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Endophyte isolations from Syzygium cordatum and a Eucalyptus
clone (Myrtaceae) reveal new host and geographical reports
for the Mycosphaerellaceae and Teratosphaeriaceae
Angelica Marsberg &Bernard Slippers &
Michael J. Wingfield &Marieka Gryzenhout
Received: 6 January 2014 /Accepted: 21 March 2014
#Australasian Plant Pathology Society Inc. 2014
Abstract Species of Mycosphaerellaceae and
Teratosphaeriaceae (Ascomycetes) cause important leaf, shoot
and canker diseases globally on a broad range of hosts, in-
cluding Eucalyptus and other Myrtaceae. Recently, species of
the Mycosphaerellaceae and Teratosphaeriaceae have been
isolated as asymptomatic endophytes. In this study, endophyt-
ic species of Mycosphaerellaceae and Teratosphaeriaceae
were isolated from samples taken from healthy native
Syzygium cordatum (Myrtaceae) and related non-native Euca-
lyptus grandis xE. camaldulensis (hybrid clone) growing in
Mtubatuba, KwaZulu Natal, South Africa. Multi-locus se-
quence analysis (MLSA) using the Internal Transcribed Spac-
er (ITS) region, the partial Large Subunit (LSU; 28S nrDNA)
of the nuclear ribosomal DNA operon and Translation Elon-
gation Factor-1α(TEF-1α) genes were used to correctly
identify the 22 resulting isolates. The isolates grouped in five
clades representing Readeriella considenianae that was iso-
lated only from the Eucalyptus hybrid clone, Mycosphaerella
marksii and M. vietnamensis from S. cordatum and
Pseudocercospora crystallina from both S. cordatum and the
Eucalyptus hybrid clone. Interestingly, the serious canker
pathogen T. zuluensis was isolated from Eucalyptus leaves,
although it is known only from stem and branch cankers. Of
the species found, R. considenianae and M. vietnamensis were
found in South Africa for the first time, while M. marksii,
M. vietnamensis and P. crystallina were shown to naturally
infect native S. cordatum for the first time. Despite the limited
number of trees sampled, the new host and distribution reports
show that more intensive sampling, especially following an
endophyte approach, will reveal more complete patterns of
host preference and geographical distribution for these fungi.
Keywords Mycosphaerellaceae .Teratosphaeriaceae .
Eucalyptus grandis .Syzygium cordatum .Native and
non-native trees .South Africa
Introduction
Species of fungi in the Mycosphaerellaceae and
Teratosphaeriaceae (Ascomycetes) previously resided in the
single genus, loosely referred to as Mycosphaerella sensu lato
(Capnodiales,Dothideomycetes). This group of fungi includ-
ed approximately 3,000 species names with diverse ecological
roles representing endophytes, pathogens and saprobes
(Corlett 1995; Crous et al. 2000;Hunteretal.2004,2006;
Crous et al. 2009c;Hunteretal.2011). The pathogens in these
genera include important leaf and shoot pathogens of a num-
ber of hosts, including Eucalyptus and other trees in the
Myrtaceae. Collectively they cause the disease known as
Mycosphaerella Diseases or Teratosphaeria Diseases (Hunter
et al. 2011) in order to accommodate the groups that were
previously defined solely as Mycosphaerella leaf blotch
[MLB] or Mycosphaerella leaf diseases [MLD] (Crous et al.
1991;Hydeetal.2007;Hunteretal.2009; Sánchez Márquez
et al. 2011). Globally, over 30 species of Mycosphaerella and
Tera tos p ha e ria , and their associated anamorph species, have
been found to cause important leaf and canker diseases of
Eucalyptus (Crous et al. 2000,2004; Crous et al. 2006; Crous
2009; Crous et al. 2009a,b).
Morphological identification is difficult for species of the
Mycosphaerellaceae and the Teratosphaeriaceae because they
typically grow slowly in culture and many also sporulate only
A. Marsberg :B. Slippers :M. J. Wingfield
Department of Genetics, Forestry and Agricultural Biotechnology
Institute, University of Pretoria, Private Bag x20, Hatfield,
0028 Pretoria, South Africa
M. Gryzenhout (*)
Department of Plant Sciences, University of the Free State, PO Box
339, Bloemfontein 9300, South Africa
e-mail: GryzenhoutM@ufs.ac.za
Australasian Plant Pathol.
DOI 10.1007/s13313-014-0290-y
on host tissue (Crous 1998; Crous et al. 2001a;Hunteretal.
2004,2006). Where morphology can be used for identifica-
tion, it is based on the associated but morphologically variable
anamorph genera (Crous 1998; Crous et al. 2001a; Hunter
et al. 2004). Thus, in recent years, DNA sequence data from
the Internal Transcribed Spacer (ITS) ribosomal DNA region
has most frequently been used to identify the species in these
twogroups(Crousetal.2001a,b; Hunter et al. 2004,2006).
Application of multi-gene analyses using sequences for the
ITS region, Large Subunit (LSU) of the nuclear ribosomal
DNA operon, Translation Elongation Factor-1α(TEF-1α)
and Actin (Act) genes showed that Mycosphaerella was poly-
phyletic (Crous et al. 2001b;Hunteretal.2006;Crous2009).
Consequently, this led to the separation of the families to
include the Cladosporiaceae (Schubert et al. 2007;Bensch
et al. 2010), Dissoconiaceae (Crous et al. 2009a),
Teratosphaeriaceae and the Mycosphaerellaceae (Crous et al.
2007,2009d). Following this taxonomic rearrangement, many
species of Mycosphaerella have been transferred to the closely
related Teratosphaeriaceae, which include pathogens of
Eucalyptus such as Teratosphaeria cryptica,T. nubilosa,T.
gauchensis and T. zuluensis amongst others (Crous et al. 2007;
Andjic et al. 2010).
In South Africa, numerous species of the
Mycosphaerellaceae and Teratosphaeriaceae are known to
infect Eucalyptus trees (Crous and Wingfield 1996; Crous
1998; Hunter et al. 2004,2011). Fewer species including
M. syzygii,M. marasasii and P. syzygiicola have been found
to be associated with the closely related native Myrtaceous
tree, Syzygium cordatum (Sutton and Crous 1997; Crous
1999; Crous et al. 2001a). Previous studies identifying the
Mycosphaerellaceae and Teratosphaeriaceae on Eucalyptus
spp. and S. cordatum followed a broad approach where trees
were sampled over a large geographic area. Furthermore, the
samples were collected from diseased trees. In the present
study, a more dense endophyte sampling approach was used
to directly compare the Mycosphaerellaceae and
Teratosphaeriaceae species endophyte assemblages from a
single Eucalyptus grandis hybrid clone and S. cordatum tree.
Samples were collected at the same time from the same
geographical location and identified using the most recently
available multi-locus sequence phylogenies.
Materials and methods
Sample collection
AS. cordatum tree was sampled in a private nature reserve in
the Mtubatuba area of KwaZulu Natal, South Africa (E32′9″
54.1; S28′29″53.0). An E. grandis xE. camaldulensis hybrid
clone was selected in an adjacent plantation approximately
500 m from the S. cordatum tree. Four leaves from each of
four twigs that originated from four branches located in
different positions, and a trunk increment core with the bark
attached, were collected from each tree. Ten disks (approx-
imately 5 mm in diameter) were cut from each leaf and 5
pieces (3 mm long) were taken from each twig and branch.
Trunk increments were approximately 5 mm in diameter.
The material was surface disinfected by first immersing
them in 10 % hydrogen peroxide (H
2
O
2
)for3–5min,
followedbytwowashesof1min each with sterilised
distilled water.
After surface disinfection, the samples from the Eucalyptus
and S. cordatum were plated onto 2 % Malt Extract Agar
(MEA) (20 g malt extract, 20 g agar, 1 L distilled water;
Biolab, Midrand, South Africa). These were incubated at
25 °C for a month to allow for slow growing endophytic fungi
to emerge. The endophytic fungi resembling the
Mycosphaerellaceae and Teratosphaeriaceae that emerged
were purified by transferring them to new MEA plates and
further incubated at 25 °C. Primary isolations were monitored
for a month to ensure that these slow growing fungi were all
captured. Approximately 50 isolates emerged of which only a
subset of these were used in the study. These cultures are
maintained in the Culture Collection (CMW) of the Forestry
and Agricultural Biotechnology Institute (FABI), Pretoria,
South Africa.
Multi-locus sequence analysis
Mycelium was scraped from the surface of cultures growing
on 2 % MEA and freeze dried for 24 h. DNAwas subsequent-
ly extracted using a modified CTAB method (Moller et al.
1992). After precipitating the DNA, the supernatant was
discarded and the DNA pellet was washed twice with 100 μl
of 70 % ethanol and centrifuged at 10,000 rpm for 5 min for
each wash step. The ethanol was removed and the pellet was
air dried after the second wash until all the ethanol evaporated.
The DNA pellet was re-suspended in 50 μl nuclease-free
water and allowed to incubate overnight at 4 °C (Moller
et al. 1992). The DNA concentration of each sample was
determined using a NanoDrop™1000 Spectrophotometer
version 3.7 (Thermo Fisher Scientific Inc., Germiston, South
Africa) and the working DNA stocks were diluted to 40 ng/μl
with nuclease-free water.
The full-length ITS region of the ribosomal operon was
amplified for each sample. PCR amplification was performed
in a 2720 Thermal Cycler (Applied Biosystems, Foster City,
CA, USA) using the V9G (de Hoog and Gerrits van den Ende
1998) and ITS4 (White et al. 1990)primerpair.Thevolume
for each reaction was 25 μlwith40ngoftemplateDNA,
10 pmoles of each primer (Integrated DNA Technologies,
Coralville, Iowa, USA), 1× NH
4
Reaction Buffer (Roche
Products Pty Ltd, South Africa), 1.5 mM MgCl
2
(Roche
Products Pty Ltd, South Africa), 75 μMdNTPs(Roche
A. Marsberg et al.
Products Pty Ltd, South Africa) and 5 units of Fast Start Taq
(Roche Products Pty Ltd, South Africa). Nuclease-free water
was added to obtain a final volume of 25 μl. The cycle
parameters were as follows: 94 °C denaturation for 5 min,
followed by 35 cycles of denaturation for 30 s at 94 °C,
annealing at 50 °C for 30 s, and elongation at 72 °C for
45 s. A final extension step followed at 72 °C for 7 min.
The primers used to amplify the TEF-1αwere EF1-728 F and
EF1-986R (Carbone and Kohn 1990)whileLROR
(Moncalvo et al. 1995) and LR7 (Vilgalys and Hester 1990)
were used to amplify the partial LSU (28S nrDNA) of the
nuclear ribosomal DNA operon. The cycling conditions for
the ITS and TEF-1αwere identical, while those for the LSU
consisted of an initial denaturation step of 96 °C for 2 min,
followed by 35 cycles of denaturation at 94 °C for 30 s,
annealing at 55 °C for 30 s, and elongation at 72 °C for
1 min with a final extension step at 72 °C for 7 min.
The PCR products were cleaned with Sephadex G-50
columns (Steinheim, Germany) to enable direct DNA se-
quencing. The cleaned PCR product was used as a template
for each sequencing reaction performed with the ABI
PRISM™Big Dye Terminator Cycle Sequencing Ready Re-
action Kit 3.1 (Applied Biosystems, Forster City, CA). For-
ward and reverse sequencing was performed with the same
primers used for the PCR amplification. However, two addi-
tional internal primers, namely LR3R (Vilgalys and Hester
1990) and LR5 (Rehner and Samuels 1995)wererequiredfor
the LSU region. The sequencing reactions were subsequently
run on an ABI PRISM™3100 automated DNA sequencer
(Applied Biosystems, Foster City, CA) and the resultant se-
quences were manually analysed with CLC Main Workbench
version 5.6 (CLC Bio A/S, Aarhus, Denmark) to assemble
contigs with both the forward and reverse sequences. The
sequences were identified by BLAST analysis on the
GenBank database housed at the National Centre for Biotech-
nology Information (NCBI; www.ncbi.nlm.nih.gov).
In order to assign the isolates to genera, the LSU sequences
were aligned with those for all species belonging to the
Mycosphaerellaceae and Teratosphaeriaceae using MAFFT
version 5.667 (Katoh et al. 2005) and a dataset with reference
sequences provided by Dr E. W. Groenewald (CBS-KNAW
Fungal Biodiversity Centre; Netherlands). The auto algorithm
was employed in MAFFT as it automatically selects the ap-
propriate strategy for the size of the dataset. A neighbour-
joining tree, with complete deletion, was produced in MEGA
version 4.0 (Tamura et al. 2007) to determine the genera in
which the isolates could be placed. Due to the large size of the
dataset (data not shown), only the species representing the
nearest neighbours to the isolates of unknown identity were
retained for further analyses.
The ITS and TEF-1αsequences of these nearest neigh-
bours were obtained from GenBank and included in the addi-
tional datasets. The ITS data set consisted of 33 taxa, which
included 22 sequences generated in this study and 11 refer-
ence sequences. The LSU dataset consisted of 34 taxa, of
which 22 were generated sequences and 12 were reference
sequences. The TEF-1αdataset was comprised of 36 taxa,
including 22generated sequences and 14reference sequences.
The sequences for the three gene regions were aligned using
the auto algorithm in MAFFT version 5.667 (Katoh et al.
2005). The combined ITS/LSU/TEF-1αdataset consisted of
33 taxa, of which 22 sequences were generated and 11 were
reference sequences.
Parsimony trees were constructed for the individual and
combined datasets using PAUP version 4.0 b10 (Swofford
2003). The heuristic search function with random sequence
additions (100) and the Tree Bisection Reconnection (TBR)
algorithm was used to obtain the equally most parsimonious
trees. All uninformative characters, including gaps and missing
data, were excluded and characters were reweighted according
to the consistency index (CI). Bootstrap values were calculated
after 1,000 replicates to determine the level of branch support
(Felsenstein 1985). Tree length (TL), consistency index (CI),
retention index (RI) and the homoplasy index (HI) were all
calculated to assess the trees for signal, noise and reliability
(Hillis and Huelsenbeck 1992). A partition homogeneity test
(PHT) consisting of 1,000 replicates was used to determine
whether the ITS, LSU and TEF-1αdatasets could be combined
(Farris et al. 1994). The trees were rooted to Neofusicoccum
ribis and Phaeobotryosphaeria visci as they are sister taxa to
the Mycosphaerellaceae and Teratosphaeriaceae in the
Dothiodeomycetes (Maxwell et al. 2005).
MrBayes version 3 was used for Bayesian analyses of the
various datasets (Ronquist and Huelsenbeck 2003).
MrModeltest version 2.2 was first used to determine the
nucleotide substitution model that would best fit the individual
datasets (Nylander 2004). The Markov Chain Monte Carlo
(MCMC) analyses of six chains from random tree topology
were made twice on 1,000,000 generations and trees were
saved every 1,000 generations. Tracer version 1.4 was used to
determine the posterior probabilities indicating the level of
branch support (Rambaut and Drummond 2007).
Results
Multi-locus sequence analysis
Sequence data were obtained for 22 isolates, of which nine
were from the S. cordatum tree and 13 from the E. grandis x
E. camaldulensis clone (Table 1). Maximum parsimony and
Bayesian analyses were done on the individual and combined
datasets (Figs. 1,2,3, and 4) with appropriate models as
determined by MrModeltest version 2.2 (Table 2). The max-
imum parsimony and Bayesian trees for the individual and
combined datasets had similar topologies, although clades
Endophytic Mycosphaerellaceae and Teratosphaeriaceae species
Tabl e 1 Details of the fungal species and reference sequences used in the phylogenetic analyses
Species Accession Host Country Genbank accession
CMW
a
CBS
b
ITS LSU TEF-1α
Mycosphaerella ellipsoidea –CBS 110843 Eucalyptus sp. South Africa AY725545 ––
4934 –Eucalyptus sp. South Africa –DQ246253 DQ235129
–CBS 110843 Eucalyptus sp. South Africa –GQ852602 –
5166 –Eucalyptus sp. South Africa ––DQ235127
Mycosphaerella endophytica –CBS 114662 Eucalyptus sp. South Africa DQ302953 GU214435 –
5225 –Eucalyptus sp. South Africa ––DQ235128
14912 CBS 111519 Eucalyptus sp. South Africa DQ267579 –DQ235131
Mycosphaerella marksii 14781 CBS 682.95 E. grandis South Africa DQ267587 DQ246249 DQ235133
5230 –E. botryoides Australia DQ267588 DQ246246 DQ235135
37697 Syzygium cordatum South Africa JQ732925 JQ732974 JQ733021
Mycosphaerella marksii 33962 S. cordatum South Africa JQ732926 JQ732975 JQ733022
37682 S. cordatum South Africa JQ732929 JQ732978 JQ733025
37694 S. cordatum South Africa JQ732930 JQ732979 JQ733026
38277 S. cordatum South Africa JQ732931 JQ732980 JQ733026
Mycosphaerella vietnamensis 23442 –E. camaldulensis Vietnam DQ632678 EU882135 –
23441 CBS 119974 E. grandis Vietnam DQ632675 JF700944 –
37695 S. cordatum South Africa JQ732923 JQ732972 JQ733019
37696 S. cordatum South Africa JQ732924 JQ732973 JQ733020
Pseudocercospora crystallina 3042 –E. bicostata South Africa DQ267578 DQ204746 DQ211662
3033 CBS 681.95 E. bicostata South Africa DQ632681 DQ204747 DQ211663
38912 Eucalyptus grandis xE. camaldulensis South Africa JQ732911 JQ732960 JQ733008
38913 S. cordatum South Africa JQ732927 JQ732976 JQ733023
Pseudocercospora crystallina 37698 S. cordatum South Africa JQ732932 JQ732981 JQ733028
Readeriella considenianae –– E. stellulata Australia GQ852792 GQ852681 –
–– E. stellulata Australia GQ852791 GQ852680 –
37671 E. grandis xE. camaldulensis South Africa JQ732893 JQ732942 JQ732990
37674 E. grandis xE. camaldulensis South Africa JQ732896 JQ732945 JQ732994
37675 E. grandis xE. camaldulensis South Africa JQ732897 JQ732946 JQ732995
37676 E. grandis xE. camaldulensis South Africa JQ732899 JQ732948 JQ732997
37678 E. grandis xE. camaldulensis South Africa JQ732901 JQ732950 JQ732999
37680 E. grandis xE. camaldulensis South Africa JQ732903 JQ732952 JQ733001
Teratosphaeria molleriana 4940 CBS 111164 E. globulus Portugal AF309620 DQ246220 DQ235104
2734 CBS 111132 E. globulus U.S.A. AF309619 DQ246223 DQ235105
Teratosphaeria zuluensis 17320; –E. grandis Zambia DQ240148 EU019296 DQ240206
15833 Eucalyptus sp. Mexico ––DQ240162
Teratosphaeria zuluensis 37669 E. grandis xE. camaldulensis South Africa JQ732891 JQ732940 JQ732988
37670 E. grandis xE. camaldulensis South Africa JQ732892 JQ732941 JQ732989
37672 E. grandis xE. camaldulensis South Africa JQ732894 JQ732943 JQ732992
37679 E. grandis xE. camaldulensis South Africa JQ732902 JQ732951 JQ733000
37690 E. grandis xE. camaldulensis South Africa JQ732915 JQ732964 JQ733012
37693 E. grandis xE. camaldulensis South Africa JQ732920 JQ732969 JQ733016
Neofusicoccum ribis 7773 –Ribes sp. U.S.A. DQ246604 DQ246263 DQ235142
Phaeobotryosphaeria visci –CBS 100163 Sphaeropsis visci Germany EU673324 DQ377870 EU673292
Genbank accessions in bold were used in the combined analyses
a
CMW = Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa
b
CBS = CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands
–= not available
A. Marsberg et al.
often collapsed for the moreconserved gene regions. The PHT
value for the combined datasets was p<0.001, which indicat-
ed that the datasets were not congruent. However, the data
were combined since no inconsistencies in the major clades
were observed, other than those mentioned above, to strength-
en the support for the congruent clades.
One of the 160 equally most parsimonious trees obtained
from a heuristic search of the combined ITS, LSU and TEF-1α
datasets was chosen for presentation (Fig. 4). Two distinct
groups of isolates were observed representing species in the
Mycosphaerellaceae (100 % bootstrap support) and
Teratosphaeriaceae (100 % bootstrap support). The isolates
grouped into five clades with strong bootstrap support,
representing M. marksii (5 isolates), M. vietnamensis (2 iso-
lates), P. crystallina (3 isolates), R. considenianae (6 isolates)
and T. zu lue nsi s (6 isolates). Of these species, P. crystallina was
isolated from both trees, whilst M. marksii and M. vietnamensis
were isolated solely from S. cordatum and R. considenianae
and T. zu lue nsis were isolated from the E. grandis clone. All the
identified species were isolated from the leaves of the two trees
sampled. No isolates were isolated from the twigs or the trunk
increments. The sequence alignments and phylogenetic trees
for each gene region as well as the combined analysis were all
deposited in TreeBASE (http://purl.org/phylo/treebase/
phylows/study/TB2:S14876?x-access-code=
ec92eb8a6492c73537799c2fe03b8a0b&format=html).
Discussion
Five species of Mycosphaerellaceae and Teratosphaeriaceae
were isolated as endophytes in this study. These fungi were
Fig. 1 One of the equally most
parsimonious trees obtained from
a heuristic search of the ITS
dataset. Bootstrap values obtained
from 1,000 replicates along with
posterior probabilities are shown
at the nodes. The tree was rooted
with Neofusicoccum ribis and
Phaeobotryosphaeria visci.
Isolates identified in this study are
indicated in bold.Treelength=
209, CI = 0.785, RI = 0.944 and
HI = 0.215
Endophytic Mycosphaerellaceae and Teratosphaeriaceae species
obtained from healthy S. cordatum and E. grandis x E.
camaldulensis trees growing in close proximity to each other,
in South Africa. These included a number of pathogens
known to occur in South Africa and also fungi that have not
previously been recorded in the country.
Even though the sampling in this study was limited,
it revealed two new geographical records for the
Mycosphaerellaceae. These include R. considenianae
and M. vietnamensis that are recorded from South Afri-
ca for the first time. R. considenianae has recently been
foundtooccuronE. considenianae in Australia, but is
not an important pathogen (Summerell et al. 2006;
Taylor et al. 2012). M. vietnamensis is known to occur
as a weak pathogen on E. camaldulensis and E. grandis
in Vietnam (Burgess et al. 2007). In the present study, it
was not only recorded in South Africa for the first time,
but it was also found on a non-native Eucalyptus host
for the first time.
This study revealed that some fungal species of
Mycosphaerellaceae and Teratosphaeriaceae co-occur on
S. cordatum and the sampled Eucalyptus clone. Based on their
identity in previous publications, other species have the po-
tential to do so. For example, P. crystallina was found in the
leaves of both S. cordatum and the Eucalyptus clone. This
fungus is known from leaf spots on E. bicostata as well as on
E. grandis xE. camaldulensis in South Africa (Crous and
Wingfield 1996). The fungus was previously known only
from South Africa, but has since been found to occur on
Eucalyptus in China (Burgess et al. 2007). Its native host is
unknown and it could have originated on Eucalyptus and
moved to native S. cordatum or vice versa. M. marksii is not
considered an important pathogen. However, it has been
Fig. 2 One of the equally most
parsimonious trees obtained from
a heuristic search of the LSU
dataset. Bootstrap values obtained
from 1,000 replicates along with
posterior probabilities are shown
at the nodes. The tree was rooted
with Neofusicoccum ribis and
Phaeobotryosphaeria visci.
Isolates identified in this study are
indicated in bold.Treelength=
229, CI = 0.782, RI = 0.947 and
HI = 0.218
A. Marsberg et al.
found to constitutively infect the leaves of Eucalyptus in
Australia, China, Ethiopia, Indonesia, Madagascar, Portugal,
South Africa and Uruguay (Crous and Wingfield 1996;Crous
1998;Hunteretal.2004; Burgess et al. 2007;Hunteretal.
2011). In this study it was identified from the leaves of
S. cordatum, but not from the leaves of the Eucalyptus clone
sampled. It seems likely that this fungus has been introduced
into South Africa and colonised S. cordatum. M. vietnamensis
has been shown to colonise the leaves of S. cordatum in this
study but is also known to occur in other Eucalyptus spp. as a
weak pathogen in South East Vietnam (Burgess et al. 2007).
The ability of species in the Mycosphaerellaceae and
Teratosphaeriaceae to co-occur on closely related hosts, such
as Eucalyptus and S. cordatum, indicates that they have the
capacity to jump to either commercially important trees or,
alternatively, they could potentially threaten native trees. As
Eucalyptus spp. are not native to South Africa, pathogens may
have been introduced that have the potential to colonise native
Myrtaceae spp., such as S. cordatum. However, the opposite
may also be true where native trees serve as inoculum sources
for fungi that are pathogenic to Eucalyptus (Pavlic et al. 2007;
Rodas et al. 2008). This study highlights that the latter may be
occurring for leaf infecting fungi but remains largely
unrecognised. The results in this study showing evidence of
possible cross infections are surprising and the consequence of
such cross infections deserves further attention. Crous and
Groenewald (2005) suggested that the phenomenon whereby
some fungal species move from one host to another in search
of their preferred host on which they cause disease, be termed
the “pogo-stick hypothesis”(Crous and Groenewald 2005).
Teratosphaeria zuluensis is a well-known and serious path-
ogen associated with E. grandis clones in South Africa, which
causes severe cankers on the stems and branches of
Eucalyptus trees (Wingfield et al. 1996; Cortinas et al.
2010). In this study, the fungus was isolated from the leaves
of the Eucalyptus clone. This was a most unexpected result as
Fig. 3 One of the equally most
parsimonious trees obtained from
a heuristic search of the TEF-1α
dataset. Bootstrap values obtained
from 1,000 replicates along with
posterior probabilities are shown
at the nodes. The tree was rooted
with Neofusicoccum ribis and
Phaeobotryosphaeria visci.
Isolates identified in this study are
indicated in bold.Treelength=
642, CI = 0.783, RI = 0.945 and
HI = 0.217
Endophytic Mycosphaerellaceae and Teratosphaeriaceae species
symptoms of infection by the fungus have never been seen on
leaves and is found only on green stem and branch tissues.
Whether the fungus can act as a pathogen of leaves is un-
known and this deserves further study. The presence of this
serious pathogen within leaves, however, represents a
previously overlooked niche that could represent possible
opportunities for it to be moved between areas, countries
and continents. This may indeed represent a means by which
it was introduced into South Africa, potentially from China
where it is thought to be native (Chen et al. 2011).
Fig. 4 One of the equally most
parsimonious trees obtained from
a heuristic search of the combined
ITS, LSU and TEF-1αdatasets.
Bootstrap values obtained from
1,000 replicates along with
posterior probabilities are shown
at the nodes. The tree was rooted
with Neofusicoccum ribis and
Phaeobotryosphaeria visci.
Isolates identified in this study are
indicated in bold.Treelength=
1091, CI = 0.775, RI = 0.941 and
HI = 0.225
Tabl e 2 Statistical results for the phylogenetic analysis
Maximum parsimony
Dataset Number of taxa Excluded characters Included characters Tree number Tree length CI RI HI
ITS 35 400 96 51 196 0.760 0.944 0.240
LSU 35 531 136 21 223 0.794 0.953 0.206
TEF-1α36 40 232 16 642 0.783 0.945 0.217
Combined 33 1023 470 160 1091 0.775 0.941 0.225
MrBayes
Dataset Model Preset state freqpr NST Rates Burnin
ITS GTR+G (1, 1, 1, 1) 6 gamma 1,000
LSU GTR+G (1, 1, 1, 1) 6 gamma 1,000
TEF-1αGTR+G (1, 1, 1, 1) 6 gamma 1,000
Combined GTR+G (1, 1, 1, 1) 6 gamma 1,000
A. Marsberg et al.
The isolates characterised in this study were derived from
single S. cordatum and Eucalyptus trees. This limited sam-
pling in terms of the host, however, revealed three new host
and two new geographical reports. It also exposed examples
of possible cross infections by potentially pathogenic fungi on
these two tree species. This suggests that although Eucalyptus
represents a well-studied tree in South Africa, the patterns of
occurrence, host preference and ecological role of the
Mycosphaereallaceae and the Teratosphaeriaceae remain
poorly understood.
Acknowledgments We thank the Thutuka Funding programme of the
National Research Foundation (NRF), members of the Tree Protection
Co-operative Programme (TPCP) and the Department of Science and
Technology (DST)/NRF Centre of Excellence in Tree Health Biotech-
nology (CTHB), South Africa for financial support. We are also grateful
to Dr Ewald Groenewald (CBS-KNAW Fungal Biodiversity Centre;
Netherlands), for his valuable assistance with the data analysis and for
helpful suggestions regarding the manuscript. We also thank Miss Kerry-
Anne Pillay and Mr Jan Nagel for technical assistance.
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