CN104805022A - Fusarium oxysporum as well as microbial inoculum and application thereof - Google Patents
Fusarium oxysporum as well as microbial inoculum and application thereof Download PDFInfo
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Abstract
The invention discloses a strain Br-2 of Fusarium oxysporum and belongs to the field of biological prevention and treatment, and the preservation number is CGMCC No.10324. The invention also discloses a microbial inoculum prepared by using the Br-2 and a preparation method of the microbial inoculum. The microbial inoculum disclosed by the invention provides a new biological prevention and treatment method for efficiently preventing and treating orobanche aegyptiaca and orobanche cernua which are parasitic on tomatoes, tobaccos and sunflowers; secondarily, the microbial inoculum disclosed by the invention is remarkable in inhibition effect on the orobanche aegyptiaca and the orobanche cernua; the control efficiency in the field is as high as 84.75%; besides, the preparation method of the microbial inoculum disclosed by the invention is simple, low in cost and easy to popularize and apply; moreover, the microbial inoculum is free of pollution, nuisanceless, environment-friendly and capable of promoting the growth of crops.
Description
Technical field
The invention belongs to field of biological control, be specifically related to a kind of Fusarium oxysporum, also relate to microbiobacterial agent utilizing this Fusarium oxysporum to produce and its preparation method and application.
Background technology
Broomrape belongs to Orobanchaceae (Orobanchaceae) Orobanche (Orobanche), is annual root Parasitic Weeds, parasitizes on some higher plant root, without chlorophyll and root system, with sucker from derive nutrients and moisture in host plant body.Host is by after parasitism, and plant shows as poor growth, dwarfing, yellow, wilting or withered etc., causes crop yield to reduce and quality decline, farm crop can be caused time serious to have no harvest.The host range of broomrape is extensive, can colonize in the roots of plants such as composite family, pulse family, Solanaceae, Curcurbitaceae, Cruciferae, Cannabaceae, flax family (Linaceae), umbelliferae and Gramineae.The reproductivity of broomrape is strong, and every strain can produce about 100,000, seed; The broomrape seed survival phase is long, and in soil, the vitality of broomrape seed can keep 8-10.Under suitable humiture, seed can be sprouted all the year round, and within the whole breeding time of crop, broomrape all can be had to be unearthed, uneven, this characteristic becomes very difficult anti-malignant weed.In China, endanger larger broomrape mainly Sunflower Receptacle broomrape, branch broomrape and Egyptian broomrape.In recent years broomrape in Hebei, Shanxi, the Inner Mongol, the big area outburst of the ground such as Xinjiang, severe field parasitic rate reaches 100%, loses huge.
The method of current control broomrape mainly adopts farming, cultivation technique measure or uses chemical agent; But because broomrape seed generation is large, the seed survival phase is long, and under suitable humiture, seed can be sprouted all the year round, within the whole breeding time of crop, broomrape all can be had to be unearthed, uneven, make the non-constant of the prevention effect of prior art measure.
Utilize the pathogenic micro-organism of weeds to develop the campelyco that specialization is strong, little to the ozone deplation beyond target weeds, negative environment effects is few, security is high, likely become the effective way of management of weeds.Some kind as Fusarium (Fusarium spp.) can infect broomrape, causes morbidity.The Yue of king (Yue etc. of king. Agriculture in Xinjiang science and technology .1981 (7): 16-17.The Yue etc. of king. Chinese biological control .1985 (1): 24-26) broomrape of naturally falling ill from field is separated to a strain sickle-like bacteria (Fusarium orobanches) and makes biological and ecological methods to prevent plant disease, pests, and erosion agent F798, with cutting stem masking liquid method control hami melon broomrape, but the method needs first the stem of broomrape to be cut off, be coated with again and spread biocontrol microorganisms liquid, operation bothers very much, it is more difficult to promote, and affects prevention effect.Fusarium oxysporum (Fusarium oxysporum) .Annals of Botany.1999.83:453-458 such as () Heiko Thomas can be infected at the underground growth phase of Sunflower Receptacle broomrape, significantly reduces sprouting and the parasitic rate of broomrape seed.Sickle-like bacteria L2 bacterial strain (Kong Lingxiao etc. Plant Pathology .2006 36 (5): 466-469) 92.4%, L2 bacterial strain Raw toxin is reached to the prevention effect of broomrape broomrape seed germination can be suppressed, and cause broomrape seed germination pipe to damage.Utilize biological and ecological methods to prevent plant disease, pests, and erosion sickle-like bacteria prevent and treat tobacco broomrape can alleviate broomrape harm (Wu Yuanhua etc. tobacco science and technology .2012 (10): 78-80), but this kind of method needs first to regulate the potential of hydrogen of soil to spread manuer in holes biological and ecological methods to prevent plant disease, pests, and erosion sickle-like bacteria with sulphur again, more bothersome, and prevention effect is limited; Patent " a kind of layer goes out Fusariumsp and microbial inoculum thereof and application " (ZL201010209217.5) discloses and utilizes layer to go out Fusariumsp control Sunflower Receptacle broomrape, and its field control effect can reach 61.7%.As can be seen from the above, sickle-like bacteria is the effective way of control broomrape, but it is limited also to there is prevention and treatment range in existing sickle-like bacteria, fermentation level is low, the prevention effect shortcoming such as larger affected by environment, need the sickle-like bacteria finding the new efficient and long-acting control broomrape of energy badly, and the preparation method of corresponding microbial inoculum is studied.
Summary of the invention
Difficult for the anti-broomrape kind of cultivation, the problems such as the contaminate environment that cultivation step prevention effect difference and chemical pesticide control bring, the object of the invention is to provide a kind of Fusarium oxysporum.This bacterium can efficient, long-acting suppression branch broomrape and bend pipe broomrape, and has promoter action to the growth of the host plant of broomrape.
The present invention second object is to provide the microbiobacterial agent utilizing above-mentioned Fusarium oxysporum to produce.
The present invention the 3rd object is the preparation method providing mentioned microorganism microbial inoculum.
The present invention the 4th object is to provide above-mentioned Fusarium oxysporum preventing and treating the purposes on branch broomrape parasitic on tomato, tobacco and Sunflower Receptacle and bend pipe broomrape.
The present invention the 5th object is to provide mentioned microorganism microbial inoculum preventing and treating the purposes on branch broomrape parasitic on tomato, tobacco and Sunflower Receptacle and bend pipe broomrape.
The present invention is achieved through the following technical solutions:
A kind of Fusarium oxysporum (Fusarium oxysporum, also known as Fusarium oxysporum) bacterial strain Br-2, oneself is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica) on January 23rd, 2015, and deposit number is CGMCC No.10324.
Utilize the microbiobacterial agent that above-mentioned Fusarium oxysporum is produced, described microbiobacterial agent contains Fusarium oxysporum Br-2 thalline and fermented liquid.
Mentioned microorganism microbial inoculum is solid preparation, and the viable count of the Fusarium oxysporum Br-2 in described solid preparation is greater than 1 × 10
8cfu/g.
The preparation method of mentioned microorganism microbial inoculum, comprises the steps:
(1) actication of culture: by the Br-2 inoculation of cryopreservation on PDA plate culture medium, at 25 DEG C of activation culture 72h, gets bacterium sheet at the punch tool of colony edge diameter 5mm, obtains Br-2 bacterium sheet;
(2) seed liquor preparation: Br-2 bacterium sheet step (1) activated is inoculated in PDB liquid nutrient medium, every 200ml PDB liquid nutrient medium inoculation Br-2 bacterium sheet 4 ~ 5, at 25 ~ 26 DEG C, shaking culture 48 ~ 72h under rotating speed 170/rpm, obtain Br-2 seed liquor;
(3) fermentation culture: add fermention medium to fermentor tank, initial pH value HCl is adjusted to 5, when temperature drops to below 25 DEG C, adopt negative pressure to be the Br-2 seed liquor of ratio access step (2) gained of 0.5 ~ 1% according to weight percent after sterilizing, temperature be 25 ~ 30 DEG C, air flow is 1:0.3 ~ 1.0, stirring velocity is that 160 ~ 200rpm condition bottom fermentation cultivates 48 ~ 96h;
(4) fermentation culture is after 48 hours, detects conidium quantity in fermented liquid, treats that in fermented liquid, conidium quantity reaches 10
8fermentation culture can be stopped; Centrifugal concentrating 10 times, gained is the liquid preparation of Br-2 bacterial strain;
(5) in the liquid bacterial agent of the Br-2 bacterial strain of step (4) gained, add auxiliary agent according to the weight ratio of 1:1, after mixing drying, be the solid preparation of Br-2 bacterial strain.
Composition and the weight percent thereof of the PDA substratum described in above-mentioned preparation method's step (1) are: potato 20%, glucose 2%, and agar 1.5%, all the other are water.
Wherein PDB liquid nutrient medium composition described in above-mentioned preparation method's step (2) and weight percent thereof are: potato 20%, glucose 2%, and all the other are water.
Composition and the weight percent thereof of the fermention medium described in above-mentioned preparation method's step (3) are: sucrose 2 ~ 4%, ammonium sulfate 0.3 ~ 0.5%, dipotassium hydrogen phosphate 0.08 ~ 0.15%, magnesium sulfate 0.03 ~ 0.06%, Repone K 0.08 ~ 0.15% and ferrous sulfate 0.001 ~ 0.01%, all the other are water.
Air flow described in above-mentioned preparation method's step (3) is preferably 0 ~ 24h 1:0.3; 24 ~ 48h1:0.6; 48 ~ 96h 1:1.
Air flow described in above-mentioned preparation method's step (3) refers to that per minute passes into the volume of air of fermentor tank and the volume ratio of fermentation cylinder for fermentation liquid.
Stirring velocity described in above-mentioned preparation method's step (3) is preferably 160 ~ 170rpm.
Fermented incubation time described in above-mentioned preparation method's step (3) is preferably 48 ~ 60h.
Auxiliary agent described in above-mentioned preparation method's step (5) is diatomite or wheat bran.
The application of above-mentioned Fusarium oxysporum (Fusarium oxysporum) bacterial strain Br-2 in control branch broomrape (Orobanche acgyptiaca Pers.) or bend pipe broomrape (Orobanche cernua Loefling).
The application of mentioned microorganism microbial inoculum in control branch broomrape (Orobanche acgyptiaca Pers.) or bend pipe broomrape (Orobanche cernua Loefling).
Described in above-mentioned application, the host of broomrape is respectively tomato, Sunflower Receptacle or tobacco etc.
Fusarium oxysporum Br-2 of the present invention does not have pathogenic phenomena to 10 kinds of crops such as 10 kinds of staple crops (comprising unifacial leaf and dicotyledons) wheat, muskmelon, watermelon, eggplant, cotton, corn, tobacco, Sunflower Receptacle, capsicum, tomatoes, shows crop safety and has growth promoting function.
The using method of Fusarium oxysporum microbiobacterial agent of the present invention: used in tomato, tobacco seedling phase soil by above-mentioned gained microbiobacterial agent, also can be used for tomato, tobacco transplant phase or Sunflower Receptacle root in sowing time and execute by above-mentioned gained microbiobacterial agent.
The separation screening process of Br-2 bacterial strain
Br-2 bacterial strain is that Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie parasitizes tobacco susceptible broomrape from Wei County, Zhangjiakou, Hebei province is separated and obtains.In September, 2010, Hebei Prov. Academy of Agricultural &. Forest Sciences's Plant Protection Institute was from the bend pipe broomrape plant gathering morbidity body in Zhangjiakou Area, Hebei Province tobacco ground, cut after surface sterilization disease plant stem tissue put on PDA plate culture medium cultivate obtain fungal bacterial strain, purifying is carried out to these bacterial strains, after the security on 10 kinds of crops detects, filter out and have the bacterial strain of growth promoting function to crop without pathogenic effects, tobacco, tomato, there is serious plot and adopt crop root soil application to screen in Sunflower Receptacle broomrape, therefrom obtain a strain has obvious prevention effect bacterial strain to branch broomrape and bend pipe broomrape, name as Br-2.
The classification of Br-2 bacterial strain:
(1) classify according to morphological specificity
Bacterial strain Br-2 is 25 DEG C of cultivations on PDA plating medium, and bacterium colony is emitting shape growth, and colony colour white, along with incubation time Growth Center mycelia becomes lavender, seldom produce macroconidium, majority is microconidium.Macroconidium is colourless in fusiform or sickleshaped, and separate 1-5, general 3, apical cell is longer, gradually point, accidental podocyte, and size is about 30-43 μm × 3.5-4.3 μm; Microconidium is colourless, oval or oval, without every or accidental 1 to separate, size is 5-17 μm × 2-5 μm, and false head and is born on the raw phialide in side, and conidiophore is short; Between chlamydospore, raw or top is born on mycelia or large shape conidium, separately or in chain, wall is transparent, smooth or coarse, diameter 5-13 μm.According to Identification of The Genus Fusarium handbook " The Fusarium Laboratory the Manual " (.Summerell such as JohnF, Blackwell Publishing, 2006), morphological specificity (see Fig. 1, Fig. 2, Fig. 3 and Fig. 4) and Fusarium oxysporum (the Fusarium oxysporum of Br-2, also known as Fusarium oxysporum) morphological specificity describe be consistent, illustrate that Br-2 belongs to Fusarium Fusarium oxysporum (Fusarium oxysporum).
(2) classify according to translation elongation factor 1-α (EF1-α) gene order
Extract Br-2 genomic dna, with Br-2 genomic dna for template, with following EF1-α F and EF1-α R for primer carries out pcr amplification, described primer sequence is: EF1-α F 5 '-ATG GGT AAG GA (AG) GAC AAG AC-3 ' (SEQ ID No.1), EF1-α R 5 '-GGA (GA) GT ACC AGT (GC) AT CAT GTT-3 ' (SEQ ID No.2); PCR reaction system is 50 μ l, wherein template DNA 2 μ l, upstream and downstream primer each 2 μ l, PCR Master Mix25 μ l and sterilized water 19 μ l.PCR reaction conditions is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 45s, 35 circulations; Last 72 DEG C extend 10min.The product of PCR detects through 1.5% agarose gel electrophoresis, result obtains the fragment (see Fig. 5) of 684bp, this pcr amplification product is served marine life engineering company limited to carry out check order (SEQ ID No.3), warp is 98% with the EF1-α sequence similarity of the comparison discovery of Genbank database and Fusarium oxysporum (Fusarium oxysporum) KF574854.1, phylogenetic tree construction (see Fig. 6), illustrate that Br-2 belongs to Fusarium oxysporum (Fusarium oxysporum) in classification, Br-2 is different from the EF1-α sequence of every other bacterial strain simultaneously, illustrate that Br-2 is a new strains.
The advantage that the present invention has and beneficial effect: (1) the invention provides and a kind ofly high-efficiency prevention and control can parasitize the fusarium oxysporum bacterial strain of branch broomrape on tomato, tobacco and Sunflower Receptacle and bend pipe broomrape, for control branch broomrape and bend pipe broomrape provide a kind of new way of biological control; (2) inhibition of the present invention to branch broomrape and bend pipe broomrape is remarkable, and field control effect can reach 84.75%; (3) microbiobacterial agent preparation method of the present invention is simple, cost is low, be easy to apply; (4) the present invention is pollution-free, nuisanceless, environmentally friendly; And to the growth of crop, there is promoter action.
Accompanying drawing explanation
Fig. 1 is Br-2 bacterium colony full face on PDA substratum.
Fig. 2 is Br-2 bacterium colony back side photo on PDA substratum.
Fig. 3 is the product spore mode photo of Br-2 microconidium.
Fig. 4 is the microconidium photo of Br-2.
Fig. 5 is the pcr amplification product electrophoretogram of the EF1-α gene order of Br-2; Wherein M is DM2000DNA Marker.
Fig. 6 is the phylogenetic tree of Br-2.
Embodiment
Explain the present invention further with specific embodiment below, but protection scope of the present invention is not construed as limiting.Experimental technique in following embodiment, if no special instructions, is ordinary method; Percentage composition in following embodiment, if no special instructions, is weight percentage.
The preparation of embodiment 1Br-2 microbiobacterial agent
Carry out as follows:
(1), actication of culture: (oneself is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 23rd, 2015 by the fusarium oxysporum bacteria strain Br-2 of cryopreservation, deposit number is CGMCC No.10324) at PDA plate culture medium, (its composition and weight percent thereof are: potato 20%, glucose 2%, agar 1.5%, all the other are water) on, 25 DEG C of activation culture 72 hours, get bacterium sheet at the punch tool of colony edge diameter 5mm, obtain Br-2 bacterium sheet.
(2), seed liquor preparation: (its moiety and weight percent thereof are: potato 20% to make PDB liquid nutrient medium according to a conventional method, glucose 2%, all the other are water), PDB nutrient solution 200mL is loaded in 500mL triangular flask, high pressure moist heat sterilization, drop to after room temperature until temperature, the Br-2 bacterial strain that step (1) has activated is accessed in every bottle, diameter is the bacterium sheet 5 of 5mm, at 25 DEG C, shaking culture 72h on shaking table under rotating speed 170rpm, gained is Br-2 seed liquor.
(3), fermentation culture: (its moiety and weight percent thereof are: sucrose 3% to load 700L fermention medium in 1000L fermentor tank, ammonium sulfate 0.4%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, Repone K 0.08% and ferrous sulfate 0.001%, all the other are water; Conventionally prepare); Inoculation step (2) gained Br-2 seed liquor 4L in fermention medium; Initial pH value HCl is adjusted to 5, temperature be 26 DEG C, stirring velocity is 170rpm, 0 ~ 24h ventilation ratio 1:0.3; 24-48h ventilation ratio 1:0.6; The condition bottom fermentation of 48-96h ventilation ratio 1:1 is cultivated.
(4), after fermentation culture 48h, sample from the fermentor tank of step (3) every 8h and carry out microscopy, conidium number reaches 10
8/ ml, stop fermentation culture, gained is fermented liquid, by fermented liquid centrifugal concentrating 10 times (abandoning part supernatant), is the liquid bacterial agent of Br-2.
(5), by the liquid preparation of the Br-2 of step (4) gained, to mix according to the weight ratio ratio of 1:1 with diatomite that to make concentration be 10
8the microbial inoculum of/g, is the solid preparation of Br-2.
Embodiment 2Br-2 suppresses simultaneous test to branch broomrape and bend pipe broomrape seed germination
Carry out as follows:
(1) after supernatant liquor (meta-bolites of the Br-2) sterilizing, in Example 1 step (4) and with sterilized water dilute 5 times, 10 times for subsequent use.
(2), seed pre-rudiment process: get the branch broomrape of parasitic tomato and the bend pipe broomrape seed of parasitic tobacco, use 75% alcohol surface sterilization 5min respectively, and clean 3 times with aqua sterilisa.The diameter put in sterilizing be 5cm culture dish in diameter be on the little filter paper of 1cm, and with the moistening filter paper of aqua sterilisa.Each filter paper emplaces when 30, seed, culture dish to be put in incubator under 25 DEG C of dark conditions preculture 7 days.
(3), by the little filter paper being placed with pre-incubated broomrape seed move into the culture dish that 2 metafiltration paper are put in bottom, adding 2ml concentration is 10
-7the moistening filter paper of manual belle stimulator GR24 of M, then adds Br-2 supernatant liquor stoste 2ml, dilution 5 times and the dilution supernatant liquor of 10 times and clear water contrast respectively, repeats for 3 times, and moisturizing to cultivate after 7 days series of observations when the germination of seed.
The inhibiting rate of result (see table 1) Br-2 fermentation supernatant stoste to bend pipe broomrape and branch broomrape seed germination can reach 93.78% and 93.58% respectively, and the supernatant liquor restraining effect of diluting 5 times reduces, the supernatant liquor diluting 10 times contrasts with clear water does not have significant difference.Through microscopic examination, Br-2 fermented supernatant fluid can cause the necrosis of broomrape seed germination pipe, reduce broomrape seed germination rate.The active substance having and suppress bend pipe broomrape and branch broomrape seed germination and parasitism is described in Br-2 fermented supernatant fluid, and namely Br-2 can produce the material suppressing bend pipe broomrape and branch broomrape seed germination, produces obvious restraining effect to broomrape seed germination.
Bud ratio (%)=(the broomrape seed amount/broomrape seed sum of sprouting) × 100
Inhibiting rate (%)=((contrast germination rate-process germination rate)/contrast germination rate) × 100
Table 1:Br-2 fermented supernatant fluid is to the inhibiting rate of broomrape seed germination
Embodiment 3Br-2 field control broomrape simultaneous test
(1) test site: Yuxian, Hebei, Inner Mongol Chifeng, Kuerle, Xinjiang
(2) host is tested: tobacco, tomato and Sunflower Receptacle
(3) test method: the tobacco of comparatively serious Yuxian, Hebei occurs at broomrape for 2014 respectively Sunflower Receptacle ground (Sunflower Receptacle sowing time) of (tobacco transplant phase), Inner Mongol Chifeng and tomato ground (tomato transplanting time) soil of Kuerle, Xinjiang apply the concentration of embodiment 1 preparation is 10
8the Br-2 solid fungicide of/g, sowing amount is 375kg/ hectare.After broomrape is unearthed, a situation arises for investigation broomrape, analyzes Br-2 to the field control effect of broomrape.
The parasitic rate preventive effect of result (see table 2) Br-2 to broomrape parasitic on tobacco is 36.21%, and parasitic degree preventive effect is 79.43%; Be 53.19% to the parasitic rate preventive effect of broomrape parasitic on Sunflower Receptacle, parasitic degree preventive effect is 30.44%; Be 69.23% to the parasitic rate preventive effect of broomrape parasitic on tomato, parasitic degree preventive effect is 84.75%.Illustrate that Br-2 bacterial strain of the present invention all has obvious preventive and therapeutic effect to the broomrape in three different tests places; Wherein
Parasitic rate (%)=(by the host of parasitism number/host's sum) × 100
Parasitism degree=broomrape sum/host's sum
Preventive effect (%)=((contrast parasitic rate or parasitic degree-process parasitic rate or parasitic degree)/contrast parasitic rate or parasitic degree) × 100
Table 2: Br-2 bacterial strain of the present invention is to different areas broomrape field control effect comparative test result
Embodiment 4 Br-2 bacterial strain of the present invention is tested the security authentication of staple crops
Carry out as follows:
(1) seed cultivation: according to the different planting of crop; in the greenhouse of Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie by crops such as tomato, tobacco, capsicum, eggplant, watermelon, muskmelon, cottons in seedling pan nursery; transplant when seedling grows to 3-4 leaf; Sunflower Receptacle, wheat, seeding corn and other crops are directly sowed in flowerpot; every basin 2 seedlings; each Crop Species 20 basin, 10 basins are inoculation process, and 10 basins are not for inoculate contrast.
(2) preparation of bacteria suspension: the Br-2 bacterial strain of cryopreservation is activated on PDA plate culture medium, by the Br-2 inoculation of activation on PDB liquid nutrient medium, shaking culture 48 ~ 72h at 25 ~ 26 DEG C, obtains Br-2 bacterium liquid, and to be diluted to concentration with aqua sterilisa be 10
6the bacteria suspension of/ml.
(3) dip in root inoculation: taken out from seedling pan by the crop seedling of 3-4 leaf phase, shake off root earth and cut off the long bottom root system of 2cm, root system is immersed in bacteria suspension prepared by step (2), take out after 20min and transplant engagement.Routine Management, institutes an inquiry sickness rate and the growing state of each crop on the 4th week.
(4) fill with root inoculation: after crop emerges, the inoculation of filling root is carried out to crops to be measured such as Sunflower Receptacle, wheat, corns, bacteria suspension 20ml prepared by every basin inoculation step (2).Routine Management, institutes an inquiry sickness rate and the growing state of each crop on the 4th week.
Result (see table 3) Fusarium oxysporum Br-2 of the present invention can not infect 10 kinds of staple crops (see table 3) such as wheat, corn, cotton, Sunflower Receptacle, tobacco and vegetables, and have growth-promoting functions in various degree to the growth of these crops, illustrate that the microbiobacterial agent made with this Br-2 can in tomato, tobacco and the safe handling of sunflower planting district.
Table 3 Fusarium oxysporum Br-2 of the present invention is to crop safety assay
Claims (10)
1. Fusarium oxysporum (Fusarium oxysporum) bacterial strain Br-2, oneself is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 23rd, 2015, and deposit number is CGMCC No.10324.
2. the microbiobacterial agent utilizing the Fusarium oxysporum described in claim 1 to produce, is characterized in that described microbiobacterial agent contains Fusarium oxysporum Br-2 thalline and fermented liquid.
3., according to microbiobacterial agent according to claim 2, it is characterized in that described microbiobacterial agent is solid preparation, the viable count of the Fusarium oxysporum Br-2 in described solid preparation is greater than 1 × 10
8cfu/g.
4. the preparation method of microbiobacterial agent according to claim 3, comprises the steps:
(1), by the Br-2 inoculation of cryopreservation on PDA plate culture medium, at 25 DEG C of activation culture 72h, get bacterium sheet at the punch tool of colony edge diameter 5mm, obtain Br-2 bacterium sheet;
(2) the Br-2 bacterium sheet, by step (1) activated is inoculated in PDB liquid nutrient medium, every 200ml PDB liquid nutrient medium inoculation Br-2 bacterium sheet 4 ~ 5; At 25 ~ 26 DEG C, shaking culture 48 ~ 72h under rotating speed 170/rpm, obtain Br-2 seed liquor;
(3), fermention medium is added to fermentor tank, initial pH value HCl is adjusted to 5, when temperature drops to below 25 DEG C, adopt negative pressure to be the Br-2 seed liquor of ratio access step (2) gained of 0.5 ~ 1% according to weight percent after sterilizing, temperature be 25 ~ 30 DEG C, air flow is 1:0.3 ~ 1.0, stirring velocity is that 160 ~ 200rpm condition bottom fermentation cultivates 48 ~ 96h; The composition of wherein said fermention medium and weight percent thereof are: sucrose 2 ~ 4%, ammonium sulfate 0.3 ~ 0.5%, dipotassium hydrogen phosphate 0.08 ~ 0.15%, magnesium sulfate 0.03 ~ 0.06%, Repone K 0.08 ~ 0.15% and ferrous sulfate 0.001 ~ 0.01%, all the other are water;
(4) fermentation culture is after 48 hours, detects conidium quantity in fermented liquid, treats that in fermented liquid, conidium quantity reaches 10
8fermentation culture can be stopped; Centrifugal concentrating 10 times, gained is the liquid preparation of Br-2 bacterial strain;
(5) in the liquid preparation of the Br-2 bacterial strain of step (4) gained, add auxiliary agent according to the weight ratio of 1:1, after mixing drying, be the solid preparation of Br-2 bacterial strain.
5., according to preparation method according to claim 4, it is characterized in that the air flow described in its step (3) is 0 ~ 24h 1:0.3; 24 ~ 48h 1:0.6; 48 ~ 96h 1:1.
6., according to preparation method according to claim 4, it is characterized in that the stirring velocity described in its step (3) is 160 ~ 170rpm.
7., according to preparation method according to claim 4, it is characterized in that the fermented incubation time described in its step (3) is 48 ~ 60h.
8., according to preparation method according to claim 4, it is characterized in that the auxiliary agent described in its step (5) is diatomite or wheat bran.
9. the application of Fusarium oxysporum according to claim 1 (Fusarium oxysporum) bacterial strain Br-2 in control branch broomrape (Orobanche acgyptiaca Pers.) or bend pipe broomrape (Orobanche cernuaLoefling).
10. the application of microbiobacterial agent according to claim 2 in control branch broomrape (Orobanche acgyptiacaPers.) or bend pipe broomrape (Orobanche cernua Loefling).
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| CN108531404A (en) * | 2018-03-17 | 2018-09-14 | 青海省农林科学院 | One plant of layer goes out cultural method and its application of Fusariumsp |
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