<p>While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.</p> <p>Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence &ndash / associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.</p>

<p>While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.</p> <p>Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence &ndash / associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.</p>

Lui Yuk Sun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 138-148). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Abberviations --- p.v / Table of Contents --- p.vii / List of Tables --- p.xiv / List of Figures --- p.xvi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Bacteriological evaluation of water --- p.1 / Chapter 1.1.1. --- Indicator organisms for water quality monitoring --- p.2 / Chapter 1.1.2. --- Properties defined for indicator organisms --- p.3 / Chapter 1.1.3. --- Example of common indicator organisms --- p.3 / Chapter 1.1.3.1. --- Total coliform group --- p.3 / Chapter 1.1.3.2. --- "Fecal coliform, Escherichia coli" --- p.4 / Chapter 1.1.3.3. --- Fecal Streptococcus --- p.5 / Chapter 1.1.3.4. --- Klebsiella --- p.5 / Chapter 1.2. --- The need for specific detection of waterborne pathogenic organisms --- p.6 / Chapter 1.3. --- Common water-borne pathogenic organisms --- p.7 / Chapter 1.3.1. --- Bacteria --- p.7 / Chapter 1.3.1.1. --- Escherichia coli 0157:H7 --- p.7 / Chapter 1.3.1.2. --- Salmonella typhimurium --- p.11 / Chapter 1.3.1.3 --- Legionella pneumophila --- p.12 / Chapter 1.3.2. --- Protozoa --- p.14 / Chapter 1.3.3. --- Viruses --- p.15 / Chapter 1.4. --- Conventional approaches for pathogens detection --- p.16 / Chapter 1.4.1. --- Examples of conventional detection methods --- p.17 / Chapter 1.4.2. --- Problems related to the conventional detection methods --- p.18 / Chapter 1.5. --- Novel approaches for pathogens detection --- p.19 / Chapter 1.5.1. --- Modifications of media --- p.19 / Chapter 1.5.2. --- Antibody-based methods --- p.20 / Chapter 1.5.3. --- Nucleic acid-based methods --- p.21 / Chapter 1.6. --- Principles of pathogens concentration by filtration and immunomagnetic separation --- p.22 / Chapter 1.7. --- Principles of pathogens detection by polymerase chain reaction --- p.24 / Chapter 1.8. --- Principles of quantitative assay of water-borne pathogens using real-time PCR --- p.26 / Chapter 1.9. --- Aims of this study --- p.28 / Chapter 2. --- Detection of water-borne bacteria by polymerase chain reaction --- p.31 / Chapter 2.1. --- Introduction --- p.31 / Chapter 2.2. --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Bacterial strains --- p.35 / Chapter 2.2.2. --- Bacterial enumeration --- p.35 / Chapter 2.2.3. --- DNA extraction and purification --- p.36 / Chapter 2.2.3.1. --- Boiling method --- p.36 / Chapter 2.2.3.2. --- Protinesae K extraction method --- p.36 / Chapter 2.2.3.3. --- Chelex extraction method --- p.37 / Chapter 2.2.4. --- Targeted sequences --- p.38 / Chapter 2.2.4.1. --- eaeA gene --- p.38 / Chapter 2.2.4.2. --- mdh gene --- p.39 / Chapter 2.2.4.3. --- flaR gene --- p.39 / Chapter 2.2.5. --- PCR amplification --- p.40 / Chapter 2.2.6. --- Gel electrophoresis --- p.41 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Optimization of the PCR --- p.42 / Chapter 2.3.2. --- Sensitivity of PCR detection --- p.42 / Chapter 2.3.2.1. --- Boiling method --- p.42 / Chapter 2.3.2.2. --- Proteinease K method --- p.43 / Chapter 2.3.2.3. --- Chelex method --- p.43 / Chapter 2.3.3. --- Specificity of PCR detection --- p.43 / Chapter 2.3.3.1. --- primers targeted uidA gene --- p.44 / Chapter 2.3.3.2. --- primers targeted mdh gene --- p.44 / Chapter 2.3.3.3. --- primers targeted flaR gene --- p.44 / Chapter 2.4. --- Discussion --- p.57 / Chapter 3. --- Concentration and separation of water-borne bacteria by two-step-filtration and immunomagnetic separation --- p.61 / Chapter 3.1. --- Introduction --- p.61 / Chapter 3.2. --- Materials and Methods --- p.66 / Chapter 3.2.1. --- Bacterial strains --- p.66 / Chapter 3.2.2. --- Bacterial enumeration --- p.66 / Chapter 3.2.3. --- Filtration --- p.67 / Chapter 3.2.4. --- Immunomagnetic separation (IMS) --- p.68 / Chapter 3.2.4.1. --- Antibodies and Magnetic beads --- p.68 / Chapter 3.2.4.2. --- Binding of antibodies to magnetic beads --- p.68 / Chapter 3.2.4.3. --- Immunomagnetic separation of bacteria in seeded samples --- p.70 / Chapter 3.2.5. --- Determine the efficiency of filtration and immunomagnetic separation --- p.70 / Chapter 3.2.6. --- DNA extraction --- p.71 / Chapter 3.2.7. --- Multiplex PCR --- p.71 / Chapter 3.2.8. --- PCR amplification --- p.72 / Chapter 3.2.9. --- Gel electrophoresis --- p.72 / Chapter 3.3. --- Results --- p.73 / Chapter 3.3.1. --- Efficiency of filtration and immunomagnetic separation --- p.73 / Chapter 3.3.2. --- Detection limit of PCR --- p.73 / Chapter 3.3.2.1. --- Filtration and immunomagnetic separation --- p.73 / Chapter 3.3.2.2. --- Influence of background flora --- p.73 / Chapter 3.3.2.3 --- Shing Mun River and Lam Tsuen River --- p.77 / Chapter 3.3.3. --- Multiplex PCR --- p.77 / Chapter 3.4. --- Discussion --- p.91 / Chapter 4. --- Quantitative assay of water-borne pathogens using real-time PCR --- p.94 / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Materials and Methods --- p.99 / Chapter 4.2.1. --- Bacteria strains --- p.99 / Chapter 4.2.2. --- Bacterial enumeration --- p.99 / Chapter 4.2.3. --- Primers and Probes --- p.100 / Chapter 4.2.3.1. --- eaeA gene --- p.101 / Chapter 4.2.3.2. --- mdh gene --- p.102 / Chapter 4.2.3.3. --- flaR gene --- p.102 / Chapter 4.2.4. --- Targeted sequences cloning and sequencing --- p.103 / Chapter 4.2.4.1. --- Amplication of targeted sequence by PCR --- p.103 / Chapter 4.2.4.2. --- Purification of PCR product --- p.104 / Chapter 4.2.4.3. --- Ligation with cloning vector --- p.105 / Chapter 4.2.4.4. --- Transformation of E.coli DH5a cells --- p.105 / Chapter 4.2.4.5. --- Plasmid DNA isolation --- p.106 / Chapter 4.2.4.6. --- DNA quantitation and sequencing --- p.107 / Chapter 4.2.5. --- Quantitation determination using real-time PCR --- p.108 / Chapter 4.3. --- Results --- p.110 / Chapter 4.3.1. --- Determination of targeted sequences --- p.110 / Chapter 4.3.2. --- Reading of fluorescence intensity and data analysis --- p.110 / Chapter 4.3.3. --- Sensitivity of real-time PCR --- p.114 / Chapter 4.3.4. --- Specificity of real-time PCR --- p.121 / Chapter 4.3.5. --- Quantitation analysis in seeded samples --- p.121 / Chapter 4.4. --- Discussion --- p.131 / Chapter 5. --- Conclusion and future perspectives --- p.133 / Chapter 6. --- References --- p.138

Bacillus thuringiensis (Bt) é uma bactéria entomopatogênica utilizada como biopesticida contra insetos, principalmente Lepidoptera, Coleoptera e Diptera, e na produção de organismos geneticamente modificados. Com a descoberta da capacidade de Bt se movimentar sistemicamente em plantas, potencialmente atingindo insetos sugadores, surge uma nova possibilidade para o controle de Diaphorina citri Kuwayama, que transmite bactérias associadas ao Huanglongbing, uma séria doença da citricultura. O objetivo do trabalho foi desenvolver sistemas de aquisição de Bt por D. citri, in vitro e in planta, para estudos de patogenicidade. Uma estirpe de Bt transformada com o gene green fluorescent protein (Btk-gfp), cultivada em meio NYSM, foi usada como marcador de aquisição pelo inseto e movimentação nas plantas. Para o sistema de aquisição in vitro, selecionaram-se dietas com base na sobrevivência e atividade alimentar de D. citri. Btk-gfp foi adicionada à dieta selecionada, composta por uma solução de sacarose a 30% em água mineral com corantes alimentícios (verde 0,1% e amarelo 0,4%). A dieta foi acondicionada em um sachê formado por duas membranas de Parafilm®, sobre a parte inferior de uma placa de Petri de 40 mm de diâmetro (gaiola de alimentação). Dez insetos foram confinados em cada gaiola para períodos de acesso à aquisição (PAA) de até 48 h, estabelecendo-se 10 gaiolas por tratamento; como controle, utilizou-se a dieta sem Btk-gfp. Para testes de aquisição in planta, suspensões de Btk-gfp foram inoculadas em ramos novos cortados e no solo, próximo à haste de seedlings de laranja doce [Citrus sinensis (L.) Osbeck] e de murta [Murraya paniculata (L.) Jack], e em diferentes concentrações, avaliando-se a movimentação da bactéria após um período de 48 h. Posteriormente, adultos e ninfas de D. citri foram confinados nestas plantas para avaliar a aquisição de Btk-gfp, usando-se plantas inoculadas apenas com água como controle. Para os bioensaios de patogenicidade a D. citri, 21 estirpes de Bt foram testadas no sistema de aquisição em ramos cortados de murta e 5 estirpes em ramos cortados de laranja para ninfas de 3º. ínstar. In vitro, testaram-se 3 estirpes contra ninfas de 3º. ínstar e 9 contra adultos. Btgfp foi absorvido pelas raízes e ramos novos cortados de seedlings de laranja doce e de murta, e translocado até as folhas, mostrando movimentação sistêmica. Esta estirpe foi adquirida por adultos e ninfas de D. citri que se alimentaram nos ramos novos cortados, mantidos em suspensão bacteriana. O sistema de alimentação in vitro permitiu a aquisição de Bt-gfp (pellet ressuspendido na dieta de sacarose com corantes) por adultos de D. citri com apenas 12 h de PAA, mas 30 h é um período ótimo para exposição do inseto ao sistema. Nenhuma das nove estirpes testadas no sistema in vitro foi patogênica aos adultos de D. citri. Das 21 estirpes testadas contra ninfas de 3º. ínstar em ramos novos de murta, cinco causaram mortalidade de 24 a 45%, em 5 dias. Essas cinco estirpes também foram testadas contra ninfas dem ramos de citros cortados, causando mortalidade de 35 a 75% em 48 h. / Bacillus thuringiensis (Bt) is an entomopathogenic bacterium widely used as a biopesticide against insect pests, mainly Coleoptera, Lepidoptera and Diptera, or for engineering genetically-modified plants. The recent finding that Bt is able to move systemically within plants, potentially targeting piercing-sucking insects, suggests that the bacterium may be effective for microbial control of Diaphorina citri Kuwayama, the vector of Huanglongbing-associated bacteria, a serious citrus disease. The goal of this research was to develop in vitro and in planta acquisition systems of Bt by D. citri for pathogenicity assays. A transformed Bt strain with the green fluorescent protein gene (Btk-gfp), grown in NYSM medium, was used as a marker to demonstrate bacterial acquisition by the insect and movement within the plants. Artificial diets were selected for the in vitro acquisition system based on insect survival and feeding activity. Btk-gfp was added to the selected diet, a 30% sucrose solution in mineral water with green (0.1%) and yellow (0.4%) food coloring. The diet was placed inside a Parafilm® membrane sachet, covering the opening of the lower half of a 40-mm diameter culture plate, and forming the feeding cage. Ten D. citri adults or nymphs were introduced in each cage and allowed acquisition access periods (AAP) of up to 48 h on the diet; as a control, a diet without Btk-gfp was used. For testing the in planta acquisition system, Btk-gfp suspensions were inoculated in young stem cuttings or in the soil, near the stem of Citrus sinensis (L.) Osbeck and Murraya paniculata (L.) Jack seedlings, as well as in different concentrations, and bacterial movement was assessed after 48 h; plants inoculated with water were used as controls. D. citri adults and nymphs were confined on inoculated plants to verify Btk-gfp acquisition. In pathogenicity assays, 21 Bt strains were tested against 3rd instars of D. citri using the acquisition system with stem cuttings of M. paniculata and 5 strains were tested using citrus stem cuttings. The in vitro acquisition system was used to test pathogenicity of 3 and 9 Bt strains against 3rd-instar nymphs and adults, respectively. Bt-gfp absorbed by roots or young stem cuttings of inoculated C. sinensis and M. paniculata was detected in the leaves, showing systemic movement. Bt-gfp was isolated from groups of nymphs and adults that were fed on inoculated stem cuttings or on artificial diets with bacterial suspension, showing that both in planta and in vitro acquisition take place. D. citri adults can acquire Bt-gfp within 12 h of AAP to the artificial diet with bacterium inoculum, but 30 h is the optimum AAP. None of the nine Bt strains assayed in vitro were pathogenic to D. citri adults. Of 21 strains tested for pathogenicity against nymphs using inoculated stem cuttings of M. paniculata, five caused mortality rates varying from 24 to 45% mortality within 5 days. These five Bt strains were also tested against D. citri nymphs using young stem cuttings of C. sinensis, causing mortality rates of 35 to 75% within 48 h.

Bacillus thuringiensis (Bt) é uma bactéria entomopatogênica utilizada como biopesticida contra insetos, principalmente Lepidoptera, Coleoptera e Diptera, e na produção de organismos geneticamente modificados. Com a descoberta da capacidade de Bt se movimentar sistemicamente em plantas, potencialmente atingindo insetos sugadores, surge uma nova possibilidade para o controle de Diaphorina citri Kuwayama, que transmite bactérias associadas ao Huanglongbing, uma séria doença da citricultura. O objetivo do trabalho foi desenvolver sistemas de aquisição de Bt por D. citri, in vitro e in planta, para estudos de patogenicidade. Uma estirpe de Bt transformada com o gene green fluorescent protein (Btk-gfp), cultivada em meio NYSM, foi usada como marcador de aquisição pelo inseto e movimentação nas plantas. Para o sistema de aquisição in vitro, selecionaram-se dietas com base na sobrevivência e atividade alimentar de D. citri. Btk-gfp foi adicionada à dieta selecionada, composta por uma solução de sacarose a 30% em água mineral com corantes alimentícios (verde 0,1% e amarelo 0,4%). A dieta foi acondicionada em um sachê formado por duas membranas de Parafilm®, sobre a parte inferior de uma placa de Petri de 40 mm de diâmetro (gaiola de alimentação). Dez insetos foram confinados em cada gaiola para períodos de acesso à aquisição (PAA) de até 48 h, estabelecendo-se 10 gaiolas por tratamento; como controle, utilizou-se a dieta sem Btk-gfp. Para testes de aquisição in planta, suspensões de Btk-gfp foram inoculadas em ramos novos cortados e no solo, próximo à haste de seedlings de laranja doce [Citrus sinensis (L.) Osbeck] e de murta [Murraya paniculata (L.) Jack], e em diferentes concentrações, avaliando-se a movimentação da bactéria após um período de 48 h. Posteriormente, adultos e ninfas de D. citri foram confinados nestas plantas para avaliar a aquisição de Btk-gfp, usando-se plantas inoculadas apenas com água como controle. Para os bioensaios de patogenicidade a D. citri, 21 estirpes de Bt foram testadas no sistema de aquisição em ramos cortados de murta e 5 estirpes em ramos cortados de laranja para ninfas de 3º. ínstar. In vitro, testaram-se 3 estirpes contra ninfas de 3º. ínstar e 9 contra adultos. Btgfp foi absorvido pelas raízes e ramos novos cortados de seedlings de laranja doce e de murta, e translocado até as folhas, mostrando movimentação sistêmica. Esta estirpe foi adquirida por adultos e ninfas de D. citri que se alimentaram nos ramos novos cortados, mantidos em suspensão bacteriana. O sistema de alimentação in vitro permitiu a aquisição de Bt-gfp (pellet ressuspendido na dieta de sacarose com corantes) por adultos de D. citri com apenas 12 h de PAA, mas 30 h é um período ótimo para exposição do inseto ao sistema. Nenhuma das nove estirpes testadas no sistema in vitro foi patogênica aos adultos de D. citri. Das 21 estirpes testadas contra ninfas de 3º. ínstar em ramos novos de murta, cinco causaram mortalidade de 24 a 45%, em 5 dias. Essas cinco estirpes também foram testadas contra ninfas dem ramos de citros cortados, causando mortalidade de 35 a 75% em 48 h. / Bacillus thuringiensis (Bt) is an entomopathogenic bacterium widely used as a biopesticide against insect pests, mainly Coleoptera, Lepidoptera and Diptera, or for engineering genetically-modified plants. The recent finding that Bt is able to move systemically within plants, potentially targeting piercing-sucking insects, suggests that the bacterium may be effective for microbial control of Diaphorina citri Kuwayama, the vector of Huanglongbing-associated bacteria, a serious citrus disease. The goal of this research was to develop in vitro and in planta acquisition systems of Bt by D. citri for pathogenicity assays. A transformed Bt strain with the green fluorescent protein gene (Btk-gfp), grown in NYSM medium, was used as a marker to demonstrate bacterial acquisition by the insect and movement within the plants. Artificial diets were selected for the in vitro acquisition system based on insect survival and feeding activity. Btk-gfp was added to the selected diet, a 30% sucrose solution in mineral water with green (0.1%) and yellow (0.4%) food coloring. The diet was placed inside a Parafilm® membrane sachet, covering the opening of the lower half of a 40-mm diameter culture plate, and forming the feeding cage. Ten D. citri adults or nymphs were introduced in each cage and allowed acquisition access periods (AAP) of up to 48 h on the diet; as a control, a diet without Btk-gfp was used. For testing the in planta acquisition system, Btk-gfp suspensions were inoculated in young stem cuttings or in the soil, near the stem of Citrus sinensis (L.) Osbeck and Murraya paniculata (L.) Jack seedlings, as well as in different concentrations, and bacterial movement was assessed after 48 h; plants inoculated with water were used as controls. D. citri adults and nymphs were confined on inoculated plants to verify Btk-gfp acquisition. In pathogenicity assays, 21 Bt strains were tested against 3rd instars of D. citri using the acquisition system with stem cuttings of M. paniculata and 5 strains were tested using citrus stem cuttings. The in vitro acquisition system was used to test pathogenicity of 3 and 9 Bt strains against 3rd-instar nymphs and adults, respectively. Bt-gfp absorbed by roots or young stem cuttings of inoculated C. sinensis and M. paniculata was detected in the leaves, showing systemic movement. Bt-gfp was isolated from groups of nymphs and adults that were fed on inoculated stem cuttings or on artificial diets with bacterial suspension, showing that both in planta and in vitro acquisition take place. D. citri adults can acquire Bt-gfp within 12 h of AAP to the artificial diet with bacterium inoculum, but 30 h is the optimum AAP. None of the nine Bt strains assayed in vitro were pathogenic to D. citri adults. Of 21 strains tested for pathogenicity against nymphs using inoculated stem cuttings of M. paniculata, five caused mortality rates varying from 24 to 45% mortality within 5 days. These five Bt strains were also tested against D. citri nymphs using young stem cuttings of C. sinensis, causing mortality rates of 35 to 75% within 48 h.