Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O‐glycosidically linked to 3,15,16‐trihydroxypalmitic acid. The sugar moiety is acylated with 2‐hydroxy‐octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl‐transferase and is lacking a gene homologous to the α‐hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl‐transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl‐transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.     

Recombinant Protein Secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a Novel Signal Peptide

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.     

Usefulness of heterologous promoters in the Pseudozyma flocculosa gene expression system

The basidiomycetous fungus Pseudozyma flocculosa represents a promising new host for the expression of complex recombinant proteins. Two novel heterologous promoter sequences, the Ustilago maydis glyceraldehyde-3-phosphate dehydrogenase (GPD) and Pseudozyma tsukubaensis alpha-glucosidase promoters, were tested for their ability to provide expression in P. flocculosa. In liquid medium, these two promoters produced lower levels of intracellular green fluorescent protein (GFP) as compared to the U. maydis hsp70 promoter. However, GPD and alpha-glucosidase sequences behaved as constitutive promoters whereas the hsp70 promoter appeared to be morphology-dependent. When using the hsp70 promoter, the expression of GFP increased proportionally to the concentration of hygromycin in the culture medium, indicating possible induction of the promoter by the antibiotic. Optimal solid-state culture conditions were designed for high throughput screening of hygromycin-resistant transformants with the hsp70 promoter in P. flocculosa.     

Establishment of a gene transfer system for Pseudozyma flocculosa, an antagonistic fungus of powdery mildew fungi

A reliable DNA-mediated transformation system has been developed for Pseudozyma flocculosa, a fungus that is antagonistic to powdery-mildew fungi. Plasmids harboring various selectable markers under the control of different promoters were tested. Molecular analyses demonstrated that successful transformation could be achieved using a plasmid that confers resistance to hygromycin B under the control of the Ustilago maydis hsp70 promoter and terminator sequences. On average, 1-40 (mean = 20) transformants were obtained per 10 microg of linearized DNA per 10(8) protoplasts. Southern analysis of the transformants revealed that, in each case, the vector had integrated in multiple tandem copies into the genome of P. flocculosa, and that integration events were random. Pulsed-field gel electrophoresis was employed to separate the genome of P. flocculosa into at least 11 chromosomes with sizes ranging from 0.55 Mb to 2.9 Mb. Hybridization with the plasmid indicated that integration of vector DNA had occurred in one to several chromosomes depending on the transformant examined.     

Elements for modelling the dynamics of tritrophic population interactions

Models of acarine systems are reviewed with particular reference to objectives for modelling, to the methods used for model building, and to the results obtained. The following elements are considered important in models of complex systems: common food acquisition/allocation functions in multitrophic models; migration and within-systems movement; and behavioral components in functional responses. The first element is illustrated with an apple tree/mite and a cassava/mite model. In mite management, the available systems models appear more useful for strategic purposes, i.e. for selecting biological control agents and for planning pest control measures, than for tactical decision making in supervised pest management programs.     

Applications for green fluorescent protein (GFP) in the study of hostpathogen interactions

The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity. To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens. The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm). Both expression vectors were found to be stable and to direct high levels of GFP synthesis. Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells. Mm expressing gfp was also visualized in infected animal tissues, gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages. In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages. Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.     

Rapid spread agents may impair biological control in a tritrophic food web with intraguild predation

The augmentation of natural enemies against agricultural pests is a common tactic undertaken to minimize crop damage without the use of chemical pesticides. Failures of this strategy may result from (i) Allee effects acting on biological control agent; (ii) trophic interactions between the released control agent and native species in the local ecosystem; (iii) excessively rapid spreading agents. To investigate the interplay of these mechanisms in pest biocontrol efficiency in the context of intraguild predation (IGP), we develop a one-dimensional dynamical model of a spatial, tritrophic food web with intraguild predation. We show that the agent’s diffusivity (i.e., agent’s dispersal speed), and intraguild predator’s addition of alternative food sources are important factors in determining the success or failure of pest biocontrol. These results are obtained for spatially explicit models by considering the speed of dispersal of the control agent and the pest. Feedback from theoretical models as the one constructed in this work can provide useful guidelines for practitioners in biological control.     

The genetics of chutes and ladders: a community genetics approach to tritrophic interactions

Community genetics research has firmly established that intraspecific genetic variation in single populations can have large extended ecological consequences for populations and entire communities of organisms. Here, we sought to understand the bottom‐up effects of plant genetic variation on herbivore preference and performance, and the top–down control of predators on herbivores and their joint effects on plant fitness and evolution. Following three ecological genetics field experiments we detected heritable variation in plant traits that influenced both the preference and performance of a specialist weevil on Oenothera biennis. However, the weevil's preference and performance were not genetically correlated among O. biennis plant genotypes. Although predators and parasitoids were abundant, predators had no detectable effect on weevil performance because high egg and larval mortality was caused by non‐predatory factors such as intraspecific competition. Finally, neither the specialist weevil nor predators influenced plant fitness. Our results suggest that the focal tritrophic community studied here is primarily shaped by the bottom–up effects of plant genetic variation on herbivores, while top–down effects have no clear impacts on O. biennis fitness or evolution. We suggest that future studies should incorporate plant intraspecific genetic variation as a fundamental part of tritrophic interactions including their eco‐evolutionary dynamics.     

Bottom-up cascading effects in a tritrophic system: interactions between plant quality and host-parasitoid immune responses

Abstract.  1. Little is known about underlying mechanisms by which plants indirectly affect parasitism success in hymenopteran endoparasitoids. The hypothesis that host-plant effects can challenge the innate immune system of an insect host was experimentally tested in this study using a model tritrophic, crucifer – lepidopteran [ Plutella xylostella (L.)] – parasitoid [ Cotesia plutellae (Kurdjumov)], system.
2. The effects of host-plant suitability on herbivore performance and parasitism were examined. The bottom-up effect of plant suitability on host-parasitoid immune responses was then evaluated using measures of cellular and humoral effectors.
3. Host-plant quality showed a significant effect on the encapsulation response of P. xylostella to first instar but not to second instar parasitoid larvae. Encapsulation was never sufficient to prevent parasitoid emergence.
4. Poor host-plant suitability suppressed phenoloxidase activity in the absence of the parasitoid. The suppressive effect of C. plutellae on phenoloxidase activity was much greater and no plant effects were detectable after insects had been parasitized.
5. Despite strong plant effects on parasitism, those on immune effectors of the host were transitory or overwhelmed by the effect of the parasitoid.
6. These results demonstrated that plant-mediated variation in parasitism success by C. plutellae were not as a result of plant nutritional status or other attributes affecting the immune function of P. xylostella , nor to host-plant effects on superparasitism.
7. In these experiments, P. xylostella was a fully permissive host to C. plutellae and host-plant-mediated effects on the innate immune response appeared to play no part in parasitoid survival within hosts.     

The interactions of neuropeptides with membrane model systems: a case study.

The interactions between the positively charged neuropeptides substance P (SP), bradykinin (BK), and zwitterionic Met-enkephalin (ME) neuropeptides, and negatively charged SDS and zwitterionic lysophosphatidylcholine (LPC) membrane model systems, have been investigated using one- and two-dimensional nmr experiments. Proton longitudinal relaxation studies were used to characterize these interactions as intrinsic or extrinsic. An extrinsic interaction are similar to those observed for extrinsic membrane proteins. An intrinsic interaction are similar to those observed for intrinsic membrane proteins, and would require that the hydrophobic residues penetrate or insert into the hydrophobic core of the membrane. The interactions between both SP and BK and SDS, based on nmr results, may be characterized as intrinsic, and the interaction between ME and SDS may be characterized as extrinsic. Two-dimensional nuclear Overhauser enhancement spectroscopy experiments proved the insertion of the phenylalanine residues on both SP and BK into the hydrophobic core of SDS micelles. The interaction between SP and BK with LPC based on nmr results are characterized as extrinsic, with the interaction between ME and SDS characterized as weakly intrinsic.     

Improving function of biocontrol agents incorporated in antifungal fruit coatings: a review

The in-field performance of microbial biocontrol agents (BCAs) against fungal pathogens in fruit is subject to considerable variability due to their sensitivity to both adverse environmental conditions and their fluctuations. Therefore, to achieve an adequate development and implementation of biological agent-based products, it is necessary to improve their resistance and ability to control fungal diseases under a wide range of conditions. In this review, an overview of the latest strategies for the enhancement of the action of BCAs is given. The combination of the antagonists with edible polymers able to form coatings is one of the approaches with the greatest potential and it is analysed in depth. This formulation approach of biocontrol products, including adequate microbial protectants, can yield stable products with high microbial viability, ready for field applications, with improved adherence and survival of the BCA once applied in plant. The most recent studies into this field are reviewed and summarised.     

Dominant network interactions are not correlated with resource availability: a case study using mistletoe host interactions

Network theory in ecology has been central to understanding species co‐occurrence patterns, specialization and community stability. However, network theory has traditionally focused on the ‘higher’ trophic level where exploitation of network ‘partners’ (i.e. individual interactions in response to resource availability) have remained underappreciated. In this study we tested how clumping and host availability influenced mistletoe–host interactions in a semi‐arid woodland, central Australia. We used a hierarchical approach that evaluated individual interactions by modifying the traditional randomization technique to simulate clumping and host exploitation. Using published literature we then compared our results with mistletoes from other genera. We found that mistletoes clump on fewer trees than predicted, even though interaction strength was no different from random expectations, and we found no evidence that common trees were heavily infected as predicted by the host availability hypothesis. The rate of host exploitation (measured as the proportion of trees infected) in semi‐arid Australia is similar to that for mistletoe genera in other parts of the world. We hypothesize that specific host trees act as a focal point for infection that facilitates the spread and overall population size of mistletoes. Overall our results indicate that resources, such as the number of trees in a mistletoe network, are less important than clumping of individual plants. We suggest that exploitation of available resources may play a similar role in other networks that extend beyond antagonistic relationships such as parasite or herbivore interactions.     

The hunt for sustainable biocontrol of oomycete plant pathogens,a case study of Phytophthora infestans

Late blight caused by the oomycete Phytophthora infestans is considered to be one of the most severe diseases of potato and tomato worldwide. Whilst current synthetic fungicides are efficient at controlling this disease, they are an environmental and economic burden. In line with EU directives to reduce the use of synthetic pesticides and increase the use of sustainable alternative disease control strategies that can form part of integrated pest management systems, practical biological control solutions are urgently needed. Despite the fact that there has been a large body of scientific research into microorganisms with potential for the biological control of late blight disease, relatively few commercial biocontrol agents, licensed to control late blight, exist. Furthermore, the practical uptake of those in Europe is lower than might be expected, suggesting that such solutions are not yet feasible, or effective. Here we review the scientific literature, focusing on the most recent developments in the hunt for efficient and sustainable biological control of late blight disease. We discuss the progress in our mechanistic understanding of mycoparasite–prey interactions, in the context of late blight and the challenges and limitations to the use of such knowledge in practical disease control within a European context.     

Compound effects of induced plant responses on insect herbivores and parasitoids: implications for tritrophic interactions

1. Induced plant responses can affect herbivores either directly, by reducing herbivore development, or indirectly, by affecting the performance of natural enemies. Both the direct and indirect impacts of induction on herbivore and parasitoid success were evaluated in a common experimental system, using clonal poplar trees Populus nigra (Salicales: Salicaceae), the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae), and the gregarious parasitoid Glyptapanteles flavicoxis (Marsh) (Hymenoptera: Braconidae). 2. Female parasitoids were attracted to leaf odours from both damaged and undamaged trees, however herbivore‐damaged leaves were three times more attractive to wasps than undamaged leaves. Parasitoids were also attracted to herbivore larvae reared on foliage and to larval frass, but they were not attracted to larvae reared on artificial diet. 3. Prior gypsy moth feeding elicited a systemic plant response that retarded the growth rate, feeding, and survival of gypsy moth larvae, however induction also reduced the developmental success of the parasitoid. 4. The mean number of parasitoid progeny emerging from hosts fed foliage from induced trees was 40% less than from uninduced trees. In addition, the proportion of parasitised larvae that survived long enough to issue any parasitoids was lower on foliage from induced trees. 5. A conceptual and analytical model is provided to describe the net impacts of induced plant responses on parasitoids, and implications for tritrophic interactions and biological control of insect pests are discussed.     

Methodologies for screening of bacteria-carbohydrate interactions: anti-adhesive milk oligosaccharides as a case study

Many studies have demonstrated the capacity of glycan-based compounds to disrupt microbial binding to mucosal epithelia. Therefore, oligosaccharides have potential application in the prevention of certain bacterial diseases. However, current screening methods for the identification of anti-adhesive oligosaccharides have limitations: they are time-consuming and require large amounts of oligosaccharides. There is a need to develop analytical techniques which can quickly screen for, and structurally define, anti-adhesive oligosaccharides prior to using human cell line models of infection. Considering this, we have developed a rapid method for screening complex oligosaccharide mixtures for potential anti-adhesive activity against bacteria. Our approach involves the use of whole bacterial cells to "deplete" free oligosaccharides from solution. As a case study, the free oligosaccharides from the colostrum of Holstein Friesian cows were screened for interactions with whole Escherichia coli cells. Reductions in oligosaccharide concentrations were determined by High pH Anion Exchange Chromatography and Hydrophilic Interaction Liquid Chromatography (HILIC-HPLC). Oligosaccharide structures were confirmed by a combination of HILIC-HPLC, exoglycosidase digestion and off-line negative ion mode MS/MS. The depletion assay confirmed selective bacterial interaction with certain bovine oligosaccharides which in previous studies, by other methodologies, had been shown to interact with E. coli. In particular, the bacterial cells depleted the following oligosaccharides in a population dependent manner: 3'-sialyllactose, disialyllactose, and 6'-sialyllactosamine. The assay methodology was further validated by studies in which we demonstrated the inhibitory activity of 3'-sialyllactose, and a mixture of bovine colostrum oligosaccharides, on E. coli adhesion to differentiated HT-29 cells.     

A case study of modified interactions with symbionts in a hybrid mediterranean orchid

? Premise of the study: Most studies on orchid hybrids examine separately the effects of hybridization on interactions with pollinators or with mycorrhizal fungi. Here, we simultaneously investigated both interactions in the mediterranean food-deceptive Orchis simia, O. anthropophora, and their hybrid (O. ×bergonii) and tested a possible breakdown of coevolution using a multidisciplinary approach. ? Methods: We compared leaf growth, seed viability, emitted scent, and mycorrhizal fungi (species and rate of infection) among these three taxa. ? Key results: We show that leaf surface is greater in adult hybrids than in the parental species, suggesting a heterosis effect for vegetative growth. We demonstrate that flowers of the two parental species emit well-differentiated bouquets of volatile organic compounds, while hybrids emit larger quantities, accumulating most compounds of the two parental species. However, hybrids fail to attract pollinators and have a 10 times lower fruit set. We determined that closely related Tulasnellales are mycorrhizal in the three taxa, suggesting that the mycorrhizal partner does not impair hybrid survival. We propose an interpretative model for O. ×bergonii compared with its parents. ? Conclusions: In hybrids, carbon resources normally devoted to reproduction may be reallocated to the mycorrhizal symbiosis as a result of the disruption of the pollination interaction in hybrids. Higher mycorrhizal infection may in turn enhance vegetative growth and scent emission. Such interplay between the two obligate biotic interactions yields new insights into hybridization among orchids.     

Epicoccum nigrum for biocontrol agents in vitro of plant fungal pathogens

Epicoccum nigrum strains were evaluated in vitro as potential biological agents for control of the growth of Fusarium avenaceum, F. graminearum, F. oxysporum and Botrytis cinerea. Individual biotic effects of five strains of E. nigrum on the various fungi were determined using the biotic series method elaborated for fungi, on potato dextrose agar (PDA - Difco) medium. Our results show that E. nigrum strains limited the growth of all isolates of Fusarium spp., but not of those of Botrytis cinerea.     

Affinity profiling using the peptide microarray technology: a case study

Little about the reliability of measurements obtained using synthetic peptide microarrays is known. We report results from a study on the quantitative reliability of microarrays manufactured by robot-supported immobilization of presynthesized peptides for different microarray platforms. Technological precision is assessed for inter- and intra-device readout comparisons. Correlations between measured signals and known dissociation constants using a phenomenological model derived from the mass action law are discussed. Special emphasis is on discussing the pitfalls of high-throughput affinity measurements. We show that the quantitative determination of binding affinities is prone to be biased toward a mean affinity of around 10(-7)M, while the classification of peptides into either "binders" or "nonbinders" provides very high prediction accuracy. The experimental requirements needed to obtain reliable binding affinity predictions are discussed.