BIOLOGICAL CONTROL

ARTICLE NO.

7, 267274 (1996)

0093

Development of Media and Automated Liquid Fermentation Methods to

Produce Desiccation-Tolerant Propagules of Trichoderma harzianum
X. JIN,1 A. G. TAYLOR,

AND

G. E. HARMAN

Departments of Horticultural Sciences and Plant Pathology, New York State Agricultural Experiment Station, Cornell University,
Geneva, New York 14456
Received June 5, 1995; accepted April 23, 1996

scale-up of the fermentation process.

A suitable medium was developed from modified
Richards medium plus V8 juice (RM8) to produce high
levels of desiccation-tolerant conidia of Trichoderma
harzianum strain 1295-22. The addition of 9% (v/v)
glycerol to RM8 improved both biomass production
and desiccation tolerance of the conidia of T. harzianum. This medium was then used in a laboratory scale
fermenter (1.5 liter) to determine optimal operating
conditions. The optimal temperature for conidial production and desiccation tolerance improvement in the
fermenter was 32C when dissolved oxygen was maintained at 50% saturation of air, and the stirring rate
was 1000 revolutions per minute. The initial water
potential of the medium (with 9% glycerol) was 23.7
MPa, the pH was 6, and neither was controlled during
fermentation. Changes in medium pH and dissolved
oxygen were associated with the stages of morphological development and conidiation. The pH of the medium decreased concurrently with germ-tube elongation and mycelium development and then increased to
6.06.2 at phialide formation. Intensive conidiation
occurred at pH 6.36.5 and reached its maximal level at
6.97.1. Changes in pH values could be used as indicators to monitor the morphological development and
conidiation of T. harzianum during fermentation. The
use of a 48-h-old culture inoculum, rather than conidial inoculum, to start fermentation reduced the
time required to complete the shift from vegetative
growth to phialide formation. Intensive conidiation
occurred immediately after the addition of culture
inoculum and reached maximum levels within 68 h of
fermentation. Dry weight of biomass increased with
the duration of fermentation and was greatest at 96 h.
However, no improvements in conidia/gram and CFU/
gram were achieved after 72 h of fermentation. The
desiccation tolerance of conidia harvested at 72 or 96 h
was significantly (P 5 0.05) greater than that of conidia harvested at 48 h of fermentation. Results obtained from this study could be used for further
1 Present address: EcoScience Corp., 10 Alvin Court, East Brunswick, NJ 08816.

r 1996 Academic Press,

Inc.

KEY WORDS: fermentation model; Trichoderma; morphological development; conidiation.

INTRODUCTION

Mass production of Trichoderma harzianum Rifai

has become a focus of research and industrial development in the search for alternatives to chemical seed
treatments to control soilborne plant pathogens (Harman, 1991; Jin et al., 1991, 1992; Papavizas, 1985;
Papavizas and Lewis, 1981). Conidial biomass was
chosen for T. harzianum because these propagules can
be produced abundantly in liquid fermentation systems
(Harman et al., 1991; Jin et al., 1991). Biomass of
fungal biocontrol agents must be produced in a timely
and cost-effective way and to survive each processing
step, such as harvesting, drying, formulation, storage,
and delivery (Jin et al., 1992).
The biomass of a biological control agent is usually
dried to avoid microbial contamination; therefore, it is
imperative that the conidial biomass of Trichoderma be
desiccation tolerant. High spore viability is also an
important aspect of the economics of production. Dead
biomass can be a nutritional source for plant pathogens
and may result in an increase in disease severity. For
these reasons, fermentation systems must produce
biomass of both high quantity and quality (Harman et
al., 1991).
A suitable medium, modified Richards medium plus
V8 juice (RM8), has been developed to produce large
quantities of conidia in submerged shake-flask cultures
(Harman et al., 1991). However, less than 10% of these
conidia retained viability after rapid drying. This disadvantage was largely overcome by producing conidia in
RM8 amended with polyethylene glycol (PEG) (Harman et al., 1991; Jin et al., 1991). The addition of PEG
to lower medium water potential to approximately 22
MPa resulted in the production of a large quantity of
conidia with good desiccation tolerance. However, the

267

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268

JIN, TAYLOR, AND HARMAN

lowered medium water potential reduced biomass dry

weight by 2530% (Jin et al., 1991) and decreased shelf
life of the resulting product (Jin et al., unpublished).
Therefore, the addition of PEG may not be a suitable
amendment to fermenter media, so other amendments
to RM8 that would improve both biomass production
and desiccation tolerance of T. harzianum conidia were
sought.
Most published studies on biomass production of T.
harzianum have not employed fermenters that mimic
those used for commercial production (Harman et al.,
1991; Jin et al., 1991; Lewis and Papavizas, 1983;
Papavizas et al., 1984). Therefore, efforts were made to
produce T. harzianum in a laboratory-scale fermenter
to optimize fermentation processes under precisely
controlled conditions and to provide relevant information for further scale-up production.
This paper describes the amendment of RM8 by the
addition of glycerol to improve both biomass production
and desiccation tolerance of T. harzianum and the
transfer of the results from shake-flask culture to a
laboratory-scale fermenter system. To optimize fermentation conditions, emphasis was placed on the incubation temperature, since the initial pH, dissolved oxygen, and stirring rates were studied in preliminary
experiments. Morphological changes and conidiation of
T. harzianum at different fermentation temperatures
associated with medium pH and dissolved oxygen were
investigated at various intervals during growth of the
fungus in the fermenter. The effect of different types of
initial inoculum (conidial versus whole-culture inoculum) on conidiation and the effect of fermentation
duration on biomass production by using whole-culture
inoculum were also studied.
MATERIALS AND METHODS

Organism and medium. Strain 1295-22 (ATCC

20847) of T. harzianum was used in this study. This
strain was prepared by protoplast fusion (Stasz et al.,
1988) and is effective in biological control of soilborne
plant pathogens in seed treatments (Harman et al.,
1989). The basic medium employed in this study to
grow T. harzianum was modified Richards medium
plus V8 juice (Harman et al., 1991). This medium was
amended with glycerol (Sigma Chemical Co., St. Louis,
MO) and adjusted to pH 6 before autoclaving.
Preparation of inoculum. Two types of inoculum
were used in this study: conidial and whole-culture
inoculum. The density of conidial inoculum was ca. 2 3
109 conidia/ml, and the preparation of this inoculum
has been described previously (Jin et al., 1991). In the
preparation of the whole-culture inoculum, 1 ml of the
conidial inoculum was added to 100 ml of fermentation
medium in a 250-ml Erlenmeyer flask. Flasks were
incubated at 30C and shaken at 150 revolutions per

min (rpm) to provide adequate aeration. The incubation

period was 48 h and the whole culture in each flask was
then used as inoculum in each fermentation run.
Inoculation and growth conditions of shake-flask
culture. RM8 amended with 0, 3, 6, 9, or 12% (v/v) of
glycerol was used for shake-flask culture experiments.
One hundred ml of RM8 plus glycerol in a 250-ml
Erlenmeyer flask was inoculated with 1 ml of a 10-fold
dilution of the conidial inoculum. The final conidial
concentration in the flask was ca. 2 3 106 conidia/ml.
Flasks were incubated at 30C and shaken at 150 rpm
for 4 days. The amount of glycerol added to RM8 that
improved both biomass production and desiccation
tolerance of conidia was then determined and this
concentration was added to RM8 to form the medium
used in the laboratory-scale fermenter.
Inoculation and growth conditions in the fermenter.
This study was performed in a laboratory-scale fermenter (Model Proteus 2000, V 5.51; Wheaton Instruments, Millville, NJ) with a working volume of 1.5
liters. The fermenter was interfaced with a Macintosh
Plus computer, and data on fermentation conditions
were recorded every 5 min. The pH of fermentation
medium was adjusted to 6 before autoclaving while
dissolved oxygen was calibrated after autoclaving. The
stir speed was 1000 rpm, which was optimal based on
our earlier studies. The fermentation temperatures
were controlled at 30, 32, 34, and 36C, respectively.
The pH of fermentation medium was not controlled
during fermentation. Dissolved oxygen was calibrated
to 0 by nitrogen input and to 100% by air to its
saturation point. During fermentation, dissolved oxygen was controlled at 50% saturation of air. Foaming
was controlled by adding Antifoam 289 (Sigma Chemical Co.) before autoclaving at a concentration of 100
mg/liter.
The total volume of fermentation medium was ca.
1.25 liters. In the study on optimizing fermentation
temperature, conidial inoculum was added to each run
to form a final conidial density of ca. 2 3 106 conidia/ml.
The 48-h-old whole-culture inoculum grown in one
shake flask at 30C and 150 rpm was used to inoculate
each fermenter run to study its effect on conidiation
and the effect of fermentation duration on biomass
production.
Harvesting and drying of biomass. Upon harvest,
cultures were ground using a Tekmar tissue homogenizer (Tekmar Co., Cincinnati, OH) at full speed for 3
min. The uniform conidial suspensions were centrifuged at 8000g for 15 min, and the supernatants were
discarded. The pellet in each centrifuge tube was
resuspended in 100 ml of distilled water and again
homogenized for 1 min. These suspensions were centrifuged again and the supernatants discarded. The pellet
was removed from each centrifuge tube, spread in a
petri dish, and dried in a vacuum dryer (Virtis Re-

269

Trichoderma FERMENTATION

were also taken at 12-h intervals after 24 h of fermentation to investigate conidiation.

Assay. Dry preparations were soaked in sterile distilled water for 2 h and ground in a Tekmar tissue
homogenizer at full speed for 3 min. The number of
conidia/gram and colony-forming units (CFU)/gram in
the dry preparation were determined as described by
Jin et al. (1991). Culture samples were also ground in
the same way for 3 min, and the numbers of conidia
were then determined by counting in a PetroffHausser
chamber. Germination percentage, as a parameter of
desiccation tolerance of conidia in a dry preparation,
was calculated as the ratio of CFU/gram divided by
conidia/gram 3 100.
To determine the optimum fermentation temperature for the growth of T. harzianum, culture samples
were also taken over time to investigate the morphological stages and conidiation in accordance with the stages
illustrated in Fig. 1. These results were then compared
with the fluctuations of pH and dissolved oxygen in the
fermenter.
Each experiment was replicated three times except
for the fermentation temperature study at 36C, which
was done twice. The statistics were done on Super
ANOVA (Gagon et al., 1989) to discern the difference
among the treatments.
RESULTS

FIG. 1. Illustration of stages in morphological development and

conidiation of Trichoderma harzianum.

search Equipment, Gardiner, NY). In studies on the

effect of initial inoculum on conidiation and the effect of
fermentation duration on biomass production by using
whole-culture inoculum, 100-ml samples of fermentation culture were examined at 48, 72, or 96 h as
described in the next paragraph. Small samples (5 ml)

Effect of the addition of glycerol in RM8 on biomass

production. The addition of glycerol to RM8 reduced
the water potential of the medium (Table 1). The
biomass dry weights were greater with the addition of
glycerol to RM8, compared with that produced in RM8
alone (Table 1). The biomass dry weight was increased
by 37, 25, 24, and 12% for medium of 22.0, 22.8, 23.7,
and 24.8 MPa, respectively, compared with that of
20.8 MPa in RM8 alone. Conidia/gram and CFU/gram
in dry biomass were also increased with the increasing
amounts of glycerol in RM8 and reached their maxi-

TABLE 1
Biomass Dry Weight, Conidia/Gram, and Colony-Forming Units (CFU)/Gram in Dried Preparations of Trichoderma
harzianum Produced from Modified Richards Medium Plus V8 Juice (RM8) with Different Concentrations of Glycerol a
Medium

Water
potential MPa

Biomass dry weight

(mg/100 ml culture)

Log (conidia/g)

Log (CFU/g)

Germination
percentage

RM8 1 0% glycerol
RM8 1 3% glycerol
RM8 1 6% glycerol
RM8 1 9% glycerol
RM8 1 12% glycerol

20.8
22.0
22.8
23.7
24.8

358 A
492 D
447 C
444 C
400 B

10.72 A
10.75 A
10.86 B
11.04 C
10.76 A

9.33 A
9.94 B
10.41 C
10.88 D
10.33 C

4.0 A
15.3 B
35.9 C
67.3 D
37.0 C

a Cultures were grown in 250-ml Erlenmeyer flasks, each containing 100 ml of RM8 with or without glycerol and shaken at 150 rpm for 4
days at 30C. Conidial inoculum was used to start cultures. Each value is the mean of four replicates. Numbers followed by dissimilar letters
within columns are significantly different at P 5 0.05. The germination percentage of conidia was calculated by using CFU/gram divided by
conidia/gram 3 100.

270

JIN, TAYLOR, AND HARMAN

mum level at 23.7 MPa (9% glycerol). The CFU/gram

and total CFU (biomass dry weight 3 CFU/g) in the dry
biomass produced at 23.7 MPa were increased 36- and
45-fold, compared with those produced in nonamended
medium. The germination of conidia so produced was
67.3%, while that from RM8 alone was only 4.0%.
Effect of temperature on biomass production. Fermentation temperature affected both biomass production and desiccation tolerance of conidia of T. harzianum (Table 2). Biomass dry weight produced at 32C
was greater by 9, 25, and 78% than those produced at
30, 34, and 36C, respectively. Conidia/gram and CFU/
gram from biomass produced at 30 or 32C were greater
than those from 34 or 36C. The germination percentage of conidia was higher in biomass produced at 32C
than in biomass produced at higher or lower temperatures.
Relationships between pH and dissolved oxygen of the
medium and morphological development of T. harzianum at different temperatures. The stages of morphological development and conidiation of T. harzianum at
different temperature levels corresponded with the
fluctuations in medium pH and dissolved oxygen in the
fermenter (Fig. 2). The overall responses of pH and
dissolved oxygen at 30, 32, or 34C were as follows.
Medium pH decreased to 5.75.9 at germ-tube elongation and reached its lowest point at mycelium development when dissolved oxygen decreased to 50% saturation of air. The medium pH increased to 6.06.2 at
phialide formation and was 6.46.5 at lateral conidiation. Eventually, the medium pH reached 6.97.1 when
intensive conidiation was completed (data not shown).

The morphological development and conidiation of T.

harzianum were affected by the fermentation temperatures and were delayed at 34 and 36C (Fig. 2).
Approximately 10 h were required for the fungus to
germinate in the fermentation medium at temperatures of 30 and 32C, compared with 12 and 10 h at 34
and 36C, respectively. The formation of phialide and
lateral conidiation were delayed to 36 and 40 h, respectively, when the fungus was incubated at 36C, compared with those at 32C. The corresponding pH levels
for each development stage were lower at 36C than at
32C and were only 6.56.7 at the end of fermentation
(data not shown).
Effects of initial inoculum types on conidiation.
When a 48-h-old culture was used to initiate fermentation at 32C, extensive conidiation occurred immediately after inoculation and reached log 8.3 conidia/ml in
24 h (Fig. 3). However, it took 60 h to reach this level of
conidial density when conidial inoculum was used. The
highest conidial density level, log 8.8 conidia/ml, was
reached within 68 h of fermentation with whole-culture
inoculum, but more than 90 h was required to reach the
same level when conidia were used as the inoculum.
Effect of fermentation duration on biomass production. Biomass dry weight increased with the fermentation duration (Table 3) and was greatest at 96 h of
fermentation. However, no improvements in conidia/
gram and CFU/gram were achieved after 72 h of
fermentation. Based on the percentage of germination,
the desiccation tolerance of conidia harvested at 72 or
96 h was significantly (P 5 0.05) greater than those
harvested at 48 h of fermentation.

TABLE 2

DISCUSSION

Effect of Fermentation Temperature on Biomass Dry

Weight, Conidia/Gram, and Colony-Forming Units (CFU)/
Gram in Dried Preparations of Trichoderma harzianum
Produced in Modified Richards Medium Plus V8 Juice (RM8)
with 9% Glycerol in a Fermenter a

Fermentation systems for biomass production must

meet a number of criteria, including the following: the
biomass produced must be of appropriate propagule
type in an appropriate physiological state so that it can
withstand drying, and it must be produced in large
quantity (Harman et al., 1991). In addition, economical
production of biomass requires a short fermentation
time, since fermentation costs are calculated on a per
day basis (Stowell, 1991).
All of these criteria are a function of the interaction
between microbial physiology and fermentation conditions. In our previous work, we reported the development of media that gave rise to high levels of conidia.
Further, lowering water potential of the growth medium with PEG or other materials resulted in conidia
with good desiccation tolerance and greater levels of
trehalose (Harman et al., 1991; Jin et al., 1991). However, the use of PEGs has disadvantages. Highmolecular-weight PEG is viscous at concentrations
giving appropriate water potential levels, which impedes oxygen transfer, and requires sufficiently high

Fermentation
temperature
(C)

Biomass
dry weight
(mg/100 ml
culture)

Log
(conidia/g)

Log
(CFU/g)

Germination
percentage

30
32
34
36

546 BC
594 C
476 B
333 A

10.81 C
10.79 C
10.61 B
9.65 A

10.47 C
10.54 C
10.27 B
9.25 A

45 A
56 B
46 A
41 A

a A Proteus 2000, V5.51 (1.5 liter) fermenter interfaced with a

Macintosh Plus computer was used. Set points for dissolved oxygen
and stir speed were 50% of saturated air and 1000 rpm, respectively.
The pH was adjusted to 6 at medium preparation. Conidial inoculum
was used to start fermentation. Each value is the mean of three
replicates, except the value obtained from 36C was replicated twice.
Numbers followed by dissimilar letters within columns are significantly different at P 5 0.05. The germination percentage was
calculated by using CFU/gram divided by conidia/gram 3 100.

Trichoderma FERMENTATION

271

FIG. 2. The relationships between medium pH, dissolved oxygen (D. O.), and morphological changes of Trichoderma harzianum strain
1295-22 at different incubation temperatures in a laboratory-scale fermenter, Proteus 2000, V5.51 (1.5 liter). The fermentation medium used
was RM8 with 9% glycerol (23.7 MPa), and conidial inoculum was used to start cultures in the fermenter. Vertical bars indicate standard
errors.

quantities to be economically unrealistic. Low-molecular-weight PEG reduced biomass yields somewhat (Jin
et al., 1991). Use of glycerol as an osmoticum largely
overcame these disadvantages and introduced several
useful new outcomes.
In the production of conidial biomass, microcycle
conidiation is a desirable feature. Microcycle conidiation can be defined as the production of conidiophores
with limited vegetative growth (Anderson and Smith,
1971; Zuber and Turian, 1981). If microcycle conidiation can be achieved, conidial development should
occur earlier in the fermentation process, and therefore
fermentation time can be reduced.
The induction of fungal sporulation frequently is
achieved by low levels of nutrients, usually by carbon or
nitrogen starvation (Mckoy and Trinci, 1987; Righelato
et al., 1968; Turian and Bianchi, 1972; Trinci and
Collinge, 1974; Zuber and Turian, 1981). Sporulation
based on starvation is not particularly useful for the
production of conidial biomass, since starvation occurs
at such low nutrient levels that biomass yields are low.

FIG. 3. The influence of two different inoculum types, conidial

and whole-culture inoculum, on the conidiation of Trichoderma
harzianum strain 1295-22 in a laboratory-scale fermenter, Proteus
2000, V5.51 (1.5 liter). The fermentation medium used was RM8 with
9% glycerol (23.7 MPa). The incubation temperature was 32C.
Vertical bars indicate standard errors.

272

JIN, TAYLOR, AND HARMAN

TABLE 3
Effect of Fermentation Duration on Biomass Dry Weight,
Conidia/Gram, and Colony-Forming Units (CFU)/Gram in
Dried Preparations of Trichoderma harzianum Produced in
RM8 Plus 9% Glycerol in a Fermenter a

Fermentation
duration (h)

Biomass
dry weight
(mg/100 ml
culture)

Log
(conidia/g)

Log
(CFU/g)

Germination
percentage

48
72
96

495 A
669 B
905 C

10.61 A
10.79 B
10.80 B

10.24 A
10.58 B
10.59 B

42 A
64 B
62 B

a A Proteus 2000, V5.51 (1.5 liter) fermenter interfaced with a

Macintosh Plus computer was used. Set points for dissolved oxygen
and stir speed were 50% of saturated air and 1000 rpm, respectively.
The pH was adjusted to 6 at medium preparation. Whole-culture
inoculum was used to start fermentation. Each value is the mean of
three replicates. Numbers followed by dissimilar letters within
columns are significantly different at P 5 0.05. The germination
percentage of conidia was calculated by using CFU/gram divided by
conidia/gram 3 100.

Alternatively, fermentation may be continued until all

nutrients are utilized, but this approach results in
unacceptably long fermentation times. Earlier, we suggested that decreased medium water potential results
in microcycle conidiation (Jin et al., 1991); Zuber and
Turian (1981) previously demonstrated a similar phenomenon using glycerol or D-arabinose as osmotica.
Use of glycerol as the osmoticum is particularly effective in initiating microcycle conidiation; at 32C only 30
h of fermentation was required to initiate the production of new conidia (Fig. 2B). If actively growing culture
inoculum was used, extensive conidiation occurred
from the beginning of the fermentation run. High levels
of conidia were produced within 24 h of fermentation
(Fig. 3) and reached their maximum level in 68 h of
fermentation. However, the dry weight of conidial
biomass continued to increase until 96 h without
further increase in conidial numbers (Fig. 3, Table 3).
Roth (1970) obtained similar results in a study on
sporulation of yeast, Saccharomyces cerevisiae, namely
that the sporulation was characterized by an increase
in dry weight without cell division, and at least 67% of
the dry-weight increase was due to the synthesis of
cellular carbohydrates consisting of trehalose and other
insoluble components. Accumulation of trehalose in
conidia of T. harzianum was correlated with desiccation
tolerance (Harman et al., 1991; Jin et al., 1991). Trehalose is responsible for stabilizing membranes of cells
during desiccation (Crowe et al., 1984). The accumulation of trehalose in conidia of T. harzianum is probably
after conidiation enters its stationary stage, since
conidial biomass harvested after 72 and 96 h of fermentation have much higher desiccation tolerance than
those harvested at 48 h (Fig. 3, Table 3).

The effect of the glycerol-based media may be a

combination of starvation and osmotic effects. The
sucrose added to the RM8 medium was utilized within
2436 h after the initiation of fermentation (data not
shown), so this sugar was apparently utilized preferentially over glycerol. Glycerol was reported not to be
assimilated by T. harzianum (Zuber and Turian, 1981),
but this was not the case with our strain, since biomass
continued to increase for several days after sucrose was
depleted. Glycerol may be, however, a poor carbon
source for our T. harzianum strain.
Glycerol also substantially increased biomass yields
without adding substantial quantities of hyphal biomass. After 96 h of fermentation, biomass dry weight
reached 905 mg/100 ml culture (Table 3). Our previous
yields from similar media with or without PEG to lower
medium water potential did not exceed 500 mg/100 ml
(Harman et al., 1991; Jin et al., 1991). Approximately
1000 mg/100 ml probably represents a maximum level
of biomass, since the culture at this level becomes so
thick that agitation is difficult. This high level of
biomass produced within a short time was achieved
without reduction in either conidia/gram or CFU/gram
compared with our earlier results with PEG-amended
media (Harman et al., 1991; Jin et al., 1991). Thus, the
conidial biomass of T. harzianum produced in glycerolamended medium has most of the features required for
a successful biocontrol product. However, we have
determined that it is essential to remove residual
glycerol from the biomass if it is to be used as a seed
treatment; otherwise the presence of this material
enhances activity of seed-rotting Pythium spp. (data
not shown).
Water potential of RM8 with 9% glycerol (23.7 MPa)
was lower than the optimal medium water potential to
induce conidiation and desiccation tolerance of conidia
by PEG (22 MPa) (Harman et al., 1991; Jin et al.,
1991). It has been shown that fungal growth and
sporulation are more affected by matric than by osmotic potentials (Cook et al., 1972; Cook and Duniway,
1981; Hoch and Mitchell, 1973; Inch and Trinci, 1987;
Manandhar and Bruehl, 1973; Sung and Cook, 1981).
PEG 6000 lowers the water potential largely by matric
forces (Steuter et al., 1981) and the osmotic potential
has a greater contribution to the total water potential
as the molecular weight of PEG decreases (Steuter et
al., 1981). In contrast, glycerol, due to its low molecular
weight, decreased the osmotic potential of a solution.
RM8 with 9% glycerol was a suitable medium for
biomass production of T. harzianum in a fermenter
under precisely controlled growth conditions. The temperaturewater potential interactions have been investigated in Fusarium culmorum, Fusarium graminearum, Fusarium oxysporum f. sp. vasinfectum,
Gaeumannomyces graminis var. tritici, and Verticillium dahliae (Cook and Christen, 1976; Manandhar

Trichoderma FERMENTATION

and Bruehl, 1973). Generally, the optimal temperature

to support maximal growth or sporulation of a certain
fungus differs significantly at different water potentials. Our results showed that when RM8 with a water
potential of 23.7 MPa (9% glycerol, v/v) was used as the
fermentation medium, the optimal fermentation temperature for T. harzianum was 32C.
A computer-controlled fermenter offered an opportunity to compare morphological changes and conidiation
at different fermentation temperatures as the medium
pH and dissolved oxygen level changed. Changes in
medium pH and dissolved oxygen values were correlated with the stages of morphological development and
conidiation. The pH value may have increased as a
consequence of utilization of anions or by production of
ammonia from nitrogenous compounds and decreased
because of the formation of organic acids or absorption
of cations (Cochrane, 1958). Therefore, the pH levels
may be used as indicators to monitor the stage of the
fermentation process.
One of the most important factors that affect the
successful development of biological control systems is
a timely and cost-effective method of production to
produce large quantities of biomass with desired properties (Harman et al., 1991; Kenney and Couch, 1981).
Studies dealing with biomass production of T. harzianum have been conducted in shake-flask cultures in
most cases (Harman et al., 1991; Jin et al., 1991; Lewis
and Papavizas, 1983; Papavizas et al., 1984). Therefore,
it is difficult not only to determine the range of operating conditions, such as the suitability of the medium,
the type of initial inoculum, fermentation temperature,
aeration, and stirring rates, but also to develop the
criteria for further pilot-scale fermentation. Results
obtained in this study could be used to provide a
reliable basis for scale-up of this fermentation process
to an industrial scale.
ACKNOWLEDGMENTS
We thank Patricia Nielsen for technical assistance. This research
was supported in part by a grant from the Eastman Kodak Co. to
Cornell University.

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