National Diagnostic Protocol

Prepared by Eric Cother and Christine McKenzie

NSW Department of Primary Industries

Agricultural Institute

Forest Road, Orange NSW 2800

Contact : [email protected]

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)

mailto:[email protected]

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


| 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


| PAGE 4

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.


| PAGE 5

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.


| PAGE 6

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.


| PAGE 7

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.


| 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).


| 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


| 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)


| 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]


| 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


| 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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National Diagnostic Protocol