CN110331112A - One plant of Pseudomonas fluorescens for preventing and treating rice blast and its application - Google Patents
One plant of Pseudomonas fluorescens for preventing and treating rice blast and its application Download PDFInfo
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- CN110331112A CN110331112A CN201910695393.5A CN201910695393A CN110331112A CN 110331112 A CN110331112 A CN 110331112A CN 201910695393 A CN201910695393 A CN 201910695393A CN 110331112 A CN110331112 A CN 110331112A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
- C12R2001/39—Pseudomonas fluorescens
Abstract
The Pseudomonas fluorescens (Pseudomonas fluorescens) for preventing and treating rice blast the invention discloses one plant and its application, belong to microorganisms technical field.Pseudomonas fluorescens (Pseudomonas fluorescens) S149 of the invention, it include: to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), the deposit date is on March 1st, 2019, deposit number was CGMCC NO.17278.Pseudomonas fluorescens provided by the invention can prevent and treat rice blast, there is good preventive effect to rice blast and has significant bacteriostasis, prevention and treatment high-efficient various plants pathogen, it has good rice blast control efficiency to the rice of south China and northern different lines, can reduce rice industry to a greater extent in nearly picking time because of production loss caused by burst disease.
Description
Technical field
The present invention relates to microorganisms technical field more particularly to the Pseudomonas fluorescens of one plant of prevention and treatment rice blast
(Pseudomonas fluorescens) and its application.
Background technique
Rice is one of cereal crops important in the world, and rice is the master that more than half population of the whole world is relied on
Food, rice yield occupy 1/4 or more of world food total output, yield of grain in China more than half.Rice blast is on rice
Most important disease, global 85 countries have generation and China South And North Rice Regions to endanger one of rice disease of most serious.Rice
Seasonal febrile diseases cause the Rice Yield Loss Caused of 20-30% every year, and the serious time reaches 50% or more, and annual production loss can support
6,000 ten thousand populations living.Rice blast (Rice Blast) is by sac fungus Magnaporthe oryzae (T.T.Hebert)
The sudden world that is strong, being easy to prevalence of one kind caused by Yaegashi& Udagawa (Invisible element: Pyricularia oryzae)
Property fungal disease.The disease can cause damages to rice throughout the year, harm spread rice each position, have seedling rice blast,
Leaf pest, pulvinus pest, section rice blast, panicle blast, branch obstruct pest and grain pest etc..
Resistant rice cultivars breeding, agronomic measures and chemical pesticide control are the upper most common measures of current production, due to
Physiological races of rice blast fungus easily makes a variation, and disease-resistant variety breeding typical time is longer；The residual of chemical pesticide is easy pollution ecology ring
Border threatens human health, and pathogen is easy to produce drug resistance.Therefore, a kind of couple of mankind and environmental-friendly and have good are found
The novel biocontrol bacterium of control efficiency is most important to rice industry sustainable development.
Pseudomonas fluorescens (Pseudomonas fluorescens) can effectively control plant disease, have phytopathy
The advantages that opportunistic pathogen is not easy to produce resistance and promotes plant growth is the important biocontrol microorganisms in rice blast prevention and treatment.Xu Yuquan etc. is from rice
The Pseudomonas fluorescens JKD-2 separated in careless stubble secretes a kind of extracellular protein, can dissolve Pyricularia oryzae mycelium and inhibit spore
Son is sprouted, and the preventive effect under greenhouse experiment reaches 60%.Liao Xiaolan etc. isolated pseudomonas aeruginosa (P.aeruginosa) SUB8
4.53cm is reached to the antibacterial bandwidth of Pyricularia oryzae, azophenlyene substance antibacterial substance is generated, has obviously to Pyricularia oryzae
Antagonism.
Using Protocols in Molecular Biology, carry out the research and application of novel fluorescence pseudomonad biological prevention and control agent, improves rice
Rice matter and high yield synergy.The research and application of Pseudomonas fluorescens prevention and treatment rice blast are the needs of Rice Production sustainable development,
It can satisfy the requirement that the United Nations's world food tissue develops about grain security, but also lack in this field to rice rice blast
Disease has wide spectrum and the high pseudomonas fluorescens strain of efficient preventive effect, antibacterial substance yield.
Summary of the invention
It is right the present invention provides one plant in order to solve above-mentioned agricultural production and the deficiencies in the prior art
Rice blast has good preventive effect and has the pseudomonas fluorescens strain of significant bacteriostasis to various plants pathogen.
For this purpose, the present invention uses following technical scheme.
In a first aspect, the present invention provide a fluorescent pseudomonads (Pseudomonas fluorescens) S149,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), the deposit date is March 1 in 2019
Day, deposit number is CGMCC NO.17278.
In second aspect, the present invention provides a kind of microbial inoculum, and it includes Pseudomonas fluorescens S149 as described in relation to the first aspect.
In the third aspect, the present invention provides a kind of method for preventing and treating rice blast comprising glimmering by as described in relation to the first aspect
Light pseudomonad S149 or the microbial inoculum as described in second aspect are applied to rice crop.
In prevention and treatment rice blast method of the invention, the microbial inoculum is the culture solution of the Pseudomonas fluorescens S149.
In prevention and treatment rice blast method of the invention, rice blast be seedling rice blast, leaf pest, pulvinus pest, section rice blast, panicle blast,
Branch stalk one or more of pest and grain pest.
In fourth aspect, the present invention provides Pseudomonas fluorescens S149 as described in relation to the first aspect or such as second aspect institute
Purposes of the microbial inoculum stated in prevention and treatment rice blast.
In prevention and treatment rice blast of the invention on the way, rice blast is seedling rice blast, leaf pest, pulvinus pest, section rice blast, fringe neck
Pest, branch obstruct one or more of pest and grain pest.
Beneficial effects of the present invention are as follows:
Pseudomonas fluorescens S149 provided by the invention has good preventive effect to rice blast and to various plants cause of disease
Bacterium has significant bacteriostasis, prevention and treatment high-efficient.
Pseudomonas fluorescens S149 provided by the invention has wide spectrum and efficiently fungi-proofing, antibacterial ability to rice blast,
It has good rice blast control efficiency to the rice of China's different lines, can reduce rice industry to a greater extent close
Picking time is because of production loss caused by burst disease.
Pseudomonas fluorescens S149 provided by the invention is preferable to rice growth-promoting, effect of promoting production.
Pseudomonas fluorescens S149 provided by the invention is insensitive to Multiple Classes of Antibiotics, administration of antibiotics drug or fertilizer
Afterwards, still it is able to maintain its control efficiency.
Pseudomonas fluorescens S149 provided by the invention carries out the induction of system to rice significant defense enzyme, makes defensive ferment
Activity significantly improves.
Preservation information
The present invention separates the Pseudomonas fluorescens (Pseudomonas fluorescens) of identification, and it is false single to be named as fluorescence
Born of the same parents bacterium (Pseudomonas fluorescens) S149, oneself is preserved in Chinese microorganism strain preservation pipe on March 1st, 2019
Reason committee common micro-organisms center (CGMCC) (address: the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Object research institute), deposit number is CGMCC N0. 17278.
Detailed description of the invention
The attached drawing for constituting a part of the invention is used to provide further understanding of the present invention, schematic reality of the invention
It applies example and its explanation is used to explain the present invention, do not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is the culture form of Pseudomonas fluorescens S149 bacterial strain of the invention on KB culture medium flat plate.
Fig. 2 is the plate face-off picture of Pseudomonas fluorescens S149 bacterial strain and Pyricularia oryzae P131 of the invention.
Fig. 3 is Pseudomonas fluorescens S149 fermentation culture of the invention and extracellular metabolism crude extract to Pyricularia oryzae P131
Plate stand facing each other picture.
Fig. 4 is that Pseudomonas fluorescens S149 fermentation culture of the invention and extracellular metabolism crude extract infect Pyricularia oryzae
The picture that structure inhibits.
Fig. 5 is the influence of Pseudomonas fluorescens S149 of the invention to rice seedling defensive ferment POD.
Fig. 6 is the influence of Pseudomonas fluorescens S149 of the invention to rice seedling defensive ferment PPO.
Fig. 7 is the influence of Pseudomonas fluorescens S149 of the invention to rice seedling defensive ferment SOD.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described, it is clear that described embodiments are some of the embodiments of the present invention, rather than
Whole embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise
Under every other embodiment obtained, shall fall within the protection scope of the present invention.It should be noted that the case where not conflicting
Under, the feature in embodiment and embodiment in the present invention can mutual any combination.
Embodiment 1
The separation of Pseudomonas fluorescens S149 bacterial strain and screening technique are as follows:
(1) sample acquires: acquisition sample is No. 48 rhizosphere soils of the peaceful round-grained rice of rice varieties, and place is flat for Ningxia Hui Autonomous Region
Sieve county, the village Sha Qu, the town Yao Fu, extracts together with the root system of rice, is installed with collection bag, is placed in ice chest and takes back laboratory point
From.
(2) separation method: weighing 1g rhizosphere soil, is put into 100mL sterile water oscillation under room temperature and mixes 1h, uses sterile water
According to 10,102With 103Gradient dilution is drawn gradient dilution liquid and is coated on KB agar medium plate, after drying up, 30
DEG C constant incubator in be inverted culture 48h.Picking single colonie, gradient scribing line obtains single again on KB agar medium plate
The single colonie of acquisition is stored in 40% glycerol by bacterium colony, is saved backup in 80 DEG C of ultra low temperature freezers of ﹣.
(3) screening of Pseudomonas fluorescens: Pyricularia oryzae P131 bacteria cake is placed in center on tomato oat plating medium,
It is inoculated with Pseudomonas fluorescens (scribing line 2cm) at 2cm in bacteria cake two sides after 1d and makees opposite culture, blank control is set, is placed in
28 DEG C of dark culturings of incubator.Every processing sets the repetition of 10 wares.Pathogen colony diameter and antibacterial band are measured after 5d, the results are shown in Table 1
And Fig. 2.
King ' s B agar medium (KB): peptone 20g, glycerol 10mL, K2HPO41.5g, MgSO4·
7H27.2 ± 0.1,121 DEG C of sterilizing 20min of O1.5g, agar 20.0g, pH.
Tomato oat medium: oatmeal 30g adds water 1000mL, and 1h is heated on boiling water bath, is added after filtered through gauze
0.4g Carbon Dioxide calcium and 150mL tomato juice, add water to supply 1000mL, add packing sterilizing after agar powder 17-20g (121 DEG C,
20min)。
Identified, the pseudomonas fluorescens strain feature is as follows: white, opaque, cell is Gram-negative bacteria, bar
Shape, (0.7~0.8) μ m (2.3~2.8) μm, has several polar flagellas, does not generate brood cell, can secrete yellow-green fluorescence pigment
And issue fluorescence.
Inventor is by this culture presevation in China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), the deposit date is on March 1st, 2019, deposit number is CGMCC No.17278.
Embodiment 2
The biological activity determination of Pseudomonas fluorescens S149 bacterial strain:
(1) bacteria inhibition assay of the Pseudomonas fluorescens S149 fermentation culture to Pyricularia oryzae: in tomato oat plate culture
Pyricularia oryzae P131 bacteria cake is placed in center on base, and bacteria cake two sides place sterile Oxford cup at 2cm, are separately added into fluorescence in cup
The fermentation liquid of pseudomonad S149 makees opposite culture, is control with sterile KB fluid nutrient medium, is placed in 28 DEG C of incubator dark trainings
It supports.Each processing sets the repetition of 10 wares.Pathogen colony diameter and antibacterial band are measured after 5d, the results are shown in Table 1 and Fig. 3.
(2) bacteria inhibition assay of the Pseudomonas fluorescens S149 Extracellular metabolism crude extract to Pyricularia oryzae: in tomato oat
Pyricularia oryzae P131 bacteria cake is placed in center on plating medium, and bacteria cake two sides are placed sterile Oxford cup at 2cm, distinguished in cup
Extracellular metabolism crude extract is added and makees opposite culture, is control with sterile KB fluid nutrient medium, is placed in 28 DEG C of dark of incubator
Culture.Each processing sets the repetition of 10 wares.Pathogen colony diameter and antibacterial band are measured after 5d, the results are shown in Table 1 and Fig. 3.
King ' s B liquid agar medium (KB): peptone 20g, glycerol 10mL, K2HPO41.5 g, MgSO4·
7H27.2 ± 0.1,121 DEG C of sterilizing 20min of O1.5g, pH.
The preparation of Extracellular metabolism crude extract: the strains tested S149 single colonie for having activated for 24 hours is seeded to dress respectively
In the 500mL triangular flask for having 100mL King ' s B culture solution, 28 DEG C, 180rpm/min shake culture for 24 hours after, as second level
Seed liquor is spare.Secondary seed solution is inoculated into the 500mL tri- equipped with 100mL King ' s B culture solution by 1% inoculum concentration again
In the bottle of angle, 28 DEG C, 180rpm/min shake culture 72h obtain the band bacteria culture fluid of bacterium bacterial strain.Finally gained is carried disease germs training
Nutrient solution removes thallus in 4 DEG C, 12000rpm/min centrifugation 15min, and supernatant is filtered (0.22 μm) degerming, obtains extracellular generation
Thank to product crude extract.
The opposite culture of table 1 Pseudomonas fluorescens S149 bacterial strain and Pyricularia oryzae
Note: the experimental data in table is duplicate average value ± standard error three times.
(3) healthy rice varieties biocontrol effect of Pseudomonas fluorescens S149 bacterial strain under the conditions of greenhouse pot culture: are selected
G19,1% liquor natrii hypochloritis sterilize 10min, and 70% alcohol impregnates 1cm, and aseptic water washing 5 times, 28 DEG C of growth cabinets are urged
Bud is seeded in plastics breaker (bucket base diameter 18cm × high 21cm × bucket upper diameter when bud grows to 1cm
24.5cm).Pseudomonas fluorescens S149 fermentation liquid to be measured is sprayed after plantation 28d, cell concentration is 1 × 108CFU/mL, Mei Geshui
Bucket sprays 20mL, 4 repetitions of each processing.It is inoculated with magnaporthe grisea spore suspension afterwards for 24 hours, spore concentration is 1 × 106 CFU/
mL.Leaf pest are investigated after 7d, and a situation arises, calculates disease index and preventive effect size, the results are shown in Table 2.
Investigation method presses " pesticide field efficacy medicine test criterion (one) ", rice blast leaf pest investigation grade scale, and 0 grade: disease-free；
1 grade: the brown point of only needle point size；3 grades: small and round so that the brown necrosis greyness slightly grown, 1~2mm of diameter；5 grades: typical
Rice blast scab, injured area is less than 10%；7 grades: typical rice blast scab, injured area are 26%~50%；9 grades: comprehensively
Blade is dead.Rice blast panicle blast investigates grade scale, and 0 grade: no panicle blast, grain pest and branch obstruct pest；1 grade: having a pest and branch stalk
Pest, empty grain number < 5%；2 grades: having a pest and branch stalk pest, empty grain number is 6%~10%；3 grades: having a pest and branch stalk pest, empty grain
Number is 11%~20%；4 grades: having a pest and branch stalk pest, empty grain number is 21%~30%；5 grades: having panicle blast, grain pest and branch stalk
Pest, empty grain number are 21%~30%；6 grades: panicle blast causes empty grain number > 80%.
Biocontrol effect calculation formula:
2 Pseudomonas fluorescens S149 of table measures the control efficiency of leaf pest under greenhouse experiment
| Processing | Disease index | Control efficiency (%) |
| S149 | 6.67±0.56b | 79.98±0.42 |
| Pyricularia oryzae P131 control | 33.61±2.46a | / |
Note: significance analysis is using minimum range method (LSD) (P≤0.05 ± standard error).
(4) biocontrol effect of Pseudomonas fluorescens S149 bacterial strain under the conditions of field trial: strains tested activates 48h, connects
Kind is in the seed culture medium of 1000mL, and 37 DEG C, 12 h of 200r/min shaken cultivation prepares seed liquor, connects by 6% inoculum concentration
Kind into fermentor, 37 DEG C, after 200r/min shake culture 48h, prepare strains tested fermentation liquid.It is carried out in the Pingluo County town Yao Fu
The field control effectiveness test of Pseudomonas fluorescens S149, it is spraying for trying microbial inoculum in 10,35,60,75 and 95d of rice growing respectively
Fermentation culture, in plantation 60d investigation leaf blast in rice, a situation arises, and in plantation 110d investigation fringe pest, a situation arises.Leaf pest
Investigation: every experimental plot takes at 3 points at random, every 20 caves of investigation, and every plant of investigation rice falls 4 leaves.Panicle blast investigation: every cell takes 5
Point, every is investigated 20 plants, totally 100 plants.Each experimental plot area is 50m2, cell random alignment, 4 repetitions of each processing.
It the results are shown in Table 3.
Control in field effect of the 3 Pseudomonas fluorescens S149 bacterial strain of table to rice blast
Note: significance analysis is using minimum range method (LSD) (P≤0.05 ± standard error).Rice varieties are peaceful round-grained rice 47.
(5) health and the consistent rice of growing way growth-promoting ability of the Pseudomonas fluorescens S149 bacterial strain to rice seedling: are chosen
Taking-up is placed on the culture dish for being covered with filter paper after seed G19 is put into the fermentation culture of Pseudomonas fluorescens S149 the 0.5h that soaks seed
Interior, at 28 DEG C, the growth cabinet culture that RH is 70%, investigation seed shows money or valuables one carries unintentionally quantity after 48h.Each processing repeats 3 wares.It takes strong
Health and the consistent rice paddy seed of growing way are seeded in rice after being put into the fermentation culture of Pseudomonas fluorescens S149 the 0.5h that soaks seed
In seedling pond, the root long and plant height of rice seedling are investigated after growth 10 days, the results are shown in Table 4.
Growth-promoting ability of the 4 Pseudomonas fluorescens S149 of table to rice seedling
Note: significance analysis is using minimum range method (LSD) (P≤0.05 ± standard error).
(6) antagonism of the Pseudomonas fluorescens S149 bacterial strain to various plants pathogen:
Fusarium oxysporum, Fusarinm solani, fusarium moniliforme, watermelon blight are placed in center on PDA medium plate
Bacterium, botrytis cinerea, Strawberry anthracnose bacterium, Disease of Phyllsticta Mali bacterium, Malus spectabilis leaf spoting bacteria, tobacco black shank bacterium, strawberry leaves are withered
The bacteria cake (1cm) of germ, wheat Rhizoctonia solani Kuhn, is inoculated with Pseudomonas fluorescens in colony edge two sides respectively at 2cm
S149 (scribing line 2cm), is placed in 28 DEG C of dark culturings of incubator, 5 repetitions.Pathogen colony diameter and antibacterial is measured after 2-5 days
Band the results are shown in Table 5.
Antagonism of the 5 Pseudomonas fluorescens S149 bacterial strain of table to various plants pathogen
| Pathogen | Opposite mycelial growth inhibition rate (%) |
| Fusarium oxysporum | 33.33±1.32 |
| Fusarinm solani | 43.33±2.25 |
| Fusarium moniliforme | 16.67±0.85 |
| Withered germ of water-melon | 23.08±1.52 |
| Botrytis cinerea | 22.22±1.40 |
| Strawberry anthracnose bacterium | 59.26±1.24 |
| Disease of Phyllsticta Mali bacterium | 55.26±1.15 |
| Malus spectabilis leaf spoting bacteria | 31.25±1.45 |
| Tobacco black shank bacterium | 46.43±2.53 |
| Strawberry leaf spoting bacteria | 57.55±1.25 |
(7) inhibiting effect of the Pseudomonas fluorescens S149 bacterial strain to Pyricularia oryzae Infection structure:
Inhibiting effect of the bacterial strain S149 fermentation culture to rice blast pathogen conidiospore sprouting, note fields: culture 72h
Bacterial strain S149 fermentation culture and Pyricularia oryzae P131 spore suspension (1 × 106CFU/mL it) is mixed, is mixed with 1: 1 ratio
Close liquid in 2mL sterile centrifugation tube 28 DEG C of opposite cultures for 24 hours when, using the mycelia of optical microphotograph sem observation Pyricularia oryzae, point
Raw spore and the metamorphosis of appresorium.It the results are shown in Table 6 and Fig. 4.
Inhibiting effect of the bacterial strain S149 sterile supernatant to rice blast pathogen conidiospore sprouting, note fields: culture 72h
Bacterial strain S149 sterile supernatant and Pyricularia oryzae P131 spore suspension (1 × 106CFU/mL it) is mixed, is mixed with 1: 1 ratio
Close liquid in 2mL sterile centrifugation tube 28 DEG C of opposite cultures for 24 hours when, using the mycelia of optical microphotograph sem observation Pyricularia oryzae, point
Raw spore and the metamorphosis of appresorium.It the results are shown in Table 6 and Fig. 4.
6 Pseudomonas fluorescens S149 of table is sprouted to magnaporthe grisea spore and the bacteriostasis of note fields
(8) sensibility of the Pseudomonas fluorescens S149 to antibiotic:
It is big mould to ampicillin, chloramphenicol, erythromycin, kanamycins, rifampin, celebrating to test Pseudomonas fluorescens S149
The sensibility of 11 kinds of common antibiotics such as element, neomycin, nalidixic acid, streptomycin sulphate, tetracycline and spectinomycin.It will
S149 bacterial strain stays overnight shake culture in 28 DEG C, 180r/min, is respectively coated on containing 15 μ g/mL, 25 μ g/mL, 50 μ g/mL, 75 μ
On the LB plate of g/mL and 100 μ g/mL various concentration antibiotic, whether observation bacterial strain grows afterwards for 24 hours, 10 weights of every processing
It is multiple.It the results are shown in Table 7.
Sensibility of the 7 Pseudomonas fluorescens S149 of table to antibiotic
Note: "+" indicates that bacterial strain S149 is insensitive to the antibiotic；"-" indicates bacterial strain S149 to the antibiotic sensitive.
(9) induction of Pseudomonas fluorescens S149 paddy disease-resistant defensive ferment:
It is respectively that cell is dense according to experimental design when greenhouse pot culture rice plant growth is to tillering stage (after planting 14d)
Degree is 1 × 108CFU/mL strains tested culture solution 50mL/ plastic casing (15 nutritive cubes) carries out at spray inoculation rice plant
Reason, later every morning sprays water (until blade has moisture film and droplet) to rice plant, and sprays water into air, keeps proving ring
There is higher humidity in border.1d, 2d, 3d, 5d and 7d after inoculation take rice leaf to carry out enzyme assay.Take each processing
Rice leaf 1.0g be put into mortar, 5mL 0.05M phosphate buffer (pH6.8) and 0.1 g polyvinylpyrrolidone is added
(PVP) and minute quantity quartz sand, it is ground into homogenate in liquid nitrogen, is ground on ice, by mixed liquor in 4 DEG C in 7000rpm
Be centrifuged 25min, the supernatant after centrifugation is crude enzyme liquid to be measured, be placed in 80 DEG C of ﹣ of refrigerator store it is spare.
When POD determination of activity, 2.65mL 0.05M phosphate buffer (pH6. 8) is contained in reaction mixture；0.1mL
0.18M guaiacol, 0.2mL 0.03M H2O2(adding when test), 0.05 mL enzyme solution react 5min, 470nm at 20 DEG C
Place's measurement OD value, enzymatic activity is using U/g/min as unit of enzyme activity.These processes all carry out on ice, prevent enzyme from inactivating.Measurement
When with ultraviolet spectrometry luminance meter, be eventually adding H2O2It measures at once, every 30s is surveyed once, as a result total 3min is shown in Fig. 5.
When PPO determination of activity, 4.4mL 0.05M phosphate buffer (pH6. 8) is added in reaction system, 150 μM of 0.5mL
0.5mL 15%H is added in pyrogallol, 0.1mL enzyme solution, 20 DEG C of reaction 10min2SO4Stopped reaction measures OD value at 420nm, whole
A operating process is all carried out at 0~4 DEG C, using U/g/min as unit of enzyme activity, as a result sees Fig. 6.
Wherein, Δ OD470: the optical density difference of light control pipe and sample cell at 470nm；△OD420: illumination at 420nm
The optical density difference of control tube and sample cell；T: the time (min) of enzyme effect；VT: sample liquid total volume (mL)；V1: sample when measurement
Product dosage (mL)；FW: sample fresh weight (g).
Superoxide dismutase object oxidizing ferment (SOD) measurement: taking the rice leaf 1.0g of each processing to be put into mortar, and 5mL is added
0.05M phosphate buffer (pH7.8) and 0.1g PVP and a small amount of quartz sand, are ground into homogenate in liquid nitrogen, in 4 DEG C,
13000rpm is centrifuged 20min, and supernatant is crude enzyme liquid to be measured.2.2mL 0.05M phosphate buffer is first drawn when measurement, then
0.1mL 6 × 10 is added-5M riboflavin, 0.3mL 0.13M methionine, 0.1mL 3 × 10-6M EDTA, 0.2mL 1.25
×10-3M NBT is eventually adding 0.1mL enzyme solution (replacement of 0.1mL phosphate buffer is added in control).Immediately after in 4 000Lux
(fluorescent lamp) 25 DEG C of reaction 25min under illumination, the lower stopped reaction of dark, measure OD value, at 560nm to inhibit NBT light
Changing reaction reduction 50% is a unit of enzyme activity, as a result sees Fig. 7.
Wherein, CK compares OD value: the OD value of light control pipe；Handle OD value: the OD value of sample cell.
(10) Pseudomonas fluorescens S149 Biocontrol Activity active material detects:
The detection of amylase: taking the single colonie newly activated to be inoculated on the LB plate containing 0.2% soluble starch, culture
48h, after forming obvious bacterium colony, Lu Geshi iodine staining 10min, which is added dropwise, on plate can generate starch with 70% ethyl alcohol board-washing
The bacterial strain of enzyme, under the background of black, at bacterium colony growth around can form colourless transparent circle, if there is transparent circle shows bacterium
Strain can produce amylase, and 3 repetitions of each processing the results are shown in Table 8.
The detection of protease: by the strain to be tested percutaneous puncture-inoculation of activation on 1% skim milk agar plate, 30 DEG C are trained
Support 24,48, the generation of peripheral transparent circle is observed after 72h, there is transparent circle and show the generation for having protease, 3 weights of each processing
It is multiple, it the results are shown in Table 8.
Dextranase detection: bacterium to be measured is inoculated on the plate containing ABP culture medium, after 30 DEG C of cultures 48,72h, observation
Whether occur resolution circle in plate, shows the generation for having dextranase, 3 repetitions of each processing, knot if thering is resolution circle to generate
Fruit is shown in Table 8.
Thermophilic iron element detection: thermophilic iron element is detected with CAS culture medium, and the bacterium to be measured of activation is inoculated in CAS detection culture medium and is put down
On plate, after 30 DEG C of culture 72h, observe in plate whether generate crocus haloing, shows there is thermophilic iron element if there is crocus
It generates, each processing is repeated 3 times, and the results are shown in Table 8.
The detection of cellulase: it will be inoculated on cellulose screening and culturing medium plate after the bacterial strain being separated to activation, 28 DEG C
Constant temperature is inverted culture 2d, disseminates 10min, then the NaCl solution decoloration 5min with 1mol/L with 0.1% congo red staining liquid.
If bacterial strain cellulase-producing, clearly transparent circle, each processing can occur in periphery of bacterial colonies and be repeated 3 times, the results are shown in Table 8.
The production of ABP culture medium: with mortar by blocky Poria cocos grind at powder, make its can by the sieve of 120 mesh,
KH2PO46.8g, K2PHO4·12H2O 17.9g, yeast extract 6.7g, Poria cocos powder (or β -1.3- glucan) 5.0g, aniline
Blue 120mg, agar powder 12g, pH value 6.8, water 1L.
The preparation of CAS culture medium: junket peptone 9g, yeast extract 5g, citric acid trisodium 10g, disodium hydrogen phosphate 1g, biphosphate
Sodium 1g, glucose 10g, agar 20g are dissolved in 1L water.
Cellulose enrichment conditions base (1L): NaCl 6g, MgSO40.1g, KH2PO40.5g, CaCl2 0.1 g,(NH4)
SO42.0g, K2HPO42.0g, agar 15g, CMC-Na 5g, pH are adjusted to 7.0；Cellulose screening and culturing medium (1L): in above-mentioned fibre
It ties up in plain enzyme enriched medium and yeast powder 1g is added.
Congo red staining liquid: Congo red, final concentration of 0.1% (w/v) is dissolved with distilled water.
Congo red destainer: the NaCI solution of final concentration of 1mol/L.
The detection of 8 Pseudomonas fluorescens S149 Biocontrol Activity active material of table
Note: data are duplicate testing result three times；"+" is detected as a result, "-" is not detect.
It should be understood that herein, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that the process, method, article or equipment comprising a series of elements not only includes those elements, and
And further include other elements that are not explicitly listed, or further include for this process, method, article or equipment institute it is intrinsic
Element.In the absence of more restrictions, the element limited by sentence " including one ... ", it is not excluded that including described
There is also other identical elements in the process, method, article or equipment of element.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations.Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (7)
1. a fluorescent pseudomonads (Pseudomonas fluorescens) S149 characterized by comprising in being preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), the deposit date is on March 1st, 2019, preservation was compiled
Number be CGMCC NO.17278.
2. a kind of microbial inoculum, which is characterized in that include Pseudomonas fluorescens S149 as described in claim 1.
3. a kind of method for preventing and treating rice blast, which is characterized in that including by Pseudomonas fluorescens S149 as described in claim 1
Or microbial inoculum as claimed in claim 2 is applied to rice crop.
4. method as claimed in claim 3, which is characterized in that the microbial inoculum is the culture of the Pseudomonas fluorescens S149
Liquid.
5. the method as claimed in claim 3 or 4, which is characterized in that the rice blast is seedling rice blast, leaf pest, pulvinus pest, section rice
Pest, panicle blast, branch obstruct one or more of pest and grain pest.
6. Pseudomonas fluorescens S149 as described in claim 1 or microbial inoculum as claimed in claim 2 are in prevention and treatment rice blast
Purposes.
7. purposes as claimed in claim 6, which is characterized in that the rice blast be seedling rice blast, leaf pest, pulvinus pest, section rice blast,
Panicle blast, branch obstruct one or more of pest and grain pest.
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| CN113151099A (en) * | 2021-05-10 | 2021-07-23 | 上海中医药大学 | Pseudomonas aeruginosa and application thereof |
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Cited By (4)
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| CN110643551B (en) * | 2019-11-18 | 2022-03-15 | 吉林大学 | Stenotrophomonas rhizophila S11 for preventing and treating rice blast and application thereof |
| CN113151099A (en) * | 2021-05-10 | 2021-07-23 | 上海中医药大学 | Pseudomonas aeruginosa and application thereof |
| CN113151099B (en) * | 2021-05-10 | 2023-01-10 | 上海中医药大学 | Pseudomonas aeruginosa and application thereof |
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