Identification, Virulence, and Distribution of Two Biovars of Pseudomonas syringae pv. actinidiae in New Zealand.

Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit, was detected for the first time in New Zealand in November 2010. Only in Bay of Plenty, one of the four regions where this pathogen had been detected, did symptoms evolve beyond leaf spots, resulting in cane die-back, wilting of canes, and canker, sometimes leading to death of the vine. Molecular analysis (cts haplotype and BOX-polymerase chain reaction [PCR] electrophoretic pattern) of strains isolated from different regions of New Zealand revealed that two biovars could be distinguished. They have been called biovar 3 and biovar 4 to differentiate them from strains from Japan (biovar 1) or Korea (biovar 2), which have a different cts haplotype or a different BOX-PCR pattern. Biovars 3 and 4 displayed different degrees of virulence, as measured by their ability to cause leaf spots on young, potted kiwifruit plants. Biovar 3, which has also been present in Italy since 2008 and in France, was found in the Bay of Plenty, where cane diebacks were observed. In contrast, no symptoms other than leaf spots have been observed in orchards where strains of biovar 4 have been isolated. We report the distribution and the disease progression of biovars 3 and 4 in New Zealand.

Radiobiological studies using synchrotron-produced ultrasoft X-rays.

Ultrasoft X-rays have been extensively used to explore radiobiological mechanisms surrounding cell killing. These studies for the most part have been linked to a small number of X-ray energies. Recently, this field of study has been broadened by the availability of synchrotron-produced ultrasoft X-rays which can be produced at any desired energy. We have taken advantage of the University of Wisconsin Synchrotron to reexamine two fundamental radiobiological questions: Dose RBE vary with different ultrasoft X-ray energies? Dose the fraction of the nuclear volume exposed to equal total X-ray energy modify cell cytotoxicity? The first study focuses on the survival of Chinese hamster V79 and mouse C3H10T1/2 cells irradiated with synchrotron-produced 273 eV and 860 eV ultrasoft X-rays. These two energies, which are available by multilayer monochromatization of the synchrotron output spectrum, exhibit equal attenuation within living cells. Such an isoattenuating energy pair allows the direct examination of how biological effectiveness varies with the energy of the ultrasoft X-rays. In comparing survival results, we find similar biological effectiveness of these two energies for both the C3H10T1/2 and the V79 cells. These results are no consistent with previous findings of increasing RBE with decreasing ultrasoft X-ray energies. In addition, after correcting for mean nuclear based on measurements of cell thickness obtained with confocal microscopy, we find no significant differences in survival between the two ultrasoft X-ray energies and 250 kVp X-rays. These results suggest that RBE does not increase with decreasing energy of ultrasoft X-ray between 860 eV and 273 eV. In a second study we introduced an method which allows partial-volume irradiation of live cells using synchrotron-produced ultrasoft X-rays and micro-fabricated irradiation masks. The masks were made by X-ray lithography at the University of Wisconsin Synchrotron Radiation Center, and they consist of 1.85-micron-wide stripes of gold 1.35 microns apart plated onto thin silicon nitrate membranes. When placed adjacent to mylar on which live cells are plated, these masks allow cells to be irradiated in a striped pattern with dimensions much smaller than the cell nuclei. Using 1340 eV synchrotron-produced X-rays, we compare the survival of cells subjected to uniform irradiation and cells subjected to partial-volume irradiation. Our results show that, at equal mean dose to the nucleus (i.e. equal total energies deposited), survival is not statistically different for the two treatments over a wide range of doses. Thus, imparting equal energies to smaller intranuclear volumes does not appear to modulate cell killing.

A comparison of cytotoxicity after whole- or partial-cell irradiation with synchrotron-produced ultrasoft X rays.

We introduce a method which allows partial-volume irradiation of live cells using synchrotron-produced ultrasoft X rays and micro-fabricated irradiation masks. The masks were made by X-ray lithography at the University of Wisconsin Synchrotron Radiation Center, and they consist of 1.85-microm-wide stripes of gold 1.35 microm apart plated onto thin silicon nitride membranes. When placed adjacent to Mylar on which live cells are plated, these masks allow cells to be irradiated in a striped pattern with dimensions much smaller than the cell nuclei. Using 1340 eV synchrotron-produced X rays, we compare the survival of cells subjected to uniform irradiation and cells subjected to partial-volume irradiation. Our results show that, at equal mean dose to the nucleus (i.e. equal total energies deposited), survival is not statistically different for the two treatments over a wide range of doses. Thus imparting equal energies to smaller intranuclear volumes does not appear to enhance cell killing.

Synchroton-produced ultrasoft X rays: equivalent cell survival at the isoattenuating energies 273 eV and 860 eV.

In this paper we report on survival of Chinese hamster V79 and mouse C3H 10T1/2 cells after irradiation with synchrotron-produced 273 eV and 860 eV ultrasoft X rays. These two energies, which are available by multilayer monochromatization of the synchrotron output spectrum, exhibit equal attenuation within living cells. Such an isoattenuating energy pair allows the direct examination of how biological effectiveness varies with the energy of the ultrasoft X rays. In comparing survival results, we find similar biological effectiveness of these two energies for both the C3H 10T1/2 and the V79 cells. These results are not consistent with previous findings of increasing RBE with decreasing ultrasoft X-ray energies. In addition, after correcting for mean nuclear dose based on measurements of cell thickness obtained with confocal microscopy, we find no significant differences in survival between the two ultrasoft X-ray energies and 250 kVp X rays. These results suggest that RBE does not increase with decreasing energy of ultrasoft X rays between 860 eV and 273 eV. The possible impact of our results on past results for ultrasoft X rays is discussed.

A double mirror W/C multilayer monochromator for radiation biology applications.

A double-mirror multilayer monochromator was developed for the purpose of irradiating live cell cultures at the Synchrotron Radiation Center, University of Wisconsin-Madison. The monochromator is designed for the soft x-ray region with photon energies between 270 and 2400 eV. Multilayer mirrors with 55 bilayers of W/C and a bilayer spacing of d = 3.0 nm are sputter deposited on Si substrates. By proper masking of the sputtering sources, variation in the bilayer spacing over the area of the mirror is minimized. The uniformity of the bilayer spacing was measured to be delta d/d < 1%, over the 75 mm x 25 mm area of the mirrors. The reflectivity was measured as a function of energy to determine the integrated reflectivity and evaluate the contribution of the specular reflection and higher orders to the monochromatic beam. The use of suitable filters with a Si(Li) detector allows determination of the spectral output of the monochromator. The output power of the monochromator between 270 and 2400 eV is measured. The resolution of the monochromator is delta lambda/lambda = 0.04. Applications of the monochromator to radiation biology are discussed.

In situ visualization of DNA double-strand break repair in human fibroblasts.

A method was developed to examine DNA repair within the intact cell. Ultrasoft x-rays were used to induce DNA double-strand breaks (DSBs) in defined subnuclear volumes of human fibroblasts and DNA repair was visualized at those sites. The DSBs remained in a fixed position during the initial stages of DNA repair, and the DSB repair protein hMre11 migrated to the sites of damage within 30 minutes. In contrast, hRad51, a human RecA homolog, did not localize at sites of DNA damage, a finding consistent with the distinct roles of these proteins in DNA repair.

Response of thermoluminescent lithium fluoride (TLD-100) to photon beams of 275, 400, 500, 600, 730, 900, 1200, 1500, and 2500 eV.

LiF:Mg,Ti (TLD-100) extruded ribbons and cleaved crystals were exposed to monoenergetic photons of 275-2550 eV energy to determine their potential usefulness as radiation dosimeters for radiobiology experiments at these energies. The radiation source was synchrotron radiation from the 1 GeV electron storage ring, Aladdin. The authors report TLD response and glow curves for He- and air-annealed dosimeters. The undesirable effects of air annealing increase with decreasing photon penetration in the dosimeter. Under certain experimental conditions, UV radiation produced anomalous bleaching of high-temperature traps. The crystals and the chips presented a supralinear response, Supralinearity factors were determined to be of the order of 1.5 for crystals, and 1.7 for the chips. The authors' results indicate that TLDs are a reliable means to monitor the total energy deposited in irradiated cells and are now used routinely for radiobiology cell irradiations.