New or less known ascomycete species associated with trunk and branches of trees in Guilan province, Iran

The isolates were morphologically characterized on Potato Dextrose Agar, Synthetic Nutrient Agar [SNA: composed of KNO₃ (one g), KH₂PO₄ (one g), MgSO₄.7H₂O (0.5 g), CMD, Cornmeal [CM: 10g cornmeal + 20 mL distilled water] (Hoegger et al. 2002, Hirooka et al. 2012). To induce sporulation of Botryosphaeriaceous species, fungi were cultured on 2 % water agar bearing double-autoclaved pine needles or poplar twig (Phillips et al. 2013).

For morphological identification, the following references were used: Roane et al. 1986; Rossman et al. 1999; Chaverri et al. 2003; Gryzenhout et al. 2006; Jaklitsch 2009; Jaklitsch 2011; Hirooka et al. 2012; Phillips et al. 2013; Jaklitsch et al. 2014).

DNA extraction, amplification and sequencing

Due to the limited budget for sequencing of all isolates, for each species, a representative was subjected to sequencing. DNA extraction was conducted by methods of Walsh et al. (1991) and Hirata and Takamatsu (1966). The rDNA internal transcribed spacers were amplified using the primer pairs ITS1 and ITS4 (White et al. 1990). PCR amplification was performed in 25 μL reaction volume containing 2.5 μL 10x PCR buffer (0.5 M KCl, 0.1 M Tris-HCl, pH 8.3, 0.015 M MgCl₂); 200 μM of each dNTPs, 0.2 μM of each primer; one unit of Taq DNA polymerase (Cinna Gen, Iran); 3–5 μL of extracted DNA solution. For the amplification, initial denaturation of 95°C for 1.5 min was followed by 30 PCR cycles consisting of 95°C for 30 s, 52°C for 30 s, 72°C for 30 s, followed by one extension period at 72°C for 6.5 min. The 5′ portions of translation elongation factor (tef1) (approximately 370–450 bp) were amplified and sequenced using EF1-728F (Carbone and Kohn 1999) and EF2R (O’Donnell et al. 1998) primers. For the amplification, an initial denaturation for 5 min at 95°C was followed by 30 PCR cycles consisting of 94°C for 45 s, 52°C for 30 s, 72°C for 90 s, followed by one extension period at 72°for 6 min (Khodaparast et al. 2020). The nucleotide sequences of polymerase chain reaction (PCR) products were obtained using direct sequencing in a Genetic Analyzer 3130XL (Applied Biosystems, USA) in Rouyan Zista Gene company (Iran, Tehran). Sequences were analyzed and edited using MEGA7.0 (Kumar et al. 2016). Similar sequences were searched with the available sequences in NCBI GenBank nucleotide database using a BLASTN search method. Several sequences (preferably type or reference sequences, if available) from GenBank were selected for comparison.

RESULTS AND DISCUSSION

During the present investigation, 20 fungal isolates belonging to Botryosphaeriaceae, Cryphonectriaceae, Hypocreaceae and Nectriaceae families (Ascomycota) were obtained from the trunk and branches of trees during 2017–2019. In this paper, we introduced some species that are new records to the mycobiota of Iran or less known from Guilan province. All specimens were deposited in Mycological Herbarium at the University of Guilan (GUM).

Microthia havanensis (Bruner) Gryzenhout & M.J. Wingf., Studies in Mycology 55: 44 (2006)

Ascostroma on natural substrate semi-immersed to superficial, pulvinate, orange. Perithecia spherical to ovoid, 82–145×57–62 μm, in groups of 2 to 12, surrounded by host tissue. Asci fusiform to cylindrical, with 8 ascospores, 20–35×4–6 μm. Ascospores two-celled, hyaline, ellipsoid to fusoid, 6–7.51.5–3 μm. Anamorphic stromata 47–75×30–35 μm, semi‑immersed to superficial, orange, uni- to multilocular, often occurring in the same stroma that contains perithecia. Conidiophores cylindrical with long paraphyses between them and 30–60×1 μm in length. Conidia hyaline, cylindrical to ellipsoid, aseptate, 2.5–3×1 μm.

On PDA, few pycnidia were produced after seven days of incubation at 25°C a. Pycnidia orange, slimy, subglobose. Conidia yellow, 4–6×1–2 μm. No pigmentation was observed on cornmeal after seven weeks at 25°C (Fig. 1).

Specimen examined. IRAN, Guilan Province, Fouman (Roudkhan castle), on the surface of branch and trunk of Pterocarya fraxinifolia (Poir.) Spach, 7 April 2017, N. Mousavi (GUM1561).

Note. This fungus was identified based on Roane et al. (1986) as Cryphonectria havanensis (Bruner) M.E. Barr. However, Gryzenhout et al. (2006) revised the genus Cryphonectria and transferred this species to Microthia based on its long paraphyses between conidiophores. Unfortunately, sequencing of ITS and tef1 genes was failed for this species. This is the first report of this species from Iran.

Lasiodiplodia hormozganensis Abdollahzadeh, Zare & A.J.L. Phillips, Persoonia 25: 6 (2010)

Ascomata not observed. Conidiomata produced on pine needles on WA within 2–4 weeks, dark brown to black, covered with dense mycelium, globose, 0.8–1.2×0.5-0.7 mm. Paraphyses hyaline, cylindrical, 43–85×2–3 μm. Conidiogenous cells holoblastic, hyaline, cylindrical, 12–19×2–3 μm. Conidia initially hyaline, aseptate, ellipsoid to cylindrical, becoming pigmented, 1‑septate with longitudinal striations, 18–23×8–15 μm.

On PDA, mycelium covering the 9 cm plate at 25°C after 72 hours, at first, the color was bright, after a week turned to light gray (Fig. 2).

Specimen examined. IRAN, Guilan Province, Sumaehsara (Sesar), on the barks of Ailanthus altissima (Mill.) Swingle, 5 August 2016, N. Mousavi; tef1 sequence GenBank: MW330392; (GUM1559).

Note. Phillips et al. (2013) revised Botryosphaeriaceae and have provided a key for species identification. Based on this key, our species was well distinguished by its dark brown conidia with longitudinal striations from closely related taxa. BLAST queries of the sequence shared 99% identity with Lasiodiplodia hormozganensis (294/296 bp with two gaps, GU945343, IRAN1500C, holotype sequence, Abdollahzadeh et al. 2010). This species has already been reported from Hormozgan province (Abdollahzadeh et al. 2010) and this is a new report from Guilan province.

Fig. 1. Microthia havanensis. a. Ascostroma on bark b. Longitudinal section of conidioma c. Conidioma paraphyses d. Asci and ascospores e. Colony morphology in 90 mm dishes after two weeks at 25 °C in the dark regime on PDA f. Colony on CM after seven weeks (Scale bars: a: 100 μm; b: 40 μm; c: 20 μm; d: 20 μm).

Fig. 2. Lasiodiplodia hormozganensis. a. Conidiomata on pine needles in culture b. conidiogenous cells and paraphyses c. Conidia with longitudinal striations d. Colony morphology on PDA after 3 days at 25 °C (Scale bars: b: 40 μm; c: 20 μm).

Lasiodiplodia parva A.J.L. Phillips, A. Alves & Crous, Fungal Diversity 28: 9 (2008)

Ascomata not seen. Conidiomata produced on pine needles and poplar twig on WA within 2–4 weeks, dark brown to black, covered with dense mycelium, globose to subglobuse, 0.7–1×0.5–0.7 mm. Paraphyses hyaline, cylindrical, usually 45–75×2–4 μm on a pine needle, but reach to 130 μm. Conidiogenous cells holoblastic, hyaline, cylindrical, 10-15×3–5 μm. Conidia initially hyaline, aseptate, ellipsoid to cylindrical, becoming pigmented, 1-septate with longitudinal striations, 19–24×12–14 μm.

On PDA, mycelium covering the 9 cm plate at 25°C after 72 hours of incubation, at first, the color was bright after a week turned into light gray (Fig 3).

Specimen examined. IRAN, Guilan Province, Fouman (Roudkhan castle), on the barks of Pterocarya fraxinifolia (Poir.) Spach, 7 April 2017, N. Mousavi; tef1 sequence GenBank: MW330393; (GUM1560)

Note. The sequence of tef1 showed 99.4 % similarity (302/304 identity) with the sequence of the type material of L. parva (EF622063, CBS456.78, Alves et al. 2008). This is a new record for mycobiota of Iran.

Fig. 3. Lasiodiplodia parva. a. Disease symptoms on stem b. Conidia with longitudinal striations c. Colony morphology on PDA after three days at 25 °C d. Conidiomata on pine needles in culture (Scale bars: 19 μm)

Thyronectria austroamericana (Speg.) Seeler, Journal of the Arnold Arboretum 21: 405 (1940)

On the natural substrate, ascomata and pycnidia were formed separately or on the same stroma. Stromata variable in size, partly immersed, reaction to KOH and lactic acid (LA) negative. Perithecia were subglobose to globose 20–48×20–33 μm, aggregated in groups of 4–160, dark brown to black. Asci narrowly clavate, 57.6–68.4×7.2–12 μm, 8-spored, ascospores subglobose to ellipsoidal, muriform, light yellow, (11–)12–19×7–10 (12) μm. Pycnidial stromata erumpent through epidermis or developing in the stroma with ascomata, orange to peach. Pycnidia dimorphic, superficial and immersed in the stroma, KOH-, LA-. Superficial pycnidia multilocular, brown, immersed pycnidia multilocular, irregular multiple chambers with shared walls, solitary or aggregated in groups of 2–4. Conidiophores 2–5 times branched, 8–13.5×1–2 μm. Sterile hyphae mixed with phialides and acicular, straight or slightly curved, unbranched, sometimes 1–2 branched, aseptate, 20–45×1–1.5 μm. Conidia light yellow, ellipsoidal to obovate, aseptate, 2–3×1–1.5 μm.

On PDA, after 7 d at 25°C, colonies 35–45 mm. Colony surface with radial growth and sometimes wavy, peach odor on PDA slightly fruity. Sporulation on SNA rare. Conidiophores abundant, unbranched, 12.5–31×1–2 μm. Conidia hyaline, KOH+, LA-, oblong or ellipsoidal, straight or slightly curved, rounded at both ends, not germinating and budding on media, 4.5–13.5(–17)×2–3(–4) μm (Fig 4).

Specimen examined. IRAN, Guilan Province, Fouman (Alian), on the branches and barks of Gleditsia caspia Desf., 30 March 2016, N. Mousavi; ITS sequence GenBank: MW325661; (GUM1544)

Note. Recently Jaklitsch and Voglmay (2014) revised Thyronectria species and provided a dichotomous key for species identification. Based on this key, the above species is well distinguished by its muriform, subglobose to ellipsoid ascospore from closely related species. Moreover, the rDNA ITS sequence of our isolate showed 100% homology (548/548 identity) with a reliable accession number KJ570691 that has been submitted by Jaklitsch and Voglmay (2014). This is the first report of the species from Iran.

Trichoderma cf. chromospermum P.Chaverri & Samuels, Studies in Mycology 48: 51(2003)

On the natural substrate, stromata scattered or gregarious, pulvinate to discoidal with a narrowly large margin, pale yellow. When dry 0.1–1.7×0.8–1.3 mm. Perithecia only rarely projecting, ostiolar dots 30–70 μm, conspicuous, numerous, brown to dark green or black when mature, orange-yellow after addition of KOH. Perithecia 60–125×20–100 μm, subglobose or flask-shaped. A cortical layer consisting of a textura angularis of light orange, thin-walled cells, (6–)7–9(–10)×4–6 μm, often only of 1–2 layers of coarse cells; uppermost layer partly collapsing. Subcortical tissue subhyaline, thin-walled cells, 7–13×4–7(–10) μm. Subperithecial tissue sub-hyaline, thin-walled cells, 10–25×7–13 μm. Asci 60-91×5–5.5 μm. Ascospores yellowish green to green, coarsely tuberculate, cells dimorphic with little difference in shape and size, distal cells subglobose, (3.6–)4.16(–5.2)×3.6–4 μm, proximal cells subglobose to oblong, 4.16–5.2×(3–) 3–4, including warts.

Fig. 4. Thyronectria austroamericana. a. Teleomorph on natural substrata b. Cross section of superficial pycnidia c. Asci d. Ascospores e. Colony morphology on PDA after 2 weeks at 22 °C f. Conidia on SNA media (Scale bars: a: 100 μm; b-f: 20 μm).

On PDA, after 72 h of incubation 40–42 mm at 25°C, 20–22 mm at 30°C after two weeks and did not grow at 35°C after a month; mycelium covering the plate after ten days. Colony dense, thin, silky, not zonate. The surface is covered by a white cottony with pale green conidia. Agar not pigmented; no distinctive odor. Autolytic excretion absent. On SNA after 72 h 34–40 mm at 25°C, 30–35 mm at 30°C after two weeks and did not grow at 35°C after a month; mycelium covering the plate after two weeks. Colony hyaline, thin, dense, with a wavy margin, hyphae with the radial arrangement, thin, with low variation in width. Autolytic activity absent. On CMD after 72 h 48–56 mm at 25°C, 25–28 mm at 30°C after two weeks and did not grow at 35°C after a month. Colony hyaline, thin, radial, shiny, little mycelium on the agar surface, yellow-green to citrine green. Autolytic activity absent. Agar not pigmented

Conidiophores short, with verrucose base,

30–60×3–4 μm, hyaline. Phialides arising in more or less narrow angles from barrel-shaped metulae on the secondary or tertiary branches, or directly from the main branch, flask-shaped or lageniform, tapering uniformly from base to tip, slender, sometimes twisted and short, formed singly or in pairs, 10–18 μm long, 3–4 μm wide at the widest point, 2–3 μm at the base. Conidia unicellular, pale green, smooth, ellipsoidal to oblong, 3–6× (2.5–)3(–3.5) μm (Fig 5).

Specimen examined. IRAN, Guilan Province, Fouman (Roudkhan castle), on the branches and barks of Pterocarya fraxinifolia (Poir.) Spach, 6 September 2017, A. Mousavi.; tef1 sequence GenBank: MW330394; (GUM1563).

Note. According to Chaverri and Samuels’s description (Chaverri and Samuels 2003), morphologically, this fungus was identified as T. chromospermum. After Blast search, the sequence of tef1 showed 98-100 % similarity to eight sequences of T. chromospermum available in GenBank (such as KF923291, KF923292, KF923287, Zhu  and Zhuang 2015.). However, it showed 96 % similarity (523/544 identity, with eight gaps) with one sequence of this species (AY737728, strain BPI 749362, Samuels 2005). Other sequences in GenBank showed less than 80 % similarity with this fungus. According to our knowledge, this is a new record for Iran mycobiota.

Trichoderma cf. longibrachiatum Rifai, Mycological papers 116; 42 (1969)

On the natural substrate, stromata scattered or gregarious, pulvinate to discoidal with a narrowly large margin, isabelline-hazel, when dry 0.1–1.7×0.8–1.3 mm. Perithecia only rarely projecting, ostiolar dots 30–50 μm, conspicuous, numerous, brown to dark green or black when mature, no change seen in 3% KOH. Perithecia 100–170×70–130 μm, subglobose or flask-shaped. Cortical layer 4–5(–6) μm thick, consisting of a textura angularis of light yellow to hyaline, thin-walled cells, subcortical tissue subhyaline, thin-walled cells 5–7×(3–)4–5 μm. Subperithecial tissue subhyaline, thin-walled cells, 11–30×5–11 μm. Asci 50–73×5–6 μm. Ascospores hyaline, coarsely tuberculate, cells 3–4×3–4 μm including warts.

On PDA, forming concentric rings of whitish mycelium, fluffy and dense, with velvety to slightly granular zones, greenish yellow to yellowish green, yellow diffusing pigment formed within 24 h at 25, 30, and 35°C, odor indistinct. Autolytic excretion absent.

On CMD, forming concentric rings of whitish, often submerged mycelia, with granular deep green zones, pustules at first white then became green to pale green, diffusible pigment not formed.

On SNA, autolytic excretion common in marginal hyphae, light yellow. Conidiophores usually consisting of a distinctive main axis, 60–80×3–4 μm, hyaline and smooth walled, sterile hairs not formed, phialides borne singly and laterally on the main axis or from side branches, divergent in small whorls of 2 to 3 phialides arising from supporting cells, less commonly solitary, cylindrical to lageniform 6–10 μm long, 2–3 μm the widest point and 1–2 μm at the base. Conidia unicellular, brown, smooth, round to elliptical, (3–)4–5(–6)×2.5–3.5 μm (Fig 6).

This species already reported from Iran (Ershad 1995), however, the sexual state of the species is recorded for the first time from Iran.