Species of the Colletotrichum acutatum complex associated with anthracnose diseases of fruit in Brazil Carlos A. D. BRAGANC ¸A a, *, Ulrike DAMM b,c , Riccardo BARONCELLI d , Nelson S. MASSOLA J UNIOR e , Pedro W. CROUS b,f,g a Universidade Federal do Rec^ oncavo da Bahia, 710, CEP 44380-000, Cruz das Almas-BA, Brazil b CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands c Senckenberg Museum of Natural History, G€ orlitz PF 300 154, 02806 G€ orlitz, Germany d Laboratoire Universitaire de Biodiversite et Ecologie Microbienne, Universite de Bretagne Occidentale, Brest, France e Departamento de Fitopatologia e Nematologia, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de S~ ao Paulo, Caixa Postal 09, CEP 13418-900, Piracicaba-SP, Brazil f Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa g Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands article info Article history: Received 23 September 2015 Received in revised form 19 January 2016 Accepted 20 January 2016 Available online 2 February 2016 Corresponding Editor: Marc Stadler Keywords: Identification Multilocus phylogeny Pathogenicity Plant pathogenic fungus Systematics abstract Although Colletotrichum acutatum was recently investigated and shown to be a species com- plex comprising about 30 species, the name is still used in its broad sense for anthracnose pathogens of fruits in Brazil. In this study, a multilocus molecular analysis was carried out based on a dataset of ITS, HIS3, GAPDH, CHS-1, TUB2 and ACT sequences of Colletotrichum strains belonging to the C. acutatum species complex from fruits collected in different re- gions in Brazil combined with sequences of ex-type and other reference strains of species belonging to this complex. The strains were revealed to belong to Colletotrichum nymphaeae, Colletotrichum melonis, Colletotrichum abscissum and one new species, namely Colletotrichum paranaense, from apple and peach. Morphological descriptions of the new species and a strain closely related to but diverging from C. melonis are provided. From the data pres- ently available, the most common species on apple fruits in Brazil is C. nymphaeae. In a pathogenicity test, strains of all four species caused lesions on detached apple, peach and guava fruits, except for strain CBS 134730 that did not infect guava fruits. ª 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Colletotrichum species are economically important plant path- ogens, especially in tropical, subtropical and temperate regions, where they affect a wide range of plant hosts (Sutton 1992). The most common symptoms associated with Colletotrichum infections are sunken necrotic lesions, on which often orange conidial masses are produced, and that are * Corresponding author. Tel.: þ55 75 3621 3273. E-mail addresses: [email protected](C. A. D. Braganc ¸a), [email protected](U. Damm), riccardobaroncelli@gmail. com (R. Baroncelli), [email protected](N. S. Massola J unior), [email protected](P. W. Crous). journal homepage: www.elsevier.com/locate/funbio fungal biology 120 (2016) 547 e561 http://dx.doi.org/10.1016/j.funbio.2016.01.011 1878-6146/ª 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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Species of the Colletotrichum acutatum complexassociated with anthracnose diseases of fruit inBrazil
Carlos A. D. BRAGANCAa,*, Ulrike DAMMb,c, Riccardo BARONCELLId,Nelson S. MASSOLA J �UNIORe, Pedro W. CROUSb,f,g
aUniversidade Federal do Reconcavo da Bahia, 710, CEP 44380-000, Cruz das Almas-BA, BrazilbCBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The NetherlandscSenckenberg Museum of Natural History, G€orlitz PF 300 154, 02806 G€orlitz, GermanydLaboratoire Universitaire de Biodiversit�e et Ecologie Microbienne, Universit�e de Bretagne Occidentale, Brest, FranceeDepartamento de Fitopatologia e Nematologia, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de
S~ao Paulo, Caixa Postal 09, CEP 13418-900, Piracicaba-SP, BrazilfDepartment of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI),
University of Pretoria, Pretoria 0002, South AfricagMicrobiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
C. walleri CBS 125472, BMT(HL)19d Coffea sp. Vietnam JQ948275 JQ948605 JQ948936 JQ949266 JQ949596 JQ949926
a CBS: Culture collection of Centraalbureau voor Schimmecultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Working collection of Pedro W. Crous, housed at CBS, Utrecht, The
Netherlands; Col: Personal collection of Nelson Massola, housed at ESALQ/USP, Department of Plant Pathology, Piracicaba, Sao Paulo, Brazil; IMI: Culture collection of CABI Europe UK Centre, Egham,
UK; BRIP: Plant Pathology Herbarium, Department of Employment, Economic, Development and Innovation, Queensland, Australia; ICMP: International Collection of Microorganisms from Plants,
Auckland, New Zealand; STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa; CPAC: Collection Cpac-Embrapa at Embrapa-Cerrados, Planaltina,
DF, Brasil; HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; PD: Plantenziektenkundige Dienst Wageningen, Nederland; RB: Personal collection of Riccardo Baroncelli,
housed at Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali, Universit�a di Pisa, Pisa, Italy.
b GenBank numbers starting with KC and KT were generated in this study.
c Baroncelli et al. (2014).
d Ex-holotype or ex-epitype cultures; Genbank numbers started with JQ and KP were published by Damm et al. (2012) and Crous et al. (2015), respectively.
550
C.A.D.Braganca
etal.
Species of the Colletotrichum acutatum complex in Brazil 551
ribosomal gene with the two flanking internal transcribed
spacers (ITS), an intron of the glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH) and partial sequences of the chitin syn-
thase 1 (CHS-1), actin (ACT), b-tubulin (TUB2) and histone 3
(HIS3) genes were amplified and sequenced using the primers
ITS-1F (Gardes & Bruns 1993) and ITS-4 (White et al. 1990),
GDF1 and GDR1 (Guerber et al. 2003), CHS-354R and CHS-79F
(Carbone & Kohn 1999), ACT-512F and ACT-783R (Carbone &
Kohn 1999), BT2Fd (Woudenberg et al. 2009) and Bt-2b (Glass
& Donaldson 1995) and CYLH3F and CYLH3R (Crous et al.
2004b), respectively. The conditions for PCR of ITS were the
same as described by Woudenberg et al. (2009), while those
for the other genes were carried out with an initial denatur-
ation step at 94 �C for 5 min followed by 40 cycles of 30 s at
94 �C, 30 s at 52 �C and 30 s at 72 �C, and a final step at 72 �Cfor 7 min. The amplicons were visualized in 1 % agarose gels
stained with GelRed� (Biotium, USA). The sequencing was
performed using the BigDye terminator sequencing kit v.3.1
(Applied Biosystems, USA) and an ABI PRISM� 3100 DNA se-
quencer (Applied Biosystems).
The forward and reverse sequences generatedwere assem-
bled using the software SeqMan v.9.0.4 (DNASTAR�, Madison/
USA). Sequences of 59 ex-type and other reference strains of
species belonging to the Colletotrichum acutatum complex as
well as Colletotrichum orchidophilum CBS 632.80 (as outgroup),
all available on GenBank, were added to the dataset and the
sequences aligned with MAFFT v.6.7 (Katoh & Toh 2008). The
multiple sequence alignment was manually edited with
MEGA v.5.2 (Tamura et al. 2011).
Evolution models were estimated in MrModeltest v.3.7
The Bayesian tree obtained in this study suggests high ge-
netic variability among the species occurring in Brazil. This
can be important knowledge for developing control strategies.
For example, the population of a plant pathogen with high ge-
netic variability can evolve rapidly, and this information can
be used for predicting how long a control measure is likely
to be effective (McDermott & McDonald 1993). Additionally,
the correct identification of the pathogen is important for its
effective control strategy based on fungicides, because some
species are more sensitive to specific groups of chemical com-
pounds than other species (Freeman et al. 1998; Sanders et al.
2000; Wong & Midland 2007). For example, C. gloeosporioides
(s. lat.) is considered highly sensitive to benomyl, whereas C.
acutatum (s. lat.) is comparatively resistant (Freeman et al.
1998). However, the individual species within these species
complexes need to be tested for their sensitivity for specific
fungicides. Furthermore, the accurate identification of the
pathogens can improve our understanding of their epidemiol-
ogy and infection strategy, and provide important knowledge
for breeding of resistant cultivars.
Conflicts of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
Acknowledgements
We thankDr LarissaMio (Federal University of Paran�a, Paran�a,
Brazil), Dr Hugo Tozze (Secretaria de Agricultura e Abasteci-
mento do Estado de S~ao Paulo, S~ao Paulo, Brazil), Dr Silvio
Alves (EMBRAPA uva e vinho, Rio Grande do Sul, Brazil), Dr
Rosa Maria Sanhueza (Proterra, Rio Grande do Sul, Brazil)
and Dr. Natalia Peres (University of Florida, USA) for providing
some cultures. This researchwas supported by CNPq (scholar-
ships processes 140455/2010-8 and 308875/2011-7), FAPESP
(process 2011/11629-1) and CAPES (scholarship process num-
ber 9809-11-2).
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