Test tube culture method of citrus huanglongbing pathogenic bacteria

A technology for citrus Huanglongbing and cultivation methods, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of poor reproducibility and rapid proliferation of pathogenic bacteria, and achieve the effect of accelerated screening

Active Publication Date: 2021-04-30
SOUTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using existing methods to cultivate the pathogenic bacteria of citrus Huanglongbing, the reproducibility is poor, and the pathogenic bacteria cannot rapidly proliferate to a high concentration level

Method used

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  • Test tube culture method of citrus huanglongbing pathogenic bacteria
  • Test tube culture method of citrus huanglongbing pathogenic bacteria
  • Test tube culture method of citrus huanglongbing pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] On June 21, 2020, the pathogenic bacteria of citrus Huanglongbing was cultivated. The specific steps are as follows:

[0046] (1) Citrus Huanglong disease source preparation;

[0047] Take out the seeds from the mature citrus (sweet orange) fruit, sow them in the nutrient soil of the seedbed in the greenhouse after cleaning and disinfection, and transplant them into the nutrient pot when the citrus seedlings grow to a height of 20 cm, and put them in the greenhouse to cultivate;

[0048] Grafting was used to inoculate Huanglongbing bacteria, and branches were taken from citrus plants infected with Huanglongbing in the field of Huanglongbing disease area as the source of the virus. Buds were cut from the branches identified as positive by PCR, and grafted to citrus fruit by abdominal grafting. On the seedlings, continue to place the seedlings in the greenhouse for cultivation, and manage them according to the conventional citrus seedling raising method;

[0049] Regula...

Embodiment 2

[0059] Molecular detection of Huanglongbing bacteria in citrus test-tube plantlets

[0060] Respectively after inoculating Huanglongbing bacteria test-tube seedlings with bacterial buds (obtained in Example 1) to germinate for 10, 15, 20, 25, 30 and 40d, cut the young shoot leaves of test-tube seedlings to extract DNA, and use PCR and real-time fluorescence quantitative PCR (qPCR ) technology to detect the infection of Huanglongbing bacteria in test-tube seedlings.

[0061] The specific steps of CLas PCR detection: Weigh 0.15g of leaf veins or germinated young shoots of test tube seedlings (if less than 0.15g, make up with citrus test tube rootstock), put them into 1.5mL Eppendorf tube; add liquid nitrogen to grind into powder, and use plant genome The DNA extraction kit (BioFlux) was used to extract the total DNA of the sample, and the method was referred to the operating instructions of the kit. The PCR reaction system is 25μL, which contains 1×PCR Buffer (TIANGEN), 150μM d...

Embodiment 3

[0075] On September 18, 2018 and June 21, 2019, two test tube cultures of citrus Huanglongbing pathogenic bacteria were carried out successively. The specific method and steps were the same as in Example 1, and according to the method of Example 2, 10, 15, and 20 days after germination were detected. , 25, 30 and 40d the concentration of Huanglongbing bacteria in the citrus test-tube plantlets, the results are shown in Table 3 and Table 4 respectively. The results of PCR detection of Huanglongbing bacteria in test-tube seedlings cultured twice for 30 days are as follows: Figure 7 and Figure 9 Shown; The Huanglongbing bacterium that obtains detects as positive test-tube seedling respectively as Figure 8 and Figure 10 shown.

[0076] Table 3 Concentration of Huanglongbing bacteria in citrus test-tube plantlets at different time points (September 2018)

[0077] Culture time after germination (d) CLas concentration (Cell·μg -1 DNA)

[0078] Table 4 Concentrat...

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Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a test tube culture method of citrus huanglongbing pathogenic bacteria. The culture method comprises a citrus huanglongbing pathogen culture method, and is characterized by comprising the following steps: (1) preparing a citrus huanglongbing virus source; (2) disinfecting the surface of the poison source material; (3) test tube stock preparation and grafting inoculation; (4) multiplication culture; the multiplication culture comprises the following steps: preparing an MS liquid culture medium, adding carbendazim into the culture medium, sub-packaging into test tubes with filter paper bridges, and sterilizing; inoculating Huanglongbing pathogenic bacteria by adopting a test tube grafting method, cutting off axillary buds of the disinfected virus source material, and grafting the axillary buds on the citrus test tube rootstock; putting the grafted seedlings into a filter paper bridge; culturing in a darkroom at 24-28 DEG C for 14-21 days, and transferring to a photoperiod of 12-16 hours for culturing when the sprouts with the bacterial buds of the test tube grafting grow to 0.5-1.0 cm. The method disclosed by the invention is good in reproducibility; the citrus huanglongbing pathogenic bacteria can be proliferated to a high-concentration level in a short time.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a test tube culture method for citrus huanglongbing pathogenic bacteria. Background technique [0002] Citrus Huanglongbing (HLB) is a devastating disease in citrus production caused by Candidatus Liberobacter sp. infection. It is currently spreading rapidly in citrus producing areas around the world, and has become a factor restricting the healthy development of citrus industry. One of the key factors ("Research progress on pathogenic culture and molecular detection technology of citrus huanglongbing", Song Xiaobing et al., Guangdong Agricultural Science, 2013 No. 23, page 65, line 1-2 of the abstract, published on December 31, 2013 ; "Progress in Research on the Pathogen of Citrus Huanglongbing", Hu Wenzhao et al., Plant Protection, 2010, Vol. 36, No. 3, Page 30, left column, paragraph 1, line 1-2, published on December 31, 2010). [0003] At present, the pathogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01G2/30C12R1/01
CPCC12N1/20A01G2/30
Inventor 雷天刚陈善春何永睿周常勇彭爱红许兰珍邹修平姚利晓李强龙琴
Owner SOUTHWEST UNIV
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