Colonisation of spruce roots by two interacting ectomycorrhizal fungi in wood ash amended substrates

FEMS Microbiology Letters 221 (2003) 81^87 www.fems-microbiology.org Colonisation of spruce roots by two interacting ectomycorrhizal fungi in wood ash amended substrates Shahid Mahmood  Department of Microbial Ecology, University of Lund, Ecology Building, 223 62 Lund, Sweden First published online 14 March 2003 Abstract Interactions between two ectomycorrhizal fungal species, Piloderma croceum Erikss. and Hjortst. and Piloderma sp. 1 (found to colonise spruce roots and wood ash granules in the field), were investigated in wood ash amended substrates. The comparative ability of these fungi to colonise roots of non-mycorrhizal spruce (Picea abies (L.) Karst.) seedlings was studied in relation to factorial combinations of wood ash and N fertilisation. Non-mycorrhizal spruce seedlings (bait seedlings) were planted together with spruce seedlings colonised by P. croceum or Piloderma sp. 1. The growth substrate was a sand^peat mixture with wood ash or no ash and supplied with two levels of N, so that four substrate combinations were obtained. Piloderma sp. 1 mycelia colonised around 60% of the fine roots of bait seedlings in ash treatments regardless of N level and around 20^26% in treatments without ash. P. croceum only colonised 8% of the root tips in the presence of ash but 56% of the root tips in the low-N treatment without ash. However, in the high-N treatment without ash the colonisation level was reduced to around 30%. Total numbers of root tips per seedling did not vary significantly between the treatments. Possible reasons for the competitive advantage of Piloderma sp. 1 in wood ash fertilised substrate are discussed. 9 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ectomycorrhizal fungi; Spruce colonisation ; Wood ash; N fertilisation; Competition ; PCR^RFLP 1. Introduction A long-term decline in pH of forest soils has been observed in southern Sweden due to atmospheric deposition of pollutants [1,2]. Increased harvesting of forest residues for bioenergy production would further intensify the prevailing acidi¢cation by depletion of base cations from forest soils and might thus have serious environmental consequences in the future. For long-term sustainability and productivity of forest soils, recycling of stabilised wood ash has been recommended to supplement the lost nutrients and to raise the pH [3^5]. Before applying wood ash on a large scale, however, there is a need to evaluate the consequences of this silvicultural practice on the forest ecosystem. Ectomycorrhizal mycelia have been found to colonise ash granules in a wood ash fertilised spruce forest in southern Sweden [6]. The mycorrhizal taxa Piloderma * Present address: Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK. Tel. : +44 (1224) 555849; Fax: +44 (1224) 555844. E-mail address : (S. Mahmood). sp. 1, Ha-96-3, and Tor-97-1 were detected on 7, 74 and 4% respectively of the examined ash granules in the abovementioned study. Piloderma sp. 1 and Ha-96-3 have been found to solubilise tricalcium phosphate and hardened wood ash in vitro [7]. The mycelia of Piloderma sp. 1 growing in intact symbiotic associations with spruce seedlings were able to colonise wood ash in laboratory microcosms, whereas mycelia of Piloderma croceum did not colonise the ash [7]. In a recent investigation [8], higher PO33 4 concentrations were found in substrates colonised by Piloderma sp. 1 mycelia in the ash treatment compared to other treatments, indicating that Piloderma sp. 1 stimulated P release from the ash. Furthermore, Piloderma sp. 1 appeared to accumulate Ca from ash in the mycorrhizal roots [8]. There are reports that mycorrhizal fungi have the ability to mobilise nutrients by weathering of primary minerals and thus contribute to uptake of nutrients essential for plant growth [9^15]. The aim of the present study was to test whether the mycorrhizal fungi which colonise ash in the ¢eld and laboratory studies also have a competitive advantage over ‘non-ash colonising fungi’ in colonising new roots in soils amended with ash. 0378-1097 / 03 / $22.00 9 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00166-6 FEMSLE 10907 2-4-03 Received 3 June 2002; received in revised form 18 February 2003; accepted 19 February 2003 82 S. Mahmood / FEMS Microbiology Letters 221 (2003) 81^87 There were ¢ve replicate pots for each treatment. The pots were arranged in a randomised design in a phytotron (with the above-mentioned environmental parameters) and the plants allowed to grow for 120 days before harvesting. 2. Materials and methods 2.1. Mycorrhiza synthesis 2.2. Preparation of plant growth pots Plastic pots (7U7U8 cm) were ¢lled with homogenised sand^peat substrate (1:1 v/v) amended with hardened wood ash (6 g l31 , +A) (Ljungbyverket, Sydkraft va«rme AB, Sweden) or left unamended (3A). This amount corresponded approximately to 6 ton ha31 , which was the highest amount applied in the experimental forest where the ectomycorrhizal isolates were collected [6^8]. Two levels of a slow-release N fertiliser (methylene urea, Kemira, Finland) (1 g l31 , low N = LN, or 2 g l31 , high N = HN) were also applied. Mycorrhizal spruce seedlings colonised by P. croceum or Piloderma sp. 1 were planted in two opposite corners of the pot. A NM spruce seedling (bait seedling) was also planted in the centre of the pot. A similar experimental design has recently been used by Wu et al. [19]. The initial level of mycorrhizal colonisation (based on visual estimation) at the start of experiment was s 75% for both P. croceum and Piloderma sp. 1 seedlings. 2.3. Harvesting and assessment of ectomycorrhizal colonisation At the end of the experiment, the plants were taken out of the substrate without damaging the root system and washed on a sieve under tap water to remove the debris. Roots from each seedling were cut into small pieces of about 2^4 cm in length and stirred together in a container ¢lled with water. Mycorrhizal root tips colonised either by P. croceum or Piloderma sp. 1 or NM were counted after randomly selecting small root fragments totalling 1 m in length. Mycorrhizas were distinguished under a dissecting microscope and relative frequencies of each morphotype were counted. P. croceum mycorrhizas were recognised due to their bright yellow mantle and yellow external mycelium. In contrast, Piloderma sp. 1 mycorrhizas had a white mantle and white external mycelium. The morphotyped mycorrhizas were further subjected to ITS-RFLP typing to con¢rm their identities (see Section 2.4). Roots apparently lacking fungal mantle, or which were shrunken or dead, were designated as NM in this study. Shoots and roots were dried in an oven at 80‡C for 24 h before weight determinations. Soils from di¡erent treatments were (...truncated)