CN109706082A - The huge Phoma cava bacterium P2 of one plant of biological and ecological methods to prevent plant disease, pests, and erosion and its application - Google Patents
The huge Phoma cava bacterium P2 of one plant of biological and ecological methods to prevent plant disease, pests, and erosion and its application Download PDFInfo
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Abstract
The invention belongs to technical field of agricultural microbiology, specifically disclose one plant of huge Phoma cava and its application, applicant separated one plant of huge Phoma cava (Phoma macrostoma) bacterial strain, deposit number is CCTCC NO:M 2018726.The growth of (sclerotinite, Botrytis cinerea, the Rhizoctonia solani Kuhn, rape black shank bacterium) mycelia of important pathogen on rape, conidium and sclerotial germination can be effectively suppressed in huge Phoma cava or its fermentation material filtrate；Also there is the antifungic action of more wide spectrum, be able to suppress most of other plant pathogenic fungi mycelia growths.In addition, bacterial strain P2 fermentation material filtrate acts on rape seed nonhazardous, fermentation material soaks by the filtrate processing rape seed, and seed can normally be sprouted, therefore can make rape seed processing, to prevent and treat soil-borne disease.Meanwhile the bacterium has very strong high temperature resistant, the ability of acid and alkali-resistance, ultra-violet radiation resisting.Therefore, there is the potentiality of development and utilization for P2 bacterial strain.
Description
Technical field
The invention belongs to technical field of agricultural microbiology, and in particular to one plant of huge Phoma cava (Phoma
Macrostoma it) and its applies.
Background technique
Phoma (Phoma) fungi can cause the important disease of plant, it can cause harm pulse family, Solanaceae, Cruciferae
And hundreds of plant such as Magnoliaceae, the diseases such as cause the tikka, stem rot and branch of plant withered.Such as Phoma destructiva
It is to lead to tomato leaf is withered and stem is withered pathogen (Boerema et al, 2004), and Phoma lingam is to cause Cruciferae
(Perfect stage is Leptosphaeria maculans/Leptosphaeria to one important pathogen of crop stem foot canker
Biglobosa two types), the rapeseed underproduction 20%~60% can be caused.P.andigena in Phoma and
P.tracheiphila is in areas such as Europe, and P.foveata is in southern US and P.macdonaldii on Australia and other places
Area is put into local quarantine pathogen respectively, has important quarantine.In China, P.exigua, P.pinodella and
P.tracheiphila is put into " People's Republic of China (PRC) enter the territory plant quarantine harmful organism register " announced in 2007.
Phoma fungi is widely present in environment, plays an important role during degradation of organic substances；In addition, one
A little Phoma fungies are also used as bio-control factors to prevent and treat biological plant disease.Such as Arie was once reported in 1999
Phoma glomerata can generate epoxydon (epoxy element) and then inhibit the generation of crop in cruciferae clubroot；This
Outside, White reported in 2000 the bacterium can also hyperparasite powdery mildew, it can generate a large amount of mitogenetic on powdery mildew cleistothecium
Spore device, to inhibit the growth of powdery mildew.Graupner once reported huge Phoma cava (Phoma in 2003
Macrostoma Novel ring tetracid (novel cyclic tetramic acid) can) be generated as herbicide to prevent and treat lawn
Upper weeds, such as Ji, the broad leaved weeds such as dandelion, clematis, but at home and abroad pathogenic is carried out using huge Phoma cava
There is not been reported for bacterium biological control.
Summary of the invention
The object of the present invention is to provide one plant of huge Phoma cava (Phoma macrostoma), the huge chamber stem points
Mould deposit number is CCTCC NO:M 2018726, classification naming: Phoma macrostoma P2.
It is another object of the present invention to provide the application of huge Phoma cava bacterium (Phoma macrostoma) P2, institutes
Stating bacterial strain can be used for preparing biocontrol agent or is prepared into vegetable seeds coating agent.
In order to achieve the above object, the present invention takes following technical measures:
Huge Phoma cava of the invention is isolated from the rape balck shank sample of Hubei Province Yunxi County acquisition, because of its morphological feature
Much like with rape black shank bacterium Invisible element Phoma lingam, which delivers Wuhan City, Hubei Province on October 30th, 2018
China typical culture collection center preservation in Wuhan University, city, applicant are named as bacterial strain P2.The huge chamber stem
The mould deposit number of point is CCTCC NO:M 2018726, classification naming: Phoma macrostoma P2.
Bacterium colony characteristic:
After cultivating 7d on OA culture medium, colony edge is neat, and in celadon, back side light green color, pycnidia is intensive
It generates in bacterium colony center, colony diameter 49.0mm；After cultivating 7d on MA culture medium, colony edge is neat, the white suede of bacterium colony
Hairy, back side crocus has no that spore device generates, colony diameter 61.3mm；After training 7d in PDA culture medium, colony edge is whole
It together, is in celadon, back side green, pycnidia is intensively scattered, colony diameter 64.3mm；P2 14d energy in PDA culture medium
See thering is a large amount of pycnidia to generate in media surface, and pycnidia has the appearance of tangerine pink colour spore mucus.It is mitogenetic
Spore device spheroidal, sepia, rough device outer wall is in keratin shape, and size is 320~333 μm；Conidiophore father-in-law's ampuliform, greatly
Small is 6.6~8.6 μm of 6.6~11.2 μ m；Conidiophore end have an ellipticity, it is smooth, it is transparent, without every, point
Raw spore includes 2-5 oil droplet, and conidium size is 3.3-4.0 μm of 7.3-9.3 μ m.
The application of huge Phoma cava (Phoma macrostoma) P2 bacterial strain, is prepared into pathogenic including the use of the bacterial strain
The control agent of bacterium, or the coating agent as vegetable seeds；The phytopathogen includes but is not limited to: melon and fruit corruption is mould
(Pythium apanidermatum), head mold (Rhizopus stolonifer), Mucor (Mucor hiemails), aspergillus flavus
(Aspergillus flavus), aspergillus niger (Aspergillus niger), Botrytis cinerea (Botrytis cinerea), water
Rice Curvularia lunata (Curvularia lunata), rape black shank bacterium (Leptosphaeria biglobosa), Lettuce Drop
Bacterium (Sclerotinia minor), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Rhizoctonia solani Kuhn
(Rhizoctonia solani)。
Compared with prior art, the invention has the following advantages that
Important pathogen nuclear disk on rape can be effectively suppressed using huge Phoma cava P2 fermentation material filtrate provided by the invention
The growth of the mycelia such as bacterium, Botrytis cinerea, the sprouting of conidium and sclerotium；P2 fermentation material filtrate also has the antibacterial work of more wide spectrum
With growth in addition to being able to suppress most plants disease fungus, moreover it is possible to the cause of disease for effectively aspergillus flavus etc. being inhibited to be harmful to the human body
Bacterium growth.In addition, P2 fermentation material filtrate acts on rape seed avirulence, fermentation material soaks by the filtrate processing rape seed, seed
Can normally sprout, it can be considered to the pouring root P2 fermentation material filtrate when preventing and treating soil disease, avoid chemical prevention to environment and
The potential adverse effect of crop safety.Finally, the bacterium has the characteristics that very strong high temperature resistant, acid and alkali-resistance, uvioresistant, can be made
The formulation of metabolite is developed.
Detailed description of the invention
Fig. 1 is the colonial morphology figure that huge Phoma cava P2 is cultivated on culture medium；
Wherein A is OA culture medium colonial morphology；B is MEA culture medium colonial morphology；C is PDA culture medium colonial morphology；D is
PDA 14d cultivates form；E is the pycnidia formed in PDA culture medium；F is pycnidia；G is conidiophore；H
For conidium.
Fig. 2 is that huge Phoma cava P2 and other Phoma bacterial strains (P1, P3 and P4) of the invention illustrate the pathogenicity of rape
Figure.
Fig. 3 is huge Phoma cava P2 and other Phoma bacterial strains (P1, P3 and P4) of the invention and pathogen
Leptosphaeria biglobosa dual test result schematic diagram.
Fig. 4 is huge Phoma cava P2 of the invention and other Phoma bacterial strains (P1, P3 and P4) fermentation material filtrate on rape
The test result schematic diagram of inhibiting effect is infected to Leptosphaeria biglobosa.
Fig. 5 is the time dynamic schematic diagram that huge Phoma cava P2 fermentation material filtrate of the invention generates antifungal substance.
Fig. 6 is that the screening of huge Phoma cava P2 fermentation material filtrate antifungal substance biological activity determination bacterium of the invention is illustrated
Figure.
Fig. 7 is huge Phoma cava P2 fermentation material filtrate of the invention (containing antifungal substance) to Leptosphaeria
Biglobosa generates inhibition zone schematic diagram；
Wherein: A, inhibition zone overall diagram；B, transparent circle boundary graph under body formula mirror；Transparent circle boundary bacterium under C optical microscopy
Silk tip figure；D, the lopsided conidium figure in transparent circle inside.
Fig. 8 is that treatment of different temperature illustrates huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) active influence
Figure.
Fig. 9 is that soda acid is handled on huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) active influence schematic diagram.
Figure 10 is that UV treatment illustrates huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) active influence
Figure.
Figure 11 is that environment pH influences schematic diagram to huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) activation plays.
Figure 12 is the huge Phoma cava P2 fermentation material filtrate of the present invention (containing antifungal substance) various concentration to 4 kinds of pathogenics
Hypha,hyphae influences schematic diagram.
Figure 13 is the huge Phoma cava 0.6%P2 fermentation material filtrate of the present invention (containing antifungal substance) to 4 kinds of pathogen mycelia
Teratogenesis schematic diagram；
0.6%P2 fermentation material filtrate refers to (containing antifungal substance)+600 μ L P2 fermentation material of the PDA culture medium filter of 90mL
Liquid.
Figure 14 is the huge Phoma cava 10%P2 fermentation material filtrate of the present invention (containing antifungal substance) to sclerotinite and grey grape
The schematic diagram that spore sclerotial germination influences；
10%P2 fermentation material filtrate refers to (containing antifungal substance) PDA culture medium+10mL P2 fermentation material filtrate of 90mL.
Figure 15 is that the huge Phoma cava P2 fermentation material filtrate of the present invention (containing antifungal substance) impregnates sclerotinite and Botrytis cinerea
After sclerotium, schematic diagram is influenced on sclerotial germination.
Figure 16 is that the huge Phoma cava P2 fermentation material filtrate of the present invention (containing antifungal substance) sprouts Botrytis cinerea conidium
Hair influences schematic diagram.
Figure 17 is the huge Phoma cava P2 fermentation material filtrate of the present invention (containing antifungal substance) to the mitogenetic spore of rape black shank bacterium
Son, which is sprouted, influences schematic diagram.
Figure 18 is for the huge Phoma cava P2 fermentation material filtrate of the present invention (containing antifungal substance) to core on living body rape leaf
Cup fungi protection effect schematic diagram.
Figure 19 is for the huge Phoma cava P2 fermentation material filtrate of the present invention (containing antifungal substance) to ash on living body rape leaf
Grape spore protection effect schematic diagram.
Figure 20 is that the antimicrobial spectrum of the huge Phoma cava P2 fermentation material filtrate of the present invention (containing antifungal substance) measures schematic diagram.
Figure 21 is the huge Phoma cava P2 fermentation material filtrate of the present invention (containing antifungal substance) to the toxicity test of rape seed
Schematic diagram.
Specific embodiment
In order to better explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but
The contents of the present invention are not limited solely to following embodiment.Technical solution involved in the embodiment of the present invention, if not otherwise specified,
It is this field conventional scheme；The reagent or material derive from commercial channel if not otherwise specified.
Embodiment 1:
The separation identification of huge Phoma cava P2 bacterial strain and biological property analysis
One, the separation of bacterial strain and its biological property analysis
Applicant chooses the invalid body with classical symptom from the rape balck shank sample that Hubei Province Yunxi County acquires,
Tissue block is sheared in the strong intersection of its disease, size is about 0.5cm × 0.5cm；It is handled respectively with 75% ethyl alcohol, 5% sodium hypochlorite
Then 30s and 5min is used sterile water continuous wash 3 times, then gone to the PDA culture medium plate center containing 25% lactic acid,
22 DEG C of constant temperature incubation 4d finally purify the fungi to grow from diseased tissues edge.In addition, this laboratory is also respectively from oil
Isolated 4 kinds of fungies (Phoma sp.) similar with rape balck shank pathogenic bacteria in dish balck shank sample, be named as P1,
P2, P3, P4 determine that this 4 kinds of Phoma bacterial strains cannot make rape cause a disease (Fig. 2) by virulence analysis, therefore plate is used to stand facing each other
Cultivation carries out preliminary biological and ecological methods to prevent plant disease, pests, and erosion measurement to P1, P2, P3 and P4 bacterial strain；It is surveyed finally, shaking training liquid using the PDB of this 4 kinds of Phoma bacterial strains
The fixed influence that it infects rape black shank bacterium (Leptosphaeria biglobosa Lb20) on rape, judges this with this
It whether there is biocontrol bacterial strain in 4 kinds of Phoma bacterial strains.
Test result find 4 kinds of Phoma bacterial strains with rape black shank bacterium (Leptosphaeria biglobosa
Lb20) during opposite culture, it is found that the mycelia growth of Lb20 is centainly inhibited, pycnidia and the CK of generation (only connect
Kind of Lb20) compared to significantly reducing (Fig. 3).The test result that 4 kinds of Phoma strain fermentation object filtrate infects Lb20 on rape
Show to infect in rape containing the pathogen Lb20 on P2 fermentation material filtrate, the fermentation of remaining 3 kinds of Phoma bacterial strain
Lb20 can infect (Fig. 4) on rape after the processing of object filtrate, and CK (clear water is added dropwise in only inoculation Lb20, vaccination) can normally be sent out
Disease, and lesion diameter reaches 8.4mm after inoculation 7d, therefore tentatively judges that P2 can produce certain antifungal substance, so as to fine
Inhibition Lb20 mycelia growth, the bacterial strain is in the China delivered in the Wuhan University of Wuhan City, Hubei Province on October 30th, 2018
Type Tissue Collection preservation, deposit number are CCTCC NO:M 2018726, classification naming: Phoma
macrostoma P2。
Two, the morphological feature of bacterial strain
(1) colony characteristics
After cultivating 7d on OA culture medium, colony edge is neat, and in celadon, back side light green color, pycnidia is intensive
It generates in bacterium colony center, colony diameter 49.0mm；After cultivating 7d on MA culture medium, colony edge is neat, the white suede of bacterium colony
Hairy, back side crocus has no that spore device generates, colony diameter 61.3mm；After training 7d in PDA culture medium, colony edge is whole
It together, is in celadon, back side green, pycnidia is intensively scattered, colony diameter 64.3mm；P2 bacterial strain is trained in PDA culture medium
After supporting 14d, it can see thering are a large amount of pycnidias to generate in media surface, and it is viscous that tangerine pink colour spore is secreted on pycnidia
Liquid (Fig. 1).
(2) microscopic findings
Pycnidia spheroidal, sepia, rough device outer wall is in keratin shape, and size is 320~333 μm；Mitogenetic spore
Son stalk father-in-law's ampuliform, size are 6.6~8.6 μm of 6.6~11.2 μ m；Conidiophore end have an ellipticity, it is smooth,
It is transparent, without every containing 2~5 oil droplets in conidium, conidium size is 3.3~4.0 μm of 7.3~9.3 μ m (Fig. 1).
Three, the categorical attribute identification of bacterial strain P2
The P2 mycelia for cultivating 4d is scraped from the PDA culture medium for be covered with glassine paper, is put to mortar, after appropriate liquid nitrogen is added
It is fully ground, the total DNA of P2 bacterial strain is extracted by CTAB method, the identification of above-mentioned fungi is based on the Internal Transcribed Spacer l
(ITS1), 5.85rDNA, the Internal Transcribed Spacer 2 (ITS2) are identified.By primer I TS1 and ITS4 to the total DNA of bacterial strain
PCR amplification is carried out, obtained sequence carries out Blast comparison in the website NCBI, and qualification result is Phoma macrostoma
I.e. huge Phoma cava.
Embodiment 2:
The purifying and fermented and cultured of huge Phoma cava P2
(1) bacterial strain activates: the P2 bacterial strain inclined-plane that 4 DEG C of refrigerators are saved is inoculated in PDA with sterile one piece of transfer needle picking
On culture medium, 3d is cultivated in 22 DEG C of insulating boxs.
(2) strain culturing: the P2 bacterial strain of culture 3d in step (1) is forwarded in fresh PDA culture medium, 22 DEG C of constant temperature
7d is cultivated in case.
(3) it ferments: the P2 bacterial strain colony edge in step (2) being beaten into several bacteria cakes, every 100mL with aseptic card punch
5 mycelia blocks, 150rpm, 22 DEG C of difference shaken cultivations 3d, 6d, 9d and 15d, after 6000rpm centrifugation are inoculated in PDB culture solution
Different number of days fermentation material filtrate is collected respectively, is used for following embodiment.
(4) above-mentioned huge Phoma cava P2 fermentation material filtrate huge Phoma cava P2 fermentation material filtrate: is placed in 6000rpm centrifugation
10min collects supernatant, and supernatant uses 0.22 μm of biofilter filtering again, and the ferment filtrate that can be obtained is used for following implementation
Example.
(5) PDB medium component: potato 200g, glucose 20g, supplement distilled water are settled to 1000mL.
Embodiment 3:
The preparation and its active measurement of antifungal substance of huge Phoma cava P2 fermentation material filtrate
One, huge Phoma cava P2 fermentation material filtrate generates the time dynamic of antifungal substance
Fermentation material filtrate is collected in 3d, 6d, 9d, 12d and 15d that P2 bacterial strain shakes training respectively, measures pH value, gained mycelia
Dry mycelial weight (table 1) is weighed after drying.After the biofilter filtering that ferment filtrate aperture is 0.22 μm, with 10% (v/v)
Ratio (in the present invention, this percentage, refer to P2 fermentation material filtrate: culture medium is 1:9, volume ratio) be added to PDA
It is uniformly mixed in culture medium, inverted plate obtains the malicious plate of band, using the PDB solution of 10% (v/v) of addition as negative control.
By diameter be the sclerotinite (Sclerotinia sclerotiorum) of 5mm, Botrytis cinerea (Botrytis cinerea), stand it is withered
Rhizoctonia (Rhizoctonia solani) and rape black shank bacterium (Leptosphaeria biglobosa) be not (as said especially
It is bright, it is involved in the present invention to sclerotinite, Botrytis cinerea, Rhizoctonia solani Kuhn and rape black shank bacterium be following bacterial strain, wherein
Botrytis cinerea (B05.10) is the type strain of ash arrhizus bacteria, sclerotinite (Ss), Rhizoctonia solani Kuhn (Rs) and rape black shank bacterium
(Lb20) obtained for the separation of this laboratory, identified consistent with open strain characteristics) fresh inoculated by hypha block arrives with poison plate
On negative control plates, 22 DEG C of constant temperature incubations measure the colony diameter of each processing, each place after negative control covers with ware
Reason 3 repetitions of setting.Bacteriostasis rate is calculated according to formula:
As a result as shown in figure 5, with the extension for shaking the training time, biomass is maximum when huge Phoma cava P2 is cultivated to 15d,
Its bacteriostatic activity is most strong, and pH value is consequently increased, and pH value rises to 8.3 when shaking training to 15d.According to this test result,
It is all made of P2 bacterial strain in follow-up test and shakes fermentation material filtrate of the training to 15d when to measure its bioactivity.
1 bacterial strain P2 fermentation material filtrate of table generates the time dynamic of antifungal substance
Note: in PDB culture solution measure difference shake training P2 ferment filtrate to sclerotinite (Ss), Botrytis cinerea (B05.10),
The influence of Rhizoctonia solani Kuhn (Rs) and the growth of rape black shank bacterium (Lb20) mycelia.Under the conditions of 22 DEG C and 150rpm, respectively
After shaking training 3d, 6d, 9d, 12d and 15d, dry mycelial weight in fermentation material is weighed, and surveys difference respectively and shakes training number of days P2 fermentation material
Filtrate pH value, using the significance of difference between minimum difference significant method (LSD) analysis processing, alphabetical identical expression in same row
Difference is not significant (P > 0.05).
Two, the screening of huge Phoma cava P2 fermentation material filtrate (antifungal substance) biological activity determination bacterium
Respectively by easy production spore and big plant pathogenic fungi aspergillus niger Y-1 (the Aspergillus niger Y- of sporulation quantity
1), (this test selects the biggish bacterial strain of conidium yield to detect it to P2 antifungal substance to Botrytis cinerea RoseBc-3
Sensibility, the color that the bigger bacterial strain of sporulation quantity displays on culture medium is deeper, and inhibition zone is more obvious, due to ash before
Grape spore type strain B05.10 sporulation quantity is small, is not suitable in this test), rape black shank bacterium Leptosphaeria
Then conidium is washed till in sterile 50mL centrifuge tube, four layers by biglobosa (Lb20) culture to spore is produced with sterile water
Conidial suspension is obtained after lens wiping paper filtering, its concentration is adjusted to 1 × 108A spore/mL.Then to every 200mL PDA
Middle addition 20mL conidial suspension is sufficiently mixed inverted plate, sensitivity testing is carried out using cylinder-plate method, then by 200 μ L
P2 fermentation material filtrate (15d) is added in the Oxford cup (the same below) that the consistent aperture of specification is 6mm, same that 200 μ L are added
PDB solution is used as negative control, 22 DEG C of constant temperature incubation 3d into Oxford cup.The result shows that the prevent plant disease, pests, and erosion application prospect that Lb20 generates P2
Matter sensibility is most strong, average diameter of inhibition zone 2.9cm, and inhibition zone is very transparent, and what sensibility was taken second place is aspergillus niger, antibacterial
Circle average diameter is 2.1cm, and inhibition zone is translucent, and RoseBc-3 is least sensitive, and no inhibition zone generates, and is compared with PDB
The plate that carries disease germs on also without inhibition zone generate (Fig. 6).Finally most transparent inhibition zone is observed with optical microscopy, transparent circle is handed over
Visible normal mycelia (C in Fig. 7) and lopsided mycelia (C in Fig. 7) at boundary, inside transparent circle visible mycelia deformity in chain or
Routed solution (D in Fig. 7).Therefore, the biological activity determination of following tests, selection Leptosphaeria biglobosa
(Lb20) it is used as strains tested.
Three, the Stability Determination of huge Phoma cava P2 fermentation material filtrate (antifungal substance)
(1) the P2 fermentation material filtrate of training 15d will thermal stability: be shaken respectively in 40 DEG C, 60 DEG C, 80 DEG C and 100 DEG C water-baths
10min, 30min and 60min are handled, is compareed as without heat treatment.Will treated solution places rapidly cooled on ice to room temperature,
Biological activity determination is carried out using cylinder-plate method, measurement bacterium is rape black shank bacterium Leptosphaeria biglobosa
(Lb20), respectively processing bioactivity result is shown in Fig. 8.The result shows that the antifungal substance that P2 is generated is handled in 100 DEG C of water-baths
After 60min, activity is kept approximately constant compared with control (not having to heat treatment), illustrates the substance high temperature resistant, and in high temperature
Under the conditions of will not lose its bioactivity.
(2) the P2 fermentation material filtrate NaOH solution of the HCl and 4mol/L of 4mol/L of training 15d will ph stability: be shaken
It adjusts fermentation material filtrate pH and is placed on 4 DEG C of refrigerators to 2,3,4,5,6,7,8,9,10,11,12 and 13 respectively, in for 24 hours afterwards will everywhere
The fermentation material filtrate pH value of reason is adjusted to normal level (pH 6) respectively, finally carries out biological activity determination using cylinder-plate method, raw
Object determination of activity bacterium is Leptosphaeria biglobosa (Lb20), and each bioactivity result that handles is shown in Fig. 9.The result shows that
After soda acid is handled, bioactivity still remains unchanged P2 fermentation material filtrate, and the antifungal substance for illustrating that P2 is generated is acidproof
Alkali.
(3) the P2 fermentation material filtrate of training 15d will ultraviolet stability: be shaken as 20cm under ultraviolet lamp (UV-C, 30W)
Place, after irradiating 0min, 1min, 5min, 15min, 20min, 30min, 40min, 60min, 80min and 100min sampling respectively
Biological activity determination is carried out using cylinder-plate method, biological activity determination bacterium is Leptosphaeria biglobosa (Lb20), respectively
Handle bioactivity the result is shown in Figure 10.The result shows that P2 fermentation material filtrate is given birth to after ultraviolet light extension exposes to 100 min
Object activity and the control group (without ultraviolet irradiation) without ultraviolet irradiation are almost the same, illustrate, the antifungal substance that P2 is generated is resistance to
It is ultraviolet.
Embodiment 4:
Influence of the environmental pH to huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) activation plays
First by PDA culture medium with 4mol/L HCl and 4mol/L NaOH adjust respectively pH value to 3.5,4.0,5.0,
6.0,7.0,8.0,9.0, wherein PDA culture medium initial pH value is 6；It is then 1 × 10 by 20mL concentration8A spore/mL rape
The conidium liquid of black shank bacterium (Lb20) is mixed with the PDA culture medium of 200mL, and the plate that carries disease germs is made, and is carried out using cylinder-plate method
200 μ L P2 fermentation material filtrates (15d) are added in Oxford cup, 22 DEG C of constant temperature incubations by biological activity determination, observe result after 3d.
The result shows that (Figure 11) P2 fermentation material filtrate generate antifungal substance in acid condition its activation plays obtain it is best, generation
Inhibition zone is maximum, but with the raising of Medium's PH Value, bioactivity be can decrease, but still active, illustrates that P2 is generated
Antifungal substance under alkaline condition its activity not as good as playing well in acid condition.
Influence of the 2 environment pH of table to P2 fermentation material filtrate (containing antifungal substance) activation plays
Note: using the significance of difference between the significant method of minimum difference (LSD) analysis processing, in same row, letter is identical indicates poor
Different not significant (P > 0.05).
Embodiment 5:
Teratogenesis of the huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) to 4 kinds of plant pathogenic fungi mycelia
Using solid plate method, by P2 fermentation material filtrate (15d) respectively with 0.1%, 0.2%, 0.3%, 0.6%,
1.3%, 2.5%, 5.0% and 10.0% ratio (v/v) is mixed with the PDA culture medium melted, and the malicious plate of band is made, with
The PDA of fermentation material filtrate is not added as negative control.Every plate center is inoculated with pathogen (the grey grape that a diameter is 5mm
Spore, sclerotinite, Rhizoctonia solani Kuhn and rape black shank bacterium) mycelia block, 3 repetitions of every processing, 22 DEG C of constant temperature incubations, to negative right
After covering with ware fastly, the colony diameter between each processing is measured, the percentage for inhibiting the growth of pathogen mycelia is calculated with this.
As a result as shown in the figure (Figure 12), P2 fermentation material filtrate is to Botrytis cinerea, sclerotinite, Rhizoctonia solani Kuhn and the black shin of rape
Germ mycelia has teratogenesis, and with the increase of P2 fermentation material filter liquor concentration, 4 kinds of pathogen mycelia growths are obviously pressed down
System, the mycelia accumulation of bacterium colony centre, aerial hyphae is flourishing, when fermentation material filter liquor concentration is only 0.6%, sclerotinite and the black shin of rape
Germ mycelia is obviously inhibited.
The influence that (containing antifungal substance), concentration grew 4 kinds of pathogen mycelia of 3 bacterial strain P2 fermentation material filtrate of table
The mycelia of the mycelia tip of picking P2 fermentation liquid final concentration of 0.6% and negative control (being free of P2 fermentation material filtrate)
Tip finds that the pathogen mycelia branch after P2 fermentation liquor treatment increased significantly, and mycelia is entangled with microexamination after the dyeing of cotton indigo plant
Bunchy, the excessive disconnected phenomenon of mycelia；However mycelia tip is than sparse for negative control (being free of P2 fermentation material filtrate), mycelia branch is just
Often, mycelia is more full (Figure 13).
Embodiment 6
The influence that huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) sprouts sclerotinite and Botrytis cinerea sclerotium
This test measures the prevent plant disease, pests, and erosion application prospect confrontation sclerotinite of P2 fermentation material filtrate generation, Botrytis cinerea using two methods
The influence of sclerotial germination.
Method one: the almost the same sclerotium of size is chosen, with 75% ethanol solution (v/v) and 5% sodium hypochlorite solution (v/v)
1min and 5min will be handled respectively for examination sclerotium, and then be embathed 3 times with sterile water, these sclerotium are placed in sterilized filter paper
On, to blot the moisture on surface, natural air drying under gnotobasis.Finally sclerotium is immersed in P2 fermentation material filtrate (15d),
Impregnating sclerotium with PDB culture solution is control, is stood at room temperature for 24 hours.Each processing sclerotium is placed on PDA plate afterwards for 24 hours, is often put down
4 sclerotium of plate, 4 repetitions of each processing, plate is placed at 22 DEG C and is cultivated, and sclerotial germination situation is observed after 5d.
Method two: sclerotium is chosen according to method one and carries out surface sterilization.By P2 fermentation material filtrate (15d) with 10% ratio
Example (v/v) is mixed with the PDA of fusing, and the malicious plate of band is made；Not add the PDA plate of P2 fermentation material filtrate as negative control,
These plates are placed at 22 DEG C and cultivate, sclerotial germination is observed after 5d by 4 sclerotium of last each plate, 4 repetitions of each processing
Situation.
As shown in the figure (Figure 14), the sclerotium of sclerotinite and Botrytis cinerea soaks one test result of method in P2 fermentation material filtrate
After steeping for 24 hours, it is seeded on PDA plate after cultivating 5d from from the point of view of result, two kinds of pathogen sclerotium can be sprouted, but sprout
Mycelia growth out relatively slowly or is suppressed, and two kinds of sclerotium that control group PDB impregnates can be just on PDA plate
It often sprouts, and mycelia energy normal growth.
Two test result of method as shown in the figure (Figure 15), send out containing 10% (v/v) P2 by sclerotinite and Botrytis cinerea sclerotium
After cultivating 5d on the malicious plate of the band of ferment object filtrate, although two kinds of pathogen sclerotium, which can be sprouted, generates mycelia, the bacterium come out is sprouted
Silk cannot normally be grown, hence it is evident that be inhibited.And it is no addition P2 fermentation material filtrate PDA plate, both bacterium
Sclerotium can normally be sprouted, and sprout the mycelia energy normal growth come out.
Embodiment 7:
Huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) is to Botrytis cinerea and rape black shank bacterium conidium
The influence of sprouting
Sectional growing spore suspension is made under sterilizing washing in Botrytis cinerea and the conidium of rape black shank bacterium respectively
Liquid, blood counting chamber measure its conidium concentration, and the conidium concentration in suspension is adjusted to 1 × 10 with sterile water6
A spore/mL, takes 200 μ L conidium drops in plate center, is smeared conidium liquid uniformly, finally with sterile spreader
Observe conidial sprouting situation, standard whether spore germination: germ tube length is considered as when being longer than the 1/2 of conidium length
The spore has been sprouted.
Using solid plate method, by P2 fermentation material filtrate (15d) respectively with 0.1%, 0.2%, 0.3%, 0.6%,
1.3%, 2.5%, 5.0% and 10.0% ratio (v/v) is mixed with WA (containing 2% glucose) culture medium of fusing, and band is made
Malicious plate.Not add the plate of P2 fermentation material filtrate as negative control；Then respectively by the Botrytis cinerea of 200 μ L
(B05.10), rape black shank bacterium (Lb20) conidium drop is in WA plate center, with sterile spreader by conidium liquid
It is uniformly coated on plate.Every kind of processing applies 3 plates, as 3 repetitions.All plates are placed at 22 DEG C and are cultivated, grey Portugal
Grape spore conidium observes conidial sprouting situation on each plate under an optical microscope after cultivating 9h, and every plate is random
100 conidiums are counted, mitogenetic spore germination rate is counted with this.Rape black shank bacterium (Lb20) conidium is in culture 20h
Conidia germination situation is observed afterwards, and counts conidial germination rate.The result shows that the conidium of Botrytis cinerea with
The raising of P2 fermentation material filter liquor concentration, the sprouting of germ tube are significantly inhibited, when the concentration of P2 fermentation material filtrate is 2.5%
When, the germ tube length of Botrytis cinerea is obviously shorter than the germ tube length of negative control, when fermentation material filter liquor concentration is 10%, spore
It sprouts the germ tube come out and is obviously suppressed (Figure 16).And the conidium of rape black shank bacterium is quicker to P2 fermentation material filtrate
Sense, when fermentation material filter liquor concentration is 0.5%, the sprouting of germ tube has just received apparent inhibition (Figure 17).
The influence that 4 bacterial strain P2 ferment filtrate of table (containing antifungal substance) sprouts Botrytis cinerea conidiospore
Influence of the 5 bacterial strain P2 ferment filtrate of table (containing antifungal substance) to rape black shank bacterium conidia germination
Embodiment 8:
Diseases prevention of the huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) on living body rape leaf to 2 kinds of pathogens
Effect
Zhongshuang 9 kind is planted indoors, the rape of 5 vernalization of sowing inside each earthen bowl (10cm × 10cm)
Seed is respectively used to inoculation two kinds of pathogens of sclerotinite and Botrytis cinerea after Brassica campestris L seedling length to 30d.P2 is used on every true leaf
The fermentation material filtrate of bacterial strain 15d is uniformly applied to surface (about 500 μ L P2 fermentation material filtrates are added dropwise on every true leaf).It is molten with PDB
The blade of liquid processing is as control, and (4 repetitions here refer to that 4 basin rapes, every basin rape are inoculated with 4 to 4 repetitions of each processing
True leaf).Then the mycelia block for taking 5 mm of diameter is beaten from the fresh sclerotinite of growth 3d and Botrytis cinerea bacterium colony edge respectively.Mycelia
Block is seeded to the both ends of every true leaf, and then inoculated Brassica campestris L seedling is placed in plastic casing, and top is sealed with preservative film, protects
Wet culture is placed in 22 DEG C of culturing room and observes pathogen infection situation after light dark (12h/12h) culture 3d.
The result shows that being inoculated with sclerotinite under PDB solution treatment conditions, which can be on rape leaf after inoculation 3d
It normally infects, and lesion diameter reaches 14.7mm, and inoculates sclerotinite with the processed rape leaf of P2 fermentation material filtrate, the bacterium
Infecting on rape leaf is considerably restricted, and lesion diameter is only 0.9mm (Figure 18).It is connect under PDB solution treatment conditions
Kind Botrytis cinerea, the bacterium can normally infect on rape leaf, and lesion diameter reaches 8.1mm, and at P2 fermentation material filtrate
The rape leaf managed inoculates Botrytis cinerea, which can hardly obviously cause to infect on rape, lesion diameter 0mm
(Figure 19).
Embodiment 9:
The antimicrobial spectrum measurement of huge Phoma cava P2 fermentation material filtrate (containing antifungal substance)
The prevent plant disease, pests, and erosion application prospect confrontation plant pathogenic fungi generated using mycelial growth rate method detection P2 fermentation material filtrate (15d)
Bacteriostasis.P2 fermentation material filtrate is added in PDA culture medium in 10% (v/v) ratio first, the malicious plate of band is made.Point
Do not take it is fresh for try pathogen mycelia block (d=diameter 5mm), be inoculated in the plate center, not add P2 fermentation material filtrate
PDA plate as control.Every processing sets 3 repetitions, and each pathogen is placed in (22 or 28 DEG C) of suitable growth temperature trainings
It supports.When growing to wait compare bacterium colony close to culture dish edge, colony diameter is measured, and calculate bacteriostasis rate, table 6 is P2 fermentation material
The antagonism for the prevent plant disease, pests, and erosion application prospect confrontation test plant pathogen that filtrate generates and condition of culture and the time of each bacterial strain, Figure 20
Effect bacterium colony figure is suppressed for strains tested.
6 bacterial strain P2 fermentation material filtrate of table (containing antifungal substance) is to the antagonism of test plant pathogen
Embodiment 10:
Huge Phoma cava P2 fermentation material filtrate (containing antifungal substance) is to the toxicity test of rape seed
In order to which whether the sprouting and growth of the clear bacterial strain P2 prevent plant disease, pests, and erosion application prospect confrontation rape seed generated have an impact, therefore this
Test by kind be in double No. 9 rape seeds first use 75% ethanol solution (v/v) and 5% sodium hypochlorite solution (v/v) to locate respectively
1 min and 5min is managed, then uses sterile water wash 3 times, seed is finally separately immersed in P2 fermentation material filtrate (15d) and sterile
In water for 24 hours, seed is placed on culture dish and (spreads sterilized filter paper on culture dish at this time) afterwards for 24 hours, moisturizing culture, point
It does not observe in 3d and 4d and counts seed between each processing and sprout and the case where growth.As a result as shown in the figure (Figure 21), P2 hair
The processed rape seed of ferment object filtrate and the processed rape seed of sterile water are long upper without significant in sprouting, seedling stem length and bud
Sex differernce (P > 0.05), two kinds handle lower seed and normally can sprout and grow.These results illustrate P2 fermentation material filtrate to oil
Colza sprouts no toxic action.Huge Phoma cava P2 of the invention and its fermentation material filtrate can be used as coating agent for seed and is used for
Prevention phytopathogen infects rape seed and seedling.
Claims (5)
1. one plant of huge Phoma cava (Phoma macrostoma), the deposit number of the huge Phoma cava is CCTCC NO:M
2018726, classification naming: huge Phoma cava (Phoma macrostoma) P2.
2. huge Phoma cava described in claim 1 (Phoma macrostoma) P2 fermentation material filtrate.
3. huge Phoma cava described in claim 1 or P2 fermentation material filtrate as claimed in claim 2 are preparing phytopathogen
Application in inhibitor.
4. huge Phoma cava P2 described in claim 1 or P2 fermentation material filtrate as claimed in claim 2 are preparing vegetable seeds
Application in coating agent.
5. application according to claim 3, the phytopathogen include: melon and fruit corruption it is mould (Pythium apanidermatum), head mold (Rhizopus stolonifer), Mucor (Mucor hiemails), aspergillus flavus
(Aspergillus flavus), aspergillus niger (Aspergillus niger), Botrytis cinerea (Botrytis cinerea), water
Rice Curvularia lunata (Curvularia lunata), rape black shank bacterium (Leptosphaeria biglobosa), Lettuce Drop
Bacterium (Sclerotinia minor), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Rhizoctonia solani Kuhn
(Rhizoctonia solani).
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20070101745A (en) * | 2006-04-12 | 2007-10-17 | 대한민국(관리부서:농촌진흥청) | Phoma macrostoma bwc05-8-3-2 having weeding efficacy against sulfonylurea resistance monochoria vaginalis and mycelia suspension prepared using this |
| CN101282651A (en) * | 2005-07-25 | 2008-10-08 | 由加拿大农业与农产品部长代表的加拿大女王陛下 | Fungal isolates and biological control compositions for the control of weeds |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101282651A (en) * | 2005-07-25 | 2008-10-08 | 由加拿大农业与农产品部长代表的加拿大女王陛下 | Fungal isolates and biological control compositions for the control of weeds |
| KR20070101745A (en) * | 2006-04-12 | 2007-10-17 | 대한민국(관리부서:농촌진흥청) | Phoma macrostoma bwc05-8-3-2 having weeding efficacy against sulfonylurea resistance monochoria vaginalis and mycelia suspension prepared using this |
Non-Patent Citations (2)
| Title |
|---|
| M. HUBBARD等: "Impact of macrocidins, produced by Phoma macrostoma , on carotenoid profiles of plants", 《BIOLOGICAL CONTROL》 * |
| 杨雪等: "葛内生真菌Ｐｈｏｍａｍａｃｒｏｓｔｏｍａ 抗菌活性物质研究", 《陕西师范大学学报（自然科学版）》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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