Weathering microenvironments on feldspar surfaces: implications for understanding fluid-mineral reactions in soils

The mechanisms by which coatings develop on weathered grain surfaces, and their potential impact on rates of fluid-mineral interaction, have been investigated by examining feldspars from a 1.1 ky old soil in the Glen Feshie chronosequence, Scottish highlands. Using the focused ion beam technique, electron-transparent, foils for characterization by transmission electron microscopy were cut from selected parts of grain surfaces. Some parts were bare whereas others had accumulations, a few micrometres thick, of Weathering products, often mixed with mineral and microbial debris. Feldspar exposed at bare grain surfaces is crystalline throughout and so there is no evidence for the presence of the amorphous 'leached layers' that typically form in acid-dissolution experiments and have been described from some natural Weathering contexts. The weathering products comprise sub-mu m thick crystallites of an Fe-K aluminosilicate, probably smectite, that have grown within an amorphous and probably organic-rich matrix. There is also evidence for crystallization of clays having been mediated by fungal hyphae. Coatings formed within Glen Feshie soils after similar to 1.1 ky are insufficiently continuous or impermeable to slow rates Of fluid-feldspar reactions, but provide valuable insights into the complex Weathering microenvironments oil debris and microbe-covered mineral surfaces.

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.

Studies on the genus Paecilomyces in China V. Taifanglania gen. nov for some monophialidic species

A rare monophialidic fungus, Taifanglania hechuanensis gen. & sp. nov., was isolated from soil oil the banks of Jialin River, Hechuan, Chongqing City during a survey of soil-borne filamentous fungi from different phytogeographical areas in China. It is described and illustrated in this paper. A further eight monophialidic species of Paecilomyces are transferred to the genus. Diagnosis features of the new genus are white, grey, straw yellow or brown to black colonies on Czapek agar. Conidiophores are always absent or simple. Phialides are solitary, consisting of a cylindrical or ellipsoidal swollen basal portion, tapering into a thin neck, directly arising on vegetative hyphae or prophialides, sometimes consisting of a whorl of 2 to 3 phialides oil simple conidiophores. Conidia arc one-celled, hyaline, smooth-walled, subglobose, ellipsoidal or fusiform, having or no the connective between conidia and being thermotolerant. The new species is characterized by pale yellow to grey-yellow colonies, solitary phialides with ail ellipsoidal or fusiform basal portion that arise directly from the vegetative hyphae, big conidia (3.1-)3.9-8.7 x ( 1.7-)2.1-4.7(-5.1) mu m with the connective, and thermotolerant growth. A molecular study based oil the nucleotidic sequences of the SSU rDNA and ITS regions support the status of T. hechuanensis as a new species and Taifanglania as a new genus.

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.

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.

Proteogenomics and in silico structural and functional annotation of the barley powdery mildew Blumeria graminis f. sp. hordei

Blumeria graminis is an economically important obligate plant-pathogenic fungus, whose entire genome was recently sequenced and manually annotated using ab initio in silico predictions [7]. Employing large scale proteogenomic analysis we are now able to verify independently the existence of proteins predicted by 24% of open reading frame models. We compared the haustoria and sporulating hyphae proteomes and identified 71 proteins exclusively in haustoria, the feeding and effector-delivery organs of the pathogen. These proteins are ‘significantly smaller than the rest of the protein pool and predicted to be secreted. Most do not share any similarities with Swiss–Prot or Trembl entries nor possess any identifiable Pfam domains. We used a novel automated prediction pipeline to model the 3D structures of the proteins, identify putative ligand binding sites and predict regions of intrinsic disorder. This revealed that the protein set found exclusively in haustoria is significantly less disordered than the rest of the identified Blumeria proteins or random (and representative) protein sets generated from the yeast proteome. For most of the haustorial proteins with unknown functions no good templates could be found, from which to generate high quality models. Thus, these unknown proteins present potentially new protein folds that can be specific to the interaction of the pathogen with its host.

A novel plant-fungus symbiosis benefits the host without forming mycorrhizal structures

Most terrestrial plants form mutually beneficial symbioses with specific soil-borne fungi known as mycorrhiza. In a typical mycorrhizal association, fungal hyphae colonize plant roots, explore the soil beyond the rhizosphere and provide host plants with nutrients that might be chemically or physically inaccessible to root systems. Here, we combined nutritional, radioisotopic (33P) and genetic approaches to describe a plant growth promoting symbiosis between the basidiomycete fungus Austroboletus occidentalis and jarrah (Eucalyptus marginata), which has quite different characteristics. We show that the fungal partner does not colonize plant roots; hyphae are localized to the rhizosphere soil and vicinity and consequently do not transfer nutrients located beyond the rhizosphere. Transcript profiling of two high-affinity phosphate (Pi) transporter genes (EmPHT1;1 and EmPHT1;2) and hyphal-mediated 33Pi uptake suggest that the Pi uptake shifts from an epidermal to a hyphal pathway in ectomycorrhizal plants (Scleroderma sp.), similar to arbuscular mycorrhizal symbioses, whereas A. occidentalis benefits its host indirectly. The enhanced rhizosphere carboxylates are linked to growth and nutritional benefits in the novel symbiosis. This work is a starting point for detailed mechanistic studies on other basidiomycete–woody plant relationships, where a continuum between heterotrophic rhizosphere fungi and plant beneficial symbioses is likely to exist.

Do arbuscular mycorrhizas or heterotrophic soil microbes contribute toward plant acquisition of a pulse of mineral phosphate?

Aims: We investigated the role of arbuscular mycorrhizal fungi (AMF) and heterotrophic soil microbes in the uptake of phosphorus (P) by Trifolium subterraneum from a pulse. Methods: Plants were grown in sterilised pasture field soil with a realistic level of available P. There were five treatments, two of which involved AMF: 1) unsterilised field soil containing a community of AMF and heterotrophic organisms; 2) Scutellospora calospora inoculum (AMF); 3) microbes added as filtrate from the field soil; 4) microbes added as filtrate from the S. calospora inoculum; 5) no additions, i.e. sterilised field soil. After 11 weeks, plants were harvested: 1 day before (day 0), 1 day after (day 2) and 7 days after (day 8) the pulse of P (10 mg kg−1). Results: There was no difference among treatments in shoot and root dry weight, which increased from day 0 to day 8. At day 0, shoots and roots of plants in the colonised treatments had higher P and lower Mn concentrations. After the pulse, the rate of increase in P concentration in the shoots was slower for the colonised plants, and the root Mn concentration declined by up to 50 % by day 2. Conclusions: Plants colonised by AMF had a lower rate of increase in shoot P concentration after a pulse, perhaps because intraradical hyphae accumulated P and thus reduced its transport to the shoots.

Dual mycorrhizal associations of jarrah (Eucalyptus marginata) in a nurse-pot system

Jarrah (Eucalyptus marginata Donn ex Sm.) plants, like many other eucalypts, can form symbiotic associations with both arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi. To study this tripartite relationship we developed a novel nurse-pot system to allow us to investigate the extent and temporal colonisation dynamics of jarrah by two AM species (Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler comb. nov. and Scutellospora calospora Nicol. & Gerd.) and two putative ECM species (Austroboletus occidentalis Watling & N.M. Greg. and Scleroderma sp.) and their potential effects on jarrah growth and nutrition. Our nurse-pot system, using jarrah as both the nurse plant and test plant, was developed to establish extraradical hyphal networks of both AM and ECM fungi that act as single or dual inoculum for test plants. Mycorrhizal colonisation was described and quantified, and growth and nutritional effects measured and analysed. Mycorrhizal colonisation increased with time for the test seedlings exposed to hyphae networks from S. calospora and Scleroderma sp. The nurse-pot system was effective at initiating colonisation of functioning AM or (putative) ECM systems separately but the ECM symbiosis was inhibited where a dual AM + ECM inoculum (R. irregularis and Scleroderma sp.) was present. The presence of S. calospora, A. occidentalis and Scleroderma sp. individually significantly increased the shoot biomass of seedlings compared with non-mycorrhizal controls. The two AM isolates had different physiological effects on jarrah plants. S. calospora improved growth and micronutrient uptake of jarrah seedlings whereas no positive response was observed with R. irregularis. In addition, as an interesting observation, the non-responsive AM fungus R. irregularis suppressed the ECM symbiosis in dually inoculated plants where ECM structures, positive growth response and nutritional effects were absent. When inoculated individually, ECM isolates dominated the growth response and uptake of P and other nutrients in this dual symbiotic plant. Despite the positive growth response in the A. occidentalis treatment, ECM structures were not observed in either nurse or test seedlings. From the effects of A. occidentalis on jarrah we hypothesise that this fungus forms a functional mycorrhizal-type partnership even without forming archetypal structures in and on the root

The diversity of arbuscular mycorrhizas of selected Australian Fabaceae

Members of the Australian native perennial Fabaceae have been little explored with regard to their root biology and the role played by arbuscular mycorrhizal (AM) fungi in their establishment, nutrition and long-term health. The ultimate goal of our research is to determine the dependency of native perennial legumes on their co-evolved AM fungi and conversely, the impact of AM fungal species in agricultural fields on the productivity of sown native perennial legume pastures. In this paper we investigate the colonisation morphology in roots and the AMF, identified by spores extracted from rhizosphere soil, from three replicate plots of each of the native legumes, Cullen australasicum, C. tenax and Lotus australis and the exotic legumes L. pedunculatus and Medicago sativa. The plants were grown in an agricultural field. The level and density of colonisation by AM fungi, and the frequency of intraradical and extraradical hyphae, arbuscules, intraradical spores and hyphal coils all differed between host plants and did not consistently differ between native and exotic species. However, there were strong similarities between species in the same genus. The three dominant species of AM fungi in rhizosphere soil also differed with host plant, but one fungus (Glomus mosseae) was always the most dominant. Sub-dominant AM species were the same between species in the same genus. No consistent differences in dominant spores were observed between the exotic and native Fabaceae species. Our results suggest that plant host influences the mycorrhizal community in the rhizosphere soil and that structural and functional differences in the symbiosis may occur at the plant genus level, not the species level or due to provenance.

Ectomycorrhizal symbiosis can enhance plant nutrition through improved access to discrete organic nutrient patches of high resource quality

It is known that roots can respond to patches of fertility; however, root proliferation is often too slow to exploit resources fully, and organic nutrient patches may be broken down and leached, immobilized or chemically fixed before they are invaded by the root system. The ability of fungal hyphae to exploit resource patches is far greater than that of roots due to their innate physiological and morphological plasticity, which allows comprehensive exploration and rapid colonization of resource patches in soils. The fungal symbionts of ectomycorrhizal plants excrete significant quantities of enzymes such as chitinases, phosphatases and proteases. These might allow the organic residue to be tapped directly for nutrients such as N and P. Pot experiments conducted with nutrient-stressed ectomycorrhizal and control willow plants showed that when high quality organic nutrient patches were added, they were colonized rapidly by the ectomycorrhizal mycelium. These established willows (0.5 m tall) were colonized by Hebeloma syrjense P. Karst. for 1 year prior to nutrient patch addition. Within days after patch addition, colour changes in the leaves of the mycorrhizal plants (reflecting improved nutrition) were apparent, and after I month the concentration of N and P in the foliage of mycorrhizal plants was significantly greater than that in non-mycorrhizal plants subject to the same nutrient addition. It seems likely that the mycorrhizal plants were able to compete effectively with the wider soil microbiota and tap directly into the high quality organic resource patch via their extra-radical mycelium. We hypothesize that ectomycorrhizal plants may reclaim some of the N and P invested in seed production by direct recycling from failed seeds in the soil. The rapid exploitation of similar discrete, transient, high-quality nutrient patches may have led to underestimations when determining the nutritional benefits of ectomycorrhizal colonization.

‘Symptomless’ infection by Botrytis cinerea

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection.