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Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolate

Cronobacter species cause infections in all age groups; however neonates are at highest risk and remain the most susceptible age group for life-threatening invasive disease. The genus contains seven species:Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite an abundance of published genomes of these species, genomics-based epidemiology of the genus is not well established. The gene content of a diverse group of 126 unique Cronobacter and taxonomically related isolates was determined using a pan genomic-based DNA microarray as a genotyping tool and as a means to identify outbreak isolates for food safety, environmental, and clinical surveillance purposes. The microarray constitutes 19,287 independent genes representing 15 Cronobacter genomes and 18 plasmids and 2,371 virulence factor genes of phylogenetically related Gram-negative bacteria. The Cronobacter microarray was able to distinguish the seven Cronobacter species from one another and from non-Cronobacter species; and within each species, strains grouped into distinct clusters based on their genomic diversity. These results also support the phylogenic divergence of the genus and clearly highlight the genomic diversity among each member of the genus. The current study establishes a powerful platform for further genomics research of this diverse genus, an important prerequisite toward the development of future countermeasures against this foodborne pathogen in the food safety and clinical arenas

Entomopathogenic fungi are being investigated as alternatives to chemical insecticides. This study explored the versatility of the entomopathogenic fungus Metarhizium anisopliae by examining its diversification, saprophytic competence and potential for genetic enhancement. M. anisopliae is a radiating species containing both generalist and specialized lineages with broad and narrow host ranges and as such provides an excellent model system to study the evolution of pathogenesis. Using 18S RNA and protease sequences, I demonstrated that strains can be selected representing evolutionary distances ranging from <1 to 8 MY and their natural molecular variation allows analysis of processes of adaptive change. M. anisopliae is particularly abundant in the rhizosphere. Germination of M. anisopliae strain 2575 was >96% in 1 mg/ml root exudate (RE) and growth in RE resulted in 29 (58%) genes being up-regulated and 21 (42%) down-regulated. The identity of these genes is helping to define the physiological requirements for rhizosphere competence. Hypothetical and orphans proteins (41.4%) were also actively expressed indicating that many previously uncharacterized genes may have functions related to survival at the soil-root interphase. Using the fungus as a delivery vehicle for foreign toxins presents a powerful approach for increasing virulence. M. anisopliae was modified to express a scorpion toxin (AaIT) in insect haemolymph and bioassayed against the coffee berry borer Hypothenemus hampei. AaIT increased mortality up to 56.6%, and reduced the medial lethal concentration (LC 50 ) by 15.7-fold and the average survival time (AST) by 20.1%. The AaIT gene and the M. anisopliae esterase gene (Mest1) were inserted into three strains of Beauveria bassiana (ARSEF 252, 8998 and 9184) with high, medium and low mortality, respectively, against the Colorado potato beetle (CPB) Leptinotarsa decemlineata. Mortality rates were strain- and dose- dependant and increased from 16.1

Fungal endophytes are quite common in nature and some of them have been shown to have adverse effects against insects, nematodes, and plant pathogens. Our research program is aimed at using fungal endophytes-mediated plant defense as a novel biological control mechanism against the coffee berry borer, the most devastating pest of coffee throughout the world. A survey of fungal endophytes in coffee plants from Hawaii, Colombia, Mexico, and Puerto Rico has revealed the presence of various genera of fungal entomopathogens, including Acremonium, Beauveria, Cladosporium, Clonostachys, and Paecilomyces. Two of these, B. bassiana and Clonostachys rosea, were tested against the coffee berry borer and were shown to be pathogenic. This paper reviews the possible mode of action of entomopathogenic fungal endophytes.