Specific Primer Designing for Quantitative PCR (qPCR) of Entomopathogenic Fungi Isaria fumosorosea from Soil Samples

https://doi.org/10.22146/jpti.77867

Syaiful Amri Saragih(1*), Shuhei Takemoto(2), Hiroaki Sato(3), Naoto Kamata(4)

(1) Department of Agrotechnology, Faculty of Agriculture, Universitas Muhammadiyah Sumatera Utara Jalan Kapten Mukhtar Basri No. 3, Medan, North Sumatra 20238 Indonesia
(2) The University of Tokyo Tanashi Forest, The University of Tokyo Forests, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Nishi-Tokyo, Tokyo, Japan 188-0002
(3) Department of Forest Entomology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan 305-8687
(4) The University of Tokyo Chiba Forest, The University of Tokyo Forests, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Kamogawa, Chiba, Japan 299-5503
(*) Corresponding Author

Abstract


Entomopathogenic fungi are important component for regulation of pests in ecosystem. Isaria fumosorosea, as one of the entomopathogenic fungi, was reported to successfully controlled the outbreaks of forest defoliators attacked larch (Larix kaempferi) plantation in Furano, Japan and beech (Fagus crenata) forest in Hachimantai, Japan. Instead of semi-cultured method, in this paper, a culture-independent method based on DNA using qPCR was developed for specific detection and quantification of I. fumosorosea directly from soil DNA extract using specific primer. The primer IFU5821F/IFU6061R was designed and found to be the best primer pair for I. fumosorosea. Standard soil DNA was obtained with strong relationship and good fitting with five levels (R2= 0.989, E = 0.58). I. fumosorosea could not be detected from all soil samples which was possibility caused by low density of the fungi. The qPCR was likely a rapid and specific method to detect the fungus from soil. 


Keywords


Isaria fumosorosea; soil DNA; soil samples; qPCR

Full Text:

PDF


References

Ayala-Zermeño, M.A., Reyes-Montes, M.R., Arroyo-Vázquez, E., Calderón-Ezquerro, M.C., Mier, T., Robledo-Retana, T., & Toriello, C. (2011). An Isaria fumosorosea SCAR Marker for Evaluation of Soil, Insect, and Airborne Samples. Biocontrol Science and Technology, 21(9), 1091-1102. https://doi.org/10.1080/09583157.2011.603822

Chemidlin Prévost-Bouré, N., Christen, R., Dequiedt, S., Mougel, C., Lelièvre, M., & Jolivet, C. (2011). Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR. PLoS ONE, 6(9), e24166. https://doi.org/10.1371/journal.pone.0024166

Dieffenbach, C.W., Lowe, T.M., & Dveksler, G.S. (1993). General Concepts for PCR Primer Design. Genome Research, 3, S30-S37.

Enkerli, J., Widmer, F., Gessler, C., & Keller, S. (2001). Strain-specific microsatellite markers in the entomopathogenic fungus Beauveria brongniartii. Mycological Research, 105(9), 1079–1087. https://doi.org/10.1016/S0953-7562(08)61970-X

Hu, Q., Liu, S., Yin, F., Cai, S., Zhong, G., & Ren, S. (2011). Diversity and Virulence of Soil-dwelling Fungi Isaria spp. and Paecilomyces spp. against Solenopsis invicta (Hymenoptera: Formicidae). Biocontrol Science and Technology, 21(2), 225–234. https://doi.org/10.1080/09583157.2010.515733

Kamata, N., Sato, H., & Shimazu, M. (1997). Seasonal Changes in the Infection of Pupae of the Beech Caterpillar, Quadricalcarifera punctatella (Motsch.) (Lep., Notodontidae), by Cordyceps militaris Link (Clavicipitales: Clavicipitaceae) in the Soil of the Siebold’s Beech Forest. Journal of Applied Entomology, 121, 17-21. https://doi.org/10.1111/j.1439-0418.1997.tb01364.x

Kobayasi, Y. (1981). Revision of the Genus Cordyceps and its Allies. 1. Bulletin of the National Science Museum Tokyo, 7, 1–13.

Landa, Z., Osborne, L., Lopez, F., & Eyal, J. (1994), A Bioassay for Determining Pathogenicity of Entomogenous Fungi on Whiteflies. Biological Control, 4(4), 341-350. https://doi.org/10.1006/bcon.1994.1043

Lozano-Contretas, M.G., Maldonado-Blanco, M.G., Elias-Santos, M., Gonzales-Hernandez, A., & Nava-Camberos, U. (2013). Application of Isaria fumosorosea Blastospores Produced in Liquid Culture for Control of Bemisia argentifolii on Cotton Plants. Southwestern Entomologist, 38(1), 57-66. https://doi.org/10.3958/059.038.0113

Luangsa, J.J., Nigel, L., Hywel-Jones, Leka, M., & Robert, A.S. (2005). On the Relationships of Paecilomyces sect. Isarioidea species. Mycological Research, 109(5), 581-589. https://doi.org/10.1017/s0953756205002741

Meyling, N.V., & Eilenberg, J. (2006). Occurrence and Distribution of Soil Borne Entomopathogenic Fungi within a Single Organic Agroecosystem. Agriculture, Ecosystems and Environment, 113, 336-341. https://doi.org/10.1016/j.agee.2005.10.011

Meyling, N. V., & Eilenberg, J. (2007). Ecology of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae in Temperate Agroecosystems: Potential for Conservation Biological Control. Biological Control, 43, 145–155. https://doi.org/10.1016/j.biocontrol.2007.07.007

Nielsen, C., Jensen, A.B., & Eilenberg, J. (2008). Survival of Entomophthoralean Fungi Infecting Aphids and Higher Flies during Unfavorable Conditions and Implications for Conservation Biological Control. In Ekesi, S., Maniania, N.K. (Eds.), Use of Entomopathogenic Fungi in Biological Pest Management (pp. 13–38). Research Sign Post, Kerala, India.

Roberts, D.W., & St. Leger, R.J.S. (2004). Metarhizium spp., Cosmopolitan Insect Pathogenic Fungi: Mycological Aspects. Advances in Applied Microbiology 54, 1–70. https://doi.org/ 10.1016/S0065-2164(04)54001-7

Saragih, S.A., Takemoto, S., Hisamoto, Y., Fujii, M., Sato, H., & Kamata, N. (2015). Quantitative Real-Time PCR (qPCR) - Based Tool for Detection and Quantification of Cordyceps militaris in Soil. Journal of Invertebrate Pathology 124, 70-72. https://doi.org/10.1016/j.jip.2014.11.002

Saragih, S.A., Kusumoto, D., Takemoto, S., Torii, M., & Kamata, N. (2021). Virulence of Fungi Isolated from Ambrosia Beetles to Acer amoenum Branches. Plant Disease, 105, 3087-3091. https://doi.org/10.1094/PDIS-11-20-2543-RE

Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30, 2725-2729. https://doi.org/ 10.1093/molbev/mst197

Zimmermann, G. (2007). Review on Safety of the Entomopathogenic Fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Science and Technology, 17, 553–596. https://doi.org/10.1080/09583150701309006

Zimmermann, G. (2008). The Entomopathogenic Fungi Isaria farinosa (Formerly Paecilomyces farinosus) and the Isaria fumosorosea Species Complex (Formerly Paecilomyces fumosoroseus): Biology, Ecology and Use in Biological Control. Biocontrol Science and Technology, 18(9), 865-901. https://doi.org/10.1080/09583150802471812

Zhou, J., Bruns, M.A., & Tiedje, J.M. (1996). DNA Recovery from Soils of Diverse Composition. Applied and Environmental Microbiology 62, 316. https://doi.org/ 10.1128/aem.62.2.316-322.1996



DOI: https://doi.org/10.22146/jpti.77867

Article Metrics

Abstract views : 1454 | views : 827

Refbacks

  • There are currently no refbacks.


Bookmark and Share


Copyright (c) 2023 Jurnal Perlindungan Tanaman Indonesia

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Jurnal Perlindungan Tanaman Indonesia ISSN 1410-1637 (print), ISSN 2548-4788 (online) is published by the Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, in collaboration with Indonesian Entomological Society (Perhimpunan Entomologi Indonesia, PEI) and Indonesian Phytopathological Society (Perhimpunan Fitopatologi Indonesia, PFI). The content of this website is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.  

View website statistics