Cross-infectivity of Ralstonia solanacearum from
marigold grown in Andaman Islands

K. Sakthivel, V. Baskaran, K. Abirami, K. Manigundan and R.K. Gautam
ICAR-Central Island Agricultural Research Institute, Port Blair - 744 101

Andaman & Nicobar Islands, India
E-mail: vbaski01@gmail.com

ABSTRACT

Bacterial wilt disease, caused by Ralstonia solanacearum, is one of the major concerns
for marigold cultivation in Andaman Islands. Cross-infectivity potential of the bacterial
wilt pathogen, isolated from marigold, was tested in other common vegetable-hosts of
the Island. Pathogen identity was confirmed by morphological identification and Biolog
based phenotypic fingerprinting. Cross-infectivity tests revealed tomato to be the most
susce ptible among the three  solanace ous hosts tested. Highest wilt incidence was
observed in tomato and marigold (100%) plants, followed by 55.6% in brinjal and 22.3%
in chilli, upon artificial soil inoculation. Our study enlightens pathogenic potential of the
bacterial wilt pathogen in important vegetable crops of Andaman Islands and can help
formulate suitable management practices for successful management of the pathogen.

Key words: Marigold, Ralstonia solanacearum, bacterial wilt, Andaman Islands

INTRODUCTION
Marigold is one of the most popular traditional

crops grown in Andaman and Nicobar Islands. It has
gained popularity due to ease in its culture, wide
adaptability, profuse flowering, short juvenility, large
blooming period, attractive color, shape, size and good
keeping quality, attracting the attention of flower
growers. Demand for marigold flowers is increasing
very rapidly among the local farmers. One of the major
constraints faced in cultivation of marigold in the Island
is wilt disease which is highly prevalent due to the
climatic conditions prevalent on the Island. Yield loss
due to the bacterial wilt disease caused by the pathogen,
Ralstonia solanacearum, is one of the major concerns
for farmers in marigold cultivation. It is a gram-
negative, soil-borne bacterium belonging to the 5 sub-
division of Proteobacteria (Yabuuchi et al, 1995) and
has been described as a ‘species complex’ due to its
complex phenotypic and genotypic diversity (Palleroni
and Doudoroff, 1971).  It has a wide host range that
includes over 450 plant species belonging to about 50
families (Hayward, 1991) and is spread worldwide in
tropical, sub-tropical and temperate regions. R.
solanacearum species were classified into five races

based on host range (Buddenhagen et al, 1962), and
six biovars based on utilization of disaccharides and
hexose alcohols (Hayward, 1964). On the molecular
basis, the species is divided into four phylotypes (Fegan
and Prior, 2005) corresponding to four, broad genetic
groups, each related to a geographic origin (Phylotype
I: Asian origin; Phylotype II: American origin;
Phylotype III: African origin, and Phylotype IV:
Indonesian/Australian origin). Each phylotype can be
further subdivided into ‘sequevars’ (sequence variants)
according to the nucleotide polymorphism of the egl
gene (Fegan and Prior, 2005). Solanaceous vegetables
are among the major horticultural crops grown in the
Island for livelihood security of farmers. Land area
available for cultivation is limited in the Island and,
hence, farmers grow flowers and vegetables as mixed
cropping. Yie ld loss is notice d in Solana ceous
vegetables due to the wilt disease. Therefore, we
attempted a study on the cross-infectivity of the
pathoge n isola te d from marigold in c ommon
solanaceous vegetables of the Island, viz., brinjal,
tomato and chillies, to ultimately help in
                   Isolation of bacteria was done by the
method of by Kelman (1954). Briefly, stem pieces

J. Hortl. Sci.
Vol. 11(2): 179-181, 2017

Short communication



180

of 2-3cm excised from infected plants were washed
five times in sterilized, deionized water and blot-
dried for 15 min on an autoclaved paper towel. Stem
pieces were then placed in test tubes containing 5
ml sterile water (for bacterial exudation) for about
5-10 min. A loopful of ooze was then streaked onto
CPG agar (gL-1, Casamino acids 1; Peptone 10;
Glucose 10; Agar 15; pH 7.2) amended with 2,3,5
triphenyl tetrazolium chloride (1%), and incubated
for 36-48 hrs at 28-30°C. Fluidal, white colonies
with a pinkish centre were picked up and maintained
in 15% glyce rol stock at -80 C for long-term
storage.
Biolog based phenotypic fingerprinting

All the isolates were subjected to Biolog-
based phenotypic fingerprinting, using the Microlog
system (Biolog, Inc., Hayward, CA). The bacterial
solution (pre pa re d as per the  ma nufa cture r ’s
instructions) was pipetted into each of the 96 wells
in the  Biolog micropla te, and the pla te s we re
incubated at 28-30 C for 16–24h and read using
an automated plate reader (Biolog, Inc.).

Cross-infectivity assay

A virulent isolate of Ralstonia solanacearum
from the  ma rigold pla nt wa s  use d for cr oss -
inoculation assay with other important solanaceous
hosts, viz., tomato (Solanum lycopersicum), brinjal
(Solanum melongena) and c hillie s ( Capsic um
annuum). Soil inoculation technique suggested by
Kumar (2006) was followed, as described earlier,
and the treated plants were incubated at 28-32°C
under glasshouse conditions. Three replications of
each crop were maintained in each pot, and, for
each crop three pots were used. Wilt incidence was
recorded at regular intervals upon inoculation with
ba cterial suspension, a nd wilt perce ntage wa s
calculated using the following formula:

W ilting percentage =       N um ber of plants wilted           x100
Total num ber of plants inoculated

Bacterial colonies isolated from infected
marigold plants were confirmed by both morphological
and biological (biochemical phenotypic) approaches.
Morphologically, the isolates were identified as R.
solanace arum by the ir typic a l fluidal c olony
morphology (Kelman, 1954) showing a spiral, pink
centre on CPG agar. Further, the identification was
confirmed using 71 carbon sources and 23 chemical
sensitivity assays using BIOLOG based phenotypic
fingerprinting, which confirmed the identity of the
virulent isolate as R. solanacearum (>0.80 similarity
coefficient). The isolate was named MgA_Rs1 and

stored as a water slant, and glycerol stock at 20°C,
for short-term and long-term storage, respectively.

Cross-infectivity assay pe rformed with
marigold Ralsonia solanacearum isolate (MgA_Rs1)
on three important solanaceous vegetables, viz., tomato,
brinjal and chilli revealed that MgA_Rs1 could infect
all three hosts, with minor variation in incubation time.
Kumar et al (2006) also reported that Ralstonia
solanacearum isolates from ginger could induce wilt
in other commercially-important zingiberaceae plants
like small cardamom, large cardamom and turmeric,
when inoculated in the greenhouse. Our result also
revealed that the virulent strain isolated from marigold
could induce wilt symptoms in tomato and marigold
plants relatively earlier (7 days post-inoculation)
compared to that in brinjal (12 dpi) or chilli (20 dpi)
upon artificial inoculation (Table 1). In marigold and

J. Hortl. Sci.
Vol. 11(2): 179-181, 2017

Sakthivel et al

Table 1. Cros s-p athogen icity of  Rals tonia
solanac e arum isolate d fr om  m ar igold on
solanaceous Vegetables

Crop
Marigold
Tomato
Brinjal
Chilli

Days to first wilt
7
7
12
20

Percent wilt incidence
100
100
55.5
22.3

tomato, average wilting percentage observed was
100%, as, it could cause wilt in all the nine plants
inoculated within two weeks of incubation, whereas,
in the case of brinjal and chilli, the wilt percentage
was 55.6% and 22.3%, respectively (Fig. 1). MgA_Rs1

could induce wilt in 5 plants out of nine brinjal plants
and in two plants in chilli at five week intervals. Earlier,
Mondal et al (2011) reported that R. solanacearum
isolated from marigold (host) could infect solanaceous
vegetables like tomato and brinjal within 12-15 days

Fig  1. Weekly p rogre ss of wi lt of mari gold  cau sed b y a
Ralstonia solanacearum isolate on various solanaceous crops

C hilli



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Hayward, A.C. 1991. Biology and epidemiology of
ba cte ria l wil t ca us ed by Ps e udomonas
solanace ar um . Annu. R ev. Phytopathol .,
29:65-87

Kelman, A. 1954. The relationship of pathogenicity in
Ps e udomonas  solanac e arum  to colony
appearance on a tetrazolium chloride medium.
Phytopathol., 44:693-695

Kumar, A. 2006. Methods for sc reening ginger
(Zingiber officinale Rosc.) for bacterial wilt
resistance. Indian Phytopathol., 59:281-286

Mondal, B., Bhattacharya, I. and Khatua, D.C. 2011.
Crop and weed host of Ralstonia solanacearim
in West Bengal. J. Crop & Weed, 7(2):195-
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Palleroni, N.J., and Doudoroff, M. 1971. Phenotypic
characterization and deoxyribonucleic acid
homologies of Pseudomonas solanacearum.
J. Bacteriol., 107:690-696

Ramesh, R., Gauri, A.A. and Gaitonde, S. 2014.
Genetic diversity of Ralstonia solanacearum
infecting solanaceous vegetables from India
reveals the existence of unknown or newer
sequevars of Phylotype I strains. Eur. J. Pln.
Pathol. Online publication. doi. 10.1007/s10658-
014-0487-5

Yabuuchi, E., Kosako, Y., Yano, I., Hotta, H. and
Nishiuc hi, Y. 1995. Tra nsfe r of  two
Burkholderia and an Alcaligenes species to
Ralstonia gen. nov: proposal of Ralstonia
pickettii (Ralston, Palleroni and Doudoroff,
1973) comb. nov., Ralstonia solanacearum
(Smith 1896) c omb. nov. a nd Ralstonia
eutropha (Davis 1969) comb. nov. Microbiol.
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period of incubation. Also, our re sults  a re in
concurrence with findings of Ramesh et al (2014) who
reported 93% of Ralstonia solanacearum isolates
collected from various hosts all over India to be
pathogenic to eggplant and tomato. The succulent habit
of tomato plants (compared to brinjal and chilli) could
be the reason for early infection in tomato. Hundred
percent wilt incidence was noticed in marigold, as, it
is the  host pla nt for this  particula r pathogen.
Percentage of wilt incidence was comparatively low
in chilli and brinjal, which could be due to the hardy
nature of these crop plants.

Solanaceous vegeta ble s and marigold are
important crops grown in the island and yield loss in
these crops may be of serious concern to farmers.
Our present finding showed that the wilt pathogen of
marigold has an equal potential of infecting important
solanaceous vegetables like tomato, chillies and brinjal.
Our finding is a preliminary study which can help
develop effective management practices for controlling
the bacterial wilt pathogen that ca uses serious
economic loss in solanaceous vegetables and marigold.

REFERENCES

Buddenhagen, I.W, Sequeira, L. and Kelman, A. 1962.
De signa tion o f r ac e s of Ps e udomonas
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Fegan, M. and Prior, P. 2005. How complex is the
‘Ralstonia solanacearum species complex’?
In: Bacterial Wilt Disease and the Ralstonia
solanacearum Species Complex. C. Allen, P.
Prior and A.C. Hayward (eds.), Ame rican
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Ha ywa r d , A. C.  19 64.  Ch a r a c te ri s ti c s  o f
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Cross-infectivity of Ralstonia solanacearum from marigold

(MS Received 30 December 2015, Revised 22 July 2016, Accepted 19 December 2016)

J. Hortl. Sci.
Vol. 11(2): 179-181, 2017