Mycosphere Doi 10.5943/mycosphere/3/2/6 Occurrence of Chaetomidium arxii on sunn pest in Iran Arzanlou M1*, Khodaei S2 and Saadati Bezdi M3 1Assistant Professor of Plant Pathology and Mycology, Plant Protection Department, Faculty of Agriculture, University of Tabriz, PO Box: 5166614766, Iran 2MSc Student of Plant Pathology, Plant Protection Department, Faculty of Agriculture, University of Tabriz, PO Box: 5166614766, Iran 2PhD Student of Entomology, Plant Protection Department, Faculty of Agriculture, University of Tabriz, PO Box: 5166614766, Iran Arzanlou M, Khodaei S, Saadati Bezdi M 2012 – Occurrence of Chaetomidium arxii on sunn pest in Iran. Mycosphere 3(2), 234-239, Doi 10.5943 /mycosphere/3/2/6 Chaetomidium arxii was recovered from dead, overwintering adults of Eurygaster integriceps in northern Iran. The species was identified based on morphological characteristics as well as sequence data from LSU and ITS-rDNA regions. This is first report on the occurrence of C. arxii on sunn pest and a new record of C. arxii for Iran. The fungus is fully illustrated and described. Key words – Ascomycetes – cephalothecoid – Chaetomiaceae – Chaetomium – entomopathogenic Article Information Received 28 March 2012 Accepted 29 March 2012 Published online 30 April 2012 *Corresponding author: Mahdi Arzanlou – e-mail – [email protected] Introduction population of the sunn pest, Eurygaster The genus Chaetomidium (Zopf) Sacc. integriceps Puton, we found a fungal colonist (Chaetomiaceae, Sordariales), was first of unknown function. Culturing and molecular described in 1882 with C. fimeti (Fuckel) Sacc. techniques were used to identify this organism as its type species (Crous et al. 2004). The as a species of Chaetomidium. Sunn pest is a genus is characterized by non-ostiolate, evenly serious problem of wheat and barley in setulose and superficial ascomata, with clavate Western and Central Asia, and Southeast and evanescent asci (Stchigel et al. 2004). Europe, causing annual yield loss of 50–90% in Morphologically related genera such as wheat and 20–30% in barley. Boothiella Lodhi & Mirza, Thielavia Zopf, Corynascella Arx & Hodges, Melanocarpus Methods Arx and Corynascus Arx are distinguishable from Chaetomidium based on morphological Sampling, isolation and morphological study features including ascospore shape, peridium Dead adults of sunn pest were collected structure, germ pore characters and anamorphic from their overwintering sites in Mianeh, East state (Stchigel et al. 2004). Members of Azerbaijan Province, Iran. Insects were surface Chaetomidium reported in previous studies are sterilized for 1 min in 70% ethanol, rinsed 2 considered to be saprobic and they are found times with sterilized distilled water, dried, and predominately on dung (Taparia & Lodha plated on acidified potato dextrose agar (PDA; 1974, Benny 1980). Apparently, they play the Merck, Germany). Pure isolates were obtained role of the decomposers in these ecosystems. by sub-culturing emergent hyphal tips. The During a study on the entomopathogenic cultures were deposited in the living Culture mycota associated with overwintering Collection of Tabriz University (CCTU), 234 Mycosphere Doi 10.5943/mycosphere/3/2/6 Tabriz, Iran. The cultural features were studied Results on potato carrot agar (PCA; 20 g L-1 potato, 20 g L-1 carrot, 20 g L-1 agar) containing one piece DNA Phylogeny of sterile filter paper, PDA, malt extract agar There are no ITS-rDNA sequences of (MEA; Merck, Germany), and oat meal agar Chaetomidium spp. in GeneBank, and (OA; 30 g L-1 oat meal, 15 g L-1 agar) after 10 homology searches using NCBI Blast search days of incubation at room temperature in the nested our isolate within the genus dark. Growth rates were recorded as maximum Chaetomium Kunze. The LSU sequence data diameter of colony. Fungal structures were were identical to sequences in GeneBank for mounted on glass slides with water for Chaetomidium arxii (accession numbers: microscopic examination. Measurements of all FJ666359, FJ666363). The ITS rDNA and parameters were made at ×1000 magnification, nucLSU sequences generated in this study were with 30 measurements per structure. All deposited in GenBank under accession structures except asci were studied in at least numbers JQ864439 and JQ864438, respect- 15-day-old colonies, asci were studied in 7-10- tively. day-old cultures. Photographs were captured with a Leica camera system. Line drawings Morphology were made using a drawing tube attached to an Olympus BX 41 microscope. Chaetomidium arxii Benny, Mycologia. 72: 832, 1980. Figs 1–12 DNA extraction, PCR and sequencing Microscopic characteristics on OA – Genomic DNA extraction was carried Aerial mycelium sub-hyaline to dark brown, out by harvesting of actively growing septate, smooth, branched, thin-walled, 2–4 µm mycelium using the method of Moller et al. diam, covering the whole colony surface; aerial (1992). The internal transcribed spacer region mycelium around the point of inoculation and (ITS) of the nuclear ribosomal RNA operon in sectors hyaline and appressed, 1–4 µm diam. and part of the large subunit 28S rRNA (LSU) homothallic. Ascomata cleistothecioid, gene were amplified from the extracted DNA superficial, densely scattered over the whole using universal fungal primer sets ITS1/ITS4 surface of the colony, developing at (White et al. 1990) and LROR (Rehner & approximately 4 days, maturing rapidly, up to Samuels 1994)/LR5 (Vilgalys & Hester 1990), 190 µm in diam, non-ostiolate, globose, respectively. PCR reaction mixture contained arostrate, dull black, opaque, without cirrhi, 1× PCR buffer, 0.5 mM MgCl2, 0.2 mM of attached to the substrate by a poorly each deoxynucleoside triphosphate (dNTPs), 5 differentiated, scanty, hyaline to dark brown pM concentration of each primer, 0.5 U of Taq tuft of hyphae. Peridium cephalothecoid, 1–2- enzyme, 10–15 ng of DNA extract as template. layered, of textura angularis, dark brown, The final volume of the reaction was adjusted composed of polygonal plates; external cells to 25 μL by adding sterile deionized water. The prismatic. Ascomata clad with hairs; lateral and thermal-cycling conditions consisted of an terminal hairs practically indistinguishable; initial denaturation step of 96°C for 5 min, both unbranched, straight, smooth, thick- which was followed by 40 cycles of 96°C for walled, regularly septate, up to 290 µm long, 30 s, 52°C for 30 s, and 72°C for 1 min, with a 3–4 µm wide basally, 1–2 µm wide apically, final extension at 72°C for 7 min. The blackish brown, gradually tapering and paling amplified DNA fragments were sequenced upwards, with blunt tips, basal cell swollen, 5– using the BigDye Terminator v3.1 (Applied 6 µm diam. Asci fasciculate, hyaline, Biosystems, Foster City, CA) Cycle irregularly club-shaped, stalked, 8-spored, Sequencing Kits and analyzed on an ABI Prism deliquescing before spore maturity. Paraphyses 3700 (Applied Biosystems, Foster City, CA) as absent. Ascospores irregularly disposed in the recommended by the vendor. A preliminary asci, one-celled, punctuate, broadly ellipsoid, search for the identity of isolates was bluntly biapiculate or umbonate, hyaline when performed in the GenBank database using the immature, darkening to greyish brown at NCBI Blast search. maturity, with numerous guttules, 12–15 × 9– 235 Mycosphere Doi 10.5943/mycosphere/3/2/6 Figs 1–10 – Chaetomidium arxii. 1–4 Colony morphology on PDA, OA, MEA and PCA containing one piece of filter paper, respectively. 5 Cleistothecium. 6 Cephalothecoid peridium. 7-8 Ascomatal hairs. 9 Asci and ascospores. 10 Mature ascospores. – Scale (5 = 50 µm, 6-10 = 100 µm). 12 µm, mature ascospores thick-walled, with a white. Colonies on PDA reaching 40 mm in prominent apical germ pore. Anamorph absent. diam after 10 days, flat, folded, circular, entire, Cultural characteristics (Figs 1–4) – yellowish white, no ascomata. Colonies on Colonies on OA growing moderately, reaching MEA reaching 35 mm in diam after 10 days, 45 mm in diam after 10 days, without exudates, flat, vigorously folded, bright white, no circular, flat, with smoke grey, cobwebby ascomata. Colonies on PCA reaching 35 mm in aerial mycelium, and 1–3 bright white, slightly diam after 10 days, flat, circular, entire, folded, felty sectors, margins entire, dirty colorless, forming ascomata in relatively 236 Mycosphere Doi 10.5943/mycosphere/3/2/6 Figs 11–12 – Chaetomidium arxii. 11 Ascomatal hair. 12 Ascus and ascospores. – Scale = 10 µm. concentrically zonate pattern in 3 sectors (Crous et al. 2004, Greif et al. 2009), six of mostly in center of the colony; the colony and which viz., C. cephalothecoides (Malloch & agar reverse, except where ascomata are Benny) Arx, C arxii, C. galaicum Stchigel & formed, remain uncolored. Guarro, C. khodense Cano, Guarro & El Shafie, C. megasporum Doveri, Guarro, Discussion Cacialli & Caroti (Stchigel et al. 2004) and C. Morphological characters of our leptoderma (Booth) Greif & Currah (Greif & specimens are in full agreement with the genus Currah 2007) have cephalothecoid peridia. Chaetomidium. The main features of the Chephalothecoid peridia are considered to be present isolates are cephalothecoid ascomatal an adaptation for cleistothecial fungi to liberate peridium, straight, smooth and relatively short the ascospores (Greif & Currah 2007). Doveri hairs, with ascospores measuring 12–15 × 9–12 et al. (1998) provided a dichotomous key for µm. These characters revealed our isolates the identification of four species of the genus belonging to C. arxii (Benny 1980). Presently, with cephalothecoid peridia (C. cephalothe- the genus Chaetomidium comprises 12 species coides, C arxii, C. megasporum and C. 237 Mycosphere Doi 10.5943/mycosphere/3/2/6 khodense). Stchigel et al. (2004) provided a and the Studienstiftung Mykologie for financial key for the identification of 11 accepted support. morphospecies of Chetomidium. Taxonomy and phylogeny of the genus Chaetomidium, References however, have been subject of some confusion. In recent years, it has been speculated that the Badali H, Chander J, Gupta A, Rani H, Punia current morphological concept of Chaetomi- RS, Hoog GS de, Meis JF. 2011 – Fatal dium is polyphyletic (Greif et al.