RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses

Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.

Garlic viral complex: identification of Potyviruses and Carlavirus in Central Brazil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virus-free garlic was evaluated.

Sequence analysis of the capsid protein gene of an isolate of Apple stem grooving virus, and its survey in Southern Brazil

Apple stem grooving virus (ASGV) is one of the most important viruses infecting fruit trees. This study aimed at the molecular characterization of ASGV infecting apple (Malus domestica) plants in Santa Catarina (SC). RNA extracted from plants infected with isolate UV01 was used as a template for RT-PCR using specific primers. An amplified DNA fragment of 755 bp was sequenced. The coat protein gene of ASGV isolate UV01 contains 714 nucleotides, coding for a protein of 237 amino acids with a predicted Mr of approximately 27 kDa. The nucleotide and the deduced amino acid sequences of the coat protein gene showed identities of 90.9% and 97.9%, respectively, with a Japanese isolate of ASGV. Very high amino acid homologies (98.7%) were also found with Citrus tatter leaf capillovirus (CTLV), a very close relative of ASGV. These results indicate low coat protein gene variability among Capillovirus isolates from distinct regions. In a restricted survey, mother stocks in orchards and plants introduced into the country for large scale fruit production were indexed and shown to be infected by ASGV (20%), usually in a complex with other (latent) apple viruses (80%).

Identificação de xanthomonas axonopodis pv. phaseoli e x. axonopodis pv. phaseoli var. fuscans através da técnica de pcr

Este trabalho teve por objetivo verificar a viabilidade de utilização da técnica de PCR, usando primers considerados específicos, para identificação de Xanthomonas axonopodis pv. phaseoli (Xap) e x. axonopodis pv. phaseoli var. fuscans (Xapf), visando criar bases para sua utilização em diagnose rotineira e em programas de certificação de sementes. Foram incluídos no estudo 42 isolados da bactéria provenientes de locais distintos, além de outros gêneros, espécies e patovares, para ser verificada a especificidade dos primers. Os resultados obtidos permitem concluir que os primers utilizados são adequados para identificação de Xap e Xapf, embora dois isolados patogênicos não tenham sido amplificados. Foi observada também a amplificação de uma banda fraca para X. axonopodis pv. vitians, com o mesmo número de pares de bases, o que sugere existir homologia entre estes patovares, na região amplificada.

Identification of rapd marker linked to blast resistance gene in a somaclone of rice cultivar Araguaia

The gene Pi-ar confers resistance to Pyricularia grisea race IB-45 in a somaclone derived from immature panicles of the susceptible rice (Oryza sativa) cultivar Araguaia. RAPD technique was used to identify molecular markers linked to this gene utilizing bulked segregant analysis. Initially, the two parental DNAs from the resistant donor SC09 and 'Araguaia' were analyzed using random primers. Of the 240 primers tested, 203 produced amplification products. The two parental DNAs along with the resistant and susceptible bulks of F2 population were screened using 48 primers that differentiated resistant and susceptible parents. Even though eight primers differentiated the resistant bulk from the susceptible bulk, as well as somaclone SC09 and 'Araguaia', only one primer, OPC02 ('GTGAGGCGTC'), was found to be tightly linked (1.7cM) to the resistance gene of somaclone SC09.

Genetic analyses of Rhizoctonia solani isolates from Phaseolus vulgaris grown in the Atlantic Rainforest Region of São Paulo, Brazil

Rhizoctonia solani isolates obtained from common beans (Phaseolus vulgaris) grown in the mountainous Atlantic Rainforest (Mata Atlântica) region of São Paulo, Brazil, were analyzed to determine their genetic diversity using internal transcribed spacer (ITS), microsatellite and telomere sequence-based PCR primers. Restriction digestion of the ITS1/5.8S/ITS2 ribosomal regions yielded unique banding patterns specific for AG4 and its subgroups. The ITS restriction digestion (ITS/RFLP), telomere and microsatellite primers identified five to 11 genotypes within the isolates of R. solani. While all isolates were pathogenic on beans, there was no correlation found between genotypic differences and pathogenicity. The different PCR primers revealed a number of isolates that were genetically similar. Some of these genetic groups were supported by more than one of the primers utilized in this study, thus confirming their relationship.

Detection and coat protein gene characterization of an isolate of Grapevine virus B from corky bark-affected grapevines in Southern Brazil

An isolate of Grapevine virus B (GVB), obtained by indexing Vitis labrusca and V. vinifera grapevines on the indicator LN33, was transmitted mechanically to several Nicotiana species. The virus was partially purified from N. cavicola and the coat protein estimated at 23 kDa by SDS-PAGE. In negatively stained leaf extracts of experimentally inoculated N. cavicola and N. occidentalis, flexuous particles with cross banding were observed, predominantly measuring 750-770 x 12 nm, with a modal length of 760 nm. Decoration indicated a clear, positive reaction against AS-GVB. In DAS-ELISA, GVB was detected in N. cavicola and grapevine extracts, and Western blots showed homologous and cross reaction of GVB and GVA antisera with GVB coat protein. Using specific primers for GVB, a fragment of 594 bp, comprising the coat protein gene coding for 197 amino acids, was amplified by RT-PCR with viral RNA extracted from GVB-infected N. occidentalis. The nucleotide and the deduced amino acid sequences of the coat protein gene showed high identities with Italian and Japanese isolates of GVB.

Análise de restrição de DNA ribossomal amplificado (ARDRA) pode diferenciar Fusarium solani f. sp. phaseoli de F. solani f. sp. glycines

Métodos moleculares têm sido utilizados para caracterizar a diversidade entre isolados de Fusarium spp. patogênicos e não patogênicos a uma cultura e, para determinar relações genéticas entre formae speciales. Testes de patogenicidade realizados em soja (Glycine max) e feijoeiro (Phaseolus vulgaris) com 17 isolados de Fusarium solani não demonstraram especificidade de hospedeiros. Utilizou-se a técnica ARDRA (Amplified Ribosomal DNA Restriction Analysis) para analisar a região ITS1 - 5,8S rDNA - ITS2, amplificada com os primers ITS5 e ITS4. Os produtos amplificados foram digeridos com as enzimas de restrição Hae III e Msp I. Os padrões de bandas gerados pela digestão com a enzima Hae III permitiram diferenciar três grupos entre os isolados de F. solani, sendo um grupo específico para isolados de F. solani f. sp. phaseoli com 100% de similaridade entre os 11 isolados. Entre os isolados de F. solani f. sp glycines foram observados dois padrões distintos de restrição. A técnica de ARDRA utilizando a enzima Hae III apresenta, portanto, potencial para utilização como um marcador para diferenciação entre as formae specialesphaseoli e glycines, dentro do complexo F. solani.

An isolate of Apple stem grooving virus associated with Cleopatra mandarin fruit intumescence

A citrus tatter leaf isolate (CTLV-Cl) of Apple stem grooving virus (ASGV) has been found to be associated with a fruit rind intumescence in Cleopatra mandarin (Citrus reshni) in Limeira (SP). The CTLV-Cl was mechanically transmitted to the main experimental herbaceous hosts of CTLV. Chenopodium quinoa and C. amaranticolor reacted with local lesions and systemic symptoms while other test plants reacted somewhat differently than what is reported for CTLV. A pair of primers designed for specific detection of ASGV and CTLV amplified the expected 801 bp fragment from the CTLV-Cl-infected plants. Typical capillovirus-like particles were observed by the electron microscope in experimentally infected C. quinoa and C. amaranticolor leaves.

Mapeamento de genes de resistência do feijoeiro à ferrugem, antracnose e mancha-angular usando marcadores RAPD

A organização de diferentes genes de resistência da cultivar Ouro Negro de feijoeiro-comum (Phaseolus vulgaris) à ferrugem, antracnose e mancha-angular foi estudada com o auxílio de marcadores moleculares. Uma população de 154 linhas endogâmicas recombinantes (RIL's) obtidas do cruzamento entre as cultivares Ouro Negro e Rudá foram inoculadas com sete raças fisiológicas de Uromyces appendiculatus, três de Colletotrichum lindemuthianum, e quatro de Phaeoisariopsis griseola. Amostras de DNA de cada uma das RIL's foram amplificadas via PCR utilizando 70 diferentes primers. A análise da segregação da resistência à ferrugem, antracnose e mancha-angular na população de 154 RIL's revelou diferentes modos de herança para a resistência a cada uma das raças fisiológicas. A análise de ligação genética revelou que os diferentes genes de resistência à ferrugem e à antracnose estão no mesmo grupo de ligação. Os genes de resistência à mancha-angular também foram mapeados juntos, porém em outro grupo de ligação. Verificou-se neste trabalho que a utilidade dos marcadores RAPD, previamente identificados como ligados a genes de resistência do feijoeiro a doenças foi restrita. Apenas cinco dos 38 marcadores moleculares testados foram validados na população de RIL's como ligados aos genes de resistência à ferrugem e à antracnose. Três novos marcadores (OBA16(669) e OBA16(583) a 10,4 cM em acoplamento e OAD9(3210) a 13,9 cM em repulsão) ligados ao bloco gênico de resistência da cultivar Ouro Negro à mancha-angular foram identificados.

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.

Uso de marcadores RAPD na classificação de isolados de Phytophthora spp. causadores da podridão parda do cacaueiro no Brasil

Objetivou-se neste trabalho propor uma metodologia para a utilização de marcadores RAPD como uma ferramenta auxiliar na classificação de isolados de Phytophthora spp. causadores da podridão-parda do cacaueiro (Theobroma cacao) no Brasil. Existe uma necessidade constante de monitorar populações de Phytophthora spp. nas regiões cacaueiras do Brasil e a tarefa de classificação dos isolados é difícil e demorada. Com base em estudos de diversidade genética de isolados de Phytophthora capsici, P. palmivora e P. citrophthora por meio de marcadores RAPD, foram escolhidos três isolados de cada espécie como padrões e dois "primers" decâmeros mais informativos na diferenciação das espécies (OPA 13 e OPH 18). O DNA genômico dos isolados padrões e de três isolados não classificados foi extraído e amplificado, utilizando-se os dois "primers" decâmeros mais informativos. Os padrões de marcadores RAPD obtidos permitiram uma diferenciação visual clara dos isolados de cada espécie e mostraram-se úteis na classificação de isolados de Phytophthora spp. A metodologia proposta já está sendo utilizada no Centro de Pesquisas do Cacau, solucionando eventuais dúvidas resultantes da caracterização morfológica dos isolados.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Hypocrea stromatica sp. nov. teleomorfo de Trichoderma stromaticum

Trichoderma stromaticum é um fungo antagonista a Crinipellis perniciosa, agente causal da vassoura-de-bruxa do cacaueiro (Theobroma cacao). Estromas periteciais de Hypocrea sp. formaram-se sobre a superfície de frutos mumificados e vassouras secas de cacaueiros em áreas onde T. stromaticum tinha sido pulverizado, experimentalmente, no controle biológico de C. perniciosa. Ascósporos obtidos dos estromas de Hypocrea sp. originaram colônias idênticas às de T. stromaticum. DNA genômico de colônias provenientes de ascósporos desta Hypocrea sp., de conídios de T. stromaticum e de Fusarium sp. (controle negativo) foi extraído e amplificado com oito "primers" decâmeros para obtenção de marcadores RAPD. Observaram-se similaridades genéticas de 0,82 a 0,96 entre os isolados ascospóricos e conidiais com base nos marcadores RAPD e de 0,06 a 0,09 entre estes e o isolado de Fusarium sp. O teleomorfo de T. stromaticum é denominado H. stromatica Bezerra, Costa & Bastos sp. nov.

Diversidade genética de isolados de Phytophthora capsici de diferentes hospedeiros com base em marcadores RAPD, patogenicidade e morfologia

Phytophthora capsici é uma espécie onívora com vários hospedeiros cultivados na Bahia. Variações morfológicas ocorrem entre isolados de cacaueiro (Theobromae cacao), seringueira (Hevea brasiliensis) e pimenta-do-reino (Piper nigrum), todos classificados como P. capsici. Utilizaram-se marcadores RAPD e reações de patogenicidade para estudar a diversidade genética dentro desta espécie. Foram analisados 22 isolados sendo, oito de cacau, oito de seringueira, três de pimentão (Capsicum annuum) (dois antigos e um recente), um de abóbora (Cucurbita moschata), um de tomate (Lycopersicon esculentum) e um de pimenta-do-reino. Os isolados de pimentão, abóbora e tomate foram obtidos em Minas Gerais, o de pimenta-do-reino no Pará e, os demais, na Bahia e Espírito Santo. O DNA genômico de cada isolado foi extraído e amplificado utilizando-se oito primers decâmeros, os quais geraram 123 marcadores RAPD. Distâncias genéticas e análises de agrupamento permitiram a diferenciação de três grupos: o primeiro formado por isolados de cacaueiro, o segundo por sete dos oito isolados de seringueira e o terceiro por dois isolados de pimentão (antigos). Os isolados de tomate, pimenta-do-reino, pimentão (recente), o de abóbora e um de seringueira mostraram-se distantes geneticamente dos demais. Inoculações em frutos de cacau, seringueira, tomate e pimentão, com e sem ferimento, mostraram que o isolado de seringueira que não agrupou com os demais foi o menos virulento dos isolados testados, não causando lesões em frutos de seringueira sem ferimento. Frutos de pimentão só foram infetados por isolados de tomate e pimentão, enquanto os frutos de cacau foram infetados por todos os isolados testados. A manutenção de todos os grupos dentro de P. capsici é discutida.