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
 Outbreaks of produce - associated
foodborne infections
 1998 Yersinia pseudotuberculosis, 47 cases, iceberg lettuce
 1999 Salmonella Newport, 78 cases, mangoes
 2003 Salmonella Muenchen, 58 cases, pre-cut melon
 2004 Salmonella Morbificans, 12 cases, alfalfa sprouts
 2004 Salmonella Javiana, 492 cases, Roma tomatoes
 2006 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

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 • Bacteria - 600 +

• Fungi - 317 • Fungi - 2,500 +
• Viruses - 208 • Viruses - 800 +
• Viroids ? • Viroids - 35 +
• Nematodes 287 • 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 Score
Phytopthora 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 - Glycosylation

Select 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

Seed Nematode Bacterial

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

 National Plant Disease Network
What does the NPDN look like?

North Eastern Plant

North Central Plant Diagnostic Network
Diagnostic Network Cornell University
Michigan State University

Western Plant Diagnostic Network

University of California, Davis

Including Alaska, Hawaii,

& US territories National Agricultural
Great Plains Pest Information System
Diagnostic Network Purdue University
Kansas State University

Southern Plant
Diagnostic Network
University of Florida

Including Puerto Rico

NPDN: Founded 2002 USDA/Homeland Security
 Immunological Methods
Polyclonal antibodies
Monoclonal 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)
 Why is real time PCR widely used?
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
Ct: Primary Signal Analysis

Threshold Cycle

Threshold Value Threshold

penetration

Threshold Line
 Real-time PCR Platforms
Machine Company Time (h)

7700 Applied Biosystems 2.0

I Cycler Bio-Rad 2.0
MX4000 Stratagene 1.5
7500 Applied Biosystems 0.5-1

Light Cycler Roche 0.5-1

R.A.P.I.D Cycler Idaho Technology 0.5-1
Smart Cycler Cepheid 0.5-1
Real-time PCR

Applied Bio
7700

R.A.P.I.D

Smart Cycler® XC
 Mature bean seed field, Idaho

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

phaseolicola)
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
 High throughput membrane PCR

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
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-PCR
Negative 0/2 0/6 0/3
Control
40 0/3 5/6 6/6
4.2 0/3 0/5 6/6
0.4 0/3 0/3 6/6
*Tox gene has only one copy per cell
 PREPARE SPOT ON
SAMPLE AGAR
AND SOAK PLATE
IN BUFFER AFTER 2
HOURS

CUT SPOT
OUT OF
AGAR AFTER REHYDRATE READY-TO-GO
OVERNIGHT BEADS WITH 24 MICROLITERS
INCUBATION OF WATER AND ADD 1
AND MICROLITER OF SOAKATE TO
VORTEX IN PCR REACTION AND RUN
WATER SMART CYCLER
Introduction of Citrus Canker
Xanthomonas citri subsp. citri
Detection at U.S. Ports of Entry
 Citrus canker
Discovered 1957, Sao Paulo.
Extensive eradication program
has reduced canker but the
disease still exists.

Xanthomonas citri subsp. citri

 Citrus Canker Eradication
Florida, 1996-2008 1

• 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

1 Citrus 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 Ralstonia
solanacearum race 3, biovar 2

Select agent

Photo by M. Ozakman
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
 Different Symptoms of HLB

Key Laboratory of
Gene Function and
 Citrus psyllid

psyllid

Key Laboratory of
Gene Function and
 Huanlongbin
Huanglongbing disease of citrus (golden dragon
disease)
 Cultures of suspected HLB-
bacterium

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

biofilm
Figure4B Figure4B path.tif.tif

Koch’s Postulates
 HLB Assays
I II
• Cut out leaf mid vein
• Cut off leaf petiole
and place in plastic
bag • Cut into 1-2 mm
• Use press to obtain sections and soak
plant juice in water for 20 to
• Extract DNA
30 min
• Purify DNA for PCR
• Use 1ul directly
for PCR
 16S-rDNA based assay for Liberibacter
TaqMan real-time PCR Classical PCR

M 1 2 3 4 5 6 7 8 9
10

Z.K. Wang, Chongqing, China

England Checks trucks with plant material

Congress is STILL debating surveillance

of containers

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
Washington, DC

Thank You