Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets.

UNLABELLED: Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field.

Biological and phylogenetic characterization of Fusarium oxysporum complex, which causes yellows on Brassica spp., and proposal of F. oxysporum f. sp. rapae, a novel forma specialis pathogenic on B. rapa in Japan.

Although the causal agent of yellows of Brassica rapa (turnip, pak choi, and narinosa) in Japan was reported in 1996 to be Fusarium oxysporum f. sp. conglutinans, this classification has remained inconclusive because of a lack of detailed genetic and pathogenic studies. Therefore, we analyzed the taxonomic position of this organism using Japanese isolates of F. oxysporum complex obtained from diseased individuals of various B. rapa subspecies. Phylogenetic analyses using partial sequences of the rDNA intergenic spacer region and the mating-type gene (MAT1-1-1alpha-box) showed that B. rapa and cabbage isolates belong to different monophyletic clades that separated at early evolutionary stages. Additionally, correlations were observed between the molecular phylogeny and the vegetative compatibility groups. Isolates from turnip, komatsuna, and narinosa (B. rapa group) did not show pathogenicity against cabbage or broccoli (B. oleracea group), although they caused severe symptoms on their original host species. In contrast, cabbage isolates had significantly higher (P = 0.05) virulence on B. oleracea than on B. rapa crops. Our results indicate that F. oxysporum complex isolates from B. rapa and B. oleracea are not only phylogenetically distinct but also differ in host specificity. Therefore, we propose a novel forma specialis, F. oxysporum f. sp. rapae, which causes yellows on B. rapa, including turnip, komatsuna, pak choi, and narinosa.

Piperazinyl oxime ethers as NK-1 receptor antagonists.

The synthesis and structure-activity relations for a new class of centrally active NK-1 receptor antagonists are described. The new compounds are based on piperazine 2 and contain an oxime ether functionality. Several new compounds have high affinity for the NK-1 receptor and show good antagonistic activity in the gerbil foot-tapping assay.

Cloning and characterization of the mating type (MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp.

Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the MAT1-2 gene of Cochliobolus heterostrophus, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MAT1-2 sequence in both directions, then primers designed to MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomorph. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to MAT loci of other loculoascomycete fungi. MAT genes were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of MAT1-1 by MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A. rabiei on chickpea is phylogenetically distant from other legume-associated Ascochyta spp. and the specific status of A. rabiei, A. lentis, A. pisi, and A. fabae was confirmed by the HMG phylogeny

Ultrastructural studies on the pheromone-producing cells in the silkmoth, Bombyx mori: formation of cytoplasmic lipid droplets before adult eclosion.

In Bombyx mori, pheromone-producing cells accumulate a number of lipid droplets in the cytoplasm preceding the production of the sex pheromone, bombykol. The process of lipid droplet formation in the pheromone-producing cells was investigated by using light and electron microscopy. Light microscopy revealed that the lipid droplets appeared from 2 days before adult eclosion and dramatic accumulation took place between 2 days and 1 day before eclosion. Electron microscopical studies revealed that smooth endoplasmic reticulum and numerous vesicles, their sizes being less than 1 microm, were detectable 2 days before eclosion, and some vesicles were fused with mitochondria at this stage. These characteristic changes in the pheromone-producing cells suggest that fatty acyl-CoA synthesis following de novo fatty acid synthesis takes place at this time. Involutions in the basal plasma membrane of the cells occurred throughout the observed period, which were extensive on the day before adult eclosion. Besides extensive basal involutions, immature lipid droplets appeared and then mature fully electron-dense lipid droplets were observed on the day of adult eclosion. These ultrastructural observations, combined with recent physiological studies suggest, that the basal involutions presumably reflect the uptake of lipidic components required for the construction of lipid droplets, the function of which is to store the bombykol precursor and to provide it for bombykol biosynthesis in response to pheromonotropic stimuli by pheromone biosynthesis activating neuropeptide (PBAN).

Molecular organization of mating type loci in heterothallic, homothallic, and asexual Gibberella/Fusarium species.

Mating type (MAT) genes were cloned from three members of the Gibberella/Fusarium complex that differ in reproductive mode: heterothallic G. fujikuroi, homothallic G. zeae, and asexual F. oxysporum. The G. fujikuroi MAT locus organization is typical of other heterothallic pyrenomycetes characterized to date; i.e., there are three genes at MAT1-1 and one at MAT1-2. G. zeae has homologues of all four genes encoded by the two G. fujikuroi MAT idiomorphs, tightly linked on the same chromosome, interspersed with sequences unique to G. zeae. Field isolates of F. oxysporum, although asexual, have either the MAT1-1 or the MAT1-2 genes found in sexual species and these genes are highly similar to those of heterothallic G. fujikuroi. RT-PCR analysis proved that the F. oxysporum MAT genes are expressed and that all putative introns found in each of the four MAT genes in G. fujikuroi and F. oxysporum are removed. Apparent failure of F. oxysporum to reproduce sexually could not be attributed to mutations in the MAT genes themselves.

Mating-type genes from asexual phytopathogenic ascomycetes Fusarium oxysporum and Alternaria alternata.

Mating-type (MAT) loci were cloned from two asexual (mitosporic) phytopathogenic ascomycetes, Fusarium oxysporum (a pyrenomycete) and Alternaria alternata (a loculoascomycete), by a polymerase chain reaction (PCR)-based strategy. The conserved high mobility group (HMG) box domain found in the MAT1-2-1 protein was used as a starting point for cloning and sequencing the entire MAT1-2 idiomorph plus flanking regions. Primer pairs designed to both flanking regions were used to amplify the opposite MAT1-1 idiomorph. The MAT1-1 and MAT1-2 idiomorphs were approximately 4.6 and 3.8 kb in F. oxysporum and approximately 1.9 and 2.2 kb in A. alternata, respectively. In both species, the MAT1-1 idiomorph contains at least one gene that encodes a protein with a putative alpha box domain and the MAT1-2 idiomorph contains one gene that encodes a protein with a putative HMG box domain. MAT-specific primers were used to assess the mating type of F. oxysporum and A. alternata field isolates by PCR. MAT genes from A. alternata were expressed. The A. alternata genes were confirmed to be functional in a close sexual relative, Cochliobolus heterostrophus, by heterologous expression.

Haematocin, a new antifungal diketopiperazine produced by Nectria haematococca Berk. et Br. (880701a-1) causing nectria blight disease on ornamental plants.

A new antifungal diketopiperazine named haematocin was isolated from the culture broth of Nectria haematococca Berk. et Br. (880701a-1) causing blight disease on ornamental plants, Phalaenopsis spp. and Doritanopsis spp. Its structure was established by spectroscopic methods. Haematocin inhibited the germ-tube elongation and spore-germination of Pyricularia oryzae at the ED50 values of 30 microg/ml and 160 microg/ml, respectively.

REMI mutagenesis and identification of pathogenic mutants in blast fungus (Magnaporthe grisea).

Blast fungus' mutant, which is very difficult to obtain, is the key substance in the study of the relation between pathogen and plant. Rice-Aichi-ashahi was used as the host and plasmid pBF101 or pCSN43 as foreign DNA fragment, and REMI mutagenesis was applied to tag the pathogenic sequence in rice blast fungus M.grisea. Three mutants (R2H65, R2H69 and R2B1565) related to pathogenicity were recovered from several hundreds of transformants. R2H69 and R2H65 produced merely strange conidia which could not form appressorium or yield abnormals, and pathogenicity test further confirmed that they were unable to cause disease. R2B1565 possesses the same phenotypes as those of the wild type except that the ability of causing disease was much reduced.

Education in geriatric psychiatry.

The UK has seen many significant developments during the last 30 years in geriatric psychiatry, with emphasis on multi-disciplinary working and on the integration of services for the elderly. Accompanying these changes has been a growing public consciousness of the psychiatric disorders of later life and of the needs of old people and their carers. With a huge increase this century in the proportion of very elderly people, care of the elderly has achieved greater recognition in the medical training of both generalists and specialists. Greater emphasis also is being given to gerontology, both social and biological. In this paper the authors examine recent developments in medical education in the UK and show how changing approaches to medical training are shaping developments in geriatric psychiatry education. They describe recent innovations in both teaching styles and assessment methods and consider how developments in Computer Aided Learning can be applied to the teaching of geriatric psychiatry.

Efficient cloning of ascomycete mating type genes by PCR amplification of the conserved MAT HMG Box.

Cloning of mating type (MAT) genes from ascomycetes has been hampered by low conservation among them. One of the pair of MAT genes, represented by MAT-2 of Cochliobolus heterostrophus (a loculoascomycete) and mt a of Neurospora crassa (a pyrenomycete), encodes a protein with a conserved DNA binding motif called the high mobility group (HMG) box. PCR with primer pairs corresponding to the borders of the C. heterostrophus and the N. crassa HMG boxes generated an approximately 0.3-kb product from genomic DNAs of MAT-2 and mt a strains, respectively, but not from MAT-1 and mt A strains. The C. heterostrophus primers amplified approximately 0.3-kb products from DNA of most loculoascomycete genera tested but not from DNA of pyrenomycete genera; this specificity was reversed with the N. crassa primers. The validity of the PCR procedure was documented by near sequence identity between the C. heterostrophus MAT-2 HMG box and PCR products from several Cochliobolus spp. and by cosegregation of the PCR product with mating type in progeny of Setosphaeria turcica and of Cryphonectria parasitica. Regions of the MAT locus flanking the HMG box were readily cloned using the TAIL-PCR procedure with a combination of random and specific primers.

Dementia and 'borderline dementia' in Britain: 8-year incidence and post-screening outcomes.

Survivors from a nationally representative sample of elderly people originally screened in 1985 were reassessed in 1989 and again in 1993. On each occasion respondents were rated as cognitively impaired, borderline impaired or unimpaired (using a brief information/orientation scale), with the validity of these ratings assessed in subsequent clinical interviews. Where follow-up screening was not possible, information was derived from death certificates and hospital case-notes. Over 8 years (1985-93) the overall incidence rate per person-year at risk was 1.58%, giving age-specific rates of 0.72, 1.32, 1.63, 3.46, 2.55 and 1.41% for the age groups 65-69, 70-74, 75-79, 80-84, 85-89 and > or = 90 respectively. Of 43 individuals classified at screening as borderline impaired in 1985 and 1989, 19 were diagnosed as demented at clinical interviews conducted within 16 weeks of screening. Four-year follow-ups among the remaining 24 showed that 15 had died, while 6 showed a worsened cognitive status. Controlling for both age and sex, aggregated 4-year mortality was significantly higher among those defined at screening in 1985 and 1989 as either impaired or borderline, when compared with the unimpaired.

Some legal aspects of mental capacity.

This article discusses some practical matters which arise when competence to make decisions is in question. Consent, testamentary capacity, powers of attorney, the Court of Protection, "living wills," and research on people with dementia are briefly considered.

Incidence of dementia in a representative British sample.

In a four-year follow-up study of 1042 elderly people (aged 65 years or older), randomly sampled from the community, levels of dementia were assessed using a two-phase case-finding procedure (screening followed by clinical interview) among survivors. Clinical information on those not reinterviewed was provided by death certificates, hospital case notes, or postal questionnaires. The weighted four-year cumulative incidence of dementia was 3.7% (95% confidence intervals: 2.4%-5.0%), with age-specific rates of 0.9%, 2.8%, 5.2%, 9.0%, and 8.7% for the age groups 65-69, 70-74, 75-79, 80-84, and 85-89 years respectively. While consistent with data from other British regions, it remains likely that these rates underestimate true incidence.

Incidence of dementia: preliminary findings from the Nottingham Longitudinal Study of Activity and Ageing.

In a 4-year follow-up of 1,042 randomly sampled elderly people (aged 65+), levels of dementia were assessed using a two-phase case finding procedure (screening followed by clinical interview) among survivors. Clinical information on those not re-interviewed was provided by death certificates, hospital case notes or postal questionnaires. The unweighted 4-year cumulative incidence of dementia was 4.3%, with age-specific rates of 0.9, 2.8, 5.2, 9.0 and 8.7% for the age groups 65-69, 70-74, 75-79, 80-84, and 85-89, respectively.