WO2021159558A1 - Drug and method for preventing and treating citrus greening - Google Patents

Drug and method for preventing and treating citrus greening Download PDF

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WO2021159558A1
WO2021159558A1 PCT/CN2020/076627 CN2020076627W WO2021159558A1 WO 2021159558 A1 WO2021159558 A1 WO 2021159558A1 CN 2020076627 W CN2020076627 W CN 2020076627W WO 2021159558 A1 WO2021159558 A1 WO 2021159558A1
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treatment
thiazoline
copper complex
citrus
disease
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Chinese (zh)
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陈文利
段经纬
邱志燏
张钧哲
李雪
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华南师范大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/26—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-nitrogen bonds
    • A01N57/32—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-nitrogen bonds containing heterocyclic radicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A01N43/80—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,2
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16—Heavy metals; Compounds thereof
    • A01N59/20—Copper
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides

Definitions

  • the invention belongs to the field of agricultural technology. Specifically, it relates to a medicine and method for preventing and treating citrus yellow dragon disease.
  • Citrus yellow dragon disease or citrus greening disease, is a serious and devastating citrus disease. Typical symptoms include yellow buds, mottled leaves, clogged veins, premature fruit, shriveled fruit, withered branches and tree death, which reduce fruit yield. And quality, while seriously impairing the growth of citrus trees.
  • the annual loss of Huanglong diseased trees is 30-100% of the total yield, and they usually lose fruiting ability or die within 2 to 5 years after being infected. If they are not treated in time, their spreading speed is very alarming, and they are called citrus "cancers". "It affects the citrus industry all over the world, and is the number one quarantine disease in the citrus industry at home and abroad.
  • the purpose of the present invention is to provide a medicine that can effectively treat citrus Huanglongbing.
  • Another object of the present invention is to provide a method that can effectively treat citrus Huanglongbing.
  • the concentration of thiazoline and copper complex are respectively: 50-90% thiazoline diluted 1000-3000 times, 100-600 ⁇ g/ml copper complex.
  • the use concentrations of thiazoline and copper complex are: 60-80% thiazoline diluted 1500-2500 times, 200-600 ⁇ g/ml copper complex.
  • the concentration of thiazoline and copper complex are respectively: 75% thiazoline diluted 2000 times, 200 ⁇ g/ml copper complex.
  • the present invention also provides a medicine that can effectively treat citrus huanglong disease, which contains thiazoline and copper complex.
  • the dosage ratio of the thiazoline and the copper complex in the medicine is calculated according to the following standard: 50-90% thiazoline diluted 1000-3000 times: 100-600 ⁇ g/ml copper complex.
  • the dosage ratio of the thiazoline to the copper complex in the medicine is calculated according to the following standard: 60-80% thiazoline diluted 1500-2500 times: 200-600 ⁇ g/ml copper complex.
  • the dosage ratio of the thiazoline and the copper complex in the drug is calculated according to the following standard: 75% thiazoline diluted 2000 times: 200 ⁇ g/ml copper complex.
  • the present invention also provides a method that can effectively treat citrus yellow dragon disease, specifically using the above-mentioned medicine for prevention and treatment.
  • the method for treating citrus yellow dragon disease is: choose a day without rain, first spray the soil at the roots of the trees with thiazoline, and apply copper ammonia to water the roots 2-5 days later.
  • the method for treating citrus yellow dragon disease is: choose a sunny day, first dilute 50-90% thiazoline 1000-3000 times and spray the root soil, 2-5 days later, apply 1-5 times 100-
  • the roots were irrigated with 600 ⁇ g/ml copper ammonia, and the irrigating time was about 30 days apart.
  • the method for treating citrus yellow dragon disease is: choose a sunny day, firstly dilute 60-80% thiazoline by 1500-2500 times and then spray the root soil, 2-5 days later, apply 1-3 times 200- The roots were irrigated with 600 ⁇ g/ml copper ammonia, and the irrigating time was about 30 days apart.
  • the method for treating citrus yellow dragon disease is: choose a sunny day, first dilute 75% thiazoline by 2000 times and then spray the root soil, 3 days later, apply 1-2 times of 200 ⁇ g/ml copper ammonia to irrigate the roots ,
  • the watering time is about 30 days apart.
  • the dosage is determined according to the degree of citrus Huanglongbing infection during the specific operation. The more serious the disease, the higher the dosage of the drug.
  • the present invention provides a new drug scheme and treatment method for effective prevention and control of Huanglong disease, in particular the combined use of thiazoline and copper hydrazine for the treatment and control of Huanglong disease.
  • the program has a significant effect on the treatment of Huanglong disease, and the treatment method can have a long duration.
  • Figure 1 is a schematic diagram of drug use and drug treatment of the roots.
  • A The specific location of the two root drug treatments. The target treatment location of copper squamousine is ultimately to kill the yellow dragon pathogen CLas in the phloem. The main action sites of thiazoline to eliminate root knot nematodes are the parenchyma and the pericylindrical sheath.
  • B Schematic diagram of treatment with two drugs, and the roots were treated with thiazoline (2000x) and copper complex (200 ⁇ g/ml).
  • Figure 2 shows the visual evaluation of HLB with different degrees of infection. 1,2,3-The middle and late stages of the disease. 4, 5, 6-Early stage of disease
  • Figure 3 shows the detection of different parts of citrus in the early stage of HLB infection.
  • Figure 4 shows the number of Huanglongbing bacteria in different samples (the number of cells per gram of plant tissue).
  • A 1-Leaves with moderate symptoms of infection, 2-Leaves with severe symptoms of infection, 3-None-Roots with phloem shedding and xylem exposed, 4-Roots with phloem shedding and xylem exposed.
  • B Ca.Las titers of Huanglongbing bacteria in different samples, each experiment was repeated 3 times. An asterisk (*) indicates a significant difference (P ⁇ 0.05; Dunnett’s test).
  • Figure 5 The antibacterial experiment of thiazoline and copper ammonium and the relative expression levels of FP1-GP235 and FP2-GP240 after treatment with copper ammonium.
  • A Antibacterial rate of thiazoline;
  • B Antibacterial rate of copper complex;
  • C Relative expression level of FP1-GP235 gene;
  • Figure 6 is a comparison of the root condition after 30 days of treatment with thiazoline and copper hydrazine alone and 30 days after combined treatment with thiazoline and copper hydrazine.
  • A Roots treated with thiazoline.
  • B Roots treated with copper complex.
  • C Roots treated with two drugs (thiazoline; copper complex). The yellow arrow indicates that the phloem is shed and the xylem is exposed, and the red arrow indicates a new root.
  • Figure 7 shows the effective control of Huanglong disease after 90 days of combined treatment with two drugs (thiazoline; copper complex).
  • A Treat the symptoms of the front leaf.
  • B Symptoms of leaves after 3 months of treatment.
  • C Root symptoms after 3 months of treatment.
  • D The change of Huanglong disease bacterial titer CLas at different time after treatment. Water was used as a control (WSC).
  • WSC water was used as a control
  • the asterisk (*) indicates that there is a significant difference between WSC and copper sulphate (P ⁇ 0.05; Dunnett’s test).
  • the solid line represents WSC, and the dashed line represents combined treatment with two drugs.
  • Figure 8 shows the changes in the roots and leaves of the samples before and after treatment with the two drugs.
  • Figure 9 shows the comparison of the yield of infected trees after the combined treatment of the two drugs with the yield of the previous year (the more severe the yellow dragon disease (HLB) infection, the more significant the effect will be after treatment); it shows that the combined application of the two drugs can be effectively treated HLB.
  • E) shows the comparison of fruit yield before and after treatment with different degrees of HLB infection: severe, normal, and moderate. Compared with the output of the previous year (2013-2014) after drug treatment in Test Field I (2014-2015), there was no difference in the yield of severe HLB citrus fruits in Test Field II without drug treatment.
  • Figure 10 shows the results of differentially expressed genes (DEGs) analysis.
  • the abscissa represents the fold change of gene expression (log2 fold change) between the treatment group and the control group, and the ordinate represents the significant level of gene expression difference between the treatment group and the control group (-log10padj).
  • Up-regulated genes are indicated by red dots.
  • Down-regulated genes are indicated by green dots. Blue indicates changes that are not significantly different. We found that 527 genes were up-regulated, 1050 genes were down-regulated, and 22270 genes were not significantly different.
  • FIG 11 shows the expression profiles of differentially expressed genes (DEGs) before and after treatment.
  • Figure 12 is a graph of GO enrichment statistics.
  • A Scatter plot of GO enrichment for comprehensive analysis of all genes and differently expressed genes. The abscissa is the ratio of the number of differential genes annotated to GO Term to the total number of differential genes, the ordinate is GO Term, the size of the dot represents the number of genes on GO Term, and the color from red to purple represents the significance of enrichment.
  • B Histogram of GO analysis. From the results of GO enrichment analysis, select the most significant 30 terms to draw a histogram. In the figure, the abscissa is GO Term, and the ordinate is the significance level of GO Term enrichment. The higher the value, the more significant it is. Different colors represent the three GO subtypes of BP, CC, and MF.
  • Figure 13 is a statistical chart of KEGG enrichment.
  • A Scatter plot of KEGG enrichment for comprehensive analysis of all genes and differently expressed genes. The abscissa is the ratio of the number of differential genes on the KEGG pathway to the total number of differential genes, the ordinate is the KEGG pathway, the size of the dot represents the number of genes on the KEGG pathway, and the color from red to purple represents the significance of enrichment. The figure shows The genes that were up-regulated after treatment than before treatment.
  • B Histogram of KEGG analysis. From the results of KEGG enrichment, select the 20 most significant KEGG pathways to draw a histogram. The abscissa is the KEGG pathway, and the ordinate is the significance level of pathway enrichment. The higher the value, the more significant.
  • Figure 14 shows the plant hormone signal transduction pathway (cic04075).
  • the red arrow indicates that the gene expression level is up-regulated after treatment, and the green arrow indicates that the gene expression level is down-regulated after treatment.
  • the vertical dashed line represents the cell membrane, and the vertical solid line represents the cell wall.
  • Figure 15 shows the secondary metabolism of phenylalanine-related metabolic pathways.
  • the red arrow indicates the up-regulation of gene expression levels after combined treatment with two drugs (thiazoline and copper ammonium).
  • the secondary metabolites represented by yellow characters produced beneficial bacteria and activated the expression of plant resistance genes.
  • PAL stands for phenylalanine ammonia lyase; C4H stands for 4-hydroxylase.
  • Figure 16 shows the results of verifying the transcriptome by qRT-PCR.
  • the relative expression of 9 differentially expressed genes involves phenylpropane metabolism and glucuronide interconversion (cic00040), flavonoid biosynthesis (cic00941) and plant hormone signaling (cic04075).
  • the gene corresponding to C4H is Ciclev10000921m
  • the gene corresponding to PAL is Ciclev10027912m
  • the gene corresponding to PGA is Ciclev10004783m
  • the gene corresponding to PR-1 is Ciclev10029459m
  • the gene corresponding to beta-glucosidase is Ciclev10028018m
  • the gene corresponding to AHP is Ciclev10029493m.
  • the genes involved in the phenylpropane biosynthetic metabolic pathway include a PAL gene (phenylalanine ammonia lyase), a C4H gene (cinnamic acid 4-hydroxylase), a ⁇ -glucosidase gene and a POD gene (peroxidase).
  • a PGA enzyme gene and a pectin esterase are involved in the metabolic pathway of pentose and glucuronate conversion.
  • a PR1 gene pathogen-related gene-1) and an AHP gene (including group Amino acid phosphate transfer protein) is involved in plant hormone signal transduction.
  • Figure 17 is a diagram of the therapeutic mechanism of thiazoline and copper complex.
  • the red arrow indicates the increase in gene expression level after treatment; the blue arrow indicates the specific treatment route; the red character PR1 indicates the increase in gene expression level after treatment.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the following examples mainly include three major experiments: (1) copper sulphate treatment experiment; (2) thiazoline and sulphate combined treatment experiment; (3) transcriptome experiment (exploration of treatment mechanism).
  • the sampling method collect a leaf at the top, collect a leaf at the bottom, and collect a leaf in the middle.
  • the sampling method collect the top three leaves, the bottom three leaves and the middle three leaves; after the collection, weigh the It is kept in a refrigerator at -80°C.
  • root tissue Collect citrus root tissue from 2-5 inches below the soil surface in four different quadrants within 2 feet from the trunk, and then place it in a paper bag at room temperature (20°C ⁇ 25°C) Air-dry for about 24 hours to facilitate the removal of excessive soil dirt by tapping with your fingers.
  • the samples were treated immediately after collection, and the root or leaf materials were kept at -80°C in the refrigerator.
  • the experiment used citrus branches (shatang oranges) with a diameter of about 0.5-0.8 cm with symptoms of Huanglong disease infection.
  • Each branch contains about 20-24 leaves, and then the branch is immersed in a 15ml test tube.
  • the test tube contains different concentrations of copper ammonia (50 ⁇ g/ml, 100 ⁇ g/ml, 200 ⁇ g/ml).
  • the outside of the test tube is made of aluminum foil. Cover, soak in water only as a control.
  • the leaf samples were collected at 1h, 5h, 12h, 1d, 3d and 5d after the treatment with copper ammonia. Each experiment was repeated at least three times.
  • the treatment test in the incubation room is divided into a combination treatment group (thiazoline and copper complex) and a control group (water treatment), with three trees in each group.
  • the combined treatment method is as follows: First, soak the roots of citrus trees with thiazoline (diluted 2000 times) (Hebei Sannong Agrochemical Co., Ltd.). After 3-5 days, spray the citrus roots with 200 ⁇ g/ml copper complex (Figure 1). Leaf samples were collected at 0, 15, 30, 60, 90, and 120 days after treatment.
  • the treatment of trees infected with Huanglong disease in the field is similar to the treatment in a culture room. Choose a sunny day, first dilute 75% thiazoline by 2000 times and spray the soil on the roots of the trees. After 3 days, water the roots with 1-2 times of 200 ⁇ g/ml copper ammonia. The irrigation time is about 30 days apart.
  • the field test field is a navel orange orchard with a demonstration area of 40,000 square meters, which started in March 2015 and ended in November 2015.
  • group A was treated with two drugs (thiazoline and copper complex), and group B was treated with water as a control.
  • group A was treated with two drugs (thiazoline and copper complex), and group B was treated with water as a control.
  • group B was treated with water as a control.
  • each group is further divided into 3 categories, such as severe infection, moderate infection, and mild infection, and then compared with the output of the previous year (2014-2015).
  • RNA concentration 200ng/ ⁇ l as a template
  • PrimeScriptTM RT Master Mix Kit (TAKARA, Liaoning Republic, China) to obtain cDNA by reverse transcription, and perform qPCR with cDNA as a template to obtain relative expression. All qPCR reactions were repeated at least 3 times.
  • the recombinant plasmid pMD-19 embedded with the 16S rDNA of the citrus Huanglong pathogen was from Guangdong Academy of Agricultural Sciences.
  • the content of Huanglongbing bacteria was determined by qPCR, using specific primers (Huanglongbing asf, Huanglongbing r and Huanglongbing-p-Table.1)-specific primers for the 16S rDNA region of Huanglongbing bacteria Huanglongbing asf and Huanglongbing r greening disease bacteria is used as a reference, the recombinant plasmid seven kinds of gradients of 16S rDNA of pMD-19 diluted to 10 -1, 10 -2, 10 -3, 10 -4, 10 -5, 10 -6, 10 - 7 and 7 gradients were used as templates, and ddH 2 O was used as a blank control.
  • the healthy citrus leaf DNA sample was used as a negative control, and the experiment was repeated more than 3 times.
  • the total volume of the reaction mixture is 20 ⁇ l: primers HLBasf and HLBr (Sangon, Shanghai, China) are 0.4 ⁇ l, probe HLBp (Takara, Liaoning) is 0.4 ⁇ l, and TaqMan mixture (TAKARA, Liaoning, China) is 10 ⁇ l , DNA template is 1 ⁇ l, water is 7.8 ⁇ l.
  • the real-time quantitative PCR program is as follows: 95°C for 5min, 95°C for 30s, 60°C for 30s; 40 cycles.
  • HLBasf a Other guanine nucleotides (in bold, underlined "G") were added to the Huanglong disease as sequence 63 based on the 16S rDNA sequence in the'Ca.Liberibacter asiaticus' Psy62 genome called HLBasf
  • the 16s rDNA of CLas was used as an internal control to detect the relative copy numbers of FP1-GP235 and FP2-GP240. All primers are listed in Table 1.
  • SYBR real-time quantitative PCR using genomic DNA as a template, each reaction contains the following components, with a total volume of 20 ⁇ l: 0.4 ⁇ l for primers, 10 ⁇ l for SYBR Mix, 1 ⁇ l for DNA, and 8.2 ⁇ l for water. All reactions were repeated three times.
  • the real-time quantitative PCR program is as follows: 95°C for 5min, 95°C for 30s, 60°C for 30s. 40 cycles, the calculation method refers to Ding (Ding, F., et al., Molecular mechanismss underlying heat or tetracycline treatments for citrus HLB control. Horticulture research, 2018.5(1): p.30).
  • each reaction contains the following components, with a total volume of 20 ⁇ l: 0.4 ⁇ l for primers, 10 ⁇ l for SYBR Mix, 1 ⁇ l for cDNA, and 8.2 ⁇ l for water. All reactions were performed in triplicate, and the real-time qPCR program was as follows: 95°C for 5 min, 95°C for 10 seconds, and 60°C for 20 seconds. 40 cycles.
  • the gene expression level of SDE1 calculation method refers to Pagliaccia D (Pagliaccia, D., et al., A pathogen secreted protein as a detection marker for Citrus Huanglongbing. Frontiers in microbiology, 2017.8: p.2041).
  • each reaction contains the following components, with a total volume of 20 ⁇ l: 0.4 ⁇ l for primers, 10 ⁇ l for SYBR Mix, 1 ⁇ l for cDNA, and 8.2 ⁇ l for water. All reactions were performed in triplicate, and the real-time qPCR program was as follows: 95°C for 5 min, 95°C for 10s, 60°C for 20s. 40 cycles.
  • GAPDH glycosylhyde-3-phosphate dehydrogenase
  • PAL phenylalanine ammonia lyase
  • C4H cinnamate 4-hydroxylase
  • 4CL 4-coumaroyl CoA ligase.
  • the content of Huanglongbing bacteria in citrus phloem shed and exposed xylem is higher than that in leaves and roots where the non-phloem shed and xylem are exposed
  • the symptoms of the leaves are the key to judging whether they are infected with Huanglong disease.
  • the inhibitory rate for Agrobacterium tumefaciens reached 50% at 400 ⁇ g/ml, and the inhibitory rate reached 100% at 800 ⁇ g/ml, similar to thiazoline.
  • the bacteriostatic rate is also very low ( Figure 5A&5B). Based on our results, we can speculate that copper complex also has a great inhibitory effect on Huanglong disease. Thiazoline mainly destroys root-knot nematodes, so it has a weak antibacterial effect on HLB.
  • a novel Huanglong disease control strategy is to enhance the endogenous transformation of Ca.Las prophages in Huanglong disease bacteria into bacteriophages (Ding, F., et al., Molecular mechanics, underlying heat or tetracycline treatments for citrus HLB control. Horticulture research, 2018.5(1):p.30).
  • citrus yellow dragon disease controlled by copper hydrazine we immersed citrus branches infected with yellow dragon disease in different concentrations of copper hydrazine, and divided them into A (water as a control) and B (50 ⁇ g/ml copper hydrazine). C (100 micrograms/ml copper complex) and D (200 micrograms/ml copper complex).
  • the Venn diagram shows that 18376 and 18565 differential genes were identified in B (before treatment) and A (after treatment) samples, respectively. Further analysis showed that there were 17,035 differential genes overlapping between samples of citrus tree (Citrus reticulata Blanco cv. Shatang Ju) before and after treatment. This means that B (after treatment) has 1341 unique differential genes, and A (after treatment) has 1530 unique differential genes (Figure 10). Further analysis we found that in the comparison of B and A, there are 527 up-regulated genes and 1050 down-regulated genes, and 22270 genes have no significant difference (Figure 10).
  • GO Gene Ontology
  • MF molecular function
  • CC cell component
  • BP biological process
  • differential genes are mainly enriched in "movement of cellular processes”; “subcellular component processes”; “microtubules based on the movement process” and “microtubules based on the process”.
  • the role of microtubules is mainly to maintain cell morphology, assist intracellular transportation, form granular centrosomes with other proteins and participate in cell wall formation (Figure 12).
  • Microtubule transport depends on the availability of kinesin, dynein, and ATP. The results showed that the intracellular transport ability of citrus roots and the formation of cell walls were enhanced after treatment.
  • “cell wall” and “external encapsulation structure” are the most important components of this category ( Figure 12).
  • “microtubule binding”; “tubulin binding” and “microtubule motor activity” are the main subcategories.
  • KEGG (Kyoto Encyclopedia of Genes and Genomes) is mainly a biological process database, including seven categories: metabolism, genetic information therapy, environmental information therapy, cell processes, biological systems, human diseases and drug development (Zhang, C. ,et al.,Seedless mutant'Wuzi Ougan'(Citrus suavissima Hort.exTanaka'seedless') and the wild type werecompared by iTRAQ-based quantitative proteomics and integrated. (1): p.106.).
  • the KEGG pathway includes pathways related to metabolic pathways.
  • RNA-seq analysis based on their differential genes, we selected a total of 9 genes (as shown in Table 4, Figure 16), which are the differential genes of the metabolic pathway of phenylpropane biosynthesis (cic00940) : 1 C4H gene (cinnamic acid 4-hydroxylase), 1 PAL gene (phenylalanine ammonia lyase), 1 ⁇ -glucosidase gene, 1 Ciclev10015700m gene (ferulyl-CoA- 6-hydroxylase), 1 POD gene (Ciclev10017908m); differential genes for the mutual conversion of pentose and glucuronate (cic00040): 1 pectinase gene (Ciclev10004719m), 1 PGA enzyme gene; plant hormone signal Differential genes transduced (cic04075): 1 PR1 gene (related to disease course-1) and 1 AHP gene (phosphotransfer protein containing histidine); the expression levels of these genes are basically based on qPCR and
  • thiazoline and copper complex eliminate part of root knot nematodes and inhibit the growth of Huanglong disease bacteria. Some new roots absorb more nutrients and water, which helps to produce more Resistance genes and secondary metabolites, thereby improving the microbial environment of the surrounding soil. In the end, it formed a virtuous circle to eliminate the remaining root knot nematodes and yellow dragon disease bacteria (Figure 17).
  • the invention provides an important direction for field research on Huanglong disease, that is, phloem shedding and xylem exposed roots are markers for diagnosing Huanglong disease, especially for early diagnosis.
  • Our results show that the combined use of thiazoline and copper hydrazine can effectively control citrus huanglong disease, which lays the foundation and important significance for the treatment of huanglong disease and the understanding of the mechanism of huanglong disease.
  • the effect of the concentration of thiazoline and copper complex on the control effect of Huanglong disease has shown that 50-90% thiazoline diluted 1000-3000 times, combined with the application of 100-600 ⁇ g/ml complex copper, can be better Control citrus yellow dragon disease.
  • the spraying method is to spray thiazoline on the root soil first, and then irrigate the roots with copper ammonia 1-3 times after 2-5 days, and the interval of each irrigation is about 30 days.

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Abstract

A drug and method for preventing and treating citrus greening. The drug contains thiazoline and cuaminosulfate. The method specifically comprises sequentially administering thiazoline and cuaminosulfate, which are used in combination for treatment, prevention, and control of citrus greening. An experimental result shows that the solution has a remarkable treatment effect on citrus greening, and the treatment method can have long durability. The solution has important application values and prospects in the treatment, prevention, and control of citrus greening. Moreover, a prevention and control mechanism has also been explored, and a new direction and idea is provided for research on the mechanism of citrus greening and the prevention and control means.

Description

一种防治柑橘类黄龙病的药物及方法Medicine and method for preventing and treating citrus yellow dragon disease 技术领域Technical field
本发明属于农业技术领域。具体涉及一种防治柑橘类黄龙病的药物及方法。The invention belongs to the field of agricultural technology. Specifically, it relates to a medicine and method for preventing and treating citrus yellow dragon disease.
背景技术Background technique
柑橘黄龙病即柑橘绿化病，是一种严重的柑橘毁灭性病害，典型症状包括芽黄、叶面斑驳、叶脉堵塞、水果早熟、果实干瘪偏小、树枝枯萎和树木死亡，降低了水果的产量和品质，同时严重损害了柑橘树的生长。黄龙病树每年损失的平均产量为总产量的30-100％，且通常在感病后2～5年内丧失结果能力或死亡，若不及时防治，其蔓延速度十分惊人，被称为柑橘“癌症”，影响着全世界的柑橘产业，是国内外柑橘产业头号检疫性病害。Citrus yellow dragon disease, or citrus greening disease, is a serious and devastating citrus disease. Typical symptoms include yellow buds, mottled leaves, clogged veins, premature fruit, shriveled fruit, withered branches and tree death, which reduce fruit yield. And quality, while seriously impairing the growth of citrus trees. The annual loss of Huanglong diseased trees is 30-100% of the total yield, and they usually lose fruiting ability or die within 2 to 5 years after being infected. If they are not treated in time, their spreading speed is very alarming, and they are called citrus "cancers". "It affects the citrus industry all over the world, and is the number one quarantine disease in the citrus industry at home and abroad.
目前，果农一般根据叶片症状诊断柑橘类黄龙病，但是这不够准确。而当前控制黄龙病的策略包括严格植物检疫、培育无毒苗、高温治疗、给树干注射药液(如抗生素、青霉素、链霉素、四环素等)、隔离传播载体柑橘木虱、及时挖除病株并妥善管理果园(如专利201910492760.1、201910508577.6等)。然而由于种种原因的限制，这些控制方法效果有限，部分相关研究尚处于初步阶段，效果的连续性也尚未得到证实，无法从根本上解决问题。At present, fruit farmers generally diagnose citrus yellow dragon disease based on leaf symptoms, but this is not accurate enough. The current strategies for controlling Huanglong disease include strict plant quarantine, cultivating non-toxic seedlings, high temperature treatment, injection of liquid medicine (such as antibiotics, penicillin, streptomycin, tetracycline, etc.) into the trunk, isolation of the transmission carrier citrus psyllid, and timely removal of the disease. Plant and properly manage the orchard (such as patents 201910492760.1, 201910508577.6, etc.). However, due to various reasons, the effects of these control methods are limited, and some related studies are still in the preliminary stage, and the continuity of the effects has not been confirmed, and the problem cannot be solved fundamentally.
目前，柑橘黄龙病尚无有效防治手段。At present, there is no effective control method for citrus yellow dragon disease.
发明内容Summary of the invention
本发明的目的是提供一种可以有效治疗柑橘类黄龙病的药物。The purpose of the present invention is to provide a medicine that can effectively treat citrus Huanglongbing.
本发明另一目的是提供一种可以有效治疗柑橘类黄龙病的方法。Another object of the present invention is to provide a method that can effectively treat citrus Huanglongbing.
本发明上述目的通过以下技术方案实现：The above objectives of the present invention are achieved through the following technical solutions:
我们研究结果表明，一颗黄龙病柑橘树中带有韧皮部脱落且木质部暴露的根部的黄龙病细菌CLas(与Ca.Las相同)含量比叶片和没有韧皮部脱落且木质部暴露的根部的黄龙病细菌含量要高，因此韧皮部脱落且木质部暴露可以作为检测柑橘类黄龙病感染程度的标志物，且韧皮部脱落且木质部暴露的根为我们提供了一种了解黄龙病机制的新方向。为了研究络氨铜控制柑橘黄龙病的可能性，我们将柑橘枝条浸泡在不同浓度的络氨铜中，发现黄龙病体内的噬菌体marker基因FP1-GP235和FP2-GP240的相对拷贝数显著增加，这表明络氨铜可能有效控制 黄龙病的作用，因此我们继续用与黄龙病亲缘关系很近的Agrobacterium tumefaciens和Sinorhizobium meliloti为材料，发现400μg/ml络氨铜处理，抑菌率分别达到50％and 100％。进一步的研究表明，噻唑啉和络氨铜联合治疗可以通过抑制黄龙病细菌CLas含量以及治疗后90天后长出新的根和叶来更有效地控制黄龙病。更进一步地，为了探索治疗的机理，我们还进行了转录组实验，结果表明PR1的基因表达水平、治疗后细胞分裂和芽萌生明显改善，以及根据转录组苯丙烷代谢途径中的上调基因可以推测出来flavonoid，scopolin，tyrosine等含量增加可能促进有益菌的生长。总而言之，我们研究显示，噻唑啉和络氨铜联合治疗可以有效控制柑橘黄龙病。Our research results show that the content of Huanglongbing bacteria CLas (same as Ca.Las) in a citrus tree with phloem sloughing and exposed xylem roots is higher than that of leaves and roots without phloem sloughing and xylem exposed roots. Therefore, phloem shedding and xylem exposure can be used as markers to detect the degree of citrus Huanglongbing infection, and the roots with phloem shedding and xylem exposure provide us with a new direction for understanding the mechanism of Huanglongbing. In order to study the possibility of controlling citrus Huanglong disease by copper citrus, we soaked citrus branches in different concentrations of copper citrus and found that the relative copy numbers of the phage marker genes FP1-GP235 and FP2-GP240 in Huanglong disease increased significantly. It shows that copper complex may effectively control Huanglong disease. Therefore, we continue to use Agrobacterium tumefaciens and Sinorhizobium meliloti, which are closely related to Huanglong disease, as materials. It is found that 400μg/ml copper complex treatment has an inhibitory rate of 50% and 100 respectively. %. Further studies have shown that the combined treatment of thiazoline and copper hydrazine can control Huanglong disease more effectively by inhibiting the content of Huanglong disease bacteria CLas and growing new roots and leaves 90 days after treatment. Furthermore, in order to explore the mechanism of treatment, we also conducted transcriptome experiments. The results showed that the gene expression level of PR1, cell division and bud sprouting after treatment were significantly improved, and the upregulated genes in the transcriptome phenylpropane metabolism pathway can be inferred Increasing the content of flavonoid, scopolin, tyrosine, etc. may promote the growth of beneficial bacteria. All in all, our research shows that the combined treatment of thiazoline and copper hydrazine can effectively control citrus yellow dragon disease.
基于此研究结果，本发明要求保护如下方案：Based on the results of this research, the present invention claims the following solutions:
噻唑啉与络氨铜联用在防治柑橘类黄龙病中的应用。Application of thiazoline and copper complex in the prevention and treatment of citrus yellow dragon disease.
噻唑啉与络氨铜联用在制备柑橘类黄龙病防治药物中的应用。Application of thiazoline and copper complex in the preparation of drugs for preventing and curing citrus yellow dragon disease.
优选地，噻唑啉与络氨铜的使用浓度分别为：50-90％噻唑啉稀释1000-3000倍，100-600μg/ml络氨铜。Preferably, the concentration of thiazoline and copper complex are respectively: 50-90% thiazoline diluted 1000-3000 times, 100-600 μg/ml copper complex.
更优选地，噻唑啉与络氨铜的使用浓度分别为：60-80％噻唑啉稀释1500-2500倍，200-600μg/ml络氨铜。More preferably, the use concentrations of thiazoline and copper complex are: 60-80% thiazoline diluted 1500-2500 times, 200-600 μg/ml copper complex.
最优选地，噻唑啉与络氨铜的使用浓度分别为：75％噻唑啉稀释2000倍，200μg/ml络氨铜。Most preferably, the concentration of thiazoline and copper complex are respectively: 75% thiazoline diluted 2000 times, 200 μg/ml copper complex.
因此本发明还提供一种可以有效治疗柑橘类黄龙病的药物，该药物含有噻唑啉与络氨铜。Therefore, the present invention also provides a medicine that can effectively treat citrus huanglong disease, which contains thiazoline and copper complex.
优选地，所述药物中，噻唑啉和络氨铜的用量比按照如下标准计：50-90％噻唑啉经稀释1000-3000倍：100-600μg/ml络氨铜。Preferably, the dosage ratio of the thiazoline and the copper complex in the medicine is calculated according to the following standard: 50-90% thiazoline diluted 1000-3000 times: 100-600 μg/ml copper complex.
更优选地，所述药物中，噻唑啉和络氨铜的用量比按照如下标准计：60-80％噻唑啉经稀释1500-2500倍：200-600μg/ml络氨铜。More preferably, the dosage ratio of the thiazoline to the copper complex in the medicine is calculated according to the following standard: 60-80% thiazoline diluted 1500-2500 times: 200-600 μg/ml copper complex.
最优选地，所述药物中，噻唑啉和络氨铜的用量比按照如下标准计：75％噻唑啉经稀释2000倍：200μg/ml络氨铜。Most preferably, the dosage ratio of the thiazoline and the copper complex in the drug is calculated according to the following standard: 75% thiazoline diluted 2000 times: 200 μg/ml copper complex.
基于此本发明还提供一种可以有效治疗柑橘类黄龙病的方法，具体是利用上述药物进行防治。Based on this, the present invention also provides a method that can effectively treat citrus yellow dragon disease, specifically using the above-mentioned medicine for prevention and treatment.
优选地，所述治疗柑橘类黄龙病的方法是：选择无雨水的日子，先用噻唑啉喷施树木根部土壤，2-5天后施用络氨铜浇灌根部。Preferably, the method for treating citrus yellow dragon disease is: choose a day without rain, first spray the soil at the roots of the trees with thiazoline, and apply copper ammonia to water the roots 2-5 days later.
更优选地，所述治疗柑橘类黄龙病的方法是：选择晴天的日子，先将50-90％噻唑啉稀释1000-3000倍后喷施根部土壤，2-5天后施用1-5次100-600μg/ml络氨铜浇灌根部，浇灌时间相隔30天左右。More preferably, the method for treating citrus yellow dragon disease is: choose a sunny day, first dilute 50-90% thiazoline 1000-3000 times and spray the root soil, 2-5 days later, apply 1-5 times 100- The roots were irrigated with 600μg/ml copper ammonia, and the irrigating time was about 30 days apart.
更优选地，所述治疗柑橘类黄龙病的方法是：选择晴天的日子，先将60-80％噻唑啉稀释1500-2500倍后喷施根部土壤，2-5天后施用1-3次200-600μg/ml络氨铜浇灌根部，浇灌时间相隔30天左右。More preferably, the method for treating citrus yellow dragon disease is: choose a sunny day, firstly dilute 60-80% thiazoline by 1500-2500 times and then spray the root soil, 2-5 days later, apply 1-3 times 200- The roots were irrigated with 600μg/ml copper ammonia, and the irrigating time was about 30 days apart.
最优选地，所述治疗柑橘类黄龙病的方法是：选择晴天的日子，先将75％噻唑啉稀释2000倍后喷施根部土壤，3天后施用1-2次200μg/ml络氨铜浇灌根部，浇灌时间相隔30天左右。Most preferably, the method for treating citrus yellow dragon disease is: choose a sunny day, first dilute 75% thiazoline by 2000 times and then spray the root soil, 3 days later, apply 1-2 times of 200μg/ml copper ammonia to irrigate the roots , The watering time is about 30 days apart.
另外具体操作时根据柑橘黄龙病感染程度决定施用量，病情越严重药物施用量相对高一些。In addition, the dosage is determined according to the degree of citrus Huanglongbing infection during the specific operation. The more serious the disease, the higher the dosage of the drug.
本发明具有以下有益效果：The present invention has the following beneficial effects:
本发明提供了一种新的有效防控黄龙病的新药物方案和治疗方法，具体是将噻唑啉和络氨铜联用用于黄龙病的治疗与控制。该方案对黄龙病治疗效果显著，且治疗方法可以有很长的持续性。The present invention provides a new drug scheme and treatment method for effective prevention and control of Huanglong disease, in particular the combined use of thiazoline and copper hydrazine for the treatment and control of Huanglong disease. The program has a significant effect on the treatment of Huanglong disease, and the treatment method can have a long duration.
同时我们对防控机制也做了探究，为黄龙病机制以及防控手段的研究提供了新的方向和思路。本发明对于黄龙病的治疗与防控具有重要的应用价值和前景。At the same time, we have also explored the prevention and control mechanism, providing new directions and ideas for the research on the mechanism and prevention and control methods of Huanglong disease. The invention has important application value and prospects for the treatment, prevention and control of Huanglong disease.
附图说明Description of the drawings
图1为药物使用和药物治疗根部示意图。(A)两种根部药物治疗的具体位置，络氨铜靶向治疗位置最终是在韧皮部杀死黄龙病菌CLas，噻唑啉通过消灭根结线虫主要作用部位是薄壁组织和中柱鞘。(B)使用两种药物进行治疗的示意图，根部用噻唑啉(2000x)和络氨铜(200μg/ml)处理。Figure 1 is a schematic diagram of drug use and drug treatment of the roots. (A) The specific location of the two root drug treatments. The target treatment location of copper squamousine is ultimately to kill the yellow dragon pathogen CLas in the phloem. The main action sites of thiazoline to eliminate root knot nematodes are the parenchyma and the pericylindrical sheath. (B) Schematic diagram of treatment with two drugs, and the roots were treated with thiazoline (2000x) and copper complex (200μg/ml).
图2为不同感染程度HLB的视觉评价。1，2，3-疾病的中晚期。4，5，6-疾病的早期阶段Figure 2 shows the visual evaluation of HLB with different degrees of infection. 1,2,3-The middle and late stages of the disease. 4, 5, 6-Early stage of disease
图3为柑桔感染HLB早期不同部位的检测。A)1-叶；2-没有-韧皮部脱落且木质部暴露的根；3-韧皮部脱落且木质部暴露的根。B)不同样品的Ca.Las滴度(每克植物组织细胞数)。C)在ERRX(韧皮部脱落且木质部暴露的根)根部检测到SDE1的表达水平远高于叶片。每个实验重复三次。Figure 3 shows the detection of different parts of citrus in the early stage of HLB infection. A) 1-leaf; 2-no-roots with phloem shed and xylem exposed; 3-roots with phloem shed and xylem exposed. B) Ca.Las titers of different samples (number of cells per gram of plant tissue). C) The expression level of SDE1 detected in the roots of ERRX (roots with phloem exfoliated and exposed xylem) is much higher than that of leaves. Each experiment was repeated three times.
图4为不同样品中黄龙病细菌的数量(每克植物组织中的细胞数)。(A)1-有中度感染症状的叶子，2-有严重感染症状的叶子，3-没有-韧皮部脱落且木质 部暴露的根，4-韧皮部脱落且木质部暴露的根。(B)不同样品的黄龙病细菌Ca.Las滴度，每个实验重复3次。星号(*)表示差异显着(P<0.05；Dunnett’s test)。Figure 4 shows the number of Huanglongbing bacteria in different samples (the number of cells per gram of plant tissue). (A) 1-Leaves with moderate symptoms of infection, 2-Leaves with severe symptoms of infection, 3-None-Roots with phloem shedding and xylem exposed, 4-Roots with phloem shedding and xylem exposed. (B) Ca.Las titers of Huanglongbing bacteria in different samples, each experiment was repeated 3 times. An asterisk (*) indicates a significant difference (P<0.05; Dunnett’s test).
图5噻唑啉和络氨铜的抑菌实验及使用络氨铜治疗后FP1-GP235和FP2-GP240的相对表达量。(A)噻唑啉的抑菌率；(B)络氨铜的抑菌率；(C)FP1-GP235基因的相对表达量；(D)FP2-GP240基因的相对表达量；数据以平均值±标准误差表示(n＝3)。我们用与黄龙病亲缘关系很近的Agrobacterium tumefaciens和Sinorhizobium meliloti为材料，发现400μg/ml络氨铜处理，抑菌率分别达到50％and 100％，这暗示络氨铜可能有抑制黄龙病菌的能力。随着络氨铜处理浓度的增加FP1-GP235和FP2-GP240的相对表达量显著增加，这表明抗黄龙病的噬菌体在增加，说明络氨铜可能有效控制了黄龙病菌的繁殖(P<0.05)。Figure 5 The antibacterial experiment of thiazoline and copper ammonium and the relative expression levels of FP1-GP235 and FP2-GP240 after treatment with copper ammonium. (A) Antibacterial rate of thiazoline; (B) Antibacterial rate of copper complex; (C) Relative expression level of FP1-GP235 gene; (D) Relative expression level of FP2-GP240 gene; data are average ± The standard error is expressed (n=3). We used Agrobacterium tumefaciens and Sinorhizobium meliloti, which are closely related to Huanglong disease, and found that the 400μg/ml tyrosine treated with tyrosine, the antibacterial rate reached 50% and 100%, respectively, which suggests that tyrosine might have the ability to inhibit tyrosine disease. . The relative expression levels of FP1-GP235 and FP2-GP240 increased significantly with the increase of the concentration of copper complex, which indicates that the phage resistant to Huanglong disease is increasing, indicating that copper complex may effectively control the reproduction of Huanglong disease bacteria (P<0.05) .
图6为分别使用噻唑啉和络氨铜单独治疗以及噻唑啉与络氨铜联合治疗30天后的根部状态比较。(A)用噻唑啉处理过的根。(B)用络氨铜处理过的根。(C)用两种药物(噻唑啉；络氨铜)联合处理过的根。黄色箭头表示韧皮部脱落且木质部暴露，红色箭头表示新的根。Figure 6 is a comparison of the root condition after 30 days of treatment with thiazoline and copper hydrazine alone and 30 days after combined treatment with thiazoline and copper hydrazine. (A) Roots treated with thiazoline. (B) Roots treated with copper complex. (C) Roots treated with two drugs (thiazoline; copper complex). The yellow arrow indicates that the phloem is shed and the xylem is exposed, and the red arrow indicates a new root.
图7为使用两种药物(噻唑啉；络氨铜)联合治疗90天后，黄龙病得到有效控制。(A)治疗前叶片的症状。(B)治疗3个月后叶片的症状。(C)治疗3个月后根部的症状。(D)治疗后不同时间的黄龙病细菌滴度CLas变化。用水作为对照(WSC)。星号(*)表示WSC和络氨铜之间有显着差异(P<0.05；Dunnett’s test)。实线表示WSC，虚线表示用2种药物联合治疗。Figure 7 shows the effective control of Huanglong disease after 90 days of combined treatment with two drugs (thiazoline; copper complex). (A) Treat the symptoms of the front leaf. (B) Symptoms of leaves after 3 months of treatment. (C) Root symptoms after 3 months of treatment. (D) The change of Huanglong disease bacterial titer CLas at different time after treatment. Water was used as a control (WSC). The asterisk (*) indicates that there is a significant difference between WSC and copper sulphate (P<0.05; Dunnett’s test). The solid line represents WSC, and the dashed line represents combined treatment with two drugs.
图8为两种药物联合治疗前后样本的根和叶的变化。A)处理前的根系生长状况。B)处理1个月后的根系生长状况。C)处理前叶片表面状态。D)叶面处理1个月后的状态。ABCD都表示同一棵树。Figure 8 shows the changes in the roots and leaves of the samples before and after treatment with the two drugs. A) Root growth status before treatment. B) Root growth status after 1 month of treatment. C) The surface state of the front blade. D) The state after foliar treatment for 1 month. ABCD all mean the same tree.
图9为两种药物联合治疗后受感染树木的产量与上一年产量的比较(受黄龙病(HLB)感染越严重的树木治疗后效果越显著)；表明两种药物联合应用可有效地治疗HLB。A)柑桔治疗前症状。B)柑桔经3个月处理后的症状。C)柑桔经5个月处理后的症状。D)柑桔经9个月处理后的症状。E)表明不同程度HLB感染的治疗前后的果实产量比较：严重、一般、中度。试验田I药物治疗后(2014-2015年)与上年产量(2013-2014年)比较，试验田II没有药物治疗的严重的HLB柑橘果实产量无差异。Figure 9 shows the comparison of the yield of infected trees after the combined treatment of the two drugs with the yield of the previous year (the more severe the yellow dragon disease (HLB) infection, the more significant the effect will be after treatment); it shows that the combined application of the two drugs can be effectively treated HLB. A) Symptoms before citrus treatment. B) Symptoms of citrus after 3 months of treatment. C) Symptoms of citrus after 5 months of treatment. D) Symptoms of citrus after 9 months of treatment. E) shows the comparison of fruit yield before and after treatment with different degrees of HLB infection: severe, normal, and moderate. Compared with the output of the previous year (2013-2014) after drug treatment in Test Field I (2014-2015), there was no difference in the yield of severe HLB citrus fruits in Test Field II without drug treatment.
图10为差异表达基因(DEGs)分析结果。A)A组和B组DEGs的维恩图A-治疗前，B-治疗后。B)两个阶段(治疗前和治疗后)的火山图。横坐标表示治疗组和对照组基因的表达倍数变化(log2倍变化)，纵坐标表示治疗组和对照组基因表达差异的显著性水平(-log10padj)。上调基因用红点表示。下调基因用绿点表示。蓝色表示的是无显著性差异的变化。我们发现有527个基因上调，1050个基因下调，22270个基因无显著差异。Figure 10 shows the results of differentially expressed genes (DEGs) analysis. A) Venn diagram of DEGs in group A and group B. A-before treatment, and B-after treatment. B) Volcano diagram of two stages (before treatment and after treatment). The abscissa represents the fold change of gene expression (log2 fold change) between the treatment group and the control group, and the ordinate represents the significant level of gene expression difference between the treatment group and the control group (-log10padj). Up-regulated genes are indicated by red dots. Down-regulated genes are indicated by green dots. Blue indicates changes that are not significantly different. We found that 527 genes were up-regulated, 1050 genes were down-regulated, and 22270 genes were not significantly different.
图11为差异表达基因(DEGs)在治疗前后的表达谱。用K-均值法对差异表达蛋白进行聚类分析的热图。颜色表示折叠变化(红色表示上调；蓝色表示下调，均为BvsA)。Figure 11 shows the expression profiles of differentially expressed genes (DEGs) before and after treatment. Heat map for cluster analysis of differentially expressed proteins using K-means method. The color indicates the change in folding (red indicates up-regulation; blue indicates down-regulation, both are BvsA).
图12为GO富集统计图。(A)GO富集的散点图，用于对所有基因和不同表达的基因进行综合分析。横坐标为注释到GO Term上的差异基因数与差异基因总数的比值，纵坐标为GO Term，点的大小代表GO Term上的基因数，颜色从红到紫代表富集的显著性大小。(B)GO分析的柱状图。从GO富集分析结果中，选取最显著的30个Term绘制柱状图。图中横坐标为GO Term，纵坐标为GO Term富集的显著性水平，数值越高越显著，不同颜色分别代表BP，CC，MF三个GO子类。Figure 12 is a graph of GO enrichment statistics. (A) Scatter plot of GO enrichment for comprehensive analysis of all genes and differently expressed genes. The abscissa is the ratio of the number of differential genes annotated to GO Term to the total number of differential genes, the ordinate is GO Term, the size of the dot represents the number of genes on GO Term, and the color from red to purple represents the significance of enrichment. (B) Histogram of GO analysis. From the results of GO enrichment analysis, select the most significant 30 terms to draw a histogram. In the figure, the abscissa is GO Term, and the ordinate is the significance level of GO Term enrichment. The higher the value, the more significant it is. Different colors represent the three GO subtypes of BP, CC, and MF.
图13为KEGG富集统计图。(A)KEGG富集的散点图，用于对所有基因和不同表达的基因进行综合分析。横坐标为KEGG通路上的差异基因数与差异基因总数的比值，纵坐标为KEGG通路，点的大小代表KEGG通路上的基因数，颜色从红到紫代表富集的显著性大小，该图显示了治疗后比治疗前上调的基因。(B)KEGG分析的柱状图。从KEGG富集结果中，选取最显著的20个KEGG通路绘制柱状图，横坐标为KEGG通路，纵坐标为通路富集的显著性水平，数值越高越显著。Figure 13 is a statistical chart of KEGG enrichment. (A) Scatter plot of KEGG enrichment for comprehensive analysis of all genes and differently expressed genes. The abscissa is the ratio of the number of differential genes on the KEGG pathway to the total number of differential genes, the ordinate is the KEGG pathway, the size of the dot represents the number of genes on the KEGG pathway, and the color from red to purple represents the significance of enrichment. The figure shows The genes that were up-regulated after treatment than before treatment. (B) Histogram of KEGG analysis. From the results of KEGG enrichment, select the 20 most significant KEGG pathways to draw a histogram. The abscissa is the KEGG pathway, and the ordinate is the significance level of pathway enrichment. The higher the value, the more significant.
图14为植物激素信号转导途径(cic04075)。红色箭头指示治疗后基因表达水平上调，绿色箭头指示治疗后基因表达水平下调。垂直虚线表示细胞膜，垂直实线表示细胞壁。Figure 14 shows the plant hormone signal transduction pathway (cic04075). The red arrow indicates that the gene expression level is up-regulated after treatment, and the green arrow indicates that the gene expression level is down-regulated after treatment. The vertical dashed line represents the cell membrane, and the vertical solid line represents the cell wall.
图15为苯丙氨酸相关代谢通路的次级代谢。红色箭头表示用两种药物(噻唑啉、络氨铜)联合处理后的基因表达水平上调，用黄色字符表示的次级代谢产物产生了有益的细菌并且激活了植物抗性基因的表达。PAL表示苯丙氨酸氨裂解酶；C4H表示4-羟化酶。Figure 15 shows the secondary metabolism of phenylalanine-related metabolic pathways. The red arrow indicates the up-regulation of gene expression levels after combined treatment with two drugs (thiazoline and copper ammonium). The secondary metabolites represented by yellow characters produced beneficial bacteria and activated the expression of plant resistance genes. PAL stands for phenylalanine ammonia lyase; C4H stands for 4-hydroxylase.
图16为通过qRT-PCR验证转录组结果。9个差异表达基因(差异基因)的相对表达涉及苯丙烷代谢和葡萄糖醛酸酯相互转化(cic00040)，类黄酮生物合成(cic00941)和植物激素信号传导(cic04075)。C4H对应的基因为Ciclev10000921m，PAL对应的基因为Ciclev10027912m；PGA对应的基因为Ciclev10004783m；PR-1对应的基因为Ciclev10029459m；beta-glucosidase对应的基因为Ciclev10028018m；AHP对应的基因为Ciclev10029493m。(参与苯丙烷生物合成代谢途径(CIC00 940)的基因包括一个PAL基因(苯丙氨酸解氨酶)、一个C4H基因(肉桂酸4-羟化酶)、一个β-葡萄糖苷酶基因和一个POD基因(过氧化物酶)。一个PGA酶基因和一个果胶酯酶参与戊糖和葡萄糖醛酸盐相互转化的代谢途径。一个PR1基因(病原相关基因-1)和一个AHP基因(含组氨酸磷酸转移蛋白)参与植物激素信号转导。)Figure 16 shows the results of verifying the transcriptome by qRT-PCR. The relative expression of 9 differentially expressed genes (differential genes) involves phenylpropane metabolism and glucuronide interconversion (cic00040), flavonoid biosynthesis (cic00941) and plant hormone signaling (cic04075). The gene corresponding to C4H is Ciclev10000921m, the gene corresponding to PAL is Ciclev10027912m; the gene corresponding to PGA is Ciclev10004783m; the gene corresponding to PR-1 is Ciclev10029459m; the gene corresponding to beta-glucosidase is Ciclev10028018m; the gene corresponding to AHP is Ciclev10029493m. (The genes involved in the phenylpropane biosynthetic metabolic pathway (CIC00 940) include a PAL gene (phenylalanine ammonia lyase), a C4H gene (cinnamic acid 4-hydroxylase), a β-glucosidase gene and a POD gene (peroxidase). A PGA enzyme gene and a pectin esterase are involved in the metabolic pathway of pentose and glucuronate conversion. A PR1 gene (pathogen-related gene-1) and an AHP gene (including group Amino acid phosphate transfer protein) is involved in plant hormone signal transduction.)
图17为噻唑啉和络氨铜的治疗机理图。红色箭头表示治疗后基因表达水平上调；蓝色箭头表示特定的治疗途径；红色字符的PR1表示治疗后基因表达水平上调。Figure 17 is a diagram of the therapeutic mechanism of thiazoline and copper complex. The red arrow indicates the increase in gene expression level after treatment; the blue arrow indicates the specific treatment route; the red character PR1 indicates the increase in gene expression level after treatment.
具体实施方式Detailed ways
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。The present invention will be further described below with reference to the drawings and specific embodiments of the specification, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
除非特别说明，以下实施例所用试剂和材料均为市购。Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.
以下实施例主要包括三大实验：(1)络氨铜治疗实验；(2)噻唑啉和络氨铜联用治疗实验；(3)转录组实验(探索治疗机理)。The following examples mainly include three major experiments: (1) copper sulphate treatment experiment; (2) thiazoline and sulphate combined treatment experiment; (3) transcriptome experiment (exploration of treatment mechanism).
实施例1Example 1
一、实验材料与方法1. Experimental materials and methods
1.1.实验植物材料1.1. Experimental plant materials
(1)实验室培养感病枝条：2016年4月，我们将感染了黄龙病的沙糖桔树嫁接到健康的2年沙糖桔幼苗上，然后将其保存在培养室中培养。10个月后，叶片上出现静脉阻塞和斑点的典型黄龙病症状。(1) Laboratory cultivation of diseased branches: In April 2016, we grafted Shatang orange trees infected with Huanglong disease to healthy 2-year-old Shatang orange seedlings, and then stored them in a culture room for cultivation. Ten months later, the typical symptoms of Huanglong disease of vein blockage and spots appeared on the leaves.
(2)野外试验田I：来自中国广东省果园-黄龙病感染的约10年的沙糖桔树。(2) Field test field I: Shatang orange trees infected with Huanglong disease from an orchard in Guangdong Province, China for about 10 years.
(3)野外试验田II：来自中国江西省赣州市果园-感染黄龙病脐橙树约十年大的树。(3) Field Test Field II: From an orchard in Ganzhou City, Jiangxi Province, China-a navel orange tree infected with Huanglong disease about ten years old.
(4)为了最大程度地减少黄龙病细菌分布不均的影响，采样方法如下：(4) In order to minimize the impact of the uneven distribution of Huanglong disease bacteria, the sampling method is as follows:
在用络氨铜浸泡离体枝条的实验中，采样方法：在顶部收集一片叶子，底部收集一片叶子，中间收集一片叶子。在两种药物(噻唑啉；络氨铜)联合治疗柑橘类黄龙病的实验中，采样方法：收集顶部的三片叶子，底部的三片叶子和中间的三片叶子；收集后称重，将其保持在-80℃的冰箱中。In the experiment of immersing the detached branches with copper complex, the sampling method: collect a leaf at the top, collect a leaf at the bottom, and collect a leaf in the middle. In the experiment of combining two drugs (thiazoline; copper complex) on the treatment of citrus yellow dragon disease, the sampling method: collect the top three leaves, the bottom three leaves and the middle three leaves; after the collection, weigh the It is kept in a refrigerator at -80°C.
另外根组织的采样方法：在距树干2英尺范围内的四个不同象限中，从土壤表面以下2-5英寸处收集柑橘根组织，然后将其在室温(20℃～25℃)的纸袋中风干约24小时，以方便用手指轻拍去除过多的土壤污垢。In addition, the sampling method of root tissue: Collect citrus root tissue from 2-5 inches below the soil surface in four different quadrants within 2 feet from the trunk, and then place it in a paper bag at room temperature (20℃～25℃) Air-dry for about 24 hours to facilitate the removal of excessive soil dirt by tapping with your fingers.
样品采集后立即进行治疗，根或者叶片材料放在冰箱中保持在-80℃。The samples were treated immediately after collection, and the root or leaf materials were kept at -80°C in the refrigerator.
对于转录组实验，我们的根纤维样品来自中国广东省广州市的沙糖桔树根。For the transcriptome experiment, our root fiber samples were from the roots of Shatang orange trees in Guangzhou, Guangdong Province, China.
1.2.实验分组与处理方法1.2. Experimental grouping and processing methods
1.2.1.络氨铜治疗1.2.1. Therapy
实验用的是有黄龙病感染症状的直径约0.5-0.8厘米的柑橘树枝(沙糖桔)。每个枝条包括约20-24片叶子，然后将枝条浸入在15ml的试管中，试管里装有不同浓度的络氨铜(50μg/ml，100μg/ml，200μg/ml)，试管外面用铝箔纸覆盖，将只用水浸泡作对照。在络氨铜治疗后1h，5h，12h，1d，3d和5d收集叶片样品。每个实验至少重复三次。The experiment used citrus branches (shatang oranges) with a diameter of about 0.5-0.8 cm with symptoms of Huanglong disease infection. Each branch contains about 20-24 leaves, and then the branch is immersed in a 15ml test tube. The test tube contains different concentrations of copper ammonia (50μg/ml, 100μg/ml, 200μg/ml). The outside of the test tube is made of aluminum foil. Cover, soak in water only as a control. The leaf samples were collected at 1h, 5h, 12h, 1d, 3d and 5d after the treatment with copper ammonia. Each experiment was repeated at least three times.
1.2.2.两种药物联合治疗(噻唑啉；络氨铜)1.2.2. Combination therapy with two drugs (thiazoline; copper complex)
(1)培养室治疗试验(1) Cultivation room treatment test
培养室中的治疗试验分为联合治疗组(噻唑啉和络氨铜两种药物联用)和对照组(水处理)，每组三棵树。The treatment test in the incubation room is divided into a combination treatment group (thiazoline and copper complex) and a control group (water treatment), with three trees in each group.
联合治疗方法如下：首先用噻唑啉(稀释2000倍)(河北三农农化有限公司)浸泡柑橘树的根。3-5天后，使用200μg/ml络氨铜喷洒柑橘根(图1)。在治疗后的第0、15、30、60、90和120天收集叶片样品。The combined treatment method is as follows: First, soak the roots of citrus trees with thiazoline (diluted 2000 times) (Hebei Sannong Agrochemical Co., Ltd.). After 3-5 days, spray the citrus roots with 200μg/ml copper complex (Figure 1). Leaf samples were collected at 0, 15, 30, 60, 90, and 120 days after treatment.
(2)野外实验：(2) Field experiment:
野外治疗受黄龙病感染的树木的方法类似于培养室治疗。选择天晴的日子，先将75％噻唑啉稀释2000倍后喷施树木根部土壤，3天后施用1-2次200μg/ml络氨铜浇灌根部，浇灌时间相隔30天左右。The treatment of trees infected with Huanglong disease in the field is similar to the treatment in a culture room. Choose a sunny day, first dilute 75% thiazoline by 2000 times and spray the soil on the roots of the trees. After 3 days, water the roots with 1-2 times of 200μg/ml copper ammonia. The irrigation time is about 30 days apart.
野外试验田为示范面积为40000平方米的脐橙果园，始于2015年3月，截至2015年11月。我们将试验分为A组和B组。A组用两种药物(噻唑啉和络 氨铜)联合治疗，B组用水处理作为对照。根据黄龙病的感染程度将每组进一步分为3类，如重度感染，中度感染，轻度感染，之后与上一年度(2014-2015年)的产量相比。The field test field is a navel orange orchard with a demonstration area of 40,000 square meters, which started in March 2015 and ended in November 2015. We divided the trial into group A and group B. Group A was treated with two drugs (thiazoline and copper complex), and group B was treated with water as a control. According to the degree of Huanglong disease infection, each group is further divided into 3 categories, such as severe infection, moderate infection, and mild infection, and then compared with the output of the previous year (2014-2015).
1.3.定量PCR实验方法1.3. Quantitative PCR experiment method
1.3.1 DNA和RNA提取1.3.1 DNA and RNA extraction
使用液氮在研钵中快速研磨-80℃收集称重的叶片中脉或纤维根组织。按照厂家提供的说明书，使用生工生物工程股份有限公司(中国上海Sangon)的基因组DNA提取试剂盒提取总基因组DNA。这些基因组DNA被用作模板，以进行qPCR检测黄龙病细菌CLas含量和FP1-GP235和FP2-GP240的相对拷贝数。按照厂家提供的说明，使用生工(中国上海Sangon)的RNA提取试剂盒提取总RNA。使用NanoDrop ND-1000测定RNA样品的含量。将RNA浓度调节至200ng/μl作为模板，使用PrimeScriptTM RT Master Mix Kit(中国辽宁省TAKARA)反转录获得cDNA，并以cDNA为模板进行qPCR以获取相对表达量。所有qPCR反应均重复至少3次。Use liquid nitrogen to quickly grind in a mortar at -80°C to collect the weighed leaf midrib or fibrous root tissue. According to the instructions provided by the manufacturer, the total genomic DNA was extracted using the genomic DNA extraction kit of Sangon Bioengineering Co., Ltd. (Sangon, Shanghai, China). These genomic DNAs were used as templates for qPCR to detect the CLas content of Huanglong disease bacteria and the relative copy numbers of FP1-GP235 and FP2-GP240. Follow the instructions provided by the manufacturer and use the RNA extraction kit from Sangon (Sangon, Shanghai, China) to extract total RNA. Use NanoDrop ND-1000 to determine the content of RNA samples. Adjust the RNA concentration to 200ng/μl as a template, use PrimeScriptTM RT Master Mix Kit (TAKARA, Liaoning Province, China) to obtain cDNA by reverse transcription, and perform qPCR with cDNA as a template to obtain relative expression. All qPCR reactions were repeated at least 3 times.
1.3.2.黄龙病细菌CLas含量测量1.3.2. Huanglong disease bacteria CLas content measurement
嵌入柑橘黄龙病原16S rDNA的重组质粒pMD-19来自广东省农业科学院。通过qPCR测定黄龙病细菌含量，使用特异性引物(黄龙病asf，黄龙病r和黄龙病-p-Table.1)-特异性针对黄龙病细菌的16S rDNA区域的引物黄龙病asf和黄龙病r被用作黄龙病细菌的参考，将7种梯度16S rDNA的重组质粒pMD-19稀释为10 -1、10 -2、10 -3、10 -4、10 -5个、10 -6、10 -7，7个梯度分别作为模板，以ddH 2O为空白对照。健康的柑橘叶片DNA样品为阴性对照，重复了3次以上的实验。实验均在qPCR仪器Applied Biosystems QuantstudionTM 6 Flex中进行，得到相应的标准曲线y＝-3.2215x+35.238，R2＝0.9965(y表示Ct值，x表示lg(病原体拷贝数)。 The recombinant plasmid pMD-19 embedded with the 16S rDNA of the citrus Huanglong pathogen was from Guangdong Academy of Agricultural Sciences. The content of Huanglongbing bacteria was determined by qPCR, using specific primers (Huanglongbing asf, Huanglongbing r and Huanglongbing-p-Table.1)-specific primers for the 16S rDNA region of Huanglongbing bacteria Huanglongbing asf and Huanglongbing r greening disease bacteria is used as a reference, the recombinant plasmid seven kinds of gradients of 16S rDNA of pMD-19 diluted to 10 -1, 10 -2, 10 -3, 10 -4, 10 -5, 10 -6, 10 - 7 and 7 gradients were used as templates, and ddH 2 O was used as a blank control. The healthy citrus leaf DNA sample was used as a negative control, and the experiment was repeated more than 3 times. The experiments were carried out in the qPCR instrument Applied Biosystems QuantstudionTM 6 Flex, and the corresponding standard curve y=-3.2215x+35.238, R2=0.965 (y represents the Ct value, and x represents the lg (pathogen copy number).
对于TaqMan实时定量PCR，反应混合物的总体积为20μl：引物HLBasf和HLBr(中国上海Sangon)分别为0.4μl，探针HLBp(辽宁Takara)为0.4μl，TaqMan混合液(中国辽宁省TAKARA)为10μl，DNA模板为1μl，水为7.8μl。实时定量PCR程序如下：95℃5min，95℃30s，60℃30s；40个循环。表中列出了所有引物参照表1，计算方法参考Li(Li,W.,L.Levy,and J.S.Hartung,Quantitative distribution of‘Candidatus Liberibacter asiaticus’in citrus plants with  citrus huanglongbing.Phytopathology,2009.99(2):p.139-144)。For TaqMan real-time quantitative PCR, the total volume of the reaction mixture is 20μl: primers HLBasf and HLBr (Sangon, Shanghai, China) are 0.4μl, probe HLBp (Takara, Liaoning) is 0.4μl, and TaqMan mixture (TAKARA, Liaoning, China) is 10μl , DNA template is 1μl, water is 7.8μl. The real-time quantitative PCR program is as follows: 95°C for 5min, 95°C for 30s, 60°C for 30s; 40 cycles. All primers are listed in the table, refer to Table 1, and the calculation method refers to Li(Li,W.,L.Levy,and JSHartung,Quantitative distribution of'Candidatus Liberibacter asiaticus'in citrus plants with citrus Huanglongbing.Phytopathology,2009.99(2) :p.139-144).
表1.实时PCR引物和探针的寡核苷酸序列Table 1. Oligonucleotide sequences of real-time PCR primers and probes
Figure PCTCN2020076627-appb-000001
Figure PCTCN2020076627-appb-000001
a其他鸟嘌呤核苷酸(粗体，带下划线“G”)被添加到黄龙病as序列63基于16S rDNA序列在‘Ca.Liberibacter asiaticus’Psy62基因组被称为HLBasf a Other guanine nucleotides (in bold, underlined "G") were added to the Huanglong disease as sequence 63 based on the 16S rDNA sequence in the'Ca.Liberibacter asiaticus' Psy62 genome called HLBasf
b 6-FAM TM在5’末端 b 6-FAM TM at the 5'end
c Iowa Black FQ在3’末端 c Iowa Black FQ is at the 3'end
1.3.3.FP1和FP2相对拷贝数1.3.3. Relative copy number of FP1 and FP2
将CLas的16s rDNA用作内参，以检测FP1-GP235和FP2-GP240的相对拷贝数。表1中列出了所有引物。对于以基因组DNA为模板的SYBR实时定量PCR，每个反应均包含以下成分，总体积为20μl：引物为0.4μl，SYBR Mix为10μl，DNA为1μl，水为8.2μl。所有反应均重复三次。实时定量PCR程序如下：95℃5min，95℃30s，60℃30s。40个循环，计算方法参考Ding(Ding,F.,et al.,Molecular mechanisms underlying heat or tetracycline treatments for citrus HLB control.Horticulture research,2018.5(1):p.30)。The 16s rDNA of CLas was used as an internal control to detect the relative copy numbers of FP1-GP235 and FP2-GP240. All primers are listed in Table 1. For SYBR real-time quantitative PCR using genomic DNA as a template, each reaction contains the following components, with a total volume of 20 μl: 0.4 μl for primers, 10 μl for SYBR Mix, 1 μl for DNA, and 8.2 μl for water. All reactions were repeated three times. The real-time quantitative PCR program is as follows: 95°C for 5min, 95°C for 30s, 60°C for 30s. 40 cycles, the calculation method refers to Ding (Ding, F., et al., Molecular mechanismss underlying heat or tetracycline treatments for citrus HLB control. Horticulture research, 2018.5(1): p.30).
1.3.4. SDE1(CLas Sec-delivery effector-1)的基因表达水平1.3.4. Gene expression level of SDE1 (CLas Sec-delivery effector-1)
检测SDE1的基因表达水平，我们将柑橘COX基因作为内参。引物见表2。对于SYBR实时PCR，以cDNA为模板，每个反应包含以下成分，总体积为20μl：引物为0.4μl，SYBR Mix为10μl，cDNA为1μl，水为8.2μl。所有反应均一式三份进行，实时qPCR程序如下：95℃5min，95℃10s，60℃20s。40个循环。SDE1计算方法的基因表达水平参考Pagliaccia D(Pagliaccia,D.,et al.,A pathogen secreted protein as a detection marker for Citrus Huanglongbing.Frontiers in microbiology,2017.8:p.2041)。To detect the gene expression level of SDE1, we use the citrus COX gene as an internal reference. The primers are shown in Table 2. For SYBR real-time PCR, cDNA is used as a template, and each reaction contains the following components, with a total volume of 20 μl: 0.4 μl for primers, 10 μl for SYBR Mix, 1 μl for cDNA, and 8.2 μl for water. All reactions were performed in triplicate, and the real-time qPCR program was as follows: 95°C for 5 min, 95°C for 10 seconds, and 60°C for 20 seconds. 40 cycles. The gene expression level of SDE1 calculation method refers to Pagliaccia D (Pagliaccia, D., et al., A pathogen secreted protein as a detection marker for Citrus Huanglongbing. Frontiers in microbiology, 2017.8: p.2041).
表2.SDE1及COX基因引物序列Table 2. SDE1 and COX gene primer sequences
Figure PCTCN2020076627-appb-000002
Figure PCTCN2020076627-appb-000002
1.4.RNA-seq数据分析过程1.4.RNA-seq data analysis process
我们将两种药物治疗后的根纤维命名为A组，将治疗前的根纤维命名为B组。从每个组中获取每个样品的三个重复样本。如上所述的采样方法。我们将样品发送到北京诺和基因技术有限公司进行转录组分析。We named the root fibers after treatment with the two drugs as group A, and named the root fibers before treatment as group B. Obtain three replicate samples of each sample from each group. Sampling method as described above. We sent the samples to Beijing Novo Gene Technology Co., Ltd. for transcriptome analysis.
1.5.通过qPCR验证RNA-seq1.5. Verify RNA-seq by qPCR
为了验证RNA-seq的结果，通过qPCR比较差异基因相对丰度变化，该变化涉及苯丙烷代谢、葡糖醛酸相互转化(cic00040)、类黄酮生物合成(cic00941)和植物激素信号转导(cic04075)(表3)，以cDNA为模板，每个反应均包含以下组分，总体积为20μl：引物为0.4μl，SYBR Mix为10μl，cDNA为1μl，水为8.2μl。所有反应均一式三份进行，实时qPCR程序如下：95℃5min，95℃10s，60℃20s.40个循环。选择GAPDH(甘油醛-3-磷酸脱氢酶)作为内部参照基因，并用Ct法(2-ΔΔCt)计算相对表达量。In order to verify the results of RNA-seq, the relative abundance changes of differential genes were compared by qPCR. The changes involved phenylpropane metabolism, glucuronic acid interconversion (cic00040), flavonoid biosynthesis (cic00941), and plant hormone signal transduction (cic04075). ) (Table 3), using cDNA as a template, each reaction contains the following components, with a total volume of 20 μl: 0.4 μl for primers, 10 μl for SYBR Mix, 1 μl for cDNA, and 8.2 μl for water. All reactions were performed in triplicate, and the real-time qPCR program was as follows: 95°C for 5 min, 95°C for 10s, 60°C for 20s. 40 cycles. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was selected as the internal reference gene, and the relative expression level was calculated by the Ct method (2-ΔΔCt).
表3.治疗前后三种途径中差异基因的变化情况列表Table 3. List of changes in differential genes in the three pathways before and after treatment
Figure PCTCN2020076627-appb-000003
Figure PCTCN2020076627-appb-000003
Figure PCTCN2020076627-appb-000004
Figure PCTCN2020076627-appb-000004
PAL,phenylalanine ammonia lyase；C4H,cinnamate 4-hydroxylase；4CL,4-coumaroyl CoA ligase.PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl CoA ligase.
1.6.数据分析1.6. Data analysis
每个实验至少重复三遍。使用GraphPad Prism 6.0软件进行统计分析。数据表示为平均值±标准偏差(SD)，p<0.05，p<0.01和p<0.001被认为具有统 计学意义。给出的数据是三个生物学重复的平均值，具有标准误。首先通过方差分析(ANOVA)分析络氨铜对FP1-GP235和FP2-GP240拷贝数的影响，然后通过Tukey的真实显着差异检验确定治疗之间的成对比较。所有统计分析均使用软件spss statistics 23进行，显着性水平设置为0.05。Repeat each experiment at least three times. Use GraphPad Prism 6.0 software for statistical analysis. Data are expressed as mean ± standard deviation (SD), p<0.05, p<0.01 and p<0.001 are considered statistically significant. The data given is the average of three biological replicates with standard errors. First, analyze the effect of copper ammonia on the copy number of FP1-GP235 and FP2-GP240 by analysis of variance (ANOVA), and then use Tukey's true significant difference test to determine the pairwise comparison between treatments. All statistical analyses were performed using the software spss statistics 23, and the significance level was set to 0.05.
二、实验结果2. Experimental results
2.1.柑橘韧皮部脱落且木质部暴露中的黄龙病细菌含量高于叶片和非韧皮部脱落且木质部暴露的根部2.1. The content of Huanglongbing bacteria in citrus phloem shed and exposed xylem is higher than that in leaves and roots where the non-phloem shed and xylem are exposed
叶子的症状是判断其是否感染了黄龙病的关键。我们研究了黄龙病不同侵染阶段的根与叶表面之间的差异，我们已经定义了在受感染的黄龙病的早期，中期和后期叶片的不同症状(图2)。The symptoms of the leaves are the key to judging whether they are infected with Huanglong disease. We have studied the differences between roots and leaf surfaces at different stages of Huanglong disease infection, and we have defined the different symptoms of leaves in the early, middle and late stages of infected Huanglong disease (Figure 2).
在早期，韧皮部脱落且木质部暴露根>非韧皮部脱落且木质部暴露根>叶的黄龙病细菌含量(每克植物组织细胞)(图3)。我们发现韧皮部脱落且木质部暴露根中SDE1的基因表达也高于叶片中的表达(图3)。In the early stage, phloem and xylem exposed roots>non-phloem shed and xylem exposed roots>leaf Huanglongbing bacteria content (per gram of plant tissue cells) (Figure 3). We found that the gene expression of SDE1 in roots with phloem shedding and exposed xylem was also higher than that in leaves (Figure 3).
同时，在黄龙病感染中后期，黄龙病细菌CLas含量的结果如下-韧皮部脱落且木质部暴露根>严重症状叶片>非韧皮部脱落且木质部暴露根>中度症状叶片(图4)。At the same time, in the middle and late stages of Huanglongbing infection, the results of the CLas content of Huanglongbing bacteria are as follows-phloem shedding and xylem exposed roots> severe symptoms leaves> non-phloem shedding and xylem exposed roots> moderate symptoms leaves (Figure 4).
这些结果表明，在整个感染期间，韧皮部脱落且木质部暴露的黄龙病细菌含量高于叶片，因此韧皮部脱落且木质部暴露根可以在野外进行肉眼诊断时用作黄龙病诊断的标志物。These results indicate that during the entire infection period, the phloem shed and xylem exposed huanglong disease bacteria are higher than leaves, so the phloem shed and the xylem exposed roots can be used as a marker for the diagnosis of huanglong disease in the field for visual diagnosis.
2.2.络氨铜有效抑制S.meliloti和Agrobacterium tumefaciens2.2. Copper complex effectively inhibits S. meliloti and Agrobacterium tumefaciens
因为到目前为止Ca.Las还没有有效的方法获取，我们根据报道选用两种菌(S.meliloti and Agrobacterium tumefaciens)与Ca.Las相近亲缘关系来进行抑菌实验(Stover,Stange et al.,2013,Hu,Akula et al.,2016)。我们发现络氨铜在100μg/ml到200μg/ml时就能有效地抑制S.meliloti的生长，抑菌率达到75％在200μg/ml,400μg/ml时抑菌率达到100％，而噻唑啉对于S.meliloti的抑菌率比较低(图5A&5B).络氨铜在400μg/ml时对Agrobacterium tumefaciens的抑菌率到达50％，在800μg/ml，抑菌率达到100％，相似的噻唑啉的抑菌率也很低(图5A&5B)。根据我们的结果，我们可以推测络氨铜对黄龙病也具有很大的抑制作用。噻唑啉主要破坏根结线虫，所以对HLB有弱的抗菌作用。Because there is no effective method to obtain Ca.Las so far, we selected two bacteria (S.meliloti and Agrobacterium tumefaciens) and Ca.Las according to the report to carry out antibacterial experiments (Stover, Stange et al., 2013). ,Hu,Akula et al.,2016). We have found that copper complex can effectively inhibit the growth of S.meliloti from 100μg/ml to 200μg/ml, and the inhibitory rate reaches 75%. When 200μg/ml, 400μg/ml, the inhibitory rate reaches 100%, while thiazoline The antibacterial rate for S.meliloti is relatively low (Figure 5A&5B). The inhibitory rate for Agrobacterium tumefaciens reached 50% at 400μg/ml, and the inhibitory rate reached 100% at 800μg/ml, similar to thiazoline. The bacteriostatic rate is also very low (Figure 5A&5B). Based on our results, we can speculate that copper complex also has a great inhibitory effect on Huanglong disease. Thiazoline mainly destroys root-knot nematodes, so it has a weak antibacterial effect on HLB.
2.3.FP1-GP235和FP2-GP240对络氨铜治疗的有效性探讨2.3. Discussion on the effectiveness of FP1-GP235 and FP2-GP240 on the treatment of copper ammonia
一种新颖的黄龙病控制策略是增强黄龙病细菌中的Ca.Las prophages向噬菌体的内源转化(Ding,F.,et al.,Molecular mechanisms underlying heat or tetracycline treatments for citrus HLB control.Horticulture research,2018.5(1):p.30)。为了研究络氨铜控制的柑橘黄龙病的有效性，我们将感染黄龙病的柑橘枝条浸入不同浓度的络氨铜中，分为A(水作为对照)，B(50μg/ml络氨铜)，C(100)微克/毫升络氨铜)和D(200微克/毫升络氨铜)。在络氨铜治疗后1、5和12小时以及在络氨铜治疗后1、3和5天分别收集叶片样品。我们的结果表明，用200μg/ml络氨铜治疗12h后，FP1-GP235的相对拷贝数增加了12倍(图5C)，治疗12h后，FP2-GP240的相对拷贝数增加了约25倍(图5D)。A novel Huanglong disease control strategy is to enhance the endogenous transformation of Ca.Las prophages in Huanglong disease bacteria into bacteriophages (Ding, F., et al., Molecular mechanics, underlying heat or tetracycline treatments for citrus HLB control. Horticulture research, 2018.5(1):p.30). In order to study the effectiveness of citrus yellow dragon disease controlled by copper hydrazine, we immersed citrus branches infected with yellow dragon disease in different concentrations of copper hydrazine, and divided them into A (water as a control) and B (50μg/ml copper hydrazine). C (100 micrograms/ml copper complex) and D (200 micrograms/ml copper complex). Leaf samples were collected at 1, 5, and 12 hours after the treatment with copper and 1, 3, and 5 days after the treatment with copper. Our results showed that the relative copy number of FP1-GP235 increased by 12 times after treatment with 200 μg/ml copper ammonium copper for 12 hours (Figure 5C), and after 12 hours of treatment, the relative copy number of FP2-GP240 increased by about 25 times (Figure 5C). 5D).
随着络氨铜浓度的增加，柑橘中FP1-GP235和FP2-GP240的相对拷贝数增加。同时，研究表明黄龙病细菌和根结线虫总是同时感染柑橘树，我们用噻唑啉或络氨铜单独治疗黄龙病并没有取得明显的效果，但是当两种药物联合使用时，其效果变得很明显。图6A示出了单独的噻唑啉治疗的有效性，其不能使韧皮部脱落且木质部暴露根(黄色箭头表示韧皮部脱落且木质部暴露)消失并且没有新的根生长(图6A)。图6B代表单独络氨铜治疗的有效性，少量新根生长(红色箭头表示新根)并且韧皮部脱落且木质部暴露根已显着减少(图6B)。至于两种药物(噻唑啉；络氨铜)的组合治疗，已经长出了许多新的根，并且没有韧皮部脱落且木质部暴露根(图6C)。With the increase of the concentration of copper complex, the relative copy number of FP1-GP235 and FP2-GP240 in citrus increased. At the same time, studies have shown that Huanglongbing bacteria and root-knot nematodes always infect citrus trees at the same time. We have used thiazoline or copper hydrazine alone to treat Huanglong disease without significant results, but when the two drugs are used in combination, the effect becomes It is clear. Figure 6A shows the effectiveness of thiazoline treatment alone, which failed to shed phloem and exposed xylem roots (yellow arrows indicate phloem shedding and exposed xylem) disappeared and no new roots grew (Figure 6A). Fig. 6B represents the effectiveness of copper hydrazine treatment alone, a small number of new roots grow (red arrows indicate new roots) and the phloem is shed and the exposed roots in the xylem have been significantly reduced (Fig. 6B). As for the combination treatment of the two drugs (thiazoline; copper complex), many new roots have grown without phloem shedding and xylem exposed roots (Figure 6C).
2.4.结合两种药物(噻唑啉；络氨铜)可以有效控制柑橘黄龙病2.4. Combining two drugs (thiazoline; copper complex) can effectively control citrus yellow dragon disease
为了确认这两种药物(噻唑啉；络氨铜)在治疗黄龙病中的有效性，我们进行了以下操作：对于培养室治疗。我们进行了两组实验，分为A和B，每组三棵树。A作为对照(用水治疗)，B组则用两种药物(噻唑啉；络氨铜)联合治疗。经过3个月的治疗，我们发现叶子从黄色变成绿色(图7A，7B)，并且长出了许多新的根，我们用标签纸标记了韧皮部脱落且木质部暴露的根部(图7C)。我们的结果表明，治疗30天后，叶片中的黄龙病细菌CLas含量显着降低，治疗90天后，黄龙病细菌CLas含量处于较低水平(图7D)。In order to confirm the effectiveness of these two drugs (thiazoline; copper complex) in the treatment of Huanglong disease, we performed the following operations: for the treatment of the culture room. We conducted two sets of experiments, divided into A and B, with three trees in each group. A was used as a control (treatment with water), and group B was treated with a combination of two drugs (thiazoline; copper complex). After 3 months of treatment, we found that the leaves changed from yellow to green (Figure 7A, 7B), and many new roots had grown. We used label paper to mark the roots where the phloem fell off and the xylem was exposed (Figure 7C). Our results showed that after 30 days of treatment, the CLas content of Huanglongbing bacteria in the leaves was significantly reduced, and after 90 days of treatment, the CLas content of Huanglongbing bacteria was at a lower level (Figure 7D).
在比较治疗前和治疗后的样品时，我们发现治疗后会长出许多新根和新芽(图8)。为了进一步确认该药物可以进行大规模治疗，我们进行了野外治疗。When comparing the samples before and after treatment, we found that many new roots and shoots will grow after treatment (Figure 8). In order to further confirm that the drug can be used for large-scale treatment, we conducted field treatment.
对于野外治疗：在野外试验I中，我们看到了树木在治疗前后的完整变化过程，叶子从黄色变为绿色，最终果实成熟(图9A-D)。通过比较治疗后和治疗 前之间不同感染状态下果树的产量，我们发现黄龙病严重感染的树木在治疗后产量显着提高。然而，对于轻度和中度感染的黄龙病树，其增加幅度略低(图9E)。For field treatment: In the field experiment I, we saw the complete change process of the trees before and after treatment, the leaves changed from yellow to green, and finally the fruit matured (Figure 9A-D). By comparing the yield of fruit trees in different infection states between after treatment and before treatment, we found that the yield of trees with severe Huanglong disease infection increased significantly after treatment. However, for lightly and moderately infected Huanglong diseased trees, the increase was slightly lower (Figure 9E).
这些结果表明，改善根的生长状况和降低柑橘树的黄龙病细菌titers对治疗黄龙病至关重要。These results indicate that improving root growth and reducing citrus tree yellow dragon disease bacterial titers are essential for the treatment of yellow dragon disease.
为了更好地了解黄龙病的治疗机制，我们将治疗前和治疗后的根样品寄给了北京诺和公司进行转录组实验。In order to better understand the treatment mechanism of Huanglong disease, we sent the root samples before and after treatment to Beijing Nuohe Company for transcriptome experiments.
2.5.RNA-seq数据分析2.5.RNA-seq data analysis
2.5.1差异基因表达分析2.5.1 Differential gene expression analysis
维恩图显示在B(治疗前)和A(治疗后)样品中分别鉴定出18376和18565差异基因。进一步的分析表明，柑桔树(Citrus reticulata Blanco cv。Shatang Ju)的治疗前和治疗后样品之间有17035个差异基因重叠。这意味着B(治疗后)具有1341个独特的差异基因，而A(治疗后)具有1530个独特的差异基因(图10)。进一步的分析我们发现，在B和A的比较中，有527个上调基因和1050个下调基因以及22270个基因无显着差异(图10)。定量分析了治疗前和治疗后的基因表达样品，使用2倍变化标准(p值<0.05)鉴定并分析了总共1578个显着差异表达的基因，通过热图，我们发现治疗前后的基因发生了显着变化(图11)。The Venn diagram shows that 18376 and 18565 differential genes were identified in B (before treatment) and A (after treatment) samples, respectively. Further analysis showed that there were 17,035 differential genes overlapping between samples of citrus tree (Citrus reticulata Blanco cv. Shatang Ju) before and after treatment. This means that B (after treatment) has 1341 unique differential genes, and A (after treatment) has 1530 unique differential genes (Figure 10). Further analysis we found that in the comparison of B and A, there are 527 up-regulated genes and 1050 down-regulated genes, and 22270 genes have no significant difference (Figure 10). Quantitative analysis of the gene expression samples before and after treatment, using the 2-fold change standard (p value <0.05) to identify and analyze a total of 1578 significantly differentially expressed genes, through the heat map, we found that the genes before and after treatment have occurred Significant changes (Figure 11).
2.5.2.GO和KEGG分析2.5.2. GO and KEGG analysis
GO(Gene Ontology)是一种国际标准化的蛋白质功能分类系统，是对许多蛋白质功能进行分类的重要工具。GO分析已广泛应用于预测许多生物体中蛋白质的功能。GO数据库由三种组成：分子功能(MF)，细胞成分(CC)和生物过程(BP)(Zhang,C.,et al.,Seedless mutant‘Wuzi Ougan’(Citrus suavissima Hort.ex Tanaka‘seedless’)and the wild type were compared by iTRAQ-based quantitative proteomics and integratedly analyzed with transcriptome to improve understanding of male sterility.BMC genetics,2018.19(1):p.106.)。在生物过程类别中，差异基因主要富集在“细胞过程的运动”；“亚细胞成分过程”；“基于移动过程的微管”和“基于过程的微管”。微管的作用主要是维持细胞形态，协助细胞内运输，与其他蛋白质形成颗粒中心体并参与细胞壁形成(图12)。微管运输取决于驱动蛋白，动力蛋白和ATP的可用性。结果表明，治疗后柑橘根的细胞内转运能力和细胞壁的形成均得到增强。至于细胞成分，“细胞壁”和“外部封装结构”是该类别的最 主要成分(图12)。就分子功能而言，“微管结合”；“微管蛋白结合”和“微管马达活性”是主要的子类别。这些差异基因可能参与了黄龙病的治疗。GO (Gene Ontology) is an internationally standardized protein function classification system and an important tool for classifying many protein functions. GO analysis has been widely used to predict the function of proteins in many organisms. The GO database consists of three types: molecular function (MF), cell component (CC) and biological process (BP) (Zhang, C., et al., Seedless mutant'Wuzi Ougan' (Citrus suavissima Hort.ex Tanaka'seedless' and the wild type were compared by iTRAQ-based quantitative proteomics and integratedly analyzed with transcriptome to improve understanding of male sterility. BMC genetics, 2018.19(1): p.106.). In the category of biological processes, differential genes are mainly enriched in "movement of cellular processes"; "subcellular component processes"; "microtubules based on the movement process" and "microtubules based on the process". The role of microtubules is mainly to maintain cell morphology, assist intracellular transportation, form granular centrosomes with other proteins and participate in cell wall formation (Figure 12). Microtubule transport depends on the availability of kinesin, dynein, and ATP. The results showed that the intracellular transport ability of citrus roots and the formation of cell walls were enhanced after treatment. As for cell components, "cell wall" and "external encapsulation structure" are the most important components of this category (Figure 12). In terms of molecular functions, "microtubule binding"; "tubulin binding" and "microtubule motor activity" are the main subcategories. These differential genes may be involved in the treatment of Huanglong disease.
KEGG((Kyoto Encyclopedia of Genes and Genomes))主要是一个的生物过程数据库，包含七个类别：新陈代谢，遗传信息治疗，环境信息治疗，细胞过程，生物系统，人类疾病和药物开发(Zhang,C.,et al.,Seedless mutant‘Wuzi Ougan’(Citrus suavissima Hort.ex Tanaka‘seedless’)and the wild type were compared by iTRAQ-based quantitative proteomics and integratedly analyzed with transcriptome to improve understanding of male sterility.BMC genetics,2018.19(1):p.106.)。KEGG途径包括与代谢途径相关的途径。KEGG ((Kyoto Encyclopedia of Genes and Genomes)) is mainly a biological process database, including seven categories: metabolism, genetic information therapy, environmental information therapy, cell processes, biological systems, human diseases and drug development (Zhang, C. ,et al.,Seedless mutant'Wuzi Ougan'(Citrus suavissima Hort.exTanaka'seedless') and the wild type werecompared by iTRAQ-based quantitative proteomics and integrated. (1): p.106.). The KEGG pathway includes pathways related to metabolic pathways.
我们的研究集中在差异最大的苯丙烷类生物合成，戊糖和葡萄糖醛酸酯的相互转化(图13)。Our research focuses on the most different phenylpropane biosynthesis, the interconversion of pentose and glucuronic acid esters (Figure 13).
2.5.2.GO和KEGG分析2.5.2. GO and KEGG analysis
植物激素信号转导对于研究植物抗性也很重要。为了更深入地了解三种代谢途径中差异基因，我们在治疗前和治疗后的途径中进行了差异基因变化的总结(表3)。我们发现，在治疗后的样品中，三个代谢途径的转录水平更高，而且在治疗后的戊糖和葡糖醛酸相互转化的途径中，所有基因均被上调(表3)，这意味着产生了更多的糖来加速柑橘的生长。关于植物激素信号转导的途径，结果表明细胞因子中的AHP(含组氨酸的磷酸转移蛋白)在治疗后显着下调，水杨酸中的PR1明显上调，这有助于细胞分裂和芽萌生以及诱导的植物抗性(图14，表4)。根据先前的研究，已发现苯丙烷生物合成途径的次生代谢产物和相关酶PAL，POD对植物病害具有抗性(Wang，Wang等人，2014)。因此，我们的研究集中在苯丙烷类生物合成的代谢途径上，结果表明，苯丙烷生物合成的代谢途径可以提高治疗后香豆素和scopolin中涉及的相关基因的表达水平，它们具有抗菌活性，并促进根际和根际中有益细菌的生长。同时抑制有害细菌(图15和16)。此外，类黄酮提高了植物的抗药性和对病虫害的抗性。Phytohormone signal transduction is also important for studying plant resistance. In order to gain a deeper understanding of the differential genes in the three metabolic pathways, we summarized the differential gene changes in the pathways before and after treatment (Table 3). We found that in the samples after treatment, the transcription levels of the three metabolic pathways were higher, and all genes were up-regulated in the interconversion pathway of pentose and glucuronic acid after treatment (Table 3), which means This produces more sugar to accelerate the growth of citrus. Regarding the pathway of plant hormone signal transduction, the results indicate that AHP (histidine-containing phosphotransfer protein) in cytokines is significantly down-regulated after treatment, and PR1 in salicylic acid is significantly up-regulated, which contributes to cell division and budding Germination and induced plant resistance (Figure 14, Table 4). According to previous studies, the secondary metabolites of the phenylpropane biosynthetic pathway and related enzymes PAL and POD have been found to be resistant to plant diseases (Wang, Wang et al., 2014). Therefore, our research focuses on the metabolic pathways of phenylpropane biosynthesis, and the results show that the metabolic pathways of phenylpropane biosynthesis can increase the expression level of related genes involved in coumarin and scopolin after treatment, and they have antibacterial activity. And promote the growth of beneficial bacteria in the rhizosphere and rhizosphere. At the same time inhibit harmful bacteria (Figures 15 and 16). In addition, flavonoids increase the resistance of plants to pesticides and pests.
表4.qPCR引物的寡核苷酸序列(用于确定本文中使用的差异表达基因)Table 4. Oligonucleotide sequences of qPCR primers (used to determine the differentially expressed genes used in this article)
Figure PCTCN2020076627-appb-000005
Figure PCTCN2020076627-appb-000005
Figure PCTCN2020076627-appb-000006
Figure PCTCN2020076627-appb-000006
2.5.3通过qRT-PCR验证RNA-seq2.5.3 Verification of RNA-seq by qRT-PCR
为了证实通过RNA-seq分析获得的结果，根据其差异基因，我们选取了共9 种基因(如表4，图16所示)，分别是苯丙烷类生物合成的代谢途径(cic00940)的差异基因：1个C4H基因(肉桂酸4-羟化酶)，1个PAL基因(苯丙氨酸氨裂合酶)，1个β-葡萄糖苷酶基因，1个Ciclev10015700m基因(阿魏酰辅酶A-6羟化酶)，1个POD基因(Ciclev10017908m)；戊糖和葡萄糖醛酸酯的相互转化(cic00040)的差异基因：1个果胶酶基因(Ciclev10004719m)，1个PGA酶基因；植物激素信号转导(cic04075)的差异基因：1个PR1基因(与病程相关-1)和1个AHP基因(含组氨酸的磷酸转移蛋白)；这些基因的表达水平基本上在qPCR和RNA-seq数据之间显示出相似的趋势，表明RNA-seq数据是可靠的(图16)。In order to confirm the results obtained by RNA-seq analysis, based on their differential genes, we selected a total of 9 genes (as shown in Table 4, Figure 16), which are the differential genes of the metabolic pathway of phenylpropane biosynthesis (cic00940) : 1 C4H gene (cinnamic acid 4-hydroxylase), 1 PAL gene (phenylalanine ammonia lyase), 1 β-glucosidase gene, 1 Ciclev10015700m gene (ferulyl-CoA- 6-hydroxylase), 1 POD gene (Ciclev10017908m); differential genes for the mutual conversion of pentose and glucuronate (cic00040): 1 pectinase gene (Ciclev10004719m), 1 PGA enzyme gene; plant hormone signal Differential genes transduced (cic04075): 1 PR1 gene (related to disease course-1) and 1 AHP gene (phosphotransfer protein containing histidine); the expression levels of these genes are basically based on qPCR and RNA-seq data They showed similar trends, indicating that the RNA-seq data is reliable (Figure 16).
2.6总结2.6 Summary
我们分别验证了使用络氨铜、噻唑啉单独治疗，以及络氨铜和噻唑啉联合治疗的有效性。我们的结果表明，单独使用络氨铜治疗后，少量新根得以生长。结合这两种药物，治疗后可以生长更多的新根，这可能是两种药物(噻唑啉和络氨铜)结合起来治疗柑橘类黄龙病的原因。We have verified the effectiveness of the use of copper sulphate, thiazoline alone, and combination treatment of sulphate copper and thiazoline. Our results show that a small amount of new roots can grow after the treatment with copper sulphate alone. Combining these two drugs can grow more new roots after treatment. This may be the reason for the combination of the two drugs (thiazoline and copper hydrazine) to treat citrus yellow dragon disease.
然后为了探索噻唑啉和络氨铜的治疗机制，我们还进行了转录组实验。根据实验结果，总结治疗的机制是噻唑啉和络氨铜消除了部分根结线虫抑制了黄龙病细菌的生长，一些新的根吸收了更多的养分和水分，这有助于产生更多的抗性基因和次生代谢产物，从而改善了周围土壤的微生物环境。最终，它形成了一个良性循环，以消除残留的根结线虫和黄龙病细菌(图17)。Then, in order to explore the therapeutic mechanism of thiazoline and copper ammonia, we also conducted transcriptome experiments. According to the experimental results, the mechanism of the treatment is summarized as thiazoline and copper complex eliminate part of root knot nematodes and inhibit the growth of Huanglong disease bacteria. Some new roots absorb more nutrients and water, which helps to produce more Resistance genes and secondary metabolites, thereby improving the microbial environment of the surrounding soil. In the end, it formed a virtuous circle to eliminate the remaining root knot nematodes and yellow dragon disease bacteria (Figure 17).
本发明为野外研究黄龙病提供了重要的方向，即韧皮部脱落且木质部暴露根是诊断黄龙病的标志物，特别是对于早期诊断。我们的结果表明，将噻唑啉和络氨铜两种药物联合使用可以有效控制柑橘类黄龙病，这对于治疗黄龙病和了解黄龙病的机理奠定了基础和重要意义。The invention provides an important direction for field research on Huanglong disease, that is, phloem shedding and xylem exposed roots are markers for diagnosing Huanglong disease, especially for early diagnosis. Our results show that the combined use of thiazoline and copper hydrazine can effectively control citrus huanglong disease, which lays the foundation and important significance for the treatment of huanglong disease and the understanding of the mechanism of huanglong disease.
另外经噻唑啉与络氨铜的使用浓度对黄龙病控制效果的影响实验显示，50-90％噻唑啉稀释1000-3000倍，结合100-600μg/ml络氨铜的施用，均可较好的控制柑橘黄龙病。施药方法为先在根部土壤喷施噻唑啉，2-5天后用络氨铜浇灌根部1-3次，每次浇灌时间相隔30天左右。In addition, the effect of the concentration of thiazoline and copper complex on the control effect of Huanglong disease has shown that 50-90% thiazoline diluted 1000-3000 times, combined with the application of 100-600μg/ml complex copper, can be better Control citrus yellow dragon disease. The spraying method is to spray thiazoline on the root soil first, and then irrigate the roots with copper ammonia 1-3 times after 2-5 days, and the interval of each irrigation is about 30 days.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (10)

  1. 噻唑啉与络氨铜联用在防治柑橘类黄龙病中的应用。Application of thiazoline and copper complex in the prevention and treatment of citrus yellow dragon disease.
  2. 噻唑啉与络氨铜联用在制备柑橘类黄龙病防治药物中的应用。Application of thiazoline and copper complex in the preparation of drugs for preventing and curing citrus yellow dragon disease.
  3. 根据权利要求1或2所述应用，其特征在于，噻唑啉与络氨铜的使用浓度分别为：50-90％噻唑啉稀释1000-3000倍，100-600μg/ml络氨铜。The application according to claim 1 or 2, characterized in that the concentration of thiazoline and copper complex are respectively: 50-90% thiazoline diluted 1000-3000 times, 100-600 μg/ml copper complex.
  4. 根据权利要求3所述应用，其特征在于，噻唑啉与络氨铜的使用浓度分别为：60-80％噻唑啉稀释1500-2500倍，200-600μg/ml络氨铜。The application according to claim 3, characterized in that the use concentrations of thiazoline and copper complex are: 60-80% thiazoline diluted 1500-2500 times, 200-600 μg/ml copper complex.
  5. 根据权利要求4所述应用，其特征在于，噻唑啉与络氨铜的使用浓度分别为：75％噻唑啉稀释2000倍，200μg/ml络氨铜。The application according to claim 4, characterized in that the concentration of thiazoline and copper complex are respectively: 75% thiazoline diluted 2000 times, 200 μg/ml complex copper.
  6. 一种可以有效治疗柑橘类黄龙病的药物，其特征在于，含有噻唑啉与络氨铜。A medicine capable of effectively treating citrus yellow dragon disease, which is characterized in that it contains thiazoline and copper complex.
  7. 根据权利要求6所述药物，其特征在于，噻唑啉和络氨铜的用量比按照如下标准计：50-90％噻唑啉经稀释1000-3000倍：100-600μg/ml络氨铜。The medicament according to claim 6, characterized in that the dosage ratio of thiazoline and copper complex is calculated according to the following standard: 50-90% thiazoline diluted 1000-3000 times: 100-600 μg/ml copper complex.
  8. 根据权利要求7所述药物，其特征在于，噻唑啉和络氨铜的用量比按照如下标准计：60-80％噻唑啉经稀释1500-2500倍：200-600μg/ml络氨铜。The medicament according to claim 7, wherein the dosage ratio of thiazoline and copper complex is calculated according to the following standard: 60-80% thiazoline diluted 1500-2500 times: 200-600 μg/ml copper complex.
  9. 一种可以有效治疗柑橘类黄龙病的方法，其特征在于，利用权利要求6-8任一所述药物进行防治。A method that can effectively treat citrus yellow dragon disease, which is characterized in that the medicine described in any one of claims 6-8 is used for prevention and treatment.
  10. 根据权利要求9所述方法，其特征在于，先用噻唑啉喷施根部土壤，2-5天后用络氨铜浇灌根部。The method according to claim 9, characterized in that the root soil is sprayed with thiazoline first, and the roots are irrigated with copper complex after 2-5 days.
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