Suillus Luteus Research Articles

Detecting mycorrhizal colonisation in Scots pine roots using electrical impedance spectra

To investigate whether root colonisation of Scots pine (Pinus sylvestris L.) seedlings with symbiotic mycorrhizal fungi (Hebeloma sp. and Suillus luteus) could be detected in situ, classification analysis of the electric impedance spectra (IS) of the root system was carried out. The seedlings were inoculated either with Hebeloma or Suillus with some left as controls. The seedlings were firstly cultivated in long-day and high temperature (LDHT) conditions. Half of the seedlings remained in LDHT and half were moved to short-day and low temperature conditions (SDLT) to acclimatise to the cold. The electrical impedance spectra of the root systems were measured at a frequency range of 5 Hz–100 kHz. The results of principal component analysis (PCA) showed that current delivery through root system, sensed by real and imaginary parts of IS, depended upon the cold acclimation and mycorrhizal treatment. Comparison of SDLT to LDHT via correlation analysis indicated a 13% and a 27% change in PCA responses for the real and imaginary parts of the impedance, respectively. When the mycorrhizal treatments were compared with a non-mycorrhizal treatment, the respective changes in the correlation coefficients were 30% for Hebeloma sp. and 39% for S. luteus in the real part and 28% and 38% in the imaginary part, respectively. These changes in the correlation coefficients appear to indicate physicochemical changes (e.g. ionic behaviour) in the roots as a result of fungal colonisation.

Iso-suillin Isolated from Suillus luteus, Induces G1Phase Arrest and Apoptosis in Human Hepatoma SMMC-7721 Cells

Iso-suillin, a natural product isolated from Suillus luteus, has been shown to inhibit the growth of some cancer cell lines. However, the molecular mechanisms of action of this compound are poorly understood. The purpose of this study was to investigate how iso-suillin inhibits proliferation and induces apoptosis in a human hepatoma cell line (SMMC-7721). We demonstrated the effects of iso-suillin on cell proliferation and apoptosis in SMMC-7721 cells, with no apparent toxicity in normal human lymphocytes, using colony formation assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Western blotting was used to examine the expression of G1 phase-regulated and apoptosis-associated protein levels in iso-suillin treated SMMC-7721 cells. The results indicated that iso-suillin significantly decreased viability, induced G1 phase arrest and triggered apoptosis in SMMC-7721cells. Taken together, these results suggest the potential of iso-suillin as a candidate for liver cancer treatment.

Influence of ectomycorrhizal inoculation on Pinus wallichiana and Cedrus deodara seedlings under nursery conditions

A study was undertaken to examine the extent of root colonization by four locally isolated ectomycorrhizal (ECM) fungi (Hebeloma theobrominum, Boletus dryophilus, Scleroderma citrinum and Suillus luteus) and their effects on seedling growth in Pinus wallichiana and Cedrus deodara under nursery conditions. Seedlings of the two conifers were inoculated with mycelium of ECM fungi and were grown in pots containing sterilized forest soil for six months. The percentage of ECM colonization of roots was 38%–52% in Pinus wallichiana and 33%–48% in Cedrus deodara. ECM colonization increased shoot height, needle number, shoot and root biomass and survival of inoculated seedlings. Among the four ECM fungi Hebeloma theobrominum was more effective with Pinus wallichiana and Scleroderma citrinum with Cedrus deodara in promoting seedling survival and overall growth. All the four ECM fungi used enhanced growth of inoculated seedlings and thus can be used in afforestation and regeneration programmes in degraded forests ecosystems.

Intraspecific aluminium response in Suillus luteus (L.) s.f. gray., an ectomycorrhizal symbiont of scots pine

Ten isolates of the ectomycorrhizal fungus <i>Suillus luteus</i> have been cultured on an aluminium containing growth medium in order to determine their <i>in vitro</i> aluminium tolerance. Five isolates originated from a site heavily polluted by acid rain with a high availability of aluminium in the soil. Five others were collected from a site free from direct pollution. Aluminium content in the sporocarps of <i>S. luteus</i> differed according to the site of origin and did not reveal symptoms of bioconcentration, although such phenomena were found when mycelium isolated from the sporocarps was submited to 100 mg/L Al in liquid culture. A clear relationship between Al accumulation <i>in vitro</i> and the site of origin of the isolate was not observed, although the highest amount of Al was found in the mycelium derived from the polluted soil. In a second experiment all isolates were grown in agar media containing 10, 100, 500 and 1000 mg/L<sup>-1</sup> Al and the colony diameter during culture and the final colony dry weight determined.<i> S. luteus</i> appeared to be very tolerant to the presence of Al in the medium. Each of the parameters used to measure the metal tolerance of the fungus ranked the isolates in a slightly different order, but those originating from the polluted area showed some superiority over the others. In polluted soils this species seems to have been submitted to a selection for higher aluminium tolerance. The results are discussed with reference to the possibilities of transformating in vitro studies to situation in the forest ecosystem.

Suillus luteus methanolic extract inhibits proliferation and increases expression of p-H2A.X in a non-small cell lung cancer cell line

Methanolic extract of Suillus luteus was previously shown to inhibit proliferation of colon cancer cells with mutant p53. The effect of the same extract was further investigated here in a wildtype (wt) p53 non-small cell lung cancer cell line. Treatment with the extract increased the levels of p-H2A.X and the number of p-H2A.X foci/cell, indicating a possible increase in deoxyribonucleic acid (DNA) damage. Nevertheless, it did not cause alterations in wt p53 levels nor in programmed cell death. The extract caused inhibition of cellular proliferation and an increase in the % of cells in the G0/G1 phase of the cell cycle. In conclusion, even though there is evidence of DNA damage being caused by this extract, there is no induction of cell death in this p53 wt cell line.

순수배양 시 외생균근균의 질소원 요구성과 선호도

국내 산림에서 수집한 외생균근균(13속 42균주)을 질소원이 서로 다른 4종류의 시험배지에 접종하고 56일간 순수 배양하여 질소원 요구성과 선호도를 조사하였다. 그 결과, 일본연지그물버섯(KFRI 1224), 비단그물버섯(KFRI 1232), 끈적쓴맛그물버섯(KFRI 1383), 젖비단그물버섯(KFRI 1997), 알버섯류(KFRI 1434) 등이 질소원 요구성이 높은 것으로 조사되었다. 검은머리그물버섯(KFRI 1362)을 비롯하여 총 시험균주의 45%는 질산염보다 암모늄 형태의 무기질소원을 더 선호하였다. 반면 큰비단그물버섯(KFRI 1125)을 비롯한 총 시험균주의 36%는 질산염 형태의 무기질소원을 더 선호하였다. The nitrogen source-requirement and preference of ectomycorrhizal fungi (42 strains belong to 13 genera) collected from Korean forests were investigated by incubating mycelial cells in four nitrogen source test media for 56 days. The nitrogen requirement was relatively high in five strains (Heimioporus japonicas (KFRI 1224), Suillus luteus (KFRI 1232), Tylopilus castaneiceps (KFRI 1383), Suillus granulates (KFRI 1997), Rhizopogon sp. (KFRI 1434)) in comparison with other strains. Forty-five percent of ectomycorrhizal strains including Boletus griseus (KFRI 1362) preferred the ammonium-form of inorganic nitrogen source for mycelial growth, whereas thirty-six percent of tested strains including Suillus grevillei (KFRI 1125) preferred the nitrate-form as an inorganic nitrogen source.

Suillus luteus methanolic extract inhibits cell growth and proliferation of a colon cancer cell line

Several edible mushrooms extracts are known to have tumor cell growth inhibitory potential. The objective of this work was to study this potential in extracts of Suillus luteus collected from the Northeast of Portugal. Various extracts were prepared and their effect on tumor cell growth was studied with the SRB assay in four human tumor cell lines: MCF-7 (breast), NCI-H460 (non-small cell lung cancer), AGS (gastric) and HCT-15 (colon). The methanolic extract of S. luteus was the most potent extract. This extract was slightly more potent in the HCT-15 cells (with mutant p53, GI50=17.8±1.6μg/mL) than in the other cell lines tested, which suggested that its effect was not p53-dependent. Indeed, in HCT-15 cells, an increase in the levels of p53 was detected but no alterations in some of the proteins transactivated by p53 (e.g. p21 or Bax) were found. The extract caused an increase in the cellular levels of p-H2A.X, which is suggestive of DNA damage. Growth inhibition in these cells was mostly due to inhibition of cell proliferation and an increase in the % of cell in the G1 phase of the cell cycle. An increase in cell death was also found, even though to very low levels. In addition, this extract was not cytotoxic to primary cultures of porcine hepatocytes (GI50>400μg/mL). Together, these results indicate that the S. luteus methanolic extract may be an interesting source of compounds that inhibit the proliferation of tumor cells but further studies should be carried out in order to understand its potential.

Physiological aspects underlying the improved outplanting performance of Pinus pinaster Ait. seedlings associated with ectomycorrhizal inoculation

Mycorrhizal inoculation of conifer roots is a key strategy to optimize establishment and performance of forest tree species under both natural and cultivated conditions and also to mitigate transplantation shock. However, despite being a common practice, inoculation in outdoor nursery conditions has been poorly studied. Here, we have evaluated effectiveness of four fungal species (Lactarius deliciosus, Lactarius quieticolor, Pisolithus arhizus, and Suillus luteus) in the production of mycorrhizal Pinus pinaster seedlings in an outdoor commercial nursery and their ability to improve seedling physiology and field performance. All inoculated seedlings showed a significant increase in growth at the end of the nursery stage and these differences remained after 3years of growth in the field. Differences observed in the content of malondialdehyde, total chlorophyll, carotenoids, anthocyanins, and phenolic compounds from needles of mycorrhizal and control seedlings may reflect a different sensitivity to photo-oxidative damage. We conclude that ectomycorrhizal inoculation improves adaptability to changeable growing conditions of an outdoor nursery and produces a higher quality nursery stock, thereby enhancing seedling performance after planting.

Cryopreservation of ectomycorrhizal fungi has minor effects on root colonization of Pinus sylvestris plantlets and their subsequent nutrient uptake capacity

The use of ectomycorrhizal (ECM) fungi for afforestation, bioremediation, and timber production requires their maintenance over long periods under conditions that preserve their genetic, phenotypic, and physiological stability. Cryopreservation is nowadays considered as the most suitable method to maintain the phenotypic and genetic stability of a large number of filamentous fungi including the ECM fungi. Here, we compared the ability of eight ECM fungal isolates to colonize Pinus sylvestris roots and to transport inorganic phosphate (Pi) and NH4 (+) from the substrate to the plant after cryopreservation for 6months at -130°C or after storage at 4°C. Overall, the mode of preservation had no significant effect on the colonization rates of P. sylvestris, the concentrations of ergosterol in the roots and substrate, and the uptake of Pi and NH4 (+). Comparing the isolates, differences were sometimes observed with one or the other method of preservation. Suillus bovinus exhibited a reduced ability to form mycorrhizas and to take up Pi following cryopreservation, while one Suillus luteus isolate exhibited a decreased ability to take up NH4 (+). Conversely, Hebeloma crustuliniforme, Laccaria bicolor, Paxillus involutus, and Pisolithus tinctorius exhibited a reduced ability to form mycorrhizas after storage at 4°C, although this did not result in a reduced uptake of Pi and NH4 (+). Cryopreservation appeared as a reliable method to maintain important phenotypic characteristics (i.e., root colonization and nutrient acquisition) of most of the ECM fungal isolates studied. For 50% of the ECM fungal isolates, the colonization rate was even higher with the cultures cryopreserved at -130°C as compared to those stored at 4°C.

Accumulation of elements by edible mushroom species II. A comparison of aluminium, barium and nutritional element contents

The aim of the study was to compare accumulation efficiency of Al, Ba and nutritional elements (Ca, Fe, K, Mg, Mn, Na) exhibited by six edible mushrooms collected in particular regions of Poland during the last 20 years. The studied mushroom species were Boletus edulis, Cantharellus cibarius, Lactarius deliciosus, Leccinum aurantiacum, Suillus luteus and Xerocomus badius. The highest and the lowest concentrations of the elements in tested mushroom species were 11 – 410, 34 – 337, 16785 – 34600, 140 – 607, 12 – 75 and 16 – 143 mg kg−1d.m., respectively. The highest average concentrations of Al, Mg and Mn were observed in Suillus luteus fruiting bodies, while for Ba, Ca, K and Na it was in Lactarius deliciosus. BCF >1 was found for K and Mg in all tested mushroom species and additionally for the highest Ca and Na concentrations of all tested mushroom species except for C. cibarius and S. luteus, respectively. For the other tested elements (Al, Ba, Fe and Mn) BCF values < 1 were recorded.

Viability of ectomycorrhizal fungi following cryopreservation

The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at −130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of a large set of ECM fungi and that further studies are necessary for the more recalcitrant ones.

Accumulation of elements by edible mushroom species: Part I. Problem of trace element toxicity in mushrooms

The aim of this study was to evaluate Cd, Co, Cu, Hg, Ni, Pb, Sr and Zn accumulation in six edible mushroom species and to assess their risk and benefits to human consumers. Mushrooms (Leccinium aurantiacum, Xerocomus badius, Lactarius deliciosus, Boletus edulis, Cantharellus cibarius and Suillus luteus) were collected from selected regions of Poland during 1990–2010. The highest diversity between studied mushroom species was observed in terms of Cu and Zn accumulation. Significant differences in the accumulation efficiency were found among the six mushroom species examined. The most efficient were Boletus edulis (Cd and Hg), Suillus luteus (Cu and Sr), and Lactarius deliciosus (Pb and Zn). In the case of Co and Ni, the most effective were Xerocomus badius and Leccinium aurantiacum, respectively. The calculated bioconcentration factor (BCF) values of Cd, Cu, Hg, Sr and Zn were > 1 for all species in this study while Co, Ni and Pb usually were bioexcluded (BCF < 1). Additionally, based on the calculated daily intake rates of trace elements determined it can be concluded that occasional consumption of fruiting bodies of L. aurantiacum, X. badius, L. deliciosus,B. edulis, C. cibarius and S. luteus collected in Poland is safe and this finding largely agrees with results from recent studies by other authors.

Spores of ectomycorrhizal fungi as inoculants for Nothofagus pumilio and exotic conifer seedlings in Patagonia, Argentina: their activity and conservation

Ectomycorrhizal (EM) fungi incorporation in nursery production is necessary for afforestation with exotic tree species and native forests restoration programs. The fact that spore use as EM inocula is inexpensive and effective, makes it an option more realistic than vegetative inoculum production for many regions around the world. To determine spore activity from EM species selected as inoculants for Nothofagus pumilio and exotic conifers planted in Patagonia, two conservation methods were applied (i.e., dried fruit bodies and spore slurries), and their change over time was assessed (over 8–9 months). Spore activity decreased significantly with time for both native and exotic EM species. Conservation methods showed no significant differences for N. pumilio EM species considered together or for exotic conifer EM species. However, spore activity of different EM species behaved differently with conservation method and over time when considered separately. Taxa which better kept spore activity over time were Austropaxillus statuum and Setchelliogaster fragilis for N. pumilio, and Inocybe kauffmanii for exotic conifers. However, considering together fruit body spore density and spore activity, the species Hallingea purpurea and Cortinarius sp. also appeared as suitable for N. pumilio and Rhizopogon roseolus, R. villosulus, Suillus luteus and S. lakei for exotic conifers. Spore density found in EM fungal fructifications were also established in order to estimate fructification weights necessary to apply a reference dose of active spores in inoculation programs.

Effect of the geographical origin, culture media, and pH on the growth dynamic of the edible ectomycohrrizal mushroom Suillus luteus

Suillus luteus is the most important wild edible ectomycorrhizal mushroom harvested and exported in Chile. This introduced species forms mycorrhizal symbiosis with Pinus radiata , the most important exotic forest tree. To obtain optimized protocols for the controlled infection of P. radiata trees with this species, it is important to determine the appropriate culture conditions. Therefore, we studied the effect of the geographical origin (three localities from the Maule Region of Chile), culture media (Modified Melin and Norkrans (MMN) and Malt Extract Agar (MEA) 2%), and pH (5.0, 5.8, and 6.5) on the in vitro growth dynamics of S. luteus over 37 days. The results showed significant differences in the growth rate and colony diameter as a consequence of its geographical origin. However, no differences were found for either pH or culture medium. Our results confirm that a non-specific, economical culture medium, such as MEA 2%, may be used to obtain a suitable amount of mycelium for both medium- and large-scale assays of inoculation with this species. Keywords: Ectomycorrhizal, edible fungi, fungi, Pinus radiata, Suillus luteus. Suillus luteus es la especie silvestre de hongo micorricico comestible mas cosechada en Chile, que tiene una importancia economica y silvicola. Es una especie silvestre introducida, que se asocia con Pinus radiata , la especie forestal economicamente mas importante del pais. Si se quisieran desarrollar metodologias de infeccion controlada en arboles de P. radiata con este hongo ectomicorricico, es importante estudiar condiciones apropiadas para su cultivo. Por ello, se estudio el efecto del origen geografico (tres localidades de la Region del Maule de Chile), del medio de cultivo (Melin y Norkrans modificado (MMN) y extracto de malta agar (MEA) al 2%), y del pH (5,0; 5,8; y 6,5), en la dinamica de crecimiento in vitro de S. luteus durante 37 dias. Los resultados indicaron diferencias significativas en la velocidad de crecimiento y diametro de la colonia, como consecuencia de la procedencia del material fungico. Sin embargo, no hubo diferencia tanto en el medio de cultivo como en el pH. Esto confirma, para las cepas chilenas analizadas, que es posible emplear un medio de cultivo economico como MEA 2%, con el proposito de producir una cantidad adecuada de biomasa miceliar para futuros programas de micorrizacion con esta especie.

Effect of the geographical origin, culture media, and pH on the growth dynamic of the edible ectomycohrrizal mushroom Suillus luteus

Effect of the geographical origin, culture media, and pH on the growth dynamic of the edible ectomycohrrizal mushroom Suillus luteus

Antioxidant capacity and mineral contents of edible wild Australian mushrooms

Five selected edible wild Australian mushrooms, Morchella elata, Suillus luteus, Pleurotus eryngii, Cyttaria gunnii, and Flammulina velutipes, were evaluated for their antioxidant capacity and mineral contents. The antioxidant capacities of the methanolic extracts of the dried caps of the mushrooms were determined using a number of different chemical reactions in evaluating multi-mechanistic antioxidant activities. These included the Trolox equivalent antioxidant capacity, ferric ion reducing antioxidant power, and ferrous ion chelating activity. Mineral contents of the dried caps of the mushrooms were also determined by inductively coupled plasma-optical emission spectroscopy. The results indicated that these edible wild mushrooms have a high antioxidant capacity and all, except C. gunnii, have a high level of several essential micro-nutrients such as copper, magnesium, and zinc. It can be concluded that these edible wild mushrooms are good sources of nutritional antioxidants and a number of mineral elements.

Association of Pinus banksiana Lamb. and Populus tremuloides Michx. seedling fine roots with Sistotrema brinkmannii (Bres.) J. Erikss. (Basidiomycotina)

Sistotrema brinkmannii (Bres.) J. Erikss. (Basidiomycotina, Hydanaceae), commonly regarded as a wood decay fungus, was consistently isolated from bareroot nursery Pinus banksiana Lamb. seedlings. S. brinkmannii was found in ectomycorrhizae formed by Thelephora terrestris Ehrh., Laccaria laccata (Scop.) Cooke, and Suillus luteus (L.) Roussel. In pure culture combinations with sterile P. banksiana and Populus tremuloides Michx. seedlings, S. brinkmannii colonized root cortical cells while not killing seedlings. Colonization by S. brinkmannii appeared to be intracellular but typical endo- or ectomycorrhizae were not formed. The fungus did not decay roots, although it was shown to produce cellulase in enzyme tests. Results suggest a unique association between S. brinkmannii and seedling roots that is neither mycorrhizal nor detrimental; its exact function remains to be elucidated.

Analysis of some aspects of the market in fruit bodies of chanterelle (Cantharellus cibarius Fr.)

This paper presents an analysis of data on the purchase of Cantharellus cibarius Fr. fruit bodies in the vicinity of Czeremcha village. The volume purchased from seven local purchasing centres between 2004-2008 was compared to temperature and precipitation data. In each of these years, the relationship between the volume purchased and the total precipitation each month was noted. The average price of the fruit bodies of C. cibarius was 10.05 PLN/kg, which was much higher than that of other fungal species: Xerocomus badius (2.44 PLN), Suillus luteus (2.47 PLN), and even Boletus edulis (6.50 PLN). In the years 2004–2008, the mass of purchased fruit bodies of C. cibarius purchased accounted for 63% of all fungi, which indicates that C. cibarius is the most important species among the forest edible mushrooms in the Polish market. The existing regulations applicable to the forest mushroom utilisation and trade are also discussed.

Toward the Antioxidant and Chemical Characterization of Mycorrhizal Mushrooms from Northeast Portugal

Mushrooms are widely appreciated all over the world for their nutritional properties and pharmacological value as sources of important bioactive compounds. Mycorrhizal macrofungi associate with plant roots constituting a symbiotic relationship. This symbiosis could influence the production of secondary metabolites, including bioactive compounds. We focused on the evaluation of antioxidant potential and chemical composition of mycorrhizal mushrooms species from Northeast Portugal: Amanita caesarea, Amanita muscaria, Amanita pantherina, Chroogomphus fulmineus, Cortinarius anomalus, Cortinarius collinitus, Cortinarius violaceus, Lactarius quietus, Lactarius volemus, Russula sardonia, Suillus luteus, and Tricholoma ustale. A similar profile of metabolites was observed in the studied species with the order sugars > fat > ascorbic acid > phenolic compounds > tocopherols. Nevertheless, the samples revealed different compositions: prevalence of sugars in L. volemus, fat and ascorbic acid in A. muscaria, phenolic compounds in C. anomalus and tocopherols, and antioxidant activity in S. luteus. Chemical characterization of 12 mycorrhizal mushrooms was achieved. They are sources of nutraceuticals, such as sugars and fatty acids, and contain bioactive compounds, such as vitamins and phenolic acids. Edible species can be incorporated in diets as sources of antioxidants, while nonedible species can be explored as sources of bioactive metabolites.

English

The objective of this study was to evaluate the influence of five different species of ectomycorrhizal (ECM) fungi on root colonization of native fungi on putatively blight resistant chestnut hybrids (Castanea dentata x C. mollissima) in a reclaimed mine site in central Ohio. The five species were Hebeloma crustuliniforme, Laccaria bicolor, Scleroderma polyrhizum, Amanita rubescens, and Suillus luteus. We used a combination of DNA sequencing of the ITS region and phylogenetic analyses to indentify fungi found on roots after 12 and 18 months in the field. Non-metric multidimensional scaling (NMDS) ordinations were used to determine if ECM community composition was influenced by the fungal inoculum used. The results of this study demonstrated that the selected ECM species do not persist on chestnut after one year in the field. In addition, these selected ECM species did not impede natural root colonization of native fungi or influence ECM community composition after two growing seasons. Although these species did not persist in the field, the presence of ECM inoculum (with the exception of Amanita) greatly contributed to the survival of hybrid chestnut seedlings. Therefore, introduced inoculum that was present in the very early stages of outplanting had persisting effects with regard to seedling establishment in the field, even if the original inoculum did not persist. ECM fungi native to the area colonized chestnuts resulting in increased growth rates. These native assemblages may contain species better able to form functional mycorrhizas under these environmental extremes. Therefore, the conservation of these species may be necessary to facilitate long-term survival of deciduous tree species historically native to these lands. Additional Keywords: root colonization of fungi, chestnut restoration.