CN109112069B - Biocontrol endophytic fungus and application thereof - Google Patents
Biocontrol endophytic fungus and application thereof Download PDFInfo
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- CN109112069B CN109112069B CN201710949882.XA CN201710949882A CN109112069B CN 109112069 B CN109112069 B CN 109112069B CN 201710949882 A CN201710949882 A CN 201710949882A CN 109112069 B CN109112069 B CN 109112069B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention provides a panax notoginseng endophytic fungus for preventing and treating panax notoginseng root rot and application thereof, belonging to the technical field of crop disease prevention and treatment. The strain can inhibit the growth of pathogenic bacteria of Notoginseng radix by producing active substances, and has good effect in preventing and treating Notoginseng radix root rot. When in use, the root is irrigated in the seedling stage or the adult stage of the pseudo-ginseng. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use. The number of the penicillium of the biocontrol strain is Z1-1031, the penicillium is obtained from the root of pseudo-ginseng, and the preservation number is CGMCC No. 14134. The bacterium is compatible with soil ecology and panax notoginseng plants, is nontoxic and free of pathogenicity, and therefore has a very wide application prospect in biological control of panax notoginseng root rot.
Description
The technical field is as follows:
the invention relates to a bio-control endophytic fungus Penicillium sp and application thereof, in particular to an endophytic fungus with a probiotic effect on growth of panax notoginseng and application thereof in prevention and treatment of panax notoginseng root rot, belonging to the technical field of crop disease prevention and treatment.
Background art:
the panax notoginseng belongs to perennial yin plants, diseases occur frequently in the growth process, wherein the root rot of the panax notoginseng is the most main factor influencing the yield of the panax notoginseng. At present, no mature and effective method for preventing and treating root rot exists, and some researches are made on occurrence and prevention of the root rot. One of the main reasons that soil-borne pathogenic bacteria cause decay of the root of panax notoginseng, the fungal pathogenic bacteria mainly comprise: the bacillus subtilis comprises the following components of cylindrosporium, fusarium oxysporum, fusarium solani, rhizoctonia solani, verticillium dahliae and phoma, and the bacterial pathogenic bacteria mainly comprise pseudomonas. In order to ensure the yield of panax notoginseng, heptanong applies a large amount of chemical fertilizer, which causes a series of adverse problems of change of soil physicochemical properties, unbalance of flora proportion in soil, serious overproof of panax notoginseng heavy metal and the like. The endophyte is a microbial resource with important economic value, has rich varieties and wide distribution, and basically contains the endophyte adapted to each plant. Endophytes contain a variety of metabolic pathways that play an important role in plant repair, element cycling, biodegradation and biotransformation, and at the same time, in severe environmental conditions, plants can also provide shelter for microorganisms and help them reduce the pressure on the surrounding environment. The panax notoginseng endophytic fungi play an important role in balancing ecological environment and promoting the growth process of panax notoginseng. At present, 89 endophytic fungi have been isolated from the roots, stems, leaves and seeds of panax notoginseng, which are distributed in 2 phyla (ascomycota, zygomycota), 12 orders, 23 genera, and 63.4% of strains having antagonistic activity against at least one pathogenic bacterium of panax notoginseng. Those skilled in the art are making efforts to study more endophytic fungi to antagonize the root rot disease of panax notoginseng.
The invention content is as follows:
the invention aims to: aiming at the problems that the root rot of panax notoginseng is seriously generated at present, the area is enlarged year by year, the prevention and treatment measures such as pesticide, crop rotation, chemical treatment of soil and the like are not ideal in effect, and environmental pollution is caused, the biocontrol bacterium for efficiently preventing and treating the root rot of panax notoginseng and the application thereof are provided.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a biocontrol endophytic fungus Z1-1031 for preventing and treating root rot of panax notoginseng, which is identified as Penicillium sp through morphological and molecular biological research.
The preparation process of the biological control endophytic fungus penicillium comprises the following steps:inoculating the strain into a PDA culture medium plate, and performing inverted culture at 28 ℃ for 7-10 days to activate the strain. The activated strain was prepared into a spore suspension using distilled water, and the spore concentration was counted under a microscope to be about 108cfu/ml. 2ml of spore suspension was inoculated into the fermentation medium PDB. Culturing at 26-28 deg.C and 120-150r/min for 7-10 days.
The application method of the biocontrol endophytic fungi in preventing and treating the root rot of the panax notoginseng comprises the following steps:
can be used by irrigating root at adult stage of Notoginseng radix, counting under microscope, and diluting spore solution to 106cfu/ml concentration, 50ml per strain. The application is continued for 2-3 times, each time with 10-15 days interval. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
The invention has the beneficial effects that:
the screened biological control endophytic fungus penicillium fermentation liquor can obviously inhibit the growth of panax notoginseng pathogenic fungi fusarium solani, cylindracea, alternaria, and the like, and promote the growth of panax notoginseng plants. Because the biological preparation is adopted, a series of problems caused by the use of chemical pesticides do not exist, and the biological preparation is greatly beneficial to reducing agricultural pollution and effectively preventing and treating the root rot of the panax notoginseng. The biocontrol endophytic fungi are separated from the roots of healthy three-year-old panax notoginseng, also exist in rhizosphere soil of panax notoginseng, are ecologically compatible with the panax notoginseng soil, and are beneficial to fully exerting the advantages of strains.
The biocontrol endophytic fungi strain has the advantages of simple culture conditions, stable heredity, easy industrial production expansion and good development and application prospects.
The Penicillium (Penicillium sp.) of the invention is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation addresses are as follows: west road No.1, north west of the morning area, beijing, institute of microbiology, china academy of sciences, date of deposit: number of deposit at 07/2017, number: CGMCC No. 14134.
Description of the drawings:
FIG. 1 shows the colony morphology of strain Z1-1031.
FIG. 2 shows the mycelium and spore morphology of strain Z1-1031.
FIG. 3 shows that the strain Z1-1031 antagonizes the activity of pathogenic bacteria of three panax notoginseng strains.
FIG. 4 shows the fermentation liquor of strain Z1-1031 and the field replanting result of the strain.
The specific implementation mode is as follows:
for a better understanding of the present invention, the following figures and examples further illustrate, but are not to be construed as limiting, the experimental procedures set forth in the following examples are conventional and are not intended to be limiting.
Example 1 isolation of Panax notoginseng endophytic fungi
The specific process is as follows:
1 Collection of samples
Healthy three-year-old panax notoginseng plants are collected from a Miao Xiang planting base in Wenshan county of Yunnan province, are subpackaged and marked, and are put into freshness protection bags to be brought back to a laboratory for later use.
2 isolation of endophytic fungi
Plant washing procedure: cleaning soil on the surface of a plant under tap water, slightly drying the soil, cutting the plant into small blocks, putting the small blocks into a clean triangular flask, ultrasonically cleaning the small blocks repeatedly until the cleaned water becomes clear, inverting the cleaned water, and emptying the cleaned water.
Plant surface disinfection procedure: 0.1% Tween 20, 1min (soak, treat with beaker, same plant with a flask, beaker require sterilization) → treatment of root sample with sodium hypochlorite solution with 5% available chlorine content 6min → treatment of sterilized 2.5% sodium thiosulfate solution 10min → sterile water rinse at least 3 times → treatment of root sample with 75% ethanol (with sterile water) 6min → sterile water rinse 5 times. The surface sterilized samples were placed in sterile petri dishes (with sterile filter paper applied) and placed on a clean bench to dry (blow dry, no water visible, overnight in clean bench).
The surface disinfection effect is detected: taking 0.2mL of water for washing the sample in the last time, coating the water on a separation culture medium to be used, placing the flat plate at 28 ℃ for more than 2 weeks, and detecting whether the colonies grow out.
Crushing plants: placing the sample in a sterile stirrer (sterilization method same as biological identification plate, soaking with 75% alcohol for 5min, ultraviolet irradiation for 30min), crushing (beating 5s and stopping 5s when crushing, shaking the stirring cup to make the plant under the blade and not adhere to the wall), placing in a sterile plate, heating at 50 deg.C for 20min, picking the sample with sterile bamboo stick, and spreading on a separation plate. After incubation at 28 ℃ and observation for 7 days, different single colonies were picked and streaked for purification. Transferring the purified strain to a PDA culture medium for culture, storing at 4 ℃ for later use, and co-separating to obtain 21 endophytic fungi.
Example 2 identification of Strain Z1-1031
1 morphological feature Observation
The strain Z1-1031 cultured for 7-10 days was opened to a petri dish lid and photographed under a black background. The colony is gray green, and the edge is regular and is grayish white. The surface was velvet-like hyphae, and yellow pigment was produced in the medium (FIG. 1). A small amount of mycelium was taken out and made into a chip, and morphological characteristics of the mycelium and spores were observed under an optical microscope. The hyphae have transverse septa, the conidiophores of the hyphae are subjected to multiple branching to generate a plurality of asymmetric small stalks, the shapes of the small stalks are like brooms, the conidiophores are spherical and smooth (figure 2), and the strain Z1-1031 is preliminarily identified as the penicillium group according to the morphological characteristic analysis.
2 ITS sequence analysis
After extracting the genomic DNA of the strain Z1-1031 by a liquid nitrogen grinding method, adopting a primer: ITS 4: 5'-TCCTCCGCTTATTGATATGC-3', respectively; ITS 5: 5'-GGAAGTAAAAGTCGTAACAAGG-3', PCR amplification was performed. After gel electrophoresis, the gel was sent to bioengineering (Shanghai) Limited company for sequencing, and the total length of the sequence was 566bp (see SEQ). The obtained sequence was submitted to GenBank database for BLAST analysis and alignment, and the strain having high homology to Z1-1031 was found to belong to the genus Penicillium, and the strain sequence number KU360251 was submitted to NCBI.
Example 3 fermentation of Strain Z1-1031 and treatment of fermentation broth
Spore suspension 10 of the above strain8cfu/mL is inoculated in PDB liquid culture medium, the inoculation amount is 1-2mL, the temperature is 26-28 ℃, the fermentation amount is 100 mL/bottle after culturing for 7-10 days at 120-150 r/min. Separating the bacterial balls from the fermentation liquor by using gauze, extracting the fermentation liquor for 3 times by using ethyl acetate with the same volume, collecting extract liquor, and performing decompression and pumping drying to finally obtain the fermentation liquor with the dry weight of 0.1-0.2 g/bottle. The dried material is dissolved with a small amount of methanol and removed to obtain hairCrude extract of the yeast liquid.
Example 4 Strain Z1-1031 metabolite bacteriostasis test
Antagonism of pathogenic bacteria of panax notoginseng (Fusarium solani, Cylindrocarpon dynum, Alternaria panax) was tested with the Z1-1031 metabolite using 96-well plate turbidimetry. The final concentration of the extract of the fermentation broth is 0.1 mg/ml. Washing spores from slant to obtain spore suspension, counting with blood count plate, and diluting to spore concentration of 107cfu/ml. 0.4ml of the diluted spore suspension is sucked and added into 40ml of liquid PDB culture medium, and the mixture is mixed evenly to form suspension. Blank Control (CK): 198. mu.l of suspension; DMSO control group: 198. mu.l suspension +2ul DMSO; positive control group: 198. mu.l suspension + 2. mu. l G418; experimental groups: 198. mu.l suspension + 2. mu.l broth Extract (Extract), three replicates per group. The 96-well plate was incubated at 28 ℃ in an incubator, and OD was measured at 600nm after 48 hours. As shown in FIG. 3, the fermentation broth extract showed a killing effect on (Fusarium solani, Cylindrocarpon dynum, Alternaria panax), which is significantly different from the blank control group. The result of the activity screening experiment shows that the fermentation liquor of the strain Z1-1031 has better antagonistic action on pathogenic bacteria of the panax notoginseng root rot.
Example 5 crude extract of fermentation broth of Strain Z1-1031 and Strain replanting experiment
The replant strain Z1-1031 is inoculated to PDA culture medium and cultured for 7 days at 28 ℃. Preparing bacterial suspension with sterile water, and diluting to 10%6cfu/mL, and 50mL of root irrigation is carried out on each panax notoginseng seedling. The crude extract of the fermentation liquor is dissolved by water to prepare a solution of 1mg/L, and each notoginseng is irrigated with 50mL of roots. Each treatment is divided into three groups, and 200 panax notoginseng seedlings are inoculated in each group and planted on new soil (0 year soil). The seedling survival rate of the panax notoginseng seedlings is recorded every 1 month, and 4 months are observed in total. Is assisted by medicinal plant research institute of Wenshan pseudo-ginseng research institute and Yunnan province agricultural science research institute. The results are shown in FIG. 4. From the first month to the fourth month, the seedling survival rate of the control group is reduced from 92% to 55%, and the application fermentation liquor group and the replant bacteria group are respectively 100% to 90% and 100% to 88%. The experimental result shows that the strain Z1-1031 fermentation liquor and thalli can obviously reduce the death rate of panax notoginseng, improve the seedling survival rate and show good application potential.
Sequence listing
<110> Shenyang university of pharmacy
<120> biocontrol endophytic fungus and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 566
<212> DNA
<213> Penicillium purpureum (Penicillium janthinellum)
<220>
<221> allele
<400> 1
tgcggaggac attaccgagt gagggccctc tgggtccaac ctcccacccg tgtttatcat 60
acctagttgc ttcggcgggc ccgccgtcat ggccgccggg gggcacccgc ccccgggccc 120
gcgcccgccg aagccccccc tgaacgctgt ctgaagattg ctgtctgagc gattagctaa 180
atcagttaaa actttcaaca acggatctct tggttccggc atcgatgaag aacgcagcga 240
aatgcgataa gtaatgtgaa ttgcagaatt cagtgaatca tcgagtcttt gaacgcacat 300
tgcgccccct ggtattccgg ggggcatgcc tgtccgagcg tcattgctgc cctcaagcac 360
ggcttgtgtg ttgggccccc gccccccggc tcccgggggg cgggcccgaa aggcagcggc 420
ggcaccgcgt ccggtcctcg agcgtatggg gcttcgtcac ccgctctgta ggcccggccg 480
gcgcccgccg gcgacccccc tcaatctttc tcaggttgac ctcggatcag gtagggatac 540
ccgctgaact taagcatatc aataag 566
Claims (7)
1. The application of the biocontrol endophytic fungus Penicillium janthinellum in preparing the pesticide for preventing and treating root rot of pseudo-ginseng or ginseng is characterized in that the preservation number is CGMCC No. 14134.
2. The use according to claim 1, wherein the pathogenic bacteria of root rot are Fusarium solani (Fusarium solani), Cylindrocarpon didymum (Cylindrocarpon didymum) or Alternaria ginseng (Alternaria panax).
3. Use according to claim 1 or 2, wherein the biocontrol endophytic fungi are prepared by: inoculating the strain into a PDA culture medium plate, performing inverted culture at 28 ℃ for 7-10 days, activating the strain, preparing the activated strain into spore suspension by using distilled water, counting the spore concentration under a microscope, inoculating the spore suspension into a fermentation culture medium PDB, and culturing for 7-10 days at 26-28 ℃ and 120-150 r/min.
4. The use as claimed in claim 3, wherein the PDA medium is: 200g of potatoes, 10g of glucose and 20g of agar, wherein the volume is determined to be 1L of distilled water, the pH is natural, the sterilization is carried out for 30min at the temperature of 121 ℃, and the PDB culture medium comprises: 200g of potato and 10g of glucose, diluting to 1L of distilled water with a constant volume, naturally adjusting the pH value, and sterilizing at 121 ℃ for 30 min.
5. The use according to claim 1 or 2, characterized in that the biocontrol endophytic fungi is applied by root irrigation at the seedling stage or the adult stage of the panax notoginseng; the application is continued for 2-3 times, each time at 15 days intervals.
6. The use according to claim 1 or 2, wherein the biocontrol endophytic fungi are used alone or in combination with organic fertilizers.
7. Use according to claim 3, wherein the fermentation process for the biocontrol of endophytic fungi is: inoculating fresh spore suspension into PDB culture medium, shake-culturing for 7-10 days, extracting the fermentation broth with equal volume of ethyl acetate for 3 times, and collecting concentrated extract.
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| CN110669677B (en) * | 2019-10-28 | 2021-04-20 | 云南省农业科学院生物技术与种质资源研究所 | Penicillium bracteatum strain and application thereof |
| CN113388529B (en) * | 2021-06-21 | 2022-03-29 | 安徽农业大学 | Endophyte Penicillium ehrlichii of tea tree and application thereof |
| CN114606139B (en) * | 2022-03-24 | 2023-11-07 | 华南理工大学 | Penicillium javanicum and fermentation product and application thereof |
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| CN1693420A (en) * | 2004-05-08 | 2005-11-09 | 郭淑华 | Pesudo-ginseng soil regulating agent |
| WO2012030986A2 (en) * | 2010-09-01 | 2012-03-08 | North Carolina State University | Use of aryl carbamates in agriculture and other plant-related areas |
| CN105462850A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic fungus SDTE-P in preventing and controlling panax notoginseng root rot |
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| WO2010055474A2 (en) * | 2008-11-13 | 2010-05-20 | Ariel-University Research And Development Company Ltd. | Antimicrobial compounds and compositions |
| CN105112307B (en) * | 2015-09-15 | 2018-07-20 | 沈阳农业大学 | A kind of fungal bacterial strain and its cultural method and application with induction of resistance |
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|---|---|---|---|---|
| CN1693420A (en) * | 2004-05-08 | 2005-11-09 | 郭淑华 | Pesudo-ginseng soil regulating agent |
| WO2012030986A2 (en) * | 2010-09-01 | 2012-03-08 | North Carolina State University | Use of aryl carbamates in agriculture and other plant-related areas |
| CN105462850A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic fungus SDTE-P in preventing and controlling panax notoginseng root rot |
Non-Patent Citations (2)
| Title |
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| New antimicrobial compounds produced by endophytic Penicillium janthinellum isolated from Panax notoginseng as potential inhibitors of FtsZ;Jun Xie et al.;《Fitoterapia》;20181003;第131卷;第35-43页 * |
| 三七病株根际土壤微生物特征研究;官会林等;《农业与技术》;20051231;第25卷(第6期);第56-70页 * |
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