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KSÜ Tarım ve Doğa Derg 24 (5): 1084-1092, 2021
KSU J. Agric Nat 24 (5): 1084-1092, 2021
DOI:10.18016/ksutarimdoga.vi.811324
The Isolation and Characterization of Phosphate Solubilizing Bacteria from the Onion
Rhizosphere and Their Effect on Onion Growth
İdris BEKTAŞ1, Mustafa KÜSEK2
1Amasya University, Suluova Vocational School, Department of Plant and Animal Production, Amasya, 2Kahramanmaras Sutcu Imam
University, Faculty of Agriculture, Department of Plant Protection, Kahramanmaras
1https://orcid.org/0000-0001-7409-4837, 2https://orcid.org/0000-0002-6320-5869
: idris.bektas@amasya.edu.tr
ABSTRACT
In this study 269 phosphate solubilizing bacteria (PSB) strains were
isolated from onions rhizosphere. The 15 most efficient PSB were
selected. Based on BIOLOG profiling and the LOPAT test system, the
strains were identified as
Enterobacter cloacae
(7),
Yersinia
intermedia
,
Pantoea agglomerans, Bacillus subtilis
(2),
Bacillus
pumilius
,
Lysinibacillus sphaericus
and
Bacillus atrophaeus
(2). The
PSB effect on onion growth was investigated under greenhouse
conditions. These PSB increased plant height (11.7 to 38.8%), the
number of leaves (3 to 70%), the bulb diameter (6.4 to 64.5%), the
average root length (2.6 to 44.7%), the fresh weight of the bulb (2.9 to
34.1%) and the dry weight of the bulb (1.5 to 40.6%) when compared
to the negative control. Among these strains the most effective
inoculants were the highest P solubizing
E.cloacae
EB-14 and
E.
cloacae
OB-169 strains, which significantly increased all parameters
compared to the commercial plant activator ISR 2000. These results
demonstrated the potential use of these PSB as inoculants for onion
growth.
Research Article
Article History
Received : 16.07.2020
Accepted : 07.01.2021
Keywords
Onion growth
Phosphate solubilizing bacteria
Plant activator
Plant growth promoting
rhizobacteria
Soğan (
Allium cepa
L.) Rizosferindeki Fosfat Çözen Bakterilerin İzolasyonu, Karakterisasyonu ve Soğan
Gelişimi Üzerine Etkisi
ÖZET
Bu çalışmada 269 fosfat çözen bakteri(PSB) izolatı soğan rizosfer
bölgesinden izole edilmiştir. İzolatlar arasından en fazla fosfat çözen
15 bakteri seçilmiştir. BIOLOG test profiline ve LOPAT test sistemine
göre seçilen izolatlar
Enterobacter cloacae
(7),
Yersinia intermedia
,
Pantoea agglomerans, Bacillus subtilis
(2),
Bacillus pumilius
,
Lysinibacillus sphaericus
ve
Bacillus atrophaeus
(2) olarak
tanılanmıştır. Bu izolatların soğan gelişi üzerine etkisi sera
koşullarında incelenmiştir. Seçilen bu izolatlar kontrole göre, bitki
boyunu %11.7-38.8, yaprak sayısını %3-70, yumru çapını %6.4-64.5,
ortalama kök uzunluğunu %2.6-44.7, soğan yumru yaş ağırlığını
%2.9-34.1 ve soğan yumru kuru ağırlığını %1.5-40.6 arasında
artırdıkları belirlenmiştir. P çözen izolatlar arasındaki
E. cloacae
EB-
14 ve
E. cloacae
OB-169 tüm paramertreleri ticari bir bitki aktivatörü
ürün olan ISR 2000 den istatistiki olarak daha fazla artırarak en etkili
izolatlar olmuşlardır. Bu sonuçlar göstermiştir ki PSB’ler soğan
gelişimi için potansiyel bir inokulant olarak kullanılabilir.
Araştırma Makalesi
Makale Tarihçesi
Geliş Tarihi : 16.07.2020
Kabul Tarihi : 07.01.2021
Anahtar Kelimeler
Soğan gelişimi
Fosfat çözücü bakteriler
Bitki aktivatörü
Bitki gelişimini teşvik edici
bakteriler
To Cite:
Bektaş İ, Küsek M 2021. The Isolation and Characterization of Phosphate Solubilizing Bacteria from the Onion
Rhizosphere and Their Effect on Onion Growth. KSU J. Agric Nat 24 (5): 1084-1092. DOI: 10.18016/
ksutarimdoga.vi.811324.
INTRODUCTION
Onion (
Allium cepa
L.), a member of Amaryllidaceae
family, is one of the most economically important
vegetables in all parts of the world due to its
nutritional and medicinal values, including
anticancer, anti-cholesterol, anti-inflammatory and
antioxidant properties (Nasri et al., 2012). At the same
time, onion contains important amounts of mineral
salts, sulphur and potassium salts, and various trace
elements, vitamins (B1, B2, C, E, K), carotene
(provitamin A), glycosides, etheric oils and plant
KSÜ Tarım ve Doğa Derg 24 (5): 1084-1092, 2021
KSU J. Agric Nat 24 (5): 1084-1092, 2021
Araştırma Makalesi
Research Article
1085
hormones like insulin (Slimestad et al., 2007). Onion’s
root system has little absorbent and penetrating
properties, there must be an abundant source of
accessible nutrients in the root area. The required
amount of easily accessible nutritients in the soil for
an optimal yield of onion growth is 60 to 140 kg of N,
60 to 120 kg of P2O5 and 60 to 180 kg of K2O per hectare
(Kumar et al., 2001).
Chemical fertilizers are usually used to provide major
nutrients to the soil plant system around the world.
But, the price and ecological problems of fertilizers are
the most significant problem in today’s agriculture,
and there is a requirement to find different methods
that can improve crop yields, maintain ecological
safety and prevention, while protecting long-term
environmental stability in the agro-ecosystem (Majeed
et al., 2015). Plant growth-promoting rhizobacteria
(PGPR) are beneficial native and soil bacteria that
colonize the plant rhizosphere (Aktan and Soylu,
2020). PGPR inoculation of seeds or crops increases the
growth and yield of plants (Kumar et al., 2014). The
mechanisms of PGPR that fix nitrogen from the
atmosphere and supply the plant can be through the
synthesis of phytohormones and also phosphorous can
be made available to plants by the solubilization of
inorganic phosphate and by mineralization of organic
phosphate (Zhang et al., 2018). Phosphorus (P) is one
of the significant major elements for biological growth
and improvement (Bhat et al., 2017), but the
concentration of soluble P in soil is generally very low
because of different chemical reactions, especially in
arid and semi-arid soils (Sharma et al., 2013). The
largest reserves of phosphorus are rocks and other
deposits like basic apatites and another primary
elements (Lompo et al., 2018). Agricultural lands hold
major reserves of phosphorus, a important part of
which has accumulated as a result of the regular use
of P fertilizers (Cruz-Paredes et al., 2017). Different
researchers have investigated the ability of various
bacterial types to solubilize otherwise insoluble
inorganic phosphate compounds, like rock phosphate,
hydroxyapatite, dicalcium phosphate and tricalcium
phosphate (Khan et al., 2010).
The number of phosphate solubilizing bacteria (PSB)
is not adequately high enough to compete with
different bacteria usually situated in the rhizosphere
(Duman and Soylu, 2019). Therefore,the volume of P
liberated by them is usually not enough for a
significant rise in plant growth. Thus, inoculation of
plants with a PSB at a very higher concentration than
can generally be found in the soil is required to take
advantage of the characteristics of phosphate
solubilization for plant crop increases (Rodrıguez and
Fraga, 1999). In this study, potential PSB were
isolated from the rhizosphere of healthy onion plants
growing in surveyed fields located in Amasya. All
strains were tested for their
in vitro
P solubilizing
potential using broth and solid National Botanical
Research Institute Phosphate (NBRIP) containing
tricalcium phosphate (TCP). The 15 highest
phosphorus solubilizing bacterial strains were
identified with biochemical tests and the BIOLOG
GEN III system. In addition to identification studies,
the strains were tested for their ability to promote
growth under greenhouse conditions in onion plants.
The effects of PGPR strains on plant-growth
parameters were statistically compared to each other
and ISR 2000 was used as a commercial plant
activator.
MATERIALS and METHODS
Isolation and Identification of PSB
Soil samples were collected from the onion plant’s
rhizosphere from certain fields of Amasya in Turkey.
The soil samples were placed on blotting paper and
kept at 25°C. From that dry soil, 10 grams were put in
250 ml erlenmeyer flasks and 90 ml of sterile pure
physiological saline solution (0.85%) was added to
them. The erlenmeyers were shaken for 30 minutes in
a shaker. Half a milliliter of suspension and 4.5 ml of
pure physiological saline solution was added to 10 ml
tube vials and shaken for 1 minute. Up to 10−7 dilution
was achieved by a serial dilution method. An 100 µl
aliquot of this suspension was spread on to plates with
solid NBRIP growth medium containing (g l-1): 10 g of
glucose, 5 g of Ca3(PO4)2, 5 g of MgCl2⋅6H2O, 0.25 g of
MgSO4⋅7H2O, 0.2 g of KCl, 0.1 g of (NH4)2SO4 and 20 g
of agar (Nautiyal, 1999). The dishes were incubated at
25°C for 1 week and P solubilizing strain colonies with
a clear zone were consired positive for phosphate
solubilization. The index of phosphate solubilization
was determined by using the following formula
(Premono et al., 1996).
SI(Solubilisation Index)= total diameter (colony + halo
zone)/colony diameter
At the same time these phosphate solubilizing strains
were tested for their
in vitro
phosphate solubilizing
features in NBRIP broth medium ((g l-1): 10.0 g of
glucose, 10.0 g of tricalcium phosphate (TCP), 5.0 g of
MgCl2.6H2O, 0.25 g of MgSO4.7H2O, 0.2 g of KCl, 0.1 g
of (NH4)2SO4). The quantitative measurement of
phosphate solubilization was carried out using 15 ml
test tubes containing 10 ml of NBRIP growth medium
freshly inoculated with 0.1 ml of each strain of PSB.
Incubation was done at 27°C in an incubator shaker at
125 rpm for 7 days. The tubes were then centrifuged at
10.000 rpm for 15 minutes and the supernatant of each
culture was analyzed for phosphate concentration in
ppm by using the by Barton (1948) method. Among the
phosphate solubilizing strains, thirteen strains with
the highest P solubizing potential in NBRIP broth
medium and two strains with the highest SI were
selected for use
in vivo
pot experiments. The 15 strains
were identified by using the BIOLOG GEN III
KSÜ Tarım ve Doğa Derg 24 (5): 1084-1092, 2021
KSU J. Agric Nat 24 (5): 1084-1092, 2021
Araştırma Makalesi
Research Article
1086
(BIOLOG 21124 Cabot Blvd.Hayward, CA 94545)
identification system.
Pathogenicity Test and Biochemical Features of PSB
The pathogenicity tests were performed according to
by Moragrega et al. (2003), with
in vivo
methods. PSB
strains grown in Nutrient broth(NB) at 27°C for 72
hours were centrifuged for 5 min. at 10.000 rpm and
the pellet was adjusted in sterile distilled water to a
concentration of approximately 1×106 CFU ml− 1.
Bacterial suspensions of about 20 μl were injected into
the onion bulb stems using a sterile needle. Sterile
water was used as a negative control and
Pectobacterium chrysanthemi
F-161
which is the
agent of soft rot for onion was used as a positive control
(Dadaşoğlu and Kotan, 2017). In both pathogenicity
tests, all treatments were carried out in triplicate.
Onion materials were kept at 27°C in a mist chamber
and disease development was assessed up to 1 weeks.
The selected 15 PSB bacterial strains were identified
by biochemical tests including LOPAT tests (levan
production, oxidase reaction, potato soft rot, arginine
dihydrolase, tobacco hypersensitivity) according to
methods previously described by Schaad et al. (2001).
Preparing the Soil and Pot Experiment
Soils for the experiment were collected in from the
Amasya region. Soil samples were taken from a depth
of 30 to 35 cm deep from barren land for phosphorus
analysis before the study. Phosphorus analysis of the
soil samples was done to determine the amount of
soluble phosphorus in the soil. The pot experiment soil
was prepared to the phosphorus-poor soil with 5% rock
phosphate, manure and sand (vol/vol 1:1:1, pH of 7.0) .
The onion seeds were inoculated with fifteen high ratio
PSB strains. The PSB strains were grown in 15 ml
tubes in nutrient broth (NB) on a rotating shaker (125
rpm) for 24 hours at 27°C. The tubes were centrifuged
at 10.000 rpm and the supernatant was removed from
the precipitate. The density of the PSB strains was
adjusted to 0.3 absorbance at a wavelength of 600 nm
with sterile physiological saline solution using a
spectrophotometer to 108 CFU(Colony Forming
Unit)/mL. One to two drops of Tween 80 were added to
the suspension to prevent the clustering of the bacteria
and to ensure a homogeneous distribution. The PSB
strains were used as onion seed treatments. The onion
seeds were surface sterilized with an 0.1% solution of
NaClO(sodium hypochlorite) for 2.5 minutes and
rinsed completely with sterile water and air dried on
sterile filter paper. The onion seeds (5 g) were
inoculated while keeping in 50 ml of each PSB strains
for 12 hours (Ramamoorthy et al., 2002). While
negative control experiment seeds were not inoculated,
the positive control was inoculated with commercial
plant activator ISR 2000 (
Lactobacillus acidophilus
+
yeast extract + plant extract + benzoic acid). For each
treatment, five replicates from one onion plant were
grown in 20 cm diameter pots. The study was done in
a greenhouse with an average temperature of 27°C,
humidity of about 55 % and a photoperiod of about 12
to 13 hours of daylight. Six months later, the onions
were collected from the pots and the bulb diameter,
root length, plant height, number of leaves, and the
weights of fresh and dry onion bulb were determined
for each application.
Statistical Analysis
The quantitative data on onion growth were
statistically compared to each other with analysis of
variance using the SPSS 20 (Statistical Package for
Social Sciences) software program. Treatment means
were compared using the Duncan’s Multiple Range
Test at p≤0.05.
RESULTS and DISCUSSION
Isolation and Identification of PSB
For use in research, 269 potential PSB strains were
obtained from a healthy onion rhizosphere region.
In
vitro
studies of the P solubilization index of PSB
strains are defined to be between 1.3 to 5.1 on NBRIP
agar medium. However, in the NBRIP broth medium a
rate of 19.3 to 382.5 µg ml−1 was detected.Among the
PSB strains, fifteen PSB strains having the highest P
solubizing potential strains (three on NBRIP agar
medium and twelve on NBRIP broth medium) were
selected to use for the
in vivo
pot experiments. The 15
bacterial strains were identified as
Enterobacter
cloacae
(7),
Bacillus atrophaeus
(2),
Bacillus subtilis
(2),
Yersinia intermedia
,
Bacillus pumilus
,
Lysinibacillus
sphaericus
and
Pantoea agglomerans
by using LOPAT
tests, Gram tests and BIOLOG profiling (Table 1).
The Pathogenicity Test and Biochemical Features of
PSB
The PSB bacterial strains isolated from the onion
rhizosphere exhibited different pathogenicity results
on sensitive Kantartopu onion accessions. The
Pectobacterium chrysanthemi
F-161
reference strain
caused typical onion rot symptoms on the onion bulbs
(watery tissue, necrotic lesions) and was the most
pathogenic of the strains tested. In the pathogenicity
tests, all selected PSB strains showed negative
pathogenicity on onion bulbs. The selected PSB
bacterial strains that were isolated from the onion
rhizosphere and classified by the LOPAT tests
exhibited diverse results. As a result of the lopat
test,seven strains of
Enterobacter cloacae, Pantoea
agglomerans
and
Yersinia intermedia
strains resulted
in LOPAT (+,−,−,+,−), while two strains of
Bacillus
subtilis, Bacillus pumilus, Lysinibacillus sphaericus
and two strains of
Bacillus atrophaeus
strains resulted
in LOPAT (−,−,−,+,−). All
Enterobacter cloacae
and
KSÜ Tarım ve Doğa Derg 24 (5): 1084-1092, 2021
KSU J. Agric Nat 24 (5): 1084-1092, 2021
Araştırma Makalesi
Research Article
1087
Pantoea agglomerans
strains were Gram negative
except for
Bacillus
sp. strain which were Gram
positive. All PSB were oxidase, potato soft rot and
tobacco hypersensitivity negative, at the same time, all
PSB showed arginine dihydrolase positive
characteristics. The results of the levan production test
showed that
Enterobacter cloacae
and
Pantoea
agglomerans
were negative while
Bacillus
sp. was
positive (Table1).
Table1.BIOLOG profiling andLOPAT tests analyses of selected P solubilizing PGPR strains
Çizelge1. Seçilen P çözücü PGPR izolatlarının BİOLOG test profili ve LOPAT test analazi
C: control (pure water), PGPR:Plant growth promoting rhizobacteria, NBRIP: National Botanical Research Institute's
phosphate growth mediums :phosphates solubilizing index, LOPAT: Levan, Oxidase, Potato soft‐rot, Arginine dehydrolase,
Tobacco hypersensitivity
Effect of PSB on Onion Growth
Onion plant height
Pot tests were performed to determine the effects of P
solubilizing strains on onion plant growth. Plant
height ranged from 16.3 to 26.1 cm. Among tested
strains, nine PSB (EB-14, OB-169, EB-15, İB-162, MK-
252, HB-204, HB-234, MK-262 and HB-228)
significantly stimulated plant growth of 14.8 to 38.8%
in comparison to the control experiment. In contrast,
five strains (AZB-64, HB-207, ZEB-94, EB-38, EB-21)
showed reducing plant height, it was not statistically
significant according to the control. The stimulation of
plant height was statistically observed, with EB-14,
OB-169 and EB-15 higher in comparison to ISR 2000
(Table 2).
Number of onion leaves
The inoculation of seeds with PSB also affected the
number of onion plant leaves. The onion leaves ranged
from 2.3 to 4.8 cm. Onion leaves were stimulated 3 to
70%. The application of AZB-64 reduced onion leaf
numbers by 2.3%, but this application did not differ
significantly according to the negative control. Among
the PSB, three strains: EB-1, OB-169 and EB-15 were
found to be more statistically significant (50 to 70%)
than other bacterial strains and the negative control
with regard to the growth in numbers of leaves.
Moreover, the EB-14 and OB-169 strains significantly
affected the number of leaves compared to ISR 2000
(p≤0.05) (Table 2).
Onion bulb diameter
The diameters of the onion bulbs was also affected by
PSB inoculation. Onion bulb diameter ranged from 3.1
to 5.1 cm. All strains stimulated onion bulb growth by
6.4 to 64.5% compared to the growth of a non-
inoculated control bulb. Eleven PSB strains (EB-14,
OB-169, EB-15, İB-162, MK-252, HB-204, HB-234,
MK-262, HB-228, HB-230, EB-21) significantly
increased onion diameter compared to the to negative
control by 25.8 to 64.5%. Additionally, six strains (EB-
14, OB-169, EB-15, İB-162, MK-252 and HB-204)
significantly stimulated onion bulb growth (45.1 to
64.5%) compared to ISR 2000 (p≤ 0.05) (Table 2).
Average root length
All PSB strains increased the onion root length
compared to the negative control inoculation. The
average onion root length ranged from 3.8 to 5.5 cm.
Root length was statistically affected by six strains
(EB-14, OB-169, EB-15, İB-162, MK-252, HB-204) in
comparison to the negative control, whereas other PSB
treatments did not show a significant increase.
However, the strains EB-14, OB-169, MK-252 and EB-
15 showed significant increases of 36.8 to 44.7% over
inoculation with ISR 2000 (p≤ 0.05) (Table 2).
PGPR
strains
(PGPR
izolatları)
BIOLOG profiling
(BIOLOG profili)
P solubising
(P çözme
potansiyeli)
ppm
PSI
Gram test
(Gram
testi)
LOPAT tests results
(LOPAT test
sonucu)
L
O
P
A
T
C-
-
-
-
-
-
-
-
-
-
OB-169
Enterobacter cloacae
382.5±2.1
1.4
-
+
-
-
+
-
EB-14
Enterobacter cloacae
337.6±0.7
1.6
-
+
-
-
+
-
EB-15
Pantoea agglomerans
321.9±4.3
1.9
-
+
-
-
+
-
EB-21
Yersinia intermedia
316.9±3.3
2.7
-
+
-
-
+
-
HB-234
Enterobacter cloacae
304.4±2.5
1.8
-
+
-
-
+
-
HB-230
Enterobacter cloacae
298.7±2.3
2.1
-
+
-
-
+
-
HB-207
Bacillus subtilis
295.8±3.7
1.7
+
-
-
-
+
-
HB-204
Enterobacter cloacae
282.1±3.2
1.7
-
+
-
-
+
-
HB-228
Bacillus pumilis
281.3±2.8
1.6
+
-
-
-
+
-
AZB-64
Lysinibacillus sphaericus
278.8±7.3
1.9
+
-
-
-
+
-
ZEB-94
Enterobacter cloacae
259.4±7.9
5.1
-
+
-
-
+
-
EB-38
Enterobacter cloacae
265.5±4.9
4.8
-
+
-
-
+
-
MK-252
Bacillus subtilis
243.8±3.0
4.5
+
-
-
-
+
-
İB-162
Bacillus atrophaeus
258.8±4.7
1.4
+
-
-
-
+
-
MK-262
Bacillus atrophaeus
247.8±2.5
1.9
+
-
-
-
+
-
KSÜ Tarım ve Doğa Derg 24 (5): 1084-1092, 2021
KSU J. Agric Nat 24 (5): 1084-1092, 2021
Araştırma Makalesi
Research Article
1088
Table 2.The effect of phosphate solubilizing PGPR strains on onion growth
Çizelge 2. Fosfat çözücü PGPR izolatlarının soğan gelişimi üzerine etkisi
C: seeds infested with water ,ISR 2000:
Lactobacillus acidophilus
+ yeast extract + plant extract + benzoic acid (Improcrop).
Values are means of five replicates, Mean values (mean ± Standard Deviation) sharing the same letter do not differ
significantly by Duncan’s Multiple Range Test at p ≤ 0.05.
Bulb fresh and dry weight
Onions inoculated with PSB strains increased the
fresh and dry weight of their bulbs at different rates.
The eight PSB strains (EB-14, OB-169, EB-15, IB-162,
MK-252, HB-204, HB-234 and MK-262) significantly
increased the growth in the fresh and dry weights of
the bulb by 16.4 to 34.1% compared to the non-
inoculated control plant. When the growth results were
compared with the ISR 2000, six strains (EB-14, OB-
169, EB-15, IB-162, MK-252 and HB-204) there was a
significant increase in fresh weight, at the same time,
strains EB-14, OB-169 and EB-15 significantly
Treatment
(Uygulamalar)
Plant height
(Bitki yüksekliği)
(cm)± SD
Effect
(Etki)
%
Number
leaves
(Yaprak
sayısı)
± SD
Effect
(Etki)
%
Bulb
diameter
(Yumu çapı)
(cm) ± SD
Effect
(Etki)
%
C(Negative control)
18.8±2.1de
-
3±0.8de
-
3.1±0.14g
-
ISR 2000(Positive control)
22.5±2.7bc
19.6
4±0.6abcd
33.3
3.9±0.3e
25.8
Enterobacter cloacae EB-14
26.1±2.4a
38.8
4.8±0.4ab
60
5.1±0.3a
64.5
Enterobacter cloacae
OB-169
26± 1.7a
38.2
5.1±0.7a
70
5.0±0.8abc
61.2
Pantoea agglomerans EB-15
25.5±2.1a
35.6
4.5±0.8abc
50
5.0±0.7ab
61.2
Bacillus atrophaeus IB-162
24.6±1.9ab
25
4.3±1.3abcd
43.3
4.5±0.4cd
45.1
Bacillus subtilis MK-252
22.6±4.1bc
20.2
4±1.09abcd
33.3
4.5±0.4cd
45.1
Enterobacter cloacae HB-204
22.3±1.8bc
18.6
3.5±1.0bcde
16.6
4.5±0.5cd
45.1
Enterobacter cloacae HB-234
22.1±2.4bc
17.5
3.3±1.2cde
10
4.0±0.3de
29
Bacillus atrophaeus MK-262
21.8±1.7bc
15.9
4.1±1.4abcd
36.6
4.0±0.4de
29
Bacillus pumilis HB-228
21.6±2.0c
14.8
3.1±0.7cde
3
3.9±0.3e
25.8
Enterobacter cloacae HB-230
21±1.7de
11.7
4.1±1.8abcd
36.6
3.8±0.4ef
22.5
Yersinia intermedia EB-21
18.6±1.7de
-1
3.5±0.5bcde
16.6
3.7±0.2ef
19.3
Enterobacter cloacae EB-38
16.8±2.1e
-10.6
3.3±1.2cde
10
3.7±0.4efg
19.3
Enterobacter cloacae ZEB-94
16.8±1.8e
-10.6
3.1±0.8de
3
3.5±0.2efg
12.9
Enterobacter cloacae HB-207
16.6±2.0e
-11.7
3.6±1.5bcde
20
3.3±0.3fg
6.4
Lysinibacillus sphaericus AZB-64
16.3±1.8e
-13.2
2.3±0.5e
-23.3
3.3±0.3fg
6.4
Treatment
(Uygulamalar)
Average Root
length
(Kök uzunluğu)
(cm) ±SD
Effect
(Etki)
%
Bulb wet
weight
(Yumru yaş
ağırlığı)
(g) ± SD
Effect
(Etki)
%
Bulb dry weight
(Yumru kuru
ağırlığı)
(
g) ± SD
Effect
(Etki)
%
C(Negative control)
3.8±0.3e
-
17±1.5e
-
12.8±0.7e
-
ISR 2000(Positive control)
4.5±0.4cd
18.4
19.1±0.9cd
12.3
15.1±0.7cd
17.9
Enterobacter cloacae EB-14
5.1±0.6ab
34
22.8±1.4a
34.1
17.6±1.3ab
37.5
Enterobacter cloacae
OB-169
5.3±0.4ab
39.4
22.3±2.5a
31.1
18±1.6a
40.6
Pantoea agglomerans EB-15
5.5±0.4a
44.7
22.1±1.1a
30
17.6±1.3ab
37.5
Bacillus atrophaeus IB-162
4.9±0.4bc
28.9
21.1±1.6ab
27
17±1.4abc
32.8
Bacillus subtilis MK-252
5.2±0.2ab
36.8
21.3±1.2ab
25.2
17±1.9abc
32.8
Enterobacter cloacae HB-204
4.8±0.5bc
26.3
21.3±1.0ab
25.2
17.8±1.3a
39
Enterobacter cloacae HB-234
4.2±0.2de
10.5
20±1.7bc
17.6
15.6±2bcd
21.8
Bacillus atrophaeus MK-262
4.4±0.4cd
15.7
19.8±0.7bc
16.4
15.5±1.6cd
21
Bacillus pumilis HB-228
4.2±0.5de
10.5
18.6±1.5cde
9.4
14.6±1.7de
14
Enterobacter cloacae HB-230
4.2±0.4de
10.5
18.3±1.7cde
7.6
13.5±2de
5.4
Yersinia intermedia EB-21
4.1±0.4de
7.8
18.1±1.1cde
6.4
13.5±1.3de
5.4
Enterobacter cloacae EB-38
4.2±0.2de
10.5
18±1.7cde
5.8
14.6±1de
14
Enterobacter cloacae ZEB-94
3.9±0.3e
2.6
17.6±1.9de
3.5
13±2e
1.5
Enterobacter cloacae HB-207
4±0.2de
5.2
17.6±2.2de
3.5
14.1±1.6de
10.1
Lysinibacillus sphaericus AZB-64
3.9±0.4e
2.6
17.5±1.8de
2.9
12.8±2.4e
0
KSÜ Tarım ve Doğa Derg 24 (5): 1084-1092, 2021
KSU J. Agric Nat 24 (5): 1084-1092, 2021
Araştırma Makalesi
Research Article
1089
increased dry weight compared to ISR 2000 (p≤0.05)
(Table 2). The effect of PSBs on all parameters on onion
growth is also given in Figure 2.
Figure 2. Influence of PSB strains (A:Negative control, B:
Bacillus subtilis MK-252
, C:
Enterobacter cloacae
(OB-169) on onion
plant height performance. Influence of PSB strains (D:
Enterobacter cloacae EB-14
, E:
Pantoea agglomerans EB-15
,
F :Negative control) on the number of onion plant leaves, Influence of PSB strains (G:Negative control, H:
Bacillus
subtilis MK-252
, J:ISR 2000) on onion bulb diameter, Influence of PSB strains (K:Negative control, L:
Bacillus
pumilus HB-228
, M:
Pantoea agglomerans EB-15
) on average onion root length
Şekil 2.PSB izolatların soğan bitki boyuna etkisi(A:Negatif kontrol, B: Bacillus subtilis MK-252, C: Enterobacter cloacae (OB-
169). PSB izolatların soğan yaprak sayısına etkisi((D: Enterobacter cloacae EB-14, E:Pantoea agglomerans EB-15,
F:Negatif kontrol. PSB izolatların soğan yumru çapına etkisi(G:Negatif control, H: Bacillus subtilis MK-252, J:ISR
2000). PSB izolatların soğan kök uzunluğuna etkisi(K:Negative control, L: Bacillus pumilus HB-228, M: Pantoea
agglomerans EB-15)
DISCUSSION
PSB colonize at the plant roots and shows useful
effects on plant growth and development through a
large diversity of methods. The specific mechanism by
which PGPR induces plant growth is not clearly
established, while different theories, such as the
production of IAA, repression of destructive organisms,
solubilization of phosphates and a rising mineral
uptake are generally believed to be related (Ludueña
et al., 2018). P is one of the important nutrients for
plants. Most of the P in the soil is insoluble and cannot
be used by the plants (Richardson, 2001). In this study,
PSB strains were evaluated for their effects on onion
growth. Phosphate solubilizing strains form a large
zone on NBRIP agar medium. However, some of the
strains did not produce large halo zones on an agar
plate. This is possibly because of the diverse diffusion
proportion of diverse organic acids secreted by the
PSB. Therefore, the PSBs were also screened in NBRIP
broth medium to calculate their phosphate solubilizing
efficiency. Thus, the phosphate solubilizing feature of
microorganisms were easily identified. Similar to the
present study, it has also been reported that phosphate
solubilization of microorganisms are different for
isolated and distinct locations. Recently endophytic
and epiphytic plant growth promoting bacteria (PGPB)
were isolated from healthy almond trees and plant
growth promoting mechanisms were characterized. By
using MALDI-TOF analyses, 19 Gram-negative
isolates, belonging to
Serratia, Pseudomonas,
Ochrobactrum
and
Enterobacter
genus and 26 Gram-
positive isolates, belonging to
Bacillus
and
Arthrobacter
genus were identified (Aktan and Soylu,
2020). In their study, 46 isolates were positive for
siderophore production, 45 isolates for ammonium
production, 35 isolates solubilized phosphorus varying
ratios, and all isolates produced IAA. For the study
were used solid and liquid medium, and the phosphate
solubilizing ability of microorganisms were detected in
a range from 80 to 100 ppm (Audipudi et al., 2012). In
another study, the isolation of PSB was done using
Pikovskaya’s agar. In this study, thirty-two strains
were inoculated on Pikovskaya’s agar plates to
evaluate the P solubilization index (PSI). Researchers
calculated the SI of PSB strains ranging from 1.8 to 5.0
(Alia et al., 2013). The beneficial effect of PSBs in
providing sufficient levels of mineral nutrients,
particularly P, in crop production has been formally
reported. Verma et al. (2015), reported on the PSB
Enterobacter, Pantoea, Azotobacter, Burkholderia,
A
B
C
D
E
G
F
H
J
K
L
M
KSÜ Tarım ve Doğa Derg 24 (5): 1084-1092, 2021
KSU J. Agric Nat 24 (5): 1084-1092, 2021
Araştırma Makalesi
Research Article
1090
Citrobacter, Pseudomonas
and
Bacillus
in wheat.
There have been investigations of bacteria belonging to
genus
Bacillus, Enterobacter, Pseudomonas, Serratia
that solubilize the otherwise insoluble P compounds
and increase plant growth (Hameeda et al., 2008;
Duman and Soylu, 2019). They observed that five
inoculated bacterial strains with PSB and other PGPR
traits increased the plant biomass by 20 to 40% under
greenhouse conditions. Suri and Choudhary (2013),
state that application of PSB improved the
productivity, protein content, and nutrient uptake in
soybean (Glycine max).Telek et al.(2019), also reported
the effects of rhizobacteria were found favorable on
such characteristics as fruit seeds fresh weight, fruit
seeds dry weight, and fruit flesh fresh weight, fruit
flesh dry weight, fruits wet weight and root length.
Biswas et al. (2018), showed that the PSB strains
Bacillus megaterium
(MF589715),
Staphylococcus
haemolyticus
(MF 589716) and
Bacillus licheniformis
(MF 589720) sterilized seeds of mung beans (
Vigna
radiata
), and indicated a greater germination ratio and
a higher growth under bacterium-enriched conditions.
Duman and Soylu (2019) recently conducted a study
for determination of plant growth-promoting traits
(such as IAA, phosphate solubilization) and
antagonistic potentials (such as siderophore and
ammonia productions) of endophytic plant growth-
promoting bacteria (PGPB) from healthy bean plants
growing in different regions of Turkey against
bacterial halo blight disease agent
Pseudomonas
syringae
pv.
phaseoli in vitro
conditions. Among the
tested antagonist bacterial isolates, 10 isolates were
positive for the production of α-amylase, 7 isolates
positive for phosphate solubilization, 29 isolates
positive for siderophore production, 11 isolates positive
for protease production. Among the bacterial isolates,
Acinetobacter calcoaceticus
produced a relatively large
amount of phosphatase by forming inhibition zones.
The increasing growth of onion caused by PSB found in
this study is in agreement with the findings of Gupta
et al., (2012). In their work, they investigated the effect
of four PSB on the growth of
Aloe barbadensis
in soil
containing tricalcium phosphate (TCP). Researchers
determined that the PSB:
Serratia marcescens,
Enterobacter hormaechei, Burkholderia gladioli
and
Pseudomonas synxantha
, increased P solubilization by
25 to 340 ppm in the liquid medium. Inoculation of PSB
increased leaf length by 39.5%, root length by 31.1%
and the total number of leaves by 48.1% in comparison
to the control plants. In this research, PSB strains
were found to be effective solubilizers of phosphates.
The ability of PSB strains to solubilize otherwise
insoluble P and convert it to a form available to plant
is an important characteristic under conditions where
P is a limiting factor for plant growth (Zhang et al.,
2012). Due to high antagonistic properties, efficient
isolates of
Bacillus
spp. may be used as biocontrol
agent against soilborne diseases as an alternative to
pesticides to promote organic and sustainable
agriculture (Soylu et al., 2020).
CONCLUSION
The study results clearly demonstrated the potential of
P solubilizers on onion plant growth. However, further
research is needed to understand the specific
mechanisms involved in the positive effects of PSB on
onion growth under field conditions.
ACKNOWLEDGMENTS
This research was supported by the project's financial
support of Kahramanmaras Sutcu Imam University
Scientific Research Projects (grant no. 2014/3-28 D) is
gratefully acknowledged. Ph.D. thesis of İdris
BEKTAŞ is partially used to produce this manuscript.
Statement of Conflict of Interest and Author’s
Contributions
Authors have declared no conflict of interest.
Author’s Contributions
The contribution of the authors is equal.
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