Journal of Microscopy and Ultrastructure 2 (2014) 28–33

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Microscopy and Ultrastructure

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Original Article

Immunofluorescence localization and ultrastructure of

Stewart’s wilt disease bacterium Pantoea stewartii in maize

and in its flea vector pulicaria

(Coleoptera: Chrysomelidae)

a,∗,1 b,2 a,3

El-Desouky Ammar , Valdir R. Correa , Saskia A. Hogenhout ,

c

Margaret G. Redinbaugh

a

Department of Entomology, The Ohio State University (OSU), Ohio Agricultural Research and Development Center (OARDC),

Wooster, OH, USA

b

Department of Horticulture and Crop Science, OSU, OARDC, Wooster, OH, USA

c

USDA-ARS, Corn and Research Unit, Wooster, OH, USA

a r t i c l e i n f o a b s t r a c t

Article history: Pantoea stewartii is the causal agent of Stewart’s wilt of sweet corn, the most serious

Received 23 December 2013

bacterial disease of sweet corn and maize in the North-Central and Eastern USA. P. stew-

Accepted 25 January 2014

artii is transmitted mainly by the corn flea beetle Chaetocnema pulicaria (Coleoptera:

Available online 13 February 2014

Chrysomelidae) and this bacterium is assumed to overwinter in its vector beetle. Using

immunofluorescence confocal and transmission electron microscopy (TEM) we localized P.

Keywords: stewartii in its host plant and vector. In infected maize, P. stewartii was found mainly

in xylem vessels of major and minor veins as well as in intercellular spaces of infected

Stewart’s wilt disease

Pantoea stewartii bacterium leaves. In the vector beetle, P. stewartii was found by immunofluorescence in the foregut,

Corn flea beetle midgut and hindgut up to 12 days post-acquisition (post-feeding on infected plants for 2

Chaetocnema pulicaria days). TEM of thin sections in the beetle’s gut four and eight days post-acquisition revealed

Immunofluorescence

bacterial cells similar to those of P. stewartii in the gut lumen, close to or associated with

the gut microvilli, as well as inside epithelial cells of the midgut. This suggests a stronger

biological relationship between this bacterium and its vector, and that the corn flea beetle

may carry P. stewartii intracellularly as well as extracellularly within the gut, which has

implications in the persistence and overwintering of this bacterium in the vector and on

the epidemiology of Stewart’s wilt disease of corn.

Published by Elsevier Ltd. on behalf of Saudi Society of Microscopes.

1. Introduction

Pantoea stewartii subsp. stewartii (syn. Erwinia stewartii)

∗ is a Gram-negative bacterium that is the causal agent of

Corresponding author. Tel.: +1 7724625899; fax: +1 7724625960.

E-mail addresses: [email protected], Stewart’s wilt and blight of sweet corn, the most

[email protected] (E.-D. Ammar). serious bacterial disease of sweet corn and maize in the

1

Present address: USDA-ARS, US Horticultural Research Laboratory,

North-Central and Eastern USA. Significant economic

Fort Pierce, FL, USA.

impact can occur in both sweet and dent corn [1]. Initial

2

Present address: Embrapa Cenargen/Dept., Fitopatologia-

infection of corn leaves causes characteristic watersoaked

Universidade de Brasília, Brasília, DF, Brazil.

3 lesions, followed by systemic infection through the xylem

Present address: Department of Cell and Developmental Biology, John

Innes Centre, Norwich Research Park, Norwich, Norfolk NR4 7UH, UK. vessels [1,2].

2213-879X/$ – see front matter. Published by Elsevier Ltd. on behalf of Saudi Society of Microscopes. http://dx.doi.org/10.1016/j.jmau.2014.01.001

E.-D. Ammar et al. / Journal of Microscopy and Ultrastructure 2 (2014) 28–33 29

P. stewartii is mechanically transmissible to corn plants SP). For transmission electron microscopy (TEM), dissec-

and can be seed transmitted, but plant-to-plant spread ted guts (two /acquisition treatment of four and

does not occur without the presence of the corn flea eight days post-feeding on infected plants, and two insects

beetle Chaetocnema pulicaria Melsheimer (Coleoptera: from healthy controls) in addition to pieces of healthy

Chrysomelidae), and disease incidence is correlated and infected maize leaves were processed as described by

directly with the numbers of corn flea present in Ammar and Nault [8]. Briefly, these specimens were fixed

corn fields [3,4]. The bacteria persist for at least 12 days in in glutaraldehyde and osmium tetroxide and embedded

the beetle’s gut [5] and apparently overwinter in the adult in Spurr’s medium. Ultrathin sections were stained with

corn flea beetle [3]. After it emerges in the spring, the uranyl acetate and lead citrate, and examined using Phips

insect deposits the bacterial cells into feeding wounds via 201 or Hitachi H-7500 electron microscopes.

its feces [3]. The nature of relationship between P. stewartii

and the corn flea beetle is unexplored in large part because 3. Results

of the difficulty of rearing this insect in the laboratory [1].

However, recent evidence suggests that a second type III 3.1. Localization of P. stewartii in maize leaves

secretion system (T3SS) is involved in insect colonization

and persistence of the bacterium in the vector insect [5]. In cross sections of maize leaves, fixed at two to four

In the present work, we further explore host and vector days post-inoculation with P. stewartii, that were immuno-

relations of this pathogen, through ultrastructural studies labeled with antisera to this bacterium, masses of bacterial

and immunolocalization of P. stewartii in both plant and aggregates (fluorescing in green with Alexa Fluor 488)

insect hosts. were detected mainly in small or large xylem vessels of

major and minor veins (Fig. 1A). Additionally, confocal

2. Materials and methods microscopy of unsectioned pieces of infected maize leaves

showed smaller aggregates of bacterial cells dispersed

Corn flea beetle adults (C. pulicaria) were collected from between mesophyll cells (Fig. 1B; compare to control in

corn and weed plants in maize fields in Wooster, OH Fig. 1C). TEM of ultrathin sections of infected maize leaves

◦ ◦

(40 46 = 3 N, 81 54 = 20 W), throughout the maize grow- also showed aggregates of bacterial cells, typical of P.

ing season. To make sure that these beetles were free from stewartii, in xylem vessels (Fig. 1D and E) or dispersed inter-

P. stewartii, collected adults were held for at least 10 days cellularly in the chamber below guard cells (Fig. 1F) or

on healthy maize seedlings (Zea mays L. var. rugosa, cv. between mesophyll cells (Fig. 1F and G). These bacterial

◦

␮

‘Seneca Horizon’) at 27 C and 50% relative humidity with a cells, with a cell wall, were rod-shaped, ca. 0.5 m wide and

␮

16-h photoperiod. These seedlings were then tested for P. up to 2 m long. Semi-opaque fibrous material was abun-

stewartii using colony forming unit tests as described ear- dantly found either attached to the bacterial cell walls or

lier [5]. Only beetles that proved to be free of P. stewartii surrounding them (Fig. 1D–G). Patches of dark (electron-

were used in this study. The bacterial strain used (wild-type opaque) material were also found especially attached to

strain DC283), its growing conditions on artificial media, the cell walls of infected xylem vessels (Fig. 1E). Some

and mechanical inoculation of corn leaves by P. stewartii, of the bacterial cells were very dark, probably degener-

were as described by Correa et al. [5]. ated (Fig. 1D–G). Also, whereas some of the mesophyll

For bacterial acquisition, corn flea beetle adults that cells looked normal, with intact plastids and other cells

proved to be free of P. stewartii as described above were organelles (Fig. 1G), some mesophyll cells were electron-

exposed for two days to maize plants infected with P. stew- opaque and seemingly degenerated (Fig. 1F).

artii then transferred to healthy maize seedlings every two

days. Individual beetles were harvested for dissections at 4, 3.2. Localization of P. stewartii in the corn flea beetle

8 and 12 days after their transfer to healthy plants. Beetles organs

were dissected under a stereomicroscope in 0.01 M potas-

sium phosphate buffer, pH 7, using two fine-tip forceps to Dissected organs of the corn flea beetles immunoflu-

extract their guts (alimentary canals) as described [5]. Diss- orescently labeled with antisera to P. stewartii and Alexa

ected guts and other organs that were connected/attached Flour 488 are shown in Fig. 2 (with Fig. 2A showing the

to them, including nerve ganglia and reproductive organs, foregut and midgut from an insect fed on healthy con-

of at least ten beetles/acquisition treatment (plus ten bee- trol plants). In beetles dissected four days post-feeding on

tles fed on healthy uninfected control plants), as well P. stewartii infected plants, large aggregates of this bac-

hand-cut cross sections of healthy and infected maize terium were found in the foregut, midgut, and hindgut,

leaves, were processed for immunofluorescence laser scan- with smaller aggregates in the Malpighian tubules (Fig. 2B).

ning confocal microscopy generally as described by Ammar But none of these bacterial aggregates were found in other

and Hogenhout [6,7]. This procedure involved fixation for organs examined including nerve ganglia and reproduc-

4 h in 4% paraformaldehyde, incubation for 3 h with 1:500 tive organs (data not shown). In organs dissected eight to

dilution of P. stewartii antiserum [5], incubation for 1 h in twelve days post-feeding on P. stewartii infected plants,

a 1:600 dilution of goat anti-rabbit IgG conjugated with smaller aggregates of bacteria were found mainly in the

Alexa Fluor 488 (Molecular Probes, Eugene, OR), followed midgut (Fig. 2C and D) and hindgut, but very little in the

by staining for 5 min with 3 nM of the nuclear stain pro- foregut (Fig. 2C), and none were found in other organs

pidium iodide (Molecular Probes). Samples were examined examined including reproductive organs and nerve gan-

using a confocal laser scanning microscope (Leica TCS glia (Fig. 2C). Virtual sections of the midgut, using confocal

30 E.-D. Ammar et al. / Journal of Microscopy and Ultrastructure 2 (2014) 28–33

Fig. 1. Localization of Pantoea stewartii bacterium in maize leaf cells by immunofluorescence confocal microscopy (A–C) and by transmission electron

microscopy of thin sections (D–G). (A) Cross section in an infected maize leaf showing dense bacterial aggregates (green) mainly in the xylem vessels of a

major vein (arrowheads) and in minor veins (arrows). (B) Topical view of a leaf piece from an infected maize plant, showing smaller aggregates of bacterial

cells (green) dispersed between mesophyll cells (arrows). (C) Topical view of a leaf piece from a healthy uninfected maize plant. (D and E) Bacterial cells

(arrows) in xylem vessels. (F and G) Bacterial cells (arrows) in intercellular spaces under guard cells (gc) or between mesophyll cells (mc), some of which

appeared degraded (dc). In D–G, arrowheads indicate darker, probably degenerated, bacterial cells. Other abbreviations: cw, cell wall; dm, dark staining

material; fm, fibrous material; n, cell nucleus; xy, xylem.

microscopy, indicated that at eight days post-acquisition, eight days post-feeding on infected plants, very few bacte-

P. stewartii was probably located in the gut wall epithelium rial cells were found in the food boluses, some were found

rather than the gut lumen (Fig. 2D). in the lumen close to, or associated with, the microvilli, but

Fig. 3 shows electron micrographs of ultrathin sections many of these bacterial cells were found inside the cyto-

in the gut of corn flea beetles that had fed on healthy maize plasm of the midgut epithelial cells (Fig. 3D and E). Also

(Fig. 3A), or those dissected at four to eight days post- here, a small vacuole/vesicle was found surrounding each

acquisition feeding on infected plants (Fig. 3B–E). At four bacterial cell (Fig. 3D and E). However, no bacterial cells

days post acquisition, aggregates of bacterial cells typical of were found in the nuclei of these infected gut cells (Fig. 3E)

P. stewartii that were found in infected plants (Fig. 1D–G) or in other tissues surrounding the gut (Fig. 3C and D). No

were observed in the food boluses in the midgut (Fig. 3B bacterial cells were found in the gut lumen or gut epithe-

and C inset). Some of these bacterial cells were also found lial cells of beetles that fed on healthy control maize plants

in the gut lumen very close to, or associated with, the (Fig. 3A).

microvilli lining the midgut (Fig. 3B), and some appeared

to be embedded in the cytoplasm of midgut epithelial cells 4. Discussion

with a small vacuole/vesicle surrounding each bacterial cell

(Fig. 3C). Some of these bacterial cells seemed to be divid- Previous ultrastructural studies on maize plants

ing (Fig. 3C inset). In the midgut of flea beetles dissected infected with P. stewartii indicated that this bacterium

E.-D. Ammar et al. / Journal of Microscopy and Ultrastructure 2 (2014) 28–33 31

Fig. 2. Immunofluorescence confocal micrographs of Pantoea stewartii bacteria (green/yellowish green) in dissected guts of its vector beetle. A. The foregut

(fg) and midgut (mg) in a beetle fed on healthy control plants, showing only the propidium iodide staining (red). B. Gut dissected 4 days post-feeding on

infected maize, showing bacterial aggregates (green) in the foregut (fg), midgut (mg), hindgut (hg) and Malpighian tubule (mt). (C and D) Guts dissected

8 days post-feeding on infected maize, showing bacterial aggregates mainly in the midgut (mg), with very few in the foregut (fg); ng, nerve ganglia; in D,

confocal longitudinal section in the midgut indicates that the bacteria is mainly associated with the gut wall (gw) rather than in the gut lumen (lu).

resides mainly, and is probably transported through the we reported, through immunofluorescence labeling, that

plant, in xylem vessels, although it can be found in intercel- this bacterium persists in the midgut and hindgut for up

lular spaces of mesophyll cells during early infections [1,2]. to 12 days post-feeding on diseased plants [5]. In the

To the best of our knowledge, the present study provides present work, TEM of dissected organs showed bacterial

the first confirmation of these findings using immuno- cells, morphologically similar to those of P. stewartii, in

fluorescence labeling in addition to ultrastructural studies the lumen close to or associated with the microvilli, as

of infected maize leaves. The semi-opaque fibrous mate- well as inside epithelial cells of the midgut 4–8 days post-

rial, abundantly found either attached to the bacterial cell feeding on P. stewartii-infected plants. That P. stewartii

walls or surrounding them in our study, was referred to bacterium may be localized intracellularly as well as extra-

by Braun [2] as fibrillar exopolysaccaride (EPS), whereas cellularly in the gut of its vector beetle is further supported

the dark staining material we found close to the wall of by immunofluorescence confocal microcopy that showed

infected xylem vessels was referred to as host derived immunolabeled bacterial cells mostly associated with the

material (HDM) by Braun [2] who suggested that it may midgut wall (epithelium) rather than the midgut lumen

function in disease resistance. Bacterial EPS, on the other eight days post-feeding on diseased plants (Fig. 2D). These

hand, are thought to play an important role in the devel- findings suggest that the corn flea beetle may not pas-

opment and pathogeneses of bacterial wilts, and that EPS sively carry P. stewartii in its gut, but may also be infected

(or slime) production is correlated with aggressiveness of by this bacterium. Recent studies indicated that, in addi-

several welt-inducing bacteria [2,9]. Once established in tion to the Hrc-Hrp T3SS (secretion system) known to be

the xylem, P. stewartii is known to grow to high cell densi- essential for maize pathogenesis, P. stewartii has a sec-

ties and establish dense biofilms encased in an EPS capsule ond T3SS (PSI-2) that is required for persistence in its flea

and slime, called ‘stewartan’, that are thought to block the beetle vector [5]. Interestingly, PSI-2 belongs to the Inv-

water flow in the xylem and lead to wilting and death of Mxi-Spa T3SS family, typically found in pathogens

the plant [1]. [5], which is consistent with our present findings. Our

So far, vector relations of P. stewartii with its flea beetle results, however, suggest that P. stewartii invades only the

vector are poorly understood. In a previous investigation gut epithelium of the vector beetle but does not seem to

32 E.-D. Ammar et al. / Journal of Microscopy and Ultrastructure 2 (2014) 28–33

Fig. 3. Transmission electron micrographs of thin sections in the midgut of beetles that fed on healthy maize (A) or those dissected either 4 days (B and

C) or 8 days (D and E) post-feeding on maize leaves infected with Pantoea stewartii. (B and C) Bacterial cells (arrows) in a food bolus (fb), in the midgut

lumen (lu) close to or associated with the gut microvilli (mv), or embedded/vesiculated in the cytoplasm (cy) of gut epithelial cells; inset in B shows higher

magnification of bacterial cells (b), one of which appears to be dividing (db). (D and E) Many bacterial cells (arrows) embedded in the cytoplasm (cy) of

gut epithelial cells, with a white space/vesicle around each cell; some bacterial cells can also be seen in the lumen (lu) especially close to the gut microvilli

(mv). Other abbreviations: mi. mitochondria; mu, muscle tissue; nu, nucleus.

infect other surrounding tissues. The occurrence of a vesi- This is the first report that suggests that P. stewartii may

cle around each intracellular bacterial cell in the beetle’s invade the gut epithelium of its flea beetle vector. How-

gut suggests that the vector beetle may have a defense ever, the related bacterium, Erwinia tracheiphila the causal

mechanism to localize the bacterial infection and to pre- agent of bacterial wilt in cucurbits persists in the gut of

vent this bacterium from spreading further into other its beetle vector Acalymma vittatum up to 30 days post-

tissues. feeding on diseased cucumber and may also invade vector

E.-D. Ammar et al. / Journal of Microscopy and Ultrastructure 2 (2014) 28–33 33

cells [10,11]. Bacterial cells similar to E. tracheiphila were [2] Braun EJ. Ultrastructural investigation of resistant and suscepti-

ble maize inbreds infected with Erwinia stewartii. Phytopathology

found by TEM inside epithelial cells of the hindgut of bee-

1982;72:159–66.

tles that had fed on diseased plants [11]. Another plant

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pathogenic bacterium, Dickeya dadantii (Erwinia chrysan- pendium of corn diseases. St. Paul, MN, USA: APS Press; 2000. p.

3–9.

themi), which causes soft rot diseases on many crops, is

[4] Menelas B, Block CC, Esker PD, Nutter FW. Quantifying the feeding

pathogenic to the pea aphid Acyrthosiphon pisum when

periods required by corn flea beetles to acquire and transmit Pantoea

acquired orally or by septic injury [12]. Furthermore, a stewartii. Plant Disease 2006;90:319–24.

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related bacterium Erwinia aphidicola, that resides in the gut

SA, et al. The bacterium Pantoea stewartii uses two different type III

of A. pisum, was reported to infect aphid guts and to prevent

secretion systems to colonize its plant host and insect vector. Applied

post-final ecdysis growth of the insect [13]. Further stud- and Environmental Microbiology 2012;78:6327–33.

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and in host plants. Annals of the Entomological Society of America

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Conflict of interest

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ezic FL. Alimentary canal of adult Acalymma vittata (Coleoptera:

None declared.

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