RESUMEN
Fusarium head blight (FHB) disease caused by Fusarium graminearum (Fg) seriously affects the yield and quality of wheat. In this study, after bacterial community analysis of two wheat rhizosphere soils, the genus Pseudomonas was shown to be enriched in normal dry farmland (maize-wheat rotation) compared to that observed nearby paddy farmland (rice-wheat rotation) with serious FHB disease. Subsequently, a P. aeruginosa strain, NF011 with the highest antagonistic activity against Fg and excellent tolerance to 8.0 % of NaCl was isolated from the wheat rhizosphere soil in the normal dry farmland. Dual culture assay results showed that NF011 is a broad-spectrum fungicide for controlling six wheat pathogenic fungi. The major antifungal compound produced by NF011 was identified as phenazine-1-carboxamide (PCN) by LC-MS and nuclear magnetic resonance. 1.0 × 108 CFU/mL of NF011 or 32 mg/L of PCN could completely inhibit Fg spore germination and resulted in the destruction of Fg hypha vacuoles. Mannitol, peanut meal, beef extract, metal ions (Mn2+, Ca2+, Fe2+, and Mg2+), and amino acids (Arg and Lys) could promote the production of PCN by NF011, moreover, the optimal pH and temperature was 6.0 and 20 °C. The PCN produced by NF011 under the optimized culture conditions reached 436.55 ± 11.06 mg/L, 4.90-fold higher than that observed under the basic culture conditions. Finally, infection experiment results showed that NF011 can effectively prevent Fg spores from infecting wheat spikes and wheat grains and suppress the production of deoxynivalenol (DON). Therefore, the salt-tolerant PCN-producing NF011 has the potential to control wheat fungal disease.
Asunto(s)
Antibiosis , Fusarium/fisiología , Fenazinas/metabolismo , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/fisiología , Tolerancia a la Sal , Triticum/microbiología , Agricultura/métodos , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Agentes de Control Biológico/metabolismo , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Pseudomonas aeruginosa/genética , RizosferaRESUMEN
Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.
Asunto(s)
Ascomicetos/genética , Sitios Genéticos , Genética Microbiana/métodos , Biología Molecular/métodos , Mutagénesis Insercional/métodos , Recombinación Genética , Expresión Génica , Succinato Deshidrogenasa/genéticaRESUMEN
Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.
Asunto(s)
Ascomicetos/fisiología , Proteínas Fluorescentes Verdes/análisis , Microscopía Fluorescente/métodos , Coloración y Etiquetado/métodos , Ascomicetos/genética , Ascomicetos/patogenicidad , Codón , Expresión Génica , Proteínas Fluorescentes Verdes/genética , VirulenciaRESUMEN
The microtubule cytoskeleton supports vital processes in fungal cells, including hyphal growth and mitosis. Consequently, it is a target for fungicides, such as benomyl. The use of fluorescent fusion proteins to illuminate microtubules and microtubule-associated proteins has led to a break-through in our understanding of their dynamics and function in fungal cells. Here, we introduce fluorescent markers to visualize microtubules and accessory proteins in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein to α-tubulin (ZtTub2), to ZtPeb1, a homologue of the mammalian plus-end binding protein EB1, and to ZtGrc1, a component of the minus-end located γ-tubulin ring complex, involved in the nucleation of microtubules. In vivo observation confirms the localization and dynamic behaviour of all three markers. These marker proteins are useful tools for understanding the organization and importance of the microtubule cytoskeleton in Z. tritici.