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

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).