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Diseases of soybean caused by Cercospora spp. are endemic throughout the world's soybean production regions. Species diversity in the genus Cercospora has been underestimated due to overdependence on morphological characteristics, symptoms, and host associations. Currently, only two species (Cercospora kikuchii and C. sojina) are recognized to infect soybean; C. kikuchii causes Cercospora leaf blight (CLB) and purple seed stain (PSS), whereas C. sojina causes frogeye leaf spot. To assess cryptic speciation among pathogens causing CLB and PSS, phylogenetic and phylogeographic analyses were performed with isolates from the top three soybean producing countries (USA, Brazil, and Argentina; collectively accounting for ~80% of global production). Eight nuclear genes and one mitochondrial gene were partially sequenced and analyzed. Additionally, amino acid substitutions conferring fungicide resistance were surveyed, and the production of cercosporin (a polyketide toxin produced by many Cercospora spp.) was assessed. From these analyses, the long-held assumption of C. kikuchii as the single causal agent of CLB and PSS was rejected experimentally. Four cercosporin-producing lineages were uncovered with origins (about 1 Mya) predicted to predate agriculture. Some of the Cercospora spp. newly associated with CLB and PSS appear to represent undescribed species; others were not previously reported to infect soybeans. Lineage 1, which contained the ex-type strain of C. kikuchii, was monophyletic and occurred in Argentina and Brazil. In contrast, lineages 2 and 3 were polyphyletic and contained wide-host range species complexes. Lineage 4 was monophyletic, thrived in Argentina and the USA, and included the generalist Cercospora cf. flagellaris. Interlineage recombination was detected, along with a high frequency of mutations linked to fungicide resistance in lineages 2 and 3. These findings point to cryptic Cercospora species as underappreciated global considerations for soybean production and phytosanitary vigilance, and urge a reassessment of host-specificity as a diagnostic tool for Cercospora.

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ß-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 ß-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 ß-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in ß-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His (6) , Glu (198) and Phe (200) , were absent from the predicted sequence of the P. pachyrhizi ß-tubulin protein.

RESUMEN

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.

RESUMEN

Euschistus heros (F.) (Heteroptera: Pentatomidae) is the most prevalent stink bug pest in Brazil, and populations can be difficult to manage using organophosphates or endosulfan. Because E. heros is difficult to rear in the laboratory, no baseline insecticide susceptibility data have been published. Therefore, we conducted dose-mortality studies using field-collected insects, and, using the results from the dose-mortality studies, we surveyed susceptibility to acephate, methamidophos, and endosulfan in populations from southern and central Brazil. In addition, esterase activity was evaluated among populations as these enzymes have been related to organophosphate resistance. Finally, newer chemistries were evaluated for E. heros population management in small-plot field studies. In the dose-mortality bioassays, variation in susceptibility to methamidophos and endosulfan among populations was significant, but the variation in susceptibility to acephate was not. The population from Londrina (at the Embrapa Soybean Research Station, northern Parana state) was among the most susceptible, whereas the population from Pedrinhas Paulista (southwestern Sao Paulo state) was among the least susceptible. Significant variation among populations was observed in esterase activity with the population from Pedrinhas Paulista having the highest esterase levels and the population from Londrina having among the lowest levels. To survey populations, we used the dose-mortality data for the Londrina population (because it was, consistently, one of the most susceptible populations) to estimate the insecticide concentration that killed 99% of the population (LC99). The corresponding doses were 5.9 microg (AI) of acephate adult(-1), 0.7 microg (AI) methamidophos adult(-1), and 1,600 microg (AI) endosulfan adult(-1). The survey data confirmed the data from the dose-mortality bioassays in that the population from Pedrinhas Paulista was identified as one of the least susceptible and the population from Londrina was one of the most susceptible. The field tests confirmed that older chemistries provided minimal control (< 50% control), whereas the products containing mixtures of pyrethroids and neonicotinoids provided better control (> 60%).

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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.

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Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is now established in all major soybean-producing countries. Currently, there is little information about the molecular basis of ASR-soybean interactions, which will be needed to assist future efforts to develop effective resistance. Toward this end, abundance changes of soybean mRNAs were measured over a 7-day ASR infection time course in mock-inoculated and infected leaves of a soybean accession (PI230970) carrying the Rpp2 resistance gene and a susceptible genotype (Embrapa-48). The expression profiles of differentially expressed genes (ASR-infected compared with the mock-inoculated control) revealed a biphasic response to ASR in each genotype. Within the first 12 h after inoculation (hai), which corresponds to fungal germination and penetration of the epidermal cells, differential gene expression changes were evident in both genotypes. mRNA expression of these genes mostly returned to levels found in mock-inoculated plants by 24 hai. In the susceptible genotype, gene expression remained unaffected by rust infection until 96 hai, a time period when rapid fungal growth began. In contrast, gene expression in the resistant genotype diverged from the mock-inoculated control earlier, at 72 h, demonstrating that Rpp2-mediated defenses were initiated prior to this time. These data suggest that ASR initially induces a nonspecific response that is transient or is suppressed when early steps in colonization are completed in both soybean genotypes. The race-specific resistance phenotype of Rpp2 is manifested in massive gene expression changes after the initial response prior to the onset of rapid fungal growth that occurs in the susceptible genotype.

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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