CN106701618A - Bacillus amyloliquefaciens resistant to powdery mildew of melon crops and high in spore yield - Google Patents
Bacillus amyloliquefaciens resistant to powdery mildew of melon crops and high in spore yield Download PDFInfo
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Abstract
The invention discloses bacillus amyloliquefaciens KN-527 which is induced through normal-pressure room-temperature plasmas to have a high spore yield, and a preservation number of the bacillus amyloliquefaciens KN-527 is CCTCC (China Center for Type Culture Collection) NO: M2015709. Fermented spore count of the bacterial strain is increased by 161% in comparison with that before mutagenesis, a spore forming rate is high, heat resistance is good, remarkable disease resistance is achieved on melon powdery mildew, and the control effect can be 95% or higher. The bacillus amyloliquefaciens KN-527 can be applied to melon crops such as cucumbers, summer squashes, pumpkins, bitter gourds and watermelons. According to a melon field test, conventional fertilization is performed on a microbial agent produced by the bacterial strain, so that plant grows vigorously, capacity of developing lateral branches is strong, and leaf color is bright green.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens), the invention further relates to a kind of microbial bacterial agent of anti-melon crop powdery mildew.
Background technology
Powdery mildew is that melon crop occurs a kind of more universal disease, and Powdery Mildew has 3 category, 6 kinds, melon list softgel shell
Bacterium and two spore powdery mildews are main pathogens, and melon list softgel shell bacterium has been reported that the multiple biological strains of discovery.Powdery mildew main harm
Blade, also infects that petiole, melon be climing and melon.Blade morbidity just produces the small mildew of subcircular white (i.e. pathogen mycelia on blade face
And conidium), after be expanded into mildew, condition is suitable, and mildew expands rapidly, and each other in flakes, white powder can be covered with whole leaf
Piece, causes blade to become fragile, and mustiness thing becomes canescence on later stage scab, and black small grain point, the i.e. cleistothecium of germ are grown thereon.Morbidity
Weight, there is irregular yellowish-brown scab in blade, and scab causes whole blade brown to dry up in flakes, but does not fall off.Petiole, melon be climing and melon
Catch an illness, also produce white amyloid plaque, after development, sick portion is covered with white powder.Powdery mildew can reduce influences of plant crown, reduce individual plant percentage of fertile fruit,
Fruit is diminished, so as to cause the underproduction to lose market value, serious economic loss is caused to grower.
China is large agricultural country, and Pesticide use is wide in variety, and consumption is big, wherein 70~80% agricultural chemicals is directly scattered in environment
In.After applied chemistry agricultural chemicals, not only plant adsorbs a large amount of agricultural chemicals in itself, and enters inside plants by osmosis, causes
Serious residual, and then pollution is produced to agricultural product.The biological noxious material that low dosage is constantly absorbed from surrounding medium or food,
And gradually accumulation concentration in vivo, in the whole ecosystem, agricultural chemicals constantly passes through the transmission of biological concentration and food chain, by
Level is concentrated, amplified step by step, and the mankind are in food chain top, aggrieved the most serious.The continuing of chemical pesticide uses, and causes soil fertilizer
Power declines, hardened etc., can cause the activity of enzyme in the violent change of edaphon system, and then influence soil, nutriment
Conversion, change the efficiency and speed of agriculture people's ecology system trophism circulation, decline Land Sustainable Productivity.For disease
Physilogical characteristics, suit the remedy to the case in good time, the biological pesticide for selecting efficient, safety, bioactivity high, are just particularly important.
Chemical prevention and control method is used current powdery mildew more, but because powdery mildew pathogenic bacteria differentiation is fast, subspecies are more, chemical reagent
It is difficult to ensure the No-harmful apple orchard of crop long-term effectively, therefore, new safe efficient, economic preventions turn into hot topic.
Study in recent years and reported substantial amounts of antagonistic microbe, including bacterium, saccharomycete, actinomyces etc..But common bacteria, ferment
Female bacterium and actinomyces resistance are poor, and growing environment requirement is high, it is impossible to batch popularization and application.Gemma resists as in whole living nature
The most strong life entity of inverse property, in heat resistanceheat resistant, the aspect such as anti-chemicals and radioresistance is very prominent.Bacillus amyloliquefaciens conduct
A kind of aerobic sporiferous Gram-positive bacillus, there is no report to cause a disease people and animals at present, and its nutritional requirement is simple, be easy to
Survival, field planting and breeding, good stability, can not only improve the yield of crop, increase resistance, but also substantially increase soil
Earth fertility, after it breeds as dominant microflora in soil, may also reach up the effect for suppressing pathogen, be well suited as biological and ecological methods to prevent plant disease, pests, and erosion
Bacterium and a large amount of production applications of bio-feritlizer.
The content of the invention
The invention aims to make up sky of the bacillus amyloliquefaciens in melon crop controlling powdery mildew application field
In vain, there is provided one plant of bacillus amyloliquefaciens and its microbial bacterial agent with gemma high yield.
The present invention, by atmospheric pressure at room plasma mutagenesis, obtains one plant of tool with bacillus amyloliquefaciens as starting strain
There is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KN-527 of gemma high yield, in November 26 in 2015
China typical culture collection center (CCTCC) preservation of day in Wuhan, China Wuhan University, its deposit number CCTCC NO:
M2015709。
The invention provides a kind of microbial bacterial agent of anti-melon crop powdery mildew, comprising above-mentioned bacillus amyloliquefaciens
KN-527。
The microbial bacterial agent can serve as promoting the bio-feritlizer of melon crop growth.
Beneficial effects of the present invention：
Bacillus amyloliquefaciens of the invention can reach 1.19E+ by after gas ions mutagenesis screening, producing gemma number
10CFU/ grams, than 7.37E+09CFU/ grams of starting strain before mutagenesis, improve 161%.Table of field test results is bright, dispenser 14
After it, KN-527 reaches more than 95% to the preventive effect of melon powdery mildew, hence it is evident that better than chemical pesticide carbendazim.The bacterial strain can have
Effect prevention cucurbits powdery mildew generation, experimental group cucumber, pumpkin, the disease index of watermelon well below control group, 0.1 hundred million cfu/
G bacillus amyloliquefaciens microbial inoculum dilutes 3000 times of bacterium solutions (dispenser 14 days) can effectively prevent and treat cucurbits powdery mildew, and disease accuses system 6%
Hereinafter, preventive effect reaches more than 90%；After dispenser 14 days, control group disease refers to and has reached more than 97%, and to strawberry, wheat, peppery
The powdery mildew preventive effect of the non-melon crop such as green pepper is relatively low.
Specific embodiment
The present invention is described in detail by the following examples.
Embodiment 1
(1) the purifying culture of bacillus amyloliquefaciens starting strain
Picking starting strain bacillus amyloliquefaciens KN-52 (from the soil of Wuhan vegetable cultivation base Natural Selection and obtain)
Single bacterium colony, accesses by peptone 1%, yeast extract 0.5%, sodium chloride 1%, in the simple culture media of pH7.2-7.5 compositions,
Cultivated 16-20 hours on constant-temperature table under conditions of 32 DEG C and 180rpm of temperature, take 10ml zymotic fluids, 4 DEG C, 6000r/min bars
It is centrifuged under part, collects bacterium slurry stand-by；
(2) primary dcreening operation：Normal temperature and pressure plasma mutagenesis
Taking the thalline of step (1) preparation 10ml SPSSs, to dissolve concussion uniform, takes 10 μ l and is applied to and is sterilizing
Metal slide glass on, slide glass is placed on objective table, adjustment objective table highly, is set after process time to solution starch gemma bar
Bacterium is irradiated.Helium is carrier, irradiation power 150W, control air flow 10SLPM, irradiation distance 2mm, irradiation time 60
~240s.Slide glass is transferred in the EP pipes equipped with 1ml SPSSs after treatment with irradiation, vibrates 1~2min, make to be attached to
Thalline on slide glass is eluted in physiological saline, forms new sensitive bacteria suspension.Sensitive bacteria suspension is diluted to 10-4、10-5、10-6Totally 3 gradients.
(3) collection of musk melon powdery mildew pathogenic bacteria, prepares pressure screening flat board
Field gathers the muskmelon blade of severe infections powdery mildew, blade surface sprinkling sterilized water, 4 DEG C of refrigerator overnights.Super
In net workbench, water agar plate is blown into ear washing bulb, is put into incubator, 20 degrees Celsius of culture 7d.
Configuration pressure screening Solid media for plates, sucrose 5%-10%, yeast extract 0.1-0.5%, sodium chloride
0.02%-0.05%, magnesium sulfate 0.02%-0.05%, calcium carbonate 0.05%-0.2%, agar powder 2%, pH7.2-7.5 compositions,
With 45 degree or so 2% powdery mildew pathogenic bacterias of addition after sterilizing；
Each gradient for the sensitive bacteria suspension that will be diluted is coated with 3 pieces of pressure screening flat boards.Flat board after coating is placed into training
30 DEG C carry out culture 16h in foster case.The big single bacterium colony of picking inhibition zone oolemma accesses slant medium, incubated at 30 DEG C
4 DEG C of Refrigerator stores of income are standby after culture is completely formed gemma in case；
Described slant medium is by peptone 1%, yeast extract 0.5%, sodium chloride 1%, agar powder 1.5%-
2%, pH7.2-7.4 are constituted；
(4) selection of resistant strain high yield strain
Slant strains in step (3) are taken, with being fitted under the aseptic washings of 25ml in the 100ml triangular flasks without cingula bead,
Concussion is well mixed after being made bacteria suspension and is put into water-bath 30min in 70 DEG C of water-baths, and taking-up cold water is down to room temperature and is followed by immediately
Entering and be transferred to fermentation shake flask after cultivate 16-24h under conditions of 32 DEG C of fermentation seed shaking flask and 180rpm, equally at 32 DEG C and 180rpm
Under conditions of cultivate 40-48h；Shake flask fermentation liquid is taken, routine smear is dyeed with alkaline carbolfuchsin, micro- with Olympus
Spore forming rate after (16 × 100 times) the observation fermentations of mirror oil mirror.
Selection has typical bacillus amyloliquefaciens gemma form, spore forming rate bacterial strain high.It is comprehensive according to the above results
Conjunction judges the resistance against diseases and spore forming rate of mutagenic strain, obtains one plant of spore forming rate bacterial strain KN-527 high.
Fermented rear CFU contrast, KN-527 gemma number up to 1.19E+10, than the starting strain CFU 7.37E+ before mutagenesis
09,161% is improved, it is shown in Table 1.
Table 1
| Bacterial strain | Numbering | Sampling amount (ml) | Test tube | 1 | 2 | 3 | Blank | CFU |
| KN-52 (starting strain) | 1-1 | 0.5 | 7 | 67 | 82 | 71 | 3 | 7.43E+09 |
| KN-52 (starting strain) | 1-2 | 0.5 | 7 | 73 | 72 | 68 | 6 | 7.30E+09 |
| KN-527 | 2-1 | 0.5 | 7 | 110 | 117 | 119 | 5 | 1.18E+10 |
| KN-527 | 2-2 | 0.5 | 7 | 120 | 118 | 119 | 2 | 1.20E+10 |
Field inhibition checkings of the bacillus amyloliquefaciens KN-527 of embodiment 2 to melon powdery mildew.
1. material
Reagent agent：Bacillus amyloliquefaciens KN-527, chemical pesticide are from traditional controlling powdery mildew medicament.
For studying thing：Muskmelon
Controlling object：Powdery mildew
Experiment sets 3 treatment altogether：
(1) control group：Clear water treatment；
(2) treatment group：Bacillus amyloliquefaciens described in 0.1 hundred million CFU/g dilute 1000,2000 and 3000 times
(3) 50% carbendazol wettable powders；
Treatment above is carried out in the muskmelon greenhouse of scientific base.Lateral bine for extracing melon before examination muskmelon dispenser, in morbidity just
The dispenser of first time phase, every being sprayed again after 7d 1 time, liquid is uniformly sprayed on plant leaf.
2. method and result
Fixed point singling investigation, 10 plants are selected per section, and every plant regards growing state and investigates 5 blades of leaf more than 5cm long, before medicine
Investigation disease index, respectively investigation once, is classified by blade occurring degree and counted for 7d, 14d after medicine.
Grade scale：By 0~9 grade of severity Scaling standard of index
0 grade：Ye Jian, disease-free spot
1 grade：Scab accounts for less than the 10% of full leaf leaf area；
3 grades：Scab accounts for the 11%~25% of full leaf leaf area；
5 grades：Scab accounts for the 16%~40% of full leaf leaf area；
7 grades：Scab accounts for the 41%~65% of full leaf leaf area；
9 grades：Scab accounts for more than the 65% of full leaf leaf area；
Drug effect computational methods：
Disease index (%)=∑ (the sick numbers of sheets at different levels × corresponding disease series) × 100%/investigation total number of sheets × highest disease level
Number；
Disease refer to growth rate=(after medicine disease refer to-medicine before disease refer to) disease refers to × 100% before/medicine；
Preventive effect=[1- (and after treatment region medicine disease refer to × check plot medicine before disease refer to)/(before treatment region medicine disease refer to × check plot medicine after
Disease refers to)] × 100%.
Field inhibition checking of the bacillus amyloliquefaciens of table 2 to melon powdery mildew
As can be seen that 0.1 hundred million cfu/g bacillus amyloliquefaciens microbial inoculums dilution 1000,2000 and 3000 from the data of table 2
Times bacterium solution (dispenser 14 days) can effectively prevent and treat melon powdery mildew, and disease accuses system below 5%, and preventive effect reaches more than 95%；Dispenser
After 14 days, control group disease refers to and has reached 98.2%, and 50% carbendazol wettable powder is 65.4% to the preventive effect of melon powdery mildew,
And the preventive effect for diluting 3000 times of bacterium solution preventing and treating melon powdery mildew can just reach 95.9%.
The bacillus amyloliquefaciens KN-527 of embodiment 3 is verified as bio-feritlizer to the field efficacy of muskmelon.
Reagent agent：Bacillus amyloliquefaciens KN-527
For studying thing：Muskmelon
Nutrient solution：The full elemental nutrient liquids of Israel Polyfood
Test method:
Muskmelon after being colonized one week is divided into two groups, and A groups apply nutrient solution+clear water, and B groups apply nutrient solution+solution starch bud
Spore bacillus wettable powder.
Plant strain growth situation, fruit growth situation and yield, quality are investigated climing phase, seat melon phase, swollen melon phase is taken out.
The bacillus amyloliquefaciens of table 3 are verified as bio-feritlizer to the field efficacy of muskmelon
Result of the test shows that bacillus amyloliquefaciens KN-527 can have bright as bio-feritlizer to Muskmelon Plants growth
Aobvious facilitation, hair tonic side shoot ability enhancing, plant is sturdy.
The bacillus amyloliquefaciens KN-527 of embodiment 4 is to cucurbits powdery mildew and the field inhibition of other crop powdery mildews
Checking.
1. material
Reagent agent：Bacillus amyloliquefaciens CCTCC NO:M2015709
For studying thing：Cucumber, pumpkin, watermelon, strawberry, wheat, capsicum
Controlling object：Powdery mildew
Experiment sets 2 treatment altogether：
(1) treatment group：Clear water processes (CK)；
(2) 0.1 hundred million 3000 times of liquid of bacillus amyloliquefaciens described in CFU/g
Treatment above is carried out in each greenhouse in scientific base.For studying thing in early stage first time dispenser, every after 7d again
Spray 1 time, liquid is uniformly sprayed on plant leaf.
2. method and result
Fixed point singling investigation, 20 plants are selected per section, and every plant regards growing state and investigates 5 blades of leaf more than 5cm long, before medicine
Investigation disease index, respectively investigation once, is classified by blade occurring degree and counted for 7d, 14d after medicine.
Grade scale：By 0~9 grade of severity Scaling standard of index
0 grade：Ye Jian, disease-free spot
1 grade：Scab accounts for less than the 10% of full leaf leaf area；
3 grades：Scab accounts for the 11%~25% of full leaf leaf area；
5 grades：Scab accounts for the 16%~40% of full leaf leaf area；
7 grades：Scab accounts for the 41%~65% of full leaf leaf area；
9 grades：Scab accounts for more than the 65% of full leaf leaf area；
Drug effect computational methods：
Disease index (%)=∑ (the sick numbers of sheets at different levels × corresponding disease series) × 100%/investigation total number of sheets × highest disease level
Number；
Disease refer to growth rate=(after medicine disease refer to-medicine before disease refer to) disease refers to × 100% before/medicine；
Preventive effect=[1- (and after treatment region medicine disease refer to × check plot medicine before disease refer to)/(before treatment region medicine disease refer to × check plot medicine after
Disease refers to)] × 100%.
The bacillus amyloliquefaciens of table 4 are verified to melon and other crop powdery mildews fields inhibition
Powdery Mildew has 3 category, 6 kinds, and melon list softgel shell bacterium and two spore powdery mildews are main pathogens.Cucumber, pumpkin, watermelon
Pathogen Deng cucurbits powdery mildew is mainly melon list softgel shell bacterium, and strawberry, wheat, the pathogen of pepper powdery mildew are mainly the garment or robe made of feathers
Careless list softgel shell bacterium, grass family dlumeria graminis, Tartar's internal thread powdery mildew.As can be seen that 0.1 hundred million cfu/g solutions from the data of table 4
Bacillus amyloliquefacienses microbial inoculum dilutes 3000 times of bacterium solutions (dispenser 14 days) can effectively prevent and treat cucurbits powdery mildew, disease accuse system 6% with
Under, preventive effect reaches more than 90%；After dispenser 14 days, control group disease refers to and has reached more than 97%, white to strawberry, wheat, capsicum
The preventive effect of powder disease is respectively 81.3%, 50.2% and 63.8%, and prevention effect is undesirable.
Claims (2)
1. bacillus amyloliquefaciens (the Bacillus of one plant of anti-melon crop powdery mildew and high yield gemma
Amyloliquefaciens) KN-527, it is characterised in that：China typical culture collection center is preserved in, deposit number is
CCTCC NO:M2015709。
2. a kind of microbial bacterial agent of anti-melon crop powdery mildew, it is characterised in that：The active component of the microbial bacterial agent is
Bacillus amyloliquefaciens described in claim 1.
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| CN112175883A (en) * | 2020-10-26 | 2021-01-05 | 上海市农业科学院 | Bacillus amyloliquefaciens with good bacteriostatic ability |
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| CN112175883A (en) * | 2020-10-26 | 2021-01-05 | 上海市农业科学院 | Bacillus amyloliquefaciens with good bacteriostatic ability |
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