CN108410757B - One plant of streptomycete NBF715 separated and its application in control of crop disease - Google Patents
One plant of streptomycete NBF715 separated and its application in control of crop disease Download PDFInfo
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Abstract
The streptomycete NBF715 separated the invention discloses one plant and its application in control of crop disease, are preserved in Chinese Typical Representative Culture Collection Center, and deposit number is CCTCC NO:M2017414.The bacterial strain is reported for the first time can be used for preventing and treating tobacco black shank, the prevention and treatment of tomato, eggplant, capsicum and ginger bacterial wilt, the prevention and treatment of Penicillium italicum, the prevention and treatment of tealeaves zonate spot, the prevention and treatment of pepper anthracnose, the late blight of potato, with existing fungicide and insecticide no interactions drug resistance.Compared with being prevented and treated at present using chemical synthetic pesticide, since it is from natural environment, has many advantages, such as efficient, less toxic, free from environmental pollution and be not likely to produce drug resistance, have broad application prospects.
Description
Technical field
The invention belongs to crops Prevention Technique fields, and in particular to one plant separation streptomycete NBF715 and its in crop
Application in disease control.
Background technique
Since the fifties in last century, pesticide plays an important role in production estimation, however chemical pesticide is extensive
Using causing environmental pollution, pesticide residue, destroy the problems such as ecological balance and germ develop drug resistance.For these reasons,
Biological pesticide increasingly attracts people's attention, environmental-friendly and be not likely to produce drug resistance etc. a little with non agricultural chemical residuum.Chain
Mould (Streptomyces) is high actinomyces, can produce Multiple Classes of Antibiotics, plant hormone isoreactivity substance, to raising plant
Disease-resistant, anti-adversity play an important role, have exploitation biological pesticide potentiality.Such as by the 5406 of Streptomyces jingyangensis development
Bacterial manure has been widely applied in the sixties in last century in China, makes tremendous contribution for crop disease prevention volume increase.
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 be from soil separate one plant to work
Object disease has the streptomycete of good control efficiency, and the invention discloses Phytophthora Phytophthora nicotianae is prevented and treated on tobacco
The application of tobacco black shank caused by (Phytophthora nicotianae), by anthrax category colletotrichum gloeosporioides Penz
The application of pepper anthracnose caused by (Colletotrichum gloeosporioides).By bacterium Raul Salmonella
Tomato caused by (Ralstonia solanacearum), eggplant, capsicum and ginger bacterial wilt, Penicillium (Penicillium
Spp. Penicillium italicum caused by) intends the application of tea zonate spot caused by Pestalotia (Pestalotiopsis.spp.).
Summary of the invention
The object of the present invention is to provide a kind of isolated streptomycete, the streptomycete is pale purple brown streptomycete
(Streptomyces enissocaesilis) NBF715, deposit number are CCTCC NO:M2017414.The streptomycete can
For tobacco black shank, the prevention and treatment of crop bacterial wilt, Penicillium italicum, tea zonate spot, pepper anthracnose, the late blight of potato.
It is another object of the present invention to provide the preparation method of pale purple brown streptomycete NBF715, the preparation side provided
Method includes the preparation method of solid fungicide and liquid fermentation liquid, and method is simple, easy, has good prospect of production.
Final object of the present invention is that providing pale purple brown streptomycete NBF715 prevents and treats crop disease drug in preparation
In application.
In order to achieve the above object, the present invention takes following technical scheme to realize.
A kind of isolated streptomycete separates from Hefeng County soil of bestowing favour, and is identified as pale purple brown streptomycete
(Streptomyces enissocaesilis) has sent to China typical culture collection center on July 7th, 2017 and has carried out
Preservation, classification naming: pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715；Deposit number is CCTCC
NO:M2017414；Place: China, Wuhan, Wuhan University.
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 in ISP-2 culture medium well-grown,
Bacterium colony is round, the smooth of the edge, surface compact.Aerial hyphae silver gray, substrate mycelium cork yellow, bacterium colony is round, non-pigment.Spore
Sub- silk is distributed in straight-chain, spore oval or ellipse (Fig. 1).
The preparation method of pale purple brown streptomycete NBF715, solid fermentation method include: by pale purple brown streptomycete
(Streptomyces enissocaesilis) NBF715 seed liquor is connected in solid medium by 5%-10% (volume ratio),
Sample-loading amount is 10%-30% (volume ratio), 28 DEG C of -30 DEG C of culture 8d-10d；
The solid fermentation culture medium, component include: by weight ratio
Wheat bran or rice or corn quarrel 50-83, glucose 1-3, potassium nitrate 1-5, dipotassium hydrogen phosphate 1-3, sodium chloride 2-3,
Calcium carbonate 2-3, appropriate amount of water；
When using wheat bran, material-water ratio 10:5-8；
When using rice or corn is cut, material-water ratio 10:8；
The material-water ratio is the parts by weight ratio of the above-mentioned total material of solid and water.
The preparation method of pale purple brown streptomycete NBF715, liquid fermentation method include:
Seed liquor is inoculated in fermentation medium by 5%-10% amount, temperature is 28 DEG C, the mixing speed 200- of fermentor
500 revs/min, ventilatory capacity 1:1-1.2, the loading amount of fermentation medium is the 60%-80% of tank volume；Fermentation 4-6 days.
The formula of the fermentation medium includes: mannitol 1-3%, soy peptone 1-3%, yeast powder 0.3-
0.5%, calcium carbonate 0.3-0.5%, sodium chloride 0.3-0.8%；PH value 6.5-7.5, remaining is water, and percentage described above is
Mass percent.
In schemes described above, it is preferred that the preparation method of the seed liquor includes: by the pale purple brown strepto- of activation
Bacterium NBF715 picking is seeded in 100 milliliters of seed culture mediums, is cultivated 3-4 days on 28 DEG C, 130-160 revs/min of shaking table；Both
Obtain seed liquor.
Seed culture based formulas are as follows: in 1L culture medium: 20g mannitol, 10g soy peptone, 0.35g potassium dihydrogen phosphate,
0.5g calcium carbonate, 20 μ L of soya-bean oil, remaining is water.
Application of the pale purple brown streptomycete NBF715 in preparation prevention and treatment crop disease drug, including the use of pale purple brown streptomycete
NBF715 is prepared into tobacco black shank, crop bacterial wilt, Penicillium italicum, tea zonate spot, pepper anthracnose, the late blight of potato
The drug of prevention and treatment.
Compared with prior art, the invention has the following advantages that
(1) the present invention provides a kind of pale purple brown streptomycete, which is reported for the first time can be used for preventing and treating tobacco black shank,
The prevention and treatment of tomato, eggplant, capsicum and ginger bacterial wilt, the prevention and treatment of Penicillium italicum, the prevention and treatment of tealeaves zonate spot, pepper anthracnose,
The prevention and treatment of the late blight of potato, with existing fungicide and insecticide no interactions drug resistance.
(2) compared with being prevented and treated at present using chemical synthetic pesticide, since it is from natural environment, have efficiently, low toxicity,
It is free from environmental pollution and the advantages that be not likely to produce drug resistance.
Detailed description of the invention
Fig. 1 NBF715 bacterial strain phylogenetic tree.
Fig. 2 NBF715 bacterial strain spore, mycelia and colonial morphology schematic diagram.
Fig. 3 NBF715 bacterial strain wheat bran fermenting agent schematic diagram.
Fig. 4 NBF715 bacterial strain rice fermentation microbial inoculum schematic diagram.
Fig. 5 NBF715 bacterial strain corn fermentation microbial inoculum schematic diagram.
Fig. 6 NBF715 bacterial strain fermentation liquor prevents and treats tobacco black shank effect diagram.
Fig. 7 NBF715 bacterial strain fermentation liquor prevents and treats ginger bacterial wilt effect diagram.
Fig. 8 NBF714 bacterial strain fermentation liquor prevents and treats tea zonate spot effect diagram.
Specific embodiment
In order to better explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but
The contents of the present invention are not limited solely to following embodiment.Technical solution of the present invention is if not otherwise specified this field
Routine techniques, the reagent or material derive from commercial channel if not otherwise specified.
Embodiment 1:
The acquisition and identification of pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715
The streptomycete of one plant of separation, from bestowing favour, Hefeng County soil is isolated, identifies through 16s rDNA combination Physiology and biochemistry
After belong to pale purple brown streptomycete (Streptomyces enissocaesilis) (Fig. 1, table 1, table 2), on July 7th, 2017
It send to China typical culture collection center and carries out preservation, classification naming: pale purple brown streptomycete (Streptomyces
enissocaesilis)NBF715；Deposit number is CCTCC NO:M2017414；Place: China, Wuhan, Wuhan University.
In the present invention, it pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 or is called for short
NBF715。
Morphological feature: bacterium colony is round, the smooth of the edge, surface compact.Aerial hyphae silver gray, substrate mycelium cork yellow,
Bacterium colony is round, non-pigment.Fibrillae of spores is distributed in straight-chain, spore oval or ellipse.
Bacterial strain physio-biochemical characteristics:
1 bacterial strain NBF715 physio-biochemical characteristics of table-enzyme activity, carbon source oxidation
+: positive reaction；: negative reaction；W: weakly positive reaction
2 bacterial strain NBF715 physio-biochemical characteristics of table-produce acid using carbon source
+: it is positive ,-: it is negative；W: weakly positive
Embodiment 2:
The fermentation of pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715:
The preparation of pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 seed liquor: including will be oblique
Face saves strain and activates in ISP-2 plate, and picking is seeded in 100 milliliters of seed culture mediums, at 28 DEG C, 160 revs/min of shaking table
Upper culture 4 days；Up to seed liquor.
Seed culture based formulas are as follows: include 20g mannitol, 10g soy peptone, 0.35g biphosphate in 1L culture medium
Potassium, 0.5g calcium carbonate, 20 μ L of soya-bean oil, remaining is water.
Solid fermentation: pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 seed liquor is pressed
10% is connected in solid medium, sample-loading amount 20%, 30 DEG C of culture 10d, the bacterium of the culture obtained in this way
Concentration is 5*1010A spore/gram.
The solid fermentation culture medium are as follows: 83% wheat bran, 3% glucose, 5% potassium nitrate, 3% dipotassium hydrogen phosphate, 3%
Sodium chloride, 3% calcium carbonate add water and stir mixing, material-water ratio 10:6 after the above formula mixing.
Liquid fermentation: pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 seed liquor is pressed 5%
Amount is inoculated in fermentation medium, and temperature is 28 DEG C, and 500 revs/min of the mixing speed of fermentor, the loading amount of fermentation medium is tank body
The 60% of volume ferments 5 days, is used for following embodiment.
The formula of the fermentation medium includes: mannitol 3%, soy peptone 3%, yeast powder 0.3%, calcium carbonate
0.5%, sodium chloride 0.5%；PH value 6.5-7.5, remaining is water.
Embodiment 3:
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 prevents and treats tobacco black shank:
(1) NBF715 bacterial strain is to Phytophthora nicotianae Antifungal Activity in Vitro
For examination tobacco black shank pathogenicbacteria separation from tobacco black shank plant, 10 DEG C to be stored in the inclined-plane V8 spare.By inclined-plane
Bacterial strain is transferred to V8 plate and is activated, and 30 DEG C of culture 6d are spare.NBF715 is spare from slant activation to ISP2 culture medium 7d.With
4mm punch, which is beaten, takes Phytophthora nicotianae Breda bacterium dish to be placed in PDA culture medium center, is then beaten with 4mm punch and NBF715 bacterium dish is taken to set
In Phytophthora nicotianae Breda dish two sides, distance 25mm.Antibacterial band is observed after 8d.By being stood facing each other in vitro it can be observed that apparent antibacterial band
(diameter of antibacterial band is 2cm) illustrates that NBF715 bacterial strain has strong bacteriostasis to Phytophthora nicotianae.
(2) NBF715 fermentation liquid and microbial inoculum prevent and treat tobacco black shank effect
Inclined-plane bacterial strain is transferred to V8 plate to activate, 30 DEG C of culture 8d are spare.Tobacco seed is planted in 32 hole hole trays,
Greenhouse routine culture takes Nicotiana tabacum leaves to test for excised leaf after about 1 month.
NBF715 liquid fermentation liquid dilutes 50 times and 100 times using sterile water, is uniformly sprayed on tobacco leaf and is extremely saturated,
It is beaten afterwards with 4mm punch for 24 hours and takes bacterium dish, be affixed on tobacco leaf, control and processing lesion diameter are measured after moisturizing culture 5d.Each
Processing repeats three times, 15 leaves of each repetition.It is raw to survey the result shows that 50 times and 100 times of NBF715 fermentation liquid dilution is black to tobacco
Shin disease preventive effect is 100%, and the results are shown in Table 3.
Pot experiment is carried out in greenhouse, and tobacco seed is planted in 32 hole hole trays, greenhouse routine culture, is transplanted after about 20d
In pot for growing seedlings, pot experiment is used for after about 40d.By Phytophthora nicotianae from slant activation to PDA plate, then expand in V8 plate
Numerous, swipe spore after 28 DEG C of illumination 10d is put into 4 DEG C of refrigerator half an hour taking-ups, is adjusted spore to 10 with blood counting chamber5Spore
Son/ml.20ml/ plants of fermentation liquid irrigating roots of NBF715 fermentation liquid, for 24 hours afterwards be inoculated with Phytophthora nicotianae spore, 3ml/.Processing sets 3 weights
Multiple, 15 seedlings of each repetition fill clear water as compareing, control and processing disease index are counted after 20, and calculate disease incidence.
Tobacco black shank disease index grade scale is referring to People's Republic of China's tobacco business standard (YC/T39-1996)
Disease index=∑ (plant number × rank at different levels)/(investigation total strain number × highest represents rank) × 100.
Control efficiency=(control disease index-processing disease index)/control disease index × 100%
The raw NBF715 fermentation broth on tobacco balck shank preventive effect as the result is shown of surveying of potting is 100% (Fig. 6, table 4).
NBF715 bacterium solid fungicide field trial is carried out in April, 2017, every young plant 3g NBF715 solid bacterium when transplanting
Agent+3g organic commercial fertilizer (cow dung) cave is applied, singly to apply 6g cow dung as control, every plot area 50m2, plant 70 plants of cigarette, cell with
Machine arrangement, 3 repetitions are counted disease index using conventional crop field tobacco cultivation management, after 3 months and calculate preventive effect, classification and
The same pot experiment of calculation method, the results are shown in Table 5.
3 excised leaf condition NBF715 fermentation broth on tobacco balck shank preventive effect of table
| Processing | Lesion diameter | Preventive effect (%) |
| 50 times of NBF715 fermentation liquid | 0 | 100 |
| 100 times of NBF715 fermentation liquid | 0 | 100 |
| Control | 36.77 | - |
4 NBF715 fermentation broth on tobacco balck shank potting control efficiency of table
| Processing | Disease index | Preventive effect (%) |
| NBF715 fermentation liquid | 0 | 100 |
| Control | 83.60 | - |
5 NBF715 fermentation broth on tobacco balck shank potting control in field effect of table
| Processing | Disease index | Preventive effect (%) |
| NBF715 microbial inoculum | 27.67 | 62.21 |
| Control | 73.22 | - |
Embodiment 4:
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 fermentation liquid prevents and treats ginger bacterial wilt:
It activates in freezing glycerol tube ralstonia solanacearum to SPA culture medium, 160r/min shaken cultivation 48h, adjusts bacterial concentration
To 1 × 108Cfu/mL is spare.NBF715 fermentation liquid is prepared according to embodiment 2, is seeded in pot for growing seedlings after soaking ginger kind about 20min
In, it uses stab inoculation afterwards for 24 hours, then pours into ralstonia solanacearum, every plant of 20mL.Each processing repeats three times, each repetition 30
Ginger kind.Statistics control and processing morbidity strain number after the onset of wait compare sufficiently, and calculate preventive effect.
Control efficiency=(control diseased plant number-processing diseased plant number)/control diseased plant number × 100%
Potting is raw to survey the result shows that NBF715 fermentation liquid is 100% (Fig. 7, table 6) to ginger bacterial wilt preventive effect
6 NBF715 fermentation liquid of table is to ginger bacterial wilt control efficiency
Embodiment 5:
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 fermentation liquid prevention and treatment citrus adopts rear mould
Disease:
By Penicillium italicum bacterium from slant activation to PDA, growth is expanded numerous after 4 days, until PDA plate, is brushed after 28 DEG C of growth 10d
Spore is taken, is adjusted to 10 with blood counting chamber5Spore/mL is spare.Citrus in the same size is bought from market, uses 75% alcohol wipe
It is spare three times with aseptic water washing after surface sterilization, take 4mm deep hole with inoculation stylus printer, then be added NBF715 fermentation liquid (according to
It is prepared by 2 liquid fermentation mode of embodiment) 10 μ L, be added 10 μ L pathogen spore suspensions after air drying, control be added 10 μ L without
Bacterium water, moisturizing culture, each processing repeat three times, 15 citruses of each repetition.Statistics control and processing lesion diameter after about 7d,
And calculate preventive effect.
Control efficiency=(control lesion diameter-processing lesion diameter)/control lesion diameter × 100%
Test result shows that NBF715 fermentation liquid leaching fruit reaches 100% (table 7) to Penicillium italicum preventive effect.
7 NBF715 fermentation liquid of table is to Penicillium italicum control efficiency
| Processing | Lesion diameter (mm) | Preventive effect (%) |
| NBF715 fermentation liquid | 0 | 100% |
| Control | 37.45 | - |
Embodiment 6:
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 fermentation liquid prevents and treats tea zonate spot:
By Pestallozzia theae from slant activation to PDA, numerous to PDA plate, swipe spore after 28 DEG C of growth 7d is expanded in growth after 4 days
Son is adjusted to 10 with blood counting chamber5Spore/mL is spare.Tealeaves blade in the same size is acquired from tea tree, sprays NBF715 fermentation liquid
(preparing according to 2 liquid fermentation mode of embodiment) to blade face is saturated, and is beaten afterwards with 4mm punch take zonate spot bacterium bacteria cake for 24 hours, is used
Stab inoculation zonate spot bacterium, subsequent moisturizing culture, each processing repeat three times, 16 blades of each repetition, statistics pair after about 8d
According to processing lesion diameter, and calculate preventive effect.
Control efficiency=(control lesion diameter-processing lesion diameter)/control lesion diameter × 100%
Excised leaf test result shows that NBF715 fermentation liquid is 100% (table 8, Fig. 8) to tea zonate spot preventive effect.
8 NBF715 fermentation liquid of table is to tea zonate spot control efficiency
| Processing | Lesion diameter (mm) | Preventive effect (%) |
| NBF715 fermentation liquid | 0 | 100% |
| Control | 18.78 | - |
Embodiment 7
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 fermentation liquid prevents and treats pepper anthracnose:
By Colletotrichum capsici from slant activation to PDA, growth is expanded numerous after 4 days, until PDA plate, is brushed after 28 DEG C of growth 9d
Spore is taken, spore count is adjusted to 10 with blood counting chamber5Spore/mL is spare.It will be used after 75% ethanol disinfection of pepper seed
Aseptic water washing is three times, subsequent with aseptic water washing is used after 2% hypochlorite disinfectant, using NBF715 fermentation liquid (according to implementation
It is prepared by 2 liquid fermentation mode of example) to pepper seed seed soaking 1h, control is soaked seed with sterile water, and it is outstanding then to access anthrax bacteria spore
Supernatant liquid, each processing repeat three times, 50 seeds of each repetition.Moisturizing culture statistics control and processing germinative number and is counted after 7d
Calculate disease incidence and preventive effect.
Control efficiency=(control disease incidence-processing disease incidence)/control disease incidence × 100%
9 NBF715 fermentation liquid of table passes anthracnose control efficiency to capsicum kind
| Processing | Disease incidence (%) | Preventive effect (%) |
| NBF715 fermentation liquid | 0 | 100 |
| Control | 100.00 | - |
Embodiment 8:
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 fermentation liquid seed soaking prevention and treatment potato evening
Epidemic disease
Field trial carries out with bestowing favour Hefeng County potato planting in Hubei Province, and potato seed potato wedge is soaked seed in NBF715
Fermentation liquid picks up after (preparing) 30min according to 2 liquid fermentation mode of embodiment, is placed in after vacant lot is dried and sows, with work of not soaking seed
For potato seed control.In triplicate, random district's groups arrange for test.Plot area 24m2, uniline plantation, line-spacing 60cm, spacing in the rows 30cm.
Late blight is investigated at the potato florescence, every cell selects at 5 points at random, and every takes 5 plants of investigation, the classification of late blight disease index
Canonical reference GB/T 17980.34-2000.
Disease index=∑ (plant number × rank at different levels)/(investigation total strain number × highest represents rank) × 100.
Control efficiency=(control disease index-processing disease index)/control disease index × 100%
10 NBF fermentation liquid of table is to late blight of potato control efficiency
| Processing | Disease index | Preventive effect (%) |
| NBF715 fermentation liquid | 34.67 | 64.32 |
| Control | 12.37 | - |
Claims (9)
1. a kind of isolated streptomycete, the streptomycete is pale purple brown streptomycete (Streptomyces enissocaesilis)
NBF715, deposit number are CCTCC NO:M2017414.
2. the fermentation process of streptomycete described in claim 1, the fermentation process includes by streptomycete described in claim 1
Seed liquor be inoculated in fermentation medium by 5%-10% amount, temperature is 28 DEG C, 200-500 revs/min of the mixing speed of fermentor,
Ventilatory capacity is 1:1-1.2, and the loading amount of fermentation medium is the 60%-80% of tank volume；Fermentation 4-6 days；
The formula of the fermentation medium includes: mannitol 1-3%, soy peptone 1-3%, yeast powder 0.3-0.5%, carbon
Sour calcium 0.3-0.5%, sodium chloride 0.3-0.8%；PH value 6.5-7.5, remaining is water, and percentage described above is quality percentage
Than.
3. the fermentation process of streptomycete described in claim 1, the fermentation process includes by streptomycete described in claim 1
Seed liquor be connected in solid medium by 5%-10%, sample-loading amount 10%-30%, 28 DEG C of -30 DEG C of culture 8d-10d；
The solid fermentation culture medium, component include: by weight ratio
Wheat bran or rice or corn are cut 50-83, glucose 1-3, potassium nitrate 1-5, dipotassium hydrogen phosphate 1-3, sodium chloride 2-3, carbonic acid
Calcium 2-3, appropriate amount of water；
When using wheat bran, material-water ratio 10:5-8；
When using rice or corn is cut, material-water ratio 10:8.
4. streptomycete described in claim 1 is preparing the application in tobacco black shank protective agents.
5. streptomycete described in claim 1 is preparing the application in ginger bacterial wilt protective agents.
6. streptomycete described in claim 1 is preparing the application in Penicillium italicum protective agents.
7. streptomycete described in claim 1 is preparing the application in tea zonate spot protective agents.
8. streptomycete described in claim 1 is preparing the application in pepper anthracnose protective agents.
9. application of the streptomycete described in claim 1 in preparation late blight of potato protective agents.
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| CN109824433A (en) * | 2019-03-18 | 2019-05-31 | 山西省农业科学院生物技术研究中心 | It is a kind of prevent and treat Strawberry anthracnose microbial organic fertilizer and application |
| CN111593008B (en) * | 2020-06-23 | 2021-06-29 | 湖北省生物农药工程研究中心 | Solid-state fermentation method and application of streptomyces lavendulae NBF715 |
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