Associated callus culture technique for in vitro growth of rust fungi
Aniket A. Kuvalekar 1& Kanchanganga R. Gandhe 2
1,2 PG Research Centre, Department of
Botany, Modern College of Arts, Science and Commerce, Shivajinagar, Pune,
Maharashtra 411005, India
Email: 1 kuaniket@gmail.com
Date
of publication (online): 26 August 2010
Date
of publication (print): 26 August 2010
ISSN
0974-7907 (online) | 0974-7893 (print)
Editor: V.B. Hosagoudar
Manuscript
details:
Ms # o2263
Received 16 July
2009
Final revised
received 11 August 2010
Finally accepted 12
August 2010
Citation: Kuvalekar, A.A. & K.R. Gandhe (2010). Associated callus culture technique for in vitro growth
of rust fungi. Journal of Threatened Taxa2(9): 1140-1143.
Copyright: © Aniket A. Kuvalekar & Kanchanganga R. Gandhe 2010.
Creative Commons Attribution 3.0 Unported License. JoTT allows unrestricted use
of this article in any medium for non-profit purposes, reproduction and
distribution by providing adequate credit to the authors and the source of
publication.
Acknowledgements: The
authors are thankful to University Grants Commission, New Delhi for providing
financial assistance and to the authorities of Modern College, Pune for
providing infrastructure facilities.
Abstract: Uromyces hobsoni, a rust fungus, infects Jasminum officinale var. grandiflorum. The infection frequently leads to
malformations in tissues, mainly leaves and stems. Disease progression can be assessed morphologically by
observing the extent of malformation and occurrence of sporulation. The rust
fungi, in general, are obligate parasites, and need a living host to complete
their life cycle. The difficulty
of in vitro propagation of rust fungi has been a major obstacle in their
detailed biochemical and molecular analysis. In this paper, we report successful in vitro culture of rust
fungi with induction of callus from infected leaves of host plants which
contain initial differentiated structures like haustoria and intercellular
hyphae. This ‘associated callus culture’ technique has opened new paths for
studying host–pathogen interactions of rust fungi.
Keywords:Associated callus culture, haustoria, hypertrophy, host-pathogen, interaction,
malformations.
For images & table – click here
Rust fungi are
obligate parasites which require a living host for growth, reproduction and
completion of their life cycle (Eckardt 2006). There have been a few reports of in vitro culturing of rust
fungi but these studies could not be repeated by other workers (Scott &
Maclean 1969; Bose & Shaw 1974; Wiethölter et al. 2003). The inability to culture rust fungi in
defined conditions has been a major impediment to studying host–pathogen
interactions and performing detailed biochemical and molecular analysis. The present study was undertaken to
investigate the competence of rust fungus Uromyces
hobsoni Vize to grow
with host tissues in artificial culture media.
Material and methods
Collection
of plant material and tissue explant preparation: The
leaves of Jasminum
officinale var. grandiflorum (L.) Bailey, both healthy and heavily infected
by rust fungus Uromyces
hobsoni Vize. (Image 1
a-d) were chosen for culture experiments. The leaves were collected from the same host plant in the field and
preserved in brown paper bags (storage in polythene bags leads to development
of moisture and hyperparasites on the tissues). Leaves were deemed infected if they contained telial
pustules. Explants were washed
thoroughly under running tap water to remove dust and other debris, further
washed with an aqueous suspension of 0.01% (v/v) Tween 20 (a liquid detergent)
for 10 minutes followed by aqueous suspension in Dettol disinfectant for 10
minutes. Further processing of
explants was carried out under aseptic conditions in a horizontal laminar air
flow cabinet. The explants were
washed with sterile distilled water, treated with 70% alcohol for 30 seconds,
surface sterilized with an aqueous solution of 0.1% mercuric chloride for two
minutes and inoculated with fungus in test tubes containing sterile Murashige
and Skoog’s growth medium (Murashige & Skoog 1962) supplemented with 2 mg/L
2, 4 – D with 3% sucrose (w/v) and 0.8% agar (w/v) at pH 5.5-5.7.
Incubation
and Measurement: Cultures were incubated at 27±2 0C
at 1200 lux fluorescent white light at 16 hours photoperiod. Cultured explants were observed
regularly for contamination and callus development. Growth of callus tissue was analyzed at an interval of four
days after the initiation of culture. Ten cultures from each experiment were used to determine the fresh
weight of callus, which was then dried in a hot air oven at 400C
until a constant dry weight was obtained. Microscopic observation of calli involved looking for fungal structures
including mycelia and haustoria via cotton blue mounting. Final development was assessed on the
32nd day post inoculation for 10 callus cultures each from healthy
and infected tissues. Each
experiment was done in triplicate and the number of cultured explants per
replicate was 25.
Results and Discussion
The study of
disease development by rust fungi is difficult due to inaccessibility to pure
cultures. Rust fungi are obligate
parasites which require the presence of a living host for their growth,
reproduction and completion of their life cycle (Savile 1976). There are limited studies of the in
vitro cultures of rust fungi, most of which could not be repeated by other
workers (Scott & Maclean 1969; Bose & Shaw 1974; Wiethölter et al.
2003). Hotson & Cutter (1951)
for the first time reported successful axenic culture of Gymnosporangium juniperi –
virginianae. They also showed that saprophytic
cultures of the rust can reinfect the host to produce characteristic fruiting
bodies. Staples et al. (1983) and
Allen et al. (1991) have reported that uredospores of various rust fungi can be
germinated on artificial membrane grooves. Hu & Amerson (1991) reported single geneotype axenic
cultures of Cronartium
quercuum f. sp. fusiformae which can be repeatedly cultured axenically
from single spores. Successful
axenic culture has also been reported for the western gall rust (Endocronartium harknessii) using the galls produced on the host (Allen
et al. 1988). Diner & Mott
(1985) reported that the in vitro generated vegetative hyphae
from the basidiospores of Cronartium
ribicola could infect the
in vitro grown propagules of western white pine and showed intracellular
haustoria. Anikster (1986) showed
the germination of teliospores from different rusts which could even occur in
water agar. Recent studies with in
vitro cultures of rust fungi have revealed that the growth and differentiation
of the structures like haustorial mother cells is dependent upon the
synergistic action of chemical and physical signals (Wiethölter et al.
2003). Pure cultures of the rust
fungi are also reported from the basidiospores and aeciospores (Diner 1999;
Moricca et al. 2000). Pei &
Gibbs (1992) have reported the continuously growing rust cultures of Peridermium pini where aeciospores were used as the starting
material. Thus there are very few
reports of successful rust cultures.
Maheshwari et al.
(1967) have earlier reported the successful growth of rust fungus on the callus
tissue of the host. They reported
that upon inoculation of rust–infected sunflower cotyledons and red cedar
galls induced by cedar–apple rust on artificial media, the host tissue
proliferated but the rust mycelium did not invade the newly formed
tissues. They also reported that
the growth of callus tissue, when kept in contact with uredospores germinated
on collodion membranes, also failed.
The main reason
for failure of the continuous growth of the rust fungus in vitro or in
association of the host might be their requirement of specific signals which
are tightly linked to development of the host plant (Staples 2000). The callus tissue itself is an
unorganized mass of cells and the rust fungus invading the cells and/or callus
may not get the required developmental signals from the host for the profuse
growth and sporulation.
Hence the report
in the present study is particularly significant as the growth of the fungal
mycelia is observed in the callus with the formation of specialized feeding
structures. In the present
studies, cultures of healthy and infected leaves showed initiation of callus in
8-10 days (Image 1 e-f). The calli
grew quite slowly and formed a mass at the site of induction. After 32 days of callus initiation,
growth of callus originating from infected leaves seized and it turned brown,
whereas calli from healthy leaves continued to grow.
Microscopic
examination of calli from healthy and infected leaves showed the presence of
irregular cell masses. Cells from
calli induced from infected leaves showed presence of haustoria and also inter
and intracellular hyphae. These
structures were clearly visible in callus initiated from the infected
leaves. The callus from infected
leaves grew faster (Table 1) and the cells appeared larger in diameter compared
to those in the callus from healthy leaves (Image 1 g-h).
In the present
study, the growth of rust was reported along with the callus in the infected
tissues of J.
officinale var. grandiflorum. The intercellular hyphae and haustoria were clearly distinguishable in
the callus cultures derived from the infected tissues.
Although axenic
cultures have been established for many rusts they remain slow-growing and the
essential nature of their in vitro growth requirements remains unknown. There may not be a special requirement
for nutrients but rust development may be tightly linked to signals from the
host plant (Staples 2000).
In the present
study, the medium used for the culturing was a plant tissue culture medium
designed to aid the growth of plant tissues. The high concentration of auxin in the medium leads to
development of callus. The fungal
hyphae already present in the infected host tissues grow and invade the freshly
developed callus. The fungal cells
may get more nutrients from such cells which are profusely growing but the lack
of developmental signals from the host tissue may lead to termination of the
fungal growth. The present
technique of ‘associated callus culture’ can be used for indefinite growth of
the rust fungi in vitro with the host allowing for availability of the tissues
grown under controlled conditions with the rust fungi. The technique has opened the way to
such type of study in a wide variety of host-rust systems. This technique may provide the easier
way for the in vitro culture of rust fungi and study of host-parasite
interaction.
With the help of
this technique, the biochemical signals which govern the growth of rust in the
living host cells needs to be addressed. Further it can be checked if the preconditioned fungal tissues are able
to grow on the defined media. The
studies to address these questions are in progress.
References
Allen, E.A., B.E. Hazen, H.C. Hoch, Y. Kwon, G.M.E.
Leinhos, R.C. Staples, M.A. Stumpf & B.T. Terhune (1991). Appressorium formation in response to
topographical signals by 27 rust species. Phytopathology81: 323-331.
Allen, E.A., P.V. Blenis & Y. Hiratsuka (1988). Axenic culture of Endocronartium harknessii. Mycologia 80(1): 120-123.
Anikster,
Y. (1986). Teliospore
germination in some rust fungi. Phytopathology76: 1026-1030.
Bose,
A. & M. Shaw (1974).Growth of rust fungi of wheat and flax on chemically-defined media. Nature 251: 646-648.
Diner,
A.M. & R.L. Mott (1985). In
vitro inoculation of
western white pine tissue culture propagules with vegetative hyphae of Cronartium ribicola. Phytopathology75(10): 1130-1131.
Diner,
A.M. (1999). Direct
mechanical dispersion and in vitro culture of fusiform rust fungus single
basidiospores. Mycologia 91(6): 1102-1103.
Eckardt,
N.A. (2006). Identification
of rust fungi avirulence elicitors. Plant
Cell 18: 1-3.
Hotson,
H.H. & V.M. Cutter (1951). The
isolation and culture of Gymnosporangium
juniperi- virgininae schw.
upon artificial media. Proceeidngs
of National Academy of Sciences USA 37:
400-403.
Hu,
A. & H.V. Amerson (1991). Single
genotype axenic cultures of Cronartium
quercuum f. sp. fusiforme. Phytopathology81: 1294-1297.
Maheshwari, R., A.C. Hilderbrandt & P.J. Allen
(1967). Factors affecting
the growth of rust fungi on host tissue cultures. Botanical Gazette 128(3-4): 153-159.
Moricca, S., A. Ragazzi & B.N. Longo (2000). In vitro growth of the aspen rust Melampsora larici-tremulae. Mycological
Research 104: 1250-1257.
Murashige,
T. & F. Skoog (1962).A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.
Pei,
M.H. & J.N. Gibbs (1992).Axenic culture of Peridermium
pini from single
aeciospores. Plant
Pathology 41(1): 91-94.
Savile,
D.B.O. (1976). Evolution of
the rust fungi (Uredinales) as reflected by their ecological problems. Evolutionary Biology 9: 137-207.
Scott,
K.J., & D.J. Maclean (1969).Culturing of rust fungi. Annual
Review of Phytopathology7: 123-146.
Staples,
R.C. (2000). Research on the
rust fungi during the twentieth century. Annual
Review of Phytopathology38: 49-69.
Staples,
R.C., H. Grambow, H.C. Hoch & W.K. Wynn (1983). Contact with membrane grooves induces wheat
stem rust uredospore germlings to differentiate appressoria but not vesicles. Phytopathology 73: 1436-1439.
Wiethölter, N., S. Horn, K. Reisige, U. Beike & B.M.
Moerschbacher (2003). In
vitro differentiation
of haustorial mother cells of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, triggered by the synergistic action of
chemical and physical signals. Fungal
Genetics and Biology3(3): 320-326.