Soil fumigation and compost amendment alter soil microbial community composition but do not improve...

Soil fumigation and compost amendment alter soil microbial

community composition but do not improve tree growth

or yield in an apple replant site

Shengrui Yaoa, Ian A. Merwina, George S. Abawib, Janice E. Thiesc,*

aDepartment of Horticulture, Cornell University, Ithaca, NY, USAbDepartment of Plant Pathology, Cornell University, Geneva, NY, USA

cDepartment of Crop and Soil Science, Cornell University, 719 Bradfield Hall, Ithaca, NY 14853, USA

Received 1 April 2005; received in revised form 9 June 2005; accepted 14 June 2005

Available online 1 August 2005

Abstract

Apple replant disease (ARD) is a disease complex that reduces survival, growth and yield of replanted trees, and is often

encountered in establishing new orchards on old sites. Methyl bromide (MB) has been the fumigant used most widely to control ARD,

but alternatives to MB and cultural methods of control are needed. In this experiment, we evaluated the response of soil microbial

communities and tree growth and yield to three pre-plant soil treatments (compost amendment, soil treatment with a broad-spectrum

fumigant, and untreated controls), and use of five clonal rootstock genotypes (M.7, M.26, CG.6210, G.30 and G.16), in an apple replant

site in Ithaca, New York. Polymerase chain reaction (PCR)—denaturing gradient gel electrophoresis (DGGE) analysis was used to

assess changes in the community composition of bacteria and fungi in the bulk soil 8, 10, 18 and 22 months after trees were replanted.

PCR-DGGE was also used to compare the community composition of bacteria, fungi and pseudomonads in untreated rhizosphere soil

of the five rootstock genotypes 31 months after planting. Tree caliper and extension growth were measured annually in November from

2002 to 2004. Apple yield data were recorded in 2004, the first fruiting year after planting. Trees on CG.6210 rootstocks had the most

growth and highest yield, while trees on M.26 rootstocks had the least growth and lowest yield. Tree growth and yield were not

affected by pre-plant soil treatment except for lateral extension growth, which was longer in trees growing in compost-treated soil in

2003 as compared to those in the fumigation treatment. Bulk soil bacterial PCR-DGGE fingerprints differed strongly among the

different soil treatments 1 year after their application, with the fingerprints derived from each pre-plant soil treatment clustering

separately in a hierarchical cluster analysis. However, the differences in bacterial communities between the soil treatments diminished

during the second year after planting. Soil fungal communities converged more rapidly than bacterial communities, with no discernable

pattern related to pre-plant soil treatments 10 months after replanting. Changes in bulk soil bacterial and fungal communities in

response to soil treatments had no obvious correlation with tree performance. On the other hand, rootstock genotypes modified their

rhizosphere environments which differed significantly in their bacterial, pseudomonad, fungal and oomycete communities. Cluster

analysis of PCR-DGGE fingerprints of fungal and pseudomonad rhizosphere community DNA revealed two distinct clusters. For both

analyses, soil sampled from the rhizosphere of the two higher yielding rootstock genotypes clustered together, while the lower yielding

rootstock genotypes also clustered together. These results suggest that the fungal and pseudomonad communities that have developed in

the rhizosphere of the different rootstock genotypes may be one factor influencing tree growth and yield at this apple replant site.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Apple replant disease; Compost; Fumigation; Malus domestica; Microbial community composition; PCR-DGGE; Rootstock

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.soilbio.2005.06.026

* Corresponding author. Tel.: C1 607 255 5099; fax: C1 607 255 8615.

E-mail address: [email protected] (J.E. Thies).

1. Introduction

Apple (Malus domestica) replant disease (ARD) is

distributed worldwide and is often encountered in establish-

ing new orchards on old sites. Smith (1995) reported that,

for Washington State, a loss of $99,000 haK1 in gross

income over 10 years could result from failure to control

replant disease. In New York State, the economic impacts of

Soil Biology & Biochemistry 38 (2006) 587–599

www.elsevier.com/locate/soilbio

S. Yao et al. / Soil Biology & Biochemistry 38 (2006) 587–599588

this soil-borne disease complex are also serious (Arneson

and Mai, 1976).

ARD is a complex plant disease syndrome and its

etiology is still unclear (Mai and Abawi, 1981; Mazzola,

1997, 1998; Merwin et al., 2001). Both biotic and abiotic

factors are involved and the causes vary from region to

region and from site to site. ARD appears to be caused

mainly by biotic factors, since it can often be controlled by

pre-plant soil fumigation (Jaffee et al., 1982; Mai and

Abawi, 1981; Stirling et al., 1995). However, soil

fumigation or steam pasteurization suppressed ARD in

only about 50% of all replanted orchards tested in a recent

study across New York State (Merwin et al., 2001),

indicating that abiotic factors are also involved in ARD.

Soil bacteria, plant parasitic nematodes, fungi, actinomy-

cetes, and oomycetes have all been implicated individually

or together as causative agents of ARD (Catska et al., 1982;

Mai and Abawi, 1981; Mazzola, 1997, 1998; Merwin et al.,

2001; Sewell, 1981; Utkhede and Li, 1989; Westcott et al.,

1987). Root lesion nematodes (Pratylenchus sp.) were

considered a main cause for ARD in some coarse-textured

soils around New York State (Mai and Abawi, 1981), while

Mazzola (1998; 2002) emphasized fungal involvement in

ARD and the important effects of Pseudomonas species in

the suppression of ARD in Washington State.

For decades, methyl bromide (MB) has been the most

effective and commonly used fumigant to control ARD

(McKenry et al., 1994), but MB is being phased out because

of environmental concerns. Thus, scientists are searching

for safer alternatives to the use of MB. Alternative

fumigants have been evaluated (Duniway, 2002; Martin,

2003), but none have provided as effective control as MB.

Cover crops have been tested for their ability to suppress

ARD in several orchards in NY State, but results were

inconsistent from site to site (Merwin and Stiles, 1988;

Merwin et al., 2001). Compost has been shown to suppress

several soil pathogens in woody ornamentals and forest tree

nurseries (Blok et al., 2002; Hoitink and Kuter, 1984;

Hoitink et al., 1991, 1997b), turf grass (Craft and Nelson,

1996) and many different vegetable crops (De Ceuster and

Hoitink, 1999). However, there are few reports document-

ing the successful use of compost in managing ARD.

Rootstocks from the Cornell–Geneva breeding program

were shown to be tolerant to ARD in bioassays conducted

using a mixture of old orchard soils from New York State

(Isutsa and Merwin, 2000). Beneficial effects of planting

trees in the old grass drive lanes of the previous orchard,

rather than in the old tree rows, as a cultural practice to

manage ARD have also been reported (Jensen and Buszard,

1988; Leinfelder, 2005).

In this experiment, we evaluated pre-plant soil treat-

ments, rootstock genotype, and changes in orchard planting

position as viable alternatives to MB for the control of ARD

in an apple replant site in Ithaca, NY. Since the positive

effects of fumigation and compost amendment rely on

suppressing harmful microbial activities, and apple

rootstocks are also known to modify the microbial

communities of their rhizosphere (Rumberger et al.,

2004), we aimed to examine how these treatments

influenced soil microbial communities, and how any

changes observed in them were related to ARD symptoms

in newly planted trees. We used a polyphasic approach

which combined microbial plate counts, soil respiration,

nematode counts and identities, and molecular soil

microbial analyses to assess changes in rhizosphere and

bulk soil communities of bacteria, fungi, Pseudomonas and

oomycetes in response to soil fumigation, compost

amendment, and use of different rootstock genotypes.

2. Materials and methods

2.1. Site preparation

The orchard study was established on a Cornell

University research farm in Ithaca, NY. The soil is a glacial

lacustrine Hudson silty clay loam (mixed mesic Udic

Hapludalf). The old apple trees were removed in Sept. 2001,

along with as much of the previous root systems as possible.

Soil samples were taken along each old tree row and grass

lane for analysis of pH, CEC (cation exchange capacity) and

available nutrients. Based on these pre-plant soil tests,

dolomitic lime was applied uniformly over the entire site at

a rate of 11,000 kg haK1 to eventually raise the soil pH to

6.5 (Stiles and Reid, 1991).

Since the old tree rows were east–west and the new trees

were planted south–north, we were able to test the legacy

effects of previous tree rows and grass lanes on the growth

and yield of the replanted trees. There were three

experimental factors: (1) five rootstock genotypes: G.16,

CG.6210, M.26, G.30 and M.7, with expected scion

dwarfing effects ranging from dwarf to semi-dwarf,

respectively. ‘Royal Empire’ was the grafted scion on all

rootstocks; (2) four pre-plant soil treatments: (a) untreated

controls, (b) fumigation only, (c) compost only and (d)

compost addition followed by fumigation; and (3) two

planting positions: new trees planted either in the old tree

row or in the old grass drive lanes of the previous orchard.

The experiment was planted in a split-block design for

planting position with five blocks; a split-plot design with

five replicates of each pre-plant soil treatment and a 5!5

Latin square of rootstocks over planting position.

2.2. Soil treatments

A commercial compost consisting of 40% (v/v) ground

leaves and wood chips, 40% supermarket vegetable culls,

and 20% pre-composted cattle and horse manure in wood

shavings (Toad Hollow Farm, Nedrow, NY) was applied in

two portions on Sept. 24, 2001. The first portion of compost

was applied to the soil surface of the compost treatment

rows in a band 1.5 m wide with a row mulcher (Millcreek

S. Yao et al. / Soil Biology & Biochemistry 38 (2006) 587–599 589

Manufacturing Co., Leola, PA), at a rate of 69 t haK1. The

entire site was then deep-plowed to invert the soil profile

and place this initial portion of compost down into the root-

zone of the future tree rows. An equal portion of the same

compost was then applied in the identical 1.5 m wide band

along the tree rows designated for compost treatment. Based

on a nutrient analysis of the compost, a mineral fertilizer

(22-4-10: N-P-K) was applied at the rate of 1142 kg haK1

treated area (about 30% of the total area) to all of the tree

rows that did not receive compost, to compensate partially

for the slow-release of macronutrients present in organic

forms in the compost treatment.

The fumigant mixture of 78% dichloropropene C17%

chloropicrin (Telone C-17e, Dow AgroSciences, Indiana-

polis, IN) was injected at 25 cm depth within the intended

tree rows at a rate of 300 l haK1, and the soil was immediately

sealed using a heavy cultipacker. Fumigation was the final

pre-plant treatment applied at the site on Oct. 5, 2001, five

weeks prior to replanting the apple trees. Trees were planted

on Nov. 13, 2001 at 2!5 m spacing. After planting, trees

were managed according to commercial orchard recommen-

dations for irrigation, pesticide spraying, pruning and other

needed cultural practices (Agnello et al., 2002).

2.3. Soil sampling

2.3.1. Bulk soil sampling

Bulk soil samples were taken from beneath the canopy of

trees on the five rootstocks in the untreated control,

fumigated and compost-amended treatments, within both

planting positions in three replicate blocks at 8 (July, 2002),

10 (Sept., 2002), 18 (May, 2003), 20 (July, 2003) and 22

(Sept., 2003) months after planting. Four, 3-cm diam soil

cores were taken 10–20 cm from the trunk base on both

sides of the trees on each rootstock (M.7, M.26, G.30,

CG.6210 and G.16) for both planting positions (old tree

rows and old grass lanes). Soil cores from the five rootstocks

within each planting position within the same row were

combined, thus yielding three replicate samples of each soil

treatment!planting position. Any root pieces were picked

out of all samples and the remaining soil was considered as

bulk soil. The soil variables tested are listed in Table 1. All

Table 1

Sampling times and variables analyzed

2002 2003

(Months after replanting) July (8) Sept. (10) May (18

Soil sampled Bulk Bulk Bulk

Soil characteristicsa X

Soil microbial community

analysis (DGGE)

X X X

Soil respiration X

Soil nematode count X

Bacterial enumeration

(plate culture)

Cloning and sequencing

a See Table 3.

soil samples were kept on ice in the field, later they were

sub-sampled for nematode analysis, before sieving through

a 2-mm sieve. After sieving, samples for plate counts and

soil respiration assays were kept at 4 8C until tested, and

samples for DNA extraction were frozen at K20 8C until

they could be analyzed.

2.3.2. Rhizosphere soil sampling

In June 2004, 31 months after tree planting, four soil

cores per tree were sampled from three replicate trees of

each of the five different rootstocks, only in the untreated

control plots in old grass lane positions to test the rootstock

effects on adherent soil, independent of pre-plant soil

treatments (Table 1).

2.4. Soil nutrient and microbial population and activity

measurements

2.4.1. Nematode counts

Nematodes in soil were extracted by the pie-pan

extraction method, which is a modified Baermann funnel

method (Yao et al., 2005).

2.4.2. Soil nutrient availability

Available nutrients and other characteristics of the

collected soil samples were analyzed at the Cornell

University Nutrient Analysis Laboratory in 2002 and

2003, using the following methods. Macro- and micro-

nutrients were extracted in Morgan’s solution [10% (w/v)

sodium acetate in 3% acetic acid, buffered to pH 4.8], using

a 1:5 (v/v) soil:solution ratio and extracts analyzed by

inductively coupled plasma (ICP) spectroscopy. Soil

organic matter was determined by loss on ignition at

550 8C for 2 h. Soil pH was determined on a 1:1 (v/v) soil:

0.01 M CaCl2 solution; and cation exchange capacity was

estimated by extraction in 1.0 N ammonium acetate at pH

7.0 (Greweling and Peech, 1965).

2.4.3. Soil respiration

Twenty grams of bulk soil were used to measure soil

respiration by a sealed jar incubation method, employing

a 0.5 M NaOH alkali CO2 trap (Alef, 1998), at 10 and 22

2004

) July (20) Sept. (22) June (31)

Bulk Bulk Rhizosphere

X

X X

X

X

Oomycetes


months after planting. Soil respiration was measured weekly

for 7 weeks. At each sampling, the jar lid was opened, the

alkali trap removed and the solution back-titrated with

0.5 M HCl, to assess CO2 released. The alkali trap was

replaced for each weekly measurement.

2.4.4. Counts of culturable soil bacteria

and Pseudomonas spp.

One gram of rhizosphere soil (root pieces and adherent

soil) from each sample was diluted in 9.5 ml of phosphate

buffer at pH 7.0. A low-nutrient R2A medium (Difco,

Becton-Dickenson, Sparks, MD) and King B medium

(Difco) were used to culture fast-growing bacteria and

Pseudomonas, respectively. Aliquots of 100 ml of each soil

dilution ranging from 10K3 to 10K5 were spread on each

prepared Petri plate and incubated for 2–3 days at room

temperature before determining the number of colony

forming units (CFUs) in each sample.

2.5. Bulk and rhizosphere soil microbial

community analysis

2.5.1. DNA extraction

For each sample, 0.5 g of bulk soil (or root pieces plus

rhizosphere soil) was used to extract soil microbial DNA

with the Bio101w FastDNAw SPIN kit for soil (Qbiogene,

Irvine, CA) according to the manufacturer’s instructions.

After extraction, DNA was quantified with ethidium

bromide in buffer, and compared with DNA standards

using a Fluor-S Multi-imager (BioRad, Hercules, CA) to

check the extraction efficiency and determine the template

amounts to be used in the subsequent PCR amplifications.

DNA extracts were stored at K20 8C until used in

subsequent analyses.

2.5.2. PCR-DGGE

The primer pair 338f-GC/518r, which targets the V3

region in the 16S rRNA gene for bacteria; ITS1/ITS2-GC,

targeting the internal transcribed spacer for fungi; and

Table 2

Primer pairs used and the PCR programs applied for these primers

Name Sequence (50/3 0)

PRBA338f ACT CCT ACG GGA GGC AGC AG

PRUN518r ATT ACC GCG GCT GCT GG

ITS1 TCC GTA GAA CCT GCG G

ITS2 GCT GCG TTC TTC ATC GAT GC

Psf GGT CTG CTG AGA GGA TGA TCA GT

1378r CGG TGT GTA CAA GGC CCG GGA ACG

Oomf GTG CGA GAC CGA TAG CGA ACA

Oomr TCA AAG TCC CGA ACA GCA ACA A

Primer pair PCR programs

PRBA338f-GC/PRUN518r 94 8C for 5 min, then 35 cycles at 94 8C f

ITS1-GC/ITS2 94 8C for 5 min, then 35 cycles at 94 8C f

Psf/1378r 94 8C for 5 min, then 35 cycles at 94 8C f

Oomf-GC/Oomr 94 8C for 5 min, then 35 cycles at 94 8C f

Oomf/Oomr-GC targeting the 28S rRNA gene of oomycetes

were used for PCR amplification (Table 2). For Pseudomo-

nas spp., a nested PCR assay was employed with Psf and

1378r as the first set of primers to amplify Pseudomonas

species, followed by 338f-GC/518r as the second set of

primers to amplify a short sequence suitable for DGGE

resolution (Table 2). A GC clamp was attached to either the

forward or reverse primer used for PCR amplification to

improve resolution of the amplicon operational taxonomic

units (OTUs) in the DGGE analysis (Muyzer et al., 1993;

Smalla et al., 2001). In each PCR reaction, 4–6 ml of 10-fold

diluted DNA extract (12–20 ng DNA) was added to the PCR

mix which was composed of 5 ml of 10! PCR buffer, 6 ml

of MgCl2 (25 mmol lK1), 1 ml dNTPs (10 mmol lK1 each),

0.5 ml of each primer (10 mmol lK1), 1 ml Taq polymerase

(2 U mlK1), and ultra pure water to a total volume of 50 ml.

The PCR amplification conditions used for each primer pair

are given in Table 2. After amplification, PCR products

were routinely verified by running them on a 1.5% agarose

gel stained with SYBR Green I (Sigma, St Louis, MO). An

8% polyacrylamide gel, with a denaturant gradient of 35–

55% for bacteria, 25–45% for fungi, and 30–45% for

oomycetes (with 7 M urea and formamide representing

100%), was run at 60 8C and 80 V for 12 h in a BioRad

DCode System (BioRad), then stained with SYBR Green I

(Sigma) and imaged with a Fluor-S Multi-imager (BioRad).

The software package Quantity One 4.2 (BioRad) was used

to detect bands and quantify band intensity.

2.5.3. Cloning and sequencing

Dominant bands of interest from the oomycete DGGE

gels from rhizosphere soil samples were excised and re-

amplified with the Oomf/Oomr primer pair without the GC-

clamp. At least two common bands from the three replicate

samples from each rootstock, and bands unique to different

rootstocks were excised and re-amplified. The pGEM-T

Easy Vector System Kit (Promega, Madison, WI) was used

for cloning re-amplified products from excised bands

according to the manufacturer’s protocol. Three to five

Length (bp) Sources

20 Ovreas et al. (1997)

17 Muyzer et al. (1993)

16 White et al. (1990)

20 White et al. (1990)

23 Widmer et al. (1998)

24 Mincer et al. (2002)

21 Arcate et al. (2005)

22 Arcate et al. (2005)

or 30 s, 55 8C for 30 s and 72 8C for 30 s, final extension at 72 8C for 10 min




Tab

le3

Soil

char

acte

rist

ics

mea

sure

dfo

llow

ing

dif

fere

nt

pre

-pla

nt

soil

trea

tmen

tsan

din

the

two

pla

nti

ng

posi

tions,

inJu

ly2002

July

20

02

P (mg

kg

K1)

K (mg

kg

K1)

Mg

(mg

kg

K1)

Ca

(mg

kg

K1)

Fe

(mg

kg

K1)

Al

(mg

kg

K1)

Mn

(mg

kg

K1)

Zn

(mg

kg

K1)

Cu

(mg

kg

K1)

pH

OM

(%)

NO

3

(mg

kg

K1)

Con

tro

l4

.3b

18

0b

30

11

02

4c

5.7

a3

6.6

a1

7.0

a1

.50

.6c

5.9

b4

.8b

33

.9b

Fu

mig

atio

n3

.6b

17

3b

31

51

04

3c

5.5

a3

8.3

a1

8.9

a1

.30

.7c

5.8

b4

.8b

72

.1a

Com

po

st1

3.8

a2

30

a2

94

21

30

b3

.0b

20

.6b

13

.9b

1.4

1.1

b6

.9a

5.6

a1

3.6

c

Fu

mC

Co

m1

6.0

a2

31

a3

14

24

91

a3

.5b

21

.3b

17

.0ab

1.7

1.3

a6

.9a

5.9

a2

1.7

c

Old

row

7.9

b2

16

a3

02

14

68

b4

.63

1.9

a1

5.3

b1

.2a

0.9

6.3

4.5

b3

6.2

Gra

ssla

ne

11

.0a

19

1b

31

01

87

6a

4.2

26

.5b

18

.1a

1.8

b0

.96

.46

.0a

34

.5

Val

ues

are

mea

ns

of

fiv

ere

pli

cate

sp

ertr

eatm

ent.

Mea

ns

foll

ow

edb

yd

iffe

ren

tle

tter

sar

esi

gn

ifica

ntl

yd

iffe

ren

tat

P!

0.0

5.


positive clones from each band were randomly selected for

sequencing with an ABI 3730 DNA sequencer at the Cornell

Biotechnology Resource Center, Ithaca, NY.

2.6. Data analysis

Soil respiration, soil chemical analyses, bacterial plate

counts (log10 transformed) and nematode data were

analyzed by analysis of variance (Minitabw 14, Minitab

Inc., State College, PA, USA). Nematode counts were

transformed as the natural log of [counts C1] for analysis of

variance. Hierarchical cluster analysis of the PCR-DGGE

fingerprints was performed with Bionumericsw software

(Applied Maths, Heistraat, Belgium) using Ward’s linkage

and Pearson correlation. Redundancy analyses (RDA) were

used to correlate rhizosphere microbial band patterns with

crop factors such as rootstock genotype, tree growth and

yield data, with Canocow software (Canoco 4.5, Ithaca,

NY). The DNA sequences were identified to probable

species by use of the NCBI GenBank database (http://www.

ncbi.nlm.nih.gov), and DNAStar software was used for

sequence editing and alignment (DNAStar, Madison, WI).

3. Results

3.1. Bulk soil characteristics in the years following

pre-plant soil treatments

Soil P content was 3–5 fold higher in compost-amended

treatments, compared to the control and fumigation

treatments (2002 data provided in Table 3). Soil K, Ca

and organic matter (OM) contents and pH values were also

significantly higher. The available soil Ca content was twice

that in soil receiving compost, as compared to other

treatments. Control and fumigation plots had more soil Fe

and Al contents than compost treatments (associated with

soil pH differences). There were no significant differences

among the pre-plant treatments for soil Mg, Zn or Cu

contents. Soil from the fumigated and untreated control

plots had similar extractable nutrient contents except for

nitrate, which was highest in the fumigated soil and lowest

in the soil receiving compost, possibly indicating some N

immobilization in this treatment. The fumigation-plus-

compost (FumCCom) and compost-only treatments had

similar plant nutrient availability. Soil in old grass lanes

generally had greater nutrient contents (except for K) during

the first year after planting in 2002; but 1 year later, in 2003,

these trends were no longer significant.

3.1.1. Bulk soil respiration

Soil amended with compost had higher respiration rates

and greater cumulative CO2 production than soil from the

other pre-plant treatments 10 months after planting

(Fig. 1A). There was no difference in soil respiration for

old tree row vs. old grass lane positions within the same

A: Sept. 2002

0.5

1.5

2.5

3.5

4.5

5.5

Days of incubation

Cum

ulat

ive

CO

2 m

g g–1

OD

W s

oil Control Compost

Fum/Com Fumigation

B: Sept. 2003

0.5

1.5

2.5

3.5

4.5

5.5

7 14 21 28 35 42 49

7 14 21 28 35 42 49

Days of incubation

Cum

ulat

ive

CO

2 m

g g–1

OD

W s

oil

Control Compost

Fumigation

Fig. 1. Cumulative respiration rates in soil from different pre-plant soil

treatments measured by a sealed jar incubation method at 22 8C. Repeated

weekly measurements were taken over a seven-week period (nZ5, values

are meanGSE).


treatments, so the data were combined for the two planting

positions in each soil treatment. In 2003, 22 months after

planting, there were no longer any differences in bulk soil

respiration rate or cumulative CO2 evolution between the

soil treatments (Fig. 1B).

3.1.2. Soil nematode populations

The soil populations of plant-parasitic nematodes in this

orchard were low and generally below the reported damage

Table 4

Nematode populations and trophic groups in July 2002 in relation to different pre

Treatments

Helicoty-

lenchus

(Spiral

nematode)

Heterodera

(Cyst

nematode)

Paratylenchus

(Pin nematode)

Pratylenchus

(Root lesion)

Control 4 12 10.1 86 a

Fumigation 0 6 0 0 b

Compost 0 6 5.3 38 ab

FumCCom 0 6 0 34 ab

Old row 0 0 b 2.7 0 b

Grass Lane 2 15 a 5.0 79 a

Values are the mean number of nematodes per 100 cc soil, for nZ5. Data were tran

significantly different at P!0.05.

thresholds for apple (Jaffee et al., 1982). Ten months after

soil treatments were applied, lesion and root-knot nema-

todes (Pratylenchus and Meloidogyne sp., respectively)

were significantly lower in the fumigated soil as compared

to the control treatment (Table 4). Counts of these

nematodes, as well as the cyst nematode (Heterodera sp.),

were higher in the old grass drive lanes of the previous

orchard as compared to the old tree rows. Free-living

nematode counts were also significantly higher in the old

grass lanes as compared to the old tree rows. No significant

differences were observed in soil populations of free-living

nematodes among the other pre-plant soil treatments

(Table 4).

3.1.3. Bulk soil microbial community analyses

3.1.3.1. Bacteria. In the first year after soil fumigation with

Telone C-17, bacterial communities in the fumigated soils

separated distinctly from those in the other soil pre-plant

treatments, based on their PCR-DGGE fingerprint patterns

(Figs. 2 and 3A,B). Bands 1 and 2 in the July 2002 samples

(Fig. 2) were dominant in the fumigation treatments,

whereas these bands were weak or non-existent in the

control and compost treatments. Bands 3 and 4 (Fig. 2) were

more prevalent in the compost treatment than in the control

and fumigation treatments. At 22 months after replanting,

PCR-DGGE fingerprint patterns had become more similar,

with no clear differences among pre-plant treatments.

Cluster analysis of the 2002 soil samples at 8 and 10

months after replanting indicated that each pre-plant soil

treatment comprised a distinct group (Fig. 3A and B).

The two fumigation treatments differed more from the

untreated control treatment than from the compost-only

treatment.

During the following year, the PCR-DGGE fingerprints

from fumigation and control treatments became more

similar relative to compost treatments even though each

treatment comprised an individual group cluster at 10

months after replanting. By May of 2003, 18 months after

replanting, the compost-treated soils still grouped closely,

and apart from soils sampled from the fumigation and

control treatments, which were intercalated to some extent

-plant soil treatments and planting positions

Tylenchor-

hynchus

(stunt

nematode)

Tylenchus Meloidogne

(Root knot)

Free

living

Herbivores/

Free living

6 4 122 a 682 0.36

0 4 10 b 694 0.03

0 16 65 ab 890 0.14

0 4 44 ab 778 0.11

3 5 11 b 434 b 0.05

0 9 110 a 1088 a 0.20

sformed to ln (xC1) before analysis. Means followed by different letters are

Control Fumigation CompostOR GL OR GL OR GL

2

3

4

1

Fig. 2. Denaturing gradient gel electrophoresis (DGGE) gel images of the

bacterial community composition in different pre-plant soil treatments in an

apple replant orchard; OR, old row; GL, grass lane. The numbers and

arrows on the gels indicate specific bands related to treatments.


(Fig. 3C). By Sept. 2003, 22 months after replanting, there

was no longer any clear clustering of the treatments

(Fig. 3D). The bacterial community composition of bulk

soil was not greatly affected by tree planting position in old

grass lanes or old tree rows.

Fig. 3. Differences in community composition of Bacteria in the bulk soil among di

cluster analysis with Ward’s linkage and Pearson correlation analysis of DGGE

CKG-control treatment, grass lane position; FO-fumigation treatment, old row pos

old row position; CG-compost treatment, grass lane position.

3.1.3.2. Fungi. Pre-plant treatments also affected soil fungal

community composition (Fig. 4). Hierarchical cluster

analysis grouped both fumigation treatments together and

separately from the compost and control treatments in July

2002, 8 months after replanting (Fig. 4A). By Sept. 2002, 10

months after replanting, there were no longer any significant

treatment groupings for fungi associated with the three soil

pre-plant treatments (Fig. 4B). The fungal community

appeared to equilibrate faster than the bacterial community

after the various pre-plant treatments were applied.

3.2. Rhizosphere soil microbial communities associated

with different rootstocks

3.2.1. Culturable bacteria and pseudomonads

Among the rhizosphere soils sampled from different

rootstocks in June 2004, 31 months after replanting, the

Malling series rootstock, M.26, had the highest number of

culturable soil bacteria and pseudomonads compared to

counts from rhizosphere soils of the three Cornell–Geneva

series rootstocks (coded ‘CG’ before and ‘G’ after

commercial release) (Table 5). Rootstock genotype M.26

also had more culturable pseudomonads than M.7, which

fferent pre-plant soil treatments at varying times as detected by Hierarchical

gels. Treatments are as follows: CKO-control treatment, old row position;

ition; FG-fumigation treatment, grass lane position; CO-compost treatment,

Fig. 4. Hierarchical cluster analysis of DGGE gel images of the fungal community composition in bulk soil of different pre-plant soil treatments and planting

positions in 2002. Treatment legends are the same as those given in Fig. 3.


had a higher number of culturable soil bacteria than

CG.6210, and higher pseudomonad counts than G.16.

There were no differences in any microbial plate counts

among the three CG rootstocks we tested. When the

rootstocks were grouped for comparison of M (Malling)

vs. CG or G (Cornell–Geneva) series, the Malling series

supported higher numbers of culturable soil bacteria and

pseudomonads than the Cornell–Geneva series rootstocks.

Table 5

Number of colony forming units [log (CFU gK1 soil)] of bacteria and

pseudomonads cultured on R2A and King’s medium, respectively, in soil

from the rhizosphere of five apple rootstocks sampled in June 2004 (nZ12

MeanGSE, PZ0.05). Means followed by different letters are significantly

different at P!0.05

Rootstocks Bacteria Pseudomonads

M.26 7.21G6.28 a 6.34G5.54 a

M.7 7.07G6.17 ab 5.22G5.38 b

G.16 6.99G6.14 bc 5.96G5.63 b

CG.30 6.97G5.97 bc 5.90G5.25 b

CG.6210 6.88G5.82 c 5.99G5.36 b

3.2.2. Soil microbial community analysis

Pseudomonas spp. and fungal community PCR-DGGE

fingerprints derived from rhizosphere soils sampled 31

months after replanting from the rootstocks CG.6210 and

G.30 clustered together in the hierarchical cluster analysis

(Fig. 5A and B), with the exception of one replicate from

CG.6210. For bacteria, G.30 and M.26 clustered together

and separately from the other rootstocks (Fig. 5C). The

other rootstocks did not show any clear grouping for

bacteria, Pseudomonas or fungal communities at this

sampling date (Fig. 5A and C).

The DGGE fingerprints of rhizosphere oomycetes

revealed a single dominant band (OTU) for all the apple

rootstock genotypes (data not shown). Twenty-two DGGE

bands were excised and cloned from the oomycetes gel.

Sixty-one individual sequences, derived from the resulting

clone library, were submitted to GenBank. Sequencing

results indicated that Pythium species were present in the

rhizosphere of all rootstocks. Phytophthora sp. also existed

in several bands, but their sequences were less frequently

recovered than Pythium species. Only a few clones in our

samples matched known species, which made it difficult to

compare the oomycete community composition in the

rhizosphere of the different rootstocks. Ten sequences of

around 286 bp in length did not match any accessions in the

NCBI database. These could be new species, or known

species, where no 28S ribosomal RNA gene sequences have

yet been deposited in the GenBank database. The GenBank

accession numbers of the 61 oomycete sequences obtained

in this study are AY748367–AY748427.

3.3. Tree growth and yield

Rootstock genotypes and planting position were the

dominant factors affecting tree growth and yield in this

experiment (Table 6) (Leinfelder, 2005). Despite the

differences observed in soil microbial communities among

pre-plant soil treatments in our study, there were no

detectable effects of pre-plant soil treatment on tree

performance. After three years of observations, trees on

the rootstock genotypes CG.6210, G.30 and M.7 had greater

cumulative trunk-diameter growth than those on the M.26

and G.16 rootstocks. Trees planted in the old grass lanes had

greater trunk diameters than those in the old tree rows. Trees

on the CG.6210 rootstocks produced more fruit in 2004 than

all other rootstocks tested; trees on the G.30 rootstocks also

had higher yields than those on M.7, G.16 and M.26, among

which there were no differences in yield.

3.4. Redundancy analyses

The redundancy analyses of tree growth and yield data

with rhizosphere microbial communities revealed that M or

CG rootstocks accounted for 13% of the total variance in

rhizosphere fungal communities. Lateral extension growth

in 2003 and flower cluster number in 2004 accounted for 14

and 11%, respectively, of the variance in rhizosphere

Fig. 5. DGGE gel images and cluster analysis of fingerprints of (A) fungi,

(B) Pseudomonas, and (C) Bacteria in the rhizosphere soil from five

different apple rootstocks in the replanted orchard in June, 2004, 31 months

after replanting.


Pseudomonas communities. No other significant relation-

ships were revealed in this analysis (data not shown).

4. Discussion

4.1. Pre-plant soil treatment effects on bulk soil

microbial communities

Soil fumigation with Telone C-17 changed the bulk soil

microbial community composition as measured by PCR-

DGGE fingerprinting, but changes in fungal and bacterial

community composition were not detectable at 10 and 22

months after replanting, respectively. One of the com-

ponents of Telone C-17 is chloropicrin, a broad spectrum

fumigant with strong fungicidal activity, that has little

activity against nematodes (Duniway, 2002). Chloropicrin

in combination with the nematicide 1,3-dichloropropene is

purported to have broad spectrum activity against soil fungi

and nematodes. In our study, Telone C-17 affected bacterial,

fungal and nematode populations, but not tree growth in the

three years following the pre-plant soil treatments or yield in

the first fruiting year. Although cool and wet soil conditions

can cause problems with fumigation in the Northeast, the

weather during autumn of 2001 at our test site was relatively

warm and dry, and soil conditions during treatment were

favorable for fumigation efficacy. The subsequent impacts

of Telone C-17 on soil fungi, bacteria and parasitic

nematodes in our experiment indicate that this fumigant

did suppress some potential ARD pathogens, but without

concomitant responses or economic benefits for tree growth

and yield. These observations, coupled with previous

studies that showed little benefit from orchard fumigation

with Telone C-17 and metam sodium (Merwin et al., 2001),

indicate that soil fumigation may not be a reliable tactic for

controlling ARD in New York orchards.

Ibekwe et al. (2001) found that microbial community

diversity was lower in fumigated than in non-fumigated soil

at 12 weeks after fumigation. In our experiment, fumigation

effects on the soil bacterial community persisted strongly for

1 year, and remained detectable two years after treatment.

As to bacterial population shifts after fumigation, some

reports have suggested that the soil bacterial community

after fumigation is dominated by Gram-negative bacteria

(Martin, 2003), while others have reported that Gram-

positive bacteria recover preferentially after fumigation

(Inagaki et al., 2002; Macalady et al., 1998). Our DGGE

fingerprint assays suggested that bacterial and fungal

communities were affected for a much longer time after

fumigation compared to most previous studies.

Soil fumigation also affected soil nematode populations

and trophic groups (Table 4). Pre-plant fumigation

suppressed some plant-parasitic nematodes in our exper-

iment; but the populations of free-living nematodes were

less affected, or recovered faster than plant-parasitic

nematodes, perhaps because non-parasitic nematodes have

shorter life cycles. Plant-parasitic nematodes were not a

dominant factor in this ARD site, and the lack of tree growth

response to soil nematode suppression was not surprising,

considering that the population counts in soils following all

treatments were below the damage threshold level reported

for apple (Jaffee et al., 1982).

The compost treatment affected soil bacterial and fungal

community composition, and increased soil microbial

activity as measured by soil respiration (Fig. 1). Some

otherwise minor bands became dominant in the DGGE

fingerprints of compost-amended soil (Figs. 2 and 3).

Various compost amendments have been reported to

suppress soilborne pathogens in previous studies (Hoitink

Table 6

Tree performance in relation to five different rootstocks, four soil pre-plant treatments and two orchard positions (Leinfelder, 2005)

Rootstocks Caliper (mm) Extension growth of central

leader (cm)

Lateral extension

growth (cm)

Flower cluster Yield 2004

2002 2003 2002 2003 2003 2004 count kg treeK1

M.26 13.6 b 22.8 c 47.9 c 54.4 b 57.2 c 134 c 58 c 10.4 c

M.7 14.1 b 25.7 b 66.4 b 73.5 a 71.7 b 152 c 63 c 11.1 c

G.16 14.4 b 24.6 b 71.5 b 50.8 b 55.6 c 168 c 72 c 12.5 c

CG.30 14.5 b 25.8 b 66.6 b 74.5 a 80.9 a 184 b 101 b 17.7 b

CG.6210 16.5 a 31.0 a 84.6 a 72.7 a 79.4 a 268 a 153 a 26.1 a

Grass lane 15.0 a 26.6 a 70.5 a 68.2 a 70.2 183 91 15.8

Old row 14.3 b 25.9 b 64.3 b 62.2 b 67.7 179 87 15.3

Control 15.3 a 26.3 70.0 64.1 67.1 ab 182 93 15.8

Fumigation 14.4 ab 25.9 68.6 64.4 64.4 b 186 86 14.9

Compost 14.3 b 25.5 64.5 66.7 72.0 a 171 88 15.5

Fum/Com 14.5 ab 26.3 66.4 65.4 70.5 ab 188 91 16.0

Means followed by different letters are significantly different at P!0.05.


et al., 1993, 1997a). For example, Lumsden et al. (1986)

successfully used composted sewage sludge to control

lettuce drop in the field caused by Sclerotina minor. Most

researchers have attributed disease suppression by compost

amendments to biological effects (Dissanayake and Hoy,

1999; van Bruggen and Semenov, 1999). Without cloning

and sequencing the amplicons comprising distinct bands in

our DGGE gels, we could not relate our observations to

changes in known species composition. However, even with

cloning and sequencing, an exact match to known species is

not commonly obtained. At best, most sequences from soil

studies can be grouped to genera, which do not provide a

good link to species function. Regardless, the soil microbial

trends and changes we observed in the compost-amended

soil relative to other pre-plant treatments had no obvious

direct effects on tree performance in our experiment.

4.2. Effect of soil pre-plant treatments on soil microbial

communities in relation to apple growth and yield

Although fumigation reduced counts of root-lesion and

root-knot nematodes in 2002, altered soil fungal commu-

nities for a year and soil bacterial communities for 2 years

following soil treatments, these changes in soil populations

were not correlated with measured differences in tree

growth or yield in our study. Others have reported that

compost and other organic amendments have been

ineffective in controlling ARD (Granatstein and Mazzola,

2001; Neilsen et al., 2004). Compared with some previous

ARD management studies in New York State (Arneson and

Mai, 1976; Mai and Abawi, 1981) and elsewhere (McKenry

et al., 1994), the tree stunting and reduced yields in our

study were relatively mild, even for the poorly performing

Malling rootstock genotypes. Although previous studies

have confirmed the beneficial effects of soil fumigants such

as MB on tree growth in replanted orchards (Covey et al.,

1979; Mai and Abawi, 1981; McKenry et al., 1994),

fumigation with Telone C-17 did not increase yield or tree

growth in our experiment. We speculate that this could be

due to the rate or formulation of the fumigant used, limited

penetration of the fumigant into the soil at this site due to its

silty clay texture, or other environmental factors singly or in

combination. Inconsistent effects of the use of soil

fumigants in 23 New York State orchards, on a range of

soil types, has been reported previously (Merwin et al.,

2001).

4.3. Rhizosphere microbial communities of the five apple

rootstock genotypes

Rumberger et al. (2004) reported that apple rootstock

genotype had a stronger effect on the rhizosphere soil

microbial community composition than did the pre-plant

soil treatments in soils sampled from this site in 2002. We

found that 2 years later, rhizosphere communities of

bacteria, fungi, and Pseudomonas still clustered roughly

together by rootstock genotype (Fig. 5). Plant species-

specific rhizosphere microbial communities have been

reported widely (Marschner et al., 2001; Miethling et al.,

2000; Westover et al., 1997) as have changes in rhizosphere

microbial communities due to intra-specific variation

(Carelli et al., 2000; Cattelan et al., 1998; Di Giovanni

et al., 1999). In our experiment, the same scion variety

(‘Royal Empire’) was grafted onto five different apple

rootstock genotypes. Thus, differences in rhizosphere

microbial community fingerprints resulted mainly from

differences in the rhizosphere environment provided by the

different rootstock genotypes. These differences may be due

to the variation in the amount and/or chemical composition

of their rhizodeposits (e.g. Grayston and Campbell, 1996;

Marschner et al., 2001; Merbach et al., 1999).

The rhizosphere of M.26 had the highest culturable soil

bacteria counts compared with the other rootstocks, and this

rootstock also produced the least tree growth and lowest

apple yields. Higher counts of culturable bacteria may

indicate that M.26 exports more carbon to its rhizosphere.

However, increased rhizodeposition could lead either to

stimulation of beneficial microbes which in turn might


stimulate tree growth, or to a greater number of deleterious

rhizobacteria or fungi, thus potentially reducing tree growth.

The CG-series rootstock genotypes generally supported

lower soil microbial populations and produced better tree

growth and yields, compared to the Malling series.

Differences in root exudate quantity or composition

among the various rootstocks, and their effects on soil

microbial community composition and tree performance are

worthwhile subjects for further investigation.

Oomycete DGGE fingerprints grouped the five root-

stocks into two separate groups, with no obvious relation to

tree performance in the field. Pythium spp. were found in all

samples, which is consistent with other reports in the

literature suggesting that Pythium is a common factor in

ARD (Sewell, 1981). The primer pair used, targeting the

28S ribosomal RNA gene, only amplified oomycetes in the

class Peronosporomycetes. The 28S rRNA gene has been

used previously to identify oomycetes. However, the

database of 28S rRNA gene sequences is still quite limited.

Some known species of oomycetes have not yet been

sequenced and included in the database. Hence, many of the

sequences of oomycetes we obtained could not be traced to

species. Thus, their identity and possible function in the

ecosystem remains unknown.

Rootstock genotype and orchard planting location were

the dominant factors influencing tree performance in this

and several other orchard replant experiments (Facteau

et al., 1996; Foote et al., 2001; Rumberger et al., 2004).

After tree planting, rootstocks can modify their soil

microenvironment and make it more or less suitable for

their own growth and development. Plant genotypes can

also influence the community composition of saprophytic

microbes in the rhizosphere (Gu and Mazzola, 2003;

Mazzola et al., 2004). Gu and Mazzola (2003) reported

that pseudomonad community composition in the rhizo-

sphere of newly planted apple trees was affected by prior

culture of wheat (Triticum aestivum) before orchard

replanting; and that the composition of the bacterial

community in the rhizosphere of apple depended on the

specific wheat cultivar cropped previously.

In our experiment, rootstock genotype strongly affected

rhizosphere microbial community composition (Fig. 5). The

cluster dendrograms of different microbes, tree performance

variables, and redundancy analyses revealed that CG.6210

and G.30, which were the best performing rootstocks, also

had similar rhizosphere fungal and Pseudomonas commu-

nity structures. This suggests that rhizosphere fungi and

Pseudomonas communities may be more influential in the

promulgation or suppression of ARD than bacteria and

oomycetes at this site. These findings are similar to those of

Mazzola that also implicated the involvement of fungi and

pseudomonads in ARD (Gu and Mazzola, 2003; Mazzola,

1997, 1998). Rootstocks were not only a main factor

contributing to observed changes microbial composition in

the rhizosphere, but were also a dominant factor for tree

growth and yield. Rootstock genotype selection is thus a

promising alternative for managing ARD.

Acknowledgements

This research was completed with partial support from

USDA-IREE Project NYC-145560, the NYS-IPM program,

and a CSREES Hatch grant NYC-145409.

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