CN103497916B - Bacillus subtilis and application in preventing fusarium graminearum - Google Patents
Bacillus subtilis and application in preventing fusarium graminearum Download PDFInfo
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- CN103497916B CN103497916B CN201310438739.6A CN201310438739A CN103497916B CN 103497916 B CN103497916 B CN 103497916B CN 201310438739 A CN201310438739 A CN 201310438739A CN 103497916 B CN103497916 B CN 103497916B
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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 in control Fusarium graminearum thereof.
Background technology
Fusarium graminearum (Fusarium graminearum) is one of important pathogen of gramineous crop, Gibberella zeae (Sch.) Petch (Fusarium Head Blight can be caused, FHB), Fusarium graminearum is a principal element (Dubin, 1997) of restriction wheat yield.China's weather condition are extremely conducive to the generation of Gibberella zeae (Sch.) Petch, and the long-term disease in 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.Due to the impact that global warming and the cropping system such as straw-returning, no-tillage cultivation change, the generation area of China's Gibberella zeae (Sch.) Petch is rapidly to northwest, North China by the Yangtze valley and expands (Zhang Hao, 2011), particularly in 2008,2010 and 2012, whole nation Gibberella zeae (Sch.) Petch occurs very serious, cause serious product grain loss and bad social influence, 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 one of main natural toxin be present in gibberellic hypha infection 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 (Rochaet al., 2005; Sobrova et al., 2010; Pestka, 2010).DON extensively exists and a large amount of pollution wheat and goods thereof, forms very large harm, serious threat China food safety to people and animals.
Therefore, strengthen Fusarium graminearum prevention and control research and become the active demand ensureing China's grain security and food safety.
For many years mainly chemical pesticide is relied on to Gibberella zeae (Sch.) Petch control, but along with the environmental problem that chemical pesticide brings, in addition medication has for many years caused the appearance (Changjun Chen et al., 2007) of pathogenic bacteria resistance Fusarium graminearum bacterial strain, and difficulty of prevention and cure is strengthened.Although drop into a large amount of energy both at home and abroad to carry out correlative study in scab resistance quality, there is no desirable high resistance wheat breed (Li Zhenghui etc., 2007) so far.
Biological control is reduced environmental impact not only, also not easily develops immunity to drugs, and the research of correlation theory and technology is day by day by people are paid close attention to.Subtilis nutrition is simple, is widespread in nature, nontoxic to people and animals, free from environmental pollution, can multiple antibiotic and enzyme be produced, there is broad spectrum antibiotic activity and extremely strong anti-adversity ability, have more the potentiality being developed as biological prevention and control microbial inoculum compared with other microorganisms.
Summary of the invention
The object of this invention is to provide a bacillus subtilis and the application in control Fusarium graminearum thereof.
A bacillus subtilis provided by the invention (Bacillus subtilis), its deposit number is CGMCCNo.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.
Above-mentioned bacterial strains suppresses the application in the product of sickle-like bacteria also to belong to protection scope of the present invention in preparation.
In above-mentioned application, described sickle-like bacteria is Fusarium graminearum.
The method of preventing and treating the microbial Plant diseases of reaping hook also belongs to a protection scope of the present invention, and the method is in growing process, carry out spraying process; Described spraying is the bacteria suspension of 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 efficiently can suppress Fusarium graminearum when slat chain conveyor, and field plot experiment in its prevention effect up to 77.4%, be 2 times of derosal 1000 times of diluent prevention effect, namely prevention effect is obviously better than the current derosal generally used.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.
Accompanying drawing explanation
Fig. 1 is opposite culture experiment.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
NB liquid nutrient medium: be made up of solvent and solute; Solute is peptone, extractum carnis and NaCl, and solvent is water; The concentration of peptone in NB liquid nutrient medium is 1%, and the concentration of extractum carnis in NB liquid nutrient medium is the concentration of 0.3%, NaCl in 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 NB liquid nutrient medium, obtain NA solid medium.
PDA substratum: potato 200g, adds a small amount of poach 20min, and three layers of filtered through gauze, get filtrate water and be settled to 1L, add 20g glucose, 15g agar, 121 DEG C of autoclaving 20min.
Sweet mung bean soup substratum: mung bean 10g, adds a small amount of poach 20min, and three layers of filtered through gauze, get filtrate water and be settled to 1L, 121 DEG C of autoclaving 20min after packing.
Fusarium graminearum (Fusarium graminearum) FG1 document " Yang Huiyong; Li Feifeng; Lu Qiongxian; Xu Jianhong; Shi Jianrong. antagonistic strain AFR0406 is to the biological activity determination of fusarium graminearum and sheath blight fungus. Jiangsu's agriculture science; 2006 (06), 142-144. " in be 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 be disclosed, the public can obtain from Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sc.
The Isolation and ldentification of embodiment 1, bacterium
One, the separation of bacterium
In October, (one) 2011, in Bechtop, the sponge gourd flower pesticide that the suburb of Beijing gathers is placed on vibration in sterile distilled water and prepares bacteria suspension in 15 minutes, shaking speed is 180rpm.
(2) be coated on after bacteria suspension sterile distilled water being carried out concentration gradient dilution on NA culture medium flat plate, cultivate 24 hours under 30 DEG C of conditions, bacterium colony is covered with whole flat board, purify with form on transfering loop picking flat board, size, color, bacterial strain plate streaking that transparency is different, the bacterial strain after point connects purifying is applied to the experiment of Fusarium graminearum opposite culture.By the strain bacterium called after SG6 the strongest to 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) method described in, carry out the qualification of morphological specificity, cultural characters and physio-biochemical characteristics to bacterial strain SG6, concrete outcome is as follows:
The morphological specificity of thalline:
In 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 :-; Glucose fermentation aerogenesis :+; Utilize Citrate trianion :+; 50 DEG C of growths :+; PH5.7 grows :+; 7%NaCl grows :+; Starch Hydrolysis :+; Decompose casein :+; Nitrate reduction :+.
(2) 16S rDNA tests
Extract the STb gene of SG6, with it for template, utilize bacterial 16 S rDNA universal primer to carry out pcr amplification, obtain the amplified production that length is about 1.4kb, reclaimed by amplified production and check order, the sequence recorded is as shown in SEQ IDNo.1.
Compare according to Gen-Bank sequence homology, bacterial strain SG6 and Bacillus subtilis strain CICC10163 (GenBank accession number DQ005496.1) homology are 100%, be 100% with Bacillus amyloliquefaciens M20J (GenBank accession number AB735995.1) homology, this bacterium of preliminary judgement is bacillus (Bacillus sp.).
Based on above feature, bacterial strain SG6 is accredited as 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, subtilis SG6 are to the antagonistic action of Fusarium graminearum
Opposite culture method is adopted to detect subtilis SG6 to the antagonism of Fusarium graminearum.
One, be that the punch tool of 5mm is beaten at cultured pathogenic fungi Fusarium graminearum (Fusariumgraminearum) FG1 colony edge and got bacterium dish with diameter, with the toothpick of sterilizing, bacterium dish chosen into PDA plate center.
Two, at the SG6 apart from PDA plate center 25mm place four long and short dash line inoculation same volume and concentration, dull and stereotyped for control group with the PDA only inoculating Fusarium graminearum bacterium dish, cultivate 4-5 days for 28 DEG C, observe and formed with or without antibacterial band.Each group do three parallel.Result as shown in figure 1 and table 1.
In Fig. 1,1: control group; 2:SG6 group
Fig. 1 shows, SG6 has very strong inhibition to Fusarium graminearum.
Table 1 to cultivate after 5 days SG6 to the restraining effect of Fusarium graminearum
(antibacterial bandwidth refers to the shortest distance of pathogenic bacteria edge to antagonistic strain edge)
Embodiment 3, SG6 are to the field control of Fusarium graminearum
One, SG6 28 DEG C of shaken overnight in NB substratum cultivated, being diluted to concentration is 2.4 × 10
8cfu/ml.Spray at the blooming stage of new 828 kinds of stone, sprayed volume is 300ml, as experimental group.
Spray respectively at the blooming stage of new 828 kinds of wheat stone with 50% derosal, 1000 times of diluents and the NB diluent that do not connect bacterium, sprayed volume is 300ml, wherein not connect the NB diluent group of bacterium as a control group simultaneously.
Two, by Fusarium graminearum FG
1with sweet mung bean soup substratum shaking culture (28 DEG C, 5 days), obtain bacteria suspension, bacterial concentration is 2 × 10
5cfu/ml.
Three, after completing above-mentioned three groups of spraying process, carry out Fusarium graminearum bacteria suspension spray inoculation immediately, sprayed volume is 300ml, and be evenly sprayed on the community often organized, the plot area (i.e. field area) often organized is 2m
2.
Often group establishes 3 repetitions, results averaged.
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 calculates according to formula 2.
In formula, CK refers to NB diluent group, and process refers to SG6 group and derosal group.
Table 2SG6 is to the field control effect of Fusarium graminearum
Prevention effect is as shown in table 2, and result shows, subtilis SG6 can prevent and treat the wheat scab that Fusarium graminearum causes very well.
Claims (12)
1. a bacillus subtilis (
bacillus subtilis), its deposit number is CGMCC No.7621.
2. the application of bacterial strain according to claim 1 in the microbial Plant diseases of biological control reaping hook;
Described sickle-like bacteria is Fusarium graminearum.
3. application according to claim 2, is characterized in that: described disease is head blight or ear rot or root rot.
4. application according to claim 2, is characterized in that: described plant is cereal.
5. application according to claim 4, is characterized in that: described cereal is wheat crops or corn.
6. application according to claim 5, is characterized in that: described wheat crops is wheat.
7. bacterial strain according to claim 1 suppresses the application in the product of sickle-like bacteria in preparation;
Described sickle-like bacteria is Fusarium graminearum.
8. preventing and treating a method for the microbial Plant diseases of reaping hook, is in growing process, carry out spraying process;
Described spraying is bacteria suspension or the fermented liquid of subtilis according to claim 1;
Described sickle-like bacteria is Fusarium graminearum.
9. method according to claim 8, is characterized in that: described disease is head blight or ear rot or root rot.
10. method according to claim 8, is characterized in that: described plant is cereal.
11. methods according to claim 10, is characterized in that: described cereal is wheat crops or corn.
12. methods according to claim 11, is characterized in that: described wheat crops is wheat.
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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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| CN104195071A (en) * | 2014-08-06 | 2014-12-10 | 江苏农林职业技术学院 | Bacillus subtilis B33 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 |
| 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 |
| 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) |
| CN115851512B (en) * | 2022-10-21 | 2023-09-05 | 山东省花生研究所 | Bacillus subtilis subspecies in desert and application thereof |
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