Epiphytic proliferation of Zymoseptoria tritici isolates on resistant wheat leaves.

The wheat pathogen Zymoseptoria tritici is capable of a long period of pre-invasive epiphytic growth. Studies have shown that virulent isolates vary in the extent, duration and growth form of this epiphytic growth, and the fungus has been observed to undergo behaviours such as asexual reproduction by budding and vegetative fusion of hyphae on the leaf surface. This epiphytic colonisation has been investigated very little during interactions in which an isolate of Z. tritici is unable to colonise the apoplast, as occurs during avirulence. However, avirulent isolates have been seen to undergo sexual crosses in the absense of leaf penetration, and it is widely accepted that the main point of distinction between virulent and avirulent isolates occurs at the point of attempted leaf penetration or attempted apoplastic growth, which fails in the avirulent case. In this work, we describe extensive epiphytic growth in three isolates which are unable or have very limited ability to invade the leaf, and show that growth form is as variable as for fully virulent isolates. We demonstrate that during certain interactions, Z. tritici isolates rarely invade the leaf and form pycnidia, but induce necrosis. These isolates are able to achieve higher epiphytic biomass than fully virulent isolates during asymptomatic growth, and may undergo very extensive asexual reproduction on the leaf surface. These findings have implications for open questions such as whether and how Z. tritici obtains nutrients on the leaf surface and the nature of its interaction with wheat defences.

The North Wyke Farm Platform: effect of temperate grassland farming systems on soil moisture contents, runoff and associated water quality dynamics.

The North Wyke Farm Platform was established as a United Kingdom national capability for collaborative research, training and knowledge exchange in agro-environmental sciences. Its remit is to research agricultural productivity and ecosystem responses to different management practices for beef and sheep production in lowland grasslands. A system based on permanent pasture was implemented on three 21-ha farmlets to obtain baseline data on hydrology, nutrient cycling and productivity for 2 years. Since then two farmlets have been modified by either (i) planned reseeding with grasses that have been bred for enhanced sugar content or deep-rooting traits or (ii) sowing grass and legume mixtures to reduce nitrogen fertilizer inputs. The quantities of nutrients that enter, cycle within and leave the farmlets were evaluated with data recorded from sensor technologies coupled with more traditional field study methods. We demonstrate the potential of the farm platform approach with a case study in which we investigate the effects of the weather, field topography and farm management activity on surface runoff and associated pollutant or nutrient loss from soil. We have the opportunity to do a full nutrient cycling analysis, taking account of nutrient transformations in soil, and flows to water and losses to air. The NWFP monitoring system is unique in both scale and scope for a managed land-based capability that brings together several technologies that allow the effect of temperate grassland farming systems on soil moisture levels, runoff and associated water quality dynamics to be studied in detail. HIGHLIGHTS: Can meat production systems be developed that are productive yet minimize losses to the environment?The data are from an intensively instrumented capability, which is globally unique and topical.We use sensing technologies and surveys to show the effect of pasture renewal on nutrient losses.Platforms provide evidence of the effect of meteorology, topography and farm activity on nutrient loss.

A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.

Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.

Stable transformation of erysiphe graminis an obligate biotrophic pathogen of barley.

Barley powdery mildew, Erysiphe graminis f.sp. hordei, is an obligate biotrophic pathogen and as such cannot complete its life cycle without a living host. The inability to transform this fungus and manipulate its genome has constrained research towards understanding its life cycle and pathogenicity. Here we describe an in planta transformation system based on delivery of DNA using a gold-particle gun and selection using benomyl or bialaphos. Using this method, we consistently obtained stable transformants with efficiencies comparable to other filamentous fungi. Stable expression of the beta-glucuronidase in E. graminis was demonstrated by co-transforming the uidA gene with the selectable markers.

Involvement of cAMP and protein kinase A in conidial differentiation by Erysiphe graminis f. sp. hordei.

Erysiphe graminis f. sp. hordei, the causal agent of barley powdery mildew, is an obligate biotroph. On arrival on the host, a primary germ tube (PGT) emerges from the conidium. An appressorial germ tube (AGT) then appears, forms an appressorium, and effects host penetration. Such developmental precision may be due to multiple, plant-derived signals and to endogenous tactile and chemical signals. The transduction mechanism remains obscure. The isolation of an expressed sequence tag (EST) homologue of the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) enabled the corresponding gene to be characterized and the transcript to be identified in conidia and in PGT and AGT stage spores. cAMP-dependent PKA activity was detected in ungerminated conidia. These data suggest that PKA and cAMP are involved in conidial development. To substantiate this we exploited the responses of developing conidia to various surfaces, including exposure to the host leaf (fully inductive to AGT formation), cellulose membrane (semi-inductive), and glass (non-inductive). Assessment of fungal development, following application of exogenous cAMP or cAMP analogues, revealed that, at different concentrations and on different surfaces, cAMP either promoted or inhibited conidial differentiation. Various PKA inhibitors were tested for their effect on PKA activity and conidial development. A negative correlation was established between PKA inhibition in vitro and fungal development in vivo. Taken collectively, these data suggest that PKA and cAMP play a role in conidial differentiation in this obligate, plant-pathogenic fungus.

Walking into the unknown: a 'step down' PCR-based technique leading to the direct sequence analysis of flanking genomic DNA.

We describe a novel and efficient PCR-based technique for walking into unknown flanking genomic DNA without recourse to protracted laborious library screening for overlapping sequences. This two component 'hot start' and 'step down' PCR method uses 6x1 microg of genomic DNA (ca 20kb in length) restricted with six different endonucleases and ligated to adaptors with the inclusion of two further restriction enzymes to prevent self-ligation. This allowed us to walk, in a single step, up to 6kb into flanking DNA and gave sufficient PCR products for up to 200 different walking experiments. This technology enabled us to clone and characterize the previously elusive 5' sequence of the barley powdery mildew chitin synthase gene, BgChs2, which includes a myosin motor-like sequence fused to a type V chitin synthase gene.

Expression and sequence analysis of the Blumeria graminis mitogen-activated protein kinase genes, mpk1 and mpk2.

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine kinases which play a pivotal role in signal transduction processes in eukaryotic cells. Using degenerate PCR primer design based on published and aligned MAP kinase sequences we have cloned and characterised two MAP kinase genes from the barley powdery mildew fungus, Blumeria graminis. We have utilised 'step down' PCR to attain the full length mildew genomic clones. The single-copy genes, named mpk1 and mpk2, encode putative proteins of 356 and 410 amino acids and carry three and four introns, respectively. Expression studies, using RT-PCR, reveal a differing pattern of tissue gene expression with mpk1 and mpk2 during germling morphogenesis and this is compared with the constitutive expression of the 'control' beta-tubulin gene.

Homologous transformation of Cephalosporium acremonium with the nitrate reductase-encoding gene (niaD).

We report the development of a homologous transformation system for Cephalosporium acremonium using the niaD gene of the nitrate assimilation (NA) pathway. Mutants in the NA pathway were selected on the basis of chlorate resistance by conventional means. Screening procedures were developed to differentiate between nitrate reductase apoprotein structural gene mutants (niaD) and molybdenum cofactor gene mutants (cnx) as wt C. acremonium, unlike most filamentous fungi, fails to grow on minimal medium with hypoxanthine as a sole source of nitrogen. Phage clones carrying the niaD gene were isolated from a C. acremonium library constructed in lambda EMBL3 using the A. nidulans niaD gene as a heterologous probe. An 8.6-kb EcoRI fragment was subcloned into pUC18, and designated pSTA700. pSTA700 was able to transform stable niaD mutants to NA at a frequency of up to 40 transformants per microgram DNA. Transformants were easily visible since the background growth was low and no abortives were observed. Gene replacements, single copy homologous integration and complex multiple integrations were observed. The niaD system was used to introduce unselected markers for hygromycin B resistance and benomyl resistance into C. acremonium by cotransformation.

Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans.

Genomic clones containing the entire crnA-niiA-niaD gene cluster of Aspergillus nidulans have been isolated, and the structures of the niiA and niaD genes have been determined by nucleotide sequence analysis. This gene cluster is required for the assimilation of nitrate in A. nidulans, and the three genes encode a product required for nitrate uptake and the enzymes, nitrite reductase and nitrate reductase, respectively. The putative coding sequences, as deduced by comparison to cDNA clones of both niiA and niaD, are interrupted by multiple small introns, and the two genes are divergently transcribed. Identification and characterization of specific mRNAs involved in nitrate assimilation indicates that only monocistronic transcripts are involved, and that the approximate sizes of these transcripts are 1.6 kb, 3.4 kb and 2.8 kb for crnA, niiA and niaD, respectively. The results also indicate that control of niiA and niaD gene expression is mediated by the levels of mRNA accumulation, in response to the source of nitrogen in the growth medium. Two types of transcripts for niiA were observed.

Bacteria associated with Stagonospora (Septoria) nodorum increase pathogenicity of the fungus.

In studies with a laboratory isolate of the fungal pathogen Stagonospora (Septoria) nodorum three different isolates of bacteria were closely associated with the fungus. Bacteria were also closely associated with fresh isolates of S. nodorum obtained from artificially and naturally infected field material. Although a range of bacteria was isolated, only one type of bacterium was found to be associated with each isolate of S. nodorum. In co-inoculation studies with pycnidiospores of the fungus on detached leaves, some of the bacterial isolates significantly increased the pathogenicity of the fungus, particularly Xanthomonas maltophilia, Sphingobacterium multivorum, Enterobacter agglomerans and Erwinia amylovora. Evidence is presented indicating that one of the ways that the 'helper bacteria' may assist in the establishment of infections is by the production of lipases that were not detected in germinating fungal spores.

Detection of fungi in sinus fluid of patients with allergic fungal rhinosinusitis.

We aim to develop a rapid, accurate and sensitive diagnostic assay with which to detect the surface antigens of fungi thought to be involved in allergic fungal rhinosinusitis (AFRS), by assessing the usefulness of immunofluorescence microscopy (IMF) and enzyme linked immuno-absorbent assays (ELISA). The age, sex, clinical symptoms and signs, imaging (CT and/or MRI), microbiological subculture data, sinus contents, blood eosinophilia, aspergillosis precipitins, radioallergoabsorbent technology (RAST) for fungal allergens and histopathology were performed on individuals undergoing endoscopic sinus surgery for suspected AFRS. Thirteen patients were examined, and five monoclonal antibodies raised to the surface washings of various fungi were found to recognize and differentiate between fungal species implicated in sinus disease, i.e. Aspergillus niger, Alternaria alternata, Cochliobolus lunata, Penicillium expansum and Cladosporium species. The IMF microscopy proved to be a useful assay to distinguish visually between the cultured fungi, but was less useful for visualization of fungi in the patient samples. However, ELISA assays with 5 monoclonal antibodies gave clear and unambiguous data as to the presence of certain fungi within the patient samples. There is good correlation between the ELISA data and the pathology findings. This preliminary study suggests that both IMF and ELISA techniques may offer an important advance in this area.

Atomic-scale structure of biogenic materials by total X-ray diffraction: a study of bacterial and fungal MnOx.

Biogenic materials are produced by microorganisms and are typically found in a nanophase state. As such, they are difficult to characterize structurally. In this report, we demonstrate how high-energy X-ray diffraction and atomic pair distribution function analysis can be used to determine the atomic-scale structures of MnO(x) produced by bacteria and fungi. These structures are well-defined, periodic, and species-specific, built of Mn-O(6) octahedra forming birnessite-type layers and todorokite-type tunnels, respectively. The inherent structural diversity of biogenic material may offer opportunities for practical applications.

Of genes and genomes, needles and haystacks: Blumeria graminis and functionality.

SUMMARY Here, we consider the barley powdery mildew fungus, Blumeria graminis (DC Speer) f.sp. hordei (Marchal), and review recent research which has added to our understanding of the biology and molecular biology which underpins the asexual life cycle of this potentially devastating pathogen. We focus on the early stages of the host-pathogen interaction and report current understanding in the areas of leaf perception, fungal signal transduction and host-imposed oxidative stress management. Through this, it is becoming increasingly clear how closely and subtly both sides of the relationship are regulated. Collectively, however, this review highlights the high degree of complexity in working with an obligate parasite. Our experiences suggest that we would make more efficient progress towards understanding the basis of susceptibility and resistance to this true obligate biotroph if its genome sequence was available.

Differential expression of two Blumeria graminis chitin synthase genes.

Abstract Two Blumeria graminis chitin synthase genes, designated BgChs1 and BgChs2 were cloned and characterized following the synthesis and use of degenerate PCR primers designed to the conserved regions of fungal chitin synthase (Chs) genes. Their sequences revealed high similarity with the Chs genes previously cloned from other fungi and placed BgChs1 and BgChs2 with the classes I and V, respectively. Each gene was present as a single copy within the barley powdery mildew genome. Semi-quantitative RT-PCR assays revealed BgChs1 to be up-regulated at both the primary germ tube (PGT) and appressorial germ tube (AGT) stages of differentiation whilst the BgChs2 transcript was up-regulated at the PGT stage. The B. graminisbeta-tubulin gene was used as a control for all RT-PCR reactions. The BgChs1 transcript was some 30 fold less abundant than the beta-tubulin transcript and BgChs2 was some 30 fold rarer than the BgChs1 transcript. The effects of the chitin substrate analogues nikkomycin Z and polyoxin D on conidial morphogenesis were assessed. These nucleoside peptide inhibitors did not affect germination but both polyoxin D and nikkomycin Z treatment led to a large population of abnormally swollen 'balloon-shaped' AGTs, whilst by 12 h after inoculation polyoxin treatment caused the swollen germ tubes to burst.

The tomato powdery mildew fungus Oidium neolycopersici.

UNLABELLED: summary Pathogen: Powdery mildew fungus; Ascomycete although sexual stage is yet to be found; an obligate biotroph. IDENTIFICATION: Superficial mycelium with hyaline hyphae; unbranched erect conidiophores; conidia, ellipsoid-ovoid or doliform, 22-46 x 10-20 microm, lack fibrosin bodies; conidia formed singly, rarely in short chains of 2-6 conidia; appressoria lobed to multilobed, rarely nipple-shaped. Pseudoidium species. HOST RANGE: Broad, reported to attack over 60 species in 13 plant families, particularly members of the Solanaceae and Curcubitaceae. SYMPTOMS: Powdery white lesions on all aerial plant parts except the fruit. In severe outbreaks the lesions coalesce and disease is debilitating. Agronomic importance: Extremely common in glasshouse tomatoes world wide but increasing in importance on field grown tomato crops. CONTROL: Chemical control and breeding programmes for disease resistance.

The barley powdery mildew protein kinase C gene, pkc1 and pkc-like gene, are differentially expressed during morphogenesis.

summary Protein kinase C agonist assays revealed the phorbol ester, phorbol 12-myristate 13-acetate, invoked germling morphogenesis and enhanced PKC activity in Blumeria graminis. No antagonist of mildew PKC activity was found but the data fuelled a hunt for powdery mildew pkc genes. Oligonucleotides, designed on the basis of conserved ATP-binding and kinase domains within the catalytic core of eukaryotic protein kinase proteins, were used as primers to amplify chromosomal and cDNA fragments from the barley powdery mildew fungus graminis. Three kinase gene fragments were isolated (pkc1, pkc-like and cpka) and the full length genomic sequences of the mildew pkc and pkc-like genes were determined by 'step down' PCR. RT-PCR transcript profiles showed the three genes to be differentially regulated during germling morphogenesis.

Gene expression in nematode-infected plant roots.

A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.

Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase.

The Neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed Aspergillus nidulans gene libraries. These clones have a common HindIII 1.85 kb fragment. This A. nidulans nucleotide stretch hybridises to a N. crassa 2.7 kb BamHI fragment of wild type DNA but not to a co-migrating fragment from the DNA of the N. crassa am132 deletion mutant. One A. nidulans clone was shown to complement the N. crasse am132 deletion strain. The N. crassa transformants show low levels (approximately 5%) of heterologous glutamate dehydrogenase activity. The A. nidulans gdhA gene was found to locate in N. crassa at both the "homologous" (i.e. am) site as well as non-homologous sites. Partial nucleotide analysis of the fragment has revealed the 5' end of the locus and considerable homology with the N. crassa am gene. We concluded that we have cloned the A. nidulans gdhA gene.

Nucleotide sequence and regulation of expression of the Aspergillus nidulans gdhA gene encoding NADP dependent glutamate dehydrogenase.

The nucleotide sequence of the Aspergillus nidulans gdhA gene encoding NADP linked glutamate dehydrogenase has been determined and Northern blot analysis used to study the regulation of expression of this gene. The gdhA gene is 1485 nucleotides long and, by comparison with the corresponding Neurospora crassa am gene, has two putative introns of 53 nucleotides and a protein encoding region of 1380 nucleotides that codes for an inferred protein of 49.63 kDa which shows regions of homology with glutamate dehydrogenase proteins from a range of organisms. mRNA analysis of wild-type mycelium grown under a variety of conditions shows that: (a) the highest levels are seen with glucose as the carbon source with inorganic nitrogen; and (b) no gdhA mRNA is detectable when cells are transferred to amino acids as sole carbon source, closely matching the observed glutamate dehydrogenase activity levels under identical conditions. The results presented strongly suggest that a good carbon source is a prerequisite for transcription, but the molecular mechanism responsible is unclear.

The pattern of plant annexin gene expression.

Peptide sequence data derived from a plant annexin, P34 [Smallwood, Keen & Bowles (1990) Biochem. J. 270, 157-161] was used to design amplimers for PCR. A unique fragment of 95 bp, amplified from tomato (Lycopersicon esculertum) genomic DNA, was used in Northern analyses and demonstrated a differential pattern of expression in vegetative tissues of tomato, potato (Solanum tuberosum) and barley (Hordeum vulgare). The tissue-specific abundance of the annexin transcript was found to correlate closely with abundance of annexin protein as revealed by their partial purification and analysis with antisera specific for annexins isolated from tomato suspension-culture cells.