Robert E. Pruitt

The elastic light-scatter (ELS) technique, which detects and discriminates microbial organisms based on the light-scatter pattern of their colonies, has demonstrated excellent classification accuracy in pathogen screening tasks. The implementation of the multispectral approach has brought further advantages and motivated the design and validation of a hyperspectral elastic light-scatter phenotyping instrument (HESPI). The newly developed instrument consists of a supercontinuum (SC) laser and an acousto-optic tunable filter (AOTF). The use of these two components provided a broad spectrum of excitation light and a rapid selection of the wavelength of interest, which enables the collection of multiple spectral patterns for each colony instead of relying on single band analysis. The performance was validated by classifying microflora of green-leafed vegetables using the hyperspectral ELS patterns of the bacterial colonies. The accuracy ranged from 88.7% to 93.2% when the classification was performed with the scattering pattern created at a wavelength within the 473–709 nm region. When all of the hyperspectral ELS patterns were used, owing to the vastly increased size of the data, feature reduction and selection algorithms were utilized to enhance the robustness and ultimately lessen the complexity of the data collection. A new classification model with the feature reduction process improved the overall classification rate to 95.9%.

An optical forward‐scatter model was generalized to encompass the diverse nature of bacterial colony morphologies and the spectral information. According to the model, the colony shape and the wavelength of incident light significantly affect the characteristics of a forward elastic‐light‐scattering pattern. To study the relationship between the colony morphology and the scattering pattern, three‐dimensional colony models were generated in various morphologies. The propagation of light passing through the colony model was then simulated. In validation of the theoretical modeling, the scattering patterns of three bacterial genera, Staphylococcus, Exiguobacterium and Bacillus, which grow into colonies having convex, crateriform and flat elevations, respectively, were qualitatively compared to the simulated scattering patterns. The strong correlations observed between simulated and experimental patterns validated the scatter model. In addition, spectral effect on the scattering pattern was studied using the scatter model, and experimentally investigated using Staphylococcus, whose colony has circular form and convex elevation. Both simulation and experiment showed that changes in wavelength affected the overall pattern size and the number of rings.

Salmonella serovars have been associated with the majority of foodborne illness outbreaks involving tomatoes, and E. coli O157:H7 has caused outbreaks involving other fresh produce. Contamination by both pathogens has been thought to originate from all points of the growing and distribution process. To determine if Salmonella serovar Typhimurium and E. coli O157:H7 could move to the mature tomato fruit of different tomato cultivars following contamination, three different contamination scenarios (seed, leaf, and soil) were examined. Following contamination, each cultivar appeared to respond differently to the presence of the pathogens, with most producing few fruit and having overall poor health. The Micro-Tom cultivar, however, produced relatively more fruit and E. coli O157:H7 was detected in the ripe tomatoes for both the seed- and leaf- contaminated plants, but not following soil contamination. The Roma cultivar produced fewer fruit, but was the only cultivar in which E. coli O157:H7 was detected via all three routes of contamination. Only two of the five cultivars produced tomatoes following seed-, leaf-, and soil- contamination with Salmonella Typhimurium, and no Salmonella was found in any of the tomatoes. Together these results show that different tomato cultivars respond differently to the presence of a human pathogen, and for E. coli O157:H7, in particular, tomato plants that are either contaminated as seeds or have a natural opening or a wound, that allows bacteria to enter the leaves can result in plants that have the potential to produce tomatoes that harbor internalized pathogenic bacteria.

Escherichia coli O157:H7 has been associated with numerous outbreaks involving fresh produce. Previous studies have shown that bacteria can be internalized within plant tissue and that this can be a source of protection from antimicrobial chemicals and environmental conditions. However, the types of tissue and cellular locations the bacteria occupy in the plant following internalization have not been addressed. In this study, immunocytochemical techniques were used to localize internalized E. coli O157:H7 expressing green fluorescent protein in germinated mung bean (Vigna radiata) hypocotyl tissue following contamination of intact seeds. An average of 13 bacteria per mm3 were localized within the sampled tissue. The bacteria were found to be associated with every major tissue and corresponding cell type (cortex, phloem, xylem, epidermis, and pith). The cortical cells located on the outside of the vascular bundles contained the majority of the internalized bacteria (61%). In addition, the bacteria were localized primarily to the spaces between the cells (apoplast) and not within the cells. Growth experiments were also performed and demonstrated that mung bean plants could support the replication of bacteria to high levels (107 CFU per plant) following seed contamination and that these levels could be sustained over a 12-day period. Therefore, E. coli O157:H7 can be internalized in many different plant tissue types after a brief seed contamination event, and the bacteria are able to grow and persist within the plant.

A fundamental tenet of classical mendelian genetics is that allelic information is stably inherited from one generation to the next, resulting in predictable segregation patterns of differing alleles. Although several exceptions to this principle are known, all represent specialized cases that are mechanistically restricted to either a limited set of specific genes (for example mating type conversion in yeast) or specific types of alleles (for example alleles containing transposons or repeated sequences). Here we show that Arabidopsis plants homozygous for recessive mutant alleles of the organ fusion gene HOTHEAD (HTH) can inherit allele-specific DNA sequence information that was not present in the chromosomal genome of their parents but was present in previous generations. This previously undescribed process is shown to occur at all DNA sequence polymorphisms examined and therefore seems to be a general mechanism for extra-genomic inheritance of DNA sequence information. We postulate that these genetic restoration events are the result of a template-directed process that makes use of an ancestral RNA-sequence cache.

Summary: The outer epidermal plant cell wall and cuticle play an important role in regulating both abiotic and biotic interactions between the plant and its environment. In addition to acting as a protective barrier that limits water loss, the effects of detrimental irradiation and invasion by pathogens, the epidermis also offers an interface that is inert to interactions between organs and ensures proper separation and expansion of organs at the growing points of the plant. Here, we describe the molecular cloning and characterization of HOTHEAD (HTH), a gene required to limit cellular interactions between contacting epidermal cells during floral development. HTH is a member of a small gene family in Arabidopsis and encodes an enzyme related to a group of FAD‐containing oxidoreductases that have been described in several other species. Characterization of 11 independently derived mutant alleles suggests that key amino acids are shared between these related groups of enzymes and identify a cluster of other functionally important residues that are highly conserved only within the Arabidopsis gene family. Our findings add this new type of enzyme to a growing list of enzymes that have been shown to be involved in regulating post‐genital organ fusion. Expression analysis of the HTH gene shows that it is expressed in all tissues tested, including roots, and is not epidermis‐specific. Furthermore, the sequence data unequivocally show that none of the alleles isolated are epigenetic alleles as suggested by genetic behavior previously observed at this locus.