CN113303332B - Pharmaceutical preparation and method for resisting Lavandula odorata mosaic virus - Google Patents

Abstract

The present invention relates to a medicinal preparation and method for resisting the Echinacea mosaic virus, the active components of which include rhamnolipid. The inventors found through experiments that rhamnolipid can effectively reduce the accumulation of TeMV on plants, thereby effectively alleviating the mosaic disease of passion fruit caused by TeMV. In addition, through the study of physiological and biochemical mechanisms, it was found that Rls can effectively alleviate the growth inhibition of passion fruit plants caused by virus infection, and can significantly improve the resistance and defense ability of passion fruit plants to TeMV. It indicates that it has potential in improving plant resistance to viruses and the prevention and control of plant virus diseases, and has broad application prospects in future agricultural production.

Description

Pharmaceutical preparation and method for resisting Lavandula odorata mosaic virus

Technical Field

The invention relates to the field of biotechnology, in particular to a pharmaceutical preparation for resisting a night-come mosaic virus and a method thereof.

Background

The passion fruit serving as a nutritional fruit is developed rapidly in the south region of China in recent years, related industries become important channels for increasing income of farmers in the areas such as Guangxi, Guangdong, Fujian and Hainan gradually, and passion fruit planting becomes an important popularization item for poverty relief in villages. However, in recent years, owing to improper planting management, passion fruit is continuously damaged by various pests, wherein virus diseases become one of serious limiting factors of passion fruit production, and particularly mosaic virus diseases seriously affect plant growth and even fruit yield and quality. Therefore, starting from the national economic development demand, by relying on a stable disease system of the passion fruit and a virus pathogen, a relevant mechanism of the passion fruit responding to mosaic virus infection is explored, a theoretical clue is found for finding a high-efficiency environment-friendly control strategy, and the method is an urgent demand for the development of passion fruit-related industries at present.

At present, more than 20 viruses capable of infecting passion fruit are reported at home and abroad, and mainly comprise the passion fruit lignification virus, cowpea aphid mosaic virus, east Asia passion fruit virus, evening primrose mosaic virus, passion fruit ringspot virus and turnip mosaic virus of potyvirus of potyviridae; cucumber mosaic virus of the genus cucumber mosaic virus of the family brome mosaic virus; euphorbia leaf curl virus of Phaseolus of geminiviridae; passion fruit yellow mosaic virus of genus Brassica of family Brassicaceae, genus Psyllium, Passion fruit green spot virus of genus Citrus Rough virus, etc. Among them, the infection of passion fruit by the night mosaic virus (TeMV) was first discovered in thailand in 2014; passion fruit virus diseases caused by TeMV infection are found in Hainan and Fujian areas of China in 2018; our related studies also show that TeMV is one of the major pathogens causing passion fruit virus disease in the Guangdong region. After the Passion fruits are infected by TeMV, disease symptoms are characterized in that the disease symptoms are scattered in a pale green area between pulses and then spread to most leaf surfaces, and usually the leaves are shown as flowers and leaves with different degrees, diseased leaves are slightly wrinkled, the fruit shape is small, the fruit peel is wrinkled, the coloring is abnormal, and the economic value is lost. It is easy to see that TeMV has become an important virus affecting the plantation and production of passion fruit, and it is an urgent task to find a green, safe and effective prevention and treatment measure for the virus disease of passion fruit.

Once the infected plant is found in the field, the infected seedling is mainly pulled out in a root-by-root manner to reduce the further expansion of the disease; for the infected plants full of a large number of fruits, in consideration of cost, the spreading of virus diseases to new vines and leaves is usually inhibited as much as possible by spraying various pesticides or biological agents capable of inactivating viruses or inducing the immunocompetence of the plants, and the method has quite high production cost and has huge potential safety hazards to people, livestock and ecological environment. Biological control of plant diseases is carried out by utilizing the biological source pesticide, various adverse effects caused by overuse of chemical pesticide are reduced, and exploration of biological control of passion fruit virus diseases has important significance for healthy development of passion fruit industry. Aiming at virus diseases, finding a safe and effective prevention and control strategy has important practical production and application values.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a pharmaceutical preparation against the Floweringworm mosaic virus.

The specific technical scheme is as follows:

a pharmaceutical preparation for resisting Lavandula odorata mosaic virus contains rhamnolipid as active ingredient.

In some embodiments, the rhamnolipid concentration in the above pharmaceutical formulation is between 0.1g/L and 1.0 g/L.

In some embodiments, the rhamnolipid concentration in the above pharmaceutical formulation is between 0.3g/L and 0.8 g/L.

In some embodiments, the rhamnolipid concentration in the above pharmaceutical formulation is between 0.4g/L and 0.6 g/L.

In some embodiments, the rhamnolipid concentration in the above pharmaceutical formulation is 0.5 g/L.

One of the purposes of the invention is also to provide the application of rhamnolipid in preventing and treating the plant nocturnal flower myrcia virus disease.

In some embodiments, the plant is passion fruit.

In some embodiments, the plant is a gold passion fruit.

One of the objects of the present invention is also to provide a method for controlling a plant nocturnal flower myrcia virus disease.

The technical scheme for realizing the purpose is as follows:

a method of controlling a plant nocturnal geranium virus disease, comprising: spraying a preparation whose active ingredient comprises rhamnolipid.

In some embodiments, the spraying part of the preparation is plant leaf and/or stem.

In some embodiments, the above-mentioned preparation is sprayed 1-2 times per day.

In some embodiments, the plant is passion fruit.

In some embodiments, the plant is a gold passion fruit.

Compared with the prior art, the invention has the following beneficial effects:

the inventor of the invention finds that the accumulation of the night fragrant mosaic virus (TeMV) on plants can be effectively reduced by spraying rhamnolipid (Rhamonolipid, Rls) with a proper concentration on passion fruit seedlings through experiments, so that the passion fruit flower and leaf disease caused by the TeMV can be effectively relieved. In addition, physiological and biochemical mechanism researches show that Rls can also effectively relieve the growth inhibition of passion fruit plants caused by virus infection, and can remarkably improve the resistance and the defense capacity of the passion fruit plants to TeMV. The method has potential in improving plant virus resistance and preventing and treating plant virus diseases, and has wide application prospect in future agricultural production.

Drawings

FIG. 1 is a diagram showing the alignment results of TeMV nucleic acid sequences in example 1.

FIG. 2 is a phenotypic observation of 0.5g/L Rls on superior leaves of gold passion fruit plants in example 1 in response to TeMV inoculation.

FIG. 3 is a graph showing the effect of 0.5g/L Rls on the TeMV content in Passion fruit plants in example 1.

FIG. 4 is a graph showing the effect of 0.5g/L Rls on leaf relative conductivity (REC) and Malondialdehyde (MDA) content of passion fruit plants in example 1 after response to TeMV.

FIG. 5 is the effect of 0.5g/L Rls on chloroplast pigment content in passion fruit plants in example 1 after response to TeMV, where Chl-a: chlorophyll a; chl-b: chlorophyll b; car: a carotenoid.

FIG. 6 is a graph of the effect of 0.5g/L Rls on leaf superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD) activity in passion fruit plants in response to TeMV in example 1.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used in the present disclosure does not necessarily refer to the same embodiment, although it may. Moreover, the phrase "in another embodiment" as used in this disclosure does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined without departing from the scope or spirit of the invention.

Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification the meaning of "a", "an" and "the" include plural referents. The meaning of "in.

The present invention will be described in further detail with reference to specific examples.

Example 1 Effect of 1 Rls on TeMV Virus

Experimental materials: rls (Sigma, purity is more than or equal to 90%), seedlings of golden passion fruit (the seedlings are bred by golden passion fruit seeds stored by the laboratory, fruits are purchased in a digital orchard of the passion fruit in Cambodia county, Guangdong province, the fruits are collected, dried in the air and aired, and then stored in the laboratory for a series of basic scientific researches), and the virus source of TeMV is identified (through sequencing analysis and comparison of PCR products of the virus sample, the target fragment obtained by PCR has extremely high similarity (the similarity is 98.43 percent as shown in figure 1) with the TeMV sequence of a known database (the GenBank accession number of the reference sequence is KJ789129.1), and the sample is confirmed to be TeMV virus).

According to the preliminary experiment, the suppression effect of 0.1g/L Rls on TeMV on gold passion fruit is not obvious, the effect is achieved within the concentration range of 0.4-1.0g/L, the concentration of 1.0g/L is not greatly different from that of 0.5g/L in the aspect of suppressing the development of pathogenic substances, and the control effect of Rls on TeMV diseases on gold passion fruit plants is not in a linear relation with the concentration of a medicament. Therefore, on the basis of ensuring the optimal drug effect, the cost is reduced, the resources are saved, and the optimal concentration of 0.5g/L is selected for detailed analysis. The relevant specific experimental steps are as follows:

the experimental steps are as follows:

1. planting the golden passion fruit in a plant illumination incubator (the optical density is 100 mu Mm < -2 > s < -1 >, the light intensity is 16-h/the dark period is 18-h, the average temperature is 27 ℃) in a soil culture mode, and after the plant grows to 4-5 leaves, using the golden passion fruit for subsequent experiments.

2. The plant leaves were sprayed with 0.5g/L Rls aqueous solution (Rls group), the control group was sprayed with water (CK group), and 6% oligosaccharide-catenin (Zhongbao) was sprayed as the efficacy reference group (OCP group), respectively. Spraying for 5 days continuously, wherein the spraying treatment is carried out once every 8 o' clock and 30 minutes in the morning.

3. And on the sixth day, adopting a mechanical friction inoculation TeMV virus source mode to inoculate each group of plant leaves in the step 2 to form TeMV infected plant groups which are Rls + V group, + V group and OCP + V group respectively, wherein + V represents that the group is subjected to TeMV mechanical friction inoculation.

4. Observing the disease development of each group of plants (not less than 6 plants in each group are tested in each time, and 4 biological repeated tests are carried out), and carrying out related detection, including the accumulation condition of viruses and physiological and biochemical indexes closely related to the response of plants to biological stress.

The experimental results are as follows:

by observing the leaf phenotype of the passion fruit seedlings in different experimental groups after responding to the TeMV infected leaves, the results are shown in figure 2, the difference of the effects on the leaves of the passion fruit seedlings among the treatments is obvious (P is less than 0.05), and the control effect of 0.5g/L Rls aqueous solution on the TeMV disease on gold passion fruit plants is more obvious. Compared with the healthy group (CK) without virus and reagent treatment, after the leaves of the passion fruit are infected with the TeMV (+ V), the leaves of the passion fruit seedlings are shrunk and the flowers and leaves are shrunk, the parts close to the veins are shrunk and the flowers and leaves are serious. Compared with + V, the traditional Chinese medicine effect reference group in the experiment: the 6% oligosaccharide-catenin + V treated passion fruit seedlings have shriveled leaves and reduced mosaic disease, and the shriveled leaves and the mosaic disease are mainly generated at the positions close to the veins. Compared with + V, the passion fruit seedling leaf treated by 0.5g/L Rls + V has a shrinkage phenomenon only at the upper part (close to the tip) of the leaf and close to the vein. Compared with the virus treatment (+ V), 6% oligosacchride-catenin + V and 0.5g/L Rls + V, the passion fruit seedling treated by 0.5g/L Rls has the lightest leaf symptom, and the 0.5g/L Rls effectively inhibits the development of TeMV on the passion fruit plant.

The accumulation of the pathogenic TeMV is detected by adopting a conventional enzyme-linked immunosorbent assay, and the statistical result is shown in figure 3, compared with the CK group, the TeMV content of the seedling disease strain is reduced by 11.6 percent after 6 percent OCP (6 percent oligosaccharide-catenin) treatment, and the TeMV content of the seedling disease strain is reduced by 44.2 percent after 0.5g/L Rls treatment. The TeMV content of the seedling disease strain after 0.5g/L Rls treatment was reduced by 22.6% compared with that of the 6% OCP (6% oligosaccharide-catenin) treatment. Therefore, 0.5g/L Rls can better inhibit the development and accumulation of TeMV on passion fruit plants.

In addition, the influence of 0.5g/L Rls on the overground part (table 1-1) and the underground part (table 1-2) of the passion fruit plants after responding to TeMV inoculation is detected, and the condition that the passion fruit plants are inhibited from growing originally due to TeMV inoculation treatment can be well relieved under the condition that 0.5g/L Rls exists.

TABLE 1-10.5 g/L Rls Effect on the aerial parts of Passion fruit plants after response to TeMV

Note that the data in the above tables are all expressed as the mean of four biological replicates. + -. standard error, and different letters after the same column number indicate significance of difference between treatments (P <0.05, LSD).

TABLE 1-20.5 g/L Rls Effect on the lower rear portion of Passion fruit plants in response to TeMV

Note that the data in the above tables are all expressed as the mean of four biological replicates. + -. standard error, and different letters after the same column number indicate significance of difference between treatments (P <0.05, LSD).

Meanwhile, after a pathogen infects a plant, cell damage is often caused, and the relative conductivity (REC) and the Malondialdehyde (MDA) content are often used as reaction indexes of the cell damage degree in a plant stress environment. The relative conductivity (REC) and Malondialdehyde (MDA) were further detected and analyzed by routine tests aiming at the experimental groups, and the results are shown in fig. 4, which indicates that 0.5g/L Rls aqueous solution can significantly reduce the plant cell damage caused by pathogen treatment. This also reflects that with 0.5g/L Rls aqueous solution, the damage to the plants by TeMV is low.

Chloroplasts are the main sites of plant photosynthesis, and chloroplast pigments are important indexes reflecting plant growth conditions. Infection by the virus tends to inhibit chlorophyll synthesis in plant leaves, and inoculation of TeMV also causes a decrease in chlorophyll content in passion fruit plants. The results of routine detection and analysis of the chlorophyll content of the plants in the experimental group are shown in fig. 5, and the experimental group treated by 0.5g/L Rls aqueous solution can well relieve the reduction of the chlorophyll content caused by pathogens, which is undoubtedly beneficial to the growth of the plants.

The antioxidant enzyme system is an important defense related factor in the process of responding pathogen infection of plants and is often positively correlated with the disease resistance of the plants, the content of representative superoxide dismutase SOD, catalase CAT and peroxidase POD in the plant antioxidant enzyme system is subjected to routine detection and analysis aiming at the experimental group, the detection and analysis result is shown in figure 6, 0.5g/L Rls is helpful for increasing the activity of antioxidant related enzymes of passion fruit plants after responding to TeMV, and the antioxidant enzyme system comprises representative superoxide dismutase SOD, catalase CAT and peroxidase POD, namely the antioxidant enzyme system of the passion fruit plants is more obviously induced and up-regulated. This indicates to some extent that Rls may be a significant improvement in the disease resistance of passion fruit plants to TeMV by improving their antioxidant capacity.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. The application of rhamnolipid in preventing and treating plant nocturnal flower geranium virus diseases is characterized in that the use concentration of the rhamnolipid is 0.4g/L-1.0 g/L.

2. Use according to claim 1, wherein the plant is a passion fruit.

3. The use of claim 2, wherein the plant is gold passion fruit.

4. A method of controlling a plant nocturnal geranium virus disease, comprising: spraying a preparation whose active ingredient comprises rhamnolipid, the concentration of rhamnolipid used is 0.4g/L-1.0 g/L.

5. The method for controlling the plant nocturnal flower geranium virus disease according to claim 4, wherein the spraying part of the preparation is the plant leaves and/or stems.

6. The method for controlling the plant nocturnal flower-leaf virus disease according to any one of claims 4 to 5, characterized in that the spraying mode of the preparation is 1 to 2 times per day.

7. The method for controlling the nocturnal mosaic virus disease of plants according to any one of claims 4-6, wherein the plants are passion fruit.

8. The method for controlling the plant nocturnal flowery knotweed leaf virus disease according to claim 7, wherein the plant is golden passion fruit.

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