Cowpea mild mottle virus (CpMMV), the causal agent of stem necrosis disease, has drawn attention of soybean producers in recent years due to yield losses in the main producing regions of Brazil. Serological methods for viral detection require the use of an antiserum of good quality to achieve specificity and sensitivity. The entire coat protein gene of a Brazilian CpMMV isolate was cloned into a bacterial expression vector and transformed into Escherichia coli BL21::DE3 for in vitro expression. The coat protein, fused to a His-tag, was purified under denaturing conditions by affinity chromatography using a Ni-NTA resin. After renaturation, the integrity and identity of the purified recombinant protein was confirmed by SDS-Page and MALDI-ToF/ToF mass spectrometer analyses. A rabbit was immunized with increasing amounts of the recombinant protein. The specificity and sensitivity of the antiserum was demonstrated by Western blot and indirect ELISA assays. The polyclonal antisera raised against recombinant coat protein proved to be a reliable tool for CpMMV detection.

Em um levantamento conduzido na região noroeste do Paraná, principal região produtora de mandioca do estado, constatou-se que o CsCMV está amplamente disseminado, infectando mais de 90% das plantas de todas as cultivares. Um isolado do CsCMV foi purificado para a produção de um anti-soro policlonal de alto título (1/1000), o qual foi utilizado na indexação por meio de PTA-ELISA de plantas produzidas via cultura de meristemas. De uma produção inicial de 110 plantas da cultivar Olho Junto, 31 permaneceram infectadas como indicou o PTA-ELISA. Visando aumentar a sensibilidade de detecção do vírus, foi estabelecido um protocolo de IC-RT-PCR. A IgG purificada, específica para o vírus, combinada com um par de oligonucleotídeos universais para Potexvirus, prontamente detectou o CsCMV nos extratos obtidos de mandioca. Produtos de IC-RT-PCR de 750 pb foram amplificados a partir de seis de um total de 35 plantas previamente testadas como negativas para o CsCMV via PTA-ELISA. Os sequenciamentos confirmaram que os produtos de IC-RT-PCR são parte do gene codificador da replicase do CsCMV, indicando assim a presença do vírus mesmo após a termoterapia combinada com a cultura de meristemas. O PTA-ELISA permitiu a triagem preliminar de plantas vírus-positivas, possibilitando a redução do número de plantas a serem analisadas por IC-RT-PCR. Embora o CsCMV seja considerado de importância menor, o fato desse vírus estar disseminado na região noroeste do Paraná recomenda a adoção de medidas efetivas para o seu controle.

A virus survey conducted in the northwest region of Paraná, the main cassava-producing region of that state, showed Cassava common mosaic virus (CsCMV) to be widespread, infecting more than 90% of plants from all cassava cultivars. A CsCMV isolate was purified and used to raise a high-titer (1/1.000) polyclonal antiserum for indexing plants produced from meristem-tip culture, using PTA-ELISA. From an initial production of 110 plants of cultivar Olho Junto, 31 remained infected as indicated by PTA-ELISA. To improve the sensitivity of virus detection, an immunocapture-RT-PCR (IC-RT-PCR) protocol was established. Virus-specific IgG, purified and combined with a primer set for the genus Potexvirus, could readily detect CsCMV in cassava crude leaf extracts. IC-RT-PCR products of 750 bp were amplified from six out of 35 plants previously tested as virus-negative by PTA-ELISA. Sequence analysis of cloned IC-RT-PCR products confirmed they were part of the CsCMV replicase gene, indicating that the virus was still present after thermotherapy and meristem-tip culture. PTA-ELISA enabled initial screening of virus-positive cassava, reducing the number of plants to be further analyzed by IC-RT-PCR. Though CsCMV has been considered of minor importance, its widespread nature, as noticed in Paraná, indicates the need for adoption of effective control measures.

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.

Em março de 2006, plantas de soja (Glycine max) doentes foram encontradas em Passo Fundo e em Coxilha, Rio Grande do Sul, Brasil. O quadro sintomatológico caracterizava-se por plantas com folhas secas e de coloração marrom; necrose (cancro) na metade inferior da haste, de coloração marrom-clara a marrom-avermelhada, envolvendo a mesma, seguindo-se murcha e morte da planta. Características culturais em batata dextrose ágar mostraram padrões idênticos àqueles publicados para Diaporthe phaseolorum varcaulivora (Dpc). Os dados de seqüenciamento de DNA do isolado brasileiro CH 40/06 (número de acesso GenBank EU622854) foram comparados às seqüências homólogas de NCBI GenBank, e alta similaridade foi verificada com outros isolados de Dpc. Árvore filogenética foi elaborada a fim de comparar EU622854 com outras seqüências de Dpc, cujo agrupamento foi altamente confirmado pelo teste bootstrap (99%). Características morfológicas foram similares às descritas para Dpc na literatura, embora os ascosporos do espécime brasileiro fossem mais estreitos que o original americanoNa safra 2006/07, Dpc foi também encontrado em outros cinco municípios do RS.

In March 2006, diseased soybean plants (Glycine max) were found in the municipalities of Passo Fundo and Coxilha, state of Rio Grande do Sul, Brazil, showing withered, brown leaves and light to reddish-brown discoloration and necrosis (canker) of the lower half of the stem. Cultural characteristics in potato dextrose agar showed identical patterns to those described for Diaporthe phaseolorum varcaulivora (Dpc). The rDNA-ITS sequence data from the Brazilian strain CH 40/06 (GenBank accession number EU622854) were compared to homologous sequences from the NCBI GenBank database, and a high similarity with Dpc strains was verified. A phylogenetic reconstruction was performed to compare EU622854 with other related sequences and grouped with Dpc. This group was highly supported by the bootstrap test (99%). Morphology of the Brazilian specimen was also similar to that described in the literature for Dpc, but the ascospores were slightly narrower than the American original material of Dpc. In field surveys performed in the 2006/07 crop season, Dpc was also found in five additional municipalities in RS.

Podridão de carvão, causada por Macrophomina phaseolina é uma das mais importantes doenças da soja. Variabilidade genética tem sido observada entre isolados. Entretanto, o efeito de especialização deste fungo, de acordo com a planta hospedeira, não foi ainda relatado. Isolados de soja, milho e girassol foram avaliados, em inoculações cruzadas, quanto à capacidade de formação de microesclerócios em raiz. A maior e menor produção significativa de microesclerócios (P<0.005) foram observadas em raízes de soja (863 microesclerócios/g de raiz) e girassol (578 microesclerócios/g de raiz), respectivamente, independente da origem do isolado. Além disso, estudou-se a variabilidade de isolados do fungo, obtidos de sistemas de rotação de culturas estabelecido há 20 anos utilizando-se RAPD. Oitenta e nove isolados, oriundos de cinco populações foram coletados em amostras de solo de quatro sistemas de rotação e uma amostra de solo virgem. Sete agrupamentos foram observados com 85% de similaridade. As análises indicaram que M. phaseolina é uma espécie com alta variabilidade genética com forte efeito do sistema de rotação. AMOVA foi realizada com os dados de RAPD. Da variabilidade genética total, 21% (P<0.0001) foi explicada pelas diferenças entre populações e, 79% foi explicada por diferenças dentro das populações ( Φst = 0.2110; P<0.0001). A média do coeficiente de diferenciação gênica (G ST) estimada entre as cinco populações foi de 27%, similar aos resultados da AMOVA ( Φst= 0.2110). A diversidade genética total mostrou que as cinco populações têm alto grau de variabilidade (H T =0.3484). Os resultados sugerem que a diferenciação genética de M. phaseolina pode ser alterada pela rotação de culturas.

Charcoal rot, caused by Macrophomina phaseolina, is one of the most important diseases of soybean. Genetic variability among soybean isolates has been observed but the effect of host specialization on genetic variability has not been reported. In this work, isolates from soybean, corn and sunflower were evaluated based on cross inoculations and number of microsclerotia/g of roots. The highest and lowest significant (P<0.005) production of microsclerotia was obtained in soybean (863 microsclerotia/g of roots) and sunflower (578 microsclerotia/g of roots), respectively, regardless of the origin of the isolate. Additionally, the effect of a 20-year crop rotation on genetic variability based on RAPD was studied. Eighty-nine isolates from five populations were obtained from soil samples under four crop rotation systems and an uncropped soil. Seven clusters were obtained considering a similarity level of 85%. Analysis indicated that M. phaseolina is a highly diverse species and also revealed a strong effect of the rotation system on genetic diversity. AMOVA was conducted for the RAPD data. From the total genetic variability, 21% (P<0.0001) could be explained by the differences between populations while 79% could be explained by differences within populations ( Φst = 0.2110; P<0.0001). The mean coefficient of gene differentiation (G ST) estimated among the five populations indicated 27% of differentiation between populations similar to the AMOVA results where Φst= 0.2110. Total gene diversity estimated indicated high levels of variability (H T =0.3484). Results suggest that genetic differentiation of M. phaseolina can be altered by crop rotation.

O oídio da soja, causado por Erysiphe diffusa, foi considerado uma doença de menor importância, desde a sua identificação no Brasil. Entretanto, após o surto na safra de 1996/97, em todas as áreas cultivadas, a doença tornou-se uma ameaça constante para os agricultores, com perdas relatadas de até 25%. A identificação de uma nova espécie (E. glycines) no Japão e a ocorrência em outras espécies de plantas comumente encontradas próximas a campos de soja (Phaseolus vulgaris,Helianthus annuus,Sonchus oleraceus,Hypochaeris brasiliensis e Bidens pilosa) levantou questões em relação à taxonomia dos oídios encontrados nas lavouras. Análises moleculares da região espaçadora interna transcrita (ITS) do rDNA foi utilizada para a identificação molecular das espécies presentes em cada planta hospedeira. Oídios isolados de soja (Glycine max) foram identificados como E. diffusa. Isolados de feijão (P. vulgaris) foram similares a E. diffusa, com 4 nt diferentes, e diferiram de E. poligony em 11 nt. Isolados de H. annuus e S. oleraceus agruparam-se com a espécie Golovinomyces cichoracearum, enquanto isolados de H. brasiliensis e B. pilosa foram similares a Podosphaera fusca e Neoerysiphe cumminsiana, respectivamente. Ao nosso conhecimento esta é a primeira identificação molecular de oídios no Brasil, através da comparação de seqüências de rDNA. Esta também é uma evidência da ocorrência de N. cumminsiana na América.

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.

Soybean plants with symptoms of bud blight were growing close to cowpea with severe symptoms of mosaic associated with blisters in the leaves. A group of plants of both species were collected and used for etiological studies. This kind of symptom in soybeans was common in certain areas of the State of Paraná, induced by tobacco streak ilarvirus. Host range, serological reaction, particle morphology and size, protein and nucleic acid analysis, and transmission by beetles from species Cerotoma arcuata Oliv. showed that the virus involved was cowpea severe mosaic comovirus. This is the first report on the occurrence of this virus in soybean plants in the State of Paraná. Results using indirect ELISA showed that in cowpea the relative virus concentration was higher in green leaf areas than in chlorotic ones. Also, virus concentration, determined through indirect ELISA was much higher in plants kept at diurnal regime of 25º C x 23º C (12 x 12 h) than at 30º C x 28º C.