CN102282988B - Method for infecting arabidopsis by utilizing bursaphelenchus spp. - Google Patents

Abstract

The invention discloses a method for infecting Arabidopsis thaliana with the nematode of the genus Schwarz. The method provided by the invention comprises the following steps: inoculating the nematodes of the genus Umbrella to Arabidopsis thaliana with wounded petioles; and the Arabidopsis is Arabidopsis thaliana 10 days after emergence. The present invention obtains a higher infection rate by screening the inoculation conditions, which proves that Arabidopsis can be used as an alternative host for the genus Schizophrenia nematode, and overcomes the complex genetic background that exists when pine trees are used as hosts to study the pathogenic molecular mechanism of pine xylophilus, Difficult problems with genetic manipulation.

Description

A method of infecting Arabidopsis thaliana with the nematode of the genus Schwarz

technical field

The present invention relates to a method for infecting Arabidopsis thaliana with the nematode of the genus Schwarz.

Background technique

Bursaphelenchus spp. includes Bursaphelenchus xylophilus, Bursaphelenchus mucronatus and the like.

Pine wood nematode is a second-class quarantine nematode in my country, and it is also an important quarantine pest recognized internationally. The pine wood nematode disease caused by it is a devastating disease of pine trees, which can harm more than 50 species of pine trees and 10 species. A variety of non-pine tree species, pine trees usually die within 2 to 3 months after being infected. Since the first discovery of pine wood nematode disease in Nanjing Zhongshan Mausoleum in 1982, the disease has spread rapidly in my country, and the damage has become increasingly serious. At present, 197 counties (cities, districts) in 15 provinces (regions, cities) have occurred in my country. The pine wood nematode epidemic has killed more than 500 million pine trees, destroyed more than 5 million mu of pine forests, and caused economic losses of hundreds of billions of yuan. Pine wood nematode disease has become a major "killer" of green pine forest resources.

Pine wood nematode disease involves many aspects such as hosts, nematodes, long beetles, fungi, bacteria and environmental factors. There is no very economical and effective control method. Breeding for disease resistance is one of the main methods to prevent and control plant diseases. Its effect is stable, the cost is low, it can reduce or avoid the pollution of pesticides to the environment, and it is beneficial to maintain ecological balance. Pine disease-resistant breeding is also an effective method to control pine xylophilus. To study the pathogenic mechanism of B. xylophilus, understand the molecular mechanism of B. xylophilus-host interaction, obtain the target genes that play a key role in the parasitism and pathogenic process of B. xylophilus, and provide specific targets for the development of B. xylophilus target proteins. Sexual drugs and line-resistant breeding provide a theoretical basis. At present, the research on the pathogenic mechanism of pine wood nematode only stays in the isolation and identification of pathogenicity-related genes. Due to the unstable effect of RNAi interference on pine wood nematode, the research and analysis of the function of disease-causing genes is in trouble. At the same time, the complex genetic background of pine trees, the weak foundation of molecular biology research and the difficulty of genetic manipulation also hinder the research on the pathogenic mechanism of pine xylophilus and the molecular mechanism of interaction with the host.

Kirst et al. (2003) carried out large-scale high-quality EST sequencing and analysis on the tissues and organs related to the formation of loblolly pine wood, and found that more than 90% of the gene sequences larger than 1100bp can find similar expression sequences in Arabidopsis; Avila and Liu The promoters of the Pinus glutamine synthetase gene and the pathogen/wound-induced PR10 gene were respectively transferred into Arabidopsis, and it was found that the genome of Pinus plants not only has a high similarity with Arabidopsis, but also the gene expression regulation mode Also very similar. Pine xylophilus is a migratory endoparasitic nematode, which moves freely in the host body and feeds. It can infect pine trees at all ages and there is no deformation of the worm body during the feeding process. The nematode mainly parasitizes in the trunk and branches. Establishing a method for B. xylophilus to infect Arabidopsis thaliana, and using Arabidopsis as an alternative host to study the relationship between B. xylophilus and plants will provide great convenience for the study of the molecular biology mechanism of B. xylophilus-host interaction , to provide a research platform for revealing the pathogenic mechanism of this nematode.

Contents of the invention

The object of the present invention is to provide a method for infecting Arabidopsis thaliana with the nematode of the genus S. thaliana.

The method for infecting Arabidopsis thaliana with Bursaphelenchus spp. provided by the invention comprises the following steps: inoculating Bursaphelenchus spp. to Arabidopsis thaliana with wounds on petioles; Arabidopsis after 10 days.

The nematode of the genus Umbrella is Bursaphelenchus xylophilus or Bursaphelenchus mucronatus.

Each strain of the Arabidopsis thaliana can be inoculated with 300-400 nematodes.

The Arabidopsis thaliana can be Arabidopsis 10 to 25 days after emergence.

The Arabidopsis can be Chi-0 ecotype Arabidopsis, C24 ecotype Arabidopsis, Col-0 ecotype Arabidopsis, Nd-1 ecotype Arabidopsis, Pa-3 ecotype Arabidopsis, Ler ecotype Arabidopsis, Est-1 ecotype Arabidopsis, Ws-2 ecotype Arabidopsis, Sha ecotype Arabidopsis or Kas-1 ecotype Arabidopsis.

The nematodes are preferably sterile nematodes.

The preparation method of described sterile nematodes may comprise the steps:

(1) the nematode is sterilized;

(2) collecting the nematode eggs obtained in step (1);

(3) the worm eggs collected in step (2) are disinfected;

(4) Cultivate the eggs obtained in step (3) into nematodes, which are sterile nematodes.

The step (1) specifically includes the following steps: washing the nematode with a sterile M9 solution, then disinfecting it with a 1g/100mL thimerosal aqueous solution for 30min (at room temperature), and then disinfecting it with solution A at 4°C for 10h; and water; the solute and its concentration in the M9 solution are as follows: 22mM KH ₂ PO ₄ , 42mMNa ₂ HPO ₄ , 85.5mM NaCl, 1mM MgSO ₄ ; the solution consists of streptomycin sulfate, actin Composition of cycloheximide and water, the concentration of streptomycin sulfate is 0.1g/100mL, and the concentration of cycloheximide is 0.02g/100mL.

The step (2) specifically includes the following steps: inoculate the nematodes obtained in the step (1) onto a culture medium (such as a PDA medium) with Botrytis cinerea colonies, observe that the eggs are produced, and use a sterile Rinse with water and collect the nematodes and disinfect with 1g/100mL thimerosal aqueous solution for 30min (room temperature); then place the nematodes in a petri dish filled with 0.9% sterile saline (ie 0.9g/100mL sodium chloride solution) and cultivate them at 25°C, Until a large number of worm eggs are observed, the solution is poured out, and the worm eggs attached to the petri dish are collected with solution B after rinsing with sterile water; the solution B is composed of Tween, NaOH and water, and the volume percentage of Tween The content is 0.05%, and the concentration of NaOH is 8mM.

The step (3) specifically includes the following steps: washing the eggs collected in the step (2) with the sterile M9 solution, then disinfecting with 1g/100mL thimerosal aqueous solution for 30min (at room temperature), and then using the solution A 4°C Disinfect for 10 hours.

The step (4) specifically includes the following steps: inoculating the eggs obtained in the step (3) on a culture medium (such as PDA medium) with Botrytis cinerea colonies and culturing them until the eggs develop into nematodes. The step (4) may also include the step of carrying out PCR identification of the ovum and the nematode; the primers used for the PCR identification are sequence 1 (F1) of the sequence listing and sequence 2 (F2) of the sequence listing composed primer pairs.

Any of the above methods can be applied to the following (a) or (b) or (c) or (d):

(a) identification of the pathogenicity of the genus Schmidt nematode;

(b) identification of the susceptibility of different ecotypes of Arabidopsis thaliana to the genus Schmidt;

(c) isolation and identification of a gene in a plant that has the function of resisting the nematode of the genus Schizophrenia;

(d) Breeding plants resistant to the spp. nematode.

The nematode of the genus Bursaphelenchus may be Bursaphelenchus xylophilus or Bursaphelenchus mucronatus.

The Arabidopsis can be Chi-0 ecotype Arabidopsis, C24 ecotype Arabidopsis, Col-0 ecotype Arabidopsis, Nd-1 ecotype Arabidopsis, Pa-3 ecotype Arabidopsis, Ler ecotype Arabidopsis, Est-1 ecotype Arabidopsis, Ws-2 ecotype Arabidopsis, Sha ecotype Arabidopsis or Kas-1 ecotype Arabidopsis.

Inoculate 400 nematodes/strain under the condition that Arabidopsis thaliana has a wound on the petiole 15 days after emergence, and the infection rate of Arabidopsis thaliana can be as high as 90%. Therefore, Arabidopsis can be used as an alternative host for B. xylophilus. follow up research. The experimental results of Example 5 show that the present invention can quickly and efficiently screen the sensitivity of different ecotypes of Arabidopsis thaliana to B. xylophilus, although there is no ecotype that is completely resistant to B. xylophilus infection among the nine ecotypes currently tested. , but with different degrees of susceptibility to pine xylophilus. The experimental results of embodiment 6 show that the present invention is also applicable to B. xylophilus, and the infection rate of B. thaliana to Arabidopsis with weak pathogenicity is not as high as that of B. xylophilus, indicating that Arabidopsis can be used for Identification of B. xylophilus and B. xylophilus species of varying virulence.

The present invention, on the basis of obtaining aseptic pine wood nematodes, establishes the inoculation of Arabidopsis thaliana by the nematodes of the genus Schizophrenia (especially pine wood nematodes) under aseptic conditions by screening the inoculation method, inoculation concentration, and inoculation period of the nematodes. The best approach to make Arabidopsis a reality as an alternative host for the genus Schizophrenia elegans.

The invention can be used to screen the Arabidopsis ecotypes resistant to the genus genus genus, further isolate and obtain the resistance gene, provide conditions for line-resistant breeding, and can also be used to identify the pathogenicity of the genus genus genus nematodes from different sources. Utilizing the Arabidopsis ecotypes screened out by the method of the present invention for resisting the genus Sclerosus nematodes can accelerate the pathogenic mechanism of the genus Sclerosus nematodes (especially pine wood nematodes) and the molecular mechanism of interaction between the genus Sclerosus nematodes and plants The study provides theoretical guidance for the control of pine wood nematode. At the same time, the genome sequencing of Arabidopsis thaliana has been completed, the mutants are easy to obtain, and the gene isolation is convenient and fast, so that Arabidopsis can be used to study the interaction between nematodes and plants of the genus A. Genes that play a key role in the interaction between nematodes and plants, clarify the pathogenic mechanism of the genus C. The present invention obtains a higher infection rate by screening the inoculation conditions, which proves that Arabidopsis can be used as an alternative host for the genus Schizophrenia nematode, and overcomes the complex genetic background that exists when pine trees are used as hosts to study the pathogenic molecular mechanism of pine xylophilus, Difficult problems with genetic manipulation.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Pine wood nematode (Bursaphelenchus xylophilus): The public can obtain it from China Agricultural University; references are: Ge Jianjun, Cao Aixin, Liu Xianbao, Chen Changfa. Application of TaqMan-MGB probes for real-time fluorescence quantitative detection of pine wood nematode.[J]. Acta Phytopathology, 2005, 35(6): 52-58.

Pine wood nematode (Bursaphelenchus mucronatus): The public can obtain it from China Agricultural University; references are: Ge Jianjun, Cao Aixin, Liu Xianbao, Chen Changfa. Research on real-time fluorescence quantitative detection technology of pine wood nematode using TaqMan-MGB probe.[J] . Phytopathological Journal, 2005, 35(6): 52-58.

Botrytis cinerea: The public can obtain it from China Agricultural University; References: Ge Jianjun, Cao Aixin, Liu Xianbao, Chen Changfa. Application of TaqMan-MGB probes for real-time fluorescence quantitative detection of pine xylophilus.[J] . Phytopathological Journal, 2005, 35(6): 52-58.

Embodiment 1, the cultivation of Arabidopsis thaliana and pine xylophilus

1. Arabidopsis culture

The Arabidopsis varieties used in Examples 2 to 7 include: Chi-0 ecotype Arabidopsis, C24 ecotype Arabidopsis, Col-0 ecotype Arabidopsis, Nd-1 ecotype Arabidopsis, Pa- 3 ecotype Arabidopsis, Ler ecotype Arabidopsis, Est-1 ecotype Arabidopsis, Ws-2 ecotype Arabidopsis, Sha ecotype Arabidopsis and Kas-1 ecotype Arabidopsis. All Arabidopsis varieties were purchased from the Arabidopsis Biological Resource Center (ABRC).

Arabidopsis thaliana of each variety was cultivated by the following method: Arabidopsis seeds were sterilized with 70% (volume ratio) ethanol aqueous solution for 5 minutes, then sterilized with 1% (g/100mL) sodium hypochlorite aqueous solution for 10 minutes, and rinsed with sterile water 3 times. The sterilized Arabidopsis seeds were sown on-demand on sterilized MS solid medium (3% sucrose, 0.7% agar; pH5.8), 10 seeds per dish, sealed with parafilm, and treated at 4°C for 3 days. Then transfer to a light incubator for cultivation (light 2500LX, temperature 25°C, photoperiod 16h/8h, incandescent light).

2. Cultivation of pine wood nematode

Botrytis cinerea was inoculated on the PDA medium, cultivated in a constant temperature incubator at 25°C for 7 days, and when the aerial hyphae basically covered the petri dish, the pine wood nematode was inoculated (the culture medium that preserved the pine wood nematode was placed in the above the PDA medium covered with aerial hyphae), cultivated in a constant temperature incubator at 25°C for 10 days, and separated the pine wood nematode by the Berman funnel method after the mycelium was basically eaten by the nematodes (for specific methods, see the literature: Viglierchio DR and Schmitt RV. On the methodology of nematode extraction from field samples: baermann funnel modifications. [J]. Journal of nematology, 1983, 15(3): 438-444.), spare.

3. The acquisition of pine xylophilus without bacterial contamination

1. Disinfection of pine wood nematode

(1) Wash the pine xylophilus obtained in step 2 with sterile M9 solution (22mM KH ₂ PO ₄ , 42mM Na ₂ HPO ₄ , 85.5mMNaCl, 1mM MgSO ₄ ) for 3-4 times (each time by centrifuging at 3000rpm to collect the worm body ).

(2) The worm body that step (1) obtains is sterilized with 1% (g/100mL) thimerosal aqueous solution at room temperature for 30min, then with solution A (composed of streptomycin sulfate, cycloheximide and water, streptomycin sulfate The concentration is 0.1g/100mL, the concentration of cycloheximide is 0.02g/100mL) sterilized at 4°C for 10h, and centrifuged at 3000rpm to collect worms.

(3) Rinse the worm body obtained in step (2) with sterile water 3 times (collect the worm body by centrifugation at 3000rpm each time), suspend the worm body with sterile water and inoculate it into the PDA with Botrytis cinerea colonies culture medium (aerial hyphae basically cover the petri dish).

(4) After a small amount of eggs are produced in the culture medium of step (3) (observed through a microscope), the nematodes are collected by washing with sterile water, and the worm bodies are collected by centrifugation at 3000 rpm.

(5) The nematodes obtained in step (4) were sterilized with 1% (g/100mL) thimerosal aqueous solution at room temperature for 30 minutes, washed with sterile water for 3 times (the worms were collected by centrifugation at 3000rpm each time), and then the nematodes were placed in a 0.9 % physiological saline (sterile) in a petri dish, culture at 25°C, after a large number of eggs are produced (12-24 hours, observed through a microscope), pour out the solution in the petri dish, and rinse the petri dish with sterile water 7 - 8 times until the bottom of the dish is free of worms, only eggs (nematodes are present in the solution, and eggs are attached to the wall and bottom of the dish).

2. Collect and disinfect eggs

(1) Collection of eggs

Use solution B (composed of Tween, NaOH and water, the volume percentage of Tween is 0.05%, the concentration of NaOH is 8mM) to wash the dish wall and the bottom of the dish in (5) of step 1, and collect worm eggs.

(2) Disinfection of eggs

Replace the worm body with worm eggs, and the others are the same as (2) in step 1.

3. Bacteria detection of eggs

Using the eggs obtained in step 2 as a template, perform PCR amplification with a primer pair composed of F1 and R1 (bacterial universal primers), and detect the PCR amplification products by electrophoresis. Those without amplified bands are eggs without bacterial contamination.

F1 (upstream primer): 5'-AGAGTTTGATCATGGCTCAG-3';

F2 (downstream primer): 5'-ACGGTTACCTTGTTACGACTT-3'.

4. Cultivation of eggs without bacterial contamination

Eggs without bacterial contamination were inoculated on PDA medium (aerial hyphae basically covered the test tube) with Botrytis cinerea colonies and cultured until the eggs developed into nematodes. Grind nematodes into a homogenate under sterile conditions as a template, use a primer pair composed of F1 and R1 (bacterial universal primers) for PCR amplification, and detect PCR amplification products by electrophoresis, and those without amplified bands are without bacterial contamination nematodes.

5. Suspend the nematodes without bacterial contamination in sterile water, count them under a microscope, adjust to the expected concentration, and carry out the experiments of Examples 2 to 5 as the nematode solution. The inoculation method in Examples 2 to 5 is to add the nematode solution dropwise to the top of the plants, and the nematode solution freely diffuses to all the plants.

Embodiment 2, the screening of inoculation method

1. Group and inoculate

Chi-0 ecotype Arabidopsis plants (emergence 14 days) were divided into three groups, 30 plants in each group, and were processed as follows:

The first group: pinch the petiole with sterile tweezers, then inoculate the nematode solution obtained in Example 1, and the inoculation concentration is 300/strain;

The second group: pinch the stem with sterile tweezers, then inoculate the nematode solution obtained in Example 1, and the inoculation concentration is 300/strain;

The third group: do not do any treatment, directly inoculate the nematode solution obtained in Example 1, the inoculation concentration is 300/strain;

The Arabidopsis thaliana after the above treatments were respectively cultured under the same conditions for 3 days, and each plant was washed with sterile water respectively.

2. Results of evaluation step 1 screening of inoculation methods

The Arabidopsis thaliana obtained in step 1 is dissected, and whether pine xylophilus infestation has occurred in the plant is checked under a microscope, and the number of plants infected with pine wood nematode is counted, and the infestation rate of Arabidopsis thaliana is calculated (that is, the internal infection nematode The number of plants accounted for the percentage of the total number of plants). The results are shown in Table 1. The results showed that the first group (with wounds on the petioles) was very favorable for the nematode infection and was the highest; while the Arabidopsis in the third group and the second group (with wounds on the stem) were extremely low infested by nematodes.

Table 1 Infection rate of Arabidopsis thaliana after different nematode inoculation methods

First group Second Group The third group Infection rate of Arabidopsis thaliana % 73.33±5.77a 13.33±5.77b 6.67±5.77b

Embodiment 3, the screening of nematode inoculum concentration

1. Group and inoculate

Chi-0 ecotype Arabidopsis plants (emergence 14 days) were divided into three groups, 30 plants in each group, and were processed as follows:

The first group: pinch the petiole with sterile tweezers, then inoculate the nematode solution obtained in Example 1, and the inoculation concentration is 200/strain;

The second group: pinch the petiole with sterile tweezers, then inoculate the nematode solution obtained in Example 1, and the inoculation concentration is 300/strain;

The third group: pinch the petiole with sterile tweezers, then inoculate the nematode solution obtained in Example 1, and the inoculation concentration is 400/strain;

The Arabidopsis thaliana after the above treatments were respectively cultured under the same conditions for 3 days, and each plant was washed with sterile water respectively.

2. Results of evaluation step 1 inoculation concentration screening

Dissect the Arabidopsis thaliana obtained in step 1, check under a microscope whether pine xylophilus infection has occurred inside the plant, count the number of plants internally infected with pine xylophilus, and calculate the infection rate of Arabidopsis thaliana. The results are shown in Table 2. The results showed that the higher the concentration of nematodes, the higher the infection rate of Arabidopsis thaliana, and the third group (400 nematodes/plant) was significantly higher than the other two groups.

Table 2 Infection rate of Arabidopsis thaliana with different nematode inoculation concentrations

First group Second Group The third group Infection rate of Arabidopsis thaliana % 56.67±5.77b 73.33±5.77b 83.33±5.77a

Embodiment 4, the screening of nematode inoculation period

1. Inoculation

Chi-0 ecotype Arabidopsis plants (respectively 5 days, 10 days, 15 days or 25 days after emergence) were treated as follows (30 plants at each emergence stage): the petiole was pinched with sterile tweezers, Then the nematode solution obtained in Example 1 was inoculated, and the inoculation concentration was 400/strain; the Arabidopsis thaliana after the above treatment were continued to be cultured under the same conditions for 3 days, and each plant was rinsed with sterile water respectively.

2. The results of the evaluation step 1 inoculation period screening

Dissect the Arabidopsis thaliana obtained in step 1, check under a microscope whether pine xylophilus infection has occurred inside the plant, count the number of plants internally infected with pine xylophilus, and calculate the infection rate of Arabidopsis thaliana. The results are shown in Table 3. The results showed that the infection rate of Arabidopsis thaliana reached the highest 15 days after emergence, and the rate of infection by nematodes was relatively low at 5 days after emergence because the leaves were small and unfavorable for nematode invasion.

Table 3 Infection rate of Arabidopsis at different nematode inoculation stages

Embodiment 5, Different ecotypes of Arabidopsis thaliana are sensitive to pine xylophilus

1. Sensitivity determination of different ecotypes of Arabidopsis thaliana to pine xylophilus

C24 ecotype Arabidopsis, Col-0 ecotype Arabidopsis, Nd-1 ecotype Arabidopsis, Pa-3 ecotype Arabidopsis, Ler ecotype Arabidopsis, Est-1 ecotype Arabidopsis Thaliana, Ws-2 ecotype Arabidopsis, Sha ecotype Arabidopsis and Kas-1 ecotype Arabidopsis (15 days after emergence) were treated as follows (30 plants for each ecotype): The petiole was pinched, and then inoculated with the nematode solution obtained in Example 1, the inoculation concentration was 400/strain; the Arabidopsis thaliana after the above-mentioned treatment were continued to be cultivated under the same conditions for 3 days, and each was rinsed with sterile water respectively. plants.

2. The results of the evaluation step 1 sensitivity test

Dissect the Arabidopsis thaliana obtained in step 1, check under a microscope whether pine xylophilus infection has occurred inside the plant, count the number of plants internally infected with pine xylophilus, and calculate the infection rate of Arabidopsis thaliana. The results are shown in Table 4. The results showed that Arabidopsis of Col-0 ecotype, Pa-3 ecotype, and Ler ecotype of Arabidopsis were more sensitive to B. xylophilus, with the highest infection rate; Nematodes were the least sensitive; Col-0 ecotype Arabidopsis also had the highest number of nematodes.

Table 4 Sensitivity of different ecotypes of Arabidopsis thaliana to B. xylophilus

Infection rate of Arabidopsis thaliana % Number of nematodes/strain C24 Ecotype Arabidopsis 36.67±5.77cd 16.36±4.74b Col-0 Ecotype Arabidopsis 83.33±5.77a 30.91±2.88a Nd-1 ecotype Arabidopsis 20.00±17.32d 11.00±5.20b Pa-3 Ecotype Arabidopsis 70.00±20.00ab 15.46±6.41b Ler ecotype Arabidopsis 70.00±10.00ab 12.73±4.09b Arabidopsis thaliana Est-1 ecotype 36.67±15.28cd 11.70±4.06b Ws-2 Ecotype Arabidopsis 60.00±20.00abc 11.44±0.42b

Sha ecotype Arabidopsis 66.67±15.23ab 30.47±17.82a Arabidopsis thaliana Kas-1 ecotype 46.67±5.77bc 37.32±4.20a

Embodiment 6, Different ecotypes of Arabidopsis thaliana are sensitive to B. xylophilus

1. The cultivation of B. xylophilus and the acquisition of B. xylophilus without bacterial contamination

Use B. xylophilus instead of B. xylophilus, and other steps are the same as Step 2 and Step 3 of Example 1.

Suspend the nematodes without bacterial contamination in sterile water, count them under a microscope, adjust to the expected concentration, and carry out the experiment in step 2 as the nematode solution. The inoculation method in step 2 is to drop the nematode solution onto the top of the plant, and the nematode solution freely diffuses to all the plants.

2. Sensitivity determination of different ecotypes of Arabidopsis thaliana to B. xylophilus

Method is the same as step 1 of embodiment 5.

The obtained Arabidopsis thaliana was dissected, and whether B. xylophilus infection occurred inside the plant was checked under a microscope, and the number of plants infected with B. xylophilus was counted to calculate the infestation rate of Arabidopsis thaliana. The results are shown in Table 5. The results showed that: Chi-0 ecotype Arabidopsis and Ler ecotype Arabidopsis were more sensitive to B. xylophilus, and the infection rate was the highest, but far lower than that after inoculation with B. xylophilus; Kas-1 ecotype Arabidopsis thaliana, the Est-1 ecotype Arabidopsis is the least sensitive to B. xylophilus; the number of B. xylophilus invaded by different ecotypes of Arabidopsis is also significantly lower than that after inoculation with B. xylophilus. The infection rate of Arabidopsis thaliana by weak B. xylophilus was also low.

Table 5 Sensitivity of different ecotypes of Arabidopsis thaliana to B. xylophilus

Claims (10)

1. one kind is infected the method for arabidopsis with Bursaphelenchus nematodes, comprises the steps: the nematode of Bursaphelenchus is inoculated in the arabidopsis that petiole has wound; Said arabidopsis is the 10 days later arabidopsiss of emerging.

2. the method for claim 1 is characterized in that: the nematode of said Bursaphelenchus is pine wood nematode (Bursaphelenchus xylophilus) or intends pine wood nematode (Bursaphelenchus mucronatus).

3. according to claim 1 or claim 2 method is characterized in that: the said nematode of the said arabidopsis inoculation 300-400 bar of every strain.

4. according to claim 1 or claim 2 method, it is characterized in that: said arabidopsis is the arabidopsis of emerging 10 to 25 days.

5. according to claim 1 or claim 2 method, it is characterized in that: said arabidopsis is the environmental arabidopsis of Chi-0, the environmental arabidopsis of C24, the environmental arabidopsis of Col-0, the environmental arabidopsis of Nd-1, the environmental arabidopsis of Pa-3, the environmental arabidopsis of Ler, the environmental arabidopsis of Est-1, the environmental arabidopsis of Ws-2, the environmental arabidopsis of Sha or the environmental arabidopsis of Kas-1.

6. according to claim 1 or claim 2 method, it is characterized in that: said nematode is aseptic nematode.

7. method as claimed in claim 6 is characterized in that: the preparation method of said aseptic nematode comprises the steps:

(1) said nematode is carried out disinfection;

(2) collect the worm's ovum of the nematode that step (1) obtains;

(3) worm's ovum of step (2) being collected carries out disinfection;

(4) worm's ovum that step (3) is obtained is cultivated to nematode, is aseptic nematode.

8. method as claimed in claim 7 is characterized in that:

Said step (1) comprises the steps: said nematode with aseptic M9 solution washing, then with 1g/100mL thimerosal aqueous solution sterilization 30min, then with 4 ℃ of sterilizations of solution first 10h; Said M9 solution is made up of solute and water; Said solute and the concentration in said M9 solution thereof are following: 22mM KH ₂PO ₄, 42mM Na ₂HPO ₄, 85.5mM NaCl, 1mM MgSO ₄Said solution first is made up of streptomycin sulphate, cycloheximide and water, and the concentration of streptomycin sulphate is 0.1g/100mL, and the concentration of cycloheximide is 0.02g/100mL;

Said step (2) comprises the steps: the nematode that step (1) obtains is inoculated on the long medium that Botrytis cinerea bacterium bacterium colony arranged and cultivates, and observes after the worm's ovum output with aseptic water washing and collects nematode and with the 1g/100mL thimerosal aqueous solution 30min that sterilizes; Then nematode is placed 25 ℃ of cultivations of culture dish that 0.9% SPSS is housed,, outwell solution, with collecting the worm's ovum that adheres on the culture dish with solution second behind the aseptic water washing until observing a large amount of outputs of worm's ovum; Said solution second is made up of tween, NaOH and water, and the volumn concentration of tween is 0.05%, and the concentration of NaOH is 8mM;

The worm's ovum that said step (3) comprises the steps: step (2) is collected is with said aseptic M9 solution washing, then with 1g/100mL thimerosal aqueous solution sterilization 30min, then with 4 ℃ of sterilizations of said solution first 10h;

Said step (4) comprises the steps: the worm's ovum that step (3) obtains is inoculated on the long medium that Botrytis cinerea bacterium bacterium colony arranged and cultivates, and grows until worm's ovum to be nematode.

9. method as claimed in claim 8 is characterized in that: also comprise in the said step (4) said worm's ovum and said nematode are carried out the step that PCR identifies; Said PCR identifies that used primer is that the primer formed of the sequence 2 of sequence 1 and the sequence table of sequence table is right.

10. arbitrary said method is at (a) as follows or (b) or (c) or the application (d) in the claim 1 to 9:

(a) pathogenicity of the nematode of evaluation Bursaphelenchus;

(b) identify the susceptibility of the arabidopsis of different ecological type to Bursaphelenchus nematodes;

(c) have the separation and the evaluation of the gene of anti-Bursaphelenchus nematodes function in the plant;

(d) cultivate anti-Bursaphelenchus nematodes plant.