Precise identification method for pine wood nematode disease resistance of masson pine in seedling stage

Abstract

The invention relates to the field of bursaphelenchus xylophilus disease identification, in particular to a method for grading masson pines through infection death frequency, which is obtained by investigating soil relative humidity, illumination intensity, inoculum size, environment temperature, seedling strain, seedling age and other related indexes to obtain conditions for the masson pines to be most easily infected with nematodes under natural conditions. Besides, a research group continuously optimizes infection conditions in the natural environment, nematode active liquid which does not damage plants is prepared from plant liquid, it is verified that after the nematode active liquid is adopted, the disease infection time of the masson pine in the seedling stage can be greatly shortened, and by optimizing the nematode active liquid, it is given that when nematodes are inoculated in the masson pine in the seedling stage, the disease infection time of the masson pine in the seedling stage is greatly shortened. According to the method for quickly grading the disease infection condition of the pinus massoniana by judging the disease time, the grading judgment time of the pinus massoniana is greatly shortened.

Description

[Technical field]

[0001] The invention relates to the field of pine wood nematode disease identification, and in particular to a method for accurately identifying Masson pine resistance to pine wood nematode disease at the seedling stage. [Background technology]

[0002] Masson pine (Pinus massoniana) is the main afforestation tree species for ecological construction and resin material in my country. It has high utilization value and wide application. It is cultivated and distributed in many places in my country. Its rosin, bark and pollen are widely used in forest chemical industry, construction, tannin, health care products and other industries. Masson pine has strong vitality and wide ecological adaptability. It is the main pioneer tree species for afforestation of barren hills and ecological forest construction. In the development of forestry economy and ecological construction in my country, masson pine plays a decisive role. In the prior art, a large number of forestry science and technology workers have carried out a lot of research on the collection, screening and preservation of masson pine germplasm resources, and bred a large number of fast-growing and high-yield excellent strains, but the germplasm resources that have been utilized are less than 30% of all resources. From the current development of masson pine industry, it is seriously affected by pests and diseases, especially the current pine wood nematode disease.

[0003] Pine wilt disease is a devastating forest disease caused by the parasitism of pine nematodes (Bursaphelenchus xylophilus) in pine trees. The disease spreads quickly and has a high mortality rate among infected plants. It is called "pine cancer". A large number of studies have shown that there are obvious differences in resistance between individual trees within a species. Even among susceptible species, there are individual trees with extremely high resistance. The selection and breeding of resistant varieties has become an effective long-term countermeasure for the prevention and control of forest diseases and insect pests. Masson pine has a long growth cycle. The main method for breeding Masson pine strains resistant to pine wilt disease is to inoculate Masson pine seedlings with pine wilt nematodes and screen for disease-resistant plants. At present, a number of fast-growing, high-yielding and disease-resistant germplasms have been selected in the main Masson pine producing areas by inoculating seedlings with nematodes. But overall, compared with highly susceptible species to pine wilt disease such as black pine and red pine, Masson pine is more difficult to inoculate, and the repeated stability of the inoculation effect is poor. According to literature reports, there are many factors that affect the success of nematode inoculation, but there is currently no complete technical system for pine wood nematode inoculation. To this end, this application has conducted research from the aspects of genetic origin of test materials, seedling type, inoculation environment conditions, and number of inoculation heads. It was found that under the premise of optimizing a series of inoculation technical parameters, the disease resistance level of Masson pine seedlings can be accurately identified, providing a scientific technical reference for the creation of disease-resistant germplasm of Masson pine.

[0004] In addition, in the actual nematode identification process, we found that due to the different varieties of pine trees, the onset time varies greatly. After testing, we determined that the resistance of Masson pine is higher than that of other pine trees, and the onset time is longer. Therefore, in order to shorten the onset time, the susceptibility of Masson pine should be determined as soon as possible and the susceptibility should be graded. How to shorten the onset time is a problem that needs to be solved in this field. [Summary of the invention]

[0005] In view of the above content, research was conducted from the aspects of genetic origin of experimental materials, seedling type, inoculation environment conditions, number of inoculations, and shortening of disease time. It was found that under the premise of optimizing a series of inoculation technical parameters, the disease resistance level of Masson pine seedlings can be accurately identified, which provides a scientific technical reference for the creation of disease-resistant Masson pine germplasm.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for accurately identifying the resistance of Masson pine to pine wood nematode disease at the seedling stage, the method comprising: cultivating Masson pine seeds by sowing or explants by tissue culture in a nursery for one year to obtain Masson pine at the seedling stage; then inoculating nematodes into Masson pine seedlings of 4 to 12 months old, with an inoculation amount of 4,000 heads / plant; then culturing under the conditions of a temperature of 30 to 35°C, a humidity of 60% to 70%, and a light intensity of 8,000 to 12,000 lx, observing and determining the susceptible death frequency of Masson pine; when the susceptible death frequency is ≤20%, the Masson pine is evaluated as resistant; when 20%<susceptible death frequency≤30%, the Masson pine is evaluated as moderately resistant; when 30%<susceptible death frequency≤40%, the Masson pine is evaluated as moderately susceptible; when 40%<susceptible death frequency≤60%, the Masson pine is evaluated as susceptible; and when the susceptible death frequency>60%, the Masson pine is evaluated as highly susceptible.

[0008] Furthermore, the Masson pine is an asexual seedling obtained through tissue culture.

[0009] Furthermore, the Masson pine seedlings are 8 months old.

[0010] Furthermore, the nematodes are activated with an activation liquid before inoculation, and the activation liquid is prepared from mulberry leaf juice, citrus peel juice and / or grapefruit peel juice.

[0011] Furthermore, the nematodes are activated by an activation solution before inoculation, and the activation solution is obtained by mixing mulberry leaf juice, citrus peel juice, and grapefruit peel juice in a volume ratio of 2:2-4:3-5.

[0012] Furthermore, the preparation method of the mulberry leaf juice, citrus peel juice and grapefruit peel juice is: mixing the corresponding raw materials and water according to a solid-liquid mass ratio of 2:1, crushing, grinding and filtering, and taking the filtrate to obtain the juice.

[0013] Furthermore, the volume ratio of the activation liquid when activating nematodes is 5%.

[0014] Furthermore, the identification method of inoculation after activation of nematodes by the activation liquid is:

[0015] Mulberry leaf juice, citrus peel juice and grapefruit peel juice were mixed in a corresponding volume ratio to prepare a nematode active liquid, and then the nematode active liquid and distilled water were mixed to prepare a solution with a volume percentage of 5%; 20,000 nematodes were inoculated to prepare 1 mL of nematode suspension, and then the Masson pine plants at the seedling stage were inoculated by the bark grafting method, and the inoculation amount was 200 μL of the nematode suspension (about 4,000 heads). After the inoculation was completed, the temperature of the planting environment was artificially controlled to be 30° C. and the relative humidity of the soil was 60%. , cultivated under the condition of light intensity of 8000lx; the onset time was counted to grade the Masson pine seedlings, and the grading scheme was as follows: when the onset time ≤8d, the Masson pine plants were judged as highly susceptible; when 8d<onset time≤10d, the Masson pine plants were judged as susceptible; when 10d<onset time≤15d, the Masson pine plants were judged as moderately susceptible; when 15d<onset time≤25d, the Masson pine plants were judged as moderately resistant; when the onset time>25d, the Masson pine plants were judged as resistant.

[0016] The present invention has the following beneficial effects:

[0017] This application obtained the conditions under which Masson pine is most susceptible to nematode infection under natural conditions by investigating relevant indicators such as soil relative humidity, light intensity, inoculation amount, ambient temperature, seedling strain, and seedling age, and obtained a method for grading Masson pine by the frequency of disease mortality. In addition, the research team continuously optimized the conditions for infection in the natural environment and used plant liquid to prepare a nematode active liquid that does not damage the plants. It has been verified that the use of the nematode active liquid can greatly shorten the susceptibility time of Masson pine seedlings. By optimizing the active liquid, a method for quickly grading the susceptibility of Masson pine by determining the time of onset when nematodes are inoculated in the Masson pine seedling stage is provided, which greatly shortens the grading and determination time of Masson pine.

Brief Description of the Drawings

[0018] Figure 1 This is the effect curve of environmental factors. [Specific implementation method]

[0019] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0020] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0021] Embodiment 1:

[0022] This example is about the infection of nematodes on various types of pine trees. The specific investigation results are as follows:

[0023] 1. Seedling breeding

[0024] The seeds of Tongmian pine in the commercial seed garden, the seeds of adult superior trees in the Tongmian pine forest and their stem buds were used as explants, and the tissue culture technology of three genetic sources, namely, provenance, family and asexual line, was obtained through organogenesis pathway tissue culture technology. When the seedlings were cultivated for one year, pine wood nematodes were inoculated in the nursery under natural conditions, with 4000 heads / plant. After inoculation, the onset time, incidence rate and mortality rate were counted. Among them, the incidence rate is the percentage of diseased plants in the total number of inoculated plants, and the mortality rate is the percentage of dead plants in the total number of inoculated plants 2 months after inoculation. The pine wood nematodes used are highly pathogenic mixed strains. Specifically, in the epidemic area of ​​pine wood nematodes seriously infected with Masson pine in Guangxi, three highly pathogenic pine wood nematode strains with strong reproductive ability, fast spread and high mortality were screened and mixed in equal proportions through the steps of separation, cultivation and virulence detection of diseased wood nematodes. Identification of susceptible plants is mainly carried out through existing conventional pine wood nematode disease detection procedures, including: visual observation to see if the plants are dehydrated, wilted, yellowed or even dead, microscopic observation to see if there are nematodes in the plants, PCR identification of nematodes, and separation of nematodes from susceptible plants and back-grafting into highly susceptible pine trees (black pine) to test virulence.

[0025] Table 1 Effect of nematode inoculation on seedlings of different genetic origins

[0026]

[0027] Note: Lowercase letters in the table represent differences among seedlings of different genetic origins. The same letters are not significant, otherwise the differences are significant (P<0.05).

[0028] As shown in Table 1, the genetic origin of seedlings has no significant effect on the average onset time, morbidity and mortality of pine wood nematodes inoculated with Masson pine. However, the standard deviations of various indicators of different genetic origins vary greatly. Compared with the seed source and family, the standard deviation of the asexual line is 1.2-2.5, which is much lower than the standard deviation of the seed source and family (seed source: 8.5-13.8, family: 7.9-14.3). This shows that when the seed source and family source tissue culture seedlings are used for inoculation, the inoculation effect fluctuates greatly. On the contrary, when the asexual seedlings are used for inoculation, the stability of the inoculation effect is greatly improved, which is more conducive to the screening of resistant germplasm.

[0029] The degree of lignification of seedlings is closely related to the effect of nematode inoculation. Generally speaking, the older the seedlings, the higher the degree of lignification of the seedlings. Masson pine tissue culture seedlings grow fast in the early stage, and the height of the seedlings can reach 50-60cm after one year of cultivation, occupying a large space. Considering that this application is mainly to achieve accurate identification of pine wood nematode disease indoors, the inoculation effect of seedlings from asexual lines of different ages was further analyzed in a nursery under natural environmental conditions with an inoculation rate of 4,000 nematodes per plant.

[0030] Table 2 Effect of nematode inoculation on seedlings of different seedling ages

[0031]

[0032] Note: Different lowercase letters in the table indicate significant differences among seedlings of different ages (P<0.05).

[0033] The results in Table 2 show that the age of seedlings has no significant effect on the onset time after nematode inoculation, but has a significant effect on the incidence and mortality of plants. Among them, under the current inoculation conditions, the inoculation effect of 8-month-old seedlings is the best.

[0034] 2. Environmental optimization

[0035] In order to eliminate the interference of external environmental factors and realize the accurate analysis of nematode inoculation and seedling disease resistance under artificial simulated environmental conditions, the effect of nematode inoculation under the influence of 16 different environmental factors was analyzed through orthogonal experiments, with environmental temperature, humidity, and light intensity as factors, and 4 levels for each factor.

[0036] Eight-month-old Masson pine clones were used as inoculation materials, and 4,000 nematodes were inoculated per plant. As shown in Table 3, different environmental conditions resulted in different nematode inoculation effects. Among them, treatments 9, 10, 13 and 14 had the best inoculation effects. After the inoculation of nematodes (about 21 days), the mortality rate of the plants was high (about 40%).

[0037] Table 3 Effect of seedling nematode inoculation under different environmental factors

[0038]

[0039]

[0040] Note: Different lowercase letters in the table indicate significant differences among different treatments (P<0.05).

[0041] From the perspective of the range values ​​(R) of various environmental factors, temperature has the greatest impact on the onset time, morbidity and mortality (R: 16.6-26.8), followed by light (R: 11.2-14.2), while humidity has a relatively small impact (R: 2.7-7.5). The effects of environmental temperature and humidity on the susceptibility of nematodes are generally recognized, but the effects of light intensity on nematode pathogenicity have not been reported. Figure 1 From the effect curves of various environmental factors, it can be seen that all three factors have an action range, namely: when the temperature is 30-35℃, the humidity is 60%-70%, and the light intensity is 8000-120001x, the seedlings will become sick quickly after inoculation with nematodes, and the mortality rate is high, which is conducive to the rapid screening of susceptible plants.

[0042] 3. Nematode inoculation

[0043] The number of nematode inoculation heads is associated with the type of inoculation material and environmental factors. Further, under the above-mentioned optimized material type and environmental conditions, the nematode inoculation amount was analyzed. The inoculation results in Table 4 show that with 8-month-old asexual tissue culture seedlings as inoculation materials, under the artificially controlled temperature of 30-35°C, humidity of 60%-70%, and light intensity of 8000-120001x, within the inoculation amount range of 1000-10000 heads / plant, as the number of inoculation heads increases, the onset time is shortened, and the incidence and mortality of plants increase. Among them, when the nematode inoculation amount is 8000-12000 heads / plant, the plant mortality rate is significantly greater than the incidence rate. Nematode inoculation is a biological stress process for seedlings, which will cause a variety of growth physiological and metabolic activities in the plant to be disordered, thereby causing the plant's resistance to stress to be weakened, and the risk of being subjected to other biological (bacterial infection) or non-biological (drought resistance, air humidity is only 60%-70%, normal 85% or more; high temperature resistance, 30-35°C, normal 28°C) and other stresses increases. Through the separation of nematodes in susceptible seedlings, it was found that the number of nematodes in the plants was not significantly different under the inoculation treatment of 4,000 to 12,000 heads per plant. This shows that as the number of nematodes inoculated increased, the number of colonized nematodes in the plants did not change, but the excessive initial nematode inoculation induced a weakening of the plant's stress resistance and an increase in mortality, which greatly interfered with the judgment of the plant's disease resistance. In view of this, the inoculation amount of 4,000 heads per plant is the most suitable.

[0044] Table 4 Effect of seedling inoculation with different nematode inoculation amounts

[0045]

[0046] Note: Different lowercase letters in the table indicate significant differences between different inoculum sizes, and capital letters indicate differences in morbidity and mortality between treatments with the same inoculum size (P<0.05).

[0047] 4. Resistance classification

[0048] At present, there is no quantitative classification of the resistance of Masson pine to pine wood nematode disease. In order to establish a scientific and intuitive disease resistance classification standard, the highly susceptible black pine and susceptible longleaf pine and loblolly pine were used as references, and the fast-growing, high-yielding Masson pine asexual lines with certain disease resistance potential that were previously preferred by the applicant of the present invention were used as research objects. Nematode inoculation was carried out under the conditions of the invention scheme of this application. The disease level is divided into 10 levels from 0 to 9, corresponding to the highly resistant to highly susceptible resistance types. From the results of the investigation of the condition of the seedlings after inoculation with nematodes, there was no highly resistant type in the test materials, that is, there was no vaccine-free wood for pine wood nematode disease, while the black pine was a highly susceptible type, and the resistance types of the longleaf pine and loblolly pine test seedlings were relatively diverse, and the preferred Masson pine asexual line seedlings had the best disease resistance, and this result was in line with objective reality.

[0049] Table 5 Survey on the classification of seedling disease

[0050]

[0051] The survey results in Table 5 reflect that the feasibility of levels 0 to 1 and 8 to 9 is slightly insufficient in actual application. In order to improve the practicality and reliability of the disease resistance evaluation standard, the disease resistance level of Masson pine seedlings is divided into five levels in the present invention, namely: resistance, medium resistance, medium sensitivity, sensitivity and high sensitivity, and the corresponding seedling mortality rates are ≤20%, 20% to 30%, 30% to 40%, 40% to 60%, and >60%, respectively. Thus, by adopting the series of inoculation steps in the invention scheme of this application, the quantitative and accurate identification of the ability of Masson pine seedlings to resist pine wood nematode disease can be achieved, which provides a strong basis for the screening and evaluation of Masson pine resistant germplasm.

[0052] Table 6 Comprehensive evaluation criteria for seedling disease resistance

[0053]

[0054] In the present application, the calculation formula of disease-susceptible death frequency (DF) is: disease-susceptible death frequency (DF) = (number of disease-susceptible and dead plants ÷ total number of investigated plants) × 100%.

[0055] Example 2

[0056] This embodiment studies how to quickly screen highly resistant and highly susceptible plants of Masson pine, and the specific method is as follows:

[0057] In the study of Example 1, we found that: under normal natural environmental conditions, the disease resistance of Masson pine seedlings to nematodes is stronger than that of other varieties of pine trees, and the screening of Masson pine plants with strong and weak disease resistance will take a relatively longer period under natural conditions. Based on this, the applicant also found in previous studies that in the prior art, some nematode active liquids can be used to stimulate and promote nematode reproduction, improve nematode activity, thereby accelerating nematode infection efficiency, and more quickly screen and distinguish plants with strong and weak disease resistance. In the screening process of nematode active liquids, according to reports in the prior art, most of them use benzoic acid, phenylacetic acid, myrcene and other chemical components, which are reported to have certain activity against nematodes. However, we found in our research that although these components can stimulate nematode growth, they may cause damage and interference to the growth of pine trees. For this reason, we consider generating nematode active liquids by squeezing juice from natural plants. Natural plants generally do not have the problem of causing damage and interference to plant growth. However, in the prior art, we know that the composition of plant liquid is quite complex, and some plant liquids have certain antibacterial properties. We do not know whether these components can promote the growth of nematodes. Therefore, it is necessary to screen the plant liquid. We preferentially select plant liquid from a large number of woody plants, specifically: Masson pine leaves, eucalyptus leaves, mulberry leaves. Secondly, we select plant liquid from discarded fruit peels, specifically: lemon (whole), citrus peel, grapefruit peel. The specific juicing scheme is: mix the corresponding raw materials and water at a solid-liquid mass ratio of 2:1, continuously crush and grind, and then filter, and take the filtrate to obtain the corresponding plant liquid. From a large number of screening experiments, this embodiment gives the determination of the effects of several different plant liquids on the vitality and fecundity of pine wood nematodes, specifically:

[0058] 1. Mix the above-mentioned plant liquids and distilled water to prepare a 5% solution by volume;

[0059] 2. Nematode vitality determination: Pine wood nematodes were isolated from dead Masson pine plants that were susceptible to the disease. Before the test, the nematodes were disinfected with thimerosal solution and then washed three times with sterile deionized water. Then, 1,000 nematodes were inoculated into the solution of step 1 to prepare a 1 mL suspension, and cultured at 25°C. After 1 day, the pine wood nematode solution was taken and placed under a dissecting microscope to determine the survival rate of the nematodes. Nematode survival rate = (number of live worms ÷ total number of nematodes) × 100%. The nematode survival was determined by acupuncture. Sterile deionized water was used as a control and each treatment was repeated 4 times.

[0060] 3. Nematode reproductive capacity determination: Pine wood nematodes were isolated from dead Masson pine plants that were susceptible to the disease. Before the test, the nematodes were disinfected with thimerosal solution and then washed with sterile deionized water for 3 times. The solution in step 1 was then sterilized with high-pressure steam for standby use. 1000 sterilized and washed nematodes were inoculated into the sterilized solution to prepare a 1 mL suspension. According to the cotton ball method, 0.2 mL of the nematode solution was absorbed onto a sterilized cotton ball in the center of a PDA plate covered with multi-hairy spores. The nematodes were cultured at 25°C for 5 days, and the nematodes were isolated and counted. Sterile deionized water was used as a control and each treatment was repeated 4 times. The results obtained are shown in Table 7:

[0061] Table 7 Effects of different plant solutions on nematode vitality and fecundity

[0062]

[0063] Note: Different lowercase letters in the table indicate significant differences among different treatments (P<0.05).

[0064] As shown in Table 7, mulberry leaves, citrus peels and grapefruit peels can significantly improve the vitality and reproductive capacity of nematodes, while Masson pine leaves have no significant effect on improving the vitality and reproductive capacity of nematodes, and eucalyptus leaves and lemons can reduce the vitality and reproductive capacity of nematodes.

[0065] From Table 7, we know that mulberry leaves, citrus peels and grapefruit peels have a promoting effect on the growth and reproduction of nematodes, while the growth-promoting effect of Masson pine leaves is not prominent. Lemon and eucalyptus leaves have different degrees of inhibitory growth effects on the vitality and reproduction of nematodes. Therefore, it is considered to prepare nematode active liquid with mulberry leaves, citrus peels and grapefruit peels to further activate the nematodes, thereby improving the inoculation ability of nematodes. The specific plan is as follows:

[0066] The plant liquids of mulberry leaves, citrus peels and grapefruit peels were mixed according to the corresponding volume ratios in Table 8 to prepare a nematode active liquid, and then the nematode active liquid and distilled water were mixed to prepare a solution with a volume percentage of 5%; 20,000 nematodes were inoculated to prepare 1 mL of a nematode suspension, and then 200 μL (about 4,000 heads) of the nematode suspension were inoculated into plants evaluated as "resistant" (more than 10% and less than 20% of the plants were susceptible to the disease, but the onset time was late and the course of the disease was long) under the natural environment by the skin grafting method. After the inoculation, the plants were cultivated under the conditions of an artificially controlled planting environment temperature of 30° C., a relative soil humidity of 60%, and a light intensity of 8000 lx; ​​the onset time (the onset time is: the time from the date of inoculation to the beginning of the onset of disease symptoms on the plant) was counted, and the incidence rate was calculated on the 30th day and the mortality rate was calculated on the 60th day. The relevant results are shown in Table 8.

[0067] Table 8 Effect of seedling nematode inoculation under different plant solution mass ratios

[0068]

[0069]

[0070] Note: Different lowercase letters in the table indicate significant differences among different treatments (P<0.05).

[0071] As can be seen from Table 8, in terms of morbidity and mortality, the morbidity of Masson pine plants is between 10% and 20%, which meets the standard of medium resistance, indicating that the nematodes infected with Masson pine after being activated by active liquid have little effect on the morbidity and mortality of Masson pine. From the perspective of the onset time, the volume ratio of active liquid in each treatment varies greatly. From the perspective of the onset time, the onset time of treatment 1 and treatment 6-9 is the longest, with no significant difference, and the onset time is about 25 days; the onset time of treatment 2 and treatment 3 ranks second, with no significant difference between the two, and the onset time is about 20 days; the onset time of treatment 4 and treatment 5 is the shortest, with no significant difference, and the onset time is about 15 days.

[0072] From the experimental results in Table 8, we know that the effect of the treated nematode liquid on the incidence and susceptibility of the plants is not much different. For plants that are resistant in the natural environment, after adding the nematode activation liquid, the incidence and susceptibility are not greatly affected, but the onset time is significantly shortened. Therefore, we preliminarily determine that the nematodes activated by the active liquid have stronger activation and reproduction capabilities and can quickly infect pine trees. Therefore, we will use the active liquid to assist in determining the susceptibility of Masson pine, as follows:

[0073] First, the resistance of Masson pine in the seedling stage was graded according to the scheme of Example 1. The specific grading standard is referred to Table 6 of Example 1. Then, these seedlings were cultivated according to the following scheme:

[0074] Control group: 20,000 nematodes were prepared into 1 mL of nematode suspension, and then the Masson pine plants at the seedling stage were inoculated by the bark grafting method, with an inoculation volume of 200 μL of the nematode suspension. After the inoculation, the plants were cultivated under the conditions of an artificially controlled planting environment temperature of 30°C, a soil relative humidity of 60%, and a light intensity of 8000 lx; ​​the onset time was counted, and the incidence rate was calculated on the 30th day, and the mortality rate was calculated on the 60th day.

[0075] Experimental group: the plant liquid of mulberry leaves, citrus peels and grapefruit peels were mixed according to the corresponding volume ratio of treatment 4 in Table 8 to prepare a nematode active liquid, and then the nematode active liquid and distilled water were mixed to prepare a solution with a volume percentage of 5%; 20,000 nematodes were inoculated to prepare 1 mL of nematode suspension, and then the Masson pine plants in the seedling stage were inoculated by the skin grafting method, and the inoculation amount was 200 μL of the nematode suspension. After the inoculation, the plants were cultivated under the conditions of artificially controlled planting environment temperature of 30°C, soil relative humidity of 60%, and light intensity of 8000 lx; ​​the onset time (onset time is: the time from the date of inoculation to the beginning of the onset of disease symptoms) was counted, and the incidence rate was calculated on the 30th day and the mortality rate was calculated on the 60th day. The results are shown in Table 9.

[0076] Table 9 Disease susceptibility of plants with different resistance performance in different groups

[0077]

[0078] As shown in Table 9, after we activated the nematodes with the nematode active liquid, the morbidity and mortality rates were not much different from those of the control group, but the onset time was significantly shortened, among which the onset time of the highly susceptible plants was shortened by about 13 days, the onset time of the susceptible plants was shortened by about 11 days, the onset time of the moderately susceptible plants was shortened by about 10 days, the onset time of the moderately resistant plants was shortened by about 15 days, and the onset time of the resistant plants was shortened by about 15 days. In terms of the onset time, the onset time from long to short is: resistant plants > moderately resistant plants > moderately susceptible plants > susceptible plants > highly susceptible plants.

[0079] For the determination of susceptible plants of Masson pine at the seedling stage, we combined the nematode active liquid and used the onset time to quickly determine the susceptibility of the plants. The determination method was as follows: the plant liquids of mulberry leaves, citrus peels and grapefruit peels were mixed according to the corresponding volume ratios of Treatment 4 in Table 8 (the volume ratio of mulberry leaf juice: citrus peel juice: grapefruit peel juice was 2:2:3) to prepare the nematode active liquid, and then the nematode active liquid was mixed with distilled water to prepare a solution with a volume percentage of 5%; 20,000 nematodes were inoculated to prepare 1 mL of nematode suspension, and then the Masson pine plants at the seedling stage were inoculated by the skin grafting method, and the inoculation amount was 100% of the nematode suspension. The seedlings were graded according to the time of onset, and the grading scheme was as follows: when the onset time was ≤8d, the plant was judged as highly susceptible; when 8d<onset time≤10d, the plant was judged as susceptible; when 10d<onset time≤15d, the plant was judged as moderately susceptible; when 15d<onset time≤25d, the plant was judged as moderately resistant; when the onset time>25d, the plant was judged as resistant.

[0080] In summary, this application has obtained the conditions under which Masson pine is most susceptible to nematode infection under natural conditions by investigating relevant indicators such as soil relative humidity, light intensity, inoculation amount, and ambient temperature. Then, by continuously optimizing the conditions for infection in the natural environment, a nematode active liquid that does not damage the plant was prepared using plant liquid. It has been verified that the use of the nematode active liquid can greatly shorten the susceptibility time of Masson pine seedlings. By optimizing the active liquid, a method is provided for quickly grading the susceptibility of Masson pine by determining the time of onset when nematodes are inoculated in the Masson pine seedling stage, which greatly shortens the grading and determination time of Masson pine.

[0081] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for accurately identifying the resistance of Masson pine to pine wood nematode disease at the seedling stage, characterized in that: The method comprises the following steps: cultivating Masson pine seeds by sowing or explants by tissue culture, and then cultivating Masson pine in a nursery for 4-12 months to obtain seedling Masson pine; then inoculating nematodes into the Masson pine seedlings of 4-12 months, with an inoculation amount of 4,000 heads / plant; then cultivating under the conditions of temperature of 30-35°C, humidity of 60%-70%, and light intensity of 8,000lx-12,000lx, observing and determining the disease-susceptibility death frequency of the Masson pine, and when the disease-susceptibility death frequency is ≤20%, the Masson pine is evaluated as resistant; when 20%<susceptibility death frequency≤30%, the Masson pine is evaluated as medium-resistant; when 30%<susceptibility death frequency≤40%, the Masson pine is evaluated as medium-susceptibility; when 40%<susceptibility death frequency≤60%, the Masson pine is evaluated as susceptible; and when the disease-susceptibility death frequency>60%, the Masson pine is evaluated as highly susceptible.

2. The accurate identification method according to claim 1, characterized in that: The Masson pine is a clone seedling obtained through tissue culture.

3. The accurate identification method according to claim 1, characterized in that: The seedling age of the Masson pine seedlings is 8 months.

4. The accurate identification method according to claim 1, characterized in that: The nematodes are activated by an activation solution before inoculation, and the activation solution is prepared from mulberry leaf juice, citrus peel juice and / or grapefruit peel juice.

5. The accurate identification method according to claim 4, characterized in that: The nematodes are activated by an activation solution before inoculation, wherein the activation solution is obtained by mixing mulberry leaf juice, citrus peel juice and grapefruit peel juice in a volume ratio of 2:2-4:3-5.

6. The accurate identification method according to claim 4, characterized in that: The preparation method of the mulberry leaf juice, citrus peel juice and grapefruit peel juice is as follows: the corresponding raw materials and water are mixed according to a solid-liquid mass ratio of 2:1, crushed, ground, filtered, and the filtrate is obtained.

7. The accurate identification method according to claim 4, characterized in that: The volume ratio of the activation liquid when activating nematodes is 5%.

8. The accurate identification method according to claim 4, characterized in that: The identification method of inoculating after the activation liquid activates the nematodes is as follows: The nematode active liquid is prepared by mixing mulberry leaf juice, citrus peel juice and grapefruit peel juice in a corresponding volume ratio, and then the nematode active liquid and distilled water are mixed to prepare a solution with a volume percentage of 5%; 20,000 nematodes are inoculated to prepare 1 mL of nematode suspension, and then the Masson pine plants in the seedling stage are inoculated by the skin grafting method, and the inoculation amount is 200 μL of the nematode suspension. After the inoculation is completed, the planting environment is maintained at 30° C., the soil relative humidity is 60%, and the light intensity is 8000 lx under artificial controlled environmental conditions. The Masson pine seedlings are graded by counting the onset time, and the grading scheme is: when the onset time is ≤8d, the Masson pine plant is judged as highly susceptible; when 8d<onset time≤10d, the Masson pine plant is judged as susceptible; when 10d<onset time When the onset time is ≤15 days, the Masson pine plants are judged as moderately susceptible; when 15 days < onset time ≤25 days, the Masson pine plants are judged as moderately resistant; When the onset time is >25 days, the Masson pine plants are judged to be resistant.

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