CN115715541A - Application of tea saponin TS in preparation of medicine for preventing and controlling apple tree fungal diseases - Google Patents

Abstract

The invention discloses application of tea saponin TS in preparation of a medicine for preventing and controlling apple tree fungal diseases induced by Valsa mali bacteria or Botryosphaeria dothidea bacteria or Alternaria Alternaria f.sp.mali bacteria, wherein the minimum inhibitory concentration of the tea saponin TS to the Valsa mali bacteria is 0.001%, the minimum inhibitory concentration to the Botryosphaeria dothidea bacteria is 0.01%, and the minimum inhibitory concentration to the Alternaria Alternaria f.sp.mali bacteria is 0.1%. The invention adopts tea saponin TS to inhibit Valsa mali bacteria, botryosphaeria dothidea bacteria and Alternaria alternata f.sp.mali bacteria to achieve good effects, and in addition, the tea saponin TS can also enhance the disease-resistant reaction of apple leaves by inducing the activities of chitinase and beta-1, 3-glucanase.

Description

Application of tea saponin TS in preparation of medicine for preventing and controlling apple tree fungal diseases

Technical Field

The invention relates to preparation of a medicament for preventing and controlling apple tree fungal diseases, in particular to application of tea saponin TS in preparation of a medicament for preventing and controlling apple tree fungal diseases.

Background

Secondary metabolites are a very important class of organic compounds in plants and play an important role in plants' response to complex environments. Secondary metabolites in plants mainly comprise three major classes, namely phenols, terpenes and nitrogenous compounds. At present, numerous studies have shown that secondary metabolites play a broad role in the interaction between plants and biotic, abiotic factors, in particular in the protection of plants from predators and microorganisms. Based on their properties in plant protection, a variety of secondary metabolites of plant origin have been developed as plant insecticides or fungicides. Tea tree is a commercial crop rich in secondary metabolites, and tea beverage is considered as a functional beverage beneficial to health because of being rich in secondary metabolites such as tea polyphenol, catechin, amino acid and the like. Tea Saponin (Tea Saponin, TS) is one of the important secondary metabolites of Tea plant, widely found in roots, stems, leaves, flowers and seeds of plants of the camellia genus. TS is mainly extracted from seeds of the camellia, and in actual production, a byproduct, namely camellia seed meal, after camellia oil is extracted from the seeds is a main source for extracting the tea saponin TS. Tea Saponin (TS) is widely used in antibacterial applications, but the effect varies with the bacteria. For example, the inhibition rate of 0.1% TS on tea gray blight fungus (Pestalotiopsis theta) is 49.28%; the inhibition of monilinia fructicola (monilinia fructicola) by TS at a concentration of 0.737% is only 46.6%: the 10% high TS concentration also inhibits tea wheel spot pathogen and tea white star pathogen only 74.58% and 88.06%.

Apple is one of the most widely planted fruits in the world and is popular due to its excellent nutritional value and unique mouthfeel. As a perennial woody plant, it is highly susceptible to a variety of pathogens, including fungi, bacteria and viruses, during the growth of the apple tree. Wherein, the pathogenic fungus Valsa mali induces apple canker, the disease symptoms of the apple canker mainly occur in trunks, branches and fruits, the canker part mainly shows ulcerative wounds, tree vigor and yield are influenced, and the death of trees can be seriously caused. Pathogenic fungi Botryosphaeria dothidea induces apple ring rot, disease symptoms mainly occur in trunks and fruits, infected fruit symptoms are soft and sunken damages, alternating tan and brown rings exist, and fruit quality and commodity value are seriously influenced. The branches infected with the ring spot show the symptoms of ulcer, damage, necrosis and the like, and inhibit the growth of tree bodies, even lead to the death of the branches. Malli is a main pathogenic factor of Alternaria leaf spot, and the disease is characterized in that small round brown or black necrotic spots are induced on leaves and gradually expand, the edges are brownish purple, and the disease causes the photosynthesis of the leaves to be weakened and even leaves to fall, and finally the yield and the quality of fruits are influenced. The three diseases are three most serious diseases in the production and cultivation of the current apples, particularly rot and ring rot which directly produce diseases in fruits, cause serious influence on the yield and quality of the apples, and are important factors for inhibiting the green and healthy development of the apple industry.

In the current production of apples, chemical agents remain the main agents for controlling fungal diseases of apples. However, excessive and long-term use of chemical agents not only seriously affects the food safety of fruits, but also has great destructive effect on the ecological environment of orchards, and indirectly affects the yield and quality of the fruits. Therefore, the development and utilization of environmentally friendly plant-derived fungicides are increasingly receiving attention. In view of the antifungal function of tea saponin TS, the present study was directed to Valsa mali or Botryosphaeria dothidea or Alternaria alternata f.sp.mali causing important diseases in apple production, investigating the in vitro and in vivo inhibitory effects of tea saponin TS on the three pathogenic fungi mentioned above.

Disclosure of Invention

Aiming at the problem that the apple fungus diseases induced by Valsa mali bacteria, botryosphaeria dothidea bacteria and Alternaria alternata f.sp.mali bacteria are rarely subjected to plant source bactericides, the invention provides application of tea saponin TS in preparing a medicament for preventing and controlling apple fungus diseases induced by Valsa mali bacteria, botryosphaeria dothidea bacteria or Alternaria alternata f.sp.mali bacteria, and the tea saponin can inhibit the growth of the fungi and achieve good effects by singly using the tea saponin.

In order to achieve the purpose, the invention provides application of tea saponin TS in preparing a medicine for preventing and controlling apple fungus diseases induced by Valsa mali bacteria or Botryosphaeria dothidea bacteria or Alternaria alternata f.

Specifically, the minimum inhibitory concentration of the tea saponin TS to Valsa mali bacteria is 0.001%; and/or

The minimum inhibitory concentration of the tea saponin TS to Botryosphaeria dothidea is 0.01%; and/or

The minimum inhibitory concentration of the tea saponin TS to Alternaria Alternaria f.sp.mali is 0.1%.

Through the technical scheme, the invention has the following beneficial effects:

the tea saponin TS is adopted to inhibit Valsa mali bacteria, botryosphaeria dothidea bacteria and Alternaria Alternaria f.sp.mali bacteria, so that a good effect is achieved. In addition, tea saponin TS can also induce and enhance the disease resistance of the apple leaves.

Drawings

FIG. 1 shows the in vitro test results of tea saponin TS on Valsa mali bacteria;

FIG. 2 shows the results of in vitro experiments of tea saponin TS on Botryosphaeria dothidea;

fig. 3 is the results of in vitro testing of theasaponin TS against Alternaria f.sp.mali;

FIG. 4 shows the results of in vivo experiments of tea saponin TS on Valsa mali;

FIG. 5 shows the results of in vivo experiments of tea saponin TS on Botryosphaeria dothidea;

fig. 6 is the results of in vivo testing of tea saponin TS against Alternaria f.sp.mali;

FIG. 7 shows the measurement results of active oxygen content and enzyme activity when apple leaves were treated with tea saponin TS.

Detailed Description

The following examples are provided to explain the present invention in detail. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

In the following examples, tea saponin TS was purchased from shanghai source leaf biotechnology limited; apple plants were from a nursery plant of Taian, shandong.

Botryosphaeria dothidea and Alternaria Alternaria f.sp.mali were stored in the laboratory of the applicant for a long time, and Valsa mali was obtained from the university of agriculture and forestry, northwest, all of which were cultured on potato dextrose agar medium (PDA) in an incubator at 28 ℃.

All data were analyzed for one-way analysis of variance (ANOVA) using windows Data Processing System (DPS) statistical software. Mean resolution was determined by Duncan multiple range test (P < 0.05).

EXAMPLE 1 in vitro antifungal assay of tea Saponin TS

Serial concentration gradient tea saponin TS was tested for hyphal growth inhibition of Valsa mali, botryosphaeria dothidea and Alternaria alternata f. A hyphal mass of about 0.55cm in diameter was excised from the hyphal rim of a 2-day culture medium for Valsa mali, a 5-day culture medium for Botryosphaeria dothidea and a 9-day culture medium for Alternaria alternata f.sp.mali by using an inoculating loop for one week and placed in the center of a PDA culture dish of 9cm in diameter. After inoculation, the cells were cultured at 28 ℃ and the difference between the experimental group and the control group was observed.

The antifungal index was calculated as follows: inhibition (%) = (Da-Db)/(Da-0.55) × 100, where Da is the diameter of plaque growth (cm) on PDA medium supplemented with tea saponin TS and Db is the radius of plaque growth (cm) on PDA medium not supplemented with tea saponin TS. The average inhibition (%) is the average of the inhibition at different times. The results are shown in FIGS. 1 to 3.

As shown in FIG. 1, the growth of Valsa mali was inhibited on PDA plates supplemented with tea saponin TS. In the control PDA plate, the hyphae grow rapidly and it takes 3 days for the Valsa mali bacteria to overgrow the control PDA plate. When the TS content in the culture medium is 0.0001%, the growth of the apple Valsa mali hyphae is not obviously inhibited. When the TS concentration is more than 0.001%, the growth of the Valsa mali bacteria is obviously inhibited. The antifungal rates at 0.001%, 0.01% and 0.05% TS were 80.5%, 90.0% and 100%, respectively.

Also, as shown in FIG. 2, the growth of Botryosphaeria dothidea was inhibited on PDA plates supplemented with tea saponin TS. On the control PDA medium, hyphae grow rapidly, and it takes 7 days for Botryosphaeria dothidea to overgrow the control PDA medium. When the medium contained 0.001% TS, the hyphal growth of Botryosphaeria dothidea was not significantly inhibited. When the TS concentration is more than 0.01%, the growth of Botryosphaeria dothidea is obviously inhibited. The antifungal rates at TS of 0.01%, 0.03% and 0.05% were 55.9%, 84.4% and 96.5%, respectively.

In contrast, as shown in fig. 3, hyphae growth was rapid on the control PDA plates, which took 12 days for the Alternaria f. When the culture medium contains 0.01% of TS, the hyphal growth of the Alternaria alternata f.sp.mali is not obviously inhibited, and the bacteriostasis rate is only 13.3%. When the TS concentration is in the range of 0.05% -1%, the inhibition rate is between 31.9% -34.9%.

EXAMPLE 2 in vivo antifungal assay for tea saponin TS

In vivo antifungal effect test was performed with tea saponin TS of different concentration gradients. Leaves that were fully developed on annual shoots were selected, washed with sterile water and air dried. Wrapping the basal part of the petiole with sterile cotton dipped with sterile water for later use. After a series of concentrations of tea saponin TS were sprayed onto the leaves and dried, a small wound was manually poked on both sides of the main vein with an inoculating needle. A plaque piece with a diameter of 0.55cm was placed over the leaf wound. The purpose of an artificial wound is to promote the infestation of pathogenic fungi.

Control leaf (CK) was treated with sterile distilled water. The leaves were stored at 28 ℃. After leaf infection, disease grades were classified as follows according to the ratio of necrotic lesion area to total leaf area: grade I, <1%; class II, 1-10%; grade III, 11-25%; grade IV, 26-40%; v grade, 41-65%; class VI, >66%. The results of the in vivo antifungal activity are shown in FIGS. 4 to 6.

As shown in FIG. 4, consistent with the results of in vitro antifungal experiments, TS can significantly inhibit the infection of Valsa mali bacteria on leaves. After inoculation of Valsa mali bacteria, the detached leaves of the apples sprayed with sterile water are severely necrotic. In contrast, the low concentration of 0.1% TS can significantly reduce the infected area of the leaf and reduce the disease grade. Furthermore, as TS concentration increases, both the area of infection and the disease grade decrease. TS at 5% concentration greatly inhibited disease development, the necrotic area in the control group was 8-fold greater than in the TS-treated group, and 5% TS-treated leaf disease grade was mild grade II.

In addition, infection by pathogens causes oxidative burst of reactive oxygen species, causing hypersensitivity reactions that in turn lead to extensive cellular necrosis. H ₂ O ₂ And Oxygen Free Radicals (OFR) were used as indicators reflecting Reactive Oxygen Species (ROS) outbreaks. With increasing TS concentration, the active oxygen content, including hydrogen peroxide and superoxide anion, decreased, corresponding to the trend of lesion area. In addition, the activities of two defense-related enzymes (chitinase and beta-1, 3-glucanase) after spraying TS are obviously higher than those of a control group, which shows that the defense response of leaves is obviously improved after spraying TS.

As shown in FIG. 5, the control leaves were severely damaged at day 6 after inoculation with Botryosphaeria dothidea, accounting for 35% -42% of the total leaf area. TS treatment significantly reduced the area of damage to apple leaves inoculated with Botryosphaeria dothidea. As the TS concentration increased, the necrotic area became smaller, 5% TS-treated necrotic area was only 3% of the control group. The control leaf had a disease rating of grade IV or above, while 5% TS treated leaves had a disease rating below grade II.

After TS treatment, the content of active oxygen substances such as hydrogen peroxide, superoxide anion and the like is obviously reduced, and cell death caused by ROS explosion is avoided. Furthermore, with increasing TS concentration, the activities of chitinase and beta-1, 3-glucanase increased significantly.

As shown in fig. 6, when TS was sprayed at a concentration of 5%, the necrotic area of the leaf was 50% of that of the control group. Despite the administration of 5% TS, the disease grade still progressed to grade III. Although the active oxygen content decreases with the increase of the TS concentration, the variation range is small. However, TS still induced chitinase and β -1, 3-glucanase activity in the presence of Alternaria f.

EXAMPLE 3 determination of active oxygen content and enzyme Activity

Using hydrogen peroxide (H) ₂ O ₂ ) And Superoxide anion (OFR) corresponding kit. The enzymatic activities were measured using kits of chitinase and beta-1, 3-glucanase, respectively (Suzhou Comin Biotechnology Co. Ltd, suzhou, china).

To determine the effect of TS on apple leaves, the effect of TS on active oxygen content and defensive enzyme activity was tested by spraying apple detached leaves with TS at concentrations of 0%, 0.1%, 0.5%, 1%, 2%, 5% without inoculation of pathogenic bacteria, and the results are shown in fig. 7. The results show that the content of active oxygen in the apple leaves in vitro does not increase sharply because the apple leaves in vitro are not infected by pathogenic bacteria. However, after spraying TS at a range of concentrations, the active oxygen content in the leaves of the in vitro apples still decreases with increasing TS concentration, which may be due to the effect of the antioxidant properties of TS. In addition, the activities of chitinase and glucanase are obviously increased along with the increase of the concentration of TS, and the TS is further proved to be capable of inducing the activities of chitinase and beta-1, 3-glucanase of the apple trees so as to enhance the disease-resistant response of the apple trees.

The preferred embodiments of the present invention have been described in detail with reference to the examples, but the present invention is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims (2)

1. An application of tea saponin TS in preparing a medicine for preventing and controlling apple tree fungal diseases induced by Valsa mali bacteria or Botryosphaeria dothidea bacteria or Alternaria Alternaria f.

2. The use of claim 1, wherein the minimum inhibitory concentration of tea saponin TS to Valsa mali bacteria is 0.001%; and/or

The minimum inhibitory concentration of the tea saponin TS to Botryosphaeria dothidea is 0.01%; and/or

The minimum inhibitory concentration of the tea saponin TS to Alternaria Alternaria f.sp.mali is 0.1%.

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