CN106011222A - Agrobacterium tumefaciens resistance identification method of peach trees and sibling species plants thereof - Google Patents
Agrobacterium tumefaciens resistance identification method of peach trees and sibling species plants thereof Download PDFInfo
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Abstract
The invention relates to an agrobacterium tumefaciens resistance identification method of peach trees and sibling species plants thereof. In a vigorous growth season of the peach trees, an annual branch of which the stem thickness is 5-15mm is selected, a wound which has the length of 8-10mm and the width of 5-8mm and deeps into xylem is cut from top to bottom on a stem part with a sharp paper knife, then, 1-2 pieces of round filter paper dipped with 109cfu.mL<-1> pathopoiesis agrobacterium bacteria solution is clamped in a parallel tangent plane in the wound, subsequently, the upper part of the cut is swathed with a sealing film, and the cut is restored. After tap water mist spray is carried out for 30 minutes, a proper pathogenic environment is manufactured. After inoculation is carried out for 30-90 days, the maximum tumor diameter, and the stem thickness of the upper end and the lower end of an inoculation locus are investigated by a vernier caliper and tumor diameter ratio is calculated out. According to the tumor diameter ratio, resistance grades are divided, and the agrobacterium tumefaciens resistance grade of the material is determined. The method is simple in operation, infection efficiency is improved by the restoration of cut cortex, resistance evaluation considers the species and growth state differences of materials to be identified, and the agrobacterium tumefaciens resistance grade of the peach tree and sibling species materials thereof can be accurately evaluated.
Description
Technical field
The present invention relates to a kind of plant disease Resistance Identification method, a kind of root knot Resistance Identification method of Fructus Persicae and sibling species plant thereof.
Background technology
Fructus Persicae and sibling species plant thereof are mainly tone fruit trees, and on producing, its root system often suffers from the root knot harm that Agrobacterium tumefaciems causes, and causes huge economic loss.Including wild Radix Fici Hirtae, Lee, Fructus Pruni, almond etc. between sibling species fruit tree generally can mutually grafting, the material therefrom screening anti-root knot is significant as stock and stock breeding material.For tone fruit trees root knot Resistance Identification, after the most how being stabbed by root or to be scratched on inoculation method, saturated inoculation Agrobacterium solution, then after Growing season terminates, dig out root system, investigation plant root incidence, or by stabbing branch stem, then injection Agrobacterium bacterium solution, wet cotton balls wraps up, creating Agrobacterium infection condition etc., these methods also exist operation complexity, and wound or depth of needling and inoculum concentration such as are difficult to control at the shortcoming；In terms of Resistant index evaluation, main employing tumor footpath size or quality, average tumor footpath size etc. are as disease scale index or the determination of resistance class, being short of inoculation planting in advance thing material species variation, the consideration of Reducing sugar difference, its result is easily subject to plant species, the impact of growth conditions difference.
Summary of the invention
For the deficiency overcoming prior art to exist, the invention provides a kind of root knot Resistance Identification method of Fructus Persicae and sibling species plant thereof, it is by simplifying inoculation operation, homogenization inoculum concentration, manufacture morbidity critical period environmental condition, using tumor Diameter growth to be used for assessment resistance index, root knot evaluation of resistance for Fructus Persicae and sibling species material thereof provides new method, can be used for the root knot Resistance Identification of the close plant of sibship, screen or evaluate resistant material.
The present invention realizes with following technical scheme:
A kind of root knot Resistance Identification method of Fructus Persicae and sibling species plant thereof, 5～JIUYUE, choose material stem thick 5 to be evaluated～15mm annotinous branch, carving 8～10mm length, 5～8mm width, the deep and wound of xylem with sharp paper knife from the top down in stem, in wound, parallel cut surface sandwiches 1～2 and is moistened with 109
cfu·mL-1The round filter paper of virulent Agrobacterium bacterium solution, then hinders top parcel with sealed membrane at quarter, wound at quarter is resetted.5 sites of every strain inoculation, if inoculate, more than inoculation site spacing 20mm on same branch.Inoculation comparison is the filter paper being moistened with sterilized water.Inoculating in latter 3 hours, docking plant carries out tap water and sprays 30 minutes, is infecting critical period manufacture good environment.After inoculation 30～90 days, with the slide gauge investigation maximum tumor footpath of each inoculation position, inoculation site upper and lower two ends stem was thick, calculate tumor footpath ratio.According to tumor footpath than the root knot resistance evaluating material.
Specifically carry out as follows:
(1) detected materials prepares: in field grafting or cultivate Fructus Persicae to be tested and sibling species material thereof, as identified in the flowerpot that can be planted in suitable volume in greenhouse, carry out soil, fertilizer, water and the prevention and control of plant diseases, pest control and management, utilize natural conditions or greenhouse light and temperature condition by its fast-growth to meeting inoculation condition；
(2) cultivation of Agrobacterium tumefaciems bacterium solution and standard are carried disease germs the preparation of filter paper: be inoculated in YEB fluid medium by Agrobacterium pathogenic strain (such as AT4-3), at 28 DEG C, and 200
About 16h cultivated by rpm/min shaking table, and cultured bacterium solution is 5000
Under the conditions of rpm/min centrifugal 10 minutes, resuspended with the sterile purified water containing 0.01% polysorbas20, and to adjust concentration be 1 × 109cfu·mL-1, it is that monolithic dispersion is placed in culture dish by the sterilizing filter paper of diameter 5mm, culture dish is poured Agrobacterium bacterium solution submergence filter paper 5 minutes into, then discard unnecessary bacterium solution with glue head straw, cover culture dish lid standby；
(3) Agrobacterium inoculation: 5～JIUYUE, choose material stem to be evaluated slightly for the annotinous branch of 5-15mm, carving 8～10mm length, 5～8mm width, the deep and wound of xylem with sharp paper knife from the top down in stem, in wound, veneer sandwiches 1～2 and is moistened with 109cfu·ml-1The round filter paper of virulent Agrobacterium bacterium solution, then hinders top parcel with sealed membrane at quarter and wound at quarter is resetted.5 sites of every strain inoculation, if inoculating on same branch, more than inoculation site spacing 20mm, compare the filter paper being moistened with sterilized water for inoculation；
(4) spraying after inoculation: inoculate in latter 3 hours, docking plant carries out tap water and sprays 30 minutes, improves ambient humidity；
(5) incidence survey: after inoculation 30～90 days, with the slide gauge investigation maximum tumor footpath (tumor adds branch diameter) of each inoculation position, inoculation site upper and lower two ends stem thick (meansigma methods is vaccination branch diameter), calculates tumor footpath ratio (vaccination tumor adds branch diameter and branch diameter ratio)；
(6) assessment resistance and evaluation: with single plant maximum tumor footpath than the root knot resistance evaluating plant material；
(7) individual grade scale and population resistance classification:
0 grade, immunity: with compare no significant difference, inoculation position projection ＜ 1.0mm, tumor footpath ratio (the maximum crown gall nodule diameter and the inoculation position branch diameter ratio that produce after inoculation) value is 1, inoculates and does not falls ill；
1 grade, high anti-: 1 ＜ tumor footpath ratio≤1.2；
2 grades, in anti-: 1.2 ＜ tumor footpath ratios≤1.5；
3 grades, susceptible: 1.5 ＜ tumor footpath ratios≤2；
4 grades, middle sense: 2 ＜ tumor footpath ratios≤3；
5 grades, high sense: tumor footpath is than ＞ 3.
For Group Evaluation, being calculated as follows of disease index:
Disease index=(∑ (susceptible Seedling numbers at different levels × corresponding sick level)/investigation Seedling sum × 5) × 100%
Population resistance classification is as follows:
Immunity, disease index is 0；
High anti-, 0 ＜ disease index≤20；
In anti-, 20 ＜ disease index≤40；
Susceptible, 40 ＜ disease index≤60；
Middle sense, 60 ＜ disease index≤80；
High sense, disease index ＞ 80；
This beneficial effect of the invention is, this authentication method is simple to operate, the cortex carving wound is utilized to be fitted in the middle of wounded tissue position by carrying the close filter paper dick of Agrobacterium tumefaciems bacterium amount, rely on plant own wound agglutination carry out moisturizing and promote infecting of cause of disease, be difficult to cause inoculation failure because of weather arid etc.；Resistance ranking method science, utilizes tumor footpath ratio to carry out assessment resistance, it is contemplated that the grading difference that vegetable material causes because of species variation, growth conditions difference.It addition, this authentication method, field or indoor growing plant are suitable for.
Detailed description of the invention
Implementation example: Fructus Persicae and the root knot evaluation of resistance of 8 individual plants of sibling species thereof
(1) detected materials prepares: at the beginning of 3 months, in greenhouse, Nutrition Soil makees substrate, in the flowerpot of 50L cultivation grow directly from seeds Radix Fici Hirtae, the Prunus davidiana that grows directly from seeds, Fa, big Kubo, deep red Fructus Persicae, Tibet 3 (Semen Orubu mirae), Amygdalus mongolicus Maxim., 8 plants such as Xinjiang Wild Almonds, make its normal growth, and carry out daily clay fertilizer water and pest management；
(2) cultivation of Agrobacterium tumefaciems bacterium solution is carried disease germs with standard the preparation of filter paper: the Agrobacterium tumefaciems pathogenic strain AT4-3 picking mixed with YEB bacterium solution by the glycerol being saved in-20 DEG C of refrigerators is a small amount of, is inoculated in equipped with 150ml on super-clean bench
In the triangular flask of YEB fluid medium, at 28 DEG C, 200
About 16h cultivated by rpm/min shaking table.Cultured bacterium solution loads centrifuge tube 5000
Under the conditions of rpm/min centrifugal 10 minutes, thalline was resuspended with the sterile purified water containing 0.05% polysorbas20, and to adjust concentration be 1 × 109cfu mL-1.It is that monolithic dispersion is placed in culture dish by the sterilizing filter paper of diameter 5mm, culture dish is poured Agrobacterium bacterium solution submergence filter paper 5 minutes into, then discard unnecessary bacterium solution with glue head straw, cover culture dish lid standby；
(3) Agrobacterium tumefaciems inoculation: at the beginning of 6 months, choosing the annotinous branch that material stem to be evaluated is slightly 5-15mm, carve long 8-10mm, wide 5-8mm, the deep and wound of xylem with sharp paper knife from the top down in stem, veneer sandwiches 1 and is moistened with 10 in wound9cfu/ml
The filter paper of AT4-3 Agrobacterium bacterium solution, then hinders top parcel with sealed membrane at quarter and wound at quarter is resetted.5 sites of every strain inoculation.Comparison is moistened with the filter paper of sterilized water for inoculation；
(4) foliar spray after inoculation: inoculate latter 1 hour, docking plant carries out tap water and sprays 30 minutes, improves ambient humidity；
(5) incidence survey: inoculate latter 60 days, with the slide gauge investigation difference test maximum tumor footpath (tumor adds branch diameter) of each inoculation position of material, inoculation site upper and lower two ends stem thick (meansigma methods is vaccination branch diameter), calculates tumor footpath ratio (vaccination tumor adds branch diameter and vaccination branch diameter ratio)；
(6) assessment resistance and evaluation: with single plant maximum tumor footpath than the root knot resistance evaluating plant material；
(7) individual grade scale and population resistance classification:
0 grade, immunity: with compare no significant difference, inoculation position projection ＜ 1.0mm, tumor footpath ratio (the maximum crown gall nodule diameter and the inoculation position branch diameter ratio that produce after inoculation) value is 1, inoculates and does not falls ill；
1 grade, high anti-: 1 ＜ tumor footpath ratio≤1.2；
2 grades, in anti-: 1.2 ＜ tumor footpath ratios≤1.5；
3 grades, susceptible: 1.5 ＜ tumor footpath ratios≤2；
4 grades, middle sense: 2 ＜ tumor footpath ratios≤3；
5 grades, high sense: tumor footpath is than ＞ 3.
For Group Evaluation, being calculated as follows of disease index:
Disease index=(∑ (susceptible Seedling numbers at different levels × corresponding sick level)/investigation Seedling sum × 5) × 100%
Population resistance classification is as follows:
Immunity, disease index is 0；
High anti-, 0 ＜ disease index≤20；
In anti-, 20 ＜ disease index≤40；
Susceptible, 40 ＜ disease index≤60；
Middle sense, 60 ＜ disease index≤80；
High sense, disease index ＞ 80；
(8) the root knot evaluation of resistance that Agrobacterium tumefaciens AT4-3 is caused by Fructus Persicae and 8 individual plants of sibling species material thereof the results are shown in Table 1.
8 kinds of materials Resistance Identification results to Agrobacterium tumefaciens AT4-3 such as table 1 Fructus Persicae
| Material | Tumor adds a footpath (maximum, mm) | Branch footpath (mm) | Maximum tumor footpath ratio | Assessment resistance | Anti-sense is evaluated |
| Grow directly from seeds Radix Fici Hirtae | 15.43 | 8.12 | 1.9 | 3 | Susceptible |
| Grow directly from seeds Prunus davidiana | 15.84 | 7.20 | 2.2 | 4 | Middle sense |
| Big Kubo | 23.42 | 7.10 | 3.3 | 5 | High sense |
| Deep red Fructus Persicae | 27.71 | 6.04 | 4.6 | 5 | High sense |
| Fa | 22.86 | 6.35 | 3.6 | 5 | High sense |
| Tibet 3 | 24.06 | 7.76 | 3.1 | 5 | High sense |
| Amygdalus mongolicus Maxim. | 8.47 | 5.64 | 1.5 | 2 | In anti- |
| Xinjiang Wild Almonds | 12.22 | 5.82 | 2.1 | 4 | Middle sense |
Claims (2)
1. the root knot Resistance Identification method of a Fructus Persicae and sibling species plant thereof, it is characterized in that: choose material stem thick 5 to be evaluated～15mm annotinous branch, carving 8～10mm length, 5～8mm width, the deep and wound of xylem with sharp paper knife from the top down in stem, in wound, parallel cut surface sandwiches 1～2 and is moistened with 109 cfu·mL-1The round filter paper of virulent Agrobacterium bacterium solution, then hinders top with sealed membrane at quarter and swathes, and wound at quarter is resetted, 5 sites of every strain inoculation, if inoculating on same branch, more than inoculation site spacing 20mm, inoculation comparison is the filter paper being moistened with sterilized water；Inoculating in latter 3 hours, docking plant carries out tap water and sprays 30 minutes, is infecting critical period manufacture good environment；After inoculation 30～90 days, with the slide gauge investigation maximum tumor footpath of each inoculation position, inoculation site upper and lower two ends stem was thick, calculate tumor footpath ratio, according to tumor footpath than the root knot resistance evaluating material.
The root knot Resistance Identification method of a kind of Fructus Persicae the most according to claim 1 and sibling species plant thereof, it is characterised in that: concretely comprising the following steps of it:
(1) detected materials prepares: in field grafting or cultivate Fructus Persicae to be tested and sibling species material thereof, as identified in the flowerpot that can be planted in suitable volume in greenhouse, carry out soil, fertilizer, water and the prevention and control of plant diseases, pest control and management, utilize natural conditions or greenhouse light and temperature condition by its fast-growth to meeting inoculation condition；
(2) cultivation of Agrobacterium tumefaciems bacterium solution and standard are carried disease germs the preparation of filter paper: be inoculated in YEB fluid medium by Agrobacterium pathogenic strain, at 28 DEG C, about 16h cultivated by 200 rpm/min shaking tables, cultured bacterium solution is centrifuged 10 minutes under the conditions of 5000 rpm/min, resuspended with the sterile purified water containing 0.01% polysorbas20, and to adjust concentration be 1 × 109cfu mL-1；It is that monolithic dispersion is placed in culture dish by the sterilizing filter paper of diameter 5mm, culture dish is poured Agrobacterium bacterium solution submergence filter paper 5 minutes into, then discard unnecessary bacterium solution with glue head straw, cover culture dish lid standby；
(3) Agrobacterium inoculation: 5～JIUYUE, choosing material stem to be evaluated is slightly the annotinous branch of 5～15mm, carving long 8-10mm, wide 5-8mm, the deep and wound of xylem with sharp paper knife from the top down in stem, in wound, veneer sandwiches 1～2 and is moistened with 109 cfu·mL-1The round filter paper of virulent Agrobacterium bacterium solution, then hinders top with sealed membrane at quarter and swathes wound reset at quarter, 5 sites of every strain inoculation, if inoculating on same branch, more than inoculation site spacing 20mm, inoculation comparison is for being moistened with the filter paper of sterilized water；
(4) spraying after inoculation: inoculate in latter 3 hours, docking plant carries out tap water and sprays 30 minutes, improves ambient humidity；
(5) incidence survey: after inoculation 30～90 days, with the slide gauge investigation maximum tumor footpath of each inoculation position, inoculation site upper and lower two ends stem is thick, calculate tumor footpath ratio；
(6) assessment resistance and evaluation: with the maximum tumor footpath of single plant than the root knot resistance evaluating plant material；
(7) individual assessment resistance standard and population resistance evaluation:
0 grade, immunity: with compare no significant difference, inoculation position projection ＜ 1.0mm, tumor footpath ratio (the maximum crown gall nodule diameter and the inoculation position branch diameter ratio that produce after inoculation) value is 1, inoculates and does not falls ill；
1 grade, high anti-: 1 ＜ tumor footpath ratio≤1.2；
2 grades, in anti-: 1.2 ＜ tumor footpath ratios≤1.5；
3 grades, susceptible: 1.5 ＜ tumor footpath ratios≤2；
4 grades, middle sense: 2 ＜ tumor footpath ratios≤3；
5 grades, high sense: tumor footpath is than ＞ 3；
For Group Evaluation, being calculated as follows of disease index:
Disease index=(∑ (susceptible Seedling numbers at different levels × corresponding sick level)/investigation Seedling sum × 5) × 100%
Population resistance classification is as follows:
Immunity, disease index is 0；
High anti-, 0 ＜ disease index≤20；
In anti-, 20 ＜ disease index≤40；
Susceptible, 40 ＜ disease index≤60；
Middle sense, 60 ＜ disease index≤80；
High sense, disease index ＞ 80.
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Cited By (1)
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