阅读说明：本技术 提高中华羊茅-内生真菌菌株pa次生代谢产物震颤素含量的方法 (Method for improving content of tremorine in secondary metabolite of Festuca sinensis-endophytic fungi strain PA ) 是由 李春杰 刘静 陈振江 魏学凯 李涛 于 2019-09-20 设计创作，主要内容包括：本发明提供提高中华羊茅-内生真菌菌株PA次生代谢产物震颤素含量的方法,包括以下步骤：(1)将中华羊茅-内生真菌菌株PA的菌丝接种到平板PDA培养基上进行黑暗培养；(2)接种至M102种子液体培养基中继续培养；(3)接种至M104T培养基中进行发酵培养,离心收集滤液；发酵培养的条件为：黑暗培养,初始pH8-10,装液量25-100mL,接种量0.5％-5％,接种时间14d-28d,转速120r/min-160r/min,温度20℃-30℃。本发明利用中华羊茅-内生真菌菌株PA的发酵液为材料,通过优化发酵条件,提高菌株代谢产物活性物质的产率,为微生物农药的研发提供基础资料。(The invention provides a method for improving the content of tremorine of a secondary metabolite of a Festuca sinensis-endophytic fungi strain PA, which comprises the following steps: (1) inoculating hypha of a Festuca arundinacea-endophytic fungi strain PA to a flat PDA culture medium for dark culture; (2) inoculating to M102 seed liquid culture medium for continuous culture; (3) inoculating to M104T culture medium for fermentation culture, centrifuging, and collecting filtrate; the conditions of fermentation culture are as follows: dark culture, initial pH8-10, liquid loading 25-100mL, inoculation amount 0.5-5%, inoculation time 14d-28d, rotation speed 120r/min-160r/min, and temperature 20-30 ℃. The invention uses the fermentation liquor of the Festuca arundinacea-endophytic fungi strain PA as a material, improves the yield of the active substances of the metabolite of the strain by optimizing the fermentation conditions, and provides basic data for the research and development of microbial pesticides.)

1. The method for improving the content of tremorine of a secondary metabolite of a Festuca sinensis-endophytic fungi strain PA is characterized by comprising the following steps: the method comprises the following steps:

(1) extracting Festuca sinensis (Festuca sinensis) -endophytic fungi

(2) inoculating to M102 seed liquid culture medium for continuous culture;

(3) inoculating to M104T culture medium, fermenting, centrifuging, and collecting filtrate;

wherein, in the step (3), the fermentation culture conditions are as follows: dark culture, initial pH8-10, liquid loading 25-100mL, inoculation amount 0.5-5%, inoculation time 14d-28d, rotation speed 120r/min-160r/min, and temperature 20-30 ℃.

2. The method of claim 1, wherein: in the step (1), the dark culture is carried out at 22 ℃ for four weeks.

3. The method of claim 1, wherein: in the step (2), the culture is carried out in the dark at 22 ℃ and the rotating speed of 200r/min for four weeks.

4. The method of claim 1, wherein: in the step (3), the fermentation culture conditions are as follows: dark culture, pH9, liquid loading 75mL, inoculation amount 3%, inoculation time 21d, rotation speed 140r/min and temperature 20 ℃.

Technical Field

The invention relates to the improvement of Festucaininensis (Festucainensis) -endophytic fungus

sp.) content of the secondary metabolite tremorine of strain PA.

Background

Endophytic fungi refer to fungi that live in plant tissues during part or all of their life history and do not cause significant disease symptoms to plants (Siegelet, 1987; Li jade articles et al, 2014). The research shows that

Endophytic fungi can produce alkaloids as secondary metabolites with biological activity, such as indole diterpenes (lndolditerpene) represented by tremorine (lolitremB), pyrrolopyrazines (pyrrolopyrazine) represented by peramine (peramine), ergolines (ergotaloids) represented by ergonovine (ergonovine) and ergotamine (ergoline), saturated pyrroles (pyrrolizine) represented by loline (loline), and the like (Gaoka and Nanzhi Scale, 2007; Xurui et al, 2012; Liqueu et al, 2015). The four major alkaloids have certain toxicity to some nematodes and herbivorous insects. Statistically, more than 77 grasses are considered to be carried45 kinds of grass endophytic fungi (madder et al, 2018) have been isolated and identified

Grass endophytic fungi (Leuchmannetal, 2014; Jinwen et al, 2015; Shymanovecheal, 2017; Mihwaetal, 2018) and which exhibit a more pronounced resistance to at least 79 species of pests (Lixiu jade et al, 2015; Xiaetal, 2018). The grass endophytic fungi has abundant resources and potential as potential biocontrol factors.

Numerous researchers have found that under pure culture conditions,

the grass endophytic fungi can also produce some secondary metabolites of alkaloids (Blakenshipetal, 2001; Yuuetal, 2000; Gaojia flowers, 2007) which have higher virulence effects on pestsIt is described by (Christensenda Latch, 1991; Faeth, 2002; Zhang Xueben et al, 2010; Schardleal., 2013) as a biocontrol resource, but in low amounts (Gao Jia, 2007).

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for improving Festucainensis-endophytic fungi

sp.) content of the secondary metabolite tremorine (LolitremB) of strain PA. The fermentation conditions are optimized through a single-factor experiment and an orthogonal experiment, so that the yield of the metabolites of the strains is improved, the fermentation time is shortened, the energy consumption is reduced, and the pollution is reduced.

In order to achieve the technical purpose, the invention adopts the technical scheme that:

the invention provides a method for improving Festucainensis (Festucaisinensis) -endophytic fungus: (

sp.) method for the content of the secondary metabolite tremorine of strain PA comprising the steps of:

(1) mixing Festucainensis (Festucaisinensis) -endophytic fungi (

sp.) inoculating the hypha of the strain PA to a flat PDA culture medium for dark culture;

(2) inoculating to M102 seed liquid culture medium for continuous culture;

(3) inoculating to M104T culture medium, fermenting, centrifuging, and collecting filtrate;

wherein, in the step (3), the fermentation culture conditions are as follows: dark culture, initial pH8-10, liquid loading 25-100mL, inoculation amount 0.5-5%, inoculation time 14d-28d, rotation speed 120r/min-160r/min, and temperature 20-30 ℃.

Preferably, in step (1), the dark culture is carried out at 22 ℃ for four weeks.

Preferably, in step (2), the culture is performed in the dark at 22 ℃ and 200 r/min.

Preferably, in step (3), the conditions of the fermentation culture are as follows: dark culture, pH9, liquid loading capacity of 75mL, inoculation amount of 3%, inoculation time of 21d, rotation speed of 140r/min, and temperature of 20 ℃.

Preferably, the grass is festuca arundinacea.

The invention takes first to utilize Festucaininensis (Festucainensis) -endophytic fungus (

sp.) the fermentation broth of the strain PA is used as a material, the fermentation conditions (pH, liquid loading amount, inoculation time, shaking table rotating speed and temperature) are optimized through a single-factor experiment and an orthogonal experiment, the yield of the metabolite active substances of the strain is improved, and basic data are provided for the research and development of microbial pesticides.

The invention obtains fermentation liquor by shaking flask fermentation; obtaining the optimal fermentation conditions (pH, liquid loading amount, inoculation time, shaking table rotating speed and temperature) of the strains through a single-factor experiment, and obtaining the optimal fermentation combination of the strains through an orthogonal experiment; and (5) fermenting according to the optimal combination, measuring the alkaloid content, and evaluating.

The invention has the beneficial effects that:

1. the pollution in the fermentation process is reduced, and the cost is low;

2. the fermentation period is shortened, and the energy consumption is low;

3. increasing the yield of active metabolites;

4. is composed of

Grass endophytic fungi have been developed to provide a basis for pesticides.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows the effect of pH on the tremorine content.

FIG. 2 shows the effect of liquid loading on the content of tremorine.

FIG. 3 is a graph showing the effect of inoculum size on tremorine content.

FIG. 4 is a graph showing the effect of inoculation time on the tremorine content.

FIG. 5 is a graph of the effect of rotational speed on the tremorine content.

FIG. 6 is the effect of temperature on the tremorine content.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.

Festucainensis (Festucaisinensis) -endophytic fungussp.) strains were provided by the grass protection institute of grassland agriculture science and technology, university of Lanzhou, under the accession number MHLZU-FSPA.

The invention improves the content of Festucaininensis (Festucainensis) -endophytic fungus

sp.) method for the content of the strain PA secondary metabolite tremorine (LolitremB) was as follows:

1. preparing seed fermentation liquor: selecting slant test tube strain Festucainensis (Festucaisinensis) -endophytic fungus ((Festucaisinensis))

sp.) the hypha of the strain PA is transferred to a flat PDA culture medium, dark culture is carried out at 22 ℃ for four weeks, after four weeks, 3 blocks of bacterial dishes with the diameter of 6mm are cut from the edges of bacterial colonies by a sterile puncher, the bacterial dishes are cut as much as possible, the bacterial dishes are transferred to a 250mL triangular flask containing 100mLM102 seed liquid culture medium, the flask is placed in a shaking table incubator with the rotating speed of 200r/min and the temperature of 22 ℃ for dark culture for four weeks to promote the growth of mycelium, 2mL of seed liquid are respectively absorbed by a disposable injector to the 250mL triangular flask containing 100mL of M104T culture medium, the flask is placed at the rotating speed of 100r/minCulturing in a shaking incubator at 22 deg.C in the dark for two weeks to promote the production of alkali by the mycelia. Under aseptic conditions, 10mL of the fermentation broth was aspirated by a disposable syringe and placed in a centrifuge tube. Centrifuging at 6000r/min for 10min, vacuum filtering, separating filtrate and mycelium, and collecting filtrate.

The formula of the plate PDA culture medium is as follows: 200g of potato, 20g of glucose, 17g of agar and 1000mL of distilled water.

The formula of the M102 seed liquid culture medium is as follows: 30g of sucrose, 20g of maltose, 1.0g of yeast extract, 2g of peptone and MgSO 2₄·7H₂O0.5g，KCl0.5g，KH₂PO₄1g, 1000mL of distilled water. (Bacon, 1988; parrott, 1994).

The formula of the M104T culture medium is as follows: 100g of sorbitol, 40g of glucose, 3g of yeast extract, 10g of glutamic acid, 0.8g of tryptophan and MgSO₄·7H₂O0.3g, 1000ml of distilled water (Bacon, 1988; parrott, 1994).

2. In vitro detection of tremorine: 200 mul of the bacterial suspension was weighed into a 2ml centrifuge tube, 1ml of dichloromethane was added, ultrasonic extraction was carried out for 5min, centrifugation was carried out for 10min at 1000rmp, 0.4ml of the supernatant was extracted through a Sep-pak column (1ml of methanol was activated), 1ml of 20% by volume methanol and 80% by volume dichloromethane were washed, then 1ml of the same solution was collected, and the collected liquid was subjected to HPLC analysis in a 1.5ml brown bottle through a 0.33mm pore size polyethylene organic filter pad (commercially available from the England corporation under the trade name NAVIGATOR) (Gallagheretal, 1985; Liujing et al, 2017).

3. Single factor fermentation screening

The M104T medium was used as a basal medium for single factor screening of fermentation conditions including initial value (pH), liquid loading (fermentation volume), inoculum size, inoculation time, shaker rotation speed and temperature.

3.1 initial value

Placing in a 250mL triangular flask 100mLM104T culture medium, wherein the inoculum size is 2%, the initial value of the fermentation broth is 8, 9, 10, 11 and 12 respectively, placing in a rotary shaker, and culturing at 22 ℃ and 100r/min for two weeks. The effect of different initial values of pH on the tremorine content was studied, with three replicates of each treatment.

3.2 liquid loading

The screened initial pH values are adopted, the liquid loading amounts are respectively set to be 25mL, 50mL, 75mL, 100mL and 125mL, the mixture is filled into a 250mL triangular flask containing M104T culture medium and is placed in a rotary shaking table to be cultured for two weeks at 22 ℃ at 100 r/min. The effect of different liquid loading on the tremorine content was studied, with each treatment repeated three times.

3.3 inoculum size

The screened initial pH and liquid loading amount are respectively 0.5%, 1%, 2%, 3%, 4% and 5%, the obtained mixture is filled into a 250mL triangular flask containing M104T culture medium, and the flask is placed in a rotary shaking table and cultured for two weeks at 22 ℃ and 100 r/min. The effect of different inoculum sizes on the tremorine content was studied, with three replicates per treatment.

3.4 inoculation time

The screened initial pH, liquid loading amount and inoculation amount are adopted, a 250mL triangular bottle is filled with M104T culture medium, the fermentation time is respectively 0d, 7d, 14d, 21d, 28d and 35d, the mixture is placed in a rotary shaking table and cultured for the required time at 22 ℃ at 100 r/min. The effect of different inoculation times on the tremorine content was studied, with three replicates per treatment.

3.5 rotational speed of rocking bed

The screened initial pH, liquid loading amount, inoculation amount and inoculation time are adopted, a 250mL triangular bottle is filled with M104T culture medium, the medium is placed in a rotary shaking table for culture, the rotating speeds are respectively set as 100r/min, 120r/min, 140r/min, 160r/min, 180r/min and 200r/min, and the medium is cultured for the required time at 22 ℃. The effect of different rotational speeds on the tremorine content was studied, with each treatment being repeated three times.

3.6 temperature of fermentation

The screened initial pH, liquid loading amount, inoculation time and rotation speed are adopted, a 250mL triangular bottle is filled with M104T culture medium, the fermentation temperatures are respectively 10 ℃, 15 ℃,20 ℃, 25 ℃, 30 ℃ and 35 ℃, and the medium is placed in a rotary shaking table for cultivation for the required time. The effect of different fermentation temperatures on the tremorine content was studied, with three replicates per treatment.

4. Design of orthogonal experiments

According to the single-factor screening result, the experiment selects the optimum initial value (pH), the liquid loading amount (fermentation volume), the inoculation amount, the inoculation time, the rotating speed of a shaking table and the temperature to carry out the 6-factor 3-level orthogonal experiment.

And (4) selecting the optimal combination to ferment according to the optimization result of the orthogonal experiment, and sucking 10mL of fermentation liquor by using a disposable needle tube under the aseptic condition and placing the fermentation liquor into a centrifuge tube. Centrifuging at 6000r/min for 10min, vacuum filtering, separating filtrate and mycelium, and collecting filtrate. The tremorine content was determined and each treatment was repeated three times.

5. Analysis of results

5.1 Single factor experiment

According to the screening results of the single-factor fermentation conditions, the selection of initial pH (8, 9 and 10), liquid loading amount (25mL, 75mL and 100mL), inoculation amount (0.5%, 3% and 5%), inoculation time (14d, 21d and 28d), rotating speed (120r/min, 140r/min and 160r/min), temperature (20 ℃, 25 ℃ and 30 ℃) are adopted to carry out 6-factor 3 level orthogonal experiments (see table 1).

TABLE 1 fermentation factors and levels of the PA strain tremorine

5.2 significance analysis

The orthogonal experiment results are shown in table 2, the influence factors of pH, liquid loading amount, inoculation time and temperature have obvious influence on the tremblin content of the PA strain (P < 0.05), the influence of rotating speed on the tremblin content is not obvious (P >0.05), and the primary and secondary relations of 6 factors are as follows: pH > liquid loading > inoculation amount > inoculation time > temperature > rotation speed. (see Table 2).

TABLE 2 significance analysis of the main body effect test of the tremolin content of the PA strains

0.961 (0.867. regulating R square)

5.3 treatment combination preference

According to the significance analysis result of each factor, the combination of the most suitable PA strain for producing the tremulin is A₂B₂C₂D₂E₂F₁. Namely pH9, liquid loading capacity of 75mL, inoculation amount of 3%, inoculation time of 21d, rotation speed of 140r/min and temperature of 20 ℃.

5.4 optimization result verification

The content of tremulin produced by the optimized PA strain fermentation liquor is increased by 3.5 times (Table 3).

TABLE 3 fermentation optimization validation results