Changes in Fungal Communities in Dairy Manure during Nine Months of Composting as Determined by Denaturing Gradient Gel Electrophoresis

Composted dairy manure has strong potential for use as a potting mix amendment, but potential users are concerned about different microorganisms that may be present in the compost. The objective of this study was to determine the changing fungal communities in dairy manure during 9 months of composting by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Dairy manure was collected 10 days (considered month 0), 3, 6, and 9 months after the composting process was started. Collected manure was homogenized and then extracted using the Mobio Powersoil (R) DNA Isolation Kit. Polymerase chain reaction (PCR) was used to amplify fungal DNA. Two universal 18S rRNA fungal primer sets, GCfung/NS1 and FR1/NS1, were used to detect fungal DNA and mitigate the effects of primer bias. PCR products were loaded onto denaturing gradient gels that contained a urea and formamide gradient from 18 to 40%. After electrophoresis, distinct DNA bands were excised, and reclaimed DNA was amplified again by PCR and submitted to a commercial sequencing company. Due to the conserved nature of the 18S rRNA region used for DNA detection, sequence identifications were made only to the taxonomic level of order or higher. Using both sets of fungal primers, eight different orders and two classes of fungi were revealed during the 9 months of composting. The fungal populations were dominated by members of the phylum Ascomycota, particularly the class Sordariomycetes, during composting. At time 0, 55% of the identified DNA sequences belonged within the phylum Ascomycota. The Ascomycota were at their highest levels in the 6 month samples and comprised over 83% of the sample sequences. Several non-fungal groups were also identified, including the order Astigmata, which contains mites, and several clades of protists, including the Alveolata, Amoebozoa and Cercozoa. This study has shown that Ascomycota, particularly the class Sordariomycetes, and various clades of protists were the main eukaryotic organisms present during composting of dairy manure.