Molecular characterization of beta-tubulin from Phakopsora pachyrhizi, the causal agent of Asian soybean rust

β-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the β-tubulin gene (Pp tubB) in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous β-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in β-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His6, Glu198 and Phe200, were absent from the predicted sequence of the P. pachyrhizi β-tubulin protein.

Evolutionary history of Phakopsora pachyrhizi (the Asian soybean rust) in Brazil based on nucleotide sequences of the internal transcribed spacer region of the nuclear ribosomal DNA

Phakopsora pachyrhizi has dispersed globally and brought severe economic losses to soybean growers. The fungus has been established in Brazil since 2002 and is found nationwide. To gather information on the temporal and spatial patterns of genetic variation in P. pachyrhizi, we sequenced the nuclear internal transcribed spacer regions (ITS1 and ITS2). Total genomic DNA was extracted using either lyophilized urediniospores or lesions removed from infected leaves sampled from 26 soybean fields in Brazil and one field in South Africa. Cloning prior to sequencing was necessary because direct sequencing of PCR amplicons gave partially unreadable electrophoretograms with peak displacements suggestive of multiple sequences with length polymorphism. Sequences were determined from four clones per field. ITS sequences from African or Asian isolates available from the GenBank were included in the analyses. Independent sequence alignments of the ITS1 and ITS2 datasets identified 27 and 19 ribotypes, respectively. Molecular phylogeographic analyses revealed that ribotypes of widespread distribution in Brazil displayed characteristics of ancestrality and were shared with Africa and Asia, while ribotypes of rare occurrence in Brazil were indigenous. The results suggest P. pachyrhizi found in Brazil as originating from multiple, independent long-distance dispersal events.

Genetic differentiation among Brazilian populations of Euschistus heros (Fabricius) (Heteroptera: Pentatomidae) based on RAPD analysis / Diferenciação genética entre populações de Euschistus heros (Fabricius) (Heteroptera: Pentatomidae) do Brasil mediante marcadores de RAPD

The Neotropical Brown Stink Bug (NBSB), Euschistus heros (Fabricius), has a wide distribution in Brazilian soybean fields, being more important in the central region of the country. The species is the main target of insecticide applications for stink bug pest control. We determined the variability among and within populations of the NBSB by random amplification of polymorphic DNA (RAPD) analysis. Samples of NBSB were collected in soybean fields from Ubiratã (PR), Londrina (PR), Centenário do Sul (PR), Cândido Mota (SP), Ponta Porã (MS) and Sapezal (MT). Genomic DNA was extracted from the head to minimize DNA contamination by endoparasites. DNA was amplified with 10 mer primers. Fifteen primers produced 246 bands. The genetic similarity matrix was obtained, based on RAPD allele frequencies, using Nei's 1972 genetic distance. NSBS collected from the same geographical region clustered together. The populations from Londrina and Centenário do Sul were genetically closer than the others and stink bugs collected in Cândido Mota were closer to Ponta Porã population. The Sapezal geographical population was the most divergent from the others. Females and males clustered separately inside each geographical population, implying that RAPD permits gender discrimination. We did not observe individuals from one region clustering together with stink bugs from another region. The number of polymorphic loci from the different populations ranged between 40.6 percent and 52.1 percent. The gene flow indexes (overall Nm = 0.8307) were lower than that observed for Anticarsia gemmatalis Hübner and Helicoverpa armigera (Hübner) suggesting that in stink bug populations gene flow is lower than in the noctuid moths

O percevejo marrom da soja, Euschistus heros (Fabricius), possui ampla distribuição na região sojicola do Brasil, sendo mais importante na região central do País. Tem sido o alvo principal das aplicações de inseticidas dirigidas contra o complexo de pentatomídeos praga. A variabilidade nos RAPD entre e dentro das populações do percevejo marrom foi determinada. Amostras da espécie foram coletadas nos campos de soja de Ubiratã (PR), Londrina (PR), Centenário do Sul (PR), Cândido Mota (SP), Ponta Porã (MS) e Sapezal (MT). O DNA genômico foi extraído da cabeça para minimizar a contaminação do DNA proveniente dos endoparasitóides e parasitas que ocorrem na hemocele. O DNA foi amplificado com iniciadores de 10 nucleotídeos. Quinze iniciadores produziram 246 bandas. A similaridade genética foi obtida com base na freqüência alélica dos RAPD utilizando-se a distância de Nei 1972. Os percevejos coletados da mesma região geográfica apresentaram a maior similaridade genética. As populações de Londrina e Centenário do Sul foram geneticamente mais próximas que as restantes. Também, percevejos coletados em Cândido Mota foram próximos das populações de Ponta Porã. A população geográfica de Sapezal foi a mais divergente das outras. As fêmeas e os machos agruparam-se em grupos diferentes dentro de cada população geográfica, o que significa que os RAPD possibilitam a diferenciação dos sexos. Não foram observados indivíduos de uma população agrupando-se com indivíduos de outra região. O número de loci polimórficos das diferentes populações variou entre 40,6 por cento e 52,1 por cento. Os índices de fluxo gênico de E. heros (Nm = 0,8307) foram menores que os observados anteriormente para Anticarsia gemmatalis Hübner e Helicoverpa armigera (Hübner) sugerindo que as populações do pentatomídeo apresentam maior isolamento geográfico que nos referidos noctuídeos, por exemplo