BioControl (2019) 64:723–735

https://doi.org/10.1007/s10526-019-09963-z (0123456789().,-volV)
(0123456789().,-volV)

Screening of endophytic bacteria isolated from domesticated

and wild growing grapevines as potential biological control
agents against crown gall disease
Susan Asghari . Behrouz Harighi . Ali Akbar Mozafari . Qassim Esmaeel .
Essaid Ait Barka

Received: 25 February 2019 / Accepted: 9 August 2019 / Published online: 16 August 2019
Ó International Organization for Biological Control (IOBC) 2019

Abstract Crown gall caused by Agrobacterium under in vitro condition. Among them, Pseudomonas
tumefaciens and Allorhizobium vitis is an important sp. Sn48, Pseudomonas sp. Ba35 and Pantoea sp.
disease of grapevine worldwide. Both pathogens were Sa14 strains significantly reduced the size of galls in
isolated from infected grapevines cultivated in Iran grapevine plantlets. To the best of our knowledge, this
and identified by 16S rRNA sequence analysis. A total study is the first report on the diversity of endophytic
of 314 endophytic bacteria were isolated from the root, bacteria of grapevines with biological control poten-
leaves, shoots and fruits of domestic and wild growing tial against crown gall disease in Iran.
grapevines collected from various locations of Kur-
distan and West Azerbaijan Provinces. Selected Keywords Agrobacterium tumefaciens 
strains further identified by nucleotide sequencing of Allorhizobium vitis  Biological control  Endophytic
the 16S rRNA gene belonged to known genera bacteria  Grapevine
including Pseudomonas, Erwinia, Pantoea, Serratia,
Enterobacter and Micrococcus. These strains were
screened for their antagonistic effects on both
pathogens in vitro as well as their ability to reduce Introduction
gall formation in planta. In total, 17 strains were able
to prevent the growth of both or one of the pathogens Grapevine (Vitis vinifera L.) is one of the most
economically important fruit crops, covering more
than 7.5 million hectares worldwide. The domestica-
Handling Editor: Jane Debode tion of V. vinifera began ca. 6000–8000 BC in the
Caucasus mountains between the Black Sea and
S. Asghari  B. Harighi (&)
Department of Plant Protection, Faculty of Agriculture, Caspian Sea (Soneji and Nageswara-Rao 2011), close
University of Kurdistan, Sanandaj, Iran to the north-western parts of Iran. Even today, wild
e-mail: [email protected] growing grapevines are present in some parts of the
north-western areas. Because of suitable geographic
A. A. Mozafari
Department of Horticultural Sciences, Faculty of conditions, Iran is one of top ten grape-producing
Agriculture, University of Kurdistan, Sanandaj, Iran countries of the world and approximately 207 thou-
sand hectares of lands are planted. Total production is
Q. Esmaeel  E. Ait Barka
estimated at over two million tons (Anonymous 2016).
Unite de Recherche Vignes et Vins de Champagne- EA
4707, SFR Condorcet, Universite de Reims Champagne- Microorganisms are naturally presented in the rhizo-
Ardenne, Reims, France sphere associated with plants. Endophytes are referred to

123
724 S. Asghari et al.

those which colonize the internal tissues of host plants including Rahnella aquatilis HX2 (Chen et al. 2007),
including the roots, stems, leaves, flowers and fruits. Serratia plymuthica IC1270 and Pseudomonas fluo-
Endophytic communities and their plant hosts strongly rescens Q8r1-96 (Dandurishvili et al. 2010) have
interact with each other, and as a result, endophytes are various inhibition effects under in vitro conditions.
involved in most aspects of plant adaptation to biotic and The causal agents of grape crown gall disease can
abiotic stresses (Hardoim et al. 2015). In grapevine, the survive systemically in grapevines and are dissemi-
diversity and ecology of the bacterial endophytes have nated in propagation tissues (Kaewnum et al. 2013).
been extensively investigated. Several recent researches Therefore, using endophytic bacteria might be an
on bacterial endophytes of grapevine have included alternative candidate for biological control of disease.
identification of nonculturable and/or culturable strains Furthermore, endophytic bacteria isolated from grape-
such as Bacillus, Pseudomonas, Enterobacter, Pantoea, vine might be better adapted to this plant and thus
Curtobacterium, Streptomyces, Staphylococcus, Mi- create greater impact on disease control. Bell et al.
crobacterium, Paenibacillus, Stenotrophomonas and (1995) identified several xylem-inhabiting bacteria of
Variovorax (Bell et al. 1995; Baldan et al. 2014; Bulgari grape cultivars with inhibitory effects against A. vitis.
et al. 2009; West et al. 2010; Compant et al. 2011). Two endophytic bacteria isolated from grapevine and
Furthermore, comparison of endophytic bacteria within belonging to the genus Curtobacterium demonstrated
various plant parts (Compant et al. 2011) and examining strong effects in reducing crown gall development
endophytic bacterial diversity in healthy compared to (Ferrigo et al. 2017). In addition, several endophytic
diseased grapevines have been investigated (Bulgari bacteria isolated from Malus domestica significantly
et al. 2011; Faist et al. 2016). Moreover, some studies reduced the population of A. vitis and gall size
have been focused on diversity in endophytic bacterial formation under greenhouse conditions (Eastwell
population of wild and domesticated grapevine (Camp- et al. 2006).
isano et al. 2015) and on the influence of organic The present study aimed to isolation and identifi-
production and integrated pest management on the cation of the causal agents of crown gall. Other
endophytic bacterial communities (Campisano et al. objective was to screening, identification and charac-
2014). Several studies have demonstrated the plant terization of endophytic bacteria isolated from the
growth promotion effects of endophytic bacteria isolated healthy domesticated and wild growing grapevine.
from grapevines (Andreolli et al. 2016; Campisano et al. Additionally, selected endophytic bacterial strains
2015). were investigated to their effects as biocontrol agents
Most endophytic bacteria colonize the same eco- for control crown gall on the grapevine.
logical niche in plants as plant pathogens and have
various mechanisms of biocontrol activity such as
competition for place or substrate, production of Materials and methods
antibacterial substances and induction of systemic
resistance in the host plants against pathogens (Com- Isolation of Agrobacterium tumefaciens
pant et al. 2005). Grapevine plants are highly suscep- and Allorhizobium vitis
tible to a large variety of phytopathogenic bacteria,
amongst which is crown gall caused by Agrobacterium Infected cultivated grapevines (Vitis vinifera L.) with
tumefaciens and Allorhizobium vitis resulting in severe symptoms similar to crown gall including irregular,
damage to vineyards, particularly in cold climatic whitish and soft gall on stems were collected from
regions. Biological control of crown gall on some vineyards in Kurdistan Province, western Iran during
plants such as stone fruit trees has been successfully June, 2015. Galls were immersed in 0.5% sodium
conducted using Rhizobium radiobacter K84 (Kerr hypochlorite solution for 3 min, and rinsed three times
1980). This strain is not effective against grapevine in sterile distilled water. Each sterilized piece was
crown gall and thus other biological control agents macerated in 3–5 ml sterile water, kept for 1 h and the
such as A. vitis E26 (Liang et al. 1990),VAR03-1 resulting suspension was streaked onto Roy and Sasser
(Kawaguchi et al. 2008) and A. vitis F2/5 (Kaewnum (RS) medium (Roy and Sasser 1983). The plates were
et al. 2013) have been investigated. A number of incubated at 26–28 °C for five days and the presump-
studies have indicated that other bacterial strains, tive colonies were further purified by streaking on the

123
Screening of endophytic bacteria isolated from domesticated and wild 725

same medium. Isolates were grown in Luria–Bertani and watered twice a week. After that, 20 ll of the
(LB) liquid medium overnight. Sterile glycerol was bacterial suspension prepared in sterile water (density
added to the final concentration of 15% and stored at of about 1 9 108 CFU ml-1) was inoculated into the
- 80 °C. stem of 4–6 leaf stage-growth of tomato plants with a
sterile syringe. Each isolate was tested on three
Molecular identification of Agrobacterium separate tomato plants. Plants were incubated in a
tumefaciens and Allorhizobium vitis greenhouse (95% RH, 25–26 °C, 16:8 L:D photope-
riod) and gall formation was recorded up to 30 days.
Genomic DNA of bacterial isolates was extracted Plantlets of Vitis vinifera cv. Chardonnay (clone 7535)
from overnight grown cultured in LB medium. Briefly, were micro-propagated by nodal explants grown on
5 ml of bacterial suspension was treated by adding 15 ml of MM VIGNE medium supplemented with 1%
SDS/lysozyme followed by three times DNA extrac- sucrose in 25 mm culture tubes as described by Ait
tion with phenol/chloroform/isoamyl alcohol (25/24/1 Barka et al. (2006). Cultures were incubated in a
volume) and once with chloroform/isoamyl alcohol growth chamber under white fluorescent light (200
(24/1). DNA was precipitated with 0.1 vol 3 M sodium lmol m-2 s-1), with a 16:8 L:D photoperiod at a
acetate (pH 4.8) and 2 vol of absolute ethanol constant temperature of 26 °C up to four weeks. After
overnight at - 20 °C. Finally, the suspension was that, plantlets were transplanted into pots containing
centrifuged at 14,000 rpm for 5 min. DNA was soil. 20 ll of overnight growth of A. tumefaciens and
washed with 70% ethanol, dried and resuspended in A. vitis (adjusted to concentration of approximately
50 ll of TE (Tris–Cl 0.1 M, EDTA 0.01 M, pH 8) 108 CFU ml-1) was inoculated into stems (between
buffer. Isolates were identified by partial nucleotide third and fourth internodes). Each isolate was tested on
sequencing of the 16S rRNA gene by PCR using two three separate grapevine plantlets. Plantlets were
universal primers, fD2 (50 -AGA GTT TGA TCA TGG incubated in a greenhouse as described above and
CTC AG-30 , position 8-27) and rP1 [50 -ACG GTT gall formation was recorded up to 30 days. Plants
ACC TTG TTA CGA CTT-30 , position 1512-1492 inoculated with sterile water were used as a control.
(Escherichia coli)] following the method described by
Weisburg et al. (1991). The PCR products were Sample collection, isolation and identification
sequenced using an ABI3730XL DNA sequencer of endophytic bacteria
(Applied Biosystems, Foster City, CA. USA). The
16S rRNA sequences obtained were aligned and Healthy roots, shoots, leaves and fruits of wild (Vitis
manually adjusted where necessary by using BioEdit vinifera subsp. silvestris) and domestic (V. vinifera cv.
Sequence Alignment Editor 7.0.9.0 software (Hall rasheh and V. vinifera cv. bidaneh sefid) growing
2011). 16S rRNA sequences obtained were further grapevines were collected from different locations of
subjected to BLAST analysis with other sequences Kurdistan and West Azerbaijan Provinces, June to
deposited in the NCBI database using the BlastN July 2015 and 2016. Samples were used immediately
program. The phylogenetic tree was constructed by the or kept in a refrigerator until used. Plant tissue samples
neighbor-joining method with Kimura’s two-parame- were washed with tap water, surface sterilized by
ter calculation model in MEGA version 6.0 (Tamura immersing in 3–5% sodium hypochlorite solution for
et al. 2013). Bootstrap analysis with 1000 replicates 3–5 min, based on different tissues and rinsed three
was conducted with the same program. times with sterile-H2O. Sterilized tissues were macer-
ated in 4 ml sterile-H2O and 50 ll of suspension was
Pathogenicity test inoculated onto nutrient agar and KB (King’s B)
media. The plates were incubated at 26–28 °C up to
The ability of bacterial isolates to induce gall produc- 14 days. Morphologically distinct colonies growing
tion was tested on tomato (Lycopersicon esculentum) on the media were selected and purified by subsequent
and grapevine (Vitis vinifera cv. chardonnay) plants. streaking onto fresh plates. To check the efficiency of
Seeds of tomato were sterilized and potted in pots sterilization, inoculation of 50 ll of the water from the
containing steam-sterilized soil (50% sand, 20% clay, final wash onto nutrient agar media was used as a
30% peat, pH 7.1). Pots were placed in the greenhouse negative control. Preliminary phenotypic properties of

123
726 S. Asghari et al.

all isolates such as Gram reaction, oxidase activity, CFU ml-1, respectively. Then, 300 ll suspensions
catalase production, arginine dihydrolase, phos- of bacterial pathogens were spread onto nutrient agar
phatase production, levan formation, potato rot and medium and maintained at room temperature for
fluorescent pigment production were investigated 5 min. Then, a sterile paper disc immersed in suspen-
according to the standard method (Schaad et al. sion of endophytic bacteria was placed in plates. The
2001). Selected isolates which showed growth inhibi- plates were kept at 26–28 °C for 72 h and the diameter
tion effects of one or both pathogens were further of the growth inhibition zone was calculated. The
identified by partial nucleotide sequencing of the 16S experiment was repeated twice with three replications.
rRNA gene as previously described for molecular
identification of bacterial pathogens. In planta biocontrol activity of endophytic bacteria

Biological control properties of endophytic The antagonistic activity of seven selected endophytic
bacteria bacteria against A. tumefaciens was evaluated in
planta. Bacterial cells of the bacterial pathogen and
All endophytic bacteria isolated were screened for endophytic bacteria grown for 24 h in LB medium at
their ability to produce protease, lipase and DNase 26–28 °C were suspended in 10 mM phosphate
according to the procedure of Sgroy et al. (2009), buffered saline (PBS) pH 7.0 to the concentration
Sierra (1957), Graham and Hodgkiss (1967), respec- of * 107 CFU ml-1, mixed and placed (10 ll) on
tively. Siderophore production was screened on CAS wound produced between the third and fourth intern-
medium using method previously described (Schwyn odes of grapevine plantlets. Inoculated plantlets were
and Neilands 1987). Ability of endophytic bacteria for incubated in a growth chamber under white fluores-
antibiotic production was investigated based on the cent light (200 lmol m-2 s-1), with a 16:8 L:D
method described by Wright et al. (2001). Overnight photoperiod at a constant temperature of 26 °C. Gall
growth of endophytic bacteria was spectrophotomet- size was measured with CellSense imaging software
rically adjusted to the density of approximately version 1.13. Inoculated plantlets with the pathogen
2 9 108 CFU ml-1 (OD600nm ^ 0.3), and a 20 ll only or sterile PBS buffer were used as positive and
was spotted directly onto yeast extract mannitol agar negative controls, respectively. The experiments were
(YMA) medium. Plates were incubated at 28 °C for carried out with four replications.
48 h, after which bacterial colonies were swept from
the medium using sterile cotton and the plates were Statistical analysis
treated with chloroform vapor for 1 h. Thereafter,
300 ll of A. tumefaciens and A. vitis strains (OD600nm- Data analysis was performed using the SPSS (version
^ 0.1 for bacterial concentration of about 1 9 108 22) program and the comparison of means was carried
CFU ml-1) were spread onto the medium. The plates out using the Student’s t test at 5% probability level for
were kept at 26–28 °C for 48 h and the diameter of the the biocontrol assays.
growth inhibition zones was calculated. Sterile water
spotted in the plates inoculated with the bacterial
pathogen was used as a control. The bioassay was Results
conducted with four replications.
Identification of bacterial pathogens
In vitro antagonistic activity of endophytic bacteria
against A. tumefaciens and A. vitis In total, eight infected plant samples were collected
from vineyards in Sanandaj and Ghorveh, Kurdistan
All endophytic bacterial isolates were investigated for Province. Two isolates were obtained from infected
their antagonistic activity against A. tumefaciens and grapevine tissues with symptoms similar to crown gall
A. vitis as per the method described by Aliye et al. disease. Isolates formed convex and slightly mucoid
(2008). Suspensions of endophytic bacteria and colonies on RS medium. Both isolates grew well and
pathogens were prepared to final concentration of produced mucoid colonies on YMA medium. Nearly
approximately 2 9 108 CFU ml-1 and 1 9 108 full length nucleotide sequencing of the 16S rRNA

123
Screening of endophytic bacteria isolated from domesticated and wild 727

gene revealed that both strains shared 100% and 99% rRNA gene sequences obtained in this study were
similarity to Agrobacterium tumefaciens and Allorhi- deposited in the GenBank database under the acces-
zobium vitis, respectively (Table 1). The phylogenetic sion numbers listed in Table 1. The nearly full length
tree constructed showed the position of strains Gh1 16S rRNA gene BLAST analysis revealed that strains
and Gh2 with high similarity to A. tumefaciens and A. had 99–100% similarity to six known genera including
vitis, respectively among the type strains of the genera Pseudomonas, Erwinia, Pantoea, Serratia, Enter-
Agrobacterium and Allorhizobium (Fig. 1). obacter from Proteobacteria and Micrococcus from
Pathogenicity test results confirmed that galls Actinobacteria (Table 1). Based on the sequencing
appeared 10–15 days after inoculation of Gh1 and results, phylogenetic trees were constructed showing
Gh2 strains into grapevine plantlets, respectively, and the position of strains within the type strains of the
ten days after inoculation of tomato plantlets. Sur- related species (Fig. 2a, b).
prisingly, the average areas of galls formed in both
grapevine and tomato inoculated-plantlets with Gh1 In vitro antagonistic potential of endophytic
was eight- to ten-fold higher than those produced by bacteria
Gh2 strain. Therefore Gh1 strain was used for in
planta assays. Approximately 53%, 70%, 25% and 86% of isolates
were able to produce protease, lipase, DNase and
Isolation and characterization of endophytic siderophore, respectively. Among them, seven isolates
bacteria were able to produce antibiotic against one or both
pathogens. Endophytic bacteria were assessed in vitro
Plant samples were collected from 32 different for their ability to inhibit the growth of A. tumefaciens
geographical locations in Kurdistan and West- and A. vitis. In total, 17 isolates were able to prevent
Azarbaijan Provinces. In total, 88 and 14 plant the growth of both or one of the pathogens (Table 2).
samples were collected from domesticated and wild The width of the inhibition zone ranged from 1 to
growing grapevines, respectively. To evaluate the 30 mm after 72 h which indicated as weak (?),
population of potentially endophytic bacteria, a total moderate (??) and strong (???) inhibition effect.
of 314 isolates were collected from the various parts of Micrococcus sp. Sv71 and Pseudomonas sp. Sn48
healthy domestic and wild growing grapevine plants. were more effective against A. tumefaciens Gh1 while
The lack of colony growth on final washes of sterilized Micrococcus sp. Sv71, Pseudomonas sp. Ba47, Pseu-
plant tissues as control confirmed that all isolates were domonas sp. Ba35, Pseudomonas sp. Ou17 and
endophyte. The majority of the bacterial isolates (269 Pseudomonas sp. Mi49 showed higher inhibition
isolates) were obtained from domestic grapevines and effects against A. vitis Gh2.
45 isolates were collected from wild growing plants.
253, 54, four and three isolates were recovered from In planta reduction of gall formation
the root, shoot, fruit and leaves, respectively. Prelim- by endophytic bacteria
inary characterization showed that 68% and 32% of
isolates were Gram-negative and Gram-positive, The biocontrol activities of seven selected endophytic
respectively. Initial identification based on biochem- bacteria against A. tumefaciens Gh1 were evaluated in
ical and physiological characteristics, such as Gram planta. Gall sizes of plantlets inoculated with endo-
reaction, oxidase reaction, catalase production, argi- phytic bacteria and pathogen were compared to
nine dihydrolase, phosphatase production, levan test, inoculated plantlets with the pathogen. Comparisons
potato soft rot and fluorescent pigment production on were done based on the average of gall size of four
KB medium revealed that isolates separated into six replications. Results of the statistical analysis indi-
and eight phenotypic groups at about 50% and 55% cated that three strains, Pseudomonas sp. Sn48
similarity level, respectively. (t = 50.11, df = 6, p \ 0.001), Pseudomonas sp.
Seventeen isolates which showed the antagonistic Ba35 (t = 66.26, df = 6, p \ 0.001) and Pantoea sp.
activity against one or both bacterial pathogens were Sa14 (t = 47.45, df = 6, p \ 0.001) were able to
selected for further molecular analysis using 16S significantly reduce the gall size to approximately
rRNA gene amplification and sequencing. The 16S 78%, 82% and 77%, respectively, compared to the

123
 Table 1 Preliminary identification of two pathogenic and 17 endophytic bacterial isolates by phenotypic properties and partial sequencing of the 16S rRNA gene
728

Strain Plant Geographic Gram Catalase Oxidase Fluorescent Arginine Phosphatase Levan Potato % Similarity (16S rRNA GenBank
tissue origin reaction dihydrolase soft sequence) accession

123
rot number

Gh1 S Ghorveh nd nd nd nd nd nd nd nd 100% with Agrobacterium MK114594

tumefaciens
Gh2 S Ghorveh nd nd nd nd nd nd nd nd 99% with Allorhizobium vitis MK114593
Ba35 R Armardeh- - ? ? ? ? - ? - 100% with Pseudomonas MK114598
Baneh kilonensis (AJ292426)
Sn48 R SalavatAbad- - ? ? ? ? - ? ? 100% with Pseudomonas MK114596
Sanandaj kilonensis (AJ292426)
Sa15 RW Banokhalaf- - ? - ? ? - - - 99% with Pseudomonas reactans MK114600
Sardasht (AF255337)
Ou17 F TazehKand- - ? - - ? - - - 99% with Pseudomonas reactans MK114599
Orumieh (AF255337)
Mi49 R YaghlanTapeh- - ? ? ? ? - ? ? 99% with Pseudomonas MK114595
Miandoab chlororaphis (DQ682655)
Ba47 RW Goil-Baneh - ? ? ? ? - ? - 99% with Pseudomonas MK114597
chlororaphis (DQ682655)
Ou81 F TazehKand- - ? ? ? ? ? ? ? 99% with Pseudomonas MK114606
Orumieh chlororaphis (DQ682655)
Ou22 R Ghotorlar- - ? ? ? ? - - - 99% with Pseudomonas koreensis MK114602
Orumieh (AF468452)
Ou65 S TazehKand- - ? - - ? - - - 99% with Erwinia aphidicola MK114615
Orumieh (AB681773)
Sv8 S Tefli-Sarvabad - ? - - ? - - - 100% with Erwinia aphidicola MK114612
(AB681773)
Sa14 SW Shalmash- - ? - - ? ? - ? 99% with Pantoea agglomerans MK114617
Sardasht (AJ233423)
Ou23 R Zeynaloo- - ? - - - - - - 99% with Enterobacter ludwigii MK114610
Orumieh (AJ853891)
Ou80 R Gharasanloo- - ? - - - - ? - 99% with Enterobacter ludwigii MK114611
Orumieh (AJ853891)
Mi10 R YaghlanTapeh- - ? - - ? ? - - 100% with Serratia ficaria MK114622
Miandoab (AJ233428)
Ou50 R Gharahasanloo- - ? - - ? - ? - 100% with Serratia ficaria MK114619
Orumieh (AJ233428)
Ba10 RW Goil-Baneh - ? - - ? - - - 100% with Serratia quinivorans MK114621
(AJ233435)
S. Asghari et al.
Screening of endophytic bacteria isolated from domesticated and wild 729

MK114616
control plantlets infected with A. tumefaciens Gh1
accession
GenBank
(Fig. 3).
number

99% with Micrococcus aloeverae

Discussion

Crown gall disease of grapevine caused by A. tume-

% Similarity (16S rRNA

faciens and A. vitis is widely distributed and has been

reported in different regions of Iran particularly in
areas with cold climates (Rouhrazi and Rahimian
(KF524364)

2012; Mafakheri et al. 2017). Bacterial pathogen

sequence)

survives systemically in symptomless grapevine and

can persist in root debris and in soil for several years
(Burr et al. 1995). Furthermore, physical treatment of
Potato

the propagation material does not guarantee complete

soft
rot

eradication of the systemic population of the pathogen

-

(Burr et al. 1996). Consequently, planting of clean

Levan

propagation material might result in crown gall

R root, W wild growing grapevine, F fruit, S stem, nd not determined, ? positive reaction, - negative reaction
-

incidence.
Phosphatase

Endophytic bacteria have the potential biocontrol

activity against pathogens. Due to adaptation of
endophytic bacteria to grapevine, such bacteria might
-

provide better effective biocontrol activity. With this

dihydrolase

hypothesis, endophytic bacteria were isolated from

Arginine

asymptomatic domesticated and wild growing grape-

vines. These bacteria were assayed for their activities
?

against A. vitis and A. tumefaciens. Bacteria belonging

Fluorescent

to the genera identified in this study had already been

identified as endophytic bacteria of grapevine (An-
dreolli et al. 2016; Baldan et al. 2014; Bulgari et al.
-

2009; Compant et al. 2011; Campisano et al. 2014;

Oxidase

West et al. 2010). However, it should be noted that the

endophytic community can be influenced by date of
-

sample collection, grapevine age, cultivar, plant

Catalase

physiological characteristics and type of culturing

methods. Therefore, bacteria reported in this research
?

might only be considered an endophytic bacterial

reaction

community under our experimental conditions.

Gram

The number of endophytic bacteria isolated from

?

the roots was higher than those isolated from the

Tefli-Sarvabad

shoots, fruits, and leaves. This might be the result of

Geographic

the close contact of roots with soil that makes it easy

for endophytic bacteria to enter the root tissues
origin

(Kobayashi and Palumbo 2000). Results obtained in

Table 1 continued

this study revealed that 86% of isolates were positive

tissue
Plant

for production of siderophore, a low molecular weight

R

iron-chelating compound. Endophytic bacteria with

Strain

siderophore production and secreting ability to the

Sv71

inside of plant tissues enhance the transport of Fe3?

123
730 S. Asghari et al.

Fig. 1 Phylogenetic tree of partial 16S rRNA gene sequences with Kimura’s two-parameter calculation model in MEGA
showing position of Agrobacterium tumefaciens Gh1 and version 6.0. The scale bar represents the number of substitutions
Allorhizobium vitis Gh2 strains (showed in bold letters) in per site. Numbers at branching points indicate bootstrap value
addition to taxonomically similar selected references strains. derived from 1000 replicates
The analysis was conducted by the neighbor-joining method

inside the plant cell which contributes to plant growth pathogens. Previous reports have indicated the great
promotion (Saha et al. 2015). biocontrol activity of this bacterial species against
In this study, the interactions of all endophytic fungal plant pathogens (Bolwerk et al. 2003). Our
bacteria with A. tumefaciens Gh1 and A. vitis Gh2 finding indicated that all Pseudomonas strains except
were evaluated in vitro. Results of the pathogenicity Ou22, Ou81 and Sa15 were able to produce antibi-
tests revealed that gall size produced by A. tumefa- otics. Although, these three strains could not produce
ciens Gh1 was significantly larger than that incited by antibiotics, they had antagonistic activity against both
A. vitis Gh2 under our experimental conditions. pathogens which could be a result of an alternative
Therefore, it was decided to investigate the ability of mechanism. Two other strains namely, Sn48 and Ba35
the selected endophytic bacterial strains for reducing with high similarity to P. kilonensis were positive for
gall formation by using only A. tumefaciens Gh1 in protease and siderophore production and also showed
planta. Several endophytic strains belonging to Pseu- both in vitro and in planta inhibition effects against the
domonas inhibited the growth of both pathogens pathogen. This suggests that both strains are more
in vitro through production of antibiotics. The pro- aggressive colonizers of plant tissues mediated by
duction of various antibiotics by Pseudomonas species biofilm formation and attachment than the A. tumefa-
is well documented and likely to be the main ciens strain. This finding is in agreement with other
mechanism of biocontrol activity (Weller 2007). reports concerning biocontrol activity of P. kilonensis
Mi49, Ou81 and Ba47 strains which had higher 16S strain JX22 during interaction with fungal and bacte-
rRNA gene sequence similar to P. chlororaphis rial plant pathogens (Xu et al. 2014).
illustrated antagonistic activity against one or both

123
Screening of endophytic bacteria isolated from domesticated and wild 731

Fig. 2 Phylogenetic tree of partial 16S rRNA gene sequences The analysis was conducted by the neighbor-joining method
showing position of identified endophytic bacterial strains with Kimura’s two-parameter calculation model in MEGA
(showed in bold letters) along with the sequences from selected version 6.0. The scale bar represents the number of substitutions
references strains. a Erwinia, Pantoea, Enterobacter and per site. Numbers at branching points indicate bootstrap value
Serratia species, b Pseudomonas sp. and Micrococcus species. derived from 1000 replicates

Surprisingly, strain Sv71 which showed the highest Although there are no reports on the biocontrol
16S rRNA gene sequence similarity with Micrococcus activity of S. ficaria species, S. quinivorans BXF1
genus had the best inhibition effects against both and some other strains such as S. plymuthica, S.
pathogens in vitro. This antagonistic potential might marcescens and S. liquefaciens have been reported as
be a result of antibiotic production or other secondary biological control agents against bacterial and fungal
metabolites as previously reported for endophytic plant pathogens and plant parasitic nematodes. In the
actinobacteria (Dinesh et al. 2017). Several research majority of cases production of antibiotics and/or
studies have indicated that members of the genus extracellular enzymes are the main biocontrol mech-
Micrococcus are endophyte with plant growth promo- anisms (Saha et al. 2012; Nascimento et al. 2016).
tion potential (Prakash et al. 2014), but antagonistic The two strains of Ou65 and Sv8 formed subclade
effects of strains against bacterial plant pathogens with Erwinia aphidicola species. Both strains had
have not been reported so far. Although, strain Sv71 strong DNase production ability and could inhibit the
showed the highest in vitro inhibition effect against A. growth of A. vitis and A. tumefaciens, respectively. E.
tumefaciens it could not significantly reduce gall aphidicola has been reported as a seed born plant
formation in planta. pathogen of bean, isolated from the pea aphid and
Three strains, Ba10, Mi10 and Ou50 which showed detected as endophyte in seeds of hybrid maize
high 16S rRNA gene sequence similarity with Serratia (Nadarasah and Stavrinides 2011). Two other strains,
quinivorans and Serratia ficaria gene sequences Ou23 and Ou80, had high 16S rRNA gene sequence
available in the NCBI GenBank database, respec- similarity with Enterobacter ludwigii. Both strains had
tively, had in vitro biocontrol activity. These strains antagonistic effects against A. tumefaciens. Results of
could not produce antibiotics but were positive for the study conducted by Lopez-Fernandez et al. (2015)
lipase, protease, DNase and siderophore production. demonstrated that grapevine colonization by

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732 S. Asghari et al.

Table 2 In vitro antagonistic properties of 17 selected endophytic bacteria isolated from domesticated and wild growing grapevine
Strain Siderophore Lipase Protease DNase Antibiotic production Antagonistic activity
production (inhibition diameter, mm)a (inhibition diameter, mm)a
A. tumefaciens A. vitis A. tumefaciens A. vitis

Pseudomonas sp. Sa15 ? ? ? - - - ?? ?

Pseudomonas sp. Ou17 ? ? ? - ?? ?? ?? ??
Pseudomonas sp. Ou22 ? ? ? - - - ? -
Pseudomonas sp. Ba35 ? ? ? - ?? ? ?? ??
Pseudomonas sp. Sn48 ? - ? - ??? ?? ??? ?
Pseudomonas sp. Ba47 ? - ? - ??? ?? ? ??
Pseudomonas sp. Mi49 ? ? ? - ? - ? ??
Pseudomonas sp. Ou81 ? ? ? - - - - ?
Serratia sp. Mi10 ? ? ? ? - - ? -
Serratia sp. Ou50 ? ? ? ? - - ? -
Serratia sp. Ba10 ? ? ? ? - - ? -
Micrococcus sp. Sv71 ? - ? ? ?? ?? ??? ??
Erwinia sp. Ou65 ? - - ? - - - ?
Erwinia sp. Sv8 ? - - ? - - ? -
Enterobacter sp. Ou23 ? - ? - - - ? -
Enterobacter sp. Ou80 ? ? ? - - - ? -
Pantoea sp. Sa14 ? ? ? - - ?? - ?
? positive reaction, - negative reaction
a
Inhibitory activity was estimated by calculating the diameter (d) of the inhibition zone (mm) for each interaction. ? 0 \ d \ 9.99,
?? 10 \ d \ 19.99, ??? d [ 20

Fig. 3 Gall size reduction

on grapevine in vitro
plantlets infected by
Agrobacterium tumefaciens
(P) and plantlets infected by
P ? selected endophytic
bacterial strains.
Comparisons were done for
each strain separately. Data
represents the mean of four
replicates ± SE. Data was
analyzed by Student’s t test
(Asterisk indicates
significant differences at 5%
probability level)

endophytic E. ludwigii strain shifts secondary meta- endophytic bacteria from grapevine and its potential
bolism which are essential for its colonization and as biocontrol agent against Botrytis cinerea empha-
activation of the plant defense pathways. Strain Sa14 sized (Bulgari et al. 2009; Compant et al. 2011; Trotel-
showed similarity to Pantoea agglomerans species. Aziz et al. 2008). Although strain Sa14 did not show
This species has been previously isolated as in vitro inhibition effect against A. tumefaciens it

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Screening of endophytic bacteria isolated from domesticated and wild 733

significantly reduced the gall size in planta. This old grapevines of Vitis vinifera cv. Corvina and their
finding implies that antibiotic production is not the potential for plant growth promotion and phytopathogen
control. Microbiol Res 183:42–52
main biocontrol mechanism and other mechanisms Anonymous (2016) FAOSTAT, statistics, crops production.
such as production of secondary metabolites or http://www.fao.org/faostat/en/#data/QC/visualize
inducing of systemic resistance are responsible for Baldan E, Nigrisa S, Populinab F, Zottinia M, Squartini A,
its biocontrol activity. However, this hypothesis needs Baldana B (2014) Identification of culturable bacterial
endophyte community isolated from tissues of Vitis vini-
further investigation. fera ‘‘Glera’’. Plant Biosyst 148:508–516
Crown gall formation by the pathogen results in the Bell CR, Dickie GA, Harvey WLG, Chan JWYF (1995)
expression of a set of genes on T-DNA which are Endophytic bacteria in grapevine. Can J Microbiol
carried on bacterial Ti plasmid. During infection, 41:46–53
Bolwerk A, Lagopodi AL, Wijfjes AHM, Lamers GEM, Chin-
T-DNA is transferred to and integrated in the plant cell A-Woeng TFC, Lugtenberg BJJ, Bloemberg GV (2003)
chromosome (Chilton et al. 1977). All three endo- Interactions in the tomato rhizosphere of two Pseudomonas
phytic bacteria with ability to reduce gall formation in biocontrol strains with the phytopathogenic fungus
inoculated plantlets were negative for DNase produc- Fusarium oxysporum f. sp. radicis-lycopersici. Mol Plant
Microbe Interact 16:983–993
tion. This finding indicated that there is no direct Bulgari D, Casati P, Brusetti L, Quaglino F, Brasca M, Daf-
relationship between DNase production by endophytic fonchio D, Bianco PA (2009) Endophytic bacterial diver-
bacterial strains and their ability to reduce gall sity in grapevine (Vitis vinifera L.) leaves described by 16S
formation in planta. rRNA gene sequence analysis and length heterogeneity-
PCR. J Microbiol 47:393–401
In conclusion, this study demonstrated that domes- Bulgari D, Casati P, Crepaldi P, Daffonchio D, Quaglino F,
ticated and wild growing grapevine harbors highly Brusetti L, Bianco PA (2011) Restructuring of endophytic
diverse endophytic bacteria. Several strains such as bacterial communities in grapevine yellows-diseased and
Pantoea sp. Sa14, Pseudomonas sp. Ba35 and Pseu- recovered Vitis vinifera L. plants. Appl Environ Microbiol
77:5018–5022
domonas sp. Sn48 with antagonistic activity against Burr TJ, Reid CL, Yoshimura M, Momel EA, Bazzi C (1995)
crown gall agents might be good candidates for use as Survival and tumorigenicity of A. vitis in living and
biocontrol agents. decaying grape roots and canes in soil. Plant Dis
79:677–682
Acknowledgements We thank Dr. Ghader Mirzaghaderi Burr TJ, Reid CL, Spittstoesser DF, Yoshimura M (1996) Effect
(Faculty of Agriculture, University of Kurdistan) for statistical of heat treatments on grape bud mortality and survival of
analysis of data. This research work was supported by the Agrobacterium vitis in vitro and in dormant grape cutting.
University of Kurdistan, Iran. Am J Enol Vitic 47:119–123
Campisano A, Antonielli L, Pancher M, Yousaf S, Pindo M,
Compliance with ethical standards Pertot I (2014) Bacterial endophytic communities in the
grapevine depend on pest management. PLoS ONE
9(11):e112763
Conflict of interest The authors declare that they have no
Campisano A, Pancher M, Puopolo G, Puddu A, Lopez-Fer-
conflict of interest.
nandez S, Biagini B, Yousaf S, Pertot I (2015) Diversity in
endophyte populations reveals functional and taxonomic
Research involving human and animal rights This research
diversity between wild and domesticated grapevines. Am J
does not involved human participants and animals.
Enol Vitic 66:12–21
Chen F, Guo YB, Wang JH, Li JY, Wang HM (2007) Biological
control of grape crown gall by Rahnella aquatilis HX2.
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Wright SA, Zumoff CH, Schneider L, Beer SV (2001) Pantoea interaction with biological control agents, impact on the plant
agglomerans strain EH318 produces two antibiotics that defense responses.
inhibit Erwinia amylovora in vitro. Appl Environ Micro-
biol 67:284–292 Ali Akbar Mozafari is an associate professor at Faculty of
Xu J, Deng P, Showmaker KC, Wang H, Baird SM, Lu SE Agriculture, University of Kurdistan, Iran. His research is in
(2014) The pqqC gene is essential for antifungal activity of plant biotechnology and plant tissue culture.
Pseudomonas kilonensis JX22 against Fusarium oxyspo-
rum f.sp. lycopersici. FEMS Microbiol Lett 353:98–105 Qassim Esmaeel is postdoctoral fellow at Université de Reims
Champagne-Ardenne, Reims, France. His research is to
characterize beneficial microorganisms that stimulate plant
Susan Asghari is a PhD student at the Faculty of Agriculture, innate immunity or with antimicrobial activities.
University of Kurdistan, Iran. She is working in Jihad-e-
Agriculture Organization of the West Azarbaijan Province.
Essaı̈d Ait Barka is a professor at Université de Reims
Champagne-Ardenne, Reims, France. His research focuses in
Behrouz Harighi is an associate professor at Faculty of plant physiology responses to abiotic and biotic stresses,
Agriculture, University of Kurdistan, Iran. His research is in impact on the plant defense mechanisms.
molecular characterization of plant pathogenic bacteria and its

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