CN104403962B - Application in terms of one plant of Te Jila bacillus and its prevention and treatment Fusarium graminearum - Google Patents
Application in terms of one plant of Te Jila bacillus and its prevention and treatment Fusarium graminearum Download PDFInfo
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Abstract
The invention discloses the applications in terms of one plant of Te Jila bacillus and its prevention and treatment Fusarium graminearum.One plant of Te Jila bacillus (Bacillus tequilensis) disclosed by the invention, deposit number are CGMCC No.9499.As the biological control material of wheat scab, new biological pesticide or biological control microbial inoculum no matter are developed, which all has a good application prospect.
Description
Technical field
The present invention relates to Te Jila bacillus, in particular to one plant of Te Jila bacillus and its prevention and treatment Fusarium graminearum
The application of aspect.
Background technique
Fusarium graminearum (Fusarium graminearum) is one of important pathogen of gramineous crop, can cause wheat
Bakanae disease (Fusarium Head Blight, FHB), Fusarium graminearum are a principal element for limiting wheat yield
(Dubin,1997).China's weather conditions are advantageous to the generation of gibberella zeaze petch of wheat and barley, and the long-term disease in In Middle And Lower Reaches of Changjiang River is made
At production loss 10%-15%, be very popular the time underproduction nearly 50% (Huang Chang etc., 2000).Due to global warming and straw
The influence that the cropping systems such as stalk returning to the field, no-tillage cultivation change, the generation area of China's gibberella zeaze petch of wheat and barley from the Yangtze river basin rapidly to
Northwest, North China extension (Zhang Hao, 2011), especially in 2008,2010 and 2012, national gibberella zeaze petch of wheat and barley occurs very tight
Weight causes serious production grain loss and undesirable social influence, seriously threatens China's grain security.
Deoxynivalenol (DON, 3,7, the 15 single-ended spores of trihydroxy -12,13 epoxy that Fusarium graminearum generates
Mould -8 ketone of -9 alkene) it is one of the main natural toxin being present in gibberellic hypha infection wheat.DON has been identified as most dangerous
One of naturally-occurring food contaminant is put into the precedence (Larsen et al., 2004) of international research.DON can cause people
Immunity degradation, anaemia, headache, abdominal pain, nausea；Activate object food refusal, growth retardation and miscarriage (Rocha et al., 2005；
Sobrova et al.,2010；Pestka,2010).DON is widely present and largely pollutes wheat and its product, constitutes to people and animals
Very big harm, seriously threatens China's food safety.
Therefore, reinforcement Fusarium graminearum prevention and control research, which has become, ensures that China's grain security and the urgent of food safety are essential
It asks.
Chemical pesticide relied primarily on to gibberella zeaze petch of wheat and barley prevention and treatment for many years, but with chemical pesticide bring environmental problem,
In addition many years medication caused pathogen drug resistance Fusarium graminearum bacterial strain appearance (Changjun Chen et al.,
2007), difficulty of prevention and cure is increased.It is ground although putting into a large amount of energy both at home and abroad in terms of scab resistance quality and carrying out correlation
Study carefully, but there is no ideal highly resistance wheat breed (Li Zhenghui etc., 2007) so far.
Biological control not only reduced environmental impact, is also less prone to develop drug resistance, the research of correlation theory and technology
It is increasingly concerned.Te Jila bacillus nutrition is simple, is widespread in nature, nontoxic to people and animals, no
Environment is polluted, a variety of antibiotic and enzyme can be generated, there is broad spectrum antibiotic activity and extremely strong anti-adversity ability, more compared with other microorganisms
It is the potentiality of biological prevention and control microbial inoculum with exploitation.
Summary of the invention
To solve the above-mentioned problems of the prior art, the purpose of the present invention is to provide one plant of Te Jila bacillus and
It prevents and treats the application in terms of Fusarium graminearum.
In order to achieve the above objectives, the technical solution of the present invention is as follows:
One plant of Te Jila bacillus (Bacillus tequilensis), deposit number are CGMCC No.9499.
Application of the above-mentioned bacterial strains in the plant disease caused by biological control sickle-like bacteria also belongs to protection scope of the present invention.
In above-mentioned application, the sickle-like bacteria is Fusarium graminearum.
In any of the above-described application, the disease is head blight or ear rot or root rot caused by sickle-like bacteria.
In any of the above-described application, the plant is cereal；The cereal is wheat crops or corn；The wheat
Crop is specially wheat.
Application of the above-mentioned bacterial strains in the product that preparation inhibits sickle-like bacteria also belongs to protection scope of the present invention.
A kind of method of plant disease caused by prevention and treatment sickle-like bacteria also belongs to protection scope of the present invention, and this method is to plant
Spraying treatment is carried out in object growth course；It is described to be produced by spraying for the bacteria suspension of above-mentioned Te Jila bacillus or fermentation liquid or metabolism
Object.
In the above method, the sickle-like bacteria is Fusarium graminearum.
In any of the above-described method, the disease is head blight or ear rot or root rot.
In any of the above-described method, the plant is cereal；The cereal is wheat crops or corn；The wheat
Crop is specially wheat.
Compared with the existing technology, the invention has the benefit that Te Jila bacillus provided by the invention is not only flat
Fusarium graminearum can efficiently be inhibited when plate culture, and its control efficiency is up to 77.4% in greenhouse experiments.It is red as wheat
No matter the biological control material of mildew, develop new biological pesticide or biological control microbial inoculum, which all has good application
Prospect.
Detailed description of the invention
Fig. 1 is opposite culture experiment.
Fig. 2 is escaping gas bacteriostatic experiment.
Specific embodiment
Technical solution of the present invention is elaborated further with reference to the accompanying drawings and detailed description:
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
NB fluid nutrient medium: it is made of solvent and solute；Solute is peptone, beef extract and NaCl, and solvent is water；Albumen
Concentration of the peptone in NB fluid nutrient medium is 1%, and concentration of the beef extract in NB fluid nutrient medium is 0.3%, NaCl in NB liquid
Concentration in body culture medium is 0.5%, and the % is quality percent by volume (g/100ml).
NA solid medium: in NB fluid nutrient medium be added agar (ratio of agar and fluid nutrient medium be 1.5g:
100ml), NA solid medium is obtained.
PDA culture medium: a small amount of boiling 20min is added in potato 200g, and three layers of filtered through gauze take filtrate water to be settled to
20g glucose, 15g agar, 121 DEG C of high pressure sterilization 20min are added in 1L.
Sweet mung bean soup culture medium: a small amount of boiling 20min is added in mung bean 10g, and three layers of filtered through gauze take filtrate water to be settled to
1L, 121 DEG C of high pressure sterilization 20min after packing.
Phosphate buffer: it is made of solvent and solute；Solute is potassium dihydrogen phosphate and magnesium chloride, and solvent is water；Phosphoric acid
The concentration of potassium dihydrogen is 0.004%, and the concentration of magnesium chloride is 0.019%, and the % is quality percent by volume (g/
100ml)。
Fusarium graminearum (Fusarium graminearum) FG1 is in document " Yang Huiyong, Li Feifeng, Lu Qiongxian, Xu Jian
Macro, Shi Jianrong antagonistic strain AFR0406 is scientific to the biological activity determination Jiangsu's agriculture of fusarium graminearum and sheath blight fungus,
2006 (06) are disclosed in 142-144. ", and the public can obtain from Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sc.
New 828 kind of wheat stone is in document " new 828 breeding of Sun Zhijun, Yang Xiaoli, Zhang Hui, Zhang Guanghui new variety of wheat stone
Process and high-yield culture technique modern agriculture are scientific and technological, 2011 (09), are disclosed in 96-97. ", the public can be from Scientia Agricultura Sinica
Processing of farm products research institute, institute obtains.
The separation and identification of embodiment 1, bacterial strain
One, the separation of bacterial strain
The wheat disease fringe that 10 fringe Jiangsu acquire is placed on the sterile steaming of 100ml in superclean bench by June, (one) 2012
It is shaken 15 minutes in distilled water and prepares bacteria suspension, then 80 DEG C water bath processing 10 minutes.
(2) it is coated on after bacteria suspension being carried out concentration gradient dilution with sterile distilled water on NA culture medium flat plate, 30 DEG C of items
It is cultivated 24 hours under part, bacterium colony is covered with entire plate, different with form, size, color, transparency on oese picking plate
Bacterial strain flat board scribing line purifying, point connect bacterial strain after purification and test applied to Fusarium graminearum opposite culture, as a result as shown in Figure 1.
The obtained one plant bacterium strong to Fusarium graminearum antagonistic ability is named as JS5L.
Two, it identifies
(1) according to " Berger bacterial identification manual " (the 8th edition) and " common bacteria system identification handbook " (east show pearl, Cai
Wonderful English etc. is write, Beijing: Science Press, 2001.2) it is special to carry out morphological feature, culture to bacterial strain JS5L for method described in
Property and physio-biochemical characteristics identification, concrete outcome is as follows:
The form and physio-biochemical characteristics of thallus:
In NA culture medium, 28 DEG C are cultivated 1 day, and thallus is rod-shaped, and diameter is about 2.0-2.5 μm of 0.6 μ m；Bacterium colony is yellowish,
Circle, smoothly, neat in edge.
Physiological and biochemical property:
Oxidizing ferment :+；Arginine dihydrolase :+；Gelatin hydrolysis :+；Starch Hydrolysis :+；Catalase :+；Three butyric acid are sweet
Grease hydrolysis :-；Nitrate reduction :+；Aesculin hydrolysis :+；Casein hydrolysis :+；Tween80 hydrolysis :+；Indoles experiment :-；Urea
Enzyme :-；Citric acid utilizes :+；H2S is generated :-；VP experiment :+；Anaerobic growth :-；50 DEG C :+；
20 DEG C :-；PH4.0:-；PH9.0:-.
It can use glycerol, D-arabinose, L-arabinose, D-ribose, D- xylose, L- xylose, adonite, D- gala
Sugar, D-Glucose, D-Fructose, D-MANNOSE, L- sorbose, L- rhamnose, inositol, mannitol, sorbierite, the Portugal Alpha-Methyl-D-
Polyglycoside, N-acetyl-glucosamine, aesculin, salicin, D- cellobiose, D-Maltose, D- lactose, D- melibiose, D- sucrose,
D- trehalose, D- raffinose, starch, xylitol, D- gentiobiose, the Toulon D- sugar, D- lyxose, D-Tag, D- fucose,
L-fucose, D-arabitol, L- arabite produce acid.Acid is produced using dulcitol, amarogentin, arbutin, synanthrin and glycogen
Ability is poor.
(2) 16S rDNA is tested
The total DNA for extracting JS5L carries out PCR amplification using bacterial 16 S rDNA universal primer, is grown using it as template
Degree is about the amplified production of 1.4kb, and amplified production is recycled and is sequenced, the sequence measured is as shown in SEQ ID No.1.
Compared according to Gen-Bank sequence homology, bacterial strain JS5L and Bacillus sp.LB159 (GenBank accession number
KJ564121.1) homology is 99%, is with Bacillus sp.JX2 (GenBank accession number KF860138.1) homology
99%, the preliminary judgement bacterium is bacillus (Bacillus sp.).
Based on features above, bacterial strain JS5L is accredited as Te Jila bacillus (Bacillus tequilensis).It should
Bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as on July 18th, 2014
CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), it protects
Hiding number is CGMCC No.9499.
Embodiment 2, Te Jila bacillus JS5L are to the antagonism of Fusarium graminearum
Using opposite culture method detection Te Jila bacillus JS5L to the antagonism of Fusarium graminearum.
One, the punch for being 5mm with diameter is in cultured disease fungus Fusarium graminearum (Fusarium
Graminearum) FG1 colony edge, which is beaten, takes bacterium disk, is chosen bacterium disk into PDA plate center with the toothpick of sterilizing.
Two, at away from PDA plate center 25mm four chain-dotted lines inoculation same volume and concentration JS5L, to be only inoculated with cereal
The PDA plate of sickle-like bacteria bacterium disk is control group, and 28 DEG C are cultivated 4-5 days, and whether there is or not antibacterial bands to be formed for observation.Each group is done three and put down
Row.The results are shown in Table 1.Show that JS5L has very strong inhibitory effect to Fusarium graminearum.
Inhibiting effect of the JS5L to Fusarium graminearum after table 1 is cultivated 5 days
(shortest distance that antibacterial bandwidth refers to pathogen edge to antagonistic strain edge)
Embodiment 3, JS5L fermentation liquid are to the inhibitory effect of Fusarium graminearum
Using opposite culture method detection Te Jila bacillus JS5L to the antagonism of Fusarium graminearum.
One, the punch for being 5mm with diameter is in cultured disease fungus Fusarium graminearum (Fusarium
Graminearum) FG1 colony edge, which is beaten, takes bacterium disk, is chosen bacterium disk into PDA plate center with the toothpick of sterilizing.
Two, at away from PDA plate center 25mm 4 points punching inoculation same volume and concentration JS5L, to be only inoculated with cereal
The PDA plate of sickle-like bacteria bacterium disk is control group, and 28 DEG C are cultivated 4-5 days, and whether there is or not antibacterial bands to be formed for observation.Each group is done three and put down
Row.The results are shown in Table 1.Show that JS5L has very strong inhibitory effect to Fusarium graminearum.
Inhibiting effect of the JS5L to Fusarium graminearum after table 2 is cultivated 5 days
(shortest distance that antibacterial bandwidth refers to pathogen edge to antagonistic strain edge)
Embodiment 4, JS5L escaping gas are to the inhibitory effect of Fusarium graminearum
The escaping gas generated using opposite culture method detection Te Jila bacillus JS5L is to the short of money of Fusarium graminearum
It is anti-.
One, the punch for being 5mm with diameter is in cultured disease fungus Fusarium graminearum (Fusarium
Graminearum) FG1 colony edge, which is beaten, takes bacterium disk, is chosen bacterium disk into PDA plate center with the toothpick of sterilizing.
Two, it is inoculated with JS5L in NA plate center, be buckled in the plate for being connected to Fusarium graminearum, to be only inoculated with Fusarium graminearum
The PDA plate of bacterium disk is control group, and 28 DEG C are cultivated 4-5 days, and whether there is or not fungistatic effects for observation.Each group is done three in parallel.As a result such as
Shown in Fig. 2.Show that JS5L can produce the escaping gas for inhibiting Fusarium graminearum.
Embodiment 5, JS5L are to the greenhouse experiments of Fusarium graminearum inhibitory effect
One, 28 DEG C of shaken overnight cultures in NB culture medium by JS5L, use phosphate buffer be diluted to concentration for 2 ×
108cfu/ml。
Two, Fusarium graminearum FG1 is obtained into bacteria suspension, using phosphorus with sweet mung bean soup culture medium shaken cultivation (28 DEG C, 5 days)
It is 2 × 10 that phthalate buffer, which dilutes bacterial concentration,5cfu/ml。
Three, by after dilution JS5L and Fusarium graminearum FG1 according to 1:1 ratio mix, final concentration is respectively 108cfu/
Ml and 105cfu/ml。
Four, be inoculated in the middle part of the wheat head in the flowering early stage of new 828 kind of stone, connecing thallus product is 10 μ l, as experimental group.
Simultaneously only to contain the phosphate buffer of Fusarium graminearum as positive controls, using be only inoculated with phosphate buffer as negative
Property control.Every group sets 10 repetitions, and experiment is repeated 3 times, and results are averaged.
After Fusarium graminearum bacterial suspension inoculation 16 days, the morbidity of the wheat head is observed, has the wheat head is withered to whiten or rubescent, internal seed
The wheat head that grain is not full, weight saving shows is the morbidity wheat head.
Wherein head blight index=disease incidence × disease index/100.
Greenhouse control efficiency of the table 3JS5L to Fusarium graminearum
The results are shown in Table 3, and Te Jila bacillus JS5L, which can prevent and treat very well Fusarium graminearum under greenhouse experiment, to be caused
Wheat scab.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
The change or replacement expected without creative work, should be covered by the protection scope of the present invention.Therefore, of the invention
Protection scope should be determined by the scope of protection defined in the claims.
Claims (10)
1. one plant of Te Jila bacillus (Bacillus tequilensis) is preserved in the micro- life of China on July 18th, 2014
Object culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.9499.
2. application of the bacterial strain described in claim 1 in the plant disease caused by biological control sickle-like bacteria；The sickle-like bacteria is
Fusarium graminearum.
3. application according to claim 2, it is characterised in that: the disease is head blight or ear rot or root rot.
4. application according to claim 2 or 3, it is characterised in that: the plant is cereal；The cereal is wheat crops
Or corn.
5. application according to claim 4, it is characterised in that: the wheat crops are wheat.
6. application of the bacterial strain described in claim 1 in the product that preparation inhibits sickle-like bacteria；The sickle-like bacteria is cereal reaping hook
Bacterium.
7. a kind of method of plant disease caused by prevention and treatment sickle-like bacteria, is to carry out spraying treatment in growing process；
Described be the bacteria suspension or fermentation liquid of Te Jila bacillus described in claim 1 by spraying；
The sickle-like bacteria is Fusarium graminearum.
8. according to the method described in claim 7, it is characterized by: the disease is head blight or ear rot or root rot.
9. method according to claim 7 or 8, it is characterised in that: the plant is cereal；The cereal is wheat crops
Or corn.
10. according to the method described in claim 9, it is characterized by: the wheat crops are wheat.
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| CN105695368B (en) * | 2016-04-11 | 2019-03-22 | 江苏省农业科学院 | One plant of Te Jila bacillus and its application |
| CN106342898A (en) * | 2016-08-29 | 2017-01-25 | 佛山市艳晖生物科技有限公司 | Compound microorganism preparation against sclerotinia rot of colza and preparing method thereof |
| CN106635903B (en) * | 2016-12-23 | 2019-08-23 | 河北省农林科学院遗传生理研究所 | The Promoting bacteria combination of heavy salinized ground salt tolerance of crop in a kind of raising |
| CN106701624B (en) * | 2016-12-29 | 2019-07-30 | 山东农业大学 | The Te Jila bacillus of one plant of antagonism fructus lycii root rot and its application |
| CN108285875A (en) * | 2017-01-10 | 2018-07-17 | 中国科学院微生物研究所 | One plant of Te Jila bacillus and its application |
| CN108998394B (en) * | 2018-08-16 | 2020-11-20 | 浙江农林大学 | Bacillus and application thereof |
| CN110402929A (en) * | 2019-08-21 | 2019-11-05 | 浙江省桐庐汇丰生物科技有限公司 | A kind of bactericidal composition and its application containing jinggangmeisu |
| 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) |
| EP4167743A4 (en) * | 2020-06-17 | 2024-07-03 | Bioconsortia Inc | Agriculturally beneficial microbes, microbial compositions, and consortia |
| CN111690566A (en) * | 2020-07-03 | 2020-09-22 | 山东第一医科大学(山东省医学科学院) | Bacillus tequilensis and application thereof |
| CN113151094B (en) * | 2021-04-27 | 2022-05-13 | 江苏师范大学 | Bacillus tequilensis for producing volatile antibacterial gas and application thereof |
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