CN113755346A - Trichoderma T6 biological agent solid fermentation culture method and solid matrix - Google Patents
Trichoderma T6 biological agent solid fermentation culture method and solid matrix Download PDFInfo
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Abstract
The invention relates to the field of biocontrol microorganism culture, in particular to a solid fermentation culture method of a trichoderma T6 biological agent and a solid matrix, wherein the matrix is obtained by crushing and mixing wheat bran, straws and fresh cabbage, and the mass ratio of the wheat bran to the straws to the fresh cabbage is 1: 1: 3, the culture temperature is 26 ℃, the humidity is 65%, the culture time is 5 d, the air permeability is 8 layers of gauze sealing, and the illumination time is 0 h. The invention takes straws, wheat bran and cabbage as raw materials, prepares different culture matrixes according to different mass ratios, screens and obtains the optimal solid culture medium formula of the trichoderma biocontrol microbial inoculum, obtains the fermentation condition of the optimal spore yield of the trichoderma T6 biological microbial inoculum through a single-factor test, and provides a certain theoretical basis for the research and development and application of the trichoderma biological microbial inoculum.
Description
Technical Field
The invention relates to the field of biocontrol microorganism culture, in particular to a solid fermentation culture method and a solid matrix for a trichoderma T6 biological agent.
Background
The agricultural waste treatment cost is high, the pollution is large, great challenges are brought to rural environment treatment, the agricultural waste is processed into the microbial agent for recycling, the huge pressure on the environment can be effectively reduced, and a new thought for harmless treatment of the agricultural waste is expanded. At present, most of research and application is to use agricultural product processing waste as a base material to process and prepare a biological agent or a bacterial fertilizer, and the development of the biological agent or the bacterial fertilizer by using the agricultural waste is reported. Meanwhile, the existing commercial trichoderma biocontrol preparation mainly takes trichoderma spore powder as a main material, but the aspects of optimization and research and development of the preparation process of the trichoderma T6 biocontrol agent are not reported.
Disclosure of Invention
In order to solve the problems, the invention provides a solid fermentation culture method and a solid matrix for a trichoderma T6 biological agent.
In order to achieve the purpose, the invention adopts the technical scheme that: a solid fermentation substrate of a trichoderma T6 biological agent is prepared by crushing and mixing wheat bran, straws and fresh cabbage, wherein the mass ratio of the wheat bran to the straws to the fresh cabbage is 1: 1: 3.
the invention also provides a solid fermentation culture method of the trichoderma T6 biological agent, which adopts the solid fermentation substrate for fermentation, wherein the culture temperature is 26 ℃, the humidity is 65%, the culture time is 5 d, the air permeability is 8 layers of gauze sealing, and the illumination time is 0 h.
Further, the solid fermentation method comprises the following steps: placing the solid fermentation substrate in a glass culture dish, sterilizing at high temperature, cooling, and adding 3 mL of Trichoderma T6 strain spore suspension (1.0 × 10)7 each.mL-1) The relative humidity is 65%, and the mixture is sealed by 8 layers of gauze and then is placed at 26 ℃ for dark culture for 5 days.
The invention takes straws, wheat bran and cabbage as raw materials, prepares different culture matrixes according to different mass ratios, screens and obtains the optimal solid culture medium formula of the trichoderma biocontrol microbial inoculum, obtains the fermentation condition of the optimal spore yield of the trichoderma T6 biological microbial inoculum through a single-factor test, and provides a certain theoretical basis for the research and development and application of the trichoderma biological microbial inoculum.
Drawings
FIG. 1 shows the influence of different culture mediums and ratios on the spore yield of trichoderma T6;
FIG. 2 is a graph showing the effect of optimal and single fermentation media on the sporulation yield of Trichoderma T6;
FIG. 3 is the effect of cultivation time on spore yield of Trichoderma T6 biological agent;
FIG. 4 influence of temperature on spore yield of Trichoderma T6;
FIG. 5 the influence of air permeability on spore yield of Trichoderma T6;
FIG. 6 influence of humidity on spore yield of Trichoderma T6;
FIG. 7 is the effect of light irradiation time on spore yield of Trichoderma T6 biological agent;
FIG. 8 is an impact factor design model significance analysis;
FIG. 9 is a significant impact factor quadratic regression fit analysis and validation;
FIG. 10 humidity and temperature interaction contour plot (left) and three-dimensional surface plot (right);
FIG. 11 humidity and incubation time interaction contour plot (left) and three-dimensional surface plot (right);
FIG. 12 temperature and incubation time interaction contour plot (left) and three-dimensional surface plot (right);
FIG. 13 effect of optimal fermentation conditions on spore production of Trichoderma T6 microbial inoculum.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Test data
1 materials and methods
1.1 materials
Test strains
Test strains of Trichoderma (Trichoderma longibrachiatum) T6 was supplied by the university of agriculture in gansu plant pathology laboratory.
1.2 test methods
1.2.1 preparation of spore suspension of Trichoderma T6 Strain
Picking hyphae of a trichoderma T6 strain stored at a low temperature by using an aseptic inoculating needle, inoculating the hyphae to a PDA culture medium for central culture for 4 d, preparing a bacterial cake by using an aseptic puncher (d =0.5 cm), and inoculating the bacterial cake to the PDA culture medium for purification culture. After culturing for 6 days, adding 1 drop of Tween-80 (Tween-80) and 5 mL of sterile water into the trichoderma T6 bacterial colony which is subjected to activation culture on a PDA (personal digital assistant) plate, fully oscillating, and uniformly dispersing the conidia in the sterile water to obtain the stock solution of the conidia suspension. Then, the spore suspension concentration was counted using a hemocytometer, and the stock spore suspension concentration was diluted to 1.0X 10 with sterile water7 each.mL-1And storing in a refrigerator at 4 ℃ for later use.
1.2.2 treatment of matrix materials
Crushing the collected corn straws and the collected tailed vegetables, and storing for later use.
1.2.3 screening of substrates prepared from Trichoderma T6 biological Agents
In the test, crushed straws, waste vegetables and wheat bran are uniformly mixed according to different mass ratios to serve as a solid fermentation culture medium (shown in table 1), and the solid fermentation culture medium is uniformly mixed and placed in a glass culture dish with the diameter of 13 cm, wherein the sample loading amount of each dish is 30 g. Then, 3 mL of spore suspension (1.0X 10) of Trichoderma T6 strain was added after sterilizing at high temperature and cooling7 each.mL-1) And adding sterile water for moisturizing, wherein the adding amount of the sterile water is 50% of the total amount of the substrate, sealing the substrate by 8 layers of gauze, and fermenting and culturing at 28 ℃ under the condition of light and dark alternation (dark/light =12/12 h). Each treatment and control was tested in 3 replicates. And after the fermentation culture is carried out for 8 d, measuring the spore yield of the trichoderma T6 strain in the fermentation culture substrate with different proportions. Reference [ Chen, Qianlin, steady lack, etc. ] utilizing response surface analysis method to optimize Trichoderma atroviride Tr16 liquid fermentation spore production culture medium research [ J]Agriculture, 2020,48(10): 117-.]The method is used for measuring the sporulation amount of the trichoderma T6 strain. Then, screening the solid fermentation culture medium with the optimal spore yield according to the spore yields of the trichoderma T6 strains in the fermentation culture mediums with different proportions. The test culture mediums were numbered A, B, C, D, E, a, B, C, D and E, respectively, and the ratios thereof are as follows (Table 1).
TABLE 1 screening of solid fermentation culture medium for trichoderma T6 biological agent
1.2.4 determination and comparative analysis of spore yield of Trichoderma T6 biological agent optimal fermentation culture substrate
The optimal and single solid fermentation medium obtained by screening were placed in glass culture dishes with a diameter of 13 cm, respectively, and the sample loading amount of each dish was 30 g. The culture substrate treatment and the inoculation culture were carried out by the method of reference 1.2.3. The test was run on a single solid matrix as a control, and each treatment and control was 3 replicates. After culturing, observing the generation conditions of hypha and spores of the trichoderma T6 strain on the surface of the culture substrate every 1d, measuring the spore production of the trichoderma T6 strain in different culture substrates by referring to a 1.2.3 method when fermenting and culturing for 7 d, and comparing and analyzing the difference of the spore production of the optimal culture substrate and the single solid fermentation culture substrate.
1.2.5 Single-factor optimization of the preparation Process of Trichoderma T6 biological agent
1.2.5.1 Effect of cultivation time on spore yield of Trichoderma T6 biological agent
The optimal solid fermentation medium obtained by screening is placed in a glass culture dish with the diameter of 13 cm, and the sample loading amount of each dish is 30 g. Then, 3 mL of spore suspension (1.0X 10) of Trichoderma T6 strain was added after sterilizing at high temperature and cooling7 each.mL-1) The relative humidity is 50%, and the mixture is sealed by 8 layers of gauze and then is placed at 28 ℃ for fermentation culture under the light-dark alternate condition (dark/light =12/12 h). After culturing, the spore concentration of the Trichoderma T6 strain in the culture medium was measured 1 time every 1d, and 7 d (1-7 d) was continuously measured. Each treatment and control was run in 3 replicates, as follows.
1.2.5.2 Effect of temperature on spore yield of Trichoderma T6 biological agent
The test takes the screened optimal culture medium as a test culture medium, the fermentation culture of the trichoderma T6 biological agent is respectively carried out under the conditions of 15 ℃,20 ℃, 25 ℃, 28 ℃, 30 ℃, 35 ℃ and 40 ℃, the spore concentration of the trichoderma T6 strain in the culture medium is measured under different temperatures after the culture is carried out for 7 days, and other culture conditions are the same as 1.2.5.1.
1.2.5.3 Effect of air permeability on spore yield of Trichoderma T6 biological agent
In the test, 2, 4, 6, 8, 10 and 12 layers of gauze are respectively used for fermentation culture sealing treatment, after the gauze is cultured for 7 days, the spore concentration of the trichoderma T6 strain in the culture medium subjected to the gauze sealing treatment with different layers is measured, and other culture conditions are the same as 1.2.5.1.
1.2.5.4 Effect of humidity on spore yield of Trichoderma T6 biological agent
The relative humidity of the optimal solid fermentation culture substrate is set to be 0%, 20%, 35%, 50%, 65% and 80% of the total mass of the substrate respectively. After culturing for 7 days, measuring the spore concentration of the trichoderma T6 strain in the culture substrate with different humidity, and the other culture conditions are the same as 1.2.5.1.
1.2.5.5 Effect of illumination time on spore yield of Trichoderma T6 biological agent
The test illumination time is set as the illumination time is dark: illumination = 24: 0; dark: illumination = 8: 16; dark: illumination = 12: 12; dark: illumination = 0: 24. after the culture for 7 d, the influence of the illumination time on the spore concentration of the trichoderma T6 strain in the culture substrate is measured, and other culture conditions are the same as 1.2.5.1.
1.2.6 screening of significant influence factors of fermentation process for preparing Trichoderma T6 microbial inoculum
A central combination test of 5 factors, 5 levels, was performed according to the single factor influencing factors (incubation time, temperature, air permeability, humidity and illumination time), and each test was repeated 3 times (table 2). And then, carrying out experimental design by using Minitab 17 software, and carrying out residual error and simulation analysis by using the sporulation amount of the trichoderma T6 strain as a determination index to obtain the most significant influence factor.
TABLE 2 Minitab test factor level design
1.2.7 Trichoderma T6 biological agent preparation Process response surface optimization
Response surface optimization is carried out by using Design-Expert 10 software, 3 factors which are obtained by an influence factor significance analysis test and have the most significant influence on the spore yield of the trichoderma T6 biological agent are set as independent variables, and the spore yield is set as a response value. 17 test treatments are designed by adopting a Box-Behnken response surface method, the preparation process of the trichoderma T6 biological agent is carried out, an optimal fermentation condition simulation equation is established, and verification is carried out.
1.2.8 data processing
Excel 2010 is adopted for data processing, SPSS 24.0 software is adopted for single-factor analysis of variance, and Duncan new repolarization method is adopted for significance test of difference (P<0.05）。
2 results and analysis
2.1 screening of substrate prepared by Trichoderma T6 biological agent
The results show that different kinds and proportions of solid fermentation media have significant influence on the spore yield of the trichoderma T6 biological agent, and that there are significant differences between different kinds and proportions of solid fermentation media (fig. 1 and table 4). Compared with the culture medium prepared from dried Chinese cabbage (fig. 1A-E), the culture medium prepared from fresh Chinese cabbage (fig. 1A-E) has the advantages that the spore yield of trichoderma T6 is obviously higher than that of the culture medium prepared from dried Chinese cabbage, and the spore yield of trichoderma T6 strain is obviously increased along with the increase of the culture time. When the culture medium C is inoculated and cultured for 2D, a large amount of dense hyphae are distributed on the surface of the culture medium C (wheat bran: dry straw: fresh cabbage = 1: 1: 3), sporulation starts at the 3 rd stage, the color of the surface of the culture medium gradually changes from white to dark green, and a large amount of trichoderma T6 spores are distributed on the surface of the culture medium after 5D (figure 1C), which is obviously higher than the hyphae and spore contents on the surfaces of other culture mediums (figures 1A-B, figures 1D-E and figures 1A-E).
Compared with the test culture medium prepared from the fresh Chinese cabbage, the yield of the trichoderma T6 spores in the culture medium C is the highest after 8 days of inoculation culture, and the spore yield of the trichoderma T6 strain is 2.02 multiplied by 109each.mL-1The spore yield is obviously higher than that of other tested culture matrixes. The spore yield of trichoderma T6 in the culture medium C is increased by 36.38%, 13.31%, 68.08% and 93.82% compared with that of the culture medium A, B, D and E respectively. Compared with the test culture medium prepared from the dried brassica oleracea, when the culture medium is inoculated and cultured for 8 days, the maximum yield of the trichoderma T6 spores in the culture medium e is 7.11 multiplied by 108 each.mL-1The sporulation yield is increased by 22.14%, 384.84%, 1042.86% and 125.35% compared with that of culture substrates a, b, c and d respectively. However, the yield of the trichoderma reesei T6 spores in the culture substrate C is significantly higher than that in the culture substrate e, and the spore yield is increased by 184.69% compared with that in the culture substrate e. Therefore, the optimal solid culture medium for screening the trichoderma biocontrol microbial inoculum and the formula mass ratio thereof are wheat bran: straw: fresh tailed vegetable = 1: 1: 3 (table 3).
TABLE 3 influence of different culture media and ratios on spore yield of Trichoderma T6 biological agent
Note: the data in the table show the spore yield when the strain is cultured for 8 days.
2.2 comparison analysis of spore yields of the optimal fermentation culture substrate of the Trichoderma T6 biological agent and a single substrate
The result shows that the screened trichoderma T6 biological agent optimal culture medium and the formula mass ratio thereof are (wheat bran: straw: fresh cabbage) 1: 1: 3, the spore yield is obviously higher than that of a single solid fermentation culture medium. When the culture medium is cultured for 2 days, a large amount of white aerial hyphae and a small amount of spores are produced on the surface of the optimal culture medium, a large amount of spores are produced at 3 days, and the color of the surface of the culture medium is gradually changed from white to dark green. After 4 days of culture, the culture medium produced a large number of spores of Trichoderma T6 strain, and the whole culture medium was covered with a large number of dark green spores (FIG. 2). However, after inoculation culture (1-7 d), the single fresh cabbage, straw and wheat bran culture medium, the trichoderma T6 strain, and particularly the single straw culture medium trichoderma T6 strain, produced significantly less hyphae and spores than the optimal culture medium (fig. 2). Compared with a single culture medium, the spore yield of the trichoderma T6 strain in the optimal culture medium is obviously higher than that of the single culture medium, and the maximum spore yield of the optimal culture medium is 2.19 multiplied by 10 when the trichoderma T6 strain is cultured for 7 d9 each.mL-1The sporulation yield is 2.8 times, 8.28 times and 2.35 times higher than that of wheat bran, straws and fresh cabbage which are single culture matrixes respectively (Table 4).
TABLE 4 Effect of optimal and Single fermentation Medium on spore production by Trichoderma T6 Biobacterial agents
Note: the data in the table show the spore yield of 7 d cultivation. The same applies below.
2.3 Single-factor optimization of fermentation culture conditions of Trichoderma T6 microbial agents
2.1 Effect of fermentation time on spore production of Trichoderma T6 biological agent
The results show that the fermentation culture time has a significant influence on the spore yield of the trichoderma T6 biological agent, and the spore yield of the trichoderma T6 biological agent tends to be stable after being rapidly increased along with the increase of the fermentation time (fig. 3 and table 5). When the culture medium is inoculated and cultured for 1-2 days, a small amount of white aerial hyphae and spores are generated on the surface of the culture medium in the initial stage, and a large amount of white aerial hyphae and spores are generated in the later stage. However, after 2 d of inoculation culture, a large amount of green spores are gradually generated on the surface of the culture medium, and as the culture time is increased, the spores gradually cover the whole culture medium, and the whole culture medium surface is full of a large amount of spores. The spore yield measurement result shows that the spore yield of the trichoderma T6 biological agent is gradually increased (1-4 d) along with the increase of the culture time, the later period is basically stable, the increase is not obvious (5-7 d), and particularly the spore yield reaches the maximum value (2.09 multiplied by 10) at the 5 d after inoculation culture (2.09 multiplied by 10)9 each.mL-1) And after 6-7 d, the product tends to be stable, and the sporulation yield is not increased obviously. Therefore, the optimal spore yield fermentation culture time of the trichoderma T6 biological agent is 5 d, so the trichoderma T6 biological agent is used as the maximum value of the culture time in the next significant influence factor analysis test.
TABLE 5 Effect of cultivation time on spore yield of Trichoderma T6 microbial inoculum
2.2 Effect of temperature on spore yield of Trichoderma T6 biological agent
The temperature has obvious influence on the sporulation quantity of a fermentation culture substrate of the trichoderma T6 biological agent, and along with the rise of the temperature (15-40 ℃),the spore yield shows a trend of increasing first and then decreasing (15-40 ℃). Meanwhile, the trichoderma T6 strain grows more slowly at 0-30 ℃ and more rapidly at higher temperature, particularly, the hypha grows most rapidly at 30 ℃ and can grow over the whole dish for 1 day. However, when the temperature reaches 40 ℃, the activity is gradually lost, and the growth and development cannot be normally carried out. After 7 days of culture, when the temperature is 25 ℃, the sporulation yield reaches the maximum value of 2.39 multiplied by 109 each.mL-1The yield of spores was significantly higher than that of other treatments, and was increased by 4.11 times, 1.2 times, 1.78 times, 1.38 times and 1.52 times, respectively, than that of culture medium at 15 deg.C, 20 deg.C, 28 deg.C, 30 deg.C and 35 deg.C (Table 6). Therefore, the optimal fermentation temperature of the trichoderma T6 biological agent is 25 ℃, so that the culture condition with the temperature of 40 ℃ is eliminated in the next analysis test of the significant influence factors.
TABLE 6 influence of temperature on sporulation yield of Trichoderma T6 microbial inoculum
2.3.3 Effect of air permeability on spore production of Trichoderma T6 biological agent
With the increase of air permeability, the spore yield of the trichoderma T6 biological agent tends to increase firstly and then decrease, and the difference between the treatments is obvious. It was observed that when the gauze was cultured in 2 layers for 1.5 days, the surface of the medium had the least hyphae, and when the gauze was cultured in 8 layers, the medium had the darkest color and the highest spore yield of 2.08X 109 each.mL-1The sporulation amount was 2.84 times, 2.21 times, 1.46 times, 1.06 times and 1.14 times higher than that in the culture medium of 2-layer, 4-layer, 6-layer, 10-layer and 12-layer gauze sealing treatment, respectively (fig. 5 and table 7). Therefore, when the culture medium is cultured for 7 days at the temperature of 28 ℃, the optimal air permeability of the trichoderma T6 biological agent culture medium is 8 layers of gauze sealing treatment.
TABLE 7 influence of air permeability on spore yield of Trichoderma T6 microbial inoculum
2.3.4 Effect of humidity on spore production of Trichoderma T6 biological agent
The results show that the trichoderma T6 strain can grow and produce spores in fermentation culture mediums with different relative humidities. And observing that when the humidity is 65%, hypha in the culture medium is thicker than other treatments after inoculation and culture for 2 d-3.5 d, the color of the culture medium is gradually deepened along with the increase of the culture time, but the color of the culture medium is not obviously changed after the culture for 5 d. When the culture medium is cultured for 7 days, the spore yield of the culture medium reaches the highest level when the relative humidity is 65 percent, and the spore yield reaches 2.25 multiplied by 109 each.mL-1The sporulation yield was increased 332.42%, 131.84%, 39.01%, 9.20% and 5.23% respectively compared to 0%, 20%, 35%, 50% and 80% relative humidity compared to other treatments (fig. 6 and table 8). Therefore, the optimal relative humidity for trichoderma T6 biological agent fermentation is 65%.
TABLE 8 influence of humidity on spore yield of Trichoderma T6 microbial inoculum
2.3.5 Effect of illumination time on spore yield of Trichoderma T6 biological agent
The results show that different illumination times have obvious influence on the sporulation amount of the trichoderma T6 biological agent, when the illumination time is increased, hypha grows faster and sporulation is faster, and when the illumination time is reduced, the hypha grows more densely. Meanwhile, the color of the culture medium is gradually deepened along with the increase of the culture time, but the color of the culture medium after the 5 th day is not obviously changed, and the color of the culture medium under the dark condition is darker than that under the illumination condition. When the culture lasts for 7 days, the spore yield is the highest under the dark condition and reaches 2.34 multiplied by 109 each.mL-1Compared with the culture substrate with the illumination time of 12 h, 16h and 24 h, the sporulation yield of the culture substrate is respectively improved by 1.12 times, 1.46 times and 1.7 times (figure 7 and table 9). Therefore, the optimal illumination condition for fermenting the trichoderma T6 biological agent is dark.
TABLE 9 influence of light irradiation time on spore yield of Trichoderma T6 microbial inoculum
2.4 significant analysis and screening of influence factors of preparation process of Trichoderma T6 biological agent
2.4.1 design of impact factor significance analysis
A 5-factor, 5-level central combination test (levels (-1 and 1) was performed based on one-factor air permeability (a), temperature (B), incubation time (C), illumination time (D), and humidity (E). Designed and analyzed by utilizing Minitab 17 software, and subjected to residual error and simulation analysis by taking the sporulation quantity of the trichoderma T6 biological agent as a response quantity (Table 10).
TABLE 10 center combination test design
2.4.2 impact factor significance analysis
The results show that the temperature (B), the culture time (C) and the humidity (E) are all significant factors influencing the sporulation quantity of the trichoderma T6 biological agent, and the temperature, the culture time, the humidity and the culture time are used as significant interactive factors influencing the sporulation quantity of the trichoderma T6 biological agent. The normalization effect pareto analysis showed that temperature, incubation time and humidity were all important factors affecting spore yield (FIG. 8I). Normalized effect normal distribution analysis found that temperature, incubation time and humidity had the most significant effect on sporulation yield (fig. 8 II). Analysis of major effect factors revealed that the slopes of temperature, incubation time and humidity were the greatest, as the most significant factors affecting sporulation yield (fig. 8 III). Interaction analysis showed that the temperature and incubation time, humidity and incubation time fit well as significant factors affecting sporulation yield (FIG. 8 IV). Among the single-factor influencing factors, the temperature (B), the culture time (C) and the humidity (E) are all significant influencing factors (A)P<0.05) (Table 11), temperature and incubation time, humidity and incubation time as significant influencing factors in the interaction: (table 11) ((P<0.05) (Table 12).
TABLE 11 impact factor design model significance analysis
2.4.3 significance impact factor design model quadratic fitting analysis and verification
Analysis of the normalized effect pareto (FIG. 9I), the normalized effect normal distribution (FIG. 9 II), the main effect influence factor (FIG. 9 III) and the interaction (FIG. 9 IV) proves that the temperature (B), the culture time (C) and the humidity (E) of the significant influence factors are all single factors and interaction influence factors which significantly influence the spore yield of the trichoderma T6 biological agent. Meanwhile, the secondary fitting analysis of the significant influence factors finds the temperature, the culture time and the humidity, the temperature and the culture time, the humidity and the culture timePAll values are less than 0.05, bending and mismatching termsPValues are all larger than 0.05, which indicates that the temperature, the culture time and the humidity are all significant influence factors, the model is not unstable, the selection is reasonable, and the optimized result is identical with the previous analysis result (Table 12).
Table 12 significant impact factor quadratic regression fit analysis and validation
2.5 optimization of response surface of preparation Process of Trichoderma T6 biological agent
2.5.1 significance influencer response surface design
Experiments a central combination experiment at 3-factor 3 level was performed with the factors affecting sporulation yield of trichoderma T6, temperature (a), humidity (B) and cultivation time (C), as 3 significant influencing factors (table 13). Design experiment is carried out by using Design Expert 10 software, the spore production amount of trichoderma T6 biological agent is used as a response value, the temperature (A), the humidity (B) and the culture time (C) are used as independent variables, a Box-Behnken method is adopted to Design a response surface combination, and each group of experiments are repeated for 3 times (Table 14).
TABLE 13 Box-Behnken test factor level design
TABLE 14 optimized design of culture conditions for Trichoderma
2.5.2 regression model analysis of variance
A regression equation model between the temperature (A), the humidity (B) and the culture time (C) of the significant influence factors and the spore yield of the trichoderma T6 biological agent is obtained according to multiple regression fitting analysis and is as follows: y =231.8+35.77A +37.53B +53.72C +25.87AB +19.4AC +42.17BC-93.91A2-75.09B2-73.6C2And Y in the equation is a predicted value of the spore production amount of the trichoderma T6 biological agent.
The analysis of variance of a prediction regression model shows that the model regression level is obvious, and the interaction of the temperature (A), the humidity (B) and the culture time (C) and the temperature (A), the humidity (B) and the culture time (C) has the most obvious influence on the spore yield of the trichoderma T6 biological agent (the method comprises the steps ofP<0.0001). At the same time, regression model equation R2And Pred R2And Adj R2The correlation is good, which indicates that the fitting performance of the prediction regression model equation is good, and the optimal sporulation quantity and the optimal fermentation condition of the trichoderma T6 biological agent can be well predicted. Meanwhile, of 3 significant factors of temperature, humidity and culture time, the culture time has the most significant influence on the sporulation quantity of the trichoderma T6 biological agent, and the humidity and the temperature are the second order (Table 15). In addition, the optimal culture conditions and the optimal spore yield are predicted based on a regression model, and the result shows that the optimal culture conditions of the trichoderma T6 biological agent culture medium are as follows: the temperature is 26.33 ℃, the humidity is 65.33%, the culture time is 4.83 d, and the optimal predicted value of sporulation quantity is 2.4587 multiplied by 109 each.mL-1。
TABLE 15 regression model ANOVA TABLE
Note: indicates significance, indicates extreme significance.
2.5.3 significant influencer interaction analysis
The response surface optimized 3D surface graph and the two-dimensional contour graph intuitively reflect the relation between the test factors and the response values and can comprehensively reflect the interaction between the two factors. The steepness of the 3D curved surface graph reflects the sensitivity of spore yield to the change of fermentation conditions, and the larger the gradient is, the more remarkable the influence on the test result is; the contour map of the response surface visually reflects the influence of each factor on the response value, and the central point of the minimum ellipse in the contour line is the highest point of the response surface. In addition, the shape of contour line can reflect the strength of interaction effect, the ellipse represents that two factors interact obviously, and the circle is opposite to the interaction effect [. The results of two-dimensional contour plot analysis show that the contour plots of A (temperature) -B (humidity) (FIG. 10), B (humidity) -C (incubation time) (FIG. 11) and A (temperature) -C (incubation time) (FIG. 12) are nearly elliptical, and the interaction between the two factors is significant; the 3D surface map analysis results show that the three-dimensional maps of A (temperature) -B (humidity) (figure 10) and B (humidity) -C (culture time) (figure 11) have larger gradient and strong interaction, have obvious influence on the spore yield of the trichoderma T6 biological agent, and have obvious influence on the spore yield of the trichoderma T6 biological agent (figure 12).
2.5.4 response surface optimal fermentation condition verification
The result of the verification of the optimal fermentation condition shows that the optimal sporulation quantity predicted value of the trichoderma T6 biological agent (2.4587 multiplied by 10)9 each.mL-1) The method is basically consistent with the measured value, and the established prediction model is proved to be reliable. When the fermentation temperature of the trichoderma T6 biological agent is 26.33 ℃, the humidity is 65.33 percent and the culture time is 4.83 d, the best measured value of the spore yield is 2.4635 multiplied by 109 each.mL-1. Meanwhile, the color of the culture medium is gradually deepened along with the increase of the culture time, dense hyphae grow over the whole culture medium after 2 d of culture, the spore production starts after 3d, and a thick layer of spore powder covers the surface of the culture medium after 4.83 d(FIG. 13). Meanwhile, compared with the spore yield of the culture medium before optimization, the spore yield is improved by 21.78%.
The invention takes straws, wheat bran and Chinese kalimeris as raw materials, different culture matrixes are prepared by different mass ratios, and the optimal solid culture medium for screening the trichoderma biocontrol agent is prepared into the trichoderma biocontrol agent with the formula mass ratio (wheat bran: straws: fresh Chinese kalimeris) of 1: 1: 3. and obtaining the optimal culture temperature of 26 ℃, the humidity of 65%, the culture time of 5 d, the air permeability of 8 layers of gauze sealing and the illumination time of 0 h through a single-factor test.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (5)
1. A solid fermentation substrate of trichoderma T6 biological agent is characterized in that: the matrix is prepared by pulverizing testa Tritici, straw, and fresh caulis et folium Brassicae Capitatae, and mixing.
2. The trichoderma T6 biological agent solid fermentation substrate of claim 1, wherein: the mass ratio of wheat bran, straw and fresh cabbage is 1: 1: 3.
3. a solid fermentation culture method of trichoderma T6 biological agent is characterized in that: fermenting with a solid fermentation substrate according to any of claims 1-2.
4. The solid fermentation culture method according to claim 3, characterized in that: the culture temperature is 26 ℃, the humidity is 65%, the culture time is 5 d, the air permeability is 8 layers of gauze sealing, and the illumination time is 0 h.
5. The solid fermentation culture method according to claim 3, characterized in that: the method comprises the following steps: placing the solid fermentation substrate of any one of claims 1-2 in a glass petri dish, cooling by autoclaving, and adding 3 mL of woodSpore suspension of the mould T6 strain (1.0X 10)7 each.mL-1) The relative humidity is 65%, and the mixture is sealed by 8 layers of gauze and then is placed at 26 ℃ for dark culture for 5 days.
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| CN102382791A (en) * | 2011-11-21 | 2012-03-21 | 滨州职业学院 | Fermentation process of trichoderma |
| CN102531720A (en) * | 2012-01-14 | 2012-07-04 | 甘肃明德伟业生物科技有限公司 | Fermented biofertilizer prepared from waste vegetables, straw and livestock and poultry feces and preparation method thereof |
| CN106348888A (en) * | 2016-08-31 | 2017-01-25 | 山东省科学院生态研究所 | Method for producing trichoderma bio-organic fertilizer by using green Chinese onion leaf waste |
| CN107446876A (en) * | 2016-11-29 | 2017-12-08 | 甘肃农业大学 | A kind of fermentation process of response phase method optimization long shoot trichoderma T6 sporulation quantities |
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| CN102382791A (en) * | 2011-11-21 | 2012-03-21 | 滨州职业学院 | Fermentation process of trichoderma |
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| CN106348888A (en) * | 2016-08-31 | 2017-01-25 | 山东省科学院生态研究所 | Method for producing trichoderma bio-organic fertilizer by using green Chinese onion leaf waste |
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