CN1884481A - Biological control bacillus subtilis for crop bacterial wilt - Google Patents
Biological control bacillus subtilis for crop bacterial wilt Download PDFInfo
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Abstract
The invention provides a crop Pythium biological control bacillus subtilis strain and belongs to the biotechnological sphere. The invention relates to a crop Pythium bacillus subtilis strain and its biological crop preserved number is CGMCC1732. The said strain can reduce the growth of Ralstonia solanacearum and the biological control experiment indicates that it can prevent and cure the Granville wilt. Simultaneously the invention also provides an antagonistic protein blend obtained from the said strain and the blend has higher application value in crop Pythium biological control.
Description
Technical field
The invention belongs to biological technical field.Further, the present invention relates to a biological control bacillus subtilis for crop bacterial wilt.Further, the present invention relates to using Bacillus subtilis bacterial strain SH7 control tobacco bacterial wilt.
Background technology
The tobacco bacterial wilt is a kind of typical vascular bundle diseases that is caused by the withered Lei Er Salmonella of green grass or young crops (Ralstonia solanacearum), the most significant symptom is withered, and withered speed is very fast, in case morbidity can cause complete stool death, is War Torn venereal disease evil on the tobacco.At present China Yangtze valley and on the south the cigarette district generally take place; Sickness rate at nonirrigated farmland, multiple lesion vega is 30%-50%, and particularly the sickness rate of nonirrigated farmland continuous cropping continuous cropping vega is more than 50%; Especially cause harm seriously indivedual time outbreak of epidemic with south China Yan Qu.In recent years, the morbidity of this disease and the scope that causes harm had the trend of the expansion of cigarette district north, and in Shandong, all there are generation in provinces such as Henan, Shaanxi and Liaoning, partial area are caused harm heavier.The loss that present this disease causes has occupied the 4th of all kinds of diseases, must pay attention to these sick preventing and controlling.Especially in recent years the compound harm of infecting of bacterial wilt, balck shank that causes of the wound that causes with the cause root knot nematode is in rising trend, causes the serious underproduction of vega.In view of pollution and the ecological balance damage of chemical prevention to environment, so the biological control of tobacco bacterial wilt more and more is much accounted of.
Tobacco is the important cash crop of China, and the major production areas of Flue-cured Tobacco in China is distributed in nearly 900 counties and cities of more than 20 provinces and regions such as Yunnan, Guizhou, Sichuan, Hubei, Hunan, Shaanxi, Henan, Anhui, Shandong, Liaoning, Jilin, Heilungkiang.Wherein, Yunnan Province is the province of China's tobacco planting area maximum.The quality of China's tobacco leaf is improving constantly, and national improved variety area reaches more than 90%, is maximum in the world tobacco producing country.
Disease is the important factor that has a strong impact on output and quality on tobacco produces.The tobacco bacterial wilt claims " cigarette pest " again, is a kind of vascular bundle diseases that is caused by Ralstonia solanacearum, mainly infects tobacco root and stem, also can infect blade; All can take place in seedbed and land for growing field crops, often cause the large stretch of withered death of cigarette strain, be a kind of destructive soil-borne disease.Indonesia in 1864 at first is reported in and causes devastating loss on the tobacco; In the U.S., found this disease first in 1880, big area was popular in 1910, and kind of a cigarette is forced to stop in some farms of the U.S. subsequently.This disease is distributed widely in damp-heat areas such as the torrid zone, the world, subtropics and temperate zone; Take place in the majorityly on the south the regional distribution the Changjiang river of China's tobacco ralstonia solanacearum, can be divided into 4 districts according to the morbidity height, multiple lesion comprises provinces and regions such as Guangdong, Jiangxi, Fujian, Hunan, Sichuan, Zhejiang, Anhui; Inferior region of disease comprises expensive thirty, Hubei; Accidental lesion comprises Yunnan, Henan, Shandong, Shaanxi, Liaoning; All the other are no lesion, and the division in each district is relative, and there is transitional zone in the interval; The trend that presents from west to east, increases gradually by north orientation south.
The host range of tobacco bacterial wilt bacterium is very wide, can infect kind of plant surplus more than 50 section 200, is the critical limitation factor of tomato, potato, tobacco, peanut, sweet potato, eggplant, ginger banana and plant production such as some costly medicines and flowers.Most of grasses and cotton, sweet potato and crudefiber crop are not infected.Germ mainly survives the winter in soil and compost, also can hide and survive the winter in seed, rhizosphere or the pin main body of growing; Under the general condition, be to invade, can not invade from pore from the root wound.The main primary infection source of bacterial wilt is the germ in soil, invalid tissue and the fertilizer, and these pathogenic bacterias are spread long-distance communications by rainwater, row irrigation, sick soil, the seedling that carries disease germs, vaccine, people and animals, means of production and insect.
Tobacco bacterial wilt all can take place in seedbed and land for growing field crops.Mainly infect cigarette strain root, stem, also can infect blade, the blade initial stage of catching an illness is still for green is suspended on the stem, so claim " bacterial wilt "; When weather was hot, the blade of diseased plant is irregular burnt, and blade becomes dry, and the edge comes off.The stem of diseased plant is vertical, and dead blade is hanging down on the stem.When soil humidity was big, the soft corruption of root was clamminess, and causes complete stool withered.
Utilize the beneficial microorganism controlling plant diseases, become one very active and begin to show the field of applications well prospect.Many treatise reports (Cook, K.J.and Baker K.F.The nature and practice of biological controlof plant pathogens.1984, APS press USA) think that biological control has critical role in the future of agriculture.A large amount of studies show that biocontrol bacteria produces multiple antagonism or emulative meta-bolites in it grows, and by direct or indirect effect, reaches the effect of obstruction or kill pathogenic bacteria.No matter be the biotype that abiogenous biological control phenomenon or people are used for biological control, the effect of bacterium is very tangible.Its main advantage is: 1) kind of bacterium and One's name is legion exist in a large number at plant rhizosphere and overground part; 2) bacterium is wider to the mode of action of pathogenic bacteria, can exert an influence to pathogenic bacteria by modes such as competition, antagonism and inducing plant generation resistances; 3) has surprising reproduction speed; 4) many bacteriums are present in plant rhizosphere and overground part, and are more suitable to the ecology of plant; 5) bacterium mostly can artificial culture, is convenient to control, in practice easy handling; 6) some bacterium can not only prevent and treat disease and also can increase crop yield (Cheng Liang etc., the progress of antagonistic bacterium [J]. Agricultural University Of Jiangxi's journal, 2003,25 (5): 732～736).Show or be considered to the beneficial bacteria kind numerous (Dviad M.Biological Control of Soilbrone Plant Pathogens in the Rizhospherewith Bacteria.Ann.Rev.Phytopathol.1988,26:379～407) of biological and ecological methods to prevent plant disease, pests, and erosion potentiality; The bacterium that has comprised many genus, the Plant diseases biocontrol bacteria of successful Application has some bacterial strains that Agrobacterium, Bacillus, Pseudomonas, Erwinia, Xanthomonas etc. belong at present.
The important mechanisms that development takes place the biocontrol bacteria controlling plant diseases is to produce antagonistic substance, roughly comprise following a few class: bacteriocin (bacteriocins), fluorescein (fluorescein), agrocin (agrocin), aldehydes matter, polypeptide antibiotics (polypeptides), protein-based antifongin and volatility inhibitory substance etc. play a part crucial in biocontrol of plant disease.The antagonistic substance kind that is produced by biocontrol bacteria is many, and the sphere of action wide spectrum can be produced by the various bacteria bacterial strain with a kind of antagonistic substance, and also can produce the antagonistic substance of multiple different structure with a kind of bacterial isolates.
Understanding antimicrobial antifungal mechanism short of money is to strengthen the important prerequisite that antagonism bacterium screening criteria was renderd a service and determined in the biological and ecological methods to prevent plant disease, pests, and erosion of antagonism bacterium, the antagonism bacterium mainly contains 3 kinds of mechanism of action: i.e. antagonistic action, competition effect (comprising the competition of nutrition, physics site, ecological site and oxygen etc.), induction of resistance and super parasitic, the mechanism of action of biocontrol bacteria is mainly former three.Since host-pathogenic bacterium-antagonism bacterium three's interaction, the coefficient result of the multiple often mechanism of the generation of each antagonism bacterium antagonistic effect.The research report is also arranged, and the antagonistic strain that has relies on number of mechanisms based on a kind of mechanism when having.
Therefore this test will be explored from the biological control aspect, the short living effective antagonistic bacterium bacterial strain of screening preventive effect, and its antifungal mechanism studied, by conventional and molecular biology approach, utilize these antagonistic bacteriums and antimicrobial substance thereof control tobacco bacterial wilt that research basis and experiment material are provided in the future.
Rhizosphere, the leaf of plant enclose and other little ecosystem in, antagonistic bacterium extensively exists, and since cultivate convenient, growth cycle is short, have very big potentiality to be exploited as new antagonism bacterium source.Tobacco bacterial wilt is one of severe diseases during tobacco produces, and the annual heavy losses of yield of tobacco and the quality of all causing descends, and is very important restraining factors during tobacco produces.The harm that how to prevent and treat effectively and alleviate tobacco bacterial wilt is the target of many researchers always.So far people do not find ideal control medicament, and breeding resistant variety is difficult to, and resistance is lost easily.Therefore constantly seeking the new tobacco bacterial wilt method of preventing and treating is a secular task.
Domestic to the existing report of tobacco bacterial wilt biological control, but most screening and pot experiment stage that only limits to biocontrol microorganisms, and it is produced the domestic not further investigation as yet of all many-sides such as diseases prevention mechanism of extraction, physio-biochemical characteristics and the biocontrol microorganisms of antimicrobial substance,
Summary of the invention
At the defective in above-mentioned field, the invention provides a biological control bacillus subtilis for crop bacterial wilt, be used for the biological control of crop bacterial wilt, for the control of bacterial wilt provides new Microbial resources.
One strain crop bacterial wilt biological and ecological methods to prevent plant disease, pests, and erosion subtilis (Bacillus subtilis) bacterial strain SH7, its biological preserving number is: CGMCC1732.
The application of described bacterial strain SH7 in control crop bacterial wilt.
Described crop is a tobacco.
The application of described bacterial strain SH7 in suppressing blue or green withered Lei Er Salmonella (Ralstonia solanacearum).
Described bacterial strain excretory antibacterial protein substance mixture is characterized in that: from bacterial strain SH7 fermented liquid, extract, and contain one under alkaline condition the albumen of stable 35kDa.
The application of described antibacterial protein mixture in control crop bacterial wilt.
The present invention is directed to tobacco ralstonia solanacearum (Ralstonia solanacearum is hereinafter to be referred as R.S), measure by indoor inhibition zone method and live body greenhouse preventive effect, filter out the antagonistic strain SH7 that under greenhouse experiment, R.S is had good prophylaxis effect, measure SEQ ID NO1 in its 16s rDNA complete sequence such as the appendix, BLAST software and the DNAMAN software used are wherein analyzed, the sequence of SH7 bacterial strain is identical with subtilis 16s rDNA partial sequence, homology 100%) (as Fig. 7) therefore identifies that the SH7 bacterial strain is subtilis (Bacillus subtilis).The culture condition of this bacterial strain is 30 ℃, common beef broth substratum, pH7.0.This bacterial strain is bacillus (Bacillus), bacillus subtilis bacterial classification, and in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date is on June 7th, 2006, protects to subtract to be numbered: CGMCC1732.Through identifying, comprise about the information of this bacterial strain: can form gemma; The excretory egg white mixture is to thermally-stabilised (seeing accompanying drawing 10), to Proteinase K, trypsinase and stomach en-insensitive (seeing accompanying drawing 14,15), to chloroform part responsive (seeing accompanying drawing 16); The SDS-PAGE electrophoresis shows that one of its action protein is about 35kDa (seeing accompanying drawing 18); Biology is measured and is shown, can suppress the growth (seeing accompanying drawing 1) of blue or green withered Lei Er Salmonella (Ralstonia solanacearum) on the beef broth flat board, and potted plant control experiment shows that preventive effect is 65.99% (seeing Table 3).
The present invention is spread bacteriostatic method by indoor flat plate, obtained the strong SH7 bacterial strain of dull and stereotyped antagonistic ability (antibacterial bandwidth 12.2mm), SH7 shows except that the prevention effect (greenhouse pot culture) of fermented liquid to tobacco bacterial wilt, through independent degerming fermentation liquor treatment with remove fermented liquid and compare the disease time of two kinds of equal deferrable tobacco bacterial wilts of processing mode with the contrast of NB substratum with ralstonia solanacearum effect 30min aftertreatment; During 29d, compare with the contrast of NB nutrient solution after inoculation, preventive effect reaches 65.99% and 53.2%, illustrates that SH7 bacterial strain excretory antimicrobial substance is the main mechanism of its prophylaxis effect.The cigarette seedling leaf look that inoculation SH7 removes behind the fermented liquid is dark green and plump, and growing way is good.
SH7 bacterial strain excretory antagonistic substance can be with 70% saturation ratio ammonium sulfate extraction.The antagonistic substance of slightly carrying is to thermally-stabilised, and the bacteriostasis rate of sample is respectively 90% and 70% (bacteriostasis rate of supposing untreated samples is 100%) behind 100 ℃ and 121 ℃ of autoclaving 20min; It is insensitive to Proteinase K, trypsinase and stomach en-, and is responsive to the chloroform part.Action activity is best at pH7.0-9.0.Through UV scanning and SDS-PAGE electrophoresis, antagonistic substance has typical albumen absorption peak at the 274.00nm place, the protein that the SH7 secretion is multiple, and the molecular weight size of one of component is about 35KD.
The biological and ecological methods to prevent plant disease, pests, and erosion bacillus subtilis strain SH7 of the antagonism ralstonia solanacearum that the present invention obtains can be applicable to the control to the crop bacterial wilt, biology is measured and is shown, can suppress the growth (seeing accompanying drawing 1) of blue or green withered Lei Er Salmonella (Ralstonia solanacearum) on the beef broth flat board, potted plant control experiment shows that preventive effect is 65.99% (seeing Table 3).Has actual using value.
The present invention has obtained antagonist protein by the broth extraction of bacterial strain SH7, and confirms that the main biocidal property material that SH7 produces is this antagonist protein.This antagonist protein Heat stability is good, insensitive to proteolytic enzyme, be the fine biological pesticide.
Culture presevation information:
Strain name: bacillus, subtilis (Bacillus subtilis)
Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center
Preservation date: on June 7th, 2006
Deposit number: CGMCC 1732
Description of drawings
Fig. 1 is the bacteriostatic activity of SH7 bacterial strain to tobacco ralstonia solanacearum.
Fig. 2 is that SH7 bacterial strain fermentation liquor supernatant is to the tobacco ralstonia solanacearum bacteriostatic activity.
Fig. 3 is the preventive effect of degerming fermentation liquor treatment cigarette seedling 14d.
Wherein, 1 is SH7 degerming fermentation liquor treatment cigarette seedling; CK is contrast.
Fig. 4 is for removing the preventive effect of fermented liquid and ralstonia solanacearum effect 30min aftertreatment cigarette seedling 14d.
Wherein, 2 for removing fermented liquid and ralstonia solanacearum effect 30min aftertreatment cigarette seedling; CK is contrast.
Fig. 5 is the preventive effect of degerming fermentation liquor treatment cigarette seedling 20d.
Wherein, 1 is SH7 degerming fermentation liquor treatment cigarette seedling; CK is contrast.
Fig. 6 is for removing the preventive effect of fermented liquid and ralstonia solanacearum effect 30min aftertreatment cigarette seedling 20d.
Wherein, 2 for removing fermented liquid and ralstonia solanacearum effect 30min aftertreatment cigarette seedling; CK is contrast.
Fig. 7 is SH7 PCR product sequence is searched for comparison in the Genbank database result.
Fig. 8 is that 70% (NH4) 2SO4 saturation ratio precipitation is to Ralstonia solanacearum bacterial strain bacteriostatic activity.
Wherein, the left side is the PBS damping fluid processing of pH value 7.6; The right side is dialysis back crude extract.
Fig. 9 is for removing the sedimentary supernatant bacteriostatic activity of 70% (NH4) 2SO4 saturation ratio gained.
Figure 10 is the bacteriostatic activity of the thick leach protein liquid of the SH7 after the treatment of different temperature to tobacco ralstonia solanacearum.
Wherein, 1 is untreated samples; 2 are 100 ℃ handles 20min; 3 are 121 ℃ handles 20min; 4 is PBS damping fluid (pH7.6).
Figure 11 is that different pH values are handled the bacteriostatic activity of SH7 thick leach protein in back to tobacco bacterial wilt.
Figure 12 is the active stability of the thick leach protein of SH7 under acidic conditions and the alkaline condition.
Wherein, 1 is raw sample; 2 are the pH5.0 precipitation; 3 is the pH5.0 supernatant; 4 are the pH3.0 precipitation; 5 is the pH3.0 supernatant
Figure 13 is the active stability of the thick leach protein of SH7 under acidic conditions and the alkaline condition.
Wherein, 1 is raw sample (pH7.0); 2 are the pH9.0 processing; 3 are the pH8.0 processing; 4 are the pH10.0 processing; 5 are the pH11.0 processing.
Figure 14 handles the antibacterial proteic stability in back for Proteinase K.
Wherein, the left side is that Proteinase K is handled; The right side is a raw sample.
Figure 15 is the stability after stomach en-and the trypsin treatment.
Wherein, 1 is untreated samples; 2 is pepsin; 3 is trypsin treatment.
Figure 16 be after chloroform is handled protein crude extract to the bacteriostatic activity of tobacco ralstonia solanacearum
Wherein, 1 is the chloroform processing; 2 is untreated samples; 3 is aqueous portion; 4 is the PBS damping fluid.
Figure 17 for behind different organic solvents the extracts thick leach protein liquid to the bacteriostatic activity of tobacco ralstonia solanacearum.
Wherein, 1 is the organic phase of the thick leach protein of extracted with diethyl ether SH7; 2 is the water of the thick leach protein of extracted with diethyl ether SH7; 3 is the organic phase of the thick leach protein of xylene extraction SH7; 4 is the water of the thick leach protein of xylene extraction SH7; 5 is the organic phase of the thick leach protein of ethyl acetate extraction SH7; 6 is the water of the thick leach protein of ethyl acetate extraction SH7; 7 are the thick leach protein that is untreated.
Figure 18 slightly carries antagonist protein SDS-PAGE electrophorogram for SH7.
Wherein, 1 thick leach protein for the chloroform processing; 2 are the thick leach protein that is untreated; 3 is Marker.
Figure 19 is the light absorption value of thick leach protein.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
1, sick experiment of screening, greenhouse pot culture control and the growth-promoting functions of 1 control tobacco bacterial wilt antagonistic bacterium SH7
Evenly adding 100 μ l concentration in the sterilization culture dish is 1 * 107cfu/ml Ralstonia solanacearum (Ralstonia solanacearum) bacteria suspension, pours the abundant mixing of fusing NA substratum 20ml that is cooled to about 50 ℃ into.After treating that substratum solidifies fully,, cultivate in 30 ℃ of incubators after 48 hours with the antagonism bacterium of inoculation circling point inoculation for examination, every ware point 8-10 tested bacteria bacterial strain, the detection inhibition zone to have that it's too late big or small.Carry out repeated test after testing the bacterial strain line purifying that antagonistic action is arranged for the first time, basic skills is the same, and every dull and stereotyped point connects 3 bacterial strains, respectively three repetitions.From dull and stereotyped fungistatic effect, the indoor flat plate good antimicrobial effect of SH7 bacterial strain, antibacterial bandwidth reaches 12.2mm, and antagonistic action is remarkable, and biocontrol bacteria has produced the antibiotics with bacteriostatic activity in the growth metabolism process.(see Table 1 and Fig. 1)
Table 1 different strains is to the restraining effect of tobacco bacterial wilt
| Strain number | The source | Tobacco ralstonia solanacearum (Ralstonia solanacearum) | |
| Antibacterial circle diameter (mm) | Antibacterial bandwidth (mm) | ||
| SH7 | Xiao Jiafang cigarette district, Fujian soil sample | 31.4 | 12.2 |
Annotate: antibacterial bandwidth is the edge of antagonism bacteria growing edge to pathogenic bacterium, and each data is the mean value of indoor flat plate diffusion preliminary survey and repetition measurement.
The bacteriostatic activity of SH7 bacterial strain fermentation liquor measure be with activatory SH7 inoculation in the NB substratum, 30 ℃ of constant temperature, 150rpm shaking culture 48 hours, 4 ℃ of centrifugal 10min remove thalline, with the aperture is 0.22 μ m filtering with microporous membrane degerming, gets 100 μ l and detect its bacteriostatic activity on flat board.(as Fig. 2)
As can be seen from Figure 2, the SH7 bacterial strain fermentation liquor is very strong to the dull and stereotyped bacteriostatic activity of tobacco ralstonia solanacearum, and inhibition zone is transparent and clear.
Measure the antagonistic bacterium that obtains with flat board and carry out potted plant test.Blank CK1 (only connecing clear water) and the every processing of morbidity contrast CK2 (only connecing ralstonia solanacearum) are established in each test, establish three repetitions, every repetition 5 young plants.Concrete experimentation is as follows:
(1) seedling is soaked root: get 5-6 sheet true leaf cigarette seedling and extract in the basin alms bowl, (concentration is in 1 * 108cfu/ml), takes out behind the 30-40min and transplants in the flowerpot that sterilization soil is housed in the examination biocontrol microorganisms bacteria suspension that supplies for preparing in advance to soak root.Contrast is soaked root in clear water.Other is according to the greenhouse Routine Management.
(2) irritate bacterium once more: behind the slow seedling, in rhizosphere inoculation antagonism bacterium bacteria suspension, concrete grammar is as follows every 5d:
Utilize the rifle head to be square and bore cave, (about deeply 5cm), totally 4 of every strains at cigarette strain rhizosphere soil.Bacteria suspension is injected the cave, the 1ml/ cave, other gets 1ml and drenches in basal part of stem, and cigarette strain rhizosphere adequately protects.As twice of above-mentioned method repeated inoculation.Contrast inserts the equivalent clear water.
(3): the inoculation morbidity: behind the 1d, will shift to an earlier date the bacteria suspension that prolific Ralstonia solanacearum is mixed with 5 * 107cfu/ml, inoculation method is the same, and inoculation position is identical, and inoculum size is the 5ml/ strain; CK1 connects the equivalent clear water.After finishing, the cigarette strain is preserved moisture with proper amount of clear water, in order to morbidity.
(4): observed and recorded: after seeing diseased plant, observed and recorded morbidity strain every day number.The visible obviously bacterial wilt symptom of plant is the morbidity strain.To inoculate pathogenic bacteria day is starting point day, observes 25-30d altogether.(seeing Table 2)
The preventive effect calculation formula:
State of an illness classification: 0 grade is asymptomatic; 1 grade is 1-2 sheet leaf withering; 2 grades are wilting of 1/3-1/2 blade or the blackening of cane base portion; 3 grades is the wilting of 2/3-3/4 blade, necrosis and cane blackening necrosis; 4 grades are whole strain death.
The potted plant test result of table 2 SH7 (20d)
| Strain number | Total strain number | The diseased plant number | Disease index | Relative control effect (%) |
| SH7 CK1 | 15 15 | 15 0 | 91.7 0 | -27.5 - |
| CK2 | 15 | 15 | 71.9 | - |
Annotate: disease index is three multiple mean values.
Detect through potted plant preventive effect, SH7 measures, and it is serious that the result contrasts the CK2 morbidity,
SH7 removes fermented liquid the preventive effect of tobacco bacterial wilt is measured: be selected to the cigarette seedling of sterile soil plantation, remove two processing of contrast peripheral hardware, handle the SH7 fermentation liquid irrigating root of a usefulness after thalline is killed in thermal treatment, the 50ml/ strain, handle once every 3d, after continuous 2 times, inoculation ralstonia solanacearum R.S; Handle dual-purpose thermal treatment kill behind the thalline the SH7 fermented liquid with mix liquid irrigating root (not inoculating R.S) after Ralstonia solanacearum bacteria suspension (concentration is 105cfu/ml) acts on 30min; The 50ml/ strain is handled once continuous 2 times every 3d.Contrast is irritated root with the NB substratum, and processing mode is the same.Processing one and contrast inoculation Ralstoniasolanacearum (R.S), bacteria suspension concentration is adjusted into 106cfu/ml, the 50ml/ strain.Every processing 8 strain cigarette seedlings repeat for 3 times, 28 ℃ of-32 ℃ of cultivation of preserving moisture, see diseased plant after, every day the observed and recorded strain number of falling ill.The visible obviously bacterial wilt symptom of plant is the morbidity strain.To inoculate pathogenic bacteria day is starting point day, observes 20-29d altogether.Preventive effect calculation formula and state of an illness classification are the same.(see Table 3 and Fig. 3,4,5,6).
Table 3 SH7 bacterial strain removes the preventive effect measurement result of fermented liquid to tobacco bacterial wilt
| Handle | The inoculation sequela time (d) | 7d | 17d | 29d | |||
| Disease refers to | Preventive effect (%) | Disease refers to | Preventive effect (%) | Disease refers to | Preventive effect (%) | ||
| SH7 removes fermented liquid SH7 and removes fermented liquid+R.S effect 30min NB substratum | 14 12 5 | 0 0 17.7 | 100 100 - | 13.5 15.6 87.5 | 84.6a* 82.2a - | 33.3 45.8 97.9 | 65.99a 53.2a - |
* represent that with the same letter on hurdle new multipole difference detects difference not remarkable (P=0.05).
From above-mentioned table 3 and figure as can be seen, through independent degerming fermentation liquor treatment with remove fermented liquid and compare with the contrast of NB substratum with ralstonia solanacearum effect 30min aftertreatment, the disease time of preceding two equal deferrable tobacco bacterial wilts of processing mode, postpone 9d and 7d respectively, 17d after inoculation, the preventive effect of two processing is respectively 84.6% and 82.2%, during to 29d, compare with the contrast of NB nutrient solution, preventive effect reaches 65.99% and 53.2%.Simultaneously, it is better than the preventive effect of removing fermented liquid and ralstonia solanacearum effect 30min aftertreatment to remove fermented liquid protection cigarette strain root with SH7 in advance.Therefore, the present invention is directed to tobacco ralstonia solanacearum (Ralstoniasolanacearum is hereinafter to be referred as R.S), measure, filter out the antagonistic strain SH7 that under greenhouse experiment, R.S is had good prophylaxis effect by indoor inhibition zone method and live body greenhouse preventive effect.
The growth-promoting functions of SH7, with the land for growing field crops loam: farm manure: vermiculite is loaded in pot for growing seedlings after the sterilization according to 7: 2: 1 ratio preparation matrix.Carefully dig out the cigarette seedling of 5-6 sheet true leaf, carefully shake off to be attached to the soil of root, and it is soaked in the SH7 biocontrol microorganisms bacteria suspension (concentration is about 108cfu/ml) that preliminary election prepares, take out behind the 30-40min, transplant respectively in the seedling alms bowl that sterilization soil is housed.Every processing 20 strains repeat for three times, random alignment, the greenhouse cultivation of preserving moisture.After treating that the cigarette seedling grows to 30d, picked at random 10 strain cigarette seedlings carefully dig out the whole strain of seedling, and flush away root earth is measured its plant height, whole strain fresh weight, root fresh weight and indexs such as dry weight and Ye Se.Dry to constant weight for 180 ℃ then, survey whole strain dry weight.(seeing Table 4)
Table 4 SH7 bacterial strain is to the growth-promoting functions measurement result of tobacco in the soil of sterilizing
| Handle | Average plant height (cm) | Whole strain fresh weight (g) | Whole strain dry weight (g) | Root fresh weight (g) | Root dry weight (g) | Root long (cm) |
| SH7 CK rate of increase (%) | 10.3 6.7 53.7 | 10.1 7.9 26.9 | 0.519 0.383 35.5 | 1.09 0.73 49.3 | 0.072 0.031 132.3 | 11.4 8.3 37.3 |
Experimental result shows, the inoculation biocontrol microorganisms is when the tobacco of sterilization soil plantation, the SH7 bacterial strain can promote tobacco growing, whole strain aquatic foods, dry weight, root aquatic foods, dry weight, root length and plant height are significantly higher than the clear water contrast, and rate of increase is 26.9%, 35.5%, 49.3%, 132.3%, 37.3% and 53.7%, and growth-promoting functions is good.Simultaneously, illustrate that also SH7 is safe to tobacco.
1, the classification position Molecular Identification of 2 SH7
Reference literature (Lin Wanming edits bacteria molecule genetic classification authentication method [M]. Shanghai, Shanghai science tech publishing house 1990) method is extracted overall dna, pcr amplification is with reference to (C.W. Dieffenbacher, G.S. the moral Vicks VapoRub is reined in work, Huang Peitang etc. translate .PCR experimental technique experiment guide [M], Beijing science tech publishing house, 2000) method is carried out.With SH7 thalline genomic dna is template, through PCR reaction amplification, detect through 0.8% agarose gel electrophoresis, obtain the above specific fragment of a 1.0kb, measure this fragment sequence (the PCR product is by the big-and-middle living bio tech ltd order-checking of Beijing China), the result shows that recording the SH7 bacterial strain is 1409bp.Submit the 16s rDNA complete sequence (seeing appendix SEQ ID NO1) that records to Genbank (www.ncbi.nlm.nlh.gov), BLAST software and the DNAMAN software used are wherein analyzed, find the sequence that the SH7 bacterial strain records identical with subtilis 16srDNA partial sequence (Fig. 7).Therefore identify that the SH7 bacterial strain is subtilis (Bacillus subtilis).
The extraction of thick leach protein and active detection the thereof: with activatory SH7 inoculation in the NB substratum, 30 ℃ of constant temperature, 150rpm shaking culture 48 hours, 4 ℃ of centrifugal 10min remove thalline, make the fermented supernatant fluid of the outer metabolite of born of the same parents.In fermented liquid, slowly add a certain amount of (NH4) 2SO4 and reach 70% saturation ratio, stir, put 4 ℃ of static spending the night of condition.
To saltout liquid in the centrifugal 20min of cold condition (12000r/min), remove supernatant liquor, sedimentable matter suspends with the PBS damping fluid (pH7.6) of 1/15 volume, with the above-mentioned suspension pre-treatment dialysis tubing (molecular weight that dams is 5kD) of packing into, two of dialysis tubing clips with dialysis clamp, place 100 times of volume PBS damping fluids (pH7.6), on magnetic stirring apparatus, dialysed 24 hours for 4 ℃, dialysed again 24 hours behind the replacing dialyzate.Crude extract matter is freezing after dialysis treatment is dissolved in the PBS damping fluid after draining, and is 0.22 μ m filtering with microporous membrane degerming with the aperture, detects its bacteriostatic activity on flat board.(as Fig. 8, Fig. 9)
As can be seen from the figure, the SH7 bacterial strain is in the NB nutrient solution behind the shaking culture 48h, the crude protein liquid that obtains after 70% (NH4) 2SO4 saturation ratio precipitation and dialysis detects through dull and stereotyped bacteriostatic activity, very strong bacteriostatic activity (as Fig. 8) is arranged, and remove 70% (NH4) 2SO4 sedimentary supernatant bacteriostatic activity of saturation ratio gained very faint (as Fig. 9); Wherein certain density salt also has germicidal action.Therefore can assert tentatively that the main bacteriostatic activity material that SH7 produces is a protein matter.
Antagonistic substance thermostability: the SH7 that obtains is slightly carried antagonist respectively handle 20min in 40 ℃, 60 ℃, 80 ℃, 100 ℃ and 121 ℃ (high pressure moist heat sterilizations), getting 100 μ l, to detect it be the bacteriostatic activity of indicator with the ralstonia solanacearum, is contrast with no thalline filtrate and PBS damping fluid without any processing; Observe the sensitivity of antagonistic substance to temperature.(see Table 5 and Figure 10)
The thick leach protein liquid of SH7 after table 5 treatment of different temperature is to the bacteriostatic activity of tobacco ralstonia solanacearum
| Temperature (℃) Tempreture (℃) | 40 | 60℃ | 80℃ | 100 | 121℃ | Untreated is untreated |
| Antibacterial circle diameter (mm) Diometer of Inhibition rings | 30 | 30 | 30 | 27 | 21 | 30 |
After handling 20min under the differing temps of setting, crude extract activity after 40 ℃, 60 ℃, 80 ℃ processing remains unchanged the thick leach protein liquid that obtains according to the 70% ammonium sulfate saturation ratio precipitator method respectively; And after temperature risen to 100 ℃ and 121 ℃ of autoclaving 20min, still keep most of bacteriostatic activity (as Figure 10) to tobacco ralstonia solanacearum, and to compare with untreated samples, antibacterial circle diameter has reduced 3mm and 9mm respectively.The bacteriostasis rate of supposing untreated samples is 100%, and then the bacteriostasis rate of sample is respectively 90% and 70% behind 100 ℃ and 121 ℃ of autoclaving 20min.This thick leach protein of deducibility is a heat resistant egg white thus, sees Table 5.
Antagonistic substance pH value stabilization: get and slightly carry antagonistic substance in right amount, and respectively that its furnishing 3,5,9,11,13 is different pH values, leave standstill 10min after, the condition of again system being recalled to pH7.0 is respectively got 100 μ l and is tested its bacteriostatic activity to tobacco ralstonia solanacearum.As Figure 11,12,13)
The result shows that the bacteriostatic activity of this antibacterial protein was the strongest when the pH value was 7.0-9.0, and antibacterial circle diameter is 20mm, the pH value be 10.0 and 11.0 o'clock antibacterial circle diameter be 18mm, and active strong than under the acidic conditions of the bacteriostatic activity under alkaline condition; This antagonist protein can produce precipitation under acidic conditions, all precipitates when the pH value is 3.0 left and right sides, and the supernatant after centrifugal does not have bacteriostatic activity.Can draw thus, antagonist protein is all stablized under neutrality or meta-alkalescence condition.
Antagonistic substance is to the stability of proteolytic enzyme: get an amount of crude extract matter and react 40min (final concentration of enzyme reaction is 1mg/ml) with Proteinase K, trypsinase and stomach en-under the suitableeest enzymatic condition respectively, to be treated to contrast, to get 100 μ l and survey its bacteriostatic activity ralstonia solanacearum without enzyme.Thick leach protein after different protease treatment is to the bacteriostatic activity and contrast untreated samples active consistent (as Figure 14,15) of tobacco ralstonia solanacearum, and this antibacterial protein of presentation of results is insensitive to proteolytic enzyme.
Antagonistic substance is to the stability of chloroform: get crude extract and chloroform balanced mix according to 1: 1 ratio, put upside down repeatedly several times, leave standstill behind the 10-15min centrifugal, treat residual chloroform volatilization after, get 100 μ l organic phases parts and aqueous portion respectively and do active detection, contrast and be equal amounts of chloroform and raw sample.The result shows, the crude extract of handling through chloroform is weaker than the crude extract that is untreated (as Figure 16), aqueous portion non-activity to the bacteriostatic activity of tobacco ralstonia solanacearum; Illustrate that chloroform can extract the antibacterial substance in this protein crude extract, and antibacterial substance is had certain influence.
Antagonistic substance different organic solvents extraction: get an amount of aseptic crude extract and add isopyknic organic solvent (dimethylbenzene, ether, ethyl acetate, acetone, Virahol and methyl alcohol therein, polarity strengthens successively) extract, after the extraction liquid layering, organic phase and water are separated, and will appoint raffinate and water after the organic solvent volatilization to filter (filter pore size is 0.22 μ m) degerming at ambient temperature, respectively get 100 μ l and measure its bacteriostatic activity, establish raw sample and organic solvent and be contrast.(see Table 6, Figure 17)
The result shows that SH7 antagonist protein crude extract is dissolved in strong polar organic solvent acetone, Virahol and the methyl alcohol; Using material that relative weakly polar organic solvent dimethylbenzene, ether and ethyl acetate be extracted into from crude extract has in various degree bacteriostatic activity to tobacco ralstonia solanacearum, and aqueous portion keeps faint bacteriostatic activity.This result shows that the little antibacterial substance composition of antibacterial substance polarity that is produced by SH7 can be extracted by weakly polar organic solvent.Contrast each extraction solvent and all do not have bacteriostatic activity.
The bacteriostatic activity of different extracts in the thick leach protein liquid of table 6 SH7
| Extraction solvent | Sample | Antibacterial circle diameter (mm) |
| Dimethylbenzene ether ethyl acetate water | The thick leach protein solution of organic phase water contrast (dimethylbenzene) organic phase water contrast (ether) organic phase water contrast (ethyl acetate) | 27 12 0 28 0 0 24 10 0 28 |
Slightly carry antibacterial substance SDS-PAGE electrophoresis: electrophoresis carries out (Wang Jiazheng Fan Ming chief editor, the protein technical manual .2000. .ISBN7-03-008329-6 of Science Press) with reference to the method for Wang Jiazheng etc., adopts 12% separation gel, and 5% concentrates glue.Kao Masi light blue R-250 dyeing.Thick leach protein liquid of the SH7 of 70% ammonium sulfate precipitation and the thick leach protein 12%SDS-PAGE electrophoresis result (as Figure 18) after chloroform is handled show, the thick leach protein of SH7 excretory has many electrophoresis bands, and the thick leach protein after chloroform is handled is compared with the protein sample that is untreated, electrophoresis band at the 35KD place disappears, and illustrates that albumen herein is that SH7 secretes one of antibacterial proteic component.
Thick leach protein light absorption value is measured: thick leach protein liquid dilution is got final product colorimetric for suitable concentration, when making sample, do blank, return to zero with blank during colorimetric.On the UV-3010 of Hitachi ultraviolet spectrophotometer, scan and write down its optical density with all wave band.Thick leach protein is the light absorption value measurement result demonstration (as Figure 19) of 220-340nm at wavelength: SH7 excretory antimicrobial substance has obvious albumen absorption peak at the 274.00nm place, is typical protein adsorption spectrum.
In a word, the antagonistic substance of the bacillus subtilis strain SH7 that the present invention obtains has the antagonistic substance that following properties (1) antagonistic bacterium SH7 bacterial strain produces can be by ammonium sulfate precipitation, can not be by the dialysis tubing of 5kD (molecular weight that dams is greater than 5kD), insensitive to protease, chloroform is handled the back bacteriostatic activity and is weakened, and can think the protein antimicrobial substance.(2) the organic solvent extraction result shows, SH7 bacterial strain secretion bacteriostatics mass-energy is extracted by weakly polar organic solvent and can not be extracted by strong polar organic solvent.(3) antibacterial substance of antagonistic bacterium SH7 bacterial strain generation is stable under alkaline condition, and the antibacterial substance precipitation is separated out under acidic conditions; When the pH value was 3.0, antibacterial substance all precipitated, and centrifugal back supernatant does not have bacteriostatic activity.Show by uv-absorbing and SDS-PAGE electrophoresis detection test-results (4), prove that further SH7 excretory antagonistic substance is albumen or polypeptides matter, and be multi-component mixture that one antibacterial protein ingredient is arranged at the 35KD place.
Attached: nucleotide sequence involved in the present invention
SEQ ID NO1 (SH7 bacterial strain 16SrDNA complete sequence):
ctggctccataaaggttacctcaccgacttcgggtgttacaaactctcgtggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcatgctgat
ccgcgattactagcgattccagcttcacgcagtcgagttgcagactgcgatccgaactgagaacagatttgtgggattggcttaacctcgcggtttcgctgccctttg
ttctgtccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttcctccggtttgtcaccggcagtcaccttagagtgcccaa
ctgaatgctggcaactaagatcaagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaaccatgcaccacctgtcactctgccc
ccgaaggggacgtcctatctctaggattgtcagaggatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggccc
ccgtcaattcctttgagtttcagtcttgcgaccgtactccccaggcggagtgcttaatgcgttagctgcagcactaaggggcggaaaccccctaacacttagcactc
atcgtttacggcgtggactaccagggtatctaatcctgttcgctccccacgctttcgctcctcagcgtcagttacagaccagagagtcgccttcgccactggtgttcct
ccacatctctacgcatttcaccgctacacgtggaattccactctcctcttctgcactcaagttccccagtttccaatgaccctccccggttgagccgggggctttcacat
cagacttaagaaaccgcctgcgagccctttacgcccaataattccggacaacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttct
ggttaggtaccgtcaaggtgccgccctatttgaacggcacttgttcttccctaacaacagagctttacgatccgaaaaccttcatcactcacgcggcgttgctccgtc
agactttcgtccattgcggaagattccctactgctgcctcccgtaggagtctgggccgtgtctcagtcccagtgtggccgatcaccctctcaggtcggctacgcatc
gtcgccttggtgagccgttacctcaccaactagctaatgcgccgcgggtccatctgtaagtggtagccgaagccaccttttatgtctgaaccatgcggttcagacaa
ccatccggtattagccccggtttcccggagttatcccagtcttacaggcaggttacccacgtgttactcacccgtccgccgctaacatcagggagcaagctcccatc
tgtccgctcgac
Claims (6)
1. strain crop bacterial wilt biological and ecological methods to prevent plant disease, pests, and erosion subtilis (Bacillus subtilis) bacterial strain SH7, its biological preserving number is: CGMCC1732.
2. the application of the described bacterial strain SH7 of claim 1 in control crop bacterial wilt.
3. application according to claim 2, described crop are tobacco.
4. the application of the described bacterial strain SH7 of claim 1 in suppressing blue or green withered Lei Er Salmonella (Ralstonia solanacearum).
5. the described bacterial strain SH7 of claim 1 excretory antibacterial protein substance mixture is characterized in that: from bacterial strain SH7 fermented liquid, extract, and contain one under alkaline condition the albumen of stable 35kDa.
6. the application of the described antibacterial protein mixture of claim 5 in control crop bacterial wilt.
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