Phaeoacremonium tuscanicum, a new fungal pathogen associated with oak decline in Zagros forests, Iran

INTRODUCTION

Zagros forests characterized as a semi-arid habitat in the west of Iran are covered by different oak species, including Quercus brantii Lindl., Q. libani Oliv. and Q. infectoria Oliv. (Heydari et al. 2013). In the last two decades, climate changes, drought and biotic agents (fungal pathogens and pests) have affected Zagros forests vegetation and dieback and decline of oak trees are the most serious disease symptoms spreading throughout the Zagros Mountains. The genus Phaeoacremonium belongs to the family Tognoniaceae (order Togniniales, Ascomycota), containing some 70 species (May 2020, www. indexfungorum.org) is a well-known genus associated with decline and dieback symptoms in different woody plants across the world (Farr & Rossman 2020). Phaeoacremonium species are known as the vascular plant pathogens causing wilt and dieback of woody hosts (Mohammadi 2012, Hashemi et al. 2017, Kazemzadeh Chakusary et al. 2017, Spies et al. 2018, Sohrabi et al. 2020). Moreover, Phaeoacremonium species are associated with humans, larvae of bark beetles (Mostert et al.2006) and soil (Crous & Gams 2000, Dupont et al.2002). This research was conducted to study the fungi associated with the oak decline in Zagros forests located in West Azarbaijan, Kurdistan, Kermanshah, Lorestan and Ilam provinces.

MATERIALS AND METHODS

During July and September 2017, samples of stems and twigs of oak trees showing dieback and decline symptoms were collected. Small pieces of infected woods showing a discoloration of xylem tissues were sterilized in 70% ethanol for 3 min, followed by washing with sterile distilled water and dried on sterile filter paper. The wood segments were plated out on potato dextrose agar (PDA, Quelab) supplemented with 100 mg/L streptomycin sulphate and ampicillin and incubated at 25°C. Fungal isolates were purified using the hyphal tip or single spore techniques on Water Agar (WA, Quelab). Fungal isolates were incubated on PDA, Oatmeal Agar (OA; 30 g boiled grinded oat, 15 g agar, 1 L distilled water) and Malt Extract Agar (MEA 2%, Quelab) at 25°C in the darkness and morphological and cultural characteristics were recorded after 8 and 16 days (Mostert et al. 2006, Essakhi et al. 2008). Type of phialids, structures and dimensions of conidiophores, and conidia recorded in 100% lactic acid. Genomic DNA of pure cultures was extracted according to the modified Raeder and Broda (1985) method as described by Abdollahzadeh et al. (2009). In this study, seventeen isolates with Phaeoacremonium morphology were obtained. Based on the ISSR fingerprinting patterns generated with M13 primer (data not shown) all isolates belonged to the same species. Therefore, one isolate was selected for phylogenetic analysis and deposited in the culture collection (IRAN) of the Iranian Research Institute of Plant Protection (Tehran, Iran). For molecular identification, partial β-tubulin gene (tub2) was amplified using primers T1 (5´-AACATGCGTGA GATTGTAAG-3´) (O’Donnell & Cigelnik 1997) and Bt2b (5´-ACCCTCAGTGTAGTGACCCTTGGC-3´) (Glass & Donaldson 1995). The PCR reaction mixtures were prepared in 25 µL volume containing 1×PCR buffer, 3 mM MgCl2, 200 µM of each nucleotide, 5 pmol of each primer, 1 U of Taq polymerase and 1 µL of template DNA (50–100 ng/µL). The PCR conditions were: primary denaturation step of 5 min at 94°C followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 90 s, with a final extension at 72°C for 7 min. The PCR products were purified and sequenced by BGI (China) via BMG (Bio Magic Gene) Co. (Karaj, Iran). The generated sequence was checked and extracted in BioEdit v. 7.0.0 (Hall 2004) and submitted to GenBank. Our isolate sequence, together with 26 Phaeoacremonium species sequences retrieved from GenBank and the outgroup species Pleurostoma richardsiae CBS 270.33, aligned with Clustal X v. 1.83 (Thompson et al. 1997). Phylogenetic analyses based on maximum parsimony and neighbor-joining algorithms were performed in PAUP v. 4.0b10 (Swofford 2003) as followed by Abdollahzadeh et al. (2009). A pathogenicity test was performed by inoculation of two-year-old Q. brantii seedlings under greenhouse conditions. Seedlings were wounded and inoculated with mycelium plugs from actively-growing cultures on PDA and controls were inoculated with sterile PDA plugs.

RESULTS AND DISCUSSION

According to tub2 phylogeny, isolate IRAN 4348C (GenBank accession no.: MZ547665) clustered in a clade containing an ex-type strain of Ph. tuscanicum (Fig. 1). So far, Ph. tuscanicum has been reported from Juglans regia (Iran), Prunus persica (Iran), Vitis sp. (Spain) and Vitis vinifera (Iran, Italy and Spain) (Farr & Rossman, 2020).

Pheoacremonium tuscnicum Essakhi, Mugnai, Surico & Crous, Persoonia 21; 119–134 (2008); Fig 2

Colonies on PDA reaching 12 mm within 7 days at 25°C, cottony and flat, with entire margin, after 16 days smoke gray to pale olivaceous gray to buff toward the margin above, reverse dark beige olive; on MEA 10 mm within 7 days at 25°C, cottony and flat, with an entire margin above, after 16 days olivaceous gray to buff toward the margin above, reverse orange; on OA 8 mm within 7 days at 25°C, appressed, fluffy in the middle with entire margin, after 16 days yellowish orange to buff toward margin above, reverse yellowish. Mycelium yellowish to pale brown, septate, branched, single or with a bundle of hyphae. Conidiophores rarely long and branched, straight, simple, septate, pale brown to sub-hyaline, (16–)20–26(–28.6) × (1.21–)1.4–1.9(–2.12) μm (mean 22.62 × 1.65 μm). Phialides mostly lateral and monophialidic, type I phialides subcylindrical, broad at the base, (3.2–)4–15(–17.8) × (1.24–)1.3–2(–2.01) μm (mean 9.17 × 1.65 μm); type II phialides narrowed at the base, widened in the middle part and tapering toward the tip, (8.03–)10–15(–18.23) × (1.2–)1.4–2(–2.16) μm (mean 12.35 × 1.73 μm); type III phialides subcylindrical, (18.3–)19–23(–24.2) × (1.67–)1.8–2(–3) μm (mean 22.62 × 2 μm). Conidia unicellular, hyaline, allantoid or sub-cylindrical, (2.01–)2.5–3.7(–5.37) × (0.8–)0.9–1.3(–2.03) μm (mean 2.94 × 1.18) μm.

Notes. Isolates collected in this study were obtained from Kurdistan (4 isolates from Q. brantii and Q. libani), Kermanshah (4 isolates from Q. brantii), Lorestan (6 isolates from Q. brantii) and Ilam (3 isolates from Q. brantii) provinces. Sequenced isolate (IRAN 4348C) was collected from Lorestan Province (N33 39.404 E48 16.855).

In the pathogenicity test, after 2 months, necrosis appeared on the leaves of inoculated seedlings. Brown lesions expanded upwards and downwards of the inoculated point on the stem internodes and no symptoms were observed on control seedlings (Fig. 3). To confirm the Kochʼs postulates, the pathogen was re-isolated from the lesions of seedlings. To our knowledge, it is the first time Ph. tuscanicum is reported from oak trees (Q. brantii and Q. libani) and confirmed as a pathogenic fungal species on Q. brantii in the world. It is important to study the pathogenicity of this new pathogen on Q. libani and Q. infectoria, the other two oak species growing in Zagros forests. Given that the phytotoxic metabolites have been characterized as pathogenicity determinants of fungi (e.g., Phaeoacremonium aleophilum) associated with the decline of woody plants (Evidente et al. 2000) to understand the interaction of Ph. tuscanicum and oak species in future studies, it is interesting to characterize the phytotoxic secondary metabolites of this fungal species and their role in pathogenicity on the hosts' trees.

Fig. 1. One of the four most parsimonious trees based on tub2 sequence data. MP/NJ bootstrap values based on 1000 pseudoreplicates are given at the nodes. The isolate sequenced in this study is in boldface. Bar = 20 changes. Pleurostoma richardsiae CBS 270.33was included as an outgroup.

Fig. 2. Sixteen-day-old colonies of Phaeoacremonium tuscanicum on a. PDA; b. MEA 2%; c. OA at 25°C; d. Bundle of hyphae; e. (1) conidiophore, (2) phialides type I; f. phialid type II; g. phialide type III; h. conidia. Scale bars: d–f and h = 7 μm, g = 5 μm.

Fig. 3. Disease symptoms caused by Phaeoacremonium tuscanicum on oak seedling in greenhouse conditions. a. inoculated plant after 3 months; b. negative control; c. necrotic lesion expanded upwards and downwards of the inoculated point on the stem internodes.