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

FEMS Microbiology Letters 221 (2003) 81^87

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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 Received 3 June 2002; received in revised form 18 February 2003; accepted 19 February 2003 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 : [email protected] (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

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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 Mycorrhizal associations were synthesised between spruce (Picea abies (L.) Karst.) seedlings and Piloderma croceum Erikss. and Hjortst. (isolate number Tor-04), or Piloderma sp. 1 (isolate number Tor-42). These ectomycorrhizal fungi were isolated in May 1997 from mycorrhizas collected in a wood ash fertilised Norway spruce forest located at Torup in southern Sweden [6,7,16]. P. croceum and Piloderma sp. 1 were identi¢ed by comparing their ITS-RFLPs (internal transcribed spacing-restriction fragment length polymorphisms) to a reference library containing RFLP pro¢les of identi¢ed ectomycorrhizal fruitbodies, axenic cultures and mycorrhizas found in our previous community structure studies at Torup and other forests located in southern Sweden [6,7,16]. Identity of Piloderma sp. 1 was further con¢rmed by sequencing the ITS region and searching for homology with known sequences for Piloderma species using an unpublished database (E. Larsson, personal communication) [6]. Mycorrhizas were synthesised using the method described by Duddridge [17] as modi¢ed by Finlay et al. [18]. In brief, root systems of 4-week-old aseptically grown spruce seedlings were enclosed in Petri dishes containing peat^vermiculite (1:4 v/v) moistened with 1/2 strength MMN solution and inoculated with three plugs of actively growing mycelium or left non-inoculated to serve as non-mycorrhizal (NM) controls. All synthesis dishes were kept in a phytotron programmed for 300 Wmol m32 s31 PAR, 80% relative humidity and an 18 h/6 h and 18‡C/16‡C day/night cycle. Both P. croceum and Piloderma sp. 1 formed mycorrhizas within 3 months of inoculation. Only well colonised ( s 75% colonised root tips) spruce seedlings were selected for the following pot-based competition experiment. 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 used for soil-solution extraction by centrifugation at 16 270Ug for 10 min. pH (H2 O) was measured in the soil solution by using a PHR-146 micro pH electrode (Lazar Research Laboratories, USA). 2.4. Molecular con¢rmation of morphotyped mycorrhizas Piloderma sp.1 and P. croceum mycorrhizas were picked randomly for DNA extraction following the procedures described by Mahmood et al. [20]. ITS1 and ITS4 primers were used for polymerase chain reaction (PCR) ampli¢cation of the fungal rDNA ITS region [21]. The concentration of reagents in the PCR reaction mixture and the thermocycling conditions were the same as described earlier by Mahmood et al. [6,7,20]. PCR-ampli¢ed products were analysed by electrophoresis on 2% agarose gels and detected by staining with ethidium bromide [22]. RFLP analysis of the PCR products was carried out with the restriction endonucleases HinfI, MboI and TaqI according to the manufacturers’ recommendations (Boehringer Mannheim). The uncut ampli¢cation products and their restriction fragments were size-fractionated using 1% agarose+1% Nusieve (FMC) gels stained with ethidium bromide [22]. The gels were documented using a Gel Doc 2000 System (Bio-Rad) and Quantity One0 software (Bio-Rad) for imaging and band analysis. Restriction fragments were compared to reference RFLPs of Piloderma sp. 1 and P. croceum [6] to con¢rm their identities.

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2.5. Statistical analysis One-way analysis of variance (ANOVA) was used to determine di¡erences (P 6 0.05) in all measured parameters between seedlings grown in variously amended substrates.

3. Results 3.1. Colonisation of bait seedlings Piloderma sp. 1 mycelia colonised around 60% of the ¢ne roots of bait seedlings in wood ash amended substrate with both levels of N (LN+A, HN+A), whereas P. croceum only colonised around 8% of the root tips in these two treatments (Fig. 1). P. croceum, on the other hand, colonised 56% of the total root tips of bait seedlings growing in pots with LN3A. However, colonisation by P. croceum declined signi¢cantly in the HN3A treatment (27%). No composite mycorrhiza formation by these two fungi was noticed. The numbers of NM root tips did not vary signi¢cantly between the treatments (Fig. 1).

Fig. 2. Percentage colonisation of P. croceum pre-inoculated seedlings when grown in the presence of Piloderma sp. 1 pre-inoculated and bait seedlings. The seedlings were grown in wood ash amended substrate (6 g l31 = +A) (or left unamended, 3A) with two levels of a slow-release N fertiliser (1 g l31 , low N = LN; 2 g l31 , high N = HN). Vertical bars represent S.E.M. of ¢ve replicates. Di¡erent letters indicate statistically di¡erent values (P 6 0.05) for P. croceum mycorrhizas. Piloderma sp. 1 or NM root tips did not vary signi¢cantly between the treatments.

3.2. Mycorrhizal status of P. croceum pre-colonised seedlings In the LN3A treatment, P. croceum mycorrhizas were found on 61% of the total ¢ne roots of pre-inoculated seedlings at the time of harvest (Fig. 2). However, the level of colonisation declined sharply in HN3A (37%), LN+A (35%) and HN+A (32%) treatments. The numbers of NM root tips were not signi¢cantly di¡erent (Fig. 2). 3.3. Mycorrhizal status of Piloderma sp. 1 pre-colonised seedlings

Fig. 1. Percentage colonisation of bait seedlings by P. croceum or Piloderma sp. 1 (or non-colonised, NM) when challenged by P. croceum and Piloderma sp. 1 pre-colonised seedlings. The seedlings were grown in wood ash amended substrates (6 g l31 = +A) (or left unamended, 3A) with two levels of a slow-release N fertiliser (1 g l31 , low N = LN; 2 g l31 , high N = HN). Vertical bars represent S.E.M. of ¢ve replicates. Di¡erent letters indicate statistically di¡erent values (P 6 0.05) for P. croceum or Piloderma sp. 1 mycorrhizas. NM root tips did not vary signi¢cantly between the treatments.

Regardless of the treatment, the colonisation level for Piloderma sp. 1 was between 49 and 65% at harvest, being lowest in the 3A and highest in the +A treatments (Fig. 3). P. croceum colonised 33% of the root tips in the LN3A treatment, but the level of colonisation declined drastically in the HN3A (5%) and in LN+A and HN+A treatments (9% and 4%, respectively). The NM root tips were in the range of 21^34% in all the treatments (Fig. 3). 3.4. PCR-RFLP analysis of Piloderma sp. 1 and P. croceum morphotypes PCR ampli¢cation success rate was 100% for the

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sampled mycorrhizas. The resultant RFLPs of Piloderma sp. 1 or P. croceum showed an exact match with the corresponding restriction fragment sizes reported previously by Mahmood et al. [6]. 3.5. Seedling growth All the seedlings grown in +A treated pots showed higher biomass compared to the 3A controls (Table 1). In contrast to the other seedlings, Piloderma sp. 1 pre-colonised seedlings responded strongly to addition of N and the biomass of these seedlings was twice as high as for other seedlings in the HN+A treatment (Table 1). Root:shoot ratios did not vary signi¢cantly between the treatments. The pH of the soil solution at the end of experiment was 4.4 and 4.5 in the substrates without ash (LN3A, HN3A), and 6.3 and 6.4 in the ash amended substrates (LN+A, HN+A) (Table 1).

Fig. 3. Percentage colonisation of Piloderma sp. 1 pre-colonised seedlings when grown in the presence of P. croceum pre-inoculated and bait seedlings. The seedlings were grown in wood ash amended substrate (6 g l31 = +A) (or left unamended, 3A) with two levels of a slow-release N fertiliser (1 g l31 , low N = LN; 2 g l31 , high N = HN). Vertical bars represent S.E.M. of ¢ve replicates. Di¡erent letters indicate statistically di¡erent values (P 6 0.05) for P. croceum mycorrhizas. Piloderma sp. 1 or NM root tips did not vary signi¢cantly between the treatments.

4. Discussion This study clearly demonstrates that Piloderma sp. 1 has a competitive advantage over P. croceum, not only in colonising bait seedlings but also in maintaining the dominance on pre-colonised seedlings when grown in a wood ash amended substrate. P. croceum has a narrow pH tolerance interval compared to many other ectomycorrhizal fungi, as indicated by earlier investigations by Erland [23]. The pH in ash amended pots was 6.4 compared to 4.4 in the controls of the present study. Erland and So«derstro«m

Table 1 Plant growth parameters for bait seedlings and P. croceum or Piloderma sp. 1 pre-colonised seedlings in wood ash amended substratesa Parameters

Total root tips per m root length Bait seedling P. croceum pre-colonised seedling Piloderma sp. 1 pre-colonised seedling Shoot wt (mg) Bait seedling P. croceum pre-colonised seedling Piloderma sp. 1 pre-colonised seedling Root wt (mg) Bait seedling P. croceum pre-colonised seedling Piloderma sp. 1 pre-colonised seedling Plant biomass (mg) Bait seedling P. croceum pre-colonised seedling Piloderma sp. 1 pre-colonised seedling pH (soil solution)

Treatments

ANOVA

LN3A

HN3A

LN+A

HN+A

P value

548 U 44 602 U 26 607 U 22

453 U 54 520 U 99 493 U 54

520 U 67 560 U 46 611 U 79

564 U 26 500 U 40 578 U 36

n.s. n.s. n.s.

83 U 10 a 126 U 32 132 U 16 a

88 U 15 a 94 U 23 142 U 12 a

146 U 20 b 193 U 21 189 U 32 ab

174 U 13 c 160 U 26 318 U 46 b

0.002 n.s. 0.006

72 U 2 a 113 U 24 122 U 16 a

65 U 7 a 71 U 17 125 U 22 a

112 U 14 b 140 U 18 183 U 21 b

130 U 16 b 135 U 17 293 U 23 c

0.006 n.s. 0.000

155 U 11 a 239 U 61 254 U 32 a 4.4 U 0.09

153 U 19 a 165 U 38 268 U 33 a 4.5 U 0.06

258 U 30 b 333 U 36 372 U 51 b 6.3 U 0.08

304 U 21 c 295 U 42 611 U 58 c 6.4 U 0.06

0.000 n.s. 0.001

a

Bait seedlings were grown together with P. croceum and Piloderma sp. 1 pre-colonised seedlings in wood ash amended substrate (6 g l31 = +A) (or left unamended, 3A) with two levels of a slow-release N fertiliser (1 g l31 , low N = LN; 2 g l31 , high N = HN) for 120 days. The table shows: total numbers of root tips per m root length, shoot weight, root weight and plant biomass for seedlings grown in variously amended substrates. pH as measured in soil solution collected from di¡erent treatments is also given. Values are means U S.E.M. of ¢ve replicates. Di¡erent letters denote di¡erences among means (P 6 0.05, ANOVA). n.s., not signi¢cant.

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[24] grew pine seedlings in limed humus (pH 4^7.5) and reported that P. croceum did not colonise at pH values higher than 6.2, and that the optimal pH for root colonisation was around 5. This may be the reason why P. croceum was unsuccessful in competing with Piloderma sp. 1 mycelia for infection sites on uncolonised seedlings treated with wood ash. It appears that high pH in the ash treated pots increased mycelial growth of Piloderma sp. 1, thereby increasing the inoculum potential to colonise new roots. Previous studies of the experimental forest at Torup (the ectomycorrhizal fungi used in the present investigation originate from this forest) have shown that there was a gradual increase in soil pH and base cations during the ¢rst 1^2 years of wood ash fertilisation as compared to the control plots, but that afterwards these di¡erences became less distinct (S. Jacobson, personal communication). In the same forest, Mahmood et al. [6] conducted an ectomycorrhizal community structure study 7 years after ash application in order to assess possible long-term e¡ects of the treatment. Although after this time interval pH di¡erences between treatments could no longer be detected, Piloderma sp. 1 was found colonising 4.3 U 3.8% (mean U S.E.M.) of the root tips in control plots and increased in abundance to 20 U 5.0% in the plots treated with 6 ton ha31 of wood ash. Most importantly, this particular species was found colonising the wood ash granules in ash fertilised plots [6]. On the other hand, P. croceum colonised 3.3 U 2.9% of root tips in the control plots and further decreased to 1.1 U 0.9% in the plots treated with 6 ton ha31 of wood ash. However, no signi¢cant di¡erences between treatment means were found in the above investigation. In an in vitro assay, Piloderma sp. 1 mycelia were able to solubilise tricalcium phosphate and hardened wood ash, as indicated by abundant formation of calcium oxalate crystals in the medium, whereas P. croceum only produced a few crystals in the same study [7]. In a microcosm study, Piloderma sp. 1 mycelia in intact symbiosis with spruce seedlings colonised wood ash patches with a thick matlike mycelium, whereas P. croceum mycelia did not colonise ash patches and distinct hollow zones appeared in the mycelium at the sites of the ash patches [7]. These reports clearly indicate that Piloderma sp. 1 mycelia have the potential to solubilise nutrients in wood ash. In this study, the higher plant biomass in Piloderma sp. 1 pre-colonised seedlings (grown in HN+A treatment), indicate that these mycelia are capable of capturing nutrients not only from the wood ash but that they may have taken advantage of the high N content in the substrate. In the LN+A treatment, plant biomass was still signi¢cantly higher than in the non-ash applied controls (LN3A, HN3A) but lower than in the HN+A, indicating that the plants had access to nutrients in the ash but that N became a (growth) limiting factor due to ash induced growth enhancement. There was no signi¢cant di¡erence in plant biomass for Piloderma sp. 1 pre-colonised seedlings grown in non-ash amended substrates (LN3A, HN3A), suggesting that N supply was

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adequate, but there was de¢ciency of other nutrients which were supplemented in the ash treatments. Improved plant growth in response to ash fertilisation has been reported both in ¢eld and laboratory studies [8,25^27]. It should be noted that P. croceum or Piloderma sp. 1 pre-inoculated seedlings had higher plant biomass compared with bait seedlings, though all seedlings were of the same age. This was due to the fact that P. croceum or Piloderma sp. 1 seedlings were inoculated three months prior to the exposure of bait seedlings to the ectomycorrhizal inoculum. According to Erland and Taylor [28] Piloderma mycorrhizas are normally associated with the organic soil layer and may have the ability to provide nutrients of organic origin to the host plant. Finlay et al. [29] demonstrated in pure culture studies that P. croceum isolates were equally e⁄cient at utilising organic N (bovine serum albumin, BSA) and ammonium. Since wood ash is highly alkaline and contains only inorganic nutrients, it is possible that high concentrations of certain trace metals in the ash may have an adverse e¡ect on the mycelial growth of P. croceum, consequently resulting in poor mycorrhizal colonisation of roots. Bramryd and Fransman [30] found a signi¢cant increase in Cu concentration in the mor layer after wood ash application to a pine forest in southern Sweden. Similarly, there was an increase in the extractable Mn concentrations in the same investigation. Elevated Cd concentrations after wood ash application can also be a risk factor to the soil environment [31,32]. High concentrations of heavy metals in the environment may be toxic to ectomycorrhizal fungi and reduce their root colonisation potential (see Ahonen-Jonnarth [33]). P. croceum mycorrhizas declined drastically at the high level of N. This might be due to sensitivity of P. croceum to high N concentration in the substrate. In a similar study of competition between Hebeloma crustuliniforme and P. croceum in N-amended substrates, a marked reduction in P. croceum mycorrhizas was noticed towards the high N concentration [34]. Although Jonsson et al. [35] reported a signi¢cant increase in the abundance of P. croceum mycorrhizas after experimental N additions to a Norway spruce forest in southwestern Sweden, most investigators report a reduced mycelial growth and colonisation potential for a majority of ectomycorrhizal fungi in high N treatments [34,36^38]. Piloderma sp. 1 was the most abundant species among ectomycorrhizal fungi found on spruce roots in a granulated wood ash fertilised forest at Torup. Moreover, this species was found colonising ash granules in the ¢eld and showed a tendency to increase in abundance at the higher dose of ash application [6]. The present study further con¢rms that ash amendments increase the colonisation potential of Piloderma sp. 1. The experiment was, however, performed in a non-sterile sand^peat substrate, where no indigenous mycorrhizal colonisation was noted. Further studies are needed to investigate the competition of Pilo-

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derma sp. 1 with indigenous mycorrhizal fungi by planting pre-colonised seedlings in forest humus and then monitoring the persistence and replacement dynamics of root infection. For such studies, it would be necessary to use DNA-based molecular methods for discrimination between inoculated and indigenous strains.

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Acknowledgements

[17]

This research was supported by the National Swedish Energy Administration (STEM). I thank Drs. Hafikan Wallander, Susanne Erland and Roger D. Finlay for their constructive and useful suggestions on this manuscript.

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