CN110367123B - Resistance identification method for sweet potato leaf curl virus disease - Google Patents

Abstract

The invention discloses a method for identifying the resistance of sweet potato leaf-rolling virus disease, comprising the following steps: after the identified varieties are cultured and molecularly detected by detoxification test-tube seedlings, the original seedlings without leaf-rolling virus disease SPLCV; High-multiplication of leaf-roll virus indicator plants; identification of virus-free tissue culture seedlings isolation, insect vector inoculation under airtight conditions; identification of plant grafting and inoculation under the condition of net room; yield comparison test planting, harvesting, yield measurement, and seed conservation ; Seed in the second year, investigate and identify the emergence rate of leaf-rolling virus of the varieties; evaluate the resistance (resistance) of the varieties to leaf-rolling virus. The invention is an identification method for resistance to sweet potato leaf roll virus disease (SPLCV), which utilizes the transmission law of the virus to carry out natural virus transmission, insect vector inoculation, grafting inoculation for the identified varieties in different periods, and through the seedbed performance of each variety in the second year , according to the grading standard, to determine the resistance, tolerance and susceptibility characteristics of varieties to sweet potato leaf roll virus disease, and to provide basic resistance sources for the selection and breeding of sweet potato leaf roll virus disease-resistant varieties.

Description

A kind of resistance identification method of sweet potato leaf roll virus disease

technical field

The present invention relates to a resistance identification method for sweet potato leaf roll virus disease.

Background technique

As the seventh largest food crop in the world, sweet potato is mainly distributed in Asia, Africa, Latin America and other regions. China's planting area accounts for about 80% of the world's total. However, due to the widespread existence of sweet potato virus disease, the yield and quality of sweet potato are seriously affected. According to some data, the incidence of sweet potato virus disease in field varieties is 100%. In East Africa and South America, the production of sweet potato has been greatly reduced or even stopped due to virus disease. Disease is an important disease in sweet potato producing areas in China, and it is an important cause of sweet potato degradation and yield reduction. The infection of virus disease can reduce the yield by 30% to 50%. The sweet potato after removing the virus can greatly improve the yield and quality. Generally, the yield is increased by more than 20% after detoxification, and the highest is as much as 200%. big difference.

Gemini virus is the only type of single-stranded circular DNA virus with twin particle shape among plant viruses, and it is also one of the largest families of plant viruses. Important commercial crops cause devastating damage. Sweet potato gemini virus disease is one of the important viral diseases on sweet potato, and the incidence is on the rise, and the damage is becoming more and more serious. The geminivirus that infects sweet potato in my country is mainly sweet potato leaf curl virus (SPLCV). Viral high temperature hidden disease is often ignored in production, and it is also easy to spread with the exchange of sweet potato germplasm resources.

The identification methods of virus resistance (tolerance) of sweet potato varieties are rarely reported in China. Xing Jiying (2002) reported the virus resistance identification of more than 1200 domestic and foreign germplasm resources stored in Xuzhou Sweetpotato Research Center. The method was based on the unit of variety plot, and the symptoms and severity of leaf virus disease were visually observed and divided into five grades. Grade 0: carefully visually inspected the leaves without typical symptoms in the whole plot; Grade 1: about 10% or less of the leaves have a small amount of systemic symptoms; Grade 2: about 20% of the leaves have typical symptoms; Grade 3: 40% to 60% of the leaves have typical symptoms ; Grade 4: more than 80% of leaves have typical symptoms. Afterwards, the symptomatic leaf position of some varieties was investigated. The leaf position takes the first fully expanded young leaf on the stem tip as the first leaf position, and below it is the second, third, ..., and nth leaf positions, and serological methods are used to detect the sweet potato pinnate in different leaf positions of each variety. The virus-carrying situation of mottle virus was determined by the lowest leaf position where it appeared positive, and the virus-free seedlings and the non-virus-free control seedlings of the same variety were cultivated in the same field to compare the production performance. Ankang (2005) grafted the identified potato seedlings onto the indicator plant Ipomoea setosa, and used the indicator plants to show virus-carrying symptoms for virus resistance identification of germplasm resources. Wang Shuang published the "Sweet potato virus disease SPVD resistance identification method and yield loss estimation" paper in the Journal of Plant Protection (2014, Vol. 41, No. 2), using SPVD-infected sweet potato vines as scions Methods After grafting and inoculation, the field was transplanted. Grafting was carried out in the evening. Relatively strong scion and rootstock were selected. The survival rate of scion was close to 100%, and a relatively uniform incidence rate was obtained after grafting. Using disease index and yield loss rate as resistance evaluation indicators, there were significant differences among the varieties, indicating that they can be used for the identification of SPVD resistance of sweet potato varieties. The above three methods have certain feasibility for the virus resistance evaluation of some viruses in sweet potato varieties, but there are many problems in the identification of sweet potato leafroll virus mainly using insects as mediators, because there are two kinds of virus resistance performance of varieties: one is the variety It has antiviral genes, and the other is to reduce insect-borne viruses and prevent diseases. Therefore, when determining the leaf-roll virus resistance characteristics of varieties, try to get as close to production reality as possible. Sweet potato is a vegetatively propagated crop. The virus has a process of gradually accumulating and developing in sweet potato. The speed of accumulation is also an important indicator for evaluating the resistance and tolerance of varieties. Grafting and yield comparison of plant high-multiplication virus seedlings finally formed a complete method for identification of sweet potato leafroll virus disease resistance.

SUMMARY OF THE INVENTION

In view of the problems existing in the above-mentioned prior art, the present invention provides a resistance identification method for sweet potato leafroll virus disease.

In order to achieve the above object, a kind of resistance identification method of sweet potato leaf roll virus disease adopted in the present invention comprises the following steps:

1) Rapid propagation of the original seedlings of the varieties to be identified that do not contain leaf-rolling virus disease: in September last year, the stem tips of the potato seedlings of the varieties to be tested were taken in the tissue culture room, and the top meristems of 0.1-0.3 mm were cut under a microscope. The plants were obtained by culturing in the medium, virus detection was carried out, and the virus-free tissue culture seedlings were obtained. The virus-free tissue culture seedlings were propagated to 150 plants in the test tube, and were transplanted into the matrix plug trays after culturing for 35 days;

2) Rapid propagation of plugs: At the beginning of February, 150 detoxified tissue culture test-tube seedlings after culturing for 35 days were taken, divided into three equal parts, and each 50 were transplanted to plugs with a diameter of 0.7 cm and pre-added substrates. medium, cultivating for 30 days for later use;

3) Leaf Roll Virus indicator plant high multiplying propagation: the indicator plant Datura datura datura or heart leaf tobacco is planted in the seeding box, and when true leaves are grown, use the Severe Sweet Potato Leaf Roll Virus strain to do scion and carry out inoculation ( Grafting or rubbing), and inoculated with a certain amount of insect vectors (whitefly or aphids), cultivating for 60 days before use;

4) Isolation of detoxified domesticated seedlings of identified varieties, and inoculation of insect vectors under airtight conditions: The plugs with 45 viable and detoxified seedlings were placed in an artificial climate chamber with a temperature of 28 ° C and a humidity of 90%, and then the cultured 500 whitefly-carrying indicator plants, Datura datura or Nicotiana serrata, were simultaneously moved into an artificial climate chamber and cultivated for 30 days;

5) Poisoning treatment under the condition of identification plant net shed: ridges are planted in net sheds above 40 meshes, the ridge spacing and plant spacing of the varieties to be identified are determined according to the actual situation of the net shed, and the plots are arranged, and 45 detoxified domesticated seedlings are planted in the plot. , 14 days later, use the cultivated poisonous indicator plant Datura datura or Citrus datura as materials for grafting or friction inoculation, and wait for use;

6) Yield comparison test Planting, harvesting, yield measurement, and seed preservation: the insect vector inoculated, grafted inoculated, and uninoculated virus-free tissue culture seedlings of each identified variety were planted into the field at the same time, with 15 plants per treatment for each variety. A plot with 3 repetitions is generally planted on June 20 and harvested on October 15. The yields of the underground and aboveground parts of each plot are recorded respectively, and stored in different plots;

7) Investigate and identify the emergence rate of leaf roll virus symptoms: in March of the second year, samples of each variety were taken from the vector-infected, grafted, and uninfected plots, and 2.5 kg were randomly taken from each plot and divided into three groups. On the 30th day of seeding, the number of symptomatic seedlings and the number of non-symptomatic seedlings of leaf roll virus strains of the cultivar were investigated and identified, and recorded respectively, the symptomatic rate was calculated, and the resistance of the cultivar was evaluated by the average symptomatic rate;

8) Evaluation of the characteristics of resistance (resistance) to leaf-rolling virus of varieties:

Resistance: the average symptomatic rate is below 5%;

Tolerance: the average symptomatic rate is 5%-15%;

Sensation: the average symptomatic rate is above 15%-30%;

High sensitivity: the average symptomatic rate is above 30%.

Compared with the prior art, the present invention is an identification method for anti-sweet potato leaf roll virus disease (SPLCV), which utilizes the transmission law of the virus to carry out natural transmission, insect vector inoculation, grafting and inoculation for the identified varieties in different periods, and through The seedbed performance of each variety in the second year is classified according to the grading standard to determine the resistance, tolerance and susceptibility of the variety to sweet potato leaf roll virus disease, and to provide basic resistance sources for the selection and breeding of sweet potato leaf roll virus disease-resistant varieties.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below. However, it should be understood that the specific embodiments described herein are only used to explain the present invention, and not to limit the scope of the present invention.

A method for identifying resistance to sweet potato leaf roll virus disease, comprising the following steps:

1) After detoxification culture and molecular detection of the varieties to be identified, it is confirmed that the original seedlings without leaf roll virus disease SPLCV are rapidly propagated

The potato seedlings of the tested varieties were detoxified and cultured in the tissue culture room in September of the previous year: take the shoot tips of the tested varieties → cut 0.1-0.3mm top meristems under the microscope → cultivate in the medium → obtain the plants → Carry out virus detection (the detection method is in accordance with the industry standard NY/T402-2016) → obtain detoxified tissue culture seedlings → propagate to 150 plants in the test tube of detoxified tissue culture seedlings, and transplant them into matrix plug trays after culturing for 35 days;

2) Quick multiplication of plugs

At the beginning of February, 150 detoxified tissue culture test-tube seedlings that had been cultivated for 35 days were divided into three parts, and each 50 plants were transplanted into plug trays with a diameter of 0.7 cm and pre-added matrix, and they were used after culturing for 30 days;

3) High multiplication of leaf-roll virus indicator plants

Plant the indicator plant Datura datura or Citrus datura in a flowerpot, and when two true leaves are produced, the collected sweet potato leaf roll virus severe strain is grafted as a scion, and inoculated with whitefly or aphids, grafting, inoculation Standby after 60 days of culture;

4) Isolation of detoxified tissue culture seedlings for identification of varieties and inoculation of insect vectors under airtight conditions

The plugs with 45 viable virus-free seedlings were placed in an artificial climate chamber (temperature 28°C, humidity 90%) (10 square meters, 100 varieties can be identified). 30 strains of louse-carrying indicator plants Datura datura or Nicotiana sinensis were simultaneously moved into an artificial climate chamber (infected by insect vectors), and cultivated for 30 days in total;

5) Identification of plants exposed to poison under the condition of net room

A small ridge was formed in a 40-mesh net room (the distance between the ridges was 75 cm, and the density was 4500 plants/mu), and 45 detoxified tissue culture seedlings of the species to be identified were planted in the net room. Fruit mandala or heart leaf tobacco is used as material for grafting or rubbing poisoning, ready to use;

6) Yield comparison test planting, harvesting, yield testing, and seed conservation

Three kinds of detoxified tissue culture seedlings of each identified variety (infected by insect vector, grafted inoculated, and not inoculated) were planted into the field at the same time, and 15 plants of each variety were treated as a plot, with 3 repetitions, usually on June 20. Planting, harvesting on October 15, recording the yields of the underground and aboveground parts of each plot, and storing them in different plots;

7) Seed in the second year, investigate and identify the emergence rate of leaf roll virus disease

In March of the second year, each variety was sampled from the vector-infected, grafted, and uninfected plots, and 2.5 kg of each plot was randomly divided into three parts for seeding. On the 30th day of seeding, the leaf-rolling virus strains of the varieties were investigated and identified. The number of symptomatic seedlings and the number of non-symptomatic seedlings were recorded separately, the symptomatic rate was calculated, and the resistance of the variety was evaluated by the average symptomatic rate;

8) Evaluation of varieties' resistance (resistance) to leaf-rolling virus

Resistance: the average symptomatic rate is below 5%;

Tolerance: the average symptomatic rate is 5%-15%;

Sensation: the average symptomatic rate is above 15%-30%;

High sensitivity: the average symptomatic rate is above 30%.

Example 1

In 2015, 2016 and 2017, the identification of resistance to leaf-rolling virus disease of some main sweet potato varieties was carried out continuously, and the variety identification was carried out according to the above-determined method for identification of resistance to sweet potato leaf-rolling virus disease (SPLCV):

Cultivation and molecular detection of virus-free test-tube seedlings of the to-be-identified varieties Original seedlings without leaf-rolling virus disease SPLCV → fast propagation in plugs → high-multiplication of leaf-rolling virus indicator plants → isolation and airtight conditions of virus-free tissue culture seedlings of identified varieties Inoculation by insect vector → identification of plant grafting and inoculation under the condition of net room → yield comparison test planting, harvesting, yield measurement, and seed preservation → seeding in the second year, investigation and identification of the emergence rate of leaf-rolling virus disease → according to leaf-rolling The average value of the virus symptom rate was used to evaluate the resistance (resistance) of the varieties to leaf-rolling virus. The identification of each main cultivar is shown in Table 1 below.

Table 1 Identification table of resistance to sweet potato leaf roll virus disease (SPLCV)

Combined with Table 1, among the 14 varieties identified, Shangshu 19, Longshu No. 9, Sushu No. 8 had no leaf rolling symptoms under the condition of natural transmission (not inoculated) and insect-borne inoculation. In the case of inoculation, only about 10% of the diseased rate of leaf rolling was found, indicating that the above varieties are relatively disease-resistant, which is very consistent with the production; In the case of no or a small amount of leaf rolling symptoms, in the case of grafting and poisoning, only about 30% of the leaf rolling symptoms are found, which is relatively disease-resistant; They are susceptible varieties. Zhenghong 22, Jihei No. 1 and Jishu 26 are highly susceptible varieties, which are in good agreement with the actual situation in production.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (1)

1. a method for identifying the resistance of sweet potato leaf roll virus disease, is characterized in that, comprises the following steps: 1) Rapid propagation of the original seedlings of the varieties to be identified that do not contain leaf-rolling virus disease: in September last year, the shoot tips of the potato seedlings to be tested were taken from the tissue culture room, and the top meristems of 0.1-0.3 mm were cut under a microscope. The plants were obtained by culturing in the medium, virus detection was carried out, and the virus-free tissue culture seedlings were obtained. The virus-free tissue culture seedlings were propagated to 150 plants in the test tube, and were transplanted into the matrix plug trays after culturing for 35 days; 2) Rapid propagation of plugs: At the beginning of February, 150 detoxified tissue culture test-tube seedlings after 35 days of culture were taken, divided into three equal parts, and each 50 were transplanted to plugs with a diameter of 0.7 cm and pre-filled with matrix medium, cultivating for 30 days for later use; 3) High-multiplication of leaf-rolling virus indicator plants: Plant the indicator plants Datura datura or Citrus datura in a seeding box, and when true leaves grow, use a severe strain of sweet potato leaf-rolling virus as a scion for inoculation. And inoculated with a certain amount of insect vectors, cultivating for 60 days after use; 4) Isolation of detoxified domesticated seedlings of identified varieties, and inoculation of insect vectors under airtight conditions: Put the plugs with 45 live detoxified seedlings in an artificial climate chamber with a temperature of 28°C and a humidity of 90%, and then cultivate the well-cultivated seedlings. 500 whitefly-carrying indicator plants, Datura datura or Nicotiana serrata, were simultaneously moved into an artificial climate chamber and cultivated for 30 days; 5) Poisoning treatment of identified plants under the condition of net shed: ridges are planted in net sheds above 40 meshes, the ridge spacing and plant spacing of the varieties to be identified are determined according to the actual situation of the net shed, and the plots are arranged, and 45 detoxified domesticated seedlings are planted in the plot. , 14 days later, use the cultivated poisonous indicator plant Datura datura or Citrus datura as materials for grafting or friction inoculation, and wait for use; 6) Yield comparison test Planting, harvesting, yield measurement, and seed preservation: plant the virus-free tissue culture seedlings of each identified variety with inoculated, grafted and uninoculated seedlings into the field at the same time, with 15 plants per treatment for each variety. One plot, set up 3 repetitions, planted on June 20, harvested on October 15, recorded the yields of the underground and aboveground parts of each plot, and stored them in different plots; 7) Investigate and identify the emergence rate of leaf roll virus symptoms: in March of the second year, samples of each variety were taken from the vector-infected, grafted, and uninfected plots, and 2.5 kg were randomly taken from each plot and divided into three groups. On the 30th day of seeding, the number of symptomatic seedlings and the number of non-symptomatic seedlings of leaf roll virus strains of the cultivar were investigated and identified, and recorded respectively, the symptomatic rate was calculated, and the resistance of the cultivar was evaluated by the average symptomatic rate; 8) Evaluation of varieties' resistance and resistance to leaf-rolling virus: Resistance: the average symptomatic rate is below 5%; Resistance: the average symptomatic rate is 5%-15%; Sensation: The average symptomatic rate is 15%-30%; High sense: the average symptomatic rate is above 30%.