1. Molecular Identification of Pathogens in Nursery Crops
Ainong Shi
Tennessee State University

2. Lilac Leaf Blight What pathogen causes this disease?
How to analyze and identify it by molecular approaches?

3. Powdery Mildew Diseases
Dogwood
Lilac
Crape myrtle
Are they caused by same pathogen?
How to analyze and identify them by molecular approaches?

4. Lilac leaf blight for example 1
How to identify a pathogen by molecular approach?
Lilac leaf blight
for example 1

5. Molecular Identification of Pathogen
ITS universal primer analysis
Specific primer analysis

6. ITS (Internal Transcribed Spacer)
(primer)
ITS1
18S rDNA
5.8S
rDNA
28S rDNA
ITS1
ITS2
ITS4
(primer)
ITS region of rDNA has been widely used in identifying pathogens for fungal diseases in plants.
Thousands of sequences of ITS regions from fungi have been published in GenBank.
Universal primer pairs can amplify ITS region for all fungi.

7. PCR Product Sequencing
ITS Universal Primer Analysis
A four-step procedure
DNA Extraction
PCR Amplification
PCR Product Sequencing
Similarity Analysis

8. DNA Extraction
Genomic DNA was extracted by use of the DNeasy Plant Mini Kit from Alternaria medium (mycelia and conidia) or directly from the lilac leaves of Alternaria leaf blight.

9. PCR Amplification ITS universal primers: ITS1: tccgtaggtgaacctgcgg *
ITS1/ITS4
ITS universal primers:
ITS1: tccgtaggtgaacctgcgg *
IST4: tcctccgcttattgatatgc
The primer pair ITS1/ITS4 produces a 570 bp fragment.
570 bp
* White, T. J., T. Bruns, S. Lee, and J. W. Taylor Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp In: PCR Protocols: A Guide to Methods and Applications.

10. PCR Product Sequence
TCCGTAGGTGAACCTGCGGAGGGATCATTACACAAATATGAAGGCGGGCTGGAACCTCTCGGGGTTACAGCCTTGCTGAATTATTCACCCTTGTCTTTTGCGTACTTCTTGTTTCCTTGGTGGGTTCGCCCACCACTAGGACAAACATAAACCTTTTGTAATTGCAATCAGCGTCAGTAACAAATTAATAATTACAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCAAAGGGCATGCCTGTTCGAGCGTCATTTGTACCCTCAAGCTTTGCTTGGTGTTGGGCGTCTTGTCTCTAGCTTTGCTGGAGACTCGCCTTAAAGTAATTGGCAGCCGGCCTACTGGTTTCGGAGCGCAGCACAAGTCGCACTCTCTATCAGCAAAGGTCTAGCATCCATTAAGCCTTTTTTTCAACTTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
The sequence above amplified from the primer pair ITS1/ITS4.
Primer Sequence (5’  3’) reversal sequence
ITS1 TCCGTAGGTGAACCTGCGG
IST4 tcctccgcttattgatatgc GCATATCAATAAGCGGAGGA

11. Similarity Analysis Query – from our data
BLAST Search
Query: 1 tccgtaggtgaacctgcggagggatcattacacaaatatgaaggcgggctggaacctctc 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 25 tccgtaggtgaacctgcggagggatcattacacaaatatgaaggcgggctggaacctctc 84
Query: 61 ggggttacagccttgctgaattattcacccttgtcttttgcgtacttcttgtttccttgg 120
Sbjct: 85 ggggttacagccttgctgaattattcacccttgtcttttgcgtacttcttgtttccttgg 144
……………………………………………………………………………………………………………………………………………………………
Query: 541 ctgaacttaagcatatcaataagcggagga 570
||||||||||||||||||||||||||||||
Sbjct: 565 ctgaacttaagcatatcaataagcggagga 594
Query – from our data
Sbject – Alternaria in GenBank
570/570 (100%) identities
Analysis indicates the sequence from our data belongs to ITS region of Alternaria.

12. Specific Primer Analysis
Develop specific primers for Alternaria genus.
Develop specific primers for A. alternata.

13. The sequence size from Al-f1 to Al-r1 is 370 bp.
Primer design for ITS region of Alternaria
TCCGTAGGTGAACCTGCGGAGGGATCATTACACAAATATGAAGGCGGGCTGGAACCTCTCGGGGTTACAGCCTTGCTGAATTATTCACCCTTGTCTTTTGCGTACTTCTTGTTTCCTTGGTGGGTTCGCCCACCACTAGGACAAACATAAACCTTTTGTAATTGCAATCAGCGTCAGTAACAAATTAATAATTACAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCAAAGGGCATGCCTGTTCGAGCGTCATTTGTACCCTCAAGCTTTGCTTGGTGTTGGGCGTCTTGTCTCTAGCTTTGCTGGAGACTCGCCTTAAAGTAATTGGCAGCCGGCCTACTGGTTTCGGAGCGCAGCACAAGTCGCACTCTCTATCAGCAAAGGTCTAGCATCCATTAAGCCTTTTTTTCAACTTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Primer Sequence (5’  3’) Reversal sequence
Al-f1 cccaccactaggacaaaca
Al-r1 gcttaatggatgctagacct aggtctagcatccattaagc
The sequence size from Al-f1 to Al-r1 is 370 bp.

14. Specific primer for Alternaria genus of ITS region
The size of band is 370 bp.
The band showed in all tested Alternaria samples.
Sequence data from the PCR amplified from the specific primer pair Al-f1/Al-r1 is identical to that in ITS region of Alternaria.
370 bp
The results indicated one kind of Alternaria was the pathogen.

15. Specific primers for Alternaria alternata (1)
Primer pair
GenBank Accession
Gene name
Sequence results
Lane
above
aa-endo-f1
aa-endo-r1
AY629233
A. alternata endopolygalacturonase gene
100% (408/408)
1 to 4
aa-gp-f1
aa-gp-r1
AF282319
A. alternata mixed-linked glucanase precursor
99% (659/664)
5 to 8

16. Specific primers for Alternaria alternata (2)
Primer pair
GenBank Accession
Gene name
Sequence results
Lane
below
aa-hsp-f1
aa-hsp-r1
AAU87808
A. alternata hsp70 mRNA
100% (237/237)
1 & 2
aa-his-f1
aa-his-r1
AF404640
A. alternata histone gene
100% (356/356)
3 & 4 M
1500 bp
1000 bp
600 bp
300 bp
100 bp

17. Alternaria A. alternata Alternaria alternata is one pathogen in
Conclusion for example 1
Alternaria alternata is one pathogen in
lilac leaf blight disease.
Specific primer pairs:
Alternaria
Al-f1/Al-r1
aa-gp-f1/aa-gp-r1
aa-hsp-f1/aa-hsp-r1
aa-hsp-f1/aa-hsp-r2
aa-his-f1/aa-his-r1
A. alternata

18. Powdery Mildew Diseases
Example two
Powdery Mildew Diseases
Dogwood
Lilac
Crape myrtle
Are they caused by same pathogen?
How to analyze and identify them by molecular approaches?

19. Molecular Identification Approaches
ITS universal primer analysis
Sequence analysis
Specific primer analysis

20. ITS universal primer analysis
Primer pair: ITS1/ITS4 M
Disease Band size Lane
Crape myrtle powdery mildew bp 1
Lilac powdery mildew bp 2
Dogwood powdery mildew bp 3

21. Sequence Analysis Disease Primer pair Sequence length CG% Pathogen
Dogwood powdery mildew
ITS1/ITS4
642 bp
54.05
Erysiphe pulchra
Lilac powdery mildew
645 bp
54.42
Microsphaera syringae-japonicae
Crape myrtle powdery mildew
666 bp
51.05
Uncinuliella australiana

22. Specific Primer Analysis
I. Erysiphe pulchra of dogwood powdery mildew
M M
Lane 1, 4, 7 are Microsphaera syringae-japonicae
Lane 2, 5, 8 are Erysiphe pulchra
Lane 3, 6, 9 are Uncinuliella australiana
Lane M are 100 bp ladder

23. Specific Primer Analysis
II Uncinuliella australiana of powdery mildew in crape myrtle
M M
The bands only for U. australiana (lane 2, 6, 10, and 14) in four
DNA groups:
Lagerstroemia indica, (2) Uncinuliella australiana,
(3) Erysiphe Pulchra, and (4) Microsphaera syringae-japonicae.
Lane M are 100 bp ladder.

24. Specific Primer Analysis
III. Microsphaera syringae-japonicae of lilac powdery mildew M
The band showed only for Microsphaera syringae-japonicae (lane 2) in three materials: 1. Erysiphe pulchra, 2. Microsphaera syringae-japonicae , and
3. Uncinuliella australiana. Lane M are 100 bp ladder

25. Eight molecular markers (specific primer pairs)
Summary for example 2
Eight molecular markers (specific primer pairs)
Pathogen
Primer pairs A B C D E F G H
Erysiphe pulchra 1
Uncinuliella australiana
Microsphaera syringae-japonicae
Primer pairs:
D=ua-f1/ua-r3
E=ua-f1/ua-r4
F=ua-f3/ua-r3
G=ua-f3/ua-r4
A=EP-f6/EP-r3
B=EP-f6/EP-r4
C=EP-f6/EP-r5
H=msj-f2/msj-r1

26. Other diseases

27. Lilac Leaf Spot Pseudocercospora bairamifera is
one pathogen in lilac leaf spot.

28. Identification of Botryosphaeria dothidea pathogen of
leaf blight disease in dogwood (Cornus florida)

29. Oak Powdery Mildew

30. Maple Powdery Mildew

31. Molecular Detection of Pathogens
DNA Extraction
Specific Primer Design
PCR Amplification
Primer Test
PCR Product Sequencing
Sequence to Verify
Similarity Analysis
Pathogen Detection

32. Face to plant diseases
How can we solve them?
This is our JOB!

33. ACKNOWLEDGEMENTS Tennessee State University:
Dr. Margaret Mmbaga, Dr. Sandra Reed,
Dr. Nick Gawel, Dr. Roger Sauve,
Dr. Suping Zhou, Dr. Emmanuel Nnodu,
Dr. Frank Mrema, and Dr. Mario Keri.

34. Thank you!
Thank all of you!