High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

SUMMARY A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented.

Spiroplasma citri, transmitted by phloem-feeding leafhoppers, moves from the gut lumen through the gut wall, hemolymph, and salivary glands and multiplies in insect tissues. Nontransmissible lines were deficient in their ability to cross these barriers. Molecular analysis revealed extensive chromosomal rearrangements between the transmissible and nontransmissible spiroplasma lines including a large chromosomal inversion and deletions of about 10 kb at each inversion border. One open reading frame of the deleted region, cloned from the transmissible strain BR3-3X, encodes an integral membrane protein of 58 kDa that shares limited sequence similarity with major adhesin proteins of two zoopathogenic mycoplasmas. Adhesion of spiroplasmas to cultured leafhopper cells was inhibited by proteases, suggesting that adherence to host cells is mediated by spiroplasma membrane protein(s). A hypothetical model for insect transmission of phytopathogenic mollicutes is presented.

Human pathogens can contaminate leafy produce in the field by various routes. We hypothesized that interactions between Escherichia coli O157:H7 and spinach are influenced by the route of introduction and the leaf microenvironment. E. coli O157:H7 labeled with green fluorescent protein was dropped onto spinach leaf surfaces, simulating bacteria-laden raindrops or sprinkler irrigation, and survived on the phylloplane for at least 14 days, with increasing titers and areas of colonization over time. The same strains placed into the rhizosphere by soil infiltration remained detectable on very few plants and in low numbers (102 to 106 CFU/g fresh tissue) that decreased over time. Stem puncture inoculations, simulating natural wounding, rarely resulted in colonization or multiplication. Bacteria forced into the leaf interior survived for at least 14 days in intercellular spaces but did not translocate or multiply. Three spinach cultivars with different leaf surface morphologies were compared for colonization by E. coli O157:H7 introduced by leaf drop or soil drench. After 2 weeks, cv. Bordeaux hosted very few bacteria. More bacteria were seen on cv. Space and were dispersed over an area of up to 0.3 mm2. The highest bacterial numbers were observed on cv. Tyee but were dispersed only up to 0.15 mm2, suggesting that cv. Tyee may provide protected niches or more nutrients or may promote stronger bacterial adherence. These findings suggest that the spinach phylloplane is a supportive niche for E. coli O157:H7, but no conclusive evidence was found for natural entry into the plant interior. The results are relevant for interventions aimed at minimizing produce contamination by human pathogens.

Two microtiter plate assays were developed to study the adherence of the plant-pathogenic mollicute Spiroplasma citri to a monolayer of cultured cells of its leafhopper vector, Circulifer tenellus. Adherence was significantly reduced by prior treatment of the spiroplasmas with proteinase K or pronase. Electrophoresis and western blotting of spiroplasma membrane proteins, before and after exposure of intact spiroplasmas to proteases, revealed the concomitant reduction in intensity of a major membrane protein (P89) and a new polypeptide of approximately 46 kDa in protease-treated preparations (P46). Triton X-114 phase partitioning demonstrated that P89 and P46 are amphiphilic, and labeling of the new polypeptide P46 with anti-P89 serum suggested that this molecule may be a breakdown product of P89. Regeneration of P89 after proteinase K treatment of spiroplasmas was directly associated with restoration of the pathogen's attachment capability. Treatment of spiroplasmas with any of several carbohydrates and glycoconjugates or with tetramethyl-urea, a compound that interferes with hydrophobic associations, had a negligible effect on attachment. These results suggest that a spiroplasma surface protein, P89, has a role in S. citri adherence to C. tenellus cells.

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Spiroplasma citri, a helical, wall-less prokaryote in the class Molli-cutes, is transmitted by the beet leafhopper, Circulifer tenellus. Invasion of leafhopper tissues and cytopathological effects by S. citri were investigated by transmission electron microscopy. All eight cell types of the principle salivary glands, as well as the adjacent muscle cells and the cells of the accessory salivary glands, were colonized by the spiroplas-mas. In both midgut epithelia and salivary gland cells, spiroplasmas usually occurred in membrane-bound cytoplasmic vesicles that often were located near the cell periphery. In several salivary gland cells, spiroplas-mas were also observed within membranous pockets apparently formed by invagination of the plasmalemma beneath intact basal lamina. These observations are consistent with spiroplasma entry into the insect cells by receptor-mediated endocytosis. Cytopathological effects of spiroplasma infection in salivary cells included loss of membrane and basal lamina integrity, presence in some cells of irregular inclusion-like structures containing dense matrices of filamentous material that labeled with anti S. citri antibodies, and apparent disorganization of the endoplasmic reticulum. Compared to the tightly aligned fiber bundles in healthy muscle cells, bundles in spiroplasma-containing muscle cells appeared fragmented and loosely arranged. Such symptoms could contribute to the reduction in longevity and fecundity that has been previously reported for S. citri-infected C. tenellus.