CN108690821B - One plant height imitates bacillus amyloliquefaciens and its microbial inoculum and application - Google Patents
One plant height imitates bacillus amyloliquefaciens and its microbial inoculum and application Download PDFInfo
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Abstract
The invention discloses plant height effect bacillus amyloliquefaciens and its microbial inoculum and applications, the wherein entitled Bacillus amyloliquefaciens of the Latin of the bacillus amyloliquefaciens, bacterial strain number is HW05, is CGMCC No.10273 in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.The efficient bacillus amyloliquefaciens of the present invention have obvious inhibiting effect to Fusarium oxysporum, phytophthora blight of pepper, Ralstonia solanacearum, grey Magnaporthe grisea, colletotrichum gloeosporioides Penz and root-knot nematode.HW05, which is administered alone, has efficient prevention and control effect to foliage disease melon powdery mildew, mango anthracnose and mulberry tree brown leaf rust, and HW05 and Paecilomyces lilacinus E16 cooperation prevention and treatment soil, which pass banana blight and banana root knot nematode disease evil, has good synergistic effect.
Description
Technical field
The present invention relates to biocontrol of plant disease field more particularly to a plant height effect bacillus amyloliquefaciens and microbial inoculum and
Using.
Background technique
By Fusarium oxysporum (Fusarium oxysporum), phytophthora blight of pepper (Phytophthora nicotianae),
Crop caused by Ralstonia solanacearum (Ralstonia solanacearum), root-knot nematode (Meloidogyne spp.) is withered
The destructiveness soil-borne disease such as disease, bacterial wilt, root rot and root knot nematode disease, and grey Magnaporthe grisea (Magnaporthe
Oryzae), monofilament shell powdery mildew (Sphaerotheca fuliginea), colletotrichum gloeosporioides Penz (Colletotrichum
Gloeosporioides), melon powdery mildew, mango anthracnose, rice rice caused by mulberry rust spore rest fungus (Aecidium mori)
The foliage diseases such as seasonal febrile diseases, mulberry tree brown leaf rust bring about great losses to China's agricultural production.The use of chemical drug can rise in a short time
To apparent control efficiency, but its there are drug resistance, agriculture is residual a series of problems, such as, can no longer meet the requirement of people, change
The application of medicine in agricultural production is increasingly restricted, and biological control is the cutting edge technology that developed recently gets up, and has safety,
The advantage of environmental protection, it has also become research hotspot.
Bacillus (Bacillus sp.) has heat-resisting, uvioresistant, resistance to electromagnetic radiation and to some kinds of chemistry
Bad condition caused by substance has the characteristics such as tolerance, therefore Biocontrol Bacillus and its preparation have in plant disease prevention and control
Have broad application prospects, but have a single function limitation since current single bacterial strain exists, is not met by production requirement, also needs
Bacterial strain wider with antimicrobial spectrum, that there is synergistic function with other biocontrol microorganisms is separated, is multiple diseases using such bacterial strain
Efficient prevention and control provide technical support.
Summary of the invention
The present invention provides a plant height and imitates bacillus amyloliquefaciens, solves bacillus amyloliquefaciens function list in the prior art
One the problem of.
The technical solution adopted by the present invention is as follows:
The present invention provides a plant height and imitates bacillus amyloliquefaciens, the entitled Bacillus of Latin
amyloliquefaciens；Bacterial strain number is HW05；Preservation mechanism: China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center；Preservation mechanism abbreviation: CGMCC；Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Preservation date: 2015 01
The moon 04；Collection is registered on the books number: CGMCC No.10273
The application of bacillus amyloliquefaciens of the invention in production thallus and/or metabolin.
The present invention also provides a kind of microbial inoculum, the microbial inoculum is prepared by bacillus amyloliquefaciens of the invention.
Further, the microbial inoculum includes at least one in the thallus and metabolite of bacillus amyloliquefaciens of the invention
Kind.
Further, the microbial inoculum is for preventing and treating the plant disease as caused by pathogen, and the microbial inoculum is for preventing and treating by disease
Plant disease caused by the original, the pathogen include Fusarium oxysporum, phytophthora blight of pepper, Ralstonia solanacearum, root knot line
Worm, grey Magnaporthe grisea, monofilament shell powdery mildew, colletotrichum gloeosporioides Penz and mulberry rust spore rest fungus, the plant disease includes banana blight, Hu
Green pepper seasonal febrile diseases, bacterial wilt of tomato, banana root knot nematode disease, rice blast, melon powdery mildew, mango anthracnose and the red rust of mulberry tree
Disease.
Further, the microbial inoculum includes for preventing and treating the plant leaf surface disease as caused by pathogen, the pathogen
Grey Magnaporthe grisea, monofilament shell powdery mildew, colletotrichum gloeosporioides Penz and mulberry become rusty spore rest fungus, the plant leaf surface disease include rice blast,
Melon powdery mildew, mango anthracnose and mulberry tree brown leaf rust.
The present invention also provides a kind of composite bacteria agent, the composite bacteria agent is by bacillus amyloliquefaciens of the invention and pale purple quasi-
Mould E16 is prepared.
Further, the composite bacteria agent include in the thallus and metabolin of bacillus amyloliquefaciens of the invention at least
One kind further includes at least one of thallus and metabolin of Paecilomyces lilacinus E16.
Further, the composite bacteria agent is for preventing and treating the plant soil-borne diseases as caused by pathogen, the pathogen
Including Fusarium oxysporum and root-knot nematode, the plant soil-borne diseases include banana blight and banana root knot nematode disease.
The present invention also provides the separation methods of the bacillus amyloliquefaciens HW05, comprising the following steps:
1) soil sample is air-dried and is mixed, 20g is weighed after sieving and is put into the triangular flask equipped with 200mL sterile water and bead
In, sufficiently vibrate, it is dilute to 1 × 10 by gradient concentration method after standing-2、1×10-3, each gradient dilution liquid is in 80 DEG C of heating water baths
15-20min；
2) preparation contains the culture medium of banana blight bacteria (i.e. Fusarium oxysporum): drawing 500 μ L Fusarium oxysporum spores
On suspension to PDA solid medium, preculture 8-12h；
3) it prepares the culture medium containing biocontrol microorganisms Paecilomyces lilacinus E16: drawing 500 μ L Paecilomyces lilacinus E16 spore suspensions
Liquid is applied on PDA solid medium, preculture 16-24h；
4) it primary dcreening operation: takes each gradient soil dilution liquid of 1mL to be coated on the culture medium containing Fusarium oxysporum, is placed in 28 DEG C
It is cultivated 2-3 days under constant incubator, the single colonie purifying that picking inhibition zone occurs is transferred to the preservation of LB or NA slant medium, makees
Secondary screening is used for for strain to be tested；
5) secondary screening: taking bacterium to be measured to be inoculated in LB culture solution, is placed in 37 DEG C of shaking table, 150r/min, cultivates 12-24h, takes fermentation
Liquid takes supernatant to be centrifuged, and through filtering with microporous membrane, takes filtrate；With punch respectively in the culture medium containing Fusarium oxysporum and
It is punched on the PDA solid medium of culture medium containing Paecilomyces lilacinus E16, is separately added into bacterium filtrate to be measured, after cultivating 2-3d,
Selection antibacterial circle diameter on the culture medium of Fusarium oxysporum is maximum, while without obvious on the culture medium of Paecilomyces lilacinus E16
The target Bacillus strain of inhibition zone, finally obtains bacillus amyloliquefaciens of the present invention.
Compared with prior art, the beneficial effects of the present invention are:
Bacillus amyloliquefaciens HW05 of the present invention is to phytopathy original Fusarium oxysporum, phytophthora blight of pepper, green withered thunder Er Shi
Bacterium, root-knot nematode, grey Magnaporthe grisea and colletotrichum gloeosporioides Penz have obvious inhibiting effect, to biocontrol microorganisms Paecilomyces lilacinus E16 without obvious
Inhibiting effect.HW05, which is administered alone to have foliage disease melon powdery mildew, mango anthracnose, mulberry tree brown leaf rust, efficiently to be inhibited to make
With for prevention and control soil biography banana blight and root-knot nematode disease with good synergy work after cooperating with Paecilomyces lilacinus E16
With being embodied in can effectively colonize in the soil, reduce the Fusarium oxysporum in soil and root-knot nematode quantity, improve soil
Respiratory rate and growth promotion to plant.
Detailed description of the invention
Fig. 1-A is the colonial morphology of the bacillus amyloliquefaciens HW05 of the embodiment of the present invention one；
Fig. 1-B is the gemma form of the bacillus amyloliquefaciens HW05 of the embodiment of the present invention one；
Fig. 2-A is the Fusarium oxysporum hypha form that clear water processing is added in the embodiment of the present invention four；
Fig. 2-B is the Fusarium oxysporum hypha form that the processing of HW05 microbial inoculum is added in the embodiment of the present invention four；
Fig. 2-C is the Fusarium oxysporum hypha form that the processing of D34 microbial inoculum is added in the embodiment of the present invention four；
Fig. 3-A is the Fusarium oxysporum spore shape that clear water processing is added in the embodiment of the present invention four；
Fig. 3-B is the Fusarium oxysporum spore shape that the processing of HW05 microbial inoculum is added in the embodiment of the present invention four；
Fig. 3-C is the Fusarium oxysporum spore shape that the processing of D34 microbial inoculum is added in the embodiment of the present invention four；
Specific embodiment
Below by specific embodiment combination attached drawing, invention is further described in detail.
Experimental method used in the embodiment of the present invention is conventional method unless otherwise specified.
Material used in the embodiment of the present invention, reagent etc., are commercially available unless otherwise specified.
NA culture solution used in the embodiment of the present invention: beef extract 3g/L, yeast extract 1g/L, peptone 5g/L, glucose
10g/L, with distilled water constant volume, pH 7.0.
LB culture solution used in the embodiment of the present invention: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L are used
Distilled water constant volume.
LB culture medium used in the embodiment of the present invention is the solid medium that agar is added in above-mentioned LB culture solution and obtains.
PDA culture solution used in the embodiment of the present invention: potato 200g/L, glucose 20g/L.
PDA culture medium used in the embodiment of the present invention: being that agar (15g/L) consolidating of obtaining is added in above-mentioned PDA culture solution
Body culture medium.
Sickle-like bacteria selective medium in the embodiment of the present invention: see document (Jing Xiaohui, Wu Lunying, Qu little Ling, Zhu Li
Woods, Xue Yuxiao, Wu Lin, a kind of selective medium [J] tropical crops journal of easy separation banana blight bacteria of Huang Junsheng,
2009,30(11):1671-1673.)。
Paecilomyces lilacinus selective medium in the embodiment of the present invention: see document (Ren Wenbin, Jiang Guifang, Zhang Shiqing, Huang
The pretty influence agro-environment science journal given birth to fermentation condition and produce chitosanase to Paecilomyces varioti E7 bacterial strain, 2006.25
(3),812-816.)
Wilt/Paecilomyces lilacinus detection method uses dilution spread flat band method, reference literature (Li Fu in soil
Kerria, Yu Ziniu, Beijing He Shaojiang agromicrobiology experimental technique [M]: Chinese agriculture publishing house, 1996:32-34,305-
306.)。
Fusarium oxysporum, phytophthora blight of pepper, Ralstonia solanacearum, the root-knot nematode, glue spore anthrax being related in embodiment
Bacterium, grey Magnaporthe grisea, bacillus subtilis BSA-6, bacillus amyloliquefaciens C10-1, bacillus amyloliquefaciens bwa7 conciliate starch
Bacillus D34 is provided by Research Institute of Environment and Plant Protection, Chinese Academy of Tropi, can be by the prior art normal
Rule method obtains.
The open source literature of bacillus subtilis BSA-6: at the beginning of Pan Jiangyu Bacillus subtillis BSA-6 preventing and controlling banana fusarium wilt
Step research [D] University Of Hainan .2013, Haikou.
The open source literature of bacillus amyloliquefaciens C10-1: the identification and biological and ecological methods to prevent plant disease, pests, and erosion of Cao Hong banana blight Antagonistic Fungi C10-1
Potential Evaluation [D] Hua Zhong Agriculture University .2013, Hubei Wuhan.
The open source literature of bacillus amyloliquefaciens bwa7: Xue Yu vertical bamboo flute bacillus amyloliquefaciens bwa7 colonizing on banana
And its Primary Study [D] the University Of Hainan .2012 of Biocontrol Mechanism, Haikou.
The open source literature of Fusarium oxysporum and bacillus amyloliquefaciens D34: Cao Zhichun constructs banana blight antagonism brood cell
Bacillus multiplex screening system and preventive effect research [D] University Of Hainan, 2016, Haikou.
The open source literature of Paecilomyces lilacinus E16: mono- plant of Huang Junsheng, Wang Jun, Liang Changcong, Deng Guoping, Ren Wenbin, Guo Lijia
Paecilomyces lilacinus and its application (ZL201210358765.3).
The separation and identification of embodiment one, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HW05
1, the separation of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HW05
From Chinese Hainan Province's Wenchang Mangrove mud soil, soil sample is acquired, soil sample is air-dried and is mixed, is claimed after sieving
20g is taken to be put into the triangular flask equipped with 200mL sterile water and bead, sufficiently oscillation 10min, it is dense by gradient after standing 5min
Degree method is dilute to 1 × 10-2、1×10-3, each gradient dilution liquid is in 80 DEG C of heating water bath 15-20min.
The culture medium that preparation contains banana blight bacteria (i.e. Fusarium oxysporum): Fusarium oxysporum spore suspension is taken (about
1.6×105A/mL), draw the PDA solid medium that 500 μ L spore suspensions are applied in the culture dish that diameter is 15cm
On, preculture 8-12h.
Preparation the culture medium containing biocontrol microorganisms Paecilomyces lilacinus E16: take Paecilomyces lilacinus E16 spore suspension (about 1.8 ×
105A/mL), it draws on the PDA solid medium that 500 μ L spore suspensions are applied in the culture dish that diameter is 15cm, it is pre- to train
Support 16-24h.
1) primary dcreening operation
It takes each gradient soil dilution liquid of 1mL to be coated on the culture medium containing Fusarium oxysporum, is placed in 28 DEG C of constant temperature training
It supports and is cultivated 2-3 days under case, the single colonie purifying that picking inhibition zone occurs is transferred to the preservation of LB or NA slant medium, as to be measured
Bacterial strain is used for secondary screening.
2) secondary screening: taking bacterium to be measured to be inoculated in LB culture solution, is placed in 37 DEG C of shaking table, 150r/min, cultivates 12-24h, takes 2mL
Fermentation liquid centrifugation (12000r/min is centrifuged 15min), takes supernatant, through 0.22 μm of filtering with microporous membrane, takes filtrate.With diameter 6mm
Punch respectively in the PDA solid culture of the culture medium containing Fusarium oxysporum and the culture medium containing Paecilomyces lilacinus E16
It is punched on base, is separately added into bacterium filtrate to be measured, observe inhibition zone transparency after cultivating 2-3d, calculated antibacterial circle diameter, repeat 3
Secondary, selection antibacterial circle diameter on the culture medium containing Fusarium oxysporum is maximum, while on the culture medium of Paecilomyces lilacinus E16
Target Bacillus strain without obvious inhibition zone, finally obtains bacillus amyloliquefaciens (Bacillus
amyloliquefaciens)HW05。
2, the identification of (Bacillus amyloliquefaciens) HW05CGMCC No.10273
(1) Morphological Identification
By the bacillus amyloliquefaciens isolated and purified (Bacillus amyloliquefaciens) HW05CGMCC
For No.10273 strain inoculated on LB culture medium, 37 DEG C of culture 8h observe colonial morphology.The bacterium colony of culture 48h is separately taken, it is appropriate dilute
In microscopically observation after releasing.Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) as the result is shown
HW05CGMCC No.10273 bacterial strain is that circle, milky, edge-smoothing are neat in LB culture medium early period, the later period be subcircular,
Faint yellow, dry tack free forms that gauffer, opaque, edge is slightly raised, such as Fig. 1-A.Gram-positive, thallus rod-short,
Produce gemma, gemma oval, such as Fig. 1-B.
(2) Molecular Identification
The 16S of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HW05CGMCC No.10273
Shown in sequence 1 (SEQ ID NO:1) in rDNA complete sequence such as sequence table, overall length 1457bp.
(3) there is the lipopeptid class functional gene of synthesis antagonism
Discovery solution starch bud is detected by polymerase chain reaction (Polymerase Chain Reaction, PCR) method
Spore bacillus HW05 has the lipopeptid class functional gene of synthesis antagonism, extracts the DNA of bacillus amyloliquefaciens HW05, referring to text
The primer for offering report, using HW05 DNA as template amplification lipopeptid class functional gene, discovery HW05 contain lipopeptide antibiotic sur3,
The biosynthesis related genes of ituA, ituB, ituC, ituD, fenB, fenE, bam.
Method preparation in the primer reference literature: Cheng Kai prevention and treatment soil passes the development of cotton verticillium wilt microbial organic fertilizer
With the Nanjing biological effect research [D]: Agricultural University Of Nanjing, 2010；Li Hui bacillus amyloliquefaciens and its biosynthesis ring grease
The Nanjing peptide antibiotic condition research [D]: Nanjing University, 2012；Athukorala S N P,Fernando W G D,Rashid
K Y.Identification of antifungal antibiotics of Bacillus species isolated
from different microhabitats using polymerase chain reaction and MALDI-TOF
mass spectrometry[J].Canadian journal of microbiology,2009,55(9):1021-1032.
Embodiment two, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HW05 CGMCC
No.10273 has the activity of sulfuric-resisting streptomysin and ampicillin
Being tested by antibiotic marker, which can induce HW05, has sulfuric-resisting streptomysin and amicillin resistance simultaneously, same
Growth can be stablized on the LB culture medium of 100 μ g/mL of 300 μ g/mL of Shi Hanyou streptomycin sulphate and ampicillin and keep antagonism
The activity of the pathogens such as Fusarium oxysporum.It, can be with antibiosis since it is with streptomycin sulphate and amicillin resistance characteristic
Element is used in conjunction with, to play the role of the germ contamination in anti-nature, be conducive to scale fermenting and producing HW05 bacterial strain and
HW05 microbial inoculum makes its antibacterial pollution of energy in applying as microbial inoculum, while being conducive to detect HW05 in the survival energy in field
Power.This is also spy of the bacillus amyloliquefaciens HW05 of the present invention better than bacterial strains such as the Biocontrol Bacillus of existing prevention and control disease
Property.
Embodiment three: the method that microbial inoculum is prepared by bacillus amyloliquefaciens HW05 CGMCC No.10273:
The method that bacillus amyloliquefaciens HW05CGMCC No.10273 prepares microbial inoculum: the solution for taking inclined-plane LB culture medium to save
Bacillus amyloliquefaciens HW05, scribing line to LB culture medium flat plate are placed in 37 DEG C, and culture 8h is chosen after growing single colonie with oese
It takes HW05 to be seeded to the triangular flask (50mL) equipped with 20mL LB culture solution Nei, is placed on shaking table, under the conditions of 37 DEG C, with
160rpm constant-temperature shaking culture 2-3h；Then the inoculum concentration access for being aseptically 1% according to volumn concentration is equipped with
In the triangular flask (500mL) of 500mL LB culture solution；Shaking flask after inoculation is trained under the conditions of 37 DEG C with the speed oscillation of 160rpm
14h is supported, bacillus amyloliquefaciens HW05 fermentation liquid, i.e. microbial inoculum are obtained.The microbial inoculum of the present embodiment contains bacillus amyloliquefaciens
HW05 thallus and metabolite, in another embodiment, microbial inoculum are the single metabolite of bacillus amyloliquefaciens HW05
Or single thallus, the invention effect of microbial inoculum selectivity antagonism pathogen of the invention can also be played, the preparation method of microbial inoculum is not
Limit the protection scope of microbial inoculum of the present invention.
Microbial inoculum prepared by example IV, embodiment three has antagonism to a variety of pathogens
The preparation method reference implementation example three of bacillus amyloliquefaciens D34 microbial inoculum.
Referring to embodiment one, prepare respectively containing Fusarium oxysporum, Phytophthora capsici, grey Magnaporthe grisea, colletotrichum gloeosporioides Penz PDA
Culture medium, the LB culture medium containing Ralstonia solanacearum utilize punch at distance center 2cm after 28 DEG C of preculture 1d
HW05 microbial inoculum, the D34 microbial inoculum of the preparation of 20 μ L embodiments three is added in punching with liquid-transfering gun respectively, adds isometric clear water conduct pair
According in 28 DEG C of incubator culture 3d, observation inhibition zone, every experiment is repeated 3 times.Its antibacterial circle diameter (the results are shown in Table 1) is measured, is sent out
Existing HW05 has antagonistic activity to a variety of opportunistic pathogen bacterium of above-mentioned disease, can be applied to the prevention and treatment of soil biography and foliage disease.
The Fusarium oxysporum silk around picking antagonism circle and spore are placed in confocal laser scanning microscope discovery simultaneously:
It is full smooth (Fig. 2-A) that clear water treated Fusarium oxysporum mycelia is added, is added HW05 microbial inoculum treated bacterium
Silk ultimate swelling, top width reduce, and have obvious teratogenesis, (Fig. 2-B) shows that HW05 microbial inoculum can obviously inhibit sharp spore reaping hook
The growth of bacterium mycelia, and the Fusarium oxysporum Mycelial growth of D34 microbial inoculum processing acts on unobvious (Fig. 2-C).
The Fusarium oxysporum spore that clear water processing is added normally sprouts (Fig. 3-A)；It is added HW05 microbial inoculum treated sharp spore
Sickle-like bacteria spore both ends are expanded, and content is degraded, and are formed micropore (Fig. 3-B)；The processing of D34 microbial inoculum is added and is mainly shown as inhibition
Sharp spore reaping hook spore germination, to spore without degradation (Fig. 2-C).
Table 1
Embodiment five: the inhibiting effect of microbial inoculum prepared by embodiment three to banana root-knot nematode
Taking 1mL concentration is the root-knot nematode egg suspension of 120/mL, and packing adds into 2mL centrifuge tube, then toward centrifuge tube
Enter the HW05 microbial inoculum of 100 μ L embodiments three preparation.
Wherein banana root-knot nematode worm egg suspension is by Meloidogyne incognita (Meloidogyne incongnita) and pawl
Root-knot nematode (Meloidogyne javanica) forms according to 1:1.
The D34 microbial inoculum that control group is then separately added into equivalent sterile water and prepared by embodiment three.Each experiment is repeated 3 times, system
Egg hatching rate is counted, the results are shown in Table 2.
Egg hatching rate (%)=[larvae number/(larvae number+do not hatch ovum grain number)] × 100
Egg hatching inhibiting rate (%)=[(control egg hatching rate one handles egg hatching rate)/control egg hatching rate] × 100
Table 2
The result shows that the egg hatching inhibiting rate that the egg hatching rate compareed when to 8d is handled up to 92.34%, HW05 microbial inoculum reaches
61.21%, better than the egg hatching inhibiting rate of D34 microbial inoculum processing.
Embodiment six: microbial inoculum prepared by embodiment three is to Paecilomyces lilacinus E16 without obvious inhibiting effect
Bacillus subtilis BSA-6 microbial inoculum, bacillus amyloliquefaciens C10-1 microbial inoculum, bacillus amyloliquefaciens bwa7 microbial inoculum,
The preparation method reference implementation example three of bacillus amyloliquefaciens D34 microbial inoculum.Bacillus subtilis BSA-6 microbial inoculum, solution starch gemma
Bacillus C10-1 microbial inoculum, bacillus amyloliquefaciens bwa7 microbial inoculum, bacillus amyloliquefaciens D34 microbial inoculum are prepared by embodiment three
The comparison microbial inoculum of HW05 microbial inoculum is prepared contained banana blight bacteria (i.e. Fusarium oxysporum) and light respectively using plate antagonism method
The PDA culture medium of purple Paecilomyces varioti E16, after 28 DEG C of preculture 10-24h, with punch successively it is equidistant it is each make a call to 5 holes, respectively
The microbial inoculum of 20 μ L embodiment tripartite's methods preparation is respectively added in 3 holes, equivalent sterile water, nothing is respectively added in remaining 2 hole
Bacterium LB culture solution is placed in 28 DEG C of incubators as control and cultivates 72h, count its antibacterial bandwidth.The results are shown in Table 3.
Wherein ,-represent unrestraint；+ represent weak antagonism, antibacterial bandwidth 1-5mm；++ stronger antagonism is represented, inhibition zone is straight
Diameter 5-10mm；+++ represent strong antagonism, antibacterial bandwidth > 10mm.
Table 3
The result shows that HW05 microbial inoculum has strong antagonism to banana blight bacteria, to E16 microbial inoculum without obvious inhibiting effect；
Comparison microbial inoculum BSA-6 microbial inoculum, C10-1 microbial inoculum, bwa7 microbial inoculum and D34 microbial inoculum have strong antagonism to banana blight bacteria, to E16
Also there is relatively strong anti-effect.To sum up, illustrate that HW05 and root-knot nematode and wilt disease biocontrol microorganisms Paecilomyces lilacinus E16 have compatibility,
Be conducive to prepare composite bacteria agent for the efficient prevention and control to a variety of soil-borne diseases such as wilt disease and root knot nematode disease.
Embodiment seven, embodiment three prepare bacillus amyloliquefaciens HW05 microbial inoculum to the preventive and therapeutic effect of melon powdery mildew
The preparation of microbial inoculum D34 microbial inoculum is compared referring to embodiment three.
It is carried out in Sanya, Hainan greenhouse microcellular, selects the Muskmelon Plants of early stage, every plot area 1m*
12m, point 3 processing, the control of (1) clear water: sprays clear water；(2) D34 is compareed: being taken 100mLD34 microbial inoculum to dilute 100 times, is sprayed；
(3) it HW05 microbial inoculum: takes 100mL HW05 microbial inoculum to dilute 100 times, sprays.Each 20 plants of processing, is repeated 3 times.Greenhouse is placed in,
Conventional water and fertilizer management investigates disease index, protection effect, plant height, pol after 7d, the results are shown in Table 4.
Melon powdery mildew occurring degree statistics is divided into following 5 standards
0 grade: disease-free；
1 grade: lesion area accounts for 20% or less entire blade area；
2 grades: lesion area accounts for the 20-50% of entire blade area；
3 grades: lesion area accounts for the 50%-70% of entire blade area；
4 grades: lesion area accounts for 70% or more of entire blade area.
Disease index=100 × ∑ (morbidity strain number × grades at different levels represent number)/(investigation total strain number × superlative degree represents
Value)
Control efficiency=(control disease index-experiment disease index)/control disease index × 100%
Table 4
The result shows that melon powdery mildew scab disappears substantially after application HW05 microbial inoculum 7d, recurrence is less, illustrates to solve starch bud
Spore bacillus HW05 has good protection effect to melon powdery mildew, and improves plant height and pol content.
Embodiment eight: embodiment three prepares bacillus amyloliquefaciens HW05 microbial inoculum and acts on the prevention and control of mango anthracnose
The preparation of microbial inoculum D34 microbial inoculum is compared referring to embodiment three.
It takes colletotrichum gloeosporioides Penz spore suspension to be sprayed on mango excised leaf surface, is placed in 28 DEG C of moisturizing cultures, it is to appear
Fragmentary scab, sprays medicament, the control of (1) clear water: point three processing spray clear water；(2) D34 microbial inoculum: 100mL D34 microbial inoculum is taken
100 times of dilution, sprays；(3) it HW05 microbial inoculum: takes 100mL HW05 microbial inoculum to dilute 100 times, sprays.20 tooth in vitro of each processing
Piece is repeated 3 times.Disease index is investigated after the above processing 3d, control efficiency is calculated, the results are shown in Table 5.Disease index and control efficiency
Statistics is referring to embodiment seven.
Table 5
The results show that the scab of mango anthracnose disappears substantially after application HW05 3d, recurrence is less, illustrates to solve starch bud
Spore bacillus HW05 has good protection effect to mango anthracnose, reaches 86.08%, is significantly better than comparison bacterial strain D34.
Embodiment nine, embodiment three prepare bacillus amyloliquefaciens HW05 microbial inoculum to the preventive and therapeutic effect of mulberry tree brown leaf rust
The preparation of microbial inoculum D34 microbial inoculum is compared referring to embodiment three.
In the mulberry tree of the mulberry tree planting site selection mulberry tree brown leaf rust early stage of Hainan Island, point three processing, (1) clear water
Control: clear water is sprayed；(2) it D34 microbial inoculum: takes 100mL D34 microbial inoculum to dilute 100 times, sprays；(3) HW05 microbial inoculum: 100mL is taken
HW05 microbial inoculum dilutes 100 times, sprays.Each 20 plants of processing, is repeated 3 times.Disease index is investigated after the above processing 3d, calculates prevention and treatment
Effect acquires the micro- sem observation scab of blade, the results are shown in Table 6.Disease index and control efficiency statistics are referring to embodiment seven.
Table 6
The result shows that the scab of mulberry tree brown leaf rust disappears substantially after application HW05 microbial inoculum 3d, recurrence is less, illustrates to solve starch
Bacillus HW05 has good protection effect to mulberry tree brown leaf rust, reaches 84.07%, is significantly better than control strain D34.
Embodiment ten: the preparation side of bacillus amyloliquefaciens HW05 microbial inoculum, Paecilomyces lilacinus E16 microbial inoculum and its composite bacteria agent
Method
The preparation of bacillus amyloliquefaciens HW05 microbial inoculum: the bacillus amyloliquefaciens HW05 for taking inclined-plane NA culture medium to save,
Scribing line is placed in 37 DEG C, culture 8h is seeded to dress with sterile toothpick picking HW05 after growing single colonie to LB culture medium flat plate
It in the triangular flask (50mL) for having 20mL LB culture solution, sets on controllable temperature shaking table, temperature is 37 DEG C, 160rpm speed, shaken cultivation
2-3h；Then aseptically according to the inoculum concentration access of volume ratio 1% equipped in the triangular flask of 200mL LB culture solution
(500mL)；Shaking flask after inoculation, with the speed oscillation culture 12-24h of 160rpm, obtains HW05 microbial inoculum under the conditions of 37 DEG C.
The preparation of Paecilomyces lilacinus E16 microbial inoculum: the Paecilomyces lilacinus E16 for taking inclined-plane PDA culture medium to save, scribing line to PDA
Culture medium flat plate is placed in 28 DEG C, and culture 48h is seeded to sterile toothpick picking E16 equipped with 20mL after growing single colonie
It in the triangular flask (50mL) of PDA culture solution, sets on controllable temperature shaking table, temperature is 28 DEG C, 150rpm, shaken cultivation 12h；Then exist
The inoculum concentration access for being 1% according to volume ratio under aseptic condition is equipped with (500mL) in the triangular flask of 200mL PDA culture solution；It connects
Shaking flask after kind, with the speed oscillation culture 48-72h of 160rpm, obtains E16 microbial inoculum under the conditions of 28 DEG C.
Composite bacteria agent: isometric HW05 microbial inoculum and the mixing of E16 microbial inoculum is taken to be formulated.
Embodiment 11: bacillus amyloliquefaciens HW05 microbial inoculum and the compound collaboration of Paecilomyces lilacinus E16 microbial inoculum prevent and treat banana
Wilt disease
HW05 microbial inoculum, E16 microbial inoculum and the composite bacteria agent that the present embodiment is selected are prepared by the method for embodiment ten respectively.
In Hainan Danzhou banana base greenhouse, prepares five each plastic flowerpots of the loading without banana blight soil, carry out 5
A experimental group.Experiment 1 is tested for blank control, is not applied banana blight bacteria (i.e. Fusarium oxysporum) spore suspension, is not applied
With HW05 microbial inoculum and E16 microbial inoculum, 200mL clear water is applied；Experiment 2 compares for banana blight bacteria, applies 100mL banana blight
Bacterium spore suspension does not apply microbial inoculum, applies 100mL clear water；Experiment 3 is tested for HW05 microbial inoculum, applies 100mL banana blight
Bacterium spore suspension takes 100mL HW05 microbial inoculum, by 105The bacterium amount of cfu/g soil accesses soil；Experiment 4 is tested for E16 microbial inoculum,
100mL banana blight bacteria spore suspension is applied, 100mL E16 microbial inoculum is taken, by 105The bacterium amount soil of cfu/g soil accesses soil；
Experiment 5 is tested for composite bacteria agent, is applied 100mL banana blight bacteria spore suspension, is taken 50mL HW05 microbial inoculum and 50mL E16
After microbial inoculum mixes, by 105The bacterium amount of cfu/g soil accesses soil.The above experiment soil treatment 5d, takes each processing soil sample using dilution
Colony counting method is detected through wilt selective medium, is compared to the banana blight bacteria quantity in microbial inoculum processing soil
After declining at least 2 orders of magnitude according to the banana blight bacteria quantity in soil, 5 leaf Brazil any of several broadleaf plants are transplanted, 50 plants of each experimental group,
It is repeated 3 times.
The above experimental plant is placed in greenhouse, conventional water and fertilizer management, after compareing and disease symptom occur, through wilt
Selective medium detects Fusarium oxysporum quantity, and Paecilomyces varioti culture medium detects Paecilomyces lilacinus E16 quantity, through containing sulfate chain
The LB culture medium of mycin and ampicillin detects HW05 quantity, and statistically upper, underground part growth indexes, soil respiration work
With, disease index and preventive effect.
Banana blight occurring degree statistics is divided into following 5 standards:
0 grade: plant is without withered and yellow symptom.
1 grade: there is slight withered and yellow symptom in plant lower blade, and tender leaf is intact, and the slight brown stain of small part root system, stem goes out
Existing water stain shape brown stain.
2 grades: there is apparent withered and yellow symptom in plant lower blade, but tender leaf is intact, and brown stain, stem and false stem occurs in root system
There is water stain shape brown stain in portion.
3 grades: there is withered and yellow symptom in whole strain, and root system brown stain is rotted, and in flakes, a small number of petioles occur red for stem and the brown stain of false stem
It is brown.
4 grades: there is withered death symptom in whole strain, and the serious brown stain of root system is rotted.
Disease index=100 × ∑ (morbidity strain number × grades at different levels represent number)/(investigation total strain number × superlative degree represents
Value)
Control efficiency=(control disease index-experiment disease index)/control disease index × 100%
The compound collaboration of bacillus amyloliquefaciens HW05 and Paecilomyces lilacinus E16 prevents and treats banana blight effect such as 7,8 institute of table
Show, disease index is lower, and control efficiency is higher.
Table 7
Table 8
The result shows that: bacillus amyloliquefaciens HW05 and Paecilomyces lilacinus E16 composite bacteria agent have very banana blight
Good protection effect and growth-promoting functions, HW05 and E16 can be colonized effectively in the soil, and had and inhibited banana blight bacterium number
Amount and the effect for improving soil respiration rates, it is quasi- using all indicators are better than single administration HW05 or pale purple after composite bacteria agent
Mould E16 illustrates that bacillus amyloliquefaciens HW05 and Paecilomyces lilacinus E16 have synergistic make to the prevention and treatment of banana blight
With.
Embodiment 12: bacillus amyloliquefaciens HW05 microbial inoculum and the compound collaboration of Paecilomyces lilacinus E16 microbial inoculum prevent and treat banana
Root-knot nematode
HW05 microbial inoculum, E16 microbial inoculum and the composite bacteria agent that the present embodiment is selected are prepared by the method for embodiment ten respectively.
Young banana base is spread in Hainan Danzhou, prepares five each plastic flowerpots of the loading without banana root knot nematode disease soil,
Carry out 5 experimental groups.Experiment 1 is tested for blank control, is not accessed banana root-knot nematode, is not applied HW05 microbial inoculum and E16 microbial inoculum,
Apply 100mL clear water；Experiment 2 compares for banana root-knot nematode, accesses 500 banana root-knot nematode second instar larvaes, does not apply bacterium
100mL clear water is applied in agent；Experiment 3 is tested for HW05 microbial inoculum, is accessed 500 banana root-knot nematode second instar larvaes, is taken 100mL
HW05 microbial inoculum, by 105The bacterium amount of cfu/g soil accesses soil；Experiment 4 is tested for E16 microbial inoculum, accesses 500 banana root-knot nematodes
Second instar larvae takes 100mL E16 microbial inoculum, by 105The bacterium amount soil of cfu/g soil accesses soil；Experiment 5 is tested for composite bacteria agent, is connect
Enter 500 banana root-knot nematode second instar larvaes, after taking 50mL HW05 microbial inoculum and 50mL E16 microbial inoculum to mix, by 105Cfu/g soil
Bacterium amount access soil.The above experiment soil treatment 5d, transplants 5 leaf Brazil any of several broadleaf plants, 50 plants of each experimental group is repeated 3 times.
Wherein the banana root-knot nematode second instar larvae of every basin access is by Meloidogyne incognita (Meloidogyne
Incongnita it) is formed with javanese root knot nematode (Meloidogyne javanica) according to 1:1.
The above experimental plant is placed in greenhouse, and conventional water and fertilizer management detects in soil after compareing and disease symptom occur
Root-knot nematode quantity, Paecilomyces lilacinus E16 quantity (being detected using Paecilomyces varioti culture medium), HW05 quantity are (through containing sulfuric acid strepto-
The LB culture medium of element and ampicillin detects), overground part and lower part growth indexes, Soil Respiration, count disease index
And preventive effect.
Banana root-knot nematode occurring degree statistics is divided into following 5 standards:
0 grade: there is no root knot or egg capsule
1 grade: 1-10 root knot or egg capsule/strain
2 grades: 10-20 root knots or egg capsule/strain
3 grades: 20-40 root knots or egg capsule/strain
4 grades: 40 or more root knots or egg capsule/strain
Disease index=100 × ∑ (morbidity strain number × grades at different levels represent number)/(investigation total strain number × superlative degree represents
Value)
Control efficiency=(control disease index-experiment disease index)/control disease index × 100%
The compound collaboration of bacillus amyloliquefaciens HW05 and Paecilomyces lilacinus E16 prevents and treats banana root-knot nematode effect such as 9 He of table
Shown in table 10, disease index is lower, and control efficiency is higher.
Table 9
Table 10
The result shows that bacillus amyloliquefaciens HW05 and Paecilomyces lilacinus E16 composite bacteria agent have banana root-knot nematode
Good protection effect and growth-promoting functions, HW05 and E16 can be colonized effectively in the soil, had and inhibited meloidogyne quantity
With improve soil respiration rates effect, using after composite bacteria agent all indicators are better than the quasi- blueness of single administration HW05 or pale purple
Mould E16 illustrates that bacillus amyloliquefaciens HW05 and Paecilomyces lilacinus E16 have synergistic make to the prevention and treatment of banana root-knot nematode
With.
Embodiment 13: bacillus amyloliquefaciens D34 microbial inoculum and the compound collaboration prevention and treatment banana of Paecilomyces lilacinus E16 microbial inoculum are withered
It withers disease
Replace bacillus amyloliquefaciens HW05 using bacillus amyloliquefaciens D34, referring to the preparation solution starch bud of embodiment ten
Spore bacillus D34 microbial inoculum, E16 microbial inoculum and composite bacteria agent 1 use bacillus amyloliquefaciens D34 bacterium referring to embodiment 11 respectively
Agent, E16 microbial inoculum and composite bacteria agent 1 carry out prevention and treatment banana blight experiment, and experimental result is as shown in table 11, and disease index is lower,
Control efficiency is higher.
Table 11
Single administration bacillus amyloliquefaciens D34 microbial inoculum or E16 microbial inoculum have preferable protection effect, multiple better than mixing application
The protection effect of combined bacteria agent 1 (D34 microbial inoculum and E16 microbial inoculum), illustrates that bacillus amyloliquefaciens D34 and Paecilomyces lilacinus E16 do not have
There is the synergistic function of prevention and treatment banana blight.
Embodiment 14: bacillus amyloliquefaciens D34 microbial inoculum and the compound collaboration of Paecilomyces lilacinus E16 microbial inoculum prevent and treat Banana Root
Tie lines worm
Replace bacillus amyloliquefaciens HW05 using bacillus amyloliquefaciens D34, referring to the preparation solution starch bud of embodiment ten
Spore bacillus D34 microbial inoculum and composite bacteria agent 1 use bacillus amyloliquefaciens D34 microbial inoculum, E16 microbial inoculum referring to embodiment 12 respectively
Prevention and treatment banana root-knot nematode experiment is carried out with composite bacteria agent 1, experimental result is as shown in table 12, and disease index is lower, control efficiency
It is higher.
Table 12
The result shows that single administration bacillus amyloliquefaciens D34 microbial inoculum or E16 microbial inoculum have preferable protection effect, it is better than
The protection effect of mixing application composite bacteria agent 1 (D34 microbial inoculum and E16 microbial inoculum), illustrates bacillus amyloliquefaciens D34 and pale purple quasi- blueness
Mould E16 does not have the synergistic function of prevention and treatment banana root-knot nematode.
The above content is specific embodiment is combined, further detailed description of the invention, and it cannot be said that this hair
Bright specific implementation is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, it is not taking off
Under the premise of from present inventive concept, a number of simple deductions or replacements can also be made.
Sequence table
<110>Research Institute of Environment and Plant Protection, Chinese Academy of Tropi
<120>one plant heights imitate bacillus amyloliquefaciens and its microbial inoculum and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1457
<212> DNA
<213>bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
<400> 1
acccatctgt ccaccttcgg cggctggctc cataaaggtt acctcaccga cttcgggtgt 60
tacaaactct cgtggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg 120
catgctgatc cgcgattact agcgattcca gcttcacgca gtcgagttgc agactgcgat 180
ccgaactgag aacagatttg tgggattggc ttaacctcgc ggtttcgctg ccctttgttc 240
tgtccattgt agcacgtgtg tagcccaggt cataaggggc atgatgattt gacgtcatcc 300
ccaccttcct ccggtttgtc accggcagtc accttagagt gcccaactga atgctggcaa 360
ctaagatcaa gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga 420
cgacaaccat gcaccacctg tcactctgcc cccgaagggg acgtcctatc tctaggattg 480
tcagaggatg tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc 540
caccgcttgt gcgggccccc gtcaattcct ttgagtttca gtcttgcgac cgtactcccc 600
aggcggagtg cttaatgcgt tagctgcagc actaaggggc ggaaaccccc taacacttag 660
cactcatcgt ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt 720
cgctcctcag cgtcagttac agaccagaga gtcgccttcg ccactggtgt tcctccacat 780
ctctacgcat ttcaccgcta cacgtggaat tccactctcc tcttctgcac tcaagttccc 840
cagtttccaa tgaccctccc cggttgagcc gggggctttc acatcagact taagaaaccg 900
cctgcgagcc ctttacgccc aataattccg gacaacgctt gccacctacg tattaccgcg 960
gctgctggca cgtagttagc cgtggctttc tggttaggta ccgtcaaggt gccgccctat 1020
ttgaacggca cttgttcttc cctaacaaca gagctttacg atccgaaaac cttcatcact 1080
cacgcggcgt tgctccgtca gactttcgtc cattgcggaa gattccctac tgctgcctcc 1140
cgtaggagtc tgggccgtgt ctcagtccca gtgtggccga tcaccctctc aggtcggcta 1200
cgcatcgtcg ccttggtgag ccgttacctc accaactagc taatgcgccg cgggtccatc 1260
tgtaagtggt agccgaagcc accttttatg tctgaaccat gcggttcaaa caaccatccg 1320
gtattagccc cggtttcccg gagttatccc agtcttacag gcaggttacc cacgtgttac 1380
tcacccgtcc gccgctaaca tcagggagca agctcccatc tgtccgctcg acttgcatta 1440
taggcacgcc gcccctt 1457
Claims (10)
1. a bacillus amyloliquefaciens, which is characterized in that the Latin of the bacillus amyloliquefaciens is entitledBacillus Amyloliquefaciens,Bacterial strain number is HW05, in China Committee for Culture Collection of Microorganisms's common micro-organisms center
Number of registering on the books be CGMCC No.10273.
2. application of the bacillus amyloliquefaciens according to claim 1 in production thallus and/or metabolin.
3. a kind of microbial inoculum, which is characterized in that the microbial inoculum is prepared by bacillus amyloliquefaciens described in claim 1.
4. microbial inoculum according to claim 3, it is characterised in that: the microbial inoculum includes solution starch bud described in claim 1
At least one of thallus and metabolite of spore bacillus.
5. microbial inoculum according to claim 3 or 4, which is characterized in that the microbial inoculum is planted as caused by pathogen for preventing and treating
Object disease, the pathogen include Fusarium oxysporum, phytophthora blight of pepper, Ralstonia solanacearum, root-knot nematode, grey Magnaporthe grisea,
Monofilament shell powdery mildew, colletotrichum gloeosporioides Penz and mulberry rust spore rest fungus, the plant disease includes banana blight, pepper seasonal febrile diseases, tomato
Bacterial wilt, banana root knot nematode disease, rice blast, melon powdery mildew, mango anthracnose and mulberry tree brown leaf rust.
6. microbial inoculum according to claim 3 or 4, which is characterized in that the microbial inoculum is planted as caused by pathogen for preventing and treating
Object foliage disease, the pathogen include grey Magnaporthe grisea, monofilament shell powdery mildew, colletotrichum gloeosporioides Penz and mulberry rust spore rest fungus, the plant
Object foliage disease includes rice blast, melon powdery mildew, mango anthracnose and mulberry tree brown leaf rust.
7. a kind of composite bacteria agent, which is characterized in that the composite bacteria agent is by bacillus amyloliquefaciens described in claim 1 and light
Purple Paecilomyces varioti E16 is prepared.
8. composite bacteria agent according to claim 7, which is characterized in that the composite bacteria agent includes described in claim 1
At least one of thallus and metabolin of bacillus amyloliquefaciens further include in the thallus and metabolin of Paecilomyces lilacinus E16
At least one.
9. composite bacteria agent according to claim 7 or 8, which is characterized in that the composite bacteria agent is for preventing and treating by phytopathy
Plant soil-borne diseases caused by the original, the pathogen include Fusarium oxysporum and root-knot nematode, the plant soil-borne diseases
Including banana blight and banana root knot nematode disease.
10. bacillus amyloliquefaciens according to claim 1, which is characterized in that isolated by the following method:
1) soil sample is air-dried and is mixed, 20g is weighed after sieving and is put into the triangular flask equipped with 200mL sterile water and bead,
Sufficiently vibrate, it is dilute to 1 × 10 by gradient concentration method after standing-2、1×10-3, each gradient dilution liquid is in 80 DEG C of heating water bath 15-
20min；
2) it prepares the culture medium containing Fusarium oxysporum: drawing 500 μ L Fusarium oxysporum spore suspensions and be applied to the training of PDA solid
It supports on base, preculture 8-12h；
3) it prepares the culture medium containing biocontrol microorganisms Paecilomyces lilacinus E16: drawing 500 μ L Paecilomyces lilacinus E16 spore suspensions and apply
On cloth to PDA solid medium, preculture 16-24h；
4) it primary dcreening operation: takes each gradient soil dilution liquid of 1mL to be coated on the culture medium containing Fusarium oxysporum, is placed in 28 DEG C of constant temperature
Cultivated 2-3 days under incubator, picking inhibition zone occur single colonie purifying, be transferred to LB or NA slant medium preservation, as to
It surveys bacterial strain and is used for secondary screening；
5) secondary screening: taking bacterium to be measured to be inoculated in LB culture solution, is placed in 37 DEG C of shaking table, 150r/min, cultivates 12-24h, take fermentation liquid with
Centrifugation, takes supernatant, through filtering with microporous membrane, takes filtrate；In the culture medium containing Fusarium oxysporum and contained respectively with punch
It is punched on the PDA solid medium of Paecilomyces lilacinus E16 culture medium, is separately added into bacterium filtrate to be measured, after cultivating 2-3d, selection exists
Antibacterial circle diameter is maximum on the culture medium of Fusarium oxysporum, while without obvious inhibition zone on the culture medium of Paecilomyces lilacinus E16
Target Bacillus strain, finally obtain bacillus amyloliquefaciens of the present invention.
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| CN109845514A (en) * | 2018-12-26 | 2019-06-07 | 云南省农业科学院农业环境资源研究所 | Beneficial microorganism banana blight preventing control method is embedded based on simulation rhizosphere soil |
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| CN105018380B (en) * | 2015-07-21 | 2018-10-26 | 中国热带农业科学院环境与植物保护研究所 | One bacillus amyloliquefaciens BR25 and its cultural method and application |
| CN105132336B (en) * | 2015-10-10 | 2018-04-13 | 中国热带农业科学院环境与植物保护研究所 | One bacillus amyloliquefaciens and its microbial inoculum and application |
| CN105647829A (en) * | 2016-01-20 | 2016-06-08 | 中国热带农业科学院环境与植物保护研究所 | Bacillus amyloliquefaciens and application thereof to prevention and treatment of sugarcane red rot |
| CN106190916A (en) * | 2016-07-22 | 2016-12-07 | 中国热带农业科学院环境与植物保护研究所 | One bacillus amyloliquefaciens JNC2 and the preparation method and application of microbial inoculum thereof |
| CN106332910A (en) * | 2016-08-23 | 2017-01-18 | 广西易多收生物科技有限公司 | Pharmaceutical composition for killing burrowing nematode of banana trees and preparation method thereof |
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