CN103468599B - Streptomycetes sp. and its application in winter jujube ring rot prevention and treatment - Google Patents
Streptomycetes sp. and its application in winter jujube ring rot prevention and treatment Download PDFInfo
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- CN103468599B CN103468599B CN201310193973.7A CN201310193973A CN103468599B CN 103468599 B CN103468599 B CN 103468599B CN 201310193973 A CN201310193973 A CN 201310193973A CN 103468599 B CN103468599 B CN 103468599B
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Abstract
The invention discloses a Streptomycetes sp. and its application in winter jujube ring rot prevention and treatment. The above strain is named as BK, and is named as Streptomycetes sp. in classification, has a preservation number of CGMCC No.7489, and is preserved in China General Microbiological Culture Collection Center on April 19, 2013. The Streptomycetes sp. is separated and screened from a Yellow River Delta shell bank soil sample for the first time in the invention, and experiments prove that the Streptomycetes sp. has a substantial antagonistic effect and a wide antimicrobial spectrum. The strain grows on a Gao solid medium, and a formed bacterium colony has the characteristics of white color, round shape, irregular edges, dry surface, non-transparency, and positive Gram staining. The invention also discloses a nucleotide sequence of the Streptomycetes sp.. The strain has an obvious inhibition effect on winter jujube ring rot pathogens and other plant pathogen fungi, for example, the strain has an obvious antagonistic effect on apple stem rot pathogens, cucumber wilt pathogens, tomato rhizoctonia rot pathogens and the like; and the strain has a very good application prospect.
Description
[technical field] the invention belongs to biotechnology and agricultural disease Prevention Technique field, and specifically, it relates to a kind of streptomycete Streptomyces sp. and the application in control winter jujube ring spot thereof.
The fresh and tender succulence of [background technology] winter jujube mouthfeel, local flavor sour-sweet is agreeable to the taste, the outstanding person eaten raw in kind, bring huge economic interests to since popularizing planting vast plantation family, but along with increasing sharply of cultivated area, the generation of winter jujube disease is more and more heavy, wherein winter jujube ring spot is comparatively common and that harm is serious fungal disease, winter jujube ring spot not only directly causes yield of winter jujubes to decline and quality reduces, and pathogenic fungi can secrete the toxin and metabolite that produce and be harmful to people and animals, great threat is formed to the health of people.At present, the control of winter jujube ring spot mainly prevents and treats by spraying chemicals, but long-term unreasonable use chemical pesticide creates problems.As: the height poison of chemical pesticide, the health of high residue to people, animal cause harm, cause severe contamination, destroy the eubiosis to soil, water body, air; The continuous enhancing of the pathogenic bacteria property of medicine, usage quantity and the usage frequency of agricultural chemicals continue to increase, and have occurred the vicious cycle that dosage and disease increase progressively mutually.Therefore develop the mankind and environmental friendliness and the good prevention effect of tool, new control of plant disease way is extremely urgent.
Along with people are to the pay attention to day by day of food safety, the needs of sustainable disease and pest control development, biological control is subject to the attention of national governments and plant protection expert.Biological control controls harmful organism based on ecology, be in Synthetical control system one must indispensable integral part.It avoid chemical pesticide use bring a series of plant protection, environment and energy aspect problem, avoid the harm of pesticide residue to people and animals.Applying biological agricultural chemicals substituting chemical pesticide becomes a kind of trend gradually.
Actinomycetes are Microbial resources that a class has Important Economic value, are the main producers of the various bioactivators such as microbiotic, zymin and immunomodulator, and the antibiosis of current agricultural and Clinical practice have 3/4ths and produced by actinomycetes.Can say that actinomycetes are that human health has made tremendous contribution.But along with extensively carrying out of actinomycetes research work, new actinomycetic discovery is more and more difficult, a large amount of actinomycetes and active result are repeatedly repeated to find and describe.To find that from common ecotope new actinomycetes are more and more difficult now.The special ecological environment samplings such as some scholars advise from ocean, animals and plants endogenous cycle border, virgin forest, salt lake, polar region, these ecotopes, due to through long-term natural selection, microbial ecological diversity is special, find that new actinomycetic possibility is larger because of the natural evolution of these environment through millions upon millions of years, microbial ecological diversity is special, there is novel bacterial probability large.New microbe bacterial classification means the existence of new gene, novel metabolic pathways, and the possibility obtaining new texture secondary metabolite is also large.
Shell bank be by the shellfish death of living in tideland after housing and chip, through wave carrying, pile up near high water mark.Wudi, Accessories during Binzhou, the Huanghe delta, Zhanhua seashore have become the shell bank formed in the process of land since nearly 5000 or six thousand years, and ecotope is unique, with the U.S. easily this Anna state and South America Suriname shell bank and be called the large Chenier in the world three.And be worldwide largest, only the old and new and the shell bank deposited.The ecotope of shell bank uniqueness imparts the distinctive Biological resources in this area, provides good resource for excavating new actinomycetes and developing biological pesticide.Accelerate research and development and the application of biological pesticide, meet the needs of China's agricultural development, for ensureing that the healthy diet of people is significant, development prospect is wide.
[summary of the invention] the object of the invention is to for the day by day serious problem of winter jujube ring spot, provides a kind of streptomycete (Streptomyces sp.) BK and the application of this bacterium in winter jujube ring spot control.
Streptomycete provided by the invention (Streptomyces sp.) BK bacterial strain, be preserved on 04 19th, 2013 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; institute of microbiology of the Chinese Academy of Sciences), its preserving number is CGMCC No.7489.
The screening of streptomycete Streptomyces sp.BK CGMCC No.7489
Get Huanghe delta shell bank soil sample 5g, 100 DEG C of dry heat treatment 1h put into the triangular flask that 50mL sterilized water is housed, add a certain amount of granulated glass sphere to put on shaking table and shake 1h, with sterilized water gradient dilution (final dilution 10000 times), (substratum forms: Zulkovsky starch 20g to coat Gause I substratum; KNO
31g; K
2hPO
40.5g; MgSO
47H
2o0.5g; NaCl0.5g; FeSO
47H
2o0.01g; Agar 20g; Water 1000mL; PH7.2 ~ 7.4), 28 DEG C of constant temperature culture 6 days.Cultivate the actinomycetes obtained to carry out classifying and numbering according to colonial morphology and color, and carry out purifying through repeatedly ruling.Be separated the bacterial strain that obtains with the winter jujube ring spot germ, cucumber fusarium axysporum germ for indicator, screened by dull and stereotyped opposite culture method, final obtain the best Streptomyces sp.BK of fungistatic effect.
Streptomycete (Streptomyces sp.) BK CGMCC No.7489 colony characteristics: cultivate the colony diameter 4 ~ 6mm of 7 days on Gause I culture medium flat plate, gas silk be white, surface compact, drying, wrinkles more, not easily provokes.
The cell morphological characteristic of streptomycete (Streptomyces sp.) BK CGMCC No.7489: gram-positive microorganism, mycelia is elongated, and branch is many, and spore ovalize smooth surface, fibrillae of spores twist.
The physiological and biochemical property of streptomycete (Streptomyces sp.) BK CGMCC No.7489: growth temperature 22 DEG C ~ 40 DEG C, pH4 ~ 10, NaCL tolerance 1% ~ 3%.The test of nitrate reduction, Starch Hydrolysis, cellulose hydrolysis test, gelatine liquefication experiment are the positive, do not solidify but peptonized milk, D-semi-lactosi, glycerine, L-rhamnosyl, maltose, glucose, D-wood sugar, sucrose, lactose, sorbose can be utilized, wood sugar, inose, L-arabinose can not be utilized.
The 16S rDNA gene sequence characteristic of streptomycete (Streptomyces sp.) BK CGMCC No.7489: CGMCC No.7489 is inoculated in Gause I substratum, 120rpm, 8 DEG C shake cultivation 24 hours, collected by centrifugation thalline, N,O-Diacetylmuramidase and SDS broken wall is added after suspension, phenol-chloroform method is adopted to extract genomic dna, with positive primer 2 7F(5 '-GAGAGTTTGATCCTGGCTCAG-3 '), with reverse primer 1541R (5 '-AAGGAGGTGATC CAGCC-3 '), pcr amplification is carried out to its 16S rDNA gene, the product of amplification is won company by Beijing three check order.PCR condition is: 94 DEG C, 10min; 94 DEG C, 45s; 55 DEG C, 45s; 72 DEG C, 90s; 30 circulations; 4 DEG C of preservations.16SrDNA gene order length is 1406bp, and the number of logging in GenBank is KF111562, is 99% with Streptomyces purpureus (HQ650808) similarity, and its 16S rDNA is as shown in sequence table.
With reference to the content of " Bergey ' s Mannual of Systematic Bacteriology " Vol. VIII, according to its morphological specificity and physiological and biochemical property, and 16S rDNA gene order result, be a new bacterium through Bacillus subtilis BK, belong to streptomyces (Streptomyces sp.BK).
Streptomycete Streptomyces sp.BK CGMCC No.7489 can at nutritional medium, as: grow in Gause I substratum, LB and nutrient agar, also can grow in the minimal medium adding glucose.
This streptomycete to the winter jujube ring spot germ and other various plants pathogenic fungi all there is obvious restraining effect.
Advantage of the present invention and positively effect:
CGMCC No.7489 bacterium provided by the invention, it is a kind of excellent biological and ecological methods to prevent plant disease, pests, and erosion streptomycete, the fermentation of this strain liquid can produce fungal diseases of plants pathogenic bacteria as winter jujube ring spot, apple stalk rot germ, cucumber fusarium axysporum germ and tomato seedling damping-off germ etc. have inhibiting active metabolite, to the winter, the biocontrol effect of jujube ring spot reaches more than 80%, has good application prospect.
[accompanying drawing explanation]:
Fig. 1 is that Streptomyces sp.BK CGMCC No.7489 bacterium is to the restraining effect of winter jujube ring spot germ;
Fig. 2 is that Streptomyces sp.BK CGMCC No.7489 bacterium is to the restraining effect of cucumber fusarium axysporum germ;
Fig. 3 is the colonial morphology of Streptomyces sp.BK CGMCC No.7489 bacterium;
Fig. 4 is the suppression (a be contrast) of Streptomyces sp.BK CGMCC No.7489 fermented liquid to winter jujube ring spot germ mycelial growth;
Fig. 5 is the impact for the treatment of of different temperature on Streptomyces sp.BK CGMCC No.7489 fermented liquid bacteriostatic activity;
Fig. 6 is the impact of different pH process on Streptomyces sp.BK CGMCC No.7489 fermented liquid bacteriostatic activity.
[embodiment]
Embodiment 1:
The separation screening of Streptomyces sp.BK bacterial strain provided by the invention.
Get Huanghe delta shell bank soil sample 5g, 100 DEG C of dry heat treatment 1h put into the triangular flask that 50mL sterilized water is housed, add a certain amount of granulated glass sphere, put on shaking table and shake 1h, with sterilized water gradient dilution (final dilution 10000 times), (substratum forms: Zulkovsky starch 20g to coat Gause I substratum; KNO
31g; K
2hPO
40.5g; MgSO
47H
2o0.5g; NaCl0.5g; FeSO
47H
2o0.01g; Agar 20g; Water 1000mL; PH7.2 ~ 7.4), 28 DEG C of constant temperature culture 6d.Cultivate the actinomycetes obtained to carry out classifying and numbering according to colonial morphology and color, and carry out purifying through repeatedly ruling.Be separated the bacterial strain that obtains with the winter jujube ring spot germ, cucumber fusarium axysporum germ for indicator, screened by dull and stereotyped opposite culture method, final obtain the best BK bacterial strain of fungistatic effect, result is as shown in Fig. 1, Fig. 2.
Embodiment 2:
The morphological specificity of Streptomyces sp.BK bacterial strain provided by the invention and physiological and biochemical property.
Test method with reference to " Bergey ' s Mannual of Systematic Bacteriology " Vol. VIII is carried out, detect its gramstaining, bacterium colony size and geometric, growth temperature, pH scope, NaCL tolerance, nitrate reduction, Starch Hydrolysis, cellulose hydrolysis, gelatine liquefication, and utilizing D-semi-lactosi, glycerine, L-rhamnosyl, maltose, glucose, D-wood sugar, lactose, sorbose, sucrose, inose, L-arabinose are tested.
Result shows, this bacterium is gram-positive microorganism, and mycelia is elongated, and branch is many, and spore ovalize smooth surface, fibrillae of spores twist.Gause I culture medium flat plate is cultivated the colony diameter 4 ~ 6mm of 7d, and gas silk is white, surface compact, drying, many wrinkles, not easily provokes (Fig. 3).Growth temperature 22 DEG C ~ 40 DEG C, pH4 ~ 10, NaCL tolerance 1% ~ 3%.The test of nitrate reduction, Starch Hydrolysis, cellulose hydrolysis test, gelatine liquefication experiment are the positive, do not solidify but peptonized milk, D-semi-lactosi, glycerine, L-rhamnosyl, maltose, glucose, D-wood sugar, sucrose, lactose, sorbose can be utilized, wood sugar, inose, L-arabinose can not be utilized.
Embodiment 3:
The pcr amplification of the 16S rDNA gene of Streptomyces sp.BK bacterial strain provided by the invention and sequencing.By this inoculation in Gause I substratum, 120rpm, 36h is cultivated in 28 DEG C of concussions, collected by centrifugation thalline, adds N,O-Diacetylmuramidase and SDS broken wall after suspension, adopt phenol-chloroform method to extract genomic dna, with positive primer 2 7F (5 '-GAGAGTTTGATCCTG GCTCAG-3 '), with reverse primer 1541R (5 '-AAGG AGGTGATCCAGCC-3 '), pcr amplification is carried out to its 16S rDNA gene, the product of amplification is won company by Beijing three and checks order.PCR condition is: 94 DEG C, 10min; 94 DEG C, 45s; 55 DEG C of 45s; 72 DEG C, 90s; 30 circulations.4 DEG C of preservations.16S rDNA gene order length is 1406bp, and the number of logging in GenBank is KF111562, is 99% with Streptomyces purpureus (HQ650808) similarity.Its result is as shown in sequence table.
Embodiment 4:
The fermentation culture of Streptomyces sp.BK bacterial strain provided by the invention.
By BK inoculation on Gause I nutrient agar, activation culture 3d at 28 DEG C, adds sterilized water, makes spore suspension.Spore suspension is inoculated in the 250mL triangular flask that 100mL Gause I liquid nutrient medium is housed by the inoculum size by 10% carries out shake flask fermentation, and temperature is 28 DEG C, and shaking speed is 200rpm, and fermentation time is 7d.By tunning through 10000r min
-1centrifugal 20min, obtains fermented supernatant fluid, for subsequent use.
Embodiment 5:
Streptomyces sp.BK bacterial strain provided by the invention is to the restraining effect of various hypha,hyphae.
Adopt containing toxic medium method, the fermented liquid supernatant (with example 4) of 2mL BK is added in 18mLPDA substratum, the flat board of falling PDA, with the pathogenic fungi mycelia block that punch tool cut-off footpath is 5mm, move into plate center place, cultivate 4d for 28 DEG C and measure mycelial growth diameter (mm), repeat 3 times, calculate bacteriostasis rate.Bacteriostasis rate=[(contrast mycelial growth diameter-test process mycelial growth diameter)/contrast mycelial growth diameter] × 100%.Result shows (table 1), and BK bacterial strain fermentation liquor has stronger restraining effect to various plants pathogenic fungi, bacteriostasis rate all more than 50%, wherein to the winter jujube ring spot germ bacteriostasis rate the highest, for 72.3%(is shown in Fig. 4).
Table 1BK bacterial strain fermentation liquor is to the restraining effect of different plant pathogenic fungi mycelial growth
Embodiment 6:
Physico-chemical property-the thermostability of Streptomyces sp.BK bacterial strain fermentation liquor provided by the invention.
BK bacterial strain fermentation liquor supernatant (with example 4) is processed 30min respectively at 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 100 DEG C, with the winter jujube ring spot germ for indicator, compare without heat treated fermented liquid, measure containing toxic medium method (with example 5) active.As shown in Figure 5, BK fermented liquid has stronger thermostability to result.50-80 DEG C respectively processes 30min, and bacteriostatic activity all maintains more than 85% of contrast, 100 DEG C of process 60min, and active is 79.6% of contrast.
Embodiment 7:
Physico-chemical property-pH the stability of Streptomyces sp.BK bacterial strain fermentation liquor provided by the invention.
Used by BK bacterial strain fermentation liquor supernatant (with example 4) 1mol/L HCl and 1mol/L NaOH process to pH value 4,5,6,7,8,9,10 respectively, static 2h, then pH is recalled to the natural ph of fermented liquid, with the winter jujube ring spot germ for indicator, fermented liquid without acid-alkali treatment compares, and measures active containing toxic medium method (with example 5).Result as shown in Figure 6, has certain bacteriostasis rate between pH5 ~ pH10, and along with the rising of pH, bacteriostasis rate is in downtrending gradually, but difference with insignificance, when pH is 6, fungistatic effect is best, reaches 72%.
Embodiment 8
The application of Streptomyces sp.BK bacterial strain provided by the invention in winter jujube experimental plot.
Can prevent and treat winter jujube ring spot by fermented liquid, detailed process is: ferment to BK, and fermented liquid dilutes 50 times of sprayings, the 1st spray pesticide after jujube tree fallen flowers, later interval dispenser in 15 ~ 20 days 1 time, totally 4 times, whole dispenser breeding time 5 times, its biocontrol effect can reach 80.3%.Fermentation culture conditions is: by BK inoculation on Gause I nutrient agar, activates 36h at 28 DEG C, and be then transferred in liquid nutrient medium by the BK bacterial strain of activation and carry out fermentation fermentation, temperature is 28 DEG C, and shaking speed is 200rpm, and fermentation time is 7d.Wherein fermention medium consists of g/L: Zulkovsky starch 20; KNO
31; K
2hPO
40.5; MgSO
47H
2o0.5; NaCl0.5; FeSO
47H
2o0.01; Water 1000mL; PH7.2 ~ 7.4.
Sequence table
Binzhou College
A kind of streptomycete and the application in winter jujube ring spot control thereof
1406bp
Streptomyces?sp.BK?CGMCC?No.7489?16S?rDNA?gene?sequence
Claims (3)
1. a streptomycete (
streptomycessp
.) BK bacterial strain, be preserved on April 19th, 2013 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number is CGMCC No.7489.
2. a streptomycete according to claim 1 (
streptomycessp
.) application of BK CGMCC No.7489, it is characterized in that, this bacterium can produce Substance, this fermented liquid can suppress the growth of winter jujube ring spot, apple stalk rot germ, cucumber fusarium axysporum germ, tomato seedling damping-off germ and cotton wilt germ, its substratum is Gause I substratum, consisting of of this substratum: Zulkovsky starch 20g; KNO
31g; K
2hPO
40.5g; MgSO
47H
2o 0.5g; NaCl 0.5g; FeSO
47H
2o 0.01g; Water 1000mL; PH 7.2 ~ 7.4.
3. streptomycete as claimed in claim 2 (
streptomycessp
.) application of BK CGMCC No.7489 bacterial strain, it is characterized in that, using the rear biocontrol effect to winter jujube ring spot can reach 80.3%; Detailed process is: carry out with Gause I substratum fermenting → fermented liquid is sprayed experimental plot → measures prevention effect.
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| CN104357369B (en) * | 2014-11-25 | 2017-10-13 | 辽宁大学 | Biological control streptomycete and its application |
| CN112746037B (en) * | 2020-12-16 | 2022-12-13 | 西南林业大学 | Streptomyces castochromogenes strain CPAT-W03 and application thereof |
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| CN1219876C (en) * | 2002-01-21 | 2005-09-21 | 云南省微生物研究所 | Streptomycete albogriseus spnov |
| CN101182483B (en) * | 2007-10-31 | 2011-05-25 | 浙江工业大学 | Streptomycete S1-5 and uses thereof |
| CN100560714C (en) * | 2007-11-19 | 2009-11-18 | 中国计量学院 | One biocontrol actinomyces-streptomyces diastatochromogenes D |
| CN101525587B (en) * | 2009-03-25 | 2011-06-01 | 中国农业大学 | Streptomyces strain and application thereof |
| CN102174440B (en) * | 2011-01-28 | 2013-01-16 | 广东省农业科学院植物保护研究所 | Streptomyces antibioticu and biological preparation prepared from active product thereof and application |
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