Application of irisin and belamcanda glycoside in the control of weeds, diseases and insect pests of crops

Abstract

The present invention discloses the application of irisin and its glycoside, belamcanda glycoside, in the prevention and control of weeds and pests of crops. The addition of irisin to the culture medium has an inhibitory effect on the growth of Arabidopsis seedlings, but has no inhibitory activity on the growth of crops corn and rice. Irisin has a significant inhibitory effect on the growth of pathogenic bacteria of crop diseases such as Rhizoctonia solani, rice white leaf blight pathogen, and rice bacterial leaf streak pathogen. The spraying of belamcanda glycoside has a significant inhibitory effect on rice sheath blight. Irisin and belamcanda glycoside both have antifeedant activity on the larvae of the agricultural pest fall armyworm. It can be seen that irisin and belamcanda glycoside can be used for the prevention and control of weeds and pests of crops. The present invention has important application value and application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of isoflavone tectoriusin and its glycosylation product tectoriusin in the prevention and control of weeds and pests of crops. Background Art

[0002] Isoflavones are a class of metabolites unique to leguminous plants. Studies have shown that isoflavones can participate in the interaction between plants and microorganisms, and can also participate in plant response to biological stress as precursors of phytoalexins. Iridogenin is a secondary metabolite of isoflavones that is abundant in Belamcanda chinensis. Belamcanda chinensis is a perennial herb of the genus Belamcanda in the family Iridaceae. As a non-leguminous plant, Belamcanda chinensis contains specific 6-methoxyisoflavones. Although the content of irisogenin and its glycoside Belamcanda glycoside is high in the rhizomes of Belamcanda chinensis, there is currently little research on their biological activity.

[0003] Crops face many biotic and abiotic stresses during their growth, among which biotic stresses such as competitive weeds, plant pathogens, and agricultural pests are the main factors affecting crop yields. Plants have evolved various metabolites during their long evolution, some of which can serve as defense compounds against these biotic stresses. Research on the activity of plant metabolites is helpful for the development of new, environmentally friendly biological control agents.

[0004] Plants release some secondary metabolites into the environment during their growth. These metabolites have certain promoting and inhibiting effects on the growth and development of surrounding plants, which is called plant allelopathic effect (Shi Xiaoping et al. 2020 Research Progress on Plant Allelopathic Effect. Biotechnology Bulletin 36(6):215-222). It is a defense mechanism for plants to cope with the environment. In addition, plant metabolites have weak autotoxicity and strong sensitivity, and can be developed as herbicides for crops, which is of great significance for reducing the invasion of competitive weeds and increasing crop yields.

[0005] Crop diseases are important factors affecting crop quality and yield, and seriously affect food security. Rice sheath blight is caused by infection with Rhizoctonia solani and is one of the main diseases of rice. The pathogen of rice sheath blight spreads in the soil, field weeds and rice seedlings in the form of sclerotia and spores through irrigation and other methods, gradually infecting the leaf sheaths, leaves, stems and panicle necks of rice, causing the leaves to wither and die, seriously affecting the normal growth and growth quality of rice. It has the characteristics of a wide range of disasters, deep impact and fast spread (Xia Hanyan. 2021 Research Progress on Prevention and Control of Rice Sheath Blight. Southern Agriculture 15(27):12-14). As rice sheath blight becomes increasingly serious, many traditional prevention and control methods are difficult to achieve the desired effect.

[0006] Bacterial leaf streak of rice and bacterial leaf blight of rice are also common diseases of rice, which seriously affect the yield and quality of rice. Both bacterial leaf streak of rice and bacterial leaf blight of rice are caused by the invasion of Xanthomonas oryzae variants. The PXO99 strain is a common pathogen of bacterial leaf blight of rice, and the HGA4 strain is a highly pathogenic pathogen of bacterial leaf streak of rice. In addition, the above three diseases are also pathogenic to other crops such as corn and wheat. Finding metabolites with growth inhibitory activity against representative pathogens of these three diseases is of great significance for improving the quality and yield of crops.

[0007] Fall armyworm is a highly destructive omnivorous pest with a wide range of host plants and feeds on a variety of crops. It is widely distributed in tropical and subtropical regions, including Asia, Africa, North America and Oceania, causing large agricultural losses worldwide (Ruixiang Tang, et al. Transcriptomics and metagenomics of common cutworm (Spodoptera litura) and fall armyworm (Spodoptera frugiperda) demonstrates differences in detoxification and development. BMC Genomics 23: 388.). The current method of controlling this pest is mainly synthetic pesticides, but chemical reagents are harmful to humans and the environment, and are prone to produce drug-resistant individuals. Exploring the anti-agricultural pest activity of natural metabolites has important research value and application prospects.

[0008] Plants produce a variety of secondary metabolites to adapt to the changing ecological environment. Exploring plant defensive metabolites is of great significance for finding biological control methods for weeds and pests of crops. Summary of the invention

[0009] In view of the above technical problems, the purpose of the present invention is to provide a new use of irisin and its glycoside belamcanda glycoside in the prevention and control of weeds and pests of crops.

[0010] The inventors discovered that irisin and belamcandarin are involved in the chemical defense of crops against various biotic stresses, including potential growth inhibition of other plants, resistance to plant pathogens, and antifeedant and growth inhibitory activity against insect pests.

[0011] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0012] In a first aspect, the present invention provides the use of irisin and its glycoside belamcandarin in the prevention and control of weeds, diseases and insect pests of crops.

[0013] Furthermore, tectoriusin and belamcanda glycoside belong to a pair of aglycones and glycosides, and belamcanda glycoside is the product of glycosylation at the 7-position of tectoriusin.

[0014] Furthermore, the weeds are other plants that have a growth competitive effect on crops.

[0015] Furthermore, the weeds include wild-type Arabidopsis thaliana.

[0016] Furthermore, the disease is caused by infection by pathogenic bacteria, which causes harm to the growth of crops.

[0017] Furthermore, the diseases include rice sheath blight, rice bacterial leaf blight and rice bacterial leaf streak.

[0018] Furthermore, the pathogens of the plant diseases include Rhizoctonia solani, the pathogen of rice bacterial leaf blight PXO99 and the pathogen of rice bacterial leaf streak HGA4.

[0019] Furthermore, the insect pests are caused by herbivorous animals that feed on crops, which affect the growth of crops.

[0020] Furthermore, the herbivorous animal includes the fall armyworm.

[0021] The present invention explores the functions of tectoriusin and belamcanda glycosides in assisting crops to resist biological stress from three aspects: weed control, disease resistance and pest control. The present invention selects representative plants, pathogens or agricultural pests in various aspects for experiments. The present invention takes wild-type Arabidopsis as an example to explore the growth inhibition effect of tectoriusin and belamcanda glycosides on weeds. The present invention takes the rice sheath blight pathogen Rhizoctonia solani, rice white leaf blight pathogen PXO99 and rice bacterial leaf streak pathogen HGA4 as examples, and explores the inhibitory effect of tectoriusin and belamcanda glycosides on pathogens through in vitro mycelium or colony growth experiments combined with sheath blight resistance experiments in rice. The present invention takes the fall armyworm as an example to explore the antifeedant activity and growth inhibition activity of tectoriusin and belamcanda glycosides on the herbivorous animal fall armyworm. Experiments show that tectoriusin and its glycoside belamcanda glycosides exhibit good weed, disease and pest control effects.

[0022] In a second aspect, the present invention provides the use of irisin as a biological pesticide for controlling weeds, diseases and pests of crops.

[0023] Furthermore, the irisin has no inhibitory activity on the growth of crops rice and corn.

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides the application of irisin and its glycoside belamcanda glycoside in weeding, resisting diseases and controlling pests of crops. The isoflavone irisin and its glycoside belamcanda glycoside have potential allelopathic effects and show significant antibacterial and anti-insect activities. At the same time, irisin and belamcanda glycoside have no inhibitory activity on the growth of crops. Irisin and its glycoside belamcanda glycoside can help crops resist various biological stresses, have the potential for the development of multifunctional biological pesticides, and have important research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A plan view of Arabidopsis seedlings grown for several days in a medium containing tectoriusin or belamcandarin.

[0027] Figure 2 The root length statistics of Arabidopsis seedlings after they were grown in the culture medium containing tectoriusin or belamcanda glycoside for several days, where CK is the control group, the same below.

[0028] Figure 3 Statistical graph of leaf spacing of Arabidopsis seedlings after growing on medium containing tectoriusin or belamcandarin for several days

[0029] Figure 4 A graphic representation of rice seedlings grown in a culture medium containing tectoriusin or tectoriusin for several days

[0030] Figure 5 The graph shows the root length of rice seedlings after they were grown in culture medium containing tectoriusin or belamcandarin for several days.

[0031] Figure 6 The graph shows the statistical growth of rice seedlings after they were grown in culture medium containing tectoriusin or belamcandarin for several days.

[0032] Figure 7 This is a plan view of corn seedlings grown in culture medium containing tectoriusin or belamcandarin for several days.

[0033] Figure 8 This is a statistical chart of the root length of corn seedlings after they were grown in culture medium containing tectoriusin or belamcandarin for several days.

[0034] Fig. 9 This is a plan view showing the growth of Rhizoctonia solani YWK196, the pathogen of rice bacterial leaf blight PXO99 and the pathogen of rice bacterial leaf streak HGA4 on culture media containing irisin and belamcandarin, respectively.

[0035] Fig.10 This is a statistical graph of the diameters of Rhizoctonia solani YWK196, the pathogen of rice bacterial leaf blight PXO99, and the pathogen of rice bacterial leaf streak HGA4 growing on a culture medium containing tectoriusin and belamcandarin.

[0036] Fig.11 This is a picture showing the lesions of rice leaf sheaths infected by Rhizoctonia solani after being pretreated with irisin and belamcanda glycosides.

[0037] Fig.12 This is a statistical chart of the lesion length of rice leaf sheaths infected by Rhizoctonia solani and pretreated with tectoriusin and belamcandarin.

[0038] Fig.13 This is a plan view of leaves after the fall armyworm feeds on leaves treated with DMSO, irisin and belamcandarin.

[0039] Fig.14 This is a statistical chart of the feeding area of ​​the fall armyworm on leaves treated with DMSO, irisin and belamcandarin.

[0040] Fig.15 The figure shows the body shape of the fall armyworm after feeding the diet supplemented with DMSO, irisin and irisin.

[0041] Fig.16 Statistical chart of body weight changes of fall armyworm after feeding feed supplemented with DMSO, irisin and irisin.

[0042] Fig.17 Schematic diagram of the biological activities of irisin and belamcanda glycosides. DETAILED DESCRIPTION

[0043] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0044] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0045] Example 1

[0046] Growth inhibition of Arabidopsis thaliana

[0047] Irisin and belamcanda glycosides (Chengdu Zhibiao Huachun Biotechnology Co., Ltd., ≥98%) were prepared into 200 mM stock solutions with DMSO and set aside.

[0048] Soak wild-type Arabidopsis seeds in 1 mL of sterile distilled water for 1 minute, sterilize with 80% ethanol for 2 minutes, and then blow with sterile distilled water 5-6 times to wash away the ethanol on the surface of the Arabidopsis seeds. Sow the sterilized Arabidopsis seeds evenly on 1 / 2MS culture medium. After sowing, vernalize in a 4°C refrigerator for 2 days, and culture in a light culture room (lighting conditions are 16h light, 8h dark, and culture temperature is 22°C) for 5-6 days.

[0049] Heat and melt 1 / 2MS culture medium, take 4mL of culture medium into a shaking tube, add 2μL and 6μL of mother solution respectively to make the final concentration of the culture medium 100μM or 300μM, add the corresponding amount of DMSO to the control group, and divide 4ml of culture medium into two wells of a six-well plate. After the culture medium cools and solidifies, use a sterilized toothpick to transplant the seedlings to the surface of the 1 / 2MS culture medium containing the corresponding compound. Transplant 10 seedlings of the same growth in each group and culture them vertically in a light culture room. After 3-4 days of cultivation, take photos to record the growth of Arabidopsis, measure the root length and leaf spacing with a ruler, and collect statistics. Figure 1 , 2 As shown in Figures 3 and 4, compared with the negative control DMSO group, 100 μM and 300 μM irisin had a significant inhibitory effect on the root growth and leaf development of Arabidopsis, and the inhibitory effect was stronger at high concentrations, while belamcanda glycosides had a weak inhibitory effect on the root growth of Arabidopsis only at a high concentration of 300 μM.

[0050] Example 2

[0051] Effects on the growth of rice and corn

[0052] Irisin and irisin were prepared into 200 mM stock solutions with DMSO respectively and set aside.

[0053] Soak the rice seeds in pure water and place them in a 37°C incubator for two days. Prepare aqueous solutions of irisin or irisin with concentrations of 100μM and 300μM respectively, and use DMSO solutions of corresponding concentrations as controls. Make up the DMSO for the low-concentration solutions according to the amount of DMSO added to the high-concentration solutions. Fold a paper towel and place it in a square culture dish (100mm×100mm), and add 10mL of a solution containing the corresponding compound to soak the paper towel. Place the rice seeds evenly on the culture dish, seal the culture dish with breathable tape, and place it in a light culture room (lighting conditions are 16h light, 8h dark, and culture temperature is 28°C), with 15 seeds of uniform size placed in each group. After 6-7 days of culture, take photos to record the growth of the rice, measure the root length with a ruler, and collect statistics. Figure 4 , 5 As shown in Figure 6, the effects of 100 μM and 300 μM tectoriusin or belamcanda glycoside on the root growth of rice are consistent with the solvent control (DMSO) treatment, indicating that 100 μM and 300 μM tectoriusin and belamcanda glycoside have no significant inhibitory activity on the root growth of rice.

[0054] Soak the corn seeds in pure water and place them in a 37°C incubator for two days. Prepare 100μM and 300μM irisin aqueous solutions and irisin aqueous solutions. The corresponding concentrations of DMSO solutions are used as controls. The low-concentration solutions are supplemented with DMSO according to the amount of DMSO added to the high-concentration solutions. Fold a paper towel and place it in a square culture dish (100mm×100mm), and add 10mL of a solution containing the corresponding compound to soak the paper towel. Place the rice seeds evenly on the culture dish, seal the culture dish with breathable tape, and place it in a light culture room (lighting conditions are 16h light, 8h dark, and culture temperature is 28°C), with 15 seeds of uniform size placed in each group. After 6-7 days of culture, take photos to record the growth of the corn, measure the root length with a ruler, and collect statistics. Figure 7 , 8 As shown, the effects of 100 μM and 300 μM tectoriusin or belamcanda glycoside on the root growth of corn were consistent with those of the solvent control (DMSO) treatment, indicating that 100 μM and 300 μM tectoriusin and belamcanda glycoside had no significant inhibitory activity on the root growth of corn.

[0055] Example 3

[0056] In vitro inhibitory effect on Rhizoctonia solani

[0057] In the clean bench, use a sterilized toothpick to pick the hyphae of R.solani AG1-IAstrain YWK196 and place it in the center of the surface of PDA medium (potato dextrose agar, 20% potato, 2% glucose, 2% agar, w / v), and invert it in a 28°C incubator for 2-3 days. Use a 6mm puncher to punch a bacterial disc at the edge of the colony and inoculate it in the center of the PDA medium containing the compound. Irisin and Belamcandarin were prepared into 200mM stock solutions with DMSO, respectively, and diluted to 100μM, 200μM and 300μM with culture medium. The low concentration solution was supplemented with DMSO according to the amount of DMSO added to the high concentration solution, and the control group was added with the corresponding amount of DMSO. After static culture in a 28°C incubator for 18-24h, the growth diameter of the hyphae was measured by the cross method. Fig. 9 , 10 As shown, compared with the solvent control (DMSO), 100μM, 200μM and 300μM tectoriusin had a significant inhibitory effect on the growth of Rhizoctonia solani, and the effect increased with increasing concentration. 200μM and 300μM tectoriusin had an inhibitory effect on Rhizoctonia solani, but the inhibitory effect was much smaller than that of tectoriusin.

[0058] Example 4

[0059] In vitro inhibitory effects on Xanthomonas oryzae PXO99 and HGA4

[0060] Resuscitate Xanthomonas oryzae pv.oryzae and Xanthomonas oryzae pv.oryzicola from cryopreserved tubes in PSA medium (Potato sucroseagar, 0.7% potato extract, 2% sucrose, 2% agar, w / v), and culture in a shaking incubator at 28°C until OD 620 =0.5. Pour the culture medium containing the compound (irisin or irisin) into a six-well plate. After the culture medium solidifies, add 2 μL of bacterial solution to the center of the six-well plate. Irisin and irisin were prepared into 200 mM stock solutions with DMSO, respectively, and diluted to 100 μM, 200 μM and 400 μM with culture medium. The low-concentration solution was supplemented with DMSO according to the amount of DMSO added to the high-concentration solution, and the control group was added with the corresponding amount of DMSO. After standing in an incubator at 28°C for 48 hours, the colony growth diameter was measured using the cross method. Fig. 9 , 10 As shown, compared with the solvent control (DMSO), 100 μM, 200 μM and 400 μM irisin had a significant inhibitory effect on the growth of PXO99 and HGA4, while 100 μM, 200 μM and 400 μM belamcanda glycosides had no significant inhibitory effect on the growth of PXO99 and HGA4.

[0061] Example 5

[0062] Inhibitory activity of rice against Rhizoctonia solani in vivo

[0063] Rice (Oryza sativa) was inoculated with Zhonghua No. 11 for rice sheath blight. The rice was grown in a greenhouse at 22°C and 60% relative humidity with a 16:8h (light:dark) cycle for about 10 weeks (before heading stage). The Rhizoctonia solani strain (R.solani AG1-IA strain YWK196) was activated on PDA medium (potato dextrose agar, 20% potato, 2% glucose, 2% agar, w / v) at 28°C. The sterilized paper pieces were placed on the PDA medium, and the activated mycelium was inoculated in the culture dish. The culture was cultured at 28°C for 2-3 days, and the paper pieces with mycelium were selected for inoculation. Eight plants containing irisin or irisin in their leaf sheaths were inoculated with Rhizoctonia solani, and plants containing DMSO were inoculated as negative controls. The length of the lesions was measured 5 days after inoculation. Fig.11 , 12As shown in the figure, compared with the solvent control (DMSO), the rice leaf sheath lesion symptoms of 300 μM irinotecan pre-treated rice were weaker and the lesion length was significantly shortened. However, the same concentration (300 μM) of irisin treatment failed to inhibit the growth of lesions and showed a similar lesion length to the control treatment.

[0064] Example 6

[0065] Antifeedant activity against fall armyworm

[0066] Irisin and irisin were prepared into 100 mM stock solutions with DMSO, respectively, and diluted to 100 μM with 0.1% (v / v) Tween-20 for later use. A 100 μM DMSO solution was prepared by diluting 0.1% (v / v) Tween-20 as a negative control. The third-instar larvae of the fall armyworm were starved for 6-12 hours. A 12 mm hole puncher was used to punch holes in tender corn leaves to obtain 12 mm leaf discs. The leaf discs were immersed in the test solution for 30 seconds and then taken out. After the solvent on the leaf surface evaporated, they were placed in a 12-well plate. One third-instar larvae was inoculated into each well and placed in a light culture room (lighting conditions: 16 hours light-8 hours dark, humidity: 85%, temperature: 26°C) for culture. The leaf feeding situation was recorded after 48 hours, and the feeding area was calculated using ImageJ software for statistical analysis. Fig.13 , 14 As shown, compared with the solvent control (DMSO), leaves treated with 100 μM tectoriusin or 100 μM belamcandarin showed significant antifeedant activity against Spodoptera frugiperda.

[0067] Example 7

[0068] Growth inhibition of fall armyworm

[0069] Irisin and Belamcanda glycosides were prepared into 100mM stock solutions with DMSO, respectively. The compound stock solutions were added to the feed to make the final concentration of 0.4μmol / g, and DMSO was used as a control. The feed was divided into 10mg and added to the wells of a 24-well plate in sequence, and filter paper pieces of the same size were placed in the well plate to keep it moist. Fifteen third-instar larvae of fall armyworm that had been starved (6 hours) were weighed in sequence and placed in a 24-well plate (the holes on the edge of the 24-well plate were left empty). After 24h, the original feed was taken out and fresh feed mixed with the compound was fed, and the process was repeated once. The fall armyworm larvae were taken out and weighed. Weight change = weight after two feedings with the compound - initial weight. As Fig.15 , 16 As shown, compared with the solvent control (DMSO), 0.4 μmol / g belamcanda glycosides showed significant inhibitory activity on the growth of Spodoptera frugiperda.

[0070] The above description is only a preferred specific implementation manner of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the scope of protection of the invention.

Claims

1. An application of irisin in controlling weeds, diseases and pests of crops, characterized in that: The crop weed is Arabidopsis thaliana, and the concentration of irisin used in the control of the crop weed is 100 μM and 300 μM; The crop diseases are caused by infection by Rhizoctonia solani, rice bacterial leaf blight pathogen PXO99 and rice bacterial leaf streak pathogen HGA4, which cause damage to the growth of crops. For Rhizoctonia solani, the concentration of irisin used in the prevention and control of the crop diseases is 100 μM, 200 μM and 300 μM; for rice bacterial leaf blight pathogen PXO99 and rice bacterial leaf streak pathogen HGA4, the concentration of irisin used in the prevention and control of the crop diseases is 100 μM, 200 μM and 400 μM; The crop pests are caused by the fall armyworm feeding on crops, which affects the growth of crops. When the fall armyworm exhibits antifeedant activity, the concentration of irisin used in the crop pest control is 100 μM.

2. A use of irisin as a pesticide for controlling weeds, diseases and pests of crops, characterized in that: The crop weeds are Arabidopsis thaliana, and the concentration of irisin used in the control of the crop weeds is 100 μM and 300 μM; The crop diseases are caused by infection by Rhizoctonia solani, rice bacterial leaf blight pathogen PXO99 and rice bacterial leaf streak pathogen HGA4, which cause damage to the growth of crops. For Rhizoctonia solani, the concentration of irisin used in the prevention and control of the crop diseases is 100 μM, 200 μM and 300 μM; for rice bacterial leaf blight pathogen PXO99 and rice bacterial leaf streak pathogen HGA4, the concentration of irisin used in the prevention and control of the crop diseases is 100 μM, 200 μM and 400 μM; The crop pests are caused by the fall armyworm feeding on crops, which affects the growth of crops. When the fall armyworm exhibits antifeedant activity, the concentration of irisin used in the crop pest control is 100 μM.

3. An application of belamcanda glycosides in the prevention and control of crop diseases and insect pests, characterized in that: The crop diseases are caused by infection by Rhizoctonia solani, which harms the growth of crops, and the concentration of belamcanda glycoside used in the prevention and treatment of the crop diseases is 200 μM and 300 μM; The crop pests are caused by the fall armyworm feeding on crops, which affects the growth of crops; when the fall armyworm exhibits antifeedant activity, the concentration of belamcanda glycoside used in the crop pest control is 100 μM; when inhibiting the growth of the fall armyworm, the concentration of belamcanda glycoside used in the crop pest control is 0.4 μmol / g.

4. An application of belamcanda glycosides as a pesticide for controlling crop diseases and insect pests, characterized in that: The crop diseases are caused by infection by Rhizoctonia solani, which harms the growth of crops, and the concentration of belamcanda glycoside used in the prevention and treatment of the crop diseases is 200 μM and 300 μM; The crop pests are caused by the fall armyworm feeding on crops, which affects the growth of crops; when the fall armyworm exhibits antifeedant activity, the concentration of belamcanda glycoside used in the crop pest control is 100 μM; when inhibiting the growth of the fall armyworm, the concentration of belamcanda glycoside used in the crop pest control is 0.4 μmol / g.

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