national diagnostic protocol
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Prepared by Eric Cother and Christine McKenzie
NSW Department of Primary Industries
Agricultural Institute
Forest Road, Orange NSW 2800
Contact : ric.cother@agric.nsw.gov.au
Phone 02 63 913 886
November 2004
The scientific and technical content of this document is current to the date published and all efforts were made
to obtain relevant and published information on the pest. New information will be included as it becomes
available, or when the document is reviewed. The material contained in this publication is produced for general
information only. It is not intended as professional advice on any particular matter. No person should act or fail
to act on the basis of any material contained in this publication without first obtaining specific, independent
professional advice. Plant Health Australia and all persons acting for Plant Health Australia in preparing this
publication, expressly disclaim all and any liability to any persons in respect of anything done by any such
person in reliance, whether in whole or in part, on this publication. The views expressed in this publication are
not necessarily those of Plant Health Australia.
National Diagnostic Protocol
Stewart’s wilt of maize
(Pantoea stewartii subsp. Stewartii)
NATIONAL DIAGNOSTIC PROTOCOLS
Table of Contents
Background .............................................................................................................................3
Symptom recognition ..............................................................................................................3
Seedling wilt ........................................................................................................................... 3
Adult leaf blight ...................................................................................................................... 5
Differentiation from other diseases .........................................................................................6
Isolation .................................................................................................................................6
Identification ..........................................................................................................................7
Additional biochemical tests ....................................................................................................7
Confirmation of diagnosis........................................................................................................8
Pathogenicity tests .................................................................................................................. 8
Molecular identification ............................................................................................................. 8
Amplification of DNA by PCR ..................................................................................................... 8
Primers .................................................................................................................................. 8
Fatty acid analysis ................................................................................................................... 9
References ............................................................................................................................ 11
APPENDIX 1: Recipes for test media ...................................................................................... 12
NATIONAL DIAGNOSTIC PROTOCOLS
| PAGE 3
Background
Stewart’s wilt is caused by the bacterium Pantoea stewartii subsp. stewartii (synonym Erwinia stewartii)
Symptom recognition
There are two main phases of Stewart’s wilt: seedling wilt and adult leaf blight.
Seedling wilt
Plants may be killed outright. Long water-soaked lesions (linear pale green to yellow streaks with
irregular wavy margins that run parallel to the veins) may extend the length of the leaf on seedlings of
susceptible plants.
Figure 1 Lesions on seedlings
Figure 2 Lesions on seedlings
Figure 3 Seedling wilt
NATIONAL DIAGNOSTIC PROTOCOLS
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When plants are infected systemically, symptoms appear on new leaves emerging from the plant whorl,
and cavities may form in the stalks near the soil line.
Figure 4 Symptoms on new leaves of plant
As the disease progresses, seedlings wilt and
eventually die. Seedlings affected by leaf blight
may continue to produce new leaves as the
older ones wither and die.
NATIONAL DIAGNOSTIC PROTOCOLS
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At advanced stages of plant growth, numerous late leaf infections may result in many small lesions
giving such leaves a scorched appearance. This type of infection is usually most severe toward the
bottom of the plant.
In severely affected plants, a rotted cavity may develop at the base of the stalk (refer Figure 5).
Figure 5 Rotted cavity at the base of the stalk
Adult leaf blight
This normally occurs after tasselling and wilting does not usually develop. The most common symptoms
are leaf lesions.
Figure 6 Leaf lesions
Multiple, coalesced lesions leaf blight caused by the
fungus Exserohilum turcicum, may resemble necrotic leaf
symptoms of the seedling wilt or the leaf blight phases of
Stewart's wilt (Pataky 2004). A simple microscopic
examination of leaf tissue for bacterial ooze can
differentiate Stewart's wilt from non-bacterial disorders
with similar symptoms.
NATIONAL DIAGNOSTIC PROTOCOLS
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Differentiation from other diseases
Stewart's Wilt lesions on plants after tasselling can be confused with lesions caused by Clavibacter
michiganensis subsp. nebraskensis (cause of Goss's Wilt). Differentiating these pathogens will require
additional tests. (Stack et al. 2002)
Table 1 Differences between Stewart’s wilt and Goss’s wilt
Stewart's wilt Goss' wilt
Inoculation of corn flea beetle hail storm
Long irregular lesions yes yes
Leaf freckle symptom no yes
Crown cavity symptom yes no
Vascular discoloration yellow orange
Pathogen Gram negative Gram positive
Symptoms of the fungal disease northern corn leaf blight can often be confused with symptoms of
bacterial wilt, especially late in the season.
Figure 7 Leaf blight lesions
During damp weather, greenish black fungal sporulation is produced in lesions caused by Exserohilum.
On hybrids carrying an Ht2 resistance gene, long yellow to tan lesions with wavy margins and no
sporulation have been observed on infected leaves.
Isolation
Transverse sections of leaf and stem tissue usually exhibit vascular discolouration and production of a
yellowish ooze. Suspend droplets of ooze in sterile water, or macerate suspect tissue in water and streak
to nutrient dextrose agar.
Leaf blight lesions (Figure 7) are long (2 to 13
cm) elliptical in shape with pointed ends,
greyish-green or tan in colour and develop first
on lower leaves. Fungal lesions differ from
bacterial wilt lesions in that they are generally
definite in shape, have greater width and do not
follow leaf veins for extended lengths. The
disease progresses upwards.
NATIONAL DIAGNOSTIC PROTOCOLS
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Identification
Colonies are slow growing (compared with other Erwinia spp.), small, yellow, flat to convex, transparent
with entire edges. If cultured from ooze, a presumptive diagnosis can be made on the basis of this and
the presence of Gram negative, short straight rods.
Additional biochemical tests
Additional tests (O/F, nitrate and gelatin) may be necessary if other pathogens (Burkholderia
andropogonis, Acidovorax avenae subsp. avenae or Erwinia chrysanthemi) are suspected.
Hugh and Leifson’s O/F medium will distinguish Erwinia and Pantoea from Burkholderia and Acidovorax.
See
Table 2 Characters useful for distinguishing between likely pathogenic isolates from wilting
maize
Pantoea
stewartii
Acidovorax
avenae subsp.
avenae
Burkholderia
andropogonis
Erwinia
chrysanthemi
Oxidation/fermentation + – – +
Nitrate reduction – + – +
Gelatin liquefaction – + – +
Pantoea stewartii is closely related to other bacteria in the Erwinia herbicola-Enterobacter agglomerans
complex. Recently, the nomenclature of the genus Erwinia was modified based on chemotaxonomic and
molecular methods, but the taxonomic complexity of this group has not been completely resolved, and a
dual system is used presently. The genus Pantoea was proposed for some strains of the Erwinia
herbicola-Enterobacter agglomerans complex, including E. stewartii, but separation of this group from
other Erwinia species is not fully supported by some methods, such as with 16S RNA sequence analysis
(Pataky, 2004).
Pantoea stewartii is a facultative anaerobic, gram-negative, non-flagellate, non-spore-forming, rod-
shaped bacterium. Culture medium affects colony colour and growth.
NATIONAL DIAGNOSTIC PROTOCOLS
| PAGE 8
Confirmation of diagnosis
Pathogenicity tests
Pathogenicity can be confirmed by stab-inoculating 1 to 2-week old seedlings of a susceptible cultivar,
through a droplet of bacterial suspension, into the basal node. Infected plants should wilt within 14 days
Molecular identification
DNA can be extracted easily from bacterial cultures using any of the commercially available DNA
extraction kits. We use and recommend the DNeasy tissue extraction kit from QIAGEN (Australia) for its
ease of use and reliability. Following the manufacturer’s recommendations, good quality DNA of sufficient
quantity can be extracted without using the optional second elution from the columns.
Amplification of DNA by PCR
Polymerase chain reactions (PCR) were conducted on a Perkin Elmer™2400 thermal cycler using primers
synthesised by Invitrogen™life technology (Mount Waverly, Australia).
All reactions were conducted in a volume of 25 µL and used final concentrations of 1× PCR buffer
supplied, 200 µM dNTPs, 1 mM MgCl2, 1µM each primer and an appropriate Unit of Taq polymerase
(Invitrogen™, Australia).
Primers
To detect Pantoea stewartii subspecies stewartii, primers based on the Coplin et al. (2002) fragment are
used. It is important to ensure that the DNA template is free of any substances that can inhibit the PCR
reaction which eliminates the possibility of a false negative.
Primer name Sequence (5’-3’) Reference
HRP1f GCA CTC ATT CCG ACC AC Coplin et al. (2002)
HRP3r GCG GCA TAC CTA ACT CC
PCR conditions to be used: 1 cycle of 94ºC for 2 mins followed by 35 cycles of denaturing at 94ºC for 30
sec, annealing at 55ºC for 30 sec and extension at 72ºC for 1 min, with an additional extension period of
72ºC for 7 mins.
PCR products were analysed in 1-2% agarose gels in 1 × TBE (90 mM Tris borate pH 8, 2 mM EDTA)
buffer, stained with ethidium bromide, and photographed using the BioRad gel documentation system
1000 under ultraviolet light (BioRad, Australia). Band sizes were determined using a 100 bp DNA ladder
(Promega, Australia).
NATIONAL DIAGNOSTIC PROTOCOLS
| PAGE 9
Fatty acid analysis
Pantoea stewartii can be reliably identified using fatty acid profiles. The predominant fatty acids are
16:0, 18:1 7c and summed feature 3 (mix of 16:1 7c/15 iso 2OH.)
Fatty acid Percent
12:0 3.37
14:0 4.26
unknown 14.502 0.62
16:0 26.86
18:1 7c 39.15
18:0 1.89
Summed Feature 2 7.48
Summed Feature 3 15.65
NATIONAL DIAGNOSTIC PROTOCOLS
| PAGE 10
Figure 8 Range and means of principal fatty acids used to discriminate Pantoea stewartii from other species
0 10 20 30 40 50
12:0
14:0
unknown 14.502
16:0
18:1 w7c
18:0
Summed Feature 2
Summed Feature 3
Pantoea-stewartii-stewartii (Erwinia stewartii)
NATIONAL DIAGNOSTIC PROTOCOLS
| PAGE 11
References
Coplin, DL, Majerczak Dr, Zhang Y, Kim W-S, Jock, S, Geider, K. (2002) Identification of
Pantoea stewartii by PCR and strain differentiation by PFGE. Plant Disease 86, 304-311.
Pataky J, Ikin R. 2003. Pest Risk Analysis - The risk of introducing Erwinia stewartii in maize
seed. The International Seed Federation, Nyon, Switzerland
Pataky JK. 2004. Stewart's wilt of corn. The Plant Health Instructor.
http://www.apsnet.org/education/LessonsPlantPath/StewartsWilt/symptom.htm
Stack J, Chaky J, Giesler L, Wright R. 2002. Stewart's Wilt of Corn in Nebraska. Nebraska
Cooperative Extension Bulletin G02-1462-A. [http://ianrpubs.unl.edu/plantdisease/nf473.htm]
NATIONAL DIAGNOSTIC PROTOCOLS
| PAGE 12
APPENDIX 1: Recipes for test media
Nutrient dextrose agar
Oxoid nutrient agar plus 10 g/L glucose
Oxidation/fermentation test
g/L
Peptone 2
NaCl 5
K2HPO4 0.3
bromothymol blue 0.03
agar 3
water 980 mL (see note)
Glucose, autoclave separately in a small amount of water to give a final concentration of 1% -
eg 10 g in 20 mL water, autoclaved and added to 980 mL of medium.
Adjust pH to 7.1. Distribute 10-15 mL to sterile test tubes or McCartney bottles. Stab inoculate
in duplicate and cover one of each pair with sterile paraffin oil or sterile molten agar. Incubate
at 25-28º C and read daily. Development of a yellow colour in the covered tube indicates
anaerobic fermentation of glucose. (The uncovered tube should also turn yellow).
Nitrate reduction
g/L
peptone 10
NaCl 5
KNO3 2
Agar 3 Adjust pH to 7
Carefully stab inoculate and incubate for 3-5 days at 27º C. Test for presence of nitrite by
adding:
1) 0.5 mL of sulphanilic acid (sulphanilamide 0.625 g in 125 mL of 1:1 water:conc. HCl).
2) 0.5 mL N (1-naphthyl)ethylenediamine HCl (0.5 g in100 mL H2O) This solution is a
BIOHAZARD
A red colour indicates the presence of nitrite. No colour indicates either no reaction (Negative)
or all the nitrate has been reduced to nitrogen gas (Positive).
Add a sprinkle of Zinc dust to the tube. A red colour indicates presence of the original nitrate
(which is reduced to nitrate by the Zn and then detected by the sulphanilic acid) and the
NATIONAL DIAGNOSTIC PROTOCOLS
| PAGE 13
reaction is read as negative. No colour development indicates the complete reduction of nitrate
(Positive).
A simpler method used in our laboratory is to use Merck Merckoquant® Nitrate test strips (Cat
No 1.10020). These have a long shelf life (years longer than on the label, if kept desiccated at
4º C) and no health hazard. A test strip is inserted into the agar mix and read after 30 sec.
Gelatin liquefaction
g/L
Beef extract 3
Peptone 5
Gelatin 120
Steam to dissolve, dispense into test tubes or McCartney bottles and autoclave. Cultures are
stab-inoculated and incubated for up to 14 days. Refrigerate before reading to ensure
liquefaction has occurred.
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