Abstract
The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.
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Acknowledgments
The authors would like to thank Dr. Yasunari Yamamoto (Mie Prefecture Livestock Research Institute, Mie Prefecture, Japan) for providing rumen fluid and Professor Kazuo Sakka (Graduate School of Mie University, Japan) for his instruction on real-time PCR. This study was supported by the Integrated Research for Developing Japanese-style Forage Feeding System to Increase Forage Self-Support Ratio of the Ministry of Agriculture, Forestry, and Fisheries of Japan.
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Lwin, K.O., Hayakawa, M., Ban-Tokuda, T. et al. Real-Time PCR Assays for Monitoring Anaerobic Fungal Biomass and Population Size in the Rumen. Curr Microbiol 62, 1147–1151 (2011). https://doi.org/10.1007/s00284-010-9843-7
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DOI: https://doi.org/10.1007/s00284-010-9843-7