Molecular Identification of Rhizosphere Trichoderma spp. and Their Antagonistic Impact Against Some Plant Pathogenic Fungi

The main aim of this study was to molecular identification and determine the antagonistic impact of rhizosphere Trichoderma spp. against some phytopathogenic fungi, including (Magnaporthe grisea) pyricularia oryzae, Rhizoctonia solani and Macrophomina phasolina. Four Trichoderma isolates were isolated from rhizosphere soils of the different host plants in different locations of Egyptian governorates. The morphological characterization of isolated Trichoderma as well as using of (ITS1-5.8S-ITS2) ribosomal gene sequence acquisition and data analyses. By comparing the results of DNA sequences of ITS region, the fungi represented one isolate were positively identified as T. asperellum (1 isolate T1) and one as T. longibrachiatum (1 isolate T2) and two as Trichoderma harzianum (2 isolates T3 and T4). The results showed similarity value of (5.8S-ITS) region sequence of the two isolates, T1 (T. asperellum) and T2 (T. longibrachiatum) of (99%, 99%), respectively. The similarity value of (5.8S-ITS) region sequence with isolates of T3, T4 (T. harzianum) of (99%). On the other side, the results of molecular identification of phytopathogenic fungi represented high similarity value of (5.8S-ITS) region sequence and were identified as P.oryzae, R. solani and M. phasolina (99, 96 and 99%) respectively. Variations and genetic relationships among 4 Trichoderma isolates were investigated by using the Rapid Amplification of Polymorphic DNA (RAPD) profiles using ten random primers. All Trichoderma isolates were assessed for their antagonistic impact on phytopathogens P. oryzae, R. solani and M. phasolina. Though T. harzianum isolates were more affects than T. longibrachiatum and T. asperellum isolates, the percent inhibitory effect among T. harzianum isolates were vary much (44.8 to 91.6%). The inhibitory effect of T. asperellum isolates ranged from 42.2 to (86.0%), while T. longibrachiatum exhibiting affect ranged between (47.5%) to (83.8%)

Cytotoxic effect of Gliotoxin from Candida spp. isolated from clinical sources against cancer and normal cell lines

Background: Invasive fungal infections have become more common during the past two decades. Candida species are the most common human fungal infections. Internal injuries characterize these infections because of virulence factors, such as gliotoxin, which is a fungal toxin that is thought to be antibacterial, antifungal, and antiviral. Objectives: To test the ability of Candida species obtained from clinical sources to produce gliotoxin as a virulence factor and investigate its cytotoxicity effects against some selected cell lines.  Materials and Methods: One hundred and ten clinical isolates of Candida species were obtained from patients attending hospitals in Baghdad from September 2021 to March 2022. They were diagnosed and characterized by routine laboratory methods and cultures. The capability of Candida isolates to secrete the gliotoxin was tested and measured by analytical methods. The cytotoxicity of produced gliotoxin was applied against normal and cancer cell lines. Results: The 110 yeast isolates were diagnosed and identified as follows: Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida Krusei, Candida kefyr, Candida lusitaniae, Candida rugosa. Twenty-eight Candida isolates showed gliotoxin production. The cytotoxicity effects of gliotoxin were reported against lymphocytes and AMGM and AMJ13 cell lines in different concentrations. The highest cytotoxic effect was noticed in the concentration of 400 µg/mL of gliotoxin. Conclusion: The results indicated that the pathogenicity of Candida was distributed among all ages, both sexes, and several types of sources of clinical isolates. Gliotoxin had an effect on normal and cancer cells. Received: Feb., 2023 Accepted: Aug. 2023 Published: Oct. 202

Prevalence and Characterization of Some Colibactin Genes in Clinical Enterobacteriaceae isolates from Iraqi Patients

                افراد العائلة المعوية تمتلك مجموعة من الجينات تدعى Polyketide synthase (pks). هذه المجموعة من الجينات تكون مسؤولة عن تصنيع الذيفان الذي يطلق عليه Colibactin والذي له دور مهم في استحثاث تكسر اشرطة الدنا المزدوجة DNA والذي يؤدي الى استحثاث ورم او ما يعرف بسرطان القولون، احد عشر من اصل ثمانية وثمانين عزلة  بكتيرية وتمثل (12.5%) كانت قد توزعت 7(8%) عزلة تعود لبكتربا E. coli، 2(2.25%) عزلة تعود لبكتريا K. pneumonia و 2(2.25%) تعود لبكتريا E. aerogenes كانت حاملة لجينات الكولبكتين قيد الدراسة. تم اختبار لتأثير السمي الخلوي لعزلتين كانت موجبة للجينات قيد الدراسة وهي E. coli and E. aerogenes تجاه خط الخلايا السرطاني المعروف بـ HeLa  بينت النائج انخفاض عدد الخلايا وحصول استطالة في انوية الخلايامقارنة بالخلايا الغير معاملة. اظهرت النتائج حصول تغيرات نسيجية في الخلايا بأستخدام صبغة AO/EBr تم ملاحظتها بأستخدام المجهر الفلورسيني: بعض هذه التغيرات تم ملاحظتها في لون كروماتين النواة ومصحوب بتكثف الدنا النووي وكذلك حصول تكسر في النوية، خلايا الـ HeLa التي ظهرت بلون اخضر ولم تحصل فيها اي تغيرات في لون المادة الكروماتينية هي خلايا حية ولم تتم معاملتها مع البكتريا الحاملة للجينات قيد الدراسة، بينما الخلايا المعاملة مع خلايا بكتيرية حاملة للجينات ظهرت انويتها بلون برتقالي داكن وهي خلايا ميتة. يستنتج من ذلك ان عزلات البكتريا المعوية المعزولة من مرضى عراقيين يمكنها ان تفرز مواد سامة (ذيفان الكولبكتين) يمكنها قتل قتل الخلايا السرطانية نوع HeLa وهذا ناتج عن تغيرات حصلت في انوية الخلايا المعرضة للبكتريا وكان واضح في كثافة وتكسر المادة الوراثية للخلايا قيد الدراسة. The members of the family of Eentrobacteriaceae harbour a gene cluster called polyketide synthase (pks) island. This cluster is responsible for the synthesis of the genotoxin colibactin that might have an important role in the induction of double-strand DNA breaks, leading to promote human colorectal cancer (CRC). Eleven out of the eighty eight isolates (12.5%) were pks+, distributed as 7 (8%) isolates of E. coli, 2 (2.25%) of K. pneumoniae and 2 (2.25%) of E. aerogenes. The cytotoxic effects of selected pks+ isolates (E. coli and E. aerogenes) on HeLa cells were represented by decreasing cell numbers and enlarged cell nuclei in comparison to the untreated cells. Cytological changes were observed when the infected HeLa cells cultures were stained with AO/EBr and visualized under fluorescent microscope. Some changes that happened in the color of the nuclear chromatin were accompanied by DNA condensation and degradation and fragmentation of nuclei. HeLa cells with green unchanged nuclear chromatin were alive while those with orange-dark and bright red nuclei were dead. It was concluded that a proportion of the Entreobacteriaceae isolates from Iraqi patients was pks+, which exerted cytotoxic effects upon using them to kill HeLa cells. In this study the microscopic observation of the cell morphology reveals the cellular response to the genotoxic insult, with reduced numbers, striking giant cells phenotype (megalocytosis) and fragmentation of nuclei due to the cell cycle arrest and cellular senescenc

Production, Characterization, and Antimicrobial Activity of Mycocin Produced by Debaryomyces hansenii

The present study was conducted to estimate the antimicrobial activity and the potential biological control of the killer toxin produced by D. hansenii DSMZ70238 against several pathogenic microorganisms. In this study, the effects of NaCl, pH, and temperature, killer toxin production, and antimicrobial activity were studied. The results showed that the optimum inhibitory effect of killer toxin was at 8% NaCl, and the diameters of clear zones were 20, 22, 22, 21, 14, and 13 mm for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pyogenes, Candida albicans, and Candida neoformans, respectively. The largest inhibition zones were observed at pH 4.5 with inhibition zone of 16, 18, 17, 18, 11, and 12 mm for the same microorganisms. The results also showed that 25°C is the optimal temperature for toxin killing activity against all targeted microorganisms. In addition, the activity of killer toxin significantly inhibited the growth of fungal mycelia for all target pathogenic fungi and the percentages of inhibition were 47.77, 48.88, 52.22, and 61.11% for Trichophyton rubrum, Alternaria alternata, Trichophyton concentricum, and Curvularia lunata, respectively. The results showed the highest growth rate of D. hansenii DSMZ70238 under condition of 8% NaCl concentration, pH 4.5, and 25°C for 72 h

PROFILING EFFUSION CELLS BY QUANTITATIVE ANALYSIS OF MORPHOLOGY AND DIFFRACTION IMAGING PATTERNS

Profiling cells in human pleural and peritoneal effusion (PPE) samples is an essential task for cytological diagnosis of cancers and patient management. Conventional PPE cytology of a PPE sample is labor intensive and its efficacy depends heavily on the experience of trained specialists in addition to low sensitivity. In this dissertation research project, we focused our effort on application of a label-free method of polarization diffraction imaging flow cytometry (p-DIFC) for quantitative profiling of cell morphology in PPE samples and their correlations to the texture features of cross-polarized diffraction image (p-DI) pairs. To establish the morphology implications of the measured (p-DI) pairs, the 3D structures of PPE cells were measured by using confocal microscopy and quantified with 27 parameters for characterization and analysis of the cellular structures by the conventional fluorescent imaging method. Furthermore, realistic optical cell models (OCM) have been developed as virtual PPE cells and used for accurate simulation of diffraction imaging process to obtain calculated p-DI pairs. This approach allows us to correlate p-DI texture feature parameters quantified by the gray-level co-occurrence matrix (GLCM) algorithm and 3D morphology parameters and investigate various approaches of morphology based cell classification. Clustering algorithms of hierarchical clustering (HC) and Gaussian mixture model (GMM) have been investigated to develop a robust classification method for profiling of the PPE cells' morphological features by the (GLCM) parameters of p-DI pairs. Correlations between the morphological feature parameters and p-DI feature parameters of the imaged PPE cells have been analyzed to gain insights on the morphology implications of image texture patterns and GLCM parameters of the measured p-DI pair data acquired from live and unstained PPE cells of patients of lung and ovarian cancers. Through this dissertation study we have utilized and developed a suite of image processing and analysis tools and obtained results that demonstrate the strong capability of the p-DIFC method to yield big data for profiling PPE cells acquired from cancer patients and the potential to detect malignant PPE cells in the future

PROFILING EFFUSION CELLS BY QUANTITATIVE ANALYSIS OF MORPHOLOGY AND DIFFRACTION IMAGING PATTERNS

Profiling cells in human pleural and peritoneal effusion (PPE) samples is an essential task for cytological diagnosis of cancers and patient management. Conventional PPE cytology of a PPE sample is labor intensive and its efficacy depends heavily on the experience of trained specialists in addition to low sensitivity. In this dissertation research project , we focused our effort on application of a label-free method of polarization diffraction imaging flow cytometry (p-DIFC) for quantitative profiling of cell morphology in PPE samples and their correlations to the texture features of cross-polarized diffraction image (p-DI) pairs. To establish the morphology implications of the measured (p-DI) pairs , the 3D structures of PPE cells were measured by using confocal microscopy and quantified with 27 parameters for characterization and analysis of the cellular structures by the conventional fluorescent imaging method. Furthermore , realistic optical cell models (OCM) have been developed as virtual PPE cells and used for accurate simulation of diffraction imaging process to obtain calculated p-DI pairs. This approach allows us to correlate p-DI texture feature parameters quantified by the gray-level co-occurrence matrix (GLCM) algorithm and 3D morphology parameters and investigate various approaches of morphology based cell classification. Clustering algorithms of hierarchical clustering (HC) and Gaussian mixture model (GMM) have been investigated to develop a robust classification method for profiling of the PPE cells' morphological features by the (GLCM) parameters of p-DI pairs. Correlations between the morphological feature parameters and p-DI feature parameters of the imaged PPE cells have been analyzed to gain insights on the morphology implications of image texture patterns and GLCM parameters of the measured p-DI pair data acquired from live and unstained PPE cells of patients of lung and ovarian cancers. Through this dissertation study we have utilized and developed a suite of image processing and analysis tools and obtained results that demonstrate the strong capability of the p-DIFC method to yield big data for profiling PPE cells acquired from cancer patients and the potential to detect malignant PPE cells in the future

Effect of leaves Extracts of Duranta repens on growth and activity of some types of Pathogenic Bacteria and Some types of Fungi

A study were conducted to examinate the effect of organic and aqueous (Hot, Cold) Extracts from leaves of Duranta repens on the growth and activities of the following types of Bacteria:- Staphylococcus aureus,Streptococcus pyogens ,Escherichia coli,Klebsilla pneumonia, in addition to the yeast Candida albicans and the fungi Aspergullis niger ,Aspergulls flavus.The result showed that gram Positive Bacteria is more sensitive to the extracts than gram negative bacteria with Minimum inhibitory concentration (MIC) value (50,25,50,100)% and Minimum Bactericidal Concentration (MBC) value (100,50,200,100)% for all types Bacteria respectively . The most active extract against A.niger ,A,flavus was cold and hot aqueous extract from the leaves with diameter growth of colony value of ( 0.93,0.37)cm for A.niger in 20 % concentration compared with organic extract (0.26)cm, and the inhibition zone value of cold and hot extract to A.flavus (0.90,0.80)cm respectively compared with organic extract (7.056)cm

Biological Control of Phytopathogenic Fungi by Kluyveromyces marxianus and Torulaspora delbrueckii Isolated from Iraqi Date Vinegar

Yeasts are distributed in all environments and have been reported as potential biocontrol agents against various phytopathogenic fungi. To investigate their enzymatic and biological activities, 32 yeasts were isolated from 15 date vinegar samples. Evaluation of the antagonistic activities of isolated yeasts against the plant pathogens Fusarium oxysporium, Sclerotinia sclerotiorum, and Macrophomina phaseolina indicated that there are two yeasts had the highest inhibitory effect against plant pathogens, these yeasts identified as Kluyveromyces marxianus and Torulaspora delbrueckii using traditional and molecular methods. These yeast isolates were tested for fungal cell wall degrading enzymes (in vitro), and results indicated that the yeasts had strong protease and amylase enzyme activity and moderate chitinase and cellulase enzyme activity. The antagonistic activities of each yeast were evaluated using a dual culture technique. The results showed that K. marxianus inhibited the mycelial growth of F. oxysporium, S. sclerotiorum, and M. phaseolina by 70.5, 57.5, and 75.5%, respectively, whereas T. delbrueckii inhibited mycelial growth of F. oxysporum, S. sclerotiorum, and M. phaseolina by 55.3%, 66.2%, and 31.11%, respectively. The biofilm production assay indicated that the tested yeast could form biofilms as a mechanism of antagonistic activity against phytopathogenic fungi

Biological Activity of Levan Produced from Rhizospheric Soil Bacterium Brachybacterium phenoliresistens KX139300

Levan is an exopolysaccharide produced by various microorganisms and has a variety of applications. In this research, the aim was to demonstrate the biological activity of levan which produced from B. phenoliresistens KX139300. These were done via study the antioxidant, anti-inflammatory, anticancer and antileishmanial activities in vitro. The antioxidant levan was shown 80.9% activity at 1250 µg/mL concentration. The efficient anti-inflammatory activity of 88% protein inhibition was noticed with levan concentration at 35 µg/mL. The cytotoxic activity of levan at 2500 µg/mL concentration showed a maximum cytotoxic effect on L20B cell line and promastigotes of Leishmani tropica. Levan has dose-dependent anticancer and antileishmanial activities. An addition to the antioxidant, anti-inflammatory and anticancer potential activities of levan, it can be concluded that levan produced from B. phenoliresistens can efficiently be applied as an antileishmanial agent