Botryosphaeriaceae from Eucalyptus and Native Myrtaceae in Uruguay

Abstract

Species of the Botryosphaeriaceae are important pathogens causing cankers and die-back on many woody plants. In Uruguay, Neofusicoccum eucalyptorum (=Botryosphaeria eucalyptorum), N. ribis (=B. ribis) and B. dothidea have previously been associated with stem cankers on plantation grown Eucalyptus globulus. These fungi also exist as endophytes in healthy Eucalyptus leaves, twigs and stems, typically causing disease after the onset of stress. There is good evidence to suggest that species of the Botryosphaeriaceae, other than those previously reported, could cause cankers on Eucalyptus spp. and native Myrtaceae trees in Uruguay. In this study, we identified the Botryosphaeriaceae present on Eucalyptus spp. and on native Myrtaceae trees, and considered the genetic diversity between isolates found on both groups of hosts. Symptomatic and asymptomatic material was collected countrywide from Eucalyptus spp. and native Myrtaceae. Monosporic cultures were identified based on conidial morphology and comparisons of DNA sequences for the ITS region of the rDNA operon. Results revealed that isolates of the N. parvum-N. ribis complex, and B. dothidea were present on both Eucalyptus spp. and native Myrtaceae. In contrast, N. eucalyptorum was found only on Eucalyptus spp. and Diplodia seriata-related (=B. obtusa) isolates were obtained only from native trees. This study expands the knowledge of the occurrence of Botryosphaeriaceae on native and introduced Myrtaceae in Uruguay.

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Agrociencia (2008) Vol XII N° 2 pág. 19 - 30
Botryosphaeriaceae from Eucalyptus and Native Myrtaceae in
Uruguay*
*This work was presented at the International Union of Forest Research Organizations (IUFRO) Conference, Working Group 2.08.03. «Eucalypts
and Diversity: Balancing Productivity and Sustainability», 22-26 October 2007,
Durban, South Africa.
Pérez, C. A.1,2; Altier, N.3; Simeto, S.3; Wingfield, M. J.4; Slippers, B.4 ; Blanchette, R. A.1
1Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall, 1991 Upper Buford Circle, MN 55108,
USA.
2Departamento de Protección Vegetal, EEMAC, Facultad de Agronomía, Universidad de la República, Ruta 3,
km 363, Paysandú, Uruguay.
3Instituto Nacional de Investigación Agropecuaria (INIA), Ruta 48, km 10, Canelones, Uruguay.
4Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.
E-mail: caperez@fagro.edu.uy
Recibido: 2/4/08 Aceptado: 30/12/08
Summary
Species of the Botryosphaeriaceae are important pathogens causing cankers and die-back on many woody plants. In
Uruguay, Neofusicoccum eucalyptorum (=Botryosphaeria eucalyptorum), N. ribis (=B. ribis) and B. dothidea have
previously been associated with stem cankers on plantation grown Eucalyptus globulus. These fungi also exist as
endophytes in healthy Eucalyptus leaves, twigs and stems, typically causing disease after the onset of stress. There
is good evidence to suggest that species of the Botryosphaeriaceae, other than those previously reported, could
cause cankers on Eucalyptus spp. and native Myrtaceae trees in Uruguay. In this study, we identified the
Botryosphaeriaceae present on Eucalyptus spp. and on native Myrtaceae trees, and considered the genetic diversity
between isolates found on both groups of hosts. Symptomatic and asymptomatic material was collected countrywide
from Eucalyptus spp. and native Myrtaceae. Monosporic cultures were identified based on conidial morphology and
comparisons of DNA sequences for the ITS region of the rDNA operon. Results revealed that isolates of the N.
parvum-N. ribis complex, and B. dothidea were present on both Eucalyptus spp. and native Myrtaceae. In contrast,
N. eucalyptorum was found only on Eucalyptus spp. and Diplodia seriata-related (=B. obtusa) isolates were obtained
only from native trees. This study expands the knowledge of the occurrence of Botryosphaeriaceae on native and
introduced Myrtaceae in Uruguay.
Key words: Botryosphaeria canker; Eucalyptus; Myrtaceae
Resumen
Botryosphaeriaceae aisladas de Eucalyptus y Mirtáceas nativas en
Uruguay
Varias species residentes en la familia Botryosphaeriaceae son importantes patógenos causantes de cancros y «die-
back» en numerosas plantas leñosas. En Uruguay, cancros del fuste observados en plantaciones de Eucalyptus globulus
han estado asociados a Neofusicoccum eucalyptorum (=Botryosphaeria eucalyptorum), N. ribis (=B. ribis) y B.
dothidea. Estos hongos también habitan como endófitos en hojas, ramas y fustes asintomáticos de Eucalyptus vol-
viéndose patógenos luego de la ocurrencia de algún estrés. Existen evidencias que sugieren que otras especies de
Botryosphaeriaceae, además de las antes mencionadas podrían causar cancros en Eucalyptus spp. y mirtáceas nati-
vas en Uruguay. En este estudio, identificamos las especies Botryosphaeriaceae presentes en Eucalyptus spp. y en

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mirtáceas nativas, y analizamos la diversidad genética entre los aislamientos aislados de ambos grupos de hospede-
ros. Muestras de material vegetal de Eucalyptus spp. y mirtáceas nativas con y sin síntomas fueron colectadas desde
diversos puntos del país. Los cultivos fúngicos monospóricos obtenidos de dichas muestras, fueron identificados en
base a la morfología de conídios y a técnicas moleculares mediante comparación de secuencias de ADN correspon-
dientes a la región ITS del ADN ribosomal. Los resultados muestran que aislamientos identificados dentro del
complejo N. parvum-N. ribis, y otros identificados como B. dothidea fueron aislados de ambos hospederos, Eucalyptus
spp. y mirtáceas nativas. Por el contrario, N. eucalyptorum fue aislado únicamente de Eucalyptus spp., mientras que
aislamientos de una especie relacionada a Diplodia seriata (=B. obtusa) fueron obtenidos únicamente de árboles
nativos. Este estudio permite expandir los conocimientos acerca de las especies Botryosphaeriaceae presentes en
mirtáceas nativas e introducidas en Uruguay.
Palabras clave: Botryosphaeria canker; Eucalyptus; Myrtaceae
Introduction
Eucalyptus is one of the most important hardwood
crops in the world (Turnbull, 2000), and it is currently
the major forest tree in Uruguay (MGAP, 2005). In the
last 10 years, the area planted to Eucalyptus in Uru-
guay has tripled. This increase is associated with
planting primarily two species, Eucalyptus globulus ssp.
globulus Labill (hereafter E. globulus) and Eucalyptus
grandis Hill ex Maid. that together make up over
480.000 ha (MGAP, 2005). Large numbers of these trees
are being generated by vegetative propagation and
stands of selected clones are commonly found.
The use of clonal material with similar genetic
characteristics over large areas of the country increases
the risk of disease outbreaks. In Uruguay, very little work
has been done on Eucalyptus pathogens and almost
nothing is known about the epidemiology and population
structure of the most important pathogens occurring on
these trees. Correct species identification and
characterization of these pathogens is a prerequisite for
effective breeding programs focused on obtaining du-
rable genetic resistance to diseases.
Eucalyptus spp. are exotic in Uruguay and pathogens
affecting these trees could also be introduced. Native
trees could also represent an important source of
Eucalyptus pathogens, as is being found elsewhere in
the world (Wingfield, 2003). Frequently, species
belonging to the Myrtaceae have been shown to be
potential hosts of pathogens that can infect Eucalyptus
spp. (Coutinho et al., 1998; Seixas et al., 2004;
Wingfield et al., 2001; Wingfield, 2003).
Severe diseases caused by fungi belonging to the
Botryosphaeriaceae have been reported on Eucalyptus
spp. worldwide. Stem cankers and die-back of
Eucalyptus spp. have been associated with
Botryosphaeria dothidea (Moug. : F.) Ces. and De Not.
(Barnard et al., 1987; Old and Davison, 2000; Smith et
al., 1994; Yuan and Mohammed, 1999), although these
reports probably refer to a number of different species
of Botryosphaeriaceae. However, very little is known
about Botryosphaeriaceae infecting exotic Eucalyptus
or native Myrtaceae trees in Uruguay. Endophytic
Botryosphaeria dothidea, Neofusicoccum eucalyptorum
(Crous, Smith ter and Wingf.) Crous, Slippers and
Phillips and N. ribis (Slippers, Crous and Wingf.) Crous,
Slippers & Phillips have been found in some Eucalyptus
spp. (Alonso, 2004; Bettucci and Alonso, 1997; Simeto
et al., 2005), while Myrceugenia glaucescens (Camb.)
Legr. and Kaus. is the only native Myrtaceae host where
a species of Botryosphaeriaceae, B. dothidea has been
found (Bettucci et al., 2004). Further investigation on
endophytic populations of native Myrtaceae and species
of Eucalyptus is very important since it is well known
that certain endophytic fungi become pathogenic in
stressed trees (Old et al., 1990; Pusey, 1989; Wene and
Schoeneweiss, 1980). Thus, the aim of this work was to
increase the knowledge of species of Botryosphaeriaceae
occurring on Eucalyptus as well as native Myrtaceae in Uru-
guay.
Materials and Methods
Fungal isolates
With the aim of isolating and identifying fungi present
on native Myrtaceae and exotic Eucalyptus species,
during 2005 and 2006, Eucalyptus plantations and na-
tural forest growing in close (<1 km) proximity to
Eucalyptus were scouted throughout Uruguay. Surveys
included the provinces of Cerro Largo, Durazno, Flori-
da, Lavalleja, Maldonado, Paysandú, Río Negro, Rive-
ra, Tacuarembó, Treinta y Tres and Rocha. A total of 21
Myrtaceae species native to Uruguay and 10 species of
Eucalyptus were examined (Table 1). Symptomatic and
asymptomatic material was collected. Endophytic
Pérez, C. A.; Altier, N; Simeto, S; Wingfield, M. J.; Slippers, B.; Blanchette, R. A.

21
microorganisms were isolated from asymptomatic fresh
material. Leaf, petiole and twig sections were
sequentially surface-sterilized in 70 % ethyl alcohol
for 1 min, immersed in 0.4 % sodium hypochlorite for
2 min, then rinsed twice in sterile distilled water and
blotted dry on sterile filter paper. Surface sterilized
plant tissue was placed on 2 % malt extract agar (MEA)
(2 % malt extract, 1.5 % agar; Oxoid, Basingstoke,
England). Plates were incubated at room temperature
(~20º C) for one week. Colonies resembling
Botryosphaeriaceae were selected for this study, and
maintained in 2 % MEA at 8º C. To verify the efficacy
of the surface sterilization and to assure the growth of
only endophytic microorganisms, imprints of sample
surfaces were made on MEA plates and observed for
one week to confirm that fungi did not grow.
To stimulate isolates to produce fruiting structures
(pycnidia) and conidia, they were grown on 1.5 % water
agar (WA) (Sigma Chemicals, St. Louis, MO) with
sterilized pine needles placed onto the medium surface.
Plates were incubated at 22º C under continuous black
light until pycnidia were observed on the pine needles
(approx. 3 weeks after plating). Monosporic cultures
were generated by plating a spore suspension taken from
two pycnidia, suspended in 300 ìl of sterile water on
WA. Germinating conidia were lifted from the agar
plates and transferred to fresh 2 % MEA.
Myrtaceae species native to Uruguay Eucalyptus species
Acca sellowiana E. camaldulensis
Agariota eucalyptides E. cinerea
Blepharocalyx salicifolius E. dunnii
Calyptranthes concinna E. ficifolia
Eugenia involucrata E. globulus
E. mansonii E. grandis
E. repanda E. maidenii
E. uniflora E. robusta
E. uruguayensis E. tereticornis
Gomidesia palustris E. viminalis
Hexachlamis edulis
Myrceugenia euosma
Myrce. glaucescens
Myrcianthes cisplatensis
Myrci. pungens
Myrciaria tenella
Myrrhinium atropurpureum var. octandrum
Psidium luridum
P. incanum
P. pubifolium
Table 1. List of species of native Myrtaceae and exotic Eucalyptus, sampled in this study.
Botryosphaeriaceae from Myrtaceae in Uruguay

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Morphology
For morphological characterization, pycnidia and
conidia produced on pine needles were mounted on
microscope slides, examined under a standard light
microscope Nikon Eclipse E600 and photographed with
a Nikon Digital Camera DXM1200F (Nikon Inc.,
Melville, NY). A total of 53 isolates with structures
resembling the Botryosphaeriaceae were obtained from
different hosts. Isolates were grouped by conidial
morphology and only one specimen per group was
further analyzed from each sampled tree, totalizing 29
isolates.
DNA extraction, PCR, sequencing and
phylogenetic analysis
For DNA extraction, the 29 isolates listed in Table 2
were grown in 2 % malt extract agar (MEA) at room
temperature for 10 days. Mycelium was scrapped
directly from the colonies on the plates and transferred
to Eppendorf tubes (1.5 ml) with 1-mm glass beads and
extraction buffer (Qiagen Inc., Valencia, CA). These
were vigorously shaken using a vortex mixer and placed
in a water bath at 60º C for 1 hr. DNA extraction was
performed using the Qiagen Plant DNeasy Mini Kit
(Qiagen Inc., Valencia, CA) following manufacturer’s
instructions.
Primers ITS1 (5’ TTC GTA GGT GAA CCT GCG G
3’) and ITS4 (5’ TCC TCC GCT TAT TGA TAT GC 3’)
(White et al., 1990) were used to amplify the internal
transcribed spacer region of the ribosomal DNA operon
(ITS). Polymerase Chain Reactions (PCR) were
performed in a 25-μl reaction mixture of 1.0 μl of 0.05%
casein, 12.5 μl of Amplitaq Gold PCR Master-Mix
(Applied Biosystems, Foster City, CA), 1.0 μl of 10 mM
ITS1, 1.0 μl of 10 mM ITS4, 8.5 μl of ddH2O and
1.0 μl of DNA template. PCR amplifications were
performed in a MJ Research PTC 200 DNA Engine
Thermal Cycler PCR (MJ Research, Reno, NV) with the
following parameters: 5 min at 94º C; 1 min at 94º C;
1 min at 50º C; 1 min at 72º C; cycle to step 2, 35 times;
5 min at 72º C; hold at 10º C.
PCR products were visualized on agarose gels,
purified and prepared for sequencing using ExoSAP-IT
PCR clean-up kit (USB Corp., Cleveland, OH) following
manufacturer’s instructions. Sequencing reactions were
performed using the same primers with the ABI Prism
Dye Terminator Cycle Sequencing Ready Reaction Kit
(Applied Biosystems, Foster City, CA) and an ABI Prism
377 automated DNA sequencer. Sequences were
obtained in both directions and assembled using
ChromasPro software version 1.33 (Technelysium Pty.
Ltd., Eden Prairie, MN). Sequences obtained in this
study were aligned with sequences of different species
in the Botryosphaeriaceae available in GenBank (Table
2). Multiple sequence alignments were made by using
Discovery Studio Gene v1.5 (Accelrys Inc., San
Diego, CA).
Phylogenetic analysis was performed using PAUP
Version 4.0b10a (Swofford, 2002). Neighbor-joining
analysis used the uncorrected «p» substitution model.
Gaps generated in the alignment process during the
comparison were treated as missing data and all
characters were treated as unordered and of equal
weight. Ties were broken randomly when found.
Maximum parsimony analysis was performed using the
heuristic search option with simple taxa additions and
tree bisection and reconstruction (TBR) as the branch-
swapping algorithm. Support for the nodes of the
shortest trees was determined by analysis of 1,000
bootstrap replicates (Hillis and Bull, 1993). Tree length
(TL), consistency index (CI), retention index (RI), and
rescaled consistency index (RC) were calculated. The
trees were rooted using the GenBank sequences of
Guignardia philoprina Berk. and Curtis and
Teratosphaeria africana (Crous and Wingf.) Crous and
Braun.
Results
Fungal Isolates
A total of 29 isolates of Botryosphaeriaceae were
obtained from different Eucalyptus spp. and native
Myrtaceae trees (Table 2). Isolates UY37 and UY88
were obtained from dead tissue of E. grandis pruning
residue, and isolates UY1050, UY1263, and UY1366
from stem canker lesions. The remaining isolates were
obtained from asymptomatic plant material. All isolates
produced conidiomata after three weeks of incubation
on water agar with pine needles. Monosporic cultures
were obtained from these structures.
Morphology and DNA sequence comparisons
Phylogenetic analysis showed that the 29 analyzed
isolates reside in the Botryosphaeriaceae. The alignment
contained 58 ingroup taxa and 2 outgroup taxa. Out of
503 total characters, 305 were constant, 74 variable
characters were parsimony-uninformative and 104 were
parsimony informative. Heuristic search analysis of the
data resulted in one tree (TL = 342 steps; CI = 0.711;
RI = 0.894; RC = 0.635; HI = 0.289) (Figure 1).
Pérez, C. A.; Altier, N; Simeto, S; Wingfield, M. J.; Slippers, B.; Blanchette, R. A.

23
Culture ID# Fungus species Host species GenBank
accession #
UY9 * Botryosphaeria dothidea Blepharocalyx salicifolius EU080907
UY16* Neofusicoccum parvum-N. ribis Blepharocalyx salicifolius EU080908
UY37* N. parvum-N. ribis Eucalyptus grandis EU080909
UY40* Neofusicoccum eucalyptorum Eucalyptus grandis EU080910
UY48* B. dothidea Eucalyptus grandis EU080911
UY52* N. parvum-N. ribis Eucalyptus grandis EU080912
UY88* N. eucalyptorum Eucalyptus grandis EU080913
UY107* Diplodia seriata -related Myrcianthes cisplatensis EU080914
UY118* N. parvum-N. ribis Eugenia uruguayensis EU080915
UY119* B. dothidea Eugenia uruguayensis EU080916
UY231* N. parvum-N. ribis Blepharocalyx salicifolius EU080917
UY339* B. dothidea Myrceugenia glaucescens EU080918
UY394* N. eucalyptorum Eucalyptus dunnii EU080919
UY543* N. parvum-N. ribis Eugenia repanda EU080920
UY587* N. eucalyptorum Eucalyptus tereticornis EU080921
UY671* D. seriata-related Hexachlamys edulis EU080922
UY672* Dothiorella iberica-related Hexachlamys edulis EU080923
UY693* D. seriata-related Eugenia uniflora EU080924
UY719* B. dothidea Myrrhinium atropurpureum var.
octandrum EU080925
UY754* N. parvum-N. ribis Eucalyptus ficifolia EU080926
UY788* D. seriata-related Blepharocalyx salicifolius EU080927
UY1050* N. parvum-N. ribis Eucalyptus globulus EU080928
UY1070* N. eucalyptorum Eucalyptus maidenii EU080929
Table 2. List of isolates used in this study, including those for which sequences downloaded from GenBank+.
Botryosphaeriaceae from Myrtaceae in Uruguay
Continúa

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UY1190* N. eucalyptorum Eucalyptus globulus EU080930
UY1225* D. seriata-related Acca sellowiana EU080931
UY1233* N. eucalyptorum Eucalyptus viminalis EU080932
UY1263* D. seriata-related Myrciaria tenella EU080933
UY1366* N. parvum-N. ribis Blepharocalyx salicifolius EU080935
CMW10122 Neofusicoccum parvum Eucalyptus grandis AF283681
CMW10125 N. eucalyptorum Eucalyptus grandis AF283686
CMW7775 D. seriata Ribes sp. AY236954
CBS115041 Do. iberica Quercus ilex AY573202
CMW6235 N. parvum Tibouchina lepidota AY615136
CMW6804 N. eucalyptorum Eucalyptus dunnii AY615139
CMW6229 Neofusicoccum eucalypticola Eucalyptus grandis AY615142
CMW6217 N. eucalypticola Eucalyptus rosii AY615143
CMW14077 Lasiodiplodia gonubiensis Syzygium cordatum AY639595
CMW13434 Pseudofusicoccum stromaticum Eucalyptus hybrid AY693974
WAC12396 Neofusicoccum ribis Eucalyptus grandis x E.
camaldulensis AY744369
WAC12398 Dichomera eucalypti Eucalyptus diversicolor AY744371
WAC12402 D. eucalypti Eucalyptus camaldulensis AY744373
WAC12399 Neofusicoccum australe Eucalyptus diversicolor AY744374
WAC12400 N. australe Eucalyptus marginata AY744375
WAC12403 Dichomera versiformis Eucalyptus camaldulensis AY744376
VPRI31988 D. versiformis Eucalyptus pauciflora AY744377
WAC12404 B. dothidea Eucalyptus calophylla AY744378
CMW15950 N. parvum Eucalyptus globulus DQ093193
CMW15948 Neofusicoccum macroclavatum Eucalyptus globulus DQ093197
Pérez, C. A.; Altier, N; Simeto, S; Wingfield, M. J.; Slippers, B.; Blanchette, R. A.
Continúa

25
Based on the analyzed DNA sequences, five different
Botryosphaeriaceae species are represented in the 29
isolates analyzed. Thus, five isolates clustered with B.
dothidea, eight isolates clustered with N. eucalyptorum,
six isolates were closely related to Diplodia seriata De
Not. (=B. obtusa (Schwein.) Shoemaker), and nine
isolates clustered with the N. parvum-N. ribis complex.
The remaining isolate is likely related to Dothiorella
iberica Phillips, Luque and Alves (=B. iberica Phillips,
Luque and Alves), but the bootstrap value was low.
Morphological comparisons confirmed the results of
the DNA sequence comparisons. Botryosphaeria
dothidea was found endophytically in Eucalyptus
grandis and in four native Myrtaceae species namely
Blepharocalyx salicifolius (Kunth) Berg, Eugenia
uruguayensis Cambess, Myrceugenia glaucescens and
Myrrhinium atropurpureum Schott var. octandrum
Benth. Isolates identified as N. parvum-N. ribis complex
were found in both Eucalyptus spp. and native
Myrtaceae. These were obtained from asymptomatic
plant tissue from Blepharocalyx salicifolius, Eugenia
uruguayensis and Eugenia repanda Berg, while the
isolate UY1366 was obtained from a stem canker on
Blepharocalyx salicifolius. On Eucalyptus, however,
they were isolated as endophyte (in E. grandis and E.
ficifolia Muell.). They were also found sporulating on
dead tissue (on E. grandis debris) and from stem cankers
(on E. globulus).
Seven isolates emerging from this study were
identified as N. eucalyptorum. Six of these were
endophytes on different Eucalyptus species (E. grandis,
E. dunnii Maiden, E. tereticornis Sm., E. maidenii
Muell, E. globulus and E. viminalis Cunn. ex Schauer),
and the remaining was found sporulating on dead E.
grandis tissue. On the other hand, Dothiorella seriata-
related isolates were obtained only from Myrtaceous
trees but not found on Eucalyptus samples. Finally, a
Dothiorella iberica-related species was found as
endophyte in Hexachlamys edulis (Berg) Kausel and
Legrand, a native Myrtaceous tree.
CMW15947 N. macroclavatum Eucalyptus saligna DQ093199
CMW14012 N. ribis Syzygium cordatum DQ316073
CMW14030 N. parvum Syzygium cordatum DQ316077
CMW13998 Neofusicoccum magniferae Syzygium cordatum DQ316081
CMW14009 B. dothidea Syzygium cordatum DQ316084
CMW14071 Neofusicoccum luteum Syzygium cordatum DQ316088
CMW14074 N. australe Syzygium cordatum DQ316089
CMW14116 Lasiodiplodia theobromae Syzygium cordatum DQ316092
CMW568 D. seriata Malus sp. DQ836726
CMW3025 Teratosphaeria africana Eucalyptus viminalis AF283690
CMW7063 Guignardia philoprina Taxus baccata AF312014
(+) Cultures marked with a (*) were sequenced in this study. Sequence data for other cultures were taken from GenBank and result from the
studies of Barber et al., 2005; Burgess et al., 2005; Gure et al., 2005; Mohali et al., 2007; Pavlic et al., 2007; Slippers et al., 2004b, 2007
Botryosphaeriaceae from Myrtaceae in Uruguay

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Figure 1. Phyogenetic relationship among the fungal isolates obtained in this study from native Myrtaceae trees and
exotic Eucalyptus plantations in Uruguay and sequences of Botryosphaeriaceae downloaded from GenBank. The
phylogenetic tree was constructed using neighbor-joining analysis, with the uncorrected «p» model on the ITS rDNA
sequences. The tree was rooted with Mycosphaerella africana and Guignardia phyloprina. Bootstrap values greater
than 50% from 1000 replications of the heuristic search are shown at the nodes.
Pérez, C. A.; Altier, N; Simeto, S; Wingfield, M. J.; Slippers, B.; Blanchette, R. A.

27
Discussion
In this study, we found five species of
Botryosphaeriaceae on Eucalyptus and native Myrtaceae
in Uruguay. Botryosphaeria dothidea was previously
reported as an endophyte infecting eucalypts (Bettucci and
Alonso, 1997; Smith et al., 1996) and also causing stem
cankers on eucalypts in Uruguay (Balmelli et al., 2004)
and other countries (Smith et al., 1994). However,
identification of species of Botryosphaeriaceae prior to
the application of DNA sequence comparisons indicates
that reference to B. dothidea probably implies a suite
of different species. Thus, some of the isolates
previously considered to be B. dothidea have
subsequently been identified as Neofusicoccum parvum
(Pennycook and Samuels) Crous, Slippers and Phillips
and N. ribis (Slippers et al., 2004b).
With the recent taxonomic concept of
Botryosphaeriaceae (Crous et al., 2006), B. dothidea
has been infrequently isolated from Eucalyptus spp. and
it has been suggested that this fungus may not be an
important pathogen of these trees (Slippers et al., 2004b;
Pavlic et al., 2007). Nevertheless, little is known about
the fungus and pathogenicity tests using different
isolates should be carried out in order to more accurately
evaluate its importance as a pathogen of Eucalyptus.
Bettucci et al. (2004) reported the presence of
endophytic B. dothidea in Myrceugenia glaucescens, a
Myrtaceous tree native to Uruguay. In this study, we
confirmed this finding and also found endophytic
infections of B. dothidea on Blepharocalyx salicifolius,
Eugenia uruguayensis, and Myrrhinium atropurpureum
var. octandrum. These results support the wide host
range for the fungus reported by Michialides et al.
(2001).These findings also emphasize the need to
consider native Myrtaceae when sampling for a
population structure study of B. dothidea in Uruguay.
Collection of isolates residing in the Neofusicoccum
parvum-N. ribis complex, was not surprising as this
fungus is known to be common on Eucalyptus. Slippers
et al. (2004a) used a multiple gene genealogy to provide
strong evidence that N. parvum and N. ribis represent
different species. They also recommend caution when
distinguishing between these two species based on
morphology or sequence data for only a single DNA
locus. Therefore, the isolates identified as belonging
to N. parvum-N. ribis complex in this study must await
more detailed comparisons using multiple gene
approach.
Neofusicoccum parvum has been found on a wide
range of hosts including certain Myrtaceae trees
worldwide (Barber et al., 2005; Burgess et al., 2005;
Gure et al., 2005; Mohali et al., 2007; Pavlic et al.,
2007; Slippers et al., 2004b). Slippers et al. (2004b)
showed that N. parvum rather than other species of the
Botryosphaeriaceae was associated with disease of
Eucalyptus in South Africa. In addition, N. parvum was
reported as an important die-back and stem canker
pathogen of Eucalyptus in Ethiopia, Republic of Con-
go, and Uganda (Gezahgne et al., 2004; Smith et al.,
1994, Roux et al., 2001). To the best of our knowledge,
this species has not previously been found in Uruguay.
However, Alonso (2004) reported the presence of N.
ribis on Eucalyptus globulus based on the morphology
and comparisons of sequence data for the ITS region of
the rDNA operon. Due to the complexity of
distinguishing between N. parvum and N. ribis, further
analyses are required to confirm this report.
Neofusicoccum ribis has a wide host range and it has
been found on certain Eucalyptus spp. (Barber et al.,
2005; Mohali et al., 2007), Myrtaceae species (Pavlic
et al., 2007) and other non-Myrtaceous hosts (Denman
et al., 2003; Zhou et al., 2001). This fungus was
associated with the death of E. radiata in Australia
(Shearer et al., 1987), and even with some concerns
raised about the species identification at that time, Pavlic
et al (2007) concluded that N. ribis is the most
pathogenic species of Botryosphaeriaceae on the
Eucalyptus clones used in their study. These reports
reinforce the need for correct identification as well as
to assess the pathogenicity of Botryosphaeriaceae
occurring on eucalypts.
Neofusicoccum eucalyptorum has previously been
reported in Uruguay as an endophyte in Eucalyptus
globulus or from bark lesions (Alonso, 2004; Bettucci,
2003). In the present study, this fungus was found on a
large number of different Eucalyptus spp. and many of
these are new host records for this fungus. It was not
found on native Myrtaceae and it might represent a non–
native pathogen accidentally introduced into Uruguay.
Smith et al. (2001) analyzed the pathogenicity of
several isolates of N. eucalyptorum, and concluded that
even when isolates of N. eucalyptorum were less virulent
than those of B. dothidea, it was clear that N.
eucalyptorum is pathogenic to eucalypts. Therefore,
pathogenicity tests are needed to assess the importance
of isolates obtained in this study. Likewise, it will be
important to confirm that the fungus does not occur on
native Myrtaceae, in which case it might also pose a
threat to these trees in Uruguay .
Diplodia seriata-related (=‘Botryosphaeria’ obtusa)
isolates were found as endophytes on five different
Botryosphaeriaceae from Myrtaceae in Uruguay

AGROCIENCIA
28
native Myrtaceae. It was also isolated from a stem canker
on Myciaria tenella. The phylogenetic tree shows that
these isolates are related to D. seriata but they formed
a defined group that suggests they could be a different
species. Since spores produced by the fungus resembled
those produced by D. seriata, multiple gene analysis is
required to resolve the identity of this group of isolates.
Diplodia seriata has been reported causing severe
disease on many different hosts (Britton and Hendrix,
1989; Brown-Rytlewski and McManus, 2000; Phillips
et al., 2007; Pusey, 1993; Swart and Botes, 1995).
However, it has not been found on Eucalyptus species
elsewhere in the world, and was also not isolated from
this host during this study. The presence of this fungus
on native Myrtaceae might indicate a potential threat to
Eucalyptus plantations. Pathogenicity tests are thus
planned to gain a better understanding of its potential
hazard to introduced Eucalyptus species.
A single isolate in this study was related to
Dothiorella iberica (=‘Botryosphaeria’ iberica). This
fungus was found as an endophyte on a member of the
Myrtaceae, Hexachlamys edulis. Dothiorella iberica has
recently been described by Phillips et al. (2007) and
the only known hosts are Malus and Quercus (Phillips
et al., 2005). Further investigation is needed to confirm
the identity of the isolate and test its pathogenicity to
Eucalyptus spp.
This study expands the knowledge of the occurrence
of Botryosphaeriaceae on native and introduced
Myrtaceae in Uruguay. It has not been experimentally
shown whether organisms that produce diseases
affecting these trees in Uruguay are native or have been
introduced. Since Uruguay has a reasonably large
number of native trees in the Myrtaceae family that are
related to Eucalyptus it could be hypothesized that these
native trees could be the hosts of some pathogens
affecting Eucalyptus spp. Results of this study evidence
B. dothidea and N. parvum-N. ribis complex occurring
in both native and introduced trees. Future work will
focus on understanding the relationships among these
fungi and their respective host. Reciprocal pathogenicity
tests and population biology studies should be
undertaken to obtain a better understanding of this
pathosystem and to better assist breeding programs
aimed at elevating resistance to diseases.
Acknowledgments
We acknowledge to Colonvade, Forestal Oriental,
Rivermol and Stora Enso for partially fund this project.
This project was also partially funded by the agreement
of cooperation FPTA221 INIA-Facultad de Agronomía.
Also thanks to Andrés Berrutti, Ana Terzaghi and Gusta-
vo Balmelli for their help during the surveys.
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