Hellenic Plant Protection Journal - EBE BENAKI PHYTOPATHOLOGICAL INSTITUTE
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Volume 13, Issue 2, July 2020
ISSN 1791-3691
Hellenic
Plant
Protection
Journal
A semiannual scientific publication of the
BENAKI
BE
E PHYTOPATHOLOGICAL INSTITUTE
Hellenic Plant Protection Journal
EDITORIAL POLICY
The Hellenic Plant Protection Journal (HPPJ) (ISSN 1791-3691) is the scientific publication of the
Benaki Phytopathological Institute (BPI) replacing the Annals of the Benaki Phytopathological Insti-
tute (ISSN 1790-1480) which had been published since 1935. Starting from January 2008, the Hel-
lenic Plant Protection Journal is published semiannually, in January and July each year.
HPPJ publishes scientific work on all aspects of plant health and plant protection referring to plant
pathogens, pests, weeds, pesticides and relevant environmental and safety issues. In addition, the
topics of the journal extend to aspects related to pests of public health in agricultural and urban
areas.
Papers submitted for publication can be either in the form of a complete research article or in
the form of a sufficiently documented short communication (including new records). Only origi-
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Editorial Board, are also published, normally one article per issue. Upon publication all articles are
copyrighted by the BPI.
Manuscripts should be prepared according to instructions available to authors and submitted in
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lished after successful completion of a review procedure by two competent referees.
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thors. The information and opinions contained herein have not been adopted or approved by the
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included in the published articles nor may be held responsible for the use to which information
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EDITORIAL BOARD
Editor: Dr F. Karamaouna (Pesticides Control & Phytopharmacy Department, BPI)
Associate Editors: Dr A.N. Michaelakis (Entomology & Agric. Zoology Department, BPI)
Dr K.M. Kasiotis (Pesticides Control & Phytopharmacy Department, BPI)
Dr I. Vloutoglou (Phytopathology Department, BPI)
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A. Karadima (Information Technology Service, BPI)
For back issues, exchange agreements and other publications of the Institute contact the Li-
brary, Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifissia, Attica, Greece, e-mail:
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This Journal is indexed by: AGRICOLA, CAB Abstracts-Plant Protection Database, INIST (Institute for
Scientific and Technical Information) and SCOPUS.
The olive tree of Plato in Athens is the emblem
of the Benaki Phytopathological Institute
Hellenic Plant Protection Journal also available at www.hppj.gr
© Benaki Phytopathological Institute
Hellenic Plant Protection Journal 13: 54-65, 2020
DOI 10.2478/hppj-2020-0006
Effect of the endophytic plant growth promoting
Enterobacter ludwigii EB4B on tomato growth
M.E.A. Bendaha1,2* and H.A. Belaouni3
Summary This study aims to develop a biocontrol agent against Fusarium oxysporum f.sp. radicis-lyco-
persici (FORL) in tomato. For this, a set of 23 bacterial endophytic isolates has been screened for their
ability to inhibit in vitro the growth of FORL using the dual plate assay. Three isolates with the most
sound antagonistic activity to FORL have been qualitatively screened for siderophore production,
phosphates solubilization and indolic acetic acid (IAA) synthesis as growth promotion traits. Antag-
onistic values of the three candidates against FORL were respectively: 51.51 % (EB4B), 51.18 % (EB22K)
and 41.40 % (EB2A). Based on 16S rRNA gene sequence analysis, the isolates EB4B and EB22K were
closely related to Enterobacter ludwigii EN-119, while the strain EB2A has been assigned to Leclercia
adecarboxylata NBRC 102595. The promotion of tomato growth has been assessed in vitro using the
strains EB2A, EB4B and EB22K in presence of the phytopathogen FORL. The treatments with the select-
ed isolates increased significantly the root length and dry weight. Best results were observed in isolate
EB4B in terms of growth promotion in the absence of FORL, improving 326.60 % of the root length and
142.70 % of plant dry weight if compared with untreated controls. In the presence of FORL, the strain
EB4B improved both root length (180.81 %) and plant dry weight (202.15 %). These results encourage
further characterization of the observed beneficial effect of Enterobacter sp. EB4B for a possible use as
biofertilizer and biocontrol agent against FORL.
Additional keywords: Biocontrol, biofertilizer, Enterobacter ludwigii, PGPR
Introduction efit the soil and plant health (Kloepper et
al., 1989). For decades, rhizobacteria bene-
The rhizospheric zone is rich in nutrients ficial to plants are often referred to as plant
compared with the neighboring bulk soil growth promoting rhizobacteria (PGPR),
due to the accumulation of a variety of or- which are characterized by at least two of
ganic compounds released by the roots the three following criteria: competitive-
through exudation, secretion and rhizode- ly root colonization, stimulation of growth
position. These organic compounds can be and reduction of disease incidence (Reddy,
used as carbon and energy sources by mi- 2013). Microbial-inoculants are being wide-
croorganisms and microbial activity is par- ly used to improve plant growth under con-
ticularly intense in the rhizosphere (Chau- trolled as well as natural field conditions
han et al., 2015). (Nadeem et al., 2013).
An alternative to increasing agricultur- Inoculants of PGPR bacteria improve
al productivity in a sustainable way is the root development through the production
manipulation of micro-organisms that ben- of certain phytohormones (Bloemberg and
Lugtenberg, 2001), such as auxins including
indole acetic acid (AIA), cytokinins and gib-
1
berellins (Vessey, 2003). Furthermore, many
Department of biology, Faculty of Nature and Life
Sciences, University Mustapha Stambouli of Mascara,
bacterial strains are able to improve the
Mascara, Algeria. health of plants by limiting the saprophyt-
2
Laboratoire de Biologie Moléculaire, Génomique et ic growth of phytopathogenic microorgan-
Bioinformatique (LBMGB), University Hassiba Ben
Bouali of Chlef, Algeria. isms, and some of them are used as biologi-
3
Laboratoire de Biologie des Systèmes Microbiens cal control agents in agriculture (Bloemberg
(LBSM), Ecole Normale Supérieure de Kouba, Algiers,
Algeria. and Lugtenberg, 2001; Whipps, 2001). The
* Corresponding author: m.a.bendaha@gmail.com great diversity of the mechanisms of action
© Benaki Phytopathological Institute
Endophytic Enterobacter ludwigii isolate on tomato 55
of these microorganisms is mainly related to of biofertilizers (Dey et al., 2004).
their great ability to produce a wide range Siderophores are low molecular weight
of secondary metabolites and to induce ISR peptides or iron chelators (Santos-Villalo-
in the host, making it less susceptible to sub- bos et al., 2012). As soon as the complex si-
sequent infection by a pathogen (Van Loon, derophore-iron enters the cytosol the cells
2007; Weller et al., 2002). through specific siderophore receptors pres-
PGPR include several bacterial endo- ent in the cell membrane, the ferric iron gets
phytes with alleged positive effects on plant reduced to a ferrous form which becomes
health and growth, which have been pur- free from the siderophore chelator complex.
sued mainly for agricultural applications to The released ferrous iron form is further used
increase yields since three decades ago (Pic- for metabolic processes (Venkat et al., 2017).
coli and Bottini, 2013). Endophytic bacterial IAA is a secondary metabolite produced
strains selected on the basis of plant growth- during the later stages of growth, after the
promoting bacteria (PGPB) characteristics are stationary growth phase (Gupta et al., 2012),
useful for formulation of inoculants to im- a phytohormone controlling many impor-
prove growth and yield (Forchetti et al., 2010). tant physiological processes in plants such
Plant growth-promoting bacterial endo- as cell division, tissue differentiation and
phytes have been successfully used to induce root initiation (Khan et al., 2014).
fungal resistance in plants (Ji et al., 2014), and Tomato, Solanum lycopersicum L. (Solan-
they are widely used in the developing areas aceae) has been considered as one of the
of forest regeneration and phytoremediation most important horticultural crop world-
of contaminated soils (Ryan et al., 2008). wide (Vos et al., 2014). However, tomato pro-
PGPR modify the rhizospheric envi- duction has shown limitations arising from
ronment by producing antagonistic mole- the use of cultivars susceptible to diseas-
cules with antibiotic or antifungal proper- es and pests causing substantial produc-
ties, or by synthesizing cell walls degrading tion losses (Dias et al., 2017). Use of biolog-
enzymes and volatile organic compounds ical control agents, such as plant growth
(VOC), which act against pathogens, disrupt- promoting rhizobacteria, can be a suitable
ing bacterial cell–cell communication (quo- approach in control of tomato diseases
rum sensing) (Grobelak et al., 2015). In addi- (Schmidt et al., 2004).
tion, many of these rhizobacterial strains can The main purpose of this study was to
also improve plant tolerance against salinity, evaluate a) the antagonistic and potential
drought, flooding and heavy metal toxicity. biocontrol activities of the endophytic bac-
Therefore, they enable plants to survive un- teria isolated from tomato roots against Fu-
der unfavorable environmental conditions sarium oxysporum f.sp. radicis-lycopersici
(Ma et al., 2011). Numerous works have re- (FORL), and b) their growth promoting abili-
ported beneficial effects of these rhizobac- ty in tomato at in vivo conditions.
teria for improving plant growth under nor-
mal as well as stressful environment (Szepesi
et al., 2009; Heidari and Golpayegani, 2012). Materials and methods
PGPR can additionally help plants by in-
creasing their uptake of nutrient elements Isolation of endophytic bacterial strains
such as Phosphate (P). The low availabili- Bacterial strains were isolated from toma-
ty of this macronutrient to plants results to roots of different hybrid varieties from Al-
from the fact that the P exists in insoluble gerian soil (Algiers locality). The roots were
forms (Kisiel and Kepczynska, 2016). Micro- washed and dried aseptically. Root sam-
bial phosphate solubilization is likely to be ples were surface-disinfected to remove ep-
involved in a better plant growth, yield and iphytes, using 95% ethanol for 30 seconds,
nutrient uptake. Thus such trait is consid- followed by a 10% sodium hypochlorite
ered as a prospective tool for development treatment for 2 min and then 75% ethanol for
© Benaki Phytopathological Institute
56 Bendaha & Belaouni
2 min. The root pieces were then rinsed three 16S rRNA gene fragments were amplified
times with sterile distilled water to remove by PCR using an Invitrogen kit. Primers were
traces of disinfectant (Evans et al., 2003; Ru- as follows: 10-30 forward: 5’-GAG TTT GAT
bini et al., 2005). In a sterile mortar, the root CCT GGC TCA-3’ and 1500 reverse: 5’-AGA
pieces were crushed with sterile distilled wa- AAG GAG GTG CAG ATC CC-3’. 5 μl of 10 x
ter, to obtain a ground product including ex- PCR buffer (Mg2+), 8 μl of deoxynucleotide
tracted bacterial cells. The enrichment was triphos-phate (200 mM of each dNTP), 100
carried out by placing 1 g of the extract in pM of each primer, 2 μl of template DNA so-
9 ml of nutrient broth. The culture was con- lution and 0.8 μl of Taq enzyme (5 U/μl). DNA
ducted at 30°C for 24 h, aiming to increase fragments were recovered and purified. Se-
the initial biomass in order to recover a max- quence determination was performed by
imum bacterial charge (Bashan et al., 1993). Beckman Coulter Genomics (United King-
0.1 ml from each enrichment tube served to dom). After 16S rRNA gene sequencing,
the isolation on nutrient agar at 30°C for 24 h. identification of phylogenetic neighbors
Once pure single colonies obtained, the con- was done using BLASTN program against
servation was achieved in glycerol at - 20°C. databases containing type strains located
at the EzBioCloud database (https://www.
Antagonistic activity of endophythic ezbiocloud.net/). Neighbor-joining method
bacterial isolates against FORL under MEGA6.0 package was used for the
The test of the antifungal activity of the construction of phylogenetic trees. The 16S
endophytic bacterial isolates against FORL rRNA gene sequences were submitted to
was carried out on PDA medium using the the NCBI GenBank database.
dual culture technique described by Lee et
al. (2010). A 6 mm fragment of FORL aged Screening endophytic bacterial isolates
of 7 days was removed and deposited in for growth promotion traits
the center of a new Petri dish containing
the PDA medium and the bacterial suspen- Phosphate solubilization
sions were adjusted to 0.5 Mac Farland (pre- Each bacterial isolate was inoculated in
pared in nutrient broth). Then 5 μl of each Pikovskaya agar containing: 10 g glucose, 5 g
tested bacterial suspension was placed at 1 tribasic calcium phosphate (Ca5HO13P3), 0.5 g
cm from the edge of the same Petri dish. The (NH4)2SO4, 0.2 g KCl, 0.1 g MgSO47H2O, trace
experimental units were incubated at 25°C of MnSO4 and FeSO4, 0.5 g yeast extract, and
for 5 to 7 days. Control units included only 15 g agar, in 1000 ml distilled water (Pikovs-
the fungus without the tested bacteria. Each kaya, 1948.). After 7 days of incubation, the
test was replicated three times. The inhibi- presence of clear halos around bacterial col-
tion rate was calculated as follows: ony was used to indicate positive Phosphate
(P) solubilizing strains (Husen, 2003).
Inhibition rate (%) = X-Y 100
X Indole-3-acetic acid production (IAA)
Where: Bacterial strains were inoculated into
nutrient broth containing: 5 g peptone, 1.5
X: Diameter of mycelium control (mm). g yeast extract, 1.5 g beef extract, and 5 g
Y: Diameter of the mycelium in the presence NaCl, in 1000 ml distilled water supplement-
of the bacterium (measured on the axis “fun- ed with L-tryptophan (500 mg/L) and incu-
gus-bacterial colony” (mm). bated at 30°C for 5 days. The supernatant of
the stationary phase culture was obtained
Identification of endophytic bacterial by centrifugation at 10000 rpm for 15 min
isolates using 16S rRNA gene analysis (Bric et al., 1991). An aliquot of 2 ml superna-
Total bacterial DNA was extracted ac- tant was transferred to a fresh tube to which
cording to the method of Liu et al. (2000). 5 ml of Salkowski’s reagent (1 ml of 0.5 M Fe-
© Benaki Phytopathological Institute
Endophytic Enterobacter ludwigii isolate on tomato 57
Cl3 in 50 ml of 35% HClO4) were added. Af- ered for sowing (Johansson et al., 2003).
ter 25 min at room temperature, the absor-
bance of pink color developed was read at Pot experiments
530 nm. Varied amounts of pure indole-3- Pot experiments were conducted from
acitic acid were used as standard (Costacur- March 2017 to April 2017. Tomato seeds
ta et al., 2006). which had undergone both bacterial and
infestation treatment were sown in pots
Siderophore production filled with a mixture of potting soil and sand
All bacterial isolates were qualitatively (v/v) (approximately 100 g per pot) (Lee et
screened for siderophore production by in- al., 2010), Twenty two seeds per strain were
oculation onto a Chrome Azurol Sulphonate used in four replicates. Pot plants were
(CAS) agar plate and incubation for 24 h at sprayed with distilled water using a spray
28°C (Schwyn and Neilands, 1987). The change gun and incubated at room temperature (20
of the medium color from bluish to yellowish- to 25°C) and photoperiod 9:15 hours L:D. Af-
orange after incubation indicates the pres- ter 31 days of culture, the evaluation of the
ence of siderophores (Dias et al., 2017). impact of fungal and bacterial treatment on
the plant growth was made by measuring
Impact of endophytic bacterial isolates the length of the stem and main root, and
on tomato plant growth the dry weight of each treated batch.
Seed treatments Statistical analysis
Seeds of tomato (Isi Sementi S.p.A., Fi- ANOVA with Duncan’s multiple range
denza «Parma» Italy) were surface steril- test was used to detect significant differenc-
ized by soaking in 70% ethanol for 1 min es of the experimental data using XLSTAT
followed by immersion in sodium hypochlo- software, version 2014.3.01 (Adinosoft).
rite (1%) solution for 10 min and rinsing at
least five times with sterile distilled water.
The seeds were germinated on sterilized fil- Results and discussion
ter paper sheets in the Petri dish (Piromyou
et al., 2011). Each seed batch was inoculated Selection of antagonistic bacteria
with one of the selected isolates, using a sus- The Dual Plate Assay revealed the pres-
pension adjusted to 108 CFU/ml to evaluate ence of isolates capable of inhibition or re-
their ability to modulate the plant response striction of the growth of FORL. The inhi-
favorably (Piromyou et al., 2011).The bacteri- bition rates obtained were: EB4B (51.51%),
al treatment of the seeds was carried out un- EB22K (51.18%) and EB2A (41.40%) (Fig. 1), in-
der aseptic conditions by putting each batch dicating that the strains possess significant
of 22 seeds sterilized in the bacterial suspen- antagonistic effect towards the pathogen.
sion for two different incubation periods (30 A relatively broader inhibition zone involves
min and 60 min). The seeds of the “negative the synthesis of relatively potent antibiotics
control” batches were inoculated with ster- (Kadir, 2008). Lee et al. (2010) were able to
ilized 0.85% NaCl solution and sown direct- isolate bacterial strains which were consid-
ly without bacterial treatment (Fallahzadeh- ered to have good antifungal potential with
Mamaghani et al., 2009; Lee et al., 2010). The inhibition rates ranging from 0 to 45%. Mi-
fungal suspension was made from fresh cul- kani et al. (2007) results in the Pseudomonas
tures of FORL, with a spore concentration of fluorescens antagonism tests showed an in-
106 conidia/ml (El Aoufir, 2001). The seeds hibition of mycelial growth with an average
were infested with FORL after the bacterial of 59.8% for the best performing strain. Bac-
treatment, by immersing the batches in box- teria can produce many antifungal metabo-
es filled with fungal suspension for a dura- lites (Weller et al., 2007) such as phenazines,
tion of 1 hour, then they have been recov- pyoluteorin, pyrrolnitrin, DAPG (2,4-diacetyl
© Benaki Phytopathological Institute
58 Bendaha & Belaouni
opportunistic human pathogenic bacteria,
which can be carried on or in plant tissue
and may enhance plant growth and health,
in particular the Enterobacteriaceae that can
invade the root tissue (Mendes et al., 2013).
All strains isolated in this study should be
further analysed using multi locus analysis
to further species clarification. Thus, we can
have a safer view of whether they are actual-
ly potential human pathogens or not.
Growth promotion traits of selected PG-
PRs
Bacterial isolates EB2A, EB4B and EB22K
Figure 1. Dual Plate Assay against Fusarium oxysporum f.sp. showed a distinct zone with the appearance
Radicis lycopersici. Inhibition rate (%) caused by Leclercia ad- of orange color indicating the production of
ecarboxylata (EB2A), Enterobacter ludwigii (EB4B) and Enter- siderophore which is beneficial to plants, via
obacter ludwigii (EB22K) isolates. Data are presented as means potential increase of iron availability. All iso-
±SE. ANOVA with Duncan’s multiple range test was used to lated strains were able to solubilize the trical-
detect significant differences. Bars with different letters with-
cium P, resulting in large clear/halo zones.
in the parameter indicate significant differences at P ≤ 0.05.
Indolic acetic acid production was not
remarkably different in the three bacteri-
phloroglucinol) and siderophores (pyover- al strains (EB2A = 140 ± 0.10 μg/mL, EB4B
dines or pseudobactins) which are the most = 152 ± 0.44 μg/mL and EB22K = 155 ± 0.78
frequently detected antifungals (Haas and μg/mL). Various plant growth promoting En-
Défago, 2005; Lemanceau et al., 2009). terobacter spp. such as Enterobacter ludwigii
have been applied for plant development
Identification of selected PGPRs (Shoebitz et al., 2009; Madhaiyan et al., 2010;
The 16S rRNA gene sequences of the Kapoor et al., 2017). Shoebitz et al. (2009)
three bacterial strains reported in this pa- and Gopalakrishnan et al. (2012) have also
per are deposited in NCBI with the GenBank, reported the exhibition of nitrogenase ac-
with the following accession numbers: EB2A tivity, phosphate solubilisation, IAA produc-
(MF693122), EB4B (MF693530) and EB22K tion and antifungal activity by a E. ludwigii
(MF706259). Based on 16S rRNA gene se- strain. Previous reports had described some
quence analysis, the isolates EB4B and EB22K Leclercia adecarboxylata as efficient PGPR
were closely related to Enterobacter ludwigii (Naveed et al., 2014; Melo et al., 2016; Kisiel
EN-119 with an homology of 99.28% and and Kepczynska, 2016). Naveed et al. (2014)
98.69%, respectively (Fig. 2).According to reported that inoculation with Leclercia ad-
16S rRNA sequence, the strain EB2A showed ecarboxylata showed statistically significant
close proximity with Leclercia adecarboxyla- greater biomass than the controls under en-
ta NBRC 102595 (99.71%) (Fig. 2). vironmental chamber conditions.
Non plant pathogenic endophytic bac- Siderophores production has been
teria can promote plant growth, improve shown (CAS positive reaction), highlighting
nitrogen nutrition, and in some cases, are the potential of the strains belonging to En-
human pathogens such as enteric bacteria terobacter genus to produce such secondary
(Tyler and Triplett, 2008). PGPRs in different metabolites. Plants are known to use vari-
field crops are considered as human oppor- ous bacterial siderophores as an iron source
tunistic pathogens (Dutta and Thakur, 2017). (Martinez-Viveros et al., 2010).
The third group of microorganisms that can All three strains were capable of sol-
be found in the rhizosphere are true and ubilizing the insoluble tricalcium phos-
© Benaki Phytopathological InstituteEndophytic Enterobacter ludwigii isolate on tomato 59
Figure 2. Phylogenetic analysis of Leclercia adecarboxylata (EB2A), Enterobacter ludwigii (EB4B) and Enterobacter ludwigii
(EB22K) based on 16s rRNA gene sequencing. Neighbor joining tree was created using MEGA 6 (1000 bootstrap replicates)
with a scale of 0.001 substitutions per nucleotide position.
phate (Ca5HO13P3). Phosphate (P)-solubiliz- pared to non-inoculated tomato seeds. The
ing bacter ia can exert a positive effect on root length measurements showed a major
tomato growth, increasing the phosphorus increase of 326.60% for EB4B and 144.33%
status (Van Veen et al., 1997) and significant- for EB22K (Fig. 3a) compared to control
ly enhance photochemical activity accom- batches. The non-treated plants resulted in
panied by an increase of chlorophyll con- lower dry weight (Fig. 4a), while the treated
tent in plants (Liu et al., 2017). exhibited an increase of 142.70% for EB4B
Production of siderophores (Loaces et and 79.94% for EB22K. After 31 days of cul-
al., 2011), solubilization of mineral phos- tivation, the plants of the “Control +” batch
phates (Ahemad et al., 2008), and synthesis (Seeds in presence of the pathogen) experi-
of indolic acetic acid (IAA) (Khan et al., 2014) enced a low development compared to the
have beneficial effects on plant growth and “Control -” batch, which demonstrates the
are considered as frequent characteristics of virulence of FORL and its incidence on to-
PGPR (Gupta et al., 2012). mato plants growth.
Treatment of tomato seeds with the en-
Effect of PGPRs on root size and plant dophytic strains in the presence of FORL re-
biomass sulted in significantly higher length of roots
The analysis of root length and dry and plant dry weight compared to con-
weight after 31 days of culture showed that trol, implying an increase of 180.81% for
a period of 30 min of bacterization led to EB4B batch (Fig. 3b) whilst the average dry
significantly better results than those with weight was enhanced by 202.15% for the
60 min and revealed that inoculation of the same treatment (Fig. 4b). Thus, a significant-
PGPRs resulted in a significant increase in ly strong protective potential against FORL
the biomass compared to the uninoculat- is indicated.
ed controls. Inoculation with the strain EB4B Overall, the strains exerted a particu-
significantly increased the root length com- larly positive effect on the root system de-
© Benaki Phytopathological Institute60 Bendaha & Belaouni
velopment by increasing in vivo root elon- early flowering, and yields of fruits and
gation and plant biomass (dry weight). The seeds (Ramamoorthy et al., 2001). The col-
effect depended on the exposure time of onization of the roots by endophytic bac-
the seeds to the inocula. A longer exposure teria introduced on the seeds is distributed
(after 60 min of contact) produced a nega- along the roots, including partial or com-
tive effect on root elongation and plant de- plete colonization of the rhizosphere: in-
velopment, while a low bacterial concentra- side the root, on the root surface and in the
tion (after 30 min of contact) increased the immediate soil of the rhizosphere (Landa et
root elongation and plant biomass. Kisiel al., 2001). Kokalis-Burelle (2002) and Vessey
and Kepczynska (2016) also reported that a (2003) reported that PGPR could increase
higher density of the inoculum produced a yield of tomato by increasing the availability
negative effect on root elongation. of nutrients in rhizosphere soil and promot-
The increase in crop yield due to a bacte- ing other beneficial plant-growth promot-
rial culture results from two main beneficial ing bacteria. PGPRs can remarkably increase
effects: the stimulation of plant growth and nutrient availability in inoculated plants
the protection of plants against soilborne in plots compared to non-inoculated ones
diseases. PGPR treatments are known to in- (Adesemoye et al., 2008) and lead to a better
crease the percentage of germination, seed- tomato growth. Karlidag et al. (2013) dem-
ling vigor, emergence, root and stem devel- onstrated that inoculation of selected PGPR
opment, total plant biomass, seed weight, increased considerably the growth, chloro-
Figure 3. Growth promotion of tomato root induced by Leclercia adecarboxylata (EB2A), Enterobacter ludwigii (EB4B) and
Enterobacter ludwigii (EB22K) isolates (30 min and 60 min of contact) a) in the absence of FORL and b) in the presence of FORL
evaluated after 31 days. Data are presented as means ±SE. ANOVA with Duncan’s multiple range test was used to detect sig-
nificant differences. Bars with different letters within the parameter indicate significant differences at P ≤ 0.05.
Figure 4. Dry weight promotion of tomato plants induced by Leclercia adecarboxylata (EB2A), Enterobacter ludwigii (EB4B)
and Enterobacter ludwigii (EB22K) isolates (30 min and 60 min of contact) a) in the absence of FORL and b) in the presence of
FORL evaluated after 31 days. Data are presented as means ±SE. ANOVA with Duncan’s multiple range test was used to de-
tect significant differences. Bars with different letters within the parameter indicate significant differences at P ≤ 0.05.
© Benaki Phytopathological InstituteEndophytic Enterobacter ludwigii isolate on tomato 61
phyll content and nutrient. stances. Based on antagonistic tests, the
The treatment of tomato seeds with bacterial isolates with remarkable beneficial
PGPR strains (Leclercia adecarboxylata and traits such as the combination of production
Enterobacter ludwigii) have led to a reduc- of IAA, siderophores and phosphate solubili-
tion in susceptibility to FORL, with a substan- zation could be a useful tool to enhance the
tial promotion of tomato growth (length of sustainable production of tomato. In vivo
root and dry weight). The protective effect trials demonstrated an efficient interaction
of the strains could be explained by their of the three isolates with the tomato plant,
ability to produce antifungal substances, promoting growth and induction of plant
as highlighted by previous antifungal po- defense against FORL. Isolate EB4B appears
tential characterization studies (bacteria to be a promising alternative for future bio-
producing salicylic acid, rhamnolipids, chi- formulation and field application.
tinase, cellulases) in addition to good com-
petitiveness for carbon and energy sources
(Haas and Défago, 2005). This work was supported by the Laboratory
In vivo tests indicate the protective ef- of Molecular Biology, Genomics and Bioinfor-
fect of the isolated PGPR against FORL in matics, University of Hassiba Ben Bouali, Chlef,
the tested tomato cultivar, but also growth Algeria. The authors gratefully acknowledge
promotion with singular rates up to 200%. Dr BEKKAR for his help and assistance.
For such reason and for other reasons, root
colonization is often considered as a limit-
ing factor for biological control in the rhizo- Literature Cited
sphere (Dekkers et al., 1997). However, selec-
Adesemoye, A.O., Torbert, H.A. and Kloepper, J.W.
tion of root colonizers has been an empirical 2009. Plant growth promoting rhizobacteria al-
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Επίδραση εφαρμογής του ενδοφυτικού βακτηρίου
Enterobacter ludwigii EB4B στην προώθηση της ανάπτυξης
φυτών τομάτας
M.E.A. Bendaha και H.A. Belaouni
Περίληψη H μελέτη αυτή στοχεύει στην ανάπτυξη ενός βιολογικού παράγοντα έναντι του Fusarium
oxysporum f.sp. radicis-lycopersici (FORL) στην τομάτα, ο οποίος παράλληλα προωθεί την ανάπτυξη των
φυτών. Για το σκοπό αυτό, εξετάστηκε ένα σύνολο 23 ενδοφυτικών βακτηριακών στελεχών για την ικα-
νότητά τους να αναστέλλουν την ανάπτυξη του FORL χρησιμοποιώντας τη δοκιμασία διπλής καλλιέρ-
γειας σε τρυβλίο. Τρία απομονωθέντα στελέχη με την πιο ανταγωνιστική δράση προς το FORL εξετά-
στηκαν επίσης ως προς την παραγωγή σιδηροφόρων, τη διαλυτοποίηση φωσφορικών αλάτων και τη
σύνθεση ινδολοξικού οξέος (ΙΑΑ) ως χαρακτηριστικά της προώθησης της ανάπτυξης. Η ικανότητα ανα-
στολής της ανάπτυξης του FORL ήταν αντίστοιχα 51,51% (ΕΒ4Β), 51,18% (ΕΒ22Κ) και 41,40% (ΕΒ2Α). Με
βάση την ανάλυση αλληλουχίας του γονιδίου 16S rRNA, τα στελέχη EB4B και EB22K κατατάσσονται φυ-
λογενετικά πλησίον του στελέχους Enterobacter ludwigii ΕΝ-119, ενώ το στέλεχος ΕΒ2Α έχει αποδοθεί
στο Leclercia adecarboxylata NBRC 102595. Η επίδραση των στελεχών ΕΒ2Α, ΕΒ4Β και ΕΒ22Κ στην προ-
ώθηση ανάπτυξης των φυτών της τομάτας εξετάστηκε in vitro παρουσία του φυτοπαθογόνου FORL. Οι
επεμβάσεις με τα επιλεγμένα στελέχη αύξησαν σημαντικά το μήκος της ρίζας και το ξηρό βάρος των
© Benaki Phytopathological InstituteEndophytic Enterobacter ludwigii isolate on tomato 65 φυτών. Τα πιο ενθαρρυντικά αποτελέσματα έδωσε το στέλεχος ΕΒ4Β απουσία του FORL αυξάνοντας το μήκος της ρίζας κατά 326,60% και το ξηρό βάρος κατά 142,70% σε σύγκριση με τους μάρτυρες. Πα- ρουσία του FORL, το στέλεχος EB4B βελτίωσε τόσο το μήκος της ρίζας (180,81%) όσο και το ξηρό βά- ρος (202,15%) των φυτών της τομάτας. Τα αποτελέσματα ενισχύουν την περαιτέρω μελέτη της παρα- τηρούμενης ευεργετικής επίδρασης του Enterobacter sp. EB4B για πιθανή χρήση του ως βιοδιεργερτι- κό παράγοντα και παράλληλα παράγοντα βιολογικής καταπολέμησης του FORL. Hellenic Plant Protection Journal 13: 54-65, 2020 © Benaki Phytopathological Institute
Hellenic Plant Protection Journal 13: 66-77, 2020
DOI 10.2478/hppj-2020-0007
Effect of the olive fruit size on the parasitism rates of Bactocera
oleae (Diptera: Tephritidae) by the figitid wasp Aganaspis
daci (Hymenoptera: Figitidae), and first field releases of adult
parasitoids in olive grove
C.A. Moraiti1, G.A. Kyritsis1 and N.T. Papadopoulos1*
Summary The olive fruit fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the major pest of olives
worldwide. The figitid wasp, Aganaspis daci (Hymenoptera: Figitidae), is a larval-prepupal endopara-
sitoid of fruit fly species, and it was found to successfully parasitize medfly larvae in field-infested figs
in Greece. To assess the potential of A. daci as a biological control agent against B. oleae, we studied
the effect of olive fruit size on parasitism rates of A. daci on 2nd and 3rd instar larvae of B. oleae, by us-
ing fruit of different size (cultivar ‘Chalkidikis’) and wild olive fruit. In addition, we conducted releas-
es of A. daci females in a pilot olive grove in Volos, Magnesia. From July to October, we released 200
A. daci females/0.1 ha/week, followed by olive fruit sampling to estimate olive fruit infestation levels
and the parasitism rates of A. daci. Laboratory trials revealed that fruit size and larvae instar were pre-
dictors of parasitism success of A. daci, with parasitism rates higher for small-size fruit of the cultivar
“Chalkidikis” and the 3rd instar larvae of B. oleae. In field trials, no A. daci adults emerged from the ol-
ive fly infested fruit.
Additional keywords: biological control, larval instar, parasitism, olive fruit fly
Introduction premature fruit drop and reduces fruit and
olive oil quantity and quality (Manousis and
The olive fruit fly Bactrocera oleae (Ros- Moore, 1987). Current management strat-
si) (Diptera: Tephritidae) is a monophagous egies for olive fruit fly populations rely pri-
species, of sub-Saharan African origin, that marily on insecticide application either
attacks fruits of African and Asian wild olive, through cover sprays or bait sprays that tar-
O. europaea ssp. cuspidate, and the commer- get adult fly. Resistance of B. oleae to insec-
cial olives O. europaea ssp. europaea (includ- ticides has been documented both for olive
ing wild olive fruit or naturalized ‘oleast- fly populations from Greece (Kampouraki et
ers’) (Hoelmer et al., 2011, Tzanakakis, 2006). al., 2018) and California (Kakani et al., 2010).
Its presence has been well documented in Thus, B. oleae poses a serious threat to the
the Mediterranean Basin, South and Central olive industry worldwide and its control is
Africa, Middle East and India, and it has re- challenging (Daane and Johnson, 2010).
cently invaded California and northwestern Over nearly 100 years, considerable ef-
Mexico (Nardi et al., 2010). The olive fruit fly forts have been made to manage the ol-
usually lays one or more eggs just below the ive fruit fly in southern Europe by introduc-
surface of the fruit, and larvae form galler- ing primarily North African populations of
ies while feeding on the pulp of the fruit. Be- Psyttalia (Opius) concolor (Szepligeti) (Hy-
sides secondary infestation from both bacte- menoptera: Braconidae), a koinobiont en-
ria and fungi, olive fruit fly infestation causes doparasitoid of the second and third larval
instar of the Mediterranean fruit fly (med-
fly), Ceratitis capitata, and the olive fly (Daane
1
Laboratory of Entomology and Agricultural Zoology, and Johnson, 2010). In Greece, initial trials in
Department of Agriculture, Crop Production and Ru- the island of Chalki gave promising results
ral Environment of Thessaly, Fytokou St., GR-384 46 N.
Ionia Volou, Magnesia, Greece. during the first year of release (18-37% par-
* Corresponding author: nikopap@uth.gr asitism rates) but parasitism rates declined
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