Rozprawy doktorskie na temat „Antifungal metabolites”

A study of the fungal ecology of Picea sitchensis stumps on mineral soils on first rotation sites in Scotland was carried out to determine fungal colonization, succession and the mechanisms of fungal interaction. Fungal and bacterial colonization of stump and buttress roots of stumps 0, 7, 28 days, 12, 16 and 48/53 months old was assessed. Melanotus proteus was found in all 12 month old stumps; Sistotrema brinkmanni was recorded most frequently in 16 and 48/53 month old stumps. A series of in vitro experiments was carried out to identify interactions occurring between pairs of P. sitchensis fungi on Norkrans agar, P. sitchensis sawdust, root blocks and billets, to determine possible modes of interaction occurring in vivo. Fungi exhibiting antagonism toward Heterobasidion annosum in vitro were noted to determine possible in vivo applications as curative/preventative biological controls against H. annosum. Sitka spruce stumps were highly receptive to H. annosum basidiospore infection up to 24 hours after felling and showed a significant level of receptivity 7 days after felling. M. proteus infection was lower in live stumps, compared to dead or moribund stumps, and was reduced or inhibited in stumps inoculated with Resinicium bicolor sawdust inoculum. In vitro experiments indicated that 5% urea prevented M. proteus basidiospore germination and hyphal growth. Treating stumps or billets with a 20% urea solution, however, had no significant effect on M. proteus colonization. Antifungal metabolites were detectable in 85% of the 25 fungal species tested representing members of the Basidiomycotina, Deuteromycotina and Ascomycotina, when bioassayed with Cladosporium cucumerinum. The production of antifungal metabolites in Sitka spruce stumps by H. annosum, R. bicolor, Stereum sanguinolentum, M. proteus and Hypholoma fasciculare was demonstrated.

Thesis (Ph. D.)--University of California, San Diego, 2007.
Title from first page of PDF file (viewed October 5, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Chemical and biological aspects of thiarubrine, a highly antifungal dithiacyclohexadiene polyine, were investigated. A tissue culture system for the production of thiarubrines was developed by culturing hairy roots of Chaenactis douglasii induced by Agrobacterium rhizogenes strain TR7. One culture line accumulated two times the levels of thiarubrines of nontransformed control root cultures, while maintaining rapid growth. The combination of fast growth and high thiarubrine accumulation could not be duplicated in controls by adding exogenous NAA to the culture medium. Hairy root cultures also produced less thiarubrine B relative to thiarubrine A compared to controls. Thiarubrine synthesis appears to be closely correlated with degree of tissue differentiation; it is suggested that it may be more practical to improve the growth rate of thiarubrine-producing root cultures by transformation rather than seek to induce synthesis in fast-growing suspension cultures. The biosynthetic relation between thiarubrines and the always co-occurring thiophenes was investigated by performing ³⁵S tracer experiments with C. douglasii hairy root cultures. It is possible that the thiophenes are not actively synthesized by the roots but rather are products of thiarubrine decomposition resulting from the extraction procedures and other manipulations of the cultures. The in vitro conversion of thiarubrine to thiophene can be induced by light, heat and other agents. No turnover of thiarubrines could be detected in the cultures in late logarithmic or stationary phases of the growth cycle. I Thiarubrines show strong light-independent antibacterial and antifungal activity. The mechanism of action of thiarubrine against E. coli and S. cerevisiae was investigated using comparative disk bioassays. A very similiar polyine from Rudbeckia hirta was as active as thiarubrine in the dark, indicating the central role of the disulfide ring in toxicity of the compounds. Visible light enhanced this activity suggesting that decomposition of the disulfide ring is important for its antibiotic effects. The photodegradation product, a thiophene, is phototoxic, probably via both type I and type II photosensitization mechanisms. The root culture extracts of Rudbeckia hirta yielded a new isomer of a known dithiacyclohexadiene polyine. MS and NMR analyses confirmed the cis configuration of this isomer.
Science, Faculty of
Botany, Department of
Graduate

The marine surface-associated bacterium Pseudoaltermonas tunicata, produces a range of compounds that inhibit fouling organisms, including invertebrate larvae, bacteria, algal spores and fungi. In addition to these antifouling compounds P. tunicata cells produce both a yellow and a purple pigment. The aim of this study was to further characterise the antifouling activities, their regulation and relationship with pigmentation, and the ecological significance of P. tunicata and related organisms. It was discovered that the anti-algal compound was extracellular, heat sensitive, polar and between 3 and 10 kDa in size. The anti-fungal compound was found to be the yellow pigment and active against a wide range of fungal and yeast isolates. Chemical analysis suggests that this compound consists of a carbon ring bound to a fatty-acid side chain. Genetic analysis supports the chemical data for the active compound as a mutant in a gene encoding for a long-chain fatty-acid CoA ligase was deficient for anti-fungal activity. To address the regulation of antifouling compounds and their relationship to pigmentation transposon mutagenesis of P. tunicata was performed. Mutants lacking the yellow pigment displayed a reduced ability to inhibit fouling organisms. Further analysis of these mutants identified genes involved with the synthesis and regulation of synthesis of pigment and antifouling compounds. One of these mutants was disrupted in a gene (wmpR) with similarity to the transcriptional regulators ToxR from Vibrio cholerae and CadC from Escherichia coli. Analysis of global protein expression using two-dimensional gel electrophoresis showed that WmpR is essential for the expression of at least fifteen proteins important for the synthesis of fouling inhibitors. The ecological significance of antifouling bacteria was addressed by assessing the antifouling capabilities of a collection of bacteria isolated from different marine surfaces. Overall, isolates from living surfaces displayed more antifouling traits then strains isolated from non-living surfaces. Five dark-pigmented strains originating from the alga Ulva lactuca were further studied. Phylogenetic and phenotypic analysis revealed that they were all members of the genus Pseudoalteromonas and were closely related to P. tunicata. Two strains represented a novel species within the genus and were taxonomically defined as P. ulvae sp. nov.

O uso de plantas na cura de enfermidades tem sido objeto de muitos estudos e desde épocas remotas está ligado ao desenvolvimento cultural de civilizações. Estudos anteriores em espécies de Myristicaceae descrevem a ocorrência da classe de lignóides como principal metabólito secundário. Variedades de estruturas químicas e de atividades biológicas são atribuídas aos lignóides. Estes metabólitos encontram-se acumulados em todas as partes da planta, principalmente nos frutos, onde os compostos predominantes são neolignanas. O presente trabalho foi realizado com Virola molissima que se encontra dispersa na Reserva Adolpho Ducke, situada nas proximidades de Manaus-AM. Os frutos foram coletados durante o mês de novembro, estação de seca na região. Não existe registro de estudo fitoquímico desta espécie. A partir de extratos de pericarpos, arilos, tegumentos e amêndoas dos frutos da Virola molissima foram isoladas por fracionamento cromatográfico as neolignanas tetrahidrofurânica, ariltetralônica e diarilbutânica. As neolignanas isoladas foram identificadas por comparação de seus dados de Ressonância Magnética Nuclear de Hidrogênio e de Carbono Treze, com aqueles descritos na literatura. A atividade antifúngica da neolignana ariltetralônica, pura ou em mistura, foi testada contra basidiomicetos Pycnoporus sanguineus, Trametes villosa e Lenzites trabeas. Estes fungos são xilófagos e causam o apodrecimento da madeira.
The use of plants in the treatment of diseases has been object of many studies, and since remote ages, the issue is linked to the cultural development of civilizations. Previous studies on Myristicaceous species described the occurrence of lignoids as its main secondary metabolites, which are known by varieties of chemical structures and biological activities. These lignoids are accumulated in all parts of the plant, mainly in the fruits, where the major constituent are neolignans. Present work was carried out on Virola molissima dispersed in Adolpho Ducke Reserve, located around Manaus, Amazon State, Brazil. Its fruits were collected during November, a month of dry season in this region. This species has not been phytochemically studied. Tetrahydrofuran, aryltetralone and dibenzylbutane neolignans were isolated from the extracts of pericarps, arils, seed coats and seeds of V. molissima fruits, by chromatographic fractionations. The structures of the isolated neolignans were elucidated through a Nuclear comparison between Magnetic Resonance of 1Hidrogen and 13Carbon data, and those described in the literature. The antifungal activity of the aryltetralone neolignan, pure or in mixture, was assayed against basidiomycetes Pycnoporus sanguineus, Trametes villosa and Lenzites trabeas. These fungi are xylophagus and they cause the decay of the wood.

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The aim of this study was to characterize the morphology, production, content, chemical composition and bioactive activity of essential oils of Lippia bromleyana, Lippia lasiocalycina, Lippia insignis and Lippia thymoides, endemic species of the Bahia semi-arid. The species were grown in the Experimental Station Horto Florestal State University of Feira de Santana (UEFS) in the city of Feira de Santana - Bahia. The essential oils are extracted from dry leaves and inflorescences, by hydrodistillation in Clevenger apparatus, and chemical composition was determined by GC/MS and GC/FID. Were carried out quantitative and qualitative morphological characterizations, in addition to agronomic characterization. The leaf anatomy, types and frequency of hair were observed in binocular microscope and electronic scanning light. The antioxidant activity was assessed by the ability of the substances present in the sample capture the free radical DPPH, using five concentrations of essential oils (2, 6, 10, 14 and 18 mg mL1) and the antifungal activity by mycelium growth in vitro testing five essential oil concentrations (0.25, 0.50, 0.75, 1.0 and 1.25 ?L mL-1). In conditions where the study was conducted, it can be concluded that there are morphological differences between species in all traits, except only the number of flowers per inflorescence; there agronomic differences for all traits with L. lasiocalycina stood out in relation to the variable oil yield, while L. insignis and L. thymoides regarding the essential oil content; were identified six types of glandular trichomes one, two and tetracelular and three types of trichomes; the species L. bromleyana presents as differential anatomical absence of trichomes on the abaxial surface; L. thymoides has glandular trichomes with irregular contours on both sides, distinguishing it from other species; the frequency of trichomes on the abaxial surface is higher in species L. insignis and L. lasiocalycina, which are more anatomically similar; the major compounds found in the samples of essential oils of L. bromleyana, L. lasiocalycina, L. insignis and L. thymoides were piperitone oxide and limonene; E-ocimenona, myrcenone, myrcene, ?-myrcene and ?-cymene; thymol, myrcenone and E-ocimenona; and ?-caryophyllene, germacrene D, respectively; L. insignis and L. bromleyana stood out in relation to the antioxidant and antifungal activity, respectively.
O objetivo desse trabalho foi caracterizar a morfologia e a produ??o, teor, composi??o qu?mica e atividade bioativa de ?leos essenciais de Lippia bromleyana, Lippia lasiocalycina, Lippia insignis e Lippia thymoides, esp?cies end?micas do semi?rido baiano. As esp?cies foram cultivadas na Unidade Experimental Horto Florestal da Universidade Estadual de Feira de Santana (UEFS), na cidade de Feira de Santana ? Bahia. Os ?leos essenciais foram extra?dos de folhas e infloresc?ncias secas, por meio da hidrodestila??o em aparelho de Clevenger e a composi??o qu?mica determinada por CG/EM e CG/DIC. Foram realizadas caracteriza??es morfol?gicas quantitativas e qualitativas, al?m da caracteriza??o agron?mica. A anatomia foliar, tipos e frequ?ncia de tricomas foram observados em microsc?pio de luz binocular e eletr?nico de varredura. A atividade antioxidante foi avaliada pela capacidade das subst?ncias presentes na amostra captarem o radical livre DPPH, utilizando cinco concentra??es dos ?leos essenciais (2, 6, 10, 14 e 18 mg mL-1) e a atividade antif?ngica pelo crescimento miceliano in vitro, testando cinco concentra??es do ?leo essencial (0,25, 0,50, 0,75, 1,0 e 1,25 ?L mL-1). Nas condi??es em que foi realizado o estudo, pode-se concluir que existem diferen?as morfol?gicas entre as esp?cies em todas as caracter?sticas avaliadas, com exce??o apenas para o n?mero de flores por infloresc?ncia; existem diferen?as agron?micas para todos os caracteres avaliados, sendo que L. lasiocalycina se destacou em rela??o ? vari?vel rendimento de ?leo, enquanto que L. insignis e L. thymoides em rela??o ao teor de ?leo essencial; foram identificados seis tipos de tricomas glandulares uni, bi e tetracelular e tr?s tipos de tricomas tectores; a esp?cie L. bromleyana apresenta como diferencial anat?mico aus?ncia de tricomas tectores na face abaxial; L. thymoides possui tricomas glandulares com contornos irregulares em ambas as faces, distinguindo-a das demais esp?cies; a frequ?ncia de tricomas tectores na face abaxial ? superior nas esp?cies L. insignis e L. lasiocalycina, as quais s?o mais semelhantes anatomicamente; os compostos majorit?rios encontrados nas amostras dos ?leos essenciais de L. bromleyana, L. lasiocalycina L. insignis e L. thymoides foram: ?xido de piperitona e limoneno; E-ocimenona, mircenona, mirceno, ?-mirceno e ?-cimeno; timol, mircenona e E-ocimenona; ?-cariofileno e germacreno D, respectivamente; L. insignis e L. bromleyana se destacaram em rela??o ? atividade antioxidante e antif?ngica, respectivamente.

O uso de bactérias promotoras de crescimento vegetal ou agentes de biocontrole como inoculantes agrícolas é uma alternativa importante e ecologicamente correta, com grandes benefícios na agricultura para substituir, ou ao menos suplementar, a excessiva utilização de fertilizantes e pesticidas. Neste trabalho avaliamos a capacidade de biocontrole e de competência rizosférica de três bactérias com características de promoção de crescimento vegetal (Plant growth promoting - PGP): Bacillus mycoides B38V, Paenibacillus riograndensis SBR5 e Burkholderia sp. 89. As três bactérias avaliadas apresentaram grande versatilidade na utilização de substratos, o que poderia lhes garantir uma vantagem competitiva no ambiente rizosférico. Porém, inconsistências foram observadas nos ensaios em câmara de crescimento, ou seja, as características de PGP e de biocontrole observadas in vitro não se refletiram em benefícios para a planta. A linhagem 89 destacou-se pela produção de um metabólito estável com ampla atividade contra fungos fitopatogênicos. Através de abordagens genômicas e de análises multilocus, descrevemos Burkholderia sp. 89 como uma nova espécie membro do complexo Burkholderia cepacia, denominada de B. catarinensis 89T. O sequenciamento de seu genoma, seguido de uma análise pela ferramenta AntiSMASH, revelou a presença de um agrupamento gênico de peptídeo sintetases não ribossomais (NRPS) relacionadas com a biossíntese do sideróforo ornibactina e um agrupamento híbrido NRPS-policetídeo sintetase responsável pela biossíntese do glicolipopeptideo cíclico com atividade antifúngica burkholdina. Como estratégia de purificação de metabólitos secundários foi utilizada a metodologia da mineração de genoma combinada com fracionamento guiado por bioensaios seguida de análises em espectrômetro de massas. Desta forma, purificamos com sucesso duas variantes de ornibactina, D e F (761 e 789 Da, respectivamente), e detectamos a variante ornibactina B (m/z= 733) e as moléculas sinalizadoras homoserina lactonas C6-HSL, 3OH-C8-HSL e C8-HSL. Análises de espectrometria de massas demonstraram a presença de um grupo de metabólitos com massas de 1240, 1254, 1268, 1216, 1244 e 1272 Da, que, provavelmente, são novas variantes do antifúngico burkoldina. Sendo assim, B. catarinensis 89T possui potencial biotecnológico com possíveis aplicações farmacêuticas e agronômicas para o biocontrole de fungos fitopatogênicos.
The use of plant growth promotion bacteria or biocontrol agents as agricultural inoculants is an important eco-friendly alternative to substitute, or at least supplement, the excessive use of fertilizers and pesticides. In this work, we evaluated the biocontrol potential and rhizosphere competence of three bacteria that had shown plant growth promotion (PGP) abilities: Bacillus mycoides B38V, Paenibacillus riograndensis SBR5 and Burkholderia sp. 89. All three bacteria presented great versatility in their substrate utilization, which could enable them to survive in a competitive rhizosphere environment. However, inconsistencies were observed in the greenhouse experiments, whereas their interesting abilities observed in vitro did not result in benefits to the plants. Strain 89 produces a stable metabolite with a wide range of antifungal activity. Genomic comparisons and multilocus sequence analysis revealed Burkholderia sp. 89 as a new species of the Burkholderia cepacia complex and we described it as B. catarinensis 89T. We sequenced its genome and analyzed it with the AntiSMASH tool. This in silico prediction revealed the presence of a nonribosomal peptide synthetase (NRPS) cluster, which is related to the production of the siderophore ornibactin. Moreover, a hybrid NRPS- polyketide synthetase cluster for the production of the antifungal cyclic glicolipopeptide burkholdin was also found. A genome mining combined with a bioassay-guided fractionation with further mass spectrometry analysis was applied for the purification of these compounds. This approach enabled us to purify and characterize two variants of the siderophore ornibactin, D and F (761 and 789 Da, respectively). Also, we could detect the variant ornibactin B (m/z= 733) and the quorum sensing molecules homoserine lactones C6-HSL, 3OH-C8-HSL and C8-HSL in the supernatant of B. catarinensis 89T. Mass spectrometry analysis showed the presence of a group of metabolites with the masses 1240, 1254, 1268, 1216, 1244 and 1272 Da, which are probably new variants of the antifungal metabolite burkoldin. Therefore, B. catarinensis 89T has a great biotechnological potential for the production of metabolites with pharmaceutical and agricultural applications for the biocontrol of phytopathogenic fungi.

Phytoalexins and phytoanticipins are antimicrobial natural products involved in plant defence pathways against plant pathogens and other stresses. Most cruciferous phytoalexins are indole containing compounds with various side chains (dithiocarbamates, isothiocyanate, isonitrile, acetonitriles etc.). Many phytoanticipins of crucifers are glucosinolates and their metabolites, which have diverse structures and precursors, including aliphatic, phenyl or indolyl containing amino acids. Indole glucosinolates are derived from tryptophan, which is also a biosynthetic precursor to cruciferous phytoalexins, however the biosynthetic relationship between cruciferous phytoalexins and indole glucosinolates has not been clarified. In this work, investigation of antifungal metabolites from wild crucifers, synthesis of antifungal metabolites and potential perdeuterated biosynthetic precursors, biosynthesis of metabolites of salt cress and that of rutabaga will be described. Investigation of the wild crucifers Brassica tournefortii, Crambe abyssinica, Diplotaxis tenuifolia and Diplotaxis tenuisiliqua for production of elicited antifungal metabolites, resulted in the discovery of a new phytoalexin, 1ꞌ,4ꞌ-dimethoxyindolyl-3ꞌ-acetonitrile, from D. tenuisiliqua. 1ꞌ,4ꞌ-dimethoxyindolyl-3ꞌ-acetonitrile is the first dimethoxy substituted phytoalexin with strong antifungal activity against plant fungal pathogens. The remaining plant species produced known phytoalexins which were initially discovered in wild and cultivated species; all of them produced arvelexin. A novel phytoalexin isocyalexin A, was isolated from rutabaga roots irradiated with UV-light; this is the first isocyanide of plant origin. The second section of the thesis deals with the biosynthesis of metabolites of salt cress (T. salsuginea) and their biosynthetic relationships with indole glucosinolates. In that regard, non-isotopically labeled compounds and perdeuterated biosynthetic intermediates such as [2,2,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H6]glucobrassicin, [2H3CS;4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H4]-1ꞌ-methoxybrassinin, L-[2ꞌ,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H5]tryptophan, [2H3CO]-1ꞌ-methoxyindolyl-3ꞌ-acetaldoxime, L-[2H3CS]methionine, [4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H4]brassinin and 1ꞌ-methoxy-2ꞌ-methylbrassinin were administered to salt cress leaves. For the first time, the biosynthetic relationship between indole glucosinolates and cruciferous phytoalexins was established. Intact incorporations of hexadeuterated glucobrassicin ([2,2,4ꞌ,5ꞌ,6ꞌ,7ꞌ-2H6]glucobrassicin) into wasalexins A, B and biswasalexins A1 and A2 were observed. Based on the feeding experiment results, for the first time a biosynthetic route that includes both indole glucosinolates (glucobrassicin and 1ꞌ-methoxyglucobrassicin) and 1ꞌ-methoxybrassinin was proposed. The third section of the thesis is about biosynthesis of metabolites of rutabaga (Brassica napus). Rutabaga produces phytoalexins that differ on their side chains. Biosynthetic origin of their side chains was investigated by administering fully labeled tryptophan (L-[U-13C11,U-15N2]Trp) and other perdeuterated precursors to rutabaga roots which revealed that the carbon and nitrogen atoms of cyclobrassinin, rapalexin A, isocyalexin A and spirobrassinin are fully derived from tryptophan, and also both rapalexin A and isocyalexin A incorporated deuterium from glucobrassicin. [4',5',6',7'-2H4]-4'-Methoxybrassinin was incorporated into 4ꞌ-methoxycyclobrassinin and 4ꞌ-methoxydehydrocyclobrassinin but not into rapalexin A, isocyalexin A and isalexin. The biosynthetic pathway that leads to isalexin, rapalexin A and isocyalexin A was further investigated using perdeuterated biosynthetic precursors such as (R,S)-[2H3CO,5',6',7'-2H3]-4'-methoxyindolyl-3'-glycine, [2H3CO,5',6',7'-2H3]-4'-methoxyindole-3'-carboxaldehyde oxime, [2H3CO,5',6',7'-2H3]desulfoglucorapassicin and etc. It has been confirmed that the pathway involves series of rearrangements that allow transformation of side chain of tryptophan into the side chains of rapalexin A and isocyalexin A without any degradations. In conclusion, cruciferous phytoalexins are derived from glucobrassicin which is a precursor for 1ꞌ- and 4ꞌ-methoxyglucobrassicins. 1-Methoxylated phytoalexins are biosynthesized through 1ꞌ-methoxyglucobrassicin via 1ꞌ-methoxybrassinin. Similarly, 4-methoxy phytoalexins are derived from 4ꞌ-methoxyglucobrassicin through two distinct pathways: via 4ꞌ-methoxybrassinin and 4ꞌ-methoxyindolyl-3ꞌ-glycine.

碩士
國立中興大學
植物病理學系所
97
Actinomycetes are widely distributed gram-positive filamentous bacteria with varied morphology. The genus Streptomyces is the main group among actinomycetes. Many reports indicate that Streptomyces can protect crops from pathogens and promote plant growth with multiple biocontrol mechanisms. The characters of numerous seconday metabolites biosynthesis, including enzymes and antibiotics, have been in commercial use in medicine, animal feeder additives, insecticides, and plant protection. This study was aimed to screen for antagonistic actinomycete strains with potential application as a biological agent. A total of 138 actinomycetes isolated from different areas in Taiwan were screened for their antagonistic activity against plant pathogens. Ten antagonistic strains were selected and identified as Streptomyces based on morphology, physiological properties and 16S rRNA. Some of them can secrete chitinase and amylase. According to the inhibition spectrum of Streptomyces sp. strains against plant pathogens, A35, A272 and A463 strains are superior in antifungal activity；A337, A377 and A454 strains have antibiotics against bacteria. The onset of antibiotic biosynthesis is determined and influenced by a variety of physiological and environmental factors. The most important medium component for growth and secondary metabolite formation is the carbon source. Investigating the influence of carbon sources on growth and antibiotic activity of Streptomyces sp. on medium and in submerged culture. According to the results, utilization of carbon sources depends on species. Morphological differeation and degrees of antifungal activity of A272 strain are varied on ISP9 basal medium and medium contained 1% arabinose, fructose, glucose, inositol, lactose, mannitol, sucrose, and xylose. One promising strain, Streptomyces sp. A272 with strong antifungal activity was selected for the further studies. After investigating the influence of initial inoculum, pH, time course, and light on the growth and antibiotic activity in liquid culture, the results revealed that the optimum condition for growth and antibiotic activity was added 106 cfu/ml inoculum in pH 5 PDB medium, and the antibiotic activity would be detected after 6 days cultured. The thermostability of the antibiotic activity in crude culture supernatant was examined. Although the antifungal activity are slightly decreased after heat treatments, the antifungal activity did not be affected between different heat treatments. A preliminary trial on the disease control efficacy indicated that the infection of R. solani AG4 and C. higginsianum on Brassica rapa L. (Chinese Group) was greatly reduced by drenching and spraying application of the broth culture. 100X-diluted A272 broth culture increased more percentage of survival of 7-days-old seedlings in infested soil；however,10X-diluted A272 broth culture was more effective on controlling damping-off disease after seed sowed. The application of 100X-diluted A272 broth culture reduced more disease index percentage of anthracnose infection than 10X-diluted broth, and the broth is much more effective than filtrate on disease controlling anthracnose and damping-off in green house. After analysis of antifungal metabolites against R. solani AG4 and C. gloeosporioides of A272 strain, the results indicated that A272 strains produced multiple water-soluble antibiotics with broad spectrum of activity, contained α-D-glucopyranoside, nucleoside antibiotics, urea, IAA, quinoline, cholestane and organic acid. Thus, the antagonistic activity of A272 strain is considered that all of secondary metabolites join in a common effect. It could decrease the risk of resistance in pathogen.

Molecular genetic and enzymological techniques have been employed to study secondary metabolite biosynthesis. These investigations have focused on two projects: the cloning and heterologous expression of biosynthetic gene clusters from unculturable marine organisms and the characterization of individual enzymes involved in the biosynthesis of the antifungal agent blasticidin S. The marine environment is proving to be a valuable source of biologically active compounds, but problems associated with sustainable harvest, laboratory culture, and organic synthesis make obtaining sufficient quantities of compounds for drug development both difficult and expensive. A method has been developed for the isolation of biosynthetic gene clusters from complex marine microbe/invertebrate associations. Using this method a mixed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene cluster has been cloned from the marine sponge Jaspis splendens. The cloned gene cluster was found to code for a PKS with three extension modules and an NRPS with three extension modules. In addition, several open reading frames (ORFs) were identified that may be involved in the biosynthesis of the PKS starter molecule. Partial characterization of catalytic domains from the NRPS was also completed. The second project centers on the characterization of enzymes involved in blasticidin S (BS) biosynthesis. Two ORFs were identified in the BS gene cluster encoding gene products predicted to be involved in the early steps of BS biosynthesis. The blsG gene product has sequence similarity to lysine 2,3-aminomutase and is believed to be involved in the formation of the β-arginine moiety of BS. A series of heterologous expression studies were undertaken to determine the function of B1sG. The product of blsM exhibits sequence homology with several nucleosidetransferases. blsM was cloned from the BS gene cluster, heterologously expressed in E. coli, and shown to catalyze the formation of cytosine using cytidine 5'- monophosphate as the preferred substrate. Point mutations were introduced in blsM to generate three B1sM mutant enzymes: S92D, E98A, and E98D. All three mutants lost cytidine 5'-monophosphate hydrolysis activity. Surprisingly, the B1sM S92D mutant exhibits cytidine deaminase activity when incubated with cytidine or deoxycytidine, resulting in the formation of uridine and deoxyuridine, respectively.
Graduation date: 2005

碩士
國立東華大學
生命科學系
99
Secondary metabolites play a pivotal role in the antagonistic activities of some biocontrol species of Trichoderma resulting in the suppression of plant pathogens. T. koningii RIS 3-8, among other 22 strains, was the best Trichoderma spp. tested against phytopathogens, Fusarium solani, F. oxysporum, Rhizoctonia solani and Botrytis cinerea in vitro. Removing proteins by heat-treatment and protease-treatment showed proteins had no role in the antagonistic tests that lead to the secondary metabolites be the primary candidates. To discern the determinants of the T. koningii RIS 3-8 secondary metabolites, antifungal oriented assays were performed. The metabolites from the 9th day chloroform extract processed best antifungal activity against pathogens. Five bioactive compounds of T. koningii RIS 3-8 were isolated chromatographically: 1-hydroxy-3-methylanthracene-9,10-dione, methyl 4-hydroxybenzoate, methyl 4-hydroxycinnamate, 4-hydroxybenzaldehyde, 4-hydroxyacetophenone that all but 1-hydroxy-3-methylanthracene-9,10-dione were firstly isolated from T. koningii. Meanwhile, [Cn-im-3OEG][Cl], amphiphilic ionic liquids and ionic liquid crystals were tested their fungicial potency. [C14-im-3OEG][Cl] possesses the best antifungal activity against R. solani with an IC50 of 130 μM. Rupture of the bioenvelop appears to be operative in the process of antifungal activity.

Nos últimos anos, a procura por novos compostos para utilização pela indústria farmacêutica conduziu a um aumento dos estudos com espécies marinhas, uma vez que a sua aplicabilidade para fins terapêuticos é conhecida por parte de comunidades costeiras desde tempos remotos. O ambiente marinho tem evidenciado uma grande biodiversidade de espécies que demonstraram possuir a capacidade de sintetizar vários compostos com elevado potencial farmacológico. Assim, a indústria farmacêutica tem investido na extração, identificação e estudos de várias moléculas que constituem metabolitos de espécies marinhas, algumas das quais não podem ser encontradas em plantas terrestres As macroalgas foram, entre outras espécies, alvo desses estudos e provaram a sua importância como fonte de compostos bioativos. Dentro das macroalgas as Phaeophyceae, macroalgas castanhas, têm vindo a demonstrar ter metabolitos como por exemplo os polissacarídeos, os florotaninos, os carotenoides e os esteróis com grande interesse para a indústria farmacêutica. Os metabolitos das macroalgas castanhas possuem várias atividades farmacológicas, tais como atividade antioxidante, anti-tumoral, antifúngica, antivírica ou antibacteriana entre outras, o que pode constituir uma mais valia no desenvolvimento de novos fármacos. Atualmente, existem no mercado produtos, principalmente suplementos e produtos cosméticos, contendo extratos de macroalgas castanhas, mas é necessária mais pesquisa para se compreender na totalidade as propriedades e aplicabilidades de todos os compostos obtidos destes organismos marinhos.
In recent years, the demand for new compounds for use by the pharmaceutical industry has led to an increase in studies with marine species, since their applicability for therapeutic purposes has been known by coastal communities since remote times. The marine environment has shown great biodiversity of species that have demonstrated the ability to synthesize various compounds with high pharmacological potential. Thus, the pharmaceutical industry has invested in the extraction, identification, and studies of various molecules that constitute metabolites of marine species, some of which cannot be found in terrestrial plants. Macroalgae were, among other species, the target of these studies and proved their importance as a source of bioactive compounds. Within the macroalgae, Phaeophyceae, brown macroalgae, have been shown to have metabolites such as polysaccharides, phlorotannins, carotenoids, and sterols with a great interest for the pharmaceutical industry. The metabolites of brown macroalgae have several pharmacological activities, such as antioxidant, anti-tumor, antifungal, antiviral or antibacterial activity, among others, which can be an asset in the development of new drugs. Currently, there are products on the market, mainly supplements and cosmetic products, containing extracts of brown macroalgae, but more research is needed to fully understand the properties and applicability of all compounds obtained from these marine organisms.

Les endophytes sont des microorganismes (généralement des bactéries et des champignons) qui vivent dans les tissus végétaux mais n'activent pas le système immunitaire/défense des plantes, contrairement aux pathogènes végétaux qui activent généralement les réponses immunitaires des plantes. Des recherches récentes ont montré que pratiquement toutes les plantes cultivées en plein champ contiennent un certain nombre d'endophytes, et que certains endophytes stimulent la croissance des plantes et renforcent la résistance contre les agents pathogènes. Les endophytes sécrètent des composés chimiques (métabolites secondaires) qui suppriment la croissance des agents pathogènes, un processus connu sous le nom de biocontrôle. En raison de ces propriétés de biocontrôle, les endophytes sont une alternative potentielle aux pesticides chimiques pour lutter contre les maladies des plantes. En conséquence, le biocontrôle est devenu un domaine de recherche important. Mon projet de recherche comportait les objectifs spécifiques suivants : (i) isoler les endophytes des plants de canneberges acquis auprès de deux producteurs commerciaux de canneberges de la variété Stevens situés au Québec, Canada (Bieler Cranberries Inc, et Gillivert Inc.) ; (ii) tester l'activité de biocontrôle des endophytes contre une collection de champignons pathogènes et ensuite inoculer les endophytes les plus actifs dans des plants de canneberges obtenus par germination de la variété Stevens (Bieler Cranberries Inc. ) et Scarlet Knight (Daniele Landreville) ; et (iii) identifier des groupes de gènes de métabolites secondaires en séquençant, assemblant et annotant le génome d'un endophyte qui présentait de fortes caractéristiques de biocontrôle. Dans le cadre de ce projet de recherche, des tests antagonistes in vitro ont été réalisés avec des endophytes de la canneberge et un champignon pathogène, qui ont montré que Pseudomonas sp. CSWB3, Pseudomonas sp. CLWB12 et la souche fongique Lachnum sp. EFK28 étaient les plus actifs et ces souches ont donc été sélectionnées pour des études plus approfondies. Des expériences de germination de semis in vitro et d'inoculation d'endophytes ont montré que les souches bactériennes Pseudomonas sp. CSWB3 et Pseudomonas sp. CLWB12 amélioraient la croissance des semis de canneberges de la variété Stevens. Comme les Pseudomonas sp. CSWB3 et Pseudomonas sp. CLWB12 ont tous deux un effet antagoniste élevé sur les champignons pathogènes, un seul (Pseudomonas sp. CSWB3) a été soumis à une analyse du génome. Le séquençage, l'assemblage, l'annotation et l'analyse du génome de Pseudomonas sp. CSWB3 a révélé que cette souche possède cinq groupes de gènes biosynthétiques de métabolites secondaires qui codent pour les protéines responsables de la biosynthèse des composés antifongiques/antimicrobiens : pyrrolnitrine, pyoluteorine, putisolvine, 2,4-diacétylephloroglucinol, bicornutine A1 et bicornutine A2. Sur la base des résultats de ces travaux, nous concluons que certains endophytes de la canneberge qui possèdent des groupes de gènes codant pour des métabolites secondaires antifongiques peuvent supprimer les pathogènes fongiques et améliorer la croissance des plantes.
Endophytes are microorganisms (typically bacteria and fungi) that live within plant tissue but do not activate the plant defense/immune system, unlike plant pathogens that typically do activate plant immune responses. Recent research has shown that virtually all plants grown under field conditions contain a number of endophytes, and that certain endophytes stimulate plant growth and enhance resistance against pathogens. Endophytes secrete chemical compounds (secondary metabolites) that suppress pathogen growth, a process known as biocontrol. Because of these biocontrol properties, endophytes are a potential alternative to chemical pesticides for combatting plant disease. Accordingly, biocontrol has become an important field of research. My research project was comprised of the following specific aims: (i) isolate endophytes from cranberry plants that were acquired from two commercial producers of cranberries of the Stevens variety located in Quebec, Canada (Bieler Cranberries Inc, and Gillivert Inc.); (ii) test the biocontrol activity of endophytes against a collection of fungal pathogens and then inoculate the most active endophytes into cranberry seedlings that were obtained by germinating Stevens (Bieler Cranberries Inc.) and Scarlet Knight (Daniele Landreville) seeds; and (iii) identify secondary metabolite gene clusters by sequencing, assembling, and annotating the genome of one endophyte that exhibited strong biocontrol characteristics. As part of this research project, in vitro antagonistic tests were conducted with cranberry endophytes and fungal pathogen, which showed that Pseudomonas sp. CSWB3, Pseudomonas sp. CLWB12, and the fungal strain Lachnum sp. EFK28 were the most active and therefore these strains were selected for further studies. In vitro seedling germination and endophyte inoculation experiments showed that the bacterial strains Pseudomonas sp. CSWB3 and Pseudomonas sp. CLWB12 enhanced the growth of cranberry seedlings of the Stevens variety. Since Pseudomonas sp. CSWB3 and Pseudomonas sp. CLWB12 both had a high antagonistic effect on fungal pathogens, only one (Pseudomonas sp. CSWB3) was subjected to genome analysis. Sequencing, assembly, annotation, and analysis of the Pseudomonas sp. CSWB3 genome revealed that this strain possesses five secondary metabolite biosynthetic gene clusters that encode proteins responsible for the biosynthesis of the antifungal/antimicrobial compounds pyrrolnitrin, pyoluteorin, putisolvin, 2,4-diacetylephloroglucinol, bicornutin A1, and bicornutin A2. Based on the results of this work, we conclude that certain cranberry endophytes that possess gene clusters encoding antifungal secondary metabolites can suppress fungal pathogens and enhance plant growth.

博士
國立中興大學
植物病理學系
91
More than 200 strains of Actinomycetes were isolated from farmland soils and agriculture wastes in Taiwan. Among those, 98% of the strains were identified as Streptomyces spp. The other 2 % strains belonged to the genus Actinomadura, Herbidospora, Microbispora, and Streptosporangium. Thirty isolates of plant fungal pathogens and nine strains of plant bacterial pathogens were used to bioassay the antagonistic ability of Streptomyces sp. PMS-702 on potato dextrose agar plates. Acremonium diospyri, A. lactucum, Alternaria brassicicola, Athelia rolfsii (Sclerotium rolfsii), Botrytis cinerea, B. elliptica, Colletotrichum dematium, C. higginsianum, C. gloeosporioides, Fusarium oxysporum f. sp. cubense, F. oxysporum f. sp. conglutinans, F. oxysporum f. sp. lactucum, F. oxysporum f. sp. lilii, F. oxysporum f. sp. niveum, F. oxysporum f. sp. raphani, F. moniliforme, F. proliferatum, F. solani, Mycosphaerella pinodes, Phellinus noxius, Penicillium digitatum, Pestalotiopsis eriobotryfolia, Phytophthora capsici, Ph. citrophthora, Ph. infestans, Ph. palmivora, Ph. parasitica, Pythium aphanidermatum, P. myriotylum, and Rhizoctonia solani AG-4. were inhibited by PMS-702 with varying degrees. However, the PMS-702 was not able to inhibit Scleroium rolfsii and nine plant bacterial pathogens. The results of spore morphology, cell wall chemotype, cultural and physiological characterization and the molecular characteristics suggested that the strain PMS-702 is identified as Streptomyces padanus Baldacci, et al. Six media, Glucose-Molasses medium (GMM), Glucose-Soybean meal-Glycerol Broth (GSG), Modified Chitin Broth (MCB), Soybean meal-Fish meal-Chitin Broth (SFC), Soybean meal-Glucose Broth (SMG) and Tryptone-Yeast extract-Glucose Broth (TYG), were evaluated for culturing Streptomyces padanus PMS-702 in shaking flasks. Higher biomasses of S. padanus PMS-702 mycelia were harvested from GSG, GMM, and SMG. Among them, the SMG broth was much more suitable for growth of S. padanus PMS-702. The culture filtrate of PMS-702 grown in SMG for 7 days was able to completely inhibit spore germination of indicator fungus, Fusarium oxysporum f. sp. cubense. In the scale-up production, S. padanus PMS-702 was grown in a 5L fermentor containing 3 L of SMG for 4 days. Then whole plants, fruits or detached leaves were used to bioassay its suppressive ability. The culture broth of S. padanus PMS-702 was effective in controlling lettuce brown spot caused by Acremonium lactucum, mango anthracnose caused by Colletotrichum gloeosporioides, Chinese cabbage anthracnose caused by C. higginsianum, peach fruit rot caused by Phytophthora citrophthora, orange green mold caused by Penicillium digitatum, cabbage downy mildew caused by Peronospora brassicae. These results suggested that S. padanus PMS-702 is a potential agent for developing a bioprotectant for controlling plant fungal diseases. Streptomyces padanus strain PMS-702 was an antagonist of Rhizoctonia solani AG-4, the causal agent of damping-off of cabbage. Treatment of cabbage seeds with the culture filtrate of S. padanus strain PMS-702 was effective in reducing incidence of damping-off of cabbage. The major active ingredient from the culture filtrate of S. padanus strain PMS-702 was purified by silica gel column chromatography and identified as the polyene macrolide, fungichromin, by one dimentional (1H NMR, 13C NMR) and two dimentional (HMQC, HMBC) nuclear magnetic resonance and Mass (FAB-MS) spectral data. Bioassay studies showed that fungichromin had a strong antifungal activity against R. solani AG-4, and its minimum inhibitory concentration (over 90% inhibition) was found to be 72 g/ml. This is the first report of fungichromin from S. padanus as an active ingredient for the control of Rhizoctonia damping-off of cabbage. Streptomyces padanus strain PMS-702 was a potential biocontrol agent of tomato late blight, caused by Phytophthora infestans. Laboratory and field tests indicated that Streptomyces PMS-702 formulation was effective in reducing tomato late blight. Four compounds, sterol glycoside, daidzein, fungichromin and unknown compound 4 were obtained from culture filtrate of S. padanus PMS-702. Among them , fungichromin was completely effective in reducing sporangial sporulation and zoospores released from zoosporangia. The advanced experiments proved that broth culture filtrate and fungichromin were significantly effective in inhibiting sporangial sporulation, sporangial germination, zoospores released from zoosporangia and cytospore germination of Ph. infestans, respectively. These effects were negatively related with the dilution rate of culture filtrate and fungichromin. The phenomena under observations of light microscope and scanning electron microscope indicated that fungichromin was able to incite substantial plasma agglutination, cell malformation and cell collapse of the treated sporangia and zoospores of Ph. infestans. It did also cause zoospore membrane rupture and plasma leakage. The minimum inhibitory concentration of fungichromin against zoospore release (over 90% inhibition) was at 5 ppm. These results suggested that fungichromin played an important role in the mode of action of PMS-702 formulation for controlling tomato late blight.

Rhododendron tomentosum is an evergreen shrub with a high content of secondary metabolites, particularly essential oils with antimicrobial effects. Diversity of endophytic fungi in this species and their possible adaptation to growth in the essential oil environment is not much explored. Therefore, the first aim of this thesis was to reveal the diversity of endophytic fungi colonising leaves of R. tomentosum on seven localities in the Czech Republic and one in Estonia. I isolated and determined (using comparison of ITS1 and ITS2 rDNA with the sequences from GenBank and morphological signs) 37 species of endophytic fungi. Among them the ubiquitous species colonising the most of the plants as endophytes were dominant. The second aim of my thesis was to explore whether the essential oil from R. tomentosum influences its endophytic fungi. The hypothesis that the strains obtained from R. tomentosum would be adapted to growth in the environment of the essential oil was postulated. I supposed that they would grow better on mediums with different concentrations of these chemical compounds added, in comparison with strains of the same species obtained from different substrates. Within four of seven species tested, the strains obtained from R. tomentosum grew better, but also on the medium without the...