A malária é uma doença grave, cujos vetores são mosquitos do gênero Anopheles. Esse gênero é composto por cerca de 500 espécies e aproximadamente 45 são responsáveis pela transmissão do parasito em todo o mundo. Alguns destes vetores são componentes de complexo de espécies crípticas como Complexo Albitarsis e Complexo Triannulatus. O Complexo Albitarsis é formado por quatro espécies: An. albitarsis s.s, An. albitarsis B, An. marajoara e An. deaneorum. O Complexo Triannulatus é composto por três espécies: An. triannulatus, An. halophylus e An. triannulatus C. Algumas técnicas moleculares são utilizadas para auxiliar na distinção destas espécies crípticas. Dessa forma, DNA de An. albitarsis s.l e An. triannulatus s.l., capturados na Usina de Porto Primavera, foram amplificados para ITS2 e para o gene ND4 do DNAmt. Todas as amostras foram submetidas à técnica de RFLP. Algumas amostras foram seqüenciadas diretamente ou clonadas para posterior seqüenciamento a fim de se confirmar a espécie. Das amostras do Complexo Albitarsis, 62,85% foram identificadas como An. deaneorum para ITS2-RFLP. As restantes foram amplificadas para ND4-RFLP e foram identificadas como An. albitarsis s.s. Observou-se, no entanto, que as amostras do Complexo Albitarsis não exibiram similaridade total com as depositadas no GeneBank sendo que para ITS2-PCR estas foram identificadas como An. albitarsis s.s. e An. deaneorum, enquanto que para o gene ND4 todas foram identificadas como An. albitarsis B. O ITS2-RFLP para o An. triannulatus s.l demonstrou polimorfismos entre as espécies. No seqüenciamento, estas amostras foram similares àquelas depositadas no GeneBank. O emprego de marcadores moleculares podem, em parte, auxiliar na distinção de espécies crípticas, porém outros marcadores devem ser avaliados a fim de se elucidar a identificação do Complexo Albitarsis. / Malaria is a severe disease whose vectors are mosquitoes belonging to the genus Anopheles. This genus contains 500 species of which approximately 45 are responsible for the worldwide transmission of these parasites. Some of these vectors belong to cryptic species such as those of the Albitarsis and Triannulatus Complex. The Albitarsis Complex is composed of four species: An. albitarsis s.s., An. albitarsis B, An. marajoara and An. deaneorum. The Triannulatus Complex contains three species: An. triannulatus, An. halophylus and An. triannulatus C. We used molecular techniques to differentiate these cryptic species. Thus, DNA of An. albitarsis s.l and An. triannulatus s.l, captured at the Porto Primavera Dam had their ITS2 and their mtDNA, ND4 genes amplified. All samples were analyzed by the RFLP technique. Some samples were directly sequenced while others were cloned for subsequent sequencing for species confirmation. Within the Albitarsis Complex, 62.85% were identified as An. deaneorum by RFLP-ITS2. The remaining were amplified by RFLP-ND4 and identified as An. albitarsis s.s. However the results of sequencing of the samples of the Albitarsis Complex did not overlap entirely with those deposited in the GeneBank since those amplified by RFLP-ITS2 were identified as An. albitarsis s.s. and An. deaneorum while those obtained by RFLP-ND4 were identified as An. albitarsis B. Also RFLP-ITS2 of A. triannulatus s.l. contained polymorphic regions among different species. By sequencing, these samples were similar to those deposited in the GeneBank. The use of molecular markers did, in some instances help to distinguish species within cryptic complexes, however other markers need to be evaluated to elucidate further identification of the Albitarsis Complex.

O Huanglongbing (HLB) é uma das mais temidas doenças da citricultura mundial, associada a bactérias do gênero Candidatus Liberibacter, que foram detectadas no Brasil em 2004. Em 2008, detectou-se outra bactéria associada a sintomas de HLB no Estado de São Paulo, que foi caracterizada como sendo um fitoplasma do grupo 16 SrIX. Fitoplasmas são molicutes fitopatógenos restritos ao floema de plantas, disseminados por insetos vetores. O presente trabalho buscou identificar cigarrinhas potencialmente vetoras do fitoplasma associado ao HLB e plantas hospedeiras desses insetos, bem como desenvolver uma técnica de quantificação de DNA desse patógeno em insetos e plantas. Amostragens de cigarrinhas foram realizadas quinzenalmente por 12 meses em dois pomares de laranja com histórico de ocorrência do fitoplasma 16SrIX na região norte do Estado de São Paulo, usando-se armadilhas adesivas amarelas em duas alturas (0,3 e 1,5 m) da copa de árvores cítricas, e rede de varredura na vegetação espontânea. Dados faunísticos identificaram uma espécie de Agalliinae (Agallia albidula Uhler) e três de Deltocephalinae, [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål) e Scaphytopius (Convelinus) marginelineatus (Stål)], como os cicadelídeos (Hemiptera: Cicadellidae) mais abundantes e frequentes nas áreas estudadas. Essas espécies predominaram na amostragem com rede de varredura e na menor altura de coleta com armadilhas adesivas, indicando comportamento de alimentação em vegetação rasteira. Com observações visuais, verificou-se associação das espécies com certas plantas invasoras, e influência da composição florística da vegetação rasteira sobre a abundância das cigarrinhas. S. marginelineatus e P. flavicosta ocorreram com maior frequência em Sida rhombifolia L. e Althernantera tenella Colla, respectivamente, enquanto que A. albidula foi predominante em Conyza bonariensis (L.) Cronq., e B. hebe ocorreu exclusivamente em gramíneas, principalmente Panicum maximum Jacq.. Plantas invasoras amostradas nas áreas foram testadas para a presença do fitoplasma 16SrIX, porém sem resultados positivos. No entanto, amostras de campo da cigarrinha S. marginelineatus foram positivas por PCR e sequenciamento para o referido fitoplasma. Indivíduos de S. marginelineatus criados em laboratório e mantidos por um período de acesso à aquisição de 72 h em citros infectado com o fitoplasma 16SrIX, foram capazes de transmití-lo para citros, após 21 dias de latência, porém com baixa eficiência (0,5%). Por meio de PCR quantitativo desenvolvido para esse fitoplasma, verificou-se baixo título do patógeno tanto em S. marginelineatus, quanto em plantas cítricas infectadas, o que pode explicar, pelo menos em parte, a baixa eficiência de transmissão pelo inseto tendo citros como fonte. Isto sugere a existência de outros hospedeiros mais adequados como fontes de inóculo para aquisição do fitoplasma por S. marginelineatus ou outro vetor ainda desconhecido. / Huanglongbing (HLB) is a severe citrus disease associated to phloemlimited bacteria in the genus Candidatus Liberibacter, which were detected in Brazil in 2004. In 2008, another bacterium was found in association with HLB symptom in the São Paulo State, and characterized as a phytoplasma belonging to 16SrIX group. Phytoplasmas are vector-borne phytopathogenic mollicutes that inhabit plant sieve elements. The goals of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants, as well as to establish a real-time PCR procedure for pathogen quantification in vectors and plants. Leafhoppers were sampled fortnightly for 12 months by yellow sticky cards placed at two heights (0.3 and 1.5 m) on the citrus tree canopy and by sweep net in the ground vegetation of two sweet orange groves with history of infection by the 16SrIX phytoplasma, in the northern region of São Paulo State. Faunistic analyses indicated 1 Agalliinae (Agallia albidula Uhler) and 3 Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål) e Scaphytopius (Convelinus) marginelineatus (Stål)] species that were the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae) in the experimental areas. These species predominated in sweep net and in sticky traps catches at 0.3 m above soil, showing that they inhabit the ground vegetation. Visual observations indicated a strong association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in the ground vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, while A. albidula was observed more often on Conyza bonariensis (L.) Cronq., and B. hebe occurred solely on grasses, more abundantly on Panicum maximum Jacq. Fourteen weed species sampled in the area were PCR tested for infection by the 16SrIX phytoplasma, but none was found infected. Nevertheless, 3 out of 30 field-collected samples (10 adults per sample) of S. marginelineatus tested positive for this phytoplasma by PCR and sequencing. Healthy lab-reared adults of S. marginelineatus were able to transmit inefficiently (0,5%) the 16SrIX phytoplasma to healthy citrus after a 72-h acquisition access period on infected citrus plants followed by a 21-day latent period on S. rhombifolia plants. By using the qPCR method developed for this phytoplasma, a very low pathogen titer was found both in S. marginelineatus and in infected citrus plants, which may explain, at least partially, the low transmission efficiency by this vector using citrus as a source plant. It also suggests that existence of alternative hosts that might be more adequate as inoculum sources for phytoplasma acquisition and spread by S. marginelineatus or another vector yet to be discovered.

A malária é uma doença grave, cujos vetores são mosquitos do gênero Anopheles. Esse gênero é composto por cerca de 500 espécies e aproximadamente 45 são responsáveis pela transmissão do parasito em todo o mundo. Alguns destes vetores são componentes de complexo de espécies crípticas como Complexo Albitarsis e Complexo Triannulatus. O Complexo Albitarsis é formado por quatro espécies: An. albitarsis s.s, An. albitarsis B, An. marajoara e An. deaneorum. O Complexo Triannulatus é composto por três espécies: An. triannulatus, An. halophylus e An. triannulatus C. Algumas técnicas moleculares são utilizadas para auxiliar na distinção destas espécies crípticas. Dessa forma, DNA de An. albitarsis s.l e An. triannulatus s.l., capturados na Usina de Porto Primavera, foram amplificados para ITS2 e para o gene ND4 do DNAmt. Todas as amostras foram submetidas à técnica de RFLP. Algumas amostras foram seqüenciadas diretamente ou clonadas para posterior seqüenciamento a fim de se confirmar a espécie. Das amostras do Complexo Albitarsis, 62,85% foram identificadas como An. deaneorum para ITS2-RFLP. As restantes foram amplificadas para ND4-RFLP e foram identificadas como An. albitarsis s.s. Observou-se, no entanto, que as amostras do Complexo Albitarsis não exibiram similaridade total com as depositadas no GeneBank sendo que para ITS2-PCR estas foram identificadas como An. albitarsis s.s. e An. deaneorum, enquanto que para o gene ND4 todas foram identificadas como An. albitarsis B. O ITS2-RFLP para o An. triannulatus s.l demonstrou polimorfismos entre as espécies. No seqüenciamento, estas amostras foram similares àquelas depositadas no GeneBank. O emprego de marcadores moleculares podem, em parte, auxiliar na distinção de espécies crípticas, porém outros marcadores devem ser avaliados a fim de se elucidar a identificação do Complexo Albitarsis. / Malaria is a severe disease whose vectors are mosquitoes belonging to the genus Anopheles. This genus contains 500 species of which approximately 45 are responsible for the worldwide transmission of these parasites. Some of these vectors belong to cryptic species such as those of the Albitarsis and Triannulatus Complex. The Albitarsis Complex is composed of four species: An. albitarsis s.s., An. albitarsis B, An. marajoara and An. deaneorum. The Triannulatus Complex contains three species: An. triannulatus, An. halophylus and An. triannulatus C. We used molecular techniques to differentiate these cryptic species. Thus, DNA of An. albitarsis s.l and An. triannulatus s.l, captured at the Porto Primavera Dam had their ITS2 and their mtDNA, ND4 genes amplified. All samples were analyzed by the RFLP technique. Some samples were directly sequenced while others were cloned for subsequent sequencing for species confirmation. Within the Albitarsis Complex, 62.85% were identified as An. deaneorum by RFLP-ITS2. The remaining were amplified by RFLP-ND4 and identified as An. albitarsis s.s. However the results of sequencing of the samples of the Albitarsis Complex did not overlap entirely with those deposited in the GeneBank since those amplified by RFLP-ITS2 were identified as An. albitarsis s.s. and An. deaneorum while those obtained by RFLP-ND4 were identified as An. albitarsis B. Also RFLP-ITS2 of A. triannulatus s.l. contained polymorphic regions among different species. By sequencing, these samples were similar to those deposited in the GeneBank. The use of molecular markers did, in some instances help to distinguish species within cryptic complexes, however other markers need to be evaluated to elucidate further identification of the Albitarsis Complex.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 µm²) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.

O Greening ou Huanglongbing (HLB) é a doença mais devastadora dos citros na atualidade. Levantamentos mostram que a enfermidade está presente em cerca de 60% dos talhões do parque citrícola paulista, demostrando sua grande capacidade de disseminação. O psilídeo-asiático-dos-citros, Diaphorina citri Kuwayama (Hemiptera: Psylidae) é um importante vetor dos agentes causais associados ao HLB. Sua capacidade de dispersão está intimamente relacionada ao progresso da doença no campo. Não se tem conhecimento dos principais fatores que possam interferir no deslocamento do inseto até plantas cítricas. Sendo assim, a presente pesquisa teve como objetivo investigar a influência de alguns fatores ambientais sobre a dispersão do psilídeo D. citri, discutindo-os em relação à epidemiologia do HLB dos citros. Inicialmente foram realizados testes em laboratório para adaptação da metodologia de marcação, liberação e recaptura usando pó fluorescente. Grupos de psilídeos adultos foram marcados e sua sobrevivência avaliada, verificando a influência de quatro cores distintas de pó fluorescente (amarelo, azul, rosa e verde). Posteriormente realizaram-se estudos de campo com quatro liberações de psilídeos, com marcadores distintos, em quatro áreas experimentais circulares e adjacentes, sendo formadas por círculos concêntricos localizados a 18, 24 e 30 m de cada centro de liberação. Os insetos foram monitorados visualmente e por armadilhas adesivas amarelas. Nas 1ª e 2ª liberações, foi verificado o efeito das coberturas vegetais milho (Zea mays L.) e capim-braquiária (Brachiaria decumbens Stapf.) na dispersão de D. citri. Já nas 3ª e 4ª liberações, foi estudado o efeito de brotações no deslocamento do inseto. A influência dos ventos foi avaliada em ambas as situações. A análise de sobrevivência indicou que não houve interferência das quatro cores de pó fluorescente em D. citri, sendo a marcação visível no corpo dos insetos durante os 48 dias de avaliação. De acordo com as análises fatoriais, não foi observada qualquer interferência da cobertura vegetal milho, medindo 2 m de altura, na dispersão do inseto. Na presença de brotações, adultos de D. citri dispersaram menos em busca de novos hospedeiros, acumulando-se nas primeiras linhas de plantas cítricas. Após 6 horas da liberação, a capacidade de dispersão máxima de D. citri foi três vezes maior na condição de ausência de brotações. A dispersão média e a área total abrangida variaram assimptoticamente com o tempo, ou seja, o inseto possui inicialmente grande velocidade de dispersão, porém esta tende a se estabilizar com o incremento do tempo. Esses resultados deixam claro que D. citri realiza movimentos dependendo das condições fenológicas de seus hospedeiros, ressaltando a grande importância de se intensificar métodos de controle regionais em épocas em que o fluxo vegetativo das plantas cítricas não é tão intenso. / Greening or Huanglongbing (HLB) is the most devastating disease of citrus today. Surveys show that the disease is present in about 60% of the park stands citrus Sao Paulo, demonstrating its great ability to spread. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psylidae) is an important vector of the causative agents associated with HLB. Its ability to spread is closely related to disease progression in the field. There is no knowledge of the main factors that can affect the movement of the insect to citrus plants. Thus, the present study aimed to investigate the influence of some environmental factors on the dispersal of psyllid D. citri, discussing them in relation to the epidemiology of HLB of citrus. Initially tests were performed in the laboratory to adapt the methodology of marking, release and recapture using fluorescent powder. Groups of adult psyllids were marked and their survival assessed by checking the influence of four different colors of fluorescent powder (yellow, blue, pink and green). Subsequent studies were conducted with four field releases psyllids, with distinct markers on four experimental areas adjacent circular and being formed by concentric circles located at 18, 24 and 30 m for each center release. The insects were monitored visually and by yellow sticky traps. In the 1st and 2nd releases, was to determine the effect of cover crops maize (Zea mays L.) and grass (Brachiaria decumbens Stapf.) in the dispersal of D. citri. In the 3rd and 4th releases, were studied the effect of shootings in insect movement. The influence of the wind has been evaluated in both situations. Survival analysis indicated that there was no interference of the four colors of fluorescent powder on D. citri, and marking was visible on the body of insects during the 48 days of evaluation. According to the factor analysis, no interference of vegetation corn (measuring 2 m in height) was observed in the dispersal of the insect. In the presence of shoots, adult D. citri less dispersed looking for new hosts and it accumulate in the first lines of citrus plants. After 6 hours of release, the D. citri maximum dispersal was three times higher in the condition of absence of shoots. The dispersal medium and the total area covered varied asymptotically with time, ie the insect has initially high speed dispersal, but this tends to stabilize with increasing time. These results clearly show that D. citri performs movements depending on the phenology of its hosts, emphasizing the importance of intensifying regional control methods at times when the flow of plant citrus is not intense.

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.

O Huanglongbing (HLB) é uma das mais temidas doenças da citricultura mundial, associada a bactérias do gênero Candidatus Liberibacter, que foram detectadas no Brasil em 2004. Em 2008, detectou-se outra bactéria associada a sintomas de HLB no Estado de São Paulo, que foi caracterizada como sendo um fitoplasma do grupo 16 SrIX. Fitoplasmas são molicutes fitopatógenos restritos ao floema de plantas, disseminados por insetos vetores. O presente trabalho buscou identificar cigarrinhas potencialmente vetoras do fitoplasma associado ao HLB e plantas hospedeiras desses insetos, bem como desenvolver uma técnica de quantificação de DNA desse patógeno em insetos e plantas. Amostragens de cigarrinhas foram realizadas quinzenalmente por 12 meses em dois pomares de laranja com histórico de ocorrência do fitoplasma 16SrIX na região norte do Estado de São Paulo, usando-se armadilhas adesivas amarelas em duas alturas (0,3 e 1,5 m) da copa de árvores cítricas, e rede de varredura na vegetação espontânea. Dados faunísticos identificaram uma espécie de Agalliinae (Agallia albidula Uhler) e três de Deltocephalinae, [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål) e Scaphytopius (Convelinus) marginelineatus (Stål)], como os cicadelídeos (Hemiptera: Cicadellidae) mais abundantes e frequentes nas áreas estudadas. Essas espécies predominaram na amostragem com rede de varredura e na menor altura de coleta com armadilhas adesivas, indicando comportamento de alimentação em vegetação rasteira. Com observações visuais, verificou-se associação das espécies com certas plantas invasoras, e influência da composição florística da vegetação rasteira sobre a abundância das cigarrinhas. S. marginelineatus e P. flavicosta ocorreram com maior frequência em Sida rhombifolia L. e Althernantera tenella Colla, respectivamente, enquanto que A. albidula foi predominante em Conyza bonariensis (L.) Cronq., e B. hebe ocorreu exclusivamente em gramíneas, principalmente Panicum maximum Jacq.. Plantas invasoras amostradas nas áreas foram testadas para a presença do fitoplasma 16SrIX, porém sem resultados positivos. No entanto, amostras de campo da cigarrinha S. marginelineatus foram positivas por PCR e sequenciamento para o referido fitoplasma. Indivíduos de S. marginelineatus criados em laboratório e mantidos por um período de acesso à aquisição de 72 h em citros infectado com o fitoplasma 16SrIX, foram capazes de transmití-lo para citros, após 21 dias de latência, porém com baixa eficiência (0,5%). Por meio de PCR quantitativo desenvolvido para esse fitoplasma, verificou-se baixo título do patógeno tanto em S. marginelineatus, quanto em plantas cítricas infectadas, o que pode explicar, pelo menos em parte, a baixa eficiência de transmissão pelo inseto tendo citros como fonte. Isto sugere a existência de outros hospedeiros mais adequados como fontes de inóculo para aquisição do fitoplasma por S. marginelineatus ou outro vetor ainda desconhecido. / Huanglongbing (HLB) is a severe citrus disease associated to phloemlimited bacteria in the genus Candidatus Liberibacter, which were detected in Brazil in 2004. In 2008, another bacterium was found in association with HLB symptom in the São Paulo State, and characterized as a phytoplasma belonging to 16SrIX group. Phytoplasmas are vector-borne phytopathogenic mollicutes that inhabit plant sieve elements. The goals of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants, as well as to establish a real-time PCR procedure for pathogen quantification in vectors and plants. Leafhoppers were sampled fortnightly for 12 months by yellow sticky cards placed at two heights (0.3 and 1.5 m) on the citrus tree canopy and by sweep net in the ground vegetation of two sweet orange groves with history of infection by the 16SrIX phytoplasma, in the northern region of São Paulo State. Faunistic analyses indicated 1 Agalliinae (Agallia albidula Uhler) and 3 Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål) e Scaphytopius (Convelinus) marginelineatus (Stål)] species that were the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae) in the experimental areas. These species predominated in sweep net and in sticky traps catches at 0.3 m above soil, showing that they inhabit the ground vegetation. Visual observations indicated a strong association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in the ground vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, while A. albidula was observed more often on Conyza bonariensis (L.) Cronq., and B. hebe occurred solely on grasses, more abundantly on Panicum maximum Jacq. Fourteen weed species sampled in the area were PCR tested for infection by the 16SrIX phytoplasma, but none was found infected. Nevertheless, 3 out of 30 field-collected samples (10 adults per sample) of S. marginelineatus tested positive for this phytoplasma by PCR and sequencing. Healthy lab-reared adults of S. marginelineatus were able to transmit inefficiently (0,5%) the 16SrIX phytoplasma to healthy citrus after a 72-h acquisition access period on infected citrus plants followed by a 21-day latent period on S. rhombifolia plants. By using the qPCR method developed for this phytoplasma, a very low pathogen titer was found both in S. marginelineatus and in infected citrus plants, which may explain, at least partially, the low transmission efficiency by this vector using citrus as a source plant. It also suggests that existence of alternative hosts that might be more adequate as inoculum sources for phytoplasma acquisition and spread by S. marginelineatus or another vector yet to be discovered.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).