Using Taq-Man based real time PCR to detect pathogen caused plant disease Jingwen Song Environment and Plant biology.

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Using Taq-Man based real time PCR to detect pathogen caused plant disease Jingwen Song Environment and Plant biology

Damage of pathogen on plants Loss of yield Wheat stem rust P. striiformis f.sp. Tritici Uredinia A great economic waste https://www.ars.usda.gov/midwest-area/stpaul/cereal-disease-lab/docs/barberry/black-stem-rust-biology-and-threat-to-wheat-growers/

Conventional detection method Biochemical tests Conidial morphology Agar plating techniques Conventional PCR based detection Time consuming Poor sensitivity Poor specificity

Taq-Man based real time PCR 5´-3´ exonuclease activity of Taq polymerase proportional to the fluorophore released and the amount of DNA template present in the PCR. https://en.wikipedia.org/wiki/TaqMan

ITS (Internal transcribed spacer) ITS is a 156 bp region on the ribosomal RNA It has pathogen specific conserved sequences

Detection and Quantification of Sporisorium scitamineum in Sugarcane Figure 3: Sensitivity test of conventional PCR based on five-fold serial dilutions of +1 leaf gDNA. M: 100 bp DNA ladder marker; Lane 1, 500 ng/μL; Lane 2, 100 ng/μL; Lane 3, 20 ng/μL; Lane 4, 4 ng/μL; Lane 5, 0.8 ng/μL; Lane 6, positive control; Lane 7, mock control; Lane 8, blank control.

Specificity of the Taq-MAN based PCR Figure 2: Sensitivity test of conventional PCR based on eight ten-fold serial dilutions of smut pbE DNA. M: 100 bp DNA ladder marker; Lane 1, 100 pg/μL; Lane 2, 10 pg/μL; Lane 3, 1 pg/μL; Lane 4, 100 fg/μL; Lane 5, 10 fg/μL; Lane 6, 1 fg/μL; Lane 7, 100 ag/μL; Lane 8, 10 ag/μL.

Detection pathogen caused sunflower leaf blight disease Leaf blight disease of sunflower caused by Alternaria helianthi Tubaki and Nishihara is a serious threat to its successful cultivation worldwide. Alternaria leaf blight reduces the average flower size, number of seeds per plant, seed yield per plant, seed weight and percentage filling of seed In India up to 90 % yield loss and 34 % oil loss have been documented due to this disease

Method Based on target pathogen ITS to design the pathogen specific primer and probe

Highly specific More sensitive Conclusion Highly specific The assay successfully detected as low as 1.0 pg fungal genomic DNA And detecting up to 1 % infection in sunflower seed lots. More sensitive

Reference Udayashankar, A. C., Nayaka, S. C., Archana, B., Anjana, G., Niranjana, S. R., Mortensen, C. N., ... & Prakash, H. S. (2012). Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower. Archives of microbiology, 194(11), 923-932. Chavhan R L, Hinge V R, Chinchole M B, et al. Rapid, specific and sensitive molecular detection assay for Alternaria helianthi, that causes leaf blight disease in sunflower[J]. European Journal of Plant Pathology, 2015, 143(4):663-675. Su, Y., Wang, S., Guo, J., Xue, B., Xu, L., & Que, Y. (2013). A TaqMan real-time PCR assay for detection and quantification of Sporisorium scitamineum in sugarcane. The Scientific World Journal, 2013. Peterson, P.D. 2001. The campaign to eradicate the common barberry in the United States. Pages 16-50 in P.D. Peterson, ed., Stem Rust of Wheat: from Ancient Enemy to Modern Foe. APS Press, St. Paul, MN.