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A novel indicator plant to test the hypersensitivity of phytopathogenic

bacteria

Article  in  Journal of Microbiological Methods · February 2008

DOI: 10.1016/j.mimet.2007.11.002 · Source: PubMed

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Sharan Umesha Patricia A Richardson

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Journal of Microbiological Methods 72 (2008) 95 – 97

www.elsevier.com/locate/jmicmeth

Note
A novel indicator plant to test the hypersensitivity of
phytopathogenic bacteria
S. Umesha, P.A. Richardson, P. Kong, C.X. Hong ⁎
Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University,
Hampton Roads Agricultural Research and Extension Center, 1444 Diamond Springs Road, Virginia Beach, VA 23455,USA
Received 8 September 2007; received in revised form 19 October 2007; accepted 2 November 2007
Available online 17 November 2007

Abstract

Hypersensitive response is an important, definitive test to separate plant pathogenic bacteria from saprophytes. The current standard protocol
uses tobacco and four o’clock plants as indicators for Gram negative and Gram positive phytopathogenic bacteria, respectively, and inoculation is
accomplished by infiltrating bacterial suspensions into intact leaves. Both plants, especially the four o’clock, have thin leaves which make
inoculation difficult, sometimes leading to inaccurate tests. Here we propose the use of Sedum hybridum as an alternative indicator plant. Sedum
plants are readily available, easy to propagate and fast growing. Their leaves are much thicker, thus infiltration of a bacterial suspension is easier
compared to tobacco and four o’clock. Additionally, sedum plants can be used universally to test both Gram negative and Gram positive
phytopathogenic bacteria.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Hypersensitivity; Indicator plant; Leaf infiltration; Phytopathogenic bacteria; Sedum

All phytopathogenic bacteria produce a hypersensitive reac-
tion (HR) in leaf mesophyll tissue. Saprophytes allegedly do not
induce this reaction, making HR a quick and useful determi-
native test to differentiate saprophytes from plant pathogens
(Braun-Kiewnick and Sands, 2001). The reaction is invaluable
for the characterization of phytopathogenic bacteria recovered
from outside their host or recovered from a host with latent
infections (Gitaitis, 1990). Most Gram negative phytopatho-
genic bacteria produce hypersensitivity in tobacco (Nicotiana
tobaccum L.) and gram positive bacteria produce hypersensi-
tivity in four o’clock (Mirabilis jalapa L.) plants. Unfortunately,
many do not respond, or like tobacco, are inconsistent in the
expression of HR when their leaves were infiltrated with
suspensions of Clavibacter michiganensis ssp. michiganensis
(Gitaitis, 1990). Injecting bacterial inoculum between the veins
of tobacco leaves is easier than in four o’clock, however, this is
a cumbersome process requiring a skillful hand. Often the
injection of a bacterial suspension into the four o’clock plant is
⁎ Corresponding author.
E-mail address: chhong2@vt.edu (C.X. Hong).
0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2007.11.002
not possible because of their extremely thin and delicate leaf
nature.
We found sedum (Sedum hybridum L. family: Crassulaceae)
plants a potentially ideal indicator for the hypersensitive test
when testing the pathogenicity of bacterial isolates recovered
from diseased sedum, iris and irrigation water retention basins.
While inoculating the bacterial suspension into leaves of sedum,
we found it very easy to infiltrate the intact leaves of sedum
plants. This study was taken up to assess the feasibility and
universality of sedum as an indicator for both Gram negative
and Gram positive phytopathogenic bacteria.
Stock sedum plants were donated by a local ornamental
nursery in eastern Virginia. Sedum plants were propagated in
controlled climatic conditions, by placing mature cuttings into
15 cm diameter plastic pots filled with a potting mix (Sungro
Metro Mix 200, WA,USA) which has excellent air porosity and
water retention. These cuttings were incubated with watering
once daily in a glasshouse at 35/25 °C and 4500 lum/ft2. All the
sedum twigs planted in the Metro mix have grown properly with
successful rooting and new leaf initiatives within 7 days. The
succulent sedum plant can easily be propagated by this asexual
method.
96 S. Umesha et al. / Journal of Microbiological Methods 72 (2008) 95–97

A total of 30 different bacterial cultures comprising of Gram
negative, Gram positive phytopathogenic and saprophytic bact-
eria (Table 1) were used to assess the feasibility and universality
of sedum as an indicator for the HR test. The Gram negative
phytopathogenic bacterial genera include Erwinia, Xanthomo-
nas, Pseudomonas, Ralstonia species along with one unknown
pathogenic bacteria. The Gram positive phytopathogenic bacte-
rial genera include Bacillus and Clavibacter along with one
unknown genus. The non-pathogenic/saprophytic Pseudomo-
nas fluorescens and one unknown bacterial saprophyte were
included in the present study. All the cultures were stored in
glycerol at − 80 °C, and bacteria were subcultured in Luria–
Bertani (LB) broth (Bactotryptone 10 g, Bactoyeast extract 5 g,
NaCl 10 g in 950 ml sterile distilled water, pH 7) and shaken for
24 h in a shaker incubator (G24, Environmental Incubator
Shaker, New Brunswick Scientific, Edison, NJ, USA). The cell
suspensions from 24 h cultures of all 30 bacterial isolates were
washed three times by centrifuging at 4000 g for 10 min and
resuspending pellet in sterile distilled water. The optical
density (OD) of the bacterial suspension was adjusted to 0.45
(A610 nm) with a UV–visible spectrophotometer (DU 800,
Beckman Coulter, USA) (Mortensen, 1999) to obtain approxi-
mately 1 × 108 cfu/ml. Similarly, 1 × 106 cfu/ml and 1 × 104 cfu/
ml bacterial suspension was prepared.
An aliquot of 200 μl bacterial suspensions were injected into
the adaxial surface of fully expanded sedum leaves using a 30 g
needle (B–D, NJ, USA) and syringe (Klement, 1963, 1983).
These plants were maintained in a glass house under climate
controlled conditions and observed for typical symptoms of
hypersensitive response in the form of necrosis, yellowing of the
infiltrated area and leaf death. All the experiments were carried
out in four replicates plants and repeated four times with the pots
arranged in a randomized block design. All known phytopatho-
genic bacteria induced necrosis in sedum plants within 24 h after
infiltration of bacterial suspension at 1 × 106 cfu/ml (Table 1).
Typical HR symptoms by Gram negative and positive bacteria
are illustrated in Fig. 1A and B. After 3–4 days the tissue
infiltrated with the pathogenic strains became entirely dry and
collapsed. However, the distilled water (diluent) did not induce
any necrosis and plants look unchanged even after two weeks
(Fig. 1C). Similarly, leaves infiltrated with saprophytic bact-
eria at 1 × 108 cfu/ml were unchanged and apparently healthy
(Fig. 1D).
The detection of pathogenicity with this method is based on
the hypersensitive response of sedum leaves. A hypersensitive
response of a similar nature has already been described for
tobacco leaves (Klement, 1963), bean pods (Klement and Lov-
rekovich, 1961) and four o’clock plant leaves (Gitaitis, 1990)

Table 1
Origin and hypersensitive response of phytopathogenic bacteria in sedum plants
Species Isolate Host Country Year of Gram's HR Supplier (Ref)
isolation reaction
Bacillus megaterium 4314 Soil USA 2003 + +
Clavibacter michiganensis ssp. michiganensis 4111 Lycopersicon esculentum India 2003 + + SU (UoM 1)
Erwinia amylovora 4317 Pyrus communis USA 2007 − +
E. amylovora 4312 Nerium oleander USA 2007 − +
E. caratovora 4211 Solanum tuberosum USA − + DN (245)
E. caratovora 4215 Water USA 2000 − + DN (250)
E. caratovora pv. Atroseptica 4212 Solanum tuberosum USA − + DN (246)
E. caratovora pv. Atroseptica 4367 Iris sp. USA 2007 − +
E. caratovora pv. Atroseptica 4365 Sedum sp. USA 2007 − +
E. chrysanthemi 4213 Chrysanthemum sp. USA − + DN (244)
E. chrysanthemi 4214 Water USA 2000 − + DN (248)
E. chrysanthemi 4313 Begonia sp. USA 2007 − +
Pseudomonas fluorescens 4114 Pennisetum glaucum India − − SU (UoM 6)
P. syringae pv. syrinage 4263 Phaseolus sp. USA 1970 − + BV (BV3 B728a)
P. syringae pv. syrinage 4368 Iris sp. USA 2007 − +
P. syringae pv. syrinage 4363 Water USA 2007 − +
P. syringae pv. Tomato 4216 Lycopersicon esculentum USA 2005 − + BV (BV40kuzzen)
P. syringae pv. Tomato 4262 Lycopersicon esculentum USA 1960 − + BV(BV 1DC3000)
Ralstonia solanacearum 4113 Solanum melangina India 2004 − + SU (UoM 5)
R. solanacearum 4316 Lycopersicon esculentum USA 2007 − +
Serratia liquefaciens 4315 Soil USA 2003 − +
Xanthomonas sp. 4217 NA USA 2005 − + BV (BV 88 L298)
Xanthomonas sp. 4218 NA − + BV (BV 87)
X. axonopodis pv. Malvacearum 4115 Gossypium sp. India 2003 − + SU (UoM 3)
X. maculogardenia 4216 Gardenia sp. − + BV (BV37)
X. vesicatoria 4112 Lycopersicon esculentum India 2003 − + SU (UoM 4)
X.oryzae pv. Oryzae 4115 Oryzae sativa India 2004 − + SU (UoM 2)
Unknown 4267 Irrigation water USA 2004 + +
Unknown 4364 Irrigation water USA 2007 − +
Unknown 4363 Irrigation water USA 2007 − −
All the bacterial cultures were subcultured in LB broth and 24 h-old bacteria were subjected for Gram's reaction by KOH solubility test, infiltrated in to adaxial surface
of sedum leaves and observed for the appearance of HR. DN = David Norman, University of Florida, Apopka, FL, USA; BV = Boris Vinatzer, Virginia Tech,
Blacksburg, VA, USA; SU = S. Umesha, Department of Applied Botany and Biotechnology, University of Mysore, Mysore, India. NA = Not Available.
 S. Umesha et al. / Journal of Microbiological Methods 72 (2008) 95–97
Fig. 1. Sedum leaves showing hypersensitive response 24 h after infiltration with (A) Gram negative and (B) Gram positive phytopathogenic bacteria. No response was
detected with infiltration of (C) diluent; and (D) saprophytic bacteria.
infected with Gram negative or Gram positive phytopathogenic
bacteria. Infiltration of four o’clock plant leaves is difficult
because of the thin leaf nature. As a succulent plant, water-
stored sedum leaf is considerably thick and bacterial cells can
easily be infiltrated. It is suspected that the microclimate in
sedum leaf mesophyll offers congenial conditions for bacterial
multiplication, which ultimately results in the expression of HR.
At this time, researchers use different indicator plants to test the
pathogenicity of Gram negative and Gram positive phytopatho-
genic bacteria. This study has shown that both Gram positive
phytopathogenic bacteria, Clavibacter michiganensis ssp mi-
chiganensis and Bacillus megaterium, and a large number of
Gram negative phytopathogenic bacterial cultures induced HR
in sedum. These plants grow rapidly and can be easily pro-
pagated. Hence, this research provides a universal system,
which until now was unavailable, that can be used to test the HR
of all phytopathogenic bacteria, irrespective of the Gram nature.
Acknowledgements
This research was supported through an Overseas Associate-
ship by Department of Biotechnology, Ministry of Science and
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97
Technology, Government of India, New Delhi to the senior
author. We thank Dr. Boris Vinatzer, Fralin Biotechnology
Center, Virginia Polytechnic Institute and State University, and
David Norman, University of Florida, Apopka, FL 32703, USA
for providing the cultures.
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