CN112725225A - Test tube culture method for pathogenic bacteria of citrus huanglongbing - Google Patents
Test tube culture method for pathogenic bacteria of citrus huanglongbing Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a test tube culture method of pathogenic bacteria of citrus huanglongbing. The culture method comprises the following steps: the method for culturing pathogenic bacteria of citrus greening disease is characterized by comprising the following steps: (1) preparing a citrus yellow dragon virus source; (2) disinfecting the surface of the poison source material; (3) preparing a test tube stock and grafting and inoculating; (4) carrying out proliferation culture; the proliferation culture comprises the following steps: preparing an MS liquid culture medium, adding carbendazim into the culture medium, subpackaging the culture medium in test tubes with filter paper bridges, and sterilizing; then inoculating yellow dragon disease pathogenic bacteria by adopting a test tube grafting method, cutting axillary buds of the disinfected poison source material, and grafting the axillary buds on citrus test tube stocks; then placing the grafted seedlings into a filter paper bridge; culturing for 14-21 days in a dark room at 24-28 ℃, and culturing for 12-16h in a photoperiod when the sprouts of the test tube grafted germ grow to 0.5-1.0 cm. The method of the invention has good reproducibility; can make the pathogenic bacteria of citrus greening disease proliferate to high concentration level in a short time.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a test tube culture method of pathogenic bacteria of citrus huanglongbing.
Background
Citrus Huanglongbing (HLB) is a devastating disease in Citrus production caused by infection with bacteria of the genus phloem (Candidatus Liberobacter sp), is currently spreading rapidly in Citrus producing areas worldwide, and has become one of the key factors that restrict the health development of the Citrus industry ("research progress on Citrus Huanglongbing pathogen culture and molecular detection technology", dawn soldier et al, guangdong agricultural science, 23 rd in 2013, lines 1-2 in summary 65, 31 th in 2013, 31 th in Citrus Huanglongbing pathogen research progress ", summons to picnic, etc., plant protection, 36 rd in 2010, 3 rd in 3 th, line 1-2 in left column 30, and 31 th in 2010, 12 th in 2010.
Currently, citrus huanglongbing pathogen is considered to be a gram-negative bacterium of alpha-proteobacteria (Alphaproteobacteria) which is a phloem-restricted parasite, and its citrus huanglongbing pathogen can be classified into asian species (canas Liberibacter asiaticus, CLas), african species (Candidatus Liberibacter africanus, claaf) and american species (Candidatus Liberibacter americanus, CLam)3 species ("research progress on culture and molecular detection technology", songrong, et al, guangdong agrscience, 23 of 2013, 2 nd paragraph 1-8 of column 65, 12 nd 31 of 2013), according to the sequence characteristics of the pathogen 16S rDNA and the sequence characteristics of the beta operon gene, as well as the propagation medium and pathogenic heat sensitivity, etc. Wherein, the CLas is distributed most widely in the global scope, the loss caused to the citrus industry is the largest, and the pathogenic bacteria of the citrus huanglongbing in China are also the species.
For a long time, researchers have been struggling to explore culture methods for citrus greening disease pathogens. However, the existing method for culturing the citrus Huanglongbing pathogenic bacteria has poor reproducibility, and the pathogenic bacteria cannot be rapidly proliferated to a high concentration level.
Disclosure of Invention
In view of the above, the present invention aims to provide a test tube culture method for pathogenic bacteria of citrus huanglongbing.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the method for culturing pathogenic bacteria of citrus greening disease comprises the following steps: (1) preparing a citrus yellow dragon virus source; (2) disinfecting the surface of the poison source material; (3) preparing a test tube stock and grafting and inoculating; (4) carrying out proliferation culture; the proliferation culture comprises the following steps: preparing an MS liquid culture medium, adding carbendazim into the culture medium, subpackaging the culture medium in test tubes with filter paper bridges, and sterilizing; then inoculating yellow dragon disease pathogenic bacteria by adopting a test tube grafting method, cutting axillary buds of the disinfected poison source material, and grafting the axillary buds on citrus test tube stocks; then placing the grafted seedlings into a filter paper bridge; culturing for 14-21 days in a dark room at 24-28 ℃, and culturing for 12-16h in a photoperiod when the sprouts of the test tube grafted germ grow to 0.5-1.0 cm.
In the invention, the pathogenic bacteria of the yellow dragon disease are Asian species.
In the invention, during the cultivation process in a dark room at 24-28 ℃, if grafted buds do not sprout after two weeks, sterile absorbent cotton is dipped in GA with the concentration of 100-200ppm3Coating bud eyes and erasing the sprouts on the rootstock.
Further, the carbendazim is used in an amount such that the final concentration is 0.15-0.3 wt%.
Further, the preparation of the citrus yellow dragon virus source comprises the following steps:
picking branches from a field plant infected with yellow dragon disease, selecting branches with partial leaf symptoms, no plant diseases and insect pests on the surface and moderate thickness, reserving 2-3 leaves on the branches, and directly using the branches with yellow dragon disease detection as positive as a virus source for test tube inoculation of pathogenic bacteria (CLas).
Further, the preparation of the citrus yellow dragon virus source comprises the following steps:
preparing citrus seedlings preserved in a greenhouse for about 1 year in advance, taking branches from field citrus plants suffering from yellow shoot, grafting double-bud branches onto the seedlings, and continuously preserving in the greenhouse for management according to a conventional citrus seedling method; and (3) detecting the pathogenic bacteria (CLas) of the citrus seedlings with transmitted toxicity about 3 months after grafting and inoculating the pathogenic bacteria of the yellow dragon disease, and taking plant branches with positive detection as test-tube plantlets to inoculate the virus source.
Further, the disease source surface disinfection treatment comprises the following steps:
taking branches from citrus plants suffering from yellow dragon disease, subtracting leaves and thorns, carefully wiping the branches by dipping cotton in detergent stock solution, washing with tap water, repeatedly wiping with detergent for 1 time and washing cleanly; rinsing with sterile water on a superclean bench for 3-5 times, soaking with alcohol solution, and rinsing with sterile water; then using HgCl2Soaking in the solution; and then washing with sterile water for 8-10 times for later use.
Further, the concentration of the alcohol solution is 75%, and the soaking time of the alcohol solution is 60s in percentage by volume.
Further, the HgCl2The concentration of the solution was 0.1 wt%, HgCl2The soaking time of the solution is 8-10 min.
Further, the test-tube rootstock preparation comprises the following steps:
taking out citrus seeds, cleaning with clear water, sterilizing, and washing with sterile water; peeling off the testa, and inoculating in sterilized MS solid culture medium; and then culturing for 2-3 weeks under the dark condition at the temperature of 26-28 ℃ to obtain the test-tube rootstock.
Further, the sterilization of the citrus seeds comprises the following steps: after surface sterilization with alcohol, the surface was sterilized with 0.1 wt% mercuric chloride or 4 wt% sodium dichloroisocyanurate for 10 minutes.
The invention has the beneficial effects that:
the method of the invention has good reproducibility.
The pathogenic bacteria of the huanglongbing cultured by the method can be rapidly proliferated.
The method can obtain the citrus test-tube plant which has no fungal pollution and carries the yellow dragon germ
The method can accelerate the screening of the citrus greening disease treatment medicament, can also be used for rapidly screening key nutritional factors of the greening disease, and is also beneficial to the research of the pathogenesis of the citrus greening disease.
Drawings
FIG. 1 shows a source of Huanglong virus inoculated with citrus tube plantlets obtained in example 1;
FIG. 2 shows the citrus test-tube plantlet rootstock obtained in example 1;
FIG. 3 shows a test tube plantlet of Citrus obtained in example 1, which has been grafted with a shoot of Pholiota citrea;
FIG. 4 shows a test tube plantlet of citrus obtained in example 1 and inoculated with Pholiota squarrosa 14 d;
FIG. 5 shows the results of PCR identification of 30d tube plantlets with germ sprouts in example 2;
FIG. 6 shows test-tube plantlets of Mandarin orange successfully inoculated with Huanglongbing in example 2;
FIG. 7 shows the result of PCR identification of citrus test-tube plantlets with germ sprouts 30d in example 3;
FIG. 8 shows test tube plantlets of Mandarin orange successfully inoculated with Huanglongbing in example 3;
FIG. 9 shows the result of PCR identification of citrus test-tube plantlets with germ sprouts 30d in example 3;
FIG. 10 shows test tube plantlets of Citrus in example 3, which were successfully inoculated with Xanthomonas campestris.
Detailed Description
The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
The PCR identification method of the following huanglongbing comprises the following steps: extracting DNA with kit (purchased from BioFlux), taking 0.1-0.15g of sample, and placing into a 1.5mL centrifuge tube; adding liquid nitrogen for grinding, adding DNA extracting solution for mixing uniformly, carrying out water bath at 65 ℃ for 20 minutes, adding 3M potassium acetate with the volume of 1/3, carrying out ice water bath for 10 minutes, centrifuging at 10000G, taking supernatant, adding isopropanol, passing through a column, washing with 75% alcohol, and eluting DNA with TE solution, wherein the specific method refers to the operation instruction water of the kit. Extracting sample DNA; PCR amplification is carried out by using detection primers OI1(5 '-GCGCGTATGCAATACGAGCGGCA-3') and OI2c (5 '-GCCTCGCGACTTCGCAACCCAT-3') (Jagoueix et al, 1994) commonly used for Asian species of Huanglongbing pathogen, and a sample with the Asian species of Huanglongbing pathogen is amplified to obtain a specific DNA fragment with the length of 1167 bp; detecting a PCR product by adopting 1.5% agarose gel electrophoresis, adding 10 mu L of 0.5mg/mL Ethidium Bromide (EB) into 100mL of gel, taking a photograph by adopting a UVP gel imaging system, wherein the electrophoresis buffer solution is 1 xTBE, and the voltage is 120-160 v for 30 minutes;
the following specific steps of the CLas PCR detection are as follows: weighing 0.1-0.15g of veins or young shoots (less than 0.1g of tender shoots supplemented with citrus test-tube stocks to 0.1g) germinated in the test-tube seedlings, and putting the tender shoots into a 1.5mL centrifuge tube; adding liquid nitrogen, grinding into powder, extracting total DNA of the sample by using a plant genome DNA extraction kit (purchased from BioFlux), and referring to the operation instructions of the kit; the PCR reaction system was 25. mu.L containing 1 XPCR Buffer (TIANGEN), 150. mu.M dNTP, 0.8U Taq enzyme, 50ng DNA, 0.4. mu.M primers, respectively; amplification was performed on a Biometra Thermocycler PCR instrument with the following amplification procedure: 5min at 95 ℃; 30s at 94 ℃, 40s at 61 ℃, 1min at 72 ℃ and 38 cycles; finally, extending for 10min at 72 ℃; detecting the PCR product by 1.5% agarose Gel electrophoresis, staining by Ethidium Bromide (EB), imaging in a UVP Gel imaging system (Gel Doc-It) and recording the result;
the specific steps of detecting the concentration of the pathogenic bacteria of the huanglongbing by the following real-time fluorescent quantitative PCR (qPCR) method are as follows: extracting sample DNA (the method is the same as PCR detection), amplifying a specific sequence of the 16S rDNA of the yellow dragon germ by utilizing qPCR, and simultaneously amplifying by taking the citrus 18S gene as an internal reference gene; collecting the CT value of the amplification of the 16S rDNA and the reference gene of the yellow dragon pathogen in the sample, and calculating the concentration of the yellow dragon pathogen in the sample by adopting a formula (Zou et al, 2017) reported by the previous people, wherein the formula is as follows:
Clas cells·μg-1 citrus DNA＝[10(-0.2718×Ct16S+10.624)/10(-0749×Ct18S+4.0531)]×103(12.7<Ct16S<32.0and 8.4<Ct18S<26.5)。
referring to a method of repairing and leveling in middle yellow Dragon disease (MDR) of a wrinkle et al (2017), the CLas 16S gene detection primers are qcla16S-f (5 '-TGAGTGCTAGCTGTTGGGTG-3') and qcla16S-r (5 '-CTGCGCGTTGCATCGAATTA-3'); the citrus 18S gene detection primers are Ct18S-f (5 '-AATTGTTGGTCTTCAACGAGGAA-3') and Ct18S-r (5 '-AAAGGGCAGGGACGTAGTCAA-3'). Each amplification contained 1 positive control and 1 negative control; 3 replicates per sample. The qPCR reaction used a 20. mu.L system containing 1 × iTaqTM Universal SYBR Green Supermix (BIO-RAD), 0.2. mu.M each primer, 50ng DNA. The amplification reaction was performed on a 7500 Fast Real-Time PCR System (AB Applied Biosystem) using the following amplification program: 5min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles. The melting curve analysis program was: 30s at 95 ℃; 1min at 60 ℃; the temperature is kept for 15s every 0.5 ℃ rise from 60 ℃, and the rise is continuously carried out for 70 times (till the temperature reaches 95 ℃).
The data collected by the real-time fluorescence quantitative PCR are analyzed by using excel 2007 software, and the concentration of the xanthomonas in the sample is calculated by referring to the method of Zou et al (2017), wherein the formula is as follows: clas cells. mu.g-1 citrus DNA＝[10(-0.2718×Ct16S+10.624)/10(-0749×Ct18S+4.0531)]×103(12.7<Ct16S<32.0 and 8.4<Ct18S<26.5)。
Example 1
Culturing the pathogenic bacteria of citrus greening disease in 21 days 6 months in 2020, comprising the following steps:
(1) preparing a citrus huanglongbing source of disease;
taking out seeds from mature citrus (sweet orange) fruits, cleaning and sterilizing, sowing in nutrient soil of a seedbed in a greenhouse, transplanting the seeds into a nutrition pot when citrus seedlings grow to the height of 20cm, and culturing in the greenhouse;
inoculating yellow dragon germs by adopting grafting and virus transmission, taking branches from citrus plants infected with yellow dragon diseases observed in a yellow dragon disease area field as a virus source, cutting bud segments from the branches identified as positive by PCR, grafting the branches onto citrus seedlings by adopting a belly grafting mode, continuously placing the seedlings in a greenhouse for culturing, and managing according to a conventional citrus seedling culture method;
regularly observing the disease condition of the plants, detecting the virus-carrying citrus seedlings by using a PCR technology about 3 months after grafting and inoculating the CLas, and taking the branches of the plants with the PCR detection as positive plants as the pathogeny of yellow dragon inoculated by the citrus test-tube plantlet (as shown in figure 1).
(2) Surface disinfection treatment of Huanglongbing bacteria source
Taking branches from citrus seedlings with yellow dragon disease stored in a greenhouse, removing leaves and thorns, carefully wiping the branches by dipping cotton in detergent stock solution, and washing the branches clean with tap water; rinsing with sterile water on an ultra-clean bench for 3 times, soaking with 75% (by volume) alcohol for 60s, and rinsing with sterile water for 3 times; then 0.1 wt% HgCl was used2Soaking the solution for 10min while stirring; washing with sterile water for 10 times, and drying water drops on the branches with sterilized filter paper; and (3) putting 5 pieces of sterilized filter paper into a sterile culture dish, soaking the sterilized filter paper by using sterile water, and putting the branches subjected to surface sterilization treatment into the sterile culture dish for later use.
(3) Test tube rootstock preparation
Taking out citrus seeds from mature citrus (sweet orange) fruits, cleaning the seeds with tap water, sterilizing the surfaces of the seeds with alcohol on a superclean bench, sterilizing the seeds with 0.1 wt% of mercuric chloride (or 4 wt% of sodium dichloroisocyanurate) for 10 minutes, and washing the seeds with sterile water for 4 times; peeling off the testa, and inoculating in sterilized MS solid culture medium; the tube stocks were obtained by dark culture at 28 ℃ for 2 weeks (as shown in FIG. 2).
The MS basic culture medium comprises the following components: (1) macroelements: NH (NH)4NO3 1650mg/L、KNO3 1900mg/L、CaCl2·2H2O 440mg/L、MgSO4·7H2O 370mg/L、KH2PO41700 mg/L; (2) trace elements: KI 0.83mg/L, H3BO36.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、Na2MnO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、FeSO4·7H2O(27.8mg/L)+Na2-EDTA·2H2O (37.3 mg/L); (3) organic components: inositol 100mg/L, nicotinic acid 0.5mg/L, pyridoxine hydrochloride (vitamin B)6)0.5mg/L thiamine hydrochloride (vitamin B)1)0.5mg/L and 2mg/L of glycine.
The MS solid culture medium is MS basic culture medium added with 8g/L of agar and 30g/L of sucrose, and the pH value is 5.8.
(4) Yellow dragon germ inoculation and culture of citrus test-tube plantlet
Preparing a filter paper bridge, preparing an MS liquid culture medium (the MS liquid culture medium is not added with agar, and other components are the same as those of an MS solid culture medium), adding carbendazim (the dosage is 0.3 wt% of the final concentration) into the culture medium, adjusting the pH value to 5.8, subpackaging the obtained product into test tubes with the filter paper bridge, and sterilizing the obtained product at high temperature and high pressure for later use; inoculating yellow dragon germs to citrus test-tube plantlets by adopting a test-tube grafting method, cutting axillary buds of the yellow dragon virus source subjected to surface disinfection treatment on a superclean bench by using a grafting knife, grafting the axillary buds to citrus test-tube stocks, and putting the citrus test-tube stocks into a filter paper bridge filled with an MS liquid culture medium (as shown in figure 3); culturing at 28 deg.C in the dark for 14d, and culturing under 16 hr photoperiod when the test tube grafted sprout with germ is 1.0cm long (as shown in FIG. 4).
Example 2
Molecular detection of citrus test-tube plantlet yellow dragon bacteria
After inoculating the test-tube plantlet with the germ (obtained in example 1) to germinate for 10 days, 15 days, 20 days, 25 days, 30 days and 40 days, cutting the germinated young shoot leaves of the test-tube plantlet to extract DNA, and detecting the infection condition of the test-tube plantlet with the yellow dragon germ by utilizing PCR (polymerase chain reaction) and real-time fluorescent quantitative PCR (qPCR) technologies.
The specific steps of the CLas PCR detection are as follows: weighing 0.15g of veins or young tips of test-tube seedlings which sprout (less than 0.15g of young tips are complemented by citrus test-tube stocks), and putting into a 1.5mL Eppendorf tube; liquid nitrogen was added and ground into powder, and total DNA of the sample was extracted using a plant genomic DNA extraction kit (BioFlux) according to the instructions of the kit. The PCR reaction was carried out in 25. mu.L containing 1 XPCR Buffer (TIANGEN), 150. mu.M dNTP, 0.8U Taq enzyme, 50ng DNA, and 0.4. mu.M primers, respectively. Amplification was performed on a Biometra Thermocycler PCR instrument with the following amplification procedure: 5min at 95 ℃; 30s at 94 ℃, 40s at 61 ℃, 1min at 72 ℃ and 38 cycles; finally, extension is carried out for 10min at 72 ℃. The PCR products were detected by electrophoresis on a 1.5% agarose Gel, stained with Ethidium Bromide (EB), imaged on a UVP Gel imaging system (Gel Doc-It) and the results recorded.
Referring to a method of Zhousheping et al (2017), the detection primers of the CLES 16S gene are qcla16S-f (5 '-TGAGTGCTAGCTGTTGGGTG-3') and qcla16S-r (5 '-CTGCGCGTTGCATCGAATTA-3'); the citrus 18S gene detection primers are Ct18S-f (5 '-AATTGTTGGTCTTCAACGAGGAA-3') and Ct18S-r (5 '-AAAGGGCAGGGACGTAGTCAA-3'). Each amplification contained 1 positive control and 1 negative control; 3 replicates per sample. The qPCR reaction used a 20. mu.L system containing 1 × iTaqTM Universal SYBR Green Supermix (BIO-RAD), 0.2. mu.M each primer, 50ng DNA. The amplification reaction was performed on a 7500 Fast Real-Time PCR System (AB Applied Biosystem) using the following amplification program: 5min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles. The melting curve analysis program was: 30s at 95 ℃; 1min at 60 ℃; the temperature is kept for 15s every 0.5 ℃ rise from 60 ℃, and the rise is continuously carried out for 70 times (till the temperature reaches 95 ℃).
The data (CT value) collected by real-time fluorescence quantitative PCR is analyzed by using excel 2007 software, and the concentration of the xanthomonas in the sample is calculated by referring to the method of Zou et al (2017), wherein the formula is as follows: clas cells. mu.g-1 citrus DNA＝[10(-0.2718×Ct16S+10.624)/10(-0749×Ct18S+4.0531)]×103(12.7<Ct16S<32.0 and 8.4<Ct18S<26.5)
The PCR result shows that the yellow dragon germ 16S gene can be amplified to obtain a corresponding target zone (as shown in figure 5), and the yellow dragon germ is determined to be a positive test-tube plantlet.
The real-time fluorescent quantitative PCR result shows that 55% of Huanglongbing PCR positive test-tube plantlets can be obtained when the citrus is germinated for 30 days after test-tube inoculation (as shown in FIG. 6). The concentrations of liberobacter asiaticum in citrus test-tube plantlets at 10, 15, 20, 25, 30 and 40 days after germination are shown in table 1.
TABLE 1 concentration of Sclerotinia chrysosporium in Citrus test-tube plantlet at different time points
| Post germination incubation time (d) | CLas concentration (Cell. mu.g)-1DNA) |
| 10day | 3.2×104±1.2×104c |
| 15day | 7.8×105±7.5×104c |
| 20day | 2.7×106±3.2×106bc |
| 25day | 5.0×107±8.3×106b |
| 30day | 1.4×108±1.6×107a |
| 40day | 1.7×108±1.1×107a |
As is clear from Table 1, the concentration of the causative bacteria of sallow's disease in the test-tube plantlet was increased with the lapse of the culture time.
Counting to 30 days of germinationThe concentration of the yellow dragon bacteria in 30 PCR positive test-tube seedlings is 107Cell·μg- 1Above DNA.
Statistics shows that the concentration of 21 PCR positive test-tube plantlets exceeds 10 when the seedlings sprout for 30 days8Cell·μg-1There were 9 strains of DNA, and the proportion reached 42.9%.
TABLE 2 concentration of Huanglongbing in 30d germinating citrus tube plantlet
| Sample numbering | CLas concentration (Cell. mu.g)-1DNA) |
| 30-1 | 4.7×107±6.9×106 |
| 30-2 | 3.2×108±4.4×107 |
| 30-3 | 5.5×107±9.3×105 |
| 30-4 | 5.9×107±3.3×106 |
| 30-5 | 1.9×108±1.5×107 |
| 30-6 | 5.7×107±8.1×106 |
| 30-7 | 3.8×108±1.7×107 |
| 30-8 | 2.2×108±2.4×107 |
| 30-9 | 2.6×108±2.3×107 |
| 30-10 | 6.7×107±5.8×106 |
| 30-11 | 3.1×107±5.3×106 |
| 30-12 | 2.0×108±2.0×107 |
| 30-13 | 1.6×107±1.5×106 |
| 30-14 | 4.3×108±4.4×107 |
| 30-15 | 9.6×107±2.1×107 |
| 30-16 | 8.6×107±1.0×107 |
| 30-17 | 1.1×108±1.2×107 |
| 30-18 | 2.0×107±8.6×106 |
| 30-19 | 6.6×107±3.7×107 |
| 30-20 | 5.1×107±3.8×106 |
| 30-21 | 1.8×108±2.7×107 |
Therefore, the method for culturing the pathogenic bacteria of the citrus greening disease can be used for rapidly proliferating the pathogenic bacteria of the citrus greening disease. Under natural conditions, the concentration of the inoculated yellow dragon germs of the oranges reaches 107Cell·μg-1About 6 months at least.
Example 3
Performing tube culture of citrus greening disease pathogenic bacteria twice in 18 days in 2018 and 18 days in 2019 and 21 days in 6 and 21 days in 2019, wherein the specific method steps are the same as those in example 1, and the concentrations of the citrus greening disease pathogenic bacteria in the citrus tube seedlings 10, 15, 20, 25, 30 and 40 days after germination are detected according to the method in example 2, and the results are respectively shown in tables 3 and 4. The PCR detection results of the test tube plantlet yellow dragon germ cultured for 30 days are respectively shown in FIG. 7 and FIG. 9; the obtained positive test-tube plantlets detected by the xanthomonas campestris are shown in fig. 8 and fig. 10 respectively.
Table 3 concentration of Sclerotinia flavipes in Citrus test-tube plantlet at different time points (2018 in 9 months)
| Post germination incubation time (d) | CLas concentration (Cell. mu.g)-1DNA) |
| 10day | 1.5×104±4.9×103c |
| 15day | 1.2×105±7.2×104c |
| 20day | 7.2×106±3.4×106bc |
| 25day | 1.8×107±1.7×106bc |
| 30day | 3.7×107±1.2×107b |
| 40day | 7.4×107±6.4×107a |
Table 4 concentration of Sclerotinia flavipes in Citrus test-tube plantlet at different time points (6 months in 2019)
| Post germination incubation time (d) | CLas concentration (Cell. mu.g)-1DNA) |
| 10day | 2.4×104±5.0×103d |
| 15day | 3.5×105±1.7×105d |
| 20day | 2.9×106±1.9×106cd |
| 25day | 7.7×106±3.0×106bc |
| 30day | 8.9×107±4.4×106b |
| 40day | 1.4×108±6.0×107a |
Therefore, the citrus greening disease pathogenic bacteria cultured by the method can be rapidly proliferated. And the method of the invention has good reproducibility.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Description of the invention
<110> test tube culture method of pathogenic bacteria of citrus greening disease
<120> university of southwest
<160>6
<210>1
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> detection primer OI1
<400>1
gcgcgtatgc aatacgagcg gca 23
<210>2
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> detection primer OI2c
<400>2
gcctcgcgac ttcgcaaccc at 22
<210>3
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> CLas 16S Gene detection primer qcla16S-f
<400>3
<210>4
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> CLas 16S Gene detection primer qcla16S-r
<400>4
<210>5
<211>23
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<213> Artificial sequence
<220>
<223> the citrus 18S gene detection primer is Ct18S-f
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aattgttggt cttcaacgag gaa 23
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<213> Artificial sequence
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<223> the citrus 18S gene detection primer is Ct18S-r
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Claims (9)
1. The method for culturing pathogenic bacteria of citrus greening disease is characterized by comprising the following steps: (1) preparing a citrus yellow dragon virus source; (2) disinfecting the surface of the poison source material; (3) preparing a test tube stock and grafting and inoculating; (4) carrying out proliferation culture; the proliferation culture comprises the following steps: preparing an MS liquid culture medium, adding carbendazim into the culture medium, subpackaging the culture medium in test tubes with filter paper bridges, and sterilizing; then inoculating yellow dragon disease pathogenic bacteria by adopting a test tube grafting method, cutting axillary buds of the disinfected poison source material, and grafting the axillary buds on citrus test tube stocks; then placing the grafted seedlings into a filter paper bridge; culturing for 14-21 days in a dark room at 24-28 ℃, and culturing for 12-16h in a photoperiod when the sprouts of the test tube grafted germ grow to 0.5-1.0 cm.
2. The culture method according to claim 1, wherein the carbendazim is used in such an amount that the final concentration thereof is 0.15 to 0.3 wt%.
3. The cultivation process according to claim 1 or 2, wherein the preparation of the citrus yellow shoot virus source comprises the steps of:
picking branches from a field plant infected with yellow dragon disease, selecting branches with partial leaf symptoms and no surface damage by plant diseases and insect pests, reserving 2-3 leaves on the branches, and directly using the branches with yellow dragon disease detection as positive as a virus source for test tube inoculation of pathogenic bacteria (CLas).
4. The cultivation process according to claim 1 or 2, wherein the preparation of the citrus yellow shoot virus source comprises the steps of:
preparing citrus seedlings preserved in a greenhouse for about 1 year in advance, taking branches from field citrus plants suffering from yellow shoot, grafting double-bud branches onto the seedlings, and continuously preserving in the greenhouse for management according to a conventional citrus seedling method; and (3) detecting the pathogenic bacteria (CLas) of the citrus seedlings with transmitted toxicity about 3 months after grafting and inoculating the pathogenic bacteria of the yellow dragon disease, and taking plant branches with positive detection as test-tube plantlets to inoculate the virus source.
5. The cultivation method according to any one of claims 1 to 4, wherein the surface sterilization treatment of the source material comprises the steps of:
taking branches from citrus plants suffering from yellow dragon disease, removing leaves and thorns, carefully wiping the branches by dipping cotton in detergent stock solution, and washing the branches clean with tap water; rinsing with sterile water on a superclean bench, soaking with alcohol solution, and rinsing with sterile water; then soaking the mixture by using HgCl2 solution; then the mixture is washed by sterile water for standby.
6. The culture method according to claim 5, wherein the concentration of the alcohol solution is 75% and the soaking time of the alcohol solution is 60s in percentage by volume.
7. The culture method according to claim 5 or 6, wherein the HgCl2 solution has a concentration of 0.1 wt% and the HgCl2 solution has a soaking time of 8-10 min.
8. The cultivation method according to any one of claims 1 to 7, wherein the tube stock preparation comprises the following steps:
taking out citrus seeds, cleaning with clear water, sterilizing, and washing with sterile water; peeling off the testa, and inoculating in sterilized MS solid culture medium; and then culturing for 2-3 weeks under the dark condition at the temperature of 26-28 ℃ to obtain the test-tube rootstock.
9. The cultivation process according to claim 8, wherein the sterilization of the citrus seeds comprises the following steps: after surface sterilization with alcohol, the surface was sterilized with 0.1 wt% mercuric chloride or 4 wt% sodium dichloroisocyanurate for 10 minutes.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114303670A (en) * | 2022-01-17 | 2022-04-12 | 湖南农业大学 | Rapid inoculation method for citrus yellow shoot |
| CN114717335A (en) * | 2022-05-10 | 2022-07-08 | 赣南师范大学 | Method for obtaining CLas infected diaphorina citri polypide body |
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| CN103891531A (en) * | 2013-12-12 | 2014-07-02 | 广西大学 | Method for cultivating citrus germplasm infected with citrus vein phloem degeneration |
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| CN103891531A (en) * | 2013-12-12 | 2014-07-02 | 广西大学 | Method for cultivating citrus germplasm infected with citrus vein phloem degeneration |
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| MARK E. HILF等: "Transmission and Propagation of ‘Candidatus Liberibacter asiaticus"by Grafting with Individual Citrus Leaves", 《PHYTOPATHOLOGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114303670A (en) * | 2022-01-17 | 2022-04-12 | 湖南农业大学 | Rapid inoculation method for citrus yellow shoot |
| CN114717335A (en) * | 2022-05-10 | 2022-07-08 | 赣南师范大学 | Method for obtaining CLas infected diaphorina citri polypide body |
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