New records of Colletotrichum species for the mycobiota of Iran

Document Type: Original Article

Authors

1 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj

2 Department of Plant Protection, Azarbaijan Shahid Madani University, Tabriz, Iran

3 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

4 Department of Botany, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

5 Senckenberg Museum of Natural History Görlitz, Germany

6 Environmental Genomics, Botanical Institute, Christian-Albrechts University of Kiel

7 Max Planck Institute for Evolutionary Biology, Plön, Germany

Abstract

Sixteen isolates of Colletotrichum were collected from leaves with anthracnose symptoms or leaf spots of twelve wild, cultivated and ornamental plant species from the Guilan, Mazandaran, and Golestan provinces in Northern Iran. Five different species, including C. aenigma, C. gigasporum, C. godetiae, C. karstii, and C. musae were identified based on the DNA sequence data (TUB2, GS, GAPDH). Four of these species, termed as C. aenigma, C. gigasporum, C. godetiae and C. karstii represent new records for the mycobiota of Iran. Moreover, this study reports many plants as new hosts of Colletotrichum species. Comprehensive morphological descriptions and illustrations are provided for the species.

Keywords

Main Subjects


INTRODUCTION

Colletotrichum species are causal agents of anthracnose and other diseases on a wide range of plant species, and belong to the economically most important fungal pathogens (Sutton 1980; Hyde et al. 2009). Recently, the genus Colletotrichum was ranked in the top 10 fungal pathogens based on perceived scientific and economic importance (Dean et al. 2012). The genus has been recorded from approximately 2200 plant species (Farr & Rossman, 2016).

Precise identification has a crucial role in order to understand the epidemiology of Colletotrichum species and to develop effective control measures of the plant diseases. Traditional identification systems in Colletotrichum relied heavily on morphological and cultural characteristics (von Arx, 1957; Sutton, 1980, 1992; Bailey & Jeger, 1992; Freeman et al. 1998). However, Sutton (1992) mentioned that morphology does not provide sufficient and informative characters for an accurate identification. The systematics of Colletotrichum has been challenging due to the lack of reliable morphological characters making it difficult to delineate species boundaries (Cai et al. 2009). To resolve this deficiency, many researchers have used DNA sequence data, physiology, secondary metabolites and pathogenicity information, as part of a multifaceted approach (e.g. Than et al. 2008; Crouch et al. 2009; Prihastuti et al. 2009). Phylogenetic analyses based on DNA sequences have been led to significant progress in the systematics of the genus, and many species were differentiated within large species complexes, named as the Gloeosporioides species complex (Prihastuti et al. 2009; Phoulivong et al. 2010; Weir et al. 2012; Liu et al. 2013; Liu et al. 2015), the Acutatum species complex (Damm et al. 2012a), the Boninense species complex (Damm et al.2012b), the Dematium species complex (Damm et al. 2009), the Orbiculare species complex (Damm et al. 2013) and the Destructivum species complex (Damm et al. 2014). Recently, two additional species complexes termed as the Caudatum species complex (Crouch, 2014) as well as the Gigasporum species complex (Liu et al. 2014) were introduced.

Accordingly, the aim of this study was to characterize Colletotrichum species gained from the infected leaves of several plant species collected from the Northern Iran around the Caspian Sea, based on morphology, cultural characteristics and phylogenetic analyses of the DNA sequence data.

 

 

MATERIALS AND METHODS

 

Sample collection

 

Samplings were conducted during 2012–2013 in three provinces in Northern Iran, the Guilan, Mazandaran and Golestan provinces. Samples were collected from hosts showing symptoms of anthracnose and spots on the leaves. Slices of plant samples (1 cm) were washed in tap water, surface-disinfected in 2% sodium hypochlorite solution for 1 min, rinsed in sterile distilled water and then moist incubated in glass petri dishes on paper towels soaked with sterile tap water. Petri dishes were kept at 20-25°C in the dark. Water agar (WA, 2%) plates supplemented with chloramphenicol (50 mg/L) were inoculated with a sterile needle dipped in conidial masses indicative of Colletotrichum that had formed on moist incubated plant tissue. Single spore or single hyphae were obtained on potato dextrose agar (PDA, Merck, Darmstadt, Germany) (Goh, 1999). Pure cultures were deposited at the Mycology Laboratory (UTFC ….) of College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. Additionally, the subcultures are preserved at Iranian Fungal Culture Collection (IRAN …C) at the Iranian Research Institute of Plant Protection, Tehran, Iran.

 

DNA extraction, PCR and sequencing

 

Fungal isolates were incubated on PDA at 25°C for 7–10 days. Genomic DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook, 2001). The primers T1 (O'Donnell & Cigelnik 1997) and Bt-2b (Glass & Donaldson 1995) were employed to amplify the partial β-tubulin (TUB2) gene. Polymerase chain reaction (PCR) was performed in a TProfessional Thermocycler (Biom- etra, Germany) in a total volume of 25 μL. The PCR mixture contained 1 μL genomic DNA, 0.2 μM of each primer, 1 × HF phusion PCR buffer (Thermo Scientific, Germany), 2 mM MgCl2, 20 μM of each dNTP, 0.75μ DMSO and 0.25 U Phusion High-Fidelity polymerase (Thermo Scientific, Germany). The PCR for TUB2 was done as following; an initial step of 5 min at 98°C, 35 cycles of 10 s at 98°C, 20 s at 65°C and 20 s at 72 °C, followed by 10 min at 
72 °C. Conditions for PCR amplification of the partial genes including glutamine synthetase (GS) and glyceraldehyde-3-phosphate dehydrogenase (GAPD H) followed by the protocols as previously described in Prihastuti et al. (2009) using the primers GSF1 & GSR1 (Stephenson et al. 1997) and GDF & GDR (Templeton et al. 1992), respectively. The PCR products were purified by Wizard Genomic DNA Purification Kit (Promega, USA) and sequenced by Macrogen Company (Amsterdam, the Netherlands) with the amplifying primers.

 

Morphology

 

Morphological experiments were performed according to Damm et al. (2012a). Both cultural and microscopic features were studied on synthetic nutrient agar (SNA) (Nirenberg 1976), and on oatmeal agar (OA) (Crous et al. 2009). Cultures were inoculated with 5-mm diameter plugs from 4–7 days old purified cultures. To enhance sporulation, isolates were transferred to SNA amended with autoclaved filter papers and double-autoclaved stems of Anthriscus sylvestris. SNA and OA cultures were incubated at 20°C under near-UV light with a 12 hours photoperiod for 10 days. Measurements and photomicrographs of fungal structures (conidia, conidiophores, conidiomata, setae, appressoria, sclerotia, perithecia, asci and ascospores) were made according to Damm et al. (2007). Appressoria were observed on the reverse side of SNA plates. Conidia were taken from acervuli. Microscopic preparations were made in clear lactic acid, with at least 30 measurements per structure and observed with a Nikon SMZ1000 dissecting microscope (DM) or with an Olympus BX51 microscope. Images were recorded with a Leica DFC320 camera on a Nikon SMZ1000 dissecting microscope (DM) or on an Olympus BX51 microscope. SNA and OA cultures were periodically examined for the development of perithecia. Ascospores were described from perithecia crushed in lactic acid. Colony characters and pigment production on SNA and OA cultures incubated for 14 days at 20°C under near-UV light with a 12 hours photoperiod were documented after 10 days. Colony color was determined according to the Rayner (1970). Growth rates were measured after 7 and 10 days.

 

Phylogenetic analysis

 

The programs Chromas Pro 1.34 and Edit Seq, parts of the DNA*Lasergene (DNAstar, Madison, WI) software package, were utilized for viewing, editing and assembling the sequences. The sequences were aligned by using MUSCLE, a multiple sequence alignment method (Edgar, 2004). The sequences of the 16 examined Iranian Colletotrichum isolates were compared with other fungal DNA sequences from NCBI GenBank database (www.ncbi.nlm.nih.gov/ genbank/) and the Q-bank Fungi database (http:// www.q-bank.eu/fungi/) through BLAST tool. Sixty sequences with high similarity were added to the alignment as reference strains (Table 1). Analyses were performed via the software MEGA v.6 (Tamura et al. 2013). Distance matrixes of the aligned sequences were calculated by the Kimura 2-parameter model (Kimura 1980), and analyzed with the maximum likelihood (ML) algorithm (Felsenstein 1985). The reliability of the inferred trees was estimated by bootstrap analyses with 1000 replicates.

In maximum parsimony (MP) analyses, the evolutionary distances were computed using the maximum composite likelihood method (Varin 2008). Gaps were treated as missing data in the pairwise sequence comparisons (Pairwise deletion option). The MP tree was obtained using the close-neighbor-interchange algorithm of Nei & Kumar (2000) with search level 1 (Felsenstein 1985, Nei & Kumar 2000) in which the initial trees were randomly obtained by the addition of sequences (100 replicates). Clade stability was assessed in a bootstrap analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa. The tree branches were drawn to scale, with lengths calculated using the average pathway method (Nei & Kumar 2000), as the unit of the number of changes over the whole sequence.

 

RESULT AND DISCUSSION

 

Phylogeny

 

The TUB2sequences of the 16 tested Colletotrichumisolates here were combined and aligned with the 60 reference sequences of 54 taxa from GenBank. The alignment consisted of 476 characters including alignment gaps, of which 244 were constant, 232 were variable and 178 were parsimony-uninfor-mative. Maximum parsimony analysis of the parsimony-informative characters resulted in 10 most parsimonious trees with a consistency index of 0.53, the retention index of 0.908486 and composite index of 0.488597 ML analysis resulted in a tree with the same topology as the MP trees. The ML tree is shown with bootstrap values of both analyses at the nodes (Fig. 1). Based on the phylogenetic analyses, the 16 isolates from various plants were grouped in four species complexes and five different Colletotrichum species named as C. gigasporum belonging to the Gigasporum species complex, C. godetiae belonging to the Acutatum species complex, C. aenigma and C. musae belonging to the Gloeosporoides species complex and C.karstii belonging to the Boninense species complex were identified (Fig. 1, Table 1). Four of these species, C. gigasporum, C. godetiae, C. karstii and C. aenigma are new records for the mycobiota of Iran.

 

Table 1. A list of Colletotrichum isolates used in this study.

Species

Strain no.1

Host

Locality

GenBank Accession number

Tub22

GAPDH3

GS4

C. aenigma

ICMP 18608*

Persea americana

Israel

JX010389

JX010044

JX010078

 

ICMP 18686

Pyrus pyrifolia

Japan

JX010390

JX009913

JX010079

 

UTFC 239, IRAN 2443 C

Vigna unguiculata

Langrood, Rasht, Guilan, Iran

KU726942

KU726946

KU726944

C. aeschynomenes

ICMP 17673*

Aeschynomene virginica

USA

JX010392

-

-

C. acutatum

CBS 112996*

Carica papay

Australia

JQ005860

-

-

C. alatae

ICMP 17919*

Dioscorea alata

India

JX010383

-

-

C. alienum

ICMP 12071*

Malus domestica

New Zealand

JX010411

-

-

C. annellatum

CBS 129826*

Hevea indica

Colombia

JQ005656

-

-

C. aotearoa

ICMP 18537*

Coprosma sp.

New Zealand

JX010420

-

-

C. asianum

CBS 130418*

Coffea arabica

Thailand

JX010406

-

-

C. arxii

CBS 132511*

Paphiopedilum sp.

Germany

KF687881

-

-

C. boninense

CBS 123755*

Crinum asiaticum var. sinicum

Japan

JQ005588

-

-

C. camelliae

LC1364*

Camellia sinensis

China

KJ955230

-

-

C. clidemiae

ICMP 18658*

Clidemia hirta

USA, Hawaii

JX010438

-

-

C. cordylinicola

ICMP 18579*

Cordyline fruticosa

Thailand

JX010440

-

-

C. dianesei

MFLU 1300058*

Mangifera indica

Brazil

KC517254

-

-

C. fioriniae

CBS 128517*

Fiorinia exter

USA

JQ949992

-

-

C. fructicola

ICMP 18581*

Coffea Arabica

Thailand

JX010405

-

-

C. fructivorum

CBS 133125*

Vaccinium macrocarpon

USA

JX145196

-

-

C. gigasporum

CBS 133266*

Centella asiatica

Madagascar

KF687866

-

-

 

CBS 124947

Theobromae cacao  

Panama

KF687871

-

-

 

UTFC 255, IRAN 2444 C

Camellia sineasis

Lahijan, Guilan Province

KR905717

-

-

 

UTFC 256

Euonymus japonicus

Namak-Abrood, Mazandaran Province

KR905718

-

-

C. gloeosporioides

IMI 356878*

Citrus sinensis

Italy

JX010445

-

-

C. godetiae

CBS 133.44*

Clarkia hybrida, cv. kelvon glory

Denmark

JQ950053

-

-

 

CBS 129913, CPC 15126

Podocarpus

South Africa

JQ950087

-

-

 

UTFC 257

Frangula alnus

Kheiroodkenar forest, Noushahr, Mazandaran Province

KR905719

-

-

 

UTFC 258, IRAN 2447 C

Acer cappadocicum

Kheiroodkenar forest, Noushahr, Mazandaran Province

KR905720

-

-

 

UTFC 259

Frangula alnus

Kheiroodkenar forest, Noushahr, Mazandaran Province

KR905721

-

-

 

ICMP 18539*

Olea europaea

Australia

JX010434

-

-

 

 

 

 

 

 

 

C. grevilleae

CBS 132879*

Grevillea sp.

Italy

KC297102

-

-

C. hebeiense

JZB330028

Vitis vinifera

China

KF288975

KF377495

 

C. henanense

CGMCC 3.17354*

Camellia sinensis

China

KJ955257

-

-

C. hipeastri

CBS 125377

Hippeastrum vittatum

China

JQ005664

-

-

C. horii

ICMP 10492*

Diospyros kaki

Japan

JX010450

-

-

C. johnstonii

CBS 128532, ICMP 12926*

Solanum lycopersicum

New Zealand

JQ950095

-

-

C. karstii

CGMCC3.14194*

Vanda sp.

China

HM585428.1

-

-

 

CBS 127536

Eucalyptus grandis

South Africa

JQ005635

-

-

 

CBS 861.72

Bombax aquaticum

Brazil

JQ005618

-

-

 

UTFC 242

Euonymus japonicus var. aureo-marginata

Amol, Mazandaran Province

KR867693

-

-

 

UTFC 243

Syringia reticulate

Sari, Mazandaran Province

KR905710

-

-

 

UTFC 244

Euonymus japonicus

Ramsar , Mazandaran Province

KR905711

-

-

 

UTFC 245

Lourus nobilis

Ramsar , Mazandaran Province

-

-

-

 

UTFC 246

Ficus benjaminia

Gorgan , Golestan Province

KR905712

-

-

 

UTFC 247

Cyperus sp.

Jelin, Golestan Province

KR905713

-

-

 

UTFC 248

Euonymus japonicus

Tonkabon, Mazandaran Province

KR905714

-

-

 

UTFC 249, IRAN 2448 C

Passiflora edulis

Tonkabon, Mazandaran Province

KR905715

-

-

 

UTFC 250

Passiflora edulis

Tonkabon, Mazandaran Province

KR905716

-

-

C. kahawae subsp. ciggaro

ICMP 18539*

Olea europaea

Australia

JX010434

-

-

C. kahawae subsp. kahawae

ICMP 17816*

Coffea Arabica

Kenya

JX010444

-

-

C. lupine

CBS 109225*

Lupinus albus

Ukraine

JQ949806

-

-

C. magnisporum

CBS 398.84*

Unknown

Unknown

KF687882

-

-

C. melanocaulon

CBS 133251*

Vaccinium macrocarpon

USA

JX145195

-

-

C. musae

ICMP 19119*

Musa sp.      

USA

HQ596280

-

-

 

ICMP 17817

Musa sapientum

Kenya

JX010395

-

-

 

UTFC 233

Musa sapientum

Gorgan, Golestan Province

KU726940

-

-

 

UTFC 234

Citrus sinensis

Gorgan, Golestan Province

KU726941

KU726945

KU726943

C. nupharicola

ICMP 18187*

Nuphar lutea subsp. polysepala

USA

JX010398

-

-

C. nymphaeae

CBS 515.78*

Nymphaea alba

Netherlands

JQ949848

-

-

C. orchidophilum

CBS 632.80*

Dendrobium sp.

USA

JQ949802

-

-

C. paxtonii

IMI 165753*

Musa sp.

Saint Lucia

JQ949936

-

-

C. phormii

CBS 118194*

Phormium sp.

Germany

JQ950097

-

-

C. phyllanthi

CBS 175.67*

Phyllanthus acidus

India

JQ005655

-

-

C. proteae

CBS 132882*

Protea sp.   

South Africa

KC297101

-

-

C. pseudomajus

CBS 571.88*

Camellia sinensis

Taiwan

KF687883

-

-

C. psidii

ICMP 19120*

Psidium sp.

Italy

JX010443

-

-

C. pyricola

CBS 128531*

Pyrus communis

New Zealand

JQ950096

-

-

C. queenslandicum

ICMP 1778*

Carica papaya

Australia

JX010414

-

-

C. radices

CBS 529.93*

Unknown

Costa Rica

KF687869

-

-

 

Table 1. Continued.

Species

Strain no.1

Host

Locality

GenBank Accession number

Tub22

GAPDH3

GS4

C. rhexiae

CBS 133134*

Rhexia virginica

USA

JX145179

-

-

C. salicis

CBS 607.94*

Salix sp.

Netherlands

JQ950111

-

-

C. salsolae

ICMP 19051*

Salsola tragus

Hungary

JX010403

-

-

C. siamense

CBS 130417*

Coffea Arabica

Thailand

JX010404

-

-

C. syzygicola

DNCL021

Syzigium Samarangense

Thailand

KF254880

-

-

C. temperatum

CBS 133122*

Vaccinium macrocarpon

USA

JX145211

-

-

C. theobromicola

ICMP 18649*

Theobroma cacao

Panama

JX010447

-

-

C. ti

ICMP 4832*

Cordyline sp.

New Zealand

JX010442

-

-

C. tropicale

ICMP 18653*

Theobroma cacao

Panama

JX010407

-

-

C. vietnamense

CBS 125478*

Coffea sp.

Vietnam

KF687877

-

-

C. viniferum

JZB330008

Vitis vinifera

China

KF288962

-

-

C. xanthorrhoeae

CBS 127831*

Xanthorrhoea preissii

Australia

JX010448

-

-

Colletotrichum sp.

CBS 159.50

Derris sp.

Indonesia

KF687867

-

-

 

CBS Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CGMCC China General Microbial Culture Collection Center; ICMP International Collection of Microorganisms from Plants, Landcare Research, Auckland, New Zealand; IRAN Iranian Fungal Culture Collection at the Iranian Research Institute of Plant Protection, Tehran, Iran; UTFC University of Tehran, Fungal Culture Collection,atmycology laboratory, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. MFLU Mae Fah Luang University, Thailand

β-tubulin

3 Glutamine synthetase

4 Glyceraldehyde-3-phosphate dehydrogenase

The isolated strains and newly generated sequences in this study are shown in bold

*indicates the ex-type cultures

 

Fig. 1. Thephylogenetic tree constructed from a maximum likelihood analysis based on the TUB2 sequence data. Bootstrap values > 50% (1000 replicates) of ML analysis is exhibited above/below the branches and those of Maximum parsimony in brackets. Our isolates are marked with a red stars, and the strain number is followed by host and the country of origin. Black square indicates the ex-type strains. Monilochaetes infuscans was used as out-group taxon.

 

Characterizations of the species

 

All characterized species are similar by having straight conidia, but they can be distinguished from each other using different phenotypic and morphological traits such as colony size and shape, conidial size and shape as well as conidial mass color. To clarify the relationship and accurate identifications of Colletotrichum isolates, we conducted phylogenetic analyses using the partial sequence of β-tubulin gene combined with morphological features. The sequences of the identified isolates of Colletotrichum were aligned against those available in Q-bank (http://www.q-bank.eu/fungi) and GenBank through blast search (Altschul et al. 1990). The sequence of the TUB2 gene could resolve the relationship of the Colletotrichum isolates, mostly in agreement with morphological characters. The five described species here including C. karstiiC. gigasporum, C. godetiae, C. aenigma and C. musae belonging to four Colletotrichum species complexes (Boninens, Giga- sporum, Acutatum and Gloeosporioides complexes). According to Damm et al. (2012 a, b) and Liu et al. (2014), C. gigasporum, C. godetiaeC. karstii and C. musae can be readily distinguished from closely related species within the Boninense, Gigasporum, Acutatum and Gloeosporioides complexes, by using the TUB2 sequence data. Similarly, C. aenigma can be distinguished from closely related taxa in the Gloeosporioides complex via the combined TUB2GS and GAPDH sequences (Weir et al. 2012; Yan et al. 2015). Therefore, we performed the partial TUB2GS and GAPDH genes sequencing for the isolates belonging to the Gloeosporioides complex. A BLASTN search of the GS and GAPDH sequences as well as phylogenetic analysis based on the combined TUB2 and GAPDH sequences (Fig. 2) revealed that the TUB2 sequence data is sufficient to precisely identify the examined species. Hence, the phylogenetic tree constructed for all tested isolates in this study is mainly based on the TUB2 sequence alignment.

All identified species here are characterized as following:

 

Colletotrichum aenigma B. Weir & P.R. Johnst., Stud. Mycol. 73: 135 (2012) = Colletotrichum populi C.M. Tian & Z. Li, Mycotaxon 120: 283 (2012) (Fig. 3).

Morphology on SNA. Ascomata and perithecia are formed after 3-4 weeks, solitary, superficial or immersed in the agar medium, non-stromatic, globose to obpyriform, ostiolate, glabrous, brown, 190–250 × 150–200 µm. Asci unitunicate, 8-spored, cylindrical to clavate or fusiform, tapering to apex and base, smooth-walled, 42–68 × 10–11 µm. Ascospores uni or biseriately arranged, initially aseptate but often septate with age, hyaline, smooth-walled, variable in shape, fusiform to ovoid, slightly curved, (12–) 13–14 (–15) × (4.88–) 5.5–6 (–6.22) µm, mean ± SD = 13.5 ± 1.18 × 5.64 ± 0.62 µm, L/W ratio = 2.39. Conidiomata absent, the conidiophores are directly formed from vegetative hyphae (Fig. 3).

Morphology on Anthriscus stem. Conidiomata acervular, Conidiophores hyaline to pale brown, aseptate or septate, branched, Conidiogenous cells are haphazardly arised from dense, tangled hyphae across agar surface, short-cylindric with a poorly differentiated conidiogenous locus. Conidia often germinate soon after release, sometimes generate appressoria, thus create thin, compact, layers of germinated, septate conidia, germ tubes, and appressoria across the central part of the colony surface. Conidia (14.5–) 15.44–16 (–18.22) × (3.18–) 3.66–4 (–4.16) µm, mean ± SD = 16.1 ± 1.52 × 3.77 ± 0.44 µm, L/W ratio = 4.26, cylindric with broadly rounded ends. Appressoria 7–16 × 5–13 µm, mean ± SD = 11.46 ± 2.32 × 7.96 ± 1.69 µm, L/W ratio=1.43, subglobose or with a few broad lobes (Fig. 3).

Cultural characteristics. Colonies on OA flat with entire margin, aerial mycelium sparse, cottony, white, the surface of agar is uniformly pale orange towards center, more or less colorless towards edge, conidia are not well associated with acervuli and no masses of conidial ooze. In reverse pale orange towards center, 85 mm diameter after 10 days (Fig. 3).

Colletotrichum aenigma was described by Weir et al. (2012). Liu et al. (2013) considered C. aenigma similar to C. populi. Colletotrichum aenigma was isolated from Pyrus pyrifolia from Japan and Persea americana from Israel (Weir et al. 2012), Vitis vinifera from China (Yan et al. 2015) and Populus sp. from China (Liu et al. 2013). Weir et al. (2012) noted that the biology of the species is more or less unknown, but as it has been found in the widely separated regions, hence, it seems that to be a geographically widespread species.

Phylogenetically, C. hebeiense is a close species to C. aenigma, but the species are morphologically different. In C. aenigma, gattulates of the conidia are equally spread, while in C. hebeiense the granular contents are mostly present at the polar ends leaving a space in the middle. Appressoria of C. hebeiense are clavate to subglobose in shape while that of C. aenigma are subglobose, sometimes with few broad ends (Yan et al. 2015).

Phylogenetic analyses based on the partial TUB2 gene (Fig. 1) grouped the studied isolate (UTFC 239) with ex-type strain ICMP 18608 from Persea americana from Israel, in a monophyletic clade with 62% bootstrap support. Additionally, in ML and MP trees, the ex-type strain of C. hebeiense (JZB330028) showed a close relationship to C. aenigma strains (Fig. 1). Colletotrichum aenigma differs from C. hebeiense by 1 base pair of ß-tubulin but they are distinguishable by the GADPH gene(Yan et al. 2015). Therefore, we employed the partial TUB2GS and GAPDH genes to sequence the isolate UTFC 239. A BLASTN search was done by the GS and GAPDH sequences and the phylogenetic analysis was performed based on the combined TUB2 and GAPDH sequences (Fig. 2) indicating the TUB2 and GAPDH genes could act as suitable markers for the identification of C. aenigma.

Colletotrichum aenigma is a new record for the mycobiota of Iran. Furthermore, it is the first report of the species on Vigna unquiculata.

 

 

Fig. 2. Thephylogenetic tree was generated by a maximum likelihood analysis based on the combined TUB2 & GAPDH sequences data. Bootstrap values > 50% (1000 replicates) of ML analysis is shown above/below the branches. Our isolate is marked with a red star and a black square indicating the ex-type strain. Colletotrichum godetiae strain CBS 133.44 was used as out-group taxon.

 

 

 

Fig. 3. Colletotrichumaenigma (UTFC 239), (IRAN 2443 C). a. Culture on OA, 10 days growth (upper); b. Ascomata and conidiomata on Anthryscus stem; c. Conidiomata on Anthryscus stem; d. Conidiomata on filter paper; e. Ascomata on SNA; f. Asci; g. Conidiomata on filter paper; h. Setae; i. Conidia; j–m. Appressoria. Scale bars — c, d, f, g = 200 μm; e= 100 µm; h–i = 20 μm; j–m =10 μm.

 

 

Colletotrichum gigasporum E.F. Rakotoniriana & Munaut, Mycol. Progr. 12: 407 (2013) (Fig. 4).

 

Morphology on Anthriscus stem. Conidiomata acervular, conidiophores and setae were formed on a cushion of angular brown cells. Setae medium to dark brown, smooth-walled to verruculose, 2–3-septate, 40–136.5 μm long, base cylindrical to inflated, 4.65–8.1 μm diameter, tip acute to obtuse. Conidiophores hyaline to brown, septate sometimes branched 27–34 × 5.5–10 μm. Conidiogenous cells hyaline to medium brown, cylindrical or clavate. Conidia hyaline, aseptate, smooth-walled, cylindrical with rounded ends (19–) 20.5–22(–23.7) × (6.65–) 7.15–7.85(–8.30) µm, mean ± SD = 21.4 ± 1.83 × 7.45 ± 0. 67 µm, L/W ratio = 2.87 (Fig. 4).

 

Morphology on SNA. Conidiomata acervular. Appressoria pale brown, aseptate, solitary, with a ellipsoidal to irregular outline and a crenate or lobed margin, 6–23 × 4.5–13.5 μm, mean ± SD = 13 ± 4.5 × 8.5 ± 2.17 μm, L/W ratio = 1.52 (Fig. 4).

Cultural characteristics. Colonies on OA flat with entire margin, surface iron-grey with a white margin, aerial mycelium lacking; reverse grey to iron-grey; colonial diameter 6.6 mm in 7 days (> 90 mm in 10 days). Colonies on SNA flat with entire margin, medium hyaline, buff around Anthryscus stem, aerial mycelium lacking; colonial diameter 6.2 mm in 7 days (> 90 mm in 10 days) (Fig. 4).

Colletotrichum gigasporum is characterized by the large conidia (22–)25–29(–32) × (6–)7–9 μm. The phylogenetic analyses based on the ITS and TUB2 sequences placed some species with large conidia in a distinct clade, which was far from the other accepted Colletotrichum species (Liu et al. 2014). Therefore, Rakotoniriana et al. (2013) considered a new complex and positioned these species in C. gigasporum species complex. Species of the C. gigasporum species complex can be easily distinguished from each other using all individual genes used by Liu et al. (2014): ITS, ACT, TUB2, CHS-1 and GAPDH (Liu et al. 2014).

A BLASTN analysis for ITS sequences of the 22 previously misidentified strains of C. crassipes, C. gloeosporioides, C. incarnatum, C. orbiculare and C. taiwanense (Glomerella septospora) originating from GenBank clustered them with the ex-type strain of C. gigasporum. Thus, Liu et al. (2014) re-identified them as C. gigasporum. The size of ascospores and conidia of C. gigasporum is similar to those of C. taiwanense, but C. gigasporum forms aseptate conidia and 0–1-septate ascospores (Rakotoniriana et al. 2013, Liu et al. 2014), whereas the conidia of C. taiwanense may become 1–5 septate with age and ascospores are mostly 3 septate and may become up to 6 or 8 septate when getting old (Sivanesan & Hsieh 1993). Likewise, C. thailandicum was morphologically similar to C. gigasporum. Phylogenetic analyses of multilocus data demonstrated that the ex-type strains of the two species group together in one strongly supported clade. Therefore, Liu et al. (2014) synonymized C. thailandicum with C. gigasporum. Moreover, C. gigasporum differs from C. incarnatum (Zimmermann 1901), the species, which was initially described from Coffea liberica in Java, by generating larger conidia (20.5–25.5 × 6–9 μm vs 14–19 × 5 μm).

Colletotrichum gigasporum enables to infect the wide range of hosts and is geographically distributed (Africa, Central and South America, Asia and New Zealand). Rakotoniriana et al. (2013) reported that C. gigasporum can ecologically occur as either endophyte or pathogen, even were isolated from human tissues (Liu et al. 2014). The studied strains by Liu et al. (2014) was isolated from Diospyros kakiSolanum betaceum, Theobroma cacao, Virola surinamensis, Coffea sp., Trichilia tuberculata, Acacia auriculiformis, Musa sp., Centella asiatica, Homo sapiens, air and stored grains, indicating it is not a host-specific isolate. C. gigasporum obtained from Camellia sinensis and Euonymus japonicus in Northern Iran. Therefore, we report C. sinensis and E. japonica as new hosts for C. gigasporum.

 

Colletotrichum godetiae Neerg., Friesia 4: 72 (1950) (Fig. 5).

 

Morphology on Anthryscus stem.Conidiomata acervular, Conidiophores hyaline, smooth-walled, simple, up to 15 µm long. Conidiogenous cells hyaline, smooth-walled, cylindrical, 5–12 × 3–5 µm, opening 1–2 µm diameter. Conidia hyaline, smooth-walled, aseptate, straight, cylindrical to fusiform with both ends acute or one end round and one end slightly acute, (8–) 10–14(–14.5) × (3.5–) 4–4.5(–5) µm, mean ± SD = 12.6 ± 4.0 × 4.4 ± 0.5 µm, L/W ratio = 2.85 (Fig. 5).

Morphology on SNA. Sexual state, chlamydospores and conidiomata absent, conidiophores are directly formed on hyphae. Setae absent. Appressoria solitary, medium brown, smooth-walled, clavate to elliptical, the edge entire or undulate 6.5–15.5 × 4.5–9 µm, mean ± SD = 9.5 ± 1.8 × 6 ± 1.1 µm, L/W ratio = 1.58 (Fig. 5).

Cultural characteristics. Colonies on SNA flat with entire margin, hyaline, with little low white aerial mycelium, SNA growth rate 5.2 mm in 7 days (70 mm in 10 days). Colonies on OA flat with entire margin; surface salmon to olive-gray, dark gray in center, no aerial mycelium, reverse salmon to gray; growth rate 40 mm in 7 days (6.2 mm in 10 days) (Fig. 5).

Colletotrichum godetiae was initially described from the seed of Clarkia (syn. Godetiahybrida cv. Kelvedon Glory by Neergard (1943). The species happens on a wide host range such as FragariaMalusPrunusRhododendron, Aeschynomene,  AgrimoniaBonzaiCeanothusCitrusFragariaJuglansLaurusMahoniaOleaParthenocissusPodocarpusPrunusRubusSambucusVitisSchinusSolanum and Ugni, 

 

Fig. 4. Colletotrichum gigasporum (UTFC 255), (IRAN 2444 C). a–b. Cultures on OA, 10 days growth, upper (a) and reverse (b); c. Setae; d. Conidiomata on Anthryscus stem; e–f. Conidiomata on SNA; g–h. Conidia; i. Conidiophores; j–m. Appressoria. Scale bars — c = 20 μm; d–f = 50 µm; g–m = 10 μm.

 

 

thespecies that is mainly present in the Europe and the Near East causing fruit, leaf, stem, cane and twig diseases (Damm et al. 2012). C. godetiae (UTFC 257, UTFC 258 and UTFC 259) obtained from Frangula alnus and Acer cappadocicum. Therefore, we report these plants as new hosts for C. godetiae. Furthermore, C. godetiae is a new record for the mycobiota of Iran.

Colletotrichum godetiae is separated from other species in the Acutatum species complex by the ITS, ACTTUB2CHS-1, GAPDH genes. The TUB2ACT and HIS3 provide more robust and precise phylogeny (Dam et al. 2012). In TUB2 ML tree UTFC 257, UTFC 258 and UTFC 259 isolates clustered with holotype strain CBS133.44 from godetia and CBS 129913 from Podocarpus that in a clade with 100% bootstrap support (Fig. 1).

 

Colletotrichum karstii Y.L. Yang, Z.Y. Liu, K.D. Hyde & L. Cai, Cryptogamie Mycologie 32: 241 (2011) (Fig. 6).

 

Morphology on Anthriscus stem. Conidiomata acervular, conidiophores hyaline to pale brown, aseptate or septate, branched, 30-80 µm long. Conidiogenous cells hyaline to pale brown, smooth-walled, cylindrical to ampulliform, sometimes extending to form new conidiogenous loci, 4.55–9.7 × 3–4.5 µm, opening 1–2 µm diameter. Conidia hyaline, smooth-walled, aseptate, straight, cylindrical, apex round, base round with a prominent hillum, the contents appearing granular, (12.85–) 13.90–14.2(–14.65) × (4.8–) 4.85–5(–5.35) µm, mean ± SD = 13.90 ± 0.76 × 5.0 ± 0.32 µm, L/W ratio = 2.75 (Fig. 6).

Morphology on SNA. Ascomata and perithecia are formed after 3-4 weeks, solitary, superficial or immersed in the agar medium, non-stromatic, globose to obpyriform, ostiolate, glabrous, brown, 96–122 × 91–111 µm. Asci unitunicate, 8-spored, cylindrical to clavate or fusiform, tapering to apex and base, smooth-walled, 39–54 × 10–12 µm. Ascospores are arranged either uni or biseriately, initially aseptate but often generate septate with age, hyaline, smooth-walled, variable in shape, fusiform to ovoid, slightly curved, (14.95–)16.8–17.2(–18.3) × (5.3–)5.7–6.15(–7.1) µm, mean ± SD = 16.8 ± 1.45 × 6.1 ± 0.75 µm, L/W ratio = 2.75. Chlamydospores and conidiomata absent, the conidiophores are directly formed from vegetative hyphae. Setae absent (Fig. 6).

 

 

 

Fig. 5. Colletotrichumgodetiae (UTFC 258), (IRAN 2447 C). a–b. Cultures on OA, 10 days growth, upper (a) and reverse (b); c. Conidiomata on SNA; d. Conidiomata on Anthryscus stem; e. Conidiophores; f. Conidia; g–k. Appressoria. Scale bars — c–d = 50 μm; e–f = 10 µm; g–k = 5 μm.

 

Fig. 6. Colletotrichum karstii (UTFC 242), (IRAN 2448 C). a–b. Cultures on OA, 10 days growth, upper (a) and reverse (b). c. Setae. d–f. Ascomata on Anthryscus stem. g. Asci h. Ascospores. i. Conidiomata on Anthryscus stem. j–l. Conidia. Scale bars — c, h, j-l = 10 μm; e–f, i = 50 µm; d = 100 μm.

 

Fig. 7. Colletotrichum musae (UTFC 234). a–b. Cultures on OA, 10 days growth, upper (a) and reverse (b); c. Conidiophore; d. Conidiomata on Anthryscus stem; e. Conidiomata on SNA; f. Conidiomata on filter paper; g. Conidiophore; h–i. Conidia. Scale bars — = 20 μm; d, f = 200 µm; = 50 μm; g–i = 20 μm.

 

 

 

 

 

Cultural characteristics. Colonies on SNA flat with entire margin, hyaline, with filter paper and Anthryscus stem covered with orange conidiomata and partly with white mycelium; reverse hyaline with grey flecks mainly under the filter paper, 59 mm in 7 days (80 mm in 10 days). Colonies on OA flat with entire margin, pale brown or buff to rosy buff to pale salmon, covered with orange to grey conidiomata in center; reverse brown to pale brown, buff, rosy buff to honey, 50 mm in 7 days (72 mm in 10 days) (Fig. 6).

Colletotrichum karstii described by Yang et al. (2011), and is known as pathogen and endophyte from the leaf and root of some plants such as Vanda sp., Calanthe argenteo-striataEria coronariaPleione bulbocodioides (Orchidaceae) (Yang et al. 2011), Bletilla ochracea (Orchidaceae) (Tao 2013) and Passiflora edulis in Brazil (Tozze et al. 2010). It occurs on many host plants and is the most common and geographically diverse species in the C. boninense complex (Damm et al. 2012).

Colletotrichum karstii is a polymorphic isolate in terms of sequence and is morphologically diverse isolate as well. Moreover, strains of the species show significant differences in conidium size and conidiomatal structures ranging from sporodochial to acervular to closed. Therefore, it is difficult or impossible to identify the species based on morphology. The conidia of C. karstii are broader than those of C. phyllanthi and smaller than those of C. hippeastri and C. dracaenae. Similarly, the asci are shorter than those of C. brassicicola and C. dracaenae. In addition, C. boninense has slightly wider and less tapered ascospores in comparation with C. karstii (Damm et al. 2012b).

Phylogenetic analyses based on the partial TUB2 gene (Fig. 1) put together the examined isolates in this study (UTFC 242, UTFC 243, UTFC 244, UTFC 246, UTFC 247, UTFC 248, UTFC 249, UTFC 250) with holotype strain CGMCC 3.14194 from Vanda sp. in a monophyletic clade with 100% bootstrap support. Furthermore, the UTFC 248 exhibited a close relationship in a sub clade with strain CBS 861.72, which was isolated from Bombax in Brazil (Fig. 1).

Colletotrichum karstii is a new record for the mycobiota of Iran. In addition, it is first report of the species on Euonymus japonicus var. aureo-marginata, Euonymus japonicus, Syringa reticulata, Laurus nobilis, Ficus benjamina and Cyperus sp. Moreover, this is the first report indicating the occurrence of C. karstii on Passiflora edulis in Iran.

 

Colletotrichum musae (Berk. & M.A. Curtis) Arx, Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2 51(3): 107. 1957 (Fig. 7).

 

Morpholgy on Anthryscus stem. Conidiophores cylindrical, tapered toward the apex, hyaline, subhyaline toward he base, up to 30 μm long. Conidia abundant, hyaline, aseptate, guttulate, oval, elliptical or cylindrical, often with a flattened base, apex obtuse, 9.5–15 × 4.35–6, mean ± SD = 13.5 ± 2.1 × 5.15 ± 1.1 µm, L/W ratio=2.62. Appressoria common, medium to dark brown, irregular in shape, often with large or deep lobes, 9-13 × 9-11.5µm (Fig. 7).

Cultural characteristics. Colonies on OA circular, with sparse to abundant, white to grey floccose aerial mycelium, conidial masses well developed, orange to cinnamon conidial mass; reverse grey-yellowish, 75 mm in 7 days. Sclerotia and Setae absent (Fig. 7).

Colletotrichum musae was originally described from the North Carolina (Berkeley 1874), and the name was epitypified by Su et al. (2011). The species is known as the pathogen of Musa sp. worldwide (Sutton 1980, Su et al. 2011, Weir et al. 2012). The TUB2 sequence separates C. musae from all other species in the Gloeosporioides complex.

Phylogenetic analyses based on the partial TUB2 gene (Fig. 1) assigned the examined isolates in this study (UTFC 233 and UTFC 234) with holotype strain ICMP 19119 from Musa sp. in a monophyletic clade with 98% bootstrap support.

Colletotrichum musae isolates (UTFC 233 and UTFC 234) obtained from Musa sapientum and Citrus sinensis. Consequently, this is the first report of C. musae on C. sinensis.

 

ACKNOWLEDGMENTS

 

We gratefully acknowledge the High Council for Research of the University of Tehran for financially supporting and providing materials under grant number 7110022/6/31. Moreover, the authors appreciate helpful comments of Petra Happel, Max Planck Institute for Terrestrial Microbiology.

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