CN102732596A - Cotton verticillium wilt resistant seedling stage identification method - Google Patents
Cotton verticillium wilt resistant seedling stage identification method Download PDFInfo
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Abstract
The invention belongs to the technical field of cotton disease resistance breeding and specifically relates to a cotton verticillium wilt resistant seedling stage identification method. The method provided by the invention comprises the following main steps of: firstly carrying out shaking culture on cotton verticillium wilt germ 6SY2-37 collected in China Center for Type Culture Collection with the collection number being CCTCC NO: M2011095 in a Czapek's liquid medium at the temperature of 25 DEG C for 5d, filtering by the use of gauze, counting by the use of a blood cell counting panel, preparing 5*106 spore/ml, pulling up seedling with a matrix when the water-planting cotton seedling grows to a stage of two leaves and one core, cutting 1cm of water root, dipping the root for 10s and inoculating, embedding wet soil into a plastic earthen bowl, inoculating pathogen, immediately watering to prevent the cotton seedling from fading, inoculating for 10 days and causing the disease. The method provided by the invention has advantages of high survival rate of the cotton seedling after inoculation, uniform inoculated pathogen, rapid morbidity, convenient operation and good stability of results. In addition, the method takes short time, requires short seedling growing time and is convenient to manage. Therefore, the method is suitable for cotton verticillium wilt resistance identification and pathogen pathogenicity differentiation determination of large-batch materials.
Description
Technical field
The invention belongs to the cotton disease resistance breeding technical field; Be specifically related to a kind of sprout period authenticating method of verticillium wilt resistance of cotton by same; The present invention relates to a kind of resistance of utilizing the seedling dish to transplant seedlings to dip in the root method to identify the cotton in seedling stage verticillium, the early stage Rapid identification of resistant material is provided for the crop disease-resistant breeding.
Background technology
Cotton verticillium wilt (Verticillium dahliae.Kleb.) is a Ragnarok property soil-borne vascular bundle disease; Have a strong impact on the yield and quality of cotton; Can infect multiple draft crop, tree crop, flowers and woody ornamentais, like strawberry, tomato, peppermint, olive, capsicum, rape, eggplant, potato, Sunflower Receptacle, chrysanthemum, rose, maple etc.
Chinese scholars generally believes, seed selection and to utilize disease-resistant variety be control cotton verticillium wilt economy and valid approach.It is breeding for disease resistance and the requisite link of disease-resistant material screening that verticillium wilt resistance of cotton by same is identified.Therefore screen antigen-like material, supply breeding work person choose reasonable parent, and then select the stable kind of resistance, supply to produce and go up that using seems presses for.The screening in anti-source, the evaluation of resistance that need carry out large-scale germplasm resource for cotton is the basis, thereby is badly in need of setting up that a kind of easy, virulence is stable fast, standard, practical disease resistance authenticate technology.
Verticillium wilt resistance of cotton by same identifies it mainly is to identify and evaluation in indoor seedling stage through the sick garden of manual work at present.Artificial sick garden identifies and is considered to actual near naturally that the most reliable disease-resistant authentication method is used the most general in the breeding for disease resistance process.The disease garden limits but this authentication method is identified, lasts oversizely, is subject to the influence of weather condition, and has stronger seasonality.The cotton in seedling stage authentication method is easy, rapid, control external environmental condition easily.Commonly used has four kinds, comprises needle punching (Gu Benkang, Li Jingyi, the real pin inoculation test [J] of verticillium dahliae, Jiangsu agricultural sciences; 1984,47.), paper pot tears end method (Shi Leiyan etc., cotton verticillium wilt sprout period authenticating method, plant protection, 1987 (1):; (1): 42.), the no end mould alms bowl bacterium liquid water the root method (simple osmanthus is good, Sun Wenji, horse is deposited, mould alms bowl bacterium liquid and water the root method at cotton verticillium wilt resistance new Identification Method-no end, the cotton journal; 2001,13 (2): 67-69), six rib polypotss quantitatively annotate bacterium liquid method (Ma Zhiying, Wang Shengfen, Zhang Guiyin; Deng. the research of Hebei province's cotton verticillium wilt bacteria pathogenic, cotton journal, 1997, (9) 1:15-20.).Wherein needle punching has the characteristics of compulsory vacination, can accurately not react the resistance of cotton seedling; Paper pot tears the end to dip in the root method is the seventies in age to the eighties greenhouse sprout period authenticating method of confirming through experimental study for many years, have advantages such as quick, accurate, easy to operate, but it is time-consuming to make paper pot, and when growing seedlings, pollutes easily.Mould alms bowl bacterium liquid and water Gen Fa and six rib polypotss quantitatively to annotate the seedling raise period of bacterium liquid method long at the no end, and it is inhomogeneous to hinder root, and qualification time is long.Four kinds of authentication methods all have certain shortcoming, this be cause seedling stage qualification result and artificial sick garden identify and have one of reason of significant difference.Set up authentication method fast and accurately,, seem very necessary to satisfy the needs that current cotton verticillium wilt fundamental research and disease resistance are identified.
Summary of the invention
The object of the invention is providing the inoculation method that a kind of qualification result is stable, workable, convenient, the suitable material in enormous quantities of evaluation carries out cotton verticillium wilt resistance evaluation fast.
Technical scheme of the present invention is following:
A kind of sprout period authenticating method of verticillium wilt resistance of cotton by same, its characteristic comprises the steps:
1) presoaking and germinating, adopting porous polyethylene foam seedling pan is carrier, is culture medium with peat, the peat composed of rotten mosses and vermiculite, each seedling-growing container is joined the 5L nutritive medium; Every 5L water adds 250ml basis nutritive medium, adds 25ml induced liquid mother liquor again, carries out under natural lighting that cotton water is floating grows seedlings, and incubation time is 28-32d, and is subsequent use;
2) the refrigerator pilot scale is guaranteed that the preserving number of depositing is that the big beautiful Verticillium 6SY2-37 of CCTCC NO:M2011095 is transferred in the petridish that fills the PDA substratum; Afterwards petridish is placed on and carries out activation in the thermostat container; Activation temperature is 25 ℃; Soak time is 10-15d, gets the bacterium piece of the about 4mm of a diameter and puts into the triangular flask that fills 50ml Czapek ' s substratum, in 25 ℃ of following shaking culture 5-6d;
3) with step 2) the germ culture filter with double gauze, add up spore count at microscopically with blood counting chamber, with sterilized water spore is transferred to 5 * 10
6The spore suspension of individual/ml treats that cotton seedling length extracts from seedling pan together with matrix wholeheartedly the time to two leaves, with clean thieving paper the moisture on the water root is blotted, and cuts off water root tip of a root 1cm with scissors then and hinders root and handle; All to immerse preserving number be CCTCC NO:M2011095 with hindering water root behind the root, and concentration is 5 * 10
6Take out immediately behind the 10s in the big beautiful Verticillium spore suspension of individual/ml, matrix is scattered, and matrix is not dipped in bacterium;
4) in polypots, spread the thick moistening sterile soil of 2.5-3.5cm in advance; Step 3) is dipped in cotton transplantation of seedlings behind the bacterium in polypots, and an alms bowl one seedling covers with moistening sterile soil again; Overburden depth is for leaving alms bowl edge 3-4cm place; Cover the exsiccant sterile soil more afterwards in case growing of other assorted bacterium places the greenhouse with polypots, culture temperature is 25-28 ℃; Water a spot of water behind the 1h, prevent here cotton plant from withering, observe the incidence of verticillium behind the 10d;
Wherein:
The described basic nutrient solution prescription of step 1) is following: saltpetre 38g/L, an ammonium nitrate 33g/L, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 7.4g/L, potassium primary phosphate 3.4g/L, calcium chloride 8.8g/L; Replenish zero(ppm) water to 1L;
The described induced liquid mother liquor of step 1) prescription is following: boric acid 1.24g/L, manganous sulfate 4.46g/L, zinc sulfate 1.7g/L, Sodium orthomolybdate 0.05g/L, copper sulfate 0.005g/L, NSC 51149 0.005g/L, ferrous sulfate 5.56g/L, EDTA Disodium 7.46g/L; Replenish zero(ppm) water to 1L;
Step 2) described Czapek ' s culture medium prescription is following: FeSO
40.02g/L, NaNO
32g/L, MgSO
41g/L, KCl1g/L, KH
2PO
41g/L, sucrose 30g/L; PH6.0; Replenish zero(ppm) water to 1L.
The applicant infects cotton verticillium wilt from the Shayang County, Hubei Province cotton plant separation obtains bacterial strain-big beautiful Verticillium (Verticillium dahliae) 6SY2-37 that a strain is exclusively used in the evaluation in seedling stage of verticillium wilt resistance of cotton by same; Deliver the Chinese typical culture collection center preservation in the Wuhan University of Wuhan City, Hubei Province on March 25th, 2011, its preserving number is CCTCCNO:M2011095.
Advantage of the present invention and beneficial effect are:
(1) postvaccinal cotton seedling morbidity is fallen ill significantly rapidly, can effectively distinguish the anti-sense type of different cotton varieties to germ, can also effectively distinguish the bacterial strain of the different bions of causing a disease
(2) hinder root, dip in bacterium one by one and carry out disease-resistant evaluation through the same one by one position of cotton seedling, it is consistent to have guaranteed to hinder the root degree, and it is consistent to connect the bacterium amount, and the inoculation sequela is even, as a result good stability.It dips in the advantage that the bacterium method compares and is with common naked; Common naked is dipped in the soil that needs the flush away root when bacterium method connects bacterium, because root system winding each other in soil is deep-rooted; In the flush away root soil, will damage root system inevitably like this, hinder the root degree and be difficult to accomplish unified.In addition, common naked cotton seedling seedling-slowing stage that dips in after the bacterium method connects bacterium and transplants probably needs the time in a week, and cotyledon withers easily and here come off, and state of an illness investigation afterwards is easy to generate error.The seedling dish cotton seedling that dips in the root method of transplanting seedlings is that water root is dipped in bacterium, carries matrix during transplanting, has kept the original appearance of root system basically, and transplanting survival rate is high, and seedling-slowing stage is short, is no more than 1d at most, here withering of cotyledon seldom occur, has guaranteed the accuracy of state of an illness investigation.
(3) inoculation method of the present invention is fast and convenient, standard is practical, and preliminary screening, the pathogenic bacteria virulence that can be used for evaluation of cotton variety disease resistance and disease-resistant material measured, the pathogenic bacteria physiological strain is differentiated.
More detailed technical scheme sees that " embodiment " is said.
Description of drawings
Fig. 1. cotton seedling incidence under the different spore liquid concentration conditions.What this figure reflected is to connect bacteria concentration gradient test-results.
Can know by Fig. 1, connect bacteria concentration 5 * 10
5Under individual/ml condition, the disease of Hubei Province cotton 24, DP410B and Yin Rui 361 refers to all very low, does not reach disease-resistant evaluation requirement.When connecing bacteria concentration is 1 * 10
6Individual/during ml, the disease of Hubei Province cotton 24 and DP410B refers to there is not significant difference, is respectively 30.6 and 30.2, silver-colored auspicious 361 disease refers to be 14.9.Susceptible contrast disease refers to also lower, can not reflect the true resistance of each kind well.When connecing bacteria concentration is 5 * 10
6Individual/during ml, the disease in Hubei Province cotton 24 refers to be 50.5, the disease of DP410B and Yin Rui 361 refers to be respectively 49.5 and 27.5.The disease of Hubei Province cotton 24 and DP410B refers to also indifference, and DP410B shows as susceptible, and silver auspicious 361 shows as anti-disease.When spore liquid concentration is 1 * 10
7Individual/ml and 5 * 10
7Individual/during ml, it is similar that Hubei Province cotton 24 and the disease of DP410B refer to, and the disease of each kind refers to all very big, all shows as susceptible.Thus it is clear that, 1 * 10
7Individual/ml and 5 * 10
7Under individual/ml concentration, bacterial concentration is excessive, should not be used for disease-resistant evaluation, therefore, and 5 * 10
6The big beautiful Verticillium spore liquid concentration of individual/ml is more suitable.
Fig. 2. difference is hindered the cotton seedling incidence of method for root.
Can be known that by Fig. 2 the disease of hindering each kind of water root 1cm refers to refer to a little less than the disease of hindering water root 1/2 each kind, but difference is not obvious, can both distinguishes anti-sense kind preferably, this shows to hinder water root 1cm and hinder water root 1/2 all can.The author's suggestion is selected for use and is hindered water root 1cm.When excising all water roots, the disease of each kind refers to all very big, all shows as susceptiblely, and anti-sense kind is distinguished not obvious.It is overweight to explain that this kind hindered root, should not be used for disease-resistant evaluation.
Fig. 3. each kind disease refers to connecing fate changing conditions behind the bacterium.
This inoculation method morbidity is rapid, so the state of an illness that assurance suits investigation is most important period.What Fig. 3 reflected is to hinder water root 1/2, spore concentration 5 * 10
6The disease of Hubei Province cotton 24 and Yin Rui 361 refers to connecing fate changing conditions behind the bacterium under individual/ml condition.After generally meeting bacterium 7d, each kind cotyledon manifests the verticillium symptom successively.The period that the disease in susceptible contrast Hubei Province cotton 24 refers to reach 45-55 is the 13-16d after connecing bacterium greatly.Revision test repeatedly shows, suitable state of an illness investigation is the 13-16d that connects behind the bacterium period.Too early disease refers on the low sidely, surpasses this time limit disease susceptible and disease-resistant material and refers to all bigger than normally, does not reach identification result.
Embodiment
One, supplies the examination material
The used cotton variety of the present invention has: Hubei Province cotton 24 (the conventional cotton variety of Hubei Province's crop varietal approval committee, by the academy of agricultural sciences's seed selection of Huanggang City, Hubei Province and seed is provided, this kind is openly promoted, referring to network address:
Http:// baike.baidu.com/view/2089471.htm), DP410B (the conventional Insect Resistant Cotton of Hubei Province's crop varietal approval committee by the seed selection of the cotton company of U.S.'s Mount Tai word, is promoted in China, sees network address:
Http:// baike.baidu.com/view/4605450.html? Tp=0_10), silver auspicious 361 (the Insect Resistant Cotton conventional variety, Shandong Province's Dezhou City auspicious Cotton Research Institute of silver and Biological Technology institute, Chinese Academy of Agricultural Sciences's associating seed selection are applied in China, seed is provided by Cotton Inst., Chinese Agricultural Academy, sees network address:
Http:// www.foods1.com/content/409310/).Wherein Hubei Province cotton 24 performance on producing is susceptible, and DP410B, silver auspicious 361 have certain disease resistance on producing.
Used big beautiful Verticillium is 6SY2-37, the defoliation bacterial strain of the strong virulence that is provided by Hua Zhong Agriculture University plant pathology laboratory.Gather the cotton verticillium wilt diseased plant from Hubei Province, Shayang County, Hubei Province assorted cotton No. 10 in October, 2007, adopts mercuric chloride to separate with the alcohol surface sterilization method.With the diseased plant branch clean, segment, in Bechtop,, use 70% alcohol disinfecting 30s again, sterile water wash 3-5 time with sterile water wash 2-3 time.Then with 0.1% mercuric chloride sterilization 2min, with sterile water wash 3-5 time, be cut into small pieces with scissors and put into the PDA substratum, 25 ℃ of constant temperature culture 3-4d carry out pathogen identification, the bacterial classification preservation is subsequent use.
Adopt dilution-plate method that the bacterial strain of screening is carried out the monospore separation.On the PDA flat board of cultivating 15d, with the aseptic washing spore of 2-3mL, process spore suspension, after 5 layers of lens wiping paper filter mycelia, observe with blood cell counting plate at microscopically, be 4 * 10 with the bacterial concentration dilution
6Individual/ml, get 0.1ml and insert on the water agar medium flat board, evenly scatter; The petridish bottom surface is divided into lattice, puts in the 24-25 ℃ of incubator microscopy behind the 12-24h, chooses the marked of having only single spore in the grid; By substratum in the aseptic technique picking grid, move on the PDA flat board then, the bacterium colony that grows is the monospore fungus strain; Move into the test tube slant, preserve subsequent use.According to the cultivation proterties of pathogenic bacteria bacterium colony, morphological specificity, and a big beautiful Verticillium conidiophore wheel shape; Every 3～4 branches of having taken turns contain the characteristics such as germ nuclear of black, and strain separated is identified.Strain identification work is with reference to Wei Jingchao, " fungi identification handbook ", Shanghai science tech publishing house, the method for 1979 editions introductions.
The biological property of big beautiful Verticillium 6SY2-37:
The long oval of conidium, unicellular few separation, size is 2.3～9.1 * 1.5～3.0um.The conidiophore wheel is dendritic, every 3～4 branches of having taken turns, and total length 110～130um, branch size 13.8～21.4 * 2.3～2.7um, metulae portion water white transparency, colony growth achromobacter filament, growth long period mycelia browning look has diaphragm diameter 2～4um.Mycelium often is the expansion shape, can form sclerotium by single mycelia or several mycelia budding.Quantity, size and proterties that different areas verticillium dahliae germ karyomorphism becomes have evident difference.
The big beautiful Verticillium 6SY2-37 of separation screening is delivered Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on March 25th, 2011, and its preserving number is CCTCC NO:M2011095.
Two, cotton water is floating grows seedlings
The floating seeding raising technology of cotton water with reference to reported method such as Chen Jinxiang (Chen Jinxiang etc. cotton floating seedlings technology [J]. Chinese cotton; 2006; 33 (11): 24-25.) adopting porous polyethylene foam seedling pan is carrier, and (mixture of peat, the peat composed of rotten mosses and vermiculite is counted the peat composed of rotten mosses according to weight ratio: peat: vermiculite=1: 1: 1 with mixed substrate; PH 6-7) for supporting; Add in each seedling-growing container with every 5L water and add 250ml basis nutritive medium, the nutritive medium water body 5L of ratio preparation that adds 25ml induced liquid mother liquor again carries out floating seedlings as the seedbed.Good cotton seed seedling rate is reached more than 90%, and the cotton shoot root system that breeds is flourishing, and vitality is strong, the growth neat and consistent, less or anosis, no weeds.Seedling pan, matrix are available from Jingzhou City agricultural rice seedling on watered bed equipment of profit ltd.The present invention is divided into two into two little seedling pans (12 vertical rows, 8 horizontally-arrangeds), hole, 12 caves of horizontally-arranged, vertical hole, 8 caves, the hole, totally 96 caves of arranging with seedling pan from the centre.The length of seedling-growing container is respectively 50cm, 35cm, 15cm.
1, presprouting of seeds
Select full seed, sophisticated foregoing cotton seeds, use vitriol oil lint.With 50 ℃ of water seed soakings, and add 50% carbendazol wettable powder (source producer: Shandong Province Qufu City Er Fu insecticide factory) to concentration be 5mg/ml, soaking the bacterium time is 10h, and seed is fully absorbed water.After seed fully absorbs water, use the flushing with clean water seed, drain redundant moisture again, be placed on the moistening double gauze, and then cover seed with moistening double gauze, be placed at last in the thermostat container, temperature is set 30 ℃, breaks chest or exposes the tip of a root until seed.
2, medium in tray
Culture medium (source producer: the sharp agricultural rice seedling on watered bed equipment in Jingzhou City ltd, composition is the peat composed of rotten mosses: peat: vermiculite=1: 1: 1, pH 6-7.) experiment is carried out before with pressure kettle 1 hour (121 ℃ of temperature, the pressure 0.6Mpa) that sterilize.Earlier that substrate with water is moistening, make water content of substrate reach 60% of own wt be advisable (it is agglomerating to be that hand is pinched, and loses flowing water between finger, and following backwardness nature scatters).Again moistening matrix is packed in the hole, cave of seedling pan, shake seedling pan up and down and make the matrix compacting in the hole, cave.Mesostroma surface, hole, cave separation disc face 1.0-1.5cm.
3, sowing
Select broken chest or root length to be equivalent to the cotton seed sowing of half granule seed length, hole, every cave is broadcast 1, and the tip of a root is broadcast the centre in the hole, cave down.Finish the back and cover and compacting with matrix, matrix is equal with the hole surface of growing seedlings.
4, the preparation of nutritive medium
Every basin is joined the 5L nutritive medium.Every 5L water adds 250ml basis nutritive medium, adds 25ml induced liquid mother liquor again.The basis nutrient solution prescription: saltpetre 38g/L, an ammonium nitrate 33g/L, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 7.4g/L, potassium primary phosphate 3.4g/L, calcium chloride 8.8g/L replenishes zero(ppm) water to 1L.Induced liquid mother liquor prescription is following: boric acid 1.24g/L, manganous sulfate 4.46g/L, zinc sulfate 1.7g/L, Sodium orthomolybdate 0.05g/L, copper sulfate 0.005g/L, NSC 51149 0.005g/L, ferrous sulfate 5.56g/L, EDTA Disodium 7.46g/L; Replenish zero(ppm) water to 1L.
5, seedbed management
Treat that the seedling dish is emerged and put into the nutritive medium of seedling-growing container after neat, grow to a leaf wholeheartedly or two leaves wholeheartedly (different) according to test until cotton seedling.Take the circumstances into consideration to spray mepiquat chloride to prevent high pin seedling according to cotton seedling growing way.
Three, cotton seedling is cultivated
Adopt the floating seeding raising technology of water to carry out the cultivation of cotton seedling.Various 2 dishes of 3 used cotton varieties.
Four, the preparation of germ cultivation and spore liquid
With the big beautiful Verticillium 6SY2-37 of cotton activation on the PDA substratum, 25 ℃ of activation temperatures, behind the dark growth 7-10d cut-off directly the bacterium piece of about 4mm put into the triangular flask that fills 50ml Czapek ' s substratum, 25 ℃ of following shaking culture 5-6d.Then, the germ culture is filtered with double gauze.Microscopically is added up spore count with blood counting chamber, dilutes and is mixed with required spore concentration.Used Czapek ' s culture medium prescription is: 1L H
2O, 0.02g FeSO
4, 2g NaNO
3, 1g MgSO
4, 1g KCl, 1g KH
2PO
4, 30g sucrose, pH=6.0.
Five, inoculation method
Treat that cotton seedling length connects matrix to two leaves wholeheartedly the time and from seedling pan, extracts lightly together, the moisture on the water root is blotted, do and hinder root and handle with the contracting current tip of a root 1cm of taking root then with clean thieving paper.Afterwards water root is all immersed and taken out (preferably do not allow matrix to be scattered, matrix is not dipped in bacterium) in the spore suspension in (10s) immediately, with being about to cotton transplantation of seedlings in polypots, an alms bowl one seedling.The prior thick moistening sterile soil in the 3cm left and right sides, shop in the polypots covers with moistening sterile soil after cotton seedling behind the bacterium is put into alms bowl dipping in again, and overburden depth is for leaving alms bowl edge 3-4cm place, covers the exsiccant sterile soil more afterwards in case the growing of other assorted bacterium.Be put into the greenhouse, temperature is controlled between 25-28 ℃.Water a spot of water after 1 hour, prevent here cotton plant from withering.Three repetitions.Observe incidence behind the 10d.
Six, incidence investigation and statistics
When treating that susceptible variety is generally fallen ill, press the morbidity grade of each experimental cultivar of table 1 standard record.The morbidity grade is divided into 0,1,2,3,4 five grade.According to morbidity rating calculation disease index, judge the resistance of each kind according to the disease index that is calculated again afterwards then to verticillium.Verticillium wilt resistance of cotton by same resistance grade scale is seen table 2.
The cotton seedling diseases mutual affection of table 1 grade standard
The disease index calculation formula is following:
Disease index=[(∑ diseased plant numbers at different levels * corresponding sick level)/(total strain number * the highest sick level)] * 100
Table 2 verticillium wilt resistance of cotton by same grade scale (national cotton is withered, verticillium integrated control research cooperative groups, 1984)
(annotate: I: immunity; HR: high anti-; R: anti-; T: anti-disease; S: susceptible)
Seven, connect the test of bacteria concentration gradient
Spore concentration is established 5 gradients altogether: 5 * 10
5, 1 * 10
6, 5 * 10
6, 1 * 10
7With 5 * 10
7Individual/ml, establish 3 repetitions altogether.Test-results shows 5 * 10
6Individual/ml is that the best connects bacteria concentration.5 * 10
6Individual/ml inoculation sequela is rapid, can distinguish the resistance to verticillium wilt of different anti-sense kinds simultaneously again.
Eight, cotton seedling is hindered the test of root degree
Connect bacterium spore concentration 5 * 10
6Individual/ml, establish 3 altogether and hinder root and handle: (1) the is dried up about 1cm of the tip of a root place of taking root; (2) 1/2 place of water root length overall; (3) cut off whole water roots.Establish 3 repetitions altogether.Test-results shows that (2) and (3) two handle no significant differences, and the disease of test material therefor refers to all bigger than normal, can not distinguish anti-sense kind well, and the disease that handle (1) refers to that size to fit can distinguish the anti-kind of feeling preferably.So more suitable at the about 1cm of the dried up tip of a root of taking root place, all cut root, cotton shoot survival percent is low, and 1/2 cuts root and 1cm cuts the root pathogenic process and does not have significant difference, so adopt the about 1cm of aquatic tip of a root place more suitable
Nine, the optimum state of an illness is investigated period
This method morbidity is rapid, so the state of an illness that assurance suits investigation is most important period.Go up according to this data presentation of two test gained, approximately meet the 14-16d behind the bacterium period that the disease in susceptible contrast Hubei Province cotton 24 refers to reach 45-55, revision test repeatedly confirms that optimum state of an illness investigation is the 14-16d that connects behind the bacterium period.Too early disease refers on the low sidely, surpasses this time limit disease susceptible and disease-resistant material and refers to all bigger than normally, does not reach identification result.
Ten, the beneficial features of present method
Postvaccinal cotton seedling survival rate is high among the present invention, and it is even to connect bacterium, and morbidity rapidly, and is easy to operate, as a result good stability.This method weak point consuming time in addition; The seedling raise period section; Convenient management; Therefore be fit to large batch of material and carry out verticillium wilt resistance of cotton by same and identify and pathogenic bacteria virulence differentiation assays, avoided mandatory shortcoming, the paper pot of needle punching to tear end method and be prone to pollute time-consuming shortcoming, the no end and mould alms bowl bacterium liquid and water the shortcoming that Gen Fa and six rib polypotss are quantitatively annotated bacterium liquid method seedling raise period length and disease time length.
Claims (3)
1. the sprout period authenticating method of a verticillium wilt resistance of cotton by same, its characteristic comprises the steps:
1) presoaking and germinating, adopting porous polyethylene foam seedling pan is carrier, is culture medium with peat, the peat composed of rotten mosses and vermiculite, each seedling-growing container is joined the 5L nutritive medium; Every 5L water adds 250ml basis nutritive medium, adds 25ml induced liquid mother liquor again, carries out under natural lighting that cotton water is floating grows seedlings, and incubation time is 28-32d, and is subsequent use;
2) the refrigerator pilot scale is guaranteed that the preserving number of depositing is that the big beautiful Verticillium 6SY2-37 of CCTCC NO:M2011095 is transferred in the petridish that fills the PDA substratum; Afterwards petridish is placed on and carries out activation in the thermostat container; Activation temperature is 25 ℃; Soak time is 10-15d, gets the bacterium piece of the about 4mm of a diameter and puts into the triangular flask that fills 50ml Czapek ' s substratum, in 25 ℃ of following shaking culture 5-6d;
3) with step 2) the germ culture filter with double gauze, add up spore count at microscopically with blood counting chamber, with sterilized water spore is transferred to 5 * 10
6The spore suspension of individual/ml treats that cotton seedling length extracts from seedling pan together with matrix wholeheartedly the time to two leaves, with clean thieving paper the moisture on the water root is blotted, and cuts off water root tip of a root 1cm with scissors then and hinders root and handle; All to immerse preserving number be CCTCC NO:M2011095 with hindering water root behind the root, and concentration is 5 * 10
6Take out immediately behind the 10s in the big beautiful Verticillium spore suspension of individual/ml, matrix is scattered, and matrix is not dipped in bacterium;
4) in polypots, spread the thick moistening sterile soil of 2.5-3.5cm in advance; Step 3) is dipped in cotton transplantation of seedlings behind the bacterium in polypots, and an alms bowl one seedling covers with moistening sterile soil again; Overburden depth is for leaving alms bowl edge 3-4cm place; Cover the exsiccant sterile soil more afterwards in case growing of other assorted bacterium places the greenhouse with polypots, culture temperature is 25-28 ℃; Water a spot of water behind the 1h, prevent here cotton plant from withering, observe the incidence of verticillium behind the 10d;
Wherein:
The described basic nutrient solution prescription of step 1) is following: saltpetre 38g/L, an ammonium nitrate 33g/L, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 7.4g/L, potassium primary phosphate 3.4g/L, calcium chloride 8.8g/L; Replenish zero(ppm) water to 1L;
The described induced liquid mother liquor of step 1) prescription is following: boric acid 1.24g/L, manganous sulfate 4.46g/L, zinc sulfate 1.7g/L, Sodium orthomolybdate 0.05g/L, copper sulfate 0.005g/L, NSC 51149 0.005g/L, ferrous sulfate 5.56g/L, EDTA Disodium 7.46g/L; Replenish zero(ppm) water to 1L;
Step 2) described Czapek ' s culture medium prescription is following: FeSO
40.02g/L, NaNO
32g/L, MgSO
41g/L, KCl1g/L, KH
2PO
41g/L, sucrose 30g/L; PH6.0; Replenish zero(ppm) water to 1L.
2. a strain is exclusively used in the big beautiful Verticillium 6SY2-37 of indoor identification cotton in seedling stage resisting verticillium, is deposited in Chinese typical culture collection center (CCTCC), and its preserving number is CCTCC NO:M2011095.
3. the described bacterial strain of claim 2 is in the cotton verticillium wilt application in identification.
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| CN102726177A (en) * | 2011-04-01 | 2012-10-17 | 华中农业大学 | Method for identifying cotton verticillium by using leaves in vitro |
| CN103355025A (en) * | 2013-06-27 | 2013-10-23 | 沈阳农业大学 | Method for rapidly identifying resistance to verticillium wilt of eggplant |
| CN105359855A (en) * | 2015-10-19 | 2016-03-02 | 中国农业科学院棉花研究所 | Improved verticillium wilt resistance identification method for cotton variety in artificial disease garden during whole growth period |
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| CN105359855A (en) * | 2015-10-19 | 2016-03-02 | 中国农业科学院棉花研究所 | Improved verticillium wilt resistance identification method for cotton variety in artificial disease garden during whole growth period |
| CN105409613A (en) * | 2015-12-05 | 2016-03-23 | 武汉市蔬菜科学研究所 | Seedling-stage root rot resistance identification method for vigna unguiculata |
| CN105925488A (en) * | 2016-05-16 | 2016-09-07 | 安徽省农业科学院园艺研究所 | Preparation method of strawberry verticillium dahlia spore suspension |
| CN106856953A (en) * | 2017-03-24 | 2017-06-20 | 合肥云都棉花有限公司 | One grows cotton cuttage root-taking matrix and its cottage method |
| CN107475350A (en) * | 2017-08-17 | 2017-12-15 | 南京农业大学 | A kind of method of seedling stage Rapid identification cotton verticillium wilt resistance |
| 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 |
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