3924 in Biosciences 8(15), Print : ISSN 0974-8,Trends

Trends
3924-3927,
in Biosciences
2015
8 (15), 2015

Novel Insights into the Phytotoxins Production of Colletotrichum

gloeosporioides Causing Anthracnose of Mango
S. PARTHASARATHY*, K. NAGENDRAN, P. NARAYANAN, J. RAJALAKSHMI,
G. THIRIBHUVANAMALA AND K. PRABAKAR
Department of Plant Pathology, Centre for Plant Protection Studies,
Tamil Nadu Agricultural University, Coimbatore -641003
email*: spsarathyagri@gmail.com

ABSTRACT
The devasting pathogen Colletotrichum
gloeosporioides produced non specific toxic
metabolites in culture filtrate. Maximum toxin content
was synthesized on 18 th day after inoculation.
Drooping and wilting were the striking symptoms
observed in most of the plant species tested in culture
filtrate but the time taken for expression of symptoms
were varied from to plant. Toxigenic culture filtrate
inhibited the seed germination of maize, sorghum,
tobacco, tomato and chilli drastically. The toxin proved
to be thermostable and retained its toxicity even after
autoclaving at 1.1kg/cm2 pressure for 15 minutes.
While, by diluting with water, its toxicity was reduced
to a greater extend. The dilution of the culture filtrate
and the time taken for symptom expression were
positively correlated.
Key words

Anthracnose, bioassay, Colletotrichum

gloeosporioides, mango, phytotoxins

It is well known fact that in many plant

diseases, toxins produced either by pathogen or by
host or by interaction of both play an important
role in pathogenesis. Basically, phytotoxins are the
secondary metabolic products of microorganisms,
which cause obvious damage to plant cells and
tissues. Phytotoxins are known to cause a number
of destructive diseases of plants (Scheffer, 1983).
Several pathogenic fungi are known to produce
host-specific toxins in culture and there is
conclusive evidence that such toxins are involved
in pathogenesis (Alam et al., 2001). In some cases
however, specificity or selectivity is related to
production and release of toxic substances with
note worthy characteristics like pathogenicity and
similar symptomatic lesion development. These are
so called host specific toxins (Pringle and Scheffer,
1964), which damage or destroy plant tissues that
are susceptible to the toxin producing pathogens,
but have little or no effect on other plants,

microorganisms or animals. Microbial products

with selective toxicity to plant have been reported
many times in past (Tanaka, 1993).
In Mango, anthracnose is incited by
Colletotrichum gloeosporioides Penz., the
symptoms of anthracnose are commonly found on
leaves, panicles and fruits (Arauz, 2000). The initial
symptoms of most Colletotrichum infected plant,
especially on leaves, starts as small irregular brown
spots, which are usually surrounded by yellow halo.
These spots later coalesce to form larger necrotic
lesion, indicating the involvement of phytotoxic
metabolites, which therefore, suggests a role for
toxic metabolite secreted by the pathogen in the
disease development. In some plant diseases,
especially with yam anthracnose, toxins often
produce a more rapid and extensive invasion by
the pathogen than would be in the case in the
absence of toxins (Amusa, 1994). Therefore, such
studies would disclose the implications in the
pathogenesis on host plant.

MATERIALS AND METHODS

Pathogen
A virulent culture of C. gloeosporioides
isolated from mango plant showing typical symptom
was used in this studies. The culture was maintained
on potato dextrose agar medium. To ascertain
growth period at which the fungus produces
maximum toxic metabolite in yeast extract broth
as basal medium. C. gloeosporioides culture
harvested at an interval of 2 days, starting from 3rd
day of incubation up to 30th day. The bioassay of
toxic metabolite was made in main host mango and
other non host plant species.

Toxicity Bioassay
To study the specificity of toxic metabolite,
main host mango cv. neelum (leaves) and non host
plants rice, maize, sorghum, chilli, tobacco, tomato

PARTHASARATHY et al., Novel Insights into the Phytotoxins Production of Colletotrichum gloeosporioides 3925

Days after
treatment

Time taken for

symptom expression in
mango leaves and fruits
(in hr)

Symptoms
expression
grade

48

44

(+)

32

12

10

++

12

++

on sapling in planta analysis and seed germination

following the methods described Naik et al., 1991.
Rice, maize, sorghum, chilli, tomato, tobacco and
coriander saplings were incubated in test tubes
containing equal volume of culture filtrate and
respective plant seeds were soaked in 16 day old
culture filtrate separately for 12 hours and later
placed in Petri dishes lined with sterile filter papers
moistened with the culture filtrate. Control was run
simultaneously with uninoculated broth and sterile
distilled water. The efficacy of toxic culture filtrate
on different saplings was evaluated immediately and
germination was counted five days after incubation
and the reduction in per cent germination over
control was calculated.

14

++

Thermostability assay

16

++

18

+++

20

+++

22

+++

24

+++

26

+++

28

+++

To study the effect of heat on culture filtrate,

an aliquot of 5ml was flamed at 50C and 70C to
get warm. Another set was heated to 100C for 10
minutes and the fourth one was autoclaved at 1.1
kg/cm2 pressure for 15 minutes. All these treatments
were assayed on host and non-host plants for
development of symptoms.

30

+++

Table 1. Production of toxic metabolites by

Colletotrichum gloeosporioides in
Richards solution at different
incubation period and its effect

-, no lesion; (+), less than 5mm; +, 5-7 mm; ++, 7-10mm; +

+ +, more than 10mm

and coriander saplings were used for differential

expression analysis of phytotoxins by following
procedures of Venkataravanappa et al., 2007. The
leaves of same size and age were tested by pin
pricking method using toxigenic culture filtrate
obtained from 16 day old C. gloeosporioides culture.
Control was run simultaneously with sterile distilled
water. The time required for symptom development
and the nature of symptoms was recorded.
Similarly, bioassay of toxic metabolite was made

Dilution assay
To study the effect of different
concentrations of culture filtrate, the mango
(leaves) and non-host plants were treated with 10ml
of differentially diluted toxic solution. The
uninoculated broth and sterile distilled water served
as control. Each treatment was replicated thrice.
Plants were observed for symptom development
at regular intervals.

RESULTS AND DISCUSSION

The mango leaves treated in the culture filtrate
obtained on second day remained apparently

Table 2. Effect of 16 day old culture filtrate of Colletotrichum gloeosporioides on seedlings of

different species
Plant species

Time taken for

expression of
symptoms (in hr)

Rice

Wilting, browning of leaves and drying

Symptoms

Maize

Drooping, browning, wilting and curling

Sorghum

Drooping, browning, wilting and curling

Tomato

Browning, drooping and curling

Tobacco

Dropping, browning, marginal curling and severe wilting

Chilli

Drooping, browning, wilting and drying

Coriander

Drooping, curling, complete wilting and brittling

3926

Trends in Biosciences 8 (15), 2015

Table 3. Effect of 16 day old culture filtrate

of Colletotrichum gloeosporioides on
seed germination of different plant
species
Plant Species
Rice
Maize
Sorghum
Tomato
Tobacco
Chilli
Coriander

Reduction in seed
germination (in per cent)
16.0
0.0
0.0
0.0
0.0
0.0
16.0

healthy, which indicates that the C. gloeosporioides

might not have produced any toxic metabolite in
that short time or the amount of toxic metabolite
produced might be insufficient to express
symptoms on leaves. Necrotizing of leaves was
evident in culture filtrate obtained from 4 th day
onwards, but the time taken for expression varied
from 2 to 48 hours (Table 1). As the age of culture
increased, due course the time required to express
symptom decreased indicating increased amount
of toxic metabolite in the culture filtrate. But the
20th day onwards time remained constant. This
clearly indicates that the maximum metabolite of
the fungus could be harvested on 22th day after
incubation.
All the non host plant species treated in the
culture filtrate showed disease syndrome like
drooping and some kind of discoloration, but the
time taken to express such symptom varied from
plant to plant. Plant seedlings of rice, maize,
sorghum and coriander took more time to express
symptoms than other plants tested (Table 2). The
study clearly indicates that necrotrophic pathogen
C. gloeosporioides produces non host selective toxic
metabolite. Sharma and Sharma (1966) noticed
similar trend in an isolate of C. gloeosporioides, a
causative of citrus die-back. The toxic metabolite
also played a significant role in reducing the seed
germination of various crop plants. Complete
reduction of seed germination was in maize,
sorghum, tobacco, tomato and chilli (Table 3).
The culture filtrate even after boiling at 100C
for 10 minutes and autoclaving at 1.1 kg/cm2
pressure for 15 minutes did not lose its toxic effect
and resulted in drooping, wilting and browning
symptoms on host tissues. Thus, the toxic principle
proved to be thermostable. The thermostable toxins
were also observed in Helminthosporium victoriae

Table 4. Effect of dilution of culture filtrate

of Colletotrichum gloeosporioides on
mango leaves

Broth Control

Time taken for

symptom
expression (in hr)
-

Crude

+++

1:1

+++

1:2.5

+++

1:5

+++

1:7.5

++

1:10

14

Dilutions
Water Control

Symptoms
-

-, no lesion; (+), less than 5mm; +, 5-7 mm; ++, 7-10mm; +

+ +, more than 10mm

(Litzenberger, 1949) and C. capsici (Bhathagar and

Kalpana, 1995). Thus the result clearly indicates
the least possibility of inactivation of toxin of C.
gloeosporioides in host tissues in nature due to
atmospheric temperature.
In the crude culture filtrate and dilutions, the
common symptoms like drooping and wilting were
noticed. In addition to drooping and wilting,
browning, curling and brittling were also seen in
the leaves kept in crude solution of culture filtrate
and these symptoms were expressed within 2 hours
of incubation (Table 4). The dilution of culture
filtrate and the time taken for symptom development
were positively related and is in agreement with
the results obtained by (Dasgupta, 1986; Gunawan
et al., 1988; Jayasankar et al., 1999; Malathi et
al., 2002; Zhang et al., 2012), in respect to toxic
metabolite produced by Colletotrichum spp. The
further analysis of the structure and function for
the active component of the toxin is under
investigation.

ACKNOWLEDGEMENT
The IDRC-CIDA-CIFSRF, Canada is highly
acknowledged for the financial support.

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Received on 18-07-2015

Accepted on 22-07-2015