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

2. Damage of pathogen on plants
Loss of yield
Wheat stem rust P. striiformis f.sp. Tritici Uredinia
A great economic waste

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

4. 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.

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

6. 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.

7. 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.

8. 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

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

10. 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

11. 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),
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):
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 The campaign to eradicate the common barberry in the United States. Pages in P.D. Peterson, ed., Stem Rust of Wheat: from Ancient Enemy to Modern Foe. APS Press, St. Paul, MN.