Identification of Fungicide Combinations for Overcoming Plasmopara viticola and Botrytis cinerea Fungicide Resistance

Abstract

Fungal diseases, including downy mildew (caused by Plasmopara viticola) and gray mold (caused by Botrytis cinerea), significantly impact the marketable yield of grapes produced worldwide. Cytochrome b of the mitochondrial respiratory chain of these two fungi is a key target for Quinone outside inhibitor (QoI)-based fungicide development. Since the mode of action (MOA) of QoI fungicides is restricted to a single site, the extensive usage of these fungicides has resulted in fungicide resistance. The use of fungicide combinations with multiple targets is an effective way to counter and slow down the development of fungicide resistance. Due to the high cost of in planta trials, in silico techniques can be used for the rapid screening of potential fungicides. In this study, a combination of in silico simulations that include Schrödinger Glide docking, molecular dynamics, and Molecular Mechanism-Generalized Born Surface Area calculation were used to screen the most potent QoI and non-QoI-based fungicide combinations to wild-type, G143A-mutated, F129L-mutated, and double-mutated versions that had both G143A and F129L mutations of fungal cytochrome b. In silico docking studies indicated that mandestrobin, famoxadone, captan, and thiram have a high affinity toward WT cytochrome b of Botrytis cinerea. Although the QoIs mandestrobin and famoxadone were effective for WT based on in vitro results, they were not broadly effective against G143A-mutated isolates. Famoxadone was only effective against one isolate with G143A-mutated cytochrome b. The non-QoI fungicides thiram and captan were effective against both WT and isolates with G143A-mutated cytochrome b. Follow-up in silico docking and molecular dynamics studies suggested that fungicide combinations consisting of famoxadone, mandestrobin, fenamidone, and thiram should be considered in field testing targeting Plasmopara viticola and Botrytis cinerea fungicide resistance.

Transcriptome Analysis of mfs2-Defective Penicillium digitatum Mutant to Reveal Importance of Pdmfs2 in Developing Fungal Prochloraz Resistance

“…Specially, the down-regulation of lipid metabolismrelating genes in the comparison NI-(Pd-d/Pd-wt), including fatty acyl-CoA oxidase-encoding gene and carnitine acetyl transferase-encoding gene (Table 2), was also reported in Pisolithusmicrocarpus and Beauveria bassiana [76,77]. And the down-regulation of energy metabolismrelating genes in the comparison NI-(Pd-d/Pd-wt), including cytochrome b-encoding gene and ATP synthase-encoding gene (Table 2), was also reported in Botrytis cinerea and Corynespora cassiicola [55,78]. Thus, even in the absence of fungicide, there has been an association of Pdmfs2 with multiple metabolisms required in the fungi adaptation to their growth environments, and such an adaptation might be one aspect of the physiological basis to develop fungicide resistance.…”

“…According to studies in the past years, more and more evidence has emerged to support comprehensive metabolism backgrounds underlying fungicide resistance. Such backgrounds include various ERG-encoding genes in fungal ergosterol biosynthesis pathways [36,41,42], acetyl-CoA carboxylase (ACCase)-encoding genes in the lipid and fatty acid oxidation path-ways [45][46][47], reactive oxygen species (ROS)-metabolizing enzyme-encoding genes in the cell wall maintenance and oxidative-stress-responsive processes [50,51], mitochondrial respiratory chain protein-encoding genes in the cellular energy metabolisms [53][54][55], ubiquitin-encoding genes in the post-translational modification processes [57][58][59], and protein kinase-encoding genes involved in mitogen-activated signal transductions [55,60,61]. In the present study, RNA-seq analysis revealed that Pdmfs2 knockout led to the down-regulation of genes involved in peroxisome (ko04146) and oxidative phosphorylation (ko00190) at no prochloraz treatment (Tables 1 and 2, and Figures 1 and 2).…”

“…These metabolisms and cellular stimuli processes in response to specific fungicide(s) have been studied, including ergosterol biosynthesis pathways, lipid and fatty acid oxidation pathways, cell wall maintenance, oxidative-stress-responsive processes, carbohydrate and amino acid metabolisms, cellular energy metabolisms, post-translational modification processes, and signal transduction pathways. All these pathways are highly dependent on many stress-responsive genes, including various ERG-encoding genes (such as erg1, erg3, erg11, erg24, erg25 and so on) [36,[40][41][42], acetyl-CoA carboxylase (ACCase)-encoding genes [43][44][45][46][47], reactive oxygen species (ROS)-metabolizing enzyme-encoding genes [48][49][50][51], mitochondrial respiratory chain protein-encoding genes [52][53][54][55], ubiquitin-encoding genes [56][57][58][59], and a series of protein kinase-encoding genes involved in mitogen-activated signal transductions [55,60,61]. As reported, MFS and ABC transporters played multiple roles in the transport of a diverse range of metabolic substrates and intermediates [62,63].…”