Although the influence of environmental factors on the microbial community in water sources is crucial, it is seldom evaluated. The seasonal relationship between microbial diversity of bacteria and fungi and environmental factors was investigated in a large drinking-water reservoir using Illumina MiSeq sequencing. Forty-one bacterial phyla and nine fungal phyla were analyzed in the Qingcaosha Reservoir, Shanghai, China. The predominant bacterial phyla were Actinobacteria, Proteobacteria, Bacteroidetes, and Cyanobacteria, with the maximum relative abundance of 46%, 36.6%, 16.1%, and 14.9%, respectively. Actinobacteria were observed to be the predominant bacterial phylum during spring and summer. The maximum relative abundance of unclassified fungi appeared in summer (98.8%), which was higher than that of Ascomycota and Basidiomycota (11.7% and 8.2%, respectively). Principal coordinate analysis (PCoA) results showed that the structural similarity in the bacterial community was greater during summer and winter; however, the fungal community exhibited a greater similarity during spring and summer. 2-Methylisoborneol (2-MIB), an olfactory compound produced by microorganisms, was detected at a concentration of 8.97 ng/L during summer, which was slightly lower than the olfactory threshold (10 ng/L). The positive correlation between Actinobacteria and unclassified fungi and 2-MIB (p < 0.05) confirmed that Actinobacteria and unclassified fungi produced 2-MIB. The chemical oxygen demand (COD) was 1.48–1.94 mg/L, and the maximum concentrations of total nitrogen (TN) and total phosphorus (TP) were 2.1 mg/L and 0.5 mg/L, respectively. Chloroflexi were negatively correlated with COD (p < 0.05) but positively correlated with TP (p < 0.01). Nitrospirae were negatively correlated with COD (p < 0.05), but positively correlated with TN (p < 0.05). Among the classified fungi, Rozellomycota, Basidiomycota (p < 0.05), and Chytridiomycota (p < 0.01) were positively correlated with TP. Therefore, the relative abundance of predominant bacteria was affected by various environmental factors; however, fungi were mainly influenced by TP.
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