CN102899386B - Method for measuring content of live spores in fungal microbial pesticide quickly - Google Patents
Method for measuring content of live spores in fungal microbial pesticide quickly Download PDFInfo
- Publication number
- CN102899386B CN102899386B CN201210434841.4A CN201210434841A CN102899386B CN 102899386 B CN102899386 B CN 102899386B CN 201210434841 A CN201210434841 A CN 201210434841A CN 102899386 B CN102899386 B CN 102899386B
- Authority
- CN
- China
- Prior art keywords
- content
- spore
- flat board
- fungal
- flat plates
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for measuring the content of live spores in a fungal microbial pesticide quickly. The method comprises the following steps of: (1) adding 0.25 to 5 weight percent of sodium deoxycholate into a fungal culture medium, and preparing flat plates; (2) soaking fungal microbial pesticide samples in sterilization water containing Tween 20 or polyethylene glycol octylphenol ether, and shaking; (3) diluting in a multiple gradient mode, coating the samples of different dilutability on the flat plate respectively and uniformly, and culturing at room temperature for 48 to 120 hours; (4) counting colony counts, wherein the flat plates on which the colony counts are 30 to 300 are effectively-counted flat plates; and (5) counting the content of the live spores, namely C is equal to T*N*10, wherein in a formula, C is the content of the fungal pesticide live spores, the unit is colony forming unit (CFU)/gram, T is dilution times corresponding to the counted flat plates, and N is average colony counts on a plurality of effectively-counted flat plates of the same dilution times. By the method, the content of the live spores of the fungal microbial pesticide can be measured simply, conveniently and quickly, and the detection accuracy is high.
Description
Technical field
The present invention relates to a kind of detection method of biological pesticide, be specifically related to a kind of for measuring the method for fungi microbe agricultural chemicals spore content alive.
Background technology
Microbial pesticide generally includes bacterium, fungi and viral micro-organisms agricultural chemicals.That natural active matter or the living organisms itself that directly utilizes bacterium, fungi and virus etc. to produce developed, to the agricultural chemicals of phytopathy Chinese caterpillar fungus undercutting row control.It is strong that this class agricultural chemicals has selectivity, to people, animal, farm crop and physical environment safety, and not task natural enemy, the difficult advantages such as resistance that produce.Bacteria containing amount (especially viable bacteria amount) in microbial pesticide is the important factor that determines quality product quality and field control effect, and therefore viable bacteria amount is the important technology index that microbial pesticide quality product detects.
Because the effective constituent of microbial pesticide is live body, can not carry out content detection by the analytical instrument of chemical pesticide.For fungi microbe agricultural chemicals, conventionally adopt blood counting chamber to detect in conjunction with the method for spore germination, first measure total spore content (sample weighing, dilution, blood counting chamber counting, result are calculated) with blood counting chamber, then measure spore germination rate (sample weighing, dilution, spore cultivation, microscopic examination spore germination situation, result are calculated), finally calculate the spore content (total spore content × spore germination rate) of living.There is following main drawback in aforesaid method: complex operation, and be not easily distinguishable under the microscope fungal spore and builder granule, and also because incubation time is short, some spores are not yet sprouted, and affect measurement result.
In recent years, some fungi microbe agricultural chemicals are as mould in beauveria bassiana, green muscardine fungus, wood, Paecilomyces lilacinus etc. obtains registration at home successively, productions in market on urgent need set up quick, easy, the quality product detection method accurately of this class agricultural chemicals.
A kind of consideration is to adopt the method for plate count directly to obtain the content of spore alive, but, according to the common the method for plate culture count for bacterial count, fungi being operated, the bacterium colony of different spores can grow together and be linked to be sheet, form large bacterium colony, affect measurement result; A bacterium colony too hour counting does not see, and can not distinguish the bacterium colony of target bacterium colony and miscellaneous bacteria.Therefore, need to find improved method.
Summary of the invention
Goal of the invention of the present invention is to provide the method for spore content alive in a kind of Fast Measurement fungi microbe agricultural chemicals, to simplify the operation, obtains detected result more accurately.
To achieve the above object of the invention, the technical solution used in the present invention is: the method for the spore content of living in a kind of Fast Measurement fungi microbe agricultural chemicals, comprises the following steps:
(1) in fungi culture medium, add 0.25 ‰～5 ‰ Sodium desoxycholate by weight, and be prepared into flat board;
(2) fungi microbe pesticide sample is soaked to 20～40min in the aqua sterilisa containing weight percent 0.05%～0.1% polysorbas20 or 0.05%～0.1% Triton X-100 (Triton X-100), then the 20～40min that vibrates;
(3) step (2) sample after treatment is carried out to multiple gradient dilution, respectively dilution difference sample is uniformly coated on the flat board of step (1) acquisition, under room temperature, cultivate 48～120h, wherein, same extent of dilution is made at least 2 repeat samples;
(4) add up the colony number on each flat board, wherein colony number is that effectively counting is dull and stereotyped at the flat board of 30～300;
(5), in fungi microbe agricultural chemicals, the content of the spore of living is:
C＝T×N×10，
In formula: C is fungal farm chemicals spore content alive, and unit is CFU/g; T is the dull and stereotyped corresponding extension rate of statistics; N is the average colony number on multiple effective counting flat board of same extension rate.
Sodium desoxycholate (Sodium deoxycholate, No. CAS: 302-95-4), Chinese another name is 3 α, 12 α-dihydroxy-5 β-ursodeoxycholic acid sodium and sodium deoxycholate, be cholate, similar bile smell, there is strong bitter taste, easily moisture absorption, soluble in water, be slightly soluble in absolute alcohol, be insoluble to ether, low toxicity, medium lethal dose (rat, per os) 1370mg/kg, chemical structural formula is as follows:
Sodium desoxycholate is ionic detergent, is a kind of application biochemical reagents more widely, and its effect mainly contains: lysing cell, soluble protein, especially can dissolve some and be insoluble in the albumen of water, as membranin etc.In addition, cholate is important bioactive molecules, can participate in many physiological processs; Be widely used in the short absorption agent of mucous membrane and percutaneous drug delivery.
The present invention adds Sodium desoxycholate in substratum to, bacterium colony expansion that can Antifungi, under lower concentration, can not allow fungal spore lethal simultaneously yet, realize the content that adopts the method for plate culture count one step directly to obtain spore alive, and can be by naked eyes or bacterial colony counting instrument statistics colony number.
In technique scheme, in step (1), described fungi culture medium is potato dextrose agar (PDA) substratum, by substratum high pressure steam sterilization, to be cooled during to 55～62 DEG C, in substratum, add Sodium desoxycholate, be prepared into flat board.
In step (2), described vibration is carried out under the condition of room temperature, 150rpm.
Preferably, in step (2), soak time is 30min, and duration of oscillation is 30min.
In step (3), adopt 10 multiple gradient dilutions, same extent of dilution is made 3 repeat samples.
Because technique scheme is used, the present invention compared with prior art has following advantages:
1. the present invention adds certain density Sodium desoxycholate in substratum, not Antifungi spore germination, but can suppress the expansion of the bacterium colony that forms after spore germination, then fungal farm chemicals spore liquid is coated on this culture medium flat plate, thereby convert and obtain the content of spore of living by the quantity of counting bacterium colony, spore content alive that can easy Fast Measurement fungi microbe agricultural chemicals.
2. due to the application of Sodium desoxycholate, the bacterium colony expansion that the present invention can Antifungi, can not allow fungal spore lethal simultaneously under lower concentration yet, thereby can be by naked eyes or bacterial colony counting instrument statistics colony number.
3. operating process of the present invention is easy, and accuracy in detection is high.
Brief description of the drawings
Fig. 1 is measuring method schema in the embodiment of the present invention.
Fig. 2 is the photo of culture plate in comparative example.
Fig. 3 is the photo of culture plate in embodiment.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described:
Embodiment: adopt the method shown in Fig. 1, respectively the spore content alive of the mould wettable powder of wood, Paecilomyces lilacinus wettable powder, Paecilomyces lilacinus liquor, the female medicine of beauveria bassiana is measured.As embodiment 1 to embodiment 4.
Determination step is as follows:
(1) potato dextrose agar powder (PDA) 38g is added in 1000mL distilled water, heated and boiled, be distributed in 500mL Erlenmeyer flask, after high pressure steam sterilization, the Sodium desoxycholate PDA flat board to preparing respectively 0.25 ‰, 0.5 ‰, 1%, 2%, 5 ‰ content behind 60 DEG C of left and right to be cooled;
(2) under aseptic technique, sample is stirred, accurately take 3.0g sample, dissolve in soaking after 30min containing in the sterilized water of 0.05%Tween20 of 27.0mL, vibration 30min, obtains diluting the sample solution of 10 times, is labeled as No. 0, then carry out successively gradient dilution, each extent of dilution is established 3 repeat samples;
(3) above-mentioned each gradient dilution liquid being drawn respectively to the L shaped glass rod of 100 μ L is uniformly coated on step (1) gained flat board, at 25 DEG C, cultivate after 48～120h, select suitable extent of dilution, colony number is that effectively counting is dull and stereotyped at the flat board of 30～300;
(4) calculate the spore content measurement result adding after different concns Sodium desoxycholate:
The spore content of living is:
C＝T×N×10
In formula: C----fungal farm chemicals spore content alive, unit is CFU/g;
T-----extension rate;
Effectively count the average colony number on flat board for N----3.
Measuring result is referring to shown in table 1 and table 2, and dull and stereotyped photo as shown in Figure 3.
Comparative example:
Take sample 1.00g, nominal is got 3 samples, carries out respectively following operation:
(1) sample is placed in respectively to the liquid holding cup of stamp mill, adds 0.05% polysorbas20 solution 100mL, slowly stir, make spore fully wetting as far as possible.
(2) pipette spore suspension 2mL with transfer pipet to test tube from solution middle part, adding distil water 8mL, about 30sec vibrates on vortex mixer.
(3) repeating step (2), prepares gradient concentration spore suspension, and the extent of dilution taking lattice spore count in 5 as 100～300 is as effective weaker concn.
(4) spore liquid is left standstill to about 30sec, draw spore suspension from solution middle part with thin head straw, drip 1 at cover glass edge, liquid is infiltrated under cover glass along edge, just be full of between cover glass and tally counting region, do not enter groove as degree taking spore liquid, should be without bubble and counting region surrounding groove absence of liquid, if any unnecessary liquid, suck with little thieving paper.
(5) with microscopic examination, choose after unsteady spore is static and count.In planning lattice district, 5 middle lattice of meter surrounding and central authorities are 80 little lattice within the scope of two-wire.Then calculate spore total content according to extension rate.
(6) potato dextrose agar powder (PDA) 38g is added in 1000mL distilled water, heated and boiled, is distributed in 500mL Erlenmeyer flask, after high pressure steam sterilization, to be cooledly to 60 DEG C of left and right, prepares respectively PDA flat board.
(7) spore suspension of D step 100 μ L are coated with on PDA flat board with L shaped glass stick, cultivate 12h at 25 DEG C after, under microscope, statistics is sprouted and is not sprouted spore count, taking the extent of dilution of lower 20 spores of microscope (400 times of object lens × eyepiece multiples) as effective number concentration.Finally calculate spore germination rate.
Result comparison:
Test result to embodiment and comparative example compares, as shown in table 1.
As can be seen from Table 1,4 embodiment adopt all methods in conjunction with spore germination a little more than conventional blood counting chamber of measurement result of method of the present invention, illustrate that the present invention is more accurate, and operation is easier.
The spore content measurement result alive comparison of table 1 blood cell plate counting process and the present embodiment method (adding 250mg/L Sodium desoxycholate)
As can be seen from Table 2, the measurement result of the Sodium desoxycholate of different concns is basically identical.
In table 2PDA substratum, add the bacteria containing amount after different content Sodium desoxycholate to measure
Claims (2)
1. a method for spore content alive in Fast Measurement fungi microbe agricultural chemicals, comprises the following steps:
(1) in fungi culture medium, add 0.25 ‰～5 ‰ Sodium desoxycholate by weight, and be prepared into flat board; Described fungi culture medium is potato dextrose agar, by substratum high pressure steam sterilization, to be cooled during to 55～62 DEG C, in substratum, adds Sodium desoxycholate, is prepared into flat board;
(2) fungi microbe pesticide sample is soaked to 20～40min in the aqua sterilisa containing weight percent 0.05%～0.1% polysorbas20 or 0.05%～0.1% Triton X-100, then the 20～40min that vibrates; Described vibration is carried out under the condition of room temperature, 150rpm;
(3) step (2) sample after treatment is carried out to multiple gradient dilution, respectively dilution difference sample is uniformly coated on the flat board of step (1) acquisition, under room temperature, cultivate 48～120h; Described multiple gradient dilution adopts 10 multiple gradient dilutions, and same extent of dilution is made 3 repeat samples;
(4) add up the colony number on each flat board, wherein colony number is that effectively counting is dull and stereotyped at the flat board of 30～300;
(5), in fungi microbe agricultural chemicals, the content of the spore of living is:
C＝T×N×10，
In formula: C is fungal farm chemicals spore content alive, and unit is CFU/g; T is the dull and stereotyped corresponding extension rate of statistics; N is the average colony number on multiple effective counting flat board of same extension rate.
2. the method for spore content alive in Fast Measurement fungi microbe agricultural chemicals according to claim 1, is characterized in that: in step (2), soak time is 30min, and duration of oscillation is 30min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210434841.4A CN102899386B (en) | 2012-11-05 | 2012-11-05 | Method for measuring content of live spores in fungal microbial pesticide quickly |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210434841.4A CN102899386B (en) | 2012-11-05 | 2012-11-05 | Method for measuring content of live spores in fungal microbial pesticide quickly |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102899386A CN102899386A (en) | 2013-01-30 |
| CN102899386B true CN102899386B (en) | 2014-06-11 |
Family
ID=47571874
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210434841.4A Expired - Fee Related CN102899386B (en) | 2012-11-05 | 2012-11-05 | Method for measuring content of live spores in fungal microbial pesticide quickly |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102899386B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148479B (en) * | 2016-08-29 | 2019-09-27 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of microorganism in grease |
| CN108085255A (en) * | 2016-11-22 | 2018-05-29 | 华中科技大学 | A kind of method for separating filamentous fungi single bacterium colony |
| CN113528346B (en) * | 2021-08-17 | 2023-01-31 | 江西新龙生物科技股份有限公司 | Preservation method of fungus biopesticide |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586144A (en) * | 2009-07-03 | 2009-11-25 | 武汉科诺生物科技股份有限公司 | Method for detecting the number of live spore in Bacillus subtilis powder of 5 hundred billion live spores/gram |
-
2012
- 2012-11-05 CN CN201210434841.4A patent/CN102899386B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586144A (en) * | 2009-07-03 | 2009-11-25 | 武汉科诺生物科技股份有限公司 | Method for detecting the number of live spore in Bacillus subtilis powder of 5 hundred billion live spores/gram |
Non-Patent Citations (9)
| Title |
|---|
| 农向群 等.真菌农药母药含孢量测定的规范化.《中国生物防治》.2007,第23卷(第2期),137-141. |
| 各课题组研究进展;江至利 等;《"生物源农药创制与技术集成及产业化开发"工作通讯》;20120916(第1期);第14页 * |
| 朱平 等.麦角菌和雀稗麦角菌原生质体的形成与再生.《真菌学报》.1992,第11卷(第4期),272-278. |
| 李平兰.第5章 微生物的生长.《食品微生物学教程》.2011,第147页. * |
| 杨民和.第6章微生物生长繁殖与控制.《微生物学》.2010,第154页. * |
| 桑国卫 等.微生物限度检测法.《中国药品检验标准操作规范 2005年版》.2005,325-334. * |
| 江至利 等.各课题组研究进展.《"生物源农药创制与技术集成及产业化开发"工作通讯》.2012,(第1期),14. |
| 真菌农药母药含孢量测定的规范化;农向群 等;《中国生物防治》;20070531;第23卷(第2期);摘要，第139页 * |
| 麦角菌和雀稗麦角菌原生质体的形成与再生;朱平 等;《真菌学报》;19921231;第11卷(第4期);摘要，第276页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102899386A (en) | 2013-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jousset et al. | Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria | |
| CN101654665B (en) | Method for preparing Bacillus subtilis | |
| CN102899386B (en) | Method for measuring content of live spores in fungal microbial pesticide quickly | |
| CN109022292A (en) | One plant of trichoderma and its application in agriculture field | |
| CN105779360B (en) | A kind of bacillus subtilis and its application | |
| CN113249243A (en) | Pseudomonas, microbial biocontrol microbial inoculum containing pseudomonas and application of microbial biocontrol microbial inoculum | |
| CN108587971A (en) | A kind of bacillus pumilus s35 and its application | |
| CN108531430A (en) | A kind of simple bacillus S62 and its application | |
| Arafa et al. | Leaf extracts from resistant wild tomato can be used to control late blight (Phytophthora infestans) in the cultivated tomato | |
| CN109792990B (en) | Application of plant essential oil in inhibition of mold in air in museum | |
| Hou et al. | Seasonal shifts in cold tolerance and the composition of the gut microbiome of Dendroctonus valens LeConte occur concurrently | |
| CN1970787B (en) | Method for detecting number of effective bacillus | |
| CN104962491B (en) | The degradation bacteria strains of D of herbicide 2,4 a kind of and its microbial inoculum and the application of production | |
| Steyaert et al. | Methods for the evaluation of the bioactivity and biocontrol potential of species of Trichoderma | |
| CN116904354A (en) | Priestia aryabhattai H2 and application thereof | |
| CN104059044B (en) | A kind of Xanthone derivative and preparation method thereof and application | |
| CN102676407A (en) | Microzyme for degrading residual lincomycin in fermented leaves and application thereof | |
| Kgosi et al. | Anti-fungal analysis of Bacillus subtilis DL76 on conidiation, appressorium formation, growth, multiple stress response, and pathogenicity in Magnaporthe oryzae | |
| Vio et al. | Multispecies Bacterial Bio-Input: Tracking and Plant-Growth-Promoting Effect on Lettuce var. sagess | |
| CN105200116B (en) | A kind of method of Paenibacillus polymyxa content in quick measure complex micro organism fungicide | |
| CN105132325B (en) | A kind of phenyl ureagroup herbicides residue degrading bacterial strain and its microbial inoculum of production | |
| CN109699683B (en) | Talcum matrix Java cordyceps militaris spore preparation | |
| CN113287621A (en) | Application of imidazole derivative A in preparation of pesticide bactericide for preventing and treating banana fusarium wilt | |
| CN106834356A (en) | Evaluation method of the grape ulcer bacterium to grape pathogenicity | |
| CN109400444A (en) | Inhibit the sesquiterpenoids and preparation method thereof of plant pathogenic fungi |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140611 Termination date: 20141105 |
|
| EXPY | Termination of patent right or utility model |