Rapid and large-scale forest disease resistance detection and disease phenotype data acquisition method

Abstract

The invention relates to the field of tree disease-resistant variety breeding, in particular to a rapid and large-scale tree disease resistance detection and disease phenotype data acquisition method. The resistance detection and phenotypic data acquisition method comprises the following steps: sterilizing the surface of a healthy forest leaf, inoculating the upper surface of the leaf with a germ block, sealing, fixing and moisturizing with a transparent adhesive tape, and puncturing and punching at multiple points of an inoculation position from the lower surface of the leaf; 5 days later, photographing, counting the form and area of a disease spot formed around each needling point of the leaf, grading the disease condition according to the area of the disease spot, calculating the morbidity and disease index, and grading the disease resistance of the forest. By adopting the method disclosed by the invention, early screening of disease resistance of breeding materials can be completed, disease phenotype data can be provided, the period of conventional forest tree breeding and modern forest tree breeding can be remarkably shortened, the breeding cost can be reduced, and disease-resistant genetic resources can be fully excavated. By taking germs as objects, the method disclosed by the invention can be used for screening pathogenicity of the germs and fully excavating genetic resources of the germs.

Description

Technical Field

[0001] The present invention relates to the field of forest disease-resistant variety breeding, and in particular to a method for rapid and large-scale forest disease resistance detection and disease phenotype data acquisition. Background Art

[0002] During the growth process, trees are invaded by pathogens such as fungi, bacteria, viruses and nematodes, which in turn cause various diseases. Fungal diseases of tree branches and trunks include different types of diseases such as ulcers, bark rot, branch dieback, dry rot, gummosis, and ring rot, which are one of the important forest biological disasters. Most of the diseases of tree branches and trunks are caused by fungal pathogens, which seriously endanger the growth and healthy development of ecological, economic and garden trees such as poplars, willows, apples, peaches, walnuts, boxwood, eucalyptus, and camphor trees. Pathogen inoculation technology is the most basic experimental technology in the pathology, physiology, and molecular biology research of plants and forest diseases, as well as disease prevention and control management. At present, the methods of pathogen inoculation for tree branch and trunk diseases include punching inoculation, scalding inoculation, bud scar inoculation, lenticel puncture inoculation, and wooden stick inoculation. The above inoculation method can meet the needs of conventional research work such as fungal disease biology, pathology, and molecular biology that require less experimental materials and less inoculation operations.

[0003] However, in some research and application fields that require disease resistance screening or testing of a large number of trees (such as the selection of disease-resistant varieties for tree branch diseases; usually it is necessary to screen or test hundreds to thousands, or even more tree species or clones), if the above-mentioned inoculation method is used for disease resistance testing, the number of tree experimental materials participating in the experiment, and then the cultivation space, time and funds for the tree experimental materials will have high requirements. Taking the selection of hybrid varieties for poplar canker as an example, usually a poplar hybrid experiment will obtain hundreds, thousands or even more hybrid clones. If the branch wound inoculation method is used to screen the disease resistance of all clones, assuming that 10 plants are inoculated for each clone (each plant is inoculated at 5 locations on the branch, and a total of 50 locations are inoculated), then the disease resistance test alone requires the cultivation of thousands to tens of thousands of poplar seedlings and takes 2 to 3 years. More importantly, branch pathogen inoculation is a destructive operation that can cause pathogen contamination, disease infection or death of valuable forest breeding materials, affecting the preservation of genetic germplasm resources and the implementation of subsequent research.

[0004] For the above reasons, there are few reports on the disease-resistant cross-breeding of forest tree branches at home and abroad. In the reported work on the selection of disease-resistant varieties of forest tree branches, small-scale disease resistance tests are also used instead of screening all hybrid clones to identify disease-resistant hybrid varieties. Taking the cross-breeding of poplar against canker as an example, the brief process is as follows: In the first year, a hybridization experiment is carried out, and hybrid poplar seedlings are obtained after cultivation; in the second to fourth years, 10-20% of the seedlings with excellent growth traits are selected for cuttage propagation (preliminary screening) and regional cultivation experiments; in the fourth to seventh years, disease resistance tests are carried out on the hybrid clones with excellent growth traits by means of field investigation, inoculation of in vitro branches of clones, etc., and disease-resistant clones with excellent traits are initially obtained. In short, the above method for selecting disease-resistant varieties sacrifices potential hybrid clone resources (preliminary screening usually eliminates 80-90% of hybrid clones, which may include some genetic resources with high disease resistance or high susceptibility but unremarkable growth traits), and it takes 10 years or more to discover and announce a new disease-resistant clone species with great application potential.

[0005] With the rapid development of high-throughput sequencing technology, new technologies such as genomic selection have been widely applied to the breeding process of the new generation of animals and plants. Genomic selection is also known as genomic prediction, which reduces the breeding cost by eliminating individuals with low potential in the early stage of breeding (for example, in the second year of poplar cross-breeding). However, genomic selection breeding requires the support of phenotypic trait data. For the selection of disease-resistant varieties, the most direct phenotypic trait data are the incidence rate, lesion area, and disease index, etc. It can be seen that the disease resistance detection of forest tree branch diseases not only restricts conventional cross-breeding but also becomes a key rate-limiting step in restricting the modern forest tree breeding process. If an early detection technology is developed, which can quickly and simply obtain disease phenotypic data and complete disease resistance evaluation in the early stage of forest tree growth (for example, in the first or second year of disease-resistant variety selection), it can significantly shorten the breeding cycle of forest tree disease-resistant varieties, reduce the consumption of breeding materials, save human resources and cultivation space, and at the same time help to fully explore and utilize forest tree disease-resistant genetic resources, which is of great significance for promoting the research on the genetic mechanism of forest tree disease resistance and the selection of disease-resistant varieties. In addition to the cross-breeding of forest tree disease-resistant varieties, this technology can also solve the problems of disease resistance detection technology in the research on the genetic differentiation of disease resistance in natural populations of forest trees propagated by seeds and the genetic differentiation of pathogenicity in pathogen populations (such as artificially induced pathogen populations, etc.). Summary of the Invention

[0006] The object of the present invention is to provide a method for rapid and large-scale detection of tree disease resistance and acquisition of disease phenotype data, so as to solve the practical technical problems of large-scale disease resistance detection required for conventional cross-breeding of disease-resistant tree varieties or pathogenicity analysis of pathogen populations, and also solve the dilemma of obtaining disease resistance phenotypic traits in modern tree breeding such as genomic selection. The disease resistance detection method of the present invention can significantly shorten the breeding cycle of disease-resistant tree varieties, reduce the consumption of tree hybrid clone materials, save experimental costs, and can fully explore and utilize the disease resistance genetic resources of tree hybrid clones. The disease phenotype trait acquisition method of the present invention can be applied to the tree breeding process for other objectives, achieve the purpose of multi-objective polymerization breeding, and further reduce breeding costs; taking the pathogen as the object, the disease resistance detection method of the present invention can quantitatively analyze the pathogenicity of the pathogen and fully explore the pathogen genetic resources.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for rapid and large-scale detection of tree disease resistance and acquisition of disease phenotype data, including the following steps:

[0009] After inoculating the pathogen mycelial block on the upper surface of the tree leaf, use transparent tape to seal the inoculated pathogen mycelial block for moisturizing; the number of mycelial blocks inoculated on each leaf is 2 - 4 pieces, and at least 3 leaves are inoculated;

[0010] In the same way as the pathogen mycelial block inoculation method, use the leaf inoculated with the sterilized water agar block as a control, and inoculate 1 leaf;

[0011] The inoculated leaves are newly mature, healthy tree leaves with the same light conditions, no pests, diseases and mechanical damage;

[0012] After inoculation, use a sterile inoculation needle to puncture the lower surface of the leaf and the pathogen mycelial block or the sterilized water agar block to form multiple puncture points, but do not pierce the transparent tape; the distance between each puncture point is more than 0.5 cm;

[0013] 3 - 7 days after inoculation, remove all the inoculated leaves, and take photos and measure the morphology and area of the necrotic spots formed around each puncture point; the inoculated leaves include the leaves inoculated with the pathogen mycelial block and the leaves inoculated with the sterilized water agar block;

[0014] According to the morphology and area of the necrotic spots on the control inoculated leaves, determine the disease occurrence standard; count the number and area of the necrotic spots formed by the mycelial block inoculation, calculate the incidence rate and disease index; according to the disease index of the lesions, judge the disease resistance of the tree to the pathogen; the disease resistance level classification standard is as shown in the following table:

[0015] Disease index Disease resistance <10.00 Highly resistant HR 10.01～20.00 Resistant R 20.01～40.00 Not susceptible NS 40.01～60.00 Susceptible S 60.01～80.00 Moderately susceptible MS 80.01～90.00 Highly susceptible HS >90.01 Very highly susceptible VHS 。

[0016] Preferably, when the pathogen mycelial block or sterilized water agar block is square, the puncture positions are located at the center and four corners of the pathogen mycelial block or sterilized water agar block, and the distance between each puncture point is more than 0.5 cm; the puncture positions at the four corners are all 1-2 mm inside the inner edge of the pathogen mycelial block;

[0017] When the pathogen mycelial block or sterilized water agar block is rectangular, the distance between each puncture point is more than 1 cm, and the distance between each puncture point is more than 1 cm. This method is suitable for the forest tree-pathogen interaction system with a relatively large expansion area of the inoculated plaque.

[0018] More preferably, solid mycelial blocks are used for inoculation; the cultured solid culture is divided into mycelial blocks with a sterile blade.

[0019] More preferably, the square is a solid mycelial block with a size of not less than 1.0 cm×1.0 cm to 1.2 cm×1.2 cm. In the specific embodiments of the present invention, mycelial blocks of 1.0 cm×1.0 cm to 1.2 cm×1.2 cm are used;

[0020] More preferably, the square solid mycelial block is a solid mycelial block with a size of not less than 1.0 cm×1.0 cm to 1.2 cm×1.2 cm; in the specific embodiments of the present invention, solid mycelial blocks with a size of 1.0 cm×1.0 cm to 1.2 cm×1.2 cm are used;

[0021] The rectangular solid mycelial block is a solid mycelial block with a size of 0.3 cm×6 cm or a longer solid mycelial block, which is suitable for the quantitative determination of weakly disease-resistant poplar clones.

[0022] The rectangle being a solid mycelial block with a size of 0.3 cm×6 cm or a longer solid mycelial block is suitable for the quantitative determination of weakly disease-resistant poplar clones.

[0023] Preferably, the pathogen contained in the pathogen mycelial block is the pathogen causing branch diseases.

[0024] Preferably, the components of the sterilized water agar block include agar and water; the mass-volume ratio of agar to water is 20 g:1000 mL.

[0025] Preferably, the forest tree leaves are the leaves of annual or perennial forest trees.

[0026] More preferably, when using 1-year-old forest tree plants, normal irrigation should be carried out within 2 weeks before inoculation, and the forest tree plants should not be subjected to drought and other environmental stresses; the inoculation objects are mature leaves that are healthy, normal in color and morphology, and free of pests, diseases and mechanical damage; when inoculating the same batch, the maturity of the inoculated leaves between different forest tree plants (species, clones, varieties) should be consistent, and the newly mature leaves (new leaves) at the 5th to 10th positions from the top of the plant are used;

[0027] When using the branches of perennial forest trees (species, clones, varieties), normal irrigation should be carried out within 2 weeks before inoculation, and the forest tree plants should not be subjected to drought and other environmental stresses; the attachment position, height, and light of the branches should be consistent; the inoculation object is the mature leaves on the current-year branches that are healthy, normal in color and morphology, and free from diseases, pests, and mechanical damage; when inoculating the same batch, the maturity of the inoculated leaves should be consistent among different forest tree plants (species, clones, varieties), and the leaves (new leaves) at the 5th to 10th positions from the top of the current-year branches should be used.

[0028] Preferably, the inoculation positions of both the pathogen mycelium blocks and the sterilized water agar blocks are 1 - 3 cm on both sides of the main vein of the forest tree leaves, and the secondary veins of the forest tree leaves should be avoided.

[0029] Preferably, the pathogen mycelium blocks are obtained after culturing for 1 day (5d) after the mycelium fills a 9-cm Petri dish.

[0030] More preferably, the thicknesses of the pathogen mycelium blocks are the same.

[0031] More preferably, conventional fungal media are used for culturing; the conventional fungal media preferably include PDA (potato dextrose agar) medium, MA (oat agar) medium, or Czapek medium, etc., and preferably also include PDA medium or MA medium, and more preferably PDA medium; the media are autoclaved at 121 °C for 20 min.

[0032] More preferably, when inoculating, the thicknesses of the media for culturing fungi are the same. The present invention preferably adds 15 mL of PDA medium into a 9-cm Petri dish.

[0033] Preferably, before inoculation, the forest tree leaves are also subjected to leaf washing and disinfection; the disinfectant used for disinfection is alcohol or mercuric chloride.

[0034] Preferably, the alcohol is an alcohol solution with a volume percentage content of 75%; the mercuric chloride is a mercuric chloride solution with a volume percentage content of 0.1%.

[0035] The present invention discloses the following technical effects: to solve the practical technical problems of large-scale disease resistance detection required for conventional cross-breeding of disease-resistant forest tree varieties or pathogenicity analysis of pathogen populations, and also to solve the dilemma of obtaining disease resistance phenotypic traits in modern forest tree breeding such as genomic selection.

[0036] The present invention provides a method for detecting the disease resistance of forest trees, which is rapid, large-scale, time-saving and easy to operate. It is a method applicable to the detection of the resistance of branches and trunks to fungal diseases in a relatively large number of forest tree hybrid breeding populations and genetic transformation populations, as well as the determination of the pathogenicity of a relatively large number of pathogenic fungal populations. It is also a rapid method for obtaining the disease resistance phenotypic traits necessary for genomic selection breeding. The advantages of the present invention are specifically reflected in the following aspects: 1. High throughput: When there are 5 puncture points for each fungal inoculum block, 10, 20 or more disease spots can be obtained simultaneously on one leaf, and multiple leaves on one plant can be inoculated simultaneously, so as to obtain more disease condition data, and the purpose of completing the disease resistance or pathogenicity test with 1 or a few clonal plants can be achieved. When 2 - 3 people cooperate, the detection of 200 - 400 hybrid clonal materials or fungal strains can be completed within one week; 2. Rapidly obtain the disease resistance detection results: The results can be obtained within 3 - 7 days for a single determination; 3. Significantly shorten the breeding cycle: When applied to the disease resistance breeding of forest tree fungal diseases, especially the disease resistance breeding of poplar branch and trunk diseases, inoculating the leaves of 1 - 2-year-old poplar seedlings (seedlings or 1-year-old cuttings) with pathogens, compared with inoculating branches and trunks by wounding (using 2 - 4-year-old cuttings as inoculation materials), the disease resistance results can be obtained 1 - 2 years or even longer in advance, so as to formulate the next breeding plan and significantly shorten the test cycle; 4. Particularly suitable for the rapid disease resistance test of rare or precious forest tree germplasm resources: Using leaf inoculation and with a relatively short experimental cycle (3 - 7 days), it can reduce or avoid the destructive impact of pathogen inoculation on hybrid clonal resources, and can be applied to the rapid disease resistance test of precious forest tree germplasm resources with fewer biological replicates, reducing or eliminating the destructive impact of pathogen inoculation on forest tree germplasm resources; 5. It can achieve the pyramiding breeding of forest trees resistant to multiple diseases: Using leaves as inoculation materials, compared with inoculating branches and trunks, the sources of inoculation materials are rich, and the pathogenic fungi of multiple different diseases can be inoculated, fully exploring breeding resources and possibly achieving the purpose of pyramiding breeding resistant to multiple diseases; 6. It can conveniently achieve the pyramiding breeding of disease resistance breeding and other breeding goals: Using leaves as inoculation materials, due to the fast disease development speed (3 - 7 days) and the small number of inoculated leaves (3 - 5 leaves for inoculating 1 kind of pathogen), compared with inoculating branches and trunks with pathogens, it does not change or less changes other phenotypic traits of the forest tree genetic population (such as the growth rate of forest trees, cold resistance, drought resistance, salt tolerance ability, etc.), so it does not affect or less affects the screening of hybrid clones based on the above traits, and can conveniently achieve the pyramiding breeding of multiple trait goals of forest trees; 7. Safe and reliable, with little possibility of pathogen spread: Using leaves as inoculation materials and completely removing the inoculated leaves after detection, since the time for the disease to infect the leaves is relatively short (3 - 7 days) and the inoculum blocks are sealed with transparent tape, the possibility of the inoculated pathogens contaminating other parts of the forest trees and the forest land is relatively small.8. Simple and easy to operate: Using the fungal mycelium culture and taking the leaves as the inoculation object, the operation is simple; 7. Wide application range: In addition to poplar, it can be applied to the large-scale detection of the disease resistance of other broad-leaved trees (such as poplar, willow, apple, peach, walnut, boxwood, eucalyptus, camphor tree, etc.) against trunk diseases (such as canker, bark canker, twig blight, dry rot, gummosis, ring rot, etc.); it can also be applied to the inoculation detection of forest leaf fungal diseases; 9. Suitable for disease resistance screening in the cross-breeding of forest trees against fungal diseases (trunk diseases and leaf diseases), especially the early disease resistance screening of trunk diseases such as canker and bark canker in cross-breeding. Compared with the trunk materials that need to be cuttaged and propagated and cultivated for 2 - 3 years, using the leaves of hybrid seedlings or 1-year-old cuttaged seedlings for disease resistance screening can significantly shorten the breeding cycle, save experimental space, manpower and material resources; at the same time, compared with the pathological effects of long-term pathogen inoculation (15 - 30 days or longer) on the trunk, the short-term leaf pathogen inoculation (5 - 7 days) has less impact on the breeding materials, and after the inoculated leaves are removed after the disease resistance screening, the pathological effects of the pathogen on the breeding materials and the potential risk of pathogen spread are eliminated, and the stability of the breeding materials can be maintained. At the same time, with the numerous leaves on forest trees as the inoculation materials, this invention can quickly and high-throughput obtain accurate data on indicators such as incidence rate and disease index, and conduct significant difference analysis, so as to obtain accurate disease resistance data of the poplar clone (plant) population; 10. Solve the pathological problem of quickly obtaining the large-scale forest disease phenotypic trait data necessary for genomic selection breeding.

[0037] The present invention can solve the technical problems of rapid and high-throughput early disease resistance screening or obtaining disease phenotypic traits of a large number of breeding materials obtained in forest tree disease resistance genetic breeding (conventional cross-breeding genetic breeding, mutagenesis breeding, and the new generation of genomic selection breeding), significantly save breeding materials, significantly reduce breeding costs and breeding cycles; it can also solve the rapid and high-throughput detection of the pathogenicity of forest tree pathogenic fungal genetic populations.

[0038] The present invention can be applied to the pathogenicity detection of different pathogen species, strains, single spore cultures, natural pathogen populations, etc. The identification method described in the present invention is suitable for inoculating artificially cultivable pathogenic fungi, especially pathogenic fungi that are not easily sporulated, and is particularly suitable for detecting the pathogenicity of pathogenic fungi associated with trunk canker diseases and bark rot diseases of forest trees (such as Botryosphaeria dothidea, the pathogen of tree canker, and Valsa sordida, the pathogen of tree bark rot) and other pathogenic fungi associated with leaf diseases of forest trees. At the same time, the present invention has no requirements for forest tree materials. Except for the poplar materials (hybrid clones of Populus deltoides - Populus nigra) involved in the present invention, this method can also be applied to large-scale disease resistance detection of other different sections of poplars, interspecific hybrid clones within the section, as well as varieties and cultivars for trunk diseases. Thus, it can be seen that the present invention can be applied to research and applications such as conventional, molecular disease resistance genetic breeding, and genetic differentiation of disease resistance in natural populations of other forest trees.

[0039] Moreover, after the experiment is completed and 5 - 7 days after the inoculation experiment, all inoculated leaves and control inoculated leaves are removed to eliminate the influence of the pathogen on the forest trees, ensuring that the present invention will not cause pathogen contamination to the breeding materials and increase the risk of pathogen transmission; the disease incidence criteria are determined according to the morphology and area of the leaf necrosis spots under the control inoculation to ensure reliable disease resistance detection results and disease phenotype trait data.

[0040] The detection method described in the present invention may be applied to the following fields and scenarios:

[0041] 1. Disease resistance testing and obtaining of disease phenotype traits for hybrid clone populations, transgenic clone populations, mutagenized populations, and seed propagation populations of forest trees formed by forest tree disease resistance genetic breeding (conventional breeding and genomic selection breeding), especially the testing of resistance to trunk canker diseases and bark rot diseases and obtaining of disease phenotype traits, as well as the testing of disease resistance to artificially cultivable leaf fungal diseases and obtaining of disease phenotype traits;

[0042] 2. Other forest tree genetic breeding processes (conventional and new generation forest tree breeding, such as genomic selection breeding) of forest trees for drought resistance, salt tolerance, cold resistance, tolerance to high CO 2 environments, and different nitrogen utilization, etc., to achieve the goal of multi-objective pyramiding breeding;

[0043] 3. Early detection or screening of disease resistance to canker diseases, bark rot diseases, and leaf fungal diseases of forest tree plants, clones, mutants, etc.;

[0044] 4. Koch's verification experiment for fungal strains isolated from forest tree tissues, and auxiliary determination of forest tree and plant pathogens;

[0045] 5. Pathogenicity detection of a large number of fungal strains obtained by separation, mutagenesis, hybridization, and genetic transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0047] Figure 1 It shows the morphology of the necrotic area after inoculating the leaves with the pathogen. Among them, A - B are the leaf lesions, C - F are the leaf necrosis spots in the control inoculation, G - I are the leaf lesions of the diseased leaves, and J - K are the formation of hyphae and pycnidia structures on the diseased leaves (only appearing in some highly susceptible clones, such as clone B246);

[0048] Figure 2 It shows the influence of leaf maturity on the development of the disease;

[0049] Figure 3 It shows the influence of light on the development of the disease;

[0050] Figure 4 It shows the influence of the pathogen culture time on the development of the disease;

[0051] Figure 5 It shows the disease resistance differentiation of 48 poplar clones;

[0052] Figure 6 It shows the normal distribution curve of the susceptibility index of 48 poplar hybrid clones;

[0053] Figure 7 It shows the pathogenicity differentiation of 12 poplar canker pathogen strains.

[0054] BIOLOGICAL DEPOSIT INFORMATION

[0055] Valsa sordida CZC was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 12, 2023. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 40575. DETAILED DESCRIPTION OF THE INVENTION

[0056] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0057] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0058] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0059] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0060] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0061] Preparation example:

[0062] Pathogenic bacterium: Valsa sordida CZC strain, which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (preservation number: CGMCC No. 40575) and the Plant Physiology Laboratory of the Institute of Ecological Protection and Restoration, Chinese Academy of Forestry.

[0063] The pathogenic bacterium is cultured using a PDA medium (20% potato, 2% glucose, 2% agar; pH 6.0; autoclaved at 121 °C for 20 minutes). The specific method is as follows: After the Valsa sordida CZC strain is activated and cultured on the PDA medium, it is then inoculated onto a new PDA medium (15 mL of the medium is added to a petri dish with a diameter of 9 cm), and cultured in the dark at 28 °C (the best mycelial block is the one after 1 day when the culture plate surface is fully covered); after the culture is completed, the solid culture of the pathogenic fungus is cut into solid mycelial blocks with a size of 1.0 cm × 1.0 cm to 1.2 cm × 1.2 cm using a sterilized scalpel and reserved.

[0064] Sterilized water agar block: 20 g of agar was added to 1000 mL of water and sterilized to obtain a sterilized water agar block.

[0065] In Examples 1-2 and Example 4, solid bacterial blocks obtained after 5 days of cultivation were used; in Example 3, solid bacterial blocks obtained after 4 days or 7 days of cultivation were used.

[0066] Effect of leaf maturity on disease development in Example 1

[0067] Using 1-year-old Populus × euramericana cv. 'Bofeng 3' as the material, the effects of inoculating leaves with different maturities (mature new leaves, located at the upper part of the branch; mature old leaves, located at the lower part of the branch) on the development of the disease were detected. Six poplar trees were evenly divided into 2 groups, with 3 trees in each group. In the new leaf group, leaves at the 5th to 8th positions from the top of the inoculated branch were inoculated. Among them, 3 leaves were inoculated with bacterial blocks of the pathogen (2 bacterial blocks per leaf), and 1 leaf was inoculated with a sterilized water agar block (2% water agar block), with 2 sterilized water agar blocks inoculated per leaf. In the old leaf group, leaves at the 15th to 18th positions from the top of the inoculated branch were inoculated, and the inoculation method was the same as that of the new leaf group. The inoculated leaves were healthy, without pest and disease infection and mechanical damage. The specific steps were as follows:

[0068] (1) One day before inoculation or on the day of inoculation, the upper and lower surfaces of the leaves were washed with clean water or tap water; on the day of inoculation, the upper and lower surfaces of the inoculated forest tree leaves were surface-sterilized with 75% (V / V) alcohol solution and air-dried naturally, and then marked for standby;

[0069] (2) On the marked leaves, avoiding the main veins and secondary veins, bacterial blocks with a size of 1.0 cm × 1.0 cm to 1.2 cm × 1.2 cm were inoculated on both sides of the main vein on the upper surface of the leaves at a position 1 to 3 cm away, with 1 bacterial block inoculated on each side, and the hyphal surface was in contact with the leaf surface, and at least 3 leaves were inoculated. A sterilized water agar block (2%) with a size of 1.0 cm × 1.0 cm to 1.2 cm × 1.2 cm was inoculated on the leaf as a control, and 1 leaf was inoculated;

[0070] (3) After inoculation, quickly cover the leaves with a 5-cm-wide household transparent tape, carefully seal the four sides to prevent the bacterial blocks (water agar blocks) from losing water, and then pierce the center and positions within 1 to 2 mm of the four corners of the 1.0 cm × 1.0 cm to 1.2 cm × 1.2 cm bacterial block (2% water agar block) from the lower surface of the leaf with a sterilized inoculation needle, but do not pierce the transparent tape, that is, 5 puncture points were formed for each bacterial block (2% water agar block);

[0071] (4) Check the sealing condition of the tape paper and the disease occurrence condition every day after inoculation. Five days after inoculation, pick the inoculated leaves to eliminate the influence of the pathogen on the forest trees. At the same time, carefully remove the transparent tape paper, fungal blocks or water agar blocks on the inoculated leaf surface, count, photograph or scan the disease occurrence condition, automatically identify the leaf necrosis spots in the ImageJ image recognition software and calculate the area of the necrosis spots (manually adjust if necessary), output the results to an Excel table, calculate the disease index, and judge the disease resistance of the forest trees to the pathogen according to the disease index and the disease index of the disease spots;

[0072] In this example, after inoculation with the pathogen, tissue necrosis formed at the puncture points in the control inoculation, and the average area of the necrotic tissue was 1.5 ± 0.5 mm 2 , and aseptic filamentous substances formed. After inoculation with the fungal blocks of the pathogen, if the area of the leaf necrosis region usually showed a significant expansion, hyphal structures and even pycnidia could be seen under a stereomicroscope ( Figure 1 ), it was determined that the leaves were diseased; otherwise, they were not diseased (Table 1). According to the average area of the control necrotic tissue above, the present invention determined that the area of the necrosis region > 2.0 mm 2 was the quantitative standard for leaf disease (Table 2). Calculate the leaf incidence rate after inoculation with the pathogen according to the number of necrosis spots formed by true disease, calculate the disease index according to the leaf disease standard in Table 1 and the disease development grading table in Table 2, and then perform a significant difference analysis on the diseased area by the T-test, chi-square test or ANOVA method. The significance test of the incidence rate was performed by the chi-square test. The calculation formulas for the incidence rate and the disease index are as follows:

[0073] Formula 1: Incidence rate (P) = number of puncture points with disease / total number of effective puncture points × 100%;

[0074] Formula 2: Disease index (DI) = ∑(number of puncture points at each level × severity at each level) / (severity at the highest level × total number of effective puncture points) × 100%.

[0075] Table 1 Criteria for judging leaf disease

[0076]

[0077] Table 2 Disease development grading table

[0078] Disease grading Black Severity No disease <![CDATA[No disease onset, necrosis area of acupuncture point is 0 - 1.99m 2 > 0 Extremely slight disease <![CDATA[Puncture necrosis area 2.00 - 3.99 mm 2 > 1 Slight disease <![CDATA[The punctate necrosis area is 4.00 - 5.99 mm 2 > 2 Moderate disease <![CDATA[The punctate necrosis area is 6.00 - 7.99 mm 2 > 3 Moderately severe disease <![CDATA[The punctate necrosis area is 8.00 - 9.99 mm 2 > 4 Severe disease <![CDATA[The punctate necrosis area is greater than 10.00 mm 2 > 5

[0079] The criteria for dividing the disease resistance level are shown in Table 3.

[0080] Table 3 Criteria for dividing the disease resistance level

[0081]

[0082]

[0083] Note: Check the closed moisture retention status of the inoculated leaves every day after inoculation. Within 3 days before inoculation, if the inoculated fungal blocks dry out, move in position, change color abnormally, or mold grows, etc., the inoculation of this fungal block is regarded as invalid inoculation; for leaf inoculation, sterile water agar blocks or sterile culture medium blocks (pH 6.0 or added with antibacterial substances) are set as controls. If the sterile water agar blocks (or sterile culture medium blocks) become moldy or change color, or obvious necrotic spots and moldy spots expand at most of the wound points (>50%) at this position, then this control is regarded as an invalid control, and further the inoculation result of the pathogen of this kind of forest tree is regarded as invalid.

[0084] The results showed that 5 days after inoculating with the strain Valsa sordida CZC of the poplar canker pathogen, the incidence rates of the upper mature new leaves and the lower mature old leaves of BF-3 poplar were 28.33% and 70.00% respectively. Chi-square test showed that the incidence rate of mature new leaves was significantly lower than that of mature old leaves (chi-square test, p < 0.05). The results of the lesion size are as Figure 2 shown. There was a significant difference in the average lesion area of mature new leaves of BF-3 poplar (T test, P < 0.05), and their sizes were 2.49 mm 2 and 2.94 mm 2 respectively, and the disease index was 5.67 and 14.67 respectively. This result indicates that the disease development of leaves with different maturities on the same branch is significantly different, and leaves with the same or similar maturity should be used during leaf inoculation. Therefore, during subsequent inoculation, the 5th to 10th mature leaves of forest trees can be used.

[0085] Example 2 Influence of light on the disease development on poplar leaves

[0086] In this example, 3 five-year-old Populus bolleana (plant spacing 1.0 m × 1.0 m) were used as plant materials, and a comparative experiment was conducted on the leaves of the illuminated-side branches and the shaded-side branches of the same poplar tree. The heights of the illuminated-side branches and the shaded-side branches on each poplar tree were basically the same. The inoculated leaves on each branch were the mature leaves at the 5th to 8th positions from the top of the branch; among them, 3 leaves were inoculated with fungal blocks, and 1 leaf was inoculated with sterile water agar blocks; 2 fungal blocks or sterile water agar blocks were inoculated on each leaf. The specific inoculation method was the same as that in Example 1. 5 days after inoculation, the disease index of the inoculated leaves and the resistance of the leaves to the strain were investigated.

[0087] The results of the lesion size are as Figure 3 shown. Light significantly affected the lesion size of the leaves. The average lesion area of the leaves on the illuminated-side branches was 3.87 mm 2 while the average lesion area of the leaves on the shaded-side branches was 7.40 mm 2(Independent sample T-test, P < 0.05); the incidence rates of the leaves on the illuminated side and the shaded side of the branches were 58.33% and 55.00% (with the lesion area greater than 2.0 mm 2 as the standard), and the disease indices were 18.00 and 26.00 respectively. The above results indicate that light has a significant impact on the leaf inoculation results, suggesting that the inoculated leaves should be those with the same light conditions, as the incidence rate of inoculated shaded leaves is higher.

[0088] Example 3 Influence of the culture time of pathogenic fungi on the development of poplar leaf diseases

[0089] Using 1-year-old Populus × euramericana cv. 'Bofeng 3' as the material, the pathogenicity of the Valsa sordida CZC strain of the poplar canker fungus cultured in the dark for 4 days and 7 days was compared. Six poplar trees were divided into 2 groups, with 3 trees in each group, and inoculated with the fungal blocks cultured for 4 days and 7 days respectively; 4 mature leaves at the 5th to 8th positions from the top of the inoculated branches of each poplar tree were inoculated, 3 leaves were inoculated with the fungal blocks, and 1 leaf was inoculated with the sterilized water agar block; 2 fungal blocks or sterilized water agar blocks were inoculated on each leaf. The specific inoculation method was the same as that in Example 1. Five days after inoculation, the disease index of the inoculated leaves and the pathogenicity of the strain were investigated.

[0090] The research results showed that the incidence rates of the leaves inoculated with the fungi cultured for 4 days and 7 days were 83.33% and 65.00% respectively, and the incidence rate of the fungus cultured for 4 days was significantly higher than that of the fungus cultured for 7 days (chi-square test, p < 0.05). As shown Figure 4 in the results, the average lesion areas of the leaves inoculated with the fungal blocks cultured for 4 days and d days were 8.51 mm 2 and 3.37 mm 2 respectively, indicating that the culture time of the fungus has a significant impact on the development of the disease (independent sample T-test, P < 0.05). With the lesion area greater than 2.0 mm 2 as the disease criterion, the incidence rates of the Valsa sordida CZC strain of the poplar canker fungus cultured for 4 days and 7 days were 83.33% and 65.00% respectively, and their disease indices were 50.67 and 13.33. This result shows that on the PDA medium, the pathogenicity of the poplar canker fungus cultured for 4 days is stronger.

[0091] Example 4 Disease resistance test of poplar hybrid clones

[0092] Plant materials: Randomly select 48 two-year-old Populus deltoides hybrid clone plants for disease resistance testing. All the poplar materials used are planted in the experimental nursery of the Chinese Academy of Forestry. The experimental poplar materials are healthy, free from pests and diseases, and are fully watered during the experiment to prevent drought stress.

[0093] For each clone, select 4 newly mature leaves with consistent light conditions (the 5th to 8th positions from the top of the branches of the current-year plants). Inoculate 3 leaves with pathogen blocks and 1 leaf with sterilized water agar; according to the leaf size, each leaf is inoculated with 2 or 4 pathogen blocks or sterilized water agar blocks of 1.5 cm × 1.5 cm; 5 puncture wounds are formed on each pathogen block or sterilized water agar block by puncturing. The specific inoculation method is the same as that in Example 1.

[0094] In this example, 2715 valid puncture point data were obtained. According to the disease criteria in Table 1, 2145 disease spot data of 48 Populus deltoides hybrid clones were determined. The investigation results of the incidence rate, diseased area and disease index are shown in Table 4, and the disease resistance grading is shown in Table 4 and Figure 5 , and the normal distribution curve of the disease index is shown in Figure 6 .

[0095] Table 4 Incidence rate and average diseased area of leaves of different poplar hybrid clones after inoculation

[0096]

[0097]

[0098] Analyze the disease occurrence of each clone. The results are shown in Table 4 and Figure 5 - Figure 6 . As shown, 5 days after inoculation with the canker pathogen, the leaf incidence rates of different poplar hybrid clones are 31.67% - 100.00% (B133, B1 and B104 respectively), the disease indexes are 7.67 - 83.67% (B133 and B135 respectively), and the diseased spot areas are 2.72 - 15.28 mm 2 (B168 and B14 respectively), indicating that there is a large differentiation in disease resistance among different poplar clones. The 48 poplar clone materials of the present invention were randomly selected for detection, and their disease indexes basically conform to the normal distribution ( Figure 6 ), indicating that the disease resistance detection method involved in the present invention can reveal the disease resistance differentiation of the poplar clone hybrid population ( Figure 5 ). According to the disease index, the 48 clones are divided into 7 disease grades (Table 3). B133 is determined as a highly disease-resistant clone, and 8 clones such as B168 are disease-resistant clones; while B135, B246 and B104 are determined as highly susceptible clones.

[0099] This example proves that the present invention can be applied to the disease resistance test of forest tree hybrid clones; the data such as the incidence rate, diseased area and disease index of the poplar clones provided in Table 4 are the most basic disease phenotype trait data necessary for genomic selection breeding. It can be inferred that the method provided by the present invention can also be applied to the analysis of disease resistance detection (screening), disease resistance heritability differentiation, etc. of large-scale population materials such as forest tree natural populations and mutagenesis breeding.

[0100] Verification of the disease resistance detection results of leaf inoculation in Example 5 (Comparison of the method in Example 4 with the method disclosed in the prior art)

[0101] The present invention provides a method for detecting the disease resistance of forest trees based on leaf inoculation, which can be applied to the evaluation and detection of the asexual resistance of hybrid offspring in the cross breeding of poplar canker and canker. Therefore, the method provided by the present invention is an alternative method for detecting the disease resistance of forest tree trunk diseases, and its effectiveness needs to be verified by the trunk inoculation disease resistance detection method.

[0102] Since there is no national or local standard in the field of disease-resistant cross breeding, there is currently no standard method. However, in the research and practice of this industry, the in vitro shoot inoculation method is adopted, and the disease index of shoot-disease incidence rate is used as the disease resistance evaluation standard. The method in the literature "Investigation and Research on Poplar Species Resistant to Canker" (Yang Junxiu, Li Wuhan, Fu Yuqin, Liu Yuanyuan. Journal of Northwest Forestry University, 1990, (04): 1-10) is the method commonly used in this industry, and the method adopted by the literature also measures the disease resistance based on the disease index of disease incidence rate. After that, this method has also been adopted by many studies (such as Yang Zixiang et al., 1991; Fu Yuqin et al., 1992; Zhang Liqin. 1993; Zhang Lu et al., 2017). It can be seen that this method has a certain authority in this industry. Therefore, in order to prove the accuracy of the present invention, the method in the literature "Investigation and Research on Poplar Species Resistant to Canker" (Yang Junxiu, Li Wuhan, Fu Yuqin, Liu Yuanyuan. Journal of Northwest Forestry University, 1990, (04): 1-10) is used to compare the effects with the method in Example 4 of the present invention. The specific experiment is as follows:

[0103] 1. Experimental method

[0104] According to the results detected by leaf inoculation, 5 poplar hybrid clones (B246, B135, B154, B76, B14; the disease index is between 60.00 and 90.00) determined as highly susceptible and moderately susceptible according to the criteria in Table 4 of the present invention, 5 poplar hybrid clones (B99, B5, B7, B1, B176; the disease index is between 40.00 and 60.00) determined as susceptible, and 5 poplar hybrid clones (B168, B145, B81, B235, B133; the disease index is between 0.00 and 20.00) determined as disease-resistant or highly disease-resistant are selected as materials, and the in vitro shoot inoculation method is used for disease resistance verification.

[0105] Select clonal branches (the criteria for branch selection are as follows: one-year-old dormant branches, free from pests and diseases, with a diameter of 2-3 cm and a length of 25-30 cm). Clean the bark surface of the branches and sterilize them with 75% alcohol solution. After air drying, seal the upper end of the branches with callus ointment to keep them moist and prevent infection by pathogens. Use an inoculation needle to form 5 inoculation points on each branch in a three-point puncture manner (i.e., each inoculation point contains 3 puncture points, and the distance between the puncture points is less than 2 mm), and inoculate with the CZC mycelial block of the canker pathogen cultured on PDA medium for 7 days. Inoculate 10 branches for each clone, and use 2 branches inoculated with sterile medium as the control. After inoculation, seal and keep moist with plastic wrap, hydroponically culture in tap water, change water once every 3 days, and keep the room temperature at 23-25°C.

[0106] Starting from the day of inoculation, observe and record the disease incidence of each inoculation point on the branches of each clone every 5 days until 40 days after inoculation. Grade according to the actual time of the appearance of lesions (Table 5) and calculate the disease index according to the formula "Disease index (DI) = ∑(number of puncture points at each level × severity at each level) / (severity at the highest level × total number of effective puncture points) × 100%".

[0107] Table 5 Severity grading standard for the disease incidence time of poplar stem segments

[0108] Disease onset time (d) ≤5 5＞～≤10 10＞～≤15 15＞～≤20 20＞～≤25 25＞～≤30 30＞～≤35 ＞35 Grade 7 6 5 4 3 2 1 0

[0109] Note: The unit of the disease incidence time is days.

[0110] 2. Experimental results

[0111] Each clone has 50 puncture inoculation points on the branches, and a total of 750 effective inoculations are obtained. Using the inoculation points without inoculating the CZC mycelial block of the canker pathogen as the control, and taking the extension of the lesion area exceeding 1 mm as the criterion for the branches to be diseased, 511 cases of diseased lesions are determined. Analyze the disease incidence of each clone, and the results are shown in Table 6.

[0112] Table 6 Verification of the resistance to inoculation on the branches by inoculation on the leaves

[0113]

[0114] The results show that: 40 days after inoculation with the canker pathogen, the disease index of the disease incidence time of 15 verified poplar clones ranges from 15.71 (B133) to 92.86 (B246) (Table 6).

[0115] After that, the inventor compared the disease index (Table 4) of leaf inoculation (the method of the present invention) with the disease index determined by branch inoculation. The results show (Table 6):

[0116] 1), Using the branch-disease development speed disease index, the results of inoculating the branches of clone B246 were consistent with those of inoculating the leaves, and it was the most severely diseased among the 15 verified clones;

[0117] 2), Similarly, the results of inoculating the branches of clone B133 were also consistent with the results of leaf inoculation detection, showing the strongest resistance among the 15 verified clones;

[0118] 3), Although the sorting results were different, among the 5 clones with the highest disease index (highly susceptible and moderately susceptible) determined by the leaf inoculation method, 4 clones were determined as the clones with the highest disease index by the branch inoculation experiment. For example, based on the branch-disease development speed disease index, the susceptibility rankings of clones B246, B135, B76, and B14 were 1, 2, 3, and 5 respectively;

[0119] 4), Similarly, except for a slight difference in sorting, the 5 clones with the lowest disease index (highly resistant and resistant) determined by the leaf inoculation method were exactly the same as the 5 clones with the lowest disease index determined by the branch-disease development speed disease index;

[0120] 5), Among the 5 diseased clones (disease index rankings from 6 to 10) determined by the leaf inoculation method, the branch-disease development speed disease index rankings of 4 clones also ranged from 6 to 10, such as B7, B99, B5, and B176.

[0121] Based on the above results, compared with the branch-lesion area disease index, this result shows that there is a high consistency (90%) between the disease resistance detection results determined by the branch-disease development speed disease index and the leaf inoculation results, indicating that the detection results of the leaf inoculation method have strong consistency with the standard method used in this field and can be applied to the selection of resistant clones in poplar cross-breeding for canker diseases.

[0122] Example 6 Pathogenicity test of leaf inoculation method for different pathogenic fungal strains

[0123] Pathogenic bacteria: The 12 strains of Botryosphaeria dothidea used in this example were collected from 3 different poplars in 9 different regions of 3 provinces (municipalities) in the country. The specific information of the strains is shown in Table 7. After activation, they were inoculated on PDA medium and cultured in the dark at 28°C for 5 days; after the culture was completed, the solid culture of the pathogenic fungi was cut into pieces of 1.0 cm × 1.0 cm to 1.2 cm × 1.2 cm with a sterilized scalpel respectively;

[0124] Table 7 Information of the pathogen strains involved in this example

[0125] Strain Pathogen classification Host plant Disease type Collection location Longitude Latitude SD47 B.dothidea Poplar Trunk canker Wangdacun, Nanzhaolou Township, Yuncheng County, Shandong Province 115.90 35.44 SD60 B.dothidea Poplar Trunk canker Huanghai Road, Yantai City, Shandong Province 119.94 37.38 CZ1218 B.dothidea Poplar Trunk canker South of Wangjiahe Village Road, Guanbei Town, Huayin City, Shaanxi Province 110.12 34.55 CZ1070c B.dothidea Poplar Trunk canker Shenzhou City, Hengshui, Hebei 115.56 37.99 CZ1070b B.dothidea Poplar Trunk canker Shenzhou City, Hengshui, Hebei 115.56 37.99 CZ843 B.dothidea Poplar Trunk canker Next to the Herbarium of Hebei Agricultural University 115.44 38.82 CZ909 B.dothidea Poplar Trunk canker Dajidian Nursery, Baoding, Hebei 115.39 38.82 CZ956b B.dothidea Poplar Trunk canker Qijiazuo Village, Tang County, Baoding City, Hebei Province 114.78 38.93 CZ1009 B.dothidea Poplar Trunk canker Hills of Ren County, Xingtai City, Hebei 114.65 37.12 CZ1010 B.dothidea Poplar Trunk canker Hills of Ren County, Xingtai City, Hebei 114.65 37.12 CZ1055 B.dothidea Chinese white poplar Trunk canker Yuanfang Village, Mengjiazhuang Town, Pingshan County, Shijiazhuang City, Hebei 113.90 38.43 CZ1068b B.dothidea Black poplar Trunk canker Shenzhou City, Hengshui, Hebei 115.56 37.99

[0126] Plant material: Hybrid poplar ‘Bofeng 3’, and all poplar materials used were planted in the experimental nursery of the Chinese Academy of Forestry. The experimental poplar materials were healthy, free from pests and diseases, and were fully watered during the experiment to prevent drought stress.

[0127] Twelve strains of pathogens were inoculated onto three mature leaves at the 5th - 9th positions from the top of one-year-old branches of ‘Bofeng 3’ poplar, and another leaf was inoculated with a sterilized water agar block; two fungal blocks or sterilized water agar blocks (ctrl) were inoculated on each leaf, sealed with transparent tape, and 10 wound points were formed by acupuncture to promote lesion formation. The specific method was the same as in Example 1. Five days after inoculation, the formation of lesions was observed, photographed, and the incidence, lesion area, and disease index were measured. The data of the lesion areas formed at the acupuncture point positions on the leaves of ‘Bofeng 3’ poplar inoculated with different strains are as Figure 7 shown.

[0128] As Figure 7 can be seen, from the results of the pathogenicity test of 12 strains of poplar canker fungi, it can be intuitively seen that there are differences in pathogenicity among the strains. The incidence of inoculation with SD47 was 90.00%. Five days after inoculation, the lesion area was 7.16 mm 2 , significantly higher than that of strain CZ1218 (p < 0.05), and it was a highly pathogenic strain. The incidences of inoculation with strains SD60 and CZ1218 were 75.00% and 77.50% respectively, and the lesion areas were 5.66 mm 2 and 3.69 mm 2 respectively. ANOVA test showed no difference (p < 0.05), and they were moderately pathogenic strains. The necrotic lesion areas of 9 strains such as CZ1070c, CZ1070b, CZ1010, and CZ1055 were almost all less than 2.0 mm 2 (the judgment criterion for leaf disease in this study), and there was no obvious difference from the necrotic lesion area of the leaves inoculated with the sterile control. Therefore, the results of this example show that these 9 strains cannot infect the leaves to cause disease and are low pathogenic strains.

[0129] The above embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for rapid and large-scale detection of forest disease resistance and acquisition of disease phenotype data, characterized in that: The following steps are involved: After inoculating the pathogen blocks onto the upper surface of the tree leaves, use transparent tape to seal the inoculated pathogen blocks to keep them moist; the number of inoculated blocks per leaf is 2 to 4, and at least 3 leaves are inoculated; The method of inoculation was the same as that of the pathogen block, with the leaves inoculated with sterile water agar blocks as the control, and one leaf was inoculated; The inoculated leaves are newly mature leaves of trees with the same light conditions, healthy, free of pests and diseases, and mechanical damage; After the inoculation is completed, use a sterile inoculation needle to puncture the lower surface of the leaf and the bacteria block or sterile water agar block to form multiple puncture points, but do not puncture the transparent tape; the distance between each puncture point is more than 0.5 cm; All inoculated leaves are removed 3 to 7 days after inoculation, and the morphology and area of ​​necrotic spots formed around each puncture point are photographed and measured; the inoculated leaves include leaves inoculated with pathogenic bacteria blocks and leaves inoculated with sterilized water agar blocks; According to the morphology and area of ​​necrotic spots on the control inoculated leaves, the disease standard is determined; the number and area of ​​necrotic spots formed by the inoculated fungus blocks are counted, and the incidence rate and disease index are calculated; according to the disease index of the disease spots, the disease resistance of the trees to the pathogen is judged; the disease resistance level classification standards are shown in the following table: 。 2. The method according to claim 1, characterized in that When the pathogen block or sterile water agar block is a square, the puncture positions are located at the center and four corners of the pathogen block or sterile water agar block, and the distance between each puncture point is more than 0.5 cm; the puncture positions of the four corners are all 1 to 2 mm from the inner edge of the pathogen block; When the pathogen block or the sterilized water agar block is rectangular, the distance between each puncture point is more than 1 cm, and the distance between each puncture point is more than 1 cm.

3. The method according to claim 1, characterized in that The pathogenic bacteria contained in the pathogenic bacteria block are pathogenic bacteria that cause branch and trunk diseases.

4. The method according to claim 1, characterized in that The components of the sterile water agar block include agar and water; the mass volume ratio of the agar to water is 20g:1000mL.

5. The method according to claim 1, characterized in that The tree leaves are leaves of annual or perennial trees.

6. The method according to claim 1, characterized in that The inoculation positions of the pathogen block and the sterilized water agar block are both 1 to 3 cm on both sides of the main veins of the tree leaves, and the secondary veins of the tree leaves should be avoided.

7. The method according to claim 1, wherein the pathogen block is obtained one day after the cultured fungal hyphae have grown to fill a 9 cm diameter culture dish.

8. The detection method according to claim 7, characterized in that: The thickness of the culture medium for culturing fungi is the same.

9. The detection method according to claim 1, characterized in that: Before the inoculation, the leaves of the trees are cleaned and disinfected; the disinfectant used for the disinfection is alcohol or mercuric chloride.

10. The detection method according to claim 9, characterized in that: The alcohol is an alcohol solution with a volume percentage of 75%; the mercuric chloride is a mercuric chloride solution with a volume percentage of 0.1%.

Images