A New Species of Melanospora on Truffles from China

Home , Ceratostomataceae, Ceratostoma (fungus), Heleococcum, Melanospora, Melanosporales, Myrothecium

Mycologia, 104(6), 2012, pp. 1433–1442. DOI: 10.3852/11-338 # 2012 by The Mycological Society of America, Lawrence, KS 66044-8897

A new species of Melanospora on truffles from China

Li Fan the Melanospora complex, and the nonsporulating Cheng-lin Hou1 Papulaspora was demonstrated to represent sterile College of Life Science, Capital Normal University, Melanospora ascomata by Davey et al. (2008). Xisanhuanbeilu 105, Haidian, Beijing 100048, China Melanospora and its relatives were placed tradition- Paul F. Cannon1 ally within the Ceratostomataceae (Sordariales) based CABI and Royal Botanic Gardens, Kew, Richmond, primarily on morphological parallels with the Chaeto- Surrey TW9 3AB, United Kingdom miaceae (e.g. Hawksworth et al. 1995). The first molecular phylogenetic studies (Rehner and Samuels Yu Li 1995, Jones and Blackwell 1998) indicated that Institute of Mycology, Jilin Agricultural University, Melanospora belonged within the Hypocreales clade. Changchun 130118, China Zhang and Blackwell (2002) and Chaudhary et al. (2006) considered the Melanospora clade to be a sister group of the Hypocreales and found that sequences Abstract: The new species Melanospora subterranea is derived from other genera placed in the Ceratostoma- described from China, based on morphological and phylogenetic analyses. This is the first record of taceae (syn. Melanosporaceae) clustered within the main Melanospora species parasitizing Chinese black and Hypocreales clade. Zhang et al. (2006) concluded that white truffles (Tuber indicum and T. huidongense), the family belonged to the Hypocreomycetidae (i.e. the and its host range indicates that the new species clade containing the Coronophorales, Glomerellales, Halo- might be a disease threat to commercially exploited sphaeriales, Hypocreales, Microascales) but that it was a European truffles including Tuber melanosporum and sister group of the Coronophorales and merited recogni- Tuber magnatum. Apparent polyphyly within the tion as the new order Melanosporales.Theorderwas Ceratostomataceae can be explained at least in part published formally by Hibbett et al. (2007). A similar by inadvertent sequencing of the host fungus instead placement was proposed by Schoch et al. (2007) in a of the parasite. study of marine fungal lineages within the Hypocreomy- Key words: fungal parasitism, phylogeny, truffles cetidae. However, the apparent paraphyly of the order Melanosporales to date has not been addressed. During our studies on the genus Tuber (i.e. the true INTRODUCTION truffles) in China, an interesting microfungus be- The Melanospora complex, a distinctive group of longing to the genus Melanospora was found within microfungi with translucent ascomata, was revised by ascomata of T. indicum (the Chinese black truffle) Cannon and Hawksworth (1982) based primarily on when we cut the fruit body into slices, with the data obtained by SEM examination of the ascospores. parasitized ascoma showing densely crowded pin-like They adopted relatively narrow generic concepts, black dots that occupied nearly all the internal space accepting Sphaerodes as emended by von Arx (1981), of the tissues. The same fungus also was found in and erected new genera including Persiciospora with ascomata of T. huidongense, an endemic species of pitted ascospores. Cannon and Hawksworth (1982) Chinese white truffle. arranged the species with smooth-walled, lemon- shaped to ellipsoidal or ellipsoidal-citriform asco- MATERIALS AND METHODS spores and sunken germ pores in Melanospora, while those with reticulate ornamentation on the ascospore Morphological studies.—Macroscopic characters were de- surface and raised tubercle-like germ pores were scribed both from fresh and rehydrated dried specimens placed in Sphaerodes. Further species were added to and microscopic characters from razorblade sections the complex by Garcı´a et al. (2002, 2004), Hawks- mounted in 3% KOH (w/v), Melzer’s reagent and 0.1% (w/v) cotton blue in lactic acid. For scanning electron worth et al. (1999), Krug (1988), Nitzan et al. (2004), microscopy (SEM), spores were scraped from the dried host Stchigel et al. (1996, 1999) and Vujanovic and Goh gleba onto doubled-sided tape, which was mounted directly (2009). Chaudhary et al. (2006) published the new on an SEM stub and coated with gold/palladium. The genus Vittatispora from India, which also belongs to treated materials were examined and photographed with a Hitachi S-4800 SEM. Submitted 13 Oct 2011; accepted for publication 2 May 2012. 1 Corresponding authors. E-mail: [email protected] & houchenglincn Molecular methods.—Total genomic DNA was extracted @yahoo.com from ascomata with PeqLab E.Z.N.A._Fungal DNA kit

1433 1434 MYCOLOGIA following the manufacturer’s protocol after the samples support was found for clades containing Persiciospora were crushed by shaking for 3 min at 30 Hz (Mixer Mill MM and Fusarium (Gibberella) species, Syspastospora para- 301; Retsch, Haan, Germany) in a 1.5 mL tube together with sitica and Isaria farinosa, and Sphaerodes retispora and one 3 mm diam tungsten carbide ball. The large subunit Trichoderma harzianum. Scopinella solani clustered (LSU) region of the nuclear ribosomal DNA (rDNA) was weakly with species of Myrothecium, and a further well amplified with PCR with primers LROR/LR5 (White et al. supported clade (Clade 1 in FIG.1)contained 1990). PCR was performed in 50 mL reactions containing DNA template 4 mL, primer (10 mM) 2 mL each, 2 3 Master sequences derived from Melanospora species but with Mix (Tiangen Biotech Co. Ltd., Beijing) 25 mL. PCR only weak support for a sister group containing reactions were run as follows: initial denaturation at 95 C Cosmospora species. for 4 min, followed by 32 cycles at 94 C for 1 min, 58 C for 1 min, 72 C for 1 min and a final extension at 72 C for TAXONOMY 10 min. The PCR products were sent to Invitrogen Biotechnology Co. Ltd. (Beijing) for purifying, sequencing Melanospora subterranea L. Fan, C.L. Hou, P.F. and editing. The other sequence data of LSU rDNA Cannon & Y. Li, sp. nov. FIGS. 2–7 included in this study were downloaded from GenBank MycoBank MB563464 (TABLE I). Ascomata globosa, immersa, 200–250 mm diam, non- Phylogenetic analyses.—DNA sequences were aligned with ostiolata, leviter vel opace fulva, perlucida; peridium MUSCLE (Edgar 2004), within the MEGA5 phylogenetic membranosum, ex cellis fuscis, perlucidis (5–)7.5–15 mm software package (Tamura et al. 2011). The aligned dataset diam. Asci clavati, 45–60 3 15–20 mm, 2-spori. Ascosporae was analyzed with maximum parsimony (MP) and maxi- 15–22.5 3 10–15(–17.5) mm, ellipsoideae-citriformes, laeves. mum likelihood (ML) analyses, also within the MEGA5 Type: CHINA, YUNNAN Province, Kunming, in package, with heuristic searches using close neighbor tissue of Tuber indicum, 23 Oct 2007, Jin-Zhong Cao interchange (CNI) on 10 initial random trees. Gaps and 121, FAN1001 (BJTC holotype, K[M] 172128 isotype). missing data were taken into consideration where . 95% Other collections examined: CHINA, YUNNAN, Baoshan, in unambiguity was encountered. One thousand bootstrap tissue of Tuber huidongense, 15 Dec 2008, Li Fan, FAN1002 replications were used. A sequence derived from Chaeto- (BJTC). sphaerella phaeostroma (AY695264) was used as outgroup. Etymology: referring to the environment of the host species. RESULTS Stromata absent. Ascomata growing in the internal tissues of Tuber ascomata, 200–250 mm diam, solitary Phylogenetic analyses.—A total of 250 characters out or gregarious, immersed, globose, cleistothecial, of 864 were found to be parsimony informative. translucent, light brown to mid brown, appearing Maximum parsimony analysis resulted in one of 20 black when the ascospores are mature. Peridium most parsimonious trees with a length (TL) of 652 membranous, composed of hyaline or light brown, steps, consistency index (CI) of 0.619632, retention translucent, polygonal to irregular pseudoparenchy- index (RI) of 0.890845 and rescaled consistency matous cells (5–)7.5–15 mm diam. Interascal tissues index (RCI) of 0.551996 (for all sites). The nrLSU absent. Asci clavate, 45–60 3 15–20 mm, thin-walled, matric is available at www.treebase (http://purl.org/ short-stalked, without apical structures, evanescent, phylo/treebase/phylows/study/TB2:S12403) 2-spored. Ascospores 15–22.5 3 10–15(–17.5) mm, The phylogenetic analysis (FIG. 1) revealed two ellipsoidal-citriform, both ends slightly apiculate, strongly supported major clades representing the guttulate and dark brown to near black at maturity, Hypocreales and the Melanosporales. The latter clade smooth-walled, with two indistinct apical germ pores. was represented by sequences from 12 taxa, with Anamorph not seen. Vittatispora coorgii included as a basal lineage within the Melanosporales, and two strongly supported DISCUSSION subclades containing four and five sequences respectively. These subclades did not appear to correlate The new species described here, with its translucent exactly with taxa as defined with morphological ascocarps and smooth-walled ellipsoidal-limoniform methods. The new species, M. subterranea, clearly spores, clearly belongs to Melanospora, and the LSU could be demonstrated as belonging within the rDNA analysis (FIG. 1) confirms this placement. Two Melanosporales clade but did not definitely cluster similar species of that genus have cleistothecial within a particular subclade. ascomata (TABLE II). Microthecium geoporae (Oberm.) Sequences derived from taxa considered as belong- Ho¨hn. (basionym Guttularia geoporae Oberm.) has ing to the Melanosporales based on morphological not been transferred formally to Melanospora. It has analysis also were found scattered throughout the much larger ascospores than those of M. subterranea, main Hypocreales clade (shaded in FIG. 1). Strong with a larger length/breadth ratio. M. zobelii appears FAN ET AL.: MELANOSPORA ON TRUFFLES 1435

TABLE I. Sources of sequences for molecular analysis

Species Voucher specimen/culture LSU

Melanospora subterranea BJTC FAN 1001 JN247804 Akanthomyces novoguineensis BCC 01423 GQ249972 Bertia moriformis SMH 5169 FJ968951 Chaetosphaerella phaeostroma SMH 4257 AY695264 Cordyceps militaris OSC 93623 AY184966 Cosmospora coccinea CBS 114050 FJ474079 Cosmospora episphaeria GJS 88-29 AY015625 Cosmospora vilior GJS96-186 AY015626 Cosmospora wegeliniana GJS 93-15 GQ506006 Fusarium oxysporum f. cubense ATCC 96285 EF590327 Fusarium polyphialidicum PP 94 FJ890384 Fusarium proliferatum PP 74 FJ890385 Gibberella sp. CBS 122679 EU552131 Isaria farinosa OSC 111005 DQ518773 Isaria farinosa OSC 111006 EF469080 Melanospora brevirostris ATCC 42427 AY015627 Melanospora fallax CBS 478.75 U17404 Melanospora fallax CBS 478.75 U47834 Melanospora singaporensis ATCC 38286 AY015629 Melanospora tiffanyae ATCC 15515 AY015630 Melanospora tiffanyae SMCD 2222 FJ748915 Melanospora zamiae ATCC 12340 AY046579 Melanospora zamiae ATCC 12340 U17405 Melanospora sp. 795 3 N01 AB470552 Myrothecium inundatum IMI 158855 AY489731 Myrothecium roridum ATCC 16297 AY489708 Papulaspora sepedonioides UAMH 10707 EU518666 Persiciospora africana ATCC 64691 AY015631 Persiciospora sp. BR439 FJ358495 Scopinella solani CBS 770.84 AY015632 Sphaerodes compressa IMI 212200 AY015633 Sphaerodes fimicola ATCC 26180 AY015628 Sphaerodes mycoparasitica SMCD 2220 FJ748916 Sphaerodes quadrangularis CBS 112764 GQ354530 Sphaerodes retispora CBS 994.72 GU205261 Syspastospora parasitica IMI 255607 AY015634 Trichoderma harzianum CBS 226.95 HM466680 Trichoderma harzianum PP 36 FJ890421 Vittatispora coorgii BICC 7817 DQ017375 to be broadly similar in most respects but has preted M. zobelii, describing instead a fungus now bilaterally flattened ascospores and as far as we can known as Melanospora tulasnei (Udagawa and Cain tell does not parasitize Tuber species. The type of M. 1969, Hawksworth and Udagawa 1977). In the light of zobelii was not indicated by Corda (1842), and these confusions and the observed difference in spore although the species was described briefly by Uda- shape between Melanospora subterranea and M. gawa and Cain (1969) there was no indication that zobelii, we think it is reasonable to accept M. they had seen the type. Even if authentic material subterranea as a new species rather than resurrect an exists it would be unlikely to be sequenced success- old species name of doubtful application. fully due to its age. Corda specified that the host Both genus and species concepts in the Melanos- fungus of M. zobelii was Rhizopogon albus, which in porales need further investigation. Ascospore orna- Corda’s sense according to Paoletti (1889), is a mentation has not been shown to be a good indicator synonym of Choiromyces meandriformis,afungus of phylogeny (Zhang and Blackwell 2002). Molecular belonging to the Ascomycota instead of the Basidio- data (Clade 2, FIG. 1) suggest that the main mycota. Most authors subsequent to Corda misinter- Melanospora/Sphaerodes clade contains at least two 1436 MYCOLOGIA

FIG. 1. Phylogenetic tree showing apparent polyphyly in the Ceratostomataceae (shaded gray). MP/ML bootstrap values are given above 50%. The sequences of Melanospora fallax (triangle) and M. zamiae (star) are derived from the same isolates.

well defined subclades that may merit generic status farinosa and Fusarium spp.) and it is likely that the but which do not appear to correlate strongly with sequences are derived from mixed samples. Sphaer- morphological characteristics of ascospores. odes retispora is not definitely known to be a fungal Based on available LSU sequences, it would appear parasite, but its clustering with a Trichoderma species at first glance that the Ceratostomataceae is highly that is itself mycoparasitic suggests that the culture polyphyletic. However, published sequences of Sy- that was sequenced might have been contaminated. spastospora parasitica and Persiciospora spp. cluster Not all the seemingly aberrant clades can be closely with their host fungi (respectively Isaria explained in this manner. Four sequences identified FAN ET AL.: MELANOSPORA ON TRUFFLES 1437

FIGS. 2–7. Melanospora subterranea (BJTC FAN1001, holotype). 2. Vertical section of Tuber indicum ascocarp colonized by Melanospora subterranea. 3. Ascomata in the ascocarp tissue of T. indicum. 4. Ascomata of M. subterranea in section, several asci and ascospores of T. indicum may also be seen. 5. Ascoma, asci and ascospores of M. subterranea observed with light microscope. 6. Asci and ascospores of M. subterranea observed with light microscope. 7. Ascospores of M. subterranea observed with scanning electronic microscope. as Melanospora species form a weakly supported sister Clade 1 are derived from host species comprising a so clade (Clade 1, FIG.1)to Cosmospora (Hypocreales, far unrecognized clade of the Hypocreales. Nectriaceae), a position that led to the former To date, host/parasite relations have not been placement of the Ceratostomataceae within the Hypo- correlated strongly with classification in Melanospora creales (Rehner and Samuels 1995). BLAST queries and its relatives. Some species (notably M. zamiae) using these sequences do not result in close matches grow easily in pure culture and frequently are isolated with possible host species, and they do not appear to from sources that are not recognized as fungal. be chimeric, but there are indications that some at Jordan and Barnett (1978) found strains of M. zamiae least are faulty. The two M. fallax sequences differ by displayed symptoms of pathogenicity when chal- five of 823 base pairs but are derived from the same lenged with a range of different fungi, but parasitism culture CBS 478.75. The culture could be genetically was defined only by production of ‘‘hook’’ or ‘‘contact’’ heterogeneous, but sequencing problems also could cells, and the effect on the host was not measured. Three account for the discrepancies. Potential sequencing species of Ceratostomataceae were investigated by Harve- errors frequently are ignored when using published son and Kimbrough (2001) for their pathogenic ability sequences as comparators but may be significant on a series of strains of Fusarium oxysporum,butwhile especially with those generated by early-generation strains of M. zamiae and Sphaerodes retispora caused equipment; see Cannon et al. (2008) for a further significant growth inhibition a strain of Persiciospora example. More significantly, M. zamiae U17405 and moreaui grew actively and sporulated in intimate AY046579 differ in no less than 180 of 853 base pairs association with F. oxysporum,withhealthofthehost and cluster in different clades but also are derived fungus unaffected. They concluded that P. moreaui is a from the same strain, in this case ATCC 12340. The mutualist (or at least a biotrophic) parasite, and it discrepancies are scattered throughout the sequence, therefore is likely to have coevolved with its host fungus. and it is difficult to conclude that they are the result It therefore seems realistic to postulate that some species of technical failings in the sequencing process. at least of Ceratostomataceae are host specific and that Further studies are necessary, but the most likely their ability to grow saprobically in pure culture in some scenario at present seems to be that the sequences in cases may mask a specialized host/parasite relationship. 48M 1438

TABLE II. Species of Melanospora possessing smooth ascospores with simple germ pores

Species Host and distribution Ascoma type Ascomata Asci Ascospore dimensions Ascospore shape Reference brevirostris on Geopora spp. and dead perithecium 125–400mm, short- 8-spored 21–34 3 11–17 mm ellipsoidal–limoniform Cannon and plant materials, widely necked, 6 glabrous Hawksworth distributed (1982) caprina on decaying fungi including perithecium 600–800 mm diam, 8-spored 16–23 3 9–16 mm ellipsoidal to Cannon and Stereum spp., Tomentella white-tomentose, limoniform Hawksworth tristisand Armillaria mellea long-necked (1982) agg., widely distributed chionea on fallen leaves and cones perithecium 200–400(–500) mm 8-spored 7.5–16 3 6–12 ellipsoidal, Cannon and of Pinus spp., also with diam, white- 3 4–7 mm discoid Hawksworth decayed Stereum sp., tomentose, (1982) common in N Europe long-necked damnosa parasitic on Fusarium spp., perithecium 150–400 mm diam, 8-spored 18–25 3 10–14 mm limoniform to Cannon and collected from cereal and sparsely tomentose, rhomboid-ellipsoidal Hawksworth potato debris, widely short-necked (1982) distributed fallax parasitic on Botrytis acinorum , perithecium 250–600 mm diam, 8-spored 21–34 3 11–17 mm ellipsoidal-limoniform, Cannon and also reported from plant bulbs, short-necked, often inaequilateral Hawksworth YCOLOGIA dung and seed, widespread in accompanied (1982) temperate regions by bulbils fimbriata on dung and isol. ex soil, perithecium 45–50 mm, unknown 18–22 3 9–11 mm limoniform, often Cannon and Greenland and Canada short-necked, inaequilateral Hawksworth glabrous (1982) fusispora parasitic on Isaria farinosa perithecium 200–300 mm diam, 8-spored 20–25 3 7-12 mm fusiform to ellipsoidal Cannon and and Beauveria bassiana glabrous, (to limoniform) Hawksworth short-necked (1982) geoporae on Geopora cooperi , Germany, cleistothecium 250–350 mm unknown 28–30 3 12–16 mm limoniform Udagawa and (Microthecium ) Sweden & USA diam, glabrous Cain (1969) lagenaria on decaying polypores includ- perithecium 600–800 mm diam, 8-spored 12–22 3 7–14 mm ellipsoidal to Cannon and ing Bjerkandera , Piptoporus , glabrous, limoniform Hawksworth Polyporus and Trametes spp. long-necked (1982) longisetosa ? parasitic on Nectria perithecium 250–400 mm diam, 4-spored 14–29 3 12–14 broadly ellipsoidal, Cannon and cinnabarina, also recorded glabrous, short- 3 8–9 mm bilaterally flattened Hawksworth from decaying leaves, UK stalked but with (1982) long coronal setae pegleri on fallen leaves of Laurus perithecium 110–130 mm diam, 8-spored (9.5–)11–12(–13) ellipsoidal to Hawksworth nobilis, Camellia and glabrous, lacking 3 4–4.5 mm plano-convex et al. (1999) Rhododendron spp.,UK a neck subterranea parasitic on Tuber indicum cleistothecium 200–250 mm diam, 2-spored 15–22.5 3 10–15 ellipsoidal to This paper and T.huidongense , China glabrous (–17.5) mm limoniform TABLE II. Continued

Species Host and distribution Ascoma type Ascomata Asci Ascospore dimensions Ascospore shape Reference

from rotting leaves perithecium 200–250 mm diam, 4-spored 25–33 3 15–17 mm ellipsoidal to ovoid, Udagawa and F theleboloides AL ET AN short-necked, asymmetrical, often Cain (1969) glabrous tapered at both ends tulasnei on Hydnocystis arenaria a perithecium 180–330 mm diam, 8-spored 21–25 3 12–16 mm limoniform, Udagawa and short-necked, rarely flattened on Cain (1969) .: M

glabrous one side ELANOSPORA washingtonensis on potato tubers perithecium 115–300 mm diam, 8-spored 10.5–12 (–13.5) 3 widely ellipsoidal Nitzan et al. long-necked, 9–10.5 (–12) 3 (2004) glabrous 7–9 mm zamiae on rotting vegetation, potentially perithecium 150–300 (–400) mm 4- or 8- 15–23 3 10–16 mm ellipsoidal to Cannon and

parasitic on a wide range diam, glabrous, spored limoniform Hawksworth TRUFFLES ON of fungi, very widespread short-necked (1982) zobelii on Choiromyces maeandriformis , cleistothecium small, globose unknown (18–)20–23(–25) 3 limoniform Hawksworth Czech Republicb (8.5–)10–12 3 and Udagawa 6–9 mm (1977)

a Placed into synonymy with M. brevirostris by Cannon and Hawksworth (1982); also reported from hymenia of Lachnea (probably Humaria sp.) and Sepultaria (5 Geopora ) sp. (Udagawa and Cain 1969). b Also reported from Coriolus flabelliformis (Papua New Guinea) and soil (Japan) (Hawksworth and Udagawa 1977). 1439 1440 MYCOLOGIA

Melanospora subterranea is known to be a parasite of leptobactrum cannot be regarded as a definite two Tuber species, T. indicum and T. huidongense. parasite of Tuber melanosporum. Melanospora sub- Thegrowthanddevelopmentofbothasciand terranea thus seems to constitute the first unequivo- ascospores of T. indicum have been greatly reduced cal record of a fungal disease of truffle species that due to colonization by M. subterranea (FIG. 3) and it is are marketed commercially. clear that the Tuber ascomata will lose their commer- T. indicum and T. huidongense belong to separate cial value after colonization. clades within Tuber (Bonito et al. 2010a, Chen et al. Commercially exploited truffles sometimes are 2011, Kinoshita et al. 2011), and so their parasite contaminated by bacteria or yeasts (Rivera et al. might be expected to have a wide host range within 2010) and truffles other than Tuber species are known the genus. T. indicum now is established in truffle to be hosts for other fungi, including several species orchards in both Europe and North America (Murat of Ceratostomataceae (Cannon and Hawksworth 1982, et al. 2008, Bonito et al. 2010b) and is considered Nikoh and Fukatsu 2000). There are few records of invasive and a potential threat to cultivation of the filamentous fungi directly associated with the asco- European black truffle T. melanosporum. Melanospora mata of Tuber species (Gams et al. 2004). Battarrina species and their relatives (Jordan and Barnett 1978, inclusa was described from ascomata of Tuber Harveson and Kimbrough 2001, Goh and Vujanovic puberulum in the UK, a cleistothecial fungus with 2010) can grow saprobically in soil and other natural cylindrical evanescent asci and small pale yellow substrata, especially in the presence of the host ascospores that to date has been placed within the fungus. Further introductions of T. indicum in the Bionectriaceae (Hypocreales; Hawker 1955, Rossman et form of mycorrhizal inoculants into regions outside al. 1999, Thoen and Schultheis 2005). Examination of its natural range therefore may pose the threat of authentic material shows that its ascospores are establishment of the new Melanospora species as a winged instead of spinose as claimed by Hawker and parasite of the highly prized European species T. Rossman and colleagues, and the genus may be melanosporum and T. magnatum. Its presence might related to Emericellopsis and Heleococcum. However, not be detected as a matter of routine, because neither of these genera is known to be mycoparasitic, infected truffle fruit bodies do not show external although H. aurantiacum has been collected from symptoms and normally are not examined internally mushroom compost (Udagawa et al. 1995, Zuccaro before sale. Biologically active metabolites have not et al. 2004). On the other hand, Battarrina may been adequately studied in Melanospora species, represent an earlier name for the genus Vittatispora, although cultural filtrates have been reported as an apparently basal member of the Melanosporales poisonous to nematodes (Zhu et al. 2008). Further clade that also has small flanged ascospores (Chaudh- evaluation of the commercial and health risks posed ary et al. 2006). Nodulisporium tuberi was isolated from by the parasite is advisable. ascomata of Tuber maculatum (Fontana and Bonfante 1971, de Hoog 1974); its ecological role is unknown. The only recent survey of fungi intimately associat- ACKNOWLEDGMENTS ed with Tuber species was published by Pacioni et al. The study was supported by NSFC (Nos. 30870008, (2007). Here seven different species of fungi were 31170019) and PHR (KZ201110028036). Bryn Dentinger isolated as endobionts from healthy ascomata of seven (Royal Botanic Gardens, Kew) is thanked for advice Tuber species. 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