Eur J Plant Pathol

https://doi.org/10.1007/s10658-020-01962-6

Characterisation of leaf spots caused by Neopestalotiopsis

clavispora and Colletotrichum siamense in macadamia
in Australia
K. Prasannath & V. J. Galea & O. A. Akinsanmi

Accepted: 18 February 2020

# Koninklijke Nederlandse Planteziektenkundige Vereniging 2020

Abstract Extensive leaf spot was observed on all leaves
of young macadamia trees planted in new orchards in
Queensland, Australia. The loss in photosynthetic ability
of these trees may contribute to their demise and poor
establishment compared to trees without symptoms. A
survey of fungal leaf spots on macadamia trees in 20
commercial orchards in Queensland revealed two distinc-
tive types of symptoms. Leaves showing circular dark
brown spots with yellow halos (Type 1) and irregular
dark brown spots (Type 2) were collected. Fungal isolates
associated with the infected leaves were identified by
morphological characteristics and DNA sequencing as
Neopestalotiopsis clavispora for Type 1 spots and
Colletotrichum siamense for Type 2 spots. Koch’s postu-
lates were fulfilled for N. clavispora and C. siamense.
Pathogenicity assays showed that both fungi caused se-
vere leaf spots, which are identical to the respective field
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10658-020-01962-6) contains
supplementary material, which is available to authorized users.
K. Prasannath (*) : O. A. Akinsanmi
Centre for Horticultural Science, Queensland Alliance for
Agriculture & Food Innovation, The University of Queensland,
Ecosciences Precinct, Dutton Park, QLD 4102, Australia
e-mail: k.prasannath@uq.net.au
K. Prasannath
Department of Agricultural Biology, Faculty of Agriculture,
Eastern University, Chenkaladi, Sri Lanka
V. J. Galea
School of Agriculture & Food Sciences, The University of
Queensland, Gatton, QLD 4343, Australia
disease symptoms. In order to clearly characterise them,
the two leaf spots were named as Pestalotiopsis leaf spot
(Type 1 spots) and Colletotrichum leaf spot (Type 2
spots). This is the first report of N. clavispora and
C. siamense causing leaf spots in macadamia in Australia.
Keywords Leaf disease . Pestalotiopsis .
Phylogenetics . Proteaceae . Tree nut
Macadamia (Macadamia integrifolia, M. tetraphylla
and their hybrids) is cultivated commercially in frost-
free tropical and subtropical regions worldwide for their
edible kernel (Trueman 2013). Australia is the centre of
origin of Macadamia spp. which originated from the
coastal southeast Queensland and northeast New South
Wales (Gross 1995). Currently, the major macadamia
producing countries are South Africa, Australia, Kenya,
USA (Hawaii), China, Guatemala, Malawi and Brazil,
and the new emerging industries include New Zealand,
Vietnam and Paraguay (International Nut and Dried
Fruit Council 2019). Global macadamia production in
2018 was valued at 224,000 t nut-in-shell (NIS), while
Australia produced 52,900 t NIS (Ziehlke 2019). In
Australia, particularly the Bundaberg region, where ex-
pansion of macadamia cultivation is taking place at a
rapid pace, produced 21,830 t NIS in 2018 (Kojetin
2019).
Major diseases of macadamia are primarily caused by
fungi and oomycetes, and include husk spot (Beilharz
et al. 2003), Phomopsis husk rot (Akinsanmi and Drenth
2017), flower blights (Akinsanmi et al. 2017;

Holtzmann 1963; Kunimoto et al. 1976; van den Berg
et al. 2008), Phytophthora trunk canker and root rot
(Zentmyer 1980), and branch dieback (Herbert and
Grech 1985; Jeff-Ego and Akinsanmi 2019). Leaf dis-
eases in macadamia such as leaf spots caused by
Pestalotiopsis versicolor (Rawal and Muniyappa
1981) and Neopestalotiopsis clavispora (Santos et al.
2019) have also been reported in India and Brazil,
respectively.
In Queensland, Australia, leaf spot with characteristic
symptoms of small circular dark brown spots with yel-
low halos were often observed on macadamia leaves in
commercial orchards and wild trees (Fig. 1a). Other than
when diseased leaves later become perforated when the
necrotic spot area drops out producing a shot hole, the
effect on tree development and yield is considered to be
very limited. In recent years, increasing incidence of the
leaf spot appears to be reducing the growth of young
trees, most likely through reduced photosynthetic abili-
ty. However, crop loss due to the disease has not been
estimated. In 2018, a variant leaf spot displaying irreg-
ular dark brown lesions was observed on nursery plants
and mature trees in the field (Fig. 2a). The increasing
occurrence and potential impact of these leaf spots is a
concern to macadamia growers.
In order to characterise the two types of leaf spots
which were preliminarily designated as Type 1 and Type
2, isolates were obtained from 20 infected leaves from
commercial orchards and macadamia nurseries in
Queensland, Australia. Samples of Type 1 leaf spot
showed small circular dark brown spots with yellow
halos (Fig. 1b, c) and Type 2 leaf spot showed irregular
dark brown lesions (Fig. 2b, c). Leaf pieces (approxi-
mately 50 mm2) were cut from the lesion margins,
surface sterilized in 2.5% (w/v) sodium hypochlorite
(NaOCl) solution for 1 min, rinsed three times in sterile
distilled water, and blot dried on sterile blotting paper.
Four pieces each were transferred onto ½-strength pota-
to dextrose agar (PDA) plates. The plates were incubat-
ed at 25 °C under 12 h/12 h light/dark conditions for
3 days and fungal colonies were sub cultured on to fresh
PDA plates. Monoconidial isolates were obtained from
pure cultures as described by Akinsanmi et al. (2017).
On PDA, the fungal colonies of Type 1 spot were
white with an irregular edge, producing a wavy surface
and black pycnidial masses (Fig. 1d). The reverse side
was pale yellow with numerous black pycnidia (Fig.
1e). Colonies reached 80 mm diameter after 7 days.
Whitish mycelia produced black smooth globular
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acervuli with slimy spore masses. Conidia were fusi-
form to clavate with 5-celled segmentation with darker
median cells and hyaline end cells and measured 23.2–
28.4 × 5.9–7.8 μm (n = 50) (Fig. 1f). Cultural and mor-
phological characteristics of these isolates matched the
genus description for Neopestalotiopsis clavispora
(Maharachchikumbura et al. 2014).
The cultures of Type 2 spot showed greyish aerial
mycelium with visible orange conidial masses (Fig. 2d)
and the reverse was dark greyish on PDA (Fig. 2e).
Conidia were single celled, hyaline, aseptate, fusiform,
and measured 10.8–15.1 × 3.7–5.9 μm (n = 50) (Fig.
2f). These morphological characteristics were consistent
with Colletotrichum siamense (Prihastuti et al. 2009).
Representative isolates were deposited in the Queens-
land Plant Pathology Herbarium (BRIP), Ecosciences
Precinct, Dutton Park, Australia (Table 1).
Genomic DNA was extracted from approximately
40 mg mycelium of each isolate grown on PDA plates.
The mycelium was homogenised using TissueLyser
(Qiagen Pty. Ltd.) for 2 min at 30 Hz, before following
the DNA extraction procedure of the BioSprint 96 DNA
Plant Kit using robotic platform (Qiagen Pty. Ltd.).
DNA concentrations were determined using a BioDrop
Duo spectrophotometer (BioDrop, Cambridge, En-
gland) and the working stock concentration was adjust-
ed to 10 ng/μl. The purified genomic DNA of the
isolates of Type 1 leaf spot served as the template for
the PCR amplifications of the internal transcribed spacer
region (ITS) using ITS4 as reverse primer and ITS5 as
forward primer (White et al. 1990), which amplify the
ITS-1, ITS-2 and the 5.8S ribosomal RNA gene regions,
and partial sequences of translation elongation factor
1-α (TEF) gene with EF1-688F and EF1-1251R primers
(Alves et al. 2008) and partial β-tubulin 2 (TUB) gene
region with primer pairs BT2A and BT2B (Glass and
Donaldson 1995). For the isolates of Type 2 leaf spot,
the PCR amplifications were performed for the ITS and
TUB gene regions, and amplification of the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
gene region with GDF and GDR primers (Templeton
et al. 1992) and partial actin (ACT) gene region with
primer pairs ACT-512F and ACT-783R (Carbone and
Kohn 1999). Each reaction was performed in a 25 μl
reaction mixture containing 5 μl of 5× reaction buffer
(Bioline, Australia), 1.5 μl of 25 mM MgCl2, 0.5 μl of
10 mM dNTPs, 1 μl each of 10 μM forward and reverse
primers, 0.125 μl of MangoTaq DNA polymerase (5 U/
μl; Bioline, Australia), 13.875 μl of nuclease free water
Eur J Plant Pathol
Fig. 1 Pestalotiopsis leaf spots of macadamia. a symptoms on
trees in a commercial orchard in Queensland, Australia, b close up
of characteristic symptoms, c symptoms on inoculated leaves in
and 2 μl of DNA template. PCR amplification was
performed in SuperCycler Thermal Cycler (Kyratec,
Australia) programmed for initial denaturation at 95 °C
for 2 min, followed by 35 cycles consisting of denatur-
ation at 95 °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 5 min. PCR amplicons were separated in 1%
(w/v) agarose gel (Bioline, Australia) in 0.5× TBE,
stained with GelRed (Biotium) and viewed under UV
light using Molecular Imager® Gel Doc™ (Bio-Rad
Laboratories Inc., Segrate, Milan, Italy). The amplicon
sizes were determined against a 1 kb HyperLadder
(Bioline, Australia) and then the targeted PCR
amplicons were purified using QIAquick PCR Purifica-
tion Kit (Qiagen Pty. Ltd.) according to the manufac-
turer’s instructions before DNA sequencing with 3730xl
DNA analyzer at Macrogen Inc. (South Korea) using the
same primers used for amplifications.
the pathogenicity test, d–f N. clavispora BRIP 70210 isolate
colony on PDA, reverse side on PDA and conidia, respectively.
Scale bar = 10 μm
Molecular Evolutionary Genetics Analysis (MEGA
X) software (Kumar et al. 2018) was used to assemble
manually the forward and reverse sequences into con-
sensus fragments. In order to provide consistency and
quality of the sequences, the chromatograms of the
sequences were checked and aligned using the MUltiple
Sequence Comparison by Log-Expectation (MUSCLE)
algorithm, and primer sequences were trimmed off at
both ends of the sequences. For each set of nucleotide
sequences, the identity of each isolate was determined
by comparison against ex-type cultures available in the
National Center for Biotechnology Information (NCBI)
GenBank database using Basic Local Alignment Search
Tool (BLAST) procedure. BLASTn analyses for each of
the ITS, TUB and TEF sequences of the isolates of Type
1 spot showed 99–100% similarity with N. clavispora
ex-type (CBS 447.73) in GenBank, while each of the
gene sequences of ITS, TUB, GAPDH and ACT of the

Fig. 2 Colletotrichum leaf spots of macadamia. a symptoms on
trees in a commercial orchard in Queensland, Australia, b close up
of characteristic symptoms, c symptoms on inoculated leaves in
isolates of Type 2 spot was 100% homologous with
C. siamense ex-type (CBS 124964) in GenBank
(Table 1). The nuclear gene sequences of the isolates
of Type 1 and 2 spots were deposited in GenBank
(Table 1). Phylogenetic analyses of the selected isolates
were performed using the maximum likelihood (ML)
method in MEGA X with the concatenated sequences of
the three gene loci (ITS + TUB + TEF) of the isolate of
Type 1 spot and four gene loci (ITS + TUB + GAPDH +
ACT) of the isolate of Type 2 spot. Sequences of the
reference strains were obtained from GenBank. The ML
phylogram was constructed in MEGA X using the Gen-
eral Time Reversible model (Nei and Kumar 2000). Tree
stability was tested by 1000 bootstrap replicates. The
results of the ML analyses of the combined sequence
data of the isolates of macadamia leaf spots together
with reference specimens showed that the isolate (BRIP
70210) of Type 1 spot shared a most recent common
ancestor with N. clavispora (CBS 447.73) and the
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the pathogenicity test, d–f C. siamense BRIP 70205 isolate colony
on PDA, reverse side on PDA and conidia, respectively. Scale
bar = 10 μm
isolate (BRIP 70205) of Type 2 spot shared a most
recent common ancestor with C. siamense (CBS
124964) (Supplementary Fig. 1 and Fig. 2).
To conduct a pathogenicity test, conidial suspension
(106 conidia/ml) of each isolate was prepared in sterile
distilled water, and sprayed until run-off onto 10 healthy
macadamia leaves of 2-year old potted plants. Control
plants were sprayed with sterile distilled water. The
inoculated plants were covered with plastic bags over-
night to maintain high relative humidity, and thereafter
remained uncovered on a bench (with the control plants)
in a shade house at mean temperature of 28 °C. The
pathogenicity tests were repeated three times. Leaf spots
identical to the respective field disease symptoms were
observed on the inoculated leaves for both Type 1 spots
(Fig. 1c) and Type 2 spots (Fig. 2c) at 7 days post
inoculation, whereas no visible symptoms appeared on
the control leaves. Koch’s postulates were fulfilled by
re-isolation and microscopic determination for the
Eur J Plant Pathol

Table 1 GenBank and culture accession numbers of Neopestalotiopsis and Colletotrichum isolates obtained from macadamia leaf spots
collected from Queensland in Australia, and of ex-type cultures of Neopestalotiopsis clavispora and Colletotrichum siamense

Species Culture accession numberb GenBank accession numbera

ITS TUB TEF GAPDH ACT

Neopestalotiopsis clavispora BRIP 70207 MN114209 MN114211 MN114210 – –

N. clavispora BRIP 70208 MN519189 MN519191 MN519190 – –
N. clavispora BRIP 70209 MN519192 MN519194 MN519193 – –
N. clavispora BRIP 70210c MN114212 MN114214 MN114213 – –
N. clavispora BRIP 70211 MN241468 MN241470 MN241469 – –
N. clavispora CBS 447.73d KM199374 KM199443 KM199539 – –
Colletotrichum siamense BRIP 70205c MN241480 MN241484 – MN241476 MN241472
C. siamense BRIP 70206 MN241482 MN241486 – MN241478 MN241474
C. siamense BRIP 70534 MN241479 MN241483 – MN241475 MN241471
C. siamense BRIP 70535 MN241481 MN241485 – MN241477 MN241473
C. siamense BRIP 70536 MN519187 MN519188 – MN519186 MN519185
C. siamense CBS 124964d KC566812 KC566234 – KC566666 KC566958
a
Abbreviations: internal transcribed spacer (ITS), β -tubulin 2 (TUB), translation elongation factor 1-α (TEF), glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and actin (ACT)
b
BRIP: Queensland Plant Pathology Herbarium, Queensland, Australia; CBS: Centraalbureau voor Schimmelcultures, Fungal Biodiversity
Centre, Utrecht, The Netherlands
c
Isolates used in pathogenicity assays
d
Ex-type cultures in GenBank homologous to study isolates

Neopestalotiopsis and Colletotrichum species. Hence,
the two leaf spots were distinguished as Pestalotiopsis
leaf spot (Type 1 spot) and Colletotrichum leaf spot
(Type 2 spot). To our knowledge, this is the first report
of N. clavispora and C. siamense causing leaf spots in
macadamia in Australia.
Due to the impact of leaf spot-pathosystems on
productivity and yield, their influence on photosyn-
thesis have been examined for a number of agricul-
tural crop species (Bergamin Filho et al. 1997;
Bourgeois and Boote 1992; Niederleitner and
Knoppik 1997). In general, decreases in leaf photo-
synthesis appear to result from a disruption of the
photosynthetic apparatus by the invading pathogens
causing leaf spots (Erickson et al. 2004). In potato,
decline in photosynthesis was attributed to stomatal
closure following pathogen infection (Bowden et al.
1990). Metabolic disruption of photosynthetic pro-
cesses and consumption of photosynthetic assimi-
lates by pathogens resulted from leaf spot diseases
(Boote et al. 1983; Farquhar and Sharkey 1982).
Neopestalotiopsis and Pestalotiopsis are relatively
important plant pathogenic genera with wide host
ranges (Maharachchikumbura et al. 2011). Several
Neopestalotiopsis spp. have been reported to cause leaf
spots in tree nut and fruit crops, including sweet almond
(Ayoubi and Soleimani 2016), macadamia (Santos et al.
2019), strawberry (Mahapatra et al. 2018) and grapevine
(Jayawardena et al. 2016). In macadamia, species in the
genus Pestalotiopsis are known to cause flower diseases
(Akinsanmi et al. 2017). Generally the pathogenic abil-
ity of these fungi depends upon their ability to produce
phytotoxins, pestalopyrones, hydroxypestalopyrones
and pestalosides (Maharachchikumbura et al. 2011).
Colletotrichum is the eighth most important phyto-
pathogenic fungal genus in the world (Dean et al. 2012),
affecting high-value fruit crops such as strawberry, man-
go, citrus, avocado and banana (Cannon et al. 2012).
Many species of Colletotrichum have been isolated as
causal agents of leaf spots from various hosts, including
cashew (Prabhakaran Nair 2010), Chinese bean tree (Fu
et al. 2013), apple (Velho et al. 2019), edible lily (Zhao
et al. 2016) and turmeric (Potdar 2006). Specifically,
C. siamense is common on tropical fruits (Sharma et al.
2014) and has recently been reported as the causal agent
of leaf spots of areca palm (Chou et al. 2019) and redbud
(Ji et al. 2019) in China. C. siamense displays a
hemibiotrophic lifestyle with intra cellular

hemibiotrophy with predominant necrotrophic life cycle
(De Silva et al. 2017). Thus, primary infection vesicles
are formed during initial infection of the epidermal cells
without killing the cells. This is followed by a
necrotrophic stage in which secondary infection hyphae
invade and kill adjacent cells (Perfect et al. 1999).
During the necrotrophic phase, they typically secrete
lytic enzymes to degrade plant components or toxins
that kill the plant tissues, resulting in development of
lesions (Gan et al. 2013). In macadamia, the causal
agent of anthracnose husk rot is often attributed to
C. gloeosporioides sensu lato (Akinsanmi and Drenth
2017). However, there is no information on whether
C. siamense infects macadamia husk. Further, the
aetiology of Colletotrichum leaf spot should be exam-
ined to determine the significance, prevalence and fac-
tors that influence this leaf spot in macadamia in
Australia.
In macadamia, dry flower disease of macadamia
is caused by Pestalotiopsis macadamiae and
Neopestalotiopsis macadamiae fungal pathogens
(Akinsanmi et al. 2017). However, it is unknown
whether the leaf spot pathogen, N. clavispora is also
associated with dry flower of macadamia or infects
the kernel. Therefore, further studies are required to
determine the contribution of Pestalotiopsis leaf spot
to dry flower epidemiology and kernel infection in
macadamia.
Acknowledgements K. Prasannath is a recipient of the Univer-
sity of Queensland Research Training Scholarship. The authors
thank macadamia growers who kindly allowed to collect leaf
samples for this study.
Funding This work was supported by grant from the Hort
Innovation using the Macadamia levy Fund, and Commonwealth
government for Project MC 16018.
Compliance with ethical standards
This research article is not submitted elsewhere for publication and
complies with the Ethical Rules applicable for this journal.
Conflict of interest The authors declare that they have no con-
flict of interest.
Animal studies and human participants This article does not
contain any studies with human participants or animals performed
by any of the authors.
Informed consent All authors consent to this submission.
Eur J Plant Pathol
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