ZINC TOLERANCE IN BETULA SPP. III. VARIATION IN RESPONSE TO ZINC AMONG ECTOMYCORRHIZAL ASSOCIATES BY HILARY J. DENNY AND D. A.

New Phytol. (1987) 106, 535-544 535 ZINC TOLERANCE IN BETULA SPP. III. VARIATION IN RESPONSE TO ZINC AMONG ECTOMYCORRHIZAL ASSOCIATES BY HILARY J. DENNY AND D. A. WILKINS Department of Plant Biology, University of Birmingham, Birmingham B15 2TT, UK {Accepted 4 March 1987) SUMMARY A survey was made of fungi forming ectomycorrhizas with Betula pendula Roth, and B. pubesce Ehrh. on zinc-contaminated mine tailings. Strai of Paxillus involutus Fr. from toxic and non-toxic soils were compared. There was no indication of fungal adaptation to zinc at either the inter- or intraspecific level. Most of the typical fungal associates of Betula on normal soils were also found on the zinc-rich soils. The ability of different strai of P. involutus to (a) grow in pure culture on agar, (b) form ectomycorrhizas with Betula, and (c) produce a beneficial growth and zinc uptake respoe in the associated plant, all in the presence of raised zinc concentratio, was not related to the zinc status of the provenance. The ameliorating influence of P. involutus on zinc toxicity to Betula seemed to be positively linked to the degree of compatibility between fungal strain and higher plant. Key words: Zinc tolerance, Betula, Paxillus involutus, mycorrhiza, compatibility. INTRODUCTION Brown & Wilki (1985a) showed that zinc toxicity to Betula pendula Roth, and B. pubesce Ehrh. was ameliorated by the presence of the ectomycorrhizal fungi Paxillus involutus Er. and Amanita muscaria Hooker; growth of Betula was promoted and the concentration of zinc in the shoots of the vascular plant was reduced. However, the suitability of these fungal species as experimental subjects in these studies was not investigated. Much evidence for succession of mycorrhizal species has recently been published (Mason et al., 1982; Deacon, Donaldson & Last, 1983; Eleming, 1983; Last et al., 1983). A. muscaria and P. involutus are characteristically associated with mature Betula, but the most crucial phase of development must be during the seedling stage. Eurther, it is not known which fungal genera and species are characteristic associates of Betula on soils contaminated with heavy metals. They may be different from those on normal soils. Therefore, a preliminary survey of the fungi found growing with Betula on toxic soils was carried out so that a suitable fungal species could be selected for inclusion in experiments to study the mechanism of ectomycorrhizal amelioration of zinc toxicity. At the intraspecific level, several authors have reported the existence of strai of both ectomycorrhizal, (Harris & Jurgeen, 1977) and vesiculararbuscular (VA) mycorrhizal fungi, (Lambert, Cole & Baker, 1980), which were specifically adapted to local edaphic conditio. Adaptation can be expressed in the present context as a differential ability of ectomycorrhizal associates of Betula to grow on agar containing raised zinc concentratio (Brown & Wilki, 1985b), 0028-646X/87/070535 + 10 $03.00/0 1987 The New Phytologist

536 H. J. D E N N Y AND D. A. W I L K I N S or of VA mycorrhizal strai to infect potential hosts in the presence of zinc (Gildon & Tinker, 1981, 1983). Krishna et al. (1985) have identified variation in respoe to VA mycorrhizal inoculation of pearl millet, although in this case the fungal strai were adapted to individual millet genotypes, rather than to the presence of a toxic metal. M A T E R I A L S AND M E T H O D S Culture of plants and fungi The aseptic culture of Betula clones was as described in Denny & Wilki (1987a). Rooted cuttings of Betula were grown in glass boiling tubes containing 15 g acid washed perlite, 10 cm^ liquid growth medium and fitted with a tralucent plastic, vented cap. The growth medium used for the culture of ectomycorrhizas was similar to that used by Brown & Wilki (1985a), but the concentration of glucose was reduced from 10 to 5 g dm~3. This was because high concentratio of soluble carbohydrate can induce abnormalities in behaviour, in both fungus and higher plant (Duddridge & Read, ). Perlite was used as the rooting substrate because it encourages better growth and mycorrhiza formation than agar, and it is cleaner and better defined than vermiculite peat. Ectomycorrhizas were synthesized as follows; viable fungal inocula, cut from the edge of colonies growing on agar, were buried just under the surface of the perlite. Rooted cuttings oi Betula were placed on top. The tubes were incubated at 20 C, under continuous illumination from four fluorescent tubes at an irradiance of 7 8 W cm"^. Mycorrhizas formed after 3 weeks. Fungal isolates were cultured in the same growth medium as the mycorrhizas, but the medium was gelled by 1 % agar in Petri dishes. The plates were incubated at 18 C in the dark. Experimental treatments comprised growth medium to which zinc sulphate had been added. The control concentration of zinc was 0*74/^mol dm~3 in all cases. Plant material was digested in concentrated nitric acid and analyzed for zinc by atomic absorption spectroscopy. Selection of fungus for intraspecific studies Three field trips to Snailbeach and Gravels, near Miterley in Shropshire, and Cwm Rheidol, near Aberystwyth, were undertaken in the autumn of. Collectio were made of fungal fruit bodies growing on lead- and zinccontaminated soils, adjacent to Betula trees, together with Betula roots growing under them. In one or two cases mycelial strands could be seen linking Leccinum fruit bodies to mycorrhizas on the Betula roots below. Fruit bodies and mycorrhizas were identified as far as possible. Some ectomycorrhizal associates could be identified only from mycorrhizal morphology (V. Fleming, pers. comm.), e.g. Cenococcum spp. and 'brown with creamy tips'. Experimental design Experiment 1. Viable inocula, cut from colonies of the 10 P. involutus strai, were each plated out onto five Petri dishes containing growth medium gelled with agar at each of five zinc concentratio, 0, 0-00075, 1-75, 3*5 and 7-0 mmol dm"^. One cube of inoculum, 5 mm square, was placed 1 cm from the edge of each plate. The plates were incubated for 5 weeks, with regular checks to eure that the

Zinc tolerance in Betula spp. Ill 537 Table 1. List of fungi thought to form ectomycorrhizas with Betula spp. on non-toxic soils (Watling, ) and their occurrence with Betula on soils contaminated with zinc and lead Genus Species Presence Evidence Hebeloma Paxillus Amanita Leccinum Boletus Lactarius Russula Inocybe Cenococcum ' Brown with creamy tips' Laccaria Thelephora Cantharellus Cortinarius Scleroderma mesophaeum involutus muscaria scabrum roseofractum melanea piperatus badius lanatus subtomentosus pubesce pulchella cibarius crocolitus, fruit body; M, mycorrhizal morphology. + + + + M + M + M M + M + M M M Table 2. Collection sites o/paxillus involutus. {Zinc contamination estimated from analysis of soil samples taken from below the fruit bodies) Fungal strain Provenance Zinc contamination Associated tree type Date F8 F9b F23 F28 F29 Pi45 Pi47 P3 F36 Pi32 Gravels, Shropshire Gravels, Shropshire Cwm Rheidol, Dyfed Cwm Rheidol, Dyfed Cwm Rheidol, Dyfed Coal Spoil, ITE, Bush, Scotland Coal Spoil, ITE, Bush, Scotland Swithland Wood, Leicestershire Cwm Rheidol by river, Dyfed Peat bog. Scotland Severe Severe Low Low Low- Nil Nil Nil Nil Nil Sitka spruce 1983 1983 1981

538 H. J. D E N N Y AND D. A. W I L K I N S colonies had not exhausted the agar. The maximum distance grown by each colony was measured. Experiment 2. One hundred growth tubes were prepared, half at 0-75/^mol dm~^ zinc and half at 3-1 mmol dm"^ zinc. There were five Betula genotypes, B109.6, Bl 10.2, B9.6, B64.2 and B64.13. B109.6 is non-tolerant of zinc; the other four are all of intermediate tolerance. Each of the 10 strai of P. involutus were paired with each of the five Betula genotypes, at both zinc concentratio. The tubes were incubated for 8 weeks before harvesting. Root tip numbers were counted for each plant. The degree of mycorrhizal infection was determined by calculating the percentage of root tips that were mycorrhizal. Experiment 3. One hundred and eighty growth tubes were prepared, each containing 7 cm^ growth medium at the control zinc concentration. All of the strai of P. involutus except F29 were included. Ten Betula genotypes of intermediate zinc tolerance were employed; B64.9, Bl 12.8, B64.4, Bl 13.2, B9.2, Bl 23.8, B64.il, B93.2, B47.3 and B1O9.5. The fungal strai and Betula genotypes were paired in all possible combinatio, with two replicates of each strain-genotype combination. The plants were incubated for 3 weeks to allow mycorrhizas to form. Sterile growth medium (2 cm^) was added to every tube to bring one of each pair of tubes up to 4 mmol dm~^ zinc, and to leave the other at 0 75 /im dm"^ zinc. The plants were incubated for a further 5 weeks. Then they were harvested, measured, dried, weighed and analyzed for zinc. RESULTS A list of fungi thought to form ectomycorrhizas with Betula on non-toxic soils, (Watling, ) and their occurrence on these toxic soils is given in Table 1. The vast majority of the ' normal' fungal associates were also found on the metalliferous soils. Therefore, it would appear that there is little adaptation to toxic metals at the interspecific level. It was noticeable that, while many of the Betula found on the tips were very small, i.e. less than 1 mm in height, by far the most prevalent fungi were typical late-stage associates such as P. involutus, Leccinum spp. and Amanita muscaria. It was decided to use strai of P. involutus in experiments to examine intraspecific variation with respect to zinc tolerance. As well as being abundant on the tips and associated with all sizes of Betula, it grows and forms mycorrhizas rapidly under aseptic culture conditio. Ten isolates of P. involutus were established from fruit bodies collected from sites ranging from toxic to non-toxic (Table 2). Experiment 1 The radial growth rates of the individual strai of P. involutus showed very highly significant differences at all five concentratio of zinc, but increasing the concentration lowered the growth rate in every case, except for F23 and F36, which showed some slight stimulation at 1-75 and 35 mmol dm~^, respectively [Fig. l(a)]. The data for radial growth were converted to tolerance ratios (Wilki, 1957) by dividing the strain mean at each concentration by the appropriate control mean value [Fig. l(b)]. Analysis of variance revealed that while individual strai differed in their tolerance of zinc, these differences were not linked to the zinc content of

Zinc tolerance in Betula spp. Ill 539 50 (a) 1-2 1-0 0 0-8 "5 k_ S 0-6 1 0-4 0-2 0 ^^ ^-.-.. ^~~^ - ^ " " '' V N (b) 1 1 1 t - - - - I 7 ^ ^ ~ ~^ Concentration of zinc in medium (mmol dm~^) Fig. 1. Growth of colonies of 10 strai of Paxillus involutus cultured on agar, at a range of zinc concentratio, for 5 weeks. Strai from zinc-contaminated soil: F23; F8; F29; F9b; F28. Strai from non-toxic soils: Pi32; Pi45; Pi47; S3; F36. (a) Mean maximum colony radius; (b) growth as a tolerance ratio. Table 3. Analysis of variance: growth of 10 strai of Paxillus involutus, five originating from zinc-contaminated soils and five from non-toxic soils, cultured on agar at a range of zinc concentratio for 5 weeks and expressed as tolerance ratios Due to df Sum of squares Mean squares F Significance Provenance + zinc Strain within provenance Zinc Error Total 1 8 3 27 39 0-0185 0-9824 1-6716 0-5427 3-2152 0-0185 0-1228 0-5572 0-0201 0-151 6-109 27-72 Probabilities: *P <0-05; **P ^ 001; P ^ OOOl;, P > 005. the soil from which they originated (Table 3). It 'was also clear that zinc tolerance was not linked to growth rates in the absence of zinc, i.e. of controls. Experiment 2 The total number of root tips, and percentage of those infected were calculated for each fungal strain, at zinc concentratio of 0*75 fivciol dnt^ and

54O H. J. DENNY AND D. A. WILKINS 200 5 100 a o N O o >. (/> Q. O o cr 100 80 60 40 20 0 LSD I II I. i III^ II F8 F9b F23 F28 F29 Pi45 Pi32 S3 Pi47 F36 Fungal strain Fig. 2. Comparison of the ability of 10 strai of Paxillus involutus, five from zinc-contaminated soils (F8, F23, F28, F29, F9b) and five from non-toxic soils (Pi32, Pi45, Pi47, S3, F36) to form mycorrhizas with Betula spp. in the presence of raised zinc concentratio, (a) Total number of root tips; (b) percentage root tips mycorrhizal. ^ Concentration of zinc in growth medium high; ^ low zinc concentration. (b) Table 4. Summary of analysis of variance : effect of fungal strain, Betula genotype and zinc concentration on the numbers of root tips and percentage of them with mycorrhizal infection Fungal strain Betula genotype Character Zinc -t-zinc -Zinc -I-Zinc Zinc No. mycorrhizal root tips No. non-mycorrhizal root tips Total no. root tips Root tips mycorrhizal (%) # **# #** # * #*# * * * ##* *#* *#* ##* Probabilities as in Table 3. 31 mmol dm ^ [Fig. 2(a), (b)]. When data were subject to analysis of variance (Table 4), it was found that while a high concentration of zinc reduced the number of root tips, it did not significantly alter the proportion infected. The total number of root tips was dependent upon the genotype of Betula, but not the fungal strain, while the degree of mycorrhizal infection was strongly influenced by the fungal

Zinc tolerance in Betula spp. Ill 541 Table 5. Analysis of variance: effect of fungal, strain, Betula genotype and zinc concentration on the extent of mycorrhizal infection Mean squares F Significance 1 3069 0339 72361 8 9045 286 337 1 1 3 557 31469 111079 8 80 99 286 337 445 393 Due to Provenance (P) Strai (S) in provenance Zinc (Z) PxZ SxZ Error Total Sum of squares df 3069 203 0-728 0-858 1132 #*# Probabilities as in Table 3. Strain, but hardly at all by the Betula genotype. Thus, it appears that while zinc may reduce the total number of root tips produced by the Betula, the different strai of P. involutus colonize a characteristic proportion of the root tips available, which is independent of the external concentration of zinc. When the percentage infection data were analyzed further (Table 5), it was clear that, while individual fungal strai differed in their ability to colonize the roots of Betula, this ability was not linked to the zinc status of their provenance. Experiment 3 Mean growth, in terms of increases in stem length, root length, shoot dry weight and root dry weight, was calculated for each strain of P. involutus at each zinc concentration [Fig. 3(a) to (d)]. The mean concentratio of zinc in the roots and shoots of Betula associated with each fungal strain were also computed [Fig. 4(a) to (b)]. Analysis of variance (Table 6) showed that, as before, zinc reduced all aspects of plant growth, but especially exteion growth. Growth was influenced by both the Betula genotype and the fungal strain. The concentration of zinc in the shoots was influenced by the genotypes but not the strai, while the concentration of zinc in roots was affected by the strai but not the genotypes. There were a number of significant interactio, mainly affecting stem length and root dry weight. Stem length was the character most seitive to zinc toxicity. Therefore, this is most likely to indicate any differential effects in the ability of fungal strai and genotypes to overcome the toxicity. Root dry weight is the character least seitive to zinc, but this is likely to be most seitive to the presence of a mycorrhizal symbiont. The zinc x genotype interaction with respect to stem length is probably indicative of differential zinc tolerance; that with respect to root dry weight may be indicative of a zinc x genotype x strain interaction, which cannot be unequivocally identified in this experiment. The strain x zinc interactio probably imply that strai differ in their ability to ameliorate zinc toxicity, and the strain x genotype interactio indicate differences in compatibility between the Betula genotypes and the fungal strai. An examination of Figures 3 and 4 shows that there is no clear link between the effects of the individual fungal strai on Betula growth or zinc uptake and the provenance of the strai. One strain, Pi32, did stand out as being atypical. This is the only strain which was isolated from a fruit body found growing with a tree

542 H. J. DENNY AND D. A. WILKINS 120 100 LSD c cu 100-80 - 60 40 20 LSD I 80 60 40 20 0 0 5 Q Fungal strain F9b F23 F29 Pi43 Pi32 S3 Pi47 F36 Fig. 3. Mean growth of 8-week-old ectomycorrhizal Betula plants associated with each of nine strai of Paxillus involutus after 5 weeks k at 0-75/^mol 0 7 5 l dm d-»» or 4-0 40 mmol l dm d-" ^ zinc, (a) Stem length; (b) root length; (c) shoot dry weight; (d) root dry weight. H High concentration of zinc; H low concentration of zinc. I 10 - "5 200 c o c a> oc o 150 100 LSD 50 0 F8 F9b F23 F29 Pi45 Pi32 S3 Pi47 F36 Fungal strain Fig. 4. Mean concentratio of zinc in (a) shoots, (b) roots of 8-week-old ectomycorrhizal Betula plants associated with nine strai of Paxillus involutus after growth at 40 mmol dm^^ zinc for 5 weeks.

Zinc tolerance in Betula spp. Ill 543 Table 6. Summary of the significance of the effects of zinc concentration, fungal strain and Betula genotype on growth and zinc uptake by Betula Main effects Interactio Character Zinc (Z) Strai (S) Genotype (G) ZxS ZxG SxG Stem length Root length Shoot d, wt Root d, wt Shoot zinc Root zinc **# #** *## *#* *# #*# * ### #** * * ** Probabilities as in Table 3. Other than Betula. This strain appears to act more like a parasite. The root dry weight values are as high as the others, which probably indicates that it was colonizing the roots as well as the others, but it produced very small increments of stem length and shoot dry weight and was also associated with unusually high concentratio of zinc in shoots. DISCUSSION The environments used in these experiments were, of necessity, artificial. Therefore, aspects of the observed respoes of the fungal strai or Betula genotypes to zinc may not be representative of the situation in the natural environment. The link between radial growth of a fungal colony on agar and growth of its mycelium in soil is particularly tenuous. Nevertheless, even though the conclusio drawn from these experiments should extrapolated to the natural situation with caution, some interesting observatio can be made. There was little indication of differential fungal adaptation to soil zinc, at either the inter- or intraspecific level. Most of the species and genera thought to be typical associates of Betula on non-toxic soils were also found growing with Betula on soils contaminated with zinc. Strai of P. involutus differed significantly in both their ability to grow on agar containing potentially toxic concentratio of zinc, and to colonize Betula roots in the presence of zinc. There were also significant differences between strai in terms of the growth and zinc uptake respoe produced by the associated Betula plant. However, none of these differences appear to be linked to the zinc content of the soil from which the strai originated. Compatibility between the fungal strain and the associated Betula would seem to be more important to the success of the relatiohip in the presence of raised zinc concentratio than fungal adaptation to the metal. Stem length was the character most indicative of tolerance or toxicity of zinc. Fungal strai differed in the stem lengths achieved by their associated Betula trees at control and high zinc concentratio. There was a significant correlation between stem length and the degree of mycorrhizal colonization achieved by the different fungal strai (r = 0-794; P < 0-01). However, this is not the whole story. The correlation coefficient between percentage infection and mycelial growth rate was not significant (r = 0-327; P >0-05). It would appear that the abihty to infect the roots of Betula is not dependent simply on the rate at which the fungus can grow along

544 H. J. D E N N Y AND D. A. WILKINS and over the roots, but rather that the success of the relatiohip depends on a more subtle, biochemical compatibility mechanism. Strain Pi32 of P. involutus colonized Betula roots very successfully, but it did not produce a correspondingly beneficial effect on Betula respoe to raised concentratio of zinc. This particular strain was isolated from a fruit body growing near a tree of Picea sitcheis in a forestry plantation. Apparently, this strain lacked the ability to form a mutualistic symbiosis with Betula, even though it formed what appeared to be normal sheathing mycorrhizas with the Betula roots. Strain F36, on the other hand, infected a relatively small proportion of Betula roots, but elicited a better than average growth respoe from the associated Betula. To summarize, the results of this investigation suggest that the ameliorating infiuence of ectomycorrhizal fungi on zinc toxicity to Betula depends more on the adaptation of the fungal strain to the higher plant associate than it does on adaptation of the fungus to zinc. The evidence points to a biochemical compatibility mechanism, rather than a simple capacity of the fungus to colonize the root surface rapidly. The indicatio are that compatibility is probably operating at the level of the species of the higher plant and may even be affected by the genotype of the higher plant. ACKNOWLEDGEMENTS We thank Professor F. T. Last and Dr Julia Wilson of ITE Bush for advice and assistance, and the Science and Engineering Research Council (UK) for financial support. REFERENCES BROWN, M. T. & WILKINS, D. A. (1985a). Zinc tolerance of mycorrhizal Betula. New Phytologist, 99, 101-106. & WILKINS, D. A. (1985b). Zinc tolerance of Amanita and Paxillus. Traactio of the British Mycological Society, 84, 367-369. DEACON, J. W., DONALDSON, S. J. & LAST, F. T. (1983). Sequences and interactio of mycorrhizal fungi on birch. Plant and Soil, 71, 257-262. DENNY, H. J. & WILKINS, D. A. (1987a). Zinc tolerance in Betula spp. I. Effect of external concentration of zinc on growth and uptake. New Phytologist, 106, 517-524. DUDDRIDGE, J. A. & READ, D. A. (). Modification of the host-fungus interface. New Phytologist, 96, 583-588. ELEMING, L. V. (1983). Succession of mycorrhizal fungi on birch: infection of seedlings planted around mature trees. Plant and Soil, 71, 263-267. GiLDON, A. & TINKER, P. B. (1981). A heavy metal tolerant strain of a mycorrhizal fungus. Traactio of the British Mycological Society, 77, 648-649. GiLDON, A. & TINKER, P. B. (1983). Interactio of vesicular-arbuscular mycorrhizal infection and heavy metals in plants. I. The eftects of heavy metals on the development of VA mycorrhizas. New Phytologist, 95, 247-263. HARRIS, M. M. & JURGENSEN, M. E. (1977). Development of S'a/t'x and PO/)U/M5 mycorrhizae in metalhc mme tailings. Plant and Soil, 47, 509-517. KRISHNA, K. R., SHETTY, K. G., DART, P. J. & ANDREWS, D. J. (1985). Genotype dependent variation in mycorrhizal colonisation and respoe to inoculation of pearl millet. Plant and Soil, 86, 113-125. LAMBERT, D. H., COLE, H. & BAKER, D. E. (1980). Adaptation of vesicular-arbuscular mycorrhizae to edaphic factors. New Phytologist, 85, 513-520. LAST, E. T., MASON, P. A., WILSON, J. & DEACON, J. W. (1983). Eine roots and sheathing mycorrhizas: their formation, function and dynamics. Plant and Soil, 71, 9-21. MASON, P. A., LAST, E. T., PELHAM, M. J. & INGLEBY, K. (1982). Ecology of some fungi associated with an ageing stand of birches {Betula pendula and Betula pubesee). Forest Ecology and Management, 4, 19-39. WATLING, R. (). Macrofungi of birchwoods. Proceedings of the Royal Society of Edinburgh, 85B, 129-140. WILKINS, D. A. (1957). A technique for the measurement of lead tolerance in plants. Nature, 180, 37-38. BROWN, M. T.