两株海藻内生真菌次生代谢产物及其生物活性研究

海洋微生物拥有丰富多样的次生代谢途径，其中海洋生物内生真菌次生代谢产物研究日益受到天然产物化学界的重视。本论文以菌丝体生物量、发酵产物重量、抗菌与细胞毒活性、薄层色谱分析结果以及高效液相色谱分析结果等为评价依据对采自青岛沿海的13株海藻内生真菌在四种液体培养基上的静置发酵产物进行了综合评价，并从中选择了黑曲霉Aspergillus niger EN-13（分离自褐藻囊藻Colpomenia sinuosa）和杂色曲霉A. versicolor EN-7（分离自褐藻鼠尾藻Sargassum thunbergii）两株真菌进行了30升规模发酵（分别采用GPYM培养基和PDB培养）和化学成分的研究，对分离得到的大部分化合物进行了初步的生物活性筛选。 发酵提取物采用常规的硅胶柱层析、反相硅胶柱层析，凝胶Sephadex LH-20柱层析、制备薄层层析、半制备高效液相色谱以及重结晶等分离手段，得到单体化合物。利用各种现代波谱技术(IR、UV、EI-MS、FAB-MS、HR-ESI-MS、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、HMBC等)并结合化学方法从两种菌株发酵提取物中鉴定了55个化合物的结构。其中从菌株A. niger EN-13分离鉴定了31个化合物，发现9个新化合物，包括2个鞘酯类化合物（AN-1~2）、3个萘并-γ-吡喃酮类化合物（AN-3~5）、3个苯乙基取代的α-吡喃酮类化合物（AN-17, AN-19~20）和1个甾体Diels-Alder加成产物（AN-21），另有1个新的天然环二肽（AN-27）被分离鉴定；从菌株A. versicolor EN-7分离鉴定了24个化合物，发现2个新化合物，为蒽醌AV-12与AV-17，另外，从前一菌株（A. niger EN-13）中鉴定的2个新鞘酯类化合物（AN-1~2）在A. versicolor EN-7中也被再次分离到。 对大部分单体化合物进行了抗菌活性、DPPH自由基清除活性和细胞毒活性测试。结果显示新化合物AN-1、AN-5和AN-20具有弱或中等强度的抑制白色念珠菌生长的活性，AN-4、AN-5、AN-21显示了弱或中等强度的抑制黑曲霉生长的活性，AV-12、AV-17显示了弱的抑制大肠杆菌生长的活性。在DPPH自由基清除活性筛选中，AN-5显示了中等强度的活性，其EC50为109.3 mM，与阳性对照BHT相近（EC50为81.8 mM）。其它部分已知化合物在抗菌和DPPH自由基清除活性的筛选中也显示了弱或中等强度的活性。在针对人肝癌细胞株SMMC-7721和人肺腺癌细胞株A549的体外细胞毒活性筛选中，所测样品均未显示显著活性。

矿物的微生物风化地球化学研究－－以磷矿石和方解石为例

微生物与矿物间的相互作用是自然界中广泛发生的一种地质作用，微生物直接参与了自然界的物质循环，微生物对矿物风化作用的过程和机理与矿物种类、菌种及环境条件密切相关。目前还缺乏对矿物风化微观过程及风化过程中微生物生理生化特征的研究资料，随着分子生物学理论与技术的快速发展，用地球化学结合微生物学与分子生物学的理论和方法来综合研究微生物的矿物风化过程已成为可能。从分子水平和微观作用过程研究其风化过程与机理，理解微生物与矿物界面之间的相互作用及其反馈机制，是表生作用过程中生物地球化学的重要研究内容。 选用胶质芽孢杆菌（Bacillus mucilaginosus）、黑曲霉（Aspergillus niger）和青霉（Penicillium sp.），以磷矿石和方解石为例，探讨实验条件下微生物对矿物的风化作用过程与机理。微生物对矿物风化作用具有能耗低、污染小和流程短等特点，充分利用这些特点是解决土壤中无效磷的有效利用、磷矿资源的开发以及减少环境污染等问题的有效途径，对矿产资源的可持续利用和建立健康的生态环境具有十分重要的意义。采用离子色谱、ICP-OES、XRD、TEM、EDS、2-DE、电子探针和MC-ICP-MS等分析测试手段，研究微生物对矿物的风化过程与机理，得到如下结果： (1) 在黑曲霉对磷矿石风化过程中，由黑曲霉生长引发的生物机械破坏作用和生物化学降解作用是对磷矿石风化的主要趋动力，包括菌丝生长对矿物的穿插作用以及机械剥蚀作用；此外，菌体及生物大分子形成生物膜覆盖在矿物表面形成易于发生生化降解的微环境，有机酸络合Ca生成次生矿物草酸钙促进了磷矿石的风化。磷矿石直接与菌体接触所发生的风化作用强于磷矿石装入透析袋中的间接风化作用。 (2) 在胶质芽孢杆菌对磷矿石的风化过程中，菌体及其胞外多糖类物质在矿粉表面形成生物膜、菌体生长、代谢产物及有机物的机械剥蚀破碎作用等是造成矿物风化的重要原因；磷矿石直接与菌体接触进行的风化作用强于磷矿石装入透析袋中的间接风化作用。 (3) 黑曲霉对磷矿石的风化作用强于胶质芽孢杆菌，两者对磷矿石进行风化时，生物物理风化作用是导致矿物风化的主要因素；胶质芽孢杆菌风化磷矿石的过程中没有草酸钙的产生。 (4) 胶质芽孢杆菌对磷矿石的风化过程中，菌体蛋白质的表达发生变化，蛋白质的量与质的变化与磷矿石的风化密切有关。磷矿石的加入导致菌体生理代谢途径改变以适应环境的变化，并分泌相关的蛋白质导致矿物的风化。 (5) 采用Sr同位素示踪研究黑曲霉与青霉对磷灰石和方解石混合物的风化差异。黑曲霉作用混合矿物的初期，培养液pH值相对较低，对其中方解石有风化作用；中后期由于pH值升高，对磷灰石有较强的风化作用,对磷灰石的风化是由黑曲霉产生的大分子有机物对磷灰石中Ca络合的结果。青霉与混合矿物发生相互作用时，在最初的很短时间内青霉对磷灰石有风化作用，但对方解石的风化作用却表现在整个风化作用时间段，相比而言，青霉对磷灰石的风化作用不明显。这种风化差异与真菌的生物学特性有关，并表现为微生物对矿物风化的选择性。 本项研究对进一步认识土壤植被生态系统中的磷循环规律和循环过程中微生物所起的作用，以及用微生物风化作用的方式促进磷矿资源开发利用具有重要理论和实际意义。

Crescimento de fungos na superfície de madeira de caixas do tipo 'K' usadas para hortaliças.

A madeira é o material mais utilizado para embalagem de hortaliças noBrasil, principalmente devido ao seu baixo custo e alta resistênciamecânica. O objetivo deste trabalho foi estimar a absorção e a perdaprogressiva de água de ripas de madeira de Pinus utilizadas namontagem de caixas do tipo "K" em três condições de umidade relativae determinar o crescimento de fungos em sua superfície. O experimentofoi conduzido no Laboratório de Pós-Colheita da Embrapa Hortaliças, emBrasília-DF, em 2003. Trinta ripas novas de madeira de Pinus (52 x 6 x0,6cm) foram pesadas individualmente, imersas em água durante 1h epesadas novamente para avaliar a absorção de água. Em outroexperimento, dez ripas foram incubadas ao acaso em cada uma das trêscâmaras úmidas (61%, 86% e 94% UR) mantidas a 25oC (±2oC). A perda progressiva de água foi avaliada por pesagens diárias das ripasindividualmente e o desenvolvimento de fungos na madeira foi avaliadocom uma escala de notas (0-3) durante oito dias. A madeira nova dePinus pode absorver até 38% de seu peso em água, e permanecerúmida durante vários dias de acordo com a condição dearmazenamento. A umidade relativa do ambiente afetou a taxa de perdade água diária da madeira, estimada em 4,7%, 2,5% e 1,0%respectivamente a 61% UR, 86% UR e 94% UR, e ao final de oito diasalcançou 37,5%, 19,9% e 7,9%, respectivamente. Os fungospredominantes foram Trichoderma harzianum e Rhizopus stolonifer, mastambém observou-se crescimento de Aspergillus sp. e Penicillium sp.

Tratamento quimico de grãos de sorgo úmidos visando o controle de fungos de armazenamento.

2000

Patologia de sementes: ilustração das estruturas dos principais fungos em soja.

Material necessário para o Blotter Test; Procedimentos; Alternaria sp.; Aspergillus sp.; Cercospora kikuchii; Cercospora sojina; Chaetomium sp.; Colletotrichum truncatum; Curvularia sp.; Diaporthe sp.; Fusarium sp.; Glomerella glycines; Helminthosporium sp.; Macrophomina phaseolina; Myrothecium sp.; Nematospora corylli; Penicillium sp.; Peronospora manshurica; Pestalotia sp.; Phomopsis sp.; Rhizoctonia solani; Rosellinea sp.; Septoria glycines; Trichothecium roseum.

Patologia e tratamento de sementes: noções gerais.

A importância da patologia de sementes reside no fato de que aproximadamente 90% das culturas utilizadas para a alimentação são propagadas por semente. Dentre essas, nove são consideradas de importância primordial: soja, trigo, arroz, milho, feijão, amendoim, sorgo, cevada e beterraba açucareira.Todas essas culturas podem ser afetadas por patógenos muito agressivos transmitidos através da semente. Assim, o teste de sanidade de semente pode ser considerado como "medicina preventiva", tanto nos programas de quarentena quanto no sistema de produção de semente certificada. Nesta publicação, em sua primeira parte, são abordados os principais aspectos da patologia de sementes, como os seus históricos no mundo e no Brasil, os diferentes métodos utilizados e os fatores que podem causar variação nos resultados dos testes. Em sua segunda parte, são discutidos, em detalhe, os principais patógenos causadores de doenças na cultura da soja que são transmitidos pela semente. Dentre esses, destacam-se, Phomopsis sp. e Fusarium semitectum, causadores de problemas de germinação no laboratório quando ocorrem chuvas durante as fases de maturação e colheita da semente (podridão de semente); Diaporthe phaseolorum f.sp. meridionalis (Phomopsis meridionalis) (cancro da haste); Colletotrichum truncatum (antracnose); Cercospora kikuchii (mancha púrpura); Cercospora sojina (mancha olho-de-rã); Sclerotinia sclerotiorum (podridão branca); Sclerotium rolfsii (tombamento e morte de plantas); Macrophomina phaseolina (podridão de carvão); Rhizoctonia solani (tombamento) e Aspergillus spp. (A. flavus) que, além de ser considerado fungo de armazenagem, é responsável pela podridão da semente no solo, quando a semeadura é feita em solos com baixa disponibilidade de água, sem o tratamento da semente com fungicida. Finalmente é discutida a importância do tratamento de semente de soja com fungicidas, cuja tecnologia, desde a safra 2001/02, vem sendo utilizada em mais de 93% da área semeada com soja no Brasil.

Identificação dos principais fungos das sementes de trigo.

Procedimento para identificação dos fungos das sementes de trigo; Descrição diagnostica dos principais fungos das sementes de trigo; Sclerotium Tode; Rhizoctonia DC; Chaetomium Kunze; Pleospora Rabenh; Sporobolomyces Kluy. & Niel; Rhodotorula Harrison; Phoma Sacc.; Septoria tritici Rob; Stagonospora nodorum (Berk.) Cas. & Germ.; Stagonospora avenae (Frank) Bisset f. sp. triticae; Colletotrichum graminicola (Ces.) Wilson; Fusarium tricinctum (Corda) Sacc; Fusarium moniliforme Sheldon; Fusarium avenaceum (Fr.) Sacc; Fusarium acuminatum Ell. & Kellerm; Fusarium equiseti (Corda) Sacc.; Fusarium graminearum Schw.; Mucor Micheli; Rhizopus Ehrenb; Aspergillus Link.; Penicillium Link.; Alternaria Nees; Epicoccum Link; Cladosporium Link; Nigrospora Zimm; Curvularia Boedijn; Drechslera tritici-repentis (Died.) Drech; Bipolaris sorokiniana (Sacc. in Sorok.) Shoem; Chave sistemática dos principais fungos de sementes de trigo; Ilustrações dos principais fungos encontrados em sementes de trigo.

Doenças de soja: diagnose, epidemiologia e controle.

Página modelo; Simbologia empregada; Doenças causadas por fungos; Míldio da soja (Peronospora manshurica); Oídio da soja (Microsphaera diffusa); Ferrugem asiática (Phakopsora pachyrhizi); Mancha parda da folha (Septoria glycines); Mancha alvo (Corynespora cassiicola); Mancha olho-de-rã (Cercospora sojina); Mancha púrpura (Cercospora kikuchi); Seca da haste e da vagem (Phomopsis spp.); Antracnose (Colletotrichum truncatum); Cancro da haste (Phomopsis phaseoli f. sp. meridionalis); Podridão parda da haste (Phialophora gregata); Podridão vermelha da raiz (Fusarium solani); Mofo branco da haste (Sclerotinia sclerotiorum); Murcha de esclerotium (Sclerotium rolfsii); Podridão da raiz e da haste (Phytophthora megasperma f. sp. glycinea); Mela da folha (Rhizoctonia solani); Tombamento (Rhizoctonia solani); Morte em reboleira (Rhizoctonia solani); Roseliniose (Dematophora necatrix); Podridão negra da raiz (Macrophomina phaseolina); Doenças causadas por nematóides; Nematóide de cisto (Heterodera glycines); Nematóide de galha (Meloidogyne incognita); Doenças causadas por vírus; Mosaico comum da soja; Queima do broto; Doenças causadas por bactérias; Pústula bacteriana (Xanthomonas axonopodis pv. glycines); Fogo selvagem (Pseudomonas syringae pv. tabaci); Crestamento bacteriano (Pseudomonas savastonoi pv. glycinea); Microorganismos que frequentemente causam a morte das sementes a campo; Aspergillus spp.; Penicillium spp.; Bacillus subtilis; Créditos fotográficos; Estádios vegetativos da planta de soja; Estádios reprodutivos da planta de soja.

Identificação dos principais fungos das sementes de trigo.

Procedimento para identificação dos fungos das sementes de trigo; Descrição diagnostica dos principais fungos das sementes de trigo; Sclerotium Tode; Rhizoctonia DC; Chaetomium Kunze; Pleospora Rabenh; Sporobolomyces Kluy. & Niel; Rhodotorula Harrison; Phoma Sacc.; Septoria tritici Rob; Stagonospora nodorum (Berk.) Cas. & Germ.; Stagonospora avenae (Frank) Bisset f. sp. triticae; Colletotrichum graminicola (Ces.) Wilson; Fusarium tricinctum (Corda) Sacc; Fusarium moniliforme Sheldon; Fusarium avenaceum (Fr.) Sacc; Fusarium acuminatum Ell. & Kellerm; Fusarium equiseti (Corda) Sacc.; Fusarium graminearum Schw.; Mucor Micheli; Rhizopus Ehrenb; Aspergillus Link.; Penicillium Link.; Alternaria Nees; Epicoccum Link; Cladosporium Link; Nigrospora Zimm; Curvularia Boedijn; Drechslera tritici-repentis (Died.) Drech; Bipolaris sorokiniana (Sacc. in Sorok.) Shoem; Chave sistemática dos principais fungos de sementes de trigo; Ilustrações dos principais fungos encontrados em sementes de trigo.

Doenças de soja: diagnose, epidemiologia e controle.

Mildio da soja (Peronospera manshurica); Oidio da soja (Microsphaera diffusa); Mancha parda da folha (Septoria glycines); Mancha alvo (Corynespora cassiicola); Mancha de alternaria (Alternaria spp.); Mancha olho-de-rã (Cercospora sojina); Mancha purpura (Cercospora kikuchii); Seca da haste e da vagem (Phomopsis spp.); Antracnose (Colletotrichum truncatum); Cancro da haste (Phomopsis phaseoli f. sp. meridionalis); Podridão parda da haste (Phialophora gregata); Podridão vermelha da raiz (Fusarium solani); Mofo branco da haste (Sclerotinia sclerotiorum); Murcha de esclerotium (Sclerotium rolfsii); Podridão da raiz e da haste (Phytophthora megasperma f. sp. glycinea); Mela da folha (Rhizoctonia solani); Tombamento (Rhizoctonia solani); Morte em reboleira (Rhizoctonia solani); Roseliniose (Dematophora necatrix); Podridão negra da raiz (Macrophomina phaseolina); Nematoide de cisto (Heterodera glycines); Nematoide de galha (Meloidogyne incognita); Mosaico comum da soja; Queima do broto; Pustula bacteriana (Xanthomonas campestris pv. glycines); Fogo selvagem (Pseudomonas syringae pv. tabaci); Crestamento bacteriano (Pseudomonas syringae pv. glycinea); Aspergillus spp.; Penicillium spp.; Bacillus subtilis; Créditos fotográficos; Estádios vegetativos da planta de soja; Estádios produtivos da planta de soja.

Biobased additives as biodegradability enhancers with application in TPU-based footwear components

Among the wide variety of materials employed in the manufacture of shoes, thermoplastic polyurethanes (TPUs) are one of the most widely used. Given its widespread use, and associated waste management problems, the development of more biodegradable and evironmentally compatible solutions is needed. In this work, a polyester-based TPU used in the footwear industry for outsoles production was modified by compounding with lignin, starch and cellulose at content of 4% (w/w). The biodegradability was evaluated by using agar plate tests with the fungi Aspergillus niger ATCC16404, the Gram-negative bacteria Pseudomonas aeruginosa ATCC9027 and an association of both (consortium), and soil tests at 37 °C and 58 °C. The obtained results evidenced a positive effect of the tested biobased additives, the most favourable results being registered with lignin. These results were corroborated by the structural modifications observed by FTIR analysis. Additionally, mechanical tests prove the suitability of using the lignin modified TPUs for footwear outsoles production.

“Green preservatives” – combating fungi in the food industry by applying antifungal lactic acid bacteria

Fungal spoilage is the most common type of microbial spoilage in food leading to significant economical and health problems throughout the world. Fermentation by lactic acid bacteria (LAB) is one of the oldest and most economical methods of producing and preserving food. Thus, LAB can be seen as an interesting tool in the development of novel bio-preservatives for food industry. The overall objective of this study was to demonstrate, that LAB can be used as a natural way to improve the shelf-life and safety of a wide range of food products. In the first part of the thesis, 116 LAB isolates were screened for their antifungal activity against four Aspergillus and Penicillium spp. commonly found in food. Approximately 83% of them showed antifungal activity, but only 1% showed a broad range antifungal activity against all tested fungi. The second approach was to apply LAB antifungal strains in production of food products with extended shelf-life. L. reuteri R29 strain was identified as having strong antifungal activity in vitro, as well as in sourdough bread against Aspergillus niger, Fusarium culmorum and Penicillium expansum. The ability of the strain to produce bread of good quality was also determined using standard baking tests. Another strain, L. amylovorus DSM19280, was also identified as having strong antifungal activity in vitro and in vivo. The strain was used as an adjunct culture in a Cheddar cheese model system and demonstrated the inhibition of P. expansum. Significantly, its presence had no detectable negative impact on cheese quality as determined by analysis of moisture, salt, pH, and primary and secondary proteolysis. L. brevis PS1 a further strain identified during the screening as very antifungal, showed activity in vitro against common Fusarium spp. and was used in the production of a novel functional wortbased alcohol-free beverage. Challenge tests performed with F. culmorum confirmed the effectiveness of the antifungal strain in vivo. The shelf-life of the beverage was extended significantly when compared to not inoculated wort sample. A range of antifungal compounds were identified for the 4 LAB strains, namely L. reuteri ee1p, L. reuteri R29, L. brevis PS1 and L. amylovorous DSM20531. The identification of the compounds was based on liquid chromatography interfaced to the mass spectrometer and PDA detector

Food preservation using antifungal lactic acid bacteria

Fungal spoilage of food and feed prevails as a major problem for the food industry. The use antifungal-producing lactic acid bacteria (LAB) may represent a safer, natural alternative to the use of chemical preservatives in foods. A large scale screen was undertaken to identify a variety of LAB with antifungal properties from plant, animal and human sources. A total of 6,720 LAB colonies were isolated and screened for antifungal activity against the indicator Penicillium expansum. 94 broad-spectrum producers were identified through 16S rRNA sequencing with the majority of the population comprising Lactobacillus plantarum isolates. Six broad-spectrum isolates were consequently characterised. Pedicococcus pentosaceous 54 displayed potent anti-mould capabilities in pear, plum and grape models and may represent an ideal candidate for use in the beverage industry. Two antifungal Lb. plantarum isolates were assessed for their technological robustness and potential as biopreservatives in refrigerated foods. Lb. plantarum 16 and 62 displayed high levels of tolerance to freeze-drying, low temperature exposure and high salt concentrations. Both lactobacilli were introduced as supplements into orange juice to retard the growth of the spoilage yeast Rhodotorula mucilaginosa. Furthermore the isolates were applied as adjuncts in yoghurt production to successfully reduce yeast growth. Lb. plantarum 16 proved to be the optimal inhibitor of yeast growth in both food matrices. To date there is limited information available describing the mechanisms behind fungal inhibition by LAB. The effects of concentrated cell-free supernatant (cCFS), derived from Lb. plantarum 16, on the growth of two food-associated moulds was assessed microscopically. cCFS completely inhibited spore, germ tube and hyphal development. A transcriptomic approach was undertaken to determine the impact of antifungal activity on Aspergillus fumigatus Af293. A variety of genes, most notably those involved in cellular metabolism, were found to have their transcription modulated in response to cCFS which is indicative of global cellular shutdown. This study provides the first insights into the molecular targets of antifungal compounds produced by LAB. The genome sequence of the steep water isolate Lb. plantarum 16 was determined. The complete genome of Lb. plantarum16 consists of a single circular chromosome of 3,044,738 base pairs with an average G+C content of 44.74 % in addition to eight plasmids. The genome represents the smallest of this species to date while harbouring the largest plasmid complement. Some features of particular interest include the presence of two prophages, an interrupted plantaricin cluster and a chromosomal and plasmid encoded polysaccharide cluster. The sequence presented here provides a suitable platform for future studies elucidating the mechanisms governing antifungal production.

Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET).

BACKGROUND: Invasive fungal infections (IFIs) are a major cause of morbidity and mortality among organ transplant recipients. Multicenter prospective surveillance data to determine disease burden and secular trends are lacking. METHODS: The Transplant-Associated Infection Surveillance Network (TRANSNET) is a consortium of 23 US transplant centers, including 15 that contributed to the organ transplant recipient dataset. We prospectively identified IFIs among organ transplant recipients from March, 2001 through March, 2006 at these sites. To explore trends, we calculated the 12-month cumulative incidence among 9 sequential cohorts. RESULTS: During the surveillance period, 1208 IFIs were identified among 1063 organ transplant recipients. The most common IFIs were invasive candidiasis (53%), invasive aspergillosis (19%), cryptococcosis (8%), non-Aspergillus molds (8%), endemic fungi (5%), and zygomycosis (2%). Median time to onset of candidiasis, aspergillosis, and cryptococcosis was 103, 184, and 575 days, respectively. Among a cohort of 16,808 patients who underwent transplantation between March 2001 and September 2005 and were followed through March 2006, a total of 729 IFIs were reported among 633 persons. One-year cumulative incidences of the first IFI were 11.6%, 8.6%, 4.7%, 4.0%, 3.4%, and 1.3% for small bowel, lung, liver, heart, pancreas, and kidney transplant recipients, respectively. One-year incidence was highest for invasive candidiasis (1.95%) and aspergillosis (0.65%). Trend analysis showed a slight increase in cumulative incidence from 2002 to 2005. CONCLUSIONS: We detected a slight increase in IFIs during the surveillance period. These data provide important insights into the timing and incidence of IFIs among organ transplant recipients, which can help to focus effective prevention and treatment strategies.

Presencia de una microflora patógena en lotes comerciales de Syngonium podophyllum

p.145-148