RESUMO
Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.
Assuntos
Entomophthorales/genética , Genes Fúngicos , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Padrões de Referência , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Fator 1 de Elongação de Peptídeos/genética , /genética , /genéticaRESUMO
The entomopathogenic fungus Beauveria bassiana (Balsamo 1835) Vuillemin is an effective alternative control agent against some agricultural pests and biological vectors of important diseases such as Chagas disease. In this work we studied an isolate of Beauveria bassiana from of the town of San Antonio Rayón, Puebla, Mexico and its entomopathogenic effects on Meccus pallidipennis (Stal 1872). Phylogenetic analysis using molecular comparison of the ITS and EF1α genes, showed that the resulting cladogram places the BUAP 04 strain with a relationship closer to the AFAO 9-6 strain, within the diversity of the B. bassiana sensu lato group. Although there was the possibility that BUAP 04 strain was a direct descendant of strains used in campaigns of biologic control, molecular study allowed us to recognize that it was a different fungus due to numerous inserts. A strain isolated from a T. dimiata was evaluated for pathogenicity against another triatoma (Meccus pallidipennis) species obtaining an LC50 of 4.16 x 10(6) spores/mL, confirming that the BUAP 04 strain is virulent for M. pallidipennis and could be a good prospect for formulations to control M. pallidipennis.
Assuntos
Animais , Beauveria/crescimento & desenvolvimento , Triatoma/microbiologia , Triatoma/fisiologia , Beauveria/classificação , Beauveria/genética , Beauveria/isolamento & purificação , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA de Helmintos/química , DNA de Helmintos/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , México , Dados de Sequência Molecular , Filogenia , Fator 1 de Elongação de Peptídeos/genética , Controle Biológico de Vetores/métodos , Análise de Sequência de DNA , Análise de Sobrevida , VirulênciaRESUMO
The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8SITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4β-acetoxy-12,13-epoxy-Δ9-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 µgmL-1. Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 µgmL-1. However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 µgmL-1). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.
Assuntos
Antifúngicos/farmacologia , Endófitos/química , Alho/microbiologia , Trichoderma/química , Tricodermina/farmacologia , Antifúngicos/isolamento & purificação , Botrytis/efeitos dos fármacos , Análise por Conglomerados , Colletotrichum/efeitos dos fármacos , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Endófitos/classificação , Endófitos/isolamento & purificação , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , Fator 1 de Elongação de Peptídeos/genética , /genética , Rhizoctonia/efeitos dos fármacos , Análise de Sequência de DNA , Trichoderma/classificação , Trichoderma/isolamento & purificação , Tricodermina/isolamento & purificaçãoRESUMO
Elongation factor 1A is a highly conserved protein that participates in translation. We report the occurrence of two genes homologous to the eukaryotic Elongation Factor 1A in Bradysia hygida and describe the partial cloning and characterization of the B. hygida eukaryotic Elongation Factor 1A-F1 (BheEF1A-F1) gene. The pattern of BheEF1A-F1 expression in the salivary gland at the end of the fourth larval instar was investigated using real-time PCR. The results showed that BheEF1A-F1 expression levels are relatively constant at the time when rapid changes in protein synthesis occur in this tissue. In situ hybridization experiments coupled to Southern blot analyses showed that the BheEF1A-F1 gene is located at position 3d of the A chromosome and a second gene homologous to eEF1A is located at position 6a of the X chromosome. Southern blot analyses showed that both the BheEF1A-F1 gene and the second gene homologous to eEF1A constitute non-amplified genes. The present results contribute to the molecular characterization of a sciarid eEF1A gene.
