Tuan Tran-An update on Ralstonia solanacearum...Tuan Tran Department of Plant Pathology University...

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4/18/2016 1 An update on Ralstonia solanacearum and Bacterial Wilt disease Tuan Tran Department of Plant Pathology University of Wisconsin - Madison Ralstonia solanacearum Gram-negative β-proteobacterium Rod-shaped, xylem inhabiting Survive long-term in water, soil and infected plant materials 2 Ralstonia solanacearum Top 10 bacterial phytopathogens Widely distributed around the globe 3 Image courtesy of B it R t 4 African daisy, 2015 Raka Mitra R. solanacearum life cycle 5 Raka Mitra R. solanacearum life cycle 6 Tuan Tran

Transcript of Tuan Tran-An update on Ralstonia solanacearum...Tuan Tran Department of Plant Pathology University...

Page 1: Tuan Tran-An update on Ralstonia solanacearum...Tuan Tran Department of Plant Pathology University of Wisconsin - Madison Ralstonia solanacearum • Gram-negative • β-proteobacterium

4/18/2016

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An update on Ralstonia solanacearum and Bacterial Wilt disease

Tuan Tran

Department of Plant Pathology

University of Wisconsin - Madison

Ralstonia solanacearum

• Gram-negative

• β-proteobacterium

• Rod-shaped, xylem inhabiting

• Survive long-term in water, soil and infected plant materials

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Ralstonia solanacearum• Top 10 bacterial phytopathogens

• Widely distributed around the globe

3Image courtesy of B it R t

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African daisy, 2015

Raka Mitra

R. solanacearum life cycle

5Raka Mitra

R. solanacearum life cycle

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Tuan Tran

Page 2: Tuan Tran-An update on Ralstonia solanacearum...Tuan Tran Department of Plant Pathology University of Wisconsin - Madison Ralstonia solanacearum • Gram-negative • β-proteobacterium

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Raka Mitra

R. solanacearum life cycle

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Bacterial Wilt disease

Jonathan Jacobs 8

Current classification systems

• Phylotype (sequence‐based)

• Race (host)

• Biovar (carbon utilization)

• Sequevar (sequence‐based)

Race 3 biovar 2

• A subgroup of Ralstonia solanacearum, R3bv2 is a select agent in the US (since 2002) and a quarantine pest in Europe and Canada.

• New regulations from USDA‐APHIS consider all R. solanacearum to be R3bv2 until proven not to be

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Phylogenetic network derived from genomic distances

Prior et al. 2016

Mass‐spec derived phylogenetic tree

Prior et al. 2016

Page 3: Tuan Tran-An update on Ralstonia solanacearum...Tuan Tran Department of Plant Pathology University of Wisconsin - Madison Ralstonia solanacearum • Gram-negative • β-proteobacterium

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Mandevilla (Dipladenia) splendens

Leaf diebackWiltingReduced top growth 

Ruhl et al. 2011 Disease Notes 95(5): 614

African daisy

Weibel et al. 2016 Plant Health Progress (accepted)

UW757 colonized a wide range of ornamental hosts

Dahl: DahliaSunC: Impatiens x hybridaNGI: Impatiens hawkeriVinc: Vinca majorPela: PelargoniumVerb: VerbenaLobe: Lobelia erinusOste: OsteospermumSute: Sutera cordataPetu: PetuniaCali: Calibrachoa

Weibel et al. 2016 Plant Health Progress (accepted) Weibel et al. 2016 Plant Health Progress (accepted)

Current detection methods

Culture‐based

‐ Enrichment in selective medium

Sequenced‐based

‐ PCR

‐ Real‐Time PCR

‐ LAMP

‐Magnetic capture hybridization

Immunological methods

‐ ELISA

‐ ImmunoStrip

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Page 4: Tuan Tran-An update on Ralstonia solanacearum...Tuan Tran Department of Plant Pathology University of Wisconsin - Madison Ralstonia solanacearum • Gram-negative • β-proteobacterium

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Loop‐mediated isothermal amplification (LAMP)

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FTA card for diagnostic of R. solanacearum

FTA card for storage of microbial DNA

• Tested for >400 bacterial strains (61 genera)

• DNA was stable for at least 3 years

• Tested on many human pathogens: Clostridium, H. influenzae, H. pylori, Salmonella, V. cholerae, etc.

Rajendram et al. 2006. Journal of Microbiological Methods. 67: 582‐592 

FTA card for diagnostic of R. solanacearum

Tran et al. 2016. Plant Disease

Field tomato samples from Virginia

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Problem: PCR inhibitors from plantSolution: evolved DNA polymerase (Kapa 3G DNA pol)

WT

M NTCWT

Molecular evolution ofKAPA3G DNA Polymerase

Gene coding for KAPA2G Robust DNA Polymerase, originally evolved for improved tolerance to common PCR inhibitors.

A library of 3 x 107 KAPA2G Robust DNA Polymerase variants is generated by mutagenesis.

Gene variants are expressed and compartmentalized in emulsion, followed by high-throughput screening in the presence of polyphenolic and polysaccharide selection pressure.

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Only improved DNA polymerase variations survive the selection.KAPA3G DNA Polymerase was evolved for improved tolerance to common plant-derived PCR inhibitors, enabling direct PCR from plant tissue and crude extracts.

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Source: Kapa Biosystem 24

Page 5: Tuan Tran-An update on Ralstonia solanacearum...Tuan Tran Department of Plant Pathology University of Wisconsin - Madison Ralstonia solanacearum • Gram-negative • β-proteobacterium

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Optimization of PCR protocol for geranium samples 

25 Tran et al. 2016 Plant Disease. 100 (3): 630‐639 

Tran et al. 2016 Plant Disease. 100 (3): 630‐639 

Practical consideration

Cost

TimeSensitivity

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Practical consideration

Cost

Time Strip < 30 min – Enrichment 48 h

Sensitivity

Strip ($6.20) vs. DP ($0.26) 

ImmunoStrip (105) vs. DP, LAMP, BIO‐PCR (101)

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Practical consideration

0 2 4 6 8

BIO-PCR48

BIO-STRIP48

Plating

BIO-PCR24

BIO-STRIP24

FTA+PCR

bFTA+PCR

FTA-Kapa

PCR

Kapa3G

LAMP

STRIP

$

*****

******

******

*****

*****

*****

0.5 h

50.5 h

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Propose pipeline for detection of Rs AcknowledgementThe Allen Lab

USDA‐Floral and Nursery Crops Research Initiative 

Department of Plant Pathology – UW Madison and CALS

Vietnam Education Foundation

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