Mycologia, 92(5), 2000, pp. 860-873. O 2000 by The Mycotogical Society ofAmerica, f,awrence, KS 6604+8897 Iactarius ectomycorrhizae on Abies alb az morphological description, molecular characteization, and taxonomic remarks Ursula Eberhardt Kq Words: ectomycorrhizal fungi, ITS scquences, Franz Oberwinkler RT'LP Speziellc Botanik, Mykolngte, [Jniaersität Tübingen, Auf der Mmgenstelle I, D 72076 Tübingen, Germany Annemieke Verbeken INTR.ODUCTION Labmatorium Plantkunde, Vahgroep Biobgie, Uniaersiteit Gent, Ledeganrhstraat 35, 8-9000 Cent, Though silver fir (Abies alba Miller) is an ecologically Belgium valuable and indigenous tree species in many Euro- pean Andrea C. Rinaldi mountain forests, verv little is known about its ectomycorrhizae (see Comandini et al i998, and ref- Dipartimento di Scienze Med,iche Internistiche, Uniuersitd, di Cagliari, I-09042 Monserrato Cagtiari, erences therein). In the present study, we describe holl the ectomycorrhizae of three Lactarius species on Ä. alba from central Italy, by standard Giovanni Pacioni morphological and anatomical parameters (Agerer Ornella Comandinir 1991): Lactarius subsericatus (Kühner & Romagn.) ex Bon, Lac. inter- Dip ar timento di Scienze Ambient ali, Llnia ersitd. rnedius (Ikombh") Berk. dell'Aquila, Via Vetoio Loc. Coppito, I-67100 & Broome , and Lac. salmom- L'Aquila, Italy icoLor R. Heim & Leclair. Mycorrhizae of the latter species were compared with an existing description (Pillukat 1996). Identification of the fungal symbi- .ll'as Abstract: To date, the ectomycorrhizae formed by onts achieved by means of sequence comparison silver fir (Abies alba), an ecologically valuable and in- between mycorrhizae and sporocarps of the expected digenous tree species in many European mountain lungal partner species, in addition to morphoana- forests, have been poorly investigated. We character- tomical analysis of mycorrhizae. ized the mycorrhizae formed by three Lactarius spe- Lactarius subsericatus (syn. Lac. ichoratus ss. Rom- cies {Lac. subsericatu,s, Lac. intermedius, Lac. salmon- agnesi) is a representative of the subgenus Russularic icolor) on silver fir, on the basis of material originat- (Fr.) Ihuffman sect. Russularia Fr. The species is ing from central Italy. The identification of the fun- closely related to tr ac. fuluissimus Romagn. from gal symbiont was achieved by means of rvhich it differs try the spore ornamentation com- morphoanatomical observations of mycorrhizae, and posed of mostll, isolated warts (more reticulate in by comparison of ITS sequences obtained fiom my- Lac. fuluissimtts).ln the field, Lac. subsericalzs is rec- corrhizae and sporocarps of putative fungal partners. ognized from the latter species by its more greasy Sequences also were obtained from specimens of the pileus surfäce, the milk which stains yeilow on white same species but from different geographic origin or iissues, the darker lamellae and an odor resembling from closely related Lactarius species. A maximum that of Lac. subdulcis (Pers.:Fr.) Gray (rubberJike, likelihood analysis of the data was performed. On the in Lac. fulaissimus much iike the odor of Lepiota cris- whole, the resultant tree is in good agreement with tata (Nb. & Schw. : Fr.) Kummer). For further differ- sporocarp and mycorrhiza morphology. RFLP pat- ences befween these hvo taxa see Schwötrel (1g79)" terns were calculated from sequence data. A discus- The insufficiently known species Lac. {trittanieusReid sion on the main morphoanatomical characters dis- also seems very closely related, if not consp ecrfic. Lac. tinguishing tlne Lactariu.r ectomvcorrhizae reported subsericatus is associated with a broad range of both in this study from those aiready described beionging conif'erous anci deciduous trees on calcareous soil in to related species, is also included. The accuracy of western Europe, but collections of broadleaved hosts diflerent methods to identify mycorrhizae formed by are <listinguished by some authors as Lac. subseticatus closely related Lactarius species on silver fir, are dis- f. pseudofulaissimus Bon (e.g., tson 1980). cussed. I-actariu,s intnmedius lnon Aga,icus intermedius F r.. = Lac. scrobiculatus (Scop.:Fr.) Fr.l fits in the sub- Accepted for publicarion April 28, 20{)0. genus Piperile.s (Fr.) Kauffman sec.t. Zo,narüQu6l. sub- i Corresponding author: emaii: [email protected] sect. Scrobiculali Hesier & A.H. Sm. it is cioseiv reiar- 860 Egl.ngenor ET AL: Ll.cr,l,tltts EcI'()NIyc()RRHIZ.\F- ()N A. ,u,n.l 861 TenLL, I. Material used for DNA sequencine Species Host Origin GenBank I-ac. subsericatus 5"7 Abies alba Italy AF140254 Lac. subscticaf.z.r ECMr' A. alba Ita11, AFI 40255 I-a,c. intermedius S A. alha Ita11, AF1 40256 Lac. intermedlzs ECM A. alba Italy Arl 40257 Lac. salmonicolor Sl A. alba Ital1' 4F140258 Lac. salmonicolorLCM A. alba Iral,v 4F140259 Lac. suh,sericatus 32 Picea abies Germany AFl 40260 Lac. subsnirulus 33 Fagus slhatica Germanl, 4F140261 I-ac. cf. fulaissimus S broadleaves France Ar204678 Lac. Jülaissimus S broadlear.es Belgium AF204679 Lac. scrobiculatus 51. P ahies Germany AFl 40262 Lac. scrobiculatus 32 P abies Germany AFI.10263 Lac. .salmonicolor 32 A. albr.t Germany AFl ,10264 Lac. salmonicolor S3 P abies, E syluatica, Germany 4F140265 Pinus sllaestris (4. alba) Lac. deterrimus Sl P abies Germany AFt,+0266 Lac. deterrimus 32 P a,bie.s G'ermanv AFt 40267 I-ac. semisanguifluus S P sllttestris (11 abies) Germanv AFt 40268 Lac. quieticolor S P sll.uestis Germany 4F140269 " S : Sporocarp. " ECM : Ectomycorrhizae. ed to Lac. scrobiculatus from which it differs by the phoanatomical features, by ITS sequences, and by paler, azonate pileus, the scarcely pubescent margin ITS PCR-RFLP, dealing rvith these closely related spe- soon becoming smooth in mature specimens, the cies. larger spores which are ornamented with finer and less connected warts and ridges, and its exclusive as- \IATERIA]-S,{\D }IETIIODS sociation with Abies,whlle Lac. scrobiculatus is knorr,n Picea (Heilmann-Clausen 1998). to grow with et al S rLmp lin g an d c h ar a c I eriz a ti o n.- I -a ct ariu s e ctomy corrhizae Lactarius intnmedius is only known from calcareous and sporocarps were collected during the growing seasons soils within the natural distribution of its host rree in 1995-1998 from two silver fir stands in the Gran Sassolaga Europe. A detailed description is given by Marchand National Park, central Ital1,: "Fonte Gelata" (artificial stand, ( 1e80). planted in early 1950s; 1000 m asl) and "Colle Pelato" (nat- Lactarius salmonicolor [syn. Z. laeticolor (S. Imai) ural stand; 1100 m asl) (T,rnr-n I). Details of the nto study Imazekil belongs to the subgenus Piperites sect. Dap- areas, including mvcocoenological, phvtosociological and pedological aspects, are reported in Comandini (1997). etes Fr. It is distinguished from the other European Soil cores rvere taken fiom beneath the sporocarps. Sub- members of this group by its exclusive association sequentl)', the samples rvere carefull,v washed in water, and with Abies species and the uniform salmon to orange ectomvcorrhizal roots were separated under a dissecting mi- color without greenish or reddish tinges. For a de- croscope (Leica Wild N,I 10) for macroscopical characteriza- tailed account on this group, we refer to Heilmann- tion, as described by Agerer (1991). Particular care was tak- Clausen et al (1998). Lactarius salmonicolor seems en to isolate mycorrhizal roots of A. alba from those of rvidely distributed in the northern hemisphere and is other host trees in the sampling sites (mainl1,.Fa-grzs syluatica reported from western and central Europe (Heil- L.). Methods to characterize ectomycorrhizae have been mann-Clausen et al 1998), Turkey (Sesli and Baydar comprehensivelv explained b,v Agerer (1986, 1987-1998, 1995), Japan (e.g., Hongo 1960 as Lac. laeticolor), 1991), and a glossary of all terms used has been published Mexico (Heim 1953); and from Abies-plantations out- by the same author (Agerer 1987-1998). Munsell (1975) has been used as reference fcrr the side the natural distributional area. description of colors of ectomycorrhizae. As the three Lactanzs species considered in this Mantle preparations of fresh ectomycorrhizae rvere fixed study all possess closely related species that may occur on slides with polwinvl lactophenol for microscopic inves- in the same area, associated with the same or differ- tigations. For light microscop,v, observations were made with ent tree species, we lvanted to find out about the a Leitz Laborlux S microscope and photographs were taken levels of resolution of different methods for identi- with Ilford Panf Plus 50 films. Cross and lonsitudinal sec- $ring mycorrhizae, i.e., identification by their mor- tions (3-5 pm thick) were cut on a cryotome (Frigocut 862 MrcoLoclq 2800, Reichert-Jrrg). \bucher specimens <if both Lactarius action volumes were reduced by half and the clcle sequenc- sporocarps and mycorrhizae collected in Italy were depos- ing reaction mix diluted 1:1 with bidistilled water. Sequence ited in AQUI (Herbarium Mycologicum Aquilanum) as editing and sequence alienment of sporocarps and their fixed material (4% ghtaraldehyde), together with slides. putative mycorrhizae for identification pr-rrposes was per- Reference specimens of the German material are kept in formed using Sequence Navigator (version 1.0, Appiied Tübingen, Institute of Botany, Spezielle Botanik, Mykologie. Biosystems, Foster Ciry', Califirrnia). The ectomycorrhizae were identified by macroscopic and Seque nces rvere aligned usins