CN107964560A - A kind of western Muskmelon Fusarium wilt and blight dis-ease seedling stage joint method for rapidly identifying resistance - Google Patents
A kind of western Muskmelon Fusarium wilt and blight dis-ease seedling stage joint method for rapidly identifying resistance Download PDFInfo
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Abstract
The present invention relates to a kind of western Muskmelon Fusarium wilt and the rapid identification method of blight dis-ease seedling stage joint resistance, will sterilize, sows after western muskmelon seeds water seed soaking to be identified first, is grown after emergence to 35 true leaves；Blade didymella bryoniae spore suspension spray inoculation is taken, seedling root is rinsed well with water, is transplanted after being immersed in wilt spore suspension in the container equipped with matrix, puts hot-house culture, 3 7d start to observe the state of an illness after inoculation.Droop and blight dis-ease incidence are investigated, the incidence of droop and the incidence of blight dis-ease and disease index are counted, according to incidence and the western muskmelon seedling stage droop of disease index classification prediction and blight dis-ease resistance.The identification method of the present invention, can Rapid identification has the western Germplasm Resources of Cucumis Melo L of droop and blight dis-ease resistance at the same time on the same plant of same kind, improve determination rates, shorten the cycle, the test materials such as seed are also saved, are adapted to a large amount of quick western muskmelon resistance screenings.
Description
Technical field
The present invention relates to a kind of western Muskmelon Fusarium wilt and the rapid identification method of blight dis-ease seedling stage joint resistance, belong to plant
Germplasm Identification technical field.
Background technology
Fusarium oxysporum (Fusarium oxysporum) infects caused droop and melon Ascochyta tini
Caused by (Ascochytacitrullina Smith, its asexual generation are to let out root Asia every spore shell Didymella bryoniae)
Blight dis-ease is two big fungal disease of melon crop, Winter-Spring heliogreenhouse and early spring, greenhouse cultivation after autumn Seedling Stage to adopting
The receipts phase can occur, and often the plant death rate up to 30%~40%, causes Severe Reduction in production, it has also become restrict melon life
The major obstacle of production.Cultivate high-quality compound disease-resistant western New melon variety be effectively control western Muskmelon Fusarium wilt, blight dis-ease it is main
Method, therefore, it is method the most cost-effective to promote the use of western muskmelon resistant variety.But Disease Resistance Identification is breeding for disease resistance
Basis, and method of resistance identification is the key of Disease Resistance Identification, makes accurate, reliable, succinct, quick identification side
Method, can improve the efficiency of breeding work, accelerate the process of breeding.
The Resistance Identification both at home and abroad to western Muskmelon Fusarium wilt, blight dis-ease was mainly that single inocalation method is identified in the past, such as withered
Sick seedling of withering phase leaching piece-root grafting kind, inoculation of being completely cured, leaf stab inoculation, and by the spray inoculation of seedling stage blight dis-ease spore suspension,
Isolated leaf inoculation etc..Single inocalation method efficiency is low, it is more to expend the test materials such as seed, and different batches experiment is unstable.
There are a small amount of report droop, blight dis-ease Mixed inoculation, i.e., connect with live body successively inoculation droop and didymella bryoniae, at present compound
Kind method while inoculating two kinds pathogen, often result in disease morbidity reciprocal effect, and disease index is higher, easily produce wrong tie
Fruit, and since wilt is fatal to plant, the substantially all death of plant after inoculation, can not carry out multiresistance seedling stage mirror
Fixed experiment, need to such as identify other disease resistances of blight dis-ease, the later stage needs to carry out independent inoculation test to sense droop material.
The content of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of western Muskmelon Fusarium wilt and blight dis-ease seedling stage connection
The rapid identification method of resistance is closed, which is adapted to a large amount of quick resistance screenings, can avoid different diseases morbidity interaction
Disease observation and statistical result are influenced, identification is quick and qualification result is more accurate.
To achieve the above object, the technical solution adopted by the present invention is：
The rapid identification method of a kind of western Muskmelon Fusarium wilt and blight dis-ease seedling stage joint resistance, includes the following steps：
(1) participate in the experiment the preparations of plant shoots：After taking western muskmelon seeds to be put into medicining liquid dipping, rinsed with circulating water and remove table
The remaining thimerosal in face, then with 50-60 DEG C of water seed soaking, stir to room temperature, place 3-6h, then take out disinfection, sowing,
Grown after emerging to 3-5 piece true leaves, it is spare；
(2) preparation of pathogen：
Sclerotium rolfsii prepares：Withered germ of water-melon is taken to be inoculated in PDA solid mediums, 26-28 DEG C is cultivated one week, then will
Cultured Sclerotium rolfsii is inoculated in PDB fluid nutrient mediums, and 26-28 DEG C, 150r/min is cultivated one week, after filtering, is added sterile
Distilled water, is configured to concentration as 5 × 105The spore suspension of spore/ml is spare；
Blight dis-ease germ prepares：Blight dis-ease germ is taken to be inoculated in PDA solid mediums, 26-28 DEG C is cultivated one week, is chosen new
The cucumber of fresh no wound, with 75% alcohol wipe surface, after drying, slightly beats after circulating water is rinsed well on cucumber surface
Epidermis is removed, the sterilization punchers of cultured bacterial strain diameter 0.7cm, which are beaten, takes mycelia block, takes mycelia block block to be placed in above cucumber,
Incubator is put into, 20 DEG C are cultivated, and are had conidium generation after 5-7d, are scraped with blade, are ground, and sterile distilled water is added after filtering,
It is configured to 5 × 105The spore suspension of spore/ml is spare；
(3) blade prepares：Take culture dish, the filter paper that bottom laying is soaked with the fresh-keeping aqueous solution of benzimidazole plant, disinfection
Afterwards, the 3-5 piece true leaves for the plant shoots being put on filter paper in step (1), it is spare；
(4) inoculation of pathogen：
Sclerotium rolfsii is inoculated with：Plant shoots in step (1) are dug out, root with blotting paper is sucked into water with after cleaning
Point, then be immersed in step (2) spore suspension, after 5min by seedling replanting in the container equipped with matrix, it is placed on 26 ± 1 DEG C
Hot-house culture, 7d starts to observe the state of an illness after inoculation；
Blight dis-ease germ is inoculated with：The climing withered spore suspension of step (2) is sprayed onto on the blade of step (3), each blade spray
500 μ 0, are then placed on culture dish in illumination box and preserve, and condition of culture is：26 ± 1 DEG C of temperature, periodicity of illumination 16h light
According to/8h dark, the state of an illness is observed after relative humidity about 90%, 3d；
(5) research and appraisal：
Droop investigation classification：Recorded according to step (4) droop disease observation result, assessment resistance is by 5 grades of marks
Quasi- investigation is recorded, i.e.,：
0 grade：Without illness；
1 grade：Cotyledon is wilted or part cotyledon is slightly wilted with true leaf：
2 grades：1 true leaf is wilted or the heavier wilting of cotyledon；
3 grades：Cotyledon and more than 60% true leaf are wilted；
4 grades：Whole strain is slightly wilted, and more than 60% is withered, but lobus cardiacus still survives；
5 grades：The serious wilting of whole strain is simultaneously withered；
Wherein, less than 2 grades are disease-resistant plant, and more than 3 grades are disease plant；Evaluation of resistance, morbidity are carried out according to incidence
Rate=(disease plant number/investigation total strain number) × 100%, result of calculation are divided into four grades, susceptible (S)：Withered rate 80%-
100%；It is gently anti-(LR)：Withered rate 51%-80%；Moderate resistance (MR)：Withered rate 21%-50%；Highly resistance (HR)：Withered rate is less than
20%；
Blight dis-ease investigation classification：It is classified according to the size of step (4) middle period on piece scab, each disease level grade scale is such as
Under：
0 grade：Blade does not have scab；
1 grade：Leaf spot lesion area≤25%；
2 grades：25% ﹤ leaf spot lesion area≤50%；
3 grades：50% ﹤ leaf spot lesion area≤75%；
4 grades：75% ﹤ leaf spot lesion area≤100%.
Disease index is calculated according to the sick level of inspection, formula is as follows：
Disease index=∑ (morbidity number × typical value at different levels at different levels)/(investigation total strain number × superlative degree represents number) ×
100%
6 grades are divided into according to result of calculation：Height sense (HS)：80.1-100；Susceptible (S)：60.1-80；Middle sense (MS)：
40.1-60；Moderate resistance (MR)：20.1-40.0；Disease-resistant (R)：10.1-20.0；Highly resistance (HR)：1-10.
In step (1), the thimerosal is 10% liquor natrii hypochloritis.
In step (2), Sclerotium rolfsii prepares with blight dis-ease germ set-up procedure, and the filtering is each meant with 4 layers of yarn
Cloth filters.
In step (3), the concentration of the fresh-keeping aqueous solution of benzimidazole plant is 11.8mg/L.
In step (4), inoculation is carried out at the same time on the same plant of same kind.
Beneficial effect：Seedling stage live body and excised leaf provided by the invention combine resistance inoculation identification method, and advantage is,
Overcome that " wilt is fatal to plant after conventional Mixed inoculation, the substantially all death of plant after inoculation, after can not carrying out
Continuous multiresistance seedling stage assay experiment, need to such as continue sense droop material to identify other disease resistances of blight dis-ease, it is necessary to carry out list
The shortcomings that only inoculation test ".Seedling stage live body and Isolated leaf inoculation joint Resistance Identification technology can realize same plant at the same time
Identify two kinds of Disease Resistances, improve determination rates, shorten the cycle, the test materials such as seed can also be saved, be adapted to a large amount of quick
Resistance screening, and the kind lethal sieve for not influencing kind and subsequently other Disease Resistances being identified susceptible on any type pathogen
Choosing.
Embodiment
Below according to specific embodiment, the invention will be further described.
Embodiment
The rapid identification method of a kind of western Muskmelon Fusarium wilt and blight dis-ease seedling stage joint resistance, includes the following steps：
(1) participate in the experiment the preparations of plant shoots：Card taking is red (anti-blight, sense blight dis-ease), and Soviet Union's honey 1 is (sense droop, climing withered
Disease), after W7 (sense droop, middle anti didymella) watermelon seed is put into liquor natrii hypochloritis's immersion 5min that concentration is 10%, use
Circulating water rinses the thimerosal for removing remained on surface, then with 50-60 DEG C of water seed soaking, stirs to room temperature, place 4h, then
Disinfection, sowing are taken out, is grown after emerging to 3-5 piece true leaves, it is spare；
(2) preparation of pathogen：
Sclerotium rolfsii prepares：Withered germ of water-melon is taken to be inoculated in PDA solid mediums, 26-28 DEG C is cultivated one week, then will
Cultured Sclerotium rolfsii is inoculated in PDB fluid nutrient mediums, and 26-28 DEG C, 150r/min is cultivated one week, with 4 layers of filtered through gauze
Afterwards, add sterile distilled water, be configured to concentration as 5 × 105The spore suspension of spore/ml is spare；
Blight dis-ease germ prepares：Blight dis-ease germ is taken to be inoculated in PDA solid mediums, 26-28 DEG C is cultivated one week, is chosen new
The cucumber of fresh no wound, with 75% alcohol wipe surface, after drying, slightly beats after circulating water is rinsed well on cucumber surface
Epidermis is removed, the sterilization punchers of cultured bacterial strain diameter 0.7cm, which are beaten, takes mycelia block, takes mycelia block block to be placed in above cucumber,
Incubator is put into, 20 DEG C are cultivated, and are had conidium generation after 5-7d, are scraped with blade, are ground, 4 layers of filtered through gauze, add sterile steaming
Distilled water, is configured to 5 × 105The spore suspension of spore/ml is spare；
(3) blade prepares：9cm diameter Petri dishes are taken, bottom is laid with fresh-keeping water-soluble with the benzimidazole plant of 11.8mg/L
The filter paper that liquid soaks, after disinfection, the 3-5 piece true leaves for the plant shoots being put on filter paper in step (1) are spare；
(4) inoculation of pathogen：
Sclerotium rolfsii is inoculated with：Plant shoots in step (1) are dug out, root with blotting paper is sucked into water with after cleaning
Point, then be immersed in step (2) spore suspension, after 5min by seedling replanting in the container equipped with matrix, it is placed on 26 ± 1 DEG C
Hot-house culture, 7d starts to observe the state of an illness after inoculation；
Blight dis-ease germ is inoculated with：The climing withered spore suspension of step (2) is sprayed onto on the blade of step (3), each blade spray
500 μ 0, are then placed on culture dish in illumination box and preserve, and condition of culture is：26 ± 1 DEG C of temperature, periodicity of illumination 16h light
According to/8h dark, the state of an illness is observed after relative humidity about 90%, 3d；
(5) research and appraisal：
Droop investigation classification：Recorded according to step (4) droop disease observation result, assessment resistance is by 5 grades of marks
Quasi- investigation is recorded, i.e.,：
0 grade：Without illness；
1 grade：Cotyledon is wilted or part cotyledon is slightly wilted with true leaf：
2 grades：1 true leaf is wilted or the heavier wilting of cotyledon；
3 grades：Cotyledon and more than 60% true leaf are wilted；
4 grades：Whole strain is slightly wilted, and more than 60% is withered, but lobus cardiacus still survives；
5 grades：The serious wilting of whole strain is simultaneously withered；
Wherein, less than 2 grades are disease-resistant plant, and more than 3 grades are disease plant；Evaluation of resistance, morbidity are carried out according to incidence
Rate=(disease plant number/investigation total strain number) × 100%, result of calculation are divided into four grades, susceptible (S)：Withered rate 80%-
100%；It is gently anti-(LR)：Withered rate 51%-80%；Moderate resistance (MR)：Withered rate 21%-50%；Highly resistance (HR)：Withered rate is less than
20%；
Blight dis-ease investigation classification：It is classified according to the size of step (4) middle period on piece scab, each disease level grade scale is such as
Under：
0 grade：Blade does not have scab；
1 grade：Leaf spot lesion area≤25%；
2 grades：25% ﹤ leaf spot lesion area≤50%；
3 grades：50% ﹤ leaf spot lesion area≤75%；
4 grades：75% ﹤ leaf spot lesion area≤100%.
Disease index is calculated according to the sick level of inspection, formula is as follows：
Disease index=∑ (morbidity number × typical value at different levels at different levels)/(investigation total strain number × superlative degree represents number) ×
100%
6 grades are divided into according to result of calculation：Height sense (HS)：80.1-100；Susceptible (S)：60.1-80；Middle sense (MS)：
40.1-60；Moderate resistance (MR)：20.1-40.0；Disease-resistant (R)：10.1-20.0；Highly resistance (HR)：1-10.
Qualification result is shown in Table 1：
1 variety of watermelon droop of table and blight dis-ease seedling stage joint Resistance Identification
As can be seen from Table 1：Soviet Union's No. 1 incidence of honey is 81.25%, is accredited as droop susceptible variety, carminum incidence
For 18.75%, droop highly resistance kind is accredited as；The climing withered resistance of variety of watermelon, Soviet Union's honey 1 are identified using watermelon seedling excised leaf
Disease index is 81.48, the high susceptible variety of blight dis-ease, and W7 disease indexs are 39.81, are blight dis-ease moderate resistance kind.Qualification result
Show that Soviet Union's honey 1 is droop susceptible variety, carminum is droop highly resistance kind, and it is the high susceptible variety of blight dis-ease to revive sweet No. 1, W7
For blight dis-ease moderate resistance kind, meet with expection.
The seedling stage live body inoculation droop and Isolated leaf inoculation blight dis-ease joint Resistance Identification method of the present invention, Neng Gou
The same plant of same kind while Rapid identification have the Watermelon Germplasm of droop and blight dis-ease resistance, obtain different west
The accurate result of melon variety source material seedling stage droop blight dis-ease Resistance Identification.
The above, is merely illustrative of the technical solution of the present invention and unrestricted, those of ordinary skill in the art are to this hair
The other modifications or equivalent substitution that bright technical solution is made, without departing from the spirit and scope of technical solution of the present invention,
It should all cover among scope of the presently claimed invention.
Claims (4)
1. the rapid identification method of a kind of western Muskmelon Fusarium wilt and blight dis-ease seedling stage joint resistance, it is characterised in that including as follows
Step：
(1) participate in the experiment the preparations of plant shoots：After taking western muskmelon seeds to be put into medicining liquid dipping, it is residual to rinse removing surface with circulating water
The thimerosal stayed, then with 50-60 DEG C of water seed soaking, stir to room temperature, place 3-6h, then take out disinfection, sowing, treat out
After seedling length is spare to 3-5 piece true leaves；
(2) preparation of pathogen：
Sclerotium rolfsii prepares：Withered germ of water-melon is taken to be inoculated in PDA solid mediums, 26-28 DEG C is cultivated one week, then will culture
Good Sclerotium rolfsii is inoculated in PDB fluid nutrient mediums, and 26-28 DEG C, 150r/min is cultivated one week, after filtering, adds sterile distillation
Water, is configured to concentration as 5 × 105The spore suspension of spore/ml is spare；
Blight dis-ease germ prepares：Blight dis-ease germ is taken to be inoculated in PDA solid mediums, 26-28 DEG C is cultivated one week, chooses fresh nothing
The cucumber of wound, with 75% alcohol wipe surface, after drying, slightly table is played on cucumber surface after circulating water is rinsed well
Skin, the sterilization punchers of cultured bacterial strain diameter 0.7cm, which are beaten, takes mycelia block, takes mycelia block to be placed in above cucumber, is put into training
Case is supported, 20 DEG C are cultivated, and are had conidium generation after 5-7d, are scraped with blade, are ground, and sterile distilled water is added after filtering, is configured to 5
×105The spore suspension of spore/ml is spare；
(3) blade prepares：Take culture dish, the filter paper that bottom laying is soaked with the fresh-keeping aqueous solution of benzimidazole plant, after disinfection,
The 3-5 piece true leaves for the plant shoots being put on filter paper in step (1), it is spare；
(4) inoculation of pathogen：
Sclerotium rolfsii is inoculated with：Plant shoots in step (1) are dug out, by root with blotting paper sucking moisture after cleaning,
It is immersed in again in step (2) spore suspension, after 5min by seedling replanting in the container equipped with matrix, is placed on 26 ± 1 DEG C of temperature
Room is cultivated, and 7d starts to observe the state of an illness after inoculation；
Blight dis-ease germ is inoculated with：The climing withered spore suspension of step (2) is sprayed onto on the blade of step (3), each blade sprays 500 μ
L, is then placed on culture dish in illumination box and preserves, and condition of culture is：26 ± 1 DEG C of temperature, periodicity of illumination 16h illumination/8h
Dark, observes the state of an illness after relative humidity about 90%, 3d；
(5) research and appraisal：
Droop investigation classification：Recorded according to step (4) droop disease observation result, assessment resistance presses 5 grades of standard tune
Record is looked into, i.e.,：
0 grade：Without illness；
1 grade：Cotyledon is wilted or part cotyledon is slightly wilted with true leaf：
2 grades：1 true leaf is wilted or the heavier wilting of cotyledon；
3 grades：Cotyledon and more than 60% true leaf are wilted；
4 grades：Whole strain is slightly wilted, and more than 60% is withered, but lobus cardiacus still survives；
5 grades：The serious wilting of whole strain is simultaneously withered；
Wherein, less than 2 grades are disease-resistant plant, and more than 3 grades are disease plant；According to incidence carry out evaluation of resistance, incidence=
(disease plant number/investigation total strain number) × 100%, result of calculation is divided into four grades, susceptible：Withered rate 80%-100%；Gently
It is anti-：Withered rate 51%-80%；Moderate resistance：Withered rate 21%-50%；Highly resistance：Withered rate is less than 20%；
Blight dis-ease investigation classification：It is classified according to the size of step (4) middle period on piece scab, each disease level grade scale is as follows：
0 grade：Blade does not have scab；
1 grade：Leaf spot lesion area≤25%；
2 grades：25% ﹤ leaf spot lesion area≤50%；
3 grades：50% ﹤ leaf spot lesion area≤75%；
4 grades：75% ﹤ leaf spot lesion area≤100%.
Disease index is calculated according to the sick level of inspection, formula is as follows：
Disease index=∑ (morbidity number × typical value at different levels at different levels)/(investigation total strain number × superlative degree represents number) × 100%
6 grades are divided into according to result of calculation：Height sense：80.1-100；It is susceptible：60.1-80；Middle sense：40.1-60；Moderate resistance：
20.1-40.0；It is disease-resistant：10.1-20.0；Highly resistance：1-10.
2. the rapid identification method of western Muskmelon Fusarium wilt and blight dis-ease seedling stage joint resistance as claimed in claim 1, its feature
It is, in step (1), the thimerosal is 10% liquor natrii hypochloritis.
3. the rapid identification method of western Muskmelon Fusarium wilt and blight dis-ease seedling stage joint resistance as claimed in claim 1, its feature
It is, in step (2), Sclerotium rolfsii prepares with blight dis-ease germ set-up procedure, and the filtering is each meant with 4 layers of gauze mistake
Filter.
4. the rapid identification method of the joint resistance of western Muskmelon Fusarium wilt and blight dis-ease seedling stage as described in claim 1 or 2 or 3,
It is characterized in that, in step (3), the concentration of the fresh-keeping aqueous solution of benzimidazole plant is 11.8mg/L.
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| CN109378031A (en) * | 2018-11-26 | 2019-02-22 | 杭州电子科技大学 | A kind of rice sheath blight disease prediction technique combined based on plant protection and weather information |
| CN109628551A (en) * | 2019-01-24 | 2019-04-16 | 广东省农业科学院蔬菜研究所 | A kind of method and its application of Rapid identification and the anti-Fusarium oxysporum wilt disease plant of screening |
| CN109750080A (en) * | 2019-03-01 | 2019-05-14 | 山东省花生研究所 | A kind of method of the anti-fruit rot germ plasm resource of quick screening peanut |
| CN110338055A (en) * | 2019-05-17 | 2019-10-18 | 扬州大学 | A kind of eggplant anti-blight rapid identification method |
| CN110506596A (en) * | 2019-09-10 | 2019-11-29 | 安徽省农业科学院烟草研究所 | The method of the tobacco mosaic virus (tmv) and Resistance to bacterial wilt of homophyletic tobacco is identified simultaneously |
| CN112369314A (en) * | 2021-01-06 | 2021-02-19 | 湖南杂交水稻研究中心 | Method for identifying phenotype of rice sheath blight at seedling stage |
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| CN109378031A (en) * | 2018-11-26 | 2019-02-22 | 杭州电子科技大学 | A kind of rice sheath blight disease prediction technique combined based on plant protection and weather information |
| CN109628551A (en) * | 2019-01-24 | 2019-04-16 | 广东省农业科学院蔬菜研究所 | A kind of method and its application of Rapid identification and the anti-Fusarium oxysporum wilt disease plant of screening |
| CN109750080A (en) * | 2019-03-01 | 2019-05-14 | 山东省花生研究所 | A kind of method of the anti-fruit rot germ plasm resource of quick screening peanut |
| CN109750080B (en) * | 2019-03-01 | 2022-01-25 | 山东省花生研究所 | Method for rapidly screening peanut fruit rot resistant germplasm resources |
| CN110338055A (en) * | 2019-05-17 | 2019-10-18 | 扬州大学 | A kind of eggplant anti-blight rapid identification method |
| CN110506596A (en) * | 2019-09-10 | 2019-11-29 | 安徽省农业科学院烟草研究所 | The method of the tobacco mosaic virus (tmv) and Resistance to bacterial wilt of homophyletic tobacco is identified simultaneously |
| CN110506596B (en) * | 2019-09-10 | 2022-03-11 | 安徽省农业科学院烟草研究所 | Method for simultaneously identifying common mosaic disease and bacterial wilt resistance of tobacco of same strain |
| CN112369314A (en) * | 2021-01-06 | 2021-02-19 | 湖南杂交水稻研究中心 | Method for identifying phenotype of rice sheath blight at seedling stage |
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