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Identifying sources of Ralstonia solanacearum race 3 biovar 2 inoculum Caitilyn Allen 1 , Jill K. Swanson 1 , Amilcar PowerPoint Presentation
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Identifying sources of Ralstonia solanacearum race 3 biovar 2 inoculum Caitilyn Allen 1 , Jill K. Swanson 1 , Amilcar Sanchez-Perez 1 and Luis Mejia 2 1 U. Wisconsin-Madison & 2 U. San Carlos of Guatemala Floral & Nursery Crops Researchers’ Workshop, Portland, OR June 12-15, 2006

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slide1

Identifying sources of Ralstonia solanacearum

race 3 biovar 2 inoculum

Caitilyn Allen1, Jill K. Swanson1,

Amilcar Sanchez-Perez1 and Luis Mejia2

1U. Wisconsin-Madison &2U. San Carlos of Guatemala

Floral & Nursery Crops Researchers’ Workshop, Portland, OR June 12-15, 2006

slide2

Pathogen Detection & Exclusion: Why Inoculum Matters

Studies of R. solanacearum in Geranium Effluent

A. Early results with small plants in potting soil

B. Field studies with large mother plants in scoria

C. Lab studies with large mother plants in scoria

D. Prospects for effluent monitoring schemes

III. Distribution of R. solanacearum R3b2 in Guatemala

A. Survey method and findings

B. Molecular characterization of Guatemalan isolates

IV. Diagnostic Challenges with Ralstonia R3b2

r solanacearum is divided into 3 races based loosely on host range
R. solanacearum is divided into 3 races based loosely on host range

Race 1: broad host-range tropical strains: tomato, peanut, eucalyptus, tobacco, etc etc.

Race 2: Moko disease of bananas and plantains.

Race 3 (=biovar 2): cool-adapted strains cause potato brown rot and Southern wilt of geranium.

slide5

Ralstonia solanacearum:

  • a wily and agile bacterium
  • - Gram-negative rod
  • - optimal growth at 28 C
  • but can survive & multiply
  • at 12-35 C
  • survives long-term in water, soil, and latent host plants
slide6

Southern wilt symptoms on geranium

-wilting, curling leaves

-yellowed leaves

-necrotic leaf wedges

-vascular browning

-eventual collapse &

death

-but long-term symptomless (latent) infections are also common

slide7

A little history…..

December, 2002:R. solanacearum Race 3 listed as a

potential bioterrorism agent in the Agricultural

Bioterrorism Protection Act of 2002, mandating strict new biosecurity and quarantine regulations.

February, 2003:R. solanacearum-infected geraniums are found in U.S. greenhouses. Regulatory actions result in destruction of ~3 million plants; cost is over $6 million.

December, 2003: Infected geraniums found in one NY greenhouse; all plants of those varieties ordered destroyed. 458 greenhouses in 41 states are affected; costs over $2 million and a major producer goes out of business.

slide8

A big lesson of the 2003-4 Ralstonia geranium outbreaks:

  • We need better methods for sampling to detect and exclude this pathogen before it gets to the US
  • Research questions:
  • Can destructive sampling be effective?
  • Approaches to large scale non-destructive sampling
  • Is R. solanacearum Race 3 common where ornamental cuttings are produced?
slide9

Is the pathogen evenly distributed throughout the plant? (for sampling individuals)

Methods

  • Plants were characterized as diseased or latently infected
  • Tissue slices cut from crown, stems, petioles, flower stalk
  • Tissue weighed & ground in water & dilution-plated on

selective medium

  • Colony counts adjusted to cfu/gm fresh weight tissue
slide12

Is the pathogen evenly distributed throughout the plant? (for sampling individuals)

No;

Sampling any one part of the plant, except for the crown, is not reliable for diagnostics.

So what about a non-destructive method based

on pooled samples?

slide13

Measuring bacteria in effluent from infected plants

  • Inoculated plant roots
  • “naturally”
  • Harvested plants at
  • various disease stages
  • H2O was poured
  • into pot; effluent was
  • collected and dilution-plated on selective medium (Sensitivity: 1x102 cfu/ml)
  • Bacterial populations in crown tissue determined by grinding & plating on selective medium
slide15

Conclusion: Testing pooled effluent from geraniums might be an efficient and sensitive way to detect latently infected mother plants.

  • But would it work in the field with large plants growing in volcanic scoria?…
  • Preliminary Field Study at USAC in Guatemala:
  • 30 Scoria-grown plants (planted two per bag) were inoculated through petiole scar
  • Effluent sampled on days 13, 14, and 15 after inoculation
  • All plants were asymptomatic
slide16

Field Results: Infected Mother Plants Did Not Always Shed Bacteria

7/14 plants gave

all false negatives

-Latent

-Latent

-Latent

-Latent

-Latent

-Latent

-Latent

-uninfected

(escape)

slide17

Do infected large geraniums reliably shed bacteria in pot effluent?

  • Repeated the experiment under controlled conditions
  • (growth chamber) using large mother plants grown in volcanic scoria.
  • Followed plants 66-75 days after inoculation, recording symptoms and measuring bacteria in effluent at intervals.
  • At end of experiment, determined bacterial population size in plant crown (lower stem)
slide20

Conclusion:

Effluent populations

from large plants

do not reliably

report infection

slide21

What R. solanacearum types are present in Guatemala?

Amilcar Sanchez-Perez with a wilting potato

slide22

Locations where R. solanacearum was isolated in Guatemala

POTATO

TOMATO

BANANA

EGGPLANT

Solanum

americanum

Geranium

Host

ATLANTIC OCEAN

MEXICO

GUATEMALA

HONDURAS

EL SALVADOR

PACIFIC OCEAN

slide23

Locations where R. solanacearum was isolated in Guatemala

POTATO

TOMATO

BANANA

EGGPLANT

Solanum

americanum

Geranium

Host

ATLANTIC OCEAN

MEXICO

GUATEMALA

HONDURAS

Jalapa

EL SALVADOR

PACIFIC OCEAN

slide24

Field Survey Method

  • Isolated from soil, waterways, and plants from all over GT
  • For each field isolate:
  • - confirm to species (immunostrip)
  • - determine biovar (biochemical growth assay)
  • - determine if Race 3 biovar 2 (PCR assay)
  • - determine phylotype (multiplex PCR reaction)
  • - determine sequevar (sequence endoglucanase gene, make phylogenetic tree)

Biovar test plate

slide25

280 bp band

from Rs

universal

primers

Potato

PHYLOTYPE

Banana

II IV I III

Tomato

Phylotype characterization using multiplex PCR

slide27

Molecular Analysis Results: Among 59 separate isolates, we found 3 very different strains of R. solanacearum:

  • Race 1 on tomatoes, weeds, & eggplant (Asian origin)
  • Race 2 on bananas and plantains (American origin)
  • Race 3 biovar 2 on potatoes, geraniums, and highland screenhouse tomatoes (American origin)
slide28

Phylotype I (Asian)

UW568

Phylotype IV

(Indonesian)

Phylotype III (African)

  • Strains in red are Guatemalan.
  • UW 573 (potato)
  • UW 566 (banana)
  • UW568 (tomato)

Phylotype II (American)

UW 566

Race 3 group

UW573

Phylogenetic tree based on endoglucanase gene sequence

Modified from: M. Fegan & P. Prior, 2006, Australasian Plant Pathology

slide29

Race 3, biovar 2 was isolated from potatoes,

geraniums, and screenhouse tomatoes at

higher elevations (900-1800 M)

slide30

Field Study Conclusions:

  • R. solanacearum is widely distributed around Guatemala
  • There is surprisingly low diversity among strains with just 3 sequevars (few introductions?)
  • Race 3 biovar 2 is widespread where potatoes are grown in the highlands
  • In addition to potatoes and geraniums, R3b2 infects screenhouse tomatoes grown in the highlands
  • New question: Can the bacterium be transmitted in fruit from infected tomato plants?
slide31

Ralstonia R3b2 continues to be difficult to diagnose

- in Fall 2004, R. solanacearum was isolated from geraniums at a

Florida facility, and from a nearby pond

- initial real time PCR tests found the bacteria belonged to

Race 3 biovar 2

- however, further study revealed that a group of Race 1 biovar 1 strains from the Caribbean give a false positive (delayed positive)

in this assay

slide32

“delayed

positive” Bv1

Classic Bv 1

Authentic

Race 3 biovar 2

slide33

Molecular Analysis:

DAR64836, Musa sp, Australia, Bv1

UW20, Banana, Venezuela, Bv1

UW566, Banana, Guatemala, Bv1

CFBP2972, Potato, Martinique, Bv1

CFBP2957,Tomato, Martinique, Bv1

CFBP2958, Tomato, Guadeloupe, Bv1

Caribbean Bv1 cluster

UW469, Potato, Brazil, Bv1

Rs125, Geranium Florida, Bv1

E81, Tomato, Guadeloupe, Bv1

Phylotype II

(Americas)

ICMP9601, Eucalyptus, Brazil, Bv1

UW154, Tobacco, Columbia, Bv1

ACH1076, Potato, Brazil, Bv1

UW134, Potato, Kenya, Bv1

Rs116, Hydrangea,USA, Bv1

UW27, Tobacco, USA, Bv1

K60,Tomato, USA, Bv1

Rs5, Tomato, USA, Bv1

Race 3

UW551, Geranium Kenya, Bv2

UW573, Potato Guatemala, Bv2

UW477, Potato, Peru, Bv2T

UW9, Heliconia, Costa Rica, Bv1

QL14, Ginger, Philippines

Rs123, Pepper, USA, Bv3

PSS160, Tomato, Taiwan, Bv3

Rs121, Pepper, USA, Bv3

JT523, Potato, Reunion Is, Bv3

Phylotype I (Asia)

CIP365, Potato, Philippines, Bv3

GMI1000, Tomato, French Guyana, Bv3

ICMP8229, Ginger, Costa Rica, Bv4

UW568, Tomato, Guatemala, Bv3

T3, Tomato, Indonesia, Bv4

CIP65, Chili pepper, Costa Rica, Bv3

R292, Morus alba, China, Bv5

ICMP9915, Clove, Indonesia, Bv1

R28, Clove, Indonesia, R. syzygii

Phylotype IV

(Indonesia)

MAFF301558, Potato, Japan, Bv2T

PSI36, Tomato, Indonesia, Bv2T

R142, Clove, Indonesia, Bv2

JT525, Pelargonium asperum, Reunion Is, Bv1

JT528, Potato, Reunion, Bv1

Phylotype III (Africa)

J25, Potato, Kenya, Bv2T

CFBP3059, Eggplant, Burkina Faso, Bv1

0.01

Phylogenetic tree based on DNA sequence of the endoglucanase gene

slide34

Conclusions:

  • Critical high-impact diagnoses of Ralstonia Race 3 bv 2 should not be based on a single assay, as no one assay is completely reliable.
  • We urgently need a second rapid, specific, sensitive test to supplement the existing PCR-based method.
  • Future work: develop a Race 3-specific antibody for diagnostics.
slide35

Acknowledgements

Geraniums:

Goldsmith Plants

Oglevee

Ball FloraPlant

Diagnostic strips:

AgDia, Inc.

Funding:

USDA-SCA Floral

Industry Task Force

Initiative

Jill Swanson