Solubilisation and colonisation of wood ash by ectomycorrhizal fungi isolated from a wood ash fertilised spruce forest

FEMS Microbiology Ecology 35 (2001) 151^161 www.fems-microbiology.org Solubilisation and colonisation of wood ash by ectomycorrhizal fungi isolated from a wood ash fertilised spruce forest Shahid Mahmood *, Roger D. Finlay b , Susanne Erland a , Ha®kan Wallander a a Department of Microbial Ecology, University of Lund, Ecology Building, S-223 62 Lund, Sweden Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S-750 07 Uppsala, Sweden Received 16 August 2000; received in revised form 11 December 2000; accepted 21 December 2000 Abstract In Sweden application of granulated wood ash has been suggested as a method to supplement nutrient loss resulting from harvesting of forest residues for bioenergy production. Mycelia of two ectomycorrhizal fungi Piloderma sp. 1 and Ha-96-3, were commonly found to colonise ash granules in a wood ash fertilised spruce forest. Thirty-eight fungal isolates were selected from 10 taxa to investigate the possible role of different ectomycorrhizal fungi in nutrient mobilisation from ash. The taxa were Cenococcum geophilum Fr., Piloderma croceum Erikss. and Hjortst., Piloderma sp. 1, Thelephora terrestris (Ehrenb.) Fr., Tylospora fibrillosa Donk, and five unidentified species, all originating from a wood ash fertilised spruce forest. The isolates were tested for their ability to solubilise tricalcium phosphate (TCP) or hardened wood ash (HWA) in vitro. Ha-96-3, P. croceum and Piloderma sp. 1 were the only taxa which solubilised TCP. Abundant calcium oxalate crystals were formed in TCP and HWA plates with Piloderma sp. 1. Ha-96-3 and two isolates of P. croceum produced intermediate amounts of crystals. Ha-96-1 and T. fibrillosa produced low amounts of crystal but no crystal formation was observed by any of the other isolates. Piloderma sp. 1 from HWA plates had significantly higher concentrations of P, compared to P. croceum or Ha-96-3. Piloderma sp. 1 and P. croceum were further tested for their ability to colonise wood ash in microcosms containing intact mycorrhizal associations. After 7 months Piloderma sp. 1 colonised ash amended patches with a dense, mat like mycelium, whereas P. croceum mycelia avoided the ash patches. Possible differences between these fungi in patterns of carbon allocation were investigated by labelling seedlings with 14 CO2 . Piloderma sp. 1 mycelia allocated significantly more 14 C to ash patches than P. croceum. P. croceum allocated relatively more 14 C to control patches than to the ash patches. The possible role of ectomycorrhizal fungi in mobilisation of nutrients from wood ash is discussed. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ectomycorrhizal fungi; PCR-RFLP ; Tricalcium phosphate; Wood ash; Solubilisation; Calcium oxalate; Spruce forest 1. Introduction Ectomycorrhizal fungi form symbiotic associations with tree roots and improve uptake of nutrients and water from soils with low fertility [1]. Mycorrhizal trees allocate carbon to mycorrhizal mycelia which take up mineral nutrients and can also play an important role in making organically bound N available to their host plants [1]. There is also convincing evidence that ectomycorrhizal fungi are able to weather primary minerals to mobilise nutrients [2^9]. Anthropogenic deposition of pollutants and increased harvesting of forest residues are known to cause acidi¢cation in coniferous forests [10^12]. Application of granu- * Corresponding author. Tel. : +46 (46) 2223756; Fax.: +46 (46) 2224158; E-mail : lated wood ash to such forest soils has been recommended to increase the pH and soil productivity through a slow supplementation of lost base cations and other nutrients essential for tree growth [13^15]. The e¡ects of ash fertilisation on the ectomycorrhizal fungi, especially the mechanisms by which certain species can capture nutrients from wood ash, have not been thoroughly investigated. In an earlier investigation [16] of the e¡ects of granulated wood ash in a spruce forest, although no signi¢cant e¡ects were reported on individual species, two mycorrhizal species (Piloderma sp. 1 and Ha-96-3) showed a tendency to increase in abundance in plots treated with ash. Moreover, Piloderma sp. 1 and Ha-96-3 mycelia were also found to colonise the ash granules. We hypothesise that these mycorrhizal fungi may play a role in mobilisation of nutrients from the wood ash. Most of the elements in wood ash are readily leachable except P which is bound in apatite like compounds with low solubilities [17]. Several investiga- 0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 1 2 4 - 0 FEMSEC 1213 29-3-01 Cyaan Magenta Geel Zwart b a; 152 S. Mahmood et al. / FEMS Microbiology Ecology 35 (2001) 151^161 2. Materials and methods 2.1. Sampling of ectomycorrhizal roots Ectomycorrhizal roots were collected from a Norway spruce (Picea abies (L.) Karst) forest planted in 1957 and located at Torup in the southwest of Sweden (56³55PN, 13³05PE). The experimental forest was treated in 1990 with 0 ton ha31 (control, C), 3 ton ha31 (low ash, LA) and 6 ton ha31 (high ash, HA) of granulated wood ash. Ectomycorrhizal roots were sampled in May, 1997. Cores from three replicates of all treatments were sampled with the aim of ¢nding representatives of all mycorrhizal taxa in the community on the roots, previously described by Mahmood [16]. 2.2. Isolation of fungi from root tips Mycorrhizas were picked from the root materials of each treatment. After surface sterilisation with 30% H2 O2 for 20^30 s and rinsing with sterile water, the roots were plated on Petri dishes ¢lled with 25 ml of 1/2 strength modi¢ed Melin^Norkrans (MMN) medium containing 30 mg l31 chlorotetracycline and 1 mg l31 Benomyl [24]. On the basis of root tip morphology, attempts were made to plate similar looking mycorrhizas on the same plate (not more than 10 per plate). The resultant axenic cultures were subsequently maintained on 1/2 MMN medium. 2.3. Molecular identi¢cation of ectomycorrhizal fungi Ninety-seven axenic cultures resembling basidiomycetes or ascomycetes were selected for molecular identi¢cation of the isolated fungi. Mycelium covering an area about 1U1/2 cm on the Petri dish was carefully removed, avoiding the agar medium. DNA was extracted using enzymatic FEMSEC 1213 29-3-01 and mechanical lysis of cell walls, SDS extraction bu¡er and phenol/chloroform steps, following the procedures described by Mahmood et al. [25]. The primers used for PCR were, ITS1, 5P-TCCGTAGGTGAACCTGCGG-3P and ITS4, 5P-TCCTCCGCTTATTGATATGC-3P designed by White et al. [26]. The 50 Wl reaction mixture for PCR contained the following ¢nal concentrations or total amounts: 0.1^10 ng total DNA, 500 pmol of each primer, 20 mM Tris (pH 8.3), 200 WM dNTP, 1.5 mM MgCl2 , 50 mM KCl a (...truncated)