Method for screening disease-resistant plukenetia volubilis strain by water culture method

Abstract

The invention discloses a method for screening a disease-resistant plukenetia volubilis strain by a water culture method. The method relates to asexual propagation of semi-lignified branches of candidate plukenetia volubilis strains, controllable root injury inoculation of pathogenic bacteria and real-time observation of the disease progress of cuttings under the hydroponic condition, and then the plukenetia volubilis strains with disease resistance are simply and rapidly screened out. The time from the hydroponic cuttage culture of candidate branches to the preliminary screening of disease-resistant strains is only about 35 days, the time for detecting the disease resistance of the branches through soil cultivation is remarkably shortened, and in the screening process of the disease-resistant strains, phenotypic changes of adventitious roots of the cuttings and the bases of the adventitious roots can be observed with naked eyes after the adventitious roots and the bases of the cuttings are infected by pathogenic bacteria. The method provided by the invention is simple to operate, low in cost and high in efficiency, can be applied to preliminary screening of disease-resistant plukenetia volubilis candidate strains, can provide early-stage technical support for cultivation of the disease-resistant plukenetia volubilis strains, and has important application value.

Description

Technical Field

[0001] The invention belongs to the field of plant disease-resistant breeding, and particularly relates to a method for screening disease-resistant Elaeagnus oleifera strains using a hydroponic method. Background Art

[0002] Plukenetia volubil is L., also known as South American oil vine and Inca Inchi, is a perennial woody vine of the Euphorbiaceae family. Its seeds are rich in oil and protein, especially the α-linolenic acid (ω-3) content in its oil is as high as 53%, which is an ideal raw material for supplementing the serious ω-3 deficiency of the vast population in my country. Its oil has a significant effect on regulating blood lipids, preventing cardiovascular diseases, and maintaining skin, and is considered to be one of the best vegetable oils in the world.

[0003] At present, the star oil vine has been planted in large quantities in Guangxi, Guizhou, Hainan, Yunnan, and surrounding Southeast Asian countries. With the expansion of the planting area and the extension of the planting period, it was found that the field of star oil vine planted for more than two years is very prone to root and stem rot disease. It was identified that the disease was caused by infection with Fusarium oxysporum FoPvo1. Fusarium oxysporum is a soil-borne pathogenic fungus with a wide host range, and there is still no effective prevention and control method. Therefore, how to quickly and effectively screen out star oil vine strains resistant to Fusarium oxysporum and obtain disease-resistant varieties is an urgent problem to be solved in star oil vine breeding.

[0004] At present, there are no reports on the screening of disease-resistant germplasm materials for Aster oleifera. Traditional field screening of disease-resistant materials takes a long time. It takes two to three years or more for Aster oleifera to preliminarily determine whether these materials are disease-resistant. In addition, due to the differences in their growth environments, field survival rates cannot be used as a criterion for judging whether they are disease-resistant. The existing root inoculation method for some other plants has a long disease cycle and the infection process of pathogens cannot be observed at any time because the plants are transplanted to the soil after root inoculation. Therefore, it is urgent to develop an efficient, low-cost, fast and simple method to screen out disease-resistant Aster oleifera strains, and provide technical support for the cultivation of disease-resistant Aster oleifera varieties. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for screening disease-resistant truncatum strains by hydroponics. The root of hydroponic rooted cuttings is inoculated with pathogens, and then transferred into a transparent plastic bottle filled with single distilled water for cultivation, and the disease progression of the cuttings is observed in real time. The survival rate of common truncatum cuttings is lower than 30% as a reference, and the strains with a survival rate of cuttings higher than 70% are preliminarily identified as disease-resistant truncatum candidate strains, which can simply and quickly screen out truncatum strains with certain disease resistance. The method is simple to operate, low in cost, and high in efficiency, and significantly shortens the time for detecting its disease resistance through soil inoculation, providing technical support for the cultivation of disease-resistant truncatum strains.

[0006] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0007] A method for screening disease-resistant Astragalus melongena strains by hydroponic method, comprising the following steps:

[0008] Selection and preparation of S1 cuttings

[0009] Rooting induction and culture of S2 cuttings

[0010] After the prepared cuttings are disinfected, they are transferred to an aqueous solution with a rooting inducer for rooting induction; adventitious roots begin to grow at the base of the cuttings after 15-20 days, and the rooting inducer is: 0.3-0.8 mg / L IBA + 0-0.2 mg / L NAA;

[0011] S3 Cultivation and infection of pathogens to be tested

[0012] When the adventitious roots of the cuttings grow to 3-5 cm, the pathogens are activated and cultured to prepare a pathogen spore suspension, and the cuttings are taken out and the root tips of each adventitious root of 1-2 cm are cut off to wound the roots; the base of the cuttings and the adventitious roots after the wounding treatment are soaked in the pathogen spore suspension for 30-40 minutes to allow the pathogens to infect the roots of the cuttings;

[0013] S4 transferred the cuttings infected by the pathogen into a culture bottle filled with single distilled water for further cutting culture. After 10-14 days of inoculation, the survival rate of the cuttings from each strain was counted. The survival rate of the cuttings of ordinary Aster oil vine materials was lower than 30% as a reference, and the materials with a cutting survival rate higher than 70% were preliminarily identified as disease-resistant Aster oil vine candidate strains.

[0014] Preferably, the rooting inducer is IBA (0.5 mg / L) + NAA (0.2 mg / L).

[0015] Furthermore, the specific method for selecting and preparing the cuttings in S1 is as follows: from March to October, collect the semi-lignified tender branches of the healthy Asteraceae plant of the year, cut the tender branches into 15-30 cm cuttings, each cutting contains 2-5 leaves, remove 1 / 2-2 / 3 of each leaf, and use a blade to cut the base of the cutting into a 1-2 cm long bevel.

[0016] Furthermore, the rooting induction condition is a culture room at 25-30°C, 16h light / 8h dark.

[0017] Furthermore, the light intensity is 100 μEm -2 s -1 , simulating the natural growth conditions of the star oil vine, which is conducive to the rapid rooting of the cuttings.

[0018] Furthermore, the branches are semi-lignified young branches of the current year of the Aster oleifera plants that have survived for more than 5 years and grown robustly in the Aster oleifera planting area.

[0019] Furthermore, the pathogen is Fusarium oxysporum.

[0020] Furthermore, the spore concentration of the pathogen suspension is 8-10×10 5 Pieces / mL.

[0021] Furthermore, the pathogen infection soaking time is 30-40 minutes.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The invention provides a method for screening disease-resistant truncatum strains by hydroponic method, which has the following beneficial effects: 1) by using the hydroponic method to screen disease-resistant truncatum strains, compared with 70-80 days required for screening disease-resistant strains from soil cutting, pathogen inoculation and soil transplantation, only 35 days are required for culturing truncatum cuttings from hydroponic cuttings to preliminarily screening disease-resistant strains, which significantly shortens the screening time; 2) the death of plants caused by damage to the root system during the soil transplanting process is reduced, and the plants that die after the inoculation can be determined to be caused by the infection of pathogens; in addition, the disease process of the cuttings can be observed in real time, and the disease resistance performance of the screened candidate materials can be predicted and evaluated in time; 3) the method is simple to operate, low in cost and high in efficiency, can effectively screen disease-resistant truncatum strains, is suitable for large-scale screening applications, and provides technical support for the cultivation of disease-resistant truncatum strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a rooting diagram of the cuttings of the star oil vine inoculated with pathogenic bacteria of the present invention.

[0025] Figure 2This is a diagram showing the death and survival of cuttings of different strains of the Aster oleifera cuttings of the present invention after they were inoculated with pathogens and hydroponically cultured for 10 days. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0027] Example 1 A method for screening disease-resistant Aster oleifera strains using a hydroponic method

[0028] 1. Selection and preparation of cuttings: Collect semi-lignified young branches of the current year of the Asteraceae plants that have survived for more than 5 years and are growing robustly in each Asteraceae planting area, and then cut the young branches into 15-30 cm cuttings, each cutting containing 2-3 leaves, remove 1 / 2-2 / 3 of each leaf, and use a blade to cut the base of the cutting into a 1-2 cm long bevel;

[0029] 2. Rooting induction and cultivation of cuttings: Soak the prepared cuttings in 1‰ carbendazim or 2‰ potassium permanganate solution for 1-2 hours, then transfer them to a plastic cup filled with IBA (0.5mg / L) + NAA (0.1mg / L) aqueous solution and place them at 23-26℃ and light for 16h (light intensity of 100μEm -2 s -1 ) / dark room for 8 hours, root induction, wait for the base of the cuttings to start growing adventitious roots, wait for the adventitious roots of the cuttings to grow to 3-5cm, then the subsequent root injury and pathogen inoculation experiments can be carried out (such as Figure 1 );

[0030] 3. Cultivation of Fusarium oxysporum and root injury treatment of cuttings: Take the Fusarium oxysporum strain stored in a -80℃ refrigerator for activation culture, then transfer the activated strain into 50mL of PDB culture medium, culture at 25℃, 150rpm for 48h, and filter and microscopically check the spore count to 1×10 6 / mL for standby; take out the cuttings and cut off the root tip of each adventitious root 1-2cm to wound the root;

[0031] 4. Infection of adventitious roots of cuttings of Aster oleifera with Fusarium oxysporum: Soak the base of the cuttings and their adventitious roots after root injury treatment in the prepared Fusarium oxysporum solution for 30 minutes;

[0032] 5. Statistics on disease resistance and survival rate of cuttings of Aster oleifera: The cuttings infected with pathogens were transferred to culture bottles filled with distilled water for continued cutting culture. 7-10 days after inoculation, the leaves of some cuttings showed obvious yellowing and wilting, and the adventitious roots began to rot and then gradually died ( Figure 2); after 10-14 days of inoculation, the survival rate of the cuttings from each strain was counted, and the survival rate of the common Aster oleifera cuttings 14 days after inoculation was lower than 30% as a reference, and the strains with a cutting survival rate higher than 70% were preliminarily identified as disease-resistant Aster oleifera candidate strains, and further field disease resistance detection can be carried out. The survival rates of different Aster oleifera strains after inoculation of pathogens at different times are shown in Table 1. Through this method, strains with certain resistance to Fusarium oxysporum can be quickly and effectively screened, providing a simple and effective method for the breeding of disease-resistant varieties of Aster oleifera.

[0033] Table 1 Survival rate of different strains of Asteraceae after inoculation with pathogens at different times

[0034]

[0035] Example 2

[0036] To simulate natural conditions, the number of pathogens inoculated was reduced to observe the disease progression of the control and resistant strains. Example 1 was repeated with the following differences: the number of spores was about 8×10 5 / mL, the infection time is about 30 minutes. The inoculated material began to develop disease in 10-12 days. 17 days after inoculation, the average survival rate of the control was 31.4±5.1%, and the survival rate of the strain resistant to 3 was 82.1±5.3%.

[0037] Example 3

[0038] Example 1 was repeated with the following differences: the cuttings infected with the pathogen were transferred to sterilized vermiculite or soil for culture, and the leaves began to wilt and become diseased 20-25 days after inoculation. The average survival rate of the control was 29.2±4.8% 30 days after inoculation, and the survival rate of the resistant strains was 78.1±5.6%.

Claims

1. A method for screening disease-resistant Aster oleifera strains by hydroponics, comprising the following steps: Selection and preparation of S1 cuttings Rooting induction and culture of S2 cuttings After the prepared cuttings are disinfected, they are transferred to an aqueous solution containing a rooting inducer for rooting induction; adventitious roots begin to grow at the base of the cuttings after 15-20 days, and the rooting inducer is: 0.3-0.8 mg / L IBA + 0.1-0.2 mg / L NAA; S3 Cultivation and infection of pathogens to be tested When the adventitious roots of the cuttings grow to 3-5 cm, the pathogens are activated and cultured to prepare a pathogen spore suspension, and the cuttings are taken out and the root tips of each adventitious root of 1-2 cm are cut off to wound the roots; the base of the cuttings and the adventitious roots after the wounding treatment are soaked in the pathogen spore suspension for 30-40 minutes to allow the pathogens to infect the roots of the cuttings; S4 transferred the cuttings infected by the pathogen into a culture bottle filled with single distilled water for further cutting culture. After 10-14 days of inoculation, the survival rate of the cuttings from each strain was counted. The survival rate of the cuttings of ordinary Aster oil vine materials was lower than 30% as a reference, and the materials with a cutting survival rate higher than 70% were preliminarily identified as disease-resistant Aster oil vine candidate strains.

2. The method for screening disease-resistant Achyranthes bidentata strains by hydroponic method according to claim 1, characterized in that: The rooting inducing agent is IBA (0.5 mg / L)+NAA (0.1 mg / L).

3. The method for screening disease-resistant Achyranthes bidentata strains by hydroponic method according to claim 1, characterized in that: The specific method for selecting and preparing the cuttings in S1 is as follows: from March to October, collect the semi-lignified tender branches of the healthy star oil vine plant of the year, cut the tender branches into 15-30 cm cuttings, each cutting contains 2-5 leaves, remove 1 / 2-2 / 3 of each leaf, and use a blade to cut the base of the cutting into a 1-2 cm long bevel.

4. The method for screening disease-resistant Achyranthes bidentata strains by hydroponic method according to claim 1, characterized in that: The rooting induction condition is a culture room at 25-30°C, 16h light / 8h dark.

5. The method for screening disease-resistant Achyranthes bidentata strains by hydroponic method according to claim 4, characterized in that: The light intensity is 100 μEm -2 s -1 , simulating the natural growth conditions of the star oil vine, which is conducive to the rapid rooting of the cuttings.

6. The method for screening disease-resistant Achyranthes bidentata strains by hydroponic method according to claim 1, characterized in that: The branches are semi-lignified tender branches of the current year of the star oil vine plants which have survived for more than 5 years and grown robustly in the star oil vine planting area.

7. The method for screening disease-resistant Achyranthes bidentata strains by hydroponic method according to claim 1, characterized in that: The pathogen is Fusarium oxysporum.

8. The method for screening disease-resistant Achyranthes bidentata strains by hydroponic method according to claim 1, characterized in that: The spore concentration of the pathogen suspension is 8-10×10 5 Pieces / mL.

9. The method for screening disease-resistant Achyranthes bidentata strains by hydroponic method according to claim 1, characterized in that: The pathogen infection soaking time is 30 minutes.

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