CN1680585A - Choice for preventing endogenic bacterial strain by potato - Google Patents
Choice for preventing endogenic bacterial strain by potato Download PDFInfo
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- CN1680585A CN1680585A CN 200510020328 CN200510020328A CN1680585A CN 1680585 A CN1680585 A CN 1680585A CN 200510020328 CN200510020328 CN 200510020328 CN 200510020328 A CN200510020328 A CN 200510020328A CN 1680585 A CN1680585 A CN 1680585A
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Abstract
A select method for strain preventing and curing endotrophic mycorrhiza in potato. Detect width of bacteriostasis braid and select strains with good effects of endotrophic mycorrhiza, do pot incubation test and field practice, detect effects of prevention and cure and fix planting ability. Detect action of the strain to pathogenetic fungi, and induct antiviral ability to potato. Advanced strain is obtained after the steps.
Description
Skill this area
The present invention relates to the long-term symbiosis of a kind of endogenetic bacteria and plant, can extenuate the defense response effect of host plant, be used for the system of selection of the endogenetic bacteria bacterial strain of the biological control of potato.
Background technology
The biocontrol microorganisms major part of Plant diseases is isolating from soil, rhizosphere or plant surface, and the bacterium that has antagonistic action at indoor measurement, when outdoor using, can not in host's body surface or body, normally grow surely mostly, be subjected to the influence of factor such as plant rhizosphere microbial population variation under changes in environmental conditions and the different ecological condition, cause their biocontrol effect instability, sometimes even invalid.
Mainly based on chemical prevention, not only medicament is difficult to long-term continuous and effective, goes back serious environment pollution in the production.Potato is important farm crop, behind the applying pesticides, not only influences quality, and the serious harm HUMAN HEALTH.
The late blight of potato is a destructive disease important on the potato, often causes serious economy loss.At present, the biological control of the late blight of potato mainly be utilize from the blade face, the isolating fungi of rhizosphere, bacterium.But its residing ecotope decision, the influence of severe environment factors such as the ultraviolet ray that is come from the outside, Storms and chemical substance.Affected by environment bigger, thereby its colonazition is poor, certain prevention effect is often arranged in the laboratory, and tend to occur the preventive effect instability in land for growing field crops production reality.
Summary of the invention
The purpose of this invention is to provide the system of selection of the biological control of a kind of potato endogenetic bacteria bacterial strain.The strain excellent that it filters out can be directly to the generation antagonistic action of pathogen with induce potato to produce resistant effect; the deciding of biocontrol microorganisms grow, affected by environment little; and be subjected to the protection of plant; stable living environment is arranged; can with the long-term symbiosis of plant; extenuate the defense response of host plant, effect is stable lasting.
The object of the present invention is achieved like this: get the pure bacterial strain of potato endogenetic bacteria, measure antibacterial bandwidth, filter out preventive effect bacterial strain preferably, get it and make pot experiment and field experiment, after measuring its biological control effect, the colonazition, potato endogenetic bacteria endogenetic bacteria bacterial strain of measuring the potato endogenetic bacteria again to the inducing anti-disease effect of potato, selected good endogenetic bacteria to the direct effect of pathogenic bacteria and potato endogenetic bacteria.
The colonazition of bacterial strain is measured, and potato set is used preventive effect endogenetic bacteria bacterial strain preferably, utilizes the antibiotic marker method to measure endogenetic bacteria and decide to grow density in breeding time different sites root, stem, leaf.
Mensuration preventive effect potato endogenetic bacteria bacterial strain preferably has following steps to the inducing anti-disease effect of potato:
A. inoculation activatory bacterial strain in nutrient solution is cultivated, and gets the sterilization of clear liquid filtering membrane and tucks in, and the potato haulm base portion is injected this liquid, the injection nutrient solution is contrast, the mould spore suspension of challenge inoculation potato epidemic disease, the dark processing of preserving moisture, keep illumination again, relative humidity is 80-90%, the investigation state of an illness;
B. the potato set of different growing is used the bacteria suspension of endogenetic bacteria, and bacteria suspension is cultivated for smear inoculating strain on flat board, adjusts concentration, and equivalent distilled water is executed in contrast.Measure the activity of peroxidase, polyphenoloxidase and phenylalanine ammonia lyase at a certain time interval, calculate the enzymic activity in every gram fresh weight tissue, analyze the changing conditions of inducing related enzyme activity in the potato body.
Advantage of the present invention is:
1. the microorganism in the microecosystem is used for biological control because itself is a composition in the microecosystem, help itself decide grow, and reduced selective pressure to pathogen, can also with the pathogen coevolution.Select the good biocontrol strain of endogenetic bacteria, because the colonazition of potato endogenetic bacteria is good, the preventive effect height.
2. select good biocontrol strains, endophytic bacterium can be distributed in the plant body, and growth and breeding is relatively stable, so good stability, is good biocontrol microorganisms resource, and some endogenetic bacteria also has growth-promoting functions.
3. utilize the beneficial microorganism controlling plant diseases, continuous and effective helps protecting environment.
4. can system be distributed in the plant tissue owing to endophytic bacterium; enough carbon sources, nitrogenous source are arranged; has good living environment; what overcome in the past from PGPR (rhizosphere is urged endophytic bacteria) screening biocontrol microorganisms decides to grow big shortcoming poor, affected by environment; be subjected to the protection of plant; than the bacterium that grows nonparasitically upon another plant that is exposed to as severe environment such as intensive daylight, UV-light, Stormss stable living environment is arranged more; can with the long-term symbiosis of plant; can extenuate the defense response effect of host plant, it is more stable lasting to act on.
5. separate in the potato body and select good biocontrol strains, meet the microecology principle, the endophyte that therefore separates from potato rhizome portion has better antibacterial and protection effect.
6. the microorganism that can play the biological and ecological methods to prevent plant disease, pests, and erosion effect in the plant microecology system except that pathogenic bacteria in addition exists, and they are by the restraining effect to pathogenic micro-organism, and the induction of resistance of plant is used for controlling disease.Be separated to probably in the potato body late blight of potato is controlled effect endogenetic bacteria preferably, and the preventive effect strain excellent can be because of the own characteristic of endogenetic bacteria, biocontrol effect more can be stablized lastingly.
Fungistatic effect preferably bacterial strain potato is also had the inducing anti-disease effect, the inoculation fungistatic effect is preferably behind the bacterial strain, peroxidase of potato and phenylalanine ammonia lyase and polyphenol oxidase activity obviously improve, and bacterial strain can directly produce antagonistic action and induce potato to produce resistant effect pathogen.Peroxidase extensively is present in the various piece of vegetable cell, but the formation of catalysis xylogen such as lubanol, and cell walls carbohydrate and protein formation covalent linkage, peroxidase participates in the synthetic of xylogen, aldehydes matter machine plant protecting chemical, cell wall structure is relevant with resistance, the weak plant in plant peroxidase plateau that plant resistance to environment stress is strong; Phenylalanine ammonia lyase is key enzyme and the rate-limiting enzyme in the phenylpropyl alcohol alkanes pathways metabolism, because it is relevant that the intermediate product aldehydes matter of this approach and materials such as end product xylogen, flavones, isoflavonoid and the defence pathogenic micro-organism of plant infect, so phenylalanine ammonia lyase is the defensive ferment of plant.
8. to the mensuration of deciding to grow density of potato endogenetic bacteria different sites, different time in potato,, endogenetic bacteria provides more strong foundation for being used for the biological control of potato disease.
9, found through experiments, the endogenetic bacteria with biological control effect also has growth-promoting functions to potato, and biocontrol of plant disease and plant growth-promoting effect have closely been connected.
The good endogenetic bacteria that adopts present method to select is used as biocontrol microorganisms, and energy Sustainable Control late blight improves biocontrol effect, the protection environment.Effect organically is joined together with the colonization ability of strain excellent, greenhouse test, land for growing field crops preventive effect, meta-bolites, inducing anti-disease effect and to plant growth-promoting, creates conditions and lays a solid foundation for further being applied to produce.
The present invention's preventive effect in greenhouse test can reach 68.23%, prevents and treats the effective of the late blight of potato than utilizing root, leaf to enclose bacterium in the past.Preventive effect can reach 58.53% in the field experiment, and the short fruit of coming into force reaches 15.10%.
Be that the present invention is described further for example to select the biological control of potato endogenetic bacteria bacterial strain to prevent and treat the late blight of potato below.
Embodiment
1. healthy potato root to be separated, stem, leaf are cleaned, dried, soak 2-5min with 75% alcohol, 0.1% mercuric chloride soaks 1-2min, cleans 4 times with aqua sterilisa successively, adds aqua sterilisa and grinds on a small quantity, leave standstill 10-15min, get bacteria suspension with the bent glass rod libation at an ancient wedding ceremony and be applied on the flat board, be coated with two flat boards continuously.Be coated with flat board with the last aqua sterilisa that cleans parting material simultaneously,, give up institute's isolated strains if grow bacterium colony on this flat board later on.Cultivate 48 hours in 28 ℃ of thermostat containers. picking list bacterium colony purifying obtains pure bacterial strain tube and preserves stand-by.
2. dull and stereotyped antagonistic effect: the potato endogenetic bacteria with the potato epidemic disease is mould relatively is seeded on the rye protein peptone culture medium flat plate, survey antibacterial bandwidth two days later.To bacterial strain screening once more on rye protein peptone culture medium flat plate that bacteriostatic action is arranged, dull and stereotyped center inoculation potato epidemic disease is mould, bacterium piece d=0.4cm, both sides symmetry inoculation endogenetic bacteria bacterial strain to be measured, the libation at an ancient wedding ceremony is got bacteria suspension and is respectively drawn a straight line at 2.5cm place, distance center, each bacterial strain repeats 3 times, surveys antibacterial bandwidth with dull and stereotyped antagonistic effect after 3 days, the record statistics.
3, make even the plate fungistatic effect better the bacterial strain (following is example with strains A tpoe) of (be with wide more, description effect is good more) do that the greenhouse pot culture control is sick tests.Executed once bacterium to be measured in preceding 18 days respectively with the inoculation pathogenic bacteria during transplanting, contrast and execute the equivalent clear water, each is handled 3 times and repeats, and each repeats 12 strains, and the mould inoculation of potato epidemic disease is a spray inoculation, and with the moistening blade of a small amount of clear water, 10ml bacterium liquid is inoculated in every strain before inoculating.20 ℃ of dark processing 24 hours, again 28 ℃ keep illumination (fluorescent lamp, 4500LX, 16h/d), relative humidity 80-90%.7 days " Invest, Then Investigate " state of an illness.According to test-results, calculate preventive effect, the big more effect of numerical value is good more, filter out preventive effect preferably bacterial strain further make pot experiment.
4, land for growing field crops amplification test: transplant, 10 times of Atpoe bacterium liquid dilutions, soaked root during transplanting 5 minutes earlier, deal with and be numbered A1; The cave is watered 500 milliliters/strain and is irritated root during 50 times of transplantings of bacterium liquid dilution, deals with and is numbered A2; Conventional production is done 3 meters interval processing and is numbered A0; Only using clear water to irritate root during transplanting compares.Except that test was handled, other production management measures were consistent with field management.
Respectively in group, budding, breeding time of three susceptible diseases in garden top investigates its late blight a situation arises, and at that time potato plant root system, growth potential and incidence are observed.
5, endogenetic bacteria Atpoe colonazition is measured: use potato endogenetic bacteria Atpoe 4-6 leaf phase potato set, through antibiotic marker (effect of mark is in order to measure its site of action), concentration is 1 * 10
8Cfu/ml.Measured it at 5 days, 10 days, 15 days respectively and decide to grow density at potato root, stem, leaf.
6, the direct mechanism of action of endogenetic bacteria Atpoe.
Cover the thick rye protein peptone substratum of the about 1mm of one deck on the sterilization slide glass, the potato epidemic disease is mould to be inoculated at a distance of 1.5cm with the Atpoe bacterial strain, is put on the sterilization culture dish introversion glass rod, adds a small amount of aqua sterilisa and preserves moisture, and it is mould that the potato epidemic disease is only inoculated in contrast.Cultivated 48 hours for 28 ℃.
Strains A tpoe to the effect of mycelia is, face-off is cultivated, and bacteriostatic action is obvious after 60 hours, and recording antibacterial bandwidth is 8mm, the fall mycelia form in forward position of microscopy potato phytophthora, the mycelia wall is cleared up, deformity obviously, the division aggravation.
Inoculation activatory strains A tpoe in the 100ml nutrient solution, shaking culture is 3 days under 28 ℃ of 120 commentariess on classics/min condition, the centrifugal 15min of 10000 commentaries on classics/min, supernatant liquor is sterilized stand-by with 0.22 μ m filtering membrane.Filtered liquid is mixed in the rye substratum in 1%, 3%, 5%, 8%, 10%, 20% ratio, contrast to not with the substratum of fermented liquid, the substratum total amount is 10ml.Dull and stereotyped center inoculation potato phytophthora silk piece (d=0.4cm), the colony growth amount is surveyed in 3 repetitions after 2 days.
The influence that the fermentation concentrated solution discharges sporocyst:
Inoculation activatory strains A tpoe in the 100mlNA nutrient solution, shaking culture is 3 days under 28 ℃ of 120r/min conditions, the centrifugal 15min of 4000r/min, supernatant liquor concentrates 8 times with Sephadex G-50, the centrifugal 15min of 10000r/min, filtration sterilization, be stored in 4 ℃ down stand-by, this is the concentrated solution of strains A tpoe fermented liquid.
Getting concentration is 1 * 10
4Individual/mould sporangia suspension of ml potato epidemic disease and above-mentioned concentrated solution are mixed by 1: 1, contrast adds meat peptone nutrient solution, repeat for 3 times, handle 20min for 8 ℃, surveyed the sporocyst release rate in following 0.5 hour, 1.5 hours, 4.5 hours, 10 hours, 22 hours respectively at 24 ℃ thereafter.
The fermentation concentrated solution is that the fermented liquid of Atpoe concentrates the obviously influence of sporocyst release nothing, handles and contrast sporocyst release rate basically identical to the influence that sporocyst discharges.
The influence that the fermentation concentrated solution moves about to zoospore:
The acquisition of zoospore, sporangia suspension is handled 20min at 8 ℃, behind 24 ℃ of following 20min, uses two-layer filtered through gauze, and adjusting zoospore concentration is 1 * 10
4Individual/ml.
Getting concentration is 1 * 10
4The zoospore suspension of individual/ml and above-mentioned concentrated solution are mixed by 1: 1, and contrast adds meat peptone nutrient solution, repeat, survey the per-cent that moves about of zoospore respectively when 24 ℃ of following 5min, 10min, 30min for 3 times.
The fermentation concentrated solution to the influence that zoosporangium moves about be, the fermentation concentrated solution can obviously suppress moving about of spore, 5min aftertreatment zoospore per-cent for the contrast about 1/2nd, 10min aftertreatment zoospore per-cent for the contrast sixth about.
The fermentation concentrated solution is to the influence of spore germination:
Getting concentration is 1 * 10
4The zoospore suspension of individual/ml and above-mentioned concentrated solution are mixed by 1: 1,5: 1, the glucose of adding 2%, contrast adds the NA nutrient solution, 3 repetitions, 24 ℃ of sprouting per-cents of surveying spore after following 4 hours respectively, the length of germ tube surpass the spore diameter length half just as spore of having sprouted.
The influence that the fermentation concentrated solution is sprouted zoospore is, surveys the sprouting per-cent of zoospore in 4 hours respectively, and concentrated solution made zoospore can not sprout 1 at 1: 1 o'clock; Made the zoospore germination rate descend about 1/2nd at 5 o'clock.
7, the inducing anti-disease mechanism of action of endogenetic bacteria Atpoe.
Inoculation activatory strains A tpoe in the 100ml nutrient solution, shaking culture is 3 days under 28 ℃ of 120 commentariess on classics/min condition, the centrifugal 15min of 4000 commentaries on classics/min, supernatant liquor is sterilized stand-by with 0.22 μ m filtering membrane.
In this liquid of the above 2-3cm of 8-10 leaf phase potato set basal part of stem place injection 0.1ml/ strain, establish injection meat peptone nutrient solution and be contrast.Each handles 3 repetitions, and each repeats 12 strains.Challenge inoculation after 6 days, the mould inoculation of potato epidemic disease is for being spray inoculation, and inoculation is preceding with the moistening blade of a small amount of clear water, every strain inoculation 10ml bacterium liquid, 20 ℃ of dark are preserved moisture and were handled 24 hours, and 28 ℃ keep illumination (fluorescent lamp again, 4500LX, 16h/d), relative humidity 80-90%.7 days " Invest, Then Investigate " state of an illness.Strains A tpoe meta-bolites has tangible inducing anti-disease effect, and preventive effect is 48.55%.
4-6 leaf phase potato set is executed strains A tpoe bacterium liquid, every strain 10ml, equivalent distilled water is executed in contrast.Bacterium liquid is to smear inoculating strain AtpAtpoe on NA (the meat peptone nutrient solution) flat board, cultivates 3d down at 28 ℃, and adjusting concentration with distilled water is 1 * 10
8Cfu/ml.Measured the activity of peroxidase, polyphenoloxidase and phenylalanine ammonia lyase respectively at 1 day, 3 days, 5 days, 7 days, 9 days, 11 days, 13 days.After executing bacterium, the activity of peroxidase, polyphenoloxidase and phenylalanine ammonia lyase, obviously comparison is according to high, and in the time of 13 days, comparison is according to 32.23,29.65,24.05 enzyme activity units that rise respectively.
Conclusion: because moving about, sprouting of the mould mycelial growth of potato epidemic disease, zoospore all is late blight of potato development of disease, popular important step, as if interrupting in any link, development of disease is popular all will to be suppressed.Potato endogenetic bacteria Atpoe to the moving about of the mould mycelial growth of potato epidemic disease, zoospore, sprout the obvious suppression effect arranged, thereby strains A tpoe directly the inhibition of potato epidemic disease is mould.And can induce potato to produce disease resistance, in case the evil of curing the disease.
The embodiment of the invention is done with potato endogenetic bacteria Atpoe and is specified, can select the good biocontrol strains of potato endogenetic bacteria to prevent and treat other potato disease with method of the present invention, equally also can select the good biocontrol strains of other endophytic bacterium to prevent and treat other Plant diseases, can reach effect of the present invention.
Claims (3)
1. the system of selection of the biological control of a potato endogenetic bacteria bacterial strain, it is characterized in that: get the pure bacterial strain of potato endogenetic bacteria, measure antibacterial bandwidth, filter out preventive effect bacterial strain preferably, get it and make pot experiment and field experiment, after measuring its biological control effect, the colonazition, potato endogenetic bacteria bacterial strain of measuring the potato endogenetic bacteria again to the inducing anti-disease effect of potato, selected good endogenetic bacteria to the direct effect of pathogenic bacteria and potato endogenetic bacteria.
2. the system of selection of the biological control of potato according to claim 1 endogenetic bacteria bacterial strain, it is characterized in that: the colonazition of bacterial strain is measured and is, potato set is used preventive effect endogenetic bacteria bacterial strain preferably, utilize the antibiotic marker method to measure endogenetic bacteria and in breeding time different sites root, stem, leaf, decide to grow density.
3. the system of selection of the biological control of potato according to claim 1 endogenetic bacteria bacterial strain is characterized in that: measure the inducing anti-disease effect of potato endogenetic bacteria bacterial strain to potato, following steps are arranged:
A. inoculation activatory bacterial strain in nutrient solution is cultivated, and gets the sterilization of clear liquid filtering membrane, and the potato haulm base portion is injected this liquid, the injection nutrient solution is contrast, the mould spore suspension of challenge inoculation potato epidemic disease, the dark processing of preserving moisture, keep illumination again, relative humidity is 80-90%, the investigation state of an illness;
B. the potato set of different growing is used the bacteria suspension of endogenetic bacteria, and bacteria suspension is cultivated for smear inoculating strain on flat board, adjusts concentration, and equivalent distilled water is executed in contrast.Measure the activity of peroxidase, polyphenoloxidase and phenylalanine ammonia lyase at a certain time interval, calculate the enzymic activity in every gram fresh weight tissue, analyze the changing conditions of inducing related enzyme activity in the potato body.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381762B (en) * | 2008-10-24 | 2011-01-26 | 福建省农业科学院植物保护研究所 | Improved measurement method of antagonistic bacteria |
| CN102666572A (en) * | 2009-09-18 | 2012-09-12 | 瓦赫宁恩大学 | Cloning and exploitation of a functional R-gene from solanum chacoense |
| CN103755433A (en) * | 2013-12-13 | 2014-04-30 | 刘美龙 | Potato late bright prevention and control agent set and a potato late bright prevention and control method |
-
2005
- 2005-01-31 CN CN 200510020328 patent/CN1289689C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381762B (en) * | 2008-10-24 | 2011-01-26 | 福建省农业科学院植物保护研究所 | Improved measurement method of antagonistic bacteria |
| CN102666572A (en) * | 2009-09-18 | 2012-09-12 | 瓦赫宁恩大学 | Cloning and exploitation of a functional R-gene from solanum chacoense |
| CN102666572B (en) * | 2009-09-18 | 2016-08-10 | 瓦赫宁恩大学 | The clone of functional r-gene and exploitation from Solanum chacoense |
| CN103755433A (en) * | 2013-12-13 | 2014-04-30 | 刘美龙 | Potato late bright prevention and control agent set and a potato late bright prevention and control method |
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| Publication number | Publication date |
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| CN1289689C (en) | 2006-12-13 |
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