ACTA AGRARIA ET SILVESTRIA

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1 POLSKA AKADEMIA NAUK ODDZIAŁ W KRAKOWIE KOMISJA NAUK ROLNICZYCH I LEŚNYCH ACTA AGRARIA ET SILVESTRIA SERIES SILVESTRIS Vol. XLV, 2007 WYDAWNICTWO ODDZIAŁU POLSKIEJ AKADEMII NAUK KRAKÓW

2 KOMITET REDAKCYJNY Władysław Filek, Andrzej Jaworski, Janusz Rząsa Jerzy Starzyk, Kazimierz Zarzycki przewodniczący REDAKTOR SERII Andrzej Jaworski ADRES REDAKCJI Katedra Szczegółowej Hodowli Lasu Akademii Rolniczej w Krakowie Kraków, al. 29 Listopada 46 REDAKTOR TOMU Krystyna Duszyk Wydanie publikacji dofinansowane przez Ministerstwo Nauki i Szkolnictwa Wyższego Copyright by Authors, Polska Akademia Nauk Oddział w Krakowie Kraków 2008 Wydawnictwo Oddziału Polskiej Akademii Nauk Kraków, ul. św. Jana 28 tel.: (012) w. 22, fax: (012) Objętość ark. wyd. 7; ark. druk. 8

3 SPIS TREŚCI CONTENTS STEFAN KOWALSKI, ELŻBIETA CHOMICZ, MAŁGORZATA CZEREMUGA, MARTA PIECHA: Selected Hebeloma species in ectomycorrhizal synthesis with scots pine (Pinus sylvestris L.) and pedunculate oak (Quercus robur L.) in container nursery Wybrane grzyby z rodzaju Hebeloma P. Kumm. w syntezie ektomikoryzowej z sosną zwyczajną (Pinus sylvestris L.) i dębem szypułkowym (Quercus robur L.) w szkółce kontenerowej JULITTA GAJEWSKA, JOANNA KOSTECKA, LESZEK BABIŃSKI: Mikroorganizmy zasiedlające współczesne drewno sosny (Pinus sylvestris L.) w glebie torfowej stanowiska archeologicznego w Biskupinie Microorganisms colonizing a contemporary pine wood (Pinus sylvestris L.) stored in a peat soil of the archeological site in Biskupin JAN KARCZMARSKI, PIOTR KOWALCZUK: Budowa, struktura i dynamika górnoreglowego boru świerkowego o charakterze pierwotnym w rezerwacie Romanka w Beskidzie Żywieckim (w okresie kontrolnym ) Structure and dynamics of upper mountane belt primeval spruce stand in the Romanka reserve Beskid Żywiecki Mountains (in the period ) GRZEGORZ DURŁO: Wieloletnia zmienność wilgotności względnej powietrza w Leśnym Zakładzie Doświadczalnym w Krynicy Zdroju The multiannual changeability of air relative humidity in Forest Experimental Station in Krynica Zdrój JÓZEF SULIŃSKI: Metoda obliczania rocznej produkcji biomasy w zbiorowisku leśnym w zależności od wysokości i wieku drzewostanu The tree-stand height and age based method for calculation of annual biomass production in a forest community JÓZEF SULIŃSKI: Maciej Sławomir Czarnowski ( ) leśnik-ekolog wspomnienie na 10. rocznicę śmierci

4 ACTA AGRARIA Series Silvestris ET SILVESTRIA Vol. XLV, 2007 PL ISSN SELECTED HEBELOMA SPECIES IN ECTOMYCORRHIZAL SYNTHESIS WITH SCOTS PINE (PINUS SYLVESTRIS L.) AND PEDUNCULATE OAK (QUERCUS ROBUR L.) IN CONTAINER NURSERY Stefan Kowalski Elżbieta Chomicz Małgorzata Czeremuga Marta Piecha Katedra Fitopatologii Leśnej Akademia Rolnicza al. 29-Listopada 46 PL Kraków ABSTRACT S. Kowalski, E. Chomicz, M. Czeremuga, M. Piechna Selected Hebeloma species in ectomycorrhizal synthesis with Scots pine (Pinus sylvestris L.) and pedunculate oak (Quercus robur L.) in container nursery. Acta Agr. Silv. ser. Silv. 45: The impact of artificial mycorrhization with 6 vegetative inocula (produced by research procedures at the Forest Pathology Department at Agricultural University in Cracow) of ectomycorrhizal fungi: Hebeloma claviceps, H. leucosarx, H. longicaudum, H. sacchariolens, H. sinapizans and H. trunkatum on following parameters: the level of mycorrhization of Scots pine and pedunculate oak seedlings, the profile of frequency and types of ectomycorrhizae on these seedlings, the influence of intervention on the growth and development characteristics of shoot and root system was studied. The results of the mycorrhization described above were compared with the mycorrhization with Polish commercial vegetative inoculum of Hebeloma crustuliniforme carried out on the seedlings of the same tree species grown in containers. The assessment revealed that all fungi species representing Hebeloma genus were very good symbionts for pine. The index of mycorrhization of the root system in each case was high and achieved level between 0,86 for H. longicaudum to 1,0 for H. sacchariolens and H. claviceps. The growth and development of oak seedlings in container culture were improved in the highest degree by H. crustuliniforme, H. leucosarx, H. sinapizans and H. sacchariolens. KEY WORDS: ectomycorrhizal synthesis, container nursery, Pinus sylvestris, Quercus robur, Hebeloma claviceps, H. crustuliniforme, H. leucosarx, H. longicaudum, H. sacchariolens, H. sinapizans, H. truncatum SŁOWA KLUCZOWE: syntezy ektomikoryzowe, szkółki kontenerowe, Pinus sylvestris, Quercus robur, Hebeloma claviceps, H. crustuliniforme, H. leucosarx, H. longicaudum, H. sacchariolens, H. sinapizans, H. truncatum

5 4 I. INTRODUCTION The quality of planting stock should be assessed not only by its growth parameters but also on the basis of the presence of proper mycorrhiza on roots. This is due to the fact that forest trees in our climatic zone form ectomycorrhizal associations to secure the optimal growth and development (Dominik 1961, Kowalski 1997, Pachlewski 1967, Rudawska 2000). Ectomycorrhizae perform important functions in protection of trees against environmental stresses such as drought or heavy metal pollution, and they also play a very important role in nutritious and physiological processes of trees, as well as in formation of their disease predisposition to pathogens (Boyd and Hellebrand 1991, Branzanti et al. 1999, Leyval et al. 1997, Marx 1972, Parke et al. 1983). In most Polish forest nurseries seedlings usually have a proper mycorrhiza. The problem occurs in situations when seedlings are grown on various substrates devoid of ectomycorrhizal fungi, or on soils with disturbed microbiological relations and devoid of fungi able to make proper ectomycorrhizal associations, specific for a given tree species. This is especially true in the case of production of planting stock in container nurseries (Kowalski 1998). On the other hand, in this kind of production there is the best chance for effective artificial mycorrhization with ectomycorrhizal fungi selected in laboratories. The artificial mycorrhization with the vegetative fungal inoculum to be effective requires a proper selection made among potential ectomycorrhizal fungi in respect of the agreement with the host and the ability to produce mycorrhizae (Pachlewski 1983). Only strains selected in such a way may be taken into account in production of the commercial vegetative inoculum. In container nurseries of State Forests the Polish technology of artificial mycorrhization of forest tree seedlings with fungus Hebeloma crustuliniforme has been implemented since 2000 (Berf 2007). Presently, there is the need to widen the spectrum of ectomycorrhizal fungi which may be used in effective artificial mycorrhization of seedlings of various tree species grown under different ecological conditions. The purpose of this study was to determine the impact of artificial mycorrhization with vegetative inocula of 6 ectomycorrhizal fungi: Hebeloma claviceps, H. leucosarx, H. longicaudum, H. sacchariolens, H. sinapizans, and H. trunkatum on the following parameters: the level of mycorrhization of Scots pine and pedunculate oak seedlings, the frequency and types of ectomycorrhizae on these seedlings, and the effect of this treatment on the growth and development characteristics. The results of the mycorrhization described above were compared with the mycorrhization of Scots pine and pedunculate oak seedlings grown in containers and inoculated with the Polish commercial vegetative inoculum of H. crustuliniforme.

6 5 II. MATERIAL AND METHODS Field studies were carried out in the Forest Container Nursery in Nędza, situated in the Rudy Raciborskie Forest District, while laboratory work was completed in the Department of Forest Pathology, Agricultural University of Cracow. Seedlings were grown on the substrate composed of highmoor peat deacidified with dolomite, and containing vermiculite (20%), pearlite (10%), and the fertilizer Osmocote (3.0 kg/1m 3 ). Peat, before addition of other components, was sterilized with hot steam (in 95 C). The introduction of inocula of various species of ectomycorrhizal fungi from the genus Hebeloma was carried out using the same method as in the case of the vegetative inoculum of H. crustuliniforme according to the Polish technology of artificial mycorrhization of forest tree seedlings (Kowalski 2007). The percentage by volume of the vegetative inoculum in the substrate varied, and it was 2.5% for H. crustuliniforme, 2.0% for H. longicaudum, 3.5% for H. truncatum, 5.0% for H. sacchariolens, 5.0% for H. sinapizans, 6.5% for H. leucosarx, and 7.0% for H. claviceps. All these inocula were used in artificial mycorrhization of seedlings of Scots pine and pedunculate oak. The germinative energy of both species was 95%. Scots pine was grown in containers V 120, and pedunculate oak in containers V 265. In each experimental variant 560 Scots pine seedlings and 364 pedunculate oak seedlings were grown. In total the experiment included 3920 Scots pine and 2548 pedunculate oak seedlings. Seeds of both tree species were sown in containers on 19 March For two months seedlings were grown in the plastic tunnel, and on 20 May 2004 containers were transferred to the field and there they were kept for next five months. Foliar fertilization of seedlings was carried out with Ekor, the fertilizer of conductivity 0.6 ms. This treatment was performed 12 times (25 June, 29 June, 13 July, 15 July, 19 July, 23 July, 26 July, 30 July, 6 August, 9 August, 16 August, and 20 August) in the case of Scots pine, and 9 times (30 June, 13 July, 15 July, 19 July, 23 July, 26 July, 3 August, 6 August, and 20 August) in the case of pedunculate oak. Prophylactic protection treatments were performed against pine needle cast and oak mildew, and control treatments against oak mildew, aphids, and liverworts. In the second week of October 2004 first measurements of seedlings were made. For this purpose 100 Scots pine seedlings were selected from each variant using the same sampling scheme. In the case of pedunculate oak 50 seedlings were selected from each variant rejecting those which originated from seeds germinating late, and which did not reach the minimum height of 12 cm. Height of seedlings was measured exact to 0.1 cm, and the root collar diameter exact to 0.01 mm. The next step was to estimate the degree of surface cover of the root ball with mycelium using a 5-degree scale (0 no

7 6 mycelium present, 1 to 25% of root ball surface covered by mycelium, 2 to 50%, 3 to 75%, 4 to 100%). The surface cover of the root ball with mycelium of respective Hebeloma species was estimated visually. Thus, this estimation was only the additional indication of fungal development within root systems of seedlings grown in containers. In the second half of October Scots pine and 14 pedunculate oak seedlings were taken from each experimental variant for the laboratory analysis. Scots pine seedlings were randomly selected from all containers, while in the case of pedunculate oak, first seedlings with higher shoot parameters were separated, and then from these seedlings a random sample was taken. Such a procedure in the case of oak aimed at limitation of characteristic variation in a sample resulting from later (even a month later) seed germination. In the laboratory, roots were washed with water, and then placed in preserving liquid in marked flasks (Kowalski 1974). In the case of each seedling, length of the main root was measured, and lateral roots of orders I, II, and III were counted. From different places of each root system twenty 5-cm-long sections of roots of the last orders were cut to determine kinds and frequency of mycorrhizae. In total the morphological analysis of ectomycorrhizae was carried out on a current meter of roots of each seedling in each experimental variant. In each section, using a binocular magnifier, living mycorrhizae and autotrophic (non-mycorrhizal) roots were counted. On the basis of the quotient of the mycorrhizal apexes and the total number of short roots of all seedlings the degree of mycorrhization of root systems was determined in each experimental variant (Aleksandrowicz-Trzcińska 2001). Individual morphological forms of mycorrhizae were counted separately, distinguishing single, dychotomic, and multiple forms in Scots pine, and single and monopodial forms in pedunculate oak. The ectomycorrhizae of morphological and anatomical features typical for the symbiosis of Scots pine and pedunculate oak with fungi from the genus Hebeloma were separated from ectomycorrhizae of other kinds. During anatomical studies the cross sections of ectomycorrhizae were examined under the microscope using magnification of 400. The Hartig's net, thickness of the mycelial mantle, and the structure of hyphae were analyzed. After completion of morphological and anatomical analyses, roots of seedlings were dried for 48 hours in temperature of 60 C in the drier, and then weighed using an electronic balance. The weight was determined in grams exact to gram. The statistical analysis was carried out using program Statistica 6.1. If variables were fulfilling conditions for using ANOVA the analysis of variance was carried out, and if not, the non-parametric Kruskala-Wallis test and the medians were used. Results are presented in tables.

8 7 III. RESULTS In the case of Scots pine seedlings, the variation of measurable parameters of over-ground portions, irrespective of fungal species used in mycorrhizal synthesis, was relatively small, but the Kruskala-Wallis test showed statistical significance of these differences. In the case of seedlings inoculated with H. crustuliniforme their height and root collar diameter were statistically significantly lower than those of seedlings inoculated with other Hebeloma species. Much greater differences were found in root structure, especially in the number of roots of order III, on which most of ectomycorrhizae occurred (Tab. 1). However, when analyzing the structure of the root system no relationship was found between the amount of the vegetative inoculum in the substrate and the structure of the root system of Scots pine seedlings investigated (Tab. 1). Taking into account the total number of roots of all orders it was possible to distinguish three groups of seedlings of similar parameters. The first group included seedlings inoculated with H. longicaudum, the second group seedlings inoculated with H. crustuliniforme, H. leucosarx, and H. claviceps, and the third group seedlings inoculated with H. truncatum, H. sacchariolens, and H. sinapizans. Seedlings of the third group were characterized by a relatively little branched root system (Table 1). However, this fact was not connected with the mean dry matter of roots which was the highest in Scots pine seedlings inoculated with H. claviceps. The differences in this respect were not significant statistically. Relatively greatest values of height and root collar diameter were found in seedlings inoculated with H. truncatum, but at the same time in this case the mean number of lateral roots and the mean root dry matter were the lowest (Tab. 1). Also no relationship between the dry matter of roots and the percentage of the vegetative inoculum in the substrate was found. However, the percentage of the vegetative inoculum in the substrate had a distinct effect on the degree of overgrowing of the root ball with mycelium of respective Hebeloma species (Tab. 1). This degree also depended on the fungal species, as it may be seen when comparing percentages of seedlings with roots overgrown with mycelium up to 100% (degree 4) in the case of H. sacchariolens and H. sinapizans, and also in the case of H. crustuliniforme and H. longicaudum (Tab. 1). This study showed that all Hebeloma species investigated were very good symbionts for Scots pine because the index of mycorrhization of the root system was in each case very high, varying from 0.86 for H. longicaudum to 1.0 for H. sacchariolens and H. claviceps (Tab. 2, Phots. 1 and 2). Taking into account values of all measurable parameters it may be assumed with a certain amount of reservation that H. claviceps, H. sacchariolens, and H. crustuliniforme had a relatively most favorable effect on growth and development of Scots pine seedlings grown in containers (Tabs. 1 and 2). This reservation mainly concerns the fact that the vegetative inoculum content in the substrate varied in individual experimental variants.

9 Table 1 Tabela 1 8 Mean values parameters of Scots pine seedlings grown in containers and inoculated with vegetative inocula of various Hebeloma fungi Średnie parametry sadzonek sosny zwyczajnej hodowanej w pojemnikach i szczepionej biopreparatem z różnymi gatunkami grzybów z rodzaju Hebeloma Percentage contents of vegetative inoculum of Hebeloma fungi in rooting medium Udział biopreperatu z grzybami z rodzaju Hebeloma w substracie hodowlanym [%] The height of seedling Wysokość sadzonki [cm] The diameter in root collar Średnica w szyi korzeniowej [mm] Lenght of main root Długość korzenia głównego [cm] The number of lateral roots of apprioprate order I III Liczba korzeni bocznych rzędu I III Dry root weight Sucha masa korzeni [g] Percentage of seedlings with root ball overgrown with mycelium in degree 0 4 Udział sadzonek z bryłką korzeniową przerośniętą grzybnią w stopniu 0 4 [%] I II III H. crustuliniforme a 2.8 a 10.7 a 43 ab 184 c 132 abc a H. claviceps bc 3.3 bc 11.6 a 37 ab 139 abc 154 bc a H. leucosarx b 3.4 bc 12.0 a 33 a 130 ab 124 ab a H. longicaudum bc 3.3 bc 11.0 a 45 b 165 bc 193 c a H. sacchariolens bc 3.3 bc 11.7 a 35 ab 110 a 94 ab a H. sinapizans b 3.1 ab 12.0 a 33 a 94 a 88 ab a H. trancatum c 3.5 c 11.0 a 33 a 105 a 77 a a Dla każdego gatunku grzyba wartości średnie w poszczególnych kolumnach oznaczone różnymi literami różnią się istotnie statystycznie (P 0,05) For each fungal treatment, means in the same column followed by a different letter are statistical significantly different (P 0.05)

10 Table 2 Tabela 2 Mean number of trophic roots in Scots pine seedlings grown in containers and inoculated with vegetative inocula of various Hebeloma fungi Średnia liczba korzeni troficznych u sadzonek sosny zwyczajnej hodowanej w pojemnikach i szczepionej biopreparatem z różnymi gatunkami grzybów z rodzaju Hebeloma Percentage contents of vegetative inoculum of Hebeloma fungi in rooting medium Udział biopreperatu z grzybami z rodzaju Hebeloma w substracie hodowlanym [%] Number of autotrophic roots Liczba korzeni autotroficznych The number of living mycorrhizae Liczba mikoryz żywych singular pojedynczych dychotomic dychotomicznych multifolded wielokrotnych total razem The number of ectomycorrhizae from infection Liczba ektomikoryz z infekcji planned with Hebeloma fungi zamierzonej grzybami z rodzaju Hebeloma accidental przygodnej Index of root mycorrhization with Hebeloma fungi Wskaźnik zmikoryzowania korzeni grzybami z rodzaju Hebeloma H. crustuliniforme H. claviceps H. leucosarx H. longicaudum H. sacchariolens H. sinapizans H. trancatum

11 10 Phot. 1. One-year Scots pine seedling with Hebeloma crustuliniforme fruit body and ectomycorrhizae on the surface of root ball Fot. 1. Roczna sadzonka Pinus sylvestris z owocnikiem grzyba Hebeloma crustuliniforme i ektomikoryzami na powierzchni bryłki korzeniowej In the case of pedunculate oak the measurable parameters of the over-ground portion and the root system of seedlings were distinctly more diversified than in Scots pine (Tab. 3). In seedlings inoculated with H. crustuliniforme height and root collar diameter were significantly greater than in seedlings inoculated with other Hebeloma species. Taking into account the mean values of measurable parameters of shoots and roots of oak seedlings it may be seen that the highest values were reached by seedlings inoculated with H. crustuliniforme, and then

12 11 Phot. 2. One-year Scots pine seedling with Hebeloma claviceps fruit body and ectomycorrhizae on the surface of root ball Fot. 2. Roczna sadzonka Pinus sylvestris z owocnikiem grzyba Hebeloma claviceps i ektomikoryzami na powierzchni bryłki korzeniowej with H. sacchariolens and H. claviceps (Tab. 3). However, when mean values of shoot parameters are compared separately from root parameters then it may be seen that the effect of artificial mycorrhization differed, although each time 3 groups of seedlings may be distinguished (Tab. 3). In both cases seedlings inoculated with H. crustuliniforme should be included in group 1, those inoculated with H. sacchariolens in group 2, and seedlings inoculated with H. leucosarx in group 3 (Tab. 3). In the case of the remaining species of fungi

13 Table 3 Tabela 3 Mean values of parameters of pedunculate oak seedlings grown in containers and inoculated with vegetative inocula of various Hebeloma fungi Średnie parametry sadzonek dębu szypułkowego hodowanego w pojemnikach i szczepionego biopreparatem z różnymi gatunkami grzybów z rodzaju Hebeloma 12 Percentage contents of vegetative inoculum of Hebeloma fungi in rooting medium Udział biopreperatu z grzybami z rodzaju Hebeloma w substracie hodowlanym [%] The height of seedling Wysokość sadzonki [cm] Mean diameter in root collar Średnica w szyi korzeniowej [cm] Lenght of main root Długość korzenia głównego [cm] The number of lateral roots of apprioprate order I III Liczba korzeni bocznych rzędu I III Dry root weight Sucha masa korzeni [g] Percentage of seedlings with root ball overgrown with mycelium in degree 0 4 Udział sadzonek z bryłką korzeniową przerośniętą grzybnią w stopniu 0 4 [%] I II III H. crustuliniforme ,2 b 7.3 c 16.2 ab 186 b 1676 b 108 ab a H. claviceps a 5.4 b 14.9 ab 143 a 924 a 196 ab a H. leucosarx a 4.9 ab 15.7 ab 129 a 985 a 140 ab a H. longicaudum a 4.8 a 15.3 ab 144 ab 1100 a 208 ab a H. sacchariolens a 5.1 ab 14.7 ab 160 ab 1304 ab 100 ab a H. sinapizans a 4.8 a 16.8 b 132 a 1350 ab 230 b a H. trancatum a 5.2 ab 14.2 a 132 a 1025 a 71 a a Dla każdego gatunku grzyba wartości średnie w poszczególnych kolumnach oznaczone różnymi literami różnią się istotnie statystycznie (P 0,05) For each fungal treatment, means in the same column followed by a different letter are statistical significantly different (P 0.05)

14 seedlings obtained higher parameters in the case of the over-ground portion or higher parameters in the case of the root system (Tab. 3). The fungal species had a considerably greater influence on the degree of cover of the root ball with mycelium than the percentage of the vegetative inoculum in the substrate. In pedunculate oak seedlings inoculated with H. claviceps, the percentage of which in the substrate was the highest (7%), the percentage of seedlings with degree 4 cover of the root ball with mycelium was only 12%, while in seedlings inoculated with H. sinapizans, the percentage of which in the substrate was smaller (5%), the percentage of seedlings with degree 4 cover of the root ball with mycelium reached the highest value, i.e. 64% (Tab. 3). This observation was also confirmed when the degree of the root ball cover with mycelium of H. sacchariolens was compared with that of H. sinapizans. In both these cases the percentage of the inoculum in the substrate was similar (5%), while the percentage of seedlings with degree 4 cover of the root ball with mycelium was distinctly different (Tab. 3). The comparison of the mean values of dry matter revealed that the highest values of this parameter were found in the case of seedlings inoculated with H. crustuliniforme and H. claviceps (Tab. 3). In the latter case, however, the index of mycorrhization of roots was distinctly smaller and amounted to 0.49 (Tab. 4). The lowest mean parameters of dry matter of roots were found for pedunculate oak seedlings inoculated with H. leucosarx and H. sinapizans, but at the same time in both these cases the index of mycorrhization of the root system was the highest, i.e and 0.81 respectively (Tabs. 3 and 4). The lowest index of root mycorrhization was found for pedunculate oak seedlings inoculated with H. longicaudum, i.e. only 0.18 (Tab. 4). However, this little satisfactory result could have been connected with low amount of the vegetative inoculum in the substrate, which in the case of this fungus was the smallest, i.e. only 2% (Tab. 4). Taking into account mean values of all measurable parameters it may be assumed that growth and development of pedunculate oak seedlings grown in containers were the most favorably affected by H. crustuliniforme, H. leucosarx, H. sinapizans, and H. sacchariolens (Tabs. 3 and 4). Within one tree species, being in symbiosis with respective Hebeloma fungi, the morphological forms of mycorrhizae were little diversified. In the case of Scots pine seedlings, beside single mycorrhizae which dominated, the percentage of complex forms (dichotomic and multiple) was relatively high ranging from 22.8% in H. truncatum to 36.6% in H. crustiliniforme, and only in H. claviceps it was distinctly lower, i.e. 11.4% (Tab. 2, Phot. 3). The young mycorrhizae were relatively short and clavate, while the older ones were elongated and more or less of equal thickness along their entire length. Some were segmented. Initially, they were light cream in color, covered with a delicate silky colorless mycelium. The ripe ones had a distinct white mycelial mantle, from which numerous colorless silky hyphae of the mycelium were growing into the 13

15 Table 4 Tabela 4 Mean number of throphic roots in pedunculate oak seedlings grown in containers and inoculated with vegetative inoculum of various Hebeloma fungi. Średnia liczba korzeni troficznych u sadzonek dębu szypułkowego hodowanego w pojemnikach i szczepionego biopreparatem z różnymi gatunkami grzybów z rodzaju Hebeloma 14 Percentage contents of vegetative inoculum of Hebeloma fungi in rooting medium Udział biopreperatu z grzybami z rodzaju Hebeloma w substracie hodowlanym [%] Number of autotrophic roots Liczba korzeni autotroficznych The number of living mycorrhizae Liczba mikoryz żywych singular pojedynczych monopodial monopodialnych total razem The number of ectomycorrhizae from infection Liczba ektomikoryz z infekcji planned with Hebeloma fungi zamierzonej grzybami z rodzaju Hebeloma accidental przygodnej Index of root mycorrhization with Hebeloma fungi Wskaźnik zmikoryzowania korzeni grzybami z rodzaju Hebeloma H. crustuliniforme H. claviceps H. leucosarx H. longicaudum H. sacchariolens H. sinapizans H. trancatum

16 15 Phot. 3. Dichotomic ectomycorrhizae of Scots pine and Hebeloma sinapizans Fot. 3. Ektomikoryzy dychotomiczne Pinus sylvestris z grzybem Hebeloma sinapizans Phot. 4. Cross section of ectomycorrhizae of Scots pine and Hebeloma longicaudum, mag. 250 Fot. 4. Przekrój poprzeczny przez ektomikoryzę Pinus sylvestris z grzybem Hebeloma longicaudum, pow. 250

17 16 substrate. They appeared as white mycelial fans and strands overgrowing the substrate (Phot. 3). The mycorrhizae of Scots pine formed by H. truncatum and H. longicaudum were distinctly different in appearance. In both these cases the mycelial mantle was meager producing few loosely intertwined hyphae of the fungus. On cross section, thickness and structure of the myceliail mantle of Scots pine mycorrhizae with respective Hebeloma species were quite diversified (Tab. 5). The surface of some mycorrhizae was covered with a loose layer of plectenchyma, from 11.9 µm to 15.9 µm thick, directly passing into mycelial fans (Phot. 4). In better developed mycorrhizae the mycelial mantle was colorless in form of compact plectenchyma, from 23.8 µm to 39.7 µm thick, with surface gradually becoming loose and passing into loose plaits of hyphae or mycelial strands (Phot. 5). In each case the Hartig's net was very well developed, encircling all layers of cells of the primary cortex parenchyma, and hyphae were colorless with diameter from 2.4 µm to 3.6 µm, having refractive walls at transversal partitions with clamp connections. In pedunculate oak single mycorrhizae dominated, while the percentage of monopodial mycorrhizae was small, varying from 5% in mycorrhizae formed by H. longicaudum to10.7% in mycorrhizae formed by H. crustuliniforme, and only in the case of H. leucosarx it was higher, amounting to 14.7% (Tab. 4). The single mycorrhizae were relatively short and slightly swollen, while the monopodial mycorrhizae were slightly elongated and sometimes segmented, light cream, and covered with a loose wooly white mycelium. Numerous colorless hyphae were growing out from the surface of mycorrhizae, forming white mycelial fans and numerous mycelial strands overgrowing the substrate (Phot. 6). Only in the case of mycorrhizae formed by H. truncatum and H. longicaudum the mycelium was very meager forming agglomerations of loose hyphae. In the latter case, within a single seedling, the mycorrhizae represented different developmental stages, from those covered with a thick layer of white mycelium to mycorrhizae poorly developed with the mycelial mantle thin or fragmentary. On cross section, there was a considerable variation in thickness and structure of the mycelial mantle in pedunculate oak mycorrhizae, depending on Hebeloma species and the developmental stage of the mycorrhiza. The thickness varied from 12.1 µm to 60.0 µm (Tab. 5). The mycelial mantle was prosenchymatic with loose surface passing into mycelial strands and compact plectenchyma, but with surface distinctly thinned down, passing into white mycelial fans and strands, as it was the case with pedunculate oak mycorrhizae with H. claviceps and H. leucosarx (Phot. 7 and 8). The Hartig's net was in each case well developed, especially in first layers of distinctly elongated cells of the primary cortex parenchyma, and it rarely reached the endoderm (Phot. 7 and 8). The hyphae were colorless, from 2.4 µm to 3.8 µm in diameter, having

18 Table 5 Tabela 5 Anatomical characteristics of ectomycorrhizae of Scots pine and pedunculate oak seedlings grown in containers and inoculated with various fungi from the genus Hebeloma Charakterystyka anatomiczna ektomikoryz sadzonek sosny zwyczajnej i dębu szypułkowego hodowanych w pojemnikach i szczepionych biopreparatem z różnymi gatunkami grzybów z rodzaju Hebeloma Percentage of inoculum of Hebeloma species in substrate Udział biopreperatu z grzybami z rodzaju Hebeloma w substracie hodowlanym [%] thickness of mentle grubość opilsni [µm] Scots pine Sosna zwyczajna diameter of hyphae średnica strzepek [µm] structure of mantle struktura opilsni thickness of mentle grubość opilśni [µm] Pedunculate oak Dąb szypułkowy diameter of hyphae średnica strzępek [µm] structure of mantle struktura opilśni H. crustuliniforme A A H. claviceps B A H. leucosarx B/A A H. longicaudum B A H. sacchariolens B B H. sinapizans B/A B H. truncatum B B A mantle of the compact prosenchyma type, with loose surface, passing to mycelial fans or mycelial strands A opilśń typu zwartej prozenchymy, o powierzchni rozluźnionej, przechodzącej w welony grzybni lub sznury grzybniowe B mantle of the loose prosenchyma type, frequently irregularly covering surface of the mycorrhiza B opilśń typu luźnej prozenchymy, często nieregularnie pokrywającej powierzchnię mikoryzy 17

19 18 Phot. 5. Cross section of ectomycorrhizae of Scots pine and Hebeloma claviceps, mag. 160 Fot. 5. Przekrój poprzeczny przez ektomikoryzę Pinus sylvestris z grzybem Hebeloma claviceps, pow. 160 Phot. 6. Singular mycorrhizae of pedunculate oak and Hebeloma leucosarx Fot. 6. Ektomikoryzy pojedyncze Quercus robur z grzybem Hebeloma leucosarx

20 19 Phot. 7 Cross section of ectomycorrhizae of pedunculate oak and Hebeloma claviceps, mag. 160 Fot. 7. Przekrój poprzeczny przez ektomikoryzę Quercus robur z grzybem Hebeloma claviceps, pow. 160 Phot. 8 Cross section of ectomycorrhizae of pedunculate oak and Hebeloma leucosarx, mag. 160 Fot. 8 Przekrój poprzeczny przez ektomikoryzę Quercus robur z grzybem Hebeloma leucosarx, pow. 160

21 20 refractive walls with prominent clamp connections at transversal partitions, which were sometimes incrusted. In pedunculate oak seedlings in synthesis with H. longicaudum, besides well developed ectomycorrhizae, there occurred perytrophic mycorrhizae within a single seedling. In that case thickness of the mycelial mantle ranged from 7.3 µm to 24.2 µm, but there was no Hartig's net in cells of the primary cortex parenchyma. IV. DISCUSSION This study showed that the spectrum of fungal species from the genus Hebeloma which could be used in the Polish technology of artificial mycorrhization of forest tree seedlings is relatively wide. In principle, all tested fungal species fulfilled the criteria necessary for the treatment of artificial mycorrhization. Only in the case of H. longicaudum there may be some doubts whether this species could be used in artificial mycorrhization of pedunculate oak seedlings. Results showed a low degree of mycorrhization of pedunculate oak roots with this fungus, but on the other hand the mycorrhizae developed on roots were characterized by a well developed Hartig's net and a relatively thick mycelial mantle. This problem should be solved during subsequent tests on mycorrhizogenic activity of this fungus in relation to pedunculate oak. Similarly H. claviceps showed in synthesis with Scots pine the highest index of root mycorrhization (1.0), while in synthesis with pedunculate oak this index was low (0.49). These two Hebeloma species seemed to show a certain higher specificity in relation to Scots pine than in relation to pedunculate oak. A diversified percentage of inoculum of different Hebeloma species in the substrate could have influenced the results concerning the degree of root mycorrhization, especially the overgrowing of the substrate ball with the mycelium. For this reason it is not quite clear to what degree the observed phenomena were the result of properties of the ectomycorrhizal fungi, and to what degree they were determined by the greater or smaller percentage of the vegetative inoculum in the substrate. It may, however, be supposed that the fungal species had a greater effect on efficiency of artificial mycorrhization than the percentage of inoculum in the substrate. This was indicated by the results obtained in the case of H. sacchariolens and H. sinapizans in synthesis with Scots pine and pedunculate oak, and also in the case of H. crustuliniforme and H. longicaudum in synthesis with pedunculate oak. In the first case the percentage of vegetative inoculum in the substrate was similar, while the index of root mycorrhization in Scots pine was similar and in pedunculate oak distinctly different. In the second case at a small difference in inoculum percentage in the substrate (0.5%) the index of root mycorrhization was highly diversified (0.43).

22 The dry matter of roots is one of the indexes of the effect of the artificial mycorrhization treatment. Results of this study did not show any considerable differences in the mean dry matter of roots between experimental variants, indicating a similar effect of H. claviceps, H. longicaudum, and H. crustuliniforme on dry matter of root systems of Scots pine and pedunculate oak seedlings. Similarly, the diversification of parameters of the over-ground portion and the root system, especially in Scots pine seedlings, was not explicitly connected with micorrhizal activities of Hebeloma species investigated during this study. This effect on measurable growth and development parameters of seedlings during the first growing season after inoculation was not clear, which was also reported in literature. Some authors found a highly positive effect of mycorrhiozal inoculation on the development of shoots and roots (Aleksandrowicz-Trzcińska 2000, Pachlewski 1983), while other did not find such a simple relationship (Molina 1982, Rinkón et al. 2001). Our study suggests that this diversification may be associated with diversification of fungal species within single genus. The degree of substrate overgrowing with mycelium was different in different experimental variants, and in the case of Scots pine seedlings it was possible to relate this diversification with the inoculum percentage in the substrate, while in the case of pedunculate oak seedlings no such a relationship was found. This would rather point to specific relationships taking place between the plant and its symbiont. However, this opinion may be burdened with error because it is based on visual observation only. In future more detailed analytical methods should be used, e.g. testing the ergosterol content. This suggestion could also find its confirmation in the lack of a clear relationship between the degree of overgrowing of the substrate with mycelium and the index of root mycorrhization calculated on the basis of microscopic analyses. In the case of Scots pine seedlings inoculated with H. truncatum and H. longicaudum the root mycorrhization index was close to the maximum, while the percentage of cover of root balls with mycelium was very small. All Hebeloma species, tested during this study, showed a high index of mycorrhization of Scots pine roots, which is a good prognosis for seedling survival in plantations (Szabla 2007). However other authors are of the opinion that for inoculation of the planting stocks in forest nurseries it would be better to choose fungi less expansive which would only partly colonize root systems of seedlings. This way seedlings would get support in the initial phase of growing in the plantation, and at the same time colonization of non-mycorrhized parts of roots with fungi naturally occurring in soil would be possible, and therefore the biodiversity of mycorrhizal fungi would be increased (Steinström et al. 1990). However, it should be remembered that planting stock grown according to the Polish technology of artificial mycorrhization is usually designated for planting on so called difficult grounds where the soil is devoid of fungi able to make proper ectomycorrhizal contacts with tree roots (Kowalski 21

23 ). Rudawska (2000) assumed that the inoculation result is positive when 50% of small plant roots form ectomycorrhizae with expected fungus. Taking this index into account, only H. longicaudum would not fulfill the requirements in respect of pedunculate oak. According to Pachlewski (1983), in order to determine mycorrhizal properties of fungi a special attention should be paid to their penetration into the primary cortex and to the structure of the Hartig's net as the index of mycorrhizal virulence. According to this criterion all fungal species tested in this study were characterized by a high mycorrhizal virulence in respect of Scots pine because their Hartig's net included the entire primary cortex, right to endodermis. In the case of pedunculate oak the Hartig's net was usually limited to the first layer of cells of the primary cortex parenchyma. This is a characteristic feature for this tree species. Also thickness of the mycelial mantle was different in mycorrhizae of both species under investigations. The mycorrhizae formed by Hebeloma species with pedunculate oak were characterized by the mycelial mantle several times thicker than in mycorrhizae formed with Scots pine. Micorrhizae in pedunculate oak seedlings showed a great developmental diversification. Some of them were incompletely developed. This is in agreement with the conclusion that some features of ectomycorrhizae, e.g. thickness of the mycelial mantle, depth of the Hartig's net penetration, or the amount of hyphae among plant cells, develop gradually and depend on the developmental stage of symbiosis (Brunner et al. 1991). Perhaps the occurrence of the peritrophic mycorrhizae in this tree species with Hebeloma fungi, tested during our study, was also the result of a gradual development of mycorrhizae because under definite ecological conditions the peritrophic mycorrhizae may be the initial stages of the ectomycorrhiza. Any way, this loose contact of fungal hyphae with roots has also its physiological, nutritional, and protective consequences (Kowalski 1998). Pachlewski (1983) pointed out that also the phenomenon of a positive effect of the ectomycorrhizal fungus on the plant may occur without anatomical relations. In the case of Scots pine the ectomycorrhizae were generally well developed, and this was in agreement with conclusion of Pachlewski (1983) that results of a positive mycorrhizal test indicate a high regularity in formation of ectomycorrhizae with Scots pine by numerous Hebeloma species. A future optimal development of seedlings in the forest plantation will be the confirmation of the positive effect of mycorrhization with fungi tested during this study. For this reason they should be monitored, also from the point of view of vitality and survival of mycorrhizae in the environment to which they have been introduced. Also the dynamics of formation of new mycorrhizae with the introduced fungus, as well as the participation of natural mycorrhization will be of importance (Kowalski 1998).

24 23 ACKNOWLEDGEMENT Authors would like to thank Dr Zofia Heinrich for collecting and identification of fructifications of Hebeloma fungi, from which cultures were isolated for subsequent experiments. V. REFERENCES Aleksandrowicz-Trzcińska M The dependence between biometric attributes of one-year-old Scots pine seedlings and the number of autotrophic roots and mycorrhizal roots. Ann. Wars. Agricult. Univ.-SGGW Forestry a. Wood Techn. 50: Aleksandrowicz-Trzcińska M Określenie stopnia zmikoryzowania systemu korzeniowego rocznych sadzonek sosny na podstawie próby. Sylwan 145, 2: Berft M Strategia Lasów Państwowych we wdrażaniu technologii mikoryzacji sadzonek drzew leśnych. [In:] Ektomikoryzy. Nowe biotechnologie w polskim szkółkarstwie leśnym. Kowalski S. (ed.). Centrum Infor. Lasów Państw., Warszawa. Boyd C. D., Hellebrand K. E Assessment of the effect of mycorrhizal fungi on drough tolerance of conifer seedlings. Can. J. Bot. 69: Branzanti M. B., Rocca E., Pisi A Effect of ectomycorrhizal fungi on chestnut ink disease. Mycorrhiza 9: Brunner I., Amiet R., Schneider B Charakteryzation of naturally grown and in vitro synthesized ectomycorrhizas of Hebeloma crustuliniforme and Picea abies. Mycol. Res. 95, 12: Dominik T Studium o mikoryzie. Folia Forest. Pol. Ser. A, 5: Kowalski S Badania nad syntezą mikoryz sosny zwyczajnej (Pinus silvestris L.) w warunkach laboratoryjnych. PTPN Wydz. Nauk Roln. i Leśn. Prace Kom. Nauk Roln. i Kom. Nauk Leśn. 38: Kowalski S Praktyczne aspekty mikotrofizmu w szkółkach leśnych. Sylwan 141, 6: Kowalski S Potrzeby i możliwości mikoryzowania sadzonek drzew leśnych w szkółkach kontenerowych. Post. Techn. w Leśn. 65: Kowalski S Zasady postępowania technologicznego przy stosowaniu polskiego biopreparatu z grzybem Hebeloma crustuliniforme w zabiegu sterowanej mikoryzacji sadzonek drzew leśnych. [In:] Ektomikoryzy. Nowe biotechnologie w polskim szkółkarstwie leśnym, S. Kowalski (ed.). Centrum Infor. Lasów Państw., Warszawa. Leyval C., Turnau K., Haselwandter K Effect of heavy metal pollution on mycorrhizal colonization and function: physiological, ecological and applied aspects. Mycorrhiza 7: Marx D.H Ectomycorrhizae as biological deterrent to pathogenic root infections. Ann. Rev. Phytopathol. 10: Molina R Use of the ectomycorrhizal fungus Laccaria laccata in forestry. I Consistence between isolates in effective colonization of containerized conifer seedlings. Canad. J. For. Res. 12, 3: Pachlewski R Mikotrofizm systemu korzeniowego. [In:] Zarys fizjologii sosny zwyczajnej, S. Białobok, W. Żelawski (ed.). PWN, Poznań Warszawa Pachlewski R Grzyby symbiotyczne i mikoryza sosny (Pinus sylvestris L.). Prace IBL 615: Parke J., Linderman R. G., Black C. H The role of ectomycorrhizal in drought tolerance of Douglas fir seedlings. New Phytol. 95: Rincón A., Alvarez I. F., Pera J Inoculation of containerized Pinus pinea L. seedlings with seven ectomycorrhizal fungi. Mycorrhiza 11:

25 24 Rudawska M. (ed.) Ektomikoryza jej znaczenie i zastosowanie w leśnictwie. Instytut Dendr. PAN, Kórnik. Stenström E., Ek M., Unestam T Variation in field response of Pinus sylvestris to nursery inoculation with four different ectomycorrhizal fungi. Can. J. For. Res. 20: Szabla K Cechy morfologiczno-rozwojowe oraz przeżywalność sadzonek różnych gatunków drzew leśnych w uprawach doświadczalnych na gruntach nieleśnych i leśnych o różnym stopniu degradacji. [In:] Ektomikoryzy. Nowe biotechnologie w polskim szkółkarstwie leśnym, S. Kowalski (ed.). Centrum Infor. Lasów Państw., Warszawa. Streszczenie S. Kowalski, E. Chomicz, M. Czeremuga, M. Piechna Wybrane grzyby z rodzaju Hebeloma P. Kumm. w syntezie ektomikoryzowej z sosną zwyczajną (Pinus sylvestris L.) i dębem szypułkowym (Quercus robur L.) w szkółce kontenerowej Badano wpływ sterowanej mikoryzacji z użyciem 6 biopreparatów z grzybnią wegetatywną grzybów ektomikoryzowych: Hebeloma claviceps, H. leucosarx, H. longicaudum, H. sacchariolens, H. sinapizans i H. trunkatum na: stopień zmikoryzowania sadzonek sosny zwyczajnej i dębu szypułkowego, kształtowanie się frekwencji i rodzajów ektomikoryz u tych sadzonek, wpływu przeprowadzonego zabiegu na parametry rozwojowo wzrostowe części nadziemnej i systemu korzeniowego jak również porównano te parametry z tymi, jakie osiągają sadzonki badanych gatunków drzew szczepione polskim biopreparatem z grzybem H. crustuliniforme, hodowane w pojemnikach. Sosnę hodowano w pojemnikach V 120 cm 3, natomiast dąb V 265 cm 3, na substracie torfowym z udziałem 20% wermikulitu i 10% perlitu oraz nawozami Osmocote w ilości 3 kg/m 3. Udział biopreparatu w substracie hodowlanym, w stosunku objętościowym był zróżnicowany i wynosił 2,5% w wypadku H. crustuliniforme oraz w wypadku: H. longicaudum 2,0%, H. truncatum 3,5%, H. sacchariolens 5,0%, H. sinapizans 5,0%, H. leucosarx 6,5% i w wypadku H. claviceps 7,0%. W przypadku sadzonek sosny zróżnicowanie mierzalnych parametrów części nadziemnych, niezależnie od gatunku grzyba użytego do zabiegu mikoryzacji, było niewielkie. Znacznie wyższe różnice stwierdzono w budowie korzeni, a zwłaszcza w liczebności korzeni III rzędu, na których znajdowała się większość ektomikoryz (tab. 1). Uwzględniając sumaryczną liczbę korzeni wszystkich rzędów można wydzielić 3 grupy sadzonek o podobnych parametrach. Do pierwszej należy zaliczyć sadzonki szczepione grzybem H. longicaudum, do drugiej szczepione grzybami: H. crustuliniforme, H. leucosarx i H. claviceps, a do trzeciej grupy szczepione: H. truncatum, H. sacchariolens i H. sinapizans. Nie można było przyczynowo powiązać wielkości parametru suchej masy korzeni z procentowym udziałem biopreparatu w substracie hodowlanym. Procentowy udział biopreparatu w substracie hodowlanym miał jednak wyraźny wpływ na stopień przerośnięcia bryłki korzeniowej grzybnią poszczególnych gatunków grzybów z rodzaju Hebeloma (tab.1). Badania wykazały, że w przypadku sadzonek sosny wszystkie badane gatunki grzybów z rodzaju Hebeloma były bardzo dobrymi symbiontami tego gatunku drzewa, ponieważ wskaźnik zmikoryzowania systemu korzeniowego w każdym przypadku był bardzo wysoki i wahał się od 0,86 u H. longicaudum do 1,0 u H. sacchariolens i H. claviceps (tab. 2). Uwzględniając średnie wartości wszystkich mierzalnych parametrów można z pewnym zastrzeżeniem przyjąć, że na wzrost i rozwój sadzonek sosny w hodowli kontenerowej stosunkowo najkorzystniej wpływały grzyby: H. claviceps, H. sacchariolens i H. truncatum.

26 W przypadku sadzonek dębu, mierzalne parametry części nadziemnej i systemu korzeniowego były wyraźnie bardziej zróżnicowane niż u sosny (tab. 3). Odmiennie jak u sadzonek sosny, znacząco większy wpływ na stopień przerośnięcia bryłki korzeniowej grzybnią miał gatunek grzyba niż procentowy udział biopreparatu w substracie hodowlanym. Najwyższe średnie parametry suchej masy korzeni stwierdzono u sadzonek szczepionych biopreparatem z grzybem H. crustuliniforme i H. claviceps (tab. 3). W tym drugim jednak przypadku wskaźnik zmikoryzowania systemu korzeniowego był wyraźnie mniejszy i wynosił 0,49 (tab. 4). Stosunkowo najniższe średnie parametry suchej masy korzeni stwierdzono u sadzonek dębu szczepionych grzybem H. leucosarx i H. sinapizans, a jednocześnie w tych dwóch przypadkach stwierdzono stosunkowo największy wskaźnik zmikoryzowania systemu korzeniowego, bo wynoszący odpowiednio 0,91 i 0,81 (tab. 3 i 4). Można więc sądzić, że wielkość parametru suchej masy korzeni nie wykazywała przyczynowego związku ze stopniem zmikoryzowania systemu korzeniowego. Uwzględniając średnie wartości wszystkich mierzalnych parametrów można przyjąć, że na wzrost i rozwój sadzonek dębu w hodowli kontenerowej stosunkowo najkorzystniej wpływały grzyby: H. crustuliniforme, H. leucosarx, H. sinapizans i H. sacchariolens. Department of Forest Pathology University of Agriculture al. 29 Listopada 46, PL Kraków 25