Pochonia chlamydosporia: Advances and Challenges to Improve Its Performance as a Biological Control Agent of Sedentary Endo-parasitic Nematodes.

The nematophagous fungus Pochonia chlamydosporia var. chlamydosporia is one of the most studied biological control agents against plant (semi-) endo-parasitic nematodes of the genera Globodera, Heterodera, Meloidogyne, Nacobbus and, more recently, Rotylenchulus. In this paper we present highlights from more than three decades of worldwide research on this biological control agent. We cover different aspects and key components of the complex plant-fungus-nematode tri-trophic interaction, an interaction that needs to be addressed to ensure the efficient use of P. chlamydosporia as a biopesticide as part of an integrated pest management approach.

High penetrance, recurrent attacks and thrombus formation in a family with hereditary coproporphyria.

Hereditary coproporphyria (HCP) is the rarest of the autosomal dominant acute porphyrias with an estimated incidence of 0.02 per 10 million per year. HCP has been considered to be mild in presentation compared with the more common acute intermittent porphyria although there is limited information comparing the subtypes. Penetrance in the acute porphyrias is low with 90% of patients with a mutation never exhibiting symptoms. We present seven members from a family with HCP with a novel mutation in whom penetrance and severity are high. In addition, they appear to have a high rate of veno-thromboembolism. Penetrance is confirmed at 57% but is suspected to be 71%. The first patient experienced life-threatening complications, four of the seven have had recurrent attacks and the development of opioid dependence has complicated management. The case series documents the impact of a new mRNA interference molecule givosiran as well as a plan for embryo selection which is not commonly used in porphyria. The use of ketamine for the treatment of acute attacks is also documented for the first time in the porphyria literature. The use of international registries would aid the characterisation and management of this very rare disease.

Trials within trials? Researcher, funder and ethical perspectives on the practicality and acceptability of nesting trials of recruitment methods in existing primary care trials.

BACKGROUND: Trials frequently encounter difficulties in recruitment, but evidence on effective recruitment methods in primary care is sparse. A robust test of recruitment methods involves comparing alternative methods using a randomized trial, 'nested' in an ongoing 'host' trial. There are potential scientific, logistical and ethical obstacles to such studies. METHODS: Telephone interviews were undertaken with four groups of stakeholders (funders, principal investigators, trial managers and ethics committee chairs) to explore their views on the practicality and acceptability of undertaking nested trials of recruitment methods. These semi-structured interviews were transcribed and analysed thematically. RESULTS: Twenty people were interviewed. Respondents were familiar with recruitment difficulties in primary care and recognised the case for 'nested' studies to build an evidence base on effective recruitment strategies. However, enthusiasm for this global aim was tempered by the challenges of implementation. Challenges for host studies included increasing complexity and management burden; compatibility between the host and nested study; and the impact of the nested study on trial design and relationships with collaborators. For nested recruitment studies, there were concerns that host study investigators might have strong preferences, limiting the nested study investigators' control over their research, and also concerns about sample size which might limit statistical power. Nested studies needed to be compatible with the main trial and should be planned from the outset. Good communication and adequate resources were seen as important. CONCLUSIONS: Although research on recruitment was welcomed in principle, the issue of which study had control of key decisions emerged as critical. To address this concern, it appeared important to align the interests of both host and nested studies and to reduce the burden of hosting a recruitment trial. These findings should prove useful in devising a programme of research involving nested studies of recruitment interventions.

Use of molecular methods for the detection of fungal spores.

Traditional methods for the isolation and identification of fungal spores can be time-consuming and laborious. DNA-based methods for fungal detection can be used to detect the spores of plant-pathogenic fungi. Air borne spores can be collected and identified by PCR allowing identification of the species.

UK research staff perspectives on improving recruitment and retention to primary care research; nominal group exercise.

BACKGROUND: Primary care studies often encounter recruitment difficulties, but there is little evidence to inform solutions. As part of a National Institute for Health Research School for Primary Care Research and UK Clinical Research Network programme, we elicited research staff perspectives on factors facilitating or obstructing recruitment. OBJECTIVE: To identify factors that experienced research staff consider important in successful recruitment and retention and their confidence in achieving them. METHODS: An iterative series of three workshops was held. The third used a modified nominal group technique to categorize whether factors related to the 'context' in which the research took place, the 'content' of the study or the recruitment 'process' and to prioritize them by their importance to success. RESULTS: Eighteen research staff participated in the prioritization workshop. They prioritized positive attitudes of primary care staff towards research and trust of researchers by potential participants as major contextual factors affecting recruitment. Studies needed to be considered safe and relevant by staff and fit with practice systems. They proposed that researchers strengthen relationships with staff and participants and minimize workload for primary care teams. Although confident in many recruitment processes, respondents remained uncertain how to achieve cultural change so that research became part of normal practice activity and how best to motivate patients to participate. CONCLUSIONS: Research workers taking part identified factors which might be important in recruitment, several of which they expressed little confidence in addressing. Understanding how to improve recruitment is crucial if current efforts to strengthen primary care research are to bear fruit.

Improving recruitment to health research in primary care.

BACKGROUND: Recruitment to health research is known to be problematic. However, evidence concerning ways of improving recruitment is sparse. OBJECTIVE: To outline the process of recruitment, factors impacting on recruitment success and key areas for further research and development. METHODS: Narrative literature review. RESULTS: This paper argues that three ways of improving recruitment should form the focus of future work: developing a repository of evidence-based techniques and methods which can be introduced by research teams; developing the infrastructure to support recruitment, especially new technologies around the electronic patient record; and increasing public engagement with research, to improve participation by both clinicians and patients. CONCLUSION: Recruitment to health research in primary care remains a major hurdle, and key research and development priorities must be addressed.

Lewia hordeicola sp. nov. from barley grain.

Lewia hordeicola with Alternaria anamorph was isolated from barley grains in Norway. The fungus is homothallic. It produces fertile ascomata on synthetic nutrient agar (SNA) after long incubation at 4 C in the dark. On PCA its anamorph resembles members of the A. infectoria species group. On SNA L. hordeicola differs from the latter in the shape and size of ascospores, the conidial sporulation patterns, and the shape, size, septation and roughness of conidia. A key to currently known Lewia species is included.

Comparison of Gaeumannomyces- and Phialophora-like fungal pathogens from maize and other plants using DNA methods.

Several DNA-based techniques, developed for identifying and differentiating fungi in the Gaeumannomyces-Phialophora complex associated with take-all diseases of cereals and grasses, were used to compare fungi from maize. Maize isolates obtained as G. graminis (Sacc.) Arx & H Olivier var. tritici Walker, from the UK, having been identified by ascospore morphology and in pathogenicity tests on wheat, were indistinguishable from isolates of the same variety obtained from wheat. Isolates of G. graminis (Sacc.) Arx & H Olivier var. maydis Yao et al., recently described as the maize take-all fungus from China, were identical in DNA tests to the anamorphic fungus Phialophora radicicola Cain and almost identical to Phialophora zeicola Deacon & Scott, whose description was originally based on isolates from South Africa and France. These species appear to represent the holomorph of the same fungus. The late wilt pathogen of maize, from India and Egypt, commonly known as Cephalosporium maydis Samra et al., but suggested as being the Phialophora anamorph of a Gaeumannomyces species, was closely related to other Gaeumannomyces species included in the tests.

Molecular diagnostics for fungal plant pathogens.

Accurate identification of fungal phytopathogens is essential for virtually all aspects of plant pathology, from fundamental research on the biology of pathogens to the control of the diseases they cause. Although molecular methods, such as polymerase chain reaction (PCR), are routinely used in the diagnosis of human diseases, they are not yet widely used to detect and identify plant pathogens. Here we review some of the diagnostic tools currently used for fungal plant pathogens and describe some novel applications. Technological advances in PCR-based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens and are now being applied to practical problems. Molecular methods have been used to detect several pathogens simultaneously in wheat, and to study the development of fungicide resistance in wheat pathogens. Information resulting from such work could be used to improve disease control by allowing more rational decisions to be made about the choice and use of fungicides and resistant cultivars. Molecular methods have also been applied to the study of variation in plant pathogen populations, for example detection of different mating types or virulence types. PCR-based methods can provide new tools to monitor the exposure of a crop to pathogen inoculum that are more reliable and faster than conventional methods. This information can be used to improve disease control decision making. The development and application of molecular diagnostic methods in the future is discussed and we expect that new developments will increase the adoption of these new technologies for the diagnosis and study of plant disease.

Ribosomal DNA analyses reveal greater sequence variation in Polymyxa species than previously thought and indicate the possibility of new ribotype-host-virus associations.

Polymyxa species transmit viruses to many important crops. They are poorly understood obligate parasites occupying a distinct position in the Tree of Life. To better understand the potential for spread of Polymyxa-vectored diseases, ribosomal DNA was analysed from isolates covering a wide range of geographical locations, virus associations and hosts. Internal transcribed spacer 2 structure analysis indicated that Polymyxa graminis isolates could represent many species and there was more sequence variation within the known subgroups (ribotypes) than previously described. In cereal crops and soils from temperate climates Polymyxa isolates were usually ribotype I or II, but their host specificities or preferences were unclear. For the first time, there was evidence that ribotype I (in addition to ribotype II) could transmit SBWMV/SBCMV. Different ribotypes often occurred together in the same soil or plant. New hosts were identified for particular ribotypes, including the first detection of the sugar beet-infecting Polymyxa betae, in wheat. Unexpectedly, ribotype III-like sequences, usually restricted to crops in the tropics, were found in wheat from the USA. P. betae isolates showed limited variation (≤ 2%) and the recent change in susceptibility of sugar beet varieties to BNYVV in the USA is unlikely to be due to changes in P. betae.

The Pochonia chlamydosporia serine protease gene vcp1 is subject to regulation by carbon, nitrogen and pH: implications for nematode biocontrol.

The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances.

Evidence that Polymyxa species may infect Arabidopsis thaliana.

Polymyxa spp. are obligate biotrophs belonging to the plasmodiophorid group, responsible for transmitting a large number of plant viruses to many crop species. Their obligate nature makes them difficult to study. Controlled environment experiments were used to investigate the potential of infection of Arabidopsis thaliana by Polymyxa spp. to provide a more tractable system. Two ecotypes of Arabidopsis, Columbia and Landsberg erecta, were grown in soils known to be infested with Polymyxa. At the end of a 2-month growth period, both ecotypes were found to harbour Polymyxa-like structures or spores. These findings were confirmed by Polymyxa-specific PCR tests and rDNA sequencing, which positively identified the presence of Polymyxa in the roots of both ecotypes of Arabidopsis. Both Polymyxa graminis and Polymyxa betae were identified. This is the first report of infection of Arabidopsis by Polymyxa spp. and shows the possibility of using this system for studies of infection biology and host-parasite interactions.

Phylogenetic relationships among Zygomycetes from soil based on ITS1/2 rDNA sequences.

New information was obtained on the phylogeny of Zygomycetes. PCR-RFLP analysis showed ITS1/2 rDNA to provide appropriate markers for genetic studies on Zygomycetes at the population and species levels. The use of several restriction enzymes allowed discrimination between genera and species of Mortierellales and Mucorales. ITS1/2 sequence analysis clearly indicated a deep, ancient and distinct dichotomy of Mortierellales and Mucorales. The data do not fully support the current concept of Mucorales, which recently included the family Umbelopsidaceae, but support the distinctiveness of the Umbelopsis group, which includes Mortierella turficola. The data support the hypothesis of polyphyly of Absidia and are consistent with the hypothesis of polyphyly of Mucor.

The use of conventional and quantitative real-time PCR assays for Polymyxa graminis to examine host plant resistance, inoculum levels and intraspecific variation.

* A real-time PCR protocol based on 18S rDNA sequences was developed to provide a specific, sensitive and quantitative assay for the root-infecting virus vector Polymyxa graminis. * The assay was calibrated with zoospore suspensions and inoculated roots and then shown to work with naturally infected plant roots and infested soil. Both the temperate P. graminis ribotypes previously described are detected but are not distinguished. DNA from related plasmodiophorids and from a range of fungi and plants was not detected. * Different genotypes of Triticum were grown in a soil infested with P. graminis and Soil-borne cereal mosaic virus (SBCMV). The genotypes differed in susceptibility to P. graminis, the least susceptible being the Triticum monococcum accession K-58505. * Conventional PCR assays and sequencing of amplified rDNA fragments showed that P. graminis isolates infecting wheat were mostly, but not exclusively, of ribotype II. Ribotype II was clearly associated with SBCMV transmission and seems to occur preferentially on wheat whereas ribotype I is mostly associated with barley.

Gaeumannomyces graminis, the take-all fungus and its relatives.

SUMMARY Take-all, caused by the fungus Gaeumannomyces graminis var. tritici, is the most important root disease of wheat worldwide. Many years of intensive research, reflected by the large volume of literature on take-all, has led to a considerable degree of understanding of many aspects of the disease. However, effective and economic control of the disease remains difficult. The application of molecular techniques to study G. graminis and related fungi has resulted in some significant advances, particularly in the development of improved methods for identification and in elucidating the role of the enzyme avenacinase as a pathogenicity determinant in the closely related oat take-all fungus (G. graminis var. avenae). Some progress in identifying other factors that may be involved in determining host range and pathogenicity has been made, despite the difficulties of performing genetic analyses and the lack of a reliable transformation system.

Polymyxa graminis and the cereal viruses it transmits: a research challenge.

UNLABELLED: SUMMARY Polymyxa graminis is a eukaryotic obligate biotrophic parasite of plant roots that belongs to a poorly studied discrete taxonomic unit informally called the 'plasmodiophorids'. P. graminis is non-pathogenic, but has the ability to acquire and transmit a range of plant viruses which cause serious diseases in cereal crop species and result in significant yield reductions. The viruses are protected from the environment within P. graminis resting spores ('cysts') that may remain dormant but viable for decades (probably until a suitable host plant is encountered). The persistent, soil-borne nature of these diseases makes the use of virus-resistant crop varieties currently the only practical and environmentally friendly means of control. USEFUL WEBSITES: http://www.rothamsted.bbsrc.ac.uk/ppi/links/pplinks/plasmod/index.html, http://www.dpvweb.net/, http://www.rothamsted.bbsrc.ac.uk/ppi/Iwgpvfv/index.html, http://www.rothamsted.bbsrc.ac.uk/ppi/links/pplinks/bymoviruses/index.html, http://oak.cats.ohiou.edu/~braselto/plasmos/