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Resistance to Xanthomonas campestris pv. pelargonii in geranium anddiagnosis of the bacterial blight using polymerase chain reactionShulu Zhang a; Rudrabhatla V. Sairam a; Doni Grefer a; James Feasel a; Mark Ferencak a; Stephen L.Goldman a

a Plant Science Research Center, The University of Toledo, Toledo, USA

First Published:December2009

To cite this Article Zhang, Shulu, Sairam, Rudrabhatla V., Grefer, Doni, Feasel, James, Ferencak, Mark and Goldman, StephenL.(2009)'Resistance to Xanthomonas campestris pv. pelargonii in geranium and diagnosis of the bacterial blight using polymerasechain reaction',Archives Of Phytopathology And Plant Protection,42:12,1109 — 1117

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Resistance to Xanthomonas campestris pv. pelargonii ingeranium and diagnosis of the bacterial blight usingpolymerase chain reaction

SHULU ZHANG, RUDRABHATLA V. SAIRAM, DONI GREFER,

JAMES FEASEL, MARK FERENCAK, & STEPHEN L. GOLDMAN

Plant Science Research Center, The University of Toledo, Toledo, USA

(Received 30 July 2007)

AbstractGeranium bacterial blight is caused by Xanthomonas campestris pv. pelargonii (Xcp). In this study, wetested the susceptibility and resistance of 17 geranium species to infection by Xcp based on both visuallyscored leaf blight symptoms and bacterial detections with Xcp-specific PCR. The initial symptomobservation indicated that most of the tested geraniums are susceptible to Xcp infection with sixexceptions of Geranium maderense, G. pratense, G. sanguineum, G. wallichianum, Pelargonium peltatum,and P. regalia. Of these, G. sanguineum showed delayed, mild symptoms and proved tolerant to Xcpinfection, whereas the other five species displayed few symptoms and are resistant to the infection. UsingPCR, the presence of Xcp bacterium could be detected in G. sanguineum only after the onset ofsymptoms but was never detected in the five resistant species. In contrast, the presence of the bacteriumcould be detected from the susceptible Pelargonium plants before the onset of symptoms. To ourknowledge, this is the first report describing the use of both symptom-based assay and PCR for screeninggeranium bacterial blight resistance.

Keywords: Diagnosis, geranium bacterial blight, PCR, Pelargonium, resistance, Xanthomonascampestris pv. pelargonii

Introduction

Pelargonium and Geranium are two major genera of the Geraniaceae family. The genus

Pelargonium contains more than 250 species commonly known as but mistakenly called

geranium, while the Geranium genus contains more than 300 species, the ‘‘true’’ geraniums

(Lis-Balchin 2002). Geraniums encompassing both Pelargonium and Geranium species have a

great diversity of flowers, scented leaves, variegated leaves and general ease of growth, making

them one of the most popular decorative plants for both indoor and outdoor use (Miller

2002). Zonal geraniums (Pelargonium hortorum) are among the most economically important

bedding plants with over 100 million plants produced and distributed commercially each

year. In addition, geraniums have also been used as herbal medicine and their compounds

incorporated into perfume and cosmetic products. However, due to poor seed produc-

tion, many geraniums are propagated vegetatively and cutting stocks are transported both

Correspondence: Shulu Zhang, Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

Tel: 225-578-5196. Fax: 225-578-2597. E-mail: [email protected]

Archives of Phytopathology and Plant Protection

December 2009; 42(12): 1109–1117

ISSN 0323-5408 print/ISSN 1477-2906 online ª 2009 Taylor & Francis

DOI: 10.1080/03235400701622378

Downloaded By: [Zhang, Shulu] At: 17:24 2 November 2009

nation- and worldwide. Hence disease management and control becomes a serious concern

for quarantine inspection and geranium production (Charlwood and Lis-Balchin 2002).

Geranium bacterial blight, also known as bacterial stem rot or bacterial leaf spot or bacterial

wilt, is caused by Xanthomonas campestris pv. pelargonii, a systemic gram-negative

phytopathogenic bacterium. The bacterial blight is the single most destructive disease of

geraniums and can cause up to 100% loss of geranium (Nameth et al. 1999). In particular,

this bacterium has been reported to infect the zonal geraniums and attempts to identify

resistant cultivars to Xcp have been frustrating (Dunbar and Stephens 1992). Testing

protocols based upon symptom expression alone are inefficient due to the fact that similar

symptoms in geranium can be caused by malnutrition and by other pathogens. This limitation

has resulted in only a small number of Pelargonium and Geranium species being screened for

resistance based on symptom expression alone (Dunbar and Stephens 1992; Nameth et al.

1999; Griesbath and Olbricht 2002). More importantly, many commercially important

geraniums are new hybrids and similarly have not yet been tested for resistance to Xcp

infection.

Some natural resistance to the bacterial blight has been observed in a few Pelargonium/

Geranium species such as Pelargonium domesticum, P. grandiflorum, and Geranium sylvaticum

(Dunbar and Stephens 1992; Griesbath and Olbricht 2002). However, such resistance can be

quite variable in expression as the physiological and environmental conditions of plant growth

are changed (Dunbar and Stephens 1992). Complicating the evaluation further is the fact that

malnutrition can cause the bacterial blight-like symptoms in geranium and the need to

discriminate mimics from infection is essential (Stephens and Tuinier 1989; Nameth et al.

1999). Thus, more reliable methods are needed in order to prevent such a devastating disease

and to identify genetic sources of the bacterial blight resistance in geraniums.

Polymerase chain reaction (PCR) has been used for detecting the presence of pathogens in

plants and has proved to be fast, sensitive and reliable (Manulis et al. 1994; Sulzinski et al.

1996; Chittaranjan and De Boer 1997; Zhang and Goodwin 1997). In this study, we report

the susceptibility and resistance of multiple geranium species to Xcp. The DNA-based, Xcp-

specific PCR detection assay used here first in combination with the symptom-based assay is

reliable and rapid. Further discussion on the use of such bacterial blight diagnosis and

resistance in geranium is also included.

Materials and methods

Plant species and growth

Seeds of Pelargonium and Geranium species (Table I) were obtained from the Goldsmith

Seeds Inc. (Gilroy, California) and from the Germania Seed Co. (Chicago, Illinois). The

seeds were germinated on filter papers wetted with tap water in a 1006 25 mm Petri dish

(Fisher Scientific USA) at room temperature. Seedlings approximately 1–3 cm long were

then transferred to a soil mixture PRO-MIX (Premier Horticulture Inc., USA) in pots in a

greenhouse and grown at 248C with 16-hour daily lighting. The plants at 3 to 6-leaf stage were

transferred to a growth chamber for inoculation and further growth at 288C with lighting of

20–40 mmol photons/m2/s.

Bacterial strains and growth

Three virulent strains of Xanthomonas campestris pv. pelargonii (Xcp), Xcp 514, Xcp 527 and

Xcp 711, were kindly donated by Ms. Margery L. Daughtrey at Cornell University (New

1110 S. Zhang et al.


York, USA). They were stored on a medium containing 5 g of yeast extract, 5 g of peptone, 11

g of glucose, and 15 g of agar per litre at 48C and sub-cultured once a month. To prepare the

bacterial cells for inoculations onto plants, the bacteria derived from a single colony were

inoculated from a plate and grown in 50 ml of TY medium containing 5 g of tryptone, 3 g of

yeast extract, and 0.9 g of CaCl2.2H2O per litre at 288C with shaking overnight. The cells

were pelleted, washed twice with 1x PBS buffer (Sambrook and Russell 2001) and diluted to a

final concentration of about 107 CFU/ml for plant inoculation. The bacterial concentration

(CFU/ml) was determined based on plating serial bacterial dilutions.

Plant infection and pathogenicity test

Two leaves of each plant were inoculated with 2–5 ml of the Xcp suspension at 107 CFU/ml

using a syringe and needle. Three to five plants for each species were inoculated with Xcp.

Pathogen-free buffer (1x PBS buffer) was inoculated into the plants as an inoculated control

and the plants with no injections were used as an un-inoculated control. All plants were then

maintained in the growth chamber for 15–30 days and the symptoms were visually examined

both on days 10 and 30 post-inoculation. Plants were assessed by visually estimating the

percentage of leaf area exhibiting disease symptoms. Assessments were rounded to the nearest

10% and recorded based on averages of 3–5 plants on days 10 and 30 post-inoculation. For

DNA extraction and subsequent PCR analysis, leaves with or without symptoms from the

Xcp-inoculated plants were sampled on days 10 and 30 after inoculation. Both the symptom

assay and PCR detection were repeated at least 2–3 times for confirmation.

For pathogenicity tests, the leaves of P. hortorum horizon coral spice plants with symptoms

were surface-sterilized with 0.5% sodium hypochlorite and ground in sterile 1x PBS

Table I. Responses of 17 species/varieties in two genera of Pelargonium and Geranium to infection by Xcp 514. Plants

were assessed by visually estimating the percentage of leaf area exhibiting disease symptoms on days 10 and 30 after

inoculation. Assessments were rounded to the nearest 10%. The percent 0% stands for no visual symptoms and the

percent 100% represents 100% leaf surface with the symptoms. The bacterium detection was performed with the Xcp-

specific PCR. The symbols ‘‘þ’’ and ‘‘7’’ stand for positive and negative PCR results, respectively. P, Pelargonium;

G, Geranium.

Disease severity (%) PCR test

Plant species Day 10 Day 30 Day 10 Day 30

G. maderense 0 0 7 7G. pratense striatum splish splash 20 10 7 7G. ripple F1 80 100 þ þG. sanguineum 10 30 7 þG. wallichianum buxtons blue 20 20 7 7P. hortorum black velvet rose 80 100 þ þP. hortorum horizon coral spice 90 100 þ þP. hortorum maverick red 60 100 þ þP. hortorum maverick violet 70 100 þ þP. hortorum multibloom bright rose 70 100 þ þP. hortorum multibloom red 80 100 þ þP. hortorum orbit appleblossom 60 100 þ þP. hortorum orbit salmon 70 100 þ þP. hortorum pinto violet 70 100 þ þP. hortorum ringo 2000 salmon 80 100 þ þP. peltatum summer showers burgundy 20 20 7 7P. regalia 10 10 7 7

Resistance and diagnosis of geranium bacterial blight 1111


containing 0.1% (v/v) Tween-20 (Sigma) using a sterile mortar and pestle. The homogenate

was plated onto XCP medium and incubated at 288C. The colonies were then formed and 10

of them were tested using PCR with Xcp-specific DNA primers (see the following section).

The bacteria from three of these colonies were also re-inoculated onto new plants and the

symptom development was re-examined.

DNA isolation and PCR detection

About 100 mg of the leaves from un-inoculated, buffer-inoculated and Xcp-inoculated plants

were excised and ground in liquid N2 in a microcentrifuge tube with a mortar and pestle.

DNA was extracted from these samples using the Plant DNAzol1 Reagent (Invitrogen)

following manufacturer’s instructions. The DNA concentrations were determined with a

DU1 640B spectrophotometer (Beckman) and the quality of the DNA was examined by

agarose gel electrophoresis. The DNA was then used as a template in a PCR to detect the

presence of Xcp in the sample. Primers for XCP1 (50-GAGTGTCCAGTGGCAAGC-30) and

XCP2 (50-GTTGCTGCCTCTTCCTGC-30) specific to a region of the hrp gene of Xcp

(Manulis et al. 1994) were used for amplification of the DNA from the inoculated plant

tissues and bacterial suspension cultures. PCR was carried out with 40 cycles of 948C for

1 min, 588C for 1 min and 728C for 2 min in a PTC-200 DNA Engine (MJ Research, Inc.,

MA). The reaction contained 1X reaction buffer, 200 mM of dNTPs, 0.6 r mole/ml of each

primer, 1.0 ml (ca. 5 ng) of the DNA sample and 0.05 unit/ml of Taq DNA polymerase

following the Invitrogen protocol. PCR products were then separated following agarose gel

electrophoresis, stained with ethidium bromide and recorded with Kodak EDAS 290 image

system (Kodak).

Results

Susceptibility and resistance of geraniums to Xcp infection

Plants of 12 Pelargonium species and 5 Geranium species as listed in Table I were leaf-

inoculated at a 3 to 6-leaf developmental stage with the strain of Xcp 514. Symptoms began to

appear between 72 and 96 hours post-inoculation under the growth conditions described.

Typical bacterial blight symptoms including leaf spots and V-shape lesions were observed on

the leaves of 11 Pelargonium species (Figure 1–A6). The most susceptible is P. hortorum plants

in which the disease severity was visually indexed to greater than 60% (Table I). As the

infection progresses the leaves gradually wilt and the stems rot. By the 10th day posti-

inoculation most parts of the Pelargonium plants died (Figure 1–A3). Interestingly, older

plants, specifically those with 4–10 leaves at the time of inoculation, could survive as long as

30 days after inoculation.

However, six geranium species showed either significantly delayed or mild or atypical

symptoms and these plants could not be discriminated from their respective controls (Figure

1–B and 1–C). In Figure 1–B, the inoculated G. maderense plant (Figure 1–B3) grew as good

as the un-inoculated and buffer-inoculated plants (Figure 1–B1 and B2) and showed no clear

blight symptoms other than alterations in leaf colour from green to dark green or light brown

(see Figure 1–B6). Using the PCR, no Xcp bacterium was detected from the leaf samples

collected on either day 10 or day 30 post-inoculation (see Table I). These observations

indicated that G. maderense species is resistant to the Xcp infection. In contrast, the inoculated

G. sanguineum plant (see Figure 1–C3) could not be discriminated from the control plants

initially (see Figure 1–C1 and C2) but mild symptoms were developed only 20–30 days after



the inoculation under the growth conditions described (Figure 1–C6). The symptoms in G.

sanguineum plants are atypical when compared to the blight symptoms expressed in

Pelargonium hortorum. Atypical indicators include small localised lesions with dark colour

and irregular shapes and sizes (Figure 1–C6). The disease spreads much slowly and no

systemic pathologies like stem or petiole rot were observed. On day 10 after inoculation, no

Xcp cells were detected from the leaves using the PCR (see Tables I and II). However, the

leaves sampled on day 30 post-inoculation were positive for the PCR detection. This indicates

that G. sanguineum is susceptible but with a considerable tolerance to infection by Xcp. Four

other species of G. pratense, G. wallichianum, P. peltatum and P. regalia all like G. maderense

express few symptoms within 30 days of the inoculation. Occasional leaf discolouring along

the edges of some leaves was seen on these plants but the presence of Xcp bacterium was not

detected by PCR. These results together indicated that most of the tested geraniums are

highly susceptible to Xcp infection but a few of them, mostly being perennial geraniums,

Figure 1. An illustration of the visual responses of three geranium species (A, B, and C) to infection by Xcp 514. A, P.

hortorum maverick red; B, G. maderense; C, G. sanguineum. 1, un-inoculated control plants; 2, buffer-inoculated

control plants; 3, Xcp 514-inoculated plants; 4, un-inoculated control plant leaves; 5, buffer-inoculated control plant

leaves; 6, Xcp 514-inoculated plant leaves. All photos were taken on day 10 after inoculation except Figure 1–C6

which was taken on day 30 post-inoculation in order to reveal the symptoms.

Table II. Responses of five geranium species to infection by three Xcp strains of Xcp 514, Xcp 527 and Xcp 711. Plants

were assessed by visually estimating the percentage of leaf area exhibiting disease symptoms on day 10 after

inoculation. Assessments were rounded to the nearest 10%. The percent 0% stands for no visual symptoms and the

percent 100% represents 100% leaf surface with the symptoms. The bacterium detection was performed with the Xcp-

specific PCR. The symbols ‘‘þ’’ and ‘‘7’’ stand for positive and negative PCR results, respectively. P, Pelargonium;

G, Geranium.

Xcp 514 Xcp 527 Xcp 711

Plant species Disease severity (%) PCR test Disease severity (%) PCR test Disease severity (%) PCR test

G. ripple 80 þ 60 þ 90 þG. sanguineum 10 7 0 7 10 7P. hortorum 90 þ 60 þ 90 þP. peltatum 20 7 10 7 20 7P. regalia 10 7 0 7 0 7



including G. maderense, G. pratense, and G. wallichianum, P. peltatum and P. regalia are

resistant to bacterial blight.

PCR detection of Xcp bacteria in leaves with or without symptoms

PCR was used to detect the presence of Xcp 514 in the geranium plants following inoculation.

All plants of P. hortorum and G. ripple gave rise to Xcp-specific 1.2 kbp PCR fragments shown

as PCR positives in Table I and in addition expressed severe symptoms. The presence of the

bacterium was also detected in the symptom-less leaves of both Pelargonium species (see

Figure 2). Alternatively, five other species including G. maderense, G. pratense, G.

wallichianum, P. peltatum, and P. regalia showed little symptoms and failed to test positive

for the 1.2 kbp fragment (Table I). The G. sanguineum plants showed significantly delayed

symptoms and the presence of bacterial DNA was not detected by PCR 10 days after

inoculation (see Tables I and II). However, on day 30 post-inoculation, the leaves became

positive in the PCR test, indicating that the number of Xcp cells in G. sanguineum plants was

increased sufficiently enough to the threshold of the PCR detection at this time. This increase

may be due to the movement and/or multiplication of the localised bacterial cells in the plants.

These results together indicated that the Xcp bacterium could be latent and exist before the

onset of the symptoms in both susceptible geraniums such as P. hortorum and G. ripple and

tolerant geraniums such as G. sanguineum.

Differential responses of geranium species to strains of Xcp bacteria

Three virulent strains (Xcp 514, Xcp 527 and Xcp 711) of Xcp bacteria were used to infect two

Pelargonium and three Geranium species. As shown in Table II, the results from the symptom

Figure 2. An ethidium bromide-stained agarose gel following electrophoresis separation of PCR products. The

presence of a 1.2 kbp PCR product indicates detection of the Xcp bacterial DNA and thus the presence of the

bacterium in those Xcp 514-inoculated plants. DNA for the PCR was extracted either from the leaves with the

symptoms (lanes 1, 3, 5, 7 and 9) or from the symptom-less leaves (lanes 2, 4, 6, 8, and 10) both sampled on day 10

post-inoculation. Lanes 1–2, G. sanguineum; Lanes 3–4, G. ripple; Lanes 5–6, P. hortorum; Lanes 7–8, P. peltatum;

Lanes 9–10, P. regalia. The DNA size markers (kbp ladders, Invitrogen) are shown on the leftmost lane.



observation and PCR analysis indicated that different geranium species respond differentially

to different Xcp strains. Among the Xcp strains screened, the bacterial blight symptoms were

consistent within a given geranium species tested. Among the five plant species, P. hortorum

horizon coral spice is the most susceptible to all three Xcp strains according to the symptom-

based indexing as shown in Table II. Similarly, both G. sanguineum and P. regalia showed

little symptoms on the day 10 post-inoculation and failed to test positive for the presence of

the bacterium at that time. As shown in Figure 1, typical bacterial blight symptoms were

observed in P. hortorum and G. ripple following the infection. In contrast, few lesion spots were

seen on the leaves of G. sanguineum, P. peltatum, and P. regalia plants. In comparing the

infectivity of the three strains, both Xcp 514 and 711 are slightly more virulent than Xcp 527

according to the symptom-based indexing as shown in Table II.

Discussion

Geraniums are one of a few top ornamental plants in the world. Among them, zonal

geraniums are the most economically important bedding plants because of a great diversity of

flowers, scented leaves, variegated leaves and general ease of growth. New hybrids are

produced through conventional breeding using compatible lines every five years or so in order

to meet the high market demand for new varieties. In this study, we chose a total of 10 such

popular geranium hybrids, one to two representatives from each of the more than five

Pelargonium hortorum subgroups. Included subgroups here are Maverick, Multibloom, Orbit,

Pinto, and Ringo. For each hybrid we examined for the first time their susceptibility and

resistance to infection by Xcp. Our results based both on disease severity and on PCR

screening demonstrated that collectively, zonal geraniums are highly susceptible to infection

by the Xanthomonas bacterium. This conclusion is in agreement with what had been reported

earlier using limited cultivars and based on symptoms alone (Strider 1982; Dunbar and

Stephens 1992; Griesbach and Olbricht 2002). However, several species, mostly perennial,

were found to have genetically encoded resistance. Of these, G. maderense, G. pratense, and G.

wallichianum were first reported here in this study while bacterial blight resistance in P.

peltatum and P. regalia has been previously reported (Stephens and Tuinier 1989; Dunbar and

Stephens 1992). This information may be particularly useful for producing bacterial blight-

resistant geraniums through both conventional and non-conventional breeding (Charlwood

and Lis-Balchin 2002).

Geranium bacterial blight caused by Xcp is the single most destructive disease of geraniums

(Nameth et al. 1999). Although this disease has been recognised since 1923 (Brown 1923), at

present there is still no cure for the disease which is a continual threat to the geranium

industry. Thus, proper detection and identification of the bacterial blight in geraniums is

extremely important and essential to prevent and control such a devastating disease. In

particular, geranium bacterial blight symptoms can be quite variable as the physiological and

environmental conditions of plant growth changes (Dunbar and Stephens 1992) and

malnutrition can cause bacterial blight-like symptoms in geranium (Stephens and Tuinier

1989; Nameth et al. 1999). More importantly, the bacterial pathogen Ralstonia solanacearum

race 3 biovar 2, which causes a serious wilt disease in several important agricultural crops such

as potatoes, tomatoes, peppers and eggplants, can also cause Southern wilt in geraniums with

symptoms almost identical to the geranium bacterial blight (Kim et al. 2002). Therefore,

symptom-based assays alone to identify geranium bacterial blight may not be reliable and the

need to discriminate mimics from infection is essential (Stephens and Tuinier 1989; Nameth

et al. 1999). For this reason, both the symptom scoring and PCR screening methods were

used in this study to identify the presence of the Xcp bacterium/disease in plants and



distinguish the susceptible geranium species from the resistant ones. To our knowledge, this is

the first report describing the disease resistance test and detection with such a combinational

method of sensitive PCR detection and traditional symptom-based diagnosis in geranium.

The method is fast and reliable and can be used for quarantine inspection and effective

disease control. The results also provide new and useful information about the susceptibility/

resistance of many horticulturally important geranium species to the geranium industry.

Polymerase chain reaction (PCR) has proved to be a fast and sensitive way to detect

various kinds of pathogens in plants (Zhang and Goodwin 1997; Lopez et al. 2003).

However, there are considerable difficulties to do so in geraniums because the disease is

highly contagious and is strictly regulated by governments and because geranium is one of

those plants being relatively difficult to work with in genetic manipulation. Previously, the

Xcp bacterium has been detected in several geranium species using various PCR-based

methods (Manulis et al. 1994; Sulzinski et al. 1996). Manulis et al. (1994) reported that the

1.2-kb DNA fragment of the hrp gene was specific to 53 strains of Xanthomonas campestris

pv. pelargonii but not to 46 other Xanthomonas bacteria tested. We have also found from our

studies (this study and Zhang et al. 2007) that the hrp gene-derived primers are highly

specific to Xcp and PCR with these primers can detect up to 10 cells of Xcp per reaction

(Zhang et al. 2007). In this study, we used the PCR to screen 17 geranium species/cultivars

for the presence of Xcp and our results further indicated that PCR can be widely used to

detect Xcp in geraniums as early as before the symptoms become visible. Given this,

geraniums may be routinely screened for the presence of the bacterium using PCR as a

predictor of infection and subsequent loss. This will greatly help geranium growers to

prevent and control bacterial blight in geraniums.

Acknowledgements

This work was supported by a UT-USDA-ARS cooperative agreement grant 58-3607-1-193.

We thank Ms. Margery Daughtrey for providing us the bacterial pathogens.

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