Cercosporoid hyphomycetes associated with Tibouchina herbacea (Melastomataceae) in Brazil

Abstract

Abstract A survey of fungal pathogens associated with cane
tibouchina, Tibouchina herbacea (Melastomataceae), was
conducted in the neotropics, concentrated in south and southeastern
Brazil and aimed at finding potential biological control
agents for this herbaceous invasive weed for introduction into
Hawaii. Numerous fungal species were found, including five
cercosporoid fungi. Four new taxa are described and illustrated
here: Passalora tibouchinae, Pseudocercospora
subsynnematosa, Pseudocercospora tibouchina-herbaceae
and Pseudocercospora tibouchinicola. A phylogenetic analysis
of the ITS and TEF-1α regions of the three new species of
Pseudocercospora was conducted and confirmed their separation
as distinct taxa as indicated by morphological and
cultural features. Additionally, Cercospora apii is reported
for the first time on T. herbacea. Among the species of fungi
described in this study, two appear particularly promising for
use in a biological control program because they were found
to cause severe disease on T. herbacea: Passalora tibouchinae
and Pseudocercospora subsynnematosa. Additional tests, including
host-specificity screening, will be required in order to
confirm this potential.

Full text

ORIGINAL ARTICLE
Cercosporoid hyphomycetes associated with Tibouchina
herbacea (Melastomataceae) in Brazil
Douglas Ferreira Parreira &Meiriele da Silva &
Olinto Liparini Pereira &Dartanha José Soares &
Robert Weingart Barreto
Received: 19 June 2010 /Revised: 5 December 2013 /Accepted: 9 December 2013
#German Mycological Society and Springer-Verlag Berlin Heidelberg 2014
Abstract A survey of fungal pathogens associated with cane
tibouchina, Tibouchina herbacea (Melastomataceae), was
conducted in the neotropics, concentrated in south and south-
eastern Brazil and aimed at finding potential biological control
agents for this herbaceous invasive weed for introduction into
Hawaii. Numerous fungal species were found, including five
cercosporoid fungi. Four new taxa are described and illustrat-
ed here: Passalora tibouchinae,Pseudocercospora
subsynnematosa,Pseudocercospora tibouchina-herbaceae
and Pseudocercospora tibouchinicola. A phylogenetic analy-
sis of the ITS and TEF-1αregions of the three new species of
Pseudocercospora was conducted and confirmed their sepa-
ration as distinct taxa as indicated by morphological and
cultural features. Additionally, Cercospora apii is reported
for the first time on T. herbacea. Among the species of fungi
described in this study, two appear particularly promising for
use in a biological control program because they were found
to cause severe disease on T. herbacea:Passalora tibouchinae
and Pseudocercospora subsynnematosa. Additional tests, in-
cluding host-specificity screening, will be required in order to
confirm this potential.
Keywords Classical biological control .Cercospora .Fungal
survey .New taxa .Passalora .Pseudocercospora
Introduction
Tibouchina herbacea (DC.) Cogn. (Melastomataceae), known
as cane tibouchina (local names in Brazil: quaresmeirinha,
quaresmeira do brejo), is a herbaceous plant of humid habitats
that is native to South America, namely Argentina, Brazil,
Paraguay and Uruguay (Wagner et al. 1999). It was introduced
into the Hawaiian Islands as an ornamental for its showy violet
flowers and has since become a noxious invasive weed. It is
presently found in the islands of Maui, Hawaii, Molokai, Oahu
and Lanai (http://www.hear.org/starr/hiplants/maps/
tibouchina_herbacea.htm). There are no native species of
Melastomataceae in Hawaii and all species in this family that
have been introduced are noxious weeds, including Clidemia
hirta (L.) D. Don and Miconia calvescens DC., which are
ranked amongst the worst weeds in that archipelago (DeWalt
et al. 2003). In its native habitat, T. herbacea reaches a maxi-
mumheightof1.5m,whereasinHawaiitcanreachupto4m,
forming dense stands in pastures and disturbed areas (Almasi
2000; Motooka et al. 2003). It is included in the State of Hawaii
noxious weed list (http://hear.org/pier/species/tibouchina_
herbacea.htm) and its management has been made mainly
through herbicide applications. However, classical biological
control has been recognized as the sole sustainable
method of control for exotic weeds invading natural
ecosystems, as is the case of the weedy melastomes in
the Pacific (Wikler and Souza 2005).
Taxonomical novelty: Passalora tibouchinae D.F. Parreira, O.L. Pereira
& R. W. Barreto Pseudocercospora subsynnematosa D.F. Parreira, D.J.
Soares & R. W. Barreto, Pseudocercospora tibouchina-herbaceae D.F.
Parreira & R.W. Barreto, Pseudocercospora tibouchinicola D.F. Parreira,
D.J. Soares & R. W. Barreto.
D. F. Parreira :M. da Silva :O. L. Pereira :D. J. Soares :
R. W. Barreto (*)
Departamento de Fitopatologia, Universidade Federal de Viçosa
(UFV), Viçosa 36570-000, MG, Brazil
e-mail: rbarreto@ufv.br
D. F. Parreira
e-mail: douglas.parreira@ufv.br
M. da Silva
e-mail: meiriele.silva@ufv.br
O. L. Pereira
e-mail: oliparini@ufv.br
D. J. Soares
e-mail: dartanha.soares@embrapa.br
Mycol Progress
DOI 10.1007/s11557-013-0952-0

Insect surveys in Brazil yielded a series of potential bio-
control agents to be deployed against T. herbacea,but
Syphraea uberabensis (Bechhyné, 1956) (Coleoptera:
Chrysomelidae) was demonstrated to have the greatest poten-
tial (Wikler and Souza 2005;Souder2008). Studies aimed at
introducing this particular insect are well advanced. Syphraea
uberabensis is now under quarantine in Hawaii for its final
evaluation (T. Johnson, personal communication). Parallel to
this, systematic field surveys of the mycobiota on T. herbacea
were also conducted. This publication represents the first
study of the mycobiota of T. herbacea in Brazil and is the first
account of fungi associated with this plant species worldwide.
No records of fungi associated with T. herbacea were found
in the literature and it was expected that a significant propor-
tion of the fungi collected on this host may represent taxo-
nomic novelties and that some could be potential biocontrol
agents for use against this host. Sixteen fungal species have
been identified, thus far. This paper concentrates on the de-
scriptions and a discussion of five cercosporoid hyphomy-
cetes. The remaining mycobiota will be covered in a separate
publication. Tibouchina herbacea is regarded as a species
complex that remains unresolved (R. Goldenberg, personal
communication), and the records provided here include fungi
collected on herbaceous forms of Tibouchina which may
belong to more than one host species. More precise informa-
tion on host identity will await further clarification of species
delimitation in this complex.
Material and methods
Prior to the field survey, a list of collecting localities from
herbarium records of T. herbacea was compiled from the
following Brazilian herbaria: Herb. IAC, Herbário
Fanerogâmico e Criptogâmico do Instituto Agronômico;
Herb. SP, Herbário Maria Eneyda P. K. Fidalgo; Herb. RB,
Instituto de Pesquisas Jardim Botânico do Rio de Janeiro;
Herb. R, Herbário do Museu Nacional do Rio de Janeiro;
Herb. VIC, Universidade Federal de Viçosa; Herb. MBM,
Museu Botânico Municipal; Herb. OUPR, Herbário José
Badini. A range of sites in southeastern and southern Brazil
was explored based on the herbarium records. Details of the
survey and the laboratory methodology can be found in
Barreto and Evans (1994).
Morphological studies
Diseased T. h e r b acea plants found in the field were
photographed (Fig. 1) and samples were collected and dried
in a plant press and taken to the laboratory for further obser-
vation. Each sample was examined under a stereomicroscope
(Olympus SZ X7). Sections containing the fungal structures
and fungal structures scraped with a scalpel from the plant
surfaces were mounted in lactophenol. Observations, mea-
surements and line drawings were prepared using an Olympus
BX 51 light microscope fitted with a drawing tube and an
Olympus E330 camera. The collections examined were de-
posited in the herbarium of the Universidade Federal de
Viçosa (VIC).
Isolates
Pure cultures were obtained by transfer of conidia from co-
nidiophore fascicles on leaves onto potato dextrose agar
(PDA) plates with a sterile fine pointed needle. The cultures
obtained were deposited in Coleção de Culturas de Fungos
Fitopatogênicos ‘Prof. Maria Menezes’(CMM) (Table 1)and
in the culture collection of the Universidade Federal de Viçosa
(COAD). Plates were seeded with a 7-mm culture disk ob-
tained from the margin of actively growing cultures and
placed in the center of each Petri plate. Colony characters
and pigment production on PDA and V8 juice-agar (V8) were
observed in colonies incubated at 25 °C under two conditions:
complete darkness and 12/12 h light/dark. Descriptions were
prepared after 20 days of incubation. Colony colors were rated
according to Rayner (1970).
Phylogenetic analysis
Mycelium was scraped from cultures grown on PDA plates,
placed in a sterile 1.5-mL microcentrifuge tube and ground
into fine powder using liquid nitrogen. Genomic DNA of the
isolates was extracted using the Wizard
®
Genomic DNA
Purification kit (Promega) according to the manufacturer’s
instructions.
Two nuclear gene regions were targeted for PCR amplifi-
cation. The internal transcribed spacer region (ITS) and the
partial gene regions of the translation elongation factor 1-
alpha (TEF-1α). These genes were amplified and sequenced
using the primer pair ITS-1 and ITS-4 (White et al. 1990)and
EF1-728 F and EF1-986R (Carbone and Kohn 1999).
PCR conditions for each 25 μL reaction were as follows:
2μL of genomic DNA (25 ng/μL), 12.5 μLofDreamTaqTM
PCR Master Mix 2X (MBI Fermentas), 1 μLofeachprimer
synthesized by Invitrogen, 1 μL of dimethyl sulfixide
(DMSO, Sigma-Aldrich), 5 μL of 100× (10 mg/ml) bovine
serum albumin (BSA; Sigma-Aldrich) and nuclease-free wa-
ter to complete the volume. The amplifications were carried
out starting with a BIO RAD C1000 (Thermal Cycler) with
initial denaturation at 95 °C for 5 min, followed by 35 cycles
of 94 °C for 60 s, annealing at 60 °C for ITS and at 56 °C for
EF for 45 s, 72 °C for 60 s and a final extension of 72 °C
at 7 min. Amplified products were visualized on 1 %
agarose gel stained with GelRed™and viewed under UV
light to check for product size and purity. PCR products
Mycol Progress

were purified and sequenced by Macrogen, South Korea
(http://www.macrogen.com).
The DNA sequences obtained from forward and reverse
primers were used to obtain consensus sequences using DNA
Dragon software (Hepperle 2011). The sequences obtained in
this study were deposited in GenBank and compared against
other sequences of ITS and TEF1-1αin NCBI’sGenBank
nucleotide (nr) database using a megablast search program to
identify their closest species and alignments were constructed
based on these results (Table 1). Sequences for relevant taxa
for this study such as Pseudocercospora dissotidis (Chupp &
Doidge) Crous & U. Braun and Pseudocercospora tamoneae
(Chupp) U. Braun & R.F. Castañeda have not been included
because there are no sequences available for these in public
databases. The hit sequences were then downloaded in
FASTA format and aligned using multiple sequence alignment
program MUSCLE (Edgar 2004). Alignments were manually
adjusted when necessary in MEGAv.5 software (Tamura et al.
2011). Bayesian inference analyses (BI) were performed with
single and concatenated gene regions (ITS and FE-1α).
MrMODELTEST 2.3 (Posada and Buckley 2004) was used
to select the models of nucleotide substitution for each gene
region and included in the BI analysis. Once the likelihood
scores were calculated, the models were selected according to
the Akaike Information Criterion (AIC) applying a SYM+I+G
for ITS and HKY+G for FE-1α. A phylogenenetic analysis of
the concatenated aligment was performed on CIPRES
webportal (Miller et al. 2010) using MrBayes v.3.1.1
(Ronquist and Huelsenbeck 2003). Analyses of four Markov
chain Monte Carlo (MCMC) were run from random trees for
10000000 generations and sampled every 1,000 generations,
resulting in 10,000 saved trees. The first 2,500 trees, which
represented the burn-in phase of the analyses, were discarded
and posterior probabilities (Rannala and Yang 1996)
Fig. 1 Selected leaf spot
symptoms on Tibo uc hin a
herbaceaeto which cercosporoids
collected in the survey were
associated: 1 Passalora
tibouchinae;2 Pseudocercospora
subsynnematosa;3
Pseudocercospora tibouchina-
herbaceae;4 Pseudocercospora
tibouchinicola
Mycol Progress

determined from the remaining trees. New sequences
generated in this study were deposited in NCBI’s
GenBank nucleotide database (www.ncbi.nlm.nih.gov;
Tab le 1). Phylogenetic trees werevisualizedinFigTree
(Rambaut 2009) and resulting alignment was deposited into
TreeBASE (TreeBASE study S14808). (www.treebase.org/
treebase/index.html).
Results
Phylogeny
For the combined analysis, DNA sequence data from the ITS
and TEF1-αgene regions were utilized. The final alignment
contained 29 ingroup taxa with a total of 812 characters and
Septoria rubi (CPC 12331; GenBank Acession: GU269865,
GU483576) served as outgroup taxon. From the combined
alignement of 812 characters, 177 were parsimony informa-
tive, 256 were variable, and 527 were conserved.
The molecular analysis (Fig. 2) confirmed that the three
Pseudocercospora species found on Tibouchina herbaceae,as
indicated by the morphology data, are distinct and warrant
recognition as separate and novel species.
Tax onomy
Cercospora apii Fresen. sensu lato, emend. Crous and Braun,
Mycosphaerella and its anamorphs: 1. Names published in
Cercospora and Passalora. CBS Biodiversity Ser. 1: 33-36
(2003).
(Fig. 3)
Tabl e 1 GenBank accession
numbers of DNA sequences of
isolates of Pseudocercospora and
Pseudocercospora-like used in
phylogenetic analyses
The specimens obtained in this
study are highlighted in bold
CBS Centraalbureau voor
Schimmelcultures, Utrecht,
Netherlands; CPC culture collec-
tion of Dr. Pedro Crous, housed at
CBS; MUCCMurdoch University
culture collection, Australia;
CMM Coleção de Culturas de
Fungos Fitopatogênicos
‘Prof. Maria Menezes’, Brazil
Spicies Isolate Genbank acession no. References
ITS EF
Mycosphaerella africana CMW3026 DQ267577 DQ235098 Hunter et al. 2006
Mycosphaerella aurantia CBS 110500 AY725531 DQ235097 Crous et al. 2004
Mycosphaerella ellipsoidea CBS 110843 AY725545 JX901653 Quaedvlieg et al. 2012
Mycosphaerella laricina CBS 326.52 GU269643 GU384361 Crous et al. 2013
Paracercospora egenula CPC 12537 GU269698 GU384414 Crous et al. 2013
Paracercospora egenula MUCC 883 GU269700 GU384416 Crous et al. 2013
Pseudocercospora cercidis-chinensis CBS14481 GU269670 GU384387 Crous et al. 2013
Pseudocercospora cordiana CBS 114685 GU269681 GU384398 Crous et al. 2013
Pseudocercospora exosporioides MUCC 893 GU269707 GU384423 Crous et al. 2013
Pseudocercospora fukuokaensis CPC 14689 GU269713 GU384429 Crous et al. 2013
Pseudocercospora fukuokaensis MUCC 887 GU269714 GU384430 Crous et al. 2013
Pseudocercospora glauca CPC 10062 GU269715 GU384431 Crous et al. 2013
Pseudocercospora kaki MUCC 900 GU269729 GU384442 Crous et al. 2013
Pseudocercospora lythracearum CPC 10707 GU269740 GU384452 Crous et al. 2013
Pseudocercospora lythracearum MUCC 890 GU269741 GU384453 Crous et al. 2013
Pseudocercospora mali MUCC 886 GU269744 GU384456 Crous et al. 2013
Pseudocercospora pancratii CBS 137.94 GU269759 GU384470 Crous et al. 2013
Pseudocercospora paraguayensis CBS 111317 JQ324978 GU384522 Crous et al. 2013
Pseudocercospora paraguayensis CBS 111286 DQ267602 JX901685 Hunter et al. 2006
Pseudocercospora pini-densiflorae MUCC 534 GU269760 GU384471 Crous et al. 2013
Pseudocercospora pyracanthigena CPC 10808 GU269766 JX901692 Quaedvlieg et al. 2012
Pseudocercospora schizolobii CBS 120029 KF251322 KF253269 Verkley et al. 2013
Pseudocercospora sp. CPC 14711 JQ324980 JQ325002 Crous et al. 2013
Pseudocercospora sp. CBS 110998 GU269778 GU384490 Crous et al. 2013
Pseudocercospora subsynnematosa CMM 2892 KF686808 KF726140 Present work
Pseudocercospora tibouchina-herbaceae CMM 2894 KF726141 Present work
Pseudocercospora tibouchinicola CMM 2813 KF686809 Present work
Pseudocercospora tibouchinigena CBS 116462 GU269822 GU384535 Crous et al. 2013
Pseudocercospora viticicola MUCC 777 GU269828 GU384540 Crous et al. 2013
Septoria rubi CPC 12331 GU269865 GU384576 Crous et al. 2013
Mycol Progress

Lesions on living leaves, necrotic, initially circular to el-
lipsoid, later coalescing to form large spots, 3.5–42×3–
25 mm, with indistinct margins, brown. Internal hyphae
1.5–8μm diam, branched, septate, brown. External hyphae
not observed. Stromata reduced to few cells in the substomatal
cavities. Conidiophores hypophyllous, arising through stoma-
ta, fasciculate, erect, subcylindrical, 48–175× 4–5.5 μm, 2–5-
septate, unbranched, brown, paler at apex, thin-walled,
smooth. Conidiogenous cells terminal and intercalary, inte-
grated, subcylindrical, proliferating sympodially, 23.5–73×
3.5–5μm, light brown. Conidiogenous loci conspicuous, 1–
3 per cell, 2.5–4μm diam., thickened, darkened. Conidia
solitary, ranging from obclavate-cylindrical to (mostly)
acicular-filiform, straight to curved, 30–115 × 2 –5μm, apex
acute, base truncate, 2–7-septate, guttulate, hyaline, thin-
walled, smooth, hila thickened and darkened.
KnowndistributiononlivingleavesofTibouchina
herbacea in Brazil: Espírito Santo, Minas Gerais, Paraná and
Rio Grande do Sul.
Material examined: Minas Gerais Viçosa, 02 Feb. 2001,
R.W. Barreto (VIC 30569); Paraná, Curitiba, 30 Mar 1998,
R.W. Barreto (VIC 30620); Rio Grande do Sul, Nova
Petrópolis, 18 Jan 2001, R.W. Barreto (VIC 30695); Rio
Grande do Sul, Nova Petrópolis, 19 Jan 2001, R.W. Barreto
(VIC 30696); Minas Gerais, Viçosa, road to Cajuri, 16 Mar
1996, R.W. Barreto (VIC 30660); Minas Gerais, Jequeri, 21
Apr 1996, R.W. Barreto (VIC 30668); Minas Gerais, Juiz de
Fora, 06 Nov 1998, R.W. Barreto (VIC 30673); Espírito
Fig. 2 Multilocus phylogenetic tree inferred from Bayesian analysis based on the combined sequences of the ITS and TEF-1α. Bayesian posterior
probabilities are indicated above the nodes. Septoria ribi represents the outgroup taxon. The species obtained in this study are in bold
Mycol Progress

Santo, Venda Nova do Imigrante, entrance of Parque Estadual
Pedra Azul, 09 Dec 2004, O.L. Pereira (VIC 30682).
Passalora tibouchinae D.F. Parreira & O.L. Pereira sp. nov.
(Fig. 4)
MycoBank: 512080.
Etymology: named in reference to the host genus.
Stromata 6.5–32.5× 7.5–45 μm. Conidiophores 15–76× 2–
5μm, 0–6-septate. Conidia solitary, acicular to obclavate, 30–
115 × 2–5μm, 2–7-septate.
Leaf spots initially small becoming circular, brown with an
outer reddish margin and light brown center, 1.5–6mmdiam.
Internal hyphae 1.5–8μm diam., branched, septate, light
brown. External hyphae absent. Stromata variable in size
and shape, immersed either reduced to few cells filling the
substomatal cavity or well developed, 6.5–32.5× 7.5–45 μm,
composed of dark brown textura angularis. Conidiophores
amphigenous, arising through stomata, fasciculate, erect,
straight to slightly curved or sinuose, subcylindrical, 15–
76× 2–5μm, 0–6-septate, unbranched, brown, becoming pal-
er at apices, thin-walled, smooth. Conidiogenous cells termi-
nal, integrated, cylindrical, conspicuously geniculate, prolif-
erating sympodially, 5–41×2.5–5μm, light brown.
Conidiogenous loci conspicuous, 1–5 per cell, 1–2μmdiam,
thickened and darkened. Conidia solitary, cylindrical to fili-
form, straight to curved, 30–115 × 2–5.0 μm, apex obtuse or
subacute, base subtruncate, 2–7-septate, guttulate, light
brown, thin-walled, smooth, hila thickened and darkened. In
culture (CMM 2816): Colonies slow-growing (10–18 mm
diam after 20 days), flat, somewhat lobate, sometimes ap-
pressed, aerial mycelium velvety, iron gray to olivaceous-
black sometimes greenish black in the center followed by an
intermediate iron gray to olivaceous-gray area and greenish
black margin, reverse olivaceous-gray; diurnal zonation ob-
served under light/darkness alternation regime; no
sporulation.
KnowndistributiononlivingleavesofTibouchina
herbacea in Brazil: Espirito Santo, Minas Gerais, Paraná,
Rio de Janeiro, Rio Grande do Sul and São Paulo.
Material examined: Espirito Santo, Ibatiba, 9 Dec 2004,
O.L. Pereira (VIC 30568, holotype; CMM 2816, ex type
culture); Espírito Santo, Venda Nova do Imigrante, entrance
of Parque Estadual da Pedra Azul, 9 Dec 2004, O.L. Pereira
(VIC 30680); Espírito Santo, Venda Nova do Imigrante, en-
trance of Parque Estadual Pedra Azul, 9 Dec 2004, O.L.
Pereira (VIC 30681); Minas Gerais, Lagoa da Prata, 5 Feb
2005, O.L. Pereira (VIC 30683); Paraná, Curitiba, Road
Fig. 3 Cercospora apii(VIC 30569) 5Acicular conidia. 6Conidiophores
arising through stomata either, solitary or fasciculate. Bar 10 μm
Fig. 4 Passalora tibouchinae (VIC 30568). 7Conidia. 8Conidiophore
fascicles arising through stomata. Bars 10 μm
Mycol Progress

Curitiba-Paranaguá Km 51, 31 Mar 1998, R.W. Barreto (VIC
30619); Rio de Janeiro, Nova Friburgo, 24 Feb 1998, R.W.
Barreto (VIC 30621); Rio de Janeiro, Vila do Grama, 24 Feb
1998, R.W. Barreto (VIC 30625); Minas Gerais, Rio Pomba,
19 Oct 1998, R.W. Barreto (VIC 30628); Minas Gerais, Santa
Barbara do Tugúrio, 21 Nov 1998, R.W. Barreto (VIC 30629);
Rio Grande do Sul, Nova Petrópolis, 19 Jan 2001, R.W.
Barreto (VIC 30696); Minas Gerais, Coronel Pacheco, 13
Mar 2002, R.W. Barreto (VIC 30641); Rio de Janeiro, Nova
Friburgo, 6 Apr 2008, R.W. Barreto (VIC 30643); São Paulo,
Bananal, Serra da Bocaina, 28 Dec 1995, R.W. Barreto (VIC
30656); Minas Gerais, Araponga, Cachoeira do Estouro, 9
Mar 1996, R.W. Barreto (VIC 30659); Minas Gerais, Alto
Caparaó, near Hotel Caparaó, river margin, 31 Mar 1996,
R.W. Barreto (VIC 30665); Rio de Janeiro, Nova Friburgo,
19 Oct 1996, R.W. Barreto (VIC 30671).
Pseudocercospora subsynnematosa D.F. Parreira & D.J.
Soares sp. nov.
(Fig. 5)
MycoBank: 512071.
Differing from all species of Pseudocercospora described
on members of Melastomataceae by having conidiophores
aggregated in short compact synnemata.
Leaf spots initially small and discrete brown surrounded by
a reddish brown margin, circular to elliptic, 2–7mmdiam,
later developing into a larger spots, light brown in the center,
with a reddish brown margin, vein-delimited. Internal hyphae
thin-walled, 1.5–3.5 μm diam, branched, septate, light brown.
External hyphae absent. Stromata epiphyllous, well-
developed, immersed to erumpent, subsphaerical, 21–55×
16–50 μm, composed of dark brown textura angularis.
Conidiophores epigenous, aggregated in dense short
synnemata, erect, straight to slightly sinuose, subcylindrical,
21–76×3–5μm, 1–5-septate, unbranched, brown, becoming
paler at the apex, thin-walled, smooth. Conidiogenous cells
terminal, integrated, cylindrical, 8.0–28.5× 2.5–5.0 μm, light
brown, smooth. Conidiogenous loci often protruding as a
short stalk, 1–2 per cell, 1.5–3μm diam, unthickened, not
darkened. Conidia solitary, obclavate-cylindrical to
subcylindrical, attenuating gradually towards the apex,
straight to slightly curved, 45–145.5×2–4μm, apex subacute
to occasionally obtuse, base either subtruncate or sometimes
protruding as a short stalk, 4–15-septate, guttulate, pale oliva-
ceous to pale brown, smooth, thin-walled, hila unthickened,
not darkened. In culture (CMM-2892): Colonies slow-
growing (9–13 mm diam. after 20 days), appressed, flat, with
lobate margins, felty, olivaceous-gray to iron gray in the center
becoming olivaceous-gray to pale olivaceous-gray towards
the margins, reverse iron gray to greenish black, with diurnal
zonation under light/dark alternation regime; sporulation
abundant on V8 and scarce to absent on PDA.
KnowndistributiononlivingleavesofTibouchina
herbacea in Brazil: Minas Gerais.
Material examined: Minas Gerais, Tabuleiro, 15 Oct 2007,
D.F. Parreira and D.J. Soares (VIC 30717). Minas Gerais,
Tabuleiro, 26 Sep 2009, D.F. Parreira and R.W. Barreto (VIC
30717, holotype; CMM-2892, COAD-276, ex type culture).
Pseudocercospora tibouchinicola D.F. Parreira & D.J.
Soares sp. nov.
(Fig. 6)
MycoBank: 512079.
Similar to Pseudocercospora dissotidis but having smaller
conidiophores (11–53× 3–5μm) and longer and narrower
conidia (16.5–195×2–3.5 μm).
Colonies on living leaves, not forming conspicuous leaf
spots, growing mainly abaxially, forming a dense olivaceous
mat composed of conidia and conidiophores, colonies some-
what vein-delimited abaxially; adaxially colonies sparse and
less commonly formed; on senescing leaves with “green
islands”forming around colonized areas, whereas the rest of
the lamina becomes necrotic and red or brown colored. Inter-
nal hyphae 1.5–3μm diam, branched, septate, hyaline, thin-
walled. External hyphae absent. Stromata absent.
Conidiophores arising through stomata, sparsely fasciculate,
erect, straight, subcylindrical, 11–53×3–5μm, 1–4-septate,
mostly unbranched, light brown, thin-walled, smooth.
Conidiogenous cells terminal, integrated, 5–22.5× 3–5μm,
pale olivaceous, smooth. Conidiogenous loci protruding,
1–2 per cell, 1–2μm diam, unthickened, not darkened.
Conidia solitary, obclavate-cylindrical to subcylindrical,
straight to curved, occasionally somewhat distorted by
one infra-median heeled cell, 16.5–195 × 2.0–3.5 μm,
apex subacute, occasionally obtuse, with a strongly pro-
truding hilum at the base, 1–16-septate, guttulate, pale
olivaceous, thin-walled, smooth, hila unthickened, not
darkened. In culture (CMM 2813): Colonies slow-
growing (10–13 mm diam. after 20 days), flat, aerial
mycelium velvety gray-olivaceous, olivaceous-gray,
smoke gray to iron gray centrally, pale olivaceous to
olivaceous-gray or greenish black at periphery, reverse
olivaceous-gray to iron gray, in the middle iron gray at
the periphery; no sporulation.
KnowndistributiononlivingleavesofTibouchina
herbacea in Brazil: Minas Gerais.
Material examined: Minas Gerais, Tabuleiro, 15 Oct. 2007,
D.F. Parreira and D.J. Soares (VIC 30564, holotype; CMM
2813, COAD-127 ex type culture).
Pseudocercospora tibouchina-herbaceae D.F. Parreira &
R.W. Barreto sp. nov.
(Fig. 7)
MycoBank: 512076.
Similar to Pseudocercospora tamoneae but having
narrower conidiophores aggregated in sporodochia, longer
and narrower conidia which are 3-11 septate and also differing
from P. subsynnematosa by the absence of synnemata and a
truncate conidial base.
Mycol Progress

Leaf spot similar to those caused by P. subsynnematosa,but
larger, 1.5–19 mm diam. Internal hyphae indistinct. External
hyphae absent. Stromata subglobose to irregular, well-
developed, sub-immersed to erumpent, 17.5–45 × 20–
62.5 μm, composed of dark brown textura angularis.
Conidiophores aggregated in sporodochia, erect, straight to
slightly curved or somewhat sinuose, subcylindrical, 8.5–
40.5× 2–4.5 μm, 0–4-septate, unbranched, light brown, thin-
walled, smooth. Conidiogenous cells terminal, integrated,
subcylindrical, 5.5–20× 2–3μm, light brown. Conidiogenous
loci inconspicuous, 1–2 per cell, 1–3μm diam, unthickened,
not darkened. Conidia solitary, cylindrical to acicular, straight
to slightly curved, sometimes distorted by one suprabasal to
infra-median curved cells, 55–202×2–3μm, apex subacute,
base truncate, 3–11-septate, guttulate, pale olivaceous, thin-
walled, smooth, hila unthickened and not darkened. In culture
(CMM-2894): Colonies slow-growing (13–18 mm diam. after
20 days), flat, sometimes ridged, aerial mycelium velvety, pale
olivaceous gray, lavender gray, gray-olivaceous to olivaceous-
black in the center with an olivaceous-gray, apricot or
olivaceous-black intermediate area and iron gray margins,
reverse olivaceous gray to pale olivaceous with iron gray
margin; coloring the medium with a pink diffusate; no
sporulation.
KnowndistributiononlivingleavesofTibouchina
herbacea in Brazil: Minas Gerais, Paraná, Rio de Janeiro,
Rio Grande do Sul.
Material examined: Rio de Janeiro, Itatiaia, Parque
Nacional do Itatiaia, 27 Dec 1995, R.W. Barreto (VIC
30653, holotype; CMM-2894, COAD-48 ex type culture);
Minas Gerais, Rio Pomba, 19 Oct 1998, R.W. Barreto (VIC
30628); Minas Gerais, Ouro Preto, Reserva Biológica do
Tripuí,1 Aug 1998, R.W. Barreto (VIC 30566); Minas Gerais,
Ouro Preto, Rio Grande do Sul, Nova Petrópolis, 18 Jan 2001,
Fig. 5 Pseudocercospora
subsynnematosa (VIC 30565). 9
Conidia (note hila varying from
subtruncate to protuberant). 10
Conidiophores aggregated in
dense, subsynnematous fascicle.
11 Close-up of conidiophores.
Bars 10 μm
Mycol Progress

R.W. Barreto (VIC 30636); Rio de Janeiro, Minas Gerais,
Manhuaçu, road Belo Horizonte-Vitória, 30 Mar 1996, R.
W. Barreto (VIC 30661); Minas Gerais, Barão de Cocais,
Santuário do Caraça, 3 May 1996, R.W. Barreto (VIC 30667).
Discussion
The cercosporoid fungi are among the most common of all
plant pathogens (Crous and Braun 2003) and several papers
have already been published dealing with fungi in this group
from Brazil (Crous et al. 1997; Braun et al. 1999;Dornelo-
Silva et al. 2007; Rocha et al. 2008). The Cercospora species
collected on T. herbacea fits well within Cercospora apii
sensu lato. Many species of Cercospora that were distin-
guished from C. apii based solely on host-association were
reduced to synonymy with the latter species (Crous and Braun
2003). There are, nevertheless, still many unresolved issues
regarding taxonomic delimitation at the species or infra-
specific level within the C. apii complex, as revealed by
later studies (Groenewald et al. 2005,2006). This rep-
resents the first report of C. apii onamemberofthe
Melastomataceae.
There are no previous reports of a fungus in the genus
Passalora on a member of the Melastomataceae, and it is
clear that Passalora tibouchinae represents a new species for
the genus.
With the new additions included in this work, there are now
19 species of Pseudocercospora recorded on members of the
Melastomataceae (Farr and Rossman 2013). Nevertheless,
only two have been reported in association with Tibouc hina
spp: [Pseudocercospora tibouchinae (Viégas) Deighton and
Pseudocercospora tibouchinigena Crous & U. Braun], and
both differ from our species by having smaller conidiophores
and conidia (Viégas 1945; Crous et al. 2013). Information on
the morphology of these taxa is given in Table 2. The three
new species of Pseudocercospora introduced here have clear
morphological differences that allow their distinction as new
and separate taxa. Pseudocercospora tibouchinicola is some-
what similar to Pseudocercopora dissotidis (Chupp &
Doidge) Crous & U. Braun by having no stromata and
forming indistinct leaf spots. All other Pseudocercospora
species on the Melastomataceae are associated with leaf spots.
Conidiophores of P. tibouchinicola are smaller and narrower
and conidia are longer and narrower than those of P. dissotidis
(Table 2). Pseudocercospora tibouchinicola grouped in a
Fig. 6 Pseudocercospora tibouchinicola (VIC 30564). 12 Conidia (note
geniculation on some conidia, wide range of sizes and shapes and
protuberant hila). 13 Conidiophores arising from stomata. Bars 10 μmFig. 7 Pseudocercospora tibouchina-herbaceae (VIC 30653). 14 Co-
nidia. 15 Conidiophores on a dense immersed stroma. Bars 10 μm
Mycol Progress

clade that accommodates species of Pseudocercospora s. l.
and is phylogenetically distant from the other two new spe-
cies, P. subsynnematosa and P. tibouchina-herbacea,bothof
which are leaf spot-causing pathogens (Fig. 5).
Pseudocercospora subsynnematosa is easily distinguished
from other Pseudocercospora on Melastomataceae, including
the newly described species, by its conidiophore arrangement
in short compact synnemata. Pseudocercospora tibouchina-
herbaceae does not produce synnemata, as in the former
species, and has longer and narrower conidia which have a
truncate base, never ending in a strongly protruding hilum as
commonly seen in P. subsynnematosa. Its morphology is
somewhat similar to that of P. tibouchinicola but it has conid-
iophores arranged in sporodochia, whereas P. tibouchinicola
does not produce sporodochia or stromata and does not cause
leaf spots on T. herbaceae. Additionally, P. tibouchina-
herbaceaconsistently produces a pink diffusate in the medium
whereas P. tibouchinicola lacks pigments and grows more
slowly. Finally, the two species occur on different forms of
T. herbacea:P. tibouchina-herbaceae being found on the most
common form of the weed, whereas P. tibouchinicola occurs
only on the more shrubby rarer form of T. herbacea. Phylo-
genetically, P. s u b s y n n e m a t o s a grouped in a clade that
includes species of Pseudocercospora s.l. close to
Pseudocercospora schizolobii (M.J. Wingf. & Crous) M.J.
Wingf. & Crous (Fig. 5). Nevertheless, P. schizolobii is a
pathogen of a distantly related host [Schizolobium parahybum
(Vell. Conc.) S. F. Blake (Fabaceae)] and has a distinct mor-
phology—shorter and narrower conidia ((30) 40–55
(80)× (2.5) 3 (3.5) μm)—and is clearly not conspecific with
the fungus on Tibouchina. Pseudocercospora tibouchina-
herbaceae is morphologically similar to P. tamoneae but dif-
fers from the latter by having narrower conidiophores and
longer and narrower conidia. It grouped in a clade close to
P. tibouchinigena which, according to Crous et al. (2013), is
not well resolved because it does not have the typical features
of Pseudocercospora s.str. It is thought that taxa in this
subclade may eventually be recognized as a distinct genus,
but until the clade has been more fully resolved, this species is
retained in Pseudocercospora.
Among the species of fungi described in this study, two
appear to have potential for introduction as classical biological
control agents against T. herbacea for being associated with
severe disease symptoms: Passalora tibouchinae and
Pseudocercospora subsynnematosa. Although the specificity
of these fungi has not yet been tested, P. tibouchinae and
Tabl e 2 Morphology of Pseudocercospora species recorded on the Melastomataceae
Species Stromata Conidiophores Conidia References
Size (μm) Size (μm) Septation Size (μm)
P. aciotidis (Chupp) U. Braun & Crous Reduced to few cells 20–85× 4–5.5 3–920–95× 3.5–5Chupp1954
P. curta (Syd.) U. Braun & Crous 60–80 diam. 10–20× 3.5–5Absent60–85×5–7Sydow1939
P. d i s s o t id i s (Chupp & Doidge) Crous & U. Braun Absent 10–150×4–61–420–65× 4.5–6CrousandBraun1996
P. erythrogena (G. F. Atk.) U. Braun Absent or reduced to
few cells
10–70×2–4.5 0–420–100×2–4Braun1999
P. leandrae (Syd.) U. Braun 35–50 diam. 30–70×3.5–4.5 1–245–140×4–5Sydow1939
P. melastomobia (W. Yamam.) Deighton 30-40 diam. 10–50×3.5–5.5 Septate 50–150×3–4HsiehandGoh1990
P. miconiae (Gonz. Frag. & Cif.) U.
Braun & Crous
Absent or reduced to
few cells
45× 6 Septate 90× 5.5 Saccardo 1972
P. miconiicola (Chupp) U. Braun & Crous 20–35 diam. 50–130×3.5–51–525–45× 4–8Chupp1954
P. miconiigena U. Braun & R. Urtiaga Absent 80–280×3–5Septate20–40× 4–7 Braun and Urtiaga 2008
P. mirandensis (Chupp) R.F. Castañeda
&U.Braun
30–75 diam. 5–25×2–3.5 Rarely 20–90× 2–3.5 Kirschner and
Piepenbring 2006
P. monochaeticola (Chupp) U. Braun & Crous 20–60 diam. 10–55×1.5–3 Rarely 30–85×1.5–3Chupp1954
P. osbeckiae (Chona, Lall & Munjal) Kamal,
M.K. Khan & R.K. Verma
18–26×15–20 or
lacking
10-80×3-4.5 Septate 40–90×3–5 Kamal and Khan 1990
P. oxysporae (A.K. Kar & M. Mandal) Deighton 10–42.2 diam. 10–83.5×3–50–630–88.5×3–4.5 Deighton 1987
P. subsynnematosa D.F. Parreira & D.J. Soares 21–55×16–50 21–76×3–51–545–145.5× 2–4presentwork
P. t a m o n e ae (Chupp) U. Braun & R.F. Castañeda 10–80 5–50× 2.5–71–220–120×2–4.5 Braun and Castañeda
1991
P. tibouchina-herbaceae D.F. Parreira &
R.W. Barreto
17.5–45×20–62.5 8.5–40.5×2–4.5 0–455–202×2–3 Present work
P. tibouchinae (Viégas) Deighton 30–35× 50–70 15–20×2–30–140–120× 2–3 Viégas, 1945
P. tibouchinicola D.F. Parreira & D.J. Soares Absent 11–53× 3–50–416.5–195 ×2–3.5 Present work
P. tibouchinigena Crous & U. Braun 50–20 15–25×3–50–315–60× 1.5–3 Crous et al. 2013
Mycol Progress

P. subsynnematosa belong to a genus that is known for in-
cluding host-specific plant pathogens. Cercospora apii is an
exception in the cercosporoids for being regarded as a polyph-
agous pathogen with a broad host range, although host-
specific populations within this complex are known to occur.
It is difficult to evaluate its biocontrol potential at this stage.
Other fungal genera that were found associated to T. herbacea
were preliminarily identified as: Asteridiella,Asterina,
Chaetophiophoma,Cladosporium,Gnomonia,Hainesia,
Leptosphaeria,Mollisia,Perisporiopsis,Pestalotiopsis, and
Septoria. They will be described and discussed in a separate
publication.
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