Auxin-like activity of extract from hypertrophied tissue of Acacia eburnea infected with Ravenelia esculenta

Abstract

Ravenelia esculenta Naras. and Thirum. is a rust, pathogenic to Acacia eburnea Willd. The infection leads to hypertrophy changing the morphology with bizarre shapes of plant organs. Healthy and infected
tissues were subjected to extraction of IAA and indole derivatives and were estimated by spectrophotometric methods. The hypertrophy
produced was presumed to be due to increase in the indole-3-acetic acid (IAA) content in the infected tissue, however, the
amount of IAA in infected tissues decreased with the progression of disease. Concomitantly, the infected tissue showed the
presence of a novel, slow migrating, indole derivative on TLC. Cultured shoot tips of Withania somnifera were dosed with the methanolic extract of the infected hypertrophied tissue (MEHT) (0.25, 0.5, 0.75, 1.00 and 1.25mg/l).
The stimulation in shoot growth along with profuse rooting was observed in a dose dependent manner with maximum at 1.00 and
1.25mg/l concentration.

Full text

RESEARCH NOTE
Auxin-like activity of extract from hypertrophied tissue
of Acacia eburnea infected with Ravenelia esculenta
Kuvalekar Aniket ÆPawar Pankaj ÆKhare Ankita Æ
Gandhe Kanchanganga ÆHarsulkar Abhay
Received: 4 March 2008 / Accepted: 18 April 2008 / Published online: 6 May 2008
ÓSpringer Science+Business Media B.V. 2008
Abstract Ravenelia esculenta Naras. and Thirum.
is a rust, pathogenic to Acacia eburnea Willd. The
infection leads to hypertrophy changing the morphol-
ogy with bizarre shapes of plant organs. Healthy and
infected tissues were subjected to extraction of IAA
and indole derivatives and were estimated by spec-
trophotometric methods. The hypertrophy produced
was presumed to be due to increase in the indole-3-
acetic acid (IAA) content in the infected tissue,
however, the amount of IAA in infected tissues
decreased with the progression of disease. Concom-
itantly, the infected tissue showed the presence of a
novel, slow migrating, indole derivative on TLC.
Cultured shoot tips of Withania somnifera were dosed
with the methanolic extract of the infected hypertro-
phied tissue (MEHT) (0.25, 0.5, 0.75, 1.00 and
1.25 mg/l). The stimulation in shoot growth along
with profuse rooting was observed in a dose depen-
dent manner with maximum at 1.00 and 1.25 mg/l
concentration.
Keywords Acacia eburnea Auxin IAA 
Ravenelia esculenta Rooting Rust Withania
somnifera
Ravenelia esculenta Naras. and Thirum. is a rust,
infecting Acacia eburnea Willd. producing 15 to
20 times hypertrophy in infected organs (Fig. 1)
changing the morphology of plant organs like thorns,
inflorescence axis, flowers with bizarre shapes (Nara-
simhan and Thirumalachar 1961). Severely infected
host parts show the presence of brown coloured aecial
cups. This host––pathogen system is of particular
interest as fungal infection causes severe hypertrophy
due to qualitative and quantitative alterations in the
hormonal contents of the infected host cells. There are
many host––pathogen systems which lead to alteration
in the hormonal content of the host during or after the
infection as in case of infection due to Colletotrichum
gloeosporioides f. sp. aeschynomene in Aeschynomene
verginica which leads to in planta production of IAA
(Maor et al. 2004). There are also bacterial and fungal
host-pathogen systems which alter the hormonal
balance in the infected host tissue (Fett et al. 1987;
Gardan et al. 1992; Grsic-Rausch et al. 2000). In light
of these studies, A. eburnea infected with R. esculenta
was screened for hormonal contents, with special
emphasis to IAA and its derivatives.
Healthy and infected host tissues were collected
from the field during the severe infection period of
K. Aniket G. Kanchanganga
Department of Botany, PG Research Centre, Modern
College of Arts, Science and Commerce, Shivajinagar,
Pune, MS 411 005, India
P. Pankaj K. Ankita H. Abhay (&)
Interactive Research School for Health Affairs, Bharati
Vidyapeeth Medical College Campus, Bharati Vidyapeeth
University, Dhankawadi, Pune, MS 411 042, India
e-mail: aharsulkar@yahoo.com
123
Plant Cell Tiss Organ Cult (2008) 94:101–104
DOI 10.1007/s11240-008-9388-7

the fungus, separately in sterile vials and immediately
stored in liquid N
2
. IAA from healthy and infected
tissues was extracted by using the method of
McDoughall and Hillman (1978). About 5 g of fresh
healthy and infected tissues were homogenized in
80% methanol and soaked overnight in 80% meth-
anol, homogenized, again after 24 h, in 100 ml 80%
methanol and filtered through Whatman no.1 filter
paper. The methanol in filtrate was removed by rotary
flash evaporation system (Buchi type) at 35°C. The
pH of the aqueous residue was adjusted to 8.0 with
NaOH and partitioned twice against equal volume of
ethyl acetate to remove phenolic impurities. The pH
of the aqueous residue was adjusted to 2.5 with HCl
and extracted twice with equal volume of ethyl
acetate. The organic phase was concentrated with
rotary flash evaporation at 35 °C and acidic ethyl
acetate fractions were dissolved in methanol for
further analysis. The extracted IAA was estimated by
using Salkowski’s reagent (Glickman and Dessaux
1995).
About 20 ll of the methanolic extract from
healthy and infected material was loaded on the
TLC plate and compared with the standard IAA. The
plate was developed in benzene:acetone:acetic acid
(13:6:1) and visualized in iodine atmosphere.
Estimations of IAA extracted from healthy and
infected tissues revealed that there is a decrease in the
amount of IAA after infection. There are several
reports indicating either increase in the IAA content
or de novo production of IAA by infecting pathogens,
resulting in increased amounts of IAA in infected
tissues of the host (Chan and Thrower 1980; Fran-
kenberger and Poth 1987; Jentschke and Godbold
2000; Kaldorf and Ludwig-Muller 2000; Li et al.
2003; Chung and Tzeng 2004; Maor et al. 2004).
However, the studies on A. eburnea–R. esculenta
system showed decrease in the IAA content in the
hypertrophied material of the host after fungal
infection. The content of IAA in healthy was
1.161 ±0.03 mg/gm which gets reduced to
0.464 ±0.02 after infection. In addition, the hyper-
trophy is a result of interaction in IAA, free GA and
bound GA in the same host–pathogen system (Gand-
he and Kuvalekar 2007). Thin layer chromatographic
analysis of the IAA extract from healthy and infected
tissues showed the presence of a slow moving, novel
indole derivative only in the extract of infected
tissues. Appearance of this novel indole derivative
has also been demonstrated by HPLC (Fig. 2).
Seeds of W. somnifera were obtained from
Mahatma Phule Agricultural University, Rahuri,
India and were germinated aseptically on paper boats
in liquid MS basal medium. MS medium with 3%
sucrose (w/v), adjusted to pH 5.5–5.7, gelled with
0.8% agar (w/v), autoclaved at 1.05 kg cm
-2
for
20 min, was used as a culture medium. To authen-
ticate that the MEHT has auxin-like activity, filter
sterilized MEHT (0.25, 0.5, 0.75, 1.00 and 1.25 mg/l)
was mixed with autoclaved MS basal medium under
sterile conditions. Sterile W. somnifera shoots were
inoculated in the medium. The tubes were incubated
at 27 ±2°C at 1200 lux fluorescent white light at
16 h. photoperiod and observed regularly for root
development. In present study, the IAA extract from
the infected material along with the novel indole
derivative showed potent auxin-like activity in the
form of root development in the W. somnifera shoot
tip explants on MS basal medium. W. somnifera
seedlings do not produce roots on MS basal medium
and there is requirement of IAA and NAA in
combination for root development in vitro (Pawar
et al. 2001). However, profuse rooting in W. somnif-
era shoot tips by supplement of the MEHT from the
Fig. 1 Progressive hypertrophy production in the tissues of
Acacia eburnea infected with Ravenelia esculenta. The
infected tissues turn succulent showing several times hyper-
trophy with appearance of aecial cups during progression in the
disease. The progressive disease stages are marked by the
extent of hypertrophy and appearance of aecial cups in the
infected tissue
102 Plant Cell Tiss Organ Cult (2008) 94:101–104
123

hypertrophied tissue of Acacia eburnea establishing
its auxin like activity was observed. Induction of
rooting in W. somnifera shoot tips, by the auxin
extract from hypertrophied tissues was observed to
have a dose dependent effect. Increasing the amount
of extract supplied, increases the number of roots
produced in vitro. Higher concentrations of extract in
the medium showed profuse rooting and improved
shoot growth in the seedlings (Fig. 3). Stirk et al.
(2006) have reported the auxin- and cytokinin-like
activity in the extracts of Puccinia thlaspeos infected
dyer’s woad (Isatis tinctoria) with mung bean
bioassay.
Further purification and characterization of the
derivative in MEHT is under progress which will give
insights into its role in development of hypertrophy
during disease progression.
Acknowledgements Authors are thankful to University
Grants Commission, New Delhi, India for financial assistance
and to authorities of Modern College and Interactive Research
School for Health Affairs for providing infrastructure facilities
to carry out the work.
Fig. 2 High Performance
Liquid Chromatograph. (1)
Standard IAA; (2) IAA
from healthy material; (3)
IAA from infected material.
Note the appearance of a
novel indole derivative in
infected tissues (arrow)
with decrease in the
amounts of IAA over
healthy material. (HPLC,
Waters make; Column,
Nucleosil 100—C18; Flow
rate, 1 ml/min; Run time,
37 min; Mobile phase,
gradient of methanol: 0.1 M
perchloric acid from 10%
methanol and 90% 0.1 M
perchloric acid till 2 min;
55% methanol and 45%
0.1 M perchloric acid till
35 min and 10% methanol
and 0.1 M perchloric acid
till 37 min)
Fig. 3 Rooting response in Withania seedlings due to supple-
ment of extract from infected material after 2 weeks. Note the
dose dependent effect in rooting response with supplement of
increasing amounts of MEHT (C: Control; 1: 0.25 mg/l
MEHT; 2: 0.5 mg/l MEHT; 3: 0.75 mg/l MEHT; 4: 1.00 mg/
l MEHT; 5: 1.25 mg/l MEHT. Each experiment was done in
triplicate and the number of cultured explants per replicate was
25)
Plant Cell Tiss Organ Cult (2008) 94:101–104 103
123

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