root endophyte communities differ between sodic and non ... · root endophyte communities differ...
TRANSCRIPT
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