Análise de uma biblioteca de mutantes de Xanthomonas citri subsp. citri quanto à patogenicidade

Pós-graduação em Microbiologia Agropecuária - FCAV

Monitoramento da expressão gênica de Xanthomonas axonopodis pv. citri em diferentes condições ambientais

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Sistema para depleção de proteínas em Xanthomonas citri subsp. citri

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressão gênica de Xanthomonas citri subsp. citri colonizando laranja doce ‘pêra rio’ (Citrus sinensis (L.) Osbeck) e lima ácida ‘galego’ (Citrus aurantifolia Swingle)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mutagênese sítio-dirigida da ORF XAC0024 de Xanthomonas citri subsp. citri e suas implicações no desenvolvimento do cancro cítrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of plant structure, cultural pratices and environmental conditions on the development of the bacterial canker of kiwifruits

Italy has a preeminent rank in kiwifruit industry, being the first exporter and the second largest producer after China. However, in the last years kiwifruit yields and the total cultivated area considerably decreased, due to the pandemic spread of the bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa). Several climatic conditions and cultural practices affect the development of the bacterial canker. This research work focused on the impact of agricultural practices and microclimate conditions on the incidence and epidemiology of Psa in the orchard. Therefore, the effect of fertilization, irrigation, use of bio-regulators, rootstock, training system and pruning were examined. The effect of different tunnel systems was analyzed as well, to study the plant-pathogen interaction. Considering the importance of insects as vectors in other pathosystems, the role of Metcalfa pruinosa in the spread of the bacterial canker was investigated in controlled conditions. In addition, quality and storage properties of fruits from infected plants were assessed. The study of all these aspects of the agronomic practices is useful to define a strategy to limit the bacterial diffusion in the orchard. Overall, excess nitrogen fertilization, water stress, stagnant water supplies, pruning before summer and the high number of Metcalfa pruinosa increased the Psa incidence. In contrast, tunnel covers may be useful for the control of the disease, with special attention to the kind of material.

Caracterização fisiológica e do perfil de expressão gênica do transporte de nitrogênio em genótipos contrastantes para processo de fixação biológica de N2 de cana-de-açúcar (Saccharum spp.)

A cana-de-açúcar é uma cultura agrícola de grande importância econômica para o Brasil, e a expansão de seu cultivo para solos marginais requer uma maior utilização de fertilizantes à base de nitrogênio (N). Na maioria dos países produtores, a adubação nitrogenada se baseia em altas doses de aplicação, enquanto, no Brasil, o seu uso é relativamente baixo devido, em parte, ao processo de fixação biológica de nitrogênio (FBN) pela ação de bactérias diazotróficas. Além da FBN, as plantas adquirem fontes de N, como amônio e nitrato, por meio de transportadores de membranas localizados nas raízes. Há evidências que a associação com microrganismos pode favorecer as plantas por meio da regulação dos genes de transportadores de N. Desta forma, este trabalho teve como objetivo caracterizar o transporte de amônio e nitrato, avaliando a expressão gênica dos principais transportadores de N em cana-de-açúcar cultivada in vitro sob o efeito da associação com bactérias diazotróficas. Também foi descrita a comunidade bacteriana de plântulas in vitro, bem como o efeito da fertilização com N e da inoculação com bactérias diazotróficas em plantas maduras. Plântulas de \'SP70- 1143\' e \'Chunee\', que contrastam para FBN, foram empregadas em ensaios in vitro sob diversas concentrações e fontes de N em associação ou não com uma estirpe de Gluconacetobacter diazotrophicus ou um mistura de bactérias diazotróficas (G. diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense e Burkholderia tropica). A caracterização do transporte de N por meio de ensaios de absorção de nitrato e amônio marcados (15N) revelou que a interação entre cana-de-açúcar x G. diazotrophicus induziu a expressão do gene do transportador de nitrato ScNRT2.1, o que levou a uma tendência no aumento no influxo de nitrato, assim como dos genes de transportadores de amônio ScAMT1.1 e ScAMT1.3, resultando em maiores influxos de amônio apenas para a cultivar \'SP70- 1143\'. Já a associação da cana-de-açúcar com a mistura de bactérias diazotróficas revelou que somente houve indução transcricional de ScAMT1.1, o que resultou na maior absorção de amônio em \'SP70-1143\'. Por sua vez, quando analisada a interação in vitro por 30 dias, a presença da bactéria, apesar de transiente, possivelmente favoreceu a expressão dos genes de transportadores de nitrato ScNRT1.1 e ScNRT2.1, e do transportador de amônio ScAMT1.1, resultando no maior acúmulo de 15N-nitrato de amônio nas plantas de \'SP70-1143\'. Foi detectada uma comunidade bacteriana associada a plântulas micropropagadas, a qual é distinta entre os genótipos \'SP70-1143\' e \'Chunee\' e se altera com a inoculação com G. diazotrophicus. Para as plantas cultivadas em campo, a comunidade bacteriana existente foi alterada pela fertilização de N, mas não pela inoculação com diazotróficas. Portanto, a inoculação com bactérias diazotróficas parece induzir a expressão dos principais genes transportadores de amônio e nitrato em plântulas do genótipo \'SP70-1143\' resultando na maior absorção de fontes inorgânicas de N.

Análise de metabólitos e proteínas totais em folhas de Eucalyptus grandis durante a infecção por Puccinia psidii

Os mecanismos moleculares envolvidos na resistência de plantas contra patógenos são um tema bastante discutido no meio acadêmico, sendo o objetivo maior dos estudos a diminuição das perdas de produtividade provocadas por doenças em plantações do mundo todo. Muitos modelos de interação patógeno-hospedeiro foram propostos e desenvolvidos priorizando plantas e culturas de rápido desenvolvimento com ciclo de vida curto. Espécies de ciclo longo, porém, devem lidar durante anos - ao menos até a idade reprodutiva - contra o ataque de bactérias, fungos e vírus, sem contar, nesse meio tempo, com recombinações genéticas e mutações que tornariam possível o escape contra as moléstias causadas por microrganismos. Assim, como alternativa aos modelos usuais, o presente trabalho estudou um diferente par de antagonistas: Eucalyptus grandis e Puccinia psidii. Apesar da contribuição de programas de melhoramento genético, o patossistema E. grandis X P. psidii ainda é pouco descrito no nível molecular, havendo poucos estudos sobre os processos e as moléculas que agem de forma a conferir resistência às plantas. Assim, buscando o melhor entendimento da relação entre E. grandis X P. psidii, o presente trabalho estudou a mudança dos perfis de proteínas e metabólitos secundários ocorrida nos tecidos foliares de plantas resistentes e susceptíveis durante a infecção pelo patógeno, com o auxílio da técnica de cromatografia líquida acoplada à espectrometria de massas. Os resultados obtidos indicam que as plantas resistentes percebem a presença do patógeno logo nas primeiras horas pós-infecção, produzindo proteínas ligadas à imunidade (HSP90, ILITYHIA, LRR Kinase, NB-ARC disease resistance protein). Essa percepção desencadeia a produção de proteínas de parede celular e de resposta oxidativa, além de modificar o metabolismo primário e secundário. As plantas susceptíveis, por outro lado, têm o metabolismo subvertido, produzindo proteínas responsáveis pelo afrouxamento da parede celular, beneficiando a absorção de nutrientes, crescimento e propagação de P. psidii. No trabalho também são propostos metabólitos biomarcadores de resistência, moléculas biomarcadoras de resposta imune e sinais da infecção por patógeno em E. grandis.

Management of plant invasions mediated by frugivore interactions

1. Some of the most damaging invasive plants are dispersed by frugivores and this is an area of emerging importance in weed management. It highlights the need for practical information on how frugivores affect weed population dynamics and spread, how frugivore populations are affected by weeds and what management recommendations are available. 2. Fruit traits influence frugivore choice. Fruit size, the presence of an inedible peel, defensive chemistry, crop size and phenology may all be useful traits for consideration in screening and eradication programmes. By considering the effect of these traits on the probability, quality and quantity of seed dispersal, it may be possible to rank invasive species by their desirability to frugivores. Fruit traits can also be manipulated with biocontrol agents. 3. Functional groups of frugivores can be assembled according to broad species groupings, and further refined according to size, gape size, pre- and post-ingestion processing techniques and movement patterns, to predict dispersal and establishment patterns for plant introductions. 4. Landscape fragmentation can increase frugivore dispersal of invasives, as many invasive plants and dispersers readily use disturbed matrix environments and fragment edges. Dispersal to particular landscape features, such as perches and edges, can be manipulated to function as seed sinks if control measures are concentrated in these areas. 5.Where invasive plants comprise part of the diet of native frugivores, there may be a conservation conflict between control of the invasive and maintaining populations of the native frugivore, especially where other threats such as habitat destruction have reduced populations of native fruit species. 6. Synthesis and applications. Development of functional groups of frugivore-dispersed invasive plants and dispersers will enable us to develop predictions for novel dispersal interactions at both population and community scales. Increasingly sophisticated mechanistic seed dispersal models combined with spatially explicit simulations show much promise for providing weed managers with the information they need to develop strategies for surveying, eradicating and managing plant invasions. Possible conservation conflicts mean that understanding the nature of the invasive plant-frugivore interaction is essential for determining appropriate management.

Host-bacteria interactions : Host cell responses and bacterial pathogenesis

Helicobacter pylori colonizes the human stomach, where it causes gastritis that may develop into peptic ulcer disease or cancer when left untreated. Neisseria gonorrhoeae colonizes the urogenital tract and causes the sexually transmitted disease gonorrhea. In contrast, Lactobacillus species are part of the human microbiota, which is the resident microbial community, and are considered to be beneficial for health. The first host cell types that bacteria encounter when they enter the body are epithelial cells, which form the border between the inside and the outside, and macrophages, which are immune cells that engulf unwanted material.       The focus of this thesis has been the interaction between the host and bacteria, aiming to increase our knowledge of the molecular mechanisms that underlie the host responses and their effects on bacterial pathogenicity. Understanding the interactions between bacteria and the host will hopefully enable the development of new strategies for the treatment of infectious disease. In paper I, we investigated the effect of N. gonorrhoeae on the growth factor amphiregulin in cervical epithelial cells and found that the processing and release of amphiregulin changes upon infection. In paper II, we examined the expression of the transcription factor early growth response-1 (EGR1) in epithelial cells during bacterial colonization. We demonstrated that EGR1 is rapidly upregulated by many different bacteria. This upregulation is independent of the pathogenicity, Gram-staining type and level of adherence of the bacteria, but generally requires viable bacteria and contact with the host cell. The induction of EGR1 is mediated primarily by signaling through EGFR, ERK1/2 and β1-integrins. In paper III, we described the interactions of the uncharacterized protein JHP0290, which is secreted by H. pylori, with host cells. JHP0290 is able to bind to several cell types and induces apoptosis and TNF release in macrophages. For both of these responses, signaling through Src family kinases and ERK is essential. Apoptosis is partially mediated by TNF release. Finally, in paper IV, we showed that certain Lactobacillus strains can reduce the colonization of H. pylori on gastric epithelial cells. Lactobacilli decrease the gene expression of SabA and thereby inhibit the binding mediated by this adhesin.

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

Adenanthera pavonina TRYPSIN INHIBITOR RETARD GROWTH OF Anagasta kuehniella (LEPIDOPTERA: PYRALIDAE)

Anagasta kuehniella is a polyphagous pest that feeds on a wide variety of stored products. The possible roles suggested for seed proteinase inhibitors include the function as a part of the plant defensive system against pest via inhibition of their proteolytic enzymes. In this study, a trypsin inhibitor (ApTI) was purified from Adenanthera pavonina seed and was tested for insect growth regulatory effect. The chronic ingestion of ApTI did result in a significant reduction in larval survival and weight. Larval and pupal developmental time of larvae fed on ApTI diet at 1% was significantly longer; the larval period was extended by 5 days and pupal period was 10 days longer, therefore delaying by up to 20 days and resulting in a prolonged period of development from larva to adult. As a result, the ApTI diet emergence rate was only 28% while the emergence rate of control larvae was 80%. The percentage of surviving adults (%S) decreased to 62%. The fourth instar larvae reared on a diet containing 1% ApTI showed a decrease in tryptic activity of gut and that no novel proteolytic form resistant to ApTI was induced. In addition, the tryptic activity in ApTI -fed larvae was sensitive to ApTI. These results suggest that ApTI have a potential antimetabolic effect when ingested by A. kuehniella. (C) 2010 Wiley Periodicals, Inc.

Postharvest harpin or Bacillus thuringiensis treatments suppress citrus black spot in `Valencia` oranges

Citrus black spot (CBS) caused by Guignardia citricarpa represents an important threat to citriculture in Brazil. Limited information is available regarding potential biological control agents and new alternative compounds that may provide protection of orange fruits against the disease. In this study, the effects of commercial products based on Bacillus thuringiensis var. kurstaki (Bt) bacterium, Bt pure isolates and Harpin protein (Messenger (R)) on the postharvest control of CBS, were evaluated in `Valencia` sweet orange fruits harvested for three consecutive years in a citrus grove. The fruits were sprayed with the following products: DiPel (R) WP (Bt, subspecies, kurstaki strain HD-1,16,000 International Units mg(-1), 32 g active ingredient kg(-1)) (1, 20 and 50 mg ml(-1)), Dimy Pel (R) WP (Bt, subspecies, kurstaki, strain HD-1, 17,600 IU mg(-1), 26 g active ingredient l(-1)) (2, 20 and 50 mg ml(-1)), Messenger (R) (3% harpin protein) (1 and 2 mg ml(-1)) and fungicide Tecto (R) Flowable SC (thiabendazole, 485 gl(-1)) (0.8g active ingredient l(-1)), besides the Bt isolates, Bt- HD-567, Bt- DiPel and Bt- Dimy (9 x 10(8) CFU ml(-1)). Ten days after treatment, the number of newly developed CBS lesions and pycnidia produced were evaluated using fifty fruits per treatment. The Dimy Pel (R) and Messenger (R) reduced the number of new developed CBS lesions on fruits in up to 67% and 62%, respectively. All applied treatments drastically decreased the number of pycnidia produced in the CBS lesions on orange fruits with 85% to 96% reductions compared to the untreated control. Volatile compounds produced by the isolates Bt- HD-567, Bt- Dimy and Bt- DiPel, reduced the number of lesions on treated fruits by 70%, 65% and 71% compared to the control, respectively. In addition, the survival of Bt isolates on orange fruit surfaces were evaluated by recovering and quantifying the number of CFU every seven days for up to 28 days. The declines in survival rates on orange fruit surfaces were drastic for the three strains of Bt in the first week. The CFU numbers of all applied isolates declined by 4 to 5 orders of magnitude after storage at room temperature for 28 days. In vitro assays revealed that the Bt isolates significantly reduced the mycelial growth of the pathogen, ranging from 32% to 51%, compared to the control, whereas no inhibitory effect was observed in the presence of Messenger (R). (C) 2010 Elsevier Ltd. All rights reserved.

Bacterial community in the rhizosphere and rhizoplane of wild type and transgenic eucalyptus

The rhizosphere is a niche exploited by a wide variety of bacteria. The expression of heterologous genes by plants might become a factor affecting the structure of bacterial communities in the rhizosphere. In a greenhouse experiment, the bacterial community associated to transgenic eucalyptus, carrying the Lhcb1-2 genes from pea (responsible for a higher photosynthetic capacity), was evaluated. The culturable bacterial community associated to transgenic and wild type plants were not different in density, and the Amplified Ribosomal DNA Restriction Analysis (ARDRA) typing of 124 strains revealed dominant ribotypes representing the bacterial orders Burkholderiales, Rhizobiales, and Actinomycetales, the families Xanthomonadaceae, and Bacillaceae, and the genus Mycobacterium. Principal Component Analysis based on the fingerprints obtained by culture-independent Denaturing Gradient Gel Electrophoresis analysis revealed that Alphaproteobacteria, Betaproteobacteria and Actinobacteria communities responded differently to plant genotypes. Similar effects for the cultivation of transgenic eucalyptus to those observed when two genotype-distinct wild type plants are compared.

Host preference of the bean weevil Zabrotes subfasciatus

It is largely known that the range of an insect diet is mostly determined by oviposition behavior, mainly in species with endophytic larvae such as Zabrotes subfasciatus. However, the proximate factors determining host choice and the subsequent steps leading to the expansion or reduction of the host number and occasional host shifts are largely unknown. We analyzed various factors determining host preference of Z. subfasciatus through the evaluation of: (i) oviposition preference of a wild population of Z subfasciatus on the usual host (bean) and unusual hosts (lentil, chickpea and soy), and the performance of the offspring; (ii) artificial selection for increasing preference for hosts initially less frequently chosen; (iii) comparison of oviposition behavior between two different populations (reared for similar to 30 generations in beans or chickpeas, respectively); (iv) oviposition timing on usual and unusual hosts; and (v) identification of preference hierarchies. We found that when using unusual hosts, there is no correlation between performance and preference and that the preference hierarchy changes only slightly when the population passes through several generations on the less frequently accepted host. We also found a positive response to artificial selection for increasing oviposition on the less preferred host; however, when the host-choice experiment involved two varieties of the usual host, the response was faster than when the choice involved usual and unusual hosts. Finally, beetles reared on an unusual host (chickpea) for 26 generations showed similar good fitness on both usual and unusual hosts, indicating that the use of a new host does not necessarily result in the loss of performance on the original host. Nevertheless, this population showed lower fitness on the usual host than that of the original population, suggesting an underlying partial trade-off phenomenon which may contribute to a broadening of diet of this insect species.