Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction

A sensitive and specific assay for detecting Xylella fastidiosa in potential insect vectors was developed. This assay involves immunomagnetic separation of the bacteria from the insect, followed by a two-step, nested polymerase chain reaction (PCR) amplification using previously developed oligonucleotide primers specific to X. fastidiosa . A total of 347 leafhoppers representing 16 species were captured and sampled from American elm ( Ulmus americana L.) trees growing in a nursery where bacterial leaf scorch caused by X. fastidiosa occurs. Two of these leafhopper species, Graphocephala coccinea and G. versuta , regularly tested positive for X. fastidiosa using this technique. These insects are therefore potential vectors of X. fastidiosa . Using immunocapture and nested PCR, it was possible to detect as few as five bacteria per sample.     

Autoradiographic study of RNA synthesis during meiotic prophase in the human oocyte

The incorporation of 3H-uridine in oogonia and oocytes during meiotic prophase I was studied in three human fetuses 13, 18, and 19 weeks old. Following a 40- or 60-min pulse, intense nuclear and nucleolar labeling was observed in oogonia. During the preleptotene chromosome condensation stage, the heteropycnotic masses were unlabeled, while numerous silver grains were seen on the filaments persisting around these masses. During leptotene, chromosomal and nucleolar RNA synthesis was significant, but less than that in the oogonia. The rate of incorporation declined rapidly during zygotene and fell to a very low level at early pachytene. Throughout pachytene no nucleolar RNA synthesis was observed. Chromosomal RNA synthesis progressively recovered during middle pachytene, was of moderate intensity at late pachytene, and increased again at early diplotene. Nucleolar RNA synthesis was very intense at early diplotene, at the same time as nucleolar size and basophilia increased.     

Effect of abscisic acid on membrane potential and transport of glucose and glycine in Lemna gibba G1

The membrane potential of Lemna gibba G1 was measured with a microelectrode; glucose and glycine uptake were measured with ¹⁴C-labeled substances. The membrane potential was increased by 85 mV on the average, after the plants had been pretreated with 10 M abscisic acid (ABA) for more than 30 min. This effect is not linked to the endogenous level of soluble sugars. The concentration of these soluble sugars was increased to more than 200% by pretreatment of the plants with ABA, however, the respiration of the plants was not affected. ABA stimulated uptake of glucose and glycine. Glucose- and glycine-dependent depolarization and repolarization of the membrane was altered: depolarization was less and repolarization was slower; during uptake of glycine, the first typical phase of repolarization was suppressed. The data suggest that ABA interferes with the primary steps of substrate uptake.Abbreviations ABA abscisic acid - FW fresh weight - IAA indole acetic acid - pd membrane potential difference - 1× perfusing solution (see methods) - H⁺ electrochemical proton gradient - pd solute-induced maximum depolarization of the membrane     

Cascading Trophic Interactions from Fish to Bacteria and Nutrients after Reduced Sewage Loading: An 18-Year Study of a Shallow Hypertrophic Lake

The effects of major reductions in organic matter, total phosphorus (TP), and total nitrogen (TN) loading on the chemical environment, trophic structure, and dynamics of the hypertrophic, shallow Lake Søbygård were followed for 18 years. After the reduction in organic matter loading in 1976, the lake initially shifted from a summer clear-water state, most likely reflecting high grazing pressure by large Daphniaspecies, to a turbid state with extremely high summer mean chlorophyll a (up to 1400 μg L^{? 1}), high pH (up to 10.2), and low zooplankton grazing. Subsequently, a more variable state with periodically high grazing rates on phytoplankton and bacteria was established. Changes in zooplankton abundance and grazing could be attributed to variations in cyprinid abundance due to a fish kill (probably as a consequence of oxygen depletion) and pH-induced variations in fish recruitment and fry survival. The results suggest strong cascading effects of fish on the abundance and size of zooplankton and phytoplankton and on phytoplankton production. A comparatively weak cascading effect on ciliates and bacterioplankton is suggested. Due to high internal loading, only minor changes were observed in lake-water TP after a reduction in external TP loading of approximately 80% in 1982; net retention of TP was still negative 13 years after the loading reduction, despite a short hydraulic retention time of a few weeks. TN, however, decreased proportionally to the TN-loading reduction in 1987, suggesting a fast N equilibration. Only minor improvement in the environmental state of the lake has been observed. We suggest that another decade will be required before the lake is in equilibrium with present external P loading.     

Preservation of fungi in water (Castellani): 20 years

Sixty-two isolates of Fusarium were obtained from pasture grass and soil from various areas of New Zealand and identified as F. anthophilum [2], F. avenaceum [17], F. crookwellense [8], F. culmorum [4], F. graminearum [1], F. nivale [3], F. oxysporum [3], F. sambucinum [17], F. semitectum [1], F. tricinctum [1] and an unidentified Fusarium spp. [5]. These isolates were grown on autoclaved rice and tested for toxicity to rats in feeding tests. Eighty two percent of the isolates were toxic, of which twenty-four percent were severely toxic and caused hemorrhages of stomach and intestine, hematuria, and finally death. Cultures of the most toxic isolates contained 0.1 to 104 ppm of deoxynivalenol, 0.7 and 7 ppm of 15- and 3-acetyldeoxynivalenol respectively, 0.2 to 4 ppm of fusarenon- X, 11 to 1021 ppm zearalenone, 40 to 272 ppm of the hemorrhagic factor (wortmannin), 2,100 to 7,200 ppm of moniliformin, 565 ppm of the cytotoxic factor (HM-8) and enniatin in substantial concentrations. F. sambucinum is reported as a moniliformin producer for the first time.     

Topoisomerase 3α and RMI1 Suppress Somatic Crossovers and Are Essential for Resolution of Meiotic Recombination Intermediates in Arabidopsis thaliana

Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3α (TOP3α) functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3α in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3α-2), which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3α-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3α, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3α is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3α mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3α and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.     

Isolation and molecular characterization of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> from coffee plants in Costa Rica

Coffee plants exhibiting a range of symptoms including mild to severe curling of leaf margins, chlorosis and deformation of leaves, stunting of plants, shortening of internodes, and dieback of branches have been reported since 1995 in several regions of Costa Rica’s Central Valley. The symptoms are referred to by coffee producers in Costa Rica as “crespera” disease and have been associated with the presence of the bacterium Xylella fastidiosa. Coffee plants determined to be infected by the bacterium by enzyme linked immunosorbent assay (ELISA), were used for both transmission electron microscopy (TEM) and for isolation of the bacterium in PW broth or agar. Petioles examined by TEM contained rod-shaped bacteria inside the xylem vessels. The bacteria measured 0.3 to 0.5 μm in width and 1.5 to 3.0 μm in length, and had rippled cell walls 10 to 40 nm in thickness, typical of X. fastidiosa. Small, circular, dome-shaped colonies were observed 7 to 26 days after plating of plant extracts on PW agar. The colonies were comprised of Gram-negative rods of variable length and a characteristic slight longitudinal bending. TEM of the isolated bacteria showed characteristic rippled cell walls, similar to those observed in plant tissue. ELISA and PCR with specific primer pairs 272-l-int/272-2-int and RST31/RST33 confirmed the identity of the isolated bacteria as X. fastidiosa. RFLP analysis of the amplification products revealed diversity within X. fastidiosa strains from Costa Rica and suggest closer genetic proximity to strains from the United States of America than to other coffee or citrus strains from Brazil.     

Habitat choice of Grevy's zebras (Equus grevyi) in Laikipia, Kenya

Characterizing habitat choice is essential for endangered species conservation. For the endangered Grevy's zebra ( Equus grevyi ), as with many widely ranging vertebrates, human activities may be an important factor affecting space use. Grevy's zebras are grazing ungulates inhabiting the savannahs of central-northern Kenya and Ethiopia. Past research on their social organization indicates that reproductive status shapes associations and movements. Here, we examine how habitat use varies across four reproductive classes: lactating and nonlactating females, bachelors and territorial males. We also test whether Grevy's zebra avoid locations close to active livestock corrals, or bomas . We find that forage quality, forage quantity and habitat openness of locations used by Grevy's zebra vary significantly depending on individual reproductive state. Lactating females and bachelors use areas with green, short grass and medium-dense bush more frequently than nonlactating females or territorial males. We hypothesize that lactating females trade off forage quantity and safety to access nutrients in growing grass. Across reproductive classes, Grevy's zebra choose locations further from active bomas than if they used the area randomly. Our results suggest that Grevy's zebra may require a range of vegetation characteristics for different reproductive classes. Further, they may need areas free from competition or disturbance by livestock.     

Long-distance signalling of abscisic acid (ABA): the factors regulating the intensity of the ABA signal

Abscisic acid (ABA) is a stress signal, which moves in the xylem from the roots to the aerial parts of the plant, where it regulates stomatal movement and the activity of shoot meristems. Root growth-promoting microorganisms in the rhizosphere, lateral ABA flows in the root cortex across apoplastic barriers, ABA redistribution in the stem, leaf apoplastic pH values, and the action of beta-glucosidases, both in the apoplast and the cytosol of the mesophyll, play an important role in the regulation of signal intensity. The significance of abscisic acid glucose ester as a long-distance stress signal is discussed.     

Differential responses of maize MIP genes to salt stress and ABA

Salt stress is known to reduce root hydraulic conductivity and growth. To examine a concomitant regulation of aquaporins, the expression of the maize MIP gene family in response to NaCl was analysed by DNA array hybridization. Plants responded differentially to 100 versus 200 mM NaCl treatments. Leaf water content was reduced rapidly and persistently after the application of 200 mM NaCl in contrast to 100 mM NaCl. Endogenous ABA strongly accumulated in roots after 2 h; it remained at a highly elevated level for 48 h after the addition of 200 mM NaCl, but rapidly declined in plants treated with 100 mM NaCl, indicating an early recovery from water deficit. Interestingly, 2 h after the addition of 100 mM NaCl, when maize regained the osmotic potential allowing water uptake, three highly expressed, specific isoforms ZmPIP1;1, ZmPIP1;5, and ZmPIP2;4 were transiently induced. They were preferentially transcribed in the outer root tissue suggesting a role in cellular water transport. None of the ZmTIP genes was altered. By contrast, after the addition of 200 mM NaCl these responses were missing. Instead, multiple ZmPIP and ZmTIP genes were repressed by 200 mM NaCl after 24 h. After 48 h, deregulations were overridden in both cases indicating homeostasis. ABA (1 muM) exogenously applied to the roots transiently induced ZmPIP2;4 similar to 100 mM NaCl as well as ZmPIP1;2. Thus, the early induction of ZmPIP2;4 by NaCl may be mediated by ABA. Previously, an increase in root hydraulic conductivity had been observed upon ABA application. By contrast, 100 muM ABA led to a complete, possibly non-specific repression of all detected ZmPIP and ZmTIP genes after 24 h.