CN112646733B - Tamarix chinensis endophytic antagonistic fungus as well as separation method and application thereof - Google Patents
Tamarix chinensis endophytic antagonistic fungus as well as separation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses tamarix chinensis endophytic antagonistic fungi as well as a separation method and application thereof, belonging to the technical field of agricultural biological control. The invention discloses tamarix chinensis endophytic antagonistic fungus HMST-1, which has a preservation number of CGMCC No.19261. The invention provides a method for obtaining tamarix chinensis endophytic antagonistic fungi by separation and screening, wherein the strain is identified as alternaria alternate, and provides a biocontrol strain for effectively preventing and controlling root rot diseases of cotton in a seedling stage; the invention provides a biocontrol function of liquid fermentation liquor of endophytic alternaria alternata of tamarix chinensis and a metabolite bacteriostatic active substance thereof, and provides effective components for creating a novel microbial pesticide preparation.
Description
Technical Field
The invention relates to the technical field of microbial biocontrol, in particular to tamarix chinensis endophytic antagonistic fungus and a separation method and application thereof.
Background
At present, in Xinjiang cotton production in China, various chemical pesticides are mainly used for preventing and treating root rot diseases, the defects of increased drug resistance of pathogenic bacteria, low pesticide utilization rate and the like exist, the problems of environmental pollution, serious pesticide residue and the like are easily caused, the survival of beneficial microorganisms is threatened, and the resistance of infected germs is induced to be enhanced, so that the prevention effect is gradually reduced. Therefore, the application technology and the product of the alternative chemical pesticide for preventing and treating the red rot of cotton are urgently needed to be searched.
The biological pesticide has the advantages of strong selectivity, low residue, high efficiency, low toxicity, difficulty in generating drug resistance and the like, wherein part of the microbial pesticide is derived from plant endophytic fungi, and the endophytic fungi can independently generate abundant secondary metabolites with various biological activities, are important sources of natural products and have important application potential in biological prevention and control of crop diseases. Therefore, the plant endophytic fungi are important microbial resources for developing novel biological pesticides, and the inhibition effect of the plant endophytic fungi or metabolites thereof on plant pathogenic bacteria is an important biocontrol way.
The Tamarix chinensis (Tamarix chinensis) habitat is special, and has strong saline-alkali resistance, drought resistance, sand resistance and disease and insect resistance.
Therefore, the problem to be solved by the technical personnel in the field is to provide the tamarix chinensis endophytic antagonistic fungus and the separation method and application thereof.
Disclosure of Invention
In view of the above, the invention provides tamarix chinensis endophytic antagonistic fungi and a separation method and application thereof, and the method has important theoretical and practical significance for mining and utilizing regional microbial biocontrol resources, promoting development of plant pathology, pesticides and other subjects, and reducing pesticide application and improving pesticide efficiency.
In order to achieve the purpose, the invention adopts the following technical scheme:
a tamarix chinensis endophytic antagonistic fungus HMST-1 has a preservation number of CGMCC No.19261, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, is called CGMCC for short, and is deposited at the institute of microbiology, china academy of sciences, no. 3, west Siro 1, north Cheng, inward, beijing, with the preservation date of 2019, 12 months and 16 days, and is classified and named as Alternaria alternata.
Further, a method for separating tamarix chinensis endophytic antagonistic fungi HMST-1 comprises the following specific steps:
selecting tamarix chinensis with special plant types and special forms in special habitats; selecting effective twig tissues, and removing oil and impurities indoors for soaking; shearing and segmenting by using a sterilizing scissors, disinfecting an implant, cleaning by using sterile water, passing flame through sterile filter paper, and performing 5-point separation culture on a PDA culture medium; selecting bacterial colony, separating monospore, culturing and screening opposite directions, and storing in test tube slant.
Further, a preparation method of tamarix chinensis endophytic antagonistic fungus HMST-1 liquid fermentation broth comprises the following specific steps:
(1) Transferring the strain HMST-1 from the inclined plane of the test tube to a PDA plate for activation, culturing at 25 deg.C for 5-7 days, selecting the strain HMST-1, and placing in 50mL sterile water to obtain a solution with a concentration of 1.0 × 10 5 Spore suspension per mL for use;
(2) Adopting PD liquid culture medium with pH of 7.0, inoculating at 10% inoculation ratio under aseptic condition to obtain inoculation concentration of 1.0 × 10 5 Adding 3.0mmol/L KH as metabolic factor into spore suspension 2 PO 4 Carrying out shake culture at 25 ℃ and 150r/min for 5d;
(3) After the strain HMST-1 liquid fermentation broth is centrifuged for 10min at 8000r/min by a high-speed refrigerated centrifuge, supernatant is taken and diluted by 5-10 times.
Further, the tamarix chinensis endophytic antagonistic fungus HMST-1 is applied to inhibiting red rot and rhizoctonia solani in cotton seedling stage.
Further, the tamarix chinensis endophytic antagonistic fungus HMST-1 liquid fermentation broth is applied to prevention and treatment of cotton seedling red rot and cotton rhizoctonia rot.
According to the technical scheme, compared with the prior art, the invention discloses and provides tamarix chinensis endophytic antagonistic fungi and a separation method and application thereof, the invention excavates and utilizes medicinal plants of special habitat in Xinjiang area, provides a method for obtaining tamarix chinensis endophytic antagonistic fungi by separation and screening, and the strain is identified as alternaria alternate, so that a biocontrol strain is provided for effectively preventing and controlling root rot diseases of cotton in seedling stage; the invention provides a biocontrol function of liquid fermentation liquor of endophytic alternaria alternata of tamarix chinensis and a metabolite bacteriostatic active substance thereof, and provides effective components for creating a novel microbial pesticide preparation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing the inhibitory effect of the strain HMST-1 of the present invention on cotton root rot;
wherein A represents that the strain HMST-1 is subjected to confrontation culture with fusarium verticillioides; b represents the independent culture of the fusarium verticillioides; c represents that the strain HMST-1 is subjected to opposite culture with rhizoctonia solani; d represents independent culture of Rhizoctonia solani.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The absolute ethyl alcohol, 50% carbendazim wettable powder, huifeng polymer, yiruitan and Junzhuang are all commercially available products, for example, the absolute ethyl alcohol is purchased from Tianjin Yongcheng fine chemical engineering Co., ltd, the 50% carbendazim wettable powder is purchased from Jiangsu Lanfeng biological chemical engineering Co., ltd, the huifeng polymer is purchased from Jiangsu Huifeng biological agricultural Co., ltd, the Yiruitan is purchased from Heilongjiang agricultural cultivation Sanlong biological science Co., ltd, the Junzhuang is purchased from Cangzhou Huayu biological science Co., and the commercially available product manufacturers used in the invention are not limited to the above manufacturers.
Example 1
A method for separating tamarix chinensis endophytic antagonistic fungi HMST-1 comprises the following specific steps:
(1) Collection of Tamarix chinensis samples of special habitat
Selecting tamarix chinensis with strong drought resistance, sand resistance, salt and alkali resistance and disease and insect resistance in West Gobi of Yizhou district, hami city, uygur autonomous region of Xinjiang in 2018, 5 months and 9 days, wherein the tamarix chinensis plant type is compact and grass green needle-shaped branches and leaves, collecting the twigs, packaging in envelopes, and storing in a refrigerator at 4 ℃ for later use.
(2) Sample processing
Collecting and storing samples, selecting effective ramulus et folium Tamaricis tissue indoors after 1-2 days, subpackaging in 250mL triangular flasks filled with distilled water, dripping 1-2 drops of high-efficiency detergent for carving cards after the ramulus et folium Tamaricis is completely immersed in water, mixing uniformly in the triangular flasks, and removing oil and impurities for soaking for 24h.
(3) Isolation of endogenous antagonistic fungi
Cutting and segmenting soaked tamarix chinensis twig tissues by using small sterilized scissors, sterilizing the plant body by using 2 percent of NaClO for 1min, then sterilizing the plant body by using 75 percent of alcohol for 30-40s, then cleaning the plant body by using sterile water for 3-5 times, then placing the cleaned plant body on sterile filter paper by using sterile forceps, absorbing the water, then lightly passing through an alcohol lamp outer flame, placing the plant body on a PDA culture medium by using the sterile forceps for 5-point separation when the filter paper and the plant body are anhydrous, carrying out dark culture in a 25 ℃ culture box for 3-4d, picking bacterial colonies, culturing the bacterial colonies on the PDA culture medium for 7-10d, carrying out single spore isolation culture after purification, and observing the morphological characteristics of the bacterial colonies; then the separated endophytic fungi and target bacteria (Rhizoctonia solani, fusarium verticillium) are subjected to opposite culture, endophytic fungi strains with stronger bacteriostatic activity are screened out, and test tube bevels are preserved, and the test tube bevels are named as HMST-1 by number.
(4) Endophytic antagonistic fungus species identification
Morphological characteristics: the strain HMST-1 grows vigorously on a PDA culture medium, the bacterial colony is relatively flat, hyphae are dense and wool-shaped, the grey color of the grass is changed into grey black, and the back of the strain is grey black. Conidiophore is brown, is nearly elliptical or inverted pear-shaped, is single-grown or chain-grown, has smooth surface, has 3 transverse septa and 1 longitudinal septa or oblique septa, and has slight constriction at the septal part; the beak is short column shaped, 0 interval; the brown conidiophores are upright or curved, have septa, cluster or single growth.
Molecular biological identification: inoculating the strain HMST-1 to glass paper paved on a PDA culture medium, culturing at a constant temperature of 25 ℃ for 5-7d, taking 0.1g of hypha, and extracting the genomic DNA of the strain HMST-1 by adopting a CTAB method. The rDNA-ITS amplification adopts universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'; SEQ ID NO. 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'; SEQ ID NO. 2), PCR amplification is carried out by a 50 muL reaction system, 2 muL PCR products are taken to carry out 2% agarose gel electrophoresis detection after the reaction is finished, the amplification products are sequenced by Beijing Ding national Changshen biotechnology limited, and are subjected to homology comparison with known sequences in NCBI GenBank by BLAST software, and then the MEGA 5.0 software is utilized to respectively construct phylogenetic trees, thereby determining the classification status of the strain HMST-1.
It was identified as Alternaria alternata (Alternaria alternata) by combining morphological characteristics and molecular biology, and the strain was preserved in China general microbiological culture Collection center (CGMCC).
Example 2
A determination method for bacteriostatic action of tamarix chinensis endophytic antagonistic fungus HMST-1 comprises the following specific steps:
the 2-point confronting method is adopted to determine the bacteriostasis. Respectively culturing 4 pathogenic fungi, namely rhizoctonia solani (R.solani), fusarium verticillium (F.verticillium), verticillium dahliae (V.dahliae), fusarium oxysporum (wilting specialization type) (F.oxysporum f.sp.vesinfectum) and endophytic fungus HMST-1 on a PDA for 5 days, then using a puncher to punch out colony edge bacterium blocks and inoculating the colony edge bacterium blocks on a PDA plate, respectively carrying out 2-point opposite inoculation on the 4 pathogenic fungi and the strain HMST-1, using the 4 pathogenic fungi to be singly cultured as a control, repeating the steps for 3 times, carrying out thermostatic culture at 25 ℃, and taking a picture until the control is full of a dish, wherein the result is shown in figure 1.
The diameter of the bacterial colony is measured by adopting a cross method, and the bacteriostasis rate is calculated by the following formula:
the results of figure 1 show that the endophytic antagonistic fungus strain HMST-1 has a good bacteriostatic action on both fusarium verticillioides and rhizoctonia solani, the bacteriostatic rate on the fusarium verticillioides is 75.33%, and the bacteriostatic rate on rhizoctonia solani is 71.11%.
Example 3
A preparation method of tamarix chinensis endophytic antagonistic fungus HMST-1 liquid fermentation broth comprises the following specific steps:
(1) Transferring the strain HMST-1 from the inclined plane of the test tube to a PDA plate for activation, culturing at 25 deg.C for 5-7 days, selecting the strain HMST-1, placing in a 50mL sterile water conical flask, and making into a solution with a concentration of 1.0 × 10 5 Spore suspension of one/mL for use.
(2) 200mL of PD liquid medium was placed in a 500mL Erlenmeyer flask at pH =7.0, and the above-mentioned solution was inoculated at a concentration of 1.0X 10% at an inoculation ratio of 10% under aseptic conditions 5 Adding 3.0mmol/L KH as metabolic factor into spore suspension 2 PO 4 The cells were cultured at 150 rpm at 25 ℃ for 5 days with shaking.
(3) And centrifuging the strain HMST-1 liquid fermentation liquor for 10min at 8000r/min by a high-speed refrigerated centrifuge, and taking supernatant to dilute by 5-10 times.
Example 4
The method for measuring the control effect of tamarix chinensis endophytic antagonistic fungus HMST-1 liquid fermentation broth on cotton red rot comprises the following specific steps:
(1) HMST-1 liquid fermentation broth dilution
Centrifuging the strain HMST-1 liquid fermentation liquor for 10min at 8000r/min by a high-speed refrigerated centrifuge, and diluting the supernatant by 10 times to obtain the strain HMST-1 liquid fermentation liquor with the concentration of 3.25 × 10 5 CFU/mL。
(2) Cotton red rot germ pot inoculation
Prepared at a concentration of 1.0X 10 8 After one/mL of the fusarium verticillioides spore suspension, root-damaging inoculation is carried out on the potted healthy cotton seedlings.
(3) Indoor control effect contrast test of HMST-1 liquid fermentation liquor on cotton red rot
When the roots of potted cotton seedlings start to develop diseases, continuously irrigating the roots for 2 times and 1 time every 5d respectively by using 10-time diluted HMST-1 liquid fermentation liquor, yeritan, terifluzam, 50% carbendazim wettable powder and sterile water, wherein a novel microbial agent product called Naofeng polymerization, yeritan, terifluzam and a conventional chemical agent, namely 50% carbendazim wettable powder and sterile water treatment are used as a contrast, each treatment is repeated for 5 times, the incidence of disease and the disease index of the cotton seedlings are counted after 2 times of root irrigation treatment for 5d, and the prevention effect is calculated, and the result is shown in table 1.
The control effect was calculated using the following formula:
TABLE 1 prevention and control effects of different microbial agents on cotton red rot
| Reagent for testing | Dilution factor | Incidence (%) | Index of disease condition | Control effect (%) |
| HMST-1 liquid fermentation broth | 10 | 15.00 | 0.96 | 85.19 |
| Huifeng JueCombination of Chinese herbs | 450 | 20.00 | 1.44 | 77.78 |
| Yiruitan | 10 | 18.00 | 1.52 | 76.54 |
| Fungus growing strong | 20 | 10.00 | 0.88 | 86.42 |
| 50% carbendazim wettable powder | 1000 | 16.00 | 1.85 | 71.45 |
| Sterile water | — | 88.00 | 6.48 | — |
The results in table 1 show that Alternaria alternata HMST-1 liquid fermentation broth has ideal prevention and treatment effects on cotton red rot, the prevention effects are superior to those of conventional chemical agents, the prevention effects are equivalent to those of novel microbial agent bacteria vertical strong products, and the use is safe.
Example 4
Qualitative and quantitative analysis of metabolites of endophytic Alternaria alternata HMST-1 liquid fermentation metabolite samples is carried out by utilizing a similar targeting metabolome (LC-MS), and 4 high-efficiency fungistatic compounds are screened and identified, wherein the compound names, structural formulas, classifications and relative quantitative values are shown in a table 2.
TABLE 2 Alternaria alternata HMST-1 liquid fermentation metabolism highly effective fungistatic compounds
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> institute of Nuclear technology and Biotechnology of academy of agricultural sciences in Xinjiang (center for Biotechnology research in autonomous region of Uygur autonomous region in Xinjiang)
<120> tamarix chinensis endophytic antagonistic fungus and separation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
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tccgtaggtg aacctgcgg 19
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
tcctccgctt attgatatgc 20
Claims (4)
1. An endophytic antagonistic fungus of tamarix chinensis (Alternaria alternata) HMST-1 is characterized in that the preservation number is CGMCC No.19261.
2. The preparation method of the tamarix chinensis endophytic antagonistic fungus alternaria alternata HMST-1 liquid fermentation broth, according to claim 1, comprises the following specific steps:
(1) Transferring the strain HMST-1 from the inclined plane of the test tube to a PDA plate for activation, culturing at 25 deg.C for 5-7 days, selecting the strain HMST-1, and placing in 50mL sterile water to obtain a solution with a concentration of 1.0 × 10 5 Spore suspension per mL for use;
(2) Inoculating with PD liquid culture medium with pH of 7.0 at inoculation ratio of 10% under aseptic condition to obtain inoculum concentration of 1.0 × 10 5 Adding 3.0mmol/L KH as metabolic factor into spore suspension 2 PO 4 Performing shaking culture at 25 deg.C and 150r/min for 5d;
(3) After the strain HMST-1 liquid fermentation broth is centrifuged for 10min at 8000r/min by a high-speed refrigerated centrifuge, supernatant is taken and diluted by 5-10 times.
3. The application of the tamarix chinensis endophytic antagonistic fungus alternaria alternata HMST-1 in inhibiting red rot and rhizoctonia solani in seedling stage of cotton.
4. The application of the tamarix chinensis endophytic antagonistic fungus alternaria alternata HMST-1 liquid fermentation broth in the prevention and treatment of cotton seedling-stage red rot and cotton rhizoctonia rot.
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