Unveiling the pathogenicity of Cunninghamella echinulata SMH-1 and its bioactive metabolites through metabolomics: Dual-action disruption of Solenopsis invicta mitochondrial bioenergetics and cuticular integrity

Abstract The invasive red imported fire ant, Solenopsis invicta, poses significant ecological and economic challenges, necessitating sustainable alternatives to conventional pest control. This study evaluates the biocontrol potential of Cunninghamella echinulata isolate SMH-1, an entomopathogenic fungus, through integrated morphological, biochemical, and metabolomic analyses. Light and scanning electron microscopy elucidated infection dynamics, revealing conidial germination, cuticular penetration, and systemic hyphal proliferation, culminating in complete cadaver colonization. At 1×10⁸ conidia/mL, SMH-1 achieved 100% mortality in S. invicta workers within 7 days, with virulence maintained across thermal gradients (10–30°C). Acetone extracts of SMH- 1 induced dose-dependent lethality, reaching 100% mortality at 1,000 mg/L. Nineteen significantly differentiated metabolites were identified from phospholipids category through UPLC/MS analysis of isolate SMH-1. The derived compounds also showed strong potential against S. invicta, among which dihydrocoumarin caused 100% mortality over 12-days of exposure. The bioactivity of isolate SMH-1 (Conidia), (extracts) and (dihydrocoumarin) against S. invicta revealed significantly differentiated metabolites as (Sulfolithocholylglycine, PE(18_1(9Z)_0_0), CE(12:0), LysoPI(18:1(9Z)/0:0) and Hydroxydestruxin B), (Tropine, lignoceric acid, 9-deoxy-9-methylene-16,16-dimethyl-PGE2, Flecainide and Jubanine B), and (3-O-alpha-D-Glucopyranuronosyl-D-xylose, Rilmakalim, 2-Amino-3-methyl-1-butanol, 2,4-Dimethyl-1,3-oxazole-5-carboxylic acid and Caffeinol), respectively. Citrate cycle (TCA) was significantly impacted by differential expression of pyruvate, pyruvic acid, succinic acid and fumaric acid. These findings underscore SMH-1’s multifaceted biocontrol potential, combining direct pathogenicity, thermo-tolerance, and metabolite-mediated toxicity. By delineating host-pathogen interactions and metabolic disruptions, this work advances C. echinulata isolate SMH-1 as a sustainable candidate for integrated S. invicta management, offering an eco-friendly alternative to synthetic insecticides. Author Summary Invasive fire ants (S. invicta) threaten ecosystems and agriculture worldwide, and current chemical controls often harm the environment. In this study, we explored a natural alternative: a soil-dwelling fungus called Cunninghamella echinulata (strain SMH-1). We discovered that this fungus can kill fire ants with remarkable efficiency. When applied directly, its spores caused 100% ant mortality within a week, even under varying temperatures. We also identified key fungal compounds, including dihydrocoumarin, which are lethal to ants. By studying how the fungus invades and disrupts the ants’ biology-such as breaking down their protective outer layers and interfering with their energy production, we revealed why SMH-1 is so effective. Our findings highlight SMH-1’s potential as a safe, eco-friendly biocontrol agent. This fungus could reduce reliance on synthetic pesticides, offering farmers and communities a sustainable tool to combat fire ant invasions while protecting biodiversity. Our work bridges lab discoveries and real-world pest management, paving the way for greener solutions in agriculture and ecosystem conservation. Competing Interest Statement The authors have declared no competing interest.