IMA FUNGUS · VOLUME 5 · NO 2: 161–172 doi:10.5598/imafungus.2014.05.02.01 Phialosimplex salinarum Eurotiomycetes , a new species of from a ARTICLE hypersaline habitat 5)1jO61$G2)3!1 14I/&"{772&) 2OJ$$WI/&"{772&)|}~ Abstract: K/ Key words: /0/[W Basipetospora & extremophile fungi &/ )0 Phialosimplex halophily common characteristics of producing conidia in chains or in heads on single phialides. Species of this U0K /I osmophily Phialosimplex salinarum sp. salt tolerance nov. Basipetospora halophila is also transferred to Phialosimplex as P. halophila comb. nov. 0K3! Article info:K}!"12!8|$}11K12!7|6}"12!7 INTRODUCTION O !"L8 0 !"L8 \3%0!"L20 Certain fungi tolerate highly unfavourable environmental Q 6 conditions such as extreme temperatures or osmotic K!"''&U pressures. The corresponding extreme habitats are $// scattered all over the world and represent attractive sites &et al.!"'8KVet al. !"""$ [ W$&U called “extremophiles” (Madigan et al. !""#$%& Halobacteriales (Archaea) !""' ) !""' * % ) !""' +/ % KV122# 012223%4122!56et al. 5 / 12!7* & et al. 1998) or salt / $ O K 56 et al 122! ) / Cimerman et al1222 56 et al 12!7 56et al.122!1228)et al.1228 1227/ &%$!""1*!""89/ U [ & $ / / )U * ; and that is formally described here. of salts in the substrate or water for growth. Strains which do !2 [ O/P )+ et al. MATERIALS AND METHODS 2006). * Sampling sites and sample collection predominantly colonized by Archaea (Norton et al. !""8 Sediment and water were sampled at three sites (numbered Denner et al. !""7 B et al. 1991). Haloarchaea O!P O1P O8P & with a broad range of salt tolerance were found in the salt OKH&P* GH 6 G I5 O*H4PI&/)7#8'a!#""bJ &)J!"!8Jet al. !8!a81!bW 0 !""8J%KH!"L!B%*/!""! / 5 !""L 0 Microorganisms can even be directly isolated from crystalline 'M!2 + © 2014 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: [ Non-commercial: No derivative works: For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. VOLUME 5 · NO. 2 161 Greiner et al. !2M12 K ! [ Experimental assays /12 Halotolerance and growth rates yr. Site 2 was a smaller pond of ca !{ 9 4$ K8[ 6O$|1!{ ARTICLE [; [8! coloured water. /!{+12U $} !22W\ OP# 4$O a boring rod 22 mm diam wrapped in aluminium foil; (b) !2 the brine free water body (“wb”) at ca. 10 cm depth using 120OK41#{82 !{3&W 4$ 3)|OP 6O$27 [ !{121{828{+27!{ 0'+ and 25 %). {87+ until fungal isolation. Substrate degradation UH Cultivation and morphological analyses OK41#{1LM1#{88 Microorganisms were isolated from two replicates per site !M8 9 [ amounts of sediment (ca. 0.1 cm8 '22 3V &et al.!"""6et al. (2009) /4$| 0!W7!{1{+ !2727!1 ' + H 0&34HSH enriched media around fungal colonies were indicative ) * 7!{1{/ V Media without salt addition (“0 % salt”) only contained trace /H0! 0 induced a change in colour to greenish blue around the !{+! colonies if positive. Crystalline coagulations indicated lipid W degradation. Diameters of the clearing zones and hyaline immediately transferred to fresh correspondingly composed areas were measured as a proxy for intensity of degradation 67+ activity. and at salt concentrations where colonies exhibited maximal growth. Molecular analyses Cultures of P. salinarum were examined morphologically [ Q$ 4 0 OJ$ 8' 15 % or 25 % salinity were examined in saline water. Strains Phialosimplex sclerotialis +&K 8LL## VHU OK4Q) +K&OJ$65U/IK$ +4++OK41#{82 21 V M|OK41#{1LOK41#{1#OK41#{1' /!20U0K3J$ OK4 1#{1" OK4 1#{8! OK4 1#{81 OK4 1#{88 U0K!{'KU0K1[ +&K5J$G9&/ KKI7"9 VKI!!9 6 et +I+&K!8'{'8M al 12!8 U0K!9 ) % & !""8 U0K7 G Table 1.) Test substrate Growth medium Detection reagent Starch 2'|2{J+|2L|!1|*2 VR Chitin 2'|2{J+|2L|!1|*2 none Cellulose 0.8 % peptone; 0.5 % NaCl; 0.6 % carboxymethyl cellulose; 1.2 % +|2{4*+ |*2 6 2'|!8|!1|*2|* none 7.2 V 2'|2{J+|!0'2|!1|*2 none V 1 % malt extract; 0.4 % yeast extract; 0.4 % glucose ; 1.2 % agar; 21 52*64| 21 5+| 21 4K4·7 *2 *2|1|$&0K!4|*2|*{ 162 IMA FUNGUS Phialosimplex salinarum sp.nov. from a hypersaline habitat Table 2.$Phialosimplex salinarum. ARTICLE ITS GenBank Acc. No. TSR1 GenBank Acc. No. DSM No. 591#7L'{ 5'{{{11 DSM 27526 591#7L'L 5'{{{18 DSM 27527 591#7L'# 5'{{{17 DSM 27528 591#7L'' 5'{{{1{ DSM 27529 591#7L'" 5'{{{1L OK41#{82 591#7L"2 5'{{{1# OK41#{8! 591#7L"! 5'{{{1' OK41#{81 591#7L"1 5'{{{1" OK41#{88 et al !""2 !1289 )$+0+$$+$+))))$$$+0+ RESULTS U0K7/00K3![ 9!{1L631787*% 9 [ 1{ K12!!6+35$6$8) few colonies of Archaea 6+3 5 & & IK$ 0 [ Trichocomaceae R Penicillium ; W /1{3V 06+3[+K& OP / OP 6+3 +I 5 U/ IK$ 18UPhialosimplex K;)$0+ salinarumOK41#{82 & V3 ; / 5H OK41#{1LM1#{1"OK41#{8!M1#{88 U0K!9 U0K7 U0K ; 31787 additional strains. 0K3! ; U0K ; )&/ Morphology 591#7L'{M591#7L"1| ; 4 Phialosimplex salinarum Phialosimplex sclerotialis+&K8LL## conformed with the traits considered to be diagnostic for the 591L#'L"0K3!; genus Phialosimplex (Sigler et al. 2010). )& 5'{{{115'{{{1" 0 +4$!{// 2). 27 Phylogenetic analysis 0 0K3! U0K ; structure. Some strains formed granular conglomerates of )& &V$K0 J+&U Q et )4$ al 1222 0 0K3! ; 12M1'!{4$!{M18 Phialosimplex salinarum / 1{4$#2!{+4$ representative species of Sagenomella Aspergillus 1!{/1' Penicillium were aligned using Muscle implemented in $ )/L&$JQ 07$ 0K3! ; cell walls. Conidiogenous cells were phialides laterally borne !M{!L{{7ML!2; + J!1!#81 Phialosimplex chlamydosporus $ ; U0K ; ; 9808 taxa with closer relationship to Phialosimplex as revealed U Experimental assays !#L!M!#L# !##1M!'28 !'2LM!'1L Salt and temperature preferences !'7'M!'{" !'L{M!'"{ !"22M!"!7 !"!LM!"8{ !"72M $ Phialosimplex salinarum exhibited 1!7" 1!{"M1182 1181M11{1 ; )!L"81L P. optimal growth on agar amended with salt concentrations of chlamydosporus$ !{1{KOK41#{1'M1#{88 64V / 112 ) % ) 1228 J 4$ support was evaluated based on 500 bootstrap replicates halophilic sensu)+et al. (2006). Strains DSM )03/ 1#{1LOK41#{1# /8#UKet [$ al. 12!2 poorly at 4 % salinity. The best growth rates among the tested Phialosimplex and Sagenomella was rooted with Eremascus conditions were in yeast malt submerged cultures with 15 % albus I!'8{"!Penicillium olsonii (JN121771) was used 1{0OK41#{82 as the outgroup in the initial Trichocomaceae tree. 2+9!!{M1{ VOLUME 5 · NO. 2 163 164 ARTICLE Table 3.6Phialosimplex spp. [data for P. caninusP. chlamydosporus, and P. sclerotialis from Sigler et al12!2Phialosimplex3/et al. (2012).] *Description of the synonymous taxa Scopulariopsis halophilica and Oospora halophila|OP Taxon Phialides Conidia Chlamydospores Sclerotia Growth Conidial Conidial width Conidial Soluble Habitat and Halotolerance lenght in heads presence presence at 35°C length [μm] [μm] shape pigment associations [μm] presence presence P. caninus 4.5–16 + + 2.2–4.0 !'M8# subglobose + & n/a Canidae (Canis lupus familiaris – zoopathogenous) P. chlamydosporus 8M!7 + + + 18M{' !#M82 / + Canidae n/a (Canis lupus familiaris – zoopathogenous) B. halophila* 8M12 n/a n/a 8{M"2 8{M'2 6 halophilic associated (Undaria [ ovoid P. salinarum 1.5–8.7 + + 2.8–4.0 2.8–4.0 Water in salt mine halophilic subglobose P. sclerotialis 5–15 + + 82M7{ 1.5–2.0 / Poaceae n/a truncate base (Lolium perenne – as Greiner fodder) P. sp. n/a n/a n/a n/a n/a n/a n/a n/a Deep sea water halophilic et al . OK41#{81U0K1 U0K! 1#{8!OK41#{88KOK41#{82 0 U0K OK4 1#{1LM1#{1"Molecular analysis OK4 of V 1#{82 OK41#{8!7+OK41#{1"OK4 OK4 1#{1# OK4OK41#{1LOK41#{81OK41#{88 1#{1' Decomposition ofstarchwasmoreheterogeneous.While $ 9 1 Substrate degradation temperature preferencesweresimilar. OK41#{826O$ 8{+"!{!11{ !{!#1{$1! 82+1!1! increasing growthratewithtemperaturesupto 4$!{1{+4$771! #2&K of / ; / U 0 7!22'7 7!22## obtained fromanarchaeologicalexcavationinChina salinarum P. U0K ; Samson (2011). Aspergillus ; Basipetospora halophila Phialosimplex 0 10 0K3! ; Phylogeny ; similarity 94%). halophila 0K3! ; "#" / !22 "{# 0 chlamydosporus P. ; /UJKO+| ; 0 ( ; U0K U0K ;BLAST search OK4 1#{1LM1#{88 to thegenus sclerotialis [ [ Phialosimplex salinarum Phialosimplex salinarum Phialosimplex Three species( 0 J!1!'!{'''/'8L *% ; Phialosimplex, Eurotiales P. caninus P. 9JLL#"78 / )&$)!L"81L Polypaecilum insolitum Phialosimplex chlamydosporus inaphylogenywithselected ) didnotexactlymatchany 9 7 0 . ) werepreviouslyassigned establishedby Sigler Phialosimplex Basipetospora IMA FUNGUS P. pisci P. et al. to P. Phialosimplex salinarum sp.nov. from a hypersaline habitat ARTICLE $`Q1 .^HI_ 1IV^R_ Fig. 1.)Phialosimplex salinarumOK41#{824$!{ 25 20 15 starch protein 10 cellulose Substrate depletion zone (mm) 5 0 4 C 15 C 25 C Fig. 2. 0Phialosimplex salinarum$/ H/OK41#{1LM1#{88 (2010). The eight strains of P. salinarum cluster together with The phylogenetically closest relatives to P. salinarum are four Trichocomaceae9{ Phialosimplex VOLUME 5 · NO. 2 165 Greiner et al. followed by P. chlamydosporus */ on vegetative hyphae. Phialides (monophialidic) discrete and isolated strains are separated from their closest relatives. mostly cylindrical to occasionally slightly broadened at the 9U0K;P. caninus cluster in a |!{M K;Sagenomella are distinguishable '{3!2M!{3 from Phialosimplex[ base to the tip. Conidia formed in long chains or occasionally ARTICLE described by Sigler et al.