CN102719364B - Trichoderma harzianum strain and application in prevention and control of phytophthora capsici Leonian thereof - Google Patents
Trichoderma harzianum strain and application in prevention and control of phytophthora capsici Leonian thereof Download PDFInfo
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Abstract
The invention discloses a trichoderma harzianum strain and application in prevention and control of phytophthora capsici Leonian thereof. The trichoderma harzianum (HAN-12) provided by the invention, the preservation number of which is CGMCC No. 5989, can be used for restraining pathogenic bacteria, promoting the growth of a hot pepper plant, and preventing and controlling the phytophthora capsici Leonian of the hot pepper plant. The trichoderma harzianum strain can have a wide application prospect in the sustainable agricultural development.
Description
Technical field
The present invention relates to a strain trichoderma harzianum strain and the application in the prevention and control capsicum epidemic disease thereof.
Background technology
Capsicum epidemic disease is a kind of crushing fungal disease that betides on the capsicum, and this disease can cause the various crop morbidity of different areas, causes a large amount of financial losses.The typical symptom characteristic of capsicum epidemic disease: plant is infected the back and rhizome portion tissue just took place in several days rots until a large amount of plant death.Up to the present, in the production also not to the good resistant variety of capsicum epidemic disease.
At present, in the control to capsicum epidemic disease, chemical prevention occupies consequence both at home and abroad.Higher and the easy contaminate environment of chemical prevention cost, and pathogen easily produces resistance even resistance to sterilant.So biological control becomes focus and focus that people pay close attention to gradually.
Summary of the invention
The purpose of this invention is to provide a strain trichoderma harzianum strain and the application in the prevention and control capsicum epidemic disease thereof.
Trichoderma harziarum provided by the invention (Trichoderma harzianum) HNA-12, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 04 11st, 2012 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.5989.Trichoderma harziarum (Trichoderma harzianum) HNA-12CGMCC No.5989 is called for short trichoderma harziarum HNA-12.
Described trichoderma harziarum HNA-12 can be used for suppressing pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum).
Described trichoderma harziarum HNA-12 can be used for promoting the pepper plant growth.
Described trichoderma harziarum HNA-12 can be used for prevention and control pepper plant generation capsicum epidemic disease.
The present invention also protects a kind of product, and its active ingredient is described trichoderma harziarum HNA-12, the product of described product for having at least a function in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
The present invention also protects the application of trichoderma harziarum HNA-12 in preparing product; The product of described product for having at least a function in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
The present invention also protects spore liquid or the fermented product of described trichoderma harziarum HNA-12.
Described spore liquid specifically can be 5 * 10
6The spore suspension of individual spore/mL.
Described fermented product can be solid fermentation thing or liquid fermentate.
The preparation method of described liquid fermentate is specific as follows: collect trichoderma harziarum HNA-12 spore, be inoculated in the fermentor tank that contains seed culture medium, obtaining concentration after the fermentation is 2.5 * 10
8The liquid fermentate of CFU/mL (seed liquor); The condition of described fermentation is: dissolved oxygen concentration about 20%, temperature 28-30 ℃, stirring velocity 200-250r/min, air flow are 10-15L/min.
Described seed culture medium (pH6.0-6.5) is made up of solute and water; The concentration of described solute in seed culture medium is as follows: wheat bran 2g/100mL, glucose 1.0g/100mL, sal epsom 0.5g/100mL, potassium primary phosphate 0.3g/100mL, calcium chloride 0.3g/100mL.
The preparation method of described solid fermentation thing is specific as follows: described liquid fermentate and solid medium are mixed (contain 1.5 * 10 in every gram mixture
6CFU trichoderma harziarum HNA-12), ferment then, air-dry to 7-10% then, obtain the solid fermentation thing; The condition of described fermentation is: temperature 28-30 ℃, and relative air humidity 95-100%, substratum thickness 5-7cm, incubation time 5-7 days.
Described solid medium mixes wheat bran and rice husk and water and obtains, and the mass ratio of wheat bran and rice husk is 7:3, and the water content of solid medium is about 70%, pH6.0-6.5; 121 ℃ of sterilization 30min.
The application of described spore liquid or described fermented product, at least a in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
The present invention also protects a kind of product, and its active ingredient is described spore liquid or described fermented product; The product of described product for having at least a function in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
The present invention also protects described spore liquid or the application of described fermented product in preparing product; The product of described product for having at least a function in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
Trichoderma harziarum HNA-12 provided by the invention, find through indoor face-off antagonistic experiment, potted plant control experiment and daejeon prevention test, this bacterial strain has good fungistatic effect to multiple pathogenic bacteria (particularly phytophthora capsici), and utilizes Trichoderma preparation controlling plant diseases to have, product non agricultural chemical residuum nontoxic to people and animals, advantage such as free from environmental pollution.The present invention has a extensive future in sustainable agriculture development.
Social benefit of the present invention: apply the biological pesticide balanced growth of can preserving the ecological environment; objectionable impurities is residual in raising quality of agricultural product and the reduction agricultural-food; strengthen the market competitiveness of agricultural-food, increase farmers' income, and then strengthen the sustainability of agriculture production.
Economic benefit of the present invention: apply the usage quantity that biotechnological formulation can reduce chemical pesticide, can improve the epidemic prevention and control effect, reduce the control cost; Can effectively realize agricultural product security production and significantly reduce the agricultural-food pesticide residue, promote the economic value added of agricultural-food, enlarge China's agricultural byproducts export markets; Advance the development of pollution-free industry, increasing farmers' income has important prograding.
Description of drawings
Fig. 1 is the dull and stereotyped form of cultivating of the PDA of bacterial strain HNA-12.
Fig. 2 is conidiophore and the conidium of bacterial strain HNA-12.
Fig. 3 is that the photo after 72 hours is cultivated in trichoderma harziarum HNA-12 and phytophthora capsici face-off.
Fig. 4 is the hyperparasitism of the phytophthora capsici of trichoderma harziarum HNA-12.
Fig. 5 is the hyperparasitism of the phytophthora capsici of trichoderma harziarum HNA-12.
Fig. 6 is that trichoderma harziarum HNA-12 volatility metabolite is to the restraining effect of phytophthora capsici on flat board.
Fig. 7 is that the non-volatile metabolite of trichoderma harziarum HNA-12 is to the restraining effect of phytophthora capsici on flat board
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Potato dextrose agar (PDA substratum): potato 200g, glucose 15g, agar 20g, distilled water 1000ml; Standby behind 121 ℃ of sterilization 20min.
Trichoderma half selective medium (TSM substratum): MgSO
4(7H
20) 0.2g, K
2HPO
40.9g, KCl 0.15g, NH
4NO
31.0g, glucose 3.0g, rose-red 0.15g, 60% fenaminosulf wettable powder, 0.3 gram, PCNB 0.2g, agar 20g, distilled water 950ml; 121 ℃ of sterilization 20min are standby.
Phytophthora infestans (Phytophthora infenstans): the public can obtain from China Agricultural University; Reference: Daayf F, Adam L and Fernando WGD (2003) Comparative screening of bacteria for biological control of potato late blight (strainUS-8) using in vitro, detached leaves, and whole plant testing systems.Canadian Journal of Plant Pathology, 25:276 – 284.
Phytophthora capsici (Phytophthora capsici): the public can obtain from China Agricultural University; Reference: Ahmed AS, Sanchez CP and Candela ME (2000) Evaluation of induction of systemic resistance in pepper plants (Capsicum annuum) to Phytophthora capsici using Trichoderma harzianum and its relation with capsidiol accumulation.European Journal of Plant Pathology 106:817 – 824.
Capsicum pine root fungus (Fusarium oxysporum): the public can obtain from China Agricultural University; Reference: Liu Liyun. Liu Xiaolin, Liu Zhiheng, etc. capsicum pine root fungus biological characteristic research. Agricultural University Of Shenyang's journal, 2007,38 (1): 54-58.
It is red that used capsicum variety is China among the embodiment: Hebei province's Dingzhou City uncle fort vegetables seed stock breeding station.
Spore suspension among the embodiment is all collected spore and is resuspended in water and obtains.
The acquisition of embodiment 1, trichoderma harziarum HNA-12 and evaluation
One, the acquisition of bacterial strain HNA-12
1, sample collecting
Sample is for gathering from top layer, Earthquake of Anyang station in Henan capsicum farm crop field rhizosphere soil.
Push the topsoil of field 3-5cm aside with the equipment that fetches earth, the root system of soil together with plant dug out, install with polyethylene plastic bag, take back the laboratory and separate.
2, the acquisition of bacterial strain HNA-12
The root system that adheres to soil is air-dry slightly, pat root system, the superincumbent soil of adhesion is come off, use the sterilized water gradient dilution, draw the 0.1mL diluent respectively and splash on the TSM culture medium flat plate, even with the L shaped spreading rod coating of sterilization, place 25-28 ℃ constant incubator to cultivate 3-4 days.The picking form is transferred to like single bacterium colony of Trichoderma carries out purifying and cultivates on the PDA flat board, number after the mirror mirror is tentatively regarded as Trichoderma, and be saved on the PDA inclined-plane.
Obtain a strain bacterial strain, with its called after HNA-12.
Two, the evaluation of bacterial strain HNA-12
20 ℃ of colony diameters of cultivating 5d are 9.0cm on the PDA substratum, and the speed of growth is very fast, and spider's thread shape is to ulotrichy, and white is green owing to produce the spore surface under the scattered light, and the back side is colourless, and the later stage is because sporulation quantity ambassador bacterium colony is green slightly; The tree-shaped conidiophore of multi-branched forms quite loose flora; The wide 4-5 μ of major branch m, side shoot is many, forms pyramid; Adnation bottle stalk can reach 5, becomes to intend colyliform arrangement or single along little side shoot irregular alignment; The contracting of slightly hanging of adnation bottle metulae portion, expand at the middle part, and upwards gradually narrow one-tenths bottle obstructs 5-7 * 3-3.5 μ m; It is relative long and very thin with atypical bottle of stalk that give birth on the top, 13-18 * 2.5-3.5 μ m; Bottle stalk mostly with wide-angle on its carrier give birth to, slightly bend towards the top sometimes; Phialospore is the spheric conidium head, the subsphaeroidal or obovate of conidium, and wall is smooth, light green, 2.8-3.2 * 2.5-2.8 μ m.Fig. 1 is the dull and stereotyped form of cultivating of the PDA of bacterial strain HNA-12.Fig. 2 is conidiophore and the conidium of bacterial strain HNA-12.
Three, the preservation of bacterial strain HNA-12
By the evaluation of step 2, bacterial strain HNA-12 belongs to trichoderma harziarum (Trichoderma harzianum).
Trichoderma harziarum (Trichoderma harzianum) HNA-12, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 04 11st, 2012 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.5989.Trichoderma harziarum (Trichoderma harzianum) HNA-12CGMCC No.5989 is called for short trichoderma harziarum HNA-12.
The restraining effect of embodiment 2, the pathogenic bacteria of trichoderma harziarum HNA-12
Experimental group: in the cultivation (vertical range at two bacterium cake centers is 3cm, and the diameter of two bacterium cakes is 5mm) that stands facing each other of same PDA flat board inoculation trichoderma harziarum HNA-12 bacterium cake and pathogenic bacteria bacterium cake; Organize in contrast with the PDA flat board of only inoculating pathogenic bacteria bacterium cake; Measure the colony diameter (longest diameter and the mean value of short diameter) of pathogenic bacteria behind 25 ℃ of dark culturing certain hours and calculate inhibiting rate.The colony diameter * 100% of inhibiting rate=(colony diameter of the colony diameter of control group pathogenic bacteria-experimental group pathogenic bacteria)/control group pathogenic bacteria.Every kind of pathogenic bacterium arrange three re-treatments, and contrast arranges three re-treatments, results averaged.
Pathogenic bacteria is respectively phytophthora capsici, phytophthora infestans and capsicum pine root fungus.
The results are shown in Table the mean value of three tests of 1().The photo that trichoderma harziarum HNA-12 and phytophthora capsici face-off were cultivated after 72 hours is seen Fig. 3.The result shows that trichoderma harziarum HNA-12 is all inhibited to each pathogenic bacteria.
Trichoderma harziarum HNA-12 is to the bacteriostasis rate (%) of each pathogenic bacteria behind each incubation time of table 1
| Pathogenic bacteria | 24 hours | 36 hours | 48 hours | 60 hours | 72 hours | 84 hours |
| Phytophthora capsici | 20.5 | 42.1 | 47.6 | 56.7 | 66.5 | 75.0 |
| Phytophthora infestans | 15.5 | 42.5 | 54.5 | 59.8 | 69.2 | 74.3 |
| The capsicum pine root fungus | 15.4 | 31.3 | 50.5 | 65.3 | 69.7 | 72.5 |
The greenhouse biocontrol effect experiment of embodiment 3, trichoderma harziarum HNA-12
Pepper seed vernalization behind 55 ℃ of 30min that hot water treatment of seeds, the chitting piece of selecting the growing way unanimity is seeded in culturing pot, 20 in every alms bowl; (25-30 ℃) cultivated and grown 4 true leaves until pepper plant in the greenhouse, and following operation (every group of 3 culturing pots) is carried out in grouping:
Experimental group: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL trichoderma harziarum HNA-12 spore suspension
6Individual/mL) (spore concentration is 5 * 10 with 10mL phytophthora capsici spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe;
Control group: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL phytophthora capsici spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe.
30 days " Invest, Then Investigate " sickness rate (capsicum epidemic disease classification investigation standard sees Table 2) of inoculating spores suspension also calculate disease index.
Table 2 capsicum epidemic disease classification investigation standard
| Sick level | Grade scale |
| 0 | Health, the rhizome place is asymptomatic, and the area that rots is less than 1%; |
| 1 | The rhizome base portion has the water soaking mode scab, or dark green little scab, and the part of rotting is around stem week 1-25%; |
| 2 | The rhizome base portion brown of rotting, the part of rotting is around stem week 26-50%; |
| 3 | The rhizome base portion rots brown to Vandyke brown, and the part of rotting has the contracting of hanging slightly around stem week 51-75%; |
| 4 | The rhizome base portion rots dark brown to black, and the part of rotting is around stem week 76-89%, the contracting of obviously hanging; |
| 5 | The rhizome base portion black that rots, the part of rotting is around stem week more than 90%, the contracting of seriously hanging. |
The results are shown in Table the mean value of three tests of 3().The result shows that the capsicum epidemic disease of trichoderma harziarum HNA-12 has significant biocontrol effect.
The greenhouse prevention effect of the capsicum epidemic disease of table 3 trichoderma harziarum HNA-12
| Handle | Diseased plant rate (%) | Disease index | Relative control effect (%) |
| Experimental group | 15.1 | 1.6 | 64.0 |
| Control group | 39.5 | 4.5 | ----- |
The short living ability of embodiment 4, the pepper plant of trichoderma harziarum HNA-12
Pepper seed vernalization behind 55 ℃ of 30min that hot water treatment of seeds, the chitting piece of selecting the growing way unanimity is seeded in culturing pot, 20 in every alms bowl; (25-30 ℃) cultivated and grown 4 true leaves until pepper plant in the greenhouse, and following operation (every group of 3 culturing pots) is carried out in grouping:
Experimental group 1: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL trichoderma harziarum HNA-12 spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe;
Experimental group 2: every strain capsicum inoculation 10mL water; Vaccination ways is for to inject rhizosphere soil with water with syringe;
Experimental group 3: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL phytophthora capsici spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe.
Inoculating spores suspension is measured plant height, dry weight and the fresh weight of pepper plant after 30 days.The results are shown in Table the mean value of three tests of 4().The result shows that trichoderma harziarum HNA-12 can promote the pepper plant growth.
The growth-promoting functions of the pepper plant of table 4 trichoderma harziarum HNA-12
The control in field effect test of embodiment 5, trichoderma harziarum HNA-12
Pepper seed carries out following processing respectively then through 55 ℃ of 30min that hot water treatment of seeds:
Experimental group 1: with seed trichoderma harziarum HNA-12 spore suspension (5 * 10
6Individual/as mL) to soak seeding and seedling raising behind the 10min, be cultured to and be transplanted to the land for growing field crops, 200 milliliters of trichoderma harziarum HNA-12 spore suspensions (5 * 10 of every strain capsicum pouring after 10 days after growing 7 true leaves
6Individual/mL);
Experimental group 2: seed is soaked seeding and seedling raising behind the 10min with thiram solution (0.02g/mL), be cultured to and be transplanted to the land for growing field crops, every strain capsicum pouring 200 milliliters of thiram solution (0.02g/mL) after 10 days after growing 7 true leaves;
Control group: with the seed seeding and seedling raising behind the 10min that is soaked in water, be cultured to and be transplanted to the land for growing field crops after growing 7 true leaves, every strain capsicum is watered 200 ml waters after 10 days.
Statistics sickness rate and disease index, method is with embodiment 3.
The mean value that the results are shown in Table three tests of 5(of first year test), the mean value that the results are shown in Table three tests of 6(of second year test).The result shows that the capsicum epidemic disease of trichoderma harziarum HNA-12 has significant biocontrol effect.
The field control effect of the capsicum epidemic disease of table 5 trichoderma harziarum HNA-12 (test of first year)
The field control effect of the capsicum epidemic disease of table 6 trichoderma harziarum HNA-12 (test of second year)
The root colonization experiment of embodiment 6, trichoderma harziarum HNA-12
Pepper seed vernalization behind 55 ℃ of 30min that hot water treatment of seeds, the chitting piece of selecting the growing way unanimity is seeded in culturing pot, 20 in every alms bowl; (25-30 ℃) cultivated and grown 4 true leaves until pepper plant in the greenhouse, and following operation (every group of 3 culturing pots) is carried out in grouping:
Experimental group 1: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL trichoderma harziarum HNA-12 spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe;
Experimental group 2: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL trichoderma harziarum HNA-12 spore suspension
6Individual/mL) (spore concentration is 5 * 10 with 10mL phytophthora capsici spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe.
Inoculating spores suspension adopts plate dilution method (TSM substratum) to detect the content of trichoderma harziarum HNA-12 in the rhizosphere soil after 30 days, the results are shown in Table the mean value that 7(tests for three times).
The content (CFU) of trichoderma harziarum HNA-12 in the every gram rhizosphere soil of table 7
| Handle | 7 days | 14 days | 28 days |
| Experimental group 1 | 4.5×10 6 | 5.5×10 5 | 3.5×10 3 |
| Experimental group 2 | 3.5×10 5 | 2.8×10 4 | 4.5×10 2 |
Embodiment 7, trichoderma harziarum HNA-12 can promote the photosynthetic fluorescent effect of pepper plant
Pepper seed vernalization behind 55 ℃ of 30min that hot water treatment of seeds, the chitting piece of selecting the growing way unanimity is seeded in culturing pot, 20 in every alms bowl; (25-30 ℃) cultivated and grown 4 true leaves until pepper plant in the greenhouse, and following operation (every group of 3 culturing pots) is carried out in grouping:
Experimental group 1: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL trichoderma harziarum HNA-12 spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe;
Experimental group 2: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL trichoderma harziarum HNA-12 spore suspension
6Individual/mL) (spore concentration is 5 * 10 with 10mL phytophthora capsici spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe;
Experimental group 3: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL phytophthora capsici spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe;
Experimental group 4: every strain capsicum inoculation 10mL water; Vaccination ways is for to inject rhizosphere soil with water with syringe.
Inoculating spores suspension is looked into the photosynthetic of pepper plant and change in fluorescence situation after 30 days.The results are shown in Table the mean value of three tests of 8().
Photosynthetic and the fluorescence influence of the pepper plant of table 8 trichoderma harziarum HNA-12
| Handle | A | g s | Fv/Fm | ΦPSII | Fv′/Fm′ | q p |
| Experimental group 1 | 15.76 | 0.77 | 0.81 | 0.34 | 0.57 | 0.63 |
| Experimental group 2 | 10.83 | 0.33 | 0.78 | 0.18 | 0.42 | 0.47 |
| Experimental group 3 | 0.80 | 0.041 | 0.71 | 0.07 | 0.34 | 0.19 |
| Experimental group 4 | 18.10 | 0.69 | 0.79 | 0.32 | 0.52 | 0.60 |
The superparasitism ability of embodiment 8, the phytophthora capsici of trichoderma harziarum HNA-12
Cultivate trichoderma harziarum HNA-12 and phytophthora capsici in PDA flat board face-off, get the mycelia piece with the punch tool of 5mm, it is inoculated in lcm * 3cm celloyarn two ends on the PDA flat board, 25 ℃ of face-off cultivations respectively.After the contact of two mycelia, under Electronic Speculum, observe the hyperparasitism of the phytophthora capsici mycelia of trichoderma harziarum HNA-12.
Face-off was cultivated after 4 days, and trichoderma harziarum HNA-12 bacterium colony can cover the phytophthora capsici bacterium colony fully, and produces green spores in a large number.Under Electronic Speculum, observe Trichoderma phytophthora capsici is had tangible supercrescence, parasitic mode is various, with twine, parallel growth and mode such as intert parasitize on the germ mycelia, and produce trichoderma conidium, make the distortion of germ mycelia, make the fracture of germ silk at last, disintegrate.The hyperparasitism of the phytophthora capsici of trichoderma harziarum HNA-12 is seen Fig. 4 and Fig. 5.
The detection of embodiment 9, trichoderma harziarum HNA-12 metabolite
One, the detection of the non-volatile metabolite of trichoderma harziarum HNA-12
Experimental group: in the double-layer sterile glassine paper diaphragm of l0cm, the inoculation diameter is the trichoderma harziarum HNA-12 bacterium cake of 5mm in dull and stereotyped central authorities in PDA plate culture medium tiling slightly larger in diameter, and 25 ℃ of constant temperature culture to colony diameters reach 5cm; Throw off the glassine paper diaphragm, the inoculation diameter is 5mm phytophthora capsici bacterium cake in dull and stereotyped central authorities, 25 ℃ of constant temperature culture;
Control group: the inoculation diameter is 5mm phytophthora capsici bacterium cake in PDA plate culture medium central authorities, 25 ℃ of constant temperature culture.
Pick up counting from inoculation, measure colony diameter and calculate inhibiting rate behind the cultivation certain hour, method is with embodiment 2.The results are shown in Table 9.
Trichoderma harziarum HNA-12 volatility metabolite is seen Fig. 6 to the restraining effect of phytophthora capsici on flat board.
Two, the detection of the volatility metabolite of trichoderma harziarum HNA-12
Experimental group: the inoculation diameter is the trichoderma harziarum HNA-12 bacterium cake of 5mm in the dull and stereotyped central authorities of PDA, and 25 ℃ of constant temperature culture to colony diameters reach 5cm; Cover with double-deck slightly larger in diameter above it in the circular aseptic glassine paper of 10cm, equal sizes of top make-up, central authorities' inoculation diameter are the phytophthora capsici bacterium cake of 5mm, 25 ℃ of constant temperature culture;
Control group: cover with double-deck slightly larger in diameter in the circular aseptic glassine paper of 10cm above the dull and stereotyped central authorities of PDA, equal sizes of top make-up, central authorities' inoculation diameter are the phytophthora capsici bacterium cake of 5mm, 25 ℃ of constant temperature culture.
Pick up counting from inoculation, measure colony diameter and calculate inhibiting rate behind the cultivation certain hour, method is with embodiment 2.The results are shown in Table 9.
The non-volatile metabolite of trichoderma harziarum HNA-12 is seen Fig. 7 to the restraining effect of phytophthora capsici on flat board.
The metabolite of table 9 trichoderma harziarum HNA-12 is to the inhibiting rate (%) of phytophthora capsici
| Handle type | 24h | 48h | 72h |
| Non-volatile metabolite | 41.00 | 46.00 | 52.14 |
| The volatility metabolite | 8.30 | 13.70 | 19.93 |
The cultivation of embodiment 10, trichoderma harziarum HNA-12
One, the preparation of solid culture
1, slant strains
Adopt solid PDA substratum, trichoderma harziarum HNA-12 is inoculated on the inclined-plane PDA substratum, in incubator, cultivated 3-4 days under 28 ℃ of conditions.
2, eggplant bottle bacterial classification
Adopt solid PDA substratum, the trichoderma harziarum HNA-12 in the test tube slant substratum is inoculated in the eggplant bottle on the PDA substratum, under 28 ℃ of conditions, cultivated 3-4 days at shaking table.
3, liquid spawn
Seed culture medium (pH6.0-6.5) is made up of solute and water; The concentration of described solute in seed culture medium is as follows: wheat bran 2g/100mL, glucose 1.0g/100mL, sal epsom 0.5g/100mL, potassium primary phosphate 0.3g/100mL, calcium chloride 0.3g/100mL; 121 ℃ of moist heat sterilization 30min.
Trichoderma harziarum HNA-12 spore in the eggplant bottle is swept away with sterilized water, be inoculated in the medium-sized fermentor tank and (contain seed culture medium), cultivating and obtaining concentration is 2.5 * 10
8The seed liquor of CFU/mL.Fermenting process keeps dissolved oxygen concentration about 20%, and temperature 28-30 ℃, stirring velocity 200-250r/min, air flow are 10-15L/min.
4, solid medium is cultivated
Solid medium mixes wheat bran and rice husk and water and obtains, and the mass ratio of wheat bran and rice husk is 7:3, and the water content of solid medium is about 70%, pH6.0-6.5; 121 ℃ of sterilization 30min.
The seed liquor that 1 parts by volume step 3 is obtained and 9 parts by volume solid mediums mix and (contain 1.5 * 10 in every gram mixture
6CFU trichoderma harziarum HNA-12), transfer to the solid culture indoor cultivation after inoculation finishes.The control of culturing room temperature is at 28-30 ℃, and relative air humidity is controlled at 95-100%, substratum thickness be 6cm(5-7cm all can), incubation time is 6 days (5-7 days all can).After cultivation finished, with the culture natural air drying, the finished product water content was controlled at 7-10%, obtains trichoderma harziarum HNA-12 solid culture.
Two, the application of solid culture
Pepper seed vernalization behind 55 ℃ of 30min that hot water treatment of seeds, the chitting piece of selecting the growing way unanimity is seeded in culturing pot, 20 in every alms bowl; (25-30 ℃) cultivated and grown 4 true leaves until pepper plant in the greenhouse, and following operation (every group of 3 culturing pots) is carried out in grouping:
Experimental group: (spore concentration is 5 * 10 for every strain capsicum inoculation 10mL solid culture solution (be dissolved in the 0.1g solid culture 10mL water obtains) and 10mL phytophthora capsici spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe;
Control group: (spore concentration is 5 * 10 to every strain capsicum inoculation 10mL phytophthora capsici spore suspension
6Individual/mL); Vaccination ways is for to inject rhizosphere soil with spore suspension with syringe.
30 days " Invest, Then Investigate " sickness rate of inoculating spores suspension also calculate disease index (method is with embodiment 3).
The sickness rate 8.5% of experimental group, disease index are 1.2.The sickness rate of control group is 45%, and disease index is 6.3.
Claims (10)
1. trichoderma harziarum (Trichoderma harzianum) HNA-12, its deposit number is CGMCC No.5989.
2. the application of the described trichoderma harziarum HNA-12 of claim 1 in suppressing pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum).
3. the application of the described trichoderma harziarum HNA-12 of claim 1 in promoting the pepper plant growth.
4. the application of the described trichoderma harziarum HNA-12 of claim 1 in prevention and control pepper plant generation capsicum epidemic disease.
5. product, its active ingredient is the described trichoderma harziarum HNA-12 of claim 1; The product of described product for having at least a function in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
6. the application of the described trichoderma harziarum HNA-12 of claim 1 in preparing product; The product of described product for having at least a function in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
7. spore liquid or the fermented product of the described trichoderma harziarum HNA-12 of claim 1.
8. the application of the described spore liquid of claim 7 or fermented product describedly is applied as at least a in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
9. product, its active ingredient is the described spore liquid of claim 7 or fermented product; The product of described product for having at least a function in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
10. the described spore liquid of claim 7 or the application of fermented product in preparing product; The product of described product for having at least a function in following (1) to (3):
(1) suppresses pathogenic bacteria; Described pathogenic bacteria is at least a in the following microorganism: phytophthora infestans (Phytophthora infenstans), phytophthora capsici (Phytophthora capsici) and capsicum pine root fungus (Fusarium oxysporum);
(2) promote the pepper plant growth;
(3) prevention and control pepper plant generation capsicum epidemic disease.
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| CN103299811B (en) * | 2013-06-18 | 2014-07-23 | 江苏省农业科学院 | Method for enhancing soil waterflooding effect to control pepper phytophthora blight |
| CN103392744A (en) * | 2013-06-24 | 2013-11-20 | 浙江大学 | Trichoderma preparation for controlling pepper phytophthora blight and field tank mix pesticide containing the same |
| US9320283B2 (en) * | 2013-08-20 | 2016-04-26 | The United States of America, as repressented by The Secretary of Agriculture | Trichoderma asperellum to remediate Phytophthora ramorum-infested soil |
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| CN101503659B (en) * | 2009-03-11 | 2011-03-23 | 中国农业大学 | Trichoderma harzianum strain and use thereof |
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Effective date of registration: 20170122 Address after: 102101 Badaling Economic Development Zone, Yanqing District, East Ring Road, No. 8, No. Patentee after: The middle peasants lvkang Biotechnology Co. Ltd. (Beijing) Address before: 100193 Beijing Old Summer Palace West Road, Haidian District, No. 2 Patentee before: Chinese Agricultural Univ. |