First Report of Neurospora on Corylus Avellana in Natural Forest of Iran
Vol. 6(4), pp. 31-36, July 2015 DOI: 10.5897/JYFR2014.0149 Article Number: 25CB15654690 ISSN 2141-2413 Journal of Yeast and Fungal Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/JYFR
Full Length Research Paper
First report of Neurospora on Corylus avellana in natural forest of Iran
Kamran Rahnama* and Roghaieh Habibi
Department of Plant Protection, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
Received 30 November, 2014; Accepted 27 May, 2015
Teleomorph stage of the fungus Neurospora udagawae was formed abundantly on the culture media of potato dextrose agar, potato carrot agar and malt extract agar. Perithecia were visible on the culture medium and plant tissues (on the bark of tree) of hazelnut tree (Corylus avellana L.) from specimens of the Northwest forest of Iran. The fungus was identified for the first time in this area. The isolate was examined for its macroscopic and microscopic features and identified as nonconidiating species of Neurospora. Growth rate of the fungus hyphae was more than twice in 30 than 10 and 20°C. There was no growth at 5 and 40°C. This fungus is recorded here for the first time from hazelnut tree of Iran and so in the world.
Key words: Neurospora udagawae, morphology, homothallic.
INTRODUCTION
The Sordariaceae (Ascomycota) encompasses approxi- ascospores, with a grooved surface with longitudinal ribs. mately 10 genera (Eriksson et al., 2003) which are Different members of this genus use one of three coprophilous, soil borne or grow on plant debris. They are different mating strategies: heterothallism, homothallism characterized by ampulliform or conical and usually or pseudohomothallism (partial self-compatibility) ostiolate ascomata; asci are commonly cylindrical with an (Perkins and Turner, 1988). However, some heterothallic apical ring; and the ascospores are generally aseptate, species are difficult to separate morphologically and can with the surface smooth, pitted, reticulate or striate, only be differentiated by mating tests. The genus sheathed or unsheathed, darkly pigmented and with one Neurospora includes five conidiating species: Neurospora or several germ pores. The largest genera are crassa, Neurospora intermedia, Neurospora sitophila, Gelasinospora Dowding (1933) and Neurospora (Shear Neurospora discreta (all heterothallic) and Neurospora and Dodge, 1927). The main difference between them tetrasperma (pseudohomothallic) studied in North lies in the ornamentation of the ascospore wall. America and as well as Europe (Jacobson et al., 2006). The generic name Neurospora was introduced by Shear N. crassa is the best known and has been used and Dodge (1927) for four species characterized by dark extensively for genetic, biochemical and molecular studies
*Corresponding author. E-mail: [email protected]. Fax: +981732251608.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 32 J. Yeast Fungal Res.
Figure 1. The geographical location where fungi strain are collected (as red boll) ranging from the Longitude 28° 25´ E and Latitude 48° 40, indicating the collection sites of Neurospora udagawae strain in Ardabil province, north west of Iran (from Google earth). Specimen Examined- Iran, East Azarbijan Prov., Ardabil city, Fandoghlo forest areas, on the healthy and fire burned twigs of Corylus avellana L. (Corylaceae), Longitude 28° 25´ E, Latitude 48° 40´,Co RAHNAMA K & Habibi R (NEUROSPORA IRAN2235C).
(Davis, 2000). The five conidiating Neurospora species 2006). But, molecular studies in Lasiosphaeriaceae, cannot be distinguished from one another on the basis of Melanospora (Hypocreales) and Onygenales also vegetative morphology or color. Morphology nonetheless suggested that generic distinction only on the basis of continue to be useful for identifying N. tetrasperma this character does not correlate with phylogenetic because this pseudohomothallic species produces relationships inferred from DNA sequences (Cano et al., perithecia with asci containing four dikaryotic and 2002; Miller and Huhndorf, 2002). binucleate spores, as opposed to the eight-spored asci Today about 3500 fungus have been extensively found in all other Neurospora species (Turner et al., identified and reported in Iran, and most reports of new 2001). records are limited to check lists with rare descriptions. The names Neurospora and Gelasinospora are Thus, fungi of Iran have received more attention in the synonymized and so far 49 species of Neurospora have past few decades. The recent indexed of fungi by Ershad been recognized in the genus by Garcia et al. (2004). (2009) and, the most reports on the fungal species from The generic diagnosis was also expanded to include different substrates indicated recently, one report of N. those with ascospores broadly fusiform, ellipsoidal or tetrasperma from fire burned twigs of plane trees nearly spherical, 1-celled, hyaline to yellowish brown or (Platanus orientalis) in the north east of Iran (Aghapour et olive-brown, becoming dark and opaque at maturity, al., 2010). In order to look at distribution of fungus in ascosporic wall with longitudinal ribs or pitted, various natural conditions, this study determines to find occasionally nearly smooth. possibility of new host for Neurospora in the distribution Anamorphs are known in only a relatively small number areas from North West of Iran. of species, belonging to Chrysonilia (Garcia et al., 2004). However, investigations on multiple-gene sequences and morphology concluded that although Gelasinospora and MATERIALS AND METHODS Neurospora are closely related, there is insufficient The plant material for this investigation was obtained from the north evidence to place currently accepted Gelasinospora and west of Iran, Ardabil province (Longitude 28° 25´E & Latitude 48° Neurospora species into the same genus (Cai et al., 40´) in the autumn of 2013 (Figure 1). At the time of sampling of Rahnama and Habibie 33
A B C
D E F
Figure 2.Figure Macroscopic 2. Macroscopic characteristics characteristics of Neurospora of Neurospora udagawae udagawae on Corylus on Corylus avellana avellana branch. branch. A~B, A- B,Artificial Artificial perithecia formationperithecia of burned formation twigs on of the burned bark twigs of Corylus on the avellanabark of Corylus (scale avellana bar: A,B=2 (scale m m)bar: A,B=2 mm); C, Formation of perithecia (small black dot) on WA 2% after 12 days; D-F, 12 days old cultures of Neurospora udagawae on PDA at 30±1°C; PCA at 20±1°C; on MEA at 20±1°C.
hazelnut trees (Corylus avellana L.), twigs with no sign of the media and different temperatures under continuous dark fungus on the surface were collected. In order to produce sexual conditions for 24 h (Table 1). There was no growth at 5 within Petri dishes and incubated at 25°C (Figure 2A and B). and 40°C temperatures after 24 h incubation. Perithecia formed after 14 days on the bark surface in the laboratory. The specimens with fruiting bodies were observed by using an Olympus light microscope (model Olysia Bio Report). Pieces of plant tissue were placed on potato dextrose agar (PDA) Colony characteristics culture medium after disinfestations then sub cultured on water agar (2%) medium and pure fungal cultures were obtained by Colonies on PDA and PCA filled the plates (9 cm) within transferring hyphal tips. The fungal isolates were grown on PDA culture medium then incubated at 25±1°C under dark condition. The 24 h at 30±1°C. Ascomata were produced abundantly macro and microscopic morphological characteristics of the fungi after 12 days, but on water agar 2% with very little were studied on potato dextrose agar (PDA), malt extract agar numbers as compared to those on PCA which was more (MEA, Oxoid) and potato carrot-agar (PCA; potato 20 g, carrot 20 g, regular and many ascomata at 20±1°C. Ascomata agar 20 g/l) at various temperatures of 5±1, 10±1, 20±1, 30±1 and produced pale brown to dark brown color on PDA, with 40±1°C (Garcia et al., 2004; Nygren et al., 2011), as well as growth scanty aerial hyphae especially at the periphery of the rates of the fungal isolates, were measured. The morphology of the ascospores, mainly the ornamentation pattern, number and plate and dark reverse (Figure 2D). Colonies on MEA placement of germ pores, and the presence or not of ostiole in the filled the plate within 48 h, with white color mycelia ascomata are the most important features for species distinction. (Figure 2F). The margins of fungal colonies on PDA and The fungal isolate were identified (based on morphological species PCA medium were smooth or partly irregular but on MEA concept) by comparison with the descriptions of Perkins and Turner medium were branchy. Colony color in PCA and MEA (1988), Turner et al. (2001) and Garcia et al. (2004). medium were white (Figure 2E and F), but subsequently,
the fungus metabolites turned culture to pale orange just RESULTS AND DISCUSSION on PDA medium at 30±1°C (Figure 2D). The colony expanded quickly and the expanding hyphae were broad, The growth rate of fungus was measured on various septate, thick-walled, hyaline and branched. 34 J. Yeast Fungal Res.
Table 1. Effect of temperature and media culture on mycelial growth of N. udagawae after 24 h incubation.
Mycelial growth in culture medium (mm/h) Temperature (°C) PDA PCA MEA 5±1 00.0 ± 00.0 00.0 ± 00.0 00.0 ± 00.0 10±1 23.0 ± 0.39 22.3 ± 0.39 9.30 ± 0.47 20±1 15.0 ± 0.08 12.1 ± 0.07 13.2 ± 0.09 30±1 45.0 ± 0.21 43.0 ± 0.20 33.7 ± 0.25 40±1 00.0 ± 00.0 00.0 ± 00.0 00.0 ± 00.0
Potato dextrose agar (PDA), potato carrot-agar (PCA), malt extract agar (MEA).
20±1 ºC; on MEA at 20±1 ºC.
A B C
D E F
G H I
J K L
Figure 3. Sexual stage of Neurospora udagawae on Corylus avellana branch. A, Globose or subglobose perithecia (scale bar: A=100 μm); B, Formation perithecia with lengthy neck; C, Ostiolate ascomata (scale bar: C=100 μm); D, Asci 8 spored (scale bar: D=20 μm); E, Uniseriate ascospores (scale bar: E=20 μm); F-G, Detail of the ascus (scale bar: F-G=40 μm); H, One celled ascospore and global ornate pits (scale bar: H=100 μm); I, Abnormal hyaline sheath (arrows) (scale bar: I=100 μm); J-K, Developing perithecia from coiled ascogonia (scale bar: J~K=100 μm); L, Irregularly and remotely septate (scale bar: L=100 μm). Rahnama and Habibie 35
Sexual stage obtained from healthy twigs and fire burned twigs in artificial condition in vitro. Homotallic species is reported The sexual stage of the fungus was formed abundantly from Hungary previously (Krug and Khan, 1991). Sexual on the WA 2% (Figure 2C) and PCA especially at 20±1°C structures were not frequently observed in the nature (Figure 2E) and the perithecia were visible on the culture because of difficulty in condition for black perithecia medium as very small black dots. Teleomorph stage was formation on the hazelnut trees and in vitro conditions. confirmed as homothallic, perithecia developing from We hope to continue our studies on phylogenetic and coiled ascogonia (Figures 3J and K), were superficial to biological species diversity among reported N. udagawae somewhat immersed covered with white hyphal-like hairs, strains in Iran in addition, with some other collected scattered (Figure 2C) to aggregated (Figure 2E), hairs strains of the fungus from other plants in the future. hyaline, flexuous, irregularly and remotely septate (Figure 3L), rarely branching with slightly thickened walls, globose or subglobose (Figure 3A) with one lengthy neck Conflict of Interests with 40-84 (63) μm in length (Figure 3B). Ascomata were ostiolate (Figure 3C), dark brown to black, smooth or The author(s) have not declared any conflict of interest. downy with loose hyphae and 226-240 (235) μm in diameter. Asci were unitunicate, hyaline, cylindrical, thin- walled having ring-like thickening at the tip, short stalked, ACKNOWLEDGEMENTS often 8-spored (Figure 3D, F and G), 103–109 (105) × 13–18 (16) μm in diameter. Ascospores were uniseriate The authors gratefully acknowledged the Iranian Plant (Figure 3E) or somewhat overlaping, initially hyaline, Protection Biodiversity and Genetic Resource Complex. becoming yellowish brown to black with maturity, one They wish to specially thank Prof. Dr. R. Zare and Dr. B. celled ellipsoidal or elongate, ascospore wall surface with Asgari, the mycologist of Plant Protection Research global ornate pits (Figure 3H), 0.7-1.6 μm in diameter and Institute of Iran for helpful suggestions during the abnormal hyaline sheath (arrows) (Figure 3I). Ascospores preparation and endorsement of species in Neurospora. were 19–26 (23) × 12–15 (14) μm in diameter and had circular and apical germ pores at each end. 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