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Detection of cabbage yellows fungus Fusarium oxysporum f. sp. conglutinans in soil by PCR and real-time PCR

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Abstract

For the specific detection of Fusarium oxysporum f. sp. conglutinans (Foc), we designed a primer set for PCR and a primer–probe set for real-time PCR based on its endopolygalacturonase gene sequence. Using the primer set, we could distinguish Foc from other pathogenic forms and nonpathogenic isolates of F. oxysporum. Moreover, we detected the fungus by real-time PCR using DNA isolated from soil. Sensitivity of real-time PCR was improved 10- to 10,000-fold by adding bovine serum albumin or by performing pre-PCR. Our method can detect Foc in the soil at a density that causes only slight yellows symptom.

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Acknowledgments

We thank Dr. M. Fujinaga (Nagano Vegetable and Ornamental Crops Experiment Station), Mr. H. Ogiso (Saku Branch, Nagano Vegetable and Ornamental Crops Experiment Station) and Dr. M. Shimosaka (Shinshu University) for providing fungal isolates. This study was partly supported by the eDNA Project (Development of Soil Diversity Analysis System with Environmental DNA) of the Ministry of Agriculture, Forestry, and Fisheries (MAFF), and Grants-in-Aid from The Japan Society for the Promotion of Sciences (JSPS) for TA and TK.

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Author notes

  1. Takeshi Kashiwa

    Present address: Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science (CSRS), Wako, Saitama, 351-0198, Japan

  2. Keigo Inami

    Present address: Research Department 1, Central Research Division, Bridgestone Corporation, Kodaira, Tokyo, 187-8531, Japan

Authors and Affiliations

  1. United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, 183-8509, Japan

    Takeshi Kashiwa

  2. Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, 183-8509, Japan

    Takeshi Kashiwa, Keigo Inami, Tohru Teraoka, Ken Komatsu & Tsutomu Arie

Authors
  1. Takeshi Kashiwa
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  2. Keigo Inami
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  3. Tohru Teraoka
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  4. Ken Komatsu
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  5. Tsutomu Arie
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Corresponding author

Correspondence to Tsutomu Arie.

Electronic supplementary material

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10327_2016_668_MOESM1_ESM.tiff

Phylogenic tree of representative Fusarium oxysporum isolates. Neighbor-joining (NJ) tree based on the pg1 sequence from 23 F. oxysporum isolates. Phylogenic analysis was done as in our previous study (Kashiwa et al. 2013). Numbers on nodes represent bootstrap values estimated from 1000 replications of the data set. The bar indicates a phylogenetic distance of 0.5%. All 8 isolates of f. sp. conglutinans formed one distinctive cluster. Supplementary material 1 (TIFF 14203 kb)

10327_2016_668_MOESM2_ESM.tiff

Primers and probe on pg1 sequence. Alignment of pg1 sequence from 10 F. oxysporum isolates. 1, f. sp. conglutinans Cong:1-1; 2, f. sp. lycopersici race 1 MAFF 305121; 3, f. sp. lycopersici race 2 JCM 12575; 4, f. sp. lycopersici race 3 Chz1-A; 5, f. sp. niveum MAFF 305608; 6, f. sp. cucumerinum Rif-1; 7, f. sp. raphani MAFF 240328; 8, f. sp. radicis-lycopersici MAFF 103044; 9, f. sp. melonis NRRL 26406; 10, f. sp. melongenae MAFF 103051. Primer sets PG1congF/PG1congR (orange) were designed for PCR. Primer–probe set Cong_PG1_F/Cong_PG1_R/Cong_PG1_Probe (blue) were designed for real-time PCR. Primers PG1congF and Cong_PG1_R were used in the pre-PCR step. Red nucleotides represent single nucleotide polymorphisms unique to the sequence of f. sp. conglutinans. Supplementary material 2 (TIFF 18953 kb)

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Kashiwa, T., Inami, K., Teraoka, T. et al. Detection of cabbage yellows fungus Fusarium oxysporum f. sp. conglutinans in soil by PCR and real-time PCR. J Gen Plant Pathol 82, 240–247 (2016). https://doi.org/10.1007/s10327-016-0668-5

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  • DOI: https://doi.org/10.1007/s10327-016-0668-5

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