CN107897179A - The effective use method of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis - Google Patents

The effective use method of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis Download PDF

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CN107897179A
CN107897179A CN201711118761.7A CN201711118761A CN107897179A CN 107897179 A CN107897179 A CN 107897179A CN 201711118761 A CN201711118761 A CN 201711118761A CN 107897179 A CN107897179 A CN 107897179A
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pts
bacillus subtilis
dilution
root
wettable powders
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乔俊卿
刘邮洲
刘永锋
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Jiangsu Academy of Agricultural Sciences
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Jiangsu Academy of Agricultural Sciences
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION 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
    • A01N25/00Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/12Powders or granules
    • A01N25/14Powders or granules wettable
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION 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
    • A01N63/00Biocides, 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

Abstract

The present invention relates to a kind of effective use method of 394 wettable powder field control Fusarium solanis of bacillus subtilis PTS, belong to biocontrol agent applied technical field.This method provides more specific more efficient method for use of 394 wettable powders of bacillus subtilis PTS in field, the use scope and prevention target of 394 wettable powders of bacillus subtilis PTS have not only further been expanded, has also provided theoretical foundation for the further marketization and field use of 394 wettable powders of bacillus subtilis PTS.

Description

The height of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis Imitate application method
Technical field
The invention belongs to biocontrol agent applied technical field, and in particular to a kind of bacillus subtilis PTS-394 wettables The effective use method of pulvis field control Fusarium solani.
Background technology
Greenhouse pepper is the major industry of increasing peasant income in Jiangsu Province's modern agriculture, reaches 100-120 ten thousand per annual planting area Mu.However, closed environment and continuous continuous cropping continuous cropping due to facility, cause soil surface characters to accumulate, soil-borne disease (is dampinged off Disease, droop, root rot, damping-off, epidemic disease, bacterial wilt etc.) frequently outburst.In recent years, the capsicum as caused by eggplant handle sickle-like bacteria Root rot progressively becomes the important disease of facility cultivation, and average attack rate reaches 10-30%, underproduction 20%-50%, seriously makes The about safety in production of greenhouse pepper and quality guarantee.
At present, facility for prevention and control Fusarium solani relies primarily on chemical pesticide in production, but prevention effect is undesirable always, root Maize ear rot still occurs more serious;In addition, chemical pesticide easily causes facility ecological environmental pollution, increase vegetables agricultural product pesticide is residual Stay, seriously endangered to mankind's health care belt.Though there is research Biocontrol microorganism to control the relevant report of Fusarium solani in recent years, But the screening of Fusarium solani biocontrol microorganisms is focused primarily upon, goes forward side by side and enters the market there has been no molding biological prevention and control agent product Demonstration And Extension .At present, (active ingredient is bacillus subtilis to the SubtilexNG of external existing Becker Underwood companies exploitation MBI 600) product, can apply or dress seed in root prevention sickle-like bacteria (Fusarium spp.), aspergillus (Aspergillus Spp.), beans, wheat class, cotton caused by Alternaria (Alternaria spp.) and Rhizoctonia (Rhizoctonia spp.) Flower and peanut root disease.Country's bacillus subtilis commercial preparation for having succeeded in developing and having put into production have hundred anti-, wheat Fengning, Line Qu Ning, obtain according to day, root-rot disappears.The microbial pesticide " hundred is anti-" that Yunnan Prov Agriculture University and China Agricultural University develop jointly (1,000,000,000/g bacillus subtilis B908 wettable powders) obtains the Ministry of Agriculture and registers, and is promoted the use of in multiple provinces, promotes About 4 667hm2 of area, mainly prevents rice sheath blight disease, notoginseng root rot, tobacco black shank.Institute of Plant Protection of academy of agricultural sciences of Jiangsu Province is old Will friendship researcher exploitation " line Qu Ning " is 10,000,000,000 living spores/ml bacillus subtilis Bs916 aquas and 2.5% jinggangmeisu Compound preparation, it is main to prevent rice sheath blight disease and false smut, be widely popularized on Jiangsu and other places.Root-rot disappears as Kunming The wettable powder compounded by bacillus subtilis and Pseudomonas fluorescens that Wo Lin bio-engineering corporations register, passes through filling Root processing prevention notoginseng root rot.
The important soil-borne disease cause of disease of facilities vegetable is carried out since 2006 in Plant Protection Inst., Jiangsu Academy of Agriculture The research of the species of bacterium, biological characteristics and occurrence regularity, it is (solanaceous vegetables bacterial wilt, withered with several important soil-borne diseases Disease, damping-off, root rot) pathogen is target, screening obtains plant height effect antagonistic strain, i.e. bacillus subtilis (Bacillus subtilis) PTS-394, China Microbiological collection bacterial strain preserving number are:CGMCCNo.8085 (application numbers For the Chinese patent of 201110339098.X).Then, by optimize its zymotechnique (Application No. 201310081080.3 Chinese patent), and successfully develop bacillus subtilis PTS-394 wettable powders.As most vegetable cultivation family is to life The receiving of the theory of thing prevention, corresponding biological pesticide effective use technology has become the key point in prevention, in order to solve In Field information, peasant household is often each by rule of thumb using biological pesticide (PTS-394 wettable powders), insecticide spraying time, medication Mode and dosage are chaotic, so that the situation of various regions field soil-borne disease prevention effect unstable (40-85%) is caused, we Early period for facility solanaceous vegetables bacterial wilt, droop, capsicum damping-off and Strawberry Root Rot field practical technique into (Chinese patent application of Application No. 201510760770.0) is screened in research of having gone.
The invention mainly relates to bacillus subtilis PTS-394 wettable powders in prevention Fusarium solani, promotion capsicum The effective use method of growth etc., is that above-mentioned PTS-394 has studied the further abundant of invention and expanded.It is of the invention main logical Cross in laboratory, it is peppery in prevention that greenhouse pot culture and field plot etc. have screened bacillus subtilis PTS-394 wettable powders Green pepper root rot, usage time, dosage and the mode etc. in chili growth of promoting, and further integrate, being formed detailed efficiently makes With technology, theoretical guarantee is provided for the field effective use and Demonstration And Extension of bacillus subtilis PTS-394 wettable powders.
The content of the invention
The purpose of the present invention is to solve existing Biocontrol microorganism pesticide because field use technical deficiency caused by preventive effect Wild effect, and provide a kind of the efficient of bacillus subtilis PTS-394 wettable powders field control Fusarium solani Application method.This method has not only further expanded use scope and the prevention of bacillus subtilis PTS-394 wettable powders Target, also provides theoretical foundation for the further marketization and field use of bacillus subtilis PTS-394 wettable powders.
The present invention adopts the following technical scheme that:
The effective use method of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis, including with Lower step:
Step 1, seed treatment:Choose full seed, soak in water, then by seed PTS-394WP dilution Middle seed soaking is overnight;
Step 2, seedling medium, which is mixed, to be applied:Before planting, the Nutrition Soil of hole plate seedling growth mixes native processing with PTS-394WP, uniformly mixes Soil simultaneously carries out program request;
Step 3, field planting processing:Before transplanting, spread fertilizer over the fields with PTS-394WP and colonize cave, a water living is poured after transplanting;
Step 4,5-7d pour seedling recovering waters after transplanting, the dilution liquid irrigating root of PTS-394WP is used after transplanting 15-20d, and is carried out Earth up root:
Step 5, according to the different plantation crops for rotation root-rot pathogenetic times, synchronous liquid manure rule, uses at interval of 15-20d The dilution of PTS-394WP carries out pouring root, until the root rot important generation phase terminates;
Step 6, in fruit expanding period, with the dilution liquid irrigating root of PTS-394WP;
Step 7, other pest and disease damages routinely schema management.
Further, step 1 is specially:Full seed is chosen, 4-5h is soaked in warm water, then seed exists Soak seed overnight in the dilution of PTS-394WP, it is PTS-394 wettable powders that PTS-394WP is stated in step 1.
Further, the temperature of warm water described in step 1 is 40-55 DEG C, and the dilution of the PTS-394WP is The dilution that PTS-394WP is obtained after being mixed with water according to 1: 1000 mass ratio, the seed soaking overnight time is 12-16h.
Further, when soil is mixed described in step 2, the ratio of PTS-394WP and Nutrition Soil is 10kg bacterium powders/m3Battalion Support soil.
Further, before being transplanted in step 3, uniformly spread fertilizer over the fields according to 8g PTS-394WP/ seedlings and colonize cave, and watered When every seedling watering 800mL.
Further, the dilution of PTS-394WP described in step 4 for PTS-394WP and water according to 1: 100 matter The dilution than being obtained after mixing is measured, and pouring root is carried out according to a 400ml dilutions/seedling during pouring root.
Further, the dilution of PTS-394WP described in step 5 for PTS-394WP and water according to 1: 100 matter The dilution than being obtained after mixing is measured, and pouring root is carried out according to a 400ml dilutions/seedling during pouring root.
Further, planted the crops for rotation root-rot pathogenetic times according to different described in step 5, synchronous liquid manure rule, Specially:Spring stubble:After capsicum is colonized (mid-March), impose 2 times;Autumn and winter stubble:2 are imposed after capsicum is colonized (mid-September) It is secondary;Overwintering stubble:Imposed 1 time after capsicum is colonized (mid-November), start April in the coming year to impose again 2 times.
Further, the dilution of PTS-394WP described in step 6 for PTS-394WP and water according to 1: 1000 matter Measure the dilution than being obtained after mixing.
Compared with prior art, the present invention its advantage is mainly realized by following approach, it is specific as follows:
The present invention is stood facing each other by tablet test first, tentatively specify that PTS-394 bacterial strain wettable powder methanolic extracts There is good inhibiting effect to Fusarium Solani (eggplant handle sickle-like bacteria);
Further Mass Spectrometer Method, finds still to contain fat peptide matters in wettable powder, but fat peptide matters Species and content have more loss.This shows that from the exploitation that screens of antagonistic strain be ripe formulation products, can be to life The fungi-proofing characteristic of itself has larger change;The assessment experiment display of the potted plant growth-promoting of Hot Pepper in Greenhouse, preventive effect etc., bacillus subtilis The concentration of PTS-394 wettable powders has an impact growth-promoting, the preventive effect of capsicum, is specifically rendered as:Low dosage has growth-promoting Effect, and higher concentration has preventive and therapeutic effect.Above result of study is bacillus subtilis PTS-394 wettable powders in field Between application, there is provided the screening of suitable concentration and usage time etc., subsequent field plot trial are further perfect The effective use methods of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis, this method is withered grass Use of the bacillus PTS-394 wettable powders in field provides more specific more detailed operating process, not only expands The use scope and prevention target of bacillus subtilis PTS-394 wettable powders, it is also wettable for bacillus subtilis PTS-394 Property pulvis it is further the marketization and field effective use provide theoretical foundation.
Brief description of the drawings
Fig. 1 is inhibitory action schematic diagrames of the bacillus subtilis PTS-394 to Fusarium Solani, and wherein PTS-394 is PTS-394 thalline;PTS-394YM is YPG zymotic fluid methanolic extracts;PTS-394WPM is PTS-394 wettable powder methanol Extract;PTS-394WPM10 dilutes 10 times for PTS-394 wettable powders methanolic extract;Control is methanol.
Embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair Bright scope.
The effective use method of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis, including with Lower step:
Step 1, seed treatment:Full seed is chosen, immersion 4-5h, then exists seed in warm water (40-55 DEG C) (12-16h) is stayed overnight in seed soaking in PTS-394WP dilutions (1: 1000);
Step 2, seedling medium, which is mixed, to be applied:Before planting, the Nutrition Soil of hole plate seedling growth mixes native processing with PTS-394WP, according to 10kg bacterium powders/m3Soil (1kg bacterium powders: 100L soil) ratio, uniformly mixes soil and carries out program request and (have anti-sprout term disease and promotion concurrently Seed sprouting acts on);
Step 3, field planting processing:Before transplanting, withered PTS-394WP is spread fertilizer over the fields according to 8g/ seedling colonizes cave, and living is poured after transplanting Water, every seedling watering 800mL, dilutes 100 times, for preventing root rot equivalent to by bacterium powder;
Step 4:7d pour seedling recovering waters after transplanting, 1: 100 times of dilution liquid irrigating root (400ml/ of bacterium powder is pressed after transplanting 15-20d Seedling), and carry out root ridging;
Step 5:According to the different plantation crops for rotation root-rot pathogenetic times, synchronous liquid manure rule, uses at interval of 15-20d PTS-394WP (by 400ml/ seedling of 1: 100 times of dilution liquid irrigating root of bacterium powder), up to the root rot important generation phase terminates the (spring Stubble:After capsicum is colonized (mid-March), impose 2 times;Autumn and winter stubble:Imposed 2 times after capsicum is colonized (mid-September);Overwintering stubble: Imposed 1 time after capsicum is colonized (mid-November), start April in the coming year to impose again 2 times);
Step 6:In fruit expanding period, PTS-394WP (1: 1000) pouring root can be used, for promoting plant and fruit to give birth to It is long;
Step 7:Other pest and disease damages routinely schema management.
Embodiment 1:The inhibitory action of bacillus subtilis PTS-394 and its wettable powder to Fusarium solani opportunistic pathogen
Experiment is with Biocontrol Bacillus subtilis PTS-394, Fusarium solani pathogenic bacteria --- eggplant handle sickle-like bacteria HALJ3-1 is equal Separated, preserved with biological control research room by Plant Protection Inst., Jiangsu Academy of Agriculture rice disease.Biological and ecological methods to prevent plant disease, pests, and erosion withered grass bud Spore bacillus PTS-394 pulvis is processed by Luyuan Biochemical Co., Ltd. Yangzhou and (processed within 2016, content is about 2 × 1010 CFU Gemma/g bacterium powders).Fusarium Solani activation culture in PDA culture medium.Bacillus subtilis PTS-394 is in improvement YPG (yeast peptone glucose) culture medium (yeast extract 5g, tryptone 5g, glucose 5g, pH 7.0~7.2, solid training Foster base need to add agar 15g) in activation culture.
Bacteriostatic test uses tablet face-off method:
1) PTS-394 is activated on YPG solid mediums, then unit cell is moved into 5mL YPG nutrient solutions, at 28 DEG C (150r/m) culture 12h is vibrated, according to 1% inoculum concentration, inoculation PTS-394 bacterium solutions enter in YPG liquid to be vibrated at 28 DEG C (150r/m) cultivates 48h, obtains zymotic fluid, while utilize hydrochloric acid precipitation YPG zymotic fluids, methanol extraction purification lipopeptid class chemical combination Thing, finally carries out methanol dissolving, 4 DEG C save backup, this methanol extract liquid is labeled as PTS-394YM according to 1: 10 ratio.By 5g Bacillus subtilis PTS-394 wettable powders are dissolved in 5mL chromatographies methanol (pH=7.0), and 4 DEG C extract the fat in pulvis overnight Peptides (about 16h), 0.22 μm of filtering, 4 DEG C save backup, and this methanol extract liquid is labeled as PTS-394WPM.
2) mycelial growth experiment is suppressed:Fusarium Solani HALJ3-1 is after 28 DEG C of activation cultures of PDA plate, 3d along bacterium Fall edge card punch (diameter 5mm) punching, bacteria cake is placed in blank PDA plate center, at bacteria cake 25mm, in " ten Word " type, instills 1 μ L or 50 μ l (drying) of methanol fat peptide matters extract of PTS-394 bacterium solutions, and often processing is repeated 3 times, and is set Control.All tablets investigate antibacterial bandwidth in 28 DEG C of constant temperature incubations when compareing tablet and covering with.
3) fat peptide matters suppress spore germination experiment:Fusarium Solani HALJ3-1 is in 28 DEG C of activation trainings of PDA plate Support, punched after 3d along colony edge card punch (diameter 5mm), bacteria cake is placed in concussion in sterile water obtains spore liquid (adjustment Concentration is to 104 spore/mL).100 μ L of root-rot bacterium spore liquid are coated with blank PDA, are beaten with plate center in " cross " type Hole, adds 50 μ l (drying) of methanol fat peptide matters extract, and often processing is repeated 3 times, and sets control.All tablets are in 28 DEG C of perseverances Temperature culture, antibacterial bandwidth is investigated when compareing tablet and covering with.Experimental result is as shown in Figure 1:
Bacillus subtilis PTS-394 thalline, hairs of the PTS-394 in YPG have detected using external tablet face-off method respectively Zymotic fluid fat peptide extract and PTS-394 wettable powder fat peptide extracts are to Fusarium Solani (eggplant handle sickle-like bacteria) spore Sprout and the Inhibition test of mycelial growth, Fig. 1 show, bacillus subtilis PTS-394 wettable powder fat peptide extracts pair The spore germination of pine root fungus all has the inhibitory action as PTS-394 culture medium zymotic fluids with mycelial growth.Antibacterial survey Amount data show that to the antibacterial bandwidth 5mm of mycelia, the sprouting antibacterial circle diameter to spore is PTS-394 wettable powders 13.33mm, and PTS-394 zymotic fluid fat peptide matters are 8.67mm to the antibacterial bandwidth of mycelia, the sprouting inhibition zone to spore A diameter of 20.67mm, result above show, fat peptide matters, but its bacteriostasis are still contained in PTS-394 wettable powders Effect than PTS-394 zymotic fluid all significantly reduces, this is probably caused by wettable powder loses in process.
Embodiment 2:The lipopeptide antibiotic detection of bacillus subtilis PTS-394 wettable powders
Fermentations and its fat peptide matters extracting method of the bacillus subtilis PTS-394 in YPG culture mediums are according to implementation Carried out described in example 1;The extraction of fat peptide matters in bacillus subtilis PTS-394 wettable powders is according in embodiment 1 Method, that is, utilize methanol (pH=7.0) extraction, -20 DEG C of leaching liquor is long-term to be preserved.Utilize Matrix assisted laser desorption ion Change-flight time mass spectrum (MALDI-TOF-MS), which is detected in PTS-394 pulvis methanol extract and PTS-394 zymotic fluids, contains fat The species of peptide matters, matrix are α-cyanogen -4- hydroxyls cinnamic acid (α-cyano-4-hydroxy-cinnamic acid).Detection knot Fruit is as shown in table 1:
The mass spectrum of 1 bacillus subtilis PTS-394 zymotic fluids of table and wettable powder methanol extract liquid peptide matters containing fat Detection
(note, "-" represent to be not detected by)
Using ion flight mass spectrum (MALDI-TOF-MS) to PTS-394YPG culture medium zymotic fluid methanolic extracts and PTS-394 wettable powder methanolic extracts are scanned detection, and wavelength is between 200-1600nm.Analyzed by comparing, can With determine PTS-394 YPG fermentation cultures and wettable powder methanolic extract in can detect Surfactin and Two lipoid peptide matters (table 1) of Fengcyin.As seen from Table 1, it can detect that molecular weight is in PTS-394 wettable powders 1021.6, the 1035.7 and 1049.6 Surfactin and Fengycin that molecular weight is 1490.8, and in PTS-394 zymotic fluids Also include 993.6 and 1007.6 Surfactin and Fengycin that molecular weight is 1476.8 and 1504.8;In addition, we It has also been found that the Mass Spectrometer Method intensity (intensity) containing fat peptide matters is only PTS-394 zymotic fluids in PTS-394 pulvis 2 ‰ -1%, this result shows that, Surfactin and Fengycin has a large amount of damages in PTS-394 pulvis process Lose, this is also the main reason for its inhibitory action declines.
Embodiment 3:Growth-promoting functions of the bacillus subtilis PTS-394 wettable powders to capsicum
1. Germination of Capsicum Seed is tested:1) by PTS-394 pulvis according to 1: 10,1: 50,1: 100,1: 200 and 1: 1000 Dilute again spare.Pepper seed soaks 5min sterilizings in 3% liquor natrii hypochloritis, and aseptic water washing, then will be peppery to tasteless Green pepper seed, which is placed in the PTS-394 bacterium solutions of different extension rates, soaks 24h, and control is soaked seed with sterile water;Seed is taken out, is dripped It is positioned over after dry bacterium solution in the culture dish containing 4 layers of aseptic filter paper, then covers sterile gauze layer 2-3, and adds sterile water 10mL, 30 are sowed per tablet, is repeated 3 times, germination situation is counted after 6d, calculates germination rate.2) by PTS-394 pulvis and hole tray Sowing Nutrition Soil mixes soil according to 1: 100,1: 200 and 1: 1000 times of volume ratio, makes 72 hole disk seedbeds, broadcasts pepper seed is dry Enter in hole tray, per 1, hole, after planting blinding waters, and is placed in greenhouse (natural lighting, 15 DEG C -28 DEG C), compares as normal nutrition Soil sowing;It is repeated 3 times, growth of cereal crop seedlings condition is checked after 15d, calculates germination rate.
2. conventional soil prepares to be used for greenhouse Biological control according to sieving nature soil, Nutrition Soil and the proportioning of vermiculite 1: 1: 1: 1. Pepper seed is first sown into conventional soil, when 41 heart of leaf of Hot Pepper Seedling, transplanting Hot Pepper Seedling is required in list according to growth-promoting relevant treatment In only basin alms bowl, heliogreenhouse growth (natural lighting, 15 DEG C -28 DEG C) is placed in.1) greenhouse pot culture plant growth-promoting is tested:If at 4 Reason, is Routine control (CK), 1: 100 times of liquid of pouring root PTS-394 pulvis processing (PTS-1: 100) respectively, pouring root PTS-394 pulvis 1: 1000 times of liquid processing (PTS-1: 1000) of 1: 200 times of liquid processing (PTS-1: 200) and pouring root PTS-394 pulvis.Often handle 30 Alms bowl, is repeated 3 times.In 3 PTS-394 experiment process, every capsicum pouring root PTS-394 Dilution for powder liquid 20mL.25d after transplanting When, plant plant height is measured, and carry out the growth-promoting effect of statistical analysis bacillus subtilis PTS-394 pulvis.Specific experiment result As shown in table 2:
Growth-promoting functions of the 2 bacillus subtilis PTS-394 wettable powders of table to capsicum
Tablet germination rate, hole tray germination rate and plant height be respectively in place after 6,15, the data of 25d.Data are average in table Number ± standard deviation.No, expression do not carry out testing in related place.Represent poor through the new multipoles of DuncanShi with different letters after column data Method examines significant difference (P < 0.05).
It is that promotion of the bacillus subtilis PTS-394 wettable powders to Germination of Capsicum Seed and plant strain growth is made in table 2 With.Tablet vernalization experiment display, the PTS-394 microbial inoculums dilution of various concentrations influence Germination of Capsicum Seed different, vernalization 6d Afterwards, the germination rate of sterile water control is 41.1%, and PTS-394 microbial inoculums 1: 1000,1: 200 and 1: 100 times of dilution processing Seed germination rate be respectively 66.7%, 58.3% and 60%, significantly promote the sprouting of seed than control;In addition, PTS-394 The seed germination rate of 1: 10 times of processing of microbial inoculum is only 24.4%, significantly suppress the sprouting of pepper seed.Greenhouse hole tray (soil) After nursery is the results show that PTS-394 microbial inoculums mix soil by 1: 100,1: 200 and 1: 1000 times of volume ratio, Germination of Capsicum Seed after 15d Rate is respectively 56.4%, 52.2% and 59.6%, and the germination rate of Routine control is only 25%.Result above shows, PTS-394 The low concentration dilution of microbial inoculum has significant facilitation effect to Germination of Capsicum Seed.In addition, capsicum greenhouse pot culture plant growth-promoting The results show that after the dilution root irrigation of 1: 1000,1: 200 and 1: 100 times of PTS-394 microbial inoculums, capsicum plant height is respectively 20.7,22.4 and 21.2cm, and it is only 17.5cm to compare plant height, and 18.3% is added respectively compared to Routine control plant height, 28.0% and the processing of 21.1%, PTS-394 pulvis difference diluted concentration significantly promote the plant height of capsicum.Integrated comparative PTS- For 394 pulvis difference diluted concentrations to Germination of Capsicum Seed, the growth-promoting effect of plant height, takes into account cost, best results principle, uses PTS-394 pulvis is according to 1000 times of processing of dilution, its seed to capsicum is sprouted and plant growth-promoting functions are the most suitable.
Embodiment 4:Bacillus subtilis PTS-394 wettable powders prevent the recruitment evaluation of potted plant Fusarium solani
By PTS-394 wettable powders according to 1: 100,1: 200 and 1: 1000 times of spare, Fusarium Solani of dilution in PDA solid mediums activate, and after 28 DEG C of culture 5d, access corn flour and sandy soil are prepared in the sick soil culture medium of (volume 2: 1), and 28 DEG C culture 20d after, according to sandy soil culture medium:Sieve nature soil, and Nutrition Soil and 1: 1: 1: 1 volume proportion of vermiculite make sick soil, accurate It is standby to carry out preventive effect isothermal chamber experiment.Greenhouse pot culture efficiency test:It is at inoculation Fusarium Solani sick soil respectively if 4 processing 1: 100 times of (HALJ), inoculation Fusarium Solani sick soil and pouring root PTS-394 pulvis liquid processing (HALJ+PTS-1: 100) are managed, It is inoculated with 1: 200 times of liquid processing (HALJ+PTS-1: 200) of Fusarium Solani sick soil and pouring root PTS-394 pulvis and inoculation capsicum 1: 1000 times of liquid processing (HALJ+PTS-1: 1000) of pine root fungus sick soil and pouring root PTS-394 pulvis.30 alms bowls are often handled, repeat 3 It is secondary.In 3 HALJ+PTS experiment process, every Hot Pepper Seedling pouring root PTS-394 Dilution for powder liquid 20mL.After transplanting during 21d, press Potted plant Fusarium solani incidence is investigated according to following Fusarium solani grade scale, and calculates bacillus subtilis PTS-394 The prevention effect of pulvis.Fusarium solani is divided into 5 grades by the morbidity order of severity, and disease scale is as follows:0 grade, plant is not wilted;1 Level, a small amount of blade performance restorability are wilted;3 grades, blade 10%~30% is wilted, and leaf is still green;5 grades, blade 30.1%~ 50% obvious wilting, yellow leaf;7 grades, plant is wilted, and blade 50.1%~80%, which turns yellow, wilts, and rhizome portion becomes brown;9 grades, Complete stool is withered, butt rot.Disease index=and ∑ (sick level strain number × represent numerical value)/(generation of strain number summation × morbidity most heavy duty Table numerical value) × 100.Protection effect (%)=(control disease index-processing disease index)/control disease index × 100.
Tested by greenhouse pot culture, entry evaluation bacillus subtilis PTS-394 wettable powder difference diluted concentrations To the prevention effect of Fusarium solani, as shown in table 3,100 times of diluted concentrations of PTS-394 wettable powders are to Fusarium solani Prevention effect is best, and preventive effect is up to 69.6%, and with the reduction of concentration, its prevention effect is gradually lowered, when dilution 1000 Times when, its preventive effect is only 22.6%.
3 bacillus subtilis PTS-394 wettable powders of table are to potted plant Fusarium solani prevention effect
Embodiment 5:Bacillus subtilis PTS-394 wettable powders prevent the effect of field Fusarium solani
Test period:2016-2017
Test site:Jiangsu Province Huai'an Huaiyin District Yu Gou towns Yang Miao villages (spring stubble) and seven groups of (autumn and winter of Wu Dun towns Wang Qiao villages Stubble) the interior progress of greenhouse pepper greenhouse;
Test variety:Local main breed:Spring stubble is millionaire kind, and autumn and winter stubble is first red No. 1;
Test medicine:Bacillus subtilis PTS-394 wettable powders;Comparison medicament is 50% carbendazim, purchased from Jiangsu Hui Feng agrochemical limited company.
Seedling medium:Seeding and seedling raising organic substrate (Huai'an Zhong Yuan gardening Development Co., Ltd).
Test method:1 heavier greenhouse of Fusarium solani natural occurrence is chosen as experiment greenhouse (spring stubble and autumn and winter Stubble, each 1 greenhouse), experiment is routinely operated with seedling, through hole plate seedling growth and two stages of field planting.By bacillus subtilis during sowing Bacterium PTS-394 wettable powders and seedling medium are mixed by 1: 100 volume ratio, conventional planting;It is being colonized during field planting After bacillus subtilis PTS-394 wettable powders being spread fertilizer over the fields in ditch according to the dosage of 8g/ (1: 100) or 4g/ (1: 200), Hot Pepper Seedling is transplanted again;800mL field planting water is poured, every 15 days after field planting, bacillus subtilis is diluted with water according to 1: 100 ratio PTS-394 wettable powders carry out pouring root, every seedling pouring root 400mL;In subsequent watering proce, according to disease, a situation arises Impose PTS-394 pulvis 1-2 times, it is each to handle about 360 seedlings of cell, it is repeated 3 times, cell random distribution;Contrast agents 50% Carbendazim is recommended by product description;Set up clear water control group at the same time.
Incidence is investigated during the capsicum morbidity of clear water check plot, and calculates preventive effect;Incidence=investigation cell morbidity strain Number/investigation total strain number × 100;The morbidity of preventive effect=(incidence of incidence-check plot of processing cell)/check plot Rate;Result of the test is as shown in table 4:
4 bacillus subtilis PTS-394 wettable powders of table are to field greenhouse pepper root rot prevention effect
By table 4 as it can be seen that using 1: 100 and 1: 200 times of diluted bacillus subtilis PTS-394 wettable powders field respectively Between be respectively 75.2-77.1% and 68.5-70.4% to Fusarium solani preventive effect, and the preventive effect of chemical agent carbendazim is 55.7-63.3%, effect and the conventional chemical medicine of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis The effect of 50% carbendazim of agent is quite even slightly higher.It can be seen from the above that reasonable, in due course, scientifical use bacillus subtilis PTS-394 Wettable powder can effectively prevent the generation of field greenhouse pepper root rot.
Embodiment 6:Bacillus subtilis PTS-394 wettable powder field control Fusarium solani effective use technology collection Into
It is simple according to greenhouse pepper field production operation by the screening and assessment of above example, low input high production Basic principle, integrate summarize bacillus subtilis PTS-394 wettable powder field control Fusarium solani effective use skills Art, specific main points are as follows:
Step 1, seed treatment:Full seed is chosen, immersion 4-5h, then exists seed in warm water (40-55 DEG C) (12-16h) is stayed overnight in seed soaking in PTS-394WP dilutions (1: 1000);
Step 2, seedling medium, which is mixed, to be applied:Before planting, the Nutrition Soil of hole plate seedling growth mixes native processing with PTS-394WP, according to 10kg bacterium powders/m3 soil (1kg bacterium powders: 100L soil) ratio, uniformly mix soil and carry out program request (have concurrently anti-sprout term disease and promote kind Sub- sprouting effect);
Step 3, field planting processing:Before transplanting, withered PTS-394WP is uniformly spread fertilizer over the fields according to 8g/ seedling colonizes cave, is poured after transplanting A water living, every seedling watering 800mL, dilutes 100 times, for preventing root rot equivalent to by bacterium powder;
Step 4:7d pour seedling recovering waters after transplanting, 1: 100 times of dilution liquid irrigating root (400ml/ of bacterium powder is pressed after transplanting 15-20d Seedling), and carry out root ridging;
Step 5:According to the different plantation crops for rotation root-rot pathogenetic times, synchronous liquid manure rule, uses at interval of 15-20d PTS-394WP (by 400ml/ seedling of 1: 100 times of dilution liquid irrigating root of bacterium powder), up to the root rot important generation phase terminates the (spring Stubble:After capsicum is colonized (mid-March), impose 2 times;Autumn and winter stubble:Imposed 2 times after capsicum is colonized (mid-September);Overwintering stubble: Imposed 1 time after capsicum is colonized (mid-November), start April in the coming year to impose again 2 times);
Step 6:In fruit expanding period, PTS-394WP (1: 1000) pouring root can be used, for promoting plant and fruit to give birth to It is long;
Step 7:Other pest and disease damages routinely schema management.
At present, the prevention of greenhouse pepper root rot is main or uses the control method of chemical agent, and this method is easily made The pollution and residual harm of paired environment, with the development of biological control theory, utilize Biocontrol microorganism prevention and control plant disease Technology has obtained researcher and has managed the concern of peasant household.Separated one plant of this research team early stage has stronger bacteriostasis Antagonism bacillus subtilis PTS-394, the bacterial strain can induce plant and produce disease resistance, to solanaceous vegetables in plant rhizosphere colonization Bacterial wilt, droop have favorable effect.With going deep into for research, we it is successfully developed for biocontrol agent it is (wettable Property pulvis), and applied related patents protect.
The present invention is stood facing each other by tablet test first, tentatively specify that PTS-394 bacterial strains and its wettable powder methanol carry Take thing that there is good inhibiting effect to Fusarium Solani (eggplant handle sickle-like bacteria), further Mass Spectrometer Method, finds wettable Still fat peptide matters are contained in pulvis, but the species and content of fat peptide matters have more loss.This shows from short of money Antibacterial strain screens exploitation as ripe formulation products, can have larger change to the characteristic of biocontrol microorganisms in itself, how keep raw Anti- characteristic, and the vital task in biological control research field.The assessment experiment of the potted plant growth-promoting of Hot Pepper in Greenhouse, preventive effect etc. is aobvious Show, bacillus subtilis PTS-394 wettable powders concentration has an impact growth-promoting, the preventive effect of capsicum, and low dosage is presented and promotees Raw effect, and higher concentration is preventive and therapeutic effect.Result above, is that bacillus subtilis PTS-394 wettable powders are peppery in field Application technology in green pepper cultivation, there is provided the screening of suitable concentration and usage time etc., is efficiently made by integrated field Demonstrated with technology and further and promoted and applied.The present invention is with biological prevention and control agent bacillus subtilis PTS-394 wettable powders Agent is object, has not only further expanded its use scope and prevention target, also for the product field using Demonstration And Extension and The further marketization is provided fundamental basis.
It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, above-described embodiment and explanation Merely illustrating the principles of the invention described in book, without departing from the spirit and scope of the present invention, the present invention also have Various changes and modifications, these changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention It is defined by the appending claims and its equivalent thereof.

Claims (9)

1. the effective use method of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis, its feature exist In comprising the following steps:
Step 1, seed treatment:Full seed is chosen, soaks, then soaks seed in the dilution of PTS-394WP in water Kind is overnight;
Step 2, seedling medium, which is mixed, to be applied:Before planting, the Nutrition Soil of hole plate seedling growth mixes native processing with PTS-394WP, uniformly mixes soil simultaneously Carry out program request;
Step 3, field planting processing:Before transplanting, spread fertilizer over the fields with PTS-394WP and colonize cave, a water living is poured after transplanting;
Step 4,5-7d pour seedling recovering waters after transplanting, the dilution liquid irrigating root of PTS-394WP is used after transplanting 15-20d, and carries out root Ridging;
Step 5, according to different plantation crops for rotation root-rot pathogenetic times, synchronous liquid manure rule, at interval of 15-20d using once The dilution of PTS-394WP carries out pouring root, until the root rot important generation phase terminates;
Step 6, in fruit expanding period, with the dilution liquid irrigating root of PTS-394WP;
Step 7, other pest and disease damages routinely schema management.
2. the height of bacillus subtilis PTS-394 wettable powders field control Fusarium solani according to claim 1 Imitate application method, it is characterised in that step 1 is specially:Full seed is chosen, 4-5h is soaked in warm water, then seed exists Soak seed overnight in the dilution of PTS-394WP, it is PTS-394 wettable powders that PTS-394WP is stated in step 1.
3. the height of bacillus subtilis PTS-394 wettable powders field control Fusarium solani according to claim 2 Imitate application method, it is characterised in that the temperature of warm water described in step 1 is 40-55 DEG C, and the dilution of the PTS-394WP is The dilution that PTS-394WP is obtained after being mixed with water according to 1: 1000 mass ratio, the seed soaking overnight time is 12-16h.
4. the height of bacillus subtilis PTS-394 wettable powders field control Fusarium solani according to claim 1 Imitate application method, it is characterised in that when soil is mixed described in step 2, the ratio of PTS-394WP and Nutrition Soil is 10kg bacterium powders/m3 Nutrition Soil.
5. the height of bacillus subtilis PTS-394 wettable powders field control Fusarium solani according to claim 1 Imitate application method, it is characterised in that before being transplanted in step 3, uniformly spread fertilizer over the fields according to 8g PTS-394WP/ seedlings and colonize cave, and poured Every seedling watering 800mL during water.
6. the height of bacillus subtilis PTS-394 wettable powders field control Fusarium solani according to claim 1 Imitate application method, it is characterised in that the dilution of PTS-394WP described in step 4 is for PTS-394WP with water according to 1: 100 The dilution obtained after mass ratio mixing, and during pouring root pouring root is carried out according to a 400ml dilutions/seedling.
7. the height of bacillus subtilis PTS-394 wettable powders field control Fusarium solani according to claim 1 Imitate application method, it is characterised in that the dilution of PTS-394WP described in step 5 is for PTS-394WP with water according to 1: 100 The dilution obtained after mass ratio mixing, and during pouring root pouring root is carried out according to a 400ml dilutions/seedling.
8. the height of bacillus subtilis PTS-394 wettable powders field control Fusarium solani according to claim 1 Imitate application method, it is characterised in that according to different plantation crops for rotation root-rot pathogenetic times, synchronous liquid manure rule described in step 5 Rule, is specially:Spring stubble:After capsicum is colonized (mid-March), impose 2 times;Autumn and winter stubble:Imposed after capsicum is colonized (mid-September) 2 times;Overwintering stubble:Imposed 1 time after capsicum is colonized (mid-November), start April in the coming year to impose again 2 times.
9. the height of bacillus subtilis PTS-394 wettable powders field control Fusarium solani according to claim 1 Imitate application method, it is characterised in that the dilution of PTS-394WP described in step 6 is for PTS-394WP with water according to 1: 1000 Mass ratio mixing after obtained dilution.
CN201711118761.7A 2017-11-09 2017-11-09 The effective use method of bacillus subtilis PTS-394 wettable powder field control Fusarium solanis Pending CN107897179A (en)

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