Self-assembly of the anti-fungal polyene amphotericin B into giant helically-twisted nanotapes

The amphiphilic polyene amphotericin B, a powerful treatment for systemic fungal infections, is shown to exhibit a critical aggregation concentration, and to form giant helically-twisted nanostructures via self-assembly in basic aqueous solution.

Determination of the impact of continuous defoliation of Lolium perenne and Trifolium repens on bacterial and fungal community structure in rhizosphere soil

To determine the effects of defoliation on microbial community structure, rhizosphere soil samples were taken pre-, and post-defoliation from the root tip and mature root regions of Trifolium repens L. and Lolium perenne L. Microbial DNA isolated from samples was used to generate polymerase chain reaction-denaturing gradient gel electrophoresis molecular profiles of bacterial and fungal communities. Bacterial plate counts were also obtained. Neither plant species nor defoliation affected the bacterial and fungal community structures in both the root tip and mature root regions, but there were significant differences in the bacterial and fungal community profiles between the two root regions for each plant. Prior to defoliation, there was no difference between plants for bacterial plate counts of soils from the root tip regions; however, counts were greater in the mature root region of L. perenne than T. repens. Bacterial plate counts for T. repens were higher in the root tip than the mature root region. After defoliation, there was no effect of plant type, position along the root or defoliation status on bacterial plate counts, although there were significant increases in bacterial plate counts with time. The results indicate that a general effect existed during maturation in the root regions of each plant, which had a greater impact on microbial community structure than either plant type or the effect of defoliation. In addition there were no generic consequences with regard to microbial populations in the rhizosphere as a response to plant defoliation.

Iron Uptake and Homeostasis in Microorganisms

Preface. Iron is considered to be a minor element employed, in a variety of forms, by nearly all living organisms. In some cases, it is utilised in large quantities, for instance for the formation of magnetosomes within magnetotactic bacteria or during use of iron as a respiratory donor or acceptor by iron oxidising or reducing bacteria. However, in most cases the role of iron is restricted to its use as a cofactor or prosthetic group assisting the biological activity of many different types of protein. The key metabolic processes that are dependent on iron as a cofactor are numerous; they include respiration, light harvesting, nitrogen fixation, the Krebs cycle, redox stress resistance, amino acid synthesis and oxygen transport. Indeed, it is clear that Life in its current form would be impossible in the absence of iron. One of the main reasons for the reliance of Life upon this metal is the ability of iron to exist in multiple redox states, in particular the relatively stable ferrous (Fe2+) and ferric (Fe3+) forms. The availability of these stable oxidation states allows iron to engage in redox reactions over a wide range of midpoint potentials, depending on the coordination environment, making it an extremely adaptable mediator of electron exchange processes. Iron is also one of the most common elements within the Earth’s crust (5% abundance) and thus is considered to have been readily available when Life evolved on our early, anaerobic planet. However, as oxygen accumulated (the ‘Great oxidation event’) within the atmosphere some 2.4 billion years ago, and as the oceans became less acidic, the iron within primordial oceans was converted from its soluble reduced form to its weakly-soluble oxidised ferric form, which precipitated (~1.8 billion years ago) to form the ‘banded iron formations’ (BIFs) observed today in Precambrian sedimentary rocks around the world. These BIFs provide a geological record marking a transition point away from the ancient anaerobic world towards modern aerobic Earth. They also indicate a period over which the bio-availability of iron shifted from abundance to limitation, a condition that extends to the modern day. Thus, it is considered likely that the vast majority of extant organisms face the common problem of securing sufficient iron from their environment – a problem that Life on Earth has had to cope with for some 2 billion years. This struggle for iron is exemplified by the competition for this metal amongst co-habiting microorganisms who resort to stealing (pirating) each others iron supplies! The reliance of micro-organisms upon iron can be disadvantageous to them, and to our innate immune system it represents a chink in the microbial armour, offering an opportunity that can be exploited to ward off pathogenic invaders. In order to infect body tissues and cause disease, pathogens must secure all their iron from the host. To fight such infections, the host specifically withdraws available iron through the action of various iron depleting processes (e.g. the release of lactoferrin and lipocalin-2) – this represents an important strategy in our defence against disease. However, pathogens are frequently able to deploy iron acquisition systems that target host iron sources such as transferrin, lactoferrin and hemoproteins, and thus counteract the iron-withdrawal approaches of the host. Inactivation of such host-targeting iron-uptake systems often attenuates the pathogenicity of the invading microbe, illustrating the importance of ‘the battle for iron’ in the infection process. The role of iron sequestration systems in facilitating microbial infections has been a major driving force in research aimed at unravelling the complexities of microbial iron transport processes. But also, the intricacy of such systems offers a challenge that stimulates the curiosity. One such challenge is to understand how balanced levels of free iron within the cytosol are achieved in a way that avoids toxicity whilst providing sufficient levels for metabolic purposes – this is a requirement that all organisms have to meet. Although the systems involved in achieving this balance can be highly variable amongst different microorganisms, the overall strategy is common. On a coarse level, the homeostatic control of cellular iron is maintained through strict control of the uptake, storage and utilisation of available iron, and is co-ordinated by integrated iron-regulatory networks. However, much yet remains to be discovered concerning the fine details of these different iron regulatory processes. As already indicated, perhaps the most difficult task in maintaining iron homeostasis is simply the procurement of sufficient iron from external sources. The importance of this problem is demonstrated by the plethora of distinct iron transporters often found within a single bacterium, each targeting different forms (complex or redox state) of iron or a different environmental condition. Thus, microbes devote considerable cellular resource to securing iron from their surroundings, reflecting how successful acquisition of iron can be crucial in the competition for survival. The aim of this book is provide the reader with an overview of iron transport processes within a range of microorganisms and to provide an indication of how microbial iron levels are controlled. This aim is promoted through the inclusion of expert reviews on several well studied examples that illustrate the current state of play concerning our comprehension of how iron is translocated into the bacterial (or fungal) cell and how iron homeostasis is controlled within microbes. The first two chapters (1-2) consider the general properties of microbial iron-chelating compounds (known as ‘siderophores’), and the mechanisms used by bacteria to acquire haem and utilise it as an iron source. The following twelve chapters (3-14) focus on specific types of microorganism that are of key interest, covering both an array of pathogens for humans, animals and plants (e.g. species of Bordetella, Shigella, , Erwinia, Vibrio, Aeromonas, Francisella, Campylobacter and Staphylococci, and EHEC) as well as a number of prominent non-pathogens (e.g. the rhizobia, E. coli K-12, Bacteroides spp., cyanobacteria, Bacillus spp. and yeasts). The chapters relay the common themes in microbial iron uptake approaches (e.g. the use of siderophores, TonB-dependent transporters, and ABC transport systems), but also highlight many distinctions (such as use of different types iron regulator and the impact of the presence/absence of a cell wall) in the strategies employed. We hope that those both within and outside the field will find this book useful, stimulating and interesting. We intend that it will provide a source for reference that will assist relevant researchers and provide an entry point for those initiating their studies within this subject. Finally, it is important that we acknowledge and thank wholeheartedly the many contributors who have provided the 14 excellent chapters from which this book is composed. Without their considerable efforts, this book, and the understanding that it relays, would not have been possible. Simon C Andrews and Pierre Cornelis

Effects of desiccation on seed germination, cracking, fungal infection and survival in storage of Sterculia foetida L

Seeds of Sterculia foetida were tested for germination following desiccation and subsequent hermetic storage. Whereas seeds at 10.3% moisture content were intact and provided 98% germination, further desiccation reduced germination substantially. The majority of seed coats had cracked after desiccation to 5.1% moisture content. Ability to germinate was not reduced after 12 months' hermetic storage at 10.3% and 7.3% moisture content at 15 degrees C or -18 degrees C, but was reduced considerably at 5.1%. Fungal infection was detected consistently for cracked seeds in germination tests and they did not germinate. However, almost all embryos extracted from cracked seeds germinated if first disinfected with sodium hypochlorite (1%, 5 minutes). In addition. 80 -100% of disinfected extracted embryos from cracked seeds stored hermetically for 28 d at -18 degrees C or -82 degrees C with 3.3% to 6.0% moisture content, and excised embryos stored in this way, were able to germinate. Hence. failure of the very dry seeds of Sterculia foetida to germinate was not due to embryo death from desiccation but to cracking increasing susceptibility to fungal infection upon rehydration. Cracking was associated negatively and strongly with relative humidity and appears to be a mechanical consequence of substantial differences between the isotherms of whole seeds compared with cotyledons and axes.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

The severity of malarial anaemia in Plasmodium chabaudi infections of BALB/c mice is determined independently of the number of circulating parasites

Background: Severe malarial anaemia is a major complication of malaria infection and is multifactorial resulting from loss of circulating red blood cells (RBCs) from parasite replication, as well as immune-mediated mechanisms. An understanding of the causes of severe malarial anaemia is necessary to develop and implement new therapeutic strategies to tackle this syndrome of malaria infection. Methods: Using analysis of variance, this work investigated whether parasite-destruction of RBCs always accounts for the severity of malarial anaemia during infections of the rodent malaria model Plasmodium chabaudi in mice of a BALB/c background. Differences in anaemia between two different clones of P. chabaudi were also examined. Results: Circulating parasite numbers were not correlated with the severity of anaemia in either BALB/c mice or under more severe conditions of anaemia in BALB/c RAG2 deficient mice (lacking T and B cells). Mice infected with P. chabaudi clone CB suffered more severe anaemia than mice infected with clone AS, but this was not correlated with the number of parasites in the circulation. Instead, the peak percentage of parasitized RBCs was higher in CB-infected animals than in AS-infected animals, and was correlated with the severity of anaemia, suggesting that the availability of uninfected RBCs was impaired in CB-infected animals. Conclusion: This work shows that parasite numbers are a more relevant measure of parasite levels in P. chabaudi infection than % parasitaemia, a measure that does not take anaemia into account. The lack of correlation between parasite numbers and the drop in circulating RBCs in this experimental model of malaria support a role for the host response in the impairment or destruction of uninfected RBC in P. chabaudi infections, and thus development of acute anaemia in this malaria model.

The population dynamical consequences of density-dependence in fungal plant pathogens

Almost all stages of a plant pathogen life cycle are potentially density dependent. At small scales and short time spans appropriate to a single-pathogen individual, density dependence can be extremely strong, mediated both by simple resource use, changes in the host due to defence reactions and signals between fungal individuals. In most cases, the consequences are a rise in reproductive rate as the pathogen becomes rarer, and consequently stabilisation of the population dynamics; however, at very low density reproduction may become inefficient, either because it is co-operative or because heterothallic fungi do not form sexual spores. The consequence will be historically determined distributions. On a medium scale, appropriate for example to several generations of a host plant, the factors already mentioned remain important but specialist natural enemies may also start to affect the dynamics detectably. This could in theory lead to complex (e.g. chaotic) dynamics, but in practice heterogeneity of habitat and host is likely to smooth the extreme relationships and make for more stable, though still very variable, dynamics. On longer temporal and longer spatial scales evolutionary responses by both host and pathogen are likely to become important, producing patterns which ultimately depend on the strength of interactions at smaller scales.

Apical leaf necrosis and leaf nitrogen dynamics in diseased leaves: a model study

Apical leaf necrosis is a physiological process related to nitrogen (N) dynamics in the leaf. Pathogens use leaf nutrients and can thus accelerate this physiological apical necrosis. This process differs from necrosis occurring around pathogen lesions (lesion-induced necrosis), which is a direct result of the interaction between pathogen hyphae and leaf cells. This paper primarily concentrates on apical necrosis, only incorporating lesion-induced necrosis by necessity. The relationship between pathogen dynamics and physiological apical leaf necrosis is modelled through leaf nitrogen dynamics. The specific case of Puccinia triticina infections on Triticum aestivum flag leaves is studied. In the model, conversion of indirectly available N in the form of, for example, leaf cell proteins (N-2(t)) into directly available N (N-1(t), i.e. the form of N that can directly be used by either pathogen or plant sinks) results in apical necrosis. The model reproduces observed trends of disease severity, apical necrosis and green leaf area (GLA) and leaf N dynamics of uninfected and infected leaves. Decreasing the initial amount of directly available N results in earlier necrosis onset and longer necrosis duration. Decreasing the initial amount of indirectly available N, has no effect on necrosis onset and shortens necrosis duration. The model could be used to develop hypotheses on how the disease-GLA relation affects yield loss, which can be tested experimentally. Upon incorporation into crop simulation models, the model might provide a tool to more accurately estimate crop yield and effects of disease management strategies in crops sensitive to fungal pathogens.

Evolution of micromorphological and chemical characters in the lichen-forming fungal family Pertusariaceae

Micromorphological characters of the fruiting bodies, such as ascus-type and hymenial amyloidity, and secondary chemistry have been widely employed as key characters in Ascomycota classification. However, the evolution of these characters has yet not been studied using molecular phylogenies. We have used a combined Bayesian and maximum likelihood based approach to trace character evolution on a tree inferred from a combined analysis of nuclear and mitochondrial ribosomal DNA sequences. The maximum likelihood aspect overcomes simplifications inherent in maximum parsimony methods, whereas the Markov chain Monte Carlo aspect renders results independent of any particular phylogenetic tree. The results indicate that the evolution of the two chemical characters is quite different, being stable once developed for the medullary lecanoric acid, whereas the cortical chlorinated xanthones appear to have been lost several times. The current ascus-types and the amyloidity of the hymenial gel in Pertusariaceae appear to have been developed within the family. The basal ascus-type of pertusarialean fungi remains unknown. (c) 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 615-626.

Antagonistic activities of selected fungal isolates against Armillaria mellea

The antagonistic activities of six selected fungal isolates against Armilloria mellea were studied on two different concentrations of three media, on fungicides-amended malt extract agar (MEA) medium, and in glasshouse pots filled with John Innes No.2 compost and natural field soil. Trichoderma hamatum isolate Tham1 was found the most effective in reducing Armillaria growths on both the low and high concentrations of malt extract, potato dextrose and V-8 juice in MEA, potato dextrose agar (PDA) and V-8 juice agar (VJA), respectively, followed by T. harzianum isolate Th2 and T. viride isolate Tv3. Neither dose rate (200 or 2000 mg l(-1)) of fenpropidin allowed any growth of Armillaria on MEA, while that of the antagonists was also completely inhibited or greatly restricted. However, both dose rates of fosetyl-A1 allowed the growth of Armillaria and almost all the antagonists. Data on colony diameters of Armillaria showed Tham1 as the most effective antagonist along with Th2, Th23 and Tv3. Tham1 was also found the most effective in protecting hazel billets from colonization by Armillaria, followed by Th2 and Th23. Compared with 7.1 colonized billets in the inoculated controls, only 1.3, 2.6 and 2.7 billets (out of ten) were colonized, respectively, when protected with these antagonists. The results indicate that the Trichoderma isolates are able to maintain their antagonistic effects on A. mellea under a variety of nutritional, chemical and edaphic regimes. More investigations are needed to develop a system of control for the disease with these potential antagonists.

The effect of carrier substrate, dose rate and time of application on biocontrol efficacy of fungal antagonists against Armillaria root rot of strawberry plants.

In a glasshouse experiment using potted strawberry plants (cv. Cambridge Favourite) as hosts, the effect of selected fungal antagonists grown on 25 or 50 g of mushroom compost containing autoclaved mycelia of Agaricus bisporus, or wheat bran was evaluated against Armillaria mellea. Another glasshouse experiment tested the effect of application time of the antagonists in relation to inoculations with the pathogen. A significant interaction was found between the antagonists, substrates and dose rates. All the plants treated with Chaetomium olivaceum isolate Co on 50 g wheat bran survived until the end of the experiment which lasted 482 days, while none of them survived when this antagonist was added to the roots of the plants on 25 g wheat bran or 25 or 50 g mushroom compost. Dactylium dendroides isolate SP had a similar effect, although with a lower host survival rate of 33.3%. Trichoderma hamatum isolate Tham 1 and T. harzianum isolate Th23 protected 33.3% of the plants when added on 50 g and none when added on 25 g of either substrate, while 66.7% of the plants treated with T. harzianum isolate Th2 on 25 g, or T viride isolate TO on 50 g wheat bran, survived. Application of the antagonists on mushroom compost initially resulted in development of more leaves and healthier plants, but this effect was not sustained. Eventually, plants treated with the antagonists on wheat bran had significantly more leaves and higher health scores. The plants treated with isolate Th2 and inoculated with Armillaria at the same time had a survival rate of 66.7% for the duration of the experiment (475 days), while none of them survived that long when the antagonist and pathogen were applied with an interval of 85 days in either sequence. C. olivaceum isolate Co showed a protective effect only, as 66.7% of the plants survived when they were treated with the antagonist 85 days before inoculation with the pathogen, while none of them survived when the antagonist and pathogen were applied together or the infection preceded protection.

Cultural techniques for improvement in biocontrol potential of fungal antagonists for biological control of Armillaria root rot of strawberry plants under glasshouse conditions

Several in vitro and in vivo experiments were conducted to develop an effective technique for culturing potential fungal antagonists (isolates of Trichoderma harzianum, Dactylium dendroides, Chaetomium olivaceum and one unidentified fungus) selected for activity against Armillaria mellea. The antagonists were inoculated onto (1) live spawn of the oyster mu shroom (Pleurotus ostreatus), (2) extra-moistened or sucrose-enriched mushroom composts containing living or autoclaved mycelia of P. ostreatus or Agaricus bisporus (button mushroom), (3) pasteurized compost with or without A. bisporus mycelium, wheat bran, wheat germ and (4) spent mushroom composts with living mycelia of A. bisporus, P. ostreatus or Lentinus edodes (the Shiitake mushroom). In one experiment, a representative antagonist (isolate Th2 of T. harzianum) was grown together with the A. bisporus mycelium, while in another one, the antagonist was first grown on wheat germ or wheat bran and then on mushroom compost with living mycelium of A. bisporus. Some of the carrier substrates were then added to the roots of potted strawberry plants in the glasshouse to evaluate their effectiveness against the disease. The antagonists failed to grow on the spawn of P. ostreatus even after reinoculations and prolonged incubation. Providing extra moisture or sucrose enrichment also did not improve the growth of Th2 on mushroom composts in the presence of living mycelia of A. bisporus or P. ostreatus. The antagonist, however, grew rapidly and extensively on mushroom compost with autoclaved mycelia, and also on wheat germ and wheat bran. Colonization of the substrates by the antagonist was positively correlated with its effectiveness in the glasshouse studies. Whereas only 33.3% of the inoculated control plants survived in one experiment monitored for 560 days, 100% survival was achieved when Th2 was applied on wheat germ or wheat bran. Growth of the antagonist alone on pasteurized or sterilized compost (without A. bisporus mycelia) and simultaneous growth of the antagonist and mushroom on pasteurized compost did not improve survival over the inoculated controls, but growth over mushroom compost with the living mycelium resulted in 50% survival rate. C. olivaceum isolate Co was the most effective, resulting in overall survival rate of 83.3% compared with only 8.3% for the inoculated and 100% for the uninoculated (healthy) controls. This antagonist gave the highest survival rate of 100% on spent mushroom compost with L. edodes. T harzianum isolate Th23, with 75% survival rate, was the most effective on spent mushroom compost with P. ostreatus, while D. dendroides isolate SP resulted in equal survival rates of 50% on all the three mushroom composts.

Comparisons between the in vitro and in vivo efficacies of potential fungal antagonists of Armillaria mellea

Seventeen fungal isolates were tested in vitro as potential antagonists of two isolates of the root rot pathogen, Armillaria mellea. Some of the isolates were also added on mushroom composts with living mycelia to the roots of Armillaria-inoculated potted strawberry plants in the glasshouse to find out if they had the same degree of efficacy against the disease. Dactylium dendroides isolate SP was the most effective in reducing mycelial growth of A. mellea isolate 1 (Am1), followed by Trichoderma harzianum isolate Th2 and T. viride isolate Tv4. Th2, Th22, Tv3 and SP grew extensively over Am1 colonies, disintegrating the rhizomorphs. Isolate Tham1 of T hamatum was the most effective in reducing mycelial growth of A. mellea isolate 2 (Am2), followed by Tv3. Th12, Th22, Tv1, Tv3 and SP inhibited the initiation and growth of rhizomorphs of Am2. Regeneration tests showed that both Am1 and Am2 attacked by Trichoderma isolates and SP were no longer viable. Th23 and SP were almost as effective in vivo as in vitro. But isolate Co of Chaetomium olivaceum, which was ineffective in vitro, was found effective in vivo. Conversely, Th2, which exhibited good antagonistic activity in vitro, performed poorly in vivo. These results show that the in vitro and in vivo efficacies of potential antagonists may not necessarily be closely correlated. Hence, there is a danger that potentially effective isolates may be discarded if decisions are made only on the basis of preliminary screening tests carried out under laboratory conditions.