Novel endophytic species of Talaromyces sect. Talaromyces associated with saffron plant to the mycobiota of Iran

INTRODUCTION

Saffron is one of the oldest and globally most expensive plant spices known as an important medicinal plant cultivated since the past (Negbi et al. 1989). According to the latest statistics for saffron trade in 2019, Iran is the world’s largest saffron producer, supplying 430 tons of the total 450 tons of saffron produced worldwide (https://behinexir.com/ global-saffron-market/). Saffron stigma has become more popular due to its versatile biological and medicinal properties, such as aphrodisiac, antispasmodic, expectorant, antidepressant, and stomachic activity (Abdullaev 1993, Richelson 1993, Wang et al. 2010). Medicinal plants are rich sources of endophytic fungi that lived within the tissues of host plants and have proven to be a wealthy pool of bioactive secondary metabolites; some of them are shared with the hosts (Yu et al. 2009, Kusari et al. 2009, Zhu et al. 2010, Kusari 2012).

The genus Talaromyces was introduced by Benjamin (1955) as a teleomorph genus related to the Penicillium species. Phylogenetic studies revealed that Penicillium was a polyphyletic and Talaromyces species, and Penicillium subgenus Biverticillium belonged to a clade distinct from Penicillium sensu stricto (Chen et al. 2016). Based on phylogenetic, phenotypic and extrolite data, and following the nomenclatural priority and single name nomenclature concepts, Samson et al. (2011) transferred the most accepted Penicillium subgenus Biverticillium species to Talaromyces. Talaromyces currently includes around 140 accepted species, grouped into seven sections, namely section Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces and Trachyspermi. One of these sections is Sect. Talaromyces. Stolk and Samson (1972) introduced Talaromyces section Talaromyces as species that produce yellow ascomata, which can occasionally be white, creamish, pinkish or reddish, and have yellow ascospores. Conidiophores are usually from the biverticillate–symmetrical type, with some species that have reduced conidiophores with solitary phialides. Phialides are usually acerose, with a minor proportion of species having wider bases (Stolk & Samson 1972). Section Talaromyces species are commonly isolated from soil, indoor environments, humans with penicilliosis and food products (Yilmaz et al., 2014).

Talaromyces atroroseum and T. minioluteus have been reported from Iran (Ershad 2009; Khodaei et al. 2015). A large number of Penicillium species have been previously reported from Iran, though after revising Talaromyces genus by Samson et al. (2011) and separating it from the genus Penicillium, these species have not yet been reviewed.

Talaromyces species have a worldwide distribution isolated from a wide range of substrates, mainly in soil (Barbosa et al. 2018, Sun et al. 2020). Talaromyces include species that are medically and industrially important (Yilmaz et al., 2014). On the other hand, species of Talaromyces are good producers of anticancer, antibacterial, and antifungal compounds (Nicoletti et al. 2018). Several species also proved to be effective as biocontrol agents against soil-borne pathogens (Sun et al. 2020).

This study aimed to identify endophytic Talaromyces species isolated from saffron, collected from different saffron producer provinces in Iran. We provide morphological descriptions using macro- and micromorphological characters. Phenotypic characteristics, combined with partial β–tubulin sequences, were applied to identify isolates.

MATERIALS AND METHODS

Sample collection and isolation

Sampling was conducted from saffron farms in Khorasan Razavi, South Khorasan, North Khorasan, Kerman, Isfahan, Fars and Yazd provinces, Iran. Plant samples were collected from corms, leaves, and flowers without disease symptoms. Isolation of endophytic fungi was performed according to the protocol reported by Petrini (1986) with some modifications. First, healthy collected samples were washed under running water for 10 minutes. Afterward, superficial disinfection was performed for one minute in 75% ethanol, two minutes in dilute sodium hypochlorite solution (NaClO) (containing 1% active chlorine) and two minutes in 70% ethanol. Disinfected samples were washed in sterile distilled water thrice. After surface-disinfection, the samples were placed on WA, at 25°C in darkness condition. The living strains were deposited in the Culture Collection of Agricultural Biotechnology Research Institute of Iran (ABRRI).

Morphological analysis

Morphological identification of Talaromyces species was performed using macroscopic and microscopic characteristics. Macroscopic characteristics such as colony diameter, texture, the color of conidia, mycelia, soluble pigments, and acid or possible base production were studied on MEA, OA, YES, CZ, CYA, and CYAS medium after one week of incubation at 25 °C in darkness. Moreover, micros–
copic characteristics, such as the number of branching points between stipe and phialides (i.e., solitary phialides to quaterverticilate), dimension, shape and texture of stipes, vesicles, metulae/branches (when present), phialides, conidia, cleistothecia, asci and ascospores (when present) on MEA medium, were measured after one week of incubation at 25 °C in darkness (Samson et al. 2019). All isolates were inoculated at the three–point method on all the media.

DNA extraction and PCR amplification

DNA extraction was performed from one–week–old colonies grown on potato dextrose agar (PDA) at 25 °C in darkness, according to Zhong & Steffenson (2001). The β–tubulin gene region was partially amplified using primer pair BT2A/BT2b (5´–GGTAACCAAA TCGGTGCTGCTTTC/ ACCCTCAGTGTAGTGACCC TTGGC–3´) (Glass & Donaldson 1995). Polymerase chain reaction (PCR) mixture was prepared in a total volume of 26 µL containing 10 µL Master Mix Red (CinnaGen, Iran), 1.5 mM MgCl₂, 1 µL of each primer (10 pmol) and 10–20 ng of genomic DNA. PCR condition for β–tubulinamplification was as follow: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 58°C for 45 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min.

DNA sequencing and phylogenetic analysis

The PCR products were purified using GeneAll Purification Kit (combo GP, 50p). The purified PCR products were sequenced in the forward direction by Cardiogenetics Research Center in Iran. The received sequences were edited by Pro Chromas Version 1.7.6 and Editseq version 5.01. The Basic Local Alignment Search Tool (BLAST) was used to compare the similarities of nucleotide sequences with GenBank database (http://www.ncbi.nlm.nih.gov/blast/). Sequences obtained from this study and the GenBank were aligned using MEGA 7 software (molecular evolutionary genetics analysis) and multiple alignments option (Muscle).Maximum likelihood analysis was performed. The best model for ML was selected based on the Akaike Information Criterion (AIC), which was calculated in MEGA. ML analysis was done by calculating an initial tree using BioNJ and the subsequent Heuristic search done with the Nearest-Neighbour- Interchange (NNI) option. The reliability for each group was evaluated by 1000 replications bootstrap.Rasamsonia aegroticola was selected as a suitable outgroup. The sequences used in this analysis were deposited in GenBank (Table 1).

RESULTS AND DISSCUSSION

Morphological identification

Based on morphological characteristics, endoph-
ytic Talaromyces isolates obtained from leaf and corm of saffron in this study, belonged to the Thalaromyces section Thalaromyces. Overall, five Talaromyces isolates were obtained and according to morphological studies, the isolates were identified as Talaromyces versatilis, T. aurantiacus, T. pinophilus, T. funiculosus and T. purpureogenus species.

Phylogenetic analysis

In this study, sequence data of the β-tubulin gene region was used to identify relationships within Talaromyces species. The β-tubulin gene generated 390- to 409-bp fragments and comprised 368 characters after alignment. The phylogenetic tree generated by β-tubulin sequences of species in sections Thalaromyces, Purpurei, Trachyspermi, Bacillispori and Islandici from GenBank (Table. 1). The results are shown in Fig. 1, which demonstrate that five isolates examined in this study are belonged to five distinct species (Fig. 1). The isolate ABRIICC 10349 is close to Talaromyces aurantiacus and form a well–supported clade with 93% bootstrap. The isolate ABRIICC 10348, form a sister group with T. versatilis (94%). Additionally, isolates ABRIICC 10350, ABRIICC 10351 and ABRIICC 10352 are close to T. pinophilus, T. funiculosus and T. purpureogenus with high bootstrap support (100%), respectively.

Phylogenetic classification using β–tubulin genes showed that β–tubulin sequence had acceptable performance in species recognition and separating the Thalaromyces section species with a higher bootstrap value.As concluded in the investigation conducted by Visagie et al. (2014), β–tubulin could successfully identify Talaromyces species. They recommended β–tubulin as an identification marker for Talaromyces (Yilmaz et al. 2014). Except from the new species, all other isolates could be reliably identified using
β–tubulin sequences (Barbosa et al. 2018).

Taxonomy

Talaromyces aurantiacus (J.H. Mill., Giddens & A.A. Foster) Samson, Yilmaz & Frisvad, Stud. Mycol. 71: 175. 2011.

Basionym:  Penicillium aurantiacum J.H. Mill., Giddens & A.A. Foster, Mycologia 49: 797. 1957.

Colonies on MEA at 25°C, 19 mm growth; margins low, plane; mycelia white and light pink; texture floccose; reverse greyish orange. On OA at 25°C, 17 mm growth; colonies low, plane; margins low, plane; mycelia white; texture floccose; conidia en masse greyish green. On YES at 25°C, 21 mm growth; Colonies raised, slightly sulcate; margins low, plane; mycelia white; texture floccose; conidia en masse greyish green. On CZ at 25°C, 10 mm growth; margins low, plane; mycelia white; texture floccose; reverse greyish green. On CYA at 25°C, 15 mm growth; sterile white aerial mycelia, pale red appearance because of reverse coloring; margins low, plane; mycelia white; texture floccose and funiculose; reverse pastel red. On CYAS at 25°C, No growth (Fig. 2).

Fig. 2. Morphological characters of Talaromyces aurantiacus, isolate ABRIICC 1349. Colonies on a. MEA, b. CYA, c. OA, d. YES, e. MEA (reverse), f. CYA (reverse), g. CZ, h. CYAS, i–k. Conidiophores, l–m. Conidia.

Fig. 4. Morphological characters of Talaromyces pinophilus, isolate ABRIICC 1350. Colonies on a. MEA, b. CYA, c. OA, d. YES, e. MEA (reverse), f. CYA (reverse), g. CZ, h. CYAS, i–l. Conidiophores, m. Conidia.