CN103053624A - Method for control of phytophthora blight of pepper by mixed application of trichoderma preparation and fungicides - Google Patents
Method for control of phytophthora blight of pepper by mixed application of trichoderma preparation and fungicides Download PDFInfo
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Abstract
The present invention discloses a method for control of phytophthora blight of pepper by mixed application of trichoderma preparation and fungicides. The method comprises: seeding after mixing pepper seeds with the trichoderma preparation during seeding, or, applying the trichoderma preparation during pepper seedling transplanting and planting; during pepper cultivation, applying the fungicides before the occurrence of the disease or in early days of the disease; wherein the active strain in the trichoderma preparation is Trichodermaasperellum Thz01, and the preservation number thereof is CGMCC No. 6422; the fungicides are one or both of eugenol and mandipropamid. The trichoderma preparation used in the method of the invention has good control effect on phytophthora blight of pepper in the fields, and can effectively reduce the primary infection source when being used alone; while the additional application of the fungicides can effectively control disease epidemics. According to the method of the invention, the application amount of the fungicides is reduced, so residues of pesticides in the soil and pepper products are decreased, thereby ensuring environmental and product safety.
Description
Technical field
The present invention relates to biological control and the Techniques For Chemical Control field of plant disease, relate in particular to the method that a kind of wooden removing mildew and bactericide are used the control capsicum epidemic disease with.
Background technology
Capsicum epidemic disease is commonly called as " dead seedling is sick ", is a kind of soil-borne disease that is caused by Phytophthora capsici (Phytophthora capsici), is a kind of destructive disease, can cause serious production loss.Because pathogen produces a large amount of zoospores in the popular phase of disease, can cause rapidly the capsicum Large Scale Death in a short time.The conventional anti-control techniques of capsicum epidemic disease has been difficult to effective control, and easily causes ecology, environmental problem.
The application for a patent for invention of publication number CN102415378A discloses a kind of bactericide and application thereof that promotes to grow and prevent and treat capsicum epidemic disease, this bactericide comprises cinnamic acid 0.1～10% according to percentage by weight, emulsifier 10～30%, azone 0.1～10%, alcohol 20～50%, all the other are water.This bactericide can be used for preventing and treating Phytophthora capsici and promotes plant growth, but it has used chemicals and plant extracts cost higher, is unfavorable for large-scale application.For many years, chemical control is the medium-term and long-term major measure of administering capsicum epidemic disease of production practices, but use in a large number chemical agent on the potential harm of human health, on the pollution of food and environment, on the impact of non-target organism and cause the problem such as the drug-fast accelerated development of pathogen to cause the growing interest of society, it is the inexorable trend of development that decrement is used chemical pesticide.
In recent years, biological prevention and control agent has been introduced in the aspect that prevents and treats of capsicum epidemic disease, and control is effective to especially wooden mould biological prevention and control agent proof to capsicum epidemic disease.We have filtered out trichoderma asperellum strain (Trichoderma asperellum) CGMCC 6422 on the basis of for many years research, the capsicum epidemic disease control efficiency is reached about 70%.Yet, although Trichoderma have saprophytic property, resistance and pathogen had lasting antagonism, but long-term surviving can reduce pathogenic soil inoculum quantity in the medium characteristic of soil, but the pathogen effect in disease popular period has been shown limitation.Therefore, the efficient medicament of screening from the bactericide of present control capsicum epidemic disease, these bactericide are to all unrestraint effects of mycelia, spore germination and Sporulation of the mould biological prevention and control agent of wood simultaneously, and this also is core content of the present invention.On the basis of wooden removing mildew control, suitably use a small amount of bactericide and less number of times in the popular phase of disease, can effectively prevent and treat capsicum epidemic disease, use thereby reach the chemical pesticide decrement.
Combine with the decrement bactericide technology report not yet so far of control capsicum epidemic disease of the mould biological prevention and control agent of wood, this technology is effectively controlled the popular of capsicum epidemic disease, thereby has been reduced loss on the basis of minimizing bactericide quantity; Simultaneously, reduced the pollution of bactericide and protected ecotope.Accordingly, the present invention proposes meter and has founded combine with the agricultural chemicals new method of Collaborative Control capsicum epidemic disease of a kind of biological control.
Summary of the invention
The invention provides a kind of wooden removing mildew and bactericide and use the method for control capsicum epidemic disease with, can not control the popular problem of disease in order to solve the simple biological control of capsicum epidemic disease, decrement is used chemical agent simultaneously.
A kind of in the pepper planting process wooden removing mildew and bactericide use the method for control capsicum epidemic disease with, the plantation of capsicum comprises sowing, grows seedlings, transplant planting, cultivate and gather; Described method comprises:
When (1) sowing, pepper seed is mixed rear sowing with wooden removing mildew; Perhaps, when the Hot Pepper Seedling transplant planting, use wooden removing mildew;
(2) during the pepper cultivation, before disease occurs or initial stage of origination use bactericide;
Wherein, the active bacterial strain trichoderma asperellum in the described wooden removing mildew (Trichoderma asperellum) Thz01, the preserving number of trichoderma asperellum Thz01 is CGMCC No.6422;
Described bactericide is one or both in eugenol and the mandipropamid.
The wooden removing mildew of trichoderma asperellum Thz01 preparation has good preventive effect in the field to capsicum epidemic disease, unites use in disease emergence period and bactericide, can effectively prevent the generation of disease; The simultaneously use of energy decrement bactericide.The wood removing mildew generally is comprised of active bacterial strain and corresponding auxiliary material, and auxiliary material is not only as carrier, and provides nutrient to strain growth, and the key of epidemic prevention is the selection of active bacterial strain.
Among the present invention, preservation of described trichoderma asperellum Thz01, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) that is positioned at Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date: on August 13rd, 2012, preserving number: CGMCCNo.6422.According to morphological feature and molecular data analysis, the Classification And Nomenclature of this bacterial strain is trichoderma asperellum (Trichoderma asperellum).
This bacterial strain is to collect from the capsicum rhizosphere soil soil of mountain vegetables garden, Linan morbidity, the biological property of this bacterial strain is: bacterium colony is 25 ℃ of lower cultivations on the PDA solid culture medium, expanded to 9cm in 3-4 days, initial stage white is sparse, mycelia is along the media surface sprawl growth, the green spore district that produces of rear formation, reverse side white, the mycelia tool every; Conidium is spherical, and is subsphaeroidal, single closely colourless, is pistac during gathering, and wall is smooth.Upright, the ampulla shape of bottle, width 3.0-5.6 μ m.The upper conidium of CMD is spherical to inferior spherical, 3.8-5.9 * 2.8-4.8 μ m.Morphological feature meets trichoderma asperellum bacterium feature.Analyze according to ITS rDNA (SEQ ID NO.1) and transcription factor 1a (tef 1-a) sequence (SEQ ID NO.2), the most consistent with the trichoderma asperellum bacterium sequence of pattern among the GenBank and confirmation.These confirm that this bacterial strain is trichoderma asperellum (Trichoderma asperellum).
Owing to need the competence exertion effect of field planting a period of time after wooden removing mildew is used, for this reason, described wooden removing mildew was used the early stage of plantation when Hot Pepper Seedling transplant planting (during sowing or), microorganism wherein is colonizated in the soil preferably, occur in order to prevent disease.
In order further to strengthen the action effect of trichoderma, during pepper cultivation, observe and predict information according to the local capsicum epidemic disease state of an illness, can before occuring, disease around the capsicum rhizosphere, use wooden removing mildew in individual month by 0.5-1.Like this, can play the effect of strengthening preventive effect.
When using, the consumption of described wooden removing mildew is preferably the 1.5-2g/ strain at every turn, and the Microorganism colonization effect is better, and can not cause the preparation waste.When using during pepper cultivation, amount of application also can a situation arises adjusts as one feels fit according to local disease.
Described wooden removing mildew can prepare by the following method: trichoderma asperellum Thz01 spore suspension is inoculated in the wheat that boils sterilization, in illumination in 12 hours and 12 hours dark alternate culture 15-20 days, makes wooden removing mildew.
Wherein, described trichoderma asperellum Thz01 spore suspension prepares by the following method: trichoderma asperellum Thz01 is inoculated in the PDA medium, in 23-28 ℃ of lower the cultivation 5-6 days; After cultivation is finished, spore is scraped in the aqua sterilisa, preparation obtains trichoderma asperellum Thz01 spore suspension.
The viable bacteria concentration of described trichoderma asperellum Thz01 spore suspension is preferably 107-108/mL.
Described wheat is the matrix that is fit to trichoderma asperellum Thz01 growth.
In every kilogram of wheat, the consumption of described trichoderma asperellum Thz01 spore suspension is preferably 2-4mL.
Incubation time behind the trichoderma asperellum Thz01 spore suspension access wheat is preferably 18 days, is convenient to produce on the matrix a large amount of spores.
Can in time stir wheat between culture period, so that fully long upper trichoderma asperellum Thz01 spore on the wheat.
Using of described bactericide can be carried out according to operation instruction, generally carries out the dispenser first time at the disease initial stage of origination, and the lasting period that chili growth disease in season development and medicament are looked in further dispenser is decided.
During the pepper cultivation, observe and predict information according to the local capsicum epidemic disease state of an illness, before disease occurs or initial stage of origination use described bactericide, can effectively prevent and treat disease.Preferably, can be before disease occurs 1-2 days or initial stage of origination field use described bactericide when fragmentary scab plant occurring.
Described bactericide can be eugenol; In every mu of ground, the consumption of eugenol active ingredient can be 0.078-0.084g; Be preferably 0.081g.Eugenol can adopt the Asia, Baoding to reach the product (active constituent content is 0.3%) that chemical industry Co., Ltd produces, and in every mu of ground, gets 26-28g, is watered 60L, obtains the medicament that active ingredient concentration is 1.3-1.4 μ g/mL, then sprays.The eugenol consumption is very few, does not have control efficiency; Consumption is excessive, and it is active to affect trichoderma asperellum Thz01, even can kill trichoderma strain.Under this consumption condition, eugenol and wooden removing mildew coupling effect are best.
Described bactericide can be mandipropamid; In every mu of ground, the consumption of mandipropamid active ingredient can be 1.8-2.1g; Be preferably 1.95g.Mandipropamid can adopt Switzerland just reaching first the product (active constituent content is 250g/L) that crop protection Co., Ltd produces; in every mu of ground; get 7.2-8.4g (being equivalent to approximately 7.2-8.4mL); be watered 30L; obtain the medicament that active ingredient concentration is 60-70 μ g/mL, then spray.Similarly, under this consumption condition, mandipropamid and wooden removing mildew coupling effect are best.
Disease can further be used bactericide according to the pepper diseases development after occuring, and strengthens preventive effect.Each amount of application is: eugenol is that active ingredient reaches 0.078-0.084g/ mu, is preferably 0.081g/ mu; Perhaps, mandipropamid is that active ingredient reaches 1.8-2.1g/ mu; Be preferably 1.95g/ mu.
Test confirms that wooden removing mildew and bactericide mix the consumption that use has effectively reduced bactericide, reach the control efficiency identical with chemical control, is safe to wooden removing mildew also simultaneously.In the production, bactericide eugenol (Asia, Baoding reaches chemical industry Co., Ltd) recommendation amount is 30g/ mu; And wooden removing mildew and eugenol mix use, and 27g/ mu eugenol usage amount is equivalent to reduce 3g/ mu with usage amount is identical separately, and namely decrement is used bactericide 10%; Inhibition test shows that wooden removing mildew is safe using under this bactericide 30g/ mu condition.Similarly, in the production, bactericide mandipropamid (Switzerland is just reaching first crop protection Co., Ltd) recommendation amount is 12g/ mu; And wooden removing mildew and mandipropamid mix use, and 7.8g/ mu mandipropamid amount of application is equivalent to reduce 4.2g/ mu with usage amount is identical separately, and namely decrement is used bactericide 35%; Inhibition test shows that wooden removing mildew is safe using under this bactericide 9.6g/ mu condition.
Among the present invention, the disease time of origin is mainly judged according to local meteorological data or the state of an illness information of observing and predicting.The disease time of the popular disease of capsicum epidemic disease is relevant with season, weather etc., but annual generation slightly changes period.The time that occurs in the plastic greenhouse of Hangzhou such as capsicum epidemic disease is about by the end of March, and open country occurs in mid-April.Different regions are because weather conditions are different, and the disease time of origin is slightly different, be generally moon mid-April such as the In Hangzhou Region of Zhe Jiang Province open country, and Ningbo area will be done sth. in advance about 10 days generally.
The present invention in suitable period, in a certain order, adopts the wooden removing mildew of appropriate dose and bactericide mixing to make to prevent and treat capsicum epidemic disease during pepper planting.
Adopt the inventive method, have following beneficial effect:
(1) the wooden removing mildew of trichoderma asperellum Thz01 preparation has good preventive effect in the field to capsicum epidemic disease, and independent wooden removing mildew can effectively reduce primary source of infection.
(2) in the use of disease emergence period increase bactericide, it is popular effectively to control disease.But using of this utilization decrement bactericide, the amount of application of bactericide does not have inhibitory action to the growth of Trichoderma, and the eugenol reducing application amount 10%, the mandipropamid reducing application amount 35%, both effectively controlled the popular of capsicum epidemic disease, reduce again residual in soil and capsicum product of agricultural chemicals, guaranteed the safety of environment and product.
(3) the inventive method is reasonable in design, and is easy and simple to handle, and input and operating cost are lower, is fit to large-scale operation.Applying of the method has facilitation for capsicum health, safety in production and sustainable agriculture development.
Description of drawings
Fig. 1 is that wooden removing mildew mixes the rear field of the use capsicum epidemic disease incidence of disease, disease index and control efficiency with the mandipropamid of various dose; Wherein, figure A-C is respectively dispenser the disease incidence of disease, disease index and control efficiency measurement result after 15 days.
Fig. 2 is that wooden removing mildew mixes the rear field of the use capsicum epidemic disease incidence of disease, disease index and control efficiency with the eugenol of various dose; Wherein, figure A-C is respectively dispenser the disease incidence of disease, disease index and control efficiency measurement result after 15 days.
Fig. 3 is that two kinds of bactericide are respectively to the inhibition of phytophthora blight of pepper PC004 and trichoderma asperellum Thz01; Wherein, figure A and figure C are respectively phytophthora blight of pepper PC004 the PDA flat board cultivation that contains 65 μ g/ml mandipropamids or the former medicine of 1.35 μ g/ml eugenols 7 days; Figure B and figure D are respectively trichoderma asperellum Thz01 and cultivated 3 days at the PDA flat board that contains 65 μ g/ml mandipropamids or the former medicine of 1.35 μ g/ml eugenols.
Embodiment
Trichoderma asperellum Thz01 bacterial strain
Trichoderma asperellum (Trichoderma asperellum) Thz01 bacterial strain is to collect from the capsicum rhizosphere soil soil of mountain vegetables garden, Linan morbidity, the biological property of this bacterial strain is: bacterium colony is 25 ℃ of lower cultivations on the PDA solid culture medium, expanded to 9cm in 3-4 days, initial stage white is sparse, mycelia is along the media surface sprawl growth, the green spore district that produces of rear formation, reverse side white, the mycelia tool every; Conidium is spherical, and is subsphaeroidal, single closely colourless, is pistac during gathering, and wall is smooth.Upright, the ampulla shape of bottle, width 3.0-5.6 μ m.The upper conidium of CMD is spherical to inferior spherical, 3.8-5.9 * 2.8-4.8 μ m.Morphological feature meets trichoderma asperellum bacterium feature.According to ITS rDNA (SEQ ID NO.1) and transcription factor 1a (tef 1-a) sequence analysis, the most consistent with the trichoderma asperellum bacterium sequence of pattern among the GenBank and confirmation.These confirm that this bacterial strain is trichoderma asperellum (Trichoderma asperellum).
The preservation of trichoderma asperellum Thz01 bacterial strain, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date: on August 13rd, 2012, preserving number: CGMCCNo.6422.
The preparation of embodiment 1 wooden removing mildew
(1) preparation trichoderma asperellum Thz01 spore suspension: trichoderma asperellum Thz01 is inoculated in the PDA medium, in 25 ℃ of lower cultivations 5-6 days; After cultivation is finished, spore is scraped in the aqua sterilisa, preparation obtains trichoderma asperellum Thz01 spore suspension, and (viable bacteria concentration is 10
8Individual/mL).
(2) prepare wooden removing mildew: trichoderma asperellum Thz01 spore suspension is inoculated in the wheat (feed wheat) that boils sterilization, in time stir between culture period, at illumination in 12 hours and 12 hours dark alternate cultures after 18 days, produce a large amount of spores on the inducing culture, spore is prepared into wooden removing mildew together with spore.
In every kilogram of wheat, the consumption of trichoderma asperellum Thz01 spore suspension is 3mL.
The screening of embodiment 2 bactericide
13 kinds of bactericide that are usually used in selecting to produce preventing and treating capsicum epidemic disease comprise for screening test: 80% phosethyl-Al (the good magnificent chemical industry in Zhejiang Co., Ltd); 250g/L mandipropamid (Switzerland is just reaching first crop protection Co., Ltd); The 250g/L Amici reaches (Britain Syngenta Co., Ltd); 72% cymoxanil mancozeb (du pont company); 50% dimethomorph (the biochemical Co., Ltd of Shijiazhuang Yi Nuo); 69% dimethomorph mancozeb (the biochemical Co., Ltd of Ke Lilong); 72% metalaxyl (Jiangsu Kai Lier biochemical corp); 50% second aluminium MnZn (favorable to the people chemical industry Co., Ltd); 46% Kocide SD (du pont company); 75% tpn (favorable to the people chemical industry Co., Ltd); 0.3% eugenol (Asia, Baoding reaches chemical industry Co., Ltd); 20% flumorph (Shenyang Kechuang chemicals Co., Ltd); The 66.8% the third gloomy iprovalicarbs (German Bayer Cropscience N.V.).
The screening of medicament is divided into following three processes:
(1) different bactericide are to the inhibitory action of trichoderma asperellum Thz01 mycelial growth
Adopt the mycelial growth rate determination method to measure different bactericide to the inhibitory action of trichoderma asperellum Thz01 mycelial growth, filter out the medicament that bactericide does not suppress trichoderma asperellum Thz01 mycelia.
Method: at first measure the concentration range that each Medicaments on Trichoderma viride mycelial growth begins inhibiting rate, then around this concentration 5 series concentration setting up and down.Reagent agent respectively with being made into certain density mother liquor, is drawn needed volume according to the concentration of having set, join in the triangular flask.Triangular flask contains the PDA medium that has melted, and usually is incorporated as suitablely about 45 ℃, pours respectively the culture dish of sterilization behind the mixing into (among the Φ=90mm), take the PDA flat board that adds the equal-volume sterile water as contrast.(Φ=5mm) cut at 2 days Trichoderma bacterium cake of PDA medium cultivation is inoculated in the PDA medium central authorities of pastille, places dark culturing in 25 ℃ of constant incubators, and each concentration is processed and repeated 3 times with card punch.Measured afterwards the colony diameter of strains tested on variable concentrations pastille medium in 3 days, calculate each chemicals treatment to the growth inhibition ratio of bacterium colony expansion with comparing, analyze more different bactericide to the impact for examination germ mycelial growth, calculate the virulence regression equation of medicament.The theoretical value of drug concentration when being 1% according to virulence regression equation calculating inhibiting rate, on the basis of this theoretical value the drug concentration gradient is set, under identical training method and condition, determine not suppress fully the measured value of mycelial growth, each concentration is processed and is repeated 3 times.
The result shows, in 13 kinds of bactericide, the relatively poor bactericide of inhibition to trichoderma asperellum Thz01 mycelial growth has phosethyl-Al, mandipropamid, cymoxanil mancozeb, dimethomorph, dimethomorph mancozeb, Kocide SD and the third gloomy iprovalicarb, and its virulence approaches or surpasses 1000 μ g/ml.To the inhibition of trichoderma asperellum Thz01 mycelial growth preferably bactericide tpn and eugenol are arranged, its EC50 value is respectively 4.15 μ g/ml and 2.62 μ g/ml (table 1).
The theoretical value of medicament is found with the drug concentration measured value that does not suppress mycelial growth fully when being 1% by comparing inhibiting rate: theoretical value and measured value are consistent substantially, if can filter out the suitable bactericide of using with, then measured value can be used as the reference value (table 2) of field test Chinese medicine application concentration.
Table 1. medicament is to the inhibition of trichoderma asperellum Thz01 mycelial growth
Table 2. does not suppress theoretical value and the measured value of trichoderma asperellum Thz01 mycelial growth
(2) different bactericide bacterium are to the inhibitory action of spore germination
Scraping prepares spore suspension at 8 days wooden mould spore of PDA medium growth, and transferring to concentration is 10
6Individual/ml.Measure the concentration range that each Medicaments on Trichoderma viride spore germination begins inhibiting rate, then around this concentration 5 series concentration setting up and down.Reagent agent is made into respectively certain density mother liquor, draws needed volume according to the concentration of having set, join in the triangular flask that contains 45 ℃ of left and right sides PDA medium.After cooling, draw 100 μ l spore suspensions, drip in the culture dish center, be coated with the rod coating evenly with triangular glass.Behind 25 ℃ of lower cultivation 12h, microscopically is observed the Germination suppression rate of also calculating.Same, draw the calculating virulence regression equation.Drug concentration when being 1% according to virulence regression equation calculating inhibiting rate arranges the drug concentration gradient on the basis of this theoretical value, under identical training method and condition, determines not suppress fully the measured value of spore germination, and each concentration is processed and repeated 3 times.
The result shows that in 13 kinds of bactericide, Kocide SD and eugenol all do not have inhibition to spore germination; Three second aluminium phosphates and mandipropamid are poor to the inhibition of trichoderma asperellum Thz01; A Ximida, dimethomorph mancozeb, second aluminium MnZn, tpn and the third gloomy iprovalicarb are obvious to the inhibition of trichoderma asperellum Thz01 spore germination, and its EC95 concentration is all below 0.05 μ g/ml (table 3).
The theoretical value of medicament is found with the drug concentration measured value that does not suppress spore germination fully when being 1% by comparing inhibiting rate: theoretical value and measured value are consistent substantially, if can filter out the suitable bactericide of using with, then measured value can be used as the reference value (table 4) of field trial Chinese medicine application concentration.
Table 3. medicament is to the inhibition of trichoderma asperellum Thz01 spore germination
Annotate: Kocide SD and eugenol do not have inhibitory action to spore germination; The Amici reaches, dimethomorph mancozeb, second aluminium MnZn, tpn and the third gloomy iprovalicarb have the strong inhibition effect to spore germination.More than 7 kinds of medicaments in table, do not list.
Table 4. does not suppress theoretical value and the measured value of trichoderma asperellum Thz01 spore germination
A represents that bactericide has strong inhibitory action to spore germination, and in the extremely low situation of concentration (c＜0.01), inhibiting rate is still more than 95%;
B represents that bactericide does not have inhibitory action to spore germination, and in the dense situation (c＞1000), inhibiting rate is still below 5%.
(3) different bactericide bacterium are to the inhibitory action of Sporulation
More than two experiments drawn respectively every kind of medicament to the mycelia of trichoderma asperellum Thz01 and the measured value of spore germination unrestraint concentration, by the comparison of these two values, select less value to make concentration and further detect bactericide produces spore to trichoderma asperellum Thz01 inhibitory action.Get the Trichoderma mycelia piece of cultivating a day, the mycelia surface is placed in the sterile petri dish up, adds the bactericide liquid until surpass mycelia piece surface, contrast processes to add the equivalent sterile water, then place 25 ℃ of incubators, behind the placement 24h, examine under a microscope and produce spore quantity.
The result shows that trichoderma asperellum Thz01 is produced the relatively poor bactericide of spore inhibition mandipropamid, metalaxyl, flumorph and eugenol, and the mycelium surface sporulation quantity of processing through these bactericide is substantially identical with contrast; To trichoderma asperellum Thz01 produce the spore inhibition preferably bactericide have that the Amici reaches, dimethomorph mancozeb, tpn, second aluminium MnZn, Kocide SD and three second aluminium phosphates, substantially do not produce spore (table 5) through the mycelium surface of these chemicals treatment.
Table 5. medicament is to the inhibition of trichoderma asperellum Thz01 Sporulation
Produce conidium on the+expression mycelium, plus sige more multilist shows that the conidium of generation is more
Above-mentioned 3 result of the tests show that mycelial growth, spore germination and product spore are different to the susceptibility of bactericide.The concentration that eugenol does not suppress trichoderma asperellum Thz01 mycelial growth is 1.5 μ g/ml, and the concentration that does not suppress to produce spore is 6 μ g/ml, and concentration is when reaching 200 μ g/ml, to spore germination also unrestraint.The concentration that mandipropamid does not suppress trichoderma asperellum Thz01 mycelial growth is 250 μ g/ml, and the concentration that does not suppress spore germination is 80 μ g/ml, and the concentration that does not suppress to produce spore is 160 μ g/ml.
Eugenol recommendation amount aborning is 30g/ mu, is watered 60L (being that concentration is 1.5 μ g/ml), this shows, directly uses the concentration of using in producing also not suppress mycelial growth, spore germination and the product spore of trichoderma asperellum Thz01.Yet, mandipropamid recommendation concentration aborning is 12g/ mu, is watered 30L (100 μ g/ml), under this concentration, mandipropamid has to a certain extent inhibition to the mycelium growth and sporulation unrestraint effect of trichoderma asperellum Thz01 to spore germination.
Screening test is used in embodiment 3 wooden removing mildews and bactericide field with
At first divide the residential quarter, field, arrange respectively blank district, medicament control treatment district and wood mould-medicament mixed processing district.During the capsicum field planting, wood mould-medicament mixed processing district is transplanted to pepper seedling in the planting hole jointly together with wooden removing mildew, the 1.5-2 gram is used in every strain; Blank district and contrast medicament district do not use wooden removing mildew.With filling with root inoculation method inoculation phytophthora spore suspension, suspension concentration is 107/ml after one month, every strain capsicum inoculation 3ml.Inoculate after 3 days, wood mould-medicament mixed processing district sprays bactericide according to following 6 amount of application gradients respectively: mandipropamid 6g/ mu, 6.6g/ mu, 7.2g/ mu, 7.8g/ mu, 8.4g/ mu, 9g/ mu, every mu is watered 30L; Eugenol 24g/ mu, 25g/ mu, 26g/ mu, 27g/ mu, 28g/ mu, 29g/ mu, every mu is watered 60L.Medicament control treatment district is according to application concentration mandipropamid 12g/ mu in producing, and every mu is watered 30L and eugenol 30g/ mu, and every mu is watered 60L and sprays bactericide.Each is processed and repeats 3 times, and each repeats 12 strains.The spray medicine is after 15 days, and the strain incidence inoculated in record.
(1) respectively records afterwards incidence in 15 days with the spray medicine at the initial stage of a disease, and calculate the incidence of disease and disease index.
Stage division: 0 grade: asymptomatic; 1 grade: the slight blackening of seedling rhizome section, blade are not wilted or restorability is wilted; 2 grades: the blackening of seedling rhizome section reaches 1-2cm, and the blade irrecoverability is wilted, and the lower blade idol has and comes off; 3 grades: the blackening of seedling rhizome section surpasses 2cm, and blade is obviously wilted or fallen leaves obviously; 4 grades: the seedling rhizome section blackening contracting of hanging, all fallen leaves or plant wither except growing point; 5 grades: plant is withered; The incidence of disease and disease index calculate by formula (1), formula (2):
Diseased plant rate (the %)=total strain numerical expression of 100 * diseased plant number/investigation (1)
Disease index=100 * ∑ (the sick numbers of sheets at different levels * typical values at different levels)/(investigating total number of sheets * highest typical value) formula (2)
(2) calculate drug effect
According to the disease index of each residential quarter, calculate medicament control treatment district and wood mould-control efficiency in medicament mixed processing district.Control efficiency is calculated by formula (3):
Control efficiency (%)=100 * (contrast disease index-processing disease index)/contrast disease index
Formula (3)
According to above result of the test, calculate wood mould-medicament mixes control efficiency and the control efficiency of bactericide use only.
Field biological and ecological methods to prevent plant disease, pests, and erosion result of the test shows, for trichoderma asperellum Thz01, when the mixing amount of application of 250g/L mandipropamid suspending agent (Switzerland is just reaching first crop protection Co., Ltd) is 7.8g/ mu, is watered 30L (active ingredient 1.95g; Active ingredient concentration 65 μ g/ml) time, control efficiency is 84.71%, is better than the preventive effect 84.06% (Fig. 1) of the field of above-mentioned independent chemicals treatment.Under this consumption, mandipropamid reduces 4.2g/ mu than normal usage amount, and namely the decrement bactericide uses 35%.Among Fig. 1, figure A is dispenser disease incidence of disease measurement result after 15 days, and wooden removing mildew+7.8 g/acre mandipropamid control efficiency is similar to independent chemical control efficiency; Figure B is dispenser disease index measurement result after 15 days, and wooden removing mildew+7.8 g/acre mandipropamid control efficiency is similar to independent chemical control efficiency; Figure C is that dispenser prevents and treats the statistical effect result after 15 days, and wooden removing mildew+7.8 g/acre mandipropamid control efficiency is similar to independent chemical control efficiency.
When 0.3% soluble eugenol solvent (Asia, Baoding reaches chemical industry Co., Ltd and produces) amount of application 27g/ mu, be watered 60L (active ingredient 0.081g; Active ingredient concentration 1.35 μ g/ml) time, control efficiency is 87.45%, is better than the preventive effect 86.82% (Fig. 2) of the field of above-mentioned independent chemicals treatment.Under this consumption, eugenol reduces 3g/ mu than normal usage amount, and namely the decrement bactericide uses 10%.Among Fig. 2, figure A is dispenser disease incidence of disease measurement result after 15 days, and wooden removing mildew+27 g/acre eugenol control efficiency is similar to independent chemical control efficiency; Figure B is dispenser disease disease index measurement result after 15 days, and wooden removing mildew+27 g/acre eugenol control efficiency is similar to independent chemical control efficiency; Figure C is that dispenser prevents and treats the statistical effect result after 15 days, and wooden removing mildew+27 g/acre eugenol control efficiency is similar to independent chemical control efficiency.
Wood removing mildew and decrement bactericide mix under the service condition, because the usage amount of two kinds of bactericide all is lower than the amount of independent use bactericide, therefore, under this disease control condition, wooden removing mildew is not subjected to the impact of bactericide.
Embodiment 4 bactericide are to the inhibition test of phytophthora PC004 and trichoderma asperellum Thz01
To contain concentration be that dull and stereotyped (Φ=90mm) is take the PDA flat board that adds the equal-volume sterile water as contrast for the PDA of 65 μ g/ml mandipropamids and 1.35 μ g/ml eugenols in preparation respectively.(Φ=5mm) contain medicament PDA medium central authorities from cultivating 2 days Trichoderma bacterium colony edge or cut the bacterium cake from the phytophthora blight of pepper bacterium colony edge of cultivating 5 days, being inoculated in places dark culturing in 25 ℃ of constant incubators with card punch.Each is processed and repeats 3 times.Measure colony diameter after trichoderma asperellum Thz013 days; Measure colony diameter after phytophthora blight of pepper PC0047 days.
The result shows, phytophthora blight of pepper PC004 is after the PDA flat board that contains the former medicine of 65 μ g/ml mandipropamids is cultivated 7 days, it is fully suppressed to grow, and the growth of trichoderma asperellum Thz01 is not suppressed fully, cultivates its bacterium colony size and contrast consistent (Fig. 3) after 3 days.Same, phytophthora blight of pepper PC004 is after the PDA flat board that contains the former medicine of 1.35 μ g/ml eugenols is cultivated 7 days, and it is fully suppressed grow, and the growth of trichoderma asperellum Thz01 is not suppressed fully, cultivate after 3 days its bacterium colony big or small with contrast consistent.
Among Fig. 3, figure A, phytophthora blight of pepper PC004 is after the PDA flat board that contains the former medicine of 65 μ g/ml mandipropamids is cultivated 7 days, and it is fully suppressed to grow; A1, A2, A3 are repetitions; CK is contrast.Figure B, trichoderma asperellum Thz01 are after the PDA flat board that contains the former medicine of 65 μ g/ml mandipropamids is cultivated 3 days, and the bacterium colony size is consistent with contrast; B1, B2, B3 are repetitions; CK is contrast.Figure C, phytophthora blight of pepper PC004 is after the PDA flat board that contains the former medicine of 1.35 μ g/ml eugenols is cultivated 7 days, and it is fully suppressed to grow; C1, C2, C3 are repetitions; CK is contrast.Figure D, trichoderma asperellum Thz01 are after the PDA flat board that contains the former medicine of 1.35 μ g/ml eugenols is cultivated 3 days, and the bacterium colony size is consistent with contrast; D1, D2, D3 are repetitions; CK is contrast.
Claims (9)
- One kind in the pepper planting process wooden removing mildew and bactericide use the method for control capsicum epidemic disease with, the plantation of capsicum comprises sowing, grows seedlings, transplant planting, cultivate and gather, and it is characterized in that described method comprises:When (1) sowing, pepper seed is mixed rear sowing with wooden removing mildew; Perhaps, when the Hot Pepper Seedling transplant planting, use wooden removing mildew;(2) during the pepper cultivation, before disease occurs or initial stage of origination use bactericide;Wherein, the active bacterial strain in the described wooden removing mildew is trichoderma asperellum (Trichodermaasperellum) Thz01, and the preserving number of trichoderma asperellum Thz01 is CGMCC No.6422;Described bactericide is one or both in eugenol and the mandipropamid.
- 2. method according to claim 1 is characterized in that, during pepper cultivation, 0.5-1 used wooden removing mildew in individual month around the capsicum rhizosphere before disease occurs.
- 3. method according to claim 1 and 2 is characterized in that, the consumption of described wooden removing mildew is the 1.5-2g/ strain.
- 4. method according to claim 1, it is characterized in that, described wooden removing mildew prepares by the following method: trichoderma asperellum Thz01 spore suspension is inoculated in the wheat that boils sterilization, in illumination in 12 hours and 12 hours dark alternate culture 15-20 days, makes wooden removing mildew.
- 5. method according to claim 4 is characterized in that, the viable bacteria concentration of described trichoderma asperellum Thz01 spore suspension is 10 7-10 8Individual/mL.
- 6. method according to claim 4 is characterized in that, in every kilogram of wheat, the consumption of described trichoderma asperellum Thz01 spore suspension is 2-4mL.
- 7. method according to claim 4 is characterized in that, in time stirs wheat between culture period.
- 8. method according to claim 1 is characterized in that, described bactericide is eugenol; In every mu of ground, the consumption of eugenol active ingredient is 0.078-0.084g.
- 9. method according to claim 1 is characterized in that, described bactericide is mandipropamid; In every mu of ground, the consumption of mandipropamid active ingredient is 1.8-2.1g.
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