CN101643772B - Method for quickly evaluating disease resistance of banana fusarium wilt - Google Patents
Method for quickly evaluating disease resistance of banana fusarium wilt Download PDFInfo
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- CN101643772B CN101643772B CN2009101921760A CN200910192176A CN101643772B CN 101643772 B CN101643772 B CN 101643772B CN 2009101921760 A CN2009101921760 A CN 2009101921760A CN 200910192176 A CN200910192176 A CN 200910192176A CN 101643772 B CN101643772 B CN 101643772B
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Abstract
The invention relates to a method of quickly evaluating disease resistance of banana fusarium wilt, belonging to the technical field of plant protection of agriculture. The invention has the following steps: transferring test-tube plantlets which grow for 2-3 weeks in a root culture medium to an improved culture medium MIS to pre-culture for 1-2 weeks; selecting plantlets with the height of 4.5-5cm, the number of blades unfolded on the upper part of plant being more than or equal to 2 and the number of roots being more than or equal to 3 to inoculate Fusarium oxysporum f. sp. cubense (Foc) ona super-clean worktable in the phase of root culture of fast in vitro propagation of banana; placing a culture vessel in a tissue culture room with the temperature of 25 plus or minus 2 DEG C to observe the condition of pathogenesis; evaluating disease grades of singleplant rooted test-tube plantlet according to the disease evaluation grade from grade 0 to grade 6 24 days after inoculating FOC. Compared with the present widely adopted pot culture systems and water culture systems, the evaluation method provided in the invention is not only able to be closely linked with the research of breeding for disease resistance of banana, but also has the characteristics of accuracy and fast speed.
Description
Technical field
The present invention relates to the disease resistance of banana fusarium wilt (Fusarium wilt) is carried out the method for Rapid identification; Especially under the condition of isolated culture; Through the improved culture medium prescription and root culture stage inoculation pathogenic bacteria; Screen disease-resistant germ plasm resource of banana and breeding material fast and effectively, improve the efficient of banana fusarium wilt resistance breeding.The invention belongs to agricultural-plant protection technology field.
Background technology
Banana fusarium wilt (claiming Panama disease, yellow leat disease again) is one of main disease that threatens in recent years China and even whole world banana production.This disease generally is considered to one of the most serious since the dawn of human civilization crushing Plant diseases, and it is caused by soil fungi Fusarium oxysporum f.sp.cubense (E.F.Smith) Snyd.&Hans (Foc).Confirmed at present 4 physiological strains of Foc, wherein No. 4 physiological strains (race 4) is crushing maximum.
More existing prophylactico-therapeutic measuress, for example chemical prevention, waterflooding are lain fallow, crop rotation and use organism are improved the soil etc. all can not control fusarium wilt effectively.In recent years, domestic and international research about the banana blight biological control mainly concentrates on antimicrobial screening short of money and indoor pot control test (Perez-vicente etc., 2003; Saravanan etc., 2003; Thangavelu etc., 2003; Cheng Liang etc., 2005; The equality of going on a spring outing, 2005; Yellow little light etc., 2006), produce the also few of reality but really can be applied to.Generally believe that at present breeding resistant variety is one of should disease effective means of control.And set up simple and effective resistance identification system is the key of carrying out the banana blight breeding for disease resistance.
Because the banana fusarium wilt is a kind of soil-borne disease, traditional field identification method requires to have big area morbidity plot comparatively uniformly, not only time-consuming taking a lot of work, and qualification result also receives the influence of envrionment conditions easily.The artificial inoculation on seedling identification method that generally adopts at present mainly is divided into 2 types: potted plant system (Matsumoto et al., 1995; Woods time lag etc., 2000; Li Minhui etc., 2007; Weber et al., 2007; Low-priced friend etc. not, 2008; Smith et al., 2008) and hydroponic system (De Ascensao and Dubery, 2000; Groenewald et al., 2006; Van den Berg et al., 2007).Inoculation method usually adopts hinders root and soaks wheat that (pourings) bacterium liquid, soil or nutrient solution inoculation bacterium liquid, usefulness inoculates Foc in advance or sorghum grain as inoculum etc.So far do not appear in the newspapers as yet big improved other resistance authentication methods are arranged.
Summary of the invention
The objective of the invention is to:, a kind of new banana fusarium wilt resistance identification system is provided to the characteristics of banana breeding for disease resistance and the deficiency of prior art.
The present invention realizes through following technical scheme:
Exsomatize the root culture stage of breeding fast at banana; With being transferred to improved culture medium (modified medium for interaction system at all test-tube plantlets of root culture basal growth 2-3; MIS) cultivate in advance, each culture vessel is inoculated 1 strain test-tube plantlet.
The composition of MIS comprises NH
4NO
3825mg/l, KNO
3950mg/l, MgSO
47H
2O 185mg/l, KH
2PO
485mg/l, CaCl
22H
2O 220mg/l, KI 0.83mg/l, H
3BO
36.2mg/l, MnSO
44H
2O 22.3mg/l, ZnSO
47H
2O8.60mg/l, Na
2MoO
42H
2O 0.25mg/l, CuSO
45H
2O 0.025mg/l, CoCl
26H
2O 0.025mg/l, NaEDTA37.26mg/l, FeSO
47H
2O 27.80mg/l and 6g/l agar.
The banana rooting tube plantlet after week, is chosen plant height 4.5-5cm at MIS substratum growth 1-2, launch the number of blade >=2, take root and count >=3 plantlet inoculation Foc in plant top.
During inoculation, on Bechtop, open the lid of culture vessel, will be immersed in Foc spore suspension (10 in advance with tweezers
6Aseptic filter paper sheet (diameter 5mm) in the individual spore/ml) is placed on the MIS media surface apart from the about 1cm of plantlet place.The last lid of building culture vessel again, and to place it in temperature be to observe incidence in 25 ± 2 ℃ the group training chamber.
24d behind the inoculation Foc carries out the disease grade identification according to the disease opinion rating of 0-6 level to the individual plant rooting tube plantlet.Wherein the growth of 0=plantlet is normal, does not have any disease symptom; The vanelets of the false basal part of stem of 1=is withered, but the color of false stem itself does not change; The zone of the false stem colour-darkening of 2=is less than or equal to 1/2 of whole false stem height; The zone of the false stem colour-darkening of 3=surpasses 1/2 of whole false stem height; 4=flavescence or withered upper blade number are less than or equal to 1/2 of plantlet top total leaf number; 5=flavescence or withered upper blade number surpass 1/2 of plantlet top total leaf number; 6=puts in order strain test-tube plantlet withered death.
The present invention has the following advantages:
(1) the existing banana Cultivar overwhelming majority is 3 times of bodies, no matter all can not get seed from flower or cross-pollination between 3 times of bodies; 3 times of bodies (female parent) also are only limited to some kind with 2 times of bodies (male parent) hybridization, and have only small quantities of seed.Therefore, the priority research areas of banana blight breeding for disease resistance is to exsomatize to select (Matsumoto etc., 1995,1999 at present; Bhagwat and Duncan, 1998a, 1998b; Smith etc., 1993,2006; Bang is auspicious etc., and 2007; Hu Yulin etc., 2008), genetic transformation (Remy etc., 1998; Pei Xinwu etc., 2005a is 2005b) with protoplastis fusion etc. (Matsumoto etc., 2002).Above-mentioned research all is that cell, tissue culture with banana is as the basis; And most of potted plant system and the required vegetable material of hydroponic system are the seedlings of heeling in about plant height 15cm; Given this, authentication method provided by the invention can more closely be connected with present banana breeding for disease resistance research.
(2) no matter potted plant system or hydroponic system, disease resistance is identified and generally in warmhouse booth, is carried out; And identification system of the present invention is in group training chamber, to observe incidence.Existing observation and research show that the activity of pathogenic bacteria Foc receives Effect of Environmental such as temperature (Liu Shaoqin etc., 2001).Because the envrionment conditions of culturing room is much stable than warmhouse booth, identification system itself of the present invention in addition is the system of a complete closed, makes the repeatability of qualification result and safety be guaranteed.
(3) potted plant system and hydroponic system respectively will be behind inoculation Foc 7-8 week with just can carry out disease 6 weeks and identify, and authentication method provided by the invention only needs 3-4 all.
Embodiment
With 6 kinds such as Brazil, GCTCV-119, Formosana, No. 1, agricultural section, No. 1, wide powder and big Jiao in Dongguan is experiment material.Above-mentioned kind is seeded to root media by the indefinite bud of inhaling the generation of bud stem apex explant: 1/2MS+1.0mg/l NAA+0.2mg/l IBA+30g/l sucrose+2g/l gac+6g/l agar.Root culture 2-3 is after week, blade is begun the unfolded rooting tube plantlet is transferred to MIS and cultivates in advance, with the 150ml triangular flask as culture vessel, every bottle graft kind 1 strain test-tube plantlet.The rooting tube plantlet of above-mentioned kind after week, is chosen plant height 4.5-5cm at MIS substratum growth 1-2, launch the number of blade >=2, take root and count >=3 plantlet inoculation Foc in plant top.During inoculation, on Bechtop, open the film that seals of triangular flask, will be immersed in the spore suspension (10 of Foc race 4 with tweezers in advance
6Aseptic filter paper sheet (diameter 5mm) in the individual spore/ml) is placed on the MIS media surface apart from the about 1cm of plantlet place.Again cover at last and seal film, and to place it in temperature be to observe incidence in 25 ± 2 ℃ the group training chamber.5 processing of each kind, each handles repetition 3 times.24d behind the inoculation Foc carries out the disease grade identification according to the disease opinion rating of 0-6 level to the individual plant rooting tube plantlet.Wherein the growth of 0 grade-plantlet is normal, does not have any disease symptom; The vanelets of 1 grade-false basal part of stem is withered, but the color of false stem itself does not change; The zone of 2 grades-false stem colour-darkening is less than or equal to 1/2 of whole false stem height; The zone of 3 grades-false stem colour-darkening surpasses 1/2 of whole false stem height; 4 grades-flavescence or withered upper blade number are less than or equal to 1/2 of plantlet top total leaf number; 5 grades-flavescence or withered upper blade number surpass 1/2 of plantlet top total leaf number; 6 grades-whole strain test-tube plantlet withered death.The qualification result following table:
Annotate: in the table behind the same column numerical value different letter representation differences reach level of signification P≤0.05.
Can know by last table, supply the average disease grade of 6 kinds of examination consistent, prove and use susceptible variety and the disease-resistant variety that authentication method provided by the invention can be distinguished the banana fusarium wilt quickly and accurately with the result of field disease evaluation.
Claims (1)
1. one kind is carried out the method for Rapid identification to the disease resistance of banana fusarium wilt, it is characterized in that:
1) exsomatize the root culture stage of breeding fast at banana, cultivate in advance being transferred to improved culture medium MIS at all test-tube plantlets of root culture basal growth 2-3, each culture vessel is inoculated 1 strain test-tube plantlet;
2) composition of MIS comprises NH
4NO
3825mg/l, KNO
3950mg/l, MgSO
47H
2O 185mg/l, KH
2PO
485mg/l, CaCl
22H
2O 220mg/l, KI 0.83mg/l, H
3BO
36.2mg/l, MnSO
44H
2O 22.3mg/l, ZnSO
47H
2O8.60mg/l, Na
2MoO
42H
2O 0.25mg/l, CuSO
45H
2O 0.025mg/l, CoCl
26H
2O 0.025mg/l, NaEDTA37.26mg/l, FeSO
47H
2O 27.80mg/l and 6g/l agar;
3) the banana rooting tube plantlet is cultivated 1-2 after week in advance at MIS, chooses the plantlet inoculation pathogenic bacteria Foc of plant height 4.5-5cm, the expansion number of blade >=2, plant top, the number >=3 of taking root;
4) when inoculation, on Bechtop, earlier the aseptic filter paper sheet of diameter 5mm being immersed in concentration is 10
6In the Foc spore suspension of individual spore/ml; Open the lid of culture vessel again; The filter paper that will soak with tweezers is placed on the MIS media surface apart from plantlet 1cm place; The last lid of building culture vessel again, and to place it in temperature be to observe incidence in 25 ± 2 ℃ the group training chamber;
5) 24d behind the inoculation Foc carries out the disease grade identification according to the disease opinion rating of 0-6 level to the individual plant rooting tube plantlet, and wherein the growth of 0 grade-plantlet is normal, does not have any disease symptom; The vanelets of 1 grade-false basal part of stem is withered, but the color of false stem itself does not change; The zone of 2 grades-false stem colour-darkening is less than or equal to 1/2 of whole false stem height; The zone of 3 grades-false stem colour-darkening surpasses 1/2 of whole false stem height; 4 grades-flavescence or withered upper blade number are less than or equal to 1/2 of plantlet top total leaf number; 5 grades-flavescence or withered upper blade number surpass 1/2 of plantlet top total leaf number; 6 grades-whole strain test-tube plantlet withered death.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101974650B (en) * | 2010-11-30 | 2012-06-27 | 中国农业科学院植物保护研究所 | Polymerase chain reaction (PCR) method for detecting fusarium oxysporum and kit |
| CN102783478B (en) * | 2012-08-30 | 2013-11-06 | 四川三甲农业科技股份有限公司 | Fruit preservative solution for red heart kiwi fruit and application method thereof |
| US9586871B2 (en) * | 2014-05-23 | 2017-03-07 | Robert Sabin | Potentiation of fixed coppers and other pesticides containing copper and supplementing plant nutrition |
| CN104160846B (en) * | 2014-07-15 | 2016-02-17 | 武汉市蔬菜科学研究所 | A kind of sprout period testifying method of cowpea anti-blight |
| CN106031352B (en) * | 2015-03-11 | 2018-12-04 | 李保华 | A set of evaluation and test fungicide is to the method for infecting disease-controlling effect from apple floral organ |
| CN105950699A (en) * | 2016-07-06 | 2016-09-21 | 广东海洋大学 | Method for identifying pathogenicity of pathogenic bacteria of banana wilt |
| 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 |
| CN107646661B (en) * | 2017-10-31 | 2020-06-09 | 广东省农业科学院果树研究所 | Banana root system in-vitro hydroponic system and culture method |
| CN110073957A (en) * | 2019-04-18 | 2019-08-02 | 广东省农业科学院果树研究所 | A kind of system and method carrying out Rapid identification using disease resistance of the cultivation technique without soil to research of fusarium wilt disesase of banana |
| CN110804645B (en) * | 2019-11-07 | 2022-06-28 | 广东省农业科学院果树研究所 | Method for evaluating disease resistance of banana fusarium wilt |
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| CN1768577A (en) * | 2004-11-02 | 2006-05-10 | 中国农业科学院生物技术研究所 | Method for breeding anti-blight banana |
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| CN1768577A (en) * | 2004-11-02 | 2006-05-10 | 中国农业科学院生物技术研究所 | Method for breeding anti-blight banana |
| CN101423870A (en) * | 2008-09-05 | 2009-05-06 | 中国热带农业科学院南亚热带作物研究所 | Method for rapid screening banana fusarium wilt resistance |
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