CN102174427A - Endophytic antagonistic strain for controlling soft rot of camellia oleifera and application thereof - Google Patents
Endophytic antagonistic strain for controlling soft rot of camellia oleifera and application thereof Download PDFInfo
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- CN102174427A CN102174427A CN 201010605101 CN201010605101A CN102174427A CN 102174427 A CN102174427 A CN 102174427A CN 201010605101 CN201010605101 CN 201010605101 CN 201010605101 A CN201010605101 A CN 201010605101A CN 102174427 A CN102174427 A CN 102174427A
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Abstract
The invention discloses an endophytic antagonistic strain for controlling soft rot of camellia oleifera and application thereof. The strain is bacillus amyloliquefaciens sp. L46, with a collection number of CCTCC M2010324. The bacillus amyloliquefaciens L46 is obtained by flat-plate antagonistic test sieving, can be used for effectively suppressing growth of soft rot pathogenic bacteria of camellia oleifera, and has the advantages of high prevention effect, high environmental safety and broad development and application prospect.
Description
Technical field
The invention belongs to the plant protection field, be specifically related to a kind of interior living antagonistic strain and application thereof that prevents and treats the oil tea soft rot, relate in particular to a kind of bacillus amyloliquefaciens L46 and application of high-efficiency prevention and control oil tea soft rot.
Background technology
Oil tea (Camellia oleifera) is the distinctive edible oil materials seeds of China, also is one of the world's four big woody oil tree species, utilization and the cultivation history in existing more than 2000 year.Its wide adaptability, strong stress resistance, suitable kind in vast red soil hills lowland area.Mainly be grown in south, based on Hunan, Jiangxi, Guangxi, wherein the Hunan Province is the oil tea place of production and the maximum tea oil consumption area of domestic maximum.
At present, the disease species of oil tea is various, and its hazard rating is also more serious.Due to illness the financial loss that does harm to every year and cause is difficult to estimate that wherein the oil tea soft rot is a kind of common oil tea disease.The oil tea soft rot is that cause of disease is oil tea umbrella shank spore bacterium (Agari-codochium camelliae Liu, Wei et Fan), belongs to imperfect fungi hyphomycetes Moniliales umbrella shank spore and belongs to.In the camellia oleifera lam of growing up, this disease is very few to threaten to full wafer tea woods, and the shedding but susceptible individual plant is fallen leaves in a large number causes very big loss.The oil tea soft rot has now become additional Quarantine Objects.
In each province, China south, the oil tea soft rot all has harm in various degree.This disease main harm oil tea blade and fruit, rotting in the position that is injured, and causes a large amount of fallen leaves and shedding, even leaf, the fruit light that falls.In the nursery, the oil tea occupation is injured quite serious, even whole strain is withered.The oil tea soft rot has now become additional Quarantine Objects.At present, to the control of oil tea soft rot mainly based on chemical prevention, but for a long time, use chemical pesticide to exist potential harm repeatedly and in a large number, it causes soil, water body and atmospheric pollution, pesticide residue increase in the agricultural byproducts, cause food safety question serious day by day, and chemical pesticide has also destroyed the eubiosis when killing pathogenic bacteria.Pathogenic fungi and resistance are constantly deepened and are enlarged simultaneously, thus the vicious cycle that can cause chemical pesticide to use.Utilizing biological control is that microbial metabolites (agricultural antibiotic) or microorganism live bacteria preparation controlling plant diseases have not only avoided chemical pesticide to bring a series of problems, has developed into to plant one of crucial effective measure in the disease control.Biological control is with its safety, efficiently reach the important channel that characteristics such as pollution-free have become control of plant disease, wherein antagonistic bacterium plays important effect in biocontrol of plant disease, its main advantage is: 1) kind and One's name is legion exist in plant phyllospheric, leaf in a large number; 2) mode of action to pathogenic bacteria is varied, comprises the generation of competition, antagonism, parasitism, inducing plant resistance etc.; 3) reproduction speed is fast; 4) many bacteriums in the leaf of plant or the phyllosphere colonazition strong; 5) bacterium mostly can artificial culture, and easy handling is convenient to production application; 6) some bacterium not only can prevent and treat disease and can increase crop yield.
Separation screening obtains biocontrol strain that the oil tea Bacteria erwinia is had antagonistic action in the oil tea plant, control to the oil tea soft rot has great importance, but correlation technique is still very weak even blank at present, so far the antagonistic bacterium that can effectively prevent and treat oil tea soft rot disease in more than 20 year does not appear in the newspapers, and does not does not prevent and treat the report of the biological pesticide of oil tea soft rot yet.
Summary of the invention
The objective of the invention is blank at prior art, a kind of interior living antagonistic strain bacillus amyloliquefaciens L46 of high-efficiency prevention and control oil tea soft rot is provided, bacillus amyloliquefaciens L46 be a kind of in the oil tea plant separation screening obtain biocontrol bacteria bacterial strain that the oil tea Bacteria erwinia is had antagonistic action, significant to the control of oil tea soft rot.
The bacillus amyloliquefaciens L46 that another object of the present invention provides described high-efficiency prevention and control oil tea soft rot is in the multiple application that prevents and treats aspect the soft rot disaster.
The bacillus amyloliquefaciens L46 of high-efficiency prevention and control oil tea soft rot of the present invention, Bacillus amyloliquefaciens sp.L46 on November 30th, 2010, has carried out preservation at China typical culture collection center, preserving number CCTCC M 2010324.Its bacterium colony on beef-protein medium is creamy white, circle or subcircular, and the edge is irregular, central uplift, dry wrinkle, opaque, chromogenesis not, the general 2～3mm of colony diameter, thalline is shaft-like, gives birth to Gram-positive in the gemma.
The bacillus amyloliquefaciens L46 of high-efficiency prevention and control oil tea soft rot of the present invention obtains by dull and stereotyped face-off experiment sieving.
The present invention also provides the application of the bacillus amyloliquefaciens L46 of described high-efficiency prevention and control oil tea soft rot, and the first is to the restraining effect of oil tea soft rot germ mycelia.
It two is teratogenesis to oil tea soft rot germ mycelia.
It three is restraining effect to oil tea soft rot germ mycelia conidia germination.
The invention has the beneficial effects as follows:
(1) bacillus amyloliquefaciens L46 is an endophytic bacterium, strong to oil tea Bacteria erwinia restraining effect, multiple pathogenic fungi had antagonistic action, has the important effect that antagonistic bacterium is played in biocontrol of plant disease, for example kind and One's name is legion exist in plant phyllospheric, leaf in a large number; The mode of action to pathogenic bacteria is varied; Advantages such as reproduction speed is fast, especially significant to the control of oil tea soft rot.
(2) the strain culturing condition is simple, preserves easily, is easy to suitability for industrialized production, has the excellent development application prospect.
Description of drawings
Fig. 1 is the colonial morphology picture of L46 bacterial strain;
Fig. 2 is the phylogenetic tree picture of L46 bacterial strain;
Fig. 3 is the flat board face-off antagonistic action picture of L46 bacterial strain to the oil tea Bacteria erwinia;
Fig. 4 be under 10 * 100 power microscopes observed bacillus amyloliquefaciens L46 to the teratogenesis picture of oil tea soft rot mycelia;
Wherein 1: the pathogenic bacteria mycelia (10 * 100) of normal mycelia; 2: early stage, mycelia was expanded outstanding (10 * 100).
Embodiment
The invention provides the new bacterial strain of bacillus amyloliquefaciens L46, it shows has stronger antagonistic activity to the oil tea soft rot.
Strains separation of the present invention is from the oil tea leaf.
Bacillus amyloliquefaciens L46 of the present invention demonstrates following form and biochemical character: bacterium colony is creamy white on beef-protein medium, circle or subcircular (see figure 1), the edge is more neat, central uplift, dry fold, opaque, chromogenesis not, the general 2～3mm of colony diameter, thalline is shaft-like, Gram-positive is given birth in the gemma.The biochemical reaction catalase of bacterial strain, glucose fermentation, aerobism are measured and are positive, V-P, clark and Lubsreaction feminine gender, its growth energy hydrolyzed starch, gelatin.
Bacillus amyloliquefaciens L46 of the present invention, through 16S rDNA sequential analysis, constructing system is grown tree, shows that itself and Bacillusamyloliquefaciens have very high affinity (see figure 2).
The present invention is described in further detail below in conjunction with embodiment, and can not limit the present invention.
The separation of embodiment 1 bacillus amyloliquefaciens L46
Get the healthy leaf of common oil tea, adopt that conventional method separates, purifying and preservation.
After oil tea leaf water is rinsed well, choose an amount of health tissues, respectively after the weighing, be cut into the tissue block of 5mm * 5mm, in 75% ethanol, soak 1min, in 0.1% mercuric chloride solution, soak 40s～70s then, put into 75% alcohol immersion 30s again, take out back aseptic water washing 4 times.In aseptic mortar, add the sterilized water of 10ml and the quartz sand of a little bacterium of going out and grind to form homogenate, add a cover and leave standstill 30min, behind 10 times of serial dilutions of homogenate, draw 0.1ml respectively to ready NA flat board with liquid-transfering gun, put into incubator after smearing evenly and cultivate, the record bacterium is measured behind the 48h, and chooses according to colonial morphology, color and size, on flat board, carry out purification storage, be divided into from 5 batches.
The screening of embodiment 2 bacillus amyloliquefaciens L46
Primary dcreening operation: adopt the face-off culture method.Endophyte antagonism pathogenic fungi is screened.With accomplishing fluently diameter is that the oil tea Bacteria erwinia bacterium cake of 6mm is put in dull and stereotyped central authorities, dip in the suspension of getting tested bacteria equidistant (anomaly plate central authorities 3cm) point and connect 2～3 kinds of endogenetic bacterias with connecing collarium then, be put in 25 ℃ of dark culturing in the incubator, the size that has that it's too late of the antibacterial band of observed and recorded repeats 3 times altogether behind the 4d.Select antibacterial bandwidth and carry out multiple sieve, be followed successively by L1, L6, L12, L19, L29, L33, L35, L39, L46, L48, carry out multiple sieve (seeing Table 1) more than or equal to 10 bacterial strains of 4mm.
Table 1 endogenetic bacteria is to the bacteriostatic action of oil tea Bacteria erwinia
Multiple sieve: adopt fermentation method.The 10 strain bacterium that preliminary screening is gone out pipette a ring and insert (250m1) in the triangular flask that the 150mlNB nutrient solution is housed with connecing collarium respectively after cultivating 2d on the NA substratum, shaking culture 48h on 28 ℃, the shaking table of 150rpm.Nutrient solution filters with 0.22 μ m biofilter and obtains culturing filtrate, pour filtrate and about 50 ℃ of PDA substratum into culture dish by 1: 19 mixing, the soft-rot bacterium bacterium cake of d=6mm is put in the cooling back in dull and stereotyped central authorities, measure the germ colony diameter behind the 4d, with the sterilized water is contrast, the fungistatic effect of L46 bacterial strain fermentation liquor is the most obvious, and inhibiting rate reaches 83.4% (seeing Table 2).
Table 2 fermented liquid is to the restraining effect of oil tea Bacteria erwinia
Embodiment 3 bacillus amyloliquefaciens L46 are to the restraining effect of oil tea soft rot mycelia
With diameter is that the oil tea Bacteria erwinia bacterium cake of 6mm is put in the dull and stereotyped central authorities of potato sucrose substratum PDA, and equidistant then 3cm point meets antagonistic bacterium L46, repeats 25～28 ℃ of cultivations, the diameter of the antibacterial band of mensuration after 4 days three times.Bacillus amyloliquefaciens L46 is strong to oil tea Bacteria erwinia bacteriostatic action as seen from Figure 3.
Embodiment 4 bacillus amyloliquefaciens L46 are to inhibition and the teratogenesis of oil tea soft rot mycelia
Glassine paper is cut into the strip of 5mm * 25mm, 121 ℃ of autoclavings, with tweezers with its lay between bacterial strain L46 and pathogenic bacteria.28 ℃ of constant temperature culture 5～7 days, tangible antibacterial band appears between bacterial strain L46 and the pathogenic bacteria mycelia and after, glassine paper is carefully taken off, place the metamorphosis of observation mycelia 10 * 40 power microscopes under, be contrast with the pathogenic bacteria that does not meet bacterial strain L46.Observe mycelia and expand outstandingly in earlier stage, the later stage obviously attenuates, and form is inhomogeneous, and mycelia has fracture, broken phenomenon (see figure 4), and wherein 1: the pathogenic bacteria mycelia (10 * 100) of normal mycelia; 2: early stage, mycelia was expanded outstanding (10 * 100).
Embodiment 5 bacillus amyloliquefaciens L46 are to the restraining effect of oil tea soft rot mycelia conidia germination
Adopt the slide glass sessile drop method.Oil tea soft-rot bacterium germ cultivated 7d in (22 ℃) incubator on the PDA flat board after, transfer in the test tube that fills aqua sterilisa with transfering loop picking sorus, vibrate be made into after shaking up spore suspension (5 * 108/m1), standby.Get 100 μ l spore suspensions, mix respectively with the fermented liquid and the 100 μ L ferment filtrates of the fermenation raw liquid of 100 μ L L46 bacterial strains and diluted 10 times, 100 times and (the bacterial classification L46 that grows on the inclined-plane to be inserted in the 50ml NA liquid nutrient medium with transfering loop, 30 ℃, 140r/min, shaking culture 24h is fermenation raw liquid, is fermented liquid after the dilution, and centrifugal back gained supernatant liquor is ferment filtrate), being added drop-wise on the depression slide, is contrast to drip sterilized water.Every processing repeats 3 times, places 28 ℃ of incubators to cultivate, and microscopy spore germination situation after the 48h is calculated spore germination rate.By table 3 result as can be known, the fermenation raw liquid of L46 all has the obvious suppression effect to the growth of conidial sprouting of Bacteria erwinia and germ tube, and wherein the inhibiting rate to spore germination reaches 93.7%, and the diluted back of fermented liquid suppresses effect and reduces gradually.The L46 ferment filtrate is also fairly obvious to the inhibition effect of spore germination, makes spore germination rate reduce by 87.7%, and the result shows that bacterial strain may produce has inhibiting meta-bolites to Bacteria erwinia in the fermentation culture process.
Table 3 endophyte strain nutrient solution is to the influence of Bacteria erwinia conidia germination
Claims (4)
1. an interior living antagonistic strain and an application thereof that prevents and treats the oil tea soft rot is characterized in that, described bacterial strain is bacillus amyloliquefaciens Bacillus amyloliquefaciens sp.L46, preserving number CCTCC M 2010324.
2. the described bacterial strain of claim 1 is applied to antagonism oil tea soft rot germ.
3. application method according to claim 2 is characterized in that, described bacterial strain is applied to suppress or the mycelia of teratogenesis oil tea soft rot germ.
4. application method according to claim 2 is characterized in that, described bacterial strain is applied to suppress the conidial sprouting of oil tea soft rot germ mycelia.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103274825A (en) * | 2013-06-09 | 2013-09-04 | 苏州仁成生物科技有限公司 | Special polyelement microorganism fertilizer for preventing celery soft rot and preparation method of special polyelement microorganism fertilizer |
| CN105219681A (en) * | 2015-11-04 | 2016-01-06 | 湖北工程学院 | A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM and preparation method and application |
| CN112322521A (en) * | 2020-10-23 | 2021-02-05 | 贵州大学 | Bacillus amyloliquefaciens GUOR0918 and application thereof in preventing and treating soft rot of kiwi fruits |
Citations (2)
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| CN1792167A (en) * | 2005-12-27 | 2006-06-28 | 云南农业大学 | Biological prepn. for preventing and controlling soft rot of konjak, and its application |
| CN101892156A (en) * | 2010-07-02 | 2010-11-24 | 湖北省农业科学院经济作物研究所 | Rapid separation method of antagonistic bacteria against pathogens of konjac soft rot disease |
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| CN1792167A (en) * | 2005-12-27 | 2006-06-28 | 云南农业大学 | Biological prepn. for preventing and controlling soft rot of konjak, and its application |
| CN101892156A (en) * | 2010-07-02 | 2010-11-24 | 湖北省农业科学院经济作物研究所 | Rapid separation method of antagonistic bacteria against pathogens of konjac soft rot disease |
Non-Patent Citations (1)
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103274825A (en) * | 2013-06-09 | 2013-09-04 | 苏州仁成生物科技有限公司 | Special polyelement microorganism fertilizer for preventing celery soft rot and preparation method of special polyelement microorganism fertilizer |
| CN105219681A (en) * | 2015-11-04 | 2016-01-06 | 湖北工程学院 | A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM and preparation method and application |
| CN105219681B (en) * | 2015-11-04 | 2018-02-13 | 湖北工程学院 | A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM and preparation method and application |
| CN112322521A (en) * | 2020-10-23 | 2021-02-05 | 贵州大学 | Bacillus amyloliquefaciens GUOR0918 and application thereof in preventing and treating soft rot of kiwi fruits |
| CN112322521B (en) * | 2020-10-23 | 2022-05-03 | 贵州大学 | Bacillus amyloliquefaciens GUOR0918 and application thereof in preventing and treating soft rot of kiwi fruits |
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