A comparison of arbuscular and ectomycorrhizal ... - Wiley Online Library

New Phytol. (1998), 140, 125–134

A comparison of arbuscular and ectomycorrhizal Eucalyptus coccifera : growth response, phosphorus uptake efficiency and external hyphal production B MELANIE D. JONES"*, D. M. DURALL"†  P. B. TINKER#‡ " Plant Mycorrhizal Unit, Natural Environment Research Council, Department of Plant Sciences, Parks Road, Oxford OX1 3PF, UK # Terrestrial and Freshwater Science Directorate, Natural Environment Research Council, Polaris House, North Star Avenue, Swindon SN2 1EU, UK (Received 21 January 1998 ; accepted 27 May 1998)  Eucalyptus coccifera Hook., a plant capable of forming both arbuscular mycorrhizas and ectomycorrhizas, was used to compare the effects of the two mycorrhizal types on phosphorus uptake and C allocation. Seedlings were grown in a P-deficient soil\sand mixture inoculated with peat\vermiculite spawn of Laccaria bicolor (Maire) Orton or Thelephora terrestris (Ehrh.) Fr. ; or with 250-µm sievings from leek colonized by Glomus caledonium (Nicol. & Gerd.) Trappe & Gerde., Glomus sp. type E3 or Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or with autoclaved spawn (non-mycorrhizal control). Before the 89-d harvest, a subset of the harvested plants was labelled with "%C (45–60-min pulse, 202-h chase). Growth promotion and the increase in seedling P content was largest in the two ectomycorrhizal treatments. Production of fluorescein diacetate-stained external hyphae was three to seven times higher by ectomycorrhizal (ECM) fungi compared with arbuscular mycorrhizal (AM) fungi and was highly correlated with P uptake and shoot weight. Phosphorus inflow rates of ECM and AM seedlings were 3n8 times, and 2n0–2n7 times those of non-mycorrhizal seedlings. Phosphorus acquisition efficiencies were similar (11n2 and 10n0 µmol P mmol−" C for T. terrestris and Glomus E3 plants, respectively) for the two mycorrhizal types, and appeared to be greater than in uninoculated plants (7.2 µmol P mmol−" C) grown at the same P level. Key words : Carbon allocation, Eucalyptus, external hyphae, mycorrhizas, phosphorus.

 The two most widespread and important types of mycorrhizal associations are the arbuscular mycorrhizas and the ectomycorrhizas. In spite of their profound morphological and taxonomic differences, some of the effects of AM fungi and ECM fungi on host physiology are similar (Tinker, Jones & Durall, 1992). Both mycorrhizal types increase the uptake of P, of other poorly mobile elements, and of water, and can increase resistance to some root pathogens (Parke, Linderman & Black, 1983 ; Allen & Allen, 1986 ; Duchesne, Peterson & Ellis, 1988 ; GarciaGarrido & Ocampo, 1988 ; Bougher, Grove & * To whom correspondence should be addressed at (present address) : Department of Biology, Okanagan University College, 3333 College Way, Kelowna, BC V1V 1V7, Canada. E-mail : mjones!okanagan.bc.ca ‡ Present address : Department of Plant Sciences, South Parks Road, Oxford OX1 3RB, UK.

Malacjzuk, 1990 ; Jones, Durall & Tinker, 1990). The effects of the two associations on host physiology are even quantitatively similar. For example, P inflow rates in AM onion and clover were found to be four to six times as high as in non-mycorrhizal (NM) plants (Sanders & Tinker, 1971 ; Smith, 1982), and three times as high in ECM willow as in NM plants (Jones, Durall & Tinker, 1991). Arbuscular mycorrhizal plants allocate c. 7–10 % more of their photosynthate to the root system than do NM plants (Lambers, 1987) ; equivalent values for ECM plants were 4–36 % (Reid, Kidd & Ekwebelam, 1983 ; Durall, Jones & Tinker, 1994). All of these can be viewed as a cost to the plant ; however, under many conditions plant photosynthesis can be sink-limited rather than source-limited and, under these conditions, the plant might be able to supply the C required by the fungus without reducing C supply to other sinks.

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126

M. D. Jones, D. M. Durall and P. B Tinker

An interesting question is how relative costs (C) and benefits (nutrient uptake) to the plant compare between ectomycorrhizas and arbuscular mycorrhizas. The question of how to calculate costs and benefits of mycorrhizas has been discussed theoretically by Koide & Elliott (1989), Koide (1991), and Tinker, Durall & Jones (1994). Two approaches have been used : (a) the calculation of a ratio between P uptake (benefit) and below-ground C allocation (cost) ; and (b) the calculation of a ratio between the extra C produced in a mycorrhizal plant (benefit) and the C costs of the association. Douds, Johnson & Koch (1988) compared the P status and C allocation in NM, AM and split-root AM\NM citrus plants. They concluded that AM citrus plants were no more efficient at taking up P than were NM plants. Jones et al. (1991) calculated that willow ectomycorrhizal with Thelephora terrestris took up more P per unit of C than did NM plants. Pearson & Jakobsen (1993) compared the efficiency of three AM fungi by measuring hyphal "%C use per unit $#P transported and below-ground "%C use per unit $#P transported. Both Tinker et al. (1994) with ECM willow and Raju et al. (1990) with AM sorghum calculated costs and benefits to the mycorrhizal plant in terms of C. Tinker et al. (1994) determined that ECM willow converted 85 % of the extra C fixed into shoot biomass ( l C-use efficiency). Raju et al. (1990) found that in a low-P soil, the percentage benefit of arbuscular mycorrhizas to the sorghum averaged 84 %, but the approach in this experiment was totally different and more related to the concepts suggested by Stribley, Tinker & Snellgrove (1980). We cannot use results such as those cited above to make any conclusions about the relative effects of ECM and AM fungi on host nutrition or C physiology. Plant species differ in their response to P addition and therefore, in their response to mycorrhization (Koide, 1991). In order to make direct comparisons between the effects of AM and ECM fungi on their phytobionts, and to be able to test hypotheses about differences between the two types of mycorrhizas, experiments must be performed on the same host. It has been known for some time that Alnus, Populus, Salix, Eucalyptus and some woody legumes can form both AM and ECM associations (Harley & Harley, 1987). Surprisingly, very few studies have made use of these species to compare the effects of the two types of mycorrhizas (Chatarpaul, Chakravarty & Subramaniam, 1989 ; Osonubi et al., 1991). By inoculating one plant species separately with both types of fungi it should be possible to ascertain which type of fungus enhances growth of that host growing in a particular soil type by the greatest degree, and whether greater growth enhancement is due to a greater increase in nutrient uptake or a smaller diversion of C to the fungus. The study reported here compared growth responses, total P uptake, P inflow rates, external hypha

production and C allocation patterns in Eucalyptus coccifera seedlings colonized by one of several ECM or AM fungi. E. coccifera is a relatively slow-growing shrub which occurs just below the snow line in Tasmania (N. Malajczuk, pers. comm.).  Experimental design The overall experimental design consisted of Eucalyptus coccifera Hook. seedlings inoculated with one of two ECM fungi, or one of three AM fungi, or left uninoculated. All of the mycorrhizal seedlings, and one group of the NM seedlings (UI-P), were grown in a substrate (see below) containing 4 mg of bicarbonate-extractable P kg−". Other NM seedlings were grown in the same substrate to which P had been added to give bicarbonate-extractable P concentrations of 6, 10, or 15 mg P kg−". Non-mycorrhizal plants were grown at several soil P concentrations so that the seedlings which most closely matched the size of the mycorrhizal plants could be used for any physiological measurements. Seedlings were grown at 20 mC\18 mC day\night, at an irradiance of 400 µmol m−# s−" in a Saxcil2 Growth Environmental Cabinet (R. K. Saxton Sax-Air Ltd., London, UK) in a completely randomized design. Plants were re-randomized every 2 wk, and five randomlyselected plants of each treatment harvested after 89 d. Subsequent harvests occurred at intervals up to 155 d but because there was evidence (reduced growth rates of larger plants) of P depletion in the pots after 89 d, growth, P uptake and C allocation data are presented from the 89-d harvest only. The older plants were used to estimate hyphal production (see below).

Soil preparation and planting Partly sterilized (2n5i10% Gy gamma irradiation) sandy loam soil (Kershope Forest, Northumberland, UK ; National Grid Reference NY 76305150) was combined 1 : 2 by volume with autoclaved sand. Calcium carbonate was added (0n8 g kg−") to adjust the pH of the final mixture (see below) to 6n0. To produce the elevated levels of soil P, 18, 61n5 or 120n4 mg KH PO kg−" dry soil were applied as a # % spray. The soil was mixed, moistened, and allowed to equilibrate for 25 d. Before planting, the soil : sand combination was mixed 6 : 1 by volume with peat\vermiculite spawn (Molina & Palmer, 1982) containing either live mycelia of Laccaria bicolor (Maire) Orton (isolate S238 from Oregon State University, OR, USA) or Thelephora terrestris (Ehrh.) Fr. (isolate 011n25 from the Institute of Terrestrial Ecology, Penicuik, UK) or autoclaved spawn which had contained live


Arbuscular and ectomycorrhizal eucalypts Laccaria proxima (Boud.) Pat. mycelia (for the AM and UI treatments). Before mixing, the spawn was rinsed with tap water for 3 h and then dried at room temperature for 24 h. To prepare the pots (10-cm diameter, 12 cm in height) for planting, the moist equivalent of 380 g dry soil\sand\spawn mix was weighed into each pot. Next, a layer of c. 5 ml of moist sand containing either 250 µm sievings from AM leeks colonized by one of three AM fungi or with sievings from NM leek (for the ECM and non-mycorrhizal treatments) was added. The pots were then filled with the moist equivalent of 170 g of dry soil mix. The three AM isolates, Glomus caledonium (Nicol. & Gerd.) Trappe & Gerde., G. mosseae (Nicol. & Gerd.) Gerde. & Trappe, and the fungus Rothamsted E3, an undescribed Glomus frequently and erroneously named as Glomus fasiculatum (C. Walker, pers. comm.), were obtained from the Rothamsted, UK culture collection. Two seeds of E. coccifera which had been surfacesterilized (20 min in 30 % H O plus Tween2 20) # # and germinated on water agar, were planted in each pot. After 28 d, the seedlings were thinned to one per pot and fertilized with 50 ml of a complete nutrient solution minus P (Mason, 1980), thereby adding 5n5 mg N per pot. Fertilization was repeated twice more before harvest at 89 d. Carbon allocation Nine d before harvest, plants colonized by T. terrestris or G. E3, and UI-P and UIjP (NM plants grown at 10 mg kg−" P) plants were pulse-labelled with "%C. These two mycorrhiza treatments were selected because they produced the largest growth responses to ECM and AM fungal inoculation. Five seedlings were randomly selected for harvest from amongst the 20 seedlings planted per treatment, then three of these five plants (the median three plants when these five seedlings were ranked in size) were used in the labelling experiment. The pots containing these plants were transferred to chambers similar to those described by Snellgrove et al. (1982) ; the shoot chambers were made of Plexiglass2 and were 0n4-m tall and 0n15 m in diameter ; the root chambers were constructed of 0n15-m diameter PVC pipe and were 0n15-m tall. Each shoot chamber contained a small fan ; air throughput was 600 ml min−". There was no air flow between the root and shoot compartments once the chambers were sealed. Leak tests were performed on each chamber just before labelling to confirm this. The techniques for labelling were the same as those used by Jones et al. (1991). Preliminary experiments were performed to determine the time for the CO concentration in the chambers to reach # the compensation point (as indicated by no further decrease in CO concentration) when the chambers #

127 were sealed. Pulse times of 45 min for the T. terrestris, G. E3 and UIjP treatments and 90 min for the UIjP treatment were selected to optimize "%C uptake, but to minimize the time spent at the CO compensation point. The seedlings were # labelled with 740 kBq "%CO (released from # NaH"%CO with lactic acid), within 2 h of each other, $ 6–7 h before the end of the photoperiod. The photosynthesis measurements (see below) and labelling were all performed in the growth chamber where the seedlings were growing (i.e., 400 µmol quanta m−# s−" and 20 mC). The chase period was 202 h. Air containing "%CO respired # from each root or shoot chamber was bubbled through 100 ml of 2  NaOH. The NaOH was sampled and replaced at c. 20, 90, 130 and 200 h after labelling. The "%C in duplicate 50-µl aliquots from each sample was quantified by liquid scintillation counting. Previous sampling showed that this method resulted in a 95 % trapping efficiency of respired "%CO . # At the end of the chase period, three labelled and two non-radioactive plants were harvested, the shoots removed, the root system washed free of soil, the tissues dried, weighed and ground, and the P content determined as described in Jones et al. (1991). The root systems were washed in the minimum amount of water possible (c. 4–5 l) in a plastic basin ; this water was combined with the soil and evaporated to dryness at 80 mC. Counts of "%C in the shoot tissue, root tissue, and in the soil were determined following the processing of triplicate subsamples (100 mg) in a Packard2 Tri-Carb Oxidizer.

Colonization A weighed subsample of roots from each plant was not dried, but instead, was stained for percent colonization. The subsample was taken by cutting the roots into 2-cm lengths and distributing them evenly in water in a glass baking dish. The subsample was taken from one quadrant of the dish, and stained according to Phillips & Hayman (1970) with lactic acid substituted for lactophenol. The grid-line intercept method (Newman, 1966 ; Giovanetti & Mosse, 1980) was used to determine the percentage of the root system (roots 0n5 mm in diameter) forming either type of mycorrhiza, and the overall root length. An intersection was scored as ECM if a recognizable mantle was present and as AM if internal arbuscular hyphae were present. This method has been used to compare the degree of AM and ECM colonization in eucalypts in earlier studies (Lapeyrie & Chilvers, 1985 ; Chilvers, Lapeyrie & Horan, 1987 ; Oliveira, Schmidt & Bellei, 1997). It is considered appropriate because in both willow and eucalyptus, ECM mantles and Hartig nets were


128


visible on long stretches of lateral root, and were not limited to regions near root tips. Harley & Smith (1983) emphasize that several hierarchies of root can be mycorrhizal and must therefore be considered when quantifying colonization.

Quantification of external hyphae A 7-mm corkborer was used to remove three soil cores midway between the stem and the edge of the pot. The three cores were combined and 1 g wet weight of soil was placed in 100 ml of tap water. A second 1-g sample was oven-dried at 70 mC to obtain a d. wt conversion factor. The soil suspension was mixed on a magnetic stir plate for 30 min. Then a 6ml aliquot was removed with a syringe and filtered through a 1n2-µm Millipore2 filter. The retained material was suspended in 0n45 ml of fluorescein diacetate (FDA) solution, prepared by diluting a 5 mg FDA ml−" acetone stock 1 in 50 by volume into pH 7n4 phosphate buffer. The FDA-soil suspension was distributed on two microscope slides and covered with 10i22-mm coverslips. Three aliquots were examined for each soil sample. Because fluorescence from FDA fades with time, each aliquot was examined for a maximum of 45 min. Owing to this time constraint, the percentage of the microscope slide examined decreased for treatments with very high hyphal production from 100 % early in the experiment to 20 % (every fifth transect) later in the experiment. The intersections between fluorescing hyphae and ocular grid lines (0n5 mm per side on the graticule) were counted at i200 magnification. Hyphal length was quantified using the method of Newman (1966). Soil for hyphal quantification was sampled at intervals (17 d on average for each treatment) from pots containing living plants. Because hyphae from only two or three pots could be examined per day, and because samples could not be stored, it was impossible to produce data points for every treatment for the same day. Instead, seven independent (i.e., samples were taken from different plants) data points, from samples collected between days 47 and 150 after planting, were used to create regression lines for hyphal length (cm g−" soil) against time in each treatment. The regression lines were then used to estimate the length of viable hyphae present at 89 d for each treatment so that the treatments could be compared. The data was log-transformed before calculation of regression equations. The regression coefficients were 0n9 or higher, except for G. mosseae where r# l 0n4 ; however, the hyphal lengths for this fungus varied little over time relative to the other fungi. The length of ‘ mycorrhizal ’ hyphae was estimated by subtracting the length of fluorescing hyphae present in soil from uninoculated seedlings from that present in inoculated pots.

Data analyses The percentage of the fixed "%C which was allocated below-ground was calculated as the sum of the "%C in root plus fungal tissue, root plus fungal respiration, and soil, divided by the "%C measured in all tissues, shoot and root respiration, and soil. Weight and below-ground "%C data, the tissue P data, and root colonization data were subjected to one-factor s to detect inoculation effects. Tissue P data were loge-transformed for normality. Fisher’s Protected Least Significant Difference Test (PLSD) was used as an a posteriori multiple comparison test when s detected significant effects (P 0n05). In addition, orthogonal comparisons (Sokal & Rohlf, 1981) were used as a priori tests for pre-planned comparisons of specific treatments or groups of treatments. For the latter, the two ECM treatments and the three AM treatments were grouped separately and compared with the UI-P and\or UIjP treatments. Phosphorus inflow values (uptake of P per unit length of root per unit time) were calculated according to the formula of Brewster & Tinker (1970). Phosphorus uptake efficiency (amount of P taken up per unit of C allocated below-ground) was calculated according to the methods used in Jones et al. (1991). Phosphorus uptake was calculated as the difference in the mean P content of seedlings of each treatment at days 28 and 89. Total below-ground supply of C between days 28 and 89 was calculated as follows ∆Cb l ∆Ws

, 0%C %C 1 BG ST

where ∆Ws is the mean increase in shoot weight between days 28 and 89, % CBG is the mean percentage of the "%C found in the below-ground compartment (including soil, root and fungal tissue, and respiration) at day 89, and % CST is the mean percentage of the "%C found in the shoot tissues at day 89. We justify the application of the % CBG\% CST ratio to the entire 28–89-d period by noting that the amounts of C translocated below-ground were much more affected by the ratio at the end of the period, when the plants were substantially larger (0n4–1n6 g d. wt at 89 d compared with 0n005 g at 28 d), than the ratio at the beginning. Moreover, the seedlings inoculated with T. terrestris and G. E3 were already well colonized by 28 d, so the influence of the fungus on C allocation would have occurred over the entire interval. Correlations were performed between several plant variables and the length of external hyphae estimated to be present at 89 d. The length of ‘ mycorrhizal ’ hyphae present was calculated by subtracting the length of hyphae present in the UIP treatment from that in the other treatments, and is


Arbuscular and ectomycorrhizal eucalypts presented as m mycorrhizal hyphae per m of colonized root.  Colonization Colonization of root systems was evaluated on seedlings removed from the pots during thinning at 28 d, and on seedlings after the harvest at 89 d. All of the fungi had begun to colonize the roots by 28 d. Hartig nets were clearly visible in roots inoculated with T. terrestris, whereas roots inoculated with L. bicolor had loose mantles only. Glomus E3 appeared to have produced the largest number of infection points and density of internal hyphae of the three AM fungi by 28 d. At 89 d, the root lengths colonized by different fungi were similar (P 0n05 ; Table 2). Growth response to inoculation The growth response of the seedlings to inoculation was greater to the two ECM fungi than to the three AM fungi (P 0n0001, planned contrast ; Fig. 1). These inoculated plants were more than three times as large as the UI-P plants and 1n5–3n5 times as large as the AM plants. Growth response to inoculation with AM fungi was less pronounced and more variable. Only seedlings colonized by G. E3 were significantly larger than UI-P plants after 89 d. Neither inoculation nor P application affected root weight fraction (P 0n05 ; means ranged from 0n21 to 0n27). There was no correlation between the proportion of the root system colonized by the different AM or ECM fungi, and the growth response of the plants (Table 1).

Seedling d. wt (mg)

2000 a

ab

a bc

1000

cd d

d

0 Tht

Lcb

GE3

Gcal Gmos UI–P Treatment

129 Phosphorus uptake Treatment effects on total P contents of the plants generally paralleled the effects on dry weight. Ectomycorrhizal plants contained five to six times as much P as UI-P plants after 89 d (Fig. 2). Mycorrhizal colonization of any kind increased the P concentrations in shoots by at least 330 µg g−" or 40 % (Table 2). Similar results were observed with root P concentrations, although the ECM plants had higher P concentrations than AM plants (P 0n0001, planned contrast). There were distinct differences in P inflow rates between the two types of mycorrhiza (Table 3). The inflow rates into ECM plants were approx. 3n8 times those of UI-P plants, whereas those of AM plants ranged from 2n0, for G. mosseae plants, to 2n7 times higher than those of UI-P plants for G. E3 plants. Thus, as expected, inflow rates could be ranked in the same order as the growth-promoting ability of the fungi (compare Table 3 and Fig. 1). Quantification of external hyphae The soil in pots of ECM plants contained substantially more FDA-stained hyphae than did that of AM plants (Fig. 3). Values for the individual ECM fungi were two to six times as high as those for individual AM fungi. The amount of hyphae produced by a mycobiont was highly correlated both with the total P taken up by the plant and with shoot weight (Table 1). Carbon allocation There was no significance treatment effect on the percentage of "%C which the seedlings allocated below-ground (soil plus below-ground respiration plus root and associated fungal tissue, T. terrestris 13n5p0.5 %, Glomus E3 12n4p1.4 %, UI-P 13n0p 3.3 %, UIjP 19n1p2n9 %) after 89 d of growth. Likewise, the distribution of "%C amongst the belowground compartments did not differ between treatments, although there was a tendency for the seedlings with higher P contents (UIjP and T. terrestris) to allocate more C to below-ground tissues and less to respiration (Table 4).

UI+P

Figure 1. Dry wt of Eucalyptus coccifera seedlings inoculated with ectomycorrhizal or arbuscular mycorrhizal fungi and grown in soil containing 4 mg kg−" P, or left uninoculated and grown at 4 (UI-P) or 10 (UIjP) mg kg−" P for 89 d. Tht, Thelephora terrestris ; Lcb, Laccaria bicolor ; GE3, Glomus ‘ E3 ’ ; Gcal, Glomus caledonium ; Gmos, Glomus mosseae. Meansp1  ; n l 5. Planned contrasts (d.f. l 1, 21) detected differences between the ectomycorrhizal and arbuscular mycorrhizal plants at P l 0n0001. Bars with different letters differed at P l 0n05 according to Fisher’s Protected Least Significant Difference.

Phosphorus uptake efficiency Phosphorus uptake efficiency, as used here, is a measure of how much P was taken up by a root system over a time interval, relative to the amount of C translocated below-ground. For the present experiment, P uptake efficiencies were calculated for the period 28–89 d (Table 3). Statistics could not be performed on these data because they were calculated from the mean values of plants harvested at the two dates ; nevertheless, the mycorrhizal (T. terrestris



130

Table 1. Correlations between shoot weight or phosphorus content and fluorescein diacetate-stained extramatrical hyphal lengths in vesiculararbuscular and ectomycorrhizal Eucalyptus coccifera after 89 d of growth. % colonization

Hyphal length (cm g−" dry soil)

Total phosphorous content of plant r# 0n186 0n985 P 0n47 0n0008 Shoot dry weight r# 0n128 0n968 P 0n55 0n0024

Seedling P content (mg)

3

a

a

a 2 b bc

1

bc

c

0 Tht

Lcb

GE3

Gcal Gmos UI–P Treatment

UI+P

Figure 2. Total phosphorus content (mg) of Eucalyptus coccifera seedlings (shoots plus roots) inoculated with ectomycorrhizal or arbuscular mycorrhizal fungi and grown in soil containing 4 mg kg−" P, or left uninoculated and grown at 4 (UI-P) or 10 (UIjP) mg kg−" P for 89 d. Tht, Thelephora terrestris ; Lcb, Laccaria bicolor ; GE3, Glomus ‘ E3 ’ ; Gcal, Glomus caledonium ; Gmos, Glomus mosseae. Meansp1  ; n l 5. Planned contrasts (d.f. l 1, 21) detected differences between the ectomycorrhizal and arbuscular mycorrhizal plants at P l 0n0001. Bars with different letters differed at P l 0n05 according to Fisher’s Protected Least Significant Difference.

and Glomus E3) plants appeared to have higher P uptake efficiencies than the non-mycorrhizal plants. The T. terrestris plants translocated 3n8 times as much C below-ground but took up six times as much P as the UI-P plants, during the above interval.

Mycorrhizal hyphal length (m m−" of mycorrhiza) 0n095 0n0048 0n956 0n011

 Although there is no information specific to E. coccifera (N. Malajczuk, pers. comm.), Eucalyptus spp., in general, can form both AM and ECM associations (Jones, Durall & Tinker, unpublished ; Malajczuk et al., 1981 ; Malajczuk, Molina & Trappe, 1982). In some systems, arbuscular mycorrhizas are the more abundant type initially, with ectomycorrhizas succeeding them over a period of months (Lapeyrie & Chilvers, 1985 ; Chilvers et al., 1987) ; however, the relative abundance of the two types of mycorrhizas can be influenced by the types of host plants previously grown on the site (Oliveira et al. 1997). Previous experiments have shown stimulation of growth and P uptake of Eucalyptus spp. by both ECM (Malajczuk, McComb & Loneragan, 1975 ; Heinrich & Patrick, 1986) and AM associations (Lapeyrie & Chilvers, 1985 ; Adjoud et al., 1996). The experiment reported here showed growth rates, total P uptake, and P inflow rates of E. coccifera to be stimulated more by colonization with either of two ECM fungi than with any of the three AM fungi used. These results agree with those of Chatarpaul et al. (1989) who found that shoot height and total d. wt was higher in ECM than in AM nodulated Alnus incana. By contrast, Lapeyrie & Chilvers (1985) concluded that most of the growth promotion of Eucalyptus dumosa growing in calcareous soils was

Table 2. Shoot and root phosphorus concentrations, total root length and percentage colonization of mycorrhizal and non-mycorrhizal Eucalyptus coccifera seedlings grown for 89 d

Laccaria bicolor Thelephora terrestris Glomus sp. type E3 Glomus caledonium Glomus mosseae Uninoculated kP Uninoculated jP

Shoot P (µg g−" d. wt)

Root P (µg g−" d. wt)

Root length (m)

% colonization

1088p75 a 1273p95 a 1163p132 a 1142p127 a 1150p67 a 810p183 b 1454p120 a

1581p25 ab 1392p240 bc 1063p48 cd 1176p79 bc 1244p98 bc 801p39 d 2144p301 a

50n1p8n5 ab 61n2p10n1 a 42n5p7n6 abc 35n5p4n5 bc 19n6p2n4 c 37n9p4n8 abc 61n0p21n9 ab

42n3p7n5 40n8p7n3 48n5p9n5 34n8p10n2 55n9p6n2 0n1p0n1 0n1p0n1

Values l meansp1 . n l 5. When values within a column are followed by a different letter, they differ at P l 0n05 according to a Fisher’s Protected Least Significant Difference Test, and a one-factor  (d.f. l 6, 28) detected differences amongst treatments at P l 0n05.


Arbuscular and ectomycorrhizal eucalypts

131

Table 3. Phosphorus inflow and phosphorus uptake efficiencies of mycorrhizal and non-mycorrhizal Eucalyptus coccifera seedlings

Thelephora terrestris Laccaria bicolor Glomus sp. type E3 Glomus caledonium Glomus mosseae Uninoculated kP Uninoculated jP

P inflow (mol m−" s−")

Phosphorus uptake efficiency (Days 28–89)

Days 28–89

∆P (µmol)*

∆Cb (mmol)†

∆P\∆Cb

14i10−"$ 15i10−"$ 10i10−"$ 8n6i10−"$ 7n2i10−"$ 3n7i10−"$ 15i10−"$

67n4 — 34n8 — — 11n3 69n4

6n0 — 3n5 — — 1n6 11n4

11n2 — 10n0 — — 7n2 6n1

* ∆P l the difference in P content between seedlings harvested at 28 and 89 d. † ∆Cb is the total amount of C allocated below-ground between days 28 and 89. ∆Cb l ∆Ws (%CBG\%CST), where ∆Ws is the mean increase in shoot weight between days 28 and 89, %CBG is the mean percentage of the "%C found in the below-ground compartment (including soil, root and fungal tissue, and respiration) at day 89, and %CST is the mean percentage of the "%C found in the shoot tissues at day 89.

‘Mycorrhizal’ FDA-stained hyphae (m m–1 colonized root)

100 80 60 40 20 0

Tht

Lcb

GE3 Treatment

Gcal

Gmos

Figure 3. Estimated length of fluorescein diacetate-stained mycorrhizal hyphae per unit of colonized root for Eucalyptus coccifera seedlings inoculated with one of two ectomycorrhizal (Tht, Thelephora terrestris ; Lcb, Laccaria bicolor) or one of three arbuscular mycorrhizal fungi (GE3, Glomus E3 ; Gcal, Glomus caledonium ; Gmos, Glomus mosseae) and grown for 89 d. Data are estimates, based on regression lines of data from seven independent samples collected between days 47 and 150. The length of ‘ mycorrhizal ’ hyphae was estimated by subtracting the length of fluorescing hyphae present in soil from uninoculated seedlings.

due to arbuscular mycorrhizas which were later succeeded by ectomycorrhizas. This conclusion is debatable, however, because the difference in size between inoculated and uninoculated plants was greater at 5 months, when ectomycorrhizas were more common, than at 2 months, when they were less common. In this experiment, the growth of the seedlings appeared to be P limited ; the seedlings grew in direct proportion to the amount of P taken up. There was no sign of luxury consumption which might indicate growth limitation by another nutrient. Given this, the difference in the amount of external hyphae produced by the ECM and AM associations might explain the difference in host response to the two mycorrhizal types. Total P uptake and the length of FDA-stained hyphae per pot were highly correlated (Table 1). By contrast, there was no difference in the percentage root length colonized by the five fungi at 89 d, and thus this variable did not correlate well with growth-promoting ability. McGonigle et al. (1990) have shown that total percentage root length colonized by a AM fungus is not a particularly good

Table 4. Percentage of below-ground "%C allocated to tissue (root plus fungus), respiration (root plus micro-organisms), and soil (extramatrical hyphae and rhizodeposition) by mycorrhizal and non-mycorrhizal Eucalyptus coccifera seedlings during a 1 h pulse\200 h chase experiment

Thelephora terrestris Glomus sp. type E3 UIkP plants UIjP plants

Tissue %

Respiration %

Soil %

42n6p4n7 31n5p2n4 32n7p6n9 44n9p6n8

45n4p5n7 58n4p0n7 57n0p4n8 48n6p5n6

12n1p1n7 10n1p1n7 10n3p2.2 6n5p1n2

Values are meansp1  for three plants. One-factor  (d.f. l 3,8) did not detect differences between treatments for any of the three components (P 0n2).


132


predictor of growth-promoting effects by that fungus. The length of external hyphae produced by a mycorrhizal fungus can be a good predictor of its relative ability to take up P (Jones et al., 1990), but in other cases, it is the average distance of extension from the root which is more important (Jakobsen, Abbott & Robson, 1992 a, b). In addition, the hyphae of different AM fungi can differ in the amount of P taken up per unit length (Jakobsen et al., 1992 a) ; thus, the total length of hyphae produced is not always a reliable indicator of the efficacy of P uptake. The final calculated lengths of hyphae were lower than those found in earlier studies for both ECM (1000–8000 m m−" (Read & Boyd, 1986) ; 100–300 m m−" (Jones et al., 1990)) and AM fungi (71–1420 m m−" (Smith & Gianinazzi-Pearson, 1988)). This is probably because most of these earlier studies were measuring total, not FDAstained, hyphal lengths. Studies evaluating AM fungal hyphal lengths using vital stains have found that after the length of time that our plants were grown, only 15–40 % of the hyphae were viable (Schubert et al., 1987 ; Hamel, Fyles & Smith, 1990). Using this conversion factor, our hyphal lengths fall within the range of those found in other AM studies. To our knowledge, no one has studied the changes in the percentage of viable ECM fungi over time. The differences in response to the two mycorrhizal types might also result from differences in the activity of the associations at different times. Arbuscular mycorrhizas can go through cycles of activity, with many arbuscules being produced during periods of growth in young roots, and vesicles predominating at other times (Douds & Chaney, 1982). Thus, P uptake efficiencies would be expected to change according to the developmental state of the mycorrhizas. Ectomycorrhizas do not possess anatomical structures which so obviously indicate changes in physiology over time. Nevertheless, microscopic observations suggest that mantle hyphae might have a limited period of activity (Downes, Alexander & Cairney, 1992 ; Hagerman & Jones, unpublished). Moreover, the ratio of P uptake to C demand appears to change as ECM root systems develop (Jones et al., 1991). Our "%C allocation experiment ran for only 9 d of the 89-d growth period and it is possible that the relative P uptake efficiencies of the two types of mycorrhiza were different earlier or later in the experiment. The increases in P inflow rates in the mycorrhizal plants (four times in ECM plants, and two to three times in AM plants) were comparable to those found previously. The values for the ECM plants were slightly higher than the 2n7 times increase that Jones et al. (1991) found for Salix viminalis mycorrhizal with the same isolate of T. terrestris. The increase in P inflow seen with AM colonization in the present experiment was slightly lower than the 3n1–4n7 times increase in inflow observed for AM onions by

Sanders & Tinker (1973), and the 4n2–6n3 times increase observed in AM Trifolium subterraneum by Smith (1982). These earlier studies cannot be used to make any firm conclusions about the relative impact of AM and ECM formation on inflow rates because they were not conducted on the same host species. In the present study, we have evidence that P inflow rates were higher in ECM than AM plants, although this could not be tested statistically. It is possible that the difference in inflow rates between the two types of mycorrhiza would have been even greater if measured earlier in the experiment. At 89 d, the ectomycorrhizal plants had extracted 82 % (L. bicolor) to 95 % (T. terrestris) of bicarbonate-extractable P estimated to be in the soil. Arbuscular mycorrhizal plants had extracted only 21 % (G. mosseae) to 50 % (G. E3). Inflow rates might thus have been decreasing in ECM plants toward the latter part of the measurement period. In contrast to earlier experiments (Snellgrove et al., 1982 ; Reid et al., 1983 ; Durall et al., 1994), we did not detect any mycorrhizal effects on the proportion of fixed C which was allocated to the root system. The plants were still quite small (Fig. 1) after 89 d of growth and had low root weight fractions (0n21–0n27). We expect that differences between mycorrhizal and NM treatments would have increased with time as root weight fraction, and thus the amount of fungal tissue, increased. Differences in below-ground C allocation increased with time between ECM and NM Salix viminalis (Durall et al., 1994) and were not significant until root weight fractions in the ECM plants exceeded 0n35. The results of this experiment confirm earlier results that both ECM (Jones et al., 1991) and AM (Pearson & Jakobsen, 1993) plants are more efficient than NM plants at acquiring P per unit of C allocated below-ground. This is to be expected given that a unit of C used in hyphal production creates more absorbing length than can be created if the same unit of C was used in root production. In this study P uptake efficiencies were calculated for only one ECM and one AM treatment ; the T. terrestris and Glomus sp. type E3 plants appeared to have very similar P uptake efficiencies, yet the ECM plants were 50 % larger. If the degree of growth response depends upon both the extent to which the mycorrhizal system can raise the inflow rate to that required for maximum growth rate, and any potential growth reduction which might result from the diversion of C from the shoot to the root system as a consequence of the mycorrhizal infection, the ability to increase P inflow seems to be the more important of the two factors in explaining the differences in the growth response to ECM and AM fungi. Similar conclusions about the relative importance of P uptake and C diversion were reached by Pearson & Jakobsen (1993) when comparing mycorrhizal effici-


Arbuscular and ectomycorrhizal eucalypts encies of three arbuscular fungi associated with Cucumis sativus. Conclusions about whether ectomycorrhizas generally have higher P uptake efficiencies than arbuscular mycorrhizas must await comparisons between larger numbers of species of each type. Obviously a considerable amount of variability, such as that found by Pearson & Jakobsen (1993), is to be expected within each of the mycorrhizal groups.                The authors wish to thank Dr Jeff Troke and Mr Dave Smith of Hoechst Inc., Milton Keynes, UK, for generously oxidizing and counting the "%C-labelled tissue and soil. Mr Kevin Ingleby and Dr David Stribley provided the fungal isolates used in this study. Helpful comments on an earlier version of this manuscript were received from Drs Chris Andersen and Iver Jakobsen.

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