Fungal Diversity
DOI 10.1007/s13225-013-0235-8
Phyllosticta capitalensis, a widespread endophyte of plants
Saowanee Wikee & Lorenzo Lombard & Pedro W. Crous &
Chiharu Nakashima & Keiichi Motohashi & Ekachai Chukeatirote &
Siti A. Alias & Eric H. C. McKenzie & Kevin D. Hyde
Received: 21 February 2013 / Accepted: 9 April 2013
# Mushroom Research Foundation 2013
Abstract Phyllosticta capitalensis is an endophyte and weak
plant pathogen with a worldwide distribution presently known
from 70 plant families. This study isolated P. capitalensis from
different host plants in northern Thailand, and determined their
different life modes. Thirty strains of P. capitalensis were isolated
as endophytes from 20 hosts. An additional 30 strains of P.
capitalensis from other hosts and geographic locations were also
obtained from established culture collections. Phylogenetic analysis using ITS, ACT and TEF gene data confirmed the identity of
all isolates. Pathogenicity tests with five strains of P. capitalensis
originating from different hosts were completed on their respective host plants. In all cases there was no infection of healthy
leaves, indicating that this endophyte does not cause disease on
healthy, unstressed host plants. That P. capitalensis is often
isolated as an endophyte has important implications in fungal
biology and plant health. Due to its endophytic nature, P.
capitalensis is commonly found associated with lesions of plants,
and often incorrectly identified as a species of quarantine importance, which again has implications for trade in agricultural and
forestry production.
S. Wikee : E. Chukeatirote : K. D. Hyde
School of Science, Mae Fah Luang University,
Chiangrai 57100, Thailand
C. Nakashima
Graduate School of Bioresources, Mie University,
Kurima-machiya 1577,
Tsu, Mie 514-8507, Japan
S. Wikee (*) : E. Chukeatirote : K. D. Hyde
Institute of Excellence in Fungal Research,
Mae Fah Luang University, Chiangrai 57100, Thailand
e-mail: wikeemammaam@gmail.com
L. Lombard : P. W. Crous
CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8,
3584 CT Utrecht, The Netherlands
P. W. Crous
Microbiology, Department of Biology, Utrecht University,
Padualaan 8, 3584 CH Utrecht, The Netherlands
P. W. Crous
Laboratory of Phytopathology, Wageningen University and
Research Centre (WUR), Droevendaalsesteeg 1,
6708 PB Wageningen, The Netherlands
Keywords Guignardia . Leaf spot . Morphology .
Molecular phylogeny . Quarantine
Introduction
Species in the genus Phyllosticta are mostly plant pathogens
of a wide range of hosts and are responsible for diseases
including leaf spots and black spots on fruits (Wulandari et
al. 2009; Glienke et al. 2011; Wang et al. 2012). There are
about 3,200 names listed for the genus Phyllosticta in Index
K. Motohashi
Electron Microscope Center, Tokyo University of Agriculture,
Sakuraoka 1-1-1, Setagaya,
Tokyo 156-8502, Japan
S. A. Alias
Institute Ocean and Earth Sciences, Institute for Postgraduate
Studies, University Malaya, Kuala Lumpur 50603, Malaysia
E. H. C. McKenzie
Landcare Research, Private Bag 92170, Auckland Mail Centre,
Auckland 1142, New Zealand
Fungal Diversity
Fungorum (http://www.indexfungorum.org/; accessed February
2013) and 3,340 names in MycoBank (http://
www.mycobank.org/; accessed February 2013). The USDA
Fungal Database lists 78 Phyllosticta records associated with
plant hosts (http://nt.ars-grin.gov/fungaldatabases/; accessed
February 2013).
Phyllosticta species may be associated with a
“Guignardia-like” sexual state (van der Aa 1973; Wikee et
al. 2011). For example, the sexual state of Phyllosticta
ampelicida (Engelm.) Aa, the black rot pathogen of grapevine is Guignardia bidwellii (Ellis) Viala & Ravaz (van der
Aa 1973; Ullrich et al. 2009). Leaf spots on Morinda
citrifolia (Rubiaceae) commonly have both ascomata and
pycnidia of P. morindae (Petr. & Syd.) Aa (Wulandari et al.
2010a, b). Likewise, both ascomata and pycnidia of P.
maculata M.H. Wong & Crous are present on banana leaves
with freckle disease (Wong et al. 2012).
Guignardia citricarpa Kiely (synonym of P. citricarpa
(McAlpine) Aa), which causes black spot of citrus (e.g. oranges), is of quarantine concern in Europe (Baayen et al. 2002;
Agostini et al. 2006), but P. citriasiana Wulandari, Crous &
Gruyter, which causes brown spot of pomelo fruit (Citrus
maxima) is not of quarantine concern as this fruit is not grown
in Europe (Wulandari et al. 2009). A few species have also been
reported as endophytes and saprobes (Van Der Aa et al. 2002;
Baayen et al. 2002; Glienke et al. 2011). Phyllosticta maculata
the cause of banana leaf freckle has also been isolated as an
endophyte from healthy grapevine leaves (Kuo and Hoch
1996). Phyllosticta capitalensis Henn. is commonly isolated
as an endophyte and is a widespread species (Glienke-Blanco et
al. 2002; Silva and Pereira 2007; Silva et al. 2008).
Phyllosticta capitalensis was described by Hennings
(1908) who found it associated with necrotic leaves of
Stanhopea sp. (Orchidaceae) collected in Brazil. The supposed sexual morph, G. mangiferae A.J. Roy was later described from Mangifera indica L. (Anacardiaceae) in India
(Roy 1968). However, there has been confusion with the
identification and naming of the P. capitalensis sexual morph.
Okane et al. (2003) stated that the teleomorph of P. capitalensis
differed morphologically from G. mangiferae and that it was, in
fact, G. endophyllicola Okane, Nakagiri & Tad. Ito. The latter
fungus was described as a pathogen of several ericaceous plants
by Okane et al. (2001). In the past there has also been confusion
between G. endophyllicola and G. citricarpa. Both sexual
names have been used for this fungus, for example, G.
endophyllicola (Okane et al. 2003; Pandey et al. 2003) and
G. mangiferae (Baayen et al. 2002; Glienke-Blanco et al. 2002;
Guo et al. 2003; Suryanarayanan et al. 2004; Devarajan and
Suryanarayanan 2006; Shaw et al. 2006). However, G.
citricarpa is a distinct species and the cause of citrus black
spot (Paul et al. 2005; Baayen et al. 2002).
Fungal endophytes colonise healthy plant host tissues but
may become pathogenic when the plant host is stressed
through environmental or biological factors (Petrini 1991;
Hyde and Soytong 2008; Purahong and Hyde 2011) that induce
the fungus to change from one life mode to another (Fisher and
Petrini 1992). As with Phyllosticta, some species of other common genera such as Bipolaris, Cladosporium, Colletotrichum,
Curvularia, Diaporthe, Fusarium, Pestalotiopsis, Phoma and
Verticillium have been isolated as endophytes (Photita et al.
2001, 2004; Anderson et al. 2011; Bensch et al. 2012; Damm
et al. 2012a, b; de Gruyter et al. 2013; Lima et al. 2012;
Orlandelli et al. 2012), and some of these are also serious
pathogens (Photita et al. 2004; Slippers and Wingfield 2007).
The present study provides an overview of the distribution and host range of P. capitalensis worldwide, through
the application of multi-gene phylogeny to illustrate its
widespread nature. Generally, Phyllosticta species are considered plant pathogens but it is still unclear whether they
are generalists or host-specific. The distinction between a
pathogen and a latent pathogen with endophytic nature is
also unclear. In this study we isolated Phyllosticta species
from northern Thailand, both as endophytes and as pathogens associated with leaf spots of various hosts. We also
obtained a range of geographically diverse isolates of P.
capitalensis from the CBS-KNAW Fungal Biodiversity
Centre. All isolates were sequenced compared with sequences downloaded from GenBank.
Material and methods
Isolates
Thirty strains of Phyllosticta were isolated from leaf
spots or as endophytes from healthy leaves of ornamental plants (Table 1). If pycnidia were present on diseased tissue then a single spore isolation procedure as
described by Chomnunti et al. (2011) was used to
obtain cultures. To obtain isolates of Phyllosticta from
diseased leaves of host plants when fruit bodies were
not present, the leaf was surface disinfected by wiping
with 70 % ethanol. Small pieces of leaf were then cut
from the interface between healthy and diseased tissue.
These were surface sterilised in 70 % ethanol, and
plated onto ½ strength potato dextrose agar (½PDA;
Crous et al. 2009). For isolation of endophytes, healthy
leaves were washed in tap water and surface disinfected
with 70 % ethanol. They were then cut into small
pieces (about 1×1 cm), suspended in 70 % ethanol (3
times for 15 min each) and washed in distilled water (3
times) before placing on ½PDA. All dishes were incubated at 27 °C for 1 week and observed daily. The
growing tips of hyphae of Phyllosticta colonies that
developed were cut out and transferred to fresh PDA
dishes. Isolates are deposited in Mae Fah Luang
Fungal Diversity
Table 1 Isolates of Guignardia and Phyllosticta used in the phylogenetic study
Strain
G. bidwellii
G. mangiferae
P. brazilianiae
P. brazilianiae
P. brazilianiae (ex-type)
Code1
Host
Mode*
Country
Gene and GenBank No.
ITS
TEF1
ACT
CBS 111645
IMI 260576
LGMF 333
LGMF 334
LGMF 330
CBS 126270
CPC 20251
CPC 20252
CPC 20254
CPC 20255
CPC 20256
CPC 20257
CPC 20258
CPC 20259
CPC 20263
CPC 20266
CPC 20268
Parthenocissus quinquefolia
Mangifera indica
Mangifera indica
Mangifera indica
Mangifera indica
P
E
E
E
E
USA
India
Brazil
Brazil
Brazil
JN692542
JF261459
JF343574
JF343566
JF343572
EU683653
JF261501
JF343595
JF343587
JF343593
JN692518
JF343641
JF343658
JF343650
JF343656
wild plant
Punica granatum
Saccharum officinarum
Arecaceae
Ophiopogon japonicus
Ficus benjamina
Ophiopogon japonicus
Orchidaceae
Magnoliaceae
Polyscias sp.
Hibiscus syriacus
P
P
E
P
P
P
P
P
E
E
E
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
KC291333
KC291334
KC291335
KC291336
KC291337
KC291338
KC291339
KC291340
KC291341
KC291342
KC291343
KC342553
KC342554
KC342555
KC342556
KC342557
KC342558
KC342559
KC342560
KC342561
KC342562
KC342563
KC342530
KC342531
KC342532
KC342533
KC342534
KC342535
KC342536
KC342537
KC342538
KC342539
KC342540
CPC 20269
CPC 20270
CPC 20272
CPC 20275
CPC 20278
CPC 20423
LC 0002
LC 0006
Ophiopogon japonicus
Tectona grandis
Orchidaceae
Polyalthia longifolia
Euphorbia milii
Philodendron ‘Xanadu’
Alocasia sp.
Dieffenbachia sp.
E
E
P
E
E
P
E
E
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
KC291344
KC291345
KC291346
KC291347
KC291348
KC291349
KC291350
KC291351
KC342564
KC342565
KC342566
KC342567
KC342568
KC342569
KC342570
KC342571
KC342541
KC342542
KC342543
KC342544
KC342545
KC342546
KC342547
KC342548
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
LC 0008
LC 0009
LC 0010
LC 0025
CBS 100175
CBS 114751
CBS 115046
CBS 115047
CBS 115049
CBS 123373
Anthurium sp.
Sansevieria hyacinthoides
Tinospora craspa
Calophyllum sp.
Citrus sp.
Vaccinium sp.
Myracrodruon urundeuva
Aspidosperma polyneuron
Bowdichia nitida
Musa paradisiaca
E
E
E
E
E
P
E
E
E
E
Thailand
Thailand
Thailand
Thailand
Brazil
New Zealand
Brazil
Brazil
Brazil
Thailand
KC291352
KC291353
KC291354
KC291355
FJ538320
EU167584
FJ538322
FJ538323
FJ538324
FJ538341
KC342572
KC342573
KC342574
KC342575
FJ538378
FJ538407
FJ538380
FJ538381
FJ538382
FJ538399
KC342549
KC342550
KC342551
KC342552
FJ538436
FJ538465
FJ538438
FJ538439
FJ538440
FJ538457
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis (ex-epitype)
P. citriasiana (ex-type)
P. citriasiana
CBS 123404
CBS 226.77
LGMF 03
LGMF 181
LGMF 219
LGMF 240
LGMF 222
LGMF 220
LGMF 358
CPC18848
CBS 120486
CBS 123370
Musa paradisiaca
Paphiopedilum callosum
Citrus lalifolia
Citrus reticulata
Citrus sinensis
Citrus sinensis
Citrus sinensis
Citrus sinensis
Mangifera indica
Stanhopea graveolens
Citrus maxima
Citrus maxima
E
P
P
P
E
E
E
E
E
P
P
P
Thailand
Germany
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Thailand
Vietnam
FJ538333
FJ538336
JF261452
JF261447
JF261448
JF261443
JF261450
JF261446
JF261449
JF261465
FJ538360
FJ538355
FJ538391
FJ538394
JF261494
JF261489
JF261490
JF261485
JF261492
JF261488
JF261491
JF261507
FJ538418
FJ538413
FJ538449
FJ538452
JF343634
JF343629
JF343630
JF343625
JF343632
JF343628
JF343631
JF343647
FJ538476
FJ538471
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
P. capitalensis
Fungal Diversity
Table 1 (continued)
Strain
Code1
Host
Mode*
Country
Gene and GenBank No.
ITS
TEF1
ACT
P. citriasiana
P. citriasiana
P. citribraziliensis (ex-type)
P. citribraziliensis
P. citricarpa
P. citricarpa
P. citricarpa (ex-epitype)
P. citricarpa
P. citricarpa
CBS 123371
CBS 123372
CBS 100098
LGMF09
CBS 102374
CBS 120489
CBS 127454
CBS 127452
CBS 127455
Citrus
Citrus
Citrus
Citrus
Citrus
Citrus
Citrus
Citrus
Citrus
maxima
maxima
sp.
sp.
aurantium
sinensis
limon
reticulata
sinensis
P
P
H
H
P
P
P
P
P
Vietnam
Vietnam
Brazil
Brazil
Brazil
Zimbabwe
Australia
Australia
Australia
FJ538356
FJ538357
FJ538352
JF261436
FJ538313
FJ538315
JF343583
JF343581
JF343584
FJ538414
FJ538415
FJ538410
JF261478
GU349053
FJ538373
JF343604
JF343602
JF343605
FJ538472
FJ538473
FJ538468
JF343618
FJ538429
FJ538431
JF343667
JF343665
JF343668
P. citrichinaensis
P. citrichinaensis
P. citrichinaensis
P. citrichinaensis
P. kerriae (ex-holotype)
P. hypoglossi
P. hypoglossi
P. hypoglossi
P. owaniana
P. owaniana
P. spinarum
P. podocarpi
ZJUCC 200956
ZJUCC 200964
ZJUCC 2010150
ZJUCC 2010152
MUCC 17
CBS 101.72
CBS 434.92
CBS 167.85
CBS 776.97
CPC 14901
CBS 292.90
CBS 111646
Citrus reticulata
Citrus maxima
Citrus maxima
Citrus sinensis
Kerria japonica
Ruscus aculeatus
Ruscus aculeatus
Ruscus hypoglossum
Brabejum stellatifolium
Brabejum stellatifolium
Chamaecyparis pisifera
Podocarpus falcatus
P
P
P
P
P
P
P
P
P
P
P
P
China
China
China
China
Japan
Italy
Italy
Italy
South Africa
South Africa
France
South Africa
JN791664
JN791662
JN791620
JN791611
AB454266
FJ538365
FJ538367
FJ538366
FJ538368
JF261462
JF343585
AF312013
JN791515
JN791514
JN791459
JN791461
KC342576
FJ538423
FJ538425
FJ538424
FJ538426
JF261504
JF343606
KC357671
JN791589
JN791582
JN791533
JN791535
AB704209
FJ538481
FJ538483
FJ538482
FJ538484
JF343644
JF343669
KC357670
1
CBS: CBS-KNAW Fungal Biodiverstiy Centre, Utrecht, The Netherlands; CPC: working collection of Pedro Crous housed at CBS; IMI:
International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, LC: culture collection of Nilam F. Wulandari, Chiangmai, Thailand.
LGMF: culture collection of Laboratory of Genetics of Microorganisms, Federal University of Parana, Curitiba, Brazil, ZJUCC: Zhejiang
University Culture Collection, Zhejiang, China
*P pathogen, E endophyte
University Culture Collection (MFLUCC) and in the
working collection of Pedro Crous (CPC) housed at
the CBS-KNAW Fungal Biodiversity Centre (CBS),
Utrecht, The Netherlands (CBS). Other fungal isolates
of representative Phyllosticta spp. were obtained from
the CBS (Table 1).
Morphology
Growth rates, cultural characteristics and morphology of the
isolates were determined on culture media prepared
according to Crous et al. (2009). All isolates were grown
at 27 °C. To induce sporulation, isolates were grown on pine
needle agar (PNA) and synthetic nutrient-poor agar (SNA),
and incubated under near UV-light. Colony colour and
growth rate were established on PDA, malt extract agar
(MEA) and oatmeal agar (OA). Culture characteristics were
assessed, and the colour of upper and lower surface of
cultures was recorded after 14 days in the dark at 27 °C.
Colony colour on MEA, OA and PDA were determined
using the colour charts of Rayner (1970).
Molecular phylogeny
DNA extraction, amplification, and sequencing
Strains were grown on MEA at room temperature for 2–
3 days, after which the mycelium was harvested. DNA was
isolated using Ultraclean™ Microbial DNA kit (Mo Bio,
Calsbad, CA, USA) following the manufacturer’s protocol.
Transcribed spacer-polymerase chain reaction (ITS-PCR)
was performed with primers V9G (De Hoog and Gerrits
van den Ende 1998) and ITS4 as described by White et al.
(1990). Part of elongation factor 1-α gene (TEF) was amplified with forward primer EF1 and reverse primer EF2.
The primers ACT-512F and ACT-783R were used to amplify part of the actin gene (ACT). Cycle sequencing of PCR
products were performed in PCR condition. PCR products
were separated by gel electrophoresis at 130 V for 20 min in
1 % agarose gel in 1× TAE running buffer and visualized
under UV light using a GeneGenius Gel Documentation and
Analysis System (Syngene, Cambridge, UK). Purified PCR
products were sequenced using both PCR primers with a
Fungal Diversity
BigDye Terminator Cycle Sequencing Kit v3.1 (Applied
Biosystems, Foster City, CA, USA) containing AmpliTag
DNA Polymerase. The amplified products were analyzed on
an automatic DNA sequencer (Perkin-Elmer, Norwalk, CN)
and aligned using MEGA v5 software. Phylogenetic analyzing was executed by Phylogenetic analyses using parsimony; PAUP v4.0b10 (Swofford 2003) and MrBayes v. 3.0b4
(Huelsenbeck and Ronquist 2001) for Bayesian analyses.
Guignardia bidwellii was chosen as outgroup for the phylogenetic tree. Representative sequences were deposited in
GenBank.
Pathogenicity testing
Attached, young healthy leaves of five plant species
(Cordyline fruticosa, Dendrobium lindleyi, Ficus sp.,
Ophiopogon japonica, Punica granatum) were washed
with distilled water, wiped with 70 % ethanol and dried
with sterile tissue paper. To complete the Koch’s postulated the inoculation methods followed Than et al.
(2008). Two to five leaves of each plant were wounded
with a total of ten wounds. The leaves were wounded
by pricking them with a pin. Both wounded and
unwounded leaves were inoculated with plugs (0.7 mm
diam) taken from the edge of 14 day-old colonies of
test fungi growing on PDA; sterile agar plugs served as
a control. All leaves were kept individually in moist
chambers for 1 week and observed for symptom expression every other day. After 7 days, if positive, the
fungus was reisolated from any tissue showing lesions
and this isolate was considered to be pathogenic; absence of symptoms on leaves classified the isolate as
non-pathogenic.
Results
Collection of Phyllosticta species
Thirty strains of Phyllosticta capitalensis were isolated from
20 host plants growing in the north of Thailand (Table 1, see
also Fig. 1). No other species of Phyllosticta were isolated.
Morphological description of Phyllosticta capitalensis
(Fig. 2)
On Punica granatum Pycnidia epiphyllous, globose, brown
or black, 120–125 μm high, 135–140 μm wide, wall 12–
15 μm thick. Conidiogenous cells lining wall of pycnidium,
phialidic, cylindrical, hyaline, 2–2.2×2.2–3 μm. Conidia
ellipsoidal, hyaline, 1-celled, smooth-walled, 8–11 × 5–
6 μm, surrounded by a mucilaginous sheath, bearing a
single apical appendage, usually 5–8 μm long.
In culture On SNA, ascomata forming on and under media
in 3 days, black, globose, 69–74×104–119 μm (x ¼ 73 2
109 5; n=10), wall composed of a single layer, 7–9 μm
thick (x ¼ 8 1; n=10), brown. Asci bitunicate, containing
6–8 ascospores, irregularly biseriate, clavate, 36–80×7–
15 μm (x ¼ 51 1 11 2, n=10). Ascospores ellipsoid
to broadly fusoid, widest in the middle, hyaline, smooth,
thin-walled, 12–22×5–10 μm (x ¼ 16 2 7 1, n=50),
1-celled, surrounded by mucilaginous sheath. On OA, colonies appear flat with an irregular margin, initially hyaline
with abundant mycelium, gradually becoming greenish after
3–4 days. On MEA, colonies appear woolly, flat, irregular,
initially white with abundant mycelium, gradually becoming
greenish to dark green after 2–3 days with white hyphae on the
undulate margin, eventually turning black; reverse dark green
to black. At 27 °C, in the dark, mycelium reached the edge of
the Petri-dish in 20 days with a growth rate of 0.4 cm per day.
On PDA, colonies appear woolly, initially white with abundant mycelium, gradually becoming greenish to dark green
after 2–3 days with white hyphae on the undulate margin,
eventually turning dark green to black; reverse black. After
10 days in the dark at 27 °C, mycelium reached the edge of the
Petri-dish with a growth rate of 0.9 cm per day.
Material examined All CPC collected by Saowanee Wikee
and LC by Nilam F. Wulandari, from June 2010 to
November 2011, Chiang Rai, Thailand. From leaf spots of
unknown wild plant CPC 20251; from leaf spots Punica
granatum, CPC 20252; from healthy leaf of Saccharum
officinarum CPC 20254; from leaf spots of Arecaceae
CPC 20255; from leaf spots of Ophiopogon japonica CPC
20256, CPC 20258 and CPC 20269; from leaf spots of
Ficus benjamina CPC 20257; from leaf spots of
Orchidaceae CPC 20259 and CPC 20272; from healthy leaf
of Magnoliaceae CPC 20263; from healthy leaf of
Codiaeum variegatum CPC 20265; from healthy leaf of
Polyscias sp. CPC 20266; from healthy leaf of Hibiscus
syriacus CPC 20268; from healthy leaf of Tectona grandis
CPC 20270; from healthy leaf of Poloalthia longifolia CPC
20275; from healthy leaf of Euphorbia milli CPC 20278;
from healthy leaf of Philodendron sp. CPC 20423; from
healthy leaf of Alocasia sp. LC 0002; from healthy leaf of
Dieffenbachia sp. LC 0006; from healthy leaf of Anthurium
sp. LC 0008; from healthy leaf of Sansevieria hyacinthodes
LC 0009; from healthy leaf of Tinospora craspa LC0010;
from healthy leaf of Calophyllum sp. LC 0025.
Phylogenetic analysis
Phylogenetic relationships among the Phyllosticta capitalensis
isolates from various hosts and locations were investigated in
this study using MP and Bayesian phylogenetic analyses. The
analysis of combined ITS, TEF and ACT genes of the
Fungal Diversity
Fig. 1 Leaf spot symptoms on living leaves of hosts and cultures
characteristic of Phyllosticta capitalensis on PDA (left), MEA (middle)
and OA (right). a Punica granatum (CPC20252; MFLUCC11-0053) b
Ficus sp. (CPC20257; MFLUCC11-0058) c. Ophiopogon japonica
(CPC20258; MFLUCC11-0059) d. Dendrobium lindleyi (CPC20259;
MFLUCC11-0064) e. Cordyline fruticosa (CPC20273; MFLUCC100135) f. Philodendron ‘Xanadu’ (CPC20423; MFLUCC 12–0232)
Phyllosticta strains newly sequenced in this study and 67
strains of Phyllosticta obtained from GenBank and Mei
University, Japan (Table 1) were aligned and used to construct
their phylogeny. The combined dataset of 64 strains (including
the out-group) consisted of 974 characters, of which 483
characters were constant, and 148 characters were variable
and parsimony-uninformative. Parsimony analysis generated
48 trees, of which the best one is shown in Fig. 3 (TL = 873,
CI = 0.804, RI = 0.963, RC = 0.774). In the parsimony tree
(Fig. 3) bootstrap values and Bayesian analysis of combined
data are given at the nodes.
In the phylogenetic tree 12 clades representing various
Phyllosticta species are evident. Guignardia bidwellii was
chosen as out-group. The representative strain of G.
mangiferae (IMI 260576) fell outside the P. capitalensis s.
str. clade. The isolates in the P. capitalensis s. str. clade were
from different hosts and different continents. Phyllosticta
brazilianiae was isolated from an orchid in Brazil; P.
citricarpa was isolated from Citrus sp. and P. citriasiana
was isolated from Citrus maxima, Vietnam; P. spinarum was
isolated from Chamaecyparis pisifera, France; P. kerriae was
isolated from Kerria japonica, Japan; P. citribraziliensis was
Fungal Diversity
Fig. 2 Phyllosticta capitalensis on Punica granatum (CPC 20252). a–c Leaf spots on host plant d–f. Vertical section through pycnidia showing
developing conidia g–k. Conidia (d, bar=20 μm, g–k bars=10 μm)
isolated from citrus, Brazil; P. hypoglossi was isolated from
Ruscus aculeatus, Italy; P. citrichinaensis was isolated from
Citrus maxima, China; P. podocarpi was isolated from
Podocarpus falcatus, South Africa and P. owaniana from
Brabejum stellatifolium, South Africa.
Pathogenicity testing with Phyllosticta capitalensis
The ability of Phyllosticta capitalensis strains isolated from
leaf spots of five hosts in Thailand to induce leaf spot symptoms on these host species was tested through inoculating
mycelium plugs onto attached wounded and unwounded living leaves. In all cases there was no infection of the young
healthy plant leaves.
Discussion
This study reviews previous data on Phyllosticta capitalensis
and provides additional data on host infection and distribution
in Thailand. Many factors such as environmental conditions,
host and non-host organisms, and plant defence mechanisms
(e.g. secondary metabolite, specific and non-specific protein
expression and hydrogen peroxide residue) play an important
role in response to microbial infection.
Phyllosticta capitalensis has been repeatedly isolated
worldwide from healthy plant tissues as an endophyte and
rarely from leaf spots as a pathogen, and has been recorded
from almost 70 plant families (Baayen et al. 2002; Okane et
al. 2003; Motohashi et al. 2009, Tables 1 and 2, Fig. 4, this
study). The fact that it is isolated so often as an endophyte
has important implications to studies of fungal biology
including plant pathology methodology, ecological results
of endophyte studies and screening for novel compounds
from endophytes.
Implications to plant pathology methodology
A standard protocol used for isolating plant pathogens
involves cutting segments from the leading edge of lesions, which are then surface sterilized and plated onto
media (Crous et al. 2009). The rationale is that the causative agent grows out from the lesions and can be isolated
as a pure culture. Testing can then be undertaken to
establish pathogenicity, while the colony can be identified
using morphology. This standard methodology (Koch’s
postulate) has been long used by plant pathologists to
determine the identity of non-sporulating pathogens ad
infinitum (Phoulivong et al. 2010; Thompson et al. 2010;
Wikee et al. 2011).
Fungal Diversity
Fig. 3 Phylogenetic tree generated from 1000 replicates bootstrap values parsimony analysis/Bayesian analysis based on combined ITS rDNA,
TEF1 and ACT sequence data. The tree is rooted with Guignardia bidwellii (CBS 111645)
Fungal Diversity
Table 2 Hosts and countries from which Phyllosticta capitalensis has been isolated, usually as an endophyte, rarely as a pathogen (P) (See also
Fig. 1)
Plant family
Plant genus
Country
Acanthaceae
Anacardiaceae
Mackaya
Anacardium
Comocladia
Loxostylis
Mangifera
South Africa
Brazil
Puerto Rico
South Africa
Brazil
Ghana
Brazil
South Africa
South Africa
Brazil
South Africa
Thailand
Brazil
Apocynaceae
Myracrodruon
Rhus
Sclerocarya
Spondias
Monanathotaxis
Polyalthia
Aspidosperma
Aquifoliaceae
Secamone
Cerbera
Nerium
Ilex
Annonaceae
Asparagaceae
Cerbera
Cussonia
Hedera
Polyscias
Schefflera
Polyscias
Alocasia
Anthurium
Dieffenbachia
Livistona
Spathiphyllum
Philodendron
Sansevieria
South Africa
Japan
Japan
USA
Japan
Japan
South Africa
South Africa
Puerto Rico
Costa Rica
Thailand
Thailand
Thailand
Thailand
Thailand
Japan
Thailand
Thailand
Boraginaceae
Calophyllaceae
Capparaceae
Chrysobalanaceae
Ophiopogon (P)*
Cordia
Calophyllum
Maerua
Parinari
Thailand
South Africa
Thailand
South Africa
South Africa
Combretum
Ipomoea
Curtisia
Davidia
Putterlickia
Cercidiphyllum
Diospyros
Euclea
Rhododendron
Enkianthus
Vaccinium
Bowdichia
South Africa
Malaysia
South Africa
Japan
South Africa
Japan
South Africa
South Africa
Japan
Japan
New Zealand
Brazil
Araliaceae
Araceae
Combretaceae
Convolvulaceae
Cornaceae (Nyssaceae)
Celastraceae
Cercidiphyllaceae
Ebenaceae
Ericaceae
Fabaceae
Reference
Glienke et al. 2011
Baayen et al. 2002
Glienke et al. 2011
Baayen et al. 2002
Present study
Glienke et al. 2011
Okane et al. 2003
Motohashi et al. 2009
Okane et al. 2003
Okane et al. 2003
Baayen et al. 2002
Present study
Present study
Present study
Present study
Present study
Motohashi et al. 2009
Present study
Present study
Present study
Present study
Present study
Baayen et al. 2002
Motohashi et al. 2009
Baayen et al. 2002
Motohashi et al. 2009
Okane et al. 2003
Okane et al. 2001
Glienke et al. 2011
Glienke et al. 2011
Fungal Diversity
Table 2 (continued)
Plant family
Fagaceae
Ginkgoaceae
Lamiaceae
Lauraceae
Lecythidaceae
Leguminosae
Loganiaceae
Lythraceae
Malvaceae
Meliaceae
Menispermaceae
Moraceae
Magnoliaceae
Menispermaceae
Euphorbiaceae
Plant genus
Country
Reference
Cercis
Lithocarpus
Ginkgo
Vitex
Cinnamomum
Ocotea
Barringtonia
Caesalpinia
Stychnos
Anthocleista
Punica (P)
Hibiscus
Japan
Japan
Japan
Malaysia
Japan
South Africa
South Africa
Japan
South Africa
South Africa
Thailand
Thailand
Motohashi et al. 2009
Motohashi et al. 2009
Motohashi et al. 2009
Present study
Okane et al. 2003
Ekebergia
Trichilia
Cocculus
Artocarpus
Ficus (P)
Morus
Michelia
Magnolia
South Africa
South Africa
USA
Thailand
Thailand
Thailand
Thailand
Thailand
USA
Thailand
South Africa
South Africa
Thailand
South Africa
Thailand
South Africa
USA
Thailand
Present study
Thailand
Indonesia, USA
Brazil, South Africa
Okane et al. 2003
Glienke et al. 2011
Glienke et al. 2011
Brazil
Japan
South Africa
USA
Japan
Thailand
Thailand
Germany
Malaysia
Brazil
Hawaii
Thailand
South Africa
Hawaii
Hawaii
Australia
Baayen et al. 2002
Motohashi et al. 2009
Flacourtiaceae
Iteaceae
Lamiaceae
Tinospora
Clutia
Croton
Codiaeum
Ctenomeria
Euphorbia
Dovyalis
Itea
Tectona
Musaceae
Musa
Myrtaceae
Eucalyptus
Psidium
Oleaceae
Ligustrum
Schrebera
Botrychium
Arundina
Coelogyne
Dendrobium
Paphiopedilium
Rhynchostylis sp.
Stanhopea
Pittosporum
Saccharum
Podocarpus
Leucospermum
Protea
Telopea
Ophioglossaceae
Orchidaceae
Orchidaceae
Pittosporaceae
Poaceae
Podocarpaceae
Proteaceae
Baayen et al. 2002
Okane et al. 2003
Present study
Present study
Baayen et al. 2002
Baayen et al. 2002
Present study
Present study
Glienke et al. 2011
Present study
Baayen et al. 2002
Present study
Present study
Okane et al. 2003
Present study
Okane et al. 2001
Williams & Liu 1976, Singh 1980
Glienke et al. 2011
Baayen et al. 2002
Present study
Fungal Diversity
Table 2 (continued)
Plant family
Plant genus
Country
Reference
Pittosporaceae
Pteridophta
Rhamanaceae
Pittosporum
Pteridophytes
Scutia
Zizyphus
Kandelia
Cliffortia
Rubus
Prunus
Eriobotrya
Canthium
Coprosma
Gardenia
Japan
Japan
South Africa
South Africa
Japan
South Africa
Japan
Japan
Japan
South Africa
Hawaii
South Africa
Motohashi et al. 2009
Okane et al. 2003
Pavetta
Rauvolfia
Rothmannia
Zanthoxylum
Citrus (P)
Smilacaceae
Solanaceae
Stangeriaceae
Fortunella
Vitex
Zanthoxylum
Allophylus
Dodonaea
Litchi
Nephelium
Paullinia cupana
Smilax
Capsicum
Stangeria
South Africa
South Africa
South Africa
Japan
Argentina, Australia, Brazil,
China, Hong Kong, New Zealand,
South Africa, Taiwan, Thailand, USA
USA
South Africa
Pueto Rico
South Africa
Hawaii
South Africa
USA
Brazil
South Africa
Dominican
South Africa
Sterculiaceae
Theaceae
Tiliaceae
Sterculia
Camellia
Grewia
Puerto Rico
USA
South Africa
Trimeniaceae
Ulmaceae
Veronicaceae
Viscaceae
Vitaceae
Xymalos
Trema
Hebe (Veronica)
Viscum
Ampelopsis
Cryphostemma
Rhoicissus
Encephalartos
Amomum
Zingiber
South Africa
South Africa
South Africa
South Africa
USA
South Africa
South Africa
South Africa
Thailand
Thailand
Rhizophoraceae
Rosaceae
Rubiaceae
Rutaceae
Sapindaceae
Zamiaceae
Zingiberaceae
Okane et al. 2003
Okane et al. 2003
Okane et al. 2003
Motohashi et al. 2009
Baayen et al. 2002
Okane et al. 2003
Glienke et al. 2011;
Wang et al. 2012
Baayen et al. 2002
Glienke et al. 2011
Baayen et al. 2002
Glienke et al. 2011
Glienke et al. 2011
Baayen et al. 2002
Baayen et al. 2002
Baayen et al. 2002
Okane et al. 2003
Okane et al. 2003
*(P) = Leaf spot
Recent studies on Phyllosticta causing freckle disease of
banana and disease of other hosts have shown that extreme
caution must be applied when using the above standard
plant pathology approach (Wong et al. 2012). Conidia of
Phyllosticta rarely germinate in culture and thus with many
species it is impossible to obtain single spore cultures
Fungal Diversity
Fig. 4 World distribution of Phyllosticta capitalensis (the dots represent countries)
(Chomnunti et al. 2011). If freckle infected banana tissues
are surface sterilized and plated on agar, P. capitalensis
invariably grows out and, therefore, is concluded to be the
pathogen, which is not the case. If these strains of P.
capitalensis are used in pathogenicity testing they may also
be weak pathogens and thus “substantiate” the record as the
causal agent. However, Wong et al. (2012) carefully dissected whole ascomata from freckle diseased banana tissues.
They then surface sterilized the ascomata and plated them
out to obtain “single ascomata cultures”. In this way they
were able to establish that freckle disease was caused by
more than one species of Phyllosticta and discerned the
causal agent of freckle in Queensland as P. cavendishii
M.H. Wong & Crous (Wong et al. 2012). Phyllosticta
citricarpa, which causes citrus black spot (CBS) is widespread in some citrus-producing countries but is absent from
EU and USA, where it is a regulated pathogen. CBS has
been often misdiagnosed on citrus fruit and many of the
lesions are, in fact, colonised by P. capitalensis. Traditional
methods of diagnosis are time consuming and involve incubation of infected material, morphological examination of
the fungus, and perhaps dissecting and plating of lesion
pieces. Misdiagnosis of CBS may result in significant financial loss to farmers and exporters. An acurate and less time
consuming method to verify and identify Phyllosticta species on citrus fruit is essential for both the producer and
regulatory authorities (Meyer et al. 2012).
Further careful research of this type in other banana
growing regions is likely to reveal other species causing
freckle disease. The above example serves to illustrate
how a Koch’s postulate can result in incorrect data
concerning the identity of causal agents of disease, particularly with Phyllosticta species. Besides banana disease we
suspect that many diseases caused by Phyllosticta (and
“Guignardia”), unless directly identified via sporulating
structures, e.g. Guignardia candeloflamma K.D. Hyde, on
a species of Pinanga in north Queensland, Australia and an
unidentified palm in Irian Jaya (Fröhlich and Hyde 1995),
may be wrongly attributed to P. capitalensis. Future studies
must take this problem of protocol into account. Whether
this phenomenon applies to other fungal genera needs future
investigation.
Endophyte study protocols
There are many definitions of an endophyte and these have
been summarized by Hyde and Soytong (2008). A standard
definition is “organisms that colonize plant organs in some
period of time in plant life cycle without causing obvious
harm on the host” (Petrini 1984; 1991). The standard methodology for isolating endophytes has been reviewed in
numerous instances (e.g. Guo et al. 1998, 2001; Photita et
al. 2004, 2005) and has been criticised for being biased
towards fast growing fungal strains (Hyde and Soytong
2007). However, in principle the method is the same as that
used by plant pathologists for isolating pathogens from
diseased tissue, albeit that endophyte researchers use
healthy leaves. The problem with the protocol mentioned
above concerning isolating P. capitalensis rather than the
Phyllosticta causal agent may also occur in endophyte
Fungal Diversity
studies. Phyllosticta capitalensis is a quick growing species;
in culture the colony covers a 9 cm Petri-dish in 10 days.
Other species grow more slowly, e.g. P. yuccae reaches 3–
5 cm diam in 15 days (Bissett 1986), while growth of P.
vaccinii can be as low as 0.4 mm/day. Four species of
Phyllosticta (P. citriasiana, P. capitalensis, P. citricarpa and
P. citrichinaensis) were recently isolated from Citrus in China
(Wang et al. 2012) and P. citrichinaensis grew at 3.8±
0.34 mm per day at 24 °C on PDA. Therefore, it is highly
likely that P. capitalensis will be isolated in endophyte studies,
while others species which are probably also endophytes, will
not be isolated. This will skew the results considerably and the
resulting endophyte lists, percentages and statistics may have
little scientific meaning.
If this phenomenon of isolating P. capitalensis for the reasons mentioned above is happening in the case of Phyllosticta it
may also be happening in other genera such as Colletotrichum,
Diaporthe, Fusarium or Pestalotiopsis (Promputtha et al.
2005; Udayanga et al. 2011; Summerell et al. 2010;
Maharachchikumbura et al. 2011; Damm et al. 2012a, b). To
determine this fact we took the common ubiquitous endophytes
Colletotrichum siamense Prihastuti, L. Cai & K.D. Hyde,
Diaporthe phaseolorum (Cooke & Ellis) Sacc., and
Pestalotiopsis adusta (Ellis & Everh.) Steyaert and blasted the
ITS sequence data from the epitype strains against GenBank
accessions and established the percentage of them that were
isolated as endophytes. Twelve strains of Colletotrichum in
GenBank had 100 % similarity with the ITS sequence data of
C. siamense (Prihastuti et al. 2009) and 50 % of these strains
were isolated as endophytes. The ITS sequence of ex-isotype of
D. phoenicicola (CBS161.64, Udayanga et al. 2012) was
subjected to a standard BLAST search in GenBank to analyze
the homology of sequences. Among the first 10 results of
highly similar sequences (100 or 99 % similarity) of retrieved
data, eight were isolated as endophytes from a wide range of
hosts. This is not surprising as Diaporthe is a commonly
isolated genus of fungal endophytes (Botella and Diez 2011;
Sun et al. 2011; Hofstetter et al. 2012). Eleven strains of
Pestalotiopsis in GenBank had 100 % similarity with the ITS
sequence data of P. adusta (Maharachchikumbura et al. 2012)
and 73 % were endophytes. Again this is not surprising as
Pestalotiopsis species are often isolated as endophytes (Aly et
al. 2010; Debbab et al. 2011, 2012; Maharachchikumbura et al.
2011). Therefore, it seems that certain taxa in these genera are
widespread endophytes and this needs further study.
Screening endophytes for novel compounds
It has been common practice to isolate endophytes from
medicinal plants using the premise that strains will be isolated
that can produce bioactive compounds similar to those produced by the plant (Krohn et al. 2007; Huang et al. 2008;
Kumaran et al. 2008; Xu et al. 2010; Zhao et al. 2010). The
fungi are thought to have obtained the mechanisms of production of natural products from the plant by so called horizontal gene transfer (Strobel et al. 2004); whether this premise
is correct or pure speculation is open to debate (Schulz et al.
2002; Selim et al. 2012) and in fact may be false (Heinig et al.
2013). The isolation of endophytes may provide a large diversity of highly creative fungi for screening (Aly et al. 2010; Xu
et al. 2011; Debbab et al. 2011; 2012). The findings of the
present study indicate that there are problems with the above
approach. It is clear in the case of Phyllosticta that P.
capitalensis will probably be the only endophyte species
isolated. Therefore, we recommend that researchers screening
for novel compounds should study the saprobes and pathogens as well as the endophytes. This will give a higher fungal
diversity and higher likelihood of isolating rare and unusual
species, and thus a higher likelihood of discovering greater
chemical diversity.
Acknowledgments We are grateful to Dhanushka Udayanga and
Sajeewa S.N. Maharachchikumbura for their assistance. This study
was financially supported by the Thailand Research Fund through the
Royal Golden Jubilee Ph. D. Program (Grant No. PHD/0198/2552) to
S. Wikee and Kevin D. Hyde. The National Research Council of
Thailand is thanked for the award of grant No 54201020004 to study
the genus Phyllosticta in Thailand.
References
Agostini JP, Peres NA, Mackenzie SJ, Adaskaveg JE, Timmer LW
(2006) Effect of fungicides and storage conditions on postharvest
development of citrus black spot and survival of Guignardia
citricarpa in fruit tissues. Plant Dis 90:1419–1424
Aly AH, Debbab A, Kjer J, Proksch P (2010) Fungal endophytes from
higher plants: a prolific source of phytochemicals and other bioactive natural products. Fungal Divers 41:1–16
Anderson CSR, Dominique G, Ana PTU, Rita TOC, Isabela SA,
Carlos RRM, Aristóteles GN (2011) Foliar endophytic fungi from
Hevea brasiliensis and their antagonism on Microcyclus ulei.
Fungal Divers 47:75–84
Baayen R, Bonants P, Verkley G, Carroll G, Van Der Aa H, De Weerdt
M, Van Brouwershaven I, Schutte G, Maccheroni W Jr, De
Blanco C (2002) Nonpathogenic isolates of the citrus black spot
fungus, Guignardia citricarpa, identified as a cosmopolitan endophyte of woody plants, G. mangiferae (Phyllosticta
capitalensis). Phytopathology 92(5):464–477
Bensch K, Braun U, Groenewald JZ, Crous PW (2012) The genus
Cladosporium. Stud Mycol 72:1–401
Bissett J (1986) Discochora yuccae sp. nov. with Phyllosticta and
Leptodothiorella synanamorphs. Can J Bot 64:1720–1726
Botella L, Diez JJ (2011) Phylogenetic diversity of fungal endophytes
in Spanish stands of Pinus halepensis. Fungal Divers 47:9–18
Chomnunti P, Schoch CL, Aguirre-Hudson B, Ko-Ko TW, Hongsanan S,
Jones EBG, Kodsueb R, Phookamsak R, Chukeatirote E, Bahkali
AH, Hyde KD (2011) Capnodiaceae. Fungal Divers 51:103–134
Crous PW, Verkleij GJM, Groenewald JZ (2009) In: Samson RA (ed)
Fungal biodiversity, vol 1, CBS laboratory manual series.
Centraalbureau voor Schimmelcultures, Utrecht
Damm U, Cannon PF, Woudenberg JHC, Crous PW (2012a) The
Colletotrichum acutatum species complex. Stud Mycol 73:37–113
Fungal Diversity
Damm U, Cannon PF, Woudenberg JHC, Johnston PR, Weir BS, Tan
YP, Shivas RG, Crous PW (2012b) The Colletotrichum boninense
species complex. Stud Mycol 73:1–36
de Gruyter J, Woudenberg JHC, Aveskamp MM, Verkley GJM,
Groenewald JZ, Crous PW (2013) Redisposition of Phoma-like
anamorphs in Pleosporales. Stud Mycol 75:1–36
De Hoog GS, Van Den Gerrits EAHG (1998) Molecular diagnostics of
clinical strains of filamentous Basidiomycetes. Mycoses 41:183–189
Debbab A, Aly AH, Proksch P (2011) Bioactive secondary metabolites
from endophytes and associated marine derived fungi. Fungal
Divers 49:1–12
Debbab A, Aly AH, Proksch P (2012) Endophytes and associated
marine derived fungi—ecological and chemical perspectives.
Fungal Divers 57:45–83
Devarajan PT, Suryanarayanan TS (2006) Evidence for the role of
phytophagous insects in dispersal of non-grass fungal endophytes.
Fungal Divers 23:111–119
Fisher PJ, Petrini O (1992) Fungal saprobes and pathogens as endophytes of rice (Oryza sativa L.). New Phytol 120:137–143
Fröhlich J, Hyde KD (1995) Guignardia candeloflamma sp. nov.
causing leaf spots of Pinanga sp. Mycol Res 99:110–112
Glienke C, Pereira O, Stringari D, Fabris J, Kava−Cordeiro V, Galli
−Terasawa L, Cunnington J, Shivas R, Groenewald J, Crous PW
(2011) Endophytic and pathogenic Phyllosticta species, with reference to those associated with citrus black spot. Persoonia 26:47–56
Glienke-Blanco C, Aguilar-Vildoso CI, Vieira MLC, Barroso PAV,
Azevedo JL (2002) Genetic variability in the endophytic fungus
Guignardia citricarpa isolated from citrus plants. Genet Mol Biol
25:251–255
Guo LD, Hyde KD, Liew ECY (1998) A method to promote sporulation in palm endophytic fungi. Fungal Divers 1:109–113
Guo LD, Hyde KD, Liew ECY (2001) Detection and taxonomic placement of endophytic fungi within frond tissues of Livistona chinensis
based on rDNA sequences. Mol Phylogenet Evol 20:1–13
Guo LD, Huang GR, Wang Y, He WH, Zheng WH, Hyde KD (2003)
Molecular identification of white morphotype strains of endophytic
fungi from Pinus tabulaeformis. Mycol Res 107(6):680–688
Heinig U, Scholz S, Jennewein S (2013) Getting to the bottom of taxol
biosynthesis by fungi. Fungal Divers. doi:10.1007/s13225-0130228-7
Hennings P (1908) Fungi S. Paulenses IV a cl. Puttemans collecti.
Hedwigia 48:1–20
Hofstetter V, Buyck B, Croll D, Viret O, Couloux A, Gindro K (2012)
What if esca disease of grapevine were not a fungal disease?
Fungal Divers 54:51–67
Huang WY, Cai YZ, Hyde KD, Corke H, Sun M (2008) Biodiversity of
endophytic fungi associated with 29 traditional Chinese medicinal
plants. Fungal Divers 33:61–75
Huelsenbeck JP, Ronquist FR (2001) MrBayes: Bayesian inference of
phylogenetic trees. Biometrics 17:754–755
Hyde KD, Soytong K (2007) Understanding microfungal diversity-a
critique. Cryptog Mycolog 28:281–289
Hyde KD, Soytong K (2008) The fungal endophyte dilemma. Fungal
Divers 33:163–173
Krohn K, Ullah Z, Hussain H, Flörke U, Schulz B, Draeger S, Pescitelli
G, Salvadori P, Antus S, Kurtán T (2007) Massarilactones E-G, new
metabolites from the endophytic fungus Coniothyrium sp., associated with the plant Artimisia maritime. Chirality 19:464–470
Kumaran RS, Muthumary J, Hur B (2008) Production of taxol from
Phyllosticta spinarum, an endophytic fungus of Cupressus sp.
Eng Life Sci 8:438–446
Kuo K, Hoch HC (1996) The parasitic relationship between
Phyllosticta ampelicida and Vitis vinifera. Mycologia 88:626–634
Lima JS, Figueiredo JG, Gomes RG, Stringari D, Goulin EH,
Adamoski D, Kava-Cordeiro V, Galli-Terasawa LV, Glienke C
(2012) Genetic diversity of Colletotrichum spp. an endophytic
fungi in a medicinal plant, Brazilian pepper tree. ISRN Microbiol.
doi:10.5402/2012/215716
Maharachchikumbura SSN, Guo LD, Chukeatirote E, Bahkali AH,
Hyde KD (2011) Pestalotiopsis—morphology, phylogeny, biochemistry and diversity. Fungal Divers 50:167–187
Maharachchikumbura SSN, Guo LD, Cai L, Chukeatirote E, Wu WP,
Sun X, Crous PW, Bhat DJ, McKenzie EHC, Bahkali AH, Hyde
KD (2012) A Multi-locus backbone tree for Pestalotiopsis, with a
polyphasic characterization of 14 new species. Fungal Divers
56:95–129
Meyer L, Jacobs R, Kotzé JM, Truter M, Korsten L (2012) Detection
and molecular identification protocols for Phyllosticta citricarpa
from citrus matter. S Afr J Sci. doi:10.4102/sajs.v108i3/4.602
Motohashi K, Inaba S, Anzai K, Takamatsu S, Nakashima C (2009)
Phylogenetic analyses of Japanese species of Phyllosticta sensu
stricto. Mycoscience 50:291–302
Okane I, Nakagiri A, Ito T (2001) Identity of Guignardia sp. inhabiting
ericaceous plants. Can J Bot 79:101–109
Okane I, Lumyong S, Nakagiri A, Ito T (2003) Extensive host range of
an endophytic fungus, Guignardia endophyllicola (anamorph:
Phyllosticta capitalensis). Mycoscience 44:353–363
Orlandelli RC, Alberto RN, Rubin Filho CJ, Pamphile JA (2012)
Diversity of endophytic fungal community associated with Piper
hispidum (Piperaceae) leaves. Genet Mol Res 11:1575–1585
Pandey AK, Reddy M, Sudhakara S, Trichur S (2003) ITS-RFLP and
ITS sequence analysis of a foliar endophytic Phyllosticta from
different tropical trees. Mycol Res 108:974–978
Paul I, Van Jaarsveld AS, Korsten L, Hattingh V (2005) The potential
global geographical distribution of citrus black spot caused by
Guignardia citricarpa (Kiely): likelihood of disease establishment in the European Union. Crop Prot 24:297–308
Petrini O (1984) Endophytic fungi in British Ericaceae: a preliminary
study. Trans Br Mycol Soc 83:510–512
Petrini O (1991) Fungal endophytes of tree leaves. In: Fokkema NJ,
van den Heuvel (eds) Microbial ecology of leaves. Cambridge
University Press, Cambridge, pp 185–187
Photita W, Lumyong S, Lumyong P, Hyde KD (2001) Endophytic
fungi of wild banana (Musa acuminata) at Doi Suthep Pui National Park, in Thailand. Mycol Res 105:1508–1513
Photita W, Lumyong S, Lumyong P, McKenzie EHC, Hyde KD (2004)
Are some endophytes of Musa acuminata latent pathogens? Fungal Divers 16:131–140
Photita W, Taylor PWJ, Ford R, Hyde KD, Lumyong S (2005) Morphological and molecular characterization of Colletotrichum species
from herbaceous plants in Thailand. Fungal Divers 18:117–133
Phoulivong S, Cai L, Chen H, McKenzie EHC, Abdelsalam K,
Chukeatirote E, Hyde KD (2010) Colletotrichum gloeosporioides
is not a common pathogen on tropical fruits. Fungal Divers
44:33–43
Prihastuti H, Cai L, Chen H, McKenzie EHC, Hyde KD (2009)
Characterization of Colletotrichum species associated with coffee
berries in northern Thailand. Fungal Divers 39:89–109
Promputtha L, Jeewon R, Lumyong S, McKenzie EHC, Hyde KD
(2005) Ribosomal DNA fingerprinting in the identification of non
sporulating endophytes from Magnolia liliifera (Magnoliaceae).
Fungal Divers 20:167–186
Purahong W, Hyde KD (2011) Effects of fungal endophytes on grass
and non-grass litter decomposition rates. Fungal Divers 47:1–7
Rayner RW (1970) A mycological colour chart. Commonwealth Mycological Institute and British Mycological Society, Kew, 34 pp
Roy AJ (1968) Some fungi from Almora. Indian Phytopathol 20:340–348
Schulz B, Boyle C, Draeger S, Römmert AK (2002) Endophytic fungi:
a source of novel biologically active secondary metabolites.
Mycol Res 106:996–1004
Selim KA, El-Beih AA, Abdel-Rahman TM, El-Diwany AI (2012)
Biology of endophytic fungi. CREAM 2:31–82
Fungal Diversity
Shaw BD, Carroll GC, Hoch HC (2006) Generality of the prerequisite
of conidium attachment to a hydrophobic substratum as a signal
for germination among Phyllosticta species. Mycologia 98:186–
194
Silva M, Pereira OL (2007) First report of Guignardia endophyllicola
leaf blight on Cymbidium (Orchidaceae) in Brazil. Australas Plant
Dis 2:31–32
Silva M, Pereira OL, Braga IF, Leli SM (2008) Leaf and
pseudobulb diseases on Bifrenaria harrisoniae (Orchidaceae)
caused by Phyllosticta capitalensis in Brazil. Australas Plant
Dis 3:53–56
Singh KG (1980) A check list of host and disease in Malaysia. Bull
Minist Agric Malays 154:280
Slippers B, Wingfield MJ (2007) Botryosphaeriaceae as endophytes
and latent pathogens of woody plants: diversity, ecology and
impact. Fungal Biol Rev 21:90–106
Strobel GA, Daisy B, Castillo U, Harper J (2004) Natural products
from endophytic microorganisms. J Nat Prod 67:257–268
Summerell BA, Laurence MH, Liew ECY, Leslie JF (2010) Biogeography and phylogeography of Fusarium: a review. Fungal Divers
44:3–13
Sun X, Guo LD, Hyde KD (2011) Community composition of endophytic fungi in Acer truncatum and their role in decomposition.
Fungal Divers 47:85–95
Suryanarayanan TS, Ravishankar JP, Venkatesan G, Murali TS (2004)
Characterization of the melanin pigment of a cosmopolitan fungal
endophyte. Mycol Res 108:974–978
Swofford DL (2003) Paup*: Phylogenetic analysis using parsimony
(*and other methods), version 4.0. Sinauer Associates,
Sunderland
Than PP, Jeewon R, Hyde KD, Pongsupasamit S, Mongkolporn O,
Taylor PWJ (2008) Characterization and pathogenicity of
Colletotrichum species associated with anthracnose on chilli
(Capsicum spp.) in Thailand. Plant Pathol 57:562–572
Thompson S, Alvarez-Loayza P, Terborgh J, Katul G (2010) The
effects of plant pathogens on tree recruitment in the Western
Amazon under a projected future climate: a dynamical systems
analysis. J Ecol 98:1434–1446
Udayanga D, Liu XX, McKenzie EHC, Chukeatirote E, Bahkali AH,
Hyde KD (2011) The genus Phomopsis: biology, applications,
species concepts and names of common phytopathogens. Fungal
Divers 50:189–225
Udayanga D, Liu XX, Crous PW, McKenzie EHC, Chukeatirote E,
Hyde KD (2012) A multi-locus phylogenetic evaluation of
Diaporthe (Phomopsis). Fungal Divers 56:157–171
Ullrich CI, Kleespies RG, Enders M, Koch E (2009) Biology of the
black rot pathogen, Guignardia bidwellii, its development in
susceptible leaves of grapevine Vitis vinifera. J Kult 61:82–90
Van Der Aa HA (1973) Studies in Phyllosticta I. Stud Mycol 5:1–110
Van Der Aa H, Vanev S, Aptroot A, Summerbell R, Verkley G (2002) A
revision of the species described in Phyllosticta. Centraalbureau
voor Schimmelcultures, Utrecht
Wang X, Chen G, Huang F, Zhang J, Hyde KD, Li H (2012)
Phyllosticta species associated with citrus diseases in China.
Fungal Divers 52:209–224
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innes MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols. A guide to methods and applications. Academic, San
Diego, pp 315–322
Wikee S, Udayanga D, Crous PW, Chukeatirote E, McKenzie EHC,
Bahkali AH, Dai DQ, Hyde KD (2011) Phyllosticta—an overview
of current status of species recognition. Fungal Divers 46:171–182
Williams TH, Liu PSW (1976) A host list of plant disease in Sabah,
Malaysia. Phytopathol Pap 19:1–67
Wong MH, Crous PW, Henderson J, Groenewald JZ, Drenth A (2012)
Phyllosticta species associated with freckle disease of banana.
Fungal Divers 56:173–187
Wulandari NF, To−Anun C, Hyde KD, Duong L, De Gruyter J, Meffert
J, Groenewald JZ, Crous PW (2009) Phyllosticta citriasiana sp.
nov., the cause of Citrus tan spot of Citrus maxima in Asia.
Fungal Divers 34:23–39
Wulandari NF, To-Anun C, Hyde KD (2010a) Guignardia morindae
frog eye leaf spotting disease of Morinda citrifolia (Rubiaceae).
Mycosphere 1(4):325–331
Wulandari NF, To-Anun C, Lei C, Abd-Elsalam KA, Hyde KD
(2010b) Guignardia/Phyllosticta species on banana. Cryptog
Mycol 31(4):403–418
Xu J, Aly AH, Guan HS, Wray V, Proksch P (2010) Pestalotiopsis a
highly creative genus: chemistry and bioactivity of secondary
metabolites. Fungal Divers 44:15–31
Xu YC, Yao DQ, Jian HW, Zheng Z, De LW, Jin DF, Bing CG (2011)
Molecular identification of endophytic fungi from medicinal plant
Huperzia serrata based on rDNA ITS analysis. World J Microbiol
Biotechnol 27:495–503
Zhao J, Zhou L, Wang J, Shan T, Zhong L, Liu X, Gao X (2010) In:
Mendez-Vilas A (ed), Current Research, Technology Education
Topics in Applied Microbiology and Microbial biotechnology:
Endophytic fungi for producing bioactive compounds originally
from their host plants. p. 567–576