CN103497916A - Bacillus subtilis and application in preventing fusarium graminearum - Google Patents
Bacillus subtilis and application in preventing fusarium graminearum Download PDFInfo
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- CN103497916A CN103497916A CN201310438739.6A CN201310438739A CN103497916A CN 103497916 A CN103497916 A CN 103497916A CN 201310438739 A CN201310438739 A CN 201310438739A CN 103497916 A CN103497916 A CN 103497916A
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Abstract
The invention discloses bacillus subtilis and an application in preventing fusarium graminearum. The preservation number of the bacillus subtilis disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No.7621. As a biological prevention material against wheat scab, the bacillus subtilis has a good application prospect in developing a new biological pesticide or biological control bacterial agent.
Description
Technical field
The present invention relates to a bacillus subtilis and the application aspect the control Fusarium graminearum thereof.
Background technology
Fusarium graminearum (Fusarium graminearum) is one of important pathogen of gramineous crop, can cause Gibberella zeae (Sch.) Petch (Fusarium Head Blight, FHB), Fusarium graminearum is a principal element (Dubin, 1997) of restriction wheat yield.China's weather condition extremely are conducive to the generation of Gibberella zeae (Sch.) Petch, and the long-term disease of In Middle And Lower Reaches of Changjiang River causes production loss 10%-15%, nearly 50% (Huang Chang etc., 2000) of the time underproduction of being very popular.The impact changed due to cropping systems such as global warming and straw-returning, no-tillage cultivations, the generation area of China's Gibberella zeae (Sch.) Petch by the Yangtze valley rapidly northwestwards, North China expansion (Zhang Hao, 2011), particularly in 2008,2010 and 2012, whole nation Gibberella zeae (Sch.) Petch occurs very serious, the serious loss of product grain and bad social influence have been caused, serious threat China grain security.
The deoxynivalenol (DON, mould-9 alkene-8 ketone of the single-ended spore of 3,7,15 trihydroxy--12,13 epoxy) that Fusarium graminearum produces is to be present in gibberellic hypha to infect one of main natural toxin in wheat.DON has been identified as one of the most dangerous naturally-occurring contaminants in food, is put into the precedence (Larsen et al., 2004) of international research.DON can cause people's immunity degradation, anaemia, headache, stomachache, feel sick; Activate thing food refusal, growth retardation and miscarriage (Rocha et al., 2005; Sobrova et al., 2010; Pestka, 2010).DON extensively exists and a large amount of wheat and goods thereof of polluting, and people and animals are formed to very large harm, serious threat China food safety.
Therefore, strengthen Fusarium graminearum prevention and control research and become the active demand that ensures China's grain security and food safety.
For many years to the main chemical pesticide that relies on of Gibberella zeae (Sch.) Petch control, but the environmental problem of bringing along with chemical pesticide, medication has for many years caused the appearance (Changjun Chen et al., 2007) of pathogenic bacteria resistance Fusarium graminearum bacterial strain in addition, and difficulty of prevention and cure is strengthened.Although carry out correlative study at a large amount of energy of domestic and international input aspect the scab resistance quality, there is no so far desirable high resistance wheat breed (Li Zhenghui etc., 2007).
Biological control is reduced environmental impact not only, also is difficult for developing immunity to drugs, and the research of correlation theory and technology is paid close attention to by people day by day.Subtilis nutrition is simple, is widespread in nature, nontoxic to people and animals, free from environmental pollution, can produce multiple antibiotic and enzyme, there is broad spectrum antibiotic activity and extremely strong anti-adversity ability, have more the potentiality that are developed as biological prevention and control microbial inoculum than other microorganisms.
Summary of the invention
The purpose of this invention is to provide a bacillus subtilis and the application aspect the control Fusarium graminearum thereof.
A bacillus subtilis provided by the invention (Bacillus subtilis), its deposit number is CGMCC No.7621.
The application of above-mentioned bacterial strains in the microbial Plant diseases of biological control reaping hook also belongs to protection scope of the present invention.
In above-mentioned application, described sickle-like bacteria is Fusarium graminearum.
In above-mentioned arbitrary described application, described disease is head blight or ear rot or root rot.
In above-mentioned arbitrary described application, described plant is cereal; Described cereal is wheat crops or corn; Described wheat crops is specially wheat.
The application of above-mentioned bacterial strains in the product of preparation inhibition sickle-like bacteria also belongs to protection scope of the present invention.
In above-mentioned application, described sickle-like bacteria is Fusarium graminearum.
A kind of method of preventing and treating the microbial Plant diseases of reaping hook also belongs to protection scope of the present invention, and the method is the processing of spraying in growing process; The bacteria suspension that described spraying is above-mentioned subtilis or fermented liquid or meta-bolites.
In aforesaid method, described process of growth is blooming stage.
In above-mentioned arbitrary described method, described sickle-like bacteria is Fusarium graminearum.
In above-mentioned arbitrary described method, described disease is head blight or ear rot or root rot.
In above-mentioned arbitrary described method, described plant is cereal; Described cereal is wheat crops or corn; Described wheat crops is specially wheat.
Subtilis provided by the invention not only can efficiently suppress Fusarium graminearum when flat board is cultivated, and in community, field experiment its prevention effect up to 77.4%, be 2 times of 1000 times of diluent prevention effect of derosal, prevention effect obviously is better than the derosal of generally using at present.As the biological control material of wheat scab, no matter develop new biological pesticide or biological control microbial inoculum, this bacterium all has good application prospect.
The accompanying drawing explanation
Fig. 1 is the face-off culture experiment.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
NB liquid nutrient medium: formed by solvent and solute; Solute is peptone, extractum carnis and NaCl, and solvent is water; The concentration of peptone in the NB liquid nutrient medium is 1%, and the concentration of extractum carnis in the NB liquid nutrient medium is that the concentration of 0.3%, NaCl in the NB liquid nutrient medium is 0.5%, and described % is quality volume percent (g/100ml).
NA solid medium: add agar (ratio of agar and liquid nutrient medium is 1.5g:100ml) in the NB liquid nutrient medium, obtain the NA solid medium.
The PDA substratum: potato 200g, add a small amount of poach 20min, three layers of filtered through gauze, get filtrate water and be settled to 1L, adds 20g glucose, 15g agar, 121 ℃ of autoclaving 20min.
The sweet mung bean soup substratum: mung bean 10g, add a small amount of poach 20min, three layers of filtered through gauze, get filtrate water and be settled to 1L, 121 ℃ of autoclaving 20min after packing.
Fusarium graminearum (Fusarium graminearum) FG1 document " Yang Huiyong; Li Feifeng; Lu Qiongxian; Xu Jianhong; Shi Jianrong. the biological activity determination of antagonistic strain AFR0406 to fusarium graminearum and sheath blight fungus. Jiangsu agricultural sciences; 2006 (06), 142-144. " in disclosed, the public can obtain from Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sc.
New 828 kinds of wheat stone document " Sun Zhijun, Yang Xiaoli, Zhang Hui; Zhang Guanghui. new 828 Breeding Process of new variety of wheat stone and high-yield culture technique. modern agriculture science and technology; 2011(09), 96-97. " in disclosed, the public can obtain from Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sc.
The separation of embodiment 1, bacterium and evaluation
One, the separation of bacterium
In October, (one) 2011, in Bechtop, the sponge gourd flower pesticide that the suburb of Beijing is gathered is placed on vibration in sterile distilled water and within 15 minutes, prepares bacteria suspension, and shaking speed is 180rpm.
(2) bacteria suspension is carried out being coated on the NA culture medium flat plate after the concentration gradient dilution with sterile distilled water, under 30 ℃ of conditions, cultivate 24 hours, bacterium colony is covered with whole flat board, with form on transfering loop picking flat board, size, color, bacterial strain plate streaking that transparency is different, purify, the bacterial strain that point connects after purifying is applied to Fusarium graminearum face-off culture experiment.By the strongest strain bacterium called after SG6 to the Fusarium graminearum antagonistic ability obtained.
Two, identify
(1) according to " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) and " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying etc. write, Beijing: Science Press, 2001.2) the middle method of describing, bacterial strain SG6 is carried out to morphological specificity, cultural characters and physio-biochemical characteristics and identify, concrete outcome is as follows:
The morphological specificity of thalline:
Be shaft-like, peritrichous, Gram-positive.
Cultural characters:
Bacterium colony is dirty white or micro-yellow, and surface irregularity is opaque, and bacterium colony is soft and moistening, homogeneous.
Physio-biochemical characteristics:
Glucose :+; Wood sugar :+; L-arabinose :+; N.F,USP MANNITOL :+; Lactose :-; The glucose fermentation aerogenesis :+; Utilize Citrate trianion :+; 50 ℃ of growths :+; The pH5.7 growth :+; The 7%NaCl growth :+; Starch Hydrolysis :+; Decompose casein :+; Nitrate reduction :+.
(2) 16S rDNA test
Extract total DNA of SG6, take it as template, utilize bacterial 16 S rDNA universal primer to carry out pcr amplification, obtain the amplified production that length is about 1.4kb, amplified production is reclaimed and checked order, the sequence recorded is as shown in SEQ ID No.1.
According to the Gen-Bank sequence homology relatively, bacterial strain SG6 and Bacillus subtilis strain CICC10163 (GenBank accession number DQ005496.1) homology are 100%, with Bacillus amyloliquefaciens M20J (GenBank accession number AB735995.1) homology be 100%, this bacterium of preliminary judgement is bacillus (Bacillus sp.).
Based on above feature, bacterial strain SG6 is accredited as to subtilis (Bacillus subtilis).This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 15th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.7621.
Embodiment 2, the subtilis SG6 antagonistic action to Fusarium graminearum
Adopt the face-off culture method to detect the antagonism of subtilis SG6 to Fusarium graminearum.
One, the punch tool that is 5mm with diameter is beaten and is got the bacterium dish at cultured pathogenic fungi Fusarium graminearum (Fusarium graminearum) FG1 colony edge, with the toothpick of sterilizing, the bacterium dish is chosen to the dull and stereotyped center into PDA.
Two, at the SG6 of 25mm place, the dull and stereotyped center of distance PDA four long and short dash line inoculation equal volume and concentration, the PDA flat board of only inoculating Fusarium graminearum bacterium dish of take is control group, cultivates 4-5 days for 28 ℃, observes and has or not antibacterial band to form.Each group do three parallel.Result is as shown in Fig. 1 and table 1.
In Fig. 1,1: control group; The 2:SG6 group
Fig. 1 shows, SG6 has very strong inhibition to Fusarium graminearum.
Table 1 is cultivated the restraining effect of SG6 to Fusarium graminearum after 5 days
(antibacterial bandwidth refers to the shortest distance of pathogenic bacteria edge to the antagonistic strain edge)
Embodiment 3, the SG6 field control to Fusarium graminearum
One, SG6 28 ℃ of shaken overnight in the NB substratum are cultivated, being diluted to concentration is 2.4 * 10
8cfu/ml.Blooming stage in new 828 kinds of stone is sprayed, and sprayed volume is 300ml, as experimental group.
With 1000 times of diluents of 50% derosal and the NB diluent that do not connect bacterium, at the blooming stage of new 828 kinds of wheat stone, sprayed respectively, sprayed volume is 300ml simultaneously, wherein with the NB diluent group that do not connect bacterium as a control group.
Two, by Fusarium graminearum FG
1with sweet mung bean soup substratum shaking culture (28 ℃, 5 days), obtain bacteria suspension, bacterial concentration is 2 * 10
5cfu/ml.
Three, after completing above-mentioned three groups of spraying processing, carry out immediately Fusarium graminearum bacteria suspension spray inoculation, sprayed volume is 300ml, evenly is sprayed on the Mei Zu community, and Mei Zu community area (being the field area) is 2m
2.
Establish 3 repetitions, results averaged for every group.
Fusarium graminearum bacteria suspension spray inoculation is after 21 days, observes the morbidity of the wheat head, has that the wheat head is withered to turn white or rubescent, and the wheat head of inner hollow kernels, weight saving performance is the morbidity wheat head.
The disease index of each group calculates according to formula 1.
The prevention effect of SG6 group and derosal group is calculated according to formula 2.
In formula, CK refers to NB diluent group, processes and refers to SG6 group and derosal group.
The field control effect of table 2SG6 to Fusarium graminearum
Prevention effect is as shown in table 2, and result shows, the wheat scab that the fine control of subtilis SG6 energy Fusarium graminearum causes.
Claims (10)
1. a bacillus subtilis (Bacillus subtilis), its deposit number is CGMCC No.7621.
2. the application of bacterial strain claimed in claim 1 in the microbial Plant diseases of biological control reaping hook.
3. application according to claim 2 is characterized in that: described sickle-like bacteria is Fusarium graminearum.
4. according to the described application of claim 2 or 3, it is characterized in that: described disease is head blight or ear rot or root rot.
5. according to the arbitrary described application of claim 2-4, it is characterized in that: described plant is cereal; Described cereal is wheat crops or corn; Described wheat crops is specially wheat.
6. the application of bacterial strain claimed in claim 1 in the product of preparation inhibition sickle-like bacteria.
7. a method of preventing and treating the microbial Plant diseases of reaping hook, be the processing of spraying in growing process; The bacteria suspension that described spraying is subtilis claimed in claim 1 or fermented liquid or meta-bolites.
8. method according to claim 7, it is characterized in that: described sickle-like bacteria is Fusarium graminearum.
9. according to the described method of claim 7 or 8, it is characterized in that: described disease is head blight or ear rot or root rot.
10. according to the arbitrary described method of claim 7-9, it is characterized in that: described plant is cereal; Described cereal is wheat crops or corn; Described wheat crops is specially wheat.
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| CN104651319A (en) * | 2015-01-27 | 2015-05-27 | 中国农业科学院植物保护研究所 | Fusarium graminearum low-toxicity virus FgHV2/JS16 and application thereof |
| CN104877932A (en) * | 2015-05-05 | 2015-09-02 | 江苏省农业科学院 | Paenibacillus polymyxa and application thereof |
| CN110200014A (en) * | 2019-05-17 | 2019-09-06 | 河南省农业科学院植物保护研究所 | The microorganism formulation and its preparation method and application for preventing and treating fusarium disease |
| CN111560327A (en) * | 2020-04-28 | 2020-08-21 | 湖北大学 | Preparation and application of alcaligenes faecalis capable of antagonizing fusarium graminearum and efficiently degrading DON (Don) |
| CN115851512A (en) * | 2022-10-21 | 2023-03-28 | 山东省花生研究所 | Bacillus subtilis subspecies deserticola and application thereof |
| CN116836856A (en) * | 2023-06-26 | 2023-10-03 | 上海市农业科学院 | Bacillus subtilis, pathogenic bacteria antagonist and application thereof |
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| CN106635929A (en) * | 2017-02-14 | 2017-05-10 | 河南工业大学 | Bacillus subtilis strain and application of antifungal metabolite in grain storage |
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Cited By (12)
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| CN104195071A (en) * | 2014-08-06 | 2014-12-10 | 江苏农林职业技术学院 | Bacillus subtilis B33 and application thereof |
| CN104651319A (en) * | 2015-01-27 | 2015-05-27 | 中国农业科学院植物保护研究所 | Fusarium graminearum low-toxicity virus FgHV2/JS16 and application thereof |
| CN104651319B (en) * | 2015-01-27 | 2017-05-24 | 中国农业科学院植物保护研究所 | Fusarium graminearum low-toxicity virus FgHV2/JS16 and application thereof |
| CN104877932A (en) * | 2015-05-05 | 2015-09-02 | 江苏省农业科学院 | Paenibacillus polymyxa and application thereof |
| CN104877932B (en) * | 2015-05-05 | 2018-04-20 | 江苏省农业科学院 | One plant of Paenibacillus polymyxa and its application |
| CN110200014A (en) * | 2019-05-17 | 2019-09-06 | 河南省农业科学院植物保护研究所 | The microorganism formulation and its preparation method and application for preventing and treating fusarium disease |
| CN111560327A (en) * | 2020-04-28 | 2020-08-21 | 湖北大学 | Preparation and application of alcaligenes faecalis capable of antagonizing fusarium graminearum and efficiently degrading DON (Don) |
| CN111560327B (en) * | 2020-04-28 | 2021-12-10 | 湖北大学 | Preparation and application of alcaligenes faecalis capable of antagonizing fusarium graminearum and efficiently degrading DON (Don) |
| CN115851512A (en) * | 2022-10-21 | 2023-03-28 | 山东省花生研究所 | Bacillus subtilis subspecies deserticola and application thereof |
| CN115851512B (en) * | 2022-10-21 | 2023-09-05 | 山东省花生研究所 | Bacillus subtilis subspecies in desert and application thereof |
| CN116836856A (en) * | 2023-06-26 | 2023-10-03 | 上海市农业科学院 | Bacillus subtilis, pathogenic bacteria antagonist and application thereof |
| CN116836856B (en) * | 2023-06-26 | 2024-06-04 | 上海市农业科学院 | Bacillus subtilis, pathogenic bacteria antagonist and application thereof |
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