Effect of Cultivation Time on Production of Antifungal Metabolite(s) by Streptomyces hygroscopicus in Laboratory‐Scale Bioreactor

Biological control agents offer one of the best alternatives to reduce the use of pesticides . Fungi from the genera Alternaria, Colletotrichum and Fusarium are listed among the most important storage pathogens of apple fruits. During storage, transport and marketing, pathogenic fungi can cause significant losses of apple fruits. This investigation studied the potential of Streptomyces hygroscopicus as a biocontrol agent against pathogenic fungi obtained from apple fruit samples expressing rot symptoms. Production of antifungal metabolites by S. hygroscopicus was carried out in 3‐l bench‐scale bioreactor (Biostat^® Aplus, Sartorius AG, Germany) during 7 days. Fermentation was carried out at 27°C with aeration rate of 0.5 vvm and agitation rate of 200 rpm. The aim was to analyse bioprocess parameters of batch biofungicide production in medium containing glucose as a carbon source and to examine at which stage of bioprocess production of antifungal metabolite(s) against six phytopathogenic fungi occurs. In vitro antifungal activity of the produced metabolites against six fungi of the genera Colletotrichum, Fusarium and Alternaria grown on potato dextrose agar were determined every 24 h using wells technique. Antifungal activity of cell‐free culture filtrate and filtrate treated with high temperature were tested. The filtrate treated with high temperature did not show any antifungal activity suggesting that active components are thermo unstable. Stationary phase of growth occurred between the third and fourth day of cultivation when production of secondary metabolites begins. Obtained results showed that maximal antifungal activity is achieved on fifth and sixth day of S. hygroscopicus cultivation under defined conditions (inhibition zone diameter higher than 30 mm for all test fungi).     

Influence of the extraction mode on the yield of hyperoside, vitexin and vitexin-2'-O-rhamnoside from Crataegus monogyna Jacq. (hawthorn)

Introduction –  The extract of Crataegus monogyna shows sedative, hypotensive, vasodilator and cardio‐tonic actions. Although several papers dealing with the extraction of metabolites from Crataegus have been published, the plant productivity in terms of bioactive compounds is not easily understandable as yet. Objective –  To investigate the influence of the extraction mode on the yield of bioactive compounds from Crataegus monogyna Jacq. in order to evaluate plant productivity. Methodology –  Samples were prepared by extraction of powdered material obtained from top branches, flowers and leaves. Soxhlet extraction, maceration and ultrasound‐ and microwave‐assisted extraction at different experimental conditions were investigated for the exhaustive extraction of hyperoside, vitexin and vitexin‐2′′‐O‐rhamnoside. The phytocomponents were identified and quantified by HPLC‐UV/PAD, comparing HPLC retention times and UV spectra of individual peaks with those of the standards analysed under the same conditions. Results –  An easy‐to‐use HPLC isocratic method suitable for the quantification of hyperoside, vitexin and vitexin‐2′′‐O‐rhamnoside in raw plant extracts was developed. The optimised HPLC methodology was applied to evaluate different extraction procedures. The ultrasound and microwave‐assisted extraction protocols showed higher extraction efficiency than the others. In particular, the optimised microwave protocol gave rise to the highest extraction efficiency with high reproducibility. Conclusions –  A microwave protocol combined with isocratic HPLC analysis is proposed for the rapid screening of plant materials collected in different environmental conditions in order to evaluate the productivity of Crataegus monogyna Jacq. and to find out the best ecological conditions to cultivate hawthorn in Northern Italy. Copyright © 2008 John Wiley & Sons, Ltd.     

Discussion of development processes in insect‐fungus association derived from the shaggy parasol fruiting on the nests of hairy wood ants

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An improved extraction method enables the comprehensive analysis of lipids,proteins, metabolites and phytohormones from a single sample of leaf tissue under water‐deficit stress

Phytohormones play essential roles in the regulation of growth and development in plants. Plant hormone profiling is therefore essential to understand developmental processes and the adaptation of plants to biotic and/or abiotic stresses. Interestingly, commonly used hormone extraction and profiling methods do not adequately resolve other molecular entities, such as polar metabolites, lipids, starch and proteins, which would be required to comprehensively describe the continuing biological processes at a systematic level. In this article we introduce an updated version of a previously published liquid:liquid metabolite extraction protocol, which not only allows for the profiling of primary and secondary metabolites, lipids, starch and proteins, but also enables the quantitative analysis of the major plant hormone classes, including abscisic acid, auxins, cytokinins, jasmonates and salicylates, from a single sample aliquot. The optimization of the method, which uses the introduction of acidified water, enabling the complete purification of major plant hormones into the organic (methyl‐tert‐butyl‐ether) phase, eliminated the need for solid‐phase extraction for sample clean‐up, and therefore reduces both sampling time and cost. As a proof‐of‐concept analysis, Arabidopsis thaliana plants were subjected to water‐deficit stress, which were then profiled for hormonal, metabolic, lipidomic and proteomic changes. Surprisingly, we determined not only previously described molecular changes but also significant changes regarding the breakdown of specific galactolipids, followed by the substantial accumulation of unsaturated fatty‐acid derivatives and diverse jasmonates in the course of adaptation to water‐deficit stress.     

Antifungal Activity in Ethanolic Extracts of <Emphasis Type="Italic">Carica papaya</Emphasis> L. cv. Maradol Leaves and Seeds

Bioactive compounds from vegetal sources are a potential source of natural antifungic. An ethanol extraction was used to obtain bioactive compounds from Carica papaya L. cv. Maradol leaves and seeds of discarded ripe and unripe fruit. Both, extraction time and the papaya tissue flour:organic solvent ratio significantly affected yield, with the longest time and highest flour:solvent ratio producing the highest yield. The effect of time on extraction efficiency was confirmed by qualitative identification of the compounds present in the lowest and highest yield extracts. Analysis of the leaf extract with phytochemical tests showed the presence of alkaloids, flavonoids and terpenes. Antifungal effectiveness was determined by challenging the extracts (LE, SRE, SUE) from the best extraction treatment against three phytopathogenic fungi: Rhizopus stolonifer, Fusarium spp. and Colletotrichum gloeosporioides. The leaf extract exhibited the broadest action spectrum. The MIC₅₀ for the leaf extract was 0.625 mg ml⁻¹ for Fusarium spp. and >10 mg ml⁻¹ for C. gloeosporioides, both equal to approximately 20% mycelial growth inhibition. Ethanolic extracts from Carica papaya L. cv. Maradol leaves are a potential source of secondary metabolites with antifungal properties.     

The nutritional landscape of host plants for a specialist insect herbivore

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Involvement of LeMRP,an ATP‐binding cassette transporter,in shikonin transport and biosynthesis in Lithospermum erythrorhizon

• Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown.
• In this study, we isolated a cDNA encoding LeMRP, an ATP‐binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real‐time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes.
• Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real‐time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up‐regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down‐regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots.
• Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.

     

Effects of plant age on performance of the tropical perennial fodder grass,Cenchrus ciliaris L. subjected to elevated ultraviolet‐B radiation

• Abiotic stress, notably high ultraviolet‐B (eUV‐B), limit growth and productivity of many crop plants, but information on response of forage grasses to eUV‐B radiation is rather limited.
• The present study was therefore conducted to increase our understanding of differential age‐related responses on growth, metabolism and fodder quality of Cenchrus ciliaris‐3108 (Buffel grass) to elevated UV‐B (eUV‐B: 7.2 kJ·m^?2·day^?1).
• Plant growth at both growth stages was notably reduced in response to eUV‐B, except for the number of nodes and tillers at vegetative and reproductive stages. At anthesis, tillering increased due to the perennial habit of this plant, but leaf senescence reduced the number of leaves per tiller. Unlike ambient UV‐B, eUV‐B at the vegetative stage resulted in diversion of photosynthate for the formation of secondary metabolites (tannins and phenolics), providing dual protection from photooxidative damage and from herbivory.
• The forage biomass as well as quality showed a marked decline under eUV‐B and relative nutritive value was reduced at both growth stages.

     

Metabolomic Tools to Assess the Chemistry and Bioactivity of Endophytic Aspergillus Strain

Endophytic fungi associated with medicinal plants are a potential source of novel chemistry and biology that may find applications as pharmaceutical and agrochemical drugs. In this study, a combination of metabolomics and bioactivity‐guided approaches were employed to isolate secondary metabolites with cytotoxicity against cancer cells from an endophytic Aspergillus aculeatus. The endophyte was isolated from the Egyptian medicinal plant Terminalia laxiflora and identified using molecular biological methods. Metabolomics and dereplication studies were accomplished by utilizing the MZmine software coupled with the universal Dictionary of Natural Products database. Metabolic profiling, with aid of multivariate data analysis, was performed at different stages of the growth curve to choose the optimized method suitable for up‐scaling. The optimized culture method yielded a crude extract abundant with biologically‐active secondary metabolites. Crude extracts were fractionated using different high‐throughput chromatographic techniques. Purified compounds were identified by HR‐ESI‐MS, 1D‐ and 2D‐NMR. This study introduced a new method of dereplication utilizing both high‐resolution mass spectrometry and NMR spectroscopy. The metabolites were putatively identified by applying a chemotaxonomic filter. We also present a short review on the diverse chemistry of terrestrial endophytic strains of Aspergillus, which has become a part of our dereplication work and this will be of wide interest to those working in this field.     

Nonconsumptive predator effects modify crayfish‐induced bioturbation as mediated by limb loss: Field and mesocosm experiments

1. We addressed the implications of limb loss and regeneration for multispecies interactions and their impacts on ecosystem engineering in freshwater stream environments.
2. We included regenerative and nonregenerative crayfish as well as fish predators in a 2 × 2 factorial design to assess the effects on water turbidity of interactions between crayfish ecosystem engineers differing in regenerative status and their fish predators.
3. We demonstrated that crayfish limb loss and predation risks lead to more turbidity in field and mesocosm conditions. Moreover, ongoing regeneration of crayfish increased turbidity, while fish presence seemed to hinder crayfish turbidity‐inducing behaviors (such as tail‐flipping and burrowing) in the mesocosm experiment.
4. We confirmed that greater numbers of crayfish produce a greater amount of turbidity in situ in streams.
5. Although mechanical burrowing crayfish capacities may depend on crayfish burrowing classification (primary, secondary, or tertiary), our work emphasizes the implication for turbidity levels of crayfish autotomy in freshwater streams.

     

Context dependency of infectious disease: the cyanobacterium Microcystis aeruginosa decreases white bacterial disease in Daphnia magna

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Complex interactions between bacteria and haemosporidia in coinfected hosts: An experiment

1. Hosts are typically coinfected by multiple parasite species whose interactions might be synergetic or antagonistic, producing unpredictable physiological and pathological impacts on the host. This study shows the interaction between Plasmodium spp. and Leucocytozoon spp. in birds experimentally infected or not infected with Mycoplasma gallisepticum.
2. In 1994, the bacterium Mycoplasma gallisepticum jumped from poultry to wild birds in which it caused a major epidemic in North America. Birds infected with M. gallisepticum show conjunctivitis as well as increased levels of corticosterone.
3. Malaria and other haemosporidia are widespread in birds, and chronic infections become apparent with the detectable presence of the parasite in peripheral blood in response to elevated levels of natural or experimental corticosterone levels.
4. Knowing the immunosuppressive effect of corticosterone on the avian immune system, we tested the hypothesis that chronic infections of Plasmodium spp. and Leucocytozoon spp. in house finches would respond to experimental inoculation with M. gallisepticum as corticosterone levels are known to increase following inoculation.
5. Plasmodium spp. infection intensity increased within days of M. gallisepticum inoculation as shown both by the appearance of infected erythrocytes and by the increase in the number and the intensity of positive PCR tests.
6. Leucocytozoon spp. infection intensity increased when Plasmodium spp. infection intensity increased, but not in response to M. gallisepticum inoculation. Leucocytozoon spp. and Plasmodium spp. seemed to compete in the host as shown by a negative correlation between the changes in their PCR score when both pathogens were present in the same individual.
7. Host responses to coinfection with multiple pathogens measured by the hematocrit and white blood cell count depended on the haemosporidian community composition. Host investment in the leukocyte response was higher in the single‐haemosporidia‐infected groups when birds were infected with M. gallisepticum.
8. A trade‐off was observed between the immune control of the chronic infection (Plasmodium spp./Leucocytozoon spp.) and the immune response to the novel bacterial infection (M. gallisepticum).

     

The mechanism of competition between two bloom‐forming Microcystis species

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Penicillium roqueforti: a multifunctional cell factory of high value‐added molecules

This is a comprehensive review, with 114 references, of the chemical diversity found in the fungus Penicillium roqueforti. Secondary metabolites of an alkaloidal nature are described, for example, ergot alkaloids such as festuclavine, isofumigaclavines A and B, and diketopiperazine alkaloids such as roquefortines A–D, which are derived from imidazole. Other metabolites are marcfortines A–C, PR‐toxin, eremofortines A–E, mycophenolic and penicillic acids, and some γ‐lactones. Also, recent developments related to the structural characteristics of botryodiplodin and andrastin are studied—the latter has anticancer properties. Finally, we discuss the enzymes of P. roqueforti, which can participate in the biotechnological production of high value‐added molecules, as well as the use of secondary metabolite profiles for taxonomic purposes.     

Influence of body mass and environmental conditions on winter mortality risk of a northern ungulate: Evidence for a late‐winter survival bottleneck

1. A relationship between winter weather and survival of northern ungulates has long been established, yet the possible roles of biological (e.g., nutritional status) and environmental (e.g., weather) conditions make it important to determine which potential limiting factors are most influential.
2. Our objective was to examine the potential effects of individual (body mass and age) and extrinsic (winter severity and snowmelt conditions) factors on the magnitude and timing of mortality for adult (>2.5 years old) female white‐tailed deer (Odocoileus virginianus [Zimmerman, 1780]) during February–May in the Upper Peninsula of Michigan, USA.
3. One hundred and fifty deer were captured and monitored during 2009–2015 in two areas with varying snowfall. February–May survival ranged from 0.24 to 0.89 (mean = 0.69) across years. Mortality risk increased 1.9% with each unit increase in cumulative winter severity index, decreased 8.2% with each cumulative snow‐free day, and decreased 4.3% with each kg increase in body mass. Age and weekly snow depth did not influence weekly deer survival. Predation, primarily from coyote (Canis latrans [Say, 1823]) and wolves (Canis lupus [L., 1758]), accounted for 78% of known‐cause mortalities.
4. Our results suggest that cumulative winter severity, and possibly to a lesser degree deer condition entering winter, impacted deer winter survival. However, the timing of spring snowmelt appeared to be the most influential factor determining late‐winter mortality of deer in our study. This supports the hypothesis that nutrition and energetic demands from weather conditions are both important to northern ungulate winter ecology. Under this model, a delay of several weeks in the timing of spring snowmelt could exert a large influence on deer survival, resulting in a survival bottleneck.

     

Insecticidal and insect growth regulatory activities of secondary metabolites from entomopathogenic fungi,Lecanicillium attenuatum

Insect growth regulators (IGRs) are effective alternatives to chemical insecticides because of their specificity and low environmental toxicity. Entomopathogenic fungi are an important natural pathogen of insects and have been developed as biological control agents. They produce a wide range of secondary metabolites such as antibiotics, pesticides, growth-promoting or inhibiting compounds and insect attracting agents. In this study, to explore novel IGR substances from entomopathogenic fungi, culture extracts of 189 entomopathogenic fungi isolated from Korean soil samples were investigated for their juvenile hormone (JH)-based IGR activities. Whereas none of the culture extracts exhibited JH agonist (JHA) activity, 14 extracts showed high levels of JH antagonist (JHAN) activity. Among them, culture extract of JEF-145 strain, which was identified as Lecanicillium attenuatum, showed the highest insecticidal against Aedes albopictus and Plutella xylostella. At liquid culture condition, JHAN activity was observed in culture soup rather than mycelial cake, indicating that substances with JHAN activity are released from the JEF-145 strain during culture. Furthermore, while extract from solid cultured JEF-145 strain showed insecticidal activities against both A. albopictus and P. xylostella, that from liquid cultured fungi showed insecticidal activity only against A. albopictus, indicating that L. attenuatum JEF-145 strain produces different kinds of secondary metabolites with JHAN activity depending on culture conditions. These results suggested that JHAN substances derived from entomopathogenic fungi could be usefully exploited to develop novel eco-friendly IGR insecticides.     

Intrapopulation variability in a functional trait: Susceptibility of Daphnia to limitation by dietary fatty acids

1. Functional traits are measurable characteristics of an organism that have an impact on its fitness. Variation in functional traits between and among species has been suggested to represent the basis for competition and selection, thus allowing for evolution in natural populations.
2. In freshwater ecosystems, the availability of essential polyunsaturated fatty acids (PUFAs), in particular ω3‐ and ω6‐PUFAs, determines the food quality of phytoplankton for the herbivorous zooplankton Daphnia, an unselective filter feeder. The content of such essential PUFAs in the phytoplankton is thus a functional phytoplankton trait affecting the trophic transfer efficiency and dynamics at the pelagic plant–herbivore interface.
3. In turn, the susceptibility of consumers to become limited by the availability of essential PUFAs is a fitness‐determining trait of Daphnia genotypes, and variability of this herbivore trait may thus affect the daphnids’ intrapopulation competition. To estimate the intrapopulation variation in susceptibility, we isolated clonal lines of Daphnia longispina from a natural population and compared the strength of their limitation by dietary PUFA availability via standardised laboratory growth assays. We used a liposome supplementation technique to enrich a PUFA‐poor green alga with essential ω3‐ and ω6‐PUFAs and determined juvenile somatic growth rate of different D. longispina genotypes as a fitness proxy.
4. As expected, D. longispina genotypes that coexisted in a natural population differed markedly in their specific patterns of susceptibility to dietary PUFA availability. On average, the D. longispina population was more strongly susceptible to limitations in the availability of the ω6‐PUFA arachidonic acid (20:4ω6) than to limitations in the availability of ω3‐PUFAs α‐linolenic acid (18:3ω3) and eicosapentaenoic acid (20:5ω3).
5. The ability to cope with PUFA limitation is thus a crucial trait that can probably affect intraspecific competition and Daphnia population structure. Therefore, we suggest that such intrapopulation variation in susceptibility to absence of dietary PUFAs might be one of the driving forces of natural selection and local adaptation among freshwater zooplankton.

     

Glass bead cultivation of fungi: Combining the best of liquid and agar media

Production of bioactive compounds and enzymes from filamentous fungi is highly dependent on cultivation conditions. Here we present an easy way to cultivate filamentous fungi on glass beads that allow complete control of nutrient supply. Secondary metabolite production in Fusarium graminearum and Fusarium solani cultivated on agar plates, in shaking liquid culture or on glass beads was compared. Agar plate culture and glass bead cultivation yielded comparable results while liquid culture had lower production of secondary metabolites. RNA extraction from glass beads and liquid cultures was easier than from agar plates and the quality was superior. The system allows simple control of nutrient availability throughout fungal cultivation. This combined with the ease of extraction of nucleic acids and metabolites makes the system highly suitable for the study of gene regulation in response to specific nutrient factors.