A method for accurately identifying pinus massoniana resistant to bursaphelenchus xylophilus at seedling stage

Abstract

The present application relates to the field of pine wood nematode disease identification, in particular to the present application, through the investigation of soil relative humidity, light intensity, inoculation amount, environmental temperature, seedling strain, seedling age and other related indexes, the conditions of the most susceptible to nematode infection of pinus massoniana under natural conditions are obtained, the method of grading pinus massoniana by disease death frequency is summarized, in addition, the research group continuously optimizes the conditions of infection under natural environment, and prepares nematode active liquid without damage to plants by using plant liquid, it is verified that after using the nematode active liquid, the disease time of pinus massoniana seedling stage can be greatly shortened, through optimizing the active liquid, the method of grading the disease of pinus massoniana by judging the disease time during the inoculation of nematode in pinus massoniana seedling stage is given, which greatly shortens the grading determination time of pinus massoniana.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pine wood nematode disease identification, in particular to a method for accurately identifying the resistance of Pinus massoniana to pine wood nematode disease at the seedling stage. BACKGROUND

[0002] Pinus massoniana is a major afforestation tree species for ecological construction and resin production in China, with high utilization value and wide application, and is cultivated and distributed in many places in China. Its pine resin, bark and pollen are widely used in forest chemical industry, construction, tannin, health products and other industries. Pinus massoniana has strong vitality and wide ecological adaptability, and is a major pioneer tree species for afforestation of barren mountains and ecological forest construction. A large number of forestry technology workers have carried out a lot of research on the collection, screening and preservation of Pinus massoniana germplasm resources in the prior art, and have bred a large number of fast-growing and high-yielding excellent lines. However, the utilized germplasm resources are less than 30% of the total resources. From the current development of Pinus massoniana industry, it is seriously affected by diseases and pests, especially the current pine wood nematode disease.

[0003] Pine wood nematode disease is a large-scale destructive forest disease caused by the parasitism of pine wood nematode (Bursaphelenchus xylophilus) in pine trees. The disease spreads quickly and has a high mortality rate, and is known as "pine cancer". A large number of studies have shown that there are obvious resistance differences between individual trees within a species, and even in susceptible tree species, there are individual trees with high resistance. Resistance variety breeding has become an effective long-term strategy for forest disease and pest control. Pinus massoniana has a long growth cycle, and screening of disease-resistant plants by inoculating seedlings with pine wood nematodes is the main method for breeding Pinus massoniana varieties resistant to pine wood nematode disease. At present, a number of fast-growing, high-yielding, and disease-resistant potential germplasms have been bred in the main production areas of Pinus massoniana through seedling inoculation with nematodes. However, overall, compared with Pinus thunbergii and Pinus densiflora, which are highly susceptible to pine wood nematode disease, Pinus massoniana is more difficult to inoculate, and the repeatability and stability of the inoculation effect are 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. Therefore, the present application studies the genetic source of test materials, seedling types, inoculation environmental conditions, and the number of inoculation heads, and finds that under the premise of optimizing a series of inoculation technical parameters, the disease-resistant level of Pinus massoniana seedlings can be accurately identified, providing a scientific technical reference for the creation of disease-resistant Pinus massoniana germplasm.

[0004] In addition, in the actual nematode identification process, we found that due to the difference in species of pine trees, the time of onset is very different, and through testing, we determined that the resistance of Pinus massoniana is relatively higher than that of other pine trees, and the time of onset is longer. In order to shorten the time of onset, determine the disease condition of Pinus massoniana as soon as possible and classify the disease, how to shorten the time of onset is a problem to be solved in the present field. SUMMARY

[0005] In view of the above, this study investigated the genetic origin of the experimental materials, seedling type, inoculation environment conditions, number of inoculated seedlings, and shortening the disease onset time. It was found that by optimizing a series of inoculation technical parameters, the disease resistance level of Masson pine seedlings can be accurately identified, providing a scientific and technical reference for the creation of disease-resistant Masson pine germplasm.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A precise identification method for the resistance of Masson pine seedlings to pine wilt disease is disclosed. The method involves: cultivating Masson pine seeds through sowing or tissue culture of explants in a nursery for one year to obtain seedlings; then inoculating 4000 nematodes per seedling at 4-12 months of age; and culturing the seedlings under conditions of 30-35℃, 60%-70% humidity, and 8000-12000 lx light intensity. The disease-affected mortality frequency of the Masson pine is observed and measured. When the mortality frequency is ≤20%, the Masson pine is evaluated as resistant; when 20% < mortality frequency ≤30%, it is evaluated as moderately resistant; when 30% < mortality frequency ≤40%, it is evaluated as moderately susceptible; when 40% < mortality frequency ≤60%, it is evaluated as susceptible; and when the mortality frequency >60%, it is evaluated as highly susceptible.

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

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

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

[0011] Furthermore, the nematodes are activated with an activation solution before inoculation. The activation solution is prepared 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 as follows: mix the corresponding raw materials and water at a solid-liquid mass ratio of 2:1, crush and grind them, filter them, and take the filtrate.

[0013] Furthermore, the volume ratio of the activating solution to the nematodes is 5%.

[0014] Furthermore, the identification method for inoculation after activating nematodes with the activation solution is as follows:

[0015] The mulberry leaf juice, citrus peel juice and grapefruit peel juice are mixed according to the corresponding volume ratio to prepare a nematode active liquid, then the nematode active liquid and distilled water are mixed to prepare a solution with a volume percentage of 5%; 20000 nematodes are inoculated to prepare a nematode suspension liquid 1 mL, then the seedling stage of Pinus massoniana is inoculated by using the skin connection method, the inoculation amount is the nematode suspension liquid 200 μL (about 4000 heads), after the inoculation is completed, the seedling stage of Pinus massoniana is cultivated under the conditions that the artificial control planting environment temperature is 30 DEG C, the soil relative humidity is 60%, and the illumination intensity is 8000 lx; the seedling stage of Pinus massoniana is classified according to the disease occurrence time, and the classification scheme is that when the disease occurrence time is less than or equal to 8 days, the Pinus massoniana plant is determined as high susceptible; when 8 days < disease occurrence time < 10 days, the Pinus massoniana plant is determined as susceptible; when 10 days < disease occurrence time < 15 days, the Pinus massoniana plant is determined as medium susceptible; when 15 days < disease occurrence time < 25 days, the Pinus massoniana plant is determined as medium resistant; and when the disease occurrence time is more than 25 days, the Pinus massoniana plant is determined as resistant.

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

[0017] The present application obtains the conditions that the Pinus massoniana is most susceptible to nematodes under natural conditions by investigating the soil relative humidity, illumination intensity, inoculation amount, environment temperature, seedling line, seedling age and other related indexes, and obtains the method for classifying the Pinus massoniana by the disease death frequency, in addition, the subject group continuously optimizes the infection conditions under the natural environment, and prepares the nematode active liquid which is harmless to plants by using the plant liquid, and it is verified that after the nematode active liquid is used, the disease occurrence time of the seedling stage of Pinus massoniana can be greatly shortened, the method for rapidly classifying the disease occurrence of the Pinus massoniana by determining the disease occurrence time is given by optimizing the active liquid when the nematode is inoculated to the seedling stage of Pinus massoniana, and the classification determination time of the Pinus massoniana is greatly shortened. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The environmental factor effect curve is shown in the figure. DETAILED DESCRIPTION

[0019] All features disclosed in this specification, and / or all steps of any methods or processes disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0020] Any feature in the present specification, unless explicitly stated to the contrary, is intended to be an example of a corresponding set of equivalents, for example, equivalent

[0021] Embodiment 1:

[0022] The infection and investigation of nematodes on various different species of pine trees are as follows:

[0023] 1. Seedling selection

[0024] The seeds of seed orchard, the seeds of adult superior trees in the stand of Jatropha curcas L., and the stems and buds of Jatropha curcas L. were used as explants, respectively, and the tissue culture seedlings of three genetic sources, i.e., seed source, family, and clone, were obtained through the organogenesis pathway. After the seedlings were cultivated for one year, the pine wood nematode was inoculated in the nursery land under natural conditions, and the inoculation amount was 4000 heads per plant. After inoculation, the onset time, incidence rate, and mortality rate were statistically analyzed. The incidence rate was the percentage of the number of diseased plants to the total number of inoculated plants, and the mortality rate was the percentage of the number of dead plants to the total number of inoculated plants after two months of inoculation. The pine wood nematode used was a high pathogenicity mixed strain. Specifically, in the serious pine wood nematode-infected area of Pinus massoniana in Guangxi Zhuang Autonomous Region, three high pathogenicity pine wood nematode strains with strong reproductive ability, fast transmission, and high mortality rate were screened through the steps of wood nematode isolation, culture, and virus detection, and then mixed in an equal ratio. The identification of diseased plants was mainly based on the existing conventional pine wood nematode disease detection regulations, including: observing whether the plants showed a dehydrated, wilted, and yellowing growth morphology or even death, observing whether there were nematodes in the plants under a microscope, PCR identification of nematodes, and isolation of nematodes from diseased plants to re-inoculate a highly susceptible pine tree (Pinus thunbergii) to detect the virulence.

[0025] Table 1 Inoculation effect of nematodes on seedlings of different genetic sources

[0026]

[0027] Note: In the table, lowercase letters represent differences between seedlings of different genetic sources. When the letters are the same, there is no significant difference, and vice versa (P < 0.05).

[0028] As shown in Table 1, the genetic source of seedlings had no significant effect on the average onset time, incidence rate, and mortality rate of P. massoniana inoculated with pine wood nematodes. However, the standard deviations of each index of different genetic sources were quite different. Compared with seed source and family, the standard deviation of clone was 1.2-2.5, which was much lower than that of seed source (8.5-13.8) and family (7.9-14.3). This indicates that when seed source and family-derived tissue culture seedlings are inoculated, the inoculation effect fluctuates greatly, whereas when clone-derived seedlings are inoculated, the stability of the inoculation effect is greatly improved, which is more conducive to the screening of resistant germplasm.

[0029] The degree of woodification of seedlings is closely related to the inoculation effect of nematodes. Generally, the older the seedlings, the higher the degree of woodification. The early growth of P. massoniana tissue culture seedlings is fast, and the seedlings can reach a height of 50-60 cm after one year of cultivation, occupying a large space. Considering that the present application is mainly for the precise identification of pine wood nematode disease in the laboratory, the inoculation effect of nematodes on clone-derived seedlings of different ages was analyzed by inoculating 4000 nematodes per plant in the nursery land under natural conditions.

[0030] Table 2 Inoculation effect of nematodes on seedlings of different ages

[0031]

[0032] Note: Different lowercase letters in the table represent significant differences between different seedling ages (P <0.05).

[0033] The results of Table 2 showed that seedling age had no significant effect on the disease onset time after nematode inoculation, but had significant effect on the plant disease incidence and mortality rate. Among them, the 8-month-old seedlings had the best inoculation effect under the current inoculation conditions.

[0034] 2. Environmental optimization

[0035] In order to exclude the interference of external environmental factors and achieve precise analysis of nematode inoculation and seedling disease resistance under artificial simulated environmental conditions, the environmental temperature, humidity and light intensity were used as factors, with 4 levels for each factor. Orthogonal test was conducted to analyze the inoculation effect under the influence of 16 different environmental factors.

[0036] The 8-month-old Pinus massoniana tissue culture seedlings were inoculated with 4000 nematodes per plant. As shown in Table 3, the inoculation effect was different under different environmental conditions. Among them, the inoculation effect of treatments 9, 10, 13 and 14 was the best, and the plant disease mortality rate was high (about 40%) after inoculation with nematodes (about 21 days).

[0037] Table 3 Nematode inoculation effect of seedlings under different environmental factors

[0038]

[0039] Note: Different lowercase letters in the table represent significant differences between different treatments (P <0.05).

[0040] From the range (R) of each environmental factor, temperature had the greatest effect on the disease onset time, disease incidence and mortality rate (R: 16.6~26.8), followed by light (R: 11.2~14.2), and humidity had relatively small effect (R: 2.7~7.5). The effects of environmental temperature and humidity on nematode pathogenicity are well recognized, but the effect of light intensity on nematode pathogenicity has not been reported. Combined with the Figure 1 The effect curves of each environmental factor showed that there was an action range for each factor, i.e. when the temperature was 30~35℃, the humidity was 60%~70%, and the light intensity was 8000~12000 lx, the seedlings had fast disease onset and high disease mortality rate after inoculation with nematodes, which was beneficial to the rapid selection of diseased plants.

[0041] 3. Nematode inoculation

[0042] The number of nematode inoculation heads is associated with the type of inoculation material and environmental factors. Further, under the above-optimized material type and environmental conditions, the nematode inoculation amount is analyzed. Table 4 inoculation results show that, under the environmental conditions of artificial control temperature 30-35 DEG C, humidity 60%-70%, light intensity 8000-12000 1x, with 8-month-old clone tissue culture seedlings as the inoculation material, in the inoculation amount range of 1000-10000 heads / plant, with the increase of the number of inoculation heads, the disease onset time is shortened, and the plant disease rate and mortality rate are increased. Among them, when the nematode inoculation amount is 8000-12000 heads / plant, the plant mortality rate is significantly greater than the disease rate. The nematode inoculation is a biological stress process for seedlings, which can cause the disorder of various growth and physiological metabolic activities in the plant body, thereby causing the weakening of the plant stress resistance and the increase of the risk of other biological (bacteria infection) or non-biological (drought resistance, air humidity is only 60%-70%, normal is more than 85%; high temperature resistance, 30-35 DEG C, normal is 28 DEG C) stress. Through the nematode separation of the diseased seedlings, it is found that the number of nematodes in the plant is not significantly different under the inoculation treatment of 4000-12000 heads / plant. This shows that, with the increase of the number of nematode inoculation, the number of nematodes colonized in the plant does not change, but the high initial nematode inoculation amount induces the weakening of the plant stress resistance and the increase of the mortality rate, which greatly interferes with the judgment of the plant disease resistance. In view of this, the inoculation amount of 4000 heads / plant is the most suitable.

[0043] Table 4 inoculation effect of seedlings with different nematode inoculation amounts

[0044]

[0045] Note: In the table, different lowercase letters represent that the difference between different inoculation amounts reaches a significant level, and capital letters represent the difference (P<0.05) between the disease rate and the mortality rate of the same inoculation amount treatment.

[0046] 4, resistance grading

[0047] At present, there is no quantitative grading of the resistance of Pinus massoniana to pine wood nematode disease. In order to establish a scientific and intuitive disease resistance grading standard, high-susceptible P. thunbergii and susceptible P. elliottii and P. taeda are taken as references, and the fast-growing and high-yield P. massoniana clones with certain disease resistance potential optimized by the applicant are taken as research objects, and the nematode inoculation is carried out under the conditions of the application scheme. The disease grade is divided into 0-9 levels, which correspond to high resistance to high susceptibility resistance types in turn. From the disease investigation results of the seedlings after inoculation of nematodes, it is found that there is no high resistance type in the test materials, that is, there is no immune seedling to pine nematode disease, P. thunbergii is a high-susceptible type, P. elliottii and P. taeda have various resistance types, and the optimized P. massoniana clone seedlings have the best disease resistance performance. The results are in line with the objective reality.

[0048] Table 5 disease grading investigation of seedlings

[0049]

[0050] The investigation results in Table 5 reflect that the 0-1 grade and the 8-9 grade are slightly insufficient in feasibility in practical application, in order to improve the practicability and reliability of the evaluation standard of disease resistance, the disease resistance grade of the Pinus massoniana seedling is divided into five grades, i.e. resistance, medium resistance, medium susceptibility, susceptibility and high susceptibility, and the corresponding seedling mortality rates are ≤20%, 20%-30%, 30%-40%, 40%-60% and >60% respectively. Therefore, by adopting the series inoculation steps in the application, the quantitative and accurate identification of the ability of the Pinus massoniana seedling to resist the pine wood nematode disease can be realized, and a strong basis is provided for the screening and evaluation of the Pinus massoniana resistant germplasm.

[0051] Table 6: Comprehensive evaluation standard of seedling disease resistance

[0052]

[0053] In the application, the calculation formula of the disease frequency (DF) is: disease frequency (DF) = (disease death plant number ÷ total plant number of investigation) x 100%.

[0054] Example 2

[0055] This example studies how to quickly screen the high-resistant and high-susceptible Pinus massoniana plants, and the specific method is as follows:

[0056] In the study of embodiment 1, we found that the pine seedlings in the conventional natural environment conditions have stronger disease resistance to nematodes than other varieties of pine trees, and the screening of the disease resistance of the pine trees in the natural conditions will be relatively longer. Based on this, the applicant also found in the previous study that some nematode active liquid can be used to stimulate, promote the reproduction of nematodes, improve the activity of nematodes, and accelerate the infection efficiency of nematodes, so as to more quickly screen and distinguish the disease resistance of the plants. In the screening process of the nematode active liquid, according to the reports in the prior art, most of the chemical components such as benzoic acid, phenylacetic acid, lauryl and the like are used, and these substances are reported to have certain activity to nematodes. However, we found in the study that these components can stimulate the growth of nematodes, but may cause damage and interference to the growth of pine trees. Therefore, we consider generating the nematode active liquid from natural plants by juicing and the like. Generally, the natural plants will not cause damage and interference to the growth of plants, but in the prior art, we know that the components of the plant liquid are quite complex, and some plant liquids have certain bacteriostatic properties. Whether these components can promote the growth of nematodes is unknown, so it is necessary to screen the plant liquid. We prefer to select the plant liquid from a large number of woody plants, specifically: pine needles, eucalyptus leaves, mulberry leaves, and then select the plant liquid from discarded peels, specifically: lemons (whole), citrus peels, and pomelo peels. The specific juicing scheme is: the corresponding raw materials and water are mixed according to the solid-liquid mass ratio of 2:1, continuously crushed, ground and filtered, and the filtrate is obtained to obtain the corresponding plant liquid. From a large number of screening experiments, this embodiment gives the determination of the influence of several different plant liquids on the activity and reproductive capacity of pine wood nematodes, specifically:

[0057] 1. The above several plant liquids and distilled water are mixed to prepare a solution with a volume percentage of 5%;

[0058] 2. Nematode activity determination: pine wood nematodes are isolated from diseased and dead pine trees, and the nematodes are sterilized with a mercuric chloride solution and then washed with sterile deionized water for 3 times before testing. Then 1000 nematodes are inoculated into the solution prepared in step 1 to prepare a suspension of 1 mL, which is cultured at 25℃. After 1 day, the pine wood nematode liquid is taken out and placed under a dissection microscope to determine the survival rate of the nematodes. The survival rate of the nematodes is (the number of living nematodes ÷ the total number of nematodes) × 100%. Whether the nematodes are alive is determined by needle puncture method. Sterile deionized water is used as a control, and each treatment is repeated 4 times.

[0059] 3. Nematode reproductive capacity determination: B. xylophilus was isolated from sick and dead Pinus massoniana plants. Before testing, the nematodes were sterilized with a mercuric chloride solution and then washed three times with sterile deionized water. The solution of step 1 was sterilized by high-pressure steam and then stored. 1000 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 Pestalosphaeria sp. The plate was incubated at 25°C for 5 days. The nematodes were isolated and counted. The sterile deionized water was used as a control and each treatment was repeated four times. The results are shown in Table 7:

[0060] Table 7. Effects of different plant liquids on the activity and reproductive capacity of nematodes

[0061]

[0062] Note: Different lowercase letters in the table indicate that the difference between different treatments reached a significant level (P < 0.05).

[0063] As shown in Table 7, mulberry leaves, citrus peels, and pomelo peels can significantly improve the activity and reproductive capacity of nematodes, while Pinus massoniana leaves have no significant effect on the activity and reproductive capacity of nematodes. Eucalyptus leaves and lemon can reduce the activity and reproductive capacity of nematodes.

[0064] From Table 7, we know that mulberry leaves, citrus peels, and pomelo peels have a promoting effect on the growth and reproduction of nematodes, while Pinus massoniana leaves have no significant effect on the growth of nematodes. Lemon and eucalyptus leaves have different degrees of inhibitory effects on the activity and reproductive capacity of nematodes. Therefore, we consider using mulberry leaves, citrus peels, and pomelo peels to prepare nematode active liquid to further activate nematodes and improve their inoculation capacity. The specific scheme is as follows:

[0065] The nematode active liquid was prepared by mixing the plant liquids of mulberry leaves, citrus peels, and pomelo peels according to the corresponding volume ratio in Table 8. Then, the nematode active liquid and distilled water were mixed to prepare a 5% solution by volume. 20000 nematodes were inoculated to prepare a 1 mL nematode suspension. Then, the skin inoculation method was used to inoculate the nematode suspension 200 μL (about 4000 individuals) into plants that were evaluated as "resistant" (10% or more of the plants were sick, but the onset of the disease was late and the disease course was long) in the natural environment. After inoculation, the plants were cultivated under the conditions of an artificial control planting environment temperature of 30°C, a soil relative humidity of 60%, and a light intensity of 8000 lx. The onset time (the time from the inoculation date to the appearance of sick symptoms) was recorded. 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 8.

[0066] Table 8. Nematode inoculation effect of seedlings treated with different plant liquid mass ratios

[0067]

[0068] Note: Different lowercase letters in the table represent significant differences (P < 0.05) between different treatments.

[0069] From Table 8, in terms of morbidity and mortality, the morbidity of Pinus massoniana plants was between 10% and 20%, meeting the standard of medium resistance, indicating that the nematodes had little effect on the morbidity and mortality of Pinus massoniana after being activated by the active liquid, and in terms of the onset time, the onset time of different volumes of active liquid was quite different. In terms of the onset time, the onset time of treatment 1, treatment 6-treatment 9 was the longest, and the difference was not significant, and the onset time was about 25 d; the onset time of treatment 2 and treatment 3 ranked the second, and the difference between them was not significant, and the onset time was about 20 d; the onset time of treatment 4 and treatment 5 was the shortest, and the difference was not significant, and the onset time was about 15 d.

[0070] From the experimental results of Table 8, we know that the nematode liquid after treatment has little effect on the morbidity and disease rate of the plants, and in the natural environment, the plants showing resistance basically have little effect on the morbidity and disease rate after adding the nematode activation liquid, but the onset time is significantly shortened. Therefore, we preliminarily determine that the activated nematodes have stronger activation and reproduction ability, and can quickly infect pine trees, so we will use the active liquid to assist in determining the disease condition of Pinus massoniana next, as follows:

[0071] First, according to the scheme of Example 1, the resistance of Pinus massoniana at the seedling stage is classified, and the specific classification standard is referred to Table 6 of Example 1, and then the seedlings are cultivated according to the following scheme:

[0072] Control group: 20,000 nematodes were prepared into a nematode suspension of 1 mL, and then the seedling stage Pinus massoniana plants were inoculated by skin grafting, and the inoculation amount was nematode suspension 200 μL. After inoculation, the plants were cultivated under the conditions of artificial control planting environment temperature of 30℃, soil relative humidity of 60%, and light intensity of 8000 lx; the onset time was counted, and the morbidity was calculated on the 30th day, and the mortality was calculated on the 60th day.

[0073] Experimental group: the nematode active liquid was prepared by mixing the plant liquids of mulberry leaves, citrus peels and grapefruit peels according to the corresponding volume ratio of Table 8 treatment 4, and then the nematode active liquid and distilled water were mixed to prepare a solution with a volume percentage of 5%; 20000 nematodes were inoculated to prepare a nematode suspension liquid of 1 mL, and then the seedling stage of Pinus massoniana was inoculated by the skin connection method, the inoculation amount was 200 μL of nematode suspension liquid, after inoculation, the plants were cultivated under the conditions of artificial control planting environment temperature of 30℃, soil relative humidity of 60%, and light intensity of 8000 lx; the incidence time (the incidence time is the time from the inoculation day to the appearance of the diseased 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.

[0074] Table 9 Disease conditions of plants with different resistance performances in different groups

[0075]

[0076] From Table 9, we can know that after activating the nematodes with the nematode active liquid, the incidence rate and mortality rate have little difference with the control group, but the incidence time is significantly shortened, among them, the incidence time of the highly susceptible plants is shortened by about 13 d, the incidence time of the susceptible plants is shortened by about 11 d, the incidence time of the moderately susceptible plants is shortened by about 10 d, the incidence time of the moderately resistant plants is shortened by about 15 d, and the incidence time of the resistant plants is shortened by about 15 d. And from the incidence time, the incidence time from long to short is in turn: resistant plants > moderately resistant plants > moderately susceptible plants > susceptible plants > highly susceptible plants.

[0077] For the determination of the diseased plants of the seedling stage of Pinus massoniana, we combined the nematode active liquid to quickly determine the disease condition of the plants by the incidence time, the determination method is: the nematode active liquid is prepared by mixing the plant liquids of mulberry leaves, citrus peels and grapefruit peels according to the corresponding volume ratio of Table 8 treatment 4 (the volume ratio of mulberry leaf juice: citrus peel juice: grapefruit peel juice is 2:2:3); then the nematode active liquid and distilled water are mixed to prepare a solution with a volume percentage of 5%; 20000 nematodes are inoculated to prepare a nematode suspension liquid of 1 mL, and then the seedling stage of Pinus massoniana is inoculated by the skin connection method, the inoculation amount is 200 μL of nematode suspension liquid, after inoculation, the plants are cultivated under the conditions of artificial control planting environment temperature of 30℃, soil relative humidity of 60%, and light intensity of 8000 lx; the incidence time is counted to classify the seedling stage of Pinus massoniana, the classification scheme is: when the incidence time is ≤8 d, the Pinus massoniana plant is determined to be highly susceptible; when 8 d < incidence time ≤10 d, the Pinus massoniana plant is determined to be susceptible; when 10 d < incidence time ≤15 d, the Pinus massoniana plant is determined to be moderately susceptible; when 15 d < incidence time ≤25 d, the Pinus massoniana plant is determined to be moderately resistant; when the incidence time is >25 d, the Pinus massoniana plant is determined to be resistant.

[0078] In summary, the present application obtains the conditions most likely to infect nematodes of Pinus massoniana under natural conditions by investigating soil relative humidity, light intensity, inoculation amount, environmental temperature and other related indexes, and then continuously optimizes the conditions under natural environment, and prepares nematode active liquid without damage to plants by using plant liquid. It is verified that after using the nematode active liquid, the disease infection time of Pinus massoniana seedling stage can be greatly shortened. By optimizing the active liquid, a method for rapidly grading the disease infection of Pinus massoniana by judging the disease infection time during the inoculation of nematodes in the seedling stage of Pinus massoniana is given, which greatly shortens the grading judgment time of Pinus massoniana.

[0079] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A precise identification method for resistance to pine wilt disease in seedling pine (Pinus massoniana), characterized in that, The method is as follows: Masson pine seeds are sown or explants are tissue cultured, and then cultivated in a nursery for 4-12 months to obtain seedlings. The 4-12 month old seedlings are then inoculated with nematodes at a rate of 4000 nematodes per seedling. The seedlings are then cultured under conditions of 30-35℃, 60%-70% humidity, and 8000-12000 lx light intensity. The disease mortality rate of the Masson pine is observed and measured. When the disease mortality rate is ≤20%, the Masson pine is rated as resistant; when 20% < disease mortality rate ≤30%, it is rated as moderately resistant; when 30% < disease mortality rate ≤40%, it is rated as moderately susceptible; when 40% < disease mortality rate ≤60%, it is rated as susceptible; and when the disease mortality rate >60%, it is rated as highly susceptible. The nematodes were activated with an activation solution before inoculation. The activation solution was prepared by mixing mulberry leaf juice, citrus peel juice and grapefruit peel juice in a volume ratio of 2:2-4:3-5. The identification method for nematode activation followed by inoculation with the activated solution is as follows: A nematode activation solution is prepared by mixing mulberry leaf juice, citrus peel juice, and grapefruit peel juice in the corresponding volume ratios. This nematode activation solution is then mixed with distilled water to prepare a 5% (v / v) solution. 20,000 nematodes are inoculated, and a 1 mL nematode suspension is prepared. Seedlings of *Pinus massoniana* are then inoculated using the skin grafting method, with an inoculation volume of 200 μL of the nematode suspension. After inoculation, the seedlings are cultivated under artificially controlled environmental conditions, maintaining a planting temperature of 30℃, a relative soil humidity of 60%, and a light intensity of 8000 lx. The seedlings are graded based on the time of disease onset: when the time of disease onset is ≤8 days, the seedlings are considered highly susceptible; when 8 days < disease onset time ≤10 days, the seedlings are considered susceptible; when 10 days < disease onset time ≤15 days, the seedlings are considered moderately susceptible; when 15 days < disease onset time ≤25 days, the seedlings are considered moderately susceptible. When the disease duration is >25 days, the Masson pine plant is considered moderately resistant; when the disease duration is >25 days, the Masson pine plant is considered resistant.

2. The precise identification method according to claim 1, characterized in that, The Masson pine mentioned is an asexual seedling obtained through tissue culture.

3. The precise identification method according to claim 1, characterized in that, The Masson pine seedlings are 8 months old.

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

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