CN108002885A - A kind of Trichoderma-jinggangmeisu granule and preparation method thereof - Google Patents
A kind of Trichoderma-jinggangmeisu granule and preparation method thereof Download PDFInfo
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- CN108002885A CN108002885A CN201610936252.4A CN201610936252A CN108002885A CN 108002885 A CN108002885 A CN 108002885A CN 201610936252 A CN201610936252 A CN 201610936252A CN 108002885 A CN108002885 A CN 108002885A
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Abstract
A kind of Trichoderma jinggangmeisu granule the invention discloses plant biological prevention and control field and preparation method thereof, the raw material weight proportioning of soup processed of the granule is：Trichoderma fermented liquid 36 45%, jinggangmeisu 3 6%, corn flour 4.5 5.5%, diatomite 35 38%, wheat bran 11 13%, zinc sulfate fertilizer 0.8 1.2%, humic acid 0.2 0.5%, nitrogen-phosphorus-potassium compound fertilizer 0.3 0.5%.Preparation process of the present invention is simple, production cost is low, is the granule using a kind of trichoderma strain and jinggangmeisu as core.The trichoderma strain is the separated wild strain of Foshan area vegetable fields, to vegetable fields microorganism species without ecological threat；Sporulation quantity is big, and fermentation medium components are simple, and technique easily optimizes, and microbial inoculum manufacturing cost is low.Two kinds of components synergistic effect in the Trichoderma jinggangmeisu granule, biocontrol effect significantly improves compared with one-component, is particularly suitable for nascent salination or continuous cropping obstacle and fungal disease is serious intensive vegetable filed, has the very high universal use value in field.
Description
Technical field
The present invention relates to biological technical field, is specifically a kind of Trichoderma-jinggangmeisu granule and preparation method thereof, special
It is not a kind of Trichoderma-jinggangmeisu granule for preventing maize sheath blight and preparation method thereof.
Background technology
As widely applied biological control microorganism in the world, Trichoderma at least belongs to 29 kinds of disease funguses to 18 to be had
Antagonism.At present, it has proved that Trichoderma can be parasitic to the plant pathogenic fungi from 12 categories, is respectively Rhizoctonia
(Rhizoctonia), sclerotium (Sclerotium), Sclerotinia (Sclerotinia), Helminthosporium
(Helminthosporium), hair disc Pseudomonas (Lachnum), Fusarium (Fusarium), Verticillium (Verticillium),
Inner seat shell category (Endothia), pythium (Pythium), a seat shell category (Diaporthe) and Fusicladium (Fusicladium)
Deng.The mechanism of Trichoderma Biocontrol Effect is mainly Competition (Competition) and hyperparasitism
(Hyperparasitism) and inducing plant resistance (Inducing plant resistance)；In addition, Trichoderma also has
Antibiosis (Antibiosis).In most cases, the biocontrol effect of Trichoderma is the coefficient knot of above-mentioned number of mechanisms
Fruit.
Jinggangmeisu (Validamycin) is the secondary metabolite of streptomyces hygroscopicus (S.hygroscopicus), is produced
Bacterium has the mutation of streptomyces hygroscopicus lemon (S.hygroscopicus var.limoneus) IFO12703 and 12704, water suction strepto-
Bacterium well ridge mutation (S.hygroscopicus var.Jinggangensis) 5008 etc..The agriculture very strong as a kind of systemic action
With antibiotic, after jinggangmeisu is contacted with the mycelium of Rhizoctonia solani Kuhn, it can quickly be absorbed and conduct in vivo, act on
In the trehalose energy metabolism system that Rhizoctonia solani Kahn highly relies on, disturb and suppress its cell normal growth and development.Jing Gang
Mycin is widely used in China, and annual output is up to 3000~4000 tons, for the use on 6~800,000,000 mu of ground, initial master
It is only rice sheath blight disease to want controlling object, is expanded to wheat, the banded sclerotial blight of seeding corn and other crops later.However, on jinggangmeisu,
At present or there are some, especially going abroad for agricultural product is much limited, and mainly China does not put into effect well so far
The research such as the national standard of ridge mycin method for detecting residue, its harm and effect on environment to people and animals is deep not enough.
Trichoderma has Rhizoctonia solani Kahn obvious competition and hyperparasitism, and most it is degraded at last.Jinggangmeisu
Can the trehalose energy metabolism system that is highly relied in Rhizoctonia solani Kahn of snap action, and then suppress its growth, but cannot will
It is degraded.In contrast, the Trichoderma prevention cycle is long, preventive effect is thorough, but it is slightly slow to take effect；Jinggangmeisu is quick, but prevents
Cycle is short.At present, maize sheath blight prevention is relied primarily on using jinggangmeisu, but since jinggangmeisu has used for many years, preventive effect
It has been declined that, and its own also has limitation, is cooperateed with for this by Trichoderma with jinggangmeisu using raising maize sheath blight
Preventive effect, reduces chemical pesticide and use, and is urgently solved the problems, such as in production.In view of the above-mentioned problems, the present invention successfully formulates out
To prevent maize sheath blight as main syllabus target Trichoderma-jinggangmeisu granule.
The content of the invention
The defects of for the problem that the prior art, cycle and timeliness, this hair are prevented particularly directed to domestic maize sheath blight
Bright to provide a kind of Trichoderma-jinggangmeisu granule and preparation method thereof, preparation process is simple, production cost is low, is with one
Kind trichoderma strain (Trichoderma asperellum, numbering GDFS1009) and the granule that jinggangmeisu is core.The wood
Trichoderma strain is the separated wild strain of Foshan area vegetable fields, to vegetable fields microorganism species without ecological threat；Sporulation quantity
Greatly, fermentation medium components are simple, and technique easily optimizes, and microbial inoculum manufacturing cost is low.In the Trichoderma-jinggangmeisu granule
Two kinds of components synergistic effect, biocontrol effect are significantly improved compared with one-component, be particularly suitable for nascent salination or continuous cropping obstacle and
Fungal disease is serious intensive vegetable filed, has the very high universal use value in field.
The present invention is achieved by the following technical solutions：
In a first aspect, the present invention provides a kind of Trichoderma-jinggangmeisu granule, the raw material weight proportioning of soup processed of the granule
For：
Raw material components, constituent content are recording a demerit by creativeness screening, if what the content of raw material was limited beyond the present invention
Scope, then the growth to corn Rhizoctonia solani Kuhn, Rhizoctonia zeae does not have obvious suppression, and the morbidity to germ also rises
Less than obvious control action.
Preferably, the raw material weight proportioning of soup processed of the granule is：
When granule is prepared with raw material proportioning, best results.
Preferably, the preparation of the trichoderma fermented liquid includes：It is 0.5% that part by weight is accessed into fluid nutrient medium
Second class inoculum, the fermentation time 7d at 28 DEG C, becomes green, i.e. trichoderma liquid zymotic fluid to zymotic fluid；Wherein, the liquid
The component of culture medium is maize flour 10kg, water 210kg, potassium dihydrogen phosphate 766g, magnesium sulfate 100g, manganese sulfate 0.5g, zinc sulfate
0.4g, sodium nitrate 284g, ammonium sulfate 220g, sodium chloride 200g；
The preparation of the second class inoculum includes：Trichoderma is accessed in PD, in rotating speed be 180 revs/min, the bar of 28 DEG C of temperature
Fermentation is 3g under part, up to second class inoculum, containing the cotton-shaped mycelia and scattered conidium liquid spawn largely dissipated.
Preferably, the formulation of the jinggangmeisu includes 5% aqua.
Preferably, the trichoderma fermented liquid refers specifically to Trichoderma asperellum (Trichoderma asperellum)
The zymotic fluid of GDFS1009 CGMCC NO.9512.
Second aspect, the present invention provide a kind of preparation method of the Trichoderma-jinggangmeisu granule, including following step
Suddenly：It is uniformly mixed by percentage by weight stock, extruder grain, 43~50 DEG C of dry 1~2h are up to Trichoderma-jinggangmeisu
Granule.
Preferably, the preparation method further includes the step of test and appraisal jinggangmeisu influences Trichoderma growth and breeding, test and appraisal
The step of absorption of the Trichoderma on jinggangmeisu, degradation rate influence.
Preferably, the step of test and appraisal jinggangmeisu influences Trichoderma growth and breeding, specifically includes as follows：With trichoderma
Bacterium is seeded in PDA plate containing jinggangmeisu as processing, the Trichoderma of jinggangmeisu PDA plate is free of to be inoculated in as control, system
Count, compare processing with compareing speed of production, form, spore output etc..
Preferably, the step of absorption of the test and appraisal Trichoderma on jinggangmeisu, degradation rate influence, specifically includes：
(1) Trichoderma conidiospore suspension is transferred in the PD culture mediums containing jinggangmeisu, constant temperature incubation, must ferment
Liquid；
(2) zymotic fluid is filtered, collected supernatant, LC-MS detects the content of jinggangmeisu, mould with initial well ridge
The PD culture mediums of cellulose content are control.
Preferably, in step (1), in the PD culture mediums containing jinggangmeisu, the concentration of jinggangmeisu is 500 μ g/mL；
In the PD culture mediums, the conidial concentration of Trichoderma is 106cfu/mL；The condition of the constant temperature incubation is：28℃、
180rpm、7d。
Preferably, in step (2), the filtering uses 0.22 μm of millipore filter.
Preferably, described be uniformly mixed is included by the way of mixer stirring.
Preferably, the granulation is included using granulation extruder.
Preferably, the drying includes using drying machine.
It is general that Trichoderma asperellum (Trichoderma asperellum) GDFS1009 CGMCC NO.9512 are preserved in China
Logical Microbiological Culture Collection administrative center (CGMCC NO.9512), has extremely strong adaptive capacity to environment, and growth is very rapid,
Relative to the rhizoctonia for triggering maize sheath blight, there is stronger Competition.The bacterial strain can produce chitinase, cellulose
The cell wall degrading enzymes such as enzyme, suppress rhizoctonia by hyperparasitism and grow, most it is degraded at last.In addition, the strain secretes
Zytase may have inducing plant resistance, increase the function of via plant immunity.
The agricultural antibiotic very strong as absorbability, after jinggangmeisu is contacted with rhizoctonia mycelium, can be inhaled by it quickly
Receive and conduct in vivo, and then the energy metabolic pathways-trehalose approach for disturbing its height to rely on, the synthesis of glucose is prevented,
Efficiently suppress its growth development, but cannot be killed or degraded.
Compared with prior art, the present invention has following beneficial effect：
(1) antibacterial experiment in vitro the result shows that, addition is mixed with jinggangmeisu by Trichoderma so that corn miliary damping-off
The increment of bacterium reduces more than 32%, and the increment of Rhizoctonia zeae reduces more than 62%.
(2) excised leaf experiment fruit shows, addition is mixed with Validacin (Takeda) by Trichoderma so that corn miliary damping-off
The increment of bacterium and Rhizoctonia zeae also substantially reduces.
(3) by applying Trichoderma-jinggangmeisu granule so that the incidence of maize sheath blight reduces by 60%, the state of an illness
Index reduces by 66%, and corn emergence rate and plant height etc. are significantly increased.
Brief description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, further feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is influence of the various concentrations Validacin (Takeda) processing to Trichoderma asperellum GDFS1009 CGMCC NO.9512；Figure
In, respectively 100 μ g/mL, 500 μ g/mL Validacin (Takeda)s and CK from left to right；
Fig. 2 is Validacin (Takeda) to Trichoderma asperellum GDFS1009 CGMCC NO.9512 Competitions and hyperparasitism
Influence；A, Competition；B, hyperparasitism；
Fig. 3 is influence of the Validacin (Takeda) to Trichoderma asperellum GDFS1009 CGMCC NO.9512 chitinase activities；
Fig. 4 is influence of the Validacin (Takeda) to Trichoderma asperellum GDFS1009 CGMCC NO.9512 cellulase activities；
Fig. 5 is that LC-MS detects degradeds of the Trichoderma asperellum GDFS1009 CGMCC NO.9512 to Validacin (Takeda)；
Wherein, A, the 7d that ferments in blank PD nutrient solutions of 500 μ g/mL Validacin (Takeda)s；B, final concentration 106Cfu/mL spine
+ 100 μ g/mL Validacin (Takeda)s of spore Trichoderma spore suspension ferment 7d in PD nutrient solutions；
It is withered that Fig. 6 for 500 μ g/mL Validacin (Takeda)s with Trichoderma asperellum GDFS1009 CGMCC NO.9512 cooperates with corn to stand
The experiment in vitro of rhizoctonia；
It is withered that Fig. 7 for 500 μ g/mL Validacin (Takeda)s with Trichoderma asperellum GDFS1009 CGMCC NO.9512 cooperates with corn to stand
The experiment in vitro of rhizoctonia；
Fig. 8 cooperates with the beautiful another name for Sichuan Province of suppression with Trichoderma asperellum GDFS1009 CGMCC NO.9512 for 500 μ g/mL Validacin (Takeda)s
The experiment in vitro of broomcorn millet rhizoctonia；
Fig. 9 is 100 μ g/mL Validacin (Takeda)s and 106Cfu/mL Trichoderma asperellum GDFS1009 CGMCC NO.9512 spores hang
Liquid collaboration suppresses the excised leaf experiment of Rhizoctonia zeae；
Figure 10 cooperates with the beautiful another name for Sichuan Province of suppression with the Trichoderma asperellum GDFS1009 CGMCC NO.9512 antagonism factors for Validacin (Takeda)
The experiment of broomcorn millet rhizoctonia；A, 100 μ g/mL Validacin (Takeda)s and 20% chitinase zymotic fluid；B, 100 μ g/mL Validacin (Takeda)s with
20% cellulase fermentations liquid；
Figure 11 is Adult plant inoculation test result figure, its middle left and right is respectively the application Trichoderma granule, blank control
Processing.
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following embodiments will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
Pathogen：Rhizoctonia solani Kahn, including：Corn Rhizoctonia solani Kuhn and Rhizoctonia zeae, are stored in Shanghai friendship
Logical university's agricultural and biological institute's agricultural environment microbial project laboratory, while be disclosed in following documents：
Ji Zhen, Lee's treasured is sincere, Bi Yuping, and Zhou Shanyue Shandong Province maize production kind and part self-mating system are to sharp eyespot resistance
Identify plant protection, 2010,36 (4):152-154；
Niu Fufang, Dong Mingming, Zhao Xinlan, Ma Yanan, Liu Aixin China some areas corn rhizoctonia form and its cause a disease
Type analysis plant protection journals, 2009,36 (4):289-294.
Corn variety：Prosperous standing grain 8 is stored in Shanghai Communications University's agricultural and the micro- raw engineering experiment of biological institute's agricultural environment
Room.
(HPLC grades) of jinggangmeisu (Validacin (Takeda)) analytical standard product are purchased from Shanghai Ke Xing bio tech ltd.Receive
He is purchased from Aladdin- Aladdin reagents (Shanghai) Co., Ltd. by mycin analytical standard product (HPLC grades).0.22 μm of millipore filter
It is purchased from Millipore companies of the U.S..3,5- dinitrosalicylic acids (DNS), xylose, sodium carboxymethylcellulose (CMC-Na, 800-
1200mPa), dimethylbenzidine (DAB) is purchased from Sinopharm Chemical Reagent Co., Ltd..Xylan is public purchased from Germany Serva
Department.
The influence that 1 jinggangmeisu of embodiment grows trichoderma
1. influence of the various concentrations Validacin (Takeda) to Trichoderma asperellum bacterium colony
(1) 28 DEG C of incubator constant temperature incubation Trichoderma 3d；
(2) 5mg and 25mg Validacin (Takeda)s are dissolved in 5mL sterile waters, 0.22 μm of millipore filter filtering, is added to 45mL
In PDA, after mixing, 100 μ g/mL and 500 μ g/mL Validacin (Takeda) plates are prepared, sterile aqueous media is control；
(3) Trichoderma bacteria cake is taken with diameter 7mm card punch, inversion is transferred to plate center；
(4) 28 DEG C of incubator constant temperature incubation 3d, measure Trichoderma colony diameter, and observe production spore situation.Each experiment is extremely
Few 3 repetitions.
Compared with the control, in the plate containing jinggangmycin A, the mycelial speed of growth of Trichoderma asperellum, form and
Spore output all has no change, be specifically shown in Fig. 1 (in figure from left to right be respectively 100 μ g/mL, 500 μ g/mL Validacin (Takeda)s and
CK)。
2. Trichoderma asperellum is competed Validacin (Takeda) and the influence of hyperparasite ability
2.1 competitions and the observation of hyperparasite ability
(1) 28 DEG C of incubator constant temperature incubation Trichoderma and Corn stalk rot pathogens PDA cultures 3d；
(2) 25mg Validacin (Takeda)s are dissolved in 5mL sterile waters, 0.22 μm of millipore filter filtering, is added to 45mL PDA
In, after mixing, prepare the Validacin (Takeda) plate of 500 μ g/mL；Sterile aqueous media is control；
(3) beaten with the card punch of 7mm and take Trichoderma and Pathogen bacterium dish, two bacterium dish are individually placed to 9cm and put down at a distance of 4cm
The both ends of ware；
(4) 28 DEG C of constant temperature incubation 4d, observe competitiveness；
(5) scalpel cuts the part that 2 kinds of bacterium colonies contact with each other, and volume size is about 0.4cm × 0.4cm × 0.1cm；
It is placed in 2.5% glutaraldehyde of precooling, 4 DEG C of fixed 12h；
(6) hyperparasite ability of the Trichoderma to pathogen is observed under light microscope (400 ×).
The results show that the Trichoderma asperellum pair the results show that after Validacin (Takeda) processing is tested with optics electron microscopic observation in face-off
The Competition and hyperparasite ability of Corn stalk rot pathogens are with compareing no difference (Fig. 2).
2.2 hyperparasitism related enzyme systems determination of activity
2.2.1 the preparation of daughter bacteria is planted
(1) Trichoderma spore by -80 DEG C of preservations is transferred in PD culture mediums, 28 DEG C, 180rpm, constant-temperature shaking culture
2d, grows prosperity to mycelium；
(2) vacuum filtration removes supernatant, precipitation i.e. kind of daughter bacteria；
(3) several pieces 1g kind daughter bacterias are weighed, for producing enzyme inducing culture of transferring；
2.2.2 the preparation of Validacin (Takeda) mother liquor
300mg Validacin (Takeda)s are dissolved in 30mL sterile waters, 0.22 μm of millipore filter, for producing enzyme Fiber differentiation of transferring
Base；
2.2.3 transfer respectively chitinase and cellulase induction culture medium
(1) 1g kind daughter bacterias are transferred in producing enzyme inducing culture, and add 5mL Validacin (Takeda) mother liquors, using sterile water as
Control；
(2) 28 DEG C, 180rpm constant-temperature shaking cultures 4d；
(3) 1mL is sampled daily, and 6000rpm centrifugation 10min, take supernatant, for enzyme activity determination.
Chitinase activity measure is at least repeated 3 times with reference to conventional method, each experiment, is shown using t-test detections difference
Work property.
The results show (Fig. 3), in fermentation 3-4d, Trichoderma asperellum chitinase yield reaches plateau.At Validacin (Takeda)
Reason Trichoderma fermentation 4d when enzyme activity be：121.481 ± 9.114U/mL, compares and is：119.837 ± 6.231U/mL, by
This is not as it can be seen that Validacin (Takeda) influences Trichoderma production chitinase.
Cellulase activity measures：(1) by 100 μ L cellulase solutions (cellulase induction nutrient solution supernatant for control) with
1.9mL CMC-Na solution (pH 4.8) mixes, 50 DEG C of water-bath 1h, using blank inducing culture as control；(2) 3mL is added
DNS agent, boiling water bath reaction 10min, OD is measured after dilution540nmLight absorption value.The foundation of glucose standard curve is with reference to the Chinese people
Republic's light industry standard (QB 2583-2003).By 50 DEG C, under the conditions of pH 4.8, every milliliter of zymotic fluid discharges per hour
The required enzyme amount of 1mg glucose is defined as an enzyme activity unit.Each experiment is at least repeated 3 times, and is detected using t-test
The significance of difference.During the 4d that ferments, Validacin (Takeda) also has no influence to the yield of Trichoderma asperellum cellulase.Hair
Ferment 3d reaches enzyme activity and reaches maximum, and the enzyme activity after Validacin (Takeda) processing is：1.717 ± 0.212U/mL, compares and is：
1.723 ± 0.112U/mL (Fig. 4).
The absorption to jinggangmeisu of embodiment 2, Trichoderma, degradation rate
1st, Trichoderma conidiospore suspension is transferred in the PD culture mediums containing 500 μ g/mL jinggangmycin As, spore is dense eventually
Spend for 106Cfu/mL, cultivates 7d by 28 DEG C, 180rpm；
2nd, 0.22 millipore filter filters, and collects fermented liquid supernatant liquid, LC-MS detects the content of jinggangmycin A, with containing 500 μ
The PD culture mediums of g/mL jinggangmycin As are control；
3rd, detection device：Waters Quattro Premier XE liquid chromatography-tandedm mass spectro-metries instrument is equipped with UPLC；
4th, chromatographic condition：Liquid chromatogram is Waters, US's ultra performance liquid chromatography system (UPLC), and chromatographic column is
(BEH) C18 columns (1.7 μm, 2.1 × 100mm), 2 μ L of sample size, 40 DEG C of column temperature, autosampler temperature maintain 4 DEG C, flowing
Phase A is ultra-pure water, and Mobile phase B is trifluoroacetic acid aqueous solution, and 0.1% (V/V) formic acid, flow velocity 0.3mL/min are added in mobile phase；With line
Property gradient elution, primary condition is 10% Mobile phase B, and 10% Mobile phase B maintains 1min, 10~90% Mobile phase Bs 1~
Next sample is gathered after 6min, 90% Mobile phase B balance 3min, 90~10% Mobile phase B 0.5min；
5th, Mass Spectrometry Conditions：Electron spray ionisation (ESI) source, negative ions ionization pattern, ion source temperature (Source
Temperature) 150 DEG C, 350 DEG C of desolventizing temperature (desolvation temperature), desolventizing nitrogen flow rate
(desolvation gas flow) 500L/h, taper hole blowback nitrogen (cone gas flow) 50L/h, cation and anion
Mode capillary tube ionization voltage is 3.0kV, and sampling taper hole voltage (sampling cone) is 20eV, extracts taper hole
(extraction cone) 2eV, 100~1500m/z of quadrupole rod scanning range；
LC-MS the results shows Trichoderma asperellum and jinggangmycin A co-fermentation 7d, Trichoderma and its zymotic fluid are mould to well ridge
The degraded of plain A is very limited, and degradation rate is only 20%, illustrates presence of the Trichoderma asperellum to jinggangmycin A also without notable shadow
Ring (see Fig. 5).
3 Validacin (Takeda) of embodiment cooperates with the experiment for suppressing corn Rhizoctonia solani Kuhn with Trichoderma asperellum
3.1 plates are tested
(1) in PDA culture medium, 28 DEG C of incubator constant temperature, cultivate Trichoderma and Rhizoctonia solani Kuhn 3d；
(2) 25mg Validacin (Takeda)s are dissolved in 5mL sterile waters, 0.22 μm of millipore filter filtering, is added to 45mL PDA
In, after mixing, prepare the Validacin (Takeda) plate of 500 μ g/mL.Using sterile aqueous media as control；
(3) after tablet solidification, beaten with the card punch of 7mm and take Trichoderma and pathogen bacterium dish, rhizoctonia bacterium bacterium dish difference
The culture dish tablet side of diameter 9cm, opposite side inoculation Trichoderma bacterium dish are placed in, two bacterium dish are stood at a distance of 4cm with what is individually cultivated
Withered silk kernel fungus are control；
(4) 28 DEG C of constant temperature incubation 4d, observe the effect of synergistic effect.Each experiment at least 3 repetitions.
After cultivating 1d, Validacin (Takeda) can be by Rhizoctonia solani Kuhn efficient absorption, bacteriostasis highly significant, rhizoctonia bacterium
It is only 1.8 ± 0.2cm to fall diameter, and control group colony diameter has reached 6.1 ± 0.3cm.Although Trichoderma asperellum growth is rapid,
Fungistatic effect is also obvious, but has obvious gap with the effect of Validacin (Takeda), and rhizoctonia colony diameter is 4.0 ± 0.2cm.
Moreover, Validacin (Takeda) and the advantage of Trichoderma asperellum synergistic effect are not shown at this time, because the rhizoctonia after collaboration processing
Colony diameter is 1.9 ± 0.3cm, compared with Validacin (Takeda) is individually handled, no significant difference.But with the extension of time,
After cultivating 4d, the inhibitory action of Validacin (Takeda) begins to decline or is gradually released from, and rhizoctonia colony diameter rises to 7.2 ±
0.4cm, control group are 9.0 ± 0.0cm.But the biological and ecological methods to prevent plant disease, pests, and erosion advantage of Trichoderma is embodied as out at this time, the silk efficiently suppressed
The only surplus 1.8 ± 0.2cm of pyrenomycetes colony diameter.Moreover, Validacin (Takeda) and Trichoderma cooperate with disease resisting effect also to be embodied as out,
Under the priority effect of the mechanism such as antibiosis, competition, hyperparasite, rhizoctonia occupied area, hence it is evident that at any type single-factor
Manage (Fig. 6).It can be seen from the above that Validacin (Takeda) acts synergistically with Trichoderma, the mutual supplement with each other's advantages of Biocontrol Mechanism can be not only realized, also
Action effective each other or the deficiency on the cycle can be made up, thus efficiently, long-term suppress corn Rhizoctonia solani Kuhn.
3.2 excised leafs are tested
1. the plantation of corn and the preparation of excised leaf
(1) by corn seed (prosperous standing grain 8) plantation in the flowerpot of 10cm bores, per 6 seeds of basin, in 25 DEG C of artificial gas
It is dark to seven leaf phases, daily 10h illumination, 14h to wait culture in room；
(2) the 4th blade of 7 leaf phase corns of clip health, is placed in the plate containing appropriate 6-BA (40mg/mL)；
(3) bacteria cake for taking corn Rhizoctonia solani Kuhn is beaten with the card punch of diameter 0.4cm, central vane is placed on and (does not place
The blade of bacteria cake is control).
2. handle corn excised leaf
(1) 100 μ g/mL Validacin (Takeda)s, 40 μ L are added in bacteria cake；
(2)106Cfu/mL Trichoderma asperellum GDFS1009 spores, 40 μ L are added in bacteria cake；
(3) 100 μ g/mL Validacin (Takeda)s+Trichoderma asperellum GDFS1009 spores/mycelium 106Cfu/mL Trichoderma asperellums
GDFS1009 spores, 40 μ L are added in bacteria cake；
(4) 40 μ L of sterile water are added in bacteria cake；
(5) 40 μ L of sterile water are added on blade；
(6) after the completion of handling, 28 DEG C of constant incubators is placed on, pay attention to moisturizing, each processing is at least repeated 3 times.
The results show that after inoculation corn Rhizoctonia solani Kuhn 12h, the growth of control group (CK-1) rhizoctonia is vigorous, and mycelium is in
Pure white, and large area is infected on maize leaf.Trichoderma single-factor effect under, rhizoctonia mycelium mycelia volume density and
Vigorous degree substantially reduces, and compared with control group (CK-1), is infected with less area on blade.Validacin (Takeda) single-factor
Under effect, there is significant change in rhizoctonia mycelium morphology, and the speed of growth is slow, and maize leaves are extended to the area of very little
On piece.Under Validacin (Takeda) and Trichoderma synergistic effect, the growth of rhizoctonia is almost stagnated, only see faint mycelium exist and
There is significant change in mycelium morphology.The maize leaf (CK-2) not being inoculated with, shows no sign of damage (Fig. 7).
4 Validacin (Takeda) of embodiment cooperates with the experiment for suppressing Rhizoctonia zeae with Trichoderma asperellum
Plate experiment-Validacin (Takeda) and Trichoderma, with embodiment 3.Excised leaf experiment-Validacin (Takeda) and Trichoderma are same
Embodiment 3.
After cultivating 1d, Validacin (Takeda) can be by Rhizoctonia zeae efficient absorption, bacteriostasis highly significant, rhizoctonia
Colony diameter is only 1.9 ± 0.1cm, and control group colony diameter has reached 5.5 ± 0.2cm.Although Trichoderma asperellum growth is fast
Speed, fungistatic effect is also obvious, but has an obvious gap with the effect of Validacin (Takeda), rhizoctonia colony diameter for 4.5 ±
0.2cm.Moreover, Validacin (Takeda) and the advantage of Trichoderma asperellum synergistic effect are not shown at this time, because after collaboration processing
Rhizoctonia colony diameter is 1.9 ± 0.1cm, compared with Validacin (Takeda) is individually handled, no significant difference.
But with the extension of time, after culture 4d, the inhibitory action of Validacin (Takeda) gradually obtains to a certain extent
Release, rhizoctonia colony diameter rises to 5.3 ± 0.2cm, and control group is 9.0 ± 0.0cm.But the biological and ecological methods to prevent plant disease, pests, and erosion of Trichoderma at this time
Advantage is embodied as out, and the rhizoctonia colony diameter being suppressed is reduced to 3.4 ± 0.1cm.Moreover, Validacin (Takeda) and trichoderma
The collaboration disease resisting effect of bacterium is also substantially embodied, under the priority effect of the mechanism such as antibiosis, competition, hyperparasite, rhizoctonia
The only surplus 2.9 ± 0.2cm of colony diameter, and occupied area, hence it is evident that handle (Fig. 8) less than any type single-factor.It can be seen from the above that
Validacin (Takeda) and Trichoderma act synergistically, and can not only realize the mutual supplement with each other's advantages of Biocontrol Mechanism, can also make up when acting on each other
Effect or the cycle on deficiency so that efficiently, suppress Rhizoctonia zeae for a long time.
After being inoculated with Rhizoctonia zeae 12h, the growth of control group (CK-1) rhizoctonia is vigorous, and large area infects maize leaves
On piece.Under the effect of Trichoderma single-factor, rhizoctonia mycelium mycelia volume density and vigorous degree substantially reduce, with control group (CK-
1) compare, infected with less area on blade.Under the effect of Validacin (Takeda) single-factor, rhizoctonia mycelium morphology occurs
Significant change, and growth is substantially suppressed, speed is extremely slow.Under Validacin (Takeda) acts synergistically with Trichoderma, rhizoctonia
Growth is fully completed.The maize leaf (CK-2) not being inoculated with, shows no sign of damage (Fig. 9).
Plate experiment-Validacin (Takeda) and Trichoderma cell wall degrading enzyme
(1) 28 DEG C of incubator constant temperature incubation Rhizoctonia zeae PDA cultures 3d.
(2) 25mg Validacin (Takeda)s are dissolved in 10mL sterile waters, 0.22 μm of millipore filter filtering, is added to 40mL PDA
In, after mixing, prepare the plate containing 100 μ g/mL Validacin (Takeda)s.
(3) by two kinds of zymotic fluids in above-mentioned 1.2.3.2 and 1.2.4.1, filtered, be added to 0.22 μm of millipore filter
In 40mL PDA, after mixing, the plate containing 20% enzyme liquid is prepared.
(4) two kinds of zymotic fluids being dissolved in 25mg Validacin (Takeda)s respectively in 10mL above-mentioned 1.2.3.2 and 1.2.4.1, are used
0.22 μm of millipore filter filtering, is added in 40mL PDA, after mixing, prepares+20% enzyme liquid Han 100 μ g/mL Validacin (Takeda)s
Plate.
(5) 10mL sterile waters are added in 40mL PDA, after mixing, prepare control plate.
(6) after tablet solidification, beaten with the card punch of 7mm and take Rhizoctonia zeae bacterium dish to be placed in plate center, 28 DEG C of perseverances
Temperature culture to bacterium colony is in contact, and the Rhizoctonia zeae bacterium colony that measurement processing group and control group are distinguished using crossing method is straight
Footpath, calculates inhibiting rate after culture respectively.Each experiment at least 3 repetitions, the significance of difference is detected using t-test.
The results show that culture 4d after, Validacin (Takeda) cooperateed with Trichoderma chitinase handle germ colony diameter be
3.0±0.2cm；The germ colony diameter of Validacin (Takeda) and Trichoderma single-factor processing be respectively 3.7 ± 0.2cm and 5.9 ±
0.3cm；In control, germ colony diameter is 9.0 ± 0.1cm.The disease resisting effect of synergistic effect is significantly higher than single-factor effect (figure
10-A).After cultivating 4d, Validacin (Takeda) and the germ colony diameter of Trichoderma cellulase synergistic processing are 3.5 ± 0.1cm；Have
Validamycin A and the germ colony diameter of Trichoderma single-factor processing are respectively 4.2 ± 0.2cm and 7.1 ± 0.2cm；In control, disease
Bacterium colony diameter is 9.0 ± 0.1cm.The disease resisting effect of synergistic effect is significantly higher than single-factor effect (Figure 10-B).
The preparation of embodiment 5, Trichoderma-jinggangmeisu granule
The present embodiment provides a kind of preparation method of Trichoderma biologic grain agent, specifically comprise the following steps：
1st, tablet strain：Trichoderma asperellum GDFS1009 is inoculated in PDA culture medium plate, in 28 DEG C of incubators, is fallen
Put 2~3d of culture；
PDA culture medium：200g potatoes are cut into 1cm2Fritter, be boiled with soft fire 30min, 4 layers of filtered through gauze, retain juice；Add
Enter 20g glucose, 20g agar powders, distilled water is settled to 1L, 121 DEG C of high pressure steam sterilization 30min.
2nd, the preparation of second class inoculum：Potato 200g, glucose 20g, water 1L are taken, is configured to second order fermentation nutrient solution, is adjusted
PH value is 6~8, is divided in 100ml in 250ml triangular flasks, and sterilize 30min at 121 DEG C, accesses tablet strain, shaking speed
For 180 revs/min, 28 DEG C, fermentation time 3d of fermentation temperature, the cotton-shaped mycelia largely dissipated and scattered conidium liquid
Body strain.
3rd, prepared by trichoderma liquid zymotic fluid：Fermentation medium components are：Maize flour 10kg, water 210kg, potassium dihydrogen phosphate
766g, magnesium sulfate 100g, manganese sulfate 0.5g, zinc sulfate 0.4g, sodium nitrate 284g, ammonium sulfate 220g, sodium chloride 200g, first by nothing
Machine salt is dissolved in water, then solid fermentation culture medium is made after compost and water are sufficiently mixed, and solid fermentation culture medium loads
In 300L fermentation tanks, sterilize 30~60min at 121 DEG C, accesses the second class inoculum of part by weight 0.5%, controls fermentation temperature
For 28 DEG C, fermentation time 7d, become green i.e. trichoderma liquid zymotic fluid to compost；
4th, prepared by Trichoderma granule：Trichoderma liquid zymotic fluid 40%, corn flour 5.2%, diatomite 37%, wheat bran
12%th, zinc sulfate fertilizer 1%, humic acid 0.4%, nitrogen-phosphorus-potassium compound fertilizer 0.4%, jinggangmeisu (5% aqua) 4%, use stirring
Machine stirs evenly, and after comminutor extruder grain, the dry 1~2h finished products of 43~50 DEG C of drying machine are Trichoderma granule.
The quality standard of the Trichoderma that this preparation method is prepared-jinggangmeisu biologic grain agent is specially：Particle diameter 0.15
~0.2cm, surface is smooth, hard, water content 10~20%, pH 5~7, spore amount 2~4 × 10 living8CFU/g, particle 6~
10min can be entirely soluble in water, and administration is mixed with chemical fertilizer suitable for mechanization.
Trichoderma-jinggangmeisu granule prepared by embodiment 6, embodiment 5 suppresses the effect of Rhizoctonia solani Kahn
Spread manuer in holes 6g Trichoderma asperellum GDFS1009 granules per basin soil before treatment group plantation, using normally plant be used as it is empty
White control, per 5 plants of basin, often handles 5 basins, typhon mouth phase potted plant basal part of stem is inoculated with pathogen germ and (hinders the inoculation of root method to take respectively
Toothpick with corn Rhizoctonia solani Kuhn), count biocontrol effect after morbidity.
1st, Trichoderma asperellum GDFS1009 investigates the biocontrol effect of corn stalk rot disease：
Diseased plant rate=diseased plant rate/investigation total strain number × 100%,
Biocontrol effect=[(diseased plant rate of control diseased plant rate-processing)/control diseased plant rate] × 100；
2nd, Trichoderma asperellum GDFS1009 investigates the biocontrol effect of maize sheath blight：
Disease index=[the representative numerical value of (the sick level strain number × disease level represents numerical value)/total strain number × morbidity most heavy duty)]
× 100% (with reference to table 1),
Biocontrol effect=(disease index of control disease index-processing)/control disease index × 100%；
Table 1
| State of an illness rank | Symptom describes |
| 1 | 4th leaf sheath and the morbidity of following leaf sheath under fruit ear |
| 3 | 3rd leaf sheath and the morbidity of following leaf sheath under fruit ear |
| 5 | 2nd leaf sheath and the morbidity of following leaf sheath under fruit ear |
| 7 | 1st leaf sheath and the morbidity of following leaf sheath under fruit ear |
| 9 | Fruit ear and its morbidity of above leaf sheath |
Table 2
Adult plant inoculation test：As shown in figure 11, its middle left and right is respectively the application Trichoderma granule, blank control
Processing；Wherein, the incidence of Trichoderma granule treatment, disease index are respectively 40%, 10, and blank control group is respectively
100%th, 29.Trichoderma granule is respectively 65.52% to the preventive effect of maize sheath blight.Trichoderma granule treatment is to corn line
Blight preventive effect is preferably (being specifically shown in Table 2).
At present, the main control method of the maize sheath blight triggered to rhizoctonia is that chemical pesticide-Validacin (Takeda) is direct
It is sprayed on basal part of stem leaf sheath.The mechanism of action of Validacin (Takeda) predominantly disturbs sheath blight fungus energetic supersession, and then suppresses its life
It is long, but cannot be degraded.Trichoderma can prevent sheath blight fungus by mechanism such as competition, hyperparasitisms, and most it is degraded at last.
In addition, Trichoderma induces resistance of the plant to sheath blight fungus, and it is obvious more than Validacin (Takeda), and Trichoderma can be in root system of plant
Colonized with the long period in stem foot cortex, form homobium with plant tissue, holding effect control Rhizoctonia solani Kahn infects.Cause
This, if Validacin (Takeda) can act synergistically with Trichoderma, it is contemplated that the prevention level to banded sclerotial blight can significantly improve；Meanwhile also favorably
In realizing the use of the minimizing to antibiotics chemical pesticide, environmental safety is improved.Validacin (Takeda) acts synergistically with Trichoderma
Pattern it is substantially as follows：(1) Validacin (Takeda) is applied to corn basal part of stem, can quickly be absorbed into sheath blight fungus body, but not
It can be killed, but be at weak state.(2) Trichoderma colonized in plant tissue further to weak germ into
Row competition and hyperparasitism, are degraded.(3) dual induction of resistance effect caused by Validacin (Takeda) and Trichoderma, can be strong
Change Resistant reaction of the corn to sheath blight fungus.
This research first combines Trichoderma asperellum GDFS1009 with Validacin (Takeda), finds the Validacin (Takeda) of various concentrations
Growth and development, production spore ability and biological and ecological methods to prevent plant disease, pests, and erosion function etc. to Trichoderma asperellum have no significant effect.In addition, during the fermentation, spine
Spore Trichoderma only has Validacin (Takeda) 20% degradation rate.This cooperates with use to establish for Trichoderma asperellum and Validacin (Takeda)
Certain theoretical foundation, illustrates the feature that there is combination biological and ecological methods to prevent plant disease, pests, and erosion to have complementary advantages.This conclusion, with both at home and abroad it is only few
Number result of study is similar, but the molecular basis research both at home and abroad to Validacin (Takeda) and the affine interaction of Trichoderma still belongs to blank at present.
In conclusion Trichoderma asperellum can cooperate with prevention maize sheath blight with Validacin (Takeda).From now on, it is necessary to explore other
Whether the Antagonistic Trichoderma of species can also act synergistically with Validacin (Takeda), to develop a variety of compound Trichoderma biological prevention and control agents
Lay the foundation.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (10)
1. a kind of Trichoderma-jinggangmeisu granule, it is characterised in that the raw material weight proportioning of soup processed of the granule is：
2. Trichoderma according to claim 1-jinggangmeisu granule, it is characterised in that the raw material weight of the granule
Measuring proportioning is：
3. Trichoderma according to claim 1 or 2-jinggangmeisu granule, it is characterised in that the trichoderma fermented liquid
Preparation include：Part by weight is accessed into fluid nutrient medium as 0.5% second class inoculum, the fermentation time 7d at 28 DEG C, extremely
Zymotic fluid becomes green, i.e. trichoderma fermented liquid；
Wherein, the component of the fluid nutrient medium is maize flour 10kg, water 210kg, potassium dihydrogen phosphate 766g, magnesium sulfate 100g,
Manganese sulfate 0.5g, zinc sulfate 0.4g, sodium nitrate 284g, ammonium sulfate 220g, sodium chloride 200g；The preparation bag of the second class inoculum
Include：By Trichoderma access PD in, in rotating speed be 180 revs/min, under conditions of 28 DEG C of temperature fermentation be 3g, up to second class inoculum.
4. Trichoderma according to claim 1 or 2-jinggangmeisu granule, it is characterised in that the agent of the jinggangmeisu
Type includes commercially available 5% aqua.
5. Trichoderma according to claim 1 or 2-jinggangmeisu granule, it is characterised in that the trichoderma fermented liquid
Refer specifically to the zymotic fluid of Trichoderma asperellum (Trichoderma asperellum) GDFS1009CGMCC NO.9512.
6. a kind of preparation method according to any one of claim 1 to the 5 Trichoderma-jinggangmeisu granule, its feature exists
In including the following steps：By the percentage by weight for raw material, it is uniformly mixed, extruder grain, 43~50 DEG C of dry 1~2h are
Obtain Trichoderma-jinggangmeisu granule.
7. the preparation method of Trichoderma-jinggangmeisu granule according to claim 6, it is characterised in that the preparation side
Method further includes the absorption to jinggangmeisu of test and appraisal jinggangmeisus the step of being influenced on Trichoderma growth and breeding, test and appraisal Trichoderma, drops
The step of solution rate influences.
8. the preparation method of Trichoderma-jinggangmeisu granule according to claim 7, it is characterised in that the test and appraisal well
The step of ridge mycin influences Trichoderma growth and breeding, specifically includes as follows：PDA plate containing jinggangmeisu is seeded in Trichoderma
For processing, using be inoculated in be free of jinggangmeisu PDA plate Trichoderma for compare, count, compare processing with compare speed of production,
Form, spore output.
9. the preparation method of Trichoderma-jinggangmeisu granule according to claim 7, it is characterised in that the test and appraisal wood
The step of absorption of the mould on jinggangmeisu, degradation rate influence, specifically includes：
(1) Trichoderma conidiospore suspension is transferred in the PD culture mediums containing jinggangmeisu, constant temperature incubation, obtains zymotic fluid；
(2) zymotic fluid is filtered, collected supernatant, LC-MS detects the content of jinggangmeisu, is contained with initial jinggangmeisu
The PD culture mediums of amount are control.
10. the preparation method of Trichoderma-jinggangmeisu granule according to claim 6, it is characterised in that the mixing is equal
It is even including using mixer stirring by the way of；The granulation is included using granulation extruder；The drying includes using drying machine.
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