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Tunisian Journal of Plant Protection 15 Vol. 12, Special Issue, 2017
Use of Endophytic Bacteria Naturally Associated with
Cestrum nocturnum for Fusarium Wilt Biocontrol and
Enhancement of Tomato Growth
Rania Aydi-Ben Abdallah, INAT, Université de Carthage, UR13AGR09-
Production Horticole Intégrée au Centre-Est Tunisien, Centre Régional des
Recherches en Horticulture et Agriculture Biologique (CRRHAB), Université de
Sousse, 4042 Chott-Mariem, Tunisia, Boutheina Mejdoub-Trabelsi, ESAK,
Université de Jendouba, 7119 Kef, Tunisia, Ahlem Nefzi, Faculté des Sciences de
Bizerte, Université de Carthage, 1054 Bizerte, Tunisia, Hayfa Jabnoun-
Khiareddine and Mejda Daami-Remadi, UR13AGR09-Production Horticole
Intégrée au Centre-Est Tunisien, CRRHAB, Université de Sousse, 4042 Chott-
Mariem, Tunisia
ABSTRACT
Aydi-Ben Abdallah, R., Mejdoub-Trabelsi, B., Nefzi, A., Jabnoun-Khiareddine, H., and
Daami-Remadi, M. 2017. Use of endophytic bacteria naturally associated with Cestrum
nocturnum for Fusarium wilt biocontrol and enhancement of tomato growth. Tunisian
Journal of Plant Protection 12: 15-40.
Three endophytic bacterial isolates, recovered from Cestrum nocturnum (night blooming jasmine)
leaves and stems, were assessed for their ability to suppress tomato Fusarium wilt disease, caused by
Fusarium oxysporum f. sp. lycopersici (FOL), and to improve growth of tomato plants. Isolates tested
had significantly decreased disease severity by 46.6-97.7% compared to FOL-inoculated and untreated
control. The isolate C4 was found to be the most effective in decreasing leaf damage by 86.6% and the
vascular browning extent by 97.7% relative to control. A significant increment by 39-41.6%, compared
to pathogen-inoculated and untreated control, was recorded in tomato growth parameters. Moreover,
the isolate C4 had significantly enhanced plant growth by 24.5-53.3% over pathogen-free and untreated
control. This isolate C4 was morphologically and biochemically characterized and identified using 16S
rDNA sequencing genes as Serratia sp. (KX197201). Screened in vitro for its antifungal activity
against FOL, Serratia sp. C4 led to 19.52% decrease in pathogen radial growth and to the formation of
an inhibition zone of 8.62 mm in diameter. Cell-free culture filtrate of Serratia sp. C4, supplemented to
PDA medium at 20% (v/v), had lowered pathogen growth by 23% as compared to 21.7 and 9.2%
recorded after heating at 50 and 100°C, respectively. Chloroform and n-butanol extracts from Serratia
sp. C4, applied at 5% (v/v), displayed antifungal potential against FOL expressed as growth inhibition
by 54.6-66.5% compared to untreated control which was higher than that achieved using two
commercial pesticides i.e. Bavistin® (50% carbendazim, chemical fungicide) and Bactospeine®
(16000UI/mg, Bacillus thuringiensis-based biopesticide). Serratia sp. C4 was found to be a chitinase-,
pectinase-, and protease-producing agent and was able to produce the indole-3-acetic acid and to
solubilize phosphate.
Keywords: Antifungal activity, Cestrum nocturnum, endophytic bacteria, Fusarium oxysporum f. sp.
lycopersici, growth promotion, secondary metabolites, tomato
____
Corresponding author: Rania Aydi Ben Abdallah
Email: raniaaydi@yahoo.fr
Accepted for publication16 March 2017
Tomato Fusarium wilt incited by
Fusarium oxysporum f. sp. lycopersici
(FOL) is a destructive disease infecting
Tunisian Journal of Plant Protection 16 Vol. 12, Special Issue, 2017
tomato. Fusarium wilt causes important
losses of tomato crops grown both in
open field and under greenhouses
(Ignjatov et al. 2012; Moretti et al. 2008).
Diseased plants exhibit yellowing and
wilting of the foliage, vascular
discoloration, stunting and eventual death
of the whole plant (Lim et al. 2006).
Control of tomato Fusarium wilt is
difficult due to the pathogen survival
structures (chlamydospores) in soil for
many years without a host and due to its
progress within vascular tissues (Ignjatov
et al. 2012). Moreover, chemical and
genetic control failed to successfully
suppress disease due to fungicide
resistance development and to emergence
of new physiological races of FOL (Ge et
al. 2004). Given the internal progress of
the pathogen within vascular tissues, the
use of endophytic fungi (Mahdi et al.
2014) and bacteria (Goudjal et al. 2014;
Kalai-Grami et al. 2014) may be effective
in biologically controlling disease.
These endophytic microorganisms
are known to colonize plant tissues
without causing any harmful effects on
their host plants. They may remain at
their entry points or spread throughout the
plant (Hallmann et al. 1997). Endophytes
are excellent plant growth promoters
and/or sources of biocontrol agents
(Strobel, 2006). Worldwide, endophytic
bacteria have been used for the control of
some pathogens causing vascular diseases
on various plants such as F. oxysporum f.
sp. vasinfectum on cotton (Chen et al.
1995) and Verticillium dahliae on
rapeseed (Alström, 2001), eggplant, and
potato (Eleftherios et al. 2004). Several
previous studies have demonstrated the
growth-promoting effect induced by
endophytic bacteria on treated plants.
Indeed, Burkholderia caribensis,
Kosakonia oryzae, Pectobacterium sp.,
Enterobacter asburiae, E. radicincitans,
Pseudomonas fluorescens, and E.
cloacae, isolated from sugarcane roots
and stems, have improved the
development of this plant (Marcos et al.
2016). Four endophytic bacteria namely
Azospirillum brasilense, Burkholderia
ambifaria, Gluconacetobacter
diazotrophicus, and Herbaspirillum
seropedicae were shown able to colonize
the internal tissues of roots, stems and
leaves of Solanum lycopersicum var.
lycopersicum and to stimulate its growth
(Botta et al. 2013). Endophytic bacteria
inhibit pathogen growth through the
production of antibiotics, cell wall-
degrading enzymes, competition for
nutrients and minerals, and/or via the
induction of systemic resistance
(Lugtenberg et al. 2013). Plant growth
promotion may be achieved through
indole-3-acetic acid (IAA) and
siderophore production, phosphate
solubilization and nitrogen fixation
(Rosenblueth and Martínez-Romero,
2006).
Several previous studies have
shown that cultivated Solanaceae species
may be useful as potential sources of
bioactive molecules i.e. Cestrum spp. (C.
parqui, C. diurnum and C.
sendtenerianum) (Ahmad et al. 1993;
Chaieb et al. 2007; Haraguchi et al. 2000)
and as biocontrol agents, especially
endophytic bacteria, i.e. Capsicum annum
(Paul et al. 2013), Solanum tuberosum
(Sturz et al. 2002), and S. lycopersicum
(Ramyabharathi and Raguchander 2014).
Cestrum nocturnum is a cultivated
Solanaceae species used as an ornamental
plant; its flowers exude a special sweet
fragrance at night, the main reason for its
folk names night cestrum, lady of the
night and night blooming jasmine (Sharif
et al. 2009). Several previous studies have
valorized this plant as natural source of
bioactive metabolites with insecticidal
(Jawale and Dama 2010; Patil et al. 2011;
Yogalakshmi et al. 2014), antibacterial,
Tunisian Journal of Plant Protection 17 Vol. 12, Special Issue, 2017
and antifungal potential (Khan et al.
2011; Prasad et al. 2013; Sharif et al.
2009). However, C. nocturnum was only
reported as source of isolation of
endophytic fungi without assessment of
its antimicrobial activity (Huang et al.
2008). Moreover, to our knowledge, this
ornamental species was not yet explored
as natural source of isolation of
endophytic bacteria.
In this study, three endophytic
bacterial isolates, recovered from surface-
sterilized stems and leaves of C.
nocturnum plants were assessed for their
antifungal potential toward FOL and for
their growth-promoting traits on tomato
plants.
MATERIALS AND METHODS
Tomato seedling preparation.
Tomato cv. Rio Grande, known to
be susceptible to FOL races 2 and 3
(Barker et al. 2005), was used in this
study. Seedlings were kept under
greenhouse with 16 h light and 8 h dark,
60-70% relative humidity and air
temperatures ranging between 20 and
30°C, and grown until reaching the two-
true-leaf growth stage. A sterilized peat®
(Floragard Vertriebs GmbH für
gartenbau, Oldenburg) was used as
culture substrate.
Pathogen culture.
F. oxysporum f. sp. lycopersici
isolate used in this study was originally
recovered from tomato stems showing
vascular discoloration (Aydi Ben
Abdallah et al. 2016a) and maintained in
the fungal culture collection at the
Laboratory of Plant Pathology, Centre
Régional des Recherches en Horticulture
et Agriculture Biologique (CRRHAB),
Chott-Mariem, Tunisia. It was cultured on
Potato Dextrose Agar (PDA) and
incubated at 25°C for 7 days before use.
Cestrum nocturnum sampling and
isolation of endophytic bacteria.
Endophytic bacteria, used in this
study, were isolated from leaves (C1 and
C2) and stems (C3 and C4) of healthy C.
nocturnum plants sampled on April 2013
from Chott-Mariem (N35°56'20.451'';
E10°33'32.028''), Tunisia.
Samples were individually
disinfected by soaking in 70% ethanol for
1 min, immersion in 1% sodium
hypochlorite for 10 min then in 70%
ethanol for 30 s. They were rinsed three
times in sterile distilled water (SDW) and
air-dried on sterile filter papers. Each
sample was checked for disinfection
process efficiency based on Hallmann et
al. (1997) protocol. In fact, 100 µl of the
SDW used in the last rinse were injected
onto Nutrient Agar (NA) medium. After
48 h of incubation at 25°C, if no
microbial growth was observed on
medium, the surface disinfection
procedure was considered as succeeded.
Twenty surface-sterilized stem and
leave pieces, of about 1 cm length, were
cut longitudinally with a sterile scalpel
and aseptically transferred onto NA
medium with the longitudinal sectioning
surface placed directly in contact with
medium. Plates were incubated at 25°C
for 48 h. For each sampled organ,
bacterial colonies exhibiting
morphological diversity were picked
separately onto NA and purified.
Before being used in the different
bioassays, bacterial stock cultures
maintained at -20°C in Nutrient Broth
(NB) supplemented with 40% glycerol
were grown on NA medium and
incubated at 25°C for 48 h.
Test of endophytic colonization ability.
The endohytic colonization ability
of the four bacterial isolates collected was
tested according to Chen et al. (1995)
method. Isolates were grown onto NA
Tunisian Journal of Plant Protection 18 Vol. 12, Special Issue, 2017
amended with streptomycin sulfate (100
µg/ml w/v) and rifampicin (100 µg/ml
w/v). Only double-resistant isolates were
selected in order to follow their presence
in tomato stems after re-isolation in NA
medium amended with these both
antibiotics and the wild-type ones (the
original isolates) were used for
inoculation of tomato. Seedlings were
dipped for 30 min into bacterial cell
suspensions adjusted to 108 cells/ml using
a hemocytometer (Botta et al. 2013).
SDW was used for treatment of control
seedlings. Five seedlings were used for
each individual treatment. Seedlings were
transplanted into individual pots (12.5 ×
14.5 cm) containing commercialized peat
and grown for 60 days under greenhouse
conditions as previously described.
Bacterial isolates were re-isolated from
tomato stems onto NA medium
supplemented with streptomycin sulfate
and rifampicin (100 µg/ml (w/v) each)
and incubated at 25°C for 48 h. Bacterial
colonies similar to the wild-type ones
were considered as endophytes
(Hallmann et al. 1997) and subjected to
further screening bioassays.
Test of plant growth-promoting ability.
Three endophytic isolates were
tested for their potential to promote
tomato growth under greenhouse
conditions. Healthy tomato seedlings (cv.
Rio Grande), at two-true-leaf stage, were
carefully removed from alveolus plates
and soaked for 30 min into water bacterial
suspensions adjusted to 108 cells/ml
(Botta et al. 2013). Control seedlings
were dipped into SDW only. Inoculated
and control seedlings were transplanted
into individual pots (12.5 × 14.5 cm)
containing sterilized peat. Five
replications were used for each individual
treatment. At 60 days post-treatment,
plants were carefully uprooted and their
roots were washed under running water to
remove peat. Growth parameters noted
were plant height, fresh weight of the
aerial parts and roots, and maximum root
length.
Test of disease-suppressive ability.
Tomato cv. Rio Grande seedlings
were treated with the three bacterial
isolates separately by drenching 25 ml of
bacterial suspensions into culture
substrate near the collar level (Nejad and
Johnson, 2000). Six days after bacterial
treatment, 25 ml of FOL conidial
suspension (106 conidia/ml) were applied
as substrate drenching (Fakhouri and
Buchenauer 2002). Negative control
seedlings were not inoculated with FOL
and treated with SDW only. Positive
control seedlings were pathogen-
inoculated and treated with SDW. Each
individual treatment was replicated five
times.
Assessment of Fusarium wilt
severity was performed, 60 days post-
inoculation (DPI), on tomato plants
inoculated with FOL based on intensity of
leaf yellowing and necrosis using the
following 0-4 scale where 0 = no disease
symptoms (healthy leaves in the whole
plant) and 4 = 76-100% of leaves with
yellowing and/or necrosis (Amini 2009).
Furthermore, wilt severity was assessed
based on the extent of the vascular
browning (from collar) after performing
longitudinal stem sectioning. Pathogen re-
isolation frequency was calculated for
five plants per individual treatment as the
percentage of FOL colonization of stem
sections on PDA (Moretti et al. 2008).
Growth parameters such as plant height
and fresh weight of whole plant were also
noted for all tomato plants challenged or
not with FOL.
The most effective isolate in
suppressing Fusarium wilt severity and in
promoting plant growth was further
subjected to morphological and
Tunisian Journal of Plant Protection 19 Vol. 12, Special Issue, 2017
biochemical characterization and
molecular identification.
Characterization of the most active
endophytic isolate.
Morphological characterization.
Colonies of the most active isolate were
morphologically characterized based on
their size, shape, margin, elevation,
texture, opacity, consistency and
pigmentation on NA medium (Patel et al.
2012). Gram's staining was performed
using light microscopy.
Biochemical characterization.
The most bioactive isolate was also
characterized using conventional
biochemical tests according to Schaad et
al. (2001) protocols. The biochemical
tests performed in this study include
catalase, Red of Methyl (RM), Vosges
Proskauer (VP), mannitol, lecithinase,
urease, indole, tryptophan deaminase,
Simmons citrate, hydrogen sulfide, nitrate
reductase, lysine decarboxylase, and
pyocyanin on King A medium.
Molecular characterization.
Molecular characterization of the selected
isolate was performed after extraction of
genomic DNA according to Chen and
Kuo (1993) for the Gram negative
bacteria. The 16S rDNA was amplified
using the universal eubacterial primers
27f (5'-AGAGTTTGATC(A/C)TGGCTC
AG-3') and 1492r (5'-TACGG(C/T)TAC
CTTGTTACGACTT-3') (Moretti et al.
2008). The PCR conditions were as
follows: one denaturing cycle at 94°C for
4 min, followed by 40 cycles of
denaturing at 94°C for 30 s, annealing at
45°C for 30 s, and polymerization at 72°C
for 45 s, then an extension cycle at 72°C
for 7 min. Amplifications were carried
out in Thermal Cycler® (CS Cleaver,
Scientific Ltd., TC 32/80).
The homology of the 16S rDNA
sequence of the given isolate was
performed using BLAST-N program from
GenBank database (http:
www.ncbi.nlm.gov/BLAST/). Alignment
of sequences was performed using the
ClustalX (1.81). The phylogenetic
analysis for the aligned sequences was
performed using the Kimura two-
parameter model (Kimura 1980). The
phylogenetic tree was constructed based
on neighbor joining (NJ) method with
1000 bootstrap sampling. The bioactive
endophytic bacterium (isolate C4)
sequence was submitted to GenBank and
assigned the following accession number:
KX197201.
Hypersensitivity test. Hyper-
sensitivity test of the selected isolate was
performed on tobacco plants. Ten
microliters (10 µl) of water bacterial cell
suspension (~108 cells/ml) were injected
to tobacco leaves using a sterile
microsyringe. Leaves injected with the
same volume of SDW were used as
negative control. All tobacco plants were
incubated at room temperature for 24 h.
After incubation, inoculated leaf areas
were checked for the presence of
chlorotic and/or necrotic zones indicating
that the tested isolate is phytopathogenic
and should be excluded from further
biocontrol trials (Nawangsih et al. 2011).
Hemolytic test. Bacterial cell
suspensions (~108 cells/ml, 100 µl) of the
selected isolate were transferred on Blood
Agar® (HiMedia, India) medium to test its
ability to degrade hemoglobin. Bacterial
cultures were incubated at 25°C for 48 h.
Positive hemolytic activity is indicated by
the formation of clear zones around
bacterial colonies (Murray et al. 2003).
Thus, the tested isolate will be considered
to be pathogenic to humans and excluded
from the following tests.
Tunisian Journal of Plant Protection 20 Vol. 12, Special Issue, 2017
Assessment of the antifungal activity of
Serratia sp. C4.
Streak method. Bacterial
suspension of Serratia sp. C4 (~108
cells/ml) were streaked across the center
and perpendicularly to the first streak on
the surface of PDA poured in Petri plates
(9 cm in diameter). Four agar plugs (6
mm in diameter), removed from 7 day-old
cultures of FOL, were placed at each side
of the streaked bacterial suspensions
(Sadfi et al. 2001). Control plates were
streaked with SDW only. Each individual
treatment was repeated four times. After 4
days of incubation at 25°C, pathogen
colony diameter was noted. The
inhibition rate (IR) of FOL mycelial
growth was calculated using Tiru et al.
(2013) formula as follows: IR% = [(D2-
D1) / D2] × 100 where D2: Diameter of
pathogen colony in control plates and D1:
Diameter of pathogen colony co-cultured
with the tested bacterial isolate.
Disc diffusion method. The
antifungal activity of the selected
bacterial isolate was also evaluated on
PDA using the disc diffusion method.
FOL was incorporated into molten PDA
and after medium solidification, 20 µl
droplets of bacterial suspensions (~108
cells/ml) were deposited on Whatman No.
1. filter paper discs (6 mm in diameter).
Four discs were used per plate (Vethavalli
and Sudha 2012). For control plates,
paper discs were treated with a same
volume of SDW. Each individual
treatment was repeated four times. After
four days of incubation at 25°C, the
diameter of the inhibition zone was noted.
Activity of cell-free culture
filtrates. Serratia sp. C4 colonies were
grown in Luria-Bertani broth (LB) at 28 ±
2°C for 3 days and under continuous
shaking at 150 rpm. Two ml of the
obtained liquid culture were centrifuged
for 10 min at 10,000 rpm. The
centrifugation was repeated three times.
Cell-free culture filtrate was sterilized by
filtration through a 0.22 μm pore size
filter. To determine the stability of
extracellular metabolites produced by this
isolate, filtrate was incubated at 50 or
100°C for 15 min (Romero et al. 2007).
Antifungal activity of cell-free cultures,
untreated and heated, used at 20% (v/v)
was assessed according to Karkachi et al.
(2010). Control cultures contained LB
filtrate only. Each individual treatment
was replicated three times. After four
days of incubation at 25°C, the diameter
of FOL colony was measured and the
mycelial growth inhibition rate was
calculated (Tiru et al. 2013).
Activity of Serratia sp. C4
organic extracts.
Extraction of secondary
metabolites. Two types of extraction were
carried out to extract the antifungal
metabolites produced by Serratia sp. C4.
The first one was performed using
chloroform (Bhoonobtong et al. 2012)
and the second one with n-butanol
(Romero et al. 2007). Sixty milliliters (60
ml) of cell-free culture filtrate of Serratia
sp. C4, prepared as described above, were
placed in a separating funnel. Then, 60 ml
of the solvent (chloroform or n-butanol)
were added carefully. The funnel was
reversed several times by degassing from
time to time. The mixture was allowed to
settle for few minutes with the cap open.
The organic phase (the lower phase for
extraction with chloroform and the upper
one with n-butanol) were collected. The
aqueous phase was replaced in the funnel
and the extraction was repeated two other
times as described. The solvent was
evaporated in a rotary evaporator at 35°C
for chloroform and at 75°C for n-butanol
with a slight rotation at 150 rpm.
Tunisian Journal of Plant Protection 21 Vol. 12, Special Issue, 2017
Testing of antifungal activity of
organic extracts. Obtained organic
extracts were assessed for their biological
activity against FOL. Each extract was
suspended in ethanol (1/1) (w/v) and
added to Petri plates containing 10 ml of
molten PDA amended with streptomycin
sulfate (300 mg/l) (w/v) at two
concentrations 2.5 and 5% (v/v). Control
cultures were treated with ethanol also
tested at 2.5 and 5% (v/v). The antifungal
activity of secondary metabolites released
by Serratia sp. C4 was compared to two
commercial products i.e. Bavistin® (50%,
chemical fungicide with carbendazim as
active ingredient) and Bactospeine®
(16000UI/mg, Bacillus thringiensis-based
biopesticide). After solidification of the
mixture, an agar plug (6 mm in diameter),
removed from FOL culture previously
grown at 25°C for 7 days, was placed at
the center of each plate. After seven days
of incubation at 25°C, FOL colony
diameter was measured and the inhibition
rate was calculated (Tiru et al. 2013).
Assessment of Serratia sp. C4
enzymatic activity.
Chitinase production. Chitinase
production ability of Serratia sp. C4 was
checked according to Tiru et al. (2013) on
minimum-medium supplemented with
chitin® (MP Biomedicals, LLC, IIIKrich,
France) by streaking bacterial
suspensions (~108 cells/ml) onto sterilized
chitin-agar medium (0.5% w/v). Chitin-
agar medium plates non-streaked with
bacterial suspensions were used as
control. Treatments were replicated
thrice. After 72 h of incubation at 28 ±
2°C, the presence of clearing zones
around bacterial colonies was noted.
Protease production. Serratia sp.
C4 was assessed for its potential to
release protease onto skim milk agar or
SMA (3% v/v) medium (Tiru et al. 2013).
Plates containing SMA only were used as
control. Treatments were performed in
triplicate. The diameter of the clear zone
formed around the bacterial spots was
measured after 48 h of incubation at 28 ±
2°C.
Pectinase production. Pectinase
production ability of Serratia sp. C4 was
detected according to Tiru et al. (2013)
method. Water bacterial suspensions
(~108 cells/ml) were streaked onto NA-
pectin® (ICN Biomedicals, Inc, Germany)
medium (0.5% w/v). Plates containing the
NA-pectin medium only were used as
control. Treatments were performed in
triplicate. After 48 h of incubation at 28 ±
2°C, the presence or the absence of clear
zones around bacterial colonies was
noted.
Phosphate solubilization.
Phosphate solubilization activity of the
selected bacterial isolate was evaluated
qualitatively according to Katzenlson and
Bose (1959) method with some
modifications. An agar plug (6 mm in
diameter) containing Serratia sp. C4
colonies, previously grown on NA during
48 h, was putted onto Pikovskaya agar
medium. Un-inoculated plates were used
as control. Experiments were performed
in triplicate. After seven days of
incubation at 28 ± 2°C, clear zones
formed around colonies, due to the
utilization of tricalcium phosphate present
in the medium, were measured.
Production of indole-3-acetic
acid (IAA).The ability of Serratia sp. C4
to produce IAA was checked using the
colorimetric method described by
Glickmann and Dessaux (1995) with
some modifications. Serratia sp. C4 was
cultivated into LB medium supplemented
with L-tryptophan (50 µg/ml) under
continuous shaking at 150 rpm for 2 days
Tunisian Journal of Plant Protection 22 Vol. 12, Special Issue, 2017
in the dark. The bacterial suspension was
centrifuged at 10,000 rpm for 10 min.
One ml of the culture supernatant was
mixed with 2 ml of Salkwoski’s reagent
and 2-3 drops of orthophosphoric acid.
Un-inoculated LB medium was used as
negative control. Absorbance was
measured daily at 530 nm. The
concentration of IAA was determined and
compared to a standard curve prepared
from IAA dilution series at 100 µg/ml in
LB medium.
Statistical analysis.
Data were subjected to a one-way
analysis of variance (ANOVA) using
Statistical Package for the Social Sciences
(SPSS) software for Windows version
16.0. For all the in vitro antifungal
potential bioassays and the in vitro tests
of enzymes and IAA production and
phosphate solubilization ability, each
treatment was replicated three to four
times. For cell-free culture filtrate tests,
data were analyzed according to a
completely randomized factorial design
with two factors (Heating and bacterial
treatment). The in vitro assay of organic
extracts was analyzed according to a
completely randomized factorial model
with two factors (treatments and
concentrations). For the remaining in
vitro bioassays, data were analyzed
according to a completely randomized
design. All the in vivo bioassays were
analyzed in a completely randomized
model and each treatment was replicated
five times. For the in vitro antifungal
activity tests using the streak and the disc
diffusion methods, means were separated
using Student t test at P ≤ 0.05. For the
remaining bioassays, means were
separated using LSD test for the in vitro
antifungal activity test of cell-free
cultures and organic extracts and using
Duncan Multiple Range test for the others
to identify significant pair-wise
differences at P ≤ 0.05. Correlations
between Fusarium wilt severity and plant
growth parameters were analyzed using
bivariate Pearson’s test at P ≤ 0.01.
RESULTS
Endophytic ability of bacterial isolates
recovered from Cestrum nocturnum.
Four bacterial isolates exhibiting
macro-morphological diversity on NA
medium were selected among twenty
others recovered from stems and leaves of
C. nocturnum plants. The four selected
isolates were found to be resistant to
streptomycin and rifampicin (100 µg/ml)
and only three were successfully re-
isolated from the internal stem tissues of
tomato cv. Rio Grande plants on NA
medium amended with these antibiotics.
These three endophytic isolates (namely
C1, C3, and C4) were further assessed in
vivo and in vitro for their antifungal
potential toward FOL and for their
growth-promoting effects on tomato
seedlings.
Plant growth-promoting ability
displayed by the selected endophytic
isolates.
The three endophytic bacterial
isolates (C1, C3 and C4) were screened
for their growth-promoting potential onto
pathogen-free tomato plants. ANOVA
analysis revealed that all plant growth
parameters (plant height, aerial part fresh
weight, maximum root length, and root
fresh weight), noted 60 days post-
treatment, varied significantly (at P ≤
0.05) depending on bacterial treatments
tested.
Data given in Table 1 revealed
that, C1- and C4-based treatments led to
significant (P ≤ 0.05) increase in plant
height by 18.7 and 31.9%, respectively,
compared to the untreated control. The
highest plant height improvement (by
Tunisian Journal of Plant Protection 23 Vol. 12, Special Issue, 2017
31.9% over control) was achieved using
C4 isolate.
The aerial part fresh weight was
significantly enhanced by 50% using C4-
based treatment compared to control
(Table 1). As estimated by the maximum
root length, a significant (P ≤ 0.05)
improvement (by 24.6% over control)
was recorded on tomato plants treated
with C4. Similar trend (53.3%) was noted
based on root fresh weight (Table 1).
Table 1. Comparative plant growth-promoting ability of endophytic bacterial isolates
recovered from Cestrum nocturnum on tomato cv. Rio Grande plants noted 60 days post-
treatment
*C1: Isolate from C. nocturnum stem; C3 and C4: Isolates from C. nocturnum leaves; NIC:
Un-inoculated with the pathogen and untreated control. For each column, values followed
by the same letter are not significantly different according to Duncan Multiple Range test at
P ≤ 0.05.
Fusarium wilt suppression by the
selected endophytic isolates.
The three selected endophytic
bacterial isolates were tested on tomato
cv. Rio Grande plants challenged with
FOL. ANOVA analysis revealed that
Fusarium wilt severity, noted on tomato
plants 60 DPI, varied significantly (P ≤
0.05) depending on bacterial treatments
tested. A significant (P ≤ 0.05) decrease
in leaf damage index (yellowing and/or
necrosis), by 46.6 to 86.6% compared to
pathogen-inoculated and untreated
control, was noted on tomato plants
already challenged with FOL and treated
using the three tested isolates (Table 2).
The reduction of the vascular browning
extent was significant, by 55.5 to 97.7%
compared to control, using C1-, C3-, and
C4-based treatments. The isolate C4 was
found to be the most effective in
suppressing leaf yellowing and wilt
symptoms (86.6%) and in reducing the
vascular browning extent (97.7%) relative
to FOL-inoculated and untreated control.
Furthermore, C4-treated plants behaved
significantly similar to the un-inoculated
(disease-free) and untreated ones based
on both disease severity parameters
(Table 2).
Growth parameters of tomato
plants (plant height and fresh weight),
noted 60 DPI with FOL, varied
significantly (P ≤ 0.05) depending on
treatments tested. The increment in plant
height ranged significantly between 12.6
and 39% over FOL-inoculated and
untreated control using C1-, C2-, and C4-
based treatments and the highest
enhancement (of about 39%) was
achieved using C4 isolate. It should be
also highlighted that tomato plants
infected with FOL and treated with C4
exhibited a significantly higher (by
12.7%) plant height than disease-free and
untreated ones.
All bacterial isolates tested had
significantly (P ≤ 0.05) increased plant
fresh weight by 23.2 to 41.7% over FOL-
inoculated and untreated control and the
highest increment (41.7%) was induced
by the isolate C4. The fresh weight of
Bacterial
treatment*
Plant
Height (cm)
Aerial part
fresh weight (g)
Maximum root
length (cm)
Root fresh
weight (g)
NIC
20 ± 0 c
8 ± 0.1 b
17.2 ± 0.6 b
4.2 ± 0.1 b
C1
24.6 ± 0.3 b
6.8 ± 0.2 b
19 ± 0.5 b
5.8 ± 0.2 b
C3
21.6 ± 0.5 c
7 ± 0.5 b
18.8 ± 0.8 b
5.8 ± 0.2 b
C4
29.4 ± 0.8 a
16 ± 0.1 a
22.8 ± 0.5 a
9 ± 0.7 a
Tunisian Journal of Plant Protection 24 Vol. 12, Special Issue, 2017
FOL-inoculated tomato plants treated
with C4 isolate was significantly similar
to that of disease-free control ones (Table
2).
FOL re-isolation frequency from
tomato stems varied depending on
bacterial treatments tested. A decrease in
pathogen isolation frequency, ranging
between40 and 90% relative to the
untreated control, was recorded from
tomato plants already infected with FOL
and treated with the three enpdophytic
isolates tested. The highest decrease in
FOL re-isolation frequency was achieved
by using the isolate C4 (90%) (Table 2).
Table 2. Effects of endophytic bacterial isolates recovered from Cestrum nocturnum on Fusarium
wilt severity, plant growth parameters and Fusarium oxysporum f. sp. lycopersici (FOL) re-isolation
frequency from tomato cv. Rio Grande plants as compared to controls
*C1: Isolate from C. nocturnum stem, C3 and C4: Isolates from C. nocturnum leaves; NIC: Un-
inoculated with the pathogen and untreated control. IC: Inoculated with FOL and untreated control.
**The re-isolation of FOL was carried out from stems of five tomato plants cv. Rio Grande at 0 -15
cm high from the collar. Ten (10) stem fragments were plated onto PDA medium and incubated at
25°C for 4 days. After incubation, the percentage of FOL colonization of stems sections was
calculated.
For each column, values followed by the same letter are not significantly different according to
Duncan Multiple Range test at P ≤ 0.05.
Correlation analysis between Fusarium
wilt severity and plant growth
parameters.
Pearson’s analysis revealed that
decreased Fusarium wilt severity as
estimated by leaf damage index (and/or
necrosis) and vascular browning extent
led to increment in all plant growth
parameters. In fact, plant height was
significantly and negatively correlated to
the leaf damage index (r = -0.874; P =
0.053) (Fig. 1A) and to the vascular
browning extent (r = -0.909; P = 0.033)
(Fig. 1B). Furthermore, the plant fresh
weight was significantly and negatively
correlated to leaf yellowing score (r = -
0.981; P =0.003) (Fig. 1C) and to the
vascular browning extent (r = -0.973; P =
0.005) (Fig. 1D).
Pearson’s analysis demonstrated
that lowered Fusarium wilt severity led to
decrease in tomato stem colonization by
FOL and consequently growth promotion
where significant and negative
correlations were also detected between
FOL re-isolation frequency, plant height
(r = -0.914; P = 0.03) (Fig. 1E), and
whole plant fresh weight (r = -0.975; P =
0.005) (Fig. 1F). Moreover, pathogen re-
isolation frequency was positively
correlated to leaf damage index (r =
0.991; P = 0.001) (Fig. 1G) and to the
vascular browning extent (r = 0.987; P =
0.002) (Fig. 1H).
The endophytic bacterial isolate
C4 shown to be effective in suppressing
Fusarium wilt severity and in promoting
growth of tomato plants inoculated or not
Bacterial
treatment*
Disease
severity (0-4)
Vascular
browning
extent (cm)
Plant height
(cm)
Plant fresh
weight (g)
FOL re-
isolation**
(%)
NIC
0 ± 0 c
0 ± 0 c
24.8 ± 0.4 b
7.05 ± 0.2 ab
0
IC
3 ± 0.1 a
9 ± 0.5 a
17.3 ± 1 d
4.2 ± 0.1 d
100
C1
1.2 ± 0.1 b
4 ± 0.1 b
21 ± 0.5 c
6.25 ± 0.1 bc
50
C3
1.6 ± 0.2 b
4 ± 0.1 b
19.8 ± 0.4 c
5.47 ± 0.3 c
60
C4
0.4 ± 0.2 c
0.2 ± 0.1 c
28.4 ± 0.3 a
7.21 ± 0.2 a
10
Tunisian Journal of Plant Protection 25 Vol. 12, Special Issue, 2017
y = -3.27x + 26.31
R= 0.76
0
10
20
30
40
0 1 2 3 4
Plant height (cm)
Disease severity (0-4)
y = -1.08x + 25.98
R= 0.82
0
10
20
30
40
0 2 4 6 8 10
Plant height (cm)
Vascular browning extent (cm)
B
y = -1.04x + 7.33
R= 0.96
0
2
4
6
8
10
0 1 2 3 4
Plant fresh weight (g)
Disease severity (0-4)
y = -0.33x + 7.17
R= 0.95
0
2
4
6
8
10
0 2 4 6 8 10
Plant fresh weight (g)
Vascular browning extent (cm)
D
y = -8.44x + 232.01
R= 0.83
0
20
40
60
80
100
010 20 30 40
FOL isolation frequency
(%)
Plant height (cm)
y = -31.78x + 235.91
R= 0.95
0
20
40
60
80
100
0246810
FOL isolation frequency
(%)
Plant fresh weight (g)
F
y = 34.21x + 1.58
R= 0.98
0
20
40
60
80
100
0 1 2 3 4
FOL isolation frequency
(%)
Disease severity (0-4)
y = 10.86x + 6.63
R= 0.97
0
20
40
60
80
100
0246810
FOL isolation frequency
(%)
Vascular browning extent (cm)
H
A
C
E
G
with FOL (Fig. 2) was selected for further
characterization, identification and
elucidation of its mechanisms of action
involved in those both effects.
Fig. 1. Correlation between Fusarium wilt severity and plant growth parameters (A, B, C, D, E, F) and between FOL
isolation frequency and Fusarium wilt severity parameters (G, H). Correlation analysis was performed using
bivariate Pearson’s test at P ≤ 0.01.
Tunisian Journal of Plant Protection 26 Vol. 12, Special Issue, 2017
NIC
+ C4
NIC
+ C4
IC FOL + C4
Fig. 2. Effect of endophytic bacterial isolate C4 recovered from Cestrum nocturnum on Fusarium
wilt severity and growth promotion of tomato cv. Rio Grande plants compared t o the untreated
controls. NIC: Un-inoculated with the pathogen and untreated control. IC: Inoculated with FOL
and untreated control; C4: Isolate from Cestrum nocturnum leaves.
Morphological, biochemical and
molecular characterization of the
selected bacterial isolate.
The colony morphology of the
selected isolate C4 showed a small size
and translucent colonies with circular
form, an entire margin and plane
elevation, smooth surface and cream
color on NA medium (Table 3). C4 was
found to be a Gram negative strain.
C4 was able to produce catalase,
lecithinase, lysine decarboxylase, nitrate
reductase, and indole. The isolate C4
cannot produce urease, tryptophane
desaminase, hydrogen sulfide, and
pyocyanin on King A medium. Simmons
citrate and mannitol were not used by C4
colonies as carbon sources. C4 cannot
ferment glucose through the mixed acid
(MR-) but by using the glycol butylene
path (VP +) (Table 3).
Blast-N analysis of sequenced 16S
rDNA gene homology and the
phylogenetic analysis based on neighbor
joining (NJ) method with 1000 bootstrap
sampling revealed that the isolate C4
belonged with 100% of similarity to
Uncultured Serratia sp. strain CTL-81 and
Serratia proteamaculans strain AP-
CMST (Table 3; Fig. 3). The accession
number of Serratia sp. strain C4 deposed
in GenBank was KX197201 (Table 3).
Tunisian Journal of Plant Protection 27 Vol. 12, Special Issue, 2017
Table 3. Characterization and molecular
identification of the selected endophytic bacterial
isolate C4 recovered from Cestrum nocturnum leaves
+: Positive test; -: Negative test. Numbers in
parenthesis indicate the percentage (in %) of sequence
homology obtained from Bla st-N analysis from
GenBank database (http:
www.ncbi.nlm.gov/BLAST/).
The nucleotide sequences used of
representative strains were obtained from
Genbank database under the following
accession numbers: FJ752236 (Serratia
proteamaculans AP-CMST), JQ798999
(Uncultured Serratia sp. CTL-81),
KU682855 (Serratia sp. PKL:12),
KJ922535 (Shewanella sp. XH39),
JX162043 (Enterobacteriaceae bacterium
Pokym2-b), NR_037112 (Serratia
quinivorans 4364), KU999993 (Serratia
liquefaciens strain ZMT-1), KM187235
(Gluconacetobacter sp. CC3H1),
KM453915 (Uncultured bacterium D05
placa2), NR_113616 (Serratia grimesii
NBRC 13537), and for the bacterial
isolate tested: KX197201 (C4). The tree
topology was constructed using ClustalX
(1.81).
Morphological characterization
Size
Small
Form
Circular
Margin
Entire
Elevation
Plane
Surface
Smooth
Opacity
Translucent
Color
Cream
Gram staining
Negative
Biochemical characterization
King A
-
Catalase
+
Urease
-
Lecithinase
+
Nitrate reductase
+
Tryptophane deaminase
-
Lysine decarboxylase
+
Mannitol
-
Simmons citrate
-
Indole
+
Red of Methyl
-
Voges-Proskauer
+
Hydrogen sulfide
-
Molecular characterization
Most related species
CTL-81, Uncultured
Serratia sp. (100)
AP-CMST, Serratia
proteamaculans (100)
Accession number
GenBank
KX197201
Hypersensivity reaction
-
Hemolytic activity
-
Tunisian Journal of Plant Protection 28 Vol. 12, Special Issue, 2017
Serratia_liquefaciens_ZMT-1
Enterobacteri aceae_bacterium_P
Serratia_grimesii_NBRC-13537
Uncultured_bacterium_D05-placa
Shewanella_sp._XH39
Gluconacetobacter_sp. _CC3H1
Serratia_sp._PKL-12
Serratia_quinivorans_4364
38
70
67
86
95
54
Serratia_proteamaculans_AP-CMS
Uncultured_Serratia_sp._CTL-81
C4
100
100
0.001
Fig. 3. Neighbor-joining phylogenetic tree of partial 16S rDNA sequences of the best antagonistic and plant
growth promoting endophytic bacterial isolate C4 recovered from Cestrum nocturnum and their closest
phylogenetic relatives.
Hypersensivity reaction and hemolytic
activity of Serratia sp. C4.
No hypersensitive reaction (HR)
(chlorotic or necrotic zone) was detected
on inoculated tobacco leaf areas as
compared to control ones after 24 h of
incubation. Thus, the isolate C4 was
found to be non phytopathogenic and was
selected for further screenings.
No hemolytic activity was
displayed by the isolate C4 expressed by
the absence of clear zones around its
colonies grown on Blood Agar medium
after 48 h of incubation at 25°C. Thus,
this isolate was nonpathogenic to humans
and it can be used in the following tests
(Table 3).
Antifungal activity of Serratia sp. C4
against Fusarium oxysporum f. sp.
lycopersici.
Activity of whole culture. The
endophytic bacterial isolate Serratia sp.
C4, tested using the streak method,
induced a significant (P ≤ 0.05) decrease,
by 19.5% compared to control, in FOL
mycelial growth noted after 4 days of
incubation at 25°C (Table 4; Fig. 4 A).
Tested using the disc diffusion
method on PDA medium, Serratia sp. C4
formed an inhibition zone of about 8.62
mm in size around FOL colony after 4
days of incubation at 25°C (Table 4; Fig.
4B).
Tunisian Journal of Plant Protection 29 Vol. 12, Special Issue, 2017
FOL+Ethanol
FOL+Ethanol
FOL+F
FOL+F
FOL+Bio-F
FOL+Bio-F
FOL+EB-C4
FOL+EC-C4 FOL+EB-C4
FOL+EC-C4
2.5%
5%
FOL FOL+FC4 FOL+FC4FOL+FC4
50C 100CUntreated filtrateControl
FOL FOL+C4 FOL+C4FOL
10 mm10 mm
10 mm
10 mm
A
D
C
B
Streak method Disc diffusion method
Table 4. Antifungal activity of Serratia sp. C4 against Fusarium oxysporum f. sp. lycopersici
(FOL)
Bacterial treatment
Diameter of FOL colony (cm)a
Inhibition zone (mm)b
Untreated control
3.71 ± 0.08
0 ± 0
Serratia sp. C4 (KX197201)
2.98* ± 0.04
8.62* ± 0.5
Values with asterisk indicate a significant difference with the control (t-test at P ≤ 0.05).
a Tested using the streak method on PDA medium and incubated at 25°C for 4 days.
b Tested using the disc diffusion method on PDA medium and incubated at 25°C for 4 days.
Fig. 4. Antifungal activity of endophytic Serratia sp. C4 against Fusarium oxysporum f. sp.
lycopersici (FOL) using whole culture (A and B), u ntreated cell-free culture and filtrates heated at
50 and 100°C (C) and organic extracts tested at two concentrations (D) as compared to controls.
C4: Whole culture of Serratia sp. C4; FC4: Cell-free culture filtrate from Serratia sp. C4; Ethanol:
Negative control; F: Positive control (Bavistin®, chemical fungicide with carbendazim as active
ingredient); Bio-F: Positive control (Bactospeine®, Bacillus thuringiensis-based biopesticide); EC-
C4: Chloroform extract from Serratia sp. C4; EB-C4: n-butanol extract from Serratia sp. C4.
Tunisian Journal of Plant Protection 30 Vol. 12, Special Issue, 2017
a
b
a
b
a
b
0
1
2
3
4
5
Control
FC4
Control
FC4
Control
FC4
Untreated filtrate
50C
100C
Colony diameter (cm)
Bacteria l treatment
Activity of cell-free culture filtrate.
Analysis of variance revealed significant
(P ≤ 0.05) variation in pathogen colony
diameter depending on treatments tested
and a significant interaction was recorded
between cell-free treatments and heating.
A significant decrease in FOL colony
diameter, 23% versus the un-inoculated
control, was induced by the untreated
cell-free culture of Serratia sp. C4 (Fig.
5). The filtrate heated at 50°C for 15 min
had also significantly (P ≤ 0.05) reduced
FOL growth by 21.7% (Fig. 5). However,
heating treatment at 100°C for 15 min
decreased the antifungal activity of the
tested cell-free culture supernatant toward
FOL where pathogen growth was
inhibited by 9.3% relative to 21.7 and
23% recorded using filtrate heated at
50°C and unheated one, respectively
(Figs. 4C and 5).
Fig. 5. Effect of heating of the cell-free culture filtrate of Serratia sp. C4 on its
antifungal activity against Fusarium oxysporum f. sp. lycopersici (FOL) noted after
4 days of incubation at 25°C as compared to controls.
FC4: Cell-free culture filtrate from Serratia sp. C4 (KX197201) isolated from
surface sterilized Cestrum nocturnum leaves. Control: Luria-Bertani broth filtrate.
LSD (Bacterial treatment × Heating): 0.33 cm at P ≤ 0.05. For each heating
treatment, bars with the same letter are not significantly different according to
Duncan Multiple Range test at P ≤ 0.05.
Activity of organic extracts. ANOVA
analysis revealed a significant (P ≤ 0.05)
variation in the mean colony diameter of
FOL depending on organic extracts
(chloroform and n-butanol extracts)
tested, concentrations used, and their
interactions. Chloroform and n-butanol
extracts from Serratia sp. C4, applied at 1
mg/ml, had inhibited FOL growth by 27.6
to 66.5% as compared to ethanol control
whatever the concentration tested (Fig. 6).
Both organic extracts from
Serratia sp. C4 were found to be more
active at the concentration 5% than at
2.5% (v/v) where pathogen growth was
inhibited by 54.6-66.5% and 27.6-31.3%,
respectively (Fig. 6). In fact, chloroform
extract from Serratia sp. C4 decreased
FOL growth by 54.6% when applied at
Tunisian Journal of Plant Protection 31 Vol. 12, Special Issue, 2017
a
b
c
b
b
a
b
bc
cd
d
0
1
2
3
4
5
6
7
8
Ethanol
F
Bio-F
EC4
EC4
Ethanol
F
Bio-F
EC4
EC4
Commercial products
Chloroform
extrac t
n-buta nol
extrac t
Commercial products
Chloroform
extrac t
n-buta nol
extrac t
2.5
5
Colony diameter (cm)
Treatments tested at two c oncentrations (% v /v)
5% (v/v), compared to 31.3% recorded at
2.5% (v/v). In addition, n-butanol extract
from this isolate, applied at 5% (v/v), had
inhibited pathogen growth by 66.5%
compared to 27.6% recorded at 2.5%
(v/v) (Figs. 4D and 6).
It should be highlighted that the
decrease in FOL growth was higher using
Serratia sp. C4 organic extracts at 5%
(v/v) (54.6-66.5%) than that achieved
using Bavistin® (31.3-39.5%) and
Bactospeine® (40.9-43.2%) whatever the
concentration used (Figs. 4D and 6).
Fig. 6. Effect of chloroform and n-butanol extracts from endophytic Serratia sp. C4 tested at two concentrations
against Fusarium oxysporum f. sp. lycopersici noted after 7 days of incubation at 25 °C as compared to controls.
EC4: Organic extract from Serratia sp. C4 (KX197201) isolated from surface sterilized Cestrum nocturnum
leaves. Control: Ethanol control. F: Bavistin® (Chemical fungicide, carbendazim); B io-F: Bactospeine® (Bacillus
thuringiensis-based biopesticide). LSD (Treatments tested × Concentrations used): 0.86 cm at P ≤ 0.05. For each
concentration, bars sharing the same letter are not significantly different according to Duncan Multiple Ra nge
test at P ≤ 0.05.
Production of chitinase, protease and
pectinase by Serratia sp. C4
Serratia sp. C4 formed clear zones
around its colonies when grown onto
chitin-, pectin- and milk-agar media. This
indicates that Serratia sp. C4 is able to
produce cell-wall degrading enzymes i.e
chitinase-, pectinase and protease,
respectively (Table 6).
Phosphate solubilization and IAA
production ability of Serratia sp. C4
Serratia sp. C4 was able to
solubilize phosphate as indicated by the
formation of a clear zone of about 10.33
mm width around its colonies when
grown on Pikovskaya agar medium
(Table 5).
The selected endophytic isolate,
Serratia sp. C4, was able to produce the
indole-3-acetic acid (IAA), involved in
plant growth promotion, estimated at
29.52 µg/ml after 48 h of incubation
compared to 0.3 µg/ml recorded after 24
h (Table 5).
Tunisian Journal of Plant Protection 32 Vol. 12, Special Issue, 2017
Table 5. Production of cell-wall degrading enzymes and plant growth -promoting compounds by
endophytic Serratia sp. C4 recovered from Cestrum nocturnum leaves
a Tested on chitin-agar (0.5 % w/v) medium and incubated at 28 ± 2°C for 72 h; +: Presence of clear
zone.
b Tested on skim milk agar (3% v/v) medium and incubated at 28 ± 2°C for 48 h; +: Presence of clear
zone (14.5 ± 0.03 mm in diameter).
c Tested on pectin-agar (0.5 % w/v) medium and incubated at 28 ± 2 °C for 48 h; +: Presence of clear
zone.
d Indole-3-acetic acid (IAA) compounds production after 24 and 48 h of incubation at 28 ± 2°C in Luria -
Bertani broth medium; +: Production of IAA compounds (0.3 ± 0 µg/ml) ++: Production of IAA
compounds (29.52 ± 0.05 µg/ml).
e Tested on Pikovskaya agar medium and incubated at 28 ± 2°C for 7 days; +: Presence of clear zone
(10.33 ± 0.01 mm in diameter).
DISCUSSION
Endophytic bacteria isolated from
cultivated plants i.e. tomato and colza
were successfully used as biocontrol
agents against Fusarium wilt of tomato
(Nandhini et al. 2012; Nejad and Johnson
2000; Ramyabharathi and Raguchander
2014). In view of previous studies, the
present investigation focused on potential
use of endophytic bacteria recovered from
C. nocturnum grown in Tunisia (Chott-
Mariem, Sousse) for controlling tomato
Fusarium wilt.
C. nocturnum was successfully
used as a natural source of bioactive
metabolites with antifungal activities. In
fact, ethanol, methanol and butanol
extracts from C. nocturnum were
explored for their antifungal activity
against Trichoderma sp. and Aspergillus
sp. (Prasad et al. 2013). Furthermore,
aqueous extracts and chloroform, ethyl
acetate and n-butanol extracts from C.
nocturnum showed an inhibitory effect
against Candida albicans, Microsporum
canis, Candida glaberata growth ranging
between 30 and 65% (Khan et al. 2011).
Few data were available on C. nocturnum
use as potent source of isolation of
microorganisms such as fungi i.e
Alternaria spp., Aspergillus spp.,
Colletotrichum spp., Fusarium spp.,
Gliocladium sp., Phoma spp., Phomopsis
spp., Phyllosticta sp., Torula sp. and
Xylariales sp. (Huang et al. 2008). To our
Knowledge, no data had previously
valorized this species as natural source of
isolation of bacterial biocontrol agents.
Hence, this is the first study reporting on
possible exploration of C. nocturnum as
potential source of endophytic bacteria
exhibiting antifungal activity against
Fusarium wilt and growth-promoting
potential onto tomato seedlings.
In the current study, four bacterial
isolates were collected among many
others based on macro-morphology
diversity onto NA medium and were
found to be resistant to streptomycin and
rifampicin (100 µg/ml). Endophytic
Isolate
Cell-wall degrading enzymes
Plant growth-promoting co mpounds
Chitinasea
Proteaseb
Pectinasec
Indole-3-acetic acid
(µg/ml)d
Phosphate
solubilizatione
24 h
48 h
Serratia sp. C4
( KX197201)
+
+
+
+
++
+
Tunisian Journal of Plant Protection 33 Vol. 12, Special Issue, 2017
progress within tomato stems was
confirmed for three isolates among the
four tested after re-isolation onto NA
medium amended with these both
antibiotics. These three bacterial isolates
recovered from C. nocturnum and
exhibiting endophytic behavior on tomato
plants, were assessed for their ability to
control Fusarium wilt disease and to
enhance growth of tomato seedlings. In
fact, assessed on seedlings un-inoculated
with FOL, the current results clearly
demonstrated that the bacterial isolate C4
had significantly stimulated the growth of
the aerial plant parts by 31.9-50% and
root development by 24.6 to 53.3%
compared to the untreated control. Hence,
the isolate C4, recovered from C.
nocturnum leaves, exhibited bio-
fertilizing properties. Similar results on
plant growth-promoting potential (PGPB)
were reported on wheat seedling treated
with Serratia marcescens KR-4, B.
thuringiensis KR-1 and Enterobacter
asburiae KR-3, originally recovered from
nodule of Pueraria thunbergiana
(Selvakumar et al. 2008a). When assessed
on tomato seedlings inoculated with FOL,
the strongest suppressive effect of disease
severity was also displayed by the isolate
C4. This ability to suppress Fusarum wilt
was expressed by more than 86.6%
decrease in leaf damage index and 97.7%
in vascular browning extent resulting in a
significant decrease in FOL colonization
of stem tissues. Nejad and Johnson (2000)
also observed a decrease by at least 75%
in tomato Fusarium wilt severity using
two unidentified endophytic isolates PA
and PF, issued from healthy cultivated
oilseed rape plants. The selected C4
isolate was also able to enhance plant
growth on tomato plants inoculated or not
with FOL. Furthermore, Pearson
correlation analysis indicates that
Fusarium wilt severity decrease was
found to be related to the registered
increment in all plant growth parameters.
Ramyabharathi and Raguchander (1994)
also found that tomato Fusarium wilt-
suppressive effect, by 68.4%, displayed
by an endophytic bacterium B. subtilis str.
EPC016, isolated from cotton plants, was
associated to promotion of plant growth
and fruit yield in tomato compared to
control. In our recent studies, a strong
decrease in Fusarium wilt severity was
also achieved using various endophytic
bacteria including Stenotrophomonas sp.
S33, Pseudomonas sp. S85, B. mojavensis
S40, S. maltophilia S37, B. tequilensis
SV104, Bacillus sp. SV101, Alcaligenes
feacalis subsp. faecalis S8, B. cereus S42
and A. faecalis S18, originally recovered
from wild Solanaceae species namely
Datura metel, D. stramonium, Solanum
elaeagnifolium, Withania somnifera, and
Nicotiana glauca, respectively. These
endophytic isolates were also shown
effective in enhancing tomato growth in
plants inoculated or not with FOL (Aydi
Ben Abdallah et al. 2016a-e).
The best antagonistic and plant
growth-promoting isolate (C4) was
macro-morphologically and
biochemically characterized and
molecularly identified by 16S rDNA gene
sequencing as Serratia sp. C4
(KX197201).Gyaneshwar et al. (2011)
used an isolate of Serratia marcescens,
showing an endophytic ability on rice
plants, which significantly stimulated
plant growth as estimated by the length of
roots and the dry weight of the whole
plant. Similarly, plant height and fresh
weight, leaf dry weight and the number of
fruits per plant were also improved in
cultivated tomato using various species of
rhizospheric and/or plant-associated
bacteria belonging to Pseudomonas
putida, P. fluorescens, S. marcescens, B.
amyloliquefaciens, B. subtilis, and B.
cereus (Almaghrabi et al. 2013).
Furthermore, S. marcescens B2, applied
Tunisian Journal of Plant Protection 34 Vol. 12, Special Issue, 2017
to cyclamen plants inoculated with
sclerotia of Rhizoctonia solani and/or
conidia of F. oxysporum f. sp. cyclaminis,
was found to be effective in suppressing
both diseases induced by these pathogens
(Someya et al. 2000).
In the current study, Serratia sp.
C4, evaluated in vitro for its antagonistic
potential toward FOL, had reduced
pathogen mycelial growth and formed an
inhibition zone. Similar effects were
reported by Patel et al. (2012) using
endophytic bacteria from cultivated
tomato, identified as Pseudomonas
aeruginosa str. HR7, for F. oxysporum
biocontrol. The cell-free culture filtrate of
Serratia sp. C4, from 3-day-old culture,
tested in the current study had also
significantly reduced FOL growth
compared to control even if heated at 50,
100°C for 15 min and/or unheated.
However, the antifungal potential of
Serratia sp. C4 cell-free culture filtrate
significantly declined, as compared to
control filtrate, after heating at 100°C for
15 min. In the same way, antagonistic
potential of culture filtrate from
Streptomyces hygroscopicus toward
Colletotrichum gloeosporioides and
Sclerotium rolfsii declined heating at
100°C for 45 min (Prapagdee et al. 2008).
However, the antifungal activity of
culture filtrate from Bulkhorderia cepacia
toward C. gloeosporioides was similar to
that expressed by the untreated filtrate
even if heated at 50 or 100°C and
autoclaved at 121°C for 20 min (Kadir et
al. 2008).
In this study, Serratia sp. C4 cell-
free culture filtrate was also subjected to
an extraction with chloroform and n-
butanol to elucidate the antifungal
potential of its extracellular metabolites.
Results showed that both organic extracts
from Serratia sp. C4, used at 1 mg/ml,
had reduced FOL growth by 27.6 to
66.5%, compared to ethanol control.
Biologically active metabolites extracted
with ethyl acetate, methanol and
chloroform from Serratia sp., isolated
from the coralline red algae Amphiroa
anceps, inhibited the mycelial growth of
several pathogenic bacteria and fungi
(Karkachi et al. 2010). The chloroform-
methanol and diethyl ether extracts from
S. marcescens S10, isolated from the gut
of the insect American cockroach showed
antimicrobial potential toward various
bacterial and fungal agents namely
Lesteria spp., Salmonella spp., Klebsiella
spp., Staphylococcus aureus, C. albicans,
Aspergillus niger, and Geotricum spp.
(Ahmed and Hassen 2013).
Chloroform and n-butanol extracts
from Serratia sp. C4 were found to be
more effective at the concentration of 5
than at 2.5% (v/v). The highest antifungal
potential toward FOL growth (66.5%)
was achieved using the n-butanol extract
from the tested isolate used at 5% (v/v)
versus the ethanol control and the two
commercial products used i.e. Bavistin®
(50%, carbendazim) and Bactospeine®
(16000UI/mg, biopesticide). These results
are in agreement, in part, with Aydi Ben
Abdallah et al. (2015) findings using
chloroform and n-butanol extracts from
six endophytic Bacillus spp. isolates,
recovered from wild Solanaceae plants
(namely D. metel, S. nigrum, S.
elaeagnifolium and N. glauca), where the
two types of organic extracts from
Bacillus spp. exhibited an interesting
antifungal potential toward FOL which
was higher than that induced by the same
two commercial products used in the
present study whatever the concentrations
used. Furthermore, chloroform and n-
butanol extracts from Bacillus spp. were
found to be more effective at the
concentration of 5 than at 2.5% (v/v)
toward FOL (Aydi Ben Abdallah et al.
2015).
Tunisian Journal of Plant Protection 35 Vol. 12, Special Issue, 2017
Serratia sp. C4 was elucidated in
vitro for its properties deployed in the
observed antifungal effect. In fact, this
isolate was shown able to produce
chitinase, protease and pectinase enzymes
as shown on chitin-, milk-, and pectin-
agar media, respectively. Several genera
of endophytic bacteria such as Serratia,
Bacillus, Burkholderia, Acinetobacter,
Pseudomonas, Enterobacter,
Stenotrophomonas, Micrococcus, and
Microbacteruim were found able to
produce chitinase, protease and β-1,3-
glucanase involved in cell-wall
degradation of various pathogens (Berg et
al. 2005; Bibi et al. 2012). Moreover,
pectinase and cellulase were previously
reported by Hallmann et al. (1997) as
essential enzymes for colonization of
plant tissues. These enzymes may be also
involved in the enhancement of tomato
growth (Baldan et al. 2003). As
metabolites involved in the recorded
increment of tomato growth relative to
the untreated control, in the present study,
the selected isolate Serratia sp. C4 was
found able to produce indole-3-acetic acid
(IAA). The IAA amount released by our
isolate Serratia sp. C4 (29.52 µg/ml, after
48 h of incubation) is interestingly higher
when compared to 11.1 µg/ml produced
by S. marcescens SRM, recovered from
flowers of Cucurbita pepo, in Selvakumar
et al. (2008b) study but lower than the
amount produced by Serratia
nematodiphila (58.9 µg/ml) isolated from
forest soil (Dastager et al. 2011).
Phosphate solubilization ability
was also assessed, in this study, and
confirmed for Serratia sp. C4. Ngamau et
al. (2012) study revealed that endophytic
bacteria such as Pseudomonas spp.,
Serratia spp., Enterobacter asburiae,
Rahnella aquatilis, Ewingella americana,
and Yokenella regensburgei were able to
solubilize the phosphate. Furthermore,
plant growth promotion attributes such as
phosphate solubilization, IAA production,
hydrogen cyanide production and N-
fixation were found in S. marcescens KR-
4 recovered from Pueraria thunbergiana
(Selvakumar et al. 2008a), S. marcescens
KiSII isolated from the rhizosphere of
coconut palms (George et al. 2013) and S.
nematodiphila issued from forest soil
(Dastager et al. 2011). The phosphatase
activity of our endophytic isolate Serratia
sp. C4 was indicated by the presence of
clear zone of about 10.33 mm. Phosphate
solubilization ability of unidentified
endophytic bacterial isolates and P.
aeruginosa HR7, recovered from tomato
plants, was also expressed by the
formation of a clear zone of about 8 to 31
mm around their colonies (Patel et al.
2012).
The chemical identification of
Serratia sp. C4 extracellular metabolites
separated in the different organic extracts
tested will give additional information on
the nature of bioactive molecules
involved in the recorded disease-
suppressive potential of this endophytic
isolate.
_____________________________________________________________________
RESUME
Aydi Ben Abdallah R., Mejdoub-Trabelsi B., Nefzi A., Jabnoun-KhiareddineH. et
Daami-Remadi, M. 2017. Utilisation de bactéries endophytes naturellement associées à
Cestrum nocturnum pour la lutte biologique contre la flétrissure fusarienne et
l'amélioration de la croissance de la tomate. Tunisian Journal of Plant Protection 12:
15-40.
Trois isolats bactériens endophytes, isolés à partir des feuilles et des tiges de Cestrum nocturnum
(jasmin de nuit), ont été évalués pour leur aptitude à supprimer la fusariose vasculaire, causée par
Tunisian Journal of Plant Protection 36 Vol. 12, Special Issue, 2017
Fusarium oxysporum f. sp. lycopersici (FOL), et à améliorer la croissance des plants de tomate. Les
isolats testés ont significativement diminué la sévérité de la maladie de 46,6 à 97,7% par rapport au
témoin inoculé par FOL et non traité. L'isolat C4 s’est révélé le plus efficace dans la réduction des
altérations foliaires de 86,6% et de la hauteur du brunissement vasculaire de 97,7% par rapport au
témoin inoculé par FOL et non traité. Une augmentation significative, de 39 à 41,6% par rapport au
témoin inoculé par le pathogène et non traité, a été enregistrée au niveau des paramètres de croissance
de la tomate. De plus, l'isolat C4 a significativement augmenté les paramètres de croissance de 24,5-
53,3% par rapport aux plants témoins non inoculés par le pathogène et non traités. Cet isolat a été
morphologiquement et biochimiquement caractérisé et identifié en utilisant le séquençage du gène 16S
ADNr comme Serratia sp. (KX197201). Criblé in vitro pour son activité antifongique contre FOL,
Serratia sp.C4 a induit une diminution de 19,52% de la croissance radiale du pathogène et la formation
d'une zone d'inhibition de 8,62 mm de diamètre. Le filtrat de culture de Serratia sp. C4, additionné au
milieu PDA à raison de 20% (v/v), a réduit la croissance radiale du pathogène de 23% comparé aux
21,7 et 9,2% enregistrés après son chauffage à 50 et 100°C, respectivement. L'extrait n-butanolique de
Serratia sp. C4, appliqué à 5% (v/v), a présenté un potentiel antifongique contre FOL traduit par une
inhibition de la croissance de 66,5% par rapport au témoin non traité et qui a été supérieure à celle
obtenue moyennant deux pesticides commerciaux, à savoir Bavistin® (carbendazime à 50%, fongicide
chimique) et Bactospeine® (16000 UI/mg, biopesticide à base de Bacillus thuringiensis). Serratia sp.
C4 s'est montré un agent producteur de chitinase, de pectinase et de protéase et capable de produire
l'acide indole-3-acétique et de solubiliser le phosphate.
Mots clés: Activité antifongique, bactéries endophytes, Cestrum nocturnum, Fusarium oxysporum f. sp.
lycopersici, métabolites secondaires, promotion de la croissance, tomate
________________________________________________________________________ Cestrum nocturnum Tunisian Journal of Plant Protection 12: 15-40.
Cestrum nocturnum
Fusarium oxysporum f. sp. lycopersici (FOL)
46,6 %97,7
FOLC4
.6%% FOL
6% 41
C424.5 %53.3
SSerratia sp. (KX197201)
FOLC4 Serratia sp.%19.52
8.62
Serratia sp. C4
PDA20%% 21.7%9.2
Serratia sp. C4
% FOL %66.5
®
Bavistin %50
arbendazimC
®
Bactospeine
16000 UI Bacillus
thuringiensisSerratia sp. C4
.
Tunisian Journal of Plant Protection 37 Vol. 12, Special Issue, 2017
Fusarium
oxysporum f. sp. lycopersiciCestrum nocturnum
_____________________________________________________________________
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