CN103409342A - Biocontrol strain YKY01 for preventing and treating aromatic tobacco black shank and anti-biological inoculant of biocontrol strain YKY01 - Google Patents
Biocontrol strain YKY01 for preventing and treating aromatic tobacco black shank and anti-biological inoculant of biocontrol strain YKY01 Download PDFInfo
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Abstract
The invention discloses a biocontrol strain YKY 01 for preventing and treating aromatic tobacco black shank and an anti-biological inoculant of the biocontrol strain YKY01, belonging to the technical field of bio-pesticides. The preservation number of the strain YKY01 is CGMCC (China general Microbiological Culture Collection) NO.7723. The anti-biological inoculant is obtained by the following steps of transplanting the strain YKY01 to a culture dish for activating, and culturing for 72 hours at 33 DEG C; transferring the activated lawn into 300ml of NA culture solution which is contained in a 500ml triangular flask, culturing for 72 hours at the rotation speed of 175 revolutions/min under the temperature of 33 DEG C to obtain primary fermentation liquor; inoculating the primary fermentation liquor into the NA culture solution in a seed tank according to an inoculation amount proportion of 1:150, thereby obtaining strain liquor for secondary fermentation after fermenting for 48 hours with the temperature of the seed tank of 32 DEG C-35 DEG C; and inoculating the strain liquor for the secondary fermentation to a fermentation tank for fermentation according to the inoculation amount proportion of 1:15, fermenting for 120 hours at the temperature of 32 DEG C-35 DEG C, and storing the strain liquor as biocontrol liquor, centrifuging for 3 minutes at 1500 rpm, and preparing with special-purpose preservative fluid to form active fungicide, so that the total concentration of viable bacteria in the fungicide is above1*1011 FU/ml. According to the biocontrol strain YKY 01 for preventing and treating aromatic tobacco black shank and the anti-biological inoculant of the biocontrol strain YKY01, the cost is low, the successive cropping obstacle of the aromatic tobacco black shank can be effectively prevented, and the yield and the quality are improved.
Description
Technical field:
The present invention relates to a kind of biocontrol strain YKY01 and biocontrol fungicide thereof of preventing and treating the Turkish tobaccos balck shank, belong to biological pesticide technical field.
Background technology:
Turkish tobaccos claim again Turkey's cigarette, east type cigarette, originate in Mediterranean country, are a kind of special tobacco types of safflower tobacco (Nicotian atobacum).Because Turkish tobaccos have strong fragrance and pure jealous quality characteristic, special using value is arranged in tobacco product, be one of important source material of production mixed type, outer odor type and oriental type cigarette and pipe tobacco.Thereby especially blended type cigarette has the strength foot, flavor is dense, materials extensive, can effectively reduce tar content, alleviate the harm characteristics of smoking to HUMAN HEALTH, deeply be subjected to smoker's favor, become the popular cigarette type in the world today.At present, Chinese Turkish tobaccos mainly carry out scale production in Baoshan, Yunnan, Xinjiang, Zhejiang, Hubei.Yunnan Province, since 1988 formally carry out the Turkish tobaccos exploitation, through test, demonstration and the large-scale promotion plantation in 10 years 12 seasons, and has set up stable Turkish tobaccos production garden in Baoshan City, Nujiang Prefecture and Lincang City, Annual planting area is stabilized in 4000 hm at present
2Left and right, year purchase tobacco leaf 6000～6500T.
The Turkish tobaccos balck shank is one of a kind of important Turkish tobaccos destructive disease caused by black shank bacterium (Phytophtora parasitica var.nicotianae).This disease is distributed in the World tobacco growing area widely, also becomes one of greatest factor of restriction China Ge Yan district leaf tobacco production
[1-2].At present, more to the research report of flue-cured tobacco black shank bacterium in the research flue-cured tobacco cultivars both at home and abroad, about the relevant research relative deficiency of Turkish tobaccos balck shank
[3-6].The Baoshan, Yunnan city is the main Turkish tobaccos plantation producing region of China, in recent years, the Turkish tobaccos balck shank seriously occurs in the Turkish tobaccos planting base, from nursery stage, has just caused cigarette seedling loss in various degree in a lot of cigarettes district, gently cigarette seedling Quality Down, germ is brought to and transplants land for growing field crops, cause the tobacco leaf underproduction, heavy total destruction causes without seedling and can plant, seriously restricted the development that Turkish tobaccos are produced, the regional Turkish tobaccos planting bases such as while ShaoXing,ZheJiang occur also comparatively general
[6-8].
The havoc of chemical farming to farmland ecosystem, pathogenic bacteria is also more and more obvious to the resistance of medicament, and chemical pesticide is difficult for degraded simultaneously, and high residue has all seriously limited the application in agriculture production in the future to the uneasy congruence of people and animals.Therefore, non-chemically class prophylactico-therapeutic measures in the Turkish tobaccos organic farming, become the core mode of preventing and treating Turkish tobaccos balck shank and soil-borne disease at present, and biological control is the most promising method.
By literature search, have no the open report identical with the present invention.
Reference:
[1] Wang Zhifa, Liu Yanrong, Xie Chengsong, Zhang Guangmin, Xing Shuhua, ginger Zhu, Chen Ruitai. Shandong Province's Races of Phytophthora Parasitica Var. Nicotianae preliminary evaluation. plant protection journal, 1985,12 (1): 51-55.
[2] Wang Zhifa, Liu Yanrong, Xie Chengsong, Zhang Guangmin, Chen Ruitai. China's Identification of Races of Phytophthora Parasitica Var. Nicotianae. Journal of Shandong agri.Univ, 1987,18 (1): 1-8.
[3]?Abdul?Wajd?S?M,?Shenoi?MMand?Rao?S?V.?Performance?of?Flue?Cured?Tobacco?Line?FCH?6005?Against?Black?Shank?Disease?in?Nursery?and?Field.?Tob.?Res.,?1988,?14(?2?)?:?102-105.
[4] Zhang Zhulin. resisting tobacco black shank kind (being) test bulletin. tobacco science and technology, 1995 (1): 38-39.
[5] Gong Longying, Zheng Xiaobo, Liu Xiang, Geng Jiguo, Lu Jiayun. Phytophthora nicotianae is to the pathogenic of tobacco and Physiological Differentiation research. Agricultural University Of Nanjing's journal, 1993,16 (4): 68-72.
[6] Yang Jianqing, Jiang Tong, Chen Xueping, Huang Jianru. the disease resistance research to tobacco black shank bacterium of different oriental tobacco varieties and strain. the Chinese tobacco journal. 2001,7 (3): 34-36.
[7] Tang Xubing, Li Guangxi, Song Yuchuan, Qu Shengbin, Lan Yinghai, pay Rong, Yang Yanli. and Turkish tobaccos balck shank cause of disease is identified and the control Chemicals. Chinese tobacco journal, 2010,16 (2): 66-69
[8] Ma Deru. Practice of Oriental Tobacco in Yunnan. Kunming: Yunnan science and technology press, 1998.
Summary of the invention:
The object of the invention is to that difficulty is large in organic farming, cost is high for Turkish tobaccos balck shank control in current agriculture production, should not use the problem that chemical pesticide and pesticide residue are high, a kind of biocontrol strain YKY01 and biocontrol fungicide thereof of preventing and treating the Turkish tobaccos balck shank is provided.
The present invention, from Baoshan, Yunnan city Longyang District Turkish tobaccos growing area Turkish tobaccos rhizosphere soil, separating and obtain bacterial strain YKY01, shows that to its qualification result this bacterium belongs to bacillus amyloliquefaciens, this bacterial strain called after bacillus amyloliquefaciens (
Bacillus amyloliquefaciens) YKY01.This bacterial strain has been kept at China Committee for Culture Collection of Microorganisms's common micro-organisms center; Address: China. No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on June 15th, 2013; Preserving number: CGMCC NO.7723.
Thalline feature of the present invention and colony characteristics: thalline is rod-short, the tool mobility, and amphimicrobian, can form gemma, and sporangium is expanded, ovalize, Gram-positive.On the NA substratum, be shallow White-opalescent bacterium colony, surface is more coarse, and colony edge is irregular, does not produce pigment.
Biocontrol fungicide of the present invention obtains through following steps:
A. bacterial strain YKY01 is transplanted in culture dish and carries out a ring activation, cultivated 72 hours for 33 ℃; NA solid culture based formulas in culture dish is: peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, agar 15.0g, distilled water 1L, pH value 7.3;
B. getting a lawn immigration encircled after activating with the bottled 300ml formula of 500ml triangle is: peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, distilled water 1L, in the NA nutrient solution of pH value 7.3,175, turn/min, cultivated 72 hours under 33 ℃, obtain one grade fermemtation bacterium liquid;
C. one grade fermemtation bacterium liquid is inoculated in to fermentation culture in NA nutrient solution identical with the b step in seeding tank in the ratio that inoculum size 1:150 trains, 32～35 ℃ of the temperature of seeding tank, fermented after 48 hours, obtains the second order fermentation strain liquid;
D. second order fermentation is inoculated in to fermentation culture in NA nutrient solution identical with the b step in fermentor tank with strain liquid in inoculum size 1:15 ratio doubly, the temperature of fermentor tank is 32～35 ℃, fermented 120 hours, gemma content is more than or equal to 88% in the fermented liquid in fermentor tank, potential of hydrogen is 6.8～7.4 o'clock, stores as biocontrol microorganisms liquid;
E. by centrifugal 3 min of biocontrol microorganisms liquid 1500rpm, thalline and the special-purpose preservative fluid got after centrifugal are made into active microbial inoculum, and making viable bacteria total concn in finished product is 1 * 10
11More than CFU/ml; The special-purpose preservative fluid weight percent of thalline formula is: neusilin 0.5%, xanthan gum 0.12%, urea 3%, SP-2728(comb type macromolecule dispersing agent) 2.3%, distilled water 94.08%.
The present invention's equipment used is the general-purpose equipment of market sale.
The invention has the advantages that:
1, biocontrol fungicide making of the present invention easily and cost is low, nontoxic, harmless, non-environmental-pollution, can carry out the organic farming of Turkish tobaccos, and the ecosystem that protects the fields, implement agriculture Sustainable development; This microbial inoculum also has the growth-promoting production-increasing function, is conducive to improve the Turkish tobaccos planting soil, increases the microorganism active in soil.
2, dull and stereotyped antagonistic experiment shows: the YKY01 microbial inoculum has obvious restraining effect to Turkish tobaccos black shank bacterium and flue-cured tobacco related diseases fungal pathogens.
4, plot experiment result in greenhouse shows: the YKY01 microbial inoculum can effectively be controlled the Turkish tobaccos black shank bacterium to the infecting of Turkish tobaccos plant, and suppresses the growth of pathogenic bacteria mycelia, reaches and controls the effect that the balck shank state of an illness occurs.In addition, the YKY01 microbial inoculum can effectively promote the growth of Turkish tobaccos plant and improve its degeneration-resistant and disease resistance.
5, greenhouse test and land for growing field crops result show: the YKY01 microbial inoculum is 80～85% to Turkish tobaccos balck shank preventive effect, volume increase 10～15%.
Embodiment:
Embodiment equipment used is the general-purpose equipment of market sale.
Embodiment 1: the preparation of biocontrol fungicide
A. bacterial strain YKY01 is transplanted in culture dish and carries out a ring activation, cultivated 72 hours for 33 ℃; NA solid culture based formulas in culture dish is: peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, agar 15.0g, distilled water 1L, pH value 7.3;
B. getting a lawn immigration encircled after activating with the bottled 300ml formula of 500ml triangle is: peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, distilled water 1L, in the NA nutrient solution of pH value 7.3,175, turn/min, cultivated 72 hours under 33 ℃, obtain one grade fermemtation bacterium liquid;
C. one grade fermemtation bacterium liquid is inoculated in to fermentation culture in NA nutrient solution identical with the b step in seeding tank in the ratio that inoculum size 1:150 trains, 33 ℃ or 34 ℃ or 35 ℃ of the temperature of seeding tank, fermented after 48 hours, obtains the second order fermentation strain liquid;
D. second order fermentation is inoculated in to fermentation culture in NA nutrient solution identical with the b step in fermentor tank with strain liquid in inoculum size 1:15 ratio doubly, 33 ℃ or 34 ℃ or 35 ℃ of the temperature of seeding tank, fermented 120 hours, gemma content is more than or equal to 88% in the fermented liquid in fermentor tank, potential of hydrogen is 6.8 or 7.4 o'clock, stores as biocontrol microorganisms liquid;
E. by centrifugal 3 min of biocontrol microorganisms liquid 1500rpm, thalline and the special-purpose preservative fluid got after centrifugal are made into active microbial inoculum, and making viable bacteria total concn in finished product is 1 * 10
11More than CFU/ml; The special-purpose preservative fluid weight percent of thalline formula is:: neusilin 0.5%, xanthan gum 0.12%, urea 3%, SP-2728(comb type macromolecule dispersing agent) 2.3%, distilled water 94.08%.
Embodiment 2: dull and stereotyped antagonistic effect
According to the flat board in Fang Zhongda " planting the disease research method " to holding the culture experiment method, the YKY01 bacterium is estimated the inhibition of Turkish tobaccos black shank bacterium, when sterile purified water control treatment group germ mycelia was covered with flat board simultaneously, statistics was as follows, and YKY01 is shown in 91.50%(table 1 to Turkish tobaccos black shank bacterium growth inhibition ratio).Illustrate that the YKY01 bacterial strain has significant restraining effect to the Turkish tobaccos balck shank.
The restraining effect of table 1 YKY01 to Turkish tobaccos black shank bacterium and relevant germ
Embodiment 3: Turkish tobaccos balck shank greenhouse efficiency test
If three processing, processing 1 is the aqua sterilisa control group, and processing 2 is YKY01 microbial inoculum diluent, and processing 3 is mycocide derosal (Nanjing Chemical Industry Science Co., Ltd of doctor nation).The transplantation of seedlings of Turkish tobaccos floating disc enters in Turkish tobaccos black shank bacterium sick soil, with 30 times of liquid of YKY01 microbial inoculum 1 ︰, dips in root during transplanting, and transplant on Zhan Gen limit, limit; Crop is transplanted after 7 days and uses 30 times of bacterium liquid irrigating roots of YKY01 microbial inoculum 1 ︰, and every liter of liquid is filled with root 10 strain crops; Crop is transplanted after 90 days and uses 30 times of bacterium liquid irrigating roots of YKY01 microbial inoculum 1 ︰, and every liter of liquid is filled with root 10 strain crops.Other clear water contrasts and derosal pesticide-treated are with above-mentioned method and consumption.Every 667m
2Use the amount of said preparation, according to crop, transplant the calculating of strain number and determine.Result shows, the YKY01 microbial inoculum reaches 89.58% to the preventive effect of Turkish tobaccos balck shank in greenhouse test, higher than the agricultural chemicals control group.This microbial inoculum can promote the growth of Turkish tobaccos plant simultaneously, and the biomass rate of increase is shown in 14.03%(table 2).Transplant Turkish tobaccos floating disc seedling plant on November 15th, 2012, investigate in May, 2013, investigate the growth of Turkish tobaccos and the situation that disease occurs.
The greenhouse test result of table 2 YKY01 to the Turkish tobaccos black shank bacterium
Embodiment 4: Turkish tobaccos balck shank field efficiency test
If 3 processing, processing 1 is the aqua sterilisa control group, and processing 2 is YKY01 microbial inoculum diluent, and processing 3 is mycocide derosal (Nanjing Chemical Industry Science Co., Ltd of doctor nation).The transplantation of seedlings of Turkish tobaccos floating disc enters in the sick soil of Turkish tobaccos black shank bacterium field, with 30 times of liquid of YKY01 microbial inoculum 1 ︰, dips in root during transplanting, and transplant on Zhan Gen limit, limit; Crop is transplanted after 7 days and uses 30 times of bacterium liquid irrigating roots of YKY01 microbial inoculum 1 ︰, and every liter of liquid is filled with root 10 strain crops; Crop is transplanted after 90 days and uses 30 times of bacterium liquid irrigating roots of YKY01 microbial inoculum 1 ︰, and every liter of liquid is filled with root 10 strain crops.Other clear water contrasts and derosal pesticide-treated are with above-mentioned method and consumption.Each processing is established 3 communities and is repeated, and each community area is 150m
2, each processes the community random alignment.Result shows, the YKY01 microbial inoculum reaches 85.36% to the preventive effect of Turkish tobaccos balck shank in field plot trial.This microbial inoculum can promote the growth of Turkish tobaccos plant simultaneously, and the biomass rate of increase is shown in 10.78%(table 3).Transplant annual Turkish tobaccos floating disc seedling plant on November 15th, 2012, investigate in May, 2013, investigate the growth of Turkish tobaccos and the situation that disease occurs.
The field sick soil test-results of table 3 YKY01 to the Turkish tobaccos black shank bacterium
In above all embodiment, YKY01 microbial inoculum diluent should all the time>=1 * 10
9The bacillus amyloliquefaciens quantity that individual/g is above.
The test-results of embodiment all shows, YKY01 microbial inoculum of the present invention is better for the prevention effect of Turkish tobaccos balck shank evil, obviously reduced the sickness rate of Turkish tobaccos balck shanks.And for growing of Turkish tobaccos stronger promoter action, economic benefit and obvious environment benefit.
Claims (2)
1. biocontrol strain YKY01 who prevents and treats the Turkish tobaccos balck shank, for bacillus amyloliquefaciens (Bacillus amyloliquefaciens), be preserved in China Microbial Culture Preservation Commission's common micro-organisms center, preserving number: CGMCC NO.7723 on June 15th, 2013.
2. the biocontrol fungicide of the control Turkish tobaccos balck shank prepared with biocontrol strain YKY01 claimed in claim 1 is characterized in that this biocontrol fungicide obtains through following steps:
A. bacterial strain YKY01 is transplanted in culture dish and carries out a ring activation, cultivated 72 hours for 33 ℃; NA solid culture based formulas in culture dish is: peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, agar 15.0g, distilled water 1L, pH value 7.3;
B. getting a lawn immigration encircled after activating with the bottled 300ml formula of 500ml triangle is: peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, distilled water 1L, in the NA nutrient solution of pH value 7.3,175, turn/min, cultivated 72 hours under 33 ℃, obtain one grade fermemtation bacterium liquid;
C. one grade fermemtation bacterium liquid is inoculated in to fermentation culture in NA nutrient solution identical with the b step in seeding tank in the ratio that inoculum size 1:150 trains, 32～35 ℃ of the temperature of seeding tank, fermented after 48 hours, obtains the second order fermentation strain liquid;
D. second order fermentation is inoculated in to fermentation culture in NA nutrient solution identical with the b step in fermentor tank with strain liquid in inoculum size 1:15 ratio doubly, the temperature of fermentor tank is 32～35 ℃, fermented 120 hours, gemma content is more than or equal to 88% in the fermented liquid in fermentor tank, potential of hydrogen is 6.8～7.4 o'clock, stores as biocontrol microorganisms liquid;
E. by centrifugal 3 min of biocontrol microorganisms liquid 1500rpm, thalline and the special-purpose preservative fluid got after centrifugal are made into active microbial inoculum, and making viable bacteria total concn in finished product is 1 * 10
11More than CFU/ml; The special-purpose preservative fluid weight percent of thalline formula is: neusilin 0.5%, xanthan gum 0.12%, urea 3%, SP-2728 comb type macromolecule dispersing agent 2.3%, distilled water 94.08%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232528A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus amyloliquefaciens preparation |
| NL2033340B1 (en) * | 2022-10-18 | 2023-06-19 | Yunnan Tobacco Company Kunming Branch | Trichosporon asahii and application thereof |
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| US6960342B2 (en) * | 2002-09-12 | 2005-11-01 | National Taiwan University | Method for inhibiting pathogenic fungi in plants using Bacillus amyloliquefaciens |
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| CN104232528A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus amyloliquefaciens preparation |
| NL2033340B1 (en) * | 2022-10-18 | 2023-06-19 | Yunnan Tobacco Company Kunming Branch | Trichosporon asahii and application thereof |
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