CN109112069A - A kind of biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus and its application - Google Patents
A kind of biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus and its application Download PDFInfo
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- CN109112069A CN109112069A CN201710949882.XA CN201710949882A CN109112069A CN 109112069 A CN109112069 A CN 109112069A CN 201710949882 A CN201710949882 A CN 201710949882A CN 109112069 A CN109112069 A CN 109112069A
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Abstract
The present invention provides the Radix Notoginseng endogenetic fungus and its application of one plant of prevention and treatment notoginseng root rot, belongs to crop disease control technical field, and the biocontrol bacterial strain is Penicillium notatum (Penicillium sp.).The bacterial strain inhibits the growth of Radix Notoginseng pathogen by generating active material, shows preferable control efficiency to notoginseng root rot.Using when Radix Notoginseng seedling stage either Adult plant pouring root use.The microbial inoculum can also be mixed and made into bacterial manure use with organic fertilizer.Biocontrol bacterial strain Penicillium notatum number of the present invention, which is Z1-1031, to be obtained from Panax notoginseng root, and deposit number is CGMCC No.14134.This bacterium is mutually melted with soil ecology and Radix Notoginseng plant harmony, nontoxic, no pathogenicity, therefore has very wide application prospect in notoginseng root rot biological control.
Description
Technical field:
The present invention relates to one plant of biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus mould (Penicillium sp.) and its applications, and in particular to one plant
The endogenetic fungus for have prebiotic effect to Panax notoginseng Growth and its application in prevention and treatment notoginseng root rot, it is anti-to belong to corps diseases
Control technical field.
Background technique:
Radix Notoginseng belongs to perennial shade plant, and disease occurs frequently in growth course, and wherein notoginseng root rot is to influence Radix Notoginseng
The main factor of yield.At present to the prevention and treatment of root rot still without mature effective method, forefathers to the generation of the disease with prevent
It controls and made some researchs.Soil-borne pathogen causes Panax notoginseng root that one of the main reason for rotting occurs, and fungal pathogens mainly wrap
Include: column spore bacterium, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani Kuhn, Verticillium sp bacterium, Phoma sp bacterium, bacterial disease opportunistic pathogen is with vacation
Based on monad.Seven agricultures apply a large amount of chemical fertilizer, soil physico-chemical property are caused to change to guarantee the yield of Radix Notoginseng, bacterium in soil
Group's proportional imbalance, a series of bad problems such as Radix Notoginseng heavy metal severe overweight.Endophyte is a kind of with Important Economic value
Microbial resources, type is abundant, widely distributed, all containing endophyte adaptable therewith substantially in each plant.Interior life
Contain multiple metabolic pathways in bacterium, to phytoremediation, element circular, biodegrade and bioconversion play an important role, simultaneously
In serious rugged environment condition, plant may be that microorganism provides protection, and microorganism is assisted to reduce ambient enviroment bring
Pressure.Radix Notoginseng endogenetic fungus plays an important role during balancing ecological environment and promoting Panax notoginseng Growth.Currently, we are from Radix Notoginseng
Root, stem, separate 89 plants of endogenetic fungus in leaf and seed, they are distributed in 2 doors (Ascomycota, Zygomycota), 12
A mesh, 23 categories, 63.4% bacterial strain have the activity of at least one plant Radix Notoginseng pathogenic bacteria of antagonism.Those skilled in the art are making great efforts
More endogenetic fungus are studied, with the root-rot disease of antagonism Radix Notoginseng.
Summary of the invention:
It is an object of the invention to: seriously occur for current notoginseng root rot, and area expands year by year, pesticide, crop rotation,
The control measures effect such as chemical treatment of soil is undesirable, while the problem of bring environmental pollution, providing an efficient prevention three
The biocontrol microorganisms of seven root rot and its application.
The purpose of the present invention can be achieved through the following technical solutions:
The present invention provides a kind of biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus Z1-1031 for preventing and treating notoginseng root rot, by morphology and molecule
Biological study is accredited as mould (Penicillium sp.).
The preparation process of the biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus Penicillium notatum is as follows: the strain inoculated is put down in PDA culture medium
In ware, 28 DEG C of inversions are cultivated 7-10 days, activated strains.Activated bacterial strain is prepared into spore suspension using distilled water, is shown
It is about 10 that spore concentration is counted under micro mirror8cfu/ml.2ml spore suspension is taken to be inoculated in fermentation medium PDB.26-28 DEG C,
120-150r/min is cultivated 7-10 days.
Application method of the biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus in prevention and treatment notoginseng root rot is as follows:
It can use in Radix Notoginseng Adult plant pouring root, be counted under microscope, dilution spore liquid to 106Cfu/ml concentration, every plant makes
Use 50ml.Continuous use 2-3 times, every minor tick 10-15 days.The microbial inoculum can also be hybridly prepared into bacterial manure use with organic fertilizer.
The invention has the advantages that:
The biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus mould fermented liquid that the present invention filters out can obviously inhibit Radix Notoginseng pathomycete Beancurd sheet sickle
The growth of spore, column spore, rod method etc., and promote Radix Notoginseng plant strain growth.Due to being biological agent, make without chemical pesticide
With bring a series of problems, it is very beneficial to reducing agricultural pollution and effectively preventing notoginseng root rot.It is raw in biological and ecological methods to prevent plant disease, pests, and erosion of the present invention
Fungi is isolated from healthy 3 years raw sangqi ginseng roots, while existing in the rhizosphere soil of Radix Notoginseng, with Radix Notoginseng soil ecology and
It is humorous compatible, be conducive to the advantage for giving full play to bacterial strain.
Biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungal bacterial strain condition of culture of the present invention is simple, inheritance stability, expansion easy to industrialized production, has good
Good development and application prospect.
Mould (Penicillium sp.) of the invention, it is commonly micro- in China Committee for Culture Collection of Microorganisms
Bio-Centers preservation, preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica,
Preservation date: on 07 07th, 2017, deposit number are as follows: CGMCC No.14134.
Detailed description of the invention:
Fig. 1 is bacterial strain Z1-1031 colonial morphology.
Fig. 2 is bacterial strain Z1-1031 mycelium and spore shape.
Fig. 3 is three plants of Radix Notoginseng pathogenic activities of bacterial strain Z1-1031 antagonism.
Fig. 4 is that bacterial strain Z1-1031 fermentation liquid and bacterial strain field are returned and plant result.
Specific embodiment:
In order to better understand the present invention, following accompanying drawings and embodiment are further elaborated the present invention, but are not pair
Limitation of the invention, experimental method described in following example are conventional method unless otherwise specified.
The separation of 1 Radix Notoginseng endogenetic fungus of embodiment
Detailed process is as follows:
The acquisition of 1 sample
Healthy 3 years raw sangqi ginseng plant are acquired from the Yunnan Province Wenshan County planting base Miao Xiang, are packed into freshness protection package after packing label
In to take back laboratory spare.
The separation of 2 endogenetic fungus
Plant cleaning procedure: in the soil of originally underwater cleaning plant surface, slightly drying, plant be cut into small pieces, and is packed into
It is ultrasonic in clean triangular flask, it cleans, until the water after cleaning becomes limpid, is inverted, empty water repeatedly.
Plant surface disinfectant program: 0.1%Tween 20,1min (are impregnated, are handled with beaker, same strain plant is with one
Flask, beaker require sterilizing) → available chlorine content be 5% liquor natrii hypochloritis handle root sample 6min → sterilizing 2.5%
Hypo solution handles 10min → sterile water wash at least 3 times → 75% ethyl alcohol (being matched with aqua sterilisa) and handles root sample
6min → sterile water wash 5 times.Sample after surface sterilization, which is placed in sterile culture dish, (is covered with sterile filter in culture dish
Paper), placement makes it dry (drying, cannot see that water, can stay overnight in super-clean bench) on the super-clean bench.
The effect of surface sterilization is detected: taking the water of last time cleaning sample of 0.2mL, is coated on stand-by separation
On culture medium, plate is placed in 28 DEG C of cultures 2 weeks or more, has detected whether that bacterium colony is grown.
Plant crushes: sample is put into sterile stirrer (sterilizing methods steep 5min with bioassay disk, 75% alcohol,
It is ultraviolet to shine 30min) it sufficiently smashes and (beats 5s when smashing and stop 5s, shake stirring cup, make plant under blade, is not adherent), it is put into
In sterilized petri dishes, then 50 DEG C of 20min with sterile bamboo stick picking samples and point are sprinkling upon on separation plate.28 DEG C of culture observations, 7
The single colonie that picking is different after it, which is crossed, to be purified.The bacterial strain of purifying is transferred in PDA culture medium and is cultivated, 4 DEG C save backup, altogether
Isolated 21 plants of endogenetic fungus.
The identification of 2 bacterial strain Z1-1031 of embodiment
The observation of 1 morphological feature
The bacterial strain Z1-1031 cultivated 7-10 days is opened into plate lid, is taken pictures under black background.Bacterium colony is celadon, side
Edge is neatly partially greyish white.Surface is in the velvet-like mycelia of swan, generates yellow pigment (Fig. 1) in culture medium.A small amount of mycelium is taken out, is made
Piece observes mycelium and spore shape feature under an optical microscope.It was found that mycelia has tabula, conidiophore is through excessive
Secondary branch generates a few asymmetric stigmas of wheel, shaped like broom, conidium spherical smooth (Fig. 2), according to the above morphological feature point
Analysis, is initially identified as Penicillium notatum monoid for bacterial strain Z1-1031.
The analysis of 2 ITS sequences
After liquid nitrogen grinding method extracts bacterial strain Z1-1031 genomic DNA, using primer: ITS4:5'-
TCCTCCGCTTATTGATATGC-3'；ITS5:5'-GGAAGTAAAAGTCGTAACAAGG-3' carries out PCR amplification.Gel electricity
The sequencing of Yong Housong bioengineering (Shanghai) Co., Ltd., sequence are 566bp (see SEQ).Gained sequence is submitted to
GenBank database carries out BLAST analyses and comparison, and discovery belongs to Penicillium with the higher bacterial strain of Z1-1031 homology, and
It is KU360251 that strain sequence number is had submitted on NCBI.
3 bacterial strain Z1-1031 of embodiment fermentation and fermentation liquor treatment technique
By above-mentioned bacterial strains spore suspension 108Cfu/ml is inoculated in PDB fluid nutrient medium, inoculum concentration 1-2ml, 26-
After 28 DEG C, 120-150r/min, culture 7-10 days, amount of fermentation is 100mL/ bottles.Using gauze by bacterium ball and separation of fermentative broth, send out
Zymotic fluid is extracted 3 times using isometric ethyl acetate, collects extract liquor decompressing and extracting, and finally obtaining fermentation liquid dry weight is 0.1-
0.2g/ bottles.It is spin-dried for object using the dissolution of a small amount of methanol, and is transferred out to obtain fermentation broth coarse extract.
The 4 antibacterial test of bacterial strain Z1-1031 metabolin of embodiment
Using 96 orifice plates-turbidimetry, by Z1-1031 metabolin to Radix Notoginseng pathogenic bacteria (Fusarium solani,
Cylindrocarpon didynum, Alternaria panax) make antagonism test.The effect of fermentation liquid extract is final concentration of
0.1mg/ml.Spore is washed down from inclined-plane and spore suspension is made by fungi, and blood counting chamber counts, and is diluted to spore concentration
107cfu/ml.It draws the good spore suspension 0.4ml of above-mentioned dilution to be added in 40ml liquid PDB culture medium, is mixed into suspension
Liquid.Blank control group (CK): 198 μ l suspensions；DMSO control group: 198 μ l suspension+2ul DMSO；Positive controls: 198 μ
+ 2 μ l G418 of l suspension；Experimental group :+2 μ l fermentation liquid extract (Extract) of 198 μ l suspension, every group three parallel.It will
96 orifice plates are placed in 28 DEG C of incubators and cultivate, and measure OD value after 48h at 600nm.As a result as shown in figure 3, fermentation liquid extract pair
(Fusarium solani, Cylindrocarpon didynum, Alternaria panax) has killing effect, with blank pair
According to group, there are significant differences.Screening active ingredients the experimental results showed that, the fermentation liquid of bacterial strain Z1-1031 has Radix Notoginseng root-rot pathogenic bacteria
There is preferable antagonism.
5 bacterial strain Z1-1031 fermentation broth coarse extract of embodiment and bacterial strain, which return, plants experiment
Plant bacterial strain Z1-1031 will be returned and be inoculated in PDA culture medium, 28 DEG C of culture 7d.Bacteria suspension is prepared with sterile water, is diluted to
106Cfu/mL, every plant of Radix Notoginseng seedling pouring root 50mL.Fermentation broth coarse extract is dissolved using water, is configured to the solution of 1mg/L, and every plant three
Seven pouring root 50mL.Each processing is divided into three groups, and 200 plants of Radix Notoginseng seedlings of every group of inoculation are planted on new native (0 year soil).Every 1
Record Radix Notoginseng seedling the moon deposits seedling rate, observes altogether 4 months.By Wenshan Sanqi Research Institute, Yunnan Province's academy of agricultural science medicine
It assists to complete with Institute of Zoology.As a result as shown in Figure 4.From first month to four month, control group deposits seedling rate by 92%
55% is dropped to, and applies zymotic fluid group and returns and plant bacterium group respectively by 100% to 90%, 100% to 88%.The experimental results showed that
The death rate of Radix Notoginseng can be significantly reduced in bacterial strain Z1-1031 fermentation liquid and thallus, and seedling rate is deposited in raising, and it is latent to show good application
Power.
Sequence table
<110>Shenyang Pharmaceutical University
<120>a kind of biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 566
<212> DNA
<213>microassembly robot (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 (8)
1. biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus Penicillium notatum (Penicillium sp.) is preparing the application in biocontrol agent.
2. biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus Penicillium notatum (Penicillium sp.) is in the pesticide of preparation prevention and treatment Radix Notoginseng or ginseng root rot
Using.
3. application as claimed in claim 1 or 2, which is characterized in that the biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus, deposit number CGMCC
No.14134。
4. application as claimed in claim 1 or 2, which is characterized in that the biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus is prepared via a method which:
By the strain inoculated in PDA culture medium plate, 28 DEG C of inversions are cultivated 7-10 days, and activated strains will be lived using distilled water
The bacterial strain changed is prepared into spore suspension, counts spore concentration under microscope, spore suspension is taken to be inoculated in fermentation medium
In PDB, 26-28 DEG C, 120-150r/min, cultivate 7-10 days.
5. application as claimed in claim 4, which is characterized in that the PDA culture medium are as follows: potato 200g, glucose 10g,
Agar 20g is settled in 1L distilled water, and PH is naturally, 121 DEG C of sterilizing 30min.The PDB culture medium are as follows: potato 200g, Portugal
Grape sugar 10g, is settled in 1L distilled water, and PH is naturally, 121 DEG C of sterilizing 30min.
6. application as claimed in claim 1 or 2, which is characterized in that it is described by biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus Radix Notoginseng seedling stage or at
Strain phase pouring root uses；Continuous use 2-3 times, every minor tick 15 days.
7. application as claimed in claim 1 or 2, which is characterized in that the biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus independent role or with it is organic
Fertilizer is used in mixed way.
8. application as claimed in claim 4, which is characterized in that the fermentation process of biological and ecological methods to prevent plant disease, pests, and erosion endogenetic fungus are as follows: use Fresh spores
It is collected dense using isometric ethyl acetate extractive fermentation liquid 3 times inoculation of suspension liquid shaking flask culture 7-10 days in PDB culture medium
Contracting extract liquor, it is spare.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110669677A (en) * | 2019-10-28 | 2020-01-10 | 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 |
| CN114606139A (en) * | 2022-03-24 | 2022-06-10 | 华南理工大学 | Beautiful millettia root endophytic fungus and fermentation product and application thereof |
| CN115521877A (en) * | 2022-01-25 | 2022-12-27 | 浙江中医药大学 | Penicillium brefeldianum strain and application thereof in preparation of brefeldin A |
| NL2029438B1 (en) * | 2021-06-21 | 2022-12-30 | Univ Anhui Agricultural | Strain of camellia sinensis endophyte penicillium ehrlichii and its application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669677A (en) * | 2019-10-28 | 2020-01-10 | 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 |
| NL2029438B1 (en) * | 2021-06-21 | 2022-12-30 | Univ Anhui Agricultural | Strain of camellia sinensis endophyte penicillium ehrlichii and its application |
| CN115521877A (en) * | 2022-01-25 | 2022-12-27 | 浙江中医药大学 | Penicillium brefeldianum strain and application thereof in preparation of brefeldin A |
| CN115521877B (en) * | 2022-01-25 | 2024-04-19 | 浙江中医药大学 | Penicillium strain and application thereof in preparation of brefeldin A |
| CN114606139A (en) * | 2022-03-24 | 2022-06-10 | 华南理工大学 | Beautiful millettia root endophytic fungus and fermentation product 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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| CN109112069B (en) | 2021-09-24 |
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