Potencial do flavedo (epicarpo) de Citrus aurantifolia cv. Tahiti no controle do bolor verde e da antracnose em citros

O Brasil é considerado o maior produtor de citros e o maior exportador de suco de laranja. Doenças de pós-colheita representam uma grande perda para a citricultura, sendo que para a exportação de frutos são rígidas as exigências com relação a isenção de resíduos químicos nos mesmos. Patógenos de importância em pós-colheita de citros incluem o Penicillium digitatum, agente causal do bolor-verde e o Colletotrichum gloeosporioides, agente causal da antracnose. Dada a importância econômica que representam estas doenças dos frutos cítricos, tanto em termos de comprometimento da qualidade e dificuldade de controle, a busca de alternativas adicionais que possam viabilizar a capacidade produtiva e garantir a obtenção de frutos com excelentes padrões de qualidade torna-se imprescindível. Portanto, estudou-se os efeitos dos extratos aquosos do flavedo de Citrus aurantifolia var. Tahiti, Lentinula edodes, Agaricus subrufescens (syn. Agaricus brasiliensis), albedo de Citrus sinensis var. Valência e do ácido jasmônico no controle póscolheita do bolor verde e da antracnose e na indução de resistência em frutos de laranjeira Valência (Citrus sinensis). Foi possível observar que o extrato aquoso do flavedo (C. aurantifolia) apresentou efeito inibitório sobre os patógenos, quando tratados em pós-colheita, em função da redução dos sintomas e esporulação. Porém, os extratos de albedo (C. sinensis), L. edodes, A. subrufescens e o ácido jasmônico não apresentaram efeitos sobre P. digitatum e C. gloeosporioides.

Elicitors of induced disease resistance in postharvest horticultural crops: a brief review

Increasing loss of conventional fungicides due to pathogen resistance and general unacceptability in terms of public and environmental risk have favoured the introduction of integrated pest management (IPM) programmes. Induction of natural disease resistance (NDR) in harvested horticultural crops using physical, biological and/or chemical elicitors has received increasing attention over recent years, it being considered a preferred strategy for disease management. This article reviews the enhancement of constitutive and inducible antifungal compounds and suppression of postharvest diseases through using elicitors. The effect of timing of pre- and/or postharvest elicitor treatment and environment on the degree of elicitation and the potential for inducing local acquired resistance, systemic acquired resistance and/or induced systemic resistance to reduce postharvest disease is discussed. The review highlights that more applied and basic research is required to understand the role that induced NDR can play in achieving practical suppression of postharvest diseases as part of an IPM approach. (C) 2003 Elsevier B.V. All rights reserved.

Utilization and optimization of a waste stream cellulose culture medium for pigment production by Penicillium spp

Aims This research sought to determine optimal corn waste stream–based fermentation medium C and N sources and incubation time to maximize pigment production by an indigenous Indonesian Penicillium spp., as well as to assess pigment pH stability. Methods and Results A Penicillium spp. was isolated from Indonesian soil, identified as Penicillium resticulosum, and used to test the effects of carbon and nitrogen type and concentrations, medium pH, incubation period and furfural on biomass and pigment yield (PY) in a waste corncob hydrolysate basal medium. Maximum red PY (497·03 ± 55·13 mg l−1) was obtained with a 21 : 1 C : N ratio, pH 5·5–6·0; yeast extract-, NH4NO3-, NaNO3-, MgSO4·7H2O-, xylose- or carboxymethylcellulose (CMC)-supplemented medium and 12 days (25°C, 60–70% relative humidity, dark) incubation. C source, C, N and furfural concentration, medium pH and incubation period all influenced biomass and PY. Pigment was pH 2–9 stable. Conclusions Penicillium resticulosum demonstrated microbial pH-stable-pigment production potential using a xylose or CMC and N source, supplemented waste stream cellulose culture medium. Significance and Impact of the Study Corn derived, waste stream cellulose can be used as a culture medium for fungal pigment production. Such application provides a process for agricultural waste stream resource reuse for production of compounds in increasing demand.

A gene encoding a new cold-active lipase from an Antarctic isolate of Penicillium expansum

Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12–C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5′- and 3′-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.

Characterisation, cloning and production of industrially interesting enzymes: gluconolactone oxidase of Penicillium cyaneo-fulvum and gluconate 5-dehydrogenase of Gluconobacter suboxydans

The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated. The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. 1.1.3.4) of A. niger was demonstrated. The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli. An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose). In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested. The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.

捷安肽素对采后柑桔青绿霉菌抑制效果及作用机理研究

捷安肽素是一种由枯草芽孢杆菌（Bacillus subtilis）ZK 产生的抗真菌多肽。本文以柑桔青霉菌（Penicillium italicum）和绿霉菌（Penicillium digitaum）为供试真菌，研究了捷安肽素的抑菌性能及作用机理，为捷安肽素开发为有效的生物杀菌剂提供理论依据。全文共分两部分：第一部分：捷安肽素对柑桔青霉菌和绿霉菌抑制效果研究。采用琼脂扩散法测定捷安肽素对柑桔青霉菌和绿霉菌的抑菌活性。53.9 µg/mL 捷安肽素对绿霉菌和青霉菌的抑菌圈直径分别为26.7mm 和24.1mm。结果表明捷安肽素能够抑制柑桔青绿霉菌的生长，柑桔绿霉菌比青霉菌对捷安肽素敏感。在柑桔果实上，研究了不同浓度、不同接入时间的捷安肽素对柑桔青霉病和绿霉病的防治效果，并与常用化学杀菌剂抑霉唑、咪鲜胺、甲基硫菌灵和多菌灵作比较。53.9 µg/mL捷安肽素处理柑桔果实，柑桔青霉病和绿霉病发病率分别为5.0 %和5.3 %,比对照低95.0 %和94.7 %；柑桔青霉病和绿霉病的病情指数分别为1.87 和2.18，比对照低73.73 和97.82。结果表明，捷安肽素能够有效地防治柑桔青绿霉病。与对照相比，捷安肽素先于或后于柑桔青绿霉菌接入时，对柑桔青绿霉菌均有抑制作用，但抑制效果随接入间隔时间的增长而降低。第二部分：捷安肽素对绿霉菌作用机理研究。首先在光学显微镜和透射电镜下观察捷安肽素处理后绿霉菌菌丝表面形态结构与菌丝体内超微结构的变化。形态观察发现，捷安肽素处理24h以内，绿霉菌菌丝结构无变化。捷安肽素作用36h后，绿霉菌菌丝不规则缢缩和膨大。48h后，在绿霉菌菌丝顶端、中部、末端的多处细胞均可发生畸形的球状结构，这种畸变结构随处理的延长而增加，致使细胞成为捻珠状。处理72 h后，畸变球形细胞开始断裂离解。处理96h后，镜下几乎无完整菌丝，成单个的球状细胞，部分细胞出现破裂。而对照菌丝表面光滑，结构完整。通过透射电镜观察发现，与对照相比，捷安肽素处理后，绿霉菌细胞壁、细胞膜轮廓模糊不清，细胞质外泄。推测捷安肽素能够使绿霉菌细胞膜通透性发生改变。进一步实验利用紫外-可见分光光度计检测捷安肽素作用后绿霉菌胞外液紫外吸光度的变化，表明捷安肽素作用于绿霉菌菌丝后，细胞内蛋白质、核酸缓慢泄漏。通过Atomscan Advantage单道扫描等离子体发射光谱仪(ICP)测定捷安肽素作用后菌丝体内K+浓度的改变，结果表明捷安肽素作用于柑桔绿霉菌1h内，菌丝体内K+含量迅速下降，为对照绿霉菌K+含量的37.53 %，1 h后菌丝体内K+含量变化趋于平缓。K+的迅速泄漏，以及蛋白质、核酸的泄漏表明捷安肽素通过迅速改变绿霉菌细胞膜通透性，使绿霉菌菌丝生长受到抑制。Jiean-peptide produced by Bacillus subtilis ZK has broad-spectrumresistance to plant pathogens. In this study, we investigated the antifungal propertyand the possible antifungal mechanism of jiean-peptide against two commonphytopathogenic fungi of citrus fruits: blue molds (P. italicum) and green molds (P.digitatum).The paper involved two parts:Part 1 is the study of the antifungal property of jiean-peptide against blue moldsand green molds of citrus fruits. The in vitro inhibition effect of jiean-peptide againstblue molds and green molds was detected by agar diffusion method. The diameters ofinhibition zones of green molds and blue molds are 26.7mm and 24.1mm respectivelyby treating with 53.9 µg/mL jiean-peptide. It shows that jiean-peptide effectivelyinhibits the both phytopathogenic fungi, and it is more effective for inhibiting greenmolds than blue molds. The effectiveness of jiean-peptde to inhibit green molds andblue molds in vivo was investigated compared with four conventional fungicides thatare imazalil, prochloraz, carbendazin and methylthiophanate. The result is that the incidences of the blue mold disease and green mold disease are 5.0 % and 5.3 %, thedisease severities are 1.87 and 2.18 respectively when citrus are inoculated with 53.9µg/ml jiean-peptide. The decay incidences and disease severities were significantlyreduced by treating with jiean-peptide compared with the control. The results indicateJiean-peptide is effective for controlling blue molds and green molds on citrus. Theoptimized inoculation time was also investigated. When inoculated with jiean-peptideat 0 h, 6 h, 12 h, 24 h and 48 h before or after pathogens’ inoculation, Jiean-peptidecan suppress the occurrence of blue molds and green molds compared with the control, but the effect of later inoculation decreases compared with the inoculation at the sametime.In Part 2, we investigated the possible antifungal mechanism against greenmolds of citrus. At first, we observed the exterior morphological changes andultrastructural changes of blue molds under light microscopy (LM) and transmissionelectron microscopy (TEM). Compared with untreated control cells which aregenerally uniform in shape, the appearances of treated hyphae change obviously. Itshows that some cells of hyphae irregularly shrink or enlarge when cultured for 36h.When the treating time of jiean-peptide increases, the aberrance of the hyphaebecomes more obvious, and hyphae exhibit the moniliform appearances. Finally, thereis no intact hypha leaved except only single cells, and some of which appear fractured.By transmission electron microscopy (TEM) observation, we find that the outline ofthe cell wall and the cell membrane of hyphae are blurry, and the cytoplasma oozesout. The observation result under LM and TEM suggests that jiean-peptide mightchange the permeability of the cell membrane. So we conducted further experiment todetect the change of permeability when the cells of blue molds were treated withjiean-peptide. And the effect of jiean-peptide on non-growing cells of blue molds wastested. By the spectrophotometer measurement, we found that compounds with lightabsorption at 260 nm and 280 nm were released and amounts increased within 12 hcompared with the control. Moreover, by the ICP measurement, the leakage of K+occurred immediately in the presence of jiean-peptide within 1 h, but with nearly nofurther change after 1 h. All these results indicate that jiean-peptide could change themembrane permeability of blue molds immediately and result in leaking nucleotides,proteins and K+ from cells.

Hualyzin, a symmetrical urea derivative isolated from Penicillium herquei isolate GA4

Penicillium herquei isolate GA4 was isolated from the infected Conchocelis of Porphyra yezoensis. A large-scale fermentation using yeast extract sucrose medium and repeated chromatography afforded a new symmetrical urea derivative, hualyzin (1). The structure was determined by detailed NMR spectroscopic investigations and MS fragmentation analysis.

Chemical Constituents of a Marine-Derived Endophytic Fungus Penicillium commune G2M

Cultivation of the endophytic fungus Penicillium commune, which was isolated from the semi-mangrove plant Hibiscus tiliaceus, afforded one new compound 1-O-(2,4-dihydroxy-6-methylbenzoyl)-glycerol (1) along with thirteen known products, including 1-O-acetylglycerol (2), N-acetyltryptophan (3), 3-indolylacetic acid methyl ester (4), 1-(2,4-dihydroxy-3,5-dimethylphenyl)ethanone (5), 2-(2,5-dihydroxyphenyl)acetic acid (6), (4R,5S)-5-hydroxyhexan-4-olide (7), thymidine (8), uracil (9), thymine (10), ergosterol (11), beta-sitosterol (12), beta-daucosterol (13), and ergosta-7,22-dien-3 beta,5 alpha,6 beta-triol (14). The structures of these compounds were established by detailed NMR spectroscopic analysis, as well as by comparison with literature data or with authentic samples.

Comparison of two marine sponge-associated Penicillium strains DQ25 and SC10: differences in secondary metabolites and their bioactivities

Two strains of Penicillium, DQ25 and SC10, isolated from marine sponge Haliclona angulata (Bowerbank) and Hymeniacidon sp. respectively, were subjected to stationary cultivation under GYP medium for 30 days. The fermentation extracts were undergone bioactivities assays against human pathogens, phytopathogenic fungi and brine shrimp (Artemia salina). Bioassays-guided compounds isolation was performed by Silica gel columns and Sephadex LH-20 chromatography. Spectroscopic methods were used to structures elucidation of the compounds. Results showed the activities of secondary metabolites of strain DQ25 were generally stronger than that of strain SC10. Major bioactive molecules isolated from strain DQ25 were a 1,4-naphthoquinone derivative and an unidentified alkaloid. The two components were not isolated from the extract of strain SC10. ITS sequences revealed that these two species have the greatest similarity with Penicillium vinaceum and Penicillium granulatum respectively.

Engineering design of a bioprocess for the production of natural red colourants by submerged fermentation of the thermophilic fungus Penicillium purpurogenum GH2

The development of a new bioprocess requires several steps from initial concept to a practical and feasible application. Industrial applications of fungal pigments will depend on: (i) safety of consumption, (ii) stability of the pigments to the food processing conditions required by the products where they will be incorporated and (iii) high production yields so that production costs are reasonable. Of these requirements the first involves the highest research costs and the practical application of this type of processes may face several hurdles until final regulatory approval as a new food ingredient. Therefore, before going through expensive research to have them accepted as new products, the process potential should be assessed early on, and this brings forward pigment stability studies and process optimisation goals. Only ingredients that are usable in economically feasible conditions should progress to regulatory approval. This thesis covers these two aspects, stability and process optimisation, for a potential new ingredient; natural red colour, produced by microbial fermentation. The main goal was to design, optimise and scale-up the production process of red pigments by Penicillium purpurogenum GH2. The approach followed to reach this objective was first to establish that pigments produced by Penicillium purpurogenum GH2 are sufficiently stable under different processing conditions (thermal and non-thermal) that can be found in food and textile industries. Once defined that pigments were stable enough, the work progressed towards process optimisation, aiming for the highest productivity using submerged fermentation as production culture. Optimum production conditions defined at flask scale were used to scale up the pigment production process to a pilot reactor scale. Finally, the potential applications of the pigments were assessed. Based on this sequence of specific targets, the thesis was structured in six parts, containing a total of nine chapters. Engineering design of a bioprocess for the production of natural red colourants by submerged fermentation of the thermophilic fungus Penicillium purpurogenum GH2.