CN109402009A - A kind of method and its application for the Azotica screening antagonism Rhizoctonia solani Kuhn - Google Patents
A kind of method and its application for the Azotica screening antagonism Rhizoctonia solani Kuhn Download PDFInfo
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Abstract
The invention discloses a kind of method and its application of Azotica for screening antagonism Rhizoctonia solani Kuhn.The present invention enables Rhizoctonia solani Kuhn and Azotica to realize in the same culture dish preferably to grow by Optimal Medium, and the interaction both realized under simulating actual conditions can observe directly the growth face-off situation of algae and Rhizoctonia solani Kuhn.Method provided by the invention in 4 sectional culture dishs can simultaneously 2 algaes of screening, using more sectional culture dishs then can simultaneously 4-8 algae of screening.1 plate compared to traditional extracting solution plated growth Inhibition test 1 algae of screening and can only must extract algae effective component and be tested.Therefore, the present invention substantially increases screening efficiency, reduces environmental pollution.Simultaneously, it is often more important that, experimental result is closer to the phycomycete interaction situation under physical condition, so the validity of screening is stronger.
Description
Technical field
The present invention relates to the biological prevention and control field of plant disease and microbe to screen field, especially a kind of screening antagonism stands withered
The method and its application of the Azotica of rhizoctonia.
Background technique
Rice sheath blight disease is a kind of soil facsimile disease caused by fungal infection.Its cause of disease is thanatephorus cucumeris(frank) donk
Thanatephorus cucumeris (Frank) Donk. belongs to Basidiomycotina fungi, Invisible element Rhizoctonia solani
K ü hn claims Rhizoctonia solani Kuhn, belongs to Deuteromycotina fungi.The sclerotium resistance that rice sheath blight disease pathogen is formed is strong, in the soil
Can be with long-term surviving, when morbidity, can also carry out lateral propagation by plant leaf blade, have both that soil passes and leaf passes ability, become one
Kind is difficult to the intractable disease (Yang Yingqing etc., 2014) prevented and treated.In recent years, with high yield, of short stem, more tillers, the rice varieties of resistance to rich water
Application, the variation of tillage method and weather conditions, it is in current Rice Production that rice sheath blight disease, which is got worse,
One of Major Diseases (thank ancestor China etc., 2012).For rice sheath blight disease controlling, plantation disease-resistant variety is relied primarily at present and is applied
Use chemical pesticide.Since pathogen host range is wide, the rice germplasm that banded sclerotial blight is immunized, resistant variety are not yet found so far
It is very rare, difficulty (Srinivasachary et al., 2011) is increased to breeding for disease resistance.And disease-resistant variety resistance is easy
It degenerates, and harvesting high-quality high-yield kind general resistance is lower.Jinggangmeisu is the specific medicament of banded sclerotial blight, is that China rice field is main anti-
Control drug, but long-term single use jinggangmeisu prevent and treat its potential ecological environment and Human Health Risk it is high (Mew et al.,
2004).European Union formally forbade jinggangmeisu to sell from 2004.Biocontrol microorganisms resource is found, using plant rhizosphere beneficial organism,
Prevention and treatment rice sheath blight disease environmental protection and cheap biological control method are explored, is of great significance to agricultural sustainable development
(Khokhar et al.,2012).In recent years, domestic and international researcher has discovered that some pairs of rice sheath blight disease pathogens-are vertical
Withered silk kernel fungus has the fungi of antagonism, bacterium and actinomyces (Nagarajan et al., 2013).But due to biocontrol microorganisms
Variability and its influenced in Field information by various complicated and biological factor, cause control efficiency unstable, hinder
Therefore its large-scale commercial application is badly in need of further research and development effect better biocontrol microorganisms.
After nineteen thirty-nine India scientist reports fertilized the soil with cyanobacteria for the first time, many countries have carried out correlative study in succession, grind
Studying carefully proof inoculation cyanobacteria in paddy field can increase production.Starting Li Shanghao the fifties in last century, part provinces and regions carry out in south China
Support the test of algae in rice field, the results showed that increasing production of rice is up to 10~30% (Li Shanghao etc., 1962).It is sought in global warming and water body richness
There is important meaning using two packing spaces sexual development rice field low-carbon agriculture and control agricultural non-point source pollution today that feedingization is got worse
Justice (Jackson et al., 2007).Biological nitrogen fixation, which is carried out, using cyanobacteria just suits this trend.Put cyanobacteria in a suitable place to breed not only in rice field
Nitrogen (fixed nitrogen) and organic matter (carbon sequestration) can be provided for crop, improve yield, reduce fertilizer amount and production cost, raising phosphoric acid
Salt solubility reduces water and soil pollution, moreover it is possible to growth regulatings such as crop root oxygen supply, secretion plant hormone, amino acid, vitamins
Agent inhibits weed growth, to can reach purpose (the Prasanna et for maintaining the ecological balance and promoting agricultural sustainable development
al.,2013；Singh et al.,2011).And Singh etc. (2014) etc. thinks that cyanobacteria may also play the part of in terms of crop protection
Key player, about blue green bloom banded sclerotial blight pathogen, there are reports, such as the laboratory of Chaudhary (2012) etc. is ground
Study carefully the culture solution for showing some kinds of nostoc and anabena or frustule extract can inhibit including R.solani very
More harmful microorganisms.
The classical way for usually judging microorganism Competition is plate face-off experiment, can be clear by face-off experiment
Observe the interaction between two or more microorganisms.But since the culture medium prescription of algae and bacterium is widely different, no
Growth face-off experiment can be carried out on the same plate.The method of existing screening antagonism pathogenic microorganism algae is generally mentioned using algae
The plate growth inhibition assay of object or mucilage secretion is taken, the method can only judge the repercussion effect of algae and microorganism indirectly.
Also, experiment needs culture algae in advance, prepares algae extract or mucilage secretion, the frond effective component extracted with different solvents
Or mucilage secretion is added in the culture medium for being suitable for pathogenic microorganism growth and prepares plate, operating process is complicated, screens flux
It is low, due also to being polluted using organic solvent extraction.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art that cannot assess the direct interaction of algae and bacterium, mentions
For a kind of method of Azotica for screening antagonism Rhizoctonia solani Kuhn.
Another object of the present invention is to provide the applications of the method for the Azotica of above-mentioned screening antagonism Rhizoctonia solani Kuhn.
The purpose of the invention is achieved by the following technical solution: a kind of side for the Azotica screening antagonism Rhizoctonia solani Kuhn
Method includes the following steps:
(1) BG11 is prepared respectively0Solid medium and PDA solid medium；It will still be trained in the PDA solid of liquid after sterilizing
Support base and BG110Solid medium mixing, obtains mixed culture medium；
(2) mixed culture medium and PDA solid medium are poured into respectively in the lattice of the sterile culture dish with lattice,
Mixed culture medium and PDA solid medium are adjacent；The height and training of mixed culture medium and PDA solid medium in culture dish
The height for supporting the interruption of formation lattice in ware is the same；
(3) it is inoculated with algae to be screened in the central area for the lattice for having mixed culture medium, is placed in growth cabinet and trains
It supports, growth is stablized in culture to algae to be screened on culture medium, then is connect in the central area for the lattice for having PDA solid medium
Kind Rhizoctonia solani Kuhn；
(4) it cultivates；Rhizoctonia solani Kuhn wait compare in culture dish is observed and is measured when covering with culture dish, such as observes
Algae can be with normal growth, and germ can be inhibited to the diffusion of algae region, that is, thinks that the algae has antagonism Rhizoctonia solani Kuhn
Ability, and antibacterial power is judged according to the size of inhibition zone, show no resistance if by the covering of miliary damping-off mycelia, such as schemes
Shown in 1.
BG11 described in step (1)0The composition of solid medium is as follows: K2HPO4.3H2O 0.04g/L、MgSO4.7H2O
0.075g/L、CaCl2.2H2O 0.036g/L, citric acid 0.006g/L, ferric citrate 0.006g/L, EDTA 0.001g/L,
Na2CO30.02g/L, microelement A5 1mL/L, 15~20g/L of agar；
The composition of microelement A5 is as follows: H3BO3 2.860g/L、NaMoO4.2H2O 0.021g/L、MnCl2.4H2O
1.810g/L、ZnSO4.7H2O 0.222g/L、CuSO4.5H2O 0.079g/L、NiSO4.6H2O 0.479g/L。
The composition of PDA solid medium described in step (1) is as follows: what the peeling chopping of 200g fresh potato was boiled mentions
Object, glucose 20g, 15~20g of agar are taken, pure water is settled to 1L, natural pH.
The condition of sterilizing described in step (1) is preferably 115~121 DEG C of 15~30min of sterilizing；More preferably 121 DEG C
Sterilize 15~20min.
PDA culture medium described in step (1) and the BG110Culture medium is by the proportion mixing of 1:2~18；More preferably
1:10 proportion mixing by volume.
Culture dish described in step (2) is preferably the culture dish that diameter is 90mm.
The inoculum concentration of algae described in step (3) preferably by formed diameter be 3~10mm algae group amount calculate；More preferably press
It forms the algae group that diameter is 5mm and measures calculating.
The condition of culture described in step (3) is preferably cultivated 3~5 days in 23~30 DEG C, 2500~3200lx of illumination；
More preferably cultivated 4 days in 25~28 DEG C, illumination 3000lx.
The inoculum concentration of Rhizoctonia solani Kuhn described in step (3) is preferably based on the Rhizoctonia solani Kuhn of inoculation 3~8mm of diameter
It calculates；More preferably calculated by the Rhizoctonia solani Kuhn of inoculation diameter 5mm.
The condition of culture described in step (4) is preferably cultivated in 23~30 DEG C, 2500~3200lx of illumination；More preferably
For in 25~28 DEG C, 3000lx culture.
Azotica of the method for the Azotica of above-mentioned screening antagonism Rhizoctonia solani Kuhn in screening antagonism Rhizoctonia solani Kuhn
In application.
The present invention has the following advantages and effects with respect to the prior art:
The microorganism that the present invention keeps two class condition of culture widely different by Optimal Medium is in the same culture dish
Realization is preferably grown, and realizes the interaction of the two under simulating actual conditions, and the method provided can observe directly algae
With the growth face-off situation of Rhizoctonia solani Kuhn.Meanwhile using this method in 4 sectional culture dishs can simultaneously 2 algaes of screening, use
More sectional culture dishs then can 4~8 algaes of screening simultaneously.1 compared to traditional extracting solution plated growth Inhibition test is flat
Ware 1 algae of screening and can only must extract algae effective component and be tested.Therefore, method provided by the invention substantially increases
Screening efficiency reduces environmental pollution.Simultaneously, it is often more important that, experimental result is mutually admired closer to the phycomycete under physical condition
Condition, so the validity of screening is stronger.
Detailed description of the invention
Fig. 1 is the principle of the present invention figure；Wherein, figure A is using four sectional culture dishs, and scheming B is to use 5 sectional culture dishs；
A1~A4Different cyanobacterias is represented, R represents Rhizoctonia solani Kuhn, and P represents PDA culture medium, and B represents BG110Culture medium.
Fig. 2 is different PDA:BG110The upgrowth situation photo figure of Rhizoctonia solani Kuhn on volume proportion culture medium.
Fig. 3 is different PDA:BG110Match the upgrowth situation photo figure of two kinds of representative Azoticas on culture medium.
Fig. 4 is Azotica and Rhizoctonia solani Kuhn face-off experimental result photo figure on mixed culture medium；Wherein, it puts down for the 1st
Ware is PDA control.
Fig. 5 is cement pit experiment photo figure.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Material used in embodiment:
Cyanobacteria: nostoc (Nostoc sp.) FACHB-85, marsh nostoc (Nostoc paludosum) FACHB-89,
Nostoc (Nostoc sp.) FACHB-131, Anabaena variabilis (Anabaena variabilis) FACHB-164 and Fish Sampled from Paddy Field
Raw meat algae (Anabaena oryzae) FACHB-604, Chinese Academy of Sciences's fresh water algae library；This laboratory isolates and purifies preservation
(deposit number is CCTCC NO:M 2018194, preservation day to raw nostoc (Nostoc piscinale) SCAU-003 in algae strain pond
Phase is on April 11st, 2018, and depositary institution is the China typical culture collection center positioned at Wuhan, China Wuhan University, is existed
It is disclosed in patent application 201810397659.3).
Rhizoctonia solani Kuhn, number GIM3.512 are purchased from Guangdong Microbes Inst strain library, cause a disease for rice sheath blight disease
Bacterium.
Embodiment 1: the screening of mixed culture medium
(1) BG11 for containing 1.5% agar is prepared respectively0Solid medium and PDA solid medium；To sterilize, (121 DEG C go out
Bacterium 15min) after still in the PDA solid medium and BG11 of liquid0Solid medium by different volumes than proportion mixing (1:2,1:
6,1:10,1:14,1:18), obtain mixed culture medium.
(2) PDA solid medium, the BG11 for obtaining step (1)0The mixed culture medium of solid medium and different ratio
Inverted plate is inoculated with microalgae and Rhizoctonia solani Kuhn respectively on plate, is cultivated under 28 DEG C, 3000lx light environment.
(3) the experimental results showed that, test discovery above-mentioned 6 kinds of cyanobacterias used can not normally give birth in pure PDA culture medium
It is long, and Rhizoctonia solani Kuhn GIM3.512 is in BG110Growth result is also poor on culture medium.PDA culture medium and BG110Culture medium
By on the mixed culture medium that proportion obtains in step (1), compared with the control, other than 1:14 and 1:18, culture can be covered
The most areas of ware is shown in Fig. 2, and PDA culture medium and mixed culture medium are in the culture dish that the diameter of 4 lattices is 9cm in Fig. 2
It successively arranges, is mixed culture medium between PDA culture medium and PDA culture medium, Rhizoctonia solani Kuhn is inoculated on PDA, control group
It (CK) is PDA culture medium in 4 lattices.The result of Fig. 2 illustrates, from the perspective of being conducive to Rhizoctonia solani Kuhn growth, PDA
With BG110The ratio of culture medium is that 1:0~10 are suitable.Simultaneously, it is contemplated that the adverse effect that PDA culture medium grows algae, to two
The typical Azotica (Nostoc sp.FACHB-131 and Anabaena variabilis FACHB-164) of kind is in different proportion
Upgrowth situation on mixed culture medium is assessed, as a result as shown in figure 3, showing on two kinds of mixed culture mediums of 1:2 and 1:6,
Two seeds algaes are grown obviously than pure BG110The control of culture medium is poor, algae growth and BG11 on the mixed culture medium of 1:100Control is suitable,
As it can be seen that for Azotica, PDA and BG110The ratio of culture medium is that 1:10~18 are suitable.Therefore, subsequent to select PDA
With BG110The ratio of culture medium is the mixed culture medium of 1:10 proportion.
Embodiment 2: the screening of the microalgae of antagonism Rhizoctonia solani Kuhn
(1) preparation of culture medium, with 1 step of embodiment (1), difference is, mixed culture medium be PDA culture medium and
BG1101:10 is mixed fluid nutrient medium by volume.
(2) with four sectional culture dishs, culture medium is poured into lattice, and mixed culture medium, 2 lattices are poured into 2 lattices
Inside pour into PDA culture medium, wherein mixed culture medium and mixed culture medium are separated by PDA culture medium.The height of culture medium in lattice
It spends consistent with the interruption height between lattice.
It (3) will be in advance in BG110The algae of solid medium culture be inoculated into step (2) plate containing mixed culture medium
Lattice central area, it is 5mm that algae, which rolls into a ball diameter, 28 DEG C, cultivate 4 days under intensity of illumination 3000lx environment.Then in step (2) plate
In lattice central area containing PDA culture medium be inoculated with activated Rhizoctonia solani Kuhn (bacteria cake that diameter is 5mm).Continue at phase
It is cultivated at a temperature of with illumination.
(4) after the mycelia wait compare in plate covers with plate, whether there is or not antibacterial bands with the close region of bacterium for observation algae.If any suppression
Cingula measures its width, and the antagonistic ability size of algae is determined by its width size.
It is final that experimental result is shown in Fig. 4, the 1st, left side ware is control (CK), other 3 be algae and Rhizoctonia solani Kuhn pair
It stands erect and tests plate, the lower-left of each ware and each algae strain of upper right；The results show that there is apparent suppression in the periphery algae SCAU-003
Bacterium circle, although and FACHB-85, FACHB-131 and FACHB-89 do not have mycelia covering without inhibition zone completely, and
FACHB-604 and FACHB-164 is then covered by Rhizoctonia solani Kuhn completely.
Embodiment 3: the cement pit confirmatory experiment of antagonism Rhizoctonia solani Kuhn Azotica
In March, (1) 2017-August, place be Agricultural University Of South China bio-farm.It is tested according to above-mentioned flat-plate experimental result
Select algae (algae strain SCAU-003, FACHB-85, FACHB-89 and FACHB-604) processing of 4 different-effects that algae is not added with 1
Compare CK, every group of three repetitions, totally 15 cement pits (1m × 1m).
(1) in use for laboratory BG1104 seeds algae of culture medium culture, yarn thin,tough silk are collected by filtration, and 500ml is diluted with water to after weighing
It is spread fertilizer over the fields when rice (Huang Huazhan is purchased from the grand embroidery done with golden or silver thread coiled evenly seed-grain industry Co., Ltd in Shenzhen) starts tiller into pond after uniformly.Each cement
Add the wet algae of 6g in pond.After algae grows 1 week, it is added and uses 12 days Rhizoctonia solani Kuhn bacterium solutions of PDB fluid nutrient medium sterile culture
50mL.PDB culture medium prescription are as follows: the extract that the peeling chopping of 200g fresh potato is boiled, glucose 20g, pure water 1L are not added
Agar, natural pH.Sick grade is investigated after Rice Heading 30 days.Experimental field is shown in Fig. 5.
Inhibit the effect of rice sheath blight disease obvious the experimental results showed that putting SCAU-003 in cement pool environment in a suitable place to breed, compares CK
Inhibiting rate 37%, and FACHB-85 and FACHB-89 have certain inhibitory effect, FACHB-604 then shows as promoting banded sclerotial blight
Development, this is all consistent with aforementioned flat-plate experimental result.This illustrates that tablet face-off method the selection result provided by the invention is reliable.
The 1 disease-resistant experimental result of cement pit Azotica of table
Note: on the basis of CK, negative number representation promotes infecting for bacterium compared with CK
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of method for the Azotica for screening antagonism Rhizoctonia solani Kuhn, it is characterised in that include the following steps:
(1) BG11 is prepared respectively0Solid medium and PDA solid medium；By after sterilizing still in the PDA solid medium of liquid
And BG110Solid medium mixing, obtains mixed culture medium；
(2) mixed culture medium and PDA solid medium are poured into respectively in the lattice of the sterile culture dish with lattice, it is adjacent
Lattice pours into a kind of culture medium respectively；Mixed culture medium and PDA solid medium are adjacent；Mixed culture medium in culture dish and
The height of PDA solid medium is as the height of interruption for forming lattice in culture dish；
(3) it is inoculated with algae to be screened in the central area for the lattice for having mixed culture medium, is placed in growth cabinet and cultivates, trains
It supports and stablizes growth on culture medium to algae to be screened, then is vertical in the inoculation of the central area for the lattice for having PDA solid medium
Withered silk kernel fungus；
(4) it cultivates；Rhizoctonia solani Kuhn wait compare in culture dish is observed and is measured when covering with culture dish, such as observes that algae can
With normal growth, and germ can be inhibited to the diffusion of algae region, that is, think the ability that the algae has antagonism Rhizoctonia solani Kuhn,
And antibacterial power is judged according to the size of inhibition zone, show no resistance if by the covering of miliary damping-off mycelia.
2. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to claim 1, it is characterised in that:
BG11 described in step (1)0The composition of solid medium is as follows: K2HPO4.3H2O 0.04g/L、MgSO4.7H2O
0.075g/L、CaCl2.2H2O 0.036g/L, citric acid 0.006g/L, ferric citrate 0.006g/L, EDTA 0.001g/L,
Na2CO30.02g/L, microelement A5 1mL/L, 15~20g/L of agar；
The composition of microelement A5 is as follows: H3BO3 2.860g/L、NaMoO4.2H2O 0.021g/L、MnCl2.4H2O 1.810g/
L、ZnSO4.7H2O 0.222g/L、CuSO4.5H2O 0.079g/L、NiSO4.6H2O 0.479g/L。
3. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to claim 1, it is characterised in that: step
(1) PDA culture medium and the BG11 described in0Culture medium is by the proportion mixing of 1:2~18.
4. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to claim 3, it is characterised in that: described
PDA culture medium and the BG110Culture medium 1:10 proportion mixing by volume.
5. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to claim 1, it is characterised in that:
The condition of sterilizing described in step (1) is 115~121 DEG C of 15~30min of sterilizing；
Culture dish described in step (2) is the culture dish that diameter is 90mm.
6. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to claim 1, it is characterised in that:
The inoculum concentration of algae described in step (3) is calculated by the algae group amount that diameter is 3~10mm is formed；
The inoculum concentration of Rhizoctonia solani Kuhn described in step (3) is calculated by the Rhizoctonia solani Kuhn of inoculation 3~8mm of diameter.
7. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to claim 6, it is characterised in that:
The inoculum concentration of algae described in step (3) is calculated by the algae group amount that diameter is 5mm is formed；
The inoculum concentration of Rhizoctonia solani Kuhn described in step (3) is calculated by the Rhizoctonia solani Kuhn of inoculation diameter 5mm.
8. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to claim 1, it is characterised in that:
The condition of culture described in step (3) is to cultivate 3~5 days in 23~30 DEG C, 2500~3200lx；
The condition of culture described in step (4) is to cultivate in 23~30 DEG C, 2500~3200lx.
9. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to claim 8, it is characterised in that:
The condition of culture described in step (3) is to cultivate 4 days in 25~28 DEG C, 3000lx；
The condition of culture described in step (4) is to cultivate in 25~28 DEG C, 3000lx.
10. the method for the Azotica of screening antagonism Rhizoctonia solani Kuhn according to any one of claims 1 to 9 is vertical in screening antagonism
Application in the Azotica of withered silk kernel fungus.
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| CN111560319A (en) * | 2020-04-29 | 2020-08-21 | 华南农业大学 | Nitrogen-fixing blue algae in rice field and application thereof in reducing toxicity of cadmium to rice |
| CN113214997A (en) * | 2021-06-08 | 2021-08-06 | 云南绿A生物工程有限公司 | Screening method of filamentous bacterium pollution resistant spirulina strain |
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| SHOBITTHAPA等: "Nutrients and host attributes modulate the abundance and functional traits of phyllosphere microbiome in rice", 《MICROBIOLOGICAL RESEARCH》 * |
| VIDHI CHAUDHARY等: "Bioefficacy of novel cyanobacteria-amended formulations in suppressing damping off disease in tomato seedlings", 《WORLD J MICROBIOL BIOTECHNOL》 * |
| 丁玥琪等: "烟田拮抗微生物的互作及其应用效果研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111560319A (en) * | 2020-04-29 | 2020-08-21 | 华南农业大学 | Nitrogen-fixing blue algae in rice field and application thereof in reducing toxicity of cadmium to rice |
| CN111560319B (en) * | 2020-04-29 | 2021-09-24 | 华南农业大学 | Nitrogen-fixing blue algae in rice field and application thereof in reducing toxicity of cadmium to rice |
| CN113214997A (en) * | 2021-06-08 | 2021-08-06 | 云南绿A生物工程有限公司 | Screening method of filamentous bacterium pollution resistant spirulina strain |
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| CN109402009B (en) | 2021-03-16 |
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