Indian Phytopath.

62 (4) : 493-498 (2009)

Standardization of production of mycoherbicidal agent

Colletotrichum dematium FGCC# 20 by solid substrate
fermentation
JAYA SINGH and A.K. PANDEY
Department of Biological Sciences, R.D. University, Jabalpur 482 001

Abstract: The aim of the present study is to select a suitable, cheaper and easily available agro-waste
substrate for mass production of mycoherbicidal agent Colletotrichum dematium FGCC# 20, which is a highly
effective bioherbicidal agent against Parthenium hysterophorous. The outcome of the study indicates that,
pretreated sugarcane bagasse supported maximum mycelia coverage while maize cob grits supported
maximum sporulation and cause maximum seedling mortality.
Key words: Mass production, agro-waste, mycoherbicide, Colletotrichum dematium

Concern about the negative effects of herbicides

have led to emphasis on alternative strategies for
weed control. There is a world wide resurgence of
interest in the use of indigenous ecofriendly and
host specific fungal pathogens as herbicides
(mycoherbicides) and a significant advance in mass
production and fermentation of some of them have
been observed (Jackson & Slininger 1993; Diagle
et al., 1998; Pfirter et al., 1998; Eilenberg et al.,
2001). However, only few organisms have been
commercially produced for large-scale field
applications (Pandey et al., 1999 and Pfirter et al.,
1999, Gressel, 2003). Non availability of low-cost
mass production technology is one of the major
hindrance in their application (Pandey et al., 2001).
This may be achieved by selecting a suitable
substrate that is simple in composition, cheaper in
price and available in large quantities and developing
a production procedure that is easy to apply with
minimum labour. Colletotrichum species are well
known for crop disease, but they are also capable
of causing disease on some weeds and have
become popular mycoherbicide for several reasons.
Colletotrichum is retalively ubiquitous in distribution
and strains can be extremely host-specific (Watson
et al., 2000). Solid substrate fermentation (SSF)
technology i.e. culturing of fungi on solid agrowastes

is extensively used in Biotechnology for production

of organisms itself and their by-products (Feng et
al., 1994; Sharma et al., 1995; Hesseltine 1997,
Diagle et al., 1998 and Pandey et al., 2000;
Pandey 1999). However no generalized conditions
have been observed. Therefore, the present
investigation which is aimed to standardize mass
production technique of Colletotrichum dematium
FGCC#20 is the most effective mycoherbicide
against the obnoxious and deadly weed Parthenium
hysterophorous L.
MATERIALS AND METHODS
Mycoherbicidal Agent
Colletotrichum dematium FGCC#20, isolated
originally from diseased seedlings of Parthenium
hysterophorous L. (Gayathri and Pandey, 1997)
was obtained from Fungal Germplasm Collection
Centre (FGCC), Department of Biological Science,
R. D. University, Jabalpur (M.P.) India. Lyophilysed
stock cultures were stored in a refrigerator (Smith
& Onions, 1994). Inocula were activated as per
Jackson and Schisler (1992, 1996). Sporulated
potato dextrose agar plates were rinsed with
sterilized distilled water and conidial suspension @
5 x 105 conidia/ml was used as inoculum.

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Indian Phytopathology

Solid Substrate Cultivation

To standardize the mass production technique
agro-based waste products procured from localmarket and farmers field were used. Initially the
substrates were soaked overnight in distilled water.
Pretreatment of hardcore substrate viz. sugarcane
bagasse, maize cobgrits and wheat straw were
carried out as per Rodriquez-vazquez et al. (1992);
Nigam and Singh (1994). Initial pH and moisture of
the substrates was 6.0-6.5 and 60% respectively.
Moisture content was determined by the formulae
given by Singh (1977).
For sterilization 5.0 gm of each mositened
substrate was placed into a 500 ml which were
thoroughly sterilized at 15 psi for 30 min Each flask
was separately seeded with 5.0 ml spore
suspension (5 x 105 conidia/ml) incubated at
280C10C in a BOD incubator (make:yorco, India)
for 10 days. After incubation, flasks were 100C10C
to avoid germination of conidia.
Extraction of Conidia
Conidia was harvested after 15 days by
suspending the substrates in 250ml sterilized
distilled water and 0.02% Tween 80. The suspension
was filtered through a double layered muslin-cloth
and numbers of conidia were determined
microscopically with a haemocytometer
(make:Reichert Brighitline, Warner- Lambert
Technologies Buffalo. N.Y.).
Bioassay
Efficacy of various infested agro-waste
substrates was evaluated through seedling bioassay
technique (Berger & Hanson, 1963 and Winder &
Watson, 1994). 3-4 week old Parthenium seedlings,
grown in the plant growth chamber (make:Yorw,
India) were sprayed to run off with conidial
suspension. The necessary saturated atmosphere
following inoculation were obtained by placing the
plants is an Environmental test chamber,
(make:Remi India) at 280l0C and 90% RH. In the
Green House necessary conditions were developed
by covering the inoculated seedlings with plastic
bag for 24 hrs suggested by Pfirter and Defago
(1998). Additional water was atomized in to the bag
to increase humidity. After the initial exposure to
dew, the plastic bags were removed and treated

[Vol. 62(4) : 2009]

seedlings were transferred to the Green House. To

evaluate the field performance, seedlings were raised
in 1x1 sq.meter protected plots and sprayed to run
off with 4 x 105 conidia/ml. Control seedlings were
sprayed to run off with distilled water only. Treated,
as well as, control sets were observed daily.
Development of the disease was monitored by
recording. Percentage seedling mortality was upto
12-14 DAT (Days after treatment).
All the experiments were carried out in
triplicates Representative data were subjected to
statistical analysis viz., Analysis of Variance and
means of the variance and means of the various
treatments were separated by Fishers LSD (Least
significant difference) as per SAS Institute (1998).
RESULTS AND DISCUSSION
Mass Production
Data recorded in Table 1 shows significant
variations in mycelial colonization of various agrowaste. Extensive mycelial coverage was observed
on all solid substrates except paddy semolina,
castor oil cake, kapas oil cake and sawdust.
Except coconut coir, wood showing, paddy straw,
kapas oil cake, caster oil cake and saw dust, all
mycelial growth was associated with acervauli,
setae and conidial formation but the number was
highly variable. Mycelial coverage was maximum
on sugarcane bagasse and Gram semolina followed
by wheat grain and wheat straw. Significant
mycelium formation was observed on other
agrowastes viz., wheat semolina, paddy gain. Host
leaves supported good fungal growth. Least mycelial
coverage was reported for paddy straw, coconut
coir and waste news paper. Wheat straw, maize
cob grits, sugarcane bagasse and pea peels
supported maximum acervuli and setae formation:
similarly maize cob grit and gram semolina
supported maximum spore production. Potato peels
failed to support acervuli and setae formation but
significantly supported spore formation. Similarly
wheat bran wheat semolina and maize flaps were
also found to be good substrates for spore
production, mustard oil cake was significantly
colonized by the fungus but failed to support spore
formation, Agricultural waste has also been
considered a good source for mass production of
various other mycoherbicidal agents.

[Vol. 62(4) : 2009]

Indian Phytopathology

495

Table 1. Screening of solid agrowaste substrate for mass production of C. dematium (FGCC # 20)
S.
No.

Name of Solid substrate

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.

Wheat Semolina
Wheat straw
Wheat bran
Wheat grain
Paddy grain
Paddy straw
Paddy semolina
Maize flaps
Maize bran
Maize cob grit
Kapas oilcake
Catton stalks
Soyabean bran
Soyabean oilcake
Mixed pulses bran
Sugarcame bagase
Caster seed
Caster oilcake
Gram bran
Gram semolina
Wood shaving
Saw dust
Groundnut hull
Groundnut oilcake
Tea waste
Musturd oilcake
Host leaves
Jowar bran
Bajara bran
Coconut coir
Pea peels
Patato peels
Mung bran
Waste newspaper
Barley straw

Natural
moisture
content
(%)
60
70
70
20
20
30
30
60
60
70
80
60
70
80
73
90
20
65
72
70
60
80
79
80
80
80
70
49
55
60
80
69
55
65
60

Mycelium
coveragea
(%)
84.00
90.00
77.00
91.00
85.67
11.33
0.00
82.67
85.00
91.00
0.00
25.33
35.00
21.00
42.33
92.33
28.00
0.00
41.00
92.33
43.67
0.00
84.00
76.33
20.67
66.33
88.00
58.67
62.00
12.33
83.67
70.67
65.00
12.67
23.00

1.00
1.00
2.00
1.00
1.15
1.53
0.00
2.08
1.00
1.00
0.00
1.53
1.00
1.73
2.52
1.53
1.00
0.00
1.00
2.08
1.53
0.00
1.73
1.53
1.15
1.53
1.00
1.53
2.00
1.53
1.53
1.15
1.00
2.08
1.00

Acervulic
formation

Seateb
formation

Spored
production
(x 105
c/ml /g)

+
+
+
++
++
++
+
++
+
+
+
+
++
+
+
++
++
+
+
+
++
+
+
++
+
+
+
+

++
+++
+
+++
+++
+
+++
+++
+
+++
+
+
+++
+
+
+++
++
++
+
+++
+
+
+++
+++
+
+
+

6.5
7.8*
6.7
8.9
7.2*
00
00
7.2*
7.6
8.14*
00
2.9
5.1
3.11
3.5
7.12*
42
00
4.2
8.5*
00
00
6.7
6.5
2.9
00
6.2
5.2
4.9
00
8.2*
6.1
4.2
2.5
1.9

Values are means SD of three observations

a - Visual rating,
b - Formation of setae determined microscopically (+++ : Abundant, ++ : Average, + : Less),
c - Formation of Acervuli determined microscopically (+ : Acervuli present, - : No Acervuli)
d - Spore counted through Heamocytometer.

Hilderband and McCain (1978) obtained

significant growth sporulation of Fusarium
oxysporoum f. sp. cannabis on wheat straw to
control Canabis sativa (Murijuma) Boyette (1982)
used oat seed infested with F. solani f. sp.
curcurbitae to control (Taxas gourd). Morin et al.
(1990) reported significant variation in growth and
sporulation in Phomopsis convolvulus, a

mycoherbicidal agent for field bindweed when grown

on various agrowaste similar observation regarding
mycelial growth, acervuli and conidial formation in
Stagonospora convoluli # LA 39 have also been
recorded by Pfirter et al. (1999) Acervuli, setae &
spores from the different substrates did not
significantly differ morphologically. The yield from 1
g of maize cob grits was equal to that from four 9

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Indian Phytopathology

[Vol. 62(4) : 2009]

cm culture plates each containing 20 ml of media.

The high yield of spores obtained with maize cob
grit may be to its large surface area, good aeration
and non-clumping stable structure. It was also
recorded that the spores did not germinate even
after 2 weeks when transferred at 100 C 1.
Similar, observation have also been made by Vimla
Devi (1994), Diagle et al. (1998) and Pfirter et al.
(1999).

It is also evident from seedling mortality (Table

2) that virulence of spores varied significantly with
production substrates (Table 1). Spores obtained
from maize cob grits caused maximum seeding
mortality nearly under all the three conditions.
Which was followed by maize flaps and wheat
straw. Spores extracted from sugarcane bagasse,
paddy grains were also responsible for more than
80% seedling mortality. Some agro-wastes viz

Table 2. Herbicidal potential of inoculum produced on different agricultural solid waste substrates
S.
No.

Name of Solid substrate

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.

Wheat Semolina
Wheat straw
Wheat bran
Wheat grain
Paddy grain
Paddy straw
Paddy semolina
Maize flaps
Maize bran
Maize cob grit
Kapas oilcake
Catton stalks
Soyabean bran
Soyabean oilcake
Mixed pulses bran
Sugarcame bagase
Caster seed
Caster oilcake
Gram bran
Gram semolina
Wood shaving
Saw dust
Groundnut hull
Groundnut oilcake
Tea waste
Musturd oilcake
Host leaves
Jowar bran
Bajara bran
Coconut coir
Pea peels
Patato peels
Mung bran
Waste newspaper
Barley straw
LSD (P=0.05)

In lab condition
53.33 2.89
92.33 2.52
33.33 10.41
76.67 2.89
82.67 2.08
0.00 0.00
0.00 0.00
89.00 6.56
65.00 4.58
94.67 1.53
22.33 4.04
0.00 + 0.00
62.33 2.52
19.67 0.58
27.67 2.52
89.67 0.58
34.00 6.93
3.33 0.58
29.33 6.03
96.00 1.73
21.00 1.00
0.00 0.00
32.33 2.52
56.33 1.53
7.67 0.58
10.67 1.15
82.00 3.00
2.33 0.58
0.83 0.76
0.00 0.00
78.00 9.85
50.67 1.15
7.67 2.52
44.67 2.52
10.67 1.15
5.923496

% Seedling mortality
In green house conditionb

In field conditionc

40.67 1.15
70.33 0.58
21.33 3.21
68.00 2.65
71.33 1.15
0.00 0.00
0.00 0.00
74.33 2.08
43.00 2.00
75.67 6.03
11.33 1.15
0.00 0.00
42.33 2.52
10.67 0.58
18.00 2.00
63.00 1.73
13.00 2.65
1.67 0.58
25.00 2.65
71.67 1.53
19.00 1.00
0.00 0.00
25.00 2.65
42.33 1.53
1.67 0.58
8.00 1.00
72.67 2.08
0.00 0.00
0.00 0.00
0.00 0.00
57.67 2.52
42.33 2.52
6.00 1.00
20.67 5.31
6.67 1.53
5.166259

25.00 0.00
53.33 2.89*
22.33 2.52
57.67 2.52
54.00 1.00*
0.00 0.00
0.00 0.00
57.67 2.52*
30.00 5.00
72.33 2.52*
1.00 0.00
0.00 0.00
21.67 2.89
1.17 0.29
12.33 2.52
51.33 1.15*
2.17 0.29
0.00 0.00
19.33 0.58
61.67 3.51*
12.00 2.65
0.00 0.00
19.33 0.58
27.33 2.08
0.00 0.00
5.00 1.00
62.33 2.52
0.00 0.00
0.00 0.00
0.00 0.00
33.67 1.53*
31.00 3.61
3.00 1.00
16.33 1.53
3.00 1.00
3.110640

Values are means SD of three observation

a. At 20 DAT, 28 10C, 90% RH, 12 hrs dew period, 4.0 x 105 c/ ml).
b. 2 to 3 c.m. water level was maintained, 35/23 50C dya/ night temperature, 12 hrs photo period and average
light intensity of 350 u Es-1.
c. In mansoon, plot size 5 x 5squre meter, 4.0 x 105 c/ml. 1 liter spore suspension / meter.

[Vol. 62(4) : 2009]

coconut coir and mustard oilcake which failed to

stimulate spore production but mycelial fragments
and chlamydospores from the substrate caused
minor damage to the weed. Variation in virulence
might be due to the chemical constituents of the
substrate. Herbicidal potential of inoculum produced
on selected substrates was also evaluated under
controlled, green house and field conditions.
It is evident from Table 2 that virulence was
highly variable and environment dependent.
Effectiveness was high under laboratory conditions,
however, declined marginally during field trails
because in laboratory due to control environmental
condition, the fungus performance is better and as
we take to the green house and field condition. The
environmental condition reduces its efficiency and
performance. Inoculum produced on maize cob grit
was highly effective in field trials. On the basis of
above finding maize cob grit is selected for further
studies.
On the basis of above findings it can be
concluded that solid substrate fermentation using
agro-waste is one of the most promising way to get
sufficient inoculum of a mycoherbicidial agent for
field application, However, Churchill 1982, Boyette
et al., 1991 and Stanbury et al., 1995, stated that
labour costs, difficulties in maintaining sterility,
lack of controls on fermentation conditions and
recovery of the spores from the substrate are the
major problems associated with solid substrate
fermentation, However, this method is highly
applicable for those agents, which do not sporulate
well in submerged to liquid fermentation as the
case of C. dematium FGCC# 20. Application of
conidia along with colonized substrate may provide
excellent opportunities for further multiplication of
the agent in field conditions. Thus, the mycoherbicide
agent can multiply on the selected substrates.
However, further studies regarding shelf-life,
formulations and stability are to be carried out
before commercial recommendation, which are
underway.
ACKNOWLEDGEMENTS
We are thankful to the Head, Department of
Biological Science, Rani Durgawati University,
Jabalpur (M.P.) India, for laboratory facilities and
University Grant Commission, New Delhi for financial
assistance.

Indian Phytopathology

497

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Received for publication May 24, 2008