Crop Protection 30 (2011) 1334e1339

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Crop Protection
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Extended shelf-life of liquid fermentation derived talc formulations

of Trichoderma harzianum with the addition of glycerol
in the production medium
Subbaraman Sriram*, Kodigehalli Panchakshari Roopa, Maheshwarappa Jeyamma Savitha
National Bureau of Agriculturally Important Insects, (Formerly Project Directorate of Biological Control), HA Farm post, Bellary Road, Hebbal, Bangalore, Karnataka 560024, India

a r t i c l e i n f o a b s t r a c t

Article history: Liquid fermentation based formulations of Trichoderma spp. are vulnerable to desiccation compared to
Received 11 February 2011 solid state fermentation based formulations. The effect of the addition of glycerol, an osmoticant in the
Received in revised form production medium on the shelf-life of Trichoderma harzianum was studied. The addition of glycerol at 3,
30 May 2011
6 or 9% reduced the water activity in the medium. Both in shaker culture and fermentor, the addition of
Accepted 5 June 2011
glycerol in production medium prolonged the shelf-life of talc formulation. The addition of glycerol at 3
and 6% extended the shelf-life (with viability of >2
106 CFU g1) to 7 and 12 months, respectively
Keywords:
compared to 4e5 months shelf-life in formulations derived without the addition of glycerol. Regression
Trichoderma harzianum
Liquid fermentation
analysis showed that there was positive correlation between colony forming unit (CFU) and water
Shelf-life activity. In bio-efcacy tests, even after storage for 12 months, formulations derived with the addition of
Formulation glycerol at 3 or 6% in the production medium could protect the tomato plants from Fusarium wilt inci-
Glycerol dence by 44e50%. The use of addition of osmoticants in production medium for enhancing the shelf-life
of liquid fermentation derived fungal formulations is discussed.
2011 Elsevier Ltd. All rights reserved.

1. Introduction developed and applied in many developing countries for mecha-

Many commercial products of Trichoderma spp. are available for
the management of soilborne plant diseases (Fravel, 2005). Among
the different propagules like conidia, chlamydospores and vegeta-
tive mycelium, the conidia have been the most widely employed in
biocontrol programs (Elad et al., 1993). Conidial biomass can be
obtained either by submerged or solid substrate cultivation tech-
niques. Conidia of Trichoderma derived from solid state fermenta-
tion are highly tolerant to abiotic stresses compared with
propagules or biomass derived from liquid fermentation
(Watanabe et al., 2006). Hence the formulations derived from liquid
fermentation based mass production methods have relatively
shorter shelf-life. Though high quality spores could be easily
produced by solid state fermentation (SSF) at bench scale, the
engineering aspects related to scaling up of SSF have not been
Abbreviations: CFU, colony forming units; SSF, solid state fermentation; PEG,
poly ethylene glycol; IMTECH, Institute of Microbial Technology, Chandigarh, India;
MTCC, Microbial Type Culture Collection; MYE, molasses yeast extract; PDA, potato
dextrose agar; ANOVA, analysis of variance; DMRT, Duncans multiple range test.
* Corresponding author. Tel.: 91 8023511982x355; fax: 91 8023411961.
E-mail address: sriram1702@gmail.com (S. Sriram).
nized or automated mass production of Trichoderma. Wettable
powder formulations based on liquid fermentation are popular in
India though they have shorter shelf-life (Singh et al., 2006). Liquid
fermentation has the advantages of control over the production
process, short time for production, requirement of less space and
labor, control over contamination level, etc. Liquid fermentation
can facilitate abundant production of conidial biomass at a shorter
period (Harman et al., 1991). Hence, there is need to extent the shelf
life of Trichoderma formulations derived from liquid fermentation.
Many factors like medium and inoculum type (Elzein et al., 2004),
method of drying, the addition of protectants (Friesen et al., 2006)
and environmental conditions during storage (Connick et al., 1996)
affect the viability of the formulation derived from liquid fermen-
tation. Different interventions can be made during fermentation or
at post-fermentation stages to extend the shelf-life of formulations.
For example, the addition of chitin as specic nutrient in produc-
tion medium or in formulation can increase the shelf life of liquid
fermentation derived Trichoderma harzianum talc formulation by
two months compared with un-amended formulation or medium
(Sriram et al., 2010). Similarly, heat shock at the end of log phase at
40  C for 30 min also induced desiccation tolerance and extended
the shelf life by 4 months.

0261-2194/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cropro.2011.06.003
 S. Sriram et al. / Crop Protection 30 (2011) 1334e1339
The osmoticum of the production medium can be adjusted by
the addition of poly-ethylene glycol or glycerol that can induce
trehalose production and provide the desiccation tolerance (Jin
et al., 1991, 1996). Since the biomass of a microbial biocontrol
agent is dried after mixing with the carrier material to avoid
possible contamination during shelf-life, the conidial biomass has
to be desiccation tolerant besides having high spore viability.
Compared with poly ethylene glycol (PEG), the addition of glycerol
was found to be more benecial since PEG addition resulted in
reduced biomass though it provided desiccation tolerance. Use of
glycerol as the osmoticant is particularly effective in initiating
micro-cycle conidiation. Accumulation of trehalose that is respon-
sible for stabilizing membranes of cells during desiccation in con-
idia of T. harzianum was correlated with desiccation tolerance (Jin
et al., 1996). In the present study, the effect of the addition of
glycerol in the production medium that was used in liquid
fermentation on the shelf-life of the talc formulations of
T. harzianum was studied. The bioefcacy of formulations obtained
from liquid fermentation with the addition of glycerol in the
production medium was tested using Fusarium wilt e tomato
system.
2. Materials and methods
2.1. Biocontrol agent
T. harzianum (NBAII-Th10, IMTECH MTCC accession number
5584), maintained at the National Bureau of Agriculturally Impor-
tant Insects, Bangalore, was used in the study. It was maintained on
potato dextrose agar (PDA) slants. The identity of the culture was
conrmed based on morphological characters and molecular
methods using oligo-nucleotide barcode (Trichokey) developed by
Druzhinina et al. (2005). T. harzianum was grown on potato
dextrose agar for 96 h and the conidia from the colonies were
selected using sterile inoculation needle and transferred to sterile
distilled water. The conidial population was adjusted to 1
107
conidia per ml using a haemocytometer and transferred to potato
dextrose broth so that the nal conidial population would be
1
105 conidia per mL and incubated in shaker culture at 28  C and
200 rpm for 48 h. The 48-h-old culture was used as inoculum for
the main culture (liquid fermentation for production purpose) at
the rate of 10 ml L1.
2.2. The effect of the addition of glycerol in production medium on
the shelf life of T. harzianum
2.2.1. Shaker culture
To study the effect of the addition of glycerol in the production
medium on the shelf life of talc formulations of Trichoderma spp.,
the production medium (molasses yeast extract (MYE) medium
containing 30 g molasses, 10 g yeast extract in 1 L of distilled water)
was amended with 3, 6, 9 and 12% glycerol (V/V). Five replications
were maintained for each treatment with ve asks for each
replication. T. harzianum grown on MYE broth without glycerol
served as control. The broth cultures were inoculated with
T. harzianum and incubated in a shaker at 28  C and 200 rpm for
a week. The biomass along with medium was mixed with talc
powder (Mesh No. 300, 53 mm size, white coloured) at 1:2 ratio
(v:w) and dried to 15% moisture content. The air-drying method
was followed at ambient conditions. The initial population and the
population of viable conidia after one month of storage were
enumerated in these formulations by plating 1 g of sample serially
diluted to 106 dilution using pour plate method using a Tricho-
derma-specic medium (Elad et al., 1981). The formulations were
stored in polypropylene covers (density 0.90 g/cc) at ambient
1335
temperature in closed storage cabinets and viability in terms of
CFUs (colony forming units) were enumerated as described earlier
at monthly intervals till the population in all treatments was less
than 2
106, the minimum recommended level of CFUs g1
prescribed by the Central Insecticides Board Act, Govt. of India. The
average maximum and minimum ambient temperatures were
23  Ce29  C during the experimental period.
2.2.2. Fermentor culture
The above experiment was conducted in fermentor conditions
with 200 rpm agitation at 28  C with glycerol amendment (3, 6 and
9%) in production medium. A Virtis fermentor of 2 L working
volume was used. The fermentor culture was maintained for 96 h.
The aeration was at 1.5 bar pressure. Production of biomass and
formulation preparation was as described earlier.
2.3. Shelf life studies
The viability of the talc formulations was checked by enumer-
ation using serial dilution technique. The water activity and percent
moisture content were also recorded during the shelf life studies.
The water activity was measured using water activity meter (HW3
model of Rotronic make) and the moisture content was recorded
using Sartorius moisture analyzer.
2.4. Bioefcacy of talc formulations
Fusarium oxysporum f. sp. lycopersici (NBAII e Fol e 01) culture
isolated from wilt infected tomato plants was used in the present
study. Fusarium isolates were stored at 4  C on PDA. Tomato
(Lycopersicon esculentum Mill.) cv Selection 22 was used in all the
experiments. Bio-peat (SG) compost that contains coconut coir-pith
(locally called as cocopeat) was used as planting medium for all the
experiments. Seeds were sown in cocopeat and the seedlings of
25e30 days were used for the bioefcacy tests.
Talc formulations of T. harzianum derived with the addition of
glycerol at 3 or 6% in the production medium were tested for their
bioefcacy in protecting the tomato plants against wilt disease
caused by F. oxysproum f. sp. lycopersici after a shelf life of 12
months. Fresh formulation obtained without the addition of glyc-
erol in the production medium that was prepared just before the
bioefcacy assays was also tested for comparison. One set of plants
were treated with these formulations and challenge inoculated
with the pathogen while another set was treated only with
T. harzianum formulation. One set of plants was treated with
pathogen alone. Control plants without any treatment served as
control.
Fifteen day-old tomato seedlings were washed with sterile
water and subjected to seedling dip with Trichoderma formulations.
The seedlings were dipped for 10 min. Talc formulations were used
at the rate of 10 g L1 for seedling dip. After treatment the seedlings
were transplanted in plastic seedling trays that contained Bio-peat
(SG) compost. The pathogen inoculation was done as spore drench
with spore suspension of F. oxysporum f. sp. lycopersici (4e6
106
spores ml1). There were ve replications each having 20 plants.
Observations were recorded for wilt symptoms for up to ve weeks.
Percent wilt incidence and plant growth parameters were recorded.
2.5. Statistical analysis
The CFU values of were log transformed (base to 10) and
analyzed by analysis of variance (ANOVA) (Gomez and Gomez,
1984). Similarly, water activity and percent moisture content data
were analyzed by ANOVA. For the post-ANOVA statistical compar-
ison, DMRT (Duncans Multiple Range Test) was followed. SYSTAT12
1336 S. Sriram et al. / Crop Protection 30 (2011) 1334e1339

software was used for chi square test and regression analysis. For
the bioefcacy tests, the data obtained were analyzed by ANOVA
using SYSTAT12.
3. Results
3.1. The effect of the addition of glycerol in production medium on
the shelf life of T. harzianum
3.1.1. Shaker culture
Addition of glycerol at 3, 6 and 9% concentration (v/v) reduced
the water activity in the production medium from 0.96 to 0.95,
0.946 and 0.933, respectively. The addition of glycerol at 3 and 6%
resulted in signicantly higher viability of T. harzianum with an
average of 8.13 and 8.06 Log CFUs g1 of formulation during the
shelf-life for up to eight months, respectively (Table 1). The reduced
viability in formulations with the addition of glycerol (9 and 12%)
was due to the increased fungal contamination during shelf life.
Chi-squared analysis was carried out to determine whether the
average CFUs in formulations derived from biomass grown on
medium amended with different concentrations of glycerol remain
constant. Since the computed c2 value (0.38) was smaller than the
corresponding tabular c2 value (44.31) at 5% level of signicance,
the hypothesis of independence between glycerol concentration in
production medium and viability during shelf life could not be
rejected, implying that ratio of differences in viability due to the
addition of glycerol remained constant in the formulations. Varia-
tion in viability due to the addition of glycerol at different
concentrations was not dependent on time of viability test.
3.1.2. Fermentor culture
Among formulations obtained from fermentor (2L), in control
that were derived from the biomass grown on the MYE medium
without the addition of glycerol, the shelf life was only 6 months
(Table 2). With the addition of glycerol at 3 or 9%, the shelf life was
nine months beyond the time when the viability was reduced. With
the addition of glycerol at 6%, the viability was more than 2
106
CFUs for up to 13 months indicating that viable propagules survived
for longer period in the formulation that was derived from biomass
grown on glycerol amended medium (6%). Though the addition of
glycerol (3%) resulted in good viability for up to nine months, there
was a sharp decline in viability of the propagules.
3.2. Effect on glycerol addition in production medium on water
activity and moisture content in the formulation
Since the formulations were packed in polypropylene bags that
allow the exchange of vapor, there was variation in denite pattern
in water activity according to the change in weather parameters
(Fig. 1). The analysis of variance revealed that there was no signif-
icant difference in water activity in formulations prepared with the
addition of glycerol 3% in production medium. In other formula-
tions derived with the addition of glycerol at 0, 6 or 9% concen-
tration, there were signicant differences in the water activity.
Regression analysis of water activity in formulations over a period
of time (shelf life) using non-linear regression method revealed
that in formulations derived with the addition of glycerol at 3, 6 or
9% in production medium, the response was reverse to that of
formulation derived with no addition of glycerol in production
medium. The addition of glycerol in the production medium at
more than 3% increased the capacity of the talc formulations of
T. harzianum to retain the water activity during the shelf life.
The percent moisture content in all formulations decreased
gradually from the original 15% at which they were packed during
shelf life (Fig. 2). There was no signicant variation in moisture
content in formulations obtained without the addition of glycerol
in the production medium during its short shelf-life while there
was signicant variation in formulation obtained with the addition
of glycerol. With the addition of glycerol at 3%, the moisture content
in formulations was reduced to 7.95% after 10 months from the
initial 15%. With the addition of glycerol at 6%, the moisture content
was retained at above 9% even after 12 months with the recovery of
propagules at 106 dilutions. With increase in the concentration of
glycerol added to the production medium, there was increased
retention of moisture in the formulation during shelf-life. The
regression analysis of the moisture content in relation to shelf life
revealed that the rate of reduction in moisture content in formu-
lation was inversely proportional to the concentration of glycerol
added in the production medium. The slope of the regression
equations tted for the three concentrations of the addition of
glycerol (3, 6 and 9%) with respect to moisture content over
a period of time was 0.81, 0.58 and 0.25, respectively, indicating
that increased glycerol content in production medium facilitated
better retention of moisture in the formulation during the shelf life.
Chi-squared analysis was carried out to test whether the ratio of
viability (in terms of CFUs g1) in formulations obtained from
biomass grown in medium amended with different concentrations
of glycerol remained the same at different water activity (aw) or
moisture content ranges. Since the computed c2 values (57.28,
73.59) were greater than the corresponding tabular c2 value (34.81,
38.93) at 1% level of signicance, the hypothesis that CFUs in
formulation obtained from biomass grown in the medium amen-
ded with different concentrations of glycerol was independent of
aw or moisture content was rejected, implying that viability of
formulations obtained from biomass grown in medium amended
with different concentrations of glycerol was dependent on mois-
ture content and water activity. Similarly, the chi-squared test to
study the relationship between aw and moisture content in

Table 1
The effect of the addition of glycerol in the production medium on the viability (Log CFUs) of talc formulation of T. harzianum (shaker culture).a

% Glycerol added to the production medium Log of CFUs during shelf life at 2-month intervalb

Initial 2 months 4 months 6 months 8 months Average

0% 7.81 (7.22) 7.51 (7.65) 7.48 (7.23) 6.30 (7.02) 5.00 (4.43) 6.82e (6.71)
3% 8.32 (8.61) 9.16 (9.12) 8.44 (8.62) 8.26 (8.36) 6.50 (5.93) 8.13a (8.13)
6% 8.09 (8.54) 9.37 (9.04) 8.25 (8.55) 8.25 (8.30) 6.36 (5.88) 8.06b (8.06)
9% 8.11 (8.11) 8.49 (8.58) 8.33 (8.12) 8.35 (7.88) 5.00 (5.59) 7.65c (7.66)
12% 8.15 (7.96) 8.31 (8.43) 8.00 (7.97) 8.13 (7.73) 5.00 (5.52) 7.52d (8.52)
Average 8.09s (8.09) 8.57p (8.56) 8.10p (8.10) 7.86q (7.85) 5.57r (7.47) 7.65 (7.62)

Values in the last column followed by the same letter do not differ signicantly at P 0.05. Similarly, s in the last row followed by the same letter do not differ signicantly at
P 0.05.
a
SE (d): Time- 0.043, Glycerol concentration e 0.043, time
glycerol concentration interaction e 0.096; CD at P 0.05: Time: 0.087; glycerol concentration e 0.088;
time
glycerol concentration interaction e 0.193.
b
Values in parentheses are expected values as per c2 analysis. Computed c2 value: 0.38, Tabular value @ (d.f 25): 44.31 at P 0.01.
 S. Sriram et al. / Crop Protection 30 (2011) 1334e1339 1337

Table 2
Effect of the addition of glycerol in the production medium on the viability (Log
CFUs) of talc formulation of Trichoderma harzianum (fermentor culture).

Time (months) Viability in terms of Log of CFUs g1 in formulation

derived
after glycerol amendment at different glycerol
concentrations

0% 3% 6% 9%
1 7.89 9.00 7.94 8.12
2 7.79 9.99 8.05 7.86
3 6.78 9.69 7.98 7.32
4 7.48 9.87 7.82 7.34
5 6.95 10.10 7.54 7.59
6 6.31 10.65 7.46 6.83
7 4.60 10.77 7.10 6.64
8 e 10.41 7.01 6.95
9 e 8.22 7.21 6.30
10 e 5.0 7.14 5.00
11 e e 7.16 4.00
12 e e 7.27 e
13 e e 6.48 e
SE(d) 0.43 0.22 0.41 0.25
Fig. 2. Effect of the addition of glycerol in the production medium on the moisture
CD @ P 0.05 0.93 0.46 0.84 0.52
content of talc formulation of Trichoderma harzianum (fermentor culture).
CV% 8.02 2.85 6.92 4.58
F 15.96 145.33 3.83 48.51
d.f. (14, 20) (20, 29) (26, 38) (22, 32)

Note: e not tested. 3.3. Bio-efcacy tests

c2 analysis to check the effect of the addition of glycerol on the viability (Log CFUs),
water activity and moisture content of talc formulation of T. harzianum amended
with glycerol in the production medium (fermentor culture) was done and the c2
values are as follows.
% glycerol in production medium vs. water activity in formulation - c2 - 57.28, table
F 28.87 @ P 0.05, df 18; % glycerol in production medium vs. moisture content
in formulation- c2 - 73.59, table F 32.66 at P 0.05, df 21; Water activity in
formulation vs. moisture content in formulation - c2 113.07, table F 43.77 at
P 0.05, d.f. 30.
formulations also revealed that both the parameters were inde-
pendent (c2 113.07, Tabular F 50.89, d.f. 30, P 0.01).
The average CFUs during shelf-life in formulations derived with
the addition of glycerol at 3% in production medium was higher
(>109 Log CFUs 9.37) while with the addition of glycerol at 6% it
was >107 (Log CFUs 7.47). The average water activity did not
differ signicantly in all the treatments (0.955e0.969). There was
a decrease in the percent moisture content during shelf life.
Seedling dip treatment of tomato seedlings with talc formula-
tions of T. harzianum derived from biomass grown on production
medium amended with different concentrations of glycerol (3, 6
and 9%) reduced wilt incidence by F. oxysporum f. sp lycopersici in
tomato seedlings by 44e50% (Table 3). The bioefcacy of these
formulations was tested for 12 months. The formulations that had
12 months shelf life were used in the bioefcacy test and compared
with freshly prepared formulations. With pathogen inoculation in
control, there was 80% infection while in plants treated with fresh
formulations or formulations derived with the addition of glycerol
at 3% there was only 45% wilt infection resulting in nearly 44%
reduction in wilt incidence. Formulations derived with the addition
of glycerol at 6% showed only 40% infection, resulting in 50%
reduction in wilt incidence. Pathogen inoculation reduced the
average root length, shoot length, root weight and shoot weight
compared with plants that were not inoculated with pathogen.
There was no signicant change in the root weight due to different
treatments. The root length was shorter in plants inoculated with
pathogen. Plants treated with different formulations of Trichoderma
were having more shoot length (14.03e15.23 cm) compared to
control plants (11.03 cm). Similarly shoot weight was also higher in
Trichoderma treated plants (0.39e0.49 g) compared with control
(0.24 g). There was no signicant difference in root length or weight
between Trichoderma treated and control, though the pathogen
inoculated plants showed reduced root weight and length
compared with plants without pathogen inoculation.

4. Discussion

In the present study the effect of the addition of glycerol in the

Fig. 1. Effect of the addition of glycerol in the production medium on the water acti-
vity(aw) of talc formulation of Trichoderma harzianum (fermentor culture).
production medium on the shelf life of talc formulation of
T. harzianum has been studied. The addition of glycerol before
drying the formulation increased the dispersal of conidia of
Penicillium oxalicum used for the biocontrol of F. oxysporum f. sp.
lycopersici (Sabuquillo et al., 2005). The addition of glycerol at 7.5%
increased the conidial viability of Epicoccum nigrum and Penicil-
lium frequentans that were used in the biological control of post-
harvest brown rot of peaches (Melgarejo et al., 2007). Recently
Kolombet et al. (2008) developed a paste formulation of Tricho-
derma asperellum with the addition of glycerol in the formulation
1338 S. Sriram et al. / Crop Protection 30 (2011) 1334e1339

Table 3
Bio-efcacy tests of talc formulations of Trichoderma harzianum grown in glycerol amended medium against Fusarium wilt of tomato.

Treatment Shoot length in cm Root length in cm Total in cm Shoot weight in g Root weight in g Total in g % wilted plants % reduction
in wilt incidence
Control 11.03b 3.64b 16.67a 0.24c 0.02 0.25c e e
3%G 15.23a 3.51b 18.74a 0.42b 0.01 0.43b e e
6% G 14.43a 3.32b 17.75a 0.39b 0.01 0.40b e e
Fresh formulation 14.03a 3.72b 17.75a 0.49a 0.01 0.51a e e
Control Fol 10.93b 2.13a 13.06b 0.20d 0.01 0.21c 80b e
3%G Fol 9.90b 1.85a 11.75b 0.19e 0.01 0.20c 45a 44.7
6% G Fol 10.18b 1.54a 11.72b 0.18d 0.00 0.19c 40a 50.0
Fresh formulation Fol 11.09b 1.82a 12.91b 0.18d 0.01 0.19c 45a 44.7

(3%G, 6%G e Talc formulation obtained from liquid fermentation with addition of 3% and 6% glycerol in production medium respectively, Control - Talc formulation obtained
from liquid fermentation without the addition of glycerol in production medium, Fol e F. oxysporum f. sp. lycopersici), Fresh formulation e formulation obtained without
glycerol addition in the production medium. Values in a column followed by same alphabets do not differ signicantly at P 0.05, by DMRT analyzed through ANOVA.

that had extended shelf life. The addition of glycerol (2%) as
carbon source promoted the biosynthesis of secondary metabolite
by Trichoderma spp. (Gogoi et al., 2008). Earlier, Hallsworth and
Magan (1995) reported that conidia of Metarhizium anisopliae
and Paecilomyces farinosus that were with high intercellular
glycerol content germinated quickly compared with normal
cultures even at low water activity. In the present study we report
the enhanced shelf-life of T. harzianum formulations in response
to the addition of glycerol in production medium (liquid
fermentation).
The addition of glycerol aided in maintaining higher moisture
content in the formulation and protected the viable propagules
from the reduced water activity during shelf-life. Both the shaker
and fermentor culture derived formulation exhibited extended
shelf-life due to the addition of glycerol compared with no addition
of glycerol in production medium. The addition of glycerol at 3 or
6% in the production medium resulted in retention of viable
propagules above 2
106 per g in formulations, which is the
minimum requirement as per the guidelines of the Central Insec-
ticide Board, Government of India. During the second month of
storage, there was increased viability in the formulation compared
with that of packing time. The packing was done at 15% moisture
content and with the available water vapor, the propagule present
in the formulations derived with the addition of glycerol, might
have germinated and resulted in increase in the viability when
plated for colony count. Water activity is a crucial factor for the
fungal propagules derived from liquid fermentation, since low
water activity would reduce the viability of the propagules. Kreditcs
et al. (2000, 2004) established that water activity in the production
medium inuenced the growth as well as extracellular enzyme
activities of the biocontrol strains of Trichoderma spp. The addition
of glycerol alters the water activity and provides the desiccation
tolerance to the propagules. In present study, the reduced water
activity in the production medium due to the addition of glycerol
resulted in desiccation tolerant formulation of T. harzianum. With
the addition of glycerol the water activity in the production
medium was reduced to 9.33, however the production of viable
propagules was not affected. The addition of glycerol at 9% resulted
in retention of moisture content at above 14% till 11th month. There
was a reduction in viability with no recovery of propagules at 106
dilutions due to fungal contamination at later stages of shelf life
probably favored by relatively higher moisture content.
In the bio-efcacy studies, even after 12 months of storage, at
ambient temperature, talc formulations of T. harzianum derived
from liquid fermentation with the addition of glycerol in produc-
tion medium were effective in controlling the Fusarium wilt inci-
dence in tomato plants and they were on par with fresh
formulations prepared without the addition of glycerol. This indi-
cates that the addition of glycerol in production medium increases
the desiccation tolerance of propagules in the formulation without
reducing the bio-efcacy of the bio-agent.
The approximate cost of glycerol (Analar grade) in Indian
currency is Rs.360 per L. Addition of glycerol at the rate of 3% would
result in increase of cost of production by approximately Rs.5.40 per
kg of talc formulation. Considering the benet of extended shelf-
life and marketability of the product, this increase in cost of
production is very negligible. The commercial units producing
Trichoderma formulations can use this simple production medium
manipulation by addition of glycerol and get extended shelf-life for
their products. This will ensure good quality and viable products in
the market available for the farmers who are the end users. Uptake
of more biocontrol agents will in turn reduce the dependence on
the chemical fungicides and cost of production also. Trichoderma
usage not only will reduce disease incidence but will help in getting
good plant growth promotion thereby indirectly helping in
increased yield parameters.
Acknowledgement
The authors sincerely acknowledge the Director, National
Bureau of Agriculturally Important Insects (formerly Project
Directorate of Biological Control), Bangalore for providing the
infrastructure and other facilities.
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