CN103224888A - Long-term preservation method of blumeria graminis strains - Google Patents
Long-term preservation method of blumeria graminis strains Download PDFInfo
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Abstract
The invention discloses a long-term preservation method of blumeria graminis strains, which comprises the steps of (1) the expanding propagation of monadic strains; (2) the collection of conidium; and (3) after silica gel is slowly dried for 5h, preservation in a refrigerator at a temperature of -70 DEG C. According to the method, the preservation time of blumeria graminis strains can be longer, is increased to 1 year from 10-12d by using conventional methods, and the method is simple, convenient and practical. The defects of the conventional preservation methods that operation is complicated, manpower and material resources consumption are seirous are overcome, so that the normal implementation of related researches on blumeria graminis strains is ensured. The method disclosed by the invention can be popularized and applied to wheat disease research and breeding mechanisms.
Description
Technical field
The present invention relates to the technical field of crop pathogens store method, relate in particular to a kind of long-term preservation method of wheat powdery mildew single-ascospore strain.
Background technology
Wheat (Triticum aestivum L.) is one of most important food crop in the world now, and its yield and quality is influenced by diseases such as bacterium, fungi, virus very easily.In wheat diseases, Powdery Mildew be occurrence scope the widest, to one of bigger disease of yield effect.After wheat is subjected to powdery mildew harm, the powdery spot appears on blade and tassel, yellow leaf is early withered when serious, and plant is short and small thin and delicate, and photosynthesis reduces, respiration is strengthened, tiller number reduces, and the percentage of earbearing tiller reduces, and thousand seed weight descends, but the general underproduction 5%～10% can make wheat yield 10%～30% when popular.
Wheat powdery mildew is that (its pathogenic bacteria is the live body bacterial parasite of specialized form, can not grow on artificial medium for Blumeria graminis f.sp.tritici, the fungal disease that Bgt) causes by powdery mildew.The breeding switching of germ all needs to carry out once (conventional store method) every about 10 days on wheat live body seedling.In addition, length consuming time is collected in conidial breeding, and workload is big, tends to produce cross staining on the other hand, causes losing of specific pathogenic bacterial strains.Therefore, setting up long-term preservation method can this germ population genetic diversity, reduced time is accelerated the experiment progress to the molecule monitoring for resistance greatly.Simultaneously, the different white powder germ pathotype inoculation wheat breeds of preservation can comprehensively, accurately be estimated the disease resistance of kind, for wheat breeding provides auxiliary.
Therefore, people urgently wish under laboratory condition, seek a kind of easy and simple to handle, time saving and energy saving, energy-conservation, and can reduce aleopation, prolong the method for wheat powdery mildew bacterial strain shelf time, so that carry out the research of the variation of wheat powdery mildew toxicity and host and pathogenic bacteria interaction.
Summary of the invention
The objective of the invention is, short, time-consuming at the former bacterium of existing wheat powdery mildew existing preservation period on the wheat live body is preserved, take a lot of work and the easy defective of generation cross staining etc. in frequent switching process, propose a kind of easy and simple to handle, time saving and energy saving, and can less different strains between cross staining, the method that can the long period preserve the wheat powdery mildew bacterial strain is to guarantee normally carrying out of wheat powdery mildew former bacterium correlative study work.
The object of the invention is achieved through the following technical solutions:
One aspect of the present invention relates to a kind of long-term preservation method of wheat powdery mildew bacteria strain, it is characterized in that comprising the steps:
(1) collection of single-ascospore strain: gather the wheat leaf blade that only has single sorus from the field, face of blade is placed on the fresh-keeping substratum of the agar that contains benzimidazolyl up, in super clean bench, smear on the blade gently with toothpick picking spore, place between growth and cultivate (17 ± 1 ℃, 17-19h illumination/sky) 8-10 days, wait to produce a large amount of fresh conidiums, standby as first numerous bacterial strain;
(2) expansion of single-ascospore strain is numerous: above-mentioned wheat plant first leaf is cut into the 2.5-3.5cm section of coming into leaves occupies and containing on the fresh-keeping substratum of benzoglyoxaline, in the inoculation tower, above-mentioned stripped leaf section is inoculated in advance the good fresh conidium of breeding just, place between growth and cultivate (17 ± 1 ℃, 17-19h illumination/sky) 8-10 days, produce a large amount of fresh conidiums, wait to collect and preserve;
(3) conidial collection: the conidium that on Bechtop (2) is produced is collected, culture dish is tipped upside down on the template, beat 2-3 time with tweezers gently, collect in the centrifuge tube, bacterial strain of every collection is changed a template, prevents the cross staining between bacterial strain;
(4) silica gel is slowly dry: diameter 3-6mm silica gel particle is placed centrifuge tube, place 23 ± 1 ℃ of dry 4-6h down, place-70 ℃ of Ultralow Temperature Freezers to preserve again with sealing film phonograph seal then.
In a preferred embodiment of the present invention, described Ultralow Temperature Freezer time of preserving is 1,3,6 or more than December.
In a preferred embodiment of the present invention, described preservation agent is formulated by the benzoglyoxaline that per hundred ml distilled waters add 0.4-0.6g agar+2-4mL0.1-0.3%.
In another preferred embodiment of the present invention, described method also comprises the resurrection step of bacterial strain, described resurrection step makes its slow intensification for the described centrifuge tube of step (4) is put into 4 ℃ of ice chests, is inoculated in the fresh leaf section according to the described method of step (1) then.
In another preferred embodiment of the present invention, the resurrection rate of described bacterial strain is more than 90%, to be preferably 100%.
The present invention can make the shelf time of wheat powdery mildew longer, extend to 1 year by the method 10-12d of routine, and method is simple and practical.Shortcomings such as conventional store method complex operation, labor intensive, material resources are bigger have been overcome, to guarantee normally carrying out of wheat powdery mildew former bacterium correlative study work.The present invention can apply in wheat diseases research and breeding agency.
Description of drawings
Fig. 1: cryopreservation is the strain pathogenic strength experimental result after December.
Embodiment
The present invention is described in further detail by following examples,
The high sense of wheat wide spectrum Powdery Mildew kind: Zheng wheat 98,
Benzoglyoxaline: chemical pure, chemical reagents corporation of traditional Chinese medicines group;
Dry silica gel: U.S. Sigma aldrich company produces
Agar: chemical pure, Beijing ancient cooking vessel state Bioisystech Co., Ltd;
Seal film: U.S. parafilm company produces;
Embodiment 1, the slow dry prolonged preservation of wheat powdery mildew bacteria strain
This method is carried out according to the following steps:
1, the collection of single-ascospore strain with expand numerous: bacterial strain gather preceding 10 days earlier sowing wide spectrum high sense wheat powdery mildew kind " Zheng wheat 98 " to a leaf wholeheartedly, standby; Gather the blade that only has single sorus from the field, it is long that preprepared wheat plant first leaf of clip interrupts 3cm, face of blade is placed on the fresh-keeping substratum of the agar that contains benzimidazolyl up, in super clean bench, smear on the blade gently with toothpick picking spore, place between growth and cultivate (17 ± 1 ℃, 18h illumination) 8-10 days, wait to produce a large amount of fresh conidiums, standby as first numerous bacterial strain.Above-mentioned wheat plant first leaf is cut into the 3cm section of coming into leaves occupies on bright substratum (the benzoglyoxaline ratio by 100mL distilled water+0.5g agar+3mL0.2% is formulated), (in 15 * 22 * 22cm) above-mentioned stripped leaf section is inoculated in advance the good fresh conidium of numerous breeding just at the inoculation tower, place between growth and cultivate (17 ± 1 ℃, 18h illumination/sky) 8-10 days, produce a large amount of fresh conidiums, wait to collect and preserve;
2, conidial collection: the conidium that on Bechtop (1) is produced is collected, culture dish is tipped upside down on the template, gently with tweezers beat culture dish lid 2-3 time, collect in the 1.5mL centrifuge tube, bacterial strain of every collection all need be changed a template, prevents the cross staining between bacterial strain.With 75% alcohol super clean bench is sterilized simultaneously.The conidium quality of collecting is advisable about with 20mg;
3, silica gel is slowly dry: the silica gel particle (diameter 3-6mm) of 3-4 piece is placed step (2) centrifuge tube, place 23 ℃ of dry 5h down, place-70 ℃ of Ultralow Temperature Freezers preservations 1,3,6 and December again with sealing film phonograph seal then;
4, conidial virulence is measured: in actual applications, the described centrifuge tube of step (3) can be put into 4 ℃ of ice chests and make its slow intensification, in order to avoid the shock heating cracking of spore is inoculated in the fresh leaf section according to the described method of step (1) then.Place between growth and cultivated (17 ± 1 ℃, 18h illumination) 8-10 days.Observe the spore virulence;
5, the resurrection rate of bacterial strain under the relatively different shelf times: carry out the comparison of bacterial strain resurrection rate under the different shelf times according to this method.Preserve 20 bacterial strains altogether, the shelf time is respectively 1,3, and 6 and December, preserve in contrast with ordinary method.
Show by the virulence detection, adopt this method bacterial strain can survive December at least, and the resurrection rate of bacterial strain is 100% (table 1 and Fig. 1), though and conventional preservation also can reach 100% resurrection rate, but to plant 1 time every switching in 10 days, consuming time taking a lot of work is not suitable for preserving on a large scale bacterial strain, and the bacterial strain that method of the present invention is preserved inoculation in 1 year once.
The comparison (20 bacterial strains) of table 1 wheat powdery mildew strain survival rate under the different shelf times
The above only is the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any variation or replacement of expecting without creative work all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (5)
1. the long-term preservation method of a wheat powdery mildew bacteria strain is characterized in that comprising the steps:
(1) collection of single-ascospore strain: gather the wheat leaf blade that only has single sorus from the field, face of blade is placed on the fresh-keeping substratum of the agar that contains benzimidazolyl up, in super clean bench, smear on the blade gently with toothpick picking spore, place between growth and cultivate (17 ± 1 ℃, 17-19h illumination/sky) 8-10 days, wait to produce a large amount of fresh conidiums, standby as first numerous bacterial strain;
(2) expansion of single-ascospore strain is numerous: above-mentioned wheat plant first leaf is cut into the 2.5-3.5cm section of coming into leaves occupies and containing on the fresh-keeping substratum of benzoglyoxaline, in the inoculation tower, above-mentioned stripped leaf section is inoculated in advance the good fresh conidium of breeding just, place between growth and cultivate (17 ± 1 ℃, 17-19h illumination/sky) 8-10 days, produce a large amount of fresh conidiums, wait to collect and preserve;
(3) conidial collection: the conidium that on Bechtop (2) is produced is collected, culture dish is tipped upside down on the template, beat 2-3 time with tweezers gently, collect in the centrifuge tube, bacterial strain of every collection is changed a template, prevents the cross staining between bacterial strain;
(4) silica gel is slowly dry: diameter 3-6mm silica gel particle is placed centrifuge tube, place 23 ± 1 ℃ of dry 4-6h down, place-70 ℃ of Ultralow Temperature Freezers to preserve again with sealing film phonograph seal then.
2. in accordance with the method for claim 1, described Ultralow Temperature Freezer time of preserving is 1,3,6 or more than December.
3. in accordance with the method for claim 1, described preservation agent is formulated by the benzoglyoxaline that per hundred ml distilled waters add 0.4-0.6g agar+2-4mL0.1-0.3%.
4. in accordance with the method for claim 1, described method also comprises the resurrection step of bacterial strain, and described resurrection step makes its slow intensification for the described centrifuge tube of step (4) is put into 4 ℃ of ice chests, is inoculated in the fresh leaf section according to the described method of step (1) then.
5. in accordance with the method for claim 4, the resurrection rate of described bacterial strain is more than 90%, to be preferably 100%.
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| CN103805521A (en) * | 2014-02-17 | 2014-05-21 | 四川农业大学 | Fast method for obtaining genetically homogenized wheat powdery mildew |
| CN105861291A (en) * | 2016-04-15 | 2016-08-17 | 湖北省农业科学院植保土肥研究所 | Simple and convenient inoculating device for blumeria graminis f.sp.tritici and application method of inoculating device |
| 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 |
| CN107460231A (en) * | 2017-08-21 | 2017-12-12 | 湖北省农业科学院植保土肥研究所 | A kind of wheat powdery mildew spray inoculation method |
| CN108641962A (en) * | 2018-03-23 | 2018-10-12 | 湖北省农业科学院植保土肥研究所 | A method of improving the wheat powdery mildew Plantlet in vitro time |
| CN108977363A (en) * | 2018-07-19 | 2018-12-11 | 河南省农业科学院植物保护研究所 | Test wheat powdery mildew culture preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103805521A (en) * | 2014-02-17 | 2014-05-21 | 四川农业大学 | Fast method for obtaining genetically homogenized wheat powdery mildew |
| CN103805521B (en) * | 2014-02-17 | 2017-02-01 | 四川农业大学 | Fast method for obtaining genetically homogenized wheat powdery mildew |
| CN105861291A (en) * | 2016-04-15 | 2016-08-17 | 湖北省农业科学院植保土肥研究所 | Simple and convenient inoculating device for blumeria graminis f.sp.tritici and application method of inoculating device |
| CN105861291B (en) * | 2016-04-15 | 2017-12-08 | 湖北省农业科学院植保土肥研究所 | The easy classification inoculation apparatus and its application method of wheat powdery mildew |
| 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 |
| CN107460231A (en) * | 2017-08-21 | 2017-12-12 | 湖北省农业科学院植保土肥研究所 | A kind of wheat powdery mildew spray inoculation method |
| 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 |
| CN107460231B (en) * | 2017-08-21 | 2020-07-10 | 湖北省农业科学院植保土肥研究所 | Wheat powdery mildew spray inoculation method |
| CN108641962A (en) * | 2018-03-23 | 2018-10-12 | 湖北省农业科学院植保土肥研究所 | A method of improving the wheat powdery mildew Plantlet in vitro time |
| CN108977363A (en) * | 2018-07-19 | 2018-12-11 | 河南省农业科学院植物保护研究所 | Test wheat powdery mildew culture preparation method |
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