A new oil-based formulation of Trichoderma asperellum for the biological control of cacao black pod disease caused by Phytophthora megakarya

Full text

A new oil-based formulation of Trichoderma asperellum for the biological
control of cacao black pod disease caused by Phytophthora megakarya
J.B. Mbarga
a,b,1
, B.A.D. Begoude
a
, Z. Ambang
b
, M. Meboma
a,b
, J. Kuate
a
, B. Schiffers
c
,
W. Ewbank
d
, L. Dedieu
e
, G.M. ten Hoopen
a,f,
⇑
a
Laboratoire Régional de Lutte Biologique et de Microbiologie Appliquée, Institut de Recherche Agricole pour le Développement (IRAD), B.P. 2067, Yaoundé, Cameroon
b
Département de Biologie et Physiologie Végétale, Faculté des Sciences, Université de Yaoundé 1, B.P. 812, Yaoundé, Cameroon
c
Gembloux Agro-Bio Tech/Université de Liège – Laboratoire de Phytopharmacie – Passage des Déportés 2, B-5030 Gembloux, Belgium
d
Ajinomoto OmniChem S.A, Rue Fonds Jean Pâques, 8 B-1435 Mont-St-Guibert, Belgium
e
CIRAD, DGD-RS – Dist, F-34398 Montpellier, France
f
CIRAD, UPR Bioagresseurs, F-34398 Montpellier, France
highlights

New oil-based dispersion with a half-
life of T. asperellum conidia of
22.5 weeks.

Complete inhibition of P. megakarya
on sprayed detached pods.

Enhanced rate and duration of
protection on sprayed cacao pods in
the field.

50% of pods protected 3.2 weeks after
spraying in the field.

Formulation suitable for the spraying
equipment of small-scale cacao
producers.
graphical abstract
Formulated conidia of Trichoderma provide similar or even better protection of cacao pods against black
pod disease than a conventional fungicide.
0
20
40
60
80
100
01234567
Protecon level (%) of cacao pods against
Phytophthora megakarya
Time (weeks)
Trichoderma asperellum conidia in oil formulaon Kocide
Oil formulaon without conidia Trichoderma asperellum conidia in water
Water only control
article info
Article history:
Received 27 February 2014
Accepted 5 June 2014
Available online 13 June 2014
Keywords:
Oil dispersion
Formulation
Phytophthora megakarya
Cacao black pod disease
Trichoderma asperellum
Biological control
abstract
In African cacao-producing countries, control of cacao black pod disease caused by Phytophthora
megakarya is a priority. Introducing biological control agents as part of a P. megakarya control strategy
is highly desirable, especially in a perspective of pesticide reduction. Trichoderma species are among
the most used biological control agents. In Cameroon, Trichoderma asperellum formulated in wettable
powder has produced positive effects against this disease. However, with this type of formulation,
shelf-life and persistence of conidia on pods are limited. Our study therefore sought to develop a new
T. asperellum formulation that would be more effective and better suited to the conditions of field appli-
cation by small-scale producers in Cameroon. We selected a soybean oil-based oil dispersion, in which
the half-life of the conidia reached 22.5 weeks, versus 5 weeks in aqueous suspension. Tested on
detached pods, the formulation completely inhibited the development of the disease. When sprayed in
the field on cacao clones highly sensitive to P. megakarya, the formulation resulted in 90% protection
of treated pods after 1 week, and 50% after 3.2 weeks. The formulations exercised a measurable effect
for up to 7 weeks, versus 2 weeks in the case of an aqueous conidial suspension and 5 weeks for that
of a conventional fungicide (Kocide). Trichoderma asperellum formulated in oil dispersion has therefore
great potential for the control of cacao black pod disease with less recourse to synthetic fungicides.
http://dx.doi.org/10.1016/j.biocontrol.2014.06.004
1049-9644/Ó2014 Elsevier Inc. All rights reserved.
⇑
Corresponding author. Address: CIRAD Afrique Centrale, B.P. 2572, Yaoundé, Cameroon.
E-mail address: tenhoopen@cirad.fr (G.M. ten Hoopen).
1
Deceased.
Biological Control 77 (2014) 15–22
Contents lists available at ScienceDirect
Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Moreover, this formulation is well adapted to the types of sprayers used by small-scale cacao producers
in Cameroon.
Ó2014 Elsevier Inc. All rights reserved.
1. Introduction
Cacao, Theobroma cacao L., is one of the most important cash
crops in numerous developing countries. World production of mer-
chantable cacao, estimated around 4.3 million tons (ICCO, 2011),
originates from 5 to 6 million producers and is essential to the live-
lihood of 30–40 million people throughout the world (World Cacao
Foundation, 2012). Africa’s two million small-scale producers
account for 75% of world production (ICCO, 2011).
Cacao black pod disease, caused by several species of the Stra-
minipile genus Phytophthora (Kroon et al., 2004; Tyler et al.,
2006) is the most common disease of cacao causing global yield
losses estimated at 30% (Guest, 2007). In Cameroon, the most
widespread species is Phytophthora megakarya Brassier & Griffin
(Mfegue, 2012), which is endemic to West Africa and present in
all four of the largest cocoa producing countries in this region,
Ivory Coast, Ghana, Nigeria and Cameroon (Guest, 2007). Cacao
pods can be attacked all year round at all development stages.
However, high-incidences occur during rainy seasons (Guest,
2007). In the absence of any crop protection measures, P. megak-
arya can sometimes cause up to 80–100% yield loss (Ndoumbe-
Nkeng et al., 2004). In African cacao-producing countries, control
of cacao black pod disease is thus both a necessity and a priority.
Current control approaches are mainly based on strategies involv-
ing a mix of cultural and chemical control means, improved agri-
cultural practices, the use of partially resistant and/or tolerant
varieties, and biological control methods. Yet, for many, spraying
of fungicides remains their principal control method.
Successes in biological control, using antagonistic agents against
plant pathogens, have led to the manufacture and registration of a
number of biological control products (Fravel, 2005; Kaewchai
et al., 2009). Several Trichoderma species, such as Trichoderma
asperellum, T. harzianum, T. polysporum, T. viride and T. virens have
already been used successfully as biological control agents against
a variety of phytopathogenic fungi (Almeida et al., 2007; Benitez
et al., 2004; Harman et al., 2004; Hermosa et al., 2013; Kaewchai
et al., 2009). Trichoderma species are also being developed as biolog-
ical control agents of cacao diseases such as witches’ broom (caused
by Moniliophthora perniciosa), frosty pod rot (caused by Monilioph-
thora roreri) and black pod disease (caused by Phytophthora spp.)
(e.g. De Souza et al., 2008; Krauss et al., 2006, 2010; Tondje et al.,
2007). In the case of Witches’ broom this has even led to the
development of a registered biological control product, called
Trichovab
Ò
(Samuels, 2004), based on Trichoderma stromaticum,
being used in a Brazilian government funded program to control
M. perniciosa.
In Cameroon, research efforts to develop a biological control
strategy against P. megakarya resulted in the selection of mycopar-
asitic strains of T. asperellum (Tondje et al., 2007). These strains
were formulated as wettable powders, using cassava flour as car-
rier, and have been tested in field trials (Deberdt et al., 2008;
Tondje et al., 2007). The results of these trials, although relatively
positive, also showed limitations of the formulation being used,
such as a high susceptibility to wash-off due to rain and fast desic-
cation of the conidia when applied to cacao pods (Deberdt et al.,
2008).
Improving the conidial formulation of T. asperellum is there-
fore an essential step towards optimization of this biological con-
trol strategy. Bateman and Alves (2000), recommend oil-based
formulations for conidial biological control formulations because
of their greater ability to adhere to the substrate. Moreover, such
formulations slow down the desiccation process under conditions
of fluctuating environmental factors such as temperature and rela-
tive humidity. Therefore, this study focused on assessing the feasi-
bility of using oil dispersions as a carrier matrix for conidial
formulations of T. asperellum. The objective is to prolong shelf-life
and persistence of the biological control agent on the biological
target and thus improve the protection of cacao pods against P.
megakarya. The findings will help to optimize the practical use of
T. asperellum in the biological control of cacao black pod disease.
2. Materials and methods
2.1. Fungal material
All experiments were performed with P. megakarya strain EL1.
This strain was isolated from a naturally-infected cacao pod,
collected in a trial plot in Eloumden (Yaoundé, Cameroon). Strain
PR11 of T. asperellum [Genebank number EF186002] was isolated
in Cameroon from a soil sample taken from within the rhizosphere
of cocoyam plants (Xanthosoma sagittifolium) with symptoms of
root rot and conserved in national and international collections
as described in Begoude et al., (2007). This strain was characterized
molecularly (Samuels et al., 2010), and its antagonistic activity in
relation to P. megakarya and Pythium myriotylum – the pathogens
respectively responsible for cacao black pod disease and cocoyam
root rot disease – was ascertained (Mbarga et al., 2012; Tondje
et al., 2007).
Mass production of T. asperellum PR11 conidia was done follow-
ing the method described by Hanada et al. (2009). Conidia were
produced using a solid state fermentation process with rice as
the substrate. Conidia were extracted from their growth substrate
using a mycoharvester, version V (http://www.dropdata.net/
mycoharvester).
2.2. Formulations
2.2.1. Carrier additives
To develop the formulation matrix, several carrier additives of
four different types were tested: (1) two vegetable oils (palm oil
and soybean oil), (2) five emulsifying-dispersing agents (Tensiofix
NTM, DB08, IW60, OC653, and Tween 20), (3) one structural agent
(Tensiofix 869) and (4) one source of carbon (glucose). The Tensio-
fix agents were provided by the agrochemical company S.A.
Ajinomoto OmniChem N.V. (www.tensiofix.com). All tested emul-
sifying-dispersing agents are liquid between 20 and 35 °C, have a
0.5% water content and are non-ionic, with the exception of
Tensiofix IW60, which is anionic. Tensiofix 869 is a bentonite clay
that comes as a powder with a maximum water content of 10%.
2.2.2. Preparation of the oil dispersions
The compositions of the different oil dispersions tested are
detailed in Table 1. They were prepared by first mixing the oil with
the emulsifying-dispersing agent, and then adding the structural
agent and the glucose and where applicable, finally the water.
The T. asperellum conidia were then incorporated progressively.
The mixture was homogenized using a Diax 900 homogenizer
(Heidolph Co., Germany) for 10 min at 5000 rpm. All formulations
16 J.B. Mbarga et al. / Biological Control 77 (2014) 15–22

had a final conidial concentration of 2.7 10
7
conidia ml
1
(Table 1). The prepared formulations were each divided over ten
test tubes and stored at 25 ± 2 °C in a room with 30% relative air
humidity.
2.2.3. Characterization of the oil dispersions
The pH and viscosity of each formulation were measured for
each of ten test tubes, every day for ten consecutive days, using a
pH-meter and a viscometer (VT-03 viscometer, Rion Co. Ltd.,
Japan).
An emulsification test was carried out to evaluate the stability
of the formulations when mixed with water. Graduated glass test
tubes were filled with 95 ml of water of moderate pH (CIPAC stan-
dard water D with 342 ppm CaCO
3
) and kept in a 30 °C bain-marie
for 24 h. Subsequently, 5 ml of a formulation was added and
homogenized manually. The homogeneity of the resulting mix-
tures and the degree of sedimentation of the conidia were assessed
every day for 10 days. Ten test tubes were used for each formula-
tion, and the experiment was replicated three times.
A formulation was considered stable if the different variables
monitored remained constant throughout the ten-day-long exper-
iment. The formulation with the best stability and lowest viscosity
was selected.
2.3. Shelf-life of T. asperellum conidia in the formulation
The viability of T. asperellum conidia in the formulation and in
aqueous suspension was assessed over a period of 35 weeks. Each
week, a sample was diluted 10
4
-fold and 1 ml was spread in five
Petri dishes containing Potato Dextrose Agar (PDA) (Difco Becton
Dickinson, Sparks, MD) culture medium. The control was a 1:10
4
dilution of the aqueous suspension of pure T. asperellum conidia
(2.7 10
7
conidia ml
1
). After 12 h at 25 ± 2 °C, three sample areas
of 1 cm
2
were delimited within each Petri dish and examined
under a light microscope. The number of germinated conidia out
of a sample of 100 conidia per sample area examined was recorded.
A conidium was considered germinated when its germ tube
exceeded the conidium’s diameter. Incubation was pursued to fur-
ther ascertain the growth potential of the formulated PR11 strain.
2.4. Effect of the carrier additives on the in vitro growth of
P. megakarya
The poisoned food technique (Shekhawat and Prasad, 1971)
was used. A V8 culture medium (200 ml V8 juice, 15 g agar, 3 g cal-
cium carbonate and distilled water qsp 1000 ml) was mixed with
10% v/v of the selected formulation, yet without T. asperellum
conidia, and used to prepare a set of Petri dishes. All formulation
constituents were sterilised separately before mixing them as
described in Section 2.2.2.
A 5 mm diameter implant from a five-days-old P. megakarya
culture was then deposited in the middle of each amended Petri
dish, with the mycelium-covered side in contact with the cul-
ture medium. A total of 30 replicate plates were incubated at
25 ± 2 °C for 7 days. Mycelial growth of P. megakarya was assessed
by measuring every day the diameter of each colony along two pre-
defined perpendicular axes. Control dishes contained P. megakarya
cultured on unamended V8 medium. The data recorded were used
to calculate the radial growth rate of each colony, expressed in
mm day
1
, based on the slope of the linear regression of the
growth curve (Begoude et al., 2007).
2.5. Protective effect of the formulation on detached cacao pods
A detached cacao pod test was carried out based on the method
for evaluating the resistance of cacao clones to Phytophthora
palmivora in the laboratory, as developed by Iwaro et al. (2000).
The test was undertaken using three cacao clones: SNK10, highly
sensitive to cacao black pod disease (Efombagn et al., 2011), and
BBK1606 and SNK630, respectively moderately sensitive and toler-
ant (unpublished data). The effectiveness of the treatment with the
selected formulation was compared with four other treatments: (i)
the selected formulation without conidia, (ii) an aqueous
suspension of conidia adjusted to a concentration of 2.7 10
7
conidia ml
1
, (iii) a water-only control treatment, and (iv) a con-
ventional fungicide treatment using Kocide 2000 (a.i. copper
hydroxide) (LDC Cameroun, BP 2368, Douala, Cameroon). The pods
of the various cacao clones were first washed in water, and each
was sprayed with one of the different treatment liquids with a
hand held pressurized sprayer (mean volume used: 5 ml of formu-
lation per pod). Treated pods were deposited in trays lined with
absorbent paper and left to dry at 25 ± 2 °C for 2 h. Each one was
then sprayed with 200
l
l of a suspension of P. megakarya
zoospores of the EL1 strain, adjusted to a concentration of 3 10
5
zoospores ml
1
. Zoospores of P. megakarya were obtained using the
protocol as described by Tondje et al. (2006). The absorbent sheet
of paper lining each tray was moistened using sterile distilled
water and the trays were covered in order to create a favorable
environment for the development of the disease. Four pods per
clone and per treatment were used, and the experiment was repli-
cated three times. The pods were left to incubate at 25 ± 2 °C for
4 days. The severity of the infection was scored according to the
0–7 scale developed by Iwaro et al. (2000): 0 – no visible lesion;
1 – 1 to 5 localized lesions; 2 – 6 to 15 localized lesions; 3 – over
15 localized lesions; 4 – 1 to 5 expanding lesions; 5 – 6 to 15
expanding lesions; 6 – over 15 expanding lesions; 7 – coalesced
lesions. Each pod was thus awarded a sensitivity score. For each
treatment, the rate of inhibition of P. megakarya development
was calculated according to the following formula:
Ið%Þ¼ 1Cn
Co

100
With Ithe mean inhibition rate in%, Cn the mean sensitivity
score of the treated pods, and Co the mean sensitivity score of
the water only control pods. This was done for individual clones
as well as for pooled data.
2.6. Protective effect of the formulation on cacao pods in the field
The trial was set up in September 2012 in a clonal cacao plot at
the IRAD Nkolbisson research station (Ten Hoopen et al., 2012),
and was monitored for a period of 7 weeks. This relatively homoge-
neous cacao plot is made up of two sub-plots. Cacao trees in each
Table 1
Composition (w/w) of the six different oil dispersions tested.
Ingredients of formulations Formulations
123456
Palm oil 0 0 74% 74% 0 71%
Soybean oil 74% 74% 0 0 71% 0
Tensiofix NTM 15% 15% 15% 15% 0 0
Tensiofix DB08 0 0 0 0 9% 9%
Tensiofix IW60 0 0 0 0 5% 5%
Tensiofix OC653 0 0 0 0 1% 1%
Tween 20 0 5% 0 5% 0 0
Tensiofix 869 5% 0 5% 0 0 0
Glucose 4% 4% 4% 4% 4% 4%
Water 0 0 0 0 24% 24%
Pure T. asperellum
Conidia 2% 2% 2% 2% 2% 2%
J.B. Mbarga et al. / Biological Control 77 (2014) 15–22 17

sub-plot were planted in 2001. Spacing between individual trees is
2.5 m 2.5 m and maximum tree height is 3 m. Separate clones
are planted along lines of approximately 30 m. Black pod disease
has not yet been recorded in this particular plot. For this trial,
500 four-months-old pods were selected from 20 cacao trees of
clone SNK10, 10 trees in each of the two sub-plots.
The effectiveness of the oil-based formulation was compared
with the other four treatments described in Section 2.5. Each treat-
ment was applied to 100 pods. Before application, each pod was
wiped clean with 70% ethanol, left to air dry and subsequently
sprayed with 10 ml of treatment liquid to incipient run-off using
a hand held pressurized sprayer. For each group, every week for
7 weeks, 10 healthy pods were chosen randomly and brought to
the laboratory to assess the protective effect against P. megakarya.
For this, a droplet of 10
l
l of a suspension of zoospores of the EL1
strain of P. megakarya adjusted to 3 10
5
zoospores ml
1
were
deposited onto the middle of the pod. After 4 days of incubation
at 25 ± 2 °C in trays lined with moistened absorbent paper, the
number of healthy pods were counted for each treatment, and
the corresponding protection rate was calculated by dividing
the number of healthy pods by the total number of pods and
multiplying by 100.
2.7. Data analysis
The evolution of the germination rate of both the formulated
and non-formulated (aqueous suspension) conidia was analyzed
using Curve Expert software, version 9.0. Data relating to the effects
of the carrier additives were arc-sine-transformed prior to statisti-
cal analyses. The pod severity scores were log(n+ 1) transformed
prior to analysis. The analyses of variance (ANOVA) were per-
formed using SAS software, version 9.1 (SAS Institute Inc., 2007).
Whenever significant, Duncan’s multiple range test was used to
differentiate between means.
3. Results
3.1. Selection of the most appropriate oil dispersion
The physico-chemical characteristics of the six oil dispersions
tested are shown in Table 2. When formulations 1 and 3 were
mixed with water, they remained homogeneous, of a creamy-
white color, and no sedimentation of the T. asperellum conidia
other than traces was recorded during the ten-day-long experi-
ment. In contrast, when the other four tested formulations were
diluted in water and homogenized, ten minutes later the phases
had separated and the T. asperellum conidia had all sedimented.
In all formulations, pH values remained constant. Formulation 1
differed from formulation 3 in that it exhibited lower viscosity.
On the basis of these physico-chemical characteristics, formulation
1 was the only one that could be considered as a good emulsion.
This formulation was therefore selected for subsequent use.
3.2. Shelf-life of T. asperellum conidia in formulation 1
The germination rate of the conidia suspended in formulation 1
was 90.6 ± 2.3% after 1 week, and 20.9 ± 2% after 35 weeks. In con-
trast, that of the conidia in aqueous suspension was 92.4 ± 2.1%
after 1 week, fell to less than 5% after just 10 weeks and was down
to zero after 17 weeks. In formulation 1, the half-life of T. asperel-
lum conidia (the time after which 50% of conidia were still able
to germinate) was estimated at 22.5 weeks (Fig. 1). For the aqueous
suspension, this point was already reached after 6 weeks. After
each germination test, the viability of the T. asperellum conidia
was further confirmed by the fact that petri-dishes became
Table 2
Characteristics of the six tested oil dispersions.
Characteristics of formulations Formulations
12 345 6
pH 6.1 5.2 5.3 5.2 6 5.4
Viscosity (cps)
a
106 ± 14 208.61 ± 10 3849 ± 8 90 ± 15 1620 ± 35 9250 ± 25
Conidial sedimentation
b
Traces Total Traces Total Total Total
a
Viscosity measured with a viscometer. Viscosity remained practically constant from day 0 to day 10. Figures shown are the means of 10 samples of formulation ± standard
deviation.
b
Conidial sedimentation was assessed every day for the 10 days that followed the preparation of the formulations.
Fig. 1. Germination level of T. asperellum conidia over time for formulated () and non-formulated conidia (N)ofT. asperellum PR11.
18 J.B. Mbarga et al. / Biological Control 77 (2014) 15–22

completely colonized. The linear regression equation that
described the decrease in germination rate of formulated conidia
over time is y= 95.1–2 x(R
2
= 0.99). The non-linear decrease in
germination rate of the conidia in aqueous suspension was best
described by the Farazdaghi-Harris’ model and was given by
y= 1/(0.01 + 5.8 10
6
X
4.3
)(R
2
= 0.99).
3.3. Effect of the carrier additives of formulation 1 on the in vitro
growth of P. megakarya
The effect of formulation components on in vitro growth of
P. megakarya is presented in Fig. 2. In the culture medium amended
with all carrier additives from formulation 1, the onset of P. megak-
arya mycelial development was delayed by 24 h. Interestingly, no
other effect on subsequent growth of the pathogen was observed.
The analysis of variance showed no significant difference
(P= 0.383) for P. megakarya growth rate, yet revealed a highly sig-
nificant difference (P=0.0001) regarding the y-intercept values of
the two regression lines. The linear regression equations that
described P. megakarya growth on the unamended and amended
growth media were y= 10.3X+ 3.8 (R
2
= 0.992) and y= 11.3X-11.4
(R
2
= 0.986), respectively.
3.4. Protective effect of formulation 1 on detached pods
Four days after inoculation with P. megakarya, all pods treated
with formulation 1 containing T. asperellum conidia were still free
of disease symptoms, regardless of the cacao clone used (Table 3).
For all other treatments, some P. megakarya lesions were found,
either restricted and localized or numerous and touching, depend-
ing on the treatment administered.
The analysis of variance regarding the severity of the symptoms
observed on the detached pods showed no differences between
replications (P= 0.394) However, significant differences between
cacao clones (P< 0.0001) as well as treatments (P< 0.0001) were
observed. Moreover, there was a significant (P< 0.001) interaction
between clone x treatment which would indicate that a treatment
would be more or less effective depending on the clone it is applied
to. Here, the interaction was due to the fact that clone BBK1606
was equally sensitive to the control treatment (water only) as
SNK10 which is considered to be more sensitive, and to the fact
that the formulation with T. asperellum conidia was 100% capable
of protecting pods, irrespective of their sensibility to black pod dis-
ease. However, overall results did indeed show that SNK10 is more
sensitive than BBK1606, and SNK630 is least sensitive, confirming
their relative susceptibility to black pod disease as mentioned in
Section 2.5. Treating the pods with formulation 1 including conidia
and carrier additives resulted in the complete inhibition of the dis-
ease (Table 3). This formulation was even more effective than the
synthetic fungicide used as positive control. The aqueous suspen-
sion of conidia as well as the carrier additives of formulation 1,
were also able to significantly inhibit the disease in comparison
with the water-only control, but formulation 1 was the only treat-
ment that fully protected the cacao pods.
Fig. 2. Effect of the carrier additives of formulation 1 on the mycelial growth of P. megakarya.
Table 3
Protective effect of formulation 1 on detached pods of three clones with differential susceptibility to P. megakarya.
Clone SNK10 Clone SNK630 Clone BBK1606 All pods
Treatment Pod sensitivity
score
a
Black Pod
inhibition
rate (%)
Pod sensitivity
score
Black Pod
inhibition
rate (%)
Pod sensitivity
score
Black Pod
inhibition
rate (%)
Pod sensitivity
score
b
Black Pod
inhibition
rate (%)
Formulation 1 with conidia 0 ± 0
a
100% 0 ± 0
a
100% 0 ± 0
a
100% 0 ± 0 a 100%
Kocide 0.8 ± 1.2
b
88.0% 0 ± 0
a
100% 0 ± 0
a
100% 0.3 ± 0.8 b 95.6%
Formulation 1 without conidia 1.9 ± 1.3
b
72.3% 0.3 ± 0.5
a
b
95% 1.0 ± 1.2
b
85.7% 1.1 ± 1.2 c 83.3%
Conidia in aqueous suspension 6.5 ± 0.7
b
c
6.0% 3.5 ± 1.6
b
30% 4.8 ± 0.5
b
32.1% 4.9 ± 1.6 d 22.0%
Water only control 6.9 ± 0.3
c
n.a.
d
5.0 ± 0
b
n.a. 7.0 ± 0
c
n.a. 6.3 ± 1.0 e n.a.
Mean clona l sensitivity score
c
3.2 ± 3.0 C n.a. 1.8 ± 2.2 A n.a. 2.0 ± 0.8 B n.a.
a
Pod sensitivity scores followed by the same greek small letter do not differ at P= 0.05 (Duncan).
b
Pod sensitivity scores followed by the same small letter do not differ significantly at P= 0.05 (Duncan).
c
Mean clonal sensitivity scores followed by the same letter do not differ significantly at P= 0.05 (Duncan).
d
n.a. = not applicable.
J.B. Mbarga et al. / Biological Control 77 (2014) 15–22 19

3.5. Protective effect of formulation 1 on cacao trees in the field
The results from the field experiment showed that 90 ± 4.5% of
the pods treated in the field with formulation 1 were still protected
1 week after spraying, with only 10 ± 5.5% of the pods exhibiting
symptoms of black pod disease. Regarding the other treatments,
after 1 week, the percentage diseased pods was 100% for the
water-only control, 73.3 ± 8.2% for the aqueous suspension of
T. asperellum conidia, 33.3 ± 8.7% for the treatment containing the
carrier additives of formulation 1 (without T. asperellum conidia),
and 16.6 ± 6.9% for Kocide 2000 (Fig. 3). The median lethal time
(LT
50
) – the time interval after which the applied product still pro-
vided a 50% protection of the pods – was approximately 3.2 weeks
for formulation 1. It was around 1.5 weeks for the carrier additives
(without conidia), and about 3 weeks for Kocide 2000. The protection
thereafter decreased progressively with time. The longest-lasting
protective effect – 7 weeks – was obtained with formulation 1.
4. Discussion
Our study is the first contribution to the development of an oil-
based formulation of T. asperellum conidia as a biological control
tool against P. megakarya, the causal agent of cacao black pod dis-
ease. The objectives of this study, to develop a formulation that
would increase shelf life and persistence on cacao pods of formu-
lated conidia and to improve protection against cacao black pod
disease have been attained. The formulation selected was com-
posed of soybean oil (74%), Tensiofix NTM (15%, an emulsifying-
dispersing agent), Tensiofix 869 (5%, a structural agent) and glu-
cose (4%). Our findings showed that the viability of T. asperellum
conidia as well as their persistence on the treated pods were
enhanced by this oil dispersion in comparison with the water sus-
pension. We also showed that the application of thus formulated
T. asperellum conidia in the field on pods of cacao clones highly
sensitive to the disease have the potential to improve both the rate
and the duration of the protection against P. megakarya in compar-
ison with the aqueous conidial suspension and with the synthetic
fungicide Kocide 2000.
About 90% of the formulations of antagonistic fungi for the bio-
logical control of plant diseases use species of Trichoderma, such as
T. harzianum, T. virens and T. viride (Kaewchai et al., 2009). In most
cases, the propagules are formulated as granules (Jin and Custis,
2011) or wettable powders (Fravel, 2005). Very few formulations
use vegetable oils. To our knowledge, invert or reverse emulsions
(of the water-in-oil type) are the only type of oil-based formula-
tions of Trichoderma spp. conidia known to have been used so far
(Batta, 2004, 2007; Wijesinghe et al., 2010, 2011). Our Trichoderma
conidia formulated as an oil dispersion is therefore very interesting.
Especially so, since it offers one important advantage compared with
reverse emulsions: it is better adapted to side level knapsack (SLK)
sprayers, which are the sprayers most commonly used by small-scale
farmers. The conidial formulation of T. asperellum as a soybean oil-
based oil dispersion mixes readily with water and the conidia remain
in stable suspension for a long time. This is not the case of the reverse
emulsions developed by Batta (2004), in which the oil and water
phases can easily be discriminated. The miscibility and stability of
the propagules in the formulation should facilitate their uniform dis-
tribution in the sprayer’s tank when the spray liquid is prepared and
the even colonization of the pods by T. asperellum during spraying.
Reverse emulsions are applied in ultra-low volumes (Batta, 2004)
with spraying equipments such as mist blowers or foggers, which
many small-scale producers do not have.
In our formulation, the half-life of T. asperellum conidia is
22.5 weeks, i.e. 18 weeks longer than conidia in aqueous suspen-
sion. This half-life figure is close to that obtained by Batta (2004)
with T. harzianum conidia formulated in reverse emulsion
(21 weeks). Several studies have found that formulated conidia
remain viable for longer periods than non-formulated conidia
(Guijarro et al., 2007; Larena et al., 2007; Wijesinghe et al., 2010,
2011). This prolonged viability is thought to be related to the spe-
cific role of the various ingredients of the formulation – in this case
glucose and soybean oil. The contribution of glucose to increasing
the viability of microbial propagules was documented by Guijarro
et al. (2007), who showed that amending a formulation with a
source of carbon such as 7.5% glucose made it possible to keep
Penicillium frequentans conidia alive for 12 months. Moreover, oils
may also help to boost conidial viability by providing the micro-
organisms with a food basis while regulating water availability
(Paau, 1998). In this respect, it has been shown that soybean oil
is among the vegetable oils that confer the best viability to
Beauveria bassiana conidia (Mola and Afkari, 2012).
In the laboratory, treating detached cacao pods with our formu-
lation completely inhibited the development of P. megakarya,
whereas lesions of various sizes were observed on pods treated
Fig. 3. Protective effect over time of formulation 1 () on cacao pods in the field compared with a fungicide (j), the formulation without conidia (N), conidia in aqueous
suspension () and a water only control ( ).
20 J.B. Mbarga et al. / Biological Control 77 (2014) 15–22

with the other products, including Kocide 2000. The carrier addi-
tives used in our formulation may contribute to this protective
effect, since they alone accounted for a 24 h delay in the onset of
P. megakarya development in vitro and for a 83% reduction of dis-
ease development in comparison with the water-only treatment.
The carrier additives included in the formulation are in theory inert
ingredients in that they exhibit no capacity to control the disease
in themselves. However, they may constitute a physical barrier
to the infectious zoospores, and they also play a role in enhancing
the effectiveness of the formulation through improving conidial
viability, target coverage and wettability (Fravel, 2005).
Even though an interaction was observed between treatment
and cacao pod sensibility to black pod disease, the results shown
here confirm findings by Ten Hoopen et al. (2003) in that biological
control and genetic disease resistance should have additive effects
when both control options are applied simultaneously.
In the field, our formulation demonstrated its protective effect
on the pods of cacao clones known for their susceptibility to the
disease. One week after the treatment, 90 ± 4.5% of the pods were
still protected. Our formulation still had 50% effectiveness after
3.2 weeks. This duration of action is practically the same as that of
the Trichoderma ovalisporum conidial formulation used to control
M. roreri, the causal agent of cacao frosty pod rot disease (Krauss
et al., 2010). It is also similar to that of the systemic fungicides com-
monly employed by cacao producers in Cameroon (3 weeks). How-
ever, it is somewhat longer than that of contact fungicides such as
Kocide 2000, which in practice demand a fortnightly application
(Sonwa et al., 2008). Persistence probably results from the ability
of Trichoderma species – when appropriately formulated – to colo-
nize the cortex of the cacao pods, even when exposed to direct sun-
light (Krauss et al., 2006; Ten Hoopen et al., 2003). However,
effectiveness was primarily determined in the laboratory using arti-
ficial inoculations with the pathogen. Determining the effectiveness
of the formulation and the persistence and viability of the T. asperel-
lum PR11 spores under actual farmer-field conditions necessitates
field trials, which are currently underway.
In conclusion, we selected an oil dispersion of T. asperellum con-
idia that is both stable and homogeneous, mainly constituted of
soybean oil. This formulation has a positive effect on the viability
and persistence of the conidia. Tested on detached pods, the for-
mulation completely inhibited the development of the disease.
When sprayed in the field on cacao tree clones highly sensitive
to P. megakarya, the formulation performed better than a synthetic
fungicide in terms of rate and duration of the protection. This
T. asperellum in oil dispersion holds therefore potential for the con-
trol of cacao black pod disease, making it possible to rely less heav-
ily on costly and environmentally hazardous synthetic fungicides.
This biological control approach is interesting from an environ-
mental perspective, but also for the small-scale cacao producers of
Cameroon, with the added advantage that our formulation is per-
fectly suited to the types of sprayers they use.
Acknowledgments
This study was carried out with the financial and logistical
support of the agrochemical company OmniChem S.A. (a division
of Ajinomoto) and CIRAD. The authors were free to publish any
or all results originating from their experiments. They wish to
thank IRAD, on whose premises the totality of the research work
took place. They also wish to thank Anya Cockle-Bétian for the
translation from French to English of this article.
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