Comparative Metagenomic Analysis of Phyllospheric Microbial Diversity in Azolla imbricata and Azolla pinnata

Comparative Metagenomic Analysis of Phyllospheric Microbial Diversity in Azolla imbricata and Azolla pinnata | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Metagenomic Analysis of Phyllospheric Microbial Diversity in Azolla imbricata and Azolla pinnata Yan-Qiu Yang, Su-Fang Deng, You-Quan Yang, Zhao-Yang Ying This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6299122/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: The aquatic fern Azolla grows rapidly, produces nutrient-rich biomass, and adapts well ecologically due to symbiotic interactions with phyllospheric microorganisms. Azolla imbricata (Aim) and Azolla pinnata (Api) are valuable for biofertilization and phytoremediation, with their ecological differences potentially influenced by species-specific microbial partnerships. This study examines their phyllospheric microbiomes under controlled conditions to explore adaptive strategies. Method and Results: High-throughput metagenomic sequencing identified 10,857 bacteria, 986 eukaryotes. Proteobacteria dominated, particularly Burkholderiales, with higher abundance in Aim. Api exhibited greater Cyanobacteria abundance, especially Nostocaceae (genus Trichormus ). Streptophyta constituted over 90% of fungal communities in both species. LEfSe analysis highlighted unique taxa in each species, including enriched Magnoliopsida and Bacillariophyta in Aim, while Api had more Marchantia and Streptophyta. KEGG pathway analysis identified ribosome, oxidative phosphorylation, photosynthesis, quorum sensing, and two-component systems among the most enriched pathways. Resistance gene analysis showed Aim had higher levels of cmlA9, qacEdelta1, tetD, and ANT3-Ii-AAC6-IId_fusion_protein, while Api had more AAC6-IIa. Conclusions: Aim’s enrichment in Proteobacteria may enhance resilience, while Api’s nitrogen-fixing Cyanobacteria suggest distinct nitrogen management. Functional and resistance gene differences highlight ecological adaptability, providing insights for optimizing Azolla’s agricultural potential. Azolla phyllosphere microorganisms metagenome Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Oct, 2025 Reviews received at journal 18 Sep, 2025 Reviews received at journal 15 Sep, 2025 Reviewers agreed at journal 14 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers invited by journal 05 Sep, 2025 Editor invited by journal 07 May, 2025 Editor assigned by journal 27 Mar, 2025 Submission checks completed at journal 27 Mar, 2025 First submitted to journal 27 Mar, 2025 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. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-6299122","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512159385,"identity":"a5589365-d3a6-4c83-a3e5-04b0c75088d1","order_by":0,"name":"Yan-Qiu Yang","email":"","orcid":"","institution":"Fujian Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yan-Qiu","middleName":"","lastName":"Yang","suffix":""},{"id":512159386,"identity":"9900a50c-b0de-43cd-ac7b-f0f5dfaab534","order_by":1,"name":"Su-Fang Deng","email":"","orcid":"","institution":"Fujian Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Su-Fang","middleName":"","lastName":"Deng","suffix":""},{"id":512159387,"identity":"933043fe-d169-4401-9372-3b155d9567e1","order_by":2,"name":"You-Quan Yang","email":"","orcid":"","institution":"Fujian Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"You-Quan","middleName":"","lastName":"Yang","suffix":""},{"id":512159388,"identity":"8ca511aa-bc61-4b17-84d9-d6c025f67230","order_by":3,"name":"Zhao-Yang Ying","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACCTiLsYGBoUJCTp5ELWcsjA0biNcC0tVWkchwgIAO+dnNDx/z1Nyxa5BIbnv4dZ5EAmMD88NHN/BoYZxzzNiY59iz5AaJxHZj2W0SeewMbMbGOXi0MEskmEnzsB1OZpBIbJOW3CZRzNjAwyaNTwubRPo3aZ5/MC1zJBIbDhDQwiORYybN23bYDqRF8mMDEVokJHKKDef2HU5g4HnYJs1wTMLYsJmAX+RnpG988ObbYXsG9vRnkj9q6uTk2YFhiE8LCDDxMDAk7r+QwMDMA+IyE1AOAow/GBjsGfgPgBmjYBSMglEwCjAAAKlER/kNPvTsAAAAAElFTkSuQmCC","orcid":"","institution":"Fujian Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Zhao-Yang","middleName":"","lastName":"Ying","suffix":""}],"badges":[],"createdAt":"2025-03-25 02:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6299122/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6299122/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91119311,"identity":"5a1c279d-6dee-4751-94c2-7446ae5beb67","added_by":"auto","created_at":"2025-09-11 18:28:26","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1072798,"visible":true,"origin":"","legend":"","description":"","filename":"ComparativeMetagenomicAnalysisofPhyllosphericMicrobialDiversityinAzollaimbricataandAzollapinnata2025.3.21.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6299122/v1_covered_00beda36-73f6-4952-94dd-f05a243f7d5e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Metagenomic Analysis of Phyllospheric Microbial Diversity in Azolla imbricata and Azolla pinnata","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Azolla, phyllosphere, microorganisms, metagenome","lastPublishedDoi":"10.21203/rs.3.rs-6299122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6299122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe aquatic fern \u003cem\u003eAzolla\u003c/em\u003e grows rapidly, produces nutrient-rich biomass, and adapts well ecologically due to symbiotic interactions with phyllospheric microorganisms. \u003cem\u003eAzolla imbricata\u003c/em\u003e (Aim) and \u003cem\u003eAzolla pinnata\u003c/em\u003e (Api) are valuable for biofertilization and phytoremediation, with their ecological differences potentially influenced by species-specific microbial partnerships. This study examines their phyllospheric microbiomes under controlled conditions to explore adaptive strategies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod and Results: \u003c/strong\u003eHigh-throughput metagenomic sequencing identified 10,857 bacteria, 986 eukaryotes. Proteobacteria dominated, particularly Burkholderiales, with higher abundance in Aim. Api exhibited greater Cyanobacteria abundance, especially Nostocaceae (genus \u003cem\u003eTrichormus\u003c/em\u003e). Streptophyta constituted over 90% of fungal communities in both species. LEfSe analysis highlighted unique taxa in each species, including enriched Magnoliopsida and Bacillariophyta in Aim, while Api had more Marchantia and Streptophyta. KEGG pathway analysis identified ribosome, oxidative phosphorylation, photosynthesis, quorum sensing, and two-component systems among the most enriched pathways. Resistance gene analysis showed Aim had higher levels of cmlA9, qacEdelta1, tetD, and ANT3-Ii-AAC6-IId_fusion_protein, while Api had more AAC6-IIa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Aim’s enrichment in Proteobacteria may enhance resilience, while Api’s nitrogen-fixing Cyanobacteria suggest distinct nitrogen management. Functional and resistance gene differences highlight ecological adaptability, providing insights for optimizing Azolla’s agricultural potential.\u003c/p\u003e","manuscriptTitle":"Comparative Metagenomic Analysis of Phyllospheric Microbial Diversity in Azolla imbricata and Azolla pinnata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 18:04:19","doi":"10.21203/rs.3.rs-6299122/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-29T06:49:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T05:25:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-15T08:08:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133222497995439455504157344760989316326","date":"2025-09-14T11:16:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244064335643756869086343676760924312039","date":"2025-09-08T03:49:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31080700516183478393449976195675337802","date":"2025-09-07T10:49:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-05T08:37:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-07T10:52:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T07:27:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-27T07:19:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-03-27T07:18:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cf0392a2-f18c-4428-8b8a-061df8be16ac","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-04T11:23:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-11 18:04:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6299122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6299122","identity":"rs-6299122","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}