RESUMO
AIMS: To investigate the effects of the endophyte Bacillus subtilisALB629 on the growth of cacao seedlings at early developmental stage and to evaluate its antimicrobial properties. METHODS AND RESULTS: Germinating cacao seeds were inoculated with ALB629, and seedlings growth was evaluated 30 days later. Significant increase (P < 0·05) was observed in the root system (up to 30%), leaf area (14%) and stem height (7·6%). ALB629 colonized the entire plant, prevailing over indigenous micro-organisms. In addition, it was tested in vitro, by pairing assays, and showed antagonistic effect against the phytopathogenic fungi Moniliophthora perniciosa, Colletotrichum sp. and C. gossypii. When tested in cacao-grafting procedure in the field, ALB629 increased the grafting success rate (24%), indicating its protective effect. In addition, this Bacillus secretes an antagonist compound, as shown by the antifungal activity of the cell-free culture. CONCLUSIONS: Bacillus subtilisALB629 promotes cacao root growth, besides promoting growth of the aerial part of cacao seedlings. It has antimicrobial properties and produces an antifungal compound. SIGNIFICANCE AND IMPACT OF THE STUDY: ALB629 presented beneficial characteristics for cacao cultivation, being a good biological control agent candidate. Furthermore, it is a potential source of antifungal compound with potential for commercial exploitation.
Assuntos
Antibiose , Bacillus subtilis/fisiologia , Cacau/crescimento & desenvolvimento , Cacau/microbiologia , Endófitos/fisiologia , Agaricales , Antifúngicos/química , Bacillus subtilis/química , Agentes de Controle Biológico , Colletotrichum , Endófitos/química , Raízes de Plantas/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimentoRESUMO
Brazil nut 2S albumins lack the essential amino acid tryptophan. In order to improve the protein's nutritional value and create a basis for structural investigations, three separate modified Brazil nut 2S albumin genes were constructed. The first mutant contains five consecutive tryptophan codons, while the other two modified genes encode proteins carrying single tryptophan residues at sites that will allow confirmation of the predicted protein structure through fluorescence quenching techniques. The modified genes, under the regulation of the CaMV 35S promoter, were introduced into Nicotiana tabacum. All three modified genes were correctly transcribed and the 2S albumin accumulated in the seeds of transgenic plants.
Assuntos
Albuminas/química , Nozes/química , Proteínas de Plantas/química , Plantas Geneticamente Modificadas/genética , Triptofano/análise , Agrobacterium tumefaciens/genética , Albuminas/genética , Sequência de Bases , Northern Blotting , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica de Plantas/genética , Imuno-Histoquímica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Proteínas de Plantas/genética , Plantas Tóxicas , Conformação Proteica , Software , Nicotiana/genética , Transformação Genética/genética , Triptofano/genéticaRESUMO
A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.
Assuntos
Hibridização de Ácido Nucleico , Vírus de Plantas/isolamento & purificação , Sondas RNA , Viroides/isolamento & purificação , Digoxigenina , Estudos de Avaliação como Assunto , Formaldeído , Frutas/virologia , Vírus de Plantas/genética , RNA Viral/análise , RNA Viral/efeitos dos fármacos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Viroides/genéticaRESUMO
The complete nucleotide (nt) sequence of the original coleus yellow viroid (CYVd) from Solenostemon scutellarioides, 'Golden Bedder', has been determined. The covalently closed single-stranded CYVd RNA molecule consists of 248 nt residues which assumes a rod-like secondary structure when folded in the model of lowest free energy. The sequence was determined by direct sequencing of RNA and from three overlapping cDNA clones. Comparison of the CYVd sequence with that of Coleus blumei viroid 1 (CbVd 1) from Germany demonstrated that they are closely related. The differences observed in the genome organization of CYVd relative to CbVd 1 were at three sites: position 25 (one U deletion), position 26 (a U was replaced by an A) and position 241 (one A insertion). The first two mutations were detected in one A-rich segment of eight nt (between positions 25 and 34). Northern blot hybridization of partially purified nucleic acids from the leaf tissue of S. scutellarioides 'Frilled Fantasy', inoculated with double-stranded cDNA, demonstrated that this fragment was infectious. These data enable CYVd to be assigned to the viroid class of plant pathogens, based on its biological properties and molecular structure. This work also gives additional support to the present classification system, in which the viroids isolated from S. scutellarioides form a distinct subgroup.