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Post
harvest
biological
control
of
apple
bitter
rot
by
soil-borne
Actinomycetes
and
molecular
identification
of
the
active
antagonist
Mahdieh
Sadeghian
a,
*,
Gholam
Hosein
Shahidi
Bonjar
b
,
Gholam
Reza
Sharifi
Sirchi
a
a
Department
of
Agricultural
Biotechnology,
College
of
Agriculture,
Shahid
Bahonar
University
of
Kerman,
Kerman,
Iran
b
Department
of
Plant
Protection,
College
of
Agriculture,
Shahid
Bahonar
University
of
Kerman,
Kerman,
Iran
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
30
September
2014
Received
in
revised
form
26
September
2015
Accepted
26
September
2015
Available
online
xxx
Keywords:
Colletotrichum
gloeosporioides
Actinomycetes
Postharvest
biological
control
16S
rRNA
gene
Amycolatopsis
sp.
A
B
S
T
R
A
C
T
Apple
bitter
rot
is
a
destructive
fruit
rot
of
apple
worldwide.
Because
of
recent
advances
in
postharvest
biological
control
as
an
eco-friendly
and
a
potent
alternative
of
fungicides,
the
present
study
evaluated
the
antagonistic
activities
of
more
than
100
Actinomycetes
isolated
from
apple
orchard
soils
of
Kerman
city
(Iran)
against
causal
agent
of
the
disease.
In
vitro
bioassays
revealed
that
six
of
the
isolates
had
significant
inhibitory
effects
against
the
mycelial
growth
of
the
pathogen.
Postharvest
in
vivo
experiments
were
performed
with
either
direct
application
of
antagonists
spore
and
mycelial
mat
or
suspensions
of
their
crude
extracts.
Statistical
results
indicated
that
antagonists
inhibited
rotting
of
apple
fruit
(p
<
0.01)
either
by
inhibition
of
disease
onset
or
preventing
further
expanse
of
diseased
fruit-
lesions.
Molecular
identification
of
the
antagonist
performed
based
on
16S
rDNA
nucleotide
sequence,
and
identified
as
Amycolatopsis
sp.
The
present
study
is
a
preliminary
step
toward
production
of
an
applicable
eco-friendly
biocontrol
product.
Future
larger
scale
postharvest
evaluations
would
reveal
such
feasibility.
ã
2015
Elsevier
B.V.
All
rights
reserved.
1.
Introduction
Postharvest
diseases
of
fruits
account
for
considerable
posthar-
vest
losses
reaching
as
much
as
20–25%
during
transportation
in
developed
countries
(El-Ghaouth
et
al.,
2004;
Droby,
2006;
Zhu,
2006;
Ippolito
and
Nigro,
2000)
and
more
in
developing
ones.
Unfortunately,
in
the
recent
decades,
use
of
synthetic
fungicides
has
been
the
primary
choice
for
reducing
postharvest
losses
to
meet
food
demands
for
an
ever-increasing
world
population
(Kelman,
1989).
Simultaneously,
increasing
constraints
on
the
application
of
fungicides,
including
their
perceived
adverse
effects
on
human
health
and
the
environment
and
the
development
of
fungicide-resistant
strains
of
postharvest
pathogens,
necessitate
the
development
of
efficient
and
safer
alternatives,
mainly
biological
controls
(Droby,
2006;
Sharma
et
al.,
2009;
Janisiewicz
and
Korsten,
2002;
Tripathi
and
Dubey,
2003;
Ray
et
al.,
2011).
In
addition,
biological
control
of
postharvest
diseases
(BCPD)
holds
advantages
of
feasibility
and
cost-effectiveness
under
controlled
and
limited
storage
conditions
(Wilson
and
Pusey,
1985;
Sharma
et
al.,
2009;
Droby,
2006;
Janisiewicz
et
al.,
2003).
Effective
microbial
antagonists
have
been
found
and
in
some
cases,
successfully
commercialized
against
major
postharvest
pathogens
of
different
fruits
(Janisiewicz
and
Korsten,
2002;
Janisiewicz
et
al.,
2003;
Ray
et
al.,
2011;
Gholamnejad
et
al.,
2009;
Vinas
et
al.,
1998).
BioSave,
Aspire,
Avogreen,
YieldPlus,
SHEMER
and
Candifruit
are
commercially
available
products
effective
against
rots
caused
by
Botrytis,Penicillium,
Rhizopus,and
Aspergilluson
strawberries,
grapes,
citrus
and
pome
fruitsthroughout
the
world
(Droby,
2006;
Janisiewicz
and
Korsten,
2002;
Sharma
et
al.,
2009;
Janisiewicz
et
al.,
2003).
Actinomycetes,
as
potential
biocontrol
agents
of
phytopathogens,
represent
a
remarkable
fraction
of
the
soil
microbial
biomass,
which
produce
a
valuable
source
of
agro-
active
compounds
(Doumbou
et
al.,
2002;
Shimizu,
2011).
Several
strains
of
Actinomycetes
have
been
found
to
protect
plants
against
a
wide
range
of
phytopathogenic
fungi
by
production
of
fungal
cell-
wall
degrading-enzymes,
antifungal
antibiotics,
plant
growth
promoters
(Bressan,
2003;
Doumbou
et
al.,
2002;
Eccleston
et
al.,
2010;
Trejo-Estrada
et
al.,
1998;
Yuan
and
Crawford,
1995;
El-Tarabily
and
Sivasithamparam,
2006;
El-Tarabily
et
al.,
2000;
Jorjandi
et
al.,
2009).
There
are
increasing
instances
of
biological
control
of
fungal
and
bacterial
plant
pathogens
by
Actinomycetes
species,
which
have
already
achieved
the
market
or
are
likely
to
*
Corresponding
author.
Fax:
+98
34
33222043.
E-mail
address:
mahdie.sadeghi@gmail.com
(M.
Sadeghian).
http://dx.doi.org/10.1016/j.postharvbio.2015.09.035
0925-5214/ã
2015
Elsevier
B.V.
All
rights
reserved.
Postharvest
Biology
and
Technology
112
(2016)
46–54
Contents
lists
available
at
ScienceDirect
Postharvest
Biology
and
Technology
journal
home
page:
www.elsevier.com/locat
e/postharvbio
develop
commercially
in
the
coming
years
(Doumbou
et
al.,
2002;
Shimizu,
2011;
Rugthaworn
et
al.,
2007;
Macagnan
et
al.,
2008;
El-
Tarabily
and
Sivasithamparam,
2006;
Prapagdee
et
al.,
2008;
Gonzalez-Franco
and
Hernandez,
2009).
Bitter
rot
of
apple,
caused
by
Glomerella
cingulata
(Stonem.)
Spauld.
and
Schrenk,
anamorph:
Colletotrichum
gloeosporioides
(Penz.)
Sacc.,
is
a
potentially
devastating
pre-
and
postharvest
disease
in
virtually
all
countries
where
apples
are
grown
(Snowdon,
2010;
Schubert,
1983;
Boyd-Wilson
et
al.,
2006).
Prior
to
harvest,
pre-harvest
quiescent
infections
establishing
as
minor
fruit
decays
in
orchards
lead
to
major
rots
during
subsequent
storage
(Gonzalez
et
al.,
2006;
Snowdon,
2010).
Under
humid
and
hot
conditions,
when
the
infective
inocula
level
is
elevated,
drenching
fruit
before
storage
may
result
in
cross-contamination
of
damaged
fruit
with
the
released
spores
from
rotting
ones,
thereby
cause
severe
epidemic
decay
in
storage
(Janisiewicz
et
al.,
2003).
In
humid
and
temperate
areas,
the
malady
can
cause
yield
losses
as
high
as
50%
(Gonzalez
and
Sutton,
2004).
The
use
of
scarce
biocontrol
agents
including
Bacillus
subtilis
B-3,
Metchnikowia
pulcherrima
T5-A2
and
some
other
yeast
isolates
to
control
this
disease
has
also
been
reported
(Boyd-Wilson
et
al.,
2006;
Pusey
and
Robins,1991;
Janisiewicz
et
al.,
2003).
The
present
study
aimed
to
determine
inhibitory
effects
of
Actinomycetes
isolated
from
soils
of
apple
orchards
on
establishment
and
extension
of
bitter
rot
on
apple
fruits
in
storage.
More
than
100
Actinomycetes
isolates
both
in
vitro
and
in
vivo
were
screened
for
bioactivity
in
search
of
efficient
biocontrol
agents
capable
of
inhibiting
or
slowing
the
progress
of
the
disease
and
protecting
pre-harvest
contaminated
apple
fruit
against
the
pathogen
during
the
postharvest
period.
In
lengthy
storage
periods,
protecting
the
produce
by
safe
biocontrol
agent
rather
than
by
the
application
of
protective
fungicides
would
address
concerns
about
fungicide
residues
in
such
freshly
produce.
Comprehensive
in
vitro
and
in
vivo
bioassays
were
performed
to
verify
some
aspects
of
the
biocontrol
processes
involved
and
the
nature
of
the
inhibitory
effects
as
a
step
towards
sustainable
biological
control.
2.
Materials
and
methods
2.1.
Fruits
The
apple
cultivar
“Golden
Delicious”
was
used
in
all
experi-
ments.
Fresh
(no
previous
storage)
fruit
were
selected
for
uniform
size
and
maturity,
having
no
wounds,
scars
and
rots
on
the
surface.
2.2.
Pathogen
A
virulent
isolate
of
C.
gloeosporioides
was
obtained
from
the
Laboratory
of
Plant
Pathology
of
the
Agricultural
Research
Center,
Ministry
of
Agriculture,
Kerman,
Iran,
and
maintained
on
Potato-
Dextrose-Agar
(PDA,
Difco)
plates.
2.3.
Antagonists
Random
rhizosphere
soil
samples
were
collected
from
several
apple
orchards
of
Kerman
province,
Iran,
as
described
by
Lee
and
Hwang
(2002).
Soil
samples
were
taken
from
a
depth
of
10–20
cm.
The
10
cm
superficial
soil
from
the
surface
was
excluded.
Samples
were
air-dried
at
ambient
temperature
for
7–10
days,
passed
through
a
0.8
mm
mesh
sieve,
and
maintained
in
polyethylene
bags
at
room
temperature
until
used.
To
isolate
Actinomycetes,
10
g
of
the
soil
was
suspended
in
90
mL
of
sterile
distilled
water,
shaken
thoroughly
for
1
h
and
allowed
to
settle
for
1
h.
Subsequently,
ten-
fold
successive
dilutions
(10
2
–10
6
)
were
prepared
in
sterile
distilled
water.
Aliquots
(1
mL)
of
10
3
–10
6
soil
dilutions
were
poured
into
autoclaved
Casein-Glycerin-Agar
plates
(25
mL)
at
50
C
with
three
replicates
for
each
dilution.
Plates
were
incubated
at
28
C
for
up
to
14
days.
Once
emerging,
Actinomycetes
colonies
were
isolated
on
CGA
Petri
plates,
incubated
at
28
C
for
one
week
and
stored
in
refrigerated
chambers
as
stock
cultures
before
use.
For
screening
studies,
more
than
100
pure
Actinomycete
isolates
were
collected
(Aghighi
et
al.,
2004).
2.4.
Screening
procedure
and
in
vitro
antifungal
bioassays
To
examine
the
antifungal
activity
of
isolated
Actinomycetes
against
the
pathogen,
bioassays
were
carried
out
via
agar
disk,
dual
culture
and
well
diffusion
methods
as
described
by
Shahidi-Bonjar
(2004)
and
Aghighi
et
al.
(2004).
Antifungal
activity
was
evaluated
by
measuring
the
diameter
of
inhibition
zones
(DIZ)
around
the
Actinomycete
agar
disks.
The
level
of
inhibition
in
dual
cultures
was
calculated
via
modified
rating
method
of
Lee
and
Hwang
(2002)
and
El-Tarabily
et
al.
(2000).
The
distance
(mm)
of
fungal
growth
towards
an
antagonist
colony
(g)
was
subtracted
from
the
fungal
growth
radius
(g
)
of
a
control
culture
to
get
Dg
=
g
g.
Where
Dg:
5–9
mm,
+
(weak
inhibition);
Dg:
10–19
mm,
++
(moderate
inhibition);
and
Dg
>
20
mm,
+++
(strong
inhibition).
2.5.
Chloroform
sensitivity
assay
of
bioactive
compounds
To
evaluate
the
stability
of
bioactive
principles
in
a
non-polar
solvent
such
as
chloroform,
spore
suspensions
(approximately
10
6
spores
mL
1
)
of
each
isolate
were
spotted
(1
mL
per
spot)
onto
15
mL
CGA
plates.
Plates
were
incubated
at
28
C
for
3
days.
Then,
emerged
colonies
were
killed
by
exposing
the
uncovered
plates
for
3
h
over
watch
glasses
containing
5
mL
of
chloroform.
Afterwards,
watch
glasses
were
removed
and
the
plates
were
aerated
in
a
fume
hood
for
30
min
to
remove
residual
chloroform.
They
were
subsequently
covered
with
15
mL
of
1%
water
agar.
After
solidification,
C.
gloeosporioides
cultured
uniformly
over
the
surface
of
the
agar
and
the
plates
were
incubated
at
28
C
for
5–
7
days.
The
appearance
or
lack
of
fungal
growth
inhibition
zones
surrounding
spotted
isolates
were
judged
as
chloroform
resistant
or
sensitive
active
principal(s),
respectively.
Three
replicates
were
considered
for
each
isolate
(Davelos
et
al.,
2004;
Jorjandi
et
al.,
2009).
2.6.
Submerged
cultures
and
preparation
of
crude
extract
The
most
active
Actinomycete
antagonists
were
grown
in
submerged
cultures
of
CG
medium
on
a
shaker
incubator
under
at
2.15
s
1
at
28
C.
For
monitoring
the
activity
change
trend
versus
post
seeding
time,
small
amounts
of
culture
filtrate
were
taken
aseptically
at
24
h
intervals
for
25
consecutive
days
and
their
activity
was
evaluated
by
the
well
diffusion
method
(Aghighi
et
al.,
2004)
whereby
the
day
of
maximum
inhibitory
effect
against
the
pathogen
was
determined.
For
preparation
of
crude
extract,
about
4–6
days
from
inoculation
(depending
on
isolate)
when
the
activity
reached
its
maximum,
spore
and
mycelia-free
culture
filtrates
were
prepared
by
filtration
through
two
layers
of
cheesecloth.
The
culture
filtrate
was
then
freeze-dried
to
a
dark
crude
(200
kPa,
40
C)
and
kept
refrigerated
before
use
(Jorjandi
et
al.,
2009).
2.7.
Determination
of
minimum
inhibitory
concentrations
(MIC)
To
measure
the
MIC
value,
two-fold
serial
concentrations
of
50,
25,
12.5,
6.25,
3.125,
1.562
and
0.781
g
L
1
of
the
crude
extract
were
prepared
in
dimethyl
sulfoxide:methanol
(1/1:v/v)
solvent
(DM
solvent)
and
antifungal
activity
was
assayed
by
the
well
diffusion
method
(Jorjandi
et
al.,
2009).
Here,
the
MIC
refers
to
the
lowest
concentration
capable
of
inhibiting
any
observable
fungal
growth.
All
data
represent
the
average
of
three
replicated
experiments.
M.
Sadeghian
et
al.
/
Postharvest
Biology
and
Technology
112
(2016)
46–54
47
2.8.
In
vivo
trails
In
vivo
studies
were
carried
out
following
two
distinct
procedures.
2.8.1.
Direct
application
of
antagonists
Experiments
were
performed
statistically
as
a
completely
randomized
design
(CRD)
with
10
replicates
for
each
treatment.
After
surface
disinfestation
(Zhou
et
al.,
2001;
He
et
al.,
2003)
of
fruit,
wounding
of
fruit
to
the
depth
of
1
mm
was
performed
with
a
sterile
needle
upon
which
appropriate
disk
plugs
were
placed
as
follows.
For
each
isolate
of
Actinomycetes,
four
treatments
were
designated:
(A)
10
wounded
apple
fruit
were
treated
with
plain
disks
of
CGA
medium,
(B)
10
wounded
apple
fruit
were
inoculated
with
6
mm
disks
of
well-grown
culture
of
Actinomycete
isolate
on
wounded
areas,
(C)
10
wounded
apple
fruit
were
inoculate
with
6
mm
disks
of
well-grown
culture
of
C.
gloeosporioides,
(D)
10
fruit
were
inoculate
with
6
mm
disks
of
well-grown
cultures
(possess-
ing
abundant
conidia)
of
both
antagonist
and
the
pathogen,
consecutively.
Inoculation
of
the
antagonist
and
pathogen
was
performed
as
follows:
(1)
All
apple
fruit
were
aseptically
needle-
wounded
as
described
above,
(2)
6
mm
plugs
of
antagonist
or
pathogen
were
placed
on
wounds
and
incubated
for
12
h,
(3)
after
this
period,
disks
in
treatment
4
were
removed
and
replaced
with
6
mm
disks
of
well-grown
culture
of
C.
gloeosporioides
(possessing
abundant
conidia).
Replacements
in
treatment
1
were
done
with
plain
agar
disks
and
in
treatment
2
with
Actinomycete
plugs.
All
treatments
were
kept
for
an
additional
12
h
under
sealed
incubation,
(4)
after
this
period,
all
disk
plugs
were
removed
from
all
treated
samples,
(5)
All
treatments
were
packed
in
new,
separate,
sealed,
plastic
bags
and
incubated
at
28
C
up
to
16
days,
(6)
Progress
of
the
disease
was
monitored
at
4
days
intervals
up
to
the
16th
day
by
measuring
the
diameter
of
rotten
areas
for
statistical
analysis.
2.8.2.
Application
of
antagonistic
crude
extracts
For
each
isolate,
a
three-fold
MIC
concentration
of
crude
extract
in
sterile
distilled
water
was
prepared
and
mixed
thoroughly
for
10
min.
Treatments
were
performed
as
described
in
the
previous
section
with
a
few
differences
as
follows:
100
mL
of
suspension
was
spread
in
a
radius
of
20
mm
around
the
wound
by
means
of
sterile
swabs.
After
air-drying
the
applied
suspension,
four
treatments
designed
for
each
crude
sample
of
Actinomycetes:
(A)
10
wounded
apple
fruit
were
treated
with
100
mL
sterile
distilled,
(B)
10
wounded
apple
fruit
were
inoculated
with
100
mL
of
crude
sample
of
Actinomycete
isolate,
(C)
10
wounded
apple
fruit
were
inoculated
with
6
mm
disks
of
well-grown
culture
of
C.
gloeospor-
ioides,
(D)
10
fruit
were
inoculated
first
with
100
mL
of
crude
sample
of
Actinomycete
isolate
followed
by
6
mm
disks
of
well-
grown
cultures
(possessing
abundant
conidia)
of
the
pathogen.
All
treatments
were
packed
in
separate,
sealed,
plastic
bags
and
incubated
at
28
C
for
16
days.
Progress
of
the
disease
was
monitored
at
4
days
intervals
by
measuring
the
diameter
of
rotten
areas
for
statistical
analysis.
2.8.3.
Statistical
analysis
Recorded
data
were
subjected
to
analysis
of
variance
(ANOVA)
with
SAS
software
(SAS
Institute,
version
9,
Cary,
NC).
Statistical
significance
was
determined
at
the
p
0.01.
Duncan’s
Multiple-
Range
Test
used
to
compare
means.
2.9.
Production
of
extracellular
lytic
enzymes
by
antagonists
To
evaluate
production
of
extracellular
lytic
enzymes
by
antagonists,
three
replicates
were
evaluated
in
each
bioassay
for
each
active
Actinomycete
isolate
in
all
enzymatic
assays
of
this
section
and
data
were
recorded
by
measuring
the
diameter
of
halo
zones
resulting
from
enzymatic
activity
of
the
antagonist
in
the
Fig.
1.
In
vitro
antifungal
activity
of
Actinomycete
isolates
against
Colletotrichum
gloeosporioides.
Center,
agar
plug
of
C.
gloeosporioides,
clockwise
from
the
bottom:
(a)
blank
agar
disk
as
control,
Actinomycete
isolates
550,
536,
534;
(b)
control,
541,
505,
521;
(c)
control,
528,
574,
560;
(d)
control,
115 ,
354,
101.
Inhibitory
effects
against
mycelial
growth
of
the
pathogen
are
noticeable
in
several
isolates.
Fig.
2.
Bioactivities
of
aqueous
culture
filtrate
of
Actinomycete
isolate
521
in
the
well
diffusion
method
against
lawn
culture
of
Colletotrichum
gloeosporioides.
Clockwise
from
the
bottom:
control
well
that
received
uninoculated
culture
filtrate,
culture
filtrate
of
Actinomycete
isolate
521
at
3rd,
4th,
and
5th
day
from
seeding
time
representing
inhibition
of
C.
gloeosporioides
growth.
48
M.
Sadeghian
et
al.
/
Postharvest
Biology
and
Technology
112
(2016)
46–54
relevant
substrate.
Results
are
presented
as
means
of
three
replicates.
2.9.1.
Chitinases
Each
of
the
active
isolates
was
spot-seeded
onto
a
minimal
1.5%
agar
medium
containing
0.4%
colloidal
chitin
to
detect
chitinases.
For
preparation
of
colloidal
chitin,
40
g
of
non-colloidal
chitin
(Sigma)
was
ground
and
the
resulting
powder
was
digested
overnight
in
37%
HCl
at
4
C.
The
suspension
was
then
neutralized
with
saturated
NaOH
to
pH
7–7.5.
The
mixture
was
centrifuged
and
the
pellet
re-suspended
in
distilled
water
and
the
process
was
repeated
3
times.
Finally,
the
obtained
colloidal
chitin
was
dried
at
Fig.
4.
Results
of
in
vivo
trails
of
biological
control
of
apple
bitter
rot
by
direct
application
of
Actinomycete
isolates.
(a)
isolate
521;
(b)
isolate
550;
(c)
isolate
505;
(d)
isolate
536;
(e)
isolate
541
and
(f)
isolate
534
at
the
12th
day
of
inoculation.
Each
picture
includes,
clockwise
from
the
left:
positive
control
treated
by
Colletotrichum
gloeosporioides;
joint
treatment
of
C.
gloeosporioides
plus
Actinomycete
antagonist;
negative
control
(no
treatment
of
pathogen
and/or
antagonist)
and
treatment
of
Actinomycete
isolate.
Fig.
3.
Diameter
of
the
inhibition
zone
caused
by
culture
filtrate
of
submerged
cultures
of
Actinomycete
isolate
521
as
indicative
of
bioactivity
against
Colletotrichum
gloeosporioides
versus
post
seeding
time
monitored
by
the
well
diffusion-method.
Maximum
inhibitory
effect
was
obtained
at
the
5th
day
of
inoculation.
Each
data
point
is
representative
of
the
mean
of
three
replicates.
M.
Sadeghian
et
al.
/
Postharvest
Biology
and
Technology
112
(2016)
46–54
49
40
C
in
oven
overnight
(Macagnan
et
al.,
2008).
Chitinolytic
activity
was
identified
as
the
appearance
of
a
clear
zone
around
the
colonies
after
5
days
of
incubation
at
28
C.
2.9.2.
Glucanases
Glucanase
production
by
each
of
the
active
antagonist
was
detected
by
transferring
a
6
mm
disk
of
well-grown
culture
onto
a
minimal
1.5%
agar
medium
containing
0.1%
lichenan
(Sigma).
After
3
days
ofincubation
at
28
C,
plates
were
stained
with
0.3%
Congo
red
(Sigma)
solution
for
15
min.
After
removal
of
residual
dye
by
thoroughly
rinsing
the
agar
surface
with
water,
the
stain
was
fixed
by
flooding
the
plate
in
1
M
NaCl
for
10
min.
Lichenan
hydrolysis
was
judged
by
appearance
of
a
clear
zone
around
glucanolytic
colonies
(Walsh
et
al.,
1995;
Sergeyenko
and
Los,
2003).
2.9.3.
Proteases
Proteolytic
activity
was
detected
by
casein
hydrolysis
on
agar
plates
containing
0.3%
of
casein,
0.1%
of
glucose,
and
1.5 %
of
agar.
Antagonists
were
transferred
as
6
mm
disks
of
their
well-grown
cultures
onto
the
medium.
Plates
were
incubated
at
28
C
for
3–
4
days.
Formation
of
a
clear
zone
around
colonies
was
indicative
of
proteolysis
activity
(Dunne
et
al.,
2000;
Pereira
Rodarte
et
al.,
2011).
2.9.4.
Lipases
Lipase
detecting
medium
composed
of
1%
peptone,
0.5%
NaCl,
0.01%
CaCl
2
and
1.5%
agar
was
prepared
and
supplemented
with
10
mL
of
separately
autoclaved
Tween-80.
Actinomycete
isolates
were
cultured
on
the
medium
as
mentioned
for
protease
detection.
Plates
were
incubated
at
28
C
for
3–4
days.
When
a
powder-like
sedimentary
halo
appeared
around
lipolytic
colonies
(Sierra,1957),
the
bioassay
was
marked
as
positive.
2.9.5.
Amylases
About
0.2%
of
soluble
starch
was
added
to
suitable
nutrient
agar
basal
medium.
After
inoculation
of
Actinomycetes
as
mentioned
above
and
incubation
at
28
C
for
3–4
days,
starch
hydrolysis
was
evaluated
by
flooding
the
plate
in
dilute
(Lugol’s)
iodine.
Absence
of
the
bluish-purple
color
was
characteristic
of
the
starch–iodine
complex
that
indicates
hydrolysis
of
starch
(Society
of
American
Bacteriologists,
1951 ).
2.10.
Molecular
identification
According
to
results
of
the
previous
assays,
Actinomycete
isolate
521
was
selected
as
the
most
efficient
antagonist.
For
Table
2
Results
of
means
comparison
for
data
(diameter
of
disease
rotting
area)
resulted
from
direct
application
of
Actinomycete
antagonist
isolates
against
Colletotrichum
gloeosporioides
after
4,
8,12
and
16
days
from
onset
of
treatments.
Similar
letters
within
columns
are
not
significantly
(p
0.5)
different
according
to
Duncan’s
Multiple-Range
Test
(SSR
test).
Despite
the
fact
that
all
antagonists
had
significant
inhibitory
effects
on
disease
progress
in
comparison
with
control,
there
are
no
significant
differences
among
Actinomycetes
in
their
inhibitory
effects.
Day
of
recording
the
diameter
of
rotting
area
16th
12th
8th
4th
Treatments
(Actinomycete
isolates)
Control
4.88
a
3.74
a
2.2
a
1.29
a
Isolate
505
1.0 1
b
0.64
b
0.2
b
0.11
b
Isolate
521
0.42
b
0.38
b
0.16
b
0.06
b
Isolate
534
0.91
b
0.53
b
0.26
b
0.09
b
Isolate
536
1.42
b
0.91
b
0.37
b
0.15
b
Isolate
550
0.41
b
0.38
b
0.14
b
0.05
b
Isolate
541
0.88
b
0.48
b
0.16
b
0.01
b
Table
1
Analysis
of
variance
of
diameter-of-disease-lesions
data
resulting
from
direct
application
of
Actinomycete
antagonists
against
Colletotrichum
gloeosporioides
4,
8,
12
and
16
days
after
onset
of
treatments.
Results
indicated
significant
differences
(p
0.01)
between
Actinomycete
treatments
and
diseased
controls.
Mean
of
squares
(MS)
Day
of
recording
(after
onset
of
treatment)
16th
12th
8th
4th
F
value
6.35
9.15
14.28
20.18
Treatment
(df
=
6)
24.5191429
*
14.84147619
*
5.69214286
*
2.11747619
*
*
Significance
level
at
p
0.01.
Fig.
5.
General
trend
in
expansion
of
bitter
rot
lesions
during
16-days
period
of
in
vivo
trials
by
direct
application
of
Actinomycete
isolates
against
Colletotrichum
gloeosporioides.
Effects
of
antagonists
slowing
down
the
disease
progress
are
noticeable.
50
M.
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et
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/
Postharvest
Biology
and
Technology
112
(2016)
46–54
molecular
identification
of
the
isolate
based
on
16S
rRNA
sequence,
genomic
DNA
was
extracted
by
the
CTAB
protocol
as
described
by
Rogers
and
Bendich
(Kieser
et
al.,
2000).
The
following
Actino-
mycetes-specific
primers
were
used
to
amplify16S
rRNA
gene;
F16SS
(5
0
-ACGGGTGAGTAACACG-3
0
)
and
R16SS
(5
0
-
TACCGCGGCTGCTGGCACG-3
0
)
(CinnaClone,
Tehran,
Iran).
PCR
amplification
was
performed
and
purified
16S
rDNA
PCR
product
was
ligated
into
vector
pTZ5R/T
by
a
Ins
T/A
Clone
TM
PCR
Cloning
Kit
(Fermentas).
Fragments
were
sequenced
in
both
directions
by
Faza-Pajhouh
company
(Tehran,
Iran)
using
M13F
(20)
and
M13R-
PUC
(40)
primers.
The
obtained
16S
rRNA
sequences
were
compared
to
all
accessible
sequences
in
the
NCBI
GenBank
database
with
the
Basic
Alignment
Search
Tool
(BLAST,
http://
www.ncbi.nlm.nih.gov/).
Fig.
6.
Results
of
in
vivo
trials
of
biological
control
of
apple
bitter
rot
by
applying
crude
extract
of
antagonistic
Actinomyceste
isolates.
(a)
Isolate
534;
(b)
isolate
536;
(c)
isolate
521;
(d)
isolate
541;
(e)
isolate
505
and
(f)
isolate
550
at
4th
day
after
inoculation
and
onset
of
treatments.
Clockwise
from
left,
each
picture
includes
positive
control
exclusively
treated
by
Colletotrichum
gloeosporioides;
joint
treatment
of
C.
gloeosporioides
plus
Actinomycete
isolate;
negative
control
(no
treatment
of
pathogen
and/or
antagonist)
and
treatment
of
Actinomycete
isolate.
M.
Sadeghian
et
al.
/
Postharvest
Biology
and
Technology
112
(2016)
46–54
51
3.
Results
3.1.
Detection
of
antifungal
capability
of
Actinomycete
isolates
In
dual
culture
assays,
eight
of
more
than
120
Actinomycete
isolates,
designated
as
isolates
354,
505,
521,
534,
536,
541,
550
and
574,
were
found
to
be
capable
of
inhibiting
C.
gloeosporioides
growth
as
indicated
in
Fig.
1.
3.2.
Chloroform
assay
Among
the
8
most
effective
Actinomycete
antagonists,
isolates
354
and
574
lost
their
antifungal
activities,
while
other
six
isolates
retained
their
antifungal
activities
after
exposure
to
chloroform
and
selection
for
subsequent
investigations.
3.3.
Antifungal
activity
of
submerged
cultures
Results
of
daily
successive
assays
using
culture
filtrates
of
active
isolates
indicated
that
for
isolates
550,
541
and
521,
antifungal
activity
reached
its
maximum
4
days
from
inoculation
time,
while
for
isolate
534,
the
peak
of
activity
was
detected
in
the
5th
and
for
isolates
505
and
536
at
the
6th
day
of
inoculation.
The
inhibitory
effect
of
culture
filtrate
and
activity
versus
post
seeding
time
in
submerged
cultures
for
Actinomycete
isolate
521
is
shown
in
Figs.
2
and
3.
These
times
were
used
to
harvest
cultures
to
prepare
crude
extract
for
further
studies.
3.4.
Determination
of
MIC
MIC
of
the
crude
extract
to
inhibit
visible
growth
of
C.
gloeosporioides
was
determined
as
low
as
6.25
g
L
1
for
isolates
534,
505,
541
and
521.
While
for
isolates
550
and
536,
concentrations
of
12.5
and
25
g
L
1
of
crude
extract
was
required
to
inhibit
fungal
growth,
respectively.
3.5.
In
vivo
trails
3.5.1.
Direct
application
of
antagonists
Complete
lack
of
disease
symptoms
was
observed
on
treat-
ments
of
uninoculated
controls
and
Actinomycete-inoculated
apple
fruit.
Symptoms
were
observed
in
the
other
two
treatments,
which
included
pathogen
inoculated
and
pathogen
plus
antago-
nist.
Data
were
analyzed
as
a
completely
randomized
design
containing
seven
treatments
including
joint
treatment
by
each
antagonist
and
Colletotrichum
as
well
as
positive
Colletotrichum-
inoculated
control.
Results
of
the
data
analysis
by
SAS
9.1
software
showed
that
treatment
of
fruit
by
Actinomycete
antagonists
had
significant
(p
0.01)
suppressing
effects
on
the
extension
of
disease
lesions
compared
to
diseased
controls.
However,
means
comparison
with
Duncan’s
Multiple-Range
Test
(SSR
Test)
(p
0.05)
showed
no
statistically
significant
differences
among
six
examined
Actinomycets
isolates
in
their
disease
controlling
effects.
Phenotypic
observations
and
results
of
statistical
analysis
have
been
indicated
in
Figs.
4
and
5
and
Tables
1
and
2
3.5.2.
Application
of
antagonistic
crude
extracts
Results
were
indicative
of
significant
reduction
in
disease
symptoms
on
crude
extract-treated
fruit
compared
with
controls
by
data
analysis
using
SAS
9.1
software
suggesting
treatment
of
fruit
by
crude
extracts
of
antagonists
had
significant
(p
0.01)
inhibitory
effects
on
extension
of
disease
lesions
compared
to
diseased
controls.
There
are
no
significant
differences
between
various
antagonist
isolates.
Fig.
6
and
Tables
3
and
4
show
symptom
expression
and
results
of
statistical
analysis,
respective-
ly.
3.6.
Production
of
extracellular
lytic
enzymes
All
Actinomycete
isolates
were
able
to
produce
various
hydrolytic
enzymes
to
different
degrees.
Results
of
related
bioassays
are
shown
in
Fig.
7.
As
the
results
indicate,
Actino-
myceste
isolate
521
was
the
most
potent
antagonist
able
to
produce
all
considered
enzymes,
but
prominently
chitinases
and
glucanases.
This
isolate
was
selected
for
16S
rDNA
nucleotide
sequence-based
identification.
3.7.
16S
rDNA
analysis
Comparison
was
made
between
the
partial
406
bp
16S
rRNA
sequence
obtained
from
Actinomycete
isolate
521
(submitted
to
GenBank
by
accession
number
of
HQ393406.1)
and
previously
existing
sequences
in
GenBank,
indicated
that
the
isolate
is
classified
in
genus
Amycolatopsis
of
family
Pseudonocardiaceae.
4.
Discussion
Due
to
public
concerns
regarding
the
safety
of
food
and
the
environment,
the
use
of
biocontrol
agents
to
manage
postharvest
diseases
of
fruits
has
emerged
as
an
alternative
to
the
use
of
Table
3
Analysis
of
variance
for
data
resulted
from
application
of
Actinomycetes’
crude
extracts
against
Colletotrichum
gloeosporioides
(diameter
of
disease
lesion)
at
4th
and
8th
day
after
onset
of
treatments
indicating
significant
differences
between
applied
treatments
and
controls.
Mean
of
squares
(MS)
Day
of
recording
8th
4th
F
value
9.47
4.88
Treatment
(df
=
6)
4.77247619
*
0.76723810
*
*
Significance
level
at
p
0.01.
Table
4
Results
of
means
comparison
for
data
resulted
from
application
of
crude
extract
of
Actinomycete
isolates
against
Colletotrichum
gloeosporioides
at
4th
and
8th
day
after
treatment
onset.
Identical
letters
within
columns
are
not
significantly
(p
0.5)
different
according
to
Duncan’s
Multiple-Range
Test
(SSR
test).
Day
of
recording
the
diameter
of
rotting
area
8th
4th
Treatments
(Actinomycetes
isolates)
Control
4.34
a
1.68
a
Isolate
505
2.48
b
0.98
b
Isolate
521
1.84
b
0.74
b
Isolate
534
1.52
b
0.6
b
Isolate
536
1.76
b
0.64
b
Isolate
550
1.66
b
0.56
b
Isolate
541
2.12
b
0.74
b
52
M.
Sadeghian
et
al.
/
Postharvest
Biology
and
Technology
112
(2016)
46–54
synthetic
fungicides.
In
the
present
research,
six
isolates
of
the
antagonistic
Actinomycetes
–
known
microbial
sources
of
more
than
90%
of
bioactive
compounds,
which
have
potential
applica-
tions
in
pharmacy,
industry,
agriculture
and
environment
–
exhibiting
biocontrol
efficiency
against
bitter
rot
of
apple
fruits
caused
by
C.
gloeosporioides
were
found.
Among
the
tested
antagonists,
isolate
521,which
subsequently
was
identified
as
Amycolatopsis,
was
the
most
effective
in
in
vitro
bioassays.
Its
bioactivity
was
prominent
in
all
bioassays
used.
Chloroform
tests
showed
that
its
antifungal
effect
persists
upon
exposure
to
chloroform.
In
antifungal
bioassays,
the
calculated
mean
for
diameter
of
inhibition
zone
against
C.
gloeosporioides
was
higher
than
that
of
its
other
counterparts.
Along
with
isolates
550
and
541,
it
reached
its
maximum
bioactivity
after
only
4
days
in
submerged
cultures.
Minimum
inhibitory
concentration
of
crude
extract
for
this
isolate
was
measured
as
low
as
6.25
g
L
1,
producing
all
of
the
considered
hydrolytic
enzymes,
especially
glucanasaes,
chitinases
and
proteases,
which
are
potentially
involved
in
biocontrol
and
probable
site
and
substrate
competition
processes
against
the
tested
pathogen.
Results
of
in
vivo
trials
were
statistically
significant.
The
percentage
of
infected
fruit
in
Actinomycetes-
containing
treatments,
in
comparison
with
controls,
showed
disease
reduction
by
40–50%
throughout
the
trial
data
recording
periods.
In
treatments
receiving
Actinomycetes,
severity
of
symptoms
and
lesions
was
significantly
lower
and
extended
slower
than
controls,
especially
for
isolate
521.The
findings
are
valuable
because
of
the
fact
that
this
one
method
to
control
bitter
rot
of
apple
is
effective
in
delaying
the
growth
of
the
rot
during
storage.
Comparing
the
two
inoculation
methods
used:
(1)
applying
antagonist
mycelial
and
spore
plugs
and
(2)
crude
extracts,
the
results
indicate
that
the
first
method
is
more
effective.
In
other
words,
application
of
active
antagonists
as
practical
formulations
seems
more
effective
compared
to
crude
extracts
used
in
the
trials.
Although
the
in
vivo
trials
for
preventing
bitter
rot
did
not
show
significant
differences
among
the
active
isolates,
based
on
its
mentioned
characteristics,
isolate
521
was
selected
for
molecular
identification
that
led
to
it
being
categorized
as
genus
Amycolatopsis.
It
is
noticeable
that
some
of
this
species
are
producers
of
valuable
bioctive
products
such
as
Rifamycin,
Vancomycin
and
new
broad-spectrum
antifungal
antibiotics
Octacosamicins
A
and
B
(Ding
et
al.,
2007;
Majumdar
et
al.,
2006).
Despite
partial
un-clarity
regarding
the
nature
and
number
of
involved
principles
in
the
biocontrol
process,
the
present
results
are
sufficiently
convincing
to
suggest
the
candidate
active
isolates
for
further
biochemical
and
molecular
investigations
as
efficient
biocontrol
agents.
Better
understanding
of
their
mechanisms
of
action
should
facilitate
their
application
not
only
for
biocontrol
of
the
present
pathogen
but
for
establishment
of
novel
technologies
to
control
a
spectrum
of
postharvest
diseases
in
fruits
and
vegetables.
Production
and
formulation
of
bioactive
metabolites
via
genetic
engineering
and
fermentation
may
result
in
safe
and
eco-friendly
fungicides.
Generally,
the
present
study
is
the
first
step
along
the
way
to
producing
commercial
formulations
for
safer
postharvest
and
pre-storage
treatments
of
fruits
to
inhibit
or
decrease
losses
of
apple
fruit
rotting
fungal
pathogens.
Acknowledgement
Thanks
to
Shahid
Bahonar
University
of
Kerman
for
financial
support.
This
research
is
part
of
MSc
Thesis
of
the
first
author
and
is
dedicated
to
Mr.
Afzalipour
and
Mrs.
Fakhereh
Saba,
the
founders
of
Universities
in
Kerman.
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