Diversity of pathogenic and endophytic Colletotrichum ...

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Received: 25 May 2017    Revised: 30 April 2018    Accepted: 3 May 2018 DOI: 10.1111/efp.12448

ORIGINAL ARTICLE

Diversity of pathogenic and endophytic Colletotrichum isolates from Licania tomentosa in Brazil Daniela O. Lisboa1 | Mariana A. Silva1 | Danilo B. Pinho2 | Olinto L. Pereira1

 | 

Gleiber Q. Furtado1 1 Departamento de Fitopatologia,  Universidade Federal de Viçosa, Viçosa, Brazil 2

Abstract Licania tomentosa (Chrysobalanaceae), also known as “oiti,” is a forest tree mainly

Departamento de Fitopatologia,  Universidade de Brasília, Brasília, Brazil

used for urban afforestation in Brazil. Although anthracnose caused by

Correpondence Gleiber Furtado, Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. Email: [email protected]

fication of the species was based on morphological characteristics only. Owing to the

Colletotrichum gloeosporioides is the main disease that threatens this tree, the identineed to use the molecular approach to pinpoint the identity of this pathogen with precision, the aim of this study was to identify endophytic and pathogenic Colletotrichum isolates from L. tomentosa based on both morphological and molecular data. For prior identification, partial sequences of the GAPDH region were obtained

Editor: J. Hantula

of all the 35 isolates (10 endophytic and 25 pathogenic). After analysis, ten isolates, representative of each clade, were selected for multilocus phylogenetic analysis (ACT, CAL, CHS-­1, GAPDH, TUB2, SOD2 and ITS). In addition, a tree based on the ApMat region was obtained for comparison with the multilocus tree. Morphological characterization (colony growth, conidial size and appressoria shape) was also performed for each species. To prove pathogenicity, L. tomentosa leaves were inoculated on the adaxial surface by mycelial plugs and conidial suspension. All isolates obtained belong to the Colletotrichum gloeosporioides complex. The Apmat tree has the same topology as the multilocus tree, allowing for the discrimination of the different species of Colletotrichum on L. tomentosa. Endophytic isolates of C. fructicola, C. queenslandicum, and C. siamense were acquired whereas pathogenic isolates were identified as C. siamense and C. tropicale, although all species were pathogenic on the wounded leaves of L. tomentosa. This is the first worldwide report of this Colletotrichum species associated with L. tomentosa and the first recording of C. queenslandicum in Brazil. KEYWORDS

anthracnose, ApMat, Colletotrichum gloeosporioides complex, forest pathology, multilocus phylogeny

1 |  I NTRO D U C TI O N

wood are recommended for construction, posts, sleepers, shipbuilding and various other applications thanks to its hard and durable

The

arboreal

species

Licania tomentosa

(Benth.)

Fritsch

quality (Lorenzi, 1992).

(Chrysobalanaceae) occurs naturally in Brazil mainly in the rainforest

Anthracnose caused by Colletotrichum on both seedlings and

and semi-­deciduous broadleaved forests. This species is widely used

adult plants is the main disease that threatens L. tomentosa. The

for urban afforestation in Brazil because its ornamental features and

symptoms are lesions in leaves and fruits, isolated or interconnected

Forest Pathology. 2018;e12448. https://doi.org/10.1111/efp.12448

wileyonlinelibrary.com/journal/efp

© 2018 Blackwell Verlag GmbH  |  1 of 11

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LISBOA et al.

2 of 11      

irregularly round in shape, and dark brown in colour with a lighter

and in cases where symptomatic tissue did not present sporulation

centre. The lesions on leaves are visible mainly on the adaxial sur-

of Colletotrichum. In this study, endophytics were defined as fungi

face, where orange-­cream spore masses are often observed in acer-

isolated from asymptomatic leaves after rigorous surface steriliza-

vuli (Ferreira, 1989).

tion (Arnold et al., 2003). For indirect isolation, leaf fragments were

In addition to the outstanding importance of Colletotrichum spp.

washed in running water, and the surface sterilized by consecutive

as a plant pathogen, there are several reports on its performance as an

immersion in 70% ethanol for 1 min, in 1% sodium hypochlorite

endophyte, epiphyte and saprophyte (Damm et al., 2013; Prihastuti,

for 3 min, and in sterile distilled water (3×) for 1 min. These frag-

Cai, Chan, McKenzie, & Hyde, 2009; Sutton, 1992). Colletotrichum

ments were transferred to a Petri dish containing PDA and incu-

species are considered to be foliar endophytes with a wide range of

bated at 25°C under fluorescent light (λ = 380–775 ηm) with a 12-­hr

hosts (Hyde et al., 2009; Lu, Cannon, Reid, & Simmons, 2004; Rojas

photoperiod.

et al., 2010) which may be dormant in the early stages but are later

To obtain single-­spore cultures, a mass of conidia observed under

parasitic, and lead to the development of lesions (Cannon, Damm,

a stereomicroscope (45x) was deposited in 10 μl of distilled water

Johnston, & Weir, 2012). However, a comprehensive understanding

under slide glass. Then, 5 μl of the suspension was transferred to a

of how these fungal species change from nonpathogenic to patho-

Petri dish containing 2% water agar (WA -­Himedia®), spread with

genic is still limited (Hyde et al., 2009; Lu et al., 2004; Rojas et al.,

a sterile Drigalski spatula and incubated at 25°C under fluorescent

2010).

light for 6 hr. Afterwards, under a stereomicroscope (45×) a single

Colletotrichum species associated with cultivated crops are usu-

germinated conidium was transferred to a Petri dish containing PDA.

ally well characterized, but their activity on ornamental and forest

Plates were maintained at 25°C under fluorescent light with a 12-­hr

trees has been understudied. Historically, C. gloeosporioides is con-

photoperiod. Isolates were stored in sterilized distilled water.

sidered to be the only aetiologic agent of this disease. This identification was based on the observation of phenotypic characteristics of a few isolates only (Farr & Rossman, 2017; Mendes & Urben, 2017). The incorporation of molecular data in taxonomy of the genus re-

2.2 | DNA extraction, PCR amplification and sequencing

vealed cryptic species and a classification based on complex spe-

Single-­ spore isolates were grown on PDA at 25°C with a 12-­ hr

cies (Cannon et al., 2012). Furthermore, most species have a wide

photoperiod for 10 days. The mycelium was scraped from the me-

range of hosts and several species of different complexes have

dium surface and placed in a sterilized 1.5 ml microcentrifuge tube.

been reported in a single host (Lima et al., 2013; Weir, Johnston, &

Mycelium was ground in liquid nitrogen to a fine powder using a

Damm, 2012). Thus, there is a need for molecular studies that will

microcentrifuge tube pestle. Crushing was continued after add-

determine the aetiology of this disease and also expand the knowl-

ing 100 μl of Nuclei Lysis Solution from the Wizard Genomic DNA

edge of the diversity of Colletotrichum species in tropical regions.

Purification Kit (Promega Corporation, WI). After the first grinding,

The objective of this study was to identify Colletotrichum species as-

another 500 μl of the above-­mentioned solution was added. The ex-

sociated with symptomatic and asymptomatic leaves of L. tomentosa

traction was continued as described by Pinho, Firmino, Pereira, and

using a polyphasic approach and to verify the potential of the Apmat

Ferreira Junior (2012). Sequences were obtained from eight genic regions, actin (ACT),

marker for discriminating the species.

calmodulin (CAL), chitin synthase 1 (CHS-­1), glyceraldehyde-­3-­ phosphate dehydrogenase (GAPDH), ribosomal internal transcribed

2 |  M ATE R I A L S A N D M E TH O DS

spacer (ITS), manganese-­superoxide dismutase (SOD2), β-­tubulin 2 (TUB2) and Apn2-­Mat1-­2 (ApMat) intergenic spacer by amplification

2.1 | Sample collection and isolation

and sequencing with the following combinations of primers for each

During December 2013 and August 2014, asymptomatic leaves of

region, respectively, ACT-­512F + ACT-­783R (Carbone & Kohn, 1999),

L. tomentosa or leaves with anthracnose symptoms were collected

CAL-­228F (Carbone & Kohn, 1999) + CAL2RD (Groenewald et al.,

from eight Brazilian states (Minas Gerais, Bahia, Espírito Santo,

2013), CHS-­79F + CHS-­345R (Carbone & Kohn, 1999), GDF + GDR

São Paulo, Rio de Janeiro, Goiás, Paraná, Alagoas) and from the

(Templeton, Rikkerink, Solon, & Crowhurst, 1992), ITS1 + ITS4

Distrito Federal. The samples were sent to the Laboratory of Forest

(White, Bruns, Lee, & Taylor, 1990), SODglo2-­ F  +  SODglo2-­ R

Pathology (Department of Plant Pathology, Federal University of

(Moriwaki & Tsukiboshi, 2009), T1 (O’Donnell & Cigelnik, 1997) +

Viçosa).

2A or 2B (Glass & Donaldson, 1995), CgDL-­F6 + CgMAT1-­F2 (Rojas

The samples with symptoms were first examined for the pos-

et al., 2010). PCR was performed with 12.5 μl of Dream Taq TM PCR

sible presence of fungal fruiting structures. From these structures,

Master Mix 2× (MBI Fermentas, Vilnius, Lithuania); 1 μl of 10 μM

direct isolations were performed. A mass of conidia was transferred

each forward and reverse primer; 1 μl of dimethyl sulfoxide; 5 μl

®

to a Petri dish containing potato dextrose agar (PDA -­Acumedia )

of 100× (10 mg/ml) Bovine Serum Albumin; 2 μl of genomic DNA

under a stereomicroscope [Motic® SMZ-­140 (20X)]. The plates were

(25 ng/μl) and 2.5 μl of nuclease-­free water.

incubated at 25°C with a 12-­hr photoperiod. Indirect isolation was

The PCR conditions for ITS were 4 min at 95°C, then 35 cycles at

performed both on asymptomatic leaves to obtain endophytic fungi

95°C for 30 s, 52°C for 30 s, 72°C for 45 s, and finally 7 min at 72°C.

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LISBOA et al.

The annealing temperatures were different for the other regions,

The HKY + G model of evolution was used for ApMat. For multilocus

with the optimum for each as follows: ACT: 58°C, CAL: 59°C, CHS-­1:

analysis, the HKY + G was used for ACT, GTR + I for CAL, SYM + I

58°C, GAPDH: 60°C, SOD2: 54°C, TUB2: 55°C, ApMat: 56°C.

for CHS-­1, GTR + I for GAPDH, SYM for ITS, HKI + I + G for SOD2

The PCR products were analyzed by electrophoresis on 2% agarose gels that were stained with GelRed™ (Biotium Inc., Hayward,

and K80 + I for TUB2. The ApMat and concatenated trees were rooted with Colletotrichum salsolae ICPM19051.

CA) in a 1× TAE buffer and visualized under UV light to check for amplification size and purity. The amplicons were purified and sequenced by Macrogen Inc., South Korea (http://www.macrogen. com).

2.4 | Morphological characterization A representative isolate from each clade identified in the multilocus phylogenetic analysis was used for morphological characterization.

2.3 | Data editing and phylogenetic analysis

The size and shape of conidia, shape of apressoria and colony morphology were all assessed.

The nucleotide sequences were edited using the BioEdit software

The isolates were grown for 10 days on plates containing PDA.

program (Hall, 2014). All of the sequences were checked manu-

They were incubated at 25°C with a photoperiod of 12-­hr with black

ally, and nucleotides with ambiguous positions were clarified using

light (λ = 320–400 ηm) to induce sporulation. The conidia were

both primer direction sequences. New sequences were deposited

transferred to glass slides containing lactic acid. Measurements

in GenBank (http://www.ncbi.nlm.nih.gov). In a preliminary analysis,

(n = 30) of the conidia were taken using a light microscope (Olympus

consensus sequences of the GAPDH gene were compared against

CX31). Images were obtained with a light microscope (Olympus

GenBank’s database using their Mega BLAST program and com-

CX31) fitted with a digital camera (Olympus Q-­COLOR5).

pared with the Q-Bank Fungi database. The GAPDH sequences of

Appressoria were produced using the slide culture technique,

all isolates obtained in this study were aligned using the multiple se-

a method focusing on the hyphae whereby 10 mm squares of ster-

quence alignment program MUSCLE® (Edgar, 2004), which is built

ile PDA were placed in an empty Petri dish, spores were deposited

into the MEGA software (Tamura et al., 2011). The alignments were

on the edge of the agar and a sterile cover slip was placed over the

checked, and manual adjustments were made where necessary. Gaps

agar. Appressoria formed across the underside of the cover slip. After

were treated as missing data. The resulting alignment was deposited

14 days, the shape and size of the appressoria were formed and the im-

into TreeBASE (http://www.treebase.org/) under accession num-

ages were obtained using a light microscope as previously described.

ber S20974. Bayesian inference (BI) analyses employing a Markov

For observation of cultural characteristics, mycelial plugs (5 mm

Chain Monte Carlo method were performed on every sequence of

in diameter) were taken from the margins of actively growing colo-

the GAPDH gene. Before launching the BI, the best nucleotide sub-

nies grown on PDA for 5 days, and transferred to the centre of a Petri

stitution model was determined with MrMODELTEST 2.3 program

dish containing PDA and were incubated at 25°C with a photoperiod

(Posada & Buckley, 2004). Once the likelihood scores were calcu-

of 12-­hr with fluorescent light (λ = 380–775 ηm). Measurements of

lated, the models were selected according to the Akaike informa-

the colony diameter were carried out at 4 and 7 days of growth. The

tion criterion (AIC). The HKY evolution model was used for GAPDH.

colour, shape and type of mycelium were evaluated after 7 days.

Phylogenetic analysis was carried out using the CIPRES web portal

Four replicates were used for each isolate.

(Miller, Pfeiffer, & Schwartz, 2010) using MrBayes program v. 3.2.3

Representative isolates of each taxon were deposited in the

(Ronquist & Huelsenbeck, 2001). Four MCMC chains were run si-

culture collection of fungi “Coleção Octávio Almeida Drummond”

multaneously, starting from random trees for 10,000,000 genera-

(COAD) at the Universidade Federal de Viçosa (UFV).

tions. The trees were sampled every 1,000th generation for a total of 10,000 trees. The first 2,500 trees were discarded as the burn-­in phase of each analysis. The posterior probabilities were determined

2.5 | Pathogenicity test

from a majority-­rule consensus tree that was generated from the re-

The isolates used for morphological characterization were tested for

maining 7,500 trees. The trees were visualized in FigTree (Rambaut,

pathogenicity on L. tomentosa leaves by mycelial plugs and conidial

2009) and exported to graphic programs.

suspension. For both methods 7-­day-­old pure cultures at 25°C with

Based on the GAPDH tree, a subset of 10 representative iso-

a 12-­hr photoperiod were used.

lates were selected for further BI analysis using alone sequences

To realize inoculation by conidial suspension, fungal cultures

from ApMat and multilocus analysis using sequences from ACT,

were flooded with 10 mL of sterilized distilled water and the surface

CAL, CHS-­1, GAPDH, ITS, SOD2 and TUB2. Sequences of selected

lightly scraped with sterilized Drigalski-­spatel. The suspension was

Colletotrichum species from GenBank and Weir et al. (2012) were

filtered through a double layer of cheesecloth and the conidia con-

included in this study (Table 1). The closest sequences were then

centration of 1 × 106 conidia mL−1was determined using Neubauer’s

downloaded in FASTA format and aligned as previously described.

counting chamber. Each species of Colletotrichum was inoculated by

The Bayesian inference (BI) analyses were performed on every se-

wounded and nonwounded methods, with two replicates for each

quence, first with ApMat gene/locus separately and then with the

method. Four control plants, two with and two without wounds,

concatenated sequences (ACT, CAL, CHS-­1, ITS, SOD2 and TUB2).

were sprayed with sterilized distilled water.

Mangifera indica Mangifera indica Mangifera indica Licania tomentosa Coffea arabica Ficus edulis V. vinifera cv. Cabernet Sauvignon V. vinifera cv. Cabernet Sauvignon Hymenocallis americana Hymenocallis americana Jasminum sambac

CMM4083

CMM3814a

CMM3740

COAD1961

ICMP 18581a

ICMP17921

JZB 330028

JZB 330024

CSSN 2

CSSN 3

ICMP 19118a

GZAAS5.09538

C. siamense (C. dianesei)

C. siamense (C. endomangiferae)

C. siamense (C. endomangiferae)

C. fructicola

C. fructicola

C. fructicola

C. hebeiense

C. hebeiense

C. siamense (C. hymenocallidis)

C. siamense (C. hymenocallidis)

C. siamense (C. jasmini-sambac)

C. siamense (C. murrayae)

JX010251

JQ247633

HM131511

GQ485601

GQ485600

KF156873

KF156863

JX010181

JX010165

MG674923

KC702978

KC702994

KC329779

JN390931

JN248669

JN248673

JN248668

KJ955082

KJ955081

JX010217

JQ247608

HM131497

GQ856759

GQ856757

KF377505

KF377495

JX009923

JX010033

MG674953

KC702954

KC702955

KC517194

KC790760

KC790737

KC790739

KC790736

KJ954783

KJ954782

JX010018

JX010028

JX009930

JQ247597

JX009713

GQ849451

GQ849463

-­

-­

JX009671

FJ917508

MG674998

KC992371

KC992372

KC517209

KF451946

-­

KF451956

KF451955

KJ954635

KJ954634

JX009664

JX009654

JX009721

JX009684

JX009683

CAL

JQ247656

HM131507

GQ856776

GQ856775

KF377542

KF377532

JX009495

FJ907426

MG675018

KC702921

KC702922

KC517298

KC790647

KC790623

KC790625

KC790622

KJ954364

KJ954363

JX009580

JX009572

JX009483

JX009519

JX009443

ACT

-­

JX009895

GQ856729

GQ856730

-­

KF289008

JX009839

JX009866

MG674943

KC598098

KC598113

KJ155469

KF451981

-­

KF451991

KF451990

-­

-­

JX009754

JX009882

JX009799

JX009789

JX009774

CH-­1

-­

-­

-­

-­

-­

-­

JX010322

JX010327

MG674988

-­

-­

-­

-­

-­

-­

-­

-­

-­

JX010307

JX010333

JX010314

JX010312

JX010311

SOD2

JQ247645

JX010415

GQ849439

GQ849438

-­

KF288975

JX010400

JX010405

MG675008

KM404169

KM404170

KC517254

KC790893

KC790870

KC790872

KC790869

KJ955231

KJ955230

JX010385

JX010411

JX010392

JX010390

JX010389

TUB2

-­

JQ899273

JQ899283

JQ807842

-­

-­

-­

JQ807838

MG674933

KJ155453

KJ155452

-­

KC790697

KC790675

KC790677

KC790674

KJ954498

KJ954497

-­

(Continues)

KM360144

KM360145

-­

KM360143

ApMat

|

Murraya sp.

Psidium guajava

Bauhinia variegata

GS02

MTCC9663

C. siamense (C. dianesei)

Citrus sp. (orange)

Citrus sp. (orange)

Ca. sinensis, pathogen

Ca. sinensis, pathogen

C. siamense (C. dianesei)

GS01

GS06

C. siamense (C. communis)

LC 1365

C. camelliae

C. siamense (C. communis)

LC 1364

C. camelliae

ICMP 18691

C. alienum

a

Malus domestica Persea americana

ICMP 12071a

C. alienum

JX010176

JX009913

Aeschynomene virginica

JX010243

Pyrus pyrifolia

ICMP 18686

ICMP 17673a

C. aenigma

C. aeschynomenes

JX010044

JX010244

Persea americana

ICMP 18608a

GAPDH

C. aenigma

ITS

Host

Isolate

Species

GenBank accession number

TA B L E   1   GenBank accession numbers of DNA sequences of Colletotrichum spp. used in the phylogenetic analysis

4 of 11      

LISBOA et al.

JX010184

JN412802

JN412804

MG674924

JN412800

JN412798

MG674954

JN412782

JQ309639

JX009722

JX009719

MG674999

FJ917505

MG675006

MG675005

MG675004

MG675003

MG675002

MG675001

MG675000

JX009696

JX009694

JX009693

JX009692

JX009691

JX009694

MG674997

JX009663

JX009661

JX009689

JX009742

JQ247596

CAL

JN412793

JN412795

JX009480

JX009489

MG675019

FJ907423

MG675026

MG675025

MG675024

MG675023

MG675022

MG675021

MG675020

JX009562

JX009490

JX009504

JX009573

JX009447

JX009490

MG675017

JX009437

JX009486

JX009432

JX009433

JQ247657

ACT

-­

-­

JX009826

JX009870

MG674944

JX009865

MG674951

MG674950

MG674949

MG674948

MG674947

MG674946

MG674945

JX009863

JX009890

JX009900

JX009759

JX009899

JX009890

MG674942

JX009835

JX009834

JX009815

JX009896

-­

CH-­1

-­

-­

JX010318

JX010329

MG674989

JX010326

MG674996

MG674995

MG674994

MG674993

MG674992

MG674991

MG674990

JX010325

JX010334

-­

-­

JX010336

-­

MG674987

JX010320

JX010319

JX010317

JX010335

-­

SOD2

Note. aEx-­t ype cultures. Isolates obtained in this study are highlighted in bold. COAD, Coleção Octávio Almeida Drummond at the Universidade Federal de Viçosa.

Vitis vinifera, cv. Hongti

GZAAS 5.08608

C. viniferum

JX010020

Vitis vinífera

JX010275

Litchi chinensis

ICMP 18672

GZAAS 5.08601a

C. tropicale

Theobroma cacao

ICMP 18653

C. viniferum

JX010007

JX010264

Licania tomentosa

COAD2083

C. tropicale

JX009924

MG674961

C. tropicale

JX010171

Coffea arabica

MG674931

Licania tomentosa

COAD2086

MG674960

MG674959

ICMP 18578a

MG674930

MG674929

MG674958

MG674955

C. siamense

Licania tomentosa

COAD2085

MG674928

MG674925

C. siamense

Licania tomentosa

COAD2084

C. siamense

C. siamense

MG674957

Licania tomentosa

MG674927

Licania tomentosa

COAD2081

COAD2082

C. siamense

Licania tomentosa

COAD2080

C. siamense

MG674956

MG674926

Licania tomentosa

COAD1962

C. siamense

C. siamense

JX009916

JX010036

Salsola tragus

JX010242

Coffea sp.

ICPM 18705

JX010185

JX009919 JX010010

ICMP 19051a

JX010186

JX009934

JX010036

MG674952

C. queenslandicum

Carica sp.

ICPM1780

JX010276

JX010185

MG674922

JX009972

JX009936

JX010189 JX010187

JX010015

JX010050

JQ247609

GAPDH

JX010142

JX010146

JQ247632

ITS

GenBank accession number

C. salsolae

Persea americana

ICPM 12564

C. queenslandicum

C. queenslandicum

Licania tomentosa

COAD1960

C. queenslandicum Carica papaya

Nuphar lutea subsp. Polysepala

ICMP 18187

C. nupharicola

Carica papaya

Nuphar lutea subsp. Polysepala

CMP 17938

C. nupharicola

ICMP 17921

Musa sapientum

ICMP 17817

ICMP 1778a

Musa sp.

ICMP 19119

C. musae

C. musae

C. queenslandicum

Murraya sp.

GZAAS5.09506a

C. siamense (C. murrayae)

C. queenslandicum

Host

Isolate

Species

TA B L E   1   (Continued)

JN412811

JN412813

JX010396

JX010407

MG675009

JX010404

MG675016

MG675015

MG675014

MG675013

MG675012

MG675011

MG675010

JX010403

JX010412

-­

-­

JX010414

JX010412

MG675007

JX010398

JX010397

JX010395

HQ596280

JQ247644

TUB2

KJ609020

-­

GU994430

KC790728

MG674934

JQ899289

MG674941

MG674940

MG674939

MG674938

MG674937

MG674936

MG674935

KC888925

-­

-­

-­

KC888928

-­

MG674932

JX145319

JX145323

KC888926

JQ899271

-­

ApMat

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The inoculation by mycelial plug method was carried out using one plug with 4-­m m diameter containing mycelia from the margins of the fungal cultures and deposited over wounds in the surfaces of healthy leaves. As a control, one PDA plug (4-­m m diameter) was placed on the wounded surfaces of leaves. In both assays, the inoculated plants remained in a moist

chamber

for

24  hr

inside

a

growth

chamber

at

25°C with a 12-­h r photoperiod. The incisions were performed on leaves with a cylindrical tool, 10 mm in diameter, containing a set of needles. The design was completely randomized. The incidence of disease was evaluated 10 days after inoculation (DAI). The leaf was considered diseased when it presented necrotic lesion.

3 | R E S U LT S 3.1 | Fungal isolation In total, 35 Colletotrichum isolates were collected, of which 25 were obtained from leaves with anthracnose and ten from asymptomatic tissues. Among the pathogenic isolates, 14 and 11 were obtained, respectively, by direct and indirect methods of isolation. In 100% of the healthy samples, it was possible to obtain endophytic isolates of the Colletotrichum. Recognition of the endophytic Colletotrichum isolates was based on cultural aspects of the fungus and on observations of the conidial morphology. Afterwards, these fungi were identified based on morphological and phylogenetic comparisons.

F I G U R E   1   Multilocus phylogenetic tree inferred from Bayesian analysis based on the combined sequences of actin (ACT); calmodulin (CAL); chitin synthase (CHS-­1); glyceraldehyde-­3-­phosphate dehydrogenase (GAPDH); β-­tubulin 2 (TUB2); manganese-­superoxide dismutase (SOD2); ribosomal internal transcribed spacer (ITS). Bayesian posterior probabilities are indicated next to nodes. The tree was rooted with Colletotrichum salsolae ICPM19051. The species of this study are highlighted in bold and ex-­t ype isolates are emphasized with asterisks

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3.2 | Phylogenetic analyses

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species belonging to the Colletotrichum gloeosporioides complex (Table 1). The concatenated sequences were composed of 2514 bp

The first analysis using partial sequence of the GAPDH region

(Figure 1). Based on ApMat tree (Figure 2), the endophytic iso-

(GenBank Accession Nos. MG674952 to MG674986) from the 35

late COAD 1961 (E) showed 100% similarity with sequences of

endophytes (E) and pathogenics (P) isolates revealed that all are

the C. fructicola, and the pathogenic isolate COAD 2083 (P) ex-

members of the C. gloeosporioides complex by means of Bayesian

hibit 100% similarity with C. tropicale. In addition, the endophytic

analyses (Treebase accession number S20974).

isolate COAD 1960 (E) was grouped with 100% similarity with

From the initial analysis of the GAPDH region, a subset of 10

Colletotrichum queenslandicum. The isolates COAD 1962 (P), COAD

pathogenic (P) and endophytic (E) isolates representing all taxa

2080 (P), COAD 2081 (E), COAD 2082 (P), COAD 2084 (E), COAD

were selected for phylogenetic analysis with partial sequences of

2085 (P), and COAD2086 (P) were grouped with Colletotrichum si-

the ApMat gene and multilocus dataset [ACT (1–230), CAL (231–

amense with high phylogenetic support. This analysis confirms the

803), CHS-­1 (804–1071), GAPDH (1072–1313), ITS (1314–1785),

result found by the phylogenetic analysis based on the multilocus

SOD2 (1786–2086), TUB2 (2087–2514)] involving sequences of

dataset (ACT, CAL, CHS-­1, GAPDH, ITS, SOD2, TUB2).

F I G U R E   2   Phylogenetic tree inferred from Bayesian analysis based on the partial sequences of the Apn2-­Mat1-­2 (ApMat) gene. Bayesian posterior probabilities are indicated next to nodes. The tree was rooted with Colletotrichum salsolae ICPM19051. The species of this study are highlighted in bold and ex-­t ype isolates are emphasized with asterisks

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3.3 | Morphological characterization

and colony colouration, respectively, of 88 mm (whitish colour) and 79 mm (colouration ranging from white to grayish), 70 mm (coloura-

The COAD 1961 (E), COAD 2083 (P), COAD 1960 (E), and COAD 1962

tion ranging from white to grayish) and 72 mm (colouration ranging

(P) isolates (previously identified by molecular data) used to morpho-

from white to pink) for the 7 days of cultivation on PDA.

logical characterization, belong to C. fructicola, C. queenslandicum, C. siamense and C. tropicale species, respectively. All the species presented subcylindrical conidia with rounded extremities. The appressoria were subglobous, clavate and fusiform (Figure 3) (Table 2).

3.4 | Pathogenicity test The isolates of C. siamense (P), C. tropicale (P), C. fructicola (E) and

As regards cultural characteristics, isolates of the C. fructicola,

C. queenslandicum (E) were pathogenic on L. tomentosa leaves when

C. tropicale, C. queenslandicum, and C. siamense showed growth (mm)

inoculated with mycelial plugs. However, only wounded leaves

F I G U R E   3   Apressoria and conidia observed on culture with seven days of growth on potato dextrose agar (PDA). a, b, c – Colletotrichum fructicola (COAD1961); d, e, f – Colletotrichum queenslandicum (COAD1960), g, h, i – Colletotrichum siamense (COAD1962), j, k, l – Colletotrichum tropicale (COAD2083)

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TA B L E   2   Morphological characteristics of Colletotrichum spp. associated with Licania tomentosa Conidia Colletotrichum species

Length (μm)

Width (μm)

Shape

Apressoria Shape

C. fructicola

13.0–14.5

4.5–5.0

Subcylindrical

Clavated/fusiform

C. queenslandicum

15.0–17.0

4.0–5.0

Subcylindrical

Subglobose/clavate

C. siamense

10.0–23.0

3.5–6.0

Subcylindrical

Subglobose

C. tropicale

15.5–17.0

3.5–4.5

Subcylindrical

Subglobose/clavate/fusiform

presented anthracnose symptoms using conidial suspension. The

and Malus sylvestris in Australia and in coffee berries in Fiji (Weir

dark brown to black lesions appeared seven DAI and were similar for

et al., 2012). Of late, this species has been reported on Citrus × lati-

all Colletotrichum species. The Colletotrichum spp. were recovered

folia in the United States (Kunta, Park, Vedasharam, Graça, & Terry,

from symptomatic leaves. Control plants remained asymptomatic.

2018). In Brazil, this was the first report of this fungus. At first, C. siamense was proposed as a complex, belonging to the subclade Musae of the C. gloeosporioides complex, comprising seven

4 | D I S CU S S I O N

species (Sharma et al., 2014): C. dianesei (Lima et al., 2013), C. endomangiferae (Vieira et al., 2014), C. hymenocallidis (Yang et al., 2009),

In this study, it was possible to identify endophytic and pathogenic

C. jasmini-sambac (Wikee et al., 2011), C. murrayae (Peng, Yang, Hyde,

isolates of Colletotrichum based on multilocus sequence analy-

Bahkali, & Liu, 2012), C. melanocaulon (Doyle, Oudemans, Rehner, &

sis, together with examination of the phenotypic characters. All

Litt, 2013) and C. communis (Sharma et al., 2014). However, results of

specimens found are members of the C. gloeosporioides complex.

new molecular analyses have proven that C. siamense s. lat. is a sin-

Therefore, they showed very similar morphological and cultural

gle species rather than a species complex (Liu, Wang, Damm, Crous,

characteristics. However, according to Weir et al. (2012), these

& Cai, 2016). In Brazil, C. siamense has been previously reported on

characteristics can vary within the same species. The phylogenetic

other hosts, such as Manihot carthaginesis and M. tomentosa (Oliveira,

analyses performed on both the Ap-­Mat region and concatenated

Silva, Diamantino, & Ferreira, 2018), Acca sellowiana (Fantinel et al.,

sequences (ACT, CAL, CHS-­1, ITS, SOD2 and TUB2) allowed for

2017), Fragria × ananassa (Capobiango, Pinho, Zambolim, Pereira, &

the identification of those isolates of Colletotrichum from L. tomen-

Lopes, 2016). In addition, it has also been reported on Diospyros kaki

tosa leaves with high phylogenetic support. Of ten isolates, seven

in Korea (Chang, Hassan, Jeon, Shin, & Oh, 2018), M. domestica and

were identified as C. siamense and the other three as C. fructicola,

Bauhinia forficata in Argentina (Fernadez et al. 2018, Larran, Vera,

C. queenslandicum, and C. tropicale. Endophytic isolates of C. fruc-

Dal Bello, Franco, & Balatti, 2015) and Juglans regia in China (Wang

ticola, C. queenslandicum, and C. siamense were obtained whereas

et al., 2017).

pathogenic isolates were identified as C. siamense and C. tropicale.

Colletotrichum tropicale, described by Rojas et al. (2010), was

The same species found in this study, with the addition of C. asianum

commonly isolated as an endophyte from leaves from a wide

and exception of C. queenslandicum, were also found endophytically

range of host species in the tropical forests of Panama, includ-

associated with mango in Brazil (Vieira, Michereff, de Morais, Hyde,

ing Theobroma cacao, Trichilia tuberculata, Viola surinamensis and

& Câmara, 2014).

Cordia aliodora, also isolated from a rotting fruit of Annona muricata.

Based on the results obtained in this study, the ApMat tree has

In Brazil, C. tropicale was reported to have caused anthracnose on

the same topology as the multilocus tree, allowing for discrimination

mango fruit (Lima et al., 2013), carnauba palm fruit (Araujo, Lima,

of the different species of the Colletotrichum on L. tomentosa. This

Rabelo Filho, Ootani, & Bezerra, 2018) and wild cassava species

information confirms the efficiency of this molecular marker for the

(Oliveira, Bragança, & Silva, 2016).

taxonomy of members of the C. gloeosporioides complex (Sharma, Pinnaka, & Shenoy, 2014).

According to the pathogenicity test, all species were able to cause disease on L. tomentosa leaves, even when they were

The C. fructicola species was originally reported to cause anthrac-

found endophytically. Therefore, according to these results,

nose on coffee fruit in Thailand (Prihastuti et al., 2009). This pathogen

a same species of Colletotrichum associated with L. tomentosa

had been previously reported on Manihot sculenta, Nopalea coche-

leaves may behave as both an endophyte and a pathogen. This

nillifera,

Mangifera indica,

suggests that endophyticism can plays an important role in the

Phaseolus lunatus and Malus domestica in Brazil (Bragança, Silva,

life cycle of Colletotrichum spp. It is possible endophytic fungi

Haddad, & Oliveira, 2016; Conforto, Lima, Garcete-­Gomez, Câmara,

may remain dormant in host tissues and when under appropriate

& Michereff, 2017; Costa et al., 2017; Lima et al., 2013; Sousa et al.,

conditions they may cause parasitism, leading to lesions (Cannon

2018; Velho, Alaniz, Casanova, Mondino, & Stadnik, 2015).

et al., 2012). This hypothesis has been corroborated by Delaye,

Annona muricata,

Annona squamosal,

The C. queenslandicum species was originally reported to have

García-­G uzmán, and Heil (2013); these authors studied the ge-

caused anthracnose in Carica papaya, Persea americana, M. indica

netic relationships between endophytic and pathogenic fungi

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and concluded that these can alternate between endophytic and necrotrophic during their evolution. On the other hand, it is possible that the tissue was already infected at the time of sample collection. However, it remained asymptomatic due to the incubation period of the anthracnose. To our knowledge, this is the first worldwide report of C. fructicola, C. siamense, C. tropicale and C. queenslandicum associated with L. tomentosa and the first recording of the last species in Brazil. This information is very useful for knowledge of the host range of Colletotrichum species which are common in the tropics, for quarantine and to determine a control strategy for anthracnose in several hosts.

ORCID Olinto L. Pereira  Gleiber Q. Furtado 

http://orcid.org/0000-0002-0274-4623 http://orcid.org/0000-0001-5842-3389

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How to cite this article: Lisboa DO, Silva MA, Pinho DB, Pereira OL, Furtado GQ. Diversity of pathogenic and endophytic Colletotrichum isolates from Licania tomentosa in Brazil. For Path. 2018;e12448. https://doi.org/10.1111/efp.12448