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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
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 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: • 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 -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) 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
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?
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
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
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)
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)
Conclusion: Effluent populations from large plants do not reliably report infection
What R. solanacearum types are present in Guatemala? Amilcar Sanchez-Perez with a wilting potato
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
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
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
280 bp band from Rs universal primers Potato PHYLOTYPE Banana II IV I III Tomato Phylotype characterization using multiplex PCR
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)
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
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 positive” Bv1 Classic Bv 1 Authentic Race 3 biovar 2
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
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