Agricultural
Communication
Biosci. Biotech. Res. Comm. 9(3): 530-538 (2016)
Screening of salt tolerant sugarcane endophytic
bacteria with potassium and zinc for their solubilizing
and antifungal activity
Mahboobeh Pirhadi
1
, Naeimeh Enayatizamir
1
, Hossein Motamedi
2
and Karim Sorkheh
3
1
Department of Soil Science, Faculty of Agriculture Shahid Chamran University of Ahvaz, Iran
2
Department of Biology, Shahid Chamran University of Ahvaz
3
Department of Anatomy and Plant Breeding, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Iran
ABSTRACT
Nowadays high consumption of fertilizers and fungicides in agriculture can increase the problem of soil salinity.
Some endobacteria survive in saline condition and induce plant resistance in harm environment. We examined
the diversity of halotolerant endophytic bacteria in the internal tissues of sugarcane roots, stems and leaves, with
zinc and potassium solubilizing ability and also their antifungal activity. Nutrient Agar medium was used to isolate
endophytic bacteria, and then they were screened in view of salinity tolerance on nutrient agar medium containing
different concentrations (100, 200, 400, 600 mM) of NaCl, CaCl
2
and MgCl
2
at ratio of 3: 2: 1. Zinc and potassium
solubilizing ability of isolates was respectively assessed using PVK medium containing 0.1% insoluble zinc com-
pound (zinc oxide) and Alexandrov agar medium containing vermiculite. The effect of superior isolates inoculation
to supply potassium for wheat was examined in greenhouse condition. As well as antifungal activity of isolates
against Fusarium sp. was determined using a dual culture technique. DNA of superior isolates was extracted and the
16S rRNA gene was partially sequenced and used for molecular identi cation. From 55 endophytic bacteria, 5 halo-
tolerant isolates which solubilize potassium and zinc selected to assay antifungal activity. The isolates were divided
into three genus were composed of Enterobacter cloacae, Bacillus pumilus, Pseodomonas sp. Inoculating Enterobacter
cloacae (R-1) with higher potassium solubilizing index into pot caused to increase uptake of K by wheat. Antifun-
gal activity of Pseodomonas sp (S-49) and Enterobacter cloacae (R-10) was higher than other isolates. These results
showed that some of isolates are integral part of sugarcane as endophytic bacteria survive in saline environment and
have antifungal activity.
KEY WORDS: BIOFERTILIZER, INHIBITION, SALINITY, DISSOLUTION
530
ARTICLE INFORMATION:
*Corresponding Author: n.enayatzamir@scu.ac.ir
Received 10
th
Sep, 2016
Accepted after revision 30
th
Sep, 2016
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007
Thomson Reuters ISI ESC and Crossref Indexed Journal
NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006
© A Society of Science and Nature Publication, 2016. All rights
reserved.
Online Contents Available at: http//www.bbrc.in/
Mahbobeh Pirhadi et al.
INTRODUCTION
Bacteria that exist within plant tissues during at least
one period of their life-cycles without any adverse in u-
ences on plant growth called endophyte. These micro-
organisms set up a mutualistic relationship with the
plants, because of ecological advantages to them. Endo-
phytic bacteria provide more bene ts than rhizospheric
bacteria because: endophytic bacteria living inside of
organs and tissues of the plant that keeps safe them from
unfavorable environmental conditions than in the rhizo-
sphere; they cannot be washed away by rainfall, runoff
or irrigation such as rhizospheric bacteria; they are less
exposed to UV radiation and there isn’t a lot of compe-
tition among them as in the rhizosphere, (Coêlho et al.
2011, Gaiero et al. 2013, Hidayati et al. 2014, Jhala et al.
2015 and Yuan et al. 2015).
Endophytic bacteria have been distributed in many
plant species and isolated from different plant organs
such as roots, stems, leaves, fruit,  owers and seeds. This
microbial community may play an important role in
agriculture by contributing plant development through
producing phytohormones, siderophores increasing
resistance to pathogens, promoting biological nitrogen
xation and antibiotic production, (O’Sullivan and Gara
1992, Pal et al. 2001, Han et al. 2005 Strobel and Daisy
2003 Karthikeyan et al. 2005 and Feng et al. 2006 and
El-Deeb et al. 2013).
Sugarcane (Saccharum of cinarum L.) is a major
crop in Iran, where it is grown for production of sugar,
bioethanol, and its waste such as bagasse and vinasse
can be used to conserve soil against erosion. Conse-
quently, sustaining and enhancing the growth and yield
of sugarcane have become a major focus of research.
The growth and performance of sugarcane in the field
are adversely affected by a number of abiotic and biotic
factors, including soil salinity and a wide range of fun-
gal and bacterial diseases. On the other hand to get sus-
tainable and organic agriculture it is necessary to use
soil potential and reduce utilization of chemical fertiliz-
ers and pesticides. Potassium is one of the major nutri-
ents, essential for plant growth. Potassium is associated
with movement of water, nutrients, and carbohydrates
in plant tissue. If potassium is de cient or not supplied
in adequate amounts, growth is stunted and yields are
reduced, (Ashley et al. 2006).
Most of the potassium in soil exists in various insol-
uble minerals, (Goldstein 1994). Microorganisms play an
important role to release potassium from minerals and
supply soluble K for plant. These bacteria are usually
known as potassium solubilizing bacteria or biological
potassium biofertilizers. Among the micronutrients, zinc
de ciency often happens in crops due to low solubil-
ity of zinc in soil, (Iqbal et al. 2010). The solubility of
Zn depends upon soil pH, cationic competition and soil
moisture, (Vasanthi et al. 2012). The majority of soils
under sugarcane cultivation in the Khuzestan province
have more than 40% of lime and their pH is greater than
7, hence are often zinc de cient. Some microorganisms
are able to dissolve the zinc-containing compounds and
release Zn, (Han and Lee 2006, Sharma et al. 2012 and
Diep and Hieu 2013).
Excessive using of pesticides causes environmen-
tal problems and adversely affects the health of living
organisms and this has prompted researchers to look
for new environment friendly solutions for controlling
plant pathogens. Recently, many studies have revealed
the potential of endophytic bacteria for biological con-
trol of fungal diseases, (Munif et al. 2012), (Szilagyi-
Zecchin et al. 2014), (Xu et al. 2007). The antifungal
properties of endophytic bacteria are attributed to their
ability to produce antibiotics, (Wang et al. 2013) or/and
hydrolytic enzymes (Bacon and Hinton 2011).
In sugarcane, most of the research on endophytic
bacteria has focused on diazotrophs (Muangthong et al
2015), (Boddey et al. 2003), (Ramos et al. 2011), and to
our knowledge there is not any report on investigation
of potassium and zinc solubilizing ability of endophytic
bacteria from sugarcane. So, the present study aimed to
isolate salt tolerance endophytic bacteria from sugarcane
with potassium and zinc solubilizing ability and anti-
fungal activity. Understanding the diversity of bene cial
endophytic bacteria and their role in plant production
has important implication in agriculture to encourage
using them as eco-friendly approaches to manage crop
production and sustain agro-ecosystem, (Schenk et al.,
2012).
MATERIAL AND METHODS
ISOLATION OF ENDOPHYTIC BACTERIA
Bacteria were isolated from the tissues of sugar cane
grown on the Debal-Khazaei agro-industrial unit
located in Ahvaz-Abadan road in the province of Khoz-
estan, Iran (latitude 31°05’; longitude 48°30’). To iso-
late endophytic bacteria, samples of roots, leaves and
stems weighing 10.0 g were washed in tap water and
surface sterilized according to the method of Marcon et
al. (2002). The inner section of the stem was removed
using a sterilized hole punch. The organs were placed in
separate sterile mortar and were well crushed into 90 ml
sterile physiologic serum until to obtain a homogeneous
suspension and serially diluted with sterile physiologic
serum. About 50μl of 10
3
-10
8
dilutions was inoculated
onto nutrient agar medium and incubated at 28ºC for 72
h. All bacterial colonies were puri ed according to their
morphology on nutrient agar.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SCREENING OF SALT TOLERANT SUGARCANE ENDOPHYTIC BACTERI 531
Mahbobeh Pirhadi et al.
SCREENING OF ENDOPHYTIC BACTERIA FOR
CHARACTERISTICS OF SALT TOLERANCE
To determine salt tolerance of isolates, from the over-
night culture of each isolates were dropped in triplicate
on nutrient agar plate containing different concentra-
tions (100, 200, 400, 600 mM) of NaCl, CaCl
2
and MgCl
2
mixture at a ratio of 3: 2: 1 and incubated at 30°C for
three days. Growth colony diameter was recorded every
day. A control without any salt addition was kept to
compare colony growth. Percentage reduction of growth
in salt amended media was calculated by using the for-
mula (100 x A-B /A), where A is colony diameter growth
in control plate in ‘mm’ of the isolate and B is colony
diameter growth in salt amended plate.
POTASSIUM SOLUBILIZATION ASSAY
Alexandrov culture medium was used to assay the isolates
potassium solubilizing ability with following ingredi-
ents: 0.5% glucose, 0.2% Ca
3
(PO
4
)
2
, 0.05% MgSO
4
.7H
2
O,
0.01% CaCO
3
, 0.0006% FeCl
2
, 0.15% K
2
HPO
4
or vermicu-
lite and Agar with pH7.0 (Aleksandrov et al. 1967). 7μl
from the overnight culture of isolates were dropped on
Alexandrov agar medium and were incubated at 30ºC
for 3 days. Colony diameter was measured at third day.
Solubility index was calculated by using the clear zone
diameter / colony diameter formula (Shanware et al.
2014).
EFFECT OF SELECTED ISOLATE ON WHEAT
PLANT GROWTH
Based on solubilizing ability, one isolate were selected
to test its effect on potassium uptake by wheat. For this,
greenhouse experiment consisted of a 2×3 factorial in
complete randomized design whit four replications was
arranged. The factors included two levels of inoculation
(with and without inoculant) and three levels of potas-
sium. The nitrogen fertilizer used 140 kg/ha urea, 90
mg/kg P as single super phosphate before seed sowing to
prevent possible effects of nutrient de ciency. Potassium
from source of potassium sulfate applied at three levels
of 120 mg/kg (K3), 60 mg/kg (K2) and without potas-
sium application (K1). The soil was completely mixed
and irrigated by distilled water to  eld capacity (70%).
Seeds were surface sterilized in 10% sodium hypochlorite
solution for 10 min, then rinsed with sterilized distilled
water and air dried (Ahmad and Haddad 2011).The seeds
were planted in pots containing 4kg of steam sterilized
clay loam soil. Overnight culture of isolate was diluted
to10
6
CFUmL
−1
and then applied under seeds. Potassium
concentration of leaves three month after growing was
analyzed after dry digestion of organ using  ame pho-
tometer (Gupta 2004). The data were analyzed statisti-
cally via SAS version 9.1. Mean comparisons was done
using Duncan test at 5%.
ZINC SOLUBILIZATION ASSAY
The isolates were examined for zinc solubilization abil-
ity by using modi ed PVK medium (Pikovskaya 1948).
The medium including: 10.0 g glucose, 1.0 g ammonium
sulphate, 0.2 g potassium chloride, 0.2 g dipotassium
hydrogen phosphate, 0.1 g magnesium sulphate, 0.2g
Yeas, 0.1% insoluble zinc from source of ZnO in1000
ml distilled water with pH 7.0. From the overnight cul-
ture of isolates 7μl were dropped on plates containing
the mentioned medium and incubated at 30ºC for 72 h.
Colony and halo zones diameter was recorded. Solubility
index was calculated by using the clear zone diameter /
colony diameter formula, (Ramesh et al. 2014).
ANTIFUNGAL ACTIVITY ASSAY
The isolates with salinity tolerance and potassium and
zinc solubilization were screened for in vitro antago-
nism against Fuzarium. sp on PDA plates using a dual
culture technique. The controls were prepared using pure
cultures of fungi. The plates were incubated at 30°C until
fungal mycelia covered the agar surface of the control
plates. Radial growth of Fuzarium. sp was measured on
the 5th days after inoculation. The inhibition percent in
the mycelial development of the pathogen fungus was
calculated by the formula: R1=(C-T)/C x 100; where RI is
the inhibition percentage of the radial mycelial growth,
C is the radial growth of the pathogen in the control
(mm), and T is the radial growth of the pathogen in dual
culture, (Ohike et al. 2013).
IDENTIFICATION OF ENDOPHYTIC BACTERIA
The superior isolates from point of characteristics
related to plant growth promotion were identi ed by
morphological,physiological and biochemical character-
istics with reference to Bergeys Manual of Systematic
Bacteriology and by sequencing the 16S rRNA, (Weis-
burg et al., 1991).
RESULTS AND DISCUSSION
In total, 55 endophytic bacteria from the root (10 isolates),
stem (21 isolates) and leaves (24 isolates) of the sugarcane
were isolated. The results of isolates response to differ-
ent level of salt concentration have presented in table 1.
Salt tolerance of microorganisms depends on the range
of external salinity over which it is able to sustain these
conditions in the cytoplasm [(Yeo 1998). The salinity tol-
erance of isolates was classi ed as very resistant (0-25%
growth inhibition), resistant (25-50% growth inhibition),
532 SCREENING OF SALT TOLERANT SUGARCANE ENDOPHYTIC BACTERI BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SCREENING OF SALT TOLERANT SUGARCANE ENDOPHYTIC BACTERI 533
Mahbobeh Pirhadi et al.
Table 1: Mean inhibition percentage of isolates growth under different level of salinity
Salinity level (mM) Situation Salinity level (mM) Situation
Isolate code Source 100 200 400 600 Isolate
code
Source 100 200 400 600
1N- R-1 Root 60.0 50.0 30.0 40.0 Resistant 29N- S-29 Stem 30.0 30.0 20.0 34.5 Resistant
2N- R-2 Root 63.6 63.6 36.4 45.0 Resistant 30N- S-30 Stem 33.3 22.2 0.0 5.0 Very resistant
3N- R-3 Root 38.5 34.6 26.9 15.0 Very resistant 33N- S-33 Stem 42.9 28.6 14.3 5.0 Very resistant
4N- R-4 Root 50.0 53.6 28.6 22.5 Very resistant 34N- B-34 Leaf 14.3 0.0 -7.1 -5.0 Very resistant
5N- R-5 Root 57.4 57.9 36.8 40.0 Resistant 35N- B-35 Leaf 50.0 40.0 30.0 35.0 Resistant
6N- R-6 Root 42.9 42.9 32.1 29.0 Resistant 36N- S-36 Stem 68.8 75.0 68.8 65.0
7N- R-7 Root 63.6 56.8 45.5 52.5 Moderate 37N- B-37 Leaf 50.0 50.0 25.0 -15.0 Very resistant
8N- R-8 Root 60.0 55.0 50.0 45.0 Resistant 38N- S-38 Stem 56.3 46.9 37.5 55.0 Moderate
9N- R-9 Root 61.4 47.7 39.5 47.5 Resistant 39N- B-39 Leaf 100 55.6 38.9 27.5 Resistant
10N- R-10 Root 56.7 47.2 50.0 35.0 Resistant 40N- S-40 Stem 100 37.5 40.6 35.0 Resistant
11N- B-11 Leaf 81.7 76.7 73.3 63.5 Moderate 41N- S-41 Stem 100 50.0 25.0 22.5 Very resistant
12N- B-12 Leaf 43.3 33.3 22.2 40.0 Resistant 42N- S-42 Stem 100 68.8 70.3 69.0 Moderate
13N- B-13 Leaf 50.0 50.0 40.0 45.0 Resistant 43N- S-43 Stem 73.4 68.8 65.6 59.5 Moderate
14N- S-14 Stem 30.0 25.0 39.5 45.0 Resistant 45N- S-45 Stem 100 66.7 58.3 57.5 Moderate
15N- B-15 Leaf 35.3 26.5 28.8 20.0 Very resistant 46N- S-46 Stem 70.3 62.5 62.5 58.0 Moderate
16N- B-16 Leaf 50.0 50.0 25.0 20.0 Very resistant 47N- B-47 Leaf 81.7 76.7 73.3 70.0 Moderate
17N- B-17 Leaf 68.1 65.6 62.5 60.0 Moderate 48N- S-48 Stem 47.4 36.8 26.3 25.0 Resistant
18N- S-18 Stem 62.5 62.5 59.4 60.0 Moderate 49N- S-49 Stem 65.0 52.5 40.0 32.5 Resistant
19N- B-19 Leaf 75.0 65.0 67.0 65.0 Moderate 50N- S-50 Stem 61.5 61.5 38.5 30.0 Resistant
20N- B-20 Leaf 100 100 73.7 69.0 Moderate 51N- B-51 Leaf 83.0 82.1 74.1 75.0 Moderate
21N- B-21 Leaf 68.8 65.6 62.5 61.0 Moderate 52N- B-52 Leaf 100 75.0 55.6 55.0 Moderate
22N- B-22 Leaf 66.7 60.0 60.0 60.0 Moderate 53N- B-53 Leaf 28.1 25.0 25.0 25.0 Resistant
23N- B-23 Leaf 45.0 45.0 49.0 35.0 Resistant 54N- S-54 Stem 100 60.0 57.5 57.5
24N- B-24 Leaf 44.4 11.1 0.0 -10.0 Very resistant 55N- B-55 Leaf 56.8 54.5 50.0 60.0 Moderate
25N- S-25 Stem 66.7 53.3 51.7 51.0 Moderate 56N- B-56 Leaf 60.0 53.3 50.0 50.0 Resistant
26N- B-26 Leaf 62.5 54.7 56.3 55.0 Resistant 57N- B-57 Leaf 100 100 37.5 30.0 Resistant
27N- S-27 Stem 64.1 53.1 50.0 49.0 Resistant 59N- S-59 Stem 100 25.0 21.9 22.5 Resistant
28N- S-28 Stem 62.5 59.4 56.3 52.0 Moderate 29N- S-29 Stem 44.4 44.4 33.3 15.0 Very resistant
534 SCREENING OF SALT TOLERANT SUGARCANE ENDOPHYTIC BACTERI BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Mahbobeh Pirhadi et al.
moderate resistant (50-75% growth inhibition), sensi-
tive (75-100% growth inhibition) and very sensitive
(100% growth inhibition). Isolates growth decreased by
increasing salt concentration in the medium. The results
revealed that increasing salt concentration induced some
isolates growth and seams they are halophile. Most of
isolates classi ed in moderate group. Microorganisms
possess multiple strategies to overcome salinity. One of
these strategies is the osmoprotectants accumulation in
the cytoplasm, (Mishra and Sharma 2012). This important
feature demonstrates the potential of endophytic bacteria
to alleviate salt stress of host plant by exopolysaccharides
production which restricts sodium adsorption by plant
(Milosevic et al. 2012) or indirectly by auxin production,
(Yaish et al. 2015).
POTASSIUM SOLUBILIZATION ASSAY
The dissolution of insoluble potassium base on halo
zone around the colonies of the isolates has shown in
table 2. Highest solubility index recorded in R-1 iso-
late (Enterobacter) in presence of both vermiculite and
K
2
HPO
4
followed by S-49 isolate (Pseudomonas).
Most of the potassium solubilizing bacteria obtained
from the plant rhizosphere [(Murali et al 2005), (Zhang
and Kong 2014)] and identi ed as Bacillus sp., Pseu-
domonas sp., Bacillus mucilaginosus [(Liu 2001), (Murali
et al. 2005), (Zhou et al. 2006), (Sugumaran and Janart-
hanam 2007)]. However, there are relatively few stud-
ies on potassium solubilizing bacteria in the sugarcane
rhizosphere. Ghevariya and Deasi (2014) identi ed Pseu-
domonas sp. capable solubilize potassium from mica.
The ability of potassium solubilizing by Enterobacter
spp. has been already reported (Zhang and Kong 2014).
Yuan et al. (2015) isolated Enterobacter spp. as endo-
phytic bacteria from rhizome, root, leaves and stem of
Moso Bamboo with the ability of dissolving potassium.
PLANT GROWTH AND POTASSIUM UPTAKE BY
WHEAT
Inoculation in uence on plant growth and potassium
concentration has been shown in  gure1.The results
showed that potassium concentration of leaves of
wheat signi cantly (P≤0.05) increased by inoculation
and increased level of potassium application. The least
potassium concentration was recorded in un-inoculated
plants without potassium application, and the highest
observed in inoculated plant and with potassium appli-
cation (K3) followed by inoculated plant with second
level of potassium application (K2) without signi cant
difference between them. From this obtained results, it is
clear that bio-fertilizer application were more effective
to increase potassium concentration.
Increasing the bioavailability of P and K in soils with
inoculation of PGPR such as Bacillus mucilaginosus, by
producing organic acids and other chemical which may
lead to increased K and P uptake and plant growth, was
reported by many researchers, (Sheng et al. 2002; Lin et
al. 2002). Nutrient uptake enhance by inoculated plants
may attribute to the production of plant growth regula-
tors by the bacteria at the root interface, which stimu-
lated root development and resulted in better absorption
of water and nutrients from the soil, (Abbasi et al. 2011).
Table 2: Colony and halo Diameter of isolates in Alexandrov medium
Isolate
code
sample Diameter
Genus
name
Vermiculite K
2
HPO
4
Colony
diameter
(mm)
Halo
diameter
(mm)
HD/CD
(mm)
Colony
diameter
(mm)
Halo
diameter
(mm)
HD/CD
(mm)
1N- R-1 Root Enterobacter 5 10 2 4 5 1.25
5N- R-5 Root Streptococcus 4 1 0.25 4 1 0.25
7N- R-7 Root Enterobacter 3.5 0.5 0.14 - - -
8N- R-8 Root Streptococcus 4 1 0.25 - - -
9N- R-9 Root Enterobacter 4 2 0.5 5 5 1
10N- R-10 Root Enterobacter 4 1 0.25 - - -
41N- S-41 Stem Arthrobacter 8 2 0.25 8 1 0.125
49N- S-49 Stem Pseudomonas 6 10 1.6 6 4 0.66
51N- B-51 Leaf Arthrobacter 5 3 0.6 8 2.5 0.31
52N- B-52 Leaf Enterobacter 5 4 0.8 10 2 0.2
56N- B-56 Leaf Bacillus -- ----
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SCREENING OF SALT TOLERANT SUGARCANE ENDOPHYTIC BACTERI 535
Mahbobeh Pirhadi et al.
FIGURE 1. Mean comparison of treatments effect Potassium level (K) and bacterium on the
dry matter of wheat and K concentration in plant Means followed by the same letters are
not signi cantly difference based on duncan at =5%.
FIGURE 2. Growth inhibition of Fusarium sp. by endophytic bacteria
ZN SOLUBILIZATION ASSAY
The bacteria were able to dissolve zinc in plates pro-
duced clear halo around them (Table 3). Five isolates
had the ability of zinc oxide dissolution. Isolates R-7
and R-10 had the highest rate of zinc dissolution among
the  ve zinc solubilizing bacteria. Both of the bacte-
ria were isolated from the roots of sugarcane and are
belonging to the genus Enterobacter. Zinc solubilizing
bacteria have been isolated from different plant rhizhos-
phere. Enterobacter aerogenes and Pseudomonas aerugi-
Table 3: Colony and halo Diameter of isolates in
medium containing ZnO
Isolate
code
Colony
diameter (mm)
Halo zone
diameter (mm)
HD/CD
(mm)
1N- R-1 7 7 1
7N- R-7 5.5 6.5 1.18
10N- R-10 5 7 1.4
22N- B-22 3 3 1
49N- S-49 4 4 1
536 SCREENING OF SALT TOLERANT SUGARCANE ENDOPHYTIC BACTERI BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Mahbobeh Pirhadi et al.
nosa from agricultural  elds (Sunithakumari et al. 2016),
Bacillus sp. from sugarcane (Vasanthi et al. 2012) and
Pseudomonas sp. and Bacillus sp. from soybean rhizo-
sphere (Sharma et al. 2012) have been reported to solu-
bilize insoluble zinc. Of the basic mechanisms for the
dissolution of zinc-containing compounds is the secre-
tion of organic acids which reduce the pH and thereby
increase the availability of zinc (Sunithakumari et al.
2016).
The biochemical information and molecular identi-
cation of isolates showed R-1, R-7, R-10 isolates are
belonging to Enterobacter cloacae under accession num-
ber of KX262849, KX262850 and KX262851 respec-
tively and B-22 and S-49 isolates were Bacillus pumilus
(
KX262852) and Pseudomonas sp. (KX262853) respec-
tively. Enterobacter isolate has the ability to withstand
different abiotic stresses such as salinity (Tantawy et al.
2009) and increased the resistance, growth and nitrogen
xing of inoculated plant under salt stress (Tantawy et
al. 2009).
IN VITRO ANTAGONISTIC ACTIVITY OF
ISOLATES
Results showed that each isolate could prevent Fusarium
growth but there was difference among them. Highest
growth inhibition was recorded by applying S-49 isolate
which followed by R-10 isolate (Figure 1). Three other
isolates ability to inhibit Fusarium growth in dual cul-
ture was less than mentioned two isolates which proves
their ef cacy in management of crop diseases. However,
it is necessary to do complementary experiment to con-
rm their antagonistic ability and also their mechanisms
to inhibit pathogens growth.It has been reported antago-
nistic activity of Pseudomonas aeruginosa, P. uores-
cens and P. putida isolated from the stalks of sugar-
cane against Colletotrichum falcatum (Viswanathan et
al. 2003), Bacillus amyloliquefaciens, B. subtilis, and B.
thuringiensis from banana against Fusarium oxysporum
f. sp cubense and Colletotrichum guaranicola, (Souza
et al. 2014).
Most bacterial species with biological control poten-
tial isolated from the soil rhizosphere, but their use is
limited because they can hardly colonize plant roots,
perhaps endophytic bacteria can be good choice for
this purpose (Chang-Qing, Zhao et al. 2008). The bio-
control potential of enophytic bacteria against Verti-
cilium dahlia has been reported by Ferrara et al. (2012).
Plant growth promoting endobacteria may induce the
plant’s defense system against pathogens or enhance
plant resistance through production of antimicrobial
compounds (Heydari and Pessarakli, 2010). The growth
inhibition of pathogen may be related to antibiotic and
toxin secretion (Wang et al. 2013), compete with patho-
gens for space and nutrients impoverishment and pH
alteration in the medium (Backman and Sikora 2008) or
cell wall degrading enzymes secretion such as chitinase
and -1, 3-glucanase (Roberts and Selitrennikoff 1988).
CONCLUSION
Salinity is one of the most widespread constraints to soil
fertility. During the latest years, a great attention has
been paid to saline soils due to the reducing arable land,
and of the increasing demand for agricultural produc-
tion of areas in uenced by secondary salinisation pro-
cesses. Our study revealed a high plasticity of bacterial
phyla that evidently possess genera and species adapt-
able to salinity conditions with plant growth promoting
properties.There is scope for use of zinc and potassium
solublizing bacteria as potential biofertilizers for recla-
mation saline soils of local area because isolates belongs
to the same soil. From 59 isolates,  ve isolate having
plant growth promotion (potassium and zinc solubiliz-
ing ability) identi ed. We also showed that Pseudomonas
sp. and Enterobacter cloacae have the ability to inhibit
the growth of Fusarium. It is interesting to investigate
which mechanisms would be related to fungal inhibition
activity of strains in our study. Our experiment demon-
strated the advantage of isolate R-1 (Enterobacter cloa-
cae) inoculation on potassium uptake by wheat. High
cost of chemical fertilizers and harmful environmental
effects of them caused to recommend using of biologi-
cal fertilizer to increase soil fertility. Future studies are
promising to test the biotechnological potential of these
strains under  eld conditions in the hope that they will
contribute as an alternative source of biological fertilizer
and biological control.
ACKNOWLEDGMENTS
We thank Shahid Chamran University of Ahvaz for aca-
demic support and Iran National Science Foundation for
nancial support under grant number of 93045179.
REFERENCES
Abbasi M., Sharif S., Kazmi M., Sultan T., Aslam M. (2011).
Isolation of plant growth promoting rhizobacteria from wheat
rhizosphere and their effect on improving growth, yield and
nutrient uptake of plants. Plant Biosystems, 145(1): 159-168.
Aleksandrov V., Blagodyr R., Ilev I. (1967). Liberation of
phosphoric acid from apatite by silicate bacteria. Mikrobiol Z
(Kiev), 29:111-114.
Araújo W.L., Marcon J., Maccheroni W., Elsas J.D., Vuurde
J.W., Azevedo J.L. (2002). Diversity of endophytic bacterial
populations and their interaction with Xylella fastidiosa in cit-
rus plants. Appl. Environ. Microbiol. 68(10): 4906-4914.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SCREENING OF SALT TOLERANT SUGARCANE ENDOPHYTIC BACTERI 537
Mahbobeh Pirhadi et al.
Ashley M .K., Grant M., Grabov A. (2006). Plant responses to
potassium de ciencies: a role for potassium transport proteins.
J. Exp. Bot. 57(2): 425–436.
Bacon C.W., Hinton D.M. (2011). Bacillus mojavensis: its
endophytic nature, the surfactins, and their role in the plant
response to infection by Fusarium verticillioides. In Bacteria in
Agrobiology: Plant Growth Responses. pp. 21-39.
Backman PA and Sikora RA (2008) Endophytes: An emerging
tool for biological control Biol.Control. 46: 1-3
Boddey R. M., Urquiaga S., Alves B. J. R., Reis V. (2003). Endo-
phytic nitrogen fixation in sugarcane: present knowledge and
future applications. Plant Soil. 252: 139–149.
Chang-Qing ZS-MS., Zhao WY-XLJ., Xian-Cheng X-YZ.
(2008). Isolation and Characterization of Antifungal Endo-
phytic Bacteria from Soybean. Microbiology, 10, 27.
Coêlho M.M., Ferreira-Nozawa M.S., Nozawa S.R., Santos A.L.
(2011). Isolation of endophytic bacteria from arboreal species
of the Amazon and identi cation by sequencing of the 16S
rRNA encoding gene. Genet Mol Biol. 34(4): 676-680.
de Santi Ferrara F. I., Machado Oliveira Z., Soto Gonzales H.
H., Segal Floh E. I., Ramos Barbosa H. (2002). Endophytic and
rhizospheric enterobacteria isolated from sugar cane have dif-
ferent potentials for producing plant growth-promoting sub-
stances. Plant Soil. 353: 409–417.
Diep C.N., Hieu T.N. (2013). Phosphate and potassium solu-
bilizing bacteria from weathered materials of denatured rock
mountain, Ha Tien, Kiên Giang province Vietnam. Am. J. Life
Sci. 1(3): 88-92.
El-Deeb B., Fayez K., Gherbawy Y. (2013). Isolation and char-
acterization of endophytic bacteria from Plectranthus tenui o-
rus medicinal plant in Saudi Arabia desert and their antimicro-
bial activities. J. Plant Interact. 8(1): 56-64.
Feng Y., Shen D., Song W. (2006). Rice endophyte Pantoea
agglomerans YS19 promotes host plant growth and affects
allocations of host photosynthates. J. Appl. Microbiol. 100(5):
938-945.
Ghevariya K.K., Desai P.B. (2014). Rhizobacteria of sugarcane:
In vitro screening for their plant growth promoting potentials.
Res. J. Rec. Sci. 3: 52-58.
Gaiero J.R., McCall C.A., Thompson K.A., Day N.J., Best A.S.,
Dun eld K.E.( 2013). Inside the root microbiome: bacterial root
endophytes and plant growth promotion. Am. J. Bot. 100(9):
1738-1750.
Goldstein AH (1994) Involvement of quino protein glucose
dehydrogenase in the solubilization of exogenous mineral by
Gram negative bacteria In: Torriani Gorini A., A Yagli, E Silver
S Eds Phosphates in Microorganisms: Cellular and Molecular
Biology ASM Press Washington DC 194-203.
Gupta P.K. (2004). Soil, Plant, Water And Fertilizer Analysis.
(Agrobios (India).
Han H-S., Lee K. (2006): Effect of co-inoculation with phos-
phate and potassium solubilizing bacteria on mineral uptake
and growth of pepper and cucumber. Plant Soil Environ. 52(3):
130-136.
Han J., Sun L., Dong X., Cai Z., Sun X., Yang H., Wang Y., Song
W. (2005).Characterization of a novel plant growth-promoting
bacteria strain Delftia tsuruhatensis HR4 both as a diazotroph
and a potential biocontrol agent against various plant patho-
gens. Syst Appl Microbiol 28(1): 66-76.
Heydari A., Pessarakli M. (2010). A review on biological con-
trol of fungal plant pathogens using microbial antagonists. J.
Biol. Sci. 10: 273–290.
Hidayati U., Chaniago I.A., Munif A., Santosa D.A. (2014). Potency
of Plant Growth Promoting Endophytic Bacteria from Rubber
Plants (Hevea brasiliensis Müll. Arg.). J. Agron. 13(3): 147-152.
Iqbal U., Jamil N., Ali I., Hasnain S. (2010). Effect of zinc-
phosphate-solubilizing bacterial isolates on growth of Vigna
radiata. Annal Microbiol 60(2): 243-248.
Jhala Y., Shelat H., Vyas R., Panpatte D. (2015). Biodiversity of
Endorhizospheric Plant Growth Promoting Bacteria. J. Biofer-
til. Biopestic. 6(1): 1-5.
Karthikeyan M., Bhaskaran R., Radhika K., Mathiyazhagan S.,
Jayakumar V., Sandosskumar R., Velazhahan R. (2005). Endo-
phytic Pseudomonas  uorescens Endo2 and Endo35 induce
resistance in black gram (Vigna mungo L. Hepper) to the patho-
gen Macrophomina phaseolina. J. Plant Interact. 1(3): 135-143.
Lin Q.M., Rao Z. H., Sun Y X., Yao J., Xing L.J. (2002). Identi -
cation and practical application of silicate dissolving bacteria.
Agr. Sci. China. 1: 81–85.
Liu G. (2001): Screening of silicate bacteria with potassium
releasing and antagonistical activity. Chin. J. Appl. Environ.
Biol. 7 (1): 66–68.
Milosevic N., Marinkovic J B., Tintor B. (2012). Mitigating abi-
otic stress in crop plants by microorganisms, Proc. Nat. Sci.
Matica Srpska Novi. Sad. 123: 17-26.
Mishra I.G., Sharma A. (2012): Exogenously supplied osmopro-
tectants confer enhanced salinity tolerance in rhizobacteria. J.
Ecobiotechnol. 4: 11-13.
Muangthong A., Youpensuk S., Rerkasem B. (2015). Isolation
and characterisation of endophytic nitrogen  xing bacteria in
sugarcane. Trop Life Sci Res, 26(1): 41–51.
Munif A., Hallmann J., Sikora R. (2012). Isolation of endo-
phytic bacteria from tomato and their biocontrol activities
against fungal disease. Microbiol. Indones. 6(4); 148-156.
Murali G., Gupta A., Nair R.V. (2005). Variations in hosting
bene cial plant associated microorganisms by root (wilt) dis-
eased and  eld tolerant coconut palms of west coast tall vari-
ety. Current Sci. 89: 1922–1927.
Ohike T., Makuni K., Okanami M., Ano T. (2013). Screening of
endophytic bacteria against fungal plant pathogens. J. Envi-
ron. Sci. 25: 122-126.
O’Sullivan DJO., Gara F. (1992): Traits of  uorescent Pseu-
domonas sp. involved in suppression of plant root pathogens.
Microbiol. Rev. 56: 662–676.
Pal K., Tilak K., Saxcna A., Dey R., Singh C. (2001). Suppres-
sion of maize root diseases caused by Macrophomina phaseo-
lina, Fusarium moniliforme and Fusarium graminearum by
538 SCREENING OF SALT TOLERANT SUGARCANE ENDOPHYTIC BACTERI BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Mahbobeh Pirhadi et al.
plant growth promoting rhizobacteria. Microbiol. Res. 156(3):
209-223.
Pikovskaya R. (1948): Mobilization of phosphorus in soil in
connection with vital activity of some microbial species. Mik-
robiologiya. 17: 362–370.
Ramesh A., Sharma S.K., Sharma M.P., Yadav N., Joshi O.P.
(2014). Inoculation of zinc solubilizing Bacillus aryabhattai
strains for improved growth, mobilization and bioforti cation
of zinc in soybean and wheat cultivated in Vertisols of central
India. Appl. Soil Ecol. 73, 87– 96.
Ramos P. L., Van Trappen S, Thompson F.L., Rocha R. C. S., Bar-
bosa H. R., De Vos P., Moreira-Filho C. A. (2011). Screening for
endophytic nitrogen- xing bacteria in Brazilian sugar cane vari-
eties used in organic farming and description of Stenotropho-
monas pavanii sp. nov. Int. J. Syst. Evol. Microbiol. 61, 926–931.
Roberts W.K., Selitrennikoff C.P. (1988). Plant and bacterial
chitinases differ in antifungal activity. Microbiology. 134(1):
169-176.
Schenk P.M., Carvalhais L.C., Kazan K. (2012). Unraveling
plant–microbe interactions: can multi-species transcriptomics
help? Trends Biotechnol. 30: 177–184.
Shanware A.S., Kalkar S.A. Trivedi M.M. (2014). Potassium Sol-
ublisers: Occurrence, Mechanism and Their Role as Competent
Biofertilizers. Int. J. Curr. Microbiol. App. Sci. 3: 622-629.
Sharma S.K., Sharma M.P., Ramesh A., Joshi O.P. (2012). Char-
acterization of zinc-solubilizing Bacillus isolates and their
potential to in uence zinc assimilation in soybean seeds. J.
Microbiol. Biotechnol. 22: 352-359.
Sheng X.F., He L.Y., Huang W.Y. (2002). The conditions of
releasing potassium by a silicate-dissolving bacterial strain
NBT. Agr. Sci. China, 1: 662–666.
Souza A., Cruz J., Sousa N., Procópio A., Silva G. (2014): Endo-
phytic bacteria from banana cultivars and their antifungal
activity. Genet. Mol. Res. 13: 8661-8670.
Strobel G., Daisy B. (2003). Bioprospecting for microbial endo-
phytes and their natural products. Microbiol. Mol. Biol. Rev.
67(4): 491-502.
Sugumaran P., Janarthanam B. (2007). Solubilization of potas-
sium containing minerals by bacteria and their effect on plant
growth. World J. Agric. Sci. 3(3): 350-355.
Sunithakumari K., Padma Devi S., Vasandha S. (2016) Zinc
solubilizing bacterial isolates from the agricultural  elds of
Coimbatore, Tamil Nadu, India. Curr. Sci.110(2): 196-205.
Szilagyi-Zecchin V.J., Ikeda A.C., Hungria M., Adamoski D.,
Kava-Cordeiro V., Glienke C., Galli-Terasawa L.V. (2014). Iden-
ti cation and characterization of endophytic bacteria from
corn (Zea mays L.) roots with biotechnological potential in
agriculture. AMB. Express 4(1): 1-9.
Tantawy A., Abdel-Mawgoud A., El-Nemr M., Chamoun Y.G.
(2009). Alleviation of salinity effects on tomato plants by
application of amino acids and growth regulators. Eur. J. Sci.
Res. 30(3): 484-494.
Vasanthi N., Saleena L. M., Raj S.A. (2012). Concurrent Release
of Secondary and Micronutrient by a Bacillus sp. Am. Eurasian
J. Agric. Environ. Sci. 12 (8): 1061-1064.
Viswanathan R., Sundar A.R., Premkumari S.M. (2003). Myco-
lytic effect of extracellular enzymes of antagonistic microbes
to Colletotrichum falcatum, red rot pathogen of sugarcane.
World J. Microbiol. Biotechnol. 19(9): 953-959.
Wang S., Wang W., Jin Z., Du B., Ding Y., Ni T., Jiao F. (2013).
Screening and diversity of plant growth promoting endophytic
bacteria from peanut. Afr. J. Microbiol. Res. 7(10): 875-884.
Weisburg W.G., Barns S.M., Pelletier D.A., Lane D.J. (1991).
16S ribosomal DNA ampli
cation for phylogenetic study. J.
Bacteriol.173(2): 697-703.
Xu D., Xia X., Xu N., An L. (20070. Isolation and identi cation
of a novel endophytic bacterial strain with antifungal activ-
ity from wild blueberryVaccinium uliginosum. Ann. Micro-
biol.57(4): 673-676.
Yaish M.W., Antony I., Glick B.R. (2015). Isolation and char-
acterization of endophytic plant growth-promoting bacteria
from date palm tree (Phoenix dactylifera L.) and their potential
role in salinity tolerance. Antonie van Leeuwenhoek, 107(6):
1519-1532.
Yeo A. (1998): Molecular biology of salt tolerance in the con-
text of whole-plant physiology. J. Exp. Bot. 49(323): 915-929.
Yuan Z-S., Liu F., Zhang G-F. (2015). Isolation of culturable
endophytic bacteria from moso bamboo (Phyllostachys edulis)
and 16S rDNA diversity analysis. Arch. Biol. Sci. 67(3): 1001-
1008.
Zhang C., Kong F. (2014). Isolation and identi cation of potas-
sium-solubilizing bacteria from tobacco rhizospheric soil and
their effect on tobacco plants. Appl. Soil Ecol. 82, 18–25.
Zhou H., Zeng X., Liu F., Qiu G., Hu Y. (2006). Screening,
identi cation and desilication of a silicate bacterium. J. Cent.
South Univ. Technol. 13: 337–341.