Detection of Clavibacter michiganensis subsp. michiganensisand Salmonella in Irrigation Water

S. A. Miller, N. Khatri, F. Baysal-Gurel and J. T. LeJeune

Department of Plant Pathology and Food Animal Research Program

30th Tomato Disease WorkshopBaton Rouge, LA4 Oct 2015

Propagation

Medium preparationSowingGraftingRaising seedlings

Greenhouse production

Medium preparationTransplantingTrainingPruning & trussingLeaning & loweringFertilizer applicationPesticide applicationHarvesting

Post-harvest treatment

GradingWashingPackingStorageTransport

Water management

Fertilizer preparation

Pest management

WasteManagement

Sanitation

People flowWorkers hygiene and behaviorWorkers health policy

InfrastructureBuildingSurroundingsEquipment

SeedReceiving

Greenhouse Production Flow

Produce-Associated Human Pathogens

Fresh fruit and vegetables increasingly associated with human disease outbreaks

Zoonotic pathogens Shiga toxin-producing E.

coli; Salmonella; Listeria

Enteric viruses Norovirus

Clavibacter michiganensissubsp. michiganensis

Systemic disease

Seed-borne

Water-borne

Long-lived in GH environment

Most economically damaging disease of greenhouse tomatoes

Pepper is a secondary host

Bacterial Canker

Three-week old seedlings clip-inoculated with bioluminescent Cmm.

Images taken every 5 days post inoculation (DPI).

5 DPI 10 DPI

15 DPI 20 DPI

83% RH 45% RH 83% RH 45% RH

Cmm Root Colonization

Salmonella on Tomatoes Salmonella does not

cause symptoms on plants

But it can:

Persist on tomato plants and fruit

Colonize seeds

Survive in water Bioluminescent Salmonella enterica serovarTyphimurium on tomato seeds and seedlings

Objectives

Verify the sensitivity and specificity of available detection assays for Cmm and Salmonella

Identify methods to concentrate Cmm and Salmonella from greenhouse water

Develop a plan for water testing going forward

Experimental Approach

Test available assays for accuracy

Re-isolating bacteria by culturing considered the Gold Standard

Select best bacterial concentration method

Determine sensitivity of detection assays with selected concentration methods

Comparison of Pathogen Detection Assays

Method Details

Culturing mCmm1medium CmmLB medium - Salmonella

Mutiplex PCR Multiplex Cmm 5/6 + PSA8/Rprimers - Cmm

Real Time PCR PTSSK TaqMan - Cmm http://www.worldseed.org/isf/ishi_vegetable.html

Lateral Flow Device -Serological

Agdia Immunostrip - Cmm

Isothermal Amplification

Envirologix Cmm DNAbleEnvirologix Salmonella DNAbleSmart-Dart Cmm LAMP (Univ. HI)Fi

eld

met

ho

ds

La

b m

eth

od

s

Assay % True +

% True

-

%False

+

% False -

qPCR 96 95 5 4

PCR Cmm5/6 48 100 0 52

PCR PSA8/R 72 100 0 28

DNAble 92 100 0 8

Immunostrip 88 55 45 12

Tested 49 tomato/5 pepper field samples

Symptomatic

Asymptomatic

Other diseases/damage

Leaves, stems, fruits

Test results compared with Cmm culturing on mCmm1 medium

Cmm Assay Evaluation-1

DNAble, Immunostrip negative on samples < 1 x 105 Cmm/g

Assay %True +

%True

-

% False

+

% False -

qPCR 100 100 0 0

PCR Cmm5/6 100 100 0 0

PCR PSA8/R 100 100 0 0

LAMP 100 100 0 0

Immunostrip 100 50 50 0

Smart-DART 100 100 0 0

Tested 12 tomato/6 pepper field samples

14 symptomatic

4 asymptomatic

Leaves, stems, fruits

Test results compared with Cmm culturing on mCmm1 medium

Cmm Assay Evaluation-2

Cmm in positive samples: 1.2 2.4 x 108 Cmm/g

Summary Cmm Detection Assays

Agdia Immunostrips: Non-specific-high rate of false positives Detection limit ~ 1 x 105 Cmm/g

Cmm DNAble: Specific-no false positives Detection limit ~ 1 x 105 Cmm/g

Smart-DART: Specific- no false positives Detection limit ~ 1 x 105 Cmm/g

Multiplex PCR: Low sens.-high rate of false negatives

qPCR: Highly specific and sensitive

Field assays

Lab assays

Salmonella Assays

Culturing on LB medium

Envirologix DNAble for Salmonella

Both require 24-hr enrichment step

Neither differentiate serovars

Culturing vs. DNAble for Salmonella Detection

Re-circulated water

Concentration of Bacteria

Concentration methods tested:

Centrifugation

Immunomagnetic separation

Filtration

20m Nalgene filters + vacuum filtration

DIF-30 Disposable Inline Filters + peristaltic pump Fast filtration 5L in approx. 20 minutes

In-filter enrichment

Excellent trapping of bacteria

Detection of Cmm, Salmonella in Water

Source or Production Water

Filter (DIF-IN30)

Enrich Direct

Bacterial Detection Tests

Detection of Cmm and Salmonella in Water

Protocol

Filter (DIF-IN30) 5L water Culture from filter Quantify

Filter water Direct test by multiplex PCR, RT qPCR, Immunostrips, DNAble, SmartDart

Filter water Enrich from filter suspension Dilution plate (24, 48, 72 hr) Test by multiplex PCR, RT qPCR, Immunostrips, DNAble, SmartDart

Spike 5L re-circulated GHwater with 102, 104 or 106

CFU bioluminescent Cmmor Salmonella

Concentration of Bacteria in Water

GH water collectedusing a Rotatecperistaltic pump

Enrichment

Concentration of Bacteria in Water

Filters disrupted using Pulsifier

Bacteria concentrated by centrifugation

Detection of Bacteria in Water

Culturing/counting

SmartDART LAMP

Real-Time qPCR

Multiplex PCR

Immunostrip

DNAble

Testing Filtration and Assays with GH Source and Production Water

Well water (cleanest)

Water with fertilizer added

Re-circulated production water

Method

CFUBioluminescentCmmAddedto5L

Water

Control Log2 Log4 Log6

ImmunoStrip --- +--/--- +++ +++

Smart-Dart --- +++/--- +++ +++

MultiplexPCR -- +++/--- +++ +++

qPCR --- Log1.5 Log3.1 Log5.0

Culturing --- Log3.9 Log6.2 +++

Direct Detection of Cmm in 5L Well Water

Detection of Cmm in 5L Well Water-Enriched

Detection of Cmm in 5L Fertilizer+ Water

Detection of Cmm in 5L Re-circulated Water

Detection of Salmonella in 5L Well Water

Detection of Salmonella in 5L Fertilizer+ Water

Detection of Salmonella in 5L Re-circulated Water

Effect of Simulated Shipping Conditions/Time on Cmm Recovery from

Water

0

1

2

3

4

5

6

Ice packs 24C 30C

Day 1

Day 3

Day 5

Log

Cm

m C

FU/5

L W

ater

Effect of Simulated Shipping Conditions/Time on Salmonella Recovery from Water

0

1

2

3

4

5

6

Ice packs 24C 30C

Day 1

Day 3

Day 5

Log

Salm

on

ella

CFU

/5L

Wat

er

Summary Filtration of water samples through disposable inline filters

superior to other methods of bacterial concentration

Real-Time quantitative PCR most sensitive and compared best to culturing Cmm:100 cells/5L

DNAble for Salmonella similar to culturing in sensitivity if enriched: 100 cells/5L

Cmm but not Salmonella sensitive to higher temperatures; filters need to be shipped on ice

Currently expensive but economies of scale and combining assays may reduce costs

Acknowledgments

Melanie Lewis Ivey

OSU Food Animal Health Research Program

Sanja Ilic, Mike Kaufman, Ana Arciniega

Dan Myhaver, EnviroLogix

Anne Alvarez, Jarred Yasuhara-Bell, Dan Jenkins, Univ. HawaiI

BCGGA/ Growing Forward 2

USDA NIFA Specialty Crop Research Initiative SCRI- 2010-600-25320