CN106867934B - One plant of Siam bacillus and its application in prevention and treatment plant powdery mildew - Google Patents
One plant of Siam bacillus and its application in prevention and treatment plant powdery mildew Download PDFInfo
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- CN106867934B CN106867934B CN201710079963.9A CN201710079963A CN106867934B CN 106867934 B CN106867934 B CN 106867934B CN 201710079963 A CN201710079963 A CN 201710079963A CN 106867934 B CN106867934 B CN 106867934B
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Abstract
The invention discloses a kind of Siam bacillus (Bacillus siamensis) X, it is CCTCC NO:M 2017008 in the deposit number of China typical culture collection center, strain of the present invention is applied in prevention and treatment plant powdery mildew, relative to existing chemical prevention medicament, control efficiency is significant, can quickly and effectively control and mitigate the state of an illness, diseased plant is set quickly to get well, and control efficiency is stable, uneasy to recur, and it is safe and nontoxic, harmless, environmentally friendly, effectively avoid chemical pesticide control bring pesticide residue and problem of environmental pollution；And product of the present invention preparation method is simple, easily operated, is suitable for industrialized production and marketing application.
Description
Technical field
The invention belongs to microorganism fields, and in particular to one plant of Siam bacillus and its prevention and treatment plant powdery mildew in
Using.
Background technique
Powdery mildew is caused by the fungi of Erysiphaceae, is common plant disease, multiple in water fruits and vegetables, predominantly
Evil aboveground vegetation part, including the positions such as leaf, stem, anthocaulus and bud, petal, fruit, it is mould that white powder shape is born on affected part surface
Layer, mildew early stage individually dispersion, bisque is thin, after be unified into a big mildew, white powder thickness and it is more, it might even be possible to cover full leaf or
Flowers and fruits or whole strain, serious person infect in flakes, seriously affect photosynthesis, eubolism is made to be interfered, and cause early ageing, subtract
It produces, ordinary circumstance lower part blade is more than upper blade, and vacuum side of blade is more than front.
Such as powdery mildew of strawberry is the Major Diseases of open country and protecting field strawberry production, rapidly, harm is serious for disease sprawling,
Its pathogenSphaerotheca aphanisBelong to Ascomycotina gang pyrenomycetes Erysiphales Erysiphaceae Sphaerotheca.Due to same
One area is planted year after year, and in recent years, powdery mildew of strawberry is in the trend gradually aggravated, especially protecting field powdery mildew of strawberry, is occurred
When serious, for sick leaf rate 45% or more, diseased fruit rate has seriously affected the yield and quality of strawberry, to Strawberry industry 50% or more
Healthy and sustainable development constitute potential threat.
Currently, production is upper mainly using chemical pesticide controls powdery mildews such as triazolone, nitrile bacterium azoles, difenoconazoles.However,
Chemical pesticide it is long-term, continuous use not only Powdery Mildew is made to produce drug resistance, cause Agro-chemicals control effect year by year under
Drop, and the serious pollution agricultural product of chemical agent residual and environment, jeopardize human and livestock health.Therefore there is an urgent need to develop efficient, low
Malicious and environmentally safe biological pesticide preparation.
Biological control not only reduced environmental impact, is also less prone to develop drug resistance, the research of correlation theory and technology
It is increasingly concerned.Beneficial microorganism screening in recent years and its separation of metabolite have become biological control research
Hot spot, it is some by Biocontrol microorganism exploitation pesticides important function is played in pest management.
Summary of the invention
The object of the present invention is to provide one plant of Siam bacillus (Bacillus siamensis) X, in January, 2017
It is preserved within 5th in China typical culture collection center, deposit number is CCTCC NO:M 2017008, preservation address: in
State, Wuhan, Wuhan University.
Another object of the present invention is to provide the new application of Siam bacillus X, i.e. its answering in prevention and treatment plant powdery mildew
With.
It is applied and is being prepared with the bacteria suspension of Siam bacillus X, culture solution, tunning or microbial inoculum containing it in
It prevents and treats in powdery mildew of strawberry formulation products, said preparation product, can also be anti-including plant disease using above-mentioned substance as active constituent
The auxiliary material being applicable in controlling, routinely dosage adds, and is prepared into suitable for practical product forms.
In use, carrying out spraying treatment in growing process；It is by spraying above-mentioned Siam bacillus X in specific culture
Fermentation liquid or its bacteria suspension or its culture solution or its zymotic fluid product in base or the microbial inoculum containing it.
Heretofore described plant is strawberry, romaine lettuce, Lettuce, cucumber, sponge gourd etc., and the powdery mildew feature is by conventional mirror
Surely be the common powdery mildew of such plant, grown on the blade of plant, petiole, flower or/and fruit very thin white hypha layer,
White powder object or/and white scab.
The culture medium that Siam bacillus X is saved and activation is used is basic culture medium, 5 .0g of beef extract, peptone
10 .0g, NaCl 5g, agar 15g are settled to 1 liter with distilled water, pH 7.2~7.4.
Control efficiency of the invention: Siam bacillus provided by the invention prevents and treats powdery mildew of strawberry in potted plant experiment
Control efficiency is up to 100.0%, and in greenhouse field trial, sprinkling is uniformly comprehensive, control efficiency up to 90.0% or more,
And the strawberry growing way for being jetted through the microbial inoculum is healthy and strong, fruit colour, size and the fruit without spraying microbial inoculum are compared redder bigger, it was demonstrated that
The microbial inoculum plays the role of promoting strawberry quality；Preventive effect reaches in the prevention and treatment of the vegetables powdery mildew such as romaine lettuce, Lettuce, sponge gourd, cucumber
75% or more, relative to traditional chemical medicament, strain control efficiency of the present invention is high and effect stability, it is uneasy to recur, highly-safe,
Environmentally friendly noresidue effectively avoids chemical pesticide control bring pesticide residue and problem of environmental pollution；Therefore strain of the present invention is made
For the biological control material of plant powdery mildew, new biological pesticide or biological control microbial inoculum no matter are developed, is all had good
Application prospect, and product of the present invention preparation method is simple, easily operated, large-scale production cost of material is low, is suitable for industrial metaplasia
Production and marketing application.
Detailed description of the invention
Fig. 1 is Antagonistic Fungus form when Siam bacillus X has found；
Fig. 2 is the form photo of Siam bacillus X after purification；
Fig. 3 is the phylogenetic tree of the 16SrDNA building based on Siam bacillus X.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and examples, but the scope of the present invention is not limited to
The content, unless otherwise specified, it is unless otherwise specified routine using reagent that routinely operation carries out to method in embodiment
Purchase reagent or the reagent prepared according to a conventional method.
Base agar culture medium used in embodiment: 5 .0g of beef extract, 10 .0g of peptone, NaCl 5g, agar
15g is settled to 1 liter with distilled water, pH 7 .2~7 .4；
Liquid basal medium: 5 .0g of beef extract, 10 .0g of peptone, NaCl 5g are settled to 1 liter with distilled water, pH
The .4 of 7 .2~7；
LB culture medium: 5 .0g of yeast extract powder, 10 .0g of peptone, NaCl 10.0g, agar 15g, with distilled water constant volume
To 1 liter, pH 7 .2~7 .4；
Potato dextrose agar (PDA): potato 200g, glucose 20g, agar 15g use distilled water
It is settled to 1 liter, natural pH.
Embodiment 1: separation, screening and the identification of Siam bacillus X
One, the separation and screening of bacterial strain
1,9 grades of infection powdery mildew of the strawberry greenhouse of Xishan District, Kunming City Guanyin Mountain highway owner-peasant villa garden are acquired
Strawberry fruit, and installed with sterilizing vinyl disc and take back laboratory；
2, on aseptic operating platform near Alcohol Flame, vinyl disc is opened, strawberry fruit is clamped with sterilizing tweezers, by white powder
Layer is slightly compressed on base agar culture medium flat plate on one side than thicker, and same method separately takes two powdery mildew of strawberry fruits point
It is not slightly compressed in PDA culture medium and LB culture medium flat plate, cultivates (about 20 DEG C) at room temperature；
3, after 6 days, several similar fungies are grown on different plates, wherein finding on a LB culture medium flat plate
While having grown several fungies, there is the growth of fungi on one plant of bacterium strong inhibition plate on side, and form will be as shown in Figure 1, will
The bacterial strain is labeled as X；
4, strain X is chosen and carries out scribing line purifying, bacterial strain after purification is used for follow-up test and preservation.
Two, the identification of bacterial strain
1, strain X morphological feature
On LB culture medium, 30 DEG C are cultivated 1 day, and bacterium colony is round, and water moistens clear viscous, and neat in edge grows bacterium two days later
Fall gradual drying, surface fold occurs and bleaches (Fig. 2).
2, the physiological and biochemical property of strain X
Siam bacillus X can be very good using dextrose and saccharose, be 30-40 DEG C than convenient growth temperature；
Than convenient pH 5.0-8.0, optimal pH 7.0；It can be grown under conditions of NaCl concentration is 0~15%, than convenient salt
Concentration is 0~5%；Other physiological and biochemical properties are shown in Table 1.
The physiological and biochemical property of 1 strain X of table
Note: "+" representative can be grown or the positive, and "-" representative is not grown or feminine gender.
3,16S rRNA is identified
The total DNA of strain X is extracted, and using the DNA as template, PCR amplification is carried out using universal primer 27f and 1492r, is expanded
Volume increase object carries out sequencing after detecting, and sequence is as shown in SEQ ID NO:1, the phylogenetic tree based on 16S rDNA sequence
As shown in Figure 3；
Primer sequence is as follows:
27f (upstream primer): 5 '-AGAGTTTGATCCTGGCTCAG-3 '；
1492r (downstream primer): 5 '-TACGGTTACCTTGTTACGACTT-3 '；
According to morphological feature, Physiology and biochemistry and Molecular Identification result and analysis, strain X is accredited as Siam bacillus
(Bacillus siamensis).
Embodiment 2: controlling experiment of Siam bacillus X to the powdery mildew of strawberry of Pot-strawberry
1, Siam bacillus X is seeded in liquid basal medium, cultivates 3d or so in 30 DEG C, 160rpm, obtains
Fermentation liquid, coating count, result 109Cfu/mL or more, it is spare.
2, pot experiment
In the strawberry greenhouse of owner-peasant villa garden, chooses powdery mildew and falls ill than heavier 15 plants of wounded in the battle, fruit strawberry (basin),
3 plants of healthy strain (basin), concentration is put together, 1-18 and labelled is numbered, while recording the powdery mildew of each plant of strawberry
Situation, including blade, the powdery mildew rank of fruit, quantity etc.；
1. number strawberry: having 7 great Ye, many white powder scabs in the back side, 3, the blade of 9 grades of powdery mildews, the leaf of 7 grades of powdery mildews
Piece 3,5 grade blades 1 have a big titbit, 2 fruitlets, and fruit does not have powdery mildew.
2. number strawberry: having 6 great Ye, many white powder scabs in the back side, whole blades are 9 grades of powdery mildews, no Hua Wuguo.
3. number strawberry: having 7 great Ye, 3, the blade of 9 grades of powdery mildews, no Hua Wuguo.
4. number strawberry: having 7 great Ye, many white powder scabs in 2 tender leaf back sides are 9 grades of powdery mildews, there is 1 big fruit, 4 small
Fruit, fruit do not have powdery mildew.
5. number strawberry: having 6 great Ye, the back side is white powder scab, all 9 grades of powdery mildews of blade entirely.
6. number strawberry: having 8 great Ye, blade does not have powdery mildew, there is powdery mildew on 1 titbit, and 1 little Hua is entirely white
Powder, 9 grades white powder disease fruit 2.
7. number strawberry: have 6 great Ye, blade powdery mildew is light, 5 grades powdery mildew disease fruit 1,3 fruitlets, 1 spend it is disease-free.
8. number strawberry: having 8 great Ye, old leaf is disease-free, there is obvious small white powder spot, 7 grades of sick grade on 2 new leaf backs.
9. number strawberry: blade has a slight powdery mildew, 9 grades powdery mildew disease fruit 2.
10. number strawberry: there are 5 great Ye, 1, the blade of 9 grades of powdery mildews, 7 grades 3,5 grades 1,7 grades disease fruit 1.
Number strawberry: having 6 great Ye, 3, the blade of 7 grades of powdery mildews, and 5 grades 3,5 grades white powder disease fruit 1, on titbit handle
There is slight white powder.
Number strawberry: having 7 great Ye, 3, the blade of 7 grades of powdery mildews, and 5 grades 1,7 grades white powder disease fruit 1.
Number strawberry: there are 8 great Ye, 3, the blade of 9 grades of powdery mildews, no Hua Wuguo.
Number strawberry: having 7 leaves, 3, the blade of 9 grades of powdery mildews, and 9 grades white powder disease fruit 1,7 grades 1.
Number strawberry: having 5 great Ye, and blade white powder is slight, and 5 grades white powder disease fruit 2.
~Number strawberry: healthy strain, blade, flower, fruit are without powdery mildew.
Spray first time fermentation liquid: the fermentation liquid 400mL of strain X is sprayed on 18 plants of strawberries by the way that spray pattern is careful,
Fermentation liquid squirts strawberry strain, including leave dual sides and flower, fruit comprehensively as far as possible；It is primary every observation in 3 days later, it observes 4 times
After find, powdery mildew does not have spreading trend, without brushy subiculum before and once trembling the conidia powder being scattered, white bacterium
Silk layer it is thinning it is shrivelled be sticked on the leaf back side or fruit, susceptible fruit mycelia is shrivelled, and fruit has a sign of rehabilitation, and shake is original
Susceptible blade and fruit is scattered without spore, normal plants.
It sprays second of fermentation liquid: carrying out within 12 days after the spray of first time fermentation liquid, added in the fermentation liquid 200mL of strain X
After the dilution of 200mL water, being sprayed in 18 plants of strawberry strains, i.e., fermentation liquid dilutes one times, and it is primary every observation in 3 days later, it observes 4 times
Afterwards, whole strawberry strains are found all without powdery mildew, leaf color is normal, and fruit is bright and beautiful, new long in addition to original white powder residuum
Floral leaf fruit out is without powdery mildew.
Embodiment 3: controlling experiment of the strain X fermentation liquid to powdery mildew of strawberry in greenhouse field trial
1, strain X is seeded in liquid basal medium, cultivates 3d or so in 30 DEG C, 160rpm, obtains fermentation liquid, apply
Cloth counts, and as a result 109Cfu/mL or more, every time with 6kg fermentation liquid to water 54kg, i.e. original fermentation liquor dilutes 10 times, for following
Test.
2, test carries out in three strawberry vinyl houses of the Xishan District, Kunming City village Fu Shan strawberry cultivating family Dong, each
The long 48m of vinyl house, wide 6m, 8 ridge of planting strawberry, about 860 plants of every ridge strawberry, than heavier, strawberry, which is in, contains fruit for powdery mildew morbidity
Phase, powdery mildew is multiple fruit and to be taken, disease index 35%；Test process: No. 1 canopy uniformly sprays the strain X of 10 times of dilution
Fermentation liquid；No. 2 canopy sprays benzene first propiconazole (difenoconazole containing 15%) compare as medicament；No. 3 canopies, which do not spray any medical fluid, to be made
For blank control；Spray second of medical fluid after 7 days, the 14th day record drug effect, the 21st day record powdery mildew recurrence be as a result as follows
Shown in table 2.
Table 2 prevents and treats powdery mildew of strawberry test result
The results show that the powdery mildew disease index of spray strain X fermentation liquid zone is 3%, the disease index of blank district is
38%, control efficiency 92.1% is significantly higher than the preventive effect of chemical agent benzene first propiconazole, and strain X fermentation liquid control efficiency
It is more stable lasting, it is uneasy to recur.Test effect shows that the disease fruit front and back sides of No. 1 canopy sprinkling fermentation liquid are widely different, front fermentation
Liquid sprays more, and fruit face restores fast, and glossiness is good, and the fermentation liquid being sprayed on one side close to film is few, and powdery mildew is still being grown,
Fruit face will rot, and after extracing similar fruit, the strawberry entirety growing way in greenhouse is restored, and fruit powdery mildew significantly reduces and no longer
Recurrence.Spray is it has been observed that the strawberry growing way for being jetted through the microbial inoculum is healthy and strong after one month, and fruit colour, size are with no sprinkling bacterium
The fruit of agent is red bigger compared to more, it was demonstrated that the microbial inoculum plays the role of promoting strawberry quality.
Embodiment 4: controlling experiment of the strain X fermentation liquid to vegetables powdery mildew in greenhouse field trial
1, strain X is seeded in liquid basal medium, cultivates 3d or so in 30 DEG C, 160rpm, obtains fermentation liquid, apply
Cloth counts, and as a result 109Cfu/mL or more, every time with 1 kg fermentation liquid to water 9kg, i.e. original fermentation liquor dilutes 10 times, for following
Test.
2, test carries out in the vinyl house at the Kunming Chenggong village Qu Dounan vegetable cultivation family, and kind is implanted with romaine lettuce, naked oats
Dish, cucumber, greenhouse each one of sponge gourd, each vinyl house area 150m2, identified blade germ is powdery mildew, the state of an illness
Index is 5%~25%；Test process: each greenhouse is divided into three parts, one third part uniformly sprays 10 times of dilution
Strain X fermentation liquid；Another one third part spray benzene first propiconazole (difenoconazole containing 15%) compares as medicament；It is remaining
One third does not spray any medical fluid as blank control；Powdery mildew situation is recorded after 10 days, as a result as shown in table 3 below；
3 strain X fermentation liquid of table prevents and treats vegetables powdery mildew test result
The results show that the vegetables powdery mildew of spray strain X fermentation liquid zone has obtained effective control, control efficiency up to 76% or more,
It is significantly higher than the anti-efficiency of chemical agent benzene first propiconazole, and strain X fermentation liquid control efficiency is more stable persistently, is not easy multiple
Hair；Vegetables powdery mildew is prevented and treated using microbial bacterial agent, environmentally friendly to vegetable safety, no chemical pesticide residual, ecological, environmental protective
It is pollution-free.
Sequence table
<110>Kunming Agriculture Technology Research Institute
<120>one plants of Siam bacillus and its application in prevention and treatment plant powdery mildew
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1360
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<213>Siam bacillus X
<400> 1
tgcagtcgag cggaagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac 60
acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct aataccggat 120
ggttgtttga accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg 180
acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc 240
gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300
cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360
tgaaggtttt cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg 420
gcggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480
taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt 540
tcttaagtct gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa 600
cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg 660
gaggaacacc agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc 720
gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780
tggttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840
agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtgggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960
tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080
ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140
tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200
caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260
gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1360
<210> 2
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agagtttgat cctggctcag 20
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tacggttacc ttgttacgac tt 22
Claims (2)
1. one plant of Siam bacillus (Bacillus siamensis) X, in the preservation of China typical culture collection center
Number is CCTCC NO:M 2017008.
2. application of Siam bacillus X described in claim 1 in prevention and treatment plant powdery mildew.
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| CN107384839B (en) * | 2017-09-05 | 2020-06-30 | 农业部沼气科学研究所 | Bacillus siamensis BERC-11 and application thereof |
| CN107624632A (en) * | 2017-09-21 | 2018-01-26 | 广西吉朋投资有限公司 | A kind of potted plant cultural method of strawberry |
| CN108060100B (en) * | 2017-12-29 | 2021-01-29 | 中国科学院南海海洋研究所 | Multifunctional Siamese bacillus and preparation and application of bioactive substance thereof |
| CN108849986B (en) * | 2018-07-09 | 2020-09-15 | 江苏师范大学 | Application of bacillus simmer in preventing and treating white rot |
| CN109362787A (en) * | 2018-12-07 | 2019-02-22 | 江苏师范大学 | Application of Siam bacillus in prevention and treatment nematode |
| CN109734486A (en) * | 2019-02-15 | 2019-05-10 | 陕西天仁雪农业科技有限公司 | A kind of kitchen garbage devil liquor recovery utilizes processing method |
| CN113439763B (en) * | 2021-07-28 | 2022-05-10 | 广西壮族自治区农业科学院 | Application of endophytic fungus strain X22 in preventing and treating powdery mildew |
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