MYCORRHIZAL COLONIZATION AND PHENOLIC 1905 NOTAS CIENTÍFICAS MYCORRHIZAL COLONIZATION AND PHENOLIC COMPOUNDS ACCUMULATION ON ROOTS OF EUCALYPTUS DUNNII MAIDEN INOCULATED WITH ECTOMYCORRHIZAL FUNGI

Compatibility between Eucalyptus dunnii and the ectomycorrhizal fungi Hysterangium gardneri and Pisolithus sp. _ from Eucalyptus spp. _, Rhizopogon nigrescens and Suillus cothurnatus _ from Pinus spp._, was studied in vitro. Pisolithus sp., H. gardneri and S. cothurnatus colonized the roots. Pisolithus sp. mycorrhizas presented mantle and Hartig net, while H. gardneri and S. cothurnatus mycorrhizas presented only mantle. S. cothurnatus increased phenolics level on roots. Pisolithus sp. and R. nigrescens decreased the level of these substances. The isolates from Eucalyptus seem to be more compatible towards E. dunnii than those from Pinus. The mechanisms involved could be related, at least in the cases of Pisolithus and Suillus, to the concentration of phenolics in roots. COLONIZAÇÃO E ACUMULAÇÃO DE COMPOSTOS FENÓLICOS EM RAÍZES DE EUCALYPTUS DUNNII MAIDEN INFECTADAS COM FUNGUS ECTOMICORRÍZICOS RESUMO Estudou-se a compatibilidade entre Eucalyptus dunnii e os fungos ectomicorrízicos Hysterangium gardneri e Pisolithus sp. _ isolados de Eucalyptus spp._, Rhizopogon nigrescens e Suillus cothurnatus _ isolados de Pinus spp._, in vitro. Pisolithus sp., H. gardneri e S. cothurnatus colonizaram as raízes. As micorrizas de Pisolithus sp. apresentaram manto e rede de Hartig; as de H. gardneri e S. cothurnatus apresentaram apenas manto. S. cothurnatus provocou aumento de fenóis nas raízes; Pisolithus sp. e R. nigrescens provocaram diminuição dessas substâncias. Os fungos isolados de Eucalyptus parecem mais compatíveis em relação a E. dunnii do que os de Pinus. A concentração de fenóis nas raízes parece estar envolvida nesse fenômeno, particularmente em relação a Pisolithus sp. e S. cothurnatus. 1 Accepted for publication on December 27, 1999. Part of B.Sc. thesis presented by the senior author to Universidade Federal de Santa Catarina (UFSC). 2 Biologist, Dep. de Microbiologia e Parasitologia, Centro de Ciências Biológicas, UFSC, Caixa Postal 476, CEP 88040-900 Florianópolis, SC, Brazil. CAPES scholar. E-mail: elvoigt@garoupa.bio.ufpr.br 3 Agronomist, Doct., Dep. de Microbiologia e Parasitologia, Centro de Ciências Biológicas, UFSC. E-mail: veturia@mbox1.ufsc.br 4 Biologist, Ph.D., Associate Professor, Dep. de Botânica, Centro de Ciências Biológicas, UFSC. E-mail: bot@ccb.ufsc.br

Specificity between ectomycorrhizal (ECM) fungi and host plants has been observed both in field (Molina & Trappe, 1982;Molina et al., 1992) and controlled conditions (Malajczuk et al., 1982(Malajczuk et al., , 1984;;Oliveira et al., 1994).Knowledge of the mechanisms controlling this phenomenon is important to understand mycorrhizal functioning and to guide the selection of isolates for inoculation programmes.Malajczuk et al. (1982Malajczuk et al. ( , 1984) ) reported that ECM fungi from eucalypts were unable to colonize Pinus radiata, while those from conifers did not colonize eucalypts.In incompatible pairings, phenolics accumulated in roots as a result of a hypersensitive reaction.In mycorrhizas of Picea abies, Larix decidua and Pinus sylvestris, a lower concentration of soluble and cell wall bound phenolics than in uninoculated roots was observed (Münzenberger et al., 1990(Münzenberger et al., , 1995(Münzenberger et al., , 1996)).Later, they observed that laccase and peroxidase activities differed between mycorrhizas and uninoculated roots of P. abies and L. decidua (Münzenberger et al., 1997).In both species, mycorrhizas contained the highest laccase activity and the lowest peroxidase activity.The high laccase activity could induce the polymerisation of soluble phenolics contributing to their decreasing.The low peroxidase activity would inhibit oxidative rigidification of cell wall.These reactions would favour root colonization by ECM fungi.
In Southern Brazil, Eucalyptus spp.and Pinus spp., even when planted in the same sites, seldom have the same fungal symbionts.This difference in fungal diversity could possibly be related to fungus-host specificity.In this sense, this phenomenon deserves further consideration.
Thus, this study was carried out with four ectomycorrhizal fungi well known by their specific occurrence in Eucalyptus or Pinus stands in Santa Catarina, Southern Brazil.The aim was to determine fungal infectivity towards Eucalyptus dunnii Maiden and its relationship with phenolic compounds accumulation on roots.
Seeds of E. dunnii were disinfected in 70% ethanol (30 seconds) and surface sterilized in 1% sodium hypochloride (20 minutes).They were placed on the surface of Modified Melin Norkrans agar (MMN) (Marx, 1969)  For mycorrhizal synthesis, five seedlings were placed concentrically on the surface of the cellophane film with their roots in contact with the fungus (Burgess et al., 1995) and kept at the same conditions described for the germination.Uninoculated controls were prepared similarly, except for the absence of fungal colony.There were ten replicates (dishes) per treatment (50 seedlings).
Five weeks later, plants were carefully removed, shoots were eliminated, and roots were placed in distilled water.Roots were observed under stereomicroscope (30 x) to determine the number of colonized root tips per plant.After that, they were divided into two parts, one for microscope observations and another for extraction of phenolic compounds.
Three samples of 100 mg of fresh roots were used per treatment for the extraction of total phenolics.Each sample was grinded in 2 mL of 70% ethanol and kept in a water bath at 60ºC before being centrifuged for 2 minutes at 700 g.The pellet was dissolved and extracted twice again by the same procedure (Phillips & Henshaw, 1977).The three extracts of the same sample were combined in order to obtain a final extract of 6 mL.
Phenolic compounds were quantified in three aliquots of 0.5 mL from each sample (three samples/treatment), according to the Folin-Denis technique (Swain & Hills, 1959).The extract was dried at 50ºC for 24 hours.The pellet was redissolved in 1 mL of distilled water and 0.5 mL of Folin-Denis reagent and 1 mL of saturated calcium carbonate solution were added.The final volume was adjusted to 10 mL with distilled water.The solution was kept at room temperature for 45 minutes.Optical density was measured at 725 nm.The results were compared with a standard curve obtained with tanic acid at different concentrations (0, 20, 40, 60, 80 and 100 mg/mL).
Data on number of colonized root tips and total phenolic compounds per plant were submitted to variance analysis and the averages compared by the t test.
Pisolithus sp., H. gardneri and S. cothurnatus colonized E. dunnii roots, whereas no colonization was observed in plants inoculated with R. nigrescens.Pisolithus sp.formed typical mycorrhizas (Fig. 1B), with a well developed mantle and a Hartig net limited to the first layer of cortical cells.H. gardneri and S. cothurnatus mycorrhizas (Figs.1A and 1D), although presenting a well developed mantle, showed no discernible Hartig net besides a few hyphae dispersed in epidermal intercellular spaces.No mantle nor Hartig net were observed on roots inoculated with R nigrescens (Fig. 1C).
According to statistical analysis, Pisolithus sp. and H. gardneri showed a higher infectivity in relation to E. dunnii _ with 15.8 and 2.8 colonized root tips per plant, respectively than S. cothurnatus and R. nigrescens which colonized only 1.4 and 0.0 root tips, in this order (Table 1).Smith & Read (1997) consider that mantle and mainly Hartig net are indicative of effective establishment of ectomycorrhizas.In this way, Pisolithus sp.(UFSC-Pt44) presents higher compatibility towards E. dunnii than the other fungi, because this fungus formed typical mantle and Hartig net structures on roots.In comparison, H. gardneri and S. cothurnatus mycorrhizas although presenting a well developed mantle, had no typical Hartig net, presenting a superficial colonization.However, other authors consider that an ectomycorrhiza is characterized by any case of a fungus forming a mantle, with or without Hartig net (Warcup, 1980).Superficial mycorrhizas in Eucalyptus spp.formed by Hysterangium spp.have previously been described by Warcup (1980) and Malajczuk et al. (1987).The former author demonstrated plant growth stimulation by this type of mycorrhizas.In this sense, H. gardneri and S. cothurnatus superficial colonizations have been considered as ectomycorrhizas as well.
Data on number of mycorrhizal root tips show that Pisolithus sp. and H. gardneri were more infective towards E. dunnii roots than S. cothurnatus and R. nigrescens, suggesting that the isolates from Eucalyptus spp.are more compatible in relation to this plant than those from Pinus spp.Species of Suillus and Rhizopogon spp.are well known by associating specifically with certain plant genera mainly of conifers (Garbaye, 1990).Plants inoculated with S. cothurnatus presented a higher accumulation of phenolics in roots, with an average of 12.3 mg/mg of fresh weight (Table 1).Those inoculated with H. gardneri did not differ from controls, with an average of 11.1 and 10.2 mg/mg of fresh weight, in this order.Conversely, roots inoculated with Pisolithus sp. and R. nigrescens presented a lower level of these substances, 9.1 and 8.8 mg/mg of fresh weight, respectively.
Métraux (1994) related phenolic accumulation in plants to defence mechanisms against pathogenes.Malajczuk et al. (1982Malajczuk et al. ( , 1984) ) related this phenomenon also to plant reaction to incompatible ECM fungi.In this study, S. cothurnatus induced a significant accumulation of phenolics in roots which coincided with a lower infectivity compared to Pisolithus sp. and H. gardneri.Roots inoculated with H. gardneri did not significantly increase the production of these substances, whereas those inoculated with Pisolithus sp. and R. nigrescens presented a lower level of phenolics than uninoculated roots.These observations indicate that only S. cothurnatus, isolated from Pinus sp., stimulated a hypersensitive reaction on E. dunnii roots.Conversely, R. nigrescens, also from Pinus, did not induce this reaction, but was unable to colonize roots.The incompatibility between this fungus and E. dunnii must be related to other mechanisms, unless the total absence of infection had prevented accumulation of phenolics.
The results suggest that the specific occurrence of Pisolithus sp. and H. gardneri and the absence of S. cothurnatus and R. nigrescens in Eucalyptus plantations could be related to their compatibility/incompatibility towards these plants.Nevertheless, these results refer only to four isolates hence the need for more studies in order to establish a full explanation for the fungus-host specificity observed in Southern Brazil plantations.

TABLE 1 .
Mycorrhizal colonization and total phenolic compounds accumulation in Eucalyptus dunnii roots inoculated with ectomycorrhizal fungi after five weeks of incubation 1 .Values in the same column followed by different letters are significantly different according to t test (P=0.05).2Values are the average of 50 replicates per treatment.3Values are the average of nine replicates per treatment. 1