Comparison of Two Coniochaeta Species (C. Ligniaria and C
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Vol. 52, 2016, No. 1: 18–25 Plant Protect. Sci. doi: 10.17221/45/2014-PPS Comparison of Two Coniochaeta Species (C. ligniaria and C. malacotricha) with a New Pathogen of Black Pine Needles – Sordaria macrospora Helena IVANOVÁ1, Peter PRisTAš 2,3 and Emília ONDRUšKOVÁ1 1Institute of Forest Ecology of the Slovak Academy of Sciences, Branch for Woody Plants Biology, Nitra, Slovak Republic; 2Institute of Biology and Ecology, Pavol Jozef šafárik University, Košice, Slovak Republic; 3Institute of Animal Physiology of the Slovak Academy of Sciences, Košice, Slovak Republic Abstract Ivanová H., Pristaš P., Ondrušková E. (2016): Comparison of two Coniochaeta species (C. ligniaria and C. malacotricha) with a new pathogen of black pine needles – Sordaria macrospora. Plant Protect. Sci., 52: 18–25. A new pathogen, Sordaria macrospora, isolated from damaged needles of black pine (Pinus nigra) causes discolouration, brown spots, blight symptoms, and necroses spoiling aesthetic value. Two species, C. ligniaria and C. malacotricha, the most common anamorphs attributed to Coniochaeta species occurring on selected conifers, and a new pathogen, Sordaria macrospora, occurring on Pinus nigra, are compared. Specific differences in spore size and anamorph mor- phology between the similar species C. malacotricha and C. ligniaria could be confirmed. Keywords: Ascomycota; morphological characteristics; phylogenetic analysis; Pinus nigra Sordariomycetes is a heterogeneous group of uni- or striate, sheathed or unsheathed. Spores are sur- tunicate pyrenomycetes with globose or flask-shaped rounded by gelatinous sheath which is sometimes solitary perithecial large ascomata, with large-celled thick and conspicuous to even difficult to detect. membraneous or coriaceous walls. These fungi are Darkly pigmented ascospores show wide variation in worldwide distributed, commonly in dung or decay- the kinds of appendages or sheaths (Alexopoulos ing plant matter, rarely on coniferous needles. These et al. 1996; García et al. 2004). species colonise whole stems and xylem of trees at In the recent literature the family Coniochaetaceae dry sites (Fisher et al. 1992, 1993; Petrini & Fisher is placed under Sordariales, but differs from them in 1988, 1990). Members of the order Sordariales can be ellipsoidal to fusoid, nearly globose ascospores with coprophiliac, fungicolous, herbicolous, lignicolous, longitudinal germ slits extending over the narrow side soil-inhabiting or grow on plant debris. Species of the (Mahoney & La Favre 1981; Hanlin 1990; Lee & family Sordariaceae have persistent cylindrical, basally Hanlin 1999; Kirk et al. 2001; Weber 2002). A dis- clustered asci in perithecial forms and clavate asci in tinguishing character of the genus Coniochaeta is the cleistothecial forms. Asci are commonly cylindrical presence of brown, usually straight and pointed setae with an apical ring. The ascus apex usually has one (Asgari & Zare 2006). Coniochaeta species have been or several germ pores and a refractile ring through reported from different substrates and in many cases which the ascospores are discharged. Sordaria species were isolated from wood samples that rarely showed has smooth-walled, dark brown ascospores, generally necrosis, and were always found in combination with aseptate, with the surface smooth, pitted, reticulate other fungi and basidiomycetes (Asgari & Zare 2006; Supported by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences – VEGA, Grants No. 2/0071/14 and No. 2/0069/14. 18 Plant Protect. Sci. Vol. 52, 2016, No. 1: 18–25 doi: 10.17221/45/2014-PPS Damm et al. 2010). Coniochaeta species are of low viru- magnification. Measurements were performed us- lence on most hosts, usually appearing on dead tissue ing the QuickPhotomicro 2.2 programme and the or as opportunistic invaders of previously infected, morphometric values were compared with previously wounded or senescent tissue (Damm et al. 2010). published data for the taxa (Petrini & Fischer During the investigation of mycoflora on a pine tree 1988; Fischer et al. 1992, 1993; Alexopoulos et (Pinus nigra) a fungus with distinctive characteristics al. 1996; Crous et al. 2009). was isolated. The aim of this work was to compare, DNA extraction, PCR amplification and sequencing. on the basis of light-microscopic morphological For DNA extraction, 7–8-day-old fungal mycelia were studies, two Coniochaeta species (C. ligniaria and used. Mycelium was scraped from MEA, ground and C. malacotricha) commonly occurring on conifer homogenised in liquid nitrogen by mortar and pestle. needles, with a newly detected fungus Sordaria mac- Total DNA was extracted according to DNeasy Plant rospora Auersw., that was confirmed by phylogenetic Mini Kit (Qiagen, Germany) protocol and stored in analysis. The similarity and the differences of these –20°C. Primer pairs ITS1F (5'-CTTGGTCATTTAGAG- fungi are discussed. GAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATT- GATATGC-3') (Gardes & Burns 1993; White et al. 1990), and LR0R (5'-ACCCGCTGAACTTAAGC-3') MatERIAL anD METHODS and LR7 (5'-TACTACCACCAAGATCT-3') (Vilgalys & Hester 1990) were used for amplification of ITS or From spring to autumn 2013 and in spring 2014, the partial large subunit ribosomal RNA (LSU) region, needles of Pinus nigra with blight symptoms were respectively. PCR amplifications were performed using collected from plants growing in private gardens a thermocycler (Biometra T Professional, Göttingen, and public greenery at several locations of Nitra, Germany) under the following conditions: 95°C/5 min, Slovakia (Nitra-Zobor, Nitra-Chrenová, Nitra-Sihoť, 95°C/15 s, 50°C/30 s, 72°C/80 s (Husson et al. 2011). Nitra-Kynek). Altogether 20 trees were studied. PCR reaction mixture (20 µl in total) contained 50 ng The age of the evaluated trees was 35–40 years. of DNA, 20 pmol of each primer, 0.2 mM dNTPs, and Samples were taken at each of these localities from 1U Hot Firepol Blend Master mix (Solis BioDyne, some sections of trees with damaged needles. Each Estonia). The PCR products were purified with Nu- sample was cultivated on 30 Petri dishes (PD) with cleoSpin Gel and PCR Clean up (Macherey-Nagel, 3% PDA medium. Occurrence of the new fungus Germany) prior to sequencing. The PCR products were S. macrospora was enumerated in % and identified sequenced with each primer used in the PCR (Sigma by microscopical analysis based on the appearance Aldrich, Germany). DNA sequences were assembled of the fruiting bodies, spore bearing organs (asci), using DNA Baser software (Heracle BioSoft SRL, Ro- reproduction organs (conidia and ascospores) and mania) and submitted to the GenBank database under on the appearance of the fungus in pure cultures. the accession No. KT013302 for S. macrospora Iv 1 Morphometric measurements of ascomata, setae, partial LSU sequence and No. KT013303 for S. mac- asci, and ascospores were made for each sample PD rospora Iv 1 ITS sequence. The DNA sequences were with the occurrence of the fungus S. macrospora. compared against GenBank database using BLASTn The main value of length and width was assessed for and BLASTx algorithms (Altschul et al. 1990). 30 conidia of each isolate. The samples of biological For analysis of phylogenetic relatedness DNA material were deposited in herbarium at the Institute sequences were aligned using Muscle algorithm of Forest Ecology of the Slovak Academy of Sciences, (Edgar 2004) and phylogenetic relationships were Branch for Woody Plant Biology in Nitra, Slovakia. constructed using the Neighbor Joining method avail- To isolate and obtain pure cultures, cultivation able in the MEGA software version 5 (Tamura et al. was performed on nutritive 3% PDA medium in a 2011). Phylogenetic robustness of trees obtained was versatile environmental test chamber MLR-351H tested by bootstrap analysis after 1000 replications. (Sanyo, Osaka, Japan) with constant temperature (24 ± 1°C), humidity (45%), under dark conditions. The needles parts cut from the diseased plants were RESULts surface-sterilized for 20 min. Fungal structures were investigated using a light clinical microscope BX41 Culture characteristics of the fungus isolated in (Olympus, Tokyo, Japan) under a 400× and 1000× our experiments from needles of Pinus nigra cul- 19 Vol. 52, 2016, No. 1: 18–25 Plant Protect. Sci. doi: 10.17221/45/2014-PPS Figure 1. Sordaria macrospora on Pinus nigra: (a–b) anamorph: (a) colony on PDA after 2 weeks; (b) vegetative hyphae; (c–o) teleo- morph: (c) ascomata with setae; (d) perithecial neck; (e–g) opening of perithecium; (h–k) rosettes of asci; (i) 8-spored ascus; (j) asco- spore sheath; (m) immature as- cospores; (n) granular content of ascospores; (o) mature ascospores. Scale bars: m = 20 µm, d, i = 50 µm, e = 100 µm tivated on PDA medium: the fungus formed sparse aseptate, cylindrical, with a non-amyloid apical thick- aerial mycelium of pale white colour, after 2 weeks of ening (Figures 1h, i, k), 8 spored (Figure 1j), 160–175 × inoculation dark pycnidia were formed (Figure 1a). 20 μm in size and formed rosettes (Figures 1h, k). Vegetative hyphae were hyaline, 4–5 μm wide, septate, Ascus was elongated to release the ascospores, which branched, lacking chlamydospores. Conidia did not were uniseriate, linearly arranged (Figure 1i), first appear (Figure 1b). After 2 weeks, black perithecia, green to pale brown coloured (Figure 1h), broadly pyriform, setose, solitary (Figure 1c) with a central ellipsoidal, later brown, smooth-walled with granu- ostiole were formed (Figure 1e), size 370–400 (500) × lar contents (Figures 1m, n) without guttules, sur- 250–300 µm (Figures 1f, g), perithecial neck was rounded by a gelatinous sheath (Figure 1j). As the positively phototropic, 35 (60)–150 µm (Figure 1d). asci mature, they swell and fill the upper part of the Setae occurred relatively scarcely, they were brown perithecium. One of the asci stretched and pushed or hyaline, smooth-walled, strait with globose or sub- through the ostiolar opening, while its base remained globose apices, 2 × 55 (68) µm (Figure 1c). Asci with attached to the perithecial wall, 50 μm in size (Fig- truncate apex and small apical rings were unitunicate, ure 1e).