Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-24T18:48:59Z No. of bitstreams: 1 texto completo.pdf: 3493058 bytes, checksum: 1aa65a58b68661df0465f55d054a91ab (MD5) / Made available in DSpace on 2017-04-24T18:48:59Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3493058 bytes, checksum: 1aa65a58b68661df0465f55d054a91ab (MD5) Previous issue date: 2003-03-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Visando entender os mecanismos envolvidos na resistência de quatro cultivares de batata à pinta preta, esse trabalho teve por objetivos: 1) quantificar os componentes epidemiológicos da resistência dos cultivares Aracy, Delta, Desirée e Bintje que apresentam diferentes níveis de resistência à pinta preta, e estudar o efeito da idade dos tecidos nesses componentes; 2) realizar a análise histológica do processo infeccioso nesses cultivares, visando identificar possíveis mecanismos estruturais e relacionados com os níveis de resistência dos cultivares, e com a resistência de tecidos jovens; 3) verificar se a expressão diferencial de genes homólogos aos genes que codificam as proteínas PR gluB (glucanase básica de tabaco) e chiB (quitinase básica de feijão) respectivamente, se correlaciona com os níveis de resistência dos cultivares analisadas, e com a maior resistência dos tecidos jovens. Os componentes epidemiológicos da resistência, período de incubação (PI), número de lesões (NL), a severidade, taxa de expansão das lesões (TEL), período latente (PL) e o número de esporos por área foliar afetada (NEAA) foram quantificados nos terços inferior, médio e superior de plantas dos cultivares Aracy (resistente), Delta (resistência intermediária), Desirée(suscetível) e Bintje (suscetível) inoculadas com Alternaria solani numa concentração de 10 3 conídios.mL -1 . No cultivar Aracy constatou-se maior PI, redução do NL, da TEL e conseqüentemente da severidade em todos os terços avaliados. Já o menor NL, TEL, e severidade no terço médio explicam a resistência moderada do cultivar Delta. O cultivar Desirée, classificado como suscetível, apresentou, no terço superior, NL e severidade similar ao cultivar Aracy. O PL e o NEAA não foram adequados para a avaliação da resistência em condições de casa de vegetação e, provavelmente, foram influenciados pela falta de molhamento foliar. Todos os componentes foram influenciados pela idade dos tecidos. Maior PI e menores valores de NL, TEL, severidade e AACPD foram observados no terço superior, comprovando a menor suscetibilidade desses tecidos. Foram efetuados estudos histológicos do processo infeccioso de A. solani nas diferentes cultivares e terços usando a técnica de diafanização de tecidos. Foi quantificado o número de conídios germinados, número de apressórios, número de penetrações diretas e número de sítios de penetração exibindo células com reação de hipersensibilidade (HR). Não se constatou mecanismos de resistência que pudessem ser associados com os níveis de resistência dos cultivares estudados, nem com a maior resistência dos tecidos jovens. Constatou-se uma relação positiva entre o número de sítios de penetração mostrando HR e a resistência do cultivar Aracy. Houve associação desse componente com a resistência dos tecidos jovens. A expressão de genes homólogos à glucanase básica de tabaco (gluB) e à chitinase básica de feijão (chiB) como resposta a infecção de A. solani, foi analisada nos terços inferior e superior dos cultivares Bintje e Aracy, mediante a técnica de "Northern Blotting". Um aumento na expressão de transcritos a gluB e chiB foi constatado no terço inferior do cultivar Aracy, mas não em Bintje. Esse padrão de expressão sugere um possível envolvimento de isoformas destes genes com a resistência de Aracy a A. solani. Essa expressão diferenciada, não foi observada em folhas do terço superior desse cultivar sugerindo um padrão de regulação gênica dependente da idade dos tecidos, e o envolvimento de outros mecanismos bioquímicos na resistência dos tecidos jovens. / The resistance components of four potato cultivars to Alternaria solani were studied in three different levels: 1) quantification of epidemiological components related to resistance in cultivars Aracy, Delta, Desirée, and Bintje, and the effect of age-related resistance in these components; 2) histological analysis of the infectious process in these cultivars, aiming at identifying possible structural mechanisms related to resistance; 3) differential expression of genes homologous to gluB (basic glucanase of tobacco) and chiB (basic chitinase of bean) that encode pathogenesis-related proteins (PR) and the correlation between gene expression levels and cultivar/tissue age resistance levels. The epidemiological components related to resistance, incubation period (PI), number of lesions (NL), early blight severity, lesion expansion rate (TEL), latent period (PL), and the number of spores produced by lesion area (NEAA) were quantified in the lower, middle, and upper parts of cultivars Aracy (resistant), Delta (intermediate resistance), Desirée (susceptible), and Bintje (susceptible) inoculated with a suspension of 103 conidia of A. solani.mL-1. Longer PI, reduced NL, low TEL, and low severity were recorded in Aracy in all parts of the plant. For 'Delta', the reduced NL, low TEL and severity on the middle part of the plant corroborates its intermediate resistance level. Desirée is a susceptible cultivar, but NL and severity on the upper part did not differ from Aracy. The PL and NEAA were not informative when assessing resistance under greenhouse conditions. Probably, PL and NEAA were influenced by the shortage or lack of leaf wetness. All components were influenced by tissue age. Longer PI and smaller values of NL, TEL, and severity were observed in the upper leaves, indicating higher resistance of these tissues. Histological analyses of the infectious process of A. solani in different cultivars were carried out by tissue discoloration. The number of germinated conidia, number of apressoria, number of direct penetrations, and number of penetration sites with cells exhibiting hypersensitive reaction (HR) were quantified. No mechanisms of resistance were associated with cultivar resistance levels or with tissue age. For cultivar Aracy there was a direct relationship between number of penetration sites showing HR and cultivar resistance. The number of sites with HR was also associated with resistance of young tissues. The expression of gluB and chiB in response to A. solani infection was analyzed in the lower and upper parts of 'Bintje' and 'Aracy' plants by Northern blotting analysis. An increase in the level of transcripts related to gluB and chiB was recorded in the lower part of Aracy, but not in Bintje. Differential expression suggests the involvement of isoforms of these genes in Aracy resistance. Differential expression was not observed in leaves of the upper part of Aracy suggesting an age-dependent gene regulation process and/or the involvement of other biochemical mechanisms associated with young tissue high resistance level. / Tese importada do Alexandria

Made available in DSpace on 2014-06-11T19:32:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-31Bitstream added on 2014-06-13T20:23:19Z : No. of bitstreams: 1 molina_lmr_dr_jabo.pdf: 480341 bytes, checksum: b838d0062518eb208c7e881bd5eb7588 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No início dos anos 90, o fungo Rhizoctonia solani emergiu como um patógeno importante associado à morte de pastagens do gênero braquiária na Colômbia. Inicialmente, esse estudo indicou que R. solani AG-1 IA predomina como patógeno associado à queima da folha da braquiária nos Llanos Colombianos. Para o estudo da estrutura genético-populacional do patógeno, um total de 198 isolados de R. solani AG-1 IA foram coletados de campos de B. brizantha cv. Toledo, de Brachiaria híbrido Mulato e do arroz. Os isolados foram genotipados usando dez loci microssatélites. Um sistema reprodutivo misto (reprodução sexuada e clones adaptados) caracterizou as populações de R. solani AG-1 IA que infectam a braquiária. A alta fração clonal e os desvios do equilíbrio de Hardy-Weinberg encontrados foram consistentes com o efeito Wahlund associado à mistura de populações. Padrões históricos de migração entre populações hospedeiro-distintas indicaram a origem provável das populações que infectam braquiária a partir de populações que infectavam arroz. Para determinar o efeito do estresse térmico na evolutibilidade para crescimento micelial, populações de R. solani AG-1 IA infectando braquiária ou arroz e R. oryzae-sativae do arroz, foram submetidas à temperatura ótima e de estresse (25 e 35°C). A herdabilidade para crescimento micelial sob condições de estresse foi considerada alta na população de R. oryzae-sativae do arroz, indicando que os patógenos têm potencial de adaptação à temperatura de 35°C / In the early '90s, the fungus Rhizoctonia solani has emerged as an important pathogen associated with the death of Brachiaria pastures in the Colombian Llanos. Initially, this study indicated that R. solani AG-1 IA was the predominant pathogen associated with the leaf blight in the Colombian Llanos. To study the population genetic structure of the pathogen, a total of 198 isolates of R. solani AG-1 IA were collected from fields of B. brizantha cv. Toledo, Brachiaria Mulato hybrid and from rice. These isolates were genotyped using ten microsatellite loci. A mixed mating system (sexual reproduction and dispersal of adapted clones) characterized the populations of R. solani AG-1 IA infecting Brachiaria. The high clonal fraction and deviations from Hardy-Weinberg equilibrium found in three out of four populations were consistent with Wahlund effect associated with the mixing of populations. Historical patterns of migration between different host-populations indicated that the likely source of the current populations infecting Brachiaria in the Colombian Llanos was from populations that originally infected rice. To determine the effect of heat stress on the evolvability for mycelial growth, two host distinct populations of Rhizoctonia solani AG-1 IA infecting either brachiaria or rice and a rice-infecting population of R. oryzae-sativae, were submitted to optimal and stress temperatures (25°C and 35°C). The heritability values for mycelial growth under heat stress was considered high for rice-infecting population of R. oryzae-sativae, indicating that these pathogen’s population have a high potential to adapt to 35°C temperature

Made available in DSpace on 2014-06-11T19:29:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-13Bitstream added on 2014-06-13T19:18:06Z : No. of bitstreams: 1 basseto_ma_me_ilha.pdf: 949120 bytes, checksum: dd3f3b104d600fa3d0cc25023fb35d18 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A mancha areolada causada por Thanatephorus cucumeris, é uma das doenças mais importantes da seringueira (Hevea brasiliensis) na região Amazônica. Apesar disso, há pouca informação disponível sobre a diversidade biológica, patogênica e genética do patógeno. Uma questão importante sobre o real posicionamento filogenético deste patógeno ainda não foi respondida. Neste estudo, foram analisadas seqüências da região ITS-5.8S do rDNA de uma população de T. cucumeris (fase anamórfica = Rhizoctonia solani AG 2-2) associado à mancha areolada da seringueira, obtida em Belém (PA), Manaus (AM) e Xapuri/Rio Branco (AC). Esta população foi também comparada filogeneticamente com membros do AG 2 descritos mundialmente. Este estudo representa um passo importante para revelar a origem, os padrões de movimento e amplificação de genótipos epidemiologicamente importantes de T. cucumeris da seringueira. Filogenéticamente, através de análise Bayesiana e de máxima parcimônia, encontramos suporte para nomear um novo grupo de anastomose associado à mancha areolada da seringueira: o AG 2-2 Hb. Este grupo constitui-se numa unidade evolucionária independente em relação aos subgrupos mundiais do AG 2-2 analisados. Na genealogia construída por análise coalescente, observou-se que a população de R. solani AG 2-2 Hb, de Belém, é relativamente mais velha que as demais populações analisadas. O ancestral comum de todas as três populações analisadas está associado com a mancha foliar do maracujazeiro (Passiflora edulis), em Belém, e tem cerca de 0,8 unidades evolucionárias coalescentes de idade. Nenhum haplótipo da região ITS-5.8S do AG 2-2 Hb, de Belém, foi observado em outras regiões. Entretanto, a população de Manaus compartilhou dois, de seus quatro haplótipos, com aqueles observados em Xapuri / Rio Branco, no Acre, indicando fluxo gênico e deriva genética. / Thanatephorus leaf spot is one of the most important diseases of rubber tree (Hevea brasiliensis) in the Amazon region, Brazil. However, there is fill information available about the biological, pathogenic and genetic diversity of the pathogen. An important question about the actual phylogenetic placement of this pathogen is not answered yet. In this study, we analyzed sequences of the ITS-5.8S rDNA region from a population of T. cucumeris (anamorphase = Rhizoctonia solani AG 2-2) associated to the rubber tree leaf spot obtained in Belém (PA), Manaus (AM) and Xapuri/Rio Branco (AC). This population was also phylogenetically compared with members of AG 2 world-widely described. This study represents an important step to reveal the origin, the patterns of movement and amplification of epidemiologically important genotypes of rubber tree-infecting T. cucumeris. Phylogenetically, through both Bayesiana and maximum parsimony analyses, we found support to nominate a new group of anastomosis associated with the rubber tree foliar spot: the AG 2-2 Hb. This group consisted of a independent evolutionary unit in relation to the world-wide sub-groups of AG 2-2 analyzed. In the gene genealogy built by coalescent analysis, was observed that the population of R. solani AG 2-2 Hb of Belém is relatively older than the other populations analyzed. The oldest most recent common ancestor of all the three populations analyzed was associated with a sample obtained from passion-fruit (Passiflora edulis) leaf blight in Belém and has about 0.8 coalescent evolutionary units of age. No AG 2- 2 Hb ITS-5.8S rDNA haplotype from Belém was observed in any other regions. However, the population from Manaus shared two, of its four haplotypes, with those observed in Xapuri/Rio Branco (Acre), indicating both gene flow and genetic drift.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-11-28T12:59:54Z No. of bitstreams: 1 Dyana de Albuquerque Tenorio.pdf: 1359840 bytes, checksum: 2d9414b172e6c51ef868267286f6ef2b (MD5) / Made available in DSpace on 2016-11-28T12:59:54Z (GMT). No. of bitstreams: 1 Dyana de Albuquerque Tenorio.pdf: 1359840 bytes, checksum: 2d9414b172e6c51ef868267286f6ef2b (MD5) Previous issue date: 2015-02-25 / The damping-off and stem rot caused by the soil pathogen Rhizoctonia solani, is considered one of the most important diseases of common bean (Phaseolus vulgaris) and cowpea (Vigna unguiculata), being one of the factors that contribute to the low productivity of these crops. This study aimed to characterize isolates of the causal agent of damping-off and stem rotof beans in the rural area of Pernambuco State, using microsatellite markers and phylogenetic analysis; and evaluate the use of yeasts in the control of the causal agent of damping-off and stem rot of beans. In bean growing areas of the southern region of Pernambuco, there were collections of plants with symptoms of damping-off and stem rot. Thirty-four fungal isolates were identified as Rhizoctonia. In the phylogenetic analysis, the sequences of ITS rDNA region of isolates were similar to at least three anastomosis groups (AG4 HG-I, AG3-PT and AG4 HG-III). In microsatellite analysis, five sets of primers (TC_AG3_1, TC01, TC06, TC11 and TC_AG3_09) amplified a total of 13 bands, showing genetic variation. Seventy R. solani were evaluated for aggressiveness, being the isolate CMM-3643 selected to the biological control trials. Seventy yeast isolates were obtained from apparently asymptomatic bean plants. From these, were chosen only isolates where the disease severity index was ≤ 25%. Experimental tests showed that the three yeast isolates (C6A, FVF10 (R1), FVC10) were selected as promising biocontrol agents of damping-off and stem rot of cowpea (cv. IPA-207), being efficient in controlling the disease caused by isolated CMM -3643. Increases in the levels of POX and CAT enzymes by treating the seeds with FVF10 yeast (R1), as well as, the absence of antibiosis mechanisms suggests that the biocontrol mechanism in question is the induction of disease resistance. In view of the results it is concluded that the AG4 HG-I is the primary anastomosis group causing damping-off and stem rot of beans in the São João municipality, Pernambuco State. In addition, this is the first report of damping-off and stem rot symptoms reduction in cowpea seedlings due to the antagonistic action of yeasts against R. solani. / A rizoctoniose, causada pelo fungo de solo Rhizoctonia solani, é considerada uma das doenças mais importantes do feijoeiro-comum (Phaseolus vulgaris) e do feijoeiro-caupi (Vigna unguiculata), sendo um dos fatores responsáveis pela baixa produtividade da cultura. O presente trabalho teve como objetivos: caracterizar isolados do agente causal da rizoctoniose do feijoeiro na região do agreste de Pernambuco, utilizando marcadores microssatélites e análise filogenética; e avaliar o uso de leveduras no controle do agente causal da rizoctoniose do feijoeiro. Em áreas de cultivo do feijoeiro da região do agreste meridional de Pernambuco, realizaram-se coletas de plantas com sintomas da rizoctoniose. Sendo obtidos 34 isolados fúngicos identificados como Rhizoctonia. Na análise filogenética, as sequências gênicas da região ITS do rDNA destes isolados foram similares a pelo menos três grupos de anastomose (AG4 HG-I, AG3-PT e AG4 HG-III). Na análise de microssatélites, cinco conjuntos de iniciadores (TC_AG3_1, TC01, TC06, TC_AG3_09 e TC11) amplificaram um total de 13 bandas, observando-se variação genética. Setenta isolados de R. solani foram avaliados quanto a agressividade, sendo selecionado o isolado CMM-3643 para o ensaio de controle biológico. Foram obtidos 70 isolados de leveduras de plantas de feijoeiro aparentemente assintomáticas, destes foram escolhidos os que proporcionaram índice de severidade da doença ≤ 25%. Ensaios experimentais demonstraram que os três isolados de leveduras (C6A, FVF10 (R1), FVC10) selecionados foram promissores como agentes de biocontrole da rizoctoniose do feijoeiro-caupi (cv. IPA-207), apresentando eficiência no controle da doença causada pelo isolado CMM-3643. Aumentos nos níveis das enzimas POX e CAT pelo tratamento das sementes com a levedura FVF10 (R1), assim como a ausência de mecanismos de antibiose, sugere que o mecanismo de biocontrole em questão é a indução de resistência da planta. Com base nas avaliações realizadas concluiu-se que o AG4 HG-I é o principal grupo de anastomose da rizoctoniose do feijoeiro no município de São João. Além disso, este é o primeiro relato da redução dos sintomas da rizoctoniose em plântulas de feijoeiro-caupi pela ação antagônica de leveduras sobre R. solani.

Memoria para optar al título profesional de: Ingeniero Agrónomo Mención: Sanidad Vegetal

Allium sativum and Tulbaghia violacea are some of the most important medicinal plants used by South African traditional healers for the treatment of flu, fever, cold, tuberculosis, asthma and many more diseases. However, growth, yield and quality are constrained by excessive and under fertilization. This study was carried out to determine, firstly, the effect of N source (ammonium sulphate and calcium nitrate) on yield and quality of A. sativum and T. violacea plants. Secondly, to determine the best season for harvesting T. violacea and lastly, to determine the antifungal effects of A. sativum and T. violacea plant extracts against plant pathogens Altenaria solani and Sclerotium rolfsii. Both plants were treated with both N sources applied as topdressing treatments at a total of 0, 50, 100, 150 and 200 kg.ha-1, divided into three applications at three week (A. sativum) and three month (T. violacea) intervals. A. sativum plants were sampled at 54, 82, 112, 140 and 175 days after planting (DAP) while, T. violacea plants were sampled monthly for ten months. Parameters recorded were growth analysis, yield and bioactivity for both plant species. Both nitrogen sources improved plant growth and yield of A. sativum and T. violacea plants. Calcium nitrate at 150 kg•ha-1 and ammonium sulphate at 200 kg•ha-1 produced the highest at 24 t•ha-1 and 27 t•ha-1, respectively. Ammonium sulphate improved bioactivity of leaves with the highest bioactivity recorded at 82 and 112 DAP. Yield obtained from the autumn harvest was not affected by N source. Ammonium sulphate and calcium nitrate at 200 kg•ha-1 produced the highest yields of 23.6 t•ha-1 and 23.5 t•ha-1, respectively. In contrast, yield obtained from the winter harvest was affected by N source at 200 kg•ha-1, with significantly better yield of 30.8 t•ha-1 with calcium nitrate compared to 27.4 t•ha-1 with ammonium sulphate. Crude extracts of T. violacea bulbs that were treated with ammonium sulphate significantly inhibited the growth of plant pathogenic fungi, whereas extracts from plants treated with calcium nitrate showed low bioactivity. Extracts from plants grown with ammonium sulphate at 100 kg•ha-1 were more effective in controlling growth of plant pathogens when compared to other N levels. The minimum inhibitory concentration (MIC) effects of A. sativum against S. rolfsii and A. solani were at 0.01 mg•mL-1. The MIC of T. violacea extracts against A. solani was at 0.006 mg•mL<Sup>-1. The MIC of T. violacea extracts were better than previously reported in literature. Therefore, A. sativum and T. violacea plant extracts can be used as fungicides against S. rolfsii and A. solani diseases for crops such as tomato and potato. / Dissertation (MInstAgrar)--University of Pretoria, 2010. / Plant Production and Soil Science / unrestricted

Early blight (Alternaria solani Sorauer)is a major foliar disease of potatoes in most growing regions of the world and is underestimated in South Africa. This project studies the epidemiology and control of the disease in South Africa. A decision support system (DSS) for early blight in South Africa was developed and evaluated in field trials. This early blight DSS is the first such system to be developed in South Africa and once incorporated with the late blight model, will represent innovative technology for use in the South African potato industry. Trends in weather variables and concentrations of airborne conidia of A. solani were monitored. Distinct seasonal variation was noted. Peaks in spore concentration coincided with periods favourable for spore formation and dispersal; most notable was diurnal periodicity and interrupted wetting periods. The results obtained from these measurements will be useful in improving early blight DSSs for southern Africa. Isolates of A. solani from various potato-growing regions in South Africa were characterized using virulence, vegetative compatibility (VC) and random amplified microsatellite (RAMS) primers. Neither the virulence assays nor VC tests sufficiently characterised the population. Analysis of RAMS profiles revealed 27% genetic diversity among the isolates. This value is similar to diversity values obtained by previous authors studying A. solani, however, it is relatively high for an asexually reproducing fungus. There was no evidence for geographical clustering of isolates, indicating that isolates are widespread across South Africa. A survey on control practices and grower perceptions of early blight in South Africa was conducted using a questionnaire. These questionnaires were distributed to growers from 10 potato-growing regions in South Africa. Results highlighted the most popular control methods and cultivars in the South African potato industry. The majority of respondents indicated that they would use an accurate, cost-effective early blight DSS, and that more research is necessary on early blight in South Africa. A survey on control practices and grower perceptions of early blight in South Africa was conducted using a questionnaire. These questionnaires were distributed to growers from 10 potato-growing regions in South Africa. Results highlighted the most popular control methods and cultivars in the South African potato industry. The majority of respondents indicated that they would use an accurate, cost-effective early blight DSS, and that more research is necessary on early blight in South Africa. Estimated crop losses ranged from 1% - 60%, with an average of approximately 20%. This is the first comprehensive epidemiological study to be conducted on early blight in South Africa and has highlighted the need for further research. / Thesis (DPhil (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Taxol (generic name paclitaxel), a plant‐derived antineoplastic agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. Obtaining taxol from this source requires destruction of trees. It has been used alone or in combination with other chemotherapeutic agents for the treatment of breast, ovarian as well as many other types of cancer, including non‐small cell lung carcinoma, prostate, head and neck cancer, and lymphoma, as well as AIDSrelated Kaposi’s sarcoma. The mode of action of taxol against a number of human cancer cells is by preventing the depolymerization of tubulin during cell division. This molecule increases microtubule stability in the cell and induces apoptosis. From yew trees, the yield of taxol is usually between 0.004 to 0.1% of the dry weight. The commercial isolation of 1 Kg of taxol requires about 6 to 7 tons of T. brevifolia bark obtained from 2000‐3000 well‐grown trees. The limited supply of the drug has prompted efforts to find alternative sources of taxol. Alternative methods for taxol production, such as chemical synthesis, tissue and cell cultures of the Taxus species are expensive and give low yields. A fermentation process involving any microorganism would be the most desirable means to lower the cost and increase availability. The first report on the isolation of taxol‐producing fungi from Taxus brevifolia appeared in 1993 (Stierle, et al., 1993). Several taxol‐producing fungi have been identified since, such as Taxomyces andreanae, Taxodium disticum, Tubercularia sp., Pestalotiopsis microspora, Alternaria sp., Fusarium maire and Periconia sp (Li, et al., 1996, Strobel, et al., 1996a, Strobel, et al., 1996b, Li, et al., 1998b, Ji, et al., 2006, Xu, et al., 2006). This thesis investigates the isolation of an endophytic fungus, isolated from the stem cuttings of Taxus celebica, which produces taxol and related taxanes. We observed morphological and cultural characteristics and analyzed the sequences of rDNA ITS from the strain. The isolated fungus grew on potato carrot agar (PCA) medium at 25 °C and the colonies were white to off‐white, floccose, with irregular margins. The reverse side of the culture was cream in color. The morphology was examined microscopically following staining with cotton blue in lactophenol. Cultures produced macroconidia on slender, 85 μm long phialides. The macroconidia were 25‐40 X 3.75 μm. Cultures also produced round or oval microconidia. Analysis of the ITS and D1/D2 26S rDNA sequence revealed 99 % identity with Fusarium solani voucher NJM 0271. Based on its morphological, cultural characteristics and 26S rDNA sequence, the fungus was identified as F. solani. This fungus is different from the previously reported endophytic taxol‐producing species of Fusarium. Taxol and baccatin III, produced by this fungus, were identified by chromatographic and spectroscopic comparison with standard compounds. The amount of taxol produced by F. solani in potato dextrose liquid medium is low (1.6 μg l‐1) (Chakravarthi, et al., 2008). We further investigated different growth media and various factors of cultivation to select the medium and conditions that maximize production of taxol and other taxanes by this fungus. F. solani was grown in five well‐defined culture media under stationary and shake conditions separately for various time intervals and the amounts of taxol, baccatin III and other taxanes produced were estimated by competitive immunoassay. The modified flask basal medium (MFBM) was shown to yield the highest production of taxol (128 μg l‐1) which is 80 times more than when grown in potato dextrose liquid medium, baccatin III (136 μg l‐1) and total taxanes (350 μg l‐1) under shake conditions. From our results the highest taxol production of F. solani was achieved when cultured in MFBM. The production in MFBM was 80 times higher than that cultured in the potato dextrose liquid medium. In conclusion, it was shown that the culture medium plays a major role in taxol and other taxanes production and fungal growth. MFBM is the best medium, among the media studied, to produce taxol and other taxanes. The higher concentrations of NH4NO3, MgSO4, KH2PO4 and FeCl3 in the FBM medium seem important for production of taxol and other taxanes. These results can be considered as starting‐point for the research directed to improve taxol and baccatin III production by F. solani via different approaches including fermentations, strain improvement and genetic engineering techniques. Finally, in order to get more insights into the mode of action of this fungal taxol and baccatin III (for the first time), their apoptotic activity on different cancer cell lines was determined. We elucidated the biochemical pathways leading to apoptotic cell death after fungal taxol‐ and baccatin III‐ treatment in different cancer cell lines. Experiments are done on various cancer cell lines namely JR4 Jurkat (T‐cell leukemia), J16 Bcl‐2 Jurkat T cells, HepG2 (hepatoma), caspase‐8‐deficient Jurkat T cells, HeLa (human cervical carcinoma), Ovcar3 (human ovarian carcinoma) and T47D (human breast carcinoma) cells. We were able to demonstrate that both fungal taxol and baccatin III can induce apoptosis in all the cell lines tested, by flow cytometric analysis. Hallmarks of apoptosis following the signaling pathway to far more upstream‐located events were investigated using biochemical and cell biological methods. It has shown that during fungal taxol‐ and baccatin III‐induced apoptosis, DNA is degraded resulting in a increased number of hypodiploid cells reaching up to 65‐70% after 48 h. Disruption of mitochondrial membrane potential was examined by flow cytometric analysis using mitochondrial membrane potential sensitive dye JC‐1 and JR4‐Jurkat cells were shown to undergo significant loss of mitochondrial membrane potential loss of mitochondrial membrane potential reaching up to 70% in 6 nM fungal taxol and 65 % in 3.5 μM baccatin III after 36 h. These results were similar to those observed with standard taxol and baccatin III. We further investigated the role of caspases in fungal taxol‐ and baccatin III‐induced apoptosis, caspase‐8‐deficient Jurkat cells, Bcl‐2‐over‐expressed J16‐Jurkat cells and caspase inhibitors were used. Results derived from caspase‐8‐deficient Jurkat cells show that caspase‐8 is not involved in fungal taxol‐ and baccatin IIIinduced apoptosis of Jurkat cells. Using the pan‐caspase inhibitor (Z‐VAD‐FMK), caspase‐9 inhibitor (Z‐LEHD‐FMK), caspase‐3‐inhibitor (Z‐DEVD‐FMK), caspase‐2‐ inhibitor (Z‐VDVAD‐FMK) and caspase 10‐inhibitor (Z‐AEVD‐FMK), it was shown that caspase‐10 is involved in fungal taxol‐ and baccatin III‐ induced apoptosis in JR4‐Jurkat cells. It was also shown that inhibitors of caspases‐9, ‐2 or ‐3 partially inhibited fungal taxol‐ and baccatin III‐ induced apoptosis, whereas the caspase‐ 10 inhibitor totally abrogated this process. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in fungal taxol‐ and baccatin III‐treated JR4‐Jurkat and HeLa cells. DNA fragmentations were shown by agarose gel electrophoresis method. Our work showed that treatment of JR4‐ Jurkat and HepG2 cells with fungal taxol and baccatin III induces apoptosis as shown by DNA ladder formation. Herein it was demonstrated that fungal taxol and baccatin III have a similar mechanism of action, but the efficacy of fungal taxol to induce apoptosis is higher. In summary, fungal baccatin III is found to be effective in inducing apoptosis similar to taxol but at higher concentration and both fungal taxol and baccatin III induce apoptosis via caspase‐10 and mitochondrial pathway in Jurkat cells. In conclusion, the present study describes isolation of a taxol‐producing endophyte F. solani IISc.CJB‐1. The growth requirements of this fungus for production of taxol, baccatin III and other taxanes were studied. The apoptotic activity of taxol and baccatin III (for the first time) was observed. In addition, our results show that the culture medium plays a major role in taxol and other taxanes production and fungal growth. Among the media studied, modified flask basal medium (MFBM) is the best to produce taxol and other taxanes. It is evident from this data that this fungal strain can be promising candidate for large‐scale production of taxol and related taxanes.

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