Mother trees of common ash (Fraxinus excelsior) disperse different sets of mycobiome through their samaras

Mother trees of common ash (Fraxinus excelsior) disperse different sets of mycobiome through their samaras | 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 Short Report Mother trees of common ash (Fraxinus excelsior) disperse different sets of mycobiome through their samaras Feng Long, James Michael Doonan, Lene Rostgaard Nielsen, Erik Dahl Kjaer, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3797020/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Objective Endophytic mycobiomes are present in all studied plant compartments, including fruits and seeds, but a transmission of the mycobiome between generations is largely unknown. Our objectives were to examine mycobiome transfer via seed wings (samaras) of European ash ( Fraxinus excelsior ), and to test whether these mycobiomes differ among trees. To achieve this, we used ITS1-based amplicon sequencing and two genotypes of F. excelsior as a model to compare the mycobiome of mother trees and their samaras. Results We profiled the mycobiome of 57 seed stalks and seed wings (samaras) collected from two genotypes of F. excelsior using three ramets of each genotype. Alpha diversity indices (Observed OTUs and ACE) suggested a higher richness of the mycobiome associated with seed wing than seed stalk within each genotype. However, there was neither significant differences in diversity between the mycobiomes from the two tissue types nor the two genotypes. PERMANOVA analysis revealed significant differences in the mycobiome composition between seed wings, but not between seed stalks, of the two genotypes. Our results suggest that Fraxinus excelsior mother trees disperse different sets of mycobiomes with their samaras, which may be important for germination and seedling establishment – especially in the light of ash dieback. Figures Figure 1 Figure 2 Introduction The plant mycobiome is the collective group of fungi co-existing with plant hosts, which play a crucial role in growth and development of the host. The mycobiome is found ubiquitously in almost all studied plant compartments, including seed and fruit [ 1 , 2 , 3 , 4 , 5 ]. Similar to other plant compartments, seed mycobiomes are formed both within the tissues (endophytic mycobiome) and on the surfaces (epiphytic mycobiome) [ 5 , 6 ]. Previous studies suggest that both vertical and horizontal factors contribute to the formation of seed mycobiomes [ 7 , 8 ]. Despite limited research outside of model plant species, a growing body of evidence supports the beneficial nature of certain fruit-associated fungi in which they directly or indirectly benefit diverse physiological processes of the hosts [ 9 , 10 ]. The fact that the seed mycobiome can affect a successive microbial recruitment of seedlings [ 9 ] has underlined the need to better understand not only composition but also function of the seed-associated mycobiome. European ash ( Fraxinus excelsior ) is a fast-growing broadleaved species distributed across most of Europe [ 11 , 12 ]. It has substantial ecological value as it grows in several forest types housing hundreds of associated organisms [ 12 ]. The dispersal unit of the species is the samara, which is the seed enclosed by a wing [ 13 ]. Samaras are developed from the ovary wall of the mother tree. In this study, we used two F. excelsior genotypes to study mycobiome transfer from mother tree to samara to reveal whether the mother trees disperse different mycobiome sets. Mycobiome dispersal is particularly important to address in the light of the ongoing extensive European ash dieback (ADB) pandemic, caused by an invasive fungus, Hymenoscyphus fraxineus . The data we provide in this study contributes to a better understanding of the vertical transfer and early dispersal of the mycobiome through seed (samara) in F. excelsior and provides a novel foundation for conservation of the species. Materials and Methods Plant materials Samaras were collected from two F. excelsior mother trees (hereafter genotype 33 and 35), grown in a former Danish clonal seed orchard located at Tuse Næs, Zealand [ 14 ]. Genotype 35 was previously found to be superior to genotype 33 in terms of susceptibility to ash dieback (ADB) [ 14 ]. Eight to ten samaras were collected randomly from three different locations of each ramet, and three ramets were used per genotype. In total, 57 samaras from six mother trees (27 from genotype 33 and 30 from genotype 35) were used in this study. Samaras were surface sterilized as follows: 95% ethanol for 1 min, 3% sodium hypochlorite for 3 min, 95% ethanol for 1 min before being rinsed with sterile deionized water twice. Finally, samples were air dried in a laminar flow and kept at -20 °C until further processing. DNA manipulation and construction of ITS1 metabarcoding libraries A fraction of the seed wing and seed stalk were homogenized at 30 Hz for 2 min twice using a RETSCH MM400 Mixer Mill (RETSCH, Germany). DNA was extracted using DNeasy PowerPlant Pro Kit (QIAGEN, Germany) following the manufacturer’s instruction and DNA concentration was determined using QUBIT3 fluorometer (ThermoFisher Scientific, USA). A two-step PCR approach was used to construct ITS1 amplicon libraries. PCR-I was performed in duplicate using five to ten nanograms of DNA, the primer pair BITS/B58S3 [ 15 ], and Phusion Hot Start II High-Fidelity PCR Master Mix (ThermoFisher Scientific, Lithuania) on a BIO-RAD T100 thermal cycler (BIO-RAD, USA). A unique combinatorial Nextera XT v.2 barcode was introduced to each sample during PCR-II using the same polymerase as PCR-I, but the cycling number was limited to 12. The PCR-II products were purified using PureLink PCR Micro Kit (Invitrogen, USA), pooled in equimolar ratio and sequenced with Illumina MiSeq platform (v3 chemistry; 2x300 bp) at Macrogen Europe (Amsterdam, the Netherlands). Bioinformatics and statistical analyses Only forward reads were used in this study. Low quality bases (Q < 20) and short reads (< 120 bases) were removed using BBduk in the BBtools suite v.38.90 ( https://jgi.doe.gov/data-and-tools/software-tools/bbtools/ ). High quality reads were then parsed to QIIME2 2022.11 [ 16 ], where primers were removed with Cutadapt [ 17 ]. VSEARCH [ 18 ] was used for de novo OTU clustering at 99% identity and chimera identification. OTUs with frequency less than 0.005% of total reads were considered spurious [ 19 ] and dislodged from further analysis. Taxonomy assignment was performed using Sklearn [ 20 ] and the UNITE ITS database v.9 [ 21 ]. All OTUs belonging to phyla other than fungi were removed from further analysis. For diversity analyses, all 57 samples were rarefied to 3,031 reads per sample and used to calculate alpha (Observed OTUs, ACE and Shannon) and beta (Bray-Curtis) diversity indices. A pairwise Kruskal-Wallis test was carried out to determine significant differences in the alpha indices between the two tissue types and the two genotypes. Bray-Curtis dissimilarity-based non-metric multidimensional scaling (NMDS) ordinations were plotted using Phyloseq [ 22 ] to explore variations between genotypes and tissue types. The Bray-Curtis index and permutational multivariate analysis of variance (PERMANOVA) with 1,000 permutations were used to compare mycobiome compositions from the two genotypes and two tissue types. All bioinformatic and statistical analyses were carried out under the QIIME2 space unless otherwise stated. Results and discussion Our study revealed a total of 435 OTUs from the two tissue types and two genotypes (Supplementary table 1 ). The majority of the OTUs (354 OTUs; 81%) were present in both genotype 33 and genotype 35 (Fig. 1 A), and 341 OTUs (78%) were shared between the two tissue types (Fig. 1 A). Only a small fraction of OTUs were present uniquely either in the seed wing or seed stalk of each genotype. There was a higher number of OTUs in genotype 35 (412 OTUs) compared to genotype 33 (377 OTUs) (Fig. 1 A) and a higher number of OTUs in seed stalk (414 OTUs) than in seed wing (362 OTUs) (Fig. 1 A). This is in contrast with previous studies where the ADB-less susceptible genotype 35 was found to have lower species richness and species diversity of endophytic mycobiome compared to the more susceptible genotypes [ 23 , 24 ]. However, those studies were based on other compartments i.e., twig and leaf, while the focus of this study was on the seed stalk and wing. We were able to assign 413 out of 435 OTUs (94.9%) at genus level, leaving only 22 OTUs (5.1%) unidentified. Fungal communities associated with seed stalk and seed wings of genotype 33 and genotype 35 consisted of 59 genera. Irrespective of genotype, the communities were largely dominated by Fusicladium , which occupied up to 94.3% of total reads and was present in both tissue types. Other less abundant taxa included species in the genus Alternaria , Erysiphe and Vishniacozyma (Fig. 1 B and 1 D). While the largest proportion of prevalent taxa were ascomycete fungi, a basidiomycete yeast from the genus Vishniacozyma was also present. One member of the genus Vishniacozyma , V. victoriae has previously been shown to be an antagonist of plant pathogens [ 25 , 26 ]. In this study, OTUs annotated to V. victoriae were recorded more abundantly in the seed wing of genotype 35 than that of genotype 33. As genotype 35 is less susceptible to ADB than genotype 33, the presence of this species may be involved with ADB tolerance. Further investigation is needed to verify this hypothesis. Diversity indices for species richness and other measures are presented in Fig. 2 . The pairwise Kruskall-Wallis test identified significant differences in alpha diversity indices (ACE and Observed OTUs) between the mycobiomes associated with seed wing of genotype 33 and 35 (ACE, P < 0.01; Observed OTUs < 0.001) and between the mycobiomes associated with seed stalk of genotype 33 and 35 (Observed OTUs < 0.01). However, we did not observe significant differences in Shannon diversity between the communities. The non-multidimensional scaling (NDMS) plot showed clear separation between the fungal communities associated with different tissue types (Fig. 1 C). Although separation between the two genotypes was less pronounced, PERMANOVA analysis based on the Bray-Curtis dissimilarity matrix revealed a significant difference between the seed wing community compositions of genotype 33 and 35 (P < 0.05), but not between seed stalks of the two genotypes. The results suggest differences in the fungal communities of the seed wing (the dispersal unit of seeds) and of different genotypes, but not between the communities of seed stalks. Conclusions The mycobiome is increasingly recognized as a crucial component of plant health and ecosystem function. Understanding the composition and dynamics of the mycobiome related to seeds (here the samara) is central to the goal of advancing understanding of plant-microbe interactions and developing sustainable agricultural practices. Here we identified differences in mycobiome composition of seeds (samaras) collected from two different genotypes of F. excelsior with variation in susceptibility to ADB. These data suggest that mother trees disperse a distinct mycobiome which is dependent on the genotype of the mother tree. Limitations This experiment suffers from a relatively small sample size. While the number of seeds per genotype is adequate, the number of total genotypes is limited to two (clone 33 and clone 35). The small number of clones limits detailed statistical analyses e.g., identification of enriched fungal species. Trees are home to fungi, bacteria and viruses. This study focuses only on stalk and seed-associated fungal communities, bacterial and viral communities remain untouched. As this study does not reflect the entire seed microbiome, care should be taken when a connection between the seed microbiome and seed health is discussed. Knowledge of fungal communities during seed germination is limited. The extent to which the seed mycobiome persists after vertical transmission is unknown. This study therefore requires follow-up experiments to further investigate the in situ roles of the mycobiome. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Data availability All sequence data is available at NCBI under BioProject PRJDB17107. Competing interests The authors declare no competing interests. Funding This work was supported by a research grant (36117) from VILLUM FONDEN. Authors contributions FL - wrote original manuscript draft (equal). Created figures; analysed the data. JMD - created figures. Revised and edited final manuscript draft. LRN - assisted in project design; revised and edited final manuscript draft. EDK - assisted in project design; revised and edited final manuscript draft. CK - funding acquisition; wrote original manuscript draft (equal). Acknowledgments Not applicable. References Adam E, Bernhart M, Muller H, Winkler J, Berg G. The Cucubita pepo seed microbiome: genotype-specific composition and implications for breeding. Plant Soil. 2018;422:35–49. Nelson EB. The seed microbiome: origins, interactions, and impacts. Plant Soil. 2018;422:7–34. 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Nian L, Xie Y, Zhang H, Wang M, Yuan B, Cheng S, et al. Vishniacozyma victoriae: An endophytic antagonist yeast of kiwifruit with biocontrol effect to Botrytis cinerea. Food Chem. 2023;411:135442. 10.1016/j.foodchem.2023.135442 . Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 25 Dec, 2023 Submission checks completed at journal 25 Dec, 2023 First submitted to journal 23 Dec, 2023 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-3797020","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":263101463,"identity":"c23d8ae0-bcb0-40d7-897f-20fe2e8e93a1","order_by":0,"name":"Feng Long","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Long","suffix":""},{"id":263101465,"identity":"825f5260-b7be-44db-87b2-ad74685153ec","order_by":1,"name":"James Michael Doonan","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"Michael","lastName":"Doonan","suffix":""},{"id":263101468,"identity":"6b248290-33af-445c-a775-f9643cd979a0","order_by":2,"name":"Lene Rostgaard Nielsen","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Lene","middleName":"Rostgaard","lastName":"Nielsen","suffix":""},{"id":263101470,"identity":"1e6e8d32-c06b-4a97-925a-869b2ffe9675","order_by":3,"name":"Erik Dahl Kjaer","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Erik","middleName":"Dahl","lastName":"Kjaer","suffix":""},{"id":263101473,"identity":"4dba5a0e-b098-4d7d-81ac-0e8dfe907760","order_by":4,"name":"Chatchai Kosawang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYDACCTYGhg8GQAYPCB8gUgvjDJK1MINUE6+Ff3Zb4mObgto8Bp4zBgxvzhBjyZ1jh41zDI4XM/D2GDDOuUGEFgOJ9DbpHINjiQ38PAbMPB+I1WJBopa0Y9IMBjWJDUCHMfMQ4zCJG2nJhj0GBxLbeI4VHJxDjPf5Z6QZPvjxpy6xnyd544M3x4jQAgWHGYCJgOEA8RoYGOpIUTwKRsEoGAUjDQAAtVIzrdjJ9q4AAAAASUVORK5CYII=","orcid":"","institution":"University of Copenhagen","correspondingAuthor":true,"prefix":"","firstName":"Chatchai","middleName":"","lastName":"Kosawang","suffix":""}],"badges":[],"createdAt":"2023-12-23 15:44:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3797020/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3797020/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49024645,"identity":"355279b0-0fc9-4d3e-a59f-fd939f6d3697","added_by":"auto","created_at":"2024-01-01 11:38:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":799284,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Venn diagram showing OTUs between different ash (\u003cem\u003eFraxinus excelsior\u003c/em\u003e) seed compartments and ash genotypes: (B) taxonomy bar plot of the fungal communities associated with seed stalk and seed wing of genotypes 33 and 35: (C) principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity of ash seed mycobiomes. The ellipses represent 95% confidence intervals for each tissue type: (D) heatmap illustrating the Z-score distribution of relative abundance of the top 15 dominant genera in seed samples.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3797020/v1/5e9e1157c7a2a11c6010d79f.png"},{"id":49024642,"identity":"15a68262-f7c3-4e85-b361-f36ba8f1d79e","added_by":"auto","created_at":"2024-01-01 11:38:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":448437,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot illustrating alpha diversity indices between tissue types and genotypes. *Denotes significance *p\u0026lt;0.05, **p\u0026lt; 0.01 and ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3797020/v1/743ec155da9e3986ade6eaf5.png"},{"id":49025116,"identity":"bf4a3ac4-5662-483d-bff7-92cca8e3bf42","added_by":"auto","created_at":"2024-01-01 11:46:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":522246,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3797020/v1/4af4e109-e02e-42d6-b597-e9397859b633.pdf"},{"id":49024641,"identity":"a27ef70c-65a4-42ab-8ac7-637223f5f155","added_by":"auto","created_at":"2024-01-01 11:38:51","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28512,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3797020/v1/d472afc32569658658c7a7e5.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mother trees of common ash (Fraxinus excelsior) disperse different sets of mycobiome through their samaras","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe plant mycobiome is the collective group of fungi co-existing with plant hosts, which play a crucial role in growth and development of the host. The mycobiome is found ubiquitously in almost all studied plant compartments, including seed and fruit [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similar to other plant compartments, seed mycobiomes are formed both within the tissues (endophytic mycobiome) and on the surfaces (epiphytic mycobiome) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Previous studies suggest that both vertical and horizontal factors contribute to the formation of seed mycobiomes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Despite limited research outside of model plant species, a growing body of evidence supports the beneficial nature of certain fruit-associated fungi in which they directly or indirectly benefit diverse physiological processes of the hosts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The fact that the seed mycobiome can affect a successive microbial recruitment of seedlings [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] has underlined the need to better understand not only composition but also function of the seed-associated mycobiome.\u003c/p\u003e \u003cp\u003eEuropean ash (\u003cem\u003eFraxinus excelsior\u003c/em\u003e) is a fast-growing broadleaved species distributed across most of Europe [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has substantial ecological value as it grows in several forest types housing hundreds of associated organisms [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The dispersal unit of the species is the samara, which is the seed enclosed by a wing [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Samaras are developed from the ovary wall of the mother tree. In this study, we used two \u003cem\u003eF. excelsior\u003c/em\u003e genotypes to study mycobiome transfer from mother tree to samara to reveal whether the mother trees disperse different mycobiome sets. Mycobiome dispersal is particularly important to address in the light of the ongoing extensive European ash dieback (ADB) pandemic, caused by an invasive fungus, \u003cem\u003eHymenoscyphus fraxineus\u003c/em\u003e. The data we provide in this study contributes to a better understanding of the vertical transfer and early dispersal of the mycobiome through seed (samara) in \u003cem\u003eF. excelsior\u003c/em\u003e and provides a novel foundation for conservation of the species.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eSamaras were collected from two \u003cem\u003eF. excelsior\u003c/em\u003e mother trees (hereafter genotype 33 and 35), grown in a former Danish clonal seed orchard located at Tuse N\u0026aelig;s, Zealand [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Genotype 35 was previously found to be superior to genotype 33 in terms of susceptibility to ash dieback (ADB) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Eight to ten samaras were collected randomly from three different locations of each ramet, and three ramets were used per genotype. In total, 57 samaras from six mother trees (27 from genotype 33 and 30 from genotype 35) were used in this study. Samaras were surface sterilized as follows: 95% ethanol for 1 min, 3% sodium hypochlorite for 3 min, 95% ethanol for 1 min before being rinsed with sterile deionized water twice. Finally, samples were air dried in a laminar flow and kept at -20 \u0026deg;C until further processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA manipulation and construction of ITS1 metabarcoding libraries\u003c/h2\u003e \u003cp\u003eA fraction of the seed wing and seed stalk were homogenized at 30 Hz for 2 min twice using a RETSCH MM400 Mixer Mill (RETSCH, Germany). DNA was extracted using DNeasy PowerPlant Pro Kit (QIAGEN, Germany) following the manufacturer\u0026rsquo;s instruction and DNA concentration was determined using QUBIT3 fluorometer (ThermoFisher Scientific, USA). A two-step PCR approach was used to construct ITS1 amplicon libraries. PCR-I was performed in duplicate using five to ten nanograms of DNA, the primer pair BITS/B58S3 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and Phusion Hot Start II High-Fidelity PCR Master Mix (ThermoFisher Scientific, Lithuania) on a BIO-RAD T100 thermal cycler (BIO-RAD, USA). A unique combinatorial Nextera XT v.2 barcode was introduced to each sample during PCR-II using the same polymerase as PCR-I, but the cycling number was limited to 12. The PCR-II products were purified using PureLink PCR Micro Kit (Invitrogen, USA), pooled in equimolar ratio and sequenced with Illumina MiSeq platform (v3 chemistry; 2x300 bp) at Macrogen Europe (Amsterdam, the Netherlands).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics and statistical analyses\u003c/h2\u003e \u003cp\u003eOnly forward reads were used in this study. Low quality bases (Q\u0026thinsp;\u0026lt;\u0026thinsp;20) and short reads (\u0026lt;\u0026thinsp;120 bases) were removed using BBduk in the BBtools suite v.38.90 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jgi.doe.gov/data-and-tools/software-tools/bbtools/\u003c/span\u003e\u003cspan address=\"https://jgi.doe.gov/data-and-tools/software-tools/bbtools/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e High quality reads were then parsed to QIIME2 2022.11 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], where primers were removed with Cutadapt [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. VSEARCH [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] was used for \u003cem\u003ede novo\u003c/em\u003e OTU clustering at 99% identity and chimera identification. OTUs with frequency less than 0.005% of total reads were considered spurious [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and dislodged from further analysis. Taxonomy assignment was performed using Sklearn [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and the UNITE ITS database v.9 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. All OTUs belonging to phyla other than fungi were removed from further analysis.\u003c/p\u003e \u003cp\u003eFor diversity analyses, all 57 samples were rarefied to 3,031 reads per sample and used to calculate alpha (Observed OTUs, ACE and Shannon) and beta (Bray-Curtis) diversity indices. A pairwise Kruskal-Wallis test was carried out to determine significant differences in the alpha indices between the two tissue types and the two genotypes. Bray-Curtis dissimilarity-based non-metric multidimensional scaling (NMDS) ordinations were plotted using Phyloseq [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] to explore variations between genotypes and tissue types. The Bray-Curtis index and permutational multivariate analysis of variance (PERMANOVA) with 1,000 permutations were used to compare mycobiome compositions from the two genotypes and two tissue types. All bioinformatic and statistical analyses were carried out under the QIIME2 space unless otherwise stated.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eOur study revealed a total of 435 OTUs from the two tissue types and two genotypes (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The majority of the OTUs (354 OTUs; 81%) were present in both genotype 33 and genotype 35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and 341 OTUs (78%) were shared between the two tissue types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Only a small fraction of OTUs were present uniquely either in the seed wing or seed stalk of each genotype. There was a higher number of OTUs in genotype 35 (412 OTUs) compared to genotype 33 (377 OTUs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and a higher number of OTUs in seed stalk (414 OTUs) than in seed wing (362 OTUs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This is in contrast with previous studies where the ADB-less susceptible genotype 35 was found to have lower species richness and species diversity of endophytic mycobiome compared to the more susceptible genotypes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, those studies were based on other compartments i.e., twig and leaf, while the focus of this study was on the seed stalk and wing.\u003c/p\u003e \u003cp\u003eWe were able to assign 413 out of 435 OTUs (94.9%) at genus level, leaving only 22 OTUs (5.1%) unidentified. Fungal communities associated with seed stalk and seed wings of genotype 33 and genotype 35 consisted of 59 genera. Irrespective of genotype, the communities were largely dominated by \u003cem\u003eFusicladium\u003c/em\u003e, which occupied up to 94.3% of total reads and was present in both tissue types. Other less abundant taxa included species in the genus \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eErysiphe\u003c/em\u003e and \u003cem\u003eVishniacozyma\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). While the largest proportion of prevalent taxa were ascomycete fungi, a basidiomycete yeast from the genus \u003cem\u003eVishniacozyma\u003c/em\u003e was also present. One member of the genus \u003cem\u003eVishniacozyma\u003c/em\u003e, \u003cem\u003eV. victoriae\u003c/em\u003e has previously been shown to be an antagonist of plant pathogens [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, OTUs annotated to \u003cem\u003eV. victoriae\u003c/em\u003e were recorded more abundantly in the seed wing of genotype 35 than that of genotype 33. As genotype 35 is less susceptible to ADB than genotype 33, the presence of this species may be involved with ADB tolerance. Further investigation is needed to verify this hypothesis.\u003c/p\u003e \u003cp\u003eDiversity indices for species richness and other measures are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The pairwise Kruskall-Wallis test identified significant differences in alpha diversity indices (ACE and Observed OTUs) between the mycobiomes associated with seed wing of genotype 33 and 35 (ACE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Observed OTUs\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and between the mycobiomes associated with seed stalk of genotype 33 and 35 (Observed OTUs\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, we did not observe significant differences in Shannon diversity between the communities. The non-multidimensional scaling (NDMS) plot showed clear separation between the fungal communities associated with different tissue types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Although separation between the two genotypes was less pronounced, PERMANOVA analysis based on the Bray-Curtis dissimilarity matrix revealed a significant difference between the seed wing community compositions of genotype 33 and 35 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not between seed stalks of the two genotypes. The results suggest differences in the fungal communities of the seed wing (the dispersal unit of seeds) and of different genotypes, but not between the communities of seed stalks.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe mycobiome is increasingly recognized as a crucial component of plant health and ecosystem function. Understanding the composition and dynamics of the mycobiome related to seeds (here the samara) is central to the goal of advancing understanding of plant-microbe interactions and developing sustainable agricultural practices. Here we identified differences in mycobiome composition of seeds (samaras) collected from two different genotypes of \u003cem\u003eF. excelsior\u003c/em\u003e with variation in susceptibility to ADB. These data suggest that mother trees disperse a distinct mycobiome which is dependent on the genotype of the mother tree.\u003c/p\u003e"},{"header":"Limitations","content":"\u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThis experiment suffers from a relatively small sample size. While the number of seeds per genotype is adequate, the number of total genotypes is limited to two (clone 33 and clone 35). The small number of clones limits detailed statistical analyses e.g., identification of enriched fungal species.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTrees are home to fungi, bacteria and viruses. This study focuses only on stalk and seed-associated fungal communities, bacterial and viral communities remain untouched. As this study does not reflect the entire seed microbiome, care should be taken when a connection between the seed microbiome and seed health is discussed.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eKnowledge of fungal communities during seed germination is limited. The extent to which the seed mycobiome persists after vertical transmission is unknown. This study therefore requires follow-up experiments to further investigate the \u003cem\u003ein situ\u003c/em\u003e roles of the mycobiome.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll sequence data is available at NCBI under BioProject PRJDB17107.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a research grant (36117) from VILLUM FONDEN.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFL - wrote original manuscript draft (equal). Created figures; analysed the data. JMD - created figures. Revised and edited final manuscript draft. \u0026nbsp;LRN - assisted in project design; revised and edited final manuscript draft. EDK - assisted in project design; revised and edited final manuscript draft. CK - funding acquisition; wrote original manuscript draft (equal).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdam E, Bernhart M, Muller H, Winkler J, Berg G. The Cucubita pepo seed microbiome: genotype-specific composition and implications for breeding. Plant Soil. 2018;422:35\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNelson EB. The seed microbiome: origins, interactions, and impacts. 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Food Chem. 2023;411:135442. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodchem.2023.135442\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2023.135442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3797020/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3797020/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eEndophytic mycobiomes are present in all studied plant compartments, including fruits and seeds, but a transmission of the mycobiome between generations is largely unknown. Our objectives were to examine mycobiome transfer via seed wings (samaras) of European ash (\u003cem\u003eFraxinus excelsior\u003c/em\u003e), and to test whether these mycobiomes differ among trees. To achieve this, we used ITS1-based amplicon sequencing and two genotypes of \u003cem\u003eF. excelsior\u003c/em\u003e as a model to compare the mycobiome of mother trees and their samaras.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe profiled the mycobiome of 57 seed stalks and seed wings (samaras) collected from two genotypes of \u003cem\u003eF. excelsior\u003c/em\u003e using three ramets of each genotype. Alpha diversity indices (Observed OTUs and ACE) suggested a higher richness of the mycobiome associated with seed wing than seed stalk within each genotype. However, there was neither significant differences in diversity between the mycobiomes from the two tissue types nor the two genotypes. PERMANOVA analysis revealed significant differences in the mycobiome composition between seed wings, but not between seed stalks, of the two genotypes. Our results suggest that \u003cem\u003eFraxinus excelsior\u003c/em\u003e mother trees disperse different sets of mycobiomes with their samaras, which may be important for germination and seedling establishment \u0026ndash; especially in the light of ash dieback.\u003c/p\u003e","manuscriptTitle":"Mother trees of common ash (Fraxinus excelsior) disperse different sets of mycobiome through their samaras","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-01 11:38:46","doi":"10.21203/rs.3.rs-3797020/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2023-12-25T08:23:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-25T08:23:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2023-12-23T15:32:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5d6c3655-75c9-49a0-ae00-c06560a5ea4f","owner":[],"postedDate":"January 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-12T10:14:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-01 11:38:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3797020","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3797020","identity":"rs-3797020","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}