CN108893520B - Method for identifying resistance of Chinese rose downy mildew - Google Patents
Method for identifying resistance of Chinese rose downy mildew Download PDFInfo
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Abstract
The invention discloses a method for identifying the resistance of Chinese rose downy mildew, which comprises the following steps: (1) collecting a typical downy mildew sample; (2) propagation of downy mildew pathogenic bacteria; (3) inoculating downy mildew pathogenic bacteria; (4) evaluation of disease resistance to Chinese rose downy mildew: counting the incidence and severity of the Chinese rose downy mildew, calculating disease indexes, and evaluating the disease resistance of the Chinese rose downy mildew according to the average disease indexes in the counting period; the disease resistance grading standard of Chinese rose downy mildew is as follows: immunization: average disease index is 0.00; high resistance: the average disease index is more than or equal to 0.01 and less than or equal to 5.00; disease resistance: the average disease index is more than or equal to 5.01 and less than or equal to 10.00; anti/intermediate feel: the average disease index is more than or equal to 10.01 and less than or equal to 20.00; the infection: the average disease index is more than or equal to 20.01 and less than or equal to 40.00; high feeling: the average disease index is more than or equal to 40.01. The identification method is simple to operate, and can be used for efficiently screening the Chinese rose variety with strong resistance to Chinese rose downy mildew.
Description
Technical Field
The invention relates to the technical field of plant disease resistance identification, in particular to a method for identifying the resistance of Chinese rose downy mildew.
Background
China rose, a plant of the genus Rosa of the family Rosaceae, has been cultivated in China for over 2000 years and is a cut flower with the largest volume of international market trading at present. Chinese rose downy mildew is an important disease of greenhouse Chinese roses and is also a devastating disease caused by infection with rose downy mildew. The oospore or mycelium of the pathogenic bacteria is remained in the diseased tissue for overwintering, and the disease is easy to occur and spread under the conditions of low air temperature, high relative humidity and water drops on the surface of a plant. Therefore, the diseases are serious when the greenhouse ventilation is poor, the plants are dense, the humidity is high and the nitrogen fertilizer is excessive. Open field cultivation often occurs in rainy seasons, mainly in greenhouses or greenhouses for 3-4 months and 9-10 months.
Chinese rose downy mildew mainly harms leaves, young shoots, flower stalks and flowers. At the initial stage, irregular light green stripes appear on the leaf surface, then the leaf surface is enlarged to be yellowish brown and purple, then the leaf surface is grayish brown, the edge color is darker, no obvious limit is formed between the leaf surface and healthy tissues, and finally leaf distortion and deformity are caused. When the weather is wet, a sparse grey frost-mildew layer can be seen at the diseased spots on the back of the leaves. The small leaves often turn yellow, some disease spots are purple red, the center is grey white, and the disease spots are similar to pesticide damage and chemical fertilizer burn. When young shoots and flowers are infected, the lesions are similar to those of leaves, but the lesions are slightly depressed on young shoots. In severe cases, leaves shrink and fall off, and young shoots die.
Compared with other diseases, the Chinese rose downy mildew has unobvious symptoms, but when more diseases are found, the prevention and control period is missed, the harm to Chinese rose production is large, and after the Chinese rose is infected, the cut flowers are completely harvested in the same year due to untimely treatment, so that the yield and the quality of the Chinese rose are seriously influenced, and the industrial development is hindered to a great extent.
According to the existing report, the Chinese rose downy mildew is mainly prevented and controlled by selecting a disease-resistant variety, reasonably and densely planting the Chinese rose downy mildew, avoiding high temperature and high humidity and applying a medicament; therefore, the disease resistance of different varieties of Chinese roses to downy mildew is researched, and varieties with high resistance are screened for planting, so that the occurrence of downy mildew can be reduced, and the development of the flower industry is promoted. However, at present, no method and technology for identifying the resistance to the Chinese rose downy mildew exist, and most Chinese roses planted in the field have downy mildew, so that the research on the identification technology for identifying the resistance to the downy mildew of different varieties has great significance in screening out Chinese rose varieties with strong disease resistance.
Disclosure of Invention
In view of the above, the invention provides a method for identifying the downy mildew resistance of Chinese roses, so as to screen out Chinese rose varieties with stronger downy mildew resistance.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method for identifying the resistance of Chinese rose downy mildew is characterized by comprising the following steps of:
(1) a typical downy mildew sample was taken: collecting Chinese rose leaves with downy mildew attack symptoms in a field;
(2) propagation of downy mildew pathogenic bacteria: placing the collected typical downy mildew sample in a culture chamber for moisture preservation culture to ensure that downy mildew pathogenic bacteria on the scab are propagated and grown;
(3) inoculating downy mildew pathogenic bacteria: when the back of a downy mildew diseased leaf is full of visible hypha and spores, preparing a spore suspension or shearing a disease spot on an ultra-clean workbench, and inoculating a plant, an isolated branch or an isolated leaf to be identified;
(4) evaluation of disease resistance to Chinese rose downy mildew: after inoculation, the disease condition and the disease severity of the Chinese rose downy mildew are investigated and counted, the disease index is calculated, and the disease resistance of the Chinese rose downy mildew is evaluated through the average disease index in the counting period;
the disease resistance grading standard of Chinese rose downy mildew is as follows:
immunization: average disease index is 0.00;
high resistance: the average disease index is more than or equal to 0.01 and less than or equal to 5.00;
disease resistance: the average disease index is more than or equal to 5.01 and less than or equal to 10.00;
anti/intermediate feel: the average disease index is more than or equal to 10.01 and less than or equal to 20.00;
the infection: the average disease index is more than or equal to 20.01 and less than or equal to 40.00;
high feeling: the average disease index is more than or equal to 40.01.
Preferably, the leaves in the step (1) are packaged by using a freshness protection bag, 1-2 sterile filter paper wetted by sterile water is placed in the bag, and the bag is blown to seal and take back, so that the subsequent propagation expanding culture is promoted.
Preferably, in the step (2):
on an ultra-clean workbench, the leaf back of each leaf is upwards placed in a sterile tray filled with double-layer sterile absorbent paper, the depth of the tray is 5-8 cm, and the absorbent paper is wetted by sterile water; spraying vaporous sterile water on the blades to ensure that the absorbent paper is wet, and when the tray is slightly inclined, the water cannot flow down in strands, but a small amount of water is accumulated at the edge of the tray; sealing the disc by using a sealing film, and culturing in a culture chamber; culture conditions in the culture chamber: the illumination intensity is 1800-3200lx, the light period is 12-16 hours, the dark period is 8-12 hours, the air relative humidity is 75-90%, and the culture is carried out for 5-10 days at the room temperature of 15-22 ℃.
Preferably, the propagation temperature is 15-20 ℃; the rapid propagation is finished through living body moisture-preserving culture, and the leaf expansion rate of scab hyphae reaches 32-56%.
Preferably, the spore suspension is prepared by the following method: on a clean bench, using sterile brush to dip sterile water, gently washing off spores on the back of downy mildew-infected leaves to obtain a solution with a concentration of 1 × 104-1×106Spore suspension of each/mL is kept for 2h at 4-10 ℃ for later use.
Preferably, in the step (3), the plants and the in vitro branches are inoculated by a spore suspension painting or spore suspension spraying mode; the detached leaf is inoculated by adopting a mode of scab grafting, spore suspension brushing or spore suspension liquid drop transplanting.
Preferably, the in vitro tender leaf has a concentration of 1 × 104Inoculating spore suspension of each/mL, and adopting 1 × 10 for in vitro middle and old leaves, in vitro branches and plants5And (4) inoculating one/mL spore suspension.
Preferably, the step (3) is performed by needling around the veins of the leaves to be inoculated, but without piercing the leaves. The needling can promote the downy mildew pathogenic bacteria to quickly infect the leaves.
Preferably, the incidence of the Chinese rose downy mildew is measured after inoculation, the incidence is expressed by an incidence rate D and is calculated according to the following formula:
D(％)＝(N/T)×100
in the formula: d-incidence in percent (%);
n is the number of diseased plants or diseased leaves, and the unit is plant or leaf;
t-investigating the total plant number or the total leaf number, wherein the unit is a plant or a leaf;
the severity of the disease is expressed by a disease index and is calculated according to the following formula:
ID＝[∑(N×R)/(M×T)]×100
in the formula: ID-disease index;
sigma-investigating the sum of the relative disease grade value of the disease and the product of the plant number or the leaf number;
n-the number of plants or leaves at a certain level of the disease, and the unit is the plant or leaf;
r is relative disease grade value of the disease;
m is the highest disease grade value of the disease;
t-investigating the total plant number or the total leaf number, wherein the unit is the plant or the leaf.
Preferably, the ex vivo leaf disease survey grading criteria:
grade 0-no disease spots on leaves;
grade 1-the lesion area accounts for 1-10% of the leaf area;
grade 3, the lesion area accounts for 11 to 25 percent of the leaf area;
grade 5, the lesion area accounts for 26 to 40 percent of the leaf area;
grade 7, the lesion area accounts for 41 to 60 percent of the leaf area;
grade 9, the disease spot area accounts for more than 61% of the leaf area or the diseased leaves die;
field or plant disease survey grading standard:
level 0-no disease spots on the whole normal leaf;
grade 1-diseased leaves account for 1-10% of the whole developed leaves, downy mildew is not obvious;
grade 3-diseased leaves account for 11-25% of the whole developed leaves, downy mildew is not obvious;
grade 5, the diseased leaves account for 26 to 50 percent of the whole developed leaves, and the downy mildew is obvious;
grade 7, diseased leaves account for 51-75% of the whole unfolded leaves, and the diseased leaves are partially withered;
grade 9-diseased leaves account for over 75% of the whole leaf or most of the leaf yellowing and withering.
Grading the disease condition according to the area of the disease spots and the number of the disease leaves, so that the calculation of the disease condition index is facilitated; simple calculation, scientific method and accurate result.
According to the technical scheme, compared with the prior art, the method for identifying the resistance of the Chinese rose downy mildew disclosed by the invention has the advantages that the collected downy mildew sample is rapidly propagated, the leaf expansion rate of scab hyphae is higher, pathogenic bacteria are inoculated to in-vitro leaves, branches or plants to be identified for culture, the disease condition and the disease severity of the Chinese rose downy mildew are counted, the disease index is calculated, and the disease resistance of the Chinese rose downy mildew can be evaluated through the average disease index in the counting period; the evaluation result is accurate and intuitive, and the identification method can be used for identifying the downy mildew resistance of different varieties of Chinese roses, so that resistant varieties can be effectively screened out.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1A sample of typical downy mildew was collected
According to different disease conditions in the field, branches can be collected from plants with serious disease, and the branches are inserted into a plastic bucket filled with water and brought back; the typical diseased leaves are collected from sporadic diseased plants, packaged in fresh-keeping bags and brought back by placing 1-2 sterile filter paper moistened with sterile water and blowing air and sealing.
The symptoms of downy mildew are as follows: at the early stage of disease occurrence, irregular light green stripes appear on leaf surfaces, then the leaf surfaces are enlarged to be yellowish brown and purple, then the leaf surfaces are grayish brown, the edge color is darker, no obvious limit exists between the leaf surfaces and healthy tissues, and finally leaf distortion and deformity are caused. When the weather is wet, a sparse grey frost-mildew layer can be seen at the diseased spots on the back of the leaves. The small leaves often turn yellow, some disease spots are purple red, the center is grey white, and the disease spots are similar to pesticide damage and chemical fertilizer burn. When young shoots and flowers are infected, the lesions are similar to those of leaves, but the lesions are slightly depressed on young shoots. In severe cases, leaves shrink and fall off, and young shoots die.
Example 2 propagation of downy mildew pathogen
Performing moisture preservation culture on the collected typical downy mildew sample to ensure that downy mildew pathogenic bacteria on the scab are propagated and grown for 5-10 days:
the branches are inserted in a culture room under the conditions of illumination intensity of 2000lx, photoperiod of 14 hours and dark period of 10 hours in a photoperiod, relative air humidity of 75-80% and room temperature of 15-22 ℃, the middle lower parts of the branches are wrapped by a wet preservative film for preserving moisture, the tops of the branches are exposed, and the water in the bottle is changed every day; 2/3 test branches wither leaves and dry leaves after 5-8 days, 1/3 test branches have insignificant lesion extension, and the branches gradually wither after continuously observing for 10-15 days, and downy mildew fails to propagate.
On an ultra-clean workbench, the back of a diseased leaf is upwards placed in a sterile tray filled with double layers of sterile absorbent paper, the depth of the tray is 5 cm, and the absorbent paper is wetted by sterile water; spraying vaporous sterile water on the blades to ensure that the absorbent paper is wet, and when the tray is slightly inclined, the water cannot flow down in strands, but a small amount of water is accumulated at the edge of the tray; the plate was sealed with a sealing film and placed in a culture chamber for culture. Culture conditions in the culture chamber: the illumination intensity is 3200lx, the light period is 16 hours, the dark period is 8 hours, the relative air humidity is 80-85%, the room temperature is 15-22 ℃, the scab of the leaf is successfully expanded after the cultivation for 5-10 days, and the hypha and the pathogenic spores are successfully propagated.
Further, in order to study the optimum propagation temperature, the propagation effect of the scab hyphae on the leaf culture for 7 days is tested within the range of 12-28 ℃ under the conditions of the illumination intensity of 3200lx, the photoperiod of 16 hours, the dark period of 8 hours and the relative air humidity of 80-85%; wherein the plaque hypha expansion leaf rate is 23-28% within the range of 12-14 ℃, 32-56% within the range of 15-20 ℃, 29-51% within the range of 21-22 ℃ and 4-21% within the range of 23-28 ℃; therefore, the suitable propagation temperature of the Chinese rose downy mildew is 15-20 ℃.
The ratio of the extended leaf of the scab hypha (the area of the scab after the end of the sample culture-the area of the scab at the time of sample sampling)/the area of the scab at the time of sample sampling is 100%.
Furthermore, on an ultra-clean workbench, four culture mediums (PDA, oat, rye and fresh rose leaf juice) are tried to culture downy mildew pathogenic bacteria, and the treatment method comprises direct attachment of Chinese rose downy mildew scabs, selection of mildew layers, attachment of healthy leaves, addition of scabs and attachment of whole diseased leaves. Each treatment was cultured in an incubator at 22. + -. 1 ℃ for 5 days and 10 days, and the results are shown in Table 1. The four culture mediums have no downy mildew pathogenic bacteria colony or mildew layer, downy mildew spores are not detected on the culture mediums, and only the mildew layer and the downy mildew spores are seen on the original scab, so that the propagation expanding method is simple and practical, and the propagation expanding effect is good.
TABLE 1 results of induced culture of downy mildew pathogens in four media
Example 3 Pre-inoculation test
When the backs of the downy mildew diseased leaves are full of visible hypha and spores, a super workbench is dipped with sterile water by using a sterile brush, the spores on the backs of the downy mildew diseased leaves are washed down lightly, the spore suspension obtained has high spore concentration, and the inoculation is carried out under the condition of no dilution, wherein the inoculation method is spraying.
Selecting fresh healthy Chinese rose branches with the same collection place and growth period, and spraying the undiluted spore suspension to the front and back surfaces of leaves; and then inserting a branch clear water bottle, wrapping a wet preservative film outside the middle lower part of the branch for moisturizing, putting the branch clear water bottle into a place with the illumination intensity of 1800lx, the photoperiod of 12 hours, the dark period of 12 hours and the relative air humidity of 85-90 percent, culturing at the temperature of 15-22 ℃, observing once every other day and recording the disease occurrence condition, and taking out the preservative film after moisturizing for the third day. The test was repeated 3 times, 5 shoots each time, with 20 leaves per shoot.
And (3) test results: no obvious disease symptoms are observed in 2 days after inoculation, slight disease symptoms of 3 leaves are observed in 4 days, 5 leaves are accumulated in 6 days, leaves on branches are gradually yellowed and withered in 8 days, and 7 leaves exist in the disease leaves. The results show that the spore suspension is moisturized for 72 hours after spray inoculation, and downy mildew pathogenic bacteria infect the leaves of the Chinese roses to cause disease attack.
EXAMPLE 4 spore suspension concentration test
Collecting diseased leaves of Shilin base according to examples 1 and 2, propagating, selecting diseased spots with many mildew layers, dipping sterile water with a brush, washing off the mildew layers, placing spore suspension under a microscope, observing the number with a blood counting chamber, adjusting the number to 1 × 10 with sterile water3、104、105、106、107Spore/ml or 1X 103、104、105、106Each spore/ml is put into a refrigerator at 4 ℃ and is placed for 2 hours for standby.
And taking the first 5 small leaves or the first 7 small leaves which are completely unfolded from the top of the Chinese rose 'shadow star' branch downwards at the same collection place and contain 5 or 7 leaves as the materials to be tested, and washing and airing the materials. On the superclean bench, the leaf back is up and put in the aseptic shallow tray that has filled with double-deck aseptic absorbent paper, and the shallow tray is 5 centimetres deep, and absorbent paper is moistened with aseptic water. Before inoculation, part of healthy leaves are slightly punctured for 2-4 times around veins without puncturing the leaves, and the other part of healthy leaves are not punctured. And shaking and uniformly mixing the spore suspension liquid with each concentration, sucking 20ul of the spore suspension liquid by using a liquid transfer gun, and dripping the spore suspension liquid on the acupuncture part of the leaf or the periphery of the vein of the leaf, wherein 1-2 drops of the spore suspension liquid are dripped on each leaf. After inoculation, spraying mist sterile water on the leaves to ensure that the absorbent paper is moist, preventing the water from flowing down in strands when the tray is slightly inclined, but a small amount of accumulated water is arranged at the edge of the tray, covering a sealing film to preserve moisture, culturing under the conditions that the illumination intensity is 1800lx, the light period is 16 hours, the dark period is 8 hours, the air relative humidity is 75-80 percent, the temperature is 15-20 ℃, observing the morbidity every 2-3 days after inoculation, recording the morbidity and disease index, and performing parallel tests, wherein the results are shown in Table 2.
The incidence D is calculated according to the following formula:
D(％)＝(N/T)×100
in the formula: d-incidence in percent (%);
n-number of diseased leaves, in leaves;
t-survey total leaf number, unit is leaf;
the disease index is calculated according to the following formula:
ID＝[∑(N×R)/(M×T)]×100
in the formula: ID-disease index;
e, searching the sum of the relative disease grade numerical value of the disease and the product of the leaf number of the disease;
n is the number of leaves of a certain level of disease, and the unit is leaf;
r is relative disease grade value of the disease;
m is the highest disease grade value of the disease;
t-survey total number of leaves, in leaves.
Wherein the grading standard of the in vitro leaf disease investigation is as follows:
grade 0-no disease spots on leaves;
grade 1-the lesion area accounts for 1-10% of the leaf area;
grade 3, the lesion area accounts for 11 to 25 percent of the leaf area;
grade 5, the lesion area accounts for 26 to 40 percent of the leaf area;
grade 7, the lesion area accounts for 41 to 60 percent of the leaf area;
grade 9-the lesion area accounts for more than 61% of the leaf area or the diseased leaves die.
TABLE 2 treatment of the incidence of needle-prick inoculation of the young leaves of the rose 'ghost' with different concentrations of downy mildew
The data in table 2 are the average of the results of two experiments. From the above data, the concentration of the spore suspension of downy mildew pathogenic bacteria was 1X 103、104、105、106、107Each dose of the drug can cause diseases, and the pathogenicity difference among different concentrations is obvious. Low concentration of 1X 103、104Per ml spore suspensionThe floating liquid has obvious disease, wherein 1X 104Outbreaks of spore suspensions per ml; high concentration of 1X 105、106、107The spore suspension per ml has light morbidity; the more severe the degree of onset is with time.
Further, diseased leaves from Anfengying base were collected and propagated as in examples 1 and 2 to prepare spore suspension, which was used at a concentration of 1X 103、104、105、106The 'ghost star' variety in vitro middle and old leaves are inoculated by each ml spore suspension at 8 months and 16 days, the first statistics are carried out at 8 months and 23 days, and the experimental results are shown in table 3.
TABLE 3 incidence of needle-prick inoculation of the old leaves in vitro of the Chinese rose variety
The data in Table 3 are the average of the results of two experiments, with disease onset at 4 different concentrations, 1X 105Outbreak of spore suspension per ml; the disease condition did not spread after five days of onset.
In conclusion, the inoculation concentration of the Chinese rose downy mildew pathogenic bacteria is 1X 104And 1X 105The disease is better between individuals/ml, and the tender leaves of the in vitro leaves are 1 multiplied by 104Inoculating spore suspension of each/ml, and inoculating in vitro middle and old leaves and plants with 1 × 105Inoculation with one/ml spore suspension gives good results.
Example 5 comparative testing of different vaccination methods
Preparation of spore suspension: selecting diseased leaves with good downy mildew propagation and multiple visible mildew layers on a superclean workbench, dipping sterile water with a sterile brush, washing the mildew layers, placing under a microscope, observing the number with a blood counting chamber, and adjusting the number to be 1 × 104Spores per ml, and placing in a refrigerator at 4 ℃ for 2 hours to promote spore production.
Making the scab: and (3) selecting diseased leaves with good downy mildew propagation and multiple visible mildew-selecting layers on a superclean workbench, and shearing diseased spot parts (avoiding shearing diseased spot parts in a withered yellow state) for later use.
Placing a plate: taking 48 leaves of 2 varieties of healthy leaves of 'Menglu' and 'fruit bubble' which are collected from Tonghai and have consistent growth period, cleaning and airing; on an ultra-clean workbench, the leaves are placed into a sterile tray in the back-up direction, each variety is provided with a tray, 6 rows of leaves are arranged on each tray, 8 leaves are arranged on each row, 4 leaves in each row are slightly needled by using a sterile inoculating needle in a centralized manner around the vein, 4 leaves are not needled, and moist sterile absorbent paper is padded in the tray.
Inoculation: on the superclean workbench, the 1 st and 2 nd rows of each variety of leaves are subjected to scab sticking treatment, each leaf has a scab, and the back of the scab is stuck to a needling part or the periphery of a vein; in lines 3 and 4, aseptic brushes are used for brushing spore suspension liquid, and only one side of the leaves is brushed; and (5) performing 20ul of transfer treatment on the spore suspension liquid drops in the 5 th and 6 th rows, and dropping one drop of the spore suspension liquid drop on the acupuncture part on the leaf or around the vein.
After inoculation, a layer of mist-shaped sterile water is sprayed on the leaves, the water absorption paper is ensured to be wet, the water does not flow down in strands when the tray is inclined, but a small amount of water is accumulated at the edge of the tray, a sealing film is covered on the tray for moisturizing, the cultivation is performed under the conditions that the illumination intensity is 3200lx, the light cycle is 14 hours of light period and 10 hours of dark period, the air relative humidity is 75-80%, the room temperature is 15-20 ℃, the cultivation is performed in a moisturizing manner, and the observation and the recording are performed once every 2-4 days, and the results are shown in Table 4.
TABLE 4 comparison of test results for different inoculation methods of Rosa chinensis Peronospora
As can be seen from the table above, the menglu variety shows disease resistance characteristics no matter whether the scab is attached, the spore suspension is coated by a brush, the spore suspension is transferred by liquid drops, and the inoculated part of the leaf shows stabbing and non-stabbing treatment; the variety with the cardiac phase is treated by the method, wherein the hair brush shows the affection after being brushed, and other methods show the high feeling.
According to the test data, the incidence of diseases of acupuncture inoculation is generally higher than that of acupuncture-free treatment; the detached leaves are inoculated by a method of inoculating scabs, brushing spore suspension liquid on a brush and inoculating spore suspension liquid drops by 20 mul, the scabs are well inoculated after 25 days, and the quantity of spores of peronospora destructor on the scabs is large; in Table 4, 2 varieties all had downy mildew with different degrees, and the disease incidence trend is shown to be increased day by day.
Example 6 plant resistance identification test
Recording the natural morbidity of the 18-month-season variety seedlings, wherein the 18-month-season variety seedlings come from the same enterprise in the open sea and are 15-25 cm high;
the incidence D is calculated according to the following formula:
D(％)＝(N/T)×100
in the formula: d-incidence in percent (%);
n is the number of diseased plants, and the unit is the plant;
t-investigating the total plant number, wherein the unit is plant;
the disease index is calculated according to the following formula:
ID＝[∑(N×R)/(M×T)]×100
in the formula: ID-disease index;
sigma-investigating the sum of the products of the relative disease grade numerical values and the plant numbers of the diseases;
n is the number of plants of a certain grade of diseases, and the unit is a plant;
r is relative disease grade value of the disease;
m is the highest disease grade value of the disease;
t-investigation of the total number of plants, the unit is plant.
Plant disease survey grading standard:
level 0-no disease spots on the whole normal leaf;
grade 1-diseased leaves account for 1-10% of the whole developed leaves, downy mildew is not obvious;
grade 3-diseased leaves account for 11-25% of the whole developed leaves, downy mildew is not obvious;
grade 5, the diseased leaves account for 26 to 50 percent of the whole developed leaves, and the downy mildew is obvious;
grade 7, diseased leaves account for 51-75% of the whole unfolded leaves, and the diseased leaves are partially withered;
grade 9-diseased leaves account for over 75% of the whole leaf or most of the leaf yellowing and withering.
The disease resistance grading standard of Chinese rose downy mildew is as follows:
immunization: average disease index is 0.00;
high resistance: the average disease index is more than or equal to 0.01 and less than or equal to 5.00;
disease resistance: the average disease index is more than or equal to 5.01 and less than or equal to 10.00;
anti/intermediate feel: the average disease index is more than or equal to 10.01 and less than or equal to 20.00;
the infection: the average disease index is more than or equal to 20.01 and less than or equal to 40.00;
high feeling: the average disease index is more than or equal to 40.01.
The results are reported in Table 5.
Natural disease of seedlings of table 518 common Chinese rose varieties
As can be seen from the table, under the natural morbidity condition, the resistance to downy mildew of different varieties of Chinese roses is obviously different, and 18 varieties of resources comprise: 7 variety resources (lip, Jinhui, Wai-awag, Douta, fruit bubble, lyssodexin and purple-clouds fairy) show immunity to downy mildew and have strong resistance; other varieties are resistant to diseases and high resistance levels. It should be noted that 7 varieties (lip, jinhui, awaking, dolosta, ocean riddle, golden flame, orange barbie) resources are infected by other diseases such as powdery mildew in 6 days of 9 months, and 3 varieties (fruit bubble, lyssodexin, and lacca) resources among 18 China rose variety resources are better resistant to downy mildew and simultaneously resistant to other diseases, and can be used as preferable resistant resources.
Further, the leaves of healthy seedling plants of each Chinese rose variety are coated with spore suspension, bagged and moisturized, sprayed, watered and moisturized, and inoculated for 4-6 days to investigate the disease condition. After the plants are inoculated, the plants are reasonably fertilized, moisturized and transparent (to moderate temperature and high humidity). If the diseases are mild, spraying pesticide on plants or cutting off leaves of the diseases which are not targeted, such as powdery mildew, gray mold, red spider and the like; if the disease is serious, the plants are moved out of the healthy plant management area and are no longer used as test plants, so that the spread of non-purpose diseases is prevented; when other diseases occur, the pesticide is not suitable to be sprayed to prevent downy mildew, and the diseased leaves are cut off or the plants are moved out of the healthy plant management area. When watering, the leaf surface is kept moist, insect pests are treated in time, and the pesticide is applied or washed by water and the like.
First batch inoculation test: spore suspension concentration is 1X 104Each variety was tested in 10 groups of 3 strains, and the results are shown in Table 6, which is the mean of 10 groups.
Inoculating downy mildew 1 x 10 on healthy leaves of the variety in the month of 618 superficies4Resistance of spore suspension/ml
The disease indexes of the variety resources of downy mildew are different in 18 months, and 5 variety resources (lip, dolosta, Maria and Pixiu 6) show immunity and high resistance; the variety of reviving, dream barbie, orange barbie, purple nepheline, magic bubble, ocean riddle, basin show 1, basin show 2, basin show 3, basin show 4 and the like shows moderate resistance level; fruit bubble and pot elegans 5 appear to be disease resistant; the golden brightness and golden flame are the manifestations of infection.
Second batch inoculation test: spore suspension concentration is 1X 105Each group had 3 strains per ml, and the results are shown in Table 7.
TABLE 712 month season healthy leaves inoculated with downy mildew 1 × 105Resistance of spore suspension/ml
The disease indexes of the variety resources of downy mildew in 12 months are different. Wherein the red lip, golden glow and golden flame are high-feeling; awakening, dolosta, Maria, dream Bobby, and Zixia fairy in the form of influenza; the mystery of orange barbie, fruit bubble, wild bubble and sea shows a feeling of feeling; the variety resources in 12 months reach the peak of disease onset 20-30 days after artificial inoculation. By combining the tests, the bubbles of the wild Albizzia julibrissin have better and stable resistance to downy mildew, namely the screened resistant variety.
In general, the downy mildew morbidity of the plant leaves inoculated with the natural morbidity and the spore suspension is different, and the morbidity of artificial inoculation is more serious; the main reason is that the number of pathogenic bacteria which are naturally attacked is limited, and the pathogenic bacteria are generally hidden in soil and are easily influenced by various factors such as soil environment, humidity, temperature and the like; the quantity of the artificially inoculated pathogenic bacteria is sufficient, the humidity of the leaves is high, and the infection of the pathogenic bacteria is facilitated; in the actual identification process, natural morbidity and artificial inoculation tests can be respectively carried out, and the relative resistance of different Chinese rose varieties to downy mildew can be obtained by combining the test results of the natural morbidity and the artificial inoculation tests, so that the varieties with higher relative resistance can be screened out.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. The method for identifying the resistance of Chinese rose downy mildew is characterized by comprising the following steps of:
(1) a typical downy mildew sample was taken: collecting Chinese rose leaves with downy mildew attack symptoms in a field;
(2) propagation of downy mildew pathogenic bacteria:
the collected typical downy mildew sample is placed in a culture room for moisture culture, so that downy mildew pathogenic bacteria on the scab are propagated and grown, and the method specifically comprises the following steps:
on a super-clean workbench, the leaf back of each leaf is upwards placed in a sterile tray filled with double-layer sterile absorbent paper, the depth of the tray is 5-8 cm, and the absorbent paper is wetted by sterile water; spraying vaporous sterile water on the blades to ensure that the absorbent paper is wet, and when the tray is slightly inclined, the water cannot flow down in strands, but the edge of the tray is accumulated with water; sealing the disc by using a sealing film, and culturing in a culture chamber; culture conditions in the culture chamber: the illumination intensity is 1800-3200lx, the light period is 12-16 hours, the dark period is 8-12 hours, the air relative humidity is 75-90 percent, and the culture is carried out for 5-10 days at the room temperature of 15-22 ℃;
(3) inoculating downy mildew pathogenic bacteria: preparing a spore suspension or shearing diseased spots after visible hypha and spores are overgrown on the backs of the downy mildew diseased leaves, and inoculating the plants, in-vitro branches or in-vitro leaves to be identified;
(4) evaluation of disease resistance to Chinese rose downy mildew: after inoculation, the disease condition and the disease severity of the Chinese rose downy mildew are investigated and counted, the disease index is calculated, and the disease resistance of the Chinese rose downy mildew is evaluated through the average disease index in the counting period;
the disease resistance grading standard of Chinese rose downy mildew is as follows:
immunization: average disease index is 0.00;
high resistance: the average disease index is more than or equal to 0.01 and less than or equal to 5.00;
disease resistance: the average disease index is more than or equal to 5.01 and less than or equal to 10.00;
anti/intermediate feel: the average disease index is more than or equal to 10.01 and less than or equal to 20.00;
the infection: the average disease index is more than or equal to 20.01 and less than or equal to 40.00;
high feeling: the average disease index is more than or equal to 40.01;
the disease incidence is expressed by the disease incidence rate D and is calculated according to the following formula:
D(％)＝(N/T)×100
in the formula: d-incidence in percent (%);
n is the number of diseased plants or diseased leaves, and the unit is plant or leaf;
t-investigating the total plant number or the total leaf number, wherein the unit is a plant or a leaf;
the severity of the disease is expressed by a disease index and is calculated according to the following formula:
ID＝[∑(N×R)/(M×T)]×100
in the formula: ID-disease index;
sigma-investigating the sum of the relative disease grade value of the disease and the product of the plant number or the leaf number;
n-the number of plants or leaves at a certain level of the disease, and the unit is the plant or leaf;
r is relative disease grade value of the disease;
m is the highest disease grade value of the disease;
t-investigating the total plant number or the total leaf number, wherein the unit is a plant or a leaf;
in vitro leaf disease investigation grading standard:
grade 0-no disease spots on leaves;
grade 1-the lesion area accounts for 1-10% of the leaf area;
grade 3, the lesion area accounts for 11 to 25 percent of the leaf area;
grade 5, the lesion area accounts for 26 to 40 percent of the leaf area;
grade 7, the lesion area accounts for 41 to 60 percent of the leaf area;
grade 9, the disease spot area accounts for more than 61% of the leaf area or the diseased leaves die;
field or plant disease survey grading standard:
level 0-no disease spots on the whole normal leaf;
grade 1-diseased leaves account for 1-10% of the whole developed leaves, downy mildew is not obvious;
grade 3-diseased leaves account for 11-25% of the whole developed leaves, downy mildew is not obvious;
grade 5, the diseased leaves account for 26 to 50 percent of the whole developed leaves, and the downy mildew is obvious;
grade 7, diseased leaves account for 51-75% of the whole unfolded leaves, and the diseased leaves are partially withered;
grade 9-diseased leaves account for over 75% of the whole leaf or most of the leaf yellowing and withering.
2. The method for identifying resistance to Chinese rose downy mildew according to claim 1, wherein in the step (1), the leaves are packaged by using a freshness protection package, 1 to 2 sterile filter papers wetted by sterile water are placed in the package, and the package is sealed and brought back by blowing air.
3. The method for identifying resistance to Chinese rose downy mildew according to claim 1, wherein the spore suspension in the step (3) is prepared by: dipping sterile water on a clean bench by using a sterile brush, gently washing spores on the backs of downy mildew-infected leaves to prepare a mixture with the concentration of 1 × 104-1×106Spore suspension of each/mL is kept for 2h at 4-10 ℃ for later use.
4. The method for identifying the resistance to Chinese rose downy mildew according to claim 3, wherein in the step (3), the plants and the in-vitro branches are inoculated by a spore suspension painting mode or a spore suspension spraying mode; the detached leaf is inoculated by adopting a mode of scab grafting, spore suspension brushing or spore suspension liquid drop transplanting.
5. The method for identifying resistance to Chinese rose downy mildew according to claim 4, wherein the concentration of the tender leaves in vitro is 1 x 104Inoculating spore suspension of each/mL, and adopting 1 × 10 for in vitro middle and old leaves, in vitro branches and plants5And (4) inoculating one/mL spore suspension.
6. The method for identifying resistance to Chinese rose downy mildew according to claim 1, wherein before inoculation in step (3), the leaf vein of the leaf to be inoculated is pricked by a needle, but the leaf is not pricked.
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