CN102747135A - Early identification method for curvularia leaf spot in maize inbred lines - Google Patents
Early identification method for curvularia leaf spot in maize inbred lines Download PDFInfo
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Abstract
The invention belongs to the field of crop breeding for disease resistance and relates to an early identification method for curvularia leaf spot in maize inbred lines. The early identification method for curvularia leaf spot in maize inbred lines includes treating leaves of three-leaf seedlings of maize inbred lines in maize curvularia lunata toxin solution, and mixing with sodium borate buffer solution to extract enzyme crude extract of phenylalnine ammonialyase, peroxidase and superoxide dismutase in the leaves; measuring enzyme activities of the phenylalnine ammonialyase, the peroxidase and the superoxide dismutase, and calculating specific activity; calculating resistance values according to a formula, and calculating average of the resistance values. The average larger than 3 is the value of resistance of the inbred line to maize curvularia leaf spot. The method is accurate and reliable. The method can be used to identify and screen indoors without limitations of external environment conditions. In addition, the method has no season limitation in seedling-stage disease resistance identification, and shortens identification time. The method is high in work efficiency.
Description
Technical field
The invention belongs to the breeding for disease resistance field, be specifically related to the early stage authentication method of corn inbred line Curvularia lunata leaf spot resistance.
Background technology
Maize curvularia leaf spot (Corn Curvularia Leaf Spot Disease) is the bigger new disease of a kind of harm that takes place in the North China the eighties middle and later periods in 20th century, and the main harm blade also endangers leaf sheath and bract.Take out male back disease rapidly expansion spread, plant is covered with scab, blade is withered ahead of time, the general underproduction 20～30%, the serious plot underproduction is more than 50%, even total crop failure.
The pathogenic bacteria of maize curvularia leaf spot is crescent Curvularia lunata (Curvularia lunata (Wakker) Boed) and does not wait curved spore mould (Curvularia inaeguacis), belongs to the Deuteromycotina Curvularia.Control to the maize curvularia leaf spot mainly is methods such as breeding for disease resistance, tillage and cultivation, chemical agent, and wherein cultivating disease-resistant variety is most economical effective means.In the breeding of anti-maize curvularia leaf spot; Evaluation to its disease resistance mainly is to adopt artificial inoculation Curvularia lunata spore suspension; Utilize then disease indexs such as scab number, sporulation quantity carry out evaluation of resistance method (Wang Hong etc. Agricultural University Of Shenyang's journal, 2000,31 (5): 476-478; Zhao Laishun etc. Agricultural University Of Hebei's journal, 1995,18 (4): 36-38), this authentication method workload is bigger, and be subject to other diseases and region influence (Diao Yi etc. southern agriculture journal, 2011,42 (2): 161-163; The cold court of a feudal ruler is auspicious etc. modern agriculture science and technology, 2010, (17): 159-161,163); Other disease resistance authentication method is to adopt Isozyme Analysis, enzyme linked immunosorbent detection etc.; Though these methods are accurate; But complex steps; Cost is higher, is difficult to identify on a large scale and screen, therefore press for seek a kind of simple and reliable and can be in the method for indoor identification corn inbred line Curvularia lunata leaf spot resistance.
Crescent Curvularia lunata (Curvularia lunata (Wakker) Boed) toxin is the main virulence factor of maize curvularia leaf spot; It is the toxic substance that plays decisive action in the plant course of disease; In the generation of maize curvularia leaf spot disease, evolution, have tangible pathogenic effects (Li Fuhua etc. plant protection; 2004,30 (6): 5-10).After crescent Curvularia lunata toxin was handled, the maize leaf weave construction obviously came to harm, the cell wall structure fracture; Membrane structure breaks, and basal granule and stroma lamella discharge, and mitochondrial inclusion is cleared up; Vacuolation takes place, thus the normal photosynthesis of serious interference blade and respiration (Zhang Xinfang etc. corn science, 2009; 17 (6): 118-120,123).
The protective reaction of plant is complicated metabolic processes; Plant receives pathogen infect after; Resist, feel phenylalanine ammonia lyase (phenylalanine ammonia-lyase abbreviates PAL as), polyphenoloxidase (polyphenol oxidase abbreviates PPO as), px (horseradish peroxidase between the kind; Abbreviate POD as), superoxide-dismutase (superoxide dismutase; Abbreviate SOD as) and the activity change of other important enzyme notable difference (.Plant Physiology such as Shoresh, 2008,147:2147-2163 are arranged; .Proteomics such as Segarra, 2007,7 (21): 3943-3952; He Zuhua etc. plant physiology journal, 2001,27 (4): 281-290).After handling corn seedling with maize curvularia leaf spot toxin, the PAL of disease-resistant variety and POD are responsive than susceptible variety to maize curvularia leaf spot toxin, the enzyme peak time of occurrence of disease-resistant variety shift to an earlier date or peak value greater than susceptible variety; And maize curvularia leaf spot toxin can stimulate that susceptible variety SOD is active to be increased, the SOD that has suppressed disease-resistant variety active (Chen Jie etc. Plant Pathology, 2002,32 (1): 43-48).Therefore, after crescent Curvularia lunata toxin-induced was handled, the variation of enzyme activity also can be used as the evaluation of corn inbred line Curvularia lunata leaf spot resistance and the index of screening in the protective reaction.
Summary of the invention
The object of the present invention is to provide a kind of early stage authentication method of corn inbred line Curvularia lunata leaf spot resistance.Utilize this method to break season limit and to reduce the miscellaneous work that field resistance is identified, increase work efficiency in the evaluation and the screening of the anti-Curvularia lunata leaf spot of indoor completion corn inbred line.
The early stage authentication method of corn inbred line Curvularia lunata leaf spot resistance provided by the present invention, carry out according to following method:
(1) the corn inbred line seedling of 3 leaf phases is cut from the rhizome place, putting into concentration is 20～100 μ g/mL corn crescent Curvularia lunata toxin soiutionses, and the 200mmHg negative pressure of utilizing vacuum pump to produce is permeated 6～48h, in 25 ℃ of thermostat containers, leaves standstill 18h then;
(2) get the 3rd leaf (upper blade) of the corn inbred line seedling of handling through corn crescent Curvularia lunata toxin in the step (1), cut off blade tip, get the blade middle and upper part; Blot water with filter paper, take by weighing blade 1.0g and silica sand 0.1g, put into mortar; Add the sodium borate buffer liquid 2.0ml of pH 8.8,0.05mol/L, grind to form homogenate, homogenate is changed in the centrifuge tube; And, change centrifuge tube over to the lingering section on the sodium borate buffer liquid 2.0mL flushing mortar, stir 5min; Centrifugal 30min under 4 ℃, 12000g gets supernatant then; The 3ml supernatant is diluted to 15ml with pH 8.8,0.05mol/L sodium borate buffer liquid, and the supernatant that must dilute is preserved down for-4 ℃; Contain 5mmol/L mercaptoethanol and 1mmo1/L EDTA in the wherein said sodium borate buffer liquid; Described supernatant is the mixing crude extract of phenylalanine ammonia lyase, px and superoxide-dismutase;
(3) enzymic activity (U) of phenylalanine ammonia lyase (PAL), px (POD) and superoxide-dismutase (SOD) in the supernatant of the dilution of the middle gained of difference determination step (2); And calculate the ratio vigor of every kind of enzyme, than vigor calculation formula be:, calculate the resistance grade numerical value x of corn inbred line disease resistance more according to the following equation respectively than vigor=enzymic activity/protein content
1, x
2And x
3,
y
1=22.13x
1+ 35.17 (formula 1);
y
2=50.17x
2 2-136.9x
2+ 222.5 (formula 2);
y
3=-4.432x
3+ 38.04 (formula 3);
Y wherein
1Ratio vigor for phenylalanine ammonia lyase (PAL); y
2Ratio vigor for px (POD); Y wherein
3Ratio vigor for superoxide-dismutase (SOD);
(4) calculate x
1, x
2And x
3MV x, i.e. x=(x
1+ x
2+ x
3The corn inbred line of x>=3 is chosen in)/3, is the self-mating system of anti-maize curvularia leaf spot.
Corn crescent Curvularia lunata toxin soiutions described in the aforesaid method step (1); According to the preparation of following method (referring to Xiao Shuqin etc. the corn science; 2006; 14 (4): 138-140): with corn crescent Curvularia lunata strain culturing (moity of PDA substratum and weight ratio thereof are: contain yam 200g and glucose 15g in every 1L zero(ppm) water) on the PDA substratum; (Φ=0.7cm) on the bacterium colony of cultivating, punch, 6 crescent Curvularia lunata bacterium sheets are connected to 100mL improvement Fries liquid nutrient medium, and (its moity and ratio thereof are: contain sucrose 20g, tartrate ammonia 5g, NH in every 1L zero(ppm) water with punch tool after cultivating 7d
4NO
31g, KH
2PO
41g, sodium-chlor 0.1g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.5g, calcium chloride 0.13g and yeast extract paste 1g, pH6.2) in, cultivate shaking culture 10～15d down in 25 ℃, 120rpm; Filter the aforesaid liquid substratum with activated carbon column (4cm * 46.5cm, granulated active carbon 30g), flow velocity is 4.2mL/min, with 300mL methanol-eluted fractions charcoal post 3 times, and 100mL at every turn; Use the 100mL distilled water wash again after each methanol-eluted fractions, mix 3 times meoh eluate, in 60～70 ℃ of water-baths, be concentrated into about 10mL; Evaporate to dryness in 25～65 ℃ of thermostat containers obtains soup compound; The gained soup compound is dissolved in the 50mL temperature in 25～37 ℃ methyl alcohol; Filter, remove insolubles, get filtrating, the chloroform that adds 3 times of volumes mixes, and 25 ℃ of following hold over night, evaporate to dryness methyl alcohol, dissolved in chloroform layer obtain the tawny crystal, are the thick toxin of crescent Curvularia lunata; The adding distil water dissolving, processing concentration is 20～100 μ g/mL crescent Curvularia lunata toxin soiutionses.
The corn inbred line seedling of 3 leaf phases described in the aforesaid method step (1) obtains according to following method: select the seed of full self-mating system, through 0.1%HgCl
2Sterilization 10min, clean with distilled water flushing, in 25 ℃ of following imbibition 24h, transfer in the white disk that is lined with 6 layers of moistening filter paper, in 28 ℃/25 ℃ (daytime/night) the dark down 72h that sprouts; Be transferred to after the sprouting and continue in the illumination box to cultivate, culture condition is 28 ℃/25 ℃ (daytime/night), and intensity of illumination is 200mmol/m
2S, every day, light application time 12h cultivated for 2～3 weeks, got the corn inbred line seedling of 3 leaf phases.
The active mensuration of phenylalanine ammonia lyase (PAL) described in the aforesaid method step (3), according to Koukol etc. (.Journal of Biology Chemistry such as Koukol J, 1961,236:2692-2698) disclosed method is carried out.Getting the supernatant 1ml of the dilution of gained in the aforesaid method step (2), is substrate with the L-phenylalanine(Phe) then, measures the enzyme of phenylalanine ammonia lyase at the 290nm place and lives; Per hour to make OD
290The enzyme amount of increase by 0.01 is decided to be enzyme unit (U) alive, calculates the ratio vigor of phenylalanine ammonia lyase.
The active mensuration of px (POD) described in the aforesaid method step (3), according to guaiacol method (referring to Zhang Zhiliang etc. plant physiology experiment instructs [M]. Beijing: Higher Education Publishing House, 2003,123-124) carry out; Get the supernatant 1ml of the dilution of gained in the aforesaid method step (2), make substrate with methyl catechol (guaiacol) then, survey the OD value at the 470nm place; With every milligram of albumen OD of PM
470The activity of increased value expressed enzyme, the ratio vigor of calculating px.
The active mensuration of superoxide-dismutase (SOD) described in the aforesaid method step (3); Get the supernatant 0.1ml of dilution in the step (2); According to nitroblue tetrazolium(NBT) (NBT) photochemical reduction method measure the SOD vigor (referring to Wang Aiguo etc. plant physiology journal, 1983,9 (1): 77-84).Find out the enzyme liquid measure that suppresses NBT 50% photoreduction, be decided to be enzyme unit (U) alive, calculate the ratio vigor of superoxide-dismutase.
The measuring method of protein content described in the aforesaid method step (3): the supernatant of getting the dilution in the step (2); Utilize Coomassie brilliant blue G-250 micromethod measure its protein content (concrete grammar is referring to Zhang Zhiliang etc. plant physiology experiment instructs [M]. Beijing: Higher Education Publishing House; 2003,159-160).
Corn inbred line Curvularia lunata tikka characteristic of disease judgement criteria (referring to Wang Hong etc. Agricultural University Of Shenyang's journal, 2000,31 (5): 476-478; Zhao Laishun etc. Agricultural University Of Hebei's journal, 1995,18 (4): 36-38) for disease-resistant: the scab type is R or M, 1 grade of severity; In anti-: the scab type is R or M, 2 grades of severities; Middle sense: scab type S or M, 3 grades of severities; Susceptible: scab type S, 4 grades of severities.
PAL, POD and SOD described in the aforesaid method step (3) is than the establishment method of the regression equation of vigor and disease resistance: with typical disease-resistant maize self-mating system BC2433; In anti-corn inbred line 178; Middle sense corn inbred line H21; Susceptible corn inbred line is tucked in 478 and is material, and the PAL, POD and the SOD that record crescent Curvularia lunata toxin processing back corn seedling according to preceding method compare energy value.With the resistance grade is x, the susceptible self-mating system resistance of 1 expression, sense self-mating system resistance in 2 expressions; Anti-self-mating system resistance in 3 expressions, 4 expression resistance self-mating system resistances are y with PAL, POD and SOD than vigor data; Utilize DAS Excel2007 to make the scatter diagram of expression x and y relation earlier, tentatively judge x and y relation, utilize DPS (Data Processing System again; It is the DPS statistical software; This software is the multi-functional statistical analysis software of experimental design and statistical study) 7.05 carry out linear regression or quadratic polynomial regression analysis according to scatter diagram, set up PAL, POD and SOD regression equation than vigor and disease resistance, the result is respectively:
y
1=22.13x
1+ 35.17 (formula 1);
y
2=50.17x
2 2-136.9x
2+ 222.5 (formula 2);
y
3=-4.432x
3+ 38.04 (formula 3);
Y wherein
1Ratio vigor for phenylalanine ammonia lyase (PAL); y
2Ratio vigor for px (POD); Y wherein
3Ratio vigor for superoxide-dismutase (SOD); X wherein
1, x
2And x
3Resistance grade numerical value for the corn inbred line disease resistance.
Whether 3 regression equations setting up set up carry out test of hypothesis, the result shows that PAL is than vigor and the extremely remarkable linear positive correlation of corn inbred line disease resistance existence, linear equation y
1=22.13x
1+ 35.17 (R
2=0.955, P＜0.01); There is curve positive correlation in POD than vigor and disease resistance, and regression equation is y
2=50.17x
2 2-136.9x
2+ 222.5 (R
2=0.996, P=0.058); There is extremely remarkable linear negative correlation in SOD than vigor and corn disease resistance, and regression equation is y
3=-4.432x
3+ 38.04 (R
2=0.961, P＜0.01).
The method of the resistance level of described estimation corn inbred line Curvularia lunata leaf spot is for going out x according to PAL, POD and SOD than the regression equation calculation of vigor and disease resistance
1, x
2And x
3, get its MV x, be expressed as in susceptible self-mating system, 2≤x＜3 expressions according to 1≤x＜2 and feel self-mating system, anti-self-mating system in 3≤x＜4 expressions, x>=disease-resistant self-mating system of 4 expressions is judged the resistance of corn inbred line.Select x >=3, be the self-mating system of anti-maize curvularia leaf spot.
Compared with prior art, the present invention has following advantage or beneficial effect: (1) the inventive method accurately, reliably.(2) the inventive method can need not to carry out disease resistance in the field and identify in the evaluation and the screening of indoor completion corn inbred line Curvularia lunata leaf spot resistance, does not receive the restriction of external environmental condition.(3) the inventive method is carried out the disease resistance evaluation in seedling stage, need not to have broken season limit, and shortened and identified the required time becoming the strain phase to identify.(4) the present invention reduces the miscellaneous work that field resistance is identified, high efficiency.
Embodiment
The preparation of embodiment 1 corn crescent Curvularia lunata toxin soiutions
1. corn crescent Curvularia lunata bacterial strain (this bacterial strain is by teacher Yan Honghai of plant pathology teaching and research room of Qingdao Agricultural University isolation identification) is cultivated on the PDA substratum (its moity and weight ratio thereof are: contain yam 200g and glucose 15g in every 1L zero(ppm) water); Cultivate behind the 7d that (Φ=0.7cm) on the bacterium colony of cultivating, punch is connected to 100mL with 6 crescent Curvularia lunata bacterium sheets and improves that (its moity and weight ratio thereof are: contain sucrose 20g, tartrate ammonia 5g, NH in every 1L zero(ppm) water in the Fries liquid nutrient medium with punch tool
4NO
31g, KH
2PO
41g, sodium-chlor 0.1g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.5g, calcium chloride 0.13g and yeast extract paste 1g, pH6.2), in 25 ℃, the following shaking culture 15d that cultivates of 120rpm; Filter the aforesaid liquid substratum with activated carbon column (4cm * 46.5cm, granulated active carbon 30g), flow velocity is 4.2mL/min, with 300mL methanol-eluted fractions charcoal post 3 times, and 100mL at every turn; Use the 100mL distilled water wash again after each methanol-eluted fractions, mix 3 times meoh eluate, in 60～70 ℃ of water-baths, be concentrated into about 10mL; Evaporate to dryness in 37 ℃ of thermostat containers obtains soup compound; The gained soup compound is dissolved in 37 ℃ of methyl alcohol of 50mL; Filtered through gauze is removed insolubles, gets filtrating, and the chloroform that adds 3 times of volumes mixes; 25 ℃ of following hold over night, evaporate to dryness methyl alcohol, dissolved in chloroform layer obtain the tawny crystal; Be the thick toxin of crescent Curvularia lunata, the adding distil water dissolving, processing concentration is 50 μ g/mL corn crescent Curvularia lunata toxin soiutionses.
The early stage evaluation simultaneous test of embodiment 2 corn inbred line Curvularia lunata leaf spot resistances
Carry out according to following method:
Test materials: 10 corn inbred lines: neat 319, Q58, prosperous 7-2, Zheng 58, U.S. 26800, red 340, Zheng 0510, Shan 814,189, red yellow 25.
Test design:
TP: (one) indoor seedling test
(1) selects the seed of full self-mating system, through 0.1%HgCl
2Sterilization 10min, clean with distilled water flushing, in 25 ℃ of following imbibition 24h, transfer in the white disk that is lined with 6 layers of moistening filter paper in 28 ℃/25 ℃ (daytime/night) the down dark 72h of sprouting; Be transferred to after the sprouting and continue in the illumination box to cultivate, culture condition is 28 ℃/25 ℃ (daytime/night), and intensity of illumination is 200mmol/m
2S, every day, light application time 12h cultivated for 2～3 weeks, got the corn inbred line seedling of 3 leaf phases.
(2) the corn inbred line seedling of 3 leaf phases of selection uniformity is cut from the rhizome place and puts into the 50mL test tube; Each test tube 5 strain; The concentration that adds preparation among the embodiment 1 is 50 μ g/mL crescent Curvularia lunata toxin soiutions 50mL; Permeate 12h under the 200mmHg negative pressure, leave standstill 18h in 25 ℃ of thermostat containers, get the 3rd leaf and carry out the detection of enzymic activity.
(3) with the 3rd leaf of the corn seedling of handling through crescent Curvularia lunata toxin soiutions in (2), cut off blade tip, get the blade middle and upper part; After blotting water with filter paper, take by weighing the 1.0g blade, add pH8.8,0.05mol/L sodium borate buffer liquid (includes the 5mmol/L mercaptoethanol; 1mmol/L EDTA) 2.0ml adds silica sand 0.1g and grinds to form homogenate, changes homogenate over to centrifuge tube; With 2.0mL sodium borate buffer liquid flushing lingering section, change centrifuge tube over to; Stir 5min, centrifugal 30min under 4 ℃, 12 000 * g gets supernatant then; Get supernatant 3ml again and be diluted to 15ml with pH8.8,0.05mol/L sodium borate buffer liquid, the supernatant that must dilute (or claiming enzyme liquid) is preserved in-4 ℃ of refrigerator-freezers.
(4) (a) phenylalanine ammonia lyase (PAL) determination of activity: get dilution supernatant 1ml in the step (3); According to (.Journal of Biology Chemistry such as Koukol J such as Koukol; 1961,236:2692-2698) described method is carried out the active mensuration of PAL, and concrete outcome is seen table 1.
Table 1 corn inbred line phenylalanine ammonia lyase determination of activity result
| Self-mating system | PAL active (U) |
| Neat 319 | 1547.06 |
| Q58 | 1591.63 |
| Prosperous 7-2 | 1438.83 |
| Zheng 58 | 1412.09 |
| U.S. 26800 | 1261.84 |
| Red 340 | 1216.00 |
| Zheng 0510 | 1258.02 |
| Shan 814 | 938.42 |
| 189 | 910.41 |
| Red yellow 25 | 957.52 |
(b) px (POD) determination of activity: the supernatant 1ml that gets the dilution in (3) makes substrate with methyl catechol (guaiacol); According to methods such as Zhang Zhiliang (Zhang Zhiliang etc. plant physiology experiment instructs [M]. Beijing: Higher Education Publishing House; 2003; 123-124) carry out the active mensuration of px (POD), concrete outcome is seen table 2.
Table 2 corn inbred line peroxidase activity is measured the result
| Self-mating system | POD active (U) |
| Neat 319 | 5855.8 |
| Q58 | 5473.9 |
| Prosperous 7-2 | 5766.69 |
| Zheng 58 | 6199.51 |
| U.S. 26800 | 4850.13 |
| Red 340 | 4684.64 |
| Zheng 0510 | 4315.47 |
| Shan 814 | 2265.94 |
| 189 | 1833.12 |
| Red yellow 25 | 2036.8 |
(c) superoxide-dismutase (SOD) determination of activity: get the supernatant 0.1ml of the dilution in (3), according to nitroblue tetrazolium(NBT) (NBT) photochemical reduction method (referring to Wang Aiguo etc. the plant physiology journal, 1983,9 (1): 77-84) measure the SOD enzyme and live, concrete outcome is seen table 3.
Table 3 corn inbred line superoxide dismutase activity is measured the result
| Self-mating system | SOD active (U) |
| Neat 319 | 254.6 |
| Q58 | 267.33 |
| Prosperous 7-2 | 280.06 |
| Zheng 58 | 296.609 |
| U.S. 26800 | 341.164 |
| Red 340 | 313.158 |
| Zheng 0510 | 325.888 |
| Shan 814 | 420.09 |
| 189 | 445.55 |
| Red yellow 25 | 381.9 |
(d) mensuration of protein contnt in the dilution enzyme liquid: accurately measure dilution supernatant 2mL utilize Coomassie brilliant blue G-250 micromethod survey protein contnt in the enzyme liquid (concrete grammar is referring to Zhang Zhiliang etc. plant physiology experiment instructs [M]. Beijing: Higher Education Publishing House; 2003; 159-160); Recording protein content is 12.73 mg (albumen)/g (fresh weight), calculates the ratio vigor of corn inbred line PAL, POD and SOD, and the result sees table 4.
Table 4 corn inbred line PAL, POD and SOD than vigor result
(5) the ratio vigor of definition corn inbred line PAL, POD and SOD is respectively y
1, y
2, y
3, according to y
1=22.13x
1+ 35.17, y
2=50.17x
2 2-136.9x
2+ 222.5 and y
3=-4.432x
3+ 38.04 calculate the disease resistance x of self-mating system
1, x
2, x
3And x.The result sees table 5.
The resistance level result of the indoor identification of table 5 corn inbred line
| Self-mating system | x 1 | x 2 | x 3 | x |
| Neat 319 | 3.90 | 3.93 | 4.07 | 3.97 |
| Q58 | 4.06 | 3.81 | 3.84 | 3.90 |
| Prosperous 7-2 | 3.52 | 3.90 | 3.62 | 3.75 |
| Zheng 58 | 3.42 | 4.03 | 3.33 | 3.59 |
| U.S. 26800 | 2.89 | 3.60 | 2.53 | 3.01 |
| Red 340 | 2.73 | 3.53 | 3.03 | 3.10 |
| Zheng 0510 | 2.88 | 3.40 | 2.81 | 3.03 |
| Shan 814 | 1.74 | 2.34 | 1.13 | 1.74 |
| 189 | 1.64 | 1.89 | 0.69 | 1.41 |
| Red yellow 25 | 1.81 | 2.13 | 1.81 | 1.92 |
(2) field resistance is identified simultaneous test
(1) the corn crescent Curvularia lunata strain culturing that will separate preservation from the milpa of dying corn crescent Curvularia lunata disease is at the PDA substratum; 28 ℃ of thermostat containers are cultivated 7-14d; With an amount of sterilized water washing (it is an amount of to add tween), filter then, process and mix the spore suspension with double gauze.Become strain phase inoculum density to be about 2.0 * 10
5Individual spore/ml.
(2) will supply that the examination material is neat 319, Zheng 58, Q58, prosperous 7-2, U.S. 26800, red 340, Zheng 0510, red yellow 25, Shan 814 and 189 is seeded in that physical features is smooth, the uniform field of fertility piece; Plantation 2 row, every row 15 caves, seeding row spacing is 0.5m * 0.8m; Seedling is stayed in two strains, 3 repetitions.By ordinary method corn inbred line is carried out field management.
(3) with manual sprayer in the trumpet period spray inoculation, tie up the 48h that preserves moisture after the inoculation, observe day by day the record incubation period later on, treat stable disease after, by strain record scab response type, scab number, scab size and severity.Investigation of each item index and measuring method are following: (1) incubation period: after the inoculation, observe each kind symptom every day and the date occurs, be seeded to the fate that manifest symptom occurs and be designated as the incubation period.(2) scab type: fixedly 1 leaf is the respondent on the fruit ear position, by strain investigation scab type.Method with reference to methods such as Zhao Laishun (Zhao Laishun etc. maize pinta research II. symptom type [J]. Agricultural University Of Hebei's journal, 1995,18 (4): 36-38).(3) severity: after treating the field stable disease, get above 3 leaves of fruit ear leaf by strain investigation severity.Grade scale with reference to methods such as Wang Hong (Wang Hong etc. corn hybrid seed is identified [J] to Curvularia lunata leaf spot resistance. Agricultural University Of Shenyang's journal, 2000,31 (5): 476-478).(4) scab number: after treating stable disease, measure the scab number on every strain 13 leaf left and right sides unit surfaces.The result sees table 6.
The field resistance qualification result of table 6 corn inbred line
Indoor seedling qualification result (seeing table 5) shows: neat 319, Zheng 58, Q58, prosperous 7-2, U.S. 26800, red 340 and Zheng 0510 indoor disease-resistant grade＞3 are disease-resistant self-mating system; Red yellow 25, Shan 814 and 189 resistance grade＜3 are susceptible self-mating system.That outdoor field qualification result (seeing table 6) shows is neat 319, the field resistance of Zheng 58, Q58 and prosperous 7-2 is disease-resistant self-mating system, U.S. 26800, red 340 and Zheng 0510 be in anti-self-mating system, red yellow 25, Shan 814 and 189 is middle sense self-mating system.
Comprehensive above-mentioned indoor seedling and field qualification result: neat 319, Zheng 58, Q58, prosperous 7-2, U.S. 26800, pellet 340 and Zheng 0510 are disease-resistant self-mating system; Pellet Huang 25, Shan 814 and 189 are susceptible self-mating system; Both qualification result basically identicals; Explain that the present invention is accurate, quick to the method for the resistance evaluation of the anti-Curvularia lunata leaf spot of corn inbred line in the indoor seedling stage; And this method do not receive season limit, and can reduce the field and identify required lot of test land used and manpower, financial resources, practices thrift cost and labour.
Claims (5)
1. the early stage authentication method of corn inbred line Curvularia lunata leaf spot resistance, carry out according to following method:
(1) the corn inbred line seedling of 3 leaf phases is cut from the rhizome place, putting into concentration is 20～100 μ g/mL corn crescent Curvularia lunata toxin soiutionses, and the 200mmHg negative pressure of utilizing vacuum pump to produce is permeated 6～48h, in 25 ℃ of thermostat containers, leaves standstill 18h then;
(2) get the 3rd leaf of the corn inbred line seedling of handling through corn crescent Curvularia lunata toxin in the step (1), cut off blade tip, get the blade middle and upper part; Blot water with filter paper, take by weighing blade 1.0g and silica sand 0.1g, put into mortar; Add the sodium borate buffer liquid 2.0ml of pH8.8,0.05mol/L, grind to form homogenate, homogenate is changed in the centrifuge tube; And, change centrifuge tube over to the lingering section on the sodium borate buffer liquid 2.0mL flushing mortar, stir 5min; Centrifugal 30min under 4 ℃, 12000g gets supernatant then; The 3ml supernatant is diluted to 15ml with pH8.8,0.05mol/L sodium borate buffer liquid, and the supernatant that must dilute is preserved down for-4 ℃; Contain 5mmol/L mercaptoethanol and 1mmol/LEDTA in the wherein said sodium borate buffer liquid; Described supernatant is the mixing crude extract of phenylalanine ammonia lyase, px and superoxide-dismutase;
(3) enzymic activity of phenylalanine ammonia lyase, px and superoxide-dismutase in the supernatant of the dilution of the middle gained of difference determination step (2); And calculate the ratio vigor of every kind of enzyme, than vigor calculation formula be:, calculate the resistance grade numerical value x of corn inbred line disease resistance more according to the following equation respectively than vigor=enzymic activity/protein content
1, x
2And x
3,
y
1=22.13x
1+ 35.17 (formula 1);
y
2=50.17x
2 2-136.9x
2+ 222.5 (formula 2);
y
3=-4.432x
3+ 38.04 (formula 3);
Y wherein
1Ratio vigor for phenylalanine ammonia lyase (PAL); y
2Ratio vigor for px (POD); Y wherein
3Ratio vigor for superoxide-dismutase (SOD);
(4) calculate x
1, x
2And x
3MV x, i.e. x=(x
1+ x
2+ x
3The corn inbred line of x>=3 is chosen in)/3, is the self-mating system of anti-maize curvularia leaf spot.
2. according to the described early stage authentication method of claim 1; It is characterized in that the corn crescent Curvularia lunata toxin soiutions described in its step (1); According to the preparation of following method: with corn crescent Curvularia lunata strain culturing on the PDA substratum; Punch on the bacterium colony of cultivating with punch tool after cultivating 7d, 6 crescent Curvularia lunata bacterium sheets are connected in the 100mL improvement Fries liquid nutrient medium, in 25 ℃, 120rpm cultivation shaking culture 10～15d down; Filter the aforesaid liquid substratum with activated carbon column, flow velocity is 4.2mL/min, with 300mL methanol-eluted fractions charcoal post 3 times, and 100mL at every turn; Use the 100mL distilled water wash again after each methanol-eluted fractions, mix 3 times meoh eluate, in 60～70 ℃ of water-baths, be concentrated into about 10mL; Evaporate to dryness in 25～65 ℃ of thermostat containers obtains soup compound; The gained soup compound is dissolved in the 50mL temperature in 25～37 ℃ methyl alcohol; Filter, remove insolubles, get filtrating, the chloroform that adds 3 times of volumes mixes, and 25 ℃ of following hold over night, evaporate to dryness methyl alcohol, dissolved in chloroform layer obtain the tawny crystal, are the thick toxin of crescent Curvularia lunata; The adding distil water dissolving, processing concentration is 20～100 μ g/mL crescent Curvularia lunata toxin soiutionses; The moity of wherein said PDA substratum and weight ratio thereof are: contain yam 200g and glucose 15g in every 1L zero(ppm) water; Described improvement Fries liquid nutrient medium moity and ratio thereof are: contain sucrose 20g, tartrate ammonia 5g, NH in every 1L zero(ppm) water
4NO
31g, KH
2PO
41g, sodium-chlor 0.1g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.5g, calcium chloride 0.13g and yeast extract paste 1g, pH6.2.
3. according to the described early stage authentication method of claim 1; It is characterized in that the active mensuration of phenylalanine ammonia lyase described in its step (3); Getting the supernatant 1ml of the dilution of gained in its step (2), is substrate with the L-phenylalanine(Phe) then, measures the enzyme of phenylalanine ammonia lyase at the 290nm place and lives; Per hour to make OD
290The enzyme amount of increase by 0.01 is decided to be enzyme unit alive, calculates the ratio vigor of phenylalanine ammonia lyase.
4. according to the described early stage authentication method of claim 1, it is characterized in that the mensuration of the peroxidase activity described in its step (3), get the supernatant 1ml of the dilution of gained in its step (2), make substrate with methyl catechol then, survey the OD value at the 470nm place; With the activity of every milligram of albumen OD470 of PM increased value expressed enzyme, calculate the ratio vigor of px.
5. according to the described early stage authentication method of claim 1; It is characterized in that the superoxide-dismutase described in its step (3)) active mensuration; Get the supernatant 0.1ml of the dilution in its step (2), measure the SOD vigor, find out the enzyme liquid measure that suppresses NBT 50% photoreduction according to nitroblue tetrazolium(NBT) photochemical reduction method; Be decided to be enzyme unit alive, calculate the ratio vigor of superoxide-dismutase.
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|---|---|---|---|---|
| CN105760871A (en) * | 2014-12-14 | 2016-07-13 | 仲恺农业工程学院 | Plant leaf spot disease resistance identification new method |
| CN110463600A (en) * | 2019-09-06 | 2019-11-19 | 温州科技职业学院 | A kind of tomato selection of resisting etiolation leaf curl viral disease |
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|---|---|---|---|---|
| CN101946693A (en) * | 2010-08-16 | 2011-01-19 | 山东省农业科学院玉米研究所 | Seed production method for hybrid corn |
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| 叶克苁等: "玉米抗弯孢菌叶斑病的快速鉴定方法", 《西北农业学报 》, vol. 20, no. 4, 31 December 2011 (2011-12-31), pages 168 - 172 * |
| 陈捷等: "玉米弯孢叶斑病菌毒素对寄主防御酶系玉米弯孢叶斑病菌毒素对寄主防御酶系玉米弯孢叶斑病菌毒素对寄主防御酶系活性的影响及诱导抗性效应", 《植物病理学报》, vol. 32, no. 1, 28 February 2002 (2002-02-28), pages 43 - 48 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105760871A (en) * | 2014-12-14 | 2016-07-13 | 仲恺农业工程学院 | Plant leaf spot disease resistance identification new method |
| CN110463600A (en) * | 2019-09-06 | 2019-11-19 | 温州科技职业学院 | A kind of tomato selection of resisting etiolation leaf curl viral disease |
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