FORESTRY IDEAS, 2016, vol. 22, No 2 (52): 113-126 NEW RECORDS AND DATA ON HYPOGEOUS ECTOMYCORRHIZAL FUNGI IN BULGARIA
Teodor Nedelin1*, Melania Gyosheva2, Kaloyan Kostov3, and Slavcho Savev1 1Department of Silviculture, Faculty of Forestry, University of Forestry, 10 St. Kliment Ohridski Blvd., 1797 Sofia, Bulgaria. *E-mail: [email protected] 2Department of Plant and Fungal Diversity and Resources, Institute of Biodiversity and Ecosystem Research, BAS, 23 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria 3AgroBioInstitute, Agricultural Academy, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria
Received: 05 June 2016 Accepted: 26 July 2016
Abstract In this article, new data on species diversity of hypogeous macrofungi in Bulgaria are re- ported. Three species: Tuber rufum, Gautieria graveolens and Lactarius stephensii were recorded for first time in the country. New localities of noteworthy fungi Tuber excavatum and Gautieria morchelliformis are also reported. A DNA sequencing for most studied hypogeous fungi and de- tailed phylogenetic analysis for T. excavatum were performed. Results confirmed that representa- tives of T. excavatum group have a very high intraspecific genetic variability. Key words: ascomycetes, basidiomycetes, Bulgaria, fungal conservation, hypogeous fungi, phylogenetic analysis.
Introduction ten reported in present studies as one of the main components of ectomycorrhizal Almost all of hypogeous macrofungi (as- associations in various forest ecosystems comycetes and basidiomycetes) are ec- under particular ecological conditions (Be- tomycorrhizal species, living in symbiotic nucci et al. 2011). Truffles generally grow relationship with roots of trees. Because in calcareous soils, but some of them are of their cryptic, subterranean fruit bodies, well adapted to slightly acidic soils (Jail- they are often overlooked and neglected lard et al. 2014, Pacioni and Comandini in different studies until now, when a rap- 1999). Most of them have a relatively idly development of molecular tools takes wide plant partners range and also grow- main part of scientific research. The de- ing conditions and only a few number is sign and construction of specific prim- restricted to particular ones. ers can be used as an easier and more The Balkan Peninsula and the western sensitive tool for the identification of truf- parts is still insufficiently explored regard- fle species in any stage of their life cycle, ing hypogeous fungi, including ’true‘ truf- including the mycorrhizal phase (Bertini et fles of the genus Tuber (Gyosheva et al. al. 1999). 2012a, Marjanović et al. 2010, Dimitrova Many hypogeous fungi, for instance and Gyosheva 2008). truffles, are pioneer species. They are of- Geographic position of Bulgaria and 114 T. Nedelin, M. Gyosheva, K. Kostov, and S. Savev respectively the great diversity of ecologi- tum and Gautieria morchelliformis Vittad cal conditions let to assume that the large are also presented. Most of fungal speci- number of hypogeous ectomycorrhizal mens are DNA sequenced. Phylogenetic fungi could be found in the country. We analyses have been done for T. excava- can expect slightly genotype difference for tum and T. fulgens Quél. Recent molecu- some known species because of particu- lar studies have shed some light on com- lar conditions in a process of adaptation to plex diversity among closely related Tuber their plant partners. taxa. Molecular and phylogenetic stud- Eleven hypogeous ascomycetes (truf- ies on well-known species T. excavatum fles and truffles-like fungi) have been are insufficient until now (Alvarado et al. published until now from Bulgaria: Choi- 2012; Bonito et al. 2010a,b,c; Zampieri et romyces meandriformis Vittad., Elapho- al. 2009; Chen and Liu 2007; Wang et al. myces granulatus Fr., Hydnotrya cerebri- 2006, etc.). formis Harkn., H. tulasnei (Berk.) Berk. & Broome, Stephensia bombycina (Vittad.) Tul., Tuber aestivum Vittad., T. brumale Materials and Methods Vittad., T. borchii Vittad., T. excavatum Vittad., T. macrosporum Vittad., T. pu- The investigated specimens were har- berulum Berk. & Broome (Dimitrova and vested in several localities in Bulgaria in Gyosheva 2008, 2009; Gyosheva et al. 2014–2015. Some information concern- 2012a,b; Lacheva 2012; Peev et al. 2015). ing their habitats was recorded – habitat There is a great species diversity of type, nearby trees, soil structure and type larger basidiomycetes in Bulgaria and (where data are available). One perma- up-to-date more than 1500 species are nent plot (3×3 m) in the area near Gradets recorded (Denchev and Assyov 2010). village was chosen on a productive site Some of them from genera Gautieria Vit- for periodical observation and ecological tad., Hymenogaster Vittad., Macowanites studies (at least 1 time per month from Kalchbr., Melanogaster Corda, Octaviania September to April). Vittad. and Rhizopogon Fr. are hypogeous Morphological observation of main and ectomycorrhizal. character features of fungal specimens Hypogeous macrofungi are with high was studied on fresh specimens using conservation value. Ten species (5 asco- Carl Zeiss Jena Citoval stereo micro- mycetes and 5 basidiomycetes) were in- scope. Micromorphological characters cluded in the Red List of Fungi in Bulgaria were observed under Amplival, Leitz (Gyosheva et al. 2006) and also in the Laborlux Phase-contrast microscope and Red Data Book of the Republic of Bulgar- Zeiss light microscope. As mounting me- ia (Peev et al. 2015). All species are from dium deionized water was used. For bet- categories Critically Endangered (CR) – 1 ter observation and for important diagnos- and Endangered (EN) – 9. ing chemical reactions some colorizing This paper presents three species reagents were used – KOH, Congo Red of hypogeous fungi: Tuber rufum Picco, in ammonia solution, Cotton Blue in lac- Gautieria graveolens Vittad. and Lactarius tophenol, and Melzer’s reagent. The mi- stephensii (Berk.) Verbeken and Walleyn, crophotographs were taken by AmScope recorded for first time in Bulgaria. New MA1000, 10 MP× camera. Measurement data for the distribution of Tuber excava- of asci, spores and peridium were per- New Records and Data on Hypogeous Ectomycorrhizal Fungi in Bulgaria 115 formed by Piximetre 5.8 software (http:// lyzed for best fit model – lowest-scoring ach.log.free.fr/Piximetre/). maximum likelihood tree. To obtain this, Scanning electron microscopy (SEM) several tests were used: hierarchical like- was carried out at the Institute of Physi- lihood ratio test (hLRT), BIC test (Bayes- cal Chemistry, Bulgarian Academy of ian information criterion) – BIC = -2ln(L) + Sciences. Specimens were examined on Kln(n), where ln(L) = loglikelihood of the JSM-5510 scanning electron microscope best tree found, K = number of parameters at 10 kV. and AIC Model test – AIC = -2ln(L)+2K, All studied specimens are kept at the where ln(L) = loglikelihood of the best tree Mycological Collection of the Institute of found and K = number of parameters. Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia (SOMF). Results and Discussion The nomenclature of hypogeous fungi follows Kirk et al. (2001). The author’s abbreviations of fungi are according to New records of hypogeous Kirk and Ansell (2004). Hypogeous fungi macrofungi for Bulgaria were identified following Riousset et al. Ascomycota (2012), Dissing (2000), Montecchi and Pezizales Sarasini (2000), Montecchi and Lazzari Tuberaceae (1993), Pegler et al. (1993), Breitenbach Tuber rufum Picco, Meleth. Bot.: 80 and Kränzlin (1984), Trappe (1979), Den- (1788) nis (1978) and Moser (1963). The threat Ascomata hypogeous or semihypo- status follows the Red List of Fungi in Bul- geous, irregular, lobed and very rare garia (Gyosheva et al. 2006). subglobose (Fig. 1a), 1.5–4 cm in diam- For DNA analysis was used only prop- eter, yellow-brown to reddish-brown, fine erly dried material (5–6 h, 55–60 °C) stored warted and areolate, cartilaginous. Warts in freezer at – 20 °C. For DNA extraction, are fine and almost pyramidal. Peridium was followed and modified Dellaporta et al. is very thick (380–480 μm) and two lay- (1983) protocol for plant tissue isolation. ers can be distinguished – outer, which PCR amplification was performed with the is composed by 3–5 rows of thick-walled primers ITS1 and ITS4 (White et al. 1990, large hyphae and inner layer, which is Gardes and Bruns 1993) for the ITS re- paler and almost white, forming pseudo- gions. After gel-electrophoresis, success- parenchymatous tissue (Fig. 1b). Gleba ful DNA fragments were visualized and of interwoven to parallel hyphae is more cleaned using GeneMATRIX Basic DNA loosely structured than the inner layer of Purification Kit. Samples were sequenced peridium, hard and solid, initially whitish, at GATC biotech laboratory in Germany. than yellowish, ochre to olive-brown or The results were compared with their olive-gray at maturity, with a close net- closest relatives obtained with BLAST al- work of whitish meandering and translu- gorithm. Phylograms are constructed for cent veins. Odor: relatively strong, not T. excavatum and T. fulgens. Sequences specific and unpleasant. Taste is mild. were first aligned in CLC Genomics work- Asci 50–70 × 80–100 μm, clavate to al- bench 8.0 and then corrected manually. most globose (depending of spore posi- Dendrogram was constructed and ana- tion and view – Fig. 1d), short-stalked 116 T. Nedelin, M. Gyosheva, K. Kostov, and S. Savev
Fig. 1. Tuber rufum (BG13AG10): a – general view; b – inner layer of pseudoparenchymatous tissue of peridium in Melzer’s reagent; c– SEM detail view of spore; d – SEM general view; e – asci with ascospores in Congo red; f – Tuber excavatum (BG13AG06) – ascospores.
(10–25 μm) (Fig. 1e), usually 3–4-spored. N = 50; C = 95 % Me = 33.9 × 25.4 µm;
Ascospores 27.1 [32.9; 34.8] 40.7 × 20.2 Qe = 1.3, broadly ellipsoid, ornamented [24.6; 26.1] 30.5 µm; Q = 1.0 [1.3; 1.4] 1.6; with conical spines (Fig. 1c), 2.5–3.5 μm New Records and Data on Hypogeous Ectomycorrhizal Fungi in Bulgaria 117 high, initially translucent, yellow – ochre p. 27, 1831. tobrownish at maturity (Fig. 1e). Basidiomata hypogeous or semi- Habitat. This species is relatively wi- hypogeous, irregularly subglobose, often despread associated with deciduous trees wrinkled, with small depressions, 2–4 cm and conifers, ascomata maturing during in diameter, ochre to reddish-brown. At the late summer and autumn (Pegler et al. base with whitish mycelial tuft of strands 1993). (hyphae). Peridium is thin (220–330 μm – Specimens examined. Western Fo- Fig. 2a) and can be seen only in immature rebalkan, near Belotintsi village, buried in stage and through the development humus layer of soil, partially on the sur- disappear. Gleba with a labyrinthine- face, near Tilia tomentosa Moench arti- like elongate or near roundish cavities, ficial plantation, 14.09.2014, leg. T. Ne- situated near ochre, reddish – brown or delin, det. M. Gyosheva (SOMF 29671; grayish branches which often enlarge BG13AG10). toward the base and forming white or Comments. Although there are sever- whitish columella. Odor very strong and al species with similar features, according unpleasant. Taste undefined. Basidia to Pegler et al. (1993) main differences 13–20 × 8–10 μm, broadly clavate, usually in close related ones can be observed in 2-spored (rare 4-spored). Cystidia clavate type and colour of peridium, its cell organi- and hyaline. Basidiospores 16–19 × zation and structure, also in consistency of 11.5–13 μm, Q range = 1.3–1.6, broadly gleba. T. rufum was also reported recently ellipsoid, brownish at maturity. Spore from Turkey (Türkoğlu and Castellano ribs are smooth and apiculus is rounded 2014). Due to its high intraspecific varia- (Fig. 2c). bility a set of specific primers were devel- Habitat. In soil, in coniferous and oped – Ru1f/Ru2f-ITS4 (Iotti et al. 2007). deciduous forests (mainly under oak), Our result of DNA sequencing matches from late summer to late autumn. 100 % for this species in GenBank, with Specimens examined. Tracian Low- standard fungal primers – ITS1/ITS4, but land, near Haskovo town, in the Park using specific primers is recommended, ’Kenana‘, buried in organic layer of soil, because the detection can be more sensi- under Quercus sp., 14.09.2014, leg. tive and it is possible for only 0.2 mg of T. Chokova and E. Nankova, det. M. this truffle to be identified successfully. It Gyosheva (SOMF 29672; BG13AG07). is especially important when mycorrhizal Comments. G. graveolens was re- tips are used, instead of fungal tissue from corded in Greece in forests of Quercus ascoma. According to the same authors frainetto Ten. (Konstantinides 2006). It is Tuber ferrugineum Vittad. is a morpho- also reported from some other European logical form of T. rufum characterized by countries and also from North America a rusty coloured peridium and spores with (Benedek and Pál-Fám 2001, Zeller and long spiny ornamentations. Dodge 1918, etc.). There is insufficient data of exact host range of tree species Basidiomycota forming ectomycorrhiza with this hypo- Gomphales geous fungus. It can be easily recognized Gomphaceae from Gautieria morchelliformis (Fig. 2b) by Gautieria graveolens Vittad., its smaller spores and clear visible peridi- Monographia Tuberacearum (Milano), um in early stages of development. 118 T. Nedelin, M. Gyosheva, K. Kostov, and S. Savev
Fig. 2. Gautieria graveolens: a – immature fruitbody with clear visible peridium (orange arrows); b – section view of Gautieria morchelliformis; c – Gautieria graveolens – spore and paraphyses; d – Gautieria morchelliformis – spores.
Russulales of peridiopellis (Fig. 3b). Gleba whitish to Russulaceae pale orange or pinkish, discoloring reddish Lactarius stephensii (Berk.) Verbeken on exposure after cutting, labyrinthic, with & Walleyn, in Nuytinck, Verbaken, Dela- irregularly elongated chambers (Fig. 3c). rue & Walleyn, Belg. JI Bot. 136 (2): 151 Columella cannot be seen. Odour very (2004) [2003] strong, specific, fruity. Taste very strong, Basidiomata hypogeous or semihypo- fruity, and specific, even in dry speci- geous 1.0–2.5 cm in diameter (Fig. 3a), mens. Basidia broadly clavate to almost mostly irregular or subglobose, lobbed globose, short-stalked, with 3–4-spores. with distinctive radial ridges at the base, Basidiospores 10.91 [12.20; 12.56] together with thin, mycelial cords arising 13.84 × 10.37 [11.36; 11.64] 12.62 from a minutely projecting sterile base. µm; Q = 0.97 [1.06; 1.09] 1.18; N = 64;
Peridium at first pale ochraceous to C = 95 %; Me = 2.38 × 11.50 µm; Qe = 1.08 orange-red, finally darkreddish-brown, (excluding ornaments) (Figures 3d, e), dull, smooth, viscid, easily detached – globular, ornamented with conical spines 150–200 μm total, two layers – 50–80 μm rounded apically with length 1.2–1.8–(2.2) New Records and Data on Hypogeous Ectomycorrhizal Fungi in Bulgaria 119
Fig. 3. Lactarius stephensii (BG13AG08): a – general view; b – two layers of peridium (green arrows) – outer is with doubled green arrows (≈80 μm); c – detail section view of mature fruit body; d, e – SEM of spores at different magnifications. 120 T. Nedelin, M. Gyosheva, K. Kostov, and S. Savev
µm, initially translucent, yellow-ochre to Comments. T. excavatum group is brownish at maturity. well recognized from other truffles be- Habitat. Usually gregarious, under cause of their usually well-developed ba- humus, with ectomycorrhizal hosts from sal cavities. This group seems to have a genera Corylus L., Carpinus L., Fagus great intraspecific diversity and a dozen L., Populus L., Quercus L., Tília L. (Vidal of varieties, subspecies and forms were 2004). described (Riousset et al. 2012, Gross Specimens examined. Western Bal- 1991). For instance the colour of peridium kan Mts., near Gradets village, buried in in monticellianum form is olive brown. Fis- organic layer of soil, near Carpinus betu- cher (1923) distinguishes varieties with lus L., 03.09.2014, leg. & det. T. Nedelin small spores (brevisporum) and other with (SOMF 29673; BG13AG08). larger spores (longisporum). Most species Comments. Two genera of seques- have been shown a very close intraspe- trate fungi related with genus Lactarius cific ITS to interspecific ITS sequence Pers. are well recognized presently: Ar- variation and sometimes overlapped gaps cangeliella Cavara and Zelleromyces can be recognized (Bonito et al. 2010c). Singer et A. H. Sm. First of both is seco- Thus requires comprehensively morpho- tioid and contrarily, Zelleromyces is non- logical and phylogenetic studies, espe- secotioid and sessile. In Europe only 6 cially in case, where degree of variability species from second are known (Vidal between examined specimens is higher 2004). and there are significant differences in ecological aspects (host plant and pe- riod of maturing in respect to geographi- cal latitude). Several samples from each New localities of hypogeous fungi T. excavatum locality were compared and in Bulgaria macro- and micro-morphologically stud- ied, and were found some slightly distinct Ascomycota features. BG13AG05 has more wrinkled Tuber excavatum Vittad. surface (Fig. 4a) and paler colours in the Specimens examined: BG13AG05 – peridium, also its basal cavities are nar- Northeast Bulgaria, near Razgrad town, rowed (Fig. 4c). In BG13AG06 the basal buried in organic layer of soil (1–5 cm un- cavities are enlarged, especially in young der litter), near T. tomentosa artificial plan- stage of development (figures 4b and 4d). tation, 13.11. 2013, leg. K. Kyose, det. It has finely papillose peridium, contrary T. Nedelin (SOMF 29674); BG13AG06 to BG13AG05 which peridium is gener- – Western Stara Planina Mts., near Gra- ally smooth. The colour of its gleba is dark dets village, buried in organic layer of soil purple to pinkish purple and dark purple (1–5 cm under litter), near Carpinus orien- to purple brown in BG13AG06 (Fig. 4d), talis Mill., 30.10.2014, leg. &. det. T. Ned- without pink tints in the last. Asci have no elin (SOMF 29675). significant differences in their morphology The species was reported previously or size, but spore ratio (Q) for Razgrad in Bulgaria also from Northeast Bulgaria samples is 1.0 [1.2; 1.3] 1.4 (Fig. 4e) and – Razgrad Hills (Dimitrova and Gyosheva respectively for Gradets village samples 2008) and from Western Rhodopes Mts., – 1.2 [1.4] 1.6 (Figures 1f and 4f). Both near Ravnogor village (Lacheva 2012). specimens (BG13AG05, BG13AG06) New Records and Data on Hypogeous Ectomycorrhizal Fungi in Bulgaria 121
Fig. 4. Tuber excavatum: a – general view (BG13AG05); b – detailed view of peridium BG13AG06); c – section (BG13AG05), d – section (BG13AG06); e – SEM, BG13AG05; f – SEM, BG13AG06. have different tree hosts and there is structures, type and climate condition for a great possibility that this might be the the ecological plasticity of T. excavatum most important factor in addition to soil group. 122 T. Nedelin, M. Gyosheva, K. Kostov, and S. Savev
Basidiomycota specific variation >9.2 %. T. excavatum is Gautieria morchelliformis Vittad. the only species from their study, which (Fig. 2d). shows lower interspecific to intraspecific Specimens examined. Western Stara variation and overlaps and we confirm Planina Mts., above the village of Buhovo, this. semihypogeous in soil, near Quercus sp., T. excavatum samples from Gradets 25.11.2014, leg. A. Goranov, det. M. Gyo- village are genetically closer to T. fulgens sheva (SOMF 29677). than to T. excavatum group I. The com- This species was reported only once parison of BG13AG05 and BG13AG06 for Bulgaria from Southern Black Sea is shown in Fig. 6. More symmetric lines coast – Ropotamo Reserve (Kuthan and correspond to more similarity in their nu- Kotlaba 1981). It is also found in Greece, cleotide basis. under Quercus ilex L. (Konstantinides 2006) and in forests of Pinus nigra Arn. (Diamandis and Perlerou 2008).
Conclusion
The data of carried out morphological Results from DNA analysis and molecular studies of Tuber excava- tum samples confirmed the great genetic According to various authors (Rioussett variability amongst this group. Using spe- et al. 2012, Montecchi and Sarasini 2000, cific primers, especially for the group of Pengler et al. 1993) the closest taxonomi- white truffles, would bring further clarity in cally species to T. excavatum is T. ful- their diversity. The dramatic and constant gens, which differs from it for its brighter, decrease of whole genome sequenc- orange-brown colour and its subglobous ing cost price since 2008 (from 100 mil- spores. The phylogram on Fig. 5 is con- lion USD in 2001 to 1000 USD in 2015 at structed for most T. excavatum and T. ful- Illumina HiSeq X10) and establishment gens samples in the GenBank, compared of the Next-Gen Sequencers (Bahassi with their identical ITS regions. It was ob- and Stambrook 2014) will make direct tained in CLC Genomic Benchmark 8.0 sequencing of the entire genome in near after (computer and then manual) align- future more accessible – up-to-date the ments analysis of parallel ITS sequences most reliable method to determine spe- after several tests for construction of best cies. All this, however, must be accompa- tree model. nied by the most possible detailed mor- Two major clades clearly emerge with phological, anatomical, physiological and several subgroups amongst them, which other studies of hypogeous fungi from can be distinguished also without molec- different habitats to determine borders of ular studies. It was found that in the first their ecological plasticity. group intraspecific variation is slightly be- All reported hypogeous fungi in the pa- low 6 % and in the second slightly above per are uncommon or threatened in Eu- 12 %. This corresponds within the borders rope and should be estimated according accepted by Bonito et al. (2010a) – for to IUCN criteria at the next update of Red intraspecific variation <14.7 % and inter- List of Fungi in Bulgaria. New Records and Data on Hypogeous Ectomycorrhizal Fungi in Bulgaria 123
Fig. 5. Phylogenetic tree of Tuber excavatum and Tuber fulgens specimens from public database.
Acknowledgements work of the Bulgarian-Swiss Research Programme, project IZEBZO_143109. This work was supported by the Swiss The work of M. Gyosheva is carried Enlargement Contribution in the frame- out within the framework of the project 124 T. Nedelin, M. Gyosheva, K. Kostov, and S. Savev
Fig. 6. Dot-plot diagram of both Bulgarian Tuber excavatum specimens (BG13AG05, BG13AG06).
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