Method for identifying disease resistance of difficult-to-culture pathogen and application thereof

Abstract

The present application relates to a kind of difficult culture pathogen resistance identification method and its application, belong to the field of agriculture and forestry science, step 1: the culture screening of toxic source plant, plant to be identified and indicator plant;Step 2: grafting transmission, the toxic source plant screened only with difficult culture pathogen, the plant to be identified without difficult culture pathogen and indicator plant are arranged in position, and transmission is carried out using grafting method;Step 3: observation and detection, according to the disease cycle of difficult culture pathogen, the disease phenotype of plant to be identified and indicator plant in step 2 is observed, and the colonization, propagation of difficult culture pathogen is detected by PCR method;Step 4: disease resistance and pathogen running speed evaluation.Effectively solve the problem that the identification result is inaccurate due to the low inoculation rate of difficult culture pathogen using transmission medium, the inconsistency of inoculation source pathogen and other reasons, the error is larger in resistance identification work, etc.

Description

Technical Field

[0001] This invention belongs to the field of agricultural and forestry science, specifically relating to a method for identifying the disease resistance of difficult-to-culture pathogens and its application. Background Technology

[0002] Difficult-to-culture pathogens are those that infect the vascular tissue of plant phloem and cannot currently be isolated and cultured. Among them, *Candidatus Liberibacter* (Ca.L.), belonging to the class α-Proteobacteria and the genus *Liberibacter*, is a Gram-negative bacterium that is an obligate parasitic organism of the phloem. The citrus Huanglongbing (HLB) it causes is one of the most serious diseases affecting citrus production worldwide. *Candidatus Liberibacter* can infect all citrus varieties and hybrids, as well as other Rutaceae plants, at all stages of the citrus growth cycle, ultimately leading to tree death. Paulownia witches'-broom disease is caused by a mycoplasma-like organism. Symptoms include abundant clustering of axillary and adventitious buds, shortened internodes, yellowing and smaller leaves, prominent veins, and persistent twigs resembling a bird's nest in winter. Severely infected plants die within the same year, while mildly infected plants may die after several years. The pathogen of jujube witches' broom disease is jujube phytoplasma, which is distributed in the phloem sieve tube cells. Infected jujube trees develop slowly, their branches and leaves wither, and often lead to the death of the whole tree or large areas.

[0003] Identification of plant pathogens that are difficult to culture from vascular tissues has always been challenging. Currently, resistance identification in host plants (the plants on which the parasite or pathogen depends for survival) is mostly conducted through indirect inoculation via vectors (infected insects or scions, etc.). However, this method suffers from low inoculation efficiency and inconsistent pathogen sources, leading to significant errors and inaccurate results in resistance identification.

[0004] In view of this, the present invention provides a method for identifying the disease resistance of difficult-to-culture pathogens and its application. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for identifying the disease resistance of difficult-to-culture pathogens and its application. The aim is to effectively solve the problems of large errors in resistance identification work and inaccurate identification results caused by low inoculation rates of transmission vectors and inconsistent inoculation source pathogens.

[0006] To address the aforementioned technical problems, the first objective of this invention is to provide a method for identifying the disease resistance of difficult-to-culture pathogens, comprising the following steps:

[0007] Step 1: Cultivation and screening of toxic plants, plants to be identified, and indicator plants

[0008] The cultured source plants, plants to be identified, and indicator plants were tested for difficult-to-culture pathogens using PCR to screen out source plants containing only difficult-to-culture pathogens, plants to be identified that do not contain difficult-to-culture pathogens, and indicator plants.

[0009] Step 2: Direct transmission of the virus

[0010] The selected plants containing only difficult-to-culture pathogens, plants to be identified and indicator plants without difficult-to-culture pathogens were arranged in a specific location, and the virus was transferred by contact grafting.

[0011] Step 3: Observation and Testing

[0012] Based on the disease cycle of the difficult-to-culture pathogen, observe the disease phenotype of the plants to be identified and the indicator plants in step 2, and detect the colonization and reproduction of the difficult-to-culture pathogen by PCR.

[0013] Step 4: Evaluation of disease resistance and pathogen translocation rate

[0014] Based on the observation and testing results in step 3, the disease resistance of the plants to be identified and the rate of pathogen transport were statistically analyzed.

[0015] The beneficial effects of this invention are as follows: This invention arranges the positions of the source plant and the plant to be identified, and uses indicator plants (plant types or crop varieties with faster disease progression and typical and obvious symptoms) as controls. On the basis of maintaining the growth of the source plant, through statistical experimental design, the layout and distribution of the plant to be identified and the indicator plant are scientifically carried out. This effectively solves the problems of low inoculation rate and inconsistent inoculation pathogens, which lead to large errors in resistance identification and inaccurate identification results.

[0016] Based on the above technical solution, the present invention can be further improved as follows.

[0017] Furthermore, the position layout described in step 2 includes a parallel contact identification layout or a series contact identification layout.

[0018] The beneficial effects of adopting the above-mentioned further scheme are: the present invention uses serial and parallel grafting of the toxin source plant and the plant to be identified, with the indicator plant as the control, to carry out resistance identification work by statistical experimental design, while maintaining the growth of the toxin source plant, and to evaluate the disease resistance and pathogen translocation speed.

[0019] Furthermore, the parallel approach grafting identification layout is: toxic plant + plant to be identified + indicator plant, or toxic plant + indicator plant + plant to be identified + indicator plant, where + indicates approach grafting; the serial approach grafting identification layout is to first arrange X indicator plants + rootstock of the plant to be identified + scion of the plant to be identified + X indicator plants, and then the toxic plant is approach grafted onto the rootstock or scion of the plant to be identified, where X is an integer between 1 and 5, where + indicates approach grafting.

[0020] The beneficial effects of adopting the above-mentioned further scheme are: the parallel approach identification layout of the toxic plant + plant to be identified + indicator plant is used for the initial selection of the resistance of the plant to be identified; the parallel approach identification layout of the toxic plant + indicator plant + plant to be identified + indicator plant is used to evaluate the pathogen transport speed in the plant to be identified; the series approach identification layout further screens the resistance of the plant to be identified.

[0021] Furthermore, the difficult-to-culture pathogens include *Citrus citrus Huanglongbing*, *Paulownia tomentosa*, or *Ziziphus jujuba*. These are pathogens that infect the phloem of plants and cannot currently be isolated and cultured.

[0022] Furthermore, the plants to be identified include plant species or varieties with unknown resistance or susceptibility characteristics among the host plant species (or closely related species) of the pathogen; the indicator plants include plant species or varieties known to be susceptible to the pathogen and showing obvious symptoms, such as Ponkan orange in the identification of resistance to Huanglongbing in citrus.

[0023] Furthermore, the specific cultivation method of the toxic plant mentioned in step 1 is as follows:

[0024] (1) Collect diseased plant branches and detect the presence of the target difficult-to-culture pathogen on the diseased plant branches by PCR. Keep the diseased plant branches that only carry the target difficult-to-culture pathogen to obtain the source branches.

[0025] (2) The obtained toxic source branches are propagated into diseased seedlings by grafting or cutting. The seedlings are then tested again by PCR to see if they carry the target difficult-to-culture pathogen. Only the plants carrying the target difficult-to-culture pathogen are retained. The diseased seedlings are then cultured to produce 3-5 lateral branches to obtain the toxic source plants.

[0026] Furthermore, the cultivation method for the plant to be identified in step 1 is as follows:

[0027] (1) Collect plant branches to be identified, and detect the presence of target difficult-to-culture pathogens on the plant branches by PCR. All plant branches carrying target difficult-to-culture pathogens are discarded to obtain plant branches for identification.

[0028] (2) The selected plant branches are propagated into seedlings to be identified by grafting or cutting, and then lateral branches are cultivated to 3-5 to obtain the plant to be identified.

[0029] Furthermore, the method for cultivating the indicator plant in step 1 is as follows:

[0030] (1) Collect indicator plant branches and detect the presence of target difficult-to-culture pathogens on the indicator plant branches by PCR. All indicator plant branches carrying target difficult-to-culture pathogens are discarded to obtain identification plant branches.

[0031] (2) The selected identification plant branches are propagated into seedlings to be identified by grafting or cutting, and then lateral branches are cultivated to 3-5 to obtain indicator plants.

[0032] Furthermore, the nucleotide sequences of the primers used for the PCR method in the detection of the difficult-to-culture pathogens are SEQ ID NO: 1 and SEQ ID NO: 2; or SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0033] The second objective of this invention is to provide an application of a method for identifying the resistance of difficult-to-culture pathogens, characterized in that it is used for identifying the resistance of plants to difficult-to-culture pathogens.

[0034] Furthermore, the plant to be identified is a citrus, paulownia, or jujube tree.

[0035] Furthermore, the citrus fruits include mandarins, tangerines, oranges, pomelos, kumquats, citrons, large-winged oranges, closely related species, and distantly related species (such as navel oranges, Shatin pomelos, Wogan tangerines, wampee, and kumquats). Attached Figure Description

[0036] Figure 1 This is a layout diagram of the parallel contact I disease resistance identification method of the present invention;

[0037] Figure 2 This is a layout diagram of the parallel contact II disease resistance identification method of the present invention. Detailed Implementation

[0038] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0039] Experimental materials

[0040] Citrus Huanglongbing is caused by Citrus phloem bacillus, a difficult-to-culture bacterium that poses a huge threat to the global citrus industry. Screening and evaluating resistant and tolerant plant materials is an important task in current citrus resistance breeding, but efficient and accurate screening and evaluation methods have always been a key limiting factor in this work.

[0041] When symptoms of Huanglongbing (HLB) are obvious in autumn, diseased citrus branches are collected from the citrus orchard. Simultaneously, PCR is used to identify branches carrying other pathogens. Only branches carrying the HLB pathogen are retained, and all others are discarded, yielding branches from the source plants. Branches from citrus varieties to be identified (e.g., navel orange, Shatang pomelo, Wogan, yellow peel, kumquat) are collected, and PCR is used to discard all branches carrying the HLB pathogen, yielding branches from the plants to be tested.

[0042] Example 1

[0043] This embodiment utilizes different grafting methods on branches of the source plant and the test plant to assess the impact on the efficiency of resistance identification. Approach grafting: Branches of the test plant and the source plant are grafted together; Budding method 1: A poisonous bud is grafted onto the test plant; Budding method 2: A bud of the test plant is grafted onto the poisonous plant.

[0044] Survival rate: The percentage of budded buds or plants that survive 3 months after budding or approach grafting out of the total number of grafted buds or plants.

[0045] Branching rate: The percentage of grafted buds or plants that have produced branches (branch length ≥ 5cm) 6 months after budding or approach grafting, out of the total number of grafted buds or plants.

[0046] Virus transmission rate: The percentage of virus-carrying test plants detected one year after budding or approach grafting out of the total number of grafted buds or plants.

[0047] Table 1. Effects of different grafting methods on resistance identification efficiency

[0048]

[0049] As shown in Table 1, the contact method is more efficient in the identification of adversity. The combination of experimental design and distribution will make the identification results more scientific and accurate.

[0050] Example 2

[0051] 1. Experimental Materials and Methods

[0052] 1.1 Cultivation of toxic citrus seedlings

[0053] Cultivation Method: When symptoms of Huanglongbing (HLB) are obvious in autumn, diseased citrus branches are collected from the citrus orchard. The presence of HLB pathogens on the branches is detected using PCR [(primers OI1: 5'-GCGCGTATGCAATACGAGCGGCA-3', SEQ ID NO: 1; OI2c-gj: 5'-TCGCGACTTCGCAACCCAT-3', SEQ ID NO: 2) or real-time fluorescent PCR (Las-F: 5'-TCGAGCGCGTATGCAATACG-3', SEQ ID NO: 3; Las-R: 5'-GCGTTATCCCGTAGAAAAAGGTAG-3', SEQ ID NO: 4; Las-P: FAM 5'-AGACGGGTGAGTAACGCG-3'BHQ, SEQ ID NO: 5) or other primers]. At the same time, the presence of other pathogens is identified using PCR. Only branches carrying HLB pathogens are retained, and all others are discarded.

[0054] The selected branches carrying citrus Huanglongbing (HLB) were propagated into diseased seedlings through grafting or cuttings. When the seedlings reached a height of 40cm, PCR was used to detect the presence of the HLB pathogen and to identify other pathogens. Only seedlings carrying the HLB pathogen were retained, and all others were discarded. By using techniques such as pinching, 3-5 lateral branches were cultivated to obtain virus-infected citrus seedlings.

[0055] 1.2. Cultivation of citrus varieties (germplasm) and indicator plants to be identified.

[0056] Cultivation Method: Citrus branches to be identified were collected. The citrus varieties to be identified included navel orange, Shatang pomelo, Wogan, yellow peel, or kumquat. PCR was used to detect the presence of citrus Huanglongbing (HLB) pathogen on the branches, and all branches carrying the pathogen were discarded. The selected branches were propagated using grafting to create seedlings and indicator plants for identification. When the seedlings and indicator plants reached a height of 40cm, 3-5 lateral branches were cultivated using techniques such as pinching.

[0057] 1.3. Design the approach test using statistical methods based on the purpose of resistance evaluation.

[0058] Based on the purpose of the resistance evaluation test, statistical methods were used to design the contact test and prepare the test plan.

[0059] 1.3.1 Parallel identification scheme:

[0060] The candidate experimental plants were tested using PCR to detect the pathogen of citrus Huanglongbing, and the seedlings and indicator plants were identified as carrying the pathogen. The seedlings to be identified and the indicator plants were confirmed to be virus-free, and the seedlings of the pathogen were confirmed to be carrying the pathogen of citrus Huanglongbing.

[0061] (1) Approach grafting method 1: Using approach grafting, the citrus fruits to be identified (navel orange, Shatin pomelo, Wogan mandarin orange, wampee, or kumquat) and indicator plants are arranged in the order of "citrus fruit to be identified n + indicator plant". Different citrus fruit units (navel orange, Shatin pomelo, Wogan mandarin orange, wampee, or kumquat) are directly approach grafted onto the toxin source seedlings to form a treatment of "toxin source + citrus fruit to be identified n + indicator plant". The same treatment is repeated 3 times, where the citrus fruit to be identified n is navel orange, Shatin pomelo, Wogan mandarin orange, wampee, or kumquat. This method can be used for the initial selection of resistant materials.

[0062] (2) Approach grafting method 2: Using approach grafting, the citrus n to be identified (navel orange, Shatin pomelo, Wogan mandarin orange, wampee, or kumquat) and the indicator plant are arranged in the order of "indicator plant + citrus n to be identified + indicator plant". Different citrus n to be identified (navel orange, Shatin pomelo, Wogan mandarin orange, wampee, or kumquat) units are directly approach grafted onto the source seedlings to form a treatment of "source of pathogen + indicator plant + citrus n to be identified + indicator plant". The same treatment is replicated 3-5 times. This method can be used to evaluate the transmission rate of pathogens in resistant materials.

[0063] (3) The control group is “toxin source + indicator plant”.

[0064] Note: When there are not enough branches of the toxic seedlings, multiple toxic seedlings can be selected to form a treatment. However, different toxic seedlings in the same treatment must have the same citrus unit to be identified. Alternatively, multiple toxic sources can be grafted together to form a single toxic source.

[0065] 1.3.2 Re-detection of plant pathogen-carrying status

[0066] According to the experimental design above, reserve citrus seedlings for virus source, identification seedlings and indicator plants were selected. Before the experiment, the pathogen status of citrus Huanglongbing pathogen in the citrus seedlings, identification seedlings and indicator plants was tested again by PCR method, and unqualified plants were eliminated.

[0067] 1.3.3. Direct contact transmission of the virus

[0068] According to the parallel identification scheme designed above, the selected source plants, plants to be identified or crop varieties (germplasm) and indicator plants are scientifically arranged in positions, and the virus is transmitted by contact method.

[0069] 1.4 Evaluation and Observation

[0070] Based on the disease cycle, regularly observe and identify the disease phenotype of seedlings and indicator plants, regularly use PCR methods to detect the colonization and reproduction of the citrus Huanglongbing pathogen, and keep records of the observation results.

[0071] 1.4.1 Evaluation of disease resistance

[0072] Based on the experimental design and observation results, and using the disease phenotype of plants with the same location and the results of pathogen detection by PCR method as references, the resistance and tolerance of the tested varieties (germplasm) to citrus Huanglongbing were statistically analyzed and evaluated.

[0073] 1.4.2 Evaluation of the transmission rate of pathogens in resistant materials

[0074] Based on the experimental protocol and observation results, the time between the onset of disease and the positive PCR results of the indicator plants before and after the test of the variety (germplasm) was statistically analyzed, and the time difference was calculated, which represents the transmission rate of pathogens in the test variety (germplasm).

[0075] 2. Test Results

[0076] Table 2. Observation of resistance to Huanglongbing by different citrus varieties and their closely related species based on parallel identification method.

[0077]

[0078] Table 3 Evaluation of the translocation rate of Huanglongbing fungus in different citrus varieties and their closely related genera based on parallel identification method

[0079]

[0080] Tables 2 and 3 show that the grafting identification had a high success rate and higher experimental efficiency. The experimental design and distribution made the identification results more scientific and accurate. The experimental results show that there are differences in the resistance of different citrus varieties and their closely related species to Huanglongbing. Navel oranges develop the disease faster, and their indicator plants are also susceptible to the disease faster. Kumquats and wampee are more resistant, and their indicator plants are more susceptible to the disease. This indicates that navel oranges are susceptible to the disease, and the pathogen spreads faster in them, while kumquats and wampee are more resistant, and the pathogen spreads slower in them.

[0081] Example 3

[0082] 1. The methods for seedling cultivation, pathogen detection, grafting for virus transmission, and observation and evaluation are the same as in Example 2.

[0083] 2. Design of a tandem identification scheme

[0084] The candidate experimental plants were tested using PCR to detect whether the seedlings carried the citrus Huanglongbing pathogen. This further confirmed that the seedlings to be identified and the indicator plant seedlings were virus-free, and confirmed that the source seedlings were seedlings carrying the citrus Huanglongbing pathogen.

[0085] (1) Using approach grafting, the citrus plants to be identified and the indicator plants were arranged in the order of "indicator plant + citrus rootstock n to be identified + citrus scion m to be identified + indicator plant" to form a treatment. The same treatment was repeated 5 times. Among them, the citrus rootstock n to be identified was trifoliate orange, sour orange, trifoliate orange, sweet orange, etc.; the citrus scion m to be identified was navel orange, Shatang pomelo, etc.

[0086] 3. Test Results

[0087] Table 3. Observation of resistance to Huanglongbing by navel orange and different rootstock combinations based on tandem identification method.

[0088]

[0089] Note: This test result is for the scion material.

[0090] Table 3 shows that the grafting method has a certain impact on the disease incidence of the scion, but the enhancement effect of the four rootstocks, including trifoliate orange, on the disease resistance of the scion varieties is not strong. However, this grafting method has a positive effect on the interaction between the rootstock and scion in terms of disease resistance and on the screening of resistant rootstocks.

[0091] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for identifying disease resistance of a fastidious pathogenic bacterium, characterized by, It comprises the following steps: Step 1: culture screening of toxic plants, plants to be identified and indicator plants The cultured toxic plants, plants to be identified and indicator plants are detected for difficult-to-culture pathogenic bacteria by PCR method, and the toxic plants containing only difficult-to-culture pathogenic bacteria, the plants to be identified not containing difficult-to-culture pathogenic bacteria and the indicator plants are screened; Step 2: grafting transmission The toxic plants containing only difficult-to-culture pathogenic bacteria, the plants to be identified not containing difficult-to-culture pathogenic bacteria and the indicator plants screened are arranged in positions, and the grafting method is used for transmission; Step 3: observation and detection According to the disease cycle of the difficult-to-culture pathogenic bacteria, the disease phenotypes of the plants to be identified and the indicator plants in step 2 are observed, and the colonization and reproduction of the difficult-to-culture pathogenic bacteria are detected by PCR method; Step 4: evaluation of disease resistance and pathogen running speed According to the observation and detection results of step 3, the disease resistance and pathogen running speed of the plants to be identified are statistically analyzed; The position arrangement in step 2 comprises parallel grafting identification arrangement or series grafting identification arrangement; The parallel grafting identification arrangement is: toxic plants + plants to be identified + indicator plants, or toxic plants + indicator plants + plants to be identified + indicator plants, wherein + is grafting; the series grafting identification arrangement is: first arrange X indicator plants + plant to be identified stock + plant to be identified scion + X indicator plants, and then graft the toxic plants on the plant to be identified stock or the plant to be identified scion, wherein X is an integer between 1 and 5, and + is grafting; The difficult-to-culture pathogenic bacteria are citrus Huanglongbing bacteria; The method is used for resistance identification of plants to be identified to difficult-to-culture pathogenic bacteria; The plants to be identified are citrus.

2. The method of claim 1, wherein the method is characterized by, The specific culture method of the toxic plants in step 1 is: (1) collecting diseased plant branches, detecting the target difficult-to-culture pathogenic bacteria carried by the diseased plant branches by PCR method, reserving only the diseased plant branches carrying the target difficult-to-culture pathogenic bacteria to obtain toxic branches; (2) using grafting or cutting method to expand the diseased seedlings to obtain toxic plants.

3. The method of claim 1, wherein the method is characterized by, The culture method of the plants to be identified in step 1 is: (1) collecting plant branches to be identified, detecting the target difficult-to-culture pathogenic bacteria carried by the plant branches to be identified by PCR method, and discarding all the plant branches to be identified carrying the target difficult-to-culture pathogenic bacteria to obtain identification plant branches; (2) using grafting or cutting method to expand the identification plant branches to obtain identification seedlings, and culturing the lateral branches to 3-5 branches to obtain the plants to be identified.

4. The method of claim 1, wherein the method is characterized by, The culture method of the indicator plants in step 1 is: (1) collecting indicator plant branches, detecting the target difficult-to-culture pathogenic bacteria carried by the indicator plant branches by PCR method, and discarding all the indicator plant branches carrying the target difficult-to-culture pathogenic bacteria to obtain identification plant branches; (2) using grafting or cutting method to expand the identification plant branches to obtain indicator plants, and culturing the lateral branches to 3-5 branches to obtain the indicator plants.

5. The method according to any one of claims 1 to 4, wherein the method is for identifying the disease resistance of a fastidious pathogenic bacterium, characterized in that, The nucleotide sequences of the primers for detecting the fastidious pathogenic bacteria by the PCR method are SEQ ID NO:1 and SEQ ID NO:2; or SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:

5. The nucleotide sequences of the primers for detecting the fastidious pathogenic bacteria by the PCR method are SEQ ID NO:1 and SEQ ID NO:2; or SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

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