Root Endophyte Communities Differ Between Sodic and Non-Sodic Soils

Root endophyte communities differ between sodic and non-sodic soils in a catena ecosystem of the Kruger National Park, South Africa Poster ID 496 Marieka Gryzenhout1, Brooke Bailey1, Antonie Kloppers. 1, Errol D. Cason2, Tonjock Rosemary Kinge1,3 1Department of Genetics, University of the Free State, Bloemfontein, P. O. Box 339, Bloemfontein 9300, Republic of South Africa; 2Department of Microbial Biochemical and Food Biotechnology, University of the Free State, Bloemfontein 3Department of Biological Sciences, Faculty of Science, The University of Bamenda, P.O. Box 39, Bambili, North West Region, Cameroon. INTRODUCTION RESULTS DISCUSSION

• Fungal communities play an important role in the • A total of 104 474 and 102 066 ITS2 sequences • The obtained metagenome sequencing data of functionality of any ecosystem. where generated for the non-sodic and sodic site, the ITS2 rDNA yielded data useful to analyse • Next Generation Sequencing (NGS) technologies respectively. fungal community composition and differences in allow for the rapid characterization of communities • Alpha Diversity rarefaction plots of Shannon based on soil pH in a catena. with a level of identification that adds insight to indices (5.51 for sodic soil and 4.95 for non-sodic • Rarefaction plots revealed that the samples interactions. soil) were significant. contained enough sequences to represent the • Using this more rapid approach, the possible • A total of 57 molecular operational taxonomic fungal community present in the roots in both usefulness of fungal communities as indicators units (MOTU) were detected (Fig. 3). sites. can be studied. • Less than 10% of sequences from both sites • Differences existed between the endophytic • A catena (Fig. 1a) is a sequence of soil types contained sequences that did not cluster with communities in the roots of the sodic and non- down a hill slope because of precipitation, MOTUs on the UNITE database. sodic sites. infiltration and runoff (Taleqani, 2008). • The majority of the MOTU’s belonged to the • For instance the most prevalent OTUs • These create diverse ecotypes, soils and Ascomycota (47 of 54) with the rest in the identified in either site were often less frequent hydrological processes. Basidiomycota. in the other site. • AIM: Study the effect of sodic vs. non-sodic soils • A number of the OTUs found in the non-sodic soil • Some taxa were missing between sites. in a catena system on endophytes associated were not identified in the sodic soil, while others • Using an environmental approach, it was shown with plant roots. found in the sodic soil w (e.g. that fungal communities within plant roots of the • An indicator plant present in both soil types were Botryosphaeriaceae) were not present in the non- same plant species differ within an certain locality chosen to negate bias based on plant species. sodic soil. based only on soil conditions. • The most abundant MOTU for non-sodic soil • For conservation purposes results are significant belonged to the Botryosphaeriaceae, making up because our approach indicated that despite the 32% of the sequence data while only making up wide spread occurrence of a plant species, 0,012% of the genera of the sodic site. differences on the microbial levels can exist that • The most abundant OTU in sodic soil was that of should be incorporated in conservation planning. an unclassified taxon in the Dothideomycetes, • Fungi can thus be useful as bioindicators. making up 26% of the sequence data, while only making up 4% of the non-sodic MOTU’s. ACKNOWLEDGMENTS a b • Some genera were showed to represent various https://www.researchgate.net/figure/258342996_fig7_Fig-1- (Taken by B Janecke) The-studied-soil-catenas-catena-1-above-catena-2-below species based on phylogenetic analyses (Fig. 4) Figure 1. (a) Representation of catena by means of terrain morphological units. (b) F. incarnatum-equiseti species complex (NRRL 26922 MLST type: 9-c) Funding was provided by the University of the Free Adjacent sodic (dotted arrow) and non-sodic (arrow) sites in a Kruger Park catena GU932675 MG11 1946 Maximum F. incarnatum-equiseti species complex (NRRL 20423 MLST type: 4-a) Fusarium F. incarnatum-equiseti species complex (NRRL 13379 MLST type: 23-b) State as part of a multi-disciplinary research system. likelihood F. incarnatum-equiseti species complex (NRRL 20722 MLST type: 27-a) incarnatum- Model JC+G 97 F. incarnatum-equiseti species complex (NRRL 13402 MLST type: 9-b) F. incarnatum-equiseti species complex (NRRL 13335 MLST type: 21-a) equiseti species project. The Kruger National Park is thanked for F. incarnatum-equiseti species complex (NRRL 20697 MLST type: 14-b) 74 EU714404 MG11 2086 complex MATERIALS AND METHODS KR909402.1 F. andiyazi strain MRC8046 survey services and support, and the Next KR020684.1 F. verticillioides strain LAPEMI 09.2015 F. oxysporum species complex (NRRL 20433 MLST type: 2)(2) Fusarium 83 F. oxysporum species complex (NRRL 20433 MLST type: 2)(3) Generation Sequencing facility of the University of 99 F. oxysporum species complex (NRRL 20433 MLST type: 2) oxysporum PREPEPARTION FOR ILLUMINA SEQUENCING HE649383 MG11 3794 86 GQ121293 MG11 137 the Free State for generating the sequence results. HM102504 MG12 12682 species complex • Selected plant: Sida cordifolia (Malvaceae), flannel JX162363.1 F. pseudograminearum strain CBS 131261 DQ459848.1 F. boothii strain NRRL29105 Dr Vincent Robert (Johanna Westerdijk Institute) is 99 JX162395.1 F. graminearum strain CBS 131778 weed, invasive. 83 DQ459854.1 F.A acaciae-mearnsii strain NRRL34207 F. chlamydosporum species complex (NRRL 28505 MLST type: 4-b) thanked for providing the Fusarium sequence EU818693 MG12 13780 Fusarium • 20 samples were collected from both the sodic and 89 F. chlamydosporum species complex (NRRL 13338 MLST type: 4-a) F. chlamydosporum species complex (NRRL 32521 MLST type: 1-e) chlamydosporum dataset. F. chlamydosporum species complex (NRRL 13444 MLST type: 2-a) F. chlamydosporum species complex (NRRL 28578 MLST type: 1-a) species complex non-sodic site within a catena at the Southern Granite F. tricinctum species complex (NRRL 34036 MLST type: 1-a) 96 F. acuminatum (NRRL 36147 MLST type: 2-a) KR909433.1 F. thapsinum strain MRC8558 Supersite, near Skukuza in the Kruger National Park. 74 KR071692.1 F. thapsinum strain CBS 130176 GU226829 MG11 2167(2) 77 GU226829 MG11 2167 • These sites were adjacent to each other (Fig. 1b) and GU226829 MG11 2167(3) Fusarium fujikuroi F. fujikuroi species complex (NRRL 13164 MLST type: none) REFERENCES 2. 81 F. fujikuroi species complex (NRRL 13164 MLST type: none)(2) species complex the total area of sampling were c. 50 m F. fujikuroi species complex (NRRL 13164 MLST type: none)(3) F. fujikuroi species complex (NRRL 13164 MLST type: none)(4) F. fujikuroi species complex (NRRL 13164 MLST type: none)(5) • Roots were surface sterilized in a water-3% bleach- F. solani species complex (CBS 475.67 MLST type: 3+4-ccc) F. solani species complex (CBS 101427 MLST type: 3+4-ddd) Fusarium solani 70 F. solani species complex (NRRL 22166 MLST type: 8-e) 70% ethanol-sterile, distilled water series. F. solani species complex (NRRL 22162 MLST type: 13-c) Kõljalg et al. (2005). New Phytologist 166: 1063– F. solani species complex (NRRL 22098 MLST type: 10-b) species complex JN786598 MG11 1515 • The Nucleospin® Plant II Kit (Machery-Nagel) was used 91 B. dimerum species complex (NRRL 34026 MLST type: ) 1068. B. dimerum species complex (NRRL 34029 MLST type: ) 100 B. dimerum species complex (NRRL 20715 MLST type: ) B. dimerum species complex (NRRL 34027 MLST type: ) Taleqane, M. 2008. Soil dictionary. Babylon to extract gDNA. 89B. dimerum species complex (NRRL 22260 MLST type: ) • The Internal Transcribed Spacer 2 region was 0.02 Information Platform. Available online at: amplified with ITS3 and ITS4 primers fitted with Fig. 4. Maximum Likelihood phylogram based on Internal Transcribed Spacer 2 http://agriculture.agriculture.science- sequences of representatives of Fusarium and Bisifusarium with bootstrap dictionary.org/Soil-Dictionary/. overhang Illumina adapters. Amplicons were pooled support values. The reads from this study are indicated with arrows. Analyses and sequenced with an Illumina MiSeq at the Next were done with Mega v. 7. 1.2 Generation Sequencing facility at the Department of Health Sciences, University of the Free State. Genus Barchart Sodic site Non-sodic site ITS2 DATA ANALYSIS 1 • Fastqc (Babraham Bioinformatics) was used to assess sequence quantities and quality of the sequences. • Quality control was performed using Prinseq-lite Rhodotorula v0.20.4 to obtain a sequence length of 240-251 bases 0.8 Sebacinales, and a mean quality score of ≤20 using a 7 nt window with a (?) nt step. unassigned 24 • Reads were merged with PEAR 0.9.6, and quality Fusarium

filtering was run in QIIME to obtain a FASTA output 0.6

file. “Lasiodiplodia” Relative Relative Abundance(%) • Identification of chimeric sequences was performed using usearch 6.1.544 against the RDP “Gold” “Rhizopycnis” database. 0.4 “Botryosphaeria” • QIIME was used to filter out all chimeras using the identify_chimeric_seqs.py and filter_fasta.py Dothideomycete commands. Botryosphaeriaceae unassigned 4 • OTU clusters and taxonomy were assigned using the 0.2 unassigned 4 pick_open_reference_otus.py scripts at a 97% sequence similarity against the UNITE database 7.0 (Kõljalg, 2013). 0 • Species level identities were investigated with MG12 MG11 Unassigned 1 Unassigned 2 Ascomycota;Unassigned 3 Maximum Likelihood analyses in MEGA v. 6, with the Ascomycota;Dothideomycetes;Unassigned 4 Ascomycota;Dothideomycetes; Botryosphaeriales;Botryosphaeriaceae;Unassigned 5 Ascomycota;Dothideomycetes;Botryosphaeriales;Botryosphaeriaceae;Botryosphaeria Ascomycota;Dothideomycetes;Botryosphaeriales;Botryosphaeriaceae;Diplodia Ascomycota;Dothideomycetes;Botryosphaeriales;Botryosphaeriaceae;Lasiodiplodia Ascomycota;Dothideomycetes;Botryosphaeriales;Botryosphaeriaceae;Microdiplodia most relevant sequences from Genbank included. Ascomycota;Dothideomycetes;Capnodiales;Unassigned 6 Ascomycota;Dothideomycetes;Capnodiales;Mycosphaerellaceae;Unassigned 7 Ascomycota;Dothideomycetes;Capnodiales;Teratosphaeriaceae;Teratosphaeria Ascomycota;Dothideomycetes;Dothideales;Dothioraceae;Aureobasidium Ascomycota;Dothideomycetes;Incertae sedis;Rhizopycnis Ascomycota;Dothideomycetes;Pleosporales;Unassigend 8 Ascomycota;Dothideomycetes;Pleosporales;Cucurbitariaceae;Curreya Ascomycota;Dothideomycetes;Pleosporales;Incertae sedis;Unassigned 9 Ascomycota;Dothideomycetes;Pleosporales;Incertae sedis;Fusculina Ascomycota;Dothideomycetes;Pleosporales;Incertae sedis;Phoma Ascomycota;Dothideomycetes;Pleosporales;Lophiostomataceae;Lophiostoma Ascomycota;Dothideomycetes;Pleosporales;Massarinaceae;Unassigend 10 Sodic Ascomycota;Dothideomycetes;Pleosporales;Pleosporaceae;Unassigned 11 Ascomycota;Dothideomycetes;Pleosporales;Pleosporaceae;Alternaria Ascomycota;Dothideomycetes;Pleosporales;Pleosporaceae;Curvularia Ascomycota;Dothideomycetes;Pleosporales;Pleosporaceae;Epicoccum Ascomycota;Dothideomycetes;Pleosporales;Unassigned 12 Ascomycota;Dothideomycetes;Unassigned 13 Ascomycota;Eurotiomycetes;Chaetothyriales;Herpotrichiellaceae;Exophiala Ascomycota;Eurotiomycetes;Chaetothyriales;Unassigned 14 Ascomycota;Orbiliomycetes;Orbiliales;Orbiliaceae;Unassigned 15 Non- Ascomycota;Sordariomycetes;Unassigned 16 Ascomycota;Sordariomycetes;Diaporthales;Unassigned 17 Ascomycota;Sordariomycetes;Diaporthales;Diaporthaceae;Diaporthe Ascomycota;Sordariomycetes;Diaporthales;Togniniaceae;Phaeoacremonium Ascomycota;Sordariomycetes;Diaporthales;Valsaceae;Phomopsis Ascomycota;Sordariomycetes;Hypocreales;Nectriaceae;Fusarium1 Sodic Ascomycota;Sordariomycetes;Hypocreales;Nectriaceae;Fusarium2 Ascomycota;Sordariomycetes;Hypocreales;Nectriaceae;Haematonectria Ascomycota;Sordariomycetes;Incertae sedis;Myrmecridium Ascomycota;Sordariomycetes;Sordariales;Chaetomiaceae;Unassigned 18 Ascomycota;Sordariomycetes;Sordariales;Chaetomiaceae;Chaetomium Ascomycota;Sordariomycetes;Sordariales;Chaetomiaceae;Humicola Ascomycota;Sordariomycetes;Sordariales;unidentified;Unassigned 19 Ascomycota;Sordariomycetes;Xylariales;Amphisphaeriaceae;Unassigned 20 Ascomycota;Sordariomycetes;Xylariales;Amphisphaeriaceae;Bartalinia Fig.2. Shannon Ascomycota;Sordariomycetes;Xylariales;Amphisphaeriaceae;Pestalotiopsis Ascomycota;Sordariomycetes;Xylariales;Amphisphaeriaceae;Truncatella Ascomycota;Sordariomycetes;Xylariales;Amphisphaeriaceae;Unassigned 21 Ascomycota;Unassigned 22 Basidiomycota;Unassigned 23 Basidiomycota;Agaricomycetes;Sebacinales;Unassigend 24 Diversity Index Basidiomycota;Agaricomycetes;Sebacinales;Unassigned 25 Basidiomycota;Incertae_sedis;Malasseziales;Unassigned 26 Basidiomycota;Microbotryomycetes;Sporidiobolales;Incertae_sedis;Rhodotorula for each plant Basidiomycota;Tremellomycetes;Filobasidiales;Filobasidiaceae;Cryptococcus Basidiomycota;Unassigned 27 Unassigend 28 tissue. Fig. 3. Graph comparing the identified MOTU clusters on genus level with a similarity of 97% to the UNITE v7.0 database as well as proportion of sequences within each cluster.