CN106399119B - Biocontrol bacterial strain HZ-9 and its application in prevention and treatment soy bean cyst roundworm - Google Patents
Biocontrol bacterial strain HZ-9 and its application in prevention and treatment soy bean cyst roundworm Download PDFInfo
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Abstract
The invention discloses a kind of biocontrol bacterial strain HZ-9 and its application in prevention and treatment soy bean cyst roundworm, which is named as hook-shaped Trichoderma HZ-9, and deposit number is CCTCC NO:M 2016207, and the preservation time is on April 19th, 2016.Biocontrol bacterial strain HZ-9 of the invention is hook-shaped Trichoderma (Trichoderma hamatum), and spore suspension has preferable control efficiency to soybean cyst nematode, when spore concentration is up to 109Cfu/ml, sporangiocyst inhibiting rate reach 87.5%.
Description
Technical field
The present invention relates to technical field of biological control more particularly to biocontrol bacterial strain HZ-9 and its in prevention and treatment soy bean cyst roundworm
In application.
Background technique
Soy bean cyst roundworm (Soybean cyst nematode, SCN) main parasitic is at soybean (Glycine maxL.)
On, this nematode and other cyst roundworms are carried out Morphological comparison for the first time in nineteen fifty-two by Ichinohe, and are named as soybean
Cyst roundworm (Heterodera glycines Ichinohe).
So far, soy bean cyst roundworm has spread to main Soybean-planting in the world, and to endanger America's economy most heavy
Plant pathogeny organism (Wrather J A, the Koenning S R.Estimates of disease effects on wanted
soybean yields in the United States 2003to 2005[J].Journal of Nematology,
2006,38 (2): 173-180), very serious (the simple perseverance plant nematology of harm in the soybean producing region of Japan and northeast China
The China Agricultyre University Press [J], 2011:89-94).
In the control measure of soy bean cyst roundworm, most basic and most economical control method is cultural control.Plantation
Disease-resistant variety is domestic and international control soy bean cyst roundworm harm, reduce soybean yield loss effective ways (De Bruin J L,
Pedersen P.Response of old and new soybean cultivars to Heteroder aglycines
Ichinohe [J] .Agronomy Journal, 2008,100 (5): 1347-1353), China has obtained some disease-resistant big at present
Beans kind, such as anti-line 1, anti-line 2, (Kong Xiangchao, Li Hongmei, Geng Tian, Huang Wenkun, Peng Deliang are big for neat Huang 25, tender rich 15
Resistance Identification [J] the plant protection of beans germ plasm resource to No. 3 and No. 4 biological strains of soy bean cyst roundworm, 2012,38 (1):
146-150；Xu Wenping, Shen Hongbo, Miao Xing are fragrant, Yao Wenqiu Indentification of Soybean Germplasm Resistant To Soybean Cyst Nematode [J] Soybean Science,
2007,26(3):377-380).But if continuously planting same disease-resistant variety in areal, SCN toxicity enhances and causes
Soybean varieties lose resistance,
Soybean Seed-coating is commonly used for preventing and treating the harm of Soybean Seedling cyst roundworm as a kind of chemical fertilizer and pesticide complexing agent
(obstacle of the II, successive soybean cropping soil pests of research of Han Xiaozeng, Xu Yanli Soybean Cropping underproduction major obstacle factor is imitated
Answer [J] Soybean Science, 1999,18 (1): 48-53；Application effect [J] of several seed coat agents of Jia Yuqin, Jiang Guangsheng, Wang Xiaotong
Agricultural modernization, 1995, (12): 10-11).Since these chemical agent general toxicities are higher, in the soil the residence time compared with
It is long, can to a series of side effect such as environment, safety of human and livestock, grain security, meanwhile, so that soy bean cyst roundworm is generated anti-medicine
Property, it is now mostly disabled.
The biological control research of Plant nematode starts to walk early, and development is slow, the research of the biological control of soy bean cyst roundworm at present
It is concentrated mainly on several fields such as fungi, bacterium and suppressive soil (Wei Wenbin, Wu Yuhuan, Huang Jianming, Zheng Zhixing soybean spore
Capsule biocontrol of nematodes progress [J] Hebei Agricultural Sciences, 2013,17 (5): 56-58+99).
Studying more about soy bean cyst roundworm at present is biocontrol fungi, since its type is more, segregative feature, one
A little biocontrol fungis become hot spot concerned by people, and are commercialized applied to crop field.
Since being separated to biocontrol fungi from soy bean cyst roundworm sporangiocyst by Morganjones et al. earliest from 1984,
People to the classification of the biocontrol fungi of the SCN filtered out and preventive effect done numerous studies (Morganjones G,
Rodriguezkabana R,Tovar J G.Fungi associated with cysts of Heteroder
aglycinesin the Cauca Valley,Colombia[J].Nematropica,1984,14(2):173-177)。
Carris etc., which is described, to be colonized in 80 kinds of fungies on soy bean cyst roundworm sporangiocyst, and the U.S. has also reported nearly hundred kinds
Nematophagous fungi (Carris L M, Glawe D A, Smyth C A, Edwards D I.Fungi associated with
populations of Heteroderaglycinesin 2illinois soybean fields[J].Mycologia,
1989,81(1):66-75)；And Chen etc. is isolated at least 168 kinds of fungi (Chen F J, Chen S from sporangiocyst
Y.Mycofloras in cysts,females,and eggs of the soybean cyst nematode in
Minnesota[J].Applied Soil Ecology,2002,19(1):35-50)。
China is separated to biocontrol fungi from the sporangiocyst of the SCN of different regions and belongs to 51 kinds for totally 30, wherein with thick wall wheel branch
Bacterium (V.chlamydosporium), Fusarium oxysporum (Fusariumoxysporum), Paecilomyces lilacinus (P.lilacinus)
Etc. frequencies of occurrences highest (lijin is flourish, Duan Yuxi, Chen Lijie, and Xue Chunsheng soybean nodulation endogenetic bacteria influences soybean cyst nematode Heterodera glycines
Study [J] Soybean Science, 2005,24 (2): 154-156；Primary Study [J] of Lin Maosong fungi autoeciousness soy bean cyst roundworm
Biological control notification, 1990,6 (1): 38-41；Liu Xingzhong, Liu Wenmin, Zhang Dongsheng are colonized in the pale purple quasi- of soybean cyst nematode Heterodera glycines
The prevention and treatment of mould biological characteristic research [J] Chinese biological, 1995,11 (2): 23-27).With in recent years to plant nematode
Biocontrol fungi research deepen continuously, have researcher that Nematophagous fungi is divided into nematode-trapping fungi, inner parasitic epiphyte, chance
(the Barron G L.NematophagousHyphomycetes-new species of such as fungi and Toxigenic fungi
Harposporium[J].Antonie Van Leeuwenhoek Journal of Microbiology and Serology,
1972,38(2):217-222)。
Summary of the invention
The present invention provides a kind of biocontrol bacterial strain HZ-9 and its application in prevention and treatment soy bean cyst roundworm, the biocontrol bacterial strains
HZ-9 can effectively prevent soybean cyst nematode.
The biocontrol bacterial strain HZ-9 is named as hook-shaped Trichoderma (Trichoderma hamatum) HZ-9, deposit number
For CCTCC NO:M 2016207, the preservation time is on April 19th, 2016, preservation address are as follows: in Wuhan University, Wuhan, China city
State's Type Tissue Collection.
The biological property of biocontrol bacterial strain HZ-9 are as follows: it is in fluffy colony, mycelia is undeveloped, and conidiospore stalk is transparent,
Wall is smooth, and main shaft is straight, and top is tapered, conidium green, ellipse, long × roomy small range greatly 3.5-4.9 μ m
2.6-3.4μm。
The ITS sequence of biocontrol bacterial strain HZ-9 is as shown in SEQ ID NO.1；The eEF1a1 sequence such as SEQ of biocontrol bacterial strain HZ-9
Shown in ID NO.2.
The present invention also provides application of the biocontrol bacterial strain HZ-9 in prevention and treatment crops cyst roundworm.
The crops are soybean.
Specifically, the kind of the soybean is to close rich 55.
The application, comprising:
(1) biocontrol agent of the biocontrol bacterial strain HZ-9 is prepared；
(2) biocontrol agent is applied on the crops.
The biocontrol agent be the spore suspension of biocontrol bacterial strain, spore suspension dilution or biocontrol bacterial strain fermentation
Liquid.
Preferably, the concentration of the biocontrol bacterial strain HZ-9 is 10 in the biocontrol agent6~109CFU/mL。
Specifically, in terms of 1 liter, the fluid nutrient medium of the fermentation liquid is corn flour 30g, glucose 20g, distilled water
1000ml。
In terms of 1 liter, the solid medium of the spore suspension is peeled potatoes 200g, glucose 20g, agar 20g,
Distilled water 1000ml.
Fermentation condition in the fermentation liquid preparation process are as follows: 28 DEG C, 100rpm shaken cultivation 10d.
Compared with prior art, the invention has the following advantages:
(1) biocontrol bacterial strain HZ-9 of the invention be hook-shaped Trichoderma, spore suspension to soybean cyst nematode have compared with
Good control efficiency, when spore concentration is up to 109Cfu/ml, sporangiocyst inhibiting rate reach 87.5% or more.
(2) after biocontrol bacterial strain HZ-9 bacterium colony of the invention and soy bean cyst roundworm contact 48h, larva loses activity；It is raw
The fermentation liquid of anti-bacterial strain HZ-9 is able to suppress the hatching of ovum in sporangiocyst or female adult.
(3) spore suspension of biocontrol bacterial strain HZ-9 of the present invention and fermentation liquid can promote the growth of soybean.
Detailed description of the invention
Fig. 1 is influence of the strain culturing filtrate to egg hatching.
Fig. 2 is the aspect graph of bacterial strain HZ-9；
A. colony characteristics；B-D. conidiophore and conidium.
Fig. 3 is the area ITS of bacterial strain HZ-9 and the electrophoretogram of the area eEF1a1 pcr amplification product；
Fig. 4 is the phylogenetic tree that bacterial strain HZ-9 is constructed based on trichoderma ITS and eEF1a1 collating sequence, number in branch
Indicate the Bootstrap test value (1000 repetitions) greater than 50%.
Fig. 5 is corrected mortality (%) of the bacterial strain spore suspension to different nematodes.
Fig. 6 is control efficiency of the spore suspension to soybean cyst nematode of various concentration.
When Fig. 7 is pot experiment 50d, development of the soy bean cyst roundworm in soybean root；
A, B: processing group；C, D: control group.
Fig. 8 is influence of the bacterial strain HZ-9 various concentration spore suspension to soybean root fresh weight and plant height.
Fig. 9 is influence of the temperature to the mycelium growth and sporulation amount of bacterial strain HZ-9.
Figure 10 is influence of the illumination to the mycelium growth and sporulation amount of bacterial strain HZ-9.
Figure 11 is influence of the pH value to bacterial strain HZ-9 mycelium growth and sporulation amount.
Figure 12 is influence of the carbon source to the mycelium growth and sporulation amount of bacterial strain HZ-9.
Figure 13 is influence of the nitrogen source to the mycelium growth and sporulation amount of bacterial strain HZ-9.
Specific embodiment
The present invention is further explained combined with specific embodiments below.
The separation and screening of 1 soy bean cyst roundworm biocontrol fungi of embodiment
One, the acquisition of soy bean cyst roundworm sporangiocyst, second instar larvae and ovum suspension
Soy bean cyst roundworm picks up from Hangzhou, Zhejiang province city, Taigu County, Shanxi Province and Xiuzhou City, Anhui Province soy bean cyst roundworm danger
The serious plot of evil.
1, soy bean cyst roundworm sporangiocyst is obtained
Collected Soybean Root and sick soil are put into and eluriated in bucket, after being sufficiently mixed, suitable water is added and eluriates uniformly, makes
Soil sample is completely dissolved, and after standing about 10-20 second, suspension is poured on the double-deck sieve (upper layer is 20 mesh, lower layer 100
Mesh), sieve is rinsed with strong water flow.The residue on 20 mesh mesh screen of upper layer is removed, with thin water flow the deposit on 100 mesh mesh screens
In silt wash away, residue with slow-flowing stream collect in a clean beaker.With tweezers therefrom picking under anatomical lens
Fresh full brown sporangiocyst, it is spare to be put into 4 DEG C of refrigerators.
The sporangiocyst of acquisition is placed in hatchery, is placed in 28 DEG C of biochemical cultivation cases and is hatched, it will be in hatchery after 5 days
Suspension by 500 meshes, can be obtained cleaner J2 suspension.
2, soy bean cyst roundworm second instar larvae and ovum suspension are obtained
The above-mentioned sporangiocyst gathered is placed in the small container of a diameter about 3cm, with glass bar bottom by sporangiocyst gently
Then crushing is poured in the double-deck mesh (upper layer is 100 mesh, and lower layer is 500 mesh) and is flushed to ovum and second instar larvae with strong water flow
Lower layer's sieve collects the liquid on lower layer's sieve into 50ml centrifuge tube, and is added appropriate 76% and shows the sucrose solution matched,
2000rpm is centrifuged 2min, and then upper layer suspension is poured in 500 mesh sieve, is gently rinsed with water flow to remove sucrose
The suspension for obtaining soy bean cyst roundworm ovum, is subsequently placed in 4 DEG C of refrigerators and saves backup (Acedo J R, Dropkin V
H.Technique for obtaining eggs and juveniles of Heterodera glycines[J]
.Journal of Nematology,1982,14(3):418-420.)。
Two, the separation of soy bean cyst roundworm biocontrol fungi
1, in rhizosphere soil soy bean cyst roundworm parasitical fungi separation
Every part of pedotheque, which weighs 1g and is added to fill in the triangular flask of 99ml sterile water, is placed in 200rpm on shaking table, 60min
Afterwards, it is prepared into soil supension, then carries out 10-2、10-4、10-6With 10-8Gradient dilution, each dilution draws 200 μ l soil
Suspension aseptically uses rubbing method to be inoculated in the PDA culture medium containing 100mg/L ammonia benzyl and streptomycin sulphate, coating
After uniformly, plate is inverted in dark culture in 25 DEG C of constant incubators.Each dilution is repeated 3 times, and is carried out after bacterium colony is grown
Isolate and purify (the separation identification of dothiorella gregaria biocontrol microorganisms in Yang Lei, Liang Jun, Zhou Guoying, Ni Yang, Lv Quan, Zhang Xingyao soil
The agriculture journal in the southwest [J], 2015,51 (4): 116-124.).
By the above method, learn when soil mixed liquor is diluted to 10-6When, single colonie can be found on PDA, picking is new
The fresh mycelia grown is transferred in new PDA culture medium, carries out purifying culture；30 bacterium isolates are obtained altogether, after being saved
It is spare.
2, on soy bean cyst roundworm sporangiocyst parasitical fungi separation
By the sporangiocyst being collected into above with after 0.5% hypochlorite disinfectant 3min, with aseptic water washing three times, sterile
A sterilized round qualitative filter paper is padded in culture dish bottom, and sporangiocyst is placed on filter paper, seals ware lid, and mark,
Moisturizing culture 3d at 25 DEG C.After 3d, choose sporangiocyst of the surface with mycelia, is placed on the PDA of composite containing streptomycin sulphate and ammonia benzyl mycin
On solid medium, 25 DEG C of cultures.After growing bacterium colony, it is flat that new culture medium is gone to the mycelium for choosing needle and cutting colony edge
In plate, 25 DEG C of purifying cultures are three times.By the above method, 34 fungal strains are filtered out altogether and are saved backup after purified culture.
3, on soy bean cyst roundworm second instar larvae and ovum parasitical fungi separation
It is clear with sterile water after the second instar larvae being above collected into and ovum first to be used to 0.5% hypochlorite disinfectant 3min
It washes three times, prepares the suspension of second instar larvae and ovum, then carry out 10 times, 100 times, 1000 times dilute.Take respectively former suspension,
10 times of dilutions, 100 times of dilutions, 1000 times of 200 μ l of dilution are uniformly coated on the PDA of composite containing streptomycin sulphate and ammonia benzyl mycin
On solid plate, the mycelia of picking single colonie is placed on new plate after 3d, and 25 DEG C of purifying cultures are three times.By the above method,
81 fungal strains are isolated to save backup after purified culture.
The fungi that above-mentioned three kinds of methods filter out, as shown in table 1 below.
The separation of 1 soy bean cyst roundworm parasitical fungi of table
4, the preservation of biocontrol fungi
The bacterial strain of picking after purification is placed on the inclined-plane PDA of composite containing streptomycin sulphate and ammonia benzyl mycin after cultivating 2 weeks and is put in 4
DEG C refrigerator carries out short-term preservation；Long-term preservation uses ultra-low temperature preservation method simultaneously, cold with 1mL is added after the sterilizing of 1.5mL cryovial
It is spare to freeze preservation liquid.It is about 0.5cm from eugonic colony edge picking2Several pieces of fungus block, be directly placed into freezen protective
Guan Zhong.
The cryovial that fungus block is added first is placed in 4 DEG C of refrigerator pre-cooling 30min, places into -20 DEG C of refrigerator 30-40min, then turn
Enter -75 DEG C of refrigerators to save backup.The formula of freezen protective liquid is following (1L): 15% glycerol (v/v), glucose 10g, and yeast extracts
Object 1g, casein hydrolysate 1g, it is spare after high pressure sterilization.
Three, the screening of biocontrol bacterial strain (HZ-9 comes from soybean rhizosphere)
1, parasitics of the bacterial strain to soy bean cyst roundworm sporangiocyst
To the bacterial strain that picking goes out, the different fungi of colonial morphology is chosen, the mirror of soy bean cyst roundworm sporangiocyst parasitics is carried out
It is fixed, by methodology above acquisition sporangiocyst, with 0.5%NaClO surface sterilization 3min, with sterile water wash three times.
Bacterial strain after purification is gone in PDA culture medium, when colony diameter it is long to culture dish 1/2-3/4 when, after disinfection
Sporangiocyst be put into the edge of fresh mycelia in PDA culture medium, every ware places 10 sporangiocysts, and three biology weights are arranged in each bacterial strain
It is multiple.It is placed in 25 DEG C of biochemical cultivation case and cultivates 10d, sporangiocyst is gently chosen and (is careful not to stave sporangiocyst), with 0.5%
NaClO surface sterilization 3min, sterile water wash three times, each sporangiocyst is put on the filter paper of a sterilizing, moisturizing culture 5d.5d
The case where observing long bacterium on filter paper afterwards, records the situation of peridium parasitism.
From 145 fungus strains filtered out, chooses colonial morphology and inconsistent 33 fungal strains of spore shape carry out
The experiment of tieback sporangiocyst measures screened bacterial strain to the parasitics of sporangiocyst.
In 33 selected fungal strains, most of fungal bacterial strain can carry out tieback sporangiocyst, and wherein infection rate reaches 100%
Bacterial strain have 7 plants, be respectively: HZ-L9, TG-9, HZ-7, HZ-L21, TG-L37, TG-3 and AH-8；Infection rate is greater than 90%
Bacterial strain has 10 plants, is respectively as follows: HZ-3, HZ-4, HZ-5, HZ-L23, HZ-L26, TG-13, TG-17, AH-3, TG-1 and HZ-9；It invades
Bacterial strain of the dye rate greater than 80% has 11 plants, is respectively as follows: HZ-L25, HZ-L18, HZ-L7, HZ-L5, AH-L16, TG-15, TG-L8,
AH-L7, AH-L6, HZ-1 and HZ-9；Infection rate has 5 plants, respectively HZ-L4 less than 80%, AH-1, TG-L5, AH-L15 and
TG-6。
2, Preliminary Determination of the bacterial strain to soy bean cyst roundworm second instar larvae toxicity
By the strain inoculated of pure culture in the PDA solid medium tablets of composite containing streptomycin sulphate and ammonia benzyl mycin (90mm
Sterile petri dish), after cultivating 2-3d in 25 DEG C of biochemical cultivation cases, 20 μ L second instar larvae suspension (about 200 are accessed in colony edge
Item), respectively at for 24 hours, microscopically observation is adhered by mycelia and the case where penetrate and be then digested after 48h, while not being connect
Also nematode as much is accessed in the PDA culture medium of bacterium as control, each processing is arranged three biology and repeats.
The pathogenic preliminary screening of soy bean cyst roundworm second instar larvae is carried out to above-mentioned bacterial strains, the results show that being measured
33 plants of bacterial strains in, wherein HZ-4, HZ-9, HZ-L9, TG-1, TG-15, TG-L37 and AH-8 bacterial strain is to soy bean cyst roundworm two
The mobility of instar larvae has an impact, and after polypide contacts mycelia, nematode freedom of action degree is reduced；HZ-L9 and HZ-9 bacterium colony and children
After worm contact, larva loses activity after 24h, and 48h is covered by mycelia later；TG-1, TG-15 and AH-8 contact worm in mycelia
Nematode polypide just starts stiff after body 48h, and other bacterial strain mycelia do not have toxic action to soy bean cyst roundworm second instar larvae, worm
Freedom of movement is remained to after body 48h.
3, influence of the bacterial strain fermentation liquor to egg hatching in sporangiocyst or female adult
By strain inoculated in the triangular flask of 250ml for filling 150ml corn flour fluid nutrient medium, 28 DEG C, 100rpm vibration
Culture 10d is swung, the sterilized double-deck lens wiping paper of culture solution is filtered to remove hypha body, and then 4 DEG C of 12000rpm are centrifuged 20min, on
Clear liquid, to get bacterial strain fermentation liquor, it is spare to be stored in 4 DEG C of refrigerators by the miillpore filter of diameter 0.22um.
The bacterial strain fermentation liquor of 1ml is added in every hole in 24 hole culture mediums, and 5 female adults are separately added, and sterile water compares, each
The fermentation liquid of bacterial strain and control are all provided with 6 repetitions, and 24 porocyte culture plates are placed in 28 DEG C of biochemical cultivation cases, dilution 0x,
Five concentration gradients of 5x, 10x, 20x, every ware are added 500 μ L, are compareed with sterile water, in triplicate, and obturaged with sealed membrane,
Prevent moisture evaporation and living contaminants.It is placed in 10d in 25 DEG C of incubators.The nematode number hatched is observed and recorded, hatching phase is calculated
To inhibiting rate.
Influence using above method detection bacterial strain fermentation liquor to soy bean cyst roundworm egg hatching, first respectively with 33 plants of bacterium
The fermentation liquid stoste of strain measures its influence to soy bean cyst roundworm egg hatching, as a result it is found that bacterial strain HZ-L9, HZ-9, HZ-
L26, AH-1, AH-L6, TG-L5, TG-16 have inhibiting effect to the hatching of ovum in soy bean cyst roundworm sporangiocyst or female adult, wherein bacterium
The fermentation liquid stoste of strain HZ-L9 and bacterial strain HZ-9 cannot make the egg hatching (Fig. 1) in sporangiocyst or female adult substantially.
5x is carried out by the fermentation liquid stoste to bacterial strain HZ-L9 and bacterial strain HZ-9,10x, 20x dilution are opposed with sterile water
According to measuring influence of its dilution to egg hatching again, find the fermentation liquid of two plants of acquired purpose bacterial strains to soybean cyst
The hatching of line eggs has certain inhibiting effect (being shown in Table 2 and 3), and the fermentation liquid of each diluted concentration and sterile water process it
Between, hatching on nematode influences significant difference, and wherein fermentation liquid stoste is handled, almost without nematode hatching；When 20x dilutes, two bacterium
Strain respectively reaches 47.75% and 45.87% to the hatching inhibiting rate of ovum in sporangiocyst or female adult, shows good inhibiting effect.
Influence of the 2 HZ-L9 fermentation liquid difference extension rate of table to soy bean cyst roundworm egg hatching
Note: different large and small English alphabets of writing respectively indicate difference extremely significant (P≤0.01) and significant difference (P≤0.05),
The identical person of letter is that difference is not significant.
Influence of table 3 hook-shaped trichoderma (HZ-9) the fermentation liquid difference extension rate to soy bean cyst roundworm egg hatching
Note: different large and small English alphabets of writing respectively indicate difference extremely significant (P≤0.01) and significant difference (P≤0.05),
The identical person of letter is that difference is not significant.
The identification of 2 biocontrol bacterial strain of embodiment
One, Morphological Identification
The bacterial strain being stored in 4 DEG C of PDA culture inclined-planes is moved on the PDA plate of diameter 90mm, 28 DEG C of biochemical cultivation cases
Middle activation 3-5d cuts fungus block in colony edge with the punch of diameter 14mm, is transferred on new PDA plate, 28 DEG C of biochemistry
Constant temperature incubation in incubator, 3 repetitions.Day by day colonial morphology, color and upgrowth situation are observed and recorded.
After above-mentioned bacterium colony culture 3d, with electron microscope (LEICA DMI 3000B, Leca) observation mycelia, divide
The morphological feature of raw sporophore, conidium etc., counts the length and width of 100 spores, calculates its length-width ratio.It is true according to trichoderma
Bacterium colonial morphology, spore feature carry out Preliminary Identification to it.
Bacterial strain HZ-9 is in fluffy colony in PDA, and mycelia is undeveloped, and conidiospore stalk is transparent, and wall is smooth, and main shaft is straight, top
End is tapered, conidium green, ellipse, long × roomy 2.6-3.4 μm of μ m of small range about 3.5-4.9 (Fig. 2).
Two, molecular biology identification
1, the extracting method of DNA
With the mycelia for having cultivated 3d on scalpel scraping PDA plate in 25 DEG C of biochemical cultivation cases, it is placed on containing stone
In the oscillating tube of sand, 800 μ l CTAB extracts are added in every pipe, shake 90s with oscillator, 65 DEG C of water-baths are incubated for 1-2h, are added
Isometric chloroform-isoamyl alcohol (24:1) mixes, and 10000rpm is centrifuged 15min, and supernatant (about 750 μ l) is taken to be transferred to 2ml centrifugation
Guan Zhong is added isometric phenol-chloroform-isoamyl alcohol (25:24:1) and mixes, and 12000rpm is centrifuged 20min, takes supernatant (about 450
μ l) into 1.5ml centrifuge tube, the isopropanol that 2/3 volume is added mixes 5min, and 12000rpm is centrifuged 10min, abandons supernatant, is added
The ethyl alcohol of 200 μ l 70% cleans, and 12000rpm is centrifuged 1-2min, and loss of gloss liquid dries precipitating, and it is molten that 30 μ l TE aqueous suspensions are added
Solution precipitating, can be obtained DNA.
2, the amplification of ITS sequence and eEF1a1 segment
Using genomic DNA as template, with the segment and primer in fungi universal primer ITS1 and ITS4 the amplification area ITS
Tef71f and tef997R expands eEF1a1 segment (Shoukoui E, Bissett J.Preferred primers for
sequencing 50end of the translation elongation factor 1-alpha gene(EF1-a1)and
subnit 2of the RNA ploymerase B gene(RPB2)ISTH available from:http://
isth.info/methods,2008.)。
It tests PCR amplification kit used and is purchased from Dalian treasured bioengineering Co., Ltd (Takara).Primer is by the English Weihe River
The synthesis of Jie Ji (Shanghai) trade Co., Ltd.
PCR amplification uses 50 μ l reaction systems, and each reagent dosage is as follows:
Primer sequence:
ITS1 5’-TCCGTAGGTGAACCTGCGG-3’
ITS4 5’-TCCTCCGCTTATTGATATGC-3’
tef71f5’-CAAA ATG GGT AAG GAG GAS AAGAC-3’
tef997R5’-CA GTA CCG GCR GCR ATR ATS AG-3’
Expand the response procedures in the area ITS: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 56 DEG C of annealing 45s, 72 DEG C extend
1min, 35 circulations；72 DEG C of extension 10min, 8 DEG C of persistences.
It expands eEF1a1 segment and uses ' touchdown ' amplification program: 94 DEG C of initial denaturation 4min first, then 4 circulations:
94 DEG C of denaturation 1min, 70 DEG C of annealing 1min30s, 72 DEG C of extension 1min30s；26 circulations: 94 DEG C of denaturation 1min, 68 DEG C of (every warps
Crossing a circulation reduces by 0.5 DEG C) annealing 1min30s, 72 DEG C of extension 1min30s；12 circulations: 94 DEG C of denaturation 1min, 55 DEG C are moved back
Fiery 1min30s, 72 DEG C of extension 1min30s；72 DEG C of extension 7min, 8 DEG C of preservations.
After taking 2 μ l PCR products that 3 μ 6 × Loding of l buffer mixing is added after the completion of PCR program, with 1.0% fine jade
(120V, 400mA, 25min) is separated by electrophoresis in sepharose.10-15min is dyed through EB after electrophoresis, is observed on Ultraviolet Detector
With take pictures.
3, the purifying of PCR product
Select Zhejiang love pursue progress biotech company (AxyGen) AxyPrep DNA gel QIAquick Gel Extraction Kit carry out PCR
Recycling, the purifying of product.
4, the connection of purified product
PCR purified product can directly be connect with pUCM-T, using 10 μ l reaction systems, be sequentially added:
12000rpm is centrifuged 10 seconds, sets 4 DEG C of connections overnight or 16 DEG C connect 4 hours.
5, the conversion of connection product
(1) 100 μ l Escherichia coli DH, 5 α competence is taken from -70 DEG C of refrigerators, 1min thaws on ice；
(2) the above-mentioned connection solution of 10 μ l is added into centrifuge tube, is gently mixed uniformly, places 20-30min on ice；
Thermal shock 90s in (3) 42 DEG C of water-baths is immediately placed in cooled on ice 5min after thermal shock；
(4) 1ml LB liquid medium (being free of Amp), 37 DEG C of shaken cultivation 40min after mixing are added into pipe；
(5) 1500rpm is centrifuged 2min, removes 800 μ l of supernatant, and piping and druming mixes remaining liq, draws 200 μ l and is coated on containing Amp,
It in X-gal and IPTG screening flat board, faces up and places half an hour, be cultured after base absorbs completely after bacterium solution and be inverted culture dish,
37 DEG C of culture 16-24h.
6, the PCR identification of recombinant plasmid
Bacterium solution is directly used in PCR identification, steps are as follows:
(1) 100 μ l ammonia benzyl mycins (100mg/ml) are added in 100ml LB liquid medium
(2) culture of the 2ml LB containing ammonia benzyl mycin is inoculated into 0.2 μ l sterile eppendorf tubes picking white single colonie
In liquid, shaken cultivation (37 DEG C, 180rpm) about 12h；
(3) 2 μ l supernatants is taken to identify (25 μ l reaction system) for PCR.
7, determined dna sequence and analysis
3 positive colonies of each plate picking send raw work biological (Shanghai) Science and Technology Ltd. sequencing.Sequencing result is used
DNAstar is edited, with trichoderma identify 7Hc/70KEY on online website (http://www.isth.info) and
Sequence is compared in TWcABLAST.
RDNA-the ITS (SEQ ID NO.1) and eEF1a1 segment (SEQ ID NO.2) of bacterial strain HZ-9 is cloned through PCR, is surveyed
Sequence, as a result such as Fig. 3, the rDNA-ITS (SEQ) and eEF1a1 clip size of bacterial strain HZ-9 is respectively 600bp and 760bp, with wood
Mould identifies that sequence is compared in 7Hc/70KEY and TWcABLAST on online website (http://www.isth.info)
Analysis is it is found that two segments of bacterial strain HZ-9 have high homology with Trichoderma hamatum.
Three, Phylogenetic Analysis
Blast comparison will be carried out in GenBank by the sequence after DNAStar software editing, according under sequence homology
Carry correlated series.The sequence of downloading carries out multisequencing connection with 6.0 software Clustal W program of Mega and matches, and distich is carried out with result
Phylogenetic Analysis constructs systematic evolution tree with adjacent method (N-J), is tested with Bootstrap to genealogical tree, 1000 weights
It is multiple.
Trichoderma belongs to the ITS sequence and eEF1a1 sequence (being shown in Table 4) for the kind announced in selection GenBank, uses
Sequence carries out clustering after 6.0 software of Mega merges rDNA-ITS and eEF1a1 segment, and respectively with hook-shaped trichoderma
Trichoderma hamatum, trichoderma reesei Hypocrea lixii are used after Clustal W matching arrangement as outer group
Mega6.0 software N-J method distinguishes phylogenetic tree construction, and bacterial strain HZ-9 and Trichoderma hamatum is same as can be seen from Figure 4
One branch, affiliation are nearest.
Table 4 is used for the Trichoderma strain of polygenes sequence analysis
In conclusion combining form and molecular biology identification are as a result, be accredited as hook-shaped trichoderma for bacterial strain HZ-9
(T.hamatum)。
Above-mentioned bacterial strains HZ-9 is subjected to culture presevation, which is named as hook-shaped Trichoderma (Trichoderma
Hamatum) HZ-9, deposit number are CCTCC NO:M 2016207, and the preservation time is on April 19th, 2016, preservation address are as follows:
Wuhan University, Wuhan, China city China typical culture collection center.
Toxic action of the 3 biocontrol bacterial strain spore suspension of embodiment to different nematodes
Meloidogyne incognita (Meloidogyne incognita) second instar larvae (juvenile 2) is for a long time on tomato
Pure culture obtains, and the egg capsule of picking tomato root Meloidogyne incognita, which is placed in incubator, is hatched, and can be obtained two days later
Then it is standby to be made into the 4 DEG C of refrigerators preservations of suspension of every 10ul containing 100 J2 for a large amount of clean Meloidogyne incognita (J2) suspension
With.
Sweet potato stem nematode (Ditylenchus destructor), Bursaphelenchus xylophilus (Bursaphelenchus
Xylophilus), aphelenchoides besseyi (Aphelenchoides bessyi) is long-term cultivation partly posting on grey staphylococcus
Nematode is seeded in the PDA culture medium for covering with grey staphylococcus by natural disposition nematode, and after one month, 5ml sterile water, which is added, into ware is
It can get clean nematode filtrate, be then made into the 4 DEG C of refrigerators of nematode suspension of every 10 μ l containing 100 J2 and save backup.
1, the preparation and counting of bacterial strain spore suspension
From save bacterial strain inclined-plane on sterilizing choose needle picking mycelium inoculation to contain streptomycin sulphate and ammonia benzyl mycin
PDA plate center, be placed in 28 DEG C of biochemical cultivation cases after cultivating 1 week, 6ml aqua sterilisa be added to PDA plate surface, use is sterilized
The mycelia of the three angle rods scraping bacterial strain of bacterium and spore, suspension obtained are filtered to remove mycelia by sterilized double gauze
Up to clean spore suspension.
Spore suspension is counted with the blood counting chamber of model XB-K-25, spore suspension is mixed well,
10 times to 100 times are diluted according to the actual situation or is not diluted, and are first dripped the dropper of the spore suspension after mixing flat in center
On platform, covered after finding grid under low power lens, is observed and is counted under conversion to high power lens, usually checks 5
Spore count in a middle lattice, 5 middle lattice are distributed in different orientation on four angles with center, the grid of counting more respectively,
Caused error can be unevenly distributed in each orientation to avoid spore.After counting, required concentration is adjusted to sterile water and is put in 4
DEG C refrigerator saves backup.
2, biocontrol bacterial strain spore suspension (concentration 108/ ml) measurement to nematode toxic action
The suspension of soy bean cyst roundworm second instar larvae is made into the suspension that every 10 μ l contains 100 J2, with sterile 24
Hole Tissue Culture Dish is added 500 μ l preprepared spore suspensions, is compareed with sterile water, 3 biology of each processing
Repeat, be added in every hole the configured J2 suspension of 10 μ l or other for trying nematode suspension, observe and calculate after 48h and knock down
Rate.
Bacterial strain HZ-9 all has certain activity to the several plant pathogenic nematode for examination, corrects to soy bean cyst roundworm
The death rate reaches 83.3%；85.6% is reached to Bursaphelenchus xylophilus corrected mortality；To sweet potato stem nematode and Meloidogyne incognita
Toxic action is relatively poor, and corrected mortality is respectively 64.0% and 58.3% (Fig. 5).
The potting efficiency test and its biological characteristics of 4 biocontrol bacterial strain of embodiment
One, experimental material
(1) bacterial strain: HZ-9；
(2) for trying soybean varieties: soy bean cyst roundworm susceptible variety closes rich 55；
(3) for trying soy bean cyst roundworm: picking up from Xiuzhou City, Anhui Province soy bean cyst roundworm and endanger serious plot, through for many years
Pure culture obtains；
(4) galactolipin, soluble starch, sucrose, mannose, fructose, maltose, glucose, sorbierite, sucrose；Chlorination
Ammonium, glutamine, tryptone, beef extract, sodium nitrate, yeast extract, proline, glycine, ammonium sulfate purchase are raw in raw work
Object (Shanghai) Science and Technology Ltd..
Two, experimental method
The spore suspension (method is with embodiment 2) for preparing biocontrol bacterial strain HZ-9, with sterile water respectively the spore of bacterial strain
Suspension is diluted to 109cfu/ml、108cfu/ml、106cfu/ml、104cfu/ml、102The different gradients of cfu/ml.
Soya seeds are put into 0.5% liquor natrii hypochloritis, are stirred 4min with glass bar, are then used sterile water cleaning down
After 3 times, soybean is impregnated to 1h in sterile water to remove remaining sodium hypochlorite, be subsequently placed in and be placed with the big of one layer of aseptic filter paper
One is covered again in culture dish (diameter 15cm), on soybean and soaks aseptic filter paper, is covered culture dish and is placed in 25 DEG C of incubators
Dark culturing.
Period uses aseptic water washing soybean daily and pays attention to moisturizing culture, and temperature setting is at 25 DEG C in incubator, 16h light
According to, 8h dark processing, humidity 60%, illumination 100%.After 5 days, as root about 4-5cm long, selects the consistent young shoot of growing way and move
It plants and fills 350cm3In the plastic cup of sterile fine sand (diameter 12cm), then all plastic cups are all put into plastic box
(550 × 350 × 90mm) culture.
After seedling grows 3 days, the spore suspension of various concentration set by 15ml is accessed in each cup.It is outstanding to be inoculated with bacterium
Nematode, 1500 ovum of every plant of inoculation are inoculated with after liquid 2d.3-4 are beaten in soybean root surrounding soil with the glass bar of diameter 0.5cm
The duck eye of 3cm depth.The prepared nematode suspension 4ml for containing 1500 ovum is injected in duck eye, and is sealed up with sand.Each bacterium
The processing of suspension concentration is repeated 4 times, and replaces bacteria suspension to compare processing with sterile water.
After being inoculated with nematode, the spore suspension (10 of primary setting concentration was poured every 7 days9cfu/ml、108cfu/ml、
106cfu/ml、104cfu/ml、102) or fermentation liquid stoste cfu/ml.Female adult quantity in soybean root and soil is counted after 50 days
And the online borer population of root, calculate sporangiocyst inhibiting rate.
After being inoculated with nematode 50d, using sodium hypochlorite-acid fuchsin colouring method, soybean root nematode is dyed.It will
Clean old complaint is put into the small beaker of 150ml, and 50ml distilled water is added in beaker, and appropriate 5.25% hypochlorous acid is added
Sodium (the tender degree of children that the dosage of sodium hypochlorite depends on root tissue), takes out root after being stirred continuously 4min with glass bar, uses water flow
45s is rinsed, root tissue is immersed in 15min in distilled water, to completely remove sodium hypochlorite, distilled water is removed, root tissue is put
In the beaker for filling 30-50ml distilled water, and the acid fuchsine solution of 1ml is added, 30s is boiled in micro-wave oven, after cooling, uses
Distilled water rinses root tissue, and root tissue is put into 20-30ml acidity glycerol (hydrochloric acid of 2-3 drop 5mol/L is added dropwise in pure glycerin)
In boil root tissue made to fade (be careful not to boil excessively, otherwise root tissue easily turns yellow), after cooling, take out root tissue in body
It is observed under stereomicroscope.
After soybean bean seedling growth 50d, the sporangiocyst quantity of soybean root is investigated, and calculates different disposal to soy bean cyst roundworm
Inhibiting rate: sporangiocyst inhibiting rate (%)=(control group sporangiocyst quantity-processing group sporangiocyst quantity)/control group sporangiocyst quantity ×
100%.
After the investigation of soybean root sporangiocyst quantity, the plant height and root fresh weight of whole plant are measured.
Test data uses one-way analysis of variance (One Way through 2013 software statistics of SAS software and Excel
ANOVA significance difference analysis) is carried out.
1, influence of the temperature to mycelium growth and sporulation amount
Using bacterium dish inocalation method, i.e., the punch for being 14mm with diameter, from the colony edge on the PDA plate for having cultivated 3d
Bacterium dish is got, bacterium dish is inoculated on new PDA plate, 15 DEG C, 20 DEG C, 25 DEG C, 28 DEG C, 30 DEG C, 35 DEG C of biochemistry are respectively placed in
Culture in incubator, each temperature are 1 processing, and 4 repetitions of each processing observe bacterium colony upgrowth situation, cross after cultivating 2d
Interior extrapolation method measures colony diameter, and its sporulation quantity is checked after 5d.
2, influence of the illumination to mycelium growth and sporulation amount
It beats after taking bacterium dish, is inoculated on PDA plate, be respectively placed in that light, 12h light 12h for 24 hours be dark, dark for 24 hours lower training at 28 DEG C
It supports, every kind of illumination condition is 1 processing, 4 repetitions of each processing.Bacterium colony upgrowth situation, crossing method are observed after cultivating 2d
Colony diameter is measured, and checks its sporulation quantity after 5d.
3, influence of the pH to mycelium growth and sporulation amount
In superclean bench by the PDA culture medium after high pressure sterilization, with l molL-1HCl and lmolL-1's
NaOH configuration pH value is respectively 4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 PDA culture medium, and the flat of 90mm is made
Plate, beats and bacterium dish is taken to be placed on plate center, and each pH value is 1 processing, 4 repetitions of each processing.Observation bacterium colony is raw after cultivating 2d
Long situation, crossing method measure colony diameter, check its sporulation quantity after 5d.
4, influence of the different carbon source to mycelium growth and sporulation amount
With czapek's medium (NaNO32.00g K2HPO41.00g, KCl 0.50g, MgSO4·7H2O 0.50g, FeSO4
0.01g, sucrose 30.00g, adds water to be settled to 1000ml) it is basic culture medium.With NaNO3For nitrogen source, galactolipin, solvable is measured
Property the influence to strain growth of starch, mannitol, fructose, maltose, glucose, sorbierite, sucrose, with no carbon source Cha Shi culture
Base is control, 4 repetitions of each processing.Bacterium colony upgrowth situation is observed after cultivating 2d, crossing method measures colony diameter, after 5d
Check its sporulation quantity.
5, influence of the different nitrogen sources to mycelium growth and sporulation amount
It is basic culture medium with czapek's medium.Using sucrose as carbon source, glycine, glutamine, proline, yeast are measured
The influence of cream, ammonium sulfate, beef extract, tryptone, ammonium chloride, potassium nitrate to strain growth, with no nitrogen source czapek's medium
For control, 4 repetitions of each processing.Bacterium colony upgrowth situation is observed after cultivating 2d, crossing method measures colony diameter, examines after 5d
Look into its sporulation quantity.
Two, experimental result
1, the control efficiency of biocontrol bacterial strain spore suspension or fermentation liquid stoste to soybean cyst nematode
After the spore suspension 50d for applying various concentration in the big bean seedlings root soil of potting, soybean root and soil are counted
White female adult and sporangiocyst quantity in earth calculate purpose biocontrol fungi to the spore of soybean cyst nematode caused by soy bean cyst roundworm
Capsule inhibiting rate；The nematode number of soybean root different larval instar, observation processing group and control group root are counted by acid fuchsin staining
Portion nematode reduces situation.
Fig. 6 is assessment of the hook-shaped trichoderma of biocontrol fungi (HZ-9) to soybean cyst nematode control efficiency.
Compared with the control, hook-shaped trichoderma (HZ-9) spore suspension concentration is 106-109When, control efficiency is preferable, and sporangiocyst
Inhibiting rate is up to 87.5%.
Fig. 7 be pot experiment 50d after, spore suspension processing with infected with the soy bean cyst roundworm of sterile water process it is big
Beans root nematode infection situation, compared with the control, nematode highest reduces 96% on root, illustrates filtered out biocontrol bacterial strain
Spore suspension is inhibited to soy bean cyst roundworm in pot experiment.
2, influence of the biocontrol bacterial strain various concentration spore suspension to soybean plant height and root fresh weight
Root fresh weight and soybean plant height to soybean after 50d is for statistical analysis, and Lai Hengliang purpose biocontrol fungi is long to soybean
The influence of gesture.
Hook-shaped trichoderma (HZ-9) various concentration spore suspension to infected soybean cyst nematode soybean root fresh weight and
The influence result (Fig. 8) of plant height.
Apply hook-shaped reesei spores suspension processing group compared with the control group, the root fresh weight and plant height meeting of soybean plant strain
There is different degrees of increase.
In short, biocontrol bacterial strain has inhibiting effect to the sporangiocyst of soy bean cyst roundworm and second instar larvae in pot experiment,
And there is certain facilitation to the growth of soybean.
3, biocontrol bacterial strain HZ-9 fermentation liquid is to the control efficiency of soybean cyst nematode and to soybean plant height and root fresh weight
Influence it is as shown in table 5, the fermentation liquid of bacterial strain HZ-9 has preferable control efficiency to soybean cyst nematode, by fermentation liquid stoste
It is applied in soybean pot experiment, sporangiocyst inhibiting rate is up to 88.9%, and highest is reduced nematode population compared with the control on root
75%, meanwhile, compared with the control, the plant height of soybean has certain increase, and processing group root fresh weight is suitable with control group.
5 biocontrol bacterial strain HZ-9 fermentation liquid of table is fresh to the control efficiency of soybean cyst nematode and to soybean plant height and root
The influence of weight
4, influence of the temperature to mycelium growth and sporulation amount
Influence of the temperature to hook-shaped trichoderma HZ-9 mycelium growth and sporulation amount, as shown in Figure 9.
Temperature has certain difference to the growth effect of bacterial strain HZ-9, wherein bacterial strain HZ-9 is 35 DEG C of Shi Busheng in temperature
It is long, it is grown when temperature is 25-28 DEG C best, but does not produce spore when temperature is 15 DEG C and 35 DEG C, temperature produces spore when being 28 DEG C
Amount is maximum.
5, influence of the illumination to mycelium growth and sporulation amount
Figure 10 is influence of the illumination condition to hook-shaped trichoderma HZ-9 mycelium growth and sporulation amount.
The influence that illumination condition grows hook-shaped trichoderma mycelia is little, but has certain influence to sporulation quantity, full exposure for 24 hours
When, sporulation quantity is maximum, reaches 2.4 × 109It is a, and when illumination and dark alternate culture, it is most disadvantageous in hook-shaped trichoderma and produces spore.
6, influence of the pH value to mycelium growth and sporulation amount
Figure 11 is influence of the different pH value to hook-shaped trichoderma HZ-9 mycelium growth and sporulation amount.
Hook-shaped trichoderma pH value be 6 when, growth and breeding reach most preferably, but the production spore of hook-shaped trichoderma to alkaline environment more
Sensitivity does not produce spore substantially when pH value is 11.0.
7, influence of the carbon source to mycelium growth and sporulation amount
Figure 12 is influence of the carbon source to the mycelium growth and sporulation amount of hook-shaped trichoderma HZ-9.
8 kinds of carbon sources mycelium growth and sporulation amount compared with carbon-free control has certain difference, and different carbon source is raw to mycelia
Long to only exist little difference, hook-shaped trichoderma is grown most fastly on the culture medium using soluble starch as carbon source, at the same with
Galactolipin is that sporulation quantity is maximum on the culture medium of carbon source, reaches 4.8 × 108It is a.
8, influence of the nitrogen source to mycelium growth and sporulation amount
Figure 13 is influence of the nitrogen source to hook-shaped trichoderma (HZ-9) mycelium growth and sporulation amount.Hook-shaped trichoderma is being with yeast extract
It is grown on the culture medium of nitrogen source and breeds best, its sporulation quantity can reach 0.4 × 10 when 5d8It is a.
Claims (7)
1. a kind of biocontrol bacterial strain HZ-9, which is characterized in that be named as hook-shaped Trichoderma HZ-9, deposit number is CCTCC NO:M
2016207, the preservation time is on April 19th, 2016.
2. biocontrol bacterial strain HZ-9 as described in claim 1, which is characterized in that the biological property of the biocontrol bacterial strain HZ-9
Are as follows: it is in fluffy colony, mycelia is undeveloped, and conidiophore is transparent, and wall is smooth, and main shaft is straight, and top is tapered, mitogenetic spore
Sub- green, ellipse, long × roomy small range are 2.6-3.4 μm of 3.5-4.9 μ m.
3. biocontrol bacterial strain HZ-9 as described in claim 1, which is characterized in that the ITS sequence such as SEQ of the biocontrol bacterial strain HZ-9
Shown in ID NO.1, eEF1a1 sequence is as shown in SEQ ID NO.2.
4. application of the biocontrol bacterial strain HZ-9 as described in claim 1 in prevention and treatment crops cyst roundworm, which is characterized in that institute
The crops stated are soybean.
5. application as claimed in claim 4, which is characterized in that the kind of the soybean is to close rich 55.
6. such as the described in any item applications of claim 4~5 characterized by comprising
(1) biocontrol agent of the biocontrol bacterial strain HZ-9 is prepared；
(2) biocontrol agent is applied on the crops.
The biocontrol agent is spore suspension, the fermentation liquid of biocontrol bacterial strain or the dilution of spore suspension of biocontrol bacterial strain.
7. application as claimed in claim 6, which is characterized in that in the biocontrol agent, the biocontrol bacterial strain HZ-9's is dense
Degree is 106~109cfu/mL。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1066959A (en) * | 1991-05-23 | 1992-12-16 | 韩国化学技术研究院 | The improvement preparation technology of imbedded microbe insecticide and the insecticide for preparing by this technology |
| CN102532247A (en) * | 2011-11-08 | 2012-07-04 | 华南农业大学 | Nematicidal compound derived from trichoderma virens as well as preparation method and application thereof |
| CN105132296A (en) * | 2015-10-19 | 2015-12-09 | 中国农业科学院蔬菜花卉研究所 | Hook-like trichoderma strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1066959A (en) * | 1991-05-23 | 1992-12-16 | 韩国化学技术研究院 | The improvement preparation technology of imbedded microbe insecticide and the insecticide for preparing by this technology |
| CN102532247A (en) * | 2011-11-08 | 2012-07-04 | 华南农业大学 | Nematicidal compound derived from trichoderma virens as well as preparation method and application thereof |
| CN105132296A (en) * | 2015-10-19 | 2015-12-09 | 中国农业科学院蔬菜花卉研究所 | Hook-like trichoderma strain and application thereof |
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