CN103555813A - Method for rapid identification of sweet potato stem rot resistance - Google Patents
Method for rapid identification of sweet potato stem rot resistance Download PDFInfo
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Abstract
The invention relates to a method for rapid identification of sweet potato stem rot resistance. The method comprises the operation steps of: trimming a sweet potato stem or side branch into a stem branch with 3-4 extended leaves, and conducting pre-culture for 2-3 days under room temperature; when fibrous roots grow out of the stem branch, placing the stem branch horizontally, stabbing a small hole by a pipettor tip between two sections in the stem branch center without penetrating the stem branch, then placing the stem branch horizontally in a sealed plastic box paved with filter paper steeped by sterile water, and making the stem branch side stabbed with the small hole upward, absorbing 15-25 microliters of a 1*10<8>cfu/mL stem rot pathogenic bacterial suspension, and injecting the suspension into the small hole for inoculated culture; and after 20-24h of culture, observing the disease condition and conducting disease grade identification on the sweet potato stem branch. The method provided by the invention has the advantages of simple and fast operation, strong practicability, strong operability, and high identification result repeatability and reliability, and has potential social and economic significance for screening excellent germplasm resources resisting sweet potato stem rot.
Description
Technical field
The present invention relates to the disease resistance of Sweet Potato maize ear rot to carry out the method for Rapid identification, especially the screening method to Sweet Potato Against stem rot germ plasm resource and breeding material, belongs to plant protection technology field.
Background technology
Sweet potato (Ipomoea batatas(L.) Lam) be one of world food, the energy and insutrial crop, China is that maximum in the world sweet potato produces country, and its cultivated area is only second to paddy rice, wheat and maize and occupies the 4th.In recent years along with the sweet potato market requirement is vigorous, peasant planting and the enthusiasm introducing new breeds increase, seedling or potato seed are flowed frequent between countries and regions, and some new diseases or traditional disease eruption and prevalence again, has thereupon appearred in unordered plantation in addition in China sweet potato main producing region.
First Sweet Potato maize ear rot is found at Georgia State, USA, and other countries are not reported in the world.First 2006 sweet potato producing region, eastern region, Nian China Guangdong Province find the new disease of this disease ，Wei China sweet potato.This disease spread is fast, endangers a plurality of cities and counties outburst such as Guangzhou, Huizhou, riverhead, Zhanjiang, Zengcheng, Haifeng county, Boluo, Huidong in serious ，Yi Guangdong Province, and the state of an illness increases the weight of year by year, may become the sweet potato disease that Guangdong Province's onset area is maximum, harm is the heaviest.The potato seedling of the field ， Cheng Kui that falls ill serious is dead, and the potato seedling in even full field is dead, and No kernels or seeds are gathered, as in a year of scarcity, has a strong impact on the production of sweet potato and sweet potato grower's interests.The pathogenic bacteria of Sweet Potato maize ear rot belongs to one of D.dadantii ，Shi China three class quarantine harmful organisms according to new categorizing system, and this germ be take potato seed or seedling, and to pass band be main mode of transmission.There is no in the world at present the method for effectively preventing and treating for Sweet Potato maize ear rot.
As everyone knows, the use of disease-resistant variety is the method for preventing and treating of economical and effective environmental protection the most, is also one of essential measure of controlling disease.In the Breeding Process of disease-resistant variety, need to carry out a large amount of Disease Resistance Identification tests, and the disease Resistance Identification evaluation method of Erecting and improving is the important prerequisite of carrying out this research.Therefore, a kind of simple to operate, fast and accurately inoculation identification method be research in indispensable.At present, the inoculation method of plant pathogenetic bacteria is more, as leaf-cutting method, soak (dipping in) root method, needle punching, spray method, rubbing manipulation, injection, perfusion soil method etc.These methods, because Micobial Disease is different with host, cause action effect also different.
First the research of Sweet Potato maize ear rot reported in the U.S..Schaad(1977) etc. utilize Koch's Postulates to adopt injection inoculation method to verify pathogenic bacteria, confirmed that pathogenic bacteria is Erwinia chrysanthemi (Schaad N W, and Brenner D.A bacterial wilt and root rot of sweet potato caused by Erwinia chrysanthemi.Phytopathology, 1977,67:302-308).Clark etc. (1989) utilize Erwinia chrysanthemi suspension to be applied to potato piece slice surface and inoculate, and result shows sweet potato root tuber inoculation pathogenic bacteria 6 days or after the longer time, between kind, sweet potato root tuber Resistant Difference is remarkable; This inoculation method reacts non-correlation (Clark with the resistance of stab inoculation Sweet Potato branch in addition, C A., Wilder-Ayers, J A., and Duarte, V.Resistance of Sweet potato to Bacterial Root and Stem Rot Caused by Erwinia chrysanthemi.Plant Disease, 1989,73:984-987).In China, Sweet Potato maize ear rot belongs to new disease, less to the research of this disease, and the evaluation of Sweet Potato maize ear rot resistance method is not had especially.Contriver attempts by American scholar at the pathogenic evaluation of Sweet Potato maize ear rot needle punching used and potato piece section bacterination process and has reported several conventional Micobial Disease inoculation methods such as leaf-cutting method, spray method, the disease symptom of finding the pathogenic sweet potato that these inoculation methods obtain is unstable, connect bacterium amount and be difficult to guarantee unanimously, be not suitable for the large-scale inoculation Resistance Identification of Sweet Potato maize ear rot.Therefore, set up a kind of screening simple to operate, that morbidity is rapid, the inoculation identification method of effect stability resists stem rot Sweet Potato Germplasm and breeding material and seem particularly important.
Summary of the invention
For the feature of Sweet Potato maize ear rot breeding for disease resistance and the deficiency of existing Sweet Potato maize ear rot resistance inoculated identification technology, the object of the present invention is to provide a kind of fast, accurately, can be applicable to the Sweet Potato maize ear rot Resistance Identification method that large sample amount is identified.
For addressing the above problem, the technical solution adopted in the present invention is as follows:
A method for Rapid identification Sweet Potato maize ear rot resistance, comprises the following steps:
(1) processing of stem branch and preculture: get growth normal, neat, without sweet potato stem or the side shoot of disease and pest, with aseptic operation cutter, sweet potato stem or side shoot are trimmed to the stem branch with 3～4 exhibition leaves, cut the rear juice that washes away clip outflow with sterilized water; The stem branch of pruning cleaning is put into sterilizing wide-necked bottle and carry out preculture, sterilizing wide-necked bottle is equipped with distilled water or the sterilized water that accounts for its volume 1/3, cultivates 2～3 days under room temperature condition;
(2) preparation of stem rot pathogenic bacteria bacteria suspension: Sweet Potato maize ear rot pathogenic bacteria bacterial strain H12 is inoculated in improvement nutrient agar, and after 28 ℃ of streak culture 40～48h, being made into concentration with sterilized water is 1 * 10
8the stem rot pathogenic bacteria bacteria suspension of cfu/mL;
(3) inoculation culture of stem branch: grow after fibrous root until stem branch, by stem branch horizontal, between stem branch mid-way and two joints, with the rifle head of pipettor, prick 1 duck eye and do not penetrate stem branch, then stem branch is flat in the airtight plastics casing that is covered with the filter paper that soaks into sterilized water, and the side that bundle is had to a duck eye upward, draw the bacteria suspension that 15～25 μ L steps (2) make, inject in stem branch duck eye and inoculate, using sterilized water as blank, after inoculation, plastics casing is built to sealing, be placed in the illumination box of 30 ℃, cultivate 20～24h;
(4) disease observation of symptoms and grade identification: cultivate after 20～24h, observe disease situation, and Sweet Potato branch is carried out to disease grade identification.
Sweet Potato maize ear rot pathogenic bacteria bacterial strain H12 of the present invention derive from the sick sample of Guangdong Province Sweet Potato maize ear rot (Huang Lifei, Luo Zhongxia, the First Report of Studies of the new disease stem rot of Fang Baiping ，Deng. China sweet potato. Plant Pathology .2011,41 (1): 18-23.).
Preferably, stem branch described in step (1) is disconnected from the incision of sweet potato leaf segment, and the length of described stem branch is 12～15cm.
Preferably, in described step (2), draw the bacteria suspension that 20 μ L steps (2) make, inject in stem branch duck eye and inoculate.
Preferably, stem scion grafting kind is placed in the illumination box of 30 ℃ in described step (2), cultivates 24h.
Preferably, the composition of described improvement nutrient agar comprises peptone 5g/L, sucrose 10g/L, and extractum carnis 3g/L, yeast extract 1g/L and agar 15g/L, the pH value of described improvement nutrient agar is 7.0～7.2.
As further technical scheme of the present invention, step is divided into 1～5 grade by the occurring degree of sweet potato in (4), and wherein 1-is asymptomatic, health; 2-duck eye occurs that black rots, but rots to be less than 1cm; 3-rots to be more than or equal to 1cm and is less than 2cm, and rots not by any one leaf segment; The rotten length of 4-is more than or equal to 2cm and is less than 3cm or rots by any one leaf segment; The rotten length of 5-is greater than 3cm or rots by 2 leaf segments; The occurring degree of sweet potato of usining is identified the disease resistance of sweet potato variety as disease judgement criteria, occurring degree=5 be susceptible variety highly, and 3≤occurring degree ﹤ 5 is susceptible variety; 1≤occurring degree ﹤ 3 is disease-resistant variety.
Compared to existing technology, beneficial effect of the present invention is:
(1) practicality, workable: the present invention can be directly used in the screening of the test of Resistance Identification in Sweet Potato maize ear rot breeding for disease resistance and Sweet Potato Against stem rot germ plasm resource;
(2) simple to operate fast: pathogenic bacteria inoculum density homogeneous, inoculum size is consistent, measures comparatively simply, is applicable to carrying out the test of large sample amount; The aobvious disease time of the rear sweet potato of inoculation is short and qualification time is short, and inoculation pathogenetic bacteria can be seen significant disease symptom after 20～24h, just can complete whole inoculated identification process in 3～5 days, has greatly alleviated the working strength of disease Resistance Identification;
(3) qualification result has good repeatability and reliability.By embodiment, confirm, the qualification result between different evaluations batch different repetition is stable, and error is less.
In sum, the present invention, as the novel method of Sweet Potato maize ear rot Resistance Identification, under the controlled condition of environment, inoculates quantitative pathogenic bacteria by Sweet Potato branch by Zha Dong, can fast and effeciently screen Some Disease-resistant Varieties in Sweet Potato resource and breeding material, improve the efficiency of Sweet Potato maize ear rot breeding; Can, for the tests such as pathogenic detection of evaluation, the breeding of Sweet Potato Against stem rot and the separated germ of the anti-stem rot of Sweet Potato Germplasm, for the inoculation method that enriches sweet potato pathogenetic bacteria, there is positive effect; Meanwhile, for screening Sweet Potato Against stem rot high-quality germ plasm resource, there is potential social and economic significance.
Embodiment
Experiment material: 98 parts of Sweet Potato Germplasms that preserve in " sweet potato resource garden, national Guangzhou ".
Experimental technique: 98 parts of Sweet Potato Germplasms (specifically in Table 1) that " sweet potato resource garden, national Guangzhou " preserved have carried out Sweet Potato maize ear rot Resistance Identification, according to the occurring degree of sweet potato, these Sweet Potato Germplasms are divided into height susceptible variety, susceptible variety, disease-resistant variety.
Experimental procedure: processing and the preculture of (1) stem branch: from sweet potato leaf segment, get growth normal, neat, without sweet potato stem or the side shoot of disease and pest, with aseptic operation cutter, sweet potato stem or side shoot are trimmed to the stem branch with 3～4 exhibition leaves, the length of stem branch is 12～15cm, cuts the rear juice that washes away clip outflow with clear water; The stem branch of pruning cleaning is put into sterilizing wide-necked bottle and carry out preculture, sterilizing wide-necked bottle is equipped with distilled water or the sterilized water that accounts for its volume 1/3, cultivates 2～3 days under room temperature condition;
(2) preparation of stem rot pathogenic bacteria bacteria suspension: Sweet Potato maize ear rot pathogenic bacteria bacterial strain H12 is inoculated in improvement nutrient agar, the composition of improvement nutrient agar comprises peptone 5g/L, extractum carnis 3g/L and agar 15g/L, pH value is 7.0～7.2, after 28 ℃ of streak culture 48h, with sterilized water, being made into concentration is 1 * 10
8the stem rot pathogenic bacteria bacteria suspension of cfu/mL;
(3) inoculation culture of stem branch: grow after fibrous root until stem branch, by stem branch horizontal, between stem branch mid-way and two joints, with pipettor gun head, prick 1 duck eye and can not penetrate stem branch, then stem branch is flat in the airtight plastics casing that is covered with the filter paper that soaks into sterilized water, and the side that bundle is had to a duck eye upward, draw the bacteria suspension that 20 μ L steps (2) make, inject in stem branch duck eye and inoculate, using sterilized water as blank, after inoculation, plastics casing is built to sealing, be placed in the illumination box of 30 ℃, cultivate 24h;
(4) disease observation of symptoms and grade identification: cultivate after 24h, observe disease situation, and Sweet Potato branch is carried out to disease grade identification.The occurring degree of sweet potato is divided into 1～5 grade, wherein 1-asymptomatic, health; 2-duck eye occurs that black rots, but rots to be less than 1cm; 3-rot to be more than or equal to 1cm to be less than 2cm, and rot not by any one leaf segment; 4-the length of rotting is more than or equal to 2cm and is less than 3cm or rots by any one leaf segment; 5-the length of rotting is greater than 3cm or rots by 2 leaf segments; The occurring degree of sweet potato of usining is identified the disease resistance of sweet potato variety as disease judgement criteria, occurring degree=5 be susceptible variety highly, and 3≤occurring degree ﹤ 5 is susceptible variety; 1≤occurring degree ﹤ 3 is disease-resistant variety.
Experimental result and analysis: the indoor inoculation that the present embodiment has been done Sweet Potato maize ear rot to 98 parts of Sweet Potato Germplasms is identified, concrete Resistance Identification the results are shown in Table 1, wherein 63 parts of Sweet Potato Germplasms are accredited as high sense, and 19 parts of Sweet Potato Germplasms are accredited as middle sense, and 16 parts of sweet potato resources are disease-resistant.In order to guarantee the accuracy of result of study, be necessary that the further field test of resistance resource to screening is observed, by field test, found that, the result that method of the present invention is identified and field test result approach, and both errors are very little.
The concrete disease-resistant type qualification result of table 1 resource
Note: state garden number is the sweet potato resource country numbering that national Germplasm Resources is preserved.
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, the variation of any unsubstantiality that those skilled in the art does on basis of the present invention and replacement all belong to the present invention's scope required for protection.
Claims (6)
1. a method for Rapid identification Sweet Potato maize ear rot resistance, is characterized in that comprising the following steps:
(1) processing of stem branch and preculture: get growth normal, neat, without sweet potato stem or the side shoot of disease and pest, with aseptic operation cutter, sweet potato stem or side shoot are trimmed to the stem branch with 3～4 exhibition leaves, cut the rear juice that washes away clip outflow with sterilized water; The stem branch of pruning cleaning is put into sterilizing wide-necked bottle and carry out preculture, sterilizing wide-necked bottle is equipped with distilled water or the sterilized water that accounts for its volume 1/3, cultivates 2～3 days under room temperature condition;
(2) preparation of stem rot pathogenic bacteria bacteria suspension: Sweet Potato maize ear rot pathogenic bacteria bacterial strain H12 is inoculated in improvement nutrient agar, and after 28 ℃ of streak culture 40～48 h, being made into concentration with sterilized water is 1 * 10
8the stem rot pathogenic bacteria bacteria suspension of cfu/mL;
(3) inoculation culture of stem branch: grow after fibrous root until stem branch, by stem branch horizontal, between stem branch mid-way and two joints, with the rifle head of pipettor, prick 1 duck eye and do not penetrate stem branch, then stem branch is flat in the airtight plastics casing that is covered with the filter paper that soaks into sterilized water, and the side that bundle is had to a duck eye upward, draw the bacteria suspension that 15～25 μ L steps (2) make, inject in stem branch duck eye and inoculate, using sterilized water as blank, after inoculation, plastics casing is built to sealing, be placed in the illumination box of 30 ℃, cultivate 20～24 h;
(4) disease observation of symptoms and grade identification: cultivate after 20～24 h, observe disease situation, and Sweet Potato branch is carried out to disease grade identification.
2. the method for Rapid identification Sweet Potato maize ear rot resistance according to claim 1, is characterized in that: stem branch described in step (1) is disconnected from the incision of sweet potato leaf segment, and the length of described stem branch is 12～15 cm.
3. the method for Rapid identification Sweet Potato maize ear rot resistance according to claim 1, is characterized in that: in described step (2), draw the bacteria suspension that 20 μ L steps (2) make, inject in stem branch duck eye and inoculate.
4. the method for Rapid identification Sweet Potato maize ear rot resistance according to claim 1, is characterized in that: in described step (2), stem scion grafting kind is placed in the illumination box of 30 ℃, cultivates 24 h.
5. the method for Rapid identification Sweet Potato maize ear rot resistance according to claim 1, it is characterized in that: the composition of described improvement nutrient agar comprises peptone 5 g/L, sucrose 10 g/L, extractum carnis 3 g/L, yeast extract 1g/L and agar 15 g/L, the pH value of described improvement nutrient agar is 7.0～7.2.
6. the method for Rapid identification Sweet Potato maize ear rot resistance according to claim 1, is characterized in that: in step (4), the occurring degree of sweet potato is divided into 1～5 grade, wherein 1-and asymptomatic, health; 2-duck eye occurs that black rots, but rots to be less than 1 cm; 3-rot to be more than or equal to 1 cm to be less than 2 cm, and rot not by any one leaf segment; 4-the length of rotting is more than or equal to 2 cm and is less than 3 cm or rots by any one leaf segment; 5-the length of rotting is greater than 3 cm or rots by 2 leaf segments; The occurring degree of sweet potato of usining is identified the disease resistance of sweet potato variety as disease judgement criteria, occurring degree=5 be susceptible variety highly, and 3≤occurring degree ﹤ 5 is susceptible variety; 1≤occurring degree ﹤ 3 is disease-resistant variety.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962605A (en) * | 2015-07-01 | 2015-10-07 | 中国农业科学院蔬菜花卉研究所 | Screening method of medicinal agents for preventing bacterial soft rot of vegetables |
| CN104996182A (en) * | 2015-07-10 | 2015-10-28 | 江苏徐州甘薯研究中心 | Resistance identification method for sweet potato scurf |
| CN106635830A (en) * | 2016-11-22 | 2017-05-10 | 江苏省农业科学院 | Culture method of alternaria brassicicola used for identifying resistance to black spot of sweet potatoes |
| CN109136327A (en) * | 2018-10-08 | 2019-01-04 | 华中农业大学 | A method of identification citrus plant Resistance To Root Rot Disease |
| CN111057741A (en) * | 2019-12-31 | 2020-04-24 | 安徽农业大学 | Method for identifying resistance of kiwi fruits to canker |
| CN112522359A (en) * | 2020-12-09 | 2021-03-19 | 广东省农业科学院作物研究所 | Resistance identification method for sweet potato scab for stem tip vegetables |
| CN112931184A (en) * | 2021-03-23 | 2021-06-11 | 中国科学院华南植物园 | Identification method of weevil-resistant sweet potato variety |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962605A (en) * | 2015-07-01 | 2015-10-07 | 中国农业科学院蔬菜花卉研究所 | Screening method of medicinal agents for preventing bacterial soft rot of vegetables |
| CN104996182A (en) * | 2015-07-10 | 2015-10-28 | 江苏徐州甘薯研究中心 | Resistance identification method for sweet potato scurf |
| CN104996182B (en) * | 2015-07-10 | 2017-07-14 | 江苏徐州甘薯研究中心 | Sweet potato scurf Resistance Identification method |
| CN106635830A (en) * | 2016-11-22 | 2017-05-10 | 江苏省农业科学院 | Culture method of alternaria brassicicola used for identifying resistance to black spot of sweet potatoes |
| CN109136327A (en) * | 2018-10-08 | 2019-01-04 | 华中农业大学 | A method of identification citrus plant Resistance To Root Rot Disease |
| CN111057741A (en) * | 2019-12-31 | 2020-04-24 | 安徽农业大学 | Method for identifying resistance of kiwi fruits to canker |
| CN112522359A (en) * | 2020-12-09 | 2021-03-19 | 广东省农业科学院作物研究所 | Resistance identification method for sweet potato scab for stem tip vegetables |
| CN112931184A (en) * | 2021-03-23 | 2021-06-11 | 中国科学院华南植物园 | Identification method of weevil-resistant sweet potato variety |
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Application publication date: 20140205 |