Improvement and application of qPCR assay revealed new insight on early warning of Phaeocystis globosa bloom

Phaeocystis globosa bloom develops from its early solitary cells, providing clues for early warning of its bloom and timely responding to possible consequences. However, the early prediction requires quantification of the solitary cells for a thorough understanding of bloom formation. Therefore, we developed an accurate, sensitive, and specific qPCR assay for this need. Results show that the accuracy of qPCR was significantly enhanced by ameliorating DNA barcode design, improving genomic DNA extraction, and introducing a strategy of internal amplification control (IAC). This approach reached a quantification limit of 1 cell/reaction, making low-abundance cells (10¹-10³ cells/L) detection possible, and we also observed a plunge in the abundance of the solitary cells before the bloom outbreak in two winters in 2019 and 2020 for the first time, which is quite unique from laboratory results showing an increase instead. The plunge in solitary-cell abundance might be associated with the attachment of solitary cells to solid matrices to form non-solitary attached aggregate, the precursor of colonies, which gains supports from other studies and needs more investigations in the future. Therefore, as the plunge in solitary-cell abundance is a sign of colony formation, it can be used as an early warning indicator to P. globosa bloom.