Development of qPCR assays to monitor the ability of Gliocladium catenulatum J1446 to reduce the cereal pathogen Fusarium graminearum inoculum in soils
A qPCR approach was developed to specifically monitor in soils Fusarium graminearum, the main agent responsible for Fusarium Head Blight, and the biocontrol agent Gliocladium catenulatum J1446 (Prestop®). For both fungi, the amplification efficacy of standard curves obtained by mixing pure fungal DNA and soil background DNA was high (qPCR efficacy>96% with R2 > 0.97) with a linear range from 10−3 ng to 10 ng/μL. Our qPCR method allowed quantifying down to 1 μg of F. graminearum and G. catenulatum J1446 mycelium per g of soil. The strong correlation observed between fungal biomass and quantified DNA (R2 = 0.9927 and 0.9356 for F. graminearum and G. catenulatum J1446, respectively) supported the use of the primers to monitor both fungi in soils. Under our experimental conditions, the ability of Prestop® to reduce F. graminearum growth was significantly higher in autoclaved soil compared to living soils, suggesting that there is an antagonistic effect of the soil microbial communities. In contrast, G. catenulatum J1446 growth was mostly not affected by the presence of F. graminearum and was able to persist in both autoclaved and living soils after 15 days of incubation. These results indicate that our qPCR approach may be used to assess the success of soil colonization by a biocontrol agent and its control efficacy by monitoring the dynamics of the BCA and the targeted pathogen in soil.
KeywordsCereal disease Fusarium graminearum Prestop® qPCR Soil Biocontrol
We gratefully thank Triskalia as project partners and the farmers for kindly giving us access to their field. We also thank the UBO Culture Collection for kindly providing us with fungal strains.
Compliance with ethical standards
Authors confirm that this study is original and has not been published previously, and is not under consideration for publication elsewhere. All authors have approved the manuscript and agree with submission to this journal. This research does not contain any conflict of interest, nor research involving humans or animals.
- Burgess, L. W., Backhouse, D., Summerell, B. A., & Swan, L. J. (2001). Crown rot of wheat. Pages 271-294 in. Fusarium: Paul E. Nelson. Memorial Symposium. American Phytopathology Society, St. Paul, MN.Google Scholar
- Davari, M., van Diepeningen, A. D., Babai-Ahari, A., Arzanlou, M., Najafzadeh, M. J., van der Lee, T. A. J., & de Hoog, G. S. (2012). Rapid identification of Fusarium graminearum species complex using rolling circle amplification (RCA). Journal of Microbiological Methods, 89, 63–70.CrossRefPubMedGoogle Scholar
- Demeke, T., Gräfenhan, T., Clear, R. M., Phan, A., Ratnayaka, I., Chapados, J., Patrick, S. K., Gaba, D., Lévesque, C. A., & Seifert, K. A. (2010). Development of a specific TaqMan® real-time PCR assay for quantification of Fusarium graminearum clade 7 and comparison of fungal biomass determined by PCR with deoxynivalenol content in wheat and barley. International Journal of Food Microbiology, 141, 45–50.CrossRefPubMedGoogle Scholar
- Fredlund, E., Gidlund, A., Olsen, M., Börjesson, T., Spliid, N. H. H., & Simonsson, M. (2008). Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels. Journal of Microbiological Methods, 73, 33–40.CrossRefPubMedGoogle Scholar
- Jiménez-Fernández, D., Montes-Borrego, M., Navas-Cortés, J. A., Jiménez-Díaz, R. M., & Landa, B. B. (2010). Identification and quantification of Fusarium oxysporum in planta and soil by means of an improved specific and quantitative PCR assay. Applied Soil Ecology, 46, 372–382.CrossRefGoogle Scholar
- Kulik, T., Ostrowska, A., Buśko, M., Pasquali, M., Beyer, M., Stenglein, S., Załuski, D., Sawicki, J., Treder, K., & Perkowski, J. (2015). Development of an FgMito assay: A highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto. International Journal of Food Microbiology, 210, 16–23.CrossRefPubMedGoogle Scholar
- Kumar, A., Karre, S., Dhokane, D., Kage, U., Hukkeri, S., & Kushalappa, A. C. (2015). Real-time quantitative PCR based method for the quantification of fungal biomass to discriminate quantitative resistance in barley and wheat genotypes to fusarium head blight. Journal of Cereal Science, 64, 16–22.CrossRefGoogle Scholar
- Lv, X.-C., Li, Y., Qiu, W.-W., Wu, X.-Q., Xu, B.-X., Liang, Y.-T., Liu, B., Chen, S. J., Rao, P. F., & Ni, L. (2016). Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine. Food Control, 66, 69–78.CrossRefGoogle Scholar
- Ratti, C., Budge, G., Ward, L., Clover, G., Rubies-Autonell, C., & Henry, C. (2004). Detection and relative quantitation of soil-borne cereal mosaic virus (SBCMV) and Polymyxa graminis in winter wheat using real-time PCR (TaqMan®). Journal of Virological Methods, 122, 95–103.CrossRefPubMedGoogle Scholar
- Wagner, A. O., Praeg, N., Reitschuler, C., & Illmer, P. (2015). Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil. Applied Soil Ecology, 93, 56–64.CrossRefPubMedPubMedCentralGoogle Scholar
- Wegulo, S. N., Bockus, W. W., Nopsa, J. F. H., Peiris, K. H. S. & Dowell, F. E. (2013). Integration of fungicide application and cultivar resistance to manage Fusarium Head Blight in wheat. In M. Nita (Ed.), Fungicides-showcases of integrated plant disease management from around the world. InTech.Google Scholar
- Woodhall, J. W., Adams, I. P., Peters, J. C., Harper, G., & Boonham, N. (2013). A new quantitative real-time PCR assay for Rhizoctonia solani AG3-PT and the detection of AGs of Rhizoctonia solani associated with potato in soil and tuber samples in great Britain. European Journal of Plant Pathology, 136, 273–280.CrossRefGoogle Scholar