Tree-distance and tree-species effects on soil biota in a temperate agroforestry system

Abstract

Aims
Cropland agroforestry systems are land-use systems with numerous environmental advantages over monoculture croplands including promotion of soil life. This study aimed to investigate tree-species and tree-distance effects on soil biota in a temperate agroforestry system.

Methods
Our study was conducted at a paired alley-cropping and monoculture cropland system. The tree rows of the agroforestry system comprised of blocks of poplar Fritzi Pauley, poplar Max 1 or black locust. Within the agroforestry system, soil microbial and earthworm communities were collected along transects spanning from the center of the tree rows into the crop rows. Archaea, bacteria, and fungi were quantified using real-time PCR. The community composition of fungi and earthworms was deciphered using amplicon sequencing and morphological identification, respectively.

Results
Tree rows promoted the abundance of bacteria and earthworms, which we attribute mainly to tree litter input and the absence of tillage. Fungal community composition was altered by the tree rows, resulting in an increased proportion of ectomycorrhizal fungi in the tree-row associated mycobiome. The proportion of Blumeria graminis, the causal agent of powdery mildew, increased with increasing distance from the trees. We suggest that enhanced microbial antagonism, increased earthworm densities and/or altered microclimate contributed to the suppression of B. graminis in vicinity of the trees. Tree-species effect had a minor influence on the abundance and composition of soil communities at our study site.

Conclusions
In comparison to monoculture cropland, agroforestry benefits the abundance, diversity, and function of soil biota and may enhance soil suppressiveness.

Full text

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https://doi.org/10.1007/s11104-023-05932-9
RESEARCH ARTICLE
Tree‑distance andtree‑species effects onsoil biota
inatemperate agroforestry system
AnnaVaupel· ZitaBednar· NadineHerwig·
BerndHommel· VirnaEstefaniaMoran‑Rodas·
LukasBeule
Received: 30 October 2022 / Accepted: 5 February 2023
© The Author(s) 2023
Abstract
Aims Cropland agroforestry systems are land-use
systems with numerous environmental advantages
over monoculture croplands including promotion of
soil life. This study aimed to investigate tree-species
and tree-distance effects on soil biota in a temperate
agroforestry system.
Methods Our study was conducted at a paired alley-
cropping and monoculture cropland system. The tree
rows of the agroforestry system comprised of blocks
of poplar Fritzi Pauley, poplar Max 1 or black locust.
Within the agroforestry system, soil microbial and
earthworm communities were collected along tran-
sects spanning from the center of the tree rows into
the crop rows. Archaea, bacteria, and fungi were
quantified using real-time PCR. The community com-
position of fungi and earthworms was deciphered
using amplicon sequencing and morphological iden-
tification, respectively.
Results Tree rows promoted the abundance of bac-
teria and earthworms, which we attribute mainly to
tree litter input and the absence of tillage. Fungal
community composition was altered by the tree rows,
resulting in an increased proportion of ectomycorrhi-
zal fungi in the tree-row associated mycobiome. The
proportion of Blumeria graminis, the causal agent of
powdery mildew, increased with increasing distance
from the trees. We suggest that enhanced microbial
antagonism, increased earthworm densities and/or
altered microclimate contributed to the suppression
of B. graminis in vicinity of the trees. Tree-species
effect had a minor influence on the abundance and
composition of soil communities at our study site.
Conclusions In comparison to monoculture crop-
land, agroforestry benefits the abundance, diver-
sity, and function of soil biota and may enhance soil
suppressiveness.
Keywords Temperate agroforestry· Alley
cropping· Earthworms· Soil microorganisms· Soil
mycobiome· Soil suppressiveness
Responsible Editor: Timothy J. Fahey.
Anna Vaupel and Lukas Beule contributed equally to this
work.
Supplementary Information The online version
contains supplementary material available at https:// doi.
org/ 10. 1007/ s11104- 023- 05932-9.
A.Vaupel· Z.Bednar· N.Herwig· B.Hommel·
L.Beule(*)
Institute forEcological Chemistry, Plant Analysis
andStored Product Protection, Julius Kühn Institute
(JKI)—Federal Research Centre forCultivated Plants,
14195Berlin, Germany
e-mail: lukas.beule@julius-kuehn.de
V.E.Moran-Rodas
Department ofCrop Sciences, Division ofAgricultural
Entomology, University ofGoettingen, Grisebachstraße 6,
37077Goettingen, Germany
/ Published online: 17 February 2023
Plant Soil (2023) 487:355–372
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Introduction
Agroforestry systems are land-use systems that com-
bine trees with crops and/or livestock. In the temper-
ate zone, alley-cropping agroforestry systems that
alternate rows of trees with rows of crops are gain-
ing popularity. Tree rows of these systems usually
consist of either fast-growing trees (e.g. poplar or
willow) for biomass production or quality hardwoods
such as cherry or walnut trees. The environmental
benefits of temperate agroforestry practice are well
recognized and include, inter alia, increased carbon
sequestration (e.g. Mayer etal. 2022), reduced nutri-
ent leaching (e.g. Allen etal. 2004; Wang etal. 2011),
reduced soil erosion, and promotion of biodiversity
(e.g. Varah etal. 2013, 2020). Certain advantages of
alley-cropping systems over monoculture croplands,
such as the complementary use of resources, are due
to interspecific interactions between the trees and the
crops (Jose et al. 2004). For example, deep-rooting
trees are able to take up nutrients leached below the
rooting zone of the crops (‘safety-net’-function of the
trees; Allen etal. 2004) and make them available to
crops via litter fall. This process was recently dubbed
as ‘nutrient pumping’ (Isaac and Borden 2019) and is
supported by findings of increased soil nutrient avail-
ability in vicinity of the tree rows of temperate agro-
forestry systems (Pardon etal. 2017).
Soil biota are known to predominantly benefit
from temperate agroforestry practice (Beule et al.
2022a; Marsden etal. 2020). Several studies on soil
microorganisms in alley-cropping systems revealed
that the tree rows promote microbial population size
and that this effect is not only limited to the tree rows
but extends gradually into the crop rows (i.e. stronger
promotion in vicinity of the trees) (e.g. Beule etal.
2022b; Guillot etal. 2021). Furthermore, soil micro-
biome studies reported that the composition of bacte-
rial and fungal communities strongly differs between
the tree and crop rows (e.g. Beule etal. 2021; Beule
& Karlovsky 2021a). Based on these studies, it
was recently reviewed that alley-cropping systems
increase microbial diversity in soil through increased
beta diversity rather than alpha diversity (Beule etal.
2022a). Recent articles showed that soil fungal com-
munities strongly respond to temperate agroforestry
practices. For example, one study found that Basidi-
omycota in soil were up to 330 times more abun-
dant in poplar tree rows of alley-cropping systems as
compared to adjacent cropland monocultures (Beule
etal. 2021). The same study used amplicon sequenc-
ing to investigate the composition of the soil fungal
community and found that certain ectomycorrhizal
fungi (Cortinarius,Geopora, andInocybe) were par-
ticularly promoted by poplar trees, which holds great
potential to improve nutrient acquisition in agro-
forestry systems (Beule etal. 2021). In the present
study, we aimed to explore the impact of different tree
species on soil fungal communities in agroforestry
systems.
In 1999, Seiter and co-workers (1999) observed
that most of the tree leaf litter in an alder–maize alley-
cropping system was pulled into the burrows by Lum-
bricus terrestris but did not provide data to substanti-
ate their field observation. Few months earlier, Price
and Gordon (1998) published an article demonstrat-
ing tree-species specific promotion of earthworms
through tree rows in an alley-cropping system. Since
then, greater densities of earthworms in tree rows of
temperate agroforestry systems as compared to bor-
dering crop rows or adjacent monoculture croplands
have been reported (Cardinael etal. 2019; D’Hervilly
et al. 2022). Yet, studies investigating earthworm
communities in temperate agroforestry systems along
fine spatial gradients from the tree rows into the crop
rows comprising more than two distances from the
tree rows are scarce. Furthermore, most temperate
agroforestry systems feature either a single tree spe-
cies or an intermixture of tree species (e.g. Cardinael
etal. 2019), thus disabling direct comparisons among
tree species within the same system. To our best
knowledge, the investigation by Price and Gordon
(1998) is the only study to assess earthworm com-
munities under different tree species within the same
temperate agroforestry system. However, the authors
did not report species identities or ecological groups
of collected earthworms. Recent studies indicated dif-
ferences in the response of different ecological groups
of earthworms to agroforestry practice (Cardinael
etal. 2019; D’Hervilly etal. 2022), highlighting the
importance of investigating them.
In the present study, we chose two poplar species,
as poplar trees are the most commonly planted short-
rotation trees in modern alley-cropping agroforestry
systems in the temperate zone. Although not com-
monly planted in temperate agroforestry systems,
we further chose black locust as it is a fast-growing,
nitrogen-fixing tree species. We expect differences
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in overall soil biota communities under different tree
species because root architecture, litter quality, and
soil-nutrient cycling are tree species-specific (e.g.
Das and Chaturvedi 2008, Aponte etal. 2013, Borden
etal. 2017).
This study aimed to investigate the impact of three
different tree species (two poplar species and black
locust) on representatives of the soil biota commu-
nity (archaea, bacteria, fungi, and earthworms) in a
temperate alley-cropping agroforestry system. Fur-
thermore, tree-distance effects on soil biota were
tested by sampling multiple locations along transects
spanning from the tree row into the crop row of the
agroforestry system. We hypothesized that tree rows
promote the abundance and alter the community
composition of soil biota. We further hypothesized
that these changes are dependent on the distance to
the trees (tree-distance effect) as well as the tree spe-
cies (tree-species effect).
Materials andMethods
Study site and sampling design
Our study site was located on a Gleyic Cambi-
sol soil (IUSS Working Group WRB 2015) near
Forst, Brandenburg, Germany (51°47′11″N,
14°38′05″E; m.a.m.s.l.: 67 m; mean annual tem-
perature: 9.6 ± 0.2 °C; mean annual precipitation:
568 ± 21 mm; Fig. 1A), which is located in the gla-
cially influenced region of the North German Plain.
General biochemical and physical soil properties
of the site were recently characterized by Schmidt
et al. (2021). According to aerial images, the study
site was under agricultural use for at least 50years
prior to conversion of cropland monoculture to alley-
cropping agroforestry. At the study site, a conven-
tionally managed alley-cropping agroforestry system
was spatially paired with a conventional monoculture
cropland system that served as a reference land use.
The agroforestry system was established in 2010 and
was 12years old during sampling. The system con-
sisted of 12m-wide rows of trees (north–south orien-
tation) that were alternated with 48 m-wide rows of
arable crops (Fig.1B). Tree rows were hand planted
and consisted of three different tree species. The tree
rows comprised four individual rows of trees (Fig.1
C, D, E). The tree rows that defined the crop row con-
sisted of three different tree species that were planted
in alternating segments at a length of approx. 165m
(Fig.1B). The different tree species were i) Populus
trichocarpa cv. Fritzi Pauley (referred to as ‘poplar
Fritzi Pauley’; Fig.1C), ii) P. nigra × P.maximowiczii
C D
E
poplar Fritzi Pauley
poplar Max 1
black locust
cropland
replicate plot
sampling location *
agroforestry cropland monoculture
cropland
BA
N
Forst
*in theagroforestrycropland, samples
were collectedinthe tree rowaswellas
in thecroprow at 1, 7, and18m
distance from thetrees.Inthe
monoculturecropland, sampleswere
collected in each replicateplot.
12 m48 m
Fig. 1 Study site and study design. Location of the study site
near Forst (federal state of Brandenburg), Germany (A), study
design (B), and photos of the three different tree species culti-
vated at the site (poplar Fritzi Pauley (C), poplar Max 1 (D),
and black locust (E)) taken in April 2022. Photo credit: Lukas
Beule
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cv. Max 1 (referred to as ‘poplar Max 1’; Fig.1D),
and iii) black locust (Robinia pseudoacacia) (referred
to as ‘black locust’; Fig.1E). The aboveground bio-
mass of the trees was harvested using a forage har-
vester in February 2015, March 2018, and February
2021. Tree harvests were conducted when the soil was
frozen to avoid soil compaction by the machinery.
The management of the crop rows was identical to
that of the monoculture cropland. The crop rotation
was maize (Zea mays) – summer barley (Hordeum
vulgare) – summer oat (Avena strigosa) – winter
wheat (Triticum aestivum) – winter barley (H. vul-
gare) (2018 – 2019 – 2020 – 2021 – 2022). In March
2022, the crop row of the agroforestry system and the
monoculture cropland received 40 – 0 – 0kg N – P
– K ha−1 in the form of mineral fertilizer. Maize and
small-grain cereal crops were harvested with a stand-
ard 24-m wide combine harvester. Crop rows and
monoculture cropland were conventionally ploughed
at a depth of 25cm.
It is well established that trees in alley-cropping
systems introduce spatial heterogeneity (e.g. Guillot
etal. 2021; Beule etal. 2020). Thus, samples in the
agroforestry system were collected along four tran-
sects (replicate plots) per tree species spanning from
the tree row into the crop row. Samples were col-
lected in the center of the tree row as well as at 1m,
7m, and 18m distance from the trees within the crop
row (3 tree species × 4 transects × 4 sampling loca-
tions within each transect = 48 samples; Fig.1B). In
the adjacent monoculture cropland, samples were col-
lected in the center of each replicate plot (4 samples;
Fig.1B).
Soil and earthworm sampling
Soil samples for the determination of the microbial
community and general soil properties (soil pH, soil
organic carbon (SOC), total N, extractable nutrients)
in the upper 5-cm topsoil were collected on 23 Feb-
ruary 2022 (prior to fertilization). Three soil samples
with a volume of 250 cm3 were collected at each sam-
pling location using stainless steel cylinders (5 cm
height), transferred toa sterile polyethylene bag, and
thoroughly homogenized. An aliquot of approx. 50g
fresh soil was transferred into a sterile 50-ml Falcon
tube and frozen at -20°C in the field. Upon arrival
at the laboratory, samples were stored at -20°C until
freeze-drying for 48h. The freeze-dried material was
finely ground using a vortexer (Beule et al. 2019a)
and stored at -20 °C until DNA extraction. The
remaining fresh soil in the polyethylene bag was used
for determination of the general soil properties. Since
earthworm activity at our site is generally low in Feb-
ruary, earthworm communities were sampled on 11
and 12 April 2022 as described below (see Extraction
of earthworms). Soil samples (250 cm3) for the deter-
mination on soil bulk density and water-filled pore
space (WFPS) were collected on all sampling dates
and sampling locations (see above).
Extraction of earthworms
Earthworms were directly extracted from soil by
applying 5 L of 0.01% (w/v) allyl isothiocyanate
(AITC) solution on an area of a quarter square meter
as described by Zaborski (2003). The extractant was
prepared in the field by adding 500mg AITC (pre-
dissolved in 50ml isopropanol (w/v)) to 4.95 L tap
water immediately before extraction. To ensure that
the extractant was applied only to a surface of 0.25
m2, it was poured into a 50 × 50 cm open metal
frame which was embedded approx. 5cm deep into
the soil (FigureS1 A). To allow better monitoring of
extracted worms, plant material within the frame was
carefully removed prior to the application of AITC.
Following the AITC application, the soil surface
within the frame was continuously monitored for at
least 30min and any surfacing earthworms were col-
lected using tweezers, thoroughly washed, and stored
in tap water (FigureS1 B). Upon arrival to the labora-
tory, earthworms were stored at 5°C in the dark until
morphological identification. Morphological identi-
fication and recording of the fresh weight (including
gut content) of each individual earthworm were car-
ried out within 48 h post sampling and earthworms
were subsequently released. Furthermore, earth-
worms were classified into three ecological groups:
anecic, endogeic, and epigeic species, which were
introduced by Bouché in 1972. Earthworm data have
been deposited at the BonaRes repository (https:// doi.
org/ 10. 20387/ bonar es- y3je- zz30).
Soil DNA extraction
DNA from soil was extracted using a CTAB-based
protocol with phenol and chloroform/isoamyl alco-
hol extraction (Beule et al. 2019a). Briefly, 50 mg
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finely ground soil was suspended in a mixture con-
taining 1 ml CTAB, 1 µL 2-mercaptoethanol, and 2
µL pronase E (30mg/ml). The mixture was incubated
at 42°C and subsequently at 65°C for 10min each
and 900 µL phenol were added. The mixture was
shaken, centrifuged at 4,600 × g at room tempera-
ture for 10min, and 800 µL of the supernatant were
transferred into a new 2mL tube. 800 µL chloroform/
isoamyl alcohol (24:1 (v/v)) were added, the mixture
was shaken, incubated on ice for 10min, and centri-
fuged at 4,600 × g at room temperature for 10min.
Following centrifugation, 700 µL of the superna-
tant were transferred into a new 2mL tube, 700 µL
chloroform/isoamyl alcohol were added, the mixture
was shaken, incubated on ice for 10min, and centri-
fuged at 4,600 × g for 10 min at room temperature.
DNA was precipitated by transferring 600 µL of the
supernatant into a new 1.5 mL tube containing 200
µL PEG 6,000 (30% (w/v)) and 100 µL 5 M NaCl.
The mixture was incubated at room temperature for
20min and centrifuged at 20,240 × g at room temper-
ature for 15min to pellet the DNA. DNA pellets were
washed with 70% (v/v) EtOH twice and dried using
a vacuum centrifuge. Dried pellets were re-dissolved
in 50 µL 1 × TE buffer (10mM Tris, 1mM ethylen-
ediaminetetraacetic acid (EDTA); adjusted to pH 8.0
with HCl) and incubated at 42°C for 2h to facilitate
re-dissolving. Extracted DNA was inspected using gel
electrophoresis on 1% (w/v) agarose gels stained with
SYBR Green Solution I (Thermo Fisher Scientific,
Waltham, MA, USA).
Real-time PCR
Real-time PCR assays for the quantification of total
bacteria, total fungi, Basidiomycota, Ascomycota,
Acidobacteria, Actinobacteria, Alpha-, Beta-, Gam-
maproteobacteria, Bacteriodetes, and Firmicutes were
performed in a Peqstar 96Q thermocycler (PEQLAB,
Erlangen, Germany). The composition of the master-
mix and the thermocycling conditions correspond to
those described by Beule etal. (2020). Total archaea
were amplified using the primer pair ARC787F and
ARC1059R (Yu et al. 2005). The reaction mixture
comprised of 3 µL mastermix (double distilled H2O,
buffer (10 mM Tris–HCl, 50 mM KCl, 2.0 mM
MgCl2, pH 8.3 at 25°C); 100μM of each deoxynu-
cleoside triphosphate (New England Biolabs, Bev-
erly, Massachusetts, USA); 0.3μM of each primer;
0.1 × SYBR Green I solution (Thermo Fisher Scien-
tific, Waltham, MA, USA); 1μg μL−1bovine serum
albumin; 0.03 u μL−1Hot Start Taq DNA Polymer-
ase (New England Biolabs, Beverly, Massachusetts,
USA)) and 1 μL of template DNA or double distilled
H2O for a negative control. Thermocycling condi-
tions were as follows. Initial denaturation at 95°C for
2min followed by 40 cycles of denaturation (95°C
for 20s), annealing (62 °C for 30s), and elongation
(68°C for 20s). Final elongation was performed at
68 °C for 5 min. Following amplification, melting
curves were generated by step-wise heating of the
PCR product from 65 to 95 °C by 0.5 °C per step
under continuous fluorescence measurement. DNA
extracts were tested for PCR inhibitors according to
Guerra etal. (2020) and diluted 1:50 (v/v) in double
distilled H2O prior to PCR to overcome PCR inhibi-
tion. Real-time PCR data have been deposited at the
BonaRes repository (https:// doi. org/ 10. 20387/ bonar
es- y3je- zz30).
Library preparation and amplicon sequencing of soil
fungi
Sequencing libraries were prepared by amplifying
the fungal ITS1 region using the primer pair ITS1-
F_KYO2 (5’-TAG AGG AAG TAA AAG TCG TAA-
3’) (Toju et al. 2012) / ITS86R (5’-TTC AAA GAT
TCG ATG ATT CA-3’) (Vancov & Keen 2009). PCR
reactions were carried out in 96-well plates in an
Eppendorf Mastercycler EP Gradient S thermocycler
(Eppendorf, Hamburg, Germany) in 25 µL reaction
volume within one PCR run using the same master-
mix for all libraries. The reaction volume comprised
18.75 µL mastermix and 6.25 µL template DNA
diluted 1:50 (v/v) in double distilled H2O or double
distilled H2O for negative a control. The master-
mix contained double distilled H2O, buffer (10mM
Tris–HCl, 50 mM KCl, 2.0 mM MgCl2, pH 8.3 at
25 °C), 100 µM of each deoxynucleoside triphos-
phate (New England Biolabs, Beverly, Massachu-
setts, USA), 0.5µM of each primer (ITS1-F_KYO2 /
ITS86R), 1mg mL−1bovine serum albumin, and 0.03
u µL−1 Hot Start Taq DNA Polymerase (New Eng-
land Biolabs, Beverly, Massachusetts, USA). Each
primer was a mixture of primer with (50%) and with-
out (50%) Illumina TruSeq adapters (5’-GAC GTG
TGC TCT TCC GAT CT-3’ for the forward primer and
5’-ACA CGA CGC TCT TCC GAT CT-3’ for the reverse
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primer) at the 5`-end. The thermocycling conditions
were as per (Beule and Karlovsky 2021a, b): initial
denaturation (95°C for 2min), 3 touch-up cycles of
denaturation (95 °C for 20 s), annealing (50 °C for
30 s), and elongation (68 °C for 60 s) followed by
25 cycles of denaturation (95°C for 20s), annealing
(58°C for 30s), and elongation (68°C for 60s), and
final elongation (68 °C for 5 min). Following ther-
mocycling, successfulness of the amplification was
confirmed by visualizing 2 µL of the PCR product on
1.7% (w/v) agarose gels stained with SYBR Green I
solution (Thermo Fisher Scientific GmbH, Dreieich,
Germany). Gel electrophoresis was performed at
4.6V cm−2for 60min. Libraries were shipped to the
facilities of LGC Genomics (Berlin, Germany) for a
second amplification step using standard i7- and i5-
sequencing adapters and Illumina sequencing. The
second PCR was performed in 20 µL reaction volume
containing 1 × MyTaq buffer (Bioline GmbH, Luck-
enwalde, Germany), 15 pmol of each forward and
reverse i7- and i5- sequencing adapters, 2µl of Bio-
StabII PCR Enhancer (Sigma-Aldrich Chemie GmbH,
Taufkirchen, Germany), and 0.075 u µL−1 MyTaq
DNA polymerase (Bioline GmbH, Luckenwalde,
Germany).The thermocycling conditions were as fol-
lows: initial denaturation (96°C for 1min), 3 touch-
up cycles of denaturation (96°C for 15s), annealing
(50°C for 30s), and elongation (68°C for 90s) fol-
lowed by 7 cycles of denaturation (96 °C for 15 s),
annealing (58°C for 30s), and elongation (68°C for
90s), and final elongation (70 °C for 2min). DNA
quantity was assessed on agarose gels and indexed
sequencing libraries were pooled. The multiplexed
libraries were sequenced using on an Illumina MiSeq
using V3 chemistry (2 × 300bp) (Illumina, Inc., San
Diego, CA, USA) at LGC Genomics, Berlin, Ger-
many. Amplicon sequencing data have been deposited
at NCBI’s Short Read Archive (PRJNA885015).
Processing of amplicon sequencing data
Raw paired-end sequencing data were demulti-
plexed using Illumina’s bcl2fast v. 2.20 (Illumina,
San Diego, CA, USA) and sorted by their barcodes.
Barcodes with more than two mismatches as well
as one-sided and conflicting barcode pairs were
excluded. Furthermore, Illumina sequencing adapt-
ers and primers (allowing three mismatches per
primer) were clipped, whereas reads shorter than
100bp were excluded. Afterwards, reads were pro-
cessed in QIIME 2 version 2022.2 (Bolyen et al.
2019). Sequence quality was evaluated utilizing
the ‘q2-demux’ plugin. Using DADA2 (Callahan
et al. 2016), forward and reverse reads were trun-
cated to 220 and 180 bp, respectively, quality fil-
tered (allowing two expected errors per read), and
merged. Moreover, chimeras and singletons were
removed. Afterwards, reads were clustered into
exact amplicon sequence variants (ASVs). ASVs
were taxonomically classified against the UNITE
database version 8.3 QIIME developer release
(Abarenkov etal. 2021) using a scikit-learn Naive
Bayes machine-learning classifier (‘q2-fit-classifier-
naive-bayes’ and ‘q2- classify-sklearn’ plugin) in
the ‘balanced’ configuration ([6,6]; 0.96) as sug-
gested by Bokulich etal. (2018). After non-fungal
ASVs were removed, 3,945,026 sequence counts
remained. Sequence counts were normalized to
41,564 counts per sample using scaling with ranked
subsampling (SRS) (Beule and Karlovsky 2020) in
the R environment v. 4.2.1 (R Core Team 2013) uti-
lizing the ‘SRS’ R package version 0.2.3 (Heidrich
etal. 2021). SRS curves were generated using the
same R package. The normalized dataset contained
2,949 fungal ASVs.
Soil properties
Soil pH was determined from sieved (< 2mm) and
air-dried soil in deionized H2O at a ratio of 1:2.5
(soil/water (w/v)). Soil bulk density was deter-
mined from 250-cm3 soil cores dried at 105°C for
24h. WFPS was determined from the same sample
assuming a particle density of 2.65g cm−3. Double
lactate-extractable phosphorus (PDL) and potas-
sium (KDL) were determined from sieved (< 2mm)
and air-dried soil as per (VDLUFA 1991a). Cal-
cium chloride-extractable magnesium (MgCaCl2)
was determined as described previously (VDLUFA
1991b). Carbonates in sieved (< 2 mm) and air-
dried soil were removed using acid fumigation as
per Harris etal. (2001) and concentrations of SOC
and total nitrogen were determined on a CNS ele-
mental analyzer (Vario EL Cube, Elementar, Ger-
many). Soil properties data have been deposited at
the BonaRes repository (https:// doi. org/ 10. 20387/
bonar es- y3je- zz30).
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Statistical analysis
For each tree species, differences in soil proper-
ties, microbial abundance, and earthworm param-
eters among the different sampling locations within
the agroforestry system (i.e. tree row, 1m, 7 m, and
18 m into the crop row) and the monoculture crop-
land were tested using one-way analysis of variance
(ANOVA) followed by Tukey honestly significant
difference (HSD). Relationships between param-
eters (i.e. soil properties, microbial abundance, and
earthworm parameters) were explored using Spear-
man’s rank correlation test. For the soil fungal com-
munity, alpha (Shannon diversity, Chao1 index, and
Pielou’s evenness) and beta diversity (Bray–Curtis
dissimilarity index) were computed using ‘vegan’ R
package v. 2.6.2 (Oksanen et al. 2013). Differences
in fungal community composition among the differ-
ent sampling locations within the agroforestry sys-
tem and the monoculture cropland were identified
using permutational multivariate analysis of variance
(PERMANOVA) based on Bray–Curtis dissimilari-
ties using the ‘vegan’ R package. Multivariatehomo-
geneity of dispersions was tested using the same R
package. Furthermore, differential abundance analy-
sis using data collapsed at genus level was performed
using the ‘metacoder’ R package v. 0.3.5 (Foster etal.
2017). The same package was utilized to generate
heat trees. For each tree species, differences in alpha
diversity and taxonomic groups of fungi among the
different sampling locations within the agroforestry
system and the monoculture cropland were tested
using one-way ANOVA followed by Tukey HSD
test or Kruskal–Wallis test followed by Dunn’s test.
All data were tested for normality of residuals (Sha-
piro–Wilk test) and homogeneity of variance (Lev-
ene’s test). Statistical significance was considered at
p < 0.05. All tests were performed in R version 4.1.2.
Results
Mean earthworm density in the tree rows of the agro-
forestry system was 2.9 to 12.3 times greater than in
the crop rows and the monoculture cropland (Fig.2A)
(p ≤ 0.0216). Likewise, mean earthworm biomass was
5.7 to 34.8 times greater in the tree rows than in the
crop rows and the monoculture cropland (Fig. 2B)
(p ≤ 0.0014). A gradual decline in earthworm den-
sity from the trees into the crop rows and monocul-
ture cropland was evident for all three tree species
(Fig. 2A). For example, earthworm density was
Fig. 2 Earthworm density (A) and biomass (B) in a paired
temperate alley-cropping agroforestry and monoculture crop-
land system in Germany. Poplar Fritzi Pauley, poplar Max 1,
and black locust are different tree species within the agrofor-
estry system. Within the agroforestry system, samples were
collected in the tree row as well as at 1m, 7m, and 18m dis-
tance from the tree row within the crop row. Bars with error
bars represent the mean and its standard deviation (n = 4). Dots
represent individual data points. Different uppercase letters
of the same font indicate statistically significant differences
(p < 0.05)
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greater at 1 m than at 18 m distance from the trees
and monoculture cropland under poplar Max 1 cul-
tivation (Fig. 2A) (p = 0.0344). The gradual decline
in population density was mainly driven by endogeic
(FigureS2 A) rather than anecic species (Figure S2
C). Across sampling locations, population density
and biomass of earthworms were strongly positively
correlated (r = 0.84; p < 0.0001). In total, five differ-
ent earthworm species of three different ecological
groups were found at our study site: Allolobophora
chlorotica, Aporrectodea caliginosa, Aporrectodea
rosea (endogeic species), Lumbricus rubellus (epi-
geic species), and Lumbricus terrestris (anecic spe-
cies). The monoculture cropland was dominated by
endogeic species with no anecic and epigeic species
present (Fig. 3). Anecic species were always pre-
sent in the tree rows of all three tree species ranging
from 13.8 to 44.9% of the total community and were
Fig. 3 Relative abundance of ecological groups of earthworms
in a paired temperate alley-cropping agroforestry and monocul-
ture cropland system in Germany. Poplar Fritzi Pauley, poplar
Max 1, and black locust are different tree species within the
agroforestry system. Within the agroforestry system, samples
were collected in the tree row as well as at 1m, 7m, and 18m
distance from the tree row within the crop row. Bars represent
individual samples (n = 4)
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occasionally recovered in the crop rows (Fig.3). In
the poplar Max 1 system, the epigeic species (i.e. L.
rubellus) was found in half of all replicate plots in the
tree row and at 1m distance from the trees as well as
in a quarter of all the replicate plots at 7m distance
(Fig. 3). Epigeic earthworms were only detected in
one replicate plot of the tree rows in the black locust
system (Fig.3).
We quantified 12 groups of soil microorganisms
(total bacteria, total fungi, total archaea, Basidi-
omycota, Ascomycota, Acidobacteria, Actinobac-
teria, Alpha-, Beta-, Gammaproteobacteria, Bacte-
riodetes and Firmicutes) and found only positive or
no effects of agroforestry on microbial abundance.
Total soil bacterial abundance was greater in the tree
rows than at 18m distance from the trees within the
crop rows (Fig.4A) (p ≤ 0.0378). In the poplar Max
1 system, gene abundance of bacteria as well as Act-
inobacteria was greater under the trees as compared
to 7 and 18m into the crop row and the monoculture
cropland (Fig.4A, C) (p ≤ 0.0288). In the same sys-
tem, greater abundance of Acidobacteria and Bacte-
roidetes was detected in the tree row as compared to
the crop row and the monoculture cropland (Fig.4B,
S3 A) (p ≤ 0.0290) and Firmicutes were more abun-
dant under the trees than at 18 m distance into the
crop row and the monoculture cropland (Figure S3
B) (p ≤ 0.0092). The abundance of Basidiomycota in
the poplar Max 1 system was greater in the tree row
than in the crop row at 7 and 18m distance and in
the monoculture cropland (FigureS4 C) (p ≤ 0.0313).
Gene abundances of total soil fungi, Ascomycota,
total archaea, Alpha-, Beta-, and Gammaproteobac-
teria did not differ among sampling locations (Fig-
ureS4 A-B, S5, S6).
The soil fungal community was dominated
by Ascomycota (51.4 ± 16.7%), Basidiomycota
(32.4 ± 22.4%), and Mor tierellomycota (5.8 ± 4.0%).
The dominant fungal classes were Sordariomycetes
(28.7 ± 14.4%), Agaricomycetes (27.4 ± 22.8%),
Dothideomycetes (6.9 ± 5.3%), and Mortierel-
lomycetes (5.8 ± 4.0%) (Fig. 5A). Acremonium
(8.6 ± 6.2%), Mortierella (4.9 ± 3.4%), Copr inellus
(4.5 ± 8.9%), Laccaria (3.2 ± 9.8%), and Agrocybe
(2.9 ± 10.7%) were the most abundant genera in the
dataset. Alpha diversity (Shannon diversity, Chao1
index, and Pielou’s evenness) did not differ among
sampling locations (i.e. tree row, 1, 7, 18 m crop
rows, and monoculture cropland) within each agro-
forestry system (i.e. poplar Fritzi Pauley, poplar Max
1, and black locust agroforestry system) (TableS1).
Sampling location significantly affected fungal com-
munity composition (TableS2) (p = 0.0001) and was
driven by the differences between the tree row and
the arable land (crop row and monoculture crop-
land) (Fig.5B). Multivariatehomogeneityof disper-
sions was given under all PERMANOVA test con-
ditions performed (p ≥ 0.32). Differential abundance
analysis was visualized by heat trees (Figure S 7) and
a total of 15 genera and species were identified whose
relative abundance was either positively or negatively
affected by agroforestry (Fig.6). Relative abundance
Fig. 4. 16S rRNA gene abundance of total bacteria (A), Aci-
dobacteria (B), and Actinobacteria (C) in soil of a paired tem-
perate alley-cropping agroforestry and monoculture cropland
system in Germany. Poplar Fritzi Pauley, poplar Max 1, and
black locust are different tree species within the agroforestry
system. Within the agroforestry system, topsoil samples were
collected in the tree row as well as at 1m, 7m, and 18m dis-
tance from the tree row within the crop row. Bars with error
bars represent the mean and its standard deviation (n = 4). Dots
represent individual data points. Different uppercase letters
of the same font indicate statistically significant differences
(p < 0.05)
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of Gamsia, Ilyonectria, Laccaria, and Preussia spp.
were generally enhanced by the tree rows, whereas
the increased relative abundance of Inocybe spp. was
specific for poplar Max 1 tree rows (Fig. 6). Rela-
tive abundance of Protomyces spp., Sporobolomyces
spp., and Blumeria graminis showed a positive trend
with increasing distance from the trees. Among these,
Blumeria graminis was absent in the tree rows. Rela-
tive abundance of Mycosphaerella tassiana as well
as Dioszegia, Itersonilia, Lectera, Leucosporidium,
Microdochium, and Neosetophoma spp. was overall
higher in the crop row or monoculture cropland com-
pared to the tree rows; however, no trends regarding
sampling distance were observed (Fig.6).
In the black locust system, SOC and total N con-
centrations in topsoil (upper 0–5 cm soil) were 54
to 97% greater in the tree rows than in the crop row
and monoculture cropland (Fig. 7) (p ≤ 0,0004).
Likewise, in the poplar Max 1 system, SOC con-
centrations in topsoil were greater in the tree rows
than in the crop row and monoculture cropland
(Fig. 7A) (p ≤ 0.0376). Tree rows of poplar Fritzi
Pauley showed greater SOC concentrations than the
crop row at 18m distance (Fig.7A) (p = 0.0093) but
similar total N concentrations among all sampling
locations (Fig.7B). Soil bulk density showed slight
differences across sampling locations, however, no
distinct spatial pattern among sampling locations
was detected (TableS3). In the black locust system,
MgCaCl2 was greater under the trees than in the crop
row (p = 0.0001 – 0.0004) (Table S3). Similarly,
MgCaCl2 in the system with poplar Fritzi Pauley was
greater in the tree row than in the crop row and mon-
oculture cropland (p = 0.0006 – 0.0086) (TableS3).
In both, the poplar Max 1 and the black locust sys-
tem, KDL increased in the tree row as compared to
the crop row and monoculture cropland (p < 0.0001
– 0.0073) (Table S3). Tree rows of poplar Max 1
showed greater PDL as compared to monoculture
cropland (p = 0.0106). Earthworm density and bio-
mass were positively correlated to SOC and total N
concentrations, KDL, MgCaCl2, and WFPS (r = 0.35
– 0.56; p < 0.0001 – 0.011) but not PDL (r = 0.04
– 0.14; p = 0.32 – 0.77) (Figure S8). Positive rela-
tionships were found between soil bacteria, Bacte-
roidetes, Acidobacteria, and Actinobacteria and SOC
and total N concentrations, KDL, MgCaCl2, and PDL
(r = 0.28 – 0.54; p < 0.0001 – 0.041) (FigureS9).
Fig. 5 Soil fungal community composition in a paired temper-
ate alley-cropping agroforestry and monoculture cropland sys-
tem in Germany. Poplar Fritzi Pauley, poplar Max 1, and black
locust are different tree species within the agroforestry system.
Within the agroforestry system, soil samples were collected in
the tree row as well as at 1m, 7 m, and 18m distance from
the tree row within the crop row (n = 4). Relative abundances
of dominant (≥ 0.5% mean relative abundance) fungal classes
(A) are shown. Beta diversity is shown by non-metric multidi-
mensional scaling (NMDS) of Bray–Curtis dissimilarities (B).
Circles, squares, and triangles represent individual data points.
AF = agroforestry
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Discussion
Improved soil fertility through agroforestry
In agreement with previous findings (e.g. Pardon etal.
2017), soil fertility (i.e. SOC, total N, and extractable
nutrients (KDL and MgCaCl2)) increased under the trees
(Fig.7, TableS3). This increase was likely due to the
input of above- and belowground tree litter-derived
nutrients, which declines with increasing distance
Fig. 6 Differentially abundant genera and species in a paired
temperate alley-cropping agroforestry and monoculture crop-
land system in Germany. Poplar Fritzi Pauley, poplar Max 1,
and black locust are different tree species within the agrofor-
estry system. Within the agroforestry system, soil samples
were collected in the tree row as well as at 1m, 7m, and 18m
distance from the tree row within the crop row (n = 4)
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from the trees (Schmidt etal. 2021). Although tree-
derived nutrient inputs can reach several meters
into the crop row (Schmidt et al. 2021), increased
soil fertility did not gradually extend into the crop
rows which we attribute to the relatively young age
(12years) of our system (cf. Pardon etal. 2017). We
expect that as our system ages and more tree litter is
deposited, the increase in soil fertility under the trees
will increasingly extend into the crop rows.
Agroforestry promotes earthworm communities
In the present study, earthworms were sampled using
AITC extraction without hand sorting. Compared to
hand sorting combined with AITC extraction (e.g. as
per ISO (2018) 23611–1), using exclusively AITC
extraction for earthworm sampling may result in reduced
efficacy for juveniles and endogeic species whereas
adult anecic earthworms are well recovered (e.g.Pelosi
et al. 2009; Čoja etal. 2008). Hand sorting, however,
is rarely feasible for large-scale studies as it requires
a substantial amount of labour and may prolong the
sampling campaign (Iannone etal. 2012). Furthermore,
if additional on-site data needs to be collected, chemical
extraction is preferred over hand sorting since it does
not physically disturb the soil (Iannone etal. 2012; Lees
etal. 2016; Tóth etal. 2020).
Unlike Price and Gordon (1998), we did not detect
tree-species effects on earthworm abundance, diver-
sity or community composition at our study site. In
line with previous studies (Cardinael et al. 2019;
D’Hervilly et al. 2022), population size of earth-
worms was greater in the tree rows as compared to
the crop rows (Fig.2A). In agreement with the results
of Cardinael etal. (2019), the increase in density was
evident for all three ecological groups (Figure S2).
Furthermore, we were able to demonstrate that earth-
worm abundance decreases with increasing distance
from the trees (Fig. 2A). This gradual decline was
driven mainly by endogeic species (cf. FigureS2 A,
C, E), which almost exclusively colonize the topsoil.
A recent study conducted in two similarly managed
alley-cropping agroforestry systems of similar age
in Germany found that tree roots in the topsoil of
the tilled crop rows are scarce (Schmidt etal. 2021).
Consequently, endogeic species in the crop row likely
benefited from tree litter input in vicinity of the trees
rather than tree root litter. To our best knowledge,
the present study is the first to investigate earthworm
communities at more than two distances from the tree
rows into the crop rows. Although a greater number
Fig. 7 Soil organic carbon (SOC) (A) and total N concentra-
tions (B) in 0–5cm topsoil in a paired temperate alley-crop-
ping agroforestry and monoculture cropland system in Ger-
many. Poplar Fritzi Pauley, poplar Max 1, and black locust are
different tree species within the agroforestry system. Within
the agroforestry system, topsoil samples were collected in the
tree row as well as at 1m, 7 m, and 18m distance from the
tree row within the crop row. Bars with error bars represent the
mean and its standard deviation (n = 4). Dots represent indi-
vidual data points. Different uppercase letters of the same font
indicate statistically significant differences (p < 0.05)
Plant Soil (2023) 487:355–372366
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of sampling locations is demanding, we believe that
such study designs are essential to improve the char-
acterization of spatial gradients within agroforestry
systems, and thus, the extent by which the tree rows
influence the crop rows and vice versa.
Earthworm communities in the tree rows were
characterized by a shift towards anecic earthworms
(Fig.3), which is in agreement with previous findings
(D’Hervilly etal. 2020, 2021). In contrast to the grad-
ual decrease of endogeic earthworms with increasing
distance from the trees, density of anecic earthworms
did not decline gradually but rapidly dropped which
is most likely due to the tillage in the crop rows. It
is well established that anecic earthworms benefit
from reduced tillage (Chan 2001; Ernst & Emmerling
2009). Unsurprisingly, the absence of tillage under
the trees has recently been identified as a main driver
for the abundance increase of anecic earthworms in
the tree rows of agroforestry systems (Cardinael etal.
2019). Since anecic earthworms feed on the soil sur-
face, litter fall and understory vegetation should also
be considered as promoting factors for deep burrow-
ing species. Additionally, Gilbert et al. (2014) were
able to show that saprophagous earthworms, includ-
ing Lumbricus terrestris, benefit from tree fine roots
and fine root associated-mycorrhizae as a feeding
source. In order to disentangle the impact of soil
management (i.e. tillage) and food resource availabil-
ity (i.e. litter, fine roots, mycorrhizae, and understory
vegetation) on the abundance of anecic earthworms,
studies in no-till agroforestry systems are required.
The sporadic detection of epigeic individuals iden-
tified as L. rubellus under as well as in close proxim-
ity to the trees (FigureS2 E, F) is in accordance with
the results of D’Hervilly etal. (2020). We argue that
this is likely due to tree litter input, as those species
require a permanent surface cover with organic mate-
rial and are therefore mostly absent in arable crop-
lands. We speculate that depending on the size of the
agroforestry system, the colonization of tree rows by
epigeic earthworms requires several years to reach a
spatially homogeneous equilibrium state.
Effects of agroforestry on soil microbial population
size
The population size of total bacteria, Acidobacte-
ria, and Actinobacteria was greater in the vicinity of
trees in all agroforestry systems (Fig.4), which is in
line with results of recent studies (Beule etal. 2020;
Guillot et al. 2021). The positive effect of trees in
agroforestry systems on the abundance of soil micro-
organisms is well-described (Banerjee et al. 2016;
Beuschel etal. 2019; Beule etal. 2020; Guillot etal.
2021; Luo etal. 2022) and is likely due to differences
in soil management (i.e. absence of tillage in the tree
rows versus tilled crop rows) and vegetation cover
(i.e. woody perennials versus annual crops) (Beule
etal. 2022a). Furthermore, some studies were able to
demonstrate that microbial abundance increases with
decreasing distance from the trees (e.g. Guillot etal.
2019, Beule etal. 2020; D’Hervilly etal. 2021; Luo
et al. 2022). At our study site, no such trends were
observed except for total bacteria (Fig.4A), Actino-
bacteria (Fig. 4C), Firmicutes (Figure SI2 B), and
Bacteriodetes (Figure SI 3C) in the poplar Max 1 sys-
tem. In contrast to previous studies where trees were
either not harvested at all (Guillot etal. 2019, 2021;
D’Hervilly etal. 2021) or harvested four to five years
(Beule etal. 2020; Luo etal. 2022) prior to soil sam-
pling, the aboveground biomass of the trees at our
study site was harvested one year prior to soil sam-
pling. Therefore, we suggest that tree harvesting tem-
porally alters soil microbial communities throughout
agroforestry systems. We relate these alterations to
changes in substrate input due to reduced tree litter
input and/or altered tree root functioning (e.g. dying
off of roots, changes in quantity and quality of root
exudation). Furthermore, this hypothesis may explain
why in contrast to previous studies (Beuschel et al.
2019; Beule etal. 2020; Guillot etal. 2021), soil fun-
gal abundance at our study site was not promoted by
agroforestry (Figure SI 3). Further studies explor-
ing temporal dynamics along tree rotation cycles are
required to investigate this hypothesis.
Agroforestry alters the soil mycobiome and putative
phytopathogen abundance
Our finding that agroforestry does not affect alpha
(TableS1) but beta diversity of soil fungi (TableS2,
Fig. 5B) agrees with previous studies on the soil
mycobiome of agroforestry systems (Beule et al.
2021, Beule & Karlovsky 2021b). Differences in
fungal community composition were mainly driven
by the tree rows (Fig.5B) which are known to exert
strong influence on the soil fungal community even
few months after tree planting (Beule & Karlovsky
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2021b). Across all three tree species, the tree-row asso-
ciated mycobiome was characterized by the promotion
of genera harbouring ectomycorrhizal fungi (EMF)
such as Laccaria and Preussia spp. (Fig.6). Notably,
affiliates of the genus Inocybe, harbouring EMF that
can associate with poplar (e.g. Long etal. 2016), were
strongly promoted under poplar Max 1 (Fig.6) which
indicates a tree species-specific tree-EMF interaction.
Colonization of roots by mycorrhiza is often advan-
tageous for plant growth as it improves nutrient and
water acquisition. Under field conditions, poplar and
black locust trees can associate with both arbuscular
(AMF) as well as ectomycorrhizal fungi (e.g. Khasa
etal. 2002; Bratek etal. 1996). For poplar trees, colo-
nization rates of AMF and EMF have been shown to be
poplar genotype-specific (Khasa etal. 2002), whereas
black locust genotype specificity of mycorrhizal asso-
ciations has not been investigated yet. Our findings on
EMF highlight the importance of investigating several
tree species (e.g. hardwood and fast-growing tree spe-
cies) at the same study site in order to disentangle tree-
species from study-site effects.
Concerns regarding an increased risk of crop dis-
eases is among the main impediments of farmers to
implement temperate agroforestry. In our study, a
gradual decrease in relative abundance of Blumeria
graminis (formerly known as Erysiphe graminis)
with decreasing distance from tree rows was observed
(Fig.6). B. graminis is the causal agent of powdery
mildew, one of the most common cereal diseases
worldwide that can reduce grain quality and cause
significant yield losses in temperate zones (e.g.
Bélanger et al. 2002; Dreiseitl 2011; Dean et al.
2012). The ability of B. graminis to cause disease
is host specific and thus forma specialis dependent
(Wyand and Brown 2003). Sequencing of short-read
amplicons (2 × 300bp) did not enable identification of
B. graminis on forma specialis level, which is not sur-
prising considering the limited taxonomic resolution
of short-read amplicon sequencing (Heidrich & Beule
2022). Furthermore, B. graminis can overwinter in
form of cleistothecia on plant residues (Zhang etal.
2005). Thus, it was not possible to relate B. graminis
to any of the host crops that were grown in the crop
rotation at our study site. Hence, we refer to B.
graminis as a putative pathogen. Still, our results pro-
vide the first evidence of a positive effect of tree rows
in temperate agroforestry systems on the reduction
of the putative pathogen B. graminis. As microbial
antagonists of B. graminis have been isolated in Ger-
many (Köhl etal. 2019), antagonistic interactions in
soil and on aboveground crop parts mayhave contrib-
uted to the suppression of B. graminis at our study
site. In addition to microbial antagonism, the abil-
ity of earthworms to suppress soil-born fungal phy-
topathogens is well recognized (e.g. Plaas etal. 2019;
Stephens etal. 1994). Thus, the gradual increase in
endogeic earthworm abundance with decreasing dis-
tance from the trees may have enhanced biological
control within the crop rows and thereby contributed
to the suppression of B. graminis.
In addition to potential biological control, tree
rows in agroforestry systems increase structural diver-
sity and act as physical barriers which is expected to
lower the spread of crop diseases through the dilu-
tion of the host crop (host dilution effect) (Beule etal.
2019b). In 2019, Kanzler etal. reported reduced wind
speed and evaporation rates at our study site under
the agroforestry as compared to the monoculture
cropland. Furthermore, reductions of wind speed and
evaporation were dependent on the distance from the
trees (Kanzler etal. 2019). Such microclimatic altera-
tions are known to affect the epidemiology of plant
diseases (e.g. Aust & von Hoyningen-Huene 1986,
Waggoner 1965). Overall, the enhanced control of B.
graminis in the agroforestry as compared to the mon-
oculture cropland system cannot be attributed directly
to one of the factors listed above. We rather expect
that the control of B. graminis was due to a combi-
nation of factors that cannot be disentangled in our
experimental setting.
As of writing, the study conducted by Beule
etal. (2019b) is the only study that investigated the
effect of temperate agroforestry on crop health.
Their results revealed that colonization of oilseed
rape plants with Verticillium longisporum and wheat
grain with Fusarium tricinctum was lower in temper-
ate agroforestry systems compared to monoculture
cropland systems. Colonization of wheat and barley
grain and oilseed rape plants with other major fungal
pathogens did not differ between agroforestry sys-
tems and monoculture systems (Beule etal. 2019b).
Furthermore, the authors observed a relationship
between abundance of a phytopathogen and distance
to the trees only for the phytopathogen Leptospha-
eria biglobosa in oilseed rape plants. Considering our
findings on B. graminis, we hypothesize that suppres-
sion of fungal phtyopathogens within agroforestry
Plant Soil (2023) 487:355–372368
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systems is limited to certain pathogens and is a func-
tion of the distance to the trees (i.e. suppressiveness
increases as the distance to the trees decreases). Stud-
ies on diseases and disease related factors in temper-
ate agroforestry systems are scarce. We suggest that
future research should investigate crop diseases in
agroforestry systems of different spatial designs (e.g.
wide versus narrow crop allays) as well as manage-
ment practices. New findings could help farmers to
optimize the design of future agroforestry systems in
order to maximize the beneficial effects of these sys-
tems on disease control.
Conclusion
The integration of tree rows into arable land (agro-
forestry) increased the abundance of soil bacteria and
earthworms (anecic, endogeic, and epigeic species)
as compared to monoculture cropland. We attribute
this mainly to the absence of tillage and the input
of large amounts of tree litter under the trees. Com-
munity composition of soil fungi was altered by the
tree rows, resulting in a tree-row associated myco-
biome, which was particularly characterized by an
increased proportion of EMF. The tree-row associ-
ated mycobiome not just enhances overall fungal
diversity of agroforestry systems but is also expected
to alter soil functions such as nutrient cycling. As the
distance from the trees decreased, the proportion of
Blumeria graminis, the causal agent of powdery mil-
dew, decreased. We suggest that enhanced microbial
antagonism, increased earthworm densities and/or
altered microclimatic conditions contributed to the
suppression of B. graminis within the agroforestry
system. Whereas distinct tree-distance effects were
observed, tree-species effects were identified as a
minor driver of the abundance and composition of
soil communities at our study site. Overall, agrofor-
estry benefits the abundance, diversity, and function
of soil biota and may enhance soil suppressiveness.
Future research should investigate crop diseases in
agroforestry systems of different spatial designs and
management practices in order to maximize the ben-
eficial effects of these systems on disease control.
Author contributions AV and LB contributed to the concep-
tion and design of the study. AV and LB performed the field
work. AV, ZB, and LB performed the laboratory work. AV,
ZB, and LB performed the statistical analysis. AV, ZB, and LB
wrote the first draft of the manuscript. NH, BH, and VEMR
contributed resources and critically revised the manuscript. All
authors read and approved the manuscript.
Funding Open Access funding enabled and organized by
Projekt DEAL. This study was supported by the German
Federal Ministry of Education and Research (BMBF) in the
framework of the Bonares-SIGNAL project (funding codes:
031A562A, 031B0510A, 031B1063A). AV was supported by
the joint project MonViA—the German Farmland Biodiver-
sity Monitoring that has been funded by the Federal Ministry
of Food and Agriculture (BMEL). The funder had no role in
study design, data collection and analysis, decision to publish,
or preparation of the manuscript. Open access funding enabled
and organized by Projekt DEAL.
Declarations
Conflict of Interests The authors declare that the research
was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of
interest.
Open Access This article is licensed under a Creative Com-
mons Attribution 4.0 International License, which permits
use, sharing, adaptation, distribution and reproduction in any
medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Crea-
tive Commons licence, and indicate if changes were made. The
images or other third party material in this article are included
in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your
intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit
http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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