CN114303670A - Rapid inoculation method for citrus yellow shoot - Google Patents
Rapid inoculation method for citrus yellow shoot Download PDFInfo
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- CN114303670A CN114303670A CN202210049665.6A CN202210049665A CN114303670A CN 114303670 A CN114303670 A CN 114303670A CN 202210049665 A CN202210049665 A CN 202210049665A CN 114303670 A CN114303670 A CN 114303670A
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Abstract
The invention discloses a rapid inoculation method for citrus huanglongbing, which comprises the following steps: (1) accelerating germination of citrus seeds; (2) sowing citrus seeds; (3) collecting branches with yellow dragon disease; (4) grafting a yellow dragon disease branch with a leaf stalk to citrus seedlings; (5) culturing and managing after citrus seedlings are inoculated; (6) regularly detecting the content of the yellow dragon germs of the citrus seedlings and observing symptoms. Aiming at the defects and shortcomings of long incubation period and unobvious symptoms of citrus huanglongbing inoculation at present, the invention shortens the time for detecting and stabilizing the appearance of symptoms after inoculation by grafting the huanglongbing branches on citrus seedlings, and innovates the huanglongbing inoculation mode. Simple and easy to implement, low in cost and space-saving.
Description
Technical Field
The invention relates to an inoculation method for citrus huanglongbing, in particular to a method for quickly inoculating and expressing symptoms of citrus huanglongbing.
Background
Citrus Huanglongbing (Citrus huanganglingbin) is a disease caused by the gram-negative bacterium, bacillus subtilis, c.las, and is not currently cultured ex vivo, and no disease-resistant germplasm has been found, mainly transmitted by grafting and diaphorinacri. Meanwhile, the pathogenic bacteria of the huanglongbing disease are unevenly distributed in the plant body, the incubation period is long, and the symptoms are various, so that the research progress is slow.
Therefore, shortening the inoculation time of pathogenic bacteria of the huanglongbing disease and stabilizing the time of expressing symptoms are effective ways for improving the research efficiency of the huanglongbing disease, and the research of researching the interaction of the pathogenic bacteria and the host, breeding disease-resistant germplasm and the like in the early stage can be realized.
Disclosure of Invention
The invention aims to overcome the defects of long inoculation time and unstable properties of pathogenic bacteria of citrus greening disease in the prior art, and provides a simple, efficient, rapid, visual and reliable-result rapid inoculation and identification method for citrus greening disease.
In order to solve the technical problems, the invention provides the following technical scheme:
the technical scheme adopted by the invention for solving the technical problems is as follows: a rapid inoculation method for citrus yellow shoot comprises the following steps:
(1) accelerating germination of citrus seeds: selecting full seeds of citrus varieties sensitive to yellow dragon disease, such as pomelos, ponkan oranges, sweet oranges and the like, and accelerating germination in a high-temperature and high-humidity environment;
(2) and (3) citrus seed sowing: selecting plump seeds grown from the germs in the step (1), and sowing the seeds in nutrient soil to obtain robust seedlings;
(3) collecting Huanglongbing branches: collecting yellow dragon disease aged branches stored in a field or a greenhouse, and taking leaves to detect the content of pathogenic bacteria of the yellow dragon disease;
(4) grafting a yellow dragon branch with a leaf stalk to citrus seedlings: grafting stem sections with leaf stalks of the branches with the yellow dragon diseases obtained in the step (3) to the citrus seedlings obtained in the step (2), simultaneously grafting healthy branches to the same batch of seedlings and the citrus seedlings which are not subjected to grafting treatment to serve as a contrast, and attaching and fixing scions and stocks by using a sealing film;
(5) culturing and managing after citrus seedling inoculation: transferring the grafted seedlings obtained in the step (4) into a constant-temperature constant-humidity culture chamber, keeping sufficient illumination and moisture, and simultaneously replenishing a water-soluble compound fertilizer into nutrient soil every week;
(6) regularly detecting the content of yellow dragon germs of citrus seedlings and observing symptoms: and (4) sampling the tender leaves of the citrus seedlings inoculated with the yellow shoot disease in the step (4) at 20, 30, 40, 50, 70, 90, 180 and 240dpi respectively to detect the content of yellow shoot pathogenic bacteria, and closely paying attention to the symptom change of the leaves.
Further, in the step (1), the product seed disinfection process is that 0.1mol/L NaOH is used for pectin removal treatment for 5min, distilled water is used for washing for 3-4 times, warm water heat treatment is carried out for 10min at 52 ℃, episperm is peeled off, 75% ETOH is used for disinfection for 30s, then the product seed is immediately placed into 1% NaClO with the pH value of 7 for disinfection for 10-15 min, and distilled water is used for washing for 3-4 times;
further, in the step (2), the seed germination accelerating condition is 25-30 ℃, and dark culture is carried out at the humidity of 60-80%.
Further, in the step (3), selecting seeds which germinate after germination in sowing, wherein the seed culture condition is that the seeds grow for 7-10 days in a constant-temperature constant-humidity culture room with the temperature of 25-28 ℃ and the humidity of 60-80%.
Further, in the step (4), the content of pathogenic bacteria of the huanglongbing branches is detected by extracting leaf DNA by a CTAB method, performing PCR amplification by using an A2/J5 primer, and performing qPCR detection on the content of pathogenic bacteria of the huanglongbing branches by using an HLBr/HLBas primer, cutting small-segment branches with leaf stalks and buds during grafting, inserting the small-segment branches on seedling stems of pomelos, ponkan oranges, sweet oranges and the like, and keeping the tender leaves of the stocks complete.
Further, in the step (5), the culture conditions of the grafted seedlings are that the temperature is 25-28 ℃ and the humidity is 60-80%.
Further, in the step (6), the detection primer is HLBr/HLBas, and qPCR tracking detection is carried out to detect the content change of the yellow dragon germ.
The invention has the following beneficial effects:
the inoculation method of the invention has the advantages and positive effects that: the method can be used for quickly and efficiently inoculating the citrus seedlings with the huanglongbing, and the seedlings under a constant-temperature culture room are inoculated with the huanglongbing pathogenic bacteria, so that the environment and the growth are proper, the influence of environmental factors can be reduced, and the stable morbidity is ensured; the required occupied space is small, the operability is strong, the test device is suitable for testing large sample capacity, and the working strength is obviously reduced; after inoculation, plants can be observed for a long time, stable and typical citrus yellow shoot resistant/tolerant symptoms can be seen, and the method has positive significance for fast and efficient yellow shoot research. The citrus seedling material is easy to obtain, the survival rate of grafted branches is higher than 90%, the seedlings grow vigorously, after 20 days of grafting, 80% -90% of roots of grafted seedlings can detect a high amount of pathogenic bacteria of the yellow dragon disease, the yellowing symptom is obvious after 40-50 days of inoculation, the vein lines are obvious after 50-70 days of inoculation, the veins on the front side of the leaves are rough and protruded, and the typical symptom of young shoots or whole-plant mottled yellowing appears after 90 days of inoculation.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a 40-day effect chart of the sand field seedlings inoculated with the yellow dragon disease pathogenic bacteria in example 1 of the invention;
FIG. 2 is a graph showing the effect of the seedling of the sand field inoculated with the pathogen of Huanglongbing for 80 days in the example 1 of the present invention;
FIG. 3 is a graph showing the effect of inoculating Dahong sweet orange seedlings with Huanglongbing pathogenic bacteria for 100 days in the example of the present invention;
FIG. 4 is a schematic illustration of an inoculation flow scheme according to an embodiment of the present invention;
FIG. 5 shows the result of measuring the content of Huanglong germs inoculated to 40dpi Shatian pomelo leaves.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Examples
A rapid inoculation method for citrus yellow shoot comprises the following steps:
(1) accelerating germination of citrus seeds: sowing Shatian pomelos and red sweet oranges with full seeds, and carrying out dark culture and germination acceleration at the temperature of 25-30 ℃ in an environment with the humidity of 60% -80%;
(2) and (3) citrus seed sowing: sowing the seeds with the germs in nutrient soil, and culturing for 7-10 days at 25-28 ℃ in an environment with 60% -80% of humidity;
(3) collecting Huanglongbing branches: collecting branches with obvious symptoms of the huanglongbing preserved in a greenhouse, and detecting leaves as positive of the huanglongbing;
(4) grafting a yellow dragon branch with a leaf stalk to citrus seedlings: grafting stem sections with leaf stalks of the yellow dragon branches to Shatian pomelo and red sweet orange seedlings, grafting healthy branches to the same batch of seedlings and grafting-treated citrus seedlings which are not subjected to grafting treatment to serve as a control, and attaching and fixing scions and stocks by using sealing films;
(5) culturing and managing after citrus seedling inoculation: transferring the grafted seedlings into a constant-temperature constant-humidity culture room, keeping sufficient illumination and moisture, and simultaneously replenishing water-soluble compound fertilizer into nutrient soil every week;
(6) regularly detecting the content of yellow dragon germs of citrus seedlings and observing symptoms: the young leaves of the citrus seedlings inoculated with the yellow dragon disease are respectively sampled at 20, 30, 40, 50, 70, 90, 180, 240dpi and the like to detect the content of the yellow dragon disease, and the symptom change of the leaves is regularly observed. The results are shown in the following table.
The result of seedling grafting and inoculation of yellow dragon disease shows: referring to fig. 1, 70-80% of the seedlings of Shatian pomelos are detected to be positive by yellow dragon disease after being inoculated for 40 days, and part of positive seedlings begin to show abnormal veins; referring to fig. 2, typical symptoms of the leaf began to be clear when the Shatian pomelo was inoculated for 80 d. Referring to fig. 3, when the red sweet orange seedlings are grafted and inoculated with the citrus greening disease for 100 days, the typical symptoms of the citrus greening disease appear in the whole plant. Wherein HLB is the seedlings which are not subjected to grafting treatment, and HLB is the seedlings which are subjected to grafting treatment.
FIG. 5 shows the content detection of Huanglongbing germs obtained by inoculating Shatian pomelo with 40dpi leaves, and the quantitative PCR result shows that Cq value is 24, which is positive.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A rapid inoculation method for citrus yellow shoot is characterized by comprising the following steps:
(1) accelerating germination of citrus seeds: selecting full seeds of Shatian pomelo, ponkan or sweet orange, and carrying out germination accelerating treatment;
(2) and (3) citrus seed sowing: selecting plump seeds grown from the germs in the step (1), and sowing the seeds in nutrient soil to obtain robust seedlings;
(3) collecting Huanglongbing branches: collecting yellow dragon disease aged branches stored in a field or a greenhouse, and taking leaves to detect the content of pathogenic bacteria of the yellow dragon disease;
(4) grafting a yellow dragon branch with a leaf stalk to citrus seedlings: grafting stem sections with leaf stalks of the branches with the yellow dragon diseases obtained in the step (3) to the seedlings obtained in the step (2), grafting healthy branches to the same batch of seedlings and citrus seedlings which are not subjected to grafting treatment to serve as a reference, and attaching and fixing scions and stocks by using sealing films;
(5) culturing and managing after citrus seedling inoculation: transferring the grafted seedlings obtained in the step (4) into a constant-temperature constant-humidity culture chamber, keeping sufficient illumination and moisture, and simultaneously replenishing a water-soluble compound fertilizer into nutrient soil every week;
(6) regularly detecting the content of yellow dragon germs of citrus seedlings and observing symptoms: and (4) periodically sampling young leaves of the citrus seedlings inoculated with the huanglongbing in the step (4) to detect the content of the huanglongbing, and closely paying attention to the symptom change of the leaves.
2. The method for rapidly inoculating citrus greening disease according to claim 1, wherein in the step (1), the sample seeds are sterilized before germination accelerating treatment, and the sterilization process comprises the following steps: 0.1mol/L NaOH is used for pectin removal treatment for 5min, distilled water is used for washing for 3-4 times, warm water at 52 ℃ is used for heat treatment for 10min, the testa is peeled off, 75% ETOH is used for disinfection for 30s, the mixture is immediately placed into 1% NaClO with the pH value of 7 for disinfection for 10-15 min, and the mixture is washed for 3-4 times by the distilled water.
3. The method for rapidly inoculating citrus greening disease according to claim 1 or 2, wherein in the step (1), the germination accelerating treatment is performed under the conditions of 25-30 ℃ and 60-80% humidity for dark culture.
4. The method for rapidly inoculating citrus greening disease according to claim 1, wherein in the step (2), seeds which just germinate after germination are selected during sowing, and the seeds are cultured in a constant-temperature constant-humidity culture chamber with the temperature of 25-28 ℃ and the humidity of 60-80% for 7-10 days.
5. The method for rapidly inoculating citrus greening disease according to claim 1 or 4, wherein in the step (3), the content of pathogenic bacteria of the greening disease branches is detected as follows: extracting leaf DNA by a CTAB method, performing PCR amplification by using an A2/J5 primer, and performing qPCR detection on the content of the yellow dragon bacteria by using an HLBr/HLBas primer.
6. The method for rapidly inoculating citrus yellow shoot according to claim 5, wherein in the step (4), the yellow shoot aged shoot with high content of pathogenic bacteria of yellow shoot is inoculated through PCR verification, small segments of the shoot with a leaf stalk and a bud are cut during grafting, and the small segments of the shoot are grafted on the stem of the seedling, so that the tender leaves of the stock are kept intact.
7. The method for rapidly inoculating citrus greening disease according to claim 1 or 6, wherein in the step (5), the culture condition of the grafted seedlings is that the temperature is 25-28 ℃ and the humidity is 60-80%.
8. The method for rapidly inoculating citrus huanglongbing according to claim 7, wherein in the step (6), the detection primer is HLBr/HLBas, and qPCR tracking detects the content change of the huanglongbing.
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