How can long-term cold storage affect the content of glomalin-related soil proteins from an arbuscular mycorrhizal inoculum?

It has been shown that cold long-term storage of Entrophospora etunicata inoculum may decrease the concentration of spore proteins. However, no studies have been carried out to understand how long-term storage under cold conditions may interfere with glomalin-related soil proteins (GRSP) concentration, thermostable glycoproteins released by arbuscular mycorrhizal fungi (AMF). To elucidate how this shelf-life factor may modulate GRSP concentration, the same isolate of E. etunicata was cultivated in 2000 (Ee2000) and 2023 (Ee2023) and stored since then under cold conditions (4-6ºC). The easily extractable fraction of GRSP (EE-GRSP) was extracted from these inocula using 0.25g of soil-inoculum, equivalent weight for 50 spores in the soil-inoculum, and a spore suspension with 50 spores. The results were expressed as the concentration in µg for each approach evaluated and as the Carbon of GRSP (C-GRSP). In the treatment of equivalent weight for 50 spores, the EE-GRSP and C-GRSP concentrations were reduced by 35% in Ee2000 in comparison to Ee2023, indicating that such a reduced concentration of GRSP and C-GRSP may occur in stored inocula at a slow rate due to cold storage. A slight decrease (9.5%) in EE-GRSP and C-GRSP was observed in Ee2000 soil-inoculum from 0.25g treatment, and no difference in protein concentration was shown in the spore suspension. It is concluded that E. etunicata inoculum has its EE-GRSP and C-GRSP negatively affected by long-term storage in cold conditions. This is the first study to report the behaviour of GRSP in cold long-term stored AMF inoculum.