A method to enhance the drought resistance of bryophytes

Abstract

This invention discloses a method for enhancing the drought resistance of bryophytes, relating to the field of environmental engineering technology. The method provided by this invention enables artificially cultivated bryophyte crusting materials, whose drought resistance needs to be enhanced, to passively receive induced VOCs to form a stress imprint, self-adjusting their physiological state to enhance drought resistance. This process consumes low material and energy costs, resulting in better plant performance under subsequent stress. It avoids the drawbacks of active methods, such as slow growth and high mortality rates after inoculation, thus exhibiting a greater survival advantage, higher propagation success rate, and greater stability. This method is effective not only during drought but also during rehydration. Furthermore, the method provided by this invention is simple and easy to implement.

Description

Technical Field

[0001] This invention relates to the field of environmental engineering technology, and more specifically, to a method for improving the drought resistance of bryophytes. Background Technology

[0002] Bryophytes are renowned for their exceptional drought tolerance, adapting to a wide range of terrestrial environments (from cold and hot deserts to tropical regions), and are frequently used as restoration species in terrestrial ecosystem restoration projects. However, the drought resistance of bryophyte materials rapidly cultivated under artificially comfortable conditions is significantly reduced, resulting in low survival rates and slow growth when directly applied to restoration projects. This greatly limits the rate of stable bryophyte crust formation and the effectiveness of restoration. Therefore, to overcome this limitation, researchers have explored various methods to mitigate the negative impacts of drought stress, primarily two: one is to provide additional support measures to improve drought stress conditions; the other is to subject artificially cultivated bryophyte materials to a drought hardening process to enhance their inherent drought resistance. However, both methods are complex, time-consuming, and costly. Therefore, there is an urgent need to develop an efficient, low-cost, and easy-to-operate method.

[0003] VOCs are a class of compounds characterized by small quantities, low boiling points, low octane numbers, high saturated vapor pressures, and large Henry's constants. VOCs can mediate plant-plant communication, including drought resistance information, enhancing the drought resistance of receiving plants, thus making it possible to utilize this important potential ecological function. Furthermore, VOCs are characterized by their wide spread and rapid sensitivity; most plants (inducers) can rapidly release large amounts of VOCs after being stimulated, and these emissions can be sensed by surrounding plants (receivers), thereby enhancing the latter's drought resistance.

[0004] There are currently no reports of using VOCs to improve the drought resistance of artificially cultivated bryophyte materials.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for improving the drought resistance of bryophytes to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows: This invention provides a method for improving the drought resistance of bryophytes, comprising the following steps: S1: Repeated precipitation-drought cycle treatment under natural light to induce bryophytes to produce VOCs in the induction chamber: The induced bryophytes belong to the same species as the bryophytes whose drought resistance needs to be improved; the induction conditions are: precipitation 0.1-3 mm, precipitation interval 4-21 days, and induction cycle 1-5 times. S2: Introduce the gas from the induction chamber into the response chamber where the bryophyte to be enhanced for drought resistance is located.

[0008] The present invention has the following beneficial effects: This invention utilizes VOCs to enhance the drought resistance of bryophytes. The induced bryophytes and the bryophytes whose drought resistance needs to be enhanced belong to the same species, thereby ensuring that the specific secondary metabolites (plant volatile organic compounds, VOCs) produced by the induced bryophytes can be received by bryophytes of the same species. In other words, through species specificity, the drought resistance of bryophytes can be specifically enhanced.

[0009] Screening the induction conditions revealed that controlling the precipitation to 0.1-3 mm, the precipitation interval to 4-21 days, and the number of induction cycles to 1-5 times helped maintain high levels of NSC (non-structural carbohydrates), soluble sugars, and starch in bryophytes whose drought resistance needs to be improved. This resulted in lower accumulation of malondialdehyde (MDA), a "cell damage indicator" for drought resistance, and maintained high superoxide dismutase activity, thereby improving the overall drought resistance of bryophytes.

[0010] The method provided by this invention enables artificially cultivated moss crust materials with enhanced drought resistance to form a stress imprint by passively receiving induced VOCs, thus adjusting their physiological state to enhance drought tolerance. This process consumes low material and energy costs, resulting in better plant performance under subsequent stress. It avoids the drawbacks of active methods, such as slow growth and high mortality rates after inoculation, thus offering a greater survival advantage, higher propagation success rate, and greater stability. This method is effective not only during drought but also during rehydration.

[0011] The method provided by this invention is simple to operate and reduces the workload of directly inducing and enhancing the drought resistance of bryophytes. It only requires exposing the bryophytes to the VOCs environment or atmosphere generated during drought induction. Compared with traditional drought induction methods, mass induction of bryophyte drought resistance via VOCs has the technical advantage of saving resources. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1The content of non-structural carbon and its components in the responders under different drought-induced modes; (A&B are terrestrial responses to *Bryum denticulatum*; C&D are responses to *Bryum denticulatum*; lowercase letters represent significant differences in the responders across different drought-induced cycles (p<0.05)). Figure 2 The statistical results of malondialdehyde content in the response groups under different drought induction methods are shown in the figure (A&B are terrestrial responses to *Bryum denticulatum*; C&D are responses to *Bryum denticulatum*; lowercase letters represent significant differences in the response groups at different drought induction cycles (p<0.05)). Figure 3 The figure shows the statistical results of superoxide dismutase activity of the response groups under different drought induction methods (A&B are the response groups of terrestrial moss to moss; C&D are the response groups of moss; lowercase letters represent the significant differences of the response groups in different drought induction cycles (p<0.05)). Figure 4 The figure shows the statistical results of physiological indicators of terrestrial responses to moss at different physical distances (the values ​​in the figure are the weighted mean ± standard error (SE); lowercase letters represent significant differences in response at different patch area ratios (p<0.05)). Figure 5 The figure shows the statistical results of physiological indicators of the responders of *Rhizoctonia solani* at different physical distances (the values ​​in the figure are the weighted mean ± standard error (SE); lowercase letters represent the significant differences of the responders at different patch area ratios (p<0.05)). Figure 6 This is a structural diagram of the culture chamber; Figure 7 The chlorophyll content of the response under different drought induction methods is shown in the figure (values ​​are the weighted mean ± standard error (SE); A is the response of terrestrial moss to *Bryum dentata*; B is the response of *Bryum dentata*; lowercase letters indicate significant differences in the response under different drought induction cycles). p <0.05)); Figure 8 Comparison of moss cover using and not using the induction method of this invention (different lowercase letters indicate significant differences within the species). Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0015] Existing technologies for enhancing the drought resistance of artificially cultivated bryophytes involve actively inducing drought tolerance through a drought-resistant training process, which often comes at the cost of high-cost carbohydrate consumption. In contrast, this invention enhances drought resistance by passively receiving and analyzing VOCs emitted by other plants, adjusting physiological states to achieve this effect at a lower cost. Using artificially cultivated bryophyte material as the receiver, and enhancing its own stress resistance by receiving VOCs generated by the inducing agent (i.e., the induced bryophyte), undoubtedly has a significant positive impact on rapidly increasing the drought resistance of artificially cultivated bryophyte material, thus providing a new approach and method to overcome the technical bottlenecks in the large-scale promotion of artificially cultivated bryophyte crust formation.

[0016] This invention provides a method for improving the drought resistance of bryophytes, comprising the following steps: S1: Repeated precipitation-drought cycle treatment under natural light to induce bryophytes to produce VOCs in the induction chamber: The induced bryophytes belong to the same species as the bryophytes whose drought resistance needs to be improved; the induction conditions are: precipitation 0.1-3 mm, precipitation interval 4-21 days, and induction cycle 1-5 times. S2: Introduce the gas from the induction chamber into the response chamber where the bryophyte to be enhanced for drought resistance is located.

[0017] By ensuring that the induced bryophytes and the bryophytes whose drought resistance needs to be enhanced belong to the same species, it is possible to ensure that the specific secondary metabolites (volatile organic compounds, VOCs) produced by the induced bryophytes can be received by bryophytes of the same species. This species-specific approach allows for the specific enhancement of the bryophytes' drought resistance. Using different species of bryophytes as the induced and drought-resistant bryophytes, respectively, will result in poor induction effects.

[0018] Screening induction conditions revealed that controlling precipitation (0.1-3 mm), precipitation intervals (4-21 days), and induction cycles (1-5 times) helps maintain high levels of NSC (non-structural carbohydrates), soluble sugars, and starch in bryophytes to enhance drought resistance. It also results in lower accumulation of malondialdehyde (MDA), a key indicator of cell damage during drought, and maintains high superoxide dismutase (SOD) activity, thus improving overall drought resistance. In drought-resistant plants, under the same drought conditions, MDA accumulation is lower and rises more slowly; SOD activity is significantly enhanced, forming an effective protective barrier that rapidly dismutates toxic superoxide anion free radicals generated under drought stress into less toxic hydrogen peroxide and oxygen; and total NSC remains stable or slightly increases, thus maintaining the energy required for plant respiration and basic metabolism. Conversely, in drought-resistant plants, MDA content increases sharply and significantly, SOD activity decreases, and total NSC decreases.

[0019] The method provided by this invention enables artificially cultivated moss crust materials with enhanced drought resistance to form a stress imprint by passively receiving induced VOCs, thus adjusting their physiological state to enhance drought tolerance. This process consumes low material and energy costs, resulting in better plant performance under subsequent stress. It avoids the drawbacks of active methods, such as slow growth and high mortality rates after inoculation, thus offering a greater survival advantage, higher propagation success rate, and greater stability. This method is effective not only during drought but also during rehydration.

[0020] Overall, the method provided by this invention is simple and easy to implement, reducing the workload of directly inducing and enhancing the drought resistance of bryophytes. It only requires exposing the bryophytes to the VOCs environment or atmosphere generated during drought induction. Compared with traditional drought induction methods, mass induction of bryophyte drought resistance via VOCs has the technical advantage of saving resources.

[0021] In a preferred embodiment of the present invention, the induced bryophyte is selected from at least one of *Bryophytum terrestris* and *Bryophytum terrestris*.

[0022] In a preferred embodiment of this invention, the induction conditions for terrestrial *Bryum dentata* are: rainfall of 0.1-3 mm, rainfall interval of 4-14 days, and induction cycles of 1-5 times; the induction conditions for *Bryum dentata* are: rainfall of 0.1-3 mm, rainfall interval of 4-14 days, and induction cycles of 1-5 times. Induction under these conditions enables the bryophytes whose drought resistance needs to be improved to exhibit higher SOD activity, higher NSC (non-structural carbohydrates), and lower MDA content.

[0023] In a preferred embodiment of the present invention, the induction conditions for terrestrial moss are as follows: precipitation of 0.1 mm, 0.2 mm, 0.245 mm, 0.3 mm, 0.39 mm, 0.4 mm, 0.5 mm, 0.535 mm, 0.68 mm, 0.825 mm, 0.97 mm, 1 mm, 1.115 mm, 1.26 mm, 1.405 mm, 1.55 mm, 1.695 mm, 1.84 mm, 1.985 mm, 2 mm, 2.13 mm, 2.275 mm, 2.42 mm, 2.565 mm, 2.71 mm, 2.855 mm, or 3 mm; precipitation interval of 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days; and induction cycles of 1, 2, 3, 4, or 5.

[0024] In a preferred embodiment of the present invention, the induction conditions for terrestrial *Tetranychus dentata* are: 0.1 mm of rainfall, a rainfall interval of 4 days, and 3 induction cycles; under these conditions, the content of NSC and its components in terrestrial *Tetranychus dentata* is higher.

[0025] The induction conditions for *Rhizoctonia solani* were: 0.2 mm of precipitation, a 7-day interval between precipitation events, and one cycle. Under these conditions, *Rhizoctonia solani* exhibited higher levels of NSCs and their components.

[0026] In the experiment, the precipitation-drought cycle treatment in the control response chamber was the same as that in the induction chamber. In other embodiments, the precipitation-drought cycle treatment in the control response chamber may also be different from that in the induction chamber.

[0027] In a preferred embodiment of the present invention, the induction conditions for terrestrial moss are: 0.2 mm of rainfall, 7-day interval between rainfalls, and 3 induction cycles; or, 1 mm of rainfall, 4-day interval between rainfalls, and 1 induction cycle. Under these conditions, the MDA content in terrestrial moss is lower, and the drought resistance of the moss is higher.

[0028] The induction conditions for *Rhizoctonia solani* were: 0.1 mm of precipitation at 7-day intervals, with 3 cycles; 3 mm of precipitation at 7-day intervals, with 1 cycle; or 0.1 mm of precipitation at 7-day intervals, with 1 cycle. Under these conditions, the MDA content in *Rhizoctonia solani* was lower.

[0029] The induction conditions for terrestrial bryophytes or bryophytes are: 0.2 mm of precipitation, 7-day interval between precipitation events, and 3 cycles, which result in higher SOD activity.

[0030] In a preferred embodiment of the present invention, the distance between the induction chamber and the response chamber is 0-60 cm, for example, 0 cm, 1 cm, 2 cm, 3 cm, 10 cm, 20 cm, 30 cm, 40 cm, 50 cm, 58 cm, 59 cm or 60 cm.

[0031] In a preferred embodiment of the present invention, the distance between the induction chamber and the response chamber is 0-30 cm. For example: 0 cm, 2 cm, 4 cm, 6 cm, 8 cm, 10 cm, 12 cm, 14 cm, 16 cm, 18 cm, 20 cm, 22 cm, 24 cm, 26 cm, 28 cm, 30 cm.

[0032] In a preferred embodiment of the present invention, when the induced bryophyte is *Bryophytum terrestris*, the distance between the induction chamber and the response chamber is 0-5 cm; when the induced bryophyte is *Bryophytum terrestris*, the distance between the induction chamber and the response chamber is 0-30 cm.

[0033] The physical transport distance of information chemicals (VOCs) has a significant impact on the physiological indicators of the responders. The SOD activity of the terrestrial sphagnum moss (responder) was significantly higher than that of the control group within a physical distance range of 0-30 cm. When the distance exceeded 30 cm, the SOD activity showed a decreasing trend, with no significant difference from the control group. The MDA content was significantly lower than that of the control group within a physical distance range of 0-5 cm, and increased within a range of 15-60 cm, with no significant difference from the control group. The soluble sugar content was significantly higher within a range of 5-30 cm than at other distance ranges.

[0034] The SOD activity of the response formula of *Erythrina variegata* was higher than that of the control group in the range of 0-60 cm; the MDA content was significantly lower than that of the control group in the range of 0-30 cm, but there was no significant difference in MDA content between the two groups at 60 cm; the soluble sugar content and the ratio of soluble sugar to starch were higher than those of the control group in the range of 0-60 cm; and the ABA content remained at a high level in the range of 5-30 cm.

[0035] In a preferred embodiment of the present invention, the flow rate of the gas from the induction chamber into the response chamber of the bryophyte whose drought resistance is to be enhanced is 200 ml / min ± 20 ml / min.

[0036] In a preferred embodiment of the present invention, the response chamber is further provided with a gas outlet connected to the induction chamber to achieve airflow circulation; the induction chamber is provided with an air inlet, and air is delivered to the air inlet by a pump.

[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0038] Example 1 This embodiment provides a method for improving the drought resistance of bryophytes, which includes the following steps: (1) According to Figure 6 The diagram shows the actual culture chamber setup, with two transparent, enclosed polyethylene culture chambers serving as the induction chamber (right) and the response chamber (left). The induction chamber induces bryophytes to produce VOCs, while the response chamber allows the bryophytes whose drought resistance needs to be enhanced to passively receive VOCs from the induction chamber. The artificially cultured bryophyte crust material (responder) requiring enhanced drought resistance is placed in the response chamber. Well-grown bryophyte crusts of the same species as the responder are selected, and litter and weeds are removed from the sample surface before being placed in the transparent, enclosed polyethylene culture chamber (induction chamber). The induction chamber and response chamber are connected by a polyethylene conduit (13 mm inner diameter * 16 mm outer diameter). The conduit length (i.e., physical distance) is controlled.

[0039] A dual-pump system is used to facilitate the transfer of volatile organic compounds (VOCs) from the induction chamber to the response chamber: one pump uses activated carbon to filter air at a rate of 270 ml / min. - ¹ A flow rate of 1 is input into the induction chamber; simultaneously, another pump delivers VOC-containing air generated in the donor chamber to the response chamber at a flow rate of 200 ml / min. Furthermore, the response chamber is also equipped with a gas outlet connected to the induction chamber, through which gas from the response chamber is pumped to the induction chamber at a flow rate of 200 ml / min, thereby achieving airflow circulation; The two chambers are sealed with a polytetrafluoroethylene (PTFE) membrane to minimize the infiltration of external air.

[0040] (2) A controlled induction chamber was used to simulate a precipitation-drought cycle under natural light: For *Bryum denticulatum*, the precipitation was 0.1 mm on the first day and 0.1 mm on the fifth day, with a precipitation interval of 4 days and 3 cycles. For *Bryum esculentum*, the precipitation was 0.2 mm on the first day and 0.2 mm on the eighth day, with a precipitation interval of 7 days and 1 cycle. After each precipitation event, the VOCs generated in the induction chamber within 48 hours were transferred to the response chamber via an air pump. In the experiment, the precipitation-drought cycle treatment in the response chamber was the same as that in the induction chamber. In other implementation methods, the precipitation-drought cycle treatment in the response chamber could be different from that in the induction chamber.

[0041] The length of the polyethylene conduit was controlled to be 5 cm. The ratio of the crust area of ​​the inducing agent (i.e., the induced bryophyte) and the responding agent (i.e., the bryophyte whose drought resistance is to be improved) was set to 1:1.

[0042] Experimental Example 1 This experiment used the dominant bryophyte species *Bryophytum terrestris* and *Bryophytum terrestris* in arid sandy areas as examples. Combining the precipitation characteristics of arid regions and the physiological characteristics of bryophytes, six precipitation gradients (0, 0.1, 0.2, 0.5, 1, and 3 mm), five precipitation time intervals (4, 7, 14, 21, and 28 days), and three drought induction cycles (1, 3, and 5 cycles) were set up in the induction chamber. A total of 90 drought induction methods were combined to control these three factors. The inducing organism was exposed to these 90 drought induction methods, and the osmotic regulation system of the inducing and responding organisms was compared to assess the degree of improvement in the physiological drought resistance of the responding organism.

[0043] The content of NSC (non-structural carbohydrates), soluble sugars, and starch was determined by the following methods.

[0044] Non-structural carbon determination: After the experimental operation was completed and the sample surface was dried, the sample was placed in an oven at 105℃ for 15 min to kill the enzymes, and then dried at 65℃ for 48 h to constant weight. The upper plant tissue and lower soil layer of the sample were carefully separated using a scalpel, ground into powder, and passed through a 100-mesh sieve (0.15 mm diameter). The powdered sample was stored at -4℃ for further chemical analysis. 0.1 g of the ground sample was weighed and extracted at 80℃ for 15 min using the extraction buffer. After cooling, it was centrifuged at 3000 g for 5 min at RT. The precipitate was collected and the starch content was measured using Solarbio's kit (BC0705) on the SpectreMax Paradigm multimodal detection platform (Molecular Devices). Another 0.1 g of the ground sample was taken, 1 mL of distilled water was added, the slurry was homogenized, extracted at 95℃ for 10 min, cooled, and centrifuged at 8000 g for 10 min at room temperature. Then, the supernatant was collected, and the soluble sugar content was measured using a plant soluble sugar content assay kit (BC0030, Solarbio, Beijing). Similarly, 0.03 g of the sample was weighed, and the starch content was determined using a plant starch content assay kit (BC0070; Solarbio, Beijing).

[0045] From three-dimensional Figure 1 It can be seen that the content of NSC (non-structural carbohydrates) and its components in the terrestrial *Pseudomonas thunbergii* is higher when the precipitation interval is 4-7 days, and lower when the precipitation interval is 14-28 days; while the contents of NSC, soluble sugar and starch are highest when the precipitation amount is 0.1 mm and the precipitation interval is 4 days. Figure 1 A) indicates that this condition is conducive to the response to regulate the non-structural carbon balance in plants. Then, a planar diagram was established under this condition. The LSD one-way ANOVA results showed that when the number of drought-induced cycles was 3, the content of NSC and its components was significantly greater than that when the number of induction cycles was 1 or 5.

[0046] The same method determined that the highest NSC and its component contents in the responder of *Rhizopus dentata* were found when the precipitation was 0.2 mm, the precipitation interval was 7 days, and the number of cycles was 1. Figure 1 (Figures C and D in the diagram).

[0047] Experiment Example 2 This experiment used the dominant bryophyte species *Bryum terreum* and *Bryum terreum* in arid sandy areas as examples. Combining the precipitation characteristics of arid regions and the physiological characteristics of bryophytes, six precipitation gradients (0, 0.1, 0.2, 0.5, 1, and 3 mm), five precipitation time intervals (4, 7, 14, 21, and 28 days), and three drought-induced cycle numbers (1, 3, and 5 cycles) were set. A total of 90 drought-induced methods were combined to control these three factors. The inducer was exposed to these 90 drought-induced methods. This experiment compared the antioxidant systems of the inducer and the responder to evaluate the degree of improvement in the physiological drought resistance of the responder.

[0048] 1. The MDA content in the respondent was detected using the following method: Malondialdehyde (MDA) content detection: Oxygen free radicals act on unsaturated fatty acids in lipids, generating lipid peroxides; these peroxides gradually decompose into a series of complex compounds, including MDA. The level of lipid oxidation can be detected by measuring the level of MDA. Under acidic and high-temperature conditions, MDA can condense with thiobarbituric acid (TBA) to form a brownish-red trimethylolpropionate (3,5,5-trimethyloxazol-2,4-dione), with a maximum absorption wavelength at 532 nm. Colorimetric analysis can estimate the MDA content in the sample. Weigh approximately 0.1 g of tissue moss sample, add 1 mL of extraction buffer, and homogenize on ice; centrifuge at 8000 g at 4℃ for 10 min, discard the supernatant, and place on ice for testing. Preheat the microplate reader for at least 30 min and zero with distilled water. Using the Solarbio MDA content detection kit (BC0025), add the MDA detection working solution, distilled water, sample, and reagent three sequentially. The mixture was incubated in a 100℃ water bath for 60 min, then cooled in an ice bath and centrifuged at 10000g at room temperature for 10 min. 200 μL of the supernatant was then transferred to a 96-well plate, and the absorbance of each sample was measured at 532 nm and 600 nm.

[0049] Experimental results: The MDA content of terrestrial mosses significantly increased under conditions of precipitation intervals of more than 7 days and precipitation amounts greater than 1 mm. Figure 2 (See Figure A in the figure). The MDA content was lower when the precipitation interval was 4 days and the precipitation amount was 0.1 mm or 1 mm; and when the precipitation interval was 7 days and the precipitation amount was 0.2 mm.

[0050] Further develop the floor plan ( Figure 2 Figure B in the figure shows that, under the above-mentioned precipitation interval and precipitation amount constraints, the MDA content of the terrestrial moss responder remained at a high level only when the precipitation interval was 4 days, the precipitation amount was 1 mm, and the number of drought-induced cycles was 3, while it remained at a low level under other induction conditions.

[0051] The MDA content in the response formula of *Rhizopus dentata* increases with the extension of the precipitation interval, and the planar surface... Figure 2 The CD plots showed that the MDA content of the response formula remained low when the precipitation interval was 4 days, the precipitation amount was 3 mm, and the number of cycles was 5; or when the precipitation interval was 7 days, the precipitation amount was 1 mm, and the induction cycle was 1 or 3 times; or when the interval was 7 days, the precipitation amount was 3 mm, and the induction cycle was 1 time.

[0052] Therefore, when considering reducing the MDA content index, the following methods were selected when improving the drought resistance of terrestrial mosses: a 7-day interval between precipitation and a precipitation amount of 0.2 mm, repeated 3 times; and a 4-day interval between precipitation and a precipitation amount of 1 mm, repeated once.

[0053] When considering reducing the MDA content index, the following methods were selected to enhance the drought resistance of *Bryophytum esculentum*: a precipitation interval of 7 days and a precipitation amount of 0.1 mm, repeated 3 times; a precipitation interval of 7 days and a precipitation amount of 3 mm, repeated once; or a precipitation interval of 7 days and a precipitation amount of 0.1 mm, repeated once.

[0054] 2. The superoxide dismutase content in the response sample was detected using the following method: Superoxide dismutase (SOD) activity assay: Freshly treated samples were temporarily stored at -80℃. For measurement, the sample was ground under liquid nitrogen, and 0.1 g of the SOD extract was weighed and added to an ice-bath homogenate. The homogenate was then centrifuged at 8000 g for 10 minutes at 4℃. The supernatant was collected, and SOD activity was measured using a superoxide dismutase assay kit (WST-8, Suzhou Keming). The detection principle is: superoxide anion (O2... - It is produced by the reaction system of xanthine and xanthine oxidase and can react with freshly ground sample (0.1g).

[0055] Since the intra-group variance of SOD activity in the response group of *Erythrina solani* is greater than that of precipitation amount and precipitation time interval under the interaction of the number of induction cycles and precipitation time interval, a stereogram is constructed with precipitation time interval and number of induction cycles as constraints.

[0056] Figure 3 The results showed that SOD activity gradually decreased with the extension of precipitation interval and the increase of drought-induced cycles. Further analysis based on the stereographic diagram revealed higher SOD activity at precipitation intervals of 4-14 days and at 1 and 3 drought-induced cycles. Through variance analysis and comprehensive consideration of MDA content, NSC and its components, higher SOD activity was observed at a precipitation interval of 7 days, a rainfall of 0.2 mm, and 3 drought-induced cycles.

[0057] Experimental Example 3 This experimental example tests the effect of the length of the polyethylene conduit (i.e., the physical distance of the conduit connecting the induction chamber and the response chamber) on the information transmission effect.

[0058] The SOD activity, malondialdehyde (MDA) content, NSC content, soluble sugar to starch ratio, and abscisic acid (ABA) content of the native *Bryophytum solani* and *Bryophytum solani* in the response chamber were measured separately. The methods for detecting SOD activity, MDA content, NSC content, and soluble sugar to starch ratio were the same as in Experiments 1-2.

[0059] The detection methods for photosynthetic pigments and abscisic acid are as follows: Detection of abscisic acid (ABA) content in plants: A competitive enzyme-linked immunosorbent assay (ELISA) was used to determine ABA content. ABA calibrator and the sample to be tested were added to a microplate pre-coated with anti-abscisic acid antibody (solid-phase antibody), followed by horseradish peroxidase (HRP)-labeled detection antigen (enzyme-labeled antigen). After incubation and thorough washing to remove unbound components, a solid-phase antibody-enzyme-labeled antigen immune complex was formed on the solid-phase surface of the microplate. 0.01% hydrogen peroxide and 0.1% tetramethylbenzidine were added as substrates. Under HRP catalysis, the substrate produced a blue product, which turned yellow under the action of a stop solution (2 mol / L). The absorbance (OD value) was measured at 450 nm using an ELISA reader. The OD value was negatively correlated with the ABA concentration in the sample. The ABA concentration in the sample could be calculated by fitting a calibration curve.

[0060] Detection of photosynthetic pigments: 0.5 g of the moss material to be tested was ground with liquid nitrogen and then extracted with 80% acetone. The absorbance (OD value) at wavelengths of 665 nm and 649 nm was measured using a UV / VIS-752N UV-Vis spectrophotometer.

[0061] Chlorophyll a (chla) = (13.95 OD665 - 6.88 OD649) × V / 1000 × FW; Chlorophyll b (chlb) = (24.96 OD649 - 7.32 OD665) × V / 1000 × FW; In the formula, V is the volume of the extract (ml); FW is the weight of the material (g); and calculate: chl (total) = chla + chlb.

[0062] The control group (CK) was treated in a way that did not involve drought induction or the reception of VOCs.

[0063] The results showed that the physical distance of information chemical substances has a significant impact on the physiological indicators of the responders. Figure 4The results showed that the SOD activity of the terrestrial response to moss was significantly higher than that of the control group within a physical distance range of 0-30 cm. When the distance was greater than 30 cm, the SOD activity showed a decreasing trend, and there was no significant difference from the control group. The MDA content was significantly lower than that of the control group within a physical distance range of 0-5 cm. The MDA content increased within a physical distance range of 15-60 cm, and there was no significant difference from the control group. The soluble sugar content was significantly higher within a physical distance range of 5-30 cm than other distance ranges.

[0064] Figure 5 The results showed that the SOD activity of the response formula of *Erythrina variegata* was higher than that of the control group in the range of 0-60 cm; the MDA content was significantly lower than that of the control group in the range of 0-30 cm, but there was no significant difference in MDA content between the two groups at 60 cm; the soluble sugar content and the ratio of soluble sugar to starch were higher than those of the control group in the range of 0-60 cm; and the ABA content remained at a high level in the range of 5-30 cm.

[0065] Changes in chlorophyll content reflect the photosynthetic regulation strategies of bryophytes under drought stress. Based on three-dimensional... Figure 7 The chlorophyll content of the terrestrial *Bryum dentata* response modulus was highest (approximately 0.6 mg / g) at a rainfall of 3 mm and a rainfall interval of 21 days; and lowest (approximately 0.1 mg / g) at a rainfall of 0 mm and a rainfall interval of 4 days. The chlorophyll content of the *Bryum dentata* response modulus was lowest at rainfall of 0.5 or 1 mm and rainfall intervals of 7 or 14 days; and lowest at rainfall of 0 or 0.1 mm and rainfall intervals of 28 days.

[0066] Experiment Example 4 Two types of moss materials (Neopteris terreus and Rhizophora dentata) were used, one with and one without this method, at a concentration of 10 g / m². 2 Crush the seeds and sow them on the surface of the sandy bottom of the hill after the first rain in autumn. Cover with 1 mm of sand and water saturated (5 mm of rainfall). Observe the coverage of moss crust (%) after 3 months. Figure 8 The results show that the method provided in Example 1 of this invention can improve the coverage of sclerotium lesions compared with the uninduced method.

[0067] In summary, the method provided by this invention has the following technical advantages: (1) From the perspective of drought resistance of bryophytes, artificially cultivated bryophyte crusting materials form a stress imprint by passively receiving induced VOCs, adjusting their physiological state to enhance drought resistance. This process consumes low material and energy costs, resulting in better performance under subsequent stress. It avoids the disadvantages of slow growth and high mortality rates commonly found in active methods, thus exhibiting a greater survival advantage, higher propagation success rate, and greater stability. This method works not only during drought but also during the rehydration stage.

[0068] (2) From the perspective of feasibility, this method is simple to operate and reduces the workload of directly inducing and enhancing the drought resistance of bryophytes. It only requires exposing the bryophytes to be induced to VOCs generated by the bryophytes during the drought induction process.

[0069] (3) In terms of resource consumption, compared with the traditional drought-inducing method, the method of enhancing the drought resistance of bryophytes by mass induction with VOCs is resource-saving.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for improving the drought resistance of bryophytes, characterized in that, It includes the following steps: S1: Repeated precipitation-drought cycle treatment under natural light to induce bryophytes to produce VOCs in the induction chamber: The induced bryophytes belong to the same species as the bryophytes whose drought resistance needs to be improved; the induction conditions are: precipitation 0.1-3 mm, precipitation interval 4-21 days, and induction cycle 1-5 times. S2: The gas in the induction chamber is introduced into the response chamber where the bryophyte whose drought resistance is to be enhanced is located.

2. The method for improving the drought resistance of bryophytes according to claim 1, characterized in that, The induced bryophytes were selected from at least one of *Bryophytum terrestris* and *Bryophytum terrestris*.

3. The method for improving the drought resistance of bryophytes according to claim 2, characterized in that, The induction conditions for terrestrial moss are: 0.1-3 mm of precipitation, 4-14 days of precipitation interval, and 1-5 induction cycles; the induction conditions for moss are: 0.1-3 mm of precipitation, 4-14 days of precipitation interval, and 1-5 induction cycles.

4. The method for improving the drought resistance of bryophytes according to claim 3, characterized in that, The induction conditions for terrestrial moss were: 0.1 mm of precipitation, 4-day interval between precipitation events, and 3 induction cycles; the induction conditions for moss rib were: 0.2 mm of precipitation, 7-day interval between precipitation events, and 1 cycle.

5. The method for improving the drought resistance of bryophytes according to claim 3, characterized in that, The induction conditions for terrestrial moss are: 0.2 mm of precipitation, 7-day precipitation interval, and 3 induction cycles; or, 1 mm of precipitation, 4-day precipitation interval, and 1 induction cycle. The induction conditions for *Rhizophora dentata* are: 0.1 mm of precipitation, 7-day interval between precipitation events, and 3 cycles; 3 mm of precipitation, 7-day interval between precipitation events, and 1 cycle; or 0.1 mm of precipitation, 7-day interval between precipitation events, and 1 cycle.

6. The method for improving the drought resistance of bryophytes according to claim 3, characterized in that, The induction conditions for terrestrial basidiomycetes or basidiomycetes are: 0.2 mm of precipitation, 7-day interval between precipitation events, and 3 cycles.

7. The method for improving the drought resistance of bryophytes according to claim 2, characterized in that, The distance between the induction chamber and the response chamber is 0-60cm; Preferably, the distance between the induction chamber and the response chamber is 0-30cm.

8. The method for improving the drought resistance of bryophytes according to claim 7, characterized in that, When the induced bryophyte is *Bryophytum terrestris*, the distance between the induction chamber and the response chamber is 0-5 cm; when the induced bryophyte is *Bryophytum terrestris*, the distance between the induction chamber and the response chamber is 0-30 cm.

9. The method for improving the drought resistance of bryophytes according to claim 7, characterized in that, The gas from the induction chamber is introduced into the response chamber of the bryophyte whose drought resistance is to be enhanced at a flow rate of 200 ml / min ± 20 ml / min.

10. The method for improving the drought resistance of bryophytes according to claim 9, characterized in that, The response chamber is also provided with a gas outlet connected to the induction chamber to achieve airflow circulation; the induction chamber is provided with an air inlet, and air is delivered to the air inlet by a pump.

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