Impacts of tillage, cover crop, and nitrogen on populations of earthworms, microarthropods, and soil fungi in a cultivated fragile soil

Applied Soil Ecology 33 (2006) 243–257 www.elsevier.com/locate/apsoil

Impacts of tillage, cover crop, and nitrogen on populations of earthworms, microarthropods, and soil fungi in a cultivated fragile soil R.D. Reeleder a,*, J.J. Miller a, B.R. Ball Coelho a, R.C. Roy b a

Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ont., Canada N5V 4T3 b Agriculture and Agri-Food Canada, P.O. Box 186, Delhi, Ont., Canada N4B 2W9 Received 10 February 2005; accepted 25 October 2005

Abstract The impacts of tillage regime, cover crop, and nitrogen on various soil organisms inhabiting a fragile sandy soil (Brunosolic Gray Brown Luvisol) were determined. Soil samples were collected between 2000 and 2003 from a long-term tillage experiment, established in 1988 to determine the effect of tillage systems on yield of corn (Zea mays), soil quality, and weed populations. Populations of several of the soil organisms studied were significantly affected by one or more agronomic treatments. A single earthworm species, Aporrectodea turgida, was found in the experimental area. Worm populations were generally low and dominated by juveniles. Spring-sampled populations were significantly higher in no-till plots than in conventionally tilled plots. Fall-sampled populations were not affected as greatly by tillage, but were generally higher in no-till plots not receiving additional nitrogen or in plots overseeded with a rye (Secale cereale) cover crop. Soil microbial biomass, as represented by extractable soil DNA, was higher in the spring than in the fall. Populations of the soilborne stramenopile Pythium were generally higher in conventionally tilled plots, and were increased by a rye cover crop. Higher rates of nitrogen increased populations of total soil fungi but nitrogen had little effect on prostigmatid or cryptostigmatid mites; prostigmatid populations were generally higher in no-till plots. Spring populations of mesostigmatid mites were higher in plots with a rye cover crop than in plots without an overwintering plant cover. Conventional tillage stimulated populations of astigmatid mites during periods of high rainfall. Collembola populations were dominated by the families Onychiuridae and Isotomidae, but neither was greatly affected by any tillage treatment. Principal component analysis showed that populations of A. turgida and soil aggregation tended to be positively associated with one another, but that variations in populations of Onychiuridae springtails, prostigmatid mites, and Pythium tended not to be associated with changes in other variables. Overall, effects of tillage treatments on soil organisms were found to differ from previous reports in several respects, suggesting that soil type may impose conditions that over-ride the impacts of agronomic cultivation systems on populations of soil organisms. Crown Copyright # 2005 Published by Elsevier B.V. All rights reserved. Keywords: Soil DNA; Pythium; Aporrectodea; Acarina; Springtails

1. Introduction No-till systems are widely used in crop production and can be particularly valuable in fragile sandy soils * Corresponding author. Tel.: +1 519 457 1470x297; fax: +1 519 457 3997. E-mail address: [email protected] (R.D. Reeleder).

subject to wind and water erosion. Reduced tillage of these fragile soils has also been shown to improve soil structural stability (Ball Coelho et al., 2000). Combining reduced tillage with use of a cover crop that reduced nitrate leaching in sandy soils resulted in a sustainable system for continuous corn (Zea mays) on fragile soils (Ball Coelho and Roy, 1997; Ball Coelho et al., 2005). Relatively little work has been done with respect to the

0929-1393/$ – see front matter. Crown Copyright # 2005 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2005.10.006

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analysis of tillage impacts on soil organisms in sandy, low organic matter soils. Long-term mixed grass plots on a sandy soil were found to have substantial populations of microarthropods (approximately 140
103 individuals m2) but earthworm (Aporrectodea tuberculata) populations were barely detectable (Tomlin and Miller, 1987). As earthworms in particular are often associated with improved soil structure (Tomlin et al., 1995a), their response to changes in tillage practices in such earthworm-impoverished soils is of interest. Several researchers have explored the effects of tillage on soil organisms. In a review of several studies exploring the effects of tillage on earthworms, it was concluded that deep ploughing and intensive tilling generally reduced earthworm populations in clay loam soils but that effects of tillage in sandy loams were variable and dependent upon several factors including the earthworm species present in the soil (Chan, 2001). Conservation tillage practices that left crop residue on the soil surface tended to support higher densities of earthworms (Mele and Carter, 1999), soil fungi (Beare et al., 1993), phytopathogens such as stramenopile Pythium spp. (Pankhurst et al., 1995), and microarthropods (Miyazawa et al., 2002). By contrast, Wardle (1995) found that populations of several mesofaunal groups (e.g. enchytraeids and astigmatid mites) were increased following tillage operations. Cultivation of bromegrass (Bromus inermus) increased microarthropod populations (particularly Acari) when compared to corn production (Winter et al., 1990). Different tillage regimes had little effect on populations of soil actinomycetes (Zaitlin et al., 2004). Increasing the rate of applied N tended to suppress endogaeic earthworm populations, although various fertilizer formulations differed markedly in this respect (Ma et al., 1990). Intensive, high-input cropping systems were found to promote populations of those taxa of Collembola and Acari that fed on detritus or fungi, whereas low-input systems promoted phytophagous Collembolan groups (Larink, 1997). Various soil organisms have been shown to suppress or consume plant pathogenic fungi (Anas and Reeleder, 1987; Lartey et al., 1994; Maplestone and Campbell, 1989). Differential responses of soil organisms to various tillage systems suggest that populations of selected beneficial organisms can be enhanced via alterations in agronomic practice (Reeleder, 1992; Sturz et al., 1997; Peters et al., 2003). The introduction of new tillage practices may alter the composition of soil communities and thereby affect suppressiveness of soil

to plant pathogens. As populations of Pythium in these sandy soils can be high (Reeleder et al., 2002), the impact of cultural practices on this plant pathogen is of interest. Although a number of previous studies have documented the impacts of agronomic systems on various soil organisms, most studies have dealt with specific groups, such as microarthropods or earthworms in loam or clay soils. Relatively few studies have examined the responses of diverse groups of soil organisms to agronomic treatments and none have previously used multivariate analyses to examine the relationships between these diverse organisms and soil physical and chemical changes arising from agronomic treatments. The main objective of this study was to determine if population changes in earthworm, microarthropod, stramenopile, or soil fungi communities in a fragile sandy soil were associated with tillage or related agronomic factors, such as the rate of applied N and presence or absence of a cover crop. In addition, effects of agronomic treatments on microbial growth were determined by using extractable soil DNA as a measure of microbial biomass. Principal component analyses were used to show relationships between various physical, chemical, and biological parameters. 2. Materials and methods 2.1. Site information A continuous corn trial was established in 1988, at the Delhi, Ontario, research farm (428470 N, 808380 W) of Agriculture and Agri-Food Canada, on Fox loamy sand (Brunisolic Gray Brown Luvisol) containing 90% sand. The experimental area was arranged in a randomized split plot design with four replications. The two main treatments, conventional tillage (CT) and no-tillage (NT), were each divided into six split plot treatments consisting of a nitrogen rate (0, 50, 100, 125, 150, or 200 kg N ha1). Note that for the purposes of the study reported here only plots receiving 0, 100, or 200 kg N ha1 were used for sample collection. N was side-dressed as urea– ammonium–nitrate. Each of the 48 plots was 22 m in length
12 m wide. This experimental design was used for 5 years. In 1993 and subsequent years, a cover crop treatment (no cover (NC) or a fall-seeded winter rye (RC) (Secale cereale) seeded at 188 kg ha1) was introduced as a split–split plot factor. Rye was overseeded manually (broadcast) onto half of each split plot in August, a seeding date that minimizes competition with corn while allowing sufficient time

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Table 1 Field operation and sample collection dates Operation

2000

2001

Sample collection Plow (CT plots) Planting (maize) Sample collection Sample collection Rye (Secale cerale) seeded Sample collection Grain harvest Sample collection

25 April—earthworm/soil fauna 28 April, 1 May 12 May – – 1–2 August – 26 October 1 November—earthworm/soil fauna

– 25 April 5 May 16 May—earthworm/soil fauna 5 June—soil fungi/soil DNA 15 August 25 October—soil fungi/soil DNA 5 November 30 October—earthworm/soil fauna

Main and split treatment plots were first established in 1988; split–split treatments were established in 1993. Field operations dates for 2000 and 2001 only are shown. Sample dates for soil fungi in 2003 were 3 July and 4 November. Operation dates for previous years are available elsewhere (Ball Coelho and Roy, 1997; Ball Coelho et al., 2005).

for the rye to establish before winter. Relationships between agronomic operations and sample collection are provided in Table 1. Details on plot establishment and analytical methods are found elsewhere (Ball Coelho and Roy, 1997; Ball Coelho et al., 2005). Soil physical and chemical variables measured in these plots were used in certain analyses below. 2.2. Microarthropod extraction and estimations Arthropod populations were collected and processed as previously described (Fox et al., 1999; Tomlin et al., 1995b). A single sample, 5 cm in diameter and 15 cm deep, was obtained at each of four sampling dates (Table 1) from a random location in each split–split plot. Soil cores were kept intact and extraction of ambulatory arthropods from soil commenced within 24 h, using modified Tullgren funnels. Extractions were terminated after 72 h and arthropods were held in vials with 70% alcohol for identification. Acari were identified to the sub-order level and Collembola to family level. 2.3. Earthworms Earthworm populations were assessed using a formaldehyde extraction technique, as previously described (Tomlin and Miller, 1987), in the fall and spring of 2000 and 2001 (Table 1). A 0.36-m2 quadrat was located randomly in each split–split plot at each sampling date. Worms that surfaced following application of formaldehyde within the quadrat were collected and preserved for later identification. 2.4. Fungi ‘‘Total’’ fungi in soil were determined using an agar medium (rose bengal agar; RBA) that supports

the growth of a number of fungal genera found in soil (Beare et al., 1993). This medium is, however, not useful for quantifying the stramenopile genus Pythium; therefore a selective agar medium (P5ARP; Jeffers and Martin, 1986)) was used for this taxon. Pythium contains a number of plant pathogenic species and many of these are able to rapidly colonize green plant material once incorporated into soil. Although stramenopiles are not true fungi, they have many characteristics of this group (Money, 1998). 2.4.1. Soil dilutions Fungal populations were determined in the spring and fall of 2001 and 2003. Ten soil cores (2 cm
10 cm) were collected from each split–split plot, using a ‘W’ sampling pattern. Cores from each plot were thoroughly mixed; three 10-g subsamples were removed and dried at 104 C for 3 days to determine soil moisture. Another 10-g sample was removed and mixed with 40 ml of a diluent (0.25% (w/ v) water agar) using a magnetic stir bar. Awide-tip pipet was used to remove 10 ml of the dilution, which was then added to an additional 90 ml of diluent. This last step was repeated resulting in a range of dilutions (1 in 5 to 1 in 1000). Selected dilutions were used to seed previously prepared plates of semi-selective media (P5ARP and RBA, respectively). Three plates of each medium were seeded for each plot at each sampling date (spring and fall 2001, 2003). Seeded P5ARP plates (1 in 50 dilution) were incubated in the dark at ambient room temperature (20  2 8C). Numbers of colonies per plate were determined after 2 days incubation. Seeded RBA plates (1 in 500 or 1 in 1000 dilution) were incubated for 2 days in the dark at room temperature then for an additional 8 days under black light prior to recording numbers of colonies. Colony counts were

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adjusted using soil moisture data and expressed as propagules of fungi per gram dry soil. Means of the colonies observed on the three plates per plot were used in all analyses.

precipitated and resuspended in buffer. After final purification using guanidine isothiocyanate (Wizard DNA Clean-up System, Promega, Madison, WI), DNA was quantified by fluorometry and expressed as mg g1 dry soil.

2.5. Soil DNA extractions 2.6. Statistical analyses Extractions of soil DNA were made as previously described (Reeleder et al., 2003) and resulting data were used as a measure of microbial biomass. Total extractable DNA per gram of soil was determined for each plot during the spring and fall of 2001. In brief, 5 g of soil from samples collected for estimation of fungal populations (see above) was added to a plastic 50 ml tube containing 5 ml of buffered extraction solution and 3.5 ml of 1.0 mm zirconium oxide beads. Tubes were shaken mechanically for 20 min, and then centrifuged to remove soil particles from the extraction solution. DNA in the extraction solution was then

Effects of treatments and their interactions on the biological variables were determined using Proc Mixed (SAS v. 8.02). Sampling date effects were found to be highly significant for all variables and subsequent analyses were carried out by date. Principal component analysis (PCA; XLStat-Pro v. 7.5) was used to determine relationships among the available variables. Input data included soil physical variables obtained from previous studies on this site (Ball Coelho et al., 2005), as well as the biological variables described above.

Table 2 Summary of significant main treatment effects and their interactions with respect to earthworm numbers and biomass, Pythium and total fungal populations, and extractable soil DNA Taxon

Date

Aporrectodea turgida b

F 00 S 01 F 01

Total number

Adult numberb

b

Juvenile number

Biomassc

Pythium spp.

Total fungi

Soil DNA a b c

S 00

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

01 01 03 03

Tillage a

Cover

0.0509

0.0605 0.0092

T
N

T
C

0.0359 0.0509

0.0605

N
C

T
N
C

0.0497

0.0494

0.0388

0.0651

0.0002 0.0010

0.0652 0.0757

0.0144

0.0596

0.0051

0.0405

0.0001 0.0001

0.0242

0.0002

0.0273

0.0678 0.0317

0.0015

0.0612 0.0212

0.0478 0.0242

0.0082

0.0612

0.0409 0.0273

0.0025 0.0042

0.0658

F 01 S 03 F 03 S 01 F 01

N rate

0.0055 0.0551 0.0191 0.0001

0.0088 0.0017

0.0613 0.0096 0.0852

0.0001

0.0760

0.0423

S 00, spring 2000; F 00, fall 2000; S 01, spring 2001; F 01, fall 2001; S 03, spring 2003; F 03, fall 2003. Number of individuals per m2. Worm biomass; g fresh wt per m2.

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3. Results and discussion 3.1. Earthworms Tillage was the dominant factor affecting earthworm populations (Table 2). Aporrectodea turgida was the only species found in the experimental area. The similar species A. tuberculata was reported previously as the only earthworm in a site immediately adjacent to the experimental area used in this study (Tomlin and Miller, 1987). Overall, populations were low, as expected in a coarse-textured, low-organic-matter soil. When the different treatments were compared with respect to the total number of worms extracted, tillage was found to provide the dominant effect during spring sampling periods (2000 and 2001), with no-till (NT) having higher populations than conventional tillage (CT) (Table 2; Fig. 1). Populations during spring sampling

Fig. 1. Populations of Aporrectodea turgida in conventional tillage (CT) and no-till (NT) plots during spring sampling periods. (A) 2000 and (B) 2001. Standard error bars are shown. P values are given for CT/NT treatment comparisons for numbers of juvenile, adult, and total worms extracted.

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were lower in CT plots irrespective of whether worms were extracted before (2000) or after (2001) spring ploughing operations. Even in NT plots, however, earthworm populations were found to be low (<15 individuals m2) when compared to those obtained in the spring from heavier-textured (loam) cultivated soils (Kladivko et al., 1997). There, 11 of 14 sites sampled had total populations more than double the maximum found here. Fall sampling generally yielded higher populations than spring sampling; however the main tillage effect was not significant in the fall sample of either year (Table 2). The mean population (total individuals) recovered in 2000 was 27.6 worms m2 from NT plots and 21.1 m2 from CT plots (P = 0.5746); corresponding values for 2001 were 28.6 and 17.4, respectively (P = 0.4124). In fall 2000, there was a tillage
N rate interaction: within NT, the lowest rate of N (0 kg ha1) resulted in significantly higher total worm populations (37.5 worms m2) than those found in CT plots receiving 0 kg N ha1 (13.2 worms m2). By contrast, higher rates of N did not differentially affect populations in CT or NT plots. Previously, it had been reported that, in turf plots harrowed once per year, low N rates supported higher populations of endogaeic earthworms (such as Aporrectodea spp.) for four of five N formulations tested (Ma et al., 1990). In fall 2001, rye cover (RC) treatments resulted in significantly higher (P = 0.0092) populations (33.1 worms m2) than found in no cover (NC) plots (12.8 worms m2). Precipitation was much higher (789 mm) during the 2000 growing season (April– September) than during 2001 (352 mm). In 2001, water availability was more limited; in CT plots, water-holding capacity was greater with RC than NC, and soil water content was higher in RC plots than in NC plots of both tillage systems (Ball Coelho et al., 2005). Worm survival and growth may have been favored in RC plots in 2001 due to increased availability of water in this treatment. The earthworm population was dominated by juveniles; for example, they contributed over 80% of the total number in spring 2000 and over 90% in spring 2001. As a result, earthworm biomass generally followed the same treatment response as juvenile populations (Table 2). Treatment effects on adults were less marked (no significant effects in spring 2000 or 2001), however, experimental effects may have been masked by the overall low number of adults present. As this experimental plot was established in 1988, the population data suggest that, while NT stimulates successful reproduction in earthworms, this low organic matter soil does not support high adult

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Table 3 Summary of significant main treatment effects and their interactions with respect to populations of Acari, Collembola and total fauna Taxon

Date

Acari (suborder) Prostigmata

Cryptostigmata

Astigmata

Mesostigmata

Total Acari

S F S F

00 a 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

Tillage

N rate

0.0143

0.0878

Cover

T
N

T
C

0.0001

N
C

T
N
C

0.0083 0.0301 0.0019

0.0859

0.0642 0.0200

0.0067 0.0761

0.0383 0.0884

0.0533

0.0446

0.0069

0.0662

0.0016

0.0133 0.0428 0.0025 0.0333 0.0414

0.0143 0.0654 0.0076

0.0971 0.0625

0.0076

0.0315 0.0982

0.0531

0.0064

b

Collembola (family) Onychiuridae

Isotomidae

Total collembolac

Total soil fauna

d

0.0157 0.0444 0.0732 0.0529 0.0279 0.0683 0.0174 0.0250 0.0823

0.0945 0.0009 0.0835

0.0656

0.0721 0.0517

0.0908 0.0898

0.0031

a

S 00, spring 2000; F 00, fall 2000; S 01, spring 2001; F 01, fall 2001; S 03, spring 2003; F 03, fall 2003. Data for families Poduridae, Entomobridae, and Sminthuridae are not shown as, overall, they contributed only 1% to the total Collembola population. c Data includes Onychiuridae, Isotomidae, Poduridae, Entomobridae, and Sminthuridae. d All fauna collected from Tullgren extraction funnels. Apart from Acari and Collembola (97% of individuals recovered), Diptera, Symphyla, Coleoptera, and Nematoda were, in decreasing order, the most common taxa recovered. b

populations regardless of the tillage system or cover crop used. Adults and juveniles may migrate into CT plots from adjacent NT plots, however, the mechanical disruption caused by tillage operations likely causes the worms to devote more energy to burrow construction than to growth and reproduction (Mele and Carter, 1999).

Earthworm populations in most North American cultivated soils are dominated by introduced species such as A. turgida (Reynolds, 1977). Introduction of species adapted to coarse-textured sandy soils may be a useful approach where more substantial earthworm populations are desired (Kladivko et al., 1997), and where data suggest that introduction will result in

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improved soil properties (Marashi and Scullion, 2003). However, it is clear from the work reported here that certain soil types will not support large worm populations regardless of the tillage system used. 3.2. Microarthropods Soil fauna populations were dominated by soil mites and springtails; they provided over 97% of the extractable soil fauna in these plots. There were few consistently significant (P < 0.10) treatment or interaction effects on total soil fauna (Table 3); however, RC plots supported significantly higher (P = 0.0009) fauna populations in spring 2000 than did NC plots. At other sampling periods, RC populations were numerically but not significantly superior. 3.2.1. Mites (Acari) Tillage and cover crop were the most important factors affecting mite populations in soil (Table 3). Total mite population was most affected by cover crop. For the first three sampling dates (spring 2000, fall 2000 and spring 2001), RC plots had significantly elevated populations compared to NC (Table 3; Fig. 2). The same trend (but non-significant) was present at the fourth sampling date. Mite populations were dominated by prostigmatid and oribatid (Cryptostigmata) mites; overall, they contributed over 70% of the population. Tillage, both as a main effect and in interactions with cover, provided most of the significant (P < 0.10) effects on individual suborders of mites (Table 3). Populations of prostigmatid mites were significantly

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increased in NT plots at two of four dates, and were numerically greater on the remaining dates. Cryptostigmatid mites were less affected by tillage; NT significantly increased populations on only one date. Where tillage
cover interactions were significant, RC stimulated populations of both suborders but only in CT plots. Presumably, in NT plots, populations were at a maximum for this soil type and therefore the rye cover crop had no additional effect. When the effects of various treatments on the proportion contributed to total mite populations by each of these two suborders were examined, it was found that, while tillage and cover effects were common, N rate had no effect on the percentage contributed by either suborder (Table 4). The proportion of total mite populations contributed by prostigmatids was significantly higher in NT than CT plots on three of four sampling dates, and was numerically superior on the remaining date. The percentage data also indicate that oribatid mites are less affected by tillage in low organic matter soils. It can be concluded that NT favors prostigmatids and that oribatid mites do not respond significantly to tillage. Neither group responded often to N rate as a main effect but significant responses to T
N or N
C were found on most sampling dates. Previous work on a clay loam soil suggested that tillage had little impact on either of these groups (Fox et al., 1999); however, studies on a volcanic ash soil indicated that reduced tillage (in combination with manure) favored total mite populations but that cover crop had little effect (Miyazawa et al., 2002). NT corn was found to increase populations of cryptostigmatids but not prostigmatids when

Fig. 2. Total Acari (mite) populations obtained using modified Tullgren funnel extractors over four sampling periods (2000–2001) from plots with (RC) or without (NC) a rye (Secale cereale) cover crop. Standard error bars are shown. P values are given for CT/NT treatment comparisons at each sampling period.

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Table 4 Summary of significant main treatment effects and their interactions with respect to percentage distribution of populations of Acari suborders and families of Collembolaa Taxon

Date

Acari (suborder) Prostigmata

Cryptostigmata

Astigmata

Mesostigmata

S F S F

00b 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

S F S F

00 00 01 01

Tillage

N rate

0.0156

Cover

T
N

T
C

0.0022

N
C

T
N
C

0.0022 0.0802

0.0251 0.0005

0.0199 0.0603 0.0104

0.0343

0.0360 0.0427

0.0513

0.0245 0.0254 0.0022 0.0329 0.0570 0.0570 0.0119

0.0070

0.0291

c

Collembola (family) Onychiuridae

Isotomidae

0.0825

0.0601

0.0257 0.0389

0.0026 0.0453 0.0277 0.0017

a

Percentages of total mite or total Collembola populations provided by each suborder or family were used in data analysis. S 00, spring 2000; F 00, fall 2000; S 01, spring 2001; F 01, fall 2001; S 03, spring 2003; F 03, fall 2003. c Data for families Poduridae, Entomobridae, and Sminthuridae are not shown as, overall, they contributed only 1% to the total Collembola population. b

compared to CT corn in a silt loam (Winter et al., 1990). No previous studies have examined interactions of N rate and tillage treatment with respect to mite populations. Astigmatid mites are reported to increase in response to tillage (House and Parmalee, 1985; Wardle, 1995); we found that this group of mites contributed less than 15% of the mite population overall and that tillage stimulated their populations on two of four sampling dates (Table 3). CT plots had higher astigmatid mite populations during the fall 2000 and spring 2001 samplings but tillage had no effect on populations in spring 2000 or fall 2001. The higher populations in fall 2000 and spring 2001 may have been partly a result of higher than normal rainfall during 2000. In years of higher rainfall, corn yield was greater in CT plots than NT and this may have resulted in increased food supplies for astigmatid mites (Ball Coelho et al., 2005). Astigmatid mites provided as much as 40% of the total

mite population in CT plots on these dates; astigmatids may therefore be favored by rainfall in low organic matter soils. By contrast, presence or absence of a rye cover crop was not a significant factor with respect to astigmatid mite populations. Mesostigmatid mites (12.9% overall of total mites extracted) responded quite differently to agronomic treatments than other suborders. These mites were rarely affected by tillage but cover crop had significant effects (Table 3). RC increased mesostigmatid populations on both spring sampling dates and, on both fall samplings, 0 kg N ha1 resulted in higher populations than 200 kg N ha1 in CT plots. These increased populations generally were reflected as higher percentages of mesostigmatids in total mite populations in these plots (Table 4). By contrast, N rate had no impact on populations in NT plots. Note that, for earthworms, low N rates within NT plots stimulated populations. The astigmatid and mesostigmatid suborder are both

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comprised mainly of predacious species; however, these groups nonetheless appear to respond differently to the agronomic treatments used here. Mesostigmatids were also more affected by the rate of applied nitrogen and cover crop than the dominant suborders Prostigmata and Cryptostigmata. No previous reports have documented the impacts of cover crop and nitrogen on mesostigmatids.

(Tomlin and Miller, 1987). When data of Tomlin and Miller (1987) from adjacent forest soil (as opposed to soil + litter layer) are examined, microarthropod populations are also found to be similar to those of the CT and NT plots reported here. Overall, the effects of cultivation on soil microarthropod populations are likely smaller than the effects of soil chemical and physical properties on these organisms.

3.2.2. Springtails (Collembola) There were few significant treatment effects on springtail populations and no clear trends (Table 2). In spring 2000, RC supported higher populations than NC. In spring 2001, an interaction between tillage and N rate was the only significant effect; within CT, the highest N rate supported the highest population. Overall, the families Onychiuridae and Isotomidae contributed 99% of the springtail population. Previous reports have documented increased populations of springtails under conservation tillage treatments in volcanic (Miyazawa et al., 2002) and silt loam soils (Winter et al., 1990). The data reported here indicate that this effect may not occur in sandy soils. Data reported here indicate that microarthropod populations in NT and CT plots are similar to those previously reported from adjacent long-term grass plots

3.3. Soil fungi 3.3.1. Pythium Populations of this stramenopile fungus-like organism were affected by agronomic treatment. Tillage
cover interactions were highly significant in three of four sampling periods (Table 2). Populations in CT plots generally were higher than in NT; however, the rye cover crop suppressed Pythium populations in NT yet stimulated populations in CT (Fig. 3). This latter effect might be expected since the incorporation of green plant tissue following disking of the rye cover crop in CT/RC plots would provide additional food supplies for organisms such as Pythium that are known to readily colonize such tissue (Garrett, 1970). Plough operations in CT systems may increase availability of simple carbohydrates utilizable by Pythium spp. and thereby

Fig. 3. Pythium populations as estimated by enumerating colonies arising on a selective agar medium (P5ARP). Each colony was assumed to have arisen from one propagule. Data from tillage/cover crop split plots are shown for each of four sampling periods. NT, no-till; CT, conventional tillage; RC, rye (Secale cereale) cover crop; NC, no cover crop. Standard error bars are shown. P values indicate the level of significance of split plot treatment differences at each sampling period.

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stimulate their populations. Nonetheless, previous studies found that Pythium populations were higher under NT than CT in red-brown clay soils (Pankhurst et al., 1995) and that incorporation of rye into loam soil suppressed Pythium populations (Lumsden et al., 1976). Impacts of cultivation systems on Pythium may be dependent upon soil type and climate. On sandy soils, a less perturbed NT environment may not have been as favorable to Pythium as a tilled soil; the combination of a large supply of simple carbohydrates with relatively few competitors would be less likely to be available under NT. NT resulted in a denser soil structure, which restricted root growth; as a result, uptake of nutrients by plants was likely reduced. Young, rapidly growing root tissue is generally regarded as being more susceptible to invasion by Pythium. Where root growth is restricted, proportionately less of the root biomass may be susceptible to invasion by Pythium; consequently, growth of Pythium is reduced due to reduced access to nutrients. In spring 2001, the highest N rate significantly (P = 0.0015) suppressed Pythium populations but this effect was not observed at subsequent sampling dates (Table 2). As a result of frequent periods of rain in 2000, yields were greatest at N200 in 2000 (Ball Coelho et al., 2005). This would result in greater crop residue incorporation in these plots that fall, since residue yields are proportional to grain yields. Higher residue (dried plant material) incorporation may have encouraged Pythium competitors or consumers. Dried plant

material would be expected to be less stimulatory to Pythium populations than green tissue (Garrett, 1970). 3.3.2. Total fungi Populations of all fungi, as estimated by the RBA medium, were also affected by treatment. However, the response was more straightforward than that of Pythium spp. In two of three sampling periods there was a significant increase in population as the amount of applied N was increased (Table 2; Fig. 4). A similar but non-significant trend was observed at the remaining sampling period. Increased N might have direct and indirect impacts on soil fungi. Higher plant biomass resulting from increased N (from both larger root systems and more residues being incorporated at the end of the growing season) would provide more food resources for fungi. As the N was soil-applied, a portion of it may have been taken up directly by fungi or other microorganisms and utilized in support of crop residue colonization (Dighton and Boddy, 1989). Such colonization would result in increased fungal populations. It should be noted, however, that the populations reported here reflect colonies arising on agar media; these may arise from spores or from viable mycelial fragments produced during the soil dilution procedure. Fungi that produce few or no spores in soil, that do not produce fragmentable hyphae, and that do not grow well on RBA will be under-represented in these assays. Alternative methods (Morgan et al., 1991; Olsson et al., 1995) of assessing fungal populations or biomass may have given

Fig. 4. Total soil fungi populations as estimated by enumerating colonies arising on rose bengal agar (RBA). Each colony was assumed to have arisen from one propagule. Data from plots receiving different amounts of nitrogen (N) are shown for each of three sampling periods (no assay was done in spring 2000). Standard error bars are shown. P values indicate the level of significance of N rate treatment differences at each sampling period.

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different results. This appears to be the first report documenting the positive relationship between nitrogen rate and populations of total fungi in field soils. The response of fungi to nitrogen may, in part, be due to the low organic matter status of this soil; nitrogen, through its impact on crop growth, has increased the amount of a limiting resource available to fungi. 3.4. Soil DNA Recent studies have shown that the amount of DNA extracted from soil is highly correlated with traditional measures of microbial biomass, as determined by chloroform fumigation–extraction and other methods (Agnelli et al., 2004; Blagodatskaya et al., 2003a,b; Marstorp et al., 2000). Correlation was poorer in a forest soil (Leckie et al., 2004); however, it has been shown that chloroform fumigation does not accurately measure microbial biomass in forest soils (Ingham et al., 1991). Soil DNA extraction, followed by quantification with fluorometry, is thus a rapid and relatively inexpensive method of assessing microbial biomass as contributed by fungi and bacteria. Further, there is evidence that methods that more directly measure biomass, such as DNA estimates, may more accurately reflect effects of environmental changes on microbial growth than does chloroform fumigation (Ingham et al., 1991). In preliminary tests, we found that removing fine roots from the soil samples did not affect DNA yield; therefore root material is not likely to be a significant contributor of DNA in these assays. Soil microarthropod DNA may also be present in the extracted DNA but the high correlations previously demonstrated between soil DNA yield and microbial biomass, as estimated with fumigation–extraction, suggest that the presence of microarthropod DNA does not appreciably affect the interpretation of soil DNA data as a useful measure of microbial biomass. Soil DNA extracted per g of dry soil was approximately 50% lower in fall 2001 than in spring 2001. This may represent overall differences in microbiological growth during these periods. Although there were no significant treatment effects during the spring sampling, both tillage and cover crop affected DNA yield at the fall sampling. In fall 2001, significantly (P < 0.0001) more DNA was obtained from NT (0.64 mg g1 of dry soil) plots than from CT (0.48 mg g1) plots. In addition, significantly (P < 0.0423) more DNA was extracted from RC (0.60 mg g1) plots than NC (0.52 mg g1) plots. Treatments that generally resulted in increases in crop growth (NT) or that possessed a cover crop (RC) might

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be expected to supply more raw materials (including root exudates) to the soil environment, resulting in increased soil microbial growth and, consequently, increased soil DNA. Treatment effects during spring 2001 may have been obscured by overall increases in microbial growth or activity. These, in turn, may have been a consequence of high levels of precipitation during the preceding year. Soil DNA quantification, as an alternative measure of microbial biomass, merits more consideration by researchers evaluating impacts of agro-ecosystems on microbial growth. With appropriate techniques, the extracted DNA may also be used to assess, quantitatively, populations of specific taxa of soil organisms (Rubio et al., 2005). 3.5. Principal component analyses Data for a number of physical and chemical variables and grain yield are available for this experimental site as a result of previous work (Ball Coelho et al., 2005). These data were collected from 1998 through 2001. As the field trial was initiated in 1988, soil physical factors were assumed to have stabilized by 1998 and data for soil bulk density (SBD) and soil aggregation (AG1 and AG2) from 1998 were included in this analysis (Fig. 5). This information was combined with data for the biological variables described above from the spring and fall sampling dates for 2000 and 2001. Significant (P < 0.10) correlations were found for a number of biological and physical variables. In spring 2000, earthworm populations were positively correlated with prostigmatid mites (r = 0.440), soil bulk density (0.531), and the percentage of mid-size (AG2) (>0.85–5.7 mm) soil particles (0.469), as determined by dry soil aggregation fraction estimation (Ball Coelho et al., 2005). By contrast, worm populations were significantly and negatively (0.490) associated with the small-size (AG1) (<0.85 mm) soil fraction. In fall 2000, worms were significantly correlated with AG1 (0.433) and AG2 (0.446) and Onychiuridae springtails were significantly correlated with SBD (0.326). In spring 2001, worms were significantly correlated with total fungi (0.399) and negatively correlated with Pythium (0.475). Extractable soil DNA was significantly and negatively correlated with Pythium (0.442), total fungi (0.616), SBD (0.969), and AG1 (0.964). Total fungi were significantly correlated with SBD (0.640). In fall 2001, DNA was again significantly and negatively associated with AG1 (0.538) but was positively correlated with AG2 (0.505), SBD (0.399) and earthworms (0.340). Worms were also significantly correlated with prostigmatids

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Fig. 5. Principal component analysis of selected biological and physical soil variables. (A) Spring (25 April) 2000 sampling. The two components shown explain 66.5% of dataset variability. (B) Fall (1 November) 2000 sampling. Components describe 62.4% of dataset variability. (C) Spring (16 May) 2001 sampling. The components shown explain a total of 61.9% of dataset variability. (D) Fall (30 October) 2001 sampling. Components describe 48.6% of the variability in the dataset. Variables: AG1, dry soil aggregation expressed as percentage of soil weight in <0.85 m size fraction in top 7.5 cm of soil; AG2, dry soil aggregation expressed as percentage of soil weight in 0.85–5.7 mm size fraction in top 7.5 cm of soil; APT, population of Aporrectodea turgida (total number of individuals m2 of sample area); CONY, Onychiuridae (Collembola) (individuals m2); DNA, extractable soil DNA (mg g1 soil); FUN, total soil fungi (propagules g1 soil); MPRO, prostigmatid mites (Acari; individuals m2); PYT, Pythium species (propagules g1 soil); SBD, soil bulk density (Mg m3). Individual observations are shown from each agronomic treatment plot (tillage/ cover crop only; N rates not shown) as symbol/number pairs, where (&) represents rye cover (RC), (^) represents no cover (NC), ‘‘1’’ represents conventional tillage (CT) and ‘‘2’’ represents no-till (NT). The combination ‘‘& 1’’ thus represents a split plot with a rye cover treatment within a main CT plot.

(0.277) and AG2 (0.417) but negatively correlated with AG1 (0.442). A series of principal component analyses were carried out on selected biological and physical variables in order to further explore the relationships among variables (Fig. 5). Two components explained more than 60% of the variability at three of four sampling periods. Small-size soil aggregation particles (AG2) and the population of A. turgida (APT) were associated in the spring and fall of 2000 and again in fall 2001. These variables tended to be higher in NT/RC plots, as can be

seen in Fig. 5A, B, and D. In the NT/RC treatment combination, dry stable aggregation increased from 8 to 12% of the 0.85–5.7 mm soil fraction, as compared to CT. Worms may contribute to this soil stabilization effect; however, the relatively low worm populations found in this sandy soil might not be sufficient to explain the observed changes in soil stabilization. Previous reports have indicated that earthworms may improve soil stabilization under certain conditions (Larink et al., 2001; Marashi and Scullion, 2003; Schrader and Zhang, 1997). Most of these studies,

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however, were not designed to exclude other possible contributors (perhaps positively correlated with worm populations) to soil aggregation. One such potential contributor is the fungal-produced glycoprotein glomalin, which has been shown to be present in greater amounts in reduced tillage sites than in conventionally tilled soils (Wright and Anderson, 2000). Total soil fungi were also associated here with increased soil aggregation (AG2) and SBD to some extent. Increases in populations of soil fungi under NT have been documented previously, and were associated with increases in soil aggregation (Beare et al., 1997). By contrast, populations of Onychiuridae springtails, prostigmatid mites and Pythium were not generally associated with the above variables; mites and Collembola in particular tended to behave similarly with respect to other variables in most cases. Collembola, as discussed previously, did not respond significantly in most cases to the agronomic treatments used here, although PCA analyses did suggest that, in 2000, there was an association between the Onychiuridae and CT/RC treatments (Fig. 5A and B). These analyses show that these groups tend to be influenced by the tillage and cover crop treatments but not are consistently associated with any other physical or biological variable examined here. Data for Pythium and soil fungi were available for 2001 and 2003 but only 2001 data were used here, in order to be consistent with other biological variables. The similarity between 2001 and 2003 with respect to Pythium response to plot treatment suggests that these responses are fairly stable over time. 4. Conclusions Soil organisms were obviously affected by the applied treatments; however, there were no treatments that affected soil organisms similarly across the board. In a number of cases, responses were found to be distinct from those reported previously from other soil types. Tillage reduced both earthworm populations and the yield of extractable soil DNA but, as a main treatment effect, had little influence on most microarthropod groups or fungi. Tillage and cover crop interactions were generally significant for Pythium, where the conventional tillage/rye cover crop combination resulted in increased populations. Cover crops also tended to stimulate mesostigmatid mite populations but, as a main effect, had little impact on other organisms studied. As a main effect, the greatest influence of nitrogen was on the population of total fungi, where higher rates

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of N resulted in increased populations. By contrast, high N rates in conventional tillage suppressed mesostigmatid mites. The factor(s) suppressing mites at high N rates is unknown but reduced consumption of fungal propagules by mites in these plots may have permitted fungal populations to increase. Grazing of fungal mycelium by Collembola, on the other hand, has been shown to stimulate fungal growth (Hedlund et al., 1991). Fragile low-organic-matter sandy soils can be valuable agricultural resources, if managed on a sustainable basis. The tillage experiment used in this study was first established in 1988 and treatment effects upon crop yields, nitrogen management, and weed populations have been previously discussed (Ball Coelho and Roy, 1997; Ball Coelho et al., 1998, 2005; Shrestha et al., 2002). NT treatments provided greater yields in dry years than did conventional tillage. This advantage was associated with improved water relations, improved soil aggregation, and greater waterholding capacity in NT plots. These soil physical characteristics were positively associated with populations of soil fungi and soil DNA, as well as earthworms. Identifying more precisely those organisms that play the dominant role in soil aggregation could result in biotechnologies useful in promoting soil aggregation and improved water relations in fragile soils. Differences between our findings (especially with respect to microarthropods) and previous studies on other soil types suggest that soil physical and chemical characteristics have over-riding impacts on soil organism populations, and that these impacts cannot always be altered through changes to tillage systems. Although a number of studies have documented relationships between tillage systems and various soil organisms, no previous work has documented such a wide range of taxa and their associations with each other, as well as with soil physical parameters. This approach may help clarify relationships between these parameters, tillage systems and soil organisms. Acknowledgements The assistance of B. Capell, L. Tomlinson, K. Henning, and Richard Muth in collecting and processing samples is appreciated. References Agnelli, A., Ascher, J., Corti, G., Ceccherini, M.T., Nannipieri, P., Pietramellara, G., 2004. Distribution of microbial communities in a forest soil profile investigated by microbial biomass, soil respira-

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tion and DGGE of total and extracellular DNA. Soil Biol. Biochem. 36, 859–868. Anas, O., Reeleder, R.D., 1987. Recovery of fungi and arthropods from sclerotia of Sclerotinia sclerotiorum in Quebec muck soils. Phytopathology 77, 327–331. Ball Coelho, B.R., Roy, R.C., 1997. Overseeding rye into corn reduces NO3 leaching and increases yields. Can. J. Soil Sci. 77, 443–451. Ball Coelho, B.R., Roy, R.C., Bruin, A.J., 2005. Long-term effects of a cover cropping system on corn grain yield and nitrogen balance. Can. J. Plant Sci. 85, 543–554. Ball Coelho, B.R., Roy, R.C., Swanton, C.J., 1998. Tillage alters corn root distribution in coarse-textured soil. Soil Till. Res. 45, 237– 249. Ball Coelho, B.R., Roy, R.C., Swanton, C.J., 2000. Tillage and cover crop impacts on aggregation of a sandy soil. Can. J. Soil Sci. 80, 363–366. Beare, M.H., Hu, S., Coleman, D.C., Hendrix, P.F., 1997. Influences of mycelial fungi on soil aggregation and organic matter storage in conventional and no-tillage soils. Appl. Soil Ecol. 5, 211–219. Beare, M.H., Pohlad, B.R., Wright, D.H., Coleman, D.C., 1993. Residue placement and fungicide effects on fungal communities in conventional and no-tillage soils. Soil Sci. Soc. Am. J. 57, 392– 399. Blagodatskaya, E.V., Blagodatskii, S.A., Anderson, T.H., 2003a. Quantitative isolation of microbial DNA from different types of soils of natural and agricultural ecosystems. Microbiology 72, 744–749. Blagodatskaya, E.V., Khokhlova, O.S., Anderson, T.-H., Blagodatskii, S.A., 2003b. Extractable microbial DNA pool and microbial activity in paleosols of Southern Urals. Microbiology 72, 847– 853. Chan, K.Y., 2001. An overview of some tillage impacts on earthworm population abundance and diversity—implications for functioning in soils. Soil Till. Res. 57, 179–191. Dighton, J., Boddy, L., 1989. Role of fungi in nitrogen, phosphorous and sulfur cycling in temperate forest ecosystems. In: Boddy, L., Marchant, R., Read, D.J. (Eds.), Nitrogen, Phosphorous and Sulphur Utilization by Fungi. Symposium of the British Mycological Society held at the University of Birmingham, April 1988. Cambridge University Press, Cambridge UK. Fox, C.A., Fonseca, E.J.A., Miller, J.J., Tomlin, A.D., 1999. The influence of row position and selected soil attributes on Acarina and Collembola in no-till and conventional continuous corn on a clay loam soil. Appl. Soil Ecol. 13, 1–8. Garrett, S.D., 1970. Pathogenic Root-Infecting Fungi. Cambridge University Press, Cambridge, UK. Hedlund, K., Boddy, L., Preston, C.M., 1991. Mycelial responses of the soil fungus, Mortierella isabellina, to grazing by Onychiurus armatus (Collembola). Soil Biol. Biochem. 23, 361–366. House, G.J., Parmalee, R.W., 1985. Comparison of soil arthropods and earthworms from conventional and no-tillage agroecosystems. Soil Till. Res. 5, 351–360. Ingham, E.R., Griffiths, R.P., Cromack, K., Entry, J.A., 1991. Comparison of direct vs fumigation incubation microbial biomass estimates from ectomycorrhizal mat and non-mat soils. Soil Biol. Biochem. 23 (5), 465–471. Jeffers, S.N., Martin, S.B., 1986. Comparison of two media selective for Phytophthora and Pythium species. Plant Dis. 70, 1038–1043. Kladivko, E.J., Akhouri, N.M., Weesies, G., 1997. Earthworm populations and species distributions under no-till and conventional tillage in Indiana and Illinois [USA]. Soil Biol. Biochem. 29, 613– 615.

Larink, O., 1997. Springtails and mites: important knots in the food web of soils. In: Benckiser, G. (Ed.), Fauna in Soil Ecosystems: Recycling Processes, Nutrient Fluxes, and Agricultural Production. Marcel Dekker, New York, USA, pp. 225–264. Larink, O., Werner, D., Langmaack, M., Schrader, S., 2001. Regeneration of compacted soil aggregates by earthworm activity. Biol. Fertil. Soils 33, 395–401. Lartey, R.T., Curl, E.A., Peterson, C.M., 1994. Interactions of mycophagous Collembola and biological control fungi in the suppression of Rhizoctonia solani. Soil Biol. Biochem. 26, 81–88. Leckie, S.E., Prescott, C.E., Grayston, S.J., Neufeld, J.D., Mohn, W.W., 2004. Comparison of chloroform fumigation-extraction, phospholipid fatty acid, and DNA methods to determine microbial biomass in forest humus. Soil Biol. Biochem. 36, 529–532. Lumsden, R.D., Ayers, W.A., Adams, P.B., Dow, R.L., Lewis, J.A., Papavizas, G.C., Kantzes, J.G., 1976. Ecology and epidemiology of Pythium species in field soil. Phytopathology 66, 1203– 1209. Ma, W.-C., Brussaard, L., de Ridder, J.A., 1990. Long-term effects of nitrogenous fertilizers on grassland earthworms (Oligochaeta: Lumbricidae): their relation to soil acidification. Agric. Ecosyst. Environ. 30, 71–80. Maplestone, P.A., Campbell, R., 1989. Colonization of roots of wheat seedlings by bacilli proposed as biocontrol agents against take-all. Soil Biol. Biochem. 21, 543–550. Marashi, A.R.A., Scullion, J., 2003. Earthworm casts form stable aggregates in physically degraded soils. Biol. Fertil. Soils 37, 375– 380. Marstorp, H., Guan, X., Gong, P., 2000. Relationship between dsDNA, chloroform labile C and ergosterol in soils of different organic matter contents and pH. Soil Biol. Biochem. 32, 879–882. Mele, P.M., Carter, M.R., 1999. Impact of crop management factors in conservation tillage farming on earthworm density, age structure and species abundance in South-Eastern Australia. Soil Till. Res. 50, 1–10. Miyazawa, K., Tsuji, H., Yamagata, M., Nakano, H., Nakamoto, T., 2002. The effects of cropping systems and fallow managements on microarthropod populations. Plant Prod. Sci. 5, 257–265. Money, N.P., 1998. Why oomycetes have not stopped being fungi. Mycol. Res. 102, 767–768. Morgan, P., Cooper, C.J., Battersby, N.S., Lee, S.A., Lewis, S.T., Machin, T.M., Graham, S.C., Watkinson, R.J., 1991. Automated image analysis method to determine fungal biomass in soils and on solid matrices. Soil Biol. Biochem. 23, 609–616. Olsson, P.A., Baath, E., Jakobsen, I., Soderstrom, B., 1995. The use of phospholipid and neutral lipid fatty acids to estimate biomass of arbuscular mycorrhizal fungi in soil. Mycol. Res. 99, 623– 629. Pankhurst, C.E., Mcdonald, H.J., Hawke, B.G., 1995. Influence of tillage and crop rotation on the epidemiology of Pythium infections of wheat in a red-brown earth of South Australia. Soil Biol. Biochem. 27, 1065–1073. Peters, R.D., Sturz, A.V., Carter, M.R., Sanderson, J.B., 2003. Developing disease-suppressive soils through crop rotation and tillage management practices. Soil Till. Res. 72, 181–192. Reeleder, R.D., 1992. Alterations of fungal communities in integrated management of plant disease. In: Carroll, G.C., Wicklow, D.T. (Eds.), The Fungal Community: Its Organization and Role in the Ecosystem. Marcel Dekker Inc., New York, pp. 869–884. Reeleder, R.D., Capell, B., Tomlinson, L., Hickey, W., 2003. The extraction of fungal DNA from multiple large soil samples. Can. J. Plant Pathol. 25, 182–191.

R.D. Reeleder et al. / Applied Soil Ecology 33 (2006) 243–257 Reeleder, R.D., Roy, R., Capell, B., 2002. Seed and root rots of ginseng (Panax quinquefolius) caused by Cylindrocarpon destructans and Fusarium spp. J. Ginseng Res. 26, 151–158. Reynolds, J.W., 1977. The Earthworms of Ontario. Royal Ontario Museum, Toronto, ON, p. 141. Rubio, M.B., Hermosa, M.R., Keck, E., Monte, E., 2005. Specific PCR assays for the detection and quantification of DNA from the biocontrol strain Trichoderma harzianum 2413 in soil. Microb. Ecol. 49, 25–33. Schrader, S., Zhang, H., 1997. Earthworm casting: stabilization or destabilization of soil structure? Soil Biol. Biochem. 29, 469–475. Shrestha, A., Knezevic, S.Z., Roy, R.C., Ball Coelho, B.R., Swanton, C.J., 2002. Effect of tillage, cover crop and crop rotation on the composition of weed flora in a sandy soil. Weed Res. (Oxford) 42, 76–87. Sturz, A.V., Carter, M.R., Johnston, H.W., 1997. A review of plant disease, pathogen interactions and micropbial antagonism under conservation tillage in temperate humid agriculture. Soil Till. Res. 41, 169–189. Tomlin, A.D., Miller, J.J., 1987. Composition of the soil fauna in forested and grassy plots at Delhi, Ontario. Can. J. Zool. 65, 3048–3055.

257

Tomlin, A.D., Shiptalo, M.J., Edwards, W.M., Protz, R., 1995a. Earthworms and their influence on soil structure and infiltration. In: Hendrix, P.F. (Ed.), Earthworm Ecology and Biogeography in North America. Lewis Publishers, Boca Raton, FL, pp. 159–183, 244 pp, pp. 159–183. Tomlin, A.D., Tu, C.M., Miller, J.J., 1995b. Response of earthworms and soil biota to agricultural practices in corn, soybean and cereal rotations. Acta Zool, Fennica 196, 195–199. Wardle, D.A., 1995. Impacts of disturbance on detritus food webs in agro-ecosystems of contrasting tillage and weed management practices. In: Begon, M., Fitter, A.H. (Eds.), Advances in Ecological Research. Academic Press, London UK, pp. 105–185. Winter, J.P., Voroney, R.P., Ainsworth, D.A., 1990. Soil microarthropods in long-term no-tillage and conventional tillage corn production. Can. J. Soil Sci. 70, 641–653. Wright, S.F., Anderson, R.L., 2000. Aggregate stability and glomalin in alternative crop rotations for the central Great Plains. Biol. Fertil. Soils 31, 249–253. Zaitlin, B., Turkington, K., Parkinson, D., Clayton, G., 2004. Effects of tillage and inorganic fertilizers on culturable soil actinomycete communities and inhibion of fungi by specific actinomycetes. Appl. Soil Ecol. 26, 53–62.