Methods for improving pine tree resistance based on stockroot-scion interaction

Abstract

The present invention relates to the technical field of pine resistance improvement. The present invention provides a method for improving pine resistance based on stock and scion interaction, comprising the following steps: using artificial inoculation to evaluate and screen the resistance of different pine species and different varieties of the same species, and clarifying the resistance level to pine wood nematodes; evaluating and screening the resistance of different pine species and different varieties of the same species, and clarifying the resistance level to pine sawtooth beetles; designing tree species / varieties with high resistance to pine sawtooth beetles and high resistance to pine wood nematodes as "scions" and "stocks" respectively; removing the top of the pine tree and retaining the lower half as the stock; selecting the semi-lignified branches of the current year as scions; grafting the scions onto the stock, and performing post-grafting management. The present invention breaks through the traditional single prevention and control technology, takes double resistance as the goal, moves the focus of prevention and control forward to the vector insects, blocks the transmission path of the pine sawtooth beetles, effectively reduces the reproduction and spread of pine wood nematodes, and greatly improves the resistance level.

Description

Technical Field

[0001] The present invention relates to the technical field of pine tree resistance improvement, and in particular to a method for improving pine tree resistance based on stock-scion interaction. Background Art

[0002] Pine wilt disease is the most serious and dangerous major forest disease to occur in my country in the past 50 years, and has been designated a major quarantine target both internally and externally. China is currently the country most severely threatened by pine wilt disease, with the number of infected areas, the area affected, and the number of trees dying from the disease rapidly increasing. Most research, both domestically and internationally, has focused on three key areas: disease quarantine and epidemic monitoring, control of infected trees, and insect vector control. However, due to the complex causes and spread of pine wilt disease, breakthroughs have yet to be achieved. Breeding for disease resistance will become a long-term strategy for pine wilt disease prevention and control.

[0003] At present, the subgenus of Pinus spp. in the two subgenera of Pinaceae in my country, the uniaxial and biaxial pine subgenera, have been affected by pine wood nematode disease. The prevention and control technology and experience focused on pine wood nematodes in the past 40 years have been seriously challenged. Since pine wood nematodes mainly parasitize in the trachea of ​​pine beetles, each beetle can carry up to 280,000 nematodes. When the adult pine beetles bite the tender branches of pine trees to supplement nutrition, the pine wood nematodes can be released through the wounds and then infected with pine wood nematode disease. The present invention starts from a new perspective of resistance breeding, deeply excavates the pathogenic mechanism and control mechanism of pine wood nematodes and their vector pine beetles. Experiments on pine wood nematodes and their vector pine beetles have confirmed that the reproduction and spread of pine wood nematodes depend on the thin-walled cells and microbial environment in the plant body, and the damaged part is the trunk, while the active feeding of pine beetles depends on the needle terpenoid substances, and the damaged part is the crown. The two completely different pathogenic pathways determine the complexity of the prevention and control strategy. Based on the above research conclusions, the project team reversed the traditional control method that focused on pine wood nematodes, shifted the focus of control to vector insects, and innovatively proposed a dual control strategy that takes into account both pine wood nematodes and pine sawyer beetles. First, based on the different pathogenic mechanisms of pine wood nematodes and pine monochamus, a comparative study was conducted on the resistance of different tree species of the subgenus Pinus. The ranking of resistance to pine wood nematodes (parasites) was loblolly pine ≈ masson pine > Pinus rapa > Pinus yunnanensis ≈ Pinus chinensis > Pinus stygosi > Pinus elliottii > Pinus caribbean ≈ Pinus elliottii; the ranking of resistance to pine monochamus (vectors) was loblolly pine ≈ Pinus rapa < Pinus yunnanensis ≈ Pinus rapa < Pinus yunnanensis ≈ Pinus stygosi < Pinus elliottii < Pinus elliottii ≈ Pinus elliottii; secondly, due to the different sites of parasitism and host action and the differences in dual resistance among tree species / varieties, based on the principle of stock / scion interaction, tree species / varieties resistant to vector insects were designed as "spikes" and tree species / varieties resistant to pine wood nematodes were designed as "stocks", and resistant varieties to longhorn beetles and nematodes were bred with the goal of dual resistance. They actively avoid or reduce the feeding of vector insects, reduce the reproduction and spread rate of pine wood nematodes, cut off the disease transmission path, and achieve the goal of dual resistance. This invention breaks through the traditional single prevention and control theory and applies the stockpile-scion interaction method to accurately design dual-resistant varieties, effectively solving the problems of passive prevention and control technology and low prevention efficiency, and achieving significant improvement in resistance. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for improving the resistance of pine trees based on the interaction between stock and scion, breaking through the traditional prevention and control technology that focuses on pine wood nematodes and ignores vector insects, shifting the focus of prevention and control forward, blocking the transmission path of pine wood beetles, greatly reducing the reproduction and spread of pine wood nematodes, and achieving the dual resistance goal.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for improving the resistance of pine trees based on stock and scion interaction, comprising the following steps:

[0007] (1) Use artificial inoculation to evaluate and screen the resistance of different pine species and different varieties of the same species to determine the resistance level to pine wood nematodes;

[0008] (2) Using forced feeding, evaluate and screen the resistance of different pine species and different varieties of the same species to Monochamus alternatus to determine the resistance level;

[0009] (3) Design tree species / varieties with high resistance to Monochamus alternatus and pine wood nematode as “ear” and “stock” respectively;

[0010] (4) Remove the top of the pine tree and keep the lower half as the rootstock;

[0011] (5) Select semi-lignified branches that have sprouted in the current year as scion;

[0012] (6) Select a smooth and straight surface 0.2 to 0.3 m above the ground, graft the scion onto the rootstock, and perform post-grafting management.

[0013] Preferably, the evaluation and screening method for high resistance to pine wood nematodes is as follows: 8,000 to 10,000 nematodes are artificially inoculated per seedling, and the seedling damage level is counted 15 to 30 days after inoculation at a temperature of about 30°C. The level evaluation index is level 0: no yellowing of needles, level I: yellowing of needles < all 1 / 2, level II: 1 / 2 ≤ yellowing of needles < 2 / 3, level III: yellowing of needles = 2 / 3, level IV: yellowing of needles > 2 / 3; the damage level is evaluated based on the percentage of level IV seedlings in the total seedlings, <10% is highly resistant, <40% is moderately resistant, and >50% is highly susceptible; highly susceptible species / variety are directly eliminated, and moderately resistant species are preserved in the germplasm resource bank as alternative materials by asexual reproduction, and the surviving plants of highly resistant species / variety are inoculated a second time, and the surviving plants are highly resistant strains that are used as hybrid parents or directly promoted and applied.

[0014] Preferably, the method for evaluating and screening the highly resistant Monochamus alternatus is as follows: 10 to 20 male and female Monochamus alternatus beetles are placed in a space sealed with a 16-18 mesh insect-proof net where the seedlings or branches to be tested are placed, and a forced feeding test is conducted;

[0015] Set up bait trees in the forest in the epidemic area about 60 days in advance, and add the bait trees to the laboratory in strict accordance with the treatment method for major quarantine objects before the pine alternating beetles emerge. Collect pine alternating beetles once every morning, and select beetles with the same male-female ratio collected on the same day for forced feeding experiments; check the feeding situation of the beetles twice a day in the morning and afternoon, replenish water once a day, remove the bitten spikelets in time, and record the feeding length, limit it to 7 days, and repeat 3 times; the tree species / variety damaged on the first day are highly susceptible, on the third day are moderately resistant, and on the 6th to 7th days are highly resistant.

[0016] Preferably, the rootstock varieties are masson pine, loblolly pine, pine and masson pine × loblolly pine hybrids that are resistant to pine wood nematodes, and masson pine, loblolly pine, pine and masson pine × loblolly pine that are highly resistant to pine wood nematodes.

[0017] Preferably, the varieties of the scion are slash pine, Caribbean pine, South Asian pine or wetland pine that are resistant to pine alternating beetles, and slash pine, Caribbean pine, South Asian pine and wetland pine that are highly resistant to pine alternating beetles.

[0018] Preferably, the rootstock in step (1) has a seedling height of 0.45 to 0.65 cm and a ground diameter of 1.0 to 1.5 cm.

[0019] Preferably, the diameter of the scion in step (2) is 0.8 to 1.3 cm, and the length is 10 to 15 cm.

[0020] Preferably, the grafting time in step (3) is from November to February of the following year or from April to May.

[0021] Preferably, the grafting method in step (3) is cleft grafting, cut grafting, bark grafting or pith cambium grafting.

[0022] Preferably, the post-grafting management method in step (3) is: the interface cannot be exposed to water within 4 to 6 days after grafting, a small incision is made at the budding bud after the spike sprouts and begins to emerge, and topping is performed 2 to 5 times according to the growth of the rootstock.

[0023] The present invention provides a method for improving the resistance of pine trees based on stock and scion interaction, comprising the following steps: (1) evaluating and screening the resistance of different pine species and different varieties of the same species by artificial inoculation, and clarifying the resistance level of pine wood nematodes; (2) evaluating and screening the resistance of different pine species and different varieties of the same species by forced feeding, and clarifying the resistance level of pine wood nematodes; (3) designing tree species / varieties with high resistance to pine wood nematodes and high resistance to pine wood nematodes as "scions" and "stocks" respectively; (4) removing the top of the pine tree and retaining the lower half as the stock; (5) selecting semi-lignified branches that shoot out in the current year as scions; (6) selecting a smooth and straight surface at 0.2 to 0.3 m above the ground, grafting the scion onto the stock, and performing post-grafting management. The present invention breaks through the traditional single control technology, takes dual resistance as the goal, shifts the focus of control to the vector insects, blocks the transmission path of pine wood nematodes, effectively reduces the reproduction and spread of pine wood nematodes, and significantly improves the resistance level. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The transmission chain of pine wood nematode disease (left: host Monochamus alternatus; right: parasitic pine wood nematode under a 10x microscope);

[0025] Figure 2Analysis of the testing steps for pine wood nematode resistance;

[0026] Figure 3 For pine wood nematode resistance testing;

[0027] Figure 4 is the resistance evaluation (blue: highly resistant strain; red: highly susceptible strain);

[0028] Figure 5 For the test of resistance to Monochamus alternatus (forced feeding on the first day);

[0029] Figure 6 To test the resistance to Monochamus alternatus (on the second day of forced feeding, the highly susceptible strain was bitten by 5 beetles);

[0030] Figure 7 For the test of resistance to Monochamus alternatus (5th day of forced feeding);

[0031] Figure 8 It is a double-resistant seedling breeding (spike: resistant to pine black nematode; rootstock: resistant to pine wood nematode);

[0032] Figure 9 It is a double-resistant seedling cultivation (spike: longhorn beetle-resistant species Pinus elliottii; rootstock: nematode-resistant species Pinus taeda). DETAILED DESCRIPTION

[0033] The present invention provides a method for improving the resistance of pine trees based on stock and scion interaction, comprising the following steps:

[0034] (1) Use artificial inoculation to evaluate and screen the resistance of different pine species and different varieties of the same species to determine the resistance level to pine wood nematodes;

[0035] (2) Using forced feeding, evaluate and screen the resistance of different pine species and different varieties of the same species to Monochamus alternatus to determine the resistance level;

[0036] (3) Monochamus alternatus harms needles, while pine wood nematodes harm tree trunks. Tree species / varieties with high resistance to Monochamus alternatus and pine wood nematodes are designed as "spike" and "stockstock" respectively. By using the interaction principle between the stockstock and the spike, as the tree body evaporates, the small molecules resistant to pine wood nematodes in the stock move upward, affecting and improving the overall resistance.

[0037] (4) Remove the top of the pine tree and keep the lower half as the rootstock;

[0038] (5) Select semi-lignified branches that have sprouted in the current year as scion;

[0039] (6) Select a smooth and straight surface 0.2 to 0.3 m above the ground, graft the scion onto the rootstock, and perform post-grafting management.

[0040] In the present invention, the evaluation and screening method for high resistance to pine wood nematodes is preferably: the evaluation and screening method for high resistance to pine wood nematodes is: 8,000 to 10,000 nematodes are artificially inoculated per seedling, and the seedling damage level is counted 15 to 30 days after inoculation at a temperature of about 30°C. The level evaluation index is level 0: no needle yellowing, level I: needle yellowing < all 1 / 2, level II: 1 / 2 ≤ needle yellowing < 2 / 3, level III: needle yellowing = 2 / 3, level IV: needle yellowing > 2 / 3; the damage level is evaluated based on the percentage of level IV seedlings in the total seedlings, <10% is highly resistant, <40% is moderately resistant, and >50% is highly susceptible; highly susceptible species / variety are directly eliminated, and moderately resistant species / variety are preserved in the germplasm resource bank as alternative materials by asexual reproduction. The surviving plants of highly resistant species / variety are inoculated a second time, and the surviving plants are highly resistant strains that are used as hybrid parents or directly promoted and applied.

[0041] In the present invention, pine wood nematodes mainly parasitize in the trachea of ​​Monochamus alternatus, and each beetle can carry up to 280,000 nematodes. Once the Monochamus alternatus bites, the pine wood nematodes are released, leading to pine wood nematode disease.

[0042] In the present invention, the method for evaluating and screening Monochamus alternatus with high resistance to pine sawdust is preferably as follows: 10 to 30 male and female Monochamus alternatus beetles are placed in a space sealed with a 16-18 mesh insect-proof net where the seedlings or branches to be tested are placed, and a forced feeding test is performed.

[0043] In the present invention, the varieties of the rootstocks are masson pine, loblolly pine, pine and masson pine×loblolly pine hybrids that are resistant to pine wood nematodes, and masson pine, loblolly pine, pine and masson pine×loblolly pine that are highly resistant to pine wood nematodes.

[0044] In the present invention, the varieties of the scion are slash pine, Caribbean pine, South Asian pine or wetland pine resistant to pine monochamus alternatus and slash pine, Caribbean pine, South Asian pine and wetland pine highly resistant to pine monochamus alternatus.

[0045] In the present invention, the seedling height of the rootstock in step (1) is preferably 0.45 to 0.65 cm, more preferably 0.55 cm, and the ground diameter is preferably 1.0 to 1.5 cm, more preferably 1.2 to 1.3 cm.

[0046] In the present invention, the diameter of the scion in step (2) is preferably 0.8 to 1.3 cm, more preferably 1.0 to 1.1 cm, and the length is preferably 10 to 15 cm, more preferably 12 to 13 cm.

[0047] In the present invention, the grafting time in step (3) is preferably from November to February of the following year or from April to May, and more preferably from December to January of the following year or from late April to early May.

[0048] In the present invention, the cultivation of resistant seedlings includes two parts: rootstock cultivation and grafting cultivation. First, different pine seedlings with a ground diameter that meets the grafting requirements are transplanted into a 14cm×20cm nutrient cup. The substrate ratio is 60% loess soil + 20% coconut bran + 15% peat soil + 5% substrate. Grafting is carried out after the seedlings have recovered and stabilized 20 to 30 days after transplantation. Post-grafting care is carried out in a centralized nursery. Seedling hardening is carried out 45 days before afforestation, and fertilizer and water control is controlled. Gradient moderate to severe drought stress treatment is carried out 25 to 30 days before afforestation to improve the afforestation survival rate.

[0049] In the present invention, the grafting method in step (3) is preferably cleft grafting, cut grafting, peeling grafting or pith cambium grafting, and more preferably pith cambium grafting.

[0050] In the present invention, the method for docking the pith cambium is preferably: select a rootstock that is 8 to 20 cm away from the ground and is relatively straight, use a grafting knife to cut a 5 to 8 cm incision, select a spike that is similar in size to the rootstock and 10 cm in length, and cut a knife downward 2 cm from the top of the spike so that the length of the knife edge is equivalent to the length of the cut of the rootstock (5 to 8 cm), place the spike close to the rootstock, with the knife edge facing the knife edge, and align the cambium on at least one side, and seal it from bottom to top with a grafting film and tie it tightly. During the tying process, the spike is constantly pressed hard by the hand to ensure that the rootstock and the rootstock are connected. The scion strips fit tightly together. It is further preferred to select a 12-16 cm above the ground and relatively straight surface of the stock and use a grafting knife to cut a 6-7 cm incision. Select a scion strip that is similar in size to the stock and 10 cm in length, and cut it 2 cm from the top of the scion strip so that the length of the blade is equivalent to the length of the cut of the stock (6-7 cm). Place the scion strip close to the stock, with the blade facing the blade and the cambium on at least one side aligned. Use a grafting film to seal it from bottom to top and tie it tightly. During the tying process, keep pressing the scion strip with your hands to ensure that the stock and the scion strip fit tightly together.

[0051] In the present invention, the method for post-grafting management described in step (3) is preferably: within 4 to 6 days after grafting, the interface cannot touch water, and a small opening is made at the sprouting bud after the fringe sprouts and begins to emerge. Topping is performed 2 to 5 times according to the stock growth. It is further preferably that the interface cannot touch water within 5 days after grafting, otherwise it seriously affects the survival rate. After about 30 days, the fringe sprouts and begins to emerge. A small opening is made near the sprouting bud with a sharp blade to help the bud elongate. The incision is as close to the bud point as possible and the smaller the opening, the better, which is conducive to avoiding rainwater from entering the interface in the grafting film and causing waterlogging and rot. In order to avoid excessive growth of the stock, the growth of the stock is suppressed. Topping is performed 2 to 5 times depending on the stock growth to cultivate the scion growth. After about 2 months, the scion extracts new buds, and the interface heals well. The grafting film is completely untied. The stock is broken within 7 days. When the scion grows well and grows vigorously, all the stock above the piercing opening is cut off. After 1 month, the grafted plant can survive stably.

[0052] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] A method for improving pine tree resistance based on stock-scion interaction, comprising the following steps:

[0055] (1) Remove the top of the Masson pine and retain the lower half of the seedling with a height of 0.45 cm and a ground diameter of 1.3 cm as the rootstock;

[0056] (2) Select semi-lignified branches of the current year's slash pine with a diameter of 1.0 cm and a length of 10 cm as scion;

[0057] (3) On November 12, the scion was grafted onto the rootstock using the cleft grafting method at a distance of 0.3 m from the ground on a smooth and straight surface. The interface was kept away from water within 4 days after grafting. After the scion sprouted and began to emerge, a small incision was made at the sprouting bud. The scion was toppled and removed according to the growth of both the rootstock and the scion for 5 times.

[0058] Example 2

[0059] A method for improving pine tree resistance based on stock-scion interaction, comprising the following steps:

[0060] (1) Remove the top of the loblolly pine and retain the lower half of the seedling with a height of 0.65 cm and a ground diameter of 1.5 cm as the rootstock;

[0061] (2) Select semi-lignified branches of Pinus truncatulae that have been grown in the current year and have a diameter of 0.8 cm and a length of 15 cm as scions;

[0062] (3) On May 3rd of the following year after the scion is selected, select a smooth and straight surface 0.2m above the ground and graft the scion onto the rootstock using the peeling-butt method. The interface should not be exposed to water within 6 days after grafting. After the scion sprouts and begins to emerge, a small incision is made at the sprouting bud. Top and remove the sprouts twice according to the growth of both the rootstock and the scion.

[0063] Example 3

[0064] A method for improving pine tree resistance based on stock-scion interaction, comprising the following steps:

[0065] (1) Remove the top of the Laya pine and keep the lower part of the seedling with a height of 0.55 cm and a ground diameter of 1.2 cm as the rootstock;

[0066] (2) Select semi-lignified branches of the current year's wet-added pine with a diameter of 1.0 cm and a length of 13 cm as scion;

[0067] (3) On February 26 of the following year after the scion was selected, a smooth and straight surface was selected at 0.25 m above the ground, and the scion was grafted onto the rootstock using the pith cambium docking method. The interface should not be exposed to water within 5 days after grafting. After the scion sprouted and began to emerge, a small incision was made at the sprouting bud. The scion was toppled and removed according to the growth of both the rootstock and the scion for 4 times.

[0068] Test example

[0069] A method for improving pine tree resistance based on stock-scion interaction, comprising the following steps:

[0070] 1. Resistance level evaluation

[0071] (1) Evaluation and screening of pine wood nematode resistance

[0072] The detection and evaluation of resistance to pine wood nematode disease adopts the artificial inoculation nematode method. The first step is to isolate pine wood nematodes: the Baermann funnel method is used to isolate nematodes. First, prepare a 20 cm wide glass funnel with a 15 cm long latex tube at the end. Place the glass funnel with the latex tube on the funnel stand and install a spring water-stop clamp on the latex tube. Slowly inject an appropriate amount of sterile water into the glass funnel to expel the bubbles at the bottom of the latex tube and observe whether the latex tube is leaking. If it is leaking, clamp it or replace the water-stop clamp. Split the infected wood into 5 cm long and 0.5 cm wide wood chips, wrap them with two layers of gauze or paper towels and put them into a funnel. Put a label on the funnel and slowly inject an appropriate amount of sterile water along the inner wall of the funnel until the sample is immersed. After soaking the infected wood for 12 hours, loosen the water stop clamp and collect 15 ml of nematode suspension in a clean small culture dish or centrifuge tube; secondly, centrifuge the nematode suspension to remove the supernatant, sterilize it with 1.5% lactic acid for 1 hour, and culture the nematodes on PDA-Botrytis cinerea flat culture medium that has been cultured at a constant temperature of 25℃ for 7 days. Inoculate 10 selected and identified female and male adults into each culture dish, culture them at a constant temperature of 27℃ for 9 days, and then use them for resistance detection; thirdly, resistance detection. Choose a sunny day with a temperature of around 30℃, use a stationery knife to cut the bark of the tender branch 3 cm long and 3 mm wide at the semi-lignified part of the seedling, cut to the phloem, mix the nematodes into a certain proportion of insect liquid, and inoculate 10,000 nematodes per plant; the fourth step is the resistance level. The damage level of seedlings was calculated starting from 30 days after inoculation with insects. The evaluation index was as follows: Level 0: no yellowing of needles, Level I: yellowing of needles < all 1 / 2, Level II: 1 / 2≤yellowing of needles < 2 / 3, Level III: yellowing of needles = 2 / 3, Level IV: yellowing of needles > 2 / 3. The damage level was evaluated based on the percentage of Level IV seedlings in the total seedlings, <10% was highly resistant, <40% was moderately resistant, and >50% was highly susceptible. Highly susceptible species / variety were directly eliminated, and moderately resistant species were preserved in germplasm resource banks as alternative materials through asexual reproduction. The surviving plants of highly resistant species / variety were inoculated with insects for a second time, and the surviving plants were regarded as highly resistant strains and used as hybrid parents or directly promoted and applied.

[0073] (2) Evaluation and screening of pine wood nematodes. Pine wood nematodes mainly parasitize in the trachea of ​​pine wood nematodes, and each beetle can carry up to 280,000 nematodes. Pine wood nematodes can be released by biting pine wood nematodes, which can then infect pine wood nematode disease. The forced feeding method is used for the evaluation and screening of pine wood nematodes. The specific steps are as follows: Step 1, the adult pine wood nematodes are paired and selected. About 60 days in advance, the bait trees are set in the forest in the epidemic area. Before the pine wood nematodes emerge, the bait trees are pulled back to the laboratory in strict accordance with the treatment methods for major quarantine objects. Pine wood nematodes are collected once every morning, and beetles with the same male and female ratio collected on the same day are selected for forced feeding experiments; Step 2, resistance evaluation. Seal the space containing the test seedlings / branches with an 18-mesh insect-proof net and introduce 30 male and female Monochamus alternatus beetles for a forced feeding test. Check the beetles' feeding activity twice daily, morning and afternoon. Replenish water once daily, promptly remove any chewed shoots, and record the feeding length. Repeat three times over a seven-day period. Tree species / variety infested on the first day are considered highly susceptible, those infested on the third day are moderately resistant, and those infested on days 6-7 are considered highly resistant.

[0074] (3) Precise design of new dual-resistance germplasm

[0075] Breaking through the traditional single-point control theory, the stockpile-scion interaction method is used to precisely design dual-resistant varieties. Based on the differences in parasitism and host action sites, as well as the differences in dual resistance between tree species / varieties, tree species / varieties resistant to vector insects are designed as "spikes" and tree species / varieties resistant to pine wood nematodes are designed as "stocks." With dual resistance as the goal, longhorn beetle-resistant and nematode-resistant varieties are cultivated. This actively avoids or reduces feeding by vector insects, slows the reproduction and spread of pine wood nematodes, cuts off the disease transmission path, and achieves the dual resistance goal. This effectively solves the problems of passive control technology and low control efficiency, and significantly improves resistance.

[0076] (4) Cultivation of stock with interactive rootstock and scion

[0077] Based on the design of the new dual-resistance germplasm, suitable rootstocks and scions are cultivated separately. First, the top of the rootstock is removed, retaining the lower half as the rootstock. Second, a semi-lignified branch that has sprouted in the current year is selected as the scion. Third, the scion is grafted onto the rootstock on a smooth, straight surface 0.3 m above the ground, and post-grafting care is performed to form a new rootstock-scion hybrid germplasm.

[0078] Results Statistics

[0079] Table 1 Comparison of resistance of new varieties with stock and scion interaction

[0080]

[0081]

[0082] As can be seen from the above embodiments, the present invention provides a method for improving the resistance of pine trees based on stock and scion interaction, comprising the following steps: (1) evaluating and screening the resistance of different pine species and different varieties of the same species by artificial inoculation, and clarifying the level of resistance to pine wood nematodes; (2) evaluating and screening the resistance of different pine species and different varieties of the same species by forced feeding, and clarifying the level of resistance to pine sawyer beetles; (3) designing tree species / varieties with high resistance to pine sawyer beetles and high resistance to pine wood nematodes as "spices" and "stocks", respectively; ( 4) Remove the top of the pine tree and keep the lower half as the rootstock; (5) Select the semi-lignified branches of the current year as the scion; (6) Select a smooth and straight surface at 0.2 to 0.3 m above the ground, graft the scion onto the rootstock, and perform post-grafting management; (7) The three new double-resistant varieties created based on the interaction between the rootstock and scion in Examples 1 to 3, namely, Pinus massoniana: Elliot pine, Pinus taeda: Torch pine, and Pinus rapa: Elliot pine, all reached the highest level of double resistance, and the double resistance target was at least 2 levels higher than the control (single resistance), with a significant resistance improvement effect. The present invention breaks through the traditional single prevention and control technology, accurately customizes new resistant germplasm with double resistance as the target, shifts the focus of prevention and control to the vector insects, blocks the transmission path of pine sawyer beetles, effectively reduces the reproduction and spread of pine wood nematodes, and greatly improves the resistance level.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for improving pine tree resistance based on stock and scion interaction, characterized in that: The steps include: (1) Use artificial inoculation to evaluate and screen the resistance of different pine species and different varieties of the same species to determine the resistance level to pine wood nematodes; (2) Use forced feeding to evaluate and screen the resistance of different pine species and different varieties of the same species to determine the resistance level to Monochamus alternatus; (3) Design tree species / varieties with high resistance to Monochamus alternatus and pine wood nematode as "ears" and "stockstocks" respectively; (4) Remove the top of the pine tree and keep the lower half as the rootstock; (5) Select semi-lignified branches that have sprouted in the current year as scion; (6) Select a smooth and straight surface 0.2-0.3 m above the ground, graft the scion onto the rootstock, and perform post-grafting management; The highly resistant pine beetle evaluation and screening method is as follows: 10 to 20 female and 10 to 20 male pine beetles are placed in a space sealed with a 16 to 18 mesh insect-proof net where the seedlings or branches to be tested are placed, and a forced feeding test is performed; About 60 days in advance, bait trees were placed in the infected forest. Before the Monochamus alternatus emerged, the bait trees were pulled back to the laboratory in strict accordance with the treatment methods for major quarantine objects. Monochamus alternatus was collected once every morning, and beetles with the same male-female ratio were selected on the same day for forced feeding experiments. The feeding status of the beetles was checked twice a day, in the morning and afternoon. Water was replenished once a day, and the chewed spikelets were promptly removed and the feeding length was recorded. This was repeated three times within a limit of 7 days. The tree species / variety damaged on the first day were highly susceptible, those on the third day were moderately resistant, and those on the sixth to seventh days were highly resistant. The rootstock varieties are pine wood nematode-resistant masson pine, loblolly pine, pine of Laya, and a hybrid of masson pine and loblolly pine. The variety of the scion is the pine alternatant-resistant loblolly pine, Caribbean pine, South Asian pine or loblolly pine.

2. The method according to claim 1, characterized in that The evaluation and screening method for highly resistant pine wood nematodes is as follows: 8,000 to 10,000 nematodes are artificially inoculated on each seedling, the temperature is around 30°C for inoculation, and the damage level of the seedlings is counted starting from 15 to 30 days, with the evaluation index being level 0: no yellowing of needles, level I: yellowing of needles < all 1 / 2, level II: 1 / 2 ≤ yellowing of needles < 2 / 3, level III: yellowing of needles = 2 / 3, level IV: yellowing of needles > 2 / 3; the damage level is evaluated based on the percentage of level IV seedlings in the total seedlings, <10% is highly resistant, <40% is moderately resistant, and >50% is highly susceptible; highly susceptible species / variety are directly eliminated, and moderately resistant species are preserved in a germplasm resource bank as alternative materials through asexual reproduction, and the surviving plants of highly resistant species / variety are inoculated a second time, and the surviving plants are highly resistant strains that are used as hybrid parents or directly promoted and applied.

3. The method according to claim 2, characterized in that The rootstock seedlings are 0.45~0.65m tall and 1.0~1.5cm in diameter at ground level.

4. The method according to claim 1, wherein The diameter of the scion is 0.8~1.3cm and the length is 10~15cm.

5. The method according to claim 1, wherein The grafting time is from November to February of the following year or from April to May.

6. The method according to claim 3, characterized in that The grafting methods are cleft grafting, cut grafting, bark grafting or pith cambium grafting.

7. The method according to claim 4, characterized in that The management method after grafting is: the interface cannot be exposed to water within 4 to 6 days after grafting. After the scion sprouts and begins to emerge, a small incision is made at the sprouting bud. Top and remove the sprouts 2 to 5 times according to the growth of both the rootstock and the scion.

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