Article
Correlations of Soil Fungi, Soil Structure and T ree
V igour on an Apple Orchard with Replant Soil
Ulrike Cavael 1 , *, Philipp T ost 1 , Katharina Diehl 2 , Frederick Büks 3 and Peter Lentzsch 1
1 Leibniz Centre of Agricultural Landscape Resear ch (ZALF), 15374 Müncheberg, Germany;
[email protected] (P .T .); [email protected] (P .L.)
2 Johann Heinrich von Thünen-Institut, 38116 Braunschweig, Germany; [email protected]
3 Department of Soil Science, Institute of Ecology , Faculty VI Planning Building Environment,
T echnische Universitat Berlin, 10623 Berlin, Germany; [email protected]
* Correspondence: [email protected] ; T el.: + 49-(0)33432-82-134
Received: 8 October 2020; Accepted: 27 November 2020; Published: 3 December 2020
     
  

Abstract:
The soil-borne apple r eplant disease (ARD) is caused by biotic agents and a ff ected by
abiotic pr operties. Ther e is evidence for the interrelation of the soil fungal population and soil
aggr egate structure. The aim of this study conducted between Mar ch and October 2020 on an
or chard in north-east Germany was to detect the corr elations of soil fungal density , soil structure
and tr ee vigour under replant conditions in a series of time intervals. By using the r eplant system as
the subject matter of investigation, we found that r eplanting had an impact on the increase of soil
fungal DNA, which corr elated with a mass decrease of lar ge macro- aggr egates and an increase of
small macr o- and large micr o-aggregates in the late summer . Increased pr oportions of water-stable
aggr egates (WS) with binding forces
≤
50 J mL
− 1
, decr eased proportions of WS > 100 J mL
− 1
and a
decr ease of the mean weight diameter of aggregates (MWD) emphasised a r eduction of aggregate
stability in r eplant soils. Corr elation analyses highlighted interactions between replant-sensitive soil
fungi ( Alternaria -gr oup), the loss of soil structure and suppr essed tr ee vigour , which become obvious
only at specific time intervals.
Keywords:
aggr egate stability; Alternaria -gr oup; apple replant disease (ARD); gr owing season;
soil aggr egates; tree vigour
1. Introduction
Replant disease describes a phenomenon of disturbed physiological and morphological reactions of
plants after r eplanting crop species at sites pr eviously used for similar crop cultur es [
1
]. Replant disease
has been r eported for several horticultural crops, including apples, peaches and cherries in nurseries
and or chards all over the world [
2
,
3
]. On apple tr ees, symptoms of replant disease include damaged
r oot systems; stunted growth above and below gr ound; and reduced fruit yields [
1
,
4
]. While the direct
cause of the soil-borne r eplant disease has not been revealed, it has been attributed to a plethora of
potential biotic and also abiotic factors. Biotic factors ar e generally believed to be the pr edominate
causal agents of r eplant disorders, since r eplant soils treated with soil fumigation, soil pasteurisation
and soil sterilisation have shown r estored r egular plant growth [
5
–
8
]. Conver gence has evolved
ar ound genera of oomycetes ( Pythium , Phytophthora ); actinomycetes or bacteria ( Bacillus , Pseudomonas );
and multiple fungal species (e.g., Cylindrocarpon -like fungi, Rhizoctonia , Fusarium ) that appear to
contribute to the complex disease [
9
–
11
]. However , a definite relation between sequence data and
r eplant disease in the microbiome of r eplant soils has not been shown yet [ 12 ].
Abiotic factors, on the other hand, are understood as influences r egulating the extent of the
symptomatic e ff ect of r eplant on tree vigour , rather than a primary cause [
1
,
4
]. Abiotic factors include
Soil Syst. 2020 , 4 , 70; doi:10.3390 / soilsystems4040070 www .mdpi.com / journal / soilsystems

Soil Syst. 2020 , 4 , 70 2 of 16
water logging, soil pH and (micro)nutrient deficiencies [
1
,
13
,
14
]. Replant-sensitivity of apple tr ees
has been found to di ff er by soil type [
13
,
15
] and soil textur e [
11
,
16
].
T ewoldemedhin et al. (2011)
,
for example, gr ouped the status of the apple replant disease (ARD) by gr owth response in
non-tr eated versus pasteurised replant soil: low ARD—status on soils of clay and loamy texture;
moderate ARD—status on soils of loamy textur e; and sever e ARD—status on soils of sandy texture [
11
].
Replant-r elated suppression of apple tr ee growth performance has been found individually
pr onounced between apple understocks and trees, r espectively [
17
,
18
]. The suppression r esults in an
uneven gr owth by heterogeneous distribution of mor e or less replant, symptomatic apple plants acr oss
r eplanted apple orchar ds. The suppr ession of tree vigour in apple tr ees, and the consequential lack of
a development of best-performing tr ees across the or chard, leads to decr eased profitability of yields
that can add up to 50% thr oughout the life cycle of replanted or chards [ 19 , 20 ].
By meta-analysis, Nicola et al. (2018) showed that the soil microbial community significantly
di ff ers under r eplant conditions [
12
]. However , shifted micr obial communities show relatively
small overlaps of micr obial constituents between geographically distantly located replant sites,
indicating site-specific r eplant e ff ects on the soil microbiome [
12
]. In field studies, ARD-symptomatic
and non-symptomatic tr ees have been associated with shifts in the density of several soil fungi,
including the class Dothideomycetes, and mor e specifically in the order Pleosporales [
18
]. The genetically
determined Alternaria -gr oup (Ag) (order Pleosporales , family Pleosporaceae ) has been identified as a
r eplant-sensitive soil fungal population which responds to r eplanting by abundance [
19
]. The pr oportion
of Ag on the total soil fungal population was found to be 2% in r eplant soil; this was found to be
10-fold gr eater compared to no-r eplant soil. Such slight shifts in the Ag population can reflect lar ger
shifts in the soil fungal community (beyond Ag) and thus be indicative for shifts in the distribution of
sieve-size fractions and aggr egate stabilities.
Most micr obial studies indicating replant-r elated or even causal agent(s) of replant disor der
have focussed on homogenized soil samples. Soil micr obial interactions, however , occur in habitats
much smaller than those generally captur ed in homogenized soil cores [
21
]. Microbial community
composition is str ongly mediated by soil structure [
22
,
23
]. The general heterogeneity of the soil
structur e supports a high diversity of microhabitats with di ff er ent physico-chemical gradients and
discontinuous envir onmental conditions [
24
,
25
], even when the overall envir onment of the soil is
constant [
23
]. Specific microbial taxa have habitat pr eferences that ar e linked to the morphological,
chemical and physical pr operties of the interior and exterior interfaces of soil aggregates [
26
,
27
].
In general, the proportion of fungi within soil aggr egates varies within aggregate size, as a gr eater
pr oportion of fungi have been associated with macro-aggr egates ( > 250
µ
m), wher eas bacteria were
mainly associated with micr o-aggregates (
≤
250
µ
m) [
21
,
28
]. The microbial community was also found
to vary within and among aggr egate fractions of the same soil under di ff erent management and tillage
practices [ 29 , 30 ].
Soil micr oorganisms e ff ect the formation and stabilisation of soil aggr egates, and thereby
significantly involve themselves in the pr ocesses of building soil structure [
31
–
33
]. Microbes r elease
excr etions, including extracellular polymeric substances, which enmesh soil particles into aggr egates.
Similarly , soil particles can also be enmeshed into aggregates by fungal hyphae [
34
]. Fungi have been
found involved in the binding of lar ger particles, and are pr edominantly responsible for stabilization
of macr o-aggregates due to their hyphae structur e [
27
,
35
,
36
]. The influence of fungi and bacteria on
aggr egate stabilization varies widely among species and depends considerably on the nature of the
available substrates [
37
]. In general, fungi ar e better correlated with aggr egate stability and lead to
str onger binding forces between soil particles than with bacteria [ 38 ].
Soil physical structur es and microbial community composition shift in short timescales (weeks)
depending on envir onmental conditions, such as (soil-)climate and related soil ecosystem conditions,
e.g., soil moistur e. The extents of the shifts in soil abiotic and biotic pr operties di ff er depending on the
cr op and the management system of the cultivation [ 39 – 41 ].

Soil Syst. 2020 , 4 , 70 3 of 16
Overall, this indicates a seasonal connection between the soil fungal population and the soil
structur e, particularly the size and mass distribution of aggregates. Our aim was to explor e this
possible corr elation between the soil fungal population and the soil structure in a case study for apple
r eplant disease. This was done by analysing the sizes and mass distributions of soil sieve-size fractions,
and their physical stability , and contrasting the results with the r eplant e ff ects on tr ee vigour . For this,
we analysed and compar ed the soils of an apple orchar d where apples wer e cultivated on initially
planted and r epeatedly planted soils in the direct vicinity and under identical cultivation management.
The data wer e collected over four time intervals in one growing season fr om Mar ch to October in 2018.
2. Materials and Methods
2.1. T est Site and Sampling Design
The study was conducted on an intensively managed commercial fr uit orchar d, located east of
Berlin in the district Märkisch Oderland (Altlandsber g, longitude: 52.62623, latitude: 13.804264)
in Brandenbur g, a state in north-eastern Germany . On this or chard, a variety of fruit tr ees,
including di ff er ent varieties of dessert apples, are cultivated on sandy br own, dry and warm diluvial
Eutric Retisols (Geoabruptic, Ar enic, Aric) and physico-chemically very similar Geoabruptic Luvisols
(
Ar enic, Aric, Cutanic
) (accor ding to W orld Refer ence Base for Soil Resources, WRB) [
42
]. W ithin the
or chard we selected two test fields, one with initial apple cultivation (no-replant, nr) and one with
r epeated apple cultivation (replant, r), both in the dir ect vicinity of one another and identically managed.
Both cultivations wer e set up in 2009 with tall spindles from apple scions ROHO 3615 EVELINA
®
cultivated on understock of M.9.
On both test ar eas we periodically collected data on tree vigour , and sampled soil cor es to analyse
soil fungal populations, starting in March 2018 with the beginning of the gr owing season. For the
analysis, we selected three tr ee rows in the r eplant ar ea and one row on the no-r eplant area. W ithin each
r ow we selected a consecutive number of 18 trees. The selected tr ees stood in parallel with a minimum
of inter -row distance of 3.5 m, in order to r educe the influence of the inherent soil-r elated spatial
variability in soil physiochemical pr operties, and thus, soil microbial structur e [
18
]. W ithin each row
we selected thr ee trees for the analysis of soil. This selection of planting spots for further soil analysis
was based on tr ee vigour , so that the three selected tr ee spots each r epresented the str ongest, a medium
and the lowest tr ee vigour of the respective 18 tr ees in line.
W e started sampling just after a strong period of gr ound frost [
43
] up to 50.0 cm soil depth,
and ended in October with vegetation dormancy . Sampling started in week 10 (5 March); was r epeated
in week 16 (19 April), week 25 (20 June) and in week 31 (2 August); and was last performed in week 43
(22 October). The sequence of sampling aligned with the annual growing season of the apples and the
cultivation plan of the farmer .
2.2. Measurement of T r ee V igour
The trunk cr oss-sectional area ( CSA ) is a practical and r obust parameter for tree vigour [
19
].
Ther efore, trunk cir cumference was measur ed by standar d folding ruler at 40.0 cm above soil surface
and a millimetr e tapeline. CSA was calculated using Equation (1):
CSA = π / 4 × ( trunk circum f erence ) 2 (1)
2.3. Soil Sampling
Soil cor es were sampled fr om the top 20.0 cm of the trees, 10.0 cm distance fr om the tree tr unk
with a Puer ckhauer sampler . Three soil cor es were collected fr om each sampling point in a distance of
15.0 cm at each sampling time. The topmost 2.0 cm of each soil cor e were r emoved. Soil cores wer e
stor ed at 4 ◦ C.

Soil Syst. 2020 , 4 , 70 4 of 16
2.4. Size Fractionation of Soil
Soil samples wer e fractionised to determine the mass distribution of soil fractions across size
classes and to quantify soil fungal densities per size fraction. Soil fractionation was performed with
dried soil material. As the pr ocess of slowly air-drying soils changes the micr obial growth and
activity [
44
,
45
], the soil samples in a moist-field state wer e dried rapidly by 70
◦
C in a pr e-heated kiln
for 2 h to minimise the e ff ect of drying by a rapid r eduction of soil moisture. Soil samples were spr ead
out in a thin layer to ensure even drying of soil material. After half time of drying, soil samples were
turned and the drying pr ocedure was continued for an additional hour .
Dried soil material was sieved by dry-sieving procedur e. Bach and Hofmockel (2014) suggest that
dry-sieving is a useful alternative to wet-sieving to mor e closely capture shorter in situ measur es of
seasonal and intra-annual soil microbial activity . Soil microor ganisms and associated activities have
been found to be sensitive to (r e-)wetting events, while dry-sieving prevents cr oss-contamination
between fractions due to “washing” [
46
] and lysis of microbes. Furthermore, di ff er ent spatial domains
of micr obial diversity can be distinguished by patterns in the adhesive forces [
46
]. W et-sieving
can either enhance or diminish these adhesive forces between aggr egate particles, and thus alter
measur ed communities.
Dried soil material > 6300
µ
m was r emoved using a hand-held flat sieve. Remaining soil
material was separated into six sieve-size fractions: 2000–6300, 1000–2000, 500–1000, 250–500,
125–250 and
≤ 125 µ m
, corresponding to W entworth’s (1922) classification scheme of soil particle
sizes, which allows for a higher resolution of soil parameters than would be observed by micr o
(
≤ 250 µ m
), small (
250–1000 µ m
), medium (1000–2000
µ
m) and lar ge macro-aggr egates ( > 2000
µ
m) [
47
].
The disaggr egation of less stable soil structural units due to mechanical stress [
48
] was lar gely avoided
by applying manual sieving, thereby imitating horizontal movements at a str oke of 20 min
− 1
. Flat sieves
wer e filled with 5 mL soil material and r otated for 3 min. Each sieve-size fraction was separately
fractionised to ensur e the same time of cycling of soil material on each sieve mesh and weighed.
Sieved soil material was locked in bags to avoid moistening during storage at 4
◦
C. As a concession
to the analysis of the micr obial community , with this method we did not separate water-stable fr om
water -labile aggregates and non-aggr egated primary particles, but measured total aggr egate masses.
The mass distribution of sieve-size fractions in soil was calculated by using the weighed masses of the
sieve-size fractions and normalising them with r espect to the total soil material sieved.
The mean weight diameter ( MWD ) of aggr egates was calculated according to V an Bavel (1949)
by Equation (2):
MW D =
n
X
i = 1
x i w i (2)
wher e x
i
is the mean diameter of any sieve-size fraction, and w
i
is the weight pr oportion of this
fraction [ 49 ]. W e used the MWD as a parameter of the soil aggregation level.
2.5. Fractionation of Aggregate-Stability Classes
2.5.1. Calibration of the Ultrasonication Device
The dispersion of soil samples was performed using an ultrasonic apparatus (Sonoplus 2070
Ultraschall-Homogenisator , BANDELIN electronics GmbH and Co. KG, Berlin, Germany) with a
V 70 T sonotr ode (Ø 13.0 mm). The power of the device was 70 W with an oscillation frequency of
20 kHz. The cavitational action of the sonotrode (J s
− 1
) was determined by measuring the heating
rate of deionized water inside a Dewar vessel [
50
]. The calibration was performed by subjecting five
r eplicates of 180 g deionized water to successive ultrasonications of 1, 2, 3, 4 and 5 min with the
r espective measurement of temperatur e. The performance of the ultrasonic device was then calculated
following Schmidt et al. (1999) [
51
]. Graf-Rosenfellner et al. (2018) demonstrated that this way of

Soil Syst. 2020 , 4 , 70 5 of 16
calibrating the power output cr eates replicable r esults when applied with di ff erent sonication devices
and pr ocedural details [ 52 ].
2.5.2. Dispersive T reatment
The pr oportions of water-stable aggr egates and their stability in the face of mechanical stress
wer e determined by consecutive applications of 0, 50 and again 50 J mL
− 1
, r espectively , for wet-sieving
and weighing. W et-sieving was performed following the operator ’s manual (Eijkelkamp Soil and
W ater , Giesbeek, the Netherlands; str oke length 1.3 cm, at 34 stroke min
− 1
), which is similar to existing
methods (e.g., Kemper and Rosenau, 1986 [
53
]). Each 3.0 g of soil material was placed into a sieve.
The mesh size was always the lower limit of the sieve-size fraction tested at the time (125, 250, 500
µ
m).
The soil samples wer e remoistened with deionized water by capillary action and then submer ged.
The soil material was wet-sieved for 3 min. Material smaller than the mesh diameter passed the sieve
and was caught in stainless steel cans.
Immediately after the wet sieving procedur e, the sieves, then containing only the water-stable
aggr egates, were placed into new cans, each filled with 70.0 g of deionized water . For subsequent
ultrasonication, the sonotrode was submer ged 1.5 cm into the sieves and 50 J mL
− 1
was applied.
The sieves and cans wer e placed together into the wet-sieving apparatus and wet-sieving was repeated
as described above. Ultrasonication and wet-sieving were then conducted once again. As a r esult,
we gained disaggr egated soil material of three distinct stability classes: the water-labile class (WL),
the water -stable class with binding forces
≤
50 J mL
− 1
(WS
≤
50 J mL
− 1
) and the water -stable class with
binding for ces
≤
100 J mL
− 1
(WS
≤
100 J mL
− 1
). A fourth stability class, of water-stable aggr egates
with binding for ces of > 100 J mL
− 1
(WS > 100 J mL
− 1
), comprised the soil sample that r emained in the
sieve and was not disaggr egated by the procedur e.
The disaggr egated soil fragments were air -dried at 105
◦
C until the weight r emained unchanged
by evaporation any further ( > 17 h). After drying, the masses of the first three stability classes wer e
weighed. The mass of the water -stable class with binding forces > 100 J mL
− 1
was calculated by
subtracting the sum of the fragments fr om the original dry-weight of the sample. The procedur e of
disaggr egation was repeated thr ee times per sampling point and sieve-size fraction. Repeated measures
wer e standardised for each 3 g of origin soil material.
2.6. Quantification of Soil Fungal Densities
T otal DNA was extracted from 0.5 g soil accor ding to the standard pr otocol of the NucleoSpin soil
kit (Macher ey-Nagel GmbH and Co. KG, Dür en, Germany). The total amounts of purified DNA wer e
assessed using a NanoDr op 1000 microvolume spectr ophotometer following the NanoDrop ND-1000
standar d protocol (Kisker Biotech GmbH and Co. KG, Steinfurt, Germany). T otal fungal DNA was
amplified using the highly conserved fungal rRNA gene primers ITS1F and ITS4 [
54
,
55
]. The total
fungal DNA in a sample was quantified by SYBR gr een fluorescence qPCR (
QuantStudio 12 K flex
,
Applied Biosystems) using 5
µ
L of template DNA in a 20
µ
L r eaction mix (qPCR HRM-mix,
Solis BioDyne, T artu, Estonia). The PCR thermal protocol consisted of an initial 15 min denaturation
step at 95
◦
C; 32 amplification cycles of 95
◦
C for 30 s, 55
◦
C for 30 s and 72
◦
C for 60 s; and a final
extension step of 72 ◦ C for 10 min.
For the quantification of the Alternaria -group, standar d curves wer e generated based on dilution
series of DNA fr om Alternaria tenuissima GH50t (e ffi ciency > 0.91 and R
2
> 0.998) (cultur e collection
of micr oorganisms of the working gr oup “Fungal Interactions” at the Leibniz Centre of Agricultural
Landscape Resear ch Müncheberg). The primers and pr obes used for detection of Ag were as described
by Grube et al. (2015) [
56
], for the detection of all genetically defined species of Ag according to
Lawr ence et al. (2013) and W oudenber g et al. (2015) [
57
,
58
]. The PCR conditions were adapted to the
qPCR mix (3 mM MgCl2, Solis BioDyne, T artu, Estonia [
59
]). Di ff er ent strains of plant-associated fungal
species wer e used as negative controls, as they were r eference strains of V erticillium (CBS 130603,

Soil Syst. 2020 , 4 , 70 6 of 16
CBS 130339, CBS 130340, DSM 12230 and CBS 447.54), Gibellulopsis (CBS 747.83), T richoderma spp.
(St365) and Fusarium [ 60 ].
2.7. Statistical Analyses
The data for all soil fungal and soil structural parameters (MWD, mass distribution of sieve-size
fractions, aggregate-stability classes) wer e analysed using ANOV A (analysis of variance) and significant
di ff er ences between no-replant soil and r eplant soil were calculated by Games–Howell post-hoc test.
p < 0.05 was accepted as significant.
As datasets of plant parameters and soil parameters did not follow a normal distribution,
Spearman’s rank corr elation coe ffi cient (
ρ
s) was calculated for corr elations between plant and soil
parameters. Significant correlations wer e accepted at p < 0.05. Subsequently , significant correlations
wer e calculated using non-linear regr ession analysis. All statistics were conducted using IBM SPSS
Statistics 22.
3. Results
3.1. Soil Fungal Densities in No-Replant Soil and Replant Soil
Densities of soil fungi exhibited seasonal dynamics and di ff er ed between no-replant soil and
r eplant soil, with stronger e ff ects by season, as demonstrated for Ag and total fungi (ITS) in Figure 1 .
The Ag density in no-replant soil di ff er ed mar ginally between sampling dates from Mar ch to August,
followed by a str ong rise with a 70-fold increase until October . Starting from similar fungal densities
on both planting ar eas after strong gr ound frosts in Mar ch, Ag density in replant soil continuously
incr eased 21-fold following a logarithmic scale (R
2
= 0.92) to a maximum in October . The di ff ering
tr ends of Ag proportion among total fungi between no-r eplant soil and replant soil wer e found most
pr onounced in August ( p ≤ 0.01) and least in October (Figure 2 ).
Soil Sys t. 2020 , 4 , x FO R P E ER R E VIEW 6 of 16

As d ata s ets of pl ant par am eters and s oil p ar amete rs di d not fo ll ow a norm al d is tr ibutio n,
Spearm an’s r ank corr el ati on coeff icien t (ρs) wa s ca l c ulated for cor rel ations bet ween pla nt an d soi l
para met er s. S ig ni fi c ant cor rel ations w er e accept ed at p < 0 .0 5. Su bs equ ent ly , sig nif i cant cor re la tion s
were c al c ulat ed usi n g non - li n ear r egre s si on an alys is . A ll stat is tics were con d uct ed usi n g IB M S PS S
Statis ti cs 22.
3. R esu lt s
3.1. Soil Fu ngal Den sities in No - Re plant So il and Re plant Soil
Densitie s o f s oil fu n gi exhi bited se as on al dyn amic s an d di ff e red b etw een no - re pla nt so il an d
repla nt so il , with stron ger eff ect s by s eason , as demo nstrated for Ag and to ta l f ungi (ITS) in Fi g ure 1.
Th e A g dens ity i n no - rep lant soi l di ffere d m ar g inally bet ween s am pling d ates f r om M ar ch to August,
followed b y a stron g ri se w ith a 70 - fo ld i ncreas e until Oct ob er. St art ing from si m i la r f ung al de nsitie s
on bo th pla nt ing ar e as aft e r stron g grou nd fro sts in Ma rch , A g de nsity in rep lant soil con tin uously
incre as ed 21 - fol d followin g a log arit hm ic scale ( R 2 = 0 . 92) to a ma ximum in Oc to ber. Th e di f feri n g
tren ds of A g pro po rtion am ong to tal f u ngi bet ween no - repl ant so i l and r epl ant s oil were fo u nd mo st
pro nounced i n Au gus t ( p ≤ 0 .0 1) a nd l e ast i n Oct ob er (F i gure 2).
In repla nt soi l, th e pro po rt ion of Ag am ong to tal fu n ga l den si ty in creased d is pr op ort iona l ly , as
th e incre ase i n to tal f ung al - dens ity w as not com pen s ated due to i ncreas ed Ag density . To ta l f ung al
density w as at a m axi m u m in M ar ch in bo th soi ls (no - repl ant/ repla nt ) . D en si ty o f to tal fu ng i
decrea sed by approx im atel y 7 5 % from Ma rch to Oct ob er in soi ls , result ing in sim ilar den s itie s in no -
repla nt soi l an d repl ant s oil in Oct ob er. In no - rep l ant so il , to ta l f ung al dens i ty decre as e d at an
exp onen tial r ate (R 2 = 0 . 50), fo ll owe d by an incr ease from Au gus t to Oct ob er. I n con trast, th e to tal
fu ng al dens ity in rep la nt s oil r api d ly d e creased from Ma rch to Apr il ( −6 6 %), foll owed by an i n crease
up to J une ( +36% ) and a f u rth er decre ase from J une t o Oct ob er ( −4 6%). Di fferen t dynam ics o f fu n gal
densiti es bet ween no - repl ant so il an d r epla nt soi l r es ul ted in incre as ed to tal f un ga l den si ty in J une ( p
> 0 .0 5) and si gnific antly in creased den sit y i n A ug ust ( p ≤ 0 .0 1) unde r repl ant con ditions .

Figure 1.
Concentration of Alternaria -group (Ag) (genome / g soil) and total fungal DNA (log (ITS))
in no-replant soil (nr) and r eplant soil (r). Significances calculated between nr and r soil.
α
= 0.05,
** p ≤ 0.01.

Soil Syst. 2020 , 4 , 70 7 of 16
Soil Sys t. 2020 , 4 , x FO R P E ER R E VIEW 7 of 16

Figure 1. Con c entration of Alt er naria - gr oup ( Ag) (gen ome/g so il ) and tota l fun gal DNA (l og (I TS )) in
no - replant so i l (nr ) and replan t so il (r). Si gn if ic ances cal cula ted be tween nr and r s oi l . α = 0 .0 5, ** p ≤
0 . 01 .

Figure 2. Re lat iv e di ff erence s (%) of prop ortion of Ag out of tota l fun gal DNA ( Ag/ITS) and tota l
fun gal DN A (I TS) betwee n no - replant s oi l a nd replant s o il.
3.2. A gg r eg a ti on Le ve l in No - Replan t an d Replant Soil
Th e soi l a ggr ega tion leve l vari e d ov er ti me and b etw een no - rep la n t soil and repl ant soi l (F i gu re
3). Bot h so ils had th e h ig h est M WD in Ma rch ; how e ver , th e ag gr ega tes ten de d to di ff e r by a lower
MWD in repl ant soi l ( p ≤ 0 .1 0). Dur ing t he fi r st ha lf o f th e grow ing season, th e aggreg ation le v el of
no - repl ant so i l dec reased to a min imum i n J une , and in turn, incr eas ed d uri n g s ec ond ha lf of g r owing
season. I n rep la nt soil th e s oil , aggre ga ti on leve l w as a t a min imu m in A pri l, an d th en rem ained at a
con stant leve l until August, which wa s fo ll owed b y an incre as e up to Oct ob er. In August, no - re pla nt
soil a n d rep lant soi l diff ere d signi fi c antl y due t o l ess soil aggre ga ti on of the repl ant so il .
Seasona l d iff erences in M WD were m ainly driven b y th e 200 0 – 63 00 µ m fr actio n. A decr ease in
MWD in no - r epla nt so il du ring th e fi r st half of th e gr owing s eason was con com i tant with an i ncreas e
of so il in fr ac tions ≤ 1000 µ m, where as it wa s con co mitant with an incre as e in soil in fr actio ns from
125 to 2000 µ m in rep la nt soil. In th e se con d ha lf of th e grow ing s ea so n , an inc rea s e in MW D wa s
driven by agg rega tion o f th e 125 to 1000 µ m f ra ct ion s i n bo th so ils. T hrougho ut, th e m as s o f th e 200 0 –
6300 µ m fr action w as lowe r, and ma sse s of th e 12 5 to 1000 µ m fr actions were hi g her in r epl ant so il
th an in no - re pla nt soi l, w i th an excep tion in Apri l ( Table 1) . Sig nif i cant di ffe rences in th e mass
dis trib ution o f s ieve - size fr actions bet we en so il s (no - r epla nt /rep l an t) were ob ser ved in A ugus t and
Oct ob er. In A ugust , s ieve - s iz e fr action 200 0 – 63 00 µ m was signi fi c an tly decre ase d by 3 6 %, w hereas
fra ct ions 12 5 – 250 µ m (+2 9%), 25 0 – 500 µ m ( +43% ) and 50 0 – 1000 µ m (+5 4%) were s ig n ifi cantly
incre as ed un der r epl ant c onditions . In Oct ob er, s iev e - s iz e fra ct io ns ≤ 125 µ m (+1 2%) , 50 0 – 1000 µ m
(+5 7%) an d 100 0 – 20 00 µ m (+1 7%) were s ig ni fi c antly i ncreas ed in re pla nt soi l co mp ar ed to no - repl ant
soil.

Figure 2.
Relative di ff erences (%) of pr oportion of Ag out of total fungal DNA (Ag / ITS) and total fungal
DNA (ITS) between no-replant soil and r eplant soil.
In r eplant soil, the proportion of Ag among total fungal density incr eased disproportionally , as the
incr ease in total fungal-density was not compensated due to increased Ag density . T otal fungal density
was at a maximum in March in both soils (no-r eplant / replant). Density of total fungi decreased by
appr oximately 75% from Mar ch to October in soils, resulting in similar densities in no-r eplant soil
and r eplant soil in October . In no-r eplant soil, total fungal density decreased at an exponential rate
(R
2
= 0.50), followed by an increase fr om August to October . In contrast, the total fungal density
in r eplant soil rapidly decreased fr om March to April (
−
66%), followed by an increase up to June
( + 36%) and a further decr ease from June to October (
−
46%). Di ff er ent dynamics of fungal densities
between no-r eplant soil and replant soil r esulted in increased total fungal density in June ( p > 0.05)
and significantly incr eased density in August ( p ≤ 0.01) under replant conditions.
3.2. Aggregation Level in No-Replant and Replant Soil
The soil aggr egation level varied over time and between no-replant soil and r eplant soil (Figure 3 ).
Both soils had the highest MWD in Mar ch; however , the aggregates tended to di ff er by a lower MWD
in r eplant soil ( p
≤
0.10). During the first half of the growing season, the aggr egation level of no-replant
soil decr eased to a minimum in June, and in turn, increased during second half of gr owing season.
In r eplant soil the soil, aggregation level was at a minimum in April, and then r emained at a constant
level until August, which was followed by an increase up to October . In August, no-replant soil and
r eplant soil di ff ered significantly due to less soil aggr egation of the replant soil.
Seasonal di ff er ences in MWD were mainly driven by the 2000–6300
µ
m fraction. A decrease in
MWD in no-r eplant soil during the first half of the growing season was concomitant with an incr ease
of soil in fractions
≤
1000
µ
m, wher eas it was concomitant with an increase in soil in fractions fr om 125
to 2000
µ
m in r eplant soil. In the second half of the growing season, an increase in MWD was driven by
aggr egation of the 125 to 1000
µ
m fractions in both soils. Thr oughout, the mass of the 2000–6300
µ
m
fraction was lower , and masses of the 125 to 1000
µ
m fractions wer e higher in replant soil than in
no-r eplant soil, with an exception in April (T able 1 ). Significant di ff erences in the mass distribution of
sieve-size fractions between soils (no-r eplant / replant) wer e observed in August and October . In August,
sieve-size fraction 2000–6300
µ
m was significantly decr eased by 36%, whereas fractions 125–250
µ
m

Soil Syst. 2020 , 4 , 70 8 of 16
( + 29%), 250–500
µ
m ( + 43%) and 500–1000
µ
m ( + 54%) wer e significantly increased under r eplant
conditions. In October , sieve-size fractions
≤
125
µ
m ( + 12%), 500–1000
µ
m ( + 57%) and 1000–2000
µ
m
( + 17%) wer e significantly increased in r eplant soil compared to no-r eplant soil.
Soil Sys t. 2020 , 4 , x FO R P E ER R E VIEW 8 of 16

Figure 3. Mea n weight di am eter (MWD) of no - replant so i l (nr ) and repl ant so il (r). S i gn ifi canc e s
cal culate d bet ween nr and r so il. α = 0 .0 5, ** * p ≤ 0 .0 01, 1 p ≤ 0 .1 0.
Table 1 . Mean m ass val ues (g) of s ie ve - si ze fr act io ns from ≤ 125 µ m to 6300 µ m in no - repl ant so il (nr )
and replant soi l (r).

Sieve - Fracti on (µ m)

Soil

March

Ap rl

June

Au gust

Oc tober

MW

SD

MW

SD

MW

SD

MW

SD

MW

SD

200 0 – 6300

nr

45. 15

1. 28

22. 34

3. 42

18. 86

5. 86

33. 28

4. 19

38. 58

6. 53

r

35. 93

7. 14

22. 39

4. 82

22. 92

5. 96

21. 24 ***

3. 52

31. 89

6. 78

100 0 – 2000

nr

11. 46

4. 88

13. 64

1. 45

10. 96

2. 04

13. 86

1. 02

8. 34

3. 14

r

8. 57

1. 05

14. 84

2. 73

14. 54

2. 53

13. 31

1. 13

12. 70 *

2. 88

50 0 – 1000

nr

6. 56

2. 76

15. 44

1. 70

12. 09

0. 85

9. 47

2. 05

6. 81

2. 08

r

7. 13

0. 59

15. 81

1. 08

13. 84

2. 72

14. 57 ***

1. 64

11. 06 *

2. 99

25 0 – 500

nr

7. 98

7. 24

15. 85

0. 46

14. 97

0. 88

10. 57

1. 93

6. 81

0. 71

r

11. 38

1. 51

14. 85

2. 34

13. 61

3. 48

15. 16 ***

1. 34

10. 90

2. 18

12 5 – 250

nr

13. 71

7. 71

18. 16

2. 34

23. 24

4. 42

16. 24

1. 25

18. 21

0. 67

r

17. 88

3. 35

17. 54

3. 23

18. 97

3. 20

20. 89 **

2. 02

17. 68

1. 24

≤ 125

nr

15. 14

7. 83

14. 56

1. 98

19. 88

3. 83

16. 59

2. 78

18. 83

2. 90

r

19. 11

3. 26

14. 58

2. 54

16. 12

1. 85

14. 83

1. 20

15. 77 *

1. 73

Si gn ifi canc e s c alc ulated betw een nr and r so il . α = 0 .0 5 , ** * p ≤ 0 .0 01, * * p ≤ 0 .0 1, * p ≤ 0 .0 5.
3.3. A gg r eg a te - St abi li t y of No - Replan t an d Replant Soil
In l ine with t he decr eased ag gr eg ate siz e in r epl ant s oil s ob s erved in A ug ust, re pla nt soi ls al s o
showed a hig hly s ig n ifica n t increase of l ess st abl e agg rega tes (W S ≤ 50 J m l −1 ) w ith sizes < 500 µm ,
and a corr esp onding decre as e of th e mo re st abl e k ind ( WS > 100 J ml −1 ), com pa r ed to no - rep l ant so il
(Ta bl e 2) . W ith 74 to 97%, th ese tw o stab il ity cl asses c ont ai n th e bu lk soi l m as s , wherea s for WL and
WS ≤ 10 0 J m l −1 neither hav e large m as s e s (except for th e coars e s an d fr action wit hin th e 50 0 – 1000 µ m
fra ct ion) no r show an y sig nif i cant e ffect s.

Figure 3.
Me an w eig ht d ia met er ( MWD ) of n o- re pl an t soi l (n r) an d r ep lan t so il (r ). Sig ni fi can ce s cal cu la ted
be tw een n r an d r so il. α = 0.0 5, * ** p ≤ 0. 00 1, 1 p ≤ 0 .10 .
T able 1.
Mean mass values (g) of sieve-size fractions fr om
≤
125
µ
m to 6300
µ
m in no-r eplant soil (nr)
and replant soil (r).
Sieve-Fraction ( µ m) Soil March April June August October
MW SD MW SD MW SD MW SD MW SD
2000–6300 nr 45.15 1.28 22.34 3.42 18.86 5.86 33.28 4.19 38.58 6.53
r 35.93 7.14 22.39 4.82 22.92 5.96
21.24 ***
3.52 31.89 6.78
1000–2000 nr 11.46 4.88 13.64 1.45 10.96 2.04 13.86 1.02 8.34 3.14
r 8.57 1.05 14.84 2.73 14.54 2.53 13.31 1.13 12.70 * 2.88
500–1000 nr 6.56 2.76 15.44 1.70 12.09 0.85 9.47 2.05 6.81 2.08
r 7.13 0.59 15.81 1.08 13.84 2.72
14.57 ***
1.64 11.06 * 2.99
250–500 nr 7.98 7.24 15.85 0.46 14.97 0.88 10.57 1.93 6.81 0.71
r 11.38 1.51 14.85 2.34 13.61 3.48
15.16 ***
1.34 10.90 2.18
125–250 nr 13.71 7.71 18.16 2.34 23.24 4.42 16.24 1.25 18.21 0.67
r 17.88 3.35 17.54 3.23 18.97 3.20 20.89 ** 2.02 17.68 1.24
≤ 125 nr 15.14 7.83 14.56 1.98 19.88 3.83 16.59 2.78 18.83 2.90
r 19.11 3.26 14.58 2.54 16.12 1.85 14.83 1.20 15.77 * 1.73
Significances calculated between nr and r soil. α = 0.05, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
3.3. Aggregate-Stability of No-Replant and Replant Soil
In line with the decr eased aggregate size in r eplant soils observed in August, replant soils also
showed a highly significant incr ease of less stable aggregates (WS
≤
50 J mL
− 1
) with sizes < 500
µ
m,
and a corr esponding decrease of the mor e stable kind (WS > 100 J mL
− 1
), compar ed to no-replant soil
(T able 2 ). W ith 74 to 97%, these two stability classes contain the bulk soil mass, wher eas for WL and

Soil Syst. 2020 , 4 , 70 9 of 16
WS
≤
100 J mL
− 1
neither have lar ge masses (except for the coarse sand fraction within the 500–1000
µ
m
fraction) nor show any significant e ff ects.
T able 2.
Mean concentration (mg) and proportion (%) of aggregate-stability classes in sieve-size
fractions from 125 to 1000 µ m in no-r eplant soil (nr) and replant soil (r) in August.
Aggregate-Stability Class Soil 125–250 µ m 250–500 µ m 500–1000 µ m
MW SD % MW SD % MW SD %
WL nr 42 17 1 280 84 12 711 200 24
r 58 14 2 279 60 9 693 140 23
WS ≤ 50 J mL − 1 nr 405 100 14 892 139 31 1203 224 40
r 679 *** 81 23 1235 *** 75 41 1369 122 46
WS ≤ 100 J mL − 1 nr 58 25 2 183 82 6 71 23 2
r 79 25 3 132 57 4 65 16 2
WS > 100 J mL − 1 nr 2494 95 83 1645 248 51 1015 146 34
r 2184 *** 90 73 1355 * 82 45 873 * 30 29
W ater labile aggr egates (WL), water-stable aggregates with binding for ces
≤
50 J mL
− 1
(WS
≤
50 J mL
− 1
),
water-stable aggr egates with binding forces
≤
100 J mL
− 1
(WS
≤
100 J mL
− 1
) and water-stable aggr egates with
binding forces > 100 J ml − 1
(WS > 100 J mL
− 1
) in sieve-size fractions: (a) 125–250
µ
m, (b) 250–500
µ
m, (c) 500–1000
µ
m.
Significances calculated between nr and r soil. α = 0.05, *** p ≤ 0.001, * p ≤ 0.05.
3.4. Correlations between Soil Fungi and Structural Parameters
W e correlated soil fungal densities in moist-field, non-sieved soil with soil str uctural parameters
(MWD and mass distribution of sieve-size fractions), as after soil drying and dry-sieving,
the quantification of soil fungal densities for sieve-size fractions did not result in r eliable data.
As a r esult, analysis showed corr elations in August (T able 3 ). Indirect corr elations between soil
fungal parameters and MWD in a logarithmic scale wer e due to di ff ering correlations between
fungal-parameters and mass distributions of sieve-size fractions. Fungal parameters were indir ectly
corr elated with fraction 2000–6300 µ m, and with fractions from 125 to 1000 µ m.
T able 3.
Correlation coe ffi cients and r egression coe ffi cients (R
2
) between soil fungal parameters and
MWD and mass distributions of sieve-size fractions.
Soil Structure
Parameter
Fungal
Parameter March April June August October
MWD
Ag / ITS − 0.442 − 0.527 − 0.147 − 0.790 ** (R 2 = 0.674) 0.238
Ag − 0.624 − 0.482 − 0.154 − 0.895 *** (R 2 = 0.825) 0.102
ITS − 0.055 − 0.082 0.154 − 0.671 * (R 2 = 0.570) − 0.140
2000–6300 µ m
Ag / ITS − 0.527 − 0.564 − 0.049 − 0.846 0.294
Ag − 0.685 * (R 2 = 0.215) − 0.500 − 0.140 − 0.951 ** (R 2 = 0.827) 0.137
ITS − 0.079 − 0.100 0.084 − 0.629 * (R 2 = 0.562) − 0.133
1000–2000 µ m
Ag / ITS − 0.336 − 0.136 − 0.294 − 0.280 − 0.483
Ag − 0.445 − 0.227 − 0.217 − 0.399 − 0.238
ITS 0.045 − 0.327 0.154 − 0.399 0.119
500–1000 µ m
Ag / ITS − 0.527 − 0.564 − 0.049 − 0.846 ** (R 2 = 0.554) 0.294
Ag 0.309 − 0.245 0.021 0.650 * (R 2 = 0.675) − 0.294
ITS 0.027 − 0.464 0.259 0.545 0.056
250–500 µ m
Ag / ITS 0.482 0.182 − 0.133 0.664 * (R 2 = 0.598) − 0.385
Ag 0.700 * (R 2 = 0.021) 0.191 0.021 0.797 ** (R 2 = 0.809) − 0.371
ITS 0.264 0.055 − 0.007 0.762 ** (R 2 = 0.688) − 0.189
125–250 µ m
Ag / ITS 0.418 0.391 0.413 0.804 ** (R 2 = 0.635) 0.154
Ag 0.564 0.373 0.308 0.867 ** (R 2 = 0.745) 0.186
ITS − 0.036 0.227 − 0.203 0.605 * (R 2 = 0.466) 0.168
≤ 125 µ m
Ag / ITS 0.427 0.409 0.476 − 0.063 0.552
Ag 0.664 * (R 2 = 0.007) 0.427 0.301 − 0.126 0.406
ITS 0.182 0.227 − 0.196 − 0.399 0.315
Significances calculated for α = 0.05, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Soil Syst. 2020 , 4 , 70 10 of 16
For fractions 125–250, 250–500 and 500–1000
µ
m, non-linear corr elations on a logarithmic scale
was observed between soil fungal parameters and WS
≤
50 J mL
− 1
and WS > 100 J mL
− 1
. Ag density
was dir ectly correlated with WS
≤
50 J mL
− 1
in fractions 125–250 and 250–500
µ
m, whereas it was
indir ectly correlated with WS > 100 J mL
− 1
in fractions 125–250 and 500–100
µ
m. T otal fungal-density
was only dir ectly correlated with WS
≤
50 J mL
− 1
in fraction 250–500
µ
m, and indirectly corr elated
with WS > 100 J mL
− 1
in fractions 125–250, 250–500 and 500–1000
µ
m. The proportion of Ag among
total fungi was only dir ectly correlated with WS
≤
50 J mL
− 1
and tended to an indir ect correlation with
WS > 100 J mL − 1 in fraction 125–250 µ m.
3.5. Correlation between T r ee V igour (CSA), Soil Fungi and Soil Structur e
The mean CSA of tr ees on replant soil of 19.0 cm
2
was significantly lower compared to the mean
CSA of r eference tr ees on no-replant soil of 38.5 cm
2
( p
≤
0.01). The range of tr ee vigour on replant soils
spanned a minimum of 5.4 cm
2
to a maximum of 33.8 cm
2
. For no-replant soil, the range of tr ee vigour
was 34.4 to 39.2 cm
2
. Di ff er ences in the CSA between no-replant soil and r eplant soil, but also within
soil variants (nr / r), wer e assumed to be reflected by soil fungal densities and r elated soil structure.
Corr elations were observed by a dir ect comparison of all data between no-replant soil and r eplant
soil (T able 3 , nr + r). Corr elation analysis of soil fungal parameters and soil structural parameters
(MWD and mass distribution of sieve-size fractions) with CSA highlights the significant correlations
between parameters in August (T able 4 ). Indirect corr elations on an exponential scale were observed
between Ag density , and proportion of Ag among total fungi and CSA. An exponential fitting of
r egression between MWD and CSA was due to dir ect correlation of CSA with the mass distribution of
fraction 2000–6300 µ m and indir ect correlation with fractions 250–500 + and 500–1000 µ m.
T able 4.
Spearman’s rank corr elation coe ffi cient (
ρ
s) among cross-sectional ar eas (CSA), soil fungal
parameters, MWD and mass distributions of sieve-size fractions.
Soil 1 March April June August October
Ag / ITS nr + r − 0.109 − 0.392 0.332 − 0.818 ** (R 2 = 0.77) 0.574
r − 0.059 − 0.228 0.197 − 0.679 0.418
Ag nr + r − 0.273 − 0.469 0.056 − 0.782 ** (R 2 = 0.70) 0.420
r − 0.301 − 0.287 0.192 − 0.429 0.117
ITS nr + r 0.118 − 0.255 − 0.242 − 0.370 − 0.018
r − 0.059 − 0.431 0.084 0.679 − 0.059
MWD nr + r 0.483 − 0.027 0.021 0.595 0.266
r 0.151 − 0.156 0.360 0.084 − 0.033
2000–6300 µ m nr + r 0.452 0.000 0.109 0.687 *(R 2 = 0.51) 0.308
r 0.101 − 0.024 0.460 0.268 − 0.117
1000–2000 µ m nr + r 0.487 − 0.328 − 0.186 0.039 − 0.447
r 0.426 − 0.359 0.209 − 0.218 0.004
500–1000 µ m nr + r 0.494 − 0.169 − 0.613 − 0.716 ** (R 2 = 0.55) − 0.550
r 0.445 − 0.539 − 0.747 − 0.387 − 0.226
250–500 µ m nr + r − 0.158 0.200 − 0.137 − 0.712 ** (R 2 = 0.62) − 0.343
r − 0.244 − 0.156 − 0.510 − 0.328 − 0.076
125–250 µ m nr + r − 0.095 0.173 0.161 − 0.487 0.028
r − 0.177 0.252 − 0.134 0.109 − 0.259
≤ 125 µ m nr + r − 0.126 0.183 0.483 0.106 0.441
r − 0.226 0.383 0.351 0.029 0.276
1 No-replant soil (nr) and r eplant soil (r), significances calculated for α = 0.05, ** p ≤ 0.01, * p ≤ 0.05.
4. Discussion
In this study we investigated correlations between the dynamics of soil fungal populations and
soil structur e (aggregates) in r elation to a gradual impact of replanting on tree vigour in a series of
time intervals over one gr owing season; we used not-replanted and r eplanted soil. Our results show
r educed aggregate size and stability , along with decr easing density of total soil fungal DNA (ITS)

Soil Syst. 2020 , 4 , 70 11 of 16
and incr easing density of Alternaria -group (Ag) for apple tr ees repeatedly planted on the same site,
which su ff er ed a loss of vigour . W e found that the density of Ag and soil structur e parameters correlate
at r eplant-indicative time intervals—in our study observed in August.
The determination of total fungal densities highlights a replant-r elated e ff ect in June and August
(and shows no r eplant-related specific behaviour of the total fungal population in Mar ch, April or
October). One replant-r esponsive soil fungal group, exemplary for indicating shifts in the soil fungal
community , the Alternaria -gr oup (Ag) (class Dothideomycetes , order Pleosporales , family Pleosporaceae ) [
19
],
continuously incr eased its density in replant soil over the gr owing season, r esulting in a distinct
di ff er ence of Ag density between soils in August. On the same site, the Ag was found to be
r eplant-indicative by density of soil fungal population two years earlier (2019) [
19
]. However , in 2016
an incr eased Ag density under replant conditions was observed in April with a two-fold gr eater Ag
density in no-r eplant soil and a four-fold gr eater Ag density in replant soil as compar ed to Ag densities
found in April 2018. Inter-annual variations have also been r eported for the date of maximum gr owth
di ff er ence between treated and non-tr eated replant soils, and for the e ff ect of soil tr eatments regar ding
combating r eplant-a ff ecting soil microbes [ 61 ].
The formation of aggr egates exhibits di ff erent dynamics between r eplant soil and no-replant soil
during the gr owing season. While the degree of aggr egation follows similar patterns, the aggregate
formation pr ocess is changed under replant conditions. The steady degr ee of aggregation between
April and August suggests that aggr egate turnover processes ar e prevented over summer under
r eplant conditions. A decreased aggr egation of replant soil has pr eviously been reported for the
r eplanting of peaches ( Prunus persica ) [
30
,
62
]. Concomitant with our results, the authors showed that
the r eplant-specific low aggregation was due to a decr eased proportion of fraction 2000–6300
µ
m and
incr eased proportions of fractions 125–250, 250–500 and 500–1000 µ m under r eplant conditions.
Our r esults show that fractions from 125 to 1000
µ
m and fraction 2000–6300
µ
m ar e replant-sensitive.
In contrast, fractions
≤
125
µ
m and 1000–2000
µ
m ar e replant-inert. The measurements of the mass
distribution of sieve-size fractions highlighted di ff er ences in the composition of soils regar ding soil
structur es (aggregates) in the r eplanted and initial planting ar ea, which are significantly pr onounced in
August and less str ong in October . Our indications of r eplant-sensitive sieve fractions in size ranges of
fine sand (125–250
µ
m), medium sand (250–500
µ
m) and coarse sand (500–1000
µ
m), though not fine
sand to silt and clay (
≤
125
µ
m), ar e consistent with several studies that state greater r eplant-related
tr ee vigour suppression in light sandy soils as compar ed to heavy clay or loamy soils [
11
,
13
,
15
,
63
].
This r esult implies that aggregated particles in size range of small and lar ge macroaggr egates (sands)
may perform as alternative micr ohabitats for increased densities of soil fungi.
Accor ding to our results, the mass distributions of fractions fr om 125 to 1000
µ
m, and fractions
2000–6300
µ
m ar e linked to increases of soil fungal densities (Ag) in August. The decrease of soil in
fractions 2000–6300
µ
m and the incr ease of soil in fractions from 125 to 1000
µ
m, correlate with an
incr ease in the density of soil fungi (Ag) under replant conditions. This observation is supported by a
distinct, though not significant r eplant-specific behaviour of soil fungi also observed in June, but this
could not be r elated to any change in the mass distributions of sieve-size fractions at that time of the
year . Nevertheless, the observations suggest an interaction between soil fungi (Ag) and the formation
of soil structur es (aggregates) during summer . Soil fungi, in particular filamentous fungi, have a
well-documented impact on soil structur e by formation or disintregration of aggr egates, especially
of macr oaggregates ( > 250
µ
m) [
64
,
65
]. The relevance of the corr elation analysis is the consideration
of potential interactions between soil and the apple under r eplant conditions over time. The ecology
of the soil fungal populations and their association with the soil structur e may be the next step in
understanding causal interlinkages r elated to replant disease. For this purpose, we understand our
case study as a first step that needs to be further tested by annual duplication in r epeated studies in
the field.
W et-sieving and ultrasonication highlighted an increased concentration of less stable soil str uctures
in fractions fr om 125 to 1000
µ
m in r eplant soils in August. Less stability of fractions 250–5000,

Soil Syst. 2020 , 4 , 70 12 of 16
500–1000 and
2000–4000
µ
m, though not in fraction 1000–2000
µ
m, has pr eviously been reported for
the r eplanting of peaches ( Prunus persica ) [
62
]. Other observations of r eplant-specific proportions
of aggr egates with di ff ering stability , notwithstanding 2 h sterilisation due to autoclaving [
30
,
62
],
showed a high persistence of aggr egate structures under r eplant conditions, even under extreme
abiotic conditions. Persistence of water-stable aggr egates for decades to centuries was already pr oven
by Jastr ow (1996) [
66
]. The potential persistence of a r eplant-sensitive aggregate stability class of
WS ≤ 50 J mL − 1
could contribute to the str ong persistence of replant-e ff ects that have been observed
also after grubbing and irr espective of catch crops [ 67 ].
Aggr egate-disintegrating processes in smaller and less stable aggr egates have been found at a
high Ag density . Increased Ag density is linked to less stable aggr egates and tr ee vigour suppression in
r eplant soil. The correlation between Ag density , soil structur e and tree vigour means that r eplant-e ff ects
can pass unnoticed for most of the vegetation period and become obvious only in specific time intervals.
This is r elevant for monitoring replant e ff ects by parameters of the soil.
A steady incr ease of replant-e ff ects on soil parameters in the summer season may potentially
match with the sensitive stage of apple nutrition by r oot performance. For apple understock M.9,
steadily incr eased growth of r oot has been reported fr om June until August [
68
,
69
]. Root flush has also
been r eported around bloom [
68
], approximately in late April to May in Germany [
70
], in line with
r eplant-specific increased Ag density observed in April 2016 [
19
]. Interestingly , the soil parameters’
r eturn to a similar density as was determined at the beginning of the growing season in Mar ch before
the beginning of dormancy season in October , and then did not di ff er between no-replant soil and
r eplant soil anymore. This suggests that a replant-e ff ect based on a shifted quantitative composition of
soil fungal population is in competition with root gr owth in soil and de facto diminishes tree vigour by
an o ff set of ontogenetic development, pr obably due to seasonally limited access to nutrients.
Our observations suggest that di ff er ences between replant and no-r eplant soils are pr onounced,
but may occur at irr egular intervals. This in turn would mean that continuous and densely gridded
monitoring of soil (and plant) during the whole gr owing season of apple would be necessary to detect
indicative parameters for apple r eplant disease. It also shows that one-time sampling of or chard test
sites and homogenised soil samples taken at few times only can be misleading in detecting interactions
of soil fungi and soil structur e (and tree vigour), depending on the time of sampling.
An analysis of interr elations between soil fungi, soil structure and apple tr ee vigour (suppression)
will r equire continuous and densely gridded monitoring of soil to detect r eplant e ff ects at indicative
time intervals, and needs to be performed on single planting spots rather than with homogenised
soil samples.
5. Conclusions
Soil structur e was found to be replant-sensitive by mass distribution of lar ge and small
macr oaggregates (2000–6300
µ
m, fr om 250 to 1000
µ
m) and lar ge microaggr egates (125–250
µ
m).
Small macr oaggregates and lar ge microaggr egates are less stable under r eplant conditions. The statistical
analyses suggest that specific r eplant-responsive soil fungi, here the Alternaria -gr oup (Ag), are involved
in r eplant-related changes in soil structur e. Hence, replant-specific aggr egate-disintegrating processes
seem to be r elated to densities of soil fungi. A correlation between soil fungi and str ucture can only be
detected at specific time intervals over the gr owing season. Pronounced di ff er ences in soil structur e
between no-r eplant soil and replant soil occur together with a selective growth of Ag densities in
late summer .
The density of r eplant-responsive soil fungi (Ag), in particular , is highly correlated with the
plant r eaction of trees in r eplant soil, so we conclude that the r eplant e ff ect is a biologically active
pr ocess. On the one hand, changes in soil structur e contribute to the functional conditions for growth
of specific soil fungi, and on the other hand, soil fungi may be involved in the formation of less stable
soil aggr egates. Our study suggests that the interaction between soil fungi and soil aggr egates may be
causally linked to interr elations between replant soil and plants.

Soil Syst. 2020 , 4 , 70 13 of 16
An analysis of the interrelations between soil fungi, soil structur e and apple tree vigour
(suppr ession) will requir e continuous and densely gridded monitoring of soil to detect replant
e ff ects at indicative time intervals, and needs to be performed on single planting spots rather than with
homogenised soil samples. In an applied context of the restoration of r eplant soil, our results pr ovide
the first indication that a potentially negative e ff ect of the Ag on soil structur e could be managed by
good soil aggr egators, e.g., mycorrhiza, to r estore soil structur e under replant conditions.
Author Contributions:
Conceptualization, U.C. and P .L.; methodology , U.C., P .T . and P .L.; validation, F .B. and
P .L.; formal analysis, U.C. and P .L.; investigation, U.C. and P .T .; r esources, U.C., P .T ., F .B. and P .L.; data curation,
U.C.; writing—original draft preparation, U.C.; writing—review and editing, K.D. and F .B.; visualization, U.C. and
P .T .; supervision, P .L.; pr oject administration, K.D.; funding acquisition, K.D. All authors have read and agr eed to
the published version of the manuscript.
Funding:
The resear ch for this paper was financed by the German Federal Ministry of Education and Research
(BMBF) within the framework of the BonaRes Initiative (FKZ:031B0025D), www .ordiamur .de .
Acknowledgments:
The authors gratefully acknowledge the work of Petra Lange. W e also thank Lutz Günzel
and his team for providing access and insights into his plantation during our r esearch cooperation.
Conflicts of Interest: The authors declare no conflict of interest.
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