A Newly Recorded Endophytic Preussia Species in China

MA Yu，KE Yang，QIANG Yi，LI Bo*

(1.Shaanxi Microbiol.Inst.，Shaanxi Acad.of Sci.，Xi＇an 710043;2.Inst.of Life Sci.，Shaanxi Normal Uni.，Xi＇an 710062)

The genus of Preussia Fckl.is closely related to Sporormia and is a member of the family Sporormiaceae，Loculoascomycetes.This genus was erected in 1866 with Preussia funiculata(Preuss)Fuckel as its type species(Cain，1961)［1］.It was initially characterized by non-ostiolate ascomata with a thin peridium without any definite cleavage area，broadly clavate，8-spored，short-to long-stipitate，irregulary disposed asci with a crozier at the base and dark brown ascospores with readily sparable transverse septa and the elongated germ-slit extending over the full length of each cell.

At present，approximately 94 species including both coprophilous and terricolous taxa have been described(http://www.indexfungorum.org/names/Names.asp)，with some being collected from plant materials from subtropical regions of the world(http://www.discoverlife.org/).In a survey of endophytic fungi associated with Chinese traditional medicinal plants in Qinling Mountain，2010 ～ 2012，Preussia dakotensis was recovered，which is reported as a new record in China herein.The morphological characteristics of the species are described and illustrated as follows.

1 Materials and methods

All strains were isolated from a Chinese medicinal plant Isodon rubescens(Hemsl.)Hara(Lamiaceae)collected in Funiu Mountain，a southern branch of Qinling Mountain in Henan Province.The fungus was grown on MEA agar and produced mature ascocarps in 14 or 15 days.The genomic DNA of the specimen was extracted from fresh cultures following the protocol of Guo et al.(2000)［2］and Wang et al［3］.The PCR amplification，DNA sequencing and subsequent analysis following the protocol of Liu et al.(2007)［4］.Morphological characteristics were examined and photographed by optical microscope(Olympus BX-41 microscope).Measurements were made from distilled water mount.The voucher specimens IR599 have been deposited at the Microbiology Institute of Shaanxi，Xi＇an，China.

2 Result

2.1 Taxonomy

Preussia dakotensis(Griffiths)Valldos.＆Guarro，Boln Soc.Micol.Madrid 14:85.1990.

=Sporormia dakotensis Griffiths，Mem.Torrey Bot.Club 11:114.1901.

=Sporprmiella dakotensis(Griffiths)S.I.Ahmed ＆ Cain，Can.J.Bot.50:439.1972

Colony was white to gray，thin，appressed，with sparse aerial hyphae，developing a slimy appearance due to turning dark with ascocarp production.Hyphae hyaline，branching，up to 6 μm in diameter，anastomosing frequently in region of spermogonia and ascocarps.Ascocarps scattered or aggregated in small groups，immersed，dark brown，obpyriform，(450～900)μm ×(300～350)μm，glabrous.Neck black，cylindrical，bare.Peridium dark brown，pseudoparenchymatous，membranous.Asci 8-spored，clavate，(70～86)μm ×(8～12)μm，rounded above，tapering gradually from the broadest part near the apex into a long stipe.Paraphyses hyaline，filiform，ventricose，measuring 3 ～4 μm in diameter.Ascospores biseriate，4-celled，olive to dark brown，cylindrical，(22～28)μm ×(3～5)μm，rounded at the ends，septa transverse，constrictions at septa broad and deep，segments easily separable;all cells nearly equal in length，surrounded by a narrow hyaline gelationous sheath.Germ slit parallel.

Preussia dakotensis was firstly discovered in Madrid(Valldos et al.，1990)［5］，and as a coprophilous species in world wide(Valldos et al.，1990;Chang et al.，2009)［5-6］，but has no occurrence reported as an endophytic and in China mainland so far.The morphological characteristics of the strain IR599 isolated in this study are similar to those originally described by Valldos(1990)and，there was no other difference observed but the respect to the substrate was plant materials.This species is similar to Preussia kansensis(Griffiths)by 4-celled ascospores with strictly parallel germ slits but those of the latter are much bigger(57～77)μm ×(9～12.5)μm(Chang et al.，2009)［6］.

Fig.1 Preussia dakotensis IR599

2.2 Molecular phylogenetics

The ITS region sequence(ITS1，partial sequence;5.8S rRNA gene，complete sequence;and ITS2，partial sequence)of IR599 was 542 bp(NCBI Accession number:KF002732).By the phylogenetic analyses of the species belong to Sporormiaceae published on GenBank，the strain IR599 shared 99%identity to the genus of Preussia(Fig.2).

Fig.2 The N-J Phylogenetic tree of strain IR599

3 Discusion

The classification of many groups of Fungi has traditionally been based on a few easily observed characteristics，which often reflect the aspects of their ecology and thus may be subject to parallel evolution or extreme specialization in highly derived groups.Sporormia，Sporormiella and Preussia are considered closely related and are easily confused by their shared morphological features in the Sporomiacea.Sporormia is characterized by globose pseudothecia，which open with an ostiole.The≥16-celled spores are typically joined together in a bundle with one common gelatinous sheath，when released from the ascus(Ahmed ＆ Cain，1972)［7］.Sporormiella the most species-rich genus in the family includes species with perithecioid ascomata and≥4-celled spores with germ slits.Preussia differs morphologically from 4-celled Sporormiella species only by the ascomata being cleistothecioid(Cain，1961［1］;Barr，2000［8］).

Substrate choice has been another important diagnostic character for separating Preussia and Sporormiella utilized by von Arx and van der Aa(1987)［9］.Preussia，in their sense，includes species on plant debris，wood or soil［10-12］，while Sporormiella is restriced to coprophilous species with peritheciold ascomata.Doveri(2004)［13］also considered the preference for a certain substrate an important feature，together with the shape of the ascus where Sporormiella should have cylindric or cylindric-claviform asci and Preussia more or less clavate asci.He also believed that Preussia in general have more superficial ascomata.

In summary，the morphological characteristics of the strain IR599 isolated in this study are identical with Preussia dakotensis(Griffiths)Valldos.＆Guarro and in conformity with the above mentioned distinctions between Preussia and Sporormiella.The molecular phylogeny further proved that strain IR353 was Preussia dakotensis.

Acknowledgements:The authors would like to thank Dr.SU Yuan-ying at the Systematic Mycology＆ Lichenology Laboratory，Institute of Microbiology，Chinese Academy of Sciences for giving her opinions about the fungus and reviewing.

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