Real-time PCR and diagnosis of plant bacterial disease

Threat Risks and Detection of Introduced Plant Pathogens

N.W. Schaad, USDA/ARS, Foreign Disease-Weed Science Research

Unit, Ft. Detrick, MD. U.S.A

Threat Risks of Introduced Plant Diseases/pathogens

• Typical and emerging plant diseases• Introduction of non-indigenous diseases• Cross-domain bacteria• Human toxin produced by a plant

nematode/bacteria complex• Rapid diagnosis of plant pathogenic bacteria

Value of US Agriculture

• Annual exports of 100 billion• Employs 17% of US workforce• Contributes 1 trillion to economy• Production is concentrated - livestock/poultry in three states - wheat in plains and Northwest - soybeans and corn in Midwest - specialty crops in CA, OR, FL. - Fresno Co. crop value > then any other state besides CA. • Crops = 79 %

Farmers face many challenges

• Weather– Frost/hail/rain– Lack of bees/pollination– Wind storms– Draught/flooding

• Natural pests and diseases- Wild animals/deer/birds/rodents

• Global trade and markets (China, apples) • Accidental or deliberate introduction of

pathogens and pests

Why Crops are Vulnerable to Biological Threats

• It’s not about a food supply• It’s about

– Harming our infrastructure

– Introducing animal and human pathogens/toxins via food sources (> enterics), organic vegetable farming

– Economic damage such as trade and markets

– Public’s lack of trust in government’s ability to ensure a supply of safe food

Threat

of fruit

laced

with biological

agent

Sept. 10, 2004

Non-indigenous Diseases are an Increasing Threat to Agriculture

• Bacterial wilt, potato• Citrus canker• Citrus Variegated

Chlororis• Huanglongbing

(HLB)• Rye Grass toxicity

Why an Increase in Non-indigenous Diseases ?

• Decline in geographic isolation due to:- Increased global travel - Free-trade agreements - Increased air cargo and sea shipping (sea

vs.rail)• Decrease in genetic diversity in crops • Increased knowledge base of plant diseases• Increase in exotic diseases and pests resulted in

Executive Order 13112 on Invasive Species signed by Pres. Clinton in 1999.

Types of Introductions

• Natural- Wind, rain, insects

• Accidental- Plant collectors (Pres. Jefferson) and breeders

• Deliberate- Self interest traveler

plant collectors, breeders, ethnic cooking- Low tech terrorists- State sponsored BW

Cross Domain Bacteria

• Burkholderia cepacia - cystic fibrosis- Originally described as an onion pathogen

B. gladiolli - clinical settings- Pathogen of onion, glads, iris, orchid

• Enteric bacteria– E.coli, Salmonella survive as epiphytes on produce– 2006 spinach outbreak in Salinas, California Source identified as livestock raised adjacent to spinach

field

1998 Yersinia pseudotuberculosis, 47 cases, iceberg lettuce1999 Salmonella Newport, 78 cases, mangoes 2003 Salmonella Muenchen, 58 cases, pre-cut melon2004 Salmonella Morbificans, 12 cases, alfalfa sprouts2004 Salmonella Javiana, 492 cases, Roma tomatoes2006 E. coli 0157:H7, spinach, Salinas, CA.

Currently:• E. coli O157:H7, 11 cases, precut Romaine lettuce• E. coli O157:H7, 32 cases, Fruit salad (melons?) • E. coli O157:H7, 12 cases, fresh apple cider

Increasingly recognizing a problem in fresh produce

Outbreaks of produce - associated foodborne infections

Robert Tauxe, CDC, American Phytopathology Society Annual Meetings July 28-Aug. 1, 2007. San Diego, CA.

Emerging Diseases

• Crops have hundreds of major diseases:- development of a molecular-based detection for every pathogen is impossible- a priority list of pathogens is necessary

• Lists (Australian, Weller et al, USDA Select List) are available but all are based on biased inputs

Human Pathogens Plant Pathogens

• Bacteria - 538• Fungi - 317• Viruses - 208• Viroids ?• Nematodes 287

• Bacteria - 600 +• Fungi - 2,500 +• Viruses - 800 +• Viroids - 35 +• Nematodes 4,500 +

Ratings for Potato Pathogens

Pathogen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 ScorePhytopthora infestans M M L L L M M L L L M H M L H H H 58.5

Rhizoctonia solani L H L M L L M L L L M M L L H L M 41.7

Heterodera rostochiensis L H L H L H L H M M M M H M M H M 56.4

R. solanacearum, bv2 H H L H H H H H H H M M H H M H H 72.5

Clavibacter sepedonicus H M L H L H H H H H M M H H M M M 57.6

Erwinia chrysanthemi H H L H H L H L H H M M L H M H M 43.9

Leaf roll virus L L L M L H M H M H M M H H M H H 55.5

Spindle tuber virus L L L M L H M H M H M M H H M H H 50

Human and Animal Toxins Produced by Plant Pathogens

Pathogen Host Toxin Action

Aspergillus flavus Peanuts, corn Aflatoxin Carcinogen

Fusarium spp. Wheat, corn Zearlenones Protein synthesis

Trichothecins Fumonisins

Pseudomonas tabaci Soybeans, tobacco Tabtoxin Glutamine Stable dipeptide synthetase

Rathayibacter toxicus Wheat, grasses Tunicaminyl - GlycosylationSelect Agent uracil toxicosis

Toxigenic bacterium

• Toxin produced by R. toxicus in plants• Not host specific• Nematode vector

Adheres to cuticle, antagonist not pathogen• Not vector specific

Adheres to Anguina spp.• Associated with bacteriophage ()

Like Corynebacterium diptheriae

R. toxicus and galls in Triticum

NematodeSeed Bacterial

Animal toxicity

Annual ryegrass toxicity Australia (Lolium rigidum)South Africa (L. rigidum)Japan (imported L. rigidum hay from Australia)

Flood plain staggersSouth Australia (Polypogon monspeliensis)New South Wales (Agrostis avenacea)

Fescue toxicity (?)Oregon, Kentucky, USA (Festuca nigrescens)

Corynetoxin poisoning

• Inhibit N-glycosylation of proteins (Uridine diphospho-N- acetylglucosamine:

dolichol-phosphate N-

acetylglucosamine-1-phosphate transferase

inhibitor)

• Often fatal neurological disease• Damage to membranes in brain• Damage to liver

Cattle poisoned by corynetoxin

Rapid Diagnosis of Plant Diseases

National Plant Disease Network

What does the NPDN look like?What does the NPDN look like?

Western Plant Diagnostic NetworkUniversity of California, Davis

Great Plains Diagnostic Network

Kansas State University

Southern Plant Diagnostic NetworkUniversity of Florida

North Central Plant Diagnostic Network

Michigan State University

North Eastern Plant Diagnostic NetworkCornell University

National AgriculturalPest Information System

Purdue University

NPDN: NPDN: Founded 2002 USDA/Homeland SecurityFounded 2002 USDA/Homeland Security

Including Alaska, Hawaii,& US territories

Including Puerto Rico

National Plant Disease Network

Immunological MethodsPolyclonal antibodiesMonoclonal antibodies

Agglutination and precipitation Enzyme-linked immunosorbant assay

(ELISA) Immunoblots Dip-stick assays

DNA-based Methods

Polymerase Chain Reaction (PCR)

Real-time PCR

Real-time PCR

• Real-time PCR is the ability to record PCR products in real time using fluorescence

– Target sequence must be amplified in the presence of a fluorescent reporter

– Increase in fluorescence is proportional to the increased amount of DNA (amplicon)

Because It eliminates post-PCR process=>Integrated reporter/probe It is specific => when using nucleic acid probes It is sensitive => 1 copy per reaction It is easy =>Dry bead formulation It is fast => 2 h or less It is quantitative => Cycle threshold + standard curves It is highly reliable => Controls It is contamination-free => Closed system It is informative => Multiplex for multiple pathogens It is flexible => Portable platforms available for field

Why is real time PCR widely used?

Threshold Line

Thresholdpenetration

Threshold Cycle

Threshold Value

Ct: Primary Signal Analysis

Real-time PCR Platforms

Machine Company Time (h)

7700 Applied Biosystems 2.0I Cycler Bio-Rad 2.0MX4000 Stratagene 1.57500 Applied Biosystems 0.5-1

Light Cycler Roche 0.5-1R.A.P.I.D Cycler Idaho Technology 0.5-1Smart Cycler Cepheid 0.5-1

Applied Bio 7700

Smart Cycler® XC

R.A.P.I.D

Real-time PCR

Brown spot (Pseudomonas syringae pv. syringae) or halo spot (P. s. phaseolicola)

Mature bean seed field, Idaho

One-hour Real-time PCR Protocol

• Remove 1-2 mm section of tissue or use plant sap

• For tissue, soak in 50ul water for 20 minutes; not longer

• Add 96ul of water to dry beads containing all PCR reagents

• Transfer 24ul to each of 4 PCR reaction tubes• Add 1ul of sample • Start PCR; 1 sec denaturation, 20 sec

annealing, 62 C • Observe PCR results in real-time (20-25min)• Total time is 45-55 minutes

Special designed 96 well membrane plate for high throughput ultra-sensitive Membrane PCR

Should work well for screening grape vines for PD during bud break using direct PCR

High throughput membrane PCR

Membrane BI0-PCR

• Add 1 ml of sample; plant extract or water to well

• Apply vacuum and place 96 well plate on soft agar

• Incubate for 12 to 24 h

• Wash wells with 50 µl buffer

• Use 1 µl for real-time PCR

• Plate 40 µl onto agar plate for viable recovery

Comparison between direct BIO PCR, and 96 well membrane BIO-PCR for Pseudomonas phaseolicola*

Direct Membrane

PSP, cfu/ml PCR BIO-PCR BIO-PCRNegative 0/2 0/6 0/3Control40 0/3 5/6 6/64.2 0/3 0/5 6/60.4 0/3 0/3 6/6

*Tox gene has only one copy per cell

PREPARE SAMPLE AND SOAK IN BUFFER

SPOT ON AGAR PLATE AFTER 2 HOURS

CUT SPOT OUT OF AGAR AFTER OVERNIGHT INCUBATION AND VORTEX IN WATER

REHYDRATE READY-TO-GO BEADS WITH 24 MICROLITERS OF WATER AND ADD 1 MICROLITER OF SOAKATE TO PCR REACTION AND RUN SMART CYCLER

Introduction of Citrus Canker Xanthomonas citri subsp. citri

Detection at U.S. Ports of Entry

Citrus canker

Xanthomonas citri subsp. citri

Discovered 1957, Sao Paulo.Extensive eradication program has reduced canker but the disease still exists.

Citrus Canker Eradication Florida, 1996-20081

• Quarantine area of 3,542 miles• All trees, including healthy ones, removed

- Residential, 1.2 million (value of $100/tree)- Commercial, 18.5 million

• Costs- $345 million

• Source of introduction- Miami International Airport suspected (fingerprints)

• - Quarantined ended in 2007 due to successful legal action against USDA

1Citrus industry valued at 8.5 billion

Mike Petrillo, APHIS National Identification Service SFO

Detecting X. citri at LAX Tissue Origin PCR(Ct) Isolation

Leaf Laos 32.8 +

Leaf Cambodia 34.6 -

Fruit India - -

Leaf Thailand 31.2 +

Leaf Cambodia 27.9 -

Leaf Vietnam 28.7 -

Leaf Thailand 33.2 -

Leaf Vietnam 32.1 -

On-site detection of citrus canker

Port PCR Isolation

LAX 7/8 2/8

SFO 15/28 2/28

Brown rot of potato caused by Brown rot of potato caused by

Ralstonia solanacearum race 3, Ralstonia solanacearum race 3,

biovar 2biovar 2

Photo by M. Ozakman

Select agent

BIO-PCR assay for R. solanacearum

• Extract core tissue from 200 tubers by soaking in buffer, 4 h• Plate 100 µl of extract onto each of 5 plates of mSMSA agar; incubate at 28°C.• Do direct real-time PCR using duplicate 10µl samples of extract (extracting DNA not necessary).• If direct PCR is negative, wash 3 plates after 28-30 h; use duplicate 10µl for direct PCR.• After 5 days, observe remaining mSMSA plates for possible colonies of R. solanacearum; confirm by PCR

Removing stem-end tissue from 200 tubers

Detecting Ralstonia.solanacearum Biovar 2 from asymptomatic potato tuber with Bio-PCR (36 cells/ml)

Detection of Huanglongbing Disease of Citrus

Canididatus Liberibacter asiaticus

C. Liberibacter africanus

C. Liberibacter americanus

Non-culturable bacterium restricted to phloem tissue; identification based upon 16S rDNA sequencing

Key Laboratory of Gene Function and Regulation at Chongqing

Different Symptoms of HLB

Key Laboratory of Gene Function and Regulation at Chongqing

Citrus psyllid

psyllid

HuanlongbinHuanglongbing disease of citrus (golden dragon disease)

Cultures of suspected HLB-bacterium

LAS on Liber A agar medium LAF in Liber A liquid mediumbiofilm

Figure4B Figure4B path.tif.tif

Koch’s Postulates

HLB Assays

• Cut out leaf mid vein and place in plastic bag

• Use press to obtain plant juice

• Extract DNA• Purify DNA for PCR

• Cut off leaf petiole• Cut into 1-2 mm

sections and soak in water for 20 to 30 min

• Use 1ul directly for PCR

I II

M 1 2 3 4 5 6 7 8 9 10

TaqMan real-time PCR Classical PCR

Z.K. Wang, Chongqing, China

16S-rDNA based assay for Liberibacter

Congress is STILL debating surveillance

of containers

England Checks trucks with plant material

Dip sticks

Conclusions

• Crops are highly vulnerable to both natural and intentional disease introductions

• Cross-domain bacteria pose an increasing threat

• National Plant Diagnosis Network is in place for detecting plant diseases and validating assay protocols

• No routine assays are required for produce in the field or packing sheds or US ports

Thank You

Washington, DC