New reports of endophytic fungi associated with cherry (Prunus avium) and sour cherry (Prunus cerasus) trees in Iran

Document Type: Original Article

Authors

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

Abstract

Endophytic fungi constitute a remarkable multifarious group of microorganisms live within plants tissues without causing any obvious negative effect. Endophytic fungi have been found in every plant species examined to date. During an investigation on endophytic fungi, 123 fungal isolates were obtained from healthy twigs and leaves of cherry (Prunus avium) and sour cherry (P. cerasus) trees in Iran. The isolates identified based on sequence data of 18S rDNA (SSU) region, as well as morphological and cultural features. As a result, four species namely Coniothyrium olivaceum, Collophora paarla, Sarocladium strictum and Quambalaria cyanescens identified. All these identified species are new reports as endophytic fungi from P. cerasus and P. avium in the world. Among them, Collophora paarla and Quambalaria cyanescens are new taxa for the mycobiota of Iran.

Keywords

Main Subjects


INTRODUCTION

Endophytic fungi are a group of fungi that colonize internal tissues of plants without causing any negative effects (Hirsh and Braun 1992). Endophytes may play many important and beneficial roles in the host plant. They also are rich source of novel bioactive compounds with huge potential for exploitation in a wide variety of medical, agricultural, and industrial areas (Tan and Ziu 2001). Endophytes have also recognized as potential sources of novel natural products for industrials, agricultural and pharmaceutical uses (Strobel and Daisy 2003).

Cherry (Prunus avium L.) and sour cherry (P. cerasus L.) from the family of Rosaceae are the most important stone fruit trees with valuable fruits worldwide (Nemati and Abdollahzadeh 2009). Stone fruit trees are a cultivated worldwide group of plants with a great economic importance which can be used as a model for endophyte studies (Pimenta et al. 2012). Fungal endophytes of fruit trees have investigated less frequently compare to other forest trees, and most of the researches have focused mainly on aerial parts, especially leaves, branches and fruits (Hortova and Novotny 2011). Haddadderafshi et al. (2011) isolated 150 endophytic fungal strains from 4500 cherry tissue segments. These isolates belonged to 25 different species of genera such as Acremonium, Alternaria, Botryotinia, Aspergillus, Chaetomium, Cladosporium, Embellisia, Epicoccum, Fusarium, Glomerella, Macrophomina, Neonectria, Phoma, Phomopsis, Pyronema, Rhizoctonia, Rhizopycnis, Rosellinia and Xylaria. Hortova and Novotny (2011) isolated some endophytic fungi from branches of sour cherry trees, however the isolates belonged to 15 fungal species. Alternaria alternata and Aureobasidium pullulans were the most frequent fungal species. Pimenta et al. (2012) obtained 163 fungal isolates and, finally 14 different species from plum (Prunus domestica) leaves, additionally they investigated their antagonistic activity against Monilinia fructicola.

The main goal of the present study was the identification and characterization of some endophytic fungi associated with cherry and sour cherry trees in different areas of Iran based on morphological and phylogenetic studies.

 

MATERIALS AND METHODS

Isolation of Endophytic Fungi

Fungal endophytes were isolated from healthy and living tissues of cherry (P. avium) and sour cherry (P. cerasus) trees that were collected from Western Azerbaijan, Eastern Azerbaijan, Ardabil, Isfahan, Ilam, Ghazvin, Hamedan, Razavi Khorasan, South Khorasan, North Khorasan and Kerman provinces of Iran during 2014–2015. Samples transferred to mycological laboratory of University of Tehran and stored at 4 °C for future use. The endophytic fungi were isolated using a method described by Refaei et al. (2011). Plant materials washed in running tap water for 10 min. After surface sterilization by immersion of plant tissues in 70% ethanol for 1 min, 2.5% sodium hypochlorite solution for 3 min, 70% ethanol for 30 s, and then rinsed with sterile water. The outer tissues of the plant materials removed with sterile scalpel and were cut in small pieces (0.5 cm2) and then placed in Petri dishes containing 2% water agar (WA). Fungal isolates were purified on PDA culture medium using hyphal tip method, and then incubated at 24±1 °C until the pure fungal colonies were appeared. For long–term storage, fungal isolates were grown on sterile filter papers placed on PDA for 7–10 days. Subsequently, colonized filter papers were taken from the surface of culture medium, and were dried at room temperature for four or five days, and then stored at –20 °C for future use.

Morphological examination

The morphological identification of endophytic fungi were performed based on the morphology of fungal colony or hyphae, the characteristics of the fruiting bodies such as conidiomata, conidiogenous cells, conidiophores and conidia (Barnett & Hunter 1998; Chen et al. 2015; Damm et al. 2010; Giraldo et al. 2015; de Hoog & de Vries 1973). After 7–14 days incubation of the pure fungal colonies, the fungi assessed by light microscope using the microscopic slides that were prepared in lacto–phenol or lacto–phenol cotton blue solutions. Morphology of the studied characteristics such as colony color, conidia and conidiophore structures measured and recorded using a light microscope. Photographs were taken using BH2 Olympus microscope. Measurements made using macro– and micro–morphological features of different recovered isolates.

Morphological studies of Coniothyrium olivaceum was performed on oatmeal agar (OMA), malt extract agar (MEA) and potato dextrose agar (PDA), and the cultures were incubated under near–ultraviolet (nUV) light (12 h light/12 h darkness). Colony diameter measured after 14 days, micro–morphological features (conidiomata, conidiogenous cells and conidia) and measurements were performed according to Chen et al. (2015).

Collophora paarla isolates identified based on colony morphology on PDA and micro–morphological characteristics such as presence of conidiomata, microcyclic conidiation or endoconidia, size and shape of conidia and conidiophores (Damm et al. 2010, Gramaje et al. 2012). The cultures were incubated at 25 °C in continuous dark condition. Colony diameter measured after 14 days.

Morphological characterization of Sarocladium strictum carried out from cultures grown on PDA. Culture incubated at 25 °C in constant dark condition. Colony diameter measured after 14 days. This species characterized based on colony morphology, conidiophores, phialides and conidia (Giraldo et al. 2015).

Morphological studies of Quambalaria cyanescens were done on PDA. The culture incubated in continuous dark condition at 25 °C. Colony diameter measured after 10 days and micro–morphological description provided based on measurements of conidiogenous cells, conidia and secondary conidia (de Hoog & de Vries 1973).

Molecular examination

After morphological identifications, one isolate of each morphotypes selected for molecular investigations. Genomic DNA of the isolates was extracted by the method of Zhong & Steffenson (2001) and partial sequence of 18S rDNA (SSU) locus was amplified and sequenced using the primer pairs NS1 and NS2 (White et al. 1990). PCR amplification carried out in a final volume of 25 µl containing 10 µl of Taq DNA Polymerase Mix Red–Mgcl2, 11 µl deionized water, 0.2 pmol of each primer and 10–30 ng.µl–1 template DNA. PCR amplification performed on Eppendorf Thermal Cycler (Mastercycler, ep gradient), with cycling conditions of 4 min at 95 °C for initial denaturation, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 50 s and a final extension at 72 °C for 10 min. PCR products were purified and directly sequenced in one direction using NS1 primer by Macrogen Company (Seoul, South Korea).

Phylogenetic analyses

In order to molecular identification and confirmation of morphological identification, sequences were evaluated by Chromas 2.6 (Technelysium Pty Ltd., South Brisbane, Australia). Obtained nucleotide sequences compared with other fungal DNA sequences which deposited in GenBank (NCBI) database (www.ncbi.nlm.nih.gov/genbank/) using BLAST search tool. Also, the relevant sequences were obtained from NCBI for phylogenetic analyses, and evolutionary trees were generated using MEGA v. 7.0 (Kumar et al. 2016) with 1000 boot–strap replicates. Multiple sequence alignment performed with Muscle using MEGA v. 7.0 software and alignment manually corrected where it was necessary. Distance matrixes of the aligned sequences calculated by the Tamura–Nei model (Tamura & Nei 1993), and analyzed with the maximum likelihood algorithm (Felsenstein 1985). Eremothecium gossypii (AY046265) selected as an out–group taxon. The newly obtained nucleotide sequences of the 18S rDNA region in this study deposited in the GenBank using the Sequin software (NCBI, USA). Detailed information of the examined isolates in this study is provided in Table 1.

 

RESULTS AND DISCUSION

From total of 123 endophytic fungal isolates 116 and seven were from branches and leaves respectively which were obtained from P. cerasus and P. avium. In total, 58 strains including 56 from branches and two from leaves were isolated from P. cerasus. Furthermore, a total of 65 strains include; 60 from branches and five from leave were isolated from P. avium.

In this study, four species including Coniothyrium olivaceum, Sarocladium strictum, Collophora paarla and Quambalaria cyanescens were identified and described based on both morphological criteria and molecular data. All four species are reported for the first time as endophytic fungi from P. cerasus and P. avium trees in the world. Furthermore, Collophora paarla and Quambalaria cyanescens are new taxa for the mycobiota of Iran.

Taxonomy

Coniothyrium olivaceumBonord., in Fuckel, Symbolae mycologicae: 377 (1869).

Specimen examined. Iran, West Azerbaijan Prov., Khoi, recovered from branch of P. cerasus, Oct. 2015, (code of the isolates; AGA6, AGA7, AGA8, AGA9), recovered from branch of P. avium. October 2015, (code of the isolates; AG2, AG3, AG4, AG5, AG6), East Azerbaijan Prov., Marand, recovered from branch of P. cerasus, Oct. 2015, (code of the isolates; ASA1, ASA3, ASA8), recovered from branch of P. avium, Oct. 2015, (code of the isolates; AS1, AS2, AS3, AS4, AS10, AS12), Ardabil Prov., Sarein, recovered from branch of P. cerasus, Oct. 2015, (code of the isolates; ARA13, ARA14, ARA17, ARA18, ARA19), recovered from branch of P. avium, Oct. 2015, (code of the isolates; AR13, AR14, AR15, AR16, AR17, AR18, AR19), Kerman Prov., Rafsanjan, recovered from branch of P. cerasus, Oct. 2014, (code of the isolate; KEA1, KEA2, KEA3, KEA6, KEA7, KEA8, KEA10, KEA11, KEA12, KEA14, KEA15, KEA16, KEA17, KEA18, KEA19, KEA20, KEA31, KEA32, KEA33, KEA34, KEA35, KEA36), recovered from branch of P. avium, Oct. 2014, (code of the isolate; KE1, KE2, KE3, KE4, KE7, KE8, KE9, KE11, KE12, KE15, KE18, KE19, KE20, KE21, KE22), Sh. Abdollahi Aghdam. Code of selected isolate: UTFC–EP23, isolated from West Azerbaijan Prov., Khoi, recovered from branch of P. cerasus, Oct. 2015.

Colonies on PDA reached to 53 mm in diameter after 14 days at 25 °C under Ultra Violet (UV) light (12 h light/12 h dark). Colonies on PDA and OMA respectively become green in center due to production of pycnidia. Colonies on MEA become brown in center after two weeks. Pycnidia reached to 0.5–2 mm in diameter, dark brown, immersed, spherical. Conidiogenous cells hyaline, smooth–walled, sub–cylindrical to ampulliform, 5–10(8.6) × 3–6(4.5) µm. Conidia 1–celled, pale brown, thick and smooth–walled, ellipsoidal to sub–cylindrical, 7–9(7.5) × 4–5(4.3) µm in diameter (Fig. 1). Morphological features of the investigated isolate were similar to description provided by Chen et al. (2015).

 

Fig. 1. Coniothyrium olivaceum,isolate UTFC–EP23.a. Colony on PDA; b. Colony on OA; c. Colony on MEA after 14 days; d. Conidiogenous cell; e., f. Conidia. — Scales bars = 10 µm.

 

Collophora paarla Damm & Crous, in Damm, Fourie & Crous, Persoonia 24: 67 (2010).

Specimen examined: Iran, Isfahan Prov., Khansar, recovered from branch of P. avium, May 2014, (code of the isolate; ES1–1, ES1–2, ES1–3), Ghazvin Prov., Boyin Zahra, recovered from branch of P. avium, July 2014, (code of the isolate; GA25, GA26), recovered from branch of P. cerasus, July 2014, (code of the isolate; GAA21), Sh. Abdollahi Aghdam. Code of selected isolate: UTFC–EP45, isolated from Isfahan Prov., Khansar, recovered from branch of P. avium, May 2014.

Conidiomata pseudopycnidial, solitary, subglobose, superficial, pale to dark brown (observed once on PDA and have not observed again). Conidiophores lining the inner conidiomatal cavity, hyaline, smooth–walled, filiform, branched, 10–45(27.5) × 2.5–4(2.9) µm in diameter. Conidiogenous cells enteroblastic, hyaline, mono–phialidic, 3–7(5.4) × 1–2.5(1.9) µm in diameter. Conidia of pseudopycnidia hyaline, aseptate, smooth–walled, cylindrical with obtuse ends, 2 × 1 µm in diameter.

Colonies on PDA reached to 32 mm in diameter after 14 days at 25°C in constant dark condition. Colonies slow growing, moist, cream, lacking aerial mycelium. Conidiophores, hyaline, branched, septate and filiform. Conidiophores mostly reduced to conidiogenous cells. Conidiogenous cells enteroblastic, intercalary, 1.5–4 (2.6) × 1–2(1.5) µm in diameter. Conidia hyaline, 1–celled, cylindrical, with both ends obtuse or with a papillate apex, smooth–walled, almost biguttulate, 5–10(7.8) × 1.5–3(2.1) µm in diameter. Endoconidia formed uniseriately within hyphae, hyaline, 1–celled, cylindrical, smooth–walled (Fig. 2). Morphological features of the investigated isolate were similar to description of Collophora paarla provided by Damm et al. (2010).

Sarocladium strictum (W. Gams) Summerb., in Summerbell, Gueidan, Schroers, Hoog, Starink, Arocha Rosete, Guarro & Scott, Stud. Mycol. 68: 158 (2011). 

Specimen examined. Iran. West Azerbaijan Prov., Urmia, recovered from branch of P. cerasus, (code of the isolate; AGA1, AGA2, AGA3, AGA5), recovered from branch of P. avium, May 2014, (code of the isolate; AG9, AG10), Isfahan Prov., Khansar, recovered from branch of P. cerasus, May 2014, (code of the isolate; ESA17, ESA18), recovered from branch of P. avium, May 2014, (code of the isolate; ES1, ES2, ES5, ES6, ES10), recovered from leaf of P. avium, May 2014, (code of the isolate; ES14, ES15, ES18), Ilam Prov., Ilam, recovered from branch of P. avium, Sept. 2015, (code of the isolate; EL3, EL4, EL5, EL8), Razavi Khorasan Prov., Kashmar, recovered from branch of P. avium, Apr. 2014, (code of the isolate; KA3, KA6, KA8), recovered from leaf of P. avium, Apr. 2014, (code of the isolate; KA9, KA26), South Khorasan Prov., Birjand, recovered from branch of P. cerasus, Sept. 2014, (code of the isolate; KJA1, KJA2, KJA3, KJA5, KJA5, KJA9), North Khorasan Prov., Esfarayen, recovered from branch of P. cerasus, May 2015, (code of the isolate; KSA8, KSA9, KSA11), recovered from leaf of P. cerasus, May 2015, (code of the isolate; KSA12, KSA13). Sh. Abdollahi Aghdam. Code of selected isolate: UTFC–EP36., isolated from West Azerbaijan Prov., Urmia, May 2014, recovered from branch of P. cerasus.

Colonies on PDA reached 41 mm in diameter in 14 days at 25­°C in the continuous dark condition. Colonies were moist to smooth, pale orange to pink. Vegetative hyphae septate, hyaline, smooth–walled, hyphal coils formed abundantly. Phialides hyaline, slender, smooth–walled, arising from vegetative hyphae, 13–40(23.5) × 2–4(3.2) µm in diameter. Conidia grouped in slimy heads, 1–celled, hyaline, straight, cylindrical or ellipsoid, 4–10(6.7) × 1–3(2.2) µm in diameter (Fig. 3). Chlamydospores not observed. Morphological features of the investigated isolate were similar to description of Sarocladium provided by Gams (1971).

 

Fig. 2. Collophora paarla, isolate UTFC–EP45. a. Colony on PDA after 14 days; b. Conidiophora in pseodopycnidia; c. Conidiogenous cell in pseodopycnidia; d. Conidia in pseudopycnidia; e, f. Hyphae and endoconidia; g, h. Conidia. — Scales bars = 10 µm.

 

Quambalaria cyanescens (de Hoog & G.A. de Vries) Z.W. de Beer, Begerow & R. Bauer, in de Beer, Begerow, Bauer, Pegg, Crous & Wingfield, Stud. Mycol. 55: 295 (2006).

 Specimen examined: Iran, Hamedan Prov., Malayer, recovered from branch of P. avium, June 2015, (code of the isolate; HA25, HA27, HA28, HA29, HA30, HA36, HA39, HA40), recovered from branch of P. cerasus, June 2015, (code of the isolate; HAA26, HAA27, HAA30, HAA31, HAA32, HAA34), Sh. Abdollahi Aghdam. Code of selected isolate: UTFC–EP47, isolated from Hamedan Prov., Malayer, June 2015, recovered from branch of P. avium.

Colonies on PDA reached to 15 mm in diameter after 10 days at 25°C in continuous dark condition. Colonies are restricted, farinose or velvety, snow–white, deep blue/violet pigment into agar. Hyphae are hyaline, smooth–walled, branched and sub–erect. Conidiogenous cells are undifferentiated, cylindrical and variable in size, with a cluster of small denticles apically, 5–25(19.6) × 1–1.5(1.3) µm in diameter. Conidia hyaline, smooth–walled, obovoid, 3–4(3.6) × 1.5–2(1.7) µm in diameter, larger conidia [4–7(5.8) × 2 µm] producing secondary conidia (Fig. 4). Morphological features of the investigated isolate were similar to description of Quambalaria provided by Smith & Batenburg–Van der Vegte (1985) and de Hoog & de Vrier (1973).

 

 Fig. 3. Sarocladium strictum, isolate UTFC–EP36. a. Colony on PDA after 14 days; b, c. Slimy heads; d. Phialides; e, f. Hyphal coils; g–i. Conidia. — Scales bars = 10 µm.

 Fig. 4. Quambalaria cyanescens, isolate UTFC–EP47. a. Colony on PDA after 10 days; b–e. Conidiophores; f, g. Conidia. — Scales bars = 10 µm.

 

Phylogenetic analysis

The phylogenetic analyses performed using twenty–two 18S rDNA nucleotide sequences including our isolates and the others obtained from GenBank including the out–group (Table 1).

 

Table 1. Fungal strains used in the phylogenetic analysis.

Fungal species

Isolate

Source (plant host)

Origin

NCBI accession no.

Reference

Sarocladium strictum

CBS 346.70 T

Triticum aestivum

Germany

HQ232211

Summerbell et al. (2011)

 

UTFC–EP36

Prunus cerasus

Iran

MF000698

 

S. bactrocephalum

CBS 749.69 T

Ustilago sp.

Canada

HQ232180

Summerbell et al. (2011)

 

KF103

 

 

KM096139

Panzer et al. 2015

S. kiliense

CBS 146.62

 

 

HQ232197

Summerbell et al. (2011)

 

CBS 122.29 T

 

 

HQ232198

Summerbell et al. (2011)

Microsphaeropsis olivacea

CBS 401.81

 

 

AY642517

Verkley et al. (2004)

 

CBS 442.83

 

 

AY642518

Verkley et al. (2004)

 

CBS 336.78

 

 

AY642519

Verkley et al. (2004)

 

CBS 116669

Cytisus scoparius

Netherlands

EU754071

de Gruyter et al. (2009)

Coniothyrium olivaceum

UTFC–EP23

Prunus cerasus

Iran

MF000696

 

C. cereale

CBS 122787

 

Germany

EU754052

de Gruyter et al. (2009)

Collophora paarla

CBS:120877 T

Prunus salicina

South Africa

GQ154634

Damm et al. (2010)

 

UTFC–EP45

Prunus avium

Iran

MF000694

 

 

CBS:121443

Prunus salicina

South Africa

GQ154633

Damm et al. (2010)

 

CBS:120878

Prunus salicina

South Africa

GQ154632

Damm et al. (2010)

C. rubra

CBS:120873 T

Prunus persica

South Africa

GQ154627

Damm et al. (2010)

 

CBS:121441

Prunus persica var. nucipersica

South Africa

GQ154628

Damm et al. (2010)

C. capensis

CBS:120879 T

Prunus salicina

South Africa

GQ154631

Damm et al. (2010)

C. africana

CBS:120872 T

Prunus salicina

South Africa

GQ154630

Damm et al. (2010)

Quambalaria cyanescens

CBS 876.73

Eucalyptus pauciflora

Australia

KF706440

Wang et al. (2014)

 

UTFC–EP47

Prunus avium

Iran

MF000697

 

 

UM 1095

Skin scraping

Malaysia

KT186108

Kuan et al. (2015)

Microstroma phylloplanum

JCM 9035

 

 

AB038131

 

 

CBS8073 T

Baksia collina

Australia

AJ496258

 

Sympodiomycopsis kandeliae

CBS 11676

 

 

KP322963

Wang et al. (2015)

S. paphiopedili

IAM 13459

 

 

D14006.1

 

 

AFTOL–ID 1772

 

 

DQ832239

 

Eremothecium gossypii

NRRL Y–1056

 

 

AY046265

 Kurtzman & Robnett 2003

 DNA sequence analysis revealed that all investigated isolates are placed in four distinct clades, corresponding to four different fungal orders including Pleosporales, Helotiales, Hypocreales in Ascomycota and Microstromatales in Basidiomycota. As shown in Fig. 5, all isolates from P. avium and P. cerasus hosts could be classified in four clades; Pleosporales (Clade I), Helotiales (Clade II), Hypocreales (Clade III),and Microstromatales (Clade IV).

Coniothyrium and Microsphaeropsis species grouped in the same cluster (Clade I) with 100% bootstrap support. Our examined isolate of Coniothyrium olivaceum (MF000696) was 100% identical to other isolates of this species in GenBank (AY642517) with 100% query coverage in BLAST search. According to Index Fungorum website, the current name of M. olivacea is C. olivaceum. The species belongs to order Pleosporales, taxonomically.

Sarocladium species grouped in the Clade II with 100% bootstrap support. A BLAST search showed that our examined isolate of S. strictum (MF000698) was 99% identical to other isolates of this species in GenBank(HQ232211) with 99% coverage. The species belongs to order Hypocrealestaxonomically.

Different Collophora species were grouped in the same cluster (Clade III) with 87% bootstrap support. The partial 18S rDNA nucleotide sequence of our examined Collophora paarla isolate (MF000694) was 100% similar to other isolates of this species in GenBank (GQ154634) with 100% coverage in a BLAST search. The species belongs to order Helotiales, taxonomically.

The isolates in the Clade IV grouped with 100% bootstrap support. A BLAST search showed that partial 18S rDNA nucleotide sequence of our examined Quambalaria cyanescens isolate (MF000697) was 99% identical to other isolates of this species in GenBank(KT186108 and KF706440) with 100% coverage. The species is belonging to order Microstromatales, taxonomically.

The isolate UTFC–EP23 identified as Coniothyrium olivaceum based on morphology and the description provided by Chen et al. (2015) as well as based on molecular data. This species differs from other Coniothyrium species in characteristics of conidiogenous cells and conidial shape and size. This species has been reported from stem of Hedera helix inAustria, needles of Pinus laricio in Franceand dead twigs and pods of Sarothamnus sp. in Netherland (Chen et al. 2015). This species was reported as causal agent of brown spine rot in Alhagi maurorum in Iran (Razaghi and Zafari 2016). Petrini and Fisher (1988), isolated M. olivacea as endophytic fungi from xylem and stems of Pinus sylvestris. Hormazabal and Piontelli (2009), isolated this species as an endophytic fungi from Chilean gymnosperms. Coniothyrium olivaceum is reported for the first time as endophytic fungus from P. cerasus and P. avium trees in the world.

 

 Fig. 5. A maximum likelihood tree inferred from 18S rDNA sequences of 29 isolates using MEGA v. 7.0 software. The numbers on the branches shows the bootstrap values of 1000 replicates. The length of branches is proportional to the number of base changes, indicated by the scale bar. Eremothecium gossypii (AY046265) was used as out–group.

The isolate UTFC–EP45 identified as C. paarla using morphological characteristics, according to the description provided by Damm et al. (2010). The genus has at least six species including Collophora aceris, C. africana, C. capensis, C. hispanica, C. paarla and C. rubra (Damm et al. 2010; Xie et al. 2013, Gramaje et al. 2012). Collophora paarla unlike the any other species of the Collophora, produceed endoconidia. Furthermore, conidiophores of this species in the conidiomata differ from other species. Unlike the five other species of the genus, C. paarla did not release any pigments into culture medium. C. paarla has been isolated from Prunus salicina in South Africa (Damm et al. 2010). Some C. paarla isolates have obtained from the dark brown necrosis symptoms in woods of P. persica (Damm et al. 2010). According to the morphological and molecular analysis, the fungus was identified as C. paarla. This is the first report of Collophora paarla as new taxon for the mycobiota of Iran. In addition, C. paarla is reported for the first time as endophytic fungus from P. cerasus and P. avium trees in the world.

Molecular data confirmed the morphological identification of UTFC–EP36 as Sarocladium strictum. Summerbell et al. (2011) have reported some Sarocladium species, such as S. strictum, segregated from Acremonium, based on phylogenetic analysis of SSU and LSU sequences. Sarocldium strictum has the morphological similarity and close relationship with S. pseudostrictum, but S. strictum, has a faster growth rate on PDA and larger phialides (Giraldo et al. 2015). S. strictum unlike S. kiliense does not formed unicellular chlamydospores (Perdomo et al. 2011). Sarocldium strictum has been reported from Vitis sylvestris, Zea mays and as fungi accompanying the scab symptoms in Iran (Ershad 2009; Ebrahimi and Fotouhifar, 2016). Species of Sarocladium have been reported as endophytes in grass species such as Spinifex littoreus and native Turkish grasses (Tunali et al. 2000; Yeh & Kirschner 2014). Here, S. strictum is reported for the first time as endophytic fungus from P. cerasus and P. avium trees in the world.

The isolate UTFC–EP47 was identified as Quambalaria cyanescens based on morphological features provided by Smith and Batenburg–Van der Vegte (1985), de Hoog and de Vrier (1973) and the molecular data. The fungus has six species including Quambalaria coyrecup, Q. cyanescens, Q. eucalypti, Q. pitereka, Q. pusilla and Q. simpsonii (Paap 2008; de Beer et al. 2006; Simpson 2000; Cheewangkoon et al. 2009). Our isolate of Q. cyanescens differs from other five Quambalaria species by the differences in colony color and conidial shape and size. Quambalaria cyanescens was isolated for the first time from human skin and was reported as Sporothrix cyanescens (Fan et al. 2014). This species is one of the rare clinical basidiomycetous pathogens and most reported species from the human in the 1990s (Fan etal. 2014). Almost all species of the genus Quambalaria are regarded as plant–pathogenic fungi, causing disease on species of Eucalyptus (de Beer et al. 2006). Quambalaria cyanescens generally regarded as a saprophyte that live or decay on different plant tissues (de Beer et al. 2006). Also, this species is associated with canker on Eucalyptus pauciflora, Corymbia calophylla, C. ficifolia, and C. citriodora in Australia (Pegg et al. 2008). Quambalaria cyanescens is new taxon for the mycobiota of Iran. Also, this speciesis reported for the first time as endophytic fungus from P. cerasus and P. avium trees in the world. It is well understood that endophytes might play important role in the growth and development of the host plant through providing of protection against various sources, and potential biological active natural products (Strobel et al. 2004). In this study, we investigated fungal endophytes associated with P. avium and P. cerasus using the morphological features and molecular data based only on the sequences of genomic SSU rDNA. This is the first study of fungal endophytes from thesehosts in Iran. Some genera and species of endophytic fungi including Coniothyrium olivaceum, Collophora paarla, Sarocladium strictum and Quambalaria cyanescens were also isolated and identified in the present study.

 

ACKNOWLEDGEMENTS

This study supported by the University of Tehran, Iran. So, the authors are pleased to appreciate the University of Tehran.

 

Barnett HL, Hunter BB. 1998. Illustrated Genera of Imperfect Fungi. APS Press, St. Paul, Minnesota, USA.

Cheewangkoon R, Groenewald JZ, Summerell BA, Hyde K.D, To–anun C, Crous PW. 2009. Myrtaceae, a cache of fungal biodiversity. Persoonia 23:55–85.

Chen Q, Jiang JR, Zhang GZ, Cai L, Crous PW. 2015. Resolving the Phoma enigma. Studies in mycology 82:137–217.

Damm U, Fourie PH, Crous PW. 2010. Coniochaeta (Lecythophora), Collophora gen. nov. and Phaemoniella species associated with wood necroses of Prunus trees. Persoonia 24:60–80.

de Beer ZW, Begerow D, Bauer R, Pegg GS, Crous PW, Wingfield MJ. 2006. Phylogeny of the Quambalariaceae fam. nov., including important Eucalyptus pathogens in South Africa and Australia. Studies in Mycology 55:289–298.

de Gruyter J, Aveskamp MM, Woudenberg JH, Verkley GJ, Groenewald JZ,  Crous PW. 2009. Molecular phylogeny of Phoma and allied anamorph genera: towards a reclassification of the Phoma complex. Mycological research 113:508–519.

de Hoog GS, de Vries GA. 1973. Two new species of Sporothrix and their relation to Blastobotrys nivea. Antonie van Leewenhoek 39:515–520.

Ebrahimi L, Fotouhifar K. 2016. Identification of some fungi accompanying the scab symptoms in Iran. Mycologia Iranica 3:25–37.

Ershad D. 2009. Fungi of Iran. Iranian Research Institute of Plant Protection, Tehran, Iran, 531 pp.

Fan X, Xiao M, Kong F, Kudinha T, Wang H, Xu, YC. 2014. A rare fungal species, Quambalaria cyanescens, isolated from a patient after augmentation mammoplasty–enviromental contaminant or pathogen? PLoS One 9:1–8.

Felsenstein J. 1985. Phylogenies and the Comparative Method. The American Naturalist 125: 1–15.

Gams W. 1971. Cephalosporium–artige Schimmelpilze (Hyphomycetes). G. Fischer, Stuttgart, Germany 262 pp.

Giraldo A, Gene J, Sutton DA, Madrid H, de Hoog GS, Cano J, Decock C, Crous PW, Guarro J. 2015. Phylogeny of Sarocladium (Hypocreales). Persoonia 34:10–24.

Gramaje D, Agustí–Brisach C, Pérez–Sierra A, Moralejo E, Olmo D, Mostert L, Damm D, Armengol J. 2012. Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain). Persoonia 28:1–13.

Haddadderafshi N, Halasz K, Posa T, Peter G, Hrotko K, Gasper K, Lukaces N. 2011. Diversity of endophytic fingi isolated from cherry (Prunus avium). Journal of Hoticulture, Forestry and Biotechnology 15:1–6.

Hirsh GU, Braun U. 1992. Communities of parasitic micro-fungi. In: Handbook of Vegetation Science: Fungi in vegetation science, Vol. 19. (ed. W. Winterhoff). Kluwer Academic, Dordecht, Netherlands, 225–250.

Hormazabal E, Piontelli E. 2009. Endophytic fungi from Chilean native gymnosperms: antimicrobial activity against human and phytopathogenic fungi. World Journal of Microbiology and Biotechnology 25(5):813–819.

Hortova B, Novotny D. 2011. Endophytic fungi in branches of sour cherry trees: a preliminary study. Czech Mycology 63:77–82.

Kuan CS, Yew SM, Toh YF, Chan CL, Lim SK, Lee KW, Na SL, Hoh CC, Yee WY, Ng KP. 2015. Identification and characterization of a rare fungus, Quambalaria cyanescens, isolated from the peritoneal fluid of a patient after nocturnal intermittent peritoneal dialysis. PLoS one 10:1–15.

Kumar S, Umar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33:1870–1874.

Kurtzman CP, Robnett CJ. 2003. Phylogenetic relationships among yeasts of the ‘Saccharomyces complex’ determined from multigene sequence analyses. FEMS yeast research 3:417–32.

Nemati H, Abdollahzadeh A. 2009. Sweet and sour cherry– Production and Utilization. Jehad University of Mashhad Press, Iran.

Paap T, Burgess TI, McComb JA, Shearer BL, Hardy GE St J. 2008. Quambalaria species, including Q. coyrecup sp. nov., implicated in canker and shoot blight diseases causing decline of Corymbia species in the southwest of Western Australia. Mycological Research 112:57–69.

Panzer K, Yilmaz P, Weiß M, Reich L, Richter M, Wiese J, Schmaljohann R, Labes A, Imhoff JF, Glöckner FO, Reich M. 2015. Identification of habitat–specific biomes of aquatic fungal communities using a comprehensive nearly full–length 18S rRNA dataset enriched with contextual data. PLoS One 10: e0134377.

Pegg GS, O'Dwyer C, Carnegie AJ, Burgess TI, Wingfield MJ, Drenth A. 2008. Quambalaria species associated with plantation and native eucalyptus in Australia. Plant Pathology 57:702–14.

Perdomo H, Sutton DA, García D, Fothergill AW, Cano J, Gene J, Summerbell RC, Rinaldi MG, Guarro J. 2011. Spectrum of clinically relevant Acremonium species in the United States. Journal of Clinical Microbiology 49: 243–256.

Petrini O, Fisher PJ. 1988. A comparative study of fungal endophytes in xylem and whole stem of Pinus sylvestris and Fagus sylvatica. Transactions of the British Mycological Society 91:233–238.

Pimenta RS, Da Silva JFM, Buyer JS, Janisiewicz WJ. 2012. Endophytic fungi from plums (Prunus domestica) and their antifungal activity against Monilinia fructicola. Journal of Food Production 75: 1883–1889.

Razaghi P, Zafari D. 2016. First report of Microsphaeropsis olivacea causing brown spine rot on Alhagi maurorum in IRAN. Journal of Plant Pathology 98: 677–697.

Refaei J, Jones EBG, Sakayaroj J, Santhanam J. 2011. Endophytic fungi from Rafflesia cantleyi: species diversity and antimicrobial activity. Mycosphere 2:429–447.

Simpson JA. 2000. Quambalaria, a new genus of eucalypt pathogens. Australasian Mycologist 19: 57–62.

Smith MT, Batenburg–Van der Vegte WH (1985). Ultrastructure of septa in Blastobotrys and Sporothrix. Antonie van Leeuwenhoek 51:121–128.

Strobel G, Daisy B. 2003. Bioprospecting for microbial endophytes and their natural products. Microbiology and Molecular Reviews 67:491–502.

Strobel G, Daisy B, Castillo U, Harper J. 2004. Natural products from endophytic microorganisms. Journal of Natural products 67:257–268.

Summerbell RC, Gueidan C, Schroers HJ, de Hoog GS, Starink M, Arocha Rosete Y, Guarro J, Scott JA. 2011. Acremonium phylogenetic overview and revision of Gliomastix, Sarocladium, and Trichothecium. Studies in Mycology 68:139–162.

Tamura K, Nei M. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10:512–526.

Tan R X, Zou WX. 2001. Endophytes: a rich source of functional metabolites. Natural Product Reports 18:448–459.

Tunali B, Shelby RA, Morgan–Jones G, Kodan M. 2000. Endophytic fungi and ergot alkaloids in native Turkish grasses. Phytoparasitica 28:375–7.

Verkley GJ, da Silva M, Wicklow DT, Crous PW. 2004. Paraconiothyrium, a new genus to accommodate the mycoparasite Coniothyrium minitans, anamorphs of Paraphaeosphaeria, and four new species. Studies in Mycology 50:323–335.

Wang QM, Theelen B, Groenewald M, Bai FY, Boekhout T. 2014. Moniliellomycetes and Malasseziomycetes, two new classes in Ustilaginomycotina. Persoonia 33:41–47.

Wang QM, Begerow D, Groenewald M, Liu XZ, Theelen B, Bai FY, Boekhout T. 2015. Multigene phylogeny and taxonomic revision of yeasts and related fungi in the Ustilaginomycotina. Studies in Mycology 81:55–83.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M.A. Inis, D.H. Gelfand, J.J. Sninsky and T. J. White (Eds.), PCR Protocols: A guide to Methods and Applications. Academic Press, San Diego, USA, pp: 315−322.

Xie J, Strobel GA, Mends MT, Hilmer J, Nigg J, Geary B. 2013. Collophora aceris, a novel antimycotic producing endophyte associated with Douglas maple. Microbial Ecology 66:784–795.

Yeh YH, Kirschner R. 2014. Sarocladium spinificis, a new endophytic species from the coastal grass Spinifex littoreus in Taiwan. Botanical Studies 55:25.

Zhong S, Steffenson BJ. 2001. Virulence and molecular diversity in Cochliobolus sativus. Phytopathology 91:469–476.