(Quercus Cerris, Q. Frainetto, Q. Robur) and the Optimization Perspective of Growth Conditions for in Vitro Propagated Plants Transplanted in the Field

Ann. For. Res. 54(1): 57-71, 2011 ANNALS OF FOREST RESEARCH www.e-afr.org

Mycorrhizal status of several Quercus species in Romania (Quercus cerris, Q. frainetto, Q. robur) and the optimization perspective of growth conditions for in vitro propagated plants transplanted in the field

E. Fodor, A. Timofte, T. Geambau

Fodor E., Timofte A., Geambau T. 2011. Mycorrhizal status of several Quercus species in Romania (Quercus cerris, Q. frainetto, Q. robur) and the optimization perspective of growth conditions for in vitro propagated plants transplanted in the eld. Ann. For. Res. 54(1): 57-71, 2011.

Abstract. There is an increasing interest for important tree species conservation in the context of climate change, anthropogenic pressure and invasion of alien tree species. A key factor in the survival of trees is represented by the mycorrhizal association. The success of micropropagated trees also depends on the acquisition of mycorrhizal mutualists. Ectomycorrhizal roots samples from several Quercus species (Q. cerris, Q. frainetto, Q. robur) were examined for mycorrhizal morphotypes’ characterization. The samples were collected during the vegetation season from stands located in Southern and North-Western Romania. 30 morphotypes of active mycorrhizae were identified with Cenococcum geophilum Fr. (Ascomycota) as dominating morphotype. Previous studies on somatic embryogenesis in Q. robur and Q. frainetto demonstrated the utility of in vitro techniques in obtaining plants from these recalcitrant seed producing species, considered at risk in various areas of the country, due to increasingly stressful conditions. The success rate of the acclimatization process depends on the mycorrhization performed either artificially, in the laboratory, either naturally, in the field. Ex situ mycorrhization solutions are considered as less costly, yet efficient alternative to improve the ex vitro survival of micropropagated plants or endangered tree species or for those with eco- nomic importance, in vitro propagation is an important conservation tool combined with the acquisition of appropriate mycorrhizal mutualists. Keywords ectomycorrhizae, morphotypes, Quercus robur, Quercus cerris, Quercus frainetto.

Author. Ecaterina Fodor, Adrian Timofte, Teodora Geambau (ecaterina fodor@ yahoo.com) - Oradea Station, 70 tefan cel Mare Street, Oradea. Manuscript received March 8, 2010; revised December 16, 2010; accepted De- cember 20, 2010; online rst February 3, 2011.

57 Ann. For. Res. 54(1): 57-71, 2011 Research articles

Introduction sociations are diffuse and non-speci c in the most associations, multi-host fungi dominating Quercus species form ectomycorrhizas with temperate terrestrial ecosystems (Selosse et al. several fungal partners. The actual associa- 2006). Playing a central role in tree nutrition tions vary in time and space, according to tree and connectivity, mycorrhizal fungi are to be and stand age, to successional moment and en- considered keystone species in forest ecosys- vironmental factors. The diversity of the fun- tems. gal partners is shedding light on biocomplex- The microenvironment of roots, especially ity - properties emerging from the interplay of of the assimilative roots, is highly heterog- behavioral, biological, chemical, physical and enous, shaping the interaction with different social interactions that affect, sustain or are mycobionts. modi ed (Michener et al. 2001) by tree-fungi Both assimilative roots and mycelia are of associations. Mycorrhizal association is to be modular nature. Their relative autonomy is a considered the elementary functional unit of partial explanation for the multiple mycobi- the forest ecosystem and one of the problems otic mutualists, relating to the same phytobi- at work in the frame of biocomplexity name- ont namely the tree. Modularity has evolved ly, the interaction roles of mycorrhizal fungi, to explore and exploit a patchy environment, plants and soil resources in carbon and nutrient fungal mycelia being organized as networks transfer (NSF-Biocomplexity 1999). Also, my- and functioning whole organisms. One of the corrhizae have been recognized as ecosystem network properties is the possibility of recon- level functional systems, involved in nutrient guration to adapt to new conditions. Plants turn-over, producing a profound alteration of are connected by mycorrhizal networks. root chemistry, architecture and biology of the The niche represented by the tree is parti- organisms living in the rhizoplane (Langley & tioned among the mycorrhizal mycobionts and Hungate 2003). other organisms, mutualists as helper bacteria From theoretical point of view, the diversity and an extended array of pathogens. Appar- of mycorrhizal morphotypes raises questions ently, the fungal species are interchangeable on the niche partition, community assemblage according to the neutral community theory and rarity vs. commonness of particular mor- (Kelly et al. 2008). photypes. From a practical point of view, the There are several approaches in studying selection of a particular morphotype, in order mycorrhizal diversity (Trudell, 2003): mush- to enhance the acclimatization success of mi- rooms surveys (the oldest method of investiga- cropropagated plants, is a major goal in ob- tion), morphotyping and the use of molecular taining quickly and ef ciently tree seedlings markers, such as DNA ngerprinting. Mush- by combining in vitro micropropagation and room surveys are still at use, although the list mycorrhization. Forests are nitrogen limited, of mycobionts in a particular environment is most of this element is organically bounded incomplete, due to the rarity of most of my- and mycorrhizal fungi get access to it (Bon- corrhizal partners, due to unpredictable fruit- fante 2003). ing or due to the fact that many mycobionts One of the possible explanations on the di- do not produce fructi cations. Morphotyping versity of the morphotypes is the insurance hy- is based on the observed fact that different pothesis (Yachi & Loreau 1999). The hypothe- combinations of plants and fungi produce dif- sis is based on the intuitive idea that increasing ferent looking ectomycorrhizas. A wide range biodiversity insures the ecosystems against of macroscopical and microscopical charac- declines and their functioning caused by envi- teristics are used to describe morphotypes. Al- ronmental uctuations. Also, mycorrhizal as- though by morphotyping actual identity of the

58 Fodor et al. Mycorrhizal status of several Quercus species in Romania ... fungal partner is not always assigned, still it for the the mycorrhization of microprapagated offers valuable information upon the actual ac- plantlets of Q, robur and Q. frainetto. tive mycorrhizas is obtained. An almost complete record of the existing mycorrhizas in a particular site is produced including also the Methods inactive mycobionts. Facing the threat of extinction for many tree Site locations. Locations of root samples species of the world, we are challenged to nd were selected in various forest stands from dif- new breeding methods to preserve plant biodi- ferent forest districts in Southern, Western and versity. In vitro micropropagation is one of al- North Western Romania (Table 1). ternatives. Maximum bene ts can be obtained Forest stands are dissimilar with respect combining the micropropagation with mycor- to site conditions and stressful factors. For rhization with an appropriate fungal partner on instance, Western and North-Western sites appropriate substrate (Azcon-Aguilar & Barea (Dobreti, Tinca, Oradea, Radna, Buteni) loca- 1997). Mycorrhization of in vitro propagated tions are assigned to hilly forest types, natural plantlets has a positive impact on them in terms as well as plantations, exception being Tinca, of post-transplanting performance (Rai 2001). situated in the high Miersig plain. The forest The aim of the present study was to: (i) assess stands investigated in Southern part (Giurgiu, mycorrhizal diversity in terms of morphotypes Comana, tefneti, Vlsia) are assigned to under different conditions (Southern, Western broadleaved mixed forests, dominated by Q. and North-Western Romania) in several Quer- cerris and Q. frainetto, occasionally with Q. cus species (Quercus cerris L., Q. frainetto robur (tefneti). Sampling was performed Ten., Q. robur L.), (ii) nd the similarities in during growing seasons, in the period 1996- mycorrhizal status (morphotypes) of trees veg- 2001. etating under different site and geographical The climate of North-Western and Western conditions, (iii) identify the most frequently Romania is of a particular type, displaying found morphotype in order to recommend it sub-Mediterranean and Atlantic in uences,

Table 1 Site locations County Forest District Longitude Latitude Ilfov Giurgiu 43° 56’ 07.50’’ N 25° 56’ 15.88’’ E Brneti 44° 42’ 52.54’’ N 26° 21’ 07.36’’ E Vlsia 44° 42’ 29.47’’ N 26° 00’ 14.42’’ E tefneti* 44° 20’ 10.75’’ N 25° 10’ 38.92’’ E Vldiceasca 44° 39’ 47.65’’ N 26° 06’ 48.25’’ E Bneasa 44° 31’ 52.92’’ N 26° 00’ 48.00’’ E Snagov 44° 42’ 52.54’’ N 26° 08’ 58.26’’ E Clrai Brganu 44° 25’ 30.68’’ N 27° 36’ 08.10’’ E Bihor Dobreti 46° 50’ 50.42’’ N 22° 08’ 42.89’’ E Oradea 47° 06’ 11.71’’ N 21° 55’ 25.94’’ E Tinca 46° 46’ 33.15’’ N 21° 55’ 25.02’’ E Arad Radna 46° 07’ 02.98’’ N 21° 40’ 37.75’’ E Buteni 46° 21’ 46.20’’ N 22° 08’ 42.89’’ E Giurgiu Mihai Bravu 44° 39’ 47.65’’ N 26° 04’ 02.44’’ E Comana 44° 09’ 47.50’’ N 26° 08’ 28.42’’ E Teleorman Slveti 44° 20’ 10.75’’ N 25° 10’ 38.92’’ E 59 Ann. For. Res. 54(1): 57-71, 2011 Research articles ed ed Hairy, Hairy, hyphae smooth smooth smooth smooth smooth smooth smooth smooth rami rami branched, right angles emerging at emerging rhizomorphs and emergent and emergent Texture of the Texture robur cerris cerris cerris cerris cerris cerris White hyphae Quercus Quercus Quercus Quercus Quercus Quercus Quercus Quercus Quercus frainetto frainetto rhizomorphs Rhizomorphs and emergent of 0.4 M3 M5 M4 M8 M2 Form (mm) Round diameter the apex, white color, texture Mantle, abundant silvery areas Smooth, with Wooly, white, Wooly, Shinning white, Cottony, silvery- Cottony, under the mantle Smooth with rare Radiating hyphae emergent hyphae, emergent Wooly, white with Wooly, greasy with cystids with brownish tinge dense wooly texture M6 White, conspicuous. hyaline rhizomorphs in forest stands from southern and north-western Romania 0.4 tive (mm) 0.2-0.4 white Wooly, M1 between Distance consecu- branches Q. frainetto 3 0.8-1 4 0.3-1.5 2 0.2 3 0.3-0.4 3.5 0.4-0.5 5-6 0.7-1 and base 1.8-9 (mm) and at base system Length of mycorrizal constricted constricted 4-5 brown axe loose uneven pinnate bent and branches Sympodial Repeatedly Sympodial, Sympodial, pinnate with Monopodial, palmetti type Not branched 0.5-3 rough M7 dichotomous, loose, clavate pinnate, loose Dichotomous, with elongated curved apexes, Coralloid, loose, with bent apexes with bent apexes Pinnate, compact, Quercus robur, Q. cerris robur, Quercus ed in ed loose orange contour dull brown purple tinge Yellow, with Yellow, Black-brown Black-brown, with rusty spots, Yellow- whitish, Yellow- White-yellowish Yellow-orange to Yellow-orange Dull brown, later, Dull brown, later, Orange-yellowish visible brown cell deep brown with a orange-brown base greasy with cystids with rusty spots and Vlsia Buteni Slveti Slveti 08.1998 06.1996 09.1996 08.1997 08.1997 06.1996 08.1997 Bneasa 08. 1996 08. 1996 Comana., tefneti Vldiceasca Vldiceasca tefnneti Oradea. et. al. robur cerris cerris cerris cerris cerris cerris Quercus Quercus Quercus Quercus Quercus Quercus Quercus Quercus Quercus Quercus frainetto frainetto frainetto Mycorrhizal morphotypes identi

M4 M6 M5 M1 M7 M8 M2 M9 M3 Code Host Location Color Branching Table 2 Table 60 Fodor et al. Mycorrhizal status of several Quercus species in Romania ... smooth Texture Texture of the rhizomorphs and emergent hyphae smooth smooth smooth ed none none none none none none noneLong emergent hyphae none Rhizomorphs and emergent hyphae Fan shaped brown rhizomorphs emerging from base to apex Brown rhizomorphs emerging near the apex Fan rami white rhizomorphs Clavate 0.3 Clavate, discolored 0.3-3 Round or acute 0.5-1 Round 0.3-0.4 Round 0-7-1 Form of the apex, diameter (mm) Round or clavate 0.3-0.4 Round and digitiform at the beginning 0.4-0.6 Round or clavate 0.3-0.4 Shinning white, dense wooly, Smooth, shinning with greasy shine Wooly, white with Wooly, silvery areas Mantle, color, texture Smooth with rough areas or velvety Shinning white, dense short wooly, Distance between consecu- tive branches (mm) 2.5 0.1-0.3 3-11 1 8-10 0.5-1 hyaline Velvety, 4 0.3 5 0.4 3 0.1-0.2 smooth Length of mycorrizal system (mm) 3 0.3-0.5 Rough, greasy 2.2-4.2 0.3 Pinnate, Monopodial, loose, pedicelated, bent apexes Pinnate, Sympodial, loose Sympodial uneven, loose (up to 80 branches/ system) Monopodial, loose, rare Sympodial, pinnate, dense Monopodial, bent apexes, bumpy Sympodial, uneven or coralloid, sympodial, bent apexes White-yellowish or pink Dirty white Shinning black with reddish tinge, constricted at base Yellow-brownish Yellow-brownish with branch base and apexes brown Dirty white Orange to brown, discolored apex, older parts brown, basal constriction Amber yellow with brown spots on apexes Chocolate brown with discolored apexes, bent Tinca 10. 1997 Brneti 06.1997 Snagov 06.1997 Tinca 04. 1997 Tinca 04. 1997 Tinca 04. 1997 Vlsia 10.1996 Dobreti 07.1998 Dobreti 07.1998, Buteni 06.2000 and (continuation) Quercus cerris Quercus cerris Quercus cerris Quercus frainetto Quercus cerris Quercus cerris Quercus cerris Quercus cerris Quercus cerris Quercus robur M15 M14 M13 M11 M12 Code Host Location Color Branching M10 M17 M16 Table 2 Table

61 Ann. For. Res. 54(1): 57-71, 2011 Research articles Texture of the Texture rhizomorphs and emergent hyphae smooth reticulated and smooth smooth hairy surface smooth and branched smooth ed Rhizomorphs and emergent hyphae Emerging hyphae, right angles Brown-black rhizomorphs noneWhite, shinning, smooth none none none Rami rhizomorphs, interconnected Orange rhizomorphs Rare emergent hyphae Round some Form of the apex, diameter (mm) Round 0.3-0.4 Round 0.4-0.6 Round to clavate 0.3 Round or relatively acute 0.2-0.4 Round 0.3-0.4 with rusty spots 0.3-0.4 Round 0.3-0.6 Round 0.2-0.3 Wooly, abundant, Wooly, dense Mantle, color, texture Smooth with sulfur yellow granulations Rough without conspicuous hyphae shinning Velvety, white, relatively rare Smooth with granular areas Smooth, rare hyphae emerging (also laticiferous hyphae) White-yellowish with silvery areas, wooly Distance between consecu- tive branches (mm) 0.2-0.3 or less Length of mycorrizal system (mm) 2 0.2 11 0.5-1 19-29 1.2-2 1-4 0.4 5 0.5-0.8 1.5-2.2 0.5 Sympodial, pinnate, sinuous apexes MonopodialSympodial, basal 3constriction 0.1-0.3 velvety Monopodial, pinnate, loose, sinuous branches Monopodial, pinnate, loose Coralloid or pinnate Monopodial, pinnate, sinuous apexes Sulfur yellow Black-purplish to jet black Rusty-black with a pinkish tinge OrangeYellow. CoralloidPale yellow with rusty spots and granulation 6 dissolving in KOH 10% yellow Orange to brown with white laticiferous hyphae Blackish brown with white tinge Mihai Bravu 08.1997 Dobreti 07.1998 Tinca 09.1998 Dobreti 07. 1998 Buteni 06.2000 Giurgiu, Mihai Bravu 08. 1997 Giurgiu, Mihai Bravu 08. 1997 Brganu 08.1997 Brganu 08.1997 (continuation) Quercus cerris + Russula virescens Quercus cerris Quercus frainetto Quercus cerris, Querus robur Quercus cerris + Russula atropurpurea Quercus frainetto+ Xerocomus chrysenteron Quercus Quercus cerris+ Lactarius quietus Quercus cerris + Scleroderma citrinum Code Host Location Color Branching M18 M19 M20 M21 M22 M23 M24 M25 Table 2 Table 62 Fodor et al. Mycorrhizal status of several Quercus species in Romania ... Texture Texture of the rhizomorphs and emergent hyphae smooth hairy smooth smooth smooth ed Rhizomorphs and emergent hyphae Smooth, rami rhizomorphs Fan shaped rhizomorphs Rare rhizomorphs emerging at right angles Bundles of short emerging hyphae Black, rigid emanating hyphae Sclerotia present: black, spherical, grainy, 2 mm, hard and brittle, coated with soil particles Form of the apex, diameter (mm) Round 0.3-0.4 Round 0.3-0.4 Round, slightly bent 0.2-0.3 Round, hyaline 0.3-0.4 Round, in older parts, black Mantle, color, texture Velvety, rare, Velvety, shinning white Granular and spiky due to cystids yellowish Stringy and shiny due to emerging black hyphae, grainy Distance between consecu- tive branches (mm) Length of mycorrizal system (mm) 0.7-1 0.2-0.4 farinaceous 0.8-1 0.3-1 dense Wooly, 4-8 0.1-1 4-6 0.2-0.4 1-2.5 Repeatedly monopodial, pyramidal aspect, bent apexes Monopodial to coralloid, dense, sometimes basal solitary, constriction Dichotomous to coralloid Pinnate pyramidal Unbranched or Monopodial pinnate Yellow-dull brown Yellow-dull White-yellowish, near hyaline Yellow-orange White-yellowish with rusty areas Jet black with sclerotia Radna 10.1998 Radna 10.1998, Oradea 2000 Dobreti 07.1998 Buteni 06.2000 Oradea 06.2000 Buteni 06. 2000, Oradea 06.2000 All locations (continuation) Quercus frainetto Quercus cerris, Quercus robur Quercus cerris + Amanita mairei, Quercus robur Quercus robur All species + Cenococcum geophilum Table 2 Table M26 M27 M28 M29 Code Host Location Color Branching M 30 63 Ann. For. Res. 54(1): 57-71, 2011 Research articles wetter and milder during the winter. In South, alphitoides (Griffon & Maubl.) U.Braun & climate is temperate continental, with pro- S.Takam and infestations produced by gall in- nounced temperature extremes and prolonged sects-Dryomyia circinans (Giraud) (Dobreti) drought during the summer months. Southern and Cynips quercus-folii (Linnaeus) (Oradea), forest stands are located in plains, frequently (v) defoliations produced mainly by Lymantria exposed to summer drought (Giurgiu - Mihai dispar (Linnaeus) (Tinca). Bravu, Brneti, Vlsia - Buriau, Experimen- Root processing protocol. Mycorrhizal tal Station tefneti, Brgan, Vldiceasca, system is de ned as a lateral rami cation and Slveti, Snagov, Comana). Comana and Mi- all its tributary apices from a sustaining suberi- hai Bravu are located an the Neajlov river ed root, total length varying between 1 and 5 delta. The stand age varied between 60 and 90 cm. During the research mycorrhizal root sys- years. tems were studied according to this de nition, Stressful conditions affecting the trees from pieces of 10-20 mm being cut and investigated different investigated locations were assigned for alive and declining mycorrhizal apices. to groups: (i) soil pollution from an oil extrac- Blocks of soil of 5 x 5 x 5 cm were exca- tion plant (Tinca), (ii) several years of recur- vated in the rhizosphere of selected tree host ring drought (Brgan), (iii) multiple sources species (Quercus spp.), in the area of horizon- of city pollution in the city of Oradea, (iv) tal crown projection, three trees of the same chronic foliar disease produced by Erysiphe species per stand. The blocks were wrapped

16 14 12 10 8 6 4

Number of morphotypes 2 0 qft qct qfa qfg qca qcs qfd qcb qcd qco qcv qfsl qcsl qcvl qcba qfmb qcmb Host species

Figure 1 Number of mycorrhizal morphotypes by host (Quercus cerris, Q. frainetto, Q. robur) and location Notation: qca - Quercus cerris, Arad; qcbr - Quercus cerris, Brgan; qcba - Quercus cerris, Bneasa; qcd - Quercus cerris, Dobreti; qcmb - Quercus cerris, Mihai Bravu, qco - Quercus cerris, F.D. Oradea, qcsl - Quercus cerris, Slveti; qcs - Quercus cerris, tefneti; qct - Quercus cerris, Tinca; qcv - Quercus cerris, Vldiceasca; qcvl - Quercus cerris, Vlsia; qfa - Quercus frainetto, Arad (Radna); qfd- Quercus frainetto, Dobreti; qfg - Quercus frainetto, F.D. Giurgiu (Comana); qfmb - Quercus frainetto, Mihai Bravu; qfsl - Quercus frainetto, Slveti; qft - Quercus frainetto, Tinca; qrb - Quercus robur, Buteni; qro - Quercus robur, city of Oradea; qcb - Quercus cerris, Buteni. 64 Fodor et al. Mycorrhizal status of several Quercus species in Romania ...

Table 3 Proportion of mycorrhizal apices in root samples (15 mycorrhizal systems selected at random) col lected during the vegetation season from several Quercus species, different locations (1996-2001) Tree species, location and date Proportion of mycorrhizal apices (%) Q. cerris, tefneti (08.1996) 43.5 Q. cerris, Mihai Bravu (10.1996) 42.0 Q. cerris, Brganu (10.1996) 85.0 Q. frainetto, Comana (10.1996) 86.0 Q. cerris, Tinca (10.1997) 20.0 Q. frainetto, Radna (10.1998) 30.0 Q. frainetto, Tinca (09.1998) 32.4 Q. cerris, Tinca (09.1998) 31.4 Q. frainetto, Mihai Bravu (10.1996) 46.0 Q. cerris, Dobreti (07.1998) 46.3 Q. frainetto, Dobreti (09.1998) 13.1 Q. cerris, Dobreti (07.1998) 67.6 Q. cerris, Oradea (09.1998) 67.6 Q. robur, Buteni (07.2000) 78.0 Q. robur, city of Oradea (06. 2001) 33.0 in paper and brought to the laboratory. The was selected to perform the speci ed statistical samples, corresponding to one tree merged analyses due to the fact that it corresponds to in one composite sample subjected to further the highest diversity of morphotypes found at processing. Roots were carefully washed in any location, at a particular moment. tap water using sieves, placed in Petri dishes The association status of different morpho- of 9 cm and observed under stereomicroscope types with Coenococcum geophilum Fr., most cleaning meanwhile the adhered soil with ne frequently encountered morphotype, was as- brushes and needles. The descriptions of the sessed using Yule coef cient of association. morphotypes are based on macroscopic char- Yule’s Q coef cient of association is a sym- acters following the adapted protocol after metric measure, based on the difference be- Agerer (1987-2002). Actual mycorrhizas were tween concordant (meaning both absences a, con rmed microscopically by the existence of and both presences, d) and discordant (mean- the Hartig net and mycorrhizal mantle (Nylund ing absence-presence data, b and presence-ab- et al., 1982). sence data, c) data pairs (Singh, 2004). Quantitative analysis. Comparison of the mycorrhizal status (relative frequencies of Q = (ad-bc)/(cd+bc) active mycorrhizal apices) in 5 root samples taken from Quercus cerris at Dobreti was The coef cient takes values between -1 and performed by means of One-Way ANOVA. A 1: -1 corresponds to a complete exclusion of previous test of variance homogeneity (Bar- the species, 0 corresponds to random associa- tlett) con rmed the lack of signi cant differ- tions and 1 to constant associations. Q statistic ences with respect to the variances among de nes null relationship as statistical inde- samples. Also Tukey test of pair-wise multiple pendence. comparisons was performed in order to detect Hierarchical cluster analysis of tree species any signi cant differences in mycorrhization and locations, with regard to mycorrhizal mor- frequency in the 5 samples set collected from photypes, was performed after the calculation Quercus cerris roots at Dobreti. This location of the Sørensen similarity index. The similar-

65 Ann. For. Res. 54(1): 57-71, 2011 Research articles

ity-dissimilarity matrix was used for clustering m30 by means of group average method. Statistical tests were performed using KyPlot program. m29 m28 m27 Results m26 There were identi ed 30 morphotypes of ecto- m25 mycorrhizae, corresponding to Quercus robur, m24 Quercus cerris and Quercus frainetto in South- m23 ern, Western and North-Western locations, presented in Table 1. The original descriptions m22 (with the exception of Cenoccocum geophilum m21 described by Agerer in 1987), are presented in m20 Table 2. m19 The investigation of the maximum number of morphotypes active a certain moment in the m18 rhizosphere of the host is 14 (Fig. 1) which is m17 the real instantaneous value of morphotype m16 richness (Q. cerris at Dobreti). The described morphotypes include only 6 m15 cases of identi ed mycobionts (Amanita mairei m14 Foley, Scleroderma citrinum Pers., Russula m13 atropurpurea (Krombh.) Britzelm, Lactarius m12 quietus (Fr.) Fr., Russula virescens (Schaeff.) Fr., Boletus chrysenteron Bull.) based on myc- m11 elial continuity between mycorrhizal man- m10 tle and the mycelium at the base of the car- m9 pophores. Easily recognizable Cenoccocum m8 geophilum Fr. is also included in morphotype descriptions. Mantles of mycorrhizas with spe- m7 cies of Lactarius as mycobionts (as examined m6 on cross sections of ne roots) exhibit charac- m5 teristic laticifers and pigments which are also m4 found in the structure of the sporocarps. Those of Rusulla spp. exhibit distinctive cystidia and m3 sulfovaniline reactive cells (Kernaghan and m2 Currah 1997). m1 The analysis of frequency distribution of the various morphotypes (Fig. 2) shows that the 01020 dominating type is Cenococcum geophilum (M30) that is present in all locations. Next Figure 2 Frequencies of mycorrhizal morpho- most frequent morphotype is M2 described in types in root samples of Quecus cerris, Q. robur and Q. cerris at Buteni, Oradea and Quercus frainetto and Quercus robur Slveti but found in almost all locations less in several locations in Southern and frequently than C.geophilum. North-Western Romania 66 Fodor et al. Mycorrhizal status of several Quercus species in Romania ...

Table 4 The Yule coef cient of association of Cenococcum geophilum with several other mycorrhizal mor photypes in Quercus cerris, Dobreti Association Yule coef cient of association C. geophilum + M10 -0.8004 C. geophilum + M17 -0.1287 C. geophilum + M8 0.2564 C. geophilum + M3 0.2467 C. geophilum + M11 -0.0943

C. geophilum associates either randomly A distinct cluster is formed by hosts located with other morphotypes in the same mycor- in forest stands from Southern Romania where rhizal system (values close to 0), or excludes these are vegetating in the plain and are ex- other morphotypes (value close to -1 in the as- posed to temperate-continental climate with sociation with M10) according to our results, a harsh winters and dry summers. Another dis- pattern consistent with its dominating position. tinct cluster is formed by hosts vegetating in It is also the most frequent and broad spectrum stands from Mihai Bravu, in the Neajlov river host associated mycorrhizal type as other au- delta, characterized by wetter soil conditions thors report (Moser et al. 2005). as compared to other Southern locations. How- The comparison of the mycorrhizal status ever, Q. frainetto from Comana, a nearby lo- (relative frequencies of active mycorrhizal api- cation, to Mihai Bravu clusters together with ces) among 5 samples of roots from Quercus other Southern locations characterized by cerris at Dobreti by of one way ANOVA re- drier soil conditions. North Western locations sulted in no signi cant differences at p > 0.05. cluster, together presents same pattern of as- Also Tukey test for pair-wise multiple compar- sociation with their mycobionts, including as isons didn’t reveal any signi cant differences location Tinca and Oradea. Quercus robur is between samples. highly dissimilar to other host species with Søresen similarity index (Table 4) calculated regard to associated mycobionts and clusters in order to compare locations and Quercus separately. Recurrent associations, in this case species (within site and between sites) show of ectomycorrhizal mophotypes, correspond maximum values in the following cases: Quer- to fundamental properties of the interaction cus frainetto at Tinca with Quercus cerris at between species and their physical (site) and Oradea (0.72), Quercus robur at Buteni with biotic environment (hosts) (Legendre & Leg- Quercus robur in the city of Oradea (0.61), endre, 1998). Quercus cerris and Quercus frainetto at Mihai Previous studies (Timofte 2007) reported the Bravu (0.57). possibility to obtain micropropagated plantlets The analysis of the dendrogram resulted of Q. robur and Q. frainetto by means of so- from the ordination of Sørensen similarity ma- matic embryogenesis initiated from acorns. trix, comparing different sites and tree hosts The embryos can be converted to plantlets with regard to identi ed morphotypes (Fig. 3), which, theoretically, can be outplanted. re ects the association between similar sites within same geographical and meso-climatic area. Within same location, different host spe- Discussion cies are highly similar with respect to their mycorrhizal associates, such as Q. cerris and Q. The energy ow through the mycorrhizal net- frainetto at Dobreti or Tinca (South-West at works represents a major food chain in the for- Mihai Bravu). est trophic web. The network represented by 67 Ann. For. Res. 54(1): 57-71, 2011 Research articles species Quercus Quercus Locations and

Distance

Figure 3 Cluster Analysis of mycorrhizal morphotypes associated with Quercus cerris, Q. frainetto and Q. robur using Sørensen Similarity Index and Average Linkage Method. Notation: 1 - Quercus robur, Buteni; 2 - Quercus robur, Oradea; 3 - Quercus cerris, Brneti; 4 - Quercus frainetto, Mihai Bravu; 5 - Quercus cerris, Mihai Bravu; 6 - Quercus frainetto, Dobreti; 7 - Quercus cerris, Dobreti; 8 - Quercus cerris, Tinca; 9 - Quercus frainetto, Tinca; 10 - Quercus cerris, Oradea; 11 - Quercus cerris, Arad; 12 - Quercus frainetto, Arad (Radna); 13 - Quercus cerris, tefneti; 14 - Quercus cerris, Vlsia; 15 - Quercus cerris, Slveti; 16 - Quercus frainetto, Slveti; 17 - Quercus cerris, Vldiceasca; 18 - Quercus cerris, Bneasa; 19 - Quercus frainetto, Comana (Giurgiu); 20 - Quercus cerris, Brgan. mycelia and assimilative roots associated in number of active morphotypes associated to a mycorrhizae operates in allocations and real- host species, in a particular location, was of 14 locations of nutrients, water, in blocking the (Quercus cerris at Dobreti), a fact that is con- potential root infection courts relying on the cordant with the hypothesis of redundant spe- diversity of mycobionts, each performing a cies as functional insurance (Yachi & Loreau, different job. The identi ed 30 morphotypes, 1999). There is a sequential mobilization of associated with species of Quercus, are as- mycorrhizal fungi from the common species signed differently, depending on geographical pool, active within a particular time window. distribution of hosts, on health status (there are The degree of mycorrhization differs among fewer root active apices in trees vegetating un- trees, parts of the root system, and phenologi- der stressful conditions) and host species. cally. Our ndings revealed a uctuation in The niche partitioning among several myco- frequencies of mycorrhizal apices between bionts is possible because the ectomycorrhizal 13.1% and 86% during the same period. fungi differ in their tolerance to water stress One of our ndings during the study is the and temperature extremes, in their ability to dominance of C. geophilum morphotype, a take up different nutrients, their resistance to result shared with other similar studies. Ger- pathogens (Jones et al. 1998). The instantane- hardt et al. (2007) found that this is the domi- ous richness value, for morphotypes active at nating morphotype in 80% of the investigated a certain moment on the same host species is Quercus rubra stands also the most abundant far less than the total richness of the existing mycorrhiza forming mycobiont on the roots mycobionts, associated with a host, as can be of Quercus garryana (Valentine et al. 2004). revealed by molecular methods. During the in- C. geophilum is a pioneer stage mycorrhizal vestigations of the present study, the maximum partner but, also a late stage, being present 68 Fodor et al. Mycorrhizal status of several Quercus species in Romania ... in seedling mycorrhizae as well as in mature in uenced the mycorrhizal status of Q. cerris trees’ roots (Piggott 1982, Kranabetter & and Q. frainetto, re ected in low frequencies Wylie 1998) associating with 122 host species of mycorrhizal apices. (Trappe 1964). The outer layer of the mantle Modern investigations on the diversity of consists of thick walled cells, resembling the mycorrhiza, associated with a particular tree gelatinous walls of many lichenicolous fungi. host, rely on DNA nger-printing. This diver- When wet, these walls become gelatinous, sity assessment takes into account all poten- providing an environment for water storage, tial mycobionts associated with a tree and not which is of capital importance during the those active at a particular moment. For active drought episodes. This pecularity gives an ad- mycorrhiza at a time snapshot, the classical as- vantage to seedlings possessing C. geophilum sessment based on morphotypes is a better ap- mycorrhiza (Piggot 1982). Being ubiquitous by proach on our opinion based on the presented nature, C. geophilum links plants in a common results. mycorrhizal network (Valentine at al. 2004). It These results lead to the idea that arti cial is an early stage and also a late stage associ- inoculation with site-adapted mycobionts ate in seral succession of the forests. Late stage would enhance plant growth and survival af- fungi, such as Lactarius spp. and Russula spp. ter outplanting, an opinion shared with other are infrequent on roots and the inoculation authors (e.g. Gerhardt et al. 2007). The myc- produces via root connections, rather than by orrhization is induced either under laboratory mycelial fragments or spores as in early stage conditions on dual host-fungus systems, either mycorrhiza (Jones et al. 1998). during the seedlings growth by inoculation of The association of different morphotypes is the fungal inoculum in nursery containers, the more or less random, with respect to closely inoculum being represented by spores, myc- related hosts in the same location and show elia or simply, fragments of appropriate carpo- relatively high similarity between locations phores (Martinez-Amores et al. 1991). The mi- related to the same host species. Still the null cropropagated plantlets of Quercus species can hypothesis of random association is to be care- be exposed to mycobionts in order to induce fully veri ed on larger sets of data. The gener- mycorrhization. In our opinion, a good candi- al trend is a variation of morphotypes, accord- date is Cenococcum geophilum, due to its large ing to geographical factors (Southern versus ecological and host range, and the capacity to North-Western Romania) and also to related induce frequent mycorrhizal tips, a hypothesis hosts allocation (Quercus spp.). There is an worth to test under experimental conditions. evidence of within site partition of the same As a consequence, it is a good candidate for morphotypes among Quercus hosts and simi- mycorrhization in dual systems (oak microcut- larity of sites from same geographical area. It tings or micropropagated plants + mycobiont) is worth to mention the dissimilarity in mor- in Petri dishes, a similar approach being pro- photype allocation in Q. robur as compared to posed by Herrmann et al. (1998). Q. cerris and Q. frainetto. Stressful conditions affect trees in urban areas, a fact re ected in their mycorrhizal status, Conclusion and in lower frequency of mycorrhizal apices. Also, carpophore production is a seldom event The investigation of mycorrhizae using the in urban areas (Danielson & Pruden 1989, Bax- classical approach of morphotype description ter et al. 1999) never observed in present sur- yielded 30 types common for Quercus robur, vey (from 1997 to 2008) in the city of Oradea. Q. cerris, Q. frainetto. The original descrip- Drought and oil pollution reported from Tinca tions of the morphotypes provide a recognition

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