MALDI mass spectrometry-based identification of antifungal molecules from endophytic Bacillus strains with biocontrol potential of Lasiodiplodia theobromae, a grapevine trunk pathogen in Peru

Abstract

Lasiodiplodia theobromae, 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 Bacillus velezensis M1 and Bacillus amyloliquefaciens M2 grapevine endophytic strains. Solid medium antagonism assays were performed confronting B. velezensis M1 - L. theobromae and B. amyloliquefaciens M2 - L. theobromae 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 m/z) and mycobacillin (1529.6 m/z) 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 B. velezensis M1 - L. theobromae. 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.

Keywords: Antifungal proteins; Endophytic bacteria; Grapevine; Lasiodiplodia theobromae; Lipopeptides; MALDI imaging mass spectrometry.

Graphical abstract

Fig. 1

Three days-old dual culture showing interactions between a) B. velezensis M1 - L. theobromae, b) B. amyloliquefaciens M2 - L. theobromae, c) B. cenocepacia - L. theobromae; d) L. theobromae control. Microorganisms were isolated from grapevine branches tissues with Botryosphaeria dieback-symptoms (L. theobromae) and asymptomatic (endophytic bacteria).

Fig. 2

MALDI TOF MS mass spectra of bacterial lipopeptides from dual culture. Masses ranges 1400–1560 (m/z) for fengycin and 1040–1110 (m/z) for iturin were proposed. a) Fengycin peaks with different intensity found in B. velezensis M1 - L theobromae and B. amyloliquefaciens M2 - L. theobromae confrontations. The fengycin isoforms protonated with H⁺ adducts [m+H⁺] were 1449.8 m/z Fgy-C₁₅, 1463.8 m/z Fgy-C₁₆ and 1477.8 m/z Fgy-C₁₇ (blue arrows), while 1487.7, 1501.8 and 1515.8 correspond to the same isoforms but protonated with K⁺ adducts [m+K⁺] (red arrows). b) Iturin peaks with different intensity found in B. velezensis M1 - L. theobromae confrontation. The iturin isoforms protonated with H⁺ adducts [m+H⁺] were 1043.5 m/z Itu-C₁₄ and 1057.5 m/z Itu-C₁₅ (blue arrows), while 1081.4 and 1095.5 correspond to the same isoforms but protonated with K⁺ adducts [m+K⁺] (red arrows). In addition, 1529.8 m/z and 1071.5 m/z peaks of mycobacillin and mycosubtilin respectively (green arrows) were found in M1 and M2 endophytic strain.

Fig. 3

MALDI Imaging MS from solid medium assays of B. velezensis M1 and B. amyloliquefaciens M2 against L. theobromae. The confrontations show the expression and spatial distribution of antifungal lipopeptides in interaction zones. Antifungal lipopeptide signals are interpreted on the color scale as relative intensity. a) Spatial distribution of mycobacillin 1529.6 m/z [higher signal of yellow dots] present in B. velezensis M1 - L. theobromae interaction (left); spatial distribution of fengycin 1463.9 m/z [higher signal of yellow dots] present in B. velezensis M1 - L. theobromae interaction (center); spatial distribution of mycobacillin 1529.6 m/z [higher signal of yellow dots] present in B. amyloliquefaciens M2 - L. theobromae interaction (right). b) Spatial distribution of same lipopeptides for M1 and M2 strains but without interaction with the pathogen (yellow signal decreased).

Fig. 4

Lipopeptides genes were confirmed by PCR. The B. velezensis M1 exhibited all lipopeptides fenD, srfAA, bmyB, ituC and bacA followed by B. amyloliquefaciens M2 (except ituC) and none for B. cenocepacia A1. MW (molecular weight marker), C+ (positive control) and C- (negative control).

Fig. 5

MALDI TOF MS/MS mass spectra of different peptide sequences from oxalate decarboxylase bacisubin protein. a) the masses of different precursor ions 1513.79, 1572.75 and 2309.06 m/z that correspond to peptide sequences (in red) of bacisubin protein are shown. b) the spectrum exhibits y⁺ fragments for sequence 2309.06 m/z (LKDDIVEGPNGEVPYPFTYR).