CN109749938A - The endogenetic fungus and its microbial inoculum of one plant of reduction notoginseng root rot disease incidence - Google Patents
The endogenetic fungus and its microbial inoculum of one plant of reduction notoginseng root rot disease incidence Download PDFInfo
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- CN109749938A CN109749938A CN201711072883.7A CN201711072883A CN109749938A CN 109749938 A CN109749938 A CN 109749938A CN 201711072883 A CN201711072883 A CN 201711072883A CN 109749938 A CN109749938 A CN 109749938A
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- microbial inoculum
- root rot
- endogenetic fungus
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- notoginseng root
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Abstract
The invention belongs to microorganisms technical fields, are related to the endogenetic fungus and its microbial inoculum of one plant of reduction notoginseng root rot disease incidence.The bacterial strain (SYPF 7101) is located away from 2 years raw healthy Radix Notoginseng stems, by being accredited as cladosporium Cladosporium tenuissimum, the bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.14126.The present invention also provides the production methods of the microbial inoculum containing the bacterial strain, are divided into 4 steps, strain preparation, culture medium preparation, microbial inoculum production and microbial inoculum application.Microbial bacterial agent produced by the invention can significantly reduce the incidence of notoginseng root rot, show good Ecological Information prospect.
Description
Technical field:
The invention belongs to microorganisms technical fields, are related to the Cladosporium endogenetic fungus of one plant of reduction notoginseng root rot disease incidence
(Cladosporium tenuissimum) further relates to the preparation of the microbial inoculum comprising the fungi.
Background technique:
Notoginseng root rot is the main disease during Panax notoginseng Growth, every year because root rot endanger the underproduction about 10%~
20%, serious up to 70% or more, root rot has become an important factor for influencing Radix Notoginseng yield.Notoginseng root rot is micro- by cause of disease
Caused by biology infects, including Mycophyta pathogen Cylindrocarpon destructans, Cylindrocarpon
didynum、Fusarium solani、Fusarium oxysporum、Fusarium moniliform
Var.intermeclium、Phytophthora cactorum、Phoma herbarum、Alternaria panax、
Alternaria tenuis and Rhizoctonia solani etc. (notoginseng root rot cause of disease research, Scientia Agricultura Sinica, 2006,
39 (7): 1371-1378；Notoginseng root rot opportunistic pathogen (Cylindrocarpon spp.) biological characteristic research, Yunnan University are learned
Report: natural science edition, 2006, (S1): 342-346) and bacterium class pathogen Pseudomonas sp. etc. (notoginseng root rot is multiple
Close the research for infecting middle pathogenetic bacteria, Yunnan Prov Agriculture University's journal, 1999,14 (2): 123-127).Since Species of Pathogens is numerous
Mostly difficulty is brought to prevention and treatment.
Notoginseng root rot mainly passes through chemical control, and Pesticide use can bring pesticide residue and heavy metal pollution to Radix Notoginseng
Deng harm, therefore Pesticide use is rationally reduced, exploitation biological source microbial inoculum plays an important role to prevention and treatment notoginseng root rot.Chinese invention
One plant of Brevibacillus laterosporus that can prevent and treat notoginseng root rot is disclosed in patent (application number: CN200410079548.6)
(Brevibacillus laterosporus) bacteria agent preparation method, seed culture and pulvis including microorganism,
The preparation of the dosage forms such as granula and wettable powder.One is disclosed in Chinese invention patent (application number: CN201310020577.4)
Kind can prevent a variety of mixed biologic bacterial preparation process of notoginseng root rot, the system of fermentation, solid fungicide including microorganism
It is standby.However, notoginseng root rot is still one of the most important problem faced in production at present, reinforcing root rot prevention and treatment is in weight
Weight.Endophyte of plant is a kind of microbial resources with important value, and exploitation derives from the microbial inoculum of endophyte of plant more
Safety.
Summary of the invention:
In order to overcome the drawbacks of the prior art, the present invention provides one plant of endogenetic fungus is isolated from healthy Radix Notoginseng stem
(SYPF 7101) cladosporium (Cladosporium tenuissimum), is used for the prevention and treatment of notoginseng root rot.The bacterial strain comes
Derived from Radix Notoginseng endogenetic fungus, pathogenic effects will not be generated to Radix Notoginseng, be a kind of safe notoginseng root rot biocontrol agent.
The present invention is achieved through the following technical solutions:
Endogenetic fungus (SYPF 7101) cladosporium (Cladosporium tenuissimum) of the invention passes through such as lower section
Method obtains:
From 2 years raw sangqi ginseng plant of Yunnan Province, the planting base Miao Xiang, Wenshan County acquisition health, ultrasonic cleaning uses nothing after clarification
The cleaning of bacterium water, drains.
Plant surface disinfectant program: 20 immersions of 0.1-0.5%Tween → sterile water wash → sterile 2.5-5.0% is thio
Metabisulfite solution processing → sterile water wash → sterile 75% alcohol treatment → sterile water wash.Sample after surface sterilization is set
In sterile culture dish, sterile drying.
Sample comminution: the sample of surface sterilization being put into sterile stirrer and is sufficiently smashed, is put into sterilized petri dishes, is done
It is dry, then picking samples and point it is sprinkling upon on separation plate.Culture observation, single colonie are transferred in PDA culture medium after growing and cultivate,
Obtain bacterial strain SYPF 7101.
Endogenetic fungus of the invention is determined as Cladosporium tenuissimum by identification:
The identification of bacterial strain SYPF 7101 uses molecular biology method: liquid nitrogen grinding method extracts 7101 gene of bacterial strain SYPF
After group DNA, using primer I TS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-
GGAAGTAAAAGTCGTAACAAGG-3' PCR amplification) is carried out.The sequencing of electrophoresis Hou Song bioengineering (Shanghai) Co., Ltd., it is two-way
Ordered sequence overall length is 537bp after splicing.Discovery SYPF 7101 and Cladosporium is compared with GenBank database
Tenuissimum similitude is up to (99%).
Endogenetic fungus (SYPF 7101) cladosporium (Cladosporium tenuissimum) of the present invention can be prepared into bacterium
Agent, the prevention and treatment for notoginseng root rot.The microbial inoculum is prepared via a method which:
(1) prepared by culture medium
Seed prepares culture medium (PDA 1L): 150-200g peeled potatoes water extract, glucose 15-20g, agar powder
17-20g, adds water to 1L, and pH is natural.It sterilizes 30 minutes with postponing in 121 DEG C.
Solid fermentation culture medium: wheat bran 300-350g, corn flour 50-100g, beancake powder 10g, KNO30.5g, K2HPO4
1.5g, NaCl 1.5g, CaCO34g adds water 400ml, and 121 DEG C sterilize 30 minutes.
(2) actication of culture and seed preparation
Actication of culture:
The fungal spore in freeze-drying pipe is taken, is inoculated on PDA plate and is cultivated in 25-28 DEG C, the picking bacterium after growing bacterium colony
It falls to be inoculated on PDA plate and continues to cultivate 5-7d.
Spore suspension preparation:
A. by 28 DEG C of trainings in cladosporium (Cladosporium tenuissimum) strain inoculated to PDA plate after activation
5-7d is supported, being configured to concentration with sterile water elution spore is 1.0 × 106-1.0×107The spore suspension of a/mL.B. it takes respectively
The spore suspension of 100 μ L is coated on PDA plate.The plate of coating is placed in upper 25-28 DEG C of culture 5-7d.With sterile water elution
Spore, being configured to concentration is 1.0-5.0 × 106The spore suspension of a/mL.
(3) prepared by microbial inoculum
Microbial inoculum solid fermentation:
The above-mentioned spore suspension of 50mL is inoculated into 400g solid matrix.25-28 DEG C is cultivated, sterile after culture 3-4d
Under the conditions of once stirred, continue after stirring cultivate 4-5d, obtain solid fermentation microbial inoculum.
Microbial inoculum is dry:
Obtained solid microbial inoculum is placed in 30-35 DEG C of drying box, 25-30h is dried in air blast, and solid fungicide is dried to and is contained
Water can achieve 1.0-5.0 × 10 in 10-20%, the microbial inoculum viable count8A/g or more.Packaging, is placed in dry place.
The method of administration of microbial inoculum of the invention is as follows:
(1) it irrigates application: microbial inoculum being placed in sink as irrigation water flows near plant, every 1000 plants of Radix Notoginseng dosages
For 500-800g.
(2) it directly spreads fertilizer over the fields: solid fungicide is spread fertilizer over the fields on Radix Notoginseng seedbed, every 1 young plant dosage is 0.5-1.0g, or by bacterium
Agent fine sand dissipates after mixing in plant rhizosphere.
Bacterial strain cladosporium (Cladosporium tenuissimum) used in the present invention is on May 27th, 2017
In China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, address: BeiChen West Road, Chaoyang District, BeiJing City 1
No. 3 Institute of Microorganism, Academia Sinica, institute, deposit number are CGMCC No.14126.
Specific embodiment:
Embodiment 1:
The acquisition of Radix Notoginseng endogenetic fungus Cladosporium tenuissimum SYPF 7101:
Acquire 3 years raw sangqi ginseng plant of health, surface sterilization program are as follows: 0.1Tween 20, immersion 1min → sterile
Water cleans 3 times → sterile 2.5% hypo solution and handles 10min → sterile water wash → sterile 75% alcohol treatment 6min
→ sterile water wash.Sample is being cultivated in coating PDA culture medium by crushing, that is, obtains bacterial strain SYPF 7101.
Embodiment 2:
The composition of the microbial inoculum includes Radix Notoginseng endophyte Cladosporium tenuissimum SYPF 7101: its preparation side
It is 1.0-5.0 × 10 that formula, which is by 50mL concentration,6The spore suspension of a/mL is inoculated into 25-28 DEG C of culture in 400g solid matrix,
It is aseptically once stirred after culture 3-4d, continues culture 4-5 days after stirring, obtain solid fermentation bacterium.By gained
Solid fungicide is placed in 30-35 DEG C of drying box, and 25-30h is dried in air blast, and packaging is placed in dry place.
Embodiment 3: the microbial inoculum of embodiment 2 is to notoginseng root rot control efficiency
It chooses and transplanted seedlings phase application solid fungicide without planting the soil of Radix Notoginseng, amount of application is 0.5-1.0g/ plants, totally 120
Strain seedling, using solid matrix or clear water as compareing；
Microbial inoculum is additional, is once added after 3 months to microbial inoculum, additional quantity is same as above；
A situation arises for observation notoginseng root rot after application 12 months, and control group root rot incidence is 55.8%, processing group
Root rot incidence is 20.8%, the results showed that processing group reduces 35% compared with control group root rot incidence.
Embodiment 4: the microbial inoculum of embodiment 2 is to continuous cropping Sedum uizoon root rot control efficiency
The 3 years stifling notoginseng soil in interval is chosen, in phase application solid fungicide of transplanting seedlings, amount of application 0.5-1.0g/
Strain, totally 200 plants of seedling, using clear water as compareing；
Microbial inoculum is additional, carries out microbial inoculum after 3 months, 6 months respectively and adds, additional quantity is same as above；
Notoginseng root rot is observed after 12 months, and a situation arises, and control group root rot incidence is 91.5%, processing group root-rot
Sick incidence is 71%, the results showed that processing group reduces 20.5% compared with control group root rot incidence.
Claims (9)
1. the endogenetic fungus of one plant of reduction notoginseng root rot disease incidence, which is characterized in that deposit number are as follows: CGMCC
No.14126。
2. the endogenetic fungus microbial inoculum of one plant of reduction notoginseng root rot disease incidence, which is characterized in that be prepared via a method which:
(1) prepared by culture medium
Seed prepares culture medium PDA 1L:150-200g peeled potatoes water extract, glucose 15-20g, agar powder 17-20g,
1L is added water to, pH is natural.It sterilizes 30 minutes with postponing in 121 DEG C.
Solid fermentation culture medium: wheat bran 300-350g, corn flour 50-100g, beancake powder 10g, KNO30.5g, K2HPO41.5g
NaCl 1.5g, CaCO34g adds water 400ml, and 121 DEG C sterilize 30 minutes.
(2) actication of culture and seed preparation
Actication of culture: the fungal spore in freeze-drying pipe is taken, is inoculated on PDA plate and cultivates, picking colony connects after growing bacterium colony
Continue to cultivate 5-7d in kind to PDA plate.
Spore suspension preparation:
It will be cultivated in cladosporium (Cladosporium tenuissimum) strain inoculated to PDA plate after activation, use is sterile
Water elution spore is configured to spore suspension, and spore suspension is taken to be coated on PDA plate, and the plate of coating is placed in 25-28 DEG C of training
It supports, with sterile water elution spore, is configured to spore suspension.
(3) prepared by microbial inoculum
Microbial inoculum solid fermentation: spore suspension is inoculated into solid matrix, 25-28 DEG C of culture, in sterile item after culture 3-4d
It is once stirred under part, continues to cultivate 4-5d after stirring, obtain solid fermentation microbial inoculum；
Microbial inoculum is dry: obtained solid microbial inoculum being placed in 30-35 DEG C of drying box, 25-30h is dried in air blast, and solid fungicide is dried
It does to water content in 10-20% hereinafter, packaging, is placed in dry place.
3. endogenetic fungus microbial inoculum as claimed in claim 2, which is characterized in that the cultivation temperature in step (2) is 25-28 DEG C.
4. endogenetic fungus microbial inoculum as claimed in claim 2, which is characterized in that in the preparation of step (2) spore suspension, described in a
Spore suspension concentration are as follows: 1.0 × 106-1.0×107The concentration of spore suspension described in a/mL, b be 1.0-5.0 ×
106A/mL.
5. endogenetic fungus microbial inoculum as claimed in claim 2, which is characterized in that in step (3) solid fermentation process, spore suspension
The inoculum density of liquid are as follows: 1.0-5.0 × 106A/mL
6. endogenetic fungus microbial inoculum as claimed in claim 2, which is characterized in that the viable count 1.0- of the microbial inoculum of step (3) preparation
5.0×108A/g.
7. the endogenetic fungus of one plant of reduction notoginseng root rot disease incidence described in claim 1 is in preparing endogenetic fungus microbial inoculum
Using.
8. the endogenetic fungus of one plant of reduction notoginseng root rot disease incidence described in claim 1 treats notoginseng root rot medicine in preparation
Application in object.
9. the endogenetic fungus microbial inoculum of one plant of reduction notoginseng root rot disease incidence as claimed in claim 2 treats Radix Notoginseng root-rot in preparation
Application in medicine.
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| CN108823104A (en) * | 2018-06-23 | 2018-11-16 | 沈阳药科大学 | With the fungi and its application for improving the effect of notoginsenoside content |
| CN114250176A (en) * | 2021-12-15 | 2022-03-29 | 云南大学 | Preparation method and application of microbial agent for preventing and treating root rot of panax notoginseng |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108823104A (en) * | 2018-06-23 | 2018-11-16 | 沈阳药科大学 | With the fungi and its application for improving the effect of notoginsenoside content |
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| CN114250176A (en) * | 2021-12-15 | 2022-03-29 | 云南大学 | Preparation method and application of microbial agent for preventing and treating root rot of panax notoginseng |
| CN114250176B (en) * | 2021-12-15 | 2023-09-15 | 云南大学 | Preparation method and application of microbial agent for preventing and treating root rot of pseudo-ginseng |
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