Egyptian Journal of Biotechnology

Biological control of necrotrophic plant pathogenic fungi: Tomato plants as a model

O A Wahid — Wed, 05 Dec 2007 00:00:00 +0000

Biocontrol agent, Bacillus subtilis, was screened in a laboratory experiment against 6 necrotrophic fungi. These fungi were isolated from diseased plant materials. This biocontrol agent exhibited different potentials. It was highly effective against Fusarium oxysporum f. sp. lycopersici which causes tomato wilt and Fusarium oxysporum f. sp. fabae which causes broad bean wilt. It inhibited radial growth (RGI) by more than 75 %. However, it was less effective against Phytophthora infestans (64.7 % RGI), Botrytis cinerea (58.3 % RGI), Sclerotinia sclerotiorum (57.4 % RGI) Rhizoctonia solani (42.7 % RGI). When B. subtilis was applied to Tomato field, it exhibited different efficiency in suppressing Fusarium wilt. Time of implementation was critical for the efficacy of B. subtilis. It strongly suppressed wilt when applied 7 days before transplanting, by showing 72.7, healthy plant percentage (HP), followed by application at cotyledon stage (69.4 % HP). Salicylic acid was comparable to B. subtilis in protecting tomato against vascular wilt (68.3 % HP). Different mechanisms may be involved in the activity of B. subtilis in field. According to the laboratory test, diffusible suppressive substances as well as lytic enzymes may be produced by this biocontrol agent.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 1-12

Identification of some actinomycete species by restrication endonuclease analysis of an amplified the 16SrRNA gene

I H Abbas, M H Awwad — Wed, 05 Dec 2007 00:00:00 +0000

Restriction fragment length polymorphisms of 16S rRNA genes of representative strains of the actinomycete species (P-100-1 Saccharomonospora halophila, A-3-4 Nocardiopsis sp., A-1 Nocardiopsis halotolerance, P-7-2 Nocradiopsis sp. and Microbispora sp.) were determined following the isolation and genes amplification. 16S rDNAs were amplified with two primers; 5\'-TCACGGAGAGTTTGATCCTG-3\' and 5\'-AGAAAGGAGGTGATC-3\'. Actinomycete 16S rRNA genes were used with eight restriction endonucleases (EcoRI, StyI, AvaI, BanI, HindIII, NaeI, AsnI and BanII) to obtain restriction fragment length polymorphisms (RFLPs). The PCR/RFLP profiles of SrRNA genes indicated that the actinomycete isolates are polyphylogenetic when digested with BanI restriction endonuclease. Also, some actinomycete strains are monophylogenetic in nature, such as N. A-3-4 and S. P-100-1. PCR-RFLPs of SrDNA is an effective marker to differentiate between the actinomycete strains in a much more accuracy than do the morphological studies alone.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 13-26

Assessment of multi-strain PGPRs biofertilization as compared to sole-strain or mineral n-fertilization on wheat plants grown in clayey soil in Egypt

G A Mekhemar, F Sh Badawi, T E Radwan, B A Hassona — Wed, 05 Dec 2007 00:00:00 +0000

A field experiment was performed at Malawi Station, Agric. Res. Center (ARC), Minia, Egypt during 2004-2005 and 2005-2006 winter seasons, to investigate the response of bread wheat (Triticum aestivum L.) cv. Sids 1, grown in clayey soil to inoculation with plant-growth promoting rhizobacteria (PGPRs) such as cyanobacteria spp; Serratia sp; Rhizobium leguminosarum bv. viciae and Bacillus polymyxa either solely or in combinations in comparison to mineral N-fertilization, Full N-dose; 75 Kg N/fed. Results revealed the superiority of Bacillus polymyxa for increasing wheat growth and productivity followed by Serratia, Cyanobacteria, and Rhizobium inoculation. Concerning combined inoculations, tetras combination treatment caused the highest increases in wheat parameters, triple combinations were the second, dual combinations were the third and the sole inoculation caused lower increases but still higher than control treatment. On the other hand, full dose of N-fertilizers recorded increases ranged between those of tetras and those of triple combinations. Also, results confirmed the necessity of combined applications with N2-fixers and its superiority on mineral N-fertilizations for wheat productivity.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 27-44

Enzymatic synthesis of oligosaccharides by recombinant Eschericia coli - expressed Candida molichiana β-glucosidase II

A A Hassan — Wed, 05 Dec 2007 00:00:00 +0000

In the present study, the biosynthetic activity of recombinant β-glucosidase from E. coli was described. The purified β-glucosidase from the recombinant E. coli – expressed C. molichiana (E. coli pBG₂₂: JM109) was obtained at the end of the fermentation process (24 h) with an enzyme activity of 180 IU/ml and the enzyme retained 93% of its initial activity in 20% (V/V) DMSO (dimethyl sulphoxide). The initial substrate concentration, the incubation time and temperature and the type of substrate was reported to influence the biosynthesis of oligosaccharides. The efficiency of the biosynthetic reaction increased to about 2-fold in the presence of DMSO. The yield of oligosaccharides was higher in the presence of sophorose than gentiobiose and cellobiose with DMSO.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 45-59

Effects of cultivation medium and conditions on the production of alpha-galactosidase by Aspergillus awamore & A/ carbonarius

UF Ali, Z M Ibrahim, G S Isaac — Wed, 05 Dec 2007 00:00:00 +0000

The α-gal production by Aspergillus awamori and A. carbonarius (local isolates) as affected by several culture conditions was studied showing that 0.5 % corn bran and 2.0 % cane baggase were used for the production of α-gal by Aspergillus awamori & Aspergillus carbonarius respectively; the best pH value, incubation temperature and incubation period for the production of α-gal were 4.8, 30º C and 6 days for both experimental fungi; raffinose at the concentration 1 % supplemented with 0.5 % corn bran and acacia gum at concentration 1 % supplemented with 2 % cane baggase were the best synthetic carbon source for the production of α-gal for Aspergillus awamori and Aspergillus carbonarius respectively; L-Asparagine (0.262 %) was the best concentration of nitrogen source for Aspergillus awamori whereas sodium nitrate (0.4 %) was the best concentration of nitrogen source for Aspergillus carbonarius and the best ratio of surface area to volume of medium was 0.525 for Aspergillus carbonarius and 2.260 for Aspergillus carbonarius.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 60-69

Stabilization of 5A1 urease by covalent attachement to wool

S A Ahmed — Wed, 05 Dec 2007 00:00:00 +0000

The investigation of five bacterial strains for urease production referred that Bacillus licheniformis 5A1 had the highest urease activity (10.3U/ml/min) after 24h. The enzyme was covalently coupled to different carriers via glutaraldehyde, and wool gave the highest immobilization yield (76.4%) and retained 85% of the original specific activity. The immobilized urease retained 62% of its activity when incubated at 50°C for 45min and was quietly stable at higher temperature. The optimum reaction temperatures of the free and the immobilized urease were 40 and 45°C respectively. The optimum pH of free urease (7.0) apparently was shifted 1.0 unit acidic region upon immobilization. . Activation energy ( EA ) of free and immobilized urease were 7.91 and 3.64 Kcal/mol respectively . Deactivation rate constant at 60°C of both free and immobilized urease were 3.2x10^-2 and 0.7x10^-2/min, respectively. Half-life time ( t1/2 ) at 50°C of immobilized urease was higher than free urease ( 58.5 and 12.3 min, respectively). The immobilized enzyme was repeated 13 times with 70% residual activity and would have promise in clinical and industrial uses. The activity of both free and immobilized urease were significantly improved by addition of CaCl^{2 (10mM) and immobilization processes protected the enzyme against inhibitors like Cu²⁺.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 70-81}

Biological activity and toxicitiy of imported and synthetic metal working fluids on Scendesmus obliquus algae

A El Shimy, N Tantwy, A El Tabei, A M Omar — Wed, 05 Dec 2007 00:00:00 +0000

Effects of imported (I) and synthetic (S) metal working fluids and different combinations formed from these fluids with each of benzyl triethyl ammonium chloride (A), benzyl triethanol ammonium chloride (B), P- methyl benzyl triethyl ammonium chloride(C) and P- methyl benzyl triethanol ammonium chloride(D) in different concentrations were studied on growth and structure of green alga Scendesmus obliquus. The toxicity of surfactants to Scendesmus obliquus are arranged in the order: imported fluid > Synthetic fluid > S+ D > I+A> S+B> I+ C> I+B > I+D > I+D >S+A > I+4. These results prove that, the toxicity of fluids depends on its chemical structure.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 82-93

Comparison between chemical and biological control of Fusarium-root rot disease on the response of some physiological aspects of Cucumis sativus seedlings

N Haikal, F A El-Daly

This work was carried out at the Girls College of Education, Jeddah, Saudi Arabia. The results revealed that treatment with the fungicide carbomar or T. harzianum as well as with B. subtilis, in presence of F. solani increased the % of healthy seedlings as well as their length , fresh and dry weight than in presence of F. solani alone but still less than the control. Coupling treatment with T. harzianum and seed coating with B. subtilis gave the best result. The photosynthetic pigments content of the leaves increased when treated with the pesticide carbomar or biological control, T. harzianum and/or B. subtilis. The same almost applied to amino acids, total nitrogen or total soluble sugar content of the seedling\'s leaves, though carbomar alone lowered it than control. Both treatments (chemical control, biological control) increased the test elements of cucumber seedling especially Zn and Fe than in presence of F. solani alone.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 94-101

Production of lovastatin by Pleurotus ostreatus

A R El-Shami, E R Hamed — Wed, 05 Dec 2007 00:00:00 +0000

Lovastatin is one of the earlier statin drugs, which is effective for the treatment of hypercholesterolemia, as an inhibitor of the HMG-CoA reductase. The present work was undertaken to determinate lovastatin production by Pleurotus ostreatus in fruiting bodies and its production on different fermentation media. The favorable medium was found to be Dox-rice. Determination of the optimum conditions for lovastatin production were studied. Results showed that the optimum condition for incubation was 10 days with aeration speed at 180 rpm, at 28ºC when pH medium was 6.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 102-110

Production, purification, and characterization of thermostable alkaline protease under solid state fermentation conditions for application in bio-detergent technology

R A Bayoumi, S S Louboudy, N M Sidkey, M A Abd-El-Rahman — Wed, 05 Dec 2007 00:00:00 +0000

Screening studies were carried out for one hundred, and fifty-three bacterial isolates with respect to their ability to produce protease(s), after growing on some industrial wastes at 55ºC, and pH 9.Two most potent thermophilic bacterial isolates concerning of alkaline thermostable protease(s) production were identified as Bacillus licheniformis B42 and Geobacillus stearothermophilus B78. Alkaline thermostable proteases productivity by the two most potent bacterial isolates was affected by substrate concentrations (solid substrate), carbon source, nitrogen source, mineral ions, vitamins, amino acid supplements, incubation temperature, incubation period, and inoculum size. Maximum both enzymes production by B. licheniformis B42 and G. stearothermophilus B78 were obtained on slaughter house wastes (SHW) concentrations,1.5,2.5 %;carbon sources; galactose, no tested sugar; nitrogen sources; diammonium hydrogen phosphate, ammonium molybdate; chelating agents; ethylene diamine tetra acetic acid (EDTA) (500 ppm), no tested ions; vitamins; thiamine pyridoxine; amino acids; arginine, DL-tyrosine at 55°C for 72 h. when inoculated by 0.5, and 2.5 ml respectively. The protease yield under all optimal conditions was increased many folds from 563.68 to 17825 U/ml (31 fold) by B. licheniformis and from 23.7 to 7096 U/ml (299 fold) by G. stearothermophilus. The purification fold of B. licheniformis B42 alkaline thermostable protease increased to 394.7 after applying Sephadex G200 and 411.9 times after Sephadex G100 column chromatography techniques. Fifteen amino acids were detected in the partially purified protease. Optimum conditions of the partially purified alkaline thermostable protease on Sephadex G200 followed by Sephadex G100 produced by B. licheniformis B42 were: incubation temperature, 50°C; thermostable between 50-70°C; pH,10; and incubation time,36 h exhibited maximal activity. The purified enzymes exhibited good stability towards chlorine. Compatibility of the purified enzyme with various commercial detergents reported that purified protease was unstable in presence of commercial detergents. Calcium chloride was able to restore and enhance the activity of the partially purified enzyme in the presence of commercial detergents. The alkaline serine proteases secreted by both strains are industrially important in respect of their abilities to act in alkaline pH and to show stability in broad pH ranges in addition to their stabilities toward sodium dodecyl sulphate(SDS), and hydrogen peroxide (H₂O₂), suggesting that it is a potentially candidate as an additive in the detergents formulations.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 111-129

Influence of gamma-radiation on the occurrence of toxigenic Pennicium strains and mycotoxins production in different feedstuffs

Z A Matter, N H Aziz — Wed, 05 Dec 2007 00:00:00 +0000

One hundred and fifty random poultry and animal feed samples were collected and analyzed for Penicillium toxins. The effect of gamma irradiation on the production of mycotoxins in feeds was studied. The results revealed the isolation of 12 Penicillium species. Out of 179 pure cultures of Penicillium species only 104 isolates (58%) were mycotoxin-producers. The isolates have the ability to produce, citrinin, patulin, ochratoxin A, rubratoxin B and cyclopiazonic acid with variable levels. Of the 150 samples examined, 70 were positive for Penicillium toxins. Analysis of feed samples revealed the occurrence of citrinin, ochratoxin A, cyclopiazonic acid and rubratoxin B. Irradiation at a dose 4.0 kGy reduced the Penicillium growth greatly relative to unirradiated controls and there was no growth and mycotoxin production at dose 5.0 kGy. Treatment of feed samples with 10 kGy detoxified citrinin by 92.5 to 97.5% and ochratoxin A by 68.8 to 78.5%. A dose of 15 kGy was sufficient for complete destruction of citrinin and by 90 to 95% for ochratoxin A. Patulin, cyclopiazonic acid and rubratoxin B were not detect at 5.0 kGy.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 130-144

Effect of Cymbopogon ctratus L. essential oil on the growth and morphogenesis of Candida albicans

E K El-Khair — Wed, 05 Dec 2007 00:00:00 +0000

Addition of 2.0 l/ml of Cymbopogon citratus L. essential oil to the Sabouraud\'s medium was inhibited growth completely of Candida albicans in liquid medium. Light microscope and scanning electron microscope observation showed morphogenic changes in the treated cells including decrease in cell size. While investigation by transmission electron microscope also showed changes in the ultrastructure of cells including: cell wall, plasma membrane and mitochondria. Oil treatment also increased leakage of ions from yeast cells and decreased in its lipid content.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 145-158

Effect of antifungal agents on the physiological activities of towo plant pathogenic fungi: Rhizoctonia solani & Fusarium oxysporum

A A El-Mehalawy

Each of the antifungal compounds extracted from each of the antagonistic microorganisms was found to has an inhibitory effect on Rhizoctonia solani and Fusarium oxysporum. Four antifungal compounds were found to be the most inhibitory than the other compounds. The first one from Eupenicillium senticosum, the second from Streptomyces aurantiacus, the third from Curtobacterium pusillum and the last one from Saccharomyces unispora respectively. The first three compounds were active against R. solani, while the last one was active against F. oxysporum. All the four compounds reduce or inhibit the growth of Rhizoctonia solani and Fusarium oxysporum. They also reduced the carbohydrate concentrations, as well as reducing the nitrogen content (total nitrogen, soluble nitrogen and protein nitrogen). Regarding their effects on the enzyme activity, the active compounds increased the activity of fumarase, malic dehydrogenase and succinic dehydrogenase, the activity of catalase enzyme of both R. solani and F. oxysporum was sharply decreased by such compounds as compared to control.

Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp.159-168