RESUMO
The antifungal activity of Serratia plymuthica CCGG2742, a bacterial strain isolated from grapes berries skin, against a phytopathogenic fungus isolated from blueberries was evaluated in vitro and in vivo. In order to characterize the wild fungal isolate, phylogenetic analysis using concatenated DNA sequences from the RPB2 and TEF1 genes and of the ITS region was performed, allowing the identification of the fungal isolate that was called Alternaria tenuissima CC17. Hyphae morphology, mycelium ultrastructure, conidia and reproductive structures were in agreement with the phylogenetic analysis. The antifungal activity of the S. plymuthica strain was dependent on the composition of the culture medium. The greatest inhibition of mycelial growth of A. tenuissima CC17 by S. plymuthica CCGG2742 was observed on YTS medium, which lacks of an easily assimilable carbon source. Fungal growth medium supplemented with 50 % of bacterial supernatant decreased the conidia germination of A. tenuissima CC17 up to 32 %. Preventive applications of S. plymuthica CCGG2742 to blueberries and tomato leaves at conidia:bacteria ratio of 1:100, protected in 77.8 ± 4.6 % and 98.2 ± 0.6 % to blueberries and tomato leaves from infection caused by A. tenuissima CC17, respectively. To the best of our knowledge, this is the first report on the antifungal activity of S. plymuthica against A. tenuissima, which could be used as a biological control agent of plant diseases caused by this fungal species. In addition, the results of this work could be a starting point to attribute the real importance of A. tenuissima as a pathogen of blueberries in Chile, which until now had been considered almost exclusively to A. alternata. Likewise, this research could be relevant to start developing highly effective strategies based on S. plymuthica CCGG2742 for the control of this important phytopathogenic fungus.
Assuntos
Alternaria , Antibiose , Filogenia , Doenças das Plantas , Serratia , Esporos Fúngicos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Alternaria/crescimento & desenvolvimento , Alternaria/genética , Serratia/genética , Serratia/fisiologia , Esporos Fúngicos/crescimento & desenvolvimento , Micélio/crescimento & desenvolvimento , Antifúngicos/farmacologia , Solanum lycopersicum/microbiologia , Hifas/crescimento & desenvolvimento , Meios de Cultura/química , Folhas de Planta/microbiologia , Vitis/microbiologiaRESUMO
The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (-ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs.
Assuntos
Genoma Viral , Isavirus/genética , Motivos de Ligação ao RNA/genética , Ribonucleoproteínas/genética , Salmo salar/virologia , Proteínas Virais/genética , Animais , GenômicaRESUMO
Eight different double-stranded RNA (dsRNA) molecules were found in the wild-type fungal strain Botrytis cinerea CCg427. The electrophoretic profile displayed molecules with approximate sizes of 1, 1.3, 1.6, 1.8, 3.3, 4.1, 6.5, and 12 kbp. Sequences analysis of the molecules in the 6.5-kbp band revealed the presence of two different dsRNA molecules (dsRNA-1 and dsRNA-2) of 6192 and 5567 bp. Each molecule contained a unique ORF (5487 and 4836 nucleotides in dsRNA-1 and dsRNA-2, respectively). The ORF of dsRNA-1 encodes a 205-kDa polypeptide that shares 58% amino acid sequence identity with the RNA-dependent RNA polymerase (RdRp) encoded by dsRNA-1 of Alternaria sp. SCFS-3 botybirnavirus (ABRV1), whereas the ORF of dsRNA-2 encodes a 180-kDa polypeptide that shares 52% amino acid sequence identity with an unclassified protein encoded by dsRNA-2 of ABRV1. Genome organization and phylogenetic analysis based on the amino acid sequences of RdRps in members of different dsRNA virus families showed that the dsRNAs in the 6.5-kbp band correspond to the genome of a new botybirnavirus that we have named "Botrytis cinerea botybirnavirus 1".
Assuntos
Botrytis/virologia , Micovírus/genética , Genoma Viral/genética , Vírus de RNA/genética , RNA Viral/genética , Sequência de Aminoácidos , Micovírus/classificação , Micovírus/isolamento & purificação , Filogenia , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genéticaRESUMO
Janthinobacterium lividum is a Gram-negative bacterium able to produce violacein, a pigment with antimicrobial and antitumor properties. Janthinobacterium lividum colonizes the skin of some amphibians and confers protection against fungal pathogens. The mechanisms underlying this association are not well understood. In order to identify the advantages for the bacterium to colonize amphibian skin we sequenced Janthinobacterium lividum strain MTR, a strain isolated from Cajón del Maipo, Chile. The strain has capnophilic behavior, with growth favored by high concentrations (5 %) of carbon dioxide. Its genome is 6,535,606 bp in size, with 5,362 coding sequences and a G + C content of 62.37 %. The presence of genes encoding for products that participate in the carbon fixation pathways (dark CAM pathways), and the entire set of genes encoding for the enzymes of the glyoxylate cycle may explain the capnophilic behavior and allow us to propose that the CO2 secreted by the skin of amphibians is the signal molecule that guides colonization by Janthinobacterium lividum.
RESUMO
BACKGROUND: In most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus does not show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA). So to analyze a large number of fungal isolates it is necessary to have a simple and rapid method to detect dsRNA. RESULTS: A rapid method to isolate dsRNA from a virus-infected filamentous fungus, Botrytis cinerea, and from a killer strain of Saccharomyces cerevisiae using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtained sufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing. CONCLUSIONS: The method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNA from any type of fungi or any biological sample that contains dsRNA.
Assuntos
Botrytis/virologia , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/isolamento & purificação , Saccharomyces cerevisiae/virologia , Cromatografia Líquida/métodos , Virologia/métodosRESUMO
The infectious salmon anemia virus (ISAV) is the causative agent of the ISA syndrome that affects mainly Atlantic salmon (Salmo salar) and has caused high mortality epidemics in Norway, Scotland, Canada, the United States and Chile. It is classified as an Orthomyxoviridae, its genome is composed of 8 single-strand RNA segments with negative polarity that code for 11 polypeptides. Through functional studies of the coded proteins it has been established that RNA segments 5 and 6 code for a fusion protein and hemagglutinin, respectively, while two polypeptides coded by segments 7 and 8 inhibit interferon induction. The functions of the rest of the possible proteins coded by the viral genome have been assigned by comparison with the corresponding ones of the influenza virus genome. As to its pathogenicity, some growth parameters such as incubation period, resistance to chemical and physical factors, establishment of the infection in other marine species, and dissemination ability among the different organs have been evaluated in several salmonids. Genomic analysis has shown (i) the existence of a high polymorphism region (HPR) in segment 6, and (ii) sequence insertion in segment 5. More than 20 HPR variants have been determined, all originating from HPR0, which is associated with low pathogenicity, while 4 different sequence insertions in segment 5 have not been related with some characteristic of the virus infection. Much progress has been made in the characterization of the virus in 20 years of study, but more detailed knowledge of the specific function of the proteins coded by all the viral genes is still missing, including the pathogenicity mechanism at the molecular level.