# Presentation: 870057

## 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**
- 1U. Wisconsin-Madison &2U. San Carlos of Guatemala
- Floral & Nursery Crops Researchers’ Workshop,   Portland, OR June 12-15, 2006

## 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 causes Southern wilt of geraniums

***R. solanacearum***** causes Southern wilt of geraniums**

## 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**.

## Ralstonia solanacearum:

_***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**

## Southern wilt symptoms on geranium

**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**
**	**

##        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.**
** **

## 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?

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

-  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

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

## Slide 10

## Slide 11

## 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?**

## 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

**Measuring bacteria in effluent from infected plants**

## Slide 14

## 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

## -uninfected

**Field Results: Infected Mother Plants Did Not Always Shed Bacteria**

**-Latent**

**-Latent**

**-Latent**

**-Latent**

**-Latent**

**-Latent**

**-Latent**

*7/14 plants gave*
*all false negatives*

***-uninfected***
***   ******(escape)***

## 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)

## Effluent Populations from Large Plants are Variable

## Slide 19

## Conclusion:

**Effluent populations**
**from large plants**
**do not reliably **
**report infection**

**Amilcar Sanchez-Perez with a wilting potato**

**What *****R. solanacearum***** types are present in Guatemala?**

## Locations where R. solanacearum was isolated in Guatemala

- EL SALVADOR

- HONDURAS

- MEXICO

- ATLANTIC OCEAN

- PACIFIC OCEAN

-    POTATO
-    TOMATO
-    BANANA
-    EGGPLANT
-    *Solanum*
*  **americanum*
-   Geranium

**GUATEMALA**

**Locations where***** R. solanacearum***** was isolated in Guatemala**

**Host**

## Locations where R. solanacearum was isolated in Guatemala

- EL SALVADOR

- HONDURAS

- MEXICO

- ATLANTIC OCEAN

- PACIFIC OCEAN

-    POTATO
-    TOMATO
-    BANANA
-    EGGPLANT
-    *Solanum*
*  **americanum*
-   Geranium

**GUATEMALA**

**Locations where***** R. solanacearum***** was isolated in Guatemala**

**Host**

**Jalapa**

## 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

## Banana

-  II    IV   I   III

-      PHYLOTYPE

**Banana**

**Potato**

**Tomato**

**Phylotype characterization using multiplex PCR**

**280 bp band **
**from Rs **
**universal  **
**primers**

## 59 R. solanacearum Strains Collected in Guatemala

| Host Plant | Phylotype | Race | Biovar | Total |
| --- | --- | --- | --- | --- |
| tomato | I | 1 | 3 | 23 |
| eggplant | I | 1 | 3 | 1 |
| S. americanum | I | 1 | 3 | 1 |
| banana | II | 2 | 1 | 11 |
| tomato | II | 3 | 2 | 4 |
| potato | II | 3 | 2 | 18 |
| geranium | II | 3 | 2 | 1 |

- 59* R. solanacearum* Strains Collected in Guatemala

## 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)

## Phylogenetic tree based on endoglucanase gene sequence

**UW573**

**UW 566**

**Phylotype II (American)**

**Phylotype III (African)**

**Phylotype I (Asian)**

**Phylotype IV**
** ****(Indonesian)**

-   **UW568**

**Phylogenetic tree based on endoglucanase gene sequence**
**Modified from: M. Fegan & P. Prior, 2006, *****Australasian Plant Pathology***

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

**Race 3 group**

## Race 3, biovar 2 was isolated from potatoes, 

**geraniums, and screenhouse tomatoes at **
**higher elevations (900-1800 M)**

## 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?

## 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

## “delayed

**“****delayed**
**  ****positive” Bv1**

**Classic Bv 1**

**Authentic **
**Race 3 biovar 2**

## Molecular Analysis:

**0.01**

- 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

- UW469, Potato, Brazil, Bv1

**Rs125, Geranium Florida, Bv1**

- E81, Tomato, Guadeloupe, Bv1

- 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**

**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

- 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*

- MAFF301558, Potato, Japan, Bv2T

- PSI36, Tomato, Indonesia, Bv2T

- R142, Clove, Indonesia, Bv2

- JT525, *Pelargonium asperum*, Reunion Is, Bv1

- JT528, Potato, Reunion, Bv1

- J25, Potato, Kenya, Bv2T

- CFBP3059, Eggplant, Burkina Faso, Bv1

**Phylotype II**
**(Americas)**

**Phylotype I (Asia)**

**Phylotype IV**
**(Indonesia)**

**Phylotype III (Africa)**

**Race 3**

**Caribbean Bv1 cluster**

**Molecular Analysis:**

- Phylogenetic tree based on DNA sequence of the endoglucanase gene

## 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.

## Acknowledgements

***Geraniums:***
**Goldsmith Plants**
**Oglevee**
**Ball FloraPlant**
***Diagnostic strips:***
**AgDia, Inc.**
***Funding:***
**USDA-SCA Floral **
**Industry Task Force **
**Initiative**

**Jill Swanson**