophialophora *Cladosporium spp.* is a dominant species of dark-colored septate endophytic fungi (DSE), with a wide host range. Current research has found that *Cladosporium spp.* Cladophialophora *Cladosporium spp.* has a growth-promoting effect on crops and also has high disease-preventing value, exhibiting good control over bacterial wilt. *Cladosporium spp.* (Guangxi) Cladophiaphora guangxiense HX2 is a superior strain screened by our research team in Guangxi. Its accession number is CGMCC NO. 41498. Previous research by our team found that this strain can effectively activate the plant's immune system, making it a biocontrol bacterium that integrates growth promotion and disease prevention effects. It has broad development prospects as a plant growth regulator and immune inducer. However, in existing technical reports, we know that *Cladosporium* primarily reproduces through conidia. Currently, there are no reports of strains of this genus reproducing via chlamydospores. To effectively improve the application of *Cladosporium* and extend product shelf life, it is necessary to develop a suitable system for *Cladosporium* species, especially those from Guangxi. Cladophiaphora guangxiense The culture medium and production method for HX2 to produce chlamydospores were developed to improve the application effect of Cladosporium. Summary of the Invention

[0005] In view of the above, it is necessary to develop a set of methods suitable for *Cladosporium* species, especially *Cladosporium guangxiense*. Cladophiaphora guangxiense The culture medium and production method for HX2 to produce chlamydospores were developed to improve the application effect of Cladosporium.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a method for producing dark-colored septate endophytic fungus Cladosporium chlamydospores by liquid fermentation, the method comprising the following steps.

[0007] (1) Inoculate the Cladosporium strain into PDA medium for activation culture.

[0008] (2) The activated bacterial blocks were transferred to PDB liquid culture medium to prepare seed culture.

[0009] (3) the seed liquid is inoculated into the liquid culture medium of the chlamydospore of the dark septate endophyte phialophora radicicola to ferment the chlamydospore agent; in the step (3), the liquid culture medium of the chlamydospore of the dark septate endophyte phialophora radicicola is composed of the following components: 10-30 g / L soybean powder, 10-30 g / L corn powder and 70-90 g / L molasses; the inoculation amount is 1%-5%; the fermentation conditions of the liquid culture medium of the chlamydospore of the dark septate endophyte phialophora radicicola fermentation are as follows: the bottle filling amount is 75-125 mL / 250 mL triangular flask, the rotation number of the shaking culture is 140-180 r / min, the culture time is 7-10 days, the temperature is 23-30 DEG C, and the original pH of the fermentation liquid is 5.12.

[0010] The phialophora radicicola strain is phialophora radicicola (Guangxi) HX2, the strain preservation information is as follows: the classification name is: Cladophiaphora guangxiense Cladophiaphora guangxiense , the Chinese classification name is phialophora radicicola (Guangxi), the preservation number is CGMCC NO.41498; the strain is preserved in the China General Microbiological Culture Collection Center, the address is No.3, Beichen West Road, Chaoyang District, Beijing, and the preservation date is September 5, 2024.

[0011] Further, the liquid culture medium of the chlamydospore of the dark septate endophyte phialophora radicicola in the step (3) is composed of the following components: 26 g / L soybean powder, 25 g / L corn powder and 84 g / L molasses; the inoculation amount of inoculation is 3.6%; the fermentation conditions of the liquid culture medium of the chlamydospore of the dark septate endophyte phialophora radicicola fermentation are as follows: the bottle filling amount is 115 mL / 250 mL triangular flask, the rotation number of the shaking culture is 170 r / min, the culture time is 7-10 days, the temperature is 28 DEG C, and the original pH of the fermentation liquid is 5.12.

[0012] Further, the chlamydospore concentration of the seed liquid is 1x10 5 / mL.

[0013] The present application has the following beneficial effects: 1, the present application finds the culture medium and culture conditions suitable for the growth of phialophora radicicola (Guangxi) HX2 through single factor, response surface optimization and other experiments, after optimization, the method greatly improves the yield of chlamydospore of phialophora radicicola, and effectively reduces the cost of producing chlamydospore; it is calculated that the yield of chlamydospore after fermentation for 7-10 days reaches 1.39x10 8 ~2.71x10 8 ​The yield of this invention is 139.8% higher than that of traditional PDB medium, while reducing costs by 47%. Its shelf life at room temperature is extended by approximately 8 months compared to inoculants fermented on traditional PDB medium. This effectively improves the utilization of the growth-promoting and disease-resistant strain HX2. The prepared chlamydospores exhibit strong stress resistance and a long shelf life, making them suitable for developing biocontrol agents to promote crop growth and disease resistance. The culture medium of this invention is simple to prepare, and the fermentation process is stable, making it suitable for large-scale production. This provides a new and effective approach for the later promotion and application of Cladophora spp. strains.

[0014] 2. The thick-walled spores of *Cladosporium guilloché* HX2 from Guangxi have a thick-walled structure and are highly resistant to adverse environments such as dryness, high temperature, and soil antibacterial effects. This solves the technical bottleneck of short shelf life of traditional conidial preparations, and the shelf life at room temperature can reach more than one year.

[0015] 3. The Guangxi Cladosporium HX2 chlamydospore preparation has significant field control efficacy against bacterial wilt of tomatoes and ginger, providing a new approach for the green control of soil-borne diseases.

[0016] 4. The culture medium of the present invention uses inexpensive agricultural by-products such as molasses and corn flour as the main raw materials, which is low in cost; the fermentation conditions (such as pH 5.12, which do not need to be adjusted) are easy to control and are suitable for large-scale promotion.

[0017] 5. This invention is the first to realize the large-scale production of thick-walled spores of *Cladosporium glomeratum* HX2 from Guangxi through liquid fermentation, filling the gap in the preparation technology of thick-walled spores of this fungus and providing new resources for the development of biocontrol agents. Attached Figure Description

[0018] Figure 1 This is the value of the analysis of variance for the Plackett-Burman experiment.

[0019] Figure 2 These are electron micrographs comparing the production of chlamydospores by strain HX2 under different culture conditions; the left side shows the conventional PDB medium, and the right side shows the liquid fermentation medium of this application.

[0020] Figure 3 This is a response surface plot of the effects of soybean flour and corn flour on the yield of chlamydospores of strain HX2.

[0021] Figure 4 This is a contour plot showing the effects of soybean flour and corn flour on the yield of chlamydospores of strain HX2.

[0022] Figure 5 This is a response surface plot showing the effect of soybean flour and molasses on the yield of thick-walled spores of strain HX2.

[0023] Figure 6 This is a contour plot showing the effects of soybean flour and molasses on the yield of thick-walled spores of strain HX2.

[0024] Figure 7 This is a response surface plot showing the effect of corn flour and molasses on the yield of thick-walled spores of strain HX2.

[0025] Figure 8 This is a contour plot showing the effect of corn flour and molasses on the yield of thick-walled spores of strain HX2.

[0026] Figure 9 This is a prediction diagram of the optimal culture components for strain HX2.

[0027] Figure 10 This is a graph showing the results of a single-factor experiment on the inoculum size of strain HX2.

[0028] Figure 11 This is a graph showing the results of a single-factor experiment on the bottling volume of strain HX2.

[0029] Figure 12 This is a graph showing the results of a single-factor experiment on the rotation speed of strain HX2.

[0030] Figure 13 This is a graph showing the results of a single-factor temperature experiment for strain HX2.

[0031] Figure 14 This is a graph showing the results of a single-factor pH experiment for strain HX2.

[0032] Figure 15 This is a response surface plot showing the effect of rotation speed and bottling volume on the yield of thick-walled spores of strain HX2.

[0033] Figure 16 This is a contour plot showing the effect of rotation speed and bottling volume on the yield of thick-walled spores of strain HX2.

[0034] Figure 17 This is a response surface plot showing the effect of rotation speed and inoculum size on the yield of thick-walled spores of strain HX2.

[0035] Figure 18 This is a contour plot showing the effect of rotation speed and inoculum size on the yield of thick-walled spores of strain HX2.

[0036] Figure 19 This is a response surface plot showing the effect of bottling volume and inoculum volume on the yield of thick-walled spores of strain HX2.

[0037] Figure 20 This is a contour plot showing the effect of bottling volume and inoculum volume on the yield of thick-walled spores of strain HX2.

[0038] Figure 21 This is a prediction diagram of the optimal fermentation conditions for strain HX2.

[0039] Information on the preservation of biological materials

[0040] The strain information preserved in this application is: *Cladosporium guangxiense* ( Cladophiaphora guangxienseHX2, which is classified as: Cladophiaphora guangxiense , and the Chinese classification name is Guangxi Cladosporium phialophora, and the preservation number is CGMCC NO.41498; the strain is preserved in the General Microbiological Center of the China Microbial Culture Collection Committee, located at No.3, Beichen West Road, Chaoyang District, Beijing, and the preservation date is September 5, 2024. DETAILED DESCRIPTION

[0041] To make the above-mentioned purposes, features and advantages of the present application more apparent and easy to understand, the specific embodiments of the present application are described in detail below. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the scope of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0042] The strain used in the embodiments of the present application is Guangxi Cladosporium phialophora (Gams) Schmiedel. Cladophiaphora guangxiense HX2, and the preservation information is as follows: the preservation number is CGMCC NO.41498, and the classification name is: Cladophiaphora guangxiense , and the Chinese classification name is Guangxi Cladosporium phialophora, and the preservation number is CGMCC NO.41498; the strain is preserved in the General Microbiological Center of the China Microbial Culture Collection Committee, located at No.3, Beichen West Road, Chaoyang District, Beijing, and the preservation date is September 5, 2024.

[0043] Example 1

[0044] The present embodiment is a method for producing chlamydospores of Guangxi Cladosporium phialophora (Gams) Schmiedel. Cladophiaphora guangxiense HX2 (hereinafter referred to as strain HX2), and the specific method is as follows.

[0045] I. Method for producing chlamydospores of strain HX2

[0046] (1) Inoculate strain HX2 on a PDA culture medium plate and cultivate at 28°C for 10 days. Cut the colonies on the PDA plate and transfer them to a PDB liquid culture medium, and place them in a constant temperature shaker at 28°C and 120 r / min for 14 days. Adjust the spore concentration to 1×10 5 / ml as a seed liquid for standby.

[0047] (2) Transfer the colonies activated in step (1) to a PDB liquid culture medium and cultivate at 28°C and 120 r / min for 14 days to prepare a seed liquid.

[0048] (3) Inoculate the seed liquid into a liquid culture medium and shake cultivate to obtain a chlamydospore fermentation broth.

[0049] The spore counting method of strain HX2 is as follows: the fermented HX2 bacterial liquid is broken by a homogenizer for 2 min, 1 mL of the bacterial suspension is taken and diluted proportionally, then 20 μL of the diluted bacterial suspension is taken and counted on a blood cell counting plate, and the above process is repeated three times. The counting method is to count the left and not the right, and to count the top and not the bottom. The counting formula is (I).

[0050]

[0051] The present applicant found in the research that in the laboratory research, the strain HX2 was cultured using PDB liquid medium, which could induce a small amount of chlamydospores, but when the strain HX2 was cultured on a large scale, the amount of chlamydospores produced by the strain HX2 could not be increased, therefore, the present applicant further researched the fermentation medium in the large-scale culture.

[0052] II. The liquid medium of step (3) is cultured under different conditions and the components of the medium are explored, and the specific method is as follows.

[0053] 1. Water is used as a carrier, and the following test carbon sources are selected: glucose, sucrose, soluble starch, corn powder, molasses, sorbitol, mannitol, glycerol, maltose, fructose, which are respectively used as the only carbon source at 50 g / L and sterilized for standby. Under the conditions of 100 mL of liquid volume in a 250 mL flask, 1% of HX2 inoculation amount, 120 r / min of rotation speed and 28℃, the best carbon source for promoting the production of chlamydospores by the strain HX2 is screened. The results are shown in Tables 1-2.

[0054]

[0055] Note: In the table, different lowercase letters indicate significant differences (P<0.05) in the same column, and the same letter indicates no significant difference (P>0.05) in the same column, and the same applies to the following table.

[0056] As shown in Table 1, among different carbon sources, the amount of chlamydospores produced by the strain HX2 from high to low is as follows: molasses > corn powder > sucrose > maltose > glycerol > glucose > mannitol > soluble starch > sorbitol, and the amount of chlamydospores produced when molasses and corn powder are used as carbon sources is the highest, which is 2.68×10 7 / mL and 1.02×10 7 / mL respectively, and when sorbitol and soluble starch are used as carbon sources, the strain HX2 cannot be induced to produce a large amount of chlamydospores, therefore, molasses and corn powder are selected as carbon sources of the medium and are screened at different concentrations, and the results are shown in Table 2.

[0057]

[0058] Table 2 shows that the highest sporulation was achieved when the molasses concentration was 70-90 g / L, and the highest sporulation was achieved when the corn flour concentration was 10-30 g / L. This was used as the basis for subsequent response surface methodology experiments. The optimal molasses concentration was 80 g / L (spore yield was 8.725 × 10⁻⁶). 7 The optimal concentration of corn flour is 30 g / L (spore yield is 2.91 × 10⁻⁶ / mL). 7 (units / mL).

[0059] 2. Using water as a carrier, the following nitrogen sources were selected for testing: soybean flour, tryptone, NH4Cl, beef meal, corn steep liquor powder, NH4NO3, and KNO3. They were inoculated at 20 g / L and sterilized. The cultures were then incubated at 100 mL, 1% HX2 inoculum, 120 r / min, and 28℃ to screen for the optimal nitrogen source. The results are shown in Tables 3 and 4.

[0060]

[0061] Table 3 shows that, among different nitrogen sources, the amount of chlamydospores produced by strain HX2, from highest to lowest, is: soybean meal > tryptone > beef meal > KNO3 > NH4NO3 > NH4Cl > corn steep liquor powder. The highest amount of chlamydospores was produced when soybean meal was used as the nitrogen source, reaching 1.31 × 10⁻⁶. 8 The concentration of spores / mL was high, but strain HX2 could not be induced to continue producing large quantities of chlamydospores when NH4NO3, NH4Cl and corn steep liquor were used as nitrogen sources. Based on the optimal carbon source (a mixture of 80 g / L molasses and 30 g / L corn flour), soybean flour was selected as the nitrogen source in the culture medium in gradients of 10, 20, 30, 40 and 50 g / L and the concentrations were screened. The results are shown in Table 4.

[0062]

[0063] As shown in Table 4, the highest spore yield was achieved when the soybean flour concentration was between 10 g / L and 30 g / L. Therefore, this concentration was chosen as the basis for subsequent response surface methodology experiments.

[0064] 3. Using water as a carrier, the following inorganic salts were selected for testing: FeSO4, NaHCO3, KH2PO4, CaCl2, MgSO4, MnSO4, and CuSO4, each at 0.2 g / L. The mixture was cultured in 250 mL Erlenmeyer flasks with a liquid volume of 100 mL, an inoculum size of 1% for HX2, a rotation speed of 120 r / min, and a temperature of 28℃. The optimal inorganic salt for promoting the production of chlamydospores by strain HX2 was screened, and the results are shown in Tables 5 and 6.

[0065]

[0066] From Table 5, in different inorganic salt screening, the amount of spore production of strain HX2 from high to low is KH2PO4>CuSO4>MnSO4>CaCl2>CK>FeSO4>NaHCO3>MgSO4, wherein the highest spore production is KH2PO4, followed by CuSO4, MnSO4, the amount of spore production is 9.36×10 7 , 9.33×10 7 , 7.8×10 7 / mL, all higher than CK and with significant difference, while when FeSO4, NaHCO3 and MgSO4 are inorganic salts, the amount of spore production of strain HX2 is lower than CK, which shows that the three inorganic salts have certain inhibitory effect on strain HX2 to continue to produce spores. Therefore, KH2PO4, CuSO4 and MnSO4 are temporarily selected as inorganic salts of the culture medium, and on the basis of the optimal carbon source (80 g / L of molasses and 30 g / L of corn powder), the concentration of the three inorganic salt ions is screened, and the concentration range is 0.2, 0.4, 0.6, 0.8, 1.0 g / L, and the results are shown in Table 6.

[0067]

[0068] From Table 6, the concentration range of KH2PO4 is 0.4-0.8 g / L, the concentration range of CuSO4 is 0.6-1.0 g / L, and the concentration range of MnSO4 is 0.2-0.6 g / L.

[0069] 4. On the basis of the foregoing single factor, the response surface optimization test of the culture medium components of strain HX2 is carried out: on the basis of the single factor test, A (soybean powder), B (corn powder) and B (molasses) are selected as three factors, the amount of spore production of strain HX2 is taken as the response value, the selected single factor is taken as the independent variable, and DesignExpert 13 software is used for response surface test design to determine the optimal composition of the fermentation medium.

[0070] (1) Plackett-Burman test: according to the results of the single factor test, the culture medium components of strain HX2 are molasses, corn powder, soybean powder, KH2PO4, CuSO4 and MnSO4. According to the results, Plackett-Burman test is carried out according to Table 7 to optimize the design of the culture medium components.

[0071]

[0072]

[0073]

[0074] As shown in Table 8 and Figure 1 three key factors affecting the test, soybean meal, corn meal, molasses, and found no significant inorganic salt ions are not obvious in the subsequent test were added, which soybean meal, corn meal, molasses are significant factor and are higher than the t value, in the test showed a positive effect, and according to the results of Table 9 variance showed that the three factors reached a significant level, R 2 = 0.9166, adj R 2 = 0.8165 can be explained by the regression equation, indicating that the model on the key factors of the medium effect.

[0075] (2) the steepest climb test: according to the selected three key factors design steepest climb test, according to the model of the coefficients of each variable to determine the key factor of the climb direction and step length, to determine the optimal range of key factors, so that the strain HX2 spore yield maximum point as the center combination design test center point for 4, according to Table 10, so as to determine the range of the key components of the medium and the range of the culture conditions.

[0076]

[0077] (3) BoxBehnken test: BoxBehnken response surface method (BBD) design 3 factor 2 level design center combination test (see Table 11), the key factors were optimized, and the amount of chlamydospore was fitted with the quadratic multiple regression equation, and the amount of chlamydospore of strain HX2 was obtained: the amount of chlamydospore = 2.062 + 0.065 A + 0.02 B - 0.05 C - 0.04 AB - 0.05 AC - 0.01 BC - 0.11 A 2 -0.056 B 2 -0.136 C 2 .

[0078]

[0079]

[0080] As shown in Table 12, R 2 = 0.9871, R 2 adj = 0.9706, the key factors on Y can be explained by the regression equation, and the regression equation variance analysis results show that the regression equation has significant (P < 0.01). The response surface and contour line of the interaction of soybean meal, corn meal and molasses are shown inFigure 3 、 Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 and Figure 8 shown, from Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 and Figure 8 can be intuitively reflected that the production of endospore of strain HX2 is affected. From Figure 9 can be known that the optimal medium component ratio is soybean powder 26 g / L, corn powder 25 g / L, molasses 84 g / L, and the test obtains endospore amount of 2.135 x 10 8 / mL, which exceeds the predicted level of 2.079 x 10 8 / mL, proving the rationality of the prediction. The following fermentation condition optimization is tested according to this component ratio.

[0081] III. Optimal reaction condition: under the condition of determining the optimal medium concentration, the fermentation reaction condition of strain HX2 is studied, which is as follows.

[0082] 1. Single factor experiment: using the optimized medium component and concentration, with inoculation amount 1%, pH 5, liquid loading amount 100 mL, temperature 28℃, rotation speed 120 r / min as the initial culture condition, the single factor experiment is carried out on different setting values of pH, fermentation temperature, rotation speed, liquid loading amount, inoculation amount, respectively, the endospore amount of strain HX2 fermentation broth is determined, the parameters of each fermentation condition are optimized, and the single factor experiment results of pH, fermentation temperature, rotation speed, liquid loading amount, inoculation amount are shown in Figure 10- Figure 14 : Figure 10 the effect of inoculation amount on endospore, Figure 11 the effect of liquid loading amount on endospore, Figure 12 the effect of rotation speed on endospore, Figure 13 the effect of temperature on endospore, Figure 14The effect of pH on chlamydospore; experimental results show that the inoculum size of 1%-5% of chlamydospore production is the highest, when the inoculum size reaches 11%, due to the inoculum size is too large, leading to mycelium knot, but affect the efficiency of sporulation; the bottle volume of 75-125 mL / 250 mL of spore production is the most, chlamydospore production decreases with the increase of bottle volume, too large bottle volume affects the shaking amplitude of the medium, but reduces the sporulation efficiency, the conclusion is that chlamydospore production decreases with the increase of bottle volume; the rotation speed of 140-180 r / min of chlamydospore production is the highest; the temperature of 28℃ of chlamydospore production is the most and the initial temperature selected is consistent, the original pH of the fermentation broth is 5.12, through the test, it is found that the pH does not need to be controlled to achieve the best state of sporulation.

[0083] 2. Orthogonal optimization experiment: according to the results of single factor experiment, the fermentation conditions, bottle volume, inoculum size and rotation speed of strain HX2 were used for response surface experiment, and the specific experimental method was as follows.

[0084] (1) Box-Behnken experiment of fermentation conditions: taking inoculum size, bottle volume and rotation speed as independent variables, the spore yield data of strain HX2 were subjected to quadratic polynomial regression fitting, and the dynamic model of key factors was obtained as follows: Y=2.356+0.0425 A+0.04625 B-0.03125 C+0.015 AB-0.01 AC-0.0225 BC-0.12925 A 2 -0.07175 B 2 -0.07675 C 2 . R 2 =0.9577, the corrected R 2 =0.9032, in the formula, Y is the predicted value of spore yield of strain HX2, which indicates that the regression equation has 90.32% probability to explain the influence of key factors on Y, and the regression equation has significance (as shown in Table 14).

[0085]

[0086]

[0087] According to the response surface graph obtained from the data in Table 14 ( Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 19 and Figure 20), it is revealed that the inoculation amount, the bottle volume and the rotation speed three key factors interact with each other on the chlamydospore yield. According to the canonical analysis of response surface, the regression model has a maximum value, when the inoculation amount is 3.6%, the bottle volume is 115 mL and the rotation speed is 170 r / min (such as Figure 21 ), after 10 days of fermentation, the predicted maximum chlamydospore yield is 2.29 ×10 8 / mL. According to the predicted fermentation conditions, the maximum spore yield is 2.71 ×10 8 / mL, which is 18.3% more than the predicted value, and 139.8% higher than the PDB medium commonly used in the laboratory (1.13 ×10 8 / mL).

[0088] Based on the above experimental results, we obtain the optimal fermentation process of chlamydospore of strain HX2 as follows.

[0089] (1) The strain HX2 is inoculated on PDA medium plate, and cultured at 28℃ for 10 days. The colony on PDA is cut and transferred to PDB liquid medium, and placed in a constant temperature shaker at 28℃ and 120 r / min for 14 days. The spore concentration is adjusted to 1 ×10 5 / mL, which is used as seed liquid.

[0090] (2) The activated fungus block in step (1) is transferred to PDB liquid medium, and cultured at 28℃ and 120 r / min for 14 days to prepare seed liquid.

[0091] (3) The seed liquid is inoculated in liquid medium, and the chlamydospore fermentation broth is obtained by shaking culture; wherein, the liquid medium of chlamydospore of the dark septate endophytic fungus Phialophora fortunii is composed of the following components: soybean powder 26 g / L, corn powder 25 g / L and molasses 84 g / L; the inoculation amount is 3.6%; the fermentation conditions of the liquid medium of chlamydospore of the dark septate endophytic fungus Phialophora fortunii are as follows: the bottle volume is 115 mL / 250 mL flask, the rotation speed of shaking culture is 170 r / min, the culture time is 7-10 days, the temperature is 28℃, and the original pH of the fermentation broth is 5.12.

[0092] In the above reaction conditions: the soybean powder is 10-30 g / L, the corn powder is 10-30 g / L, and the molasses is 70-90 g / L; the inoculation amount is 1%-5%; the bottle volume is 75-125 mL / 250 mL flask, the rotation speed of shaking culture is 140-180 r / min, the culture time is 7-10 days, and the temperature is in the range of 23-30℃, all of which can make the yield of strain HX2 optimal.

[0093] The comparison chart of the chlamydospore produced by the fermentation strain HX2 using the above optimal method and the chlamydospore produced by the strain HX2 only induced by PDB medium is shown in Figure 2. Figure 2 As shown in the figure, the density of the chlamydospore under the microscope after liquid fermentation culture is much greater than that of the PDB medium. It is detected that under the reaction condition, after 10 days of fermentation, the maximum spore yield is 2.71 x 10 8 / mL, which is 18.3% higher than the predicted value (2.29 x 10 8 / mL) and is 139.8% higher than the PDB medium commonly used in the laboratory. It is found that only the liquid fermentation medium (soybean powder 10-30 g / L, corn powder 10-30 g / L, and molasses 70-90 g / L) described in the application can produce a large amount of chlamydospore, and the induction culture in other liquid induction media will inhibit the production of chlamydospore by the strain HX2.

[0094] Example 2

[0095] This example mainly studies the cost comparison between the conventional PDB medium and the liquid fermentation medium of the application, as follows.

[0096] 1. The PDB medium is composed of 200 g / L of potato and 20 g / L of glucose.

[0097] The liquid fermentation medium is composed of 84 g / L of sugarcane molasses, 26 g / L of soybean powder, and 25 g / L of corn powder. The price comparison of the above two components is shown in Table 15.

[0098]

[0099] As shown in Table 15, the price of 1 ton of fermentation using the PDB medium is 849.6 yuan, while the price of 1 ton of fermentation using the liquid fermentation medium of the application is only 453.08 yuan, which is reduced by about 47%. Therefore, the cost of preparing chlamydospore of the strain HX2 using the medium of the application is significantly reduced, which is very suitable for large-scale promotion.

[0100] Example 3

[0101] This example mainly studies the shelf life of chlamydospore, as follows.

[0102] Experimental method: compare the shelf life of the bacterial agent product of the strain HX2 under the fermentation conditions of the traditional PDB medium and the optimal fermentation conditions of Example 1. The shelf life is measured by counting the viable bacteria on the same day every month from the first to the twentieth month after the culture, and then calculating the viable bacteria concentration and spore germination rate. The calculation method of the viable bacteria concentration is as follows.

[0103] Viable cell concentration (CFU / g or CFU / mL) = plate colony number x dilution factor / inoculation volume (mL).

[0104] The viable cell counting method is as follows: after the liquid bacterial agent is gradient diluted to a suitable multiple, 100 ul of the sample diluted to a suitable multiple is taken for coating, the number of colonies is counted after 7 days, and the viable cell quantity is calculated, with 3 repetitions for each.

[0105] The spore germination rate determination method is as follows: the PDA plate is placed under a microscope, the number of spore germination is counted, 5 repetitions are set for each treatment, the results with large differences are eliminated, 3 repetitions are selected to take their average value as the result analysis, and 100 coating numbers per plate are taken as the base, and the germination rate is expressed by percentage.

[0106] The initial spore production of the PDB culture medium when the production of the two methods is completed is 1.13 x 10 8 / mL, the initial spore production of the best fermentation condition (liquid culture medium) of example 1 is 2.54 x 10 8 / mL; the viable cell quantity and germination rate results measured every month from the first month to the 20th month are shown in table 16.

[0107]

[0108] As shown in table 16, the viable cell quantity and germination rate of the bacterial agent prepared by the two methods decrease with the increase of time, the viable cell quantity of the bacterial agent prepared by the method of example 1 is of the order of 10 10 in the first month, and the germination rate is as high as 95%; the viable cell quantity of the bacterial agent cultured by the PDB routine method is of the order of 10 7 in the first month, and the germination rate is as high as 97%, and the difference in germination rate is not large; when entering the second month, the germination rate of the bacterial agent prepared by the method of example 1 is significantly higher than that of the bacterial agent cultured by the PDB routine method; from the viable cell quantity, the bacterial agent prepared by the method of example 1 still maintains a high viable cell quantity of the order of 10 9 in the 20th month, but the viable cell quantity of the bacterial agent cultured by the PDB routine method is less than 10% in the 12th month at room temperature, and the order of magnitude of the viable cell quantity cannot reach 10 7 ; it is shown that the shelf life of the bacterial agent prepared by the method of example 1 is much better than that of the bacterial agent cultured by the PDB routine method, and the shelf life is prolonged by about 8 months than that of the bacterial agent cultured by the PDB routine method.

[0109] Example 4

[0110] This example mainly studies the induction of spores of other strains of the same genus by liquid fermentation medium, and the specific process is as follows.

[0111] The optimal fermentation process of the chlamydospore of the strain HX2 of Example 1 was used to ferment the same genus of the strain of the fungus.

[0112] (1) The strain HX2, DH3, LC4 and LC83 (the strains DH3, LC4 and LC83 are strains screened by the research group and belong to the same genus and have been disclosed in the literature “Research on the Diversity and Ecological Function of Two Habitats of Dark Septate Endophytic Fungi in Guangxi”) were inoculated on PDA medium plates and activated and cultured at 28°C for 10 days. The colonies on the PDA were cut and transferred to PDB liquid medium and placed in a constant temperature shaker at 28°C and 120 r / min for 14 days. The spore concentration was adjusted to 1×10 5

[0113] (2) The colonies activated in step (1) were transferred to PDB liquid medium and cultured at 28°C and 120 r / min for 14 days to prepare the seed liquid.

[0114] (3) The seed liquid was inoculated in the liquid medium and shaken to obtain the chlamydospore fermentation liquid; the liquid medium was composed of soybean powder 26 g / L, corn powder 25 g / L and molasses 84 g / L; the fermentation conditions of the liquid fermentation were as follows: inoculation amount 3.6%, bottle filling amount 115, rotation speed 170 r / min; and the chlamydospore yield obtained is shown in Table 17.

[0115]

[0116] As shown in Table 17, the strain HX2 can effectively produce chlamydospores under the optimized process of the application, and the spore yield is significantly higher than that of the strains of the same genus. The strains DH3 and LC83 can produce chlamydospores, but the yield is significantly lower than that of the strain HX2 of the application. The strain LC4 cannot produce chlamydospores. The above experiments prove that the production of chlamydospores has strong specificity, which is inseparable from the characteristics of chlamydospore induction requiring extreme environment. Different strains have different requirements for extreme environment, and the reaction conditions and reaction medium need to be accurately controlled.

[0117] In summary, the culture medium and culture conditions suitable for the growth of the strain HX2 were found out by strain screening, single factor, response surface optimization and the like. After optimization, the reaction conditions greatly improved the yield of chlamydospores produced by the strain HX2, and effectively reduced the cost of producing chlamydospores by the strain HX2. It is calculated that the yield of chlamydospores is increased by 139.8% compared with the PDB medium, and the cost is reduced by 47%. The use of the strain HX2 which promotes growth and resists diseases is effectively improved, the shelf life of the strain is improved, and a new effective idea for the later popularization and application of the strain HX2 is provided.

[0118] ​The several embodiments of the present application are described more specifically and in greater detail herein, but are not to be construed as limiting the scope of the present application. It should be noted that, for one of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these should all fall within the scope of the present application. Therefore, the scope of the present application should be defined by the appended claims.

Claims

1. A method for liquid fermentation production of dark-septate endophytic fungal conidia of Phialophora richardsiae, characterized by, The method comprises the following steps: (1) inoculate the strain of Cladophialophora into PDA culture medium for activation culture; (2) transfer the activated fungus block into PDB liquid culture medium to prepare seed liquid; (3) inoculate the seed liquid into the liquid culture medium of chlamydospores of the dark septate endophytic fungus Cladophialophora to obtain chlamydospore fungicide through fermentation; In the step (3), the liquid culture medium of chlamydospores of the dark septate endophytic fungus Cladophialophora is composed of the following components: soybean powder 10-30 g / L, corn powder 10-30 g / L and molasses 70-90 g / L; The inoculation amount in the step (3) is 1%-5%; The fermentation conditions of the liquid culture medium of chlamydospores of the dark septate endophytic fungus Cladophialophora in the step (3) are as follows: the bottle filling amount is 75-125 mL / 250 mL triangular flask, the rotation number of shaking culture is 140-180 r / min, the culture time is 7-10 days, the temperature is 23-30℃, and the original pH of the fermentation liquid is 5.12; Cladophiaphora guangxiense The *Cladosporium* strain is *Cladosporium guilloché* (from Guangxi). In the step (3), the liquid culture medium of chlamydospores of the dark septate endophytic fungus Cladophialophora is composed of the following components: soybean powder 26 g / L, corn powder 25 g / L and molasses 84 g / L; HX2, with accession number CGMCC NO.41498.

2. The method of claim 1, wherein, The inoculation amount in the step (3) is 3.6%; The fermentation conditions of the liquid culture medium of chlamydospores of the dark septate endophytic fungus Cladophialophora in the step (3) are as follows: the bottle filling amount is 115 mL / 250 mL triangular flask, the rotation number of shaking culture is 170 r / min, the culture time is 7-10 days, the temperature is 28℃, and the original pH of the fermentation liquid is 5.

12. ​ 3. The method of claim 1, wherein, The concentration of chlamydospores in the seed liquid is 1 x 10 5 per mL.

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