CN102808015B

CN102808015B - Method for identifying resistance of sclerotinia by inoculating in-vitro stalk of plant

Info

Publication number: CN102808015B
Application number: CN201210229952.1A
Authority: CN
Inventors: 钱伟; 梅家琴; 丁一娟; 魏大勇
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2012-07-04
Filing date: 2012-07-04
Publication date: 2014-10-15
Anticipated expiration: 2032-07-04
Also published as: CN102808015A

Abstract

The invention relates to a method for identifying the resistance of sclerotinia by inoculating an in-vitro stalk of a plant. The method comprises the following steps of: cutting the stalk or branches of the plant down, and putting the stalk or the branches into closed environment in which temperature and humidity are constant to inoculate sclerotinia sclerotiorum; and recording the extension lengths of bacterial plaques to measure the strength of disease resistance of the material. Compared with the conventional method for identifying the resistance of the stalk with sclerotinia, the method has the advantages that the method is performed under the condition of controllable environment, the pathogenetic condition is consistent, and inoculating identification is stable; the method is simple, short in period and high in inoculating efficiency; and inoculated parts are flexible, and a single plant can be identified repeatedly, so the method is suitable for the large-scale inoculating identification of the material.

Description

A kind of method of Vitro Plant cane inoculated identification resistance to sclerotinia sclerotiorum

Technical field

The invention belongs to plant pest Resistance Identification method, be specifically related to a kind of method of Vitro Plant cane inoculated identification resistance to sclerotinia sclerotiorum.

Background technology

Sclerotium disease can endanger 400 various plants, such as rape, and annual harm China rape underproduction 10 – 80% because of sclerotium disease.Cultivating disease-resistant variety is to prevent and treat sclerotium disease to endanger most economical approach.Simple, resistance to sclerotinia sclerotiorum authentication method is the key of the disease-resistant material of screening accurately.

Resistance to sclerotinia sclerotiorum qualification at present has blade Resistance Identification and cane Resistance Identification, blade Resistance Identification to comprise Isolated leaf inoculation, axil inoculation, petiole inoculation, cotyledon inoculation etc.Although these methods are simple to operate, the resistance of blade can not reflect the resistance of cane completely.Sclerotium disease is contaminated after cane, will cause output seriously to reduce.Cane Resistance Identification is had to field cane toothpick inoculation and cane mycelia piece binding method, and two kinds of methods are simple, are applicable to large-scale inoculation qualification, but are easily subject to the impact of land for growing field crops envrionment conditions, and inoculated identification is unstable.Such as running into the dry weather condition that are unfavorable for sclerotium disease morbidity that wait, the failure of land for growing field crops inoculated identification will be caused.

Summary of the invention

The object of the present invention is to provide a kind of method of simple, stable cane inoculated identification resistance to sclerotinia sclerotiorum.Present method, under indoor controlled condition, is created the temperature and humidity environment that suitable sclerotium disease is contaminated, and in vitro cane is carried out to inoculated identification, weighs the power of the anti-sclerotium disease of material according to the length of bacterial plaque.

Technical scheme of the present invention is as follows:

A method in vitro cane inoculated identification resistance to sclerotinia sclerotiorum, first, is paved with the edge punching of the PDA substratum of sclerotinite mycelia with punch tool edge, obtain PDA mycelia piece of the same size, inoculates in vitro cane as inoculum; Then intercept cane and in vitro cane is transferred to indoor, on cane, manufacture with the wound of inoculum same shape after, cover inoculum, in the constant indoor environment of temperature and humidity, carry out sclerotinite inoculation; Record bacterial plaque extension length, weigh the power of anti-sclerotium disease.

Advantage of the present invention: this inoculation method is that onset condition is consistent under the controllable environment condition of laboratory, inoculated identification is stable, method is simple, and inoculation efficiency is high, and the cycle is short, inoculation position is flexible, can carry out repetitive identified to individual plant, is applicable to material to carry out large-scale inoculation qualification.

Brief description of the drawings

Fig. 1 is the length of the 3rd day bacterial plaque after inoculation rape cane.

Embodiment

Below to identify a kind of embodiment of Btassica cane sclerotium disease disease resistance as the inventive method, but be not the restriction to the inventive method, any conversion not surpassing from flesh and blood of the present invention, must belong to protection scope of the present invention.

Embodiment 1

1. the preparation of inoculum

1) pathogenic bacteria is collected in experimental plot, rape engineering center, Chongqing City, its purge process is: the sclerotium of collection is inoculated in potato glucose substratum (PDA), in 22 DEG C of dark culturing 2 days, access new PDA substratum from edge picking mycelia, after 2 days, again the mycelia at edge is transferred, so repeatedly, until the mycelia growing in substratum does not contain other miscellaneous bacteria.Wherein being prepared as of PDA substratum: 200g potato, 20g sucrose and 15g agar powder, constant volume is to 1L.

2) isolated experiment inoculum (PDA mycelia piece) preparation: get bacterial classification one fritter after activation, be inoculated in the sterile petri dish central authorities that PDA substratum is housed, under 22 DEG C, 85% humidity condition, cultivate 2 days, evenly be paved with after media surface until mycelia, with the punch tool of diameter 6mm, along the punching of culture dish edge, the PDA mycelia piece of acquisition can be used in vitro cane inoculation.

2. in vitro cane is seeded in the whole florescence, at the stem of growing apart from the about 30cm of intercepting of 20cm place, ground 2 side shoots that intercept the long lowermost end of about 30cm, by preservative film winding (to prevent moisture loss) for cane two ends.Carry out sclerotium disease inoculated identification as an example of 17 parts of brassica plants example.These materials comprise 3 portions of Chinese cabbages (B.rapa), 2 parts of wild cabbages (B.oleracea), and 6 parts of swede type rapes (B.napus), 4 parts of mustard type rapes (B.juncea) and 2 parts of Ethiopia leaf mustard (B.carinata), its concrete material is in table 1.

Inoculation is to carry out in the transfer room of approximately 20 square metres of areas, temperature controllable.First by 1 × 2m ²three-ply-wood be positioned over and have a due proportion of, on the aluminum alloy frame of high 40cm, spread wet towel at three-ply-wood, pad one deck filter paper above.The cane of fetching is placed on the filter paper of getting ready, the punch tool that is 4mm with diameter is made a call to 2 holes on cane surface, thereby causes surface injury, between 2 holes, is spaced apart 10cm.The PDA mycelia piece face of carrying disease germs that is 6mm by diameter is close to site of injury (Fig. 1).After inoculation, above three-ply-wood, place again the aluminum alloy frame of a formed objects, finally with plastic film, the aluminum alloy frame of three-ply-wood and top is together airtight, and keep the humidity of airtight to maintain a constant humidity in 90-95%.Room temp is controlled at a steady temperature in 22-24 degree Celsius.After in vitro inoculation 3 days, measure the length of bacterial plaque, find that the bacterial plaque size of storeroom is variant obviously, its concrete manifestation is in table 1.There is significant positive correlation (r=0.657, P=0.911) in the size of stem and side shoot bacterial plaque.There is not significant correlation (stem: r=0.056, P=0.824 in the bacterial plaque length of stem and side shoot and the diameter of cane; Side shoot: r=-0.021, P=0.935).

The present embodiment result is as table 1.

The analytical results of this example unanimously shows, in vitro cane inoculated identification is that one can large-scale operation, and envrionment conditions is controlled, good stability, and inoculation efficiency is high, and flexibility ratio is high, can realize the method for the repetitive identified of individual plant.

Claims

1. a method for Vitro Plant cane inoculated identification resistance to sclerotinia sclerotiorum, first, is paved with the edge punching of the PDA substratum of sclerotinite mycelia with punch tool edge, obtain PDA mycelia piece of the same size, as inoculum; Then intercept plant stem, bar stem two ends are wound around with preservative film, and in vitro cane is transferred to indoor, on cane, manufacture with the wound of inoculum same shape after, inoculum in covering, the closed environment of contaminating in constant, the suitable sclerotium disease of temperature and humidity, carries out sclerotinite inoculation; Record bacterial plaque extension length, weigh the power of anti-sclerotium disease; The environment that described suitable sclerotium disease is contaminated is, sets a steady temperature within the scope of 22-24 DEG C, sets a constant humidity in 90-95% humidity range;

The cane of described in vitro inoculation is side shoot;

Described inoculation is to carry out in the transfer room of approximately 20 square metres of temperature controllables of an area, first by 1 × 2m ²three-ply-wood be positioned over and have a due proportion of, on the aluminum alloy frame of high 40cm, spread wet towel at three-ply-wood, pad one deck filter paper above; Then cane is placed on the filter paper of getting ready, the punch tool that is 4mm with diameter is made a call to 2 holes on cane surface, thereby causes surface injury, between 2 holes, is spaced apart 10cm; The PDA mycelia piece face of carrying disease germs that is 6mm by diameter is close to site of injury; After inoculation, above three-ply-wood, place again the aluminum alloy frame of a formed objects, with plastic film, the aluminum alloy frame of three-ply-wood and top is together airtight, and keep the humidity of airtight to maintain constant humidity.

2. according to the method for the Vitro Plant cane inoculated identification resistance to sclerotinia sclerotiorum described in claim 1, it is characterized in that: described plant is rape.

3. according to the method for the Vitro Plant cane inoculated identification resistance to sclerotinia sclerotiorum described in claim 1, it is characterized in that: within the 3rd to 4 days after inoculation, measure the length of bacterial plaque growth.