CN105647829A - Bacillus amyloliquefaciens and application thereof to prevention and treatment of sugarcane red rot - Google Patents

Bacillus amyloliquefaciens and application thereof to prevention and treatment of sugarcane red rot Download PDF

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CN105647829A
CN105647829A CN201610035467.9A CN201610035467A CN105647829A CN 105647829 A CN105647829 A CN 105647829A CN 201610035467 A CN201610035467 A CN 201610035467A CN 105647829 A CN105647829 A CN 105647829A
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twc2
bacillus amyloliquefaciens
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梁艳琼
吴伟怀
雷照鸣
贺春萍
李锐
郑肖兰
易克贤
习金根
郑金龙
唐文
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CATAS Environment and Plant Protection Institute
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Abstract

The invention relates to the technical field of microorganisms and fermentation, in particular to bacillus amyloliquefaciens and application thereof to prevention and treatment of sugarcane red rot. The bacillus amyloliquefaciens TWC2 is preserved in China General Microbiological Culture Collection Center on 19th October, 2015, and the preservation number is CGMCC No.11515. The bacillus amyloliquefaciens TWC2 is separated out of tropical grass Taiwan grass leaves, and an environment-friendly, simple and effective way is provided for prevention and treatment of the sugarcane red rot. The bacillus amyloliquefaciens TWC2 is a strain with good biocontrol application prospects, and due to the fact that the strain is separated out of the surfaces of plants, the strain has natural harmonious compatibility with natural ecology. Compared with chemical pesticide, no toxic or side effect is caused to soil ecology, no residue exists, and therefore potential business development and application value is achieved in disease biological control practice.

Description

A kind of bacillus amyloliquefaciens and the utilization in preventing and treating red rot of sugar cane thereof
Technical field
The present invention relates to microorganism and fermentation technical field, be especially a kind of bacillus amyloliquefaciens and the utilization in preventing and treating red rot of sugar cane thereof.
Background technology
Caulis Sacchari sinensis is most important sugar crop, and sucrose accounts for world's sugar total output more than 70%, accounts for China's sugar and always produces more than 90%. Caulis Sacchari sinensis also becomes one of most important regenerative resource crop because it has the feature such as high-biomass and high microsteping. Sugarcane Disease is one of key factor affecting sugarcane yield and sugar. The red rot of sugar cane caused by sickle type anthrax ColletotrichumfalcatumWent is one of disease important in world's sugarcane production process. Yield is not only impacted by this disease, and its pathogen can also promote sucrose inversion, thus reducing sugarcane juice purity and reducing sucrose sugar.
The preventing and treating of red rot of sugar cane tradition relies primarily on chemical agent, and red rot of sugar cane there is no efficient chemical pesticide at present, causes that abusing high poison highly toxic pesticide phenomenon yet suffers from. Along with socioeconomic development and growth in the living standard, the Consciousness of food security of people strengthens day by day, to problem growing interests such as the pollutions having non agricultural chemical residuum and other harmful substance in Caulis Sacchari sinensis. Antagonistic microbe preventing and treating Sugarcane Disease is utilized to be expected to become the safe and nontoxic and effective new method of one replacing chemical agent. Antagonistic microbe is possible not only to diseases prevention, and can also promote plant growing, to improving woodland ecosystems, keeps woodland bio-diversity, and safeguards that woods field ecological balance realizes forestry sustainable development and will play an important role. The yield of whole world biological pesticide is with the speed increase of annual 10%-20%. At present, whole world biological pesticide product alreadys more than kind more than 100, and wherein 90% is microbial insecticide, and microbial bactericide is relatively fewer. Prevent and treat red rot of sugar cane especially with solution starch bud pole bacterium have not been reported.
Summary of the invention
It is an object of the invention to there is the defect in red rot of sugar cane preventing and treating for above-mentioned technology, it is provided that bacillus amyloliquefaciens and the utilization in preventing and treating red rot of sugar cane thereof.
For achieving the above object, the technical solution used in the present invention is: a kind of bacillus amyloliquefaciens TWC2, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 19th, 2015, and deposit number is CGMCCNo.11515.
The present invention provides bacillus amyloliquefaciens TWC2 utilization in preventing and treating red rot of sugar cane, utilizes the biological preparation that the preparation of this bacterial strain prevents and treats red rot of sugar cane to be applied to Caulis Sacchari sinensis.
The present invention provides the cultural method of a bacillus amyloliquefaciens TWC2 fermentation liquid, by TWC2 inoculation in LB culture medium flat plate, 37 DEG C of constant temperature culture 24h, obtain the TWC2 bacterial strain list bacterium colony of activation, then picking list colony inoculation is in LB culture medium, 37 DEG C, 200rpm/min when shaken cultivation 16h obtain TWC2 seed liquor, bacillus amyloliquefaciens TWC2 seed liquor is accessed in LB culture medium by the inoculum concentration by 7%, 34 DEG C, 200rpm/min, shaking table is cultivated 48h and is obtained bacillus amyloliquefaciens TWC2 fermentation liquid.
Further, cultivating pH is 5.0��9.0.
Further, cultivating pH is 6.0.
Further, described culture medium consists of: maltose 5g/L, fructose 10g/L, peptone 10g/L, yeast leach 10g/L, calcium chloride 1g/L.
The method have the benefit that: bacillus amyloliquefaciens TWC2 of the present invention is isolatable from tropical pasture Taiwan blade of grass sheet, the preventing and treating for red rot of sugar cane provides an environmental protection, approach simple, effective; The bacillus amyloliquefaciens TWC2 of the present invention is the bacterial strain that a strain has good biological and ecological methods to prevent plant disease, pests, and erosion application prospect, because this bacterial strain is to separate from plant surface to obtain, with natural, ecological, there is natural harmonious blending, compared with chemical pesticide, soil ecology is had no side effect, noresidue, therefore, in the Biological control of disease is put into practice, there is potential business development and using value.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the premise not paying creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
Fig. 1: the opposite culture experimental result of bacillus amyloliquefaciens TWC2 and red rot of sugar cane bacterium is (wherein, left figure is the face-off result of red rot of sugar cane bacteria strain SL121 and TWC2, and right figure is the face-off result of red rot of sugar cane bacteria strain SL120 and TWC2);
Fig. 2: the bacillus amyloliquefaciens TWC2 aspect graph (left figure is the TWC2 colony growth front at LB flat board, and right figure is the TWC2 colony growth back side at LB flat board) on LB flat board;
Fig. 3: bacillus amyloliquefaciens TWC2 growth curve chart;
Fig. 4: the mensuration of bacillus amyloliquefaciens TWC2 lethal temperature is (wherein, respectively 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 99 DEG C of left figure processes TWC2 colony growth situation on rear plate, in right figure respectively 90��98 DEG C process after the colony growth situation of TWC2);
Fig. 5: bacillus amyloliquefaciens TWC2 antibiotic resistance figure (being followed successively by comparison, cephamycin, erythromycin, streptomycin, ampicillin, rifampicin, kanamycin, tetracycline, chloromycetin from left to right);
Fig. 6: bacillus amyloliquefaciens TWC2 with the antagonistic effect figure of tropical crops encountered pathogenic fungus (wherein, 1��5 represents withered germ of water-melon respectively, red rot of sugar cane bacterium, Colletotrichum gloeosporioides Penz in Mango, anthracnose of rubber trees bacterium, imperatoria Phoma sp leaf spot and TWC2 opposite culture);
Fig. 7: bacillus amyloliquefaciens TWC2 to the prevention effect figure of red rot of sugar cane excised leaf, (CK is clear water comparison; Process 1: first spray TWC2, inoculate pathogen afterwards; Process 2: first inoculate pathogen, after spray TWC2; Process 3: only connect pathogen).
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments. Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, broadly fall into the scope of protection of the invention.
The screening of 1.TWC2
TWC2 bacterial strain is to provide from Hainan Province of China Danzhou City forage seeds to be separated to the Taiwan grass plant leaf of resource garden, concrete grammar: clip diameter is about the leaf tissue of 5mm �� 5mm size respectively on aseptic superclean bench, surface sterilization 30s is carried out with the mercuric chloride of 75% ethanol and 0.1%, after sterile water wash 3 times, it is transplanted in potato dextrose agar plate culture medium, cultivating in 28 DEG C of incubators, the bacterial strain that picking colony form is different, line purification storage is standby further.
By 5 bacterination process, the red rot of sugar cane bacterium disease of 2 strain High pathogenicity is accessed PDA culture medium flat plate central, biocontrol microorganisms is connected to pathogen surrounding, the bacterial strain that screening bacteriostasis is the strongest, result obtains a strain to the inhibited bacterial strain of red rot of sugar cane, and its preventive effect reaches more than 70%, being TWC2 by this Strain Designation, antagonism result is as shown in Figure 1.
The qualification of 2.TWC2 bacterial strain
(1) morphological observation and qualification: TWC2 bacterial strain is cultivated 2d in LB culture medium at 28 DEG C, observes its colonial morphology (shape, color gloss, surface whether bending, edge shape etc.);
(2) biochemical reactions is observed: by TWC2 inoculation in the test media such as salt tolerance, citrate utilization, gelatin liquefaction, Starch Hydrolysis, catalase, C.I. 13020., V_P, cellulose hydrolysis, indole experiment, observing response situation;
(3) molecular biology identification: extract the genomic DNA of bacterial strain, with bacterial universal primers 27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492R:5 '-TACGGCTACCTTGTTACGACTT-3 ', with the genomic DNA of TWC2 bacterial strain for template, through electrophoresis detection after pcr amplification, order-checking, sequence is such as shown in SEQNO:1.
Adopt customary physiological biochemical and TWC2 bacterial strain is identified by 16sDNA sequence analysis, it is determined that this bacterial strain is bacillus amyloliquefaciens. The Basic Biological Character of this bacterial strain is in Table 1.
The Basic Biological Character of table 1.TWC2 bacterial strain
Oxidase is tested - C.I. 13020. is tested +
Indole is tested - Gelatin liquefaction is tested +
Citrate growth experiment + Starch Hydrolysis +
V-p tests + Cellulose hydrolysis -
Catalase is tested + Colony characteristics Bacterium colony is white, subcircular, and surface folding is opaque
7%NaCl + 10%NaCl -
"+", is positive, and maybe can grow, utilize; "-" is negative reaction, maybe cannot grow, utilize.
Bacterial strain is white in LB culture medium, subcircular, surface wettability, has fold, opaque (see picture 2). Basis of microscopic observation, bacterial strain thalline is shaft-like, produces spore, and Gram-reaction is positive. The mensuration of bacterial strain TWC2 some biochemical indexes is according to " common bacteria system identification handbook ", and TWC2 bacterial strain physiological and biochemical property is all identical with bacillus amyloliquefaciens.Therefore by its called after bacillus amyloliquefaciens (Bacillusamyloliquefaciens) TWC2, bacillus amyloliquefaciens (Bacillusamyloliquefaciens) TWC2, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 19th, 2015 (to be called for short: CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101, phone: 010-64807355). Preserving number: CGMCCNo.11515.
With the genomic DNA of bacillus amyloliquefaciens TWC2 for template, using antibacterial 16srDNA sequence universal primer as primer, the product fragment of the 1.5kb that pcr amplification goes out. Sequencing result shows that sequence length is that the sequence Blast software of mensuration is carried out tetraploid rice with the known 16srDNA in GenBank by 1511bp (see sequence table :), finds that with Bacillusamyloliquefaciens (accession number: JN382250, CP007165 and CP010556) homology be 99% by comparison.
3. the preparation of bacillus amyloliquefaciens TWC2 seed liquor and best inoculation time are determined
By TWC2 inoculation in LB culture medium flat plate, 37 DEG C of constant temperature culture 24h, obtaining the TWC2 bacterial strain list bacterium colony of activation, then picking list colony inoculation is in LB culture medium, 37 DEG C, 200rpm/min when shaken cultivation 16h namely obtain TWC2 seed liquor.
Bacillus amyloliquefaciens TWC2 seed liquor is accessed in LB fluid medium by the inoculum concentration by 1%, and 37 DEG C, the cultivation of 200rpm/min shaking table, every 1h sampling and measuring OD600, METHOD FOR CONTINUOUS DETERMINATION 36h, draws growth curve chart to determine the best inoculation time of seed liquor.
TWC2 culture fluid is taken, with its OD of spectrophotometric determination every 1h600, METHOD FOR CONTINUOUS DETERMINATION 36h has drawn a series of OD numerical value, and these numerical value are made growth curve of bacteria figure (such as Fig. 3). Being in exponential phase at 0��24h, thalli growth power is vigorous, and cell concentration increases rapidly, and TWC2 was exceedingly fast with geometrical progression and increased this period, OD during 24h600Maximum is 2.335; 24��36hTWC2 enters stable phase, and growth flora sum is in flat phase, OD600Value fluctuates between 2.100��2.400. Consider cell concentration and fermentation costs, choose the TWC2 bacterium solution seed liquor as best inoculation time of cultivation 24h.
4. bacillus amyloliquefaciens TWC2 optimal culture conditions
4.1 strain growth optimum mediums
TWC2 seed liquor is accessed in LB fluid medium, 200rpm/min shaken cultivation 24h, it is respectively connected in following several culture medium according to the inoculum concentration of 1%: NA culture medium (glucose sugar 10g, peptone 5g, yeast powder 1g, Carnis Bovis seu Bubali cream 3g, distilled water 1000mL), basal fermentation medium (glucose sugar 10g, peptone 10g, KH2PO40.2g, MgSO40.2g, distilled water 1000mL), LB culture medium (peptone 10g, yeast powder 5g, sodium chloride 10g, distilled water 1000mL), YSP culture medium (yeast extract 5g, sucrose 20g, peptone 10g, distilled water 1000mL), NYBD culture medium (beef extract 8g, yeast extract 5g, glucose 10g, distilled water 1000mL), Preliminary fermentation culture medium (Carnis Bovis seu Bubali cream 3g, peptone 5g, glucose 10g, distilled water 1000mL), YPG culture medium (tryptone 5g, glucose 5g, yeast extract 5g, distilled water 1000mL) and BY culture medium (Carnis Bovis seu Bubali cream 5g, sodium chloride 5g, yeast extract 5g, glucose 5g, distilled water 1000mL). 28 DEG C, 180rpm/min shaken cultivation 24h, with OD600It is index with fermentation liquid bacteriostatic activity, detects bacterial strain optimum medium.It is shown that basal fermentation medium bacterium solution OD600Being worth the highest, be 0.255, bacteriostasis rate is 56.50%, and the value of NA culture medium is minimum, is 0.140, and bacteriostasis rate is 42.005% (result is in Table 2).
The table 2TWC2 bacterial strain growing state when different culture media
Note: do not represent that difference reaches extremely notable (p < 0.01) and significant level (p < 0.05) with the mother stock that writes of different size after column data, lower together.
The optimization of 4.2 culture medium
Basal fermentation medium: glucose sugar 10g, peptone 10g, KH2PO40.2g, MgSO40.2g, distilled water 1000mL.
4.2.1 optimum carbon source
With under the premise of components unchanged original in basal fermentation culture fluid, xylose with 1%, sucrose, soluble starch, fructose, mannitol, maltose, lactose, starch, glucose, sorbitol are carbon source, in 28 DEG C, 48h cultivated by the shaking table of 180rpm/min, measures its OD600With fermentation liquid bacteriostatic activity. It is shown that from the OD value of TWC2 bacterial strain, the OD value of various carbon sources differs greatly, wherein, the OD of maltose600Maximum, it is 0.812, the OD of glucose600Minimum; From bacteriostasis rate, when fructose is carbon source, the bacteriostasis rate of TWC2 bacterial strain is maximum, is 55.00%, and other are followed successively by maltose, mannitol, starch, sorbitol, glucose, lactose, xylose, sucrose, soluble starch (result is in Table 3). Therefore, select fructose and maltose as the compounded carbons of Optimal Medium.
The table 3TWC2 bacterial strain growing state when different carbon source
4.2.2 optimum nitrogen source screening
Respectively with the wheat bran of 1% (m/V) content, tryptone, Carnis Bovis seu Bubali cream, yeast powder, carbamide, ammonium chloride, ammonium nitrate, yeast powder: tryptone=(1:1), yeast powder: tryptone: ammonium chloride=(2:2:1), peptone: yeast extract powder: 10 kinds of nitrogenous sources such as ammonium nitrate=(2:2:1), replacing the peptone in basal fermentation medium, other conditions are ibid. Test shows, when nitrogenous source is yeast powder: during tryptone=(1:1), and the OD of TWC2 bacterial strain600Value and bacteriostasis rate are all the highest, respectively reach 0.433,48.210%, secondly nitrogenous source is peptone: yeast extract powder: ammonium chloride=(2:2:1), wheat bran, beef extract, peptone: yeast extract powder: when ammonium nitrate=(2:2:1), yeast extract powder and peptone, the OD of TWC2 bacterial strain600Value and bacteriostasis rate are also higher, but when nitrogenous source is carbamide, ammonium chloride, ammonium sulfate, OD600Value and bacteriostasis rate are minimum, are 0 (result is in Table 4). Therefore, peptone is selected: yeast extract powder=1:1 is as the nitrogenous source of Optimal Medium.
The table 4TWC2 bacterial strain growing state when different nitrogen sources
4.2.3 best metal ion screening
KH is added respectively by 0.1% (m/V) content2PO4��FeSO4��ZnSO4��NaCl��MnSO4��MgSO4��CaCl2And CuSO4Deng 8 kinds of inorganic salts, compareing as the culture medium without inorganic ion, other conditions are ibid. Test shows, with comparison without compared with the culture medium of inorganic ion, as the OD adding calcium chloride culture medium600Value and bacteriostasis rate all dramatically increase, and respectively 2.224 and 60.780%. When metal ion is magnesium sulfate, OD600Value and bacteriostasis rate also significantly improve, when metal ion is manganese sulfate, sodium chloride, and its OD600Value relatively compares and reduces, but its bacteriostasis rate relatively compares and improves. Add FeSO4And ZnSO4Culture medium, TWC2 bacterial strain does not grow, and the growth of both metal ion strong inhibition TWC2 is described. Therefore calcium chloride is as the best metal ion of Optimal Medium.
Table 5TWC2 bacterial strain growing state under different metal ionic conditions
4.2.4 nutrient media components ratio optimization
The selection result according to optimum carbon source, nitrogenous source and metal ion, by orthogonal, selects the 5 horizontal L of factor 416(45) orthogonal table determines the optimum proportioning of each component, to determine suitable TWC2 strain growth and to produce the best of breed of Optimal Medium of antibiotic substance.
It is optimized by the orthogonal optimum carbon source to screening, nitrogenous source and metal ion, in conjunction with TWC2 bacteriostatic activity, finally determines the best medium of TWC2. Its constituent content is: maltose 5g/L, fructose 10g/L, peptone 10g/L, and yeast leaches 10g/L, calcium chloride 1g/L.
The table 6TWC2 bacterial strain growing state when different culture media
4.2.5 the screening of optimal culture condition
After determining best medium, further the optimal culture condition of TWC2 is screened from aspects such as pH, temperature, liquid amount, time, inoculum concentration and rotating speeds..
1. pH value
0.1mol/LHCl and 0.1mol/LNaOH is adopted to regulate Optimal Medium pH value. Respectively the Optimal Medium 100ml that pH value is 4.0,5.0,5.5,6.0,6.5,7.0,7.5,8.0,9.0,10.0 is loaded in 250ml triangular flask, 121 DEG C of sterilizing 20min, after cooling inoculate 1mLTWC2 seed liquor, 37 DEG C, 180rpm/min cultivate 48h.
It is shown that when pH value is 5.0��9.0, the OD value of TWC2 bacterial strain and bacteriostasis rate difference are not notable, and pH value is all unfavorable for the growth of bacterial strain and the generation of antibacterial substance less than 4.0 or more than 10.0. Therefore, cultivating optimum range is 5.0��9.0, and optimum pH is 6.0 (result is in Tables 7).
Table 7TWC2 bacterial strain is at the growing state of different pH
2. strain growth optimum temperature
Filling Optimal Medium in 250ml triangular flask is 100mL, 121 DEG C of sterilizing 20min. Inoculum concentration is inoculation 1mLTWC2 seed liquor in each shaking flask, respectively at 10 DEG C, 15 DEG C, 20 DEG C, 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C, 34 DEG C, 37 DEG C, 40 DEG C, cultivates 48h when 180rpm/min. Test shows, this bacterial strain is at 10��40 DEG C of equal energy growths, significant difference. When temperature range is 26��40 DEG C, bacterial strain OD600 value is 0.648��0.846, and bacteriostasis rate is 61.814��67.303. Optimum temperature is 34 DEG C, and its OD value and bacteriostasis rate are maximum, respectively reach 0.985,69.928% (result is table 8 such as).
Table 8TWC2 bacterial strain is at the growing state of condition of different temperatures
3. ventilation
250mL triangular flask is respectively charged into the Optimal Medium that volume is 20mL, 40mL, 60mL, 80mL, 100mL, 120mL, 140mL, 160mL, 121 DEG C of sterilizing 20min. Inoculum concentration be in each shaking flask inoculation 1mLTWC2 seed liquor, in 37 DEG C, 180rpm/min when cultivate 48h. It is shown that when liquid amount is 40mL, strain growth is best, OD600Value is 1.126, and bacteriostasis rate is 56.038% (result is in Table 9). Liquid amount is more or less than 40mL, and its OD value and bacteriostasis rate all decrease. Therefore, when shake flask fermentation, liquid amount is too much or the generation of the very few growth being all unfavorable for bacterial strain and antibacterial substance, and in 250mL triangular flask, best liquid amount should be 40mL.
Table 9TWC2 bacterial strain is at the growing state of different ventilation conditions
4. incubation time
Filling Optimal Medium in 250mL triangular flask is 100mL, 121 DEG C of sterilizing 20min. Inoculum concentration is inoculation 1mLTWC2 seed liquor in each shaking flask, in 37 DEG C, the incubation time of TWC2 bacterial strain is adjusted to 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 120h respectively under 180rpm/min. It is shown that growth is best when incubation time is 48h, OD600Value 0.590, bacteriostasis rate is 67.064 (result is in Tables 10), along with the increase of incubation time, OD600Value first increases and reduces afterwards, it may be possible to owing to incubation time is long, and the nutrient consumption of culture fluid is too much, so that OD value reduces.
Table 10TWC2 bacterial strain is at the growing state of different incubation time conditions
5. inoculum concentration
Filling Optimal Medium in 250mL triangular flask is 100mL, 121 DEG C of sterilizing 20min. The inoculum concentration (V/V) of TWC2 seed liquor is adjusted to 1%, 3%, 5%, 7%, 9% respectively, in 37 DEG C, 180rpm/min when cultivate 48h. It was found that OD600Value size is followed successively by 7% > 9% > 5% > 3% > 1%, bacteriostasis rate size is followed successively by 7% > 3% > 5% > 9% > 1%, it follows that when inoculum concentration is 7%, strain growth and produce antibacterial substance best (result is in Table 11).
Table 11TWC2 bacterial strain is at the growing state of different vaccination amount condition
6. rotating speed
Filling Optimal Medium in 250mL triangular flask is 100mL, 121 DEG C of sterilizing 20min. Inoculum concentration is inoculation 1mLTWC2 seed liquor in each shaking flask, at 37 DEG C, cultivates 48h when shaking speed is adjusted to 120rpm/min, 160rpm/min, 180rpm/min, 200rpm/min, 220rpm/min respectively. It is shown that when bacterial strain is when rotating speed is 200rpm/min, strain growth and bacteriostasis rate are the best (result is in Table 12).
Table 12TWC2 bacterial strain is at the growing state of different rotating speeds condition
6. the mensuration of lethal temperature
TWC2 bacterial strain is accessed fermentation medium, 180rpm/min, cultivate 24h for 28 DEG C, then it is sub-packed in test tube, often pipe 10mL, it is respectively placed in 10min in the water-bath of temperature 50 C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 99 DEG C (water-bath temperature does not reach 100 DEG C), is then coated with flat board, after 24h, observe colony growth situation. Result shows, bacterial strain is at 50��90 DEG C of equal energy normal growths, and 99 DEG C can not grow. According to the above results, then set one group of temperature: 91 DEG C, 92 DEG C, 93 DEG C, 94 DEG C, 95 DEG C, 96 DEG C, 97 DEG C, 98 DEG C, after 24h, observe colony growth situation. It is shown that the lethal temperature of this bacterial strain is 98 DEG C, 10min. (see Fig. 4).
5. the antibiotic resistance of bacillus amyloliquefaciens TWC2
Determine TWC2 to ampicillin, tetracycline, streptomycin, kanamycin, rifampicin, cephamycin, the Resistant reaction of chloromycetin. Above-mentioned antibiotic is dissolved in distilled water or ethanol, is configured to the solution of debita spissitudo, standby after Filtration sterilizing. Bacterial strain is accessed LB culture medium and makes containing bacterium flat board, take containing the antibiotic scraps of paper on flat board with aseptic nipper, put 34 DEG C of incubators and cultivate, after 24 hours, observe strain growth situation. It was found that above-mentioned antibiotic is respectively provided with resistance (see Fig. 5) by TWC2 bacterial strain
6. the antagonism of tropical crops encountered pathogenic fungus is detected by bacillus amyloliquefaciens TWC2
TWC2 is detected the antagonism of this bacterium with various pathogenic bacteria co-cultivation respectively. Opposite culture: by 5 bacterination process at the good PDA culture medium flat plate central authorities' different types of pathogen of access (this experiment preservation), TWC2 is inoculated in flat board surrounding, being placed in 28 DEG C of opposite culture 5��7d, result shows that pathogen is had stronger antagonism (see Fig. 6) by this strain antibacterial that screening obtains. Measuring antimicrobial spectrum width and the suppression ratio of TWC2 and each pathogen, result is in Table 13.
The table 13TWC2 antagonism to tropical plants pathogen
7. the bacillus amyloliquefaciens TWC2 antagonistic effect to red rot of sugar cane bacterium excised leaf
The preparation of 7.1 bacillus amyloliquefaciens TWC2
By TWC2 inoculation in LB culture medium flat plate, 37 DEG C of constant temperature culture 24h, obtaining the TWC2 bacterial strain list bacterium colony of activation, then picking list colony inoculation is in LB culture medium, 37 DEG C, 200rpm/min when shaken cultivation 16h namely obtain TWC2 seed liquor.Bacillus amyloliquefaciens TWC2 seed liquor is accessed in LB culture medium by the inoculum concentration by 7%, 34 DEG C, 200rpm/min, and 48h cultivated by shaking table, takes culture fluid and measures its OD600=2.3 (bacterial concentration crosses the dilution of High Availabitity sterilized water), standby. Every blade sprays 2mL bacterium solution.
Prepared by 7.2 pathogen
The red rot of sugar cane pathogen being stored in slant medium is transferred in PDA culture medium, after 28 DEG C of cultivation 3d, punches with 5mm card punch, standby.
Take light green Sugarcane Leaves, stab with needle point method. Concrete process is as follows: 1. first spray TWC2 bacterium solution in Sugarcane Leaves vein. Connect pathogen in acupuncture place after air-dry at once; 2. pathogen bacterium cake is first connect in acupuncture place, rear sprinkling TWC2 bacterium solution; 3. pathogen is only connect in acupuncture place; 4. blank, only spray clear water is in blade surface. Observed result after 3d, surveys lesion diameter by decussation method and obtains area, being calculated as follows scab suppression ratio. Scab suppression ratio (%)=(the comparison scab gross area-process scab gross area)/comparison scab gross area. Result shows, the lesion area of process 1 is minimum, and bacteriostasis rate is the highest, next to that process 2, is finally process 3 (result is shown in Fig. 6). Result shows that first biocontrol microorganisms is shone in spray, can produce disease resistance by inducer blade. Thus reaching prevention effect.
Table 14TWC2 is to the antagonism to red rot of sugar cane excised leaf
Those skilled in the art of the present technique are appreciated that unless otherwise defined, all terms used herein (include technical term and scientific terminology) and have with the those of ordinary skill in art of the present invention be commonly understood by identical meaning. Should also be understood that in such as general dictionary, those terms of definition should be understood that have the meaning consistent with the meaning in the context of prior art, and unless defined as here, will not explain by idealization or excessively formal implication.
One of ordinary skill in the art will appreciate that: the parts in device in embodiment can describe in the device being distributed in embodiment according to embodiment, it is also possible to carries out respective change and is disposed other than in one or more devices of the present embodiment. The parts of above-described embodiment can merge into parts, it is also possible to is further split into multiple subassembly.
It should be noted last that: above example is only in order to illustrative not limiting technical scheme, although the present invention being described in detail with reference to above-described embodiment, it will be apparent to an ordinarily skilled person in the art that: still the present invention can be modified or equivalent replacement, without deviating from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of scope of the presently claimed invention.

Claims (6)

1. a bacillus amyloliquefaciens TWC2, deposit number is CGMCCNo.11515.
2. bacillus amyloliquefaciens TWC2 utilization in preventing and treating red rot of sugar cane according to claim 1, it is characterised in that utilize the biological preparation that the preparation of this bacterial strain prevents and treats red rot of sugar cane to be applied to Caulis Sacchari sinensis.
3. the preparation method of bacillus amyloliquefaciens TWC2 fermentation liquid according to claim 2, it is characterized in that, by TWC2 inoculation in LB culture medium flat plate, 37 DEG C of constant temperature culture 24h, obtain the TWC2 bacterial strain list bacterium colony of activation, then picking list colony inoculation is in LB culture medium, 37 DEG C, 200rpm/min when shaken cultivation 16h obtain TWC2 seed liquor, bacillus amyloliquefaciens TWC2 seed liquor is accessed in LB culture medium by the inoculum concentration by 7%, 34 DEG C, 200rpm/min, shaking table is cultivated 48h and is obtained bacillus amyloliquefaciens TWC2 fermentation liquid.
4. the preparation method of bacillus amyloliquefaciens TWC2 fermentation liquid according to claim 3, it is characterised in that cultivating pH is 5.0��9.0.
5. the preparation method of bacillus amyloliquefaciens TWC2 fermentation liquid according to claim 4, it is characterised in that cultivating pH is 6.0.
6. the preparation method of bacillus amyloliquefaciens TWC2 fermentation liquid according to claim 3, it is characterised in that described culture medium consists of: maltose 5g/L, fructose 10g/L, peptone 10g/L, yeast leach 10g/L, calcium chloride 1g/L.
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CN108690821A (en) * 2017-12-07 2018-10-23 中国热带农业科学院环境与植物保护研究所 One plant height imitates bacillus amyloliquefaciens and its microbial inoculum and application
CN113774002A (en) * 2021-11-02 2021-12-10 浙江珲达生物科技有限公司 Bacillus amyloliquefaciens culture medium and application thereof
CN113930364A (en) * 2021-11-08 2022-01-14 怀化学院 Bacillus and application and use method thereof in preventing and treating polygonatum sibiricum stem-point leaf spot disease
CN113930364B (en) * 2021-11-08 2023-07-07 怀化学院 Bacillus and application and use method thereof for preventing and treating leaf spot of rhizoma polygonati phoma

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