Applied Soil Ecology 92 (2015) 4753

Contents lists available at ScienceDirect

Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Molecular prole of microbiota of Finnish commercial compost

suppressive against Pythium disease on cucumber plants
a

D. Yu , A. Sinkkonen
a ,d ,
M. Romantschuk
*

a,d

, N. Hui , J.M. Kurola , S. Kukkonen , P. Parikka , M. Vestberg ,

University of Helsinki, Department of Environmental Sciences, Niemenkatu 73, FI-15140 Lahti, Finland
Natural Resources Institute Finland (Luke), Green Technology, Antinniementie 1, FI-41330 Vihtavuori, Finland
c
Natural Resources Institute Finland (Luke), Natural Resources and Bioproduction, FI-31600 Jokioinen, Finland
d
Institute of Environmental Sciences, Kazan Federal University, 420008 Kazan, Russia
b

A R T I C L E

I N F O

A B S T R A C T

Article history:
Received
Received
Accepted
Available

4 September 2014
in revised form 12 February 2015
13 March 2015
online 1 April 2015

Keywords:
Compost
Pythium wilt suppression
Actinobacteria
Acidobacteria Gp14
Cystobasidiomycetes
454 sequencing

1. Introduction

The use of compost of varied origins for the management of plant disease has been reported all
over the
world for decades, and microorganisms are suggested to be one of the driving forces. In t
his study,
composts produced during two successive years were mixed with peat at 1:4 v/v ratios, and scr
eened for
cucumber Pythium disease suppression in two experiments in 2009. Nine composts were select
ed based
on their suppressive effects against Pythium on cucumber plants, and thereafter were su
bjected to
molecular analyses. The aim of this study was to compare the bacterial and fungal communitie
s present
in strongly-suppressive and non- or weakly-suppressive composts, in an effort to identify
bacterial and
fungal groups associated with Pythium disease suppression. The total genomic DNA from the c
omposts
was extracted and amplied using primers targeting 16S rRNA and ITS rRNA genes of bacteria a
nd fungi,
respectively, prior to 454 sequencing. Sequencing results indicated the potential roles o
f bacterial
Acidobacteria Gp14 and fungal Cystobasidiomycetes in the suppression of Pythium wil t diseas
e, due to
their presence and absence in composts with strong and lower/absent disease suppressi
on ability,
respectively. The abundance of Actinobacteria was associated positively with suppr
essiveness.
Phosphorus concentration was the only abiotic factor that was associated with suppressi
veness: a
negative correlation was found in both experiments. These results indicate that certain bac
terial and
fungal groups can potentially suppress Pythium wilt while the abiotic conditions might be less i
mportant.
2015 Elsevier B.V. All rights reserve
d.

1997; Noble and Coventry, 2005; Pugliese et al., 2011; Mehta

et al.,
2014). Various mechanisms have been suggested to explai
n the
microbial

Composting is an aerobic
driven process
that
degrades organic waste into a stable, sanitary, humus-like
material

that can be safely returned into the environment. I

t is also
considered as an alternative to pesticides in means of con
trolling

soil borne plant diseases while generally boosting plant healt disease suppression by compost and its microbiota. Among t
h and
hese
productivity (Hadar and Mandelbaum, 1992; Neher et al., are competition for nutrients and space among mi
2013).
crobial
Reports on the use of composts to suppress soil-borne populations (Dinez et al., 2005; Vestberg et al., 2014), inhib
plant
ition
disease can be traced back almost half a century (Hoitink on pathogen growth by antibiotics produced by microbiota (Me
hta
et al.,
1977). Since then, this concept has been extensively revie et al., 2014), and the hyperparasitism where a microorg
wed in
anism
numerous publications (Hoitink and Fahy, 1986; Hoitink directly attacks a pathogenic microorganism and k
ills it
et al.,
(de Bertoldi, 2010). Studies have shown that heating or autocl
aving
eliminates the disease suppression capacity of the compost,
* Corresponding author at: University of Helsinki, Department of Environm
while
ental
this capacity can be recovered by mixing in natural compost
Sciences, Niemenkatu 73, FI-15140, Lahti, Finland. Tel.: +358 9 191 20334;
with
fax: +358 9 191 20331.
the heated or sterilized composts (Nelson and Hoitink,
E-mail address: martin.romantschuk@helsinki. (M. Romantschuk).
1983;
Hoitink and Fahy, 1986). In addition to the presumed activi
ttp://dx.doi.org/10.1016/j.apsoil.2015.03.005
0929-1393/ 2015 Elsevier B.V. All rights reserved.
ty of
antagonistic microorganisms, abiotic factors such as nitrogen (
total
and available), C:N ratio, pH, heat, moisture, as well as degr
ee of
compost maturity have been suggested to be associated with
the
suppression capacity (Kuter et al., 1988; Hadar and Gorecki,
1991;
Hoitink and Grebus, 1997).
48

D. Yu et al. / Applied Soil Ecology 92 (2015) 4753

Pythium is one of the most studied soil borne plant pathog

ens.
Various species of Pythium cause damping-off and wilting in a
very
large number of plant species. Both naturally occurrin
g and
amended composts suppressive against Pythium plant dis
eases
have been described. Composts made with peat and bark h
ave a
low indigenous suppressiveness against pathogens su
ch as
Pythium spp. (Bonanomi et al., 2010), and have t
herefore
commonly been used as substrates to produce compost mixt
ures.
Chen et al. (1988) and Hagn et al. (2008) reported the signi
cance
of compost microorganisms in amended composts suppre
ssive
against Pythium dumping-off. The microbial communi
ty in
compost depends on both compost feedstock (e.g., ingred
ients,
pH, nutrients, C:N, NH 4 /NO3 ) and composting process
(e.g.,
temperature, oxygen, ammonia, moisture, microbial act
ivity).
The microbial community and its metabolic activity h
as an
important role in affecting the disease suppression abil
ity of
compost (McKellar and Nelson, 2003; Hagn et al., 2008;
Neher
et al., 2013). However, knowledge about how microbes contro
l soil
borne pathogens is still scarce.

This is a continuation study of Vestberg et al. (2011, 2014),)

, in
which twenty-one Finnish commercial mature composts of
varied
origins (e.g., sewage sludge, biowaste, manure) were mixed w
ith
sterilized peat and screened for their ability to suppress cucum
ber
wilt caused by Pythium in two experiments in 500 ml pots with
ve
replicates. To inoculate the cucumber plants, Pythium mycel
ium
and the growth media (PDA potato dextrose agar) were grou
nd
into pulp. Five grams of Pythium inoculum was added to each p
ot at
planting depth. The cucumber plants were grown in a greenho
use
at temperatures of 24 (day) and 18 C (night) and a 16-hour
day
(Vestberg et al., 2011). Thereafter, nine compost mixtures
that
showed suppressive ability or were neutral/conducive tow
ards
cucumber Pythium disease were divided into two groups
for
microbial and statistical analyses in the present study. The ai
m of
this study was to investigate the connections between
the
microbial communities and the disease suppression abilit
y of
compost against the soil-borne pathogen. To the best of
our
knowledge this is the rst study that at the genus-level ta
rgets

both bacteria and fungi suppressive against Pythium dampin Table 1

Compost mixtures with strong disease suppression ability (Group A) and compo
g-off.
st
Speci cally, the objectives were to (a) characterize the mi mixtures with low or absent disease suppression ability (Group B) in
crobial
two
community composition, (b) identify the speci c bacteria experiments. Two Group A compost mixtures were omitted from each experime
nt
l and
fungal groups (e.g., at genus-level) associated with the su due to their low sequence reads. Raw materials of composts: 1 poultry manure
,3
ppresbiowaste, 5 horse manure/paper mill sludge, 6 cattle manure/garden waste,
siveness against cucumber Pythium wilt, and (c) exami 7
ne the
biowaste, 13 biowaste, 14 sludge, 17 sludge/biowaste, 20 biowaste.
connection between relative abundance of bacteria and fungi
Group A compost mixtures
Group B compost mixtures
and
a
Bacteria 1stB
5
14
20
1
3
6
the disease suppression ability of composts.
7
b

1stE
a
2ndB
b
2ndE

2. Material and methods

2.1. Suppressiveness of selected compost
Fungi

1stB

5
14
14
a

14
17
17
14

17
20
20
20

1
1
1

3
3
3
1

6
6
6
3

7
7
7
6

Nine composts of different sources (Table 1) produced in

7
the
b
1stE
5
14
20
1
3
6
7
successive years 2008 and 2009 were selected for the
a
2ndB
14
17
20
1
3
6
7
b
present
2ndE
14
17
20
1
3
6
7
microbial diversity study based on their respective suppre a
B: beginning of the suppressiveness experiment.
ssiveb
E: end of the suppressiveness experiment.
ness characteristics during two large screening exper
iments
Sinkkonen et al. (2013). Suppressiveness capacity value
against cucumber Pythium wilt, which were carried out i s of
n two
composts that signicantly (at p < 0.05) differed from the cont
periods: from February to March 2009 (1st experiment, begin
rol
ningtreatment (steam sterilized peat), were regarded as suppres
end, Vestberg et al., 2011), and from August to September sive
2009
(Vestberg et al., 2014). Based on the results of Vestberg et al. (2
(2nd experiment, beginning end, Vestberg et al., 2014). Pr 011),
ior to
among nine selected composts, seven (numbers 3, 5, 7, 13, 14,
both suppressiveness experiments, composts were mixed 17,
with a
20 in Table 1) were suppressive to different extents, one (numb
mixture of 75% steam sterilized natural Sphagnum peat an er
d 25%
6 in Table 1) was neutral, and one (number 1 in Table 1)
sand at 1:4 v/v ratios called compost mixtures in the p was
resent
conducive to Pythium wilt disease in the rst suppressive
study. The steam sterilized and natural non-sterilized peat ness
-sand
experiment using composts of 2008. Similar observations w
mixtures without composts were applied as controls in ere
both
obtained in the second suppressiveness experiment using co
experiments.
mSuppressiveness capacity was calculated as the % differen posts of 2009 (Vestberg et al., 2014).
ce in
In the present study, three compost mixtures with str
shoot dry weight (DW) between non-inoculated and Py ong
thium
suppression ability were categorized as Group A the suppressi
inoculated cucumber plants. DW was measured as describ
ve
ed by
group, and the remaining compost mixtures with lower or abse
nt
disease suppression ability were categorized as Group B
the
other group (Table 1). The natural peat (number 23) was wea
kly
suppressive against wilt disease, while the steam sterilized
peat
(number 22) was neutral.
2.2. DNA extraction and quanti cation
To study the bacterial and fungal communities, total genom
ic
DNA was extracted and ampli ed using primers targeting 16S rR
NA
and ITS rRNA genes of bacteria and fungi, respectively. Sampli
ng

was performed as described by Vestberg et al. (2014). Total Oligomer (Helsinki, Finland). PCR ampli cations were conduct
DNA
ed
was extracted from 0.3 g of compost mixtures and pure peat at two stages. At the rst stage, the bacterial 16S rDNA target g
from
ene
each suppressiveness experiment using a FastDNA 1 SPIN Kiwas ampli ed using primers 341F (5 0- CCT ACG GGA GGC AGC
t for
AG
soil (Qbiogene Inc., Carlsbad, USA) according to the manufactu -3 0) (Muyzer et al., 1993) and 907R (5 0- CCG TCA ATT CMT TTG A
GT
rer s
0
instructions. The concentration of DNA was measured TT -3 ) (Ishii et al., 2000). PCR mixtures included 1 ml of
uorogenomic
metrically using PicoGreen 1 dsDNA Quantitation Reagent and DNA; 1 ml of each primer (10 mM); 5 ml of 10 DyNAzyme Bu
Kits
ffer,
(Molecular Probes Inc., Eugene, OR, USA).
1 ml of dNTP (10 mM), 0.5 ml of DyNAzymes II DNA polymer
ase
2.3. Ampli cations of bacterial 16S and fungal 18S ITS target g (2 U/ l) from Finnzymes, Thermo Scienti c, Finland; 0.5
m

ml of
enes
bovine serum albumin (BSA) (20 mg/ml) from Fermentas, Ther
mo
using 454 sequencing
Scienti c, Finland; and 40 ml of sterile water. Cycling conditi
All primers used in the present study were purchased ons
from
were 5 min at 94 C followed by 20 cycles of 20 s at 94 C, 20
s at
D. Yu et al. / Applied Soil Ecology 92 (2015) 4753

57 C, 30 s at 72 C, and a nal elongation of 5 min at 72

C. The
fungal ITS target gene was ampli ed using primers ITS1F (5 0
-CTT
GGT CAT TTA GAG GAA GTA A-30) (Gardes and Bruns,
1993) and
ITS2R (5 0 -GCT GCG TTC TTC ATC GAT GC-30) (White et al.,
1990).
PCR mixture ingredients were similar to that of bacteri
a with
modi cations in the BSA (1 ml), polymerase (1 ml), and
sterile
water (39 ml). Cycling conditions were 8 min at 95 C follow
ed by
20 cycles of 1 min at 95 C, 1 min at 55 C, 1 min at 72 C, an
d a nal
elongation of 10 min at 72 C. The rst stage PCR ampli catio
n was
performed in PTC-100 Thermo Cycler (MJ Research Inc.,
Walthman,
MA, USA). In the rst round three replicates per sampl
e were
pooled and puri ed using Agencourt AMPure XP PCR puri ca
tion
kit (Beckman Coulter, Beverly, MA, USA) prior to the second r
ound.
At the second stage, the ampli cations of both bacteri
a and
fungi were completed with 10 additional cycles with s
ample
speci c tag sequences and linker A (5 0 -CGT ATC GCC TCC CTC
GCG
CCA TCA G-3 0) attached to the forward primer (adapter A
), and
linker B (5 0 -CTA TGC GCC TTG CCA GCC CGC TCA G-3 0)
attached to
the reverse primer (adapter B). PCR mixtures included 1 ml
of PCR
products from the rst round,1 ml of each Adapter (10 mM),1
0 ml of
5 Phusion GC Buffer, 0.4 ml of dNTP (10 mM), 0.5 ml of P
husion
DNA Polymerase (2 U/ml), 1.25 ml of DMSO, and 34.85 ml of
sterile
water. Cycling condition for bacterial 16S rDNA ampli cations
was

49

30 s at 98 C followed by 10 cycles of 10 s at 98 C, 30 s at
65 C, 10 s
at 72 C, and a nal elongation of 5 min at 72 C. Wi
th fungal
adapters the annealing temperature was 55 C. The seco
nd stage
PCR ampli cation was performed in PTC-225 Thermo Cy
cler (MJ
Research Inc., Walthman, MA, USA). The PCR produ
cts were
puri ed and quanti ed as described by Koskinen et al. (20
11) prior
to 454 sequencing. The sequencing was performed u
sing the
454 GS FLX protocol, yielding read length of about 250 bp (
454 Life
Sciences, Roche Diagnostics, CT, USA).
2.4. Sequence data processing
The bacterial sequence data were analyzed using MOT
HUR (v.
1.35.0, 64-bit for Linux; Schloss et al., 2009) according to a
standard
operating procedure with modi cations (Schloss et al.
, 2011),
which were (i) due to majority of our sequences were short
er than
200 bP, we pre-clustered sequences to remove sequences
within a
distance of 1 bp (pre.cluster, diffs = 1); (ii) we clustered th
e distance
matrix at 95% similarity as previously suggested by
Hui et al.
(2012). The fungal sequence data were analyzed using
MOTHUR
following the same method as described by Brown et al.
(2013).
This generated a matrix that consisted of 4537 bacterial O
TUs and
648 fungal OTUs (excluding singletons OTUs with o
nly one
sequence), and this matrix was used in diversity analys
es made
with R statistical software (http://www.r-project.org).
Thereafter,

bacterial and fungal OTUs were classi ed using Rib 2.5.2. Suppressiveness analyses
Components of the SPSS (IBM SPSS Statistics, Armonk,
osomal
Database Project (RDP; Wang et al., 2007) and UNITE ITS dat New
York, U.S.) program package were used in statistical an
abase
(Koljalg et al., 2013), respectively. To explore organismal cov alyses.
Paired samples t-tests were used to analyze if time a
erage
among pathogen suppressive composts, rarefaction curves ffected
Pythium disease suppression % (i.e., suppressiveness) and
were
microcalculated using EstimateS (Colwell, 2011).
bial diversity. Pearson correlation analysis was used to co
mpare
2.5. Statistical analyses
suppressiveness in the rst vs. the second experiment comp
osts.
Mann Whitney U tests were used to analyze the assoc
2.5.1. Diversity analyses
Species diversity indices were calculated as described b iation
y Hui
between suppressiveness and the relative abundances bet
et al. (2012). Total number of OTUs was used as observed sp ween
bacteria and fungi. Pearson correlation analysis was us
ecies
richness of bacteria and fungi. Total species richness of bactered to
associate suppressiveness with diversity indices and bacteria
ia and
fungi was estimated using Chao1 index (Chao 1984; Hughes l and
fungal abundances (both as percentages and as reads)
et al.,
2001). Shannon diversity and Simpson s indices were u at the
phylum and class levels. Linear regression analysis was u
sed to
estimate diversity of bacterial and fungal communities (Cha sed to
determine whether microbial abundances (%, at the e
o and
nd of
Shen, 2003).
experiment) affected suppressiveness. The most abundant b
acterial phyla (Proteobacteria, Bacteroidetes, Actinobacteria,
Acidobacteria, Flavobacteria and Firmicutes) were included i
n the
regression analysis.
In the statistical analyses, each experiment and time
point
(either the beginning or the end of an experiment) was a
lways
analyzed separately, i.e., the values were not combined or tr
eated
as (pseudo) replicates. Composts of low sequence reads
were
excluded from all statistical analyses. As bacterial taxo
nomic
groups in the beginning of composting and fungal taxon
omic
groups were not associated with suppressiveness in any o
f our
attempts to perform a predictive or descriptive statistical anal
ysis,
we show only the results of bacterial correspondence analys
es in
the end of the experiments.
2.6. Quantitative PCR ampli cation
To determine the ratio of bacteria (16S rDNA) and fungi
(ITS
rDNA), quantitative PCR (qPCR) was done using a DNA E
ngine
OPTICON 2 (Continuous Fluorescence Detector, MJ Research
Inc.,
Waltham, MA, USA) in a nal volume of 20 ml containing 1 10
ng of
TM
total DNA. A DyNAmo
HS SYBR 1 Green qPCR kit (Finnzy
mes,
Thermo Scienti c, Finland) was used in all PCR runs. Bacteria
l and

fungal DNA was ampli ed using primers pairs pE and pF, ITS3 a that varied between 26.71 486.97 mg l 1 in different co
nd
mposts
ITS4 as described earlier (Yu et al., 2014)
(Table 1 in Supplementary material) was associated
with
3. Results and discussion
suppressiveness in the rst (r s = 0.82, n = 11, p = 0.007)
and the
3.1. Compost properties and disease suppression
second (r s = 0.72, n = 11, p = 0.13) experiments. Other corr
elations
Compost of biowaste and sludge sources suppressed disea between abiotic variables and suppressiveness did not exist.
se
symptoms of cucumber wilt caused by Pythium (Vestberg et
3.2. Species richness and disease suppression
al.,
2014). First experiment suppressiveness correlated with sec
None of the rarefaction curves for bacterial and
ond
fungal
experiment suppressiveness (r = 0.67, n = 9, p = 0.049), and a sequences reached the asymptote (data not shown), indi
verage
cating
suppressiveness was the same regardless of the production y that coverage of the richness of bacteria and fungi in co
ear
mpost
(t = 1.2, df = 8, p = 0.26). Direct bivariate correlations be mixtures studied was insuf cient. However, the num
tween
ber of
suppressiveness and abiotic variables (described in Table 1 bacterial OTUs increased during cucumber growth in the
in
rst
Supplementary material) were rare. Phosphorus concentrati experiment (t = 3.7, df = 6, p = 0.010) and was indicativ

on
e in the
50

D. Yu et al. / Applied Soil Ecology 92 (2015) 4753

second experiment (t = 2.2, df = 6, p = 0.07), demonst

rating a
possible better coverage of bacterial community richness i
n the
end of both suppressiveness experiments, or a more
diverse
bacterial community. The number of OTUs or the change i
n the
number of OTUs, however, did not correlate with suppressive
ness
at any time point (r
0.46, n = 6, p ! 0.29). Chao1 index,
Shannon
diversity index and Simpson s diversity index did not cor
[(Fi
relate
with
g._ suppressiveness at any time point (p ! 0.11).

Samples with exceptionally low number of sequences (162

1215)
were excluded from statistical analysis. Forty-six classes
belonging
to 20 bacterial phyla were detected and the most dominant clas
ses
were ranked according to their sequence abundance orders (Fig
. 1).
As expected, Proteobacteria comprised the majority of
the
sequences in all composts (Danon et al., 2008), accountin
g for
49 64% and 34 61% of the total sequences in the rst and
second
suppressiveness experiments, respectively; while its subp
hyla,
3.3. Microbial pro ling of composts
Gammaproteobacteria, Alphaproteobacteria, and Betaproteob
acThe biotic component has been documented to play a cr teria were the most abundant groups. The most dominant clas
s in
ucial
the bacterial community shifted from Gammaproteobacteria (
role in disease suppression, but very few studies have des 1st
cribed
experiment beginning: 18 48%, 2nd experiment begin
both bacterial and fungal diversities in plant disease suppre ning:
ssive
composts (Bonanomi et al., 2010). In general, the major bac
terial
and fungal groups found in the present study using 454 seque
ncing
are similar to previous studies that have used other sequ
encing
approaches (e.g., Illumina MiSeq sequencing, Sanger sequen
cing)
to describe microbial communities in composts of simila
r raw
materials (Neher et al., 2013).
3.3.1. Bacterial diversity overview and disease suppression
A total of 237,710 quality- ltered sequences were
identi ed as
bacteria by RDP classi er. The number of 16S rDNA seq
uences
obtained per sample varied from 2284 to 11749 (median =
5893).

6 43%) to Alphaproteobacteria (1st experiment end: 2 chemical and physical properties. Within those classes Sphin
3 46%,
go2nd experiment end: 34 50%) during both suppressiv bacteria (5 42%), and Flavobacteria (0.1 19%) were numer
eness
ous.
experiments. Previously, Alpha- and Gammaproteobacteria Actinobacteria were correlated positively with suppressiveness
have
in
been documented as typical compost bacteria (Michel et al., 2 both experiments (r ! 0.82, n = 7, p
0.029). In backward r
002;
egresHagn et al., 2008; Partanen et al., 2010). Gammaproteoba sion, the abundances (%, at the end of experiment) of Act
cteria,
inoespecially Pseudomonas, were described as typical bioc bacteria affected suppressiveness in both experiments (MS = 36
ontrol
00,
organisms that protects plant from the oomycete Pythium ulti F = 9.2, df = 1, 6, p = 0.029 in the rst and MS = 2610, F = 23.2,
mum
df = 1, 6,
(Boehm et al., 1993; Rezzonico et al., 2005) and attain p = 0.005 in the second experiment). The effects were positive,
ed the
i.e.,
highest percentage of positive correlation with disease sup high actinobacterial abundance increases Pythium wilt dise
presase
siveness (Bonanomi et al., 2010). However, in the current suppression. It has been well documented that Actinobacteria h
study,
ave
Gammaproteobacteria sequence reads dropped during a positive impact on plant disease suppression due to its str
both
ong
disease suppression experiments. A possible explanation for ability to produce antibiotic compounds (Cross, 1982). A rec
this
ent
observation could be the trophic interaction between the fu study by McKellar and Nelson (2003) also supported
ngal
this
pathogen (Pythium) and bacteria (Gammaproteobacteria) in statement by demonstrating a more dominant presence
the
of
root environment (rhizosphere) (Frey-Klett et al., 2011),
Actinobacteria in disease suppressive consortia than in dise
therefore
ase
causing the replacement of Gammaproteobacteria with Alp conductive or neutral consortia. However, after expl
haporing
roteobacteria.
2423 cases derived from 252 studies on disease manage
Phyla Bacteroidetes, Acidobacteria, and Firmicutes were c ment
omusing organic amendments, Bonanomi et al. (2010) concluded t
monly distributed in all composts regardless of their maturity hat
and

Fig. 1. The abundance of major bacterial classes (log 10 scale) in Group A and Group B compost mixtures in the rst experiment. The rank of bacterial classe
s in the second
experiment was exactly the same as in the rst experiment. In spite of the potential roles of Acidobacteria Gp14 in the suppression of Pythium wilt diseas
e, its sequence
abundance was too low to be visible in this gure. (a) Beginning of experiment, (b) end of experiment.
D. Yu et al. / Applied Soil Ecology 92 (2015) 4753

51

Actinobacteria were only directly correlated with disease sup 3.3.2. Fungal diversity overview and disease suppression
presThe number of ITS rDNA sequences obtained per sample va
sion in a limited number of experimental cases. Actinobacteri ried
a are
from 818 to 7171 (median = 2143). Thirteen classes belongin
known to have a major role in decomposition of organic mate g to 3
rials
phyla were detected (121,783 quality- ltered sequences
particularly for degradation of macromolecules such as cellu across all
lose,
samples), and the abundance rank order of the most dom
hemicellulose, lignin and chitin (Steger et al., 2003). Therefo inant
re the
classes are shown in Fig. 2. As expected, Ascomycota represe
differences in these observations could be explained by diff nted
erent
the largest phylum accounting for 85 97% of the total seq
experimental conditions such as compost origin, compost ch uence
emireads across all compost mixtures in the rst suppressi
cal and physical properties, compost maturity, com veness
posting
experiment. Ascomycota classes Eurotiomycetes, Dothide
method, soil-borne pathogens, and test plants in the omyabovecetes, Sordariomycetes, and Saccharomycetes were the
mentioned studies. Also, the experimental conditions fa most
voring
predominant groups. The results of Langarica-Fuentes
Actinobacteria might be fortuitously detrimental to soil- et al.
borne
(2014) and Hultman et al. (2010) were in agreement wi
pathogens with no direct cause-effect relationship, and th th our
ereby
ndings that Ascomycota was the most abundant phylum i
not be based on competition between similar microbes. In all n the
, the
compost fungal community. During the second suppressive
presence and dominance of Actinobacteria appears to b ness
e case
experiment, Ascomycota maintained its overwhelming domin
speci c and prone to large variations.
ance
On the conducive side, constant trends were not detected. in all Group A composts (37 97%) as well as in the majo
The
rity of
huge taxonomic variability at narrower taxonomic levels
Group B composts. However, exceptions were found in two Gr
(e.g.,
oup
genus) may be a reason why straightforward patterns could n B members (Compost 1 and 3) of poultry manure and bio
ot be
waste
detected.
origin, in which Zygomycota (Zygomycota class incertae
Results from OTU analysis using 454 sequencing data rev sedis)
ealed
replaced Ascomycota as the most dominant group and accou
that Acidobacteria are present in all composts studied. No nted
tably,
for up to 54 66% of the total sequence reads. The nding
Acidobacteria Gp14 was detected only in Group A co
supports
mpost
the previous statement that Zygomycota are more
mixtures i.e., composts with strong disease suppression a closely
bility
associated with composts of manure origin than compos
(0.011 0.018%), which indicated its potential role in the
ts of
suppresother origins (Neher et al., 2013). In contrast, in t
sion of Pythium wilt disease. Acidobacteria have previously he rst
been
suppressiveness experiment Zygomycota comprised only 0.
detected in waste water, sludge, compost and biofertilize
2 1%
r that
of the total sequence reads in Composts 1 and 3. The total am
associated with banana Fusarium wilt disease suppression (Z ounts
hang
of nutrients in compost mixtures used in both suppressiv
et al., 2012; Fracchia et al., 2006; Shen et al., 2014), bu eness
t not its
experiments did not differ in a signicant way from each
subgroup Acidobacteria Gp14. To our knowledge, this is the
other.
very
However in the second experiment, Compost 3 was found
rst study to note the potential relevance of Acidobacteria Gp to be
14 in
immature according to its NH 4 -N/NO3 -N ratio and Rotte
Pythium disease suppression. The results obtained her grad
e are,
measurement, while, Compost 1 had clearly lower ele
however, not suf cient to declare Acidobacteria Gp14 ctrical
as the
conductivity (EC) in comparison with Compost 1 in th
reliable indicator of Pythium disease suppressive capability. e
rst
More
experiment (Vestberg et al., 2014). The relationship be
comprehensive research on its association with disease sup tween
prescompost maturity and its microbial community is yet too com
sion needs to be conducted.
plex

[(Fig._2)TD$IG]

to be identi ed. Therefore, more studies are needed to con r

m the

Fig. 2. The abundance of major fungal classes (log10 scale) in Group A and Group B compost mixtures in the rst experiment. The rank of fungal class
es in the second
experiment was exactly the same as in the rst experiment. (a) Beginning of 1st experiment, (b) end of 1st experiment.
52

D. Yu et al. / Applied Soil Ecology 92 (2015) 4753

putative relationship between compost maturity/EC and rel

ative
abundances of Zygomycota and Ascomycota.
454 sequencing results of the rst suppressiveness experi
ment
indicated that Basidiomycota class Cystobasidiomycetes
could
have contributed to the suppression of Pythium wilt disease,
due
to its presence (0.056 0.36%) in all Group A compost
mixtures and
its absence in Group B compost mixtures. In the
second
suppressiveness
experiment
no
Cystobasidiomycetes
were
detected in any compost studied. Cystobasidiomycetes have r
arely
been mentioned in previous studies. For example, Cystoba
sidiomycetes have not been reported as potential indicators
of any
plant disease suppression by compost, although certain speci
es of
the class, such as the parasitic fungus Rhodotorula (Singh
et al.,
2012), have been detected in the early phase of mu
shroom
composting. However, it is commonly accepted that
some
members of the same higher taxonomic level may have
similar
activity in plant microbe soil interactions, while others may
not
have. To improve understanding the potential disease suppres
sion
abilities of Cystobasidiomycetes and its members, further st
udies
are needed. Previously, some benign fungi that are
closely

related to plant pathogenic fungi (e.g., Fusarium spp.) have

been
reported to suppress to plant disease apparently due to t
heir
competition for space and resources in the same plant envi
ronment (Fravel et al., 2003; Bonanomi et al., 2010). Howev
er, no
benign fungi related to pathogenic Pythium were found in
the
current study.
Fungal abundances (%) at the class level of any compost var
ied
extensively between experiments, for example signicant di
fferences were found in Dothideomycetes, Tremellomycetes,
and
Saccaromycetes (paired samples t-test t ! 2.9, df = 8, p
0.02
9). The
abundance of the above-mentioned groups seem to depend
more
on local environment than the compost per se.
3.4. Relative abundance of bacteria and fungi using quantitative
PCR
Results calculated from qPCR standard curves indicated that
in
spite of low sequence reads in some composts, the copy num
ber
ratios of bacterial 16S and fungal ITS rDNA uctuated within
the
ranges of 7 90 throughout both screening experiments in
all
composts (24 22.6 and 36 27.7 in Group A and Gr
oup B
compost mixtures, respectively). There was no clear associ
ation

between Pythium disease suppression and the relative abund Acknowledgements

ances
We wish to thank the composting plants for giving us compos
between bacteria and fungi (U ! 3, Z ! 1.5, n = 9, p ! 0.1
ts.
2).
This study was mainly supported by the Academy of Finland (Gr
ant
No. 121574). Yu s work was partially supported by the Fin
4. Conclusions
nish
Composts made from biowaste and sludge suppressed dis Cultural Foundation (Central Fund and Pijt-Hme Regional
Fund)
ease
caused by Pythium. Actinobacteria enhanced disease suppre and the EnSTe graduate school (Finnish Doctoral Programme
in
ssion
in both experiments. Phosphorus concentration was asso Environmental Science and Technology).
ciated
negatively with disease suppression in both experiment Appendix A. Supplementary data
s. The
Supplementary data associated with this article can be found
studied compost mixtures contained abundant and d
, in
iverse
http://dx.doi.org/10.1016/j.
microbial communities, with Proteobacteria and Ascom the online version, at
apsoil.2015.03.005.
ycota
being the dominant bacterial and fungal representatives, re
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