The Eucalyptus Cuticular Waxes Contribute in Preformed Defense Against Austropuccinia psidii

Austropuccinia psidii, the causal agent of myrtle rust, is a biotrophic pathogen whose growth and development depends on the host tissues. The uredospores of A. psidii infect Eucalyptus by engaging in close contact with the host surface and interacting with the leaf cuticle that provides important chemical and physical signals to trigger the infection process. In this study, the cuticular waxes of Eucalyptus spp. were analyzed to determine their composition or structure and correlation with susceptibility/resistance to A. psidii. Twenty-one Eucalyptus spp. in the field were classified as resistant or susceptible. The resistance/susceptibility level of six Eucalyptus spp. were validated in controlled conditions using qPCR, revealing that the pathogen can germinate on the eucalyptus surface of some species without multiplying in the host. CG-TOF-MS analysis detected 26 compounds in the Eucalyptus spp. cuticle and led to the discovery of the role of hexadecanoic acid in the susceptibility of Eucalyptus grandis and Eucalyptus phaeotricha to A. psidii. We characterized the epicuticular wax morphology of the six previously selected Eucalyptus spp. using scanning electron microscopy and observed different behavior in A. psidii germination during host infection. It was found a correlation of epicuticular morphology on the resistance to A. psidii. However, in this study, we provide the first report of considerable interspecific variation in Eucalyptus spp. on the susceptibility to A. psidii and its correlation with cuticular waxes chemical compounds that seem to play a synergistic role as a preformed defense mechanism

Bactérias associadas a plantas de cana-de-açúcar : diversidade genética e promoção de crescimento vegetal

As BPCV podem estimular o crescimento da planta, reduzir a ocorrência de infecções causadas por fitopatógenos, e ainda diminuir os efeitos de estresses bióticos ou abióticos sob as plantas. Logo, o objetivo geral deste trabalho foi identificar os mecanismos diretos de promoção de crescimento vegetal em milho (Zea mays L.) e em sorgo forrageiro (Sorghum vulgare), e estudar a diversidade genética de bactérias associadas a plantas de cana-de-açúcar e mandioca. A seleção de isolados bacterianos com mecanismos de promoção de crescimento vegetal foi realizada através dos testes de fixação biológica de nitrogênio (FBN) na ausência e presença de 2,5% de NaCl; síntese de ácido indol acético (AIA) sob a presença de diferentes fatores abióticos; expressão da molécula quorum sensing (ALH) e produção de exopolissacarídeos (EPS). O estudo da diversidade genética foi realizado através das técnicas de BOX-PCR e REP-PCR; e, a presença de isolados pertencentes ao gênero Burkholderia por meio de PCR específica. As bactérias positivas para os diversos mecanismos de promoção foram submetidas ao perfil de ácidos graxos e sequenciamento parcial do gene 16S rRNA. Em seguida, foi feita a inoculação de BPCV em sementes de sorgo forrageiro (Sorgum vulgare, var. Ipa 467-4-2) e milho (Zea mays, var. São José) para avaliar o desempenho de tais bactérias sobre a promoção vegetal, inclusive sob condições de estresse salino. Os resultados mostraram que todas as bactérias foram positivas para a FBN e síntese de AIA; 87,04% dos isolados foram positivos para a produção de quorum sensing (ALH), enquanto que 92,30% das linhagens bacterianas pertencentes ao gênero Burkholderia, foram positivas ao teste; 7,40% das bactérias foram negativas à produção de EPS, nenhum isolado bacteriano apresentou pouca produção, 20,37% dos isolados tiveram média e 72,22% tiveram alta produção. Em relação às linhagens Burkholderia, 38,46% das linhagens avaliadas foram negativas ao teste; apenas a linhagem UAGC131 obteve halo de produção menor que 10 mm de diâmetro (pouca produção), o que corresponde a 3,85% da frequência relativa; 19,23% tiveram média e 38,46% apresentaram alta produção de EPS. A PCR especifica revelou que dos isolados avaliados, apenas UAGF01 e UAGF09, tiveram a presença de banda, sendo, portanto, pertencentes ao gênero Burkholderia. A técnica de BOX-PCR evidenciou a presença de 41 grupos (clusters), com alta variabilidade genética entre os isolados bacterianos avaliados. O coeficiente de similaridade de Jaccard, através da técnica de REP-PCR, evidenciou a presença de 17 grupos (cluters), com alta variabilidade genética entre as linhagens avaliadas, a 30% de similaridade. O estudo do perfil de ácidos graxos e a identificação genética a partir do sequenciamento parcial do gene 16S rRNA, mostrou a presença de diversos gêneros bacterianos de grande importância nos estudos de promoção de crescimento vegetal, como Burkholderia, Enterobacter, Klebisiella e Pantoea. As bactérias avaliadas promoveram aumento de pelo menos uma característica seja por meio do IVG, ou PC, ou comprimento de radícula, parte aérea e total, ou fitomassa total fresca e seca das plântulas de sorgo ou milho, inclusive sob condições de estresse salino. Esses resultados mostraram que as bactérias avaliadas possuem potencial biotecnológico para promoção de crescimento vegetal em condições laboratoriais, necessitando, portanto, de testes em ambiente protegido e em campo para confirmar o potencial dessas bactérias.The BPCV can stimulate the growth of the plant, reduce the occurrence of infections caused by plant pathogens and still reduce the effects of biotic or abiotic stresses in plants. Therefore, the goal of this work was to identify the direct mechanisms of plant growth promotion in maize and sorghum, and study the genetic diversity of bacteria associated with cane sugar plants and manioc. The selection of bacterial isolates of plant growth promoting mechanisms were performed using biological nitrogen fixation test (BNF) in the absence and presence of 2.5% NaCl; indole acetic acid synthesis (IIA) in the presence of different abiotic factors; expression of quorum sensing molecule (HLA) and exopolysaccharide (EPS). The genetic diversity study was conducted through the BOX- PCR and REP-PCR techniques; and the presence of isolates belonging to the genus Burkholderia by specific PCR. Positive bacteria for different promotion mechanisms were submitted to the fatty acids profile and partial sequencing of the 16S rRNA gene. Then, the BPCV inoculation was done in sorghum seeds (Sorgum vulgare var. Ipa 467-4-2) and corn (Zea mays var. St Joseph) to assess the performance of such bacteria on promoting plant, even under salt stress. The results showed that all bacteria were positive for IAA and BNF synthesis; 87.04% of the isolates tested positive for the production of quorum sensing (HLA), while 92.30% of bacterial strains belonging to the genus Burkholderia, the test was positive; 7.40% of the bacteria were negative for EPS production, no bacterial isolate showed little EPS production, 20,37% of the isolates had average production of EPS and 72,22% had high production of EPS. In relation to Burkholderia lineages, 38.46% of tested strains were negative to the test; UAGC131 strain had only minor production of halo 10 mm diameter (low output), which corresponds to 3.85% of relative frequency; 19.23% had average production of EPS and 38.46% had high production of EPS. The specific PCR revealed that the isolates, only UAGF01 and UAGF09, had the presence of band, and therefore belonging to the genus Burkholderia. The BOX-PCR revealed the presence of 41 groups (clusters) with high genetic variability among the evaluated bacterial isolates. The Jaccard similarity coefficient by REP-PCR, revealed the presence of 17 groups (cluters) with high genetic variability between strains evaluated, 30% similarity. The fatty acids profile of the study and the genetic identification from the partial sequencing of the 16S rRNA gene showed the presence of several bacterial genera of great importance in the promotion of studies of plant growth, as Burkholderia, Enterobacter, Pantoea and Klebisiella. The bacteria assessed promoted increase of at least one feature or through the IVG, or PC, or radicle, shoot and total, or total fresh and dry phytomass of seedlings of sorghum or maize, even under salt stress. These results showed that the bacteria have evaluated biotechnological potential for plant growth promotion in laboratory conditions, requiring therefore tests in greenhouse and field to confirm the potential of these bacteria

Desvendando a patogênese e o papel das interações moleculares precoces entre Eucalyptus spp. x Austropuccinia psidii

Austropuccinia psidii, the causal agent of myrtle rust, is a biotrophic pathogen, and therefore its growth and development depend on the host tissues. The uredospores of A. psidii infect Eucalyptus by engaging in close contact with the host surface and interacting with the leaf cuticle that provides important chemical and physical signals to trigger the infection process. Due to the inherent characteristics of the Eucalyptus cuticle, it was hypothesized that the preformed mechanism, comprised mostly by cuticular waxes, plays a crucial role in Eucalyptus resistance against A. psidii and its ability to modulate the expression of genes associated to the pathogenicity of A. psidii during the early stage of infection. In chapter 2, the cuticular waxes of Eucalyptus spp. were analyzed to determine their composition or structure and then correlated to susceptibility/resistance to Austropuccinia psidii. Twenty-one Eucalyptus spp. in the field were classified as resistant or susceptible. From these, the resistance/susceptibility level of six Eucalyptus spp. was evaluated in controlled conditions using qPCR, revealing that the pathogen can germinate on the eucalyptus surface of some species without multiplying in the host. CG-TOF-MS analysis detected 26 compounds in the Eucalyptus spp. cuticle and led to the discovery of the role of hexadecanoic acid in the susceptibility of E. grandis and E. phaeotricha to A. psidii. The scanning electron microscopy check revealed differences in A. psidii germination during host infection. It was found a correlation between epicuticular morphology and the resistance to A. psidii. In chapter 3, we investigated gene expression of A. psidii through bioassays in vitro containing cuticular waxes from E. grandis (E. g), E. urograndis (E. ug) and E. urophylla (E. u). Mineral oil (MO) treatment was used to all comparative analysis (negative control). The presence of cuticular waxes from E. g induced the expression of genes encoding proteins related to growth and colonization of A. psidii such as binding proteins (peptidylprolyl isomerase and ribosomal) and cell wall degrading proteins (beta-xylanase). However, other pathogenic proteins were repressed in presence of cuticular wax of E. g, for instance, triosephosphate isomerase, family 18 glycoside hydrolase, mitochondrial ATP carrier, and glutamine-dependent NAD synthetase. The E. ug x MO analysis resulted in DEGs associated with proteins related to membrane transporters and receptors, DNA repair and glycine dehydrogenase. As to the cuticular wax of E. u, it up-regulated the expression of genes encoding proteins associated with pheromone, cutinases, and prefoldin. Thus, for the first time, it was demonstrated a considerable interspecific variation in Eucalyptus species on the susceptibility to A. psidii and its correlation with cuticular waxes chemical compounds that seem to play a synergistic role as a preformed defense mechanism. We also demonstrated that Eucalyptus spp. cuticular waxes may modulate the A. psidii gene expression, suggesting the importance of early plant-pathogen molecular interaction to the development of myrtle rust.Austropuccinia psidii é o agente causal da ferrugem das mirtáceas com crescimento biotrófico, ou seja, o patógeno depende dos tecidos do hospedeiro para crescer e se desenvolver. Os uredósporos de A. psidii infectam Eucalyptus por meio do contato inicial com a superfície do hospedeiro e também pela interação com a cutícula da folha que por sua vez fornece importantes sinais químicos e físicos capaz de desencadear o processo de infecção. Devido às características inerentes à cutícula de Eucalyptus, consideramos as hipóteses de que o mecanismo pré-formado, composto principalmente pelas ceras cuticulares, desempenha um papel crucial na resistência de Eucalyptus spp. contra A. psidii, e, também, é capaz de modular a expressão fúngica de genes associados a patogenicidade durante o estágio inicial de infecção de A. psidii. No capítulo 2, as ceras cuticulares de Eucalyptus spp. foram analisadas para determinar a composição/estrutura e sua correlação com suscetibilidade/resistência de A. psidii. Vinte e uma espécies de Eucalyptus foram classificadas em campo como resistentes ou suscetíveis. A análise de qPCR de seis Eucalyptus spp. revelou que o patógeno pode germinar na superfície de algumas espécies de eucaliptos sem se multiplicar no tecido hospedeiro. Foram identificados 26 compostos presentes na cutícula de Eucalyptus spp. e descobrimos o papel do ácido hexadecanóico na suscetibilidade de E. grandis e E. phaeotricha à ferrugem. Por meio da microscopia eletrônica de varredura encontramos uma correlação entre a morfologia epicuticular e a resistência contra A. psidii. No capítulo 3 para compreender a expressão gênica de A. psidii realizamos bioensaios (in vitro) contendo as ceras cuticulares de E. grandis (E. g), E. urograndis (E. ug) e E. urophylla (E. u). O tratamento com óleo mineral (MO) foi utilizado em todas as análises comparativas como controle negativo. A presença de ceras cuticulares de E. g induziu a expressão de genes que codificam proteínas relacionadas ao crescimento e colonização de A. psidii, como proteínas de ligação (peptidylprolyl isomerase e ribosomal) e proteínas de degradação da parede celular (beta xilanase). No entanto, outras proteínas patogênicas foram reprimidas na presença da cera cuticular de E. g, por exemplo, triosephosphate isomerase, family 18 glycoside hydrolase, mitochondrial ATP carrier e glutamine-dependent NAD synthetase. A análise de E. ug x MO resultou na ativação de proteínas associadas a transportadores e receptores de membrana, reparo de DNA e glycine dehydrogenase. Já a cera cuticular de E. u induziu a expressão de genes que codificam proteínas associadas a feromônios, cutinases e prefoldin. Pela primeira vez, está sendo apresentado a considerável variação interespecífica em espécies de Eucalyptus quanto à suscetibilidade a ferrugem, e, sua correlação com os compostos químicos de ceras cuticulares, os quais parecem ser um importante mecanismo de defesa pré-formado. Também foi revelado que as ceras cuticulares de Eucalyptus spp. são capazes de modular a expressão gênica de A. psidii, evidenciando o papel da interação molecular planta-patógeno precoce no desenvolvimento da ferrugem das mirtáceas

Diazotrophic bacteria isolated from Brachiaria spp.: genetic and physiological diversity

Resumen Pastos del género Brachiaria spp. predomina en pasturas con suelos poco fértiles. Este escenario resalta la importancia de la asociación con microorganismos para fomentar el cultivo de plantas. Este estudio tuvo como objetivo evaluar la variabilidad genética e identificar los mecanismos de promoción del crecimiento de las plantas, in vitro, de bacterias asociadas con Brachiaria decumbens Stapf. y Brachiaria humidicola (Rendle.) Schweickerdt en Pernambuco, Brasil. Evaluamos 20 aislamientos de bacterias diazotróficas obtenidas de la comunidad de endofitas o rizosfera. Las características genéticas se determinaron mediante la secuenciación de la región 16S rRNA, lo que permitió identificar diez géneros bacterianos diferentes: Bacillus sp., Burkholderia sp., Enterobacter sp., Klebsiella sp., Microbacterium sp., Pantoea sp., Ralstonia sp., Rhizobium sp., Sinomonas sp., y Sphingomonas sp., con una especificidad del género Rhizobium sp. a Brachiaria decumbens Stapf.. Las características fenotípicas y funcionales revelaron que el 100% de las cepas bacterianas producían ácido indol-3-acético (AIA) con la adición de L-triptófano y el 60% presentaban producción de AIA independientemente de la vía de L-triptófano. También detectamos que el 70% de las bacterias aisladas poseían la capacidad de solubilizar el fósforo. El análisis de la producción enzimática reveló que el 30% de los aislados producían celulasa, 60% pectato liasa, 15% poligalacturonasa y el 30% producía amilasa. También detectamos la producción de N-acyl homoserine lactones en el 65% de las cepas bacterianas. En resumen, las plantas de B. decumbens Stapf. y B. humidicola (Rendle.) Schweickerdt interactuó con diferentes géneros de bacterias capaces de promover el crecimiento de la planta.Abstract Grass from the genus Brachiaria spp. predominates in pastures with low fertile soils. This scenario highlights the importance of the association with microorganisms to foster plant growth, which becomes essential to the successful establishment of this forage in such environments. This study aimed to evaluate the genetic variability and identify the mechanisms of plant growth promotion, in vitro, of bacteria associated with Brachiaria decumbens Stapf. and Brachiaria humidicola (Rendle.) Schweickerdt in Pernambuco, Brazil. We evaluated 20 isolates of diazotrophic bacteria obtained from the endophyte or rhizosphere communities. The genetic characteristics were determined via sequencing the 16S rRNA region, which allowed us to identify ten different bacterial genera: Bacillus sp., Burkholderia sp., Enterobacter sp., Klebsiella sp., Microbacterium sp., Pantoea sp., Ralstonia sp., Rhizobium sp., Sinomonas sp., and Sphingomonas sp., with a specificity of the genus Rhizobium sp. to Brachiaria decumbens Stapf.. The phenotypic and functional characteristics revealed that 100% of the bacterial strains produced indol-3-acetic acid (IAA) with the addition of L-tryptophan, and 60% presented IAA production independent of the L-tryptophan pathway. We also detected that 70% of the isolated bacteria possessed the capacity to solubilize phosphorus. The analysis of the enzymatic output revealed that 30% of the bacterial isolates produced cellulase, 60% produced pectate lyase, 15% produced polygalacturonase, and 30% produced amylase. We also detected the production of N-acyl homoserine lactones in 65% of bacterial strains. In summary, our results showed that plants of B. decumbens Stapf. and B. humidicola (Rendle.) Schweickerdt interacted with different bacterial genera capable of promoting plant growth

Revealing the high variability on nonconserved core and mobile elements of Austropuccinia psidii and other rust mitochondrial genomes.

Mitochondrial genomes are highly conserved in many fungal groups, and they can help characterize the phylogenetic relationships and evolutionary biology of plant pathogenic fungi. Rust fungi are among the most devastating diseases for economically important crops around the world. Here, we report the complete sequence and annotation of the mitochondrial genome of Austropuccinia psidii (syn. Puccinia psidii), the causal agent of myrtle rust. We performed a phylogenomic analysis including the complete mitochondrial sequences from other rust fungi. The genome composed of 93.299 bp has 73 predicted genes, 33 of which encoded nonconserved proteins (ncORFs), representing almost 45% of all predicted genes. A. psidii mtDNA is one of the largest rust mtDNA sequenced to date, most likely due to the abundance of ncORFs. Among them, 33% were within intronic regions of diverse intron groups. Mobile genetic elements invading intron sequences may have played significant roles in size but not shaping of the rust mitochondrial genome structure. The mtDNAs from rust fungi are highly syntenic. Phylogenetic inferences with 14 concatenated mitochondrial proteins encoded by the core genes placed A. psidii according to phylogenetic analysis based on 18S rDNA. Interestingly, cox1, the gene with the greatest number of introns, provided phylogenies not congruent with the core set. For the first time, we identified the proteins encoded by three A. psidii ncORFs using proteomics analyses. Also, the orf208 encoded a transmembrane protein repressed during in vitro morphogenesis. To the best of our knowledge, we presented the first report of a complete mtDNA sequence of a member of the family Sphaerophragmiacea