Mitigation of legacy effects in invaded soil using alien plant-derived biochar

Mitigation of legacy effects in invaded soil using alien plant-derived biochar | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitigation of legacy effects in invaded soil using alien plant-derived biochar Weitao Li, Hui Ning, Yangping Li, Mingzhu Zhang, Ruifang Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3663026/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims The introduction of non-native plant species often leads to extensive colonization of land and poses a threat to the already limited resources of arable land. Restoring invaded land and improving soil fertility are therefore essential. Methods In this study, biochar from invader Chromolaena odorata and fungicide were jointly used to improve soil invaded by this species. Results The results show that fungicide increase the total biomass of the subsequent plants grown in the invaded soil. The soil fungicide reduces the occurrence of pathogenic fungi in invaded soil and at the same time promotes the recruitment of mycorrhizal fungi and saprophytic fungi by the plants. The addition of biochar to the invaded soil has a negative legacy effect on subsequent invasive plants, while it exerts a positive enhancement effect on native plants. The simultaneous application of biochar and fungicide to soils invaded by alien plants can effectively diminish the competitive advantage of invasive species, while biochar can markedly increase the competitive advantage of native plants. Conclusions This study proposes a remediation method for legacy effect after alien plant invasion that not only effectively controls the spread of alien plants, but also improves soil fertility and increases economic benefits. Alien invasive plant biochar soil legacy effect soil pathogenic fungi Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction As the process of global economic integration accelerates and human interactions increase, the invasion of alien species becomes more serious. After the introduction of alien plants, soil properties are altered to promote successful invasion. Even after removal of the alien plants, the remaining soil environment can continue to influence the growth of subsequent plants, referred to as soil legacy effects (Jing et al. 2022 ). Soil legacies caused by alien plant invasion pose serious ecological risks and serve as important mechanisms for understanding the successful invasion of alien plants. Currently, there are also many problems with alien plant removal measures, such as large-scale herbicide use and burning, which not only damage the soil's "sink" function but also increase carbon emissions. There is an urgent need to take environmentally friendly measures to remove alien plants. After the removal of alien plants, the removal of soil legacy effects is also particularly urgent. On the one hand, when alien plants invade a new habitat, they recruit some beneficial microorganisms, such as mycorrhizal fungi and phosphate-solubilizing bacteria, which help the plants to absorb minerals. It has been found that the invasive plant Mikania micrantha significantly increases its content of available phosphorus in the rhizosphere compared to the two native plants Polygonum chinense and Paederia scandens , and promotes its own invasion through the accumulation of phosphate-solubilising bacteria ( Pseudomonas and Enterobacter ) (Yin et al. 2020 ). On the other hand, alien plants can rapidly accumulate pathogens of native plants in the invaded area. These pathogens often have a significant inhibitory effect on native plants but not on the invasive plants, affecting competition between invasive and native plants (Eppinga et al. 2006 ). For example, the invasive plant Chromolaena odorata increases the abundance of the pathogenic fungus Fusarium semitectum in the area it has invaded, which can significantly inhibit the growth of two native plants but has no apparent effect on C. odorata , thereby promoting C. odorata invasion (Mangla et al. 2008). The interaction between plants and soil microorganisms is an important mechanism for their successful invasion. The changes in soil microorganisms caused by previous plant stages can influence subsequent plant growth through interactions between plants and soil microbes. The interaction between plants and the rhizosphere microorganisms living with them leads to heritable changes in plant traits (Jacquiod et al. 2022 ). Research has shown that fungi left in the soil from previous extreme droughts can influence root architecture and biomass through a negative feedback between plant and soil (Lozano et al. 2022 ; Shi et al. 2021 ). The structure of microbial communities in the rhizosphere is closely related to the input of organic carbon from the plant (rhizodeposites and litter) and soil properties, which in turn can influence plant phenology and alter the distribution of photosynthetic carbon in the different plant components (Te Beest et al. 2009 ; Van Nuland et al. 2021 ). After Lasthenia californica was planted for three months in soil invaded by alien plants ( Aegilops triuncialis ), the flowering time of L. californica was delayed, the aboveground biomass was significantly reduced and the composition of the microbial community in the soil was significantly altered (Batten et al. 2008 ). Invasive alien plants become hosts for pathogens and create a microbial legacy in the soil that affects the growth of subsequent plants. Urgent action is needed to prevent the spread of invasive plants and the transmission of pathogenic microorganisms that affect the establishment of local plant communities. By influencing the microbial legacy in the soil, subsequent plant communities can recruit new microbial communities and establish a positive plant-soil feedback, restoring native ecosystems. Research has shown that the application of fungicide to soil can disrupt the existing network of soil pathogens, allowing subsequent plants to recruit new microbial networks and restore soil biological activity (Dominguez-Begines et al. 2021 ; Maron et al. 2013 ). The introduction of soil fungicide inevitably affects nutrient cycling involving soil microorganisms in the early stages of plant establishment, so nutrient supplements are needed to compensate for nutrient deficiencies. Measures must also be taken to prevent pollution from harmful fungicide runoff. Biochar has a wide range of applications in soil remediation, including adsorption of organic and inorganic pollutants in the soil and binding of chemical substances (Cao and Harris 2010 ; Harindintwali et al. 2023 ). In addition, the ash content in biochar facilitates nutrient uptake by plants. The combination of soil fungicide and biochar can block the effects of pathogenic microorganisms on later plant growth, while biochar can provide the nutrients needed for early plant growth. The effects of soil remediation by combination of fungicide and biochar can be evaluated through plant-soil interactions. Chromolaena odorata is native to North, Central and South America. It invaded tropical regions in Asia and Africa in the mid-19th century and poses a serious threat to the growth, biodiversity and ecological security of native plants in the invaded areas due to its large seed bank, rapid spreading ability. In the tropical regions of China with abundant water and heat, C. odorata can quickly accumulate a large biomass and high seed yield. Research has shown that preparing alien plants into biochar can effectively inactivate the seeds produced by the plants and their rhizomes, controlling the spread risk of the alien plants to some extent (Feng et al. 2021 ). We hypothesize that 1) the introduction of alien plants strongly affects soil properties, which significantly affects the subsequent growth of the plants; 2) these effects can be suppressed by the application of fungicide; 3) the impairment of plant growth caused by fungicide can be alleviated by the addition of biochar, which has a positive effect on subsequent plant growth. We collected soil severely invaded by C. odorata in the field, eliminated the influence of the original pathogen community in the soil by fungicide and prepared C. odorata into biochar by high temperature pyrolysis. The application of biochar effectively promotes early plant establishment (Xu et al. 2023 ), achieving the goal of soil remediation. This technology not only controls the spread of alien plants, but also increases the yield of above-ground plants, which benefits the stability of the entire ecosystem. 2. Materials and methods 2.1. Preparing biochar by using invasive plant Chromolaena odorata In the area where Chromolaena odorata had invaded, we collected the biomass, dried it in the sunlight and processed it into biochar. We used a carbonization furnace to produce the biochar by the anaerobic dry distillation method with a temperature setting of 3 ℃/min heating from room temperature to 500℃ and maintained this temperature for 2 hours. The resulting biochar was sieved through a 60 mesh screen and used for the pot experiments. 2.2. Soil sampling and sterilization Excavate soil from two typical invasive areas: Menglun Town, Mengla County (21°91′ N, 101°26′E) and Shiliangzi, Xiangming Township, Mengla County (22°12′ N, 101°16′E). Use non-invasive soil as control, and select three sampling points for each sampling area with a distance of 20–50 meters between point repetitions. Excavate surface soil (0–20 cm), sieve it through a 5 cm sieve, and was fumigated with Dazomet fungicide. 2.3. Pot experiment In each pot, there was intra-specific competition among plants of the same species, as well as interspecific competition between Chromolaena odorata , an invasive species, and Triumfetta rhomboidea , a native plant. The total number of pots was calculated as follows: 2 (invaded areas) × 2 (invaded soils) × 3 (soil repetitions) × 3 (species combinations) × 2 (biochar) × 2 (fungicide) = 144. We conducted regular weeding, pest control, and watering to minimize interference from pests, diseases, and weeds. (1) Measurement of plant height: At mid-growth, the height of each plant was measured with a tape measure. (2) Measurement of specific leaf area: At mid to late plant growth, 5 complete leaves were taken from each pot and the total leaf area of the plant leaves was measured with a leaf area metre. The leaves were then dried (70°C, 36 h). Plant-specific leaf area = total leaf area/total dry weight. (3) Biomass determination: The biomass and leaf, stem and root biomass of each pot were harvested at the later stages of plant growth. The total biomass of each pot was calculated, as well as the proportion of the different components in the biomass. 2.4. Illumina sequencing analysis of 18S gene amplicons Genomic DNA was extracted from 0.5 g of fresh soil samples using a FastDNA™SPIN kit (MP Biomedicals, Santa Ana, CA, USA) following the manufacturer's protocol. The universal primers SSU0817F (5’-TTAGCATGGAATAATRRAATAGGA-3’) and 1196R (5’-TCTGGACCTGGTGAGTTTCC-3’) were used to amplify the 18S genes. The amplicons were subjected to paired-end sequencing on an Illumina MiSeq platform following a standard protocol at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Paired-end reads obtained from the original DNA fragments were merged using FLASH (Magoc and Salzberg 2011 ) and subsequently assigned to each sample using unique barcodes. The sequences were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) software package (Caporaso et al. 2010 ), and in-house Perl scripts were employed to analyze beta (between sample) diversity. Initially, the reads underwent filtration using QIIME quality filters. Subsequently, we utilized pick_de_novo_otus.py to select operational taxonomic units (OTUs) and generate an OTU table. Sequences exhibiting ≥ 97% similarity were assigned to the corresponding OTU. A representative sequence was selected for each OTU, and the taxonomic information for eukaryotic microbes was annotated using a subset of the SILVA 119 database ( http://www.arb-silva.de/download/archive/qiime/ ). The sequencing data were categorized into three functional groups, namely pathogenic fungi, AM fungi, and saprotrophic fungi, using the available data from FUNGuild (Nguyen et al. 2016 ). The sequencing data for the fungal genomes were deposited in the NCBI Sequence Read Archive ( http://trace.ncbi.nlm.nih.gov/Traces/sra/ ) under the accession number PRJNA1002838. 2.5. Data analysis Soil biota effect (SBE) = ln(P Live /P Sterile ), where P Live refers to the total biomass of plants in pots without sterilization treatment, and P Sterile refers to the growth traits of plants in sterilized pots. SBE > 0 indicates a positive soil biota effect, while SBE < 0 indicates a negative soil biota effect. Competitiveness index (CI) of plants = ln(P inter /P intra ), where P inter and P intra refer to the total biomass of plants in pots with interspecific and intraspecific competition, respectively. These formulas were based on the algorithm proposed by Lekberg et al. ( 2018 ). Linear mixed models (LMMs) were used for data analysis, with the response variables being growth traits of plants (total biomass, root biomass, root to shoot ratio, plant height and specific leaf area). The fixed factors were invasive plant presence, microbial treatment (sterilization or not), and biochar addition. Soil source (different invasion areas) were treated as random factors. The effects of soil biota legacy on the growth of invasive and native plants after invasion were analyzed, as well as the restorative effect of biochar on the soil biota legacy. The LMMs analysis was performed using the "lmerTest" package in R software (ver. 3.6.2). 3. Results 3.1. The subsequent plants were improved by the addition of fungicide and biochar Soil fertilization significantly increased the total biomass and plant height of subsequent plants in the invaded soil (Fig. 2 B, S1B). The addition of soil fungicide resulted in a significant reduction in the root-to-shoot ratio of native plants (Fig. 2 F) and a concurrent decrease in the specific leaf area of invasive plants (Fig. S1 D). The addition of biochar to the invaded soil resulted in a noteworthy enhancement in the root-to-shoot ratio and specific leaf area of indigenous plant species (Fig. 2 E, S1C). 3.2. Eukaryotic microbial community in response to soil sterilization and biochar addition Soil sterilization and the addition of biochar, as well as their interaction, have significant effects on the structure of soil eukaryotic microbial community (Fig. 3 A). Soil sterilization led to a 20% increase and a 13% decrease in eukaryotic microbial abundance, respectively. However, the impact of biochar application on the eukaryotic microbial community was only 0.7% (Fig. 3 C, D). Biochar application leads to an increase in pathogenic fungi in the soil while suppressing the abundance of mycorrhizal fungi and saprophytic fungi (Fig. S2A, C, E). The soil fungicide reduces the presence of pathogenic fungi in invaded soil and at the same time promotes the recruitment of mycorrhizal fungi and saprophytic fungi by the plants (Fig. S2B, D, F). Conversely, in non-invaded soil, the fungicide promotes the occurrence of soil-borne pathogenic fungi and mycorrhizal fungi, while significantly reducing the abundance of saprophytic fungi. 3.3. Mitigation of biochar on biological legacy effects The presence of a legacy in the soil following the invasion of exotic plants hampers the biomass accumulation in subsequent invasive plants, resulting in a negative legacy effect (Fig. S3A). The application of biochar to invaded soil mitigates the negative residual effects of soil biota on native plants, while intensifying the negative suppression effect on invasive plants (Fig. S3B). The inclusion of biochar can enhance the competitiveness of native plants while suppressing that of invasive plants (Fig. S4A). The application of biochar enhances the residual effects of eukaryotic microorganisms, and there is a significant negative correlation between the impact of soil residual effects on root systems and the diversity of soil microorganisms (Fig. 4 ). The utilization of biochar diminishes the impact of residual effects from soil microorganisms on roots (Fig. 5 ). 4. Discussion 4.1. Soil legacy effects after invasion of alien plants The remaining soil biota after invasion by alien plants has an inhibitory effect on the biomass accumulation of subsequent plants. Following the introduction of alien plants, their apparent competitive advantage due to the absence of above- and below-ground natural enemies resulted in greater impacts on the soil environment, including soil nutrients, enzyme activity, microbial community structure and diversity, and soil animals (Lau and Lennon 2012 ). The impact of invasive plants on the soil forms a soil "memory" that continues to influence the growth of subsequent plants. The aim of this study was to analyze the effects of retained soil biota on subsequent plant growth by performing soil sterilization. Soil sterilization reduces the feeding of soil organisms on plant roots and the uptake of root secretions by microorganisms, indirectly reducing the downward secretion of photosynthetic carbon in plants and investing more biomass in the formation of aboveground plant components (Li et al. 2023 ). In addition, our research has shown that the use of fungicide inhibits the germination of seed banks in the soil, thereby reducing weed competition and indirectly promoting plant growth. The invasive plant Chromolaena odorata is known for its high seed production. These huge seeds can germinate rapidly after removal of the invasive plant, hindering the establishment of native plant communities. However, the fungicide used in this study effectively inhibits the germination of the seed bank, creating favourable conditions for the subsequent restoration of the local plant community. 4.2. The eukaryotic microbial community assembly in response to the addition of fungicide and biochar In our research we have found that fungicides, biochar and their interactions significantly alter eukaryotic microbial communities in soil, with fungicides having the strongest effects (Fig. 3 ). Soil microbial memory is eliminated by the fungicide and plants then recruit new microbial communities. The soil fungicide impaired the occurrence of pathogenic fungi but promoted the recruitment of mycorrhizal fungi and saprophytic fungi by the plants (Fig. S2). The structure and function of plant rhizosphere microbial community are closely related to the input of plant-derived organic carbon (rhizosphere sedimentary carbon and litter) and soil properties (Zhang et al. 2019 ). Conversely, soil microorganisms can affect the phenology of aboveground plant leaves and the accumulation of biomass through their effects on nutrient cycling and availability. Although the use of soil fungicides weakens microbial memory, it directly affects the turnover of nutrients in the soil, which is detrimental to subsequent nutrient uptake by the plants. The addition of biochar replenishes early nutrients, facilitating nutrient uptake and utilization by plants (Hue 2020 ). Biochar can also absorb some chemicals in the soil and promote plant growth. The combined use of biochar and fungicides exhibits evident interactive effects that have a positive impact on the stability of the subsequent plant, which is due to the stable interaction between plant and soil. 4.3. The remedial effect of biochar on soil legacy effects The introduction of invasive plants alters the composition of root exudates and litter, selectively stimulating certain soil microorganisms, affecting microbial community structure and function, and influencing the growth of subsequent plants through the "memory" of the soil microbial pool. In addition, invasive plants can also alter the physico-chemical properties of the soil, such as pH, organic matter content and nutrient availability, which in turn can affect microbial communities and the soil biota. Biochar contains some ash elements that can be added directly to plant growth. In addition, the addition of biochar can improve the physical structure of the soil, increase the permeability of the soil, improve the soil environment and indirectly promote plant growth. In addition, biochar has a relatively large specific surface area that can adsorb chemically sensitive substances in the soil, which indirectly promotes plant growth. Overall, our study highlights the importance of considering the legacy of invasive plants on the soil environment and subsequent plant growth. The soil "memory" created by invasive plants can significantly affect the growth and survival of subsequent plants and have long-term consequences for ecosystem functioning. It is therefore important to take measures to prevent the introduction and spread of invasive plants and to restore and rehabilitate invaded ecosystems to minimize their impact on the soil environment and subsequent plant growth. Converting invasive plants into biochar can completely destroy their seeds and rhizomes, which is beneficial for controlling the spread of invasive plants. The processed biochar provides nutrients, adsorbs chemicals in the soil and improves soil aggregation, which directly promotes plant growth. Additionally, the utilization of biochar effectively alleviates the initial deficiencies of nutrients and root damage caused by fungicides, while simultaneously remediating the environmental pollution resulting from fungicide use. In conclusion, the combined use of biochar and fungicides effectively controls legacy effects caused by invasive plant invasions and has significant practical benefits for invasive plant control and land use. Conclusions We used the invasive species C. odorata to produce biochar, which was then combined with a fungicide to alleviate the legacy remained in the invaded soil. The soil fungicide reduces the occurrence of pathogenic fungi in invaded soil and at the same time promotes the recruitment of mycorrhizal fungi and saprophytic fungi by the plants. The incorporation of biochar and fungicide into the soil can significantly reduce the competitive ability of non-native plants while improving the competitive ability of native plants. The application of biochar can effectively mitigate the legacy in the invaded soil and underlines the great importance of soil remediation for the restoration of native plants diversity. Declarations Acknowledgements The authors would like to thank Wen-bian Bo for taking care of the potted plants and Long Li for their assistance in collecting the biomass data. This work was supported by the National Key R&D Program of China (2022YFF1302402) and Yunnan Fundamental Research Projects (202201AT070609), the National Natural Science Foundation of China (32171660, 32071661) and the 14th Five-Year Plan of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (E3ZKFF3B, E3ZKFF9B) and the “Yunnan Revitalization Talent Support Program” in Yunnan Province. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Weitao Li, Hui Ning and Yulong Zheng. The first draft of the manuscript was written by Weitao Li and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Batten KM, Scow KM, Espeland EK (2008) Soil microbial community associated with an invasive grass differentially impacts native plant performance. Microb Ecol 55:220-228. Cao X, Harris W (2010) Properties of dairy-manure-derived biochar pertinent to its potential use in remediation. Bioresour Technol 101: 5222-5228. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335-336. 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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-3663026","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":259412276,"identity":"b5997a8a-23c3-4b34-a7bd-d5ff74a3c775","order_by":0,"name":"Weitao Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIie3RsQrCMBCA4ZNAs0SzRhR9hZaALtJnuSA4ia6Cg0LBZ/AxBKGuhUK71N3RLs6KDhmN4uJi4yaYH3JTPjgSAJfrB2sQM9AcDuDZEe9JjGkuiS15TkP8xJpQFhxLHXbkYZqVNwi7QPdJxWJM+ohD2StSKtswDJZsghWkHgtEouI88loCCIJgfhXZacSF2kbkQRZWJDYvlqoNjbzmGVIbwq8CR7kURdqX4OfBio0/E84zddGDeYev1anUs3mX0+IzeYs8VrL8nVc1/dV1l8vl+pfujN02dYpumEAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9778-7513","institution":"Xishuangbanna Tropical Botanical Garden Chinese Academy of Sciences Key Laboratory of Tropical Forest Ecology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Weitao","middleName":"","lastName":"Li","suffix":""},{"id":259412277,"identity":"144345c6-381b-47a1-b0e6-a27d556e7912","order_by":1,"name":"Hui Ning","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Ning","suffix":""},{"id":259412278,"identity":"69054cb2-91a4-4128-be8a-32b9b33a1aa1","order_by":2,"name":"Yangping Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yangping","middleName":"","lastName":"Li","suffix":""},{"id":259412279,"identity":"9a468dcd-a127-4a61-8442-d5e9f2c1f7a6","order_by":3,"name":"Mingzhu Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingzhu","middleName":"","lastName":"Zhang","suffix":""},{"id":259412280,"identity":"2058007d-745c-408a-9b1b-dd7c67faec4a","order_by":4,"name":"Ruifang Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruifang","middleName":"","lastName":"Wang","suffix":""},{"id":259412281,"identity":"e8991cfb-11c7-4288-991b-9f701abdbe83","order_by":5,"name":"Yulong Zheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yulong","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2023-11-25 10:00:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3663026/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3663026/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":48299393,"identity":"8171d5b6-65f7-4629-9be5-6a39049fc0d5","added_by":"auto","created_at":"2023-12-15 21:33:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":367480,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of experimental layout.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3663026/v1/476804981beafb8178e4a0df.png"},{"id":48299392,"identity":"62f4bb46-486c-4d78-a766-132364f2cc60","added_by":"auto","created_at":"2023-12-15 21:33:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":168142,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of fungicide treatment and biochar addition on biomass and allocation in subsequent plants. Panels A-B compare the effects of fungicide and biochar addition (Y) and non-addition (N) on subsequent plant biomass. Panels C-D focus on root biomass, while panels E-F examine the root-to-shoot ratio.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3663026/v1/c6f4f74c21c805abdab597ce.png"},{"id":48299391,"identity":"c8a0c370-93b1-4f42-a411-d688cb2c73bb","added_by":"auto","created_at":"2023-12-15 21:33:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":254589,"visible":true,"origin":"","legend":"\u003cp\u003eEukaryotic microbial community in response to the addition of biochar and fungicide. Panels A-B examine the eukaryotic microbial community. Panels C-D investigate the degree of changes induced by the addition of fungicide and biochar in the eukaryotic microbial community. *, **, and *** indicate statistical significance at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, and \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, respectively.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3663026/v1/87c3961f80ca012f6aed50b0.png"},{"id":48299764,"identity":"06f5efba-f416-4a8d-af60-719b6b05e7a6","added_by":"auto","created_at":"2023-12-15 21:41:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":152780,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of eukaryotic microbial communities on plant biomass and its distribution following biochar addition. * indicate statistical significance at p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3663026/v1/e4176d1010fb0ebf677861aa.png"},{"id":48299395,"identity":"59262771-a7a1-4f8f-b879-d693973ae2bc","added_by":"auto","created_at":"2023-12-15 21:33:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":497129,"visible":true,"origin":"","legend":"\u003cp\u003eDepicts the concept of remediating eukaryotic microbial legacy effects through the addition of biochar and fungicide.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3663026/v1/4e9054047ffcb019d82664f1.png"},{"id":57169662,"identity":"2b418cd7-4ab9-440b-9f5e-7bc1094a8320","added_by":"auto","created_at":"2024-05-26 20:23:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2031885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3663026/v1/d6297c15-b0f8-4a20-ad76-720de1320afb.pdf"},{"id":48299394,"identity":"a0b5ade8-8026-4f5e-a160-c8024718a077","added_by":"auto","created_at":"2023-12-15 21:33:11","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1108828,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-3663026/v1/b5722635335dc65f1964944d.docx"}],"financialInterests":"","formattedTitle":"Mitigation of legacy effects in invaded soil using alien plant-derived biochar","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs the process of global economic integration accelerates and human interactions increase, the invasion of alien species becomes more serious. After the introduction of alien plants, soil properties are altered to promote successful invasion. Even after removal of the alien plants, the remaining soil environment can continue to influence the growth of subsequent plants, referred to as soil legacy effects (Jing et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Soil legacies caused by alien plant invasion pose serious ecological risks and serve as important mechanisms for understanding the successful invasion of alien plants. Currently, there are also many problems with alien plant removal measures, such as large-scale herbicide use and burning, which not only damage the soil's \"sink\" function but also increase carbon emissions. There is an urgent need to take environmentally friendly measures to remove alien plants. After the removal of alien plants, the removal of soil legacy effects is also particularly urgent.\u003c/p\u003e \u003cp\u003eOn the one hand, when alien plants invade a new habitat, they recruit some beneficial microorganisms, such as mycorrhizal fungi and phosphate-solubilizing bacteria, which help the plants to absorb minerals. It has been found that the invasive plant \u003cem\u003eMikania micrantha\u003c/em\u003e significantly increases its content of available phosphorus in the rhizosphere compared to the two native plants \u003cem\u003ePolygonum chinense\u003c/em\u003e and \u003cem\u003ePaederia scandens\u003c/em\u003e, and promotes its own invasion through the accumulation of phosphate-solubilising bacteria (\u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eEnterobacter\u003c/em\u003e) (Yin et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the other hand, alien plants can rapidly accumulate pathogens of native plants in the invaded area. These pathogens often have a significant inhibitory effect on native plants but not on the invasive plants, affecting competition between invasive and native plants (Eppinga et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). For example, the invasive plant \u003cem\u003eChromolaena odorata\u003c/em\u003e increases the abundance of the pathogenic fungus \u003cem\u003eFusarium semitectum\u003c/em\u003e in the area it has invaded, which can significantly inhibit the growth of two native plants but has no apparent effect on \u003cem\u003eC. odorata\u003c/em\u003e, thereby promoting \u003cem\u003eC. odorata\u003c/em\u003e invasion (Mangla et al. 2008). The interaction between plants and soil microorganisms is an important mechanism for their successful invasion.\u003c/p\u003e \u003cp\u003eThe changes in soil microorganisms caused by previous plant stages can influence subsequent plant growth through interactions between plants and soil microbes. The interaction between plants and the rhizosphere microorganisms living with them leads to heritable changes in plant traits (Jacquiod et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Research has shown that fungi left in the soil from previous extreme droughts can influence root architecture and biomass through a negative feedback between plant and soil (Lozano et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The structure of microbial communities in the rhizosphere is closely related to the input of organic carbon from the plant (rhizodeposites and litter) and soil properties, which in turn can influence plant phenology and alter the distribution of photosynthetic carbon in the different plant components (Te Beest et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Van Nuland et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). After \u003cem\u003eLasthenia californica\u003c/em\u003e was planted for three months in soil invaded by alien plants (\u003cem\u003eAegilops triuncialis\u003c/em\u003e), the flowering time of \u003cem\u003eL. californica\u003c/em\u003e was delayed, the aboveground biomass was significantly reduced and the composition of the microbial community in the soil was significantly altered (Batten et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Invasive alien plants become hosts for pathogens and create a microbial legacy in the soil that affects the growth of subsequent plants. Urgent action is needed to prevent the spread of invasive plants and the transmission of pathogenic microorganisms that affect the establishment of local plant communities.\u003c/p\u003e \u003cp\u003eBy influencing the microbial legacy in the soil, subsequent plant communities can recruit new microbial communities and establish a positive plant-soil feedback, restoring native ecosystems. Research has shown that the application of fungicide to soil can disrupt the existing network of soil pathogens, allowing subsequent plants to recruit new microbial networks and restore soil biological activity (Dominguez-Begines et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Maron et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The introduction of soil fungicide inevitably affects nutrient cycling involving soil microorganisms in the early stages of plant establishment, so nutrient supplements are needed to compensate for nutrient deficiencies. Measures must also be taken to prevent pollution from harmful fungicide runoff. Biochar has a wide range of applications in soil remediation, including adsorption of organic and inorganic pollutants in the soil and binding of chemical substances (Cao and Harris \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Harindintwali et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, the ash content in biochar facilitates nutrient uptake by plants. The combination of soil fungicide and biochar can block the effects of pathogenic microorganisms on later plant growth, while biochar can provide the nutrients needed for early plant growth. The effects of soil remediation by combination of fungicide and biochar can be evaluated through plant-soil interactions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eChromolaena odorata\u003c/em\u003e is native to North, Central and South America. It invaded tropical regions in Asia and Africa in the mid-19th century and poses a serious threat to the growth, biodiversity and ecological security of native plants in the invaded areas due to its large seed bank, rapid spreading ability. In the tropical regions of China with abundant water and heat, \u003cem\u003eC. odorata\u003c/em\u003e can quickly accumulate a large biomass and high seed yield. Research has shown that preparing alien plants into biochar can effectively inactivate the seeds produced by the plants and their rhizomes, controlling the spread risk of the alien plants to some extent (Feng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We hypothesize that 1) the introduction of alien plants strongly affects soil properties, which significantly affects the subsequent growth of the plants; 2) these effects can be suppressed by the application of fungicide; 3) the impairment of plant growth caused by fungicide can be alleviated by the addition of biochar, which has a positive effect on subsequent plant growth. We collected soil severely invaded by \u003cem\u003eC. odorata\u003c/em\u003e in the field, eliminated the influence of the original pathogen community in the soil by fungicide and prepared \u003cem\u003eC. odorata\u003c/em\u003e into biochar by high temperature pyrolysis. The application of biochar effectively promotes early plant establishment (Xu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), achieving the goal of soil remediation. This technology not only controls the spread of alien plants, but also increases the yield of above-ground plants, which benefits the stability of the entire ecosystem.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparing biochar by using invasive plant Chromolaena odorata\u003c/h2\u003e \u003cp\u003eIn the area where \u003cem\u003eChromolaena odorata\u003c/em\u003e had invaded, we collected the biomass, dried it in the sunlight and processed it into biochar. We used a carbonization furnace to produce the biochar by the anaerobic dry distillation method with a temperature setting of 3 ℃/min heating from room temperature to 500℃ and maintained this temperature for 2 hours. The resulting biochar was sieved through a 60 mesh screen and used for the pot experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Soil sampling and sterilization\u003c/h2\u003e \u003cp\u003eExcavate soil from two typical invasive areas: Menglun Town, Mengla County (21\u0026deg;91\u0026prime; N, 101\u0026deg;26\u0026prime;E) and Shiliangzi, Xiangming Township, Mengla County (22\u0026deg;12\u0026prime; N, 101\u0026deg;16\u0026prime;E). Use non-invasive soil as control, and select three sampling points for each sampling area with a distance of 20\u0026ndash;50 meters between point repetitions. Excavate surface soil (0\u0026ndash;20 cm), sieve it through a 5 cm sieve, and was fumigated with Dazomet fungicide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Pot experiment\u003c/h2\u003e \u003cp\u003eIn each pot, there was intra-specific competition among plants of the same species, as well as interspecific competition between \u003cem\u003eChromolaena odorata\u003c/em\u003e, an invasive species, and \u003cem\u003eTriumfetta rhomboidea\u003c/em\u003e, a native plant. The total number of pots was calculated as follows: 2 (invaded areas) \u0026times; 2 (invaded soils) \u0026times; 3 (soil repetitions) \u0026times; 3 (species combinations) \u0026times; 2 (biochar) \u0026times; 2 (fungicide)\u0026thinsp;=\u0026thinsp;144. We conducted regular weeding, pest control, and watering to minimize interference from pests, diseases, and weeds.\u003c/p\u003e \u003cp\u003e(1) Measurement of plant height: At mid-growth, the height of each plant was measured with a tape measure. (2) Measurement of specific leaf area: At mid to late plant growth, 5 complete leaves were taken from each pot and the total leaf area of the plant leaves was measured with a leaf area metre. The leaves were then dried (70\u0026deg;C, 36 h). Plant-specific leaf area\u0026thinsp;=\u0026thinsp;total leaf area/total dry weight. (3) Biomass determination: The biomass and leaf, stem and root biomass of each pot were harvested at the later stages of plant growth. The total biomass of each pot was calculated, as well as the proportion of the different components in the biomass.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Illumina sequencing analysis of 18S gene amplicons\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from 0.5 g of fresh soil samples using a FastDNA\u0026trade;SPIN kit (MP Biomedicals, Santa Ana, CA, USA) following the manufacturer's protocol. The universal primers SSU0817F (5\u0026rsquo;-TTAGCATGGAATAATRRAATAGGA-3\u0026rsquo;) and 1196R (5\u0026rsquo;-TCTGGACCTGGTGAGTTTCC-3\u0026rsquo;) were used to amplify the 18S genes. The amplicons were subjected to paired-end sequencing on an Illumina MiSeq platform following a standard protocol at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Paired-end reads obtained from the original DNA fragments were merged using FLASH (Magoc and Salzberg \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and subsequently assigned to each sample using unique barcodes. The sequences were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) software package (Caporaso et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and in-house Perl scripts were employed to analyze beta (between sample) diversity. Initially, the reads underwent filtration using QIIME quality filters. Subsequently, we utilized pick_de_novo_otus.py to select operational taxonomic units (OTUs) and generate an OTU table. Sequences exhibiting\u0026thinsp;\u0026ge;\u0026thinsp;97% similarity were assigned to the corresponding OTU. A representative sequence was selected for each OTU, and the taxonomic information for eukaryotic microbes was annotated using a subset of the SILVA 119 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.arb-silva.de/download/archive/qiime/\u003c/span\u003e\u003cspan address=\"http://www.arb-silva.de/download/archive/qiime/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The sequencing data were categorized into three functional groups, namely pathogenic fungi, AM fungi, and saprotrophic fungi, using the available data from FUNGuild (Nguyen et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The sequencing data for the fungal genomes were deposited in the NCBI Sequence Read Archive (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://trace.ncbi.nlm.nih.gov/Traces/sra/\u003c/span\u003e\u003cspan address=\"http://trace.ncbi.nlm.nih.gov/Traces/sra/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under the accession number PRJNA1002838.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Data analysis\u003c/h2\u003e \u003cp\u003eSoil biota effect (SBE)\u0026thinsp;=\u0026thinsp;ln(P\u003csub\u003eLive\u003c/sub\u003e/P\u003csub\u003eSterile\u003c/sub\u003e), where P\u003csub\u003eLive\u003c/sub\u003e refers to the total biomass of plants in pots without sterilization treatment, and P\u003csub\u003eSterile\u003c/sub\u003e refers to the growth traits of plants in sterilized pots. SBE\u0026thinsp;\u0026gt;\u0026thinsp;0 indicates a positive soil biota effect, while SBE\u0026thinsp;\u0026lt;\u0026thinsp;0 indicates a negative soil biota effect. Competitiveness index (CI) of plants\u0026thinsp;=\u0026thinsp;ln(P\u003csub\u003einter\u003c/sub\u003e/P\u003csub\u003eintra\u003c/sub\u003e), where P\u003csub\u003einter\u003c/sub\u003e and P\u003csub\u003eintra\u003c/sub\u003e refer to the total biomass of plants in pots with interspecific and intraspecific competition, respectively. These formulas were based on the algorithm proposed by Lekberg et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLinear mixed models (LMMs) were used for data analysis, with the response variables being growth traits of plants (total biomass, root biomass, root to shoot ratio, plant height and specific leaf area). The fixed factors were invasive plant presence, microbial treatment (sterilization or not), and biochar addition. Soil source (different invasion areas) were treated as random factors. The effects of soil biota legacy on the growth of invasive and native plants after invasion were analyzed, as well as the restorative effect of biochar on the soil biota legacy. The LMMs analysis was performed using the \"lmerTest\" package in R software (ver. 3.6.2).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. The subsequent plants were improved by the addition of fungicide and biochar\u003c/h2\u003e \u003cp\u003eSoil fertilization significantly increased the total biomass and plant height of subsequent plants in the invaded soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, S1B). The addition of soil fungicide resulted in a significant reduction in the root-to-shoot ratio of native plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) and a concurrent decrease in the specific leaf area of invasive plants (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). The addition of biochar to the invaded soil resulted in a noteworthy enhancement in the root-to-shoot ratio and specific leaf area of indigenous plant species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, S1C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Eukaryotic microbial community in response to soil sterilization and biochar addition\u003c/h2\u003e \u003cp\u003eSoil sterilization and the addition of biochar, as well as their interaction, have significant effects on the structure of soil eukaryotic microbial community (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Soil sterilization led to a 20% increase and a 13% decrease in eukaryotic microbial abundance, respectively. However, the impact of biochar application on the eukaryotic microbial community was only 0.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Biochar application leads to an increase in pathogenic fungi in the soil while suppressing the abundance of mycorrhizal fungi and saprophytic fungi (Fig. S2A, C, E). The soil fungicide reduces the presence of pathogenic fungi in invaded soil and at the same time promotes the recruitment of mycorrhizal fungi and saprophytic fungi by the plants (Fig. S2B, D, F). Conversely, in non-invaded soil, the fungicide promotes the occurrence of soil-borne pathogenic fungi and mycorrhizal fungi, while significantly reducing the abundance of saprophytic fungi.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Mitigation of biochar on biological legacy effects\u003c/h2\u003e \u003cp\u003eThe presence of a legacy in the soil following the invasion of exotic plants hampers the biomass accumulation in subsequent invasive plants, resulting in a negative legacy effect (Fig. S3A). The application of biochar to invaded soil mitigates the negative residual effects of soil biota on native plants, while intensifying the negative suppression effect on invasive plants (Fig. S3B). The inclusion of biochar can enhance the competitiveness of native plants while suppressing that of invasive plants (Fig. S4A). The application of biochar enhances the residual effects of eukaryotic microorganisms, and there is a significant negative correlation between the impact of soil residual effects on root systems and the diversity of soil microorganisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The utilization of biochar diminishes the impact of residual effects from soil microorganisms on roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Soil legacy effects after invasion of alien plants\u003c/h2\u003e \u003cp\u003eThe remaining soil biota after invasion by alien plants has an inhibitory effect on the biomass accumulation of subsequent plants. Following the introduction of alien plants, their apparent competitive advantage due to the absence of above- and below-ground natural enemies resulted in greater impacts on the soil environment, including soil nutrients, enzyme activity, microbial community structure and diversity, and soil animals (Lau and Lennon \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The impact of invasive plants on the soil forms a soil \"memory\" that continues to influence the growth of subsequent plants. The aim of this study was to analyze the effects of retained soil biota on subsequent plant growth by performing soil sterilization. Soil sterilization reduces the feeding of soil organisms on plant roots and the uptake of root secretions by microorganisms, indirectly reducing the downward secretion of photosynthetic carbon in plants and investing more biomass in the formation of aboveground plant components (Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, our research has shown that the use of fungicide inhibits the germination of seed banks in the soil, thereby reducing weed competition and indirectly promoting plant growth. The invasive plant \u003cem\u003eChromolaena odorata\u003c/em\u003e is known for its high seed production. These huge seeds can germinate rapidly after removal of the invasive plant, hindering the establishment of native plant communities. However, the fungicide used in this study effectively inhibits the germination of the seed bank, creating favourable conditions for the subsequent restoration of the local plant community.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2. The eukaryotic microbial community assembly in response to the addition of fungicide and biochar\u003c/h2\u003e \u003cp\u003eIn our research we have found that fungicides, biochar and their interactions significantly alter eukaryotic microbial communities in soil, with fungicides having the strongest effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Soil microbial memory is eliminated by the fungicide and plants then recruit new microbial communities. The soil fungicide impaired the occurrence of pathogenic fungi but promoted the recruitment of mycorrhizal fungi and saprophytic fungi by the plants (Fig. S2). The structure and function of plant rhizosphere microbial community are closely related to the input of plant-derived organic carbon (rhizosphere sedimentary carbon and litter) and soil properties (Zhang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Conversely, soil microorganisms can affect the phenology of aboveground plant leaves and the accumulation of biomass through their effects on nutrient cycling and availability.\u003c/p\u003e \u003cp\u003eAlthough the use of soil fungicides weakens microbial memory, it directly affects the turnover of nutrients in the soil, which is detrimental to subsequent nutrient uptake by the plants. The addition of biochar replenishes early nutrients, facilitating nutrient uptake and utilization by plants (Hue \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Biochar can also absorb some chemicals in the soil and promote plant growth. The combined use of biochar and fungicides exhibits evident interactive effects that have a positive impact on the stability of the subsequent plant, which is due to the stable interaction between plant and soil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3. The remedial effect of biochar on soil legacy effects\u003c/h2\u003e \u003cp\u003eThe introduction of invasive plants alters the composition of root exudates and litter, selectively stimulating certain soil microorganisms, affecting microbial community structure and function, and influencing the growth of subsequent plants through the \"memory\" of the soil microbial pool. In addition, invasive plants can also alter the physico-chemical properties of the soil, such as pH, organic matter content and nutrient availability, which in turn can affect microbial communities and the soil biota. Biochar contains some ash elements that can be added directly to plant growth. In addition, the addition of biochar can improve the physical structure of the soil, increase the permeability of the soil, improve the soil environment and indirectly promote plant growth. In addition, biochar has a relatively large specific surface area that can adsorb chemically sensitive substances in the soil, which indirectly promotes plant growth.\u003c/p\u003e \u003cp\u003eOverall, our study highlights the importance of considering the legacy of invasive plants on the soil environment and subsequent plant growth. The soil \"memory\" created by invasive plants can significantly affect the growth and survival of subsequent plants and have long-term consequences for ecosystem functioning. It is therefore important to take measures to prevent the introduction and spread of invasive plants and to restore and rehabilitate invaded ecosystems to minimize their impact on the soil environment and subsequent plant growth. Converting invasive plants into biochar can completely destroy their seeds and rhizomes, which is beneficial for controlling the spread of invasive plants. The processed biochar provides nutrients, adsorbs chemicals in the soil and improves soil aggregation, which directly promotes plant growth. Additionally, the utilization of biochar effectively alleviates the initial deficiencies of nutrients and root damage caused by fungicides, while simultaneously remediating the environmental pollution resulting from fungicide use. In conclusion, the combined use of biochar and fungicides effectively controls legacy effects caused by invasive plant invasions and has significant practical benefits for invasive plant control and land use.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe used the invasive species \u003cem\u003eC. odorata\u003c/em\u003e to produce biochar, which was then combined with a fungicide to alleviate the legacy remained in the invaded soil. The soil fungicide reduces the occurrence of pathogenic fungi in invaded soil and at the same time promotes the recruitment of mycorrhizal fungi and saprophytic fungi by the plants. The incorporation of biochar and fungicide into the soil can significantly reduce the competitive ability of non-native plants while improving the competitive ability of native plants. The application of biochar can effectively mitigate the legacy in the invaded soil and underlines the great importance of soil remediation for the restoration of native plants diversity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Wen-bian Bo for taking care of the potted plants and Long Li for their assistance in collecting the biomass data. This work was supported by the National Key R\u0026amp;D Program of China (2022YFF1302402) and Yunnan Fundamental Research Projects (202201AT070609), the National Natural Science Foundation of China (32171660, 32071661) and the 14th Five-Year Plan of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (E3ZKFF3B, E3ZKFF9B) and the \u0026ldquo;Yunnan Revitalization Talent Support Program\u0026rdquo; in Yunnan Province.\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Weitao Li, Hui Ning and Yulong Zheng. The first draft of the manuscript was written by Weitao Li and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBatten KM, Scow KM, Espeland EK (2008) Soil microbial community associated with an invasive grass differentially impacts native plant performance. Microb Ecol 55:220-228.\u003c/li\u003e\n\u003cli\u003eCao X, Harris W (2010) Properties of dairy-manure-derived biochar pertinent to its potential use in remediation. Bioresour Technol 101: 5222-5228.\u003c/li\u003e\n\u003cli\u003eCaporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335-336.\u003c/li\u003e\n\u003cli\u003eDominguez-Begines J, Avila JM, Garcia LV, Gomez-Aparicio L (2021) Disentangling the role of oomycete soil pathogens as drivers of plant-soil feedbacks. Ecology 102:e03430.\u003c/li\u003e\n\u003cli\u003eEppinga MB, Rietkerk M, Dekker SC, De Ruiter PC, Van der Putten WH (2006) Accumulation of local pathogens: a new hypothesis to explain exotic plant invasions. Oikos 114:168-176.\u003c/li\u003e\n\u003cli\u003eFeng Q, Wang B, Chen M, Wu P, Lee X, Xing Y (2021) Invasive plants as potential sustainable feedstocks for biochar production and multiple applications: A review. Resour Conserv Recycl 164: 105204.\u003c/li\u003e\n\u003cli\u003eHarindintwali JD, He C, Xiang L, Dou Q, Liu Y, Wang M, et al (2023) Effects of ball milling on biochar adsorption of contaminants in water: A meta-analysis. The Science of the total environment 882:163643.\u003c/li\u003e\n\u003cli\u003eHue N (2020) Biochar for Maintaining Soil Health. In: Giri B, Varma A (ed) Soil health. Springer, Cham, pp 21-46.\u003c/li\u003e\n\u003cli\u003eJacquiod S, Spor A, Wei S, Munkager V, Bru D, Sorensen SJ (2022) Artificial selection of stable rhizosphere microbiota leads to heritable plant phenotype changes. Ecol Lett 25:189-201.\u003c/li\u003e\n\u003cli\u003eJing J, Cong W-F, Bezemer TM (2022) Legacies at work: plant\u0026ndash;soil\u0026ndash;microbiome interactions underpinning agricultural sustainability. Trends Plant Sci 27:781-792.\u003c/li\u003e\n\u003cli\u003eLau JA, Lennon JT (2012) Rapid responses of soil microorganisms improve plant fitness in novel environments. PNAS 109:14058-14062.\u003c/li\u003e\n\u003cli\u003eLekberg Y, Bever JD, Bunn RA, Callaway RM, Hart MM, Kivlin SN, et al (2018) Relative importance of competition and plant-soil feedback, their synergy, context dependency and implications for coexistence. Ecol Lett 21:1268-1281.\u003c/li\u003e\n\u003cli\u003eLi WT, Bi XT, Zheng YL (2023) Soil legacy effects on biomass allocation depend on native plant diversity in the invaded community. Sci Prog 106:1-12.\u003c/li\u003e\n\u003cli\u003eLozano YM, Aguilar-Trigueros CA, Ospina JM, Rillig MC (2022) Drought legacy effects on root morphological traits and plant biomass via soil biota feedback. New Phytol 236:222-234.\u003c/li\u003e\n\u003cli\u003eMangla S, Inderjit, Callaway RM (2008) Exotic invasive plant accumulates native soil pathogens which inhibit native plants. J Ecol 96:58-67.\u003c/li\u003e\n\u003cli\u003eMagoc T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957-2963.\u003c/li\u003e\n\u003cli\u003eMaron JL, Waller LP, Hahn MA, Diaconu A, Pal RW, Muller-Scharer H, et al (2013) Effects of soil fungi, disturbance and propagule pressure on exotic plant recruitment and establishment at home and abroad. J Ecol 101:924-932.\u003c/li\u003e\n\u003cli\u003eNguyen NH, Song Z, Bates ST, Branco S, Tedersoo L, Menke J, et al (2016) FUNGuild: An open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecol 20:241-248.\u003c/li\u003e\n\u003cli\u003eShi X, Li WT, Zheng YL (2021) Soil legacy effect of extreme precipitation on a tropical invader in different land use types. Environ Exp Bot 191:104625.\u003c/li\u003e\n\u003cli\u003eTe Beest M, Stevens N, Olff H, Van Der Putten WH (2009) Plant\u0026ndash;soil feedback induces shifts in biomass allocation in the invasive plant Chromolaena odorata. J Ecol 97:1281-1290.\u003c/li\u003e\n\u003cli\u003eVan Nuland ME, Ware IM, Schadt CW, Yang Z, Bailey JK, Schweitzer JA (2021) Natural soil microbiome variation affects spring foliar phenology with consequences for plant productivity and climate-driven range shifts. New Phytol 232:762-775.\u003c/li\u003e\n\u003cli\u003eXu W, Huang X, Yuan J, Wang Y, Wu M, Ni H, et al (2023) The potential for synthesized invasive plant biochar with hydroxyapatite to mitigate allelopathy of Solidago canadensis. Ecol Appl e2833.\u003c/li\u003e\n\u003cli\u003eYin LJ, Liu B, Wang HC, Zhang Y, Wang S, Jiang F, et al (2020) The Rhizosphere Microbiome ofMikania micranthaProvides Insight Into Adaptation and Invasion. Front Microbiol 11:1462.\u003c/li\u003e\n\u003cli\u003eZhang P, Li B, Wu JH, Hu SJ (2019) Invasive plants differentially affect soil biota through litter and rhizosphere pathways: a meta-analysis. Ecol Lett 22:200-210.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alien invasive plant, biochar, soil legacy effect, soil pathogenic fungi","lastPublishedDoi":"10.21203/rs.3.rs-3663026/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3663026/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eThe introduction of non-native plant species often leads to extensive colonization of land and poses a threat to the already limited resources of arable land. Restoring invaded land and improving soil fertility are therefore essential.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, biochar from invader \u003cem\u003eChromolaena odorata\u003c/em\u003e and fungicide were jointly used to improve soil invaded by this species.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results show that fungicide increase the total biomass of the subsequent plants grown in the invaded soil. The soil fungicide reduces the occurrence of pathogenic fungi in invaded soil and at the same time promotes the recruitment of mycorrhizal fungi and saprophytic fungi by the plants. The addition of biochar to the invaded soil has a negative legacy effect on subsequent invasive plants, while it exerts a positive enhancement effect on native plants. The simultaneous application of biochar and fungicide to soils invaded by alien plants can effectively diminish the competitive advantage of invasive species, while biochar can markedly increase the competitive advantage of native plants.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study proposes a remediation method for legacy effect after alien plant invasion that not only effectively controls the spread of alien plants, but also improves soil fertility and increases economic benefits.\u003c/p\u003e","manuscriptTitle":"Mitigation of legacy effects in invaded soil using alien plant-derived biochar","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-15 21:33:06","doi":"10.21203/rs.3.rs-3663026/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"400b4fe6-ca6c-489e-b676-d875a36321e0","owner":[],"postedDate":"December 15th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-26T20:14:54+00:00","versionOfRecord":[],"versionCreatedAt":"2023-12-15 21:33:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3663026","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3663026","identity":"rs-3663026","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}