CN115044643A - Evaluation method for camellia disease resistance - Google Patents
Evaluation method for camellia disease resistance Download PDFInfo
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Abstract
The invention provides an evaluation method of camellia rot disease resistance, belonging to the technical field of plant disease evaluation, and the evaluation method comprises the following steps: inoculating calyx shaped strain of Camellia japonica to the wound of petal of Camellia japonica, and culturing; measuring the lesion area and the total petal area after inoculation for 72h to obtain a relative area ratio; and dividing disease grades according to the obtained relative area ratio and giving range values of all grades. And calculating the disease index according to the grade assignment and the occurrence number. And evaluating the disease resistance of camellia rot according to the obtained disease index. The method provided by the invention can be used for obtaining the resistance level of the camellia to be identified to the flower rot for 72h, and the evaluation period is shortened.
Description
Technical Field
The invention belongs to the technical field of plant disease evaluation, and particularly relates to a camellia rot disease resistance evaluation method.
Background
Camellia blossom blight is a fungal disease caused by camellia leaf california (Ciborinia camellia), the pathogenic bacteria mainly damage flowers of camellia, initial symptoms are expressed as brown spots, and then the pathogenic bacteria spread to wither whole flowers, so that the maturing rate is reduced, and the fertility, ornamental value and economic value of camellia are seriously influenced. The flower infected by the camellia calyx can still keep the shape and the hardness after falling off, and sclerotium can be generated at the base of the petal at the later stage. The sclerotium is in a dormant state in summer and autumn, the sclerotium germinates along with the rise of the air temperature in spring to form sexual generation structures such as an ascospore disc and an ascospore, and the ascospore is emitted from the ascospore disc after being mature and is spread to healthy flowers by wind power to carry out a new round of infection. The disease is discovered in Japan as early as 1919, and is discovered in the United states, New Zealand and European partial areas successively later, and China has sporadic reports. In view of the severity of its hazards, and the prevalence of its spread and spread, goblet camellia has been listed in 2003 by the European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest category a2, and in 2013 as a national entry Plant quarantine pest list.
At present, the main methods for identifying the resistance of camellia blossom blight include an ascospore suspension inoculation method and an artificial simulated air spore inoculation method. The ascospore inoculation method can simulate the natural infection process to a certain extent: the ascospores are ejected from the ascospores to be attached to the petals for infection. The inoculation quantitative effect of the ascospore suspension is good, but in the evaluation process, the ascospore is collected indoors after the ascospore plate is collected in spring attack seasons generally, so that the collection time is long, the operation is complicated, and the induction rate of the ascospore obtained by artificially inducing sclerotium is low.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for evaluating disease resistance of camellia blossom blight, and by using the method provided by the present invention, the flower rot resistance level of camellia to be identified can be known within 72 hours.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a camellia rot disease resistance evaluation method, which comprises the following steps:
1) inoculating calyx shaped strain of Camellia japonica to the wound of petal of Camellia japonica, and culturing;
2) measuring the lesion area and the total petal area after inoculation for 72h to obtain a relative area ratio;
the relative area ratio is the percentage of the lesion spot area to the total area of the petals;
3) and dividing disease grades according to the obtained relative area ratio and giving range values of all grades. Calculating disease indexes according to the assignment and the occurrence number of each grade; evaluating the disease resistance of camellia rot according to the obtained disease index;
the disease grade and range value are as follows:
no disease spot at grade 0;
the percentage of the 1-grade lesion area in the total area of the petals is less than 15 percent;
the percentage of the 3-grade lesion area in the total area of the petals is more than or equal to 15 percent and less than or equal to 25 percent;
the area of the 5-grade scab accounts for more than 25 percent and less than or equal to 40 percent of the total area of the petals;
the percentage of the 7-grade lesion area in the total area of the petals is more than 40 percent and less than or equal to 75 percent;
the percentage of the 9-grade lesion area in the total area of the petals is more than 75 percent.
Disease grades 0, 1, 3, 5, 7 and 9, with values of 0, 1, 3, 5, 7 and 9, respectively;
the calculation formula of the disease index is as follows:
when the disease index is 0, the resistance level of camellia rot is judged as immunity;
when the disease index is more than 0 and less than or equal to 5, the resistance level of the camellia rot is judged as high resistance;
when the disease index is more than 5 and less than or equal to 10, the resistance level of the camellia rot is judged to be resistant;
when the disease index is more than 10 and less than or equal to 25, the resistance level of the camellia rot is judged to be neutral;
when the disease index is more than 25, the resistance level of camellia rot is judged as high-feeling.
Preferably, the california camellia sinensis in the step 1) is inoculated in the form of a california camellia sinensis block, and the diameter of the california camellia sinensis block is 5 mm.
Preferably, the preparation method of the calix camellia sinensis bacterial block comprises the following steps: inoculating California camellia leaf to PDA solid culture medium, and culturing at 20 deg.C under 12h illumination and 12h dark culture for 15 d.
Preferably, the central part of the camellia petal is pricked by using a sterile insect needle to obtain wounds of the camellia petal, wherein the total number of the wounds is 5-6.
Preferably, the conditions for the culture in step 1) include: the temperature is 20 ℃, the humidity is 85%, the illumination intensity is 4000Lx, and the light cycle is 12h illumination and 12h darkness.
Preferably, the lesion area and total petal area are measured using Image J software.
Preferably, the camellia comprises 'luojis', yun county camellia and monomeric camellia.
The invention has the beneficial effects that:
(1) resistance identification is carried out by adopting indoor in-vitro petals, so that a large amount of test materials are easily obtained;
(2) the hypha blocks of the camellia california are used as an inoculum, hyphae can grow and culture on a PDA culture medium, a large number of hypha blocks are easy to obtain for pathogenic test, and the problems of large collection of ascospores in the field and large difficulty in artificial induction when the ascospores of the camellia california are used as the inoculum in the traditional resistance identification are solved;
(3) the method has short identification period, and the resistance level of the material to be identified can be obtained 72 hours after inoculation;
(4) the inoculum is easy to store, and the hypha of the camellia leaf cupule can be stored for about 5 months in a refrigerator at 4 ℃ and can be stored for a long time in a refrigerator at-80 ℃;
(5) the test conditions have strong controllability: the resistance identification work is carried out in an artificial climate box, the artificial climate box can accurately control the temperature, the humidity, the illumination intensity and the photoperiod in the resistance identification process, and the controllability of test conditions is strong;
(6) the method has the advantages that the area of the lesion spots and the area of the petals are measured by adopting Image processing software (Image J), an operator does not need to contact the petals which are infected with diseases when using the method, the method is clean and sanitary, the operator does not need to calculate the area manually, and the measuring precision and the efficiency are high.
Drawings
FIG. 1 is a brown lesion on 96h 'Rogers' petal after inoculation of California camellia leaves;
FIG. 2 is the spot observation at different time points after the petals of 2 kinds of camellia are inoculated in vitro, the upper half part is camellia you county, and the lower half part is monomer camellia;
FIG. 3 shows the relative lesion area ratio distribution of 56 camellia rot diseases.
Detailed Description
The invention provides a camellia rot disease resistance evaluation method, which comprises the following steps:
1) inoculating calyx shaped strain of Camellia japonica to the wound of petal of Camellia japonica, and culturing;
2) measuring the lesion area and the total petal area after inoculation for 72h to obtain a relative area ratio;
the relative area is the percentage of the lesion spot area in the total area of the petals;
3) dividing disease grades according to the obtained relative area ratio, giving range values of all grades, and calculating disease indexes according to assignment and occurrence quantity of all grades; evaluating the disease resistance of camellia rot according to the obtained disease index;
the disease grade and range value are as follows:
no disease spot at grade 0;
the percentage of the 1-grade lesion area in the total area of the petals is less than 15 percent;
the percentage of the 3-grade lesion area in the total area of the petals is more than or equal to 15% and less than or equal to 25%;
the percentage of the area of the 5-grade scab in the total area of the petals is more than 25 percent and less than or equal to 40 percent;
the percentage of the 7-grade lesion area in the total area of the petals is more than 40% and less than or equal to 75%;
the area of the 9-grade scab accounts for more than 75 percent of the total area of the petals;
grades 0, 1, 3, 5, 7 and 9 are assigned values of 0, 1, 3, 5, 7 and 9, respectively;
the calculation formula of the disease index is as follows:
when the disease index is 0, the resistance level of camellia rot is judgment immunity;
when the disease index is more than 0 and less than or equal to 5, the resistance level of the camellia rot is judged as high resistance;
when the disease index is more than 5 and less than or equal to 10, the resistance level of the camellia rot is judged to be resistant;
when the disease index is more than 10 and less than or equal to 25, the resistance level of the camellia rot is judged to be neutral;
when the disease index is more than 25, the resistance level of camellia rot is judged as high-feeling.
The camellia japonica calix is inoculated to the wound of the in vitro petal of the camellia japonica and cultured. The source of the California camellia sinensis is not particularly limited, conventional strains are adopted, and in the specific embodiment of the invention, the California camellia sinensis with the number of CBS101527, which is commercially available from the microbial culture Collection (CBS) of Utremula under the Netherlands, is preferably adopted. In the invention, the california camellia sinensis is preferably inoculated in the form of a california camellia sinensis block, and the diameter of the california camellia sinensis block is preferably 5 mm. In the present invention, the preparation method of the calix camellia sinensis bacterial block preferably comprises: inoculating California camellia sinensis to PDA solid culture medium, culturing at 20 deg.C under 12h illumination and 12h dark culture for 15d, and punching out the California camellia sinensis block with diameter of 5mm with a puncher. In the present invention, the relative humidity of the culture is preferably 85%, and the light intensity is preferably 4000 Lx. The method preferably uses sterile insects to prick the central part of the camellia petals to obtain wounds of the camellia petals, and the total number of the wounds is 5-6. In the present invention, the conditions for the culture preferably include: the temperature is 20 ℃, the humidity is 85%, the illumination intensity is 4000Lx, and the light cycle is 12h illumination and 12h darkness. In the invention, the inoculated camellia petals are preferably placed in a culture dish of 3 pieces of moist sterile filter paper, and the culture dish is placed in an artificial climate box for culture. In the present invention, the camellia preferably includes 'lujesk', yuxian camellia and monomeric camellia.
Measuring the lesion area and the total petal area after inoculation for 72 hours to obtain a relative area ratio; the relative area is the percentage of the lesion spot area in the total area of the petals. The invention preferably uses Image J software to measure lesion area and total petal area.
Dividing disease grades according to the obtained relative area ratio and giving range values of all grades; calculating disease indexes according to the assignment and occurrence number of each grade and the disease grading standard, and evaluating the disease resistance of camellia rot according to the obtained disease indexes;
the disease grade and range value are as follows:
no disease spot at grade 0; the percentage of the 1-grade lesion area in the total area of the petals is less than 15 percent; the percentage of the 3-grade lesion area in the total area of the petals is more than or equal to 15% and less than or equal to 25%; the area percentage of the 5-grade lesion spots accounts for more than 25 percent and less than or equal to 40 percent of the total area percentage of the petals; the percentage of the 7-grade lesion area in the total area of the petals is more than 40% and less than or equal to 75%; the percentage of the 9-grade lesion area in the total area of the petals is more than 75 percent.
Disease grades 0, 1, 3, 5, 7 and 9, with values of 0, 1, 3, 5, 7 and 9, respectively;
the calculation formula of the disease index is as follows:
when the disease index is 0, the resistance level of camellia rot is judgment immunity;
when the disease index is more than 0 and less than or equal to 5, the resistance level of the camellia rot is judged as high resistance;
when the disease index is more than 5 and less than or equal to 10, the resistance level of camellia rot is judged to be resistant;
when the disease index is more than 10 and less than or equal to 25, the resistance level of the camellia rot is judged to be neutral;
when the disease index is more than 25, the resistance level of camellia rot is judged as high-feeling.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A camellia rot disease resistance evaluation method comprises the following steps:
(1) selection of the inoculation material: petals of a flower which is just opened by using a non-damaged and healthy camellia germplasm material are used as inoculation materials, 30 petals are taken from each germplasm material, and the germplasm materials are equally divided into 3 parts to be used for 3 times of repetition.
(2) Preparation of a culture medium: 200 g of potato, 18 g of glucose and 18 g of agar, and adding distilled water to reach the constant volume of 1 liter. Cleaning 200 g of fresh potatoes, peeling, dicing, putting into a pot, adding 1L of distilled water, boiling for 15-20 minutes, filtering with four layers of gauze to obtain filtrate, adding distilled water to a constant volume of 1L, adding 18 g of agar, cooking with slow fire while stirring until the agar is completely melted. 18 g of glucose was added and stirred until the glucose was completely dissolved. Pouring into a conical flask, sealing, and sterilizing at 121 deg.C under high temperature and high pressure.
(3) Preparation of inoculum: california camellia (strain number: CBS101527) was inoculated on PDA solid medium, cultured for 15 days at 20 ℃ under 12 hours of light and 12 hours of dark conditions, and then a hypha block was punched out from the edge of the PDA plate with a punch having a diameter of 5mm as an inoculum.
(4) Inoculation: prick 5 ~ 6 apertures with aseptic inoculation needle at petal central authorities, with the inoculum inoculation to petal central authorities, the petal after the inoculation is arranged in the culture dish that contains 3 moist aseptic filter paper, and the culture dish is arranged in the artificial climate case and is cultivateed, and the setting of artificial climate case is: the temperature is 20 ℃, the humidity is 85%, the illumination intensity is 4000Lx, and the light cycle is 12D: 12L.
(5) And (3) resistance evaluation: 72h after inoculation of 56 germplasm, each was repeated 3 times. Photographing by a camera, measuring the area of the disease spots and the total area of petals by adopting Image J software, calculating the area ratio of the disease spots, grading the disease grades according to the area ratio of the disease spots, dividing the camellia rot disease grades into 6 grades and giving range values of the grades (figure 1). Disease grades 0, 1, 3, 5, 7 and 9 were assigned with values of 0, 1, 3, 5, 7 and 9, respectively, and disease indices were calculated according to the following formula. And establishing a resistance judgment standard according to the disease index, and judging the disease resistance of the camellia germplasm resources.
TABLE 1 disease grading and Range values for Camellia flower rot
TABLE 2 Camellia rot resistance level grading Table
| Rank of | Level of resistance | Index range of disease condition |
| 1 | Immunization (I) | Disease index of 0 |
| 2 | High Resistance (HR) | 0<Disease index is less than or equal to 5 |
| 3 | Moderate (MR) | 5<Disease index is less than or equal to 10 |
| 4 | Middle Sense (MS) | 10<Disease index is less than or equal to 25 |
| 5 | High Sensitivity (HS) | 25<Index of disease condition |
Example 2
By adopting the evaluation method of the embodiment 1, red Camellia variety 'Rogers' (Camellia japonica 'Bea Rogers') is selected to determine the pathogenicity of the hypha block of the Camellia japonica Chaaiji, scabs are generated 96h after 10 petals are inoculated, the incidence rate of the scabs reaches 100%, the relative area percentage of the scabs is 20% -99%, and the hypha of the Camellia japonica Chaaiji has pathogenicity (figure 2).
Example 3
Using the evaluation method of example 1, 0h, 24h, 48h, 72h and 96h after inoculation of Camellia yuhsiennsis (C.yuhsienensis) and Camellia sinensis (C.uraku) as a monomer were observed (FIG. 3). No scab is observed in 0-96 h. No disease spots are observed in the monomeric red camellia after 0-24 h of inoculation. At 48h, the scab begins to obviously expand at and near the center of the petal inoculation circle, and the relative area ratio is 13.23% on average. And at 72h after inoculation, the disease spots further expand to occupy nearly half of the petals, the relative area ratio is 50.82%, and at 96h, the disease spots of the monomer camellia japonica extend to nearly half of the petals, even the whole petals, and the relative area ratio is nearly 60%. The relative area ratio of the lesion spots of different germplasms reaches obvious difference 72 hours after inoculation. Combining the above study results, a suitable time point for resistance evaluation 72h after inoculation was determined.
Relative area ratio of disease spots of different time points after in vitro inoculation of petals of table 32 camellia
| | |
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48h | 72h | 96h | ||
| You Xian tea | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | ||
| Monomer red camellia | 0.00±0.00 | 0.00±0.00 | 13.23±2.89 | 50.82±21.43 | 58.64±22.45 |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. The camellia rot disease resistance evaluation method is characterized by comprising the following steps of:
1) inoculating calyx shaped strain of Camellia japonica to the wound of petal of Camellia japonica, and culturing;
2) measuring the lesion area and the total petal area after inoculation for 72h to obtain a relative area ratio;
the relative area ratio is the percentage of the lesion spot area to the total area of the petals;
3) dividing disease grades according to the obtained relative area ratio, giving range values of all grades, and calculating disease indexes according to value assignment and occurrence quantity of all grades; evaluating the disease resistance of camellia rot according to the obtained disease index;
the disease grade and range value are as follows:
no disease spot at grade 0;
the percentage of the 1-grade lesion area in the total area of the petals is less than 15 percent;
the percentage of the 3-grade lesion area in the total area of the petals is more than or equal to 15 percent and less than or equal to 25 percent;
the area of the 5-grade scab accounts for more than 25 percent and less than or equal to 40 percent of the total area of the petals;
the percentage of the 7-grade lesion area in the total area of the petals is more than 40 percent and less than or equal to 75 percent;
the percentage of the 9-grade lesion spot area in the total area of the petals is more than 75 percent;
grades 0, 1, 3, 5, 7 and 9 are assigned values of 0, 1, 3, 5, 7 and 9, respectively;
the calculation formula of the disease index is as follows:
when the disease index is 0, the resistance level of camellia rot is judged as immunity;
when the disease index is more than 0 and less than or equal to 5, the resistance level of the camellia rot is judged as high resistance;
when the disease index is more than 5 and less than or equal to 10, the resistance level of the camellia rot is judged to be resistant;
when the disease index is more than 10 and less than or equal to 25, the resistance level of the camellia rot is judged to be neutral;
when the disease index is more than 25, the resistance level of camellia rot is judged as high-feeling.
2. The evaluation method according to claim 1, wherein the calix camellia sinensis of step 1) is inoculated in the form of a pellet of calix camellia sinensis, the pellet of calix camellia sinensis having a diameter of 5 mm.
3. The evaluation method according to claim 2, wherein the production method of the goblet-camellia-fungus block comprises: inoculating California camellia leaf to PDA solid culture medium, and culturing at 20 deg.C under 12h illumination and 12h dark culture for 15 d.
4. The evaluation method according to claim 1, wherein wounds of the petals of the camellia are obtained by pricking the central part of the petals of the camellia in vitro with a sterile insect needle, and the total number of the wounds is 5-6.
5. The evaluation method according to claim 1, wherein the conditions for the culture of step 1) include: the temperature is 20 ℃, the humidity is 85%, the illumination intensity is 4000Lx, and the light cycle is 12h illumination and 12h darkness.
6. The evaluation method according to claim 1, wherein the lesion area and the total petal area are measured using Image J software.
7. The evaluation method according to claim 1, wherein the camellia comprises 'lujesk', yuxian camellia and monomeric camellia.
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| CN105191682A (en) * | 2015-10-27 | 2015-12-30 | 河南省农业科学院植物保护研究所 | Disease resistance identification method for phoma arachidicola |
| CN107090487A (en) * | 2017-03-30 | 2017-08-25 | 云南省农业科学院生物技术与种质资源研究所 | A kind of method for identifying buckwheat black spot resistance |
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