First Report of Bituminaria Witches'-Broom in Australia Caused by a 16SrII Phytoplasma.

Bituminaria bituminosa (L.) Stirt. is a perennial legume known as Arabian pea that is used as a forage in arid areas and for stabilization of degraded soils. It is widely distributed in the Mediterranean Basin with wider adaptation across the Canary Islands (4). In July 2010, during a survey for phytoplasma, some Canary Island B. bituminosa plants with typical phytoplasma symptoms, including stunted growth with small leaves, shortened internodes, and bushy growth, were found in seed multiplication nurseries at Medina, Perth, Western Australia (115°48.5'E; 32°13.2'S). Two samples from plants with clear disease symptoms and two visibly healthy plants were collected and total DNA was extracted with the Illustra DNA extraction kit Phytopure (GE Healthcare) according to the manufacturer's instructions. Direct and nested PCR were used to test the presence of phytoplasma 16S rDNA in samples with universal primers P1/P7 and R16F2n/R16R2, respectively (1,3). The PCR amplifications from all diseased samples yielded an expected product of 1.8 kb by direct and 1.2 kb by nested PCR, but not from the healthy plant samples. The direct PCR product was used as a template DNA in sequencing and the DNA sequence was deposited in the NCBI GenBank (Accession No. HQ404357). Sequence homology analysis indicated there was a perfect match between the two isolates. BLAST search of the NCBI GenBank revealed that B. bituminosa phytoplasma shares >99% sequence identity with Crotalaria witches'-broom phytoplasma (Accession No. EU650181.1), pear decline phytoplasma (Accession No. EF656453.1), and Scaevola witches'-broom phytoplasma (Accession No. AB257291.1). On the basis of BLAST analyses of 16S rRNA gene sequences, B. bituminosa phytoplasma in Western Australia appears to belong to the peanut witches'-broom group (16SrII-D) of phytoplasma. Restriction fragment length polymorphism analysis was also performed on nested PCR products of two samples of B. bituminosa phytoplasma by separate digestion with HaeIII, Hind6I, HpaII, MboI, RsaI, Tru9I, and T-HB8I restriction enzymes. Samples yielded patterns similar to alfalfa witches'-broom phytoplasma (Accession No. AF438413) belonging to subgroup 16SrII-D (2). To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group infecting B. bituminosa in Australia and should be referred to as "Bituminaria witches'-broom phytoplasma" (BiWB). This report also indicates that the occurrence of the phytoplasma in B. bituminosa may be widespread in the Canary Islands and other species of Bituminaria might be susceptible to infection by Bituminaria witches'-broom phytoplasma. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) A. J. Khan et al. Phytopathology 92:1038, 2002. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (4) P. Mendez et al. Grassland Sci. Eur. 11:300, 2006.

Visceral inputs to neurons in the anterior hypothalamus including those that project to the periaqueductal gray: a functional anatomical and electrophysiological study.

The present study was designed to examine peripheral, in particular noxious visceral, inputs to neurons in the hypothalamus that project to the midbrain periaqueductal gray. The induction of Fos protein was used to localize hypothalamic neurons that were activated by noxious visceral stimulation. This was combined with retrograde transport of fluorescent latex microspheres from identified "pressor" and "depressor" sites in the dorsolateral/lateral or ventrolateral columns of the periaqueductal gray. A second series of electrophysiological experiments examined the receptive field characteristics, including the incidence of viscerosomatic convergence, of neurons in the ventral part of the anterior hypothalamus. Noxious visceral stimulation (intraperitoneal acetic acid) induced Fos-like immunoreactivity in significantly more neurons in the hypothalamus than control stimuli (intraperitoneal saline and intravenous phenylephrine). Particularly high numbers of Fos-positive neurons were found in the paraventricular nucleus, the supraoptic nucleus and ventral regions of the anterior hypothalamus. When combined with retrograde tracing from "depressor" sites in the ventrolateral periaqueductal gray, the highest numbers of double-labelled neurons were localized in the paraventricular nucleus and the lateral area of the anterior hypothalamus. However, the regions that contained the greatest proportions of Fos-positive neurons that projected to "depressor" sites in the ventrolateral periaqueductal gray were the lateral area of the anterior hypothalamus and its rostral extension, the lateral preoptic area. Fewer double-labelled neurons were localized in the hypothalamus after retrograde transport from sites in the dorsolateral/lateral periaqueductal gray compared to the results obtained from injections of tracer in the ventrolateral periaqueductal gray. Furthermore, the numbers of Fos-positive hypothalamic neurons that projected to the dorsolateral/lateral periaqueductal gray were very similar in experimental and control animals. The electrophysiological study confirmed that a large proportion of neurons in and around the lateral area of the anterior hypothalamus can be driven by noxious visceral stimulation and demonstrated a high incidence of viscerosomatic convergence in these cells (66% of cells driven from somatic structures were also driven by electrical stimulation of the splanchnic nerve). Somatic receptive fields of these neurons were generally large, often including all four limbs and the face. The results of the functional anatomical and electrophysiological studies have identified neurons in an area of the ventral anterior hypothalamus that are a focus of nociceptive visceral input and which project to the midbrain periaqueductal gray, in particular to its ventrolateral column. These results are discussed in relation to the roles of the anterior hypothalamus and the different longitudinal columns of the periaqueductal gray in co-ordinating autonomic and sensory functions in response to visceral pain.

Improving searching skills and evidence retrieval.

OBJECTIVE: To evaluate the effect of a three-hour training session in formulating questions and searching databases. DESIGN: A randomised controlled trial and before and after study, with blinded outcome assessment. SETTING: Oxford University Medical School, first clinical year. SUBJECTS: Altogether 108 medical students were randomly assigned to an experimental group (54) or a control group (54), and all were given the task of searching for evidence around an ulcer related problem or a cardiac problem. Students in the experimental group were randomly allocated to research one of the two problems before training and the remaining problem afterwards. Control students received no training and were randomly allocated to search for evidence around either of these problems. MAIN OUTCOME MEASURES: Searching performance; the quality of evidence retrieved; student satisfaction. RESULTS: Training improved the students' search performance and the quality of evidence retrieved. Students' satisfaction with the training was high. CONCLUSIONS: A three-hour interactive training session improved the students' ability to search databases and retrieve evidence and was well received by the students.

Responses of neurones in the medullary raphe nuclei to inputs from visceral nociceptors and the ventrolateral periaqueductal grey in the rat.

The ventrolateral periaqueductal grey matter (PAG) is believed to have a role in mediating cardiovascular responses to noxious visceral stimuli. The present study was carried out as a first stage in establishing whether the ventrolateral PAG may exert these influences after a relay in the caudal medullary raphe nuclei (nucleus raphe obscurus and nucleus raphe pallidus). Single unit extracellular recordings were made from neurones in the caudal raphe nuclei and, for comparison, in the more rostral nucleus raphe magnus in Saffan-anaesthetized and paralysed rats. Neurones in the mid-line medulla were tested for their responses to electrical stimulation at chemically identified depressor sites in the ventrolateral PAG and to noxious visceral stimuli (distensions of the urinary bladder and electrical stimulation of the greater splanchnic nerve). Fifty-two per cent of caudal and 74% of rostral mid-line neurones gave short latency excitatory responses to stimulation of depressor sites in the ventrolateral PAG. Of the neurones that were also tested with noxious visceral stimuli, 5% of the caudal and 47% of the rostral neurones responded to bladder distension, while 33 and 35%, respectively, of caudal and rostral neurones responded to splanchnic nerve stimulation. These results indicate that many mid-line medullary neurones receive inputs from both the ventrolateral PAG and visceral nociceptors and may, therefore, be part of the output pathway by which the ventrolateral PAG produces integrated physiological responses to noxious visceral stimuli.

Circadian rhythm of neuronal activity in suprachiasmatic nucleus slices from the vasopressin-deficient Brattleboro rat.

In vitro extracellular recordings were made from tissue slices of suprachiasmatic nucleus from homozygous Brattleboro rats which are deficient in vasopressin. A high proportion (56%) of neurons were excited by application of exogenous vasopressin, indicating that the V1 receptors expressed by these neurons were functional. Basal activity of these vasopressin-sensitive neurons showed a marked circadian variation (higher during the subjective light phase) while vasopressin-insensitive neurons showed no significant variation, suggesting the presence of the V1 receptor identifies a population of highly circadian neurons. Suprachiasmatic neurons from both homozygous rats and their heterozygous (vasopressin-containing) litter mates displayed a circadian rhythm of spontaneous (basal) activity, with firing rates declining during the subjective dark phase, indicating that the endogenous pacemaker driving the circadian rhythm was not dependent upon the presence of vasopressin. However, the peak of spontaneous activity displayed during the subjective light phase was significantly lower in the vasopressin-deficient animals. These data show that the presence of endogenous vasopressin within the suprachiasmatic nucleus is not necessary for the generation of the circadian pattern of activity. However, vasopressin does function to amplify the rhythm by its excitatory effect during the light phase.

Glutamatergic projections from the rostral hypothalamus to the periaqueductal grey.

Retrograde transport of fluorescent latex microspheres was combined with immunocytochemistry for glutamate to determine the organization of the projections from glutamate-containing neurones in the rostral hypothalamus to the different subdivisions of the periaqueductal grey (PAG). Double-labelled neurones, i.e. neurones immunoreactive for glutamate and projecting to the PAG, were found throughout the rostral hypothalamus. There were no apparent differences, however, in the origins of presumed glutamatergic projections from the rostral hypothalamus to the different subdivisions of the PAG.