Orchard floor management practices that maintain vegetative or biomassgroundcover stimulate soil microbial activity and alter soil microbialcommunity composition

Shengrui Yao1, Ian A. Merwin1, George W. Bird2, George S. Abawi3 & Janice E.Thies4,51Department of Horticulture, 21 Plant Science Building, Cornell University, Ithaca, NY 14853, USA.2Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA. 3Departmentof Plant Pathology, Cornell University, Geneva, NY 14456, USA. 4Department of Crop and Soil Sciences,719 Bradfield Hall, Cornell University, Ithaca, NY 14853, USA. 5Corresponding author*

Received 3 June 2004. Accepted in revised form 22 September 2004

Key words: apple, bacteria, fungi, groundcover management system, microbial community composition

Abstract

Groundcover management systems (GMS) are important in managing fruit-tree orchards because oftheir effects on soil conditions, nutrient availability, tree growth and yields. We employed a polypha-sic approach, incorporating measures of soil microbial abundance, activity and community composi-tion, to study the long-term effects of different GMS on biotic and abiotic factors in an orchardsoil. Four GMS treatments – Pre-emergence residual herbicides (Pre-H), post-emergence herbicide(Post-H), mowed-sod (Grass), and hardwood bark mulch (Mulch) – were established in 2-m-widestrips within tree rows in an apple orchard in 1992, and have been maintained and monitored annu-ally until the present. We have measured soil water and nutrient availability, tree growth, and yieldsannually from 1993 to 2003. Soil nematode numbers and trophic groups were evaluated in July andOct. 2001, and Sept. 2003. Numbers of culturable bacteria and fungi, soil respiratory activity, eubac-terial and fungal community composition were determined in May and Sept. 2003. The Pre-H treat-ment soil had the fewest culturable bacteria, while the Grass treatment had the largest population ofculturable fungi. Soil nematode population size and diversity were also affected by GMS treatments;the Pre-H treatment had the lowest ratio of (bacteriovores + fungivores) to plant parasitic nema-todes. Soil respiration rates were higher in the Mulch than in other treatments during a 40-day incu-bation period. Hierarchical cluster dendrograms of denaturing gradient gel electrophoresis (DGGE)fingerprints for eubacterial community 16S rRNA genes indicated that Post-H and Grass treatmentsclustered together and separately from the Pre-H and Mulch treatments, which were also groupedtogether. The influence of GMSs on the fungal community, as assessed by PCR-DGGE of the inter-nal transcribed spacer (ITS) region, was not as pronounced as that observed for bacteria. Soil fungalcommunity composition under the Mulch differed from that under other treatments. The effects ofGMS on soil microbial community abundance, activity, and composition were associated withobserved differences in soil organic matter inputs and turnover, nutrient availability, and apple treegrowth and yields under the different GMS treatments.

* FAX No: 1-607-255-8615.E-mail: jet25@cornell.edu

Plant and Soil (2005) 271: 377–389 � Springer 2005DOI 10.1007/s11104-004-3610-0

Abbreviations: DGGE – denaturing gradient gel electrophoresis, GMS – groundcover managementsystem, ITS – internal transcribed spacer, PCR – polymerase chain reaction

Introduction

In deciduous tree-fruit production, herbicide-treated tree rows with mowed grass drive lanesis the most widely used groundcover manage-ment system (GMS) in North America and Eur-ope. This GMS has proven to be a relativelyefficient and inexpensive system for commercialorchards (Hogue and Neilsen, 1987; Merwin,2003). With increased pressure to reduce herbi-cide applications, and new interests in organicfarming, alternative approaches for orchardweed suppression are needed (Merwin et al.,1996; Walsh et al., 1996b; Goh et al., 2001).Extensive research has been conducted andreported on different GMS, including the use ofmulches (biomass, plastic and geotextiles), covercrops and tillage (Hogue and Neilsen, 1987;Merwin, 2003). The various types of GMS caninfluence soil physical, chemical and biologicalproperties differently (Garland and Mills, 1991;Walsh et al., 1996a; Werner, 1997; Neilsen et al.,2003). Fruit-tree growth, fruit quality, and yieldsare also affected by GMS practices (Merwin andStiles, 1994; Neilsen et al., 2003), but GMSeffects on soil microbial communities aroundtree roots, and their implications for orchardproductivity and sustainability are not wellunderstood or characterized.

Soil microorganisms are involved in criticalecosystem processes such as organic matterdecomposition, soil aggregation and humus for-mation, nutrient cycling and retention, and vari-ous symbioses and parasitic relationships withplants (Paul and Clark, 1996). Groundcovermanagement systems for perennial crops mustsupport and enable soil biota to carry out keybelow-ground nutrient retention and decomposi-tion-related processes required for long-term soilfertility and crop production (Wardle et al.,2001).

The GMS experiment for our study is locatedin an apple (Malus domestica) orchard nearIthaca, NY, and has been ongoing for 12 years,in order to investigate diverse long-term effects ofGMS treatments in an orchard agroecosystem.Our objective in the present study was to evalu-

ate differences in nematode, bacterial, and fungalpopulations in soil around the roots of fruit treesafter 12 years under different GMSs.

The traditional method used to analyze soilmicrobial communities has been serial dilutionand culturing of soil samples on various selectivemedia. A major limitation of this method is thatonly a small portion of soil microbes can be cul-tured on laboratory media (Amann et al., 1995).Culture-independent molecular methods are nowwidely used in microbial community analyses,and such methods can broaden our view ofmicrobial communities in soil and other ecosys-tems, providing a useful tool to investigatemicrobial diversity (von Wintzingerode et al.,1997).

Denaturing Gradient Gel Electrophoresis(DGGE) is one method used to study the dynam-ics and structure of complex bacterial and fungalcommunities (Muyzer and Smalla 1998; Muyzer1999; Heuer et al., 2002; Marschner et al., 20012002). In this technique, soil community DNA isextracted from samples and amplified with prim-ers targeting taxonomic groups of interest. Theamplified DNA is then denatured in a gradientgel, and differences in the resultant banding pat-terns are used to discriminate between communi-ties (Muyzer et al., 1993). We used this techniquein combination with culturing and soil respira-tion measurements to study the microbial com-munities present in soil under different GMStreatments that varied in their surface vegetationand groundcover biomass, soil organic inputs,and effects on fruit-tree nutrition, growth andproductivity.

Materials and methods

Orchard site and GMS treatments

Apple trees (cv. Empire on M9/MM111 root-stocks) were planted at 3 · 6 m spacing in April1992 at a site on the eastern shore of CayugaLake near Ithaca, NY (Merwin et al., 1996). Thesoil was a silty clay loam (mixed mesic GlosaquicHapludalf) with pH ranging from 6.8 to 7.2 and

378

organic matter content of 4.7 to 5.3% at theonset of our study. Three replicates of four GMStreatments were randomly assigned to 12 plots.Each GMS plot is 9-m wide and 25-m long, con-taining 20 trees within four parallel rows, each ofwhich is separated by 4-m-wide turfgrass drivelanes between the GMS treatment areas beneaththe trees. The four GMS treatments were set upand have been maintained continuously since1992 in 2-m-wide strips within tree rows as fol-lows: 1) Pre-H: Pre-emergence residual herbicidestreatment, a combination of three herbicides(glyphosate, norflurazon and diuron) tank-mixedat 2.0, 3.0 and 2.5 kg active ingredient (a.i.) ha)1,respectively, applied in mid-May each year; 2)Post-H: Post-emergence herbicide treatment,glyphosate applied at a rate of 2 kg a.i. ha)1 inmid-May and July each year; 3) Grass: A red fes-cue (Festuca rubra) turfgrass originally seeded in1991, now a mixture of about 25 herbaceousgrass and broadleaf species, mowed monthly dur-ing the growing season each year; 4) Mulch: A15-cm layer of shredded hardwood (a mixture ofAcer, Quercus, Juglans, Fraxinus, and Tilia sp.)bark mulch first applied in May, 1992 andrenewed in May every two years since then.Glyphosate herbicide was applied in mid-May ofthe year following each biennial mulch renewal(e.g. 1999, 2001, 2003), to suppress emergentperennial weeds in the Mulch treatment.

Soil sampling and analytic procedures

All soil samples in this study were taken beneaththe canopy of replicate trees in the GMS plots,removing them intact with stainless steel extrac-tion cores. Rock fragments and surface debriswere removed from cores by hand; the sampleswere then bagged and taken to the laboratory,where they were coarse-sieved through a 2 mmmesh screen. During sieving, adherent soil wasshaken from tree and/or groundcover vegetationroot pieces that were present in all samples, andadded back to the sieved soil. For nematodecounts, fine root fragments were retained in soilsamples for subsequent analyses, so that endo-parasitic nematodes could be quantified. For allother analyses of root-zone bacterial and fungalorganisms and their activity, visible root piecesand un-decomposed organic matter (such asthatch in the Grass treatment samples) were sep-

arated from adherent soil and excluded from sub-sequent microbial analyses.

In mid July and early Oct. 2001, two samplesfor nematode analyses were extracted with a 5.5-cm-diam core to 15-cm depth beneath trees ineach plot. The samples were kept on ice in thefield and held at 4 �C while being shipped to thesoil nematode analysis facility of Dr George Birdat Michigan State University in East Lansing,MI. Nematodes were extracted from soil by thepie-pan method, which is a modified Baermannfunnel method (Kable and Mai, 1968). Eachsample was thoroughly mixed and sub-sampledprior to sieving; then two sub-samples of 50 ccsoil volume were spread on a milk filter paperover coarse sieves nested within the stainless-steelpie-pans. Water was added to cover the soil andthe samples were incubated for 4 days at 22 �C.Nematodes were then sequentially sieved fromthe pie-pan water into a counting plate, and iden-tified at 50· magnification to families, generaand trophic group levels. The nematode analysesfor soil sampled in Sept. 2003 were completed inthe laboratory of Dr George Abawi at CornellUniversity using similar methods; but free-livingnematodes were not identified to trophic groupsin these final samples.

Soil samples for culturing, molecular and res-piration analyses were taken in mid May and lateSept. 2003, extracted in metal cores (3-cm diam)to a depth of 15 cm within GMS plots beneathtrees at a distance of 30–80 cm from the treebase. These samples were composites of 8–10 soilcores (sub-samples) per plot, and were kept onice in the field and sieved through 2-mm mesh atthe laboratory on the same day. Sub-samples forsoil DNA extraction were taken after sieving,and stored at )20 �C until later use. Samples formicrobial plate counts and soil respiration werekept in a 4 �C cooler and processed the day aftersampling. The soil samples taken in May 2003were used only for molecular microbial commu-nity analysis. For the soil sampled in Sept. 2003,plant nutrient availability, soil respiration rates,soil bacterial and fungal culture plate counts, andnematode counts were also determined.

Soil nutrient availability and conditions

Available nutrients and physical conditions insoil samples were analyzed at the Cornell Univer-

379

sity Nutrient Analysis Laboratory, using the fol-lowing methods. Macro and micronutrients wereextracted in Morgan’s solution (10% (w/v)sodium acetate in 3% acetic acid, buffered to pH4.8), using a 1:5 (v/v) soil : solution ratio. Soilorganic matter was determined by loss on igni-tion at 550 �C for two hours. Soil pH was deter-mined on a 1:1 (v/v) soil : 0.01 M CaCl2solution; and cation exchange capacity was esti-mated by extraction in 1.0 N ammonium acetateat pH 7.0 (Greweling and Peech,1965).

Soil respiration

Twenty grams of soil from three replicate sam-ples from each of the 12 plots were used to mea-sure soil respiration by a sealed jar incubationmethod, employing a 0.5 M NaOH alkali CO2

trap (Alef 1998). Soil respiration was measuredweekly for a period of 6 weeks. The jar lid wasopened and the alkali trap sampled and replacedfor each weekly measurement.

Counts of culturable soil bacteria and fungi

One gram of soil from each sample was dilutedin 9.5 mL of phosphate buffer at pH 7.0. A low-nutrient R2A medium (Difco, Becton-Dickenson,Sparks, MD), and 10% Potato-Dextrose-Agar(PDA, Difco, Becton-Dickenson, Sparks, MD)were used to culture fast-growing bacteria andfungi, respectively. One hundred microlitre aliqu-ots of a soil dilution series from 10)2 to 10)5

were spread on each prepared Petri plate andincubated for 2–3 days.

Soil microbial community analysis

DNA extraction. For each sample, 0.5 g of soilwas used to extract soil microbial DNA with theMoBio UltraClean Soil DNA extraction kit (Mo-Bio, Solana Beach, CA). After extraction, sam-ples were quantified with ethidium bromide inbuffer, and compared with standard DNA usinga Fluor-S Multi-imager (BioRad, Hercules, CA)to check the efficiency of our extractions anddetermine the template amount to be used in thesubsequent PCR amplification. The DNA yieldsranged from 5 to 10 lg g)1 soil.

PCR-DGGE. The primer pair 338f-GC/518r(Muyzer et al., 1993), which targets the V3

region in the 16S rRNA gene for eubacteria, andITS1F/ITS2–GC (Taylor and Bruns, 1997), tar-geting the internal transcribed spacer (ITS) forfungi, were used for PCR amplification. In thePCR reaction, 4 lL of 10-fold diluted DNAextraction was added to the PCR mix, whichwas composed of 5 lL of 10X PCR buffer, 6 lLof MgCl2 (25 mmol L)1), 1 lL dNTPs(10 mmol L)1 each), 0.5 lL of each primer(10 mmol L)1), 1 lL Taq polymerase (2U lL)1),and ultra pure water to a total volume of 50 lL.The PCR conditions were as follows: denaturingfor 5 min at 94 �C, then 35 cycles of denaturingat 94 �C for 30 sec, annealing at 55 �C (56 �Cfor fungi) for 30 sec, and then extension at 72 �Cfor 30 sec, and a final extension at 72 �C for10 min. After amplification, PCR products wereverified by running them on a 1.5% agarose gelstained with SYBR Green I (Sigma, Saint Louis,MO). An 8% polyacrylamide gel, with denatur-ant gradients of 35–55% for bacteria and 25–45% for fungi (with 7 M urea and formamiderepresenting 100%), was run at 60 �C and 80 Vfor 12 h in a BioRad DCode System (BioRad),and then stained with SYBR Green I (Sigma,Saint Louis, MO) and imaged with a Fluor-SMulti-imager (BioRad). Quantity One 4.2 soft-ware (BioRad) was used to detect bands andquantify band intensity.

Data analysis

Plate culture, soil nutrient analyses, soil respira-tion data, tree growth and yields were analyzedwith one-way analysis of variance (Minitab� 14,Minitab Inc. State College, PA, USA). Hierarchi-cal cluster analysis of the PCR-DGGE finger-prints was performed with Minitab� 14 softwareusing Ward’s linkage and correlation coefficientdistance. Nematode counts were transformed asthe natural log of (counts + 1) for analysis ofvariance, and treatment mean separation wasbased upon Tukey’s HSD test.

Results

Soil characteristics

Soil P and Ca availability, soil cation exchangecapacity (CEC), soil pH and organic matter

380

(OM) content were significantly greater in theMulch treatment compared with the other threeGMSs (Table 1). The availability of soil P andCa under the Mulch treatment was more thandoubled compared with the other treatments. SoilOM content under the Mulch treatment was80% higher than the average of the other treat-ments. The other soil variables measured did notdiffer significantly among treatments (Table 1).

Soil nematode populations and trophic groups

In samples from July, 2001, the Mulch and Post-H treatments had substantially more nematodefungivores than the Grass and Pre-H treatments,and the Mulch treatment had more bacterivoresthan the Pre-H and Grass treatments (Table 2).In Oct. 2001 samples, there were more lesionnematodes (Pratylenchus sp.) in the Grass thanin the other treatments, and the Post-H treatmenthad fewer omnivore nematodes than the Grasstreatment. The Grass and Post-H treatments hadhigher numbers of free-living nematodes than thePre-H and Mulch treatments. The Pre-H treat-ment had a somewhat lower ratio of (bacteri-vores + fungivores) to herbivores [(B + F)/H]compared with the other three treatments in2001. The Sept. 2003 samples had generallyhigher plant parasitic nematode counts comparedto 2001 samples, but the populations were extre-mely variable, and treatments differed only forfree-living (non-plant parasitic) nematodes, whichwere more numerous in the Post-H and Grasstreatments.

Culturable bacteria and fungi

The Mulch, Grass and Post-H treatments hadmore soil bacterial colony forming units (CFUs)than the Pre-H treatment, but CFU counts weresimilar among the first three treatments (Fig-ure 1a). In contrast, the Grass treatment hadhigher CFU counts for soil fungi compared tothe other three GMS treatments (Figure 1b).

Soil respiration

Apart from the first measurements 1 week aftersoil samples were placed into the incubation jars,the Mulch treatment had consistently higher soilrespiration rates and cumulative CO2 respiredT

able

1.Plantnutrientavailabilityand

physicalconditionsin

an

orchard

soil

after

12years

ofdifferentgroundcover

managem

entsystem

s(G

MS),

inSept.

2003(n

=3,

meansseparatedbyTukey’sHSD

test)

Treatm

ent

P (mgkg

)1)

K (mgkg)1)

Mg

(mgkg

)1)

Ca

(mgkg)1)

Fe

(mgkg))

Mn

(mgkg

)1)

Al

(mgkg)1)

Cu

(mgkg)1)

pH

OM

(%)

CEC

(cmolkg

)1)

Grass

0.56bA

168

447

1102b

1.5

17.0

13.1

0.30

6.5

b5.1

b16.8

b

Post-H

0.67b

184

411

957b

2.5

17.2

19.1

0.63

6.3

b4.7

b16.2

b

Pre-H

0.60b

159

420

1058b

1.5

16.8

14.7

0.70

6.4

b4.5

b15.3

b

Mulch

1.57a

168

481

2630a

1.7

24.3

8.1

0.77

7.2

a8.6

a22.5

a

Criticaldifference

0.64

36

105

438

1.8

8.7

10.7

0.58

0.4

2.0

4.6

AMeansfollowed

bydifferentlettersweresignificantlydifferentatP=

0.05.

381

Table

2.Differencesin

nem

atodepopulationsanddiversity

insoilunder

differentGMStreatm

ents

Lesion

(Pratylenchus)

Dagger

(Xiphinem

a)

Sheath

(Hem

icycliophora)

Spiral

(Heliotylenchus)

Total

Herbivores

(H)

Fungivores

(F)

Omnivores

Carnivores

Bacterivores

(B)

Free

living

(Fr)

Fr/H

(B+F)/H

July

2001

Grass

1.7

9.0

7.7

30.3

50.7

27.3

bA

29.0

2.0

73.3b

132ab

2.6

3.2

Post-H

6.7

9.0

0.0

9.7

18.7

85.6

a41.7

1.0

116.7

ab

245ab

13.1

13.1

Pre-H

2.7

9.3

25.0

61.6

98.0

20.3

b10.7

2.3

41.7

b75b

0.8

1.3

Mulch

0.7

14.3

0.0

6.7

21.0

76.3

a21.7

1.7

205.0

a305a

14.5

36.3

SEB

3.5

6.8

13.0

14.3

26.0

9.8

11.6

1.5

39.4

54

12.1

13.1

October

2001

Grass

14.7

a14.0

7.0

37.3

73.0

28.3

40a

10.0

362

440

6.0

10.5

Post-H

0.0

b18.3

7.0

7.7

28.3

11.7

13b

3.3

58

87

3.1

2.8

Pre-H

0.0

b21.7

35.0

42.3

75.7

15.0

23ab

6.7

63

108

1.4

2.0

Mulch

0.7

b10.0

4.0

14.7

26.7

30.0

25ab

0.0

195

250

9.4

12.6

SEB

3.3

5.0

6.2

15.7

21.7

12.0

5.7

3.1

147

153

7.9

5.2

September

2003

Grass

93

00

120

373

987a

2.6

Post-H

13

00

187

213

880a

4.1

Pre-H

013

27

120

160

400b

2.5

Mulch

26

13

093

159

493b

3.1

SEB

28

9.4

13.3

55

69

90

2.2

AMeansfollowed

bydifferentlettersweresignificantlydifferentatP=

0.05.

BSEofmeanfrom

untransform

edcountdata.Values

are

themeannumber

ofnem

atodes

per

100cc

soil,forn=

3.

Data

weretransform

edto

ln(x+

1)before

analysis,andlog-transform

edmeanswereseparatedbyTukey’sHSD

test.

382

than the other treatments (Figure 2). Cumulativerespiration peaked after 2 weeks (on 8 Oct.) andthen leveled off in the Post-H and Pre-H treat-ments, whereas for the Grass treatment, cumula-tive respiration continued to increase slowly overthe course of the incubation. In the Mulch sam-ples, respiration rates were higher, and continuedat a relatively constant higher rate throughoutthe 6 weeks of incubation.

Soil microbial community analyses

In May 2003, the hierarchical cluster dendro-grams of DGGE fingerprints indicated that bac-terial communities in the Grass and Post-H soilgrouped separately from those in the Pre-H andMulch treatments, with one outlier in the Post-Hreplicates (Figure 3a). In Sept. 2003, there weretwo distinct groupings – one containing the Pre-

H and Mulch plots interspersed, and anotherincluding the Grass and Post-H bacterial groups(Figure 3b). Comparing the bacterial and fungalDGGE fingerprints in May 2003 (Figures 3cversus 4c) there were fewer bands for fungi thatwere associated exclusively with GMS treatments,and more bands that were unique to individualfield plots, indicating greater differences in local-ized fungal populations among treatment repli-cates. In the cluster dendrograms for May 2003,fungal communities grouped into two separatebranches for Grass and Mulch, with no apparentgrouping of the Post-H replicates (Figure 4a).The Pre-H and Grass treatments were all locatedon one main branch, and two of three replicatesgrouped separately for Pre-H. In Sept. 2003, theclustering was different from that of the Maysamples, with no obvious groupings among treat-ments (Figure 4b).

Tree health, nutrition, and fruit yields

The survival of apple trees in each GMS systemwas similar (100%) during the course of thisstudy, and there were no obvious symptoms ofroot disease observed on any trees. In excava-tions of one entire tree and its root system fromeach plot in Apr. 2000, there was more fine fee-der-root biomass per tree in the Mulch, Pre-Hand Post-H trees, compared with the Grass treat-ment. We also observed that feeder roots wereconcentrated at the diffuse interface between thedecomposing Mulch and the underlying mineral

Bac

teria

(C

FU

/g s

oil D

W)

4e+6

6e+6

8e+6

1e+7

Grass Mulch Post-H Pre-H

Fun

gi (

CF

U/g

soi

l DW

)

5e+5

1e+6

1.5e+6

2e+6

2.5e+6

Grass Mulch Post-H Pre-H

a a a b

a bbb

(a)

(b)

Figure 1. Number of soil bacteria and fungi cultured (CFU)on R2A and PDA media, respectively, in the different GMStreatments. The rectangular box shows the interquartile range,the median is shown as the horizontal bar inside boxes. Thefirst and last quartiles are shown as vertical lines above andbelow boxes. The line through each box indicates the treat-ment mean. Means above or below different letters were sig-nificantly different at P ¼ 0.05, n ¼ 12, based upon Tukey’sHSD test.

0

0.5

1

1.5

2

2.5

3

3.5

8-Oct 15-Oct 22-Oct 29-Oct 5-Nov 12-Nov

Date

Cu

mu

lati

ve C

O2

(mg

g-1

soil

DW

)

Pre-H Post-H Grass Mulch

Figure 2. Soil respiration rates in soil from different GMStreatments, measured by a sealed jar incubation method at22 oC. Repeated weekly measurements were taken over a6-week period (n ¼ 3, mean ± SE). The jars were set up onOct. 1 and the first measurement was taken a week later onOct. 8, 2003.

383

soil (Merwin, unpublished). Soil N content hasbeen higher during most growing seasons in theMulch and Pre-H treatments, compared withGrass and Post-H treatments (Merwin, 2004).Leaf N content has been greater most years intrees in the Mulch and both herbicide treatments,compared to the Grass treatment. Trunk cross-sectional area (at 40 cm above graft unions) oftrees after 11 years was about 15% greater in theMulch and Post-H compared with the Grass andPre-H treatments. Substantial differences in fruityields among the GMSs were observed in mostyears, including 2001 and 2003 (Figure 5). Treesin the Grass plots have generally been least pro-

ductive; yields were greater on trees in the Mulchand Post-H treatments compared with Grass in2001, and on the Post-H trees compared withGrass in 2003. Yields were unusually low in alltreatments during 2002, because of a severe frostduring bloom.

Discussion

The Mulch treatment significantly increased soilP, Ca, CEC, OM, and pH compared to the othertreatments. Even though the bark mulch was notmixed into the soil, the decomposed mulch at the

Figure 3. Differences in bacterial community composition between GMS treatments as detected by Hierarchical cluster analysiswith Ward’s linkage and correlation coefficient distance for DGGE gels. (a) May 2003; (b) Sept. 2003; and (c) DGGE gel of PCRamplified bacterial DNA from Sept. 2003. The denaturing gradient was 35–55% and the primer pair used was 338f-GC and 518r.

384

mineral soil interface was gradually incorporatedinto the soil and doubled soil organic matter con-tent over the 12 years of treatments. With thebark mulch application rate of 13.5 kg m)2 yr)1

dry weight, a total of 162 kg m)2 (DW) of mulchhas been applied in this GMS over the 12-yearduration of our experiment. The N content (DW)of our bark mulch material averaged 0.47%, sothis GMS treatment represents a very largeannual N input of 0.63 tonnes ha)1. Six years ofmonitoring the runoff and leaching of nitrate-Nfrom the four GMS treatments in this orchardhave demonstrated substantially lower losses of N

from the Grass and Mulch plots, relative to thetwo herbicide treatments (Merwin et al., 1996;Merwin, 2004). We attributed the greater N reten-tion in Mulch plots to its high C:N ratio (98:1)and microbial cycling and retention of the N min-eralized from decomposing Mulch residues, whichwas consistent with the greater soil microbial res-piration observed in that GMS (Figure 2).

In the Pre-H treatment, the soil surfacebeneath trees is weed-free year-round; hencethere is little soil organic input in this GMS,other than apple leaf litter, root exudates andturnover (Merwin, 2003, 2004). In a study of

Figure 4. Differences in fungal community composition between GMS treatments as detected by Hierarchical cluster analysis withWard’s linkage and correlation coefficient distance for DGGE gels. (a) May 2003, (b) Sept. 2003. (c) DGGE gel of PCR amplifiedfungal DNA from May 2003. The denaturing gradient was 25–45% and the primer pair used was ITS1F/ITS2-GC.

385

M9 apple root density, depth and turnover ratesat a nearby orchard on a similar soil, abouttwo-thirds of all tree roots were within the upper20 cm of soil in the tree rows, and the averagehalf-life for secondary and fine feeder roots dur-ing two growing seasons was 22 days (Psarraset al., 2000). This suggests that most of theapple root profile was included in our soil sam-pling, and that apple roots alone would providea limited amount of organic carbon and nitrogento the surrounding soil during root turnover.However, there would be substantially more Cand N inputs where the upper soil profileincluded herbaceous groundcover roots anddecomposing bark Mulch inputs in addition toapple roots.

In the Post-H treatment, a sparse weedgroundcover of summer annuals and biennials,and a nearly complete surface layer of moss cov-ers the soil surface from late August until midMay the following year (Merwin, 2004). Afterthe glyphosate herbicide applications kill thisaccumulated surface vegetation in May and Julyeach year, there is a flush of organic inputs tothe soil as above and below-ground weed bio-mass residues decompose. These conditions pro-vide more organic inputs to the soil in the Post-H than in the Pre-H treatment, and may explainthe differences we observed in microbial commu-nity composition between these two herbicidesystems. It is also possible that there is somedirect chemical influence of the two residual her-bicides (norflurazon and diuron) included in thePre-H treatments that has altered the microbial

communities in this treatment during the past12 years (Tu 1996).

In the mowed Grass treatment, a dense layerof thatch has established over the past decade,and grass litter, root exudates, and root biomassturnover are all incorporated into the soil. Thistreatment has more organic input from surfacevegetation biomass compared with the two herbi-cide treatments, but less than the Mulch treat-ment. These conditions were reflected in thecumulative respiration rates during our soil incu-bations (Figure 2). By the end of the incubation,respiration rates were beginning to level off inthe Pre-H and Post-H soils, whereas the rate con-tinued to increase at a low level in the Grass soil,and at a high level in the Mulch soil, where thecumulative respiration rate remained nearly con-stant, with no evidence of substrate limitationafter six weeks of incubation. Considering thatsoil OM content was not significantly differentamong the Grass, Pre-H and Post-H samples(Table 1), these observations indicate that miner-alization rates of groundcover biomass residueswere probably higher for Grass than for the weedresidues of herbicide treated plots.

Although the trends were complex amongtreatments and over time in serial samples fromyear to year and month to month, the GMStreatments obviously affected soil bacterial andfungal populations and diversity. It is well knownthat soil microorganisms are often carbon lim-ited, and that variation in plant biomass inputsacross soil treatments influences soil biota(Wardle et al., 2001). The greater bacterial popu-lations observed in the Mulch, Grass and Post-Htreatments versus the Pre-H treatment suggestedthat soil bacterial populations were more depen-dant than soil fungi upon soil carbon inputs(Figure 1a–b), which is consistent with otherrecent studies (Marschner et al., 2003).

The DGGE fingerprinting and cluster analysesindicated that GMS treatments also influencedmicrobial species composition in soil, althoughthese differences were qualitative and variedbetween May and September, 2003. Soil condi-tions in upstate New York are very different inMay and September, with lower temperaturesand higher water potentials earlier in the growingseason (Merwin et al., 1994). Fruit-tree andgroundcover root growth and turnover also differearly and late in the growing season, so that soil

Figure 5. Average yields (kg fruit tree)1) from 1994 to 2003,for trees in each GMS treatment. Significant differencesamong GMS treatment means (n ¼ 3) are denoted by aster-isks (*P ¼ 0.10; ** P ¼ 0.05) for each year.

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carbon and root exudates should differ fromMay to September. These trends may explain thedifferences we observed in serial samples of nem-atodes, bacteria and fungi.

Soil microbial community composition andpopulations are affected by a range of edaphicfactors, plant species, soil management practices,and climate (Hooper et al., 2000; Marschneret al., 2001). Root exudates and OM decomposi-tion in soil are important sources of energy andnutrients for soil microflora (Saetre and Baath,2000). Merbach et al. (1999) have suggested thatplant species-specific microbial community com-positions are linked to the chemical compositionof the corresponding rhizo-deposition. Apple treeroots were common to all treatments in ourstudy, but root exudates from grasses or broad-leaf weeds may favor certain bacteria, resultingin different bacterial community composition rel-ative to treatments without rooted ground-covervegetation.

For fungi, it appeared that the Mulch treat-ment had a different fungal community composi-tion than the other treatments (Figure 4a–b). Thehardwood bark mulch had a high C:N ratio(98:1) and likely contained high lignin and poly-phenol contents (Melillo et al., 1982), all ofwhich may favor colonization by some fungalspecies over others (Paul and Clark, 1986). Thehigh C:N indicates that the mulch would be moredifficult to degrade than organic materials withlower ratios, whereas the chemical complexity oflignin and polyphenols requires specializedenzymes for their degradation and hence wouldbe colonized by fungi capable of degrading thesesubstrates, thus leading to a distinct grouping inthe cluster analysis. Higher abundance of fungiin the Grass treatment is likely related to theconstant turnover of grass residues in the thatchlayer, which would provide a constant C reser-voir. Results of the respiration analysis bear thisout in that the slope of the cumulative CO2 respi-ration curve (Figure 2) did not level off in the6 weeks of incubation, yet continued to increaseat a low level.

Soil microbial respiration is an indicator ofsoil microbial activity. The Mulch treatment hadthe highest soil respiration rate, coupled with themost OM, and highest CFU counts for bacteria.Similar results were reported in a comparison ofsoil from organic and conventional orchard plots

in California (Werner, 1997). Higher microbialactivity reflects higher resource availability andoften higher microbial biomass, but it doesn’tnecessarily imply significant changes in commu-nity composition (Wardle et al., 2001). Thehigher soil respiration rates and soil bacterialpopulations in the Mulch treatment especially ascompared to the Pre-H treatment suggest thatavailable resources for microorganisms – i.e. thetotal amount of organic matter inputs –increased bacterial populations and activity inthese soils.

Nematode populations and trophic diversitywere influenced by GMS treatments in our study(Table 2). Others have proposed that ratios ofplant parasitic to non-parasitic nematodes canprovide useful indices of soil ‘‘health’’ or ‘‘qual-ity’’ (Ferris et al., 2001). Despite the substantialdifferences in soil OM, soil bacterial and fungalpopulations, soil nutrient availability, and soilmicrobial respiration in our study – there wereno significant differences in the relative popula-tion densities of (bacterivore + fungivore) toherbivore nematodes in any samples. Free-livingand bacterivorous nematode populations differedamong GMS treatments on some sampling dates,but not others. Nematode trophic group popula-tions were generally inconsistent over timeamong GMS treatments, and populations of par-asitic nematodes such as Pratylenchus and Xiphi-nema remained below damage thresholds forapple (Jaffee et al., 1982). In a previous study offruit-crop GMSs, treatments with higher organicinputs were associated with greater diversity ofnematodes and higher populations of collembola(Wardle et al., 2001), or protozoans and bacteriv-orous nematodes (Forge et al., 2003). However,our observations did not indicate a consistentlinkage between soil groundcover or biomassinputs and nematode community composition inthis orchard soil.

Despite the observed differences in soil micro-bial respiration, community composition andnematode populations, the trees in all GMS treat-ments in our experiment showed no obvious symp-toms of root disease. However, there have beendifferences in fruit yields, tree growth, nutrientuptake, and off-site N and P losses to leaching andrunoff during the course of this study(Merwin et al., 1996; Merwin, 2004). The observeddifferences in soil microbial community composi-

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tion and relative dominance of bacteria, fungi, ornematodes in each GMS appear to be within therange to which apple tree roots can adapt success-fully over time. Differences in microbial commu-nity composition and activity do not necessarilylead to strong changes in soil conditions for theapple roots. Rather, the nature of the root envi-ronment may be the stronger driver for microbialcommunity dynamics. In a study of the effects ofsoil management, rootstock genotype and plantingposition on apple replant disorder, rhizospherebacterial communities were most strongly affectedby rootstock genotype and planting position (oldrow versus grass lane) than by soil management(compost addition or fumigation) (Rumbergeret al., 2004). While it can be argued that in the12 years of this experiment there may no longer besoil beneath the trees that is not ‘root-affected’, wedid not specifically sample the rhizosphere soil ofactive apple roots as these were assumed to be aconstant across the experiment. The conditions inthe rhizosphere of currently active apple rootsmay well differ substantially from those of the‘bulk’ soil sampled. Differences we have observedbetween the treatments in this study may be lesspronounced in apple rhizosphere soil due to thedominant effect of the apple root – common to alltreatments – on these microbial communities. Fur-ther studies of this type could provide useful infor-mation about the relative importance and effectsof different soil microflora and microfauna onfruit-tree physiology and soil ecological processesin orchards that may help improve orchard man-agement practices in the future.

Acknowledgments

This work was supported by a graduate stipendto S. Yao from the Department of Horticulture,Cornell University and materials and equipmentsupport from the Department of Horticultureand the Department of Crop and Soil Sciences,Cornell University, Ithaca, NY. We thank thereviewers for their helpful comments on thismanuscript.

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