Application of β-pinene in promoting growth and inducing resistance of Panax notoginseng and Bletilla striata

Abstract

The present invention relates to the technical field of Chinese medicine planting, and specifically, to the use of β-pinene in promoting the growth of Panax notoginseng and Bletilla striata and inducing resistance. The present invention has found that β-pinene treatment can promote the early germination of Panax notoginseng seeds, and its effect is better than α-pinene. In addition, the present invention has also found that β-pinene treatment can induce disease resistance in Bletilla striata. β-pinene is a natural product with environmental protection, low toxicity, and sustainable development effects. It is abundant in source and low in acquisition cost, and therefore can be widely used in the planting of Panax notoginseng and Bletilla striata.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine planting, and in particular to the application of beta-pinene in promoting the growth of Panax notoginseng and Bletilla striata and inducing resistance. Background Art

[0002] Yunnan Province, located on the Yunnan-Guizhou Plateau on the Tropic of Cancer, has a wide range of tropical, subtropical, temperate, and cold-temperate climates due to its high altitude and low latitude. The north-south Hengduan Mountains, dry and hot river valleys, and warm, humid air currents from the South Pacific create ideal temperatures and humidity levels for the growth of Panax notoginseng and Bletilla striata. Over centuries of cultivating traditional Chinese medicine, people have continuously explored and developed suitable cultivation methods for Panax notoginseng and Bletilla striata. For example, shade nets are constructed over fields of Panax notoginseng and Bletilla striata, and a layer of pine needles is applied to the soil after planting or transplanting Panax notoginseng. This not only provides warmth and moisture, but also allows for leaching through rainwater or natural evaporation, positively impacting the growth of these two plants and their disease control.

[0003] However, when Panax notoginseng is continuously planted on the same plot of land, it can lead to poor growth, severe pests and diseases, reduced yields, and poor quality due to continuous cropping problems. Similarly, the high-yield cultivation model for Bletilla striata in large fields can also lead to reduced efficacy and quality, and frequent pests and diseases. These conditions have severely restricted the sustainable development of the Panax notoginseng and Bletilla striata related industries. Therefore, there is a continued need for low-toxicity, pollution-free cultivation methods for Panax notoginseng and Bletilla striata that can effectively improve their quality.

[0004] Studies have shown that pine needles play a crucial role in the cultivation of traditional Chinese medicine. Compared to the current practice of using plant growth regulators and chemical pesticides to increase the germination rate of Chinese medicinal materials, inhibit pathogens, and promote their growth, the abundant active ingredients in pine needles that promote plant growth and induce resistance are all natural products that are naturally degradable and do not cause environmental pollution. Mulching with pine needles is an effective cultivation method for Panax notoginseng. The inventors previously discovered that α-pinene promotes Panax notoginseng growth and induces resistance in their research on the active ingredients in pine needles (see CN113303328A).

[0005] This invention is a continuation of previous work, exploring the applicability of β-pinene in promoting the growth of Panax notoginseng and Bletilla striata and inducing resistance. It scientifically explains why pine needles should be covered in Panax notoginseng and Bletilla striata fields, providing direction for the future development of environmentally friendly biogenic resistance inducers and providing a scientific basis for the ecological cultivation of Panax notoginseng and Bletilla striata. The inventors found that β-pinene has a better effect than α-pinene in promoting the growth of Panax notoginseng and inducing resistance. In addition, the inventors also unexpectedly found that β-pinene also has the effect of promoting the growth and inducing resistance of Bletilla striata.

[0006] There is no report on relevant research results in the prior art. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides an application of β-pinene in promoting the growth of Panax notoginseng and Bletilla striata and inducing resistance.

[0008] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention is to provide the use of β-pinene in promoting the germination of Panax notoginseng seeds.

[0010] In one embodiment, the applying comprises spraying Panax notoginseng seeds with a Panax notoginseng growth promoter / β-pinene, wherein the Panax notoginseng growth promoter comprises β-pinene.

[0011] The second aspect of the present invention is to provide a method for promoting the germination of Panax notoginseng seeds, which comprises spraying the Panax notoginseng seeds with a Panax notoginseng growth promoter / β-pinene every 2 days, wherein the Panax notoginseng growth promoter contains β-pinene.

[0012] In one embodiment, the concentration of the β-pinene aqueous solution during spraying is 10-200 μl / L; in a further embodiment, the concentration of the β-pinene aqueous solution during spraying is 10-50 μl / L.

[0013] The third aspect of the present invention is to provide the application of β-pinene in promoting the growth of Panax notoginseng; the application comprises spraying β-pinene on the planted Panax notoginseng plants.

[0014] The fourth aspect of the present invention is to provide a method for promoting the growth of Panax notoginseng, which comprises: spraying β-pinene on the planted Panax notoginseng plants every 15 days.

[0015] In one embodiment, to promote the growth of Panax notoginseng, the concentration of β-pinene during spraying is 1-200 μl / L of aqueous solution; in a further embodiment, the concentration of β-pinene during spraying is 1, 10, 50, 100, or 200 μl / L of aqueous solution. In an even further embodiment, spraying with 1, 10, or 100 μl / L of β-pinene aqueous solution can promote the accumulation of fresh and dry matter in the underground part of Panax notoginseng; and 1, 100, or 200 μl / L of β-pinene aqueous solution can promote the accumulation of fresh matter in the aboveground part of Panax notoginseng.

[0016] In another embodiment, the amount of the β-pinene aqueous solution used during spraying is 1-10 L / m 2 ; preferably 5-10L / m 2 More preferably, 6.7 L / m 2 .

[0017] The fifth aspect of the present invention is to provide the use of β-pinene in inducing resistance in Bletilla striata.

[0018] In one embodiment, the use comprises spraying Bletilla striata seeds with β-pinene.

[0019] The sixth aspect of the present invention provides a method for inducing resistance in Bletilla striata, which includes the application of spraying Bletilla striata seeds with β-pinene.

[0020] In one embodiment, the concentration of the sprayed β-pinene aqueous solution is 1-50 μl / L; more specifically, the concentration of the sprayed β-pinene aqueous solution is 1, 10 or 50 μl / L.

[0021] In another embodiment, the induction time is 1-3 days.

[0022] In another embodiment, the induced resistance refers to inducing disease resistance in Bletilla striata, including rust and anthracnose.

[0023] The sixth aspect of the present invention is to provide the use of β-pinene in promoting the growth of Bletilla striata.

[0024] The seventh aspect of the present invention is to provide a method for promoting the growth of Bletilla striata, which comprises spraying β-pinene on Bletilla striata plants, wherein the β-pinene is a β-pinene aqueous solution with a concentration of 0.1-10 μl / L.

[0025] In one embodiment, the Bletilla striata plants are sprayed once every 15 days.

[0026] In the present invention, a certain amount of cosolvent may be added to the β-pinene aqueous solution in order to improve the solubility of β-pinene. The cosolvent is preferably Tween-80.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention discovered that β-pinene can promote early germination of Panax notoginseng seeds, accelerate growth, and increase the fresh and dry weight of both above- and below-ground parts. Furthermore, the present invention also discovered that β-pinene can induce disease resistance in Bletilla striata and promote its growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Effects of β-pinene on seed germination of Panax notoginseng. Data are mean ± standard error. Error bars represent standard error of the mean (n = 3). Different colored lines represent different treatment concentrations of pine needle allelopathic β-pinene.

[0030] Figure 2:The effect of β-pinene on the growth of Panax notoginseng seeds. Figure 2 A and Figure 2 B shows the effects of β-pinene on the fresh weight of the aboveground and underground parts of Panax notoginseng, respectively. Data are mean ± standard error. Error bars represent standard error of the mean (n = 3).

[0031] Figure 3 :The effect of β-pinene on the biomass of Panax notoginseng. Figure 3 A and Figure 3 B represents the effects of β-pinene on the fresh weight and dry weight of aboveground parts; Figure 3 C and Figure 3 D shows the effect of β-pinene on the dry weight and fresh weight of aerial parts. Error bars represent the standard error of the mean (n=3).

[0032] Figure 4 :Comparison of the effects of β-pinene and α-pinene on the germination of Panax notoginseng seeds. Figure 4 A and Figure 4 B represents the effects of β-pinene and α-pinene on the germination rate of Panax notoginseng seeds.

[0033] Figure 5 :Comparison of the effects of β-pinene and α-pinene on the biomass of Panax notoginseng. Figure 5 A and Figure 5 B represents the effects of α-pinene on the fresh weight and dry weight of underground parts, respectively; Figure 5 C and Figure 5 D represents the effects of α-pinene on the fresh weight and dry weight of aboveground parts, respectively; Figure 5 E and Figure 5 F represents the effects of β-pinene on the fresh weight and dry weight of aboveground parts, respectively; Figure 5 G and Figure 5 H represents the effect of β-pinene on the dry weight and fresh weight of the aboveground part, respectively. Error bars represent the standard error of the mean (n=3).

[0034] Figure 6 : Different induction treatment groups of pine needle allelopathic substance β-pinene on Bletilla striata.

[0035] Figure 7 : Effects of β-pinene induction for 1 day and 3 days on the resistance of Bletilla striata to leaf anthracnose. Figure 7 A is the relative lesion area of ​​Bletilla striata leaves induced by different concentrations of β-pinene for 1 day. Figure 7 B is the relative lesion area of ​​Bletilla striata leaves induced by different concentrations of β-pinene for 3 days. Error bars represent the standard error of the mean (n=3). Different lowercase letters indicate significant differences among treatments (P<0.05).

[0036] Figure 8Effects of 10 μL / L β-pinene on the resistance of Bletilla striata to leaf anthracnose. Error bars represent standard error of the mean (n=3). Different lowercase letters indicate significant differences among treatments (P<0.05).

[0037] Figure 9 :The effect of 10 μL / L β-pinene on SOD activity of Bletilla striata after 1-day induction.

[0038] Figure 10 :The effect of β-pinene on the fresh weight and dry weight of Bletilla striata. Figure 10 A. Figure 10 B. Figure 10 C. Figure 10 D. Figure 10 E. Figure 10 F represents the effect of β-pinene on fresh weight of roots, fresh weight of tubers, fresh weight of leaves, dry weight of roots, dry weight of tubers, and dry weight of leaves, respectively. Error bars represent the standard error of the mean (n=3). Different lowercase letters indicate significant differences among treatments (P<0.05).

[0039] Figure 11 :The effect of β-pinene on rust and anthracnose of Bletilla striata. Figure 11 A, Figure 11 B(represents the effect of β-pinene on the disease index of white rust and anthracnose, respectively. Error bars represent the standard error of the mean (n=3). Different lowercase letters indicate significant differences among treatments (P<0.05). DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0042] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0043] β-pinene: It is a terpene organic compound with a chemical formula of C10H16 and a molecular weight of 136.234. It is mainly used as an intermediate in the manufacture of flavors and fragrances. Its specific structural formula is as follows:

[0044]

[0045] Example 1: Effect of β-pinene on the germination and growth of Panax notoginseng seeds

[0046] Panax notoginseng seeds were planted in sealed bottle sand culture method, and β-pinene was sprayed in the planted sand culture bottles to explore the effect of β-pinene on the germination and growth of Panax notoginseng seeds, as well as the optimal application concentration.

[0047] First, weigh 100g of sterile quartz sand and place it in a glass tissue culture bottle with a volume of 250mL and sterilize it again in an oven at 141℃ for 4h. After cooling, evenly place 10 healthy and disease-free Panax notoginseng seeds with consistent morphology and cover them with a lid. After planting in January 2021, they were uniformly placed in a greenhouse (temperature 25℃, light and dark time 12h / 12h). After that, open the lid and spray 10mL of β-pinene with different concentrations every 2d, and the spraying concentrations were 0μl / L, 1μl / L, 10μl / L, 50μl / L, 100μl / L, and 200μl / L, respectively. The germination rate of Panax notoginseng seeds was measured for the first time after 40d, and then the germination rate of Panax notoginseng seeds was measured regularly every 5d until the 75th day. The biomass was measured for the last time.

[0048] (1) Determination of germination rate

[0049] Seedling emergence rate (%) = (number of seedlings emerging / total number of sowings) * 100%

[0050] (2) Biomass determination

[0051] Fresh weight: Rinse the Panax notoginseng plants with tap water and wipe dry with absorbent paper. Weigh using an electronic balance. The aboveground fresh weight is the weight of a single fresh leaf and stem; the underground fresh weight is the weight of a single fresh root.

[0052] Dry weight: Use oven to fix at 105℃ for 5 minutes, then dry at 50℃ until constant weight is the weight after drying. The dry weight of the aboveground part is the dry weight of the leaves and stems of a single plant; the dry weight of the underground part is the weight of a single root after drying.

[0053] The seeds of Panax notoginseng were sown in January 2021. After sowing, different gradients of pine needle allelopathic substance β-pinene were used for the first spraying. After opening the cover, the spraying was carried out every 2 days. The emergence rate of each treatment was investigated and counted in March of the same year.

[0054] The results showed that treatments with β-pinene at concentrations of 10, 50, 100, and 200 μL / L could promote early germination of Panax notoginseng seeds within 40-45 days after seedling emergence. The germination rates of Panax notoginseng at 10 and 50 μL / L increased by 66.67% at 40 days, which was significantly different from the control. The treatments also showed a trend of promoting seed germination within 75 days of planting. However, the germination-promoting effect of treatments with 100 and 200 μL / L concentrations weakened with the increase of planting time ( Figure 1 ).

[0055] β-pinene showed a trend of increasing the accumulation of fresh matter in the aboveground part of Panax notoginseng at concentrations of 1, 10, 50, and 200 μL / L, with 1 μL / L showing the best effect, increasing the accumulation by 12.53% ( Figure 2 A); Underground fresh matter showed an accumulation trend under 1, 10, and 50 μl / L treatments, with the 50 μl / L treatment increasing by 8.65% ( Figure 2 B).

[0056] Example 2: Effect of β-pinene on the growth of Panax notoginseng

[0057] This experiment was conducted in a greenhouse at the Daheqiao Modern Experimental Base of Yunnan Agricultural University in Xundian County, Kunming City, Yunnan Province (25°31′8″N, 103°16′41″E, 1980m above sea level). The greenhouse was equipped with a shade net and integrated water and fertilizer facilities to ensure the healthy growth of Panax notoginseng. The experimental plot was divided into 1.0×1.5m 2 The plots were divided into small and large groups. According to the concentration gradient in Example 1, a total of 5 treatments of the single-factor experiment were set, 3 groups of repetitions were repeated for each treatment, and 6 repetitions of the control treatment were set, for a total of 21 plots. Tween 80 was used as a cosolvent, and a 10L aqueous solvent was prepared and sprayed in the test plot. The test was planted in January 2021, and the first application began after planting. It was then sprayed every 15d until harvested. Statistics on the emergence of seedlings began in March of the same year, and sampling began in May. 30 trees were randomly taken from each plot using a five-point sampling method to measure the growth indicators of the samples.

[0058] (1) Field management

[0059] After sowing, spraying treatment is carried out every 15 days, and the rest of the management is routine.

[0060] (2) Determination of growth indicators

[0061] ① Seedling emergence rate survey: Seedling emergence rate (%) = (number of seedlings emerging / total number of sowings) * 100%;

[0062] ② Disease survey: Survey index: Incidence rate (%) = (number of diseased plants surveyed / total number of plants surveyed) * 100%;

[0063] ③ Seedling survival rate statistics: Seedling survival rate (%) = (number of surviving seedlings / total number of sowings) * 100%

[0064] ④Biomass:

[0065] Fresh weight: leaf weight is the weight of a single fresh leaf; root fresh weight is the weight of a single fresh root; stem fresh weight is the weight of a single fresh stem.

[0066] Dry weight: Leaf dry weight is the weight of a single leaf after drying; root dry weight is the weight of a single root after drying; stem dry weight is the weight of a single stem after drying.

[0067] β-pinene was applied by spraying. The first spraying was carried out after sowing, and then sprayed every 15 days. The seedling survival rate was counted at the end of October 2021, for a total of 20 sprayings. The experimental results showed (Table 1) that when the β-pinene concentrations were 1, 10, 50, 100 and 200 μL / L, the seedling survival rate had no significant difference compared with the control.

[0068] Table 1: Effect of β-pinene on the survival rate of Panax notoginseng seedlings

[0069]

[0070]

[0071] The results showed that the concentrations of 1, 10, and 100 μL / L of β-pinene spraying could promote the accumulation of fresh matter and dry matter in the underground part of Panax notoginseng, with fresh matter increasing by 9.35%, 3.58%, and 16.26%, and dry matter increasing by 23.12%, 21.11%, and 32.30% ( Figure 3 A, Figure 3 B). 1, 100 and 200 μL / L of β-pinene can promote the accumulation of fresh matter in the aboveground parts, increasing by 4.93%, 3.44% and 6.16% respectively; β-pinene at 100 μL / L can promote the accumulation of dry matter in the aboveground parts of Panax notoginseng, increasing by 32.79% ( Figure 3 C, Figure 3 D).

[0072] Example 3: Comparative Study on the Effects of β-pinene and α-pinene on the Germination and Growth of Panax notoginseng

[0073] To investigate the effects of β-pinene and α-pinene on the germination of Panax notoginseng seeds, this experiment first weighed 100g of sterile quartz sand and placed it in a 250mL glass tissue culture bottle. The jar was then oven-dried at 141°C for 4h to sterilize it. After cooling, 10 healthy, disease-free, and morphologically consistent Panax notoginseng seeds were evenly distributed. After planting in January 2021, the plants were uniformly placed in a greenhouse (temperature 25°C, 12h / 12h light / dark cycle). Subsequently, 10mL of β-pinene and α-pinene were sprayed at different concentrations: 0μl / L, 1μl / L, 10μl / L, 50μl / L, 100μl / L, and 200μl / L. Each treatment was replicated 12 times. The germination rate of Panax notoginseng seeds was first measured after 40d, and then regularly measured every 5d until the 75th day.

[0074] In order to explore the effects of β-pinene and α-pinene on the growth of Panax notoginseng, this experiment was carried out in a greenhouse at the Xundian Daheqiao Modern Experimental Base of Yunnan Agricultural University, Xundian County, Kunming City, Yunnan Province (25°31′8″N, 103°16′41″E, 1980m above sea level). The greenhouse is equipped with a shade net and integrated water and fertilizer facilities to ensure the healthy growth of Panax notoginseng. The experimental site was divided into small plots of 1.0×1.5m2, and different concentrations of β-pinene and α-pinene were configured according to the concentration gradient in Example 1, with 3 replicates for each treatment. Tween 80 was used as a cosolvent, and a 10L water solvent was prepared and sprayed in the experimental plot. The experiment was planted in January 2021, and the first application began after planting, and then sprayed every 15d until harvest. Sampling began in May, and 30 trees were randomly selected from each plot using a five-point sampling method to measure the growth indicators of the samples.

[0075] (1) Field management

[0076] After sowing, spraying treatment is carried out every 15 days, and the rest of the management is routine.

[0077] (2) Determination of growth indicators

[0078] Biomass:

[0079] Fresh weight: Leaf weight is the weight of a single fresh leaf; root fresh weight is the weight of a single fresh root.

[0080] Quantity; fresh stem weight refers to the weight of fresh stems of a single plant.

[0081] Dry weight: Leaf dry weight is the weight of a single leaf after drying; root dry weight is the weight of a single root after drying; stem dry weight is the weight of a single stem after drying.

[0082] In order to explore the difference in the effects of β-pinene and α-pinene on the germination of Panax notoginseng, this experiment investigated the emergence rate of Panax notoginseng seeds by spraying them. The results showed that after treatment with β-pinene at concentrations of 10, 50, 100 and 200 μL / L, the germination of Panax notoginseng seeds could be promoted early within the 40th to 45th day after seedling emergence. Among them, the 10 and 50 μL / L treatments increased the emergence rate of Panax notoginseng by 66.67% at 40 days, which was significantly different from the control ( Figure 4 A). However, α-pinene only had a good germination promoting effect at a concentration of 10 μL / L, increasing the effect by 24.18%, but there was no significant difference compared with the control ( Figure 4 B) Therefore, β-pinene can promote the germination of Panax notoginseng seeds more than α-pinene.

[0083] To investigate the differences in the effects of β-pinene and α-pinene on the growth of Panax notoginseng, this study sprayed field-grown Panax notoginseng for five months. The results showed that spraying α-pinene at concentrations of 1 and 10 μL / L promoted the accumulation of fresh and dry matter in the underground part of Panax notoginseng, with fresh weight increasing by 5.88% and 16.83%, respectively, and dry weight increasing by 22.12% and 32.55%, respectively. Figure 5 A, Figure 5 B); 50μL / L α-pinene treatment promoted the accumulation of fresh and dry weight of the aboveground part of Panax notoginseng, increasing by 18.99% and 17.12% ( Figure 5 C, Figure 5 D). The β-pinene spraying treatments at concentrations of 1, 10, and 100 μL / L promoted the accumulation of fresh matter and dry matter in the underground part of Panax notoginseng, with fresh matter increasing by 9.35%, 3.58%, and 16.26%, and dry matter increasing by 23.12%, 21.11%, and 32.30% ( Figure 5 E, Figure 5 F). 1, 100 and 200 μL / L of β-pinene can promote the accumulation of fresh matter in the aboveground parts, increasing by 4.93%, 3.44% and 6.16% respectively; 100 μL / L of β-pinene can promote the accumulation of dry matter in the aboveground parts of Panax notoginseng, increasing by 32.79% ( Figure 5 G, 5H). Comprehensive evaluation found that β-pinene could better promote the accumulation of fresh and dry matter in the underground part of Panax notoginseng than α-pinene.

[0084] Example 4: Effect of β-pinene on resistance induced by Bletilla striata

[0085] 4.1 β-pinene-induced resistance concentration screening test

[0086] In order to find out whether β-pinene can induce resistance of Bletilla striata and help it resist the invasion of pathogens, different concentrations of β-pinene were sprayed on Bletilla striata potted plants to induce resistance. In May, 4 annual Bletilla striata seedlings with consistent growth were selected and transplanted into a pot. After one month of seedling acclimatization, the weeds on the surface of the soil were removed and the soil was irrigated and left for one day. The pot was then covered with a fresh-keeping bag so that only the above-ground part of the Bletilla striata could come into contact with volatiles to avoid the influence of allelopathic substances on the soil. Afterwards, the pine needle allelopathic substance β-pinene was sprayed on the front and back sides of the Bletilla striata leaves of the Bletilla striata potted plants with consistent growth according to the concentrations in Table 2. The induction time was set to 1d and 3d respectively. After the Bletilla striata seedlings were induced with pine needle allelopathic substances, the leaves were pierced with a sterilized needle, and a 0.5mm punch was used to punch out a Bletilla striata anthracnose cake of the same age. The cake was placed upside down on the wound with the mycelium facing down (one leaf of each Bletilla striata seedling was selected for inoculation, and one cake was inoculated on each leaf with a needle), and then moisturized culture was carried out (sprayed with sterilized water once in the morning, noon, and evening). After inoculation, the diseased leaves were collected and the area of ​​the diseased leaves (S) was scanned using a scanner. The optimal induction concentration and the optimal induction time were obtained.

[0087] Table 2: Application table of pesticides for Bletilla striata potted plant resistance test

[0088]

[0089] 4.2 β-pinene-induced resistance test

[0090] (1) Induced resistance treatment and sample collection

[0091] The induction method is as described above. Based on the optimal induction concentration screened in the early stage, the concentration of β-pinene sprayed on the leaves was 10μL / L. The treatment time was 1 day. The pine needle allelopathic substances of the above optimal induction concentration were sprayed once in the morning, noon and evening to induce Bletilla striata. The plants were divided into 3 groups ( Figure 6 ), as shown below:

[0092] 1) Induction vs. non-induction treatment: 48 pots of Bletilla striata were placed in 12 transparent plastic culture boxes (4 pots per box). 24 of the Bletilla striata were induced with β-pinene at a concentration of 10 μL / L. After treatment, all plant materials were divided into 3 parts, each with 8 pots. The first part was taken, and Bletilla striata leaves were collected (3 replicates), quickly quenched with liquid nitrogen and moved to a -80°C refrigerator; the other 24 pots were sprayed with clean water, and after 1 day of induction, they were also divided into 3 parts, each with 8 pots. The first part was taken, and Bletilla striata leaves were collected, quickly quenched with liquid nitrogen and moved to a -80°C refrigerator ( Figure 6 , A sampling).

[0093] 2) Induction + inoculation vs. no induction + inoculation: Take the second batch of Bletilla striata (8 pots) after the treatment in 1), inoculate the leaves with Bletilla striata anthracnose pathogen (select one leaf for inoculation from each Bletilla striata seedling, and inoculate one fungus cake on each leaf), then move to a transparent plastic incubator for moisturizing culture. After 4 days of culture, the diseased leaves were collected, the lesion area of ​​the diseased leaves was scanned with a scanner, and the induction effect (%) was calculated; the leaves not inoculated with the pathogen were collected, quickly quenched with liquid nitrogen and moved to a -80℃ refrigerator ( Figure 6 In addition, the second batch of materials sprayed with clean water were also inoculated with pathogens as in the induction + inoculation group and samples were collected and kept for later use ( Figure 6 , B sampling).

[0094] 3) Induction + no inoculation vs. no induction + no inoculation: Take the third batch of Panax notoginseng materials (8 pots) after treatment in 1), move them directly into a transparent plastic incubator without inoculating pathogens, and culture them in a moisturizing incubator. Take samples at the same time as treatment 2), quickly quench them with liquid nitrogen, and move them to a -80℃ refrigerator ( Figure 6 c Sampling). In addition, the third batch of materials sprayed with clean water were not inoculated with pathogens as in the induction + non-inoculation group and samples were collected and kept for later use ( Figure 6 , sampling C).

[0095] (2) Effect of β-pinene induction on the lesion area of ​​Bletilla striata leaves

[0096] The results showed that the lesion area of ​​Bletilla striata was significantly reduced by 28.27%, 22.32% and 23.48% after 1 day of induction with β-pinene at the induction concentrations of 1μL / L, 10μL / L and 50μL / L compared with CK. Figure 7 A); β-pinene at induction concentrations of 1 μL / L, 10 μL / L, and 50 μL / L significantly reduced the lesion area of ​​Bletilla striata after 3 days of induction compared with CK, and the lesion area was significantly reduced by 31.19%, 51.50%, and 36.43% ( Figure 7 B).

[0097] After inducing resistance with β-pinene at a concentration of 10 μL / L for 1 day, the induced disease resistance effect was further studied. The leaf lesion area of ​​Bletilla striata was reduced by 39.09% ( Figure 8 ).

[0098] (3) Determination of induction-related enzyme activities after β-pinene induction

[0099] Superoxide dismutase (SOD) can effectively catalyze the decomposition of superoxide anions, inhibiting oxidative burst and preventing cell death. The WST-8 assay was used to measure SOD activity in leaves of six treatment groups that were not inoculated with pathogens. The results showed that β-pinene induction of Bletilla striata followed by inoculation with anthracnose pathogens increased SOD activity. The SOD activity of the 10 μL / L β-pinene treatment group, induced and inoculated, did not differ significantly from that of the non-induced and inoculated group, but was 14.87% higher than that of the non-induced and inoculated group ( Figure 9 ).

[0100] Example 5: Effect of β-pinene on the growth of Bletilla striata

[0101] Before planting the Bletilla striata seedlings, base fertilizer was spread at a rate of 14 kg per plot. The planting method was to dig a planting hole 4-5 cm in diameter and 5 cm deep, place the bulbs of the selected Bletilla striata seedlings with similar growth into the hole, cover the bulbs and all roots with soil, compact it, and water it thoroughly. Fifty plants were planted in each plot, with a planting spacing of 20 cm × 25 cm, and then watered regularly. After the Bletilla striata successfully survived and established for 2 months, β-pinene was applied at concentrations of 0, 0.1, 1, 10, 50, 100, and 200 μl / L, with 4 replicates and 1 blank control, for a total of 28 plots. The experimental plots were randomly distributed. The first application was made 2 months after the Bletilla striata seedlings established, and then it was applied every 15 days. A total of 10 applications were made. In December of the same year, Bletilla striata samples were collected, and the biomass and disease conditions were investigated.

[0102] Fresh weight: Rinse the Bletilla striata plants with tap water and wipe off the surface moisture with absorbent paper, and weigh them with an electronic balance.

[0103] Dry weight: Use oven to fix at 105℃ for 5 minutes, then dry at 50℃ to constant weight.

[0104] Disease index = ∑ (number of diseased plants × representative value of that level) / (total number of plants surveyed × representative value of the highest disease level) × 100%;

[0105] The results showed that spraying Bletilla striata with β-pinene at different concentrations could increase the fresh weight and dry weight of Bletilla striata roots, tubers and leaves to a certain extent. When the concentration was 1μl / L, the fresh weight of Bletilla striata roots could be increased by 9.90% compared with CK. Figure 10 A); the concentration of 0.1μl / L can effectively increase the fresh weight of Bletilla striata tubers, increasing by 15.55% ( Figure 10 B); when the concentration was 10μl / L, it could significantly increase the dry weight of Bletilla striata leaves, and could increase the dry weight of leaves by 42.55% compared with CK ( Figure 10F). In addition, different concentrations of β-pinene can reduce the rust and anthracnose of Bletilla striata to a certain extent, among which the concentration of 10μl / L has the best effect and can reduce the field disease index of Bletilla striata rust and anthracnose to the greatest extent ( Figure 11 ).

[0106] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. The application of β-pinene in inducing resistance to Bletilla striata is characterized by: The application is to spray bletilla striata seeds with beta-pinene, and the concentration of the sprayed beta-pinene aqueous solution is 1-50 μl / L.

2. The use according to claim 1, characterized in that: The concentration of the sprayed β-pinene aqueous solution was 1, 10 or 50 μl / L.

3. A method for inducing resistance in Bletilla striata, characterized by: The method comprises spraying bletilla striata seeds with beta-pinene; the concentration of the sprayed beta-pinene aqueous solution is 1-50 μl / L.

4. The method according to claim 3, wherein: The concentration of the sprayed β-pinene aqueous solution was 1, 10 or 50 μl / L.

5. The application of β-pinene in promoting the growth of Bletilla striata is characterized by: The application is to spray β-pinene on Bletilla striata plants, wherein the β-pinene is a β-pinene aqueous solution with a concentration of 0.1-10 μl / L.

6. A method for promoting the growth of Bletilla striata, characterized in that: The method comprises spraying beta-pinene on Bletilla striata plants, wherein the beta-pinene is a beta-pinene aqueous solution with a concentration of 0.1-10 μl / L.

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