CN104630086A - Bacillus amyloliquefaciens RL263 capable of preventing and controlling rice blast - Google Patents

Bacillus amyloliquefaciens RL263 capable of preventing and controlling rice blast Download PDF

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CN104630086A
CN104630086A CN201410671030.5A CN201410671030A CN104630086A CN 104630086 A CN104630086 A CN 104630086A CN 201410671030 A CN201410671030 A CN 201410671030A CN 104630086 A CN104630086 A CN 104630086A
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rice blast
pest
bacillus amyloliquefaciens
rice
blast
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CN104630086B (en
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王�琦
李双东
李燕
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China Agricultural University
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION 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
    • A01N63/00—Biocides, 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N63/00—Biocides, 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
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Abstract

The invention discloses bacillus amyloliquefaciens RL263 capable of preventing and controlling rice blast, as well as an inoculant containing the bacillus amyloliquefaciens RL263, a method for preventing and controlling rice blast by using the bacillus amyloliquefaciens RL263 and application of the bacillus amyloliquefaciens RL263 in prevention and control of rice blast. The bacillus amyloliquefaciens has an effective prevention and control effect on rice blast.

Description

A kind of bacillus amyloliquefaciens RL263 preventing and treating rice blast
Technical field
The present invention relates to microorganism field, particularly relate to a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and the application thereof that prevent and treat rice blast.
Background technology
Paddy rice is one of topmost food crop in world wide, and in China, the cultivated area of paddy rice is about 1/4 of arable land, the whole nation, and annual production accounts for the half of national total output of grain.In Rice Production process, disease problem (especially rice blast) is one of the significant obstacle factor of restriction rice high yield stable yields (Dong Jingao etc., 2008).
Rice blast is the fungal disease (Sun Taihong, 2009) caused by rice ovum sorosphere chamber bacterium Magnaprthe grisea (Hebert) Barr.Rice blast is one of the most extensive, harm the is the most serious fungal diseases of plants that distributes.Difference according to occurrent time and site of pathological change can be divided into seedling pest, leaf pest, joint pest, panicle blast, branch stalk pest, grain pest etc., wherein common with leaf pest, panicle blast, endangers larger.In China, South And North Rice Regions has this disease generation in various degree and popular every year.Its region of disease is widely distributed, especially occurs heavier with mountain area, knob; State of an illness velocity of diffusion is fast, once condition is suitable for, easily popularly to cause disaster; Cause damage serious, in the plant disease epidemic time, general underproduction 10%-20%, serious reached at 40%-50%, even No kernels or seeds are gathered, as in a year of scarcity for the field of especially severe.As can be seen here, rice blast is the important disease having a strong impact on Rice Production, thus carries out control tool to rice blast and is of great significance.
Chemical prevention, as one of topmost prophylactico-therapeutic measures of this disease, has longer Historic Evolution, and the advantage of chemical prevention is instant effect, and low, easy to use, the many disease of cost is double to be controlled.Shortcoming is a large amount of to it, irrational use for a long time, and preventive effect can be caused to reduce, and the characteristic of its high poison, high residue not only works the mischief to the health of people, animal, and causes severe contamination to soil, water body, air, destroys the eubiosis.Meanwhile, because germ resistance constantly strengthens, usage quantity and the usage frequency of agricultural chemicals continue to increase, although chemical prevention is originally very effective, along with the development of time, phytopathogen there will be resistance in various degree.
Along with the improvement of control of plant disease technology, the mankind to the great attention of environment, seek a kind of to the mankind and environmental friendliness and the control strategy of the good preventive effect of tool-utilize beneficial microorganism to prevent and treat rice blast to have become the task of top priority (Liu Guoquan, 2004).At present, the beneficial microorganism monoid preventing and treating rice blast mainly contains actinomycetes, fungi, bacterium and mycovirus etc.Bacterium at present for biological control mainly contains genus bacillus and Pseudomonas, and genus bacillus is the focus (Mu Changqing, 2006) of researcher research in recent years.
What the kind for preventing and treating rice blast in genus bacillus had been reported mainly contains subtilis (Bacillus subtilis), bacillus pumilus (Bacillus pumilis) and bacillus cereus (Bacillus cereus) etc.Peng Huaxian etc., utilize From The Rhizosphere of Rice to be separated bacillus cereus Ma-32 and the subtilis Xi-55 of acquisition, carry out field efficiency test, within continuous 2 years, preventive effect is more than 50% (Peng Huaxian, 2002).Mu Changqing etc. study discovery, and genus bacillus concentration is at 1x10 8during c fu/mL, reach 57.2% to the preventive effect of panicle blast, stimulation ratio reaches 9.6% (Mu Changqing, 2006).The research such as Gao Xuewen finds, the outer material of the born of the same parents that Bacillus subtilis B 2 Strain produces, to rhizoctonia cerealis and Pyricularia oryzae mycelial growth all inhibited (Gao Xuewen etc., 2003).Zhu Guimei, Chen Hongzhou etc., utilize bacillus pumilus TW-2 bacterial strain to prevent and treat Rice blast, result shows: TW-2 not only has strong restraining effect to Pyricularia oryzae, more than 80% (Zhu Guimei etc., 2007) are also reached to the preventive effect of panicle blast.
At present, the domestic research utilizing bacillus amyloliquefaciens to prevent and treat rice blast is less.Chen Cheng etc. study discovery, and the meta-bolites of bacillus amyloliquefaciens HN-06, after separation and purification, has significant bacteriostatic action (old one-tenth, 2010) to Pyricularia oryzae.Lu Fan etc. study discovery, and bacillus amyloliquefaciens T553 has proteolytic enzyme, cellulase activity and produces addicted to iron element ability, and to the restraining effect of rice blast fungus obvious (Lu Fan, 2011).In the research of the bacillus amyloliquefaciens control rice blast reported, the research for laboratory fungistatic effect is more, but less for the research of field control effect, production application.So, to rice blast, there is wide spectrum and preventive effect, the needs of new Bacillus amyloliquefaciens strain that can effectively increase production efficiently for also still existing in this area.Need to improve screening mode, change the conventional screening methods that in vitro bacterial strain bacteriostasis detects, be based upon high-throughout screening method rice leaf directly carrying out bacteriostatic and disease prevention detection, obtain a large amount of probiotics, and the field efficacy of bacterial strain is fully verified, accurate evaluation and Devoting Major Efforts To Developing are carried out to its production application potentiality.
Summary of the invention
The object of the present invention is to provide the Bacillus amyloliquefaciens strain that a kind of tool has stable and efficient preventive effect to rice blast, can effectively increase production.
For reaching this object, the present invention by the following technical solutions:
In first aspect, the invention provides a kind of bacillus amyloliquefaciens RL263 (Bacillus amyloliquefaciens), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation date is on May 21st, 2014, and deposit number is CGMCC No.9191.
In second aspect, the invention provides a kind of microbial inoculum, it comprises bacillus amyloliquefaciens RL263 as described in relation to the first aspect.
In the third aspect, the invention provides a kind of method of preventing and treating rice blast, it comprises bacillus amyloliquefaciens RL263 as described in relation to the first aspect or the microbial inoculum as described in second aspect is applied to rice crop.
In the method for control rice blast of the present invention, described microbial inoculum is the nutrient solution of described bacillus amyloliquefaciens RL263.
In the method for control rice blast of the present invention, described rice blast is seedling rice blast, leaf pest, pulvinus pest, joint rice blast, panicle blast, fringe pest, branch stalk pest and/or grain pest.Wherein, preferably, described rice blast is leaf pest and/or panicle blast.
In fourth aspect, the invention provides bacillus amyloliquefaciens RL263 as described in relation to the first aspect or the purposes of the microbial inoculum as described in second aspect in control rice blast.
In the purposes of control rice blast of the present invention, described rice blast is seedling rice blast, leaf pest, pulvinus pest, joint rice blast, panicle blast, fringe pest, branch stalk pest and/or grain pest.Wherein, preferably, described rice blast is leaf pest and/or panicle blast.
Advantageous Effects of the present invention is:
The bacillus amyloliquefaciens RL263 that the present invention obtains, is separated from rice plant blade interior, adapts to the micro-ecological environment in rice plant leaf portion, well surely can grow existence in blade interior, effectively can extend it and prevent and treat validity period in field.
This bacterial strain is that the excised leaf control effect testing result screening of direct basis laboratory obtains, and verifies through the field efficacy of the continuous 2 years different rice varieties in different location, and its field efficacy is stablized, and has higher practical application potentiality.
This strains separation is inner from rice plant, but not other ecosystems, and its Environmental compatibility with rice terrace better, can better keep rice terrace ecotope.
Accompanying drawing explanation
Fig. 1 is Bacillus amyloliquefaciens strain RL26316S rDNA sequencing cluster analysis collection of illustrative plates.
Fig. 2 is the gyrA sequencing cluster analysis collection of illustrative plates of Bacillus amyloliquefaciens strain RL263.
Fig. 3 is that bacillus amyloliquefaciens RL263 bacterial strain is to the inhibition of Pyricularia oryzae colony growth.
Fig. 4 be bacillus amyloliquefaciens RL263 bacterial strain to the blank of Pyricularia oryzae colony growth inhibition test, in culture medium flat plate, namely only inoculate the colony growth effect of Pyricularia oryzae.
Fig. 5 is the inhibition test result of bacillus amyloliquefaciens RL263 bacterial strain fermentation liquor to rice blast fungus spore germination.
Fig. 6 is that bacillus amyloliquefaciens RL263 bacterial strain fermentation liquor is to the inhibition of rice blast fungus spore germination.
Fig. 7 is that the rice blast fungus spore of clear water process is normally sprouted and forms appressorium.
Embodiment
Technical scheme of the present invention is further illustrated by embodiment below in conjunction with accompanying drawing.
Embodiment 1: the screening of bacillus amyloliquefaciens RL263 bacterial strain
(1) rice material: the sample adopted is rice leaf, kind is Liaoxing No.1, and collecting location is Donggang City of Liaoning Province.
(2) substratum used:
1) extractum carnis substratum (NA): extractum carnis 3g, soy peptone 7g, NaCl 5g, agar 20g, 1L dH 2o, pH value is 7.2.
2) extractum carnis liquid nutrient medium (NB): extractum carnis 3g, soy peptone 7g, NaCl 5g, 1L dH 2o, pH value is 7.2.
3) Tryptones liquid nutrient medium (LB): Tryptones 10g, yeast powder 5g, NaCl 10g, 1L dH 2o, pH value is 7.2.
4) tomato oat medium: oat 40g, tomato juice 150mL, agar 20g, CaCO 30.6g, 1L dH 2o.
(3) separation of genus bacillus: (a) rice leaf is rinsed well through clear water, 1% hypochlorite disinfectant 8min, rinsed with sterile water 4 times, gets 100 μ l the 4th rinse water and is coated with extractum carnis culture medium flat plate cultivation 3d, aseptic dropping out shows, and shows that rice leaf surface sterilization is clean; B (), by the segmentation of surface sterilization rice tissue, about 2g puts into sterilizing mortar, add appropriate sterilizing quartz sand, 10mL sterilized water fully grinds, get obtained juice 1mL, add in the test tube containing 9mL sterilized water, concussion mixes.Gradient dilution to 10 -3concentration, by 10 -1, 10 -2, 10 -3diluent water-bath 30min in the water temperature of 80 DEG C of concentration, gets 100 μ l respectively and is coated with extractum carnis culture medium flat plate, 30 DEG C of cultivations.C () is carried out single bacterium colony purifying to the bacterium colony obtained according to colonial morphology and is cultivated; The bacterial strain list bacterium colony of picking purifying, is placed in the glycerine of 15% ,-80 DEG C of Excised Embryos.
(4) screening of rice blast genus bacillus is prevented and treated:
1) preparation of Bacillus strain fermented liquid: bacterial strain is taken out from-80 DEG C of refrigerators, be seeded on extractum carnis culture medium flat plate and cultivate activation 24h, in the beef extract-peptone nutrient solution of access 100mL (triangular flask of 500mL), 28 DEG C, 180r/min, cultivate 24h, obtain bacterial strain seed liquor.Get gained seed liquor, the inoculum size by 2% joins in the beef extract-peptone nutrient solution of 100mL (triangular flask of 500mL), 28 DEG C, 180r/min, cultivates 2d, obtains bacterial strain fermentation liquor.
2) genus bacillus excised leaf control effect testing: water intaking rice four leaf phase functional leaf, put ultrasonic washing instrument cleaning 5min, aseptic water washing 3 times, puts Bechtop and dries, and cuts as 8cm long segment, and RL263 fermented liquid soaks (1.0 × 10 8cfumL-1) process 0.5h, put Bechtop and dry.In culture dish, pad the moistening filter paper moisturizing of sterilizing, parallelly put into the bamboo toothpick of sterilizing, blade is placed on the toothpick in culture dish, blade and filter paper are not come in contact, every ware 4 blades, the moistening absorbent cotton in blade two ends is wound around moisturizing, by rice blast fungus spore suspension (1 × 10 after 24h 5cfumL-1) hanging drop is on blade, 4, every blade, often drips 20 μ L, proceed to 16h illumination after 28 DEG C of dark treatment 24h: 8h dark photoperiod, if be only treated to contrast 1 with sterilized water, the process only connecing rice blast fungus is contrast 2, observe after 5d, statistics morbidity result, calculates scab inhibiting rate:
Detect through excised leaf preventive effect, the scab inhibiting rate that screening obtains the highest bacterial strain RL263 of preventive effect is 66.56%, and leaf spot lesion propagation rate is obviously slow than the contrast only connecing rice blast fungus.Show that RL263 bacterial strain has significantly suppression infection process to form scab at paddy rice in vitro to leaf pest, and delay the effect of disease development, suppression Spot expansion.
(5) qualification of bacillus amyloliquefaciens: check order and sequence alignment qualification through comprehensive 16S rDNA and gyrA, result shows: the 16S rDNA gene order of RL263 and bacillus amyloliquefaciens homology are 99%, and to gather with bacillus amyloliquefaciens (GU688203), bacillus amyloliquefaciens (FJ844828), subtilis (Gu888127), subtilis (HQ327128) be a large class (see Fig. 1).Bacillus amyloliquefaciens is the sibling species of subtilis, because they have closely similar microscopic features and physicochemical characteristics, conventional authentication method is adopted to be difficult to accurately distinguish, and utilize gyrA gene effectively to distinguish, result shows, it is two large classes (see Fig. 2) that bacillus amyloliquefaciens and subtilis gather respectively.By 16S rDNA and gyrA Phylogenetic analysis, bacterial strain RL263 is bacillus amyloliquefaciens.
Contriver by this culture presevation in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation date is on May 21st, 2014, and deposit number is CGMCC No.9191.
Embodiment 2: the biological activity determination of bacillus amyloliquefaciens RL263 bacterial strain:
(1) bacillus amyloliquefaciens RL263 is to rice leaf pest greenhouse pot culture efficiency test: respectively with Lijiang xintuanheigu, Liaoxing No.1 rice varieties for experiment material, in paddy rice four leaf one heart stage, blade spraying 1% ammoniumsulphate soln, after 3d, chooses the consistent plant of growth and carries out biological and ecological methods to prevent plant disease, pests, and erosion test.Preparation 1 × 10 5the rice blast fungus spore suspension of cfumL-1, carries out spray inoculation to RL263 bacterial strain, keeps dark 12h, humidity 90% after inoculation.After 2h, spray with the rice plant of RL263 bacterial strain fermentation liquor to inoculated pathogenic bacteria, concentration is 1.0 × 10 8cfumL-1, often processes 30 strains, and with the process of clear water spraying for contrasting 1, the process only inoculating Pyricularia oryzae is contrast 2.Condition is asked in " Invest, Then Investigate " leaf pest morbidity in 1 week, calculates disease index and preventive effect (see table 1).
Table 1 bacterial strain RL263 is to the greenhouse pot culture efficiency test of rice leaf pest
Note: in table, data are the mean value repeated for more than 3 times, significance of difference analysis spss software Duncan ' s multiple comparisons test acquired results, α=0.05
(2) bacillus amyloliquefaciens RL263 is to the field controling test of rice blast: within 2012, carried out the field efficiency test of continuous 2 years with 2013.Test site is Liaoning Province's Panjin City and rice test field, Donggang City of Liaoning Province, and rice varieties is respectively salt rich No. 47 and Liaoxing No.1.Adopt blade spraying RL263 bacterial strain fermentation liquor to prevent and treat, the cell concentration of RL263 fermented liquid is 1.0 × 10 8cfu/mL, with 75% tricyclazole wettable powder (600g/hm 2) as medicament contrast, and using clear water as blank, randomized block design is taked in test, often processes 3 repetitions, amounts to 9 communities, 80m 2/ community, arranges 1m protection row between every community.Respectively prevent and treat once at paddy rice jointing stage, tillering regularity and cut phase, full heading time, before spray bacterium, after 14d and spray bacterium, 9d investigates disease index.Investigation method: every 5, community sampling, often some investigation 10 caves, record the disease severity of susceptible leaf (fringe) number and correspondence thereof, the disease severity method of classification represents, grade scale is as follows.After paddy rice maturation, measure every community actual output, calculate per mu yield.
Leaf (fringe) pest grade scale:
Leaf pest (in units of leaf);
0 grade: anosis
1 grade: the circular slightly long little scab of grey, edge brown, diameter is 1 ~ 2mm about
2 grades: diameter is the brown point of 1mm
3 grades: typical fusiform scab, long 1 ~ 2cm, is often confined between two master pulses, area less than 2% of causing harm
4 grades: typical scab, area 2.1 ~ 10% of causing harm
5 grades: typical scab, area 10.1 ~ 25% of causing harm
6 grades: typical scab, area 25.1 ~ 50% of causing harm
7 grades: typical scab, area 50.1 ~ 75% of causing harm
8 grades: blade is all withered
Panicle blast (in units of fringe);
0 grade: anosis
1 grade: every fringe loss less than 5% (morbidity of indivedual branch stalk)
3 grades: every fringe loss 6 ~ 10% (about 1/3rd branch stalk morbidities)
5 grades: every fringe loss 21% ~ 50% (ear stem or main shaft morbidity, grain half is flat)
7 grades: every fringe loss 51% ~ 70% (ear stem is fallen ill, most of shrivelled kernel)
9 grades: every fringe loss 71% ~ 100% (ear stem is fallen ill, and causes dead ears)
According to following formulae discovery disease index and preventive effect index, disease index=∑ (diseased plant number × relative disease progression at different levels)/(investigating total strain number × the highest sick progression) × 100; Prevention effect=(check plot disease index-control district disease index)/check plot disease index × 100%.
Result shows, and RL263 fermented liquid has remarkable preventive effect (see table 2) to rice leaf pest, in the continuous efficiency test of 2 years two places, all suitable with the preventive effect of tricyclazole chemicals treatment, and it can reach more than 80% to the preventive effect of leaf pest.Meanwhile, after RL263 bacterial strain and chemicals treatment, panicle blast disease index declines all to some extent, and all in significant difference compared with contrasting with clear water, preventive effect is up to 84% (see table 3).Experimental plot determination of yield result shows, RL263 bacterial strain and the rear output of medicament (tricyclazole) process all increase, compared with contrasting with clear water respectively, all in significant difference, (see table 4).Field efficiency test shows that RL263 bacterial strain fermentation liquor has significant biocontrol effect to rice blast, can significantly improve rice yield.
Table 2 bacterial strain RL263 field control leaf blast in rice test-results
Note: in table, data are the mean value repeated for more than 3 times, significance of difference analysis spss software Duncan ' s multiple comparisons test acquired results, α=0.05
Table 3 bacterial strain RL263 field control Rice blast test-results
Note: in table, data are the mean value repeated for more than 3 times, significance of difference analysis spss software Duncan ' s multiple comparisons test acquired results, α=0.05
Table 4 bacterial strain RL263 field test yield result
Note: in table, data are the mean value repeated for more than 3 times, significance of difference analysis spss software Duncan ' s multiple comparisons test acquired results, α=0.05
(3) bacillus amyloliquefaciens RL263 is on the impact of Pyricularia oryzae colony growth: on tomato oat medium flat board, face-off inoculation Pyricularia oryzae and bacterial strain RL263 (see Fig. 3), only to inoculate Pyricularia oryzae for contrast (see Fig. 4); Cultivate 7h in 28 DEG C, observe Growth of Magnaporthe Grisea situation, measure the antibacterial bandwidth (shortest distance of Pyricularia oryzae bacterium colony and RL263 colony edge) of RL263.The antibacterial bandwidth of result display bacterial strain RL263 is average 1.5cm (see Fig. 3).
(4) bacillus amyloliquefaciens RL263 forms the impact of appressorium on rice blast fungus spore germination: bacterial strain RL263 fermentation liquor 8000r/min is centrifugal, and 10min gets supernatant liquor, through the biofilter filtering thalline that diameter is 0.22 μm, obtain aseptic fermented supernatant fluid.
Being diluted by aseptic for biocontrol microorganisms fermented supernatant fluid sterilized water is four concentration (original content, 5 times of diluents, 10 times of diluents, 40 times of diluents), get fermented supernatant fluid (four the concentration) diluent of 100 μ L and the rice blast fungus spore suspension mixing of 100 μ L respectively, put in 48 porocyte culture plates and cultivate in 28 DEG C of incubators, observe once every 4h sampling, statistics note fields rate.
Result shows, it is obvious that bacterial strain RL263 original content fermented liquid forms appressorium inhibition to rice blast fungus spore germination, and its 40 times of diluents are after 12h, and inhibiting rate spore germination being formed to appressorium still reaches more than 60% (see Fig. 5).With the rice blast fungus spore after bacterial strain RL263 fermentation liquor treatment, major part can not normally be sprouted (see Fig. 6); Magnaporthe grisea spore through clear water process normally can be sprouted and form appressorium (see Fig. 7).
(5) bacillus amyloliquefaciens RL263 Biocontrol Activity active substance detects
Biomembranous detection: biocontrol microorganisms LB nutrient solution is cultivated 16h, get fermented liquid to dilute in the ratio LB nutrient solution of 1:1000, getting 1.5mL transfers in sterile centrifugation tube, 30 DEG C of quiescent culture 48,60, add the violet staining 15min of 0.1% (w/v) after 72h, centrifuge tube is rinsed clear to washing fluid with clear water, the microbial film be colored being attached to tube wall can be seen, add the ethanol rinse 5min of 1.5mL 95%, washing lotion is surveyed light absorption value under 570nm.The results are shown in Table 5.
Diastatic detection: the single colony inoculation getting new activation, in containing on the LB flat board of 0.2% Zulkovsky starch, is cultivated 48h, after forming obvious bacterium colony, flat board dripped Lu Geshi iodine staining, wash plate with 70% ethanol, observe the generation whether having transparent circle.If bacterial strain can produce amylase to have transparent circle to show, each process three repetition.The results are shown in Table 5.
Detection addicted to iron element: make and detect dull and stereotyped addicted to iron element, by the bacterial strain percutaneous puncture-inoculation of activation on flat board, 28 DEG C cultivate 24,48, observe the generation whether bacterium colony periphery has yellow halo after 60h, due to the iron ion addicted to EDTA chelating in iron element competition substratum, make substratum by blue yellowing, therefore the large I of periphery of bacterial colonies yellow halo represents the output addicted to iron element.3 repetitions are established in each process.The results are shown in Table 5.
The detection of proteolytic enzyme: by the bacterial strain percutaneous puncture-inoculation of activation on skimmed milk agar plate, 28 DEG C cultivate 24,48, observe the generation of the peripheral transparent circle of bacterium colony after 60h, occur that transparent circle shows the generation of proteolytic enzyme.3 repetitions are established in each process, the results are shown in Table 5.
The detection of chitinase: chitinase tobacco brown spot pathogen substratum detects, the bacterial strain percutaneous puncture-inoculation of activation is detected on culture medium flat plate in chitinase, 28 DEG C cultivate 24,48, observe the generation of the peripheral transparent circle of bacterium colony after 60h, occur that transparent circle shows to have the generation of chitinase.3 repetitions are established in each process, the results are shown in Table 5.
Dextranase detects: inoculation is in the LB flat board containing lichenstarch being substrate, after 30 DEG C of cultivation 48h, with 1% congo red staining 5min, use 1mol/L NaCL wash-out subsequently, observe in flat board and whether occur transparent circle, if there is transparent circle, show the generation having dextranase.Each process three repetition, the results are shown in Table 5.
The detection of cellulase: by the bacterial strain percutaneous puncture-inoculation of activation on Cellulose and congo red differential medium flat board, 28 DEG C cultivate 24,48, observe the generation of periphery of bacterial colonies redness hydrolysis circle after 60h, occur that red hydrolysis circle shows the generation having cellulase.3 repetitions are established in each process, the results are shown in Table 5.
The detection of bio-surfactant: the uniform oil dripping that drips drips (10W-40Castorl) on 96 orifice plates, the aseptic fermented supernatant fluid of bacterial strain is dripped again after drying, if drop caves in, show that this bacterium can produce tensio-active agent, if drop becomes globule shape, show that this bacterium does not produce tensio-active agent.The results are shown in Table 5.
The detection of HCN: activated strains on the KMB substratum containing 4.5g/L glycine, the filter paper of a sterilizing is put in the face of covering of culture dish, filter paper soaks with containing the picric acid of 1% and the sodium carbonate mixed solution of 2%, sealing culture dish, 48h is cultivated at 30 DEG C, observe filter paper colour-change, if filter paper becomes by redness the generation that sorrel shows to have HCN.Each process three repetition.The results are shown in Table 5.
Table 5 different strains biological and ecological methods to prevent plant disease, pests, and erosion related substances detects
Note: "+" represents positive; "-" represents negative.
Applicant states, the present invention illustrates detailed features of the present invention and method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method, does not namely mean that the present invention must rely on above-mentioned detailed features and method could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the increase of accessory, the concrete way choice etc. of condition selected by the present invention, all drops within protection scope of the present invention and open scope.

Claims (9)

1. prevent and treat the bacillus amyloliquefaciens RL263 (Bacillus amyloliquefaciens) of rice blast for one kind, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation date is on May 21st, 2014, and deposit number is CGMCC No.9191.
2. a microbial inoculum, it comprises bacillus amyloliquefaciens RL263 as claimed in claim 1.
3. prevent and treat a method for rice blast, it comprises bacillus amyloliquefaciens RL263 as claimed in claim 1 or microbial inoculum as claimed in claim 2 is applied to rice crop.
4. method as claimed in claim 3, it is characterized in that, described microbial inoculum is the nutrient solution of described bacillus amyloliquefaciens RL263.
5. the method as described in claim 3 or 4, is characterized in that, described rice blast is seedling rice blast, leaf pest, pulvinus pest, joint rice blast, panicle blast, fringe pest, branch stalk pest and/or grain pest.
6. method as claimed in claim 5, it is characterized in that, described rice blast is leaf pest and/or panicle blast.
7. bacillus amyloliquefaciens RL263 as claimed in claim 1 or the purposes of microbial inoculum as claimed in claim 2 in control rice blast.
8. purposes as claimed in claim 7, is characterized in that, described rice blast is seedling rice blast, leaf pest, pulvinus pest, joint rice blast, panicle blast, fringe pest, branch stalk pest and/or grain pest.
9. purposes according to claim 8, is characterized in that, described rice blast is leaf pest and/or panicle blast.
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CN105936880A (en) * 2016-05-19 2016-09-14 哈尔滨亿之隆生物科技开发有限公司 Bacillus amyloliquefaciens and application thereof
CN106282049A (en) * 2016-08-08 2017-01-04 湖南农业大学 Te Jila bacillus cereus JN369 and application thereof
CN106282049B (en) * 2016-08-08 2019-07-12 湖南农业大学 Te Jila bacillus JN369 and its application
CN106566870A (en) * 2016-08-31 2017-04-19 泰山医学院 Method for high-throughput rapid screening of fungi antagonistic bacteria
CN108220180A (en) * 2016-12-14 2018-06-29 河南科技学院 A kind of bacillus amyloliquefaciens Substance extracting method and its application as microbial inoculum in rice blast biological and ecological methods to prevent plant disease, pests, and erosion
CN107574137A (en) * 2017-10-23 2018-01-12 四川农业大学 Bacillus amyloliquefaciens Rdx5 and its application
CN107828689A (en) * 2017-11-22 2018-03-23 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） The bacillus amyloliquefaciens S170 of one plant of preventing and treating rice blast
CN107828689B (en) * 2017-11-22 2019-12-31 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） Bacillus amyloliquefaciens S170 for preventing and treating rice blast
CN111172081A (en) * 2020-02-24 2020-05-19 广东省农业科学院水稻研究所 Rice leaf endophytic bacillus amyloliquefaciens and biological agent and application thereof
CN111172081B (en) * 2020-02-24 2022-01-14 广东省农业科学院水稻研究所 Rice leaf endophytic bacillus amyloliquefaciens and biological agent and application thereof

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