CN101323875B - Wheat fusarium stalk rot seedlings inoculation identification method - Google Patents
Wheat fusarium stalk rot seedlings inoculation identification method Download PDFInfo
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- CN101323875B CN101323875B CN2008100228351A CN200810022835A CN101323875B CN 101323875 B CN101323875 B CN 101323875B CN 2008100228351 A CN2008100228351 A CN 2008100228351A CN 200810022835 A CN200810022835 A CN 200810022835A CN 101323875 B CN101323875 B CN 101323875B
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Abstract
The invention relates to an inoculation and identification method for crown rot caused by fusarium graminearum of wheat at seedling stage, which pertains to the technical field of plant protection and is used for fast identifying and screening wheat germplasm resources with resistance to the crown rot caused by fusarium graminearum from wheat resources in China. After activation, fusarium graminearum is inoculated in a liquid nutrient medium of 0.6 percent of green bean soup which is sterilized with high pressure at a temperature of 121 DEG C, and cultured in a shaker for 72h under a temperature of 25 DEG C, and then fusarium graminearum suspension of 10 <5> spores/ml is obtained; wheat seeds are sprouted after disinfection and are sowed in sterilized earth after germination; inoculation is started 10 days after seedling emergence; the crown rot inspection of wheat stem base is carried out 35 days after inoculation. The inoculation and identification method has the advantages of setting up the inoculation and identification method for crown rot caused by fusarium graminearum of wheat at seedling stage, providing a basis for the research of the crown rot caused by fusarium graminearum, being capable of fast screening wheat resistance resources and improving the utilization of the wheat resources.
Description
Technical field
The present invention relates to wheat Fusarium graminearum stem rot quick inoculation identification method in (Crown Rot) seedling stage,, belong to the plant protection technology field especially to the screening method of the anti-stem rot resource material of wheat.
Background technology
Wheat sickle-like bacteria (Fusarium) is one of main pathogenic bacterium of harm improving yield of wheat, stable yields.Sickle-like bacteria can cause two kinds of main wheat diseases: i.e. head blight (wheat scab) and stem rot (crown rot), since the later stage eighties, because the influence of Global climate change and cropping system, wheat scab frequently takes place on Europe, the U.S., Canada, China, South America and other places, wheat yield and quality have been caused serious loss, people have carried out research extensively and profoundly to wheat scab for this reason, and have obtained significant achievement.China is the country of global wheat scab injured area maximum, main pathogenic bacterium are Fusarium graminearum (Fusarium graminearum), the middle and lower reach of Yangtze River and south China winter wheat district and northeast spring wheat district east wheat scab are particularly serious, and the generation area of annual wheat scab has surpassed 7,000,000 hm
2, account for 1/4 of the national wheat total area.But the research by the microbial wheat stem rot of reaping hook is not still drawn attention in China.And the wheat stem rot is fallen ill seriously in Australian, wheat planting districts, ground such as the North America is Argentinian, Italian, South Africa, Turkey, has caused local wheat breeding and pathologists' extensive concern.U.S.'s wheat stem rot mainly betides the warmer idaho of weather, Oregon and each state of northwest Pacific such as Washington, the state of Colorado, the Wyoming State and Texas, studies show that, many sickle-like bacteria that cause wheat scab such as fusarium culmorum, fusarium avenaceum, Fusarium graminearum, fusarium acuminatum (F.culmorum, F.avenaceum, F.graminearum and F.acuminatum) also can cause the wheat stem rot, as finding to take place simultaneously in same field piece wheat scab and stem rot in Australia.But compare with the research of wheat scab, the research of wheat stem rot resistance is less, and evaluation of wheat stem rot resistance and genetics of resistance mechanism thereof are made slow progress.
China Fusarium graminearum accounts for 94.5% of reaping hook bacterial classification or mutation, spread all over 21 provinces (city), be to cause present wheat scab popular advantage reaping hook bacterial classification, the head blight hazard area enlarges vast winter wheat districts such as having involved the Yellow River and Huai He River sea and northwest year by year, therefore be subjected to extensive concern and research, simultaneously find that also wheat scab grain germination rate is low, infected seed also can cause the withered and basal part of stem of seedling to rot.And foreign study has shown, many sickle-like bacteria (F.grantinearun, F.culmorum, F.avenaceun and F.nivale) can cause wheat stem rot (crown rot), and are wherein especially pathogenic the strongest with Fusarium graminearum.Also there is report in China to the wheat root disease, possesses the pathogenetic condition of wheat stem rot in China wheat scab popular some areas.But China's wheat stem rot of causing by Fusarium graminearum of play-by-play not as yet so far, and the authentication method of relevant Fusarium graminearum stem rot report not more.Serious and northern spreading trend is arranged in view of the microbial wheat scab morbidity of China's wheat reaping hook in recent years, therefore, the authentication method of setting up wheat Fusarium graminearum stem rot is imperative to problems such as screening resistance resource and research wheat stem rot resistance mechanisms.
Summary of the invention
Technical problem the objective of the invention is to: at the blank of China's wheat fusarium stalk rot seedlings authentication method, provide a kind of quick seedling stage the efficient method of identifying wheat resource material resistance.
The object of the present invention is achieved like this for technical scheme: the wheat fusarium stalk rot seedlings authentication method, it is characterized in that: behind the Fusarium graminearum actication of culture in the sterilization 0.6% sweet mung bean soup substratum (mung beanagar, MBA) 25 ℃ of shaking culture 72h, the multilayer filtered through gauze is regulated bacterial concentration to 10
5Individual spore/mL suspension.Wheat seed is used 75% alcohol disinfecting 5min, aseptic water washing 3-4 time again behind 0.1% mercuric chloride sterilization 10min.Vernalization in culture dish, be seeded in behind the seed germination in 8 * 8cm nutrition pot and grow, soil through 121 ℃ of autoclaving 40min is housed in the nutrition pot, vegetative period, temperature kept 20-25 ℃/10-15 ℃ (daytime/night), 10d (after planting about 13-14 days) twined a small amount of wetted medical absorbent cotton ball at every strain seedling basal part of stem apart from the about 0.5-1.0cm of soil table place after wheat was emerged, adjusted the spore liquid of concentration at the contact position inoculation 0.5mL of the inboard wheat of cotton balls stem, the inoculation back covers with double-deck sunshade net, and adopt the atomizing system will inoculate seedling to place 48h under the nearly saturated relative humidity, then remove sunshade net seedling normal growth and manage.Inoculation back 35d carries out the stem rot investigation to the wheat stalk base portion.Disease index=∑ state of an illness rank * this grade strain number/(investigating total strain number * the highest sick level)] * 100%.Disease index＜10% is disease-resistant; Disease index 10.01-20.0% is anti-in being; Disease index 20.01-30.0% is middle sense; Disease index＞30.01% is susceptible.Qualification test is established three repetitions, every processing 15 strains.
Beneficial effect the invention has the advantages that: take the lead in having set up wheat Fusarium graminearum stem rot resistance seedling new Identification Method in China.The present invention to China's part wheat breeding resource and draw from Australian wheat Fusarium graminearum anti-/ the sense material identifies and obtained satisfactory result.Show that this method can be used for the wheat germplasm resource that rapid screening China wheat resource has Fusarium graminearum stem rot resistance, widens the wheat resource utilization.
Embodiment
The Fusarium graminearum bacterial classification picking one fritter mycelium inoculation on Bechtop deposited of going bail for was gone up activation culture 3 days at the PDA substratum (potato dextrose agar) for preparing.Then in the 0.6% sweet mung bean soup liquid nutrient medium of picking one fritter mycelium inoculation after sterilization (mung bean agar, MBA).180r/min shaking culture 72h on 25 ℃ of temperature control shaking tables, bacterial concentration to 10 is counted, regulated to the multilayer filtered through gauze at microscopically with blood counting chamber
5Individual spore/mL suspension, 4 ℃ of preservations are standby.
Wheat seed is used 75% alcohol disinfecting 5min, aseptic water washing 3-4 time again behind 0.1% mercuric chloride sterilization 10min.Under aseptic technique,, fill up one deck filter paper in the culture dish with seed vernalization in culture dish.
Be seeded in behind the seed germination in 8 * 8cm nutrition pot and grow, soil process autoclave sterilization is 40 minutes in the nutrition pot, cooling back dress nutrition pot.Vegetative period, temperature kept 20-25 ℃/10-15 ℃ (daytime/night).
10d (after planting about 13-14 days) twined a small amount of wetted medical absorbent cotton ball at every strain seedling basal part of stem apart from the about 0.5-1.0cm of soil table place after wheat was emerged, adjusted the spore liquid of concentration with the contact position inoculation 0.5mL of wheat stem in the cotton balls inboard, the inoculation back covers with double-deck sunshade net, and adopt atomizing system or atomizer at the online atomizing of sunshade, the inoculation seedling is placed 48h under the nearly saturated relative humidity, then remove the management of sunshade net seedling normal growth.
Inoculation back 35d carries out the stem rot investigation to the wheat stalk base portion.Disease index=∑ state of an illness rank * this grade strain number/(investigating total strain number * the highest sick level)] * 100%.Disease index＜10% is disease-resistant; Disease index 10.01-20.0% is anti-in being; Disease index 20.01-30.0% is middle sense; Disease index＞30.01% is susceptible.Qualification test is established three repetitions, every processing 15 strains.Test is respectively with Australian wheat breed Sunco and Kennedy (V.Mitter, M.C.Zhang, C.J.Liu et al, .A high-throughput glasshouse bioassay to detect crown rot resistance in wheatgermplasm, Plant Pathology, 2006,55,433-441) as anti-/ sense check variety.
According to the degree of disease investigation result, wheat resource is estimated according to disease classification and disease index.
As in this test, part material qualification result such as table 1, in 35 parts of materials identifying, dependency remarkable (r=0.83) shows that this method qualification result is reliable between the different times qualification result.Qualification result shows, 5 parts of material disease indexs less than 20% be in anti-material; 8 parts of material disease indexs are felt material between 20-30% in belonging to, all the other 22 material disease indexs are susceptible material greater than 30%.Do not find the high anti-material of Fusarium graminearum stem rot.This shows needs further to enlarge germ plasm resource screening scope in wheat stem rot resistance resource is identified, for anti-stem rot breeding provides material foundation.
Table 1 wheat part material Fusarium graminearum stem rot evaluation of resistance
Annotate: disease-resistant (R); In anti-(MR); Middle sense (MS); Susceptible (S).
Claims (1)
1. the inoculation identification method in seedling stage of the resistance of a grow wheat Fusarium graminearum stem rot is characterized in that:
1) Fusarium graminearum of Bao Cuning (Fusarium graminearum) bacterial classification on the Bechtop on the PDA substratum activation culture, the picking mycelium inoculation is in the autoclaved mass ratio 0.6% sweet mung bean soup liquid nutrient medium through 121 ℃ after 3 days, 25 ℃ of following 180 rev/mins of shaking table shaking culture 72h of condition, filter, regulate bacterial concentration to 10
5Individual spore/mL obtains Fusarium graminearum suspension;
2) wheat seed is sterilized in mass ratio 0.1% mercuric chloride behind the 10min with behind the volume ratio 75% alcohol disinfecting 5min, uses aseptic water washing 3-4 time again;
3) sprout down in 25 ℃ in culture dish wheat seed sterilization back, be seeded in behind the seed germination in 8 * 8cm nutrition pot and grow, soil through 121 ℃ of autoclaving 40min is housed in the nutrition pot, vegetative period, temperature kept 10-15 ℃ of 20-25 ℃/night in daytime, wheat emerge back 10 days at every strain seedling basal part of stem apart from soil table 0.5-1.0cm place winding wetted medical absorbent cotton ball;
4) be 10 in winding cotton balls inboard and wheat stem contact position inoculation 0.5mL concentration
5The Fusarium graminearum suspension of individual spore/mL, inoculation back covers with double-deck sunshade net, and adopts the atomizing device, and the inoculation seedling is placed 48h under the nearly 100% saturated relative humidity, then removes the sunshade net, and the seedling normal growth is managed;
5) inoculation was carried out the stem rot investigation to the wheat stalk base portion after 35 days, was divided into the 0-5 level by the seriously ill index of stem rot, i.e. the 0=non-evident sympton; 1=first leaf sheath is brown withered less than leaf sheath length 10%; The brown withered leaf sheath length 11-25% that accounts for of 2=first leaf sheath; The brown withered leaf sheath length 25-50% that accounts for of 3=first leaf sheath; 4=second leaf sheath has obviously brown withered; 5=the 3rd leaf sheath has obviously brown withered or complete stool is withered; With disease index as the disease evaluation index:
Disease index=∑ (state of an illness rank * this grade strain number)/(investigating total strain number * the highest sick level) * 100%
Disease index＜10% is disease-resistant; Disease index 10.01-20.0% is anti-in being; Disease index 20.01-30.0% is middle sense; Disease index＞30.01% is susceptible.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101990819B (en) * | 2010-09-02 | 2011-11-30 | 江苏省农业科学院 | Fusarium graminearum caused rootstock rot seedling-stage resistance identification method |
| CN102061330B (en) * | 2010-11-30 | 2012-09-05 | 中国农业科学院油料作物研究所 | Method for identifying pathogenicity of sesame stem blight and blast pathogenic bacteria |
| CN104770175B (en) * | 2015-04-15 | 2017-08-15 | 广东省农业科学院作物研究所 | A kind of method of pathogenic wheel branch Fusariumsp indoor inoculation fresh edible maize |
| CN105850726B (en) * | 2016-04-08 | 2018-04-06 | 常州润达科技信息咨询有限公司 | A kind of ginkgo breeding of new variety method of anti-stem rot |
| CN106434521A (en) * | 2016-12-23 | 2017-02-22 | 河北省农林科学院植物保护研究所 | Culture base for inducing fusariumgraminearum schw to massively generate conidium |
| CN108220385A (en) * | 2017-05-08 | 2018-06-29 | 湖北省农业科学院粮食作物研究所 | A kind of method of Rapid identification wheat corn stalk rot caused by Fusarium |
| CN113403365A (en) * | 2021-07-21 | 2021-09-17 | 云南省农业科学院生物技术与种质资源研究所 | Identification method for resistance to damping-off of buckwheat |
| CN114480246A (en) * | 2022-02-14 | 2022-05-13 | 山东省农业科学院作物研究所 | Method for inducing fusarium graminearum to produce spores in large quantity and preparing spore liquid |
| CN114766354A (en) * | 2022-05-17 | 2022-07-22 | 洛阳农林科学院 | Method for identifying disease resistance of wheat |
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Non-Patent Citations (3)
| Title |
|---|
| Mitter V, et al..A high-throughput glasshouse bioassay to detect crown rot resistance in wheat germplasm.《Plant Pathology》.2006,第55卷433–441. * |
| Wallwork H, et al..Resistance to crown rot in wheat identified through an improved method for screening adult plants.《Australasian Plant Pathology》.2004,第33卷1-7. * |
| 李洪连, 徐敬友主编.实验实习二 小麦病害田间病情调查与品种抗病性.《农业植物病理学实验实习指导（植保 农学 园艺等专业用）》.中国农业出版社,2001,(第1版),25. * |
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