Biological Control 104 (2017) 28–34

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Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Evaluation of biological control of Erwinia amylovora, causal agent

of fire blight disease of pear by antagonistic bacteria
Mohamad Sharifazizi, Behrouz Harighi ⇑, Amin Sadeghi
Department of Plant Protection, College of Agriculture, University of Kurdistan, Sanandaj, Iran

h i g h l i g h t s

 Pseudomonas and Pantoea strains showed potential biocontrol activity against fire blight on detached plant organs.
 We investigate the biocontrol potential of Pantoea agglomerans strains against Erwinia amylovora.
 Antibiotic production of Pseudomonas strains were studied under laboratory conditions.

a r t i c l e i n f o a b s t r a c t

Article history: Fire blight caused by Erwinia amylovora is one of the most important diseases of pear and apple trees in
Received 4 May 2016 Kurdistan province, Iran. To develop an effective biocontrol method against the pathogen, a total of 22
Revised 27 September 2016 bacteria were isolated from above parts of pear trees and screened for in vitro antagonistic activity.
Accepted 24 October 2016
Ten isolates inhibited the growth of the pathogen were identified by biochemical tests and partial
Available online 25 October 2016
sequencing of 16s rRNA or recA genes as Pantoea agglomerans (Pa9, Pa10, Pa21), fluorescent
Pseudomonas sp. (Ps117, Ps170, Ps49, Ps89), Enterobacter sp. (En23, En113) and Serratia sp. (Se111).
Keywords:
These strains were selected for biological control efficacy on immature fruits, detached flowers and leaves
Erwinia amylovora
Fire blight of pear
under laboratory conditions. All antagonists were able to reduce the disease severity on fruit and flowers.
Biocontrol On immature fruits assay, isolates Pa21 and En23 with 83% and 25%, respectively had the highest and
Pseudomonas sp. lowest effects on disease incidence compared to the control. On flowers, isolates Ps170 with 92% and
Enterobacter sp. En23, Ps89 and Se111 with 25% reduction of infection, respectively, had the highest and lowest effects
under condition tested. Among strains tested, Ps170 produced amplified fragments corresponding to
biosynthesis genes to produce antibiotics PCA, DAPG, Pyrrolnitrin and Pyoluteorin. Based on results
obtained in this study, Ps170, Ps117, En113 and Pa21 strains have potential to be used for fire blight con-
trol, although their efficacy needs to be evaluating under natural field conditions.
Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction all commercial pear cultivars are susceptible to E. amylovora (Van

der Zwet and Beer, 1995). Effective control of fire blight can be
Pyrus with twelve species is one of the most important fruits achieved through using of the streptomycin and oxytetracycline
growing in temperate regions of Iran. Among species, Pyrus com- but this approach has a risk of pathogenic strains resistant devel-
munis known as common pear had cultured at about 18,000 hec- opment (McManus et al., 2002). Chemical control by application
tares (Ministry of Agriculture-Jahad of Iran, 2014). Fire blight of copper-based bactericides is effective but it has possible risk
disease caused by Erwinia amylovora is a destructive disease of of phytotoxicity and pathogen resistance development (Sholberg
pear, apple and other rosaceous plant species and has been world- et al., 2001). The use of chemical inducer of systemic acquired
wide distribution. In Iran, fire blight was first reported in Karaj resistance such as Acibenzolar-S-methyl has moderate protection
region (Zakeri and Sharifnabi, 1991). Since then, it has been wide- effect from the pathogen (Brisset et al., 2000; Sparla et al., 2004).
spread and is one of the most important diseases of pear and Due to the high risk of phytotoxicity and resistance of the causal
apple trees in several parts of Iran including Kurdistan province agent to chemical and antibiotics, biological control method is of
(Mollaei and Harighi, 2013; Rahnama and Mazarei, 2002). Nearly particular alternative (Vanneste et al., 2004). Various bacteria
such as Bacillus subtilis, Lactobacillus plantarum, Pseudomonas fluo-
⇑ Corresponding author. rescens, Pantoea agglomerans, Rahnella aquatilis, yeasts and plant
E-mail address: [email protected] (B. Harighi). extracts have used as biocontrol agents against fire blight

http://dx.doi.org/10.1016/j.biocontrol.2016.10.007
1049-9644/Ó 2016 Elsevier Inc. All rights reserved.
 M. Sharifazizi et al. / Biological Control 104 (2017) 28–34 29

(Zeller, 2006; Rosello et al., 2013). Among them most numerous pear trees with fire blight symptoms in Kurdistan province was
studies have been made using P. agglomerans and P. fluorescens used in all experiments (Mollaei and Harighi, 2013).
strains (Kenneth and Stochwell, 2000). Some of them became
commercially available for fire blight management. P. fluorescens
A506, P. agglomerans E325, P. agglomerans P10c, P. vagans C9-1 2.2. Screening of potential antagonists against bacterial pathogen in
and B. subtilis QST713 formulated as BlightBan A506 (Johnson dual culture assay
and Stockwell, 1998), Bloomtime FD (Pusey et al., 2008), Blossom
Bless (Vanneste et al., 2002), BlightBan C9-1 (Johnson and Three hundred lL of bacterial pathogen suspension (about
Stockwell, 2000) and Serenade (Aldwinckle et al., 2002), respec- 2  108 CFU mL 1) was poured into the plates containing nutrient
tively have been registered as biological products for fire blight agar medium and maintained at room temperature for 5 min. After
control. Some experiments showed that reducing of disease inci- which, paper disc (about 10 mm) was immersed in each suspen-
dence by using antagonist is similar to treatment with antibiotic sion of antagonistic bacteria spectrophotometrically adjusted
(Johnson and Temple, 2013). Several mechanisms such as antibio- (OD600nm) to concentration of about 108 CFU mL 1 and was spotted
sis, nutrient competition and colonization have been proposed for at the pathogen-inoculated plates. The plates were incubated at
inhibition of E. amylovora and reduction of fire blight incidence. 27 °C for 48–72 h and the width of the inhibition zones was mea-
Production of various antibiotics including phenazine, pantocin sured. Sterile water spotted in the plates with the pathogen was
A and pantocin B is a common feature of P. agglomerans strains used as control.
with potential biocontrol activity for fire blight (Giddens et al.,
2002; Wright et al., 2001; Stockwell et al., 2002). The most com-
mon mode of actions of other bacterial antagonists such as Pseu-
domonas species are competition for space and nutrients 2.3. Antibiotic, protease, hydrogen cyanide and siderophore production
(Vanneste et al., 2004; Cabrefiga et al., 2007; Lindow and by antagonistic bacteria
Suslow, 2003). In a tritrophic system involving a plant host, bio-
control agent, and a pathogen all three components are subject to Screening of antibiotics by antagonistic bacteria was done as
environmental conditions. Therefore, develop new biological con- previously described (Vanneste et al., 1992). Suspension of bacteria
trol agents adopted to specific region and using it instead of com- were adjusted to concentration of about 108 CFU mL 1 and spotted
mercially available agents might be has a better biological control into plates containing NA medium. Plates were incubated at 28 °C
efficacy. In the case of fire blight, the efficacy of biological control for 48 h, then bacterial colonies were discarded from the plates
products available is generally low, variable and needs to be used using sterile cotton before exposure to chloroform vapor for 1 h.
in combination with antibiotics (Ngugi et al., 2011). Therefore, it Subsequently, 300 ll of pathogen suspension (about 2  108 -
is still a need to finding new strains with possible novel mecha- CFU mL 1) was poured into the plates. The plates were incubated
nisms of biological control action. The first aim of this study at 28 °C for 48–72 h and the width of the inhibition zones was
was to characterize epiphytic bacteria isolated from above parts measured. Sterile water spotted in the plates with the pathogen
of pear trees. The second objective was to evaluate the potential was used as control.
of bacterial isolates as antagonist of E. amylovora by testing both Bacterial isolates were tested for production of protease on
in vitro and in sito on detached plant organs. skim milk (SKM) agar medium according to method previously
described (Smibert and Krieg, 1994). The isolates were spot inocu-
lated into SKM medium and kept at 28 °C for 48 h. Clear halo zone
production around the bacterial colony was taken as evidence of
2. Materials and methods extracellular protease production. Production of Hydrogen cyanide
(HCN) by antagonistic bacteria was detected according to the
2.1. Isolation and identification of Bacterial antagonist method described by Alstrom and Burns (1989). Briefly, 100 lL of
bacterial suspension was spread on nutrient agar medium, sealed
The potential bacterial antagonists were isolated from the leaf with parafilm and incubated in an inverted position at 26–28 °C
and blossom surface of healthy pear trees from different locations with picric acid indicator papers (5% picric acid/2% Na2CO3 solu-
of Kurdistan province during May to September 2011 and 2012. tion) placed inside the lids. A color change of indicator from yellow
Samples were collected, placed in plastic bags and used immedi- to brown or reddish brown was recorded as HCN production. Side-
ately or kept in refrigerator for short period of time. Small tissue rophore production test was done using CAS agar assay according
pieces were macerated in 2–3 ml of sterile-distilled water for to a method previously described (Schwyn and Neilands, 1987).
30 min and the suspension was streaked onto nutrient agar (NA) The isolates were spot inoculated into CAS agar medium. Plates
(Merck) or King’s Medium B Agar (KMB) plates (King et al., were incubated at 27–28 °C, and positive results were recorded
1954). The plates were incubated at 26–28 °C for 48–72 h. Identifi- as a halo zone formation around the colonies.
cation of bacteria was done by phenotypic properties and partial
nucleotide sequencing of 16S rRNA using PCR with rP1/fD2 primers
(Weisburg et al., 1991) or recA genes (Waleron et al., 2002). The 2.4. Detection of biosynthetic gene targets for known antimicrobials
PCR products were sequenced using an ABI3730XL DNA sequencer
(Applied Biosystems) and the sequences were compared with NCBI The presence of gene targets for antibiotics phenazine-1-
nucleotide sequence database using blast program. Nucleotide carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) was
sequences were aligned using the ClustalW program (Ramu et al., screened by PCR using specific primers PCA2a/PCA3b and Phl2a/
2003) available in BioEdit Sequence Alignment Editor 7.0.9.0 soft- Phl2b, respectively, according to method described by
ware (Hall, 2011). Alignments were manually adjustment where Raaijmakers et al. (1997). Screening for pyoluteorin and pyolute-
necessary. Neighbor-joining phylogenetic analysis was performed orin biosynthetic loci were done by PCR using oligonucleotide pri-
on datasets by using MEGA version 6.06 (Tamura et al., 2013). A mers PrnCf/PrnCr and PltBf/PltBr, respectively. Cycling program
phylogenetic tree was constructed using the neighbor-joining was as described by Mavrodi et al. (2001). The amplification prod-
methods (bootstrap analysis with 1000 replicates was conducted) ucts were electrophoresed on 1% agarose gels in 1 TAE buffer at
with the same program. Erwinia amylovora originally isolated from 70 V, stained with 0.5 lg/mL ethidium bromide and photographed.
30 M. Sharifazizi et al. / Biological Control 104 (2017) 28–34

2.5. Evaluation of antagonistic activity of strains in detached pear fruits were placed in sterile desiccators and incubated at 27 °C.
organs Positive results were recorded when bacterial exudates or necrosis
were detected in the well. Assays on detached leaves and flowers
All experiments were conducted under controlled laboratory were done according to methods described by Rosello et al.
conditions on detached leaves, flowers and immature fruits to (2013). Detached leaves were surface-sterilized by 70% ethanol fol-
evaluation the effect of antagonistic bacteria against E. amylovora lowed by sterile water. Each leaves were wounded by a single inci-
infection. Inhibition of E. amylovora in immature pear fruits was sion transverse to the midrib, then leaves were treated by
tested as previously described (Vanneste et al., 1992). Immature immersion in the suspension of bacterial antagonist for 1 min. All
pear fruits were surface sterilized with 70% ethanol, then 50 lL leaves were maintained in a desiccator at 27 °C and after 24 h
of bacterial antagonist suspension (about 1  108 CFU mL 1) was wounds were inoculated with 10 lL of a suspension of E. amylovora
placed into about 5 mm deep well. When bacteria were absorbed (about 2  108 CFU mL 1). The area of leaf blight was measured
by the fruits, 50 ll of a suspension of E. amylovora strain with using area measurement system (Delta-T, light abox model, UK)
the same inoculums dose was introduced into the same well. The and compared with control after 7 days. Detached flowers were

Fig. 1. Phylogenetic tree of (a) partial 16S rRNA and (b) recA gene sequences of antagonistic bacteria along with the sequences from selected references strains. The analysis
was conducted with MEGA v. 6.06 using neighbor-joining method.
 M. Sharifazizi et al. / Biological Control 104 (2017) 28–34 31

rRNA gene sequence of Se111 was closely related to Serratia ply-

muthica. The recA gene sequences of Pa9, Pa10 and Pa21 strains
had homology with Pantoea agglomerans. Phylogenetic trees con-
structed using the partial 16S rRNA and recA gene sequences of
the putative bacterial isolates and representative type strain bacte-
ria of related taxa generated by neighbor-joining are showing in
Fig. 1a and b.

3.2. In vitro antagonistic activity of bacteria against E. amylovora

Among ten bacterial antagonists, Pseudomonas sp. Ps170 fol-

lowed by Enterobacter sp. En113 inhibit growth of the pathogen
significantly with mean inhibition diameter of 32 and 27 mm,
respectively. Enterobacter sp. En23 and Pseudomonas sp. Ps89 were
weak inhibitors of the pathogen, with mean inhibition diameter of
11 and 10 mm, respectively (Fig. 2).

3.3. Antibiotic, protease, hydrogen cyanide and siderophore production

by antagonistic bacteria
Fig. 2. Inhibition of growth of E. amylovora by antagonistic bacteria.

All strains were able to produce antibiotic. Among strains

maintained in sterile desiccators with the peduncle submerged in tested, Pseudomonas sp. Ps170, Pseudomonas sp. Ps117 and Pseu-
10% sucrose. Flowers were sprayed with suspension of bacterial domonas sp. Ps49 were produced siderophore in CAS agar medium.
antagonists using sterile sprayer, then placed in a desiccators at Also strains Pseudomonas sp. Ps170, Pseudomonas sp. Ps49 and
27 °C. After 24 h, flowers were inoculated with suspension of E. Pseudomonas sp. Ps89 produced a clear halo zone on SKM medium,
amylovora (about 2  108 CFU mL 1) and symptoms were recorded indicating extracellular protease production. Among strains tested
daily up to 5 days. The experimental design consisted of three for Hydrogen cyanide production, Pseudomonas sp. Ps170 and Pseu-
(leaves), five (flowers) and six (fruits) replications per treatment domonas sp. Ps117 revealed a positive reaction (Table 1).
with two independent experiments. In all treatments plant materi-
als inoculated with sterile water or bacterial pathogen were 3.4. Detection of antibiotics produced by antagonistic bacteria
included as negative and positive control, respectively. The inci-
dence of infection was calculated as percentage of infected plant The presence of biosynthesis genes in antagonistic bacteria are
organs. Statistical analysis for all experiments was carried out with summarized in Table 2. Primers PCA2a/PCA2b and primers Phl2a/
a factorial arrangement in a completely randomized design. Data Phl2b amplified the predicted 1150-bp and 745-bp fragments,
analysis was performed using the SPSS software program and com- respectively from DNA of strains Pseudomonas sp. Ps117 and Pseu-
parison of means was done with Duncan test at 5% level. domonas sp. Ps170 indicating the presence of biosynthetic genes
for production of antibiotics phenazine-1-carboxylic acid and 2,4-
diacetylphloroglucinol. Strains Enterobacter sp. En113, Pseu-
3. Results domonas sp. Ps117, Pseudomonas sp. Ps170, Pseudomonas sp. Ps49
and Pseudomonas sp. Ps89 were able to produce expected DNA
3.1. Identification of potential antagonistic bacteria

A total of 22 bacteria were isolated from the leaf surface of pear Table 2
trees. During preliminary screening of strains, 10 with inhibiting Presence of biosynthesis genes for production of Prn, Phl, PCA and Plt antibiotics.
effects of the pathogen in vitro were selected, six strains were fur- Strain Prn Phl PCA Plt
ther identified by partial sequencing of 16s rRNA and recA genes. Enterobacter sp. En113 + +
Based on results obtained, nucleotide sequencing of the 16S rRNA Pseudomonas sp. Ps117 + + +
and recA genes from all isolates possessed 98–99% similarity with a Pseudomonas sp. Ps170 + + + +
species already described in the GenBank database. Sequence anal- Pseudomonas sp. Ps49 +
Pseudomonas sp. Ps89 + +
ysis of nearly full length of 16S rRNA gene indicates that strain
Serratia sp. Se111 +
En113 and En23 have homology with Enterobacter sp. The 16S

Table 1
Production of antibiotics, protease, hydrogen cyanide and siderophore by antagonistic bacteria.

Strain Protease HCN Antibiotic Siderophore

production production production production
Enterobacter sp. En113 +
Pseudomonas sp. Ps117 + + +
Pseudomonas sp. Ps170 + + + +
Pseudomonas sp. Ps49 + + +
Pseudomonas sp. Ps89 + +
Serratia sp. Se111 +
Pantoea agglomerans Pa9 +
Pantoea agglomerans Pa10 +
Pantoea agglomerans Pa21 +
Enterobacter sp. En23 +
32 M. Sharifazizi et al. / Biological Control 104 (2017) 28–34

amplified fragment of about 791-bp by PCR using primers PltBf/

PltBr confirming the presence of gene corresponding to pyolute-
orin production. Also DNA from strains Enterobacter sp. En113,
Pseudomonas sp. Ps170, Pseudomonas sp. Ps89 and Serratia sp.
Se111 were produced predicted 719-bp DNA amplified by using
primers PrnCf/PrnCr corresponding gene to production of
pyrrolnitrin.

3.5. Efficacy of bacterial antagonists against E. amylovora infection on

detached organs

E. amylovora-inoculated fruits, leaves and flowers treated with

bacterial antagonists were compared with non-treated control.
Strains Pseudomonas sp. Ps170, Pseudomonas sp. Ps117, Enterobac-
ter sp. En113 and Pantoea agglomerans Pa21 with 77%, 76%, 73% and
71% reduction of leaf infection, respectively were the most effec- Fig. 5. Effect of bacterial antagonists against E. amylovora infection on detached
tive antagonists (Fig. 3). Strain Pseudomonas sp. Ps170 with 92% immature fruits.
biocontrol efficacy against E. amylovora in pear detached flowers
was the most effective antagonist followed by Enterobacter sp. effects than other strains (Fig. 5). In most of the experiments there
En113, Pseudomonas sp. Ps117 and Pantoea agglomerans Pa21 were statistically significant differences between En113, Pseu-
(Fig. 4). Also strain Pantoea agglomerans Pa21 with 83% reduction domonas sp. Ps117, Pseudomonas sp. Ps170 and Pantoea agglomer-
of immature fruit infection showed the highest biocontrol activity ans Pa21 with the other strains tested.
against bacterial pathogen, after which Enterobacter sp. En113 and
Pseudomonas sp. Ps170 with 75% reduction of infection had higher
4. Discussion

In this study, we investigated the inhibition effects of bacterial

antagonists against E. amylovora, the causal agent of fire blight dis-
ease of pear tree under in vitro and in sito conditions. The effective-
ness of strains in the inhibition of infection was determined in
different plant organs. Among bacterial strains isolated from
healthy organs of plant, eleven strains belonging to genera Pantoea,
Pseudomonas, Serratia and Enterobacter had shown potential antag-
onistic activity against E. amylovora. There was a direct relation-
ship between in vitro and in sito experiment results. Strains
Pseudomonas sp. Ps170, Pseudomonas sp. Ps117, Enterobacter sp.
En113 and Pantoea agglomerans Pa21 showed the highest
inhibition effects on pathogen in vitro and the highest reduction
of infection in plant organs in in sito experiments. There are several
reports about Pseudomonas spp. as effective antagonists of E. amy-
lovora (Pusey et al., 2009; Stockwell and Stack, 2007). Pseudomonas
fluorescens strain A506 was the first commercially antagonist for
Fig. 3. Effect of bacterial antagonists against E. amylovora infection on detached fire blight management under the name Blight Ban A506 (Zeller,
leaf.
2006). The mechanism of action of Pseudomonas spp. as biocontrol
agents against E. amylovora was proposed as competition or
antibiosis (Cabrefiga et al., 2007; Wilson and Lindow, 1993;
Temple et al., 2004). Several strains of fluorescent Pseudomonas
spp. with their ability to produce antibiotics and to be effective
antagonists against plant pathogens have been reported. However,
majority of such bacteria were isolated from soil and are active
against soilborne plant pathogens (Mousa and Raizada, 2015).
Results presented in this study showed various strains were able
to produce different antibiotics. The results were confirmed by
using specific primers corresponding to phenazine-1-carboxylic
acid (PCA), 2,4-diacetylphloroglucinol (Phl), pyoluteorin (Plt) and
pyrrolnitrin (Prn) antibiotics. Pseudomonas sp. Ps170 strain was
able to produce all above antibiotics, had the highest inhibition
effect and reduction of infection in vitro and in sito, respectively.
Pseudomonas sp. Ps117 strain with significant efficacy against bac-
terial pathogen was able to produce PCA, Phl and Plt antibiotics.
Pseudomonas sp. Ps170 strain produced HCN, protease and sidero-
phore and Pseudomonas sp. Ps117 strain produced HCN and sidero-
phore in in vitro experiments. These results showed that both
Fig. 4. Effect of bacterial antagonists against E. amylovora infection on detached strains were able to use different mechanisms such as antibiosis
flowers. and competition against bacterial pathogen. Pseudomonas sp.
 M. Sharifazizi et al. / Biological Control 104 (2017) 28–34 33

Ps49 and Pseudomonas sp. Ps89 showed lower antagonistic efficacy Ps170 strain with 92% biocontrol efficacy in pear detached flower
against E. amylovora than Pseudomonas sp. Ps170 and Pseudomonas assay is the most effective strain. Compared to this, Cabrefiga
sp. Ps117. The main difference of Pseudomonas sp. Ps170 and Pseu- et al. (2007) had reported reduction of infection by 60.4% (Confer-
domonas sp. Ps117 with Pseudomonas sp. Ps49 and Pseudomonas sp. ence cultivar) and 84.1% (Doyenne du Comice cultivar) when using
Ps89 was the lack of required genes for the synthesis of PCA antibi- P. fluorescens EPS62e in detached flowers assay under laboratory
otic in later strains. Compared to our results, Cabrefiga et al. (2007) conditions.
have been reported that P. fluorescens strain EPS62e does not carry In summary, this study provided information relating identifi-
the corresponding PCA, Phl and Prn genes. Production of sidero- cation of bacteria isolated from the leaf and blossom surfaces of
phore and differences in growth potential were mechanisms of pear trees. In vitro and in sito assays show that Pseudomonas sp.
antagonistic activity of EPS62e strain against E. amylovora. The Ps170, Pseudomonas sp. Ps117, P. agglomerans Pa21 and Enterobac-
mean reduction in the percentage of infection on detached organs ter sp. En113 strains are suitable candidates for development as
observed in experiments using P. agglomerans Pa9, Pa10 and Pa21 biological control agents of fire blight disease. However, their effi-
was lower than that experiments using Pseudomonas sp. Ps117 and cacy needs to be evaluating in orchards and under different agri-
Pseudomonas sp. Ps170. Among the P. agglomerans strains tested, P. cultural conditions. Blossoms of host plants are the primary sites
agglomerans Pa21 has the highest inhibition effect and reduction of where the causal agent of fire blight becomes established and ini-
infection both in in vitro and in sito experiments. P. agglomerans tiates infection. Development of formula based on bacterial antag-
Pa9, P. agglomerans Pa10 and P. agglomerans Pa21 did not produce onists identify in this study to improvement of their fitness and
siderophore, HCN or protease, whereas it produced antibiotic enhancement their biocontrol efficacy on blossoms under natural
in vitro. All that suggests the main mechanism of these antagonistic environment might be future study.
bacteria against E. amylovora is antibiosis. These results are in
agreement with previous studies about the role of antibiotic pro- Acknowledgment
duction by various strains of P. agglomerans that inhibit the growth
of E amylovora in culture, and also antibiosis as an important This work was supported by University of Kurdistan, Iran.
mechanism of biocontrol in orchards (Stockwell et al., 2002;
Vanneste et al., 1992; Wright et al., 2001). There are several reports
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