Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, Laboratory of Plant Pathology, POB 269, 54124 Thessaloniki, Greece.
Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, Laboratory of Plant Pathology, POB 269, 54124 Thessaloniki, Greece.
Int J Food Microbiol. 2020 Dec 16;335:108896. doi: 10.1016/j.ijfoodmicro.2020.108896. Epub 2020 Sep 29.
Penicillium expansum is the most common apple fruit postharvest spoilage agent that causes a disease known as Blue Mold. Disease control is based on fungicide use. However, development of resistance to fungicides hampers the success of this control method. Fungicide sensitivity monitoring studies in Greece revealed the presence of pathogen strains exhibiting simultaneous resistance to different chemically unrelated compounds (multidrug resistance, MDR). This study was initiated aiming primarily to test the hypothesis that the MDR phenotype is associated with overexpression of efflux transporter genes and to determine the fitness of the MDR isolates. The monitoring study (n = 264) and the measurements of sensitivity in terms of EC values to 9 different compounds revealed that almost 5% of the population was of the MDR type. In the selected MDR isolates, the highest resistant factors were calculated for fludioxonil and pyraclostrobin, while the same isolates were moderately resistant to cyprodinil, thiophanate methyl and fluxapyroxad. In the resistant strains no target site mutations were detected in the target genes of each fungicide class, while in addition, a synergistic activity was observed between fungicides and the drug transporter modulator verapamil in some isolates. To obtain a direct insight on the resistance mechanism, the transcriptome of 2 MDR and 1 sensitive isolates was sequenced using Illumina HiSeq 2500 and differences in efflux transporter gene expression profile were figured out. Gene expression profiling analysis was performed before and after the exposure of fungal mycelia to fludioxonil. This analysis revealed the up-regulation of several MFS transporter genes and a limited number of ABC transporter genes either before or after the exposure to fludioxonil in the MDR isolates. Expression results for genes with the highest expression levels were verified by qRT-PCR assays. Fitness components measurements revealed that MDR isolates were of lower mycelial growth and pathogenicity compared to sensitive strains but they were producing higher number of conidia. The above mentioned data represent the first report of MDR in P. expansum associated with overexpression of drug efflux transporters and contribute to our knowledge in the mechanisms associated with fungicide resistance development in this fungal species.
扩展青霉是最常见的苹果果实采后腐烂剂，可导致一种称为“青霉病”的疾病。病害防治基于使用杀菌剂。然而，杀菌剂抗性的发展阻碍了这种防治方法的成功。在希腊进行的杀菌剂敏感性监测研究表明，存在同时对不同化学上无关的化合物（多药耐药，MDR）表现出耐药的病原菌菌株。这项研究的主要目的是检验这样一个假设，即 MDR 表型与外排转运蛋白基因的过度表达有关，并确定 MDR 分离株的适应性。监测研究（n=264）和对 9 种不同化合物 EC 值的敏感性测量表明，近 5%的人群为 MDR 型。在所选择的 MDR 分离株中，氟吡菌胺和吡唑醚菌酯的最高耐药因子被计算出来，而相同的分离株对环丙嘧啶、硫菌灵和氟环唑有中度耐药性。在耐药菌株中，未在每种杀菌剂类别的靶基因中检测到靶位基因突变，此外，在一些分离株中，还观察到杀菌剂与药物转运蛋白调节剂维拉帕米之间的协同活性。为了直接了解耐药机制，使用 Illumina HiSeq 2500 对 2 个 MDR 和 1 个敏感分离株的转录组进行了测序，并确定了外排转运蛋白基因表达谱的差异。在将真菌菌丝暴露于氟吡菌胺之前和之后，对 2 个 MDR 和 1 个敏感分离株的转录组进行了测序，并确定了外排转运蛋白基因表达谱的差异。在 MDR 分离株中，无论是在暴露于氟吡菌胺之前还是之后，都有多个 MFS 转运蛋白基因和少数 ABC 转运蛋白基因的上调。具有最高表达水平的基因的表达结果通过 qRT-PCR 测定进行了验证。适应性成分测量表明，与敏感菌株相比，MDR 分离株的菌丝生长和致病性较低，但它们产生的分生孢子数量更多。上述数据代表了与药物外排转运蛋白过度表达相关的 P. expansum 中 MDR 的首次报道，并有助于我们了解该真菌物种中与杀菌剂抗性发展相关的机制。
