CN104357333A - Fusarium oxysporum single spore isolation method for soybean root rot - Google Patents
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Abstract
The invention discloses a fusarium oxysporum single spore isolation method for the soybean root rot. The fusarium oxysporum single spore isolation method is provided for solving the problems that a soybean fusarium oxysporum single spore is small, is in light color and is not easy to isolate. The fusarium oxysporum single spore is enlarged in size after germinating, then dilution is carried out until a spore suspension with only 1-2 spores in average every 10 <Mu>L is obtained, and isolated conidium is further confirmed in a round hole grid in a 96-mesh cell culture plate cover through an inverted microscope to be a single spore. Compared with other single spore isolation methods, the method is good in operability, high in accuracy, high in speed, excellent in isolation effect, and simple in equipment. The method is accurate, simple and feasible, and a large batch of fusarium oxysporum single spores can be isolated within a short period of time in a lab. Therefore, the method is suitable for promotion and application.
Description
Technical field
The present invention relates to the method that the strain of fungi monospore is separated, particularly relate to the method for a kind of pathogen of soybean root rot point sickle spore conidium single spore separation.
Background technology
Soybean sickle spore root rot (soybean
fusariumroot rot) be by soybean point sickle spore (
f. oxysporumschl.) causing for dominant strain infects, is that a kind of distribution is wide, the worldwide disease of the heavy and control difficulty of harm.The manufacturing soybean time limit more of a specified duration plot morbidity heavier, output is lower.Root rot general area of falling ill can make soybean underproduction 10%-30%, and grave illness area can the underproduction 60%, and even total crop failure.Root rot main harm plant root, thus affect the absorption of root system to moisture and nutrient, cause plant strain growth slow, production declining.Soybean breeding for disease resistance be control soybean sickle spore root rot the most directly, the most effective approach.Carry out the prerequisite that large-scale Chinese People's Anti-Japanese Military and Political College beans sickle spore root rot Screening of Germplasm is breeding for disease resistance.But the prerequisite of resistance screening obtains a large amount of and representational monospore strain.At present, the separation method of monospore strain is more, basic skills adopts to thank preciousness (1997), Wang Shurong (1989) and Gong Guoshu (2010) etc., mainly choose by choosing pin, then be placed on agar, then carry out picking again by microscopic examination, then determine monospore, carry out purifying and cultivation, obtain monospore strain.Although apply a lot of year, also there is it not enough: these methods are better to megaspore separating effect, and sharp sickle spore conidium is little, colourless, in order to upper method inferior separating effect and difficulty is large; Meanwhile, the probability that these methods obtain monospore strain in implementation process is little, and workload is large, and expend time in length.The prior art of particularly carrying out single spore separation for pathogen of soybean root rot point sickle spore conidium did not occur, also there are no the report of this respect.
Patent publication No. is CN103320376A, and publication date is a kind of single spore separation method that the Chinese patent on September 25th, 2013 discloses uncinula necator bacterium.The method, the Powdery Mildew sample that induction gathers regenerates conidium, the conidium acupuncture needle that is single or single string newly produced is transferred on the aseptic grape leave to uncinula necator bacterium sensitivity of 1% clorox process, marking pen mark vaccination, the pure culture of the uncinula necator bacterium be separated to.Patent publication No. is CN103834572A, and publication date is the separation method that the Chinese patent on June 4th, 2014 discloses a kind of strong parasitic disease fungal pathogens.After using the kapillary punching of diameter 1 mm instead, enormously simplify operating process, make the operability of single spore separation stronger.Patent publication No. is CN103333808A, and publication date is the monospore expanding propagation method that the Chinese patent on October 2nd, 2013 discloses a kind of Plasmopara viticola.The method, first the oidium sample of collection is induced to regenerate zoosporangium, the single zoosporangium newly produced or single sporangiophore acupuncture needle are transferred to the aseptic on the grape leaf disk of Plasmopara viticola sensitivity of 1% clorox process, the Plasmopara viticola pure culture be separated to.
Summary of the invention
The technical problem to be solved in the present invention is to provide the method that the strain of a kind of pathogen of soybean root rot point sickle spore monospore is separated.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, and wherein the conidium of sharp sickle spore is made into spore suspension, 26 DEG C of dark culturing 24 h impel conidia germination.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, only contains the suspension of 1-2 spore when wherein the germination conidia sterilized water of soybean point sickle spore being diluted to 10 μ L.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, and wherein prepare the water agar moist heat sterilization of 3%, the temperature of process is 120-125 DEG C, and pressure is 103-104KPa, and the time is 20 min, for subsequent use.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, wherein 96 porocyte culture plate lids is put into 70% alcohol and soaks 12 h sterilizings.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, wherein the water agar of 15 ml 3% is laid in sterilized Tissue Culture Plate and covers.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, is wherein dripped by the conidial suspension of preparation on each circular hole lattice that the 96 porocyte culture plate lids with water agar are corresponding, every lattice 10 μ L spore suspension.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, wherein the Tissue Culture Plate lid dripping good spore suspension is put on inverted microscope Stage microscope, find in 10 × 10 visuals field and determine in corresponding circular hole lattice, only have a spore to exist, recording its place coordinate.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, and wherein preparation contains on the potato dextrose agar flat board of penicillin (20 μ g/mL) and Streptomycin sulphate (40 μ g/mL), for subsequent use.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, monospore in wherein said determination Tissue Culture Plate lid circular hole to be struck agar one circle with moving the limit of bacterium hook along roundlet lattice, then being taken off by substratum with transfering loop puts on above-mentioned potato dextrose agar flat board, and every ware puts a monospore.
The present invention is applied to the separation of pathogen of soybean root rot point sickle spore conidium monospore, 26 DEG C of thermostat container dark culturing 48 h are put in the wherein above-mentioned culture dish upset being placed with monospore strain, the mycelia at picking edge is purified in test tube, saves backup, the monospore strain namely obtained.
The present invention be directed to pathogen of soybean root rot point sickle spore conidium little, of light color, be not easy to be separated, provide a kind of method that sharp sickle spore conidium monospore is separated.The present invention, by after sharp sickle spore conidia germination, increases its volume, then carries out being diluted to a certain degree, the circular hole lattice covered by 96 porocyte culture plates, and what guarantee further to be separated to is monospore strain.Compared with other single spore separation method, operation possibility of the present invention is high, accuracy is high, speed is fast, good separating effect, and equipment is simple.Of the present invention method is simple, can carry out sharp sickle spore in enormous quantities conidial monospore the short period of time and be separated in laboratory.
Embodiment
Below in conjunction with embodiment and testing data, to above-mentioned being described in more detail with other technical characteristic and advantage of the present invention.
The conidial monospore of soybean point sickle spore is separated concrete operations
The present embodiment with pathogen of soybean root rot point sickle spore (Xing An, Wen Jingzhi, Lu Kingdom is loyal, Sun Xiaodong. the Isolation and ldentification of Fusariumsp on Heilongjiang Province soybean root rot diseased plant, Northeast Agricultural University's journal, 2009,40 (8): 5-9; Wang Jiansheng, Wang Jiamei, Li Xiao, Dong Shameng, Zhang Zhengguang, Zheng little Bo. the rapid molecular of pinch outs (Fusarium oxysporum) detects. Plant Pathology, 2013,43 (3): 318-322).96 hole regular growth culture plates are that Shanghai Ling Chu enviromental protection instrument company limited produces, and concrete operations are as follows:
(1) acquisition of soybean point sickle spore conidium mother liquor
The bacterial strain that will be separated the strain of sharp sickle spore monospore is transferred to potato dextrose medium (PDA) flat board from test tube, 26 DEG C of thermostat container dark culturing 4 d.Then, in aseptic operating platform, get 20 mL sterilized waters with sample injector and be placed in the culture dish fallen with pinch outs, with the bacterium hook that moves of sterilizing, mycelia is stirred gently.Then with sterilized four layers of gauze spore suspension crossed and filter mycelia in another aseptic empty culture dish, obtain monospore strain mother liquor for subsequent use.
(2) conidial sprouting and dilution
Mother liquor is placed on 26 DEG C of dark culturing 24 h in incubator, after spore 70% is sprouted, the spore suspension getting 10 μ L is placed in the empty triangular flask (25 mL) gone out, and adds sterilized water and is diluted to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6deng, obtain the spore suspension diluting rear different concns, examine under a microscope till only containing 1-2 spore when suspension concentration on average often gets 10 μ L.
(3) conidial single spore separation
96 porocyte culture plate lids are put into alcohol-pickled 12 h of 70%, take out to put on aseptic operating platform and dry.Then, get 3% water agar that 15 mL are sterilized, spread equably thereon after dissolving, after cooling, Tissue Culture Plate covers the spore suspension 10 μ L that correspondence in each circular hole lattice adds above-mentioned dilution.Then lie on inverted microscope Stage microscope, find in microscope 10 × 10 visual field and determine the small sircle hole lattice only containing 1 spore, write down the transverse and longitudinal coordinate of circular hole lattice at culture plate lid, then the single spore in other small sircle hole lattice is looked for again, after reaching the monospore quantity of needs, with moving the edge standardized circle of bacterium hook along roundlet lattice, then will take off on the potato dextrose agar flat board put into penicillin (20 μ g/mL) and Streptomycin sulphate (40 μ g/mL) with monosporous substratum with transfering loop, every ware puts a monospore strain, then overturn culture dish and put into 26 DEG C of thermostat container dark culturing 2 d, the mycelia at picking edge is purified in test tube, save backup.
The mensuration of the virulence of embodiment 1, the strain of soybean point sickle spore monospore
The present embodiment bacterial strain used be soybean point sickle spore (Xing An, Wen Jingzhi, Lu Kingdom is loyal, Sun Xiaodong. the Isolation and ldentification of Fusariumsp on Heilongjiang Province soybean root rot diseased plant, Northeast Agricultural University's journal, 2009,40 (8): 5-9; Wang Jiansheng, Wang Jiamei, Li Xiao, Dong Shameng, Zhang Zhengguang, Zheng little Bo. the rapid molecular of pinch outs (Fusarium oxysporum) detects. Plant Pathology, 2013,43 (3): 318-322), utilize aforesaid method to carry out monospore strain to same sharp sickle spore to be separated, obtain 15 strain monospore strains, then s-generation monospore strain separation is carried out to No. 12 monospore strain and obtain 15 strain two generation monospore strains.The present embodiment soybean varieties used is black agriculture 53, eastern agriculture 54 is bought by market.
Experimental technique is as follows:
(1) inoculation of soybean pinch outs
The black agriculture of soybean varieties 53, eastern agriculture 54 through 10% clorox process 10 min, aseptic water washing is clean, for subsequent use.
The sharp sickle spore monospore strain 15 obtained utilizing above-mentioned single spore separation method cultivates 4 d respectively on PDA flat board, the bacterium dish beating cut-off footpath 7 mm is inoculated into (sorghum grain boiling water 20 min in jowar grain substratum, then reap in 250 ml triangular flasks, every bottle of 125 g, then moist heat sterilization 121 DEG C of 20 min), 5 bacterium dish are inoculated in each triangular flask, dark culturing in 26 DEG C of incubators, shake triangular flask every day period 1 time, treat that mycelia is covered with each jowar grain surface and namely can be used for inoculation, approximately need 5 d.Inoculation method is: in diameter 8 cm culturing pot, put into sterile vermiculite to culturing pot 2/3 place, every alms bowl evenly accesses 10 sorghum grains that carry disease germs afterwards, soybean varieties is sowed after being covered with about 0.5 cm sterile vermiculite, every alms bowl 10 seeds, finally cover seed-coat with the aseptic dry vermiculite of 2 cm, put into Stainless Steel Disc, compare not inoculate pathogenic bacteria, each process repeats for 3 times, test repetition 2 times.Be placed on 25 DEG C, dark (the 12 h/12 h) alternate culture (in greenhouse) of light after watering sufficient water bottom Stainless Steel Disc, regularly water and keep vermiculite moistening, 10 d " Invest, Then Investigate " results.
(2) the pathogenic analysis of sharp sickle spore monospore strain
Pass through observation of symptoms, analyze the Symptoms in sharp sickle spore and soybean Interaction, and with reference to (1980) and Bai Liyan etc. (2009) such as Burpee, the grade scale that Fusariumsp infects slightly is adjusted, and investigate sharp sickle spore monospore strain according to this grade scale and 2 soybean varieties make sequela situation mutually, and calculate disease index.
0 grade: do not fall ill, root normal growth;
1 grade: the substantially constant or slight browning of main root, the long and vegetative point browning of fibrous root, plant strain growth is normal;
3 grades: main root blackening, main root by infection court continued growth, the blackening of the fibrous root tip of a root, plant strain growth is normal;
5 grades: the serious blackening of main root, not by infection court continued growth, fibrous root obviously reduces or does not have, overground part poor growth, and plant strain growth is short and small;
7 grades: butt rot, can not normal growth or emerge, part cotyledon rot or plant dead.
Disease index formula is:
The mensuration of virulence after table 1 sharp sickle spore conidium first-generation single spore separation
As can be seen from Table 1, point sickle spore first-generation monospore strain Pathogenic Tests, there is bigger difference for the different monospore strain virulence of the same bacterial strain of susceptible variety (black agriculture 53), disease index is from 18.47 to 62.12, but most monospore strain disease index, from 39.65 to 46.83, accounts for 53.33% of total strain number; For disease-resistant variety (eastern agriculture 54) disease index 18.31 to 49.19, but most monospore strain disease index is from 18.31 to 35.66.From interpretation of result, there is significantly differentiation in the virulence of sharp sickle spore monospore strain, illustrates that the Disease-causing gene of sharp sickle spore is polygenic.
S-generation monospore strain Pathogenic Tests is carried out in No. 12, first-generation point sickle spore monospore strain, the results are shown in Table 2.
Pathogenic Tests after table 2 sharp sickle spore monospore strain No. 12 s-generation single spore separation
As can be seen from Table 2, No. 12, first-generation point sickle spore monospore strain is carried out the strain of s-generation monospore and is separated and carries out its Pathogenic Tests, bigger difference is existed for the different monospore strain virulence of the same bacterial strain of susceptible variety (black agriculture 53), disease index is from 13.49 to 45.49, but most monospore strain disease index, from 16.27 to 28.57, accounts for 53.33% of total strain number; For disease-resistant variety (eastern agriculture 54) disease index 11.69 to 39.98, but most monospore strain disease index is from 13.39 to 23.70, and what account for total strain number accounts for 53.33% of total strain number.From interpretation of result, still there is obvious differentiation in the s-generation monospore strain virulence of point sickle spore, and the virulence of s-generation monospore strain is the virulence lower than this monospore strain of the first-generation, regardless of enantiopathy kind or susceptible variety, its virulence obviously weakens, further illustrating sharp sickle spore is that polygene causes a disease, and when Disease-causing gene quantity is fewer, virulence is more weak.
Embodiment 2, different soybean varieties are to the mensuration of sharp sickle spore disease resistance
14 soybean varieties of the present embodiment are produce upper general types, and pathogenic bacteria is the sharp sickle spore monospore strain that different sharp sickle spore (the pathogen of soybean root rot point sickle spore of random choose) is separated.
Carry out single spore separation with the soybean pinch outs strain of 12 random chooses by above-mentioned method, then do mutually with 14 soybean varieties, carry out 14 soybean varieties resistance screening by method described in embodiment 1.
Experimental result is as shown in table 3.
Table 3 different varieties shows the disease resistance of sharp sickle spore monospore strain
As can be seen from Table 3, the monospore strain that the sharp sickle spore of 12 random chooses is separated to and 13 kinds are made result mutually and are shown, there is notable difference in the disease resistance of different varieties to pinch outs, the disease index of monospore strain No.1 to black agriculture 44 as F2 is 21.33, and No. 5, the monospore strain of A41 is to the disease index 64.55 of black agriculture 44; The involutory disease index of rich 55 of No. 2, monospore strain of point sickle spore B8 is 19.93, and the involutory disease index of rich 55 55.71 of No. 5, monospore strain of A41.This illustrates that the disease resistance of same kind to difference sharp sickle spore is different, carries out resistance screening for different geographic regions, and it is feasible that application disease-resistant variety prevents and treats this disease; Meanwhile, different varieties is also different to the strain of same sharp sickle spore monospore, but difference is less, and the virulence can reacting sickle spore monospore full to the brim strain is relatively stable.
In a word, operation possibility of the present invention is high, accuracy is high, speed is fast, good separating effect, and equipment is simple.Of the present invention method is simple, can carry out sharp sickle spore in enormous quantities conidial monospore the short period of time and be separated, then carry out the screening of Pathogenic Tests and Resistance resource in laboratory.
Claims (10)
1. the method for a pathogen of soybean root rot point sickle spore monospore strain separation.
2. method according to claim 1, is characterized in that: described method is mixed with spore suspension after numerous for the pinch outs strain of separation expansion, and sprout 24 h after utilizing 4 layers of sterile gauze to filter.
3. method according to claim 2, is characterized in that: the conidial suspension sterilized water after sprouting is diluted to when on average often getting 10 μ L only containing till 1-2 spore.
4. method according to claim 3, is characterized in that: prepare the water agar of 3% in described method, and moist heat sterilization process 20 min, then get 15 ml and be laid in while hot on the lid of 96 aseptic porocyte culture plates.
5. the method according to claim 3-4, is characterized in that: the spore suspension will diluted in described method, gets the spore suspension diluted and drips to the circular hole Ge Nei center that the Tissue Culture Plate with water agar covers, add 10 μ L in every aperture lattice.
6. method according to claim 5, is characterized in that: will be placed on the Stage microscope of inverted microscope by Tissue Culture Plate lid in described method, finds and to determine in 10 μ L spore suspensions in single circular hole lattice only 1 spore under 10 × 10 visuals field.
7. method according to claim 6, it is characterized in that: with transfering loop Tissue Culture Plate covered in the PDA plate that substratum in the circular hole lattice being defined as single spore moves to penicillin (20 μ g/mL) and Streptomycin sulphate (40 μ g/mL) in described method, after 48 h, purifying causes in PDA test tube, namely obtains monospore strain.
8., according to the arbitrary described method of claim 2-7, it is characterized in that: the operation in described method all needs aseptic technique, carry out in aseptic operating platform.
9. the method according to claim 2 or 7, is characterized in that: described culture temperature is 26 DEG C, and described cultivation does not need illumination.
10. the application of the arbitrary described method of claim 1-9 in pathogen of soybean root rot point sickle spore conidium monospore is separated.
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Cited By (4)
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| CN105886451A (en) * | 2016-04-26 | 2016-08-24 | 东北农业大学 | Single spore isolation method of phytophthora capsici zoospores |
| CN107446824A (en) * | 2017-08-21 | 2017-12-08 | 湖北省农业科学院植保土肥研究所 | A kind of single spore separation method of wheat powdery mildew |
| CN110029083A (en) * | 2019-04-01 | 2019-07-19 | 中国农业科学院蔬菜花卉研究所 | A method of rape plasmodiophora brassicae monospore is separated based on methylene blue agarose method |
| CN113061565A (en) * | 2021-04-12 | 2021-07-02 | 东北农业大学 | Rapid formation method of fusarium graminearum conidia |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886451A (en) * | 2016-04-26 | 2016-08-24 | 东北农业大学 | Single spore isolation method of phytophthora capsici zoospores |
| CN105886451B (en) * | 2016-04-26 | 2020-07-10 | 东北农业大学 | Single spore separation method of phytophthora capsici zoospores |
| CN107446824A (en) * | 2017-08-21 | 2017-12-08 | 湖北省农业科学院植保土肥研究所 | A kind of single spore separation method of wheat powdery mildew |
| CN107446824B (en) * | 2017-08-21 | 2019-12-17 | 湖北省农业科学院植保土肥研究所 | Single spore isolation method of erysiphe graminis |
| CN110029083A (en) * | 2019-04-01 | 2019-07-19 | 中国农业科学院蔬菜花卉研究所 | A method of rape plasmodiophora brassicae monospore is separated based on methylene blue agarose method |
| CN110029083B (en) * | 2019-04-01 | 2020-08-18 | 中国农业科学院蔬菜花卉研究所 | Method for separating plasmodiophora brassicae monospores based on methylene blue agarose method |
| CN113061565A (en) * | 2021-04-12 | 2021-07-02 | 东北农业大学 | Rapid formation method of fusarium graminearum conidia |
| CN113061565B (en) * | 2021-04-12 | 2023-03-24 | 东北农业大学 | Rapid formation method of fusarium graminearum conidia |
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Application publication date: 20150218 |