Open Access Original Research Article

Molecular Analysis of Genetic Diversity of Pectobacterium carotovorum Subsp Carotovorum Isolated in Morocco by PCR Amplification of the 16S-23S Intergenic Spacer Region

M. Amdan, H. Faquihi, M. Terta, M. M. Ennaji, R. Ait Mhand

Biotechnology Journal International, Page 102-110
DOI: 10.9734/BBJ/2015/16045

Aims: Pectobacterium carotovorum is a ubiquitous bacterium that causes soft rot in different crop plants throughout the world. In Morocco, approximately 95% of the Strains isolates from potato plants with tuber soft rot are P. carotovorum. In this study, we test whether PCR ribotyping can be used to distinguish strains of Pectobacterium carotovorum isolated from soft rot potato and to differentiate among strains from different geographic regions.

Place and Duration of Study: Laboratory of Virology, Microbiology and Quality / Ecotoxicology and Biodiversity, department Biology, Faculty of Sciences and Techniques, University Hassan-II Mohammedia Casablanca.

Methodology: Eighty-three pectolytic enterobacteria were collected from potatoes rotten in Morocco, the strains were isolated in the Cristal Violet Pectate (CVP) medium and were purified in LPGA agar (yeast extract, peptone, glucose and agar). After purification, strains were identified by physiological and biochemical tests. The confirmation of species was performed by PCR using primers Y1 and Y2. The genetic diversity of Pectobacterium carotovoum was investigated by PCR ribotyping using primers G1/L1, which are complementary to conserved regions of the rRNA operon. Furthermore, the profiles obtained were compared by the Unweighted Pair Group Method.

Results: The biochemical and physiological analysis demonstrated that the predominant pectolytic enterobacterium present in Morocco is Pectobacterium carotovorum subsp carotovorum. The specific confirmation of species P. carotovorum by PCR has yielded a 434 bp DNA fragment of the pelY gene with all isolates. Further, PCR amplification of the 16S-23S Intergenic spacer Region (ITS-PCR) has presented a specific pattern made of 2-6 fragments ranging from 300 bp to 800 bp. The UPGMA tree has shown that there is considerable genetic diversity in P. carotovorum strains, which can be divided into four distinct groups.

Open Access Original Research Article

Tolerance Potentials of Cocoa (Theobroma cacao) in Hydrocarbon Polluted Soils

R. B. Agbor, J. E. Asor, I. U. Bassey, Chibuzor Obianwa, E. O. Ude, A. Ipoh

Biotechnology Journal International, Page 111-121
DOI: 10.9734/BBJ/2015/13863

In the present study the tolerance potentials of T. cacao in hydrocarbon polluted soil was evaluated. Top soil (0-25 cm depth) was collected from three points, bulked to form composite soil sample. Eight kilograms of the soil sample each were weighed into twenty five (25) perforated bags. The bags were polluted with 0 ml/kg, 50 ml/kg, 100 ml/kg, 150 ml/kg and 200 ml/kg of crude oil respectively with five replicates for each concentration and allowed for 2 weeks before planting. Three seeds of cocoa were sown in each of the polythene bags. Water was applied every three days to keep the soil moist.  The results showed that cocoa plants at 8 weeks after planting (WAP) had significantly higher height (P<0.05) than those of 4 weeks after planting (WAP). The plant height, petiole length, number of vine had no significant difference (P>0.05) at the different concentrations of crude oil. While the leaf length, leaf area, number of leaves, leaf width and vine length shows significant difference (P<0.05) and in treatment dependent manner. The results also showed that the concentrations of Cu, Ni, Cr and Zn in the polluted soils were treatment dose dependent but did not significantly affect the plant tolerance at P<0.05. The pH, phosphorus, nitrogen content, and other physicochemical properties of the soil were not significantly affected by the crude oil treatment. The T. cacao has been found to be tolerant to hydrocarbon polluted soil environment, its usage in oil spill impacted area should be encourage.

Open Access Original Research Article

Regeneration Procedure for Three Arachis hypogaea L. Botanicals in Uganda through Embryogenesis

D. K. Okello, L. B. Akello, P. Tukamuhabwa, S. M. Ochwo, T. L. Odong, J. Adriko, C. Mwami, C. M. Deom

Biotechnology Journal International, Page 122-133
DOI: 10.9734/BBJ/2015/17418

Aims: A procedure was developed for embryogenesis from embryo explants derived from mature seeds of freshly harvested Serenut 4T, Serenut 1R and Acholi-white groundnut cultivars representing the three broad groundnut botanical classifications.

Methodology: This study explored the use of mature embryo axes as explants for somatic embryogenesis, and determined the factors that affect regeneration of three Ugandan groundnut cultivars. Freshly harvested mature seeds of the three groundnut cultivars were collected and the embryo explants were initiated on 3 media namely; Murashige and Skoog (MS) basal media with varying concentrations of the growth regulator 2,4-Dichlorophenoxy acetic acid (2,4-D); Chu N6 basal medium with vitamins (N6); and Callus Induction Medium (CIM). The shoot formation and elongation medium contained MS basal medium supplemented with indolebutyric acid (IBA) and 6-Benzylamminopurine (BAP) in isolation, and BAP in combination with a-naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). For root induction, elongated shoots were transferred to MS medium supplemented with various combinations of NAA with IBA, BAP and a combination of IBA and Kinetin.

Results and Conclusion: Different concentrations of 2,4-D elicited different callogenesis responses in the cultivars with Acholi white (Valencia botanical) and Serenut 4T (Spanish botanical) giving the optimal response at 5mg/l whereas Serenut 1R (Virginia botanical) showed best response at a concentration of 30 mg/l. N6 and CIM supported callogenesis in Acholi white (AW) and Serenut 4T only. In all cultivars, maximum root production was gained when using MS medium supplemented with NAA- 1 mg/l and IBA -2.0 mg/l. On the other hand, for Serenut 1R and Serenut 4T, BAP 2.5 mg/l; NAA 0.5 mg/l combination yielded higher shoot regeneration percentage whereas for AW BAP 3 mg/l; NAA 0.5 mg/l supported maximum shoot production.This is the first ever report of successful regeneration of the three groundnuts botanicals in Uganda. These results are likely to facilitate genetic transformation of three preferred Ugandan groundnut varieties.

Open Access Original Research Article

Screening and Production of Biodegradable Polyester Poly(3-hydroxybutyrate) by Bacteria Endophytic to Brassica nigra L.

Rituparna Das, Arundhati Pal, Sukhendu Mandal, A. K. Paul

Biotechnology Journal International, Page 134-146
DOI: 10.9734/BBJ/2015/16750

Aims: Poly(3-hydroxybutyrate) [P(3HB)], the microbially produced biodegradable thermoplastics has find wide range of applications in recent years. Development of low cost production strategies utilizing novel organisms is a crucial challenge. Present study is aimed to isolate and screen bacterial endophytes of Brassica nigra L. for the production of P(3HB).

Place and Duration of Study: The experiments were performed in the Microbiology Laboratory, Department of Botany, University of Calcutta, Kolkata during 2012-2014.

Methodology: Culturable bacterial endophytes were isolated from surface sterilized healthy tissues of B. nigra L. and screened for P(3HB) production in mineral salts medium. The chloroform extracted dried polymer was treated with H2SO4 and quantified spectrophotometrically at 235 nm.

Results: About 78% of the bacterial endophytes recovered from surface sterilized B. nigra L. tissues showed different degrees of P(3HB) accumulation. Isolates (9) showing P(3HB) accumulation exceeding 10% of the cell dry weight (CDW) were characterized and tentatively identified as members of Bacillus, Pseudomonas, Xanthomonas, Alcaligenes and Acetobacter. The most potent isolate, BNL 06 identified as Bacillus pumilus BNL 06 (GenBank Accession No. KP202723), accumulated P(3HB) accounting 18% of CDW with an yield of 0.55 g/l. Finally the nature of the polymer was further confirmed by FTIR analysis.

Conclusion: Exploration of the endophytic bacterial diversity of B. nigra L. have clearly revealed the potential of Bacillus pumilus BNL 06 for P(3HB) production as an alternative source of thermoplastics.

Open Access Original Research Article

Isolation and Identification of Cellulytic Fungi from Agrowastes and Sawmill Soils

Ekundayo Temitope Cyrus, Arotupin Daniel Juwon

Biotechnology Journal International, Page 147-159
DOI: 10.9734/BBJ/2015/17575

Five agricultural wastes and sawmill soil were investigated for fungi capable of producing cellulase. Isolation was carried using serial dilution techniques and isolates were screened for cellulase production on carboxymethyl cellulose-containing agar plates. Copious isolates were further subjected to submerged cultivation for quantitative evaluation of cellulase biosynthesis. Highest fungal load was 6.67±0.33×106 sfu/g (Sawmill soil) and the least was 2.33±0.33×106 sfu/g (yam peels). A total of sixty seven fungi were identified from the samples. The isolates were of fifteen fungal species namely Mucor mucedo, Aspergillus niger, A. repens, A. flavus, A. parasiticus, Articulospora inflata, Gonatobotrys simplex, Gyrothrix circinata, Dendrospora erecta, Penicillium notatum, P. italicum, Varicosporium elodea, Gonatobotryum apicolatum, Mucor racemosus and Rhizopus nigricans. Aspergillus had the highest occurrence (36.11%), while the least occurrence was Varicosporium and Gonatobotryum (2.78%). Highest cellulase activity ratio was exhibited by Aspergillus niger (1.90) and the least by Aspergillus repens (1.04) on plate screening. The highest cellulase producer among selected isolates under submerged growth was Gonatobotrys simplex (1.2143±0.02 U/ml), followed by Aspergillus niger (1.1429±0.01 U/ml) and the least by Aspergillus parasiticus (0.8265±0.01 U/ml). Submerged protein content was highest in Aspergillus niger culture and least in Penicillium notatum. The study has revealed array of cellulytic fungi from agricultural wastes and sawmill soil.