CN101381762B - Improved measurement method of antagonistic bacteria - Google Patents
Improved measurement method of antagonistic bacteria Download PDFInfo
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- CN101381762B CN101381762B CN2008100719870A CN200810071987A CN101381762B CN 101381762 B CN101381762 B CN 101381762B CN 2008100719870 A CN2008100719870 A CN 2008100719870A CN 200810071987 A CN200810071987 A CN 200810071987A CN 101381762 B CN101381762 B CN 101381762B
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Abstract
The invention provides an improved bioassay method for an antagonistic bacterium, namely a double-layer culture medium filter paper sandwich method, which belongs to the technical field of a bioassay method for a strain to be tested which is required to be adopted for biological pesticide development. The antibacterial activity of the antagonistic bacterium on plant pathogenic fungi is determinedthrough the simple and easy procedures of manufacture of a sterile filter paper sheet die, manufacture of an upper culture medium and a lower culture medium, shift-in and shift-out of the sterile filter paper die, culture of the antagonistic bacterium to be tested and the target pathogenic fungi, measurement of experimental results and so on, and the antagonistic bacterium with application value can be reasonable screened out. The simple, convenient and quick novel method for implementing bioassay of the antagonistic bacterium has technological innovation. The method can truly reflect the antibacterial ability of metabolites of the strain, has the advantages of good test stability, good repeatability, cheap materials, simple and convenient operation, short experimental period and so on, can realize large-scale determination of a plurality of antagonistic bacteria on a plurality of the pathogenic fungi, and has good function on research of the biological and ecological stability of theantagonistic bacteria.
Description
Technical field
The technology of the present invention field belongs to the agronomy class, and the plant pathology secondary subject that plant protection one-level subject is divided into more specifically relates to necessary antagonistic bacterium biological activity determination method in a kind of biological pesticide development and the exploitation.
Background technology
1, more and more important effect is being brought into play in biological control in the development of sustainable agriculture.An important content of biological control utilizes the microbial control disease exactly, be used in the beneficial microorganism to study the control fungal diseases of plants more be bacterium, some kinds as bacillus (Bacillus sp) and false monospore Bacillaceae (Pseudomonas sp), people are referred to as antagonistic bacterium or biocontrol bacteria usually, numerously studies have shown that some bacteriums have stronger restraining effect to plant pathogenic fungi growth, cultivate with the germ face-off, can show tangible antibacterial band, its metabolite mass-energy cause the germ mycelia clear up with cell in molten, can suppress germ mycelial growth and spore germination, in disease control, demonstrate biocontrol effect preferably.
2, in the screening process of antagonistic bacterium, antagonistic bacterium has crucial effects to the bioassay method of pathogenic bacteria, and it is the biological pesticide development and develops a requisite important step.The bioassay method that adopts has dull and stereotyped face-off culture method between antagonism bacterium and the germ usually at present; Thalline directly acts on assay method, Sanming City therapy; Antagonistic bacterium does not have fermented liquid (biofilter filtration, high-temperature sterilization) antibacterial ability assay method, comprises again that wherein fermented liquid mixes inhibition germ colony growth assay method, fermented liquid inhibition spore germination assay method, fermented liquid cup-plate method, fermented liquid filter paper method etc. in substratum.We through for many years experimental studies have found that these methods in use exist tangible limitation.
(1) dull and stereotyped face-off method, dull and stereotyped face-off culture method between antagonistic bacterium and the germ, usually between can form a tangible antibacterial band (about 3mm～18mm), this method operation is easier, the primary dcreening operation that is suitable for antagonistic bacterium, but the antibacterial band size that antagonistic bacterium forms is not only relevant with antagonistic bacterium self excretory meta-bolites bacteriostatic activity, also relevant with bacterial strain meta-bolites diffusibility on substratum, people adopt dull and stereotyped face-off method usually with the index of antibacterial bandwidth value as evaluation antagonistic strain antibacterial ability, and keep the big bacterial strain of antibacterial bandwidth value, eliminate the little bacterial strain of antibacterial bandwidth value.We can lose thus one poor because of the excretory meta-bolites in the substratum diffusibility, and the strong good bacterial strain of meta-bolites bacteriostatic activity.The development of biological pesticide usually is the antimicrobial characteristic that utilizes the bacterial strain meta-bolites in the reality.Therefore adopt this method, can not really reflect antagonistic bacterium potential antibacterial ability.Moreover with this method test stability, poor repeatability, after same antagonistic bacterium point connect, the bacterium colony size was not just the same, antibacterial bandwidth value thereby yet difference can occur.
(2) thalline directly acts on assay method, normally on flat board, smear antagonistic bacterium bacterium liquid (fermented liquid of lawn diluent or mycetome), or utilize antagonistic bacterium bacterium liquid and the mixed flat board of substratum, containing access germ piece on the antimicrobial flat board short of money, measure germ colony growth diameter then, adopt this method, bacterium can capture dull and stereotyped culture space fast, very big to the germ growth effect, give birth to survey DeGrain, can't actual response go out the anti-microbial activity of opsonigenous substances.
(3) Sanming City therapy is the mixture of one deck substratum and antagonism bacterium bacterium liquid (containing the viable bacteria body) in the middle of two-layer substratum.In the substratum of upper strata, insert the germ piece then, but this method makes easily the thalline of middle layer substratum bring in the process of making the upper strata substratum, thereby influence the biological activity determination of antagonistic bacterium the germ bacterium colony.And antagonistic bacterium is to grow under the condition of middle level substratum shortage oxygen in culturing process, certainly will influence the normal secretion of opsonigenous substances.
(4) antagonistic bacterium does not have fermented liquid antibacterial ability assay method, comprise that fermented liquid mixes inhibition germ colony growth in substratum, fermented liquid suppresses the germ spore germination, fermented liquid filter paper method, fermented liquid cup-plate method etc., usually estimate the anti-microbial activity of antagonistic bacterium fermented liquid with this method, but there is not the normal biofilter filtration acquisition bacteria-free filtrate that adopts in the fermented liquid process at the preparation antagonistic bacterium, inconvenient in operation, easily pollute, and expense is higher, and the bacteria-free filtrate that adopts the high temperature sterilization preparation may cause the metabolite inactivation of part non-refractory in the fermented liquid, influences the performance of the true antibacterial ability of antagonism fermented liquid.In addition, adopt antagonistic bacterium not have that fermented liquid antibacterial ability assay method also is subjected in antagonistic bacterium fermentation culture culturing process, inoculum is cultivated all multifactor restrictions such as sterile filtration and detection, do not reach the purpose of fast big flow measurement.Fermented liquid filter paper method, action effect is not obvious, easily pollutes when filter paper is air-dry.The fermented liquid that adds different concns contains the fermented liquid medium therapy, owing to can influence the nutrition and the solidifiability of substratum to a certain extent, thereby influence the normal growth of bacterium in substratum.The fermented liquid cup-plate method, the application of sample amount is few, and the effect of the managed end and tube wall can influence the dispersal direction and the diffusing capacity of ferment product to a certain extent, and it is irregular to give birth to the antibacterial spot that forms when surveying, thereby influences accuracy of experimental results, complexity, consumption wealth consuming time in the operation.
Summary of the invention
Purpose of the present invention provides a kind of improved measurement method of antagonistic bacteria, solve and overcome deficiency in other method of antagonistic bacterium biological assay, as operate complicated, consumption wealth consuming time, the test verity, stability, repeatability, problems such as poor accuracy, according to characteristics such as antagonistic bacterium and metabolic secretion products thereof, and provide a kind of easy and simple to handle, fast, material is cheap, convenient sources, experimental period is short, the test verity, stability, the antagonistic bacterium biological assay novel method of good reproducibility---double-deck substratum filter paper sandwich assay, can reach the purpose of true rational evaluation antagonistic bacterium biocontrol effect, present method is applicable to the research of multiple antagonistic bacterium antimicrobial spectrum, can be to multiple pathogenic bacteria (as wilt, Pyricularia oryzae, anthrax bacteria, the corn sheath blight fungus, the bacterial wilt of peanut bacterium, citrus ulcer bacterias etc.) carry out biological activity determination, (normal temperature is deposited to research antagonistic bacterium metabolic secretion product biological and ecological methods to prevent plant disease, pests, and erosion stability, uviolizing) also has good effect.Novel method of the present invention has the using value biocontrol strain to have important effect to biocontrol effect, the screening of rational evaluation antagonistic bacterium.
Improved measurement method of antagonistic bacteria of the present invention, adopt double-deck substratum filter paper sandwich assay, finite concentration inoculum or lawn diluent evenly are applied in the substratum of upper strata, antagonistic bacterium can evenly be diffused in lower floor's substratum by filter paper layer at the metabolic secretion thing that substratum growth in upper strata produces, the upper strata substratum of removing filter paper and carrying, lower floor's substratum is the flat board that contains antagonistic bacterium metabolic secretion product, is easy to carry out the biological assay to multiple pathogenic fungi mycelial growth, spore germination and bacterial growth.
Remarkable advantage of the present invention is: the present invention utilizes antagonistic bacterium metabolic secretion product, and (antagonistic bacterium metabolic secretion product is generally 3～18mm) in the diffusion length that contains nutrient agar, utilizes cheap filter paper good penetration, utilizes the bacterium thalline in principles such as substratum growth opacity, carries out antagonistic bacterium the plant pathogenic fungi bioassay method is improved and innovation in the diffustivity of substratum.This method mensuration cycle is about 7 days, utilize the naturally even diffustivity of antagonistic bacterium metabolic secretion product in the mensuration process at substratum, the germ bacterium colony is grown regular on the substratum that contains antagonistic bacterium metabolic secretion product, has avoided bringing because of bacterial strain meta-bolites problems such as diffusibility difference, bacterial strain fermentation liquor cultivation and filtration on substratum the error of test-results.The examination material amount that adopts double-deck substratum filter paper sandwich assay to expend in testing process is few, and the antagonistic strain bioassay results of method acquisition is comparatively identical with the evaluation result that the aseptic ferment filtrate of antagonistic bacterium (being added in the germ substratum with 20%～50% dosage) that utilizes biofilter to obtain carries out the biological assay acquisition thus.Implement double-deck substratum filter paper sandwich assay energy actual response bacterial strain meta-bolites antibacterial ability, have that test good stability, good reproducibility, material are cheap, convenient sources, advantage such as easy and simple to handle, be applicable to the research of multiple antagonistic bacterium antimicrobial spectrum, can carry out biological activity determination to multiple pathogenic bacteria (as wilt, Pyricularia oryzae, anthrax bacteria, corn sheath blight fungus, bacterial wilt of peanut bacterium, citrus ulcer bacteria etc.), research antagonistic bacterium metabolic secretion product biological and ecological methods to prevent plant disease, pests, and erosion stability (normal temperature is deposited, uviolizing) is also had good effect.Novel method of the present invention has the using value biocontrol strain to have important effect to biocontrol effect, the screening of rational evaluation antagonistic bacterium.And method of the present invention can be finished the antimicrobial spectrum of a plurality of antagonistic bacteriums simultaneously and measure, and has novelty, practicality and creativeness, as a kind of new measurement method of antagonistic bacteria, has the potential using value.
Description of drawings
Fig. 1 is a utilisation technology route schema of the present invention.
Fig. 2 is a technical schematic diagram of the present invention.
Fig. 3 is several antagonistic bacterium of the present invention to the banana blight bacteria bacteriostatic activity biological assay procedure chart that exsomatizes, wherein:
A partly is: annotate the molded work of aseptic filter paper: with diameter is that the filter paper (as high speed 101 types) of 12cm is tiled in diameter for cutting in the 8.9cm glass culture dish, the filter paper edge that exceeds the about 1.5cm of width makes it gauffer, and placing the sterilization of plate mesohigh, the dull and stereotyped contour forming of aseptic filter paper is standby;
B partly is: annotate and moving into the filter paper mould on lower floor's substratum: at diameter is the lower floor's substratum that adds 10ml in the 8.9cm glass culture dish, thickness is 3～4mm, after solidifying, above lower floor's substratum, move into the aseptic filter paper mould, fold place is affixed on culture dish ware wall, and flat board locates to soak and be close to substratum;
C partly is: annotate at system upper strata substratum on the filter paper mould: the bacteria culture medium that adds 10ml on the aseptic filter paper mould is the upper strata substratum, and is stand-by after solidifying, and the upper strata substratum is separated by filter paper mould and lower floor's substratum;
D partly is: annotate and cultivate antagonistic bacterium to be measured: draw 100 μ l bacterium liquid (finite concentration contains the bacterium bacterial culture fluid) and evenly be applied in the upper panel, under the condition of suitable bacterial growth, cultivate (as 28 ℃, dark), after cultivating 48h, on mark upper strata substratum on lower floor's substratum, evenly cover with the scope of antagonistic bacterium;
E partly is: pour down a layer culture medium culturing banana blight bacteria: after antagonistic bacterium is cultivated 48h, bacterium excretory meta-bolites can evenly spread to lower floor's substratum by filter paper layer, shift out the filter paper that contains the upper strata substratum, lower floor's substratum is as the biological assay examination material to the target germ, insert germ to be measured in the substratum central authorities of lower floor that contain bacterium excretory meta-bolites, under the condition of germ suitable growth, cultivate.Behind the suitable fate of germ growth, in test lower floor substratum, measure the pathogenic fungi colony diameter.The authentic assessment antagonistic bacterium is surveyed effect to giving birth to of germ, correctly filters out the antagonistic strain of good antimicrobial effect.
Fig. 4 shows subtilis, bacterial strain 1,2,3,4,5 and banana blight bacteria face-off cultivation (7d) figure.
Fig. 5 is banana blight bacteria growing state (7d) figure in the PDA flat board that contains 33% bacterial strain, 1,2,3,4,5 culturing filtrates.
Fig. 6 is banana blight bacteria growing state (7d) figure in the PDA flat board that contains bacterial strain 1,2,3,4,5 meta-bolitess.
Embodiment
(1) makes the lower floor's substratum of suitable pathogenic fungi growth and the upper strata substratum of suitable antagonistic bacterium growth.
(2) make the dull and stereotyped mould of aseptic filter paper: with diameter is that to be tiled in diameter be in the 8.9cm glass culture dish for the filter paper (as high speed 101 types) of 12cm, the filter paper edge that exceeds the about 1.5cm of width makes it gauffer, and placing the sterilization of plate mesohigh, the dull and stereotyped contour forming of aseptic filter paper is standby.
(3) make lower floor's substratum: at diameter is the lower floor's substratum that adds 10ml in the 8.9cm glass culture dish, and thickness is 3～4mm, stand-by after solidifying.
(4) move into the aseptic filter paper mould: move into the aseptic filter paper mould above lower floor's substratum, fold place is affixed on culture dish ware wall, and flat board locates to soak and be close to substratum.
(5) make the upper strata substratum: the bacteria culture medium that adds 10ml on the aseptic filter paper mould is the upper strata substratum, and is stand-by after solidifying, and the upper strata substratum is separated by filter paper mould and lower floor's substratum.
(6) cultivate antagonistic bacterium to be measured: draw 100 μ l bacterium liquid (finite concentration contains the bacterium bacterial culture fluid) and evenly be applied in the upper panel, under the condition of suitable bacterial growth, cultivate (as 28 ℃, dark), after cultivating 48h, on mark upper strata substratum on lower floor's substratum, evenly cover with the scope of antagonistic bacterium.
(7) shift out the aseptic filter paper mould: after antagonistic bacterium was cultivated 48h, bacterium excretory meta-bolites can evenly spread to lower floor's substratum by filter paper layer, shifts out the filter paper that contains the upper strata substratum, and lower floor's substratum is as the biological assay examination material to the target germ.
(8) training objective pathogenic fungi: insert germ to be measured in the substratum central authorities of lower floor that contain bacterium excretory meta-bolites, under the condition of germ suitable growth, cultivate.
(9) test-results is measured: behind the suitable fate of germ growth, measure the pathogenic fungi colony diameter in test lower floor substratum.Be treated to blank not inoculate antimicrobial short of money, calculate the pathogenic bacteria growth inhibition ratio.
(10) screening antagonistic bacterium: the antibacterial ability of antagonistic bacterium is compared in interpretation of result by experiment, finishes the antagonistic bacterium biological activity determination, and the authentic assessment antagonistic bacterium is surveyed effect to giving birth to of germ, correctly filters out the antagonistic strain of good antimicrobial effect.
See also Fig. 1, Fig. 2, shown in Figure 3, the method of the embodiment of the invention 1 comprises in regular turn: obtain and cultivate antagonistic bacterium to be measured, obtain and the training objective pathogenic fungi, make the dull and stereotyped mould of aseptic filter paper, make the PDA of lower floor substratum, move into the aseptic filter paper mould, make upper strata NA substratum, cultivate antagonistic bacterium, shift out the aseptic filter paper mould, the training objective pathogenic fungi, test-results is measured, the screening antagonistic bacterium, wherein obtain and cultivate antagonistic bacterium to be measured, acquisition and training objective pathogenic fungi belong to conventional plant pathology organon, no longer describe at this, it is characterized in that:
(1) makes suitable pathogenic fungi growth medium (the PDA substratum with routine is an example) and suitable antagonistic bacterium growth medium (the NA substratum with routine is an example).
(2) make the dull and stereotyped mould of aseptic filter paper: with diameter is that to be tiled in diameter be in the 8.9cm glass culture dish for the filter paper (as high speed 101 types) of 12cm, the filter paper edge that exceeds the about 1.5cm of width makes it gauffer, and placing the sterilization of plate mesohigh, the dull and stereotyped contour forming of aseptic filter paper is standby.
(3) making the PDA of lower floor substratum, is that the PDA substratum that adds 10ml in the 8.9cm glass culture dish is lower floor's substratum at diameter, and thickness is 3～4mm, and it is stand-by to solidify the back.
(4) move into the aseptic filter paper mould: move into the aseptic filter paper mould above lower floor's substratum, fold place is affixed on culture dish ware wall, and flat board soaks and is close to substratum.
(5) make upper strata NA substratum: the NA substratum that adds 10ml on the aseptic filter paper mould is the upper strata substratum, and is stand-by after solidifying, and the NA substratum is separated by filter paper mould and PDA substratum.
(6) cultivate antagonistic bacterium to be measured: draw 100 μ l bacterium liquid (finite concentration contains the nutrient solution of bacterium thalline) and evenly be applied in the upper panel, under the condition of suitable bacterial growth, cultivate (as 28 ℃, dark), after cultivating 48h, on mark upper strata substratum on lower floor's substratum, evenly cover with the scope of antagonistic bacterium.
(7) shift out the aseptic filter paper mould: after antagonistic bacterium is cultivated 48h, bacterium excretory meta-bolites can evenly spread to lower floor's PDA substratum by filter paper layer, the filter paper mould that contains the upper strata substratum that shifts out keeps the lower floor's substratum that contains bacterium excretory meta-bolites and tries material as biological assay.
(8) training objective pathogenic fungi: the PDA of the lower floor substratum central authorities that contain bacterium excretory meta-bolites insert the germ bacterium cake to be measured (diameter is 5mm) of cell age unanimity, cultivate (as 28 ℃, dark) under the condition of suitable germ growth.
(9) experimental result is measured: measure colony diameter behind germ growth 4～7d, be treated to blank not inoculate antimicrobial short of money, establish a plurality of repetitions, calculate the germ mycelial growth inhibition rate.
(10) screening antagonistic bacterium: the antibacterial ability of antagonistic bacterium is compared in interpretation of result by experiment, finishes the antagonistic bacterium biological assay, and the authentic assessment antagonistic bacterium is surveyed effect to giving birth to of germ, correctly filters out the antagonistic strain of good antimicrobial effect.
Below be specific embodiments of the invention, further specify the present invention, but the present invention is not limited only to this.
Embodiment
Antagonistic bacterium to the banana blight bacteria bacteriostatic activity relatively
One, materials and methods
1, the dull and stereotyped face-off of antagonistic bacterium is to the antagonistic action of germ
Adopt the dull and stereotyped face-off of PDA growth method, the activation bacterium that some reception is surveyed (has preliminary bacteriostatic activity, derive from soil) 5 strains, and be 0.5cm banana blight bacteria (No. 4 microspecies) mycelia piece at a distance of 2cm place access diameter, after 4d and 7d are cultivated in 28 ℃ of dark face-offs, measure the antibacterial bandwidth of each antagonistic bacterium, compare the bacteriostatic activity and the stability thereof of each bacterial strain, 4 repetitions of every processing.
2, the antagonistic bacterium fermented liquid is to the restraining effect of germ mycelial growth
Wash antagonism bacterium bacterium colony to be measured with sterilized water, drawing 100 μ l bacterium liquid behind the mixing transfers cultivate behind the 3d (28 ℃ in the 250ml triangular flask that fills 150ml NB nutrient solution, 140rpm), make flat board in pouring in the culture dish that diameter is 9cm behind 1:2 ratio and PDA substratum (45 ℃) mixing after getting ferment product sterile filtration.Insert diameter 0.5cm germ piece in dull and stereotyped central authorities after the condensation, put 28 ℃ of dark culturing.With the NB liquid nutrient medium that adds same amount is contrast, 4 repetitions of every processing.After cultivating 4d, 7d, measure the germ colony diameter, arithmetic average diameter, the relatively bacteriostatic activity of each bacterial strain with the right-angled intersection method.
3, antagonistic bacterium is cultivated the restraining effect of meta-bolites to the germ mycelial growth
Adopting double-deck substratum filter paper sandwich assay, is lower floor's substratum prior to pouring the 10mlPDA substratum in the culture dish into, treat the substratum condensation after.Preprepared diameter 12cm aseptic filter paper (101 types at a high speed) is close on the PDA flat board gently, and pouring the 10mlPDA substratum again on filter paper into is the upper strata substratum.Drawing 100 μ l bacterium liquid evenly is applied in the upper panel, after cultivating 48h under 28 ℃ of dark, tear the upper strata substratum filter paper that contains antagonistic bacterium off, and at substratum central authorities of lower floor access germ piece, after cultivating 4d, 7d down, 28 ℃ of dark conditions measure the germ colony diameter again with the right-angled intersection method, arithmetic average diameter, the relatively bacteriostatic activity of each bacterial strain.Not inoculate the contrast that is treated to of antagonism fermented liquid, 4 repetitions of every processing.
Two, result and analysis
Adopt dull and stereotyped face-off culture method, the antibacterial bandwidth that 5 strain bacteriums (bacterial strain 1～5) to be measured and germ face-off are cultivated behind the 4d is respectively 0.83cm, 0.86cm, 0.81cm, 0.98cm and 0.75cm, with bacterial strain 4 antibacterial bandwidth value maximums, antibacterial bandwidth value is 4〉2〉1〉3〉5 from large to small successively, has 2 significant difference levels; Both are respectively 0.25cm, 0.50cm, 0.23cm, 0.48cm and 0.48cm at the antibacterial bandwidth of cultivating behind the 7d that stands facing each other, with bacterial strain 2 antibacterial bandwidth value maximums, antibacterial bandwidth value is 2〉4=5〉1〉3 from large to small successively, 2 significant difference levels are arranged, this is owing to the prolongation along with the face-off incubation time, germ continues to show as antibacterial bandwidth and constantly reducing towards the growth of antagonism bacterium direction.With flat board face-off method screening antagonistic bacterium, advantage is easy and simple to handle, and the time is short, and 4d is PRELIMINARY RESULTS as can be seen, is fit to the preliminary screening of antagonistic bacterium.But adopt this method to measure antibacterial bandwidth at different time, the result difference of acquisition is bigger, the test-results instability, and the difference that shows between bacterial strain is not obvious.
The dull and stereotyped face-off method of table 1 is measured antagonistic bacterium to the banana blight bacteria growth effect
Adopt the base band poison method of cultivating, cultivate 4d in the PDA flat board of banana blight bacteria in containing 5 strain bacteriums (bacterial strain 1～5) cultivation and fermentation liquid to be measured, the germ colony diameter is respectively 2.10cm, 1.69cm, 1.70cm, 1.96cm and 1.21cm, the bacteriostatic activity of each bacterial strain is respectively 5 from large to small〉2〉3〉4〉1,3 significant difference levels are arranged; Cultivate 7d in the PDA flat board of banana blight bacteria in containing 5 strain bacteriums (bacterial strain 1～5) cultivation and fermentation liquid to be measured, the germ colony diameter is respectively 3.27cm, 2.60cm, 3.08cm, 3.12cm and 2.08cm, the bacteriostatic activity of each bacterial strain is respectively 5 from large to small〉2〉3〉4〉1,4 significant difference levels are arranged.Adopt and cultivate base band poison method, the bacteriostatic activity of bacterial detection cultivation and fermentation filtrate is a kind of comparatively ideal method, anti-microbial activity size between the energy actual response bacterial strain, but comparatively loaded down with trivial details in the process of the test, the test period is longer, and the bacterial strain differences that has is not obvious.
Table 2 is cultivated base band poison method and is measured antagonistic bacterium to banana blight bacteria growth inhibitory effect (contain antagonistic bacterium and do not have fermented liquid PDA substratum)
Adopt double-deck substratum filter paper sandwich assay, banana blight bacteria is cultivated 4d in the PDA flat board that contains 5 strain bacteriums (bacterial strain 1～5) meta-bolites to be measured, the germ colony diameter is respectively 1.10cm, 0.81cm, 0.88cm, 1.00cm and 0.70cm, the bacteriostatic activity of each bacterial strain is respectively 5 from large to small〉2〉3〉4〉1,5 significant difference levels are arranged; Banana blight bacteria is cultivated 7d in the PDA flat board that contains 5 strain opsonigenous substancess to be measured, the germ colony diameter is respectively 1.51cm, 0.98cm, 1.29cm, 1.50cm and 0.88cm, each bacterial strain differences is obvious, the bacteriostatic activity of each bacterial strain is respectively 5 from large to small〉2〉3〉4〉1,4 significant difference levels are arranged.Since antagonistic bacterium metabolic secretion product in substratum by the natural force effect, even diffustivity, the germ bacterium colony grow regular on the substratum that contains antagonistic bacterium metabolic secretion product, twice measuring result unanimity.The test-results error is little, and is stable, and convenient in operation, each bacterial strain differences is obvious, and 4～7d is comparatively identical with table 2 result as can be seen in test.
The double-deck substratum filter paper of table 3 sandwich method for determining antagonistic bacterium is to the banana blight bacteria growth effect
Three, several measuring method effect assessments of antagonistic bacterium
1, dull and stereotyped face-off method: antagonism bacterium and germ are cultivated altogether needs 5～7d, easy and simple to handle, but the bacteriostatic activity instability, testing error is big, and the difference that shows between antagonistic strain is least obvious.
2, cultivate base band poison method: the antagonism fermented liquid is cultivated 2～3d, and after the centrifugate sterile filtration, aseptic detection needs 2～3d, and system is with malicious substratum, observes to have or not infectation of bacteria to need 2～3d, cultivates germ 4～7d, and the difference that shows between antagonistic strain is comparatively obvious.But filtering membrane price height is operated consumption wealth the most consuming time, and test failure is not easily led in the improper and aseptic detection of filter operation sometimes comprehensively.
3, double-deck substratum filter paper sandwich assay: the antagonism bacterium is cultivated 1～2d, cultivates germ 4～7d, and the difference that shows between antagonistic strain is the most obvious.Easy to operate, and filter paper is cheap in the test materials.Deposited 1 month under lower floor's substratum normal temperature to be tested, the anti-microbial activity of antagonistic bacterium meta-bolites is still stable in the flat board, can detect multiple antagonistic bacterium to multiple pathogenic fungi bacteriostatic activity, is fit to the research of multiple antagonistic bacterium antimicrobial spectrum.
Claims (5)
1. improved measurement method of antagonistic bacteria, it is characterized in that: adopt double-deck substratum filter paper sandwich assay, finite concentration inoculum or lawn diluent evenly are applied in the substratum of upper strata, antagonistic bacterium can evenly be diffused in lower floor's substratum by filter paper layer at the metabolic secretion thing that substratum growth in upper strata produces, the upper strata substratum of removing filter paper and carrying, lower floor's substratum is the flat board that contains antagonistic bacterium metabolic secretion product, is easy to carry out the biological assay to multiple pathogenic fungi mycelial growth, spore germination and bacterial growth;
Cultivated on the basis antagonism bacterium and pathogenic bacteria, carried out according to following steps:
1) makes the conventional substratum of suitable pathogenic fungi growth and the conventional substratum of suitable antagonistic bacterium growth according to the kind of pathogenic fungi and antagonistic bacterium;
2) make the dull and stereotyped mould of aseptic filter paper: with diameter be the filter paper of 12cm to be tiled in diameter be in the 8.9cm glass culture dish, the filter paper edge that exceeds width 1.5cm makes it gauffer, and places the sterilization of plate mesohigh, aseptic filter paper flat board contour forming is standby;
3) make being fit to lower floor's substratum of pathogenic fungi growth, is that the conventional substratum that adds the suitable pathogenic fungi growth of 10ml in the 8.9cm glass culture dish is lower floor's substratum at diameter, and thickness is 3～4mm, and it is stand-by to solidify the back;
4) move into the aseptic filter paper mould: move into the aseptic filter paper mould above lower floor's substratum, fold place is affixed on culture dish ware wall, and flat board locates to soak and be close to substratum;
5) making is fit to the upper strata substratum of antagonistic bacterium growth: the conventional substratum that adds the suitable antagonistic bacterium growth of 10ml on the aseptic filter paper mould is the upper strata substratum, and is stand-by after solidifying, and the upper strata substratum is separated by filter paper mould and lower floor's substratum;
6) cultivate antagonistic bacterium to be measured: draw bacterium liquid and evenly be applied in the upper panel, under the condition of suitable bacterial growth, cultivate, behind the cultivation 48h, evenly cover with antagonistic bacterium on the conventional substratum of the suitable antagonistic bacterium growth in upper strata;
7) shift out the aseptic filter paper mould: after antagonistic bacterium is cultivated 48h, bacterium excretory meta-bolites can evenly spread to the conventional substratum that lower floor is fit to the pathogenic fungi growth by filter paper layer, the filter paper mould that contains the upper strata substratum that shift out this moment, the lower floor's substratum that contains bacterium excretory meta-bolites can be used as biological assay examination material;
8) training objective pathogenic fungi:, under the condition of suitable germ growth, cultivate at the central germ bacterium cake to be measured that inserts the cell age unanimity of conventional substratum that the lower floor that contains the bacterial metabolism secretory product is fit to the pathogenic fungi growth;
9) experimental result is measured: measure the germ colony diameter behind germ growth 4～7d to be measured; Not inoculate the blank that is treated to of antagonistic bacterium, establish a plurality of repetitions;
10) screening antagonistic bacterium: the bacteriostasis of antagonistic bacterium is compared in interpretation of result by experiment, finishes the antagonistic bacterium biological assay, and the authentic assessment antagonistic bacterium is surveyed effect to giving birth to of germ, correctly filters out the antagonistic strain of good antimicrobial effect.
2. improved measurement method of antagonistic bacteria according to claim 1 is characterized in that: the conventional substratum of described suitable pathogenic fungi growth is conventional PDA substratum.
3. improved measurement method of antagonistic bacteria according to claim 1 is characterized in that: the conventional substratum of described suitable antagonistic bacterium growth is conventional NA substratum.
4. improved measurement method of antagonistic bacteria according to claim 1 is characterized in that: described diameter is that the filter paper of 12cm adopts 101 types at a high speed.
5. improved measurement method of antagonistic bacteria according to claim 1 is characterized in that: the diameter of the germ bacterium cake to be measured in the described step 8) is 0.5cm.
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| CN1680585A (en) * | 2005-01-31 | 2005-10-12 | 重庆工学院 | Choice for preventing endogenic bacterial strain by potato |
| WO2007043771A1 (en) * | 2005-10-07 | 2007-04-19 | Kyu Jin Yum | Compositions for preventing plant disease comprising bacillus subtilis kccm 10639 or kccm 10640 and methods of preventing plant disease by using them |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1680585A (en) * | 2005-01-31 | 2005-10-12 | 重庆工学院 | Choice for preventing endogenic bacterial strain by potato |
| WO2007043771A1 (en) * | 2005-10-07 | 2007-04-19 | Kyu Jin Yum | Compositions for preventing plant disease comprising bacillus subtilis kccm 10639 or kccm 10640 and methods of preventing plant disease by using them |
Non-Patent Citations (1)
| Title |
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| 李小俊 等.拮抗菌抗菌谱及发酵液拮抗能力测定的新方法.《生物技术》.2007,第17卷(第1期),55－58. * |
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