Method for culturing downy mildew bacteria and application
By controlling humidity and temperature in a confined space, and inoculating the rose plants with natural or spore inoculation, the problem of difficult culture of pathogenic bacteria in downy mildew research has been solved, and efficient infection and stable acquisition of downy mildew bacteria has been achieved. Suitable for the cultivation of cut roses in Yunnan.
Patent Information
- Application Number
- CN202410026675.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-09
- Publication Date
- 2024-12-20
AI Technical Summary
Downy mildew occurs rapidly under the conditions of the cut rose facility, and live nutritional fungi are difficult to cultivate through traditional culture media, resulting in a lag in research and a lack of stable pathogenic bacterial materials.
In a confined space with air humidity greater than 80%, healthy rose plants are inoculated with downy mildew bacteria through natural inoculation method or spore inoculation method, and the humidity and temperature are controlled by facility greenhouses and sprinkler irrigation devices to achieve efficient infection and culture of downy mildew bacteria.
This method can stably obtain sensory plants and pathogenic bacteria, simplify management, and is suitable for the research on cut rose downy mildew in Yunnan. It provides material guarantees for disease identification and drug screening, and is simpler and more efficient than ex vivo culture method.
Smart Images
Figure CN119161987A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of flower disease research, and particularly relates to a method for culturing downy mildew fungi and an application thereof. Background Art
[0002] Yunnan is the main production area of cut roses. Under the conditions of cut rose facility cultivation, the occurrence of downy mildew is very accidental. As long as the humidity conditions are met, the disease and spread are extremely fast. Within one or two days, all the cut roses in the entire greenhouse will be infected, and the loss rate of the current flowers can reach 70-100%. In the rainy season, the humidity in the greenhouse cannot be discharged, and the incidence rate is particularly high. Downy mildew is a biotrophic fungal disease and cannot be cultured through culture media. It is extremely difficult to have a stable pathogen for scientific research, so the research on downy mildew is relatively lagging behind other diseases. To solve the above problems, it is necessary to create a suitable and simple method to cultivate pathogens, so as to carry out follow-up research on downy mildew, provide protection for the facility cultivation of cut roses in Yunnan, and reduce the economic losses caused by downy mildew. Summary of the invention
[0003] The purpose of the present invention is to provide a method and application for culturing downy mildew fungi, which can solve the problem that biotrophic fungi are difficult to cultivate, solve the problem that disease conditions are uncontrollable, and can efficiently infect roses with downy mildew fungi, thereby obtaining susceptible plants and pathogens.
[0004] The invention provides a method for culturing downy mildew fungi, comprising the following steps: inoculating downy mildew fungi on healthy roses in a closed space with air humidity greater than 80%.
[0005] Preferably, the enclosed space includes a facility shed, and a sprinkler device is connected inside and outside the facility shed;
[0006] The bottom of the facility greenhouse is not in direct contact with the ground, and a material that promotes the formation of steam pressure in the facility greenhouse is laid.
[0007] Preferably, stone materials are laid on the ground inside the facility greenhouse, or the facility greenhouse is directly placed on a cement ground to avoid contact with soil.
[0008] Preferably, the inoculation method includes a natural inoculation method, which includes planting rose plants infected with downy mildew and healthy rose plants at the same time in the confined space, and the healthy rose plants are evenly distributed radially around the rose plants infected with downy mildew.
[0009] Preferably, the crowns of two adjacent healthy rose plants are in natural contact.
[0010] Preferably, the inoculation method includes a spore inoculation method, which includes spraying healthy rose plants with spore liquid of downy mildew fungi.
[0011] Preferably, the method for preparing the spore liquid of the downy mildew fungus comprises collecting rose leaves with downy mildew spores, cutting off the infected parts and mixing them with water, passing the mixed liquid through a 40-mesh cell sieve, and collecting the filtrate to obtain the spore liquid.
[0012] Preferably, the working solution concentration of the spore solution is 1×10 5 conidia / mL.
[0013] Preferably, the cultivation is carried out using natural light, and also includes spraying water after 4 pm every day until thick water droplets form on the leaves and the ground.
[0014] The present invention also provides an application of the above method, comprising at least one of the following:
[0015] (1) Identify the resistance of roses to downy mildew;
[0016] (2) Screening for drugs against downy mildew;
[0017] (3) Develop drugs to resist downy mildew.
[0018] Beneficial effects: The present invention provides a method for culturing downy mildew fungi. Cultivating downy mildew fungi in a closed microenvironment can not only maintain sufficient humidity, but also identify downy mildew diseases of different varieties of rose seedlings, and provide material guarantee for basic research on rose downy mildew. Compared with the in vitro culture method, the management is simpler, and extensive management and large-scale spore production can be achieved. In terms of materials, plastic greenhouses are inexpensive, and the plastic films used are widely available and cheap, and even obsolete plastic greenhouse plastic films can be used. This method is suitable for rose downy mildew cultivation in Yunnan.
[0019] By using the method of the present invention, compared with the commonly used leaf disc method and water agar culture method, the rose downy mildew fungus can be obtained more stably by using the greenhouse microenvironment culture method, which is not only convenient and easy to obtain, but also can be stored for a long time. It is the best culture method for cultivating downy mildew fungus. In the process of cultivating rose downy mildew fungus by using the greenhouse microenvironment culture method, it is found that after the rose downy mildew fungus infects the rose leaves, the rose leaves will show various symptoms: the leaves turn green and large yellow blocks appear; small purple-black spots appear on the leaf surface and are distributed on the leaves, the spots are uneven in size, and the shapes range from dots to blocks; the leaf surface turns green unevenly, resulting in uneven distribution of leaf surface color, with dark green dots or blocks appearing on the leaf surface; the leaves sometimes appear slightly curled, and large black patches appear on the leaves, with traces of being burned by fire. Secondly, the spore production on the back of the leaves is greatly related to humidity. When the humidity reaches more than 80%, it is easy to observe the spore production on the back of the leaves, and most of them appear on new leaves. In addition to leaves that are easily infected, new and tender parts such as pedicels are also easily infected. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a schematic diagram of the arch shed style and the placement of plants in the shed;
[0021] Figure 2 This is a symptom site picture of rose downy mildew. ABCD shows the diseased state on the front of the leaf, E shows the diseased state on the back of the leaf with a sparse spore layer, and F shows the diseased state on the pedicel.
[0022] Figure 3 Figures 1 and 2 are symptom pictures of the leaf disc method and the water agar culture method. A and C are the disease states of the leaves after 7 and 14 days of cultivation by the whole leaf culture method, respectively. B is the disease symptoms of the leaves after 6 days of cultivation by the water agar culture method, with sparse spores produced and accompanied by unknown pathogens at the tip of the leaves; D is a microscopic picture of the pathogen on the surface of a leaf picked after 14 days of cultivation by the leaf disc method; E is a microscopic picture of the surface spores picked by the water agar culture method. DETAILED DESCRIPTION
[0023] The invention provides a method for culturing downy mildew fungi, comprising the following steps: inoculating downy mildew fungi on healthy roses in a closed space with air humidity greater than 80%.
[0024] The enclosed space of the present invention preferably includes a facility shed, and a sprinkler device is connected inside and outside the facility shed; the bottom of the facility shed is not in direct contact with the ground, and a material that promotes the formation of steam pressure in the facility shed is laid. The facility shed of the present invention preferably includes a plastic shed, or a newly built shed, such as the outer arch shed in the embodiment, which is built with an arched thickened plastic-coated steel pipe and a thickened shed film, with an arch height of 1.8m, a side height of 1.0m, and a width of 1m. The shed length can be set according to the actual amount of the infected material; the door is opened in the front, and the door is 0.5m wide and 0.8m high.
[0025] The rose downy mildew described in the present invention is a low-temperature and high-humidity disease, and its spore germination has strict requirements on environmental control. It can only develop rapidly at a temperature of 15 to 20°C and a humidity of more than 80%. Downy mildew is a biotrophic bacterium, and the culture of the fungus source can only be carried out on living plants under suitable environmental conditions. The enclosed space described in the present invention can provide a small environment in the shed for the spore germination and efficient cultivation of rose downy mildew, provide suitable environmental control conditions, and efficiently cultivate rose downy mildew, providing a continuous source of pathogenic bacteria materials for the prevention and control of rose downy mildew and mechanism research.
[0026] The greenhouse of the present invention is preferably not in direct contact with the ground, but needs to be padded with stone materials in the greenhouse, or the greenhouse is directly placed on the cement ground to avoid contact with the soil, so as to facilitate strict control of the humidity in the greenhouse; the greenhouse is connected to a sprinkler device inside and outside to water evenly. The arrangement of the present invention can facilitate the cultivation of downy mildew in southern regions, especially in Yunnan Province, China, so that the sun-exposed ground can allow the sprayed water to evaporate and form vapor pressure, thereby regulating the environmental humidity.
[0027] The present invention can inoculate downy mildew fungi through natural inoculation or spore inoculation. The natural inoculation method requires the rose plants infected with downy mildew to be cultivated with healthy plants at the same time and space, while the spore inoculation method only plants healthy plants, and there is no special limitation on the arrangement of the plants.
[0028] The natural inoculation method of the present invention preferably includes planting rose plants infected with downy mildew and healthy rose plants in the enclosed space at the same time, and the healthy rose plants are evenly distributed around the rose plants infected with downy mildew in a radial shape, that is, a diseased plant is placed in the center, and healthy plants are evenly arranged around the diseased plant in a radial shape, and the crowns of two adjacent healthy rose plants are in natural contact. The diseased plant of the present invention can be in natural contact with the crowns of the surrounding healthy plants, or a distance can be kept without direct contact.
[0029] The spore inoculation method of the present invention preferably includes spraying healthy rose plants with spore liquid of downy mildew fungi. The preparation method of the spore liquid of downy mildew fungi preferably includes collecting rose leaves with downy mildew spores, cutting off the infected parts and mixing them with water, passing the mixed liquid through a 40-mesh cell sieve, and collecting the filtrate to obtain the spore liquid. The working solution concentration of the spore liquid of the present invention is preferably 1×10 5 conidia / mL.
[0030] The cultivation of the present invention preferably adopts natural light, and also includes spraying water after 4 pm every day until thick water droplets form on the leaves and the ground, and the humidity in the greenhouse reaches more than 80%. The light is natural light, and the water droplets form a high humidity environment in the greenhouse in the afternoon, so that the spores will germinate quickly, and the spores will be spread to the back of the leaves of other plants through the transpiration water potential of the ground. Yellow-green water stains can be seen in 4 days, and a white downy mildew layer can be observed in 6 days.
[0031] The present invention also provides an application of the above method, comprising at least one of the following:
[0032] (1) Identify the resistance of roses to downy mildew;
[0033] (2) Screening for drugs against downy mildew;
[0034] (3) Develop drugs to resist downy mildew.
[0035] The present invention adopts a greenhouse microenvironment culture method to obtain rose downy mildew more stably. It is not only convenient and easy to obtain, but also can achieve long-term storage. It is the optimal culture method for cultivating downy mildew. It can identify downy mildew diseases of rose seedlings of different varieties, and can also provide a continuous source of pathogenic bacteria materials for rose downy mildew prevention and control and mechanism research. It can also screen and / or develop drugs against downy mildew.
[0036] In order to further illustrate the present invention, a method for culturing downy mildew fungi and applications provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0037] Example 1
[0038] 1) Shed construction: The outer arch shed is built with arched thickened plastic-coated steel pipes and thickened shed film. The arch height is 1.8m, the side height is 1.0m, and the width is 1m. The length of the shed can be set according to the actual needs of the amount of infected materials. The door is opened in the front, with a width of 0.5m and a height of 0.8m ( Figure 1 ).
[0039] 2) Equipment inside the shed: Stones are placed inside the shed, or the shed is placed directly on the cement floor to avoid contact with the soil, so as to strictly control the humidity inside the shed and allow the sprayed water to evaporate and form steam pressure on the sun-exposed ground. Sprinklers are connected inside and outside the shed to evenly water the shed.
[0040] 3) There are two methods of inoculation:
[0041] Spore inoculation method: fresh rose leaves with downy mildew spores were picked and placed in a 4°C refrigerator for temporary storage (no more than 4 days). Then, the leaf tissue with spores was cut and placed in a centrifuge tube. 10 ml of distilled water was added to the tube. The tube was fully shaken using a vortex oscillator and then filtered using a 40-mesh cell strainer (Biosharp). The filtrate was placed on a hemocytometer to observe the concentration and adjusted to 1×10 5 Spray healthy plants with molecular spore leaves.
[0042] Natural inoculation method: The plants in the greenhouse are placed in a ring. One pathogenic plant is placed in the middle, and healthy plants and pathogenic plants are placed around them at equal distances, forming a ring. The distance between the pathogenic plant and the surrounding plants is 0, and it is best to allow the crowns of the plants to naturally contact each other. After the plants are placed, close the greenhouse door.
[0043] 4) Processing method:
[0044] At 4 pm every day, when the temperature is high, use the sprinkler to spray water on the leaves of diseased and healthy plants until thick water droplets form on the leaves and the ground. At this time, the humidity in the greenhouse reaches more than 80%. The light is natural light, and the water droplets form a high humidity environment in the greenhouse in the afternoon. The spores will germinate quickly and spread to the back of the leaves of other plants through the transpiration of the ground. Yellow-green water stains can be seen in 4 days, and white downy mildew can be observed in 6 days.
[0045] In the process of cultivating rose downy mildew using the greenhouse microenvironmental culture method, it was found that after the rose downy mildew infected the rose leaves, the rose leaves would show various symptoms: the leaves turned green, large yellow patches appeared ( Figure 2 Middle A); small purple-black spots appear on the leaf surface, distributed on the leaves, the spots are uneven in size, and the shapes range from dots to blocks ( Figure 2 Middle B); uneven chlorosis of the leaves, resulting in uneven distribution of color on the leaf surface, with dark green spots or blocks appearing on the leaf surface ( Figure 2 Middle C); the leaves sometimes curl slightly, and large black spots appear on the leaves, which look like burns ( Figure 2 Secondly, spore production on the back of leaves is closely related to humidity. When the humidity reaches above 80%, spore production can be easily observed on the back of leaves, and most of them appear on new leaves ( Figure 2 In addition to leaves that are easily infected, new and tender parts such as pedicels are also susceptible to infection ( Figure 2 Middle F).
[0046] Different varieties of rose seedlings were cultivated in a small environment in a greenhouse using the natural inoculation method. The experiment was conducted under four different humidity conditions (humidity below 70% 70-80%; 80-90%; 90-100%), with 100 roses in each group. The incidence rate was counted on the 6th day. The results are as follows: the incidence rate of the five varieties reached 100% under the condition of temperature of 80-90%; when the humidity was below 70%, the incidence rate was extremely low and most varieties did not get sick.
[0047] Table 1 Disease incidence of different rose varieties cultured for 6 days under different humidity conditions
[0048]
[0049]
[0050] Comparative Example
[0051] Leaf disc method and water agar culture method:
[0052] The rose downy mildew pathogen was cultured using the leaf disc method and the water agar culture method. It was found that the spore production effect of the diseased leaves cultured using the leaf disc method was not ideal, the spore production of the diseased leaves was small, and most of the spores on the diseased leaves were basically gray mold pathogens after microscopic identification ( Figure 3 Middle C), in the late culture period (about 14 days), the diseased leaf tissues basically rotted and turned yellow, and stuck to the filter paper, making it difficult to remove, resulting in downy mildew. Compared with the intact leaf method, the spore production effect of the water agar culture method is relatively stable, and most of the microscopic identification results are downy mildew. The leaves are intact and easy to remove, but a small number of leaves have spores that are gray mold after microscopic identification, which is difficult to distinguish from downy mildew. In general, the above two culture methods have large differences in spore production between leaves. Some leaves have produced spores, while others have not, so the culture time is difficult to control. Due to the difference in spore production time among leaves, it is difficult to find the right time to collect spores. If the waiting time is too long, the leaves will rot and turn into gray mold. If the time is too short, only a few leaves will produce a small amount of spores, which makes observation very difficult. In addition, the spores of downy mildew are very similar to the spores of gray mold, and are almost the same to the naked eye. It is difficult to judge whether the spores produced by the leaves belong to downy mildew or gray mold by naked eye observation, so it is necessary to observe with the help of a microscope, which undoubtedly increases the difficulty of collecting materials.
[0053] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for culturing downy mildew, characterized in that: The method comprises the following steps: inoculating downy mildew fungi on healthy roses in a closed space with air humidity greater than 80%.
2. The method according to claim 1, characterized in that: The enclosed space includes a facility shed, and a sprinkler device is connected inside and outside the facility shed; The bottom of the facility greenhouse is not in direct contact with the ground, and a material that promotes the formation of steam pressure in the facility greenhouse is laid.
3. The method according to claim 2, characterized in that: Pad the floor of the greenhouse with stones, or place the greenhouse directly on the cement floor to avoid contact with soil.
4. The method according to claim 1, characterized in that: The inoculation method includes a natural inoculation method, which includes planting rose plants infected with downy mildew and healthy rose plants in the closed space at the same time, and the healthy rose plants are evenly distributed radially around the rose plants infected with downy mildew.
5. The method according to claim 4, characterized in that: The crowns of two adjacent healthy rose plants are naturally touching.
6. The method according to claim 1, characterized in that: The inoculation method includes a spore inoculation method, and the spore inoculation method includes spraying healthy rose plants with spore liquid of downy mildew fungi.
7. The method according to claim 6, characterized in that: The method for preparing the spore liquid of downy mildew fungus comprises collecting rose leaves with downy mildew spores, cutting off the infected parts and mixing with water, passing the mixed liquid through a 40-mesh cell sieve, and collecting the filtrate to obtain the spore liquid.
8. The method according to claim 6 or 7, characterized in that: The working solution concentration of the spore solution is 1×10 5 conidia / mL.
9. The method according to claim 1, characterized in that: The cultivation adopts natural light and also includes spraying water after 4 pm every day until thick water drops are formed on the leaves and the ground.
10. The use of the method according to any one of claims 1 to 9, comprising at least one of the following: (1) Identify the resistance of roses to downy mildew; (2) Screening for drugs against downy mildew; (3) Develop drugs to resist downy mildew.