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Physiological and Molecular Assessment of Sesbania Root Nodules Bacteria from Different Iraqi Areas for Salt Tolerance.

Nitrogen fixation using biological organisms is a powerful source of nitrogen in the biosphere (200-300 kg N/ha/year), it plays a significant role in nitrogen enhancement of the earth. Leguminous plants by their symbiotic association with certain gram-negative soil bacteria, commonly known as Rhizobia, promote to fix atmospheric nitrogen. Most of rhizobial strains isolated from wild legumes were classified as members of the genera Rhizobium (1).

In corporation with rhizobia, Sesbania sesban is a plant growing in various environments, even in salinity areas, drought and arid infertility (2). Salinity influence the survival and multiplication of Rhizobium spp. In rhizosphere and soil, as well as reducing plant growth, photosynthesis, yet rhizobial populations are known to differ in their tolerance to important environment factors (3). In 2009 Ali and his coworker (4) indicated that inoculation with salt tolerant strains would improve nitrogen fixing ability and nodulation of the leguminous plants subjected to saline conditions. Also in 2017 Yanand his coworker (5) reported that bacteria associated with Sesbania cannabina could grow weakly in the presence of 5.0% w/v NaCl. Ali4 revealed that rhizobia isolated from tree legumes Leucaenaleucocephala were tolerant up to 2.5-3.5% salinity.

Many Iraqi areas had under gone harmful environmental conditions in the last decades, such like salinity and drought due to miss usage of efficient irrigation systems and low rainfalls. In 2013 Sharma and his coworker (6) pointed that the naturally occurring soil rhizobia nodulating legumes plants lived in the desert areas are expected to have higher tolerance to common adverse conditions such as salt stress. While N-fixing legumes tolerant of environmental stresses represent an important procedure to improve agricultural productivity, Rhizobia with genetic potentiality for stress tolerance are evenly vital for efficient nodulation and increase productivity of the host plants (1).

MATERIALS AND METHODS

Different geographical agricultural field sites of Iraqi regions of host plant Sesbania nodule sample were collected. Thirty two isolates were obtained from these regions, the isolates were isolated from nodules after surface sterilization according to Vincent (7), and cultured on yeast extract mannitol agar (YEMA) media containing Congored dye, petri dish plates were incubated at 28[degrees]C in the dark. The bacterial isolates were examined for morphological characteristics and gram-staining reaction as described by Somasegaran and Hoben (8). Rhizobia authenticity for host specificity was studied as demonstrated by Engelkeand his coworker (9). The effect of salt on isolates were studied by inoculating one loopfull of each isolate on YEMA media in triplicate plates containing 1, 2, 3, 4, 5 w/v% NaCl, growth was compared with control(no NaCl) and evaluated qualitatively according to Somasegaran and Hoben (8). Enzymes activity were examined for Catalase, Urease and Gelatinase enzymes as displayed by Ronald and his coworkers (10), appearance of gas bubble indicate the presence of Catalase enzyme. Intrinsic antibiotics resistance (IAR) were examined by taking freshly prepared, filter sterilized (0.22 [micro]m) solution of antibiotics and added to cooled, molten YEMA media to give the following concentration [micro]g/ml: 10, 25 and 50 of Ampicillin, Erythromycin and Kanamycin. The control treatment was consisted of YEMA plates without antibiotics. Isolates showing growth were scored as positive. Triplicate plates for each antibiotic were incubated at 28 R"C for 7 days, and scored for growth. For the evaluation of exo polysaccharide production (EPS), a loop full of each Sesbania isolates were inoculated into conical flasks containing 100 ml of yeast extract mannitol broth media. The flasks were incubated at 28 R"C on revolving shaker at 200 rpm for 72h. After incubation, the broth was centrifuged 3500xg and the supernatant was mixed with two volumes of chilled acetone. The crude polysaccharide developed was collected by centrifugation at 3500 xg for 30 min. EPS then Was washed with distilled water and acetone alternatively, then transferred to a filter paper and weighed after overnight drying at 105[degrees]C.

DNA Extraction of Sesbania isolates was done using Wizard genomic DNA purification kit (promega), DNA purity was identified using Shimadzu spectrophotometer, DNA concentration was calculated using the following equation:

ds DNA con.= O.D 260 nm x dilution factor x 50[micro]g /ml (Sambrooke/ al., 1989). DNA fingerprinting of Sesbania isolates by RAPD-PCR was applied using Master Mix reaction kit and three random primers from Bioneer Coporation (South Korea). Sequence of the primers and the reaction conditions were described in table-1. Amplification products were separated and electrophoresed on 1.5% w/v agarose gel. Total band number were calculated according to Sahiand his coworkers (11). Primer efficiency and primer discriminatory power was measured using the formulas:

Primer efficiency=total number of bands amplified by a primer / total number of bands amplified by all primers x 100.

Primer discriminatory power= the total of polymorphic bands amplified by a primer / total number of polymorphic bands amplified by all primers x 100 (12).

Statistical analysis for EPS production was done using ANOVA. Significant differences were identified by the least significant difference (L.S.D) multiple mean comparison test at p d"0.05 (Genestat program software, 2008, VSN International Ltd), as for physiological traits, comparison was performed quantitavelly on the basis of growth + or no growth--for each isolate. PCR fingerprints pattern were converted into a two-dimensional binary matrix (1, presence of a band, 0, absence of a band) and analyzed using statistic software package (version 1.92; past software, Ohammer, 2009).

RESULTS AND DISCUSSION

Morphological characterization

All thirty two Sesbania isolates were comparable in form with translucent gummy glistening, entire margin and circular rounded with diameter of 2.5-3.5mm, except of the isolates( Ses4, Ses5, Ses30 and Ses23) which were little translucent, milky and about 1-2mm in diameter after two days growth. The isolates were tested on Congo red as indicator incorporated with YEMA media, the data showed that all isolates did not absorb Congo red under dark conditions except Ses8, Ses9and Ses19 that appeared to be pink as a result of taking Congo red. Table- 2 shows the nomination and geographical origin and soil description. All isolates were examined under microscope and it revealed that the isolates were rod, motile and gram-negative cells.

Physiological characterization Salt stress response

Viability of Rhizobia isolates grown under various salt concentration using NaCl was measured. Table-3 displayed a high level of variety between isolates, data showed that 53.12% of Sesbania isolates were highly tolerant to salinity, tolerated from 4-5%w/v NaCl and that 18.75% of isolates were salt sensitive, tolerated up to 1.0% NaCl.

enzymes activity

Data in table-4 showed that all isolates gave strong positive reaction to Catalase enzyme except for Ses19, Ses28 and Ses29 were negative for Catalase test. As for Urease the table cleared that 88.23% of salt tolerant isolates were negative for this enzyme comparing to 50% of salt sensitive isolates. In regards to Gelatinase enzyme, results indicated that 70.58% of salt tolerant isolates were negative for Gelatinase production.

Antibiotic sensitivity

All thirty two isolates were tested against three kinds of antibiotics in 10, 25, 50 [micro]g[ml.sup.-1] concentration. Table-5 showed that all isolates were highly resistant to Ampicilin at 50 [micro]g[ml.sup.-1] concentration.

Exopolysaccharide (EPS) production

Table-6 showed a significant difference between Sesbania isolates regarding EPS production, the results revealed that salt tolerant isolates gave higher amount of EPS production in compared to sensitive isolates, this is agreed with Freitasand his coworkers as well as Saritha and her coworkers in addition to Alroomi (13-16). The highest production of EPS was recorded for the isolate Ses21 producing 981.2 mg/g EPS, and the least production was found in Ses17 producing 296.6 mg/g EPS. Cluster analysis based on phenotypical and physiological traits divided the isolates into two divergent groups, the first one included one isolate Ses10, which was salt moderate tolerant, and the second main group included the rest of Sesbania isolates which splits into two subgroups with 6% similarity, the first subgroup comprised all sensitive isolates plus one salt moderate (Ses9), and the second subgroup included all salt tolerant and moderate isolates.

Molecular characterization

Molecular methods used in this study was applied on eight representative isolates, salt tolerant (Ses2, Ses13, and Ses20), salt sensitive (Ses17 and Ses28) and salt moderate tolerant (Ses7, Ses18, and Ses32). DNA purity ranged from 1.3 _1.7 O.D. The RAPD-PCR amplification products comprised different bands (fig 2,3, and 4), table-6 cleared that most efficient and highest discriminatory power was 35.4% and 37% respectively for the primer OPA-10, also the cluster analysis based on RAPD-PCR products showed two divergent groups with 15% similarity, the first group contained all salt sensitive isolates, while the second group included all salt moderate and tolerant isolates, this group subdivided into two subgroups with 44% similarity, the first subgroup included all salt tolerant isolates which show 100% similarity between them, and the second subgroup comprised the salt moderate tolerant.

The study showed a wide variability for salt tolerance between Iraqi isolates, these results was consistent with Elboutahiriand her coworkers as well as Sharma and his coworkers (6, 17). Probable cause for genetic diversity could be the short soil moisture perhaps have resulted in genetic adaptations of the strains. Thus, variations between different strains of diverse origins suggested that there is genetic potential to improve tolerance to environmental stress such as low soil moisture.

CONCLUSIONS

This study demonstrated that we could isolate and purify salt tolerant Sesbania isolates from Iraqi soils, the cluster based on physiological and phenotypical traits shows that these isolates represents divers populations and this could offer selection advantage in survival and adaptation to harsh environment conditions. RAPD technique was effectively utilized to discriminate between Sesbania isolates. And the genetic potential for increased tolerance to salinity could improve production of high tolerant inoculum strains for legume plants.

http://dx.doi.org/10.13005/bpj/1652

(Received: 14 November 2018; accepted: 18 January 2019)

REFERENCES

(1.) Zahran, H. Rhizobium- legume symbiosis and nitrogen fixation under sever conditions and in arid climates. Microbiol Mol. Biol. R. 63: 968-989 (1999).

(2.) Rao, D. and Gill. H. Biomass and biofertilizer production by Sesbania cannabania in alkaline soil. Bioresour Technol 53: 169-172 (1995).

(3.) Wei, G. Yang, X. Zhang, Z. Yang. Y. and Lindstrom, K. Strain Mesorhizobium sp. CCNWGX035, a stress tolerant isolate from Glycyrrizaglabra displaying a wide host range of nodulation. Pedosphere, 18: 102-112 (2008).

(4.) Ali,S. Rawat, S. Meghvansi, M. and Mahna, S. Selection of stress-tolerant rhizobial isolates of wild legumes growing in dry regions of Rajasthan, India. ARPN J. Agric. Biol. Sci. 4: 13-18 (2009).

(5.) Yan, J. Li, Y. Yan, H. Chen, W. Zhang, X. Wang, E. Han, X. and Xie, Z. Agrobacterium salinitolerance sp. Nov., a saline--alkaline-tolerant bacterium isolated from root nodule of Sesbania cannabina. Int. J Syst Evol Microbiol 67: 1906-1911 (2017).

(6.) Sharma, S. Rao, N. Gokhale, T. and Ismail, S. Isolation and characterization of salt-tolerant rhizobia native to the desert soils of United Arab Emirates. Emir. J. Food Agric. 25(2): 102-108 (2013).

(7.) Vincent, j. A manual for the practical study of the root-nodule bacteria International Biological Program. Oxford UK. Blackwell Scientific (1970).

(8.) Somasegaran, P. and Hoben, H. Handbook of Rhizobia methods in legume-rhizobium technology. Springer-verlag. New York, U.S.A (1994).

(9.) Engelke, T. Jagadish, M. and Puhler, A. Biochemical and genetical analysis of Rhizobia melilotimutants defective in C4-dicarboxylate transport. J. Gen. Microbiol 133: 19-29 (1987).

(10.) Ronald, M. Lawrence, C. and Alfred, E. Laboratory manual of experimental microbiology. Mosby--yearbook Inc. 87-95 (1995).

(11.) Sahi, J. Akeel, H. Hadeel, A. Application of the random amplified polymorphic DNA (RAPD) marker to analyze the genetic variability in species of the fungus Ahernaria. J. Raf. Sci. 22(1): 1-16 (2011).

(12.) Grudman, H. Schneider, C. Hartung, D. Daschner, F. and Pith, T. Discriminatory power of three DNA typing techniques for P. aeruginosa. J. Clin. Microbiol 3: 28-32 (1995).

(13.) Freitas, A. Vieira, C. Santos, C. Stamford, N. and Lyra, M. Characterization of rhizobia isolates cultivated in saline soil. Bragantia 66: 497-505 (2007).

(14.) Saritha, B. Raghu, M. and Mallaiah, K. Studies on exopolysaccharide and indole acetic acid production by rhizobium strains from Indigofera. African J. Microbiol. Res. 3(1): 0-14 (2009).

(15.) Al-roomi, R. A. Genetic variations of Sinorhizobiummeliloti Iraqi isolates differing in their ability to drought tolerance, DNA diversity and phenotypic characterization. Ph.D. Thesis submitted to Ibn-Al-Haithem, University of Baghdad (2014).

(16.) Elboutahiri N, Thami-Alami I, El-Houssine Z, Udupa S.M, Physiological and Genetic Diversity in Rhizobium sullaefrom Morocco. Biomedical and life Sciences. 85-88 (2010). https://doi. org/10.1007/978-90-481-8706-5_10

(17.) Hameed, R. Hussein, N. and Al-jabouri, A. Phenotypic characterization of indigenous Iraqi Sinorhizobiummeliloti isolates for a biotic stress performance. J. OF Life Sciences 8(1): 1-9 (2014).

Rana A. Hameed

Al-Mustansiriyah University, College of Science, Dep. of Biology, Iraq.

* Corresponding author E-mail: alroomir@yahoo.com

Caption: Fig. 1. Dendrogram showing similarity levels between Sesbania isolates based on phenotypical and physiological characterization

Caption: Fig. 2. RAPD fingerprint of Sesbania isolates generated by primer OPA-10. 1= Ses17; 2-Ses28; M= 1kb DNA ladder; 3=Ses7; 4=Ses18; 5=Ses32; 6=Ses2; 7=Ses13; and 8= Ses20

Caption: Fig. 3. RAPD fingerprint of Sesbania isolates generated by primer OPC-16. M= 1kb DNA ladder 1= Ses2; 2-Ses13; 3=Ses20; 4=Ses17; 5=Ses28; 6=Ses7; 7=Ses18; and 8= Ses32

Caption: Fig. 4. RAPD fingerprint of Sesbania isolates generated by primer OPN-16. M= 1kb DNA ladder 1= Ses17; 2-Ses28; 3=Ses7; 4=Ses18; 5=Ses32; 6=Ses13; 7=Ses20; and 8= Ses2

Caption: Fig. 5. Dendrogram of Sesbania isolates derived from RAPD fingerprints generated using three different primers (0PA-10, OPC-16, and OPN-16)
Table 1. RAPD primers and
PCR reaction conditions

Primer                        Sequence from 5' to 3' end
OPA-10                        GTGATCGCAG
OPC-16                        CACACTCCAG
OPN-16                        AAGCGACCTG
PCR reaction condition
Initial denaturizing 5 min, 94[degrees]C
Denaturizing 1 min, 94 [degrees]C--
Annealing 1 min, 32 [degrees]C | 34cycle
Extension 1 min, 72 [degrees]C--
Final extension 1 min, 72 [degrees]C

Table 2. Nomination, Geographical origin and soil description of
Sesbania isolates

Isolate        Geographical         EC        Soil

Name              Origin                  description
Ses1         Amryia-Fallujah 1      5.3       Arid
Ses2         Amryia-Fallujah 2      4.6       Arid
Ses3            Abu-ghreb1          7.7       Arid
Ses4            Abu-ghreb2          7.1       Arid
Ses5            Abu-ghreb3          5.8     Semiarid
Ses6         Khaluss 1, Diyala      5.0     Semiarid
Ses7         Khaluss 2, Diyala      4.6   g. irrigate
Ses8         Khaluss 3, Diyala      4.5   g. irrigate
Ses9          Baquba 1, Diyala      3.8   g. irrigate
Ses10         Baquba 2, Diyala      3.0   g. irrigate
Ses11        Baquba 3, Diyala       4.3     Semiarid
Ses12         Balad 1, Diyala       5.1     Semiarid
Ses13         Balad 2, Diyala       5.4     Semiarid
Ses14       Seqlawea 1, Baghdad     1.3   v.g.irrigate
Ses15       Seqlawea 2, Baghdad     1.7   v.g.irrigate
Ses16       Seqlawea 3, Baghdad     2.0   v.g.irrigate
Ses17     Mustansiryia,1 Baghdad    2.2   g. irrigate
Ses18     Mustansiryia 2, Baghdad   1.7   g. irrigate
Ses19        Rashdiya, Baghdad      1.2   g. irrigate
Ses20       Zafrania 1, Baghdad     6.1       arid
Ses21       Zafrania 2, Baghdad     5.7       arid
Ses22        Nasir, Nasiriyah       3.0   g. irrigate
Ses23       Fajir 1, Nasiriyah      4.9     Semiarid
Ses24        Fajir 2, Nasiriyah     5.4     Semiarid
Ses25       Mahmodea 1, Baghdad     5.2     Semiarid
Ses26       Mahmodea 2, Baghdad     4.7     Semiarid
Ses27        Saydia 1, Baghdad      3.4   g. irrigate
Ses28        Saydia 2, Baghdad      3.2   g. irrigate
Ses29        Saydia 3, Baghdad      4.0   g. irrigate
Ses30        Mahawel 1, Babel       5.6     Semiarid
Ses31        Mahawel 2, Babel       5.7     Semiarid
Ses32        Mahawel 3, Babel       6.0     Semiarid

Table 3. Salinity tolerating levels of
Sesbania isolates

Isolate.            Highest NaCl con.
No                   tolerated (%)

Ses1                     4
Ses 2                    5
Ses 3                    4
Ses 4                    5
Ses 5                    5
Ses 6                    4
Ses 7                    3
Ses 8                    3
Ses 9                    2
Ses 10                   2
Ses 11                   4
Ses 12                   4
Ses 13                   5
Ses 14                   2
Ses 15                   1
Ses 16                   1
Ses 17                   1
Ses 18                   3
Ses 19                   1
Ses 20                   5
Ses 21                   5
Ses 22                   2
Ses 23                   4
Ses 24                   4
Ses 25                   5
Ses 26                   4
Ses 27                   3
Ses 28                   1
Ses 29                   1
Ses 30                   4
Ses 31                   4
Ses 32                   2

Table 4. Enzymes reaction pattern of Sesbania isolates

Isolate no.   Catalase   Urease   Gelatinase

Ses1             ++        -          -
Ses2             ++        -          +
Ses3             ++        -          -
Ses4             ++        +          -
Ses5             ++        -          -
Ses6             ++        -          +
Ses7             ++        +          -
Ses8             ++        -          -
Ses9             +         -          -
Ses10            +         -          -
Ses11            ++        -          -
Ses12            ++        -          -
Ses13            ++        -          +
Ses14            +         +          -
Ses15            -         -          +
Ses16            -         -          +
Ses17            +         -          +
Ses18            ++        +          -
Ses19            +         +          -
Ses20            +         -          -
Ses21            ++        -          -
Ses22            +         -          -
Ses23            +         -          +
Ses24            ++        -          -
Ses25            +         -          -
Ses26            +         -          +
Ses27            ++        -          +
Ses28            -         +          +
Ses29            +         +          +
Ses30            +         +          -
Ses31            +         -          -
Ses32            +         -          +

Table 5. Antibiotic resistance of thirty
two Sesbania isolates

Antibiotic             % Resistance of isolates

              10[micro]gm[L.sup.-4]   25[micro]gm[L.sup.-4]

Erthromycin            71                      42
Kanamycin              56                      44
Ampicilin              86                      85

Antibiotic    % Resistance of isolates

               50[micro]gm[L.sup.-4]

Erthromycin              33
Kanamycin                24
Ampicilin                83

Table 6. EPS production in Sesbania
isolates(mg/g)

Isolate   EPSmg/g   Isolate   EPS mg/g

Ses 1      900.1    Ses 17     296.6
Ses 2      678.3     Ses18     746.3
Ses 3      720.8     Ses19     361.0
Ses 4      923.6     Ses20     867.3
Ses 5      892.6     Ses21     981.2
Ses 6      763.3     Ses22     324.6
Ses 7      522.1     Ses23     656.8
Ses 8      564,9     Ses24     654.0
Ses 9      497.9     Ses25     566.3
Ses 10     488.9     Ses26     786.2
Ses 11     656.6     Ses27     678.2
Ses 12     745.6     Ses28     401.0
Ses 13     824.2     Ses29     325.3
Ses 14     325.6     Ses30     697.4
Ses 15     433.3     Ses31     548.9
Ses 16     312.9     Ses32     300.4

Table 7. Fragments amplified by three random primers in eight
Sesbania isolates and the efficiency and discriminatory power of
each primer

Primer   No. of bands amplified      Primer           Primer
            in all isolates      efficiency (%)   discriminatory

          Total    Polymorphic                      power (%)

OPA-10     11          10             35.4             37.0
OPC-16     10           8             32.2             29.6
OPN-16     10           9             32.2             33.3
Total      31          27              --               --
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Author:Hameed, Rana A.
Publication:Biomedical and Pharmacology Journal
Date:Mar 1, 2019
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