Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol14.Issue2.Apr2000:

(1)

The inconsistent effect of soil disturbance on colonization of roots by

arbuscular mycorrhizal fungi: a test of the inoculum density hypothesis

Terence P. McGonigle

∗

_{, Murray H. Miller}

Department of Land Resource Science, University of Guelph, Guelph, Canada N1G 2W1 Received 28 April 1999; accepted 30 December 1999

Abstract

Reducing the tillage of agricultural soils can increase early-season crop-P uptake. Consistent increases in plant-P have been found in both field- and laboratory-systems with undisturbed (U) compared to disturbed (D) soil. A concomitant stimulatory effect on colonization of roots in U soil by arbuscular mycorrhizal (AM) fungi has been found in some cases, but in others the colonization has been similar in U and D treatments. Disruption of the extraradical mycelium that remains from the previous crop is the mechanism by which soil disturbance restricts mycorrhizally mediated P uptake for the subsequent crop, with a tandem change in colonization not necessary, but sometimes seen. Nonetheless, a complete account of these processes will need an understanding of the conditions under which the extent of colonization is affected. Soil-P does not explain when a difference in colonization will appear. Among ecosystems in Western Australia, high inoculum density in a pasture was reported previously to preclude the appearance of a difference in colonization in response to soil disturbance, whereas for other ecosystems with lower inoculum densities a difference in colonization was seen. Here, we determined if a similar mechanism operates for an agricultural soil collected mid-season during the growth of a maize (Zea maysL.) crop in Ontario, Canada. Blending various proportions of pasteurized and non-pasteurized soil gave a range of inoculum densities. Maize was taken through two 3-week growth cycles in pots, and for the D treatment the soil was passed through a 5 mm sieve between cycles. All plants became colonized with AM fungi. Reducing the inoculum density served to limit colonization to similar low levels in both U and D soils. Stimulation of colonization and of shoot-P uptake in the U-compared with the D-treatment was greater for plants under the higher inoculum conditions tested. We conclude that the inoculum density during crop growth of the soil studied here is moderate, and that this density makes it possible, if other conditions are met, for a reduction of colonization of roots in response to soil disturbance. Whether or not a difference in colonization will appear following disturbance of a soil such as the one studied here probably depends on the interaction between the environment and the plant. Possible interactions are discussed. The high inoculum density of ecosystems such as the pasture studied in Australia likely overrides any effect of soil disturbance and ensures roots of all plants become well-colonized by AM fungi. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:Biomass carbon; Maize; Mycorrhiza; Pasteurization; Tillage; Undisturbed soil

∗_{Corresponding author. Present address: Faculty of Agriculture,}

Gifu University, Gifu 501-1193, Japan. Tel.: +81-58-293-2842; fax: 81-58-293-2842.

E-mail address:tmcgonig@cc.gifu-u.ac.jp (T.P. McGonigle)

1. Introduction

Reduced tillage has recently been adopted exten-sively for field crops in North America in order to conserve soil water and reduce soil erosion (Tisdale et al., 1993). Reduced tillage causes cooler conditions 0929-1393/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.

(2)

for seedlings because of increased residue cover, and it changes soil structure and soil organic matter (Blevins et al., 1985).

Tillage was shown in the 1980s to reduce colo-nization of roots by arbuscular mycorrhizal (AM) fungi (Mulligan et al., 1985) and early-season P up-take (O‘Halloran et al., 1986) by crops. Subsequent growth-chamber studies involving g-irradiation of soil (O‘Halloran et al., 1986), non-mycorrhizal crops (Evans and Miller, 1988), a fungicide (Evans and Miller, 1988), and soil pasteurization (McGonigle and Miller, 1996a) established that it is reduced ef-fectiveness of the AM symbiosis in disturbed (D), as compared to undisturbed (U) soil, that leads to reduced P uptake.

Greater P uptake from U than D soil has been consistent in four types of experimental systems. First, tillage experiments in the field (McGonigle and Miller, 1993, 1996b; Gavito and Miller, 1998). Sec-ond, growth-chamber studies with U cores collected from the field (Evans and Miller, 1988; Miller et al., 1995). Third, studies in pots using initially D soil that was taken through various growth cycles (Fairchild and Miller, 1988, 1990). Last, experiments with hy-phae developed in root-free soils within nylon mesh, which were then disturbed or not (Evans and Miller, 1990; Addy et al., 1994, 1997; McGonigle and Miller, 1999).

Soil disturbance affects plant P by breaking up the extraradical mycelium of the AM fungi that are asso-ciated with the roots of the previous crop (Jasper et al., 1989; Evans and Miller, 1990). Available data are consistent with the following interpretation. In U soil, the extraradical mycelium provides effective P acqui-sition as soon as roots become connected, but if this mycelium is disrupted a new one must then be de-veloped from colonized roots, which themselves must first be initiated by spores, or by fragments of either hyphae or colonized roots.

Colonization of roots by AM fungi is sometimes reduced for plants in the D soil, while in other exper-iments the U- and D-treatments have similar and rela-tively high colonization. Examples are as follows. At a single field site, colonization was reduced for maize in conventionally tilled plots compared to no-till plots in one study (McGonigle and Miller, 1996b) but not in another (McGonigle et al., 1990a). Soil-P levels could not explain this difference in outcome

(McGo-nigle and Miller, 1996b). U cores collected from one established sand dune had reduced colonization fol-lowing disturbance, but no difference was found for another site that was both close and similar (Koske and Gemma, 1997). With D soil initially, disturbance of soil between growth cycles in pots reduced coloniza-tion in some trials (Fairchild and Miller, 1988, 1990) but not others (McGonigle et al., 1990a; Miller and McGonigle, 1992). Again, soil-P was not the expla-nation (Fairchild and Miller, 1990). In another series of experiments, mesh pouches were used to produce root-free zones with hyphae either in situ in the field (McGonigle and Miller, 1999), or in growth rooms followed by burial in the field (Addy et al., 1994, 1997). Pouches were removed from the field at several times, and the colonization of bioassay plants grown in the pouch soil in the growth chamber was deter-mined. In one study (Addy et al., 1994), disturbance reduced colonization in a bioassay conducted prior to transferring pouches into the field in November, but not in those conducted on pouches collected from the field the following spring. In a subsequent experiment (Addy et al., 1997), disturbance reduced colonization of bioassay plants for pouches collected at all times from November of 1 year to May of the next. In a third experiment, (McGonigle and Miller, 1999), dis-turbance did not reduce colonization of bioassay plants in pouches collected in August, October, or May, but it did for those collected in April. Thus, month of the year for collection of pouches could not explain when it is possible for a difference in colonization to appear following disturbance.

A hypothesis that has been proposed to explain the conditional effects of disturbance on colonization is based on inoculum density. Jasper et al. (1991) raised subterraneum clover (Trifolium subterraneum L.) bioassay plants in U soil cores collected from various established ecosystems. Decreased coloniza-tion for bioassay plants was found after disturbance of soils taken from a forest and from a heathland, while colonization was high for bioassay plants in a soil from a pasture irrespective of whether the soil was U or D (Jasper et al., 1991). The interpretation made was that for the pasture a high inoculum density prevented a decrease in colonization following distur-bance (Jasper et al., 1991). Among experiments in our laboratory, variability in inoculum density may have occurred and therefore contributed to the inconsistent

(3)

effects of disturbance on colonization that have been found.

Tillage of soil can itself affect the inoculum den-sity in soil during the growth of the subsequent crop. The densities in soil of numbers of AM spores, and of lengths of structurally and metabolically stained hy-phae, was lower in the top 5 cm of plots in Quebec, Canada, following conventional tillage as compared to no-till (Kabir et al., 1998). No difference in coloniza-tion of maize roots in the field by AM fungi was found (Kabir et al., 1998). However, a difference in inoculum density following tillage may have led to a difference in colonization earlier in the growth season. Soil and roots were sampled during grain filling (Kabir et al., 1998), whereas differences for colonization of maize by AM fungi in response to tillage are typically seen only up to the six-leaf stage (McGonigle and Miller, 1993, 1996b).

Our aim was to evaluate the role that inoculum den-sity plays in determining the impact of disturbance on colonization of roots by AM fungi in agricultural soils. We used maize and the soil of the Elora Re-search Station in Ontario, Canada, which we have studied extensively. The hypothesis was as follows: colonization of roots in D soil will be reduced, rela-tive to that for plants sown in U soil, but only when the inoculum potential of the soil is low. On this basis, high inoculum should produce well-colonized roots in both U and D soils. The approach was to select a batch of soil for which a high inoculum density was expected, and to lower it by manipulation. Soil was collected mid-season from the rooting zone in a maize field. Plant growth and the colonization of roots by AM fungi after two cycles of growth in the laboratory were determined, with or without distur-bance between cycles. As an exploratory exercise, soil biomass-C was estimated for available samples using the fumigation–extraction technique, in order to see if it would reflect the behavior of the mycorrhizal fungi, and by doing so aid our understanding of mycorrhizal relationships.

2. Materials and methods 2.1. Preparation of soil

Soil was collected in early July 1995, from directly below plants in an actively growing maize field at the

Elora Research Station (43◦₃₁′_{N, 80}◦₁₄′_{W), Ont.,} Canada. This soil is a Conestogo silt loam (300 g kg−1 sand, 500 g kg−1_{silt, 200 g kg}−1_{clay), and it is} clas-sified as an Aquic Hapludalf (Ketcheson, 1980). The soil has a pH of 7.5 in a saturated soil–water paste. Soil was taken from the upper 15 cm of the profile and air-dried to approximately 0.18 g H2O g−1 dry soil. The soil was passed through a 2 cm sieve, using scissors to cut the roots as necessary, and mixing them back into the soil. This soil was then confirmed to have abundant colonized root on the basis of the following values (mean±S.D.): root-length density was 1.8±0.2 cm g−1; the proportions of root length colonized with arbuscules and hyphae (McGonigle et al., 1990b) were 43.1±_{8.6 and 67.1}±_14.5%,

re-spectively. This root length density and extent of colonization are close to the seasonal maximums for maize in this region (McGonigle and Miller, 1993).

The soil was mixed and divided into two parts, one of which was then pasteurized in an electrical soil-heating unit. Pasteurization involved heating the soil slowly to 80◦_{C, maintaining at this} tempera-ture for 2 h, and allowing it to cool. Pasteurized and non-pasteurized soils were analyzed using quadru-plicate sub-samples for extractable concentrations of macronutrients. The only differences (p_<0.05) between the nutrients of the two soils were for ex-tractable NH4–N and NO3–N. The pasteurized soil had NH4–N and NO3–N concentrations extracted with 2.0 M KCl of 33 and 24 mg kg−1_; correspond-ing values for the non-pasteurized soil were 4 and 18 mg kg−1_{. Overall values (means}_±_S.D.; _n₌_{8) for} other nutrients were as follows: P extracted in 0.5 M NaHCO3at pH 8.5 was 5.4±0.7 mg kg−1; using neu-tral 1.0 M ammonium acetate for the extraction, the soil had 75±13 mg K kg−1_{, 450}_±_{16 mg Mg kg}−1_, and 2.8±0.2 g Ca kg−1_.

The pasteurized and non-pasteurized fractions were then blended to give four inoculum-density treatments, which contained 10, 35, 65 or 90% of pasteurized soil by volume. The soil blends were fertilized with 50 mg N kg−1as KNO3powder, and 50 mg P kg−1as calcium monophosphate (Ca(H2PO4)2·H2O) powder. The soil blends were packed to a bulk density of 1.1 g dry soil cm−3_{in pots so that each pot had 4730 g dry} soil. The diameter of a pot at the soil surface was 20.5 cm, and the soil depth was 15 cm. A 300 g layer

(4)

of clean sand was added to the surface of all soils to restrict evaporation.

2.2. Plant growth

All pots were taken for two successive cycles of maize growth. Plants were kept in a growth chamber with a 17 h day at 28◦_{C, a 7 h night at} 23◦_{C, and with daytime irradiation of approximately} 400mmol cm−2s−1. Pots were watered by mass every 1 or 2 days to keep them close to but not exceeding 0.24 g H2O g−1dry soil.

The first growth cycle was as follows. After soaking for 48 h in aerated water, six maize seeds were planted per pot at a depth of 2 cm on 21 July 1995. Thinning was to three seedlings per pot at the two-leaf stage. The leaf-stage counting system used here excluded the spike leaf within the whorl. The shoots were cut at the soil surface and removed from all pots on 10 August 1995, when they were at the seven-leaf stage. The soil was then broken up by hand and passed through a 5 mm sieve to give the D treatment, or left intact to give the U treatment. In the D treatment, scissors were used to cut roots so they could pass through the 5 mm sieve, and the roots were mixed back into the soil. Disturbance was in three 5 cm depth fractions, which were handled separately and returned in order to each pot. For the second growth cycle, seeds were sown on 16 August 1995, and maize was grown in all pots as for the first growth cycle. While thinning at the two-leaf stage, root samples were taken by loosening gently the surface soil below a seedling and withdrawing the roots that were connected to the plants being removed. Root samples were taken from all pots at the three-, four-, and five-leaf stages using a 33 mm diameter soil corer, which was inserted midway between adjacent plants to the full pot depth. Holes made by coring at the three- and four-leaf stages were plugged using wooden dowels. Shoots were harvested at the five-leaf stage on 31 August 1995.

2.3. Laboratory measurements

All procedures refer to samples taken in the second growth cycle. Roots collected at the two-leaf stage were rinsed in tap water and stored in formyl-acetic alcohol (FAA), which consists of an 18:1:1 mixture of 95% ethanol, 28% w/v formaldehyde, and glacial

acetic acid. All soil cores were handled separately on their day of harvest as follows. The soil in each core was passed through an 8 mm sieve, cutting the roots as before. Each sample was well mixed, the fresh weight taken, and a 50 g fresh weight sub-sample was put to one side. All roots in the remainder of the sample were collected by rinsing against a 0.5 mm screen. The length of root was determined (Tennant, 1975) immediately, and the roots were then stored in FAA. The soil in the 50 g sub-samples was passed through a 2 mm sieve, brushing soil from the surfaces of roots and discarding those roots. The sieved soils were then stored in plastic bags for up to 3 days at 5◦C in the dark.

Shoots were rinsed in deionized water and then dried in a forced-air oven at 65◦_{C. Dried shoots were} weighed and then ground to a fine powder. Shoot-P and shoot-N concentrations were determined follow-ing the method of Thomas et al. (1967).

Stored roots were rinsed free of FAA and sub-sampled as necessary for assessment of coloniza-tion by AM fungi. A sub-sample was taken in each case by stirring roots in 3 l of water in a pitcher, and collecting the sub-sample in a 500 ml beaker plunged quickly in and out of the water. Roots were cleared by autoclaving in 10% KOH for 15 min. at 121◦_{C, and stained as described by Brundrett et al.} (1984). The percentage of root length with arbus-cules, which is the arbuscular colonization (AC), was determined as described by McGonigle et al. (1990b).

Stored 50 g soil samples at the three-, four-, and five-leaf stages were used for determination of moisture content and of soil biomass-C using the fumigation–extraction method (Vance et al., 1987) using an efficiency factor of 0.25 (Voroney et al., 1993) in the calculation.

2.4. Statistical design and analysis

Based on the soil batch used for handling dur-ing preparation, and based on position in the growth-chamber, the pots were divided into four blocked replicates in a randomized complete block design. Each replicate had eight pots, one for each combination of the four soil-blend treatments and the two soil-disturbance treatments. Data were evaluated using separate analyses of variance for each time,

(5)

with separation of means using Tukey’s test (Steel and Torrie, 1980).

3. Results

Shoot dry mass at the end of the experiment (Fig. 1a) was greater for plants in the U treatment compared to the D treatment at the two highest inoculum levels. Corresponding values for shoot-P concentration (Fig. 1b) were higher for plants in the U treatment at all in-oculum levels. Although there was no effect of inocu-lum density on shoot-N concentration (p=0.60) and no interaction with disturbance (p=0.27), shoot-N con-centration was higher (p_<0.001) at 29.4 mg g−1 _for plants in the U soil, compared to in the U soil where it was 25.9 mg g−1_.

Root-length density was not affected by inocu-lum density or soil disturbance. Overall values (mean±s.d.;n=32) at the three-, four-, and five-leaf

Fig. 1. Effect of soil disturbance on (a) shoot dry mass per plant and (b) shoot-P concentration at the five-leaf stage of the second growth cycle, in relation to the percentage of the soil blend that was pasteurized soil. Asterisks indicate where the members of a pair of means for the undisturbed (U) and disturbed (D) treatments were significantly different at the 0.05 probability level;n=4.

Fig. 2. Effect of soil disturbance on the percentage of root length with arbuscules for plants at the two-, three-, four-, and five-leaf stages in the second growth cycle, and in relation to the percentage of the soil blend that was pasteurized soil. Asterisks indicate where the members of a pair of means for the undisturbed (U) and disturbed (D) treatments were significantly different at the 0.05 probability level;n=4.

stages were 3.7±1.3, 4.1±1.3, and 6.3±0.9 cm−3_, respectively.

The inoculum density treatments successfully gen-erated a range of colonization (Fig. 2). Arbuscular col-onization was as high as 39 and 36% in the U treatment at the four- and five-leaf stages in the two soil blends with the highest inoculum density, but in the two soil blends with the most pasteurized soil the highest ar-buscular colonization reached was 16% (Fig. 2). In general, soil disturbance led to reduced levels of colo-nization, although some variability was found (Fig. 2). The more pronounced effects of disturbance on colo-nization were seen consistently under the higher, and not the lower, inoculum conditions (Fig. 2).

Fig. 3. Biomass carbon in soil following root dusting and removal at the three-, four-, and five-leaf stages in the second growth cycle, and in relation to the percentage of the soil blend that was pasteurized. Means have been averaged across disturbance treatments, for which there were no significant effects;n=8.

(6)

Soil biomass-C decreased in proportion with in-creases in content of pasteurized soil in the soil blends (Fig. 3), but it was not affected by soil disturbance. Considering each of the inoculum density treatments separately, soil biomass-C levels were similar at the three-, four-, and five-leaf stages (Fig. 3). For the data as in Fig. 3 but pooled across soil-blend treatments and times, extrapolation gave a biomass carbon value of 338 mg kg−1_{for non-pasteurized soil: biomass} car-bon (mg kg−1₎₌_{338–2.2107 pasteurized soil (%), with} r2=0.48 andn=96.

4. Discussion

Gi-gaspora margarita Becker and Hall and grew them

in a sub-soil in pots in the open for 6 months. Soils were then either broken up by hand or left intact, and pigeon pea (Cajanus cajunL. Huth.) was grown for 90 days. The root length colonized for plants in the uninoculated pots was not affected by soil distur-bance, with mean values of 16 and 17% in the U-and D-treatments, respectively. Thus, there must have been a low level of some indigenous AM fungi in

Table 1

Length densities in soil of roots colonized and not colonized by AM fungi for ecosystems studied in Western Australia (WA) and for the soil from Ontario (ON), Canada, as used here but prior to pasteurization

Site Land use Length density of root (cm 100 g−1_{dry soil)} _Reference

Not colonized Colonized

Jarrahdale, WA Jarrah forest 61 26 Jasper et al., 1991

Eneabba, WA Heathland 135 8 Jasper et al., 1991

Capel, WA Pasture 711 585 Jasper et al., 1991

Elora, ON Field crops 59 121 Present study

the sub-soil at the time of wheat planting, or some fungi must have invaded during the experiment. For the soils that were inoculated, colonization of plants in the U treatment was 56%, which was significantly higher than that of the plants in the D treatment at 41% (Yano et al., 1998). Thus, high inoculum density acted to facilitate, rather than obscure, the develop-ment of a difference in colonization in response to soil disturbance.

The effect of soil disturbance on shoot P and growth in our experiment was successively greater as the pro-portion of pasteurized soil was reduced. This pattern can be seen clearly in terms of the values correspond-ing to 90, 60, 35, and 10% pasteurized soil for the ratios of U/D of shoot-P content (mg per plant/mg per plant): namely, 1.45, 1.83, 3.34, and 3.66, respec-tively. Although at a relatively low level, colonization did develop in the treatment with 90% of pasteurized soil, and so an extraradical mycelium can be expected to have been present in all treatments at the end of the first growth cycle. In previous work with this soil, 100% pasteurized soil produced zero arbuscules and no response of shoot-P to soil disturbance (McGonigle and Miller, 1996a).

With the low inoculum densities of the treatments with 90 and 65% pasteurized soil used here, coloniza-tion was in most cases slight in both U and D soil. For the pasture studied by Jasper et al. (1991), in-oculum density was sufficiently high that AM fungi extensively colonized roots in both U and D soils, whereas for the heathland and forest it was lower, and reduced colonization of roots was seen in re-sponse to disturbance. Compared to the pasture and the other ecosystems of Jasper et al. (1991), the soil used here appears to have an intermediate inoculum density (Table 1). The appearance or not, following

(7)

distur-bance, of a difference in colonization of roots by AM fungi in the Elora soil with its intermediate inoculum density probably depends on the interaction between the environmental conditions and the type of plants and fungi involved.

It is plant-P status rather than soil-P level that is generally thought to have the greater regulating effect on colonization (Sanders, 1975; Menge et al., 1978). More specifically, there is evidence that the P status of the root may be the controlling factor (de Miranda et al., 1989; Braunberger et al., 1991). Although in-creases in plant-P status have generally resulted in de-creases in colonization, there are reports of inde-creases in colonization with increased shoot P when the ini-tial level is very low (Amijee et al., 1989; de Miranda et al., 1989). Such an effect could have played a part in the study we report here. The shoot-P concentra-tions for plants in the D soil here were consistently less than 1.0 mg g−1_{, while those for plants in the U} soil ranged from about 1.5 mg g−1 _{with the lowest} inoculum density, to more than 2.0 mg g−1 _{with the} highest. In this way, greater colonization in the U treat-ment at the two higher inoculum densities could have been related to relief from severe P shortage. While we cannot reject this explanation for the results of this study, it cannot explain the data of a related study that used the same soil as here. Fairchild and Miller (1990) observed a consistently greater colonization of roots by AM fungi in U compared to D soil, even though the shoot-P concentration in the plants in the D treatment increased from about 1.5 mg g−1_{to more} than 4.0 mg g−1 _{in response to P fertilizer. Averaged} across disturbance treatments, colonization decreased continually through this range of increasing P fertility (Fairchild and Miller, 1990).

The increase in shoot-N concentration in plants in the U treatment was small relative to those seen for shoot-P, and it was seen only as a main effect of dis-turbance. Thus, it is likely that this response of N was a secondary effect caused by improved plant-P nutrition.

Although any given isolate of AM fungus can be ex-pected to colonize any AM plant that it is challenged with in culture (Mosse et al., 1975), a picture has re-cently emerged of a degree of specificity in AM asso-ciations. Various combinations between a selection of AM fungi and rice varieties generated a wide range of extents of colonization of roots (Dhillion, 1992). From

among the pool of AM fungal species available in soil, certain combinations of fungus and plant were seen to develop more extensively than others in the mycor-rhizae of a grassland (McGonigle and Fitter, 1990) and a forest (Merryweather and Fitter, 1998b). Moreover, species of AM fungi differ in the ways in which they distribute their mycelia, both between the inside and outside of the roots (Abbott et al., 1992), and for the extraradical mycelium, with increasing distance from the rhizoplane (Jakobsen et al., 1992). In turn, different pairings of type of AM fungus and plant species can lead to widely different plant growth responses (van der Heijden et al., 1998). There is a good chance that different pairings of plant and AM fungus have oc-curred among the various treatments and experiments on soil disturbance conducted through the 1980s and 1990s in our laboratory and others. In addition, dif-ferences in types of colonist AM fungi may have con-tributed to the variability seen in effects of soil dis-turbance on extent of colonization. Certainly, differ-ent AM fungi simultaneously colonizing the same root systems can respond in different ways to soil distur-bance (Merryweather and Fitter, 1998a). However, as yet the role of the selection of different plant-fungus pairings in generating the variability seen in this ef-fect is not well understood. Indeed, in one experiment (Addy et al., 1994) the use of particular AM fungal inocula as the sole source of colonization did not elim-inate variability in the occurrence from one trial to the next of a difference in colonization between U- and D-treatments.

Values for biomass-C in the soil blend with the lowest proportion of pasteurized soil was typical com-pared to those for agricultural soils (Martens, 1995). Biomass-C was reduced in proportion to the amount of pasteurized soil, but it did not respond to soil dis-turbance, and it did not increase in association with a 70% increase of root-length density from the three- to the five-leaf stage in the second growth cycle. Even though AM hyphae and spores develop extensively in this soil even when pasteurized (McGonigle and Miller, 1999) and densities of hyphae in this soil can respond to disturbance (McGonigle and Miller, 1996a), biomass-C was unaffected by anything other than the initial pasteurization. It therefore appears that soil biomass-C reflects microbial populations in the bulk soil and not those associated with rhizo-sphere processes. Biomass-C measurement using the

(8)

fumigation-extraction method is not recommended for future studies on extraradical hyphae related to rhizosphere processes.

5. Conclusion

In contrast to previous work for established ecosys-tems (Jasper et al., 1991), higher inoculum density in an agricultural soil acted to facilitate rather that ob-scure a difference in colonization of roots by AM fungi between plants in U and D soils. Inoculum density may be important when it is the ability of the fungus to colonize that limits the extent of colonization, such as with low inoculum density, or when inoculum density is so high that other factors are overridden. When the plant regulates colonization, such as in a soil with in-oculum density that is moderate in broad terms as here, a range of colonization is possible. The appearance of a difference in colonization in response to disturbance of such soils depends on the interaction between the environmental conditions and the symbiotic partners involved. Future studies to try to reveal the factors that determine the extent a plant becomes colonized in U and D soils should consider the identity of the fungi.

Acknowledgements

Thanks to Ranee Pararajasingham for valuable tech-nical assistance. The Natural Sciences and Engineer-ing Research Council of Canada supported this work.

References

Abbott, L.K., Robson, A.D., Jasper, D.A., Gazey, C., 1992. What is the role of VA mycorrhizal hyphae in soil? In: Read, D.J., Lewis, D.H., Fitter A.H., Alexander, I.J. (Eds.), Mycorrhizas in Ecosystems. CAB International, Oxford, pp. 37–41. Addy, H.D., Miller, M.H., Peterson, R.L., 1997. Infectivity of the

propagules associated with extraradical mycelia of two AM fungi following winter freezing. New Phytol. 135, 745–753. Addy, H.D., Schaffer, G.F., Miller, M.H., Peterson, R.L., 1994.

Survival of the external mycelium of a VAM fungus in frozen soil over winter. Mycorrhiza 5, 1–5.

Amijee, F., Tinker, P.B., Stribley, D.P., 1989. The development of endomycorrhizal root systems. VII. A detailed study of effects of soil phosphorus on colonization. New Phytol. 111, 435–446. Blevins, R.L., Frye, W.W., Smith, M.S., 1985 The effects of conservation tillage on soil properties. In: D’Itri, F.M. (Ed.),

A Systems Approach to Conservation Tillage. Lewis, Chelsea, MI, pp. 99–110.

Braunberger, P.G., Miller, M.H., Peterson, R.L., 1991. Effect of phosphorus nutrition on morphological characteristics of vesicular–arbuscular mycorrhizal colonization of maize. New Phytol. 111, 435–446.

Brundrett, M.C., Piche, Y., Peterson, R.L., 1984. A new method for observing the morphology of vesicular–arbuscular mycorrhizae. Can. J. Bot. 62, 2128–2134.

de Miranda, J.C.C., Harris, P.J., Wild, A., 1989. Effects of soil and plant phosphorus concentrations on vesicular–arbuscular mycorrhiza in sorghum plants. New Phytol. 112, 405–410. Dhillion, S.S., 1992. Host-endophyte specificity of

vesicular–arbuscular mycorrhizal colonization ofOryza sativa L. at the pre-transplant stage in low or high phosphorus soil. Soil Biol. Biochem. 24, 405–411.

Evans, D.G., Miller, M.H., 1988. Vesicular–arbuscular mycorrhizas and the soil-disturbance-induced reduction of nutrient absorption in maize. I. Causal relations. New Phytol. 110, 67– 74.

Evans, D.G., Miller, M.H., 1990. The role of the external mycelial network in the effect of soil-disturbance on the vesicular–arbuscular mycorrhizal colonization of maize. New Phytol. 114, 65–71.

Fairchild, G.L., Miller, M.H., 1988. Vesicular–arbuscular mycorrhizas and the soil-disturbance-induced reduction of nutrient absorption in maize. II. Development of the effect. New Phytol. 110, 75–84.

Fairchild, G.L., Miller, M.H., 1990. Vesicular–arbuscular mycorrhizas and the soil-disturbance-induced reduction of nutrient absorption in maize. III. Influence of soil P amendments. New Phytol. 114, 641–650.

Gavito, M.E., Miller, M.H., 1998. Changes in mycorrhiza development in maize induced by crop management practices. Plant Soil 198, 185–192.

Jakobsen, I., Abbott, L.K., Robson, A.D., 1992. External hyphae of vesicular–arbuscular mycorrhizal fungi associated with Trifolium subterraneum. Spread of hyphae and phosphorus inflow into roots. New Phytol. 120, 371–380.

Jasper, D.A., Abbott, L.K., Robson, A.D., 1989. Soil disturbance reduces the infectivity of external hyphae of vesicular–arbuscular mycorrhizal fungi. New Phytol. 112, 93– 99.

Jasper, D.A., Abbott, L.K., Robson, A.D., 1991. The effect of soil disturbance on vesicular–arbuscular mycorrhizal fungi in soils from different vegetation types. New Phytol. 118, 471–476. Kabir, Z., O’Halloran, I.P., Widden, P., Hamel, C., 1998. Vertical

distribution of arbuscular mycorrhizal fungi under corn (Zea maysL.) in no-till and conventional tillage systems. Mycorrhiza 8, 53–55.

Ketcheson, J.W., 1980. Effect of tillage on fertilizer requirements for corn on a silt loam soil. Agron. J. 72, 540–542.

Koske, R.E., Gemma, J.N., 1997. Mycorrhizae and succession in plantings of beachgrass in sand dunes. Am. J. Bot. 84, 118–130. Martens, R., 1995. Current methods for measuring microbial biomass in soil: potentials and limitations. Biol. Fertil. Soils 19, 87–99.

(9)

McGonigle, T.P., Fitter, A.H., 1990. Ecological specificity of vesicular-arbuscular mycorrhizal associations. Mycol. Res. 94, 120–122.

McGonigle, T.P., Evans, D.G., Miller, M.H., 1990a. Effect of degree of soil disturbance on mycorrhizal colonization and phosphorus absorption by maize in growth chamber and field experiments. New Phytol. 116, 629–636.

McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990b. A new method which gives an objective measure of colonization of roots by vesicular–arbuscular mycorrhizal fungi. New Phytol. 115, 495–501.

McGonigle, T.P., Miller, M.H., 1993. Mycorrhizal development and phosphorus absorption in maize under conventional and reduced tillage. Soil Sci. Soc. Am. J. 57, 1002–1006. McGonigle, T.P., Miller, M.H., 1996a. Development of fungi below

ground in association with plants growing in disturbed and undisturbed soils. Soil Biol. Biochem. 28, 263–269.

McGonigle, T.P., Miller, M.H., 1996b. Mycorrhizae, phosphorus absorption and yield of maize in response to tillage. Soil Sci. Soc. Am. J. 60, 1856–1861.

McGonigle, T.P., Miller, M.H., 1999. Winter survival of extraradical hyphae and spores of arbuscular mycorrhizal fungi in the field. Appl. Soil Ecol. 12, 41–50.

Menge, J.A., Steirle, D., Bagyaraj, D.J., Johnson, F.L., Leonard, R.T., 1978. Phosphorus concentrations in plants responsible for inhibition of mycorrhizal infection. New Phytol. 80, 575– 578.

Merryweather, J., Fitter, A.H., 1998a. Patterns of arbuscular mycorrhiza colonisation of the roots of Hyacinthoides non-scripta after disruption of soil mycelium. Mycorrhiza 8, 87–91.

Merryweather, J., Fitter, A.H., 1998b. The arbuscular mycorrhiza ofHyacinthoides non-scripta. II. Seasonal and spatial patterns of fungal populations. New Phytol. 138, 131–142.

Miller, M.H., McGonigle, T.P., 1992. Soil disturbance and the effectiveness of arbuscular mycorrhizae in an agricultural ecosystem. In: Read, D.J., Lewis, D.H., Fitter A.H., Alexander, I.J. (Eds.), Mycorrhizas in Ecosystems. CAB International, Oxford, pp. 157–163.

Miller, M.H., McGonigle, T.P., Addy, H.D., 1995. Functional ecology of VA mycorrhizas as influenced by P fertilization

and tillage in an agricultural ecosystem. Crit. Rev. Biotech. 15, 241–255.

Mosse, B., 1975. Specificity in VA mycorrhizas. In: Sanders, F.E., Mosse, B., Tinker, P.B. (Eds.), Endomycorrhizas. Academic Press, London, pp. 469–484.

Mulligan, M.F., Smucker, A.J.M., Safir, G.F., 1985. Tillage modifications of dry edible bean root colonization by VAM fungi. Agron. J. 77, 140–144.

O‘Halloran, I.P., Miller, M.H., Arnold, G., 1986. Absorption of P by corn (Zea maysL.) as influenced by soil disturbance. Can. J. Soil Sci. 66, 287–302.

Sanders, F.E., 1975. The effect of foliar-applied phosphate on mycorrhizal infection of onions. In: Sanders, F.E., Mosse, B., Tinker, P.B. (Eds.), Endomycorrhizas. Academic Press, London, pp. 261–276.

Steel, R.G., Torrie, J.H., 1980. Principles and Procedures of Statistics. A Biometrical Approach, 2nd Edition. McGraw-Hill, New York, 633 pp.

Tennant, D., 1975. A test of a modified line intersect method of estimating root length. J. Ecol. 63, 995–1001.

Thomas, R.L., Sheard, R.W., Moyer, J.R., 1967. Comparison of conventional and automated procedures for nitrogen, phosphorus, and potassium analysis of plant material using a single digestion. Agron. J. 59, 240–243.

Tisdale, S.L., Nelson, W.L., Beaton, J.D., Havlin J.L., 1993. Soil Fertility and Fertilizers, 5th Edition. Macmillan, New York, 634 pp.

Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 19, 703–707.

van der Heijden, M.G.A., Klironomos, J.N., Ursic, M., Moutoglis, P., Streitwolf-Engel, R., Boller, T., Wiemken, A., Sanders, I.R., 1998. Mycorrhizal fungal diversity determines plant biodiversity ecosystem variability and productivity. Nature 396, 69–72. Voroney, R.P., Winter, J.P., Beyaert, R.P., 1993. Soil microbial

biomass C and N. In: Carter, M.R. (Ed.), Soil Sampling and Methods of Analysis. Lewis, Boca Raton, pp. 277–286. Yano, K., Yamauchi, A., Iijima, M., Kono, Y., 1998. Arbuscular

mycorrhizal formation in undisturbed soil counteracts compacted soil stress for pigeon pea. Appl. Soil Ecol. 10, 95– 102.

(1)

of clean sand was added to the surface of all soils to restrict evaporation.

2.2. Plant growth

All pots were taken for two successive cycles of maize growth. Plants were kept in a growth chamber with a 17 h day at 28◦_{C, a 7 h night at}

23◦_{C, and with daytime irradiation of approximately}

400mmol cm−2s−1. Pots were watered by mass every 1 or 2 days to keep them close to but not exceeding 0.24 g H2O g−1dry soil.

2.3. Laboratory measurements

Shoots were rinsed in deionized water and then dried in a forced-air oven at 65◦_{C. Dried shoots were}

weighed and then ground to a fine powder. Shoot-P and shoot-N concentrations were determined follow-ing the method of Thomas et al. (1967).

(1984). The percentage of root length with arbus-cules, which is the arbuscular colonization (AC), was determined as described by McGonigle et al. (1990b).

2.4. Statistical design and analysis

(2)

with separation of means using Tukey’s test (Steel and Torrie, 1980).

3. Results

Shoot dry mass at the end of the experiment (Fig. 1a) was greater for plants in the U treatment compared to the D treatment at the two highest inoculum levels. Corresponding values for shoot-P concentration (Fig. 1b) were higher for plants in the U treatment at all in-oculum levels. Although there was no effect of inocu-lum density on shoot-N concentration (p=0.60) and no interaction with disturbance (p=0.27), shoot-N con-centration was higher (p_<0.001) at 29.4 mg g−1 for plants in the U soil, compared to in the U soil where it was 25.9 mg g−1.

Root-length density was not affected by inocu-lum density or soil disturbance. Overall values (mean±s.d.;n=32) at the three-, four-, and five-leaf

stages were 3.7±1.3, 4.1±1.3, and 6.3±0.9 cm−3, respectively.

(3)

Soil biomass-C decreased in proportion with in-creases in content of pasteurized soil in the soil blends (Fig. 3), but it was not affected by soil disturbance. Considering each of the inoculum density treatments separately, soil biomass-C levels were similar at the three-, four-, and five-leaf stages (Fig. 3). For the data as in Fig. 3 but pooled across soil-blend treatments and times, extrapolation gave a biomass carbon value of 338 mg kg−1for non-pasteurized soil: biomass car-bon (mg kg−1)=338–2.2107 pasteurized soil (%), with

r2=0.48 andn=96.

4. Discussion

In contrast to the prediction of our hypothesis, the impact of soil disturbance on colonization was great-est under the highgreat-est of the inoculum densities tgreat-ested here. On the basis of our results we reject our hypoth-esis, and conclude that high inoculum density does not prevent a difference in colonization from occur-ring between U and D systems, at least for the soil studied here. The present study is in agreement with the data of Yano et al. (1998), who also reported a stronger impact of soil disturbance under conditions of higher inoculum density. Yano et al. (1998) inoc-ulated wheat (Triticum aestivum L.) plants with Gi-gaspora margarita Becker and Hall and grew them in a sub-soil in pots in the open for 6 months. Soils were then either broken up by hand or left intact, and pigeon pea (Cajanus cajunL. Huth.) was grown for 90 days. The root length colonized for plants in the uninoculated pots was not affected by soil distur-bance, with mean values of 16 and 17% in the U-and D-treatments, respectively. Thus, there must have been a low level of some indigenous AM fungi in

Table 1

Site Land use Length density of root (cm 100 g−1_{dry soil)} _Reference

Not colonized Colonized

Jarrahdale, WA Jarrah forest 61 26 Jasper et al., 1991

Eneabba, WA Heathland 135 8 Jasper et al., 1991

Capel, WA Pasture 711 585 Jasper et al., 1991

Elora, ON Field crops 59 121 Present study

(4)

It is plant-P status rather than soil-P level that is generally thought to have the greater regulating effect on colonization (Sanders, 1975; Menge et al., 1978). More specifically, there is evidence that the P status of the root may be the controlling factor (de Miranda et al., 1989; Braunberger et al., 1991). Although in-creases in plant-P status have generally resulted in de-creases in colonization, there are reports of inde-creases in colonization with increased shoot P when the ini-tial level is very low (Amijee et al., 1989; de Miranda et al., 1989). Such an effect could have played a part in the study we report here. The shoot-P concentra-tions for plants in the D soil here were consistently less than 1.0 mg g−1, while those for plants in the U soil ranged from about 1.5 mg g−1 with the lowest inoculum density, to more than 2.0 mg g−1 with the highest. In this way, greater colonization in the U treat-ment at the two higher inoculum densities could have been related to relief from severe P shortage. While we cannot reject this explanation for the results of this study, it cannot explain the data of a related study that used the same soil as here. Fairchild and Miller (1990) observed a consistently greater colonization of roots by AM fungi in U compared to D soil, even though the shoot-P concentration in the plants in the D treatment increased from about 1.5 mg g−1_{to more}

than 4.0 mg g−1 _{in response to P fertilizer. Averaged}

across disturbance treatments, colonization decreased continually through this range of increasing P fertility (Fairchild and Miller, 1990).

(5)

fumigation-extraction method is not recommended for future studies on extraradical hyphae related to rhizosphere processes.

5. Conclusion

Acknowledgements

Thanks to Ranee Pararajasingham for valuable tech-nical assistance. The Natural Sciences and Engineer-ing Research Council of Canada supported this work.

References

propagules associated with extraradical mycelia of two AM fungi following winter freezing. New Phytol. 135, 745–753. Addy, H.D., Schaffer, G.F., Miller, M.H., Peterson, R.L., 1994.

Survival of the external mycelium of a VAM fungus in frozen soil over winter. Mycorrhiza 5, 1–5.

A Systems Approach to Conservation Tillage. Lewis, Chelsea, MI, pp. 99–110.

Brundrett, M.C., Piche, Y., Peterson, R.L., 1984. A new method for observing the morphology of vesicular–arbuscular mycorrhizae. Can. J. Bot. 62, 2128–2134.

vesicular–arbuscular mycorrhizal colonization ofOryza sativa

L. at the pre-transplant stage in low or high phosphorus soil. Soil Biol. Biochem. 24, 405–411.

Evans, D.G., Miller, M.H., 1988. Vesicular–arbuscular mycorrhizas and the soil-disturbance-induced reduction of nutrient absorption in maize. I. Causal relations. New Phytol. 110, 67– 74.

Evans, D.G., Miller, M.H., 1990. The role of the external mycelial network in the effect of soil-disturbance on the vesicular–arbuscular mycorrhizal colonization of maize. New Phytol. 114, 65–71.

Gavito, M.E., Miller, M.H., 1998. Changes in mycorrhiza development in maize induced by crop management practices. Plant Soil 198, 185–192.

Jakobsen, I., Abbott, L.K., Robson, A.D., 1992. External hyphae of vesicular–arbuscular mycorrhizal fungi associated with

Trifolium subterraneum. Spread of hyphae and phosphorus inflow into roots. New Phytol. 120, 371–380.

Jasper, D.A., Abbott, L.K., Robson, A.D., 1989. Soil disturbance reduces the infectivity of external hyphae of vesicular–arbuscular mycorrhizal fungi. New Phytol. 112, 93– 99.

distribution of arbuscular mycorrhizal fungi under corn (Zea maysL.) in no-till and conventional tillage systems. Mycorrhiza 8, 53–55.