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S
TUDIES IN
M
YCOLOGY
50:
313–322.
2004.
313
Lasiodiplodia gonubiensis sp. nov., a new Botryosphaeria anamorph from
native Syzygium cordatum in South Africa
Draginja Pavlic
*
, Bernard Slippers, Teresa A. Coutinho, Marieka Gryzenhout and Michael J. Wingfield
Forestry and Agricultural Biotechnology Institute (FABI), Department of Microbiology and Plant Pathology, Faculty of
Natural and Agricultural Sciences, University of Pretoria, Pretoria, 0002, South Africa
*Correspondence: Draginja Pavlic, draginja.pavlic@fabi.up.ac.za
Abstract: Botryosphaeria spp. are common and widely distributed pathogens on many economically important crops, includ-
ing forest tree species. These fungi cause a wide variety of symptoms on trees of all ages, but are mostly associated with
canker and die-back of branches and main stems. As disease agents, Botryosphaeria spp. are often encountered in their
anamorph state, namely species of Fusicoccum, Diplodia or Lasiodiplodia. During a recent survey of botryosphaeriaceous
fungi from native Syzygium cordatum in South Africa, an unfamiliar Lasiodiplodia sp. was isolated. The aim of this study
was to compare this apparently undescribed species with other species of Botryosphaeria using morphological characteristics
and DNA sequence data of the rDNA internal transcribed spacers, ITS1 and ITS2. Based on sequence data, the isolates from
S. cordatum were more closely related to B. rhodina (anamorph Lasiodiplodia theobromae) than to other Botryosphaeria
spp., but also phylogenetically distinct from this species. Conidia of the species from S. cordatum were also different to those
of L. theobromae. We conclude that the isolates from S. cordatum represent an undescribed Lasiodiplodia sp. and provide the
name Lasiodiplodia gonubiensis for it.
Taxonomic novelty: Lasiodiplodia gonubiensis Pavlic, Slippers & M.J. Wingf. sp. nov.
Key words: Botryosphaeria, Diplodia, endophyte, Fusicoccum, Lasiodiplodia, systematics.
INTRODUCTION
Botryosphaeria Ces. & De Not. (Dothideales) con-
tains species that have a cosmopolitan distribution and
wide host range, including gymnosperms and angio-
sperms (von Arx & Müller 1954, Barr 1972). These
fungi are common endophytes and latent, opportunis-
tic pathogens on many woody plants such as Eucalyp-
tus spp. (Fisher et al. 1993, Smith et al. 1996a, b).
Typical disease symptoms associated with Botryos-
phaeria spp. are canker and die-back, followed by
kino exudation, and in severe cases tree death (Davi-
son & Tay 1983, Webb 1983, Sharma 1984, Shearer et
al. 1987, Smith et al. 1994, Old & Davison 2000,
Roux et al. 2000, 2001, Smith et al. 2001a).
Eucalyptus belongs to one of the oldest plant
families, namely the Myrtaceae (Johnson & Briggs
1981). It is largely a Southern Hemisphere family with
more than 3000 species and is particularly well repre-
sented in the tropical and temperate regions of Aus-
tralasia and Central and South America (Johnson &
Briggs 1981). Myrtaceous species are also an integral
part of Southern African indigenous flora (Palgrave
1977). The most common and widely distributed
myrtaceous tree in South Africa is Syzygium cordatum
Hochst. (Palgrave 1977).
Most Eucalyptus spp. are native to Australia
(Poynton 1979), but they are the most widely grown
trees in exotic plantations in other parts of the world
(Ciesla et al. 1996). These exotic plantations are often
planted in close association with native myrtaceous
trees that are closely related to Eucalyptus (Burgess &
Wingfield 2001). A danger in such cases is that patho-
gens from either of these related native or introduced
hosts could cross-infect the other host group and cause
serious diseases (Crous & Swart 1995, Wingfield
1999, Burgess & Wingfield 2001). An example of this
is the rust fungus Puccinia psidii G. Winter that
occurs on native Myrtaceae in South America, and has
become one of the most important pathogens on exotic
Eucalyptus in this region (Coutinho et al. 1998).
Because of its wide distribution, and the fact that
this tree often grows alongside plantations of Eucalyp-
tus, we conducted a survey of botryosphaeriaceous
fungi occurring on native Syzygium cordatum in South
Africa. This survey resulted in isolates of a Lasiodip-
lodia sp. Lasiodiplodia spp. are anamorphs of Bot-
ryosphaeria and a very common species, particularly
in tropical areas, is Lasiodiplodia theobromae (Pat.)
Griffon & Maubl. (von Arx 1974, Punithalingam
1976, 1979), teleomorph B. rhodina (Berk. & M.A.
Curtis) Arx (von Arx 1974). The fungus from S.
cordatum is similar to L. theobromae but has dis-
tinctly larger conidia and no teleomorph has been
found. The aim of this study was to identify the un-
known Lasiodiplodia sp. using both morphological
characteristics and comparisons of DNA sequence
P
AVLIC ET AL
.
314
data of the Internal Transcribed Spacer region (ITS) of
the rDNA operon.
MATERIALS AND METHODS
Isolates
Isolates of an unknown Lasiodiplodia sp. were col-
lected in the Eastern Cape Province, South Africa in
July 2002 (Table 1). Isolations were made from asym-
ptomatic twigs and leaves of naturally growing S.
cordatum. Leaf and twig portions (5 cm in length)
were washed in running tap water, surface disinfected
by submerging them for 1 min sequentially in 96 %
ethanol, undiluted bleach (3.5–5 % available chlorine)
and 70 % ethanol, and rinsed in sterile water. The
disinfected twig portions were halved and pieces from
the pith tissue (2 mm) and segments of the leaves (3
mm were placed on 2 % malt extract agar (MEA) (2
% malt extract, 1.5 % agar; Biolab, Midrand, Johan-
nesburg, S.A.). Plates were incubated at 20 °C under
continuous near-UV light for two weeks and colonies
resembling Botryosphaeria spp. were selected. These
colonies were maintained on 2 % MEA at 25 °C and
stored at 5 °C. Isolates are maintained in the Culture
Collection (CMW) of the Forestry and Agricultural
Biotechnology Institute (FABI), University of Preto-
ria, Pretoria, South Africa and in the collection of the
Centraalbureau voor Scimmelcultures (CBS), Utrecht,
The Netherlands.
Morphology and cultural characteristics
To induce sporulation, isolates were grown on 2 %
water agar (WA) (Biolab, S.A.) with sterilized pine
needles placed onto the medium, at 25 °C under near-
UV light. Herbarium specimens were also sought for
L. theobromae to compare with the fungus from S.
cordatum. In the original descriptions of the species
(Patouillard 1892) and genus (Clendinin 1896), no
reference is made to type material. CBS, ATCC and
IMI do not have cultures from the original host and
location (Theobroma cacao L. in Ecuador), and no
herbarium material from the same origin could be
located in BPI. Until original material can be located
or an epitype specimen assigned, it is necessary to rely
on descriptions from the literature. For comparative
purposes, we thus compiled a table from previous
descriptions, to provide conidial dimensions for this
species, as well as many species that have been re-
duced to synonymy with L. theobromae (Table 2).
Released conidia and squash mounts of pycnidia
formed on the pine needles, were mounted in lacto-
phenol on microscope slides and examined micro-
scopically. Sections of pycnidia were made by hand
and mounted in lactophenol to observe conidiophore
morphology. Fifty measurements were taken of
pycnidia, conidia, conidiogenous cells and paraphyses
for each isolate, and the ranges and averages were
computed. Measurements and digital photographs
were made using a HRc Axiocam digital camera and
accompanying Axiovision 3.1 software (Carl Zeiss
Ltd., München, Germany).
Colony growth rate for isolates CMW 14077 and
CMW 14078 were studied at temperatures ranging
from 5 to 35 °C, at 5 ° intervals in the dark. Mycelial
plugs, 6 mm diam, were transferred to 2 % MEA in 90
mm diam Petri dishes from the edges of 7-d-old,
single-conidial cultures. Four plates were used for
each isolate at each temperature. Two perpendicular
measurements were taken of the colony diameter daily
until the mycelium of the fastest growing isolates had
covered the plates. Average colony diameter of each
isolate was calculated from the eight readings per
isolate. Colony morphology and colour were deter-
mined from cultures grown on 2 % MEA at 25 ºC in
the dark. Colony colours (upper surface and reverse)
were described by comparison with the colour charts
of Rayner (1970).
DNA extraction and ITS rDNA amplification
For DNA extraction, single conidial cultures were
grown on 2 % MEA for 7 d at 25 °C in the dark. The
mycelium was scraped directly from the medium and
transferred to Eppendorf tubes (1.5 mL). DNA was
extracted using a modified phenol:chloroform DNA
extraction method of Raeder & Broda (1985). The
resulting DNA pellets were resuspended in 50 L
sterile SABAX water. RNAse (1 mg/mL) was added
to DNA samples and incubated overnight at 37 °C to
degrade residual protein or RNA. DNA was separated
by electrophoresis on a 1.5 % agarose gel, stained
with ethidium bromide and visualized under ultra-
violet light. DNA concentrations were estimated
against standard size markers.
Using the primer pair ITS1 and ITS4 (White et al.
1990), the ITS1 and ITS2 regions, and the 5.8S gene
of the ribosomal RNA (rRNA) operon were amplified
using the PCR protocol of Slippers et al. (2004). PCR
products were separated as described above and sizes
of PCR products were estimated against a 100 bp
molecular weight marker XIV (Roche Diagnostics,
Johannesburg, S.A.). The PCR products were purified
using a High Pure PCR Product Purification Kit
(Roche Diagnostics, Mannheim, Germany).
DNA sequencing and analysis
The purified PCR products were sequenced in both
directions using the same primers used for the PCR
reactions. The ABI PRISM Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin-Elmer, War-
rington, U.K.) was used for sequencing reactions as
specified by the manufacturers.
B
OTRYOSPHAERIA FROM
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315
Table 1. Isolates of Botryosphaeria, Guignardia and Mycosphaerella species considered in the phylogenetic study.
Culture no.
1
Other no.
1
Species Host Location Collector GenBank
no.
CMW 9081 ICMP 8003 Botryosphaeria parva Populus nigra New Zealand G.J. Samuels AY236943
CMW 10124 BOT 681 Heteropyxis na-
talensis KwaZulu-Natal, S.
Africa H. Smith AF283676
CMW 9075 ICMP 8019 Botryosphaeria dothidea P. nigra New Zealand G.J. Samuels AY236950
CMW 8000 Prunus sp. Crocifisso, Switzerland B. Slippers AY236949
CMW 10125 BOT 24 Botryosphaeria eucalypto-
rum Eucalyptus grandis Mpumalanga, S. Africa H. Smith AF283686
CMW 10126 BOT 16 E. grandis Mpumalanga, S. Africa H. Smith AF283687
CMW 992 KJ 93.52 Botryosphaeria lutea Actinidia deliciosa New Zealand G.J. Samuels AF027745
CMW 9076 ICMP 7818 Malus domestica New Zealand S.R. Pennycook AY236946
CMW 7774
Botryosphaeria obtusa Ribes sp. New York, U.S.A. B. Slippers & G.
Hudler AY236953
KJ 93.56 Hardwood shrub New York, U.S.A. G.J. Samuels AF027759
KJ 93.27 Botryosphaeria rhodina Quercus sp. California, U.S.A. E. Hecht-Poinar AF027761
ZS 96-112 Pinus radiata S. Africa W. Swart AF243401
ZS 96-172 Theobroma cacao Sri Lanka E. Müller AF243400
CMW 10130 BOT 977 Vitex donniana Uganda J. Roux AY236951
CMW 9074
Pinus sp. Mexico T. Burgess AY236952
CMW 7060 CBS 431 Botryosphaeria stevensii Fraxinus excelsior Netherlands H.A. van der Aa AY236955
ZS 94-6 Malus pumila New Zealand N. Tisserat AF243407
CBS 112545
Botryosphaeria corticola Quercus ilex Spain M.A. Sanchez &
A. Trapero AY259089
CBS 112551
Q. suber Portugal A. Alves AY259101
CBS 418.64 Botryosphaeria tsugae Tsuga heterophylla Canada A. Funk AF243405
KJ 94.07 Diplodia pinea Pinus resinosa Wisconsin, U.S.A. D.R. Smith AF027758
CMW 14077 CBS 115812
Lasiodiplodia gonubiensis Syzygium cordatum Eastern Cape, S. Africa
D. Pavlic AY639595
CMW 14078 CBS 116355
S. cordatum Eastern Cape, S. Africa
D. Pavlic AY639594
CMW 3025 Mycosphaerella africana Eucalyptus viminalis
Stellenbosch, S. Africa P.W. Crous AF 283690
CMW 7063 CBS 447 Guignardia philoprina Taxus baccata Netherlands H.A. van der Aa AY236956
1
Culture collections: BOT and CMW = Tree Pathology Co-operative Programme, Forestry and Agricultural Biotechnology
Institute, University of Pretoria; KJ = Jacobs & Rehner (1998); CBS = Centraalbureau voor Schimmelcultures, Utrecht,
Netherlands; ICMP = International Collection of Microorganisms from Plants, Auckland, New Zealand; ZS = Zhou & Stanosz
(2001).
Sequence reactions were run on an ABI PRISM
3100 automated DNA sequencer (Perkin-Elmer,
Warrington, U.K.). The nucleotide sequences were
analyzed using Sequence Navigator v. 1.0.1. (Perkin-
Elmer Applied BioSystems, Inc., Foster City, Califor-
nia) software and manually aligned by inserting gaps.
Sequence data for isolates of the unknown species
have been deposited in GenBank (Table 1).
The DNA sequences of the isolates of the unknown
species were compared with those of other Botryos-
phaeria spp. These included twenty one ITS rDNA
sequences of B. parva Pennycook & Samuels, B.
dothidea (Moug. : Fr.) Ces. & De Not., B. eucalypto-
rum Crous, H. Smith & M.J. Wingf., B. lutea A.J.L.
Phillips, B. obtusa (Schwein.) Shoemaker, B. stevensii
Shoemaker, B. tsugae Funk, Diplodia pinea (Desm.) J.
Kickx (= Sphaeropsis sapinea (Fr. : Fr.) Dyko & B.
Sutton), B. corticola A.J.L. Phillips, Alves & Luque
and B. rhodina obtained from GenBank (Table 1),
arising from previous studies (Jacobs & Rehner 1998,
Smith et al. 2001a, Zhou & Stanosz 2001, Alves et al.
2004, Slippers et al. 2004). The trees were rooted
using the GenBank sequence of Guignardia philo-
prina (Berk. & M.A. Curtis) Aa and Mycosphaerella
africana Crous & M.J. Wingf.
The DNA sequence data were manually aligned in
PAUP version 4.0b10 (Swofford 1999) by insertion of
gaps. Gaps were treated as missing data and all char-
acters included in the analyses were unordered and of
equal weight. Most parsimonious trees were found
using the heuristic search function with 1000 random
addition replicates and tree bisection and reconstruc-
tion (TBR) selected as branch-swapping algorithm.
P
AVLIC ET AL
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316
Table 2. Conidial size and septation for Lasiodiplodia theobromae described under different synonyms.
Species Host Origin Conidia size
No. of
septa
Reference
Diplodia gossypina Cooke Gossypium sp. India 22 ×12 m – Cooke 1879
Botryodiplodia theobromae Pat. Theobroma cacao Ecuador 25–35 × 12–15 m 1 Patouillard & De Lager-
heim 1892
Macrophoma vestita Prill. & Delacr. T. cacao Equatorial
America 25–28 × 13 m 1 Prillieux & Delacroix 1894
Lasiodiplodia tubericola Ellis & Everh. Ipomoea batatas Java 18–22 ×11–14 m 1 Clendinin 1896
Diplodia cacaoicola P. Henn. T. cacao Kamerun 22–28 × 12–14 m 1 Hennings 1897
Botryodiplodia gossypii Ellis & Barthol. Gossypium her-
baceum U.S.A. 15–22 × 12 m 1 Ellis & Bartholomew 1902
Lasiodiplodia nigra K.R. Appel & Laubert T. cacao, Carica
p
apaya Samoa 28–32 × 18–21 m 1 Appel & Laubert 1907
Lasiodiplodia theobromae (Pat.) Griffon &
Maubl. T. cacao Equatorial
America 20–30 × 11–15 m 1 Griffon & Maublanc 1909
Diplodia rapax Massee Hevea brasiliensis Singapore, Ghana
32–35 × 15–16 m 1 Massee 1910
Diplodia natalensis Pole-Evans Citrus sp. South Africa 24–15 m 1 Pole Evans 1910
Lasiodiplodia triflorae B.B. Higgins Prunus sp. U.S.A. 22–25 × 13–16.5 m
1 Higgins 1916
Diplodia maniothi Sacc. Manihot utilissima – 16–22 × 10–12 m 1 Sydow et al. 1916
Diplodia musae Died. Musa sapientium – 17–20 ×10–13 m 1 Sydow et al. 1916
Diplodia ananassae Sacc. Ananas sativus Philippines 23–25 ×11–12 m 1 Saccardo 1917
Diplodia theobromae (Pat.) W. Nowell T. cacao – 25–30 × 12–15 m 1 Nowell 1923
Branches of zero length were collapsed and all multi-
ple, equally parsimonious trees were saved. Branch
support was determined using 1000 bootstrap repli-
cates (Felsenstein 1985). The data set was also ana-
lysed by distance analyses using the Kimura-2 pa-
rameter (Kimura 1980). The sequence alignment and
phylogenetic tree have been deposited in TreeBASE
as S1133, M1944.
RESULTS
Morphology and cultural characteristics
The isolates from S. cordatum produced anamorph
structures on the pine needles on WA within 2–3 wk.
No sexual (teleomorph) structures were observed
during this study. The conidia (Figs 3, 4) were similar
to those described for L. theobromae in shape, colour
and striation (Clendinin 1896, Punithalingam 1976,
Sivanesan 1984). These isolates, however, differed
from L. theobromae in having markedly longer and
wider conidia (28−)32−36(−39) × (14−)16−18.5(−21)
m, while those of L. theobromae are mostly 18−30 ×
10−15 m (Table 2). Furthermore, aging conidia of
the strains from S. cordatum become 1–3-septate,
(Figs 4, 5, 11), which is different to the single septate
conidia that are typical of L. theobromae (Table 2).
DNA sequence comparisons
PCR products of approximately 560 base pairs (bp)
were amplified. Unreliable sequence data from the
ends of sequences were excluded. Alignment of the
sequences resulted in a total of 534 characters, of
which 386 uninformative characters were excluded,
and 148 parsimony informative characters were used
in the analyses. The parsimony analysis (using heuris-
tic searches) produced six most parsimonious trees of
318 steps (CI = 0.758, RI = 0.869) that only differed
in the length of the internal branches, and one of these
trees was chosen for presentation (Fig. 12). A boot-
strap search of 1000 replicates (Fig. 12) and distance
analyses produced a tree of the same topology as the
most parsimonious trees.
The species included in this comparison formed
eleven terminal groupings, designated as groups I to
XI (Fig. 12). Groups I to IV include Botryosphaeria
spp. with Fusicoccum-like anamorphs: B. parva, B.
lutea, B. eucalyptorum and B. dothidea. Groups V to
IX (Fig. 12) include Botryosphaeria spp. with Diplo-
dia-like anamorphs: B. obtusa, Diplodia pinea, B.
stevensii, B. tsugae and B. corticola. Isolates of the
unnamed species from S. cordatum grouped most
closely to B. rhodina (anamorph L. theobromae)
(group X), but also resided in a clearly distinct group
(group XI) with 95 % bootstrap support (Fig. 12).
These two groups were more closely related to isolates
that have Diplodia-like anamorphs (groups V to IX),
but also clearly separated from them with a 78 %
bootstrap value.
B
OTRYOSPHAERIA FROM
S
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317
Figs 1–8. Micrographs of fruiting structures of Lasiodiplodia gonubiensis. 1. Pycnidium formed in culture on pine needles,
covered with mycelium. 2. Paraphyses (arrows). 3. Conidia. 4. Brown conidium with one septum. 5. Brown conidium with two
septa. 6. Conidium attached to conidiogenous cell. 7, 8. Conidia, conidiogenous cells and paraphyses. Bars 1 = 500 m; 2–8 =
10 m.
P
AVLIC ET AL
.
318
Taxonomy
Based on morphological characteristics and DNA
sequence comparisons, we conclude that the fungus
isolated from native S. cordatum in South Africa is
distinct from L. theobromae and other Botryosphaeria
anamorph spp. examined in our study. Our data also
indicate that this fungus should reside in Lasiodiplo-
dia as a new taxon. We provide the following descrip-
tion for this new species.
Lasiodiplodia gonubiensis Pavlic, Slippers &
M.J. Wingf., sp. nov. MycoBank MB500079.
Figs 1–11.
Etymology: Referring to the town Gonubie, South
Africa from where the fungus was collected.
Pycnidia subimmersa, solitaria, globosa, papillata,
atroplumbea, mycelio tecta, usque ad 460 µm diametro.
Paraphyses cylindricae, non septatae, hyalinae. Cellulae
conidiogenae holoblasticae, cylindricae, hyalinae. Conidia
primaria hyalina, unicellulares, ellipsoidea vel obovoidea,
parietibus crassitunicati, contentu granulari, apice
rotundata, interdum basi truncata. Conidia senia
cinnamomescentia vel brunnescentia, longitudinaliter
striata, unum ad tria septa formantia.
Pycnidia (formed on WA on sterilized pine needles
within 7−21 d) semi-immersed, solitary, globose,
papillate, leaden-black, covered by mycelium, up to
460 m diam (Fig. 1). Paraphyses cylindrical, asep-
tate, hyaline, (14−)26.5−47(−65) × (1.5−)2–2.5(−3)
m (Figs 2, 7, 9). Conidiogenous cells holoblastic,
cylindrical, hyaline, (6.5−)10−15(−18) ×
(1−)2−4(−4.5) m (Figs 7–9). Conidia initially hya-
line, unicellular, ellipsoid to obovoid, thick-walled
with granular content, rounded at apex, occasionally
truncate at base (Figs 3, 6, 7, 9, 10). Aging conidia
becoming cinnamon to sepia with longitudinal stria-
tions, forming one to three septa, (28−)32−36(−39) ×
(14−)16−18.5(−21) m (av. 33.8 × 17.3 m, n = 100,
l/w 1.9) (Figs 4, 5, 11).
Typus: PREM 58127 holotype, fruiting structures induced
on needles of Pinus sp. on WA, South Africa, Eastern Cape
Province, Gonubie, Syzygium cordatum, Jul. 2002, D.
Pavlic (culture ex-type CMW 14077 = CBS 115812).
Cultural characteristics: Cultures initially white to
smoke-grey with fluffy, aerial mycelium, becoming
olivaceous-grey on the surface after 3−4 d, with thick
aerial mycelium, margins slightly irregular; reverse
side of the colonies dark slate-blue. Optimum tem-
perature for colony growth 25 °C, covering the me-
dium surface (90 mm diam Petri dishes) after 5 d in
the dark. Isolates growing at 35 °C produced a coral-
red pigment within 4 d.
Substrate: Symptomless leaves and branches of S.
cordatum.
Distribution: Eastern Cape Province (Gonubie), South
Africa.
Specimens examined: South Africa, Eastern Cape Prov-
ince, Gonubie, Syzygium cordatum, Jul. 2002, D. Pavlic,
holotype PREM 58127, fruiting structures induced on
needles of Pinus sp. on WA; culture ex-type CMW 14077 =
CBS 115812; Eastern Cape Province, Gonubie, Syzygium
cordatum, Jul. 2002, D. Pavlic, paratype PREM 58128,
fruiting structures induced on needles of Pinus sp. on WA,
culture ex-paratype CMW 14078 = CBS 116355.
Figs 9–11. Line drawings of Lasiodiplodia gonubiensis. 9.
Conidia, conidiogenous cells and paraphyses. 10. Aseptate
conidia. 11. 13-septate conidia. Bar = 10 m.
DISCUSSION
In this study we have identified and described the new
species Lasiodiplodia gonubiensis, that grows endo-
phytically on native S. cordatum in South Africa.
Based on its phylogenetic relationships, we expect that
the teleomorph of this fungus will be a species of
Botryosphaeria. Despite careful examination of dead
branches and twigs of S. cordatum, we have not been
able to find a sexual state for this fungus. Ideally, we
would provide a name in Botryosphaeria for it, but
this is not recommended by the ICBN (Art. 59.2,
Greuter et al. 2000).
Lasiodiplodia gonubiensis was identified as a
species of Lasiodiplodia based on conidial shape and
striation, which are characters typical for this genus
(von Arx 1974).
B
OTRYOSPHAERIA FROM
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319
B. parva CMW 9081
B. parva CMW 10124
B. lutea CMW 9076
B. lutea CMW 992
B. eucalyptorum CMW 10125
B. eucalyptorum CMW 10126
B. dothidea CMW 8000
B. dothidea CMW 9075
B. obtusa CMW 7774
B. obtusa KJ 93.56
Diplodia pinea KJ 94.07
B. stevensii CMW 7060
B. stevensii SZ 94-6
B. tsugae CBS 418-64
B. corticola CBS 112545
B. corticola CBS 112551
B. rhodina CMW 9074
B. rhodina ZS 96-172
B. rhodina CMW 10130
B. rhodina KJ 93.27
B. rhodina ZS 96-112
L. gonubiensis CMW 14078
L. gonubiensis CMW 14077
Guignardia philoprina CMW 7063
Mycosphaerella africana CMW 3025
5 changes
61
24
15
9
9
16
8
1
6
38
2
1
87
10
9
11
5
2
1
4
3
1
7
1
9
5
3
1
3
11
1
4
35
26
100
98
99
87
100
90
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
92
68
75
60
100
90
95
78
5100
62
77
Fig. 12. Phylogram showing relationships amongst Botryosphaeria spp. based on parsimony analysis of the ITS1, 5.8 S and
ITS2 rDNA sequence data (tree length = 318 steps, CI = 0.758, RI = 0.869). The tree is rooted to the outgroups Guignardia
philoprina and Mycosphaerella africana. Bootstrap values (1000 replicates) are indicated below the internodes, and branch
lengths, proportional to the number of steps, are indicated above the internodes.
Conidia of L. gonubiensis are similar in appearance to
those of L. theobromae (Clendinin 1896, Griffon &
Maublanc 1909, Goos 1961, Punithalingam 1976,
1979, Sivanesan 1984). However, L. gonubiensis can
be distinguished from L. theobromae by its substan-
tially larger and multiseptate conidia. These conidial
characters have also been useful to distinguish other
closely related Botryosphaeria anamorphs, such as B.
ribis and B. parva (Slippers et al. 2004).
Lasiodiplodia gonubiensis grouped separately from
other Botryosphaeria spp. based on comparison of
partial nrDNA ITS sequence data. The results of the
phylogenetic study further showed that L. gonubiensis
was closely related, but clearly distinct from isolates
of L. theobromae. This is another example where ITS
rDNA sequence data were useful to distinguish a new
botryosphaeriaceous species. Recent studies have used
this region extensively, combined with morphological
data, to describe new Botryosphaeria spp. and to re-
evaluate the placement of their anamorphs (Jacobs &
Rehner 1998, Denman et al. 1999, 2000, Smith et al.
2000b, Smith & Stanosz 2001, Zhou & Stanosz 2001,
Denman et al. 2003, Slippers 2003, Alves 2004).
Despite the general phylogenetic usefulness of this
region of the genome, there are cryptic species that
cannot be separated based solely on ITS rDNA se-
P
AVLIC ET AL
.
320
quence data (De Wet et al. 2003, Slippers et al. 2004).
In these cases sequence data of multiple gene regions
have revealed the cryptic species.
The teleomorph of L. gonubiensis was not ob-
served in this study. Lasiodiplodia spp. are, however,
well-known as anamorphs of Botryosphaeria. This is
confirmed in this study, because L. gonubiensis
groups significantly more closely to other Botryos-
phaeria spp. than even the closely related genus
Guignardia. Due to the rarity of Botryosphaeria
teleomorphs and their overlapping morphological
features, species are often identified based on morpho-
logical characteristics of associated anamorphs
(Shoemaker 1964, Laundon 1973, Sivanesan 1984,
Jacobs & Rehner 1998, Slippers 2003). This has been
true for L. gonubiensis, which could easily be distin-
guished from other closely related species based on
conidial morphology.
In this study, L. gonubiensis and L. theobromae
(teleomorph B. rhodina) grouped together as a sub-
clade, within a greater clade that contains Botryos-
phaeria spp. with anamorphs in Diplodia. Previous
phylogenetic re-evaluations have shown that Botryos-
phaeria anamorphs can be separated into two groups,
namely Diplodia-like anamorphs with ellipsoid, thick-
walled dark conidia, and Fusicoccum-like anamorphs
with hyaline conidia (Denman et al. 2000, Zhou &
Stanosz 2001). Lasiodiplodia has, however, always
grouped separately within the Diplodia clade (Den-
man et al. 2000, Zhou & Stanosz 2001, Slippers 2003,
Slippers et al. 2004), as was the case in our study. It
has been proposed that all Botryosphaeria anamorphs
might either be placed in Fusicoccum or Diplodia,
with Lasiodiplodia residing in Diplodia (Denman et
al. 2000). Because it is morphologically distinct,
especially based on its obvious and unique conidial
striations (von Arx 1974), there seemed little reason
from our data to change the name of this important
tree pathogen. Lasiodiplodia has also not formally
been reduced to synonymy with Diplodia and we have
thus chosen to assign the new species from S. corda-
tum to Lasiodiplodia rather than Diplodia.
Lasiodiplodia gonubiensis is the first species in
this genus to be found on native trees in South Africa.
The closely related L. theobromae is an important
opportunistic pathogen recorded from more than 500
host plants, mostly in tropical and subtropical regions
(Punithalingam 1976). Lasiodiplodia theobromae has
not been reported from native trees in South Africa,
but it occurs on exotic Acacia, Eucalyptus and Pinus
spp. in South Africa (Cilliers et al. 1993, Crous et al.
2000, Burgess et al. 2003).
Lasiodiplodia gonubiensis was discovered as an
endophyte in asymptomatic twigs and leaves of S.
cordatum. Other Botryosphaeria spp. are common
endophytes and latent, opportunistic pathogens on
Eucalyptus (Fisher et al. 1993, Smith et al. 1996a, b).
For these fungi, disease symptoms typically develop
when trees are exposed to unfavourable environmental
conditions. Lasiodiplodia gonubiensis might thus also
be a latent pathogen although we have not found it in
association with disease symptoms.
Lasiodiplodia gonubiensis could become a patho-
gen of commercial Eucalyptus spp. in South Africa.
Both S. cordatum and Eucalyptus reside in the Myrta-
ceae and they are sufficiently related that they could
share pathogens. This would be consistent with the
fact that B. parva has been shown to infect both hosts
(Smith et al. 2000a, Slippers et al. 2004). Although B.
parva has been found as a pathogen on Eucalyptus, its
pathogenicity on S. cordatum is not known. Future
studies will consider the pathogenicity and potential
threat of L. gonubiensis and other Botryosphaeria spp.
to both Syzygium and Eucalyptus spp.
ACKNOWLEDGEMENTS
We thank the National Research Foundation (NRF), mem-
bers of Tree Protection Co-operative Programme (TPCP)
and the THRIP initiative of the Department of Trade and
Industry, South Africa for financial support. We also thank
Dr Treena Burgess (Murdoch University, Australia) who
reviewed the manuscript and Mr. Wilhelm Z. de Beer who
provided relevant literature. Dr H.F. Glen (Natal Herbar-
ium, P.O. Box 52099, Berea Road, Durban, 4007, South
Africa) provided the Latin description. We are grateful to
Julia Kreiss who did the drawings for Figs 9–11.
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