CN102487630B - Identification method of special type pathogenicity of fusarium oxysporum sesame - Google Patents
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Abstract
The invention relates to an identification method of pathogenicity of fusarium oxysporum sesame special type bacterial strain to host sesame, which comprises the following steps: (1) pretreatment of seeds; (2) bacterial inoculation culture of the seeds: conidium suspending liquid is taken and poured into sterile vermiculite with the volume to be two times that of the conidium suspending liquid, vermiculite mixed with pathogenic bacteria and sterile soil are mixed evenly according to the volume ratio of 1:3 and charged in a split mode, sterile sesame seeds exposing white are sown in a paper bowl, 10 particles are evenly placed in each bowl, each variety is repeated and planted for three times, mixed soil for mixing bacteria is lightly covered on the surface of the seeds and placed into an artificial climate box for light and dark alternated culture for 12 hours per day at 25 DEG C and at the humidity of about 80%, and other culture conditions are the same with equal-volume sterile water as comparison; and (3) performing pathogenicity identification. The identification method is simple, convenient and easy to implement, pathogenicity of the pathogenic bacteria is stable, results are reliable, repeatability is good, and the fact that whether fusarium oxysporum sesame special type bacterial strain has pathogenicity on sesame seedlings and pathogenic strength of the fusarium oxysporum sesame special type bacterial strain can be accurately reflected.
Description
Technical field
The present invention relates to a kind of method that adopts indoor paper pot to cultivate Characteristics of Sesame to identify and evaluate fusarium oxysporum sesame strain pathogenic strength, belong to biological technical field.
Background technology
Fusarium oxysporum sesame is a kind of pathogenic fungus that causes sesame generation wilt disease, serious to sesame growth harm.In China's sesame is produced,, can be reached more than 30% when serious in 15% left and right by the long-term incidence of disease of this microbial fusarium wilt, larger on yield and quality impact.For verifying the pathogenic feature of fusarium oxysporum sesame pathogen, we have carried out sesame wilt germs strain collection work, and have set up China's fusarium oxysporum sesame strain storehouse.The pathogenic identification research of carrying out the main bacterial strain of strain storehouse is illustrate sesame Pathogen of Fusarium Wilt pathogenesis crucial and basic.But due to sesame disease Research foundation weakness, there is not yet so far the bibliographical information of the indoor qualification of fusarium oxysporum sesame pathogen pathogenicity and evaluation method, also only adopt sick garden, land for growing field crops to carry out to the qualification of sesame fusarium wilt disease resistance both at home and abroad.The method of utilizing bacterium cake to infect sesame germination seed qualification sesame Pathogen of Fusarium Wilt is declared in the existing patent of China, but because sesame radicle growth is slow, plant vertical growth is subject to experimental condition impact, the sustainable phase of material is short and connect the uneven first-class problem of bacterium, add that Fusarium oxysporum has the advantages that the cycle of infecting is long, morbidity is slower, the method is not suitable for the pathogenicity for qualification and evaluation fusarium oxysporum sesame pathogen.In addition, because sesame root system is undeveloped, hinder root processing plant strain growth is affected to the factors such as excessive, test also proves conventional filling root bacterination process, hinders the method such as root bacterination process, radicle bacterination process and be not suitable for sesame Pathogen of Fusarium Wilt Assessment of Virulence, more cannot perform well in pathogen analysis of genetic diversity and Race Identification research.Research definite fusarium oxysporum sesame Pathogenic characterization and evaluation method have become the bottleneck problem of sesame fusarium wilt research.
Summary of the invention
The object of the invention is to provide a kind of authentication method of special type pathogenicity of fusarium oxysporum sesame.
To achieve these goals, technical scheme of the present invention has adopted a kind of authentication method of special type pathogenicity of fusarium oxysporum sesame, realizes by following steps:
(1) seed pretreatment: choose the healthy full seed respectively with 2 disease-resistant and susceptible sesame varieties, after clear water rinses, on superclean bench, first process 30s, aseptic water washing 1 time with 70% alcohol; Then process 15min with 3% clorox, use aseptic water washing 3-5 time, afterwards at 120r/min, shaken overnight under 25 DEG C of conditions, shows money or valuables one carries unintentionally seed with vernalization;
(2) seed connects bacterium cultivation: get conidial suspension, pour in the aseptic vermiculite of 2 times of volumes and mix and mix thoroughly, then by the volume ratio of 1: 3, the vermiculite and the sterile soil that are mixed with pathogen are mixed, packing, the aseptic sesame seed that step (1) has been showed money or valuables one carries unintentionally is sowed in paper pot, every alms bowl is evenly put 10,3 repetitions of each kind plantation, gently cover the surface of the seed with the mixed soil of mixing bacterium, put to climatic cabinate, 25 DEG C, the dark alternate culture of the 12h/ daylight, humidity remains on 70-90%; Taking same volume sterile water as contrast, other condition of culture are identical;
(3) carry out pathogenic qualification.
Step (2) conidial suspension preparation method is: take out the fusarium oxysporum sesame inoculation of preserving in PDB liquid nutrient medium, at 160r/min, under 28 DEG C of conditions, shake bacterium 3d, with two-layer sterile gauze filtration, prepare Fusarium oxysporum conidial suspension, being mixed with concentration with sterile water dilution is 1 × 10
6the conidial suspension of individual/mL, in order to inoculation.
In step (2), the concentration of conidial suspension is 1 × 10
6individual/mL.
Step (3) concrete steps are: after 1) connecing bacterium, make regular check on emerging and growing state of every alms bowl seedling, guarantee that growth of seedling environment is normal; 2) connect the incidence of the 21st day every alms bowl seedling of " Invest, Then Investigate " of bacterium, record morbidity strain number and morbidity grade.
Method of the present invention is mixing in bacterium processing procedure, utilize the strong non-caking feature of the loose water imbibition of vermiculite, use sterilizing vermiculite to mix with certain volume bacterium liquid, can make bacterium liquid be distributed in uniformly vermiculite granule surface by stirring, after mixing with sterile soil, can ensure that germ spore is mixed in aseptic soil equably, bacterial classification is uniformly distributed in soil, guarantees that Pathogenicity is reliable, accurate; In addition, use the seed after sterilizing vernalization to test, avoid the experimental error causing because seed germination is irregular: adopt method of the present invention to carry out prior to seeding soil and connect bacterium processing, sesame root system is not hindered to root processing, guarantee Characteristics of Sesame is grown in the raw, avoid connecing mid-term bacterium and operated the mechanical damage that causes to plant, water stain appearance of the problem such as coercing, guaranteed that result of study is reliable, accurately; Method of the present invention adopts indoors artificial climate box to grow seedlings, and be 3 weeks qualification cycle, can meet infecting period of fusarium oxysporum sesame, can guarantee that the performance of infection process process is complete.Of the present invention method is simple, and plant morbidity result is stable, and reproducible, result of the test is reliable, can reflect exactly the Difference in Pathogenicity of fusarium oxysporum sesame on different sesame varieties, also can reflect the pathogenicity difference between bacterial strain.
Characteristics of Sesame fusarium wilt grading standard: 0 grade: grow fine without illness, overground part cauline leaf is normal, and rhizome portion vascular bundle is normal.I level: diseased plant cauline leaf is wilted, rhizome portion vascular bundle bronzing.II level: diseased plant blade dries up to complete stool death.
The computing formula of disease index is:
Disease index=∑ (morbidity grade × corresponding morbidity strain number)/total strain number
This exponential number is larger, shows the pathogenic stronger of this germ.
Assessment of Virulence: judge according to disease index that each bacterial strain is pathogenic and have or not, and pathogenicity grade.Fusarium oxysporum sesame strain pathogenic strength grade classification is level Four: 0 grade (had no pathogenicity): average disease refers to be 0; 1 grade (weak pathogenicity): average disease refers to 0.1～20; 2 grades (medium pathogenicity): average disease refers to 20.1～35; 3 grades (strong pathogenicity): average disease refers to 35.1～100.
Brief description of the drawings
Fig. 1 pours cultured conidial suspension in aseptic vermiculite into and mixes thoroughly;
Fig. 2 mixes vermiculite and the sterile soil mixed after bacterium;
Fig. 3 is filled to the bacterium soil point of mixing thoroughly in paper pot;
Fig. 4 is that the seed that vernalization after sterilizing is showed money or valuables one carries unintentionally is sowed in the sterile soil that is connected to Fusarium oxysporum;
Fig. 5 connects bacterium sesame situation of emerging after 3 days;
Fig. 6 connects 3 weeks rear 2 contrast growing states of bacterium, and Fig. 6 A is Henan sesame 11 (disease-resistant), and Fig. 6 B is Ji 9014 (susceptible);
Fig. 7 connects the processing incidence of strong Pathogenicity Strains after 3 weeks, and Fig. 7 A is Henan sesame 11 (disease-resistant), Fig. 7 B Ji 9014 (susceptible);
Fig. 8 connects the processing incidence of medium Pathogenicity Strains after 3 weeks, and Fig. 8 A is Henan sesame 11 (disease-resistant), and Fig. 8 B is Ji 9014 (susceptible);
Fig. 9 connects the processing incidence of weak Pathogenicity Strains after 3 weeks, and Fig. 9 A is Henan sesame 11 (disease-resistant), Fig. 9 B Ji 9014 (susceptible).
Embodiment
Embodiment 1
The present embodiment is pathogenicity qualification and the evaluation method of fusarium oxysporum sesame bacterial strain No.HSFO 10012:
1) PDB medium preparation: take 200g potato, clean peeling is cut into small pieces, add water well-done, boil 20-30min, can be poked by glass bar, by four layers of filtered through gauze to beaker, add glucose 20g, supply moisture to 1000ml, stirring and evenly mixing, be sub-packed in triangular flask 121 DEG C of sterilizings 20 minutes;
2) No.HSFO 10012 bacterial strain conidial suspension preparations: take out the No.HSFO10012 bacterial strain of preserving, the a small amount of bacterium liquid of picking is inoculated in PDB liquid nutrient medium, at 160r/min, shaken cultivation 3d under 28 DEG C of conditions, then filter with two-layer sterile gauze, prepare No.HSFO 10012 bacterial strain conidial suspensions, be mixed with concentration 1 × 10 with sterile water
6individual/mL conidial suspension, in order to inoculation;
3) seed pretreatment: the full healthy seed of choosing Henan sesame 11 and 2 sesame varieties of Rong County, Sichuan Semen sesami nigrum, on superclean bench, first process 30s with 70% alcohol, process 15min with 3% clorox again, then use aseptic water washing 3-5 time, at 120r/min, shaken overnight under 25 DEG C of conditions, shows money or valuables one carries unintentionally seed with vernalization afterwards;
4) seed connects bacterium and cultivates: (concentration is 1 × 10 to get the No.HSFO 10012 bacterial strain conidial suspensions of certain volume
6individual/mL), pour in the aseptic vermiculite of 2 times of volumes and mix and mix (Fig. 1) thoroughly, then by the volume ratio of 1: 3, the vermiculite and the sterile soil that are mixed with pathogen are mixed to (Fig. 2), be sub-packed in (Fig. 3) in each paper pot, the aseptic sesame seed having showed money or valuables one carries unintentionally is sowed in paper pot (Fig. 4), every alms bowl is evenly put 10, 3 repetitions of each kind plantation, gently cover the surface of the seed with the mixed soil of mixing bacterium, put to climatic cabinate, 25 DEG C, the dark alternate culture of the 12h/ daylight, humidity remains on 90%, taking same volume sterile water as contrast, other condition of culture are identical,
5) the pathogenic investigation of bacterial strain: make regular check on emerging and growing state (Fig. 5) of every alms bowl seedling after connecing bacterium, guarantee that growth of seedling environment is normal;
6) connect the incidence (Fig. 7) of the 21st day every alms bowl seedling of " Invest, Then Investigate " of bacterium, record morbidity strain number and morbidity grade, calculate disease index.Data analysis demonstration, for Henan sesame 11 kinds, the average disease index of this bacterium is 38.3; For Ji 9014 kinds, the average disease index of this bacterium is 73.3;
7) Assessment of Virulence: fusarium oxysporum sesame strain pathogenic strength grade classification is level Four: 0 grade (had no pathogenicity): average disease refers to be 0; 1 grade (weak pathogenicity): average disease refers to 0.1～20; 2 grades (medium pathogenicity): average disease refers to 20.1～35; 3 grades (strong pathogenicity): average disease refers to 35.1～100.
According to the pathogenicity criteria for classifying, No.HSFO 10012 bacterial strains all belong to strong Pathogenicity Strains for above-mentioned disease-resistant variety and susceptible variety.
Embodiment
Embodiment 2 is pathogenicity qualification and the evaluation method of fusarium oxysporum sesame bacterial strain No.HSFO 10008:
1) PDB medium preparation: take 200g potato, clean peeling is cut into small pieces, add water well-done, boil 20-30min, can be poked by glass bar, by four layers of filtered through gauze to beaker, add glucose 20g, supply moisture to 1000ml, stirring and evenly mixing, be sub-packed in triangular flask 121 DEG C of sterilizings 20 minutes;
2) No.HSFO 10008 bacterial strain conidial suspension preparations: take out the No.HSFO10008 bacterial strain of preserving, the a small amount of bacterium liquid of picking is inoculated in PDB liquid nutrient medium, at 160r/min, shaken cultivation 3d under 28 DEG C of conditions, then filter with two-layer sterile gauze, prepare No.HSFO 10008 bacterial strain conidial suspensions, be mixed with concentration 1 × 10 with sterile water
6individual/mL conidial suspension, in order to inoculation;
3) seed pretreatment: the full healthy seed of choosing Henan sesame 11 and 2 sesame varieties of Rong County, Sichuan Semen sesami nigrum, on superclean bench, first process 30s with 70% alcohol, process 15min with 3% clorox again, then use aseptic water washing 5 times, at 120r/min, shaken overnight under 25 DEG C of conditions, shows money or valuables one carries unintentionally seed with vernalization afterwards;
4) seed connects bacterium and cultivates: (concentration is 1 × 10 to get the No.HSFO 10008 bacterial strain conidial suspensions of certain volume
6individual/mL), pour in the aseptic vermiculite of 2 times of volumes and mix and mix (Fig. 1) thoroughly, then by the volume ratio of 1: 3, the vermiculite and the sterile soil that are mixed with pathogen are mixed to (Fig. 2), be sub-packed in (Fig. 3) in each paper pot, the aseptic sesame seed having showed money or valuables one carries unintentionally is sowed in paper pot (Fig. 4), every alms bowl is evenly put 10, 3 repetitions of each kind plantation, gently cover the surface of the seed with the mixed soil of mixing bacterium, put to climatic cabinate, 25 DEG C, the dark alternate culture of the 12h/ daylight, humidity remains on 70%, taking same volume sterile water as contrast, other condition of culture are identical,
5) the pathogenic investigation of bacterial strain: make regular check on emerging and growing state (Fig. 5) of every alms bowl seedling after connecing bacterium, guarantee that growth of seedling environment is normal;
6) connect the incidence (Fig. 8) of the 21st day every alms bowl seedling of " Invest, Then Investigate " of bacterium, record morbidity strain number and morbidity grade, calculate disease index.Data analysis demonstration, for Henan sesame 11 kinds, the average disease index of this bacterium is 16.7; For Ji 9014 kinds, the average disease index of this bacterium is 26.7;
Assessment of Virulence: fusarium oxysporum sesame strain pathogenic strength grade classification is level Four: 0 grade (had no pathogenicity): average disease refers to be 0; 1 grade (weak pathogenicity): average disease refers to 0.1～20; 2 grades (medium pathogenicity): average disease refers to 20.1～35; 3 grades (strong pathogenicity): average disease refers to 35.1～100.
According to the pathogenicity criteria for classifying, No.HSFO 10008 bacterial strains belong to weak Pathogenicity Strains for above-mentioned disease-resistant variety, belong to medium Pathogenicity Strains for susceptible variety.
Embodiment 3 is pathogenicity qualification and the evaluation method of fusarium oxysporum sesame bacterial strain No.HSFO 10012:
1) PDB medium preparation: take 200g potato, clean peeling is cut into small pieces, add water well-done, boil 20-30min, can be poked by glass bar, by four layers of filtered through gauze to beaker, add glucose 20g, supply moisture to 1000ml, stirring and evenly mixing, be sub-packed in triangular flask 121 DEG C of sterilizings 20 minutes;
2) No.HSFO 08005 bacterial strain conidial suspension preparation: take out the No.HSFO08005 bacterial strain of preserving, the a small amount of bacterium liquid of picking is inoculated in PDB liquid nutrient medium, at 160r/min, shaken cultivation 3d under 28 DEG C of conditions, then filter with two-layer sterile gauze, prepare No.HSFO 08005 bacterial strain conidial suspension, be mixed with concentration 1 × 10 with sterile water
6individual/mL conidial suspension, in order to inoculation;
3) seed pretreatment: the full healthy seed of choosing Henan sesame 11 and 2 sesame varieties of Rong County, Sichuan Semen sesami nigrum, on superclean bench, first process 30s with 70% alcohol, process 15min with 3% clorox again, then use aseptic water washing 3-5 time, at 120r/min, shaken overnight under 25 DEG C of conditions, shows money or valuables one carries unintentionally seed with vernalization afterwards;
4) seed connects bacterium and cultivates: (concentration is 1 × 10 to get the No.HSFO 08005 bacterial strain conidial suspension of certain volume
6individual/mL), pour in the aseptic vermiculite of 2 times of volumes and mix and mix (Fig. 1) thoroughly, then by the volume ratio of 1: 3, the vermiculite and the sterile soil that are mixed with pathogen are mixed to (Fig. 2), be sub-packed in (Fig. 3) in each paper pot, the aseptic sesame seed having showed money or valuables one carries unintentionally is sowed in paper pot (Fig. 4), every alms bowl is evenly put 10,3 repetitions of each kind plantation, gently cover the surface of the seed with the mixed soil of mixing bacterium, put to climatic cabinate, 25 DEG C, the dark alternate culture of the 12h/ daylight, humidity remains on 80%.Taking same volume sterile water as contrast, other condition of culture are identical;
5) the pathogenic investigation of bacterial strain: make regular check on emerging and growing state (Fig. 5) of every alms bowl seedling after connecing bacterium, guarantee that growth of seedling environment is normal;
6) connect the incidence (Fig. 9) of the 21st day every alms bowl seedling of " Invest, Then Investigate " of bacterium, record morbidity strain number and morbidity grade, calculate disease index.Data analysis demonstration, for Henan sesame 11 kinds, the average disease index of this bacterium is 6.7; For Ji 9014 kinds, the average disease index of this bacterium is 20.0.
Assessment of Virulence: fusarium oxysporum sesame strain pathogenic strength grade classification is level Four: 0 grade (had no pathogenicity): average disease refers to be 0; 1 grade (weak pathogenicity): average disease refers to 0.1～20; 2 grades (medium pathogenicity): average disease refers to 20.1～35; 3 grades (strong pathogenicity): average disease refers to 35.1～100.
According to the pathogenicity criteria for classifying, No.HSFO 08005 bacterial strain all belongs to weak Pathogenicity Strains for above-mentioned disease-resistant variety and susceptible variety.
Claims (2)
1. an authentication method for special type pathogenicity of fusarium oxysporum sesame, is characterized in that: realize by following steps:
(1) seed pretreatment: choose the healthy full seed respectively with 2 disease-resistant and susceptible class sesame varieties, after clear water rinses, on superclean bench, first process 30s, aseptic water washing 1 time with 70% alcohol; Then process 15min with 3% clorox, use aseptic water washing 3-5 time, afterwards at 120r/min, shaken overnight under 25 DEG C of conditions, shows money or valuables one carries unintentionally seed with vernalization;
(2) seed connects bacterium cultivation: get conidial suspension, pour in the aseptic vermiculite of 2 times of volumes and mix and mix thoroughly, then by the volume ratio of 1:3, the vermiculite and the sterile soil that are mixed with pathogen are mixed to packing, the aseptic sesame seed that step (1) has been showed money or valuables one carries unintentionally is sowed in paper pot, every alms bowl is evenly put 10, and 3 repetitions of each kind plantation, use the mixed soil of mixing bacterium gently to cover the surface of the seed, put to climatic cabinate, 25 DEG C, the dark alternate culture of the 12h/ daylight, humidity remains on 70-90%;
(3) carry out pathogenic qualification;
Wherein step (2) conidial suspension preparation method is: take out the fusarium oxysporum sesame inoculation of preserving in PDB liquid nutrient medium, at 160r/min, under 28 DEG C of conditions, shake bacterium 3d, with two-layer sterile gauze filtration, prepare Fusarium oxysporum conidial suspension, being mixed with concentration with sterile water dilution is 1 × 10
6the conidial suspension of individual/ml, in order to inoculation.
2. the authentication method of special type pathogenicity of fusarium oxysporum sesame according to claim 1, is characterized in that: step (3) concrete steps are: after 1) connecing bacterium, make regular check on emerging and growing state of every alms bowl seedling, guarantee that growth of seedling environment is normal; 2) connect the incidence of the 21st day every alms bowl seedling of " Invest, Then Investigate " of bacterium, record morbidity strain number and morbidity grade.
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| CN106967783B (en) * | 2017-02-23 | 2020-05-19 | 河南省农业科学院芝麻研究中心 | Method for identifying and evaluating resistance level of sesame wilt disease |
| CN106834418A (en) * | 2017-03-02 | 2017-06-13 | 浙江省农业科学院 | The method that seedling stage water planting is inoculated with Rapid identification tomato neckrot Resistance To Root Rot Disease plant |
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| CN102061330A (en) * | 2010-11-30 | 2011-05-18 | 中国农业科学院油料作物研究所 | Method for identifying pathogenicity of sesame stem blight and blast pathogenic bacteria |
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