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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
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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
LISBOA et al. 5 of 11
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LISBOA et al.
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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