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Thèses de doctorat - رسائل الدكتوراة

Thèses de doctorat - رسائل الدكتوراة

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  • BECHELAGHEM, NADIA (South Asian J Exp Biol; 5 (4): 143-150; 2015ISSN: 2230-9799 Vol. 5, Issue 4, Page 143-150 http://www.sajeb.org, 2017-11-09)
    ملخص تعتبر الفلورا الطبيعية عنصرا هاما في المحافظة على صحة الإنسان. فمنذ أن وصف العالم Doderlein بكتيريا حمض اللاكتيك المهبلية و الدراسات حول طبيعتها المعقدة وترابطها مع عناصر أخرى من المضيف تتزايد بشكل كبير. وقد أدت ...
  • DAHOU, Abdelkader EL-Amine (Advances in Bioresearch, 2017-11-02)
    Résumé : La fabrication des fromages industriels implique l'utilisation d'un microbiote diversifié composé d'une population microbienne endogène naturelle apportée par le lait et l'autre exogène par un ensemencement ...
  • BERBER, Nadia (Advances inEnvironmental Biology, 10(12) December 2016, Pages: 55-61 AENSI Journals Advances inEnvironmental Biology ISSN-1995-0756 EISSN-1998-1066 Journal home page: http://www.aensiweb.com/AEB/ Copyright © 2016 by authors and Copyright,American-Eurasian Network for Scientific Information (AENSI Publication). Isolation and Molecular Identification (PCR-Delta and PCR-RFLP-ITS) of the yeast from Black muscat grape cultived in El malah (Wilaya of Ain Temouchent, Algeria) 1Nadia BERBER,2Rachid AISSAOUI, 1Ahmed Mohamed Ali BEKADA, 3Morvan COARER 1Technology of Food and Nutrition Laboratory (University of Abdelhamid Ibn Badis, Mostaganem), 27000, ALGERIA. 2Bioconversion Laboratory (University of Mustapha STAMBOULI, Mascara), 29000, ALGERIA. 3Microbiology and Biotechnology Laboratory (French Institute of Vine and Wine of Nantes), 44000, France. Address For Correspondence: Nadia BERBER, Technology of Food and Nutrition Laboratory (University of Abdelhamid Ibn Badis, Mostaganem), 27000, ALGERIA. E-mail: nadia.berber27@gmail.com This work is licensed under the Creative Commons Attribution International License (CC BY).http://creativecommons.org/licenses/by/4.0/ Received 16 November 2016; Accepted 17 December 2016; Available online 22 December 2016 ABSTRACT The grape is an exemplary product of microbial diversity, it is considered as home to many wild microorganisms (yeast). In this context, we explored in this study, the divergence of the indigenous yeast flora in the vineyards of the region El Malah (Wilaya of Ain Temouchent, Algeria) by collecting varietal grape samples (Black Muscat). A high molecular diversity of the flora has been demonstrated, using two molecular techniques, where one is interested in the study of variability of rDNA, more precisely the region 5,8S rRNA ITS1-ITS2 by PCR-RFLP-ITS and the region Delta by PCR-Delta is an additional study which helped sort the yeast Saccharomyces and non Saccharomyces.So 6 yeast species belonging to the 11 studied at 4 different types were characterized according to their molecular profiles. Thus the strains studied were characterized by two restriction enzymes (HinfI and HaeIII). Meanwhile, microscopic and macroscopic studies of classical identification of yeast were able to strengthen these results. Among the yeast species identified: Torulaspora delbrueckii, Torulaspora pretoriensis, Candida solani, Candida pseudointermedia and Saccharomyces cerevisiae. This variety is an excellent tank of non Saccharomyces yeasts as evidenced by the results.However, the tools of molecular biology have brought a notorious revolution in precise yeast identification tests. The PCR-RFLP-ITS is one of the most widely used methods of identification. KEYWORDS:Grape, Yeast, Black Muscat, PCR-Delta, PC-RFLP-ITS, HinfI, HaeIII. INTRODUCTION Yeasts are naturally present on soils, plant surfaces, especially grape berries, or in wine-growing areas [3,23]. Their dissemination is assured by wind, insects and humans through its various interventions on the environment [1,13].The yeast species present on the surface of the grape berries are significantly limited in number. The density of the yeasts evolves during the maturation of the grapes. The evolution of yeast populations may be related to the increase in the area of the bay and the availability of nutrients: during the maturation of the bunch, the berries grow, the nutrient content Surface area increases, sugar concentration increases and acidity decreases [4,9].The representation of the different yeasts varies during the development of the grape berry according to several parameters: grape variety, climatic conditions, soil, wine practices, age of vines, health status of the cluster and the degree of maturity of the grape [8,10,33]. Black Muscat is a black table grape varietal. It is characterized by beautiful black cohosh, juicy flesh, staining grain is sometimes clear. This variety is fairly resistant to drought and likes in sunny areas. The grape 56 Nadia BERBER et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 55-61 whether table or tank is the habitat of many wild microorganisms since it is considered the kingdom of yeasts. However, the high sugar content of the Muscat grape must (300 g / l) promotes the growth of yeast, these microorganisms play an essential role in the food industry. They participate in the development of many food products (bread, dairy, brewery) and the production of metabolites (lipids, proteins, enzymes), but also the upgrading of agricultural and industrial waste. However, many yeast species produce a very important primary metabolite namely ethanol, used as fuel or for other industrial purposes [17]. Two strains of bacteria were isolated from the gut of termite in different media. It was observed that these isolates have produced ethanol from rice straw (7.52 ± 0.5 to 9.33 ± 0.4 g/L) followed by corn stove (6.35 ±0.6 to 6.95 ±0.5 g/L) having theoretical yields of ethanol 43.31 % (rice straw) and 39.62 % ( corn stove) [16]. The aim of this study is to isolate, characterize microscopically and identify the indigenous species of yeasts found in the grape variety (Black Muscat) grown in the region of El Maleh (Ain Temouchent, Algeria) to develop and express the diversity of the phylogenetic heritage based on the two molecular techniques (PCR-Delta and PCR-RFLP-ITS). Our work is an initiative for the creation of a "Souchier" typical yeast our country Algeria. MATERIAL AND METHODS Grape picking Source (Black Muscat): The samples are taken randomly in the month of September 2014 in the vineyards of the city El Malah, a town located 11 Km from Ain Temouchent. We collected with sterile scissors, about 500 g healthy grapes, and it was collected in sterile bags. A laboratory arrival, they are scuffed and crushed to obtain a mash and ferment for one day at 25 ° C in order to increase the viability and quantityof the desired yeast [30]. Isolation, purification and conservation of cultures: We conducted a series of seeding by the method streaks on plates of agar cultures (YPG + Gentamicin) which aims to have pure cultures. The operation is repeated in each time taking random an isolated colony. Next, the purified strains are placed in a glycerol solution (sterile) with approximately 25% and stored in the freezer at -18 ° C. Microbiological identification: Study of characters crop: After incubating the cultures for 3 days at 25-28 ° C on agar medium (YPG + Gentamicin), macroscopic observation can describe the appearance of colonies (size, pigmentation, contour, viscosity ...) [17]. The cellular characteristics: Microscopic observation to define the shape, arrangement and mode of cell division. These characteristics are observed on microscopic slides to fresh (40 x objective) [25]. Molecular identification: DNA extraction: The isolation of DNA from biological samples is a crucial step in the process of DNA-based molecular biological assays [27]. Before extracting genomic DNA from strains levuriennes, these have been cultured in the medium (liquid YPG + Gentamicin), then a small amount of culture was taken and centrifuged to separate the cells. After the supernatant was removed and the lysis of the resulting pellet of cells was performed by addition of a volume of 660 μl of SDS-50TE mix (lysis buffer).Then the mixture was incubated at 65°C for 10 minutes. To neutralize the medium, a volume of 340 μl of potassium acetate was added to the mixture, the latter is placed in the refrigerator for 30 minutes until the suspension became semi-solid. Then a second centrifugation was performed for 10 minutes and a volume of 750 μl of supernatant was mixed with 750 μl of isopropanol followed by a final centrifugation to precipitate DNA. Then, the collected DNA pellet was rinsed gently with approximately 100 μl of 95% ethanol, then suspended in a volume of 300 μl of TE1x (storage buffer) and incubated at 65 ° C for 15 minutes. Finally, the DNA was stored refrigerated 4°C until use. (According to IFV Nantes, 2012). Amplifying the target region by the two molecular techniques (PCR-Delta and PCR-RFLP-ITS): PCR-Delta (Delta 12 / Delta 21): Typing of strains of Saccharomyces cerevisiae is performed by PCR using the method developed by [18] with the primers: δ 12 (5'-TCAACAATGGAATCCCAAC-3 ') δ 21 (5'-CATCATTAACACCGTATATGA-3 ').The reaction mixture is summarized in Table 1. 57 Nadia BERBER et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 55-61 Table 1: The composition of the reaction mixture used for PCR-Delta according to (IFV) Products Volume (μl ) Volumes (μl) H2O 1220 1230 Tampon 10X 160 160 dNTP 64 64 Delta 1 or 12 8(40pmol ̸ reaction) 8 Delta 2 or 21 8(40pmol ̸ reaction) 8 Mgcl2 80 80 PCR-RFLP-ITS: The sequence ITS1 5.8S rRNA ITS2 present a conserved region in the majority of yeast species and the variable regions. The primersITS1 (5'-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') described as universal primers by [35]. The reaction mixture is summarized in Table 2. Then, in order to identify the different species of yeast, the products of PCR-ITS are treated with two restriction enzymes HinfI and HaeIII [3]. Table 2: The composition of the reaction mixture used for PCR-ITS according to (IFV) Products Volume (μl) Volume (μl) H2O 1220 1230 Tampon 10X 160 160 dNTP 64 64 ITS1 12,8 (40pmol ̸ reaction) 12,8 ITS4 12,8 (40pmol ̸ reaction) 12,8 Mgcl2 80 80 DMSO 31(2%) 31(2%) Purification of a DNA fragment (PCR-Delta +PCR-RFLP-ITS) double-strand is performed by electrophoresis on Agarose gel. The gel is mixed with ethidium bromide and cast hot in the electrophoresis tank. 20 μl contained in each tube PCR, mixed with 5 μl of loading buffer containing bromphenol blue, are deposited on the wells. Fragments of known molecular weight DNA called markers are also deposited: they are used to correlate the migration distance of the DNA fragments to their size (in base pairs). In this study, the number and length of the fragments were compared to the size marker (100 bp). according to (IFV).Then, applying a voltage of 120V / 30 minutes until the migration of the fragments [5].The nucleotide different bands resulting from the electrophoresis are shown on the gel under UV light (254 nm) and photographed with a camera UV. RESULTS AND DISCUSSION Isolation and purification by successive subcultures made after collection of yeasts in September 2014 from the Black Muscat grapes grown in the El Maleh area (Wilaya of Ain Temouchent). This allowed us to get a collection of 11 yeast isolates. Study cultivation of isolated yeasts: Macroscopic examination of cultures "levuriennes" after incubation at 25 ° C for 4-5 days shows generally well isolated colonies are whitish, cream or sometimes yellowish opaque and irregular outline, they have an intense smell. Microscopic observation has allowed us to identify cell shape of isolated isolates and vegetative reproduction modes. Of the 11 individual isolates, 10 are spherical or cylindrical, elongated or short form and are budding as vegetative reproduction mode. The remaining isolate is characterized by an oval shape and has a monopolar budding, it has a smell of yeast. The uniformity of cells confirms the purification of isolates studied. According to [17], yeasts are in the form of independent free single cell or combined in pairs with characteristic morphology for example: spherical, ovoid, cylindrical, apiculé, bottled, pyramidal. Molecular study of isolated yeasts: DNA extraction: DNA extraction has allowed us to observe a small white mass rushed to the microtube bottom, then it was inoculated in a buffer prior TE1x and must keep to the time of use. PCR-Delta: This technique allows to distinguish between the species Saccharomyces and Non Saccharomyces our results, the graph (Fig.1) shows that the first 10 isolates of the species Non Saccharomyces since no amplification profile is observed on their columns, so they belong to other species or they will be identified by 58 Nadia BERBER et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 55-61 PCR-RFLP-ITS. Regarding the last isolate (isolate 11), we see the emergence of three bands on the different molecular size column (300 + 350 + 800 bps), then we can predict that this isolate belongs to the species Saccharomyces. The study of the ecology of Non Saccharomyces yeast known significant growth since the last few years, we focus not only on the distribution of species found on grapes but also their succession during alcoholic fermentation [1,6,20,30,32]. Many of the Non Saccharomyces yeast can colonize the surface of grape berries. Indeed, school supports the idea that although minority, Saccharomyces yeasts from the surface of the grapes, and that their presence or absence exchange of a cluster to another, within the same plot [9,19,28].[21]do consider that grape berry on 1000 carries Saccharomyces cerevisiae. However, these same authors found that damaged grapes (naturally in the vineyard) are significant populations with Bay 1 of 4 carriers of Saccharomyces cerevisiae. However, according to other authors, Saccharomyces cerevisiae is undetectable on grapes [9,28]. In our study, major yeast species were represented by Non Saccharomyces yeast (10 isolates) while only one isolate was identified as Saccharomyces(isolate 11), so our work highlights the predominance of Non-Saccharomyces species or sound samples isolated from the grape must of Muscat black grape. During a spontaneous fermentation with native flora, Non-Saccharomyces yeasts predominate in the must during the pre-fermentation stage and at the beginning of the alcoholic fermentation before the yeast S. cerevisiae colonize the middle to complete the fermentation [36,12,30]. However, Non-Saccharomyces yeasts are very present, even the majority, in the grape must (Black Muscat) compared to Saccharomyces yeasts. Fig. 1: Profils PCR-Delta of 11 isolates. Lanes M correspond to molecular size standards (100-bp DNA ladder from IFV) PCR-RFLP-ITS: A total of 11 isolates of yeasts isolated from the Black Muscat grape juice were analyzed. To identify these yeasts, the region of the rRNA repeat unit comprises two non-coding regions referred to as internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were amplified and digested by two enzymes of restriction (HinfI and HaeIII). The profiles obtained from each isolate were compared with reference strains in the determination of the IFV Guide (2012). The results of this study are summarized in Table 3. The size of the PCR products and restriction fragments of the major species identified in this study are shown in Table 4. The species of yeasts isolated from the grape (black Muscat): 11 yeast isolates were identified as belonging to 4 genera and 6 different species: Torulaspora (05 strains), Hanseniaspora (03 strains), Candida (02 strains), Saccharomyces (01 strain) (Table 3). These different kinds of yeast are well documented in the literature as present on grapes and the start of alcoholic fermentation [36,1]. Table 3: Frequency of yeast species isolated from the grape “Black Muscat” Isolation source Species Frequency of isolation(Number of strains) Black Muscat grape variety Torulaspora delbrueckii Hanseniaspora uvarum Candida solani Candida pseudointermedia Torulaspora pretoriensis Saccharomyces cerevisiae 04 03 01 01 01 01 59 Nadia BERBER et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 55-61 Table 4: Size in bp of the PCR products and the restriction fragments obtained with two different endonucleases (Hinf I and HaeII)of the major species identified in this study Species Amplified product (pb) Restriction fragments (pb) Hinf I HaeII Torulaspora delbrueckii (Strains 1, 2, 3,4) 800 410+380 800 Hanseniaspora uvarum (Strains 5, 6,7) 770 350+190+160 750 Candida solani (Souche 8) 600 280+300 400 Candida pseudointermedia (Strain 9) 400 200+110 400 Torulaspora pretoriensis (Strain 10) 800 380+200+190 800 The review of the results indicates that 5 selected yeast strains (strain 1, 2, 3, 4.10) belong to the genus Torulaspora (Table 4), according to the determination of IFV guide (2012), while 4 strains were identified as belonging to the species Torulaspora delbrueckii and the remaining strain (strain 10) belongs to the species Torulaspora pretoriensis. The molecular profile of three yeast strains (strain 5, 6, 7) indicates that they look like the species Hanseniaspora uvarum, according to the determination of IFV guide (2012). However, the molecular characterization and micro-macroscopic characteristics show that the strains (8) and (9) belonging to the species Candida solani and Candida pseudointermedia respectively. Furthermore, the combination of the two results (morphological and molecular studies) indicated that the strain (11) belongs to the species Saccharomyces cerevisiae. Grape berries are the primary source of yeast during the fermentation of the must [11](Fig 2, 3, 4). In different wine regions in the world, insulation works and identification of yeasts showed that Pichia,Candida, Metschnikowia, Kluyveromyces, Cryptococcus, Rhodotorula, Debaryomyces, Issatchenkia, Zygosaccharomyces, Saccharomycodes, Torulaspora Dekkera, Schizosaccharomyces and Sporidiobolus are most frequently found [31,12,26,30,22]. Other species Non-Saccharomyces as Candida zemplinina, Torulaspora delbrueckii and Hanseniaspora spp are also an important part of the diversity of the community of the bay and are present during the fermentation, in particular during the stages pre fermentative [36]. Fig. 2: Viewing the amplified region (rRNA 5,8S ITSI-ITSII) in 11 isolates M 1 2 3 4 5 6 7 8 9 10 11 M 100 pb Fig. 3: Viewing the region (rRNA 5, 8S ITSI-ITSII) digested by HinfI in 11 isolates Fig. 4: Viewing the region (rRNA 5, 8S ITSI-ITSII) digested by HaeII in 11 isolates 60 Nadia BERBER et al, 2016 Advances in Environmental Biology, 10(12) December 2016, Pages: 55-61 In our study, the grape must (Black Muscat) is an excellent reservoir of Non-Saccharomycesyeasts as evidenced by the results obtained, we met different fermentative species such as T. delbrueckii is a non-Saccharomyces yeast naturally present in the must and the grape berries which has been described in the literature for its positive impact on the quality and complexity of wines[6,7,15] and for the purity its fermentation with especially low production of volatile acidity, acetaldehyde, diacetyl and acetoin[2,24,34]. This yeast is also described as cryophile and osmotolerant [14].Among the Non-Saccharomyces yeasts isolated from our grape must, we have the species Hanseniaspora uvarum, Candida solani and Candia pseudointermedia. Indeed yeast fermentation activity low, mainly as the species of the genus Hanseniaspora and to a lesser degree the genera Candida predominate on grapes and in the early stages of fermentation. These results are in agreement with the literature. According to[2,30,1], at the earliest stages, the Basidomycetes species are dominant, and the increasing number of Ascomycetes species, especially those that have fermentation capacity is observed at maturity stages (Metschnikowia, Hanseniaspora, Candida , Pichia). This study highlights the variability of species in each genus of yeast especially in Torulaspora genres (T.delbrueckii, T. pretoriensis) and Candida (C.Solani, C.psendointermedia). However, only one species of Saccharomyces cerevisiae was isolated from the must. This is the main agent of fermentation. Fermentations are initiated by the growth of various species of Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces, Torulaspora, and Zygosaccharomyces. Their growth is generally limited to the first two or three days of fermentation, after which they die off. Subsequently, the most strongly fermenting and more ethanol tolerant species of Saccharomyces take over the fermentation [11]. Conclusion: This study is based on the evaluation of a grape variety grown in Algeria (Black Muscat) .A genetic approach has been developed in this work and has achieved an identification of 11 isolates belonging to 4 genera and 6 different species: Torulaspora, Hanseniaspora,Candida and Saccharomyces. This variety is an excellent tank of Non Saccharomyces yeasts as evidenced by the results. In terms of our study, we strongly encourage investigations to characterize biotechnologically these identified strains, and to encourage all studies concerned with the identification and characterization of other grape varieties in all regions of Algeria. However, yeasts open up avenues of research for the future. Constantly improved, production systems make it possible to envisage the production of very different proteins, for the human or veterinary pharmacy. The final objective, drawn in the short and long term, is set in the development of the yeasts identified and then selected to serve mainly the agro-food domains (bread, dairy, brewery...) and new biotechnologies. REFERENCES [1] Barata, A. et al., 2012. The microbial ecology of wine grape berries. International Journal of Food Microbiology, 153(3): 243-259. [2] Bely, M., P. Stoeckle, I. Masneuf-Pomarède and D. Dubourdieu, 2008. Impact of mixed Torulaspora delbrueckii–Saccharomyces cerevisiae culture on high-sugar fermentation. 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Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits.Food Microbiology, 25:778-785. [35] White, T.J., T. Bruns, S. Lee and J. Taylor, 1990. Analysis of phylogenetic relationships by amplification and direct seq uencing of ribosomal RNA genes tiré de PCR Protocols: A guide to Methods and Applications. M. A. Innis, D. H. [36] Zott, K., C. Miot-Sertier, O. Claisse, A. Lonvaud-Funel and I. Masneuf-Pomarede, 2008. Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking.International Journal of Food Microbiology, 125: 197-203., 2017-10-31)
    RESUME Le raisin est un fruit exemplaire de la diversité microbienne, il est considéré comme l’habitat de multiples micro-organismes parmi les quels les levures. Dans ce cadre, cette étude consiste à isoler des souches ...
  • CHIBANI, HIBA RAHMAN (Malaysian Journal of Microbiology, Vol 13(2) June 2017, pp. 124-131 Malaysian Journal of Microbiology Published by Malaysian Society for Microbiology (In since 2011) 124 ISSN (print): 1823-8262, ISSN (online): 2231-7538 *Corresponding author Screening and characterization of plant growth promoting traits of phosphate solubilizing bacteria isolated from wheat rhizosphere of Algerian saline soil Chibani Hiba Rahman1*, Bouznad Ahcene1, Bellahcene Miloud2, Djibaoui Rachid1 1Laboratory of Microbiology and Plant Biology, Faculty of Natural and Life Sciences, University of Mostaganem, Algeria. 2University Center of Ain Temouchent, Algeria. Email: hiba.chibani@univ-mosta.dz Received 16 August 2016; Received in revised form 10 September 2016; Accepted 7 November 2016 ABSTRACT Aims: The capacity of some soil microorganisms to solubilize in soil is an important activity exhibited by plant growth promoting rhizobacteria (PGPR) to increase plant performance. This study aimed at isolation and selection of phosphate solubilizing bacteria from saline soil and in vitro evaluation of their plant growth promoting traits. Methodology and results: Phosphate solubilizing bacteria isolated from wheat rhizosphere, of saline soil in western region of Algeria were tested for their plant growth promoting traits such us indole acetic acid (IAA), hydrogen cyanide (HCN), siderophore and ammonia production and their ability to fix nitrogen. Among 104 bacterial isolates, 41 were selected for their phosphate solubilizing activity using tricalcium phosphate (TCP) as a sole phosphorus source. IAA production was shown by almost all the bacterial isolates. Twelve isolates were recorded positive for HCN production, 32 produced siderophore and 31 were able to fix nitrogen. The most dominant phosphate solubilizing bacteria found were identified as Pseudomonas followed by Aeromomas hydrophila Bacillus sp. and Burkholderia cepacia. Conclusion: Phosphate solubilizing bacteria that were isolated from saline soil showed a high potential in to producing growth promoting traits and can be used as inoculants to increase the phosphorus uptake by plants. Key words: Phosphate solubilising bacteria, saline soil, tricalcium phosphate, plant growth promoting traits INTRODUCTION Phosphorus is considered to be the most important element in plant nutrition of, after nitrogen. It is an essential component in all main metabolic procedures in plants for example energy transfer, photosynthesis, signal transduction and respiration (Khan et al., 2010). Inorganic phosphorus is found in soils, mostly in insoluble mineral complexes such as tricalcium phosphate Ca3 (PO4)2, ion phosphate FePO4 and aluminium phosphate AlPO4 (Barber, 1995), which appear after repeated applications of chemical fertilizers. Plants have not the capacity to absorb these insoluble forms besides only 0.1% of total phosphorus is in soluble form and it is available for plant nutrition (Zhou et al., 1992). It is for this reason that the available phosphorus levels have to be supplemented in most agricultural soil by adding chemical phosphorus fertilizers. The frequent and imprudent applications of chemical phosphorus fertilizers lead to the decrease of soil fertility by perturbing microbial population thus reduce crops yield (Gyaneshwar and Naresh, 2002). Among chemical environmental stress soil salinity is the most important stress factor for plants (Idikut et al., 2012). Salinity leads to osmotic stress and causes the formation of reactive oxygen species (ROS) thus disturbs cellular structures which and consequently damage mitochondria and chloroplast (Mittler, 2002). Soil salinity considerably reduces absorption of mineral nutrients, particularly phosphorus for the reason that phosphate ions precipitate with calcium ions in saline soil and become inaccessible to plants (Grattan and Grieve, 1999). Utilization of phosphate solubilizing bacteria to solve this problem for raison of their ability to solubilize phosphate in soil is supported by many researchers (Khan et al., 2007). Mechanisms like organic acid production, chelation, and ion exchange reactions are implemented by these bacteria to solubilize inaccessible phosphorus forms and make them available to plants (Vessey, 2003). Phosphate solubilizing bacteria (PSB) are part of plant growth promoting rhizobacterias (PGPRs) and are capable of solubilizing inorganic phosphate from different compounds, such as dicalcium phosphate, tricalcium phosphate and rock phosphate. Moreover, PSMs may also showed plant growth promoting traits such as indole acetic acid (IAA), cytokinins, gibberellic acid and ethylene production, hydrogen cyanide (HCN) and siderophore Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 125 ISSN (print): 1823-8262, ISSN (online): 2231-7538 production, nitrogen fixation and resistance to soil pathogens etc (Cattelan et al., 1999). The main objectives of this study were the isolation and screening of phosphate solubilizing bacteria from wheat rhizosphere of salt affected soil and in vitro assessment of their plant growth promoting activities. MATERIALS AND METHODS Soil sampling Saline soil samples were collected randomly from the rhizosphere regions of wheat plants growing at different sites at Relizane (western Algeria). All samples were kept in plastic bags and transported to the laboratory and stored at 4 °C. Total of fifteen soil samples were air-dried, ground and passed through 2 mm sieve before chemical analyses pH, soil moisture and conductivity of the soil samples were measured. Isolation of total phosphate solubilizing bacteria Isolation of PBS bacteria was carried out by serial dilution method. Ten grams of rhizosphere soil was dissolved in 90 mL of saline phosphate buffer, then shook for 30 min. One mL of rhizosphere soil suspension was added to 9 mL of sterile saline water to obtain a suspension with a 10-2 dilution. Subsequently 0.1 mL of the suspension diluted to 10-5 was grown on Pikovskaya (Pikovskaya 1948) PVK agar supplemented by 5 g of tricalcium phosphate (TCP) as a sole phosphorus source. Inoculated plates were incubated for 7 days at 28± 2 °C. Appearance of clear halo zone on Pikovskaya’s agar plates indicates positive phosphate solubilization ability. Bacterial colonies surrounded by a transparent halo on PVK agar were picked off, checked for purity and classified as putative P-solubilizers. Quantitative Estimation of Phosphate Solubilization in Culture Broth The isolated bacteria presenting halo zones on solid PVK medium were used to measure phosphate solubilization in liquid medium. The in vitro phosphate solubilizing capacity of each strain was determined on National Botanical Research Institute’s Phosphate growth medium (NBRIP) (Nautiyal, 1999) supplemented by 5 g of TCP as sole phosphorus source. The flasks containing 50 mL of NBRIP medium was inoculated with 1 mL of bacterial suspension (2×109 cfu/mL) in triplicates and incubated at 28±2 °C on a rotary shaker 180 rpm for 7 days. The cultures were harvested by centrifugation at 6000 rpm for 30 min. The phosphorus in supernatant was estimated by vanado-molybdate-yellow colour method (Jackson, 1973). The total soluble phosphorus was calculated from the regression equation of standard curve. The values of soluble phosphate liberated were expressed as μg ml-1 over control. The pH of culture supernatants were also measured using a pH Meter. Production of indole acetic acid (IAA) Production of auxin indole-3-acetic acid (IAA) by bacteria was tested using Lauria Bertani (LB) and Salkowski reagent. Fifty milliliter of Lauria Bertani (LB) containing (0.1g/L of L-tryptophan was inoculated with 1 mL of bacterial suspension (approximately 109cfu/mL) in triplicates and incubated in incubator Shaker at 28± 2 °C and 180 rpm for 4 days in dark. The bacterial cultures were centrifuged at 6,000 rpm for 30 min. Estimation of indole-3-acetic acid (IAA) in the supernatants was done using colorimetric assay (Brick et al., 1991). From a standard curve prepared with known concentration of IAA, the quantity in the culture was determined and expressed as μg/mL. Hydrogen cyanide production Screening of bacterial isolates for hydrogen cyanide (HCN) production was carried out following the method described by Bakker and Schippers (1987). Bacterial cultures were streaked on nutrient agar medium containing 4.4 g/L of glycine. A Whatman filter paper No. 1 soaked in 0.5% picric acid solution (in 2% sodium carbonate) was placed inside the plate lid. Plates were sealed with parafilm and incubated at 28±2 °C for 4 days. HCN production was evaluated according to the colour change, ranging from yellow to orange. Siderophore production Siderophore production by rhizobacterial isolates was detected according to Schwyn and Neilands (1987). Autoclaved CAS agar medium was poured in each Petri dish. The bacterial inoculum was placed in the centre of a Petri dish. The plates were incubated in the dark at 28± 2 °C for 5 days. The change of CAS agar colour from blue to orange around bacterial colony was considered as positive production of siderophore. Nitrogen fixing activity The visual detection of nitrogen-fixing activities of the selected isolates were observed by using nitrogen-free malate-mannitol medium (NF-MM) (Herman et al., 1994), containing bromothymol blue (BTB) as an indicator. The medium was inoculated by the bacterial isolates and incubated at 28± 2 °C for 24 h. The blue coloured zone producing isolates were marked as nitrogen fixers in the solid culture conditions. The colouring zone was calculated by deducting the colony diameter from the colouring zone diameter (Dobereiner and Day, 1976). Ammonia production All the bacterial isolates were tested for the production of ammonia as described by Cappuccino and Sherman (1992). Overnight grown bacterial cultures were inoculated in 10 mL of peptone broth and incubated at 28± 2 °C for 48 h on a shaker. After incubation 0.5 mL of Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 126 ISSN (print): 1823-8262, ISSN (online): 2231-7538 Nessler’s reagent was added. The development of faint yellow to dark brown colour indicated the production of ammonia. Biochemical identification of bacterial isolates The bacterial isolates were further characterized by Gram staining, catalase, oxidase, starch hydrolysis activity and motility followed by biochemical identification using API 20NE kit (BioMérieux, France). API 20 NE is a standardized system for the identification of non-fastidious, non-enteric Gram-negative rods combining 8 conventional tests, 12 assimilation tests. The results were interpreted with the API WebTM software (version 7.0). Statistical analysis The data obtained in this study was subjected to analysis of variance (ANOVA) and comparisons of means were performed by Newman and Keuls test at p ≤ 0.05 using statbox. The correlation between phosphate solubilization and pH was explored by Excel. RESULTS The pH of soil samples ranged from 7.82 to 8.46, moisture content from 10.52 to 18.6% and electrical conductivity from 6.1 to 13.3 ds/m. A total of 104 phosphate solubilizing isolates were obtained from different saline soil sites at Relizane (western Algeria). Out of these isolates 41 were selected for their phosphate solubilizing activity. These were screened for their plant growth promoting activities such as indole acetic acid (IAA), Siderophore production, HCN production, nitrogen fixing ability and ammonia production. Phosphate solubilization ability is marked by the formation of transparent halos around bacterial colony on solid Pikovskaya medium supplemented with tricalcium phosphate (Ca3(PO4)2) as a only source of phosphorus (Figure 1). All the 104 isolates were able to produce transparent halo around the colony with different diameter which indicates a positive phosphate solubilisation ability and they were also capable to solubilize inorganic phosphorus (Ca3(PO4)2) in liquid medium. The results showed that the phosphate solubilizing ability in NBRIP liquid medium of test isolates varied from 24.29 to 738.57 μg/mL using TCP as a source of insoluble P (data of all PSB are not given). The PSB showing the highest amount of solubilized phosphorus are chosen to be tested for their plant growth promoting traits (41 isolates). The isolate that showed the best capability in solubilizing phosphate was PSB91 (738.57μg/mL). pH values of the bacterial cultures decreased from initial value of 7.0 to values ranged from 3.9 to 4.61. A highly significant negative (r = -0, 86) correlation was observed between the amounts of solubilized P and pH values (Table 1). Figure 1: Phosphate solubilisation by bacterial isolate. Table 1: Phosphate solubilization by selected bacterial isolates. Isolate Concentration of phosphate (μg/ml) Final pH of P solubilization PSB3 605.34±15.91d 4.11±0.15 PSB5 525.60±14.73j 4.35±0.26 PSB8 652.83±25.28b 3.90±0.56 PSB11 515.90±12.78j 4.38±0.89 PSB13 584.17±13.78e 4.27±0.21 PSB16 553.62±24.17h 4.32±0.72 PSB18 555.86±16.60gh 4.31±0.41 PSB22 642.49±22.74c 3.92±0.46 PSB24 587.05±19.65e 4.18±0.27 PSB27 566.17±17.14fg 3.97±0.82 PSB29 562.90±21.92fgh 4.18±0.63 PSB31 410.89±16.62p 4.35±0.51 PSB34 453.28±16.95n 4.26±0.32 PSB36 564.74±11.5fg 4.26±0.65 PSB38 402.58±15.71pq 4.47±0.12 PSB41 610.96±13.87d 4.12±0.43 PSB43 571.22±18.70f 4.22±0.74 PSB44 541.59±23.67i 4.32±0.75 PSB45 601.85±22.60d 4.11±0.95 PSB49 561.33±12.03fgh 4.35±0.31 PSB52 370.04±10.61r 4.53±0.45 PSB56 570.63±16.51f 4.22±0.15 PSB58 398.00±11.76q 4.57±0.61 PSB64 487.71±15.35l 4.44±0.34 PSB66 502.25±14.83k 4.46±0.85 PSB67 481.71±16.49l 4.42±0.22 PSB70 568.05±25.89f 4.25±0.12 PSB71 447.20±17.94n 4.33±0.41 PSB72 466.94±18.60m 4.34±0.33 PSB74 435.52±20.87o 4.50±0.23 PSB78 348.05±15.24s 4.55±0.29 PSB82 327.50±16.50t 4.61±0.57 PSB85 398.70±12.42q 4.52±0.45 PSB87 538.37±12.70i 4.23±0.11 PSB89 521.9±14.66j 4.30±0.15 PSB91 738.56±16.07a 4.01±0.77 PSB93 455.13±13.18n 4.35±0.67 PSB95 410.35±22.16p 4.42±0.81 PSB97 521.77±23.66j 4.27±0.36 PSB99 407.67±15.57pq 4.47± 0.37 PSB102 604.37±17.85d 4.14±0.41 r -0.86 Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 127 ISSN (print): 1823-8262, ISSN (online): 2231-7538 Phosphate solubilizing bacteria were assayed for their capacity to produce IAA in LB medium supplemented with L-tryptophan as precursor (1%) by the appearance of pink colour after addition of Salkowski reagent to the culture (Figure 2). IAA production was revealed by almost all the bacterial isolates and IAA quantities of estimated ranged from 10.90 to 63.35 μg/mL as shown in Table 2. A relatively higher content of IAA was found in the culture of bacterial isolate PSB45 followed by PSB85 and PSB91 with 37.43 and 32.85 μg/mL respectively compared to other isolates. Figure 2: IAA production by bacterial isolates. Among the 41 isolates, only twelve between them were recorded positive for HCN production by changing filter paper from yellow to orange (Table 2, Figure 3). The ability of the tested bacterial isolates to produce siderophores in vitro was assessed qualitatively using the CAS-agar plate assay. Of the 41 bacterial isolates tested, 32 strains produced siderophores. Siderophore production capacity of the isolates was evaluated according to the diameter of the orange halo around bacterial colonies as shown in Figure 5, as weak (4-10 mm), moderate (11- 20 mm) and strong (higher than 21mm). Amongst the siderophore producing isolates, 18 were weak siderophore producers, 10 isolates were moderate siderophore producers while 7 isolates were strong siderophore producers (Table 2). Figure 3: HCN production by isolate. Phosphate solubilizing bacteria were also tested for their ability to fix nitrogen on nitrogen free medium (NF-MM) supplemented by BTB used as colour indicator to detect the release of ammonium in the culture as shown in Figure 5. Among the 41 bacterial isolates 31 were able to fix nitrogen by changing medium colour from green to blue colour. The diameter of coloration zone ranged from 7 to 26 mm. The highest value of diameter of coloration zone was obtained with the isolate PSB41 (Table 2). All our selected isolates were capable to produce ammonia (Table 2). Table 2: Characterization of bacterial isolates for multiple plant growth promoting traits. Isolate Concentration of IAA (μg/ml) HCN production SDR production Zone diameter of N fixation Ammonia production PSB3 13.80±2.63p + +++ 17 + PSB5 14.37±3.15n - - 14 + PSB8 14.84±1.12l - - 16 + PSB11 18.05±3.83g - + 11 + PSB13 11.59±4.35vw - + 09 + PSB16 12.62±3.16s - + 13 + PSB18 17.54±5.10h - ++ 12 + PSB22 11.57±2.36vw - + - + PSB24 11.48±1.94w - - 07 + PSB27 11.56±3.57vw - - 12 + PSB29 10.90±4.67z + +++ 11 + PSB31 10.77±2.45z - - 11 + PSB34 11.91±3.65u - + 10 + PSB36 16.90±3.05i + + - + PSB38 14.74±2.05m - - - + PSB41 22.67±3.55d - + 26 + PSB43 12.36±3.69t + +++ 25 + PSB44 22.73±2.54cd + + 11 + PSB45 63.35±5.28a + ++ 07 + PSB49 11.95±3.54u - + 12 + PSB52 11.66±3.45v - + - + PSB56 11.22±2.66x - + - + PSB58 13.60±3.67q - + 21 + PSB64 13.84±3.54p - + 18 + PSB66 15.49±3.05k + ++ 17 + PSB67 22.81±7.69c - + 10 + PSB70 20.21±6.76f - - 16 + PSB71 18.05±4.11g - ++ 17 + PSB72 15.45±3.84k - + - + PSB74 14.02±3.58o + ++ 18 + PSB78 20.60±5.84e + ++ 13 + PSB82 12.95±4.64r + +++ 22 + PSB85 37.43±2.57a - + 14 + PSB87 27.26±3.38ab - + 11 + PSB89 27.10±1.99b - - - + PSB91 32.85±2.32a - - - + PSB93 16.08±4.56j - ++ 07 + PSB95 16.12±4.03j + ++ 09 + PSB97 12.31±5.54t + ++ - + PSB99 11.05±2.69y - ++ 13 + PSB102 12.64±2.76s - + - + +, positive; ++, moderately positive; +++, strongly positive; -, negative A total of forty one isolates of rhizobacteria obtained were biochemically identified by several tests. 20 bacterial isolates were identified as genus Pseudomonas which is the most dominant isolate found including Gram-negative, rod shaped Pseudomonas luteola and Pseudomonas fluorescens, followed by Aeromomas hydrophila (10 isolates). Some of these are Gram-positive spore forming bacteria identified as Bacillus sp. (8 isolates). Only 3 isolates were Gram-negative and rod shaped identified as B. cepacia (Table 3). Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 128 ISSN (print): 1823-8262, ISSN (online): 2231-7538 Figure 4: Siderophore production by bacteria. In this study the highest amounts of released phosphorus were solubilized by Pseudomonas with 24.39% followed by Bacillus sp. (12.19%), Aeromomas (9.75%) and finally Burkholderia with 0.41%. Bacteria producing the highest concentration of IAA were isolates belonging to the genus Pseudomonas (24.39%) followed by Aeromonas (9.75%), Bacillus (8.33%) and Burkholderia with 0.41%. For HCN production Aeromonas was the higher producer (14.63%) followed by Pseudomonas (12.19%) Burkholderia (0.41%), and finally Bacillus with 0%. Isolates belonging to Pseudomonas were able to produce siderophore with 48. 78% followed by Aeromonas (14.63 %), Burkholderia (7.31 %) and Bacillus with 0%. The majority of isolates belonging to genus Pseudomonas were able to fix nitrogen with Table 3: Identification of selected bacterial isolates. Isolate Gram’s staining Shape Catalase Oxydase Amylase Motility ID PSB3 - rod + - + + P. luteola PSB5 + rod + - + + Bacillus sp. PSB8 + rod + + + + Bacillus sp. PSB11 - coccibacilli + - - + A. hydrophila PSB13 - rod + - + + P. fluorescens PSB16 - rod + - + + P. luteola PSB18 - rod + - - + P. luteola PSB22 - rod + + + + B. cepacia PSB24 + rod + - + + Bacillus sp. PSB27 + rod + + + + Bacillus sp. PSB29 - coccibacilli + + - + A. hydrophila PSB31 + rod + - + + Bacillus sp. PSB34 - rod + - + + P. luteola PSB36 - rod + - + + P. luteola PSB38 + rod + + + + Bacillus sp. PSB41 - rod + - + + P. luteola PSB43 - coccibacilli + + - + A. hydrophila PSB44 - coccibacilli + + - + A.hydrophila PSB45 - rod + - + + P. luteola PSB49 - rod + - + + P. luteola PSB52 - rod + - + + P. luteola PSB56 - rod + - + + P. luteola PSB58 - rod + - + + P. luteola PSB64 - rod + - + + B. cepacia PSB66 - rod + + + + P. fluorescens PSB67 - rod + - + + P. luteola PSB70 + rod + - + + Bacillus sp. PSB71 - rod + - + + P. luteola PSB72 - coccibacilli + + - + A. hydrophila PSB74 - Rod + + + + B. cepacia PSB78 - coccibacilli + + - + A.hydrophila PSB82 - rod + + - + A.hydrophila PSB85 - rod + - + + P. luteola PSB87 - rod + - + + P. luteola PSB89 - coccibacilli + + - + A. hydrophila PSB91 + Rod + - + + Bacillus sp. PSB93 - rod + - + + P. luteola PSB95 - rod + - + + P. fluorescens PSB97 - coccibacilli + + - + A. hydrophila PSB99 - rod + + - + A.hydrophila PSB102 - rod + - + + P. luteola Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 129 ISSN (print): 1823-8262, ISSN (online): 2231-7538 19.51% followed by Aeromonas and Bacillus (19.51% and 12.19% respectively) and finally Burkholderia with 4.87% Figure 5: Nitrogen fixation by bacterial isolates. DISCUSSION Plant growth promoting rhizobacteria (PGPR) are a group containing a large number of bacteria number found in the rhizosphere, at root exteriors and in association with them, which can enhance directly and/or indirectly yield and growth of agricultural crops. The capability of some rhizospheric microorganisms to solubilize soil phosphate and make it available for plants by converting it into accessible forms like orthophosphate, is an important activity in a PGPB for improving plant growth (Chen et al., 2006). In our study 104 phosphate solubilizing bacterial isolates were isolated from wheat plant rhizosphere of salt affected soil; these bacteria are more effective in phosphorus solubilization than fungi as reported by Alam et al. (2002). Our rhizobacterial isolates were biochemically identified as Pseudomonas, A. hydrophila, Bacillus sp. and B. cepacia. Srinivasan et al. (2012) also isolated Pseudomonas and Bacillus as phosphate solubilizers from salt affected soil. Moreover A. hydrophila and Bacillus sp. were isolated from the rhizospheric soil of wheat grown in saline soil by Ashraf et al. (2004). In quantitative estimation, all isolates showed diverse levels of phosphate solubilizing activity. The higher concentration of released phosphorus in cultures was exhibited by genus Pseudomonas followed by Bacillus. The same results were described by Ahmad et al. (2008) who reported that solubilization of phosphate was commonly detected in the isolates of Bacillus and Pseudomonas. Prior reports defined some Burkholderia strains as competent phosphate solubilizers (Peix et al., 2001; Caballero-Mellado et al., 2007). pH of bacterial cultures dropped significantly compared to the control. Similar results were observed by Mardad et al. (2013) and Alam et al. (2002). A negative correlation (r = −0.86) was detected between the amounts of solubilized phosphorus of bacterial cultures and their pH values. The same negative correlation was reported by Xiao et al. (2009). Mainly, the mode of action of phosphate solubilising bacteria in soil is by the secreting of acids into the medium (Khan et al., 2014; Oteino et al., 2015). Hence, various genus of bacteria including Bacillus, Pseudomonas, Enterobacter, Serratia, and Azotobacter sp. employ many solubilization reactions, such as acidification, exchange reactions, chelation and production of acids to release phosphorus from mineral complexes (Pandey and Maheshwari, 2007). Recently many researchers have studied production of phytohormones by PGPR (Rajkumar and Freitas, 2008; Ahmad and Khan, 2012). The most important plant growth regulators produced by phosphate solubilizing microorganisms are auxins (Oves et al., 2013). 75% of bacteria isolated from saline soil have the capacity to produce IAA which concluded that saline soil is a rich source of IAA producing bacteria. A high level of IAA production by Pseudomonas was noted in our study and by other researchers (Ahmad et al., 2008; Kumar et al., 2012). Other IAA producing bacteria belongs to Aeromonas (Halda-Alija, 2003) and Bacillus (Swain et al., 2007) were also reported. HCN is a volatile, secondary metabolite that suppresses the development of microorganisms. An important role of HCN produced by bacteria from rhizosphere in biological control of pathogens has been described (Siddiqui et al., 2006). In the current study isolates belonging to genus of Aeromonas, Burkholderia and Pseudomonas were able to produce HCN. To date various bacterial genera are able to produce HCN, including Pseudomonas, Aeromonas, Alcaligenes and Rhizobium (Devi et al., 2007; Ahmad et al., 2008). Siderophores produced by PGPR can promote plant growth either directly or indirectly, using radiolabeled ferric siderophores (Sujatha and Ammani, 2013). Our results showed that isolates belonging to genus Pseudomonas and Burkholderia were the best siderophore producers but isolates belonging to Bacillus sp. were unable to produce them. Also García-Gutiérrez et al. (2012) found that all Pseudomonas strains isolated from soil were able to produce siderophore, while only one strain among Bacillus was able to produce such compounds. Luvizotto et al. (2010) reported that B. cepacia exhibit a high levels of siderophore production. The presence of nitrogen-fixing bacteria in soil along with its isolation and conversion into PGPR biofertilizer is an important strategy reducing the use of expensive chemical fertilizers especially in nutrient poor and degraded soils (Cakmakci et al., 2006). The majority of our rhizobacterial isolates have the ability to fix nitrogen. Currently there are evidences that plant stimulation effect by PGPR such as Azoarcus sp., Burkholderia sp., Gluconacetobacter is related to their ability to fix nitrogen (Vessey, 2003). In the study of Cakmakci et al, (2006), the quality and yield of wheat, spinach and sugar beet was improved using nitrogen fixing bacterial isolates Pseudomonas RC06 and Bacillus OSU-142 as biofertilizers. Zhang et al. (1996) reported that A. hydrophila have the ability to fix atmospheric nitrogen. Ammonia production is an essential trait exhibited by PGPR, which can effect indirectly plants growth (Yadav et al., 2010). All our isolates were able to produce ammonia. These results are similar with those of Ahmed et al. (2008) Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 130 ISSN (print): 1823-8262, ISSN (online): 2231-7538 who revealed the production of ammonia commonly detected in all the isolates of Pseudomonas, Bacillus and Azotobacter. Similarly, all bacteria identified as Bacillus and Pseudomonas isolated by Yadav et al. (2010) from chickpea rhizosphere in India have the ability to produce ammonia. CONCLUSION Phosphate solubilizing bacteria isolated from wheat rhizosphere from saline soil located in western region of Algeria showed a high potential to produce growth promoting traits. Isolates belonging to the Bacillus and Pseudomonas showed a high phosphate solubilisation. Pseudomonas sp. was the highest producer of IAA and siderophore and had the capacity to fix nitrogen. A. hydrophila and B. cepacia showed high potential produce HCN and other PGP traits also. 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Effect of plant growth promoting rhizobacteria on seed germination and plant growth chickpea (Cicerarietinum L.) under in vitro conditions. Biological Forum An International Journal 2(2), 15-18. Zhang, F., Dashti, N., Hynes, R. K. and Smith, D. L. (1996). Plant growth promoting rhizobacteria and soybean [Glycine max (L.) Merr.] nodulation and nitrogen fixation at suboptimal root zone temperatures. Annals of Botany 77, 453-459., 2017-11-02)
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