CN1790018A - Tomato bacterial wilt resistance identification method - Google Patents
Tomato bacterial wilt resistance identification method Download PDFInfo
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- CN1790018A CN1790018A CN 200510062017 CN200510062017A CN1790018A CN 1790018 A CN1790018 A CN 1790018A CN 200510062017 CN200510062017 CN 200510062017 CN 200510062017 A CN200510062017 A CN 200510062017A CN 1790018 A CN1790018 A CN 1790018A
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Abstract
The invention discloses a tomato green-dry disease resistance identification method in the vegetable disease resistance identification technique domain, which comprises the following stages: tomato seeding period identification, size and distribution detection of false monospore bacillus flora in the plant and plant disease resistance identification, wherein the size and distribution detection of false monospore bacillus flora integrates the bacteria infectious group size of tomato green-dry disease and extension inhibition ability of false monospore bacillus flora organically. The invention proceeds synthetic assessment and divides the resistance level, which improves the accurate level of tomato green-dry disease.
Description
Technical field
The invention belongs to vegetable disease-resistant authenticate technology field, especially belong to a kind of method that the bacterial wilt of tomato resistance is identified.
Background technology
Tomato (Lycopersicon esculentum Mill.) originates from South America and Mexico, is one of most important solanaceous crops in the world.Because the influence of long-term continuous cropping and soil acidification, bacterial wilt (Ralstoniasolanacearum E.F.Smith) has seriously limited the production of tomato in the torrid zone and subtropical zone, therefore, the seed selection of tomato disease-resistant variety is one of important measures to the bacterial wilt integrated control with introducing, but because bacterial wilt pathogenic bacteria-false monospore bacillus (Ralstonia solanacearum E.F.Smith) diversity of its gene and the popularity of host range, and tomato material or strain are carried out resistance authentication method itself also have defective, this has limited the control to bacterial wilt to a certain extent.Therefore, the resistance authentication method is vital to the evaluation of tomato resistant material and the seed selection of disease-resistant variety accurately and effectively.
In the evaluation of bacterial wilt resistant material in the past, generally adopt the inoculation in seedling stage to identify and/or sick garden resistance is identified, mainly according to bacterial wilt the morbidity illness---the wilted percent of plant is determined the resistance level of material.Wherein, sick garden resistance identifies that being mainly used in tomato becomes strain to identify, its qualification result is subject to the homogeneity and the climate effect thereof of plot bacterial concentration, cause the qualification result between the different year to have certain difference, and the evaluation of sick garden is also limited because of area, can't carry out resistance to large batch of material the same period and identify; Seedling stage inoculation identifies and overcome the limitation that identify in sick garden to a certain extent, but in application process because inoculation seedling age, inoculation method, the strain system of germ and the difference of culture environment, qualification result is often also inconsistent; Above-mentioned two kinds of qualification results have just reflected tomato seedling or have become the tolerance of strain phase to ralstonia solanacearum, and be not to be the true resistance level of this kind or material, dye phenomenon because in the planting process of tomato, have the sexuality of diving toward contact, be the wound of Ralstonia solanacearum (false monospore bacillus) by the plant root, in vascular bundles such as cane, constantly expand numerous extension behind the invasion plant, have only that false monospore bacillus colony number surpasses 10 in plant
8Cfu, plant just shows the wilting symptom; Simultaneously, to expand the ability of numerous extension also be different to different resistant materials to suppressing false monospore bacillus, so the plant that does not show the wilting symptom not necessarily just not infects false monospore bacillus, so showing, simple dependence plant illness judges, also can't authentic assessment go out the resistance level of different materials.
Summary of the invention
The present invention seeks to overcome the weak point of above-mentioned prior art, propose a kind of tomato seedling in the past with become the strain phase to bacterial wilt tolerance evaluation of foundation on, interaction further combined with bacterial wilt latent infection and fungus strain and host plant, it is the testing result of false monospore bacillus quantity and population distribution in the plant body, the disease-resistant authentication method of comprehensive evaluation tomato material resistance to bacterial wilt level, improve the order of accuarcy that resistance is identified, with the seed selection of quickening tomato resistance to bacterial wilt new varieties or the screening of introducing disease-resistant variety with apply.
The object of the invention is achieved by following technical solution:
A kind of tomato bacterial wilt resistance identification method, carry out according to the following steps:
(1) the tomato seedling inoculation is identified:
1. adopt the conventional seedbed system method, cultivate the tomato seedling of growth basically identical, standby during to the 5-6 leaf;
2. expand numerous cultivation with the false monospore bacillus of LB culture medium inoculated (Ralstonia solanacearum E.F.Smith); To be diluted to concentration with sterilized water be 10 with expanding false monospore bacillus after numerous
8The bacterial suspension of cfu/ml; The OD of this suspending liquid (A600) value equals 0.4;
3. tomato seedling is hindered the root inoculation, and every strain meets bacterial classification suspending liquid 30ml;
4. inoculate seedling at temperature 25-30 ℃, cultivating and managing under the relative moisture of the soil 60% above condition;
5. inoculate back 7 days and institute an inquiry statistics plant wilted percent, investigation in per 7 days is once investigated 4 times altogether, carries out tomato seedling resistance characterization and evaluation after gathering;
(2) the false monospore bacillus colony and the detection that distributes thereof in the tomato plant:
1. after inoculating resistance seedling stage and identify finishing, will not wilt plant after flushing, wiping away dried, 75% alcohol disinfecting, the stem section will be intercepted into 0-2,2-4,4-6,6-8,8-10cm length;
2. respectively each stem section is placed the 10ml sterilized water, under 30 ℃ of conditions, cultivate, obtain bacterial suspension behind the 2h;
3. the colonies typical that each stem section bacterial suspension is diluted to this vacation monospore bacillus can be clear obvious on nutrient culture media, be easy to statistics and be standard, therefrom get the 0.1ml dilution and select to push into flat board on the nutrient culture media,, cultivate 24-48h under the RH=60% condition at 30 ℃ in TZC;
4. add up the red bacterium colony that forms on each nutrient culture media, draw the size and the distribution situation thereof of false monospore bacillus, the evaluation of resistance of the material of participating in the experiment after gathering each tomato examination material plant in-group;
(3) sick garden resistance evaluation:
1. the disease garden is identified as bacterial wilt in the disease garden of bacterial wilt over the years being fallen ill;
2. the seed of the disease-resistant material that step (2) is obtained is colonizated in the disease garden after cultivating into the tomato seedling of growth unanimity;
3. investigation statistics plant wilted percent carries out the resistance of tomato growth fruiting period and identifies;
(4) respectively try the result that material (1) is inoculated evaluation and the interior false monospore bacillus group size of (2) plant and distribute detection and the evaluation of (3) sick garden resistance seedling stage according to tomato, carry out comprehensive evaluation, the delimitation resistance level.
It is 50mg/L that described TZC selects the concentration of TZC in the nutrient culture media, and surplus is the LB nutrient culture media.
The present invention the inoculation of traditional tomato seedling identify with the basis that becomes the resistance evaluation of the sick garden of strain on, scientifically between two stages, increased the detection of false monospore bacillus group size in the plant and distribution thereof, make tomato seedling or become the strain phase to the degree of the tolerance of ralstonia solanacearum external manifestation and plant inherence bacteria infection and suppress the ability that false monospore bacillus expands numerous extension in vivo and organically combine, carry out comprehensive evaluation, delimit resistance level, thereby significantly improve accurate level (seeing Table 1) the anti-bacterial wilt of tomato of material of participating in the experiment.
Description of drawings
The change curve of the different strains of the false monospore bacillus of Fig. 1 TTS microspecies inoculation back tomato, the false monospore bacillus of different stem section bacterium colony colony
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing.
Embodiment 1:
85254,51255,47254,7585,51198,519230,1466EA, 203 and 85198 totally 9 kinds test material: tomato variety:; Wherein 1466EA and 203 is respectively as disease-resistant contrast and susceptible contrast.
Bacterial wilt of tomato bacterium source: false monospore bacillus (Ralstonia solanacearum E.F.Smith) TT4 microspecies; (the isolated bacterial wilt tomato pathogenic bacteria of academy of agricultural sciences, Zhejiang Province vegetables belong to false monospore bacillus race1, and biovar 3).
TZC selects culture medium prescription: LB nutrient culture media+TZC 50mg/l.
LB culture medium prescription: sucrose 10g, peptone 10g, yeast extract 5g, sodium chloride 5g, agar 15g, distilled water 1L.
Tested year July in November, 2002-2003 and carry out in academy of agricultural sciences, Zhejiang Province vegetables institute test base;
A kind of tomato bacterial wilt resistance identification method, carry out according to the following steps:
(1) the tomato seedling inoculation is identified:
1. adopt the conventional seedbed system method, seed is sowed at the nutritive cube of the 10cm that nutrient matrix is housed, cultivate the tomato seedling of growth basically identical, standby during to the 5-6 leaf;
2. expand numerous cultivation with the false monospore bacillus of LB culture medium inoculated (Ralstonia solanacearum E.F.Smith); The false monospore bacillus of expanding after numerous is obtained bacterial suspension with the sterilized water dilution, and according to the concentration of the OD value adjusting bacterium of bacterial suspension, (bacterial concentration is equivalent to 10 to make OD (A600) value of the bacterial suspension of final acquisition equal 0.4
8Cfu/ml);
When 3. inoculating from foundation portion 2cm place with cutter from top to bottom to matrix inscribe 2 cuttves, injure the part root system, in each nutritive cube, pour the bacterial suspension (OD (A600)=0.4) of 30ml again into, to guarantee equal bacterial concentration;
4. inoculate seedling at temperature 25-30 ℃, cultivation management under the relative moisture of the soil 60% above condition;
5. inoculate back 7 days and institute an inquiry statistics plant wilted percent, investigation in per 7 days is once investigated 4 times altogether, and tabulate statistics disease index and plant wilted percent carry out tomato seedling resistance characterization and evaluation;
(2) the false monospore bacillus colony and the detection that distributes thereof in the tomato plant:
1. after inoculating resistance seedling stage and identify finishing, will not wilt plant after flushing, wiping away dried, 75% alcohol disinfecting, the stem section will be intercepted into 0-2,2-4,4-6,6-8,8-10cm length;
2. respectively each stem section is placed the 10ml sterilized water, under 30 ℃ of conditions, cultivate, obtain bacterial suspension behind the 2h;
3. the colonies typical that each stem section bacterial suspension is diluted to this vacation monospore bacillus can be clear obvious on nutrient culture media, be easy to statistics and be standard, therefrom get the 0.1ml dilution and select to push into flat board on the nutrient culture media,, cultivate 24-48h under the RH=60% condition at 30 ℃ in TZC;
4. add up the red bacterium colony that forms on each nutrient culture media, draw size and the distribution situation thereof of false monospore bacillus each tomato examination material plant in-group, wherein the contained false monospore bacillus quantity of 0-2 stem section is promptly represented the group size of the false monospore bacillus of plant, 0-2,2-4,4-6,6-8, the change curve that the contained respectively false monospore bacillus quantity of 8-10 stem section constitutes constitutes the distribution of false monospore bacillus in plant, the evaluation of resistance of the material of participating in the experiment after gathering;
(3) sick garden resistance evaluation:
1. the disease garden is identified as bacterial wilt in the disease garden of bacterial wilt over the years being fallen ill;
2. the seed of the disease-resistant material that step (2) is obtained is cultivated into the tomato seedling of growth unanimity, is colonizated in the disease garden during to the 7-8 leaf;
3. 1 week instituted an inquiry after the field planting, and investigation in per 10 days once coextensive continuing 3 months, and statistics plant wilted percent, is carried out the resistance of tomato growth fruiting period and identified;
(4) respectively try the result that material (1) is inoculated evaluation and the interior false monospore bacillus group size of (2) plant and distribute detection and the evaluation of (3) sick garden resistance seedling stage according to tomato, carry out comprehensive evaluation, the delimitation resistance level.
Identify that with sick garden resistance this three phases carries out evaluation of resistance to 9 different resistant materials in the detection of plant in-group and distribution thereof by above-mentioned tomato seedling inoculation evaluation, false monospore bacillus.Seedling stage inoculation is identified and field resistance is identified from plant (external) of germ showed the resistance difference of estimating between the strain; The detection of false monospore bacillus colony and distribution has embodied germ expanded numerous and extension ability plant (inherent resistance) body in the plant, that is different materials is to the difference inhibition ability of identical germ, estimates resistance difference between the strain from the angle of germ.
Identify by seedling stage inoculation 9 resistant materials carried out Preliminary Identification, think 203 in 9 resistant materials resistance level minimum, 85198 take second place, other 7 material resistance levels are identical, all belong to high anti-material, the plant wilted percent is 0 (table 1).
But detect (table 1) by the false monospore bacillus of 0-2 stem section clump count, think 203 with 85198 resistance levels and seedling stage to inoculate qualification result consistent, and there is certain resistance difference in 7 materials such as 51198 grades; Further false monospore bacillus is distributed the tomato plant in-group and detect, from its result (Fig. 1), can think in these 9 resistant materials 85254,51255, the resistance of 47254 and 75854 strains is the strongest, 51198 take second place, 519230 and the resistance level of 1466EA belong to tri-layer, and resistance level the poorest be 203 and 85198.
On the result of above-mentioned two stages evaluation, these 9 resistant materials are carried out disease garden resistance identify, verify that these 9 resistant materials become the tolerance of strain to bacterial wilt.Qualification result (table 1) shows that 85254,51255,47254 and 7585 pairs of bacterial wilt resistances are the strongest, 519230 and 1466EA stronger, 51198 time, and 203 and 85198 still be that resistance against diseases is the poorest.
Comprehensive evaluation through several authentication methods, can carry out evaluation of resistance to these 9 resistant materials exactly, think in these 9 strains, there is following resistance gradient (high resisting-susceptible): (51255,47254)-(85254,7585)-(519230,1466EA)-(51198)-(203,85198).
The inoculation in seedling stage of the different strain of table 1 tomato, false monospore bacillus are at plant 0-2 stem segmented population size detection, sick garden qualification result relatively
| Strain | Inoculate qualification result (wilted percent %) seedling stage | The false monospore bacillus of 0-2 stem section clump count (* 10 4cfu) | Sick garden qualification result (wilted percent %) |
| 85198 | 16.7b | 43.35ab | 100.00A |
| 203 | 33.3a | 505.82a | 94.00A |
| 51198 | 0c | 81.85ab | 60.00B |
| 1466EA | 0c | 2.12bc | 16.00BC |
| 519230 | 0c | 2.57bc | 14.0BC |
| 47254 | 0c | 0.05c | 10.00BC |
| 7585 | 0c | 0.51bc | 4.00C |
| 85254 | 0c | 0.65bc | 4.00C |
| 51255 | 0c | 0.23c | 0C |
Annotate: letter representation Duncan ' s The result of multiple comparisons in the table has different lowercase alphabet differentials different significantly (P＜0.05) between the same row.There are different capitalizations to represent difference extremely significantly (P＜0.01) between the same row.
Claims (2)
1, tomato bacterial wilt resistance identification method, its feature is carried out according to the following steps:
(1) the tomato seedling inoculation is identified:
1. cultivate the tomato seedling of growth basically identical, standby during to the 5-6 leaf;
2. expand numerous cultivation with the false monospore bacillus of LB culture medium inoculated (Ralstonia solanacearumE.F.Smith); To be diluted to concentration with sterilized water be 10 with expanding false monospore bacillus after numerous
7The bacterial suspension of cfu/ml;
3. tomato seedling is hindered the root inoculation, and every strain meets bacterial classification suspending liquid 30ml;
4. inoculate seedling at temperature 25-30 ℃, cultivating and managing under the relative moisture of the soil 60% above condition;
5. inoculate back 7 days and institute an inquiry statistics plant wilted percent, carry out the tomato seedling resistance and identify;
(2) the false monospore bacillus colony and the detection that distributes thereof in the tomato plant:
1. after inoculating resistance seedling stage and identify finishing, will not wilt plant after flushing, wiping away dried, 75% alcohol disinfecting, the stem section will be intercepted into 0-2,2-4,4-6,6-8,8-10cm length;
2. respectively each stem section is placed the 10ml sterilized water, under 30 ℃ of conditions, cultivate, obtain bacterial suspension behind the 2h;
3. the colonies typical that each stem section bacterial suspension is diluted to this vacation monospore bacillus can be clear obvious on nutrient culture media, be easy to statistics and be standard, therefrom get the 0.1ml dilution and select to push into flat board on the nutrient culture media,, cultivate 24-48h under the RH=60% condition at 30 ℃ in TZC;
4. add up the red bacterium colony that forms on each nutrient culture media, draw the size and the distribution situation thereof of false monospore bacillus, the material evaluation of resistance of participating in the experiment each tomato examination material plant in-group;
(3) sick garden resistance evaluation:
1. the disease garden is identified as bacterial wilt in the disease garden of bacterial wilt over the years being fallen ill;
2. the seed of the disease-resistant material that step (2) is obtained is colonizated in the disease garden after cultivating into the tomato seedling of growth unanimity;
3. investigation statistics plant wilted percent carries out the resistance of tomato growth fruiting period and identifies;
(4) respectively try the result that material (1) is inoculated evaluation and the interior false monospore bacillus group size of (2) plant and distribute detection and (3) sick garden evaluation seedling stage according to tomato, carry out comprehensive evaluation, the delimitation resistance level.
2, method according to claim 1 is characterized in that it is 50mg/L that described TZC selects the concentration of TZC in the nutrient culture media, and surplus is the LB nutrient culture media.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102523907A (en) * | 2012-02-10 | 2012-07-04 | 江西省农业科学院植物保护研究所 | Method for identifying resistance of asparagus to wilt disease in seedling stage |
| CN108377893A (en) * | 2018-02-13 | 2018-08-10 | 东北农业大学 | A kind of rapid detection method of eggplant verticillium wilt disease resistance |
| CN108739147A (en) * | 2018-06-13 | 2018-11-06 | 山东省潍坊市农业科学院 | A method of the anti-clubroot Chinese cabbage germplasm of initiative |
| CN112626093A (en) * | 2020-12-25 | 2021-04-09 | 浙江大学 | Tomato bacterial wilt resistance gene Sl alpha-KGDH E2 and application thereof |
| CN113125643A (en) * | 2021-04-02 | 2021-07-16 | 宁波市农业科学研究院 | Method for rapidly identifying tomato bacterial wilt seedling stage resistance by injection inoculation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2000166561A (en) * | 1998-12-03 | 2000-06-20 | Canon Inc | New nucleic acid fragment and detection of strain tb64 using the same |
| CN1117874C (en) * | 2000-09-08 | 2003-08-13 | 广东省农业科学院蔬菜研究所 | Water culture bacterination process to determine bacterial wilt resistance of plant |
| CN1515666A (en) * | 2000-10-11 | 2004-07-28 | 常州兰陵制药有限公司 | Method for screening pseudomonads for controlling blight |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102523907A (en) * | 2012-02-10 | 2012-07-04 | 江西省农业科学院植物保护研究所 | Method for identifying resistance of asparagus to wilt disease in seedling stage |
| CN108377893A (en) * | 2018-02-13 | 2018-08-10 | 东北农业大学 | A kind of rapid detection method of eggplant verticillium wilt disease resistance |
| CN108739147A (en) * | 2018-06-13 | 2018-11-06 | 山东省潍坊市农业科学院 | A method of the anti-clubroot Chinese cabbage germplasm of initiative |
| CN108739147B (en) * | 2018-06-13 | 2020-09-01 | 山东省潍坊市农业科学院 | Method for creating clubroot-resistant Chinese cabbage germplasm |
| CN112626093A (en) * | 2020-12-25 | 2021-04-09 | 浙江大学 | Tomato bacterial wilt resistance gene Sl alpha-KGDH E2 and application thereof |
| CN112626093B (en) * | 2020-12-25 | 2022-08-23 | 浙江大学 | Tomato bacterial wilt resistance gene Sl alpha-KGDH E2 and application thereof |
| CN113125643A (en) * | 2021-04-02 | 2021-07-16 | 宁波市农业科学研究院 | Method for rapidly identifying tomato bacterial wilt seedling stage resistance by injection inoculation method |
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