Method for producing dark septum endophytic fungus cladosporium chlamydospore through liquid fermentation

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a method for producing dark isolated endophytic fungus cladosporium chlamydospore through liquid fermentation. The culture medium and culture conditions suitable for growth of the Cladosporium kwangsiensis HX2 are found out through experiments such as single factor and response surface optimization, the yield of the chlamydospore produced by the Cladosporium kwangsiensis is greatly improved through optimization, the production cost of the chlamydospore is effectively reduced, and the shelf life of the fungicide is prolonged; through measurement and calculation, compared with a traditional PDB culture medium, the yield of chlamydospore is increased by 139.8%, the cost is reduced by 47%, and the shelf life is prolonged by about 8 months. The culture medium is simple in preparation, stable in fermentation process and suitable for large-scale production, the prepared chlamydospore preparation is high in stress resistance and long in shelf life, and a new effective thought is provided for industrialization of bottle cladosporium strains in the later period.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a method for liquid fermentation to produce chlamydospores of the dark septate endophytic fungus Cladosporium cladosporioides Background Art

[0002] Chlamydospores ( Chlamydospore ) are thick-walled spores formed by the breaking of hyphal cells or the thickening of cell walls. Their thick walls are composed of chitin, glucan, and polysaccharide substances, and some outer layers contain substances similar to melanin, which enhance stress resistance. They are usually round, spindle-shaped, rectangular, or barrel-shaped, often in chains or clusters, and may divide into single cells for independent germination after maturity. It is a special dormant structure formed by fungi under specific conditions, mainly used to resist adverse environments and maintain survival ability. In the prior art, we know that chlamydospore production is relatively difficult and has obvious specific requirements for strains, mainly manifested as follows: The reproductive mode of most fungi is mainly through the growth of conidia. The production of chlamydospores requires induction under extreme environments and has a strong dependence on adversity. Most chlamydospores need to be formed under nutrient deficiency (such as carbon / nitrogen ratio imbalance), high temperature (such as Trichoderma spp. require 35-40 °C), extreme pH (such as Gliocladium virens requires pH 3), or oxidative stress (H2O2 treatment). The precision of condition control is required to be high. The core difficulties in the induced production methods of different strains lie in strong strain specificity, complex induction conditions, and unstable large-scale processes; and there are obvious strain specificities: for example, the chlamydospores of Trichoderma spp. require staged regulation of "first acidification and then alkali supplementation", while Metarhizium anisopliae requires low-temperature stimulation, and the induction logics of different strains vary greatly; in addition, the sporulation mechanism of chlamydospores is not clear: the molecular regulatory network is complex: multiple signal pathways such as the TOR signaling pathway, ROS (reactive oxygen species) signal, and MAPK pathway jointly regulate sporulation, and the functions of key genes (such as Tvhyd1 and Tvstp1 of Trichoderma) have not been fully resolved; and the irreversibility of cell differentiation: once chlamydospores are formed, they may enter a dormant state, and additional steps to break dormancy (such as specific temperature or enzyme treatment) are required; bottlenecks in large-scale production, for example, the fermentation process is unstable, traditional solid-state fermentation (such as wheat bran matrix) is easily contaminated by miscellaneous bacteria, and liquid deep fermentation is easily damaged by shear force to the hyphal structure. These conditions limit the production and formation of chlamydospores.

[0003] Since chlamydospores are rich in chitin and melanin in their cell walls and are usually formed under stress conditions such as nutrient deficiency, extreme temperature, high salt concentration, or abnormal pH, chlamydospores can tolerate adverse environments such as drought, high temperature, and chemical substances for a long time, survive after the death of the mycelium, and germinate into new mycelia when conditions are suitable. The thick-walled structure enables them to be spread by wind, water, or animals, expanding the distribution range of fungi. Therefore, developing fungal biocontrol agents with chlamydospores as the core is regarded as the key direction to break through the bottleneck of the existing technology.

[0004] Phialophora ramosa ( Cladophialophora spp.) is a dominant species of dark septate endophytes (DSE) with a wide host range. Research findings in the prior art show that Phialophora ramosa ( Cladophialophora spp.) can promote the growth of crops, has high disease prevention value, and has good control ability against bacterial wilt. Phialophora guangxiensis ( Cladophiaphora guangxiense ) HX2 is a dominant strain screened by the research group in Guangxi. The preservation number of this strain is: CGMCC NO.41498. The previous research of the research group found that this strain can effectively activate the immune system of plants. It is a biocontrol bacterium that combines the effects of promoting growth and preventing diseases, and can be developed into a plant growth regulator and immune inducer, with broad development prospects. However, in the reports of the prior art, we know that the spore reproduction mode of Phialophora ramosa is mainly conidia, and there is no relevant report on the growth of this genus of strains by chlamydospore reproduction mode. In order to effectively improve the application of Phialophora ramosa and extend the shelf life of products, it is necessary to develop a set of culture media and production methods suitable for this genus of strains, especially Phialophora guangxiensis ( Cladophiaphora guangxiense ) HX2 to produce chlamydospores, so as to improve the application effect of Phialophora ramosa. Summary of the Invention

[0005] In view of the above, it is necessary to develop a set of culture media and production methods suitable for this genus of strains, especially Phialophora guangxiensis ( Cladophiaphora guangxiense ) HX2 to produce chlamydospores, so as to improve the application effect of Phialophora ramosa.

[0006] To achieve the above object, the technical solution adopted by the present invention is: A method for liquid fermentation to produce chlamydospores of dark septate endophyte Phialophora ramosa, the method comprising the following steps.

[0007] (1) Inoculate the Phialophora ramosa strain on a PDA medium for activation culture.

[0008] (2) Transfer the activated bacterial mass to a PDB liquid medium to prepare a seed solution.

[0009] (3) Inoculate the seed liquid into a liquid medium for fermentation of the chlamydospores of the dark septate endophytic fungus Cladosporium cladosporioides to obtain a chlamydospore agent; in the step (3), the liquid medium for the chlamydospores of the dark septate endophytic fungus Cladosporium cladosporioides is composed of the following components: 10 - 30 g / L of soybean powder, 10 - 30 g / L of corn flour, and 70 - 90 g / L of molasses; the inoculation amount is 1% - 5%; the fermentation conditions for the fermentation of the liquid medium of the chlamydospores of the dark septate endophytic fungus Cladosporium cladosporioides are: the filling volume of the flask is 75 - 125 mL / 250 mL Erlenmeyer flask, the rotation speed of shaking culture is 140 - 180 r / min, the culture time is 7 - 10 days, the temperature is 23 - 30 °C, and the original pH of the fermentation broth is 5.12.

[0010] The Cladosporium cladosporioides strain is Cladosporium guangxiense ( Cladophiaphora guangxiense ) HX2, and its strain preservation information is: the taxonomic name is: Cladophiaphora guangxiense , the Chinese taxonomic name is: Cladosporium guangxiense, and the preservation number is CGMCC NO.41498; the strain is preserved in the China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is September 5, 2024.

[0011] Further, in the step (3), the liquid medium for the chlamydospores of the dark septate endophytic fungus Cladosporium cladosporioides is composed of the following components: 26 g / L of soybean powder, 25 g / L of corn flour, and 84 g / L of molasses; the inoculation amount is 3.6%; the fermentation conditions for the fermentation of the liquid medium of the chlamydospores of the dark septate endophytic fungus Cladosporium cladosporioides are: the filling volume of the flask is 115 mL / 250 mL Erlenmeyer flask, the rotation speed of shaking culture is 170 r / min, the culture time is 7 - 10 days, the temperature is 28 °C, and the original pH of the fermentation broth is 5.12.

[0012] Further, the chlamydospore concentration of the seed liquid is 1×10 5 cells / mL.

[0013] The present invention has the following beneficial effects: 1. Through experiments such as single - factor and response - surface optimization, the present invention has found the medium and culture conditions suitable for the growth of Cladosporium guangxiense HX2. After optimization, this method greatly improves the yield of chlamydospores of Cladosporium cladosporioides and effectively reduces the cost of producing chlamydospores; after measurement, the yield of chlamydospores reaches 1.39×10 8 ~ 2.71×10 8The number reached [X] CFU / mL, which was 139.8% higher than that of the traditional PDB medium. The cost was reduced by 47%, and its shelf life at room temperature was extended by about 8 months compared with the bacterial agent fermented by the traditional PDB medium. It effectively improved the utilization of the growth-promoting and disease-resistant strain HX2. The chlamydospores prepared had strong stress resistance and a long shelf life, and could be used to develop biocontrol agents for crop growth promotion and disease resistance. The medium of the present invention is simple to prepare, the fermentation process is stable, and it is suitable for large-scale production, providing a new and effective idea for the later popularization and application of the Cladosporium cladosporioides strain.

[0014] 2. The chlamydospores of Cladosporium cladosporioides HX2 from Guangxi have a thick-walled structure and strong tolerance to adverse environments such as drying, high temperature, and soil antibacterial effects, solving the technical bottleneck of the short shelf life of traditional conidial preparations, and the room temperature storage period can reach more than 1 year.

[0015] 3. The chlamydospore preparation of Cladosporium cladosporioides HX2 from Guangxi has a significant field control effect on tomato and ginger bacterial wilt, providing a new way for the green prevention and control of soil-borne diseases.

[0016] 4. The medium of the present invention uses cheap agricultural and sideline products such as molasses and corn flour as the main raw materials, with low cost; the fermentation conditions (such as pH 5.12 without adjustment) are easy to control and are suitable for large-scale promotion.

[0017] 5. The present invention first realized the large-scale liquid fermentation production of chlamydospores of Cladosporium cladosporioides HX2 from Guangxi, filling the blank of the chlamydospore preparation technology of this bacterium and providing a new resource for the development of biocontrol agents. Description of the Drawings

[0018] Figure 1 is the Value value of the variance analysis of the Plackett-Burman test.

[0019] Figure 2 is the electron microscope comparison diagram of the chlamydospore production of strain HX2 under different culture conditions; the left side is the conventional PDB medium, and the right side is the liquid fermentation medium of the present application.

[0020] Figure 3 is the response surface diagram of the influence of soybean powder and corn flour on the chlamydospore yield of strain HX2.

[0021] Figure 4 is the contour diagram of the influence of soybean powder and corn flour on the chlamydospore yield of strain HX2.

[0022] Figure 5 is the response surface diagram of the influence of soybean powder and molasses on the chlamydospore yield of strain HX2.

[0023] Figure 6 is the contour diagram of the influence of soybean powder and molasses on the chlamydospore yield of strain HX2.

[0024] Figure 7 It is a response surface plot of the effects of corn flour and molasses on the chlamydospore yield of strain HX2.

[0025] Figure 8 It is a contour plot of the effects of corn flour and molasses on the chlamydospore yield of strain HX2.

[0026] Figure 9 It is a prediction plot of the optimal culture components of strain HX2.

[0027] Figure 10 It is a plot of the results of the single-factor experiment on the inoculum amount of strain HX2.

[0028] Figure 11 It is a plot of the results of the single-factor experiment on the bottle filling amount of strain HX2.

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

[0030] Figure 13 It is a plot of the results of the single-factor experiment on the temperature of strain HX2.

[0031] Figure 14 It is a plot of the results of the single-factor experiment on the pH of strain HX2.

[0032] Figure 15 It is a response surface plot of the effects of rotation speed and bottle filling amount on the chlamydospore yield of strain HX2.

[0033] Figure 16 It is a contour plot of the effects of rotation speed and bottle filling amount on the chlamydospore yield of strain HX2.

[0034] Figure 17 It is a response surface plot of the effects of rotation speed and inoculum amount on the chlamydospore yield of strain HX2.

[0035] Figure 18 It is a contour plot of the effects of rotation speed and inoculum amount on the chlamydospore yield of strain HX2.

[0036] Figure 19 It is a response surface plot of the effects of bottle filling amount and inoculum amount on the chlamydospore yield of strain HX2.

[0037] Figure 20 It is a contour plot of the effects of bottle filling amount and inoculum amount on the chlamydospore yield of strain HX2.

[0038] Figure 21 It is a prediction plot of the optimal fermentation conditions of strain HX2.

[0039] Biological material preservation information

[0040] The information of the strain preserved in this application is: Cladosporium cladosporioides var. guangxiense ( Cladophiaphora guangxiense) HX2, whose classification name is: Cladophiaphora guangxiense , the Chinese classification name is: Cladophialophora guangxiensis, and the preservation number is CGMCC NO.41498; this strain is preserved in the China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is September 5, 2024. Specific embodiments

[0041] To make the above objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention 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 connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0042] The strain used in the embodiments of this application is: Cladophialophora guangxiensis ( Cladophiaphora guangxiense ), and its preservation information is: the preservation number is CGMCC NO.41498, and the classification name is: Cladophiaphora guangxiense , the Chinese classification name is: Cladophialophora guangxiensis, and the preservation number is CGMCC NO.41498; this strain is preserved in the China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is September 5, 2024.

[0043] Example 1

[0044] This example is a method for producing chlamydospores of Cladophialophora guangxiensis ( Cladophiaphora guangxiense ) HX2 (hereinafter referred to as strain HX2) as follows.

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

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

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

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

[0049] Among them, the method for counting the spores of strain HX2 is as follows: After the fermented HX2 bacterial liquid is broken by a homogenizer for 2 min, 1 mL of the bacterial suspension is taken and diluted in equal proportion, and then 20 μL is taken and counted on a hemocytometer, repeating three times. The counting method is to count the upper ones but not the lower ones, and count the left ones but not the right ones. The calculation formula is carried out according to (I).

[0050]

[0051] During the research, the applicant found that in laboratory research, when strain HX2 was cultured using PDB liquid medium, a small amount of chlamydospores could be induced to produce. However, when strain HX2 was cultured on a large scale, the amount of chlamydospores produced by strain HX2 did not reach a high yield. Therefore, the applicant further studied the fermentation medium in large-scale culture.

[0052] Second, explore the culture conditions and medium components of the liquid medium in step (3), and the specific method is as follows.

[0053] 1. Using water as a carrier, the following test carbon sources are selected respectively: glucose, sucrose, soluble starch, corn flour, molasses, sorbitol, mannitol, glycerol, maltose, fructose, and are respectively sterilized and reserved as the sole carbon source by adding at 50 g / L. They are cultured under the conditions of a liquid volume of 100 mL in a 250 mL Erlenmeyer flask, an inoculation amount of 1% of HX2, a rotation speed of 120 r / min, and 28 °C to screen the best carbon source for promoting the production of chlamydospores by strain HX2; the results obtained are shown in Table 1 - Table 2.

[0054]

[0055] Note: Different lowercase letters in the table indicate significant differences in the data in the same column (P < 0.05), and the same letters indicate no significant differences in the data in the same column (P > 0.05). The same applies to the following tables.

[0056] As can be seen from Table 1, among different carbon sources, the amount of chlamydospores produced by strain HX2 from high to low is molasses > corn flour > sucrose > maltose > glycerol > glucose > mannitol > soluble starch > sorbitol. The highest amounts of chlamydospores produced when molasses and corn flour are used as carbon sources are 2.68×10 7 cells / mL and 1.02×10 7 cells / mL respectively. When sorbitol and soluble starch are used as carbon sources, strain HX2 cannot be induced to continue to produce a large amount of chlamydospores. Therefore, molasses and corn flour are selected according to the gradient: the concentration range of corn flour is 10, 20, 30, 40, 50, 60, and the concentration range of molasses is 50, 60, 70, 80, 90, 100, 110, 120 g / L; they are used as the carbon sources of the medium and the concentration screening is carried out respectively. The results are shown in Table 2.

[0057]

[0058] As can be seen from Table 2, when the molasses concentration is determined to be 70 - 90 g / L, the spore production is the highest; when the corn flour concentration is determined to be 10 - 30 g / L, the spore production is the highest, which is used as the basis for subsequent response surface experiments; among them, the optimal concentration of molasses is 80 g / L (spore yield is 8.725×10 7 cells / mL), and the optimal concentration of corn flour is 30 g / L (spore yield is 2.91×10 7 cells / mL).

[0059] 2. Using water as the carrier, the following test nitrogen sources were selected respectively: soybean powder, tryptone, NH4Cl, beef powder, dry corn steep liquor, NH4NO3, KNO3, which were inoculated at 20 g / L after sterilization, and cultured under the conditions of a filling volume of 100 mL, an inoculation amount of HX2 of 1%, a rotation speed of 120 r / min, and 28°C to screen the optimal nitrogen source; the results obtained are shown in Tables 3 and 4.

[0060]

[0061] As can be seen from Table 3, among different nitrogen sources, the amount of chlamydospores produced by strain HX2 from high to low is soybean powder > tryptone > beef powder > KNO3 > NH4NO3 > NH4Cl > dry corn steep liquor. When soybean powder is used as the nitrogen source, the amount of chlamydospores produced is the highest, which is 1.31×10 8 cells / mL. When NH4NO3, NH4Cl, and dry corn steep liquor are used as nitrogen sources, strain HX2 cannot be induced to produce a large amount of chlamydospores continuously. On the basis of the optimal carbon source (a mixture of 80 g / L molasses and 30 g / L corn flour), soybean powder was selected as the nitrogen source of the medium at gradients of 10, 20, 30, 40, and 50 g / L for concentration screening, and the results are shown in Table 4.

[0062]

[0063] As can be seen from Table 4, when the concentration of soybean powder is in the range of 10 g / L - 30 g / L, the spore yield is the highest. Therefore, this concentration is selected as the basis for subsequent response surface experiments.

[0064] 3. Using water as the carrier, the following test inorganic salts were selected respectively: FeSO4, NaHCO3, KH2PO4, CaCl2, MgSO4, MnSO4, CuSO4, which were inoculated at 0.2 g / L, and cultured under the conditions of a filling volume of 100 mL in a 250 mL Erlenmeyer flask, an inoculation amount of HX2 of 1%, a rotation speed of 120 r / min, and 28°C to screen the optimal inorganic salts for promoting chlamydospore production by strain HX2, and the results obtained are shown in Tables 5 - 6.

[0065]

[0066] As can be seen from Table 5, in the screening of different inorganic salts, the amounts of chlamydospores produced by strain HX2 ranked from high to low as KH2PO4>CuSO4>MnSO4>CaCl2>CK>FeSO4>NaHCO3>MgSO4. Among them, the highest spore production was for KH2PO4, followed by CuSO4 and MnSO4, with the amounts of chlamydospores produced being 9.36×10 7 cells / mL, 9.33×10 7 cells / mL, 7.8×10 7 cells / mL, all of which were higher than CK and had significant differences. However, when FeSO4, NaHCO3, and MgSO4 were used as inorganic salts, the amounts of chlamydospores induced in strain HX2 were even lower than CK, indicating that these three inorganic salts had a certain inhibitory effect on the continuous large production of chlamydospores by strain HX2 in the enlarged culture medium. Therefore, KH2PO4, CuSO4, and MnSO4 were tentatively selected as the inorganic salts for the culture medium. Based on the optimal carbon source (a mixture of 80 g / L molasses and 30 g / L corn flour), the concentrations of the three inorganic salt ions were screened: the concentration ranges were 0.2, 0.4, 0.6, 0.8, 1.0 g / L, and the results are shown in Table 6.

[0067]

[0068] As can be seen from Table 6, the highest spore production of KH2PO4 occurred in the concentration range of 0.4 - 0.8 g / L, that of CuSO4 occurred in the concentration range of 0.6 - 1.0 g / L, and that of MnSO4 occurred in the concentration range of 0.2 - 0.6 g / L.

[0069] 4. On the basis of the above single factors, a response surface optimization experiment was carried out on the culture medium components of strain HX2: On the basis of the single factor experiment, A (soybean powder), B (corn flour), and B (molasses) were selected from the culture medium components as three factors. Taking the amount of chlamydospores produced in the fermentation broth of strain HX2 as the response value, the selected single factors were used as independent variables, and DesignExpert 13 software was used for the response surface experimental design to determine the optimal composition of the fermentation medium.

[0070] (1) Plackett-Burman experiment: Based on the results of the single factor experiment, the culture medium components of strain HX2 were molasses, corn flour, soybean powder, KH2PO4, CuSO4, and MnSO4. According to the results, the Plackett-Burman experiment was 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 it is shown that three key factors affecting the experiment, soybean powder, corn powder, and molasses, are found, and the significance of inorganic salt ions is not obvious and they are not added in subsequent experiments. Among them, soybean powder, corn powder, and molasses are significant factors and all are higher than the t-value, showing a positive effect in the experiment. According to the variance results in Table 9, the three factors reach a significant level, R 2 = 0.9166, adj R 2 = 0.8165 can be explained by the regression equation, indicating that the model has an effect on the key influencing factors of the culture medium.

[0075] (2) Steepest ascent experiment: According to the three key factors screened out, the steepest ascent experiment is designed. The climbing direction and step size of the key factors are determined according to the coefficients of the variables in the model, and the optimal range of the key factors is determined. In this way, the maximum point of the spore yield of strain HX2 is obtained as the center point of the central composite design experiment, which is 4. As shown in Table 10, the range of the addition mass ratio of the key components of the culture medium and the range of the culture conditions are thus determined.

[0076]

[0077] (3) Box - Behnken experiment: A central composite experiment with 3 factors and 2 levels is designed by the Box - Behnken response surface method (BBD) (see Table 11), and the key factors are optimized. Then, the amount of chlamydospores produced is fitted with a quadratic multiple regression equation, and the quadratic multiple regression equation for the amount of chlamydospores produced by strain HX2 is obtained: Amount of chlamydospores produced = 2.062 + 0.065A + 0.02B - 0.05C - 0.04AB - 0.05AC - 0.01BC - 0.11A 2 - 0.056B 2 - 0.136C 2 .

[0078]

[0079]

[0080] As can be seen from Table 12, R 2 = 0.9871, R 2 adj = 0.9706. 97.06% of the influence of the key factors on Y can be explained by the regression equation, and the variance analysis result of the regression equation shows that the regression equation is significant (P < 0.01). The response surface and contour lines of the interaction effects of soybean powder, corn powder, and molasses are drawn as followsFigure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown in Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 , the influence on the chlamydospore production of strain HX2 can be visually reflected. It is known from Figure 9 that the optimal medium component ratio is 26 g / L of soybean powder, 25 g / L of corn flour, and 84 g / L of molasses. Through experiments, the chlamydospore amount obtained is 2.135×10 8 cells / mL, exceeding the predicted level of 2.079×10 8 cells / mL, which proves the rationality of this prediction. All subsequent fermentation condition optimizations were tested according to this component ratio.

[0081] III. Optimal reaction conditions: Under the condition of determining the optimal medium concentration, the fermentation reaction conditions of strain HX2 were studied as follows.

[0082] 1. Single-factor experiments: Using the optimized medium components and concentration, with an inoculation amount of 1%, pH 5, filling volume of 100 mL, temperature of 28 °C, and rotation speed of 120 r / min as the initial culture conditions, single-factor experiments were successively conducted on different setting values of 5 factors including pH, fermentation temperature, rotation speed, filling volume, and inoculation amount to measure the chlamydospore amount in the fermentation broth of strain HX2, so as to optimize the parameters of each fermentation condition. The results of the single-factor experiments on 5 factors including pH, fermentation temperature, rotation speed, filling volume, and inoculation amount are as Figures 10 - 14 shown: Figure 10 shows the influence of the inoculation amount on the chlamydospore, Figure 11 shows the influence of the filling volume on the chlamydospore, Figure 12 shows the influence of the rotation speed on the chlamydospore, Figure 13 shows the influence of the temperature on the chlamydospore, Figure 14Effect of pH on chlamydospores; the experimental results show that: the chlamydospore yield is the highest when the inoculation amount is 1% - 5%. When the inoculation amount reaches 11%, due to the too large inoculation amount, mycelial clumping occurs, which instead affects the sporulation efficiency; the spore yield is the highest when the bottle filling amount is 75 - 125 mL / 250 mL. The chlamydospore yield decreases with the increase of the bottle filling amount. A too large bottle filling amount affects the oscillation amplitude of the culture medium, which instead reduces the sporulation efficiency. The conclusion is that the chlamydospore yield decreases with the increase of the bottle filling amount; the amount of chlamydospores produced is the highest when the rotation speed is 140 - 180 r / min; the amount of chlamydospores produced is the largest at 28°C, which is consistent with the initially selected base temperature. The original pH of the fermentation broth is 5.12. Through experiments, it is found that the best sporulation state can be achieved without adjusting the pH.

[0083] 2. Orthogonal optimization experiment: Based on the results of the single-factor experiment, we used the fermentation conditions, bottle filling amount, inoculation amount, and rotation speed of strain HX2 to conduct a response surface test. The specific experimental method is as follows.

[0084] (1) Box - Behnken test for fermentation conditions: Taking the inoculation amount, bottle filling amount, and rotation speed as three key factors as independent variables, quadratic polynomial regression fitting was performed on the spore yield data of strain HX2, and the key factor dynamic model was obtained as Y = 2.356 + 0.0425A + 0.04625B - 0.03125C + 0.015AB - 0.01AC - 0.0225BC - 0.12925A 2 - 0.07175B 2 - 0.07675C 2 where R 2 = 0.9577, adjusted R 2 = 0.9032. In the formula, Y is the predicted value of the spore yield of strain HX2, indicating that this regression equation has a 90.32% probability of explaining the influence of key factors on Y, showing that the regression equation is significant (as shown in Table 14).

[0085]

[0086]

[0087] The response surface diagrams obtained from the data in Table 14 ( Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 、 Figure 19 and Figure 20), revealing the effects of the pairwise interactions of three key factors, namely inoculum size, bottling volume, and rotation speed, on the chlamydospore yield. According to the canonical analysis of the response surface, the regression model has a maximum value. When the inoculum size is 3.6%, the bottling volume is 115, and the rotation speed is 170 r / min (as Figure 21 ), after 10 days of fermentation, the predicted maximum chlamydospore yield is 2.29 × 10 8 per mL. Verification based on the predicted fermentation conditions yielded a maximum spore yield of 2.71×10 8 per mL, exceeding the predicted value by 18.3% and being 139.8% higher than the commonly used PDB medium in the laboratory (1.13×10 8 per mL).

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

[0089] (1) Inoculate strain HX2 on a PDA medium plate and activate it at 28 °C for 10 days. Transfer the colony blocks on PDA to a PDB liquid medium and incubate them in a constant temperature shaker at 28 °C and 120 r / min for 14 days. Adjust the spore concentration to 1×10 5 per mL and reserve it as the seed liquid.

[0090] (2) Transfer the activated bacterial blocks from step (1) to a PDB liquid medium and incubate them at 28 °C and 120 r / min for 14 days to prepare the seed liquid.

[0091] (3) Inoculate the seed liquid into a liquid medium and shake-culture to obtain the chlamydospore fermentation broth; among them, the liquid medium for the chlamydospores of the dematiaceous septate endophytic fungus Cladosporium cladosporioides consists of the following components: 26 g / L of soybean powder, 25 g / L of corn flour, and 84 g / L of molasses; the inoculum size for inoculation is 3.6%; the fermentation conditions for the liquid medium fermentation of the chlamydospores of the dematiaceous septate endophytic fungus Cladosporium cladosporioides are: the bottling volume is 115 mL / 250 mL Erlenmeyer flask, the rotation speed for shaking culture is 170 r / min, the culture time is 7 - 10 days, the temperature is 28 °C, and the original pH of the fermentation broth is 5.12.

[0092] Among the above reaction conditions: soybean powder is in the range of 10 - 30 g / L, corn flour is in the range of 10 - 30 g / L, and molasses is in the range of 70 - 90 g / L; the inoculum size is 1% - 5%; the bottling volume is 75 - 125 mL / 250 mL Erlenmeyer flask, the rotation speed for shaking culture is 140 - 180 r / min, the culture time is 7 - 10 days, and the temperature is in the range of 23 - 30 °C, all of which can optimize the yield of strain HX2.

[0093] The comparison diagram of chlamydospores of strain HX2 produced by fermentation using the above-mentioned optimal method and chlamydospores of strain HX2 induced only by PDB medium is as Figure 2 shown. As can be seen from the figure, after liquid fermentation culture, the density of chlamydospores under the microscope is much greater than that of PDB medium. After detection, under this reaction condition, after 10 days of fermentation, the maximum spore yield obtained is 2.71×10 8 cells / mL, exceeding the predicted value (2.29 ×10 8 cells / mL) by 18.3%, and is 139.8% higher than that of the commonly used PDB medium in the laboratory. It is also found that only by using the liquid fermentation medium described in the present invention (10-30 g / L of soybean powder, 10-30 g / L of corn flour, 70-90 g / L of molasses) can a large amount of chlamydospores be produced, while the induction culture in other liquid induction media will inhibit the production of chlamydospores by 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 present 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 cane molasses, 26 g / L of soybean powder and 25 g / L of corn flour; the price comparison of the above two components is shown in Table 15.

[0098]

[0099] As can be seen from Table 15, the price of fermenting 1 ton using the PDB medium is 849.6 yuan, while the price of fermenting 1 ton using the liquid fermentation medium of the present application is only 453.08 yuan, and the cost price is reduced by about 47%. Thus, it can be seen that the cost of preparing chlamydospores of strain HX2 using the main raw materials of the medium of the present application is significantly reduced, and it is very suitable for large-scale promotion.

[0100] Example 3

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

[0102] Experimental method: Compare the shelf life of the microbial inoculum products of strain HX2 under the fermentation conditions of the traditional PDB medium and under the optimal fermentation conditions of Example 1. The measurement method of the shelf life is to measure the viable bacteria count by the viable bacteria counting method on the same day of each month from the 1st to the 20th month after the end of the culture, and then calculate 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) = Number of colonies on the plate × Dilution factor / Inoculation volume (mL).

[0104] The viable cell counting method is as follows: After diluting the liquid microbial agent to an appropriate multiple by gradient dilution, take 100 μL of the sample diluted to an appropriate multiple for coating. After 7 days, count the number of colonies to calculate the viable cell quantity, with 3 replicates for each.

[0105] The method for measuring the spore germination rate is as follows: Place the PDA plate under the microscope and count the number of germinated spores. Set 5 replicates for each treatment, eliminate the results with large differences, select 3 replicates and take their average as the result for analysis. Take 100 coated spores per plate as the base number, and the germination rate is expressed as a percentage.

[0106] When the two methods are completed, the initial spore production of the PDB medium is 1.13×10 8 cells / mL, and the initial spore production under the optimal fermentation conditions (liquid medium) of Example 1 is 2.54×10 8 cells / mL; The results of the viable cell quantity and germination rate measured monthly from the 1st to the 20th month are shown in Table 16.

[0107]

[0108] As can be seen from Table 16, for the microbial agents prepared by the two methods, both the viable cell quantity and the germination rate decrease with the increase of time. For the microbial agent prepared by the method of Example 1, the viable cell quantity reaches the order of magnitude of 10 10 in the 1st month, and the germination rate is as high as 95%; for the microbial agent prepared by conventional PDB culture, the viable cell quantity reaches the order of magnitude of 10 7 in the 1st month, and the germination rate is as high as 97%. The difference in germination rate between the two is not significant; when it comes to the 2nd month, the germination rate of the microbial agent prepared by the method of Example 1 is significantly higher than that of the microbial agent prepared by conventional PDB culture; in terms of the viable cell quantity, the microbial agent prepared by the method of Example 1 still maintains a relatively high viable cell quantity order of magnitude of 10 9 in the 20th month, but for the microbial agent prepared by conventional PDB culture, its germination rate is lower than 10% at room temperature in the 12th month, and the viable cell quantity order of magnitude cannot reach 10 7 ; It shows that the shelf life of the microbial agent prepared by the method of Example 1 is far better than that of the microbial agent prepared by conventional PDB culture, and the shelf life is extended by about 8 months compared with that of the microbial agent prepared by conventional PDB culture.

[0109] Example 4

[0110] This example mainly studies the induction of chlamydospores of other strains in the same genus by the liquid fermentation medium, as follows.

[0111] The best fermentation process of the chlamydospores of strain HX2 in Example 1 was used to ferment Cladophialophora of the same genus.

[0112] (1) Cladophialophora strains HX2, DH3, LC4, and LC83 (strains DH3, LC4, and LC83 were self-screened by the research group and molecularly identified as strains of the same genus, and have been published in the literature "Studies on the Diversity and Ecological Functions of Dark Septate Endophytic Fungi in Two Habitats in Guangxi") were inoculated on PDA medium plates and cultured at 28 °C for 10 days for activation. The colony blocks on PDA were transferred to PDB liquid medium and placed in a constant temperature shaker at 28 °C and 120 r / min for 14 days, and the spore concentration was adjusted to 1×10 5 cells / ml and used as the seed liquid for standby.

[0113] (2) The activated fungal blocks 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 into the liquid medium and cultured with shaking to obtain the chlamydospore fermentation broth; among them, the liquid medium consisted of 26 g / L of soybean powder, 25 g / L of corn flour, and 84 g / L of molasses; the fermentation conditions for liquid fermentation were: inoculation amount 3.6%, bottle filling amount 115, rotation speed 170 r / min; the chlamydospore yields obtained are shown in Table 17.

[0115]

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

[0117] In summary, through methods such as strain screening, single factor, and response surface optimization, this application found the culture medium and culture conditions suitable for the growth of strain HX2. After optimization, these reaction conditions greatly increased the chlamydospore yield of strain HX2, and at the same time effectively reduced the cost of producing chlamydospores by strain HX2; after calculation, the chlamydospore yield increased by 139.8% compared with the PDB medium, and the cost decreased by 47%; it effectively improved the utilization of the growth-promoting and disease-resistant strain HX2, extended the shelf life of the strain, and provided a new and effective idea for the later promotion and application of strain HX2.

[0118] Several embodiments of the present invention are described in a relatively specific and detailed manner, but this should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A method for liquid fermentation to produce chlamydospores of the dematiaceous septate endophytic fungus Cladosporium cladosporioides, characterized in that, The method comprises the following steps: (1) Inoculating Cladophiaphora guangxiense strain on PDA medium for activation culture; (2) Transferring the activated fungal blocks into PDB liquid medium to prepare a seed solution; (3) Inoculating the seed solution into a liquid medium for fermenting the chlamydospores of Cladophiaphora guangxiense, a dark septate endophytic fungus, to obtain a chlamydospore agent; In the step (3), the liquid medium for the chlamydospores of Cladophiaphora guangxiense, a dark septate endophytic fungus, consists of the following components: 10 - 30 g / L of soybean powder, 10 - 30 g / L of corn flour, and 70 - 90 g / L of molasses; In the step (3), the inoculation amount is 1% - 5%; In the step (3), the fermentation conditions for the liquid medium for the chlamydospores of Cladophiaphora guangxiense, a dark septate endophytic fungus, are as follows: the filling volume of the flask is 75 - 125 mL / 250 mL Erlenmeyer flask, the rotation speed of shaking culture is 140 - 180 r / min, the culture time is 7 - 10 days, the temperature is 23 - 30 °C, and the original pH of the fermentation broth is 5.12; The Cladosporium cucumerinum strain is Cladosporium cucumerinum from Guangxi ( Cladophiaphora guangxiense ), with the preservation number of CGMCC NO.41498.

2. The method according to claim 1, wherein In the step (3), the liquid medium for the chlamydospores of Cladophiaphora guangxiense, a dark septate endophytic fungus, consists of the following components: 26 g / L of soybean powder, 25 g / L of corn flour, and 84 g / L of molasses; In the step (3), the inoculation amount is 3.6%; In the step (3), the fermentation conditions for the liquid medium for the chlamydospores of Cladophiaphora guangxiense, a dark septate endophytic fungus, are as follows: the filling volume of the flask is 115 mL / 250 mL Erlenmeyer flask, the rotation speed of shaking culture is 170 r / min, the culture time is 7 - 10 days, the temperature is 28 °C, and the original pH of the fermentation broth is 5.

12.

3. The method according to claim 2, wherein The chlamydospore concentration of the seed solution is 1×10 5 cells / mL.

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