Abstract Plant growth-promoting rhizobacteria strains from special formulations have been used to optimize eucalyptus cutting production. To undertake quality control for the formulated products, the rhizobacterial strains should be characterized to assess their purity and authentication. In the present study, we characterized nine strains of rhizobacteria, including three Bacillus subtilis (S1, S2 and 3918), two Pseudomonas sp. (MF4 and FL2), P. putida (MF2), P. fulva (Ca), Frateuria aurantia (R1), and Stenotrophomonas maltophilia (CIIb). The strains were differentiated by colony morphology after 24 h of incubation in three different solid state culture media (glucose-nutritive agar, 523 medium and yeast extract-mannitol agar), sensitivity to a panel of 28 antibiotics (expressed according to the formation of inhibition halos of bacterial growth in the presence of antibiotics), and PCR-RFLP profiles of the 16S rDNA gene produced using nine restriction enzymes. It was possible to differentiate all nine strains of rhizobacteria using their morphological characteristics and sensitivity to antibiotics. The molecular analysis allowed us to separate the strains CIIb, FL2 and R1 from the strains belonging to the genera Bacillus and Pseudomonas. By using these three methods concomitantly, we were able to determine strain purity and perform the authentication.

The main areas for pepper production in Cuba were surveyed during 2006-2010 to determine the molecular diversity of the begomoviruses in this crop. Six hundred leaf samples with typical symptoms of viral diseases were collected, and from them, 50 samples per region were randomly selected for their evaluation. The total DNA extracted from each sample was previously tested by conventional PCR using begomovirus universal primers (Palv 1978/PARc496). The positive samples were used as a template for the full-length circular genome amplification by Rolling Circle Amplification. The amplified products were characterized by restriction fragment length polymorphism analysis (RFLP) with the enzymes Sac I, BamH I, Hind III, Cla I, Kpn I, Sal I, Pst I.Begomovirus infection was detected in 100% of the samples evaluated by conventional PCR, suggesting the presence of these viruses in all the regions of the country. The enzymes Sac I, Bamh I, Sal I and Pst I produced unique cuts in unique sites in the isolated genomes liberating a unique fragment with the expected length (2.6-2,7kb), and detecting four polymorphic profiles in these samples. The obtained results show the possible presence of different begomoviruses species circulating in the crop, the occurrence of viral recombinants of previously informed or the presence of the two viral components in these samples. These results could also be associated with the presence of the two viral components in these samples. It was made evident the molecular diversity in these species of begomoviruses in the pepper crop in Cuba.

El objetivo de este trabajo fue determinar la diversidad molecular de begomovirus asociados al cultivo del pimiento en Cuba. Para ello, en el periodo 2006- 2010 se realizó una prospección de begomovirus en las principales áreas de producción de pimiento en Cuba. Se colectaron 600 muestras foliares de plantas que mostraron síntomas típicos de enfermedades virales y de estas se seleccionaron al azar 50 por región para su evaluación. El ADN total extraído se evaluó mediante una PCR con cebadores universales a begomovirus (Palv1978/PARc496). A partir de muestras positivas se amplificó su ADN genómico circular completo mediante la amplificación en círculo rodante. Los productos se caracterizaron mediante análisis de restricción, utilizando las enzimas Sac I, BamH I, Hind III, Cla I, Kpn I, Sal I, Pst I. Se detectó infección por begomovirus en el 100% de las muestras evaluadas mediante PCR con cebadores universales, sugiriendo la presencia de estos virus en áreas de este cultivo de las tres regiones del país. Los enzimas Sac I, Bamh I, Sal I y Pst I produjeron un corte en sitios únicos en los ADN circulares obtenidos, liberando un único fragmento lineal de la talla esperada (2.6-2.7 Kb) en estas muestras. Los resultados obtenidos muestran la posible presencia de diferentes especies de begomovirus circulando en el cultivo, la ocurrencia de recombinantes virales de los informados previamente o la presencia de los dos componentes genómicos en estas muestras. Se evidenció diversidad molecular de estas especies en el cultivo del pimiento en Cuba.

O mamão (Carica papaya) é uma fruta de grande importância econômica em todo o Nordeste brasileiro, região responsável por 60% da produção nacional. Mamoeiros nos estados do Ceará e Rio Grande do Norte são afetados pela doença denominada amarelo letal do mamoeiro, causada pelo papaya lethal yellowing virus (PLYV). Trabalhos anteriores indicaram que o PLYV é um possível sobemovírus. A fim de estimar a variabilidade genética do PLYV, amostras foliares de mamoeiro foram coletadas em outubro de 2008 e de 2009 em regiões produtoras do Ceará, e Rio Grande do Norte. Oligonucleotídeos específicos baseados na sequência de um isolado de PLYV previamente caracterizado foram utilizados para a amplificação via RT-PCR de um fragmento com 900 pb, correspondente à região central do genoma viral. Fragmentos de 21 isolados virais foram clonados e sequenciados. A análise das sequências de nucleotídeos indicou >97% de identidade entre os isolados, entre 94-100% com o isolado de PLYV previamente sequenciado, e uma identidade baixa, porém significativa, com sobemovírus (43-48,5%). Estes resultados sugerem um baixo grau de variabilidade genética entre isolados de PLYV, e estão de acordo com a classificação provisória do PLYV como membro do gênero Sobemovirus. Conclusões de ordem taxonômica, no entanto, só podem ser tiradas após a determinação da sequência genômica completa.

The papaya (Carica papaya) is a fruit crop of great economic importance throughout the Brazilian northeast, which is responsible for 60% of the national output. Papayas in the states of Ceará and Rio Grande do Norte are affected by lethal yellowing disease, caused by papaya lethal yellowing virus (PLYV). Previous work suggested that PLYV is a putative sobemovirus. To assess the genetic variability of PLYV, foliar samples were collected in October 2008 and October 2009 in commercial fields from Ceará and Rio Grande do Norte states, and total RNA was extracted. Specific primers based on the sequence of a previously characterized PLYV isolate were used for the RT-PCR-based amplification of a 900 bp fragment corresponding to the central region of the viral genome. Fragments from 21 viral isolates were cloned and sequenced. Sequence analyses indicated >97% nucleotide sequence identity among the isolates, 94-100% identity with the previously sequenced PLYV isolate, and a lower but significant identity with sobemoviruses (43-48.5%). These results suggest a low genetic variability among PLYV isolates, and are in agreement with the provisional placement of PLYV in the genus Sobemovirus. Definitive taxonomic conclusions, however, can only be drawn after the determination of the full-length genomic sequence.