Isolation, identification and determination of biocontrol potentials of antagonistic actinobacteria against fungi causing storage rot in citrus

Actinobacteria are Gram-positive bacteria that inhabit environments such as soil and water and possess the capacity to produce numerous bioactive compounds and intracellular/extracellular enzymes. Recently, numerous studies have been conducted on the potential of actinobacteria to produce antibacterial, antifungal, and other various active compounds, as well as the possibilities for using these compounds in industries such as agriculture, food, pharmaceuticals, and cosmetics. In this study, the antifungal activities of actinobacteria and their metabolites isolated from different habitats were evaluated for the control of the phytopathogens Penicillium digitatum and P. italicum, which cause post-harvest storage rot in citrus and are the causative agents of green and blue mould disease, respectively. A total of 186 actinobacteria were isolated from soil samples collected from Manisa and its surroundings. The initial antifungal activity of the isolates was tested using phytopathogenic moulds isolated from mouldy fruits and vegetables as target organisms. In primary antifungal tests, 44 of the 186 actinobacteria isolates were found to be effective, and the 10 isolates with the best activity were selected and their metabolites obtained through fermentation were used in secondary antifungal tests. The Liquid Chromatography-High-Resolution Electrospray Ionisation Mass Spectrometry (LC-HRESIMS) chromatograms and secondary antagonistic test results of the 10 isolates from which bioactive extracts were obtained through fermentation were evaluated, and isolate GA-09 was determined to have the highest activity. Following fermentation, fractionation was performed using the Kupchan liquid-liquid partitioning method, followed by subfractionation using Medium Pressure Liquid Chromatography (MPLC). The antifungal compounds were purified from the obtained subfractions using High Performance Liquid Chromatography (HPLC). The in vitro and in vivo antifungal activities of pure metabolites were tested against P. digitatum and P. italicum. The physicochemical properties (pH, Brix, browning index, and texture parameters, including firmness) of the oranges used in the in vivo tests were measured before and after incubation. The structural characterisation of the purified metabolites was determined by 1H-NMR and 13C-NMR. Six metabolites produced by GA-09 (Streptomyces pseudovenezuelae) and thought to be antifungal were identified in the study: Rutamycin A (M-1), Rutamycin B (M-2), Unknown 1 (M-3), Unknown 2 (M-4), Desferrioxamine D2 (M-5) and Desferrioxamine E (M-6). When compared to previously identified compounds in the literature, M-1 and M-2 are known antifungal compounds, while M-3 and M-4 are reported for the first time in this study as potentially new compounds. M-5 and M-6 are iron-chelating compounds known as siderophores. M-1 and M-2 are derivatives of each other and showed the highest antifungal effect in in vitro and in vivo analyses. Furthermore, it was found that the firmness value was maintained in oranges supplemented with M-1 and M-2 during a 5-day incubation period. M-3 and M-4 showed antifungal activity close to that of the positive control. The morphological, cultural characteristics and molecular identification of 16 different microorganisms were performed. Among the selected and studied Actinobacteria, 6 Streptomyces sp. (AK-01, AK-13, AK-14, DT-06, DT-10 and DT-11), 1 Amycolatopsis sp. (SP-03) and 1 Lentzea sp. (UZ-72) were identified at the genus level, and 2 species as Streptomyces netropsis (DT-02) and Streptomyces pseudovenezuelae (GA-09) were identified. The phytopathogenic moulds identified at the species level were Penicillium italicum, Penicillium digitatum, Penicillium chrysogenum, Alternaria alternata, Aspergillus niger, and Fusarium oxysporum, respectively. According to the analyses performed and the results obtained, this research has taken other similar studies in the literature to a higher level and has revealed the commercialisation potential of the actinobacteria metabolites obtained.