The effect of earthworms and arbuscular mycorrhizal fungi on growth of and 32P transfer between Allium porrum plants

Soil Biology & Biochemistry 34 (2002) 1027±1036

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The effect of earthworms and arbuscular mycorrhizal fungi on growth of and 32P transfer between Allium porrum plants F. Tuffen a, W.R. Eason b, J. Scullion a,* a

b

Department of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, SY23 3DE, UK Plant Microorganism Interaction Group, Institute of Grassland and Environmental Research, Aberystwyth SY23 3HF, UK Received 31 May 2001; received in revised form 8 February 2002; accepted 13 February 2002

Abstract To measure P transfers, two small pots, placed 1 cm apart and each containing one leek (Allium porrum L.) plant, were embedded within a larger sand volume. Mesh windows on the facing pot sides allowed the penetration of hyphae but not roots. The effect of earthworms (Aporrectodea caliginosa Sav.) in the hyphal compartment and of arbuscular mycorrhizal fungi (AMF) on plant growth and 32P transfer between plants was investigated. In addition, the effect of mechanical disruption of the hyphal compartment on mycorrhizal plants was studied. AMF did not affect plant growth, although infection levels were high. Earthworms generally increased shoot and root growth; this effect did not appear to be related to location of earthworm activity. 32P transfer from the dying root system of labelled `donor' plants to unlabelled `receiver' plants was increased where earthworm activity was evident between pots, whereas mechanical disruption eliminated the effect of AMF on 32P transfer between mycorrhizal plants. Magnitude of transfer was only related to AMF colonisation levels in the donor plant in the absence of earthworms or mechanical disruption. These results suggest that movement of 32P into the hyphal compartment via the donor AMF mycelium was more important in in¯uencing transfer magnitude than direct transfer via AMF hyphal interconnections between the two plants. Uptake of 32P by the receiver AMF hyphae appears to have been increased by earthworm activity in the hyphal compartment; it is proposed that earthworm-mediated mobilisation of 32P, partly contained within the donor AMF mycelium, led to enhanced 32P availability in the hyphal compartment. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Arbuscular mycorrhizas; Hyphal interconnections; Interplant transfer; 32P; Earthworms

1. Introduction Arbuscular mycorrhizal fungi (AMF) are almost ubiquitous symbionts of herbaceous plants, including most agricultural crops, and can form symbioses with 80% of all plant genera (Smith and Read, 1997). The extraradical mycelium of the AMF increases the absorptive capacity of the root system by extending the soil volume exploited. The primary bene®t to the host plant is often increased P supply (Marschner and Dell, 1994; Clark and Zeto, 2000), whilst the heterotrophic fungus secures a ready supply of carbohydrates. The AM symbiosis can also enhance uptake of other nutrients such as N, Cu and Zn (Marschner and Dell, 1994), protect plants against the effects of pathogens (Newsham et al., 1995) and increase plant tolerance of toxic metals such as Al and Mn (Clark and Zeto, 2000). The mycorrhizal growth response of host plants re¯ects the relative magnitudes of stimulatory effects of enhanced P uptake * Corresponding author. Tel./fax: 144-1970-622307. E-mail address: [email protected] (J. Scullion).

and detrimental effects of the fungal drain on the host plant's photosynthate (Harley, 1975). Earthworms are also important components of the rhizosphere ecosystem, capable of enhancing plant growth (Lee, 1985; Baker et al., 1999) by improving soil physical and chemical conditions. Earthworm enhancement of soil nutrient availabilities by incorporating organic matter and promoting microbial mineralisation is well documented (Springett and Syers, 1979; Lee, 1985). Sharpley and Syers (1977) reported that surface casts contained around four times more plant-available inorganic P and twice as much plant-available organic P as the underlying soil. AMF are capable of forming hyphal interconnections between mycorrhizal plants, through which nutrients can be transferred (Newman, 1988). Although interplant nutrient transfer occurs in non-mycorrhizal plants, this transfer can be substantially increased by AMF infection (Newman, 1988; Newman and Eason, 1989). Interplant nutrient transfer can be intraspeci®c (Newman, 1988; Newman and Eason, 1993) or interspeci®c (Eason et al., 1991; Fischer et al., 1996).

0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(02)00036-6

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A large number of interconnecting hyphae may be required for signi®cant interplant nutrient transfer (Newman, 1988; Newman et al., 1992). The direction of resource transport between plants linked by a common AMF mycelium may be governed by source±sink relationships; for example, Francis and Read (1984) reported that C transfer was in¯uenced by shading of the receiver plant. Although the movement of P between mycorrhizal plants may be too slow to substantially in¯uence the nutrient status of shoots (Newman and Eason, 1993), a rapid and unidirectional transfer of P may occur where a physiological imbalance exists between plants, for example, when dying and living roots are intermingled (Ritz and Newman, 1985; Newman and Eason, 1989). A net transfer of nutrients as a result of such an imbalance may be important where one plant dies or is differentially stressed, for example, in grassland pasture as a result of trampling or selective grazing (Newman and Eason, 1989; Eason et al., 1991), and in plant communities comprising a range of plant ages and sizes. If the AMF mycelium is important in interplant nutrient transfer, it is likely that this process is affected by the activities of the soil faunal community, particularly fungivores. Larsen and Jakobsen (1996) found that the collembolan Folsomia candida caused a slight reduction in the transfer of 32P from a hyphal compartment into mycorrhizal plants. Earthworms have been found to exhibit a feeding preference for fungi associated with plant remains (St John et al., 1983; Bonkowski et al., 2000), and for fungal mycelia (Bonkowski et al., 2000), much of which is likely to be mycorrhizal (Kabir et al., 1996). Disturbance of the AMF mycelium was implicated in a transient negative effect of high densities (the equivalent of 500 m 22) of the earthworm Aporrectodea trapezoides on colonisation of Trifolium subterraneum by Glomus intraradices reported by Pattinson et al. (1997). Although earthworms may damage the AMF mycelium, earthworm-mediated improvements in soil nutrient availabilities may outweigh any effects on the host plant of AMF mycelium damage, and may potentially reduce mycorrhizal dependency. Earthworm disturbance of the AMF mycelium may also in¯uence the transfer of nutrients between mycorrhizal plants through disruption of hyphal interconnections that occur between plants. Hyphal interconnections form when non-mycorrhizal roots are invaded by hyphae from an established mycelial network, and can extend for 2 cm (Schuepp et al., 1987). Even without selective feeding, earthworm activity in the rhizosphere is likely to partially disrupt AMF hyphal interconnections. Large populations of earthworms are capable of processing an amount of soil equivalent to the surface 10 cm every 5 years (Edwards and Bohlen, 1996). This processing, plus collateral damage, may have signi®cant effects on the dynamics of AMF hyphal networks. Our study investigated the effect of earthworms and AMF

on plant growth and transfer of 32P between mycorrhizal Allium porrum plants. Interplant 32P transfer served as an indicator of the potential ecological signi®cance of earthworm disruption of P transfer between mycorrhizal plants, and also as a sensitive measure of earthworm effects on the AMF mycelium. The host species was selected as it is known to form extensive AMF infection and have a high mycorrhizal dependency (Plenchette et al., 1983). The earthworm Aporrectodea caliginosa was chosen because its abundance and ecological strategy as a soil feeder (endogee, BoucheÂ, 1969) made it a species likely to affect the AMF mycelium. Ap. caliginosa feeds predominantly in the top 70 mm soil in temporary horizontal burrows (Sims and Gerard, 1985), and has been found to feed extensively on fungal mycelia (Bonkowski et al., 2000), so making it suitable for the experimental system used in this study. In addition we examined separately the effect of mechanical disruption of the soil between mycorrhizal plants. This allowed comparison of effects of earthworm activity on interplant 32P transfer and maximum expected effects if earthworms severed all AMF hyphal interconnections.

2. Materials and methods 2.1. Experimental design The earthworm/AMF experiment was designed to maximise the opportunities for AMF and earthworm bene®ts to plants, for transfer of 32P between plants and for earthworm disruption of this transfer. In each replicate two plants were separated by a small distance (1 cm) to encourage establishment of AMF hyphal interconnections (Fig. 1). Single `donor' and `receiver' plants were used to simplify transfer pathways and because of the limited volume available for their growth. Holes (3 £ 4 cm 2) were cut in facing pot sides, and a 37 mm pore size nylon mesh (Estal, Switzerland) was glued over these holes to form `windows' through which fungal hyphae but not roots could penetrate. A separate mechanical disruption experiment, in which the continuity of any hyphal interconnections was periodically broken, was run concurrently with the main experiment to allow comparison between earthworm effects on plant growth and interplant 32P transfer and maximum expected effects if earthworms were to sever all AMF hyphal interconnections. In both experiments, plants were grown in a glasshouse for 13 weeks before 32P labelling donor plants. Donor leaves remained in the labelling solution for 7 days, suf®cient time for 32P to become distributed throughout the plant (Newman and Eason, 1993). After 14 weeks donor plants were killed by removal of their shoots. Killing of the labelled donor plant encouraged rapid 32P transfer from the dying root system to the unlabelled receiver plant by creating an exaggerated physiological imbalance between the two plants (Newman and Eason, 1989). Donor plant roots remained

F. Tuffen et al. / Soil Biology & Biochemistry 34 (2002) 1027±1036

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Fig. 1. Growth chamber design for earthworm and mechanical disruption experiments.

in position for 28 days to ensure maximum 32P transfer (Eason et al., 1991). Receiver plants were harvested at 18 weeks. 2.1.1. Earthworm/AMF experiment A factorial design was used, with four treatments: non-mycorrhizal control (NM), mycorrhizal (M), nonmycorrhizal 1 earthworms (Ew) and mycorrhizal 1 earthworms (EwM). Each treatment initially comprised 10 replicates; immediately prior to labelling this number was reduced to seven replicates whose donor and receiver plants were healthy and of roughly equal size. In Ew and EwM treatments, a high density of Ap. caliginosa (11 l 21) was introduced into the hyphal compartment at the start of the experiment.

2.2. Plant species and growing conditions Leek (A. porrum L., variety Musselburgh) seedlings were transplanted into Terragreen in 0.2 l pots (Plantpak Ltd, UK) embedded within a larger volume of sand (Fig. 1). Terragreen (Oil-Dri Ltd, UK) is an inert attapulgite clay which has been used in previous AMF studies (Boddington et al., 1999). It contains very low levels of available P, which encourages AMF development. Terragreen was washed through a 1 mm sieve to remove ®ne particles. All units were placed in a glasshouse, with a minimum temperature of 20 8C and a 16 h photoperiod. Plants were watered daily, and units were repositioned randomly on a weekly basis. At 4 weeks, test cores were taken from the root compartments of ®ve replicates using a 1 cm diameter

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corer for AMF assessment, to check that adequate AMF colonisation had become established. After 10 weeks growth all units were transferred to a glasshouse without supplementary lighting and heating, designated for use with radioisotopes. To encourage plant growth, 10 ml of Vitax Vitafeed 102 (2 g l 21 dissolved in water, a P free fertilizer, Vitax Ltd, UK) were applied weekly to each plant at this stage. 2.3. AMF The AMF inoculum consisted of roots of Allium cepa (L.), with a mean AMF colonisation of 33% …SEM ˆ 7:02† root length, infected by a mixed population of AMF spores isolated from soil used in previous research and found to be effective on leek (Scullion et al., 1998; Eason et al., 1999). Approximately 100 mg fresh weight of infected roots were placed in the planting hole prior to transplanting the A. porrum seedlings. All treatments received 50 ml root washings, which had been ®ltered, in order to introduce background non-mycorrhizal micro¯ora to all treatments. 2.4. Earthworms Earthworms (Ap. caliginosa Sav.) were hand-sorted from soil in the ®eld, identi®ed using the taxonomic key of Sims and Gerard (1985), and pretreated to destroy any infective AMF propagules present in the digestive tract or adhering to the skin. The procedure described by Alphei et al. (1996) was used, as it was found to be effective whilst having no adverse effect on the survival of previously healthy Ap. caliginosa (Tuffen F, 2000, PhD Thesis, UWA). Earthworms were subjected to two cycles of washing in 0.01% formalin followed by transfer to autoclaved sawdust for 4 days. The earthworms were then ready for experimental use. The hyphal compartment of each appropriate unit received 14 healthy adult earthworms, selected for vigour. This density (approximately 650 m 22 surface area) was considered adequate to encourage an earthworm effect, without being excessively high compared with reported densities of 300±400 m 22 of Ap. caliginosa in the ®eld (Baker et al., 1999). Earthworms were introduced into 10 cm deep holes in the sand, and holes were then ®lled over with sand. The sand particle size range was mainly 125±250 mm, suitable for ingestion by earthworms. 2.5. 32P labelling and plant harvesting Labelling was carried out 1 week after moving the units into the radiation glasshouse, when seedlings were 14 weeks old. Immediately before labelling, any surface earthworm casts in the area between the two small pots were removed and discarded. This was done to ensure that any casts present in this area at the ®nal harvest had been produced since labelling, indicating earthworm activity in the interplant area and thus potential disruption of 32P transfer. Each

randomly designated donor plant was labelled by dipping the ends of three leaves into an aqueous, carrier-free orthophosphate solution of 20 kBq 32P (Amersham International plc, UK) over a period of 1 week. This method had been used successfully in similar previous studies (Newman and Eason, 1993) and avoids problems of variability between plants in 32P uptake. Also, since it did not affect the P status of donor plants, the labelling procedure had no in¯uence on source±sink relationships between plants nor the AMF status of donor plants. After labelling, leaf tissue in contact with the solution was removed and a polythene sheath was placed over each donor plant to prevent accidental transfer of the isotope from donor to receiver plants. After 7 days, the labelled donor shoot was severed at the stem base and retained for dry weight and 32P content determination. After 18 weeks, receiver shoots were severed at the stem base. Donor and receiver plant roots were recovered, removing Terragreen from the root mass without washing. Root samples (0.5 g) were removed, placed in 50% ethanol, and stored for 1 month to allow for radioactive decay before AMF assessment. Remaining shoot and root material was used for dry weight and 32P determination. 2.6. Measurements AMF assessment was carried out using the method described by Brundrett et al. (1996). Roots were stained in trypan blue, and AMF colonisation levels determined using the gridline intersect method of Giovanetti and Mosse (1980). Total 32P in shoots and roots was determined by Cerenkov counting in acid digests (MAFF, 1986) in a liquid scintillation counter with corrections for colour quenching, background radiation and decay (Wang et al., 1975). All counts were carried out over a 30 min period. Receiver plant samples had relatively low 32P contents. Nevertheless, counts for all shoot samples exceeded 95% con®dence levels and all but six root samples also met this criterion; four of these six samples satis®ed a 90% con®dence level as calculated following manufacturers recommendations (Appendix C: Statistics of Count Measurements. Operation Manual for Packard Instrument Company, UK Tri-Carb 1500 Liquid Scintillation Analyzer). Interplant transfer may be affected by `source' and `sink' relationships (Francis and Read, 1984). A larger receiver plant might be expected to act as a greater sink than a smaller one. In order to take plant size into account, plant weight data were used to calculate a transfer coef®cient ( 32P concentration in receiver plant/ 32P content in donor plant), which provided a less plant-weighted measure of 32P transfer. All plant dry weights were determined after drying to constant weight at 90 8C. The presence or absence of surface earthworm casts in the strip between donor and receiver plant pots of each replicate was recorded, as an indicator of earthworm activity in the interpot area.

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Table 1 Effect of AMF inoculation and earthworms on plant growth and AMF colonisation (all plant data g dry weight per pot. All percentage data were transformed p (% 1 1) for statistical analyses. Asterisks denote con®dence limits. Probability: *P ˆ 95%, **P ˆ 99% and ***P ˆ 99.9%) AMF inoculation 2

Earthworms

Interaction (P)

1

P

2

1

P

Donor plants at 14 weeks Total 2.04 Shoot 1.33 Root 0.71 Root/shoot ratio 0.60 % AMF 1

1.96 1.37 0.59 0.45 80

0.404 0.985 0.176 0.095 0.000***

1.66 1.02 0.64 0.64 41

2.34 1.66 0.68 0.41 40

0.007** 0.000*** 0.391 0.008** 0.388

0.667 0.434 0.518 0.422 0.881

Receiver plants at 18 weeks Total 4.13 Shoot 2.69 Root 1.45 Root/shoot ratio 0.55 % AMF 0

3.88 2.39 1.49 0.65 75

0.882 0.847 0.900 0.364 0.000***

3.22 1.95 1.27 0.66 39

4.61 3.13 1.67 0.53 38

0.051 0.061 0.016* 0.331 0.711

0.275 0.465 0.156 0.891 0.700

2.6.1. Mechanical disruption experiment It was not appropriate for mechanical disruption to be included in the factorial design, due to the harm this disruption would cause the earthworms. Instead a concurrent experiment was conducted, sharing NM and M treatments with the main experiment and also including a mechanically disrupted AMF treatment (MDis). In this treatment, the continuity of the hyphal compartment was completely broken, at weekly intervals throughout the 18 week long experiment, by means of one vertical movement of a taut, 1 mm diameter steel wire, parallel and equidistant (0.5 cm) to each of the two mesh windows (Fig. 1). This distance was chosen because it was within the range found to reduce AMF bene®t in a previous experiment (Tuffen, 2000, PhD Thesis, University of Wales, Aberystwyth). The MDis treatment was otherwise identical to the M treatment. Experimental details, including environmental conditions and duration, host plant, AMF inoculation treatments, and 32P labelling, were identical to those described for the earthworm/AMF experiment. Similarly, assessments described in Section 2.6 were made concurrently with this experiment. 2.7. Statistical analysis Minitab Version 13.0 (Minitab, PA, USA) was used for all statistical analyses. Four analyses were used: (i) Two-way analysis of variance on factorial data from the earthworm/AMF experiment. (ii) t-Tests to compare replicates with and without surface earthworm casts in the interpot area in the earthworm experiment. (iii) One-way analysis of variance on non-factorial data from the mechanical disruption experiment. (iv) Correlations to determine AMF±plant growth and AMF± 32P transfer relationships in both the earthworm/

AMF experiment and the mechanical disruption experiment.

3. Results 3.1. Earthworm/AMF experiment Plants were healthy and grew well. At the time of labelling, donor and receiver plants were of similar size. Receiver plant weights were approximately double those of donor plants in all treatments 4 weeks after the killing of labelled donor plants (Table 1). When root samples were assessed for AMF colonisation at 10 weeks prior to labelling, a mean AMF colonisation of 22% …SEM ˆ 5:6† root length was found in M and EwM treatments, whilst NM and Ew replicates were uncolonised. AMF colonisation increased rapidly between 10 and 14 weeks, but had stabilised between 14 and 18 weeks at .70%; both donor and receiver mycorrhizal (M and EwM) plants had similar, high levels of AMF colonisation at their ®nal harvests despite their 4 week age difference (Table 1). However, AMF inoculation had no signi®cant effect on donor or receiver plant growth, although donor root/shoot ratios were reduced by a third (Table 1). Upon dismantling units at the ®nal harvest, 58% …SEM ˆ 6:2† and 54% …SEM ˆ 5:6† of the original earthworm numbers were recovered in Ew and EwM treatments, respectively. At least some of the reduction in earthworm numbers was attributable to escape from growth pots. Donor total and shoot weights were increased, donor root/shoot ratio reduced, and receiver root weight increased in earthworm treatments (Table 1). Although earthworm effects on plant growth were greater in donor plants at 14 weeks than in receiver plants at 18 weeks, increases in receiver shoot and total weights with earthworms were close to signi®cance.

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Table 2 Effect of AMF inoculation and earthworms on 32P in plant material, and 32P transfer between donor and receiver plants (Bq: becquerels, Bq g 21: becquerels g 21 dry weight plant material, Trans. coeff.: transfer coef®cient. % Transfer ˆ (receiver 32P content/donor 32P activity) £ 100. Transfer coef®cient ˆ [ 32P concentration in receiver plant (Bq g 21)/ 32P content in donor plant (Bq)] £ 100. Asterisks denote con®dence limits. Probability: *P ˆ 95%, **P ˆ 99% and ***P ˆ 99.9%) AMF inoculation 2

Earthworms

Interaction (P)

1

P

2

1

P

2613 2366 247

0.970 0.588 0.094

2600 2224 376

2569 2397 173

0.851 0.199 0.034*

0.971 0.836 0.929

0.004** 0.000*** 0.016*

0.539 0.302 0.156

0.118 0.052 0.067

0.661 0.305 0.791

0.064 0.838 0.052 0.007** 0.231

0.325 0.411 0.690 0.143 0.490

32

Donor plant P content (Bq) Total 2556 Shoot 2255 Root 303 Receiver plant 32P content (Bq) Total 7.9 Shoot 6.2 Root 1.7 Donor plant 32P concentration (Bq g 21) Total 1314 Shoot 1868 Root 470 Receiver plant 32P concentration (Bq g 21) Total 1.9 Shoot 2.3 Root 1.1 % 32P transfer 0.31 Trans. coeff. 0.074

11.8 8.0 3.8 1361 1776 422 3.0 3.3 2.6 0.46 0.115

0.069 0.022* 0.092 0.608 0.388 0.267 0.059 0.187 0.021* 0.043* 0.132

AMF inoculation did not affect donor plant 32P contents or concentrations, but signi®cantly increased receiver shoot 32 P content and root 32P concentration (Table 2). AMF increased both % 32P transfer and the transfer coef®cient, but due to high variability only the former was statistically signi®cant. In earthworm treatments, donor root 32P content was reduced and receiver total, shoot and root 32P contents were increased (Table 2). Earthworm effects on 32P concentrations in donor and receiver plants were similar to those for contents, but differences were not statistically signi®cant. Both percentage 32P transfer and the transfer coef®cient were greater in earthworm treatments, but only the former was statistically signi®cant. Comparison of earthworm replicates with and without surface casts in the interpot zone revealed signi®cantly greater percentage 32P transfer and transfer coef®cients in replicates with surface casts (Table 3). There was no indication that earthworm activity in the interplant area was affected by AMF treatment, as surface casts were found in four and ®ve replicates in Ew and EwM treatments, respectively. Given the small number of full replicates without casting, comparisons of casting effects did not differentiate between mycorrhizal treatments. 3.2. Mechanical disruption experiment AMF inoculated donor and receiver plants attained high levels of AMF colonisation, and mechanical disruption had no signi®cant effect on these colonisation levels (Table 4).

6.8 5.0 1.8 1572 2198 597

12.9 9.1 3.8 1104 1447 295

2.1 2.6 1.4 0.26 0.081

2.8 3.0 2.3 0.50 0.109

Table 3 Comparison of 32P transfer between donor and receiver plants in presence or absence of surface earthworm casts in interplant area in Ew and EwM treatments (% 32P transfer ˆ (receiver 32P content/donor 32P activity) £ 100. Transfer coef®cient ˆ [ 32P concentration in receiver plant (Bq g 21)/ 32P content in donor plant (Bq)] £ 100. Bq ˆ becquerels. Asterisks denote con®dence limits. Probability: **P ˆ 99%. n ˆ 4 and 9 for 2/1 cast data, respectively. Data means are shown only for parameters where a signi®cant difference was found between 2 and 1 cast replicates)

% 32P transfer Transfer coef®cient

Casts absent

Casts present

0.32 0.066

0.76 0.164

P 0.008** 0.005**

Donor plant Total weight Shoot weight Root weight % AMF Total 32P content Shoot 32P content Root 32P content Total 32P concentration Shoot 32P concentration Root 32P concentration

2.78 1.94 0.84 41 3172 3017 215 1186 1608 289

2.08 1.50 0.58 50 2233 2194 150 1120 1467 275

0.060 0.085 0.073 0.728 0.219 0.296 0.247 0.843 0.764 0.907

Receiver plant Total weight Shoot weight Root weight % AMF Total 32P content Shoot 32P content Root 32P content Total 32P concentration Shoot 32P concentration Root 32P concentration

4.87 2.95 1.93 36 10 8 2 2 3 1

4.75 3.23 1.51 42 17 10 7 3 3 3

0.877 0.612 0.174 0.845 0.060 0.130 0.109 0.086 0.312 0.056

F. Tuffen et al. / Soil Biology & Biochemistry 34 (2002) 1027±1036 Table 4 Effect of AMF inoculation and mechanical disruption on plant growth and AMF colonisation (NM: control, M: AMF inoculated, MDis: AMF inoculated 1 mechanical disruption. All plant data g dry weight per pot. Within each column, common letters superceding data means indicate values which are not signi®cantly different (P # 0.05). LSDs are also shown because some differences between data means are close to signi®p cance (P # 0.05). All percentage data were transformed (% 1 1) for statistical analyses) NM

M

MDis

LSD

1.61a 0.92a 0.69a 0.75a 0b

1.70a 1.12a 0.58a 0.52b 81a

1.80a 1.05a 0.75a 0.71ab 75a

0.325 0.208 0.183 0.207 ±

Receiver plants at 18 weeks Total 3.24a Shoot 2.07a Root 1.18a Root/shoot ratio 0.57a % AMF colonisation 0b

3.19a 1.82a 1.36a 0.75a 75a

4.00a 2.60a 1.41a 0.54a 75a

1.400 1.430 0.331 0.408 ±

Donor plants at 14 weeks Total Shoot Root Root/shoot ratio % AMF colonisation

In donor plants, AMF inoculation tended to increase shoot growth and reduce root growth, re¯ected in the reduced root/shoot ratio in the M treatment compared with NM and MDis treatments. AMF inoculation had no effect on growth of receiver plants, which were harvested 4 weeks later than donor plants. AMF signi®cantly increased 32P content and concentration of receiver roots, but had no effect on 32P content and concentration of donor plants (Table 5). Although AMF increased the percentage 32P transfer between plants and the transfer coef®cient, high variability meant that neither of these effects was statistically signi®cant (Table 5). Mechanical disruption did not signi®cantly affect growth or AMF colonisation of donor or receiver plants (Table 4), but did affect 32P transfer. The general pattern was that 32P transfer was reduced in the MDis treatment compared with the M treatment, although still higher than the NM treatment (Table 5). These differences were signi®cant for 32P content and concentration of receiver roots, and close to signi®cance for the transfer coef®cient. 3.3. Relationships between plant growth or transfer indices and AMF colonisation There were no direct interactions between AMF and earthworms on plant growth, AMF colonisation or 32P transfer parameters (Table 1). In the absence of earthworms, plant weights tended to be correlated negatively with AMF colonisation, although these relationships were signi®cant at 14 weeks only within the MDis treatment and at 18 weeks only in the M treatment (Table 6). With earthworms, there was some evidence of a positive AMF±plant growth relationship, but this was signi®cant only for root weight at 18 weeks.

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Table 5 Effect of AMF inoculation and mechanical disruption on 32P in plant material and 32P transfer between donor and receiver plants (NM: control, M: AMF inoculated, MDis: AMF inoculated 1 mechanical disruption. % transfer ˆ (receiver 32P content/donor 32P activity) £ 100. Transfer coef®cient ˆ [ 32P concentration in receiver plant (Bq g 21)/ 32P content in donor plant (Bq)] £ 100. Bq ˆ becquerels. Within each column, common letters superceding data means indicate values which are not signi®cantly different (P # 0.05). LSDs are also shown because some differences between data means are close to signi®cance) NM

M

MDis

LSD

2633a 2257a 376a

2344a 2046a 298a

428.6 739.5 390.1

Donor plant 32P concentration (Bq g 21) Total 1594a 1549a Shoot 2380a 2015a Root 545a 648a

1302a 1949a 397a

713.3 609.7 307.9

Receiver plant 32P content (Bq) Total 5.2b Shoot 4.7a Root 0.5b

8.3a 5.3a 3.0a

7.4ab 5.8a 1.6ab

2.56 1.73 1.49

Receiver plant 32P concentration (Bq g 21) Total 1.6a 2.6a Shoot 2.3a 2.9a Root 0.5b 2.2a

1.8a 2.3a 1.1ab

1.02 1.74 1.26

% 32P transfer Transfer coef®cient

0.32a 0.077a

0.137 0.052

Donor plant 32P content (Bq) Total 2566a Shoot 2190a Root 376a

0.20a 0.062a

0.32a 0.099a

Table 6 Correlation coef®cients between donor and receiver plant growth parameters or 32P transfer indices and % AMF colonisation in AMF inoculated treatments (M: AMF inoculated, EwM: AMF inoculated 1 earthworms, MDis: AMF inoculated 1 mechanical disruption. % transfer ˆ (receiver 32 P content/donor 32P activity) £ 100. Correlation coef®cients are only shown where a signi®cant relationship between the parameter and levels of AMF colonisation was found in at least one of the subgroups. Asterisks denote con®dence limits. Probability: *P ˆ 95%, n ˆ 7) M Donor plants at 14 weeks Total plant dry weight Root dry weight Shoot dry weight % 32P transfer

20.466 20.713 20.102 0.815*

Receiver plants at 18 weeks Total plant dry weight 20.864* Root dry weight 0.248 Shoot dry weight 20.828* 0.385 % 32P transfer

EwM

MDis

0.187 0.128 0.136 0.356

20.788* 20.717 20.846* 0.133

0.174 0.788* 20.034 0.248

0.704 0.675 0.539 0.111

Percentage 32P transfer and AMF colonisation were positively correlated for donor plants only in the absence of earthworms and mechanical disruption (Table 6). Percentage 32P transfer was not signi®cantly related to receiver plant AMF colonisation in any treatment (Table 6).

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4. Discussion 4.1. Effects of AMF on plant growth and 32P transfer AMF had no direct effect on plant growth at either harvest (Table 1), even though leeks are considered highly responsive to mycorrhizal infection (Plenchette et al., 1983). In the present trial, mycorrhizal growth responses re¯ected the relative magnitudes of stimulatory effects of enhanced P supply (other nutrients were non-limiting) and detrimental effects of the fungal drain of the host plant's photosynthate (Harley, 1975). Although light levels may not have been optimal during later phases of the experiment, rapid plant growth and maintenance of high AMF root colonisation between 14 and 18 weeks suggests that plants were not C limited over this period; in any case, environmental conditions would not have affected relative treatment responses. Allocation of C to rapidly developing mycorrhizal structures between 10 and 14 weeks, giving high levels of root infection (mean infection rates 22% at 10 weeks and .70% at 14 weeks), probably offset any bene®ts for plant nutrition. Signi®cant negative correlations between plant growth and % root AMF infection (Table 6) at 14 weeks are consistent with mycorrhizal plants being at a net disadvantage under the prevailing experimental conditions. The only source of additional P in the hyphal compartment was from hay (total P 60 mg), available for uptake only when decomposed. Much of this P would have been beyond the ranges (4±9 cm) generally reported for hyphal extension (Camel et al., 1991; Friese and Allen, 1991). The more accessible interpot volume may have been rapidly depleted of P limiting the potential bene®t from AMF infection. Mechanical disruption of mycorrhizal plants had few signi®cant effects on plant indices, but did tend to produce an allocation of resources between root and shoot in such plants that was more typical of non-mycorrhizal plants (Table 4). Disruption would have destroyed the integrity of the extraradical AMF mycelium network, consistent with effects on 32P transfer (Table 5). Disruption appeared to strengthen the negative association between % root AMF infection and plant growth at 14 weeks (donor plants, Table 6), but not at 18 weeks (receiver plants, Table 6). At 14 weeks, disruption would have damaged a well-established hyphal network, whereas at 18 weeks prior disruption would have limited the extent of hyphal development. Repeated mechanical disruption may also have stimulated re-growth of active hyphae, which can senesce over periods of 5±8 weeks (Schubert et al., 1987). AMF increased 32P transfer to receiver plants by approximately a third in this study (Table 2); this effect did not result from AMF effects on plant size (Table 1) and associated changes in source±sink relationships (Francis and Read, 1984). The transfer effect was considerably less than the 2±4-fold increases reported by Newman and Eason (1989), probably because of the greater distance between plant roots. As a consequence, the experiment may not

have been sensitive to some of the less pronounced effects of treatments on transfer processes. Further investigations will aim to increase the 32P concentration of donor plants in order to increase the potential for transfer and for detecting earthworm effects on this transfer. Non-AMF mediated transfer must also have occurred, as 32P released from non-mycorrhizal donor root systems moved across the interpot zone for uptake by receiver root systems. Increases in receiver plant 32P contents and concentrations with AMF, were reduced to non-signi®cance when AMF was mechanically disrupted (Table 5). This suggests some direct interplant 32P transfer via AMF hyphae, since such links would have been most vulnerable to disruption. AMF-mediated release of 32P from the donor root system into the interpot zone, enabling increased interplant 32P transfer, would have been less susceptible to disruption. As hyphae can extend at rates between 0.8 and 3.5 cm week 21 (Mosse et al., 1981; Camel et al., 1991), the interval between disruptions would have been suf®cient for AMF recolonisation of the hyphal compartment. Transfer mediated by the AMF mycelium, other than via direct hyphal interlinkages, has been reported to contribute to total interplant resource transfer (Martins, 1993). In the present study, AMF-mediated 32P release from the dying donor root system was limited by infection rates whereas AMF-mediated 32P uptake by the receiver plant did not show any such relationship (Table 6). The absence of any similar correlation where AMF were disrupted indicates the importance of a viable, intact hyphal system, suggesting that hyphae acted as a conduit for 32P transfer between plant roots. Release of 32P from decomposing hyphae would presumably have been enhanced by disruption. 4.2. Effects of earthworms on plant growth and 32P transfer Earthworms increase plant growth (Lee, 1985; Baker et al., 1999) by enhancing nutrient availability and improving soil physical properties. In the present study, all nutrients other than P were non-limiting and earthworms could not affect directly physical conditions in the rhizosphere. Thus, plant responses to earthworms (Table 1) were almost certainly due to increased availability of P. Earthworm faeces are richer in extractable inorganic P than the parent material (Sharpley and Syers, 1977; Lee, 1985), a result of preferential ingestion of organic matter and increased microbial activity, but these faeces were not directly accessible by roots. Earthworms increased growth irrespective of the presence of AMF. Uptake of P, mineralised within the hyphal compartment, must have occurred with similar ef®ciency in non-mycorrhizal and mycorrhizal plant roots. The contribution of earthworm bodies to this P effect, given the reduction from input to recovered earthworm numbers, is likely to have been small (maximum potential input approximately 10 mg total P per pot). The positive effect of earthworms on plant growth was more consistent at 14 than at 18 weeks, perhaps because much of the P in the

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hyphal compartment became available for plant uptake prior to 14 weeks. Earthworm activity in the interpot area, as indicated by the presence of surface casting, did not affect plant growth at 14 or 18 weeks (Table 3); if anything, growth tended to be less where interpot casting occurred. Therefore, assuming casting activity between 14 and 18 weeks was representative of the overall trial period, activity speci®cally in the interpot area did not determine the general growth responses attributable to introduced earthworms. Earthworms increased 32P transfer (Table 2) but, in contrast to their effects on growth, surface casting in the interpot area appeared to enhance the transfer process (Table 3). As both the transfer coef®cient and the percentage 32 P transfer were increased, and presence of casts did not affect plant growth, any effects of earthworm activity on 32P distribution were not determined by associated effects on plant size. Overall, earthworm mediated increases in receiver plant 32P were in proportion to associated losses in donor root 32P (Table 2). Although earthworm activity in the interpot area did not signi®cantly affect donor root losses or other 32P indices, donor plant indices were generally lower in replicates where casting was observed. There are several possible explanations for these ®ndings. Increased 32P transfer may be an indirect effect of earthworms increasing microbial populations in the donor pot and interpot media. Microbial activity may have promoted losses of 32P from the donor root system and reduced the immobilisation of this 32P, thereby enhancing transfer between plants; P in earthworm excreta has increased solubility (Sharpley and Syers, 1977). The low organic matter contents of the growing media used in this study, may have accentuated the earthworm effect on microbial activity and associated nutrient turnover (Blair et al., 1994). If damage to the hyphal network by earthworms led to compensatory growth of younger, more active hyphae, this might have enhanced P transfer. There may also have been some direct transfer of 32P enriched materials as earthworms moved soil within the interpot zone. Overall, it appeared that earthworms encouraged a net mobilisation of 32P and facilitated source±sink processes. Initial earthworm densities in our study were comparable with high ®eld population densities (Baker et al., 1999) of a single, geophagous species; actual densities were probably lower for much of the experiment due to early escape or ongoing mortality. It is not clear whether species with different feeding strategies would have a similar effect. Earthworm-mediated increases in nutrient supply may be even more bene®cial to plants where earthworms and plant root systems occupy the same soil volume. Preferential plant root growth in zones of high earthworm activity (Springett and Gray, 1997) would further enhance these bene®cial effects. 4.3. AMF±earthworm interrelationships No signi®cant AMF±earthworm interaction effects on

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plant growth or 32P dynamics were found. This is consistent with the view that P made available by earthworms was equally accessible to roots and hyphae. However, it is likely that the bene®ts of increased availability of P and its absorption by AMF hyphae was offset by earthworm disruption to AMF mycelium. Earthworm activity in the interpot area may have damaged mycelium and this may offer part explanation of the tendency towards lower growth where such activity was observed. Earthworm effects on relationships between plant growth and AMF root colonisation levels provide some further evidence of potential interrelationships between AMF and earthworms. Earthworms either eliminated signi®cant negative AMF±plant growth relationships or resulted in signi®cant positive relationships (Table 6). Earthworms may have increased the potential for P uptake by AMF hyphae and therefore the bene®ts of high root infection. For receiver plants, these earthworm effects showed some similarity to those of mechanical disruption. As noted earlier, there is evidence of hyphal grazing by earthworms (Bonkowski et al., 2000), although this evidence relates to saprotrophic fungi. Earthworms also eliminated the signi®cant positive correlation between 32P transfer and AMF colonisation in donor plants (Table 6). It may be the case that processes favoured by an intact hyphal network are inhibited by earthworm disruption whereas those favoured by active hyphal growth are enhanced. Direct earthworm effects on fungal propagule distribution and on hyphal development within the root zone were precluded in our study. Both processes have been found to in¯uence AMF root colonisation rates (McIlveen and Cole, 1976; Pattinson et al., 1997). It may therefore be the case that some potential AMF±earthworm interactions were limited by the experimental design. 4.4. Conclusions Under the given experimental conditions, earthworms but not AMF enhanced plant growth. AMF and earthworms both enhanced 32P transfer, but these effects were minor and largely independent. Earthworms seemed to in¯uence growth and transfer responses in mycorrhizal plants to differing levels of AMF infection. The location of earthworm activity appeared to be important in the earthworm effect on 32P transfer but not plant growth. These results raise interesting questions about the relative importance to plants of earthworms and AMF, and their interactions, in nutrient acquisition and cycling. Acknowledgements This project was funded by a studentship from the Institute of Biological Sciences, University of Aberystwyth. Thanks also to Dafydd Rhys Jones for help with radioisotope work, and to John Toler for the care and maintenance of plants.

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