molecular identification of pathogens in nursery crops ainong shi tennessee state university
TRANSCRIPT
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)
18S rDNA ITS15.8SrDNA
28S rDNAITS2
ITS1
ITS4(primer)
(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.
A four-step procedure
PCR Product Sequencing
ITS Universal Primer Analysis
Similarity Analysis
PCR Amplification
DNA Extraction
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.
ITS universal primers:
PCR Amplification
570 bp
ITS1: tccgtaggtgaacctgcgg *IST4: tcctccgcttattgatatgc
The primer pair ITS1/ITS4 produces a 570 bp fragment.
ITS1/ITS4
* 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
TCCGTAGGTGAACCTGCGGAGGGATCATTACACAAATATGAAGGCGGGCTGGAACCTCTCGGGGTTACAGCCTTGCTGAATTATTCACCCTTGTCTTTTGCGTACTTCTTGTTTCCTTGGTGGGTTCGCCCACCACTAGGACAAACATAAACCTTTTGTAATTGCAATCAGCGTCAGTAACAAATTAATAATTACAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCAAAGGGCATGCCTGTTCGAGCGTCATTTGTACCCTCAAGCTTTGCTTGGTGTTGGGCGTCTTGTCTCTAGCTTTGCTGGAGACTCGCCTTAAAGTAATTGGCAGCCGGCCTACTGGTTTCGGAGCGCAGCACAAGTCGCACTCTCTATCAGCAAAGGTCTAGCATCCATTAAGCCTTTTTTTCAAC
TTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Primer Sequence (5’ 3’) reversal sequenceITS1 TCCGTAGGTGAACCTGCGGIST4 tcctccgcttattgatatgc GCATATCAATAAGCGGAGGA
The sequence above amplified from the primer pair ITS1/ITS4.
Query: 1 tccgtaggtgaacctgcggagggatcattacacaaatatgaaggcgggctggaacctctc 60 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 25 tccgtaggtgaacctgcggagggatcattacacaaatatgaaggcgggctggaacctctc 84Query: 61 ggggttacagccttgctgaattattcacccttgtcttttgcgtacttcttgtttccttgg 120 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 85 ggggttacagccttgctgaattattcacccttgtcttttgcgtacttcttgtttccttgg 144 …………………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………………… Query: 541 ctgaacttaagcatatcaataagcggagga 570 ||||||||||||||||||||||||||||||Sbjct: 565 ctgaacttaagcatatcaataagcggagga 594
BLAST Search
Query – from our dataSbject – Alternaria in GenBank
Similarity Analysis
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.
TCCGTAGGTGAACCTGCGGAGGGATCATTACACAAATATGAAGGCGGGCTGGAACCTCTCGGGGTTACAGCCTTGCTGAATTATTCACCCTTGTCTTTTGCGTACTTCTTGTTTCCTTGGTGGGTTCGCCCACCACTAGGACAAACATAAACCTTTTGTAATTGCAATCAGCGTCAGTAACAAATTAATAATTACAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCAAAGGGCATGCCTGTTCGAGCGTCATTTGTACCCTCAAGCTTTGCTTGGTGTTGGGCGTCTTGTCTCTAGCTTTGCTGGAGACTCGCCTTAAAGTAATTGGCAGCCGGCCTACTGGTTTCGGAGCGCAGCACAAGTCGCACTCTCTATCAGCAAAGGTCTAGCATCCATTAAGCCTTTTTTTCAACTTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Primer design for ITS region of Alternaria
Primer Sequence (5’ 3’) Reversal sequenceAl-f1 cccaccactaggacaaacaAl-r1 gcttaatggatgctagacct 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.
370 bp
The results indicated one kind of Alternaria was the pathogen.
Primer pair
GenBank Accession
Gene name Sequence results
Laneabove
aa-endo-f1aa-endo-r1
AY629233 A. alternata endopolygalacturonase gene
100% (408/408)
1 to 4
aa-gp-f1aa-gp-r1
AF282319 A. alternata mixed-linked glucanase precursor
99% (659/664)
5 to 8
Specific primers for Alternaria alternata (1)
Primer pair
GenBank Accession
Gene name Sequence results
Lanebelow
aa-hsp-f1aa-hsp-r1
AAU87808 A. alternata hsp70 mRNA
100% (237/237)
1 & 2
aa-his-f1aa-his-r1
AF404640 A. alternata histone gene
100% (356/356)
3 & 4
Specific primers for Alternaria alternata (2)
1500 bp
1000 bp
600 bp
300 bp
100 bp
1 2 3 4 M
Alternaria alternata is one pathogen in lilac leaf blight disease.
Specific primer pairs:
aa-gp-f1/aa-gp-r1aa-hsp-f1/aa-hsp-r1aa-hsp-f1/aa-hsp-r2aa-his-f1/aa-his-r1
A. alternata
Al-f1/Al-r1 Alternaria
Conclusion for example 1
Powdery Mildew Diseases
Dogwood Lilac Crape myrtle
• Are they caused by same pathogen? • How to analyze and identify them by molecular approaches?
Example two
Molecular Identification Approaches
ITS universal primer analysis
Sequence analysis
Specific primer analysis
ITS universal primer analysis
--------------------------------------------------------------Disease Band size Lane
----------------------------------------------------Crape myrtle powdery mildew 666bp 1Lilac powdery mildew 645bp 2Dogwood powdery mildew 642bp 3-----------------------------------------------------------------------------
Primer pair: ITS1/ITS4M 1 2 3
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 syringae-japonicae
Crape myrtle powdery mildew
ITS1/ITS4 666 bp 51.05 Uncinuliella australiana
Sequence Analysis
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 pulchraLane 3, 6, 9 are Uncinuliella australianaLane M are 100 bp ladder
Specific Primer Analysis
II Uncinuliella australiana of powdery mildew in crape myrtle
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.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M
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
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 syringae-japonicae
0 0 0 0 0 0 0 1
A=EP-f6/EP-r3B=EP-f6/EP-r4C=EP-f6/EP-r5
Eight molecular markers (specific primer pairs)
Summary for example 2
D=ua-f1/ua-r3E=ua-f1/ua-r4 F=ua-f3/ua-r3G=ua-f3/ua-r4
Primer pairs:
H=msj-f2/msj-r1
Other diseases
Pseudocercospora bairamifera is one pathogen in lilac leaf spot.
Lilac Leaf Spot
Identification of Botryosphaeria dothidea pathogen of leaf blight disease in dogwood (Cornus florida)
Arthrocladiella-mougeotiiErysiphe-cichoracearum
Erysiphe-galeopsidisErysiphe-gracilis
Typhulochaeta-japonicaBrasiliomyces-trina
Uncinula-moriBlumeria-graminis
Uncinula-septataCystotheca-w rightii
Cystotheca-lanestrisSaw adaea-polyfidaSaw adaea-sp.
Phyllactinia-fraxiniPhyllactinia-kakicola
Leveillula-tauricaPodosphaera-longiseta
Podosphaera-tridactylaSphaerotheca-fusca
Sphaerotheca-aphanisPodosphaera-leucotricha
Uncinuliella-australianaUncinuliella-ustraliana
Uncinula-necatorMicrosphaera-pulchra
Erysiphe-aquilegiaeOidium-neolycopersici
Erysiphe-symphoricarpiOak-pow dery-mildew
Erysiphe-pisiMicrosphaera-trifolii
Erysiphe-heracleiMicrosphaera-friestii
Erysiphe-cruciferarumMicrosphaera-pseudolonicerae
Oidium-heveaeMicrosphaera-alphitoides
Erysiphe-elevataErysiphe-syringae
Microsphaera-platani0.1
Oak Powdery Mildew
Maple Powdery Mildew
Similarity Analysis
Molecular Detection of Pathogens
PCR Product Sequencing
PCR Amplification
DNA Extraction
Sequence to Verify
Primer Test
Specific Primer Design
Pathogen Detection
Face to plant diseases
How can we solve them?
This is our JOB!
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!