Condé Thiago Oliveira, Dorigan Adriano Francis, Moreira Silvino Intra, da Silveira Patricia Ricardino, Alves Eduardo
Department of Plant Pathology, Federal University of Lavras-UFLA, Lavras, Minas Gerais, Brazil.
Department of Biology, Federal University of Lavras-UFLA, Lavras, Minas Gerais, Brazil.
Microsc Res Tech. 2025 Feb;88(2):407-415. doi: 10.1002/jemt.24710. Epub 2024 Oct 10.
Quinone outside inhibitor (QoI) fungicide resistance in Alternaria alternata populations was reported in Brazil for the first time in 2019, in São Paulo orchards, and the mutation G143A in cytochrome b (cytb) was found in resistant isolates. Our study investigated the infectious process, production of reactive oxygen species (ROS), and fungal cell death in resistant (QoI-R) and sensitive (QoI-S) A. alternata pathotype tangerine (Aapt) isolates. Morphological characterization of Aapt isolates was performed using confocal laser scanning microscopy (CLSM). Alternaria brown spot (ABS) symptoms were produced by Aapt isolates on tangelo cv. BRS Piemonte. Germination of QoI-R conidia and production of germ tubes on tangelo leaflets treated with 100 μg mL of pyraclostrobin 18 h after inoculation (hai) was observed using scanning electron microscopy (SEM). At the same time, QoI-S conidial germination was inhibited on tangelo leaflets treated with pyraclostrobin. ROS production and cell death in Aapt isolates at high fungicide concentrations were observed using CLSM. QoI-S conidia exhibited high ROS production, indicating high oxidative stress. When dyed with propidium iodate (PI), QoI-S conidia emitted red fluorescence, showing cell death and confirming their sensitive phenotype. In contrast, QoI-R conidia neither produced ROS nor exhibited red fluorescence, indicating no cell death and confirming their resistant phenotype. Therefore, our findings evidence that microscopic techniques may help characterize events during fungi-plant interactions, ROS production, cell death, and Aapt phenotypes resistant and sensitive to QoIs using fluorometric protocols.
2019年，巴西首次在圣保罗果园报道了链格孢菌种群对醌外抑制剂（QoI）类杀菌剂产生抗性的情况，并且在抗性分离株中发现了细胞色素b（cytb）中的G143A突变。我们的研究调查了抗性（QoI-R）和敏感（QoI-S）的橘生链格孢致病型（Aapt）分离株的感染过程、活性氧（ROS）的产生以及真菌细胞死亡情况。使用共聚焦激光扫描显微镜（CLSM）对Aapt分离株进行形态学表征。Aapt分离株在红橘品种BRS Piemonte上产生了链格孢褐斑（ABS）症状。接种18小时后（hai），使用扫描电子显微镜（SEM）观察了在经100μg/mL唑菌酯处理的红橘叶片上QoI-R分生孢子的萌发及芽管的产生情况。同时，在经唑菌酯处理的红橘叶片上，QoI-S分生孢子的萌发受到抑制。使用CLSM观察了高浓度杀菌剂处理下Aapt分离株中ROS的产生和细胞死亡情况。QoI-S分生孢子表现出高ROS产生，表明存在高氧化应激。用碘化丙啶（PI）染色时，QoI-S分生孢子发出红色荧光，表明细胞死亡，证实了它们的敏感表型。相比之下，QoI-R分生孢子既不产生ROS也不发出红色荧光，表明没有细胞死亡，证实了它们的抗性表型。因此，我们的研究结果证明，显微镜技术可能有助于利用荧光检测方法来表征真菌与植物相互作用过程中的事件、ROS产生、细胞死亡以及Aapt分离株对QoIs的抗性和敏感表型。
