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

, Naeimeh Enayatizamir

, Hossein Motamedi

and Karim Sorkheh

Department of Soil Science, Faculty of Agriculture Shahid Chamran University of Ahvaz, Iran

Department of Biology, Shahid Chamran University of Ahvaz

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

and MgCl

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

Sep, 2016

Accepted after revision 30

Sep, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

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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

-10

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

and MgCl

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

(PO

)

, 0.05% MgSO

.7H

0.01% CaCO

, 0.0006% FeCl

, 0.15% K

HPO

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

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

HPO

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

HPO

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.

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