Selected Ectomycorrhizal Fungi of Black Spruce - Lakehead University

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associated phenolic compounds, and (2) to test whether these ECM fungi can ..... (1995) reported that growth of one isolate of P. involutus (NF4) was stimulated ...

J Chem Ecol (2006) 32: 1473–1489 DOI 10.1007/s10886-006-9063-6

Selected Ectomycorrhizal Fungi of Black Spruce (Picea mariana) can Detoxify Phenolic Compounds of Kalmia angustifolia Ren Sen Zeng & Azim U. Mallik

Received: 17 December 2004 / Revised: 15 January 2006 / Accepted: 21 January 2006 / Published online: 23 May 2006 # Springer Science + Business Media, Inc. 2006

Abstract Allelopathy has been implicated as a factor contributing toward failure of black spruce (Picea mariana) regeneration in Kalmia angustifolia-dominated sites in eastern Canada. Several phenolic acids of Kalmia origin inhibit primary root growth of black spruce. We tested the hypothesis that some well-adapted conifer ectomycorrhizae can degrade and detoxify water-soluble phenolic compounds produced by Kalmia and use the degraded products as a carbon source to stimulate growth. We found that hyphal growth of Paxillus involutus, a common ectomycorrhizal fungus of black spruce, was stimulated by water leachates of Kalmia leaf and litter. An equimolar mixture of three phenolic acids (ferulic, o-coumaric, and ohydroxyphenylacetic acid), commonly found in Kalmia, had no negative effects on fungal growth at 1 mM concentration. The o-hydroxyphenylacetic (o-HPA) acid, which is known to be toxic to black spruce, was found to stimulate the growth of Laccaria laccata, L. bicolor, and P. involutus (isolates 211804 and 196554) by 38.4, 29.3, 25.0, and 18.9%, respectively, at 1 mM. Pure ferulic, o-coumaric, and o-HPA acids were degraded by 100, 98, and 79.5%, respectively, within 10 d in the presence of P. involutus 211804. However, L. laccata could not tolerate high concentrations of the Kalmia leachates. P. involutus and L. bicolor used o-HPA acid as a carbon source when cultured in noncarbon nutrient medium. The 0.5 and 0.2 mM o-HPA acid inhibited the root growth of black spruce. However, after solutions had been exposed to a culture of P. involutus, they had no significant effect on seedling growth of black spruce. We concluded that some ectomycorrhizal fungi, such as P. involutus and L. bicolor, are able to degrade Kalmia phenolics. Our findings point to R. S. Zeng : A. U. Mallik (*) Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada P7B 5E1 e-mail: [email protected] R. S. Zeng Institute of Tropical and Sub-Tropical Ecology, South China Agricultural University, Wushan, Guangzhou 510642, China

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a mechanism by which ectomycorrhizal species can control species interactions in higher plants by changing the rhizosphere chemistry. Keywords Ectomycorrhizal fungi . Phenolic compounds . Detoxification . Picea mariana . Kalmia angustifolia

Introduction Naturally regenerating and planted conifers in nutrient-poor boreal forests of eastern Canada often suffer from growth inhibition due to ericaeous understory shrubs (Mallik, 1987, 2001; Mallik and Newton, 1988; Thiffault et al., 2004). Following clear cutting and fire, sheep laurel (Kalmia angustifolia L. var. angustifolia, hereafter referred to as Kalmia), a common ericaceous understory plant in eastern Canada, can seriously hinder conifer regeneration, especially black spruce [(Picea mariana (Mill.)]. One cause has been attributed to allelopathic effects of Kalmia phenolics (Mallik, 1987; Thompson and Mallik, 1989; Zhu and Mallik, 1994). Phenolic acids are released into the soil as leaf and litter leachates, root exudates, or as decaying plant residues (Siqueira et al., 1991; Gallet and Pellissier, 1997). Several are reported to act as signal molecules, and some may influence signal transduction pathways in symbiotic systems (Lynn and Chang, 1990). Fries et al. (1997) reported that certain phenolic acids stimulate arbuscular mycorrhizal (AM) colonization, which may promote growth of host plants. The mechanism of conifer growth inhibition in the presence of ericaceous plants is complex and the role of Kalmia phenolics in black spruce growth inhibition is unclear (Wallstedt et al., 2002). Reduced ectomycorrhizal (ECM) colonization of Norway spruce [Picea abies (L.) Karst] by Cenococcum graniforme, L. laccata (Pellissier 1993), and Hebeloma crustuliniforme (Souto et al., 2000) in the presence of Vaccinium myrtillus L. (hereafter referred to as Vaccinium) has been suggested as a cause of its regeneration failure in subalpine forests in Europe. Handley (1963) found few mycorrhizal fungi associated with Norway spruce root systems when grown in the presence of heather (Calluna vulgaris L.). Yamasaki et al. (1998) reported significantly lower stem height, root–shoot biomass, and reduced mycorrhization of black spruce grown near (1 m) from Kalmia under field conditions. Kalmia leaf leachates have been found to be inhibitory to mycorrhizal formation and mycelial growth of some ectomycorrhizal fungi of black spruce (Mallik et al., 1998). Ectomycorrhizal fungi often experience chemical stress from forest floor phenolics (Bending and Read, 1997). The role of phenolic acids in ectomycorrhizal associations is not well understood. Black spruce–Kalmia forests of eastern Canada and Norway spruce–Vaccinium forest of subalpine France and Fennoscandia produce large amounts of litter on the forest floor (Inderjit and Mallik, 1999; Berg and Dise, 2004). Ericaceous plants, such as Kalmia and Vaccinium, produce large amounts of phenolic acids, which may accumulate on the forest floor through leaching from leaves (Zhu and Mallik, 1994), litter, humus (Pellissier 1994), and possibly root exudates. Many of these phenolic acids inhibit primary root growth in black spruce (Zhu and Mallik, 1994), seed germination and seedling growth in

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Norway spruce, and mycorrhizal fungal growth (Pellissier 1993). Several authors have reported inhibitory effects of ericaceous litter on growth and respiration of spruce mycorrhiza (Boufalis et al., 1994; Boufalis and Pellissier, 1994). Souto et al. (2000) showed that humus phenolics of Vaccinium were more toxic to the spruce mycorrhizal fungus, H. crustuliniforme, than to the ericoid (Vaccinium) mycorrhizal fungus, Hymenoscyphus ericae. In ectomycorrhizal symbioses, plant roots and fungi function together as a unit. Formation of ECMs in plants often allows them to establish in habitats that neither symbiont may be able to occupy individually (Nehls et al., 2000). Ectomycorrhizae are able to alleviate toxic effects of heavy metal cations in host plants (Denny and Wilkin, 1987; Colpaert and Van Assche, 1993). They may have additional or related mechanisms to deal with phenolic allelochemicals on the forest floor. From a controlled experiment, Mallik et al. (1998) showed that the growth inhibitory effects of Kalmia leachate on black spruce could be overcome by inoculating black spruce with selected mycorrhizal fungi. Because mycorrhizal fungi are located at the interface between the soil-allelochemical reservoir and roots of the host plants, we hypothesized that if certain spruce ECM fungi can degrade Kalmia phenolics, and use the degraded product(s) as a carbon source, then these ectomycorrhizal fungi would be able to protect black spruce against Kalmia phenolics and enhance their growth. The objectives of our study were (1) to identify certain ECM fungi that can withstand the toxicity of Kalmia leaf, litter, and humus leachates and their associated phenolic compounds, and (2) to test whether these ECM fungi can degrade and detoxify the phenolic acids of Kalmia.

Methods and Materials Plant and Ectomycorrhizal Fungal Materials Black spruce seeds were obtained from Hills Nursery, Thunder Bay, Canada. P. involutus (Batsch. Ex Fr.) Fr. 211804, 196554, and L. laccata (Scop.:Fr.) Cooke 211651 were obtained from the Canadian Collection of Fungal Cultures, Ottawa. Pisolithus tinctorius Coker and Couch 99132 was obtained from Prof. Mingqin Gong at the Research Institute of Tropical Forestry in Guangzhou, Forestry Academy of China. Laccaria bicolor (Maire) P.D. Orton 229559 was collected from Thunder Bay. P. tinctorius 99132 was used to compare responses of ECM fungi from ericaceous-dominated and nonericaceous-dominated areas. Chemicals Ferulic, o-coumaric, and o-hydroxyphenylacetic (o-HPA) acids were purchased from Sigma (St. Louis, MO, USA). Malt extract and granulated agar were bought from Becton Dickinson Co. All solvents used were analytical or HPLC grade. Seed Surface Sterilization Black spruce seeds were surface-sterilized with 1% NaClO3 for 10 min and then rinsed with sterile water  5 to remove surface borne pathogens. Sterilized seeds were dried with autoclaved filter paper and kept at 4-C for subsequent use.

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ECM Fungal Cultures Modified Melin–Norkrans’ nutrient solution (MMN) was used for growing the ectomycorrhizal fungi (Marx, 1969). Petri dishes containing the MMN, Kalmia leachates, and phenolic acids were inoculated with 4-mm-diam agar plugs cut from the edge of 2-wk-old ectomycorrhizal fungal colonies. The inoculated plates were incubated at room temperature (20–25-C). Diameters of the fungal colonies were measured every 5 d. All experiments consisted of at least three replicates. Kalmia Leachates Kalmia fresh leaves (mature, 1-yr-old), litter (dry leaves on the ground), and humus (0–10 cm) were collected from 10 random locations in a Kalmia-dominated heath originating from a natural fire 25 yr ago near the village of Terra Nova just outside the boundary of Terra Nova National Park, Newfoundland. Composite samples of fresh leaves, litter, and humus were made by mixing the 10 samples. Coarse roots and branches were removed from the humus samples prior to mixing. Twenty g of fresh leaves and litter were soaked separately in 100-ml distilled water for 24 hr at room temperature (20–25-C). The concentrations obtained for leaf and litter leachates were 0.2 g FW equivalent of leaves or litter per ml of leachate. Kalmia humus leachate was obtained by draining 200 ml distilled water at a slow rate for 24 hr through 100 g fresh humus in a glass chromatography column (30 mm) at room temperature. The filtrate of humus leachate was adjusted to 100 ml, and its concentration was 1 g FW equivalent of humus per ml of leachate. The water leachates of leaves, litter, and humus were passed through Whatman No. 42 filter paper and then sterilized by passing through two layers of 0.45-mm Millipore filters. Experiment 1: Effects of Kalmia Water Leachates on ECM Fungi We measured the growth responses of two fungal isolates, P. involutus 211804 and L. laccata 211651, to three concentrations of leaf and litter leachates and two concentrations of humus leachate. The 0.1 g FW/ml leachate was prepared by adding 50 ml original leachate (0.2 g FW/ml) into 50 ml autoclaved MMN medium containing 1.5 g agar at 50–60-C. The 0.05 g FW/ml leachate was prepared by adding 25 ml original leachate and 25 ml sterilized water into 50 ml autoclaved MMN agar medium. The 0.01 g FW/ml leachate was prepared by adding 5 ml original leachate and 45 ml sterilized water into 50 ml autoclaved MMN medium. Concentrations of 0.25 and 0.05 g FW/ml of humus leachate were used. The original leachates of humus (25 ml, 1 g FW/ml) and 25 ml sterilized water were mixed with 50 ml autoclaved MMN medium containing 1.5 g agar at 50–60-C to obtain 0.25 FW/ml of humus leachate. The 0.05 g FW/ml humus leachate was prepared by adding 5 ml original leachate and 45 ml sterilized water into 50 ml autoclaved MMN agar medium. The control was made from half-strength MMN medium. The medium was poured into 9-cm-diam Petri dishes (20 ml medium in each dish). A disk of inoculum (diam 4 mm) from each of the five ECM fungi was cut from the edge of a 2- to 3-wk-old colony and inoculated in a Petri dish. After they were cultured for 28 d at 20–25-C, diameters of the fungal colonies were measured.

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Experiment 2: Effect of Pure Phenolics on ECM Fungi o-HPA, ferulic, and o-coumaric acids were the major phenolic acids in Kalmia leaf leachates (Zhu and Mallik, 1994) and humus (Mallik, unpublished data). Therefore, we measured the responses of five ECM fungal isolates, P. involutus (211804, 196554), L. laccata 211651, P. tinctorius 99132, and L. bicolor 229559 to these compounds. oHPA acid was directly dissolved in sterile water to obtain a 2-mM solution. Ferulic and o-coumaric acids were dissolved in sterile hot water (80-C). Phenolic solutions were sterilized by passing through two layers of 0.45-mm Millipore filters. Equivalent volumes of a 2-mM phenolic solution and a double concentration MMN agar were mixed to obtain a medium with 1 mM phenolic acid and full-strength MMN. The equimolar mixture of the three phenolic acids was obtained by mixing equivalent volumes of their respective 1 mM media for a final concentration of 0.33 mM for each acid. The control consisted of only MMN medium. Petri dishes containing the above-mentioned media were inoculated with the ectomycorrhizal fungi employed in Experiment 1, with four replicates for each treatment. The inoculated plates were incubated at 20–25-C and the diameters of the fungal colonies measured after 20 d. Experiment 3: Phenolic Acids as Carbon Source for ECM Fungi In this experiment, we investigated whether P. involutus 211804 and L. bicolor 229559 could grow with o-HPA acid as the only source of carbon. To prepare fungal inocula, P. involutus 211804 and L. bicolor were cultured on MMN agar for 16–20 d. Mycelial disks (diam 10 mm) were cut from the edges of fungal colonies and transferred to dishes containing water agar. Large-diameter inocula (10 mm as opposed to standard 4 mm) were used to provide nutrition for fungal growth. Water agar was used to exclude the possible nutrient effect of inoculated plugs on noncarbon nutrient experiment. These fungi were cultured in water agar for 22 d. After 22 d, a plug of inoculum (diam 4 mm) cut from the colony edge on water agar was inoculated on the noncarbon MMN medium (MMN agar medium without sucrose and malt extracts) without o-HPA acid (control) and noncarbon MMN medium with 1 mM o-HPA acid. There were four dishes for each fungus and treatment combination. The experiment was conducted at room temperature (20– 25-C). Diameters of the fungal colonies were measured after 25 d. Biomass Assay in Liquid Media Because mycelial biomass in the noncarbon medium was very small, half strength of MMN liquid medium was used to culture P. involutus 211804 and L. bicolor. Each Petri dish (9 cm) contained 12 ml liquid medium with or without 1 mM o-HPA acid, prepared as described above. There were 10 dishes for each treatment. After autoclaving, a U-shaped glass rod (diam 2 mm) wrapped with a piece of 4  4 cm unbleached paper towel was added to each dish and was inoculated with two plugs (diam 4 mm) of P. involutus 211804 inocula on the surface of the unbleached paper. The glass rod and unbleached paper towel facilitated the culturing of the ECM fungi in liquid media and subsequent collection of mycelia. Five ml of sterile water were added after 15 d. After 30 d culture, the mycelia were harvested from the unbleached paper. Ten dishes were divided into five groups, and mycelia from two dishes were combined, dried at 60-C for 24 hr, and weighed to T0.1 mg.

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Experiment 4: Phenolic Reduction by ECM Fungi in Pure Culture We tested whether P. involutus 211804 could metabolize phenolics. P. involutus 211804 was cultured on MMN agar medium. The Petri dish was inoculated at the center with one piece of 4 mm mycelial plug. Cultures were maintained at 20–23-C for 20 d. Then two U-shaped glass rods (diam 2 mm) were placed on the surface of the medium. Acetone solutions of the phenolic compounds (10 mM, 200 ml) were spotted on a piece of Whatman No. 3 filter paper (diam 12 mm). When the acetone had completely evaporated, 200 ml sterile water were added to the filter paper. Filter papers were placed over each rod. Each treatment consisted of three replicates. The filter papers were taken out 10 d later and they were soaked in 5 ml methanol for 24 hr. The methanol extracts were concentrated to 2.5 ml at 40-C under reduced pressure and passed through Whatman No. 1 filter paper. Phenolic compounds in the filtrates were analyzed by using a Varian Prostar HPLC equipped with a Chrompak column (250  4.6 mm) and PDA detector (Model 330) monitoring the absorbance of the elution at 280 nm. The solvent system was as follows: 0–9 min, 10% methanol and 90% water containing 2.5% formic acid; 9–20 min, 40% methanol and 60% water containing 2.5% formic acid; 20–25 min, 60% methanol and 40% water containing 2.5% formic acid; 25 min, 100% methanol. Flow rate was 1.5 ml/min, and temperature was 32-C. Pure compounds were used as standards, and phenolic acids were identified by comparison of retention times and UV spectrum. Experiment 5: Detoxification Bioassay We investigated whether inoculation of spruce seedlings with ECM fungus reduced the phytotoxicity of o-HPA acid. Because o-HPA acid was the most toxic phenolic acid in Kalmia leaf leachates affecting black spruce seedlings (Zhu and Mallik, 1994), it was used to conduct further detoxification studies. Since full-strength MMN medium itself inhibits seedling growth of black spruce, all media used were halfstrength MMN. P. involutus 211804 was cultured in MMN liquid medium (half strength) containing 0.2 and 0.5 mM o-HPA acid. Each Petri dish (10 cm) contained 15 ml liquid medium. P. involutus 211804 was inoculated and cultured as described in Experiment 4. Noninoculated culture was used as the control. Plates were incubated at room temperature (20–23-C). Five ml sterile water were added after 15 d. The culture solution was decanted from the dishes after 30 d and passed through Whatman No. 42 filter paper. Filtrates were adjusted with sterile water to initial volume and used for the phytotoxicity test with black spruce seedlings. Each dish contained 20 surface-sterilized black spruce seeds placed on a piece of Whatman 3 filter paper to which 5 ml culture solution were added. The half-strength MMN medium was used as negative control, and half-strength MMN medium with 0.2 and 0.5 mM o-HPA acid were used as positive controls. The bioassay was conducted at 20–25-C with a 16-hr photoperiod. Two ml sterile water were added to each Petri dish during incubation. The lengths of black spruce primary root and shoot were measured after 15 d. Experiment 6: Phenolic Degradation by Inoculated Black Spruce Black spruce seedlings were inoculated with P. involutus 211804 following the method described by Marx and Kenney (1982) with some modifications to

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determine whether the ECM inoculated seedlings are able to degrade o-coumaric, ferulic, and o-HPA acids and enhance the root–shoot growth of black spruce. ECM Inoculum Preparation Vermiculite (approximately 600 ml) and MMN liquid medium (300 ml) were added to 1-l glass jars. The jar lids had holes tightly plugged with cotton. After autoclaving for 30 min, each jar was inoculated with 10 mycelial disks. After 45 d incubation, the inoculum was wrapped with four layers of cheesecloth and irrigated with tap water for 5 min to remove any remaining nutrient. This material was used for black spruce seedling inoculation for the following experiment. Seedling Inoculation Autoclaved (121-C for 30 min) vermiculite and peat/moss mixture (1:1) was added to multipots (120 ml/cell) for seedling culture. Pregerminated black spruce seedlings (5 d) developed from surface-sterilized seeds were sown in multipots and kept in a growth incubator maintained at 26-C with a 16-hr photoperiod, 150 Md/m2/s PAR and 60% relative humidity. Seedlings were watered daily and fertilized once a week with 20–20–20 NPK. After 60 d incubation, the seedlings were removed to new, larger multipots (250 ml/cell) that were filled with the mycorrhizal fungal inocula and vermiculite/peat moss mixture at the ratio 1:15. Each cell contained one seedling. Inoculated seedlings were grown in the same incubator and under the same environmental conditions as in the other experiments. The roots of inoculated seedlings were surrounded by dense mycelia after 135 d inoculation. Noninoculated (control) seedlings were prepared and grown in the same manner without inoculation. No mycorrizal infection was found in control seedlings. At this point, 30 ml of 1 mM phenolic acid solution were added to each cell. There were six seedlings per treatment. Five d later, the growing media (peat/vermiculite mixture) of seedlings were extracted with 300 ml distilled water  3. The water extracts were pooled and partitioned against hexane  3 to remove lipids, then extracted with ethylacetate  3. Ethylacetate extracts were combined and concentrated to dry form at 40-C under reduced pressure, and then dissolved with 5 ml methanol/ethyl acetate (4:1). The phenolic acids were analyzed with HPLC. Statistical Analysis All data were normally distributed. One-way analysis of variance (ANOVA) was used, and treatment differences among means were tested at P = 0.01 with Duncan’s multiple range test.

Results Experiment 1: ECM Fungal Response to Kalmia Leachates Growth response of the ECM fungi to Kalmia leachates depended on the type of leachate (leaf, litter, or humus), its concentration, and the species of ECM fungus. Higher concentrations of leaf leachates and lower concentrations of litter leachates

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had stimulatory effects on P. involutus. The 0.1 and 0.05 g FW/ml leaf leachates significantly stimulated the growth of P. involutus by increasing the colony diameter of the fungus by 101.0 and 60.1%, respectively (Table 1). However, these two concentrations inhibited L. laccata, whose colony diameters were 12.6 and 11.4% smaller with 0.1 and 0.05 g FW/ml leaf leachates, respectively, relative to controls. The 0.1 g FW/ml litter leachate had no effect on colony diameter of P. involutus. However, the 0.05 and 0.01 g FW/ml litter leachates stimulated colony diameter by 72.6 and 49.0%, respectively. The 0.1 and 0.05 g FW/ml litter leachate had little effect on colony diameter of L. laccata, but with 0.01 g FW/ml litter leachate, there was a increase of 8% compared to control. The 0.25 and 0.05 g FW/ml humus leachate had no effects on colony diameter of either P. involutus or L. laccata (Table 1). Experiment 2: ECM Response to Pure Phenolic Acids The response of fungal isolates to pure phenolic acids also differed depending on the type of phenolic acid and its concentration. P. involutus 211804 was the most tolerant fungal isolate to the three phenolic acids and their equimolar mixture (Fig. 1). Indeed, the colony diameter of this isolate was increased by 25% in the presence of 1.0 mM o-HPA acid, and the mixture of the three phenolic acids did not inhibit growth at either concentration. However, P. involutus 211804 was inhibited by ferulic acid at 1 mM. Both the 1.0 and 0.1 mM phenolic mixtures inhibited colony diameter of P. involutus 199554 and L. laccata 211651. Colony diameter of L. bicolor 229559 was reduced by 41.7% on a 1 mM phenolic mixture, but it was increased (by 17.8%) in the presence of the 0.1 mM mixture. Colony diameter of both L. bicolor and L. laccata was increased by 1.0 mM o-HPA acid. Colony diameter of L. laccata was also increased by 0.1 mM o-HPA acid. P. tinctorius was the most sensitive of the fungal isolates to the phenolic mixture at 1.0 mM, and its colony diameter was inhibited by 80.5% on the 1.0 mM mixture. With respect to the effect of individual phenolic compounds on the mycorrhizal fungi, ferulic acid was the most toxic, strongly inhibiting growth of all tested ECM fungi at 1 mM. The decrease in colony diameter of P. involutus 211804, 199554, and

Table 1 Effect of Kalmia leaf, litter, and humus leachates on colony diameter of P. involutus 211804 and L. laccata 211651

Values are mean T standard error (N = 4), means followed by the same letter(s) in the same column are not significantly different (P < 0.01)

Leachates

Concentrations (g FW/ml)

Colony diameters [mm (mean T SE)] P. involutus 211804

Control Leaf Leaf Leaf Litter Litter Litter Humus Humus

0 0.1 0.05 0.01 0.1 0.05 0.01 0.25 0.05

39.8 80.0 63.7 33.3 36.7 68.7 59.3 33.3 36.0

T T T T T T T T T

2.8c 0.0a 4.0b 3.2c 0.6c 6.7b 11.4b 0.6c 1.7c

L. laccata 211651 50.0 43.7 44.3 49.0 48.3 47.3 54.0 48.0 51.3

T T T T T T T T T

1.0bc 1.2e 1.5de 1.7bc 0.6bc 1.5cd 1.0a 1.7c 0.6ab

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80 Control Phenolic mixture o-Hydroxyphenylacetic acid Ferulic acid Coumaric acid

a

Colony diameter (mm)

60

ab a ab

a a a

a

40

c

a a b

b ab

ab a

b

b

c

a a

d e

20

b

b

c

0

b

Colony diameter (mm)

60

a

Control Phenolic mixture o-Hydroxyphenylacetic acid Ferulic acid Courmaric acid

a

b

a

ab bc c

40

b

c

a a

b c

20

c c

c

c

b

d c

d d

c

d

e

0 P. involutus 211804

P. involutus 199554

L. bicolor 229559

L. laccata 211651

P. tinctorius 99132

ECM fungal isolate Fig. 1 Effect of three common Kalmia phenolic acids and their equimolar mixture on colony diameter of five ectomycorrhizal fungi at (a) 0.1 mM and (b) 1 mM. Values are mean T standard error (N = 4). Significant differences (P < 0.01) among treatments in each group are indicated by different letters

P. tinctorius was 95.0, 72.5, and 92.5%, respectively (Fig. 1). The compound also inhibited growth of P. involutus 199554 and P. tinctorius 99132 at 0.1 mM, but it stimulated the growth of L. laccata at this concentration. The o-HPA acid stimulated the colony diameter of L. laccata, L. bicolor, and the two isolates of P. involutus by 38.4, 29.3, 25.0, and 18.9%, respectively, at 1.0 mM; it also stimu-

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18

a

a

16

Control o-Hydroxyphenylacetic acid

Colony diameter (mm)

14 b

12 10

a

8 6 4 2 b

0

b

Mycelial dry biomass (mg)

20

a

a b

15

b

10

5

0 P. involutus

L. bicolor

ECM fungal isolate Fig. 2 Effects of 1 mM o-hydroxyphenylacetic acid on (a) colony diameter and (b) mycelial dry biomass of P. involutus 211804 and L. bicolor 229559 in carbon-free liquid MMN medium. Values are mean T standard error (N = 5). Significant difference (P < 0.01) among treatments in each group are indicated by different letters above bars

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lated the colony growth of P. involutus 199554 and L. laccata at 0.1 mM. It did not stimulate colony growth of P. tinctorius. Coumaric acid inhibited the colony growth of ECM fungi except P. involutus 211804 at 1 mM. At 0.1 mM, coumaric acid did not inhibit any fungus, and stimulated the growth of P. involutus 199554 (Fig. 1). Experiment 3: Phenolic Acids as Carbon Source for ECM Fungi When P. involutus 211804 was cultured on noncarbon MMN agar medium, its colony diameter increased by 41.7% in the presence of 1 mM o-HPA acid (Fig. 2a). The hyphal density of the fungus in the medium with o-HPA acid was much higher than that of the control. Control plates had little mycelia. L. bicolor did not grow at all on the noncarbon MMN medium, but did in the presence of 1 mM o-HPA acid. L. laccata did not grow either on control or o-HPA acid medium. The mycelial biomass of P. involutus 211804 and L. bicolor was increased by 20.1 and 33.9%, respectively when the two fungi were cultured separately in MMN liquid medium containing 1 mM o-HPA acid (Fig. 2b). Experiment 4: Phenolic Degradation by ECM Fungus in Pure Culture The concentrations of all three phenolic compounds, ferulic, coumaric, and o-HPA acids were reduced in the presence of P. involutus 211804 in pure culture. While the recovery of coumaric and o-HPA acids was reduced by 97.9 and 79.5%, respectively, we could not detect any ferulic acid following exposure to the fungal culture on the medium (Fig. 3).

Concentration (mM)

2.5 2.0

Control P. involutus

a

a

1.5 a

1.0 .5 b

b

b

0.0 o-Coumaric acid

o-Hydroxyphenylacetic acids

Ferulic acid

Phenolic acids Fig. 3 Concentrations of o-coumaric, o-hydroxyphenylacetic, and ferulic acids in filter paper extract without (control) or with P. involutus 211804 culture. Values are mean T standard error (N = 4). Significant differences (P < 0.01) among treatments in each group are indicated by different letters above bars

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12

a

Control P. involutus

10

a a

Root length (mm)

a

8

6 b

4 c

2

0

b 20

a

a a

a

Shoot length (mg)

a

15

10

5

0 0

0.2 Concentration (mM)

0.5

Fig. 4 Phytotoxic effects of o-hydroxyphenylacetic acid at concentrations of 0.2 and 0.5 mM with and without P. involutus culture on (a) root length and (b) shoot length of black spruce. Values are mean T standard error (N = 15). Significant differences (P < 0.01) among treatments are indicated by different letters above bars

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Concentration (mg/L)

Uninoculated Inoculated

a

100 80 60 40 20 0

a a b o-Coumaric acid

b

b o-Hydroxyphenylacetic acids

Ferulic acid

Phenolic acids Fig. 5 Concentrations of o-coumaric, o-hydroxyphenylacetic, and ferulic acids in growing media containing uninoculated (control) and inoculated black spruce seedling with P. involutus 211804. Values are mean T standard error (N = 3). Significant differences (P < 0.01) among treatments in each group are indicated by different letters above bars

Experiment 5: Detoxification of o-HPA acid by ECM Fungi Root growth of black spruce seedlings was inhibited by 82.0 and 63.9%, respectively, in the presence of o-HPA acid at 0.5 and 0.2 mM (Fig. 4a). However, no significant effect of o-HPA acid on root and shoot length was found when seedlings were grown with P. involutus 211804 culture broths (Fig. 4a,b). Experiment 6: Phenolic Degradation by Inoculated Black Spruce The concentrations of phenolic acids in peat/vermiculite mixture with mycorrhiza inoculated black spruce were much lower than those containing noninoculated black spruce (Fig. 5). Five d after adding o-HPA acid to the growing medium containing the ECM inoculated black spruce, less than 3% of the compound was recovered. Similarly, the recovery of o-coumaric acid and ferulic acid was only 13. 3 and 10.4%, respectively, in the peat–vermiculite mixture that had P. involutus 211804inoculated black spruce.

Discussion Our results show that the different ECM fungal isolates had differential growth response to Kalmia leachates and phenolic compounds. L. laccata was less resistant than P. involutus. The growth of P. involutus 211804 was significantly stimulated by leaf and litter leachates even in the presence of full-strength leaf leachate (0.1 g FW/ ml). This growth enhancement may be caused either by additional nutrients in the leaf and litter leachate and/or the degradation products of Kalmia phenolics in the

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leachate that the ECM fungi can use as a carbon source. Earlier, Mallik and Zhu (1995) reported that growth of one isolate of P. involutus (NF4) was stimulated in the presence of Kalmia leaf leachate. Cote´ and Thibault (1988) found an isolate of P. involutus (0077) to be more sensitive to leaf leachate toxicity of raspberry (Rubus idaeus) compared to other ECM fungi. In this study, water leachate of Kalmia humus showed no significant effect on colony diameter of P. involutus and L. laccata. Phenolic profiles of leaf and litter leachates were different from that of humus. It is possible that by the time leaf and litter were decomposed into humus, many of the influential alleochemicals were degraded by microorganisms. It is also possible that degradation and use of humus phenolics is more difficult for ECM fungi than those of leaf and litter. Because different isolates of the same species of ECM fungus responded differently to a particular phenolic acid, our results suggest that coadaptation may influence ECM fungal tolerances to phenolics. P. tinctorius was the most sensitive fungus to the phenolic mixture; its colony diameter was inhibited by 80.5% in the presence of 1.0 mM mixture. This isolate was collected from the subtropical region of Guangdong, South China, whereas the other four fungal isolates were collected from boreal forests of Canada. In South China, we know of no report of high concentrations of phenolic acids in soils, presumably because of high precipitation, high litter quality, rapid decomposition, and little organic matter accumulation. Therefore, this isolate may not have mechanisms to deal with high concentrations of phenolic compounds, whereas the fungi collected from Canada may possess this adaptation (Callaway and Ridenour, 2004; Vivanco et al., 2004). In the noncarbon growing medium, both P. involutus and L. bicolor grew significantly better with respect to colony diameter and mycelial biomass in the presence of o-HPA acid than those without the phenolic acid (Fig. 2). Since o-HPA acid was the only carbon source in the medium, we conclude that the fungus must have used o-HPA acid as a carbon source to increase its biomass. Our phenol reduction experiment (Experiment 4) conducted with three pure phenolic acids confirmed the idea that P. involutus is able to reduce or oxidize phenolic acids quite easily within a short time (Fig. 3). However, high concentration (1 mM in the media) of ferulic acid inhibits the growth of P. involutus (Fig. 1b). After P. involutus 211804 was cultured in 0.5 and 0.2 mM o-HPA, its phytotoxicity disappeared (Fig. 4). o-HPA acid, at 0.2 mM, was toxic to black spruce seedlings in the absence of P. involutus, but the 0.5 mM o-HPA acid solution became nontoxic in the presence of the fungal culture. From this result, we conclude that the concentration of 0.5 mM o-HPA acid was reduced to less than 0.2 mM, as a result of fungal culture. In other words, P. involutus detoxified the 0.5 mM o-HPA acid to a level lower than 0.2 mM, and allowed the seedlings to grow. Zhu and Mallik (1994) have shown that black spruce seedlings can grow in the presence of ferulic and coumaric acids but cannot tolerate high concentrations of o-HPA acid. From this study, we suggest that the symbiotic ECM fungi of black spruce can not only tolerate high concentrations of o-HPA, but can also degrade the compound and use it as a carbon source. Most of the added phenolic acids in the peat/vermiculite mixture containing P. involutus inoculated black spruce disappeared within 5 d, whereas substantial amounts of the added acids were recovered from the growing medium of the noninoculated (control) seedlings. We suggest that the P. involutus-inoculated

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seedlings are able to degrade Kalmia phenolics in the potting medium. However, the incomplete recovery of o-coumaric and ferulic acid in the uninoculated (control) medium suggests that preinoculation of black spruce seedlings with suitable ectomycorrhizal fungi may promote conifer regeneration in Kalmia dominated sites. The problem of Kalmia-induced growth inhibition in black spruce is complex and the relationship between Kalmia phenolics and mycorrhizae is still unclear (Wallstedt et al., 2002). From a field study that showed poor growth, foliar N and P concentrations and mycorrhization of black spruce seedlings growing closer to Kalmia than those growing away, Yamasaki et al. (1998) concluded that Kalmia limits the growth of black spruce in ways other than direct nutrient competition. They suggested that since mycorrhizae and plant nutrition are closely related, fewer mycorrizal short roots in black spruce growing near Kalmia means less nutrient acquisition due to poor mycorrhizal symbiosis, thus resulting into poorer growth in these seedlings. Another cause of poor growth was attributed to the susceptibility of black spruce near Kalmia to a potential pathogenic fungus, Phialocephala dimorphospora. Yamasaki et al. (1998) suspect that higher amount of mycorrhizal short roots in seedlings growing away from Kalmia may have protected them from the pathogenic fungus and consequently allowed better growth. One could also suspect long-term dominance of Kalmia in the absence of an appropriate host (in this case, black spruce) that the beneficial ECM would be reduced in soil inoculum. Our previous laboratory studies have shown that out of 51 fungal isolates tested, the growth of 41 isolates was reduced in the presence of Kalmia leaf leachate in liquid MMN agar. Yamasaki et al. (1998) suspect that Kalmia compounds could affect the survival of fungal inoculum in soil or the fungal symbionts in black spruce roots. Robinson (1972) proposed a similar hypothesis in explaining the growth inhibition of Sitka spruce [Picea sitchensis (Bong.) Carriere] seedlings in the presence of another heath-forming ericaceous shrub, C. vulgaris. Response of ECM to ericaceous compounds can be negative, positive, or neutral depending on the species and strains of the ECM (Mallik and Zhu, 1995). In this study, we found that certain black spruce ECM (particularly P. involutus) can respond positively to Kalmia phenolics with increased biomass. This, in turn, enhances seedling growth under laboratory and greenhouse conditions. Given the fact that biophysical conditions in the field can be very different from those in the controlled laboratory and greenhouse, the logical next step of this research is to test if black spruce seedlings preinoculated with P. involutus out planted in Kalmia dominated sites can overcome the growth inhibition by maintaining the symbiotic relationship. We can draw several conclusions from this study. First, our results prove that certain ECM fungi can not only offer protection to host plants against phenolic allelochemicals released from neighboring plants, but they can also use them as a carbon source. This, in turn, can benefit the host plant. Our findings have important ecological significance in the sense that they point to a mechanism by which ectomycorrhizal fungal species can control species interactions in higher plants by changing the rhizosphere chemistry. Second, the ability of ECM fungi to degrade and detoxify phenolic allelochemicals is not only species-specific, but also specific to different strains of the same fungal species. Third, tree seedlings preinoculated with beneficial ECM fungi can degrade plant phenolics in peat/vermiculite mixture. This offers some hope toward overcoming phenolic-induced conifer growth inhibition in

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ericaceous-dominated sites by outplanting tree seedlings preinoculated with carefully selected beneficial ECM fungi. Acknowledgments We acknowledge the financial support from the Natural Science and Engineering Research Council (NSERC) discovery grant (to AUM) and the National Natural Science Foundation of China (30270230, 30370246), Guangdong Natural Science Foundation of China (039254, 04105977), the National 973 project of China (2006CB100200), Program for New Century Excellent Talents in University (to RSZ). We thank Dr. Christine Gottardo, Debbie Leach, and Andrea Aguirre of the Chemistry Department, Lakehead University, and Haihong Bi of the Department of Ecology, South China Agricultural University for their help in HPLC analyses, and Dr. Ed Setliff of the Faculty of Forestry and the Forest Environment, Lakehead University for his cooperation during the inoculation experiments. Comments of two anonymous reviewers, the journal editor, Drs. Robert D. Guy and Jenelle Curtis (Faculty of Forestry, University of British Columbia), Leonard Hutchison, and Brian McLaren (Faculty of Forestry and the Forest Environment, Lakehead University) were helpful in revising the manuscript.

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