Vol. 22, February, 2006

1/22 ALTERATIONS OF OUTER MEMBRANE PROTEINS IN MULTIPLE-DRUG RESISTANCE MUTANTS OF PROTEUS MIRABILIS SELECTED BY LEVOFLOXACIN

M.A. Yassien

Faculty of Pharmacy, King Abdul Aziz University, Jeddah, Saudi Arabia

A total of 6 spontaneous levofloxacin-resistant mutants were isolated from Proteus mirabilis clinical isolate on agar plates containinglevofloxacin(6 times MICs). Five of these mutants (PM1-PM3, PM5, and PM6) are characterized by cross-resistance to other fluoroquinolones (norfloxacin, and ofloxacin), β-lactam antibiotics (cephalothin, cefamandole, cefoperazone, and cefotaxime), aztreonam, and chloramphenicol. While the sixth mutant (PM4) is characterized by selective resistance to the tested fluoroquinolones. The outer membrane proteins (OMPs) analysis showed a significant reduction ( in PM3 and PM5 mutants) or disappearance (in PM1 and PM2 mutants) of 39 KDa OMP andexpression of 48 and 52 KDa OMPs in PM6 mutant as compared to the parent isolate. In case of mutant PM4, no significant change in the OMP profile was observed. The five multiple-drug resistant mutants (PM1-PM3, PM5, and PM6) had about 2.7- to 4.4-time lower rate of norfloxacin uptake than that of the parent isolate, while no significant change in norfloxacin uptake was observed in case of PM4 mutant. Addition of 0.25 mM carbonyl cyanide m-chlorophenylhydrazone increased the norfloxacin uptake by only PM6 mutant, while no change was observed in case of the other mutants.   The results of β-lactamase induction and DNA gyrase assay showed no difference between the selected mutants and their parent, with exception for PM4 mutant in which a significant decrease in the DNA gyrase sensitivity to levofloxacin was observed. These data suggest that reduction or disappearance of 39 KDa and expression of 48 and 52 KDa OMPs in P. mirabilis isolate is associated with cross-resistance to fluoroquinolones, β-lactam antibiotics , aztreonam, and chloramphenicol.

2/22 HERBICIDAL SUBSTANCE FROM STREPTOMYCES LAVENDULAE WITH ANTIBIOTIC, INSECTICIDAL AND MITOGENIC ACTIVITIES

M.M. Aly, S.M. El-Sabbagh* and A.R. El Shanshoury**

Biology Department, Faculty of Education, Kafr El-Sheikh, Tanta University, Egypt.

*Botany Department, Faculty of Science, Minoufia University, Egypt.

**Botany Department, Faculty of Science, Tanta University, Egypt.

Of twenty five strains of actinomycetes isolated from marine water of Mediterranean sea (Alexandria, Egypt), on starch nitrate agar containing 25mg/ml nystatin and streptomycin. Screening of microbial fermentation broths for herbicidal activity against Medigaco sativa led to the discovery of new secondry metabolite, from an actinomycete isolate. The isolate was recognized as spore former with aerial mycelia containing LL-DAP (diaminopimelic acid) and glycine. The vegetative hyphae are not fragmented and the spores are borne on sporophores. These criteria in addition to other physiological and biochemical characteristics for identification suggested that the isolate could be affiliated to the genus Streptomyces and the name Streptomyces lavendulae was suggested. The herbicidal substance in the cell free extract of this isolate proved to be active against different weeds associated with wheat plants. It was extracted and purified using differnt column chromatography. The purified material has excellent antibacterial activities against both Gram positive and Gram negative bacteria with MIC ranging from 25-75 mg/ml. It has no antifungal or antitumor activities. Insecticidal activity against ants, lices and house fly was demonstrated. It showed also mitogenic activity with human cell lymphoma. Physical characters and chemical structure of the purified bioactive substance, in addition to its toxicity were elucidated and discussed.

3/22 EFFECT OF SOME GROWTH INDUCERS AND BENOMYL ON THE INFECTION MODE OF R. SOLANI AND SOME PHYSIOLOGICAL TRAITS OF PHASEOLUS VULGARIS.

S.M. Amer, S.M. Abou El Souod, A.A. Ismail*, S.W. Assawah and. H.M. Moubark

Department of Botany, Faculty of Science,. Tanta University. Tanta, Egypt.

*Plant pathology research institute, Agriculturel research center, Giza, Egypt.

The effect of four growth inducers (Ecophotoplus, photophore plus, Starner and Copprous KZ) and one fungicide (benomyl) and their combinations on Phaseolus vulgaris seedlings infected by R. solani was studied under laboratory and greenhouse conditions. In vivo benomyl was the most toxic on the shoot and root systems compared to growth inducers.. The root system was more sensitive to the tested compunds than shoot system. Copprous KZ was the most stimulatory on the growth of shoot and root systems. When benomyl was added to Copprous KZ, it   gave a stimulatory effect   on the shoot system and gave slight toxic effect on the   root system. At greenhouse, it was noticed that most growth inducers increased the percentage of survival plants. Whereas benomyl + Ecophote Plus   (combination) was the best treatment   for increasing the percentage of survival plants. The growth inducers improved the height, fresh and dry weight of the plant and increased chlorophyl, polysaccharides, Potassium, total nitrogen and phosphorus content. In the soil infested by R. solani, the present work showed that seed treatment by benomyl, Ecophote plus, Photophore plus, Starner, Copprous KZ and their combinations induced the formation of simple types of infection cushion by the fungus (sun like structure, parallel like structure, tree like structure, dome like structure and broom like structure) more than complicated types (top fan like structure , complex parallel like structure, complex dome like structure, complex broom like structure). In case of treating the hypocotyl by Ecophote, Photophore, Starner and by combinations with benomyl, the cushion was formed up on the epidermis and the infection stopped at this stage. In case of treatment of the hypocotyl by Copprous KZ a simple dome cushion was formed on the epidermis and browning of xylem vessels took place. In case of root infection all the treatments prevented   further development of the disease and the mycelia remained at   the piliferous layer.

4/22 PHYSIOLOGICAL RESPONSE OF COMBINED CONTINUOUS WATER DEPRIVATION AND NITROGEN STARVATION ON TWO CONTRASTING PEA (PISUM SATIVUM L.) CULTIVARS.

II- YIELD AND ITS COMPONENTS.

N.M. Abd El Rahman

Agricultural Botany and Plant Pathology Department,

Faculty of Agriculture, Zagazeg University

Pea plants (Pisum sativum L.) cvs "Little Marvel" and "Meteor" were grown in sandy soil to investigate the effectiveness of supplementary Nitrogen (N) applied into control nutrient solution in plants grown under water stress. Treatments were (1) well – water and N-Supply i.e WWN, (2) well – water and N-starvation i.e (WWns), (3) water stress and N-supply i.e (wsN) and (4) water – stress and N-starvation i.e (wsns). The results showed that, pea yield, and its components and some chemical compositions of pea seed (carbohydrate and nitrogen as percent of dry weight) increased significantly with increasing water and nitrogen supplies i.e control plants (WWN), conversely, all parameters studied were strongly and significantly declined with increasing water shortage combined with nitrogen starvation i.e treatment 4 (wsns). Supplementary nitrogen ameliorated and eliminated the negative and the harmful effects of drought stress on yield, yield components and chemical composition of pea seed such as carbohydrate and nitrogen contents. Varial differences in all parameters investigated except mineral N% and protein % were confirmed and the highest significant values were recorded by Meteorcultivar.

5/22 THE PHYSIOLOGICAL INTERACTION BETWEEN POTASSIUM SUPPLY AND SODIUM ON WHEAT PLANTS GROWN UNDER DIFFERENT LEVELS OF NACL SALINITY

II YIELD AND ITS CORRELATED COMPONENTS

N.M. Abd – El Rahman

Agricultural Botany and Plant Pathology Department,

Faculty of Agriculture, Zagazeg University

A greenhouse pot experiments were conducted to study the indirect effects of salinity as NaCl combined with potassium supply on wheat plant var. Giza – 168 grown under saline conditions. In these experiments, three Na Cl and three K treatments were imposed. The Na+ concentrations were O, 100 and 200 mM NaCl and K+ concentrations were either 0, 1.51 and 10mM. Yield, yield components, total carbohydrates and the minerals of K & Ca and Na were determined in grain. The results of these experiments clearly demonstrate, that yield and yield components decreased significantly with increasing salinity. Moreover the harmful effect of salinity on grain minerals such as K and Ca is very Clear, in addition salinity decrease significantly the total carbohydrates of grain. The addition of potassium increased the yield and its components of wheat plants grown under different saline conditions. Calcium and potassium in grains decreased with the increased of sodium chloride, and they are increased with the increase of potassium. The K+:Na+ and Ca+2 : Na+ increased with the increase of potassium conversely, sodium decreased with the increase of potassium. The addition of 10mM potassium was considered to be the best in overcoming of the undesirable effect of salinity on the yield and its components. Consequently, application of additional K corrected K, Ca and P deficiencies caused by harmful effect of salinity.

6/22 STUDIES ON THE BACTERIAL INDICATORS OF WATER POLLUTION AND BIOREMEDIATER ISOLATES FOR Cu2+, Fe2+ AND Zn2+ IN ROSETTA BRANCH, RIVER NILE, EGYPT

S.Z. Sabae; M.M. Hazaa*; S.A.Abdel-Halem*; N. Awny** and S.M. Daboor

National Institute of Oceanography and Fisheries, *Faculty of Science, Benha University and **Faculty of Science, Zagazig University

The study included some physico-chemical characteristics, bacteriological analyses, the characterization and identification of two bacterial isolates that are able to bioremediate the tested heavy metals (Cu+2, Fe+2 and Zn+2). The results revealed that, the water temperature ranged between 16.5°C and 30.5°C at Sobil drain area, while at Kaffr El-Zayyat it was varied from 15.9°C to 30.9°C.The (pH) values fluctuated between 7.1 and 8.5 at Sobil drain and between 6.85 and 8.1 at Kaffr El-Zayyat area. The cupper content in the water ranged between 3.8 and 88.0 µg/l at Sobil drain and between 4.8 and 74.0 µg/l at Kaffr El-Zayyat. The iron content varied from 0.2 to 1.1 mg/l and from 0.31 to 2.14 mg/l at Sobil drain and Kaffr El-Zayyat areas respectively. Zinc content values were in the range of 18.0-140.0 µg/l and 6.0-134.0 µg/l, respectively. The bacterial numbers of water samples were carried out at 22°C and 37°C, the log numbers of the total count of viable bacteria varied from 7.36 to 9.48 and from 7.48 to 10.18 at 22°C and 37°C, respectively at Sobil drain. At Kaffr El-Zayyat their values were in the range of 7.0-9.3 and 7.11-9.0/ml,at 22°C and 37°C, respectively. The bacterial indicators of faecal contamination showed that, the highest log numbers of total coliform were recorded at the mixing point at Sobil drain, ranged between 4.32 to5.18/100ml and between 2.6 to 4.38/100ml at Kaffr El-Zayyat area. The log numbers of faecal coliforms could not be detected during May at Sobil drain and during March and May at Kaffr El-Zayyat. also,the log numbers of faecal streptococci fluctuated between 0.0-4.46/100ml and between 0.0-4.36/100ml at Sobil drain and Kaffr El-Zayyat areas, respectively.Abut twenty six bacterial isolates were grown in presence of 200.0mg/l of the tested heavy metals.The two isolates No.215 (Gram positive) andNo.225(Gram negative)were the most active isolates that grew on media amended with 200.0mg/l of the tested heavy metals,which were identified as Bacillus subtilis D215 and Pseudomonas putida biovarA D225 respectively.

7/22 THE PHYSIOLOGICAL RESPONSES OF COMBINED CONTINUOUS WATER DEPRIVATION AND NITROGEN STARVATION ON TWO CONTRASTING PEA (PISUM SATIVUM L.) CULTIVARS.

1-                  GROWTH, GROWTH PARAMETERS AND SOME CHEMICAL COMPOSITION OF PEA PLANTS.

N.M. Abd-El Rahman

Agricultural Botany and Plant pathology Department,

Faculty of Agriculture, Zagzeg University.

Vegetative plant growth, physiological growth parameters and chemical composition, could be used to differentiate and understanding apparent drought combined with nitrogen shortage among the cultivars. Two pea (Pisum sativum L) cultivars (Little Marvel and Meteor) were grown in a greenhouse, subjected to water stress combined with nitrogen starvation. Four treatments for both two cultivars studied with no water stress and N-supply (WWN) i.e 1st treatment, no water stress and N-deprivation (WWns) i,e 2nd treatment, water stress and N-supply (ws N) i.e 3rd treatment and water stress and N-deprivation (wsns) i.e 4th treatment. Vegetative plant growth physiological growth parameters the results showed that, water – stress combined with N-deprivation caused significant and greater negative differences for vegetative growth parameters such as plant height, capacity of branching, number of leaves, leaf area and root, stems, leaves dry weights, moreover chlorophyll a & b, reducing sugar, total carbohydrates, photochemical activity. Moreover proline concentration and antioxidant enzymes such as catalase and peroxidase, were also large significantly negative affected. Meanwhile nitrogen supply increased significantly all parameters studied of pea plants grown under, water shortage. Moreover, varietals differences in all parameters investigated were confirmed statistically and the highest significant values were recorded by Meteor cultivar. Finally, nitrogen supply is promising method to increase pea yield and yield components under severe drought stress.

8/22 GROWTH AND ALOIN PRODUCTION IN ALOE SPP. AS INFLUENCED BY SALT AND FERTILIZERS

S.A. Sheteawi

Botany department, Women’s college, Ain Shams Univ.

A green house experiment was conducted to investigate the effect of different treatments of fertilizers in combination with salts on the growth and aloin production of Aloe grandidentata, A. arborescens and A. spinosissima.The various adopted treatments for Aloe grandidentata were 3g NaCl/L, 3g NaCl + 0.32g KNO3/L, 3g NaCl + 0.37g Ca(NO3)2/L, 0.32g KNO3/L, 0.37g Ca(NO3)2 /L, 3g NaCl + 0.27g CaSO4/L, 0.27g CaSO4/L and organic green manure of Acacia saligna (0.25 kg/pot at the start of plantation), in addition to the control treatment. The effect of salinity treatments compared to control were studied on the other two species A. arborescens and A. spinosissima. The highest growth of A. grandidentata were obtained with 0.37g Ca (NO3)2/L treatment, followed by 3g NaCl + 0.37g Ca(NO3)2/L treatment, then with 0.27g/L CaSO4 and the organic green manure. Aloe arborescens gave favorable growth with salt than A. spinosissima being more salt tolerant and could be irrigated with brackish water. Maximum aloin production of Aloe grandidentata was obtained with 3g NaCl + 0.37g Ca (NO3)2/L irrigation treatment, followed by 3g NaCl + 0.27g CaSO4/L treatment. A. grandidentata could be cultivated in reclaimed land irrigated with brackish water when calcium nitrate is added. This enhanced more production of the beneficial compound aloin. Aloe arborescens could be also cultivated in reclaimed desert land and irrigated by brackish water as it shows a degree of salt tolerance.

9/22 THE PHYSIOLOGICAL INTERACTION BETWEEN POTASSIUM SUPPLY AND SODIUM ON WHEAT PLANTS GROWN UNDER DIFFERENT LEVELS OF NACL SALINITY.

1- GROWTH, PHYSIOLOGICAL GROWTH PARAMETERS AND SOME CHEMICAL COMPOSITION OF WHEAT PLANTS

N.M Abd – El Rahman

Agricultural Botany and Plant Pathology Department,

Faculty of Agriculture, Zagazeg University

A greenhouse pot experiments were conducted to study the indirect effects of salinity as NaCl combined with potassium supply on wheat plant var. Giza- 168. In experiments three Na Cl and three K treatments were conducted. The Na+ concentrations were 0, 100 and 200mM NaCl and K+ concentrations were 0, 1.51 and 10mM. Response to treatments was determined by analysis of growth parameters such as root and shoot fresh and dry weights, plant height, leaf area, shoot : root ratio. Moreover the concentration of minerals such as (K, Na, Ca, P and Zn) and carbohydrate, photochemical activity, proline concentration, catalase and peroxidas activity were taken in the leaves. Generally K supply in the absence of salinity significantly increased all parameters studied. Meanwhile all parameters studied were significantly depressed and declined with increasing   salinity in plants lacking additional k+ i.e in plants not supplied with additional K+ and subjected to saline conditions. An important finding of this study was that supplemented K+ had an eliminated the harmful effect of NaCl stress. On the other hand additional K allow to avoid the adverse effects of salinity on all parameters studied, such as root, shoot weights, leaf area, plant height and K+ : Na+, Ca2+ : Na+ ratios, thereby photochemical activity, moreover the activities of catalase and peroxidase enzymes. It is worth to mention that, the results of these experiments reveal that, K supply as 10 mM was better than other treatments of K supply to eliminate the harmful and bad effects of salinity.

10/22 MUTAGENESIS AND IMMOBILIZATION OF KLUYVEROMYCES MARXIANUS FOR ENHANCED INULIN HYDROLYSIS

W.A. Bazaraa and A.A. Khattab*

Food Science Department, Faculty of Agriculture, Cairo University, Giza, Egypt.

* Genetic and Cytology Department, National Research Center, Cairo, Egypt.

Kluyveromyces marxianus (NRRL-2415) is a constitutive inulinase producer. This yeast was treated with the mutagenic agent, ethyl methane sulfonate and mutants resistant to catabolite repression were isolated using 2-deoxy-D-glucose. Out of the 78 selected mutants, 45 exhibited higher inulinase activites (from 100.9 to 234.6%) than the original strain. Only 2 mutants (E-51 and E-57) showed inulinase activities (25.1 U ml-1, each) higher than 2-folds that obtained with the original strain (10.7 U ml-1). During fermentation, the mutant E-57 exhibited the highest productivity (5.6 U ml-1 day-1) among the tested strains. Crude inulinase and cells of the mutant E-57 were successfully immobilized within Ca-alginate (1.5%, w/w) beads. The immobilized preparations were utilized to hydrolyze inulin in batch system. A 90% hydrolysis was achieved in 1 h, when 10 g of these preparations (12.6 U g-1) were used. The immobilized preparations were also repeatedly used for 9 consecutive batch cycles. Such preparations were stable and the percent of hydrolysis was constant (90%) during the first 6 cycles and decreased after that to reach 30% after 9 cycles. In case of the immobilized cells, it was possible to partially regain their activity and reuse them again in inulin hydrolysis.

11/22 PHYTOREMEDIATION OF THE OIL POLLUTED DESERT SOIL OF KUWAIT BY USING THE WILD DESERT PLANTS SALSOLA IMBRICATA AND CYPERUS CONGLUMERATUS

R. Al-Abdulla, A. Diab and A. Al-Hilali

Science Department, College of Basic Education

The Public Authority for Applied Education and Training

P.O. Box 27810, Safat 13139, Kuwait

The present study revealed that the roots of the wild desert plants Salsola imbricata and Cyperus conglumeratus were densly associated with hydrocarbon-degrading microorganisms such as fungi and other groups of bacteria. Generally, more reduction of the oil pollutants was observed in the oil-polluted rhizosphere soil than in the non-rhizosphere soil. Salsola roots were more active in removing TPH, total aromatics and total PAHs (47%, 45.5% and 31.3 – 34.9% respectively) than Cyperus roots (31.6%, 40.3%, and 4.2 – 10.2% respectively). The hardly degradable compounds resins and asphaltenes were highly degraded in the rhizosphere soil of Salsola (44.6% and 64.1% respectively). At the same time no reduction of resins and only a reduction of 18.4% of asphaltenes could be detected in Cyperus rhizosphere. All microbial groups investigated in this study were of highly positive response to the oil pollutants in the rhizosphere of the two plants, while in the soil apart from roots these groups were negatively responded (as indicated from their R/S ratio of less than one). The present results clearly demonstrate successful phytoremedeation of the oil polluted desert soil by applying wild desert plants, and we suggest that this process may include the following: A survey of several contaminated sites in the desert of Kuwait must be conducted to determine which plant species were growing in these sites or in their immediate vicinity. To accelerate the phytoremediation process by applying land-farming, contaminant – degrading bacteria, plant growth promoting rhizobacteria and the selected contaminant – tolerant plants.

12/22 MOLECULAR CHARACTERIZATION OF MORGANELLA MORGANII STRAINS USING ARBITRARY PRIMER PCR FINGERPRINTING ANALYSIS

M.S. Mansy

Microbiology and Immunology Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City, Cairo.

A simple, rapid and stable method for typing of Morganella morganii strains is described. An arbitrary primer-polymerase chain reaction (also termed random amplified polymorphic DNA or RAPD) based DNA fingerprinting method has been developed to distinguish among various strains of Morganella morganii. Genomic DNA's from 32 well defined isolates of Morganella morganii from different sources, were examined by PCR-based arbitrary primer analysis using twelve base oligonucleotide with a 75% G+C content to prime DNA synthesis from genomic DNA's sites. Strain-specific arrays of DNA fragments were generated, which allowed us to distinguish closely related strains of Morganella morganii. This approach was compared with the phenotypic and genotypic typing methods such as antimicrobial susceptibility pattern, biotyping, PCR– based RFLP profile, protein pattern and ribotyping. Sixteen profiles and discriminatory power with diversity indices of 0.95 were obtained by AP-PCR genomic fingerprinting from the 32 strains of Morganella morganii. The results showed that AP-PCR would be a sensitive and rapid discrimination method for molecular characterization of strains belonging to the genus Morganella and may be valuable for monitoring transmission of this pathogen.  

13/22 KARYOMORPHOLOGICAL STUDIES OF SOME MELILOTUS MILL. SPECIES IN EGYPT

H.Y. Hijazy

Botany Department, Faculty of Science, Zagazig University, Egypt

Detailed karyomorphology of wild six taxa of genus Melilotus namely M. albus, M. indicus, M. segetalis and M. messanensis have been studied. They are collected from two different floristic locations. All the studied species are diploid with 2n=16, except one taxon of M. messanensis which found to be aneuploid cytotype with chromosome number 2n=17. In common, the studied species shared many karyotype features. In finer details of karyomorphology, some recognizable differences were noticed among the six taxa such as total chromosome length, total form percent (TF %), ratio of the longest chromosome to shortest chromosome (L/S), number and distribution of sat-chromosomes. The minor differences noticed in the karyotype in the different populations of M. indicus and M. messanensis give reasons to consider these taxa as infraspecific cytotypes (varieties or forms) from a taxonomic point of view. Also the observed minor differences in the karyotype with the absence of interspecific hybrids in nature or produced experimentally in the genus Melilotus, lead to suggest that gene mutation or cryptic structural changes of chromosomes may have played a role in the evolution and diversification of Melilotus. Also the obtained karyomorphological results support the earlier classification of genus Melilotus into distinct subgenera.

14/22 SCREENING THE GENE FAMILY ENCODED RIBOSOME INACTIVATING PROTEINS IN CERTAIN PLANT SPECIES

A. Bahieldin; A. Barakat* and Z.A. Rashad*

Genetic Department, Faculty of Agriculture, Ain Shams University, Egypt.

*Microbiology Department, Faculty of Science, Ain Shams University, Egypt.

The polymerase chain reaction (PCR) was successfully employed to synthesize gene-specific primes suitable for genomic analysis and for screening gene(s) family of ribosome inactivating proteins (RIPs). The RIP-gene(s) in the genomic DNA of six plant species belonging to different taxonomical families (Caryophyllaceae, Chenopodiaceae, Cucurbitaceae, Euphorbiaceae, Nyctaginaceae) was also identified. The RIP genes of such species were investigated according to the alignment derived from 3 RIP-genes available in the GeneBank for Mirabils jalapa and Beta vulgaris vulgaris with the accession numbers d10227, d10569 and x85967. Two conserved motifs are selected to design oligonucleotide primers on the basis of the data available in the GeneBank. PCR revealed seven different RIP genes in our tested species. Among these genes, those of Beta vulgaris cicla (one RIP gene) and Spinacia oleracea (2 RIP genes) are novel. The partial length of RIP gene isolated from Beta vulgaris cicla was sequences and compared with published sequences of M.jalapa RIP genes showing a homology of 96%. The structure of the deduced amino acid sequences of the novel RIP gene as indicated by DNA Star Computer analysis, indicated that the open reading frame-3 (ORF3) might be the right ORF of the novel gene with the presence of a very short intron within it.

15/22 USE OF RANDOMLY AMPLIFIED POLYMORPHIC DNA TO CHARACTERIZE TWO EGYPTIAN STREPTOMYCES ISOLATES: STREPTOMYCES CLAVULIGERUS ANDSTREPTOMYCES EXFOLIATES

K.A. Elhlafawy; H.A. Hamza*; A.A. Hemeida**; A.F. Elbaz*** and R.A. Abbas*

Department of Molecular Biotechnology, *Department of Microbial Biotechnology, **Department of Bioinformatics and ***Department of Industrial Biotechnology,

Genetic Engineering and Biotechnology Research Institute, Menufyia University.

Two local Streptomyces isolates; Streptomyces exfoliates 29 and S. clavuligerus 6; were able to produce b-lactamase inhibitors as detected by using iodometric assay .These isolates were subjected to genetic analysis by randomly amplified polymorphic DNA (RAPD) technique. Nineteen randomly primers were selected and the amplification PCR products of the two local isolates, in addition to the references strains S. exfoliates NRRL B-1237and S. clavuligerus NRRL 3585 were electrophoresed and the results were analyzed. It was observed that the similarity between isolate 29 and S. exfoliates NRRL B-1237was 96.9%, while that between isolate 6 and S. clavuligerus NRRL 3585 was. 94.4 %.

16/22 THE INDUCED RESISTANT YOGHURT STARTER (STR.SALIVARIUS SUBSP. THERMOPHILUS AND LB. DELBRUECKII SUBSP. BULGARICUS) WHICH WERE APPLIED TO YOGHURT CONTAINING STERPTOMYCIN.

M.A. Degheidi, R.B.A. El-Tantawy, N.A.H. Elewa and H.M. El-Garhi

Department of Dairy Science, Faculty of Agric. Al-Fayoum University

Yoghurt was manufactured as follows: 1-From milk free of streptomycin and parent starter cultures (Str.salivarius subsp. thermophilus and Lb. delbrueckii supsb.bulgaricus (1:1). This was dealt with as a control. 2-From milk treated with 100μg/ml streptomycin along with induced resistant cultures (Str.salivarius subsp. thermophilus and Lb.delbrueckii subsp. bulgaricus (1:1) .This was referred to as (IRSs). parent cultures. 3-Milk with100μg/ml streptomycin and parent starter cultures (Str.salivarius subsp. thermophilus and Lb.delbrueckii subsp. bulgaricus (1:1) which served as (PSs). It was observed that the acidity was higher in the induced cultures than the control during four hours of incubation .The pH values were affected in a contrary way. The same results were obtained during 15 days of manufacturing the yoghurt. The acetaldehyde content got increased during storage for 10 days .While the diacetyl got increased after 4 days up to 14 days then started to decrease gradually. In case of counting the total bacterial counts and Str. thermophilus and Lb.bulgaricus counts, the control was higher than the treatments. The use of induced resistant starter culture could be benificial in avoiding the harmful antibiotic residues in milk used in fermented milk products.

17/22 BIODIVERSITY ASSESSMENT AMONG SOME SORGHUM CULTIVARS USING MOLECULAR MARKERS

S.H. Abdel-Aziz

Botany Department, Faculty of Science, Benha University

Genetic Biodiversity of eight different sweet sorghum cultivars, which were obtained from Sugar Crops Research Institute, ARC, Giza, Egypt, were assessed and their fingerprints were developed using Randomly Amplified Polymorphic DNA (RAPD) and simple sequences repeat (SSR). Fifteen arbitrary 10-mer primers were used for RAPD analysis and fifteen SSR pairs of primers were used for SSR analysis. The banding patterns were scored and statistically analyzed using computer program NTSYS PC-1.7 in order to determine the genetic distances between these cultivars and their genetic relationships. Dendrogram trees for each of RAPD, SSR and their combined patterns were generated. The data showed different levels of variation among these sorghum cultivars.


18/22 BLIP GENE OF EGYPTIAN STREPTOMYCES CLAVULIGERUS ISOLATE: ISOLATION AND CLONING

K.A. Elhlafawy; H.A. Hamza*; A.A. Hemeida**; A.F. Elbaz*** and R.N. Abbas*

Department of Molecular Biotechnology, *Department of Microbial Biotechnology, **Department of Bioinformatics and ***Department of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, Menufyia University.

A streptomyces sp. was isolated from an Egyptian soil sample and was previously classified as S. clavuligerus, using both traditional and molecular identification methods (Elhalafawy et al., 2006 and Abbas 2006). Bioassay disk method showed that this isolate produced two different β-lactamase inhibitors. About 37.5% of the inhibitors activity referred to the presence of β-lactamase inhibitor protein (BLIP).The blip gene was detected using PCR technique and the 580bp amplified   fragment containing this gene was cloned into the plasmid pPCRblue-Skript. The cloned plasmid was transformed into Escherichia coli Dh5ά. The detection of cloned blip gene in transformed cells was achieved by PCR technique.

19/22 GENETIC POLYMORPHISM OF SEMI-DRAY DATE PALM

(Phoenix dactylifera L.) CULTIVARS IN EGYPT

Kh.A. Soliman, R.M. Rizk* and S. El Sharabasy**

Genetics Dept, Fac. of Agrc., Ain Shams University, Ain Shams Center for Genetic Engineering and Biotechnology (ACGEB), Faculty of Agric. Branch. Cairo, Egypt.

*National Gene Bank, Agriculture Research Center, Giza, Egypt.

**The Central Laboratory of Date Palm Research & Development, Agricultural Research Center, Giza, Egypt.

Ten cultivars representing all semi-dry date palm in Egypt are subjected to identification, characterization and documentation using electrophoretic analysis. DNA profiles using RAPD analysis for 5 primers (A-07,A-12, L-13, L-16 &L-20) and ISSR analysis for 5 primers (HB-10, HB-11, HB-13, HB-15 & 17899B) as well as protein profile are conducted for establishing genetic markers in order to discriminate among the semi-dry date palm cultivars in Egypt. Degllet Nour cultivar could be separate from the Egyptian semi-dry date palm by presence of specific protein of molecular weight 45.00 KD. Deglet Nour, imported from the North West Africa, and Ashbeer which cultivated in far West in Siwa Oasis are closely related. DNA profiles using RAPD analysis for primer A-07 and L-16 are the most efficient to distinguish Deglet Nour from the other semi-dry date palm cultivars. Primer A-12 of RADD analysis and HB-13 of ISSR Analysis are less effective to discriminate the morphological identical cultivars Sewi from Saidi, but the rest 8 primers are more efficient to separate the two cultivars. So, Sewi from Saidi could be considered as two separate cultivars. This data could be enhance the information of date palm gene bank of the Central Laboratory of Date Palm Research & Development (CLDPRD), Agricultural Research Center, Egypt.


20/22 IDENTIFICATION AND CHARACTERIZATION OF SALT-REGULATED GENES IN BARLEY

A.Z.E. Abdel Salam, F.M. El-Domyati*, Kh.A. Soliman and G.H.M. El Nady

Ain-Shams Center for Genetic Engineering and Biotechnology

* Genetic Department, Faculty of Agriculture, Ain-Shams University

A complementary DNA (cDNA) sequence corresponding to AP2 domain was cloned from a salt-acclimated barley landrace cDNA (Etienne, Leger, and Winthrop) library. The isolated gene (Bsrf41) was very much related to the ethylene responsive element. Northern analysis revealed that Bsrf41 transcript accumulation is rapid and strongly up regulated by salt stress (300 mM NaCl). This accumulation was tissue-specific for roots and crowns. In addition, 20 random clones from this cDNA library were tested for its function under salt stress. Selected clones of cDNA were analyzed and sequenced and the resulted data were compared with the published sequences, which showed 80%, 92.6%, and 96.6% similarities with Bsrf41. Dhn2, and Dhn9 genes, respectively. The Bsrf41-cDNA encodes a polypeptide with a calculated molecular mass of 41.2 KDa. The expression cDNA of two of them (Dhn2, Dhn7) proved that they are dehydrin like genes. The expected polypeptide, encoded by Dhn2 has molecular weight of 20.97 KDa. Meanwhile the Dhn9 encodes a polypeptide of molecular weight of 28.04 KDa.

21/22 ISOLATION, PRODUCTION, PURIFICATION AND CHARACTERIZATION OF ACTIVE SUBSTANCE PRODUCED BY STREPTOMYCES MISIONENSIS

B.M. Refaat

Botany and Microbiology Department, Faculty of Science,

Al-Azhar University, Madenit Nasr, Cairo, Egypt

Eleven actinomycete isolates of the white and gray colour series were isolated from soil of Kuwait. Among these isolates only one isolate showed high antagonistic activity against different Gram negative and Gram positive bacteria. The cultural, morphological and physiological characteristics of this isolate indicated that it is likely similar to Streptomyces misionensis. Some physicochemical properties of the active metabolite produced by this organism was also studied.

22/22 GENETIC DIVERSITY OF SOME LILIACEAE FAMILY BULBS USING BIOCHEMICAL, MOLECULAR AND MORPHOLOGICAL CHARACTERSTICS

S.A.A. Ibrahiem, Kh.A. Soliman, N.A.K.F. Rashed*and R.M.A. Khalil*

Dept. of Genetics, faculty of Agriculture, Ain Shams Univ., Egypt.

*Plant Genetic resources Dept. Desert Research center. Matariya, Cairo, Egypt.

Asphodelus fistulosus L. and Urginea maritima L. with its two forms (white and red bulbs) are members of pharmaceutical Liliaceae family. Despite limited knowledge of the levels of genetic diversity of their populations, their utilization as a source of some medicinal component such as cardiac glycosides is widespread. In order to facilitate reasoned scientific decisions on their conservation and prepare for a selective breeding program. Genetic analysis of nine populations of A. fistulosus L. and U. maritima L. with its two forms were performed using morphological, SDS-protein, isozymes and random amplified polymorphic DNA (RAPD) markers. The morphological characters revealed considerable morphological variations among studied populations. In addition, the morpholgy of these plants is largely influenced by environmental factors. SDS-protein electrophoresis of total proteins showed genetic variations with seven monomorphic bands out of thirteen bands. These bands are common or major bands for all studied populations. In addition, A. fistulosus L. was characterized by other major bands at 73, 69, 65 and 47 KD. U. maritima L. had specific band at 78 KD. Five isozyme systems used α and βEst (esterases), Prx (peroxidase), Acp (acid phosphatase), Adh (alchol dehydrogenase) and Mdh (malate dehydrogenase) revealed 26 electrophoretic bands 74 % of them were polymorphic. Also, 10 random 10-mer Primers were used to amplify DNA via polymerase chain reaction (PCR) and 100 RAPDs were generated. Out of the total amplified bands 76% were polymorphic. Similarity matrix using Nei and Li's (1979) coefficient indicated that the two populations of U. maritma L. with red bulbs forms attained the highest similarity value 97%, whereas the similarity value between U. maritina L. with white bulbs population of EL-Diaka and A. fistulosus L. of Sad EL-karam was the lowest (60%). Dendogram generated by UPGMA analysis showed three obvious clusters, the first clustered A. fistulosus L. Populations, the second clustered U. maritina L. with white bulbs form populations and the last is U. maritima L. with red bulbs forms populations. In addition, the combined analyses (SDS- protein, isozymes and RAPD) dendrogram had the same previous trend. Meanwhile, all genetic data revealed environmental effects on different studied Liliaceae populations (A. fistulosus L. and U. maritima L. with its two forms white and red bulbs). All populations of Liliaceae tested under different environmental selection pressure generally show phenotypic differences. Such phenotypic differences could be attributed to phenotypic plasticity and genetic diversification.

23/22 COMPARATIVE STUDY ON THE MUTAGENIC EFFECTS OF A CHEMICAL FERTILIZER (CROP-FINISHER) AND A BIOFERTILIZER (CEREALIN) ON VICIA FABA PLANT

E. Abd El-Haleim, N.M. George and R.S. Shehata

Botany Department, Faculty of Science, Zagazig University

The mutagenic potentiality of crop finisher fertilizer and cerealin biofertilizer on mitosis, meosis, yield production (M1) and the changes in seed storage proteins banding pattern of Vicia faba plants have been studied. The two fertilizers have the ability to increase the mitotic activity of Vicia faba root tip meristems but the biofertilizer cerealin being more effective than crop finisher. The two biofertilizers induced a number of chromosomal aberrations in both meristematic and germinal cells. The M1 progeny showed a considerable percentage of abnormal cells and an increase in the productivity. The aberrations produced in mitosis meiosis and M1 were nearly similar. Stickiness comprise the most common abnormality where disturbed phases lagging bridges and fragment present with variable percentages. The electrophoretic proteins patterns revealed that the most effective concentration used was (2RD) after treatment with crop-finisher. This treatment is characterized by the presence of a new polypeptide of 34 KD, while the concentration (½RD) was the most effective when cerealin was used, and this treatment is characterized by the presence of 8 new polypeptides (126.15, 107.72, 94.90, 77.99, 73.36, 63.84, 37.48 and 35.83 KD). The changes of protein banding patterns may be correlated with the cytological abnormalities which lead to the loss of some of genetic materials, appearance of new characteristic band and changes in band intensity.

24/22 STUDIES ON ASSOCIATED FUNGI OF HEALTHY STUDENTS OF ASWAN GAVERNORATE, EGYPT

S.A. Elzayat and M.S. Massoud

Botany Department, Faculty of Science, South Valley University, Aswan, Egypt

Thirty-eight fungal species and one variety representing 18 fungal genera were recovered from hair, nails, external ears, nasal cavities and teeth of 100 healthy students in Aswan, South Valley University. Mycoflora of male and female samples were basically similar, most of them Tricophyton (3 species) and Microsporum (one species) were the only recovered dermatophytes in addition to well-known Keratinophilic genus Chrysosporium (3 species). Several saprophytes and cyclohexemide resistant fungi were recovered but with different frequency and these belonged to Alternaria, Aspergillus, Cladosporium, Emericella, Mucor, Penicillium, Scopulariopsis and Talaromyces. The frequency of Keratinophilic fungi found in examined samples ranged from 38 to 92% where hair samples yielded the highest number of species (92%) while, the least dominating sample was teeth samples (38.4%). Eighteen fungal isolates of dermatophytes and other moulds were tested for proteolytic and lipolytic activities, many of them were able to produce protease and lipase in varying amounts. Using the disc diffusion method, 5 methanol extracted ofSolenestemma argel, Pergularia tomentosa, Leptadinia pyrotechnica, Triblus pentandus and Astragallus eromophillsweretested at 5 – 15 % concentrations against 18 strains of dermatophyte and cyclohexamide resistant fungi.

25/22 PURIFICATION AND CHARACTERIZATION OF AN ANTIBACTERIAL AGENT FROM (ORIGANUM VULGARE) AGAINST OF SOME PATHOGENIC BACTERIA ISOLATED FROM FISHES

E.Y. Tohamy; H.M. Atta*; M. Hazah**; M.E. Abou El Atta***and D.A. El Arabe***

Botany and Microbiology Dept., Faculty of Science. Zagazig University, Egypt.; *Botany and Microbiology Dept., Faculty of Science (Boys); Al-Azhar University, Cairo, Egypt.; **Botany and Microbiology Dept., Faculty of Science. Banha University, Egypt.; ***Fish Diseases Dept., Central Laboratory For Aquaculture Research ( CLAR) .Abbassa Abou Hammad , Sharkia, Egypt.

Local bacterial cultures could be isolated from 200 Oreochromis niloticus and were caught as random samples from EL-Abassa, Abou- Hammad, Sharkia, Egypt. Fish farms with an average body weight 40±5g. Suffered From signs of septicemia as hemorrhages on several parts of the body surface (mouth) base of fins, abdominal part, opercula, and around the anal opening turbidity of the eyes and slight exophthalmia roughness of scales and sometime scale losses, postmortemally showing, heaptomegaly, spleenomegaly, congestion of gills, kidney and accumulation, of   bloody fluid in abdominal wall.     Four bacterial strains are Gram negative bacteria were isolated from fish. From the taxonomic features, the bacterial isolates was likely belonging to Pseudomonas aeruginosa-1, Pseudomonas fluorescence-2, Salmonella arizonae-3 and Citrobacter baraakii-4 in it's morphological, physiological and biochemical characters and confirmation by using APIZOE system. The active extract using bioactivity-guided technique of aqueous and organic extracts of Origanum vulgare. The separation of the active ingredient and its purification was performed using both thin layer chromatography (TLC) and column chromatography techniques. The physico-chemical characteristics of the purified antibacterial agent viz. color, melting point, solubility, elemental analysis, spectroscopic characteristics and assay of total phenolics have been investigated. This analysis indicates a suggested empirical formula of C11 H19 O8N. The biological activities i.e. MICs of the purified antibacterial agent were also determined.

26/22 GROWTH PROMOTION OF SOME ORNAMENTAL PLANTS INOCULATED WITH MANGROVE RHIZOSPHERE BACTERIA IN ENVIRONMENTALLY SENSITIVE AREAS – A CASE STUDY FROM THE RED SEA

A. Misk

Hurghada Faculty of Education, South Valley University, Hurghada, Red Sea.

Inoculation of four ornamental plant species Petunia hybrida, Vinca rosea, Lathyrus odoratus, and Matthiola incana with twelve species of osmotolerant bacteria isolated from mangrove rhizosphere and grown in the green landscaping of Hurghada Marriott Hotel, resulted in significant plant growth promotion by the end of the growing season. Statistical analysis demonstrated that inoculation with a mixture of four Azospirillum brasilense strains, four Azotobacter sp. and four Paenibacillus polymyxa strains significantly increased plant height (14.8-219 %), the number of branches (20.7-437 %) and flowering (160-533%) over uninoculated plants. At the end of the growing season the N and P content of shoot of all plants were significantly high compared to uninoculated ones. This study demonstrated the feasibility of using bacterial inoculation to promote the growth of ornamental plants (beautifying touristic resorts along the coast) cultivated in sensitive areas where the natural resources (i.e. coral reefs, mangroves, etc…) can be severely affected by the use of mineral fertilizers and chemical pesticides.

27/22 ANALYSIS AND CONSERVATION OF ARCHAEOLOGICAL LIMESTONE RELIEF FROM FUNGAL DETERIORATION USING CAPTAN FUNGICIDE (CASE STUDY)

H.A.M. Afifi and N.S.I. Geweely•

Faculty of Archaeology, Conservation Department, Cairo University, Giza, Egypt.

Faculty of Science, Botany Department (Microbiology), Cairo University, Giza, Egypt.

The conservation of works of art exhibited inside museums is influenced considerably by the indoor environment. Air pollutants, metabolic actions and excretions of microorganisms play an important role in deterioration. In this study, samples were collected from deteriorated parts of archeological limestone relief panel exhibited in Koom Oshem museum, Fayoum city, Egypt. Analyses and investigations by using X-ray diffraction analyses (XRD) followed by Energy Dispersive X-ray analysis (EDX) and scanning electron microscope (SEM) were used to reveal the deterioration factors and aspects. The data show that calcium carbonate was the major component of panel with minor halite and gypsum. Soil in which the stone was buried was analyzed. Fifteen and eight fungal species was isolated from bury soil and relief panel, respectively. Alternaria alternata followed by Penicillium chrysogenum were the most dominant species in both soil and relief. Based on fungal sensitivity to Captan fungicide, two different resistance levels were observed for Alternaria alternata, Fusarium solani and Verticillium lateritium (HR high resistance) and Aspergillus fumigatus, A. niger and Trichoderma viride (LR low resistance), respectively. The S (S Sensitive) isolates were A. versicolor and Penicillium chrysogenum. The maximum production of citric acid was shown by LR isolates (4.43 g/50 ml for A. niger). pH sensitivity of the tested fungal species was determined.
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