ABSTRACT
Shoot tips and in vitro grown proliferating buds of banana cv. Rasthali (Silk, AAB) were treated with various concentrations and durations of chemical mutagens viz., EMS, NaN3 and DES. LD50 for shoot tips based on 50% reduction in fresh weight was determined as 2% for 3 h, 0.02% for 5 h and 0.15%for 5 h, while for proliferating buds, they were 0.6% for 30 min, 0.01% for 2 h and 0.06% for 2 h for the mutagens EMS, NaN3 and DES, respectively. Subsequently, the mutated explants were screened in vitro against fusarium wilt using selection agents like fusaric acid and culture filtrate. LD50 for in vitro selection agents calculated based on 50% survival of explants was 0.050 mM and 7% for fusaric acid and culture filtrate, respectively and beyond which a rapid decline in growth was observed. This was followed by pot screening which led to the identification of three putative resistant mutants with an internal disease score of 1 (corm completely clean, no vascular discolouration). The putative mutants identified in the present study have also been mass multiplied in vitro.
ABSTRACT
Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R0 and R1 progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R1 lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.
ABSTRACT
Objective To investigate the situation and change of antifungal resistance in clinical Candida and other fungal iso- lates from 5 hospitals in diverse geographic region of China.Methods Antimicrobial susceptibility testing of 8 000 fungat iso- lates collected during 2001 and 2005 were carried out with 25?g fluconazole disk and 1?g voriconazole disk using disk diffusion method as recommend by CLSI/NCCLS M44-A.Disk test plates were automatically read and results were recoded with the BIOMIC Image Analysis System.The equivalent MICs were automatically calculated by the BIOMIC System software.Results The proportion of Candida atbicans and non-Candida albicans (e.g.Candida glabrata) in the total fungal isolates did not change significantly from 2001 to 2005.The susceptibility rate of C.albicans to fluconazole and vorieonazole were stable during 2001 and 2005.However, the resistance to fluconazole and voriconazole increased variably in C.glabrata and other non-Can- dida albicans fungal isolates during the same period.Conclusions The voriconazole demonstrated higher activity against all yeast species in comparison with fluconazole.The increasing resistance to fluconazole and voriconazole in non C.albicans fungal isolates including C.glabruta suggests the importance of surveillance of fungal resistance in Candida isolates.
ABSTRACT
O surgimento da resistência antifúngica aos tratamentos convencionais tem proporcionado o desenvolvimento de novas modalidades terapêuticas para o tratamento da candidose bucal. Nesse contexto, a utilização da PDT vem sendo sugerida como método alternativo para a inativação de microrganismos patogênicos. Este estudo avaliou a efetividade da PDT na inativação de C. albicans e C. glabrata, ATCC e resistente a fluconazol, por meio da utilização do agente fotossensibilizador Photogem® e da iluminação com LEDs de comprimento de onda azul. Inicialmente, os microrganismos avaliados foram inoculados em tubos de ensaio contendo meio de cultura líquido e incubados overnight a 37ºC. Em seguida, foram obtidas suspensões celulares das espécies de Candida avaliadas. Essas suspensões foram transferidas para placas de 96 orifícios, tratadas com cinco diferentes concentrações de Photogem® (2,5; 5; 10; 25 e 50mg/L) e expostas a quatro doses de luz (10,5; 18; 25,5 e 37,5J/cm2). Suspensões adicionais foram tratadas somente com as cinco concentrações do fotossensibilizador ou apenas com as quatro doses de luz. Cada condição experimental foi realizada três vezes. Após a realização desses experimentos, foram obtidas diluições seriadas de cada amostra (10-1 a 10-3), e alíquotas de 25 µL dessas diluições foram plaqueadas, em triplicatas, em Sabouraud Dextrose Agar. Adicionalmente, alíquotas de 25 µL foram removidas das cavidades das placas de orifícios e transferidas diretamente para um quadrante da placa de Petri, sem a realização de diluição. As placas foram incubadas a 37ºC por 48 horas. Após a incubação, foi realizada a contagem das colônias viáveis (ufc/mL), e os valores obtidos foram analisados com o teste t de Student (p < 0,05). Os 16 resultados demonstraram que a inativação de Candida spp. ocorreu de forma concentração/dose dependente, que resultou na completa inativação desses microrganismos em determinadas condições experimentais. A dose de luz mínima que promoveu a completa inativação das duas origens de C. albicans foi 18 J/cm2, em associação com 50mg/L de Photogem® . Após a aplicação de 25,5 e 37,5 J/cm2, baixas concentrações de fotossensibilizador foram requeridas para a inativação total dessa espécie, sendo que diferenças estatisticamente significantes foram apontadas entre os valores obtidos para as duas origens de C. albicans. Também foram observadas diferenças estatisticamente significantes na obtenção da inativação total das duas origens de C. glabrata. Para a ATCC, não houve crescimento de colônias viáveis após o tratamento com 10, 25 e 50 mg/L de Photogem® seguido de iluminação a 37,5 J/cm2 . No entanto, somente as concentrações de 25 e 50 mg/L foram capazes de eliminar a C. glabrata resistente a fluconazol, nas doses de 25,5 e 37,5 J/cm2. Assim, a fotoativação do Photogem® pela luz do LED demonstrou efetividade na inativação das duas espécies de Candida avaliadas, ATCC e resistente a fluconazol
Oral candidosis is an opportunistic infection that affects a significant percentage of the population. The oral infection caused by Candida spp. is usually treated with topical and systemic antifungal drugs. However, the widespread use of these agents has resulted in an alarming increase in the rate of antifungal resistance. Recently, the photodynamic therapy (PDT) has been studied as an alternative modality of killing microorganisms, including viruses, fungi and bacteria. The aim of this study was to determine whether Candida albicans and C. glabrata, ATCC and fluconazole-resistant strains, could be photosensitized by Photogem® in combination with blue Light Emitted Diode (LED). Suspensions of each Candida strain, containing viable cells per milliliter, were treated with five concentrations of Photogem® (2.5, 5, 10, 25 and 50 mg/l), followed by LED irradiation in four light doses (10.5, 18, 25.5 and 37.5 J/cm2). Each experimental condition was carried out in triplicate and repeated tree times. From each sample, serial dilutions were obtained and aliquots of 25 µl of each dilution were plated on Sabouraud Dextrose Agar. All plates were incubated at 37°C for 48 hours. After incubation, colonies were counted (CFU/ml) and the data were statistically analyzed by the Student's t test (p < 0.05). The results demonstrated a concentration/dose-dependent pattern of inactivation, that resulted in complete elimination of all Candida evaluated. The minimal light dose for the complete inactivation of both C. albicans source was 18 J/cm2 , in conjunction with 50 mg/l of Photogem®. After 25.5 and 37.5 J/cm2 , a lower concentration of Photogem® was required to totally inactivate C. albicans ATCC (5 and 2.5 mg/l) in comparison with C. albicans fluconazole-resistant (10 and 5 mg/l). There were statistically significant differences in the log (CFU/ml) for the minimal light dose to complete inactivation of both C. glabrata sources. For C. glabrata ATCC, no viable cells were detected after treatment with 10, 25.5 or 50 mg/l of Photogem® followed by 37.5 J/cm2 , while the association of 25 mg/l with 25.5 J/cm2 was sufficient to totally inactivate the C. glabrata fluconazole-resistant. The photoactivation of Photogem® by blue LED light proved to be effective for the inactivation of fluconazole-resistant and ATCC strains of C. albicans and C. glabrata