Genotypic diversity in a localized population of Ralstonia solanacearum as revealed by random amplified polymorphic DNA markers.

AIMS: To assess genotypic diversity within Ralstonia solanacearum isolates of a single field. METHODS AND RESULTS: A total of 44 field isolates and 22 in vitro generated clones of R. solanacearum were studied for genotypic diversity by random amplified polymorphic DNA (RAPD) technique. Genomic DNA of these isolates and clones was extracted by proteinase-K-SDS lysis mini-prep method. RAPD analysis was done with 30 decamer primers. The data were analysed using NTSYSpc 2.02h software. Forty-two out of 44 field isolates and all the clonal isolates were identified as distinct genotypes at 70% similarity level. CONCLUSION: Very high level of genome variability was observed within the field and clonal isolates of R. solanacearum. This might be a reason for the wide host range of this bacterium and for quick breakdown of wilt resistance in host plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that it would be difficult to design specific diagnostic protocol for R. solanacearum even for a localized population and to breed cultivars with broad-spectrum resistance.

Genetic diversity and differentiation of Indian isolates of Phytophthora infestans as revealed by RAPD analysis.

Sixty-seven isolates of Phytophthora infestans collected from Himalayan hill regions and subtropical planes of India were characterized by RAPD markers to assess diversity and differentiation based on location of origin. Ten random decamer primers generated 161 polymorphic fragments. Association of P. infestans isolates on the dendrogram and PCO plot revealed two clear grouping based on geographical location of origin-hill isolates and plane isolates. Quantification of diversity by Shannon index of diversity analysis demonstrated that most of the diversity was present with a particular population (hill or plane) of P. infestans isolates, with 85% variation being within and 15% being between hill and plane isolates. Subtropical plane isolates of P. infestans exhibited higher variability compared to hill isolates and they were more dispersed on the PCO plot. No clear differentiation of isolates based on mating type was reflected on the dendrogram and PCO plot.

Recovery of optical parameters in multiple-layered diffusive media: theory and experiments.

Diffuse photon density waves have lately been used both to characterize diffusive media and to locate and characterize hidden objects, such as tumors, in soft tissue. In practice, most biological media of medical interest consist of various layers with different optical properties, such as the fat layer in the breast or the different layers present in the skin. Also, most experimental setups consist of a multilayered system, where the medium to be characterized (i.e., the patient's organ) is usually bounded by optically diffusive plates. Incorrect modeling of interfaces may induce errors comparable to the weak signals obtained from tumors embedded deep in highly heterogeneous tissue and lead to significant reconstruction artifacts. To provide a means to analyze the data acquired in these configurations, the basic expressions for the reflection and transmission coefficients for diffusive-diffusive and diffusive-nondiffusive interfaces are presented. A comparison is made between a diffusive slab and an ordinary dielectric slab, thus establishing the limiting distance between the two interfaces of the slab for multiple reflections between them to be considered important. A rigorous formulation for multiple-layered (M-layered) diffusive media is put forward, and a method for solving any M-layered medium is shown. The theory presented is used to characterize a two-layered medium from transmission measurements, showing that the coefficients of scattering, mu'(s) , and absorption, mu(a) , are retrieved with great accuracy. Finally, we demonstrate the simultaneous retrieval of both mu;(s) and mu(a).

Characterization of genetic diversity of some serovars of Bacillus thuringiensis by RAPD.

RAPD based fingerprinting of 21 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 19 random decamer primers. A total of 172 polymorphic fragments, ranging in size from 161-2789 bp, were amplified from 13 of the 19 primers. Pairwise genetic similarity analysis revealed very low similarity values, ranging from 3-68%, among the serovars of Bt, indicating high genetic divergence. Nineteen serovars of Bt fell in two major clusters and remaining two formed solitary clusters in the dendogram. Clustering of Bt strains established genetic relatedness between serovars and serotypes. It has been suggested that RAPD analysis can be used for genotypic characterization of Bt to complement flagellar serotyping.

Near-field diffraction tomography with diffuse photon density waves.

An angular spectrum algorithm is presented for fast, near-field diffraction tomographic imaging with diffuse photon density waves in highly scattering media. A general relation in K space is derived that connects the spatial variations of the optical properties of heterogeneities to the spatial spectra of the measured scattered diffuse photon density waves. The theory is verified experimentally for situations when boundary effects can be neglected. We further describe how to reconstruct absorption and scattering properties simultaneously, and how to incorporate boundary conditions into this angular spectrum algorithm for a turbid medium of finite size (e.g., the slab medium). Limitations and potential improvements of the near-field diffraction tomography are also discussed.

Inactivation of sunflower NADH:nitrate reductase by white light-activated rose bengal.

Chemical modification of purified nitrate reductase (NR) from sunflower leaves by white light-irradiated rose bengal was studied. NADH:NR activity was inhibited by light-activated rose bengal in both a concentration- and time-dependent manner. MV:NR activity was less sensitive to inhibition than NADH:NR activity, especially when the enzyme was preincubated with NADH. Preincubation of the enzyme with FAD protected inhibition of NADH:NR activity but not the MV:NR activity. These results suggest that sunflower NR contains sensitive histidine residue which interacts with reduced FAD during catalytic electron transfer. Most importantly, NADH-reduced NR was more sensitive to the irradiated dye, indicating that conformation of the oxidized and reduced enzyme forms were different.

Diffuse optical 3D-slice imaging of bounded turbid media using a new integro-differential equation.

A new integro-differential equation for diffuse photon density waves (DPDW) is derived within the diffusion approximation. The new equation applies to inhomogeneous bounded turbid media. Interestingly, it does not contain any terms involving gradients of the light diffusion coefficient. The integro-differential equation for diffusive waves is used to develop a 3D-slice imaging algorithm based the on angular spectrum representation in the parallel plate geometry. The algorithm may be useful for near infrared optical imaging of breast tissue, and is applicable to other diagnostics such as ultrasound and microwave imaging.

Algorithms for 3D localization and imaging using near-field diffraction tomography with diffuse light.

We introduce two filtering methods for near-field diffuse light diffraction tomography based on the angular spectrum representation. We then combine these filtering techniques with a new method to find the approximate depth of the image heterogeneities. Taken together these ideas improve the fidelity of our projection image reconstructions, provide an interesting three dimensional rendering of the reconstructed volume, and enable us to identify and classify image artifacts that need to be controlled better for tissue applications. The analysis is accomplished using data derived from numerical finite difference simulations with added noise.

Diffraction tomography for biochemical imaging with diffuse-photon density waves.

The spatial structure of optically heterogeneous turbid media is probed with diffusive light. Projection images are obtained experimentally by deconvolution of the scattered diffuse-photon density waves on a planar boundary by use of a fast Fourier transform. The method is very fast, permitting object localization and characterization in ~1000 volume-element samples on subsecond computational time scales. The optical properties of slice-shape inhomogeneities are accurately determined.