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D. Yu , A. Sinkkonen
a ,d ,
M. Romantschuk
*
a,d
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.
Composting is an aerobic
driven process
that
degrades organic waste into a stable, sanitary, humus-like
material
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
1stE
a
2ndB
b
2ndE
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
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
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
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]
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
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