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Molecular Identification of Pathogens
in Nursery Crops
Ainong Shi
Tennessee State University
Lilac Leaf Blight
What pathogen causes this disease?
How to analyze and identify it by molecular approaches?
Powdery Mildew Diseases
Dogwood
Lilac
Crape myrtle
• Are they caused by same pathogen?
• How to analyze and identify them by molecular approaches?
How to identify a pathogen by molecular approach?
Lilac leaf blight
for example 1
Molecular Identification of Pathogen
ITS universal primer analysis
Specific primer analysis
ITS (Internal Transcribed Spacer)
ITS1 (primer)
18S rDNA
ITS1
5.8S
rDNA
ITS2
28S rDNA
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.
ITS Universal Primer Analysis
A four-step procedure
DNA Extraction
PCR Amplification
PCR Product Sequencing
Similarity Analysis
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.
PCR Amplification
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. 1990. Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322 In: PCR
Protocols: A Guide to Methods and Applications.
PCR Product Sequence
TCCGTAGGTGAACCTGCGGAGGGATCATTACACAAATATGAAGGCGGGCTGGAACCTCTCGGGGTTACAGCCTTGCTGA
ATTATTCACCCTTGTCTTTTGCGTACTTCTTGTTTCCTTGGTGGGTTCGCCCACCACTAGGACAAACATAAACCTTTTGTAATTG
CAATCAGCGTCAGTAACAAATTAATAATTACAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAAT
GCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCAAAGGGCATG
CCTGTTCGAGCGTCATTTGTACCCTCAAGCTTTGCTTGGTGTTGGGCGTCTTGTCTCTAGCTTTGCTGGAGACTCGCCTTAAAGT
AATTGGCAGCCGGCCTACTGGTTTCGGAGCGCAGCACAAGTCGCACTCTCTATCAGCAAAGGTCTAGCATCCATTAAGCCTTTTT
TTCAACTTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
The sequence above amplified from the primer pair ITS1/ITS4.
Primer
ITS1
IST4
Sequence (5’  3’)
reversal sequence
TCCGTAGGTGAACCTGCGG
tcctccgcttattgatatgc
GCATATCAATAAGCGGAGGA
Similarity Analysis
BLAST Search
Query: 1
tccgtaggtgaacctgcggagggatcattacacaaatatgaaggcgggctggaacctctc
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 25 tccgtaggtgaacctgcggagggatcattacacaaatatgaaggcgggctggaacctctc
Query: 61 ggggttacagccttgctgaattattcacccttgtcttttgcgtacttcttgtttccttgg
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 85 ggggttacagccttgctgaattattcacccttgtcttttgcgtacttcttgtttccttgg
……………………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………………
Query: 541 ctgaacttaagcatatcaataagcggagga 570
||||||||||||||||||||||||||||||
Sbjct: 565 ctgaacttaagcatatcaataagcggagga 594
Query – from our data
Sbject – Alternaria in GenBank
60
84
120
144
570/570 (100%) identities
Analysis indicates the sequence from our data belongs to ITS
region of Alternaria.
Specific Primer Analysis
 Develop specific primers
for Alternaria genus.
 Develop specific primers
for A. alternata.
Primer design for ITS region of Alternaria
TCCGTAGGTGAACCTGCGGAGGGATCATTACACAAATATGAAGGCGGGCTGGAACCTCTC
GGGGTTACAGCCTTGCTGAATTATTCACCCTTGTCTTTTGCGTACTTCTTGTTTCCTTGG
TGGGTTCGCCCACCACTAGGACAAACATAAACCTTTTGTAATTGCAATCAGCGTCAGT
AACAAATTAATAATTACAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAA
CGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGA
ACGCACATTGCGCCCTTTGGTATTCCAAAGGGCATGCCTGTTCGAGCGTCATTTGTACCC
TCAAGCTTTGCTTGGTGTTGGGCGTCTTGTCTCTAGCTTTGCTGGAGACTCGCCTTAAAG
TAATTGGCAGCCGGCCTACTGGTTTCGGAGCGCAGCACAAGTCGCACTCTCTATCAGCAA
AGGTCTAGCATCCATTAAGCCTTTTTTTCAACTTTTGACCTCGGATCAGGTAGGGATA
CCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Primer Sequence (5’  3’)
Al-f1
cccaccactaggacaaaca
Al-r1
gcttaatggatgctagacct
Reversal sequence
aggtctagcatccattaagc
The sequence size from Al-f1 to Al-r1 is 370 bp.
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.
The results indicated one kind of Alternaria was the pathogen.
370 bp
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 99%
glucanase precursor
(659/664)
5 to 8
Specific primers for Alternaria alternata (2)
Primer
pair
GenBank
Accession
Gene name
Sequence
results
Lane
below
aa-hsp-f1 AAU87808
aa-hsp-r1
A. alternata hsp70
mRNA
100%
(237/237)
1&2
aa-his-f1
aa-his-r1
A. alternata
histone gene
100%
(356/356)
3&4
AF404640
1 2 3 4 M
1500 bp
1000 bp
600 bp
300 bp
100 bp
Conclusion for example 1
Alternaria alternata is one pathogen in
lilac leaf blight disease.
Specific primer pairs:
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
Alternaria
A. alternata
Example two
Powdery Mildew Diseases
Dogwood
Lilac
Crape myrtle
• Are they caused by same pathogen?
• How to analyze and identify them by molecular approaches?
Molecular Identification Approaches
ITS universal primer analysis
Sequence analysis
Specific primer analysis
ITS universal primer analysis
Primer pair: ITS1/ITS4
-------------------------------------------------------------Disease
Band size
Lane
---------------------------------------------------Crape myrtle powdery mildew
666bp
1
Lilac powdery mildew
645bp
2
Dogwood powdery mildew
642bp
3
-----------------------------------------------------------------------------
M 1 2
3
Sequence Analysis
Disease
Primer
pair
Sequence
length
CG%
Pathogen
Dogwood powdery
mildew
ITS1/ITS4
642 bp
54.05
Erysiphe pulchra
Lilac powdery mildew
ITS1/ITS4
645 bp
54.42
Microsphaera syringaejaponicae
Crape myrtle powdery
mildew
ITS1/ITS4
666 bp
51.05
Uncinuliella australiana
Specific Primer Analysis
I. Erysiphe pulchra of dogwood powdery mildew
M 1 2 3 4 5 6 7 8 9 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
Specific Primer Analysis
II Uncinuliella australiana of powdery mildew in crape myrtle
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M
The bands only for U. australiana (lane 2, 6, 10, and 14) in four
DNA groups:
(1) Lagerstroemia indica, (2) Uncinuliella australiana,
(3) Erysiphe Pulchra, and (4) Microsphaera syringae-japonicae.
Lane M are 100 bp ladder.
Specific Primer Analysis
III. Microsphaera syringae-japonicae of lilac powdery mildew
1 2 3 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
Summary for example 2
Eight molecular markers (specific primer pairs)
Pathogen
Primer pairs
A
B
C
D
E
F
G H
Erysiphe pulchra
1
1
1
0
0
0
0 0
Uncinuliella australiana
0
0
0
1
1
1
1 0
Microsphaera syringaejaponicae
0
0
0
0
0
0
0 1
Primer pairs:
A=EP-f6/EP-r3
B=EP-f6/EP-r4
C=EP-f6/EP-r5
D=ua-f1/ua-r3
E=ua-f1/ua-r4
F=ua-f3/ua-r3
G=ua-f3/ua-r4
H=msj-f2/msj-r1
Other diseases
Lilac Leaf Spot
Pseudocercospora bairamifera is
one pathogen in lilac leaf spot.
Identification of Botryosphaeria dothidea pathogen of
leaf blight disease in dogwood (Cornus florida)
Oak Powdery Mildew
Arthrocladiella-mougeotii
Erysiphe-cichoracearum
Erysiphe-galeopsidis
Erysiphe-gracilis
Typhulochaeta-japonica
Brasiliomyces-trina
Uncinula-mori
Blumeria-graminis
Uncinula-septata
Cystotheca-w rightii
Cystotheca-lanestris
Saw adaea-polyfida
Saw adaea-sp.
Phyllactinia-fraxini
Phyllactinia-kakicola
Leveillula-taurica
Podosphaera-longiseta
Podosphaera-tridactyla
Sphaerotheca-fusca
Sphaerotheca-aphanis
Podosphaera-leucotricha
Uncinuliella-australiana
Uncinuliella-ustraliana
Uncinula-necator
Microsphaera-pulchra
Erysiphe-aquilegiae
Oidium-neolycopersici
Erysiphe-symphoricarpi
Oak-pow dery-mildew
Erysiphe-pisi
Microsphaera-trifolii
Erysiphe-heraclei
Microsphaera-friestii
Erysiphe-cruciferarum
Microsphaera-pseudolonicerae
Oidium-heveae
Microsphaera-alphitoides
Erysiphe-elevata
Erysiphe-syringae
Microsphaera-platani
0.1
Maple Powdery Mildew
Molecular Detection of Pathogens
DNA Extraction
Specific Primer Design
PCR Amplification
Primer Test
PCR Product Sequencing
Sequence to Verify
Similarity Analysis
Pathogen Detection
Face to plant diseases
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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.
Thank you!
Thank all of
you!