Application of slash pine rhizosphere soil water extract in growth and development of turpinia arguta

Abstract

The invention is applicable to the technical field of plant growth and development, provides application of slash pine rhizosphere soil water extract in growth and development of turpinia acuminata, analyzes the growth and development process of the turpinia acuminata regulated and controlled by slash pine based on allelopathy, and particularly comprises the following steps: irrigating the turpinia acuminata with the slash pine rhizosphere soil water extract, so as to improve the growth and development of the turpinia acuminata. The seed germination and seedling process can be regulated and controlled, so that a new thought can be provided for increasing the yield of the turpinia arguta.

Description

Technical Field

[0001] This invention belongs to the field of plant growth and development technology, and in particular relates to the application of water extract of rhizosphere soil from slash pine in the growth and development of *Pinus thunbergii*. Background Technology

[0002] Allelopathy is the beneficial or harmful effect that plants have on the growth and development of neighboring plants by releasing specific secondary chemicals into the environment. Currently, research on allelopathy is developing rapidly around the world, and researchers have found that some chemicals released by plants can also have beneficial effects on other plants and promote their growth and development.

[0003] Ruijianshan Xiangyuan has important medicinal and ecological value, with effects such as clearing heat and detoxifying, relieving sore throat and reducing inflammation, promoting blood circulation and relieving pain, and antibacterial and anti-inflammatory properties. It has significant efficacy in preventing and treating tonsillitis, pharyngitis, upper respiratory tract infections, and chronic bronchitis, with no obvious side effects. However, most current research focuses on the chemical composition, pharmacological activity, and clinical medical trials of Ruijianshan Xiangyuan. Research on the regulation of Ruijianshan Xiangyuan's growth and development by plant allelopathic mechanisms is relatively lacking. Therefore, there is an urgent need to provide a solution to address these issues. Summary of the Invention

[0004] The purpose of this invention is to provide the application of rhizosphere soil water extract of slash pine in the growth and development of *Pinus thunbergii*, aiming to solve the problems mentioned in the background art.

[0005] The present invention is implemented as follows: the application of slash pine rhizosphere soil water extract in the growth and development of *Pinus thunbergii* var. *rhizosphereus*, the growth and development of *Pinus thunbergii* var. *rhizosphereus* is regulated by irrigating with slash pine rhizosphere soil water extract, specifically including regulating seed germination and regulating seedling growth; The method for preparing the aqueous extract of rhizosphere soil from slash pine includes the following steps: After the rhizosphere soil of slash pine was air-dried and sieved, it was mixed with water and placed on a shaker for extraction to obtain a mixed solution. The mixture was filtered to obtain a water extract of the rhizosphere soil of pine.

[0006] Another objective of this invention is to provide a growth and development regulator for *Pinus thunbergii*, the regulator comprising rhizosphere soil extract of *Pinus thunbergii*.

[0007] This invention analyzes the regulation of the growth and development of *Pinus slashii* based on allelopathy. Specifically, by irrigating *Pinus slashii* with a water extract from the rhizosphere soil, the germination of its seeds and the seedling process are regulated, thus providing a new approach to increasing the yield of *Pinus slashii*. Attached Figure Description

[0008] Figure 1 This invention relates to the effect of different concentrations of water extracts from the rhizosphere soil of *Pinus slashii* on the germination rate of *Rhizophora stylosa* seeds over time, as provided in an embodiment of the invention. Figure 2 The effects of different concentrations of water extracts from the rhizosphere soil of *Pinus stropharia* on the germination rate, germination potential, and germination index of *Pinus stropharia* seeds provided in this embodiment of the invention; Figure 3 The effects of different concentrations of slash pine rhizosphere soil water extract provided in this embodiment of the invention on seedling height, seedling root length, seedling fresh weight and seedling dry weight of *Pinus thunbergii* seedlings. Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0010] The preparation method of the rhizosphere soil aqueous extract of slash pine includes the following steps: S1. The rhizosphere soil of slash pine (soil 0-10 cm away from the roots of slash pine) is air-dried and sieved (using a sieve with a mesh size of 40-60). It is then mixed with water (the ratio of soil to water is 1:1) and placed on a shaker for extraction. The temperature is 25℃, the rotation speed is 150r / min, and the time is 12h to obtain a mixture. S2. After filtering the mixture with quantitative filter paper, filter it with a 0.22 μm microporous membrane to obtain a water extract of the rhizosphere soil of pine.

[0011] The rhizosphere soil of slash pine used in this embodiment of the invention was collected from Jiangxi Academy of Forestry Sciences, and the seeds of Ruijianshan Xiangyuan were collected from Jiulian Mountain in Ganzhou City, Jiangxi Province. The experimental site was located at Jiangxi Academy of Forestry Sciences.

[0012] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0013] Example 1: Application of rhizosphere soil aqueous extract of slash pine in the growth and development of *Pinus thunbergii* var. *rhizoma*: 1. Preparation of aqueous extract of rhizosphere soil from slash pine: 1.1 Soil within 10 cm of the roots of wetland pine trees was collected using the five-point sampling method as rhizosphere soil. The collected soil was mixed and placed in a sterile bag, placed in a dark place to air dry naturally, and then impurities were removed and the soil was passed through a 40-mesh sieve. 500 g of rhizosphere soil and 500 mL of distilled water were added to an Erlenmeyer flask, sealed with sealing film, and placed on a shaker at 25 ℃ and 150 r / min for 12 h for extraction. 1.2 After initial filtration of the supernatant using quantitative filter paper, the supernatant is then sterilized by vacuum filtration through a 0.22 μm microporous membrane to prepare a 1 g / ml stock solution. This stock solution is then diluted with distilled water to prepare working solutions of 0.1 g / mL, 0.25 g / mL, and 0.5 g / mL pine rhizosphere soil water extract.

[0014] 2. Effects of water extract from rhizosphere soil of *Pinus stropharia* on seed germination and seedling growth of *Pinus stropharia*: After soaking the seeds of *Pinus thunbergii* in distilled water for 24 hours, they were disinfected with 5% NaClO solution for 10 minutes and rinsed 5 times with distilled water. The substrate (peat soil: carbonized rice husks or peat moss: perlite: organic fertilizer in a 4:4:1:1 ratio) disinfected with carbendazim was packed into container bags and compacted. Three seeds were sown in each container bag and covered with about 2 cm of substrate. A total of 12 bags were sown, with 3 bags forming a group. Each group was irrigated with 9 mL of 0.1 g / mL, 0.25 g / mL, and 0.5 g / mL rhizosphere soil water extract (experimental group) and distilled water (control group), respectively. Germination was observed daily and appropriate amounts of water extract were added to each corresponding treatment as needed. The container bags were placed on the seedbed and a 50 cm high arched shed was built with agricultural film. The treatment lasted for 41 days. On day 31, seed germination potential was calculated. The germination experiment concluded on day 41, and germination rate and germination index were calculated. Simultaneously, seedling height, root length, fresh weight, and dry weight were measured using vernier calipers and a 0.01% balance. Results are as follows: Figure 1 , Figure 2 and Figure 3 As shown; Wherein, germination rate (%) GR = total number of germinated seeds / number of tested seeds × 100%; Germination potential (%) GP = Number of seeds germinated in the first 31 days / Number of seeds tested × 100%; Germination index GI = ∑(Gt / Dt) (Gt represents the number of seeds that germinate on day t, and Dt represents the corresponding number of germination days). according to Figures 1 to 3 It can be seen that the rhizosphere soil aqueous extract of slash pine promotes seed germination and seedling growth. At the same time, the allelopathic effect of the rhizosphere soil aqueous extract of slash pine on *Pinus thunbergii* is concentration-dependent. The allelopathic promoting effect on seed germination rate, germination potential, germination index, seedling height and longest root length is strongest when the concentration is 0.25 g / mL, while the allelopathic promoting effect on seedling fresh weight and dry weight is strongest when the concentration is 0.5 g / mL.

[0015] Performance testing: 1. Chemical composition analysis of water extract from rhizosphere soil of slash pine (LC-MS): 1 mL of the aqueous extract prepared in Example 1 was mixed with 21 mL of the extract in methanol-acetonitrile solution (v:v=1:1), vortexed for 60 s, and then sonicated at low temperature for 30 min. The mixture was then centrifuged at 12000 rpm and 4 ℃ for 10 min. The supernatant was collected, and the precipitated protein was placed at -20 ℃ for 1 h, centrifuged at 12000 rpm and 4 ℃ for 10 min, and the supernatant was freeze-dried and reconstituted in 100 μL of 30% ACN. The mixture was then centrifuged at 12000 rpm and 4 ℃ for 10 min. The supernatant was then analyzed, and the top 25 allelochemicals by relative content were recorded. The results are shown in Table 1. Table 1

[0016] The data acquisition system mainly includes ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometry (Q Exactive HFX, Thermo, USA); the chromatographic column is Waters HSS T3 (100*2.1mm, 1.8μm); the mobile phase is 0.1% formic acid-water solution in phase A and 0.1% formic acid-acetonitrile in phase B; the flow rate is 0.3ml / min; the column temperature is 40℃; the injection volume is 2 uL; the elution gradient is as follows: 0.0 min-1.0 min, phase B is maintained at 0%; 1.0 min-12.0 min, phase B changes linearly from 0% to 95%; 12.0 min-13 min, phase B is maintained at 95%; 13.0 min-13.1 min, phase B changes linearly from 95% to 0%; 13.10 min-17.0 min, phase B is maintained at 0%; the sample is placed in an autosampler at 4℃ throughout the analysis; and the electrospray ionization source (Electrospray) is used. Ionization (ESI) conditions are as follows: sheath gas 40 arb; auxiliary gas 10 arb; ion spray voltage 3000 V / -2800 V; temperature 350 ℃; ion transmission tube temperature 320 ℃; scanning mode is Full-ms-ddMS2 mode; scanning method is positive ion / negative ion; primary scan range (scan m / z range): 70-1050 Da, primary resolution 70000, secondary resolution 17500.

[0017] 2. Evaluation results and analysis of allelopathic effects: The allelopathic effect index RI (response index) was calculated using the method of Williamson et al. When T≥C, RI=1-C / T; when T<C, RI=T / C-1. Where C represents the control group data, T represents the experimental group data, RI≥0 indicates a promoting effect, RI<0 indicates an inhibiting effect, and the absolute value indicates the strength of the allelopathic effect. The allelopathic effect index SE is the arithmetic mean of all measured indicators RI of *Ligusticum striatum* under the same treatment, and the calculation formula is as follows: ; The results of the allelopathic combined effect analysis are shown in Table 2: Table 2

[0018] As shown in Table 2, the allelopathic effect of the rhizosphere soil aqueous extract of Pinus slashis on Rhizophora stylosa exhibits a concentration effect. As the concentration of the rhizosphere soil aqueous extract of Pinus slashis increases from low to high, its comprehensive allelopathic effect index on Rhizophora stylosa shows a trend of first increasing and then decreasing. A high comprehensive allelopathic effect score indicates a good allelopathic promoting effect.

[0019] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The application of water extract of rhizosphere soil from slash pine in the growth and development of *Pinus thunbergii*, characterized in that... The growth and development of *Pinus thunbergii* was regulated by irrigating the rhizosphere soil with a water extract, specifically by regulating seed germination and seedling growth. The method for preparing the aqueous extract of rhizosphere soil from slash pine includes the following steps: After the rhizosphere soil of slash pine was air-dried and sieved, it was mixed with water and placed on a shaker for extraction to obtain a mixed solution. The mixture was filtered to obtain a water extract of the rhizosphere soil of pine.

2. The application according to claim 1, characterized in that, The rhizosphere soil of the slash pine is the soil 0-10 cm away from the roots of the slash pine tree; The sieve used for sieving is 40-60 mesh; The ratio of material to water in the rhizosphere soil of slash pine is 1-1.2:1; The temperature during shaking is 23-28℃, the rotation speed is 140-160 r / min, and the extraction time is 11-13 h; The filtration process involves first filtering with quantitative filter paper, and then filtration with a microporous membrane.

3. The application according to claim 1, characterized in that, The concentration of the aqueous extract of the rhizosphere soil of the slash pine is 0.25-0.5 g / mL.

4. The application according to claim 1, characterized in that, The regulation of seed germination includes the effects on seed germination rate, germination potential, and germination index.

5. The application according to claim 1, characterized in that, The regulation of seedling growth includes the effects on seedling height, root length, fresh weight, and dry weight.

6. A growth and development regulator for *Syzygium serratum*, characterized in that, The preparation includes rhizosphere soil extract of slash pine.

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