Physiological and Molecular Plant Pathology 116 (2021) 101692

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Physiological and Molecular Plant Pathology

journal homepage: www.elsevier.com/locate/pmpp

Isolation and identification of endophytic bacteria from potato tissues and

their effects as biological control agents against bacterial wilt
Kambiz Bahmani a, Nader Hasanzadeh a, *, Behrouz Harighi b, Alireza Marefat c
a
Department of Plant Protection, Faculty of Agricultural Sciences and Food Industries, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Department of Plant Protection, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran
c
Department of Plant Protection, Faculty of Agriculture, Razi University, Kermanshah, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Ralstonia solanacearum, as a causative agent of potato wilt disease, is one of the deadliest diseases worldwide, and
Ralstonia solanacearum also in the Kurdistan province, Iran. Given the importance of the disease, the aim of the present study was to
Potato isolate endophytic bacteria from potato plant tissues such as the tuber, root, stem, and leaf, to investigate their
Biological control
antagonistic effects on R. solanacearum. In this regard, 236 endophytic bacteria were isolated and screened in
Endophytic bacteria
16S rRNA
vitro. As a result, 31 isolates were found as potential antagonists against potato wilt pathogen. For phenotypic
Kurdistan and genotypic characterization, biochemical and pathogenicity tests as well as 16S rRNA gene sequence analyses
Iran were performed. 11 isolates as representatives of different taxa were selected for further investigations. These
were identified as: Bacillus pumilus Bp91, B. pumilus Bp1, B. pumilus Bp49, B. licheniformis Bl17, Paenibacillus
peoriae Pa86, Pseudomonas brassicacearum Psb101, P. brassicacearum Ps169, P. putida Ps52, Chryseobacterium
indologenes Ch54, C. lathyri Chl98, and Microbacterium phyllosphaerae Mi41. Notably, the maximum inhibitory
effects were observed by Pseudomonas brassicacearum Psb101, P. brassicacearum Ps169, Paenibacillus peoriae Pa86,
Pseudomonas putida Ps52, and Bacillus licheniformis Bl17, by forming 17.6, 17.4, 17.3, 15.5, 15.2 mm diameter
inhibition zones against R. solanacearum on nutrient agar medium, respectively. Based on the results of the
greenhouse test, all 11 selected isolates simultaneously reduced the disease by 27–55% and also significantly
increased plant growth.
Among these, the five strains Pseudomonas brassicacearum Psb101, P. brassicacearum Ps169, P. putida Ps52,
Paenibacillus peoriae Pa86, and Bacillus licheniformis Bl17 were introduced hereby as the most effective antago­
nists and growth enhancers under both laboratory and greenhouse conditions.

1. Introduction eucalyptus [3–6]. Also, in Iran, it is responsible for substantial economic

losses to potato (Solanum tuberosum) and tomato (Lycopersicon escu­
Annual estimations have suggested that about 22% of potato crops lentum) crops in different parts of the country [7]. Another ability of this
are destroyed by fungal, bacterial, and viral plant diseases as well as bacterium is leading to disease under different climate conditions such
plant pests, which is equivalent to an annual loss of 65 million tons [1]. as tropical, subtropical, warm temperate, and cool climates regions of
Among these, bacterial wilt/brown rot disease caused by the world [8,9]. Based on the host range, R. solanacearum can be divided
R. solanacearum (Smith) [2] was shown to lead to the most damages due into 5 races, and in terms of biochemical traits, it can be divided into 6
to various reasons including a wide range of host plants such as sola­ biovars. So, the race 3 is generally equivalent to biovar 2. In this regard,
naceous plants, leguminous plants, a few monocotyledons, trees, shrub the association among other races and some pathovars is unquestionable
hosts, and certain ecotypes of the Arabidopsis thaliana model plant [3]. [4,10].
Accordingly, these host plants include 54 plant families as well as over Potato (Solanum tuberosum L.) is known as the fourth most important
450 plant species. Moreover, some of the important economic hosts of food crop worldwide after wheat (Triticum aestivum), rice (Oryza sativa),
this pathogen are tomato, pepper, potato, tobacco, banana, eggplant, and maize (Zea mays) [1,10]. Moreover, it is one of the most important
cowpea, chili, peanut, ginger, cashew, groundnut, papaya, olive, and agricultural crops in Iran, with about 173,000 ha of agricultural land

* Corresponding author.
E-mail addresses: [email protected], [email protected] (N. Hasanzadeh).

https://doi.org/10.1016/j.pmpp.2021.101692
Received 19 December 2020; Received in revised form 12 July 2021; Accepted 20 July 2021
Available online 10 August 2021
0885-5765/© 2021 Elsevier Ltd. All rights reserved.
K. Bahmani et al. Physiological and Molecular Plant Pathology 116 (2021) 101692

annually allocated to potato cultivation [11]. Since it requires no special conditions.

conditions for growth and has long been considered as a major crop in
temperate or warmer regions [1], so it is one of the most widely used 2. Material and methods
plants and its annual rate of expansion in developing countries is esti­
mated as 2.8% [12]. Nowadays, this product is considered as a valuable 2.1. Isolation and confirmation of bacterial pathogen
food source by playing a strategic role for the human population [13], as
it is a high energy supply and quality protein, as well as having signif­ Potato plants with typical bacterial wilting symptoms were sampled
icant amounts of vitamins and minerals [10]. Also, it is one of the major and then transported to the laboratory. Afterward, the stem, root, and
products in the direct consumption and food industries [14]. It is note­ tuber samples were washed with tap water and surface sterilized with
worthy that in the 16th century, a variety of commercial varieties from 0.5% sodium hypochlorite solution for 30 s. These treated samples were
all over the world were imported to Europe from a limited number of then washed twice with distilled water and small fragments of the
potato clones from South America, and this has led to a low genetic base samples were macerated in 2–3 ml of distilled water. Subsequently, each
and limited resistance to plant diseases. Due to this reason, tuber func­ individual plant suspension was cultured on TZC medium [23]. By
tion and quality have ultimately reduced. passing three days from incubation at 26–28 ◦ C, typical colonies with
Some of the effective methods of disease control are as follows: crop fluidal texture and a pale red center were re-cultured on the same cul­
rotation, field sanitation, plant breeding, the use of bactericides and ture medium for purification. The phenotypic characteristics of the
application of resistant varieties, transgenic resistant plant, soil solari­ isolates were also determined in terms of the standard methods [24,25].
zation, soil amendments, the use of bio-fumigants, plant growth pro­ Pathogenicity tests were fulfilled by injecting the bacterial suspension
moting rhizobacteria, and the use of SAR inducers. Correspondingly, into the stem area near the crown [26] and through drenching the
each one of these methods has its own limitations [3,8,15]. Perhaps one bacterial suspension into the soil near the root [27]. The preliminary
of the reasons for the lack of effective control of disease is the existence identification of the most bacterial isolates were confirmed by PCR
of diversity in the strains of the pathogen, which in turn, can cause analysis using a species-specific 759/760 primer pair [28].
instability of resistance in several resistant cultivars in different coun­
tries [11]. The use of the chemical elicitor acibenzolar-S-methyl can 2.2. Isolation and identification of endophytic bacteria
reduce the incidence of bacterial wilt in some plants such as tomatoes in
moderately resistant cultivars; however, it was not effective on suscep­ Potato plants were sampled during several surveys performed on
tible cultivars [16]. In fact, chemical control of this disease is not effi­ potato fields in different parts of Kurdistan province, located in the west
cient and soil fumigation also has a little effect [17]. Therefore, other of Iran, from July to September 2016 & 2017. The samples were
alternative methods like biological control have been proposed, and transferred to the laboratory after placing in clean bags and the
several studies have been done [9,15]. Plant growth-promoting rhizo­ geographical characteristics of the sampling site were recorded. In order
bacteria (PGPR) as one of the most important biocontrol agents, are to isolate endophytic bacteria, different parts of plant organs including
present in the rhizosphere of many plants, which colonize the root leaf, stem, root, and tuber were washed with distilled water. Disinfection
surface. These bacteria alone or in mixture have their potentials because followed by using 2% sodium hypochlorite for 3–4 min and 70% ethanol
they are naturally occurring and an environmentally friendly ap­ alcohol for 1 min. The plant samples were then rinsed with 0.02 M
proaches for sustainable agriculture and improved crop production phosphate potassium buffer (pH = 7) for 4 times, in order to remove the
[12]. Although PGPRs are usually effective under in vitro and green­ disinfectant. To ensure the effectiveness of disinfectants, 50 μl buffer
house conditions, they often fail in the field to provide effective condi­ from the final wash was streaked on NA or King’s B agar media as
tions to prevent the disease. control [29]. Samples showing growth in last wash plating, were elim­
Moreover, there is another group of bacteria living inside plants inated. The samples were then cut into small pieces via a sterile blade.
called endophytic bacteria, which can be considered as effective bio­ One gram of sample tissue were macerated in sterile mortar containing
logical control agents. Accordingly, the use of endophytic bacteria for 5 ml of sterile distilled water, and after 30 min, 50 μl of the obtained
biological control of R. solanacearum is a new alternative control suspension was cultured on NA and King’s B medium. The petri dishes
methods to support sustainable agriculture. This can be due to the fact were incubated for 14 days at 28 ◦ C. Moreover, some suspected colonies
that endophytic bacteria in the apoplast are not competing for nutrition were selected, purified, and then identified by key phenotypic charac­
and/or niche, so they can become better biocontrol agents compared to teristics such as gram reaction, catalase, oxidase activity, aerobic and
rhizosphere bacteria [18,19]. Endophytic bacteria are colonized in anaerobic growth, production of levan, urease, arginine dihydrolase,
healthy plants with no visible damage or symptoms, which can also be nitrate reduction, growth at different temperatures, and production of
isolated by surface disinfection or extract of the plant [20,21]. Corre­ fluorescent pigment on King’s B agar medium [24]. The PCR was also
spondingly, they have been isolated from various plants including performed to amplify 16S rRNA gene using rP1/fD2 primer pair [30].
agronomic crops and prairie plants, tomato varieties, potato varieties, The PCR products were checked by agarose gel electrophoresis and
sugar beet, sugarcane, and trees like Eucalyptus spp [19]. Although the products with shape bands were sent to sequencing company for per­
role of endophytic bacteria is not well understood yet [21], various forming sequencing (Korean Macrogen sequencing company). Raw fast
studies have previously indicated that endophytic bacteria may play sequence data were processed using BioEdit v. which were then
beneficial roles such as promoting growth or biological control in their compared with the NCBI nucleotide sequence database using the BLAST
host plants [21]. There are hypotheses that host plants benefit from program. Phylogenetic analysis was performed on the dataset using
endophytic bacteria in various ways such as nutrition, pollutant catab­ MEGA version 6.06 software by applying Neighbor-joining method [31].
olism, and enhancing the plant’s defensive response to environmental In this regard, the phylogenetic tree was then constructed using the
stresses or attacking pathogens [22]. neighbor-joining methods (bootstrap analysis with 1000 replicates).
Other studies have also shown that endophytic bacteria increase
plant growth by producing siderophores and phytohormones, as well as 2.3. In vitro assay for antagonistic activities of endophytics bacteria
increasing resistance to pathogens [19].
According to the above-mentioned findings, the purpose of this study 2.3.1. Disk diffusion testing
was to isolate endophytic bacteria from tubers, roots, stems, and leaves The test was performed as described earlier [32]. About 300 μl of the
of potato plants and then to determine their characteristics, evaluate R. solanacearum suspension containing 2 × 108 CFU/mL was spread over
their effects as biological control agents against R solanacearum, and petri plates containing NA culture medium, which was then placed in
increase plant biomass under both laboratory and greenhouse the laminar hood for 5 min. After drying the petri surfaces, one paper

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K. Bahmani et al. Physiological and Molecular Plant Pathology 116 (2021) 101692

disk of 10 mm suspended in endophytic bacteria suspension (108 and root fresh weights) were measured by passing 60 days from
CFU/mL) was placed on the center of each culture medium. All petri planting. Then, the plants were cut from the crown and dried in oven for
plates were incubated at 28 ◦ C. Also, after 48–72 h, the width of the 48 h at 75 ◦ C, and then the shoot and root dry weights were measured
inhibition zones around the discs were measured. In addition, the [12]. This test was conducted in a completely randomized design with
experiment was done in triplicate. Notably, distilled water was used as a three replications. The relative calculation of disease’s incidence rate
negative control. and biocontrol effectiveness were done using the following formula [38,
39].
2.3.2. Screening of endophytes for production of inhibitory compounds
A suspension of each one of the endophytic bacteria was prepared in % Disease rate = (Number of the withered leaves in pot / Total number of
DH2O with an approximate concentration of 108 CFU/mL and then
leaves in pot) × 100
spotted on NA medium. In this experiment, sterile distilled water was % Disease reduction = [(Incidence of disease in control pots - Incidence of
used as a negative control. The Petri plates were kept for 48 h at 28 ◦ C, disease in pots treated with antagonist) / Incidence of disease in control] × 100.
after which the bacterial colonies were cleared by sterile alcohol-soaked
cotton from the culture medium. Afterward, a piece of chloroform-
impregnated cotton was put on each Petri’s lid and the petri plates
were kept for 1 h upside down. After the removal of cotton, 300 μl of 2.5. Statistical analysis
R. solanacearum suspension (about 2 × 108 CFU/mL) was dispersed on
culture media by glass loop. After incubation of the cultures for 48–72 h The greenhouse experiment was conducted in a completely ran­
at 28 ◦ C, the inhibitory zone diameter was measured [12,33]. domized design along with factorial design in three replications. SAS
software was used to analyze the obtained data and the means were
2.3.3. Biocontrol assay tests on endophytic bacteria compared with DMRT significance level at the 5% level.

2.3.3.1. Protease. Protease production assay is one of the known 3. Results

biocontrol mechanisms. This test is performed using skim milk agar
medium. To do this, in this study, bacterial isolates were cultured in 3.1. Identification of bacterial pathogen
Petri-containing skim milk agar (SKM) medium and then incubated for
24–48 h at 28 ◦ C. It is noteworthy that the formation of colorless halo A total of four isolates of R. solanacearum were characterized at this
around the bacterial colony indicates the activity of protease production stage. The isolates had typical colonies with fluidal texture and a pale
[34,35]. red center in TZC medium. All were positive to oxidase and nitrate
reduction tests, negative to levan, arginine dihydrolase and gelatin hy­
2.3.3.2. Hydrogen cyanide. To perform this part, the method of Alstrom drolysis. They were also unable to grow at 4 and 41 ◦ C. Utilization of
& Burns [36], was used. 100 μl suspension of each bacterial isolate was mannitol, sorbitol, dolcitol and trehalose was negative and oxidation of
spread on petri plates containing NA culture medium. Afterward, a lactose, maltose and D(+) cellobiose were positive. All these isolates
sterile filter paper was impregnated with reagent (containing 2% sodium were amplified with the expected 281 bp DNA fragment using specific
carbonate and 5% picric acid), which was inserted into the Petri lid and primer pair 759/760. The pathogenicity tests of all four strains on potato
the plates were then blocked with parafilm to prevent the escape of any plants were confirmed by causing severe wilting symptoms. They were
volatile metabolites. Subsequently, plates were inversely incubated for identified as R. solanacearum race 3 (biovar 2) based on their phenotypic
one week at 26–28 ◦ C. If HCN is produced by the bacteria, the color of and molecular characteristics. The strain coded KOB was used for all
the filter paper impregnated with the reagent solution would be changed subsequent studies.
from yellow to orange, light brown, dark brown, brick, and cream.
3.2. Identification of antagonist endophytic bacteria
2.3.3.3. Siderophore production. The siderophore production assay was
performed using CAS agar in terms of the above-mentioned method During performing this study, 236 isolates of endophytic bacteria
[37]. In this method, the isolates were cultured on both Petri and agar were isolated from tubers, roots, stems, and leaves of healthy potato
medium. The Petri plates were then incubated at 26–28 ◦ C. Notably, the plants (Table 1). Based on their phenotypic characteristics including
positive reaction was characterized by the formation of halo zone morphological, physiological, and biochemical properties, as well as
around the growing colonies. their degree of inhibition against R. solanacearum in laboratory tests, 11
isolates suspected to belong different genera/species were selected for
PCR analysis (Table 2). The strains were grouped as members of the
2.4. Greenhouse experiment genera Pseudomonas, Paenibacillus, Bacillus, Chryseobacterium, and
Microbacterium (Fig. 1).
Overall, 11 antagonistic endophytic isolates were tested in green­
house experiments. A mixture containing field soil, sand, and compost 3.3. In vitro antagonistic activity of endophytic bacteria against R.
(1.25:1.25:0.5) was prepared and autoclaved according to the standard solanacearum
protocol. Thereafter, the sterilized soil was transferred to pots dis­
infected with 1% sodium hypochlorite. The potato tubers were then The antagonistic activities of 11 endophytic bacteria isolates have
washed with distilled water and disinfected with 5% sodium hypo­ revealed that Pseudomonas brassicacearum Psb101, Pseudomonas brassi­
chlorite for 10 min. After rinsing the tubers again and air-drying them, cacearum Ps169, and Paenibacillus peoriae Pa86 could significantly (p =
they were suspended in the antagonistic endophytic bacterial suspen­ 0.05) inhibit pathogen growth with the mean inhibition diameters of
sion for approximately 1 h (~2 × 108 CFU/mL) and then sown in the 17.6, 17.4, and 17.3 mm, respectively. Moreover, Pseudomonas putida
pots. 24 h later, 200 ml of pathogenic bacterial suspension was added to Ps52, Bacillus licheniformis Bl17, Bacillus pumilus Bp1, Bacillus pumilus
each pot soil with an approximate concentration (108 CFU/mL). Bp91, and Bacillus pumilus Bp49 were also found with the mean inhibi­
Notably, in this study, the sterilized diets in sterile distilled water were tion diameters of 15.5, 15.2, 11.5, 11.2, and 11.1 mm that exhibited
used as negative controls and the dialyzed diets in bacterial suspension moderate inhibitory effect on pathogen growth, respectively. Finally,
were used as positive controls. The greenhouse conditions were adjusted the third group consisted of Microbacterium phyllosphaerae Mi41,
at 26–28 ◦ C, RH ~30%, 12 h/12 h photoperiod. Growth factors (shoot Chryseobacterium lathyri Chl98, and Chryseobacterium indologenes Ch54,

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K. Bahmani et al. Physiological and Molecular Plant Pathology 116 (2021) 101692

Table 1
Sample collection sites, number of isolates related to each organ and geographical coordinates of the cities.
Sampling areas Number of farms sampled in Number of endophytic bacteria isolated in Number of endophytic bacteria isolated Geographical coordinates of
(cities) each area each area from each part of potato plants in different the Cities
areas

Leaf Stem Root Tuber N E

(L) (S) (R) (T) Latitude Longitude

Sanandaj 2 25 8 6 10 1 35◦ 18ʹ02ʺ 46◦ 59ʹ12ʺ E

N
Dehgolan 9 53 3 16 22 12 35◦ 17ʹ38ʺ 47◦ 25ʹ05ʺ E
N
Qorveh 9 46 20 12 14 – 35◦ 09ʹ02ʺ 47◦ 48ʹ47ʺ E
N
Kamyaran 5 27 – 12 7 8 34◦ 47ʹ59ʺ 46◦ 56ʹ16ʺ E
N
Mariwan 5 17 – 11 6 – 35◦ 30ʹ37ʺ 46◦ 09ʹ37 ʺ
N E
Saqqez 12 64 13 25 17 9 36◦ 13ʹ40ʺ 46◦ 18ʹ05ʺ E
N
Divandarreh 4 4 – 1 3 – 35◦ 54ʹ17ʺ 47◦ 02ʹ24ʺ E
N
Total 236 44 83 79 30

Table 2
The code and number of the superior endophytes, the part of the potato from which they were isolated, the exact geographical address of the farms and identification of
antagonistic endophytic bacteria by partial sequencing of 16s rRNA.
Code and number The part of the City of Altitude of Exact geographical coordinates GenBank 16s rRNA % Similarity
of selected potato plant from sampling the sampling of the sampling farm (degrees/ Accession no. sequence (5ʹ
endophytic which endophytic farm minute/second) to 3ʹ)
bacteria bacteria have
been isolated

La S R T N E
Latitude Longitude

S1L2 (Bp91) * Sanandaj 1605 35◦ 26ʹ0.759ʺ 46◦ 57ʹ34.285ʺ MN326732 Range: 30 100% with Bacillus pumilus
to1383 ATCC 7061T (AY876289.1)
Sq1BS1 (Bp1) * Saqqez 1474 36◦ 16ʹ53.184ʺ 46◦ 28ʹ45.403ʺ MN326725 Range: 28 to 100% with Bacillus pumilus
1384 ATCC 7061T (AY876289.1)
Q1R1 (Bp49) * Qoeveh 1838 35◦ 14ʹ16.214ʺ 47◦ 32ʹ34.954ʺ MN326728 Range: 30 to 100% with Bacillus pumilus
1385 ATCC 7061T (AY876289.1)
Q3L1 (Bl17) * Qorveh 1893 35 09ʹ5.609ʺ
◦
47 50ʹ25.622ʺ
◦
MN326726 Range: 80 to 100% with Bacillus
1432 licheniformis ATCC 14580T
(NR_074923.1)
D2L1 (Pa86) * Dehgolan 1897 35◦ 20ʹ04.497ʺ 47◦ 16ʹ31.977ʺ MN326731 Range: 59 to 100% with Paenibacillus
1418 peoriae DSM 8320T
(AB073186.1)
D7R2 (Psb101) * Dehgolan 1875 35◦ 13ʹ01.376ʺ 47◦ 21ʹ52.007ʺ MN326734 Range: 55 to 100% with Pseudomonas
1426 brassicacearum DBK11; CFBP
11706T (NR_024950.1)
Sq6S1 (Ps169) * Saqqez 1619 36◦ 11ʹ14.894ʺ 46◦ 32ʹ39.318ʺ MN326735 Range: 75 to 100% with Pseudomonas
1417 brassicacearum DBK11; CFBP
11706T (NR_024950.1)
Q8R1 (Ps52) * Qoeveh 1932 35◦ 11ʹ30.298ʺ 47◦ 44ʹ2.336ʺ MN326729 Range: 81 to 99% with Pseudomonas
1418 putida_IAM 1236T
(NR_043424.1)
Q5L4 (Ch54) * Qoeveh 1871 35◦ 06ʹ47.665ʺ 47◦ 55ʹ51.350ʺ MN326730 Range: 33 to 100% with Chryseobacterium
1354 indologenes ZYF120413-7T
(KF017580.1)
D9S1 (Chl98) * Dehgolan 1849 35◦ 15ʹ0.304ʺ 47◦ 23ʹ16.719ʺ MN326733 Range: 72 to 100% with Chryseobacterum
1390 lathyri RBA2-6T
(NR_115853.1)
Q7S2 (Mi41) * Qoeveh 1935 35◦ 11ʹ7.227ʺ 47◦ 45ʹ31.009ʺ MN326727 Range: 63 to 99% with Microbacterium
1385 phyllosphaerae P 369/06T
(NR_025405.1)
a
L = Leaf, S= Steam, R = Root, T = Tuber.

whose mean inhibition rates were weaker than the two previous groups. 3.4. Protease, hydrogen cyanide, and siderophore production by
Correspondingly, they had average inhibition zones of 9.5, 8.5, and 8.5 endophytic bacteria
mm, respectively (Table 3 & Fig. 2).
The performed experiments in this study revealed that all strains
were capable of producing siderophore in a medium containing 1000 μM
FeCl3. Also, it was shown that all isolates except Paenibacillus peoriae
Pa86, are able to produce protease by creating a clear halo region in the

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K. Bahmani et al. Physiological and Molecular Plant Pathology 116 (2021) 101692

Fig. 1. The phylogenetic tree shows the position of antagonistic endophytic bacteria among the type strains of the species of the genera Pseudomonas, Paenobacillus,
Bacillus, Chryseobacterium and Microbacterium.

Table 3
In vitro inhibition of growth of R. solanacearum by antagonistic endophytic bacteria from potato through the production of siderophore, hydrogen cyanide and protease.
Strains Mean diameter of inhibition Diameter of halo (mm) in siderophore Diameter of halo (mm) in protease Hydrogen cyanide
zone (mm)a production test production test productionb

Bp91 11.2c ± 0.17 10 17 –

Bp1 11.5c ± 0.05 10 18 –
Bp49 11.1c ± 0.44 10 17 –
Bl17 15.2b ± 0.14 9 12 –
Pa86 17.3a ±0.06 14 0 –
Psb101 17.6a ±0.28 19 26 –
Ps169 17.4a ±0.12 19 26 –
Ps52 15.5b ± 0.26 21 14 +
Ch54 8.5e ± 0.20 15 34 –
Chl98 8.5e ± 0.34 15 34 –
Mi41 9.5d ± 0.28 15 14 –
R.solanacearum + Sterile –
distilled water
a
Values in the same column with the same letter(s) are not significantly different as determined by the DMRT test (P = 0.05).
b
+ = positive, - = negative.

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K. Bahmani et al. Physiological and Molecular Plant Pathology 116 (2021) 101692

The letter R in the above sentences stands for Ralstonia solanacearum.

4. Discussion

According to a research conducted by Ref. [40]; the causative agent

of potato bacterial wilt disease in Kurdistan province is Ralstonia sol­
anacearum race 3 biovar 2. This strain, which has been introduced in
several parts of Iran, is distributed in all regions of the world [41,42].
Control of this disease has been challenged worldwide due to a number
of reasons such as the strain diversity and wide host range. At the same
Fig. 2. In vitro inhibition growth of R. solanacearum by certain antagonistic time, several effective biological methods have been applied and also
endophytic bacteria from potato. a: Testing of production of inhibitory com­ recommended for a better management of the disease including the use
pounds, b: Agar disk diffusion testing. of bacteria isolated from the rhizosphere or endophytic of potato plants.
Correspondingly, the latter one was considered as an alternative
SKM medium. Moreover, Pseudomonas putida Ps52 was the only isolate approach for the disease’s management. Since endophytic bacteria do
that could be produced cyanide hydrogen. (Table 3 & Fig. 3). not compete for nutrition or niche in the apoplast, they have the po­
tential to become better biocontrol agents compared to rhizosphere
bacteria [19]. It is notable that endophytic bacteria have the ability to
3.5. Greenhouse experiment reduce or prevent the harmful effects of a number of pathogens, so they
appear to have their beneficial effects on their host plants through
The findings showed that there was a significant difference (p = several mechanisms described by rhizosphere-associated bacteria [43].
0.05) between the treatments in all parameters. The highest effect on Moreover, various types of endophytes have been reported to exhibit
fresh and dry weight of shoots and roots, as well as shoots and roots antagonistic activity against bacterial and fungal pathogens [44]. Up to
height, was found to be related to the Pseudomonas brassicacearum now, although many endophytic bacteria have been isolated from po­
Psb101. In this respect, Microbacterium phyllosphaerae Mi41 showed the tatoes in various studies, in culture-based surveys conducted on endo­
least effect on prompting plant growth (Table 4 & Fig. 4). Simulta­ phytic bacteria, the number of genera is often <20 and the predominant
neously, the role of antagonist bacteria in reducing disease was also
bacteria are generally related to genera Bacillus, Pseudomonas, Entero­
determined in this study. The strains P. brassicacearum Psb101, bacter, and Agrobacterium [45]. Currently, little is known about the role
P. brassicacearum Ps169, P. putida Ps52, Paenibacillus peoriae Pa86, and
of endophytic bacteria in the control of R. solanacearum. Previous
Bacillus licheniformis Bl17 were determined as the most successful studies have emphasized on the role of antagonistic Rhizobacteria species
antagonist in reducing the disease by 55.19%, 55.03%, 54.41%, 44.41%,
such as Bacillus subtilis, Paenibacillus macerans, Serratia marcescens, Ba­
and 41.31%, respectively. On the other hand, Microbacterium phyllos­ cillus pumilis, and Pseudomonas fluorescens, in the control of
phaerae Mi41 had been shown to have the least effect on disease control
R. solanacearum race 3 under both in vitro and in vivo conditions [4]. In
(Table 5 & Fig. 4).

Fig. 3. Results related to the production of siderophore, hydrogen cyanide and protease production by potential antagonistic endophytic bacteria. a: siderophore
production, b: protease production, c: hydrogen cyanide production.

Table 4
The effect of antagonistic endophytic bacteria on growth promotion and increase of potato biomass in greenhouse conditions.
Treatment Shoot height (cm) Root height (cm) Fresh weight of shoot (g) Fresh weight of root (g) Dry weight of shoot (g) Dry weight of root (g)

Bp91+ R. solanacearum 44.26ca ± 0.25 22.69cd ± 0.51 35.29bcd ± 0.15 4.36de ± 0.07 4.92bcd ± 0.03 0.91de ± 0.01
Bp1+ R. solanacearum 44.17c ± 0.03 22.56cde ± 0.40 34.85cd ± 0.23 4.27ef ±0.03 4.85cde ± 0.02 0.89ef ± 0.00
Bp49+ R. solanacearum 43.64c ± 0.06 21.44def ±0.54 33.91de ± 0.21 4.15f ±0.04 4.72de ± 0.03 0.86f ± 0.00
Bl17+ R. solanacearum 44.91b ± 0.16 23.39bc ± 0.61 35.75bc ± 0.34 4.50cd ± 0.07 4.97bc ± 0.04 0.94cd ± 0.01
Pa86+ R. solanacearum 43.78c ± 0.16 22.47cde ± 0.56 34.77cd ± 0.03 4.16f ± 0.03 4.84cde ± 0.00 0.86f ± 0.00
Psb101+ R. solanacearum 46.19a ±0.15 25.32a ±0.55 37.73a ±0.55 4.98a ±0.07 5.25a ±0.08 1.041a ±0.01
Ps169+ R. solanacearum 46.17a ±0.15 24.87ab ± 0.34 37.29a ±0.59 4.78b ± 0.03 5.20a ±0.08 1.00b ± 0.00
Ps52+ R. solanacearum 45.35b ± 0.14 24.13abc ±0.80 36.68ab ± 0.75 4.56c ± 0.03 5.11ab ± 0.11 0.95c ± 0.00
Ch54 + R. solanacearum 42.76d ± 0.26 19.93fg ± 0.24 33.27e ± 0.36 3.88gh ± 0.04 4.64e ± 0.05 0.81gh ± 0.00
Chl98+ R. solanacearum 42.95d ± 0.28 20.85efg ±0.61 33.32e ± 0.77 3.94g ± 0.07 4.65e ± 0.10 0.82g ± 0.01
Mi41+ R. solanacearum 42.76d ± 0.26 19.79fg ± 0.43 28.52f ± 0.61 3.73h ± 0.02 3.98f ± 0.09 0.78h ± 0.00
R. solanacearum 28.24f ± 0.19 14.43h ± 0.97 14.78g ± 0.44 2.3j ± 0.02 2.06g ± 0.06 0.49j ± 0.00
Control (No bacterization)b 40.73e ± 0.26 19.59g ± 0.28 28.93f ± 0.32 3.44i ± 0.04 4.02f ± 0.05 0.72i ± 0.00
a
Values in the same column with the same letter(s) are not significantly different as determined by the DMRT test (P = 0.05).
b
Sterile distilled water only.

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K. Bahmani et al. Physiological and Molecular Plant Pathology 116 (2021) 101692

Fig. 4. The seedlings treated with antagonistic endophytic bacteria show drastically wilt disease reduction and plant growth promotion in greenhouse conditions. a:
Psb101 + R, b: Ps169 + R, c: Ps52 + R, d: Bl17 + R, e: Bp91 + R, f: Bp1+R, g: Pa86 + R, h: Bp49 + R, i: Chl98 + R, j: Ch54 + R, k: Mi41 + R, l: Control+, m:
R. solanacearum).

shown to be different from the other species, because it was able to

Table 5
produce all three metabolites of siderophore, protease, and hydrogen
The effect of antagonistic endophytic bacteria on disease biocontrol and
cyanide. The antagonistic ability of P. fluorescens and P. putida species
reduction of disease symptoms in greenhouse condition.
against R. solanacearum reported in the present study is consistent with
Treatment Disease incidence (%)
previous reports [4,11,12,49]. Bacillus licheniformis Bl17 was another
Bp91+ R. solanacearum 48.06da ± 0.27 bacterium that could significantly control the disease. The role of
Bp1+ R. solanacearum 48.11d ± 0.12 bacteriocin-like substance was also reported in antagonistic activity of
Bp49+ R. solanacearum 48.24d ± 0.27
Bacillus licheniformis P40 against soft rot disease in potato [1,50]. Defi­
Bl17+ R. solanacearum 44.14c ± 0.43
Pa86+ R. solanacearum 41.81b ± 0.32 nitely, there has been no report on there R. solanacearum control by
Psb101+ R. solanacearum 33.70a ±0.38 Bacillus licheniformis, so this study could be considered as the first report
Ps169+ R. solanacearum 33.82a ±0.39 of the antagonistic effect of Bacillus licheniformis on R. solanacearum.
Ps52+ R. solanacearum 34.29a ±0.35
It is noteworthy that significant differences were observed between
Ch54+ R. solanacearum 52.72e ± 0.37
Chl98+ R. solanacearum 52.30e ± 0.09
the two isolates of Pseudomonas brassicacearum Psb101 and Ps169 on
Mi41+ R. solanacearum 54.41f ± 0.11 one side, and between the isolates of Pseudomonas putida Ps52 and Ba­
R. solanacearum 75.22g ± 0.28 cillus licheniformis Bl17 on the other side. This is most likely due to the
a
Values in the same column with the same letter(s) are not significantly production of more inhibitors. Moreover, it is notable that a significant
different as determined by the DMRT test (P = 0.05). difference was observed between the two isolates of P. brassicacearum
Psb101 and Ps169 on the one hand, and also between the isolates of
other studies, the role of Pseudomonas spp. Was also specified [46]. Pseudomonas putida Ps52 and Bacillus licheniformis Bl17 on the other
Based on the obtained data, the current research was conducted to hand. Accordingly, this may be due to the production of other inhibitors.
isolate some potential antagonists from some different areas of Kurdi­ As it was mentioned earlier, the Bacillus pumilus Bp91, Bp1, Bp49; and
stan province. At the initial screening stage of the study, 31 endophytic Chryseobacterium indologenes Ch54, Chryseobacterium lathyri Chl98, and
bacteria with promising antagonistic activities were isolated. Among Microbacterium phyllosphaerae Mi41 showed moderate to weak inhibi­
these, the highest growth inhibition zones were observed to be belonged tion effects in laboratory bioassays. Although this is the first time that
to Pseudomonas brassicacearum Psb101, Pseudomonas brassicacearum the genus Microbacterium and Chryseobacterium have been reported as
Ps169, Paenibacillus peoriae Pa86, Pseudomonas putida Ps52, and Bacillus endophytes of potatoes, to the best of our knowledge, Microbacterium
licheniformis Bl17. have already been reported from the corn and barley by Ref. [51].
Most of these isolates were able to produce siderophore, hydrogen In greenhouse experiment, the three isolates of P. brassicacearum
cyanide, and protease. Two strains of Pseudomonas brassicacearum Psb101, Ps169, and P. putida Ps52 had the highest degrees of disease
Psb101 and Ps169, as superior bacteria, have definitely used at least two reduction. This is corresponded with in vivo antagonistic activity of
of these mechanisms to control the disease. To the best of our knowl­ P. brassicacearum against R. solanacearum reported by Ref. [47].
edge, this is the first time that P. brassicacearum has been reported as Besides the inhibitory effects of P. brassicacearum Psb101 and Ps169
endophytic bacteria from potato. Moreover, many Pseudomonas spp. as well as P. putida Ps52, in disease incidence reduction by 55.19%,
Have been already reported including the strain P. brassicacearum J12 55.03%, and 54.41%, respectively, they have also increased the plant
with biocontrol activity against bacterial wilt of tomato. The latter one biomass (fresh and dry weights of shoot and root) compared to the
could also produce hydrogen cyanide, siderophore, and protease, but controls. In this regard, the role of P. brassicacearum in increasing plant
the main active compound against R. solanacearum is known to be 2,4- biomass of tomato has been previously reported by Ref. [47]. There are
diacetylphloroglucinol [47]. Some other isolates such as Paenibacillus many reports on a significant increase in plant biomass by fluorescent
peoriae Pa86, Pseudomonas putida Ps52, and Bacillus licheniformis Bl17 pseudomonads strains. Therefore, this could be considered as a result of
also had the ability of controlling R. solanacearum under laboratory introducing active systemic resistance, production of siderophores, and
conditions. It is noteworthy that, in this study, there was no significant the advantage of competition for root colonization by antagonists [10,
difference among the two Pseudomonas brassicacearum Psb101 and 52].
Ps169 strains and Paenibacillus peoriae Pa86 in the laboratory results. Subsequently, Paenibacillus peoriae Pa86 and Bacillus licheniformis
These results are consistent with the results of several other studies, Bl17 have significantly reduced the disease incidence rate by 44.41%
which indicate the antagonistic activity of Paenibacillus sp. Strains and 31.41%, respectively. In addition, both strains have also increased
against fungi and bacteria including R. solanacearum [11,12,15,48]. plant biomass compared to the control. The effect of Paenibacillus strains
Paenibacillus macerans could specifically inhibit R. solanacearum race 3 on increasing plant biomass under greenhouse conditions has been re­
growth under both in vitro and in vivo conditions [10,12]. However, the ported in the studies by Ref. [12]; and Kheirandish and Harighi, (2015).
role of protease production in Paenibacillus peoriae strain NRRL BD-62 Similar results have also been reported on tomato plants [15]. In addi­
should not be overlooked [48]. The species Pseudomonas putida was tion, some Bacillus strains have been found to increase potato yields by

7
K. Bahmani et al. Physiological and Molecular Plant Pathology 116 (2021) 101692

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