Effects of endophytic fungus Y118 on physiological and biochemical indexes and quality improvement of Zanthoxylum nitidum (Roxb.) DC

Effects of endophytic fungus Y118 on physiological and biochemical indexes and quality improvement of Zanthoxylum nitidum (Roxb.) 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DC Zebi XIE, Rong FAN, Kaiping Lai, Zining LIANG, Ning Song, Long Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5014790/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 May, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract To understand the effects of endophytic fungus Y118 on the physiological characteristics of Zanthoxylum nitidum (Roxb.) DC after co-culture with its host Zanthoxylum nitidum , we measured the plant growth index (dry root weight) of Zanthoxylum nitidum at different growth stages using the solid fermentation medium containing endophytic fungus Y118. Physiological and biochemical indexes (defensive enzyme activity) were determined by chromatographic analysis of the contents of nitidum chloride and chelidonine, the main active ingredients in Zanthoxylum nitidum root. At 3, 6, 9 and 12 months, the root dry weight of Y118 treatment group was significantly increased by 1123.53%, 51.08%, 56.40% and 47.12% compared with the control group, respectively. At the 3-month mark, superoxide dismutase (SOD) activity exceeded that of the control. After 9 months of cultivation, peroxidase (POD) activity in the treatment group showed a significant difference at a 0.05 significant level. At the 3-month mark, phenylalanine ammonia lyase (PAL) activity in the treatment group was significantly higher than that in the control group, increasing by 187.21%. After 12 months of cultivation, the contents of chlorophyll a and chlorophyll b significantly increased by 45.28% and 28.87%, respectively, compared with the control group. After 9 and 12 months of co-culture, the contents of nitidine chloride in Zanthoxylum nitidum in the Y118 treatment groups were 0.783 and 0.904 mg/g, respectively, showing a significant increase of 42.11% and 44.18% compared with the control group; the contents of chelidonine were significantly increased by 40.73% and 26.57% compared with the control group. Inoculation with the endophytic fungus Y118 significantly improved the physiological tolerance of Zanthoxylum nitidum and promoted the growth of Zanthoxylum nitidum , thereby enhancing the quality of Zanthoxylum nitidum . This implicates Y118 as a potential artificial fertilizer for Zanthoxylum nitidum . Biological sciences/Microbiology Biological sciences/Plant sciences Zanthoxylum nitidum endophytic fungus quality improvement physiological and biochemical indexes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Zanthoxylum nitidum (Roxb.) DC. is a potent herb and an authentic medicinal material commonly used by the Yao people in Guangxi, China. It is predominantly found in Guangxi, Guangdong, Yunnan and other provinces in China 1 , 2 . This plant, belonging to the genus Zanthoxylum nitidum in the family Rutaceae, is also known as double needling, double back needle, Mountain tiger. It has provided evidence of the significant anti-inflammatory, antibacterial, and anticancer properties 3 . Unfortunately, the natural habitat of Zanthoxylum nitidum has suffered due to recent deforestation and the establishment of economic fruit forests. The medicinal parts of Zanthoxylum nitidum are the root and stem bark, often leading to the harvesting of the entire plant. This indiscriminate harvesting, combined with excessive mining, has resulted in a sharp decline in the number of breeding individuals in the wild. Consequently, this has adversely affected the production of proprietary Chinese medicines and related industries relying on it as a raw material. The current artificial cultivation areas are insufficient to meet market demands 4 . Addressing this issue, recent studies have explored the role of endophytes in mitigating the impact on Zanthoxylum nitidum . Endophytes, microorganisms forming a symbiotic relationship with the host plant without inducing tissue lesions 5 , 6 , have demonstrated the ability to promote the growth and development of host plants, induce beneficial secondary metabolites, and enhance physiological responses to environmental changes, thereby improving the soil microenvironment 7 , 8 . Notably, endophytic fungi have shown promise in the scientific and environmentally friendly prevention and control of soil-borne diseases in crops, as evidenced by studies on monk fruit 9 . Study by Li et al 10 showed remarkable growth-promoting effects of the endogenous control bacterium NQ8GⅡ4 on Lycium barbarum seedlings, suggesting potential application in agriculture. Similarly, Shen et al 11 highlighted the potential of the Penicillium fungus J10469 as an anti-aluminum specific bacterial fertilizer by screening endophytic fungal strains improving nutrient utilization efficiency in Vernicia montana Lour’s seedlings under aluminum stress. While microbial fertilizers have shown great potential in increasing the yield of Chinese medicinal materials and enhancing plant physiological tolerance, there is a notable gap in the literature regarding the application of the endophytic fungus Y118 ( Phomopsis sp.) as a microbial fertilizer for the management of Zanthoxylum nitidum plantations. This study aims to comprehensively evaluate the induction of stress-related resistance enzymes in Zanthoxylum nitidum plants by the endophytic fungus Y118 at different culture stages. The findings of this study are expected to provide theoretical basis for the application of Y118 as a microbial fertilizer in the cultivation and management of Zanthoxylum nitidum and other medicinal plants. Materials and Methods Plant material collection All experimental materials were collected in the Plant Culture Room of Guangxi University of Traditional Chinese Medicine (China) between April 2021 and June 2022. Seedlings for the pot experiments were obtained through successful in-house cultivation. The endophytic fungus Y118 of Zanthoxylum nitidum belongs to the genus Phomopsis sp. and was isolated from early-stage leaves of Zanthoxylum nitidum plants in Guangxi 12 . Test method Preparation of solid fermentation medium The solid medium was prepared with 85% cornmeal and 15% rice as the carrier, with additional components including maltose (2.5%), peptone (1.5%), KH 2 PO 4 (0.2%), and MgSO 4 (0.15%). The pH was adjusted to 5.5–6.5 (solid:liquid = 1:1) according to the method described by Qin et al 13 .The endophytic fungus Y118 was revived and cultured on PDA medium for 3–4 days. Subsequently, one endophytic fungus Y118 (size: 1.76625 cm²) was added to every 60 g solid culture medium, which was then fermented at 25°C for 40 days to produce solid fermentation medium for use. Co-cultures Uniform Zanthoxylum nitidum seedlings, each with a plant height of approximately 5.0 cm and 3-5 leaves, were planted individually in pots (15.5 cm inner diameter and 14 cm high). Each plant in the treatment group was cultured with 25 g solid fermentation medium, while each plant in the control group (CK) was cultured with 25 g solid fermentation medium without the addition of fungus. Three replicates were prepared for each treatment, with 10 pots per replicate. The seedlings were watered regularly every day during maintenance, and the humidity was maintained at approximately 80%. Output measurement methods After the experimental treatment, Zanthoxylum nitidum seedling growth was observed, and samples were collected at 3 months, 6 months, 9 months and 12 months for the measurement of growth indexes. For each sample, soil was rinsed from the surface of Zanthoxylum nitidum plants, and excess water was absorbed with gauze. After drying, Zanthoxylum nitidum samples were separated into above- and underground parts. Upon drying in an oven at 60 °C to constant weight, the dry weight of the root sample was recorded. Leaves in the middle and upper part of the Zanthoxylum nitidum plant, with more uniform light access, were selected for the determination of physiological and biochemical indexes. Chlorophyll content was determined using the plant chlorophyll content detection kit. Soluble protein content was determined using the Coomassie brilliant blue method 14 . Superoxide dismutase (SOD) activity was determined using the nitrogen blue tetrazole photochemical reduction method 15 . Peroxidase (POD) activity was determined by the guaiacol method 15 . phenylalanine ammonolyase (PAL) activity was determined using an enzyme detection kit with absorbance measured at 290 nm. Malondialdehyde (MDA) content was determined using a plant MDA kit. Zanthoxylum nitidum roots were obtained at 3, 6, 9 and 12 months after treatment. After the roots were dried and passed through a 60 mesh (0.3 mm), nitidum chloride and chelidonine contents were determined by high-performance liquid chromatography (HPLC). Statistical analysis The experimental data were sorted using Excel 2003, and statistical analysis was performed with SPSS 23.0. Comparisons among multiple groups were performed using one-way analysis of variance. A significance level of P <0.05 was considered to indicate a statistically significant difference, while P <0.01 was considered to indicate an extremely significant difference. Results Effects of endophytic fungus Y118 on the growth of Zanthoxylum nitidum The changes in the root dry weight of Zanthoxylum nitidum showed significant differences at different culture stages(Fig. 1 and Fig. 2 ). At 3, 6, 9 and 12 months, the root dry weights of the Y118 treatment group significantly increased by 1123.53%, 51.08%, 56.40% and 47.12%, respectively, compared with the control group ( P < 0.01). These results indicated that treatment with the endophytic fungus Y118 promotes root growth, resulting in enhanced yield of Zanthoxylum nitidum . Effects of endophytic fungus Y118 on the activity of antioxidant enzymes in Zanthoxylum nitidum SOD and POD play crucial roles in removing excessive H 2 O 2 , contributing significantly to the anti-aging and metabolic processes of plants 16 . The effects of the endophytic fungus Y118 on SOD activity in Zanthoxylum nitidum showed significant differences at different culture stages(Fig. 3 ). No significant difference in SOD activity was observed between treatment group and the control group at different culture stages post Y118 treatment. The impact of the endophytic fungus Y118 on POD activity in Zanthoxylum nitidum showed significant differences at different culture stages(Fig. 4 ). After 9 months of cultivation, POD activity in Zanthoxylum nitidum was significantly higher compared with the control group ( P < 0.05). Effects of endophytic fungus Y118 on MDA content of Zanthoxylum nitidum Oxidative stress in plants leads to lipid peroxidation in the cell membrane, commonly assessed by measuring MDA as a marker 17 . Under the same culture conditions, lower MDA levels reflect reduced lipid peroxidation in the cell intima, indicating less damage to the cell membrane 18 . The effects of the endophytic fungus Y118 on MDA content of Zanthoxylum nitidum showed significant differences at different culture stages(Fig. 5 ). Compared with the control group, the molar mass concentration of MDA in Zanthoxylum nitidum co-cultured with Y118 decreased in the range of 10.64–16.60% as the co-culture progressed from 3 to 12 months. The most significant decrease (16.60% compared with the control) in the molar mass concentration of MDA in Zanthoxylum nitidum co-cultured with Y118 was observed at 6 months. However, no significant differences were observed between the two groups at any of the four time-points. Effects of endophytic fungus Y118 on PAL activity of Zanthoxylum nitidum PAL activity has been shown to be positively correlated with disease resistance and stress resistance in plants, serving as a key index to assess these characteristics 19 . The effects of the endophytic fungus Y118 on PAL enzyme activity in Zanthoxylum nitidum showed significant differences at different culture stages(Fig. 6 ). After 3 months, PAL activity in Zanthoxylum nitidum co-cultured with Y118 was significantly higher (187.21%) than that of the control group ( P < 0.05). However, after 6 months, no significant difference was observed between the treatment and control groups. Notably, PAL activity decreased at 9 and 12 months compared with the control, potentially influenced by local drought, cold autumn, and winter conditions during these periods, which are not conducive to mycelial growth. Effects of endophytic fungus Y118 on soluble protein content of Zanthoxylum nitidum Soluble proteins are essential nutrients that play a key role in osmoregulation in plant cells. They serve as a protective layer for biological membranes 20 and represent an important standard for a ssessing the healthy metabolism of plants 21 . Increased soluble protein content has been linked to improved metabolic intensity in plants 22 . The effects of Y118 on the soluble protein content of Zanthoxylum nitidum showed significant differences at different culture stages(Fig. 7 ). Throughout the 3, 6, 9, and 12-month co-cultures of the endophytic fungus Y118 with Zanthoxylum nitidum on solid fermentation medium, the soluble protein content in the treatment group showed an increase compared with the control group. However, these changes did not reach the threshold of statistical significance. Effects of endophytic fungus Y118 on chlorophyll content of Zanthoxylum nitidum Chlorophyll is the key photosynthetic pigment in plant photosynthesis, and serves as an important indicator of plant growth and development 23 . The effects of Y118 on the contents of chlorophyll a, chlorophyll b and total chlorophyll in Zanthoxylum nitidum leaves showed significant differences at different culture stages(Figs. 8 , 9 and 10 ), respectively. The contents of chlorophyll a, chlorophyll b and total chlorophyll in the treatment group were lower than those in the control group after 3 months and 6 months of co-culture. By the end of 9 months, the contents of chlorophyll a, chlorophyll b and total chlorophyll in the treatment group were higher than those in the control group, although the differences were not statistically significant. After 12 months, the contents of chlorophyll a and chlorophyll b significantly increased (by 45.28% and 28.87%, respectively) in Zanthoxylum nitidum leaves co-cultured with Y118 compared to the contents in the control group ( P < 0.05), while there was no significant difference in the total chlorophyll contents compared to that in the control group. Determination of nitidine chloride and chelidonine contents in Zanthoxylum nitidum roots Nitidine chloride and chelidonine are the main active components of nitidine and used as important indexes to evaluate the growth and quality of Zanthoxylum nitidum 24 . The effects of the endophytic fungus Y118 on nitidine chloride content of Zanthoxylum nitidum showed significant differences at different culture stages(Fig. 11 ), respectively. The nitidine chloride content in the root of Zanthoxylum nitidum co-cultured with Y118 increased compared with the control group, although the differences were not statistically significant at 3 and 6 months. At the 9-month mark, the nitidine chloride content in the root of Zanthoxylum nitidum treated with Y118 reached 0.783 mg/g, marking a 42.11% increase compared with the control group. After 12 months, the nitidine chloride content in the root of Zanthoxylum nitidum co-cultured with Y118 rose to 0.904 mg/g, reflecting a notable 44.18% increase compared with the control group. The effects of the endophytic fungus Y118 on chelidonine contents of Zanthoxylum nitidum showed significant differences at different culture stages(Fig. 12 ), respectively. The chelidonine content in the root of Zanthoxylum nitidum co-cultured with Y118 was higher than that in the control group at 3 and 6 months, although the differences were not statistically significant. At the 9-month mark, the chelidonine content in the root of Zanthoxylum nitidum co-cultured with Y118 reached 3.704 mg/g, marking a 40.73% increase compared with the control group. After 12 months, the chelidonine content in the root of Zanthoxylum nitidum co-cultured with Y118 rose to 4.201 mg/g, reflecting a notable 26.57% increase compared with the control group. Discussion Ecologically functional endophytes establish mutual symbiotic relationships with their host plants during co-evolution, thereby promoting host growth, alleviating the negative effects of stress, enhancing the accumulation of bioactive components, and playing a pivotal role in responding to stress conditions such as cold and drought 25 , 26 . In this study, we demonstrated a significant increase in the root dry weight of Zanthoxylum nitidum over a 12-month period when co-cultured with the endophytic fungus Y118, indicating a positive impact on Zanthoxylum nitidum yield. The Y118 treatment group showed a promotion in the growth of Zanthoxylum nitidum plants. These effects align with findings from Xiong et al 27 , who reported the influence of different microbial fertilizer treatments on the yield and quality of Zanthoxylum nitidum . It can be speculated that the target fungi optimized the soil microbial population structure, activated soil enzyme activity, accelerated organic matter decomposition, and facilitated the conversion of fixed nutrients into available forms in the soil. Moreover, it is conceivable that Y118 either inhibited pathogenic microorganisms or, alternatively, promoted root growth by producing plant hormones, subsequently enhancing the growth of Zanthoxylum nitidum plants. The observed benefits may also stem from the decomposition of secondary metabolic toxins by Y118, inhibiting the growth of pathogenic microorganisms or inducing plant hormones production to promote root growth. Our study also revealed that Y118 treatment enhanced the activity of defense enzymes in the leaves across all growth stages. In response to drought and other stresses, plants have evolved a variety of secondary metabolic pathways that generate corresponding secondary metabolites to alleviate stress 28 . After inoculation with Y118, there was no significant change in SOD activity between the treatment and control groups. However, the POD enzyme activity in Zanthoxylum nitidum leaves was significantly higher in the inoculated group than in the non-inoculated group. This suggests that the seedlings maintained higher POD activity under the same culture conditions after inoculation with Y118, reducing the damage caused by intracellular reactive oxygen species to the cell membrane. Moreover, PAL enzyme activity also demonstrated an enhancement in the disease resistance and stress resistance of Zanthoxylum nitidum after inoculation with Y118. Therefore, it can be speculated that endophytic fungi alleviated the stress on Zanthoxylum nitidum to a considerable extent, inducing an increase in antioxidase activity in the host. The potential of endophytic fungus Y118 to enhance the growth of Zanthoxylum nitidum might not solely be attributed to the reduction in lipid peroxidation levels and the augmentation of metabolic intensity in response to drought or cold conditions. The initial two culture stages in the treatment group did not show an obvious promotion of chlorophyll synthesis, possibly due to the unstable invasion and colonization of Y118, affecting the histocytes of Zanthoxylum nitidum . However, compared with the control group, the chlorophyll content of Zanthoxylum nitidum in the treatment group increased after 9 months, coinciding with the weakened light and cold climate in the local region in January. After 12 months of co-cultivation, the contents of chlorophyll a and chlorophyll b in the treatment group significantly increased, despite the wet and cold climate. These findings suggest that Y118 may enhance the resilience of Zanthoxylum nitidum in adverse environments, although further studies are needed to confirm this. It is worth noting that nitidine chloride and chelidonine, the main active components of Zanthoxylum nitidum , have anti-inflammatory, analgesic and antibacterial effects 4 . In this study, we observed significant increase in the accumulation of nitidine and chelidonine chloride in Zanthoxylum nitidum roots after 9 and 12 months of co-culture with Y118, indicating growth promotion effects. These findings align with similar observations in [ Pseudostellaria heterophylla (Miq.)] 29 . Therefore, we posit that the inoculation of the endophytic fungus Y118 may enhance the synthesis of active components, nitidum chloride, and chelidonine in Zanthoxylum nitidum during its artificial cultivation through two primary mechanisms: Firstly, by augmenting the root biomass of Zanthoxylum nitidum , thereby increasing the total production of effective alkaloids. Secondly, by elevating the content of soluble proteins and increasing the chlorophyll levels in plant leaves, leading to enhanced plant photosynthesis and heightened antioxidant enzymes activity. Thus, the inoculation of the endophytic fungus Y118 provides a promising approach for cultivating Zanthoxylum nitidum with high yield and quality. However, the full extent of Y118’s impact on Zanthoxylum nitidum requires further assessment through field trials. In addition, a comprehensive understanding of its role in reducing the dependence on chemical fertilizers and pesticides, as well as the underlying growth promotion mechanism, remains essential for maximizing the overall benefits of employing the endophytic fungus Y118 in Zanthoxylum nitidum production. Conclusions Inoculation with the endophytic fungus Y118 significantly improved the physiological tolerance and growth of Zanthoxylum nitidum plants. The increased root dry weight of Zanthoxylum nitidum , along with the elevated contents of nitidine chloride and chelidonine in the root, suggests comprehensive benefits of employing Y118 in the cultivation of Zanthoxylum nitidum crops. Declarations Data availability The authors confrm that the data supporting the findings of this study are available within the article. Author contributions Conceptualization, Z-NL. and L-C.; Methodology, Z-NL.; Software, Z-BX.and N-S.; Validation, R-F., K-PL . and Z-BX.; Formal Analysis, C-FW. And N-S.; Investigation, C-FW.; Resources, Z-NL.; Data Curation, Z-BX. and N-S.; Writing – Original Draft Preparation, Z-NL.,Z-BX.; Writing – Review & Editing, Z-NL.; Visualization, R-F.; Supervision, Z-NL.; Project Administration, Z-NL. and N-S.; Funding Acquisition, Z-NL. All authors contributed to the manuscript and approved the submitted version. Competing interests Te authors declare no competing interests. Funding This research work was supported by the Project of Guangxi Science and Technology Department (grant No. 2024GXNSFAA010350),the Project of Guangxi Science and Technology Department (grant No. 2020GXNSFAA259043), Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine (2014, grant No. 32), National Traditional Chinese Medicine Education Letter (2022, No. 226), the Collaborative Innovation Center of Zhuang and Yao Ethnic Medicine (2013, No. 20) and zyyzdxk-2023165, College students' innovative training project (S202410600137). References National, P. C. 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Cite Share Download PDF Status: Published Journal Publication published 08 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Oct, 2024 Reviews received at journal 06 Oct, 2024 Reviewers agreed at journal 30 Sep, 2024 Reviews received at journal 19 Sep, 2024 Reviewers agreed at journal 19 Sep, 2024 Reviewers invited by journal 18 Sep, 2024 Editor assigned by journal 13 Sep, 2024 Editor invited by journal 13 Sep, 2024 Submission checks completed at journal 12 Sep, 2024 First submitted to journal 01 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5014790","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":366589135,"identity":"2a8fd081-fbee-428e-a048-471d7c566317","order_by":0,"name":"Zebi XIE","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zebi","middleName":"","lastName":"XIE","suffix":""},{"id":366589136,"identity":"c343948c-1603-4557-8ea0-7aa5cef0307b","order_by":1,"name":"Rong FAN","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"FAN","suffix":""},{"id":366589137,"identity":"5b3d175b-a259-4a45-b757-2112e67fa3a1","order_by":2,"name":"Kaiping Lai","email":"","orcid":"","institution":"Guangxi Chemical Research Institute Co. LTD","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kaiping","middleName":"","lastName":"Lai","suffix":""},{"id":366589138,"identity":"354f4592-9ebd-432a-bcaa-c4192b9ee228","order_by":3,"name":"Zining LIANG","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACPmYILYcQOkBACxtUizEDAzOxWqB0YgPxWtiZnz388udO+vyI/IOfbrYxyPHdSGD8XIDXYWzmxjI8z3I3njnMLJ3bxmAseSOBWXoGXi0MZtISEodzN7Y3szEDtSRuuJHAxsyDVwv7N2kJg8Pphs3MYC31RGjhMZP8kHA4QZ4dYkuCARFayqQZDhw23MBz2Fg655yE4cwzD5ul8Wnh5z++TfLHn8Py8jMSH37OKbOR5zuefPAzPi0gAHaGwQEwWwKIGRsIaAAq+QEk5AmrGwWjYBSMgpEKAEr8QsFR0j8fAAAAAElFTkSuQmCC","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zining","middleName":"","lastName":"LIANG","suffix":""},{"id":366589139,"identity":"78c67ae4-ac64-4143-8c07-1ecf125fa325","order_by":4,"name":"Ning Song","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Song","suffix":""},{"id":366589140,"identity":"48515c41-5d16-4830-a8ca-af707fecf6fe","order_by":5,"name":"Long Chen","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Chen","suffix":""},{"id":366589141,"identity":"ab2f4a62-441d-424f-ab95-6008af0141d1","order_by":6,"name":"Chunfeng Wu","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunfeng","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-09-02 01:59:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5014790/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5014790/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-85271-0","type":"published","date":"2025-05-08T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68901044,"identity":"146b0f77-3e4a-4404-8ee9-3add23a0752d","added_by":"auto","created_at":"2024-11-13 09:42:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14362,"visible":true,"origin":"","legend":"\u003cp\u003eRoot dry weight of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e at different culture stages. Different capital letters on the columns indicate significant differences between treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/fdf2297e6895c48457a404a7.png"},{"id":68900073,"identity":"0d576833-d536-4367-8b67-777640b5cd63","added_by":"auto","created_at":"2024-11-13 09:34:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":337196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eZanthoxylum nitidum\u003c/em\u003e root growth morphology. A, B, C and D show the root growth morphology of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e at 3, 6, 9 and 12 months post-treatment, respectively; CK indicates the control.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/54a8a0db5f999e987eb30c92.png"},{"id":68900072,"identity":"a0354e12-7992-4712-8b66-d03437df8414","added_by":"auto","created_at":"2024-11-13 09:34:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15905,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on SOD activity of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/126d2076360ae9a90f31efd1.png"},{"id":68901043,"identity":"b0e60eeb-80ec-4ffd-9de4-84dad89c5f30","added_by":"auto","created_at":"2024-11-13 09:42:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15070,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on POD activity of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/ba1cf65c2b4b88ef06ade578.png"},{"id":68900068,"identity":"6a608bea-c74a-4bdb-86e0-7aca9087064d","added_by":"auto","created_at":"2024-11-13 09:34:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":15631,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the endophytic fungus Y118 on MDA content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/d11169f01fccc03f13d9f40d.png"},{"id":68901045,"identity":"16411909-23b4-43a1-bd8d-80fb42502c08","added_by":"auto","created_at":"2024-11-13 09:42:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":15584,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on PAL activity of\u003cem\u003e Zanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/0c1a8c9eb8ceeaa2b74127d6.png"},{"id":68900076,"identity":"f915cfb9-7345-4645-ad15-f0cab60ba323","added_by":"auto","created_at":"2024-11-13 09:34:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":16679,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on soluble protein content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/f0f07f82fd8bbe3ee840f6e1.png"},{"id":68901048,"identity":"26a4dcbd-df42-4afd-8979-3df4cfab3047","added_by":"auto","created_at":"2024-11-13 09:42:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":16218,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on chlorophyll a content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/fd75e101ef06407b63641686.png"},{"id":68901193,"identity":"30536d56-3661-43ef-a9db-5f50dac7e87c","added_by":"auto","created_at":"2024-11-13 09:50:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":16669,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on chlorophyll b content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/57f00bf39f44f2ba7e271567.png"},{"id":68900079,"identity":"d9e3836d-8a36-4a42-b3d8-04e087eb2a3a","added_by":"auto","created_at":"2024-11-13 09:34:16","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":16898,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on the total chlorophyll content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/fca02962fac22550a0c8066e.png"},{"id":68900078,"identity":"53a2c157-6d24-498c-a192-764e7191af26","added_by":"auto","created_at":"2024-11-13 09:34:16","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":15702,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on nitidine chloride content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/6b3bca027ec3e6eca854d1bc.png"},{"id":68901047,"identity":"7e24b3b5-65f2-48b2-bc1c-fff97eb7d90b","added_by":"auto","created_at":"2024-11-13 09:42:16","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":14757,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of endophytic fungus Y118 on chelidonine content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. Different lowercase letters indicate a significant difference between different treatments (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/8c05d28eb93250c660a6083e.png"},{"id":82537623,"identity":"94a2fd4c-6a8b-40fe-a8bd-2b0c7e320ac2","added_by":"auto","created_at":"2025-05-12 16:09:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1301140,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5014790/v1/648dc2ff-e50c-48b7-ae79-41b4e84897dc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of endophytic fungus Y118 on physiological and biochemical indexes and quality improvement of Zanthoxylum nitidum (Roxb.) DC","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eZanthoxylum nitidum \u003c/em\u003e(Roxb.) DC. is a potent herb and an authentic medicinal material commonly used by the Yao people in Guangxi, China. It is predominantly found in Guangxi, Guangdong, Yunnan and other provinces in China\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e. This plant, belonging to the genus \u003cem\u003eZanthoxylum\u003c/em\u003e \u003cem\u003enitidum\u003c/em\u003e in the family Rutaceae, is also known as double needling, double back needle, Mountain tiger. It has provided evidence of the significant anti-inflammatory, antibacterial, and anticancer properties\u003csup\u003e3\u003c/sup\u003e. Unfortunately, the natural habitat of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e has suffered due to recent deforestation and the establishment of economic fruit forests.\u003c/p\u003e\n\u003cp\u003eThe medicinal parts of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e are the root and stem bark, often leading to the harvesting of the entire plant. This indiscriminate harvesting, combined with excessive mining, has resulted in a sharp decline in the number of breeding individuals in the wild. Consequently, this has adversely affected the production of proprietary Chinese medicines and related industries relying on it as a raw material. The current artificial cultivation areas are insufficient to meet market demands\u003csup\u003e4\u003c/sup\u003e. Addressing this issue, recent studies have explored the role of endophytes in mitigating the impact on \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eEndophytes, microorganisms forming a symbiotic relationship with the host plant without inducing tissue lesions\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e6\u003c/sup\u003e, have demonstrated the ability to promote the growth and development of host plants, induce beneficial secondary metabolites, and enhance physiological responses to environmental changes, thereby improving the soil microenvironment\u003csup\u003e7\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e8\u003c/sup\u003e. Notably, endophytic fungi have shown promise in the scientific and environmentally friendly prevention and control of soil-borne diseases in crops, as evidenced by studies on monk fruit\u003csup\u003e9\u003c/sup\u003e. Study by Li et al\u003csup\u003e10\u003c/sup\u003e showed remarkable growth-promoting effects of the endogenous control bacterium NQ8GⅡ4 on \u003cem\u003eLycium barbarum\u003c/em\u003e seedlings, suggesting potential application in agriculture. Similarly, Shen et al\u003csup\u003e11\u003c/sup\u003ehighlighted the potential of the \u003cem\u003ePenicillium\u003c/em\u003e fungus J10469 as an anti-aluminum specific bacterial fertilizer by screening endophytic fungal strains improving nutrient utilization efficiency in \u003cem\u003eVernicia montana\u003c/em\u003e Lour\u0026rsquo;s seedlings under aluminum stress.\u003c/p\u003e\n\u003cp\u003eWhile microbial fertilizers have shown great potential in increasing the yield of Chinese medicinal materials and enhancing plant physiological tolerance, there is a notable gap in the literature regarding the application of the endophytic fungus Y118 (\u003cem\u003ePhomopsis \u003c/em\u003esp.) as a microbial fertilizer for the management of\u003cem\u003e Zanthoxylum nitidum\u003c/em\u003e plantations. This study aims to comprehensively evaluate the induction of stress-related resistance enzymes in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e plants by the endophytic fungus Y118 at different culture stages. The findings of this study are expected to provide theoretical basis for the application of Y118 as a microbial fertilizer in the cultivation and management of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e and other medicinal plants.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant material collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental materials were collected in the Plant Culture Room of Guangxi University of Traditional Chinese Medicine (China) between April 2021 and June 2022. Seedlings for the pot experiments were obtained through successful in-house cultivation. The endophytic fungus Y118 of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e belongs to the genus \u003cem\u003ePhomopsis\u003c/em\u003e sp. and was isolated from early-stage leaves of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e plants in Guangxi\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of solid fermentation medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe solid medium was prepared with 85% cornmeal and 15% rice as the carrier, with additional components including maltose (2.5%), peptone (1.5%), KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (0.2%), and MgSO\u003csub\u003e4\u003c/sub\u003e (0.15%). The pH was adjusted to 5.5–6.5 (solid:liquid = 1:1) according to the method described by Qin et al\u003csup\u003e13\u003c/sup\u003e.The endophytic fungus Y118 was revived and cultured on PDA medium for 3–4 days. Subsequently, one endophytic fungus Y118 (size: 1.76625 cm²) was added to every 60 g solid culture medium, which was then fermented at 25°C for 40 days to produce solid fermentation medium for use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUniform\u003cem\u003e Zanthoxylum nitidum \u003c/em\u003eseedlings, each with a plant height of approximately 5.0 cm and 3-5 leaves, were planted individually in pots (15.5 cm inner diameter and 14 cm high). Each plant in the treatment group was cultured with 25 g solid fermentation medium, while each plant in the control group (CK) was cultured with 25 g solid fermentation medium without the addition of fungus. Three replicates were prepared for each treatment, with 10 pots per replicate. The seedlings were watered regularly every day during maintenance, and the humidity was maintained at approximately 80%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutput measurement methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the experimental treatment, \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e seedling growth was observed, and samples were collected at 3 months, 6 months, 9 months and 12 months for the measurement of growth indexes. For each sample, soil was rinsed from the surface of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e plants, and excess water was absorbed with gauze. After drying, \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e samples were separated into above- and underground parts. Upon drying in an oven at 60 °C to constant weight, the dry weight of the root sample was recorded.\u003c/p\u003e\n\u003cp\u003eLeaves in the middle and upper part of the \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e plant, with more uniform light access, were selected for the determination of physiological and biochemical indexes. Chlorophyll content was determined using the plant chlorophyll content detection kit. Soluble protein content was determined using the Coomassie brilliant blue method\u003csup\u003e14\u003c/sup\u003e. Superoxide dismutase (SOD) activity was determined using the nitrogen blue tetrazole photochemical reduction method\u003csup\u003e15\u003c/sup\u003e. Peroxidase (POD) activity was determined by the guaiacol method\u003csup\u003e15\u003c/sup\u003e. phenylalanine ammonolyase (PAL) activity was determined using an enzyme detection kit with absorbance measured at 290 nm. Malondialdehyde (MDA) content was determined using a plant MDA kit.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eZanthoxylum nitidum\u003c/em\u003e roots were obtained at 3, 6, 9 and 12 months after treatment. After the roots were dried and passed through a 60 mesh (0.3 mm), nitidum chloride and chelidonine contents were determined by high-performance liquid chromatography (HPLC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental data were sorted using Excel 2003, and statistical analysis was performed with SPSS 23.0. Comparisons among multiple groups were performed using one-way analysis of variance. A significance level of \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was considered to indicate a statistically significant difference, while \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 was considered to indicate an extremely significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffects of endophytic fungus Y118 on the growth of\u003c/b\u003e \u003cb\u003eZanthoxylum nitidum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe changes in the root dry weight of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e showed significant differences at different culture stages(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 3, 6, 9 and 12 months, the root dry weights of the Y118 treatment group significantly increased by 1123.53%, 51.08%, 56.40% and 47.12%, respectively, compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicated that treatment with the endophytic fungus Y118 promotes root growth, resulting in enhanced yield of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of endophytic fungus Y118 on the activity of antioxidant enzymes in\u003c/b\u003e \u003cb\u003eZanthoxylum nitidum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSOD and POD play crucial roles in removing excessive H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, contributing significantly to the anti-aging and metabolic processes of plants\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effects of the endophytic fungus Y118 on SOD activity in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e showed significant differences at different culture stages(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No significant difference in SOD activity was observed between treatment group and the control group at different culture stages post Y118 treatment. The impact of the endophytic fungus Y118 on POD activity in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e showed significant differences at different culture stages(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). After 9 months of cultivation, POD activity in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e was significantly higher compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of endophytic fungus Y118 on MDA content of\u003c/b\u003e \u003cb\u003eZanthoxylum nitidum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOxidative stress in plants leads to lipid peroxidation in the cell membrane, commonly assessed by measuring MDA as a marker\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Under the same culture conditions, lower MDA levels reflect reduced lipid peroxidation in the cell intima, indicating less damage to the cell membrane\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effects of the endophytic fungus Y118 on MDA content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e showed significant differences at different culture stages(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Compared with the control group, the molar mass concentration of MDA in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e co-cultured with Y118 decreased in the range of 10.64\u0026ndash;16.60% as the co-culture progressed from 3 to 12 months. The most significant decrease (16.60% compared with the control) in the molar mass concentration of MDA in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e co-cultured with Y118 was observed at 6 months. However, no significant differences were observed between the two groups at any of the four time-points.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of endophytic fungus Y118 on PAL activity of\u003c/b\u003e \u003cb\u003eZanthoxylum nitidum\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePAL activity has been shown to be positively correlated with disease resistance and stress resistance in plants, serving as a key index to assess these characteristics\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effects of the endophytic fungus Y118 on PAL enzyme activity in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e showed significant differences at different culture stages(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). After 3 months, PAL activity in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e co-cultured with Y118 was significantly higher (187.21%) than that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, after 6 months, no significant difference was observed between the treatment and control groups. Notably, PAL activity decreased at 9 and 12 months compared with the control, potentially influenced by local drought, cold autumn, and winter conditions during these periods, which are not conducive to mycelial growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of endophytic fungus Y118 on soluble protein content of\u003c/b\u003e \u003cb\u003eZanthoxylum nitidum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSoluble proteins are essential nutrients that play a key role in osmoregulation in plant cells. They serve as a protective layer for biological membranes\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and represent an important standard for a ssessing the healthy metabolism of plants\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Increased soluble protein content has been linked to improved metabolic intensity in plants\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effects of Y118 on the soluble protein content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e showed significant differences at different culture stages(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Throughout the 3, 6, 9, and 12-month co-cultures of the endophytic fungus Y118 with \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e on solid fermentation medium, the soluble protein content in the treatment group showed an increase compared with the control group. However, these changes did not reach the threshold of statistical significance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of endophytic fungus Y118 on chlorophyll content of\u003c/b\u003e \u003cb\u003eZanthoxylum nitidum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eChlorophyll is the key photosynthetic pigment in plant photosynthesis, and serves as an important indicator of plant growth and development\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effects of Y118 on the contents of chlorophyll a, chlorophyll b and total chlorophyll in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e leaves showed significant differences at different culture stages(Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), respectively. The contents of chlorophyll a, chlorophyll b and total chlorophyll in the treatment group were lower than those in the control group after 3 months and 6 months of co-culture. By the end of 9 months, the contents of chlorophyll a, chlorophyll b and total chlorophyll in the treatment group were higher than those in the control group, although the differences were not statistically significant. After 12 months, the contents of chlorophyll a and chlorophyll b significantly increased (by 45.28% and 28.87%, respectively) in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e leaves co-cultured with Y118 compared to the contents in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while there was no significant difference in the total chlorophyll contents compared to that in the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of nitidine chloride and chelidonine contents in\u003c/b\u003e \u003cb\u003eZanthoxylum nitidum\u003c/b\u003e \u003cb\u003eroots\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNitidine chloride and chelidonine are the main active components of nitidine and used as important indexes to evaluate the growth and quality of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effects of the endophytic fungus Y118 on nitidine chloride content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e showed significant differences at different culture stages(Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e), respectively. The nitidine chloride content in the root of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e co-cultured with Y118 increased compared with the control group, although the differences were not statistically significant at 3 and 6 months. At the 9-month mark, the nitidine chloride content in the root of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e treated with Y118 reached 0.783 mg/g, marking a 42.11% increase compared with the control group. After 12 months, the nitidine chloride content in the root of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e co-cultured with Y118 rose to 0.904 mg/g, reflecting a notable 44.18% increase compared with the control group.\u003c/p\u003e \u003cp\u003eThe effects of the endophytic fungus Y118 on chelidonine contents of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e showed significant differences at different culture stages(Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e), respectively. The chelidonine content in the root of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e co-cultured with Y118 was higher than that in the control group at 3 and 6 months, although the differences were not statistically significant. At the 9-month mark, the chelidonine content in the root of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e co-cultured with Y118 reached 3.704 mg/g, marking a 40.73% increase compared with the control group. After 12 months, the chelidonine content in the root of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e co-cultured with Y118 rose to 4.201 mg/g, reflecting a notable 26.57% increase compared with the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEcologically functional endophytes establish mutual symbiotic relationships with their host plants during co-evolution, thereby promoting host growth, alleviating the negative effects of stress, enhancing the accumulation of bioactive components, and playing a pivotal role in responding to stress conditions such as cold and drought\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In this study, we demonstrated a significant increase in the root dry weight of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e over a 12-month period when co-cultured with the endophytic fungus Y118, indicating a positive impact on \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e yield. The Y118 treatment group showed a promotion in the growth of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e plants. These effects align with findings from Xiong et al\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, who reported the influence of different microbial fertilizer treatments on the yield and quality of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. It can be speculated that the target fungi optimized the soil microbial population structure, activated soil enzyme activity, accelerated organic matter decomposition, and facilitated the conversion of fixed nutrients into available forms in the soil. Moreover, it is conceivable that Y118 either inhibited pathogenic microorganisms or, alternatively, promoted root growth by producing plant hormones, subsequently enhancing the growth of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e plants. The observed benefits may also stem from the decomposition of secondary metabolic toxins by Y118, inhibiting the growth of pathogenic microorganisms or inducing plant hormones production to promote root growth.\u003c/p\u003e \u003cp\u003eOur study also revealed that Y118 treatment enhanced the activity of defense enzymes in the leaves across all growth stages. In response to drought and other stresses, plants have evolved a variety of secondary metabolic pathways that generate corresponding secondary metabolites to alleviate stress\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. After inoculation with Y118, there was no significant change in SOD activity between the treatment and control groups. However, the POD enzyme activity in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e leaves was significantly higher in the inoculated group than in the non-inoculated group. This suggests that the seedlings maintained higher POD activity under the same culture conditions after inoculation with Y118, reducing the damage caused by intracellular reactive oxygen species to the cell membrane. Moreover, PAL enzyme activity also demonstrated an enhancement in the disease resistance and stress resistance of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e after inoculation with Y118. Therefore, it can be speculated that endophytic fungi alleviated the stress on \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e to a considerable extent, inducing an increase in antioxidase activity in the host.\u003c/p\u003e \u003cp\u003eThe potential of endophytic fungus Y118 to enhance the growth of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e might not solely be attributed to the reduction in lipid peroxidation levels and the augmentation of metabolic intensity in response to drought or cold conditions. The initial two culture stages in the treatment group did not show an obvious promotion of chlorophyll synthesis, possibly due to the unstable invasion and colonization of Y118, affecting the histocytes of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. However, compared with the control group, the chlorophyll content of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e in the treatment group increased after 9 months, coinciding with the weakened light and cold climate in the local region in January. After 12 months of co-cultivation, the contents of chlorophyll a and chlorophyll b in the treatment group significantly increased, despite the wet and cold climate. These findings suggest that Y118 may enhance the resilience of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e in adverse environments, although further studies are needed to confirm this. It is worth noting that nitidine chloride and chelidonine, the main active components of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e, have anti-inflammatory, analgesic and antibacterial effects\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we observed significant increase in the accumulation of nitidine and chelidonine chloride in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e roots after 9 and 12 months of co-culture with Y118, indicating growth promotion effects. These findings align with similar observations in [\u003cem\u003ePseudostellaria heterophylla\u003c/em\u003e (Miq.)]\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, we posit that the inoculation of the endophytic fungus Y118 may enhance the synthesis of active components, nitidum chloride, and chelidonine in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e during its artificial cultivation through two primary mechanisms: Firstly, by augmenting the root biomass of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e, thereby increasing the total production of effective alkaloids. Secondly, by elevating the content of soluble proteins and increasing the chlorophyll levels in plant leaves, leading to enhanced plant photosynthesis and heightened antioxidant enzymes activity. Thus, the inoculation of the endophytic fungus Y118 provides a promising approach for cultivating \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e with high yield and quality. However, the full extent of Y118\u0026rsquo;s impact on \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e requires further assessment through field trials. In addition, a comprehensive understanding of its role in reducing the dependence on chemical fertilizers and pesticides, as well as the underlying growth promotion mechanism, remains essential for maximizing the overall benefits of employing the endophytic fungus Y118 in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e production.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eInoculation with the endophytic fungus Y118 significantly improved the physiological tolerance and growth of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e plants. The increased root dry weight of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e, along with the elevated contents of nitidine chloride and chelidonine in the root, suggests comprehensive benefits of employing Y118 in the cultivation of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e crops.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confrm that the data supporting the findings of this study are available within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Z-NL. and L-C.; Methodology, Z-NL.; Software, Z-BX.and N-S.; Validation, R-F., K-PL . and Z-BX.; Formal Analysis, C-FW. And N-S.; Investigation, C-FW.; Resources, Z-NL.; Data Curation, Z-BX. and N-S.; Writing \u0026ndash; Original Draft Preparation, Z-NL.,Z-BX.; Writing \u0026ndash; Review \u0026amp; Editing, Z-NL.; Visualization, R-F.; Supervision, Z-NL.; Project Administration, Z-NL. and N-S.; Funding Acquisition, Z-NL. All authors contributed to the manuscript and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was supported by the Project of Guangxi Science and Technology Department (grant No. 2024GXNSFAA010350),the Project of Guangxi Science and Technology Department (grant No. 2020GXNSFAA259043), Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine (2014, grant No. 32), National Traditional Chinese Medicine Education Letter (2022, No. 226), the Collaborative Innovation Center of Zhuang and Yao Ethnic Medicine (2013, No. 20) and zyyzdxk-2023165, College students\u0026apos; innovative training project (S202410600137).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNational, P. C. 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Effects of seed dressing with bacterial fertilizer on growth and quality of Pseudostellariae pseudostellariae. \u003cem\u003eJiangsu Agricultural Sci.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 116\u0026ndash;120 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Zanthoxylum nitidum, endophytic fungus, quality improvement, physiological and biochemical indexes","lastPublishedDoi":"10.21203/rs.3.rs-5014790/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5014790/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo understand the effects of endophytic fungus Y118 on the physiological characteristics of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e (Roxb.) DC after co-culture with its host \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e, we measured the plant growth index (dry root weight) of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e at different growth stages using the solid fermentation medium containing endophytic fungus Y118. Physiological and biochemical indexes (defensive enzyme activity) were determined by chromatographic analysis of the contents of nitidum chloride and chelidonine, the main active ingredients in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e root. At 3, 6, 9 and 12 months, the root dry weight of Y118 treatment group was significantly increased by 1123.53%, 51.08%, 56.40% and 47.12% compared with the control group, respectively. At the 3-month mark, superoxide dismutase (SOD) activity exceeded that of the control. After 9 months of cultivation, peroxidase (POD) activity in the treatment group showed a significant difference at a 0.05 significant level. At the 3-month mark, phenylalanine ammonia lyase (PAL) activity in the treatment group was significantly higher than that in the control group, increasing by 187.21%. After 12 months of cultivation, the contents of chlorophyll a and chlorophyll b significantly increased by 45.28% and 28.87%, respectively, compared with the control group. After 9 and 12 months of co-culture, the contents of nitidine chloride in \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e in the Y118 treatment groups were 0.783 and 0.904 mg/g, respectively, showing a significant increase of 42.11% and 44.18% compared with the control group; the contents of chelidonine were significantly increased by 40.73% and 26.57% compared with the control group. Inoculation with the endophytic fungus Y118 significantly improved the physiological tolerance of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e and promoted the growth of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e, thereby enhancing the quality of \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e. This implicates Y118 as a potential artificial fertilizer for \u003cem\u003eZanthoxylum nitidum\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Effects of endophytic fungus Y118 on physiological and biochemical indexes and quality improvement of Zanthoxylum nitidum (Roxb.) 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