Álvaro M. R. Almeida

Distinct Biphasic mRNA Changes in Response to Asian Soybean Rust Infection

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.

Genotypic diversity among brazilian isolates of Macrophomina phaseolina revealed by RAPD

Macrophomina phaseolina has been considered one of the most prevalent soybean (Glycine max) pathogens in Brazil. No genetic resistance has been determined in soybean and very little is known about the genetic diversity of this pathogen in tropical and sub-tropical regions. Fifty-five isolates from soybean roots were collected in different regions and analyzed through RAPD for genetic diversity. The UPGMA cluster analysis for 74 loci scored permitted identification of three divergent groups with an average similarity of 99%, 92% and 88%, respectively. The three groups corresponded to 5.45%, 59.95% and 34.6%, respectively of all isolates used. A single plant had three different haplotypes, while 10.9% of the analyzed plants had two different haplotypes. In another study the genetic similarity was evaluated among isolates from different hosts [soybean, sorghum (Sorghum bicolor), sunflower (Helianthus annuus), cowpea (Vigna unguiculata), corn (Zea mays) and wheat (Triticum aestivum)] as well as two soil samples from native areas. Results showed that more divergent isolates originated from areas with a single crop. Isolates from areas with crop rotation were less divergent, showing high similarity values and consequently formed the largest group. Amplification of the ITS region using primers ITS1 and ITS4 produced only one DNA fragment of 620 bp. None of the isolates were differentiated through PCR-RFLP. Our results demonstrated genetic variability among Brazilian isolates of M. phaseolina and showed that one single root can harbor more than one haplotype. Moreover, cultivation with crop rotation tends to induce less specialization of the pathogen isolates. Knowledge of this variation may be useful in screening soybean genotypes for resistance to charcoal rot.

Detection and partial characterization of a carlavirus causing stem necrosis of soybean in Brazil

No ano agricola de 2000/2001, ocorreu um surto de nanismo e necrose da haste em soja (Glycine max) plantada em duas areas do Brasil Central e na safra seguinte, esta anomalia foi constatada em outras regioes produtoras, mesmo distantes mais de 2.000 km de onde fora inicialmente constatada. Estudos envolvendo ensaios de transmissao (enxertia, mecânica e insetos vetores), microscopia eletronica, purificacao, sorologia e ensaios moleculares indicaram que a enfermidade foi causada por um carlavirus transmitido por mosca branca, possivelmente relacionado ao Cowpea mild mottle virus (CpMMV).

Survival of pathogens on soybean debris under no-tillage and conventional tillage systems

n†A study was conducted in the subtropical area of Southern Brazil to determine the survival of pathogens in soybean residues under conventional and no-tillage cultivation systems from March to September of 1998 and 1999. The pathogens most frequently isolated were Colletotrichum truncatum, Phomopsis†spp., Cercospora kikuchii, Fusarium†spp., Macrophomina phaseolina, and Rhizoctonia solani. Other fungi isolated were Myrothecium roridum, Penicillium†sp., Chaetomium sp., Epicoccum†sp., Corynespora cassiicola and Trichoderma†sp. The percent of survival of each pathogen varied accord- ing to the month and the year. Survival of C.†truncatum, Phomopsis†spp. and C.†kikuchii were signifi- cantly reduced (p<0.05) from the first to the last evaluation either on buried debris or maintained on the soil surface. On†the other hand, M.†phaseolina and Fusarium†spp. were either not affected or fa- vored by burying the debris. The frequency of recovery of Fusarium†spp. increased specially in debris kept under the soil. The loss of biomass, measured by debris weight along the period of this study, showed a reduction of 44.4% in the conventional system and 34.9% in the no-tillage system in†1998, when rain was better distributed. In 1999, the reduction was 48.2% and 39.0% for the conventional and no-tillage system, respectively.

Pathogenicity, molecular characterization, and cercosporin content of Brazilian isolates of Cercospora kikuchii

Cercospora kikuchii esta envolvido na desfolha da soja (Glycine max), normalmente em associacao com Septoria glycines, no final do ciclo da cultura. Setenta e dois isolados, obtidos principalmente de sementes com mancha purpura e oriundas de diferentes regioes do Brasil, mostraram variabilidade fenotipica. O teor de cercosporina e a velocidade de crescimento de colonias foram bastante variaveis entre os isolados. Uma forte correlacao foi identificada entre o teor de cercosporina e virulencia. Diferencas geneticas, entre e dentro da populacao analisada, foram observadas por RAPD com a analise de 86 loci. As analises de RAPD permitiram agrupar os isolados em sete grupos. Nenhuma relacao foi identificada entre os grupos de RAPD e a origem geografica ou teor de cercosporina. A sequencia da regiao espacadora do DNA ribossomal (ITS1-5,8S-ITS2) foi determinada em 13 isolados escolhidos nos diferentes agrupamentos. A similaridade das sequencias obtidas comparadas as sequencias de C. kikuchii depositadas no GenBank (AY266160, AY266262, AY152577 e AF291708) variou de 97 a 100%. Este trabalho demonstrou que os isolados brasileiros de C. kikuchii de diferentes origens sao variaveis quanto a virulencia, aos padroes de RAPD e ao teor de cercosporina. O teor de cercosporina pode ser um bom parâmetro na escolha de um isolado adequado para selecao de cultivares tolerantes ou resistentes a esse patogeno. Considerando que ele e facilmente transmitido por sementes nao e surpresa encontrar os mesmos haplotipos em diferentes regioes. A migracao poderia ser favorecida por sementes infetadas como demonstrado na analise de RAPD.

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.

Insecticide Susceptibility of Euschistus heros (Heteroptera: Pentatomidae) in Brazil

ABSTRACT 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 µg (AI) of acephate adult-1, 0.7 µg (AI) methamidophos adult-1, and 1,600 µg (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%).

Characterization of powdery mildews strains from soybean, bean, sunflower, and weeds in Brazil using rDNA-ITS sequences.

Soybean powdery mildew (Erysiphe diffusa) was considered a minor disease in Brazil in the decades immediately after its identification. However, since the outbreak in 1996/97 in all cultivated areas the disease has become a constant threat to farmers and losses of up to 25% have been reported. The report of a new species, E. glycines, infecting soybean in Japan, and the occurrence of the disease in other plant species (Phaseolus vulgaris, Helianthus annuus,Sonchus oleraceus,Hypochaeris brasiliensis, and Bidens pilosa) commonly found growing nearby soybean fields, raised questions in relation to the taxonomy of the powdery mildew strains found in or around soybean fields in Brazil. Analysis of the internal transcribed sequence (ITS) of the rDNA was undertaken to ascertain the pathogen species associated to each of the hosts. Powdery mildew strains isolated from Glycine max were identified as E. diffusa. Strains from P. vulgaris were very similar to E. diffusa, with 4 nt differences, and differed from Erysiphe poligony by 11 nt. Strains from H. annuus and S. oleraceus grouped with the species Golovinomyces cichoracearum, while strains from H. brasiliensis and B. pilosa were similar to Podosphaera fusca and Neoerysiphe cumminsiana, respectively. To our knowledge this is the first molecular identification of powdery mildew in Brazil based on rDNA sequence comparison. In addition, this study presented evidence for the occurrence of N. cumminsiana in America.

Biological and molecular characterization of an isolate of Tobacco streak virus obtained from soybeans in Brazil

ABSTRACT A virus was isolated from soybean ( Glycine max ) plants with symptoms of dwarfing and bud blight in WenceslauBraz County, Parana, Brazil. The host range and properties resembled those of Tobacco streak virus (TSV). The purifiedvirus showed three peaks in a frozen sucrose gradient. Antiserum was produced and the virus was serologically related toTSV. Electron microscopy detected 28 nm spherical particles. Coat protein (CP) had a Mr of 29.880 Da. A fragment of 1028nt was amplified, cloned and sequenced. One open reading frame with 717 nt was identified and associated to the CP. TheCP gene shared 83% identity with the sequence of TSV CP from white clover ( Trifolium repens ) (GenBank CAA25133).This is the first report of the biological and molecular characterization of TSV isolated from soybeans. It is proposed thatthis isolate be considered a strain of TSV named TSV-BR. Additional keywords : nucleotide sequence, Elisa, host range. RESUMOCaracterizacao biologica e molecular de um isolado de

Diaporthe endophytica and D. terebinthifolii from medicinal plants for biological control of Phyllosticta citricarpa

The citrus industry is severely affected by citrus black spot (CBS), a disease caused by the pathogen Phyllosticta citricarpa. This disease causes loss of production, decrease in the market price of the fruit, and reduction in its export to the European Union. Currently, CBS disease is being treated in orchards with various pesticides and fungicides every year. One alternative to CBS disease control without harming the environment is the use of microorganisms for biological control. Diaporthe endophytica and D. terebinthifolii, isolated from the medicinal plants Maytenus ilicifolia and Schinus terebinthifolius have an inhibitory effect against P. citricarpa in vitro and in detached fruits. Moreover, D. endophytica and D. terebinthifolii were transformed by Agrobacterium tumefaciens for in vivo studies. The transformants retained the ability to control of phytopathogenic fungus P. citricarpa after transformation process. Furthermore, D. endophytica and D. terebinthifolii were able to infect and colonize citrus plants, which is confirmed by reisolation of transformants from inoculated and uninoculated leaves. Light microscopic analysis showed fungus mycelium colonizing intercellular region and oil glands of citrus, suggesting that these two new species are capable of colonizing citrus plants, in addition to controlling the pathogen P. citricarpa.