Mechanisms of viability loss in dehydrated Metarhizium anisopliae blastospores

The commercial use of Metarhizium anisopliae blastospores is significantly limited by their poor storage stability, mainly due to viability loss over time. Understanding the mechanisms underlying this decline is therefore essential to improve blastospore-based products. In this study, we investigated factors associated with viability loss in blastospores dried to two water activity levels, a_w = 0.1 (BS01) and a_w = 0.3 (BS03), representing more severely and moderately desiccated states, respectively. Samples were stored at 28 °C for 90 days and analyzed every 30 days using viability assays, flow cytometry, protein oxidation analysis, GC-MS, HPLC, and Raman spectroscopy. Viability declined progressively by approximately three orders of magnitude during storage, with BS01 showing a markedly faster reduction than BS03 during the first 30 days. Early storage stages were characterized by high oxidative stress, including elevated reactive oxygen species, lipid peroxidation, and impaired membrane integrity, all of which declined over time as metabolic activity decreased. In contrast, protein carbonylation increased continuously, reaching 11.02 ± 2.23 nmol/mg protein after 90 days. Metabolic analysis of BS01 revealed disrupted cellular homeostasis, including trehalose depletion and glucose accumulation, suggesting impaired metabolic capacity. Raman spectroscopy confirmed progressive molecular alterations in proteins, lipids, and polysaccharides. Together, these findings indicate that cumulative oxidative damage, particularly irreversible protein carbonylation, combined with progressive metabolic dysfunction, is a central driver of blastospore viability loss during storage.