CN110241029B - Coptis chinensis soil ferulic acid degrading bacterium and application thereof - Google Patents
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Abstract
The invention discloses a coptis chinensis soil ferulic acid degrading bacterium and application thereof, belonging to the field of microorganisms. A strain of Penicillium dentatum (Penicillium daleae) has efficient degradation effect on soil allelochemicals ferulic acid, has antibacterial effect on rhizoctonia solani, rust rot and black spot and other plant pathogenic bacteria, and has repairing and biocontrol effects on continuous cropping soil. The strain is obtained by separating and purifying five-year-old coptis chinensis soil in Shizhu county, after the strain is determined to have a degradation effect on ferulic acid, the species is identified by ITS, the degradation efficiency of the strain on the ferulic acid under different addition amounts of the ferulic acid and different fermentation times is tested, the optimal fermentation time is finally determined to be 96 hours, and the degradation efficiency is obviously reduced when the ferulic acid is added into a culture medium in an amount of more than 800 micrograms/ml. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 5 and 9 months, and the preservation number is CGMCC No. 15675.
Description
Technical Field
The invention relates to a coptis chinensis soil ferulic acid degrading bacterium and application thereof, belonging to the field of microorganisms.
Background
Coptidis rhizoma is perennial herb of Ranunculaceae, and its rhizome is in the form of yellow beaded. As a traditional Chinese medicine, it is commonly used for treating gastrointestinal tract diseases such as gastrointestinal damp-heat and emesis. The berberine has the functions of reducing total cholesterol, triglyceride and low-density lipoprotein cholesterol in serum, and has the effects of reducing blood sugar and protecting cardiovascular.
The rhizome medicinal materials including coptis chinensis, radix pseudostellariae and rehmannia glutinosa have continuous cropping obstacles caused by the change of soil physicochemical properties and the imbalance of soil microecology. For example, the coptis chinensis can be planted again only after the soil in which a round of coptis chinensis is planted needs to be taken down for 5-10 years, which seriously limits the development of the coptis chinensis industry. One of the major causes of continuous cropping obstacles is the accumulation of allelochemicals secreted by plants in the soil as the planting age increases. The high-concentration phenolic acid allelochemicals inhibit key enzymes required by seed germination, inhibit the growth of coptis chinensis seedlings, interfere the photosynthesis of plants, interfere the absorption of mineral nutrient elements by the plants, and thus influence the normal growth of the plants. Ferulic acid is one of important factors causing continuous cropping obstacle of crops, and leads the activity of protective enzyme in plants to be sharply reduced so as to strongly inhibit the growth of seedlings roots.
In recent years, researches on alleviating continuous cropping obstacles by artificially managing soil microorganisms are increasingly deep, and the phanerochaete chrysosporium with a good phenolic acid degradation effect efficiently degrades phenolic acid in a soil environment; the arbuscular mycorrhizal fungi are reciprocal symbionts with microbial characteristics and plant roots in a soil ecosystem, and can effectively relieve continuous cropping obstacles. Therefore, allelochemically degrading bacteria are screened from indigenous microorganisms, and the soil microbial community structure is artificially managed, so that the repair efficiency of continuous cropping obstacles can be accelerated, and the method is favorable for planting medicinal materials.
Disclosure of Invention
The invention aims to solve the first technical problem of providing a coptis chinensis soil ferulic acid degrading bacterium.
The second technical problem to be solved by the invention is to provide the application of the coptis chinensis soil ferulic acid degrading bacteria.
In order to achieve the purpose, the invention adopts the following technical scheme:
chongqing Shizhu county is the first coptis chinensis high-yield demonstration base passing national GAP certification, and the annual yield of the Chongqing Shizhu county is 2000 tons, which accounts for more than 60% of the national coptis chinensis yield. The research separates multiple strains of fungi which have degradation effect on ferulic acid from the planting soil of the coptidis rhizoma with the stone pillar, and selects the strain with the best degradation effect to carry out identification and degradation efficiency test, so that ferulic acid degradation bacteria resources from the coptidis rhizoma soil are enriched, and a foundation is laid for repairing coptidis rhizoma continuous cropping obstacles.
A coptis chinensis soil ferulic acid degrading bacterium F-0056 with the preservation number of CGMCC No. 15675.
The strain F-0056 is Penicillium dentatum (Penicillium daleae), is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 5 months and 9 days, and has a preservation number of CGMCC No. 15675. And (4) storage address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
On the other hand, the invention provides the application of the coptis chinensis soil ferulic acid degrading bacterium F-0056 with the preservation number of CGMCC No. 15675.
The application is to prepare a soil ferulic acid degradation agent or a biological antibacterial agent. The penicillium dentatum has bacteriostatic effects on root rot, rhizoctonia solani, rust rot and black spot, so that the penicillium dentatum can be used as a bio-control fungicide to be applied in the process of planting medicinal materials.
The soil is planting soil of Chinese herbal medicine coptis, or planting soil of rhizome Chinese herbal medicines such as pseudo-ginseng, radix pseudostellariae and the like, or planting soil of crops such as apples, strawberries and the like. The phenolic acid degrading microbial inoculum can relieve continuous cropping obstacles in the planting process of the traditional Chinese medicinal materials.
On the other hand, the invention provides a method for degrading coptis chinensis soil ferulic acid, which is characterized by comprising the following steps: the coptis root soil ferulic acid degrading bacterium F-0056 with the preservation number of CGMCC No.15675 as claimed in claim 1 is added into soil for culture.
The culture conditions are as follows:
the effective number of bacteria is 1 multiplied by 109cfu/g of F-0056 spore suspension is dissolved in an amount of 100 g/mu and then irrigated to soil after one round of planting in the positive stubbles, so that phenolic acid accumulated in the soil can be rapidly degraded, and the aim of soil remediation is fulfilled.
On the other hand, the invention provides an ecological preparation for degrading coptis chinensis soil ferulic acid, which contains coptis chinensis soil ferulic acid degrading bacteria F-0056 with the preservation number of CGMCC No. 15675.
The preparation is prepared by mixing the spore powder of coptis chinensis soil ferulic acid degrading bacteria F-0056 with the preservation number of CGMCC No.15675, organic fertilizer, preservative, trichoderma harzianum and bacillus subtilis.
The preparation contains rhizoma CoptidisThe effective viable count of the ferulic acid degrading bacteria F-0056 is 1 × 109cfu/g. The ecological preparation is dry powder, wettable powder or solution.
Accumulation of phenolic acid allelochemicals in soil is one of important factors causing continuous cropping obstacles of traditional Chinese medicinal materials, and separation and purification of phenolic acid degrading bacteria from coptis chinensis soil are necessary for relieving the continuous cropping obstacles. In the research, a strain F-0056 with the best degradation effect is selected from a plurality of separated ferulic acid degrading bacteria for species identification, the degradation efficiency of the strain is tested under different fermentation time and different ferulic acid addition amount, and the ferulic acid is completely degraded when the fermentation time reaches 96 hours, wherein the ferulic acid addition amount is 200 mu g/ml-1(μ g/ml) the highest degradation efficiency.
The invention has the advantages that: the bacterial strain CGMCC No.15675 in the soil environment can still effectively decompose ferulic acid in the soil and slow down the accumulation of phenolic acid in the planting soil. The work enriches ferulic acid degrading bacteria resources from coptis chinensis soil, and lays a foundation for screening microorganisms capable of effectively repairing coptis chinensis continuous cropping obstacles. The invention has simple and stable fermentation process, easy preparation of fungal spores, more convenient preservation condition of the spores than that of mycelia, and normal-temperature transportation.
The invention is described in further detail below with reference to the figures and the specific examples, without limiting the invention. All equivalent substitutions in the art made in accordance with the present disclosure are intended to be covered by the present patent.
Drawings
FIG. 1 shows the obtained ferulic acid-degrading bacteria
FIG. 2 colony morphology in solid culture at different times
FIG. 3 conidiophores of Strain F-0056
FIG. 4 ITS sequence-based F-0056 phylogenetic tree
FIG. 5 degradation efficiency of F-0056 with addition of ferulic acid at various concentrations
FIG. 6 shows the degradation effect of F-0056 on ferulic acid during different fermentation times
FIG. 7 shows the degradation effect of F-0056 on ferulic acid in soil environment
Detailed Description
Example 1
1. Materials and methods
1.1 sample Collection and processing
The sample is rhizosphere soil of five-year-old coptis chinensis, collected from a coptis chinensis planting base (108 ° 23 'E, 30 ° 15' N) in shizhu county of Chongqing in 2016 at 10 months, and brought back to the laboratory by a sterile bag for the current day treatment.
1.2 isolation and purification of microorganisms in soil
Removing impurities such as roots, fallen leaves and stones in the soil sample, sieving the soil sample by a 20-mesh sieve, adding 5 g of the soil sample into 50ml of sterile water, diluting the soil sample by 10000 times, and uniformly coating the soil sample on a Potato Dextrose (PDA) solid culture medium added with chloramphenicol (25 mg/ml). Culturing for 3-5 days at 25 ℃ to form visible bacterial colonies, and transferring to a non-resistance PDA solid culture medium for purification and culture.
1.3 phenolic acid degrading bacteria screening
Selecting single colony of the separated and purified fungus, coating the single colony on a PDA solid culture medium, culturing the single colony for 4 to 5 days at 25 ℃ until a large number of spores are generated, and washing the spores with sterile water to prepare the fungus with the effective number of 1 multiplied by 107cfu/ml spore suspension. Inoculating the spore suspension into 50ml of inorganic salt culture medium with the inoculation amount of 1%, respectively adding 200 micrograms/ml of ferulic acid as a unique carbon source, and culturing for 3 days at the rotating speed of 180rpm under the condition of 28 ℃. Collecting thallus, drying to constant weight, and weighing thallus. And (3) uniformly mixing the fermentation liquor with ethyl acetate with the same volume, carrying out ultrasonic treatment for 30 minutes, standing overnight, taking the upper layer of ethyl acetate, carrying out rotary evaporation to dryness, and dissolving the ethyl acetate back in 2ml of methanol. Chromatographic conditions are as follows: the separation column was a symmetey C18 column from Waters, the detection wavelength was 280nm, the column temperature was 30 deg.C, the sample volume was 20. mu.l, the mobile phase consisted of methanol and water (the pH of the ultrapure water in the mobile phase was adjusted to 2.8 with analytically pure glacial acetic acid), and the volume flow was 1 ml/min-1. Gradient elution setup: at 12 minutes, the volume ratio of methanol to water was 40: 60, and at other times (0, 0.1, 20 minutes) the volume ratio of methanol to water was 30: 70.
Inorganic salt culture medium formula (1L)
1.4 genomic DNA extraction
Scraping a proper amount of hyphae cultured for 4 days, adding 600 microliters of lysis buffer and 10 glass beads, and shaking for 80 seconds by using a homogenizer; after adding 275. mu.l of 7 mol ammonium acetate, incubation was carried out at 65 ℃ for 10 minutes, followed by 5 minutes on ice, 500. mu.l of chloroform was added, vortexed for 1 minute and centrifuged at 13400 rpm for 5 minutes. Sucking 700 microliter of supernatant, mixing with isopropanol of the same volume, incubating at-20 ℃ for 30 minutes, centrifuging at 13400 rpm for 7 minutes, discarding the supernatant, washing the precipitate with 70% ethanol for 3 times, discarding the ethanol, dissolving the precipitate with sterile water after completely volatilizing the ethanol.
Lysis buffer formulation:
1M Tris(pH＝8.0) 10％
0.5M EDTA 10％
10％ SDS 10％
1.5 Gene spacer sequence (ITS) identification
The extracted genome is used as a template, and fungus ITS universal primers (27F: AGAGTTTGATCMT GGCTCAG/1525R: AAGGAGGTGWTCCARCC, sequence tables SEQ ID NO: 1 and SEQ ID NO: 2) are used for carrying out PCR reaction under the following conditions: pre-denaturation at 95 ℃ for 5 min, denaturation at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, and extension at 72 ℃ for 45 sec for 30 cycles. And (4) carrying out electrophoresis detection on the amplified fragment, and then sending the fragment to Huada Gene company for sequencing. The obtained sequence information is aligned and analyzed by BLAST, reliable reference sequences similar to the target sequence are selected, and phylogenetic analysis is carried out by using software MEGA 7.0.
1.6 exploration of fermentation conditions for different concentrations of Ferulic acid
Respectively adding 200 mug/ml of inorganic salt culture medium into 50ml of inorganic salt culture medium-1、400μg·ml-1、600μg·ml-1、800μg·ml-1And 1000. mu.g.ml-1The effective bacteria number of the ferulic acid is 1 multiplied by 107cfu/ml of fungal F-0056 spore suspension was inoculated at an inoculum size of 1%, and an equivalent amount of sterile water was added as a control, and cultured at 28 ℃ at 180rpm for 3 days. And (4) the fermentation liquor is dissolved in 2ml of methanol after being extracted, and HPLC detection is carried out, wherein the detection condition is 1.3.
1.7 exploration of different fermentation time conditions
Adding ferulic acid 200 mug/ml into inorganic salt culture medium-1And 1% of the effective number of bacteria is 1X 107cfu/ml fungal F-0056 spore suspension was incubated at 28 ℃ and 180rpm for 24 hours, 48 hours, 72 hours and 96 hours, respectively. And (4) the fermentation liquor is dissolved in 2ml of methanol after extraction, and HPLC detection is carried out, wherein the detection condition is 1.3.
1.8 monitoring the degradation effect of F-0056 on ferulic acid in soil environment
Digging appropriate amount of forest soil, and collecting the soil with effective bacteria number of 1 × 107Inoculating cfu/ml F-0056 spore suspension into 1 kg soil per pot at a ratio of 10: 1, and adding 1 mg/ml exogenous ferulic acid as control-1. Taking 50g of soil every 6 days, extracting with 150ml of ethyl acetate, and detecting the content change of the ferulic acid by HPLC, wherein the detection condition is 1.3.
2. Results and analysis
2.1 screening of degrading bacteria
46 fungi are obtained by co-separation from five-year-old coptis chinensis soil, 10 fungi capable of growing in a culture medium with ferulic acid as a unique carbon source are obtained by primary screening, and the growth conditions of the fungi under the condition with ferulic acid as a unique carbon source are shown in figure 1. According to the degradation condition and spore production efficiency of ferulic acid, the strain F-0056 is finally selected for further study.
2.2 identification of Strain F-0056
F-0056 bacterial colony is white filamentous when cultured for 2 days, the edge is neat, green spores begin to generate after 3 days, and the morphology of the bacterial colony cultured on PDA for different times is shown in figure 2; the conidiophores and spore chains unique to Penicillium notatum were examined microscopically, as shown in FIG. 3.
The strain F-0056 is subjected to ITS fragment amplification and then sequenced, and the obtained sequence is as follows, wherein the GenBank sequence number is SUB 4477629F-0056 MH 817153:
＞F-0056
after the ITS sequences are subjected to BLAST comparison, the similarity of the F-0056 strain and the ITS sequences with GenBank numbers of KF313087.1, AF033442.1 and KM357338.1 of three strains of Penicillium dentatum reaches 99 percent, so that the strain is identified as Penicillium dentatum (Penicillium daleae), and a phylogenetic tree is constructed based on the ITS sequence adjacency method as shown in FIG. 4.
The strain F-0056 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC) No.15675 in 2018, 5 and 9 months. And (4) storage address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
2.3 degradation efficiency of Strain F-0056 when different concentrations of Ferulic acid were added
The phenolic acid degradation efficiency of the strain F-0056 was tested using inorganic salt medium supplemented with different amounts of exogenous ferulic acid, and it was found that when exogenous ferulic acid was added to the inorganic salt medium in an amount of 200. mu.g/ml-1When the bacterial strain is used, the degradation efficiency is highest, the degradation effect of the bacterial strain F-0056 on the ferulic acid is gradually reduced along with the increase of the addition amount of exogenous ferulic acid, and the addition amount of the ferulic acid is increased to 800 mu g/ml-1The degradation efficiency was significantly reduced, see fig. 5.
2.4 degradation effect of the strain F-0056 on ferulic acid during different fermentation times
When the amount of the added exogenous ferulic acid is 200 mug/ml-1And (3) testing the degradation efficiency of the ferulic acid when F-0056 spore suspension and the same amount of sterile water are added as a control for fermentation for 24 hours, 48 hours, 72 hours and 96 hours, compared with the control group, the degradation rate of the ferulic acid after the F-0056 is added is increased along with the prolonging of the fermentation time, and the ferulic acid is almost completely degraded at 96 hours, which is shown in figure 6.
2.5 degradation Effect of the Strain F-0056 on Ferulic acid in soil Environment
As can be seen from FIG. 7, the degradation effect of ferulic acid is obvious after the degrading bacteria F-0056 is added into the soil, and the degradation rate of ferulic acid in the soil reaches 71.8% on day 6, which is obviously higher than that of ferulic acid in the soil without the degrading bacteria. The ferulic acid degrading bacteria can be proved to be capable of rapidly and effectively decomposing ferulic acid in the soil environment and reducing the content of the ferulic acid in the soil.
EXAMPLE 2 Ferulic acid-degrading bacteria Single component preparation
The preparation method of the F-0056 spore powder comprises the following steps:
the deposited F-0056 strain was streaked on PDA solid medium and activated by culturing at 25 ℃ for 2 days. And (3) picking a single colony of the activated fungus on a PDA solid culture medium, culturing for 3-4 days at the temperature of 25 ℃, and washing spores on the culture medium with sterile water. Washing the spores with sterile water for 2 times, and freeze-drying to obtain spore powder.
The effective number of bacteria can be 1 × 109cfu/g of F-0056 spore powder dissolved in an amount of 100 g/mu is poured into soil after one round of planting in the positive stubble, so that phenolic acid accumulated in the soil can be rapidly degraded, and the purpose of soil remediation is achieved;
the preparation can be used as a single preparation for degrading the soil ferulic acid, and can also be mixed into other soil preparations as a soil repairing additive.
EXAMPLE 3 Ferulic acid-degrading bacteria combination preparation
The single-component preparation of the ferulic acid degrading bacteria prepared in the embodiment 2 is mixed with other biocontrol bacteria such as trichoderma harzianum, bacillus subtilis and the like and organic fertilizer to prepare a mixed preparation, and the dosage form is powder. The effective viable count of the coptis root soil ferulic acid degrading bacteria F-0056 in the mixed preparation is 1 multiplied by 109cfu/g。
The combined preparation comprises the following components: 80-85% of straw powder, 10-15% of compound fertilizer, 1% of adhesive and 2% of formula bacteria.
Before the coptis chinensis seedlings are transplanted, the soil is stirred by a combined preparation containing F-0056 effective bacteria at an application amount of 100 g/mu, so that root diseases caused by rhizoctonia solani, rust rot and black spot bacteria can be effectively inhibited during the growth of the coptis chinensis, and the accumulation of phenolic acid allelopathic substances in the soil can be slowed down.
Sequence listing
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Claims (10)
1. Coptis chinensis Franch ferulic acid degrading bacterium F-0056 and classification life thereofNamed as blue tooth spore mold (Penicillium daleae) The preservation number is CGMCC No. 15675.
2. The use of the coptis chinensis soil ferulic acid degrading bacterium F-0056 with the preservation number of CGMCC No.15675 as claimed in claim 1; the application is to prepare a soil ferulic acid degrading agent or a biocontrol microbial inoculum, and the biocontrol microbial inoculum inhibits root diseases caused by rhizoctonia solani, rust rot and black spot and slows down the accumulation of phenolic acid allelopathy substances in soil.
3. Use according to claim 2, characterized in that: the soil is crop planting soil.
4. Use according to claim 3, characterized in that: the crops are rhizome Chinese herbal medicines, apples or strawberries.
5. Use according to claim 4, characterized in that: the rhizome Chinese herbal medicine is pseudo-ginseng, radix pseudostellariae or coptis chinensis.
6. A method for degrading coptis chinensis soil ferulic acid is characterized by comprising the following steps: the coptis root soil ferulic acid degrading bacterium F-0056 with the preservation number of CGMCC No.15675 as claimed in claim 1 is added into soil for fermentation culture.
7. The method for degrading coptis chinensis soil ferulic acid according to claim 6, wherein the culture conditions are as follows: the effective number of bacteria is 1 multiplied by 109 cfu/g (colony forming unit/g) of F-0056 spore powder is dissolved in an amount of 100 g/mu and then irrigated to the soil after one round of planting in the positive stubbles, so that phenolic acid accumulated in the soil can be rapidly degraded, and the aim of repairing the soil is fulfilled.
8. An ecological preparation for degrading coptis chinensis soil ferulic acid is characterized in that: contains coptis root soil ferulic acid degrading bacteria F-0056 with the preservation number of CGMCC No. 15675.
9. Ecological formulation according to claim 8, characterized in that: the preparation is prepared by mixing the spore powder of coptis chinensis soil ferulic acid degrading bacteria F-0056 with the preservation number of CGMCC No.15675, organic fertilizer, preservative, trichoderma harzianum and bacillus subtilis.
10. Ecological formulation according to any one of claims 8 to 9, characterized in that: the ecological preparation is dry powder, wettable powder or solution.
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| "两株阿魏酸降解菌的分离及其特性的研究";张月;《中国优秀硕士学位论文全文数据库（电子期刊）》;20180115;第1-63页 * |
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