Bacterial volatile organic compounds induce adverse ultrastructural changes and DNA damage to the sugarcane pathogenic fungus Thielaviopsis ethacetica

Abstract

Due to an increasing demand for sustainable agricultural practices, the adoption of microbial volatile organic compounds (VOCs) as antagonists against phytopathogens has emerged as an eco-friendly alternative to the use of agrochemicals. Here, we identified three Pseudomonas strains that were able to inhibit, in vitro, up to 80% of mycelial growth of the phytopathogenic fungus Thielaviopsis ethacetica, the causal agent of pineapple sett rot disease in sugarcane. Using GC/MS, we found that these bacteria produced 62 different VOCs, and further functional validation revealed compounds with high antagonistic activity to T. ethacetica. Transcriptomic analysis of the fungal response to VOCs indicated that these metabolites downregulated genes related to fungal central metabolism, such as those involved in carbohydrate metabolism. Interestingly, genes related to the DNA damage response were upregulated, and micro-FTIR analysis corroborated our hypothesis that VOCs triggered DNA damage. Electron microscopy analysis showed critical morphological changes in mycelia treated with VOCs. Altogether, these results indicated that VOCs hampered fungal growth and could lead to cell death. This study represents the first demonstration of the molecular mechanisms involved in the antagonism of sugarcane phytopathogens by VOCs and reinforces that VOCs can be a sustainable alternative for use in phytopathogen biocontrol.

The power of the smallest: The inhibitory activity of microbial volatile organic compounds against phytopathogens

“…The same study reported that the transverse and longitudinal septae completely disappeared in hyphae of Alternaria alternata, while conidiophores became vegetative and stunted in Cladosporium oxysporum (Chaurasia et al, 2005). Yet, other structural effects such as cell wall granulation, reduced number of mitochondria, destruction of organelles and internal cell darkening can be caused by VOCs, as it was observed in T. ethacetica (Freitas et al, 2022).…”

“…( Ye et al, 2020 ). An increase in vacuolization processes (i.e., enlargement of vacuoles in mycelia, conidiophores or hyphae) has also been observed in some fungi inhibited by microbial VOCs or the respective synthetic compounds ( Chaurasia et al, 2005 ; Tenorio-Salgado et al, 2013 ; Giorgio et al, 2015 ; Medina-Romero et al, 2017 ; Huang et al, 2018 ; Freitas et al, 2022 ). According to Macías-Rubalcava et al (2018) , these changes can compromise the respiration of microorganisms and affect cell membrane permeability, as was observed in the endophytic fungus Hypoxylon anthochroum ( Macías-Rubalcava et al, 2018 ).…”

“…Other effects on membranes were also observed when fungi were exposed to VOCs, such as strongly retracted plasma membrane in F . oxysporum, S. sclerotiorum ( Wu et al, 2015 ) and Thielaviopsis ethacetica ( Freitas et al, 2022 ), congregated and browned protoplasms in S. sclerotiorum ( Liu et al, 2008 ), abnormal morphology of appressoria, an alteration that could affect the pathogen infection ability, in Peronophythora litchi ( Xing et al, 2018 ) and M. fructicola ( Zhou et al, 2019 ). Additionally, it was also shown that the intracellular components in M. fructicola were destroyed and an empty shell was formed, corroborating the idea that VOCs can destroy the barrier function of the cell wall and cell membrane ( Zhou et al, 2019 ).…”

“…digitatum presented phosphatidylserine externalization and DNA fragmentation after treated with the isooctanol. Recently, Freitas et al (2022) showed that VOCs from Pseudomonas strains also caused DNA damage on the sugarcane pathogenic fungus Thielaviopsis ethacetica . Diverse genes involved in DNA damage response were upregulated, in comparison with phytopathogen cultivated in the absence of VOCs, and, interestingly, DNA cleavage and chromatin fragmentation were indicated via Fourier-transform infrared microspectroscopy, corroborating with the transcriptomic analysis.…”

“…For instance, it was showed that VOCs from Pseudomonas sp. caused DNA damaged to the sugarcane pathogen Thielaviopsis ethacetica ( Freitas et al, 2022 ). Structural damage on hyphae and down-regulation in the expression of virulence factors in Ralstonia solanacearum are other mechanisms by which VOCs act ( Raza et al, 2016a , b , c ).…”