CN107475350A - A kind of method of seedling stage Rapid identification cotton verticillium wilt resistance - Google Patents
A kind of method of seedling stage Rapid identification cotton verticillium wilt resistance Download PDFInfo
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Abstract
The invention discloses a kind of method of seedling stage Rapid identification cotton verticillium wilt resistance, and verticillium wilt pathogen is seeded into cotton seedling, determines the SPAD values of cotton leaf after being inoculated with and calculates verticillium wilt stress index, then coercing index according to verticillium wilt judges cotton verticillium wilt resistance.This method neither damages cotton leaf, is not also disturbed by artificial subjective judgement factor, and measurement result is relatively reliable compared with disease index method, relatively determines the active simpler and more direct of cotton seedling verticillium wilt relevant enzyme, testing index is unique.The detection method can quick nondestructive check the disease resistance of each growing stage cotton verticillium wilt, accelerate verticillium wilt resistance of cotton by same breed breeding process, directly can be applicable in production.
Description
Technical field：
The invention belongs to Cotton disease resistance detection technique field, and in particular to a kind of seedling stage Rapid identification cotton verticillium wilt resists
The method of property.
Background technology：
Verticillium wilt is one of seven big soil-borne diseases in the world, has wide distribution, harm weight, infects the characteristics of strong, it is extremely difficult to root
Control, be referred to as " cotton cancer ".It is preventing and treating verticillium wilt measure the most cost-effective to generally acknowledge plantation disease-resistant variety in the world at present,
And identify that the disease resistance of cotton verticillium wilt is the important prerequisite of verticillium wilt resistance of cotton by same breeding and preventing and controlling.
Verticillium wilt can all occur for cotton whole breeding time, and general 3~5 leaf phase starts aobvious disease, largely fallen ill after buddingging, sick leaf
Mottled in palmate, leaf margin crimps downwards, and sick leaf from bottom to top gradually comes off, and only surplus top few leaflet, cotton buds and bolls are rare when serious.
Because occurrent time and soil verticillium wilt pathogen pathogenicity are strong and weak different, through hidden disease is often presented after cotton morbidity (i.e. blade does not show disease)
Phenomenon.
Domestic general Method for Identification of Cotton Verticillium Wilt Resistance --- (Resistance Strain of Cotton disease pest evaluates skill to disease index method at present
The part of art specification the 5th：Verticillium wilt, GB/T22101.5-2009；A kind of sprout period authenticating method of verticillium wilt resistance of cotton by same,
CN201110081889.7), comprehensive descision is carried out with the order of severity according to the blade incidence of disease, if but the hidden disease of cotton plant appearance, using disease
Feelings index method there is larger limitation.According to (one in the method for the activity judgment cotton seedling disease resistance of relevant enzyme after inoculation
The detection method of kind cotton verticillium wilt disease resistance, application number：CN201510111867.9), the morbidity of cotton seedling can both be examined without aobvious disease
Survey, measurement result has certain accuracy, but the determination of activity process of enzyme is complicated, time-consuming longer, is not appropriate for batch kind
The Rapid identification of greensickness-resistance.
To ensure that qualification process is convenient and swift and the accuracy of qualification result, science are established one kind and more quick and precisely identified
The detection method of verticillium wilt resistance of cotton by same is necessary.
The content of the invention：
The present invention provides a kind of detection method of quick, simple and direct, accurate cotton in seedling stage resistance to verticillium wilt, is sent out without cotton seedling
The aobvious disease of disease, you can quickly judge verticillium wilt resistance of cotton by same.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method of seedling stage Rapid identification cotton verticillium wilt resistance, verticillium wilt pathogen is seeded to cotton seedling, after measure inoculation
The SPAD values of cotton leaf simultaneously calculate verticillium wilt stress index, and then coercing index according to verticillium wilt judges cotton verticillium wilt resistance.
The condition of culture of above-mentioned cotton seedling is：Photostage, 24 DEG C, light application time 14h of temperature, intensity of illumination are
20000Lux；Dark phase, temperature are 20 DEG C, and interlunation 10h, whole humidity is 85%.
The method that verticillium wilt pathogen is seeded to cotton seedling is：The leaf phase of cotton 2, every cotton seedling inject verticillium wilt bacterium solution to rhizosphere leech
Shi Zhong, strain are High pathogenicity strain V08DF1.
The spore concentration of the verticillium wilt bacterium solution is not less than 2 × 107Individual/m, injection rate are 10~20ml.
As a kind of optimal technical scheme, after cotton seedling is inoculated with verticillium wilt strain 3d, determine the SPAD values of 1 leaf, calculate yellow
Disease of withering coerces index；Verticillium wilt coerces index calculation method：Verticillium wilt stress index=(be not inoculated with verticillium wilt strain cotton seedling and fall 1
Leaf SPAD values-be inoculated with verticillium wilt strain cotton seedling fall 1 leaf SPAD values)/are not inoculated with verticillium wilt strain cotton seedling and fall 1 leaf SPAD value × 100.
The criterion of the resistance to verticillium wilt is：
Verticillium wilt stress index≤1.3 are immune material；
1.3 ＜ verticillium wilt stress index≤4.0 are high anti-material；
4.0 ＜ verticillium wilt stress index≤6.6 are disease-resistant material；
6.6 ＜ verticillium wilt stress index≤10.6 are resistance to sick material；
Verticillium wilt stress index ＞ 10.6 is susceptible material.
The detailed process of the method for seedling stage Rapid identification cotton verticillium wilt resistance of the present invention is：
(1) full cotton seed is selected, with 70% (v/v) alcohol-pickled 5min, 5% (w/w) H2O22h is soaked, it is sterile
Water rinses 2~3 times, then with sterilized water Steeping and budding 10h.
(2) the above-mentioned cotton seed handled well is laid on the gauze of moistening, is positioned in controllable growth cabinet, treats cotton seed
After showing money or valuables one carries unintentionally, the basically identical seed of root long, seed size is selected, sow in seedling-raising cup (length × width × height=8cm × 8cm ×
15cm), 4 are sowed for every glass, per 40 glasss of kind.Seedling medium is the vermiculite sterilized, and nutrient solution is sterilized water.
(3) when cotton seedling cotyledon is open and flat, 1 plant consistent of cotton seedling of growing way, nutrient solution hoagland ' are selected and remain for every glass with kind
S nutrient solutions, change 1 time weekly.
(4) when the leaf of cotton seedling the 2nd is open and flat, every glass is drawn verticillium wilt bacterium solution 15ml injection cotton seedling rhizosphere leech with injector for medical purpose
Shi Zhong, bacterium solution spore concentration are 2 × 107Individual/ml, strain are High pathogenicity strain V08DF1, are inoculated with 20 glasss altogether；To be inoculated with 15ml
Sterilized water, which is used as, to be compareed, totally 20 glasss.
(5) be inoculated with after 3d, determine all cotton plants fall 1 leaf SPAD values and calculate verticillium wilt coerce index.
(6) table of comparisons 1, the disease resistance of kind is judged according to verticillium wilt stress index.
Table 1 is with respect to disease index and the verticillium wilt stress index table of comparisons
Wherein：
Cotton seedling cultural method described in step (2)：Whole process is carried out in growth cabinet, photostage, temperature 24
DEG C, light application time 14h, intensity of illumination 20000Lux；Dark phase, temperature are 20 DEG C, interlunation 10h.It is whole wet
Spend for 85%.
The sterilization method of step (2) described vermiculite：High-pressure sterilizing pot sterilizes, and temperature is 121 DEG C, sterilization time 1h.
The preparation method of step (4) verticillium wilt pathogen spore suspension：By the verticillium wilt pathogen of (- 80 DEG C) preservations of ultra low temperature freezer
Kind (V08DF1, from Plant Protection Inst., Jiangsu Academy of Agriculture, the strain is conventional bacterial classification, people in the art
Member can be obtained by conventional meanses) at 25 DEG C on CheckShi solid mediums activation culture 10d, then from colony edge picking
A little fungus block, it is transferred in the triangular flask for filling 300ml CheckShi fluid nutrient mediums, training 10d is shaken in constant temperature (25 DEG C) shaking table, with 4
Layer filtered through gauze bacterium solution, blood counting chamber calculate spore concentration, then the concentration of spore suspension is adjusted to 2 with sterilized water ×
107Individual/ml.
Step (5) SPAD values determination methods：Determine the SPAD values of 1 leaf using SPAD-502 chlorophyll meters quick nondestructive,
2 points are respectively surveyed per rib both sides, and calculate its average value.
Verticillium wilt coerces index calculation method：
Verticillium wilt stress index (VDSI)=(be not inoculated with verticillium wilt strain cotton seedling fall 1 leaf SPAD values-is inoculated with verticillium wilt strain
Cotton seedling falls 1 leaf SPAD values)/be not inoculated with verticillium wilt strain cotton seedling and fall 1 leaf SPAD value × 100.
As control, 7d (sense verticillium wilt check variety disease index reaches 50 or so) starts Disease investigation simultaneously after inoculation
Relative disease index is calculated, the disease resistance of kind is judged according to relative disease index.The incidence investigates standard, with reference to Zhu
The seedling stage grade scale of lotus qin：
0 grade：Plant health, disease-free leaf, growth are normal.
1 grade：Complete stool 1-2 pieces cotyledon shows symptom, cotyledons turn yellow, and true leaf does not show disease.
2 grades：Cotyledon and 1 true leaf performance symptom.
3 grades：2 true leaves show symptom.
4 grades：Whole blades show symptom, leaf abscission when serious, it is withered to push up the heart.
Disease index (DI)=∑ (corresponding series × corresponding strain number)/(4 × total strain number) × 100
With respect to the disease index of disease index (RDI)=susceptible check variety in disease index × 50/.
The measurement of SPAD values is convenient and swift in technical solution of the present invention, and SPAD values have been reacted crop leaf chlorophyll and contained relatively
Measure, under identical soil property, extremely significant correlation is presented in the change of cotton verticillium wilt occurrence degree and cotton leaf SPAD values.Cause
This, can utilize the resistance of the change Rapid identification cotton verticillium wilt of cotton leaf SPAD values.
Beneficial effects of the present invention：
The present invention, using verticillium wilt pathogen spore suspension root-pouring method, ensure that cotton on the premise of cotton root system is not damaged
The uniformity of flower inoculation.The culture of cotton seedling carries out in growth cabinet, needed for artificial regulatory cotton verticillium wilt occurs
Temperature and humidity, hence it is evident that shorten verticillium wilt and the time used occur.Utilize SPAD-502 chlorophyll meters measure cotton leaf SPAD
Value, measure is rapid, can determine within 2 seconds 1 SPAD value, can complete within 10 seconds the measure of 1 cotton leaf SPAD value, neither damage cotton
Leaf, also do not disturbed by artificial subjective judgement factor, measurement result is relatively reliable compared with disease index method, relatively determines cotton seedling verticillium wilt
The activity of relevant enzyme is simpler and more direct, and testing index is unique.The detection method can quick nondestructive check that each growing stage cotton yellow withers
The disease resistance of disease, accelerate verticillium wilt resistance of cotton by same breed breeding process, directly can be applicable in production.
Embodiment
With reference to instantiation, the invention will be further described.
1st, material to be tested
Middle plant cotton No. 2, nasal mucus cotton No. 3, Xu cotton No. 21, nasal mucus are anti-No. 1, Shandong cotton grind No. 28, Shandong cotton grind No. 36, (sense of Ji cotton No. 11
Verticillium wilt check variety), agricultural university 601.
2nd, implementation steps
(1) above-mentioned full cotton seed is selected, with 70% (v/v) alcohol-pickled 5min, 5% (w/w) H2O2Soak 2h,
Aseptic water washing 2~3 times, then with sterilized water Steeping and budding 10h.
(2) the above-mentioned cotton seed handled well is laid on the gauze of moistening, is positioned in controllable growth cabinet, treats cotton seed
After showing money or valuables one carries unintentionally, the basically identical seed of root long, seed size is selected, sow in seedling-raising cup (length × width × height=8cm × 8cm ×
15cm), 4 are sowed for every glass, per totally 40 glasss of kind.Seedling medium is the vermiculite that high-pressure sterilizing pot sterilized, and nutrient solution is sterile
Water.The whole process of the cotton seedling incubation is carried out in growth cabinet, and photostage, temperature is 24 DEG C, and light application time is
14h, intensity of illumination 20000Lux；Dark phase, temperature are 20 DEG C, interlunation 10h.Whole humidity is 85%.It is described
The sterilization method of vermiculite：High-pressure sterilizing pot sterilizes, and temperature is 121 DEG C, sterilization time 1h.
(3) when cotton seedling cotyledon is open and flat, 1 plant consistent of cotton seedling of growing way, nutrient solution hoagland ' s are selected and remain for every glass with kind
Nutrient solution, change 1 time weekly.
(4) when the leaf of cotton seedling the 2nd is open and flat, every glass is drawn verticillium wilt bacterium solution 15ml injection cotton seedling rhizosphere leech with injector for medical purpose
Shi Zhong, verticillium wilt bacterium solution spore concentration are 2 × 107Individual/ml, strain are High pathogenicity strain V08DF1, are inoculated with 20 glasss altogether；To connect
Kind 15ml sterilized waters, which are used as, to be compareed, totally 20 glasss.
The preparation method of the verticillium wilt pathogen spore suspension：By the verticillium wilt strain of (- 80 DEG C) preservations of ultra low temperature freezer
V08DF1 (derives from Plant Protection Inst., Jiangsu Academy of Agriculture, the strain is conventional bacterial classification, and those skilled in the art are equal
Can be obtained by conventional meanses) at 25 DEG C on CheckShi solid mediums activation culture 10d, it is then a little from colony edge picking
Fungus block, it is transferred in the triangular flask for the CheckShi fluid nutrient mediums for filling 300ml, training 10d is shaken in constant temperature (25 DEG C) shaking table, with 4 layers
Filtered through gauze bacterium solution, blood counting chamber calculates spore concentration, then the concentration of spore suspension is adjusted into 2 × 10 with sterilized water7
Individual/ml.
(5) 3d after being inoculated with, determine each cotton plant and fall the SPAD values of 1 leaf, and calculate verticillium wilt and coerce index；After inoculation
7d starts incidence and investigates and calculate with respect to disease index, and the disease resistance conduct pair of kind is judged according to relative disease index
According to.
SPAD values determination methods are：Determine the SPAD values of 1 leaf using SPAD-502 chlorophyll meters quick nondestructive, per leaf master
2 points are respectively surveyed in arteries and veins both sides, and calculate its average value.
Verticillium wilt coerces index calculation method：Verticillium wilt stress index (VDSI)=(be not inoculated with verticillium wilt strain cotton seedling and fall 1
Leaf SPAD values-be inoculated with verticillium wilt strain cotton seedling fall 1 leaf SPAD values)/are not inoculated with verticillium wilt strain cotton seedling and fall 1 leaf SPAD value × 100.
The incidence investigates standard, with reference to Zhu Heqin seedling stage grade scale：
0 grade：Plant health, disease-free leaf, growth are normal.
1 grade：Complete stool 1-2 pieces cotyledon shows symptom, cotyledons turn yellow, and true leaf does not show disease.
2 grades：Cotyledon and 1 true leaf performance symptom.
3 grades：2 true leaves show symptom.
4 grades：Whole blades show symptom, leaf abscission when serious, it is withered to push up the heart.
Disease index (DI)=∑ (corresponding series × corresponding strain number)/(4 × total strain number) × 100
With respect to the disease index of disease index (RDI)=susceptible check variety in disease index × 50/.
(6) index is coerced according to verticillium wilt and judges the disease resistance of each tested variety, and product are judged according to relative disease index
The disease resistance of kind is shown in Table 1 as control, disease resistance criterion.
The criterion of the resistance to verticillium wilt is：
Verticillium wilt stress index≤1.3 are immune material；
1.3 ＜ verticillium wilt stress index≤4.0 are high anti-material；
4.0 ＜ verticillium wilt stress index≤6.6 are disease-resistant material；
6.6 ＜ verticillium wilt stress index≤10.6 are resistance to sick material；
Verticillium wilt stress index ＞ 10.6 is susceptible material.
3rd, the SPAD values of this example measure, verticillium wilt stress index and relative disease index are shown in Table 2.
Table 2 is respectively participated in the experiment material seedling stage SPAD values, verticillium wilt stress index and relative disease index
Note：Ji cotton No. 11 is sense verticillium wilt check variety.
4th, index is coerced according to verticillium wilt and relative each tested variety resistance to verticillium wilt of state of an illness exponent pair is identified respectively, as a result
Completely the same (table 3).
The test material resistance to verticillium wilt result of table 3
Note：S：It is susceptible, T：Resistance to disease, R：It is disease-resistant.
The detection method can quick nondestructive check the disease resistance of each growing stage cotton verticillium wilt, accelerate verticillium wilt resistance of cotton by same
Breed breeding process, directly it can be applicable in production.
Claims (6)
- A kind of 1. method of seedling stage Rapid identification cotton verticillium wilt resistance, it is characterised in that：Verticillium wilt pathogen is seeded to cotton seedling, surveyed The SPAD values of cotton leaf and verticillium wilt stress index is calculated after fixed inoculation, cotton verticillium wilt is then judged according to verticillium wilt stress index Resistance.
- 2. according to the method for claim 1, it is characterised in that：The condition of culture of the cotton seedling is：Photostage, temperature 24 DEG C, light application time 14h, intensity of illumination 20000Lux；Dark phase, temperature are 20 DEG C, interlunation 10h, whole wet Spend for 85%.
- 3. according to the method for claim 1, it is characterised in that：The method that verticillium wilt pathogen is seeded to cotton seedling is：The leaf of cotton 2 Phase, every cotton seedling inject verticillium wilt bacterium solution into rhizosphere vermiculite, and strain is High pathogenicity strain V08DF1.
- 4. according to the method for claim 3, it is characterised in that：The spore concentration of the verticillium wilt bacterium solution is not less than 2 × 107 Individual/m, injection rate are 10~20ml.
- 5. according to the method for claim 1, it is characterised in that：After cotton seedling inoculation verticillium wilt strain 3d, determine 1 leaf SPAD values, calculate verticillium wilt stress index；Verticillium wilt is coerced index=(be not inoculated with verticillium wilt strain cotton seedling fall 1 leaf SPAD values-and connect Kind of verticillium wilt strain cotton seedling falls 1 leaf SPAD values)/be not inoculated with verticillium wilt strain cotton seedling and fall 1 leaf SPAD value × 100.
- 6. according to the method for claim 1, it is characterised in that：The criterion of the resistance to verticillium wilt is：Verticillium wilt stress index≤1.3 are immune material；1.3 ＜ verticillium wilt stress index≤4.0 are high anti-material；4.0 ＜ verticillium wilt stress index≤6.6 are disease-resistant material；6.6 ＜ verticillium wilt stress index≤10.6 are resistance to sick material；Verticillium wilt stress index ＞ 10.6 is susceptible material.
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| CN112630184A (en) * | 2020-12-10 | 2021-04-09 | 新疆农业大学 | Method for identifying phenotype of cotton verticillium wilt disease |
| CN113670862A (en) * | 2021-08-11 | 2021-11-19 | 新疆农业科学院经济作物研究所 | Method for rapidly screening verticillium wilt-resistant cotton varieties |
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| CN113670862A (en) * | 2021-08-11 | 2021-11-19 | 新疆农业科学院经济作物研究所 | Method for rapidly screening verticillium wilt-resistant cotton varieties |
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