Bacillus subtilus can nodulate legume Phaseo luscoccineus grown in two different regions of Eastern Algeria.

ABSTRACT

The bacteria associated with legume Phaseoluscoccineus grown in two different regions of eastern Algeria, Guelma and Skikda are characterized by morphological study through two reference strains: Rhizobium sullae and Mesorhizobiumciceri; especially for the search of the Enzymes'; production that is needed to root nodules formation. A test of nodulation in controlled microbiological circumstances is ensured by highlighting the ability of isolates to nodulate the roots of the host plant. A nodule formation is proven; this indicates that our isolat are infective confirming isolates the symbiotic relationship between the host plant and the micromicro-symbion, which is a specific relationship. A molecular characterization was carried out using target sequences rDNA16S that proving that the T8 strain Bacillussubtilis was brought up.

KEYWORDS: Phaseoluscoccineus, nodules, Rhizobium sullae, Mesorhizobium ciceri, symbiotic relationship., symbiotic

INTRODUCTION

The Rhizobium-legume relationship leads to a biological fixation of atmospheric nitrogen (N2) that provide effective natural ammonia fertilizer to improve the performance of agriculture, especially in soils with nitrogen deficiency [18; 22].

For its economic and ecological importance, the symbiotic Legume-Bacteria Relationship nodulating roots (LBR) is considered the best combination found between the microbes and eukaryotes, since these bacteria have the ability to fix atmospheric nitrogen by establishing symbiotic molecular signals. These guide the symbiosis [20] in an easily identifiable manner causing nodules, the seat of the binding.ausingbinding. [2].

LBR are freely living bacteria in the soil belonging to diverse genre within the Alphaproteobacteria and Betaproteobacteria, which have the ability to form nitrogen fixing symbiosis with legumes.

However, the sequencing of a small number of available genomes of these bacteria does not currently reflect the phylogenetic diversity of GNI, or the variety of symbiotic mechanisms.

Phaseolus or beans is a legume which includes approximately 5 genera, with about 70 spec with the majority are grown in the north of Mexico and central America where it has been domesticated for over 8000 years [7; 11;12;14]

The geographical distribution of the beans in the world is very diverse, so many climate perspectives as soil perspectives made a suitable crop for very varied agroagro-farming systems[4;31].

Phaseolus has an interest in improving food production; indeed, the incorporation of the flour of these seeds with that of wheat triticum durum produces noodels of a better qualiquality[6]

Considering the agro-food situation in Algeria which is primarily based on supply and demand, only two species are grown. The objective of this study is to achieve a morphological characterization and a molecular identification of isolates nodulating the legume Phaseoluscoccineus grown in two different regions of eastern Algeria.

MATERIALS AND METHODS

The nodules collection:

This study was conducted on a specie of the genus Phaseolus; a Phaseoluscoccineus which is grown in two different regions of the east: one semi arid zone (MdjazAmmar, Guelma) and the other subsemi-aridsub-humid (Tamalous El Kol, Skikda) by way of making the collection of nodules in two different populations for each sampling site.

We notice that the period during which the collection of nodules was performed is between the end of the flowering stage until the appearance of the pods using the advocated technics by [25;30].

In fact, it is about 15 cm to dig around the plant, and 20 cm into the ground to extract the plant and its root system. Manually, we get rid of the earth at the roots without damaging the nodules; then gently washed frfrom earth remains with tap water.

Conservation and sterilization of nodulesnodules:

Nodules dried in filter paper are placed in the refrigerator at 4[degrees]C until 48 hours for immediate use. For long storage, it is recommended to use special dryer: calcium chloride (Ca [Cl.sub.2]) [30].

If the nodules are preserved in a drying agent, they are first put in water in a refrigerator overnight. Nodules are immersed 5 to 10 seconds in absolute ethanol and then transferred into a solution of mercuric chloride (HgCl2) acidified at 0.1% (w/v) for 3 minutes, then rinsed 10 times with sterile distilled water [30].

Isolation and characterization of bacteria culture:

The isolation of the bacteria is performed according to the method of[25;30]. The sterile nodules are crushed individually in a drop of distilled sterile water in a PfPetri sterile box.

The nodule juice is spread out on a Petri dish containing a specific environment, YeastodulePetriYeast-Mannitol-Agar (YMA)[30] supplemented with Congo red and incubated 2 days at 28[degrees]C [24]. This is to highlight the very few strains absorbing the dye and then inoculated these YMA boxes (Yeast-Mannitol-Agar), and on the BTB to determine their growth rate[16; 25;30 This step is followed by Gram staining.

Research of specific enzymes research for nodulation:

The goal is to look for the presence of certain enzymes that play a role in the infection process(nodulation) of the roots by bacteria, especially polygalacturonase, cellulase, nitrate reductase, a small degree of urease, and the presence of a [beta]-galactosidase, by growing isolates on specific or selective environments.

Nitrates reduction:

The bacteria are cultured on a liquid medium TryptoneTryptone-Yeast-Agar (TY) [5]containing 0.1% KNO3 (p/v) for 4containing days with shaking. After the incubation, reagent nitrate reductase 1 (sulfanilic acid in acetic 5M) and nitrate reductase 2 (a-naphthylamine in acetic acid 5M) are added in each tube. The appearance of a red color indicates the reduction of nitrate to nitrite. A negative result requires the addition of a pinch of metal zinc and observing the obtained color after a few minutes.

Cellulosic activity [26]:

Determining the presence of an endoglucanase activity is carried out according to the modified method of Congo red [27], a compound capable of binding stably to a non-degradable molecule of carboxymethylcellulose (CMC). A bacterial colony is cultured for 5 days on a BIII medium box [9] containing 0.25% CMC. After incubation at 30[degrees]C, the dishes are gently rinsed with running water and then filled with a Congo red solution (1mg/ml) and incubated for 30 min at 30[degrees]C. The Congo red solution is replaced by a solution of 1M NaCl; the boxes are subsequently let at room temperature and emptied. A yellow-orange halo around colonies indicates the presence of the endoglucanase activity.

Pictinolyticactivity:

Bacterial strains are grown on YMA medium which mannitol is replaced by inositol 0.1% supplemented with 0.2% pectin (polyglacturonate-Na), and the incubated at 28[degrees]C for 7 days. After incubation, the dishes were rinsed gently with tap water and then with ruthenium red solution 0.05% for 30 minutes.

Bleached halo around colonies indicates polyglacturonic activity.

Hydrolysis of urea (25):

To highlight the presence of urease isolates and controlled strains were grown on YMA medium containing 2% (w/v) urea and 0.012g of phenol red as PH indicator at 30[degrees]C for 48 hours. The urea solution is sterilized by filtration (0,20[micro]mfilter) and added to sterile medium maintained at 45[degrees]C. The positive reaction is indicated by the presence of colonies basifying or acidifying the medium.

Nodulation capacity of bacteria:

The ability of microorganisms to nodulation analyzed and nitrogen fixation with the host plant is an important and practical character for rhizobia or B.N.L. (Bacterial nodulating legumes) and must be ananalyzed in details[15]. Nodulation tests must be conducted in traditional jars of Leonard [30]in the presence of sprouted seeds sterile of legume.

After isolation and morphological, cultural, and enzyme characteristics of the bacteria, a first approach to identify isolates capacity and ability to form nodules with the host plant Phaseoluscoccineus, in microbiologically controlled conditions.

Molecular identification of isolates:

The best performing isolates to infect the roots of the plants have been subjected to molecular identification, the laboratory of exploiting biomass and production of proteins in eukaryotes, at the center CBS research Sfax, Tunisia.

Genomic DNA Extraction:

Vortex grated cells, about 1[cm.sup.2] cultivatedfort 24 to 48hon YMA medium, in 467[micro]l TE (10mMTris + 1mM EDTA) in an Eppendorf sterile tube, then added 30[micro]l of SDS (10%)and 2-3 [micro]l proteinase K(20mg/l)after stirring, incubation is carried out for 1h at 37[degrees]C.Phenol/chloroform extraction is performed by adding an equal volume to the previous mixture (v/v) :500[micro]l.

After centrifugation at 1200 rpm/t, the aqueous phase was gently transferred into another sterile Eppendorf tube and actually in which a 50 [micro]l of AcNa (3M) and 1 ml of Ethanol or 600[micro]l of Isopropanol were added, gently mixed and left on a bench top for 10min.

Recentrifugedpdt 10 after centrifugation at 1200 rmp for 10 mn, then recovers the pellet by is washed it with 70% of Ethanol and centrifuged for 1mn. The DNA pellet is solubilized in 50ml of sterile water.

The DNA extracted was checked by measuring the optical density (OD) at 260nm and by migration on agarose gel (0.8%); 10[micro]l of DNA plus 2[micro]lloading buffer (Promega) are deposited in the wells of the gel and were migrated in TBE buffer (Tris borate buffer) for 1h at 80 volts using Mupid (ex-Intelligent power supply unit AC 100-240 Japan). The gel was strained in Ethidium bromide (0.1%) and viewed under UV (366).

Amplification of genetic material by PCR and sequencing:

The two primers used for the amplification of the 16S rDNA are fD1 (AGAGTTTGATCCTGGCTCAG) and rD1(AAGCTTAAGGAGGTGATCCAGCC). The PCR amplificationis reaction is performed using a thermocycler Bio-Gener type GE in a final volume of 50[micro]l containing 1Uof Taq polymerase (Dream Taq),1[micro]lof ADN, 1[micro]l of dNTP,1[micro]l of rD1, and fD1,5[micro]l MgS[O.sub.4](20mM), and 5[micro]l of Taq buffer, with an amplification program of 50 cycles: 30 seconds denaturation step at 94[degrees]C, 30 seconds annealing at 55[degrees]C and 30 seconds extension at 72[degrees]C.

The DNA fragments were sequenced using a sequencedsequencer Big Dyes Terminator v3.1 cycle sequencing Kit in the ABI PRISM 3100 Genetic Analyzer.

The 16s rDNAr sequences were compared with sequences stored in the databasesdatabases[1] using NCBI web site (https://www.ncbi.nlm.nih.gov/) and aligned using Clustall and Bioedit facilities.

RESULTS AND DISCUSSION

Morphological and crop appearance isolates and growth rate:

Isolates from colonies after 48 hours incubation at 28[degrees]C on YMA medium, slightly elevated circular smooth, opaque and mucilaginous, with whitish translucent texture. Microscopic observation revealsnegative Gram rods. [23;28] confirmed the same results. The study of morpholog and Gram straining reveled the bacterial isolates are essential for possible identification [13].

The strains isolated from nodules of Phaseoluscoccineus absorb very little Congo red on YMA medium RC. This was observed in the majority of rhizobia [16;30]. This property is shared with the Bradyrhizobium; on the other hand the genus Agrobacteriumand the contaminating forms strongly absorb this dye [16].

In addition, ours trains belong to kind of fastfast-growing bacteria since the" YMA medium+ blue bromothymol" is acidified after 24 hours of incubation (turn yellow pH indicator). [8;16; 27;30].

During this study, 18 strains were isolated from nodules of the plant cultivated on the site of TamalousSkikda and 16 isolates of the site from MdjazAmmar, Guelma, in the presence of two ethnic references (Table1).

Specific enzymes of research:

All our isolates in the presence of control strains (R. sullae, Mesorhizobiumciceri) gave positive reactions for enzymes related to nodulation process (Polygalacturonase, endoglucanase) or associated with nitrogen metabolism soil (urease, nitrate reductase).

- Nitrate-reductase:

the growth of strains and controlled isolates on KN[O.sub.3] based medium and after addition of reagents of the nitrate reductase, show the presence of the nitrate reductese activity enzyme[17;26].

- Urease:

al strains produce a specific urease on medium supplemented with urea. Its presence is manifested by alkalization of the medium (production of NH3).

- Cellulosic activity:

the appearance of a yellow-orange halo around the colony of the isolates indicates the presence of an endoglucanase activity (cellulase). All strains give a positive reaction [17]. We notice that this enzyme (endoglucanase) is required to establish the first symbiotic infections of legumesroots by Rhizobium in degrading the cell wall of root hairs of the host plant without damaging the cells of the latter[22].

- Pectinolytic activity:

after the addition of ruthenium red and rinsing with water, a clear halo was observed around the colonies indicating a positive reaction. All isolates have exhibited polygalacturonase activities.

Nodulation test :

The nodulation test leads to positive results: the formation of nodules indicates that our isolates are infective.: This formation also confirms the specific symbiotic relationship between the host plant and the micromicro-symbion.

After ten weeks in a bacteriologically controlled chamber, this test shows the ability of isolates T8, T18, M3, M4 and M5 to form nodules (two to six) on the roots of the legume PhaseoluscoccineusPhaseoluscoccineus 2 to 6mm generally shaped grape cluster, whose white color, Hawevre the activity of the nodule is indicated by red or pink color that is explained by the presence of leghemoglobin, a hemoprotein fixing of oxygen required in the nitrogen fixation [21].

The isolation of bacteria, their cultivation and biochemical characterization (production of enzymes required for nodulation) and the establishment of nodulation effect are proving the membership of these (bacteria) to the Rhizobiumgenus[29].

Molecular identification:

The analysis of nucleotide sequences of the infective strain T8 revealed of its belonging to aBacillus subtilisspecies with a similarity of 98%.Moreover, [19] explained that the coexistence of the Bacillusgenus in the nodules, has the ability to produce spores when growing conditions are not favorable, and can also produce antibiotics against other co-existing microorganisms .so it is particularly competitive.

Sequence called "10" T8 fragment using the primer RD1:

ACGAATTCACCCCATCATCTGTCCCACCTTCGGCGGCTGGGTCCTAAAAGGTTACCTCACACGAATTCACCCCATCATCTGTCCTAAAAGGTTACCTCACCGACTT CGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC CATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTGAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGAT AGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGT GTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTTCTCCGTTTGGTCACCGGGGCATGATGATTTGACGTCATCCCCACCTTTCTCCGTTTG CGGTCACCTTAGAGTGCCCAACTGAATGCTGGACTAAGATCAAGGGTTGGGCTCGTTGCGGGACTTAAACTGAATGCTGGACTAAGATCAAGGGTTGGGCTCGTTGCG CCCAACATCTCA

Bacillus subtilis strain ITBCC1 16S ribosomal RNA gene, partial sequence

Sequence ID: gb|KM115537.1|Length: 1467Number of Matches: Length: 1

Related Information

Range 1: 1040 to 1460GenBankGraphicsNext MatchPrevious Match

Alignmentstatistics for match #1

Conclusion:

In this study we tried to identify bacteria isolated from nodules of legume Phaseoluscoccineus grown in two different regions of eastern Algeria in the presence of two witnesses' strains: Mesorhizobiumciceri and Rhizobium sullae. We conducted isolation and characterization according to conventional techniques specific to rhizobia according to[16;25; 30]. Phenotypic studies, including colony morphology on YMA, growth rate, growing strains on YMA RC, the presence of specific enzymes in the nodulation process,... we do believe that isolates correspond to a description of the Rhizobium type. We tested he availability of enzymes involved for hydrolysis of the plant wall is tested (cellulose, pectinase), for the same for the enzymes related the nodulation process (pectinase) and nitrogen metabolism (urease, nitrate reductase). All strains and references we are endowed of urease activity, polygalacturonase, endoglucanase (the latter two are involved in the infection process of the legume's roots by the isolates). Furthermore an enzyme (nitrate reductase) involved in the process of fixation and transformation of atmospheric nitrogen is found in all strains [3]. Symbiotic delivery is successful to the extent that the nodulation test under controlled conditions gave true nodules. At in taxonomic level, molecular identification brings out the membership of the T8 strain Bacillus subtilis with 98% of similarity. So, (28)proved that the genus of Bacillus can be nodulate legumes.

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(1) Mouna Saoudi, (2) Ali Gargouri, (2) Azza Hadj Sassi, (2) Lamia Jmal, (2) Awatef Taktak, (1) Yacine Benhizia

(1) Laboratory of molecularand cellular biology University Freres Mentouri, Route Ain el Bey, Constantine 25017, Algeria,

(2) LaboratoryofMolecular Biotechnology of Eucaryotes Centre of biotechnology, University of Sfax, Tunisia.

Address For Correspondence:

Mouna Saoudi, Laboratory of molecularand cellular biology, University Freres Mentouri, Route Ain el Bey, Constantine 25017, Algeria

This work is licensed under the Creative Commons Attribution International License (CC BY).

https://creativecommons.org/licenses/by/4.0/

Received 28 August 2016; Accepted 18 October 2016; Available online 22 October 2016

Table 1: Strains and witnesses' strains used

      Strains               Host plant           Geographicregion
A6    Rhizobium sullae A6   Hedysarum            Constantine,
                            Coronarium           Algerie
CIII  Mesorhizobium ciceri  Cicer arietinum      Constantine, Algeriee

T1    Study object          Phaseolus coccineus  Skikda, Algerie
T2    Study object          Phaseolus coccineus  Skikda, Algerie
T3    Study object          Phaseolus coccineus  Skikda, Algerie
T4    Study object          Phaseolus coccineus  Skikda, Algerie
T5    Study object          Phaseolus coccineus  Skikda, Algerie
T6    Study object          Phaseolus coccineus  Skikda, Algerie
T7    Study object          Phaseolus coccineus  Skikda, Algerie
T8    Study object          Phaseolus coccineus  Skikda, Algerie
T9    Study object          Phaseolus coccineus  Skikda, Algerie
T10   Study object          Phaseolus coccineus  Skikda, Algerie
T11   Study object          Phaseolus coccineus  Skikda, Algerie
T12   Study object          Phaseolus coccineus  Skikda, Algerie
T13   Study object          Phaseolus coccineus  Skikda, Algerie
T14   Study object          Phaseolus coccineus  Skikda, Algerie
T15   Study object          Phaseolus coccineus  Skikda, Algerie
T16   Study object          Phaseolus coccineus  Skikda, Algerie
T17   Study object          Phaseolus coccineus  Skikda, Algerie
T18   Study object          Phaseolus coccineus  Skikda, Algerie
M1    Study object          Phaseolus coccineus  Guelma, Algerie
M2    Study object          Phaseolus coccineus  Guelma, Algerie
M3    Study object          Phaseolus coccineus  Guelma, Algerie
M4    Study object          Phaseolus coccineus  Guelma, Algerie
M5    Study object          Phaseolus coccineus  Guelma, Algerie
M6    Study object          Phaseolus coccineus  Guelma, Algerie
M7    Study object          Phaseolus coccineus  Guelma, Algerie
M8    Study object          Phaseolus coccineus  Guelma, Algerie
M9    Study object          Phaseolus coccineus  Guelma, Algerie
M10   Study object          Phaseolus coccineus  Guelma, Algerie
M11   Study object          Phaseolus coccineus  Guelma, Algerie
M12   Study object          Phaseolus coccineus  Guelma, Algerie
M13   Study object          Phaseolus coccineus  Guelma, Algerie
M14   Study object          Phaseolus coccineus  Guelma, Algerie
M15   Study object          Phaseolus coccineus  Guelma, Algerie
M16   Study object          Phaseolus coccineus  Guelma, Algerie

      Source

A6    A.Benguedouar- Cne

CIII  S. Dekkiche- Cne

T1    Current study
T2    Current study
T3    Current study
T4    Current study
T5    Current study
T6    Current study
T7    Current study
T8    Current study
T9    Current study
T10   Current study
T11   Current study
T12   Current study
T13   Current study
T14   Current study
T15   Current study
T16   Current study
T17   Current study
T18   Current study
M1    Current study
M2    Current study
M3    Current study
M4    Current study
M5    Current study
M6    Current study
M7    Current study
M8    Current study
M9    Current study
M10   Current study
M11   Current study
M12   Current study
M13   Current study
M14   Current study
M15   Current study
M16   Current study

Article Details

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Author:	Saoudi, Mouna; Gargouri, Ali; Sassi, Azza Hadj; Jmal, Lamia; Taktak, Awatef; Benhizia, Yacine
Publication:	Advances in Environmental Biology
Article Type:	Report
Geographic Code:	6ALGE
Date:	Oct 1, 2016
Words:	3883
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