Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase

The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol-producing host plant, but Bclcc2 replacement mutants were as virulent as the wild-type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant-pathogen interactions and the phytoalexin concept.     

葡萄细胞悬浮培养生产白藜芦醇

以巨峰葡萄果皮为外植体,在添加2.0 mg/L 6-苄基嘌呤（6-BA）和0.1 mg/L 2,4-二氯苯氧基（2,4-D）的B5培养基上诱导葡萄愈伤组织; 以50 g/L的初始接种量在添加1.0 mg/L 6-BA和0.05 mg/L 2,4-D的B5液体培养基上建立葡萄悬浮培养体系。在25～27 ℃下,摇床振荡暗培养（120～130 r/min）18 d后,葡萄细胞生物量和白藜芦醇含量达到最大值（16.17 g/L、95.69 μg/g干质量）。在培养第12天时,向培养基中添加100 μmol/L茉莉酸甲酯（MeJA）,经过6 d处理,细胞中白藜芦醇含量达235.73 μg/g干质量。     

Putative mycotoxin-producing fungi isolated from alpataco (Prosopis flexuosa) fruits

Fungi contaminant of alpataco (Prosopis flexuosa) fruits from La Pampa province (Argentina) were identified. Alternaria alternata and Sphaeropsis sapinea were the dominant species. Phoma sp., Nigrospora sp., Preussia minima, Cladosporium sp., Pithomyces chartarum, Epicoccum nigrum, Aspergillus niger and Aspergillus speluneus were also isolated but with less frequency. Twelve strains of Alternaria alternata, the toxigenic species with higher incidence, were screened for alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production. Since one isolate was able to produce AME, six isolates produced AOH and AME and two isolates produced AOH, AME and TA, these results indicate a potential risk of contamination with Alternaria toxins in this substrate.     

Substrates and enzyme activities related to biotransformation of resveratrol from phenylalanine by Alternaria sp. MG1

To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO₄, and 0.2 g/L CaSO₄ and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 μg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously.     

Endophytic fungi in rose (Rosa hybrida) in Bogota, Colombia

We have investigated the presence of endophytic fungi associated with rose plants (Rosa hybrida) in Colombia. Endophytic fungi were isolated from healthy leaves of ten ornamental roses plants from gardens cultured in malt extract, peptone, yeast extract agar plates (MPY). We sampled 560 leaves fragments, 56 per sample. Endophytic fungi comprised 92 isolates (16.4%); of these isolates, 41 were classified as sterile mycelium (without reproductive structures that allowed their identification), 31 isolates were identified to genus or to species, and 20 isolates could not be identified at all. The identified endophytic fungi were as follow: Nigrospora oryzae, Aureobasidium spp, Acremonium spp. The fungi Nodulisporium sp, Gliocladium virens, Cladosporium sp, Alternaria sp, Phoma sp and Chaetomium globosum were represented by one isolate each. Since the endophytic fungi are known for their capacity to produce metabolites with biological activity, it is possible that the microorganisms found in this study have potential as antagonist of rose pathogens.     

Fungal endophytes from Acer ginnala Maxim: isolation, identification and their yield of gallic acid

Aims:  The aim of the study was to isolate the endophytic fungi from Acer ginnala and screen isolates rich in gallic acid.
Methods and Results:  After epiphytic sterilization, 145 fungal endophytes were isolated from the stem, annual twig and seed of Acer ginnala . The endophytes were grouped into ten different taxa, Phomopsis sp., Neurospora sp., Phoma sp., Epicoccum sp., Penicillium sp., Alternaria sp., Fusarium sp., Trichoderma sp., Cladosporium sp. and a species of Pleosporales Incertae Sedis , by their morphological traits and ITS-rDNA sequence analysis. The content and yield of gallic acid of 141 isolates were determined by HPLC. On average, the species of Pleosporales Incertae Sedis had the highest content and yield of gallic acid (13·28 mg g⁻¹ DW; 119·62 mg l⁻¹), while Alternaria sp. had the lowest.
Conclusions:  Of 141 fungal endophytes from A. ginnala , Phomopsis sp. isolate SX10 showed both the highest content and the highest yield of gallic acid (29·25 mg g⁻¹ DW; 200·47 mg l⁻¹).
Significance and Impact of the Study:  Endophytic fungi isolated from A. ginnala may be used as potential producers of gallic acid and other compounds with biological activities, or functioned as elicitors to produce natural compounds.     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

Detection of fungal infectous agent of wheat grains in store-pits of Markazi province, Iran

Wheat is an economic and important crop that provides approximately 20% of food calorie in the world. It is first crop in Iran and cultivated in the most areas of this country. Store-pit fungi make undesirable changes in quality and appearance of wheat grains. Even, some fungi produce different mycotoxins which are toxic to human and livestock's that use wheat grains as source of food. In this study, several samples were randomly collected from each of five store-pits located in different areas of Markazi Province including: Arak, Mahallat, Khomein, Saveh and Sarband. Grains were treated on PDA, and blotter, agar and washing test also used for isolating and detection of fungi. At least 100 grains per each sample were randomly used for each test and treatment. The fungi that determined in this study were Cochliobolus australiensis, Cladosporium herbarum, Epicoccum sp., Tilletia leavis, Aspergillus flavus, A. niger, A. fumigatus, Alternaria alternata, Alternaria sp., Penicillium italicum, P. digitatum, Fusarium sp., Rhizopus sp., Ustilago tritici, Scytalidium sp. Among these fungi the most isolates were belonged to Cladosporium, Alternaria, Rhizopus and Fusarium. Cladosporium herbarum was the most common in different sampling areas. Tilletia laevis and Ustilago tritici were just recovered in washing test. This study revealed that different fungi are associated with wheat grains in store-pits in Markazi Province. Some of them like Aspergillus flavus normally produce aflatoxin, a very toxic and carcinogenic mycotoxin that is harmful for human.     

Relationship between mitochondrial DNA Copy Number and SIRT1 Expression in Porcine Oocytes

The present study assessed the effect of resveratrol on the expression of SIRT1 and mitochondrial quality and quantity in porcine oocytes. Supplementing the maturation medium with 20 µM resveratrol increased the expression of SIRT1, and enhanced mitochondrial functions, as observed from the increased ATP content and mitochondrial membrane potential. Addition of resveratrol also improved the ability of oocytes to develop into the blastocyst stage following activation. The effects of resveratrol on mitochondrial number were examined by comparing the mitochondrial DNA copy number (Mt number) between group of oocytes collected from the same donor gilt ovaries. Supplementing the maturation medium with only resveratrol did not affect the Mt number in the oocytes. However, supplementing the maturation medium with 10 µM MG132, a proteasome inhibitor, significantly increased the amount of ubiquitinated proteins and Mt number by 12 and 14%, respectively. In addition, when resveratrol was added to the medium containing MG132, the Mt number increased significantly by 39%, this effect was diminished by the addition of the SIRT1 inhibitor EX527. Furthermore, supplementing the medium with MG132 and EX527 did not affect Mt number. The mean SIRT1 expression in 20 oocytes was significantly and positively correlated with the Mt number in oocytes collected from the same donor. This study suggests that the expression of SIRT1 is associated with the Mt number in oocytes. In addition, activation of SIRT1 by resveratrol enhances the biosynthesis and degradation of mitochondria in oocytes, thereby replenishing and improving mitochondrial function and the developmental ability of oocytes.     

Somatic embryos cultures of <Emphasis Type="Italic">Vitis amurensis</Emphasis> Rupr. in air-lift bioreactors for the production of biomass and resveratrol

Resveratrol are the most important bioactive compounds found in Vitis amurensis. In this study, a somatic embryo induction system for V. amurensis was established in air-lift bioreactors for the production of biomass and resveratrol. The somatic embryos biomass growth was low on solid medium (69.60 g L^?1) compared to in liquid medium in bioreactor (329.45 g L^?1). Bioreactor cultures were found to be superior compared with solid medium culture not only in terms of biomass but also resveratrol productivity. Various culture parameters, including culture method, inoculum density, carbon source, and organic compounds were optimized. An inoculum density of 20 g L^?1 embryogenic calli was found suitable for the accumulation of biomass and resveratrol production, whereas 10 g L^?1 embryogenic calli increased the amount of resveratrol per fresh weight in somatic embryos. For bioreactor culturing, sucrose was an optimum carbon source and 500 mg L^–1 casein hydrolysate acid was conducive to the biomass and resveratrol production. This result indicates that an efficient protocol for the large-scale production of resveratrol can be achieved by bioreactor culturing of V. amurensis somatic embryos and can be used as a source of medicinal raw materials.     

Fungal degradation of aflatoxin B1

A number of fungal cultures were screened to select an organism suitable to be used in the detoxification of aflatoxin B1. They were co-cultured in Czapek-Dox-Casamino acid medium with aflatoxin B1 producing Aspergillus flavus. Several fungal cultures were found to prevent synthesis of aflatoxin B1 in liquid culture medium. Among these Phoma sp., Mucor sp., Trichoderma harzianum, Trichoderma sp. 639, Rhizopus sp. 663, Rhizopus sp. 710, Rhizopus sp. 668, Alternaria sp. and some strains belonging to the Sporotrichum group (ADA IV B14(a), ADA SF VI BF (9), strain 720) could inhibit aflatoxin synthesis by > or =90%. A few fungi, namely ADA IV B1, ADA F1, ADA F8, also belonging to the Sporotrichum group, were less efficient than the Phoma sp. The Cladosporium sp. and A. terreus sp. were by far the least efficient, registering <10% inhibition. The cultures which prevent aflatoxin biosynthesis are also capable of degrading the preformed toxin. Among these, Phoma sp. was the most efficient destroying about 99% of aflatoxin B1. The cell free extract of Phoma sp. destroyed nearly 50 microg aflatoxin B1 100 ml(-1) culture medium (90% of the added toxin), and this was more effective than its own culture filtrate over 5 days incubation at 28+/-2 degrees C. The degradation was gradual: 35% at 24 h, 58% at 48 h, 65% at 72 h, 85% at 96 h and 90% at 120 h. The possibility of a heat stable enzymatic activity in the cell free extract of Phoma is proposed.     

Mixotrophic and photoautotrophic cultivation of 14 microalgae isolates from Saskatchewan, Canada: potential applications for wastewater remediation for biofuel production

Northern regions are generally viewed as unsuitable for microalgal biofuel production due to unfavorable climate and solar insolation levels. However, these conditions can potentially be mitigated by coupling microalgal cultivation to industrial processes such as wastewater treatment. In this study, we have examined the biomass and lipid productivity characteristics of 14 microalgae isolates (Chlorophyta) from the Canadian province of Saskatchewan. Under both photoautotrophic and mixotrophic cultivation, a distinct linear trend was observed between biomass and lipid productivities in the 14 SK isolates. The most productive strain under cultivation in TAP media was Scenedesmus sp.-AMDD which displayed rates of biomass and fatty acid productivities of 80 and 30.7?mg?L^?1?day^?1, respectively. The most productive strain in B3NV media was Chlamydomonas debaryana-AMLs1b which displayed rates of biomass and fatty acid productivities of 51.7 and 5.9?mg?L^?1?day^?1, respectively. In 11 of the isolates tested, secondary municipal wastewater (MCWW) supported rates of biomass productivity between 21 and 33?mg?L^?1?day^?1 with Scenedesmus sp.-AMDD being the most productive. Three strains, Chlamydomonas debaryana-AMB1, Chlorella sorokiniana-RBD8 and Micractinium sp.-RB1b, showed large increases in biomass productivity when cultivated mixotrophically in MCWW supplemented with glycerol. High relative oleic acid content was detected in 10 of the 14 isolates when grown mixotrophically in media supplemented with acetate. There was no detectable effect on the fatty acid profiles in cells cultivated mixotrophically in glycerol-supplemented MCWW. These data indicate that biomass and lipid productivities are boosted by mixotrophic cultivation. Exploiting this response in municipal wastewater is a promising strategy for the production of environmentally sustainable biofuels.     

Potato Cultivar, Pathogen Isolate and Antagonist Cultivation Medium Influence the Efficacy and Ranking of Bacterial Antagonists of Fusarium Dry Rot

The process of selecting biological control agents for further development frequently does not involve conducting bioassays of strain effectiveness on a range of pathogen isolates or host cultivars. Additionally, though previous studies have demonstrated that the medium used to produce biomass of an antagonist can alter its efficacy, this factor is also rarely considered when selecting for the most effective antagonist. Host cultivar, pathogen isolate, and the cultivation medium used to produce the antagonists' biomass were examined as factors of potential importance for assessing the relative effectiveness of bacterial biocontrol strains accurately. Five bacterial antagonists that control Fusarium dry rot on stored potato tubers were assayed for effectiveness against 10 isolates of Gibberella pulicaris . All antagonists reduced disease severity (35-81%) regardless of the specific assays conducted. However, when the antagonists' biomass were produced on two media that differed both in nutrient composition and phase, the efficacy ranking of antagonist Enterobacter sp. S11:P:08 varied from first to fourth most effective. For the antagonists studied, the phase of a nutritionally identical medium had little impact on the efficacy ranking of the five antagonists. Four of the five antagonists had efficacy rankings that ranged from first to last depending on the isolate of the pathogen used to conduct the bioassay. The cultivar of the host also caused variations in the efficacy ranking of the antagonists. These results indicate that bioassays should be conducted using a range of liquid culture production media, pathogen isolates, and host cultivars in order to choose an antagonist that has the highest likelihood for commercial development as an effective biological control product.     

Fusarin C production by North American isolates of Fusarium moniliforme.

A liquid culture medium was developed to screen North American isolates of Fusarium moniliforme Sheldon and Fusarium subglutinans (Wollenw. and Reink.) Nelson, Toussoun, and Marasas for their ability to produce fusarin C. Parameters which were important for the optimal biosynthesis of fusarin C included pH (3.0 to 4.0), aeration, and sugar concentration (30 to 40%). Of seven sugars tested, sucrose and glucose were the best carbohydrate sources for mycotoxin production, resulting in levels of fusarin C of greater than 60 ppm (greater than 60 micrograms/g) in liquid culture (28 degrees C; 7 days). A time-course study of fusarin C production was done over a 21-day period, during which time pH values, glucose concentrations, nitrogen levels, and fungal biomass were determined. Of the two Fusarium spp. studied, 13 of 16 isolates of F. moniliforme produced fusarin C in liquid medium (14 of 16 in corn), while none of the 15 isolates of F. subglutinans studied was found to produce the compound. Levels of fusarin C produced by Fusarium sp. isolates growing on corn ranged from 18.7 to 332.0 micrograms/g.     

Fusarin C production by North American isolates of Fusarium moniliforme

A liquid culture medium was developed to screen North American isolates of Fusarium moniliforme Sheldon and Fusarium subglutinans (Wollenw. and Reink.) Nelson, Toussoun, and Marasas for their ability to produce fusarin C. Parameters which were important for the optimal biosynthesis of fusarin C included pH (3.0 to 4.0), aeration, and sugar concentration (30 to 40%). Of seven sugars tested, sucrose and glucose were the best carbohydrate sources for mycotoxin production, resulting in levels of fusarin C of greater than 60 ppm (greater than 60 micrograms/g) in liquid culture (28 degrees C; 7 days). A time-course study of fusarin C production was done over a 21-day period, during which time pH values, glucose concentrations, nitrogen levels, and fungal biomass were determined. Of the two Fusarium spp. studied, 13 of 16 isolates of F. moniliforme produced fusarin C in liquid medium (14 of 16 in corn), while none of the 15 isolates of F. subglutinans studied was found to produce the compound. Levels of fusarin C produced by Fusarium sp. isolates growing on corn ranged from 18.7 to 332.0 micrograms/g.     

Pathway and Molecular Mechanisms for Malachite Green Biodegradation in Exiguobacterium sp. MG2

Malachite green (MG), N-methylated diaminotriphenylmethane, is one of the most common dyes in textile industry and has also been used as an effective antifungal agent. However, due to its negative impact on the environment and carcinogenic effects to mammalian cells, there is a significant interest in developing microbial agents to degrade this type of recalcitrant molecules. Here, an Exiguobacterium sp. MG2 was isolated from a river in Yunnan Province of China as one of the best malachite green degraders. This strain had a high decolorization capability even at the concentration of 2500 mg/l and maintained its stable activity within the pH range from 5.0 to 9.0. High-pressure liquid chromatography, liquid chromatography-mass spectrometry and gas chromatography–mass spectrometry were employed to detect the catabolic pathway of MG. Six intermediate products were identified and a potential biodegradation pathway was proposed. This pathway involves a series of reactions of N-demethylation, reduction, benzene ring-removal, and oxidation, which eventually converted N-methylated diaminotriphenylmethane into N, N-dimethylaniline that is the key precursor to MG. Furthermore, our molecular biology experiments suggested that both triphenylmethane reductase gene tmr and cytochrome P450 participated in MG degradation, consistent with their roles in the proposed pathway. Collectively, our investigation is the first report on a biodegradation pathway of triphenylmethane dye MG in bacteria.     

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.     

三株丝状真菌分解樟子松凋落物酶活性

该研究以在樟子松凋落物层中高频出现的3株丝状真菌Alternaria sp.、Penicillium sp.和Pestalotiopsis sp.为供试菌株, 以樟子松新鲜落叶为作用底物, 通过发酵纯培养的方法, 测定了底物有机物质质量损失及发酵过程中产生的漆酶(Laccase)、锰过氧化物酶(MnP)、羧甲基纤维素酶(CMCase)和滤纸酶(FPA)酶活性变化, 并验证了酶活性与底物降解的关系。结果表明, Alternaria sp. 引起底物总有机物质质量损失最大, 且产生的漆酶、羧甲基纤维素酶和滤纸酶活性都较高; Penicillium sp. 产生的锰过氧化物酶活性最高。3株丝状真菌同属于真菌功能群中的木质纤维素分解者。