Physiological and Molecular Assessment of Sesbania Root Nodules Bacteria from Different Iraqi Areas for Salt Tolerance.
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
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.
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.
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 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.
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.
(Received: 14 November 2018; accepted: 18 January 2019)
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Rana A. Hameed
Al-Mustansiriyah University, College of Science, Dep. of Biology, Iraq.
* Corresponding author E-mail: firstname.lastname@example.org
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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