Saucedo-Bazalar Manuel, Masias Pedro, Nouchi-Moromizato Estefanía, Santos César, Mialhe Eric, Cedeño Virna
Departamento de Biología y Bioquímica, Universidad Nacional de Tumbes, Av. Universitaria s/n, Pampa Grande, Tumbes, Peru.
Programa de Maestría en Biotecnología Molecular, Escuela de Posgrado, Universidad Nacional de Tumbes, Av. Universitaria s/n, Pampa Grande, Tumbes, Peru.
Curr Res Microb Sci. 2023 Sep 9;5:100201. doi: 10.1016/j.crmicr.2023.100201. eCollection 2023.
a grapevine trunk pathogen, is becoming a significant threat to vineyards worldwide. In Peru, it is responsible for Botryosphaeria dieback in many grapevine-growing areas and it has spread rapidly due to its high transmissibility; hence, control measures are urgent. It is known that some endophytic bacteria are strong inhibitors of phytopathogens because they produce a wide range of antimicrobial molecules. However, studies of antimicrobial features from endophytic bacteria are limited to traditional confrontation methods. In this study, a MALDI mass spectrometry-based approach was performed to identify and characterize the antifungal molecules from M1 and M2 grapevine endophytic strains. Solid medium antagonism assays were performed confronting M1 - and M2 - for antifungal lipopeptides identification. By a MALDI TOF MS it was possible identify mass spectra for fengycin, iturin and surfactin protoned isoforms. Masses spectrums for mycobacillin and mycosubtilin were also identified. Using MALDI Imaging MS we were able to visualize and relate lipopeptides mass spectra of fengycin (1463.9 ) and mycobacillin (1529.6 ) in the interaction zone during confrontations. The presence of lipopeptides-synthesis genes was confirmed by PCR. Liquid medium antagonism assays were performed for a proteomic analysis during the confrontation of M1 - . Different peptide sequences corresponding to many antifungal proteins and enzymes were identified by MALDI TOF MS/MS. Oxalate decarboxylase bacisubin and flagellin, reported as antifungal proteins, were identified at 99 % identity through peptide mapping. MALDI mass spectrometry-based identification of antifungal molecules would allow the early selection of endophytic bacteria with antifungal features. This omics tool could lead to measures for prevention of grapevine diseases and other economically important crops in Peru.
一种葡萄树干病原体正成为全球葡萄园的重大威胁。在秘鲁,它是许多葡萄种植区葡萄座腔菌属溃疡病的病因,且因其高传播性而迅速蔓延;因此,控制措施刻不容缓。已知一些内生细菌是植物病原体的强力抑制剂,因为它们能产生多种抗菌分子。然而,关于内生细菌抗菌特性的研究仅限于传统的对峙方法。在本研究中,采用基于基质辅助激光解吸电离质谱(MALDI)的方法来鉴定和表征M1和M2葡萄内生菌株的抗真菌分子。进行固体培养基拮抗试验,使M1和M2对峙以鉴定抗真菌脂肽。通过MALDI-TOF MS能够鉴定丰原素、伊枯草菌素和表面活性素质子化异构体的质谱。还鉴定了分枝杆菌素和真菌枯草菌素的质谱。使用MALDI成像质谱,我们能够在对峙过程中的相互作用区域可视化并关联丰原素(1463.9)和分枝杆菌素(1529.6)的脂肽质谱。通过PCR确认了脂肽合成基因的存在。进行液体培养基拮抗试验以在M1对峙期间进行蛋白质组分析。通过MALDI-TOF MS/MS鉴定了对应于许多抗真菌蛋白和酶的不同肽序列。通过肽图谱分析,以99%的同一性鉴定出了作为抗真菌蛋白报道的草酸脱羧酶芽孢菌素和鞭毛蛋白。基于MALDI质谱的抗真菌分子鉴定将有助于早期筛选具有抗真菌特性的内生细菌。这种组学工具可能会带来预防秘鲁葡萄病害和其他经济上重要作物病害的措施。