Root endophyte communities differ between sodic and non-sodic soils in

a catena ecosystem of the Kruger National Park, South Africa

. Marieka Gryzenhout1, Brooke Bailey1, Antonie Kloppers1, 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

• Fungal communities play an important role in the

functionality of any ecosystem.

• Next Generation Sequencing (NGS) technologies

allow for the rapid characterization of communities

with a level of identification that adds insight to

interactions.

• Using this more rapid approach, the possible

usefulness of fungal communities as indicators

can be studied.

• A catena (Fig. 1a) is a sequence of soil types

down a hill slope because of precipitation,

infiltration and runoff (Taleqani, 2008).

• These create diverse ecotypes, soils and

hydrological processes.

• AIM: Study the effect of sodic vs. non-sodic soils

in a catena system on endophytes associated

with plant roots.

• An indicator plant present in both soil types were

chosen to negate bias based on plant species.

MATERIALS AND METHODS

PREPEPARTION FOR ILLUMINA SEQUENCING• Selected plant: Sida cordifolia (Malvaceae), flannel

weed, invasive.• 20 samples were collected from both the sodic and

non-sodic site within a catena at the Southern GraniteSupersite, near Skukuza in the Kruger National Park.

• These sites were adjacent to each other (Fig. 1b) andthe total area of sampling were c. 50 m2.

• Roots were surface sterilized in a water-3% bleach-70% ethanol-sterile, distilled water series.

• The Nucleospin® Plant II Kit (Machery-Nagel) was usedto extract gDNA.

• The Internal Transcribed Spacer 2 region wasamplified with ITS3 and ITS4 primers fitted withoverhang Illumina adapters. Amplicons were pooledand sequenced with an Illumina MiSeq at the NextGeneration Sequencing facility at the Department ofHealth Sciences, University of the Free State.

ITS2 DATA ANALYSIS• Fastqc (Babraham Bioinformatics) was used to assess

sequence quantities and quality of the sequences.• Quality control was performed using Prinseq-lite

v0.20.4 to obtain a sequence length of 240-251 basesand a mean quality score of ≤20 using a 7 nt windowwith a (?) nt step.

• Reads were merged with PEAR 0.9.6, and qualityfiltering was run in QIIME to obtain a FASTA outputfile.

• Identification of chimeric sequences was performedusing usearch 6.1.544 against the RDP “Gold”database.

• QIIME was used to filter out all chimeras using theidentify_chimeric_seqs.py and filter_fasta.pycommands.

• OTU clusters and taxonomy were assigned using thepick_open_reference_otus.py scripts at a 97%sequence similarity against the UNITE database 7.0(Kõljalg, 2013).

• Species level identities were investigated withMaximum Likelihood analyses in MEGA v. 6, with themost relevant sequences from Genbank included.

RESULTS

Fig.2. Shannon Diversity Index for each plant tissue.

• A total of 104 474 and 102 066 ITS2 sequences

where generated for the non-sodic and sodic site,

respectively.

• Alpha Diversity rarefaction plots of Shannon

indices (5.51 for sodic soil and 4.95 for non-sodic

soil) were significant.

• A total of 57 molecular operational taxonomic

units (MOTU) were detected (Fig. 3).

• Less than 10% of sequences from both sites

contained sequences that did not cluster with

MOTUs on the UNITE database.

• The majority of the MOTU’s belonged to the

Ascomycota (47 of 54) with the rest in the

Basidiomycota.

• A number of the OTUs found in the non-sodic soil

were not identified in the sodic soil, while others

found in the sodic soil w (e.g.

Botryosphaeriaceae) were not present in the non-

sodic soil.

• The most abundant MOTU for non-sodic soil

belonged to the Botryosphaeriaceae, making up

32% of the sequence data while only making up

0,012% of the genera of the sodic site.

• The most abundant OTU in sodic soil was that of

an unclassified taxon in the Dothideomycetes,

making up 26% of the sequence data, while only

making up 4% of the non-sodic MOTU’s.

• Some genera were showed to represent various

species based on phylogenetic analyses (Fig. 4)

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.

DISCUSSION

• The obtained metagenome sequencing data of

the ITS2 rDNA yielded data useful to analyse

fungal community composition and differences in

based on soil pH in a catena.

• Rarefaction plots revealed that the samples

contained enough sequences to represent the

fungal community present in the roots in both

sites.

• Differences existed between the endophytic

communities in the roots of the sodic and non-

sodic sites.

• For instance the most prevalent OTUs

identified in either site were often less frequent

in the other site.

• Some taxa were missing between sites.

• Using an environmental approach, it was shown

that fungal communities within plant roots of the

same plant species differ within an certain locality

based only on soil conditions.

• For conservation purposes results are significant

because our approach indicated that despite the

wide spread occurrence of a plant species,

differences on the microbial levels can exist that

should be incorporated in conservation planning.

• Fungi can thus be useful as bioindicators.

ACKNOWLEDGMENTS

Funding was provided by the University of the Free

State as part of a multi-disciplinary research

project. The Kruger National Park is thanked for

survey services and support, and the Next

Generation Sequencing facility of the University of

the Free State for generating the sequence results.

Dr Vincent Robert (Johanna Westerdijk Institute) is

thanked for providing the Fusarium sequencedataset.

REFERENCES

Fig. 4. Maximum Likelihood phylogram based on Internal Transcribed Spacer 2

sequences of representatives of Fusarium and Bisifusarium with bootstrap

support values. The reads from this study are indicated with arrows. Analyses

were done with Mega v. 7.

Kõljalg et al. (2005). New Phytologist 166: 1063–

1068.

Taleqane, M. 2008. Soil dictionary. Babylon

Information Platform. Available online at:

http://agriculture.agriculture.science-dictionary.org/Soil-Dictionary/.

Sodic

Non-

Sodic

Maximum

likelihood

Model JC+G

F. incarnatum-equiseti species complex (NRRL 26922 MLST type: 9-c)

GU932675 MG11 1946

F. incarnatum-equiseti species complex (NRRL 20423 MLST type: 4-a)

F. incarnatum-equiseti species complex (NRRL 13379 MLST type: 23-b)

F. incarnatum-equiseti species complex (NRRL 20722 MLST type: 27-a)

F. incarnatum-equiseti species complex (NRRL 13402 MLST type: 9-b)

F. incarnatum-equiseti species complex (NRRL 13335 MLST type: 21-a)

F. incarnatum-equiseti species complex (NRRL 20697 MLST type: 14-b)

EU714404 MG11 2086

KR909402.1 F. andiyazi strain MRC8046

KR020684.1 F. verticillioides strain LAPEMI 09.2015

F. oxysporum species complex (NRRL 20433 MLST type: 2)(2)

F. oxysporum species complex (NRRL 20433 MLST type: 2)(3)

F. oxysporum species complex (NRRL 20433 MLST type: 2)

HE649383 MG11 3794

GQ121293 MG11 137

HM102504 MG12 12682

JX162363.1 F. pseudograminearum strain CBS 131261

DQ459848.1 F. boothii strain NRRL29105

JX162395.1 F. graminearum strain CBS 131778

DQ459854.1 F.A acaciae-mearnsii strain NRRL34207

F. chlamydosporum species complex (NRRL 28505 MLST type: 4-b)

EU818693 MG12 13780

F. chlamydosporum species complex (NRRL 13338 MLST type: 4-a)

F. chlamydosporum species complex (NRRL 32521 MLST type: 1-e)

F. chlamydosporum species complex (NRRL 13444 MLST type: 2-a)

F. chlamydosporum species complex (NRRL 28578 MLST type: 1-a)

F. tricinctum species complex (NRRL 34036 MLST type: 1-a)

F. acuminatum (NRRL 36147 MLST type: 2-a)

KR909433.1 F. thapsinum strain MRC8558

KR071692.1 F. thapsinum strain CBS 130176

GU226829 MG11 2167(2)

GU226829 MG11 2167

GU226829 MG11 2167(3)

F. fujikuroi species complex (NRRL 13164 MLST type: none)

F. fujikuroi species complex (NRRL 13164 MLST type: none)(2)

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)

F. solani species complex (CBS 475.67 MLST type: 3+4-ccc)

F. solani species complex (CBS 101427 MLST type: 3+4-ddd)

F. solani species complex (NRRL 22166 MLST type: 8-e)

F. solani species complex (NRRL 22162 MLST type: 13-c)

F. solani species complex (NRRL 22098 MLST type: 10-b)

JN786598 MG11 1515

B. dimerum species complex (NRRL 34026 MLST type: )

B. dimerum species complex (NRRL 34029 MLST type: )

B. dimerum species complex (NRRL 20715 MLST type: )

B. dimerum species complex (NRRL 34027 MLST type: )

B. dimerum species complex (NRRL 22260 MLST type: )89

100

8399

96

70

91

89

81

77

74

99

86

83

74

97

0.02

Fusarium fujikuroi

species complex

Fusarium solani

species complex

Fusarium

chlamydosporum

species complex

Fusarium

oxysporum

species complex

Fusarium

incarnatum-

equiseti species

complex

0

0.2

0.4

0.6

0.8

1

1.2

MG12 MG11

Rel

ativ

e A

bu

nd

ance

(%

)

Unassigned 1 Unassigned 2 Ascomycota;Unassigned 3

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

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

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

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

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

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

Basidiomycota;Agaricomycetes;Sebacinales;Unassigned 25 Basidiomycota;Incertae_sedis;Malasseziales;Unassigned 26 Basidiomycota;Microbotryomycetes;Sporidiobolales;Incertae_sedis;Rhodotorula

Basidiomycota;Tremellomycetes;Filobasidiales;Filobasidiaceae;Cryptococcus Basidiomycota;Unassigned 27 Unassigend 28

Genus Barchart

“Botryosphaeria”

“Lasiodiplodia”

Dothideomycete

unassigned 4

“Rhizopycnis”

Botryosphaeriaceae

unassigned 4

Fusarium

Rhodotorula

Sebacinales,

unassigned 24

Figure 1. (a) Representation of catena by means of terrain morphological units. (b)Adjacent sodic (dotted arrow) and non-sodic (arrow) sites in a Kruger Park catenasystem.

(Taken by B Janecke)https://www.researchgate.net/figure/258342996_fig7_Fig-1-

The-studied-soil-catenas-catena-1-above-catena-2-below

ba

Sodic site Non-sodic site

Poster

ID 496