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Culture conditions for jasmonic acid and

biomass production by Botryodiplodia
theobromae in submerged fermentation
ARTICLE in PROCESS BIOCHEMISTRY SEPTEMBER 1998
Impact Factor: 2.52 DOI: 10.1016/S0032-9592(98)00035-1

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Process Biochemistry' Vol. 33, No. 7, pp. 715-7211. 1998

C~ 1998 Published by Ir".lsc'~'icrScience l.td, All rights reserved
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ELSEVIER
PII:

S 0 0 3 2 - 9 5 9 2

98 )000035-

Culture conditions for jasmonic acid and

biomass production by Botryodiplodia
theobromae in submerged fermentation
F. Eng, ~' M. G u t i 6 r r e z - R o j a s b a n d E. F a v e l a - T o r r e s h*
"Divisi6n de Biotecnologia, lnstituto Cubano de lnvestigaciones de los Dcrivados de la Carla de Azficar, Vfa Blanca y Carrelera
Central 8(16, A.P. 4026, Ciudad Habana, Cuba
"Departamento de Biotecnologia, Universidad Aut6noma Metropolitana, Unidad lztapalapa, A.P. 55535, C.P. 09340,
M6xico D.F., Mdxicc~
(Received 12 January 1998; accepted 1 March 1998)

Abstract

Jasmonic acid (JA) is a plant growth regulator produced by Bottyodiplodia theobromae in submerged
fermentation. Eight strains of B. theobromae isolated from Cuban orange waste peel were screened fl~r JA
production. Strain 2434 was selected for its higher JA production (914 rag/I) and productivity (c)1.4mg/I
day). Studies carried out at different temperatures showed that the highest JA production (925 rag/I) was
obtained at 30-32C. Although biomass production was enhanced under agitation (up to 100 rpm), JA
production was negatively affected when the agitation was increased. Maximum JA production (900 rag/l)
was obtained under non-agitated conditions. Studies concerning the effect of different carbon and nitrogen
somces showed that fructose, glucose and sodium nitrate were the best sources for JA production. 1998
Elsevier Science Ltd. All rights reserved

K(vwords." Jasmonic acid, Bot~odiplodia theobromae, biosynthesis, growth, submerged fermentation,

phytohormone.

Many studies concerning the biosynthesis of JA in

plants have been published [7-13]. However, knowledge about JA production by microorganisms is still
limited. JA is a secondary metabolite synthesized and
secreted in the late growth phase or the stationary
phase after 5-10 days fermentation. Bot~odiplodia theobromae [ 14-16], mutants of Gibberella fujikuroi [ 17],
Collihya conffuens, Coprinus alkalinus and Mvcena tintinabulum [3] have been reported as JA producers.
Nevertheless, little information concerning JA production conditions is available since most of lhe studies
carried out are published as patents or abstracts. JA
production by B. theobromae D7/2 was higher at agitation rates below 190 rpm [15, 16] and at temperatures
of cultivation from 27C to 30C.
Species of the genus Botryodiplodia arc able to grow
in minimum defined media [16]. JA production by B.
theobrornae D7/2 increased with sucrose and glucose as

Introduction

Jasmonie acid (JA) [3-oxo-2-(2'-cis-pentenyl)-ciclopentane-l-acctate and their derivatives are a group of

native plant growth regulators called jasmonates, their
major representatives are isomers cis [(+)-7-iso-JA]
and trans [(--)-JA] [1]. These compounds are widely
distributed in higher plants [2] and microorganisms
[3,4] showing phytohormone action and playing
important roles as inhibitors of plant growth, inducers
of tuberization on potato, Jerusalem artichoke and yam
and in senescence promotion of detached leaves,
among others [5]. These compounds also induce the
expression of several defensive genes in plants against
pathogen attack or wounding such as the proteinase
gene inhibitors in tomato and the vegetative storage
protein genes in soybean [5,6].
*To whom correspondence should be addressed.
715

E Eng. M. GutiOrrez-Rojas, E. Favela-Torres

716

Table 1. Screening of Botryodiplodia theobromae strains for

jasmonic acid and biomass production after 10 days of
culture
Strain

Biomass (g/L)

JA (mg/l)

1
1F
2
2F
489
1119
1368
2434

9.13
6.95
16.06
9.84
4.35
6.95
14.83
11.10

457.49
ND*
ND
2.2O
ND
ND
ND
914.10

*ND: not detected.

carbon sources [16]. The effect of the nitrogen source

is not clear since JA production by B. theobromae D7/2
was higher with sodium nitrate, potassium nitrate or
calcium nitrate [16], whilst JA production by Lasiodiplodia theobromae $22L was similar when organic or
inorganic nitrogen sources were used [18].
The objective of this work was to study the effect of
temperature, agitation rate and carbon and nitrogen
sources on JA and biomass production by B. theobromae in submerged fermentation.

lactose, sorbitol, starch and starch:sucrose (l:l)] and

nitrogen sources (NH4CI, (NH4)zHPO4, NH4NO~,
NaNO3, (NH4)2SO4, urea and yeast extract) on growth
and jasmonic acid production was carried out using
21.0 g carbon/I and 1.24 g nitrogen/l, respectively. Cultures were carried out over a 10 days period in static
submerged cultures at 30C.

Analytical methods
Biomass concentration was determined by dry weight
after broth filtration on filter paper Whatman 41
followed by drying at 60C for 24 h. Glucose and fructose were determined by HPLC (Binary LC Pump 250,
Perkin Elmer) with a refraction index detector (LC-30
RI, Perkin Elmer) [19]. Sulphuric acid 30 mM at flow
rate of 0.6 ml/min was used as mobile phase through a
Rezek Organic Acid (Phenomenex) column. Glucose
(Sigma) and Fructose (Supelco) were used as standards. JA concentration was determined by HPLC as
described by Koda [4]. Determinations were made with
an ultraviolet detector (UV/Vis Spectrometric Detector
LC 290, Perkin Elmer) using methanol: acetic acid
(60:0.1) as mobile phase at 0.85ml/min through a
Spherisorb ODS-2 (Phase Sep) column. ( + ) - J A
(Sigma) was used as standard.

Materials and methods

Microorganisms

Results and discussion

Eight strains of B. theobromae (Table 1) from the lnstituto Nacional de Investigaciones Fundamentales de la
Agricultura Tropical (Cuba) isolated from Cuban
Citrus cinensis Osbeck cv Valencia, were used. The
strains were stored on malt agar extract slants at 4C
and subcultured every 2 months.

Strain screening

Culture techniques
A sample of the stock culture was transferred to malt
agar extract plates and incubated for three days at
30C. Five loops of mycelium (7 mm diameter) were
used for inoculation of 25 ml of culture medium in
100ml Erlenmeyer flasks and incubated at different
temperatures
(25-35C)
and
agitation
rates
(0-150 rpm) as indicated.

Media composition
Culture media with the following basal salt composition were used (in g/l): sucrose, 50; NaNO3, 7.5;
KHzPO4, 2.0; KCI, 0.3; MgSO4.7H20,
0.6;
FeSO4.7H20, 0.6; ZnSO4.7H20, 0.03; MnSOa.7H20,
0.003; CuSO4.7H20, 0.003; NazMoO4.2H20, 0.003;
yeast extract, 1.0. After autoclaving initial pH was
adjusted to 5.5-5.6 with NaOH (1 M). The effect of
different carbon sources [sucrose, glucose, fructose,

Botryodiplodia theobromae is a phytopathogenic fungus

common in tropical countries, capable to produce JA
and JA-like substances at commercial levels [3]. Eight
strains of B. theobromae isolated from cuban citrus peel
were screened for their capacity to produce JA on
submerged static culture at 30C. JA and biomass production are shown in Table 1. Although all the strains
tested grew at significant levels (4.3-16.1 g/l) JA was
only produced by strains 1, 2F and 2434. Strains with
lower growth (489 and 1119) and strains with higher
growth (2 and 1368) did not produce JA. Since strain
2434 produce the highest titers of JA after 10 days of
culture (Table 1) it was selected for further studies.
Previous studies also showed that JA production by
different B. theobromae strains was strain dependent [2,
14] under the same culture conditions. Maximal JA
concentrations of 500 [2] and 800mg/l [14] were
reported.

Effect of temperature
Temperature is an important factor for growth and
secondary metabolites production by microorganisms.
However, the maximum temperature for growth does

Jasmonic acid production hv B. theobromae

1000

. . . . . . . . . . . . . . .

800
,~

600

"-~ 4 0 0
200
0

25

27

29
Temperature

31

33

35

( o C)

Fig. 1. Effect of temperature on jasmonic acid production bv

Bot~odiplodia theobromae strain 2434 after 10 days of
culture.

717

glucose and fructose and completely depleted after

three days of cultivation (data not shown). In all cases,
JA production started once that growth reached the
statkmary phase [Fig. 2(b)]. Two different profiles of
JA production were observed. In the static submerged
culture (0 rpm) JA production started after 4 days of
cultivation reaching a maximum value of 900 mg/I after
12 days o! cultivation. Agitated cultures presented a
different JA production profile. JA concentration
reached maximal values of 351}, 300 and 200 mg/l at 50,
11}0 and 151}rpm, respectively after 8 days of culture
then JA concentrations strongly decreased at concentrations lower than 51) mg/I at 12 clays. Reduction in JA
concentration might be related to the consumption of
JA due to carbon limitation or to changes on pH in thc
culture medium (see below). However, since maximum
biomass concentration at 0 and 150 rpm was similar,

2O

not always correspond to the maximum for secondary

metabolitcs production [2[)]. Fig. 1 presents the
maximum JA concentration values obtained at different cultivation temperatures (25-35C). JA production occurred at all range of temperature assayed.
However, maximal JA concentrations were produced at
30-32C. Studies carried out on surface culture
demonstrated that the maximum growth of B. theobromae 2434 was attained at 30C (results not shown).
Therefore, maximal biomass and JA production was
obtained at the same temperature (30C). This result
agrees with previous studies. JA production by B theobromae D7/2 was maximum at 27-30C [16], whilst
maximal biomass and JA production were obtained at
25C by L. theohromae cultured in surface culture [18].

Even though the effect of aeration-agitation is pronounccd in JA production in submerged culture,

kinetic studies related to the JA production are not
available [ 15, 16]. A typical time course cultivation of B.
theobromae 2434 in shake flasks at different agitation
rates (0-150 rpm) for biomass, JA production and pH
evolution is shown in Fig. 2. In all cases, growth
reached a stationary phase after 4 days of culture,
followed by a decrease in the biomass concentrations
[Fig. 2(a)]. Biomass production increased with agitation rate up to 100 rpm. At 150 rpm maximum biomass
concentration (11 g/l) corresponded to the 65% of the
biomass produced at 100rpm. The reduction in
biomass concentration after 4 days of culture was
enhanced bv the stirring speed. Although biomass concentrations at 4 days of culture were strongly
dependent on agitation (12.5 g/1_+26%), similar values
(10.84g/1+12%) were obtained at 12 days of cultivation. The decrease in biomass concentration was considerably low at () and 15(I rpm. Sucrose was invcrtcd to

"
O

s
i

__.AAA

1000

B
0

800

Effect ql" agitation

.:

.=

<

6 O0
0

400

200

10
c

0
0

I~,a
A

4
i
i
2

0
0

Time

(days)

10

12

Fig. 2. Kinetics of cell growth (A), jasmonic acid production

(B) and pH (C) by Botryodiplodia theobromae strain 2434
grown in shake flasks at different agitation rates: 0 rpm ( ) ,
5(I rpm (tt), 1!)0rpm (A) and 1511rpm (e).

E Eng, M. Guti&rez-Rojas, E. Favela-Torres

718

disappearance of JA from the culture broth could be

related to final pH. Profiles of pH at different agitation
rates are shown in Fig. 2(c). pH decreased from 5.5 to
3.75 after 2 days of cultivation. Thereafter it slowly
increased throughout the cultivation up to 6 and 7 for
culture with agitation and to 8.8 for culture without
agitation. Increase of pH could be related to the depletion of nitrate ion by B. theobromae 2434 in the culture
medium generating an alkaline pH by ionization of
sodium ion. B. theobromae only biosynthesizes the cis
(+)-7-iso-JA isomer, 3 however, at pH values higher
than 7 the cis ( +)-7-iso-JA isomerizes to the trans( - ) JA isomer, resulting in an equilibrium of about 95:5
[trans(-)-JA: cis( +)-7-iso-JA] [5]. Additionally, in the
cis(+)-7-iso-JA the side-chains are oriented being
more unstable than the trans-oriented isomer. Thus, at
pH values below 7 the cis( + )-7-iso-JA might be assimilated by B. theobromae. This approach might be of
interest for JA production in bioreactor under controlled conditions (pH, stirring and aeration rate)
because biomass production can be favoured during
the first step of the culture (high aeration rate) and JA
production enhanced by reducing the aeration rate and
controlling pH at alkaline values to maintain the transoriented isomer.
The effect of agitation rate on biomass yield production (Yx/s, g biomass/g sucrose) and JA production
related to biomass (Y,~A/x, g JA/g biomass) is shown in
Fig. 3. Yx/s reached a maximum value of 0.32 g/g at
100 rpm. However, it decreased below 0.22 g/g at 150.
YJA/x decreased from 0.018 to 0.006g/g at agitation
rates ranging from 0 to 150 rpm, attaining a minimum
of 0.004g/g at 100rpm. Reduction in the Yx/s at
150rpm might be related to the production of an
extracellular polysaccharide produced at high agitation
rates, probably due to high dissolved oxygen concentrations. Increase in the viscosity of the broth in the agitated cultures was also observed in this work, Gfinther

0.40

0.02

0.016
0.30
0.012
0.20
0.008

t3

0.10

0.004

0.00
0

50

tO0

0
150

rpm

Fig. 3. Effect of the agitation rate on y,.~, (I) and YJA/x (D) in
jasmonic acid production during the growth of Botryodiplodia
theobromae strain 2434 in shake flasks.

Table 2. Effect of different carbon sources on jasmonic acid

production, final pH and biomass concentration by Botryodiplodia theobromae strain 2434
Carbon source

Biomass (g/l)

JA (mg/l)

Final pH

Dextrose
Fructose
Lactose
Sorbitol
Starch
Starch + sucrose
Sucrose

10.73
15.54
13.06
23.23
17.38
13.40
9.06

1136.6
1273.2
125.6
173.6
403.7
914.0
910.0

8.40
7.51
5.96
6.79
7.99
7.63
7.21

et al. [15, 16] reported that JA production with b. theobromae D7/2 was reduced when the culture was agitated above 190 rpm by the simultaneous synthesis of
an extracellular polysaccharide. The decrease of Y~/s at
150 rpm might also be related to the synthesis of other
metabolites such as indoles [2], curcubic acid,
(+)-9,10dihydro-7-isojasmonic acid and (+)-ll,12-didehydro7-isojasmonic acid [21].
Effect of carbon sources
The influence of different carbon sources on the production of biomass and JA is shown in Table 2. All the
carbon sources tested were used for growth by B. theobromae 2434 and JA was produced at different levels.
There was an inverse correlation between biomass and
JA production. Low growth and high JA production
was obtained with glucose and sucrose as carbon
sources. The lowest JA production was obtained with
carbon sources (sorbitol and starch) that allowed better
growth. The highest and lowest JA production were
obtained with fructose and lactose as carbon source. It
was previously reported that glucose or sucrose could
be used as sole carbon source for JA production with
B. theobrornae D7/2 [16]. Moreover, Broadbent et al.
[18] reported that sucrose, glucose, glycerol or a
mixture of these as carbon sources allowed higher JA
production by L. theobromae strain $22L.
The high JA production observed in this work with
fructose and dextrose could be related to the fact that
in order to be assimilated, these substrates do not
require previous enzymic hydrolysis as required for
sucrose, starch or lactose assimilation. Novaratnam et
al. [22] working with B. theobromae IMI 334891 in
manioc starch medium supplemented with salts found
that maximum glucoamylase activity was obtained in
shake flasks at 160 rpm and pH 6.0. Nevertheless, a
combination of two carbon sources with different
uptake rate as starch/sucrose yielded higher JA concentration. Cultures with final pH values below 7.0
(lactose and sorbitol) exhibited the lowest JA production (125.6 and 173.6 g/I, respectively).

Jasmonic acid production hv B. theobromae

1000

Table 3. Effect of nitrogen sources on jasmonic acid production, final pH and biomass concentration by Botryodiplodia
theobromae strain 2434
Nitrogen source
NH4CI
(NH~)2H PO4
N H4NO~

(NH4)2804
NaNO~
Urea
Yeas! extract

Biomass (g/l)
6.73
6.54
I0.18
4.66
9.06
11.73
10.51

JA (rag/l)
1.8
2.2
28.2
ND*
910.0
75.0
405.0

Final pH
1.95
2.31
5.72
2.10
7.21
5.49
6.56

71tt

A
800

g
g
<_,

600

400

200

--

--

4.

--

10

Effect ()1~nitrogen sources

12

.X.._._.._._._4,------.---

Different nitrogen sources were tested for biomass and

JA production in submerged cultures of B. theobrornae
2434 (Table 3). Ammonium salts, as sole nitrogen
sources, [NH4CI, (NH4)2HPO4 and (NH4)2SO4] produced the lowest final biomass and JA concentrations.
Depletion of ammonium ion by B. theobromae strain
2434 generated an acidic pH by ionization of CI
HPO~ and SO,] that might be responsible for low
growth and JA production.
The use of NH4NO3 as nitrogen produced a similar
final biomass concentration (10.18g/l) that the
obtained with urea (11.73g/1) and yeast extract
(1(t,51 g/I). The effect of final pH on final biomass and
JA concentrations is presented in Fig. 4. JA production
was strongly affected by the final pH [Fig. 4(a)]. At pH
values higher than 6, JA concentration was considerably higher than the obtained at lower pH values. The
lack of stability of the cis (+)-7-iso-JA isomer was
discussed earlier. Organic nitrogen sources as yeast
extract and urea did not produce better JA production
than sodium nitrate. Gfinther et al. [16] reported that
inorganic salts as sodium, potassium or calcium nitrate
were the best sources for JA production with B. theobromae strain D7/2 allowing JA concentration up to
800mg/l with sucrose or glucose as carbon source.
However, Broadbent et al. [18] found that JA production was independent of the nitrogen source used.
Thus, it appears that the effect of pH on JA production is more important than the nature of the nitrogen
source used. Studies with a bioreactor under controlled
conditions must be carried out in order to evaluate the
effect of pH, at constant values, on JA production.
Final biomass concentration was slightly affected by
final pH values [Fig. 4(b)]. Higher biomass concentrations were obtained at pH values above 5. It has been
rcported that B. theobromae shows good growth in the
pH range 3-10. Hewitt et al. [23] observed two maxima
of growth (4.5-5 and 7.1) in a strain of B. theobromae
in Czapek medium supplemented with yeast extract.
Gabr et al. [24] isolated two strains of B. theobromae
growing in a pH range of 3-9 with maximum growth at
pH 4-8. More recently, Yaguchi and Nakamura [25]

=.
E
.o

0
4

Final pH

Fig. 4. Effect of final pH on (A) jasmonic acid and (B)

biomass production in media with different nitrogen sources.
NH4CI (m), (NH4)2HPOa (4,), NH4NO~.(x), NaNO~ (e), Urea
(A) and yeast extract (+).

reported growth of L. theobromae without significant

differences in potato dextrose agar medium at pH
values ranging from 4 to 10.

Conclusions
Highest JA production was observed at temperature
values from 30 to 32C. JA concentration decreased
and biomass production increased when the culture
media was agitated from 0 to 150rpm, the highest
biomass production was obtained at 100 rpm. JA production was higher with fructose and dextrose as
carbon sources and sodium nitrate as nitrogen source.
However, studies of JA production must be carried out
in completely controlled bioreactors in order to stablish
the effect of culture conditions (pH, aeration and stirring) on growth and JA production yield and
productivity.

Acknowledgements
This work was supported in part by a project of
Consejo Nacional de Ciencia y Tecnologfa (Mexico)
and a grant to F. Eng by Third World Academy of
Sciences. We grateful to Dr Rafael Castafieda (lnsti-

720

E Eng, M. Guti6rrez-Rojas, E. Favela-Torres

tuto Nacional de Investigaciones Fundamentales de la

Agricultura Tropical) for providing us with strains of B.
theobromae.
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