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Автор: Grafiati

Опубліковано: 30 травня 2022

Оновлено: 31 травня 2022

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Dergham, Yasmine, Pilar Sanchez-Vizuete, Dominique Le Coq, Julien Deschamps, Arnaud Bridier, Kassem Hamze, and Romain Briandet. "Comparison of the Genetic Features Involved in Bacillus subtilis Biofilm Formation Using Multi-Culturing Approaches." Microorganisms 9, no. 3 (March 18, 2021): 633. http://dx.doi.org/10.3390/microorganisms9030633.

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Surface-associated multicellular assemblage is an important bacterial trait to withstand harsh environmental conditions. Bacillus subtilis is one of the most studied Gram-positive bacteria, serving as a model for the study of genetic pathways involved in the different steps of 3D biofilm formation. B. subtilis biofilm studies have mainly focused on pellicle formation at the air-liquid interface or complex macrocolonies formed on nutritive agar. However, only few studies focus on the genetic features of B. subtilis submerged biofilm formation and their link with other multicellular models at the air interface. NDmed, an undomesticated B. subtilis strain isolated from a hospital, has demonstrated the ability to produce highly structured immersed biofilms when compared to strains classically used for studying B. subtilis biofilms. In this contribution, we have conducted a multi-culturing comparison (between macrocolony, swarming, pellicle, and submerged biofilm) of B. subtilis multicellular communities using the NDmed strain and mutated derivatives for genes shown to be required for motility and biofilm formation in pellicle and macrocolony models. For the 15 mutated NDmed strains studied, all showed an altered phenotype for at least one of the different culture laboratory assays. Mutation of genes involved in matrix production (i.e., tasA, epsA-O, cap, ypqP) caused a negative impact on all biofilm phenotypes but favored swarming motility on semi-solid surfaces. Mutation of bslA, a gene coding for an amphiphilic protein, affected the stability of the pellicle at the air-liquid interface with no impact on the submerged biofilm model. Moreover, mutation of lytF, an autolysin gene required for cell separation, had a greater effect on the submerged biofilm model than that formed at aerial level, opposite to the observation for lytABC mutant. In addition, B. subtilis NDmed with sinR mutation formed wrinkled macrocolony, less than that formed by the wild type, but was unable to form neither thick pellicle nor structured submerged biofilm. The results are discussed in terms of the relevancy to determine whether genes involved in colony and pellicle formation also govern submerged biofilm formation, by regarding the specificities in each model.

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Savin, P. S. "Technology of growing infectious material of Claviceps purpurea under submerged culturing conditions." Russian Agricultural Sciences 33, no. 2 (April 2007): 94–96. http://dx.doi.org/10.3103/s1068367407020085.

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Bebök, Zsuzsanna, Albert Tousson, Lisa M. Schwiebert, and Charles J. Venglarik. "Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers." American Journal of Physiology-Cell Physiology 280, no. 1 (January 1, 2001): C135—C145. http://dx.doi.org/10.1152/ajpcell.2001.280.1.c135.

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Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl− secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl− current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O2 produced similar improvements in Cl− current and CFTR protein as air-liquid interface culture, while increasing Po 2 from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking.

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Lai, Long-Shan T., Tai-Her Tsai, Te Chi Wang, and Tsung-Yao Cheng. "The influence of culturing environments on lovastatin production by Aspergillus terreus in submerged cultures." Enzyme and Microbial Technology 36, no. 5-6 (April 2005): 737–48. http://dx.doi.org/10.1016/j.enzmictec.2004.12.021.

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Guo-zhang, ZU, GUAN Yuan-liang, HOU Guan-jun LI Hai-yang, CHEN Yu, TUO Ji-liang, and ZHOU Guo-zhi. "Utilization and Protection of the Submerged Plant Resources by Pen-crab Culturing in Nushan Lake." Journal of Lake Sciences 11, no. 1 (1999): 91–96. http://dx.doi.org/10.18307/1999.0115.

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Domingos, Marcelo, Priscila Brasil de Souza-Cruz, André Ferraz, and Arnaldo Márcio Ramalho Prata. "A new bioreactor design for culturing basidiomycetes: Mycelial biomass production in submerged cultures of Ceriporiopsis subvermispora." Chemical Engineering Science 170 (October 2017): 670–76. http://dx.doi.org/10.1016/j.ces.2017.04.004.

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Protsenko, M. A., N. E. Kostina, and T. V. Teplyakova. "Selection of Nutrient Media for Submerged Culturing of Wood-Destroying Mushroom of Daedaleopsis tricolor (Bull.) Bondartsev et Singer." Biotekhnologiya 34, no. 1 (2018): 45–51. http://dx.doi.org/10.21519/0234-2758-2018-34-1-45-51.

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GHAZANFAR, MISBAH, MUHAMMAD IRFAN, HAFIZ ABDULLAH SHAKIR, MUHAMMAD KHAN, MUHAMMAD NADEEM, and AHMAD IRFAN. ""CELLULASE PRODUCTION OPTIMIZATION BY BACILLUS AERIUS THROUGH RESPONSE SURFACE METHODOLOGY IN SUBMERGED FERMENTATION "." Cellulose Chemistry and Technology 56, no. 3-4 (May 5, 2022): 321–30. http://dx.doi.org/10.35812/cellulosechemtechnol.2022.56.28.

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The objective of the present study was to get better production of FPase from Bacillus aerius (MG597041) by optimizing different process parameters under submerged fermentation through the statistical approach. Optimization of physical factors of culturing medium by one factor at a time (OFAT) revealed optimum incubation time of 24 h, inoculum size of 1%, pH 5.5, and substrate concentration of 4%. Plackett-Burman design (PBD) was performed to identify the significant nutritional influence of cellulase production. Among the nine parameters screened, peptone, yeast extract, FeSO4 and K2HPO4 were found significant. CCD of significant parameters revealed maximum FPase activity (127.4 IU/mL/min) at the optimum concentration of yeast extract of 0.5 g/L, peptone of 0.5 g/L, FeSO4 of 0.2 g/L, and K2HPO4 of 0.02 g/L. ANOVA was used to analyze these results. The analysis of the results showed an F-value of 8.74 and a p-value 0.00. Maximum hydrolysis of 10% of raw Bombax ceiba seed pods using this indigenous cellulase was obtained after 24 h. Also, the study explored the potential of the obtained cellulase to be applied in denim biostoning finishing. The findings demonstrated the efficient use of the obtained enzyme in saccharification of raw Bombax ceiba seed pods, which can be of interest for production of biofuel, and in biostoning treatment of denim fabrics.

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Petre, Alexandru, Mihaela Ene, and Emanuel Vamanu. "Submerged Cultivation of Inonotus obliquus Mycelium Using Statistical Design of Experiments and Mathematical Modeling to Increase Biomass Yield." Applied Sciences 11, no. 9 (April 30, 2021): 4104. http://dx.doi.org/10.3390/app11094104.

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Submerged culturing of mycelium is an efficient technique used to increase biomass yields, more so when employed with naturally slow-growing species of mushrooms. This paper is concerned with optimizing nutrient broth components used in Inonotus obliquus cultures for achieving high biomass yields. We modeled the effect of seven biotechnological parameters (six broth ingredients and the initial pH of nutritive broth) on mycelial biomass and predicted an optimum broth formula using response surface methodology. An analysis of variance showed that the elaborated model is significant (F-value of 2.76 and p-value of 0.0316). We used bioreactor cultures to confirm the model’s optimum prediction and to compare these results with a general-purpose mycology medium, namely potato dextrose broth (PDB). The optimized bioreactor culture yielded 4.37 g/L (93.36% of the dry weight prediction), while the PDB bioreactor culture yielded 2.084 g/L, after 15 days of cultivation. The optimized formula was: 2.15299 g malt extract, 3.99296 g yeast extract, 11.0041 g fructose, 17.4 g soluble starch, 0.1 g MgSO4, and 0.05 g CaCl2 per liter of broth.

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Nguyen Thi, Minh Huyen, Hoa Tran Thi, Tuyet Lan Ninh Thi, Le Pham Thi, Men Do Thi, and Hien Tran Thi. "Evaluation of microbiological and heavy metal safety assessment of Phellinus linteus mass powder from submerged fermentation." Heavy metals and arsenic concentrations in water, agricultural soil, and rice in Ngan Son district, Bac Kan province, Vietnam 3, no. 1 (March 22, 2020): 20–28. http://dx.doi.org/10.47866/2615-9252/vjfc.99.

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Phellinus linteus is known to be a medicinal mushroom which is very good to human health. They are used as medicinal mushroom in many Asian countries such as Japan, Korea, China, Thailand and Vietnam. Because of long time growth and over-havest by humans in natural conditions, they are artificially cultured in these countries. There were advances of liquid culture of mushroom such as time saving and scaleable in culture in comparison with culturing of mushroom in decaying wood. In this study, Phellinus linteus is cultured in optimum liquid medium, lyophilized, ground into fine powder and evaluated for microbiological and total of Asen and Lead to ensure the biomass product will meet safety standards for users. Results are as follows: Total of anaerobic bacteria 2,2 × 104 CFU/g, Coliforms< 1,0 × 101 CFU/g, Coagulase positive with Staphylococci <1,0×101 CFU/g, total amount of yeast-mold < 1,0 × 101 CFU/g, not detected of E.coli, Clostridium perfringens and Salmonella spp.. Furthermore, we did not find any Asen and Lead which are supposed to remain in our sample.

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Senevirathna, J. D. M., Gayantha R. L. Kodikara, and D. H. N. Munasinghe. "Analysis of habitat characteristics of the scalloped spiny lobster Panulirus homarus (Linnaeus, 1758) in their home range along the southern coast of Sri Lanka." Indian Journal of Fisheries 64, no. 1 (March 31, 2017): 1. http://dx.doi.org/10.21077/ijf.2017.64.1.47483-01.

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The present study was aimed to investigate the quality of home range habitat characteristics of the scalloped spiny lobster Panulirus homarus (Linnaeus, 1758) in southern coastal region of Sri Lanka. Four sites were selected from south-east (Patanagalle, Godawaya) and south-west (Weligama, Hikkaduwa) regions of the southern coast of Sri Lanka (SCSL). The bottom water quality data and benthic substrate types of their home range habitats were monitored and noted in a 25 x 25 m area covering 16 subsampling points with locality information. With the use of geographical information system (GIS) tools, the spatial distribution maps of environmental parameters were created and submerged bottom substrate types of the four sites were graphed. Salinity, temperature and dissolved oxygen correlated well with depth. Hikkaduwa site was found rich in corals with less number of scalloped spiny lobsters. Sites of south-east region of the SCSL (Patanagalle, Godawaya) were found less polluted having rocks and sandy bottom with high occurrence of scalloped spiny lobsters. Results of the study showed that Patanagalle site (south-east of SCSL) could be suggested as the most suitable site for culturing scalloped spiny lobsters.

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Kalamaras, Georgios, Maria Kloukinioti, Maria Antonopoulou, Ioanna Ntaikou, Dimitris Vlastos, Antonios Eleftherianos, and Stefanos Dailianis. "The Potential Risk of Electronic Waste Disposal into Aquatic Media: The Case of Personal Computer Motherboards." Toxics 9, no. 7 (July 12, 2021): 166. http://dx.doi.org/10.3390/toxics9070166.

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Considering that electronic wastes (e-wastes) have been recently recognized as a potent environmental and human threat, the present study aimed to assess the potential risk of personal computer motherboards (PCMBs) leaching into aquatic media, following a real-life scenario. Specifically, PCMBs were submerged for 30 days in both distilled water (DW) and artificial seawater (ASW). Afterwards, PCMBs leachates were chemically characterized (i.e., total organic carbon, ions, and trace elements) and finally used (a) for culturing freshwater (Chlorococcum sp. and Scenedesmus rubescens) and saltwater (Dunaliella tertiolecta and Tisochrysis lutea) microalgae for 10 days (240 h), (b) as the exposure medium for mussel Mytilus galloprovincialis (96 h exposure), and (c) for performing the Cytokinesis Block Micronucleus (CBMN) assay in human lymphocytes cultures. According to the results, PCMBs could mediate both fresh- and marine algae growth rates over time, thus enhancing the cytotoxic, oxidative, and genotoxic effects in the hemocytes of mussels (in terms of lysosomal membrane impairment, lipid peroxidation, and NO content and micronuclei formation, respectively), as well as human lymphocytes (in terms of MN formation and CBPI values, respectively). The current findings clearly revealed that PCMBs leaching into the aquatic media could pose detrimental effects on both aquatic organisms and human cells.

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Dinil, Aparna, and Anu Jacob. "Valorization of Agro-industrial Discards in Fermentation for the Production of Cellulase Enzyme." Journal of Pure and Applied Microbiology 16, no. 1 (February 10, 2022): 347–54. http://dx.doi.org/10.22207/jpam.16.1.25.

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Cellulases are commercially important enzymes with application in various industries such as biofuel, detergent, food processing, brewery, pulp and paper. To make its production cost-effective, a preferred method is to use solid-state fermentation and with use of inexpensive substrates. Solid-state fermentation is an alternative culturing method and yields higher enzymes compared to submerged fermentation. In the current study, Aspergillus niger was isolated and further developed as inoculum for solid-state fermentation. Agroindustrial discards like banana pseudostem, jackfruit waste were used as the substrates. The substrates were pretreated by acid and were characterized by FTIR analysis to confirm the presence of cellulosic content. Different concentrations of the substrates were attempted for fermentation and the yield of the enzyme was compared. The solid-state fermentation was stable for enzyme production as well as microbial growth. The cellulase activity per gram of the substrate (U/g) was obtained maximum for jackfruit waste-based media (17±1.1 U/g). Both the lignocellulosic substrates were potential substrates for the production of cellulase enzyme. With further optimization and scale-up, this could be a cheap and sustainable process. This study has validated agro-industrial waste’s bioconversion into value-added products that have a remarkable environmental and economic advantage.

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Idrees, Ayesha, Inge Schmitz, Alice Zoso, Dierk Gruhn, Sandra Pacharra, Siegfried Shah, Gianluca Ciardelli, Richard Viebahn, Valeria Chiono, and Jochen Salber. "Fundamental in vitro 3D human skin equivalent tool development for assessing biological safety and biocompatibility – towards alternative for animal experiments." 4open 4 (2021): 1. http://dx.doi.org/10.1051/fopen/2021001.

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Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals and cosmetics in vitro testing and for the development of complex skin wound therapeutics. In vitro three-dimensional (3D) dermal-epidermal based interfollicular, full-thickness, human skin equivalent (HSE) was here developed, recapitulating skin morphogenesis, epidermal differentiation, ultra-structure, tissue architecture, and barrier function properties of human skin. Different 3D cell culture conditions were tested to optimize HSE maturation, using various commercially available serum/animal component-free and/or fully defined media, and air-liquid interface (ALI) culture. Optimized culture conditions allowed the production of HSE by culturing normal human dermal fibroblasts (NHDFs) for 5–7 days in CELLnTEC-Prime Fibroblast (CnT-PR-F) medium and then culturing normal human epidermal keratinocytes (NHEKs) for 3 days in CELLnTEC-Prime Epithelial culture (CnT-PR) medium on them. Co-culture was then submerged overnight in CELLnTEC-Prime-3D barrier (CnT-PR-3D) medium to stimulate cell-cell contact formation and finally placed at ALI for 15–20 days using CnT-PR-3D medium. Histological analysis revealed uniform distribution of NHDFs in the dermal layer and their typical elongated morphology with filopodia. Epidermal compartment showed a multi-layered structure, consisting of stratum basale, spinosum, granulosum, and corneum. NHDFs and keratinocytes of basal layer were positive for the proliferation marker Kiel 67 (Ki-67) demonstrating their active state of proliferation. The presence of typical epidermal tissue proteins (keratins, laminins, filaggrin, loricin, involucrin, and β-tubulin) at their correct anatomical position was verified by immunohistochemistry (IHC). Moreover, transmission electron microscopy (TEM) analyses revealed basement membrane with lamina lucida, lamina densa, hemidesmosomes and anchoring fibers. The epidermal layers showed abundant intracellular keratin filaments, desmosomes, and tight junction between keratinocytes. Scanning electron microscopy (SEM) analyses showed the interwoven network of collagen fibers with embedded NHDFs and adjacent stratified epidermis up to the stratum corneum similar to native human skin. HSE physiological static contact angle confirmed the barrier function. The developed HSE represents a fundamental in vitro tool to assess biocompatibility of biomaterials, pharmacotoxicity, safety and effectiveness of cosmetics, as well as to investigate skin biology, skin disease pathogenesis, wound healing, and skin infection.

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Darijani, Mojdeh, and Ashraf Kariminik. "Screening of cellulase producing bacteria from tomato waste materials for lycopene extraction." International Journal of Life Sciences 9, no. 2 (February 10, 2015): 43–47. http://dx.doi.org/10.3126/ijls.v9i2.12055.

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Cellulase is one of the industrially important enzymes that has the ability to degrade cellulose. This enzyme is produced by a variety of microorganisms. It has numerous commercial applications like malting, wood processing, and preparation of denim fabrics in textile industries, maceration of protoplasts from plant tissues and de-inking process in recycling of printed papers. The aim of the present study was the isolation, identification and screening of extracellular cellulase producing bacteria with high cellulase activity from tomato waste materials and their identification by phenotypic, biochemical and molecular tests. Cellulose degrading bacteria were isolated after serial dilution preparation from tomato waste matraials and surface culturing on CMC agar medium. 40 bacteria were isolated. Bacteria were further identified by morphological, biochemical and molecular tests. All of the isolates were Gram positive endospore forming rods, thus they were identified as Bacillus sp. The obtained isolates were screened for cellulase production and lycopene extraction in submerged fermentation process. The best isolate was Bacillus axarquiensis strain CHMS1B6 based on molecular analysis. The bacteria was subjected to different optimum conditions include pH, temperature and time of incubation. The highest cellulase activity was obtained in pH 7 and the optimum temperature at 30°C after 72 hours incubation period. Our findings indicate that the tomato waste materials are as attractive sources for the study of novel cellulolytic bacteria and effective enzymes for cellulose biodegradation and lycopene extraction.DOI: http://dx.doi.org/10.3126/ijls.v9i2.12055 International Journal of Life Sciences 9 (2) : 2015; 43-47

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Dedjell, Abdallah, and Sophie Cliquet. "Media and culturing protocol using a full 25 factorial design for the production of submerged aggregates by the potential bio-herbicide Plectosporium alismatis against weed species of Alismataceae." Biocontrol Science and Technology 29, no. 4 (February 13, 2019): 308–24. http://dx.doi.org/10.1080/09583157.2018.1560393.

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Ye, Zhang, Dina M. Silva, Daniela Traini, Paul Young, Shaokoon Cheng, and Hui Xin Ong. "An adaptable microreactor to investigate the influence of interfaces on Pseudomonas aeruginosa biofilm growth." Applied Microbiology and Biotechnology 106, no. 3 (January 11, 2022): 1067–77. http://dx.doi.org/10.1007/s00253-021-11746-5.

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Abstract Biofilms are ubiquitous and notoriously difficult to eradicate and control, complicating human infections and industrial and agricultural biofouling. However, most of the study had used the biofilm model that attached to solid surface and developed in liquid submerged environments which generally have neglected the impact of interfaces. In our study, a reusable dual-chamber microreactor with interchangeable porous membranes was developed to establish multiple growth interfaces for biofilm culture and test. Protocol for culturing Pseudomonas aeruginosa (PAO1) on the air–liquid interface (ALI) and liquid–liquid interface (LLI) under static environmental conditions for 48 h was optimized using this novel device. This study shows that LLI model biofilms are more susceptible to physical disruption compared to ALI model biofilm. SEM images revealed a unique “dome-shaped” microcolonies morphological feature, which is more distinct on ALI biofilms than LLI. Furthermore, the study showed that ALI and LLI biofilms produced a similar amount of extracellular polymeric substances (EPS). As differences in biofilm structure and properties may lead to different outcomes when using the same eradication approaches, the antimicrobial effect of an antibiotic, ciprofloxacin (CIP), was chosen to test the susceptibility of a 48-h-old P. aeruginosa biofilms grown on ALI and LLI. Our results show that the minimum biofilm eradication concentration (MBEC) of 6-h CIP exposure for ALI and LLI biofilms is significantly different, which are 400 μg/mL and 200 μg/mL, respectively. These results highlight the importance of growth interface when developing more targeted biofilm management strategies, and our novel device provides a promising tool that enables manipulation of realistic biofilm growth. Key points • A novel dual-chamber microreactor device that enables the establishment of different interfaces for biofilm culture has been developed. • ALI model biofilms and LLI model biofilms show differences in resistance to physical disruption and antibiotic susceptibility.

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WÜSTENBERG, ARNE, YVONNE PÖRS, and RUDOLF EHWALD. "Culturing of stoneworts and submersed angiosperms with phosphate uptake exclusively from an artificial sediment." Freshwater Biology 56, no. 8 (February 28, 2011): 1531–39. http://dx.doi.org/10.1111/j.1365-2427.2011.02591.x.

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Leite, Juliana Abigail, Nathalia Gonsales da Rosa-Garzon, Helen Julie Laure, José Cesar Rosa, Octavio Luiz Franco, Cristina Maria de Souza Motta, and Hamilton Cabral. "Proteomic Analysis of Intra- and Extracellular Proteins of Aspergillus niveus during Submerged Bioprocess Culturing under Different pH Conditions." Current Proteomics 17 (December 2, 2020). http://dx.doi.org/10.2174/1570164617999201202120657.

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Background: Proteomics facilitates understanding of the complexity of molecular and physiological mechanisms involved in the metabolic and biological fungal adaptations to pH changes. Proteomics enables the identification of enzymes and fungal proteins involved in these adaptations. This approach may be used to investigate such fungi as Aspergillus niveus, whose proteome has not yet been analyzed, changes the intra- and extracellular protein profiles in response to extracellular pH. Objective: In the current study, we used two-dimensional gel electrophoresis (2DE) and mass spectrometry to evaluate the response of A. niveus to grow at pH 5, 6, 7, and 8 for 96 hours submerged bioprocess culturing. Methods: This study evaluated the response of A. niveus to grow at pH 5, 6, 7, and 8 for 96 h submerged bioprocess culturing, by analysis of two-dimensional gel electrophoresis (2DE), of the intracellular proteomes and the secretome, protein spots of interest were submitted to tryptic digestion and analyzed by matrix-assisted laser desorption/ionization time-offlight tandem mass spectrometry (MALDI-TOF/TOF-MS). Results: This approach revealed substantial differences between the functions of intra- and extracellular proteins of A. niveus. The data suggested that pH-modulated global proteins are involved in important, mainly metabolic, processes, in the pentose phosphate pathway, protein regulation, cell wall maintenance, and others. Moreover, the change in extracellular pH could have altered the availability of nutrients, and induced the production of enzymes that respond to oxidative and other stresses. Conclusion: Proteomic facilitates understanding of the complexity of molecular and physiological mechanisms involved in the metabolic and biological adaptations of fungi to pH changes.

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Junaid, Muhammad, and DAVID GUEST. "Modified culture assay to obtain a diversity of hyphal structures of Ceratobasidium theobromae-VSD pathogen on cocoa." Biodiversitas Journal of Biological Diversity 22, no. 4 (March 26, 2021). http://dx.doi.org/10.13057/biodiv/d220434.

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Abstract. Junaid M, Guest D. 2021. Modified culture assay to obtain a diversity of hyphal structures of Ceratobasidium theobromae, VSD pathogen on cocoa. Biodiversitas 22: 1879-1886. Ceratobasidium theobromae, a causal agent of vascular streak dieback (VSD) disease, is a fastidious Basidiomycete fungus devastating cocoa in Southeast Asia Melanesia. One sought-after in vitro technique is pathogen subculture. As an obligate parasite, growing the pathogen onto an artificial medium is always problematic. In this paper, a new design is demonstrated to obtain a single colony of the pathogen with hyphal diversity. Putative C. theobromae is obtained from infected leaves and petioles of cocoa using rigorous surface sterilization before culturing on standard water agar (WA) medium. Once the fungus grows out from the tissue, the infected plant tissue is then removed carefully, leaving mycelium's uninterrupted growth. About 200 mL of liquid Corticium culture medium (CCM) is injected to submerge the solid medium layer, nurture hyphae development, and allow generative structure formation. Molecular amplification with a pair primer of ITS 1B (forward) and 4B (reverse) specific for fungal Basidiomycete DNA with a touchdown undertook thermal cycler program. PCR product amplification successfully confirmed the presence of C. theobromae DNA collected from the samples with VSD symptomatic lesions and identified a diversity of hyphal and branching formations.

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