CN107760628B - The bacillus pumilus S9 of one plant of prevention and treatment rice blast - Google Patents

The bacillus pumilus S9 of one plant of prevention and treatment rice blast Download PDF

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CN107760628B
CN107760628B CN201711171896.XA CN201711171896A CN107760628B CN 107760628 B CN107760628 B CN 107760628B CN 201711171896 A CN201711171896 A CN 201711171896A CN 107760628 B CN107760628 B CN 107760628B
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bacillus pumilus
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沙月霞
王昕�
沈瑞清
刘炜
石东岭
方秋香
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Institute of Plant Protection of Ningxia Academy of Agriculture and Forestry Sicience
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Abstract

The invention discloses a kind of bacillus pumilus (Bacillus pumilus) for preventing and treating rice blast and its applications.Bacillus pumilus of the invention has the wide spectrum to rice blast and efficient preventive effect, can be used for the rice blast prevention and treatment of various strain rice, the especially prevention and treatment of leaf pest and panicle blast.

Description

The bacillus pumilus S9 of one plant of prevention and treatment rice blast
Technical field
The present invention relates to microorganism field more particularly to the bacillus pumilus (Bacillus of one plant of prevention and treatment rice blast Pumilus it) and its applies.
Background technique
Rice is cereal crops important in the world, and yield occupies 1/4 or more of world food total output, occupies Chinese grain Eat total output more than half, be the staple food that more than half population of the whole world is relied on.Rice blast (Rice Blast) is by son Capsule bacterium Magnaporthe oryzae (T.T.Hebert) Yaegashi&Udagawa (Invisible element: Pyricularia oryzae) The sudden worldwide fungal disease that is strong, being easy to prevalence of caused one kind.The disease can cause damages to rice throughout the year, Harm spreads each position of rice, there is seedling rice blast, leaf pest, pulvinus pest, section rice blast, panicle blast, branch stalk pest and grain pest etc..Rice Seasonal febrile diseases are widely distributed, and global 85 countries have generation and China South And North Rice Regions to endanger one of rice disease of most serious.Rice Seasonal febrile diseases cause 10~30% Rice Yield Loss Caused in the whole world every year, and annual production loss can support 6 million people of the whole world Mouthful.
The breeding and chemical prevention of disease-resistant variety are mainly used to the prevention and treatment of the Pyricularia oryzae at present, chemical agent makes for a long time It also will cause ecological environment with the pesticide residue for not only increasing agricultural product seriously to pollute, destroy ecosystem balance, threaten food Safety.Bacillus (Bacillus) can not only effectively control plant disease, while be not easy to safety of human and livestock, phytopathogen The advantages that generating strong resistance, resistance and promotion plant growth is the important biocontrol microorganisms in rice blast prevention and treatment.Bacillus colonizes On rice plant, the growth that antibacterial substance inhibits Pyricularia oryzae is generated, inducing paddy rice generates disease resistance, to rice plant With growth-promoting functions, Rice Yield Loss Caused can be retrieved.Bacillus can prepare biological prevention and control agent and be used to prevent and treat south China rice The harm of the rice blast of area and north paddyfield has guidance meaning to the biological control of rice blast in the sustainable development of rice industry Justice.Bacillus can prevent and treat the burst harm of rice blast in the period of chemical pesticide cannot be applied, and mitigate rice blast to greatest extent Production loss caused by disease not will increase rice noxious material residual, not pollute the environment more.
Magnaporthe grisea spore be it is main infect organ, generate germ tube after spore is mature, appresorium is attached to surface, germ tube with Natural aperture is directly entered with infecting bolt from rice epidermis.Wherein, the formation of appresorium be the committed step that successfully invades it One, it must generate enough pressure can mechanical penetration host's cuticula complete invasion procedure, and melanin is exactly to generate Depositing black element in the premise of appresorium pressure, appresorium and the host surfaces contact very small region in side, eventually forms attached Hilum, complete invasion procedure.Bacillus pass through successfully colonize to plant rhizosphere, body surface or in vivo, with Pyricularia oryzae compete The nutrition of surrounding plants, secretion antibacterial material inhibit the growth of pathogen mycelia, conidial sprouting, keep appresorium lopsided and light Change melanin, while inducing paddy rice system of defense resists the invasion of Pyricularia oryzae, to achieve the purpose that biological and ecological methods to prevent plant disease, pests, and erosion.Bacillus can Pyricularia oryzae hyphal cell wall is promoted to dissolve to generate chitinase, sugar de- enzyme, cell wall degrading enzyme etc., especially those agings Even be easier to dissolve close to dead mycelia.Equality of going on a spring outing (2001；2002；2008) Bacillus subtillis is screened And bacillus pumilus, the inhibiting rate of conidia germination and note fields to two kinds of mating types of Pyricularia oryzae are respectively 82.5% and 100%, research finds that bacillus pumilus generates 3 kinds of polypeptide active substances, and it is mitogenetic to significantly inhibit Pyricularia oryzae Spore germination, and PAL, PO and PPO enzymatic activity can increase in inducing paddy rice plant body.Fan Sha etc. (2013) is from sweet osmanthus fruit Middle isolated bacillus pumilus osm-1 is 47% to the inhibiting rate that Pyricularia oryzae mycelia grows.Rong Songhao etc. (2016) is from wood The bacillus pumilus Bp6221 separated in sweet-scented osmanthus fruit, when fermentation liquid concentration is 0.025mL/mL in culture medium, Pyricularia oryzae Growth size be only positive control half.
Using enzyme engineering and protein engineering, carry out crops nearly picking time using protein as the novel of effective component The research and application of bacillus biological prevention and control agent can retrieve rice industry in nearly picking time because of burst disease to the maximum extent Caused by production loss.The research and application of bacillus prevention and treatment rice blast are the needs of Rice Production sustainable development, can be with Meet the requirement that the United Nations's world food tissue develops about grain security, but also lacks in this field and have to rice blast There are wide spectrum and the high bacterial strain of bacillus of efficient preventive effect, antibacterial substance yield.
Summary of the invention
The purpose of the present invention is to provide the rice blast of a kind of pair of different cultivars rice, especially leaf pest and panicle blasts to have Preferable preventive and therapeutic effect, and have both the strain of i (bacillus) pumilus of growth-promoting, effect of promoting production.
To achieve this purpose, the present invention adopts the following technical scheme:
In being preserved in a first aspect, the present invention provides a kind of bacillus pumilus (Bacillus pumilus) S9 State's Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), the deposit date is on May 22nd, 2017, preservation Number is CGMCC No.14178.
In second aspect, the present invention provides a kind of microbial inoculums, and it includes bacillus pumilus S9 as described in relation to the first aspect Or the culture bacillus pumilus S9 sterile supernatant obtained.
In the third aspect, the present invention provides a kind of methods for preventing and treating rice blast comprising will as described in relation to the first aspect Bacillus pumilus S9 or the microbial inoculum as described in second aspect are applied to rice crop.
In the method for prevention and treatment rice blast of the invention, the microbial inoculum is the culture solution of the bacillus pumilus S9.
In the method for prevention and treatment rice blast of the invention, the rice blast is leaf pest and/or panicle blast.
In fourth aspect, the present invention provides bacillus pumilus S9 as described in relation to the first aspect or as described in second aspect Microbial inoculum prevention and treatment rice blast in purposes.
In prevention and treatment rice blast of the invention on the way, the rice blast is leaf pest and/or panicle blast.
Advantageous effects of the invention are as follows:
1, compared with the bacillus pumilus of existing prevention and treatment rice blast, bacillus pumilus prevention and treatment efficiency of the invention is more Height, this, which is embodied in, can be used less brood cell's amount to obtain similar or better preventive effect.
2, compared with the bacillus pumilus of existing prevention and treatment rice blast, bacillus pumilus of the invention has more wide spectrum Control efficiency.
3, compared with the bacillus pumilus of existing prevention and treatment rice blast, bacillus pumilus of the invention is to leaf blast in rice It is preferable with the control efficiency of panicle blast.
4, compared with the bacillus pumilus of existing prevention and treatment rice blast, bacillus pumilus of the invention promotees rice Life, effect of promoting production are preferable.
Detailed description of the invention
Fig. 1 is culture form of the bacillus pumilus S9 bacterial strain on NA culture medium flat plate.
Fig. 2 is bacillus pumilus S9 bacterial strain and the plate face-off picture of Pyricularia oryzae P131.
Fig. 3 is bacillus pumilus S9 strain fermentation culture solution and the plate face-off picture of Pyricularia oryzae P131.
Fig. 4 is the electron microscopic picture that bacillus pumilus S9 bacterial strain inhibits Pyricularia oryzae P131 Infection structure, and upper figure is rice The control of seasonal febrile diseases bacterium P131 mycelia, the following figure are the electron microscope after bacillus pumilus S9 inhibits Pyricularia oryzae P131 Infection structure.
Fig. 5 is the electron microscopic picture that bacillus pumilus S9 bacterial strain sterile supernatant inhibits Pyricularia oryzae Infection structure, on Figure is the control of Pyricularia oryzae P131 mycelia, and the following figure is bacillus pumilus S9 sterile supernatant to Pyricularia oryzae P131 Infection structure The electron microscope of conidium and mycelia after inhibition.
Fig. 6 is bacillus pumilus S9 Biocontrol Activity active material detection picture.
Fig. 7 is suppression result of the volatile materials of bacillus pumilus S9 bacterial strain generation to Pyricularia oryzae P131, left figure For the control of Pyricularia oryzae P131 mycelia, right figure is suppression of the volatile materials of bacillus pumilus S9 generation to Pyricularia oryzae P131 Result processed.
Specific embodiment
To further illustrate the technical scheme of the present invention below with reference to the accompanying drawings and specific embodiments.
Embodiment 1: the screening and fermented and cultured of bacillus pumilus S9 bacterial strain
(1) sample acquires: acquisition sample is No. 48 rhizosphere soils of the peaceful round-grained rice of rice varieties, and place is flat for Ningxia Hui Autonomous Region Sieve county town Yao Fu.It extracts together with the root system of rice, is installed with polyethylene plastic bag, take back laboratory separation.
(2) bacillus pumilus separates: weighing 1g soil, it is 1 small to be put into 100mL sterile water oscillation mixing under room temperature When, with sterile water according to 10,102With 103Gradient dilution, micropipettor take gradient dilution liquid 200mL/ ware to be coated in spreading rod NA solid medium tablets, after 37 DEG C of incubator culture 48h, transfer needle picking single colonie uses nothing in NA solid medium tablets The scribing line of bacterium toothpick gradient, obtains bacterium single colonie after purification, single colonie is stored in 40% glycerol in -80 DEG C of ultralow temperature ices It is saved backup in case.
(3) screening of bacillus pumilus: choosing healthy rice leaf, and clip 5cm comes into leaves section, is put into bacillus pumilus In S9 fermentation liquid, oscillation colonizes 1h, obtains colonizing rear blade；It is laid with filter paper in culture dish bottom, then is put on wet filter paper Sterilizing toothpick is set, is placed on toothpick and colonizes rear blade, after colonizing culture 20h, gently pierces leaf section surface with new sterilizing toothpick, no Leaf section cuticula is injured, each leaf section stabs 5 points, then draws magnaporthe grisea spore suspension with liquid-transfering gun and be seeded in thorn respectively On 5 points of wound, obtain inoculation rear blade, growth cabinet culture for 24 hours, vaccination rice blast scab is investigated after 48h and 72h Quantity and area, selection vaccination disease-free spot or the bacillus cryo-conservation that scab is of light color and area is small are spare.
Broth bouillon (NA): peptone 10.0g, powdered beef 3.0g, sodium chloride 5.0g, agar 15.0g, pH 7.3 ± 0.1,121 DEG C of sterilizing 20min.
LB liquid medium: yeast extract 5g, peptone 10g, sodium chloride 5g, water 1000mL, pH7.4~7.6,121 DEG C sterilizing 30min.
Oat medium: oatmeal 30g, 17~20g of agar add water 1000mL, and 1h, filtered through gauze are heated on boiling water bath Afterwards plus water supplies 1000mL, packing sterilizing (121 DEG C, 20min) after adding agar to melt.
Identified, the strain of i (bacillus) pumilus feature is as follows: white, colonial morphology have semitransparent type and transparent type, have Gauffer and random.The bacterium colony cultivated on NA culture medium is smooth, gradually becomes light yellow.Cell is rod-shaped [(0.6~0.7) μm × (2.0~3.0) μm], it is in ellipsoid or cylinder that Gram-positive or variable, which moves by periflagellum, and forms brood cell, in Raw, other life or proximal end are raw, do not form vegetative cell, no film.
Inventor is by this culture presevation in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), the deposit date is on May 22nd, 2017, deposit number is CGMCC No.14178.
Embodiment 2: the biological activity determination of bacillus pumilus S9 bacterial strain:
(1) bacteria inhibition assay of the bacillus pumilus S9 bacterial strain to Pyricularia oryzae: central on tomato oat plating medium Pyricularia oryzae P131 bacteria cake is placed, bacteria cake two sides after 4d is grown and is inoculated with single colonie bacillus bacteria cake at 2cm and oppose training of standing erect It supports, is control with sterile water, is placed in 28 DEG C of dark culturings of incubator.Every processing sets 3 repetitions.It is straight that pathogen bacterium colony is measured after 2d Diameter and antibacterial band, the results are shown in Table 1.
(2) bacteria inhibition assay of the bacillus pumilus S9 sterile supernatant to Pyricularia oryzae: in tomato oat plating medium Pyricularia oryzae bacteria cake is placed in upper center, is placed sterile Oxford cup at 2cm in bacteria cake two sides, is separately added into bacillus in cup Fermentation liquid make opposite culture, be respectively control with sterile water and sterile LB liquid medium, be placed in 28 DEG C of incubator dark trainings It supports.Each processing sets 3 repetitions.Pathogen colony diameter and antibacterial band are measured after 5d, the results are shown in Table 1.
The opposite culture of table 1 bacillus pumilus S9 bacterial strain and Pyricularia oryzae
Note: the experimental data in table is duplicate average value three times.
(3) bacillus pumilus S9 excised leaf control effect testing: choosing healthy rice leaf, and clip 5cm comes into leaves section, is put into In bacillus pumilus S9 fermentation liquid, 200rpm/min, 28 DEG C of oscillations colonize 1h, obtain colonizing rear blade；In culture dish bottom It is laid with filter paper, then places 5 sterilizing toothpicks on wet filter paper, is placed on toothpick and colonizes rear blade, colonizes culture 20h Afterwards, leaf section surface is gently pierced with new sterilizing toothpick, does not injure leaf section cuticula, each leaf section stabs 5 points, then is inhaled with liquid-transfering gun It takes magnaporthe grisea spore suspension to be seeded on 5 points stabbed respectively, inoculation rear blade is obtained, in growth cabinet culture For 24 hours, vaccination rice blast Lesion number and area are investigated after 48h and 72h, calculate control efficiency.Culture dish after inoculation is placed on Growth cabinet culture, parameter setting are 28 DEG C, RH (humidity) 70%, daily light application time 4h, intensity of illumination 4400Lux, It the results are shown in Table 3.
Grade scale is investigated in rice blast leaf pest room: pressing International Rice institute rice blast resistance Assessment for classification standard (table 2)
Table 2 is that grade scale is investigated in rice blast leaf pest room
Biocontrol effect calculation formula:
3 bacillus pumilus S9 of table is to Pyricularia oryzae excised leaf control effect testing
Note: significance analysis is using minimum range method (LSD) (P≤0.05 ± standard error).
(4) biocontrol effect of bacillus pumilus S9 bacterial strain under the conditions of greenhouse pot culture: selecting healthy rice varieties D10, 1% liquor natrii hypochloritis disinfection 10min, 75% alcohol rinse 3 times, aseptic water washing 5 times, 28 DEG C of growth cabinet vernalization, to Bud is seeded in plastics breaker (bucket base diameter 18cm × high 21cm × bucket upper diameter 24.5cm) when growing to 1cm. Bacillus fermentation liquid to be measured is sprayed after plantation 28d, brood cell's concentration is 1 × 108CFU/mL, each bucket spray 20mL, each Handle 4 repetitions.It is inoculated with magnaporthe grisea spore suspension afterwards for 24 hours, spore concentration is 1 × 106CFU/mL.Leaf pest hair is investigated after 7d Raw situation, calculates disease index and preventive effect size.
4 bacillus pumilus S9 of table measures the control efficiency of leaf pest under greenhouse experiment
Processing Disease index Control efficiency (%)
S9 3.77±1.16bB 76.56±0.85bB
Tricyclazole 2.98±0.14bB 81.51±0.69aA
Clear water control 16.09±0.43aA /
Note: significance analysis is using minimum range method (LSD) (P≤0.05 ± standard error).
(5) the control in field effect test of bacillus pumilus S9 bacterial strain
It is carried out in Ningxia Hui Autonomous Region, the town Yao Fu, Pingluo County Ningxia He Yin modern agriculture Co., Ltd's Rice Cropping base The field control effectiveness test of bacillus pumilus S9, rice varieties are peaceful round-grained rice 43, test bacillus brood cell amount is 1 × 108CFU/ml, average each experimental plot area is 50m2, bacillus pumilus S9 is investigated to leaf pest in 4 replicated plots respectively With the control efficiency of panicle blast.Investigation method presses " pesticide field efficacy medicine test criterion ", and rice blast leaf pest investigation grade scale is: 0 grade: disease-free；1 grade: the only brown point of needle point size；3 grades: small and round so that the brown necrosis greyness slightly grown, 1~2mm of diameter；5 Grade: typical rice blast scab, injured area is less than 10%；7 grades: typical rice blast scab, injured area are 26%~50%；9 Grade: comprehensive blade is dead.Panicle blast investigation method is shown in Table 5, the results are shown in Table 6 and table 7.
Table 5 is that rice blast panicle blast investigates grade scale
Series Investigation standard
0 No panicle blast, grain pest and branch obstruct pest
1 There are a pest and branch stalk pest, empty grain number < 5%
2 There are a pest and branch stalk pest, empty grain number is 5~10%
3 There are a pest and branch stalk pest, empty grain number is 10~20%
4 There are a pest and branch stalk pest, empty grain number is 20~30%
5 Having panicle blast, grain pest and branch stalk pest, empty grain number is 20~30%
6 Panicle blast causes empty grain number > 80%
Biocontrol effect calculation formula:
Control in field effect measuring of the 6 bacillus pumilus S9 bacterial strain of table to leaf pest
Note: significance analysis is using minimum range method (LSD) (P≤0.05 ± standard error).
Control in field effect measuring of the 7 bacillus pumilus S9 bacterial strain of table to panicle blast
Processing Disease index Preventive effect/%
S9 13.27±0.29bcB 71.55±0.31bBC
Greenery patches health 12.25±0.73cB 76.25±0.37aA
Tricyclazole 12.06±0.51bcB 74.18±1.08aAB
Clear water control 46.86±3.06aA /
Note: significance analysis is using minimum range method (LSD) (P≤0.05 ± standard error).
(6) health and the consistent rice seed of growing way growth-promoting ability of the bacillus pumilus S9 bacterial strain to rice seedling: are chosen It takes out and is placed in the culture dish for being covered with filter paper after seed soaking 1h in No. 43 fermentation cultures for being put into bacillus pumilus S9 of sub peaceful round-grained rice, At 28 DEG C, the growth cabinet culture that RH is 70%, investigation seed shows money or valuables one carries unintentionally quantity after 48h.Each processing repeats 3 wares.Take health And the consistent rice paddy seed of growing way is put into the fermentation culture of bacillus pumilus S9 and is seeded in raising rice seedlings pond after seed soaking 1h In, the root long and plant height of rice seedling are investigated after growth 1 month, the results are shown in Table 8.
Growth-promoting ability of the 8 bacillus pumilus S9 of table to rice seedling
Note: data are that duplicate average value, significance analysis use minimum range method (LSD) (P≤0.05 ± standard three times Accidentally).
(7) health and the consistent rice paddy seed of growing way promote production ability of the bacillus pumilus S9 bacterial strain to rice plant: are taken It is seeded in raising rice seedlings pond after seed soaking 1h in No. 43 fermentation cultures for being put into bacillus pumilus S9 of peaceful round-grained rice, respectively in rice transplanting Phase, tillering stage, full heading time, the stage of yellow ripeness are sprayed the fermentation culture of bacillus pumilus S9, and rice plant is connected root system when harvesting It pulls up together, investigates number of productive ear, spike length, blighted grain number, total weight, sterile grain rate and the mass of 1000 kernel of rice plant, the results are shown in Table 9. Influence of the bacillus pumilus S9 bacterial strain to rice quality is detected, the results are shown in Table 10.
Growth-promoting ability of the 9 bacillus pumilus S9 of table to rice plant
Processing Number of productive ear/cave Spike length (cm) Sterile grain rate (%) Mass of 1000 kernel (g)
S9 10±0.81aA 193.33±1.33bAB 5.53±0.21bA 28.45±0.24aA
Greenery patches health 12±0.70aA 224.17±9.95aA 5.87±0.29bA 27.82±0.46aA
Tricyclazole 10±0.72aA 186.14±1.18bB 5.43±1.27bA 29.45±0.03aA
Clear water control 9±0.72aA 180.47±0.57bB 7.39±0.95aA 27.96±0.31aA
Note: data are that duplicate average value, significance analysis use minimum range method (LSD) (P≤0.05 ± standard three times Accidentally).
Influence of the 10 bacillus pumilus S9 of table to rice quality
Processing Coarse Rice Rate Polished rice rate Head rice rate Chalky grain rate Chalkiness degree
S9 87.74±0.24abA 81.26±0.27aA 70.01±0.94aA 7.20±0.31aA 3.21±1.32aAB
Tricyclazole 88.28±0.34aA 81.43±0.17aA 70.87±0.38aA 5.10±1.04bB 1.63±1.03cC
Greenery patches health 88.12±0.26aA 81.30±0.15aA 69.87±0.58aA 7.30±1.21aA 2.63±0.54bB
Clear water 87.13±0.29bA 81.63±0.47aA 68.83±1.42aA 7.40±1.17aA 3.57±0.45aA
Note: data are that duplicate average value, significance analysis use minimum range method (LSD) (P≤0.05 ± standard three times Accidentally).
(8) bacillus pumilus S9 bacterial strain observes the SEM that Pyricularia oryzae Infection structure inhibits:
Bacterial strain S9 observes the SEM of Pyricularia oryzae Mycelial growth: activate Pyricularia oryzae on PDA solid medium, 28 DEG C Culture 5~7 days, breaks into bacteria cake on the Pyricularia oryzae plate of activation with the punch of diameter 5mm.Using Pyricularia oryzae as target Bacterium, the Pyricularia oryzae bacteria cake that inoculation diameter is 5mm at PDA plate center, tested bacteria are inoculated in away from target bacteria cake 2cm, In It is cultivated 5~7 days in 28 DEG C of insulating boxs, when the Pyricularia oryzae mycelia of blank control covers with entire culture dish, takes opposite culture flat The mycelia (cutting together with culture medium) of antibacterial region hypha,hyphae edge and corresponding position blank control (only long fungi) on plate, adopts With the antibacterial region of scanning electron microscopic observation, Fig. 4 is as a result seen.
Bacterial strain S9 sterile supernatant observes the SEM of conidium and Mycelial growth: cultivate 48h strains tested it is sterile on Clear liquid and Pyricularia oryzae P131 spore suspension (1 × 106CFU/mL it) is mixed with 1: 1 ratio, mixed liquor is in 2mL sterile centrifugation In pipe 28 DEG C of opposite cultures for 24 hours when, after high speed refrigerated centrifuge, sediment fixes 2h or more with 2.5% glutaraldehyde, using phosphoric acid Buffer flushes three times, and after 1% starves the fixed 2h of acid, is flushed three times again with phosphate buffer, 30%, 50%, 70%, 80%, Three times, LEICAEM CPD critical point drying instrument is dried 90% and 100% Gradient elution using ethanol, EIKO IB-3 metal spraying, most Afterwards using Hitachi S-3400N scanning electron microscopic observation strains tested to the influence on rice blast pathogen conidiospore and hypha form, knot Fruit sees Fig. 5.
The preparation of bacterial strain sterile supernatant: the strains tested S9 single colonie for having activated for 24 hours is seeded to the training of LB liquid respectively It supports in base, 37 DEG C, after 200rpm/min shaken cultivation 48h, fermentation culture is in 4 DEG C, 10000rpm/min centrifugation 20min removal Thallus, supernatant are filtered (0.22 μm) degerming to get sterile supernatant.
(9) bacillus pumilus S9 Biocontrol Activity active material detects:
The detection of biomembrane: picking single colonie is inoculated on LB liquid medium, and 30 DEG C, 180rpm shakes training overnight, by 1: 1000 ratios are added in the 12 hole tissue culturing plates containing Msgg culture medium, are cultivated at 23 DEG C, and biomembrane shape is observed after 3 days At situation, it the results are shown in Table 11.
The detection of amylase: taking the single colonie newly activated to be inoculated on the LB plate containing 0.2% soluble starch, culture 48h, after forming obvious bacterium colony, Lu Geshi iodine staining 10min, which is added dropwise, on plate can generate starch with 70% ethyl alcohol board-washing The bacterial strain of enzyme, under the background of black, at bacterium colony growth around can form colourless transparent circle, if there is transparent circle shows bacterium Strain can produce amylase, and 3 repetitions of each processing the results are shown in Table 11.
The detection of protease: by the strain to be tested percutaneous puncture-inoculation of activation on 1% skim milk agar plate, 30 DEG C are cultivated For 24 hours, there is transparent circle and shows the generation for having protease, 3 weights of each processing in the generation that peripheral transparent circle is observed after 48h, 72h It is multiple, it the results are shown in Table 11.
Dextranase detection: bacterium to be measured is inoculated on the plate containing ABP culture medium, after 30 DEG C of cultures 48h, 72h, observation Whether occur resolution circle in plate, shows the generation for having dextranase, 3 repetitions of each processing, knot if thering is resolution circle to generate Fruit is shown in Table 11.
Thermophilic iron element detection: thermophilic iron element is detected with CAS culture medium, and the bacterium to be measured of activation is inoculated in CAS detection culture medium and is put down On plate, after 30 DEG C of culture 72h, observe in plate whether generate crocus haloing, shows there is thermophilic iron element if there is crocus It generates, each processing is repeated 3 times, and the results are shown in Table 11.
The detection of cellulase: it will be inoculated on cellulose screening and culturing medium plate after the bacterial strain being separated to activation, 28 DEG C Constant temperature is inverted culture 2d, disseminates 10min, then the NaCl solution decoloration 5min with 1mol/L with 0.1% congo red staining liquid.If Bacterial strain cellulase-producing then clearly transparent circle, each processing can occurs in periphery of bacterial colonies and be repeated 3 times, the results are shown in Table 11.
The detection of volatile materials: the Pyricularia oryzae of a piece of diameter 1cm is inoculated in tomato oat medium plate center P131 fungus block, the coating culture S9 bacterial strain on another an equal amount of NA culture medium flat plate, by the former left-hand thread on the latter, Two ware contact positions are sealed with sealed membrane, make blank control with the NA plate of uncoated S9 bacterial strain, at 28 DEG C after constant temperature incubation 9d The Pyricularia oryzae colony diameter of measurement coating S9 bacterial strain and control culture respectively, calculates relative inhibition.5 repetitions of every processing, It the results are shown in Table 11 and Fig. 7.
The preparation of Msgg culture medium: 0.1M Phosphate Buffer 50mL/L, 0.5M MOPS (pH 7.0) 200mL/ L, 0.1M MgCl220mL/L, 0.01M MnCl25mL/L, 1mM ZnCl21mL/L, 10mM Thiamine 0.2mL/L, 10mg/mL Phe 5mL/L, 10mg/mL Trp 5mL/L, 10mg/ml Thr 5mL/L, 50%glycerol 10mL/L, After filtration sterilization, the 10mL 5mM FeC1 of sterilizing is added in 10%Glutamate 50mL/L, deionized water 32mL/L3And 7mL 0.1M CaC12, saved backup in 4 DEG C.
The preparation of ABP culture medium: with mortar by blocky Poria cocos grind at powder, make its can by the sieve of 120 mesh, KH2PO46.8g, K2PHO4·12H2O 17.9g, yeast extract 6.7g, Poria cocos powder (or β -1.3- glucan) 5.0g, aniline Blue 120mg, agar powder 12g, pH value 6.8, water 1L.
The preparation of CAS culture medium: junket peptone 9g, yeast extract 5g, citric acid trisodium 10g, disodium hydrogen phosphate 1g, biphosphate Sodium 1g, glucose 10g, agar 20g are dissolved in 1L water.
Cellulose enrichment conditions base (1L): NaCI 6g, MgS04 0.1g,KH2P04 0.5g,CaC12 0.1g,(NH4)S04 2.0g,K2HP042.0g, agar 15g, CMC-Na 5g, pH are adjusted to 7.0；Cellulose screening and culturing medium (1L): in above-mentioned cellulose Yeast powder 1g is added in enzyme enriched medium.
Congo red staining liquid: Congo red, final concentration of 0.1% (w/v) is dissolved with distilled water.
Congo red destainer: the NaCI solution of final concentration of 1mol/L.
The detection of 11 bacillus pumilus S9 Biocontrol Activity active material of table
Note: data are duplicate testing result three times；"+" is detected as a result, "-" is not detect
The Applicant declares that the present invention is explained by the above embodiments detailed features and method of the invention, but this hair It is bright to be not limited to above-mentioned detailed features and method, that is, do not mean that the present invention must rely on above-mentioned detailed features and method It could implement.It should be clear to those skilled in the art, any improvement in the present invention, to condition selected by the present invention Equivalence replacement and increase, the selection of concrete mode of accessory etc., all fall within protection scope of the present invention and open model Within enclosing.

Claims (7)

1. a kind of bacillus pumilus (Bacillus pumilus) S9, is preserved in China Committee for Culture Collection of Microorganisms Common micro-organisms center (CGMCC), the deposit date is on May 22nd, 2017, deposit number was CGMCC No.14178.
2. a kind of microbial inoculum, it includes the bacillus pumilus S9 as described in claim 1 or culture bacillus pumilus S9 Sterile supernatant obtained.
3. a kind of method for preventing and treating rice blast comprising by bacillus pumilus S9 as described in claim 1 or as right is wanted Microbial inoculum described in asking 2 is applied to rice crop.
4. method as claimed in claim 3, which is characterized in that the microbial inoculum is the culture solution of the bacillus pumilus S9.
5. the method according to claim 3 or 4, which is characterized in that the rice blast is leaf pest and/or panicle blast.
6. bacillus pumilus S9 as described in claim 1 or microbial inoculum as claimed in claim 2 are in prevention and treatment rice blast Purposes.
7. purposes according to claim 6, which is characterized in that the rice blast is leaf pest and/or panicle blast.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101962623A (en) * 2010-01-19 2011-02-02 四川农业大学 Bacillus stain for preventing and controlling rice blast and rice sheath blight
CN102296036A (en) * 2011-06-01 2011-12-28 中国农业科学院烟草研究所 Bacillus pumilus and application in disease prevention and growth promotion thereof
CN104195069A (en) * 2014-07-24 2014-12-10 中国农业大学 Bacillus subtilis 2012SYX04 for preventing and treating rice blast

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KR20060019708A (en) * 2004-08-30 2006-03-06 주식회사 제노바이오텍 An agent of environmentally friendly anti-rice blast disease that use antifungal micorbe and plant extract

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101962623A (en) * 2010-01-19 2011-02-02 四川农业大学 Bacillus stain for preventing and controlling rice blast and rice sheath blight
CN102296036A (en) * 2011-06-01 2011-12-28 中国农业科学院烟草研究所 Bacillus pumilus and application in disease prevention and growth promotion thereof
CN104195069A (en) * 2014-07-24 2014-12-10 中国农业大学 Bacillus subtilis 2012SYX04 for preventing and treating rice blast

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